OA21037A

OA21037A - Mcl-1 inhibitor antibody-drug conjugates and methods of use.

Info

Publication number: OA21037A
Application number: OA1202100526
Authority: OA
Inventors: Olivier Geneste; Andras Kotschy; Szabolcs SIPOS; Matthew T. Burger; Maia Chanrion; Frédéric COLLAND; Marton Csekei; Lea DELACOUR; Patrice Desos; Jean-Michel Henlin; Vesela KOSTOVA; Ana Leticia MARAGNO; Eric MCNEILL; Mark G. Palermo; Francesca ROCCHETTI; Jérôme STARCK; Bing Yu; Qiang Zhang; Zhuoliang Chen; Katsumasa Nakajima
Original assignee: Novartis Ag; Les Laboratoires Servier
Priority date: 2019-05-20
Filing date: 2020-05-19
Publication date: 2023-10-09

Abstract

Anti-CD74 antibody-drug conjugates are disclosed. The anti-CD74 antibody-drug conjugates comprise an Mcl-1 inhibitor dnig moiety and an anti-CD74 antibody or antigenbinding fragment thereof that binds an antigen target, e.g.. an antigen expressed on a tumor or other cancer cell. The disclosure further relates to methods and compositions for use in the treatment of cancers by administering the antibody-drug conjugates provided herein. Linkerdrug conjugates comprising an Mcl-1 inhibitor drug moiety and methods of making same arc also disclosed.

Description

MCL-1 INHIBITOR ANT1BODY-DRUG CONJUGATES AND METHODS OF USE

RELATED APPLICATION

This application daims the benefit of and priority to the filing date under 35 U.S.C. § 119(e) of US Provisional Application No. 62/850,098 filed on May 20, 2019, the entire content of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically and is hereby incorporated by reference in its entirety.

FIELD OFTHE INVENTION

The present disclosure relates to antibody-drug conjugales (ADCs) comprising an Mcl-1 inhibîtor and an anti-CD74 antibody or antigen-binding fragment thereof that binds an antigen target, e.g., an antigen expressed on a tumor or other cancer cell. The disclosure further relates to methods and compositions useful in the treatment and/or diagnosis of cancers that express the target antigen CD74 and/or are amenable to treatment by modulatîng Mcl-1 expression and/or activity, as well as methods of making those compositions. Linker-drug conjugates comprising an Mcl-1 inhîbitor drug moiety and methods of making same are also disclosed.

BACKGROUND OF THE INVENTION

Apoptosis, or programmed cell death, îs a physiological process that is crucial for embryonic development and maintenance of tissue homeostasis. Apoptotic-type cell death generally involves morphological changes such as condensation of the nucléus and DNA fragmentation, as well as biochemical changes such as the activation of caspases that can cause damage to key structural components of the cell. Régulation of apoptosis is complex and typically involves the activation or repression of several intracellular signaling pathways (Cory et al. (2002) Nature Review Cancer 2:647-656).

Deregulation of apoptosis is associated with certain pathologies. For instance, increased apoptosis is associated with neurodegenerative diseuses such as Parkinson’s disease, Alzheimer's disease, and ischemia. Conversely, déficits in apoptosis can play a rôle in the development of cancers and ch emo résistance, auto immune diseases, inflammatory diseases, and viral infections. The absence of apoptosis is one of the phenotypic signatures of cancer (Hanahan et al. (2000) Cell 100:57-70). Anti-apoptotic proteins of the BcI-2 family are associated with numerous types of cancer, such as colon cancer, breast cancer, small cell lung cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, prostate cancer, chronic lymphoid leukemia, lymphoma, myeloma, and pancreatîc cancer.

Myeloid cell leukemia 1 (Mcl-1), an anti-apoptotic Bcl-2 family member, is a regulator of cell survival. Amplification of the Mcl-1 gene and/or overexpression of the Mcl-1 protein has been observed in multiple cancer types and is commonly implicated in tumor development (Beroukhim et al. (2010) Nature 463(7283):899-905). Mcl-1 is one of the most frequently amplified genes in human cancer and is also a critical survival factor that has been shown to médiate drug résistance to a variety of anti-cancer agents.

Mcl-1 is believed to promote cell survival by binding to and neutralizing the deathinducing activities of pro-apoptotic proteins such as Bim, Noxa, Bak, and Bax. Inhibition of Mcl-1 releases these pro-apoptotic proteins, often leading to the induction of apoptosis in tumor cells dépendent on Mcl-1 for survival. Therapeutically targeting Mcl-1 or proteins upstream and/or downstream of it in an apoptotic signaling pathway, therefore, may represent promising strategies to treat various malignancies and to overcome drug résistance in certain human cancers.

CD74 (DHLAG) is an established and attractive target for antibody drug conjugales due to its restricted expression on normal tîssues and signîficant upregulation in a range of hematological malignancies. CD74 fonctions as a chaperone that is necessary for the assembly and trafficking of MHC class II complexes as well as a receptor for macrophage migration inhibitory receptor (MIF). In oncology, it has been well established that CD74 is sîgnificantly upregulated at both the RNA and protein level in a range of B-cell and myeloid cell malignancies includîng acute myeloid leukemia, multiple myeloma, and diffuse large B-cell lymphoma. Additîonally CD74 is known to rapidly internalize upon antibody engagement and traffic to the lysosome as well as to to be rapidly repopulated on the surface of tumor cells following intemalization. Antibodîes and antibody drug conjugales targeting CD74 hâve been shown previously to demonstrate anti-tumor activity in preclinical models of cancer.

SUMMARY OF THE INVENTION

In some embodiments, the présent disclosure provides, in part, novel antibody-drug conjugate (ADC) compounds with biological activity against cancer cells. The compounds may slow, inhibit, and/or reverse tumor growth in mammals, and/or may be useful for treating human cancer patients. The présent disclosure more specifically relates, in some embodiments, to ADC compounds that are capable of binding and killing cancer cells. Jn some embodiments, the ADC compounds disclosed herein comprise a linker that attaches an Mcl-I inhibitor to a full-length anti-CD74 antibody or an antigen-binding fragment. In some embodiments, the ADC compounds are also capable of intemalizing into a target cell after binding.

In some embodiments, ADC compounds may be represented by Formula (l):

Ab-(L-D)_p (l) wherein Ab is an anti-CD74 antibody or an antigen-binding fragment thereof that targets a cancer cell;

D is an Mcl-l inhibitor;

L is a linker that covalently attaches Ab to D; and p is an integer from l to 16.

In some embodiments, p is an integer from l to 8. In some embodiments, p is an integer from l to 5. In some embodiments, p is an integer from 2 to 4. In some embodiments, p is 2. In some embodiments, p is 4. In some embodiments, p is determined by liquid chromatographymass spectrometry (LC-MS).

In some embodiments, the linker (L) comprises an attachment group, at least one spacer group, and at least one cleavable group. In some cases, the cleavable group comprises a pyrophosphate group and/or a self-immolative group. In spécifie embodiments, L comprises an attachment group; at least one bridging spacer group; and at least one cleavable group comprising a pyrophosphate group and/or a self-îmmolative group.

In some embodiments, the antibody-drug conjugate comprises a linker-drug (or “linkerpayload”) moiety -(L-D) is of the formula (A):

(A), wherein R¹ is an attachment group, Li is a bridging spacer group, and E is a cleavable group.

In some embodiments, the cleavable group comprises a pyrophosphate group. In some embodiments, the cleavable group comprises:

O O Il II .^P^P O f O ।

OH OH

In some embodiments, the bridging spacer group comprises a polyoxyethylene (PEG) group. In some cases, the PEG group may be selected from PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEGS, PEG9, PEG10, PEG11, PEG12, PEG 13, PEG 14, and PEG 15. In some embodiments, the bridging spacer group may comprise: -CO-CH2-CH2-PEG12-, In other embodiments, the bridging spacer group comprises a butanoyl, pentanoyl, hexanoyl, heptanoyl, or octanoyl group. In some embodiments, the bridging spacer group comprises a hexanoyl group.

In some embodiments the attachment group is formed from at least one reactive group selected from a maleimide group, thiol group, cyclooctyne group, and an azîdo group. For example, maleimide group may hâve the structure:

The azido group may hâve the structure: -N=N⁺=N.

The cyclooctyne group may hâve the structure:

1-12 _Or

, and wherein * is a bond to the antibody.

In some cases, the cyclooctyne group has the structure:

, and wherein * is a bond to the antibody.

In some embodiments, the attachment group has a formula

the antibody.

^H or

, and wherein * is a bond to

In some embodiments, the antibody isjoined to the linker (L) by an attachment group selected from:

wherein — * is a bond to the antibody, and wherein \ is a bond to the bridging spacer group.

In some embodiments, the bridging spacer group is joined to a cleavable group.

In some embodiments, the bridging spacer group is -CO-CH2-CH2-PEGI2-.

In some embodiments, the cleavable group is -pyrophosphate-CH2-CH2-NH2-.

In some embodiments, the cleavable group is joined to the Mcl-1 inhibitor (D).

In some embodiments, the cleavable group is joined to the Mcl-1 inhibitor (D) group through a phenyl-pyrimîdinyl group.

In some embodiments, the linker comprises: an attachment group, at least one bridging spacer group, a peptide group, and at least one cleavable group.

In some embodiments, the antibody-drug conjugate comprises a linker-drug moiety, -(L-D), is of the formula (B):

Ar^G-lp-eAsJ-d) wherein R¹ is an attachment group, Li is a bridging spacer, Lp is a peptide group comprisîng 1 to 6 amino acid residues, E is a cleavable group, L2 is a bridging spacer, m is 0 or 1 ; and D is an Mcl-1 inhibitor. In some cases, m is 1 and the bridging spacer comprises:

In some embodiments, the at least one bridging spacer comprises a PEG group. In some cases, the PEG group is selected from, PEG1, PEG2, PEG3, PEG4, PEG5, PEG6, PEG7, PEG8, PEG9, PEG 10, PEG11, PEG 12, PEG 13, PEG 14, and PEG 15. In some cases, the at least one bridging spacer is selected from *-C(O)-CH2-CH₂-PEGl-**, *-C(O)-CH2-PEG3-**, *-C(O)CH2-CH2-PEGI2**, *-NH-CH2-CH2-PEGl-**, a polyhydroxyalkyl group, and *-C(O)5

N(CH₃)-CH2-CH2-N(CH₃)-C(O)-**, wherein ** indicates the point of direct or indirect attachment of the at least one bridging spacer to the attachment group and * indicates the point of direct or indirect attachment of the at least one bridging spacer to the peptide group..

In some embodiments, L> is selected from *-C(O)-CH2-CH₂-PEGl-**, *-C(O)-CH₂PEG3-**, *-C(O)-CH₂-CH2-PEGl2**, *-NH-CH2-CH2-PEGl~**, and a polyhydroxyalkyl group, wherein ** indicates the point of direct or indirect attachment of Lj to R¹ and * indicates the point of direct or indirect attachment of Lj to Lp..

In some embodiments, m is 1 and L₂is -C(O)-N(CH₃)-CH₂-CH₂-N(CH₃)-C(O)-.

In some embodiments, the peptide group comprises 1 to 12 amino acid residues. In some embodiments, the peptide group (Lp) comprises 1 to 10 amino acid residues. In some embodiments, the peptide group (Lp) comprises 1 to 8 amino acid residues. In some embodiments, the peptide group (Lp) comprises 1 to 6 amino acid residues. Tn some embodiments, the peptide group comprises 1 to 4 amino acid residues. In some embodiments, the peptide group comprises 1 to 3 amino acid residues. In some embodiments the peptide group comprises 1 to 2 amino acid residues. In some cases, the amino acid residues are selected from L-glycine (Gly), L-valîne (Val), L-citrulline (Cit), L-cysteic acid (sulfo-Ala), L-lysine (Lys), Lisoleucîne (Ile), L-phenylalanine (Phe), L-methionine (Met), L-asparagine (Asn), L-proline (Pro), L-alanine (Ala), L-leucîne (Leu), L-tryptophan (Trp), and L-tyrosine (Tyr). For example, the peptide group may comprise Vai-Cit, Val-Al a, Val-Lys, and/or suIfo-Ala-Val-Ala. In some

embodiments, the peptide group (Lp) comprises 1 amino acid residue linked to a group. In some embodiments, the peptide group (Lp) comprises a group selected from:

In some cases, the peptide group comprises a group selected from:

In some embodiments, the self-immolative group comprises para-aminobenzyl carbamate, para-aminobenzyl-ammonium, para-amino-(sulfo)benzyl-ammonium, para-amino (sulfo)benzyl-carbamate, para-amino-(alkoxy-PEG-alkyl)benzyl-carbamate, para-amino (polyhydroxycarboxyteirahydropyranyl)alkyl-benzyl-carbamate, or para-amino(polyhydroxycarboxytetrahydropyranyl)aikyl-benzyl-ammonium.

In some embodiments, m is 1 and the bridging spacer comprises

In some embodiments, the linker-drug moiety, -(L-D), is formed from a compound selected from:

NH

O^NH₂

O

In some embodiments, the antibody-drug conjugate comprises the îinker-drug group, -(LD), which comprises a fonnula selected from:

o

wherein * is a bond to the antibody.

In some embodiments, the antibody-drug conjugale comprises the linker drug group,

-(L-D), which is of the formula (C):

Lp—G^^-A-D ^L3—R² (C),

wherein: R¹ is an attachment group, Li is a bridging spacer; L_p is a peptide group comprising 1 to 6 amino acids; D is an Mcl-I inhibitor; Gi-L₂-A is a seif-immolative spacer; L₂ is a bond, a

O * 4-o-p-l· methylene, a neopentylene or a C2-C3 alkenylene; A is a bond, -OC(=O)-*, θΗ , 0 0^. O 0 0^,*

11 YA

-O-P-O-P^

OH OH OH OH OH

OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or -OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH₃)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyi, and C₃-Cg cycloalkyl and the * of A indicates the point of attachment to D; L₃ is a spacer moiety; and R² is a hydrophi fie moiety. In some embodiments, the antibody-drug conjugale comprises the linker drug group,

-(L-D), which is of the formula (D):

wherein: R¹ is an attachment group; Li is a bridging spacer; Lp is a peptide group comprising 1

to 6 amino acids; A is a bond, -0C(=0)-*, OH , OH OH , OH

O O . . 11 11

Yo-p-o-pOH OH

-OC(=O)N(CH₃)CH₂CH2N(CH₃)C(=O)-* or -OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyi, and C₃-Cs cycloalkyl and the * of A indicates the point of attachment to D; L₃ is a spacer moiety; and R² is a hydrophilic moiety.

In some embodiments, Li comprises;

O

*-CH(OH)CH(OH)CH(OH)CH(OH)-**,wherein each n is an integer from 1 to 12, wherein the * of Li indicates the point of direct or indirect attachment to Lp, and the ** of L] indicates the point of direct or indirect attachment to R¹.

In some embodiments, Li is ⁿ , and n is an integer from l to 12 wherein the * of Li indicates the point of direct or indirect attachment to Lp, and the ** of Li indicates the point of direct or indirect attachment to R¹.

O

In some embodiments, Li is ⁿ , and n is 1, wherein the * of Lj indicates the point of direct or indirect attachment to Lp, and the ** of L] indicates the point of direct or indirect attachment to R¹.

O

In some embodiments, Li is ⁿ , and n is 12, wherein the * of Li indicates the point of direct or indirect attachment to Lp, and the ** of Li indicates the point of direct or indirect attachment to R¹.

In some embodiments, Li is

, and n is an integer from 1 to 12, wherein the * of Li indicates the point of direct or indirect attachment to Lp, and the ** of Li indicates the point of direct or indirect attachment to R¹.

OH OH ** *

In some embodiments, Li comprises OH OH , wherein the * of Li indicates the point of direct or indirect attachment to Lp, and the ** of Li indicates the point of direct or indirect attachment to R¹.

In some embodiments, Li is a bridging spacer comprising:

*-C(=O)(CH2)mO(CH₂)_m-**; *-C(=O)((CH₂)mO)t(CH2)n-**; *-C(=O)(CH2)m-**;

*-C(=O)NH((CH₂)_mO)_t(CH₂)n-**;

*-C(=O)O(CH2),nSSC(RWH2)mC(=O)NR^J(CH₂)nW³C(=O)(C^^ *-C(=O)O(CH₂)_mC(=O)NH(CH₂)m-**;*-C(=O)(CH2)mNH(CH2)_m-**;

*-C(=O)(CH2)_mNH(CH2)„C(rt))-*V *-C(=O)((CH₂)_mO)_t(CH2)_nXi(CH₂)_n-**; *-C(=O)(CH₂)_mNHC(=O)(CH2)_n-**;

*-C(=O)((CH₂)mO)_t(CH2)_nNHC(=OX^ *-C(=O)(CH₂)_mNHC(=O)(CH₂)nXi(CH₂)^ *-C(=O)((CH₂)_mO)_t(CH2)_nNHC(=O)(CH2)nXi(CH2)^ *-C(=O)((CH₂)_mO)[(CH₂)nC(=O)NH(CH₂)m-**; *-C(=O)(CH₂)_inC(R³)₂-** or *-C(=O)(CH2)mC(=O)NH(CH2)m-**, where the * of Li indicates the point of direct or indirect attachment to Lp, and the ** of Li indicates the point of direct or indirect attachment to R¹,

cach m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; and each t is independently selected from 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 and 30.

In some embodiments, R² is a hydrophilic moiety comprising polyethylene glycol, polyalkylene glycol, a polyol, a polysarcosine, a sugar, an oligosaccharide, a polypeptide, or C₂-

-f-Q-P-OH < 10 Cô alkyl substituted with 1 to 3 OH or OH ,Α/ίΛ 0 O'' '^Οχ^'θΗ , HO,C. ,Ο. 1 - HO γ γ’ T HO^J O. Il 1 Π Ί HO'' jOH HO'' Y ° HO^O , OH , Ot O^O H 0. ’ ί H ’ ^H2°3% 0 ^H l 0 h_:o₃p'°·^^^ , ctoh OH ho /U-<1 / yT'OunY’X oULoh oh °TVo ni ^H0V A n ^H0 < > OH^ ^0H U ΛΛΑΛ V· H । 0 Ο °ν^Ν'^^Λ'Ν© HO-P^-^O ^{1 ÔH} Λ 'V Us OH ' ¹ ï

bp-QH OH groups. In some embodiments, R“ is OH ^ΗΟγ\^·οΗ ,ν ho' θ^Η J j °^{H H0}'._ H Ο p'^O'-'-^U ^OH U ^H 'Ν'γ^ΝγθΖ ^H L S o^oh or ^^0H ₀ O^OH /üy^1,2Ÿ >H , OH , wherein n is an interger between 1 and 6,

In some embodiments, the hydrophilîc moiety comprises a polyethylene glycol of

formula: ⁿ or ^m , wherein R is H, -CH₃, CH₂CH₂NHC(=O)OR_a, -CH₂CH₂NHC(=O)R_a, or -CH₂CH₂C(=O)OR_a, R’ is OH, -OCH₃, CH₂CH₂NHC(=O)OR_a, -CH₂CH₂NHC(=O)R_a, or -OCH₂CH₂C(=O)OR_a, in which R_a is H or Cm alkyl optionally substiltuted with either OH or Cm alkoxyl, and each of ni and n is an integer between 2 and 25 (e.g. between 3 and 25).

In some embodiments,

the hydrophilîc moiety comprises OH

In some embodiments, the hydrophilîc moiety comprises a polysarcosine, e.g., with the following moiety

CH₂CH₂C(=O)OH.

, wherein n is an integer between 3 and 25; and R is H, -CH₃ or In some embodiments, L₃ is a spacer moiety having the structure

-|-w—x-h wherein:

W is -CH₂-, -CH₂O-, -CH₂N(R^b)C(=O)O-, -NHC(=O)C(R^b)2NHC(=O)O-, -NHC(=O)C(R^b)2NH-, -NHC(=O)C(R^b)2NHC(=O)-, -CH2N(X-R²)C(=O)O-, -C(=O)N(X-R²)-, -CH2N(X-R²)C(=O)-, -C(=O)NR^b-, -C(=O)NH-, -CH2NR^bC(=O)-, -CH2NR^bC(=O)NH-, -CH₂NR^bC(=O)NR^b-, -NHC(=O)-, -NHC(=O)O-, -NHC(=O)NH-, -OC(=O)NH-,

-S(O)₂NH-, -NHS(O)₂-, -C(=O)-, -C(=O)O- or -NH-, wherein each R^b is independently selected from H, Ci-Ci,alkyl, and C₃-Cs cycloalkyl; and

X is a bond, triazolyl, or -CH₂-triazolyl-.

In some embodiments, L₃ is a spacer moiety having the structure ^Xwherein:

W is -CH₂-, -CH2O-, -CH₂N(R^b)C(=O)O-, -NHC(=O)C(R^b)2NHC(=O)O-, -NHC(=O)C(R^b)2NH-, -NHC(=O)C(R^b)2NHC(=O)-, -CH2N(X-R²)C(=O)O-, -C(=O)N(X-R²)-, -CH2N(X-R²)C(=O)-, -C(=O)NR^b-, -C(=O)NH-, -CH2NR^bC(=O)-, -CH2NR^bC(=O)NH-, -CH2NR^bC(=O)NR^b-, -NHC(=O)-, -NHC(=O)O-, -NHC(=O)NH-, -OC(=O)NH-, -S(O)2NH-, -NHS(O)₂-, -C(=O)-, -C(=O)O- or-NH-, wherein each R^b is independently selected from H, Cj-Céalkyl, and C₃-Cs cycloalkyl; and

X îs -CH₂-triazolyl-Ci^ alkylene-OC(O)NHS(O)₂NH-,

-C4-6 cycloalkylene-OC(O)NHS(O)₂NH-, -(CH2CH₂O)_E-C(O)NHS(O)2NH-, -(CH₂CH₂O)_n-C(O)NHS(O)₂NH-(CH₂CH₂O)_n-, or

-CH₂-triazolyl-Cb₄ alkylene-OC(O)NHS(O)₂NH-(CH₂CH2O)_n-, wherein each n independently is 1, 2, or 3.

In some embodiments, the attachment group is formed by a reaction comprising at least one reactive group. In some cases, the attachment group îs formed by reacting: a first reactive group that is attached to the linker, and a second reactive group that îs attached to the antibody or is an amino acid residue of the antibody.

In some embodiments, at least one of the reactive groups comprises:

a thiol, a maleimide, a haloacetamide, an azide, an alkyne, a cyclcooctene, a triaryl phosphine, an oxanobomadiene, a cyclooctyne, a diaryl tetrazine, a monoaryl tetrazine, a norbomene, an aldéhyde, a hydroxyl amine, a hydrazine, NH₂-NH-C(=O)-, a ketone, a vinyl sulfone, an aziridine,

-SSR⁴, -S(=O)₂(CH=CH2), -(CH₂)₂S(=O)₂(CH=CH₂), -NHS(=O)₂(CH=CH₂),

R³

-NHC(=O)CH₂Br, -NHC(=O)CH₂I, O

-C(O)NHNH₂,

wherein:

each R³ is independently selected from H and Ci-Csalkyl;

each R⁴ is 2-pyridyl or 4-pyridyl;

each R^s is independently selected from H, Ci-Côalkyl, F, Cl, and -OH;

each R⁶ is independently selected from H, Ci-Côalkyl, F, Cl, -NH₂, -OCH₃, -OCH₂CH₃, N(CH₃)2, -CN, -NO₂ and-OH;

each R⁷ is independently selected from H, Crealkyl, fluoro, benzyloxy substituted with C(=O)OH, benzyl substituted with -C(=O)OH, Ci-4alkoxy substituted with -C(=O)OH and Ci-4alkyl substituted with -C(=O)OH.

In some embodiments, the first reactive group and second reactive group comprise: a thiol and a maleimide, a thiol and a haloacetamide, a thiol and a vinyl sulfone, a thiol and an aziridine, an azide and an alkyne, an azide and a cyclooctyne.

an azide and a cyclooctene, an azide and a triaryl phosphine, an azide and an oxanobornadiene, a diaryl tetrazîne and a cyclooctene, a monoaryl tetrazîne and a nonbomene, an aldéhyde and a hydroxylamine, an aldéhyde and a hydrazine, an aldéhyde and NH₂-NH-C(=O)-, a ketone and a hydroxylamine, a ketone and a hydrazine, a ketone and NH2-NH-C(=O)-,

a CoA or CoA analogue and a serine residue.

In some embodiments, the attachment group comprises a group selected from:

amide;

|o ? \

disulfide, wherein:

R³² is H, Ci-4 alkyl, phenyl, pyrimidine or pyridine;

R³⁵ is H, Ci-6alkyl, phenyl or Ci-4 alkyl substituted with 1 to 3 -OH groups;

each R⁷ is independently selected from H, Ci-6 alkyl, fluoro, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, Ci-₄ alkoxy substituted with -C(=O)OH and Ci₄ alkyl substituted with -C(=O)OH;

R³⁷ is independently selected from H, phenyl and pyridine; q is 0, 1, 2 or 3;

R⁸ is H or methyl; and

R⁹ is H, -CH₃ or phenyl.

In some embodiments, the peptide group (Lp) comprises 1 to 6 amino acid residues. In some embodiments, the peptide group (Lp) comprises 1 to 4 amino acid residues. In some embodiments, the peptide group comprises 1 to 3 amino acid residues. In some embodiments, the peptide group comprises 1 to 2 amino acid residues. In some embodiments, the amino acid residues are selected from L-glycine (Gly), L-valine (Val), L-citrulline (Cît), L-cysteic acid (sulfo-Ala), L-lysine (Lys), L-isoleucine (Ile), L-phenylalanine (Phe), L-methionine (Met), Lasparagine (Asn), L-proline (Pro), L-alanine (Ala), L-leucine (Leu), L-tryptophan (Trp), and Ltyrosine (Tyr). In some embodiments, the peptide group comprises Vai-Cit, Phe-Lys, Val-Al a. Val-Lys, Leu-Cit, sulfo-Ala-Val, and/or sulfo-Ala-Val-Ala. In some embodiments, Lp is selected from:

In some embodiments, the linker-drug group -(L-D) comprises or is formed from a compound of fonnula:

N-N

H N h,n , wherein:

R is H, -CH₃ or -CH₂CH2C(=O)OH;

O * 0 0*

Ht . Il il,⁷'

A is a bond, -OC(=O)-*,

OH

OH OH

OH

OH OH

-OC(=O)N(CH3)CH₂CH₂N(CH3)C(=O)-* or -OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CHî)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C3-Cs cycloalkyl and the * of A indicates the point of attachaient to D; and D is an Mcl-1 inhibitor.

ln some embodiments, the linker-drug group -(L-D) comprises or is formed from a compound of fonnula;

h₂n o , wherein:

R is H, -CH₃ or -CH₂CH₂C(=O)OH;

ο *

-|-ο-ρ4~

Α is a bond, -OC(=O)-*,

Ο Ο * Ο , ιι ιι , . ιι *

4-ο-ρ-ο-ρα -Ι-ο-ρ-ο^

ΟΗ ΟΗ , ΟΗ

Ο Ο . Il II 7

-|-Ο-Ρ-Ο-Ρ^

ΟΗ ΟΗ

-OC(=O)N(CH3)CH2CH₂N(CH₃)C(=O)-* or OC(=O)N(CH3)C(R^a)2C(R^a)₂N(CH₃)C(=O)-*, wherein each R^a is independently selected from H, Ci-C& alkyl, and C3-C8 cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-1 inhibitor.

In some embodiments, the linker-drug group -(L-D) comprises or is formed from a compound of formula:

R is H, -CH3 or -CH₂CH₂C(=O)OH;

O * O O . O <_ H s ,11 u ,* , 11 *

-|-o-p-j- -I-o-p-o-pA -I-o-p-oa^

A is a bond, -OC(=O)-*, OH , OH OH , OH ⁰ O * , Il II

-f-o-p-o-p—⁷

OH OH

-OC(=O)N(CH3)CH₂CH₂N(CH₃)C(=O)-* or OC(=O)N(CH3)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C3-Cs cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-1 inhibitor.

In some embodiments, the linker-drug group -(L-D) comprises or is fonned from a compound of formula:

each R is independently selected from H, -CH₃, and -CH₂CH2C(=O)OH;

O * , a b

4-o-p-h

A is a bond, -OC(=O)-*, OH

OH OH , -OC(=O)N(CH₃)CH₂CH₂N(CH3)C(=O)-* or ~OC(=O)N(CH3)C(R^a)2C(R^a)₂N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-C& alkyl, and C3-Q cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-l inhibitor.

each R is independently selected from H, -CH3, and -CH₂CH2C(=O)OH;

A is a bond, -OC(=O)-*, OH , OH OH , OH . h h 7^.

-f-o-p-o-p—^/

OH OH , -OC(=O)N(CH3)CH₂CH₂N(CH3)C(=O)-* or -OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Cj-Cô alkyl, and C₃-Cs cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-l inhibitor.

Xa îs -CH₂-, -OCH₂-, -NHCH₂- or -NRCH₂- and each R independently is H, -CH₃ or

-CH₂CH₂C(=O)OH;

A is a bond, -OC(=O)-*, OH , OH OH _; OH ,

O O , * , Il II

-t-o-p-o-p—'

OH OH , -OC(=O)N(CH3)CH2CH₂N(CH3)C(=O)-* or -OC(=O)N(CH3)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and Cs-Cs cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-l inhibitor.

R is H, -CH₃ or -CH₂CH₂C(=O)OH;

O * O O * O < Ils , II i i s Il *

-|-o-p-o-p-l· 4ο-ρ-ο<^

A is a bond, -OC(=O)-*, OH , OH OH , OH ,

O O / * . Il II W

-j-o-p-o-p—'

OH OH , -OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or -OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C3-Cg cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-l inhibitor.

Xb is -CH₂-, -OCH₂-, -NHCH₂- or -NRCH₂- and each R independently is H, -CH₃ or -CH₂CH₂C(=O)OH;

A is a bond, -OC(=O)-*, OH _; OH OH , OH _j

O O , * <, Il II G -l-o-p-o-p—⁷

OH OH , -OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C3-Cs cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-l inhibitor.

. Il II

-Vo-p-o-p^

OH OH , -OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH₃)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and Ci-Cs cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-1 inhibitor.

. Il II 7^, -i-o-p-o-p—⁷

OH OH , -OC(=O)N(CH₃)CH₂CH2N(CH₃)C(=O)-* or -OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C3-Cs cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-1 inhibitor.

, Il II Ή,

-|-ο-γο-ρ—'

OH OH , -OC(=O)N(CH3)CH2CH₂N(CH₃)C(=O)-* or

-OC(=O)N(CH3)C(R^a)2C(R^a)₂N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C₃-Cs cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-l inhibitor.

O O „ , *

OH OH , -OC(=O)N(CHî)CH2CH₂N(CH₃)C(=O)-* or -OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C3-Cs cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-1 inhibitor.

A is a bond, -OC(=O)-*, OH , OH OH

OH

4-O-P-O-P-⁷

OH OH , -OC(=O)N(CH₃)CH2CH₂N(CH₃)C(=O)-* or -OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH₃)C(=O)-*, wherein each R^a is independently selected from H, Ci-G, alkyl, and C₃-Cs cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-1 inhibitor.

, wherein:

O *

-ï-o-p-H

A is a bond, -OC(=O)-*, OH

OH OH

O

Il *

-O-P-O^y

OH s ¹¹ II ZW

-i-o-p-o-p—^/

OH OH , -OC(=O)N(CH₃)CH₂CH2N(CH₃)C(=O)-* or -OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cs alkyl, and C₃-Cs cycloalkyl and the * of A indicates the point of attachment to D; and D is an Mcl-1 inhibitor.

co₂h

, wherein:

O

OH

A is a bond, -OC(=O)-*, O O *

OH

OH OH , -OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or -OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C3-Cs cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-1 inhibitor.

R

HjN each R independently is H, -CH₃ or -CH₂CH₂C(=O)OH;

, wherein:

A is a bond, -OC(=O)-*,

ΟΗ

ΟΗ ⁰ 0 * < ii h AL

-|-o-p-o-p^

OH OH , -OC(=O)N(CH3)CH₂CH₂N(CFh)C(=O)-* or

-OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH₃)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyi, and C₃-C₈ cycloalkyl and the * of A îndicates the point of attachment to D; and

D îs an Mcl-1 inhibitor.

In some embodiments, A is a bond.

In some embodiments, R is -CH₃.

In some embodiments, the Mcl-1 inhibitor (D) comprises a compound of Formula (I):

wherein :

Ring Do is a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group,

Ring Eo is a fîiryl, thienyl or pyrrolyl ring,

Xoi, X03, Χολ and X05 independently of one another is a carbon atom or a nitrogen atom,

X02 îs a C-Ro26 group or a nitrogen atom, < J means that the ring is aromatic,

Yo is a nitrogen atom or a C-Roa group,

Zy is a nitrogen atom or a C-Rq₄ group,

Roi is a halogen atom, a linear or branched (Cj-CôJalkyl group, a linear or branched (C₂-Cô)alkenyl group, a linear or branched (C2-C&)alkynyl group, a linear or branched (Ci-Cô)haloalkyl group, a hydroxy group, a hydroxy(Ci-C&)alkyl group, a linear or branched (Ci-Cô)alkoxy group, -S-(Ci-C6)alkyl group, a cyano group, a nitro group, -Cyos, -(Co-Côlalkyl-NRoiiRon’, -0-(Ci-C&)alkyl-NRoiiRoii\ -0-(Ci-C6)alkyl-Roi2, -C(0)-ORou, -0-C(0)-Roii, -C(O)-NR₀i_fR₀n’, -NR_ou-C(0)-Ron’, -NRoii-C(O)-OR₀ii’, -(Ci-Côjalkyl-NRo11-C(O)-R₀u -SO₂-NR_0t ।Roi i ’, or -SO₂-(C 1 -C₆)alkyl,

Ro2, Roî, Rü4 and R05 independently of one another are a hydrogen atom, a halogen atom, a linear or branched (Ci-Cô)alkyl group, a linear or branched (C₂-C&)alkenyl group, a linear or branched (C₂-Cô)alkynyl group, a linear or branched (Ci-C6)haloalkyl, a hydroxy group, a hydroxy(Cj-C6)alkyl group, a linear or branched (Ci-Cô)alkoxy group, a -S-(Ci-C6)alkyl group, a cyano group, a nitro group, -(Co-Côjalkyl-NRonRoii’, -O-Cyoi, -(Co-C6)alkyl-Cy₀₁, -(C₂-C₆)alkenyl-Cyoi, -(C₂-C6)alkynyl-Cyoi,-0-(Ci-C6)alkyl-NRonRoii’,-O-(Ci-C₆)alkyl-R₀ji, -0-(Ci-C₆)alkyl-Roi2, -C(O)-OR₀h, -0-C(0)-Roii, -C(0)-NRoi 1R011’, -NRoii-C(0)-Ron’, -NR01 i-C(0)-ORoi 1 -(Ci-C₆)alkyl-NRoi i-C(0)-Roi Γ, -SO2-NR011 Roi 1 or -SO₂-(Ci-C₆)alkyl, or the pair (Roi, R02), (R02, R03), (R03, Roî), or (Roî, R05) together with the carbon atoms to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains 1 to 3 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted by 1 or 2 groups selected from halogen, linear or branched (Ci-Cô)alkyl, (Co-Céjalkyl-NRoiiRüii’, -NR013R013’, -(Co-Cf>)alkyl-Cyoi or oxo,

Roû and R07 independently of one another are a hydrogen atom, a halogen atom, a linear or branched (Ci-Ccflalkyl group, a linear or branched (C₂-C6)alkenyl group, a linear or branched (C₂-Cô)alkynyl group, a linear or branched (Ci-Cfi)haloalkyl, a hydroxy group, a linear or branched (Ci-Cfi)alkoxy group, a -S-(Ci-Cô)alkyl group, a cyano group, a nitro group, -(Co-Côjalkyl-NRonRoii’, -0-(Ci-C6)alkyl-NRonRon’, -O-Cyoi, -(Co-C6)alkyl-Cyoi, -(Cz-Côjalkenyl-Cyoi, -(C₂-C₆)alkyny1-Cyoi, -0-(Ci-C₆)alkyl-Roi2, -C(O)-ORn, -0-C(O)-Roii, -C(0)-NRonRoii’, -NRh-C(O)-R₀ii’, -NR₀u-C(O)-OR₀ii’, -(Ci-C₆)alkyl-NRoii-C(0)-R₀ii’,-S0₂-NR_oliRon’,or -SO₂-(Ci-C₆)alkyl, or the pair (Roô, R07), when fused with the two adjacent carbon atoms, together with the carbon atoms to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains l to 3 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted by a linear or branched (Ci-Côjalkyl group, -NR013R013’, -(Co-Céjalkyl-Cyoi or an oxo,

Wo is a -CH2- group, a -NH- group or an oxygen atom,

R08 is a hydrogen atom, a linear or branched (Ci-Cs)alkyl group, a -CHRoaRob group, an aryl group, a heteroaryl group, an aryl(Ci-Cô)alkyl group or a heteroaryl(CiCé)alkyl group,

R09 is a hydrogen atom, a linear or branched (Ci-Cô)alkyl group, a linear or branched (C₂-C6)alkenyl group, a linear or branched (C₂-Cô)alkynyl group, -Cyo₂, -(Ci-C6)alkyl-Cyo3, -(C₂-C6)alkenyl-Cyo2, -(C2-C6)alkynyl-Cyo₂, -Cyo₂-Cyo3, -(C2-C6)alkynyl-0-Cyo₂, -Cyo₂-(Co-C6)alkyl-0-(Co-C6)alkyl-Cyo₃, a halogen atom, a cyano group, -C(0)-Roi4, or -C(0)-NRoi4Roi4’,

Roio is a hydrogen atom, a linear or branched (Ci-Cé)alkyl group, a linear or branched (C₂-Cé)alkenyl group, a linear or branched (C2-Cé)alkynyl group, an aryl(Ci-C&)alkyl group, a (Ci-Céjcycloalkylalkyl group, a linear or branched (Ci-C6)haloalkyl, or -(Ci-Cô)alkyl-0-Cyo4, or the pair (R09, Roio), when fused with the two adjacent carbon atoms, together with the carbon atoms to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains 1 to 3 heteroatoms selected from O, S and N,

Roi । and Ron’ independently of one another are a hydrogen atom, an optionally substituted linear or branched (Ci-Cô)alkyl group, or -(Co-Cé)alkyl-Cyoi, or the pair (Roi 1, Roi 1 ’) together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains, in addition to the nitrogen atom, 1 to 3 heteroatoms selected from O, S and N, wherein the nitrogen may be substituted by 1 or 2 groups selected from a hydrogen atom and a linear or branched (Ci-Cô)alkyl group, and wherein one or more of the carbon atoms of the linear or branched (Ci-Cé)alkyl group is optionally deuterated,

R012 îs -Cyos, -Cyo5-(Co-C6)alkyl-0-(Co-Cô)alkyl-Cyo6, -Cyo5-(Co-C6)alkyl-Cyo6, -Cyo5-(Co-C6)alkyl-NRon-(Co-C6)alkyI-Cyo6, -Cyû5-Cyo6-0-(Co-C&)alkyl-Cyo7, -Cyo5-(Co-C6)alkyl-0-(C₀-C₆)alkyl-Cyo9, -Cyo₃-(C_o-C₆)alkyl-Cy_O9, -NH-C(O)-NH-R_0l 1, -Cyo5-(Co-Cô)alkyl-NRoi i-(Co-Cô)alkyl-Cyo9, -C(0)-NRoiiRou’, -NR011R011’, -OR011, -NRon-C(0)-R_oli’, -0-(Ci-C6)alkyl-ORou,-S0₂-Ron,and -C(O)-OR₀n, containing 5 to 7 ring members, which optionally contains l to 3 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted by a linear or branched (Ci-Cô)alkyl group, -NR013R013’, -(Co-C_&)alkyl-Cyoi or an oxo,

Wo îs a -CH2- group, a -NH- group or an oxygen atom,

Ros is a hydrogen atom, a linear or branched (Ci-Cs)alkyl group, a -CHRoaRob group, an aryl group, a heteroaryl group, an aryHCi-Csjalkyl group or a heteroaryl(CiCô)alkyl group,

R09 is a hydrogen atom, a linear or branched (Ci-Cû)alkyl group, a linear or branched (C₂-Cô)alkenyl group, a linear or branched (C2-C&)alkynyl group, -Cyo₂, -(Ci-C6)alkyl-Cyo2, -(C2-C₆)alkenyl-Cyo2, -(C2-C₆)alkynyl-Cy₀₂, -Cy₀2-Cy_ü3, -(C₂-C6)alkynyl-0-Cyo2, -CyQ₂-(Co-C6)alkyl-0-(Co-Cô)alkyl-Cyo3, a halogen atom, a cyano group, -C(0)-Roi4, or -C(0)-NRoi₄Roi4’,

Roio is a hydrogen atom, a linear or branched (Ci-Cô)alkyl group, a linear or branched (C₂-Cô)alkenyl group, a linear or branched (C₂-Cfi)alkynyl group, an aryl(Ci-Cô)alkyl group, a (Ci-Côjcycloalkylalkyl group, a linear or branched (Ci-C₆)haloalkyl, or -(Ci-C₆)alkyl-0-Cyo4, or the pair (Rot, Roio), when fused with the two adjacent carbon atoms, together with the carbon atoms to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains I to 3 heteroatoms selected from O, S and N,

Rom and Roi i ’ independently of one another are a hydrogen atom, an optionally substituted linear or branched (C । -Co)alkyl group, or -(Co-C6)alkyl-Cyo ।, or the pair (Ro11, Roi 1’) together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains, in addition to the nitrogen atom, 1 to 3 heteroatoms selected from O, S and N, wherein the nitrogen may be substituted by 1 or 2 groups selected from a hydrogen atom and a linear or branched (Ci-Cô)alkyl group, and wherein one or more of the carbon atoms of the linear or branched (Ci-Cô)alkyl group is optionally deuterated,

R012 is -Cyos, -Cyo5-(Co-C6)alkyl-0-(Co-C6)alkyl-Cyoô, -Cyo5-(Co-C6)alkyl-Cyo6, -Cyo5-(Co-C6)alkyl-NR_Oii-(Co-C6)alkyl-Cyo6, -Cyo5-Cyo6-0-(Co-C₆)alkyl-Cyo7, -Cyo5-(Co-C6)alkyl-0-(C₀-C6)alkyl·^ -Cyo₅-(Co-C₆)alkyl-Cyo9, -NH-C(0)-NH-Roh, -Cyo5-(C_o-C6)alkyl-NRoi i-(Co-C₆)alkyl-Cyo9, -C(O)-NR₀i i RoiΓ, -NR011 Ro 11 -ORoj 1, -NRoii-C(0)-R()n’, -O-(Ci-C₆)alkyl-OR₀n, -SO2-R011, and -C(O)-OR₀n,

Roi3, R013’, Roi4 and Rom’ independently of one another are a hydrogen atom or an optionally substituted linear or branched (Ci-C&)alkyl group,

Rüa is a hydrogen atom or a linear or branched (Ci-Cô)alkyl group,

Rob is a -0-C(0)-0-Roc group, a -0-C(0)-NRo_cRoc’ group, or a -0-P(0)(ORoc)₂group,

Roc and Ro_c’ independently of one another are a hydrogen atom, a linear or branched (Ci-Cg)alkyl group, a cycloalkyl group, a (Ci-Cû)alkoxy(Ci-C6)alkyl group, or a (C i -C6)alkoxycarbonyl(C । -Cô)alkyl group, or the pair (R_Oc, Ro_c’) together with the nitrogen atom to which they are attached form a non-aromatic ring composed of from 5 to 7 ring members, which may contain in addition to the nitrogen atom from 1 to 3 heteroatoms selected from oxygen and nitrogen, wherein the nitrogen is optionally substituted by a linear or branched (Ci-Cô)alkyl group,

Cyoi, Cyoz, Cyos, Cyo4, Cyos, Cyoô, Cyo7, Cyos and Cyoïo independently of one another, represent a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group, each of which is optionally substituted,

or Cyo9 is a heteroaryl group which is substituted by a group selected from -0-P(0)(ORo2o)₂; -Ο-Ρ(Ο)(Ο-Μ’^η)₂; -(CH₂)po-0-(CHRoi8-CHR₀j9-0)_q0-Ro₂o; hydroxy;

hydroxy(Ci-C6)alkyl; -(CH₂),o-Uo-(CH₂)sO-heterocycloalkyl; and -Uo-(CH₂)qo-NRo2iRo₂i’,

Roi5 is a hydrogen atom; a -(CH₂)po-0-(CHRoi8-CHRoi9-0)_qo-Ro20 group; a linear or branched (Ci-C6)alkoxy(Ci-Cô)alkyl group; a -Uo-(CH₂)qO-NRo2iRo₂r group; or a -(CH₂)io-Uü-(CH₂)sO-heterocycloalkyl group,

Roiô is a hydrogen atom; a hydroxy group; a hydroxy(Ci-Cô)alkyl group; a -(CH2)rtrOo-(CH₂)_so-heterocycloalkyl group; a (CH2)tO-üo-Vo-0-P(0)(ORo2o)₂ group; a -O-P(O)(O'M⁺)2 group; a -0-S(0)₂ORo₂o group; a -S(0)₂ORo₂o group; a -(CH₂)_Po-0-(CHRoi8-CHRoi9-0)_qo-Ro20 group; a -(CH₂)pû-0-C(0)-NRo2₂Ro23 group; or a -Uo-(CH₂)_qo-NRo2iRo2i’ group,

Roi7 is a hydrogen atom; a -(CH2)_Po-0-(CHRoi8-CHRoi9-0)qo-Ro20 group; a -CH₂-P(0)(ORo2o)2 group, a -O-P(O)(OR₀20)₂ group; a -Ο-ΡίΟχΟΜΑ group; a hydroxy group; a hydroxy(Ci-C₆)alkyl group; a -(CH2)₁a-Uo-(CH₂)_so-heterocycloalkyl group; a -Uo-(CH2)qD-NRo2iRo2i’ group; or an aldonic acid,

M⁺ is a pharmaceutically acceptable monovalent cation,

Uo is a bond or an oxygen atom,

Vo is a -(CH₂)_so- group or a -C(O)- group,

Rois îs a hydrogen atom or a (Ci-C6)alkoxy(Ci-C6)alkyl group,

Roi9 is a hydrogen atom or a hydroxy(Ci-Cô)alkyl group,

R020 is a hydrogen atom or a linear or bran ch ed (Ci-Cô)alkyl group,

Ro2i and R021’ independently of one are a hydrogen atom, a linear or branched (Ci-Cô)alkyl group, or a hydroxy(Ci-C_b)alkyl group, or the pair (R021, R021’) together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains, in addition to the nitrogen atom, l to 3 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted by a hydrogen atom or a linear or branched (Ci-Cô)alkyl group,

R022 is a (Ci-Cô)alkoxy(Ci-Cô)alkyl group, a -(CH₂)_po-NRo24Ro24’ group, or a -(CH₂)pO-0-(CHRoi8-CHRoi9-0)_qo-Ro20 group,

R023 is a hydrogen atom or a (Ci-C6)alkoxy(Ci-Cô)alkyl group, or the pair (R022, R023) together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5 to 18 ring members, which optionally contains, in addition to the nitrogen atom, l to 5 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted by a hydrogen atom, a linear or branched (Ci-Cô)alkyl group or a heterocycloalkyl group,

R024 and R024’ independently of one another are a hydrogen atom or a linear or branched (Ci-Cô)alkyl group, or the pair (R024, R024’) together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring composed of from 5 to 7 ring members, which may contaîn in addition to the nitrogen atom from l to 3 heteroatoms selected from O, S and N, and wherein the resulting ring îs optionally substituted by a hydrogen atom or a linear or branched (Ci-Cô)alkyl group,

R025 is a hydrogen atom, a hydroxy group, or a hydroxy(Ci-C6)alkyl group,

Ro2ô is a hydrogen atom, a halogen atom, a linear or branched (Ci-Cô)alkyl group, or a cyano group,

R027 is a hydrogen atom or a linear or branched (Ci-Côjalkyl group, R028 is a -O'P(O)(O’)(O’) group, a -0-P(0)(0 )(ORo3o) group, a

-0-P(0)(ORo3o)(OR_ü3o’) group, a -(CH₂)p0-O-SO2-O’ group, a -(CH₂)_po-S0₂-0· group, a

-(CH₂)_Po-0-S02-ORo3o group, -Cyoïo, a -(CH2)_Po-S0₂-OR(i30 group, a -O-C(O)-Rc29 group, a -O-C(O)-ORü29 group or a -0-C(0)-NRû29Ro29^! group;

R()29 and R029’ independently of one another represent a hydrogen atom, a linear or branched (Ci-Cô)alkyl group or a linear or branched amino(Ci-Cô)alkyl group,

R03O and Rojo’ independently of one another are a hydrogen atom, a linear or branched (Ci-Côjalkyl group or an aryl(Ci-Cô)alkyl group,

exists as a zwîtterionic form or has a monovalent anîonic counterion, no is an integer equal to 0 or l, po is an integer equal to Ο, I, 2, or 3, qo is an integer equal to ], 2, 3 or 4, ro and so are independently an integer equal to 0 or 1 ;

wherein, at most, one of the R03, Ro9, or R012 groups, if présent, is covalently attached to 20 the linker, and wherein the valency of an atom is not exceeded by virtue of one or more substituents bonded thereto, or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait of any of the foregoîng.

In some embodiments, Cyoi, Cyo₂, Cyoj, Cyo₄, Cyos, Cyo&, Cyo?, Cyos and Cyoïo, independently of one another, is a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group, each of which is optionally substituted by one or more groups selected from halo; -(Ci-Cô)alkoxy; -(Ci-C₆)haloalkyl; -(Ci-C₆)haloalkoxy; -(CH2)_Po-0-S0₂-ORo3o;

-(CH2)_Po-S02-ORo3o; -O-P(O)(ORq20)2; -O-P(O)(O'M⁺)₂; -CH₂-P(0)(ORo2o)2;

-(CH2)_Po-0-(CHRoi8-CHRoj9-0)_qo-Ro2o; hydroxy; hydroxy(Ci-C₆)alkyl;

-(CH2)iû-Uo-(CH₂)_so-heterocycloalkyl; or -Uo-(CH2)_qo-NRo2iRo2i’.

In some embodiments, D comprises a compound of Formula (Π):

wherein:

Zo is a nitrogen atom or a C-Rq4 group.

Roi is a halogen atom, a lînear or branched (Ci-Cô)alkyl group, a linear or branched (C₂-Cô)alkenyl group, a linear or branched (C₂-Cô)alkynyl group, a linear or branched (Ci-Cô)haloalkyl group, a hydroxy group, a linear or branched (Ci-Cô)alkoxy group, a -S-(Ci-Cô)alkyl group, a cyano group, -Cyog, -NRohRou’,

Ro2, R03 and Rü4 independently of one another are a hydrogen atom, a halogen atom, a linear or branched (Ci-Cô)alkyl group, a linear or branched (C2-Cô)alkenyl group, a linear or branched (C2-Cô)alkynyl group, a linear or branched (Ci-Cô)haloalkyl, a hydroxy group, a linear or branched (Ci-Cô)alkoxy group, a -S-(Ci-Cô)alkyl group, a cyano group, a nitro group, -(Co-CéJalkyl-NRoiiRmi’, -O-Cyoi, -(Co-Céjalkyl-Cyoi, -(C2-Cô)alkenyl-Cyoi, -(C₂-Cô)alkynyl-Cyoi, -0-(Ci-Cô)alkyl-NRoiiRoii’, -0-(Ci-Cô)alkyl-Ro3i, -C(0)-ORoii, -O-C(O)-R₀₁i, -C(0)-NRoiiRoii’, -NRoii-C(0)-Ron’, -NRou-C(O)-ORoi) -(Ci-C&)aIkyl-NRoii-C(0)-Roi i -SO2-NR011 Roi 1’, or -SO₂-(Ci-C₆)alkyl, or the pair (R02, R03) or (R03, R04) together with the carbon atoms to which they are attached form an aromatic or non-aromatîc ring containing 5 to 7 ring members, which optionally contains l to 3 heteroatoms selected from O, S and N, wherein the ring is optionally substituted by a group selected from a linear or branched (Ci-C6)alkyl, -NR013R013’, -(Co-Cô)alkyl-Cyoi and oxo,

R06 and Ro? independently of one another are a hydrogen atom, a halogen atom, a linear or branched (Ci-Cô)alkyl group, a linear or branched (C₂-Cô)alkenyl group, a linear or branched (C₂-Cô)alkynyl group, a linear or branched (Ci-Cô)haloalkyl, a hydroxy group, a linear or branched (Ci-Côjalkoxy group, a -S-(Ci-C&)alkyl group, a cyano group, a nitro group, -(Co-C6)alkyl-NRonRoi i’, -O-Cyoi, -(Co-Cojalkyl-Cyoi, -(C2-Cô)alkenylCyoi, -(C₂-Cô)alkynyl-Cyoi, -O-(Ci-C₆)alkyl-R₀i2, -C(0)-ORoii, -O-C(O)-R₀ii, -C(O)NRonRon’, -NRoirC(0)-Ro!i’, -NRon-C(0)-ORon’, -(Ci-C₆)alkyl-NRoii-C(0)-Roii’, -SO₂NRonRoii’, or -SO₂-(Ci-Cô)alkyl, or the pair (Roé, R07), when fused with two adjacent carbon atoms, together with the carbon atoms to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains l to 3 heteroatoms selected from O, S and N, and wherein the resulting ring is optionally substituted by a group selected from a linear or branched (Ci-C&)alkyl group, -NR013R013’, -(Co-C6)alkyl-Cyoi and an oxo,

Ros is a hydrogen atom, a linear or branched (Ci-Cs)alkyl group, an aryl group, a heteroaryl group, an aryl-(Ci-C6)alkylgroup, or a heteroaryl(Ci-C6)aIkyl group,

R09 is a linear or branched (Ci-Cô)alkyl group, a linear or branched (C₂-Cô)alkenyl group, a linear or branched (C2-C&)alkynyl group, -Cyo₂, -(Ci-Cô)alkyl-Cyo2, -(C₂-C6)alkenyl-Cyo2, -(C₂-Cô)alkynyl-Cyo₂, -Cyo₂-Cyo3, -(C2-Cô)alkynyl-0-Cyo₂, -Cyo2-(Co-Cô)alkyl-0-(Co-C6)alkyl-Cyo3, a halogen atom, a cyano group, -C(0)-Roi4, -C(0)-NRoi₄Roi4’₅

Roii and Rou’ independently of one another are a hydrogen atom, an optionally substituted linear or branched (Ci-Cs)alkyl group, or -(Co-Céjalkyl-Cyoi, or the pair (Roi i, Roi i’) together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains, in addition to the nitrogen atom, l to 3 heteroatoms selected from O, S and N, wherein the N atoin is optionally substituted by a linear or branched (Ci-C6)alkyl group, and wherein one or more of the carbon atoms of the linear or branched (Ci-C6)alkyl group is optionally deuterated,

Roi2 représente -Cyos -Cyo5-(Co-C6)alkyl-Cyoô, -Cyo5-(Co-C₆)alkyl-0-(Co-C6)alkybCyo6, -Cyo5-(C₀-C6)alkyl-NRoi i-(Co-C₆)alkyl-Cyo6, -Cyo5-Cyo6-0-(Co-C6)alkyl-Cyo7, -Cyos-iCo-Cô)alkyl-Cyiw, -NH-C(0)-NH-Roi i, -C(0)-NRonRoii’, -NRoiiRon’, -ORon, -NRoh-C(0)-Roii’_s -O-(Ci-C6>alkyl-ORnii, -S0₂-Ron,or -C(0)-ORoii,

Roi3, Roi3’, Roi4 and Roi4’ independently of one another are a hydrogen atom, or an optionally substituted linear or branched (Cj-C6)alkyl group,

Cyoi, Cy₀₂, Cym, Cy₀5, Cyo6, Cyo? and Cyos independently of one another, are an optionally substituted cycloalkyl group, an optionally substituted heterocycloalkyl group, an optionally substituted aryl group or an optionally substituted heteroaryl group,

Rq15

Cy₀₉ is

Rois

, wherein R₀₁₅, R_0]6, and Ro₍₇ are as defined for fonnula (I),

R^ is

are as defined for formula fl) w

herein, at most, one of the Roi.Rog, or Roi2 groups, if present, is covalently attached to the linker, or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait of any ofthe foregoing.

In some embodiments, D comprises a compound of Formula (III):

wherein:

Roi is a linear or branched (Ci-Cô)alkyl group, R03 îs -0-(Ci-C6)aIkyl-NRoi 1R01Γ,

R 027

wherein Ron and Roi i’ independently of one another are a hydrogen atom, an optionally substituted linear or branched (Ci-Cô)alkyl group, or -(Co-C&)alkyl-Cyoi;

or the pair (Roi i. Roi 1 ’) together with the nitrogen atom to which they are attached form an aromatîc or non-aromatic ring containing 5 to 7 ring members, which optionally contains, in addition to the nitrogen atom, 1 to 3 heteroatoms selected from O, S and N, wherein the N atom may be substituted by 1 or 2 groups selected from a hydrogen atom or a linear or branched (Ci-Cé)alkyl group, and wherein R027 is a hydrogen atom and Ross Îs a -(CH₂)p0-O-SO₂-O^_ group or a -(CH₂)_Po-S02-ORo3o group;

Roo is a linear or branched (C₂-C&)alkynyl group or -Cyo₂,

R012 is -Cyos, -Cyo5-(Co-C6)alkyl-Cyoû, or -Cyo5-(Co-C6)alkyl-Cyo9,

Cyoi, Cyo₂, Cyos and Cyoô independently of one another, are a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group, each of which is optionally substituted,

Cyo^ is

Roiî, Roié, and Roi? are as defmed for formula (I), wherein, at most, one of the Rqî, Ro% or Roii groups, if présent, îs covalently attached to the linker, or the enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait of any of the foregoing.

In some embodiments, Cyoi, Cyo₂, Cyos, Cyos, independently of one another, is a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group, each of which is optionally substituted by one or more groups selected from halo; -(Ci-Cô)alkoxy;

- (Ci-Cô)haloalkyl; -(Ci-Côjhaloalkoxy; -(CH₂)_pü-0-S0₂-ORo3o; -(CH₂)_po-S0₂-ORoîo;

- 0-P(0)(ORo2o)₂; -O-P(O)(O’M⁺)₂; -CH₂-P(0)(OR₀₂o)₂;

- (CH₂)po-0-(CHRoi8-CHRoi9-0)_qo-Ro2o; hydroxy; hydroxy(Ci-C₆)alkyl;

- (CH₂)iû-Uo-(CH₂)so-heterocycloalkyl; or -Uo-(CH₂)qo-NRo₂iRo₂r.

In some embodiments, Roi is methyl or ethyl.

In some embodiments, Rm is -0-CH₂-CH₂-NRoiiRoir in which Rem and Rou’ form, together with the nitrogen atom carrying them, a piperazinyl group which may be substituted by a substituted by a hydrogen atom or a linear or branched (Ci-Cô)alkyl group.

In some embodiments, R03 comprises the formula:

wherein Ro₂7 is a hydrogen atom and R028 is a

-(CH₂)pQ-0-S0₂-ORo3û group, po is an integer equal to 0, 1, 2, or 3; and wherein R030 represents a hydrogen atom, a linear or branched (Ci-Cs)alkyl group or an aryl(Ci-Cô)alkyl group.

In some embodiments, Rt» comprises the formula:

In some embodiments, Cyoi, Cyos, Cyos, Cyo4, Cyos, Cyo6, Cyo?, Cyos and Cyoïo independently of one another, are an optionally substituted cycloalkyl group, an optionally substituted heterocycloalkyl group, an optionally substituted aryl group or an optionally substituted heteroaryl group, wherein the optional substituents are selected from optionally substituted linear or branched (Ci-Cû)alkyl, optionally substituted linear or branched (C₂-Cô)alkenyl group, optionally substituted linear or branched (C2-C&)alkynyl group, optionally substituted linear or branched (Ci-Côjalkoxy, optionally substituted (Ci-C₆)alkyl-S-, hydroxy, oxo (or N-oxide where appropriate), nitro, cyano, -C(0)-ORo’, -0-C(0)-Ro’, -C(0)-NRo’Ro”, NRo’Ro”, -(C=NRo’)-ORo”, linear or branched (Cj-C₆) haloalkyl, trifluoromethoxy, or halogen, wherein Ru’ and Ro” are each independently a hydrogen atom or an optionally substituted linear or branched (Ci-Cô)alkyl group, and wherein one or more of the carbon atoms of linear or branched (Ci-Cô)alkyl group is optionally deuterated.

In some embodiments, R09 is a Cyo2 group, preferably an aryl group, more preferably a phenyl group. In some embodiments, Cyoz is an optionally substituted aryl group.

In some embodiments, Cyos comprises a heteroaryl group selected from a pyrazolyl group and a pyrimidinyl group.

In some embodiments, Cyos is a pyrimidinyl group.

In some embodiments, Cyos is a pyrimidinyl group and Cyoô is phenyl group.

In some embodiments, the linker (L) is attached to D by a covalent bond from L to R03 of formulas (I), (II), or (III). In some embodiments, the linker (L) is attached to D by a covalent bond from L to R09 of formulas (I), (II), or (III).

In some embodiments, D comprises:

an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or a pharmaceutically acceptable sait of any of the foregoing.

In some embodiments, -(L-D) is formed from a compound selected from Table A or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait thereof. For compounds in Table A, depending on their electronîc charge, these compounds can contain one pharmaceutically acceptable monovalent anionic counterion ΜΓ. In some embodiments, the monovalent anionic counterion Mf can be selected from bromide, chloride, iodide, acetate, trifluoroacetate, benzoate, mesylate, tosylate, triflate, formate, or the like. In some embodiments, the monovalent anionic counterion Mf is trifluoroacetate or formate.

Table A. Exemplary Linker Drug Groups

ΊΊ

In some embodiments, the antibody-drug conjugate has a formula according to any one of the structures shown in Table B.

Table B. ADC Structures

100

101

102

103

L77-P1

L78-P1

L79-P1

104

L86-P1

Ο = antî-CD74 antibody or an antigen-binding fragment thereof

The ADCs depicted above can also be represented by the following formula: Ab-tL-D)^ wherein is an anti-CD 74 antibody or an antigen-binding fragment thereof covalently linked to the linker-payload (L/P) depicted above; p is an integer from l to 16. In some embodiments, p is an integer from 1 to 8. In some embodiments, p is an integer from 1 to 5, In some embodiments,/? is an integer from 2 to 4. In some embodiments,/? is 2. In some embodiments,p is 4. In some embodiments,p is determined by liquid chromatography-mass spectrometry (LC-MS).

As used herein, “L/P” refers to the linker-payloads, linker-drugs, or lînker-compounds disclosed herein and the tenus “L#-P#” and “L#-C#” are used interchangeably to refer to a spécifie linker-drug disclosed herein, while the codes “P#” and “C#” are used interchangeably to refer to a spécifie compound unless otherwise specified. For example, both “Ll-Cl” and “LlPl” refer to the same linker-payload structure disclosed herein, while both “Cl” and “PI” indicate the same compound disclosed herein, including an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait of any of the foregoing.

In some embodiments, the antibody or antigen-binding fragment binds to the target antigen CD74 on a cancer cell. In some embodiments, CD74 is a human CD74 isofonn. In some

105 embodiments, the human CD74 isoform is isoform 1 (NP_001020330.1) having an amino acid sequence of:

MHRRRSRSCREDQKPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTL LLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKPPKPVSKMRMATPLLMQ ALPMGALPQGPMQNATKYGNMTEDHVMHLLQNADPLKVYPPLKGSFPENLRHLKNT METIDWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKVLTKCQEEVSHIPAVHPGSFRP KCDENGNYLPLQCYGSIGYCWCVFPNGTEVPNTRSRGHHNCSESLELEDPSSGLGVTKQ DLGPVPM (SEQ ID NO:61).

In some embodiments, the human CD74 isofonn is isoform 2 (NP_004346.1) having an amno acid sequence of: MHRRRSRSCREDQRPVMDDQRDLISNNEQLPMLGRRPGAPESKCSRGALYTGFSILVTL LLAGQATTAYFLYQQQGRLDKLTVTSQNLQLENLRMKLPKPPKPVSKMRMATPLLMQ ALPMGALPQGPMQNATKYGNMTEDHVMHLLQNADPLKVYPPLKGSFPENLRHLKNT METIDWKVFESWMHHWLLFEMSRHSLEQKPTDAPPKESLELEDPSSGLGVTKQDLGPV PM (SEQ ID NO:62).

Also provided herein, in some embodiments, are compositions comprising multiple copies of an antibody-drug conjugale (e.g., any of the exemplary antibody-drug conjugales described herein). In some embodiments, the average p of the antibody-drug conjugales in the composition is from about 2 to about 4.

[01] Also provided herein, in some embodiments, are pharmaceutîcal compositions comprising an antibody-drug conjugale (e.g., any of the exemplary antibody-drug conjugales described herein) or a composition (e.g., any of the exemplary compositions described herein), and a pharmaceutically acceptable carrier.

Further provided herein, in some embodiments, are therapeutic uses for the described ADC compounds and compositions, e.g., in treating a cancer. In some embodiments, the present disclosure provides methods of treating a cancer (e.g., a cancer that expresses the CD74 antigen targeted by the antibody or antigen-binding fragment of the ADC). In some embodiments, the present disclosure provides methods of reducing or slowing the expansion of a cancer cell population in a subject. In some embodiments, the present disclosure provides methods of determining whether a subject having or suspected of having a cancer will be responsive to treatment with an ADC compound or composition disclosed herein.

106

An exeniplary embodiment is a method of treating a subject having or suspected of having a cancer, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions disclosed herein). In some embodiments, the cancer expresses the target antigen CD74. . In some embodiments, the cancer is a tumor or a hematological cancer. In some embodiments, the cancer is a breast cancer, multiple myeloma, plasma cell myeioma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the cancer is a lymphoma or gastric cancer.

Another exemplary embodiment is a method of reducing or inhibiting the growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions disclosed herein). In some embodiments, the tumor expresses the target antigen CD74. . In some embodiments, the tumor is a breast cancer, gastric cancer, bladder cancer, brain cancer, cervical cancer, colorectal cancer, esophageal cancer, hepatocellular cancer, melanoma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the tumor is a gastric cancer. In some embodiments, administration of the antibody-drug conjugate, composition, or pharmaceutical composition reduces or inhibits the growth of the tumor by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%.

Another exemplary embodiment is a method of reducing or slowing the expansion of a cancer cell population in a subject, comprising administering to the subject a therapeutically effective amount of an antibody-drug conjugate, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugates, compositions, or pharmaceutical compositions disclosed herein). In some embodiments, the cancer cell population expresses the target antigen CD74. . In some embodiments, the cancer cell population is from a tumor or a hematological cancer. In some embodiments, the cancer cell population is from a breast cancer,

107 multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follîcular lymphoma, lymphoid malîgnancies of T-cell or B-cell orîgin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the cancer cell population is from a lymphoma or gastric cancer. In some embodiments, administration of the antibody~drug conjugale, composition, or pharmaceutical composition reduces the cancer cell population by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%. In some embodiments, administration of the antibody-drug conjugale, composition, or pharmaceutical composition slows the expansion of the cancer cell population by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%.

Another exemplary embodiment is an antibody-drug conjugale, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugales, compositions, or pharmaceutical compositions disclosed herein) for use in treating a subject having or suspected of having a cancer. Jn some embodiments, the cancer expresses the target antigen CD74. . In some embodiments, the cancer is a tumor or a hematoiogical cancer. In some embodiments, the cancer îs a breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follîcular lymphoma, lymphoid malîgnancies of T-cell or B-cell orîgin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the cancer is a lymphoma or gastric cancer.

Another exemplary embodiment is a use of an antibody-drug conjugale, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugales, compositions, or pharmaceutical compositions disclosed herein) in treating a subject having or suspected of having a cancer. In some embodiments, the cancer expresses the target antîgen CD74. In some embodiments, the cancer is a tumor or a hematoiogical cancer. In some embodiments, the cancer

108 is a breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastîc leukemia, follicular lymphoma, lymphoid malignancies of T-celi or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-smalî cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the cancer is a lymphoma or gastric cancer.

Another exemplary embodiment is a use of an antibody-drug conjugale, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugales, compositions, or pharmaceutical compositions disclosed herein) in a method of manufacturing a médicament for treating a subject having or suspected of having a cancer. In some embodiments, the cancer expresses the target antigen CD74. In some embodiments, the cancer is a tumor or a hematological cancer. In some embodiments, the cancer is a breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastîc leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the cancer îs a lymphoma or gastric cancer.

Another exemplary embodiment is a method of determining whether a subject having or suspected of having a cancer will be responsive to treatment with an antibody-drug conjugale, composition, or pharmaceutical composition (e.g., any of the exemplary antibody-drug conjugales, compositions, or pharmaceutical compositions disclosed herein) by provîding a biological sample from the subject; contacting the sample with the antibody-drug conjugale; and detecting binding of the antibody-drug conjugale to cancer cells in the sample. In some embodiments, the cancer cells in the sample express the target antigen CD74. In some embodiments, the cancer expresses the target antigen CD74. . In some embodiments, the cancer is a tumor or a hematological cancer. In some embodiments, the cancer is a breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastîc leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin,

109 melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the cancer is a lymphoma or gastric cancer. In some embodiments, the sample is a tissue biopsy sample, a blood sample, or a bone marrow sample.

Methods of producing the described ADC compounds and compositions are also disclosed. An exemplary embodiment is a method of producing an antibody-drug conjugate by reacting an antibody or antigen-binding fragment with a cleavable linker joîned to an Mcl-1 inhibitor under conditions that allow conjugation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. IA shows in vitro activity of CD74-L7-P1 and PI payload in DLBCL cell lines (CTG 72h).

FIG. IB shows in vitro activity of CD74-L7-P1, IgG-L7-Pl and PI payload in DLBCL cell lines (CTG 72h).

FIG. 2 shows in vitro activity of various CD74 ADCs and corresponding payloads in Monomacl cell line (CTG 72h).

FIG. 3 shows the in vitro activity of CD74 MCL-1 antibody drug conjugate CD74-L7Pl, Isotype IgG-L7-Pl ADC, and MCL-1 free payload PI against endogenous cancer cell lines including KMS-21BM, KMS-20, M0N0-MAC-1, NOMO-1, Kasumi-6, and EOL-1.

FIG. 4 shows the in vitro activity of CD74 MCL-1 antibody drug conjugate CD74-L5Pl, Isotype IgG-L5-Pl ADC, and MCL-1 free payload PI, alone or in combination with venetoclax, against MONO-MAC-1, NOMO-1, and EOL-1.

FIG. 5 shows the in vitro activity of CD74 MCL-1 antibody drug conjugate CD74-L5Pl, Isotype IgG-L5-Pl ADC, and MCL-1 free payload PI, alone or in combination with Compound Al, against MONO-MAC-I, NOMO-1, and EOL-1.

FIG. 6 shows tumor volume (mm³) of Nomol-grafted female SCID mice upon treatment with IgGl-Linker-Payload Fc silent, anti-CD74_CysmAb Fc silent and antî-CD74_CysmAb Fc silent_L5-Pl (30 mg/kg, administered once IV), alone or in combination with venetoclax (50mpk, administered 4ON/3OFF/4ON PO) (n=6). Triangle and dîamond shapes indicate treatment schedules of venetoclax and antibodies, respectively.

FIG. 7 shows % of body weight loss of Nomol-grafted female SCID mice upon treatment with IgGl-Linker-Payload Fc silent, anti-CD74_CysmAb Fc silent and anti-

110

CD74_CysmAb Fc silent_L5-P l (30 mg/kg, adminîstered once IV), alone or in combination with venetoclax (50mpk, adminîstered 4ON/3OFF/4ON PO) (n=6).

FIG. 8 shows tumor volume (mm³) of Karpas422-grafted female NSG mice upon treatment with IgGl-Lînker-Payload Fc silent, anti-CD74_CysmAb Fc si lent and antiCD74_CysmAb Fc silent_L5-Pl (30 mg/kg, adminîstered once IV), alone or in combination with venetoclax (50mpk, adminîstered 4ON/3OFF/4ON PO) (n=6). Triangle and diamond shapes indicate treatment schedules of venetoclax and antibodies, respectiveîy.

FIG. 9 shows % of body weight loss of Karpas422-grafted female NSG mice upon treatment with IgGl-Linker-Payload Fc silent, anti-CD74_CysmAb Fc silent and antiCD74_CysmAb Fc silent_L5-Pl (30 mg/kg, adminîstered once IV), alone or in combination with venetoclax (50mpk, adminîstered 4ON/3OFF/4ON PO) (n=6)

FIG. 10 shows tumor volume (mm³) of Monomac 1-grafted female SCID mice upon treatment with IgGl-Lînker-Payload Fc silent, anti-CD74_CysmAb Fc silent and antiCD74_CysmAb Fc silent_L5-Pl (30 mg/kg, adminîstered once IV), alone or in combination with venetoclax (50mpk, adminîstered 2ON/2OFF/5ON/2OFF/4ON PO) (n=6). Triangle and diamond shapes indicate treatment schedules of venetoclax and antibodies, respectiveîy.

FIG. 11 shows % of body weight loss of Monomac 1-grafted female SCID mice upon treatment with IgGl-Linker-Payload Fc silent, anti-CD74_CysmAb Fc silent and antiCD74_CysmAb Fc silent_L5-Pi (30 mg/kg, adminîstered once IV), alone or in combination with venetoclax (50mpk, adminîstered 2ON/2OFF/5ON/2OFF/4ON PO) (n=6).

FIG. 12 shows tumor volume (mm³) of EOL1 human acute myeloid (éosinophilie) leukemia xenografted femal SCID mice upon treatment with CD74-L5-PI Fc silent in combination with venetoclax along with varions control groups.

FIG. 13 shows % of body weight loss of EOL1 human acute myeloid (éosinophilie) leukemia xenografted femal SCID mice upon treatment with CD74-L5-P1 Fc silent in combination with venetoclax along with varions control groups.

FIG. 14 shows tumor volume (mm³) of EOL1 human acute myeloid (éosinophilie) leukemia xenografted femal SCID mice upon treatment with CD74-ADCs with different linker payloads (L5-P1, L30-P1, L31-P1, L32-P1, L33-P1 andL34-Pl).

FIG. 15 shows % of body weight loss of EOL1 human acute myeloid (éosinophilie) leukemia xenografted femal SCID mice upon treatment with CD74-ADCs with different linker payloads (L5-P1, L30-P1, L31-P1, L32-P1, L33-P1 and L34-P1).

111

FIG. 16 shows an exemplary site-specific antibody conjugaiion using bacterial transglutaminase (BTG).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The disclosed compositions and methods may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure.

Throughout this text, the descriptions refer to compositions and methods of using the compositions. Where the disclosure describes or claims a feature or embodiment associated with a composition, such a feature or embodiment is equally applicable to the methods of using the composition. Likewise, where the disclosure describes or claims a feature or embodiment associated with a method of using a composition, such a feature or embodiment is equally applicable to the composition.

When a range of values is expressed, it includes embodiments using any particular value within the range. Further, reference to values stated in ranges includes each and every value within that range. Ail ranges are inclusive of their endpoints and combinable. When values are expressed as approximations, by use of the antécédent “about,” it will be understood that the particular value fonns another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictâtes otherwise. The use of “or” will mean “and/or” unless the spécifie context of its use dictâtes otherwise. Ail references cited herein are incorporated by reference for any purpose. Where a reference and the spécification conflict, the spécification will control.

Unless the context of a description indicates otherwise, e.g., in the absence of Symbol s indicating spécifie point(s) of connectivity, when a structure or fragment of a structure is drawn, it may be used on its own or attached to other components of an ADC, and it may do so with any orientation, e.g., with the antibody attached at any suitable attachment point to a Chemical moiety such as a linker-drug. Where indicated, however, components of an ADC are attached in the orientation shown in a given formula. For example, if Formula (1) is described as Ab-(L-D)_p

Dj and the group “-(L-D)” is described as ' /, then the elaborated structure of

Ab-j-R¹-^—E-d) Ab-FD—E—L-rJ

Formula (l)is ' . It is not 'P.

112

It is to be appreciated that certain features of the disclosed compositions and methods, which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed compositions and methods that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination.

As used throughoui this application, antibody drug conjugales can be identified using a naming convention in the general fonnat of “target antigen/antibody-linker-payload”. For example only, if an antibody drug conjugate is referred to as “Target X-L0-P0”, such a conjugate would comprise an antibody that binds Target X, a linker designated as LO, and a payload designated as PO. Alternative!y, if an antibody drug conjugate is referred to as “anti-Target XLO-PO”, such a conjugate would comprise an antibody that binds Target X, a linker designated as LO, and a payload designated as PO. In another alternative, if an antibody drug conjugate is referred to as “AbX-L0-P0”, such a conjugate would comprise the antibody designated as AbX, a linker designated as LO, and a payload designated as PO. An control antibody drug conjugate comprising a non-specific, isotype control antibody may be referenced as “isotype control IgGlLO-PO” or “IgGl-L0-P0”.

Any formula given herein is also intended to represent unlabeled forms as well as îsotopically labeied forms of the compounds. Isotopically labeied compounds hâve structures depicted by the formulae given herein except that one or more atoms are repiaced by an atom having a selected atomic mass or mass number. Isotopes that can be incorporated into compounds of the invention include, for example, isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, such as ³H, ^HC, ¹³C, ^l4C, ^l5N, ¹⁸F, and ³⁶C1. Accordingly, it should be understood that the présent disclosure includes compounds that incorporate one or more of any of the aforementioned isotopes, including for example, radioactive isotopes, such as ³ H and ¹⁴C, or those into which non-radioactive isotopes, such as ²H and ¹³C are présent. Such isotopically labelled compounds are useful in metabolic studies (with ^l4C), reaction kinetic studies (with, for example ²H or ³H), détection or imaging techniques, such as positron émission tomography (PET) or single-photon émission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients. In particular, an ^,8F or labeied compound may be particularly désirable for PET or SPECT studies. Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art, e.g., using an appropriate isotopically-labeled reagents in place of the non-labeled reagent previously employed.

113

Définitions

Various terms relatingto aspects ofthe description are used throughout the spécification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the définitions provîded herein.

As used herein, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictâtes otherwise. The terms “comprisîng”, “having”, “being of ’ as in “being of a Chemical formula”, “including”, and “containing” are to be construed as open tenns (i.e., meaning “including but not limited to”) unless otherwise noted. Additionaily whcnever “comprisîng” or another open-ended terni is used in an embodiment, it is to be understood that the same embodiment can be more narrowly claimed using the intermediate term “consisting essentially of ’ or the closed tenn “consisting of’.

The term about or approximately, when used in the context of numerical values and ranges, refers to values or ranges that approximate or are close to the recited values or ranges such that the embodiment may perform as intended, as is apparent to the skilled person from the teachîngs contained herein. In some embodiments, about means plus or minus 20%, 15%, 10%, 5%, 1%, 0.5%, or 0.1% of a numerical amount. In one embodiment, the term “about” refers to a range of values which are 10% more or less than the specified value. In another embodiment, the terni “about” refers to a range of values which are 5% more or less than the specified value. In another embodiment, the term “about” refers to a range of values whîch are 1% more or less than the specified value.

The terms “antibody-drug conjugate,” “antibody conjugale,” “conjugale,” “immunoconjugate,” and “ADC” are used interchangeably, and refer to one or more therapeutic compounds (e.g., an Mcl-1 inhibitor) that is linked to one or more antibodies or antigen-binding fragments. In some embodiments, the ADC is defined by the generic formula: Ab-fL-D)^ (Formula 1), wherein Ab = an antibody or antigen-binding fragment, L = a linker moiety, D = a drug moiety (e.g., an Mcl-1 inhibitor drug moiety), and p = the nuinber of drug moieties per antibody or antigen-binding fragment. In ADCs comprisîng an Mcl-1 inhibitor drug moiety, “p” refers to the number of Mcl-1 inhibitor compounds linked to the antibody or antigen-binding fragment.

The term antibody is used in the broadest sense to refer to an immunoglobulin molécule that recognizes and specifically binds to a target, such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen récognition

114 site within the variable région of the immunoglobulin molécule. An antibody can be polyclonal or monoclonal, multiple or single chain, or an intact immunoglobulin, and may be derived from natural sources or from recombinant sources. An “intact” antibody is a glycoprotein that typically comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable région (abbrevîated herein as VH) and a heavy chain constant région. The heavy chain constant région comprises three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable région (abbreviated herein as VL) and a light chain constant région. The light chain constant région is comprised of one domain, CL. The VH and VL régions can be further subdivided into régions of hypervariability, termed compleinentarity determinîng régions (CDR), interspersed with régions that are more conserved, termed framework régions (FR). Each VH and VL îs composed of three CDRs and four FRs arranged from amino-terminus to carboxyl-terminus in the following order: FRI, CDR1, FR2, CDR2, FR3, CDR3, FR4, The variable régions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant régions of the antibodies may médiate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Cl q) of the classical complément system. An antibody can be a monoclonal antibody, human antibody, humanized antibody, camelised antibody, or chimeric antibody. The antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), or subclass. An antibody can be an intact antibody or an antigenbinding fragment thereof.

The tenu “antibody fragment” or “antigen-binding fragment” or “functional antibody fragment,” as used herein, refers to at least one portion of an antibody that retains the ability to specifically interact with (e.g., by binding, steric hinderance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen (e.g., CD74). Antigen-binding fragments may also retain the ability to întemalize into an antigen-expressing cell. In some embodiments, antigen-binding fragments also retain immune effector activity. The ternis antibody, antibody fragment, antigenbinding fragment, and the like, are intended to embrace the use of binding domains from antibodies in the context of larger macromolecules such as ADCs. It has been shown that fragments of a full-length antibody can perfonn the antigen binding function of a full-length antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv fragments, scFv antibody fragments, disulfide-Iinked Fvs (sdFv), a Fd fragment consisting of the VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (either VL or

115

VH), camelid VHH domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments Jinked by a disulfide bridge at the hinge région, and an isolated CDR or other epitope binding fragments of an antibody. An antigenbînding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, bispecific or multispecific antibody constructs, ADCs, v-NAR and bis-scFv (see, e.g., Holliger and Hudson (2005) Nat Biotechnol. 23(9): 1126-36). Antigen-binding fragments can also be grafted into scaffblds based on polypeptides such as a fibronectin type III (Fn3) (see US Patent No. 6,703,199, which describes fibronectin polypeptide minibodies). The term “scFv” refers to a fusion protein comprising at least one antigen-binding fragment comprising a variable région of a light chain and at least one antigen-binding fragment comprising a variable région of a heavy chain, wherein the light and heavy chain variable régions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, an scFv may hâve the VL and VH variable régions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL. Antigen-binding fragments are obtained using conventional techniques known to those of skill in the art, and the binding fragments are screened for utility (e.g., binding affinity, internai ization) in the saine manner as are intact antibodies. Antigen-binding fragments, for example, may be prepared by cleavage of the intact protein, e.g., by protease or Chemical cleavage.

The term “complementarity determining région” or “CDR,” as used herein, refers to the sequences of amino acids within antibody variable régions which confer antigen specificity and binding affinity. For example, in general, there are three CDRs in each heavy chain variable région (e.g., HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable région (LCDR1, LCDR2, and LCDR3). The précisé amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, includîng those described by Kabat et al. (1991) “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al. (1997) J Mol Biol. 273(4):927-48 (“Chothia” numbering scheme); ImMunoGenTics (IMGT) numbering (Lefranc (2001) Nucleic Acids Res. 29( l):207-9; Lefranc et al. (2003) Dev Comp Immunok 27(1):55-77) (“IMGT” numbering scheme); or a combination thereof. In a combined Kabat and Chothia numbering scheme for a given CDR région (for example, HC

116

CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, or LC CDR3), in some embodiments, the CDRs correspond to the amino acid residues that are defined as part of the Kabat CDR, together with the amino acid residues that are defined as part of the Chothia CDR. As used herein, the CDRs defined accordîng to the “Chothia” number scheme are also sometimes referred to as “hypervariable loops.”

In some embodiments, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDRl) (e.g., insertion(s) after position 35), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDRl) (e.g., insertion(s) after position 27), 50-56 (LCDR2), and 89-97 (LCDR3). In some embodiments, under Chothia, the CDR amino acids in the VH are numbered 26-32 (HCDRl) (e.g., insertion(s) after position 31), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDRl) (e.g., insertion(s) after position 30), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR définitions of both Kabat and Chothia, in some embodiments, the CDRs comprise or consist of, e.g., amino acid residues 26-35 (HCDRl), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDRl), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. In some embodiments, under IMGT, the CDR amino acid residues in the VH are numbered approximately 26-35 (CDRl), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residues in the VL are numbered approximately 27-32 (CDRl), 50-52 (CDR2), and 89-97 (CDR3). In some embodiments, under IMGT, the CDR régions of an antibody may be detennined using the program IMGT/DomainGap Align.

The tenn monoclonal antibody, as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antîbodies comprising the population are identîcal except for possible naturally occurring mutations that may be présent in minor amounts. Monoclonal antibodies are highly spécifie, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody préparations typically include a multitude of antibodies directed against (or spécifie for) different epitopes. The modifier monoclonal indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the présent disclosure may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be made by recombinant DNA methods (see, e.g., US Patent No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody

117 libraries using the techniques described in Clackson et al. (1991) Nature 352:624-8, and Marks et al. (1991) J Mol Biol. 222:581-97, for example. The term also includes préparations of antibody molécules of single molecuîar composition. A monoclonal antibody composition displays a single binding specîficity and affinity for a parti cular epitope.

The monoclonal antibodies described herein can be non-human, human, or humanized. The tenu specifically includes chimeric antibodies, in which a portion of the heavy and/or light chain is identîcal with or homologous to corresponding sequences in antibodies derived from a particular specîes or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they specifically bind the target antigen and/or exhibit the desired biological activity.

The tenu “human antibody,” as used herein, refers an antibody produced by a human or an antibody having an amino acid sequence of an antibody produced by a human. The terni includes antibodies having variable régions in which both the framework and CDR régions are derived from sequences of human orîgin. Furthermore, if the antibody contains a constant région, the constant région is also derived from such human sequences, e.g., human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for exampie, as described in Knappik et al. ((2000) J Mol Biol. 296(1):57-86). The structures and locations of immunoglobulin variable domains, e.g., CDRs, may be defined using well known numbering schemes, e.g., the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia, and/or ImMunoGenTics (IMGT) numbering. The human antibodies of the invention may include amino acid residues not encoded by human sequences (e.g., mutations întroduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or a conservative substitution to promote stability or manufacturing). However, the term “human antibody,” as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, hâve been grafted onto human framework sequences.

The term “recombinant human antibody,” as used herein, refers to a human antibody that is prepared, expressed, created, or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell

118 transformed to express the human antibody, e.g., from a transfectoma, antibodies isolated from a recornbinant, combinatorial human antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of ail or a portion of a human immunoglobulin gene, sequences to other DNA sequences. Such recombinant human antibodies hâve variable régions in which the framework and CDR régions are derived from human germline immunoglobulin sequences. In some embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences îs used, m vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL régions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline répertoire in vivo.

The tenn “chimeric antibody,” as used herein, refers to antibodies wherein the amino acid sequence of the immunoglobulin molécule is derived from two or more species. In some instances, the variable régions of both heavy and light chains correspond to the variable régions of antibodies derived from one species with the desired specificity, affinity, and activity while the constant régions are homologous to antibodies derived from another species (e.g., human) to minimize an immune response in the latter species.

As used herein, the tenn humanized antibody refers to fonns of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies are a type of chimeric antibody which contain minimal sequence derived from nonhuman immunoglobulin. In general, the humanized antibody will comprise substantially ail of at least one, and typically two, variable domains, in which ail or substantially ail of the hypervariable loops correspond to those of a non-human immunoglobulin and ail or substantially ail of the framework (FR) régions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant région (Fc), typically that of a human immunoglobulin. The humanized antibody can be further modified by the substitution of residues, either in the Fv framework région and/or within the replaced non-human residues to refîne and optimize antibody specificity, affinity, and/or activity.

The terni “Fc région,” as used herein, refers to a polypeptide comprising the CH3, CH2 and at least a portion of the hinge région of a constant domain of an antibody. Optionally, an Fc région may include a CH4 domain, présent in some antibody classes. An Fc région may comprise the entire hinge région of a constant domain of an antibody. In some embodiments, an

119 antibody or antigen-binding fragment comprises an Fc région and a CHl région of an antibody. In sonie embodiments, an antibody or antigen-binding fragment comprises an Fc région CH3 région of an antibody. In some embodiments, an antibody or antigen-binding fragment comprises an Fc région, a CH l région, and a kappa/lambda région from the constant domain of an antibody. In some embodiments, an antibody or antigen-binding fragment comprises a constant région, e.g., a heavy chain constant région and/or a light chain constant région. In some embodiments, such a constant région is modified compared to a wild-type constant région. That is, the polypeptide may comprise alterations or modifications to one or more of the three heavy chain constant domains (CHI, CH2, or CH3) and/or to the light chain constant région domain (CL). Example modifications include additions, délétions, or substitutions of one or more amino acids in one or more domains. Such changes may be încluded to optimize effector function, half-life, etc.

“Internalizing” as used herein in reference to an antibody or antigen-binding fragment refers to an antibody or antigen-binding fragment that is capable of being taken through the cell· s lipid bilayer membrane to an internai compartment (i.e., “internalized”) upon binding to the cell, preferably înto a degradative compartment in the cell. For example, an internalizing anti-HER2 antibody is one that is capable of being taken into the cell after binding to HER2 on the cell membrane. In some embodiments, the antibody or antigen-binding fragment used in the ADCs disclosed herein targets a cell surface antigen (e.g., CD74) and is an internalizing antibody or internalizing antigen-binding fragment (i.e., the ADC transfers through the cellular membrane after antigen binding). In some embodiments, the internalizing antibody or antigen-binding fragment binds a receptor on the cell surface. An internalizing antibody or internalizing antigenbinding fragment that targets a receptor on the cell membrane may induce receptor-mediated endocytosis. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment is taken into the cell via receptor-mediated endocytosis.

“Non-internalizing” as used herein in reference to an antibody or antigen-binding fragment refers to an antibody or antigen-binding fragment that remains at the cell surface upon binding to the cell. In some embodiments, the antibody or antigen-binding fragment used in the ADCs disclosed herein targets a cell surface antigen and is a non-internalizing antibody or noninternalizing antigen-binding fragment (i.e., the ADC remains at the cell surface and does not transfer through the cellular membrane after antigen binding). In some embodiments, the noninternalizing antibody or antigen-binding fragment binds a non-internalizing receptor or other cell surface antigen. Exemplary non-internalizing cell surface antigens include but are not

120 limited to CAI25 and CEA, and antibodies that bind to non-intemalizing antigen targets are also known in the art (see, e.g., Bast et al. f 1981 ) J Clin Invest. 68(5): 1331-7; Scholler and Urban (2007) Biomark Med. 1(4):513-23; and Boudousq et al. (2013) PLoS One 8(7):e69613).

The term “hindi ng specificity,” as used herein, refers to the ability of an individual antibody or antigen binding fragment to preferentially react with one antigenîc déterminant over a different antigenîc déterminant. The degree of specificity indicates the extent to which an antibody or fragment preferentially binds to one antigenîc déterminant over a different antigenîc déterminant. Also, as used herein, the term spécifie, specifically binds, and binds specifically refers to a binding reaction between an antibody or antigen-binding fragment (e.g., an anti-CD74 antibody) and a target antigen (e.g., CD74) in a heterogeneous population of proteins and other biologics. Antibodies can be tested for specificity of binding by comparing binding to an appropriate antigen to binding to an irrelevant antigen or antigen mixture under a given set of conditions. If the antibody binds to the appropriate antigen with at least 2, 5, 7, 10 or more times more affmity than to the irrelevant antigen or antigen mixture, then it is considered to be spécifie. A “spécifie antibody” or a “target-specific antibody” is one that only binds the target antigen (e.g., CD74), but does not bind (or exhibits minimal binding) to other antigens. In some embodiments, an antibody or antigen-binding fragment that specifically binds a target antigen (e.g., CD74) has a Kd of less than 1 xlO^-6 M, less than IxlO'⁷ M, less than 1x10'^s M, less than IxlO^-9 M, less than 1x10’¹⁰ M, less than 1x10¹¹ M, less than 1 xlO^-12 M, or less than 1x10'¹³M. In some embodiments, the Kd is 1 pM to 500 pM. In some embodiments, the Kd is between 500 pM to 1 μΜ, 1 μΜ to 100 nM, or 100 mM to 10 nM.

The terni “affmity,” as used herein, refers to the strength of interaction between antibody and antigen at single antigenîc sites. Without being bound by theory, within each antigen binding site, the variable région of the antibody “arm” interacts through weak non-covalent forces with the antigen at numerous sites; the more interactions, typically the stronger the affmity. The binding affmity of an antibody is the sum of the attractive and répulsive forces operating between the antigenîc déterminant and the binding site of the antibody.

The term k_on or ”k_a refers to the on-rate constant for association of an antibody to the antigen to form the antibody/antigen complex. The rate can be determined using standard assays, such as a surface plasmon résonance, biolayer inferometry, or ELISA assay.

The term koff or kd refers to the off-rate constant for dissociation of an antibody from the antibody/antigen complex. The rate can be determined using standard assays, such as a surface plasmon résonance, biolayer inferometry, or ELISA assay.

121

The tenu Kd refers to the equilibrium dissociation constant of a particular antibodyantigen interaction. Kd is calculated by k_a/kd. The rate can be determined using standard assays, such as a surface plasmon résonance, biolayer inferometry, or ELI SA assay.

The terni “epitope” refers to the portion of an antigen capable of being recognized and specifically bound by an antibody (or antigen-binding fragment). Epitope déterminants generally consist of chemically active surface groupings of molécules such as amino acids or carbohydrate or sugar side chains and can hâve spécifie three-dimensional structural characteristics, as well as spécifie charge characteristics. When the antigen is a polypeptide, epitopes can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of the polypeptide. An epitope may be “linear” or “conformational.” Confonnational and linear epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. The epitope bound by an antibody (or antigen-binding fragment) may be identified using any epitope mapping technique known in the art, including X-ray crystallography for epitope identification by direct visualization of the antigen-antibody complex, as well as monitoring the binding of the antibody to fragments or mutated variations of the antigen, or monitoring solvent accessibility of different parts of the antibody and the antigen. Exemplary strategies used to map antibody epitopes include, but are not lîmited to, array-based oligo-peptide scanning, limited proteolysis, site-directed mutagenesîs, high-throughput mutagenesîs mapping, hydrogen-deuterium exchange, and mass spectrometry (see, e.g., Gershoni et al. (2007) BioDrugs 21:145-56; and Hager-Braun and Tomer (2005) Expert Rev Proteomics 2:745-56).

Compétitive bindmg and epitope binning can also be used to détermine antibodies sharing identical or overlapping epitopes. Compétitive binding can be evaluated using a crossblocking assay, such as the assay described in “Antibodies, A Laboratory Manual,” Cold Spring Harbor Laboratory, Harlow and Lane (l^st édition 1988, 2^nd édition 2014). In some embodiments, compétitive binding is identified when a test antibody or binding protein reduces binding of a reference antibody or binding protein to a target antigen such as CD74 (e.g., a binding protein comprising CDRs and/or variable domains selected from those identified in Tables 3-5), by at least about 50% in the cross-blocking assay (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or more, or any percentage in between), and/or vice versa. In some embodiments, compétitive binding can be due to shared or similar (e.g., partîally overlapping) epitopes, or due to steric hindrance where antibodies or binding proteins bind at nearby epitopes (see, e.g., Tzartos, Methods in Molecular Biology (Morris, ed. (1998) vol. 66, pp. 55-66)). In some

122 embodiments, compétitive binding can be used to sort groups of binding proteins that share similar epitopes. For example, binding proteins that compete for binding can be “binned” as a group of binding proteins that hâve overlapping or nearby epitopes, while those that do not compete are placed in a separate group of binding proteins that do not hâve overlapping or nearby epitopes.

As used herein, the tenus peptide, polypeptide, and protein are used interchangeably to refer to a polymer of amino acid résidu es. The tenus encompass amino acid polymers comprising two or more amino acids joined to each other by peptide bonds, amino acid polymers in which one or more amino acid residues is an artifîcial Chemical mimetic of a corresponding naturally-occurring amino acid, as well as naturally-occurring amino acid polymers and non-naturally-occurring amino acid polymers. The tenus include, for example, biologîcally active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, dérivatives, analogs, fusion proteins, among others. The tenus also include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof. Unless otherwise indicated, a particular polypeptide sequcnce also implicitly en compassés conservatively modified variants thereof.

A recombinant” protein refers to a protein (e.g., an antibody) made using recombinant techniques, e.g., through the expression of a recombinant nucleic acid.

An isolated protein refers to a protein unaceompanied by at least some of the material with which it is normally associated in its natural State. For example, a naturally-occurring polynucleotide or polypeptide présent in a lîving organism is not isolated, but the same polynucleotide or polypeptide separated from some or ail of the coexisting materials in the living organism, is isolated. The définition includes the production of an antibody in a wide variety of organisms and/or host cells that are known in the art.

An isolated antibody, as used herein, is an antibody that has been identifïed and separated from one or more (e.g., the majorîty) of the components (by weight) of its source environment, e.g., from the components of a hybridoma cell culture or a different cell culture that was used for its production. In some embodiments, the séparation is performed such that it sufficiently removes components that may otherwise interféré with the suitability of the antibody for the desired applications (e.g., for therapeutic use). Methods for preparing isolated antibodies are known in the art and include, without limitation, protein A chromatography, anion exchange chromatography, cation exchange chromatography, virus retentive filtration, and ultrafiltration.

123

As used herein, the term “variant” refers to a nucleic acid sequence or an amino acid sequence that differs from a reference nucleic acid sequence or amino acid sequence respectively, but retains one or more biological properties of the reference sequence. A variant may contain one or more amino acid substitutions, délétions, and/or insertions (or corresponding substitution, délétion, and/or insertion of codons) with respect to a reference sequence. Changes in a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid sequence, or may resuit in amino acid substitutions, additions, délétions, frisions, and/or truncations. In some embodiments, a nucleic acid variant disclosed herein encodes an identica] amino acid sequence to that encoded by the unmodified nucleic acid or encodes a modified amino aeid sequence that retains one or more functional properties of the unmodified amino acid sequence. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the unmodified peptide and the variant are closely similar overall and, in many régions, identical. In some embodiments, a peptide variant retains one or more functional properties of the unmodified peptide sequence. A variant and unmodified peptide can differ in amino acid sequence by one or more substitutions, additions, délétions in any combination.

A variant of a nucleic acid or peptide can be a naturally-occurring variant or a variant that is not known to occur naturally. Variants of nucleic acids and peptides may be made by mutagenesis techniques, by direct synthesis, or by other techniques known in the art. A variant does not necessarily require physical manipulation of the reference sequence. As long as a sequence contains a different nucleic acid or amino acid as compared to a reference sequence, it is considered a “variant” regardless of how it was synthesized. In some embodiments, a variant has high sequence identity (i.e., 60% nucleic acid or amino acid sequence identity or higher) as compared to a reference sequence. In some embodiments, a peptide variant encompasses polypeptides having amino acid substitutions, délétions, and/or insertions as long as the polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% amino acid sequence identity with a reference sequence, or with a corresponding segment (e.g., a functional fragment) of a reference sequence, e.g., those variants that also retain one or more fonctions of the reference sequence. In some embodiments, a nucleic acid variant encompasses polynucleotides having amino acid substitutions, délétions, and/or insertions as long as the polynucleotîde has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at

124 least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% nucleîc acid sequence identity with a référencé sequence, or with a corresponding segment (e.g., a functional fragment) of a reference sequence.

The tenu “conservât!vely modified variant” applies to both amino acid and nucleic acid sequences. For nucleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU ail encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each co don in a nucleic acid (except AU G, which is ordinarily the only codon for méthionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molécule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence. For polypeptide sequences, conservatively modified variants include individual substitutions, délétions, or additions to a polypeptide sequence which resuit in the substitution of an amino acid with a chemically similar amino acid. Conservative substitutions providing functionally similar amino acids are well known in the art.

The term “conservative sequence modifications,” as used herein, refers to amino acid modifications that do not significantly affect or alter the binding characteristics of, e.g., an antibody or antigen-binding fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and délétions. Modifications can be introduced into an antibody or antigen-binding fragment b y standard techniques known in the art, such as, e.g., site-dîrected mutagenesis and PCR-mediated mutagenesîs. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains hâve been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, méthionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and

125 aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, in some embodiments, one or more amino acid residues within an antibody can be replaced with other amino acid residues from the saine side chaîn family and the altered antibody can be tested using the functional assays described herein.

The tenu “homologous” or “identity,” as used herein, refers to the subunit sequence identity between two polymeric molécules, e.g., between two nucleic acid molécules, such as, two DNA molécules or two RNA molécules, or between two polypeptide molécules. When a subunit position in both of the two molécules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molécules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions. For example, if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are matched or homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.

Percentage of “sequence identity” can be determined by comparing two optimally aligned sequences over a comparison window, where the fragment of the amino acid sequence in the comparison window may comprise additions or délétions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or délétions) for optimal alignaient of the two sequences. The percentage can be calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yieid the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplyîng the resuit by 100 to yieid the percentage of sequence identity. The output is the percent identity of the subject sequence with respect to the query sequence. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignaient of the two sequences. Generally, the amino acid identity or homology between proteins disclosed herein and variants thereof, including variants of target antigens (such as CD74) and variants of antibody variable domains (including individual variant CDRs), is at least S0% to the sequences depicted herein, e.g., identities or homologies of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, almost 100%, or 100%.

The comparison of sequences and détermination of percent identity between two sequences can be accomplîshed using a mathematical algorithm. In some embodiments, the

126 percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J Mol Biol. 48:444-53) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, ï4, 12, 10, 8, 6, or 4 and a length weight of l, 2, 3, 4, 5, or 6. In some embodiments, the percent identity between two nucléotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of l, 2, 3, 4, 5, or 6. An exemplary set of parameters is a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucléotide sequences can also be determined using the algorithm of Meyers and Miller ((l989) CABIOS 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The tenu “agent” is used herein to refer to a Chemical compound, a mixture of Chemical compounds, a biological macromolecule, an extract made from bîologîcal materials, or a combination of two or more thereof. The term “therapeutîc agent” or “drug” refers to an agent that is capable of modulating a biological process and/or has biological activity. The Mcl-1 inhibitors and the ADCs comprising them, as described herein, are exemplary therapeutîc agents.

The term chemotherapeutic agent or “anti-cancer agent” is used herein to refer to ail agents that are effective in treating cancer (regardless of mechanism of action). Inhibition of metastasis or angiogenesis is frequently a property of a chemotherapeutic agent. Chemotherapeutic agents include antibodies, biological molécules, and small molécules, and encompass the Mcl-1 inhibitors and ADCs comprising them, as described herein. A chemotherapeutic agent may be a cytotoxic or cytostatic agent. The term “cytostatic agent” refers to an agent that inhibits or suppresses cell growth and/or multiplication of cells. The term cytotoxic agent refers to a substance that causes cell death primarily by interfering with a cell’s expression activity and/or functioning.

The term “myeloid cell leukemia 1” or “Mcl-I,” as used herein, refers to any native form of human Mcl-1, an anti-apoptotic member of the B cl-2 protein family. The term encompasses full-length human Mcl-1 (e.g., UniProt Reference Sequence: Q07820; SEQ ID NO:63), as well as any form of human Mcl-1 that may resuit from cellular processing. The term also encompasses functional variants or fragments of human Mcl-1, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologie functions of human Mcl-1 (i.e., variants and fragments are encompassed unless the context indicates that the tenu is

127 used to refer to the wild-type protein only). Mcl-l can be isolated from human, or may be produced recombinantly or by synthetic methods.

The tenu inhibit or inhibition or “inhîbiting,” as used herein, means to reduce a biological activity or process by a measurable amount, and can include but does not require complété prévention or inhibition. In some embodiments, “inhibition” means to reduce the expression and/or activity of Mcl-l and/or one or more upstream modulators or downstream targets thereof.

The term “Mcl-l inhibitor,” as used herein, refers to an agent capable of reducîng the expression and/or activity of Mcl-l and/or one or more upstream modulators or downstream targets thereof. Exemplary Mcl-l modulators (includîng exemplary inhibitors of Mcl-1 ) are described m WO 2015/097123; WO 2016/207216; WO 2016/207217; WO 2016/207225; WO 2016/207226; WO 2017/125224; WO 2019/035899, WO 2019/035911, WO 2019/035914, WO 2019/035927, US 2019/0055264, WO 2016/033486, WO 2017/147410, WO 2018/1 83418, and WO 2017/182625, each of which are incorporated herein by reference as exemplary Mcl-l modulators, includîng exemplary Mcl-1 inhibitors, that can be included as drug moieties in the disclosed ADCs. For example, exemplary Mcl-l inhibitors that can be included as drug moieties

in the disclosed ADCs are those of fonnula:

wherein each variable is defined as in WO2019/035911; WO 2019/035899; WO 2019/035914;

or WO 2019/035927. Spécifie examples include, e.g.,

128

conjugated to an antibody or a linker via the nitrogen atom of the N-methyl in piperazinyl functional group of the compound. As used herein, the terms dérivative” and analog when referring to an Mcl-l inhibitor, or the like, means any such compound that retains essentially the same, similar, or enhanced biological fonction or activity as compared to the original compound but has an altered Chemical or biological structure.

As used herein, a “Mcl-l inhibitor drug moiety”, “Mcl-l inhibitor”, and the like refer to the component of an ADC or composition that provides the structure of an Mcl-l inhibitor compound or a compound modified for attachment to an ADC that retains essentially the same, similar, or enhanced biological fonction or activity as compared to the original compound. In some embodiments, Mcl-l inhibitor drug moiety is component (D) in an ADC of Formula (l ).

The term “cancer,” as used herein, refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled prolifération, immortality, 15 metastatic potential, rapid growth and prolifération rate, and/or certain morphological features.

Often, cancer cells can be in the form of a tumor or mass, but such cells may exist alone within a subject, or may circulate in theblood sircam as independent cells, such as leukemic or lymphoma cells. The term cancer” includes ail types of cancers and cancer métastasés.

129 including hematological cancers, solid tumors, sarcomas, carcinomas and other solid and nonsolid tumor cancers. Hematological cancers may include B-cell malignancies, cancers of the blood (leukemias), cancers of plasma cells (myelomas, e.g., multiple myeloma), or cancers of the lymph nodes (lymphomas). Exemplary B-cell malignancies include chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, and diffuse large B-cell lymphoma. Leukemias may include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), acute monocytic leukemia (AMoL), etc. Lymphomas may include Hodgkin's lymphoma, non-Hodgkin's lymphoma, etc. Other hématologie cancers may include myelodysplasia syndrome (MDS). Solid tumors may include carcinomas such as adenocarcînoma, e.g., breast cancer, pancreatic cancer, prostate cancer, colon or colorectal cancer, lung cancer, gastric cancer, cervical cancer, endométrial cancer, ovarian cancer, cholangîocarcinoma, glioma, melanoma, etc. In some embodiments, the cancer is a breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the cancer is a lymphoma or gastric cancer.

In some embodiments, the cancer is a hematological cancer, e.g., a leukemia, a lymphoma, or a myeloma. For example, an combination described herein can be used to treat cancers malignancies, and related disorders, including, but not limited to, e.g., an acute leukemia, e.g., B-cell acute lymphoid leukemia (B ALL), T-cell acute lymphoid leukemia (TALL), acute myeloid leukemia (AML), acute lymphoid leukemia (ALL); a chronic leukemia, e.g., chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); an additional hématologie cancer or hématologie condition, e.g., B cell prolymphocytic leukemia, blastic plasmacytoîd dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, Follicular lymphoma, Hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliférative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin’s lymphoma, plasmablastic lymphoma, plasmacytoîd dendritic cell neoplasm, Waldenstrom macroglobulinemia, myelotîbrosis, amyloid light chain amyloidosis, chronic neutrophilie

130 leukemia, essential thrombocythemia, chrome éosinophilie leukemia, chronic myelomonocytic leukemia, Richter Syndrome, mixed phenotrype acute leukemia, acute biphenotypic leukemia, and “preleukemia” which are a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells, and the like.

As used herein, the term “tumor” refers to any mass of tissue that results from excessive cell growth or prolifération, either benign or malignant, includîng precancerous lésions. In some embodiments, the tumor is a breast cancer, gastric cancer, bladder cancer, brain cancer, cervical cancer, colorectal cancer, esophageal cancer, hepatocellular cancer, melanoma, oral cancer, ovarian cancer, non-smal) cell lung cancer, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the tumor is a gastric cancer.

The tenus “tumor cell” and “cancer cell” may be used interchangeably herein and refer to individual cells or the total population of cells derived from a tumor or cancer, includîng both non-tumorigenic cells and cancer stem cells. The terrns “tumor cell” and “cancer cell” will be modified by the term “non-tumorigenic” when referring solely to those cells lacking the capacity to renew and differentiate to distinguish those cells from cancer stem cells.

The term “target-negative,” “target antigen-négative,” or “antigen-négative,” as used herein, refers to the absence of target antigen expression by a cell or tissue. The term “targetpositive,” “target antigen-positive,” or “antigen-positive” refers to the presence of target antigen expression. For example, a cell or a cell line that does not express a target antigen may be described as target-negative, whereas a cell or cell line that expresses a target antigen may be described as target-positive.

The tenus “subject” and “patient” are used interchangeably herein to refer to any human or non-human animal in need of treatment. Non-human animais include ail vertebrates (e.g., mammals and non-mammals) such as any mammal. Non-limiting examples of mammals include hum ans, chimpanzees, apes, monkeys, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rats, mice, and guinea pigs. Non-limiting exampies of non-mammals include birds and fish. In some embodiments, the subject îs a human.

The tenu “a subject in need of treatment,” as used herein, refers to a subject that would benefit biologically, medically, or in quality of life from a treatment (e.g., a treatment with any one or more of the exemplary ADC compounds described herein).

As used herein, the term “treat,” “treating,” or “treatment” refers to any improvement of any conséquence of disease, disorder, or condition, such as prolonged survival, less morbidity, and/or a lessenîng of sîde effects which resuit from an alternative therapeutic modality. In some

131 embodiments, treatment comprises delaying or amélioraiing a disease, disorder, or condition (î.e., slowing or arrestîng or reducing the development of a dîsease or at least one of the clinical symptoms thereof). In some embodiments, treatment comprises delaying, alleviating, or ameliorating at least one physical parameter of a disease, disorder, or condition, including those which may not be discernible by the patient. In some embodiments, treatment comprises modulating a disease, disorder, or condition, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. In some embodiments, treatment comprises administration of a described ADC compound or composition to a subject, e.g., a patient, to obtain a treatment benefit enumerated herein. The treatment can be to cure, heai, alleviate, delay, prevent, relieve, alter, remedy, ameliorate, pallîate, improve, or affect a disease, disorder, or condition (e.g., a cancer), the symptoms of a disease, disorder, or condition (e.g., a cancer), or a prédisposition toward a disease, disorder, or condition (e.g., a cancer). In some embodiments, in addition to treatîng a subject having a disease, disorder, or condition, a composition disclosed herein can also be provided prophylacticaily to prevent or reduce the likelihood of developing that disease, disorder, or condition.

As used herein, the term “prevent”, “preventîng, or “prévention” of a disease, disorder, or condition refers to the prophylactic treatment of the disease, disorder, or condition; or delaying the onset or progression of the disease, disorder, or condition.

As used herein, a pharmaceutical composition refers to a préparation of a composition, e.g., an ADC compound or composition, in addition to at least one other (and optionally more than one other) component suitable for administration to a subject, such as a pharmaceutically acceptable carrier, stabilizer, diluent, dispersing agent, suspending agent, thickening agent, and/or excipient. The pharmaceutical compositions provided herein are in such form as to permit administration and subsequently provide the intended biological activity of the active ingredient(s) and/or to achîeve a therapeutic effect. The pharmaceutical compositions provided herein preferably contain no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

As used herein, the terms pharmaceutically acceptable carrier and physiologically acceptable carrier, which may be used interchangeably, refer to a carrier or a diluent that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered ADC compound or composition and/or any additional therapeutic agent in the composition. Phannaceutically acceptable carriers may enhance or stabilize the composition or can be used to facilitate préparation of the composition. Pharmaceutically

132 acceptable carriers can include solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonie agents, absorption deiaying agents, salts, preservatives, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289- 1329). Except insofar as any conventional carrier is incompatible with the active ingrédient, its use in the therapeutic or pharmaceutical compositions is contemplâted. The carrier may be selected to minimize adverse side effects in the subject, and/or to minimize dégradation of the active ingredient(s). An adjuvant may also be included in any of these formulations.

As used herein, the term excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingrédient. Formulations for parentéral administration can, for example, contaîn excipients such as stérile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, or hydrogenated napthalenes. Other exemplary excipients include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose dérivatives, gelatîn, ethyl ene-vinyl acetate co-polymer partiel es, and surfactants, including, for example, polysorbate 20.

The term “pharmaceutical 1 y acceptable sait,” as used herein, refers to a sait which does not abrogate the biological activity and properties of the compounds of the invention, and does not cause signifïcant irritation to a subject to which it is adminisiered. Examples of such salts include, but are not limited to: (a) acid addition salts formed with inorganic acids, for example, hydrochlorîc acid, hydrobromîc acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesidfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (b) salts formed from elemental anions such as chlorine, bromine, and iodine. See, e.g., Haynes et al., “Commentary: Occurrence of Phannaceutically Acceptable Anions and Cations in the Cambridge Structural Database,” J. Pharmaceutical Sciences, vol. 94, no. 10 (2005), and Berge et al., “Pharmaceutical Salts,” J. Pharmaceutical Sciences, vol. 66, no. 1 (1977), which are incorporated by reference herein.

133

In some embodiments, depending on their electronic charge, the antibody-drug conjugates (ADCs), linkers, payloads and linker-payloads described herein can contain a monovalent anionic counterion Mf. Any suitable anionic counterion can be used. In certain embodiments, the monovalent anionic counterion is a pharmaceutically acceptable monovalent anionic counterion. In certain embodiments, the monovalent anionic counterion Mf can be selected from bromide, chloride, iodide, acetate, trifluoroacetate, benzoate, mesylate, tosylate, triflate, formate, or the like. In some embodiments, the monovalent anionic counterion Mf is trifluoroacetate or formate.

As used herein, the term “therapeutically effective amount” or “therapeutically effective dose,” refers to an amount of a compound described herein, e.g., an ADC compound or composition described herein, to effect the desired therapeutic resuit (i.e., réduction or inhibition of an enzyme or a protein activity, amelioration of symptoms, alleviation of symptoms or conditions, delay of disease progression, a réduction in tumor size, inhibition of tumor growth, prévention of metastasis). In some embodiments, a therapeutically effective amount does not induce or cause undesirable side effects. In some embodiments, a therapeutically effective amount induces or causes side effects but only those that are acceptable by a treating clinician in view of a patient’s condition. In some embodiments, a therapeutically effective amount is effective for détectable killing, réduction, and/or inhibition of the growth or spread of cancer cells, the size or number of tumors, and/or other measure of the level, stage, progression and/or severity of a cancer. The term also applies to a dose that will induce a particular response in target cells, e.g., a réduction, slowing, or inhibition of cell growth. A therapeutically effective amount can be detennined by first administering a low dose, and then incrementally increasing that dose until the desired effect is achieved. A therapeutically effective amount can also vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and âge of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The spécifie amount may vary depending on, for example, the particular pharmaceutical composition, the subject and their âge and existing health conditions or risk for health conditions, the dosing regimen to be followed, the severity of the disease, whether it is administered in combination with other agents, timing of administration, the tissue to which it is administered, and the physical delivery System in which it is carried. In the case of cancer, a therapeutically effective amount of an ADC may reduce the number of cancer cells, reduce

134 tumor size, inhibit (e.g,, slow or stop) tumor metastasis, inhibit (e.g., slow or stop) tumor growth, and/or relieve one or more symptoms.

As used herein, the term “prophylactically effective amount” or “prophylactîcally effective dose,” refers to an amount of a compound disclosed herein, e.g., an ADC compound or composition described herein, that is effective, at dosages and for periods of time necessary, to achieve the desired prophylactic resuit. Typically, since a prophylactic dose îs used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. In some embodiments, a prophylactically effective amount can prevent the onset of disease symptoms, including symptoms associated with a cancer.

The term “p” or “drug loading” or “drug:antibody ratio” or “drug-to-antibody ratio” or “DAR” refers to the number of drug moieties per antibody or antigen-binding fragment, i.e., drug loading, or the number of -L-D moieties per antibody or antigen-binding fragment (Ab) in ADCs of Formula (1). In ADCs comprising an Mcl-1 inhibitor drug moiety, “p” refers to the number of Mcl-1 inhibitor compounds linked to the antibody or antigen-binding fragment. For example, if two Mcl-1 inhibitor compounds are linked to an antibody or antigen-binding fragment,/? = 2. In compositions comprising multiple copies of ADCs of Formula (1), “average p refers to the average number of -L-D moieties per antibody or antigen-binding fragment, also referred to as “average drug loading.” Antibodv-Drug Conjugales

The antibody-drug conjugate (ADC) compounds of the présent disclosure include those with anti-cancer activity. In particular, the ADC compounds include an antibody or antigenbinding fragment conjugated (i.e., covalently attached by a linker) to a drug moiety (e.g., an Mcl1 inhibitor), wherein the drug moiety when not conjugated to an antibody or antigen-binding fragment has a cytotoxic or cylostatic effect. In some embodiments, the drug moiety when not conjugated to an antibody or antigen-binding fragment is capable of reducing the expression and/or activity of Mcl-1 and/or one or more upstream modulators or downstream targets thereof. Without being bound by theory, by targeting Mcl-1 expression and/or activity, in some embodiments, the ADCs disclosed herein may provide potent anti-cancer agents. Also, without being bound by theory, by conjugating the drug moiety to an antibody that binds an antigen associated with expression in a tumor cell or cancer, the ADC may provide improved activity, better cytotoxic specîficity, and/or reduced off-target killing as compared to the drug moiety when administered alone.

135

In some embodiments, therefbre, the components of the ADC are selected to (i) retain one or more therapeutic properties exhibited by the antibody and drug moieties in isolation, (ii) maintain the spécifie binding properties of the antibody or antigen-binding fragment; (iii) optimize drug loading and drug-to-antibody ratios; (iv) allow delivery, e.g., intracellular delivery, of the drug moiety via stable attachment to the antibody or antigen-binding fragment; (v) retain ADC stability as an intact conjugale until transport or delivery to a target site; (vi) minimize aggregation of the ADC prior to or after administration; (vii) allow for the therapeutic effect, e.g., cytotoxic effect, of the drug moiety after cleavage or other release mechanism in the cellular environment; (viii) exhibit in vivo anti-cancer treatment efficacy comparable to or superior to that of the antibody and drug moieties in isolation; (ix) minimize off-target killing by the drug moiety; and/or (x) exhibit désirable pharmacokinetic and pharmacodynamies properties, formulatability, and toxicologîc/immunologic profiles. Each of these properties may provide for an improved ADC for therapeutic use (Ab et al. (2015) Mol Cancer Ther. 14:1605-13).

The ADC compounds of the present disclosure may selectively deliver an effective dose of a cytotoxic or cytostatic agent to cancer cells or to tumor tissue. In some embodiments, the cytotoxic and/or cytostatic activity of the ADC îs dépendent on target antigen expression in a cell. In some embodiments, the disclosed ADCs are particularly effective at killing cancer cells expressing a target antigen while minimizing off-target killing. In some embodiments, the disclosed ADCs do not exhibit a cytotoxic and/or cytostatic effect on cancer cells that do not express a target antigen.

Provided herein, in certain aspects, are ADC compounds comprising an antibody or antigen-binding fragment thereof (Ab) which targets a cancer cell, an Mcl-1 inhibitor drug moiety (D), and a linker moiety (L) that covaiently attaches Ab to D. In some embodiments, the antibody or antigen-binding fragment is able to bind to a tumor-assocîated antigen (e.g., BCMA, CD33, PCAD, or HER2), e.g., with high specificity and high affinity. In some embodiments, the antibody or antigen-binding fragment is internalized into a target cell upon binding, e.g., into a degradative compartment in the cell. In some embodiments, the ADCs internalize upon binding to a target cell, undergo dégradation, and release the Mcl-1 inhibitor drug moiety to kill cancer cells. The Mcl-1 inhibitor drug moiety may be released from the antibody and/or the linker moiety of the ADC by enzymatic action, hydrolysis, oxidation, or any other mechanism.

An exemplary ADC has Formula (1):

Ab-(L-D)_p (1)

136 wherein Ab = an antibody or antigen-binding fragment, L = a linker moiety, D = an Mcl-l inhibitor drug moiety, and p = the number of Mcl-l inhibitor drug moieties per antibody or antigen-binding fragment.

Antibodies

The antibody or antigen-binding fragment (Ab) of Formula (l) includes within its scope any antibody or antigen-binding fragment that specifically binds to a target antigen on a cancer cell. The antibody or antigen-binding fragment may bind to a target antigen with a dissociation constant (Kd) of <l mM, <100 nM or <10 nM, or any amount in between, as measured b y, e.g., BIAcore® analysis. In some embodiments, the Kd is 1 pM to 500 pM. In some embodiments, the Kd is between 500 pM to I μΜ, 1 μΜ to 100 nM, or 100 mM to 10 nM.

In some embodiments, the antibody or antigen-binding fragment is a four-chain antibody (also referred to as an immunoglobulin or a full-length or intact antibody), comprising two heavy chains and two light chains. In some embodiments, the antibody or antigen-binding fragment is an antigen-binding fragment of an immunoglobulin. In some embodiments, the antibody or antigen-binding fragment is an antigen-binding fragment of an immunoglobulin that retains the ability to bind a target cancer antigen and/or provide at least one function of the immunoglobulin.

In some embodiments, the antibody or antigen-binding fragment is an internalizing antibody or internalizing antigen-binding fragment thereof. In some embodiments, the internalizing antibody or internalizing antigen-binding fragment thereof binds to a target cancer antigen expressed on the surface of a cell and enters the cell upon binding. In some embodiments, the Mcl-l inhibitor drug moiety of the ADC is released from the antibody or antigen-binding fragment of the ADC after the ADC enters and is présent in a cell expressing the target cancer antigen (i.e., after the ADC has been internalized), e.g., b y cleavage, by dégradation of the antibody or antigen-binding fragment, or by any other suitable release mechanism.

Amino acid sequences of exemplary antibodies of the présent disclosure, in addition to exemplary antigen targets, are set forth in Tables C, D and E.

Table C. Amino acid sequences of mAb CDRs

Ab	SEQ ID NO	IgG chain	Amino acid sequence
mîlatuzumab	SEQ ID NO:1 (Combined)	HCDR1	GYTFTNYGVN
	SEQ ID NO:2 (Combined)	HCDR2	WINPNTGEPTFDDDFKG

137

	SEQ ID NO: 3 (Combined)	HCDR3	SRGKNEAWFAY
	SEQ ID NO:4 (Kabat)	HCDR1	NYGVN
	SEQ ID NO:2 (Kabat)	HCDR2	WINPNTGEPTFDDDFKG
	SEQ ID NO: 3 (Kabat)	HCDR3	SRGKNEAWFAY
	SEQ ID NO: 5 (Chothia)	HCDRi	GYTFTNY
	SEQ ID N0:6 (Chothia)	HCDR2	NPNTGE
	SEQ ID N0:3 (Chothia)	HCDR3	SRGKNEAWFAY
	SEQ ID NO:7 (IMGT)	HCDRI	GYTFTNYG
	SEQ ID N0:8 (IMGT)	HCDR2	INPNTGEP
	SEQ ID NO:9 (IMGT)	HCDR3	SRSRGKNEAWFAY
	SEQ ID NO: 16 (Combined)	LCDRI	RSSQSLVHRNGNTYLH
	SEQ ID NO:70 (Combined)	LCDR2	TVSNRFS
	SEQ ID NO: 18 (Combined)	LCDR3	SQSSHVPPT
	SEQ ID NO: 16 (Kabat)	LCDRI	RSSQSLVHRNGNTYLH
	SEQ ID NO:70 (Kabat)	LCDR2	TVSNRFS
	SEQ ID NO: 18 (Kabat)	LCDR3	SQSSHVPPT
	SEQ ID NO: 19 (Chothia)	LCDRI	SQSLVHRNGNTY
	SEQ ID NO:20 (Chothia)	LCDR2	TVS
	SEQIDN0;21 (Chothia)	LCDR3	SSHVPP
	SEQ ID NO:22 (IMGT)	LCDRI	QSLVHRNGNTY
	SEQ ID NO:20 (IMGT)	LCDR2	TVS
	SEQ ID NO: 18 (IMGT)	LCDR3	SQSSHVPPT
Mil_HC x hzVkl a	SEQ IDNO:1 (Combined)	HCDRI	GYTFTNYGVN
	SEQ ID N0:2 (Combined)	HCDR2	WINPNTGEPTFDDDFKG
	SEQ ID NO:3 (Combined)	HCDR3	SRGKNEAWFAY
	SEQ ID NO:4 (Kabat)	HCDRI	NYGVN
	SEQ ID N0:2 (Kabat)	HCDR2	WINPNTGEPTFDDDFKG

138

	SEQ 1D NO:3 (Rabat)	HCDR3	SRGRNEAWFAY
	SEQ ID N0;5 (Chothia)	HCDR1	GYTFTNY
	SEQ ID NO:6 (Chothia)	HCDR2	NPNTGE
	SEQ ID NO:3 (Chothia)	HCDR3	SRGRNEAWFAY
	SEQ ID NO:7 (IMGT)	HCDR1	GYTFTNYG
	SEQ ID NO:8 (IMGT)	HCDR2	INPNTGEP
	SEQ ID NO:9 (IMGT)	HCDR3	SRSRGRNEAWFAY
	SEQ ID NO: 16 (Combined)	LCDR1	RSSQSLVHRNGNTYLH
	SEQ ID NO:70 (Combined)	LCDR2	TVSNRFS
	SEQ IDN0:18 (Combined)	LCDR3	SQSSHVPPT
	SEQ ID NO: 16 (Rabat)	LCDR1	RSSQSLVHRNGNTYLH
	SEQ ID N0:7Q (Rabat)	LCDR2	TVSNRFS
	SEQ ID N0:18(Kabat)	LCDR3	SQSSHVPPT
	SEQ ID NO: 19 (Chothia)	LCDR1	SQSLVHRNGNTY
	SEQ ID NO:20 (Chothia)	LCDR2	TVS
	SEQ ID NO:21 (Chothia)	LCDR3	SSHVPP
	SEQ ID NO:22 (IMGT)	LCDR1	QSLVHRNGNTY
	SEQ ID NO:20 (IMGT)	LCDR2	TVS
	SEQ ID NO: 18 (IMGT)	LCDR3	SQSSHVPPT
Hcmil x LCmil_NQ	SEQ ID NO: 1 (Combined)	HCDR1	GYTFTNYGVN
	SEQ ID NO:2 (Combined)	HCDR2	WINPNTGEPTFDDDFKG
	SEQ ID NO:3 (Combined)	HCDR3	SRGRNEAWFAY
	SEQ ID NO:4 (Rabat)	HCDR1	NYGVN
	SEQ ID NO:2 (Kabat)	HCDR2	WINPNTGEPTFDDDFRG
	SEQ ID N0:3 (Rabat)	HCDR3	SRGRNEAWFAY
	SEQ ID N0:5 (Chothia)	HCDR1	GYTFTNY
	SEQ ID N0:6 (Chothia)	HCDR2	NPNTGE
	SEQ ID NO:3 (Chothia)	HCDR3	SRGRNEAWFAY

139

	SEQ ID NO:7 (IMGT)	HCDR1	GYTFTNYG
	SEQ ID NO:8 (IMGT)	HCDR2	INPNTGEP
	SEQ ID NO:9 (IMGT)	HCDR3	SRSRGKNEAWFAY
	SEQ ID NO:35 (Combined)	LCDR1	RSSQSLVHRNQNTYLH
	SEQ ID NO:70 (Combined)	LCDR2	TVSNRFS
	SEQ ID NO:18 (Combined)	LCDR3	SQSSHVPPT
	SEQ ID NO:35 (Kabat)	LCDR1	RSSQSLVHRNQNTYLH
	SEQ ID NO:70 (Kabat)	LCDR2	TVSNRFS
	SEQ ID NO: 18 (Kabat)	LCDR3	SQSSHVPPT
	SEQ ID NO:71 (Chothia)	LCDR1	SQSLVHRNQNTY
	SEQ ID NO:20 (Chothia)	LCDR2	TVS
	SEQ ID NO:21 (Chothia)	LCDR3	SSHVPP
	SEQ ID NO: 17 (IMGT)	LCDR1	QSLVHRNQNTY
	SEQ ID NO:20 (IMGT)	LCDR2	TVS
	SEQ ID NO: 18 (IMGT)	LCDR3	SQSSHVPPT
VHmil x VKlaNQ	SEQ ID NO:1 (Combined)	HCDR1	GYTFTNYGVN
	SEQ ID NO:2 (Combined)	HCDR2	WINPNTGEPTFDDDFKG
	SEQ ID NO:3 (Combined)	HCDR3	SRGKNEAWFAY
	SEQ ID NO:4 (Kabat)	HCDR1	NYGVN
	SEQ ID NO:2 (Kabat)	HCDR2	WINPNTGEPTFDDDFKG
	SEQ ID NO:3 (Kabat)	HCDR3	SRGKNEAWFAY
	SEQ ID NO:5 (Chothia)	HCDR1	GYTFTNY
	SEQ ID NO:6 (Chothia)	HCDR2	NPNTGE
	SEQ ID NO:3 (Chothia)	HCDR3	SRGKNEAWFAY
	SEQ ID NO:7 (IMGT)	HCDRI	GYTFTNYG
	SEQ ID NO:8 (IMGT)	HCDR2	INPNTGEP
	SEQ ID NO:9 (IMGT)	HCDR3	SRSRGKNEAWFAY
	SEQ ID NO:35	LCDR1	RSSQSLVHRNQNTYLH

140

	(Combined)
	SEQ ID NO:70 (Combined)	LCDR2	TVSNRFS
	SEQ ID NO: 18 (Combined)	LCDR3	SQSSHVPPT
	SEQ ID NO:35 (Kabat)	LCDRl	RSSQSLVHRNQNTYLH
	SEQ ID NO:70 (Kabat)	LCDR2	TVSNRFS
	SEQ ID NO: 18 (Kabat)	LCDR3	SQSSHVPPT
	SEQ IDN0:71 (Chothia)	LCDRl	SQSLVHRNQNTY
	SEQ ID NO:20 (Chothia)	LCDR2	TVS
	SEQ IDNO:21 (Chothia)	LCDR3	SSHVPP
	SEQ ID NO: 17 (IMGT)	LCDRl	QSLVHRNQNTY
	SEQ ID NO:20 (IMGT)	LCDR2	TVS
	SEQ ID NO: 18 (IMGT)	LCDR3	SQSSHVPPT
VHmil x VKlbNQ	SEQ ID NO:1 (Combined)	HCDRl	GYTFTNYGVN
	SEQ ID NO:2 (Combined)	HCDR2	WINPNTGEPTFDDDFKG
	SEQ ID N0:3 (Combined)	HCDR3	SRGKNEAWFAY
	SEQ ID NO:4 (Kabat)	HCDRl	NYGVN
	SEQ ID N0:2 (Kabat)	HCDR2	WINPNTGEPTFDDDFKG
	SEQ ID N0:3 (Kabat)	HCDR3	SRGKNEAWFAY
	SEQ ID NO:5 (Chothia)	HCDRl	GYTFTNY
	SEQ ID N0:6 (Chothia)	HCDR2	NPNTGE
	SEQ ID N0:3 (Chothia)	HCDR3	SRGKNEAWFAY
	SEQ ID N0:7 (IMGT)	HCDRl	GYTFTNYG
	SEQ ID NO:8 (IMGT)	HCDR2	INPNTGEP
	SEQ ID NO:9 (IMGT)	HCDR3	SRSRGKNEAWFAY
	SEQ ID NO:35 (Combined)	LCDRl	RSSQSLVHRNQNTYLH
	SEQ ID NO:70 (Combined)	LCDR2	TVSNRFS
	SEQ ID NO: 18	LCDR3	SQSSHVPPT

141

(Combined)
SEQ ID NO:35 (Kabat)	LCDRI	RSSQSLVHRNQNTYLH
SEQ IDNO:70 (Kabat)	LCDR2	TVSNRFS
SEQ ID N0:18 (Kabat)	LCDR3	SQSSHVPPT
SEQ ID NO:71 (Chothia)	LCDRI	SQSLVHRNQNTY
SEQ ID NO:20 (Chothia)	LCDR2	TVS
SEQ ID NO:21 (Chothia)	LCDR3	SSHVPP
SEQ ID NO: 17 (IMGT)	LCDRI	QSLVHRNQNTY
SEQ ID NO:20 (IMGT)	LCDR2	TVS
SEQ ID NO: 18 (IMGT)	LCDR3	SQSSHVPPT

Table D. Amino acid sequence and nucleic acid sequnces of mAb variable régions

Ab	SEQ ID NO	IgG chain	Amino acid sequence
milatuzumab	10	VH	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSS
	11	DNA VH	CAGGTTCAGCTCCAGCAGTCTGGCAGCGAG CTGAAAAAACCTGGCGCCTCCGTGAAGGTG TCCTGCAAGGCTTCTGGCTACACCTTTACCA ACTACGGCGTGAACTGGATCAAGCAGGCCC CTGGACAAGGCCTCCAATGGATGGGCTGGA TCAACCCCAATACCGGCGAGCCCACCTTCGA CGACGATTTCAAGGGCAGATTCGCCTTCAGC CTGGACACCTCTGTGTCCACAGCCTACCTCC AGATCAGCAGCCTGAAGGCCGATGATACCG CCGTGTACTTCTGCTCCAGAAGCCGGGGAAA GAACGAGGCTTGGTTTGCCTATTGGGGCCAG GGCACACTGGTCACCGTTAGCTCT
	23	VL	DIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNG NTYLHWFQQRPGQSPRLLIYTVSNRFSGVPDR

142

			FSGSGSGTDFTLKJSRVEAEDVGVYFCSQSSHV PPTFGAGTRLEIK
	24	DNA VL	GACATTCAGCTGACACAGAGCCCTCTGAGCC TGCCTGTTACACTGGGACAGCCTGCCAGCAT CAGCTGTAGAAGCAGCCAGAGCCTGGTGCA CAGAAACGGCAACACCTACCTGCACTGGTTC CAGCAGAGGCCTGGCCAGTCTCCTAGACTGC TGATCTACACCGTGTCCAACAGATTCAGCGG CGTGCCCGATAGATTTTCCGGCAGCGGCTCT GGCACCGACTTCACCCTGAAGATTAGCAGA GTGGAAGCCGAGGACGTGGGCGTGTACTTC TGTAGCCAGTCTAGCCACGTGCCACCTACCT TTGGCGCCGGAACCAGACTGGAAATCAAG
Mil_HC x hzVkla	10	VH	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSS
	SEQ ID NO: 11	DNA VH	CAGGTTCAGCTCCAGCAGTCTGGCAGCGAG CTGAAAAAACCTGGCGCCTCCGTGAAGGTG TCCTGCAAGGCTTCTGGCTACACCTTTACCA ACTACGGCGTGAACTGGATCAAGCAGGCCC CTGGACAAGGCCTCCAATGGATGGGCTGGA TCAACCCCAATACCGGCGAGCCCACCTTCGA CGACGATTTCAAGGGCAGATTCGCCTTCAGC CTGGACACCTCTGTGTCCACAGCCTACCTCC AGATCAGCAGCCTGAAGGCCGATGATACCG CCGTGTACTTCTGCTCCAGAAGCCGGGGAAA GAACGAGGCTTGGTTTGCCTATTGGGGCCAG GGCACACTGGTCACCGTTAGCTCT
	SEQ ID NO:27	VL	DIVMTQTPLSLPVTPGEPASISCRSSQSLVHRN GNTYLHWYLQKPGQSPQLLIYTVSNRFSGVPD RFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSH VPPTFGQGTKLEIK

143

	SEQ ID NO:28	DNA VL	GACATTGTGATGACACAGACCCCTCTGAGCC TGCCTGTGACACCTGGCGAACCTGCCAGCAT CAGCTGTAGAAGCAGCCAGAGCCTGGTGCA CAGAAACGGCAACACCTACCTGCACTGGTA TCTGCAGAAGCCCGGCCAGTCTCCTCAGCTG CTGATCTACACCGTGTCCAACAGATTCAGCG GCGTGCCCGATAGATTTTCCGGCAGCGGCTC TGGCACCGACTTCACCCTGAAGATCTCCAGA GTGGAAGCCGAGGACGTGGGCGTGTACTTC TGTAGCCAGTCTAGCCACGTGCCACCTACCT TTGGCCAGGGGACCAAGCTGGAAATCAAG
Mil-HC x hzVklb	SEQ ID NO:10	VH	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSS
	SEQ ID NO: Il	DNA VH	CAGGTTCAGCTCCAGCAGTCTGGCAGCGAG CTGAAAAAACCTGGCGCCTCCGTGAAGGTG TCCTGCAAGGCTTCTGGCTACACCTTTACCA ACTACGGCGTGAACTGGATCAAGCAGGCCC CTGGACAAGGCCTCCAATGGATGGGCTGGA TCAACCCCAATACCGGCGAGCCCACCTTCGA CGACGATTTCAAGGGCAGATTCGCCTTCAGC CTGGACACCTCTGTGTCCACAGCCTACCTCC AGATCAGCAGCCTGAAGGCCGATGATACCG CCGTGTACTTCTGCTCCAGAAGCCGGGGAAA GAACGAGGCTTGGTTTGCCTATTGGGGCCAG GGCACACTGGTCACCGTTAGCTCT
	SEQ ID NO:31	VL	DVVMTQSPLSLPVTLGQPAS1SCRSSQSLVHRN GNTYLHWYQQRPGQSPRLLIYTVSNRFSGVPD RFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSH VPPTFGQGTKLEIK
	SEQ ID NO:32	DNA VL	GATGTGGTTATGACACAGAGCCCTCTGAGCC TGCCTGTGACACTTGGACAGCCTGCCAGCAT

144

			CAGCTGCAGATCTAGCCAGAGCCTGGTGCA CAGAAACGGCAACACCTACCTGCACTGGTA TCAGCAGAGGCCCGGACAGTCTCCCAGACT GCTGATCTACACCGTGTCCAACAGATTCAGC GGCGTGCCCGATAGATTTTCCGGCAGCGGCT CTGGCACCGACTTCACCCTGAAGATCTCCAG AGTGGAAGCCGAGGACGTGGGCGTGTACTT CTGTAGCCAGTCTAGCCACGTGCCACCTACC TTTGGCCAGGGCACCAAGCTGGAAATCAAG
Hcmil x LCmil_NQ	SEQ ID NO:10	VH	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSS
	SEQ ID NO: 11	DNA VH	CAGGTTCAGCTCCAGCAGTCTGGCAGCGAG CTG A AAAAAC CTGGCGCCTCCGTGAAGGTG TCCTGCAAGGCTTCTGGCTACACCTTTACCA ACTACGGCGTGAACTGGATCAAGCAGGCCC CTGGACAAGGCCTCCAATGGATGGGCTGGA TCAACCCCAATACCGGCGAGCCCACCTTCGA CGACGATTTCAAGGGCAGATTCGCCTTCAGC CTGGACACCTCTGTGTCCACAGCCTACCTCC AGATCAGCAGCCTGAAGGCCGATGATACCG CCGTGTACTTCTGCTCCAGAAGCCGGGGAAA GAACGAGGCTTGGTTTGCCTATTGGGGCCAG GGCACACTGGTCACCGTTAGCTCT
	SEQ ID NO:36	VL	DIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNQ NTYLHWFQQRPGQSPRLLIYTVSNRFSGVPDR FSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHV PPTFGAGTRLEIK
	SEQ ID NO:37	DNA VL	GACATTCAGCTGACACAGAGCCCTCTGAGCC TGCCTGTTACACTGGGACAGCCTGCCAGCAT CAGCTGTAGAAGCAGCCAGAGCCTGGTGCA CAGAAACCAGAACACCTACCTGCACTGGTTC

145

			CAGCAGAGGCCTGGCCAGTCTCCTAGACTGC TGATCTACACCGTGTCCAACAGATTCAGCGG CGTGCCCGATAGATTTTCCGGCAGCGGCTCT GGCACCGACTTCACCCTGAAGATTAGCAGA GTGGAAGCCGAGGACGTGGGCGTGTACTTC TGTAGCCAGTCTAGCCACGTGCCACCTACCT TTGGCGCCGGAACCAGACTGGAAATCAAG
VHmil x VKlaNQ	SEQ ID NÛ:10	VH	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSS
	SEQ ID NO:11	DNA VH	CAGGTTCAGCTCCAGCAGTCTGGCAGCGAG CTGAAAAAACCTGGCGCCTCCGTGAAGGTG TCCTGCAAGGCTTCTGGCTACACCTTTACCA ACTACGGCGTGAACTGGATCAAGCAGGCCC CTGGACAAGGCCTCCAATGGATGGGCTGGA TCAACCCCAATACCGGCGAGCCCACCTTCGA CGACGATTTCAAGGGCAGATTCGCCTTCAGC CTGGACACCTCTGTGTCCACAGCCTACCTCC AGATCAGCAGCCTGAAGGCCGATGATACCG CCGTGTACTTCTGCTCCAGAAGCCGGGGAAA G AACGAGGCTTGGTTTGCCTATTGGGG CC A G GGCACACTGGTCACCGTTAGCTCT
	SEQ ID NO:4Ü	VL	DIVMTQTPLSLPVTPGEPASISCRSSQSLVHRN QNTYLHWYLQKPGQSPQLLÏYTVSNRFSGVPD RFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSH VPPTFGQGTKLEIK
	SEQ ID NO:41	DNA VL	GACATTGTGATGACACAGACCCCTCTGAGCC TGCCTGTGACACCTGGCGAACCTGCCAGCAT CAGCTGTAGAAGCAGCCAGAGCCTGGTGCA CCGGAACCAGAATACCTACCTGCACTGGTAT CTGCAGAAGCCCGGCCAGTCTCCTCAGCTGC TGATCTACACCGTGTCCAACAGATTCAGCGG

146

			CGTGCCCGATAGATTTTCCGGCAGCGGCTCT GGCACCGACTTCACCCTGAAGATCTCCAGAG TGGAAGCCGAGGACGTGGGCGTGTACTTCT GTAGCCAGTCTAGCCACGTGCCACCTACCTT TGGCCAGGGGACCAAGCTGGAAATCAAG
VHmil x VKlbNQ	SEQ ID NO: 10	VH	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSS
	SEQ ID NO: 11	DNA VH	CAGGTTCAGCTCCAGCAGTCTGGCAGCGAG CTGAAAAAACCTGGCGCCTCCGTGAAGGTG TCCTGCAAGGCTTCTGGCTACACCTTTACCA ACTACGGCGTGAACTGGATCAAGCAGGCCC CTGGACAAGGCCTCCAATGGATGGGCTGGA TCAACCCCAATACCGGCGAGCCCACCTTCGA CGACGATTTCAAGGGCAGATTCGCCTTCAGC CTGGACACCTCTGTGTCCACAGCCTACCTCC AGATCAGCAGCCTGAAGGCCGATGATACCG CCGTGTACTTCTGCTCCAGAAGCCGGGGAAA GAACGAGGCTTGGTTTGCCTATTGGGGCCAG GGCACACTGGTCACCGTTAGCTCT
	SEQ ID NO:44	VL	DVVMTQSPLSLPVTLGQPASISCRSSQSLVHRN QNTYLHWYQQRPGQSPRLLIYTVSNRFSGVPD RFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSH VPPTFGQGTKLEIK
	SEQ ID NO:45	DNA VL	GATGTGGTTATGACACAGAGCCCTCTGAGCC TGCCTGTGACACTTGGACAGCCTGCCAGCAT CAGCTGCAGATCTAGCCAGAGCCTGGTGCA CCGGAACCAGAACACATACCTGCACTGGTA TCAGCAGAGGCCCGGACAGTCTCCCAGACT GCTGATCTACACCGTGTCCAACAGATTCAGC GGCGTGCCCGATAGATTTTCCGGCAGCGGCT CTGGCACCGACTTCACCCTGAAGATCTCCAG

147

AGTGGAAGCCGAGGACGTGGGCGTGTACTT CTGTAGCCAGTCTAGCCACGTGCCACCTACC TTTGGCCAGGGCACCAAGCTGGAAATCAAG

Table E. amino acid and nucleic acid sequences of full length mAb IgG chains (ail Heavy Chain sequences include E152C and S375C mutations for site-directed conjugation)

Ab	SEQ ID NO	IgG chain	Amino acid sequence
milatuzumab	SEQ ID NO: 12	Heavy Chain (Wild Type Fc)	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPCDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK
	SEQ ID NO: 13	DNA Heavy Chain (Wild Type Fc)	CAGGTTCAGCTCCAGCAGTCTGGCAGCGAG CTGAAAAAACCTGGCGCCTCCGTGAAGGTG TCCTGCAAGGCTTCTGGCTACACCTTTACCA ACTACGGCGTGAACTGGATCAAGCAGGCCC CTGGACAAGGCCTCCAATGGATGGGCTGGA TCAACCCCAATACCGGCGAGCCCACCTTCGA CGACGATTTCAAGGGCAGATTCGCCTTCAGC CTGGACACCTCTGTGTCCACAGCCTACCTCC AGATCAGCAGCCTGAAGGCCGATGATACCG CCGTGTACTTCTGCTCCAGAAGCCGGGGAAA

148

GAACGAGGCTTGGTTTGCCTATTGGGGCCAG GGCACACTGGTCACCGTTAGCTCTGCTAGCA CCAAGGGCCCAAGTGTGTTTCCCCTGGCCCC CAGCAGCAAGTCTACTTCCGGCGGAACTGCT GCCCTGGGTTGCCTGGTGAAGGACTACTTCC CCTGTCCCGTGACAGTGTCCTGGAACTCTGG GGCTCTGACTTCCGGCGTGCACACCTTCCCC GCCGTGCTGCAGAGCAGCGGCCTGTACAGC CTGAGCAGCGTGGTGACAGTGCCCTCCAGCT CTCTGGGAACCCAGACCTATATCTGCAACGT GAACCACAAGCCCAGCAACACCAAGGTGGA CAAGAGAGTGGAGCCCAAGAGCTGCGACAA GACCCACACCTGCCCCCCCTGCCCAGCTCCA GAACTGCTGGGAGGGCCTTCCGTGTTCCTGT TCCCCCCCAAGCCCAAGGACACCCTGATGAT CAGCAGGACCCCCGAGGTGACCTGCGTGGT GGTGGACGTGTCCCACGAGGACCCAGAGGT GAAGTTCAACTGGTACGTGGACGGCGTGGA GGTGCACAACGCCAAGACCAAGCCCAGAGA GGAGCAGTACAACAGCACCTACAGGGTGGT GTCCGTGCTGACCGTGCTGCACCAGGACTGG CTGAACGGCAAAGAATACAAGTGCAAAGTC TCCAACAAGGCCCTGCCAGCCCCAATCGAA AAGACAATCAGCAAGGCCAAGGGCCAGCCA CGGGAGCCCCAGGTGTACACCCTGCCCCCC AGCCGGGAGGAGATGACCAAGAACCAGGTG TCCCTGACCTGTCTGGTGAAGGGCTTCTACC CCTGTGATATCGCCGTGGAGTGGG AG AGCA ACGGCCAGCCCGAGAACAACTACAAGACCA CCCCCCCAGTGCTGGACAGCGACGGCAGCTT CTTCCTGTACAGCAAGCTGACCGTGGACAAG TCCAGGTGGCAGCAGGGCAACGTGTTCAGC TGCAGCGTGATGCACGAGGCCCTGCACAAC

149

		CACTACACCCAGAAGTCCCTGAGCCTGAGCC CCGGCAAG
SEQ ID NO:14	Heavy Chain (Fc Silenced DAPA)	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVAVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALAAPIEKT1SKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPCDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK
SEQ ID NO: 15	Heavy Chain (Fc Silenced DANAPA)	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVAVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYASTYRVVSVLTVLHQDW LNGKEYKCKVSNKALAAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPCDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK
SEQ ID	Light	DIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNG

150

	NO: 25	Chain	NTYLHWFQQRPGQSPRLLIYTVSNRFSGVPDR FSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHV PPTFGAGTRLEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC
	SEQ ID NO:26	DNA Light Chain	GACATTCAGCTGACACAGAGCCCTCTGAGCC TGCCTGTTACACTGGGACAGCCTGCCAGCAT CAGCTGTAGAAGCAGCCAGAGCCTGGTGCA CAGAAACGGCAACACCTACCTGCACTGGTTC CAGCAGAGGCCTGGCCAGTCTCCTAGACTGC TGATCTACACCGTGTCCAACAGATTCAGCGG CGTGCCCGATAGATTTTCCGGCAGCGGCTCT GGCACCGACTTCACCCTGAAGATTAGCAGA GTGG AAGCCGAG GACGTGGGCGTGTACTTC TGTAGCCAGTCTAGCCACGTGCCACCTACCT TTGGCGCCGGAACCAGACTGGAAATCAAGC GTACGGTGGCCGCTCCCAGCGTGTTCATCTT CCCCCCCAGCGACGAGCAGCTGAAGAGTGG CACCGCCAGCGTGGTGTGCCTGCTGAACAAC TTCTACCCCCGGGAGGCCAAGGTGCAGTGG AAGGTGGACAACGCCCTGCAGAGCGGCAAC AGCCAGGAGAGCGTCACCGAGCAGGACAGC AAGGACTCCACCTACAGCCTGAGCAGCACC CTGACCCTGAGCAAGGCCGACTACGAGAAG CATAAGGTGTACGCCTGCGAGGTGACCCAC CAGGGCCTGTCCAGCCCCGTGACCAAGAGC TTCAACAGGGGCGAGTGC
MiQHC x hzVkla	SEQ ID N0:12	Heavy Chain (WÎId Type Fc)	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPV

151

			TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTY1CNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPCDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK
	SEQ ID NO: 13	DNA Heavy Chain (Wild Type Fc)	CAGGTTCAGCTCCAGCAGTCTGGCAGCGAG CTGAAAAAACCTGGCGCCTCCGTGAAGGTG TCCTGCAAGGCTTCTGGCTACACCTTTACCA ACTACGGCGTGAACTGGATCAAGCAGGCCC CTGGACAAGGCCTCCAATGGATGGGCTGGA TCAACCCCAATACCGGCGAGCCCACCTTCGA CGACGATTTCAAGGGCAGATTCGCCTTCAGC CTGGACACCTCTGTGTCCACAGCCTACCTCC AGATCAGCAGCCTGAAGGCCGATGATACCG CCGTGTACTTCTGCTCCAGAAGCCGGGGAAA GAACGAGGCTTGGTTTGCCTATTGGGGCCAG GGCACACTGGTCACCGTTAGCTCTGCTAGCA CCAAGGGCCCAAGTGTGTTTCCCCTGGCCCC CAGCAGCAAGTCTACTTCCGGCGGAACTGCT GCCCTGGGTTGCCTGGTGAAGGACTACTTCC CCTGTCCCGTGACAGTGTCCTGGAACTCTGG GGCTCTGACTTCCGGCGTGCACACCTTCCCC GCCGTGCTGCAGAGCAGCGGCCTGTACAGC CTGAGCAGCGTGGTGACAGTGCCCTCCAGCT CTCTGGGAACCCAGACCTATATCTGCAACGT GAACCACAAGCCCAGCAACACCAAGGTGGA CAAGAGAGTGGAGCCCAAGAGCTGCGACAA GACCCACACCTGCCCCCCCTGCCCAGCTCCA

152

			GAACTGCTGGGAGGGCCTTCCGTGTTCCTGT TCCCCCCCAAGCCCAAGGACACCCTGATGAT CAGCAGGACCCCCGAGGTGACCTGCGTGGT GGTGGACGTGTCCCACGAGGACCCAGAGGT GAAGTTCAACTGGTACGTGGACGGCGTGGA GGTGCACAACGCCAAGACCAAGCCCAGAGA GGAGCAGTACAACAGCACCTACAGGGTGGT GTCCGTGCTGACCGTGCTGCACCAGGACTGG CTGAACGGCAAAGAATACAAGTGCAAAGTC TCCAACAAGGCCCTGCCAGCCCCAATCGAA AAGACAATCAGCAAGGCCAAGGGCCAGCCA CGGGAGCCCCAGGTGTACACCCTGCCCCCC AGCCGGGAGGAGATGACCAAGAACCAGGTG TCCCTGACCTGTCTGGTGAAGGGCTTCTACC CCTGTGATATCGCCGTGGAGTGGGAGAGCA ACGGCCAGCCCGAGAACAACTACAAGACCA CCCCCCCAGTGCTGGACAGCGACGGCAGCTT CTTCCTGTACAGCAAGCTGACCGTGGACAAG TCCAGGTGGCAGCAGGGCAACGTGTTCAGC TGCAGCGTGATGCACGAGGCCCTGCACAAC CACTACACCCAGAAGTCCCTGAGCCTGAGCC CCGGCAAG
	SEQ ID NO:14	Heavy Chain (Fc Silenced DAPA)	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVWAVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALAAPIEKTISKAKGQPRE

153

		PQVYTLPPSREEMTKNQVSLTCLVKGFYPCDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK
SEQ ID NO: 15	Heavy Chain (Fc Silenced DANAPA)	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGW1NPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVAVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYASTYRVVSVLTVLHQDW LNGKEYKCKVSNKALAAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPCDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK
SEQ ID NO:29	Light Chain	DIVMTQTPLSLPVTPGEPASISCRSSQSLVHRN GNTYLHWYLQKPGQSPQLLIYTVSNRFSGVPD RFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSH VPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO30	DNA Light Chain	GACATTGTGATGACACAGACCCCTCTGAGCC TGCCTGTGACACCTGGCGAACCTGCCAGCAT CAGCTGTAGAAGCAGCCAGAGCCTGGTGCA CAGAAACGGCAACACCTACCTGCACTGGTA TCTGCAGAAGCCCGGCCAGTCTCCTCAGCTG CTGATCTACACCGTGTCCAACAGATTCAGCG GCGTGCCCGATAGATTTTCCGGCAGCGGCTC

154

			TGGCACCGACTTCACCCTGAAGATCTCCAGA GTGGAAGCCGAGGACGTGGGCGTGTACTTC TGTAGCCAGTCTAGCCACGTGCCACCTACCT TTGGCCAGGGGACCAAGCTGGAAATCAAGC GTACGGTGGCCGCTCCCAGCGTGTTCATCTT CCCCCCCAGCGACGAGCAGCTGAAGAGTGG CACCGCCAGCGTGGTGTGCCTGCTGAACAAC TTCTACCCCCGGGAGGCCAAGGTGCAGTGG AAGGTGGACAACGCCCTGCAGAGCGGCAAC AGCCAGGAGAGCGTCACCGAGCAGGACAGC AAGGACTCCACCTACAGCCTGAGCAGCACC CTGACCCTGAGCAAGGCCGACTACGAGAAG CATAAGGTGTACGCCTGCGAGGTGACCCAC CAGGGCCTGTCCAGCCCCGTGACCAAGAGC TTCAACAGGGGCGAGTGC
Mil_HC x hzVklb	SEQ ID NO:12	Heavy Chain (Wild Type Fc)	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPCDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK
	SEQ ID NO: 13	DNA Heavy Chain	CAGGTTCAGCTCCAGCAGTCTGGCAGCGAG CTGAAAAAACCTGGCGCCTCCGTGAAGGTG TCCTGCAAGGCTTCTGGCTACACCTTTACCA

155

(Wild Type Fc)

ACTACGGCGTGAACTGGATCAAGCAGGCCC CTGGACAAGGCCTCCAATGGATGGGCTGGA TCAACCCCAATACCGGCGAGCCCACCTTCGA CGACGATTTCAAGGGCAGATTCGCCTTCAGC CTGGACACCTCTGTGTCCACAGCCTACCTCC AGATCAGCAGCCTGAAGGCCGATGATACCG CCGTGTACTTCTGCTCCAGAAGCCGGGGAAA GAACGAGGCTTGGTTTGCCTATTGGGGCCAG GGCACACTGGTCACCGTTAGCTCTGCTAGCA CCAAGGGCCCAAGTGTGTTTCCCCTGGCCCC CAGCAGCAAGTCTACTTCCGGCGGAACTGCT GCCCTGGGTTGCCTGGTGAAGGACTACTTCC CCTGTCCCGTGACAGTGTCCTGGAACTCTGG GGCTCTGACTTCCGGCGTGCACACCTTCCCC GCCGTGCTGCAGAGCAGCGGCCTGTACAGC CTGAGCAGCGTGGTGACAGTGCCCTCCAGCT CTCTGGGAACCCAGACCTATATCTGCAACGT GAACCACAAGCCCAGCAACACCAAGGTGGA CAAGAGAGTGGAGCCCAAGAGCTGCGACAA GACCCACACCTGCCCCCCCTGCCCAGCTCCA GAACTGCTGGGAGGGCCTTCCGTGTTCCTGT TCCCCCCCAAGCCCAAGGACACCCTGATGAT CAGCAGGACCCCCGAGGTGACCTGCGTGGT GGTGGACGTGTCCCACGAGGACCCAGAGGT GAAGTTCAACTGGTACGTGGACGGCGTGGA GGTGCACAACGCCAAGACCAAGCCCAGAGA GGAGCAGTACAACAGCACCTACAGGGTGGT GTCCGTGCTGACCGTGCTGCACCAGGACTGG CTGAACGGCAAAGAATACAAGTGCAAAGTC TCCAACAAGGCCCTGCCAGCCCCAATCGAA AAGACAATCAGCAAGGCCAAGGGCCAGCCA CGGGAGCCCCAGGTGTACACCCTGCCCCCC AGCCGGGAGGAGATGACCAAGAACCAGGTG

156

		TCCCTGACCTGTCTGGTGAAGGGCTTCTACC CCTGTGATATCGCCGTGGAGTGGGAGAGCA ACGGCCAGCCCGAGAACAACTACAAGACCA CCCCCCCAGTGCTGGACAGCGACGGCAGCTT CTTCCTGTACAGCAAGCTGACCGTGGACAAG TCCAGGTGGCAGCAGGGCAACGTGTTCAGC TGCAGCGTGATGCACGAGGCCCTGCACAAC CACTACACCCAGAAGTCCCTGAGCCTGAGCC CCGGCAAG
SEQ ID NO:14	Heavy Chain (Fc Silenced DAPA)	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVAVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALAAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPCDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK
SEQ ID NO: 15	Heavy Chain (Fc Silenced DANAPA)	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVAVSHEDPEVKFNWYVDGV

167

		EVHNAKTKPREEQYASTYRVVSVLTVLHQDW LNGKEYKCKVSNKALAAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPCDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK
SEQ ID NO:33	Light Chain	DVVMTQSPLSLPVTLGQPASISCRSSQSLVHRN GNTYLHWYQQRPGQSPRLLIYTVSNRFSGVPD RFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSH VPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:34	DNA Light Chain	GATGTGGTTATGACACAGAGCCCTCTGAGCC TGCCTGTGACACTTGGACAGCCTGCCAGCAT CAGCTGCAGATCTAGCCAGAGCCTGGTGCA CAGAAACGGCAACACCTACCTGCACTGGTA TCAGCAGAGGCCCGGACAGTCTCCCAGACT GCTGATCTACACCGTGTCCAACAGATTCAGC GGCGTGCCCGATAGATTTTCCGGCAGCGGCT CTGGCACCGACTTCACCCTGAAGATCTCCAG AGTGGAAGCCGAGGACGTGGGCGTGTACTT CTGTAGCCAGTCTAGCCACGTGCCACCTACC TTTGGCCAGGGCACCAAGCTGGAAATCAAG CGTACGGTGGCCGCTCCCAGCGTGTTCATCT TCCCCCCCAGCGACGAGCAGCTGAAGAGTG GCACCGCCAGCGTGGTGTGCCTGCTGAACA ACTTCTACCCCCGGGAGGCCAAGGTGCAGT GGAAGGTGGACAACGCCCTGCAGAGCGGCA ACAGCCAGGAGAGCGTCACCGAGCAGGACA GCAAGGACTCCACCTACAGCCTGAGCAGCA CCCTGACCCTGAGCAAGGCCGACTACGAGA AGCATAAGGTGTACGCCTGCGAGGTGACCC

158

			ACCAGGGCCTGTCCAGCCCCGTGACCAAGA GCTTCAACAGGGGCGAGTGC
H cm il x LCmil_NQ	SEQ ID NO: 12	Heavy Chain (Wild Type Fc)	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL M1SRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPCDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK
	SEQ ID NO: 13	DNA Heavy Chain (Wild Type Fc)	CAGGTTCAGCTCCAGCAGTCTGGCAGCGAG CTGAAAAAACCTGGCGCCTCCGTGAAGGTG TCCTGCAAGGCTTCTGGCTACACCTTTACCA ACTACGGCGTGAACTGGATCAAGCAGGCCC CTGGACAAGGCCTCCAATGGATGGGCTGGA TCAACCCCAATACCGGCGAGCCCACCTTCGA CGACGATTTCAAGGGCAGATTCGCCTTCAGC CTGGACACCTCTGTGTCCACAGCCTACCTCC AGATCAGCAGCCTGAAGGCCGATGATACCG CCGTGTACTTCTGCTCCAGAAGCCGGGGAAA GAACGAGGCTTGGTTTGCCTATTGGGGCCAG GGCACACTGGTCACCGTTAGCTCTGCTAGCA CCAAGGGCCCAAGTGTGTTTCCCCTGGCCCC CAGCAGCAAGTCTACTTCCGGCGGAACTGCT GCCCTGGGTTGCCTGGTGAAGGACTACTTCC CCTGTCCCGTGACAGTGTCCTGGAACTCTGG

159

			GGCTCTGACTTCCGGCGTGCACACCTTCCCC GCCGTGCTGCAGAGCAGCGGCCTGTACAGC CTGAGCAGCGTGGTGACAGTGCCCTCCAGCT CTCTGGGAACCCAGACCTATATCTGCAACGT GAACCACAAGCCCAGCAACACCAAGGTGGA CAAGAGAGTGGAGCCCAAGAGCTGCGACAA GACCCACACCTGCCCCCCCTGCCCAGCTCCA GAACTGCTGGGAGGGCCTTCCGTGTTCCTGT TCCCCCCCAAGCCCAAGGACACCCTGATGAT CAGCAGGACCCCCGAGGTGACCTGCGTGGT GGTGGACGTGTCCCACGAGGACCCAGAGGT GAAGTTCAACTGGTACGTGGACGGCGTGGA GGTGCACAACGCCAAGACCAAGCCCAGAGA GGAGCAGTACAACAGCACCTACAGGGTGGT GTCCGTGCTGACCGTGCTGCACCAGGACTGG CTGAACGGCAAAGAATACAAGTGCAAAGTC TCCAACAAGGCCCTGCCAGCCCCAATCGAA AAGACAATCAGCAAGGCCAAGGGCCAGCCA CGGGAGCCCCAGGTGTACACCCTGCCCCCC AGCCGGGAGGAGATGACCAAGAACCAGGTG TCCCTGACCTGTCTGGTGAAGGGCTTCTACC CCTGTGATATCGCCGTGGAGTGGGAGAGCA ACGGCCAGCCCGAGAACAACTACAAGACCA CCCCCCCAGTGCTGGACAGCGACGGCAGCTT CTTCCTGTACAGCAAGCTGACCGTGGACAAG TCCAGGTGGCAGCAGGGCAACGTGTTCAGC TGCAGCGTGATGCACGAGGCCCTGCACAAC CACTACACCCAGAAGTCCCTGAGCCTGAGCC CCGGCAAG
	SEQ ID NO:I4	Heavy Chain (Fc Silenced DAPA)	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSSASTK

160

		GPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVAVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKE YKC KVSNKA LAAPIEKTISKAKGQP RE PQVYTLPPSREEMTKNQVSLTCLVKGFYPCDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK
SEQ ID NO: 15	Heavy Chain (Fc Silenced DANAPA)	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNW1KQAPGQGLQWMGW1NPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVAVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYASTYRVVSVLTVLHQDW LNGKEYKCKVSNKALAAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPCDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK
SEQ ID NO:38	Light Chain	DIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNQ NTYLHWFQQRPGQSPRLLIYTVSNRFSGVPDR FSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHV PPTFGAGTRLEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC

161

	SEQ ID NO:39	DNA Light Chain	GACATTCAGCTGACACAGAGCCCTCTGAGCC TGCCTGTTACACTGGGACAGCCTGCCAGCAT CAGCTGTAGAAGCAGCCAGAGCCTGGTGCA CAGAAACCAGAACACCTACCTGCACTGGTTC CAGCAGAGGCCTGGCCAGTCTCCTAGACTGC TGATCTACACCGTGTCCAACAGATTCAGCGG CGTGCCCGATAGATTTTCCGGCAGCGGCTCT GGCACCGACTTCACCCTGAAGATTAGCAGA GTGGAAGCCGAGGACGTGGGCGTGTACTTC TGTAGCCAGTCTAGCCACGTGCCACCTACCT TTGGCGCCGGAACCAGACTGGAAATCAAGC GTACGGTGGCCGCTCCCAGCGTGTTCATCTT CCCCCCCAGCGACGAGCAGCTGAAGAGTGG CACCGCCAGCGTGGTGTGCCTGCTGAACAAC TTCTACCCCCGGGAGGCCAAGGTGCAGTGG AAGGTGGACAACGCCCTGCAGAGCGGCAAC AGCCAGGAGAGCGTCACCGAGCAGGACAGC AAGGACTCCACCTACAGCCTGAGCAGCACC CTGACCCTGAGCAAGGCCGACTACGAGAAG CATAAGGTGTACGCCTGCGAGGTGACCCAC CAGGGCCTGTCCAGCCCCGTGACCAAGAGC TTCAACAGGGGCGAGTGC
Vhmil x VKlaNQ	SEQ ID NO: 12	Heavy Chain (Wild Type Fc)	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQ1SSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCWVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPRE

162

			PQVYTLPPSREEMTKNQVSLTCLVKGFYPCDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK
	SEQ ID NO:13	DNA Heavy Chain (Wild Type Fc)	CAGGTTCAGCTCCAGCAGTCTGGCAGCGAG CTGAAAAAACCTGGCGCCTCCGTGAAGGTG TCCTGCAAGGCTTCTGGCTACACCTTTACCA ACTACGGCGTGAACTGGATCAAGCAGGCCC CTGGACAAGGCCTCCAATGGATGGGCTGGA TCAACCCCAATACCGGCGAGCCCACCTTCGA CGACGATTTCAAGGGCAGATTCGCCTTCAGC CTGGACACCTCTGTGTCCACAGCCTACCTCC AGATCAGCAGCCTGAAGGCCGATGATACCG CCGTGTACTTCTGCTCCAGAAGCCGGGGAAA GAACGAGGCTTGGTTTGCCTATTGGGGCCAG GGCACACTGGTCACCGTTAGCTCTGCTAGCA CCAAGGGCCCAAGTGTGTTTCCCCTGGCCCC CAGCAGCAAGTCTACTTCCGGCGGAACTGCT GCCCTGGGTTGCCTGGTGAAGGACTACTTCC CCTGTCCCGTGACAGTGTCCTGGAACTCTGG GGCTCTGACTTCCGGCGTGCACACCTTCCCC GCCGTGCTGCAGAGCAGCGGCCTGTACAGC CTGAGCAGCGTGGTGACAGTGCCCTCCAGCT CTCTGGGAACCCAGACCTATATCTGCAACGT GAACCACAAGCCCAGCAACACCAAGGTGGA CAAGAGAGTGGAGCCCAAGAGCTGCGACAA GACCCACACCTGCCCCCCCTGCCCAGCTCCA GAACTGCTGGGAGGGCCTTCCGTGTTCCTGT TCCCCCCCAAGCCCAAGGACACCCTGATGAT CAGCAGGACCCCCGAGGTGACCTGCGTGGT GGTGGACGTGTCCCACGAGGACCCAGAGGT GAAGTTCAACTGGTACGTGGACGGCGTGGA GGTGCACAACGCCAAGACCAAGCCCAGAGA

163

		GGAGCAGTACAACAGCACCTACAGGGTGGT GTCCGTGCTGACCGTGCTGCACCAGGACTGG CTGAACGGCAAAGAATACAAGTGCAAAGTC TCCAACAAGGCCCTGCCAGCCCCAATCGAA AAGACAATCAGCAAGGCCAAGGGCCAGCCA CGGGAGCCCCAGGTGTACACCCTGCCCCCC AGCCGGGAGGAGATGACCAAGAACCAGGTG TCCCTGACCTGTCTGGTGAAGGGCTTCTACC CCTGTGATATCGCCGTGGAGTGGGAGAGCA ACGGCCAGCCCGAGAACAACTACAAGACCA CCCCCCCAGTGCTGGACAGCGACGGCAGCTT CTTCCTGTACAGCAAGCTGACCGTGGACAAG TCCAGGTGGCAGCAGGGCAACGTGTTCAGC TGCAGCGTGATGCACGAGGCCCTGCACAAC CACTACACCCAGAAGTCCCTGAGCCTGAGCC CCGGCAAG
SEQ ID NO:14	Heavy Chain (Fc Silenced DAPA)	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVAVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALAAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPCDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK
SEQ ID NO: 15	Heavy Chain (Fc	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD

164

	Silenced DANAPA)	DDFKGRFAFSLDTSVSTAYLQÏSSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVAVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYASTYRVVSVLTVLHQDW LNGKEYKCKVSNKALAAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPCDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTV D KS R WQQGNVF S CS VMHE A LHNH YTQK SLSLSPGK
SEQ ID NO:42	Light Chain	DIVMTQTPLSLPVTPGEPASISCRSSQSLVHRN QNTYLHWYLQKPGQSPQLLIYTVSNRFSGVPD RFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSH VPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO:43	DNA Light Chain	GACATTGTGATGACACAGACCCCTCTGAGCC TGCCTGTGACACCTGGCGAACCTGCCAGCAT CAGCTGTAGAAGCAGCCAGAGCCTGGTGCA CCGGAACCAGAATACCTACCTGCACTGGTAT CTGCAGAAGCCCGGCCAGTCTCCTCAGCTGC TGATCTACACCGTGTCCAACAGATTCAGCGG CGTGCCCGATAGATTTTCCGGCAGCGGCTCT GGCACCGACTTCACCCTGAAGATCTCCAGAG TGGAAGCCGAGGACGTGGGCGTGTACTTCT GTAGCCAGTCTAGCCACGTGCCACCTACCTT TGGCCAGGGGACCAAGCTGGAAATCAAGCG TACGGTGGCCGCTCCCAGCGTGTTCATCTTC CCCCCCAGCGACGAGCAGCTGAAGAGTGGC

165

			ACCGCCAGCGTGGTGTGCCTGCTGAACAACT TCTACCCCCGGGAGGCCAAGGTGCAGTGGA AGGTGGACAACGCCCTGCAGAGCGGCAACA GCCAGGAGAGCGTCACCGAGCAGGACAGCA AGGACTCCACCTACAGCCTGAGCAGCACCCT GACCCTGAGCAAGGCCGACTACGAGAAGCA TAAGGTGTACGCCTGCGAGGTGACCCACCA GGGCCTGTCCAGCCCCGTGACCAAGAGCTTC AACAGGGGCGAGTGC
Vhmil x VKlbNQ	SEQ ID NO:12	Heavy Chain (Wild Type Fc)	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVP S S S LGTQTYICNVNHKP SN TKVDKRVEP K SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPCDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK
	SEQ ID NO: 13	DNA Heavy Chain (Wild Type Fc)	CAGGTTCAGCTCCAGCAGTCTGGCAGCGAG CTGAAAAAACCTGGCGCCTCCGTGAAGGTG TCCTGCAAGGCTTCTGGCTACACCTTTACCA ACTACGGCGTGAACTGGATCAAGCAGGCCC CTGGACAAGGCCTCCAATGGATGGGCTGGA TCAACCCCAATACCGGCGAGCCCACCTTCGA CGACGATTTCAAGGGCAGATTCGCCTTCAGC CTGGACACCTCTGTGTCCACAGCCTACCTCC AGATCAGCAGCCTGAAGGCCGATGATACCG

166

CCGTGTACTTCTGCTCCAGAAGCCGGGGAAA GAACGAGGCTTGGTTTGCCTATTGGGGCCAG GGC AC ACTGGTC ACC GTTAGCTCTGCTAGCA CCAAGGGCCCAAGTGTGTTTCCCCTGGCCCC CAGCAGCAAGTCTACTTCCGGCGGAACTGCT GCCCTGGGTTGCCTGGTG A AGGACTACTTC C CCTGTCCCGTGACAGTGTCCTGGAACTCTGG GGCTCTGACTTCCGGCGTGCACACCTTCCCC GCCGTGCTGCAGAGCAGCGGCCTGTACAGC CTGAGCAGCGTGGTGACAGTGCCCTCCAGCT CTCTGGGAACCCAGACCTATATCTGCAACGT GAACCACAAGCCCAGCAACACCAAGGTGGA CAAGAGAGTGGAGCCCAAGAGCTGCGACAA GACCCACACCTGCCCCCCCTGCCCAGCTCCA GAACTGCTGGGAGGGCCTTCCGTGTTCCTGT TCCCCCCCAAGCCCAAGGACACCCTGATGAT CAGCAGGACCCCCGAGGTGACCTGCGTGGT GGTGGACGTGTCCCACGAGGACCCAGAGGT GAAGTTCAACTGGTACGTGGACGGCGTGGA GGTGCACAACGCCAAGACCAAGCCCAGAGA GGAGCAGTACAACAGCACCTACAGGGTGGT GTCCGTGCTGACCGTGCTGCACCAGGACTGG CTGAACGGCAAAGAATACAAGTGCAAAGTC TCCAACAAGGCCCTGCCAGCCCCAATCGAA AAGACAATCAGCAAGGCCAAGGGCCAGCCA CGGGAGCCCCAGGTGTACACCCTGCCCCCC AGCCGGGAGGAGATGACCAAGAACCAGGTG TCCCTGACCTGTCTGGTGAAGGGCTTCTACC CCTGTGATATCGCCGTGGA GTGGG A GAGC A ACGGCCAGCCCGAGAACAACTACAAGACCA CCCCCCCAGTGCTGGACAGCGACGGCAGCTT CTTCCTGTACAGCAAGCTGACCGTGGACAAG TCCAGGTGGCAGCAGGGCAACGTGTTCAGC

167

		TGCAGCGTGATGCACGAGGCCCTGCACAAC CACTACACCCAGAAGTCCCTGAGCCTGAGCC CCGGCAAG
SEQ TD NO:14	Heavy Chain (Fc Silenced DAPA)	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVAVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALAAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPCDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK
SEQ ID NO: 15	Heavy Chain (Fc Silenced DANAPA)	QVQLQQSGSELKKPGASVKVSCKASGYTFTN YGVNWIKQAPGQGLQWMGWINPNTGEPTFD DDFKGRFAFSLDTSVSTAYLQISSLKADDTAV YFCSRSRGKNEAWFAYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPCPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCWVAVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYASTYRVVSVLTVLHQDW LNGKEYKCKVSNKALAAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPCDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK

168

	SEQ ID NO:46	Light Chain	DVVMTQSPLSLPVTLGQPASISCRSSQSLVHRN QNTYLHWYQQRPGQSPRLLIYTVSNRFSGVPD RFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSH VPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWK.VDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC
	SEQ ID NO:47	DNA Light Chain	GATGTGGTTATGACACAGAGCCCTCTGAGCC TGCCTGTGACACTTGGACAGCCTGCCAGCAT CAGCTGCAGATCTAGCCAGAGCCTGGTGCA CCGGAACCAGAACACATACCTGCACTGGTA TCAGCAGAGGCCCGGACAGTCTCCCAGACT GCTGATCTACACCGTGTCCAACAGATTCAGC GGCGTGCCCGATAGATTTTCCGGCAGCGGCT CTGGCACCGACTTCACCCTGAAGATCTCCAG AGTGGAAGCCGAGGACGTGGGCGTGTACTT CTGTAGCCAGTCTAGCCACGTGCCACCTACC TTTGGCCAGGGCACCAAGCTGGAAATCAAG CGTACGGTGGCCGCTCCCAGCGTGTTCATCT TCCCCCCCAGCGACGAGCAGCTGAAGAGTG GCACCGCCAGCGTGGTGTGCCTGCTGAACA ACTTCTACCCCCGGGAGGCCAAGGTGCAGT GGAAGGTGGACAACGCCCTGCAGAGCGGCA ACAGCCAGGAGAGCGTCACCGAGCAGGACA GCAAGGACTCCACCTACAGCCTGAGCAGCA CCCTGACCCTGAGCAAGGCCGACTACGAGA AGCATAAGGTGTACGCCTGCGAGGTGACCC ACCAGGGCCTGTCCAGCCCCGTGACCAAGA GCTTCAACAGGGGCGAGTGC

169

In some embodiments, the antibody or antigen-binding fragment of an ADC disclosed herein may comprise any set of heavy and light chain variable domains listed in the tables above or a set of six CDRs from any set of heavy and light chain variable domains listed in the tables above. In some embodiments, the antibody or antigen-binding fragment of an ADC disclosed herein may comprise amino acid sequences that are conservatively modified and/or homologous to the sequences listed in the tables above, so long as the ADC rctains the ability to bind to its target cancer antîgen (e.g., with a Kd of less than 1x10⁸M) and retains one or more fùnctional properties of the ADCs disclosed herein (e.g., ability to intemalize, bind to an antigen target, e.g., an antigen expressed on a tumor or other cancer cell, etc.).

In some embodiments, the antibody or antigen-binding fragment of an ADC disclosed herein further comprises human heavy and light chain constant domains or fragments thereof. For instance, the antibody or antigen-binding fragment of the described ADCs may comprise a human IgG heavy chain constant domain (such as an IgGl) and a human kappa or lambda light chain constant domain. In some embodiments, the antibody or antigen-binding fragment of the described ADCs comprises a human immunoglobulin G subtype l (IgGl) heavy chain constant domain with a human Ig kappa light chain constant domain.

In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDRl (HCDRl) consisting of SEQ ID NO:l, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:2, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; light chain CDRl (LCDRl) consisting of SEQ ID NO: 16, light chain CDR2 (LCDR2) consisting of SEQ ID NO:70, and light chain CDR3 (LCDR3) consisting of SEQ ID NO: IS.

In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDRl (HCDRl) consisting of SEQ ID NO:4, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO;2, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; light chain CDRl (LCDRl) consisting of SEQ ID NO:l6, light chain CDR2 (LCDR2) consisting of SEQ ID NO:70, and light chain CDR3 (LCDR3) consisting of SEQ ID NO:18.

In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDRl (HCDRl) consisting of SEQ ID NO:5, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:6, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; light chain CDRl (LCDRl)

170 consisting of SEQ ID NO:I9, light chain CDR2 (LCDR2) consisting of SEQ ID NO:20, and light chain CDR3 (LCDR3) consisting of SEQ ID NO:21.

In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDRl (HCDR1) consisting of SEQ ID NO:7, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:8, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:9; light chain CDRl (LCDRl) consisting of SEQ ID NO:22, light chain CDR2 (LCDR2) consisting of SEQ ID NO:20, and light chain CDR3 (LCDR3) consisting of SEQ ID NO: 18.

In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDRl (HCDRl) consisting of SEQ ID NO: 1, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:2, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; light chain CDRl (LCDRl) consisting of SEQ ID NO:35, light chain CDR2 (LCDR2) consisting of SEQ ID NO:70, and light chain CDR3 (LCDR3) consisting of SEQ ID NO:18.

In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDRl (HCDRl) consisting of SEQ ID NO:4, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:2, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; light chain CDRl (LCDRl) consisting of SEQ ID NO:35, light chain CDR2 (LCDR2) consisting of SEQ ID NO:70, and light chain CDR3 (LCDR3) consisting of SEQ ID NO: 18.

In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDRl (HCDRl) consisting of SEQ ID NO:5, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:6, heavy chain CDRS (HCDR3) consisting of SEQ ID NO:3; light chain CDRl (LCDRl) consisting of SEQ ID NO:71, light chain CDR2 (LCDR2) consisting of SEQ ID NO:20, and light chain CDR3 (LCDR3) consisting of SEQ ID NO:21.

In some embodiments, the antî-CD74 antibody or antigen-binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDRl (HCDRl) consisting of SEQ ID NO:7, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:8, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:9; light chain CDRl (LCDRl) consisting of SEQ ID NO: 17, light chain CDR2 (LCDR2) consisting of SEQ ID NO:20, and light chain CDR3 (LCDR3) consisting of SEQ ID NO: 18.

171

In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprises a heavy chain variable région comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable région comprising the amino acid sequence of SEQ ID NO:23. In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprises the heavy chain variable région amino acid sequence of SEQ ID NO: 10 and the light chain variable région amino acid sequence of SEQ ID NO:23, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof has a heavy chain variable région amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10 and/or a light chain variable région amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:23.

In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprises a heavy chain variable région comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable région comprising the amino acid sequence of SEQ ID NO:27. In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprises the heavy chain variable région amino acid sequence of SEQ ID NO: 10 and the light chain variable région amino acid sequence of SEQ ID NO:27, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof has a heavy chain variable région amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10 and/or a light chain variable région amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ IDNO:27.

In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprises a heavy chain variable région comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable région comprising the amino acid sequence of SEQ ID NO:31. In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprises the heavy chain variable région amino acid sequence of SEQ ID NO: 10 and the light chain variable région amino acid sequence of SEQ ID NO:31, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof has a heavy chain variable région amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10 and/or a light chain variable région amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:31.

172 ln some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprises a heavy chain variable région comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable région comprising the amino acid sequence of SEQ ID NO:36. In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprises the heavy chain variable région amino acid sequence of SEQ ID NO: 10 and the light chain variable région amino acid sequence of SEQ ID NO:36, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof has a heavy chain variable région amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10 and/or a light chain variable région amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:36.

In some embodiments, the antî-CD74 antibody or antigen-binding fragment thereof comprises a heavy chain variable région comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable région comprising the amino acid sequence of SEQ ID NO:40. In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprises the heavy chain variable région amino acid sequence of SEQ ID NO: 10 and the light chain variable région amino acid sequence of SEQ ID NO:40, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof has a heavy chain variable région amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO; 10 and/or a light chain variable région amino acid sequence that îs at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:40.

In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprises a heavy chain variable région comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable région comprising the amino acid sequence of SEQ ID NO:44. In some embodiments, the anti-CD74 antibody or antigen-binding fragment thereof comprises the heavy chain variable région amino acid sequence of SEQ ID NO: 10 and the light chain variable région amino acid sequence of SEQ ID NO:44, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the antî-CD74 antibody or antigen-binding fragment thereof has a heavy chain variable région amino acid sequence that îs at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10 and/or a light chain variable région amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:44.

173 ln some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 12 or a sequence that is at least 95% identical to SEQ ID NO: 12, and the light chain amino acid sequence of SEQ ID NO:25 or a sequence that is at least 95% identical to SEQ ID NO:25. In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 12 and the light chain amino acid sequence of SEQ ID NO:25, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD74 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:25. In some embodiments, the antî-CD74 antibody is milatuzumab (see US Patent No. 7931903), or an antigen-binding fragment thereof. In some embodiments, the anti~CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 14 or a sequence that is at least 95% identical to SEQ ID NO: 14, and the light chain amino acid sequence of SEQ ID NO:25 or a sequence that is at least 95% identical to SEQ ID NO:25. In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 14 and the light chain amino acid sequence of SEQ ID NO:25, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD74 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:25.

In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 15 or a sequence that is at least 95% identical to SEQ ID NO: 15, and the light chain amino acid sequence of SEQ ID NO:25 or a sequence that is at least 95% identical to SEQ ID NO:25. In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 15 and the light chain amino acid sequence of SEQ ID NO:25, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD74 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ IDNO:25.

In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 12 or a sequence that is at least 95% identical to SEQ ID NO: 12, and the light chain amino acid sequence of SEQ ID NO:29 or a sequence that is at least 95%

174 identical to SEQ ID NO:29. In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 12 and the light chain amino acid sequence of SEQ ID NO:29, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD74 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:29.

In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 14 or a sequence that is at least 95% identical to SEQ ID NO: 14, and the light chain amino acid sequence of SEQ ID NO:29 or a sequence that is at least 95% identical to SEQ ID NO;29. In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 14 and the light chain amino acid sequence of SEQ ID NO:29, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD74 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:29.

In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 15 or a sequence thaï is at least 95% identical to SEQ ID NO: 15, and the light chain amino acid sequence of SEQ ID NO:29 or a sequence that is at least 95% identical to SEQ ID NO:29. In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 15 and the light chain amino acid sequence of SEQ ID NO:29, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD74 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:29.

In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 12 or a sequence that is at least 95% identical to SEQ ID NO: 12, and the light chain amino acid sequence of SEQ ID NO:33 or a sequence that is at least 95% identical to SEQ ID NO:33. In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 12 and the light chain amino acid sequence of SEQ ID NO:33, or sequences that are at least 95% identical to the disclosed sequences. In some

175 embodiments, the anti-CD74 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:33.

In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 14 or a sequence that is at least 95% identical to SEQ ID NO: 14, and the light chain amino acid sequence of SEQ ID NO:33 or a sequence that is at least 95% identical to SEQ ID NO:33. In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 14 and the light chain amino acid sequence of SEQ ID NO:33, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD74 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO;33.

In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 15 or a sequence that is at least 95% identical to SEQ ID NO; 15, and the light chain amino acid sequence of SEQ ID NO:33 or a sequence that is at least 95% identical to SEQ ID NO:33. In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 15 and the light chaîn amino acid sequence of SEQ ID NO:33, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD74 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 15 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:33.

In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 12 or a sequence that is at least 95% identical to SEQ ID NO: 12, and the light chain amino acid sequence of SEQ ID NO:38 or a sequence that îs at least 95% identical to SEQ ID NO:38. In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 12 and the light chain amino acid sequence of SEQ ID NO;38, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD74 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12 and a light chain

176 amino acid sequence that is at least 96%, at least 97%, at least 98%, or ai least 99% identical to SEQ ID NO:38.

In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 14 or a sequence that is at least 95% identical to SEQ ID NO: 14, and the light chain amino acid sequence of SEQ ID NO:38 or a sequence that is at least 95% identical to SEQ ID NO:38. In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 14 and the light chain amino acid sequence of SEQ ID NO:38, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD74 antibody has a heavy chain amino acid sequence that is at least 96%, ai least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 14 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:38.

In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 15 or a sequence that is at least 95% identical to SEQ ID NO: 15, and the light chain amino acid sequence of SEQ ID NO:38 or a sequence that is at least 95% identical to SEQ ID NO:38. In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 15 and the light chain amino acid sequence of SEQ ID NO:38, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD74 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:15 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:38.

In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 12 or a sequence that is at least 95% identical to SEQ ID NO: 12, and the light chain amino acid sequence of SEQ ID N0:42 or a sequence that is at least 95% identical to SEQ ID NO:42. In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 12 and the light chain amino acid sequence of SEQ ID NO:42, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD74 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:42.

177

In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:l4 or a sequence that is at least 95% identîcal to SEQ ID NO: 14, and the light chain amino acid sequence of SEQ ID N0:42 or a sequence that is at least 95% identîcal to SEQ ID NO:42. In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 14 and the light chain amino acid sequence of SEQ ID NO:42, or sequences that are at least 95% identîcal to the disclosed sequences. In some embodiments, the anti-CD74 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identîcal to SEQ ID NO: 14 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identîcal to SEQ ID NO:42.

In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 15 or a sequence that is at least 95% identîcal to SEQ ID NO: 15, and the light chain amino acid sequence of SEQ ID N0:42 or a sequence that is at least 95% identîcal to SEQ ID NO:42. In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 15 and the light chain amino acid sequence of SEQ ID N0:42, or sequences that are at least 95% identîcal to the disclosed sequences. In some embodiments, the anti-CD74 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identîcal to SEQ ID NO: 15 and a light chain amino acid sequence that is at least 96%, at least 97%, ai least 98%, or at least 99% identîcal to SEQ ID N0:42.

In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:12 or a sequence that is at least 95% identîcal to SEQ ID NO: 12, and the light chain amino acid sequence of SEQ ID NO:46 or a sequence that is at least 95% identîcal to SEQ ID NO:46. In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 12 and the light chain amino acid sequence of SEQ ID NO:46, or sequences that are at least 95% identîcal to the disclosed sequences. In some embodiments, the anti-CD74 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identîcal to SEQ ID N0:12 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identîcal to SEQ ID NO:46.

In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 14 or a sequence that is at least 95% identîcal to SEQ ID NO: 14, and the light chain amino acid sequence of SEQ ID NO:46 or a sequence that is at least 95%

178 identîcal to SEQ ID NO:46. In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:l4 and the light chain amino acid sequence of SEQ ID NO:46, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD74 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: I4 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:46.

In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO: 15 or a sequence that is at least 95% identical to SEQ ID NO: 15, and the light chain amino acid sequence of SEQ ID NO;46 or a sequence that is at least 95% identical to SEQ ID NO:46. In some embodiments, the anti-CD74 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:15 and the light chain amino acid sequence of SEQ ID NO:46, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD74 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:15 and a light chain amino acid sequence that îs at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:46.

Residues in two or more polypeptides are said to “correspond” if the residues occupy an analogous position in the polypeptide structures. Analogous positions in two or more polypeptides can be determined by aligning the polypeptide sequences based on amino acid sequence or structural simîlarities. Those skilled in the art understand that it may be necessary to introduce gaps in either sequence to produce a satisfactory alignment.

In some embodiments, amino acid substitutions are of single residues. Insertions usually will be on the order of from about 1 to about 20 amino acid residues, although considerably larger insertions may be tolerated as long as biological funciion is retained (e.g., binding to a target antigen). Délétions usually range from about 1 to about 20 amino acid residues, although in some cases délétions may be much larger. Substitutions, délétions, insertions, or any combination thereof may be used to arrive at a final dérivative or variant. Generally, these changes are done on a few amino acids to minimize the alteration of the molécule, parti cul arly the immunogenîcity and specificity of the antigen binding protein. However, larger changes may be tolerated in certain circumstances. Conservative substitutions can be made in accordance with the foliowing chart depîcted as Table 1.

179

Table 1
Original Residue Ala Arg	Exemplary Substitutions Ser Lys
5 Asn	Gin, His
Asp	Glu
Cys	Ser
Gin	Asn
Glu	Asp
10 Gly	Pro
His	Asn, Gin
Ile	Leu, Val
Leu	Ile, Val
Lys	Arg, Gin, Glu
15 Met	Leu, Ile
Phe	Met, Leu, Tyr
Ser	Thr
Thr	Ser
Trp	Tyr
20 Tyr	Trp, Phe
Val	Ile, Leu

In some embodiments where variant antibody sequences are used in an ADC, the variants typically exhibit the same qualitative biological activity and will elicit the same immune response, although variants may also be selected to modify the characteristics of the antigen binding proteins as needed. Alternatively, the variant may be desîgned such that the biological activity of the antigen binding protein is altered. For example, glycosylation sites may be altered or removed.

The immun o conjugal es of the invention may comprise modified antibodies or antigen binding fragments thereof that further comprise modifications to framework residues within VH and/or VL, e.g. to improve the properties of the antibody. In some embodiments, the framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to “back-mutate” one or more framework residues to the corresponding germline

180 sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To retum the framework région sequences to their germline configuration, the somatic mutations can be “back-mutated” to the germline sequence by, for example, site-directed mutagenesis. Such “back-mutated” antibodies are also intended to be encompassed by the invention.

Another type of framework modification in volves mutating one or more residues within the framework région, or even within one or more CDR régions, to remove T-cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as “deimmunization” and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al.

In addition or in the alternative to modifications made within the framework or CDR régions, antibodies of the invention may be engineered to include modifications within the Fc région, typically to alter one or more functional properties of the antibody, such as sérum halflife, complément fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicîty (ADCC). Furthermore, an antibody of the invention may be chemically modîfied (e.g., one or more Chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody. Each of these embodiments is described in further detail beiow.

In one embodiment, the hinge région of CH1 is modified such that the number of cysteine residues in the hinge région is altered, e.g., increased or decreased. This approach is described further in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues în the hinge région of CH 1 is altered to, for example, facilitaie assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In some embodiments, the antibody or antibody fragment disclosed herein include modified or engineered amino acid residues, e.g., one or more cysteine residues, as sites for conjugation to a drug moiety (Junutula JR, et al., Nat Biotechnol 2008, 26:925-932). In one embodiment, the invention provides a modified antibody or antibody fragment comprising a substitution of one or more amino acids with cysteine at the positions described herein. Sites for cysteine substitution are in the constant régions of the antibody or antibody fragment and are thus applicable to a variety of antibody or antibody fragment, and the sites are selected to provide stable and homogeneous conjugates. A modified antibody or fragment can hâve one,

181 two or more cysteine substitutions, and these substitutions can be used in combination with other modification and conjugation methods as described herein. Methods for inserting cysteine at spécifie locations of an antibody are known in the art, see, e.g., Lyons et al., (1990) Protein Eng., 3:703-708, WO 2011/005481, WO2014/124316, WO 2015/138615. In certain embodiments, a modified antibody comprises a substitution of one or more amino acids with cysteine on its constant région selected from positions 117, 119, 121, 124, 139, 152, 153, 155, 157, 164, 169, 171, 174, 189, 191, 195, 197, 205, 207, 246, 258, 269, 274, 286, 288, 290, 292, 293, 320, 322, 326, 333, 334, 335, 337, 344, 355, 360, 375, 382, 390, 392, 398, 400 and 422 of a heavy chain of the antibody, and wherein the positions are numbered according to the EU system. In some embodiments a modified antibody or antibody fragment comprises a substitution of one or more amino acids with cysteine on its constant région selected from positions 107, 108, 109, 114, 129, 142, 143, 145, 152, 154, 156, 159, 161, 165, 168, 169, 170, 182, 183, 197, 199, and 203 of a light chain of the antibody or antibody fragment, wherein the positions are numbered according to the EU system, and wherein the light chain is a human kappa light chain. In certain embodiments a modified antibody or antibody fragment thereof comprises a combination of substitution of two or more amino acids with cysteine on its constant régions wherein the combinations comprise substitutions at positions 375 of an antibody heavy chain, position 152 of an antibody heavy chain, position 360 of an antibody heavy chain, or position 107 of an antibody light chain and wherein the positions are numbered according to the EU system. In certain embodiments a modified antibody or antibody fragment thereof comprises a substitution of one amino acid with cysteine on its constant régions wherein the substitution is position 375 of an antibody heavy chain, position 152 of an antibody heavy chain, position 360 of an antibody heavy chain, position 107 of an antibody light chain, position 165 of an antibody light chain or position 159 of an antibody light chain and wherein the positions are numbered according to the EU system, and wherein the light chain is a kappa chain. In particular embodiments a modified antibody or antibody fragment thereof comprises a combination of substitution of two amino acids with cysteine on its constant régions wherein the combinations comprise substitutions at positions 375 of an antibody heavy chain and position 152 of an antibody heavy chain, wherein the positions are numbered according to the EU system. In particular embodiments a modified antibody or antibody fragment thereof comprises a substitution of one amino acid with cysteine at position 360 of an antibody heavy chain, wherein the positions are numbered according to the EU system. In other particular embodiments a modified antibody or antibody fragment thereof comprises a substitution of one amino acid with cysteine at position 107 of an antibody light

182 chain and wherein the positions are numbered according to the EU system, and wherein the light chain is a kappa chain.

In additional embodiments antibodies or antibody fragments (e.g., antigen binding fragment) useful in immunoconjugates of the invention include modified or engineered antibodies, such as an antibody modified to introduce one or more other reactive amino acid (other than cysteine), includîng Pci, pyrrolysine, peptide tags (such as S6, Al and ybbR tags), and non-natural amino acids, in place of at least one amino acid of the native sequence, thus providing a reactive site on the antibody or antigen binding fragment for conjugation to a drug moiety or a linker-drug moiety with complementary reactivity. For example, the antibodies or antibody fragments can be modified to incorporate Pci or pyrrolysine (W. Ou, et al., (2011) PNAS 108 (26), 10437-10442; WO2014124258) or unnatural amino acids (J.Y. Axup, et al., Proc Natl Acad Sci U S A, 109 (2012), pp. 16101-16106; for review, see C.C. Liu and P.G. Schultz (2010) Annu Rev Biochem 79, 413-444; C.H. Kim, et al., (2013) Curr Opin Chem Biol. 17, 412-419) as sites for conjugation to a drug. Similarly, peptide tags for enzymatic conjugation methods can be introduced into an antibody (Strop P., et al., Chem Biol. 2013, 20(2): 161-7; Rabuka D., Curr Opin Chem Biol. 2010 Dec;14(6):790-6; Rabuka D, et al., Nat Protoc. 2012, 7(6): 1052-67). One other example is the use of 4’-phosphopanteiheinyl transferases (PPTase) for the conjugation of Co-enzyme A analogs (WO2013184514), and (Grünewald et al., (2015) Bioconjugate Chem. 26 (12), 2554-62). Methods for conjugating such modified or engineered antibodies with payloads or linker-payload combinations are known in the art.

In another embodiment, the Fc hinge région of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface région of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.

In yet other embodiments, the Fc région is altered by replacing at least one amino acid residue with a different amîno acid residue to alter the effector functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc

183 receptor or the Cl component of complément. This approach is described in, e.g., U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another embodiment, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complément dépendent cytotoxicity (CDC). This approach is described in, e.g., U.S. Patent Nos. 6,194,551 by Idusogie et al.

In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complément. This approach is described in, e.g., the PCT Publication WO 94/29351 by Bodmer et al. Allotypie amino acid residues include, but are not limited to, constant région of a heavy chain of the IgGl, IgG2, and IgG3 subclasses as well as constant région of a light chain of the kappa isotype as described by Jefferis et al., MAbs. 1:332338 (2009).

In some embodiments, the antibodies comprise mutations that médiate reduced or no antibody-dependent cellular cytotoxicity (ADCC) or complément-dépendent cytotoxicity (CDC). In some embodiments, these mutations are known as Fc Silencing, Fc Silent, or Fc Silenced mutations. In some embodiments, amino acid residues L234 and L235 of the IgG 1 constant région are substituted to A234 and A235 (also known as “LALA”). In some embodiments, amino acid residue N297 of the IgGl constant région is substituted to A297 (also known as “N297A”). In some embodiments, amino acid residues D265 and P329 of the IgGl constant région are substituted to A265 and A329 (also known as “DAPA”). Other antibody Fc silencing mutations may also be used. In some embodiments, the Fc silencing mutations are used in combination, for example D265A, N297A and P329A (also known as “DANAPA”).

In another embodiment, one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complément. This approach is described in, e.g., the PCT Publication WO 94/29351 by Bodmer et al. In a spécifie embodiment, one or more amino acids of an antibody or antigen binding fragment thereof of the présent invention are replaced by one or more allotypie amino acid residues. Allotypie amino acid residues also include, but are not limited to, the constant région of the heavy chain of the IgGl, IgG2, and IgG3 subclasses as well as the constant région of the light chain of the kappa isotype as described by Jefferis et al., MAbs. 1:332-338 (2009).

In still another embodiment, the glycosylation of an antibody is modified. For example, an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for “antigen.” Such

184 carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that resuit in élimination of one or more variable région framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylatîon may increase the affinity of the antibody for antigen. Such an approach is described in, e.g., U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

In another embodiment, the antibody is modified to increase its biological half-life. Varions approach es are possible. For ex amp le, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375 to Ward. Altematively, to increase the biological half-life, the antibody can be altered within the CH1 or CL région to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc région of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 by Presta et al.

Linkers

In some embodiments, the linker in an ADC is stable extracellularly in a sufficient mariner to be therapeutically effective. In some embodiments, the linker is stable outside a cell, such that the ADC remains intact when présent in extracellular conditions (e.g., prier to transport or delivery into a cell). The term “intact,” used in the context of an ADC, means that the antibody or antigen-binding fragment remains attached to the drug moiety (e.g., the Mcl-1 inhibitor).

As used herein, “stable,” in the context of a linker or ADC comprising a linker, means that no more than 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than about 3%, or no more than about 1% of the linkers (or any percentage in between) in a sample of ADC are cleaved (or in the case of an overall ADC are otherwise not intact) when the ADC is présent in extracellular conditions. In some embodiments, the linkers and/or ADCs disclosed herein are stable compared to altemate linkers and/or ADCs with altemate linkers and/or Mcl-1 inhibitor payloads. In some embodiments, the ADCs disclosed herein can remain intact for more than about 48 hours, more than 60 hours, more than about 72 hours, more than about 84 hours, or more than about 96 hours.

Whether a linker is stable extracellularly can be determined, for example, by including an ADC in plasma for a predetermined time period (e.g., 2, 4, 6, 8, 16, 24, 48, or 72 hours) and then quantifying the amount of free drug moiety présent in the plasma. Stability may allow the ADC

185 time to localize to target cancer cells and prevent the prématuré release of the drug moiety, which could lower the therapeutîc index of the ADC by indiscriminately damaging both normal and cancer tissues. In some embodiments, the linker is stable outside of a target cell and releases the drug moiety from the ADC once inside of the cell, such that the drug can bind to its target. Thus, an effective linker will: (î) maintain the spécifie binding properties of the antibody or antigen-binding fragment; (ii) allow delivery, e.g., intracellular delivery, of the drug moiety via stable attachment to the antibody or antigen-binding fragment; (iii) remain stable and intact until the ADC has been transported or delivered to its target site; and (iv) allow for the therapeutîc effect, e.g., cytotoxic effect, of the drug moiety after cleavage or altemate release mechanism.

Lînkers may impact the physico-chemical properties of an ADC. As many cytotoxic agents are hydrophobie in nature, linking them to the antibody with an additional hydrophobie moiety may lead to aggregation. ADC aggregates are insoluble and often limit achievable drug loadîng onto the antibody, which can negatively affect the potency of the ADC. Protein aggregates of biologics, in general, hâve also been linked to increased immunogenicity. As shown below, lînkers disclosed herein resuit in ADCs with low aggregation levels and désirable levels of drug loadîng.

A linker may be “cleavable” or “non-cleavable” (Ducry and Stump (2010) Bioconjugate Chem. 21:5-13). Cleavable lînkers are designed to release the drug moiety (e.g., an Mcl-1 inhibitor) when subjected to certain environment factors, e.g., when intemalized into the target cell, whereas non-cleavable lînkers generally rely on the dégradation of the antibody or antigenbinding fragment itself.

The term “alkyi”, as used herein, refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation. The terni “Ci-Côalkyl”, as used herein, refers to a straight or branched hydro carbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to six carbon atoms, and which is attached to the rest of the molécule by a single bond. Nonlimîting examples of “Ci-Côalkyl” groups include methyl (a Cialkyl), ethyl (a Csalkyl), 1methylethyl (a C₃alkyl), π-propyl (a C₃alkyl), isopropyl (a C₃alkyl), n-butyl (a C₄alkyl), isobutyl (a C₄alkyl), sec-butyl (a C₄alkyl), tert-butyl (a C₄alkyl), n-pentyl (a Csalkyi), isopentyl (a Csalkyi), neopentyl (a Csalkyi) and hexyl (a C&alkyl).

The term “alkenyl”, as used herein, refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond. The term “Ci-Cgalkenyl”, as used herein, refers to a straight or branched hydrocarbon

186 chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, having from two to six carbon atoms, which is attached to the rest ofthe molécule by a single bond. Non-limiting examples of “C₂-Côalkenyl” groups include ethenyl (a C₂alkenyl), prop-l-enyl (a C₃aikenyl), but-l-enyl (a C4alkenyl), pent-l-enyl (a Csalkenyl), pent4-cnyl (a Csalkenyl), penta-l,4-dienyl (a Csalkenyl), hexa-1-enyl (a Csalkenyl), hexa-2-enyl (a Côalkenyl), hexa-3-enyl (a Csalkenyl), hexa-1 -,4-dienyI (a Cûalkenyl), hexa-l-,5-dienyl (a Côalkenyl) and hexa-2-,4-dienyl (a Csalkenyl). The term “C2-C₃alkenyl”, as used herein, refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, having from two to three carbon atoms, which is attached to the rest of the molécule by a single bond. Non-limiting examples of “C₂-C₃alkenyr groups include ethenyl (a Csalkenyl) and prop-l-enyl (a Csalkenyl).

The term “alkylene”, as used herein, refers to a bivalent straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms and containing no unsaturation. The term “Ci-Csalkylene”, as used herein, refers to a bivalent straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to six carbon atoms. Non-limiting examples of “Cj-Csalkylene” groups include methylene (a Cialkylene), ethylene (a Cjalkylene), 1-methylethylene (a C₃alkylene), n-propylene (a C₃alkylene), isopropylene (a C₃alkylene), n-butylene (a C₄alkylene), isobutylene (a C4alkylene), sec-butylene (a C4alkylene), tert-butylene (a C₄alkylene), n-pentylene (a Csalkylene), isopentylene (a Csalkylene), neopentylene (a Csalkylene), and hexylene (a C&alkylene).

The term “alkenylene”, as used herein, refers to a bivalent straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms and containing at least one double bond. The ienn “Cz-Csalkenylene”, as used herein, refers to a bivalent straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, and having from two to six carbon atoms. Non-limiting examples of “Ca-Csalkenylene” groups include ethenylene (a C₂alkenylene), prop-1-enylene (a C₃alkenylene), but-l-enylene (a C4alkenylene), pent-1-enylene (a Cs alkenylene), pent-4-enylene (a Csaikenylene), penta-1,4-dienylene (a Csalkenylene), hexa-1-enylene (a Csalkenylene), hexa2-enylene (a Csalkenylene), hexa-3-enylene (a Csalkenylene), hexa-1-,4-dienylene (a Csalkenylene), hexa-I-,5-dienylene (a Csalkenylene) and hexa-2-,4-dienylene (a Csalkenylene). The term “C₂-C6alkenylene”, as used herein, refers to a bivalent straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at

187 least one double bond, and having from two to thee carbon atoms. Νοπ-limiting examples of “C₂-C₃alkenylene” groups include ethenylene (a C₂alkenylene) and prop-l-enylene (a C₃alkenylene).

The term “cycloalkyl,” or “C₃-Cscycloalkyl,” as used herein, refers to a saturated, monocyclic, fused bicyclic, fused tricyclic or bridged polycyclic ring system. Non-limîting examples of fused bicyclic or bridged polycyclic ring Systems include bicyclofl .1 .IJpentane, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane, bicyclo[3.1.1]heptane, bicyclo[3.2.1]octane, bicyclo[2.2.2]octane and adamanianyl. Non-limiting examples monocyclic C₃-C₈ cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl groups.

The term “haloalkyl,” as used herein, refers to a linear or branched alkyl chain substituted with one or more halogen groups in place of hydrogens along the hydrocarbon chain. Examples of halogen groups suitable for substitution in the haloalkyl group include Fluorine, Bromine, Chlorine, and lodine. Haloalkyl groups may include substitution with multiple halogen groups in place of hydrogens in an alkyl chain, wherein said halogen groups can be attached to the same carbon or to another carbon in the alkyl chain.

As used herein, the alkyl, alkenyl, alkynyl, alkoxy, amino, aryl, heteroaryl, cycloalkyl, and heterocycloalkyl groups may be optionally substituted by 1 to 4 groups selected from optionally substituted linear or branched (Ci-Cô)alkyl, optionally substituted linear or branched (C₂-Cô)alkenyl group, optionally substituted linear or branched (C₂-Cé)alkynyl group, optionally substituted linear or branched (Cj-Côjalkoxy, optionally substituted (Ci-Cô)alkyl-S-, hydroxy, oxo (or N-oxide where appropriate), nitro, cyano, -C(0)-ORo’, -0-C(0)-Ro\ -C(0)-NRo’Ro”, NRo’Ro”, -(C=NRo’)-ORq”, linear or branched (Ci-Cô) haloalkyl, trifluoromethoxy, or halogen, wherein Ro’ and Ro” are each independently a hydrogen atom or an optionally substituted linear or branched (Ci-Cô)alkyl group, and wherein one or more of the carbon atoms of linear or branched (Ci-Cô)alkyl group is optionally deuterated.

The term “polyoxyethylene”, “polyethylene glycol” or “PEG”, as used herein, refers to a linear chain, a branched chain or a star shaped configuration comprised of (OCH₂CH₂) groups. In certain embodiments a polyethylene or PEG group is -(OCH₂CH₂)_t*-, where t is 4-40, and where the indicates the end directed toward the self-immolative spacer and the indicates the point of attachment to a terminal end group R’ where R’ is OH, OCH₃ or OCH₂CH₂C(=O)OH. in other embodiments a polyethylene or PEG group is -(CH₂CH₂O)t*-, where t is 4-40, and where the indicates the end directed toward the self-immolative spacer

188 and the indicates the point of attachment to a terminal end group R” where R” is H, CH₃or CH2CH₂C(=O)OH. For example, the tenn “PEGI2” as used herein means that t is 12.

The term “polyalkylene glycol”, as used herein, refers to a linear chain, a branched chain or a star shaped configuration comprised of (O(CH₂)m)n groups. In certain embodiments a polyethylene or PEG group is -(O(CH2)_m)i*-, where m is 1-10, t is 4-40, and where the indicates the end dîrected toward the self-immolative spacer and the indicates the point of attachment to a tenninal end group R’ where R’ is OH, OCH₃ or OCH₂CH₂C(=O)OH. In other embodiments a polyethylene or PEG group is -((CHzjmOlt*-, where m is 1-10, t is 4-40, and where the indicates the end directed toward the self-immolative spacer and the indicates the point of attachment to a tenninal end group R” where R” is H, CH₃ or CH₂CH₂C(=O)OH.

The term “reactive group”, as used herein, is a functional group capable of fonning a covalent bond with a functional group of an antibody, an antibody fragment, or another reactive group attached to an antibody or antibody fragment. Non lîmiting examples of such functional groups include reactive groups of Table 2 provîded herein.

The tenn “attachment group” or “coupling group”, as used herein, refers to a bivalent moiety which links the bridging spacer to the antibody or fragment thereof. The attachment or coupling group is a bivalent moiety formed by the reaction between a reaction group and a functional group on the antibody or fragment thereof. Non limiting examples of such bivalent moieties include the bivalent Chemical moieiies given in Table 2 and Table 3 provîded herein.

The term “bridging spacer”, as used herein, refers to one or more lînker components which are covalently attached together to form a bivalent moiety which links the bivalent peptide spacer to the reactive group, links the bivalent peptide space to the coupling group, or links the attachment group to the at least one cleavable group. In certain embodiments the “bridging spacer” comprises a carboxyl group attached to the N-terminus of the bivalent peptide spacer via an amide bond.

The term “spacer moiety”, as used herein, refers to one or more lînker components which are covalently attached together to form a moiety which links the self-immolative spacer to the hydrophilic moiety.

The term “bivalent peptide spacer”, as used herein, refers to bivalent lînker comprising one or more amino acid residues covalently attached together to form a moiety which links the bridging spacer to the self immolât!ve spacer. The one or more amino acid residues can be an residue of amino acids selected from alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys),

189 leucine (Leu), méthionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), cîtrulline (Cit), norvaline (Nva), norieucune (Nie), selenocysteine (Sec), pyrrolysine (Pyl), homoserine, homocystéine, and desmethyl pyrrolysine.

In certain embodiments a “bivalent peptide spacer” is a combination of 2 to four amino acid residues where each residue is independently selected from a residue of an amino acid selected from alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu),méthionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), citruliine (Cit), norvaline (Nva), norieucune (Nie), selenocysteine (Sec), pyrrolysine (Pyl), homoserine, homocystéine, and desmethyl pyrrolysine, for example -VaJCit*; -CitVal*; -AlaAla*; -AlaCit*; -CitAla*; -AsnCit*; -CitAsn*; -CitCit*; -ValGlu*; -GluVal*; -SerCit*; -CitSer*; -LysCit*; -CitLys*; -AspCit*; CîtAsp*; -AlaVal*; -ValAla*; -PheAla*; -AlaPhe*; -PheLys*; -LysPhe*; -ValLys*; -LysVal*; AlaLys*; -LysAla*; -PheCit*; -CitPhe*; -LeuCit*; -CitLeu*; -IleCit*; -Citlle*; -PheArg*; ArgPhe*; -CifTTp*; -TrpCit*; -PhePheLys*; -LysPhePhe*; -DphePheLys*; -DlysPhePhe*; GlyPheLys*; -LysPheGly*; -GlyPheLeuGly- [SEQ ID NO:69]; -GlyLeuPheGly- [SEQ ID NO:64]; -AlaLeuAlaLeu- [SEQ ID NO:65], -GlyGlyGly*; -GlyGlyGlyGly- [SEQ ID NO:66]; GlyPheValGly- [SEQ ID NO:67]; and -GlyValPheGly- [SEQ ID NO:68], wher the indicates the point of attachment to the bridging spacer and the indicates the point of attachment to the self-immolative spacer.

The tenu “linker component”, as used herein, refers to a Chemical moiety that is a part of the linker. Examples of linker components include: an alkylene group: -(CH2)n- which can either be linear or branched (where in this instance n is l -18); an alkenylene group; an alkynylene group; an alkenyl group; an alkynyl group; an ethylene glycol unit: -OCH2CH2- or -CH2CH2O-; an polyethylene glycol unît: (-CH₂CH₂O-)_A (where x in this instance is 2-20); -O-; -S-; a carbonyl: -C(=O); an ester: C(=O)-O or O-C(=O); a carbonate: -OC(=O)O-; an amine: -NH-; an tertîary amine; an amide: -C(=O)-NH-, -NH-C(=O)- or -C(=O)N(Ci-6alkyl); a carbamate: OC(=O)NH- or -NHC(=O)O; a urea: -NHC(=O)NH; a sulfonamide: -S(O)₂NH- or -NHS(O)₂;an ether: -CH₂O- or -OCH?-; an alkylene substituted with one or more groups independently selected from carboxy, sulfonate, hydroxyl, amine, amino acid, saccharide, phosphate and phosphonate); an alkenylene substituted with one or more groups independently selected from carboxy, sulfonate, hydroxyl, amine, amino acid, saccharide, phosphate and phosphonate); an

190 alkynylene substituted with one or more groups independently selected from carboxy, sulfonate, hydroxyl, amine, amino acid, saccharide, phosphate and phosphonate); a Ci-Cioalkylene in which one or more methylene groups is replace by one or more -S-, -NH- or -O- moieties; a ring Systems having two available points of attachment such as a divalent ring selected from phenyl (including 1,2- 1,3-and 1,4- di-substituted phenyls), a C5-C6 heteroaryl, a C₃-Cg cycloalkyl (including 1,1-disubstituted cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, and 1,4disubstituted cyclohexyl), and a C4-C8 heterocycloalkyl; a residue of an amino acid selected from alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu),méthionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), arginine (Arg), serine (Ser), threonine (Thr), valîne (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), norvaline (Nva), norleucune (Nie), selenocysteine (Sec), pyrrolysine (Pyl), homoserine, homocystéine, and desmethyl pytTolysine; a combination of 2 or more amino acid residues where each residue is independently selected from a residue of an amino acid selected from alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), lysine (Lys), leucine (Leu),methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), arginine (Arg), serine (Ser), threonine (Thr), valîne (Val), tryptophan (Trp), tyrosine (Tyr), citrulline (Cit), norvaline (Nva), norleucune (Nie), selenocysteine (Sec), pyrrolysine (Pyl), homoserine, homocystéine, and desmethyl pyrrolysine, for ex ample Val-Cit; Cit-Val; Ala-Al a; Ala-Cît; CitAla; Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp; Ala-Val; Val-Ala; Phe-Lys; Lys-Phe; Val-Lys; Lys-Val; Ala-Lys; Lys-Ala; Phe-Cit; Cit-Phe; Leu-Cit; Cit-Leu; Ile-Cit; Cit-Ile; Phe-Arg; Arg-Phe; Cit-Trp; and Trp-Cit; and a selfimmolative spacer, wherein the self-immolative spacer comprises one or more protecting (triggering) groups which are susceptible to acid-induced cleavage, peptidase-înduced cleavage, esterase-induced cleavage, glycosidase induced cleavage, phosphodiesterase induced cleavage, phosphatase induced cleavage, protease induced cleavage, lipase induced cleavage or disulfide bond cleavage.

Non-limiting examples of such self-immolative spacers include:

191

PG is a protecting (triggering) group;

X_a is O, NH or S;

X_b is O, NH, NCH₃ or S;

X_c is O or NH;

Y_a is CH₂, CH₂0 or CH₂NH;

Yb is CH₂, O or NH;

Y_c is a bond, CH₂, O or NH, and

LG is a leaving group such as a Drug moiety (D) of the Linker-Drug group of the invention.

Additional non-limiting exemples of such self-immolative spacers are described in Angew. Chem. Int. Ed. 2015, 54, 7492 - 7509.

In addition, a linker component can be a Chemical moiety which is readily formed by reaction between two reactive groups. Non-limiting examples of such Chemical moieties are given in Table 2.

Table 2

Reactive Group 1 (RG1)	Réactivé Group 2 (RG2)	Chemical Moiety
a thiol	a thiol	-S-S-
a thiol	a maleimide	^f 0

192

Reactive Group 1 (RG1)	Reactive Group 2 (RG2)	Chemical Moiety
a thiol	a haloacetamide	0 Ky H H - -S
an azide	an alkyne	YnGn In or t N
an azide	a tri aryl phosphine	Ph G LG
an azide	a cyclooctyne	/¾ G’²ÎWW \· -- / A 1 o / /-\/^Zo/ O
an azide	an oxanobomadiene	O ^N/ GA NA HN . -Hr x
a tri aryl phosphine	an azide	Ph P pCph /À /° -γΗ ΗΝγ
an oxanobomad i ene	an azide	o_v ,Ν, Μ '^Ν „ΝΗ / T. ^ΛΛΛΛΛΖ*
an alkyne	an azide	1 ο
a cyclooctyne	azide	1 ΛΧΤΙΑΛΓ· Μ ο ρ\ S χ

193

Reactive Group 1 (RG1)	Reactive Group 2 (RG2)	Chemical Moiety
		^4 \ / \ ^zfv /Jv/o I O
a cyclooctene	a diaryl tetrazine	F' ν-ω I W·² o I WWW ό ή v-i*¹ ,1 Tz ²
a diaryl tetrazine	a cyclooctene	70 / À w / \ O I I Q 1—J λ'ύc? / \ o 1 1
a monoaryl tetrazine	a norbornene	>gV!2\o n^F 7Ή+ nA ^h R 37
a norbornene	a monoaryl tetrazine	° ZjVJX Xn ^R37
an aldéhyde	a hydroxylamine	H^N /
an aldéhyde	a hydrazine	hn-L x¹ / ⁵ H
an aldéhyde	NH2-NH-C(=O)-	H -^νλΛ· N Λ ⁰
a ketone	a hydroxylamine	r_3S
a ketone	a hydrazine	V ^HNX ^R3₅

194

Reactive Group 1 (RG1)	Reactive Group 2 (RG2)	Chemical Moiety
a ketone	NH₂-NH-C(=O)-	H , N °
a hydroxylamine	an aldéhyde	i Y N ^M
a hydroxylamine	a ketone	-Uo / H
a hydrazine	an aldéhyde	Ynh * \ V ^NA H
a hydrazine	a ketone	\ Z T LO en ¹
NH₂-NH-C(=O)-	an aldéhyde	V Y^NHNO
NH₂-NH-C(=O)-	a ketone	^RV 0
a haloacetamide	a thiol	1 JXTLTUWI. ογΑ ZI
a maleimide	a thiol	1 tWVW ω 1 ΛΛΛΑΛ 1
a vinyl sulfone	a thiol	/ CO O=cp-O ΛΛΛΛΙ 1
a thiol	a vinyl sulfone	0 Il !. /— S-r

195

Reactive Group 1 (RG1)	Reactive Group 2 (RG2)	Chemical Moiety
an aziridine	a thiol	H H x -l^N-\ x '—S or ^X—s
a thiol	an aziridine	H H X /—N—x x /— nI- s—^x or S—/
0 Λ X^{s S}Z	hydroxyl amine	I AWWA A Λ X^{s 5}Ά
0 Λ	hydroxylamine	1 Νθ AV? AW1
pl3---( 1 ^R V-NH Ογ	O i Y π < S	Ô O
.-x R¹⁴	r¹³AA YNH ο^γ	h χ¹ / SH Ci O
0_x XQ O S0₃’ Na+ / ° τΆ AY ο F - ^F- Z\.-F ⁰ V T γ_ογζ F F °^fA F	-nh₂,	amide

196

Reactive Group 1 (RG1)	Reactive Group 2 (RG2)	Chemical Moiety
ο r Y , i L U Cl
-nh₂,	A 0 ₀ S0₃- Na+ o 0 F Α\/ J XX Αγο γ F F A γ ⁰ A ^F F ^Ck^^SO,· γ XX -Wt Cl	amide
CoA or CoA analogue	Serine residue	. H H ?^H i? Nγχ/ΝΑγ. P Y h J> A oh 0 0 Ak H H ?^H 9 A P ° ôh° ^vO 0 4-0.. * N H H OH O ï J A OH A il H il V V ^{H H} à_H HO'Y -A N 0 . , ΐ jA—- AL^c, A - N Y -' V ^{ς H} OH H0^J Y

197

Reactive Group 1 (RG1)	Reactive Group 2 (RG2)	Chemical Moiety
		H ?^H ? J A oh U _u OH O ri ni 11 _ ^^rAAA H H ?^H 9 - A A A oh 0 0
pyridyldithiol	thiol	disulfide

where: R?² in Table 2 is H, C1-4 alkyi, phenyl, pyi'imidine or pyridine; R³⁵ in Table 2 is H, Ci^alkyl, phenyl or Csalkyi substituted with l to 3 -OH groups; each R⁷ în Table 2 is independently selected from H, Ci-ôalkyl, fluoro, benzyioxy substituted with -C(=O)OH, 5 benzyl substituted with -C(=O)OH, Ci^aikoxy substituted with =C(=O)OH and Ci-4alkyl substituted with -C(=O)OH; R³⁷ in Table 2 is independently selected from H, phenyl and pyridine; q in Table 2 is 0, l, 2 or 3; R⁸ and R¹³ in Table 2 are each H or methyl; and R⁹and R¹⁴ in Table 2 are each H, -CH3 or phenyl; R in Table 2 is H or any suitable substituent; and R³⁰ in Table 2 is H.

In addition, a linker component can be a group lîsted in Table 3 below.

Table 3.

0 χΑχ·	A 0	x4⁺0	0 r	0 0
i—L	0^ y--ξ	--y /O	0^ y--\ NH '---'	y-y / HN—’y
0 Aⁿ⁺0	O Fa 0	0 0	0 p26 II 5 H	0 0
0 xA 0	N AA \ j	A JA An	m-^N N - \ s 1 n4 AAn^z	νΛ F '^N

198

A / γ 0 N. Az Y ^Z	^h Y ο γ\ \5h'ⁿ H	^18 HN^Z H '—_N .NJ Ά	R¹⁸ H NH \ ^N vrA—' ^Λ ï V	_R!2 HN^Z H vs Y A Λ o
ZI ωϊ χ l /m ' Z T	R¹⁸ H o	R< H 0	^R¹² Y H 0	Y ZI ω 1 1
0 7 \ Y s./ 0 /-	0 ^H°VAj ws Y / Λ o	-A z? Lj y \ }Ay7 °^H 0	V/^s+ 0	, n-n (Rz)_q ^Z<^J<
(Rzk /-N , ΧΑ-ύχ-		0	0 (CH 1-^/ (GH2)\| ?	0 A TyLA(CH₂)₀._s 0
R^aa I H Ά 0	Υ^Ν'Ύ o^A	, n-n.	tpi (R₇)q	TA N^ V oX
\xx Λ N R²⁵^	ίο^Νγ'ί R²⁵	-AAoV R²⁵	JJy^y
i A^z -j-S HN^?	-^-NH S—^- o⁷	-^-NH d	-i-NH c--_eA 5 \ / A o^z	V .^R12/y o !% Az H ^Z
J H V^N>	1 N- X^{s s}yt	1 •zvyAzvvvx A	Ph O %Ph , ÂA° h Cu	Έ X/ X ° AT, t—Ty y cAi Z Ά
/ R³² AyX_N LJV	Ar33 nA'x nAT R³² °A	R³² \ 4 ^N^-y^NH /	3 HN'^Nx._R32 A^r33'YJ7	A» A-y R³²v yY ^''y n Z

199

each R⁷ is independently selected from H, Ci-6alkyl, fluoro, benzyloxy substituted with C(=O)OH, benzyl substituted with -C(=O)OH, Ci^alkoxy substituted with -C(=O)OH and Ci-4alkyl substituted with -C(=O)OH;

each R¹² is independently selected from H and Ci-Côalkyl

R⁸ is H or methyl;

R⁹ îs H, -CH3 or phenyl;

each R²⁵ is independently selected from H or C1.4 alkyl;

each R¹⁸ is independently selected from a Ci-Côalkyl, a Ci-Ci,alkyl which is substituted with azido and a Ci-Côalkyl which is substituted with 1 to 5 hydroxyl;

q is 0, 1,2 or 3;

is 1,2, 3, 4, 5 or 6;

200

As used herein, when a partial structure of a compound is illustrated, a wavy line ( '^/vw ) indicates the point of attachment of the partial structure to the rest of the molécule.

The terms “self-immolative spacer” and “self-immolative group”, as used herein, refer a 5 moiety comprising one or more triggering groups (TG) which are activated by acid-induced cleavage, peptidase-induced cleavage, esterase-înduced cleavage, glycosidase induced cleavage, phosphodiesterase induced cleavage, phosphatase induced cleavage, protease induced cleavage, lipase induced cleavage or disulfide bond cleavage, and after activation the protecting group is removed, which generates a cascade of disassembling reactions leading to the temporally

201 sequential release of a leaving group. Such cascade of reactions can be, but not limited to, 1,4-,

1,6- or 1,8- élimination reactions.

Non-limiting examples of self-immolative spacer or group include:

wherein:

wherein such groups can be optionally substituted, and

TG is a triggering group;

Xa is O, NH or S;

Xb is O, NH, NCH₃ or S;

X_c is O or NH;

Ya is CH₂, CH₂O or CH₂NH;

Yb is CH₂, O or NH;

Y_c is a bond, CH₂, O or NH, and

Additional non-limiting examples of self-immolative spacers are described in Angew. Chem. Int. Ed. 2015, 54, 7492 - 7509.

In certain embodiment the self-immolative spacer is moiety having the structure

202

D

, where Lp is an enzymatically cleavable bivalent peptide spacer and

A, D, Lj and R² are as defined herein.

In preferred embodiments, the self-immolative spacer is moiety having the structure ^P'N

H

D

, where Lp is an enzymatically cleavable bivalent peptide spacer and

D, L₃ and R² are as defined herein. In some embodiments, D is a quaternized tertiary amine containingMCI! inhibitor.

In other preferred embodiments, the self-immolative spacer is moiety having the structure

O

, where Lp is an enzymatically cleavable bivalent peptide spacer and

D, L₃ and R² are as defined herein.

The tenu “hydrophilic moiety”, as used herein, refers to moiety that is has hydrophilic properties which increases the aqueous solubility of the Drug moiety (D) when the Drug moiety (D) is attached to the linker group of the invention. Examples of such hydrophilic groups include, but are not limited to, polyethylene glycols, polyalkylene glycols, sugars,

O

-|-O-P-OH oligosaccharides, polypeptides a C2-C&alkyl substituted with 1 to 3 OH groups.

Drug Moieties

In some embodiments, an intermediate, which is the precursor of the linker moiety, is reacted with the drug moiety (e.g., the Mcl-1 inhibitor) under approprîate conditions. In some embodiments, reactive groups are used on the drug and/or the intermediate or linker. The product of the reaction between the drug and the intermediate, or the derivatized drug (drug plus linker), is subsequently reacted with the antibody or antigen-binding fragment under conditions that facilitate conjugation ofthe drug and intermediate or derivatized drug and antibody or antigen-binding fragment. Alternatively, the intermediate or linker may first be reacted with the

203 antibody or antigen-binding fragment, or a derivatized antibody or antigen-binding fragment, and then reacted with the drug or derivatized drug.

A number of different reactions are available for covalent attachment of the drug moiety and/or linker moiety to the antibody or antigen-binding fragment. This is often accomplished by reaction of one or more amino acid residues of the antibody or antigen-binding fragment, including the amine groups of lysine, the free carboxylic acid groups of glutamic acid and aspartic acid, the sulfhydryl groups of cysteine, and the various moieties of the aromatic amino acids. For instance, non-specific covalent attachment may be undertaken using a carbodiimide reaction to link a carboxy (or amino) group on a drug moiety to an amino (or carboxy) group on an antibody or antigen-binding fragment. Additionally, bifunctional agents such as dialdéhydes or imidoesters may also be used to link the amino group on a drug moiety to an amino group on an antibody or antigen-binding fragment. Also available for attachment of drugs (e.g., an Mcl-l inhibitor) to binding agents is the Schiff base reaction. This method in volves the periodate oxidation of a drug that contains glycol or hydroxy groups, thus forming an aldéhyde which is then reacted with the binding agent. Attachment occurs via formation of a Schiff base with amino groups of the binding agent. Isothiocyanates may also be used as coupling agents for covalently attaching drugs to binding agents. Other techniques are known to the skilled artisan and within the scope of the présent disclosure. Ex amples of drug moieties that can be générât ed and linked to an antibody or antigen-binding fragment using various chemistries known to in the art include Mcl-l inhibitors, e.g., the Mcl-l inhihitors described and exemplified herein.

Suitable drug moieties may comprise a compound of the formulas (I), (II), (III), or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or addition sait thereof with a phannaceutically acceptable acid or base. Additionally, the drug moiety may comprise any compounds of the Mcl-l inhibitor (D) described herein.

As used herein, “atropisomers,” are stereoisomers arising because of hindered rotation about a single bond, where energy différences due to steric strain or other contributors create a barrier to rotation that is high enough to allow for isolation of individual confonners (Bringmann et al. Angew. Chem. Int, Ed. 2005, 44, 5384-5427). For example, for compounds of formula (II) according to the invention, atropisomers may be as follows:

204

For example, a preferred atropisomer may be (5S_a), also named (5a5).

A drug moiety of the disclosure may be any one of the compounds disclosed in International Patent Application Publication Nos. WO 2015/097123; WO 2016/207216; WO

2016/207217; WO 2016/207225; WO 2016/207226; WO 2017/125224; WO 2019/035899; WO

2019/035911; WO 2019/035914; WO 2019/035927; WO 2016/033486; WO 2017/147410; WO 2018/183418; and WO 2017/182625, and U.S. Patent Application Publication No. 2019/0055264, each of which is incorporated herein by reference in its entirety.

In some embodiments, a drug moiety of the disclosure may comprise a compound of

Fonnula (I);

wherein:

Ring Eo is a furyl, thienyl or pyrrolyl ring.

205

Xoi, Xoî, X04 and X05 independently of one another are a carbon atom or a nitrogen atom,

X02 is a C-R026 group or a nitrogen atom, ( means that the ring is aromatic,

Yo is a nitrogen atom or a C-R03 group, Zo is a nitrogen atom or a C-Rû4 group, Roi is a halogen atom, a linear or branched (Ci-Côjalkyl group, a linear or branched (CF-CôJalkenyl group, a linear or branched (C₂-C6)alkynyl group, a linear or branched (Ci-Cô)haloalkyl group, a hydroxy group, a hydroxy(Ci-C&)alkyl group, a linear or branched (Ci-Cô)alkoxy group, -S-(Ci-Cô)alkyl group, a cyano group, a nitro group, -Cyos, -(Co-Célalkyl-NRonRoii’, -O-(Ci-C6)alkyl-NR₀ijR₀ii’, -O-(Ci-C₆)alkyl-R₀i2, -C(0)-ORoi i, -0-C(0)-Ron, -C(O)-NR₀iiR_0Ii’, -NRoi i-C(O)-R₀ii’, -NRoii-C(O)-OR₀n’, -(Ci-Cô)alkyl-NRo11-C(O)-R₀11 ’, -SO2-NR011Ro11 or -SO₂-(Ci-C₆)alkyl,

R02, Roî, R04 and Ro? independently of one another are a hydrogen atom, a halogen atom, a linear or branched (Ci-Cô)alkyl group, a linear or branched (C₂-C6)alkenyl group, a linear or branched (C₂-Cô)alkynyl group, a linear or branched (Ci-Côjhaloalkyl, a hydroxy group, a hydroxy(Ci-Cô)alkyl group, a linear or branched (Ci-Côjalkoxy group, a -S-(Ci-Cô)alkyl group, a cyano group, a nitro group, -(Co-Cô)alkyl-NRoiiRoii’, -O-Cyoi, -(Co-Cô)alkyl-Cyoi, -(C₂-Cô)alkenyl-Cyoi, -(C₂-C₆)alkynyl-Cy₀i, -O-(Ci-C₆)alkyl-NRoi i Roi i ’, -O-(C।-CQalkyl-Ros], -O-(Ci-C6)alkyl-R₀|₂, -C(O)-ORtni, -O-C(O)-R₀ii, -C(O)-NR₀i।Roii -NR_Oii-C(0)-Roii’, -NRoii-C(O)-OR₀ii’, -(CrCôÎalkyl-NRoi _rC(O)-R₀ii’, -S0₂-NRoi iRoii or -SO2-(Ci-C₆)alkyl, or the pair (Roi, R02), (R02, R03), (R03, R04), or (Ro₄, R05) together with the carbon atoms to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains l to 3 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted by l or 2 groups selected from halogen, linear or branched (Cj-CôJalkyl, (Co-Cô)alkyl-NRoiiRoii’, -NR013R013’, -(Co-Cé)alkyl-Cyûi or oxo,

Roè and R07 independently of one another are a hydrogen atom, a halogen atom, a linear or branched (Ci-Cô)alkyl group, a linear or branched (C₂-C6)alkenyl group, a linear or branched (C₂-Cô)alkynyl group, a linear or branched (Ci-Céjhaloalkyl, a hydroxy group, a linear or branched (Ci-Côjalkoxy group, a -S-(Ci-Cô)alkyl group, a cyano group, 206 a nitro group, -(Co-Céjalkyl-NRoiiRoii’, -0-(Ci-C6)aikyl-NRoiiRoi i’, -O-Cyoi, -(Co-C6)alky1-Cyoi, -(C₂-Cô)alkenyl-Cyob -(C₂-Cô)alkynyl-Cyoi, -O-(Ci-C6)alkyl-Rcu₂, -C(0)-ORoi i, -O-C(O)-R₀ii, -C(0)-NRoiiRoiΓ, -NRon-C(0)-Roii’, -NRoii-C(O)-OR₀ii’, -(Ci-C₆)alkyl-NRoii-C(0)-Ron’, -S0₂-NRoiiRqii’, or-SO₂-(Ci-C₆)alkyl, or the pair (Roô, R07), when fused with the two adjacent carbon atoms, together with the carbon atoms to which they are attached fonn an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains I to 3 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted by a linear or branched (Ci-C6)alkyl group, -NR013R013’, -(Co-Côjalkyl-Cyoi or an oxo, Wq is a -CH₂- group, a -NH- group or an oxygen atom, Ros is a hydrogen atom, a linear or branched (C i -Csjalkyl group, a -CHRoaRob group, an aryl group, a heteroaryl group, an aryl(Ci-C&)alkyl group, or a heteroaryI(CiCe)alkyl group,

Ro9 is a hydrogen atom, a linear or branched (Ci-Cejalkyl group, a linear or branched (C₂-Cû)alkenyl group, a linear or branched (C₂-Cô)alkynyl group, -Cyo₂, -(Ci-Côjalkyl-Cyoz, -(C₂-Ci,)alkenyl-Cyo₂, -(C₂-Cô)alkynyl-Cyo₂, -Cy_O2-Cyo3, -(C₂-Cb)alkynyl-0-Cyo2, -Cyo2-(Co-C6)alkyl-0-(Cû-C6)alkyl-Cyo3, a halogen atom, a cyano group, -C(O)-Rqi4, or-C(0)-NRoi4Rü!4^J,

R010 is a hydrogen atom, a linear or branched (Ci-Cô)alkyl group, a linear or branched (C₂-Cb)alkenyl group, a linear or branched (C₂-Cb)alkynyl group, an aryl(Ci-Cô)alkyl group, a (Ci-Côjcycloalkylalkyl group, a linear or branched (Cj-Côjhaloalkyl, or -(Ci-C&)alkyl-0-Cyo4, or the pair (R09, Roio), when fused with the two adjacent carbon atoms, together with the carbon atoms to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains 1 to 3 heteroatoms selected from O, S and N,

Roi 1 and Rou’ independently of one another are a hydrogen atom, an optionally substituted linear or branched (Ci-Cô)alkyl group, or -(Co-Cô)alkyl-Cyoi, or the pair (Roi 1, Roi 1 ’) together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains, in addition to the nitrogen atom, 1 to 3 heteroatoms selected from O, S, and N, wherein the N atom may be substituted by 1 or 2 groups selected from a linear or

207 branched (Ci-Cô)alky] group, and wherein one or more of the carbon atoms of the linear or branched (Ci-Cô)alkyl group is optionally deuterated,

R012 is -Cyos, -Cyos-(Co-C6)aIkyl-0-(Co-C6)alkyl-Cyo6, -Cyo5-(Co-C6)alkyl-Cyo6, -Cyo5-(Co-C6)alkyl-NRoi i-(Co-Cû)alkyl-Cyo6, -Cyo5-Cyo6-0-(Co-C6)alkyl-Cyo7, -Cyo₅-(Co-C6)alkyl-0-(Co-C6)alkyl-Cyo9, -Cy_O5-(Co-C6)alkyI-Cyo9, -NH-C(O)-NH-R_0l i, -Cyo5-(Co-C6)alkyl-NRoi i-(Co-Cô)alkyl-Cyo9, -C(0)-NRonRoi Γ, -NRoi iRoi i -ORoi i, -NRoii-C(0)-Roii\-0-(Ci-C6)alkyl-ORüii,-SO^^^^

Roi3, Roi?’, Roi4 and R014’ independently of one another are a hydrogen atom, or an optionally substituted linear or branched (Ci-C&)alkyl group,

Roa is a hydrogen atom or a linear or branched (Ci-Cô)alkyl group,

Rob is a -0-C(0)-0-Roc group, a -0-C(0)-NRo_cRoc’ group, or a -0-P(0)(ORo_c)2 group,

Roc and Ro_c’ independently of one another are a hydrogen atom, a linear or branched (Ci-Cs)alkyl group, a cycloalkyl group, a (Ci-C6)aIkoxy(Ci-C6)alkyl group, or a (Ci-C6)alkoxycarbonyl(C i-Cé)alkyl group, or the pair (Ro_c, Ro_c’) together with the nitrogen atom to which they are attached form a non-aromatic ring composed of from 5 to 7 ring members, which may contain in addition to the nitrogen atom from 1 to 3 heteroatoms selected from oxygen and nitrogen, wherein the nitrogen is optionally substituted by a linear or branched (Ci-C6)alkyl group,

Cyoi, Cyo2, Cyos, Cyo4, Cyos, Cyoô, Cyo₇, Cyos and Cyoïo independently of one another, are an optionally substituted cycloalkyl group, an optionally substituted heterocycloalkyl group, an optionally substituted aryl group or an optionally substituted heteroaryl group,

or Cyo9 is a heteroaryl group which is substituted by a group selected from -0-P(OXORo2o)₂; -O-P(O)(O-MQ₂; -(CH₂)_Po-0-(CHRoi8-CHRoi9-0)_qo-R₀₂₀; hydroxy;

hydroxy(Ci-C6)alkyl; -(CH₂)ifl-Uo-(CH2)so-heterocycloalkyl; and -Uo-(CH₂)_qo-NRo₂iRo2i’,

Roi5 is a hydrogen atom; a-(CH2)_Po-0'(CHRoi8-CHRoi9-0)_qo-Ro2o group; a linear or branched (Ci-C6)alkoxy(Cj-C6)alkyl group; a-Uo-(CH₂)_qo-NRo₂iRo2i’ group; or a

208

-(CH2)iO-Uo-(CH?)so-heierocycloalkyl group.

Rois is a hydrogen atom; a hydroxy group; a hydroxy(Ci-Cô)alkyl group; a -(CH2)io-Uo-(CH2)so-heterocycloalkyl group; a (CH₂)tO-Uo-Vo-0-P(0)(ORo2o)2 group; a -O-P(O)(OM⁺)₂ group; a -O-S(O)2ORq20 group; a -S(0)₂ORû₂o group; a -(CH₂)po-0-(CHRois-CHRoi9-0)_qo-R(EO group; a-(CH2)po-0-C(0)-NRo₂₂Ro₂3 group; or a -Uo-(CH₂)qo-NR_O2iRo2i’ group,

Roi? is a hydrogen atom; a -(CH₂)_po-0-(CHRoi8-CHRon-0)qo-Ro₂o group; a -CH2-P(0)(ORo2o)₂ group, a -0-P(0)(ORo?q)₂ group; a -O-P(ΟχΟ’ΜΎ group; a hydroxy group; a hydroxy(Ci-C6)alkyl group; a -(CH₂)to-Uo-(CH₂)so-heteiOcycloalkyl group;a -Uo-(CH2)_qo-NRo2iRo2i’ group; or an aldonic acid,

M⁺ is a phannaceutically acceptable monovalent cation,

Uo is a bond or an oxygen atom,

Vq is a -(CH₂)sû- group or a -C(O)- group,

Rois is a hydrogen atom or a (Ci-C6)alkoxy(Ci-Cô)alkyl group,

Row is a hydrogen atom or a hydroxy(Ci-Cû)alkyl group,

Ro2O is a hydrogen atom or a linear or branched (Ci-Cfi)alkyl group,

R021 and Rosi ’ independently of one are a hydrogen atom, a linear or branched (Ci-Cô)alkyl group, or a hydroxyCCi-CQalkyl group, or the pair (Ro?i, R021 ’) together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains, in addition to the nitrogen atom, 1 to 3 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted by a hydrogen atom or a linear or branched (Cj-Cô)alkyl group,

Ro?? is a (Ci-C6)alkoxy(Ci-Cô)alkyI group, a -(CH₂)po-NRo24Ro24’ group, or a -(CH₂)pO-0-(CHRo 1 ₈-CHRo 19-0)_qo-Ro20 group,

R023 is a hydrogen atom or a (Ci-Cô)alkoxy(Ci-C6)alkyl group, or the pair (R022, R023) together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5 ίο 18 ring members, which optionally contains, in addition to the nitrogen atom, 1 to 5 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted by a hydrogen atom, a linear or branched (Cj-Cô)alkyl group or a heterocycloalkyl group,

R024 and R024’ independently of one another are a hydrogen atom or a linear or

209 branched (Ci-Cô)alkyl group, or the pair (R024, R024’) together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring composed of from 5 to 7 ring members, which may contain in addition to the nitrogen atom from l to 3 heteroatoms selected from O, S and N, and wherein the resulting ring is optionally substituted by a hydrogen atom or a linear or branched (Ci-CQalkyl group,

R025 is a hydrogen atom, a hydroxy group, or a hydroxy(Ci-Cô)aIkyl group,

Ro26 is a hydrogen atom, a halogen atom, a linear or branched (Cj-C6)alkyl group, or a cyano group,

R027 is a hydrogen atom or a linear or branched (Ci-Cô)alkyl group,

Rü28 is a -O-P(O)(O’)(O’) group, a -0-P(0)(0 )(ORo3o) group, a -0-P(0)(ORo3o)(ORoîo’) group, a-(CH2)_P0-O-SO2- group, a -(CH2)_po-S02-0· group, a -(CH2)po-0-S02-ORo3o group, -Cyoïo, a-(CH₂)_po-S02-ORo30 group, a -0-C(0)-Ro₂₉group, a -0-C(0)-ORo29 group or a -0-C(0)-NRo₂₉Ro₂₉’ group;

Ro₂₉ and Ro29’ independently of one another are a hydrogen atom, a linear or branched (Ci-C&)alkyl group or a linear or branched ammo(Ci-Cô)alkyl group,

Rqîo and Roîo’ independently of one another are a hydrogen atom, a linear or branched (Ci-Cô)alkyl group or an aryl(Ci-Cô)alkyl group,

as a zwitterionic form or has a monovalent anionic counterion, no is an integer equal to 0 or l, po is an integer equal to 0, l, 2, or 3, qo is an integer equal to l, 2, 3 or 4, ro and so are independently an integer equal to 0 or l ;

210 wherein, at most, one of the Roj, Ro% or Roi2 groups, if present, is covalently attached to the linker, and wherein the valency of an atom is not exceeded by virtue of one or more substituents bonded thereto, or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait of any of the foregoing.

In some embodiments, a drug moiety of the disclosure may comprise a compound of

Formula (II):

wherein:

Zq is a nitrogen atom or a C-R04 group,

Roi is a halogen atom, a linear or branched (Ci-Cô)alkyl group, a linear or branched (C2-C&)alkenyl group, a linear or branched (C2-C6)alkynyl group, a linear or branched (Ci-Co)haloalkyl group, a hydroxy group, a linear or branched (Ci-CQalkoxy group, a-S-(Ci-C6)alkyl group, a cyano group, -Cyos, -NRohRoh’,

R02, Roî and Rq4 independently of one another are a hydrogen atom, a halogen atom, a linear or branched (Cj-C6)alkyl group, a linear or branched (C₂-Cô)alkenyl group, a linear or branched (C2-C&)alkynyl group, a linear or branched (Ci-C6)haloalkyl, a hydroxy group, a linear or branched (Ci-Cô)aikoxy group, a -S-(Ci-Cû)alkyl group, a cyano group, a nitro group, -(Co-Cô)alkyl-NRonRo! Γ, -O-Cyoi, -(Co-Côjalkyl-Cyoi, -(C2-C<0alkenyl-Cyoi, -(C2-C6)alkynyl-Cyoi, -0-(Ci-C₆)alkyl-NRoi 1R011 ’, -0-(Ci-C₆)alkyI-Ro3i, -C(O)-OR_0li, -O-C(O)-R_0U, -C(G)-NRoiiRoii -NRon-C(0)-Roii’, -NRoii-C(0)-ORon’, -(CrC₆)alkyl-NR₀ii-C(O)^ -SO2-NR011R011’, or

211

-SO₂-(Ci-C₆)alkyl, or the pair (R02, R03) or (R03, R04) together with the carbon atoms to which they are attached fonn an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains 1 to 3 heteroatoms selected from O, S and N, wherein the ring is optionally substituted by a group selected from a linear or branched (Ci-Cô)alkyl, -NR013R013’, -(Co-Cô)alkyl-Cyoi and oxo,

R06 and R07 independently of one another are a hydrogen atom, a halogen atom, a linear or branched (Ci-Côjalkyl group, a linear or branched (C₂-C&)alkenyl group, a linear or branched (C₂-Cô)alkynyl group, a linear or branched (Ci-Cô)haloalkyl, a hydroxy group, a linear or branched (Cj-Cô)alkoxy group, a -S-(Ci-C6)alkyl group, a cyano group, a nitro group, -(Co-Cô)alkyl-NRoiiRoii’, -O-Cym, -(Co-Côjalkyl-Cyoi, -(C₂-C6)alkenylCyoi, -(C₂-C&)alkynyl-Cyoi, -0-(Ci-C6)alkyl-Roi₂, -C(0)-ORon, -0-C(0)-Roii, -C(O)NRoi 1R011 -NRon-C(0)-Ron’, -NRon-C(0)-ORon’, -(C_l-C₆)alkyl-NRoii-C(0)-Roii’, -SO2NR011R011 *, or —SO₂-(Ci-Cô)alkyl, or the pair (Roô, R07), when fused with two adjacent carbon atoms, together with the carbon atoms to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains 1 to 3 heteroatoms selected from O, S and N, and wherein the resulting ring is optionally substituted by a group selected from a linear or branched (Ci-Cô)alkyl group, -NR013R013’, -(Co-Céjalkyl-Cyoi and an oxo,

Ros is a hydrogen atom, a linear or branched (Ci-Cs)alkyl group, an aryl group, a heteroaryl group, an aryl-(Ci-C6)alkylgroup, or a heteroaryhCi-Côjalkyl group,

Ro9 is a linear or branched (Ci-Cô)alkyl group, a linear or branched (C₂-C6)alkenyl group, a linear or branched (C₂-Cô)alkynyl group, -Cyo₂, -(Ci-C6)a1kyl-Cyo2, -(C₂-Cô)alkenyl-Cyo₂, -(C₂-C6)alkynyl-Cyo₂, -Cyo₂-Cyo3, -(C₂-C6)alkynyl-0-Cyo₂, -Cyo₂-(Co-Cô)alkyl-0-(Co-C6)alkyl-Cyo3, a halogen atom, a cyano group, -C(0)-Roi₄, -C(0)-NRoi4Roi4’,

Roi 1 and Roh’ independently of one another are a hydrogen atom, an optionally substituted linear or branched (Ci-Cô)alkyl group, or -(Co-Céjalkyl-Cyoi, or the pair (Roi 1, R011’) together with the nitrogen atom to which they are attached fonn an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally

212 contai ns, in addition to the nitrogen atom, l to 3 heteroatoms selected from O, S and N, wherein the N atom îs optionally substituted by a linear or branched (Ci-Cô)alkyl group, and wherein one or more of the carbon atoms of the linear or branched (Ci-Cô)alkyl group is optionally deuterated,

Roi2 is -Cyos, -Cyo5-(Co-Cô)alkyl-Cyo6, -Cyo5-(Co-C6)alkyl-0-(C₀-C6)alkyl-Cyoô,

-Cyo5-(Co-C6)alkyl-NRoii-(Co-C₆)aIkyl-Cyo6, -Cyo_S-Cyo6-0-(Co-C6)alkyl-Cyo7,

-Cy₀5-(Co-C6)alkyl-Cyo9, -NH-C(0)-NH-Ron, -C(0)-NRoiiRoii-NRonRon’, -ORoii,

-NRon-C(0)-Roir, -0-(Ci-C₆)alkyl-ORon, -SO2-R011, or -C(O)-OR₀n,

Roi3, R013’, Roi4 and R014’ independently of one another are a hydrogen atom, or an optionally substituted linear or branched (Ci-CQalkyl group,

Cyoi, Cyos, Cyos, Cyos, Cyo6, Cyo? and Cyos independently of one another, are an optionally substituted cycloalkyl group, an optionally substituted heterocycloalkyl group, an optionally substituted aryl group or an optionally substituted heteroaryl group, ^R01S ^R015

Cy₀₉ is

^^R017 , wherein R015, Ro^, and R017 are as defined for formula (I),

. wherein R_ü27 and R_U2$ are as defmed for formula (I) w

herein, at most, one of the Ros, R09, or R012 groups, if présent, is covalently attached to the linker, or an enantiorner, diastereoisomer, atropisomer, deuterated dérivative, and/or phannaceutically acceptable sait of any of the foregoing.

In some embodiments, a drug moiety of the disclosure may comprise a compound of Fonnula (III):

213

^R012

wherein:

Roi is a linear or branched (Ci-Cé)alkyl group, Roi is -0-(Ci-C6)alkyl-NRouRoi । ’, ^R027

wherein Roi । and Roi i ’ independently of one another are a hydrogen atom, an optionally substituted linear or branched (Ci-Cô)alkyl group, or-(Co-Cô)alkyl-Cyoi;

or the pair (Roi i, Roij’) together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains, in addition to the nitrogen atom, 1 to 3 heteroatoms selected from O, S and N, wherein the N atom may be substituted by 1 or 2 groups selected from a hydrogen atom or a linear or branched (Ci-Cé)alkyl group, and wherein R027 is a hydrogen atom and R028 is a -(CH₂)p0-O-SO₂-O’ group or a -(CH2)p0-SO₂-OR₀30 group;

5 R09 is a linear or branched (C₂-Cô)alkynyl group or -Cyo₂,

R012 is -Cyos, -Cyo5-(Co-C6)alkyl-Cy_O6, or -Cyoi-tCo-CQalkyl-Cyog,

Cyoi, Cyo2, Cyo5 and Cyoé independently of one another, are a cycloalkyl group, a

214 heterocycloalkyl group, an aryl group or a heteroaryl group, each of which is optionally substituted,

pO, Rois, Roiô, and Ron are as defîned for formula (I), wherein, at most, one of the Ræ, R09, or R012 groups, if présent, is covalently attached to the linker, or the enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait of any of the foregoing, in some embodiments, Cyoi, Cyo2, Cyo₃₎ Cyo4, Cyos, Cyo&, Cyo?, Cyos and Cyoïo independently of one another, are an optionally substituted cycloalkyl group, an optionally substituted heterocycloalkyl group, an optionally substituted aryl group or an optionally substituted heteroaryl group, wherein the optional substituents are selected from optionally substituted linear or branched (Ci-C&)alkyl, optionally substituted linear or branched (C2-Cô)alkenyl group, optionally substituted linear or branched (Ci-Côjalkynyl group, optionally substituted linear or branched (Ci-Cô)alkoxy, optionally substituted (Ci-Cô)alkyl-S-, hydroxy, oxo (or N-oxide where appropriate), nitro, cyano, -C(0)-ORo’, -0-C(0)-Ro’, -C(0)-NRo’Ro”, NRo’Ro”, -(C=NRq’)-ORo”, linear or branched (Ci-Côjhaloalkyl, trifluoromethoxy, or halogen, wherein Ro’ and Ro” are each independently a hydrogen atom or an optionally substituted linear or branched (Ci-Cô)alkyl group, and wherein one or more of the carbon atoms of linear or branched (Ci-Cô)alkyl group is optionally deuterated.

In some embodiments, the drug moiety (D) comprises:

215

216

atropisomer, deuterated dérivative, and/or a pharmaceutically acceptable sait of any of the foregoing.

Additionally, a drug moiety of the disclosure may comprise any one of the

217

In some embodiments, the linker-drug (or “linker-payload”) moiety -(L-D) may comprise a compound selected from Table A..

Drug Loadîng

Drug loadîng is represented by p, and is also referred to herein as the drug-to-antibody ratio (DAR). Drug loadîng may range from l to 16 drug moieties per antibody or antîgen10 binding fragment, in some embodiments, p is an integer from 1 to 16. In some embodiments, p is an integer from 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, I to 4, l to 3, or 1 to 2. In some embodiments, p is an integer from 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In some embodiments,/? is an integer from 1 to 16. In some embodiments, p is an integer from 1 to 8. In some embodiments, p is an integer from 1

218 to 5. In some embodiments,/? îs an integer from 2 to 4, In some embodiments,/? is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments,/? is 2. In some embodiments,/? is 4.

Drug loadîng may be limited by the number of attachment sites on the antibody or antigen-binding fragment. In some embodiments, the linker moiety (L) of the ADC attaches to the antibody or antigen-binding fragment through a chemically active group on one or more amino acid residues on the antibody or antigen-binding fragment. For example, the linker may be attached to the antibody or antigen-binding fragment via a free amino, imino, hydroxyl, thiol, or carboxyl group (e.g., to the N- or C-terminus, to the epsilon amino group of one or more lysine residues, to the free carboxylic acid group of one or more glutamic acid or aspartic acid residues, or to the sulfhydryl group of one or more cysteine residues). The site to which the linker is attached can be a natural residue in the amino acid sequence of the antibody or antigenbinding fragment, or it can be introduced into the antibody or antigen-binding fragment, e.g., by DNA recombinant technology (e.g., by introducing a cysteine residue into the amino acid sequence) or b y protein biochemistry (e.g., by réduction, pH adjustment, or hydrolysis).

In some embodiments, the number of drug moieties that can be conjugated to an antibody or antigen-binding fragment is limited by the number of free cysteine residues. For example, where the attachment is a cysteine thiol group, an antibody may hâve only one or a few cysteine thiol groups, or may hâve only one or a few sufficiently reactive thiol groups through which a linker may be attached. Generally, antibodies do not contain many free and reactive cysteine thiol groups that may be linked to a drug moiety. Indeed, most cysteine thiol residues in antibodies are involved in either interchain or intrachain disulfïde bonds. Conjugation to cysteines can therefore, in some embodiments, require at least partial réduction of the antibody. Over-attachment of linker-toxin to an antibody may destabilize the antibody by reducing the cysteine residues availabie to form disulfide bonds. Therefore, an optimal drug:antibody ratio should increase potency of the ADC (b y increasing the number of attached drug moieties per antibody) without destabilizing the antibody or antigen-binding fragment. In some embodiments, an optimal ratio may be 2, 4, 6, or 8. In some embodiments, an optimal ratio may be 2 or 4.

In some embodiments, an antibody or antigen-binding fragment is exposed to reducing conditions prior to conjugation in order to generate one or more free cysteine residues. An antibody, in some embodiments, may be reduced with a reducing agent such as dîthiothreitoi (DTT) or tris(2-carboxyethyl)phosphine (TCEP), under partial or total reducing conditions, to generate reactive cysteine thiol groups. Unpaired cysteines may be generated through partial réduction with limited molar équivalents of TCEP, which can reduce the interchain disulfide

219 bonds which link the iight chain and heavy chain (one pair per H-L pairing) and the two heavy chains in the hinge région (two pairs per H-H pairing in the case of human IgG l ) while leaving the intrachain disulfide bonds intact (Stefano et al. (2013) Methods Mol Biol. 1045:145-71). In embodiments, disulfide bonds within the antibodies are reduced electrochemically, e.g., by employing a working électrode that applies an altemating reducing and oxidizing voltage. This approach can allow for on-line coupling of disulfide bond réduction to an analytical device (e.g., an electroChemical détection device, an NMR spectrometer, or a mass spectrometer) or a Chemical séparation device (e.g., a liquid chromatograph (e.g., an HP LC) or an electro pho resis device (see, e.g., US 2014/0069822)). In some embodiments, an antibody is subjected to denaturing conditions to reveal reactive nucleophihc groups on amino acid residues, such as cysteine.

The drug loading of an ADC may be controlled in different ways, e.g., by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody; (ii) limiting the conjugation reaction time or température; (iii) partial or limiting reductîve conditions for cysteine thiol modification; and/or (iv) engineering by recombinant techniques the amino acid sequence of the antibody such that the number and position of cysteine residues is modified for control of the number and/or position of linker-drug attachments.

In some embodiments, free cysteine residues are introduced into the amino acid sequence of the antibody or antigen-binding fragment. For example, cysteine engineered antibodies can be prepared wherein one or more amino acids of a parent antibody are replaced with a cysteine amino acid. Any form of antibody may be so engineered, i.e. mutated. For example, a parent Fab antibody fragment may be engineered to form a cysteine engineered Fab referred to as a “ThioFab.” Similariy, a parent monoclonal antibody may be engineered to form a “ThioMab.” A single site mutation yields a single engineered cysteine residue in a ThioFab, whereas a single site mutation yields two engineered cysteine residues in a ThioMab, due to the dimeric nature of the IgG antibody. DNA encoding an amino acid sequence variant of the parent polypeptide can be prepared by a variety of methods known in the art (see, e.g., the methods described in WO 2006/034488). These methods include, but are not limited to, préparation by site-directed (or oligonucleotide-mediated) mutagenesîs, PCR mutagenesîs, and cassette mutagenesîs of an earlier prepared DNA encoding the polypeptide. Variants of recombinant antibodies may also be constructed by restriction fragment manipulation or by overlap extension PCR with synthetic oligonucleotides. ADCs of Formula (l) include, but are not limited to, antibodies that hâve l, 2, 3, or 4 engineered cysteine amino acids (Lyon et al. (2012) Methods Enzymol. 502:123-38). In

220 some embodiments, one or more free cysteine residues are already présent in an antibody or antigen-binding fragment, without the use of engineering, in which case the existing free cysteine residues may be used to conjugate the antibody or antigen-binding fragment to a drug moiety.

Where more than one nucleophilic group reacts with a drug-linker intermediate or a linker moiety reagent followed by drug moiety reagent, in a reaction mixture comprising multiple copies of the antibody or antigen-binding fragment and linker moiety, then the resulting product can be a mixture of ADC compounds with a distribution of one or more drug moietîes attached to each copy of the antibody or antigen-binding fragment in the mixture. In some embodiments, the drug loading in a mixture of ADCs resulting from a conjugation reaction ranges from 1 to 16 drug moieties attached per antibody or antigen-binding fragment. The average number of drug moieties per antibody or antigen-binding fragment (i.e., the average drug loading, or average p) may be calculated by any conventional method know^rn in the art, e.g., by mass spectrometry (e.g., liquid chromatography-mass spectrometry (LC-MS)) and/or highperformance liquid chromatography (e.g., HIC-HPLC). In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is determined by liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is from about 1.5 to about 3.5, about 2.5 to about 4.5, about 3.5 to about 5.5, about 4.5 to about 6.5, about 5.5 to about 7.5, about 6.5 to about 8.5, or about 7.5 to about 9.5. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is from about 2 to about 4, about 3 to about 5, about 4 to about 6, about 5 to about 7, about 6 to about 8, about 7 to about 9, about 2 to about 8, or about 4 to about 8.

In some embodiments, the average number of drug moieties per antibody or antigenbinding fragment is about 2. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is 2.

In some embodiments, the average number of drug moieties per antibody or antigenbinding fragment is about 4. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment is about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, or about 4.5. In some embodiments, the average number of drug moieties per antibody or antigen-binding fragment îs 4.

221

In some embodiments, the tenn “about,” as used with respect to the average number of drug moieties per antibody or antigen-binding fragment, means plus or minus 20%, 15%, 10%, 5%, or 1%. In one embodiment, the tenu “about” refers to a range of values which are 10% more or less than the specified value. In another embodiment, the tenu “about” refers to a range of values which are 5% more or less than the specified value. In another embodiment, the term “about” refers to a range of values which are 1% more or less than the specified value.

Individual ADC compounds, or “species,” may be identified in the mixture by mass spectroscopy and separated by, e.g., UPLC or HPLC, e.g. hydrophobie interaction chromatography (HIC-HPLC). In some embodiments, a homogeneous or nearly homogenous ADC product with a single loading value may be isolated from the conjugation mixture, e.g., by electrophoresis or chromatography.

In some embodiments, higher drug loading (e.g., p > 16) may cause aggregation, insolubility, toxicity, or loss of cellular penneability of certain antibody-drug conjugates. Higher drug loading may also negatively affect the pharmacokinetics (e.g., clearance) of certain ADCs. In some embodiments, lower drug loading (e.g., p < 2) may reduce the potency of certain ADCs agaînst target-expressing cells. In some embodiments, the drug loading for an ADC of the present disclosure ranges from about 2 to about 16, about 2 to about 10, about 2 to about 8; from about 2 to about 6; from about 2 to about 5; from about 3 to about 5; from about 2 to about 4; or from about 4 to about 8.

In some embodiments, a drug loading and/or an average drug loading of about 2 is achieved, e.g., using partial réduction of intrachain disulfides on the antibody or antigen-binding fragment, and provides bénéficiai properties. In some embodiments, a drug loading and/or an average drug loading of about 4 or about 6 or about 8 is achieved, e.g., using partial réduction of intrachain disulfides on the antibody or antigen-binding fragment, and provides bénéficiai properties. In some embodiments, a drug loading and/or an average drug loading of less than about 2 may resuit in an unacceptably high level of unconjugated antibody species, which can compete with the ADC for binding to the target antigen CD74 and/or pro vide for reduced treatment efficacy. In some embodiments, a drug loading and/or average drug loading of more than about 16 may resuit in an unacceptably high level of product heterogeneity and/or ADC aggregation. A drug loading and/or an average drug loading of more than about 16 may also affect stability of the ADC, due to loss of one or more Chemical bonds required to stabilize the antibody or antigen-binding fragment.

222

The présent disclosure includes methods of producing the described ADCs. Bnefly, the ADCs comprise an antibody or antigen-binding fragment as the antibody or antigen-binding fragment, a drug moiety (e.g., an Mcl-l inhibitor), and a linker that joins the drug moiety and the antibody or antigen-binding fragment. In some embodiments, the ADCs can be prepared using a linker having reactive fLinctionalîties for covalently attaching to the drug moiety and to the antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment îs functionalized to préparé a functional group that is reactive with a linker or a druglinker intermediate. For example, in some embodiments, a cysteine thiol of an antibody or antigen-binding fragment can form a bond with a reactive functional group of a linker or a druglinker intermediate to make an ADC. In some embodiments, an antibody or antigen-binding fragment is prepared with bacterial transglutaminase (BTG) - reactive glutamines specifically functionalized with an amine containing cyclooctyne BCN (jV-[( IR,8S,9s)-Bicyclo[6.1.0]non-4yn-9-ylmethyloxycarbonyl]-l,8-diammo-3,6-dioxaoctane) moiety. In some embodiments, sitespecific conjugation of a linker or a drug-linker intermediate to a BCN moiety of an antibody or antigen-binding fragment is performed, e.g., as described and exemplified herein. The génération of the ADCs can be accomplished by techniques known to the skilled artisan.

In some embodiments, an ADC is produced by contacting an antibody or antigen-binding fragment with a linker and a drug moiety (e.g., an Mcl-l inhibitor) in a sequential manner, such that the antibody or antigen-binding fragment is covalently linked to the linker first, and then the pre-formed antibody-linker intermediate reacts with the drug moiety. The antibody-linker intermediate may or may not be subjected to a purification step prior to contacting the drug moiety. In other embodiments, an ADC is produced by contacting an antibody or antigenbinding fragment with a linker-drug compound pre-formed by reacting a linker with a drug moiety. The pre-formed linker-drug compound may or may not be subjected to a purification step prior to contacting the antibody or antigen-binding fragment. In other embodiments, the antibody or antigen-binding fragment contacts the linker and the drug moiety in one reaction mixture, allowing simultaneous formation of the covalent bonds between the antibody or antigen-binding fragment and the linker, and between the linker and the drug moiety. This method of producing ADCs may include a reaction, wherein the antibody or antigen-binding fragment contacts the antibody or antigen-binding fragment prior to the addition of the linker to the reaction mixture, and vice versa. In some embodiments, an ADC îs produced by reacting an antibody or antigen-binding fragment with a linker joined to a drug moiety, such as an Mcl-l inhibitor, under conditions that allow conjugation.

223

The ADCs prepared according to the methods described above may be subjected to a purification step. The purification step may involve any biochemical methods known in the art for purifying proteins, or any combination of methods thereof, These include, but are not limîted to, tangential flow filtration (TFF), affmity chromatography, ion exchange chromatography, any charge or isoelectric point-based chromatography, mixed mode chromatography, e.g., CHT (ceramic hydroxyapatite), hydrophobie interaction chromatography, size exclusion chromatography, dialysis, filtration, sélective précipitation, or any combination thereof. Therapeutic Uses and Compositions

Disclosed herein are methods of using the compositions described herein, e.g., the disclosed ADC compounds and compositions, in treating a subject for a disorder, e.g., a cancer. Compositions, e.g., ADCs, may be adminîstered alone or in combination with at least one additional inactive and/or active agent, e.g., at least one additional therapeutic agent, and may be adminîstered in any phannaceutically acceptable formulation, dosage, and dosing regimen. Treatment efficacy may be evaluated for toxicity as well as indicators of efficacy and adjusted accord!ngly. Efficacy measures include, but are not iimited to, a cytostatic and/or cytotoxic effect observed in vitro or in vivo, reduced tumor volume, tumor growth inhibition, and/or prolonged survival.

Methods of determining whether an ADC exerts a cytostatic and/or cytotoxic effect on a cell are known. For example, the cytotoxic or cytostatic activity of an ADC can be measured by, e.g., exposing mammalian cells expressing the target antigen CD74 of the ADC in a cell culture medium; culturing the cells for a period from about 6 hours to about 6 days; and measuring ce!! viability (e.g., using a CellTiter-Glo® (CTG) or MTT cell viability assay). Cell-based in vitro assays may also be used to measure viability (prolifération), cytotoxicity, and induction of apoptosis (caspase activation) of the ADC.

For detennining cytotoxicity, necrosis or apoptosis (programmed cell death) may be measured. Necrosis is typically accompanied by increased penneability of the plasma membrane, swelling of the ce!!, and rupture of the plasma membrane. Apoptosis can be quantitated, for example, by measuring DNA fragmentation. Commercial photometric methods for the quantitative in vitro détermination of DNA fragmentation are availabie. Examples of such assays, including TUNEL (which detects incorporation of labeled nucléotides in fragmented DNA) and ELISA-based assays, are described in Biochemica ( 1999) 2:34-7 (Roche Molecular Biochemicals).

224

Apoptosis may also be determined by measuring morphological changes in a cell. For example, as with necrosis, loss of plasma membrane integrity can be determined by measuring uptake of certain dyes (e.g., a fluorescent dye such as, for example, acridine orange or ethidîum bromide). A method for measuring apoptotic cell number has been described by Duke and Cohen, Current Protocols in Immunology (Coligan et al., eds. (1992) pp. 3.17.1-3.17.16). Cells also ean be labeled with a DNA dye (e.g., acridine orange, ethidium bromide, or propidium îodide) and the cells observed for chromatin condensation and margination along the inner nuclear membrane. Apoptosis may also be determined, in some embodiments, by screening for caspase activity. In some embodiments, a Caspase-Glo® Assay can be used to measure activity of caspase-3 and caspase-7. In some embodiments, the assay provides a luminogenic caspase3/7 substrate in a reagent optimized for caspase activity, lucîferase activity, and cell lysis. In some embodiments, addîng Caspase-Glo® 3/7 Reagent in an “add-mix-measure” format may resuit in cell lysis, foliowed by caspase cleavage of the substrate and génération of a “glow-type” luminescent signal, produced by lucîferase. In some embodiments, luminescence may be proportional to the amount of caspase activity présent, and can serve as an indicator of apoptosis. Other morphological changes that can be measured to détermine apoptosis include, e.g., cytoplasmic condensation, increased membrane blebbing, and cellular shrinkage. Détermination of any of these effects on cancer cells indicates that an ADC is useful in the treatment of cancers.

Cell viability may be measured, e.g., by detennining in a cell the uptake of a dye such as neutral red, trypan blue, Crystal Violet, or ALAMAR™ blue (see, e.g., Page et al. (1993) Intl J Oncology 3:473-6). In such an assay, the cells are încubated in media containing the dye, the cells are washed, and the remaining dye, reflecting cellular uptake of the dye, is measured spectrophotometrically.

Cell viability may also be measured, e.g., by quantifying ATP, an indicator of metabolically active cells. In sonie embodiments, in vitro potency and/or cell viability of prepared ADCs or Mcl-l inhibitor compounds may be assessed using a CellTiter-Glo® (CTG) cell viability assay, as described in the examples provided herein. In this assay, in some embodiments, the single reagent (CellTiter-Glo® Reagent) is added dîrectly to cells cultured in serum-supplemented medium. The addition of reagent results in cell lysis and génération of a luminescent signal proportional to the amount of ATP présent. The amount of ATP is dîrectly proportional to the number of cells présent in culture

Cell viability may also be measured, e.g., by measuring the réduction of tétrazolium salts. In some embodiments, in vitro potency and/or cell viability of prepared ADCs or Mcl-I inhibitor

225 compounds may be assessed using an MTT cell viability assay, as described in the examples provided herein. In this assay, in some embodiments, the yellow tétrazolium MTT (3-(4, 5dimethylthiazolyl-2)-2,5-diphenyltetrazolîum bromide) is reduced by metabolically active cells, in part by the action of dehydrogenase enzymes, to generate reducing équivalents such as NADH and NADPH. The resulting intracellular purple formazan can then be solubilized and quantified by spectrophotometric means.

In certain aspects, the present disclosure features a method of killing, inhîbiting or modulating the growth of a cancer cell or tissue by disrupting the expression and/or activity of Mcl-l and/or one or more upstream modulators or downstream targets thereof. The method may be used with any subject where disruption of Mcl-l expression and/or activity provides a therapeutic benefit. Subjects that may benefit from disrupting Mcl-l expression and/or activity include, but are not limîted to, those having or at risk of having a cancer such as a tumor or a heniatological cancer. In some embodiments, the cancer is a breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the cancer is a lymphoma or gastric cancer.

Exemplary methods include the steps of contacting a cell with an ADC, as described herein, in an effective amount, i.e., an amount sufficient to kill the cell. The method can be used on cells in culture, e.g., in vitro, in vivo, ex vivo, or in situ. For example, cells that express CD74 (e.g., cells collected by biopsy of a tumor or metastatic lésion; cells from an established cancer cell line; or recombinant cells), can be cultured in vitro in culture medium and the contacting step can be affected by adding the ADC to the culture medium. The method will resuit in killing of cells expressing CD74, including in particular cancer cells expressing CD74. Alternative!y, the ADC can be adminîstered to a subject by any suitable administration route (e.g., intravenous, subcutaneous, or direct contact with a tumor tissue) to hâve an effect in vivo.

The in vivo effect of a disclosed ADC therapeutic composition can be evaluated in a suitable animal model. For example, xenogeneic cancer models can be used, wherein cancer expiants or passaged xenograft tissues are introduced into immune compromised animais, such as nude or SCID mice (Klein et al. ( 1997) Nature Med. 3:402-8). Efficacy may be predicted

226 using assays that measure inhibition of tumor formation, tumor régression or metastasis, and the lîke.

In vivo assays that evaluate the promotion of tumor death by mechanisms such as apoptosis may also be used. In some embodiments, xenografts from tumor beanng mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.

Further provided herein are methods of treating a disorder, e.g., a cancer. The compositions described herein, e.g., the ADCs disclosed herein, can be administered to a nonhuman mammal or human subject for therapeutic purposes. The therapeutic methods include administering to a subject having or suspected of having a cancer a therapeutically effective amount of a composition comprising an Mcl-1 inhibitor, e.g., an ADC where the inhibitor îs linked to a targeting antibody that binds to an antigen (1) expressed on a cancer cell, (2) is accessible to binding, and/or (3) is localized or predomînantly expressed on a cancer cell surface as compared to a non-cancer cell.

An exemplary embodiment is a method of treating a subject having or suspected of having a cancer, comprising administering to the subject a therapeutically effective amount of a composition disclosed herein, e.g., an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions disclosed herein). In some embodiments, the cancer expresses the target antigen CD74. . In some embodiments, the cancer is a tumor or a hematological cancer. In some embodiments, the cancer is a breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chrome lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the cancer is a lymphoma or gastric cancer.

Another exemplary embodiment is a method of delivenng an Mcl-1 inhibitor to a cell expressing CD74, comprising conjugating the Mcl-1 inhibitor to an antibody that immunospecifically binds to a CD74 epitope and exposing the cell to the ADC. Exemplary

227 cancer cells that express CD74 for which the ADCs of the présent disclosure are indicated include multiple myeloma cells.

In certain aspects, the présent disclosure further provides methods of reducing or inhibiting growth of a tumor (e.g., a CD74-expressing tumor), comprising administering a therapeutically effective amount of an ADC or composition comprising an ADC. In some embodiments, the treatment is sufficient to reduce or inhibit the growth of the patient’s tumor, reduce the number or size of metastatic lestons, reduce tumor load, reduce primary tumor load, reduce invasiveness, prolong survival time, and/or maintain or împrove the qualîty of life. In some embodiments, the tumor is résistant or refractory to treatment with the antibody or antigenbinding fragment of the ADC (e.g., an anti-CD74 antibody) when administered alone, and/or the tumor is résistant or refractory to treatment with the Mcl-l inhibitor drug moiety when administered alone.

An exemplary embodiment is a method of reducing or inhibiting the growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions disclosed herein). In some embodiments, the tumor expresses the target antigen CD74. In some embodiments, the tumor is a breast cancer, gastric cancer, bladder cancer, brain cancer, cervical cancer, colorectal cancer, esophageal cancer, hepatocellular cancer, melanoma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the tumor is a gastric cancer. In some embodiments, administration of the ADC, composition, or pharmaceutical composition reduces or inhibits the growth of the tumor by at least about 10%, ai least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%, as compared to growth in the absence of treatment.

Another exemplary embodiment is a method of delaying or slowing the growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions disclosed herein). In some embodiments, the tumor expresses the target antigen CD74. In some embodiments, the tumor is a breast cancer, gastric cancer, bladder cancer, brain cancer, cervical cancer, colorectal cancer, esophageal cancer, hepatocellular cancer, melanoma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the tumor is a

228 gastric cancer. In some embodiments, administration of the ADC, composition, or pharmaceutical composition delays or slows the growth of the tumor by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%, as compared to growth in the absence of treatment.

In certain aspects, the présent disclosure further provides methods of reducing or slowing the expansion of a cancer cell population (e.g., a CD74-expressing cancer cell population), comprising administering a therapeutically effective amount of an ADC or composition comprising an ADC.

An exemplary embodiment is a method of reducing or slowing the expansion of a cancer cell population in a subject, comprising administering to the subject a therapeutically effective amount of an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions disclosed herein). In some embodiments, the cancer cell population expresses the target antigen CD74. In some embodiments, the cancer cell population is from a tumor or a hematoiogical cancer. In some embodiments, the cancer cell population îs from a breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follîcular lymphoma, lymphoid malîgnancies of T-cell or B-cell orîgin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer. In some embodiments, the cancer cell population is from a lymphoma or gastric cancer. In some embodiments, administration of the ADC, composition, or pharmaceutical composition reduces the cancer cell population by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%, as compared to the population in the absence of treatment. In some embodiments, administration of the ADC, composition, or pharmaceutical composition slows the expansion of the cancer cell population by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%, as compared to expansion in the absence of treatment.

229

Also provided herein are methods of determining whether a subject having or suspected of having a cancer will be responsive to treatment with the disclosed ADCs and compositions. An exemplary embodiment is a method of determining whether a subject having or suspected of having a cancer will be responsive to treatment with an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions disclosed herein) by providing a biological sample from the subject; contacting the sample with the ADC; and detecting binding of the ADC to cancer cells in the sample. In some embodiments, the sample is a tissue bîopsy sample, a blood sample, or a bone marrow sample. In some embodiments, the method comprises providing a biological sample from the subject; contacting the sample with the ADC; and detecting one or more markers of cancer cell death in the sample (e.g., increased expression of one or more apoptotic markers, reduced expansion of a cancer cell population in culture, etc.).

Further provided herein are therapeutic uses of the disclosed ADCs and compositions. An exemplary embodiment is an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions disclosed herein) for use in treating a subject having or suspected of having a cancer (e.g., a CD74-expressing cancer). Another exemplary embodiment is a use of an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions disclosed herein) in treating a subject having or suspected of having a cancer (e.g., a CD74expressing cancer). Another exemplary embodiment is a use of an ADC, composition, or pharmaceutical composition (e.g., any of the exemplary ADCs, compositions, or pharmaceutical compositions disclosed herein) in a method of manufacturing a médicament for treating a subject having or suspected of having a cancer (e.g., a CD74-expressing cancer). Methods for identifying subjects having cancers that express the target antigen CD74 are known in the art and may be used to identify suitable patients for treatment with a disclosed ADC compound or composition.

Moreover, ADCs of the present disclosure may be administered to a non-human mammal expressing an antigen with which the ADC is capable of binding for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of the disclosed ADCs (e.g., testing of dosages and time courses of administration).

The therapeutic compositions used in the practice of the foregoing methods may be formulated into pharmaceutical compositions comprising a pharmaceutically acceptable carrier

230 suitable for the desired delivery method. An exemplary embodiment is a pharmaceutical composition comprisîng an ADC of the présent disclosure and a phannaceutically acceptable carrier, e.g., one suitable for a chosen means of administration, e.g., intravenous administration. The pharmaceutical composition may also comprise one or more additional inactive and/or therapeutic agents that are suitable for treating or preventing, for example, a cancer (e.g., a standard-of-care agent, etc.). The phannaceutical composition may also comprise one or more carrier, excipient, and/or stabilizer components, and the like. Methods of formulating such phannaceutical compositions and suitable formulations are known in the art (see, e.g., “Remington’s Phannaceutical Sciences,” Mack Publishing Co., Easton, PA).

Suitable carriers include any material that, when combined with the therapeutic composition, retains the anti-tumor fonction ofthe therapeutic composition and is generally nonreactive with the patient’s immune system. Phannaceutically acceptable carriers include any and ail solvents, dispersion media, coatings, antibacterial and antifongal agents, isotonie and absorption delaying agents, and the like that are physiologically compatible. Examples of phannaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, éthanol, mesylate sait, and the like, as well as combinations thereof. In many cases, isotonie agents are included, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Phannaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wettîng or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the ADC.

A phannaceutical composition of the présent disclosure can be administered by a variety of methods known in the art. The route and/or mode of administration may vary dependîng upon the desired results. In some embodiments, the therapeutic formulation is solubilized and administered via any route capable of delivering the therapeutic composition to the cancer site. Potentially effective routes of administration include, but are not limited to, parentéral (e.g., intravenous, subeutaneous), intraperitoneal, intramuscular, intratumor, intradennal, intraorgan, orthotopic, and the like. In some embodiments, the administration is intravenous, subeutaneous, intraperitoneal, or intramuscular. The phannaceutically acceptable carrier should be suitable for the route of administration, e.g., intravenous or subeutaneous administration (e.g., by injection or infusion). Depending on the route of administration, the active compound(s), i.e., the ADC and/or any additional therapeutic agent, may be coated in a material to protect the compound(s) from the action of acids and other natural conditions that may inactivate the compound(s). Administration can be either systemic or local.

231

The therapeutic compositions disclosed herein may be stérile and stable under the conditions of manufacture and storage, and may be in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The form dépends on the întended mode of administration and therapeutic application. In some embodiments, the disclosed ADCs can be incorporated into a pharmaceutical composition suitable for parentéral administration. The injectable solution may be composed of either a liquid or lyophilized dosage form in a flint or amber vial, ampule, or pre-filled syringe, or other known delivery or storage device. In some embodiments, one or more ofthe ADCs or pharmaceutical compositions is supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the approprîate concentration for administration to a subject.

Typîcally, a therapcutically effective amount or efficacious amount of a disclosed composition, e.g., a disclosed ADC, is employed in the pharmaceutical compositions of the présent disclosure. The composition, e.g., one comprising an ADC, may be formulated into a pharmaceutically acceptable dosage form by conventional methods known in the art. Dosages and administration protocols for the treatment of cancers using the foregoing methods will vary with the method and the target cancer, and will generally dépend on a number of other factors appreciated in the art.

Dosage regimens for compositions disclosed herein, e.g., those comprising ADCs alone or in combination with at least one additional inactive and/or active therapeutic agent, may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus of one or both agents may be administered at one time, severai divided doses may be administered over a predetermined period of time, or the dose of one or both agents may be proportionally increased or decreased as indicated by the exigencies of the therapeutic situation. In some embodiments, treatment involves single bolus or repeated administration ofthe ADC préparation via an acceptable route of administration. In some embodiments, the ADC is administered to the patient daily, weekly, monthly, or any time period in between. For any particular subject, spécifie dosage regimens may be adjusted over time according to the individuaFs need, and the professional judgment of the treating clinician. Parentéral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrète units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active

232 compound calculated to produce the desired therapeutic effect in association with the required phannaceuticai carrier.

Dosage values for compositions comprising an ADC and/or any additional therapeutic agent(s), may be selected based on the unique characteristics of the active compound(s), and the particular therapeutic effect to be achieved. A physician or veterinarian can start doses of the ADC employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, effective doses of the compositions of the présent disclosure, for the treatment of a cancer may vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other médications administered, and whether treatment is prophylactic or therapeutic. The selected dosage level may also dépend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the présent disclosure employed, or the ester, sait, or amide thereof, the route of administration, the time of administration, the rate of excrétion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the âge, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors. Treatment dosages may be titrated to optimize safety and efficacy.

Toxicîty and therapeutic efficacy of compounds provîded herein can be determîned b y standard phannaceuticai procedures in cell culture or in animal models. For example, LD50, ED50, EC50, and IC50 may be determîned, and the dose ratio between toxic and therapeutic effects (LD50/ED50) may be calculated as the therapeutic index. The data obtained from in vitro and in vivo assays can be used in estimating or formulating a range of dosage for use in humans. For example, the compositions and methods disclosed herein may initially be evaluated in xenogeneic cancer models (e.g., an NCI-H929 multiple myeloma mouse model).

In some embodiments, an ADC or composition comprising an ADC is administered on a single occasion. In other embodiments, an ADC or composition comprising an ADC is administered on multiple occasions. Intervals between single dosages can be, e.g., daily, weekly, monthly, or yearly. Intervals can also be irregular, based on measuring blood levels of the administered agent (e.g., the ADC) in the patient in order to maintain a relatively consistent plasma concentration of the agent. The dosage and frequency of administration of an ADC or composition comprising an ADC may also vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage may be

233 administered at relatively infrequent intervais over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutîc applications, a relatively higher dosage at relatively shorter intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complété amelioration of one or more symptôme of disease. Thereafter, the patient may be administered a lower, e.g., prophylactic régime.

The above therapeutîc approaches can be combined with any one of a wide variety of additional surgi cal, chemotherapy, or radiation therapy regimens. In some embodiments, the ADCs or compositions disclosed herein are co-formulated and/or co-administered with one or more additional therapeutîc agents, e.g., one or more chemotherapeutic agents, one or more standard-of-care agents for the particular condition being treated.

Kits for use in the therapeutîc and/or diagnostic applications described herein are also provided. Such kits may comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate éléments to be used in a method disclosed herein. A label may be présent on or with the contaîner(s) to indicate that an ADC or composition within the kit is used for a spécifie therapy or non-therapeutic application, such as a prognostic, prophylactic, diagnostic, or laboratory application. A label may also indicate directions for either in vivo or în vitro use, such as those described herein. Directions and or other information may also be included on an insert(s) or label(s), which is ineluded with or on the kit. The label may be on or associated with the container. A label may be on a container when letters, numbers, or other characters forming the label are molded or etched into the container itself. A label may be associated with a container when it is présent within a réceptacle or carrier that also holds the container, e.g., as a package insert. The label may indicate that an ADC or composition within the kit is used for diagnosing or treating a condition, such as a cancer a described herein.

In some embodiments, a kit comprises an ADC or composition comprising an ADC. In some embodiments, the kit further comprises one or more additional components, including but not limited to: instructions for use; other reagents, e.g., a therapeutîc agent (e.g., a standard-ofcare agent); devices, containers, or other materials for preparing the ADC for administration; pharmaceutically acceptable carriers; and devices, containers, or other materials for administering the ADC to a subject. Instructions for use can include guidance for therapeutîc applications including suggested dosages and/or modes of administration, e.g., in a patient

234 having or suspected of having a cancer. In some embodiments, the kit comprises an ADC and instructions for use of the ADC in treating, preventing, and/or diagnosing a cancer.

COMBINATION THERAPIES

In some embodiments, the présent disclosure provides methods of treatment wherein the antibody-drug conjugales disclosed herein are administered in combination with one or more additional therapeutic agents. Exemplary combination partners are disclosed herein.

In certain embodiments, a combination described herein comprises a PD-l inhibitor. In some embodiments, the PD-l inhibitor is chosen from PDR005 (Novartis), Nivolumab (BristolMyers Squibb), Pembrolizumab (Merck & Co), Pidilizumab (CureTech), MEDI0680 (Medimmune), REGN2810 (Regeneron), TSR-042 (Tesaro), PF-06801591 (Pfizer), BGB-A317 (Beigene), BGB-108 (Beigene), INCSHR1210 (Incyte), or AMP-224 (Amplimmune). In some embodiments, the PD-l inhibitor is PDROOl. PDROOl is also known as Spartalizumab.

In certain embodiments, a combination described herein comprises a LAG-3 inhibitor. In some embodiments, the LAG-3 inhibitor is chosen from LAG525 (Novartis), BMS-986016 (Bristol-Myers Squibb), orTSR-033 (Tesaro).

In certain embodiments, a combination described herein comprises a T1M-3 inhibitor. In some embodiments, the TIM-3 inhibitor is MBG453 (Novartis), TSR-022 (Tesaro), LY-3321367 (Eli Lily), Sym23 (Symphogen), BGB-A425 (Beigene), INCAGN-2390 (Agenus), BMS-986258 (BMS), RO-712I661 (Roche), or LY-34I5244 (Eli Lilly).

In certain embodiments, a combination descdribed herein comprises a PDLl inhibitor. In one embodiment, the PDLl inhibitor is chosen from FAZO53 (Novartis), atezolizumab (Genentech), durvalumab (Astra Zeneca), or avelumab (Pfizer).

In certain embodiments, a combination described herein comprises a GITR agonist. In some embodiments, the GITR agonist is chosen from GWN323 (NVS), BMS-986156, MK-4166 or MK-1248 (Merck), TRX518 (Leap Therapeutics), INCAGN1876 (Incyte/Agenus), AMG 228 (Amgen) or INBRX-l 10 (Inhibrx).

In some embodiments, a combination described herein comprises an IAP inhibitor. In some embodiments, the IAP inhibitor comprises LCL161 or a compound disclosed in International Application Publication No. WO 2008/016893.

In an embodiment, the combination comprises an mTOR inhibitor, e.g., RAD001 (also known as everolimus).

In an embodiment, the combination comprises a HDAC inhibitor, e.g., LBH589. LBH589 is also known as panobînostat.

235 in an embodiment, the combination comprises an IL-17 inhibitor, e.g., CJM112.

In certain embodiments, a combination described herein comprises an estrogen receptor (ER) antagonist. In some embodiments, the estrogen receptor antagonist is used in combination with a PD-1 inhibitor, a CDK4/6 inhibitor, or both. In some embodiments, the combination is used to treat an ER positive (ER+) cancer or a breast cancer (e.g., an ER+ breast cancer).

In some embodiments, the estrogen receptor antagonist is a sélective estrogen receptor dégrader (SERD). SERDs are estrogen receptor antagonists which bind to the receptor and resuit în e.g., dégradation or down-regulation of the receptor (Boer K. et al., (2017) Therapeutic Advances in Medical Oncology 9(7): 465-479). ER is a hormone-activated transcription factor important for e.g., the growth, development and physiology of the human reproductive system. ER is activated by, e.g., the hormone estrogen (17beta estradiol). ER expression and signaling is implicated in cancers (e.g., breast cancer), e.g., ER positive (ER+) breast cancer. In some embodiments, the SERD is chosen from LSZ102, fulvestrant, brilanestrant, or elacestrant.

In some embodiments, the SERD comprises a compound disclosed in International Application Publication No. WO 2014/130310, which is hereby incorporated by reference in its entîrety.

In some embodiments, the SERD comprises LSZ102. LSZ102 has the Chemical name: (E)-3-(4-((2-(2-(l,l-difluoroethyl)-4-fluorophenyl)-6-hydroxybenzo[b]thiophen-3yl)oxy)phenyl)acrylic acid. In some embodiments, the SERD comprises fulvestrant (CAS Registry Number; 129453-61-8), or a compound disclosed in International Application Publication No. WO 2001/051056, which is hereby incorporated by reference in its entirety. In some embodiments, the SERD comprises elacestrant (CAS Registry Number: 722533-56-4), or a compound disclosed in U.S. Patent No. 7,612,114, which is incorporated by reference in its entirety. Elacestrant is also known as RAD1901, ER-306323 or (6R)-6-{2-[Ethyl({4-[2(ethylamino)ethyl]phenyl}methyl)amino]-4-methoxyphenyl}-5,6,7,8-tetrahydronaphthalen-2-ol. Elacestrant is an orally bioavailable, non-steroidal combined sélective estrogens receptor modulator (SERM) and a SERD. Elacestrant is also disclosed, e.g., în Garner F et al., (2015) Anticancer Drugs 26(9):948-56. In some embodiments, the SERD is brilanestrant (CAS Registry Number: 1365888-06-7), or a compound disclosed in International Application Publication No. WO 2015/136017, which is incorporated b y reference in its entirety.

In some embodiments, the SERD is chosen from RU 58668, GW7604, AZD9496, bazedoxifene, pipendoxifene, arzoxifene, OP-1074, or acolbifene, e.g., as disclosed in McDonell et al. (2015) Journal of Médicinal Chemistry 58(12) 4883-4887.

236

Other exemplary estrogen receptor antagonists are disclosed, e.g., in WO 2011/156518, WO 2011/159769, WO 2012/037410, WO 2012/037411, and US 2012/0071535, ail of which are hereby incorporated by reference in their entirety

In certain embodiments, a combination described herein comprises an inhibitor of Cyclin-Dependent Kinases 4 or 6 (CDK4/6). In some embodiments, the CDK4/6 inhibitor is used in combination with a PD-1 inhibitor, an estrogen receptor (ER) antagonist, or both. In some embodiments, the combination is used to treat an ER positive (ER+) cancer or a breast cancer (e.g., an ER+ breast cancer). In some embodiments, the CDK4/6 inhibitor is chosen from ribociclib, abemaciclib (Eli Lilly), or palbociclib.

In some embodiments, the CDK4/6 inhibitor comprises ribociclib (CAS Registry Number: 1211441-98-3), or a compound disclosed in U.S. Patent Nos. 8,415,355 and 8,685,980, which are incorporated by reference in their entirety.

In some embodiments, the CDK4/6 inhibitor comprises a compound disclosed in International Application Publication No. WO 2010/020675 and U.S. Patent Nos. 8,415,355 and 8,685,980, which are incorporated by reference in their entirety.

In some embodiments, the CDK4/6 inhibitor comprises ribociclib (CAS Registry Number: 1211441-98-3). Ribociclib is also known as LEE011, KISQALI®, or 7-cyclopentyiN,N-dimethyL2-((5-(piperazin-l-yl)pyridin-2-yl)amino)-7H-pyrrolo[2,3-d]pyrimidine-6carboxamide.

In some embodiments, the CDK4/6 inhibitor comprises abemaciclib (CAS Registry Number: 1231929-97-7). Abemaciclib is also known as LY835219 or N-[5-[(4-Ethyl-lpiperazinyl)methyl]-2-pyridinyl]-5-fluoro-4-[4-fluoro-2 -methyl-1-(1 -methylethyl)-1Hbenzimidazol-6-yI]-2-pyrimidinamme. Abemaciclib is a CDK inhibitor sélective for CDK4 and CDK6 and is disclosed, e.g., in Torres-Guzman R et al. (2017) Oncotarget 10.1863 2/onco target. 17778.

In some embodiments, the CDK4/6 inhibitor comprises palbociclib (CAS Registry Number: 571190-30-2). Palbociclib is also known as PD-0332991, IBRANCE® or 6-Acetyl-8cyclopentyl-5-methyl-2-{[5-(l-piperazinyl)-2-pyridinyl]amino}pyrido[2,3-d]pyrimidin-7(8H)one. Palbociclib inhibits CDK4 with an IC50 of 1 InM, and inhibits CDK6 with an IC50 of 16nM, and îs disclosed, e.g., in Finn et al. (2009) Breast Cancer Research 11(5):R77.

In certain embodiments, a combination described herein comprises an inhibitor of chemokine (C-X-C motif) receptor 2 (CXCR2). In some embodiments, the CXCR2 inhibitor is

237 chosen from 6-chloro-3-((3,4-dioxo-2-(pentan-3-ylamino)cy cl obut-1-en-l-yl)amino)-2-hydroxyN-methoxy-N-methylbenzenesulfonamide, danirixin, reparixin, or navarixin.

In some embodiments, the CSF-1/1R bindîng agent is chosen from an inhibitor of macrophage colony-stimulating factor (M-CSF), e.g., a monoclonal antibody or Fab to M-CSF (e.g., MCS110), a CSF-1 R tyrosine kinase inhibitor (e.g., 4-((2-(((1 R,2R)-2hydroxycyclohexyl)amino)benzo[d]thiazol-6-yI)oxy)-N-methylpicolinamide or BLZ945), a receptor tyrosine kinase inhibitor (RTK) (e.g., pexidartinib), or an antibody targeting CSF-1 R (e.g., emactuzumab or FPA008). In some embodiments, the CSF-1/1R inhibitor is BLZ945. In some embodiments, the CSF-1/1R bindîng agent is MCS110. In other embodiments, the CSF1/1R bindîng agent is pexidartinib.

In certain embodiments, a combination described herein comprises a c-MET inhibitor. C-MET, a receptor tyrosine kinase overexpressed or mutated in many tumor cell types, plays key rôles în tumor cell prolifération, survival, invasion, metastasis, and tumor angiogenesis. Inhibition of c-MET may induce cell death in tumor cells overexpressing c-MET protein or expressing constitutively activated c-MET protein. In some embodiments, the c-MET inhibitor is chosen from capmatinib (INC280), JNJ-3887605, AMG 337, LY2801653, MSC2156119J, crizotinib, tivantinib, or golvatinîb.

In certain embodiments, a combination described herein comprises a transforming growth factor beta (also known as TGF-β ΤΟΕβ, TGFb, or TGF-beta, used interchangeably herein) inhibitor. In some embodiments, the TGF-β inhibitor is chosen from fresolimumab or XOMA 089.

In certain embodiments, a combination described herein comprises an adenosine A2a receptor (A2aR) antagonist (e.g., an inhibitor of A2aR pathway, e.g., an adenosine inhibitor, e.g., an inhibitor of A2aR or CD-73). In some embodiments, the A2aR antagonist is used in combination with a PD-1 inhibitor, and one or more (e.g., two, three, four, five, or ail) of a CXCR2 inhibitor, a CSF-I/1R bindîng agent, LAG-3 inhibitor, a GITR agonist, a c-MET inhibitor, or an IDO inhibitor. In some embodiments, the combination is used to treat a pancreatic cancer, a colorectal cancer, a gastric cancer, or a melanoma (e.g., a refractory melanoma). In some embodiments, the A2aR antagonist is chosen from PBF509 (NIR178) (Palobiofanna/Novartis), CPI444/V81444 (Corvus/Genentech), AZD4635/HTL-1071 (AstraZeneca/Heptares), Vipadenant (Redox/Juno), GBV-2034 (Globavir), AB928 (Arcus Biosciences), Theophylline, Istradefylline (Kyowa Hakko Kogyo), Tozadenant/SYN-115 (Acorda), KW-6356 (Kyowa Hakko Kogyo), ST-4206 (Leadiant Biosciences), or

238

Preladenant/SCH 420814 (Merck/Schering). Without wishing ίο be bound by theory, it is believed that in some embodiments, inhibition of A2aR leads to upregulation of IL-lb.

In certain embodiments, a combination described herein comprises an inhibitor of indoleamine 2,3-dioxygenase (IDO) and/or tryptophan 2,3-dioxygenase (TDO). In some embodiments, the IDO inhibitor îs used in combination with a PD-l inhibitor, and one or more (e.g., two, three, four, or ail) of a TGF-β inhibitor, an A2aR antagonist, a CSF-1/1R binding agent, a c-MET inhibitor, or a GITR agonist. In some embodiments, the combination is used to treat a pancreatic cancer, a colorectal cancer, a gastric cancer, or a melanoma (e.g., a refractory melanoma). In some embodiments, the IDO inhibitor is chosen from (4E)-4-[(3-chloro-4fluoroanilino)-nitrosomethylidene]-l,2,5-oxadiazol-3-amine (also known as epacadostat or INCB24360), indoximod (NLG8189), (1-methyl-D-tryptophan), a-cyclohexyl-5H-Imidazo[5,la]isoindole-5-éthanol (also known as NLG919), indoximod, BMS-986205 (formerly F001287).

In certain embodiments, a combination described herein comprises a Galectin, e.g., Galectin-1 or Galectin-3, inhibitor. In some embodiments, the combination comprises a Galectin-1 inhibitor and a Galectin-3 inhibitor. In some embodiments, the combination comprises a bîspecific inhibitor (e.g., a bispecific antibody molécule) targeting both Galectin-1 and Galectin-3. In some embodiments, the Galectin inhibitor is used in combination with one or more therapeutic agents described herein. In some embodiments, the Galectin inhibitor is chosen from an anti-Galectin antibody molécule, GR-MD-02 (Galectin Therapeutics), Galectin-3C (Mandai Med), Anginex, or OTX-008 (OncoEthix, Merck).

In some embodiments, a combination described herein comprises a MEK inhibitor. In some embodiments, the MEK inhibitor is chosen from Trametînib, selumetinib, AS703026, BIX 02189, BIX 02188, CI-1040, PD0325901, PD98059, U0126, XL-518, G-38963, or G02443714. In some embodiments, the MEK inhibitor is Trametînib.

In one embodiment, a combination described herein includes an interleukin-1 beta (IL- i β) inhibitor. In some embodiments, the IL-1 β inhibitor is chosen from canakinumab, gevokizumab, Anakinra, or Rilonacept.

In certain embodiments, a combination described herein comprises an IL-15/IL-15Ra complex. In some embodiments, the IL-l5/IL-15Ra complex is chosen from NIZ985 (Novartis), ATL-803 (Altor) or CYP0150 (Cytune).

In certain embodiments, a combination described herein comprises a mouse double minute 2 homolog (MDM2) inhibitor. The human homolog of MDM2 is also known as HDM2.

239

In some embodiments, an MDM2 inhibitor described herein is also known as a H DM2 inhibitor. In some embodiments, the MDM2 inhibitor is chosen from HDM201 or CGM097.

In an embodiment the MDM2 inhibitor comprises (S)-1 -(4-chlorophenyl)-7-isopropoxy6-methoxy-2-(4-(methyl(((lr,4S)-4-(4-methyl-3-oxopiperazin-lyl)cyclohexyl)methyl)amino)phenyl)-î ,2-dihydroisoquînolin-3(4H)-one (also known as CGM097) or a compound disclosed in PCT Publication No. WO 2011/076786 to treat a disorder, e.g., a disorder described herein). In one embodiment, a therapeutic agent disclosed herein is used in combination with CGM097.

In some embodiments, a combination described herein comprises a hypomethylating agent (HMA). In one some embodiments, the HMA is chosen from decitabinc or azacitidine.

In certain embodiments, a combination described herein comprises an inhibitor acting on pro-survival proteins of the Bcl2 family. In certain embodiments, a combination described herein comprises a Bel-2 inhibitor. In some embodiments, the Bel-2 inhibitor is venetoclax:

venetoclax)

In one embodiment, the Bcl-2 inhibitor is selected from the compounds described in WO 2013/110890 and WO 2015/011400. In some embodiments, the Bcl-2 inhibitor comprises navitoclax (ABT-263), ABT-737, BP 1002, SPC2996, APG-1252, obatoclax mesylate (GX15070MS), PNT2258, Zn-d5, BGB-11417, or oblimersen (G3139). In some embodiments, the Bcl2 inhibitor is (S)-5-(5-chloro-2-(3-(inorpholinomethyl)-l,2,3,4-tetrahydroisoquinoline-2carbonyl)phenyl)-N-(5-cyano-l,2-dimethyl-lH-pyrrol-3-yl)-N-(4-hydroxyphenyl)-l,2-dimethyllH-pyrrole-3-carboxamide), compound Al :

240 empound Al). In some embodiments, the Bcl-2 inhibitor is

N-(4-hydroxyphenyl)-3-[6-[(3S)-3-(morpholinomethyl)-3,4-dihydro-lH-isoquinoline-2carbonyl]-l,3-benzodioxol-5-yl]-N-phenyl-5,6,7,8-tetrahydroindolizine-l-carboxamide, compound A2:

(compound A2).

In one embodiment, the antibody-drug conjugates or combinations disclosed herein are suitable for the treatment of cancer in vivo. For example, the combination can be used to inhibit the growth of cancerous tumors. The combination can also be used in combination with one or more of: a standard of care treatment (e.g., for cancers or infectious disorders), a vaccine (e.g., a therapeutic cancer vaccine), a cell therapy, a radiation therapy, surgery, or any other therapeutic agent or modality, to treat a disorder herein. For example, to achieve antigenspecific enhancement of immunity, the combination can be adminîstered together with an antigen of interest. A combination disclosed herein can be adminîstered in either order or simultaneously.

ADDITIONAL EMBODIMENTS

241

The disclosure provides the foliowing additional embodiments for linker-drug groups, antibody-drug conjugales, linker groups, and methods of conjugation.

Linker-Drug Group

In some embodiments, the Linker-Drug group of the invention may be a compound having the structure of Formula (A’), or a phannaceutically acceptable sait thereof:

>L₂—A—D

FAQ—Lp-G l₃-r^z

Formula (A’) wherein:

R¹ is a reactive group;

Li is a bridging spacer;

Lp is a bivalent peptide spacer;

G-L2-A is a self-immolative spacer;

R² is a hydrophilic moiety;

L2 is a bond, a methylene, a neopentylene or a C₂-C₃alkenylene;

⁰ 0 * , Il II

Ao-p-o-p—⁷

OH OH

-OC(=O)N(CH3)CH₂CH₂N(CH₃)C(=O)-* or OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH₃)C(=O)-*, wherein each R³ is independently selected from H, Ci-Cî alkyl, and C3-C8 cycloalkyl and the * of A indicates the point of attachment to D;

L₃ is a spacer moiety; and

D is a Drug moiety that is capable of inhibiting the activity of the MC1-1 protein when, e.g., released from the Antibody Drug Conjugales or immunoconjugates disclosed herein.

Certain aspects and examples of the Linker-Drug group of the invention are provided in the foliowing listing of enumerated embodiments. It will be recognized that features specified in each embodiment may be combined with other specified features to provide further embodiments of the présent invention.

242

Embodiment 1. The compound of Fonnula (A’), or pharmaceutically acceptable sait thereof, wherein:

R¹ is a reactive group;

Li is a bridging spacer;

Lp is a bivalent peptide spacer comprising two to four amino acid residues;

G-L₂-A is a self-immolative spacer;

R² is a hydrophilic moiety;

L₂ is a bond, a methylene, a neopentylene or a C₂-C₃alkenylene;

O * O O * O , Ils . Il II 5 s ¹¹*

-bo-p-o-p-l·

A is a bond, -OC(=O)-*, ^0H , OH OH , OH ⁰O * _ς IIII

-bo-p-o-A OH OH

-OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C₃-Cg cycloalkyl and the * of A indicates the point of attachment to D;

L₃ îs a spacer moiety; and

D is a Drug moiety as defined herein, e.g., a MC1-1 inhibitor.

Embodiment 2. The compound of Formula (A’), or pharmaceutically acceptable sait thereof, wherein:

R¹ is a reactive group;

Li is a bridging spacer;

Lp is a bivalent peptide spacer comprising two to four amino acid residues;

_s A-A the L₃-R² group is selected from:

point of attachment to D (e.g., to an N or a O of the Drug moiety), the *** of indicates the point of attachment to Lp;

243

R² is a hydrophilic moiety;

L? is a bond, a methylene, a neopentylene or a C2-C₃alkenylene;

_λ ii n Th

4-o-p-o-p—⁷

OH OH

-OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C3-Cs cycloalkyl and the * of A indicates the point of attachment to D;

L₃ is a spacer moiety; and

D is a Drug moiety as defined herein, e.g., a MC1-1 inhibitor.

Embodiment 3. The compound of Formula (A’), or pharmaceutically acceptable sait thereof, having the structure of Formula (B’):

Formula (B’) wherein:

R¹ is a reactive group;

Li is a bridging spacer;

Lp is a bivalent peptide spacer comprising two to four amino acid residues;

R² is a hydrophilic moiety;

A is a bond, -OC(=O)-*, OH , OH OH _} OH , ⁰ O , * < Il II

4ογο-ρ-⁷

OH OH

-OC(=O)N(CH3)CH₂CH2N(CH₃)C(=O)-* or OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH₃)C(=O)-*, wherein each R^a is independently

244 selected from H, Ci-Cô alkyi, and C₃-C₈ cycloalkyl and the * of A indicates the point of attachment to D;

L₃ is a spacer moiety; and

D is a Drug moiety as defined herein and comprising an N or an O, wherein D is connected to A via a direct bond from A to the N or the O of the Drug moiety.

Embodiment 4. The compound of Formula (A’) or of any one of Embodiments 1 to 3, or pharmaceutically acceptable sait thereof, wherein:

F F

-SSR⁴, -S(=O)₂(CH=CH₂), -(CH₂)₂S(=O)2(CH=CH₂), -NHS(=O)₂(CH=CH₂), -

245

Li is *-C(=O)(CH₂)_mO(CH₂)_m-**;

*-C(=O)((CH₂)_mO)_t(CH₂)_n-* * ;

*-C(=O)(CH₂)_m-**;

*-C(=O)NH((CH₂)mO)t(CH2)n-**;

*-C(=O)O(CH₂)_mSSC(R³)2(CH2)mC(=O)NR^î(CH2)_mNR³C(=O)(CH₂)m-**;

*-C(=O)O(CH₂)_mC(=O)NH(CH₂)m-**;

*-C(=O)(CH₂)_mNH(CH₂)_m-**; *-C(=O)(CH₂)_mNH(CH₂)_nC(=O)-**;

*-C(=O)(CH₂)_mXi(CH₂)m-**; *-C(=O)((CH2)mO)t(CH₂)_nXi(CH₂)n-**;

*-C(=O)(CH2)_mNHC(=OXCH₂)_n-* * ;

*-C(=O)((CH₂)_ltlO)_l(CH₂)nNHC(=O)(CH₂)n-**;

*-C(=O)(CH₂)mNHC(=O)(CH₂)_nXi(CH2)_n-** ;

*-C(=O)((CH₂)_mO)_t(CH₂)_nNHC(=O)(CH₂)_nXi(CH₂)_n-**;

*-C(=O)((CH₂)_mO)_t(CH₂)_nC(=O)NH(CH₂)_m-**;

*-C(=O)(CH₂)_mC(R³)₂-**; or *-C(=O)(CH₂)mC(=O)NH(CH₂)_m-**,

246 where the * of Li indicates the point of attachment to Lp, and the ** of Li indicates the point of attachment to R¹ ;

R² is a hydrophilic moiety selected from polyethylene glycol, polyalkylene glycol, a sugar, an oligosaccharide, a polypeptide or C₂-Côalkyl substituted with 1 to 3 o

—|-O—P-OH

OH groups;

each R³ is independently selected from H and Ci-Côalkyl;

R⁴ îs 2-pyridyl or 4-pyridyl;

each R⁵ is independently selected from H, Ci-Côalkyl, F, Cl, and -OH;

each R⁶ is independently selected from H, Ci-Côalkyl, F, Cl, -NH₂, -OCH₃, OCH2CH3, -N(CHî)₂, -CN, -NO2 and -OH;

each R⁷ is independently selected from H, Ci-ôalkyl, fluoro, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, Cj.₄alkoxy substituted with -C(=O)OH and C].₄alkyl substituted with -C(=O)OH;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, S, 9 and 10;

each n is independently selected from 1,2, 3, 4, 5, 6, 7, 8, 9 and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is a bivalent peptide spacer comprising an amino acid residue seiected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, méthionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine;

O * OO* O

-|-θ-ργ- 4Ό-Ρ-Ο-Ρ4- -f-Ο-Ρ-Ο^γ

A is a bond, -OC(=O)-*, ^OH , OH OH , OH

OH OH

-OC(=O)N(CH3)CH2CH2N(CHj)C(=O)-* or OC(=O)N(CH3)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyi, and C3-Cs cycloalkyl and the * of A indicates the point of attachment to D;

247

L₃ is a spacer moiety having the structure * , where

W is -CH₂O-**, -CH₂N(R^b)C(=O)O-*% -NHC(=O)C(R^b)₂NHC(=O)O** 3

-NHC(=O)C(R^b)2NH-**,-NHC(=O)C(R^b)2NHC(=O)-**,

-CH₂N(X-R²)C(=O)O-**, -C(=O)N(X-R²)-**, -CH₂N(X-R²)C(=O)3

-C(=O)NR^b-**, -C(=O)NH-**, -CH2NR^bC(=O)-**, CH2NR^bC(=O)NH-**, -CH2NR^bC(=O)NR^b-**, -NHC(=O)-**, NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, NHS(O)₂-**, -C(=O)-, -C(=O)O-** or

-NH-, wherein each R^b is independently selected from H, Ci-Côalkyl or C₃-C8cycloalkyl and wherein the ** of W indicates the point of attachment to X;

X is a bond, triazolyl or ***-CH₂-triazolyl-*, wherein the *** of X indicates the point of attachment to W and the * of X indicates the point of attachment to R²; and the * of L₃ indicates the point of attachment to R²;

and

D is a Drug moiety as defined herein and comprisîng an N or an O, wherein D is connected to A via a direct bond from A to the N or the O of the Drug moiety.

Embodiment 5. The compound of Formula (A’) or of any one of Embodiments l to 4, or phannaceutically acceptable sait thereof, wherein:

L, is *-C(=O)(CH₂)mO(CH₂)_m-**; *-C(=O)((CH₂)_mO)_t(CH₂)_n-**; *-C(=O)(CH₂)_m-**;

or *-C(=O)NH((CH₂)mO)t(CH₂)n-, where the * of Li indicates the point of attachment to Lp, and the “ of Li indicates the point of attachment to R¹;

248 each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 1Û;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is a bivalent peptide spacer selected from ° ^NH2 (ValCit),

NH (ValLys) and ^{0 NH}2(LeuCit), where the * of Lp indicates the attachment point to Li and the ** of Lp indicates the attachment point to the NH- group of G;

L₃ is a spacer moiety having the structure * ^? , where

W is -CH2O-**, -CH₂N(R^b)C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -NHC(=O)CH2NH-**, -NHC(=O)CH2NHC(=O)-**, -CH2N(XR²)C(=O)O-**, -C(=O)N(X-R²)-**, -CH2N(X-R²)C(=O)-**, -C(=O)NR^b-**, -C(=O)NH-**, -CH2NR^bC(=O)-**, CH2NR^bC(=O)NH-**, -CH2NR^bC(=O)NR^b-**, -NHC(=O)-**, NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, NHS(O)₂-**, -C(=O)-, -C(=O)O-** or

-N H-, wherein each R^b is independently selected from H, Ci-Csalkyl or C₃-Cscycloalkyl and wherein the ** of W indicates the point of attachment to X;

249

X is a bond, triazolyl or ***-CH2-triazolyl-*, wherein the *** of X indicates the point of attachment to W and the * of X indicates the point of attachment to R²;

and the * of La indicates the point of attachment to R²;

R² is a hydrophilic moiety selected from polyethylene glycol, polyalkylene glycol, a sugar, an oligosaccharide, a polypeptide or C2~Cf>alkyl substituted with l to 3

O

-|-O-P-OH

OH groups;

O * O O * O , Il S ς ¹¹ H 3 S ¹¹ *

4-o-p-r 4o-p-o-pr -|-ο-ρ-ο<γ

A is a bond, -OC(=O)-*, θΗ , OH OH ₅ OH ,

-OC(=O)N(CH₃)CH2CH2N(CH3)C(=O)-* or

-OC(=O)N(CH3)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Ce alkyl, and Cj-Cs cycloalkyl and the * of A indicates the point of attachment to D; and

Embodiment 6. The compound of Formula (A’) or of any one of Embodiments 1 to 5, or pharmaceutically acceptable sait thereof, wherein:

O

-φ

R¹ is O ;

L| is *-C(=O)(CH2),nO(CH2)₁n-**; *-C(=O)((CH₂)_mO)_t(CH₂)n-**; *-C(=O)(CH2)_m-**; or *-C(=O)NH((CH₂)mO)t(CH₂)n-, where the * of Li indicates the point of attachment to Lp, and the ** of Li indicates the point of attachment to R¹;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each n is independently selected from 1,2, 3, 4, 5, 6, 7, 8, 9 and 10;

each t is independently selected from 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 and 30;

250

Y h 9 ** sv-y H O < nh

Lp is a bivalent peptide spacer selected from ° ^NH2 (ValCit), where the * of Lp indicates the attachment point to Li and the ** of Lp indicates the attachment point to the -NH- group of G;

Lj is a spacer moiety having the structure * ^? , where

W is -CH₂O-*X -CH₂N(R^b)C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(X-R²)C(=O)O-**, -C(=O)N(X-R²)-**, -CH2N(X-R²)C(=O)**, -C(=O)NR^b-**, -C(=O)NH-**, -CH2NR^bC(-O)-**, CH2NR^bC(=0)NH-**, -CH2NR^bC(=0)NR^b-**, -NHC(=0)-**, NHC(-O)O-**, -NHC(=O)NH-**, -OC(=O)NH-*L -S(O)2NH-**, NHS(O)₂-**> -C(=O)-, -C(=O)O-** or

-NH-, wherein each R^b is independently selected from H, Ci-Côalkyl or Cs-Cscycloalkyl and wherein the **ofW indicates the point of attachment to X;

X is a bond, triazolyl or ***-CH₂-triazolyl-*, wherein the *** of X indicates the point of attachment to W and the * of X indicates the point of attachment to R²;

and the * of L₃ indicates the point of attachment to R²;

R² is a hydrophilic moiety selected from polyethylene glycol, polyalkylene glycol, a sugar, an oligosaccharide, a polypeptide or Cs-Côalkyl substituted with 1 to 3 o -l-O-P-OH ^? 1

OH groups;

O . 1'

4-o-pA is a bond,-OC(=O)-*, OH , OH OH , OH

O O , * . H H γί,

-bo-P-O-P—⁷

OH OH , -OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-*^: or -OC(=O)N(CH3)C(R^a)2C(R^a)₂N(CH₃)C(=O)-*, wherein each R^a is independently

251 selected from H, Ci-C& alkyl, and C₃-Cs cycloalkyl and the * of A indicates the point of attachment to D;

and

D îs a Drug moiety as defined herein and comprising an N or an O, wherein D is connected to A via a direct bond from A to the N or the O of the Drug moiety.

Embodiment 7. The compound of Formula (A’) or of any one of Embodiments 1 to 6, or pharmaceutically acceptable sait thereof, wherein:

+4

R is O ;

L1 is *-C(=O)(CH2)n₁O(CH2),n-**; *-C(=O)((CH₂)mO)_Î(CH2)_!1-^:i:*; <C(=O)(CH2)_m-^Ÿ* or *-C(=O)NH((CH₂)mO)t(CH₂)n-, where the * of Li indicates the point of attachment to Lp and the ** of Li indicates the point of attachment to R¹;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each t is independently selected from 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21,22, 23, 24,25, 26,27, 28, 29 and 30;

* V H 9 ** ^H Ο γ

Λ

Lp is a bivalent peptide spacer selected from ^{0 NH}2 (ValCit), where the * of Lp indicates the attachment point to Li and the ** of Lp indicates the attachment point to the -NH- group of G;

4-w—x-p

L₃ is a spacer moiety having the structure ^? , where

W is-CHsO-**, -CH₂N(R^b)C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(X-R²)C(=O)O-**, -C(=O)N(X-R²)-**, -C(=O)NR^b-*% C(=O)NH-**, -CH2NR^bC(=O)-**, -CH2NR^bC(=O)NH-**, CH2NR^bC(=O)NR^b-**, -NHC(=O)-**, -NHC(=O)O-**, or -NHC(=O)NH-**, wherein each

252

R^b is independently selected from H, Ci-Côalkyl or Cj-Cgcycloalkyl and wherein the ** of W indicates the point of attachment to X;

and the * of Lî indicates the point of attachment to R²;

R² is a hydrophilîc moiety selected from polyethylene glycol, polyalkylene glycol, a sugar, an oligosaccharide, a polypeptide or C2-C6alkyl substituted with 1 to 3 o

-|-O-P-OH

OH groups;

A is a bond or -OC(=O)*, in which * indicates the attachment point to D;

and

D is a Drug moiety as defmed herein and comprising an N or an O, wherein D is connected to A via a direct bond from A to the N or the O of the Drug moiety.

Embodiment 8. The compound of Formula (A’) or of any one of Embodiments 1 to 7, or pharmaceutically acceptable sait thereof, wherein:

Φ

R¹ is O ;

Li is *-C(=O)(CH₂)_mO(CH₂)_m-**; *-C(=O)((CH₂)_raO)_t(CH₂)_n-**; *-C(=O)(CH₂)_m-**; or *-C(=O)NH((CH₂)mO)t(CH₂)n-, where the * of Li indicates the point of attachment to Lp and the ** of Li indicates the point of attachment to R¹; each m îs independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each n is independently selected from 1,2, 3, 4, 5, 6, 7, 8, 9 and 10;

253

Y H O ** ^H ο l

Λ

Lp is a bivalent peptide spacer selected from ^{0 NH}a (ValCit), where the * of Lp indicates the attachment point to Li and the ** of Lp indicates the attachment point to the -NH- group of G;

S Ά

L3 is a spacer moiety having the structure * , where

W is -CH2O-**, -Œ₂N(R^b)C(=O)O-**, -NHC(=O)CH₂NHC(=O)O-**, -CH₂N(X-R²)C(=O)O-**, or -C(=O)N(X-R²)-**, wherein each R^b is independently selected from H, Ci-Côalkyl or Cs-Cscycloalkyl and wherein the ** of W indicates the point of attachment to X;

X îs ***-CH₂-triazolyl-*, wherein the *** ofX indicates the point of attachment to W and the * of X indicates the point of attachment to R²;

and the * of L3 indicates the point of attachment to R²;

R² is a hydrophilic moiety selected from polyethylene glycol, polyalkylene glycol, a sugar, an oligosaccharide, a polypeptide or C₂-Côalkyl substituted with l to 3

O

-l-O-P-OH ' I oh groups;

A is a bond or -OC(=O)* in which * indicates the attachment point to D; and

D is a Drug moiety as defined herein and comprising an N or an O, wherein D is connected to A via a direct bond from A to the N or the O of the Drug moiety, Embodiment 9. The compound of Formula (A’) or of any one of Embodiments 1 to 8, or pharmaceutically acceptable sait thereof, wherein R¹ is a reactive group selected from Table 2.

Embodiment 10. The compound of Formula (A’) or of any one of Embodiments 1 to 9, or pharmaceutically acceptable sait thereof, wherein:

254

-SH, -SR³, -SSR⁴, -S(=O)₂(CH=CH₂),

-(CH₂)₂S(=O)2(CH=CH2), -NHS(=O)₂(CH=CH₂), -NHC(=O)CH₂Br, -NHC(=O)CH₂I,

255

Embodiment 11. The compound of Formula (A⁷) or of any one of Embodiments l to 9, or pharmaceutically acceptable sait thereof, wherein:

-(CH₂)₂S(=O)₂(CH=CH₂), -NHS(=O)2(CH=CH₂), -NHC(=O)CH₂Br, -NHC(=O)CH₂I,

Embodiment 12. The compound of Formula (A’) or of any one of Embodiments l to 9, or pharmaceutically acceptable sait thereof, wherein:

256

Embodiment 13. The compound of Formula (A’) or of any one of Embodiments 1 to 9, or phannaceutically acceptable sait thereof, wherein:

Embodiment 14. The compound of Formula (A’) or of any one of Embodiments 1 to 9, or

O

-φ pharmaceutically acceptable sait thereof, wherein R¹ is O

Embodiment 15. The compound of Fonnula (A’) or of any one of Embodiments 1 to 9, or 10 pharmaceutically acceptable sait thereof, wherein R^! is -ONFb.

Embodiment 16. The compound of Fonnula (A’) or of any one of Embodiments 1 to 9, or pharmaceutically acceptable sait thereof, wherein: R¹ is

Embodiment 17. The compound of Formula (A’) or of any one of Embodiments 1 to 9, or pharmaceutically acceptable sait thereof, wherein:

F

257

Embodiment 18. The compound of Formula (A’) or of any one of Embodiments I to 9, or pharmaceutically acceptable sait thereof, having the structure:

, where

R is H, -CH₃ or -CH₂CH2C(=O)OH.

Embodiment 19. The compound of Fonnula (A’) or of any one of Embodiments 1 to 9, or pharmaceutically acceptable sait thereof, having the structure:

h₂n o , where

R is H, -CH₃ or-CH₂CH2C(=O)OH.

Embodiment 20. The compound of Formula (A’) or of any one of Embodiments 1 to 9, or pharmaceutically acceptable sait thereof, having the structure:

R is H, -CH₃ or-CH₂CH₂C(=O)OH.

Embodiment 21. The compound of Formula (A’) or of any one of Embodiments 1 to 9, or 15 pharmaceutically acceptable sait thereof, having the structure:

258 each R is independently selected from H, -CH3 or -CH₂CH₂C(-O)OH.

Embodiment 22. The compound of Formula (A’) or of any one of Embodiments l to 9, or pharmaceutically acceptable sait thereof, having the structure:

each R is independently selected from H, -CH3 or -CH₂CH₂C(=O)OH.

Embodiment 23. The compound of Formula (A’) or of any one of Embodiments 1 to 9 or pharmaceutically acceptable sait thereof, having the structure:

H2N t=4 to 25 , where

Xa is -CH2-, -OCH₂-, -NHCH₂- or -NRCH₂- and each R independently is H, -CH3 or

CH₂CH₂C(=O)OH.

Embodiment 24. The compound of Formula (A’) or of any one of Embodiments 1 to 9, or pharmaceutically acceptable sait thereof, having the structure:

R is H, -CH3 or-CH₂CH₂C(=O)OH.

Embodiment 25. The compound of Formula (A’) or of any one of Embodiments 1 to 9, or pharmaceutically acceptable sait thereof, having the structure:

259

t=4 to 25 h₂n o where

Xb is -CH?-, -OCH?-, -NHCH2- or-NRCH₂- and each R independently is H, -CH₃ or

-CH₂CH₂C(=O)OH.

Embodiment 26. The compound of Formula (A’) or of any one of Embodiments 1 to 9, or pharmaceutically acceptable sait thereof, having the structure:

Embodiment 27. The compound of Formula (A’) or of any one of Embodiments 1 îo 9, or pharmaceutically acceptable sait thereof, having the structure:

Embodiment 28. The compound of Formula (A’) or of any one of Embodiments 1 to 9, or pharmaceutically acceptable sait thereof, having the structure:

Embodiment 29. The compound of Formula (A’) or of any one of Embodiments 1 to 9, or pharmaceutically acceptable sait thereof, having the structure:

260

Embodiment 30. The compound of Fonnula (A’) or of any one of Embodiments l to 9, or phannaceutically acceptable sait thereof, having the structure:

Embodiment 31. The compound of Fonnula (A’) or of any one of Embodiments 1 to 9, or phannaceutically acceptable sait thereof, having the structure of a compound in Table A.

Embodiment 32. A linker of the Linker-Drug group of Fonnula (A’) having the structure of

Formula (C’), s ^1-2

Lp”G ⁵

Formula (C’) wherein

Li is a bridging spacer;

Lp is a bivalent peptide spacer;

G-L₂-A is a self-immolative spacer;

R² is a hydrophilic moiety;

L₂ is a bond, a methylene, a neopentylene or a C₂-C3alkenylene;

O * 0 0* O _s Ils _s n 'i s s II * <ο-ρ-ο-ρ-Ι- -φο-γο,γA is a bond,-OC(=O)-*, OH , OH OH , OH , O O , *

S H il XX

4o-p-o-p^

OH OH

-OC(=O)N(CH3)CH₂CH2N(CHî)C(=O)-* or OC(=O)N(CH3)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-C& alkyl, and C3-C8 cycloalkyl and the * of A indicates the point of attachment to D,

261 and

L₃ is a spacer moiety.

Embodiment 33. The linker of Embodiment 32, wherein:

Lj is a bridging spacer;

Lp is a bivalent peptide spacer comprising two to four amino acid residues;

G-L2-A is a self-immolative spacer;

R² is a hydrophilic moiety;

L₂ îs a bond, a methylene, a neopentylene or a C2-C₃alkenylene;

O * 00* O , lis , II II . ^w < Il*

-χΟ-Ρ^ lo-p-o-p-l· -ξ-Ο-ΡΌ^

A is a bond, -OC(=O)-*, θΗ , OH , OH , ⁰O * < Il II YA

OH OH

-OC(=O)N(CH₃)CH₂CH2N(CH3)C(=O)-* or OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-C& alkyl, and C3-Cs cycloalkyl and the * of A indicates the point of attachment to D, and

L₃ is a spacer moiety.

Embodiment 34. The linker of Embodiment 32 or 33, wherein:

Li is a bridging spacer;

Lp is a bivalent peptide spacer comprising two to four amino acid residues;

/L₂—A-|r^G the L₃-R² group is selected from;

γγΑζ A* *** fi x T ^A ,l₂-a4₂ A ^l3 ^R , wherein the * of ^L3~^R indicates the point of attachment to D (e.g., to an N or a O of the Drug moiety), the *** of /L₂-a-|-

- |-G

L₃~R² indicates the point of attachment to Lp;

R² is a hydrophilic moiety;

L2 is a bond, a methylene, a neopentylene or a C2-C₃alkenylene;

262 ο *

A is a bond, -OC(=O)-*, OH

OH OH

Il *

-O-P-_O<vOH

Il II 7

-O-P-O-P—'

OH OH

-OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C3-Cs cycloalkyl and the * of A indicates the point of attachment to D, and

L₃ is a spacer moiety.

Embodiment 35. The linker of any one of Embodiments 32 to 34, wherein:

L! is *-C(=O)(CH₂)mO(CH₂)m-**; *-C(=O)((CH₂)_mO)t(CH₂)_n-**; *-C(=O)(CH₂)_in-**:

*-C(=O)NH((CH₂)mO)_t(CH₂)_n-**;

*-C(=O)O(CH2)mSSC(R³)2(CH2)mC(=O)NR³(CH2)mNR³C(=O)(CH2)m-**;

*-C(=O)O(CH₂)_mC(=O)NH(CH₂)_m-**; *-C(=O)(Œ₂)_mNH(CH₂)_m-**;

*-C(=O)(CH₂)n₁NH(CH2)nC(=O)-**; *-C(=O)(CH₂)_raXi(CH₂)_m-**;

*-C(=O)((CH₂)_mO)_t(CH₂)nXi(CH₂p*; *-C(=O)(CH₂)_mNHC(=O)(CH₂)_n-**;

*-C(=O)((CH₂)_mO)_t(CH₂)_nNHC(=O)(Œ^ *-C(=O)(CH₂)_nJ4HC(=O)(CH₂)_nXi(CH^^ *-C(=O)((CH₂)_mO)_t(CH₂)_nNHC(=O)(CH2)_nXi(CH₂)n-**;

*-C(=O)((CH₂)_mO)_t(CH₂)_nC(=O)NH(CH₂)_l *-C(=O)(CH₂)mC(R³)₂-** or *-C(=O)(CH₂)_mC(=O)NH(CH₂)_m-**, where the * of Li indicates the point of attachment to Lp;

R² is a hydrophilic moiety selected from polyethylene glycol, polyalkylene glycol, a sugar, an oligosaccharide, a polypeptide or C₂-C&alkyl substituted with 1 to 3

O

-^-O-P-OH

OH groups;

each R³ is independently selected from H and Ci-C&alkyl;

263 each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

Lp is a bivalent peptide spacer comprising an amino acid residue selected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, méthionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine;

O * O O * O . H ς , Il II A , Il *

-|-Ο-Ρ-Ο-Ρ-φ -|-O-P-O^y

A is a bond,-OC(=O)-*, OH , OH OH _} OH ,

-OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C3-C₈ cycloalkyl and the * of A indicates the point of attachment to D;

S s *

L₃ is a spacer moiety having the structure ' , where

W is-CH₂O-**, -CH₂N(R^b)C(=O)O-**} -NHC(=O)C(R^b)2NHC(=O)On ?

-NHC(=O)C(R^b)2NH-**, -NHC(=O)C(R^b)2NHC(=O)-**, -CH₂N(XR²)C(=O)O-**, -C(=O)N(X-R²)-**, -CH2N(X-R²)C(=O)-**, -C(=O)NR^b-**, -C(=O)NH-**, -CH2NR^bC(=O)-**, -

CH₂NR^bC(=O)NH-**, -CH2NR^bC(=O)NR^b-**, -NHC(=O)-**, NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, NHS(O)₂-**> -C(=O)-, -C(=O)O-** or

-NH-, wherein each R^b is independently selected from H, Ci-Côalkyl or C₃-C₈cycloalkyl and wherein the ** of W indicates the point of attachment to X;

and

264 the * of Lî indicates the point of attachment to R².

Embodiment 36. The linker of any one of Embodiments 32 to 35, wherein:

Li is *-C(=O)(CH2)inO(CH2)_in-**; *-C(=O)((CH₂)_mO)t(CH₂)_n-**; *-C(=0)(CH₂)m-**;

or *-C(-O)NH((CH₂)_mO)t(CH₂)n-, where the * of Lj indicates the point of attachment to Lp;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is a bivalent peptide spacer selected from ^{0 NH}2 (ValCit),

W (ValLys) and ° ^1H₂ (LeuCit), where the * of Lp indicates the attachment point to Li;

L₃ îs a spacer moiety having the structure

Yw-x-p where

W is -CH2O-**, -CH₂N(R^b)C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -NHC(=O)CH2NH-**, -NHC(=O)CH2NHC(=O)-**, -CH2N(XR²)C(=O)O-**, -C(=O)N(X-R²)-**, -CH2N(X-R²)C(=O)-**₅-C(=O)NR^b-**, -C(=O)NH-**, -CH2NR^bC(=O)-**, CH2NR^bC(=O)NH-**, -CH2NR^bC(=O)NR^b-**, -NHC(=O)-**, NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**,

265

NHS(O)2-**, -C(=O)-, -C(=O)O-** or-NH-, wherein each R^b is independently selected from H, Ci-Côalkyl or C₃-Cscycloalkyl and wherein the ** of W indicates the point of attachment to X;

and the * of L₃ indicates the point of attachment to R²;

R² is a hydrophi lie moiety selected from polyethylene glycol, polyalkylene glycol, a sugar, an oligosaccharide, a polypeptide or C₂-Côalkyl substituted with 1 to 3

O

-l-O-P-OH ' I

OH groups;

and

s ^(t II tA

-|-o-p-o-p^

OH OH

-OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyi, and C3-Cs cycloalkyl and the * of A indicates the point of attachment to D.

Embodiment 37. The linker of any one of Embodiments 32 to 36, wherein:

L₁ is *-C(=O)(CH₂)_mO(CH₂)_m-**; *-C(=O)((CH₂)mO)_t(CH₂)_ll-**; *-C(=O)(CH₂)_m-**; or *-C(=O)NH((CH₂)_mO)t(CH₂)_n-, where the * of Li indicates the point of attachment to Lp;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each t îs independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

266

Lp is a bivalent peptide spacer selected from

O NH₂ (ValCit), where the * of Lp indicates the attachment point to Li and the ** of Lp indicates the attachment point to the -NH- group of G;

r ., . . . _{t +} 4-w-x4*

L₃ ts a spacer moiety having the structure ' , where

Wis-CH₂O-**, -CH2N(Rb)C(=O)O-**, -NHC(=O)CH₂NHC(=O)O-**, -CH₂N(X-R²)C(=O)O-**, -C(=O)N(X-R²)-**, -CH2N(X-R²)C(=O)**, -C(=O)NR^b-**, -C(=0)NH-**, -CH2NR^bC(=O)-**, CH₂NR^bC(=O)NH-**, -CH2NR^bC(=O)NR^b-**, -NHC(=O)-**, NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, NHS(O)₂-**, -C(=O)-, -C(=O)O-** or

-NH-, wherein each R^b is independently selected from H, Ci-Côalkyl or Cs-Cgcycloalkyl and wherein the ** of W indicates the point of attachment to X;

and the * of L₃ indicates the point of attachment to R²;

R² is a hydrophilic moiety selected from polyethylene glycol, polyalkylene glycol, a sugar, an oligosaccharide, a polypeptide or C₂-Côalkyl substituted with l to 3 o

-|-O-p-oh

OH groups;

and

A is a bond,-OC(=O)-*, OH , OH OH _; OH ⁰ O * . n n -|-o-p-o-p^

OH OH , -OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or

267

-OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH₃)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and Cî-Cs cycloalkyl and the * of A indicates the point of attachment to D.

Embodiment 38. The linker of any one of Embodiments 32 to 37, wherein:

Li is *-C(=O)(CH₂)_mO(CH₂)m-**; *-C(=O)((CH₂)mO)_t(CH₂)_n-**; *-C(=O)(CH₂)_m-**; or *-C(=O)NH((CH₂)_mO)t(CH₂)_n-, where the * of Li indicates the point of attachment to Lp;

each ni is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each t is independently selected from l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 i, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 and 30;

* H θ ** ^H O L

Ah

Lp is a bivalent peptide spacer selected from ⁰ (ValCit), where the * of Lp indicates the attachment point to Li;

t ' · _t 1 ₊₁ , ₊ 4-w-xAL₃ is a spacer moiety having the structure < < , where

W is -CH2O-**, -CH2N(Rb)C(=O)O-**, -NHC(=O)CH₂NHC(=O)O-**₅-CH₂N(X-R²)C(=O)O-*% -C(=O)N(X-R²)-**, -C(=O)NR^b-**, C(=O)NH-**, -CH₂NR^bC(=O)-**, -CH2NR^bC(=O)NH-**, CH2NR^bC(=O)NR^b-**, -NHC(=O)-**, -NHC(=O)O-**, or -NHC(=O)NH-**, wherein each R^b is independently selected from H, Ci-Cealkyl or C₃-Cscycloalkyl and wherein the ** of W indicates the point of attachment to X;

X îs a bond, triazolyl or ***-CH₂-triazolyl-*, wherein the *** of X indicates the point of attachment to W and the * of X indicates the point of attachment to R²;

and the * of L₃ indicates the point of attachment to R²;

268

R² îs a hydrophi lie moiety selected from polyethylene glycol, polyalkylene glycol, a sugar, an oligosaccharide, a polypeptide or Cî-Cealkyl substituted with l to 3

O —|-O—P-OH ' I OH groups;

and

A is a bond or -OC(=O)* in which * indicates the attachment point to D.

Embodiment 39. The linker of any one of Embodiments 32 to 38, wherein:

Li is *-C(=O)(CH₂)_mO(CH₂)_m-**; *-C(=O)((CH₂)_mO)_t(CH2)n-**; *-C(=O)(CH₂),n-**;

or *-C(=O)NH((CH₂)_mO)t(CH₂)_n-, where the * of Li indicates the point of attachment to Lp;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, II, 12, 13, 14, 15, 16,

17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

Lp is a bivalent peptide spacer selected from ° ^NH2 (ValCit), where the * of Lp indicates the attachment point to Li;

t ' . n fl _{t t} -hw-X-lLî is a spacer moiety having the structure ^e < , where

Wis—CH₂O-**, -CH2N(Rb)C(=O)O-**, -NHC(=O)CH₂NHC(=O)O-**, -CH₂N(X-R²)C(=O)O-**, or -C(=O)N(X-R²)-**, wherein each R^b is independently selected from H, Ci-Céalkyl or Cj-Cseycloalkyl and wherein the ** of W indicates the point of attachment to X;

X is ***-CH₂-triazolyl-*, wherein the *** of X indicates the point of attachment to W and the * of X indicates the point of attachment to R²;

and the * of Lj indicates the point of attachment to R²;

269

R² îs a hydrophilic moiety selected from polyethylene glycol, polyaikylene glycol, a sugar, an oligosaccharide, a polypeptide or C₂-Côalkyl substituted with 1 to 3

O

-O-P-OH I

OH groups;

and

A is a bond or -OC(=O)* in which * indicates the attachment point to D.

Embodiment 40. The linker of Formula (C’) having the structure having the structure of

Formula (D’),

Formula (D’) wherein

Li is a bridging spacer;

Lp is a bivalent peptide spacer;

R² is a hydrophilic moiety;

O *

-|-o-pY

A îs a bond, -OC(=O)-*, OH n n .

-O-P-O-Prt

OH OH

O

Il *

-o-P-o^y

OH

OH OH

-OC(=O)N(CH₃)CH₂CH2N(CH3)C(=O)-* or

-OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C3-Cs cycloalkyl and the * of A indicates the point of attachment to D, and

L₃ îs a spacer moiety.

Embodiment 41. The linker of Embodiments 40, wherein:

Li is a bridging spacer;

Lp is a bivalent peptide spacer comprising two to four amino acid residues;

R² is a hydrophilic moiety;

270

A is a bond, -OC(=O)-*, OH , OH OH , OH O O , * , n η

-bo-p-o-p-⁷OH OH t

-OC(=C9N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or

-OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH₃)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and Cj-Cs cycloalkyl and the * of A indicates the point of attachment to D, and

La is a spacer moiety,

Embodiment 42. The lînker of Embodiment 40 or 41, wherein;

Li is *-C(=O)(CH₂)_mO(CH₂)_m-**; *-C(=O)((CH₂)·„O)₍(CH₂)_n-**; *-C(=O)(CH₂)_m-**;

*-C(=O)NH((CH₂)_mO)_t(CH₂)_n-**;

*-C(=O)O(CH₂)_mSSC(R³)2(CH2)mC(=O)NR³(CH2)_inNR³C(=O)(CH₂)_m-**;

*-C(=O)O(CH₂)_mC(=O)NH(CH2)m-**;*-C(=O)(CH2)_mNH(CH₂)_m-**;

*-C(=O)(CH2)_mNH(CH₂)nC(=O)-**; *-C(=O)(CH₂)_mXi(CH₂)m-**;

*-C(=O)((CH₂)_roO)_t(CH₂)_nXi(CH2)n-**;*-C(=O)(CH₂)mNHC(=O)(CH₂)_n-**;

*-C(=O)((CH₂)_mO)_l(CH₂)_nNHC(=O)(CH₂)_n-**;

*-C(=O)(CH₂)_mNHC(=O)(CH₂)_nXi(CH₂)_n-**;

*-C(=O)((CH2)mO)t(CH2)nNHC(=O)(CH2)nXi(CH₂)_n-**;

*-C(=O)((CH₂)_mO)_t(CH₂)_nC(=O)NH(CH₂)_m-**; *-C(=O)(CH₂)_mC(R³)₂-** or *-C(=O)(CH₂)_mC(=O)NH(CH₂)m-**, where the * of Li indicates the point of attachment to Lp;

O

-l-O-P-OH ^? I

OH groups;

each R³ is independently selected from H and Ci-Côalkyl;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

271 each n is independently selected from l, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each t is independently selected from l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, I7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

O * O O * O . H s i II II . ^w , Il *

-|-Ο-Ρ-γ -|-O-P—Ο-Ρ-Χ 4θ-Ρ-Ο<φ

A is a bond,-OC(=O)-*, OH , OH OH , OH

Il II

-o-p-o-p I I

OH OH

-OC(=O)N(CH3)CH₂CH₂N(CH3)C(=O)-* or

-OC(=O)N(CH3)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C3-C8 cycloalkyl and the * of A indicates the point of attachment to D;

L₃ is a spacer moiety having the structure ⁵ , where

W is -CH₂O-**, -CH2N(Rb)C(=O)O-**, -NHC(=O)CH₂NHC(=O)O-**, -CH2N(X-R²)C(=O)O·**, -C(=O)N(X-R²)-**, -CH2N(X-R²)C(=O)**, -C(=O)NR^b-**}-C(=O)NH-**,-CH₂NR^bC(=O)-**, CH₂NR^bC(=O)NH-**, -CH2NR^bC(=O)NR^b-**, -NHC(=O)-**, NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, NHS(O)₂-**, -C(=O)-, -C(=O)O-** or

-NH-, wherein each R^b îs independently selected from H, Ci-C/alkyl or Cj-Cscycloalkyl and wherein the ** of W indicates the point of attachment to X;

and the * of L3 indicates the point of attachment to R².

Embodiment 43. The linker of any one of Embodiments 40 to 42, wherein;

272

Li is *-C(=O)(CH₂)_mO(CH₂)_m-**; *-C(=O)((CH2)_inO)_t(CH₂)_n-**; *-C(=O)(CH₂)_m-** or *-C(=O)NH((CH₂)_mO)_t(CH2)_n-, where the * of Li indicates the point of attachment to Lp;

each m is independently selected from l, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each n is independently selected from l, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

Lp is a bivalent peptide spacer selected from θ HH₂ (ValCit),

Ά. _μ o **

H Ο γ

Ah (ValLys) and where the * of Lp indicates the attachment point to Li and the ** of Lp indicates the attachment point to theNH- group of G;

L₃ is a spacer moiety having the structure * ⁸ , where

Wis-CH₂O-**, -CH2N(Rb)C(=O)O-**, -NHC(=O)CH₂NHC(=O)O-**, -CH₂N(X-R²)C(=O)O-**, -C(=O)N(X-R²)-**, -CH2N(X-R²)C(=O)**, -C(=O)NR^b-**, -C(=O)NH-**, -CH2NR^bC(=O)-**, CH2NR^bC(=O)NH-**, -CH2NR^bC(=O)NR^b-**, -NHC(=O)-**, NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)2NH-**, NHS(O)₂-**, -C(=O)-, -C(=O)O-** or

-NH-, wherein each R^b îs independently selected from H, Ci-Cealkyl

273 or Cs-Cscycloalkyl and wherein the ** of W indicates the point of attachment to X;

X is a bond, triazolyl or ***-CH₂-triazolyl-*, wherein the of X indicates the point of attachment to W and the * of X indicates the point of attachment to R²;

and the * of L₃ indicates the point of attachment to R²;

R² îs a hydrophilic moiety selected from polyethylene glycol, polyalkylene glycol, a sugar, an oligosaccharide, a polypeptide or C₂-C&alkyl substituted with l to 3

O

-|-O-P-OH

OH groups;

and

OH OH , -OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or -OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH₃)C(=O)-*, wherein each R^a îs independently selected from H, Cj-Cô alkyl, and C₃-Cg cycloalkyl and the * of A indicates the point of attachment to D.

Embodiment 44. The linker of any one of Embodiments 40 to 43, wherein:

Li is *-C(=O)(CH₂)inO(CH₂)in-**; *-C(=O)((CH₂)_mO)t(CH₂)_n-**; *-C(=O)(CH₂)_m-**;

or *-C(=O)NH((CH₂)mO)t(CH₂)_n-, where the * of Li indicates the point of attachment to Lp;

each m îs independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each n îs independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16,

17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

274

X A h 9 ** WrV ^H Ο γ

X

Lp is a bivalent peptide spacer selected from ° (ValCît), where the * of Lp indicates the attachment point to Li and the ** of Lp indicates the attachment point to the -NH- group of G;

t ♦ _l m ♦ ♦ 4-W-X-lLj is a spacer moiety having the structure ^{< ΐ} , where

Wis-CH₂O-**, -CH₂N(Rb)C(=O)O-**, -NHC(=O)CH₂NHC(=O)O-**, -CH₂N(X-R²)C(=O)O-**, -C(=O)N(X-R²)-**, -CH₂N(X-R²)C(=O)**, -C(=O)NR^b-**, -C(=O)NH-**, -CH2NR^bC(=O)-**, CH2NR^bC(=O)NH-**, -CH2NR^bC(=O)NR^b-**, -NHC(=O)-**, NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)₂NH-**, NHS(O)₂-**, -C(=O)-, -C(=O)O-** or

-NH-, wherein each R^b is independently selected from H, Ci-Q.alkyl or C₃-Cscyclo alkyl and wherein the ** of W indicates the point of attachment to X;

and the * of L₃ indicates the point of attachment to R²;

R² îs a hydrophilic moiety selected from polyethyiene glycoi, polyalkylene glycol, a sugar, an oligosaccharide, a polypeptide or C₂-Ci>alkyl substituted with 1 to 3 o

-|-O-P-OH

OH groups;

and

A is a bond, -OC(=O)-*, OH , OH OH , OH

275

-OC(=O)N(CH₃)CH₂CH₂N(CHî)C(=O)-* or -OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C3-C₈ cycloalkyl and the * of A indicates the point of attachment to D.

Embodiment 45. The linker of any one of Embodiments 40 to 44, wherein:

Li is *-C(=O)(CH₂)mO(CH2)_m-**; *-C(=O)((CH2)_mO)_l(CH₂)_n-**; *-C(=O)(CH₂)_m-**; or *-C(=O)NH((CH₂)mO)t(CH2)n-, where the * of Li indicates the point of attachment to Lp;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each t is independently selected from 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

V H O ** ^H Ο x ^lNH

Lp is a bivalent peptide spacer selected from ° ΝΗ₂ (ValCit), where the * of Lp indicates the attachment point to Li and the ** of Lp indicates the attachment point to the -NH- group of G;

t * i, λ _t f 4-w-x-p

L₃ is a spacer moiety having the structure < < , where

W is -CH2O-**, -CH2N(Rb)C(=O)O-**, -NHC(=O)CH₂NHC(=O)O-**, -CH₂N(X-R²)C(=O)O-**, -C(=O)N(X-R²)-**, -C(=O)NR^b-**, C(=O)NH-**, -CH₂NR^bC(=O)-**, -CH2NR^bC(=O)NH-**, CH2NR^bC(=O)NR^b-**,

-NHC(=O)-**, -NHC(=O)O-**, or -NHC(=O)NH-**, wherein each R^b is independently selected from H, Ci-Côalkyl or C₃-C₈cycloalkyl and wherein the ** of W indicates the point of attachment to X;

and

276 the * of La indicates the point of attachment to R²;

R² is a hydrophilic moiety selected from polyethylene glycol, polyalkylene glycol, a sugar, an oligosaccharide, a polypeptide or C₂-C&alkyl substituted with l to 3 o

4-o-p-oh

OH groups;

and

A is a bond or -OC(=O)* in which * indicates the attachment point to D.

Embodiment 46. The linker of any one of Embodiments 40 to 45, wherein:

L| is *-C(=O)(CH₂)mO(CH2)m-**; ^C^OXtCfü^OMCHQn-**; *-C(=O)(CH₂)_m-**; or *-C(=O)NH((CH₂)_1OO)t(CH2)_n-, where the * of Lj indicates the point of attachment to Lp;

each m is independently selected from 1,2, 3, 4, 5, 6, 7, 8, 9 and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each t is independently selected from i, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

sW ^H O Y jh

Lp îs a bivalent peptide spacer selected from ^{0 NH}2 (ValCit), where the * of Lp indicates the attachment point to Li and the ** of Lp indicates the attachment point to the -NH- group of G;

t · ₊ i. n t t -Xw-x4Li is a spacer moiety having the structure ⁱ s , where

Wis-CH₂O-**, -CH₂N(Rb)C(=O)O-**, -NHC(=O)CH₂NHC(=O)O-**, -CH₂N(X-R²)C(=O)O-**, or -C(=O)N(X-R²)-**, wherein each R^b is independently selected from H, Ci-Côalkyl or Cî-Cgcycloalkyi and wherein the ** of W indicates the point of attachment to X;

and the * of L₃ indicates the point of attachment to R²;

277

R² is a hydrophilic moiety selected from polyethylene glycol, polyalkylene glycol, a sugar, an oligosaccharide, a polypeptide or C₂-C6aikyl substituted with 1 to 3 o

-|-O-P-OH

OH groups;

and

A is a bond or -OC(=O)* in which * indicates the attachment point to D.

Embodiment 47. The linker of any one of Embodiments 32 to 46, having the structure:

R is H, -CH₃ or -CH₂CH₂C(=O)OH.

Embodiment 48. The linker of any one of Embodiments 32 to 46, having the structure:

R is H, -CH₃ or -CH₂CH₂C(=O)OH.

Embodiment 49. The linker of any one of Embodiments 32 to 46, having the structure:

R is H, -CH₃ or -CH₂CH2C(=O)OH.

Embodiment 50. The linker of any one of Embodiments 32 to 46, having the structure:

278

each R is independently selected from H, -CH3 or-CH₂CH2C(=O)OH.

Embodiment 51. The linker of any one of Embodiments 32 to 46, having the structure:

N, ,N N

Y .-jA , where each R is independently selected from H, -CH3 or -CH₂CH₂C(=O)OH.

The linker of any one of Embodiments 32 to 46, having the structure:

Embodiment 52.

'R t=4 to 25 , where

Xa is -CH2-, -OCH2-, -NHCH2- or -NRCH₂- and each R independently is H, -CH3 or CH₂CH₂C(=O)OH.

Embodiment 53. The linker of any one of Embodiments 32 to 46, having the structure:

R is H, -CH3 or -CH₂CH₂C(=O)OH.

, where

279

Embodiment 54. The linker of any one of Embodiments 32 to 46, having the structure:

Xb îs -CH₂-, -OCH2-, -NHCH2- or -NRCH₂- and each R independently is H, -CH3 or CH₂CH₂C(=O)OH.

Embodiment 55. The linker of any one of Embodiments 32 to 46, having the structure:

Embodiment 56. The linker of any one of Embodiments 32 to 46, having the structure:

Embodiment 57. The linker of any one of Embodiments 32 to 46, having the structure:

Embodiment 58. The linker of any one of Embodiments 32 to 46, having the structure:

Embodiment 59. The linker of any one of Embodiments 32 to 46, having the structure:

280

For illustrative purposes, the general reaction scheines depicted herein provide potential routes for synthesizing the compounds of the présent invention as well as key intermediates. For 5 a more detailed description of the individual reaction steps, see the Examples section below, Although spécifie starting materials and reagents are depicted in the schemes and discussed below, other starting materials and reagents can be easily substituted to provide a variety of dérivatives and/or reaction conditions. In addition, many of the compounds prepared by the methods described below can be further modified in light of this disclosure using conventional chemistry well known to those skilled in the art.

B y way of example, a general synthesis for compounds of Formula (B’) is shown below in Scheme 1.

Scheme 1

n-n

281

Antibody Drug Conjugates of the Invention

The présent invention provides Antibody Drug Conjugates, also referred to herein as immunoconjugates, which comprise linkers which comprise one or more hydrophilic moieties.

The Antibody Drug Conjugates of the invention hâve the structure of Fonnula (E’):

Fonnula (E’) wherein:

Ab is an antibody or fragment thereof;

R^IOÜ is a coupling group;

Li is a bridging spacer;

Lp is a bivalent peptide spacer;

G-L2-A is a self-immolative spacer;

R² is a hydrophilic moiety;

L2 is a bond, a methylene, a neopentylene or a C2-C₃alkenylene;

OH OH

-OC(=O)N(CH₃)CH2CH2N(CH₃)C(=O)-* or

-OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C3-Cs cycloalkyl and the * of A indicates the point of attachment to D;

L₃ is a spacer moiety;

D is a Drug moiety as defined herein, e.g., a MC1-1 inhibitor, and may comprise an N or an O, wherein D can be connected to A via a direct bond from A to the N or the O of the Drug moiety, and y is 1, 2, 3, 4, 5, 6, 7, S, 9, 10, 11, 12, 13, 14, 15 or 16.

282

Certain aspects and examples of the Antibody Drug Conjugates of the invention are provided in the following listing of enumerated embodiments. It will be recognized that features specified in each embodiment may be combined with other specified features to provide further embodiments of the présent invention.

Embodiment 60. The immunoconjugate of Formula (E’) wherein:

Ab îs an antibody or fragment thereof;

R^,t)0 is a coupling group;

Li is a bridging spacer;

Lp is a bivalent peptide spacer comprising two to four amino acid residues;

G-L?-A îs a self-immolative spacer;

R² is a hydrophilic moiety;

L₂ is a bond, a methylene, a neopentylene or a C₂-C₃alkenylene;

O * 4-0-p-H A îs a bond, -OC(=O)-*, DH

OH OH

O n *

OH n n

Ό-Ρ-Ο-Ρ I I

OH OH , -OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or

-OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C3-C₈ cycloalkyl and the * of A indicates the point of attachment to D;

L₃ is a spacer moiety;

D is a Drug moiety as defined herein wherein D is connected to A via a direct bond from A to D (e.g., an N or O of the Drug moiety), and y is 1,2, 3,4, 5,6,7,8,9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 61. The immunoconjugate of Formula (E’) or Embodiment 60, wherein: Ab is an antibody or fragment thereof;

R¹⁰⁰ is a coupling group;

Li is a bridging spacer;

Lp is a bivalent peptide spacer comprising two to four amino acid residues; rt₂—A-;>the ^l3~R² group is selected from:

283

wherein the * of ^L3^_R indicates the point of attachment to D (e.g., to an N or a O of the Drug moiety), the *** of , /R^-A-|“ ’Κ a ^l3 ^r indicates the point of attachment to Lp;

R² îs a hydrophilic moiety;

L₂ is a bond, a methylene, a neopentylene or a C₂-C₃ alkenylene;

A is a bond,-OC(=O)-*, OH , OH OH _} OH ⁰ O . il ii -i-o-p-o-p^

OH OH

-OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or

-OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH₃)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cs alkyl, and C₃-C₈ cycloalkyl and the * of A indicates the point of attachment to D;

L₃ is a spacer moiety;

D îs a Drug moiety as defined herein and comprising an N or an O, wherein D is connected to A via a direct bond from A to the N or the O of the Drug moiety, and yis l,2, 3,4, 5,6, 7, 8,9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 62. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to having the structure of Fonnula (F’),

Formula (F’) wherein:

Ab is an antibody or fragment thereof; R^IOÛ is a coupling group;

284

Li is a bridging spacer;

Lp is a bivalent peptide spacer comprisîng two to four amino acid residues;

R² is a hydrophilic moiety;

O

A is a bond,-OC(=O)-*, θΗ , OH OH _} OH ,

O O _z * , ¹¹ n YA

-Lo-p-o-pOH OH _f -OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or -OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C3-C« cycloalkyl and the * of A indicates the point of attachment to D;

L₃ is a spacer moiety;

D is a Drug moiety as defined herein and comprisîng an N or an O, wherein D is connected to A via a direct bond from A to the N or the O of the Drug moiety, and yis 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 63. The immunoconjugate of Formula (D’) or any one of Embodiments 60 to 62, wherein:

Ab is an antibody or fragment thereof;

***, -S(=O)₂CH₂CH₂-***, -(CH₂)₂S(=O)₂CH₂CH2-***, -NHS(=O)₂CH₂CH₂.***,

-NHC(=O)CH₂CH₂-***, -ch₂nhch₂ch₂-***, -NHCH₂CH₂-***,

285

attachment to Ab;

286

Li is *-C(=O)(CH2)_mO(CH₂)_m-**; *-C(=O)((CH₂)_raO)_t(CH₂)_n-**; *-C(=O)(CH₂)_m-** *-C(=O)NH((CH₂)mO)_t(CH₂)_n-* * ;

*-C(=O)O(CH₂)_mSSC(R³)2(CH2)inC(=O)NR³(CH2)I11NR³C(=O)(CH₂)_m-**;

*-C(=O)O(CH₂)_mC(=O)NH(CH₂)_m-**; *-C(=O)(CH₂)_mNH(CH₂)_m-**;

*-C(=O)(CH2)_mNH(CH₂)nC(=O)-**;*-C(=O)(CH2)_inXi(CH2)_m-**;

*-C(=O)((CH2)mO)t(CH₂)nXi(CH₂)_n-**; *-C(=O)(CH₂)_mNHC(=O)(CH₂)_n-**;

*-C(=O)((CH₂)_mO)t(CH₂)_nNHC(=O)(CH2)_n-**;

*-C(=O)(CH₂)_mNHC(=O)(CH₂)_nXi(Œ₂)_n-** ;

*-C(=O)((CH2)mO)_t(CH₂)_nNHC(=O)(CH₂)^ *-C(=O)((CH2)mO)t(CH₂)nC(=O)NH(CH₂)m-**; *-C(=O)(CH₂)_mC(R³)₂-** or *-C(AJKCH2)_mC(AJ)NH(CH₂)in-**, where the * of Li indicates the point of attachment to Lp, and the ** of Li indicates the point of attachment to R¹⁰⁰;

R² is a hydrophihc moiety selected from polyethylene glycol, polyalkylene glycol, a sugar, an oligosaccharide, a polypeptide or Cz-Côalkyl substituted with 1 to 3

O

-l-O-P-OH ^? I OH groups;

each R³ is independently selected from H and Ci-C&alkyl;

R⁴ is 2-pyridyl or4-pyridyl;

each R⁵ is independently selected from H, Ci-C&alkyl, F, Cl, and -OFI;

each R⁶ is independently selected from H, Ci-Csalkyl, F, Cl, -NH₂, -OCH₃, -OCH₂CH₃, -N(CH₃)₂, -CN, -NO2 and -OH;

each R⁷ is independently selected from H, Ci-6alkyl, fluoro, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, Cj^alkoxy substituted with -C(=O)OH and Ci-4alkyl substituted with -C(=O)OH;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

287

Lp is a bivalent peptide spacer comprising an amino acid residue selected from valine, citrulline, lysine, isoleucine, phenylalanine, méthionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine;

< H II 7/

Z-o-p-o-p—¹

OH OH , -OC(=O)N(CH3)CH₂CH₂N(CH3)C(=O)-* or -OC(=O)N(CH3)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-C& alkyl, and C3-C8 cycloalkyl and the * of A indicates the point of attachment to D;

L3 îs a spacer moiety having the structure ⁵ , where

Wis-CH₂O-**, -CH₂N(Rb)C(=O)O-**, -NHC(=O)C(R^b)₂NHC(=O)O?

-NHC(=O)C(R^b)2NH-**, -NHC(=O)C(R^b)2NHC(=O)-**, -CH₂N(XR²)C(=O)O-**, -C(=O)N(X-R²)-**, -CH₂N(X-R²)C(=O)-**, C(=O)NR^b-**, -C(=O)NH-**, -CH2NR^bC(=O)-**, CH2NR^bC(=O)NH-**, -CH2NR^bC(=O)NR^b-**, -NHC(=O)-**, NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)₂NH-**, NHS(O)₂-**, -C(=O)-, -C(=O)O-** or—NH-, wherein each R^b îs independently selected from H, Ci-Cealkyl or Cî-Cgcycloalkyl and wherein the ** of W indicates the point of attachment to X;

and the * of L3 indicates the point of attachment to R²;

D is a Drug moiety as defined herein and comprising an N or an O, wherein D is connected to A via a direct bond from A to the N or the O of the Drug moiety, and y is 1,2,3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

288

Embodiment 64. The immunoconjugate of Formula (D’) or any one of Embodiments 60 to

63, wherein:

Ab is an antibody or fragment thereof;

point of attachment to Ab;

Li is *-C(=O)(CH₂)_mO(CH₂)_m-**; *-C(=O)((CH₂)_mO)_t(CH₂)_n-**; *-C(=O)(CH₂)_m-**;

or *-C(=O)NH((CH₂)inO)t(CH₂)_n-, where the * of Li indicates the point of attachment to Lp, and the ** of Li indicates the point of attachment to R^l0°;

each m is independently selected from l, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

(ValLys) and ⁰ (LeuCit), where the * of Lp indicates the

289 attachment point to Li and the ** of Lp indicates the attachment point to the NH- group of G;

t · _{f u t} h -i-w-x-iL₃ is a spacer moiety havmg the structure ' ⁸ , where

Wis-CH₂O-**, -CH₂N(Rb)C(=O)O-**, -NHC(=O)CH₂NHC(=O)O-**, -NHC(=O)CH₂NH-**, -NHC(=O)CH₂NHC(=O)-**, -CH₂N(XR²)C(=O)O-**, -C(=O)N(X-R²)-**, -CH₂N(X-R²)C(=O)-**, C(=O)NR^b-**,

-C(=O)NH-*% -CH₂NR^bC(=O)-**, -CH2NR^bC(=0)NH-**, CH2NR^bC(=O)NR^b-**, -NHC(=O)-**, -NHC(=O)O-**, NHC(=O)NH-**,

-OC(=O)NH-**, -S(O)₂NH-**, -NHS(O)₂-**, -C(=O)-, -C(=O)O-** or

-NH-, wherein each R^b is independently selected from H, Ci-Cgalkyl or C₃-C₈cycloalkyl and wherein the ** of W indicates the point of attachment to X;

and the * of L₃ indicates the point of attachment to R²;

R² is a hydrophilic moiety selected from polyethylene glycol, polyalkylene glycol, a sugar, an oligosaccharide, a polypeptide or C₂-Côalkyl substituted with 1 to 3

O

-l-O-P-OH

OH groups;

A is a bond, -OC(=O)-*, OH , OH OH , OH , , 9 9 a?

-Lo-p-o-p--⁷

OH OH *

-OC(=O)N(CH3)CH₂CH₂N(CH₃)C(=O)-* or

-OC(=O)N(CH₃)C(R^a)2C(R^a)₂N(CH₃)C(=O)-*, wherein each R^a is independently

290 selected from H, Ci-Cô alkyl, and Cj-Cs cycloalkyl and the * of A indicates the point of attachment to D;

D is a Drug moiety as defined herein and comprising an N or an O, wherein D is connected to A via a direct bond from A to the N or the O of the Drug moiety, and yis l, 2, 3,4, 5,6, 7, 8,9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 65. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to 64, wherein:

Ab is an antibody or fragment thereof; o

R^IOü is O , where the *** of R¹⁰⁰ indicates the point of attachment to Ab;

Lf is *-C(=O)(CH₂)_mO(CH₂)_n,-**; *^-C(⁼O)((CH2)mO)t(CH2)n-**; *-C(=O)(CH₂)_ni-**; or *-C(=O)NH((CH₂)_mO)_l(CH₂)n-, where the * of Li indicates the point of attachment ίο Lp, and the ** of Li indicates the point of attachment to R¹⁰⁰;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

* 'Y' LJ O SL· *

H O < ^kNH

Lp is a bivalent peptide spacer selected from (ValCit), where the * of Lp indicates the attachment point to Li and the ** of Lp indicates the attachment point to the -NH- group of G;

Lj is a spacer moiety having the structure $ , where

W is -CH2O-**, -CH₂N(Rb)C(=O)O-**, -NHC(=O)CH₂NHC(=O)O-**, -CH₂N(X-R²)C(=O)O-**, -C(=O)N(X-R²)-**, -CH₂N(X-R²)C(=O)**, -C(=O)NR^b-**, -C(=O)NH-**, -CH2NR^bC(=O)-**, CH2NR^bC(=O)NH-**, -CH₂NR^bC(=O)NR^b-**, -NHC(=O)-**, 291

NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)₂NH-**, NHS(O)2-**, -C(=O)-, -C(=O)O-** or-NH-, wherein each R^b is independently selected from H, Ci-C^alkyl or Ci-Cscycloalkyl and wherein the ** of W indicates the point of attachment to X;

and the * of L₃ indicates the point of attachment to R²;

R² is a hydrophilic moiety selected from polyethylene glycol, polyalkylene glycol, a sugar, an oligosaccharide, a polypeptide or C₂-Céalkyl substituted with 1 to 3 A is

O * Û

Ils s n* p-b -|-O-P-O^y

OHOH a bond, -OC(=O)-*, OH , OH ⁰ O * s ¹¹ II X

-Ιό-ρ-0-ρ—'

OH OH »

-OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or

D is a Drug moiety as defined herein and comprising an N or an O, wherein D is connected to A via a direct bond from A to the N or the O of the Drug moiety, and y is 1, 2,3,4,5,6, 7, 8,9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 66. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to

65, wherein:

Ab is an antibody or fragment thereof;

where the *** of Rⁱ⁰° indicates the point of attachment to Ab;

Li is *-C(=O)(CH₂)_mO(CH₂)_m-**; *-C(=O)((CH₂)_mO)_t(CH₂)_n-**; *-C(=O)(CH₂)_in-**;

or *-C(=O)NH((CH₂)_mO)t(CH₂)_n-, where the * of Li indicates the point of attachment to Lp and the ** of Li indicates the point of attachment to R¹⁰⁰;

292 each m îs independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each t is independently selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 and 30;

V^H ? ** ^H o k ^NH

Lp is a bivalent peptide spacer selected from ° ^NH2 (ValCit), where the * of Lp indicates the attachment point to Li and the ** of Lp indicates the attachment point to the —NH- group of G;

r · _t 1 · m . _t 4-W-X-P

L₃ is a spacer moiety having the structure ' , where

Wis-CH₂O-**, -CH₂N(Rb)C(=O)O-**, -NHC(=O)CH₂NHC(=O)O-**, -CH₂N(X-R²)C(=O)O-**, -C(=O)N(X-R²)-**, -C(=O)NR^b-**, -C(=O)NH-**, -CH2NR^bC(=O)-**, -CH2NR^bC(=O)NH-**, -CH₂NR^bC(=O)NR^b-**, -NHC(=O)-**, -NHC(=O)O-**, orNHC(=O)NH-**, wherein each R^b is independently selected from H, Ci-C(,alkyl or Cs-Cscycloalkyl and wherein the ** of W indicates the point of attachment to X;

and the * of L₃ indicates the point of attachment to R²;

R² îs a hydrophilic moiety selected from polyethylene glycol, polyalkylene glycol, a sugar, an oligosaccharide, a polypeptide or C₂-C6alkyl substituted with 1 to 3

O

-l-O-P-OH ' i OH groups;

A is a bond or -OC(=O)* in which * indicates the attachment point to D;

D îs a Drug moiety as defined herein and comprising an N or an O, wherein D is connected to A via a direct bond from A to the N or the O of the Drug moiety, and

293 yis 1,2, 3,4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 67. The immunoconjugale of Formula (E’) or any one of Embodiments 60 to

66, wherein:

Ab is an antibody or fragment thereof;

R¹⁰⁰ is O , where the *** of R¹⁰⁰ indicates the point of attachment to Ab;

Li is *-C(=O)(CH₂)mO(CH₂)_m-**; *-C(=O)((CH₂)_mO)i(CH₂)n-**; *-C(=O)(CH₂)_m-**;

or *-C(=O)NH((CH₂)_niO)Î(CH₂)_n-, where the * of Li indicates the point of attachment to Lp and the ** of Li indicates the point of attachment to R¹⁰⁰;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

Y H O ** WY ^H O V ^lnh

L₃ is a spacer moiety having the structure ' ? , where

Wis-CH₂O-**, -CH₂N(Rb)C(=O)O-**, -NHC(=O)CH₂NHC(=O)O-**, -CH₂N(X-R²)C(=O)O-**, or -C(=O)N(X-R²)-**, wherein each R^b is independently selected from H, Ci-Côalkyl or Cj-Cscycloalkyl and wherein the ** of W indicates the point of attachment to X;

and the * of L₃ indicates the point of attachment to R²;

294

O

-|-O-P-OH

OH groups;

A is a bond or -OC(=O)* in which * indicates the attachment point to D;

D îs a Drug moiety as defined herein and comprising an N or an O, wherein D is connected to A via a direct bond from A to the N or the O of the Drug moiety, and yis l,2, 3,4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 68. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to 63, wherein

0 0 +φ₇ +.’1 R ⁰⁰ is 0 , 0 s H *** ( J ^NAT' r oj X^s *** OH ? ***,-S(=O)₂CH₂CH₂- 15 -NHCÎ=O)CH₂CH₂-« s I 5 R⁶ i A R 7. Y / YÎAiA R⁶ X

s A? X ' A _} ° OH ^HY, ° OH k AWsW l r° [] 1 0 *** ΑΛ , H , -s-, -C(=O)-, -ON=***, -NHC(=O)CH₂- ***, -(CH₂)₂S(=O)₂CH₂CH₂-***, -NHS(=O)2CH2CH2.***, *** ★ Γ N J N A <^N X-CH2NHCH2CH3-***, -NHCH2CH2-***, , m As-R⁶ AA J _{q 2}nAA ^R 5 A X I ) vn^/v”‘ Aa, a Y ,Ν χΑ Ο ή An , _Re R⁶ A AY^R6 î Λ Λαυ _t xⁿX>⁷a aj U- YYa ma A d * Re-AA A Ü-“ _, β ΓΧ jn. *** 'r S r6 , Λ 0 '

295

where the *** of R^l0° indicates the point of

Embodiment 69. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to

63, wherein

attachment to Ab.

Embodiment 70. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to

63, wherein

to Ab.

Embodiment 71. The immunoconjugate of Fonnula (E’j or any one of Embodiments 60 to having the structure:

296

R is H, -CH₃ or-CH₂CH₂C(=O)OH and y is 1, 2, 3, 4, 5, 6, 7, S, 9, 10, 11, 12, 13, 14, 15 or

16,

Embodiment 72. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to having the structure:

Ris H, -CH₃ or-CH₂CH₂C(=O)OH and y is 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15or 16.

Embodiment 73. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to having the structure:

Ris H, -CH₃ or-CH₂CH₂C(=O)OH and y is 1,2,3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

297

Embodiment 74. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70 having the structure:

each R is independently selected from H, -CH₃ or -CHsCHzCAOjOH and y is 1,2,3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 75. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70 having the structure:

each R is independently selected from H, -CH₃ or -CHiCFbCAOjOH and y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 76. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70 having the structure:

298

Xa is -CH₂-, -OCH₂-, -NHCH?- or -NRCH₂- and each R is independently H, -CH₃ or -CH₂CH₂C(=O)OH and y is l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 77. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to 5 70 having the structure:

^k / V, where

Ris H,-CH₃ or-CH₂CH₂C(=O)OH and y is 1,2, 3,4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 78. The immunoconjugate of Fonnula (E’) or any one of Embodiments 60 to

70 having the structure:

Xb is -CH₂-, -OCH₂-, -NHCH₂- or -NRCH₂- and each R independently is H, -CH₃ or — CH₂CH₂C(=O)OH andyis 1,2, 3,4, 5, 6, 7,8,9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 79. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to 15 70 having the structure:

299

2, 3,4, 5,6, 7, 8, 9, ΙΟ, 11, 12, 13, 14, I5or 16.

Embodiment 80. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to having the

l, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 81. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70 having the structure:

8, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 82. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70 having the structure:

300

4, 5, 6, 7, S, 9, 10, 11, 12, 13, 14, 15 or 16.

Embodiment 83. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70 having the structure:

10, 11, 12, 13, 14, 15 or 16.

Certain aspects and examples of the Linker-Drug groups, the Linkers and the Antibody Drug Conjugales of the invention are provided in the following listing of addîtional enumerated embodiments. It will be recognized that features specifîed in each embodiment may be combined with other specifîed features to provide further embodiments of the présent invention.

Embodiment 84. The compound of Formula (A’) or any one of Embodiments 1 to 2, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 39, and the immunoconjugate of Formula (E’) or any one of

Embodiments 60 to 61, wherein:

301

G is , where the * of G indicates the point of attachment to L₂, and the ** of G indicates the point of attachment to L₃ and the *** of G indicates the point of attachment to Lp.

Embodiment 85. The compound of Formula (A’) or any one of Embodiments l to 2, or phannaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 39, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 61, wherein:

i *

Embodiment 86. The compound of Formula (A’) or any one of Embodiments l to 17, or phannaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, wherein:

Li is *-C(=O)(CH₂)_inO(CH₂)i₁₁-^ï:*; *-C(=O)((CH₂)_mO)_t(CH₂)n-**; *-C(=O)(CH₂)_m-**;

*-C(=O)NH((CH₂)mO)_t(CH₂)_n-**;

*-C(=O)O(CH₂)_lliSSC(R³)2(CH2)inC(=O)NR^î(CH2)_mNR³C(=O)(CH₂)_m-**;

*-C(=O)O(CH₂)_mC(=O)NH(CH₂)m-**; <C(=O)(CH₂)_mNH(CH₂)m-**;

*-C(=O)(CH₂)_mNH(CH₂)_nC(=O)-**; *-C(=O)(CH₂)_mX_l(CH₂)_m-**;

*-C(=O)((CH₂)_mO)_l(CH2)nXi(CH₂)_n-**;*-C(=O)(CH₂)_mNHC(=O)(CH₂)_n-**;

*-C(=O)((CH2)mO)_t(CH2)nNHC(=O)(CH₂)_n-**; *C(=O)(CH₂)mNHC(=O)(CH₂)„Xi(CH₂)_n-**; *C(=O)((CH₂)mO)i(CH₂)nNHC(=O)(CH₂)nXi(CH₂)n-**;

*-C(=O)((CH₂)_mO)i(CH₂)_nC(=O)NH(CH₂)_m-**; *-C(=O)(CH₂)mC(R³)2-** or *-C(=O)(CH₂)niC(=O)NH(CH₂)in-**, where the * of Li indicates the point of attachment to Lp, and the ** of Li indicates the point of attachment to R¹ if présent or the ** of Li indicates the point of attachment to R¹⁰⁰ if présent.

302

Embodiment 87. The compound of Fonnula (A’) or any one of Embodiments I to I7,or pharmaceutically acceptable sait thereof, the linker of Fonnula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Fonnula (E’) or any one of Embodiments 60 to 70, wherein:

Li is *-C(=O)(CH2)_mO(CH₂)_ra-**; *-C(=O)((CH₂)_raO)_t(CH₂)_n-**; *-C(=O)(CH₂)_m-**;

*-C(=O)NH((CH₂)_mO)_l(CH2)_n-**; *-C(=O)(CH₂)_mNH(CH2)_m-**:

*-C(=O)(CH₂)mNH(CH₂)nC(=O)-**; *-C(=O)(CH₂)nJG-IC(=O)(CH2)n-**;

*-C(=O)((CH2)_mO)_t(CH₂)_nNHC(=O)(CH2)n-**; *C(=OX(CH₂)_mO)t(CH₂)nC(=O)NH(CH₂)_m-**; ^CYOXCFhYQRV** or *C(=O)(CH₂)_mC(=O)NH(CH₂)m-**, where the * of Li indicates the point of attachaient to Lp, and the ** of Li indicates the point of attachment to R¹ if présent or the ** of Li indicates the point of attachment to R¹⁽¹° if présent.

Embodiment 88. The compound of Fonnula (A’) or any one of Embodiments 1 to 17, or phannaceutically acceptable sait thereof, the linker of Fonnula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, wherein:

Li is *-C(=OXCH₂)_mO(CH₂)m-**; *-C(=O)((CH₂)_mO)_t(CH₂)_n-**; *-C(=OXCH₂)_m-**;

*-C(=O)NH((CH₂)_mO)_t(CH₂)_n-**; *-C(=O)(CH₂)_mNH(CH₂)_m-**; *C(=O)(CH₂)_raNH(CH₂)„C(=O)-**; or *-C(<)XCH₂)_mNHC(=O)(CH₂)_n-**, where the * of Li indicates the point of attachment to Lp, and the ** of Li indicates the point of attachment to R¹ if présent or the ** of Li indicates the point of attachment to R¹⁰⁰ if présent.

Embodiment 89. The compound of Formula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, wherein:

Li is *-C(=O)(CH₂)₁nO(CH2)_m-**; *-C(=O)((CH₂)_mO)_t(CH₂)_n-**; *-C(=OXCH₂)_m-** or *-Ε(=Ο)ΝΗ((ΕΗ₂)ι_υΟ)ι(€Η2)η-**, where the * of Li indicates the point of attachment to Lp, and the ** of Li indicates the point of attachment to R^! if présent or the ** of Li indicates the point of attachment to R¹⁰⁰ if présent.

Embodiment 90. The compound of Fonnula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Fonnula (E’) or any one of

303

Embodiments 60 to 70, wherein Li is *-C(=O)(CH2)_mO(CH2)m-**, where the * of Li indicates the point of attachment to Lp, and the ** of Li indicates the point of attachment to R? if présent or the ** of L] indicates the point of attachment to R^1W if présent.

Embodiment 91. The compound of Formula (A’) or any one of Embodiments I to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, wherein Li is *-C(=O)((CH2)mO)_t(CH2)n-**, where the * of Li indicates the point of attachment to Lp, and the ** of Li indicates the point of attachment to R¹ if présent or the ** of Li indicates the point of attachment to R^lt)0 if présent.

Embodiment 92. The compound of Formula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, wherein Li is *-C(=O)(CH2)_m-**, where the * of Li indicates the point of attachment to Lp, and the ** of Li indicates the point of attachment to R¹ if présent or the ** of Li indicates the point of attachment to R¹⁰⁰ if présent.

Embodiment 93. The compound of Formula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, wherein Li is *-C(=O)NH((CH2)mO)t(CH₂)n-**, where the * of Li indicates the point of attachment to Lp, and the ** of Li indicates the point of attachment to R¹ if présent or the ** of Li indicates the point of attachment to R¹⁰⁰ if présent.

Embodiment 94. The compound of Formula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 93, wherein Lp is an enzymatically cleavable bivalent peptide spacer.

Embodiment 95, The compound of Formula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 94, wherein Lp îs a bivalent peptide spacer comprising an amino acid residue selected from glycine, valine, citrulline, lysine, isoleucine, phenylalanine, méthionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine.

304

Embodiment 96. The compound of Fonnula (A’) or any one of Embodiments l to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 95, wherein Lp is a bivalent peptide spacer comprising two to four amino acid residues.

Embodiment 97. The compound of Formula (A’) or any one of Embodiments 1 to 17, or phannaceutîcally acceptable sait thereof, the linker of Fonnula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Fonnula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 96, wherein Lp is a bivalent peptide spacer comprising two to four amino acid residues each independently selected from glycine, valine, cîtrulline, lysine, isoleucine, phenylalanine, méthionine, asparagine, proline, alanine, leucine, tryptophan, and tyrosine.

Embodiment 98. The compound of Fonnula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Fonnula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 97, wherein:

Ά H O **

H O < Ah A

Lp is a bivalent peptide spacer selected from ^{0 NH}2 (ValCit),

O NH₂(LeuCit), where the * of Lp indicates the attachment point to Li and the ** of Lp indicates the attachment point to the -NH- group of Formula (B’) or the ** of Lp indicates the attachment point to the G of Formula (A’).

Embodiment 99. The compound of Formula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of

305

Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of

Embodiments 60 to 70, or any one of Embodiments 84 to 98, wherein:

Ύ H O ** wA^H O v ^lnh

Lp is ° ^nh2 (ValCit), where the * of Lp indicates the attachment point to Li and the ** of Lp indicates the attachment point to the-NH- group of Formula (B’) or the ** of Lp indicates the attachment point to the G of Formula (A’).

Embodiment 100. The compound of Formula (A’) or anyone of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of

Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of

Embodiments 60 to 70, or any one of Embodiments 84 to 98, wherein:

Lp is NH₂ (PheLys), where the * of Lp indicates the attachment point to Li and the ** of Lp indicates the attachment point to the -NH- group of Formula (B’) or the ** of Lp indicates the attachment point to the G of Formula (A’).

Embodiment 101. The compound of Formula (A’) or any one of Embodiments l to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of

Embodiments 60 to 70, or any one of Embodiments 84 to 98, wherein:

VV*

Lp is ^H o (ValAla), where the * of Lp indicates the attachment point to Li and the ** of Lp indicates the attachment point to the-NH- group of Formula (B’) or the ** of Lp indicates the attachment point to the G of Formula (A’).

Embodiment 102. The compound of Formula (A’) or any one of Embodiments l to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 98, wherein:

306 * Y H O **

WY

H o

Lp is NH₂ (ValLys), where the * of Lp indicates the attachment point to Lj and the ** of Lp indicates the attachment point to the -NH- group of Formula (B’) or the ** of Lp indicates the attachment point to the G of Formula (A’).

Embodiment 103. The compound of Formula (A’) or any one of Embodiments 1 to 17, or phannaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of

Embodiments 60 to 70, or any one of Embodiments 84 to 98, wherein:

μι O ** *7~_n\n^

Λ

Lp îs θ NH₂(LeuCit), where the * of Lp indicates the attachment point to Li and the ** of Lp indicates the attachment point to -NH- group of Fonnula (B’) or the ** of Lp indicates the attachment point to the G of Fonnula (A’).

Embodiment 104. The compound of Formula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Fonnula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Fonnula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 103, wherein L₂ is a bond, a methylene, or a C₂-C₃alkenylene.

Embodiment 105. The compound of Fonnula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Fonnula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Fonnula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 104, wherein L₂ is a bond or a methylene.

Embodiment 106. The compound of Fonnula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Fonnula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 105, wherein L₂ is a bond.

Embodiment 107. The compound of Formula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of

307

Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 105, wherein L₂ is a methylene.

Embodiment 108. The compound of Formula (A’) or any one of Embodiments 1 to 30, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 83, or any one of Embodiments 84 to 107, wherein: A is a bond, -OC(=O)-, -OC(=O)N(CH₃)CH₂CH2N(CH₃)C(=O)- or -OC(=O)N(CH₃)C(R^a)2C(R^a)₂N(CH₃)C(=O)-, wherein each R^a is independently selected from H, Ci-Côalkyl or a Cj-Cg cycloalkyl.

Embodiment 109. The compound of Formula (A’) or any one of Embodiments 1 to 30, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 83, or any one of Embodiments 84 to 107, wherein A is a bond or —

OC(=O).

Embodiment 110. The compound of Formula (A’) or any one of Embodiments 1 to 30, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 83, or any one of Embodiments 84 to 109, wherein A is a bond.

Embodiment 111. The compound of Formula (A’) or any one of Embodiments 1 to 30, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of

Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of

Embodiments 60 to 83, or any one of Embodiments 84 to 109, wherein A is -OC(=O).

Embodiment 112. The compound of Formula (A’) or any one of Embodiments 1 to 30, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of

Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of

Embodiments 60 to 83, or any one of Embodiments 84 to 107, wherein:

i „ g > _s II II ς

A is °H OH OH .11 s Ί H V

-l-O-P-O^y -f-O-P-O-P—' OH or OH OH

Embodiment 113. The compound of Formula (A’) or any one of Embodiments 1 to 30, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 83, or any one of Embodiments 84 to 107, wherein:

308

A is -OC(=O)N(CH₃)CH2CH2N(CH₃)C(=O)- or OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH₃)C(=O)-, wherein each R^a is independently selected from H, Ci-Côalkyl or a C₃-Cscycloalkyl.

Embodiment 114. The compound of Formula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 113, wherein:

-kw—x-l*

L₃ is a spacer moiety having the structure * ⁸ , where

W is -CH₂O-**, -CH₂N(R^b)C(=O)O-**, -NHC(=O)C(R^b)2NHC(=O)O-**, -NHC(=O)C(R^b)2NH-**, -NHC(=O)C(R^b)2NHC(=O)-**, -CH?N(X-R²)C(=O)O-

- C(=O)N(X-R²)-**, -CH2N(X-R²)C(=O)-**, -C(=O)NR^b-**, -C(=O)NH-**, -CH2NR^bC(=O)-**, -CH2NR^bC(=O)NH-**, -CH₂NR^bC(=O)NR^b-**, -NHC(=O)-

- NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)₂NH-**, -NHS(O)₂- ï

- C(=O)-, -C(=O)O-** or—NH-, wherein each R^b is independently selected from H, Ci-Côalkyl or C₃-C₈cycloalkyl and wherein the ** of W indicates the point of attachment to X;

and the * of L₃ indicates the point of attachment to R².

Embodiment 115. The compound of Formula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 114, wherein:

i _{t k t} , -1-W-xAL₃ is a spacer moiety having the structure ^; , where

W is -CH₂O-**, -CH₂N(R^b)C(=O)O-**, -NHC(=O)CH₂NHC(=O)O-**, -NHC(=O)CH₂NH-**, -NHC(=O)CH₂NHC(=O)-**, -CH₂N(X-R²)C(=O)O-**,

309

- C(=O)N(X-R²)-**, -CH2N(X-R²)C(=O)-**, -C(=O)NR^b-**, -C(=O)NH-**, -CH2NR^bC(=O)-**, -CH2NR^bC(=O)NH-**, -CH2NR^bC(=O)NR^b-**, -NHC(=O)- î

- NHC(=O)O-**, -NHC(=O)NH-**, -OC(=O)NH-**, -S(O)₂NH-**, -NHS(O)₂£

- C(=O)-, -C(=O)O-** or-NH-, wherein each R^b is independently selected from H, Ci-C&alkyl or Cb-Cgcycloalkyl and wherein the **ofW indicates the point of attachment to X;

X is a bond; and the * of L₃ indicates the point of attachment to R².

Embodiment 116. The compound of Formula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 115, wherein:

i · . U m . . 4~W-X-P

L₃ is a spacer moiety havmg the structure ' < , where

W is -CH₂O-**, -CH₂N(R^b)C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -NHC(=O)CH2NH-**, -NHC(=O)CH2NHC(=O)-**, -CH2N(X-R²)C(=O)O-**, -C(=O)N(X-R²)-*% -CH2N(X-R²)C(=O)-**, -C(=O)NR^b-**, -C(=O)NH-**, CH₂NR^bC(=O)-**, -CH2NR^bC(=O)NH-**, -CH2NR^bC(=O)NR^b-**, -NHC(=O)-

- NHC(=O)O>*, -NHC(=O)NH-**, -OC(=O)NH>*_ÿ -8(Ο)₂ΝΗ-**> -NHS(O>

- C(=O)-, -C(=O)O-** or -NH-, wherein each R^b is independently selected from H, Ci-Côalkyl or Ca-Cgcycloalkyl and wherein the ** of W indicates the point of attachment to X;

X is a triazolyl, wherein the *** of X indicates the point of attachment to W and the * of X indicates the point of attachment to R²;

and the * of L₃ indicates the point of attachment to R².

Embodiment 117. The compound of Formula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of

310

Embodiments 32 to 46, and the immunoconjugale of Fonnula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 115, wherein:

S r¹

L₃ is a spacer moiety having the structure * , where

W is-CHiO-**, -CH₂N(R^b)C(=O)O-**, -NHC(=O)CH₂NHC(=O)O-**, -NHC(=O)CH₂NH-**, -NHC(=O)CH₂NHC(=O)-**, -CH₂N(X-R²)C(=O)O-**, -C(=O)N(X-R²)-**, -CH2N(X-R²)C(=O)-**, -C(=O)NR^b-**, -C(=O)NH-**, -CH2NR^bC(=O)-**, -CH2NR^bC(=O)NH-**, -CH2NR^bC(=O)NR^b-**, -NHC(=O)?

- NHC(=O)O-**, ^NHC(=O)NH-**, -OC(=O)NH-*X -S(O)₂NH·**, -NHS(O)₂-

- C(=O)-, -C(=O)O-** or -NH-, wherein each R^b is independently selected from H, Ci-Côalkyl or C₃-Cscycloalkyl and wherein the ** of W indicates the point of attachment to X;

X is ***-CH₂-triazolyl-*, wherein the *** of X indicates ihe point of attachment to W and the * of X indicates the point of attachment to R²;

and the * of L₃ indicates the point of attachment to R².

Embodiment 118. The compound of Fonnula (A’) or any one of Embodiments 1 to 17, or phannaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugale of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 115, wherein:

S S

L₃ is a spacer moiety having the structure * ' , where

W is -CH₂O-**, -CH₂N(R^b)C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR²)C(=O)O-**5 -C(=O)N(X-R²)-**, wherein each R^b is independently selected from H, Ci-Côalkyl or C₃-C8cycloalkyl and wherein the ** of W indicates the point of attachment to X;

and the * of L₃ indicates the point of attachment to R².

311

Embodiment 119. The compound of Formula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 115, wherein:

r · , _u ♦ ₊ -i-W-X-p

L₃ is a spacer moiety having the structure * , where

W is -CH₂O-**, -CH₂N(R^b)C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR²)C(=O)O-**, -C(=O)N(X-R²)-**, wherein each R^b is independently selected from H, Ci-C&aikyl or C3-C8cycloalkyl and wherein the ** of W indicates the point of attachment to X;

X is a bond ; and the * of L₃ indicates the point of attachment to R².

Embodiment 120. The compound of Formula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 115, wherein:

S S Ά

L₃ is a spacer moiety having the structure ' , where

W is -CH2O-**, -CH₂N(R^b)C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR²)C(=O)O-**, -C(=O)N(X-R²)-**, wherein each R^b is independently selected from H, Ci-Côalkyl or C3-Cscycloalkyl and wherein the ** of W indicates the point of attachment to X;

and the * of L₃ indicates the point of attachment to R².

Embodiment 121. The compound of Formula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 115, wherein:

312

L₃ is a spacer moiety having the structure ⁵ , where

W is -CH₂O-**, -CH₂N(R^b)C(=O)O-**, -NHC(=O)CH2NHC(=O)O-**, -CH2N(XR²)C(=O)O-**, -C(=O)N(X-R²)-**, wherein each R^b is independently selected from H, Ci-Côalkyl or C3-Cscycloalkyl and wherein the ** of W indicates the point of attachment to X;

X is ***-CH2-triazolyl-*, wherein the *** of X indicates the point of attachment to W and the * of X indicates the point of attachment to R²;

and the * of L₃ indicates the point of attachment to R².

Embodiment 122. The compound of Fonnula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Fonnula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 121, wherein R² is a hydrophilic moiety selected from polyethylene glycol, polyalkyîene glycol, a sugar, an oligosaccharide, a

O -|-O-P~OH polypeptide or C₂-C&alkyl substituted with 1 to 3 groups..

Embodiment 123. The compound of Fonnula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Fonnula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 122, wherein R² is a sugar.

Embodiment 124. The compound of Fonnula (A’) or any one of Embodiments 1 to 17, or phannaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 122, wherein R² is an oligosaccharide.

Embodiment 125. The compound of Formula (A’j or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Fonnula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 122, wherein R² is a polypeptide.

Embodiment 126. The compound of Fonnula (A’) or any one of Embodiments 1 to 17, or phannaceutically acceptable sait thereof, the linker of Formula (C’) or any one of

313

Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 122, wherein R² is a polyalkylene glycol,

Embodiment 127. The compound of Formula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 122, wherein R² is a polyalkylene glycol having the structure -(O(CH2)_m)tR’, where R’ is OH, OCH₃ or OCH₂CH2C(=O)OH, m is 1-10 and t is 4-40.

Embodiment 128. The compound of Formula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 122, wherein R² is a polyalkylene glycol having the structure -((CHijmOjtR”-, where R” is H, CH₃ or CH2CH₂C(=O)OH, m is 1-10 and t is 4-40.

Embodiment 129. The compound of Formula (A’) or any one of Embodiments l to I7,or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 122, wherein R² is a polyethylene glycol.

Embodiment 130, The compound of Formula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 122, wherein R² is a polyethylene glycol having the structure-(OCHzC^jtR’, where R’ is OH, OCH₃ or OCH2CH₂C(=O)OH and t is 4-40,

Embodiment 131. The compound of Formula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’j or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 122, wherein R² is a polyethylene glycol having the structure -(CH2CH₂O)_tR”-, where R” is H, CH3 or CH2CH2C(=O)OH and t is 4-40.

314

Embodiment 132. The compound of Fonnula (A’) or any one of Embodiments 1 to 17, or phannaceutically acceptable sait thereof, the linker of Fonnula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Fonnula (E’) or any one of

Embodiments 60 to 70, or any one of Embodiments 84 to 122, wherein:

OH

, where the * of R² indicates the point of attachment to X or L₃.

Embodiment 133. The compound of Formula (A’) or any one of Embodiments 1 to 17, or phannaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Fonnula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 122, wherein:

315

OH

, where the * of R² indicates the point of attachment to X or L₃.

Embodiment 134. The compound of Fonnula (A’) or any one of Embodiments 1 to 17, or phannaceutically acceptable sait thereof, the linker of Fonnula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Fonnula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 122, wherein:

h₂o₃p^_o ,Ό ,0 / * HjOjP-.

R² îs XOX , h₂o₃p' _or o , where the * of R² indicates the point of attachment to X or L₃.

Embodiment 135. The compound of Fonnula (A’) or any one of Embodiments l to 17, or phannaceutically acceptable sait thereof, the linker of Fonnula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Fonnula (E’) or any one of

Embodiments 60 to 70, or any one of Embodiments 84 to 122, wherein:

R² is

, where the * of R² indicates the point of attachment to X or L₃.

Embodiment 136. The compound of Fonnula (A’) or any one of Embodiments 1 to 17, or phannaceutically acceptable sait thereof, the linker of Fonnula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Fonnula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 135, wherein:

316

Embodiment 137. The compound of Formula (A’) or any one of Embodiments 1 to 17, or phannaceutically acceptable sait thereof' the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 135, wherein:

Embodiment 138. The compound of Formula (A’) or any one of Embodiments 1 to 17, or phannaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 137, wherein: each m is independently selected from 1, 2, 3, 4, and 5.

Embodiment 139. The compound of Formula (A’) or any one of Embodiments I to 17, or phannaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 137, wherein: each m is independently selected from 1, 2 and 3.

Embodiment 140. The compound of Formula (A’) or any one of Embodiments 1 to 17, or phannaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Fonnula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 139, wherein: each n is independently selected from 1,2, 3, 4 and 5.

Embodiment 141. The compound of Fonnula (A’) or any one of Embodiments 1 to 17, or phannaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Fonnula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 139, wherein: each n is independently selected from I, 2 and 3.

Embodiment 142. The compound of Fonnula (A’) or any one of Embodiments 1 to 17, or phannaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 141, wherein:

317 each t is independently selected from 2, 3, 4, 5, 6, 7, S, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30.

Embodiment 143. The compound of Formula (A’) or any one of Embodiments l to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 141, wherein:

each t is independently selected from 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21,22, 23,24 or 25.

Embodiment 144. The compound of Formula (A’) or any one of Embodiments 1 to 17, or pharmaceutically acceptable sait thereof, the linker of Formula (C’) or any one of Embodiments 32 to 46, and the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 141, wherein:

each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18.

Embodiment 145. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 144, wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

Embodiment 146. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 144, wherein y is 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

Embodiment 147. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 144, wherein y is 1,2, 3, 4, 5, 6, 7, 8, 9 or 10.

Embodiment 148. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 144, wherein y is i, 2, 3, 4, 5, 6, 7 or 8.

Embodiment 149. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 144, wherein y is 1, 2, 3, 4, 5 or 6.

Embodiment 150. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to

70, or any one of Embodiments 84 to 144, wherein y is l, 2, 3 or 4.

Embodiment 151. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to

70, or any one of Embodiments 84 to 144, wherein y is 1 or 2.

Embodiment 152. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to

70, or any one of Embodiments 84 to 144, wherein y is 2.

Embodiment 153. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to

70, or any one of Embodiments 84 to 144, wherein y is 4.

318

Embodiment 154. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 144, wherein y is 6.

Embodiment 155. The immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 144, wherein y is 8.

Embodiment 156. The compound of Formula (A’) or any one of Embodiments 1 to 30, or pharmaceutically acceptable sait thereof, the immunoconjugate of Formula (E’) or any one of Embodiments 60 to 70, or any one of Embodiments 84 to 155, wherein D is a MC1-1 inhibitor when released from the immunoconjugates.

Other Linker Groups

Other examples of linker groups that are suitable for making ADCs or immunoconjugates of a MC1-1 inhibitor disclosed herein includes those disclosed in international application publications such as WO2018200812, WO2017214456, WO20172I4458, WO2017214462, WO2017214233, WO2017214282, WO2017214301, WO2017214322, WO2017214335, WO2017214339, WO2016094509, WO2016094517, and WO2016094505, the contents of each of which are incorporated by reference in their entireties.

For example, the immunoconjugates of MC1-1 inhibitors disclosed herein can hâve a linker-payload (“-L-D”) structure selected from:

J^—(Lc)x^—Ce~A ? (>-c)_x Ce (L_c)y -R(L_C)_X-C_E)_p (L_c)y^-C_E ^—D, wherein:

Le is a linker component and each Le is independently selected from a linker component as disclosed herein;

x is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20;

y is an integer selected from 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20;

p is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

D is a MCI-1 inhibitor disclosed herein;

and each cleavage element (Ce) is independently selected from a self-immolative spacer and a group that is susceptible to cleavage selected from acid-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, glycosidase induced cleavage, phosphodiesterase induced cleavage, phosphatase induced cleavage, protease induced cleavage, lipase induced cleavage or disulfide bond cleavage.

319

In some embodiments, L has a structure selected from the following, or L comprises a

320

321

322

In some embodiments, the hnker L comprises a linker component that is selected from: -**C(=O)O(CH₂)_mNR^l 'C(=O)(CH2)m-; -**C(=O)O(CH2)mNR' ^lC(=O)(CH2)_InO(CH₂)m-; -**C(=O)O(CH₂)i_nNR’^lC(=O)XiaX_2aC(=O)(CH₂)_m-;

-**C(=O)OC(R^l2)2(CH2)mNR^,lC(=O)XiaX_2aC(=O)(CH₂)_in-;

-**C(=O)O(CH2)mNRC(=O)Xi_aX2_aC(=O)(CH₂)_mO(CH2)_in-;

-**C(=O)O(CH₂)mNR^llC(=O)Xi_aX2_aC(=O)(CH₂)_inO(CH2)mC(=O)-;

-**C(=O)O(CH2)mNR^llC(=O)X4C(=O)NR^1I(CH2)mNR^1!C(=O)(CH₂),nO(CH₂)m-;

-**C(=O)O(CH₂)_mNR^llC(=O)X5C(=O)(CH2)mNR^IlC(=O)(CH2)_m-;

-^C(=O)X₄C(=O)NR' ‘(CHzLNR¹ 'C(=O)(CH₂)_mO(CH₂)m-;

-**C(=O)(CH₂)_mNR^liC(=O)Xi_aX_2aC(=O)(CH₂)m-;

-**C(=O)O(CH₂)_mX6C(=O)Xi_aX_2aC(=O)(CH₂)m-;

-*Y(=O)(CH₂)mNR^1,C(=O)((CH₂)_inO)n(CH₂)_in-**C(=O)O(CH₂)_mX₆C(=O)(CH₂)_m-; -**C(=O)O(CH₂)_mX6C(=O)(CH₂)_mO(CH₂)_m-;

-**C(=O)O(CH2)_mX6C(=O)Xi_aX2_aC(=O)(CH₂)_m-;

-**C(=O)O(CH₂)_mX6C(=O)Xi_aX_2aC(=O)(CH₂)_mO(CH₂)_m-;

-**C(=O)O(CH₂)_mX₆C(=O)Xi_aX_2aC(=O)(CH₂)_mO(CH₂)_niC(=O)-;

-**C(=O)O(CH₂)mX6C(=O)X4C(=O)NR^ll(CH2)1nNR^IIC(=O)(CH2)ltlO(CH₂)₁₁₁-;

-**C(=O)X4C(=O)X6(CH₂)_mNRC(=O)(CH2)_mO(CH2)_m-;

-**C(=O)(CH₂)_mX₆C(=O)Xi_aX_2aC(=O)(CH₂)_m-;

-**C(=O)O((CH₂)₁nO)n(CH₂)₁₁₁NR^,lC(=O)X₅C(=O)(CH₂)_m-;

-**C(=O)O((CH2)_mO)n(CH₂)_inNR^HC(=O)X5C(=OXCH2)mNR^llC(=O)(CH2)_m-;

-**C(=O)O((CH₂)_inO)_n(CH2)_mNR^IIC(=O)X5C(=OXCH₂)_mX3(CH2)m-;

-**C(=O)O((CH₂)_mO)n(CH₂)_lIlNR^llC(=O)X5C(=O)((CH2)_mO)_n(CH₂)_1Il-;

323 **C(=O)O((CH2)_mO)n(CH2)_mNRⁿC(=O)X5C(=O)((CH₂)_mO^

2)m-;

-**C(=O)O((CH2)mO)n(CH₂)_mNR^l,C(=O))X₅C(=O)((CH₂)_mO)n(CH2)₁nX3(CH2)_m-;

-^0(=0)0((0^00(0^^^^4=0^50(=0)(0^)1^^(=0)((0^ **C(=O)O((CH₂)_mO)ntCH2)_mNRC(=O)X5C(=O)(CH2)niNR^HC(-O)((CH₂)_mO)_tl(CH₂)_ltlX₃(CH ₂)m-;

-**C(=O)O((CH₂)_inO)_il(CH₂)_mNR^llC(=O)X5(CH₂)_1I1X3(CH₂)_m-;

-**C(=O)O((CH2)inO)_li(CH2),₁iNR^llC(=O)X5((CH₂)_mO)n(CH₂)₁i₁-;

-**C(=O)O((CH₂)_mO)_n(CH2)mNR¹¹C(=O)X5((CH2)mO)11(CH2)mNR^llC(=O)(CH2)1n-;

-**C(=O)O((CH₂)_mO)n(CH₂)mNR^{l i}C(=O)X5((CH2)mO)n(CH2)rnNR^1,C(=O)(CH2)_lnX3(CH₂)m-;

-**C(=O)O((CH₂)mO)n(CH₂),i₁NR^liC(=O)X₅((CH₂)_mO)n(CH₂)₁nXî(CH₂)_m-;

-**C(=O)O((CH₂)_mO)n(CH₂)₁nNR^llC(=O)X5(CH2)mNR^ll((CH2)_mO)n(CH₂)_m-;

-**C(=O)O((CH₂)mO)n(CH₂)_InNR^1,C(=O)X5C(=O)(CH2)mNR^ll((CH2)ii₁O)n(CH₂)mX₃(CH2)m-;

-**C(=O)O((CH₂)_niO)n(CH₂)mNR^{1 l}C(=O)X5(CH₂)_m-;

-**C(=O)O((CH₂)_mO)n(CH₂)₁nNR^llC(=O)X5C(=O)((CH₂)_tiiO)_l,(CH₂)ni-;

-**C(=O)O((CH₂)_mO)n(CH2),nNR'^lC(=O)X5(CI-I₂)_mX3(CH₂)_m-; -**C(=O)O(CH₂)_lir;

-**C(=O)O((CH₂)mO)n(CH₂)_m-; -**C(=O)O(CH₂)_inNR^! ‘(CH₂)^

-**C(=0)0(CH₂)mNR^{1 l}(CH₂)i₁iC(=O)X2aXlaC(=O)-;

-**C(=O)O(CH₂)inX3(CH₂)_m-; -**C(=O)O((CH₂)_mO)n(CH₂)_mX3(CH₂V

-**C(=O)O((CH₂)_mO)₁₁(CH₂)_lnNR^llC(=O)(CH2)m-; **C(=O)O(CH2)mNR^,1C(=O(CH2)_mX3(CH2)_m-;

-**C(=O)O((CH2)_mO)_n(CH2)mNRC(=O)(CH₂)_mX₃(CH₂)_m-;

-**C(=O)O((CH₂)_mO)_nX₃(CH2)m-; -**C(=O)O((CH₂)_mO)n(CH₂)_mX₃(CH₂)_m-;

-**C(=O)0((CH₂)niO)_tl(0H₂)_inC(=O)NR^ll(CH₂)m-; -**C(=O)O(CH₂)_inC(R^I2)2-;

.**C(=O)OCH₂)₁₁iC(R¹²)2SS(CH2)niNR^!1C(=O)(CH2)1n-_iand

-**C(=O)O(CH₂)_mC(=O)NR^{l l}(CH₂)_m-, where: ** indicates point of attachment to the drug moiety (D) and the other end can be connected to R¹⁰⁰, i.e., the coupling group as described herein;

wherein:

324

or , where the * indicates the point of attachment to

X_2a;

; where the * indicates the point of attachment to Xj_a;

X4 is -O(CH2)nSSC(R^,2)2(CH2)n- or -(CH2)nC(R^l2)2SS(CH₂)nOs

325

indicates orientation toward the Drug moiety;

F FF cf₃ o— ** i--1 ** >--/ ** <—¥ ** >--( ** y—Y

A? A? V·? A? -A·³

Xô is ++, _s or , where the ** indicates orientation toward the Drug moiety;

each R¹¹ is independently selected from H and Ci-Céalkyi;

each R¹² is independently selected from H and Ci-C/alkyl;

each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, and each n is independently selected from 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17 and 18.

Methods of Conjugation

The présent invention provides various methods of conjugating Linker-Drug groups of the invention to antibodies or antibody fragments to produce Antibody Drug Conjugales which comprise a linker having one or more hydrophilic moieties.

A general reaction scheme for the formation of Antibody Drug Conjugates of Fonnula (E’) is shown in Scheme 2 below:

Scheme 2

where: RG₂ is a reactive group which reacts with a compatible R¹ group to form a corresponding R¹⁰⁰ group (such groups are illustrated in Table 2 and Table 3). D, R¹, Li, Lp, Ab, y and R¹⁰⁰ are as defined herein.

Scheme 3 further illustrâtes this general approach for the formation of Antibody Drug Conjugates of Formula (E’), wherein the antibody comprises reactive groups (RG₂) which react with an R¹ group (as defined herein) to covalently attach the Linker-Drug group to the antibody

326 via an R¹⁰⁰ group (as defined herein). For illustrative purposes only Scheme 3 shows the antibody having four RG₂ groups.

Scheme 3

(Ab1) L₂-A-D (Ab2) where LÛ is Lp—G l₃-r²

In one aspect, Linker-Drug groups are conjugated to antibodies via modified cysteine residues in the antibodies (see for example WO2014/124316). Scheme 4 illustrâtes this approach for the formation of Antibody Drug Conjugates of Formula (E’) wherein a free thiol group generated from the engineered cysteine residues in the antibody react with an R¹ group (where R¹ is a maleimide) to covalently attach the Linker-Drug group to the antibody via an R¹⁰⁰ group (where R^10ü is a succinimide ring). For illustrative purposes only Scheme 4 shows the antibody having four free thiol groups.

Scheme 4

D D

L L

327

In another aspect, Linker-Drug groups are conjugated to antibodies via lysine residues in the antibodies. Scheme 5 illustrâtes this approach for the formation of Antibody Drug Conjugates of Formula (E’) wherein a free amine group from the lysine residues in the antibody react with an R¹ group (where R¹ is an NHS ester, a pentafluorophenyl or a tetrafluorophenyl) to 5 covalently attach the Linker-Drug group to the antibody via an R^l0t) group (where R¹⁰⁰ is an amide). For illustrative purposes only Scheme 5 shows the antibody having four amine groups.

Scheme 5

>₂-a-d and where LD is -(-L|—Lp—G

L₃-R^j

In another aspect, Linker-Drug groups are conjugated to antibodies via formation of an oxime bridge at the naturally occurring disulfïde bridges of an antibody. The oxîme bridge is formed by initial l y creating a ketone bridge b y réduction of an interchain disulfïde bridge of the antibody and re-bridging using a 1,3-dihaloacetone (e.g. 1,3-dichloroacetone). Subséquent reaction with a Linker-Drug group comprising a hydroxyl amine thereby form an oxime linkage (oxime bridge) which attaches the Linker-Drug group to the antibody (see for example

WO2014/083505). Scheme 6 illustrâtes this approach for the formation of Antibody Drug Conjugates of Fonnula (E’).

328

Scheme 6

(Ab 2) (4 interchain disulfïde modified (Ab!))

A general reaction scheme for the formation of Antibody Drug Conjugates of Formula (F’) is shown in Scheme 7 below:

Scheme 7

where: RG2 is a reactive group which reacts with a compatible R¹ group to form a corresponding

R¹⁰⁰ group (such groups are illustrated in Table 2 and Table 3). D, R¹, Li, Lp, Ab, y and R¹⁰⁰ are as defined herein.

Scheme 8 further illustrâtes this general approach for the formation of Antibody Drug

Conjugates of Formula (F’), wherein the antibody comprises reactive groups (RG2) which react

329 with an R¹ group (as defined herein) to covalently attach the Linker-Drug group to the antibody via an R¹⁰⁰ group (as defined herein). For illustrative purposes only Scheme 8 shows the antibody having four RG? groups.

In one aspect, Linker-Drug groups are conjugated to antibodies via modified cysteine residues in the antibodies (see for example WO2014/124316). Scheme 9 illustrâtes this approach for the formation of Antibody Drug Conjugales of Formula (F¹) wherein a free thiol group generated from the engineered cysteine residues in the antibody react with an R¹ group (where R¹ is amaleimide) to covalently attach the Linker-Drug group to the antibody via an R^lü0 group (where R¹⁰⁰ is a succinimide ring). For illustrative purposes only Scheme 9 shows the antibody having four free thiol groups.

Scheme 9

330

In another aspect, Linker-Drug groups are conjugated to antibodies via lysine residues in the antibodies. Scheme 10 illustrâtes this approach for the formation of Antibody Drug Conjugates of Formula (F’) wherein a free amine group from the lysine residues in the antibody react with an R¹ group (where R¹ is an NHS ester, a pentafluorophenyl or a tetrafluorophenyl) to 5 covalently attach the Linker-Drug group to the antibody via an R¹⁰⁰ group (where R¹⁰⁰ is an amide). For illustrative purposes only Scheme 10 shows the antibody having four amine groups.

In another aspect, Linker-Drug groups are conjugated to antibodies via formation of an oxime bridge at the naturally occurring disulfide bridges of an antibody. The oxime bridge is formed by inîtially creating a ketone bridge by réduction of an interchain disulfide bridge of the antibody and re-bridging using a 1,3-dihaloacetone (e.g. 1,3-dichloroacetone). Subséquent reaction with a Linker-Drug group comprising a hydroxyl amine thereby form an oxime linkage (oxime bridge) which attaches the Linker-Drug group to the antibody (see for example

5 WO2014/083505). Scheme 11 illustrâtes this approach for the formation of Antibody Drug Conjugates of Formula (F’).

331

Scheme 11

Provided are also protocols for some aspects of analytical methodology for evaluating antibody conjugates of the invention. Such analytical methodology and results can demonstrate that the conjugates hâve favorable properties, for example properties that would make them easier to manufacture, easier to administer to patients, more efficacious, and/or potentially safer for patients. One example is the détermination of molecular size by size exclusion chromatography (SEC) wherein the amount of desired antibody species in a sample is determined relative to the amount of high molecular weight contaminants (e.g., dimer, multimer, 10 or aggregated antibody) or low molecular weight contaminants (e.g., antibody fragments, dégradation products, or individual antibody chains) present in the sample. In general, it is désirable to hâve higher amounts of monomer and lower amounts of, for example, aggregated antibody due to the impact of, for example, aggregates on other properties of the antibody sample such as but not limited to clearance rate, immunogenicity, and toxicity. A further example is the détermination of the hydrophobicity by hydrophobie interaction chromatography (HIC) wherein the hydrophobicity of a sample is assessed relative to a set of standard antibodies of known properties. In general, it is désirable to hâve low hydrophobicity due to the impact of hydrophobicity on other properties of the antibody sample such as but not limited to aggregation, 332 aggregation over time, adhérence to surfaces, hepatotoxicity, clearance rates, and pharmacokinetic exposure. See Damle, N.K., Nat Biotechnol. 2008; 26(8):884-885; Singh, S.K., Pharm Res, 2015; 32(11 ):3541-71. When measured by hydrophobie interaction chromatography, higher hydrophobicity index scores (i.e. elution from HIC column faster) reflect lower hydrophobicity of the conjugates. As shown in Examples below, a majority of the tested antibody conjugates showed a hydrophobicity index of greater than 0.8. In some embodiments, provided are antibody conjugates having a hydrophobicity index of 0.8 or greater, as determined by hydrophobie interaction chromatography.

EXAMPLES

The following examples provide illustrative embodiments of the disclosure. One of ordinary skill in the art wîll recognize the numerous modifications and variations that may be performed without altering the spirit or scope of the disclosure. Such modifications and variations are encompassed within the scope of the disclosure. The examples provided do not in any way limit the disclosure.

Example 1. Synthesis and Characterization of Lînkers, Linker-Payloads, and Precursors thereof.

Exemplary lînkers, linker-payloads, and precursors thereof were synthesized using exemplary methods described in this example.

Abbreviations:CuI cupper (I) iodide

DCC dicyclohexyl carbodiimîde

DCM dîchloromethane

DEA N-ethylethanamine

DIPEA: Ν,Ν-Dîisopropylethylamine

DMF: dimethylformamide

DMSO: dimethylsulfoxyde

EEDQ ethyl 2-ethoxy-2H-quinoline-l-carboxylate

Fmoc-Cit-OH (2S)-2-(9H-iluoren-9-ylmethoxycarbonylamino)-5-ureido-pentanoic acid HBTU : (2-( 1 //-benzotriazol-1 -yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

HOAt: l-Hydroxy-7-azabenzotriazole

THF tetrahydrofuran

333

MgSO₄	magnésium sulfate
NH4CI	ammonium chloride
NMP	N-methylpyrrolidone
Pd(PPh3)2Cl₂	dichloro-tri (triphenylphosph i ne)pal ladî um
PBr₃	tribromophosphane
Pt/C 10%	platinum over carbon 10%
SOCI2	thionyl chloride
TBAI	tetrabutyl ammonium, iodide
TFA	trifluoroacetic acid

Materials, Methods & General Procedures:

Ail reagents obtained from commercial sources were used without further purification. Anhydrous solvents were obtained from commercial sources and used without further drying. Flash chromatography was performed on CombiFlash Rf (Teledyne ISCO) with pre-packed s il îca-gel cartridges (Macherey-Nagel Chromabond Flash). Thin layer chromatography was conducted with 5x10 cm plates coated with Merck Type 60 F254 silica-gel. Microwave heating was performed in CEM Discover® instrument.

'H-NMR measurements were performed on 400 MHz Broker Avance or 500 MHz Avance Neo spectrometer, using DMSO-ώί or CDCh as solvent. 'H NMR data is in the form of Chemical shift values, given in part per million (ppm), using the residual peak of the solvent (2.50 ppm for DMSO-c/ή and 7.26 ppm for CDCh) as internai standard. Splitting patterns are designated as: s (singlet), d (doublet), t (triplet), q (quartet), quint (quintet), m (multiplet), br s (broad singlet), br t (broad triplet) dd (doublet of doublets), td (triplet of doublets), dt (doublet of triplets), ddd (doublet of doublet of doublets). IR measurements were performed on a Broker Tensor 27 equipped with ATR Golden Gâte device (SPECAC). HRMS measurements were performed on a LTQ OrbiTrap Vélos Pro mass spectrometer (ThermoFisher Scientific). Samples were dissolved in CH₃CN/H2O (2/1 :v/v) at a concentration range from 0.01 to 0.05 mg/mL approximately and introduced in the source by an injection of 2 pL in a flow of 0.1 mL/min. ESI ionization parameters were as follow: 3.5 kV and 35O°C transfer ion capîllary. Ail the spectra were acquired in positive ion mode with a resolving power of 30,000 or 60,000 using a lock mass.

HRMS measurements were performed on an LTQ OrbiTrap Vélos Pro mass spectrometer (ThermoFisher Scientific GmbH, Bremen, Germany). Samples were dissolved in CH3CN/H2O (2/1 :v/v) at a concentration range from 0.01 to 0.05 mg/mL approximately and

334 introduced in the source by an injection of 2 pL in a flow of 0.1 mL/min. ESI ionization parameters were as follows: 3.5 kV and 350°C transfer ion capillary. Ail the spectra were acquired in positive ion mode with a resolving power of 30 000 or 60 000 using a lock mass.

UPLC®-MS:

UPLC®-MS data were acquired using an instrument with the following parameters (Table 4);

Table 4. ÜPLC®-MS Parameters

Instrument(s)	Waters Aquîty A-class with diode array UV detector “PDA” and “ZQ detector 2” mass de vice and Mass Links software.
ZQ detector 2	MS scan from 0.15 to 6 min and from 100 to 2372 Da
PDA detector	from 190 to 400 nm Aquity UPLC ®BEH column Cl8, 1.7 pm,
Columns	130 Â, 2.1x50 mm Column used at 40°C with a flowrate of 0.6mL/min
Solvent A	water + 0.02% TFA
Solvent B	acetonitrile + 0.02% TFA
gradient	from 2% B to 100% B in 5 min, then 0.3 min washing with 100% B and 0.5 min équilibration at 2% B for the next injection (total gradient of 6 min).

Preparative-HPLC:

Préparaiive-HPLC (“Prep-HPLC”) data were acquired using an instrument with the following parameters (Table 5):

335

Table 5. Prep-HPLC Parameters

Instrument(s)	Columns Waters X-Bridge 5 or 10 pm with sizes (flowrate) of: 19x50 mm (12 ml/min), 19x100 mm (12 ml/min), 30x100 mm (30-50 ml/min), 30x250 mm (30-50 ml/min), 50x250 mm (80-150 ml/min); Interchîm Puriflash 4100 with a maximum of 100 bars and a maximum flowrate of 250 ml/min, or Interchîm Puriflash 4250 with a maximum of 250 bars and a maximum flowrate of 250 ml/min; Quatemary solvent pump with the possibility to use 4 solvents at the same time in a gradient
UV	2 wavelengths for the collection between 200 and 400 nm
Columns Collection	Waters Xbridge 10pm 8 ml or 32 ml tubes

Three Prep-HPLC methods were used:

a. TFA method: solvent: A water + 0,05 % TFA, B acetonitrîle + 0,05 % TFA, gradient from 5 to 100% B in 15 to 30 CV

b. NH₄HCOî method: solvent: A water + 0,02 M NH4HCO3, B acetonitrile/water S0/20 + 0.02 M NH4HCO3, gradient from 5 to 100 % B in 15 to 30 CV c, Neutral method: solvent: A water, B acetonitrile, gradient from 5 to 100% B in 15 to 30 CV

Ail the fractions containîng the pure compound were combined and directly freeze-dried to afford the compound as an amorphous powder.

Préparative SFC purification:

Préparative chiral SFC was performed on a PIC solution Prep200 System. The sample was dissolved in éthanol at a concentration of 150 mg/mL. The mobile phase was held isocratically at 40% ethanol/CCb. The înstniment was fitted with a Chiralpak IA column and a loop of 3 mL, The ABPR (automatic back-pressure regulator) was set at 100 bars.

Préparation of L23-P3:

336 (2 K )-2-[ (5AVr)-5-[ 4-] 2-[4-114-1 |(2S)-2-[[(2S)-2-[|2-(2-azîdoethoxy)acetyl]amino]-3-niethylbutanoyi]amino]-5-ureido-pentanoyl|amino]phenyl]methoxycarbonyl]piperazin-lyl]ethoxy]-3-chloro-2-methyl-phenyI]-6-(4-fliiorophenyl)thieno[2,3-d]pyrimidin-4-yl|oxy-3[2-[[2-(2-methoxyphenyi)pyrimidm-4-yl]methoxy]phenyl]propanoic acid

Step I: (2S)-2-[](2S)-2-]]2-[2-[2-(2-azidoethoxy)ethoxy] ethoxy] acetyl] amino]-3-methylbiUanoyl] amino] -N-]4-(hydroxymethyl)phenyl] -5-ureido-pentanamide

To a solution of 2-[2-[2-(2-azidoethoxy) ethoxy] ethoxy] acetic acid (purchased from Broadphann, 1.4 g, 6 mmol) in THF (20 mL) was added l-hydroxypyrrolidine-2,5-dione (690 mg, 6 mmol) and N,N’-Dicyclohexylcarbodiimide (1.2 g, 6 mmol). The reaction mixture was stirred at room température ovemight. The precipitate was filtered off and the fïltrate was concentrated to afford (2,5-dioxopynOlidin-l-yl) 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetate (l .9g, 6 mmol), used immediately without further purification.

To a solution of (2,5-dioxopyrrolidin-l-yl) 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetate (1.6 g; 4.85 mmol) in DMF (15 mL) was added (2S)“2-[[(2S)-2-amino-3-methylbutanoyl]amino]-N-[4-(hydroxymethyl)phenyl]-5-ureido-pentanamide (1.96 g; 5.17 mmol). The mixture was stirred at room température for 2 h and concentrated. The residue was diluted in water (20 mL) and acetonitrile (5 mL) and stirred at room température ovemight. The mixture was purified by reverse phase CI8 chromatography using the neutral method to afford (2S)-2[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl] amino]-N-[4(hydroxymethyl)phenyl]-5-ureido-pentanamide (1.07g, 1.8 mmol). ^[H NMR (400 MHz, dmsod6): δ 9.95 (s, 1H), 8.3 (d, 1H), 7.55 (d, 2H), 7.46 (d, 1H), 7.22 (d, 2H), 5.98 (t, III), 5.4 (s, 1H), 5.08 (t, 1H), 4.43 (d, 2H), 4.4 (q, 1H), 4.33 (dd, 1H), 3.95 (s, 2H), 3.6 (m, 10H), 3.38 (t, 2H), 3 (m, 2H), 2 (m, 1H), 1.7/1.6 (2m, 2H), 1.5-1.3 (m, 2H), 0.89/0.82 (2d, 6H).

337

Step 2: [4-[[(25)-2-][(23)-2-][2-[2-[2-(2-azidoethoxy)etho>y] ethoxy] acetyl] aminol-3-methylbutanoyl] amino]-5-ureido-pentanoyl] amino]phenyl]methyl (4-mtrophenyl)carbonate

To a solution of (2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]3-methyl-butanoyl]amino]-N-[4-(hydroxymethyl)phenyI]-5-ureido-pentanamide (100 mg, 0.168 mmol) in DMF (30 mL) was added DIPEA (32 pL, 0.179 mmol) and bis(4-nitrophenyl) carbonate (100 mg, 0.329 mmol). The mixture was stirred at room température for 4 h and concentrated to dryness. The residue was purified by silica gel chromatography (gradient of methanol in dichloromethane) to afford 4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2azidoethoxy)ethoxy] ethoxy] acetyl ]amino]-3-methyl-butanoyl] amino]-5-ureidopentanoyl]amino]phenyl]methyl (4-nitrophenyl)carbonate (65 mg, 0.088 mmol).'H NMR (400 MHz, dmso-d6): δ 9.95 (s, 1 H), 8.3 (d, 1 H), 7.55 (d, 2H), 7.46 (d, IH), 7.22 (d, 2H), 5.98 (t, III), 5.4 (s, IH), 5.08 (t, IH), 4.43 (d, 2H), 4.4 (q, IH), 4.33 (dd, IH), 3.95 (s, 2H), 3.6 (m, 10H), 3.38 (t, 2H), 3 (m, 2H), 3.02-2.95 (m, 2H), 2 (m, IH), 1.7 (m, IH), 1.6 (m, IH), 0.89 (d, 3H), 0.82 (d, 3H).

Step 3: (2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[]2-[2-[2-(2azidoethoxy)ethoxy]ethoxy] acetyl] amino]-3-methyl-butanoyl]amino] -5-ureidopentanoyl] amino]phenyl] methoxycarbonyl]piperazin-l-yl] ethoxy]-3-chloro-2methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid L23-P3

To a solution of ((2R)-2-[(55_a)-5-[3-chloro-2-methyI-4-(2-piperazin-l-ylethoxy)phenyl]6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yI]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4yl]methoxy]phenyl]propanoic acid P3 (147 mg, 0.17 mmol) in DMF (16 mL) were successively added DIPEA (85 pL, 0.51 mmol), 4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2az idoethoxy)ethoxy] ethoxy] acetyl] ami no] -3 -methyl -b utano yl] amino] -5-ureidopentanoyl] amino] phenyl ]methyl (4-nitrophenyl)carbonate (136 mg, 0.179 mmol), 2,6-lutidine (99 pL, 0.85 mmol) and HOAt (7 mg, 0.05 mmol). The mixture was stirred at room température ovemight and purified by Cl8 reverse phase prep-HPLC by direct deposit of the reaction mixture on the Xbridge column and using the NH4HCO3 method to afford L23-P3 (110 mg, 0.074 mmol). Ή NMR (400 MHz, dmso-d6): δ 10.05 (s, IH), 8.87 (d, 1 H), 8.59 (s, l H), 8.32 (d, IH), 7.67 (brs, IH), 7.59 (d, 2H), 7.52 (dd, IH), 7.45 (td, IH), 7.44 (d, lH),7.36(dl, IH), 7.29 (m, 2H), 7.27 (d, 2H), 7.2 (t, 2H), 7.19 (d, IH), 7.14 (d, IH), 7.13 (t, IH), 7.03 (t, IH), 6.99 (d, IH), 6.71 (t, IH), 6.24(dl, IH), 5.99 (t, IH), 5.48 (dd, 1 H), 5.41 (brs, IH), 5.23 (m, 2H), 4.97 (s, 2H), 4.39 (m, IH), 4.32 (dd, 1H),4.21 (m, 2H), 3.95 (m, 2H), 3.75 (s, 3H), 3.65-3.50 (m, 10H), 338

3.34 (m, 2H), 3.02/2.95 (m, 2H), 2.73 (t, 2H), 2.49/2.3 (m,2H), 2.45 (m, 4H), 2.3 (m, 4H), 2 (m, IH), 1.82 (s, 3H), L7/L59 (m, 2H), 1.44/1.37 (m, 2H), 0.87 (d, 3H), 0.82 (d, 3H). ^I3CNMR(l00 MHz, dmso-d6): δ 158.3, 152.9, 131.6, 131.6, 131.3, 131.3, 131, 129, 128.8, 121, 120.8, 119.5, 116.4, 116.1, 112.8, 112.4, 111.2, 74.5, 70.1, 69.3, 67.7, 66.4, 57, 56.7, 56.2, 53.7, 53.2, 50.4, 43.6, 39, 32.8,31.6, 29.6, 27.3, 19.3, 17.7. IR Wavelength (cm¹): 3500-2500, 2106, 1656. HRESI+: m/z [M+H]+ = 1479.5422 / 1479.5405 (measured/theoretical).

Préparation of L24-P1:

(2R)-2-U5S_a)-5-[4-[2-[4-(|4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2azidoethoxy)ethoxy]etlioxy] acetyl] aniÎno]-3-methyl-butanoyl]amino]-5-ureidopentanoyl] amino] phenyl]methyI|-4-methyl-piperazm-4-ium-l-yl]ethoxy]-3-chloro-2methyi-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyplienyl)pyrimidin-4-yl]methoxy]phenyI]propanoic acid; 2,2,2-trifluoroacetate;

Step 1: (25)-2-][(25)-2-/[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methylbutanoyl] amino] -N-[4-(bromomethyl)phenyl]-5-ureido-pentanamide

To a solution of (2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]3-methyl-butanoyl]amino]-N-[4-(hydroxymethyl)phenyl]-5-ureido-pentanamide (330 mg, 0.55 mmol; obtained according to Step 1 of the synthesis of L23-P3) in THF (10 mL) was added dropwise at 0°C a solution of phosphorus tribromide 1 M in dichloromethane (1 mL, 1 mmol). The mixture was stirred at 0°C for 1 h and ftnely grounded NaHCO₃ (100 mg) was added. After 10 min of stirring, the reaction was diluted with ethyl acetate and filtered. The organic layer was dried over Magnésium sulfate and concentrated. The residue (2S)-2-[[(2S)-2-[[2-[2-[2-(2azidoethoxy)ethoxy] ethoxy] acetyl] amino] -3 -methyl-butanoyl] amino] -N- [4339 (bromomethyI)phenyl]-5-ureido-pentanamide (283 mg, 0.43 mmol) was used without further purification. HR-ESI+: m/z [M+H]+ = = 595.3200 / 595.3198 (measured/theoretical).

Step 2: ((2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]-5-ureidopentanoyl] amino]phenyl] methyl]-4-methyl-piperazin-4-ium-1-yl] ethoxy]-3-chloro-2methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl] oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2~ trifluoroacetate; 2,2,2-trifluoroacetic acid L24-P1

To a solution of ethyl (2R)-2-[(5S_a)-5-[3-chIoro-2-methyl-4-[2-(4-methylpiperazin-lyl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy] phenyl] propanoate dichlorhydrate (PI) (345 mg, 0.355 mmol) în DMF (1 mL) were successively added (2S)-2-[[(2S)-2-[[2-[2-[2-(2azidoethoxy)ethoxy] ethoxy] acetyl] amino] -3 -methyl -b utanoyl] amino] -N - [4(bromomethyl)phenyl]-5-ureido-pentanamide (233 mg, 0.355 mmol) and DIPEA (50 pL, 0.304 mmol). The mixture was stirred at room température ovemight. A solution of lithium hydroxide monohydrate (15 mg, 3.55 mmol) in water (0.5 mL) was added and the reaction was stirred at room température for 24 h. The reaction mixture was purified by C18 reverse phase prep-HPLC by direct deposit of the reaction mixture on the Xbridge column and using the TFA method to afford L24-P1 (80 mg, 0.054 mmol). ‘H NMR (400 MHz, dmso-d6): δ 13.2 (m, III), 10.25 (m, 1H), 8.88 (d, 1H), 8.6 (s, 1H), 8.36 (d, 1H), 7.72 (d, 2H), 7.63 (d, 1H), 7.52 (dd, 1H), 7.46 (t, 1H), 7.44 (m, 1H), 7.43 (m, 2H), 7.37 (d, 1H), 7.3 (dd, 2H), 7.21 (t, 2H), 7.2 (d, 1H), 7.15 (d, IH), 7.15 (t, 1H), 7.03 (t, 1H), 7 (t, lH),6.72(t, 1H), 6.22 (d, 1H), 6 (t, 1H), 5.52 (m, 2H), 5.49 (dd, 1 H), 5.25 (dd, 2H), 4.5 (br s, 2H), 4.39 (m, 1 H), 4.32 (m, 1H), 4.25 (m, 2H), 3.95 (br s, 2H), 3.76 (s, 3H), 3.4/3.24 (m, 4H), 3.35 (m, 2H), 3.28/2.51 (m, 2H), 3.04/2.83 (m, 4H), 3.02/2.96 (m, 2H), 2.92 (m, 2H), 2.87 (s, 3H), 1.99 (m,l H), 1.83 (s, 3H), 1.69/1.61 (m, 2H), 1.46/1.38 (m, 2H), 0.88/0.82 (m, 6H). ¹³CNMR(125 MHz, dmso-d6): Ô 134.2, 131.4, 131.3, 131.3, 131.2, 130.7, 128.7, 120.9, 120.5, 119.2, 116.3, 115.8, 112.7, 112.3, 111, 74, 70.2, 69.6, 67.8, 58.9, 56.9,56.1, 55.4, 54, 50.5, 46.6, 44.9,39, 32.7, 31.6, 29.8, 27.5, 19.7/18.4, 18. IR Wavelength (cin¹): 3700-2200, 3000-2000, 2109, 1662, 1250-1050. HR-ESI+: m/z [M+Na]+ = 1473.5656 / 1473.5628 (measured/theoretical).

340

Préparation of L13-C4:

(2R)-2-[(5Sa)-5-[4-[2-(4-[[4-(|(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]etho xy]propanoylamino]-3-methylbutanoyl] amino] propanoyl] amino] phenyl] methoxycarbonyl]piperazin-l-yI]ethoxy]-3chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-I2-[[2-[4(phosphonomethyl)phenyi|pyrimidin-4-yl]methoxy]phenyl]propanoic acid

Step 1: Synthesis of (2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2azidoethoxy)ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethox y] ethoxy]propanoylamino] -N-](1 S)-2-[4-(hydroxymethyl)amlino]-1 -methyl-2-oxoethyl] -3-methyl-butanamide

To a solution of (2S)-2-amino-N-[(lS)-2-[4-(hydroxymethyl)anilino]-l-methyl-2-oxoethyl]-3-methyl-butanamide (0.9 g, 3.07 mmol; obtaîned according to Step 3 of the synthesis of L18-C3) and 3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] propanoic acid (purchased from Broadpharm, 2 g, 3.07 mmol) in DMF (20 mL) were successively added DIPEA (1 mL, 6.13 mmol), 3-(ethyhminomethyleneamino)propyl-dimethylammonium; chloride (EDC) (0.65 g, 3.37 mmol) and [dimethylarnino(triazolo[4,5-b]pyridm-3yloxy)methylene]-dimethyl-ammonium; hexafluorophosphate (HATU) (1.28 g, 3.37 mmol). The mixture was stirred at room température overnight and purified by C18 reverse phase prepHPLC by direct deposît of the reaction mixture on the Xbridge column and using the NH4HCOÎ method to afford (2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2azidoethoxy)ethox y] ethox y] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] propanoylamino]-N-[(l S)-2-[4-(hydroxymethyl)anîlîno]-1 -methyl-2-oxo-ethyl]-3-methylbutanamide (1.64 g, 1.81 mmol). *H NMR (400 MHz, dmso-d6): δ 9.82 (m, III), 8.14 (d, IH), 7.87 (d, IH), 7.54 (d, 2H), 7.23 (d, 2H), 5.08 (t, IH), 4.43 (d, 2H), 4.39 (m, IH), 4.2 (m, IH), 3.65-3.44 (m, 48H), 3.39 (t, 2H), 2.50-2.30 (m, 2H), 1.97 (m, IH), 1.31 (d, 3H), 0.87/0.84 (m, 6

341

H). IR Wavelength (cm'¹): 3600-3200, 3287, 2106, 1668, 1630, 1100. HR-ESI+: m/z [M+H]+ = 919.5265 /919.5234 (measured/theoretical).

Step 2: [4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2azidoethoxy/ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethox y] ethoxy]propanoylamîno] -3-methylbutanoyl] amino]propanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate

To a solution of (2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]eihoxy]ethoxy]ethoxy]ethoxy] propanoylamino]-N-[(lS)-2-[4-(hydroxymethyl)anilino]-l-methyl-2-oxo-ethyl]-3-methylbutanamide (210 mg, 0.228 mmol) in a mixture of THF and dichloromethane (respectively 5 and 2.5 mL) were successively added pyridine (30 pL, 0.479 mmol) and 4-Nitrophenyl chloroformate (97 mg, 0.479 mmol). The reaction was stirred at room température for 3h and other portions of 4-Nîtrophenyl chloroformate (40 mg, 0.197 mmol) and pyridine (30 pL, 0.479 mmol) were added. The reaction mixture was stirred at 0°C for 55h and evaporated to dryness. The residue was purified by silica-gel chromatography (gradient of MeOH in dichloromethane) to afford [4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]eÎhoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] propanoylamino]-3-methyl-butanoyl] amino]propanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate (118 mg, 0.110 mmol). 'HNMR (400 MHz, dmso-d6): δ I0.00(s, 1 H), 8.31 (d, 2H), 8.19 (d, IH), 7.S8 (d, IH), 7.64 (d, 2H), 7.58 (d, 2H), 7.41 (d, 2H), 5.25 (s, 2H), 4.39 (m, IH), 4.21 (m, IH), 3.63-3.47 (m,48H), 3.39 (t, 2H), 2.50-2.35 (m, 2H), 1.98 (m, IH), 1.31 (d, 3H), 0.89/0.85 (m, 6H). IR Wavelength (cm'¹): 3278, 2108, 1763, 1633, 1526, 1525, 1350, 1215, 1110.

342

Step 3: (2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2~[2(2azidoethoxy)ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethox y] ethoxy]propanaylami.no] -3-methylbutanoyl] amino]propanoyl] amino]phenyl] methoxycarbonyl]piperazin- 1-yl] ethoxy] 3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2[[2-[4-(phosphonomethyl)phenyl]pyrimidin-4-yl] methoxy]phenyl]propanoic acid (L13-C4)

To a solution of [4-[[(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] propanoylammo]-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate (52 mg, 47.6 pmol) in DMF (5 mL) were successively added (2R)-2-[(5S_a)-5-[3chloro-2-methyl-4-(2-piperazin- 1-yl ethoxy)ph en yl]-6-(4-fluoro phenyl )thieno [2,3-d]pyrimidin-4yl]oxy-3-[2-[[2-[4-(phosphonomethyl)phenyl]pyrimidin-4-yl]methoxy]phenyl]propanoic acid C4 (36.7 mg, 39.7pmol) and DiPEA (26pL, 108 pmol). The reaction was stirred at room température for 1 h and purified by C18 reverse phase prep-HPLC by direct deposit of the reaction mixture on the Xbridge column and using the NH4HCO3 method to afford L13-C4 (36 mg, 19 pmol). Ή NMR (400 MHz, dmso-d6): δ 10.1 (br s, IH), 8.81 (br s, 1 H), 8.55 (m, 1 H), 8.32 (brs, IH), 8.19 (d,2H), 8.02 (brs, 1 H), 7.66 (m, IH), 7.58 (d, 2H), 7.37 (d, IH), 7.29 (dd, 2H), 7.28 (d, 2H), 7.25 (d, 2H), 7.19 (t, 2H), 7.17 (d, IH), 7.08 (t, IH), 6.96 (d, 1 H), 6.68 (t, IH), 6.21 (d, IH), 5.5 (m, IH), 5.22 (m, 2H), 4.96 (s, 2H), 4.4 (m, IH),4.2 (dd, IH), 4.18 (m, 2H), 3.62/3.41 (m, 24H), 3.5 (m, 4H), 3.38 (m, 2H), 3.28 (m, 4H), 2.87 (m, 2H), 2.7 (m, 2H), 2.48/2.36 (m, 2H), 2.41 (m, 4H), 1.99 (m, 1 H), 1.79 (s, 3H), 1.3 (d, 3H), 0.87/0.83 (m, 6H). ^,3C NMR (100 MHz, dmso-d6): δ 130.7, 130.7, 130.6, 130.3, 129, 128.4, 127.4, 121, 119.6, 116.3, 116.1, 112.1, 70.2/67.3,69.5, 67.5,66.4, 58.2, 56.4, 53.2, 50.3,49.6, 43.8, 36.3, 31, 19, 18.5, 17.8. ^l9F NMR (376 MHz, dmso-d6): δ -112.4. ³¹P NMR (200 MHz, dmso-d6): δ 17.8. IR Wavelength (cm’¹): 3290, 2102, 1698, 1651, 1237, 1094, 833, 756. HR-ESI+: m/z [M+H]+ = 1867.7129 / 1867.7154 (measured/theoretical).

343

Préparation of L19-C3:

(2R)-2-[(5Sa)-5-[4-[2-|4-||4-[|(2S)-2-[[(2S)-2-[[2-[2-[2-(2 azidoethoxy)ethoxy]ethoxyJacetyl]amino]-3-methylbutanoyl|amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-l-yl]ethoxy]-3chloro-2-methyl-phenyl]-6-(4-fluorophenyi)thieno[2,3-d]pyriniidm-4-yi]oxy-3“[2-[[2-(2 methoxyphcnyl)pyrimid in-4-yl] methoxy] phenyl] propanoic acid

Step 1: [4~[[(2S)-2-[[(2S)-2-(9H-fluoren-9~ylmethoxycarbonylamino)~3-methyl~ butanoyl] amino]propanoyl] amino]phenyl] methyl (4~nitrophenyl) carbonate

To a suspension of 9H-fluoren-9-ylmethyl N-[( 1 S)- !-[[(! S)-2-[4(hydroxymethyl)anilino]-l-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate ( 1 g, 1.66 mmol) in a THF/Dichloromethane mixture (respectively 100 and 30 mL), were successively added pyridine (269 pL, 3.32 mmol) and 4-Nitrophenyl chloroformate (670 mg, 3.30 mmol). The reaction was stirred at room température ovemight and another portion of 4-Nitrophenyl chloroformate was added (335 mg, 1.66 mmol). The reaction was stirred at room température for 3h, concentrated and the residue was purified by silica gel chromatography (gradient of ethyl acetate in heptane) to afford [4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3m ethyl-butanoyl] ami no]propanoyl] ami no] phenyl] methyl (4-nitro phenyl) carbonate (658 mg, 0.97 mmol). Ή NMR (400 MHz, dmso-d6): S 10.07 (m, IH), 8.31 (d, 2H), 8.19 (d, IH), 7.89 (d, 2H), 7.74 (t, 2H), 7.64 (d, 2H), 7.57 (d, 2H), 7.41 (m, 2H), 7.41 (d, 2H), 7.4 (m, IH), 7.32 (t, 2H), 5.24 (s, 2H), 4.43 (m, 1 H), 4.36-4.19 (m, 3H), 3.92 (dd, IH), 2 (m, IH), 1.32 (d, 3H), 0.9/0.87 (m,6H). IR Wavelength (cm¹): 3350-3200, 1760, 1690, 1670, 1630, 1523, 1290.

344

Step 2; (2R)-2-[(5S_a)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9ylmethoxycarbonylamino)-3-methylbutanoyl] amino]propanoyl] amino]phenyl] methoxycarbonyl]piperazin-l-yl]ethoxyj2-methyl-phenyl]-6-(4-fliiorophenyl)thieno[2.3-d]pyrimidin-4-yl]o>y-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

To a solution of (2R)-2-[(5S_a)-5-[3-chloro-2-methyl-4-(2-piperazin-l-ylethoxy)phenyl]6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4yl] methoxy] phenyl]propanoic acid C3 (100 mg, 0.116 mmol) in DMF (1 mL) were successively added [4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-yhnethoxycarbonylarmno)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate (87 mg, 0.128 mmol) and DIPEA (38 pL, 0.232 mmol). The reaction mixture was stirred at room température ovemight and concentrated. The residue was taken up in water, filtered affording (2R)-2-[5-[3chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-l-yl]ethoxy]-2-methylphenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (110 mg, 0.078 mmol) used without further purification in the next step. ’H NMR (400 MHz, dmso-d6): δ 10.05 (br s, 1 H), 8.88 (d, IH), 8.57 (s, IH), 8.23 (d, IH), 7.88 (d, 2H), 7.75 (m, IH), 7.74 (2d, 2H), 7.58 (d, 2H), 7.53 (dd, IH), 7.45 (m, IH), 7.45 (d, IH), 7.41 (m, IH), 7.4 (m, 2H), 7.31 (m, 2H), 7.29 (m, 2H), 7.26 (d, 2H), 7.2 (t, 2H), 7.18 (m, IH), 7.14 (d, IH), 7.11 (t, IH), 7.03 (t, IH), 6.98 (d, IH), 6.69 (t, IH), 6.2 (d, IH), 5.46 (d, IH), 5.22 (m, 2H), 4.97 (s, 2H), 4.42 (t, IH), 4.26 (m, 2H), 4.21 (m, IH), 4.2 (m, 2H), 3.91 (m, IH), 3.75 (s, 3H), 3.35/2.45 (m, 2H), 3.29 (m, 4H), 2.73 (t, 2H), 2.44 (m,4H), 1.99 (m, IH), 1.8 (s, 3H), 1.29 (d, 3H), 0.88/0.85 (m, 6H). ¹³C NMR (100 MHz, dmsod6): δ 158.3, 152.7, 131.6, 131.4, 131.3, 131.1, 131.1, 128.9, 128.5, 128, 127.6, 125.8, 120.9, 120.5, 120.5, 119.4, 116.4, 116, 112.7, 112.2, 111.1,69.4, 67.8,66.5, 66.1,60.7, 56.8, 56.1, 53.2, 49.6, 47.1, 43.8, 33.3, 30.9, 19.7, 18.9, 18.1.

Step 3: (2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)~2-amino-3-methylbutanoyl/ amino]propanoyl] amino]phenyl/ methoxycarbonyl]piperazin -1 -yl]ethoxy] 3-chloro-2-methyl-phenyl]-6-(4-Jluorophenyl)thieno]2,3-d]pyrimidin-4-yl] oxy-3-[2[[2-(2-methoxyphenyl)pyrimidin-4-yl] methoxy]phenyl]propanoic acid

To a solution of (2R)-2-[(5S_a)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9ylmethoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-l-yl]ethoxy]-2-methyl345 phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl] methoxy] phenyl] propanoîc acid (176 mg, 0.125 mmol) in DMF (3 mL) was added dropwise at 0°C piperidine (300 pL, 1.25 mmol). The réaction mixture was stirred at room température for 1 h and concentrated. The residue was purified by Cl 8 reverse phase prep-HPLC by direct deposit of the reaction mixture on the Xbridge column and using the NH4HCO3 method to afford (2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-l-yl]ethoxy]-3-chloro-2methyl-phenyl]-6-(4-fluorophenyl)thieno [2,3-d]pyrimidin-4-yl]oxy-3-[ 2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl] propanoîc acid (130 mg, 0.11 mmol). *H NMR (400 MHz, dmso-d6): δ 10.2 (s, IH), 8.9 (d, IH), 8.6 (dl, IH), 8.55 (s, IH), 7.85 (d, IH), 7.6 (d, 2H), 7.55 (dd, IH), 7.45 (m, 2H), 7.25 (d, 2H), 7.25 (m, 4H), 7.2 (m, 3H), 7.15 (d, IH), 7.1 (t, IH), 7.05 (t, IH), 6.95 (d, 1H), 6.65 (t, IH), 6.15 (d, IH), 5.4 (dd, IH), 5.2 (m, 2H), 4.95 (s, 2H), 4.45 (m, 1 H), 4.2 (m, 2H), 3.75 (s, 3H), 3.4/2.35 (m, 2H), 3.3 (m, 5H), 2.6 (t, 2H), 2.4 (m, 4H), 2 (m, 3H), 1.8 (s, 3H), 1.3 (d, 3H), 0.9/0.85 (m, 6H). IR Wavelength (cm⁴): 3600-2500, 1678.

Step4:(2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2azîdoethoxy)ethoxy] ethoxy] acetyl] amino]-3-methylbutanoyl]amino]propanoyl] amino]phenyl] methoxycarbonyl]piper azin-l-yl] ethoxy] 3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl] oxy-3-[2][2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy] phenyl]propanoîc acid L19-C3

To a solution of2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyI]amino]propanoyl]amiiio]phenyl]methoxycarbonyl]pîperazin-l-yl]ethoxy]-3-chloro-2methyLphenyl]-6-(4-f]uorophenyl)thieno[2,3-d]pyrimidin-4-yI]oxy-3-[2-[[2-(2m ethoxyp heny 1 )pyrimidin-4-yl] methoxy] phenyl] propanoîc acid (50 mg, 0.042 mmol) in DMF (0.3 mL) were successively added DIPEA (14pL, 0.085 mmol) , [dimethylamino-(2,5dioxopyrro3idin-l-yl)oxy-methyiene]-dimethyl-ammonium; tetrafluoroborate (14 mg, 0.046 mmol) and a solution of 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetic acid (28 mg, 0.12 mmol) in DMF (0.5 mL). The reaction mixture was stirred at room température for 2h and purified by C18 reverse phase prep-HPLC by direct deposit of the reaction mixture on the Xbridge column and using the NH4HCO3 method to afford L19-C3 (22 mg, 0.016 mmol). Ή NMR (400 MHz, dmso-d6): δ 10.02 (s, IH), 8.88 (d, IH), 8.4 (d, IH), 7.72 (br s, IH), 7.58 (s, IH), 7.58 (d, 2H), 7.53 (d, IH), 7.45 (d, IH), 7.45 (t, IH), 7.38 (d, IH), 7.29 (dd, 2H), 7.27 (d, 2H), 7.2 (t, 2H), 7.18 (d, IH), 7.14 (d, IH), 7.11 (t, IH), 7.03 (t, IH), 6.98 (d, IH), 6.7 (t, IH), 6.21 (d, IH), 5.46 (dd, IH), 5.23 (m, 2H), 4.97 (s, 2H), 4.4 (m, IH), 4.29 (dd, IH), 4.22 (m, 2H), 3.94 (s, 2H), 3.75 346 (s, 3H), 3.65-3.53 (m, 10H), 3.35 (m, 2H), 3.3 (m, 4H), 3.3/2.5 (m, 2H), 2.73 (t, 2H), 2.44 (m, 4H), 2 (m, 1H), 1.81 (s, 3H), 1.3 (d, 3H), 0.88/0.82 (m, 6H). ¹³C NMR (100 MHz, dmso-d6): δ 158, 152.7, 131.4, 131.4, 131.3, 131.1, 131.1, 128.9, 128.6, 120.9, 120.7, 119.5, 116.2, 112,5, 112.1, 111.1,70.4, 70.4, 69.7, 67.5, 66.2, 56.8, 56.7, 56.1, 53.3, 50.4, 49.5, 43.8, 31.7, 19.5, 0.82, 18.3, 18.2. ^,9F NMR (376 MHz, dmso-d6): δ -112.3. IR Wavelength (cm⁴): 3294, 2104, 1697, 1663, 1288, 1238, 1120, 1076, 1051, 1020, 833,755. HR-ESI+: m/z [M+H]+= 1395.5083 / 1395.5070 (measured/theoretical).

Préparation of L15-C5:

azidoeth oxy)ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy ]etho xy]propanoylamino]ethoxy-hydroxy-phosphoryl]oxy-hydroxyphosphoryl]oxymethyI|phenyl]pyrimidlin-4-yI]niethoxy]phenyl]-2-[(5Sa)-5-[3-chloro-2methyI-4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3

d]pyrimidin-4-yl]oxy-propanoic acid

Step 1: (2R)-2-[(5S_a)-5-[3~chloro-2-methyl-4-[2-(4-methylpiperazin-î-yl)ethoxy]phenyl]-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-[3(phosphonooxymethyl)phenyl]pyrimidin-4-yl]methoxy]phenyl]propanoic acid; bis 2,2,2-trifluoroacetic acid

To a solution of ethyl (2R)-2-[(5S_a)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazm-lyl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-[3(hydroxymethyl)phenyl]pyrimidin-4-yl]methoxy]phenyl]propanoate (110 mg, 0.123 mmol; prepared accordîng to WO 2016/207216) in THF (0.5 mL) was added dropwise at -40°C under argon diphosphoryl chloride (51 pL, 0.368 mmol). The réaction mixture was stirred at -40°C for 30 min. Another portion of diphosphoryl chloride (10 pL, 0.074 mmol) was added at -40°C and

347 the reaction was stirred at -40°C for 20 min, quenched by addition of an aqueous saturated solution of potassium carbonate (0.1 mL) and allowed to warm to room température. The pH was adjusted to 10 by addition of potassium carbonate (powder) and the reaction was stirred for 20 min at room température. The reaction mixture was acidified to pH 2 by slow addition of aqueous 2 M HCl solution at 0°C, extracted with dichloromethane (4 times). The combined organic layers were concentrated, diluted with dioxane (3 mL) and a solution of lithium hydroxide monohydrate (17 mg, 0.403 mmol) in water (0.3 mL) was added. The reaction mixture was stirred at room température for 4 days, neutralized by an aqueous 4 M HCl solution (0.4 mL, 0.4 mmol), and evaporated. The residue was purified by C18 reverse phase prep-HPLC by direct deposit of the reaction mixture on the Xbridge column and using the TFA method to afford (2R)-2-[(5S_a)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1 -yl)ethoxy]phenyl]-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-[3(phosphonooxymethyl)phenyi]pyrimidin-4-yl]methoxy]phenyl]propanoic acid;2,2,2trifluoroacetic acid as a 2TFA sait (41 mg, 43 pmol). MS (ESI) m/z [M + 2H]/2+ = 487.5.

Step 2: 2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2azidoethoxy/ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethox y] ethoxy]propanoylami.no]ethyl dihydrogen phosphate

To a solution of3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2azidoethoxy)ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethox y] ethoxy] ethoxy] ethoxy] propanoic acid (200mg, 0.311 mmol) in dichloromethane (2 mL) were added 1hydroxypyrrolidine-2,5-dione (79 mg, 0.684 mmol), 3-(ethyHminomethyleneamino)propyldimethyl-ammonium; chloride (107 mg, 0.56 mmol). The reaction mixture was stirred at room température overnight, diluted with dichloromethane, partitioned with a saturated aqueous solution of NaHCO₃ and extracted with dichloromethane. The combined organic layers were washed with brine, dried over Magnésium sulfate and concentrated to approximately 1 mL. The residue was diluted with DMF (1 mL), 2-aminoethyl dihydrogen phosphate (30 mg, 0.214 mmol) was added and the reaction mixture was stirred at 80°C overnight, diluted with dichloromethane, washed with water. The aqueous layer was separated and freeze-dried to afford 2-[3-[2-[2-[2-[2[2-[2-[2-[2-[2-[2-[2-(2azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] propanoylamino]ethyl dihydrogen phosphate (165 mg, 0.2 mmol). ^!H NMR (400 MHz, dmsod6): δ 3.45-3.65 (m, 53H), 3.26-3.39 (m, 2H), 3.12 (m, 2H), 2.27 (t, 2H). HR-ES1+: m/z [M+H]+ = 767.3697 / 767.3686 (measured/theoretical).

348

Step 3: (2R)~3-[2-[[2-[3-[[[2-[3-[2-[2-[2-[2-[2-[2~[2-[2~[2-[2-[2-(2azidoethoxy)ethoxy]ethoxy] ethoxy]ethoxy]ethoxy] ethoxy] ethoxy]ethoxy] ethoxy] elhox y] ethoxy]propanoylamino] ethoxy-hydroxy-phosphoryl]oxy-hydroxyphosphoryl] oxymethyl]phenyl]pyrimldin-4-yl] methoxy]phenyl]-2-[(5S_a)-5-[3-chloro2-methyl-4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl]-6~(4-jluorophenyl)thieno[2,3d]pyrimldin-4-yl] oxy-propanoic acid (L15-C5)

To a solution of 2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2azidoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] propanoylamino]ethyl dîhydrogen phosphate (49 mg, 0.064 mmol) in DMF (0.2 mL) were successively added di(îmidazoM-yl)methanone (l l mg, 0.066 mmol), triethylamine (I7pL, 0.066 mmol) and 4Â molecular sieves (50 mg). The reaction was stirred at room température for 2 h. The solid was removed by filtration and the filtrate was treated with Zinc chioride (23 mg, 0.172 mmol) and (2R)-2-[(5S_a)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-lyl)ethoxy]phenyl]'6-(4-fluorophenyl)thieno[2,3-d]pyriniidin-4-yl]oxy-3-[2-[[2-[3(phosphonooxymethyl)phenyl]pynmidin-4-yl]methoxy]phenyl]propanûic acid; bis 2,2,2triiluoroacetic acid (41 mg, 0.043 mmol). The mixture was heated to 50°C overnight. The reaction mixture was purified by Cl 8 reverse phase prep-HPLC by direct deposit of the réaction mixture on the Xbridge column and using the NH4HCO3 method to afford L15-C5 (11 mg, 6pmol). HR-ESI+: m/z [M+H]+ = 1703.5962 / 1703.5959 (measured/theoretical).

Préparation of L17-C3:

(2R)-2-[(5Sa)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[(2S,3R,4S,5R)-6-azido-2,3,4,5-tetrahydroxyhexyl]amino]-3-methylhiitanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyi]piperazin-l-yl]ethoxy]-3chloro-2“methyl-phenyl]-6-(4-nuorophenyl)thieno[2,3-d]pyrimidin“4-yl]oxy-3-[2-n2-(2methoxyphenyl)pyrîmidin-4-yl]niethoxy]phenyl|propanoic acid; 2,2,2-trifluoroacetic acid

OH OH

349

To a solution of (2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyI]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-l-yl]ethoxy]-3-chloro-2methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]piOpanoic acid (230 mg, 0.I94 mmol; obtained accordîng to Step 5 of the préparation of L19-C3) and 6-deoxy-6-azido-D-galactose (l 20 mg, 0.584 mmol; obtained accordîng to Ekholm et al., ChemMedChem 2016, 11, 2501-2505) in a mixture of DMSO/water 80/20 containing 1 % of DIPEA (20 mL) was added at room température sodium cyanoborohydride (24 mg, 0.389 mmol). The reaction mixture was heated at 65 °C for 48h. Another portion of sodium cyanoborohydride (24 mg, 0.389 mmol) and 6-deoxy6-azido-D-galactose (120 mg, 0.584 mmol) were then added at room température. The reaction mixture was heated at 65°C for an additional 48h and was purified by C18 reverse phase prepHPLC by direct deposit of the reaction mixture on the Xbridge column and using the TFA method to afford L17-C3 (38 mg, 28 pmol). 'H NMR (400 MHz, dmso-d6): δ 13.2 (br s, 1H), 10.2 (s, 1H), 8.88 (d, 1H), 8.85 (d, 1H), 8.62 (s, 1H), 8.53 (brs, 1H), 7.63 (d, 1H), 7.59 (d, 2H), 7.52 (d, 1H), 7.45 (t, 1H), 7.42 (d, 1H), 7.33 (dd, 2H), 7.33 (d, 2H), 7.27 (d, 1H), 7.27 (d, 1H), 7.21 (t, 2H), 7.15 (t, 1H), 7.04 (t, IH), 7.01 (d, 1H), 6.73 (t, 1H), 6.21 (d, 1H), 5.51 (d, 1H), 5.28/5.22 (m, 2H), 5.04 (br s, 2H), 4.52 (m, 1H), 4.49 (m, 2H), 4.12 (m, 1H), 3.89 (m, 1H), 3.78 (m, 1H), 3.76 (s, 3H), 3.63 (m, 6H), 3.42/3.21 (m, 2H), 3.38 (m, 1H), 3.37 (m, IH), 3.28/2.52 (m, 2H), 3.22 (m, 4H), 2.96 (m, 2H), 2.21 (m, 1 H), 1.86 (s, 3H), 1.36 (d, 3H), 1.03/0.94 (m, 6H). ^l3CNMR(125 MHz, dmso-d6): δ 157.8, 152.5, 131.4, 131.3, 131.3, 130.6, 129.1, 129, 128.8, 120.8, 120.6, 119.4, 116.2, 116.1, 112.3, 111.3, 111.3, 74.2,71.3, 70.4, 69.5, 69.2, 67.1,65.6, 64.5, 64.5, 56.2, 54.8, 54.2, 51.9, 50.3, 49.9, 32.7, 29.4, 19.3, 18.9, 18. ^!9FNMR(470 MHz, dmso-d6): δ-74.4, -112.1. IR Wavelength (cm'j: 2200-3500, 2104, 1669, 1181, 1132, 798, 758, 720. HR-ESI+; m/z [M+H]+= 1369.4918/ 1369.4913 (measured/theoretical).

Préparation of L24-P7:

(2R)-2-|(5Ss)-5-|4-[2-(4-|[4-[|(2S)-2-[|(2S)-2-[[2-[2-[2-(2azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]phenyl]methyl]-4-methyl-pîperazin-4-ium-l-yl]ethoxy]-3-chloro-2-ethylphenyl]-6-prop-l-ynyl-thieno[2,3-d]pyriniidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yI]methoxy]phenyI]propanoic acid; 2,2,2-trifluoroacetate; 2,2,2-trifluoroacetic acid

350

To a solution of (2S)-2-[[(2S)-2-[[2-(2-azidoethoxy)acetyl]amino]-3-methylbutanoyl]amino]-N-[4-(bromomethyl)phenyl]-5-ureido-pentanamide (72 mg, 0.109 mmol) in THF (5 mL) were successively added (2R)-2-[(5S_a)-5-[3-chloro-2-ethyl-4-[2-(4inethylpiperazin-l-yl)ethoxy]phenyl]-6-prop-l-ynyI-thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid P7 (30 mg, 0.036 mmol) and DIPEA (19 pL, 0.108 mmol). The reaction mixture was stirred ovemight at room température and was purified by C18 reverse phase prep-FIPLC by direct deposît of the reaction mixture on the Xbridge column and using the TFA method to afford L24-P7 (25 mg, 18pmol). 'H NMR (400 MHz, dmso-d6): δ 10.25 (s, 1H), 8.85 (d, 1H), 8.62 (s, 1H), 8.35 (d, 1H), 7.72 (d, 2H), 7.6 (d, 1H), 7.5 (d, 1H), 7.45 (t, 1H), 7.43 (d, 2H), 7.4 (d, 1H), 7.22 (d, 1H), 7.17 (m, 1H), 7.15 (m, 1H), 7.13 (d, 1 H), 7.02 (t, 1H),7 (d, 1H), 6.78 (t, 1H), 6.3 (d, 1H), 5.98 (br s, 1H),5.5 (dd, 1H), 5.4 (br s, IH), 5.28/5.2 (m, 2H), 4.5 (br s, 2H), 4.38 (m, 1H), 4.3 (dd, 1H), 4.25 (m, 2H), 3.94 (br s, 2H), 3.74 (s, 3H), 3.70/3.50 (m, 10H), 3.50 (m, 8 H), 3.35 (t, 2H), 3.22/2.5 (m, 2 H), 3.0 (m, 2H), 2.95 (t, 2H), 2.9 (br s, 3H), 2.55/2.4 (m, 2H), 2.0 (s, 3H), 1.98 (m, 1H), 1.70/1.30 (m, 4H), 0.88/0.82 (m, 6H), 0.72 (t, 3H). IR Wavelength (cm*¹): 3321,2111, 1660, 1188, 1124, 798,756,719. HR-ESI+: m/z [M+H-CF3COOH]+ = 1409.59077 / 1409.5903 (measured/theoretical).

Préparation of L24-P6:

(2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2azidoetlioxy)ethoxy]ethoxy|acetyl]amino]-3-methyl-butanoyl]amino]-5-ureidopentanoyl] amino] phenyl]methyl]-4-methyl-piperazin-4-ium-l-yl]ethoxy]-3-chloro-2methyl-pbenyI]-6-(4-fluorophenyI)thÎeno[2,3-d]pyrimidin-4-yI]oxy-3-[2-[[2-[2(hydroxymethyl)phenyl|pyrimidin-4-yl|methoxy]phenyl]propanoic acid; 2,2,2trifluoroacetate; 2,2,2-trilluoroacetic acid

351

To a solution of (2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]3-methyl-butanoyl]amino]-N-[4-(bromomethyl)phenyl]-5-ureido-pentanamîde (55.3 mg, 84 pmol) in DMF (l mL) were successively added ethyl (2R)-2-[(5S_a)-5-[3-chloro-2-methyl-4-[2(4-methylpiperazin-l-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3[2-[[2-[2-(hydroxymethyl)phenyI]pyrimidin-4-yl]methoxy]phenyl]propanoate (53.2 mg, 59 pmol; synthesized according to EP 2 886 545) and DIPEA (44 pL, 0.252 mmol). The reaction mixture was stirred at room température for l h and concentrated under reduced pressure. The residue was diluted with dioxane (1 mL) and a solution of lithium hydroxîde monohydrate (14 mg, 0.0334 mmol) in water (0.3 mL) was added. The reaction mixture was stirred at room température ovemight, neutralized by addition of an aqueous 1 M HCl solution (0.33mL, 0.33 mmol), concentrated under reduced pressure. The crude product was purified by Cl8 reverse phase prep-HPLC b y direct deposit of the reaction mixture on the Xbridge column and using the TFA method to afford L24-P6 (47 mg, 32 pmol). Ή NMR (400 MHz, dmso-d6): δ 10.27 (s, IH), 8.94 (d, IH), 8.61 (s, IH), 8.38 (d, IH), 7.93 (d, IH), 7.73 (d, 2H), 7.68 (t, IH), 7.66 (d, IH), 7.5 (t, IH), 7.45 (d, IH), 7.43 (d, 2H), 7.38 (d, IH), 7.37 (m, IH), 7.3 (dd, 2H), 7.21 (d, IH), 7.2 (t, 2H), 7.16 (t, IH), 7.02 (d, IH), 6.72 (t, IH), 6.21 (d, IH), 6.01 (m, IH), 5.5 (d, IH), 5.4 (m, IH), 5.3 (m, 2H), 4.8 (s, 2H), 4.39 (m, IH), 4.32 (dd, IH), 4.25 (m, 2H), 3.95 (s, 2H), 3.57 (m, 16H), 3.42/3.26 (m, 2 H), 3.36 (m, 2H), 3.29/2.51 (m, 2H), 3.11/2.92 (m, 8H), 2.98 (m, 2H), 2.97 (m, 2H), 1.99 (m, IH), 1.83 (s, 3H), 1.68/1.62 (m, 2H), 1.45/1.39 (m, 2H), 0.88/0.82 (m, 6H). ^l3C NMR (100 MHz, dmso-d6): δ 158.2, 152.1, 134.2, 131.4, 131.3, 130.9, 130.8, 130.2, 128.7, 128.1, 127, 120.8, 119.3, 116.3, 115.7, 112.2, 111,74, 70.5, 70.1, 69.5,67.7, 62.3, 58.8, 57.2, 55.5, 54.1, 50.5, 46.6, 38.9, 32.5, 31.5, 29.6, 27.6, 19.6, 18.6, 18.3. ¹⁹F NMR (376 MHz, dmso-d6): δ -74.6, -112.5. IR Wavelength (cm'¹): 3303, 2104, 1730, 1662, 1182, 1124, 833,796,761. HR-ESI+: m/z [M+2H]/2+ = 726.2957 / 726.2941 (measured/theoretical).

Préparation of L20-C6:

352 (2R)-3-[2-[[2-[2-[[2-||4-[|(2S)-2-[[(2S)-2-[[2-[2-[2-(2azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butaiioyl]amino]-5-ureidopentanoyl]aniino]phenyl]methoxycarbonyl-methyl-amino]ethyl-methylcarbamoyl]oxyniethyl]phenyI]pyrimidin-4-yl]methoxy]phenyi|-2-[(5Sa)-5-[3-chloro-2methyl-4-[2-(4-methylpi per azin-1-y l)ethoxy] phenyl]-6-(4-fluorophenyl)thieno [2,3d]pyrimidin-4-yl]oxy-propanoic acid

Step 1: ethyl (2R)-3-[2-[[2-[2-[[2-[tert-butoxycarbonyl(melhyl)amino]ethyl-methylcarbamoyl] oxymethyl]phenyl]pyrimidin-4-yl] methoxy]phenyl]-2-[(5S_a)-5-[3-chloro2-methyl-4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl] -6-(4-fluorophenyl)thieno[2,3d]pyrimidin-4-yl] oxy-propanoate

To a solution of (ethyl (2R)-2-[(5S_a)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-lyl)ethoxy]phenyl]-6-(4“fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-[2(hydroxymethyl)phenyl]pyrimidin-4-yl] methoxy] phenyl]propano aie (50 mg, 55 pmol; synthesized according to EP 2 886 545) in dîchloromethane (0.5 mL) were successively added 4Nitrophenyl chloroformate (19 mg, 94 pmol) and DIPEA (69 pL, 0.5 mmol). The reaction mixture was stirred at room température for 1 h and tert-butyl N-methyl-N-[2(methylammo)ethyl]carbamate (54 mg, 0.287 mmol) was added. The mixture was stirred at room température ovemight, concentrated under reduced pressure. The residue was purified by silica gel chromatography (gradient of methanol in dîchloromethane) to afford ethyl (2R)-3-[2-[[2-[2[[2-[tert-butoxycarbonyl(methyl)amino]ethyl-methyl-carbamoyl]oxymethyl]phenyl]pyrimidin-4yI]methoxy]phenyl]-2-[(5S_a)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-lyl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yI]oxy-propanoate (30 mg, 27 pmol). Ή NMR (500 MHz, dmso-d6): δ 9.00 (d, IH), 8.58 (s, IH), 7.98 (m, IH), 7.61 (d, IH), 7.51 (t, IH), 7.48 (d, IH), 7.45 (t, 1 H), 7.31 (dd,2H),7.31 (d, IH), 7.22 (t, 2H), 7.18 (t, IH), 7.17 (d, IH), 7.02 (d, 1 H), 6.76 (t, 1 H), 6.32 (d, 1 H), 5.52 (dd, IH), 5.47 (br s, 2H), 5.26 (m, 2H), 4.2 (m, 2H), 4.07 (m, 2H), 3.24/3.17 (2m, 4H), 3.17/2.6 (2m, 2H), 2.77/2.64 (m, 6H), 2.7 (m, 2H), 2.49/2.28 (m, 8H), 2.12 (br s, 3H), 1.87 (s, 3H), 1.3 (3s, 9H), 1.07 (t, 3H). ^I3C NMR 353 (125 MHz, dmso-d6): δ 158.2, 152.4, 131, 130.1, 130.1, 129, 128.3, 128.2, 121.5, 121.4, 120.9, 116.3, 115.8, 112, 111.1,74.1, 69.2,68.1,65.6,61.2, 56.8, 55.2, 53.1,46.5,45.9, 34.5,32.4, 28.3, 17.4, 14.9.¹⁹FNMR (470 MHz, dmso-d6): δ -112.2. IR Wavelength (cm'¹): 1750, 1693, 1221/1160/1120, 834/756.

Step 2: 2R)-3-[2-[[2-[2-[[2-[[4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2azidoethoxy)ethoxy] ethoxy]acetyl]amino] -3-methyl-butanoyl] amino]-5-ureidopentanoyl] amino]phenyl] methoxycarbonyl-methyl-amino] ethyl-methylcarbamoyl] oxymethyl]phenyl]pyrimidin-4-yl] methoxy]phenyl]-2-[(5Sa)-5-[3-chloro2-methyl-4-[2-(4-methylpiperazin-1 -yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-4-yl] oxy-propanoic acid L20-C6

To a solution of ethyl (2R)-3-[2-[[2-[2-[[2-[tert-butoxycarbonyl(methyl)amino]ethylmethyl-carbamoyl]oxymethyl]phenyl]pyriinidin-4-yl]methoxy]phenyl]-2-[(5Sa)-5-[3-chloro-2methy 1-4-[2-(4-methylpiperazin-l-yI)ethoxy] phenyl]-6-(4-(1 uorophenyl)thieno [2,3-d]pyrimidin4-yl]oxy-propanoate (25 mg, 22 pmol) in dichloromethane (0.5 mL) was added at 0°C tritluoroacetic acid (35 pL, 447 mmol). The reaction mixture was stirred at room température for 6h and concentrated under reduced pressure. The residue was diluted with DMF (0.5 mL) and [4[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy] acetyl] amino]-3-methylbutanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl (4-nîtrophenyl)carbonate (20 mg, 22 pmol; obtaincd according to Step 3 of the préparation of L23-P3) and DIPEA (78 pL, 0.447 mmol) were successively added. The reaction mixture was stirred at room température ovemight, concentrated under reduced pressure, diluted with dioxane (0.5 mL) and a solution of lithium hydroxide monohydrate (3.7 mg, 89 pmol) in water (0.3 mL) was added. The reaction was stirred at room température ovemight, neutralîzed at 0°C by a dropwise addition of an aqueous IM HCl solution until pH7 and concentrated under reduced pressure.

The crude product was purified by C18 reverse phase prep-HPLC by direct deposit of the reaction mixture on the Xbridge column and using the NH4HCO3 method to afford L20-C6 (13 mg, 8 pmol). ^lH NMR (500 MHz, dmso-d6): δ 8.88 (m, IH), 8.54 (s, 1 H), 7.97 (d, IH), 7.77 (m, IH), 7.6 (d, 2H), 7.5 (m, IH), 7.47 (m, IH), 7.46 (m, IH), 7.41 (d, IH), 7.29 (dd, 2H), 7.21 (t, 2H), 7.19 (d, IH), 7.18 (m, 2H), 7.12 (t, IH), 6.97 (d, IH), 6.7 (t, IH), 6.19 (d, IH), 5.49 (d, IH), 5.45 (m, 4H), 5.23 (m, 2H), 4.89 (m, 2H), 4.4 (m, IH), 4.32 (dd, IH), 4.22 (m, 2H), 3.94 (s, 2H), 3.56 (m, 10H), 3.39/2.44 (m, 2H), 3.34 (t, 2H), 3.28 (m, 4H), 2.99 (m, 2H), 2.75/2.7 (m, 6H), 2.73 (m, 2H), 2.5/2.37 (m, 8H), 2.18 (s, 3H), 2.04 (m, IH), 1.81 (s, 3H), 1.74/1.62 (m, 2H), 1.46/1.38 (m, 2H), 0.86/0.8 (m, 6H). ¹³C NMR (125 MHz, dmso-d6): δ 158.3, 152.9, 131.5, 354

I31.4, 131.3, I3l, 130, 128.3, 128.3, 128, 127.7, 120.8, 119.3, 116.2, 115.6, 112.1, 111.1,75.3, 70.5, 70.2, 69.2, 67.6, 66.6, 65.4, 57.2, 56.7, 55.1/52.9, 54, 50.5, 46.5, 45.1, 39.1, 34.4, 31.5, 29.6,27.4, 19.9, 18.2, 18. ¹⁹F NMR (470 MHz, dmso-d6): δ-112.5. IR Wavelength (cm-'): 3323, 2106, 1691, 1660, 1220, 1120, 1051, 759. HR-ESI+: m/z [M+H]+= 1609.6517/ 1609.6500 (measured/theoretical ).

Préparation of L22-C1:

(2R)-2-[(5Sa)-5-[3-chloro-4-[2-|4-|[4-[|(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-lyi)ethoxy]propanoylamino|-3-niethyl-butanoyI]aniino]propanoyl|amino]phenyl]methyl]-4niethyl-piperazin-4-ium-l-yl]ethoxy]-2-methyl-phenyI]-6-(4-fluorophenyI)tliieno[2,3d]pyrimidin-4-yl|oxy-3-|2-[[2-(2-methoxyphcnyl)pyriniidin-4-yl]methoxy]phenyl]propanoîc acid;2,2,2-trifluoroacetate;2,2,2-trifluoroacetic acid

Step 1: (2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyl]amino]propanoyl]amino]phenyl] methyl]-4-methyl-piperazin-4-ium-I yl] ethoxy] -3-chloro-2-methyl-phenyl]-6-(4-fliiorophenyl)thieno[2,3-d]pyrimîdin-4yl] oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl] methoxy]phenyl]propanoic acid;2,2,2-trifluoroacetate; bis-2,2,2-trifluoroacetic acid

To a solution of 9H-fluoren-9-ylmethyl N-[(lS)-l-[[(IS)-2-[4-(hydroxymethyI)anilino]l-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate (200 mg, 0.388 mmol) in DMF (20 mL) were successively added triphenylphosphine (152 mg, 0.581 mmol) and NBromosuccinimide (103 mg, 0.581 mmol). The reaction mixture was stirred at room température overnight and (2R)-2-[(5Sa)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl]6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4yi]methoxy]phenyl]propanoic acid Cf (302 mg, 345 mmol) and DIPEA (120 pL, 0.691 mmol) were added. The reaction was stirred at room température for 2h and diethylamine (49pL, 486 mmol) was added. The reaction was stirred at room température for 24h , concentrated under reduced pressure and purified by Cl8 reverse phase prep-HPLC by direct deposit of the reaction

355 mixture on the Xbrîdge column and using the TFA method to afford (2R)-2-[(5S_a)-5-[4-[2-[4[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl]-4methyI-piperazin-4-ium-l-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate; bis-2,2,2-trifluoroacetic acid (253 mg, 0.220 mmol). 'H NMR (400 MHz, dmso-d6): δ 10,4 (s, IH), 8.89 (d, IH), 8.75 (d, IH), 8.61 (s, IH), 8.08 (large, 3H), 7.72 (d, 2H), 7.63 (d, IH), 7.52 (d, IH), 7.46 (t, IH), 7.45 (d, 2H), 7.39 (d, IH), 7.31 (dd,2H),7.2l (d, IH), 7.21 (t, 2H), 7.15 (d, IH), 7.15 (t, IH), 7.04 (t, IH), 7.01 (d, IH), 6.72 (t, IH), 6.22 (d, IH), 5.5 (dd, IH), 5.25 (m, 2H), 4.53 (m, 2H), 4.52 (m, IH), 4.28 (m, 2H), 3.76 (s, 3H), 3.62 (m, IH), 3.43/3.29 (m, 4H), 3.28/2.5 (m, 2H), 3.13/2.94 (m, 4H), 3.01 (m, 2H), 2.9 (br s, 3H), 2.07 (m, IH), 1.84 (d, 3H), 1.36 (d, 3H), 0.95 (d, 6H). ¹³C NMR (125 MHz, dmso-d6): δ 253, 158.2, 134.3, 131.5, 131.4, 131.4, 131.3, 131, 128.9, 121.1, 120.6, 119.5, 116.3, 115.9, 113, 112.3, 111.1,74.1,69.8,67.5, 58.7,57.9, 56.5,55.4,49.8,46.5,45.2, 32.9,30.4, 18.6, 18.4, 18.3. ^I9F NMR (470 MHz, dmso-d6): δ -74, -112.6.

Step 2: (2R)-2-[(5S_a)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-lyl)ethoxy]propanoylamino]-3-methylbutanoyl]amino]propanoyl] amino]phenyl] methyl]-4-methyl-piperazin-4-iiim-1 yl] ethoxy] -2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl] oxy-3-[2[[2-(2-methoxyphenyl)pyrimidin-4-yl] methoxy]phenyl]propanoic acid; 2,2,2trifluoroacetate; bis-2,2,2-trifluoroacetic acid L22-CI

To a solution of(2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyI]amino]propanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-l-yl]ethoxy]-3chloro-2-methyl-phenyI]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate; bis-2,2,2trifluoroacetic acid (150 mg, 0.130 mmol) in DMF (0.4 mL) was added (2,5-dîoxopyrrolidin-lyl) 3-[2-(2,5-dioxopyrrol-l-yl)ethoxy]propanoate (60 mg, 194 mmol). The reaction mixture was stirred at room température for 3h, concentrated under reduced pressure and purified by C18 reverse phase prep-HPLC by direct deposit of the reaction mixture on the Xbridge column and using the TFA method to afford L22-C1 (67 mg, 37 pmol). *H NMR (400 MHz, dmso-d6): δ 10.14 (s, IH), 8.88 (d, IH), 8.61 (s, IH), 8.22 (d, IH), 7.84 (d, IH), 7.73 (d, 2H), 7.63 (d, IH), 7.52 (dd, IH), 7.45 (td, IH), 7.44 (d, 2H), 7.38 (d, IH), 7.31 (dd, 2H), 7.21 (d, IH), 7.21 (t, 2H), 7.15 (t, IH), 7.14 (d, IH), 7.02 (t, IH), 7.01 (d, IH), 7 (s, 2H), 6.71 (t, IH), 6.21 (d, IH), 5.5 (dd, 1H), 5.25 (m, 2H), 4.53 (br s, 2H), 4.38 (m, IH), 4.25 (m, 2H), 4.19 (dd, IH), 3.76 (s, 3H), 3.58 356 (m, 2H), 3,54 (t, 2H), 3,48 (m, 2H), 3,43/3.3 (m, 4H), 3.28/2.51 (m, 2H), 3,16/2.98 (m, 4H), 3.04 (ni, 2H), 2.91 (br s, 3H), 2.43/2.33 (m, 2H), 1.93 (m, IH), 1.84 (s, 3H), 1.31 (d, 3H), 0.87/0.82 (m, 6H). ^,3CNMR(100 MHz, dmso-d6): δ 158, 152.8, 135.2, 134, 131.4, 131.3, 131.3, 131.2, 131, 128.9, 120.8, 120.6, 119.3, 116.3, 115.8, 112.4, 112.3, 111.1, 74.2, 69.6, 67.4, 67.4, 67.1, 67, 58.4, 57.9, 56.2,55.2, 49.7, 46.5,45.1,37.1, 36.3,32.7, 30.9, 19.6, 18.5, 18.2, 18.2. ¹⁹F NMR (376 MHz, dmso-d6): δ -74.6, -112.2. IR Wavelength (cm·¹): 2000-3500, 1760/1705, 1733, 1668, 1180/1128, 829/798/758/720/696. HR-ESI+: m/z [M+H]+= 1345.4944/ 1345.4954 (measured/theoretical)

Préparation of L9-C9:

3-[4-[[2-[(2R)-2-carboxy-2-[(5Sa)-5-|3-chloro-4-[2-[4-[{4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5 dioxopyrrol-l-yl)ethoxy]propanoylammo]-3-methyl-biitanoyl]aniino]“5-ureidopentanoyl]amino]phenyl]methyl]-4-methyl-pipcrazin-4-ium-l-yl]etlioxy]-2-methyl-phenyl]6-(4-nuorophenyl)thieno[2,3-d]pyrimidni-4-yl]oxy-ethyl]phenoxy]methyl]pyrimidin-2 yljbenzenesulfonate; 2,2,2-trifluoroacetate; 2,2,2-trifluoroacetic acid

Step 1 : (2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-1 -yl)ethoxy]propanoylamino] -3-methylbutanoyl] amino] -N-[4-(hydroxymethyl)phenyl]-5-ureido-pentanamide

To a solution of 3-[2-(2,5-dioxopyrrol-l-yl)ethoxy]propanoic acid (855 mg, 4.01 mmol) in THF (42 mL) were added N,N’-dicyclohexylmethanediimine (1.05 g, 5.08 mmol) and 1hydroxypyrroIîdine-2,5-dione (510 mg, 4.43 mmol). The reaction mixture was stirred at room température for 20 h. The precipitate was removed by filtration and the filtrate added to a solution of (2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-N-[4-(hydroxymethyl)phenyl]-5ureido-pentan amide (1.27 g, 3.35 mmol) in DMF (42 mL). The réaction mixture was stirred at room température for 20 h, diluted with diethyl ether (250 mL). The solid was recovered by filtration to afford (2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-l-yl)ethoxy]propanoylamino]-3

357 methyl-butanoyl]amino]-N-[4~(hydroxymethyi)phenyl]-5-ureido-pentanamide ¢1.81 g ; 3.15 mmol) as a white solid. ^JH NMR (400 MHz, dmso-d6); Ô 9.87 (s, 1H), 8.05 (d, 1H),7.82 (d, 1H), 7.53 (d, 2H), 7.21 (d, 2H), 7.00 (s, 2H), 5.95 (t, 1H), 5.39 (s, 2H), 5.07 (t, 1H), 4.41 (d, 2H), 4.34-4.40 (m, IH), 4.18-4.22 (m, 1H), 3.42-3.65 (m, 4H), 2.88-3.02 (m, 2H), 2.73 (s, 2H), 2.282.45 (m, 2H), 1.91-1.99 (m, 1H), 1.53-1.75 (m, 2H), 1.30-1.147 (m, 2H), 0.85 (d, 3H), 0.81 (d, 3H). ^I3C NMR (125 MHz, dmso-d6): δ 171.05, 170.83, 170.32, 170.09, 158.82, 137.49, 137.37, 134.50, 126.88, 118.81, 66.66, 66.53, 62.57, 57.49, 53.06, 36.74, 35.76, 30.51, 29.31, 26.79, 25.20, 19.16, 18.07. MS (ESI) m/z [M + H]+= 575.2.

Step 2: (2S)-N-]4-(bromomethyl)phenyl] -2-[[(2S)-2-[3-[2-(2,5~dîoxopyrrol-lyl)ethoxy]propanoylamino]-3-methyl~butanoyl] amino]-5-ureido-pentanamide

To a solution of (2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-l-yl)ethoxy]propanoylamino]-3methyl-butanoyl]amino]-N-[4-(hydroxymethyl)phenyl]-5-ureido-pentanamide (37.2 mg, 65 pmol) in THF (1 mL) was added dropwise at 0°C under argon phosphores tribromide (45 pL, 97 mmol). The reaction was stirred at 0°C for 1 h and at room température for 2h. The progress of the reaction was followed by UPLC-MS: an aliquot was treated by a large excess of morpholine in acetonitrile, following the formation of the corresponding morpholine adduct. The reaction was diluted with THF (3 mL), quenched by addition of 2 drops of a saturated solution of NaHCO₃, stirred for 5 min at room température, dried over Magnésium sulfate and filtered. The residue, containing the crude (2S)-N-[4-(bromomethyl)phenyl]-2-[[(2S)-2-[3-[2-(2,5dioxopynOl-l-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentanamide (45 mg, 65pmol theoretical) was used immediately in the next step. MS (ESI) m/z [M + H]+ =662.62 (morpholine adduct)

Step 3: 3-]4-[[2-[(2R)-2-carboxy-2-](5S_a)-5-[3-chloro-4-[2-[4-]]4-][(2S)-2-]](2S)-2-[3-]2(2,5-dioxopyrrol-l-yl)ethoxy]propanoylamino]-3-methyl-butanoyl] amino] -5-ureidopentanoyl] amino]phenyl] methyl] -4-methyl-piperazin-4~ium~l -yl] ethoxy]-2-methylphenyl]-6-(4-fluorophenyl)thieno]2,3-d]pyrimidin-4-yl]oxyethyl]phenoxy]methyl]pyrimidin-2-yl]benzenesulfonate L9-C9

To a solution of (2R)-2-{[(55₀)-5-{3-chloro-2-methyl-4-[2-(4-methyIpiperazin-lyl)ethoxy]phenyl}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2-(3sulfophenyl)pyrimidîn-4-yl]methoxy}phenyl)propanoic acid C9 (15 mg, 16 mmol) in DMF (0.8 mL) was added a solution of (2S)-N-[4-(bromomethyl)phenyI]-2-[[(2S)-2-[3-[2-(2,5dioxopyrrol-1 -yl)ethoxy]propanoyl amino]-3-methyl-butanoyl] amino]-5-ureido-pentanamide (45

358 mg crude, 65 pmol theoretical from step 2) în THF (l mL) and DIPEA (l4pL, 81 pmol). The reaction was stirred at room température heated at 50°C for 2h. The crude product was purified by C18 reverse phase prep-HPLC by direct deposit of the reaction mixture on the Xbridge column and using the TFA method to afford L9-C9 (5.2 mg, 3.5 pmol). HR-ESI+: m/z [Μ+Η]+ = 1481.4917/ 1481.4896 (measured/theoretical).

Préparation of L9-C13:

(2R)-2-]6-(3-amÎno-4,5-difluoro-phenyl)-(5Sa)-5-[3-chloro-4-[2-[4-][4-][(2S)-2-[[(2S)-2-[3-[2(2,5-dioxopyrrol-l-yI)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureidopentanoyl] amino] phenyl]methyl]-4-methyl-piperazin-4-ium-l-yl|ethoxy]-2-inethylphenyl]thieno|2,3-d]pyrimidin-4-yI]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4yl]methoxy|phenyl]propanoic acid;2,2,2-trifluoroacetate;2,2,2-tnfluoroacetic acid

The procedure is as in the process of synthesis of L9-C9, replacing C9 used in Step 3 by (2R)-2-{[(55,,)-6-(3-amino-4,5-difluorophenyl)-5-{3-chloro-2-inethyl-4-[2-(4-methylpiperazin-1 yl)ethoxy]phenyl}thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2-(2-methoxyphenyl)pyrimidin-4yl]methoxy}phenyl)propanoic acid C13 and using TFA method for purification. 'H NMR (400 MHz, dmso-d6): Ô 10.2 (s), 8.9 (d, IH), 8.6 (s, 1H), 8.12 (d), 7.8 (d), 7.7 (d, 2H), 7.6 (d, 1H), 7.5 (d, 1H), 7.45 (d, 2H), 7.42 (m, 1H), 7.32 (d, 1H), 7.2 (d, 1H), 7.18 (m, lH),7.18(m, 1H), 7.02 (d, 1H), 7 (s, 2H), 7 (m, 1H), 6.75 (t, 1H), 6.65 (d, 1H), 6.25 (d, 1H), 6.15 (dd, 1H), 5.98 (m, 1H), 5.48 (dd, 1H), 5.4 (br s, IH), 5.24 (dd, 2H), 4.51 (br s, 2H), 4.38 (m, 1H), 4.28 (m, 2H), 4.22 (m, IH), 3.80-3.40 (m, 8H), 3.75 (s, 3H), 3.26 (m, 4H), 3.1 (m, 2H), 2.98 (m, 4H), 2.9 (br s, 3H), 2.9/2.5 (2m, 2H), 2.43/2.3 (2m, 2H), 1.92 (m, IH), 1.88 (s, 3H), 1.70-1.30 (m, 4H), 0.82 (2d, 6H). ¹⁹F NMR (470 MHz, dmso-d6): δ -74.3, -139.3, -160.4. HR-ESI+: m/z [M+H]+ = 1464.5482 / 1464.5449 (measured/theoretical).

Préparation of L14-C3:

359 (2S,3S,4R,5R,6S)-6-[2-[(5Sa)-5-|[(2S)-2-[[(2S)-2-[[2-(2-azidoetlioxy)acetyl]amino]-3-methylbutanoyl] amino] propanoyl]amino]-2-[(4-[2-[4-[4-|(lR)-l-carboxy-2-[2-[[2-(2methoxyphenyI)pynmidin-4-yl|methoxy]phenyl]ethoxy]-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl]-2-chloro-3-methyl-phenoxy]ethyI]piperazine-lcarbonyl]oxymethyl]pheiiyl]ethyl]-3,4,5-trihydroxy-te1:rahydropyran-2-carboxyIic acid

OH OH

Step 1: 2-iodo-4-mtro-benzoic acid

To a solution of 2-amino-4-nitro-benzoic acid (10.0 g, 54.90 mmol) in acetonitrile (280 mL) was added p-toluenesulfonic acid monohydrate (32.0 g, 168.2 mmol). The mixture was stirred at room température for 15 min, then a solution of sodium nitrite (8.00 g, 115.9 mmol) and potassium iodide (24.0 g, 144.6 mmol) in solution in water (140 mL) were added dropwise in 15 min. The reaction mixture was stirred for 19 h. After completion of the reaction, the mixture was quenched with sodium thiosulfate (13.02 g, 82.36 mmol) and acidified with an aqueous solution of hydrogen chloride 3 M (25 mL). The aqueous layer was extracted with ethyl acetate (2 x 250 mL) and the combined organic layers were washed with an aqueous solution of hydrogen chloride 1 Μ (100 mL), dried over sodium sulfate, filtered and concentrated to dryness. The resulting residue was taken up in dichloromethane (1 L) and was washed with an aqueous solution of HCl 1 M (100 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to dryness to afford 2-iodo-4-nitro-benzoîc acid (15.0 g, 51.2 mmol) as an orange powder. Ή NMR (400 MHz, dmso-d6): δ 13.8 (brs, IH), 8.64 (s, IH), 8.27 (d, IH), 7.86 (d, IH).

Step 2: (2-iodo-4-nitro-phenyl)methanol

To a solution of 2-iodo-4-nîtro-benzoic acid (5.0 g, 17.06 mmol) in THF (70 mL) was added a solution of borane 1 M in THF (85 mL, 85.0 mmol). The reaction mixture was stirred at 65°C for 4 h. After the completion of the reaction, the reaction mixture was cooled to room température and was quenched with the addition of methanol (200 mL). The mixture was stirred at room température for 30 min, then was concentrated to dryness. The crude product was

360 purified by silica gel chromatography (gradient of ethyl acetate in cyclohexane) to afford (2iodo-4-nitro-phenyl)methanol (3.38 g, 12.11 mmol) as a yellow solid. ^lH NMR (400 MHz, dmso-d6): δ 8.54 (d, IH), 8.29 (dd, IH), 7.70 (d, IH), 5.82 (t, IH), 4.47 (d, 2H).

Step 3: (4-amino-2-iodo-phenyl)methanol

To a solution of (2-iodo-4-nitro-phenyl)methanol (3.70 g, 13.26 mmol) in éthanol (100 mL) and water (25 mL) were successively added iron (3.70 g, 66.25 mmol) and ammonium chloride (800 mg, 14.96 mmol). The reaction mixture was stirred for 3 hours at 80°C. After completion of the reaction, the reaction mixture was filtered over Celite®, washed with éthanol and concentrated to dryness. The resulting residue was taken up in ethyl acetate (100 mL) and washed with a saturated solution of sodium hydrogen carbonate (100 mL). The organic layer was dried over sodium sulfate, filtered and concentrated to dryness to afford (4-amino-2-iodophenyl)méthanol (2.48 g, 9.95 mmol) as a yellow oil. 'H NMR (400 MHz, dmso-d6): δ 7.02-7.10 (m, 2H), 6.57 (d, IH), 5.16 (s, 2H), 4.97 (t, IH), 4.28 (d, 2H).

Step 4: 4-][tert-butyl(dimethyl)silyl]oxymethyl]-3-iodo-aniline

To a solution of (4-amino-2-iodo-phenyl)methanol (3.51 g, 13.37 mmol) in dichloromethane (150 mL) was added imîdazole (0.95 g, 13.95 mmol). The mixture was cooled to 0°C, then a solution of tert-butyl-chioro-dimethyl-silane (2.40 mL, 13.85 mmol) in dichloromethane (150 mL) was added dropwise over 15 minutes. The ice bath was removed, and the reaction mixture was stirred at room température for 16 h. After completion of the reaction, the reaction mixture was quenched with methanol (20 mL) and concentrated to dryness. The crude product was purified by silica gel chromatography (gradient of ethyl acetate in cyclohexane) to afford 4-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-iodo-anilme (3.64 g ; 10.03 mmol ; 75%) as a yellow oil. ^lH NMR (400 MHz, dmso-d6): δ 7.05 (s, IH), 7.03 (d, IH), 6.55 (d, IH), 5.24 (s, 2H), 4.46 (s, 2H), 0.88 (s, 9H), 0.06 (s, 6H).

Step 5: (2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl] amino]propanoic acid

To a solution of (2S)-2-aminopropanoîc acid (3.22 g, 36.09 mmol) in water (90 mL) were successively added sodium carbonate (7.29 g, 68.74 mmol) and a solution of (2,5dioxopyrrolidin-l-yl) (2S)-2-(9H-fluoren-9-ylmethoxycarbonyIamino)-3-methyl-butanoate (15.0 g, 34.37 mmol) in dimethoxyethane (90 mL). The reaction mixture was stirred for 16 h at room température. After completion of the reaction, the mixture was acidîfied until pH=l with an aqueous solution of hydrogen chloride 1 M, then the aqueous layer was extracted with ethyl 361 acetate (3 x 500 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated to dryness to afford the crude mixture which was triturated with diethyl ether (50 mL) to afford (2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylaniîno)-3-methyΙΟ utanoyl] amino] propanoîc acîd (11.25 g, 27.41 mmol) as a white solid. ^!H NMR (400 MHz, dmso-d6) δ 12.48 (s, IH), 8.21 (d, IH), 7.89 (d, 2H), 7.72-7.79 (m, 2H), 7.28-7.46 (m, 5H), 4.154.32 (m, 4H), 3.90 (t, IH), 1.90-2.02 (m, IH), 1.28 (d, 3H), 0.86-0.90 (m, 6H).

Step 6: 9H-Jluoren-9-ylmethyl N-[(lS)-l-[](lS)-2-[4-[[tert-butyl(dimethyl)silyl]oxymethyl]-3iodo-anilino] -1-methyl-2-oxo-ethyl] carbamoyl] -2-methyl-propyl] carbamate

To a solution of (2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl]amino]propanoîc acid (1.50 g, 3.65 mmol) in dichloromethane (18 mL) and methanol (18 mL) were successively added 4-[[tert-butyI(dimethyl)silyI]oxymethyi]-3-iodo-aniline (1.33 g, 3.65 mmol) and ethyl 2-ethoxy-2H-quînolîne-l-carboxylate (1.36 g, 5.48 mmol). The colorless suspension was stirred for 16 h at room température. After concentration to dryness, the crude product was purified by silica gel chromatography (gradient of ethyl acetate in cyclohexane) and then by Cl 8 chromatography (gradient of methanol in water) to afford 9Hfluoren-9-ylmethyl N-[(] S)-l-[[(l S )-2-[4-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-iodo-anilino] l-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate (1.18 g, 1.56 mmol) as a white solid. Ή NMR (400 MHz, dmso-d6): δ 10.05 (s, IH). 8.16-8.24 (m, 2H), 7.88 (d, 2H), 7.71-7.77 (m, 2H), 7.55 (d, IH), 7.37-7.48 (m, 3H), 7.27-7.37 (m, 3H), 4.56 (s, 2H), 4.38 (t, IH), 4.18-4.33 (m, 3H), 3.91 (t, IH), 2.08-2.20 (m, IH), 1.30 (d, 3H), 0.83-0.95 (m, 15H), 0.06 (s, 6H).

Step 7: (3R,4S,5R,6R)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran-2-one

A suspension of (3R,4S,5R,6R)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran2-ol (30.0 g, 55.49 mmol) in DMSO (120 mL) was stirred for 30 min at room température (until full solubilisation) then acetic anhydride (90 mL) was added dropwise at room température over 15 min. The beige solution was stirred for 16 h then was cooled to 0°C and an aqueous solution of hydrogen chloride 1 M (100 mL) was siowly added. The reaction mixture was stirred for 20 ni in at room température then acetic acid was evaporated. The resulting residue was diluted with water (200 mL) and ethyl acetate (200 mL). The aqueous layer was extracted with ethyl acetate (2 x 200 mL) and the combined organic layers were washed with water (2 x 500 mL), with a saturated solution of sodium hydrogen carbonate (2 x 500 mL), then dried over sodium sulfate, filtered and concentrated to dryness to afford the crude mixture. The crude product was purified by silica gel chromatography (gradient of ethyl acetate in cyclohexane) to afford (3R,4S,5R,6R)362

3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran-2-one (25.05 g, 46.51 mmol) as a colorless oil. Ή NMR (400 MHz, dmso-d6): δ 7.19-7.39 (m, 20H), 4.85 (d, 1H), 4.57-4.72 (m, 5H), 4.46-4.56 (m, 3H), 4.36 (d, lH), 3.98-4.05 (m, 1H), 3.84-3.92 (m, 1H), 3.65-3.76 (m, 2H).

Step 8: (3R,4S,5R,6R)-3,4,5-tribenzyloxy-6-(benzyloxymethy 1)-2-(2trimethylsilylethynyl)tetrahydropyran-2-ol

To a solution of trimethylsilylacetylene (24 mL, 168.6 mmol) in THF (325 mL) was added in 20 min at -78°C a solution of butyllithium 2.5 M in hexane (59.41 mL, 148.5 mmol). The colorless solution was stirred for 45 min at -78°C and then 45 min at 0°C. The réaction mixture was cooled to -78°C and a solution of (3R,4S,5R,6R)-3,4,5-tribenzyloxy-6(benzyloxymethyl)tetrahydropyran-2-one (25.0 g, 46.41 mmol) in THF (325 mL) was added dropwise over 45 min. The reaction mixture was stirred for 4 h at this température then was quenched with water (200 mL). The aqueous layer was extracted with ethyl acetate (2 x 200 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated to dryness to afford (3R,4S,5R,6R)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)-2-(2tnmethylsilylethynyl)tetrahydropyran-2-ol (29.56 g, 46.41 mmol) as a beige oil containing the two diastereoisomers in a ratio 4/6. ^lH NMR (400 MHz, dmso-d6): δ 7.13-7.43 (m, 20H), 4.874.99 (m, 1H), 4.65-4.83 (m, 4H), 3.43-3.57 (m, 3H), 3.70-3.85 (m, 2H), 3.55-3.68 (m, 3H), 3.433.53 (m, 2H), 0.11-0.22 (m, 9H).

Step 9: trimethyl-[2-[(2S, 3S, 4R, 5R, 6R)-3,4,5-tribenzyloxy-6(benzyloxymethyl)tetrahydropyran-2-yl] ethynyl] silane

To a solution of (3R,4S,5R,6R)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)-2-(2trimethylsilylethynyl)tetrahydropyran-2-ol (29.56 g, 46.42 mmol) in acetonitrile (83 mL) and dichloromethane (193 mL) were added in 20 min at -15°C a solution of triethylsilane (44.98 mL, 278.5 mmol) in a mixture of acetonitrile/dichloromethane (37 mL/18 mL) followed by a solution of boron trifluoride diethyl etherate (23.53 mL, 185.7 mmol) in acetonitrile (37 mL) over 30 min at -15°C. The colorless solution was stirred for 5 h at the same température, then was diluted with water (500 mL). The aqueous layer was extracted with ethyl acetate (2 x 500 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated to dryness to afford trimethyl-[2-[(2S,3S,4R,5R,6R)-3,4,5-tribenzyloxy-6-(benzyloxymethyl)tetrahydropyran2-yl]ethynyl]silane (28.82 g, 46.41 mmol) as a brown oil. ^lH NMR (400 MHz, dmso-d6): δ 7.107.44 (m, 20H), 4.93 (d, 1H), 4.67-4.86 (m, 4H), 4.43-4.57 (m, 3H), 4.16-4.28 (m, 1H), 3.42-3.68 (m, 6H), 0.15 (s, 9H).

363

Step 10: (2R, 3 R, 4R,5S, 6S)-3,4,5-tribenzyloxy-2-(benzyloxymethyl)-6-elhynyl-tetrahydropyran

To a solution of trimethyl-[2-[(2S,3S,4R,5R,6R)-3,4,5-tribenzyloxy-6(benzyloxymethyl)tetrahydropyran-2-yl]ethynyl]silane (28.80 g, 46.39 mmol) in methanol (1.12 L) and dichloromethane (240 mL) was added an aqueous solution of sodium hydroxide 1 M (80 mL). The beige solution was stirred for 1 h at room température then was acidified until pH = 1 with an aqueous solution of hydrogen chloride 1 M and diluted with water (500 mL). The methanol was evaporated and then the aqueous layer was extracted with ethyl acetate (2 x 1 L). The combined organic layers were dried over sodium sulfate, fîltered and concentrated to dryness. The crude product was purified by silica gel chromatography (gradient of ethyl acetate in cyclohexane) to afford (2R,3R,4R,5S,6S)-3,4,5-tribenzyloxy-2-(benzyloxymethyl)-6-ethynyltetrahydropyran (20.00 g, 36.45 mmol) as a coiorless oil. 'H NMR (400 MHz, dmso-d6): δ 3.423.67 (m, 7H), 4.17 (d, IH), 4.44-4.56 (m, 3H), 4.67-4.86 (m, 4H), 4.90 (d, IH), 7.15-7.40 (m, 20H).

Step 11 : (2S, 3R, 4R, 5S, 6R)-2-ethynyl-6-(hydroxymethyl)tetrahydropyran-3,4,5-triol

To a solution of (2R,3R,4R,5S,6S)-3,4,5-tribenzyloxy-2-(benzyloxymethyl)-6-ethynyJtetrahydropyran (20.00 g, 36.45 mmol) in ethanthiol (400 mL) was added dropwise at room température over 5 min, boron trîfluorîde diethyl etherate (147.8 mL, 1166 mmol). The beige solution was stirred for 16 h at room température, then was cooled to 0°C and equipped with a gas trap containing an aqueous saturated solution of sodium hypochlorite. A saturated aqueous solution of sodium hydrogen carbonate (500 mL) was added dropwise at 0°C in 1 h (formation of carbon dioxide). After concentration to dryness, the crude product was purified by silica gel chromatography (gradient of methanol in dichloromethane) to afford (2S,3R,4R,5S,6R)-2ethynyl-6-(hydroxymethyl)tetrahydropyran-3,4,5-triol (4.05 g, 21.52 mmol) as a white solid. 'H NMR (400 MHz, dmso-d6): δ 5.28 (d, IH), 4.99 (d, IH), 4.91 (d, IH), 4.52 (t, IH), 3.77 (d, IH), 3.60-3.69 (m, IH), 3.35-3.43 (m, IH), 3.32 (s, IH), 2.97-3.13 (m, 4H).

Step 12 : methyl (2S, 3S, 4R, 5R, 6S)-6-ethynyl-3,4,5-trihydroxy-tetrahydropyran-2-carboxylate

To a solution of (2S,3R,4R,5S,6R)-2-ethynyl-6-(hydroxymethyl)tetrahydropyran-3,4,5triol (4.05 g, 21.52 mmol) in a saturated aqueous solution of sodium hydrogen carbonate (81 mL) and THF (81 mL) was added (2,2,6,6-tétraméthylpipéridin-l-yl)oxyl (168 mg, 1.08 mmol). The yellow suspension was cooled to 0°C and 1,3-dibromo-5,5-dimethyl-imidazolidine-2,4dîone (12.31 g, 43.04 mmol) was added portionwise in 30 min. The reaction mixture was stirred for 4 h at 0°C then was quenched with the addition of methanol (40 mL). After 30 min stirring at

364 this température, a saturated aqueous solution of potassium carbonate (10 mL) and dîchloromethane (l 00 mL) were added. The organic layer was extracted with water (2 x 200 mL) then the combined aqueous layers were acidified until pH = l with an aqueous solution of hydrogen chloride 3M and concentrated to dryness. The resulting residue was taken up in methanol (100 mL) and in an aqueous solution of hydrogen chloride 3M (20 mL). The mixture was concentrated to dryness and co-evaporated several times with methanol (4 x 100 mL). The crude product was purified by silica gel chromatography (gradient of methanol in dîchloromethane Cérium developer) to afford methyl (2S,3S,4R,5R,6S)-6-ethynyl-3,4,5trihydroxy-tetrahydropyran-2-carboxylate (3.00 g, 13.88 mmol) as a beige solid. 'H NMR (400 MHz, dmso-d6): δ 5.46 (d, IH), 5.32 (d, IH), 5.18 (d, IH), 3.93-4.00 (m, IH), 3.75 (dd, IH), 3.65 (s, 3H), 3.40-3.44 (m, IH), 3.31 (s, IH), 3.09-3.19 (m, 2H).

Step 13: methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-ethynyl-tetrahydropyran-2-carboxylate

To a solution of methyl (2S,3S,4R,5R,6S)-6-ethynyl-3,4,5-trihydroxy-tetrahydropyran-2carboxylate (3.00 g, 13.88 mmol) in DMF (37.5 mL) and pyridine (12.5 mL) was added N,Ndimethylpyridin-4-amine (84.8 mg, 0.693 mmol). The reaction mixture was cooled to 0’C then acetic anhydride (20.0 mL, 213 mmol) was added dropwise in 5 min. The colorless solution was stirred for 3 h at room température then was diluted with an aqueous solution of hydrogen chloride 1 M (200 mL). The aqueous layer was extracted with ethyl acetate (2 x 200 mL). The combined organic layers were washed with an aqueous solution of hydrogen chloride 1 M (2 x 200 mL), tbllowed with a saturated aqueous solution of potassium carbonate (200 mL), then dried over sodium sulfate, fïltered and concentrated to dryness to afford the crude mixture. The crude product was purified by silica gel chromatography (gradient of ethyl acetate in cyclohexane cérium developer) to afford methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-ethynyltetrahydropyran-2-carboxylate (4.60 g, 13.44 mmol) as a white solid. ^lH NMR (400 MHz, dmsod6): Ô 5.33 (t, IH), 4.93-5.01 (m, 2H), 4.70 (d, IH), 4.44 (d, IH), 3.67 (s, IH), 3.64 (s, 3H), 2.02 (s, 3H), 1.94-2.01 (m, 6H).

Step 14: methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-[[tertbutyl(dimethyl)silyl] oxymethyl]-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9ylmethoxycarbonylamino)-3-methyl~ butanoyl]amino]propanoyl] amino]phenyl]ethynyl] tetrahydropyran-2-carboxylate To a solution of methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-ethynyl-tetrahydropyran-2carboxylate (496 mg, 1.45 mmol) in DMF (7.3 mL) were successïvely added 9H-fluoren-9365 ylmethyl N-[( I S)-1 -[[(l S)-2-[4-[[tert-butyl(dirnethyI)silyl]oxymethyl]-3-iodo-anilino]-1 -methyl2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate (730 mg, 0.966 mmol), DTPEA (738 pL, 4.47 mmol), copper iodide ( 18.4 mg, 96.6 mmol) and dichloro-bis(triphenylphosphine)palladium(n) (67.8 mg, 96.6 mmol). The yellow solution was flushed with Argon then was stirred for 16 h at room température. After dilution with water (100 mL), the aqueous layer was extracted with ethyl acetate (2 x 100 mL). The combined organic layers were washed with water (2 x 200 mL), and with a saturated aqueous solution of ammonium chloride (2 x 200 mL), then dried over sodium sulfate, filtered and concentrated to dryness. The crude product was purified by silica gel chromatography (gradient of ethyl acetate in cyclohexane) to afford methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-[[tert-butyl(dimethyI)silyl]oxymethyl]5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]ethynyl]tetrahydropyran-2-carboxylate (782 mg, 0.806 mmol) as a yellow solid. Ή NMR (400 MHz, dmso-d6): Ô 10.09 (s, IH). 8.20 (d, IH), 7.89 (d, 2H), 7.70-7.78 (m, 3H), 7.55 (d, IH), 7.32-7.46 (m, 4H), 7.27-7.32 (m, 2H), 5.41 (t, IH), 4.965.14 (m, 3H), 4.67 (s, 2H), 4.51 (d, IH), 4.36-4.44 (m, IH), 4.16-4.32 (m, 3H), 3.88-3.95 (m, IH), 3.64 (s, 3H), 1.94-2.07 (m, 10H), 1.30 (d, 3H), 0.84-0.93 (m, 15H), 0.08 (s, 6H).

Step 15: methyl (3S,4R, 5S, 6S)-3,4,5-triacetoxy-6-[2-[2-[[tert-butyl(dimethyl)silyl] oxymethyl] 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl] amino]propanoyl] amino]phenyl] ethyl] tetrahydropyran-2-carboxylate

A solution of methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-[[tertbutyl(dimethyl)silyl]oxymethyl]-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3methyl-butanoyl]amino]propanoyl]amino]phenyl]ethynyl]tetrahydropyran-2-carboxylate (750 mg, 0.773 mmol) in THF (15 mL) was flushed with Argon. Dry Platinum 5% on carbon (75 mg, 50% ^w/w) was added. The reaction mixture was successively flushed with argon, with H2 and was stirred for 16 h at room température under H2 atmosphère (P atm). The reaction mixture was filtered through a Celite® pad, washed with THF then concentrated to dryness. The complété sequence, (addition of dry platinum 5% on carbon (75 mg, 50% ^w/w), stirring for 16 h at room température under H₂ atmosphère (1 bar) and filtration through a Celite® pad), was perfonned 4 more times. The crude product was purified by silica gel chromatography (gradient of ethyl acetate in cyclohexane) to afford methyl (3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-[[tertbutyl(dimethyl)silyl]oxymethyl]-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3methyl-butanoyl]amino]propanoyI]amino]phenyl]ethyl]tetrahydropyran-2-carboxylate (470 mg, 0.483 mmol) as a white solid. *H NMR (400 MHz, dmso-d6): δ 9.90 (s, IH), 8.16 (d, IH), 7.89 366 (d, 2H), 7.70-7.7S (m, 2H), 7.37-7.49 (m, 4H), 7.27-7.32 (m, 3H), 7.23 (d, IH), 5.29 (t, IH), 4.95 (t, IH), 4.78 (t, IH), 4.60 (s, 2H), 4.34-4.44 (m, 2H), 4.16-4.32 (m, 3H), 3.88-3.95 (m, IH), 3.723.79 (m, IH), 3.64 (s, 3H), 2.69-2.78 (m, IH), 2.50-2.60 (m, IH), 1.92-2.03 (m, l OH), 1.55-1.75 (m, 2H), 1.30 (d, 3H), 0.84-0.93 (m, 15H), 0.05 (s, 6H).

Step 16: methyl (3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-[[(2S)-2-[[(2S)-2-(9H-Jluoren-9ylmethoxycarbonylamino)-3-methyl-butanoyl] amino]propanoyl] amino]-2(hydroxymethyl)phenyl] ethyl] tetrahydropyran-2-carboxylate

To a solution of methyl (3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-[[tertbutyl(dimethyl)silyl]oxymethyl]-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3methyl-butanoyl]amino]propanoyl]amino]phenyl]ethyl]tetrahydiOpyran-2-carboxylate (470 mg, 0.483 mmol) in THF (540 pL) and water (540 pL) was added acetic acid ( 1.6 mL, 28.28 mmol). The colorless solution was stirred for 16 h at room température then diluted with water (100 mL). The aqueous layer was extracted with ethyl acetate (2 x 100 mL). The combined organic layers were washed with water (2 x 200 mL), and with a saturated aqueous solution of sodium hydrogen carbonate (200 mL), then were dried over sodium sulfate, filtered and concentrated to dryness. The crude product was purified by silica gel chromatography (gradient of ethyl acetate în cyclohexane) to afford methyl (3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-[[(2S)-2-[[(2S)-2-(9Hfluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]-2(hydroxymethyl)phenyl]ethyl]tetrahydropyran-2-carboxylate (354 mg, 0.412 mmol) as a white solid. Ή NMR (400 MHz, dmso-d6): δ 9.87 (s, 1 H), 8.16 (d, IH), 7.89 (d, 2H), 7.70-7.78 (m, 2H), 7.37-7.50 (m, 4H), 7.27-7.37 (m, 3H), 7.25 (d, IH), 5.29 (t, IH), 4.91-4.98 (m, 2H), 4.78 (t, IH), 4.34-4.44 (m, 4H), 4.16-4.32 (m, 3H), 3.88-3.95 (m, IH), 3.72-3.79 (m, IH), 3.64 (s, 3H), 2.64-2.73 (m, IH), 2.50-2.60 (m, IH), 1.92-2.03 (m, 10H), 1.69-1.79 (m, IH), 1.52-1.65 (m, IH), 1.30 (d, 3H), 0.84-0.93 (m, 6H).

Step 17: methyl (3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-]5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9ylmethoxycarbonylamino)-3-methyl-biitanoyl] amino]propanoyl] amino]-2-](4nitrophenoxy)carbonyloxymethyl]phenyl] ethyl] tetrahydropyran-2-carboxylate

To a solution of methyl (3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-[[(2S)-2-[[(2S)-2-(9Hfluoren-9-ylmethoxycarbonylammo)-3-methyl-b utanoyl] ami no]propanoyl] amino]-2(hydroxymethyl)phenyl]ethyl]tetrahydropyran-2-carboxylate (310 mg, 0.361 mmol) in THF (7.75 mL) were successively added pyridine (146 pL, 1.80 mmol) and 4-Nitrophenyl chloroformate (182 mg, 0.901 mmol). The white suspension was stirred for 16 h at room

367 température then was concentrated to dryness to afford the crude mixture. The crude product was purified by silica gel chromatography (gradient of ethyl acetate in dichloromethane) to afford methyl (3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9ylmethoxycarbonylamino)-3-methyl-butanoyI]amino]propanoyl]aniino]-2-[(4mtrophenoxy)carbonyloxymethyl]phenyl]ethyl]tetrahydropyran-2-carboxylate (257 mg, 0.251 mmol) as a white solid. Ή NMR (400 MHz, dmso-d6): δ 10.04 (s, IH), 8.31 (d, 2H), 8.20 (d, IH), 7.89 (d, 2H), 7.66-7.78 (m, 2H), 7.56 (d, 2H), 7.28-7.52 (m, 8H), 5.31 (t, IH), 5.25 (s, 2H), 4.96 (t, IH), 4.79 (t, IH), 4.40 (d, 2H), 4.16-4.32 (m, 3H), 3.88-3.95 (m, IH), 3.74-3.83 (m, IH), 3.61 (s, 3H), 2.74-2.84 (m, IH), 2.60-2.71 (m, IH), 1.90-2.03 (m, 10H), 1.72-1.83 (m, IH), 1.581.71 (m, IH), 1.30 (d, 3H), 0.82-0.94 (m, 6H). LC-MS: MS (ESI) m/z [M+Na]+= 1047.6.

Step 18: (2R)-2-[(5S_a)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9ylmethoxycarbonylamino)-3-methyl-butanoyl] amino]propanoyl] amino] -2-[2] (2SR, 3SR, 4 RS, 5SR, 6SR)-3,4,5-triacetoxy-6-methoxycarbonyl-tetrahydropyran-2yl] ethyl]phenyl] methoxycarbonyl]piperazin-1 -yl] ethoxy]-2-methyl-phenyl]-6-(4duorophenyljthieno]2,3-d]pyrimidin-4-yl] oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin4-yl]methoxy]phenyl]propanoic acid

To a solution of (2R)-2-[(5Sa)-5-[3-chloro-2-methyl-4-(2-piperazin-l-ylethoxy)phenyl]6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yI]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4yl]methoxy]phenyl]propanoic acid (C3) (118 mg, 0.121 mmol) in dimethylformamide (3.0 mL) were successively added a solution of methyl (3S,4R_}5S,6S)-3,4_s5-triacetoxy-6-[2-[5-[[(2S)-2[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyI]amino]2-[(4-nitrophenoxy)carbonyloxymethyl]phenyl]ethyl]tetrahydropyran-2-carboxylate (130 mg, 0.127 mmol) in diméthyl formamide (3.0 mL) and DIPEA (60 pL, 0.363 mmol). The reaction mixture was stirred at room température for 2 h. (2R)-2-[(5S_a)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2[[(2S)-2-(9H-fIuoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]2-[2-[(2SR,3SR,4RS,5SR,6SR)-3,4,5-triacetoxy-6-methoxycarbonyl-tetrahydropyran-2yl]ethyl]phenyl]methoxycarbonyl]piperazin-l-yl]ethoxy]-2-methyl-phenyl]-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyI)pyrnnidin-4yl]methoxy]phenyl] propanoic acid was obtained as a solution in diméthylformamide and was used like this in the next step. UPLC-MS: MS (ESI) m/z [M+H]+ = 1745.6+1747.6.

Step 19: (2R)-2-[(5S_a)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9ylmethoxycarbonylamino)-3-methyl-butanoyl] amino]propanoyl] amino] -2-[2368

[(2SR, 3 SR, 4RS, 5 SR, 6SR)-3,4,5-triacetoxy-6-methoxycarbonyl-tetrahydropyran-2yl] ethyl]phenyl] methoxycarbonyl]piperazin-1 -yl] ethoxy]-2-methyl-phenyl]-6-(4~ fluorophenyl)thieno[2,3-d]pyrimidin-4-yl] oxy-3-[2-[[2~(2-methoxyphenyl)pyrimidin4-yl] methoxy]phenyl]propanoic acid

To the solution of2SR,3SR,4RS,5RS,6SR)-6-[2-[(5S_a)-5-[[(2S)-2-[[(2S)-2-amino-3methyl-butanoyl]amino]propanoyl]amino]-2-[[4-[2-[4-[4-[(l R)-1 -carboxy-2-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]ethoxy]-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl]-2-chloro-3-methyl-phenoxy]ethyl]piperazine-lcarbonyl]oxym ethyl] phenyl] ethyl]-3,4,5-tri hydroxy-tetrabydropyran-2-carboxylic acid (0.121 mmol) in DMF (3.0 mL) from step 1S were successively added methanol (2 mL) and lithium hydroxide monohydrate (64.0 mg, 1.52 mmol) in solution in water (2 ml). The reaction mixture was stirred at room température for 1 h. The crude product was purified by Cl 8 reverse phase prep-HPLC by direct deposit of the reaction mixture on the Xbridge column and using the NH4HCO3 method to afford (2R)-2-[(5S_a)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren9-ylmethoxycarbonyIamino)-3-methyl-butano yl] amino] propanoyl] amino]-2-[2[(2SR,3SR,4RS,5SR,6SR)-3,4,5-triacetoxy-6-methoxycarbonyl-tetrahydropyran-2yl]ethyl]phenyl]methoxycarbonyl]piperazin-l-yl]ethoxy]-2-methyl-phenyl]-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methüxyphenyl)pyriinidin-4yl]methoxy]phenyi]propanoic acid (124 mg, 0.0895 mmol) as a white powder. UPLC-MS: MS (ESI) m/z [M+H]+ = 1384.3+1386.3.

Step 20: (2,3,4,5,6-pentafluoroph.enyl) 2-[2-[2-(2-azidoethoxy)ethoxy] ethoxy] acetate 2-[2-[2(2-azidoethoxy)ethoxy]ethoxy]acetic acid

To a solution of 2-[2-[2-(2-azîdoethoxy)ethoxy]ethoxy]acetic acid (75 mg, 0.342 mmol) in solution in THF (500 pL) were added a solution of 2,3,4,5,6-pentafluorophenol (75.5 mg, 0.410 mmol) in THF (500 pL) and a solution of N,N’-dicyclohexylmethanediimine (84.7 mg, 0.410 mmol) in THF (500 pL). The reaction mixture was stirred for 15 h at room température and the progress of the reaction was followed by UPLC-MS. The (2,3,4,5,6-pentafluorophenyl) 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetate was obtained as a THF solution by simple filtration of the suspension on a small disposable frit. This solution was used without further purification in the next step. UPLC-MS: MS (ESI) m/z [M-N2+H]+ = 372.3.

Step 21: (2SR,3SR,4RS,5RS,6SR)-6-[2-[(5S_a)-5-[[(2S)-2-[[(2Sfi2-[[2-[2-[2-(2azidoethoxy)ethoxy] ethoxy] acetyl] amino]-3-methyl369 butanoyl] amino]propanoyl] amino]-2-[[4-[2 -[4-[4-[(lR)-l-carboxy-2-[2-[[2-(2methoxyphenyl)pvrimidin-4-yl]methoxy]phenyl]ethoxy]-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-2-chloro-3-methylphenoxv] ethyl]piperazine-1-carbonyl] oxymethyl]phenyl] ethyl]-3,4,5-trihydroxytetrahydropyran-2-carboxylic acid L14-C3

To the solution of (2R)-2-[(5S_a)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9ylmethoxycarbonylamino)-3-methyl-butanoyl] amino] propanoyl] amino]-2-[2[(2SR,3SR,4RS,5SR,6SR)-3,4,5-triacetoxy-6-methoxycarbonyl-tetrahydropyran-2yl]ethyl]phenyl]methoxycarbonyl]piperazin-l-yl]ethoxy]-2-methyl-phenyl]-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4yl]m ethoxy] phenyl ]propanoic acid (118 mg, 0.085 mmol) in DMF (500 pL) were successively added the solution of (2,3,4,5,6-pentafluorophenyl) 2-[2-[2-(2azîdoethoxy)ethoxy]ethoxy]acetate (0.342 mmol) în THF from step 20 and DIPEA (42.2pL, 0.256mmol). The réaction mixture was stirred for 1 h at room température and the progress of the reaction was followed by UPLC-MS. The crude product was purified by C18 reverse phase prep-HPLC by direct deposit of the réaction mixture on the Xbrîdge column and using the NH4HCO5 method to afford L14-C3 as a white powder. UPLC-MS: MS (ESI) m/z [M+H]+ = 1599.0+1601.2. IR Wavelength (cm⁴): 3263, 2105, 1652, 1600, 1284/1240/1089, 756. Ή NMR(400 MHz, dmso-d6): δ 9.98 (s), 8.85 (d, IH), 8.52 (s, LH), 8.38 (d, IH), 7.93 (d, IH), 7.56 (d, IH), 7.5 (t, IH), 7.49 (d, IH), 7.47 (d, IH), 7.44 (d, IH), 7.42 (s, IH), 7.3 (dd, 2H), 7.22 (d, IH), 7.2 (t, 2H), 7.19 (t, IH), 7.13 (d, IH), 7.08 (t, IH), 7.02 (t, IH), 6.95 (d, IH), 6.65 (t, IH), 6.11 (d, IH), 5.43 (d, IH), 5.27/5.2 (m, 2H), 4.93 (brs, 2H), 4.38 (m, IH), 4.35/4.2 (2m, 2H), 4.3 (m, IH), 3.94 (s, 2H), 3.75 (s, 3H), 3.58 (m, 10H), 3.57 (m, IH), 3.51/2.29 (2dd, 2H), 3.35 (m, 2H), 3.25 (m, 4H), 3.2 (m, IH), 3.2 (m, IH), 3.06 (m, IH), 2.96 (m, IH), 2.75 (m, 2H), 2.72/2.5 (m, 2H), 2.41 (m, 4 H), 2 (m, IH), 1.99/1.6 (m, 2H), 1.8 (s, 3H), 1.3 (d, 3H), 0.88/0.82 (2d, 6H). ^!3C NMR (100 MHz, dmso-d6): δ 158.2, 152.7, 131.9, 131.4, 131.4, 131.3, 131.1, 131, 127.8, 120.6, 120.5, 120.1, 116.8, 116.3, 116, 112.6, 112, 111.6, 79.6, 79.6, 78.5, 76.8, 74.2, 73.1, 70.3, 70.3, 69.3, 66.3, 65.1, 56.8, 56.3, 56.1, 52.6, 50.3, 49.4, 43.8, 34.2, 33.5, 31.7, 28, 19.6/18.4, 18.2, 18. ¹⁹F NMR (376 MHz, dmso-d6): δ -112.5. HR-EST+: m/z [M+H]+ = 1599.5724 ( 1599.5704) (measured/theoretical)

Préparation of L18-C3:

370 (2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-|| (2S)-2-[ [(2 R)-2-[ [2-(2-azidoethoxy)acetyl] amino]-3sulfo-propanoyl]amino]-3-methyIbutanoyl]amino]propanoyl]amino] phenyl] methoxycarbonyl]piperazin-l-yl]ethoxy]-3chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]py^r>nûdin-4-yl]ûxy-3-[2-[(2-(2methoxyphenyI)pyrimidin-4-yl] methoxy] phenyl] propanoic acid

Step 1: 9H-fluoren-9-ylmethyl N-[(lS)-l-[[(lS)-2-[4-(hydroxymethyl)amlino]-l-methyl-2oxo-ethyl]carbamoyl] -2-methyl-propyl] carbamate

To a solution of (2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl]amino]propanoic acid (6.0 g, 14.6 mmol; obtaîned according to Step 5 of the préparation of L14-C3) in dichloromethane (70 mL) and methanol (30 mL) were successively added (4-aminophenyl)methanol (2.16 g, 17.5 mmol) and ethyl 2-ethoxy-2H-quînolîne-lcarboxylate (5.42 g, 21.93 mmol). The red solution was stirred at room température for 16 h (précipitation after few minutes). After completion of the reaction, the réaction mixture was diluted with diethyl ether (70 mL). The resulting precipitate was fïltered off and dried to afford 9H-fluoren-9-ylmethyl N-[(IS)-l-[[(lS)-2-[4-(hydroxymethyl)anilîno]-l-methyl-2-oxocth yl] carbamoyl ]-2-methyl-propyl] carbamate (5.16 g, 10.01 mmol) as a beige solid. “H NMR (400 MHz, dmso-d6): δ 9.91 (s, IH), 8.15 (d, IH), 7.89 (d, 2H), 7.70-7.78 (m, 2H), 7.53 (d, 2H), 7.38-7.46 (m, 3H), 7.29-7.35 (m, 2H), 7.23 (d, 2H), 5.08 (t, IH), 4.37-4.50 (m, 3H), 4.16-4.34 (m, 3H), 3.91 (t, IH), 1.92-2.02 (m, IH), 1.30 (d, 3H), 0.83-0.91 (m, 6H).

Step 2: (2S)-2-amino-N-[(lS)-2-[4-(hydroxymethyl)amlino]~I-methyl-2-oxo-ethyl]-3-methylbutanamide

To a solution of 9H-fluoren-9-ylmethyl N-[(lS)-l-[[(lS)-2-[4-(hydroxymethyl)anilino]l-methy!-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate (5.16 g, 10.01 mmol) in DMF (120 mL) was added piperidîne (52 mL, 525mmol). The reaction mixture was stirred for 2 h at 371 room température then the piperidine was evaporated and the resulting solution was diluted with water (500 mL). The resulting solid was filtered off and the filtrate was washed twice with diethyl ether (2 x 500 mL). The aqueous layer was concentrated to dryness to afford the crude reaction mixture. The crude product was purified by silica gel chromatography (gradient of methanol (containing 7M ammonia) in dichloromethane) to afford (2S)-2-amino-N-[(l S)-2-[4(hydroxymethyl)anilino]-l-methyl-2-oxo-ethylJ-3-methyl-butanamide (2.02 g, 6.89 mmol) as a beige solid. Ή NMR (400 MHz, dmso-d6): δ 10.0 (s, IH), 8.17 (s, IH), 7.53 (d, 2H), 7.23 (d, 2H), 5.12 (t, IH), 4.39-4.52 (m, 3H), 2.96-3.02 (m, IH), L86-L97 (m, IH), 1.70 (br s, 2H), l .29 (d, 3H), 0.88 (d, 3H), 0.78 (d, 3H).

Step 3: [(2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-oxo-3-sodiooxypropyl] sulfonyloxysodium

To a solution of [(2R)-2-amino-3-oxo-3-sodiooxy-propyl]sulfonyloxysodium monohydrate (3.00 g, 12.98 mmol) in water (127 mL) was added sodium carbonate (4.13 g, 38.94 mmol). A solution of 9H-fluoren-9-ylmethyl carbonochloridate (3.69 g, 14.28 mmol) in dioxane (127 mL) was added dropwise in 15 min at room température. The mixture was stirred at this température for 4 h. After completion of the reaction, the mixture was neutralized to pH = 7 with an aqueous solution of HCl 1 M, diluted with a saturated aqueous solution of sodium hydrogenocarbonate (50 mL) and concentration to dryness. The crude product was purified by Cl 8 reverse phase chromatography using the neutral method to afford [(2R)-2-(9H-fluoren-9ylmethoxycarbonylamino)-3-oxo-3-sodiooxy-propyl]sulfonyloxysodium (4.4 g, 10.11 mmol) as a white solid. ^lH NMR (400 MHz, dmso-d6): δ 7.88 (d, 2H). 7.70 (d, 2H), 7.39-7.44 (m, 2H), 7.29-7.36 (m, 2H), 6.71 (s, IH), 3.84-4.25 (m, 4H), 2.73-2.91 (m, 2H).

Step 4: [(2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-[[(l S)-!-[[(! S)-2-[4(hydroxymethyl)anilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl] amino] 3-oxo-propyl] sulfonyloxysodium

To a solution of (2S)-2-amino-N-[(lS)-2-[4-(hydroxymethyl)anilino]-l-methyl-2-oxoethyl]-3-methyI-butanamide (1.19 g, 4.04 mmol) in DMF (395 mL) were successively added [(2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-oxo-3-sodiooxy-propyl]suIfonyloxysodium (4.40 g, 10.11 mmol), DIPEA (6.01 mL, 36.38 mmol) and HBTU (3.83 g, 10.11 mmol). The white suspension was stirred for 22 h at room température and then cooled to 0°C. Dilution with water (1.5 L), with a saturated solution of sodium carbonate (20 mL) and with solid sodium chloride, gave a white émulsion that was filtrated and the filtrate concentrated to dryness to

372 afford the crude mixture. The crude product was purified by reverse phase Cl 8 chromatography (gradient of methanol in water) to afford [(2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3[[( l S)-1 -[[( l S)-2-[4-(hydroxymethyl)anilino]-l -methyl-2-oxo-ethyl] carbamoyl]-2-methylpropyI]amino]-3-oxo-propyl]sulfonyloxysodium (936 mg, Î.36 mmol) as a beige solid. ’H NMR. (400 MHz, dmso-d6): δ 9.39 (s, IH). 8.25-8.31 (m, IH), 8.11-8.17 (m, IH), 7.89 (d, 2H), 7.70 (d, 2H), 7.64 (d, 2H), 7.50-7.55 (m, IH), 7.38-7.46 (m, 2H), 7.29-7.35 (m, 2H), 7.20 (d, 2H), 5.07 (s, 1H), 4.51 (s, IH), 4.42 (s, 2H), 4.19-4.33 (m, 4H), 4.01 (s, IH), 2.90-3.10 (m, 2H), 2.08-2.20 (m, IH), 1.31 (d, 3H), 0.8-0.93 (m, 6H).

Step 5: (2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-[[(lS)-2-methyl-l-[[(lS)-I-methyl2-[4-[ (4-nitrophenoxy)carbonyloxymethyl] aniline]-2-oxoethyl] carbamoyl]propyl]amino]-3-oxo-propane-l-sulfonate

To a suspension of[(2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-[[(l S)-!-[[(! S)-2[4-(hydroxymethyl)anilino]-l-meÎhyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]amino]-3-oxopropyl]sulfonyloxysodium (600 mg, 0.87 mmol) in THF (24 mL) were added DIPEA (432 pL, 2.61 mmol), followed by 4-Nitrophenyl chloroformate (439 mg, 2.17 mmol). The mixture was stirred at room température for 4 h. Additional 4-Nitrophenyl chlorofonnate (439 mg, 2.17 mmol) was added and the reaction mixture was stirred at room température for 16 h more. Additional 4-Nitrophenyl chlorofonnate (439 mg, 2.17 mmol) was added. After 5 h stirring at room température the mixture was concentrated to dryness and purified by silica gel chromatography (gradient of ethyl acetate in cyclohexane) and then by reverse phase C18 chromatography using the neutral method to afford (2R)-2-(9H-fluoren-9ylmethoxycarbonylamino)-3-[ [(15)-2-methyl-!-[[(! S)- I-meth yl-2-[4-[(4nitrophenoxy)carbonyloxymethyl]anilino]-2-oxo-ethyl]carbamoyl]propyl]amino]-3-oxopropane-1-sulfonate (303 mg, 0.32 mmol) as a white solid. 'H NMR (400 MHz, dmso-d6): δ 9.52 (s, IH), 8.25-8.37 (m, 3H), 8.06-8.24 (m, 4H), 7.89 (d, 2H), 7.76 (d, 2H), 7.70 (d, 2H), 7.49-7.61 (m, 3H), 7.35-7.45 (m, 4H), 7.26-7.35 (m, 2H), 5.23 (s, 2H), 4.48 (s, IH), 4.20-4.33 (m, 4H), 4.01 (s, IH), 3.57-3.66 (m, 2H), 3.10-3.18 (m, 2H), 2.90-3.10 (m, 2H), 2.08-2.20 (m, IH), 1.33 (d, 3H), 1.21-1.26 (m, 15H), 0.86-0.92 (m, 6H). UPLC-MS: MS (ESI) m/z [M-H]-: 830.5.

Step 6: (2R)-2-[(5S_a)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[(2S)-2-(9H-fluoren-9ylmethoxycarbonylamino)butanoyl] amino]-3-methylbutanoyl] amino]propanoyl] amino]phenyl] methoxycarbonyl]piper azin-1-yl] ethoxy] 373

2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrîmidin-4-yl]oxy-3-[2-][2-(2methoxyphenyl)pyrimidin-4-yl/methoxy]phenyl]propanoic acid

To a solution of (2R)-2-[(5S_a)-5-[3-chloro-2-methyl-4-(2-piperazin-l-ylethoxy)phenyl]6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4yl] methoxy] pheny] ]propanoic acid ; 2,2,2-trifluoroacetic acid (C3) (l 28 mg, 0.149 mmol) in DMF (1.5mL) were successively added a solution of (2R)-2-(9H-fluoren-9ylmethoxycarbonylamino)-3-[[(lS)-2-methyl-l-[[(l S)-1-methyl-2-[4-[(4nitrophenoxy)carbonyloxymethyl]anilino]-2-oxo-ethyl]carbamoyl]propyl]amino]-3-oxopropane-1-sulfonate (150 mg, 0.156 mmol) in DMF (1.5mL) and DIPEA (77 μΐ, 0.468 mmol). The reaction mixture was stirred for 2 h at room température and the progress of the reaction was followed by UPLC-MS. (2R)-2-[(5S_a)-5-[3-chIoro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[(2S)-2-(9HfIuoren-9-ylmethoxycarbonylamino)butanoyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-l-yl]ethoxy]-2-methylphenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl] methoxy] phenyl]propanoic acid was obtained as a solution in dimethylformamide that was used like this in the next step. UPLC-MS: MS (ESI) m/z [M+H]+ = 1553.2+1555.3.

Step 7: (2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[(2S)-2-aminobutanoyl]amino]-3methyl-butanoyl]amino]propanoyl] amino]phenyl]methoxycarbonyl]piperazin-Iyl] ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4yl]oxy-3-[2-][2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

To the solution of(2R)-2-[(5S_a)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[(2S)-2-(9Hfluoren-9-ylmethoxycarbonylamino)butanoyl]amino]-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-l-yl]ethoxy]-2-methyIphenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoie acid (0.156 mmol) in dimethylformamide (3 mL) obtained in the previous step was added piperidine (30.6 pL, 0.312 mmol). The reaction mixture was stirred at room température for 15 h and the progress of the reaction was followed by UPLC-MS. The crude product was purified by Cl8 reverse phase prepHPLC by direct deposit of the reaction mixture on the Xbridge column and using the NH4HCO3 method to afford(2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[(2S)-2-aminobutanoyl]amino]3-methyl-butanoyl]aniino]propanoyl]amino]phenyl]methoxycarbonyl]piperazin-l-yl]ethoxy]-3chl oro-2-methyl -phenyl]-6-(4-fl uorophenyl)thieno [2,3 -d]pyrimidin-4-yl]oxy-3-[2-[[2-(2374 methoxyphenyl)pyrimidin-4~yl]methoxy]phenyl]propanoic acid (148 mg - 0.H 1 mmol) as a white powdcr. UPLC-MS: MS (ESI) m/z [M+H]+ = 1331.4+1333.5.

Step 8: (2R)-2-[(5Sa)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2azidoethoxy)ethoxy] ethoxy] acetyl] amino] butanoyl] amino]-3-methylbutanoyl] amino] propanoyl] amino] phenyl]methoxycarbonyl]piperazin-l-yl] ethoxy]3-chloro-2-methyl-phenyl]-6~(4-fluorophenyl)thieno]2,3-d]pyrimidin-4-yl]oxy-3-]2][2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid;2,2,2trifluoroacetic acid L18-C3

To the solution of(2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[(2S)-2aminobutano yl] amino]-3-methylbuÎanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl]piperazîn-l-yl]eîhoxy]-3-chIoro-2methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]proanoic acid (148 mg, 0.111 mmol) in DMF (1.5 mL) were successively added the solution of (2,3,4,5,6-pentafluorophenyl) 2-[2-[2-(2· azidoethoxy)ethoxy] ethoxy] acetate (0.596 mmol; obtained according to Step 20 of the préparation of L14-C3) in THF (1 mL) and DIPEA (74 pL, 0.447 mmol). The reaction mixture was stirred for 1 h at room température and the progress of the reaction was followed by UPLCMS. The crude product was purified by Cl 8 reverse phase prep-HPLC by direct deposit of the reaction mixture on the Xbridge column and using the NH4HCO3 method to afford L18-C3 (60 mg, 0.0389mmol) as a white powder. IR Wavelength (cm¹): 3288, 2101, 1659, 1237,1039, 833,755. ‘H NMR(400 MHz, dmso-d6): S (m, 10H), 9.42 (s, 1H), 8.88 (d, 1H), 8.58 (s, 1H), 8.32 (d, 1H), 8.18 (d, 1H), 8.12 (d, 1H), 7.71 (m, 1H), 7.7 (d, 2H), 7.54 (dd, 1H), 7.46 (td, 1H),7.39 (d, 1H), 7.29 (dd, 2H), 7.25 (d, 2H), 7.21 (t, 2H), 7.18 (d, 1H), 7.15 (d, 1H), 7.13 (t, 1H), 7.04 (t, 1H), 6.99 (d, 1H), 6.71 (t, lH),6.22(d, 1H), 5.47 (m, 1H), 5.23 (AB, 2H), 4.98 (s,2H),4.71 (q, 1H), 4.3 (m, 1H), 4.24/4.19 (2m, 2H), 3.97 (dd, 1H), 3.92 (m, 2H), 3.76 (s, 3 H), 3.37 (t, 2H), 3.31 (m, 4H), 3.12/2.97 (2dd, 2H), 2.74 (t, 2H), 2.45 (m, 4H), 2.15 (m, 1H), 1.81 (s, 3H), 1.33 (d, 3H), 0.91 (2d, 6H). ¹³C NMR (100 MHz, dmso-d6): Ô 157.9, 152.3, 131.3, 131.2, 131.1, 131, 130.7, 128.9, 128.6, 120.7, 120.4, 119.6, 116.4, 112.7, 112, 111.3, 70.6, 70.3, 69.4, 67.5, 66.3, 59.7, 56.7, 56.1,53.4, 52.5, 50.8, 50.4,49.9,44, 29.9, 19.6, 17.8, 17.6. ^I9FNMR(376 MHz, dmso-d6): δ ppm 112.3. HR-ESI+: m/z [M+H]+ = 1546.503 (1546.5009) (measured/theoretical).

Préparation of L16-C3:

375 (2R)-2-[(5Sa)-5-|4-[2-[4-[[4-l[(2S)-6-ammo-2-[|(2S)-2-n2-(2-azidoethoxy)acetyl]amino]-3methyl-butanoyl] amino] hexanoyl] amino |phenyl]inethoxycarbonyl]piperazin-l-yl]ethoxy|3-chloro-2-methyl-phenyl]-6-(4-nuorophenyI)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2 methoxyphenyI)pyrimidin-4-yI] methoxy] phenyl] propan oie acid

Step 1: (2S)-6-(tert-butoxycarbonylamino)-2-[[2-(9H-fluoren~9-ylmethoxycarbonylamino)-3methyl-butanoyl]amino] hexanoic acid

To a solution of (2S)-2-amino-6-(tert-butoxycarbonyiamino)hexanoic acid (2.96 g, 12 mmol) and sodium hydrogéné carbonate (1.01 g, 12 mmol) in water (30 mL) was added a solution of (2,5-dioxopyrrolidin-l-yl) 2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl butanoate (5.0 g, 11.5 mmol) in dimethoxyethane (30 mL), THF (15 mL) was added to improve the solubility, The reaction mixture was stirred at room température for 16 h. An aqueous solution of hydrochloric acid 1 M (15 mL) was added and the aqueous layer and was extracted with ethyl acetate (3 x 75 mL). The combined organic layers were dried over sodium sulfate, fïltered and concentrated to dryness to afford the crude compound. Trituration in dichloromethane/pentane with sonication led to (2S)-6-(tert-butoxycarbonylamino)-2-[[2-(9Hfluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]hexanoic acid (4.9 g, 8.63 mmol) as a white solid. Ή NMR (400 MHz, dmso-d6): 6 12.48 (s, IH), 7.89 (d, 2H), 7.74 (t, 2H), 7.287.44 (m, 6H), 6.73 (s, IH), 4.10-4.33 (m, 5H), 3.9 (t, IH), 2.82-2.90 (m, 2H), 1.52-1.73 (ni, 2H), 1.34 (s, 9H), 1.22-1.31 (m, 4H), 0.83-0.91 (m, 6H).

Step 2: 9H-fluoren-9-ylmethyl N-[l-[[(IS)-5-(tert-butoxycarbonylamino)-l-[[4(hydroxymethyl)phenyl] carbamoyl]pentyl] carbamoyl] -2-methyl-propyl] carbamate

To a solution of (2S)-6-(tert-butoxycarbonyIamino)-2-[[2-(9H-fluoren-9ylmethoxycarbonylamino)-3-methyl-butanoyl] amino] hexanoic acid (1.5 g, 2.64 mmol) in dîchloromethane (19 mL) and methanol (9.5 mL) was added (4-aminophenyl)methanol (651.0 mg, 5.28 mmol) in methanol (1.5 mL). Ethyl 2-ethoxy-2H-quinoline-l-carboxylate (1.31 g, 5.28 376 mmol) was then added. The reaction mixture was stirred at room température for 16 h then concentrated to dryness. The crude product was purified by silica gel chromatography (gradient of methanol in dichloromethane) to afford 9H-fluoren-9-ylmethyl N-[l-[[(l S)-5-(tertbutoxycarbonylamino)-l-[[4-(hydroxymethyl)phenyl]carbamoyl]pentyl]carbamoyl]-2-methylpropyl]carbamate (544 mg, 0.80 mmol) as a pale red solid. ’H NMR (400 MHz, dmso-d6): Ô 9.93 (s, IH). 8.01 (d, IH), 7.89 (d, 2H), 7.74 (t, 2H), 7.52 (d, 2H), 7.37-7.45 (m, 3H), 7.32 (t, 2H), 7.22 (d, 2H), 6.71 (s, IH), 5.08 (br s, IH), 4.43 (d, 2H), 4.21-4.40 (m, 4H), 3.92 (t, IH), 2.83-2.91 (m, 2H), 1.94-2.01 (m, IH), 1.55-1.74 (m, 2H), 1.21-1.42 (m, 4H), 1.33 (s, 9H), 0.87 (t, 6H).

Step 3: [4-[[(2S)-6-(tert-butoxycarbonylamino)-2-[[2-(9H-fluoren-9yhnethoxycarbonylamino)-3-methyl-butanoyl] amino] hexanoyl] amino]phenyl] methyl (4-nitrophenyl) carbonate

To a solution of 9H-fluoren-9-ylmethyl N-[l-[[(lS)-5-(tert-butoxycarbonylamino)-l-[[4(hydroxymethyl)phenyl]carbamoyl]pentyl]carbamoyl]-2-methyl-propyl]carbamate (600.0 mg, 0.892 mmol) in THF (19 mL), were added pyridine (361 pL, 4.46 mmol) then 4-Nitrophenyl chloroformate (448 mg, 2.22 mmol). The mixture was stirred at room température for 16 h then concentrated to dryness. The crude product was purified by silica gel chromatography (gradient of ethyl acetate in cyclohexane) to afford [4-[[(2S)-6-(tert-butoxycarbonylamino)-2-[[2-(9Hfluoren-9-ylmethoxycarbonyl ami no)-3-methyl-butanoyl] amino] hexanoyl] amino] phenyljmethyl (4-nitrophenyl) carbonate (524 mg; 0.62 mmol; 70%) as a pale pink solid. ^!H NMR (400 MHz, dmso-d6): δ 10.13 (s, IH), 8.31 (d, 2H), 8.1 (d, IH), 7.89 (d, 2H), 7.74 (t, 2H), 7.63 (d, 2H), 7.57 (d, 2H), 7.28-7.45 (m, 7H), 6.72 (s, IH), 5.24 (s, 2H), 4.35-4.42 (m, IH), 4.27-4.33 (m, IH), 4.22 (s, 2H), 3.92 (t, IH), 2.83-2.91 (m, 2H), 1.96-2.00 (m, IH), 1.58-1.73 (m, 2H), 1.20-1.30 (m, 4H), 1.33 (s, 9H), 0.86 (t, 6H). ^l3CNMR(100 MHz, dmso-d6): Ô 171.22, 170.67, 156.1, 155.5, 155.27, 151.92, 145.15, 143.87, 143.75, 140.68, 139.34, 129.43, 129.31, 127.6, 127.03, 125.38, 125.32, 122.58, 120.07, 119.11, 77.28, 70.23,65.67, 60.11, 54.89, 53.43, 46.67, 3 1.69, 30.39, 29.22,28.23,22.74, 19.19, 18.26. LC-MS: MS (ESI) m/z [M+Na]+ = 837.4.

377

Step 4: (2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-6-(tert-butoxycarbonylamino)-2-[[(2S)-2-(9Hfluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl] amino] hexanoyl] amino]phenyl] methoxycarbonyl]piperazin-l-yl] ethoxy]-3chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno ]2,3-d]pyrimidin-4-yl]oxy-3-[2[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

To a solution of (2R)-2-[(5Sa)-5-[3-chloro-2-methyl-4-(2-piperazin-l-ylethoxy)phenyl]6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4yl]methoxy] phenyl] propanoic acid C3 (166.3 mg, 0.170 mmol) in DM F (1.5 mL) were successively added a solution of [4-[[(2S)-6-(tert-butoxycarbonylamino)-2-[[2-(9H-fluoren-9ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]hexanoyl]amino]phenyI]methyl (4nitrophenyl) carbonate (150 mg, 0.179 mmol) in DMF (1.5 mL) and DIPEA (85 μΐ, 0.510 mmol). The reaction mixture was stirred for 1 h at room température and the progress of the reaction was followed by UPLC-MS. The crude product was purified by CI 8 reverse phase prepHPLC by direct deposit of the reaction mixture on the Xbrîdge column and using the NH4HCO3 method to afford(2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-6-(tert-butoxycarbonylamino)-2-[[(2S)-2(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl]aTmno]hexanoyl]amino]phenyl]methoxycarbonyl]piperazin-l-yl]ethoxy]-3-chIoro-2methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidm-4-yl]methoxy]phenyl]propanoic acid (134mg, 0.0859mmol) as a white powder. UPLC-MS: MS (ESI) m/z [M+H]+= 1559.1+1561.3, [M+Na]+: 1581.0+1583.2. IR Wavelength (cnV¹): 3309, 1698, 1238, 1162, 757, 744. 'H NMR (400 MHz, dmso-d6): δ 10.05 (s, IH), 8.87 (d, 1H), 8.6 (m, IH), 8.06 (d, IH), 7.88 (d, 2H), 7.74 (2d, 2H), 7.64 (m, IH), 7.57 (d, 2H), 7.52 (dd, IH), 7.44 (t, IH), 7.43 (d, IH), 7.4 (t, 2H), 7.35 (d, IH), 7.3 (t, 2H), 7.3 (dd, 2H), 7.26 (d, 2H), 7.2 (t, 2H), 7.18 (d, IH), 7.14 (d, IH), 7.12 (t, 1 H), 7.03 (t, IH), 6.99 (d, IH), 6.72 (t, IH), 6.71 (m, IH), 6.24 (d, IH), 5.49 (dd, IH), 5.23 (m, 2H), 4.97 (s, 2H), 4.38 (m, IH), 4.29/4.23 (m, 2H), 4.22 (m, IH), 4.2 (m, 2H), 3.92 (dd, IH), 3.74 (s, 3H), 3.29 (m, 4H), 3.29/2.5 (2dd, 2H), 2.87 (m, 2H), 2.74 (t, 2H), 2.45 (m, 4H), 1.99 (m, IH), 1.82 (s, 3 H), 1.68/1.6 (2m, 2H), 1.36/1.28 (2m, 4H), 1.32 (s, 9H), 0.86 (2d, 6H). ^!3C NMR (100 MHz, dmso-d6): δ 158, 131.4, 131.2, 131.2, 131, 130.8, 128.9, 128.5, 127.9, 127.5, 125.6, 120.8, 120.5, 120.4, 119.3, 116, 115.9, 112.4, 112.2, 111.2, 74.1, 69.2,67.9, 66.7,66.2,60.6, 56.6, 56.2, 53.8, 53, 47.1,43.7, 40,32.6, 32.2, 30.6, 29.8/23.2,28.5, 18.8, 18.1. ¹⁹F NMR (376 MHz, dmso-d6): δ112.

378

Step 5: (2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-6(tert-butoxycarbonylamino)hexanoyl] amino]phenyl] methoxycarbonyl]piperazîn-1yl]ethoxy] -3-chloro-2-methyl-phenyl]-6~(4-fluorophenyl)thieno[2,3-d]pyrimidin-4yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

To the solution of (2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-6-(tert-butoxycarbonylamino)-2[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl]amino]hexanoyl]amino]phenyl]methoxycarbonyl]piperazin-l-yl]ethoxy]-3-chloro-2methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (134 mg, 0.0859 mmol) in diinethylfonnamide (3 mL) was added piperidine (17 pL, 0.172 mmol). The reaction mixture was stirred at room température for 18 h and the progress of the reaction was followed by UP LCMS. The crude product was purified by C18 reverse phase prep-HPLC by direct deposit of the reaction mixture on the Xbridge column and using the NH4HCO3 method to afford (2R)-2[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-6-(tertbutoxycarbonylamino)hexanoyl]amino]phenyl]methoxycarbonyl]piperazin-l-yl]ethoxy]-3chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyriniidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (88 mg, 0.0658 mmol) as a white powder. UPLC-MS: MS (ESI) m/z [M+H]+ = 1337.4+1339.4, [M+Na]+= 1359.4+1361.4. IR Wavelength (cm'¹): 3307, 1683, 1290, 1238, 1162, 835, 754. Ή NMR (400 MHz, dmso-d6) S ppm 10.23 (s, IH), 8.88 (d, IH), 8.53 (m, IH), 8.47 (br, IH), 7.86 (d, IH), 7.58 (d, 2H), 7.54 (d, IH), 7.48 (d, IH), 7.45 (t, IH), 7.27 (dd, 2H), 7.25 (d, 2H), 7.19 (ί, 2H), 7.18 (d, IH), 7.14 (d, IH), 7.08 (t, IH), 7.03 (t, IH), 6.96 (t, IH), 6.72 (t, IH), 6.67 (t, IH), 6.14 (d, IH), 5.42 (d, IH), 5.21 (m, 2H), 4.97 (s, 2H), 4.4 (m, IH), 4.21 (m, 2H), 3.75 (s, 3H), 3.42/2.35 (m, 2H), 3.29 (m, 4H), 3.24 (m, IH), 2.87 (q, 2H), 2.72 (t, 2H), 2.43 (m, 4H), 1.99 (m, IH), 1.78 (s, 3H), 1.7/1.61 (2m, 2H), 1.36 (m, 2H), 1.34 (s, 9H), 1.26 (m, 2H), 0.89/0.82 (2d, 6H). ^I3CNMR (100 MHz, dmso-d6): δ ppm 158.4, 131.3, 131.2, 131.1, 131, 128.4, 128, 120.8, 120.6, 120.4, 119.7, 1 16.1, 115.9, 112.7, 111.7, 111.2, 76.2, 69.2,67.4, 66.3, 59.2, 56.6, 56.3, 53.6, 53.1,43.8,40.1,33.2, 32.4, 3 1.3, 29.3, 28.8, 22.9, 19.7/17.5, 17.9. ¹⁹F NMR (376 MHz, dmso-d6): δ ppm -112.4.

379

Step 6: (2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3-methyl-butanoyl]amino]-6-(tertbutoxycarbonylamino)hexanoyl] amino]phenyl] methoxycarbonyl]piperazin-lyl] ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin~4yl]oxy-3-[2-]]2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl] propanoic acid

To a solution of(2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyl]amino]-6-(tertbutoxycarbonylamino)hexanoyl]amino]phenyl]methoxycarbonyl]piperazîn-l-yl] ethox y]-3chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (82 mg, 0.0613 mmol) in DMF (500 pL) were successively added the solution of (2,3,4,5,6-pentafluorophenyl) 2-[2-[2-(2azidoethoxy)ethoxy]ethoxy]acetate (0.245 mmol; obtaîned according to Step 20 of the préparation of L14-C3) in THF and DIPEA (30.4 pL, 0.184 mmol). The reaction mixture was stirred for Ih at room température and the progress of the reaction was followed by UPLC-MS. The crude product was purified by Cl8 reverse phase prep-HPLC by direct deposit of the réaction mixture on the Xbridge column and using the NH4HCO3 method to afford (2R)-2[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetyl]amino]-3methyl-butanoyl]amino]-6-(tertbutoxycarbonylamino)hexanoyl]amîno]phenyl]methoxycarbonyl]piperazin-l-yl]ethoxy]-3chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2_J3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (60 mg, 0.0386 mmol) as a white powder. UPLC-MS: MS (ESI) m/z [M+H]+= 1552.2+1554.2, [M+Na]+= 1574.1 + 1576.3.

Step 7: (2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-6-amino-2-[[(2S)-2-[[2-[2-[2-(2azidoethoxy)ethoxy] ethoxy]acetyl] amino] -3-methylbutanoyl] amino] hexanoyl] amino]phenyl] methoxycarbonyl]piperazin-l-yl] ethoxy]-3chloro-2-methyl-phenvl]-6-(4-fluorophenyl)thieno [2,3-d]pyrimidin-4-yl] oxy-3-[2~ []2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid L16-C3

To a solution of (2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[2-[2-[2-(2azidoethoxy)ethoxy] ethoxy] acetyl] amino]-3-methyl-butano yl] amino]-6-(tertbutoxycarbonylamino)hexanoyl]amino]phenyl]methoxycarbonyl]piperazin-l-yl]ethoxy]-3chloro-2-methyi-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidîn-4-yl]methoxy]phenyl]propanoic acid (23 mg, 0.0148 mmol) in dichloromethane (3 mL) was added 2,2,2-trifluoroacetic acid (400 pl, 4.57 mmol). The reaction 380 mixture was stirred for 2h at room température and the progress of the reaction was followed by UPLC-MS. The crude product was purified by Cl 8 reverse phase prep-HPLC by direct deposît of the reaction mixture on the Xbridge column and using the NH4HCO3 method to afford L16C3 (5 mg, 0.00344 mmol) as a white powder. UPLC-MS: MS (ESI) m/z [M+H]+ = 1552.4+1554.5, [M+Na]+ = 1574.4+1576.4. IR Wavelength (cm'¹): 3250, 2250-3500, 2102, 1660, 1288, 1238, 1121, 833, 755. ^lH NMR(400 MHz, dmso-d6): δ ppm 10.24 (s, IH), 8.85 (d, IH), 8.49 (s, IH), 8.49 (d, IH), 7.98 (d, IH), 7.6 (d, 2H), 7.55 (d, IH), 7.51 (d, IH), 7.5 (d, IH), 7.46 (t, IH), 7.26 (d, 2H), 7.25 (dd, 2H), 7.18 (t, 2H), 7.17 (d, IH), 7.14 (d, IH), 7.05 (t, IH), 7.02 (t, IH), 6.89 (d, IH), 6.6 (t, IH), 6.05 (d, IH), 5.32 (d, IH), 5.21/5.15 (m, 2H), 5.02/4.96 (m, 2H), 4.36 (q, IH), 4.31 (dd, IH), 4.2 (m, 2H), 3.94 (s, 2H), 3.77 (s, 3 H), 3.58 (m, 10 H), 3.46/2.28 (d+t, 2H), 3.34 (t, 2H), 3.29 (m, 4 H), 2.8 (m, 2H), 2.67 (t, 2H), 2.43 (m, 4 H), 2.01 (m, 1 H), 1.75 (s, 3 H), I.69/1.6 (2m, 2H), 1.51 (m, 2H), l .31 (m, 2H), 0.86/0.8 (2d, 6 H). ¹³C NMR (100 MHz, dmso-d6): δ ppm 157.9, 153.7, 131.4, 131.4, 131.3, 131.1, 130.9, 129.3, 127.6, 120.9, 120.4, 119.8, 116.2, 116.1, 112.6, 111.8, 111.8, 78.1, 70.5, 70.4, 69.3, 66.6, 66.6, 56.9, 56.5, 56.3, 54, 52.3, 50.4, 44, 39, 33.8, 32.2, 31.8, 28.2, 23.1, 19.7/18.1, 183. ¹⁹F NMR (376 MHz, dmso-d6): δ ppm -ï 12.6. HR-ESI+: m/z [M+H]+ - 1452.5661 (1452.5648) (measured/theoretical).

Préparation of L21-C1:

(2S,3S,4R,5R,6S)-6-[2-[2-[[4-[2-[4-i4-[(lR)-l-carboxy-2-[2-[[2-(2methoxyplienyl)pyrinMdin-4-yl]methoxy]phenyI]ethoxy]-6-(4-nuorophenyl)thieno[2,3d]pyrimidin-5-yl]-2-chloro-3-nietliyl-phenoxy]ethyl]-l-methyl-piperazin-l-ium-lyl]methyl]-5-[[(2S)-2-[[(2S)-2-|3-[2-(2,5-dioxopyrrol-l-yl)ethoxy]propaiioyIainino]-3niethyI-butanoyl]amino]-5-ureido-pentanoy)]amino]phenyl]ethyl]-3,4,5-trihydroxytetrahydropyran-2-carboxylic acid;2,2,2-trifluoroacetate

OH OH

381

Step 1 : tert-butyl·[(2-iodo-4-nitro-phenyl)methoxy]-dimethyl-silane

To a solution of (2-iodo-4-mtro-phenyl)methanol (172 g, 61.64 mmol; obtained according to Step 2 ofthe préparation of L14-C3) in dichloromethane (300 mL) was added imidazole (5.04 g, 73.97 mmol). The mixture was cooled to 0°C, then a solution of tert-butylchloro-dimethyl-silane (1 L15 g, 73.97 mmol) in dichloromethane (300 mL) was added dropwise in 15 min. The ice bath was removed and the reaction mixture was stirred at room température for 16 h. After completion of the reaction, the reaction mixture was quenched with methanol (20 mL) and concentrated to dryness. The crude product was purified by silica gel chromatography (gradient of ethyl acetate in cyclohexane) to afford tert-butyl-[(2-iodo-4-nitro-phenyl)methoxy]dimethyl-silane (19.65 g, 49.96 mmol) as a white solid. ‘H NMR (400 MHz, dmso-d6): δ 8.57 (s, IH), 8.31 (d, IH), 7.66(d, 1H),4.67 (s, 2H), 0.92 (s, 9H), 0.14 (s, 6H).

Step 2: methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-[[tertbutyl(dimethyl)silyl]oxymethyl]-5-nitro-phenyl]ethynyl]tetrahydropyran-2carboxylate

To a solution of tert-butyl-[(2-iodo-4-nitro-phenyl)methoxy]-dimethyl-silane compound (3.0 g, 7.63 mmol) in DMF (55 mL) were successively added methyl (2S,3S,4R,5S,6S)-3,4,5triacetoxy-6-ethynyl-tetrahydropyran-2-carboxylate (3.39 g, 9.92 mmol; obtained according to Step 13 ofthe préparation of L14-C3), DIPEA (5.80 mL, 35.09 mmol), copper iodide (145 mg, 0.763 mmol) and dichloro-bis-(triphenylphosphine)palladium(ll) (535 mg, 0.763 mmol). The yellow solution was flushed with Argon and stirred for 16 h at room température. After dilution with water (300 mL), the aqueous layer was extracted with ethyl acetate (2 x 300 mL). The combined organic layers were washed with water (2 x 300 mL) then were dried over sodium sulfate, filtered and concentrated to dryness. The crude product was purified by silica gel chromatography (gradient of ethyl acetate in cyclohexane) to afford methyl (2S,3S,4R,5S,6S)3,4,5-triacetoxy-6-[2-[2-[[tert-butyl(dimethyl)silyl]oxymethyl]-5-nitrophenyl]ethynyl]tetrahydropyran-2-carboxylate (4.01 g, 6.60 mmol) as a beige solid. ’H NMR (400 MHz, dmso-d6): δ 8.32 (dd, IH), 8.19 (d, IH), 7.75 (d, IH), 5.45 (t, IH), 5.16(t, IH), 5.025.07 (m, 2FI), 4.82 (s, 2H), 4.55 (d, 1 H), 3.65 (s, 3H), 1.98-2.07 (m, 9H), 0.92 (m,9H), 0.14 (s, 6H).

382

Step 3: methyl (2S, 3S,4R,5S, 68)-3,4,5-triacetoxy-6-[2-[2-(hydroxymethyl)-5-nitrophenyl] ethynyl] tetrahydropyran-2-carboxylate

To a solution of methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-[[tertbutyl(dimethyl )silyl]oxymethyl]-5-nitro-phenyl] ethynyl] tetrahydropyran-2-carboxylate (4.01 g, 6.60 mmol) in THF (48 mL) and water (48 mL) was added acetic acid (193 mL, 3.36 mol). The coiorless solution was stirred for 2 days at room température then diluted with water (300 mL). The aqueous layer was extracted with dichloromethane (2 x 300 mL). The combined organic layers were washed with water (2 x 300 mL), and with a saturated aqueous solution of sodium hydrogen carbonate (400 mL), then dried over sodium sulfate, fîltered and concentrated to dryness. The crude product was purified by silica gel chromatography (gradient of ethyl acetate in cyclohexane) to afford methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-(hydroxymethyl)-5nitro-phenyl]ethynyl]tetrahydropyran-2-carboxylate (2.67 g, 5.41 mmol) as a white solid. ’H NMR (400 MHz, dmso-d6): S 8.29 (dd, IH), 8.15 (d, IH), 7.79 (d, IH), 5.68 (t, IH), 5.45 (t, IH), 5.16 (t, IH), 5.02-5.07 (m, 2H), 4.62 (d, 2H), 4.55 (d, IH), 3.65 (s, 3H), 1.98-2.07 (m, 9H).

Step 4: methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-amino-2(hydroxymethyl)phenyl] ethyl] tetrahydropyran-2-carboxylate

A solution of methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-(hydroxymethyl)-5-nitrophenyl]ethynyl]tetrahydropyran-2-carboxylate (2.67 g, 5.41 mmol) in THF (59 mL) was flushed with Argon. Platinum on carbon 5% dry ( 1.34 g, 50% w/w) was added. The reaction mixture was successively flushed with argon, with H₂ and was stirred for 2 days at room température under H₂ atmosphère (P atm). The reaction mixture was fîltered through a Celite® pad, washed with a solution of ethyl acetate/methanol 9/1 (500 mL), then concentrated to dryness. Ail the sequence, (addition of platinum on carbon 5% dry (1.34 g, 50% w/w), stirring for 16 h at room température under H? (P atm) and filtration through a Celite® pad), was repeated to allow a complété conversion. The crude product was purified b y silica gel chromatography (gradient of ethyl acetate in cyclohexane) to afford methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-amino-2(hydroxymethyl)phenyl]ethyl]tetrahydropyran-2-carboxylate (1.12 g, 2.40 mmol) as a white solid. Ή NMR (400 MHz, dmso-d6): δ 6.93 (d, IH). 6.67-6.33 (m, 2H), 5.30 (t, IH), 4.96 (t, IH), 4.88 (s, 2H), 4.81 (t, IH), 4.61 (t, IH), 4.39 (d, IH), 4.29-4.24 (m, 2H), 3.78-3.72 (m, IH), 3.65 (s,3H), 2.65-2.54 (m, 2H), 2.07-1.98 (m, 9H), 1.79-1.68 (m, IH), 1.63-1.52 (m, IH).

383

Step 5: methyl (2 S, 3 S, 4 R, 5S, 6S)-3,4,5-triacetoxy-6-[2-[5-[[(2S)-2-(tertbutoxycarbonylamino)-5-ureido-pentanoyl]amino]-2(hydroxymethyl)phenyl] ethyl] tetrahydropyran-2-carboxylate

To a solution of methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-amino-2(hydroxymethyl)phenyl] ethyl] tetrahydropyran-2-carboxylate (1.00 g, 2.14 mmol) in DMF (21 mL) were successively added (2S)-2-(tert-butoxycarbonylamino)-5-ureido-pentanoic acid (589 mg, 2.14 mmol), DIPEA (707 μΐ, 4.28 mmol) and HBTU (1.22 g, 3.21 mmol). The reaction mixture was stirred for 72 hours at room température. After completion of the reaction, the mixture was diluted with water (100 mL) and was concentrated to dryness. The crude product was purified b y silica gel chromatography (gradient of methanol in dichloromethane) to afford methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-[[(2S)-2-(tert-butoxycarbonylamino)-5-ureidopentanoyl]amino]-2-(hydroxymethyl)phenyl]ethyl]tetrahydropyran-2-carboxylate (1.05 g, 1.45 mmol) as a beige solid. 'H NMR (400 MHz, dmso-d6): Ô 9.82 (s, IH), 7.35-7.42 (m, 2H), 7.24 (d, IH), 6.95 (d, IH), 5.94 (t, IH), 5.37 (s, 2H), 5.30 (t, IH), 4.91-4.99 (m, 2H), 4.79 (t, IH), 4.36-4.42 (m, 3H), 4.01-4.08 (m, IH), 3.76 (t, IH), 3.65 (s, 3H), 2.95-3.04 (m, 2H), 2.54-2.65 (m, 2H), 1.98-2.07 (m, 9H), 1.68-1.79 (m, IH), 1.49-1.63 (m, 3H), 1.30-1.42 (m, 11 H).

Step 6: methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9vlmethoxycarbonylamino)-3-methyl-butanoyl] amino] ~5-ureido-penlanoyl] amino]-2(hydroxymethyl)phenyl] ethyl] tetrahydropyran~2-carboxylate

To a solution of compound methyl (2S,3S,4R,5S,6S)-3_}4,5-triacetoxy-6-[2-[5-[[(2S)-2(tert-butoxycarbonylamino)-5-ureido-pentanoyl]amino]-2(hydro xym ethyl )phenyl]ethyl]tetrahydropyran-2-carboxylate (950 mg, 1.31 mmol) in dichloromethane (7.5 mL) was added, at 0°C, trifluoroacetic acid (1.9 mL, 25.6 mmol). The réaction mixture was stirred at room température for 3 h. After completion of the reaction, the reaction mixture was concentrated to dryness and was coevaporated with toluene (2 x 50 mL) to afford the crude compound.

To this crude in solution in DMF (13 mL) were successively added (2S)-2-(9H-fluoren-9ylmethoxycarbonylamino)-3-methyl-butanoic acid (467 mg, 1.38 mmol), DIPEA (867 μΐ, 5.24 mmol) and HBTLJ (845 mg, 2.23 mmol). The reaction mixture was stirred for 16 h at room température. After completion of the reaction, a saturated aqueous solution of hydrogeno carbonate (20 mL) was added, the mixture was stirred at room température for l h, was diluted with water (100 mL) and was concentrated to dryness. The crude product was purified by silica gel chromatography (gradient of methanol in dichloromethane) and then b y 384 reverse phase C18 chromatography using the neutral method to afford methyl (2S,3S,4R,5S,6S)3,4,5-triacetoxy-6-[2-[5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl] amino]-5-ureido-pentanoyl] amino]-2-(hydroxymethyl)phenyl] ethyl] tetrahydropyran-2carboxylate (680 mg, 0.720 mmol) as a white solid. LC-MS: MS (ESI) m/z [M+H]+ = 946.3. ^lH NMR (400 MHz, dmso-d6): δ 9.90 (s, IH). 8.07 (d, 2H), 7.89 (d, 2H), 7.74 (t, 2H), 7.44-7.38 (m, 3 FI), 7.36-7.28 (m, 3H), 7.24 (d, IH), 5.94 (t, IH), 5.37 (s, 2H), 5.30 (t, IH), 4.99-4.92 (m, 2H), 4.79 (t, IH), 4.42-4.36 (m, 4H), 4.32-4.19 (m, 3H), 3.94-3.90 (m, IH), 3.76 (t, IH), 3.65 (s, 3H), 2.99-2.94 (m, 2H), 2.65-2.54 (m, 2H), 2.07-1.98 (ni, 10H), 1.70-1.55 (m, 4H), 1.46-1.36 (m, 2H), 0.89-0.84 (m, 6H). ^lîC NMR (100 MHz, dmso-d6): δ 171.19, 170.33, 169.58, 169.45, 169.27, 167.77, 158.81, 156.12, 143.89, 143.76, 140.69, 139.48, 137.54, 134.88, 128.44, 127.62, 127.06, 125.35, 120.08,119.42, 116.65, 75.78,74.61,72.65,71.20,69.49, 65.68, 60.49, 60.10, 53.14, 52.40, 46.68, 32.32, 30.43, 29.54, 27.19, 26.77, 20.39, 20.34, 20.24, 19.22, 18.25.

Step 7: methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-(bromomethyl)-5-[[(2S)-2-[[(2S)-2(9H-Jhioren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureidopentanoyl] amino]phenyl] ethyl] tetrahydropyran-2-carboxylate

To a solution of compound methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2’[5-[[(2S)-2[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]-2-(hydroxymethyl)phenyl]ethyl]tetrahydropyran-2-carboxylate (154 mg, 0.163 mmol) in THF (8.2 mL) was successively added triphenylphosphine (85.4 mg, 0.326 mmol) and l-bromopyrrolidine-2,5-dione (58.0 mg, 0.326 mmol). The reaction mixture was stirred for 2h at room température. The progress of the reaction was followed by UPLC-MS: an aliquot was treated by a large ex ces of MeOH, following the formation of the corresponding methyl ether. The expected bomide dérivative was stable in UPLC-MS conditions. After 5h were added triphenylphosphine (85.4 mg, 0.326 mmol) and l-bromopyrrolidine-2,5-dîone (58.0 mg, 0.326 mmol) and the reaction mixture was stirred for 15h at room température. The obtained crude methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-(bromomethyl)-5-[[(2S)-2-[[(2S)-2-(9Hfluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl] amino]-5-ureidopentanoyl]amino]phenyl]ethyl]tetrahydropyran-2-carboxylate was used like this in the next step. UPLC-MS: MS (ESI) m/z [M+Ome-Br+H]+ = 960.7.

Step 8: (2R)-2-[(5S_a)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino] -2[2-[(2S,3S, 4R, 5S, 6S)-3,4,5-triacetoxy-6-methoxycarbonyl-tetrahydropyran-2

385 yl] ethyl]phenyl] methyl]-4-methyl-piperazin-4-ium-l-yl] ethoxy]-2-methyl-phenyl]-6(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl] oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

To the solution of methyl (2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-[2-[2-(bromomethyl)-5[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonyIamino)-3-methyl-butanoyl]amino]-5-ureidopentano yl] amino] phenyl] ethyl] tetrahydropyran-2-carboxylate (O.l67mmol) in DMF from the previous step (step 7) was successively added (2R)-2-[(5Sa)-5-[3-chloro-2-methyl-4-(2piperazin-l-ylethoxy)phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy] phenyl] propanoic acid (Cl) (143 mg, 0.163 mmol) and DIPEA (114 pL, 0.652 mmol) The reaction mixture was stirred for 15 h at room température and the progress of the reaction was followed by UPLC-MS (aliquot was treated by a large exces of MeOH). The crude product was purified by Cl 8 reverse phase prep-HPLC by direct deposit of the reaction mixture on the Xbridge column and using the TFA method to afford (2R)-2-[(5Sa)5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-y1methoxycarbonylamino)-3-methylbutanoyI]amino]-5-ureido-pentanoyl]ammo]-2-[2-[(2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6methoxycarbonyl-tetrahydropyran-2-yl]ethyl]phenyl] methyl ]-4-methyl-piperazin-4-ium-lyl]ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (21.3 mg, 0.0111 mmol) as a white powder. UPLC-MS: MS (ESI) m/z [M+H]+ = 1802.9+1804.9. IR Wavelength (cm'¹): 1755, 1672, 1226,1201,1130. 'H NMR (400 MHz, dmso-d6) δ ppm 13.3 (br s, 1H), 10.2 (s, 1H), 8.88 (d, 1H), 8.61 (s, 1H), 8.14 (d, 1H), 7.88 (d, 2H), 7.73 (dd, 2H), 7.65 (d, 1H), 7.63 (d, 1H), 7.62 (m, 1H), 7.54 (br s, 1H), 7.51 (dd, 1H), 7.45 (t, 1H), 7.4 (t, 2FI), 7.38 (m, 1H), 7.32 (t, 2H), 7.3 (dd, 2H), 7.2 (d, 1H), 7.2 (t, 2H), 7.15 (t, 1H),7.15 (d, 1H), 7.03 (t, 1H), 7.01 (dd, 1H), 6.72 (t, 1H), 6.22 (d, 1H), 6 (br s, 1H),5.51 (dd, 1H), 5.34 (t, 1H),5.3 (br s, 2H), 5.27/5.21 (m, 2H), 4.98 (t, 1H), 4.85 (t, 1H), 4.57/4.49 (m, 2H), 4.39 (m, 1H), 4.35 (d, 1H), 4.27 (m, 2H), 4.26 (m, 2H), 4.23 (m, 1H), 3.93 (t, 1H), 3.76 (s, 3H), 3.71 (m, IH), 3.64 (s, 3H), 3.4 (m, 4H), 3.29/2.51 (2dd, 2H), 3.13/2.94 (2m, 4H), 3 (m, 2H), 2.98 (m, 2H), 2.93 (br s, 3 H), 2.81 (m, 2H), 1.99/1.95 (3s, 9H), 1.98 (m, 1H), 1.84 (s, 3H), 1.77/1.59 (2m, 2H), 1.64 (2m, 2H), 1.41 (2m, 2H), 0.88/0.85 (2d, 6H). ^I3C NMR (100 MHz, dmso-d6): δ ppm 158.1, 152.9, 135.6, 131.5, 131.4, 131.3, 131.2, 131, 128.9, 128.1, 127.5, 125.7, 120.9, 120.6, 120.4, 120.3, 117, 116, 116, 112.8, 112.2, 111.2, 75.6, 74.9, 73.8, 72.9, 71.4, 69.6, 69.4, 67.5, 66.1, 60.4, 58.3, 56, 55.4, 53.9, 52.8, 47.2,46.2,44.3, 39,32.8, 32.8,31, 29.6, 27.4, 27.2,21.1, 19.5/18.7, 18.1. ⁱ⁹F NMR (376 MHz, dmso-d6) δ ppm -74, -112.

386

Step 9: (2S, 3S, 4R, 5 R, 6S)-6-[2-[(5S_a)-5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5ureido-pentanoyl]amino]-2-[[4-[2-[4-[ 4-[(lR)-l-carboxy-2-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]ethoxy]-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-2-chloro-3-methyl-phenoxy]ethyl]-lmethyl-piperazin-l-ium-1-yl] methyl]phenyl] ethyl]-3,4,5-trihydroxy-tetrahydropyran2-carboxylic acid

To a solution of (2R)-2-[(5S_a)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureido-pentanoyl]amino]-2-[2[(2S,3S,4R,5S,6S)-3,4,5-triacetoxy-6-methoxycarbonyl-tetrahydropyran-2yl] ethyl] phenyl] methyl]-4-methyl-piperazin-4-ium-1-yl] ethoxy]-2-methyl-phenyl]-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4yl]methoxy]phenyl]propanoic acid (21.3 mg, 0.0111 mmol) in methanol (6.0 mL) was added a solution of Lithium hydroxide monohydrate (4.66 mg pL, 0.111 mmol) in water (4 ml). The réaction mixture was stirred at room température for 60 h and the progress of the reaction was followed by UPLC-MS. The crude product was purified by Cl8 reverse phase prep-HPLC by direct deposit of the reaction mixture on the Xbridge column and using the TFA method to afford (2S,3S,4R,5R,6S)-6-[2-[(5S_a)-5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5ureido-pentanoyl]amino]-2-[[4-[2-[4-[4-[(lR)-l-carboxy-2-[2-[[2-(2-methoxyphenyl)pyrimidin4-yl]methoxy]phenyl]ethoxy]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-2-chloro-3meth yl-phenoxy] ethyl]-1-methyl-p iperazin-l-ium-1-yl] methyl]phenyl] ethyl]-3,4,5-trihydroxytetrahydropyran-2-carboxylic acid (47.8 mg, 0.029 mmol) as a white powder. UPLC-MS: MS (ESI) m/z [M+H]+ = 1440.6+1442.6.

Step 10: (2S, 3S, 4R, SR, 6S)-6-[2-[2-[[4-[2-[4-[4-[(1R)-1 -carboxy-2-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl] methoxy]phenyl] ethoxy]-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-2-chloro-3-methyl-phenoxy] ethyl]-1methyl-piperazin-l-ium- 1-yl]methyl] -5-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-lyl)ethoxy]propanoylamino] -3-methyl-butanoyl] amino]-5-ureidopentanoyl] amino]phenyl]ethyl]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid;2,2,2-trifluoroacetate L21-C1

To a solution of(2S,3S,4R,5R,6S)-6-[2-[(5S_a)-5-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyl]amino]-5-ureido-pentanoy]]amino]-2-[[4-[2-[4-[4~[(lR)-l-carboxy-2-[2-[[2-(2methoxyph enyl)pyrimidin-4-yl]methoxy]phenyl] ethoxy]-6-(4-fluoro phenyl )thieno [2,3d] pyrimidin-5 -yl] -2- chloro-3 -methyl -phenoxy] ethyl ] -1 -methyl-piperazin-1 - i um-1 387 yl] methyl] phenyl] ethyl]-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid (47.1 mg, 0.0282 mmol) in DMF (1.5 mL) were successively added the solution of (2,5-dioxopyrrolidin-l-yl) 3[2-(2,5-dîoxopyrrol-l-yl)ethoxy]propanoate (Purshased from Broadphann, 13.1 mg, 0.0423 mmol) in DMF (500 pL) and DIPEA (17.2 pL, 0.0988 mmol).

The reaction mixture was stirred for Ih at room température and the progress of the reaction was followed by UPLC-MS. The crude product was purified by Cl 8 reverse phase prepHPLC by direct deposit of the reaction mixture on the Xbridge column and using the TFA method to afford L21-C1 (65 mg, 0.0310 mmol) as a white powder. HR-ESI+: m/z [M+H]+ = 1635.6093 (1635.6068) (measured/theoretîcal).

Préparation of L9-C1:

(2R)-2-[(5Sa)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrroI-lyl)ethoxy]propanoyiamino]-3-methyl-butanoyI]amino]-5-ureidopentanoyl] amino] phenyl] methyl)-4-methyl-piperazin-4-ium-l-yl]ethoxy]-2-methyl-phenyl]6-(4-fluorophenyl)thieno[2,3-d|pyrimidin-4-yl|oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4yl]niethoxy]phenyi]propanoic acid;2,2,2-trifluoroacetate;2,2,2-trifluoroacetic acid

Step 1: 9H-fluoren-9-ylmethyl N-[( IS)-1-[[(1 S)-l-[[4-(bromomethyl)phenyl] carbamoyl]-4ureido-butyl] carbamoyl]-2-methyl-propyl] carbamate

A solution of 9H-fluoren-9-ylmethyl N-[( l S)-1 -[[( 1 S)-1 -[[4(bromomethyl)phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]carbamate (150 mg, 0.249 mmol) in THF (3.8 ml) was cooled to 0°C. At 0°C was added dropwise tribromophosphane (1 M in dichloromethane) (374 pL, 0.249 mmol). The reaction was stirred 5 min at 0°C and Ih at room température. The progress of the reaction was followed by UPLC-MS (aliquot was treated by a large excess of MeOH). The reaction mixture was diluted with ethyl acetate (3 ml) and washed with an aqueous saturated solution of sodium hydrogen carbonate (Ix 6ml). The organic layer was dried over magnésium sulfate, fdtered. Add DMF (10 ml) and

388 evaporate the ethyl acetate and the THF. The obtained solution of 9H-fluoren-9-ylmethyl N[(lS)-l-[[(lS)-I-[[4-(bromomethyl)phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methylpropyl]carbamate is used like that in the next step. UPLC-MS: MS (ESI) m/z [M+Na]+ = 686.5+688.6.

Step 2: (2R)-2-[(5S_a)-5-[3-chloro-4-]2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureidopentanoyl] amino]phenyl] methyl]-4-methyl-piperazln-4-ium-l-yl] ethoxy]-2-methylphenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl] methoxy]phenyl]propanoic acid

To the solution of 9H-fluoren-9-ylmethyl N-[(lS)-l-[[(lS)-l-[[4(bromomethyl)phenyl]carbamoyl]-4-ureido-butyl]carbamoylj-2-methyl-propyl]carbamate (0.249 mmol) in DMF from the previous step (step l) was successively added DMF (l 0 ml), (2R)-2[(5Sa)-5-[3-chloro-2-methyl-4-(2-piperazin-l-ylethoxy)phenyl]-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (Cl) (218 mg, 0.249 mmol) and DIPEA (130 pL, 0.748 mmol). The reaction mixture was stirred for 15 h at room température and the progress of the réaction was followed by UPLC-MS (aliquot was treated by a large excess of MeOH). The obtained solution of (2R)-2-[(5Sa)-5-[3chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl]ammo]-5-ureido-pentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-lyl]ethoxy]-2-methyl-phenyI]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yI]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]Tnethoxy]phenyl]propanoic acid in DMF was used like that in the next step. UPLC-MS: MS (ESI) m/z [M+Na]+= 1458.7+1460.7.

Step 3: (2R)-2-[(5S_a)-5-[4-[2-[4~[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl~butanoyl]amino]-5ureido-pentanoyl] amino]phenyl] methyl] -4-methyl-piperazin-4-ium-1 -yl] ethoxy]-3chloro-2-methvl-phenyl]-6-(4-]luorophenyl)thieno [2,3-d]pyrimidin-4-yl] oxy-3-[2[[2-(2-methoxyphenyl)pyrimidin~4-yl]methoxy]phenyl]propanoic acid; 2,2,2trifluoroacetate; bis 2,2,2-triJluoroacetic acid

To the solution of(2R)-2-[(5S_a)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9yImethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-l-yl]ethoxy]-2-niethyl-phenyl]-6(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4yl]methoxy]phenyl]propanoic acid (0.249 mmol) in dimethylformamide (3 mL) obtained in the

389 previous step (step 2) was added piperidine (49.3 pL, 0.499 mmol). The reaction mixture was stirred at room température for 5 h and the progress of the reaction was foliowcd by UPLC-MS. The crude product was purified by C18 reverse phase prep-HPLC by direct deposit of the reaction mixture on the Xbridge column and using the TFA method to afford (2R)-2-[(5S_a)-5-[4[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5-ureidopentanoyl] amino] phenyl]methyl]-4-methyl-piperazin-4-ium-l-yl]ethoxy]-3-chloro-2-m ethylphenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimîdin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl] methoxy] phenyl] propanoic acid; 2,2,2-trifluoroacetate; bis 2,2,2trifluoroacetic acid (31.2 mg = 0.0213 mmol) as a white powder. UPLC-MS; MS (ESI) m/z [M+Na]+= 1236.7+1238.7.

Step 4: (2R)-2-[(5Sa)-5-[3-chloro-4-[2-[4-[]4-]](2S)-2-[[(2S)-2-]3-]2-(2,5-dioxopyrrol-lyl)ethoxy]propanoylamino]-3-methyl-butanoyl] amino] -5-ureidopentanoyl]amino]phenyl] methyl] -4-methyl-piperazin-4-ium-l -yl] ethoxy]-2-methylphenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2trifluoroacetate; 2,2,2-lrifluoroacelic acid L9-C1

To a solution of (2R)-2-[(5Sa)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyl]ammo]-5-ureido-pentanoyl]amino]phenyl]methyI]-4-methyl-piperazin-4-ium-lyl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyI)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (3L2 mg, 0.0213 mmol) in DMF (1.5 mL) were successively added the solution of (2,5-dioxopyrrolidin-l-yl) 3-(2-(2,5dioxopyrrol-l-yl)ethoxy]propanoate (23.8 mg, 0.0768 mmol) in DMF (500 pL) and DIPEA (31.2 pL, 0.179 mmol). The reaction mixture was stirred for 15h at room température and the progress of the reaction was followed by UPLC-MS. The crude product was purified by Cl 8 reverse phase prep-HPLC by direct deposit of the reaction mixture on the Xbridge column and using the TFA method to afford L9-C1 (6 mg, 0.00303 mmol) as a white powder. HR-ESI+: m/z (M]+= 1431.5437 (143 L5433) (measured/theoretical).

390

Préparation of L9-C8:

(2R)-2-[(5Sa)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-l yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureidopentanoyl] amino] phenyl jmethylI-4- methyl-piperazin-4-ium-l-yl| ethoxy]-2-methyl-phenyl]6-(4-fluorophenyl)thieno[2,3-d]pyrimidm-4-yl]oxy-3-(2-[[2-(3-sulfooxyphenyl)pyrimidm-4yljmethoxy] phenyl]propanoic acid;2,2,2-trifluoroacetate;2,2,2-trifluoroacetic acid

Step 1 : (2R)-2-[(5Sa)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fhioren-9ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureidopentanoyl] amino]phenyl] methyl]-4-methyl-piperazin-4-ium-l -yl] ethoxy] -2-methylphenyl]-6-(4-fluoropheny l)thieno]2,3-d]pyrimidin-4-yl] oxy-3-[2-[[2-(3sulfooxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

To the solution of 9 H-fluoren-9-yl methyl N-[(lS)-l-[[(lS)-l-[[4(bromomethyI)phenyl]carbamoyl]-4-ureido-butyI]carbamoyl]-2-methyl-propyl]carbamate (0.0230 mmol; obtained according to Step 1 ofthe préparation of L9-C1) in DMF (3 mL) was successively added DMF (5 ml), ammonium; [3-[4-[[2-[(2R)-2-carboxy-2-[(5Sa)-5-[3-chloro-2methyl-4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin4-yl]oxy-ethyl]phenoxy]methyl]pyrimidin-2-yl]phenyl] sulfate (C8) (22 mg, 0.0230 mmol) and DIPEA (12 pL, 0.069 mmol). The réaction mixture was stirred for 15 h at room température and the progress of the reaction was followed by UPLC-MS (aliquot was treated by a large exces of MeOFI). The obtained solution of (2R)-2-[(5Sa)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9Hfluoren-9-ylmethoxycarbonyl amino)-3 -methyl -butanoyl] amino]-5-ureidopentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-l-yl]ethoxy]-2-methyl-phenyl]-6(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4yl]methoxy]phenyl]propanoic acid in DMF was used in the next step. UPLC-MS: MS (ESI) m/z [M-SO₃H]+= 1444.8+1446.7.

391

Step 2: (2R)-2-[(5S_a)-5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5ureido-pentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-l-yl]ethoxy]~3chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2[[2-(3-sulfooxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; bis 2,2,2trifluoroacetic acid

To the solution of (2R)-2-[(5Sa)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]-5-ureidopentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-l-yl]ethoxy]-2-methyl-phenyl]-6(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(3-sulfooxyphenyl)pyrimidin-4yl]methoxy]phenyl]propanoic acid (0.0230 mmol) in dimethylfonriamide (3 mL) obtained in the prevîous step (step 1) was added piperidine (9 pL, 0.0920 mmol). The reaction mixture was stirred at room température for 5 h and the progress of the réaction was followed by UPLC-MS. The crude product was purified by C18 reverse phase prep-HPLC b y direct deposit of the reaction mixture on the Xbrîdge column and using the TFA method to afford [3-[4-[[2-[(2R)-2[5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5-ureidopentanoyl ] amino] phenyl ] methyl ]-4-m ethyl-piperazin-4-i um-1-yl ] ethoxy]-3-chl oro-2-m ethylphenyl]-6-(4-fluorophenyl )thieno[ 2,3-d]pyrimidin-4-yl]oxy-2-carboxyethyl]phenoxy]methyl]pyrimidin-2-yl]phenyl] sulfate; bis 2,2,2-trifluoroacetic acid (10.0 mg = 0.00633 mmol) as a white powder. UPLC-MS: MS (EST) m/z [M-SO₃]+ = 1224.12.

Step 3: (2R)-2-[(5S_a)-5-]3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-lyl)ethoxy]propanoylamino]-3-methyl-butanoyl] amino]-5-ureidopentanoyl] amino]phenyl] methyl]-4-methyl-piperazin-4-ium-l-yl] ethoxy]-2-methylphenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(3sulfooxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid;2,2,2trifluoroacetate;2,2,2-trtfluoroacetic acid

To a solution of (2R)-2-[(5S_a).5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl]-4-methyl-piperazin-4-ium-lyl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2[[2-(3-sulfooxyphenyl)pyrimidin-4-yl]methoxy] phenyl] propanoic acid (10 mg, 0.00653 mmol) in DMF (1 mL) were successively added the solution of (2,5-dioxopyrrolidin-l-yl) 3-[2-(2,5dioxopyrrol-l-yl)ethoxy]propanoate (3.1 mg, 0.00980 mmol) in DMF (500 pL) and DIPEA (4 pL, 0.0229 mmol). The reaction mixture was stirred for I5h at room température and the progress of the reaction was followed by UPLC-MS. The crude product was purified by Cl8 392 reverse phase prep-HPLC by direct deposit of the reaction mixture on the Xbridge column and using the TFA method to afford L9-C8 (6 mg, 0.00401 mmol) as a white powder. UPLC-MS: MS (ESI) m/z [M+Na]+ = 1519.5+1521.2, [M+H-SO3]+ 1417.7+1419.6. HR-ESI+; m/z [M+H]+ = 1497.486 (1497.4845) (measured/theoretical).

Préparation of L9-C10:

(2R)-2-[(5Sa)5-[3-chloro-4-[2-|4-[[4-[[(2S)-2-[[(2S)-2-[3-|2-(2,5-dioxopyrrol-lyl)ethoxy|propanoylamino]-3-methyl-butanoyl]amino]-5-ureidopentanoyl| amino] phenyl ]methyl]-4-methyl-piperazin-4-ium-l -yl] ethoxy ]-2-methyl-phenylJ6-[4-fluoro-3-(2,2,2-trifluoroethoxy)phenyi]thieno[2,3-d|pyrimidin-4-yi]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid; 2,2,2-trifluoroacetate;

2,2,2-trifluoroacetic acid

The procedure is as in the process of synthesis of L9-C9, replacing C9 used in Step 3 by (2R)-2-[((5Sa)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl}-6-[4-fiuoro3-(2,2,2-trifluoroethoxy)phenyl]thieno[2,3-d]pyrimidin-4-yl)oxy]-3-(2-{[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy}phenyl)propanoic acid CIO and using TFA method for purification. HR-ESI+: m/z [M+H]+ = 1529.543 / 1529.5413 (measured/theoretical).

Préparation of L9-C11 :

(2R)-2-|(5Sa)-5-[3-chloro-4-[2-|4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-lyl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureidopentanoyI]aniino|phenyl]methyl]-4-methyl-piperazin-4-îum-l-yl]ethoxy]-2-methyl-phenyl]6-(4-fluorophenyl)thieno[2,3-d]pyriniidin-4-yl]oxy“3-[2-[[2-(4-methoxyphenyl)pyrimidin-4yl]methoxy] phenyl] propanoic acid; 2,2,2-trifluoroacetate; 2,2,2-trifluoroacetic acid.

393

The procedure is as in the process of synthesis of L9-C9, replacîng C9 used in Step 3 by (2R)-2-{[(5Sa)-5-{3-chloro-2-methyI-4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl )-6-(4fl uorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2-(4-methoxyphenyl)pyrimidin-45 yl]methoxy]phenyl)propanoic acid Cil and using TFA method for purification. HR-ESI+: m/z [M+H]+ = 1431.5442 / 14.31.5433 (measured/theoretical).

Préparation of L9-C12:

(2R)-2-[(5Sa)-5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[|(2S)-2-[3-[2-(2,5-dioxopyrrol-lyI)etlioxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureido10 pentanoyl] amino] phenyI]methyl]-4-methyl-piperazin-4-iuin-l-yl|ethoxy)-2-niethyI-phcnyI|6-(4-nuorophenyI)thieno|2,3-d]pyriniidin-4-yl]oxy-3-|2-[[2-(2,2,2-trifluoroethyl)pyrazol-3yljmethoxy]phenyl]propanoic acid;2,2,2-trifluoroacetate;2,2,2-trifluoroacetic acid

The procedure is as in the process of synthesis of L9-C9, replacing C9 used in Step 3 by 15 (2R)-2-{[(5Sa)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl )-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2- {[1-(2,2,2-tri fl uoroethyl)-! H-pyrazol-5yl]methoxy}phenyl)propanoic acid C12 using TFA method for purification. HR-ESI+: m/z [M+H]+= 1395.5048 / 1395;5045 (measured/theoretical).

Préparation of L9-C14:

394 (2R)-2-|5-|3-ehloio-4-|2-|4-||4-{|(2S)-2-||(2S)-2-|3-|2-(2.5-dioxopyrrol-l-yl)ethoxy| propanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl] amino] phenyl]methyl]-4methyl-piperazin-4-ium-l-yl]ethoxy]-2-m€thyi-phenyl]-6-(4-fluorophenyl)thieno[2,3-d] pyrimidin-4-yl]oxy-3-[2-[(2-(3-liydroxy-2-niethoxy-phenyl)pyriinidin-4yl]methoxy]phenyl]propanoic acid;2,2,2-trîfluoroacetate

Step 1: (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl]-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxy-3-sulfooxy-phenyl)pyrimidin4-yl] methoxy]phenyl]propanoic acid

500 mg ethyl (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-lyl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[(2-chloropyrimidin4-yl)methoxy]phenyl]propanoate (0.60 mmol, WO2016/207216 Préparation 1) and 202 mg (3hydroxy-2-methoxy-phenyl)boronic acid (1.20 mmol) were dissolved in 9 mL l_s4-dioxane, then 42 mg Pd(PPhî)2Ch (0.06 mmol), 588 mg Cs₂CO₃ (1.80 mmol) and 9 mL water were added and the mixture was stirred under N2 atmosphère at 70°C until complété conversion. Then it was diluted with water, neutralîzed with 2 M aqueous HCl solution, and extracted with DCM. The combined organic phase was dried over Na₂SO₄, filtered and the filtrate was concentrated under reduced pressure. The crude ester was purified via flash chromatography using heptane, EtOAc and 0.7 M NH₃ solution in MeOH as eluents to obtain a mixture of diastereoisomers. Then it was dissolved in 23.6 mL pyridine, 0.97 mL SO3^xpyrimidine (5.98 mmol) was added and the mixture was stirred at 70°C until complété conversion. Then it was concentrated under reduced pressure, and dissolved in 2 mL dioxane, then 200 mg KOH (3.57 mmol) and I mL water were added. The mixture was stirred at rt until complété hydrolysis. Then it was neutralîzed with 2 M aqueous HCl solution, and directly injected on prep-RP-HPLC, using 25 mM aqueous NH4HCO3 solution and MeCN as eluents. The diastereoisomer eluting later was collected as product of the title. ^lH NMR (500 MHz, DMSO-d6) Ô: 8.92 (d, IH), 8.63 (s, IH), 7.68 (dd, IH), 7.63 (d, IH), 7.34 (d, IH), 7.30 (dd, 2H), 7.29 (d, IH), 7.20 (t, 2H), 7.16 (t, IH), 7.15 (d, IH), 7.10 (t, IH), 7.02 (d,

395

IH), 6.73 (t, lH), 6.38 (d, IH), 5.50 (dd, IH), 5.29/5.23 (d+d, 2H), 4.21/4.16 (m+m, 2H), 3.84 (s, 3H), 3.25/2.55 (dd+dd, 2H), 3.18-2.75 (m, 10H), 2.65 (brs, 3H), 1.82 (s, 3H).HRMS calculated for C47H44N6O10S2CIF: 970.2233; found 971.2297 (M+H).

Step 2: (2R)-2-[5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-l-yl)ethoxy] propanoylami.no] -3-methyl-butanoyl] amino] -5-ureido-pentanoyl] amino]phenyl] methyl]-4methyl-piperazin-4-ium-l-yl] ethoxy]-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d] pyrimidin-4-yl]oxy-3-[2-[[2-(3-hydroxy-2-methoxy-phenyl)pyrimidin-4yl] methoxy]phenyl]propanoîc acid;2,2,2~trifluoroacetate L9-C14

To a solution of (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-lyl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxy-3suifooxy-phenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (20.0 mg; 0.0206 mmol) in DMF (309 pL), were successively added (2S)-N-[4-(chloromethyl)phenyl]-2-[[(2S)-2-[3-[2-(2,5dioxopynOl-l-yl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentanamide (17.5 mgL; 0.0206 mmol), DIPEA (10.8 pL; 0.0618 mmol) and TB AI (1 mg; 0.0027 mmol). The reaction mixture was stirred at 70°C for 18 hours. The crude product was purified by Cis reverse phase prep-HPLC by direct deposit of the reaction mixture on the X-Bridge column and using the TFA method to afford L9-C14 (10.5 mg; 0.00725 mmol) as a white powder. HR-ESI+: m/z [M+H]+= 1448.5437 / 1448.5466 [measured/theoretical].

Préparation of L9-P15:

(HR,20R)-23,26-dichloro-20-|[4-H4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dÎoxopyrrol-l-yl)ethoxy] propanoylamino]-3- methyl-butanoyl]amino]-5-ureido-pentanoyl] amino] phenyl] methyl]-4methyl-piperazin-4-ium-l-yl]metliyl]-3-(4-fluorophenyl)-14-[[2-(2-methoxyphenyI) pyrimidin-4-yl]methoxy]-24,25-dimethyl-10,18,21-trioxa-4-thia-6,8diazapentacyclo[20.2.2.12,5*113,17.09,28] octacosa-1 (24),2,5(28),6,8,13,15,17(27),22,25decaene-ll-carboxylic acid;2,2,2-trifluoroacetate

396

To a solution of (1 lR,20R)-23,26-dichioro-3-(4-fluorophenyl)-14-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]-24,25-dimethyl-20-[(4-methylpiperazin-l-yl)methyï]10,18,2 l-trioxa-4-thia-6,8-diazapentacyclo[20.2.2.12,5.113,17.09,28]octacosa1(25),2,5(28),6,8,13,15,17(27),22(26),23-decaene-11 -carboxylic acid P15 (obtained according to WO 2019/035914; 10.0 mg; 0.0105 mmol) in DMF (630 pL), were successively added (2S)-N[4-(bromomethyl)phenyl]-2'[[(2S)-2-[3-[2-(2,5-dioxopyrroM-yl)ethoxy]propanoylamino]-3methyl-butanoyl]amino]-5-ureido-pentanamide (10.0 mg; 0.0158 mmol), DIPEA (5.5 pL; 0.0315 mmol) and TBAI (0.5mg, 0.001 Ommol). The reaction mixture was stirred at room température for 0.5 hour. The crude product was purified by Cig reverse phase prep-HPLC by direct deposit of the reaction mixture on the X-Bridge column and using the TFA method to afford L9-P15 (11.9 mg, 0.00733 mmol) as a white powder. HR-ESI+: m/z [M-CF₃CO₂]+ = 1507.5183 / 1507.5155

Préparation of L9-P16:

(HR,20R)-23,26-dichloro-14-[[2-[4-[[(2S)-l,4-dioxan-2-yl]methoxymethyl]-4-fluorocyclohexyl]pyriniidÎn-4-yl]methoxy|-20~||4-[[4-[[(2S)-2-[|(2S)-2-[3-[2-(2,5-dioxopyrrol-lyl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureido-pentanoyl] amino] phcnyI]methyi]-4-methyi-piperazin-4-ium-1-yl)methyi]-3-(4-nuorophenyl)-24,25-dimethylI0,18,21-trioxa-4-thia-6,8-diazapentacyclo|20.2.2.12,5.113,17.09,28]octacosa1(25),2,5(28),6,8,13,15,17(27),22(26),23-decaene-U-carboxylic acid;2,2,2-trifluoroacetate o

To a solution of (HR,20R)-23,26-dichloro-14-[[2-[4-[[(2S)-l,4-dioxan-2yl]methoxyrnethyl]-4-fluoro-cyclohexyl]pyrirnidin-4-'yI]mcthoxy]-3-(4-fluorophenyI)-24,25dimethyl-20-[(4-methylpiperazin-l-yl)methyI]-10,18,21-trioxa-4-thia-6,8dîazapentacyclo[20.2.2.12,5.113,17.09,28]octacosa-l (24),2,5(28),6,8,13,15,17(27),22,25decaene-11-carboxylic acid P16 (obtained according to WO 2019/035911; 14.7 mg; 0.0137 mmol) in DMF (1 mL), were successively added (2S)-N-[4-(brornomethyl)phenyl]-2-[[(2S)-2[3 - [2-(2,5-dioxopyrrol-1-yl)ethoxy]propanoylamino]-3-methyl-butanoyl ] amino]-5-ureido397 pentanamide (13.1 mg; 0.0205 mmol) and DIPEA (7.1 pL; 0.0410 mmol). The reaction mixture was stirred at room température for 2 hours. The crude product was purified by direct deposit of the reaction mixture on the X-Bridge column in using the TFA method to afford L9-P16 (7.9 mg; 0.00420 mmol) as a white powder. IR (cm^-1): 3327, 1768/1706, 1666, 1199/1118, 831/798. HR-ESI+: m/z [M-CF₃CO₂]+ = 1631.6071/1631.6054 [measured/theoretical]

Préparation of L9-P17: (llR,20R)-23,26-dichloro-14-[[2-[4-[Î(2S)-l,4-dioxan-2-yi]niethoxy]cyclohexyl]pyrÎmidîn-4y l|methoxy]-20-[[4-[[4-[|(2S)-2-[[(2S)-2-(3-(2-(2,5-dioxopyrroI-l-yl)ethoxy] propanoylaniino]-3-methyl-butanoyl(amino]-5-ureido-pentanoyl|amino]phenyl|niethyl]-4inethyl-piperazin-4-Îum-l-yl] methyl]-3-(4-fluorophenyl)-24,25-dimethy 1-10,18,2 l-trioxa-4thia-6,8-diazapentacyclo[20.2.2.12,5.113,17.09,28]octacosa1(24),2,5(28),6,8,13,15,17(27),22,25-decaene-ll-carboxyIic acid;2,2,2-trifluoroacetate_

To a solution of (1 lR,20R)-23,26-dichloro-14-[[2-[4-[[(2S)-l,4-dioxan-2yl]methoxy]cyclohexyl]pyrimidin-4-yl]methoxy]-3-(4-fluorophenyl)-24,25-dimethyl-20-[(4methylpiperazin-1 -yl )methy 1 ] -10,18,21 -trioxa-4-thia-6,8diazapentacyclo[20.2.2.12,5.1 13,17.09,28]octacosa-l(24),2,5(28),6,8,13,15,17(27),22,25decaene-11-carboxylic acid P17 (obtaîned according to WO 2019/035911; 14.5 mg; 0.0139 mmol) in DMF (1 mL), were successively added (2S)-N-[4-(bromomethyl)phenyI]-2-[[(2S)-2[3-[2-(2,5-dioxopyrrol-l-yI)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]-5-ureidopentanamide (13.3 mg; 0.0208 mmol) and DIPEA (7.3 pL; 0.0417 mmol). The reaction mixture was stirred at room température for 8 hours. The crude product was purified by direct deposit of the reaction mixture on the X-Bridge column and using the TFA method to afford L9-P17 (7.0 mg, 0.00437 mmol) as a white powder. IR (cm'¹): 3700-2400, 1771/1738/1705, 1665, 1194/1128. HR-ESI+: m/z [M-CF₃CO₂]+ = 1599.6013 / 1599.5992. HR-ESI+: m/z [MCF₃CO₂+H]2+ = 800.3049 / 800.3035 [measured/theoretical]

398

Préparation of L25-P1:

2-[[4-[2-]4-[4-[(lR)-l-carboxy-2-|2-{[2-(2-methoxyphenyl)pyrimidin-4-yl|methoxy]phenyl] ethoxy]-6-(4-fliiorophenyl)thieno[2,3-d]pyriinidin-5-yl]-2-chloro-3-methyI-phenoxy] ethyl]l-methyl-piperazin-l-ium-l-yJ]nlethyl]-5-l[(2S)-2-[[l-l2-[2-(2,5-dioxopyrrol·lyl)ethoxy]ethylcarbamoyl]cyclobutanecarbonyl]amino]-5-ureidopentanoyl]amino]benzenesulfonate;2,2,2-trifluoroacetic acid

Step 1: tert-butyl l-[2-[2-(2,5-dioxopyrrol-J-yl)ethoxy]ethylcarbamoyl] cyclobutanecarboxylate To a solution of 1-tert-butoxycarbonylcyclobutanecarboxylic acid (58.6 mg; 0.293 mmol) in DCM (5.85 ml), were successively added l-[2-(2-aminoethoxy)ethyl]pyrrole-2,5-dione (53.9 mg; 0.293 mmol), EDC (84.2 mg; 0.439 mmol), HOBt (59.3 mg; 0.439 mmol), and DIPEA (204 pL; 1.17 mmol). The reaction mixture was stirred at room température for 18 hours. The progress of the reaction was monitored by UPLC-MS. The reaction mixture was concentrated to dryness and solubilized in DMF (1 ml) and the solution was purified by X-Brîdge column Cis by direct deposit of the reaction mixture on the column and in using the TFA method to afford the title compound (57.3 mg; 0.156 mmol). IR (cm'^!): 3390, 1697/1666. ’H NMR (400 MHz, dmsod6) 6 ppm 7.5 (t, IH), 7.02 (s, 2H), 3.55/3.5 (2t, 4H), 3.38 (t, 2H), 3.17 (q, 2H), 2.33 (m, 4H), 1.77 (m, 2H), 1.38 (s, 9H). UPLC-MS: MS(ESI): m/z [M+Na]+ = 389.26 [M+H-tBu]+ = 311.22

Step 2: 1 -[2-[2-(2,5-dioxopyrrol-l -yl)ethoxy]ethylcarbamoyl]cyclobutanecarboxylic acid

To a solution of tert-butyl l-[2-[2-(2,5-dioxopyrrol-l-yl)ethoxy]ethylcarbamoyl] cyclobutanecarboxylate (7 mg; 0.0191 mmol) in DCM (0.175 mL), was added TFA (51.2 pL; 0.668 mmol). The reaction mixture was stirred at room température for 3.5 hours, then was concentrated to dryness to obtain the title compound (5.8 mg; 0.0187 mmol) as a colorless oü. The crude product was used in the next step. UPLC-MS: MS(ESI): m/z [M+H]+ = 311.35, [M+Na]4- = 333.37

399

Step 3: (2,3,4,5,6-pentafluorophenyl) 1 -[2-]2-(2,5-dioxopyrrol-1 yl)ethoxy] ethylcarbamoyl] cyclobutanecarboxylate

To a solution of 1 -[2-[2-(2,5-dioxopyrrol-1 yl)ethoxy]ethylcarbamoyl]cyclobutanecarboxylîc acid (18.2 mg; 0.0587 mmol) in THF (3 mL), were successively added 2,3,4,5,6-pentafluorophenol (13.0 mg; 0.0704 mmol) and DCC (14.5 mg; 0.0704 mmol). The reaction mixture was stirred at room température for 15 hours and the progress ofthe reaction was monitored by UPLC-MS. The reaction mixture was a suspension, the precipitate is fîltered off and washed with THF (1 ml) to afford a solution of (2,5dioxopyrrolidin-l-yl) l-[2-[2-(2,5-dioxopyrrol-lyl)ethoxy]ethylcarbamoyl]cyclobutanecarboxylate in THF. The crude product solution was used m step 9. UPLC-MS: MS(ESI): m/z [M+H]+ = 477.28, [M+Na]+ = 499.23

Step 4: methyl (2R)-2-[5-]3-chloro-2-methyl-4-[2-(4-meihylpiperazin-I-yl)ethoxy]phenyl]-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4yl] methoxy]phenyl] propanoate

To a solution of (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-lyl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoîc acid PI (5.0 g; 5.712 mmol) in DCM (25 mL) and methanol (25 mL), was added dropwise a solution of diazomethyï(trimethyl)silane (2 M in EtsO) (5.712 mL; 11.42 mmol). The reaction mixture was stirred at room température for 2 hours and the progress of the reaction was monitored b y UPLC-MS. After complet! on the reaction was quenched by a slow addition of acetic acid until the yellow color tum to red and was concentrated to dryness to afford the crude mixture. The crude product was purified by silica gel chromatography (gradient of methanol in DCM) to afford the title compound (4.52 g; 5.082 mmol). UPLC-MS: MS(ESI): m/z [M+H]+ = 889.27+891.6, [M+Na]+ = 911.31, [M+2H]2+= 445.59. IR (cm'¹): 1753, 1238/1053. ^lH NMR (400 MHz, dmso-d6) δ ppm 8.6 (s, IH), 8.45 (d, IH), 7.6 (d, IH), 7.52 (dd, IH), 7.45 (td, IH), 7.3 (m, 3H), 7.25-7.1 (m, 5H), 7.02 (t+d, 2H), 6.78 (t, 1 H), 6.31 (dd, IH), 5.52 (dd, IH), 5.25 (AB, 2H), 4.2 (m, 2H), 3.78/3.65 (2s, 6H), 3.2/2.58 (2dd, 2H), 2.71 (t, 2H), 2.5/2.3 (2ml, 8H), 2.12 (s, 3H), 1.88 (s, 3H).

Step 5: 5-[[(2S)-2-(9H-Jluoren-9-ylmethoxycarbonylamino)-5-ureido-pentanoyl]amino]-2(hydroxymethyl)benzenesulfonic acid

400

To a solution of Fmoc-Cit-OH (2.224 g; 5.596 mmol) in DCM (22.2 mL) and methanol (22.2 mL), were successively added sodium 5-amino-2-(hydroxymethyI) benzenesulfonate (l .89 mg; 8.395 mmol) and EEDQ (2.768 g; 11.19 ml). The reaction mixture was stirred at room température for 25 hours, then was concentrated to dryness. The crude product was purified by silica gel chromatography (gradient of methanol in DCM) to afford the title compound (2.81 g; 4.823 mmol) as white powder. IR (cm'¹): 3700-3000, 1660(large), 1180. 'H NMR (400 MHz, dmso-d6) δ ppm 10.02 (s, IH), 7.88 (m, 3H), 7.76 (2t, 2H), 7.7 (dd, IH), 7.61 (d, IH), 5.99 (t, IH), 5.38 (m, 2H), 5.03 (t, 1 H), 4.72 (d, 2H), 4.3-4.2 (m, 3H), 4.15 (m, IH), 3.06-2.90 (m, 2H), 1.75-1.30 (m, 4H). UPLC-MS: MS(ESI): m/z [M+H]+ = 583.42, [M+Na]+ = 565.31.

Step 6: 2-(chloromethyl)-5-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5-ureidopentanoyl] amino]benzenesulfonic acid

To a solution of 5-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5-ureidopentanoyl]amino]-2-(hydroxymethyl)benzenesulfonie acid (543.6 mg; 0.933 mmol) in NMP (5 mL) were added at room température a solution of SOCb (68.1 pL; 0.933 mmol) in NMP (200 pL). The reaction mixture was stirred at room température for 15 min and the progress of the reaction was monitored b y UPLC-MS. To achieve a complété conversion, the SOCb addition (68 pL) has to be done 7 more times. The excess SOCb was evaporated under vaccum, and the residue was purified by direct deposit of the reaction mixture on an Oasis column in using the TFA method to afford the title compound (362 mg; 0.512 mmol) as a white solid. UPLC-MS: MS(ESI): m/z [M+H]+= 601.19+603.23 [M+Na]+= 622.93

Step 7: 2-[[4-[2-[2-chloro-4-[6-(4-fluorophenyl)-4-[(IR)-2-methoxy-l-[[2-[[2-(2methoxyphenyl)pyrimidin-4-yl] methoxy]phenyl] methyl]-2-oxo-ethoxy] thieno[2,3-d]pyrimidin-5ylj-3-methyl-phenoxy] ethyl]-1 -methyl-piperazin-1 -ium-1 -yl] methyl]-5-[[(2S)-2-(9H-]luoren-9ylmethoxycarbonylamino)-5-ureido-pentanoyl] amino] benzenesulfonate

To a solution of 2-(chloromethyl)-5-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5ureido-pentanoyl] amino] benzenesulfonic acid (195.6 mg; 0.277 mmol) from step 6 in solution in NMP (10 mL), were successively added methyl (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4methylpiperazin-l-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2[[2-(2-methoxyphenyl)pyrimidiii-4-yl]methoxy]phenyl]propanoate (123 mg; 0.138 mmol) from step 4, DIPEA (385 pL, 2.213 mmol) and TB AI (10 mg, 0.027 mmol). The reaction mixture was stirred at 70°C for 12 hours and the progress of the reaction was monitored by UPLC-MS. The

401 crude product in solution in NMP was directly used in the next step. UPLC-MS: MS(ESI): m/z [M+H]+ = 1231.12-h1233.45, [M+2H]2+ = 616.34+617.37

Step 8: 5-[[(2S)-2-amino-5-ureido-pentanoyl] amino] -2-[[4-[2-[4-[4-[(IR)-l-carboxy-2-[2-[[2(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl] ethoxy]-6-(4-fluorophenyl)thieno [2,3d]pvrimidin-5-yl] -2-chloro-3~methyl-phenoxy] ethyl]-1-methyl-piperazin-1-ium-lyl] methyl] benzenesulfonate ;2,2,2-trifluoroacetic acid

To the previous solution of2-[[4-[2-[2-chloro-4-[6-(4-fluorophenyl)-4-[(lR)-2-methoxyl-[[2-[[2-(2-methoxypheny])pyrimidin-4-yl]methoxy]phenyl]methyl]-2-oxo-ethoxy|thieno[2,3d]pyrimidin-5-yl]-3-methyl-phenoxy]ethyl]-1 -methyl-piperazin-l-ium-l-yl]methylJ-5-[[(2S)-2(9H-fluoren-9-ylmethoxycarbonylamino)-5-ureido-pentanoyl]amino]benzenesulfonate in NMP, was added a solution of lithium hydroxyde mono hydrate (82.2 mg; 1.106 mmol) in water (4 mL). The reaction mixture was stirred at room température for 1.5 hours and the progress of the reaction was monitored by UPLC-MS. The crude product solution was purified by direct deposit of the reaction mixture on a X-Bridge column in using the TFA method to afford the title compound (45.6 mg; 0.0374 mmol) as a white powder. UPLC-MS: MS(ESI): m/z [M+H]+ = 1217.46, [M+Na]+= 1241.16, [M+2H]2+= 609.61

Step 9: 2-[]4-[2-[4-]4-[(lR)-l-carboxy-2-]2-[]2-(2-methoxyphenyl)pyrimidin-4yl] methoxy]phenyl] ethoxy]-6-(4-]luorophenyl)thieno[2,3-d]pyrimidin-5-yl]-2-chloro-3-methylphenoxy] ethyl] -1 -methyl-piperazin-1 -ium-1 -yl] methyl]-5-[[(2S)-2-[[ 1 -[2-[2-(2,5-dioxopyrrol-1 yl)ethoxy] ethylcarbamoyl] cyclobutanecarbonyl] amino]-5-ureidopentanoyl]amino] benzenesulfonate L25-P1

To a solution of 5-[[(2S)-2-amino-5-ureido-pentanoyl]amino]-2-[[4-[2-[4-[4-[(lR)-lcarbo xy-2- [2- [ [2-(2-metho xypheny 1 )pyrimidin-4-y1 ] methoxy] phenyl]ethoxy]-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-2-chloro-3-methyl-phenoxy]ethyl]-l-methylpiperazin-l-ium-l-yl]methyl]benzenesulfonate (22.6 mg; 0.0186 mmol) in DMF (1.4 mL), were successively added a THF solution of (2,3,4,5,6-pentafluorophenyl) l-[2-[2-(2,5-dioxopyrrol-lyl)ethoxy]ethylcarbamoyl]cyclobutanecarboxylate (from step 3) (26.8 mg; 0.0562 mmol) and DIPEA (12.9 pL; 0.0742 mmol). The reaction mixture was stirred at room température for 2 hours. The crude product solution was purified by direct deposit of the reaction mixture on a XBridge column and in using the TFA method to afford L25-P1 (7.5 mg; 0.0050 mmol) as a white powder. IR (cm'¹): 3321, 1705/1624, 1666, 1581, 1180/1124, 833/798/756/719/696. Ή NMR

402 (400/500 MHz, dmso-d6) δ ppm 10.4 (s), 8.88 (d, IH), 8.61 (s, IH), 8.I3 (df, 1H), 7.92 (dd, iH), 7.78 (d), 7.74 (t), 7.63 (d, IH), 7.52 (dd, IH), 7.47 (d, IH), 7.46 (t, l H), 7.38 (d, IH), 7.3 (dd, 2H), 7.23 (d, IH), 7.21 (t, 2H), 7.16 (t, IH), 7.14 (d, IH), 7.03 (t, IH), 7.01 (d, IH), 7 (s, 2H), 6.73 (t, IH), 6.22 (d, IH), 5.99 (m), 5.55 (si), 5.5 (dd, IH), 5.25 (AB, 2H), 5.1 (si, 2H), 4.37 (m, IH), 4.33 (m, 2H), 3.76 (s, 3H), 3.7 (m, 10H), 3.55 (m, 2H), 3.5 (m, 2H), 3.42 (m, 2H), 3.28/2.52 (2dd, 2H), 3.21 (m, 2H), 3.04 (si, 3H), 2.97 (m, 2H), 2.4 (m, 4H), 1.85 (w, 3H), 1.74/1.62 (2m, 2H), 1.73 (m, 2H), 1.43/1.35 (2m, 2H). ¹³C NMR (400/500 MHz, dmso-d6) δ ppm 157.5, 152.8, 135.4, 134.9, 131.5, 131.4, 131.4, 131.2, 131.1, 128.7, 121, 120.6, 119.2, 119.2, 116.3, 116, 112.8, 112.2, 111,74, 69.5, 68.9, 67.4, 66.6, 56.2, 55.3/46.5, 54.1, 45.7, 39.4, 39.2, 37.2, 32.9, 29.7, 29.7, 27.3, 18, 16. ^I9F NMR (400/500 MHz, dmso-d6) δ ppm-74.6,-112.2. HR-ESI+: m/z [M+H]+ = 1509.4867 / 1509.4851 [measured/theoretical]

Préparation of L26-P1:

(2R)-2-l5-[3-chloro-4-[2-l4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-l-yl)ethoxy] propanoylamino]-3-methyl-butanoylIamino|-5-ureidO“pentanoyI]amino]-2-[3“[2-[2-[2-[2-[2[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy] ethoxy] ethoxy]ethoxy]propyl| phenyl]methyl|-4-methyI-piperazm-4-ium-l-yl]ethoxy|-2-Inethyl·phenyl]-6-(4fluorophenyl)thieno[2,3-d]pynniidin-4-yI|oxy-3-[2-[[2-(2-methoxypheiiyl)pyrÎmidin-4yl]methoxy]phenyl]propanoate;2,2,2-trifluoroacetate_

Step 1: 3-]2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy] ethoxy]prop-1-yne

To a solution of 2-[2-[2-[2-[2-[2-[2-(2methoxyethoxy)ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy]éthanol (1.95 g; 6.50 mmol) in THF (25.0 mL), was added at 0°C sodium hydride (260.0 mg; 6.57 mmol). After 5 minutes, a solution of 3-bromoprop-l-yne in toluene (1.42 mL; 13.14 mmol) was added. The reaction mixture was stirred at 0°C for 1 hour and 2 days at room

403 température and the progress of the reaction was monitored by UPLC-MS. Then, the reaction mixture was filtered and the filtrate was concentrated to dryness, and purified by silica gel chromatography (gradient DCM in methanol) to afford the title compound (1.74 g; 4.12 mmol) as a colorless oil. ‘H NMR (CDCb): 2.43 (t, IH, J = 2.4 Hz), 3.37 Q, 3H), 3.53-3.55 (m, 2H), 3.64-3.70 (m, 30H), 4.20 (d, 2H, 7= 2.4 Hz).

Step 2: 9H-fluoren-9-ylmethyl N-[(1S)-1 -[[4-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-iodophenyl] carbamoyl] -4-ureido-butyl] carbamate

To a solution of [[tert-butyl(dîmethyl)silyl]oxymethyl]-3-iodo-aniline (10.0 g; 27.52 mmol) in methanol (70 mL) and DCM (140 mL), were successively added Fmoc-Cit-OH (12.0 g; 30.28 mmol) and EEDQ (8.17 g; 33.03 mmol). The reaction mixture was stirred for 14 hours at room température. After the completion of the reaction, the resulting residue was purified by column chromatography on silica gel using DCM / methanol (100/0 to 88/12) as eluent to afford the title compound (17.09 g; 21.97 mmol) as a white solid. ’H NMR (DMSO): δ 0.09 (5, 6H), 0.91 (s, 9H), 1.38-1.48 (m, 2H), 1.59-1.68 (m, 2H), 2.93-3.05 (m, 2H), 4.06-4.15 (m, 2H), 4.204.29 (m, 3H), 4.56 Q, 2H), 5.41 (y, 2H), 5.98 (t, IH, J= 5.5 Hz), 7.30-7.43 (m, 5H), 7.55 (dd, IH, J =8.8, 2.1 Hz), 7.69 (d, IH, J =7.8 Hz), Ί.Ί4 (dd, 1 H, 7=7.2, 3.4 Hz), 7.89(7, 1 H, 7=7.5 Hz), 8.25 (d, 1H,7= 1.5 Hz), 10.12 (5, IH).

Step 3: (2S)-2-amino-N-[4-[[tert-butyl(dimethyl)silyl] oxymethyl] -3-iodo-phenyl]-5-ureidopentanamide

To a solution of 9H-fluoren-9-ylmethyl N-[(lS)-l-[[4-[[tertbutyl(dimethyl)silyl]oxymethyl]-3-iodo-phenyl]carbamoyl]-4-ureido-butyl]carbamate (17.08 g; 23.00 mmol) in THF (120 mL), was added dimethylamine 2M in THF (44.5 mL; 89.00 mmol). The reaction mixture was stirred for 15 hours at room température. After concentration to dryness, the resulting residue was purified by column chromatography on Cie using water / acetonitrile (98/02 to 0/100) as eluent to afford compound the title compound (5.47 g; 10.50 mmol) as a white solid. Ή NMR (DMSO): Ô 0.0 (s, 6H), 0.81 (5, 9H), 1.27-1.38 (m, 3H), 1.471.53 (m, IH), 2.83-2.89 (m, 2H), 3.16-3.19 (m, 1H),4.46 (s, 2H), 5.26 (5, 2H), 5.82 (t, 1H,7 = 5.6 Hz), 7.24 (d, 1H,7= 8.5 Hz), 7.50 (dd, IH, 7= 8.3, 2.0 Hz), 8.17 (d, 1H,7=2.O Hz).

Step 4: 9H-fluoren-9-ylmethyl N-[(lS)-l~[[(lS)-l-[[4-[[tert-butyl(dimethyl)silyl]oxymethyl]-3iodo-phenyl] carbamoyl]-4-ureido-butyl] carbamoyl]-2-methyl-propyl] carbamate

404

To a solution of (2S)-2-amino-N-[4-[[tert-butyl(dimethyl)silyl]oxymethyl]-3-iodophenyl]-5-ureido-pentanamide (3.00 g; 5.76 mmol) in 2-methyltetrahydrofuran (240 mL), were successîvely added Fmoc-Val-Osu (8.65 g; 8.65 mmol) and DIPEA (1.90 mL; 11.53 mmol). The reaction mixture was stirred for 15 hours at room température. The reaction mixture was fîltered through a sintered funnel and the recovered solid was washed with 2-methyltetrahydrofuran (2 x 250 mL), then dried under high vacuum to afford the title compound (3.57 g; 4.24 mmol) as a white solid. ‘H NMR(DMSO): δ 0.10 (s, 6H), 0.83-0.95 (m, 15H), 1.27-1.52 (m, 2H), 1.52-1.75 (m, 2H), 1.93-2.07(m, IH), 2.88-3.09 (m, 2H), 3.93 (t, IH, J = 8.0 Hz), 4.17-4.49 (m, 4H), 4.56 Q, 2H), 5.40 A 2H), 5.96 {t, IH, J= 5.6 Hz), 7.27-7.37 (m, 3H), 7.37-7.48 (m, 3H), 7.54 (d, IH, J = 8.0 Hz), 7.74 (t, 2H, J= 7.2 Hz), 7.88 (d, 2H, J= 7.6 Hz), 8.13 {d, IH, J= 7.6 Hz), 8.22 (s, IH), 10.11 (5, IH).

Step 5 : 9H-fluoren-9-ylmethyl N-[(lS)-l-[[(lS)-l-[[4-[[tert-butyl(dimethyl)silyl]oxymethyl]-3[3-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy] ethoxy] ethoxy] ethoxy] ethoxy]ethoxy] prop-1 -ynyi]phenyl] carbamoyl] -4-ureido-butyl] carbamoyl]-2-methyl-propyl] carbamate

To a solution of 9H-fluoren-9-ylmethyl N-[(1S)-1 -[[(1 S)-l-[[4-[[tertbutyl(dîmethyl)silyl] oxymethyI]-3-iodo-phenyI]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]carbamate (1.23 g; 1.46 mmol) in dimethylformamide (15.40 mL), were added successîvely 3-[2-[2-[2-[2[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]prop-l-yne (930.0 mg; 2.20 mmol) and DIPEA (2.47 mL; 14.92 mmol). After 3 purges with argon, Pd(PPh₃)2C12 (220 mg; 0.307 mmol) and Cul (68.0 mg; 0.36 mmol) were added and the reaction mixture was purged with argon 3 times. The reaction mixture was stirred for 3 hours at room température and the progress of the reaction was monitored by UPLC-MS. The mixture was diluted with isopropyl acetate (200 mL) and washed with brine (3 x 150 mL). The combined organic layers were dried over sodium sulfate, fîltered and concentrated to dryness. The crude product was purified by C18 reverse phase prep-HPLC by direct deposit of the réaction mixture on the X-Bridge column ad using neutral method to afford the title compound (790.0 mg; 0.70 mmol) as a pale yellow gum. ^!H NMR(DMSO): δ 0.08 (i, 6H), 0.85-0.90 (m, 15H), 1.36-1.45 (m, 2H), 1.58-1.71 (m, 2H), 1.97-2.00 (m, 1 H), 2.93-3.03 (m,2H), 3.23 (5, 3H), 3.40-3.43 (m, 2H), 3.49-3.52 (m, 25H), 3.56-3.58 (m, 2H), 3.63-3.66 (m, 2H), 3.93 {dd, IH, J= 8.9, 6.9 Hz), 4.23-4.32 {m, 3H), 4.37-4.43 {m, 3H), 4.75 (5, 2H), 5.39 Q, 2H), 5.97 (/, IH, J=6A Hz), 7.30405

7.43 (m, 6H), 7.51-7.54 (m, IH), 7.72-7.78 (m, 3H), 7.88(/, 2H7=7.5 Hz), 8.12 (7, 2H, J=1A Hz), 10.10(5, IH).

Step 6: 9H-fluoren-9-ylmethyl N-[(lS)-l-[[(lS)-l-[[4-(hydroxymethyl)-3-[3-[2-[2-[2-[2-[2-[2[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]prop-lynyl]phenyl] carbamoyl]-4-ureido-butyl] carbamoyl]-2-methyl-propyl] carbamate

To a solution of 9H-fluoren-9-ylmethyl N-[( 1 S)-1 -[[(1 S)-l -[[4-[[tertbutyi(dimethyl)silyl] oxymethyl]-3-[3-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy]ethoxy]prop-l-ynyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methylpropyl]carbamate (452 mg; 0.40 mmol) in tetraliydrofuran (0.60 mL) and water (0.90 mL), was added acetic acid (4.17 mL; 72.78 mmol). The reaction mixture was stirred for 22 hours at room température and the progress of the réaction was monîtored by UPLC-MS. After concentration to dryness, the crude product was purified by Cl8 reverse phase prep-HPLC by direct deposit of the reaction mixture on the X-Bridge column ad using neutrai method to afford the title compound (327 mg, 0.32 mmol) as a white gum. 'H NMR (DMSO): Ô 0.87 (dd, 6H, J = 11.7, 6.8 Hz), 1.36-1.45 (m, 2H), 1.58-1.71 (m, 2H), 1.97-2.00 (m, 1 H), 2.93-3.02 (m, 2H), 3.23 (5, 3H), 3.31 (5, 5H), 3.40-3.43 (m, 2H), 3.48-3.53 (m,21H), 3.54-3.64 (m, 6H), 3.91-3.95 (m, l H), 4.234.42 (m, 4H), 4.56 (d, 2H, 7= 5.5 Hz), 5.19 (t, IH, 7= 5.6 Hz), 5.39 (5, 2H), 5.96 (t, IH, 7= 5.8 Hz), 7.30-7.34 (m, 2H), 7.39-7.43 (m, 4H), 7.50-7.52 (m, 1 H), 7.72-7.76 (5, 3H), 7.88 (d, IH J = 7.5 Hz), 8.12 (/, 2H, 7 = 7.4 Hz), 10.06 (5, IH).

Step 7; 9H-fluoren-9-ylmethyl N-[(lS)-l-][(lS)-l-[[4-(hydroxymethyl)-3-[3-[2-[2-[2-]2-[2-[2[2-(2-methoxyethoxy)ethoxy]ethoxy] ethoxy] ethoxy]ethoxy] ethoxy] ethoxy]propyl]phenyl] carbamoyl]-4-ureido-butyl] carbamoyl] -2-methyl-propyl] carbamate

To a solution of 9H-fluoren-9-ylmethyl N-[( 1 S)-1 -[[( 1 S)-1 -[[4-(hydroxymethyl)-3-[3-[2[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]prop1 -ynyl] phenyl] carbamoyl]-4-ureido-butyl] carbamoyl]-2-methyl-propyl] carbamate (327.0 mg; Ü.32 mmol) in THF (3.7 mL), was added acetic acid (0.37 mL). After 3 purges with argon, Pt/C 5% (195 mg) was added and after 3 more purges with argon, the reaction mixture was placed under hydrogen atmosphère and stirred for 18 hours at room température and the progress of the reaction was monîtored by UPLC-MS. The mixture was fïltered through PTFE and the fîltrate was concentrated to dryness, then triturated in dichloromethane/pentane (1/4 mixture, 50 mL).

406

The precipitate was recovered by filtration to afford, after drying, the title compound ( 130 mg; 0.13 mmol) as a white solid. 'HNMR (DMSO): 5 0.85-0.89 (m, 6H), 1.23-1.46 (m, 2H), 1.56l.76(m,4H), 1.97-2.02 (m, IH), 2.56-2.60 (m, 2H), 2.91-3.04 (m, 2H), 3.23 (s, 3H), 3.38-3.43 (m,4H), 3.48-3.54 (m, 30H), 3.93 (dd, 1H,J=8.9, 6.9 Hz), 4.21-4.31 (m, 3H), 4.38-4.41 (m, 11-1),4.45 (d, 2H, J =5.3 Hz), 4.94 (t, IH, J =5.3 Hz), 5.37 (s, 2H), 5.95 (t, IH, J=5.8 Hz), 7.25 (d, IH, J =3.3 Hz), 7.30-7.34 (m, 2H), 7.39-7.43 (s, 5H), 7.72-7.76 (m, 2H), 7.88 (d, IH J =1.5 Hz), 8.06 (ri, 2H, J= 7.6Hz), 9.88 (5, IH). UPLC-MS: MS (ESI) m/z [M+H]+= 1026.52

Step 8: 9H-fluoren-9-ylmethyl N-](lS)-l-[[(lS)-l-][4-(bromomethyl)-3-[3-[2-[2-[2-[2-]2-[2-[2(2-methoxyethoxy)ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy]propyl]phenyl] carbamoyl]-4-iireido-butyl] carbamoyl] -2-methyl-propyl] carbamate

To a solution of 9H-fluoren-9-ylmethyl N-[(lS)-l-[[(lS)-l-[[4-(hydroxymethyl)-3-[3-[2[2-[2-[2-[2-[2-[2-(2methoxyethoxy)ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] propyl] phenyl] carbamoyl]-4-ureîdo-b ut yl]carbamoyl}-2-methyl-propyl] carbamate (60 mg; 0.0584 mmol) in THF (6.6 mL), was added dropwise at 0°C PBr₃ (IM solution in THF) (0.0877 mL; 0.0877 mmol). The solution was then stirred 3 hours at room température. The progress of the reaction was monitored by UPLC-MS after addition în an ali quoi morpholine to react the bromo expected compound. The reaction was worked up with an aqueous saturated NH4CI solution (50 pL). After 5 minutes the mixture was dryed over MgSCL, filtered and washed with THF (2 ml) to afford the bromo title compound as a THF solution used crude in the next step. UPLC-MS anajysis is done after methanol and morpholine addition.

Step 9: (2R)-2-[5-[3-chloro-4-]2-[4-[]4-[](2S)-2-[[(2S)-2-(9H-fluoren-9ylmethoxycarbonylamino)-3-methyl-butanoyl] amino] -5-ureido-pentanoyl] amino]-2-[3-[2-[2-[2[2-[2-[2-]2-(2-methoxyethoxy)ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy]propyl] phenyl] methyl] -4-methyl-piperazin-4-ium-l-yl] ethoxy] -2-methyl-phenyl]-6-(4fluorophenyl)thieno]2,3-d]pyrimidin-4-yl] oxy-3-[2-][2-(2-methoxyphenyl)pyrimidin-4yl]methoxy]phenyl]propanoic acid;2,2,2-trifluoroacetate

To the THF solution of 9H-fluoren-9-ylmethyl N-[(lS)-l-[[(lS)-l-[[4-(bromomethyl)-3[3-[2-[2-[2-[2-[2-[2-[2-(2methoxyethoxy) ethoxy] ethoxy] ethoxy] ethoxy]ethoxy] ethoxy] ethoxy] propyl] phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]carbamate from the previous

407 step (0.0584 mmol), were successively added DMF (1.5 mL), (2R)-2-[5-[3-chloro-2-methyl-4[2-(4-methylpiperazin-l-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid PI (46.1 mg; 0.0527 mmol) and DIPEA (0.173 mL; 0.995 mmol). The reaction mixture was stirred 20 hours at room température and the progress of the reaction was monitored by UPLC-MS. The crude mixture containing the expected title compound and the Fmoc-deprotected one (expected in step 10) îs used in the deprotective next step. UPLC-MS: MS (ESI) m/z [M-Fmoc+H+H]+ = 1660.99

Step 10: (2R)-2-[5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5-ureidopentanoyl] amino]-2-[3-[2-[2-[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy]propyl]phenyl] methyl]-4-methyl-piperazin-4-ium-l-yl] ethoxy]-3-chloro-2methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl] methoxy]phenyl]propanoic acid;2,2,2-trifluoroacetate;2,2,2trifluoroacetic acid

To the crude mixture obtained in the previous step in DMF was added piperidine (11.6 pL; 0.117 mmol). The reaction mixture was stirred at room température for 15 hours and the progress of the reaction was monitored by UPLC-MS. After compietion of the reaction, the crude product was purified by Cis reverse phase prep-HPLC b y direct deposît of the reaction mixture on the X-Bridge column in using the TFA method to give the title compound (29.2 mg; 0.0155 mmol) as a white powder. IR: 3600-2300, 1672, 1602, 1541 + 1516. HR-ESI+; m/z [MCFîCOO]+ = 1660.7574 (1660.7575 theoretical)

Step 11: (2R)-2-[5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-lyl)ethoxy]propanoylamino] -3-methyl-butanoyl] amino]-5-ureido-pentanoyl] amino] -2-[3-[2-[2[2-[2-[2-[2-[2-(2-methoxyethoxy)ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy]propyl] phenyl] methyl]-4-methyl-piperazin-4-ium-l-yl] ethoxy]-2-methyl-phenyl]-6-(4fluorophenyl)thieno]2,3-d]pyrimidin-4-yl]oxy-3-[2-][2-(2-methoxyphenyl)pyrimidin-4yl]methoxy]phenyl]propanoate;2,2,2-trifluoroacetate L26-P1

To a solution of (2R)-2-[5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyI]amino]-5-urcido-pentanoyl]amino]-2-[3-[2-[2-[2-[2-[2-[2-[2-(2methoxyethoxy)ethoxy] ethoxy ] ethoxy] ethoxy] ethoxy] ethoxy] ethoxy]propyl]phenyl]methyl]-4-methyl-piperazin-4-ium-l-yl]ethoxy]-3chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2

408 methoxyphenyl)pyrimidin-4-yl]methoxy]phenyI]propanoic acid ;2,2,2-trifl uoroacetate;2,2,2trifluoroacetic acid (42.5 mg; 0.0225 mmol) in DMF (1.28 mL), were successively added a solution of (2,5-dioxopyrrolidin-l-yl) 3-[2-(2,5-dioxopyrrol-l-yl)ethoxy]propanoate (Brodpharm 21854) (7.71 mg; 0.0247 mmol) and DIPEA ( 19.6 pL; 0.112 mmol). The reaction mixture was stirred at room température for 15 hours and the progress of the reaction was monitored by UPLC-MS. The crude product was purified by Cis reverse phase prep-HPLC by direct deposit of the reaction mixture on the X-Bridge column and using the TFA method to afford L26-P1 (28 mg; 0.0151 mmol) as a white powder. HR-ESI+: m/z [M-CF₃COO]+ = 1855.8105 (1855.8106 theoretical)

Préparation of L27-P1: (2R)-2-[5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-||(2S)-2-[3-[2-(2,5-dioxopyrrol-lyllethoxylpropanoylaminol-S-methyl-butanoyllammol-S-methyl-S-ureidopentanoyl]amino]-2-sulfo-phenyl] methyl |-4-methyl-piperazin-4-ium-l-yl] ethoxy |-2-methylphenyl|-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid o^nh₂

Step I: 2-(chloromethyl)-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl] amino] -5-ureido-pentanoyl] amino] benzenesulfonate

5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]5-ureido-pentanoyl] ami no]-2-(hydroxymethyl)benzenesulfonic acid (300 mg; 0.4263 mmol) was dissolved in anhydrous NMP (6 mL) at room température. In parallel, a solution of SOCh (206 pL) in NMP (6 mL) was prepared. To the reaction, were added 6 times over a 75minutes period, a solution 900 pL of the SOCh solution. After the last addition, the réaction mixture was stirred at room température for 15minutes. The crude product was purified by direct deposit of the reaction mixture on an Oasis column in using the TFA method to afford the title compound (138 mg; 0.1971 mmol) as a white powder. ^lH NMR (400 MHz, dmso-d6) δ ppm

409

10.15 +8. i +7.42+6.0 (s+2d+m, 4H), 7.9 (m,HH), 7.75 (m, 3H), 7.42+7.31 (2m, 5H), 5.23 (s, 2H), 4.4 (m, IH), 4.3-4.2 (m, 3H), 3.95 (dd, IH), 3.0 (m, 2H), 2.0 (m, IH), 1.7 + 1.6 (2m, 2H), 1.48 + 1.37 (2m, 2H), 0.88 (2d, 6H). HR-ESI+ : m/z [M+H]+ = 700.2199 / 700.2202 [measured/theoretical]

Step 2: 5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl] amino]-5-ureido-pentanoyl]amino]-2-[[4[2-[2-chloro-4-[6-(4-fluorophenyl)-4-[(lR)-2-methoxy-l-[[2-[]2-(2-methoxyphenyl)pyrimidin-4yl] methoxy]phenyl] methyl]-2-oxo-ethoxy] thieno[2,3-d]pyrimidin-5-yl]-3-methyl-phenoxy] ethyl]}-methyl-piperazin-1-ium-1-yl] methyl] benzenesulfonic acid

To a solution of 2-(chloromethyl)-5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9ylmethoxy carbonyl amino)-3 -methyl-bu tanoyl] amino] -5-ureidopentanoyl ] amino] benzenesulfonate (82.4 mg; 0.1177 mmol) in anhydrous NMP (2.5 mL), was added at room température DIEA (94 pL; 0.540 mmol) followed by methyl (2R)-2-[5-[3-chloro2-methyl-4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-4-yi]oxy-3-[2-[[2-(2-mcthoxyphenyl)pyrimidm-4-yl]methoxy]phenyl]propanoate (60 mg; 0.067 mmol) and TB AI (12.4 mg; 0.034 mmol). The reaction was stirred at 80°C for 4 hours and overnight at room température. Then, 2-(chloromethyl)-5-[[(2S)-2-[[(2S)-2-(9Hfl uoren-9-ylmethoxycarbonyl amino)-3-methyl-butanoyl ] amino]-5-ureidopentanoyl]amino]benzenesulfonate was again added (14 mg; 0,017 mmol) followed by TBAI (17 pL; 0.0337 mmol) and the reaction was stirred ai 80°C for 4 hours and then overnight at room température. The Fmoc deprotection step was realized in adding DEA (53 pL; 0.515 mmol) to the reaction and stirring at room température overnight. Purification was realized by direct injection of the mixture on Oasis eluted with a gradient of a solution A :H2O/CH₃CN/NH4HCO₃(1960 ml/40/3.16 g) to a solution B: CfhCN/FbO/NFLHCCh (1600 ml/400 ml/3.16 g) to afford the title compound (17 mg; 0.009 mmol). UPLC-MS: MS (ESI) m/z [M]+ = 1329

Step 3 : (2R)-2-[5-[4-[2-[4-][4-[[(2S)-2-[](2S)-2-amino-3-methyl-butanoyl] amino]-5-ureidopentanoyl] amino] -2-stdfo-phenyl] methyl] -4-methyl-piperazin-4-ium-l-yl] ethoxy]-3-chloro-2methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl] oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl] methoxy]phenyl]propanoic acid

To a mixture of 5-[[(2S)-2-[[(2S)-2-amino-3-methyl-butanoyl] amino]-5-ureidopentanoyl] amino]-2-[[4-[2-[2-chloro-4-[6-(4-fluorophenyl)-4-[( 1 R)-2-methoxy-1 -[[2-[[2-(2410 methoxyphenyl)pyrirmdin-4-yl]methoxy]phenyI]methyi]-2-oxo-ethoxy]thieno[2,3-d]pyrimidin5-yl]-3-methyl-phenoxy] ethyl]-1-methyl-piperazin-l-ium-l-yl] methyl] benzenesulfonic acid (18 mg; 0.014 mmol) in dioxane/water (1 mL/Ι mL) was added LiOH H₂O (2.3 mg; 0.054 mmol) and the reaction was stirred at room température for 4 hours. The solution was adjusted to pH 67 by addition of HCl IN and dioxane was evaporated under reduced pressure. Purification was realized by direct injection of the mixture on Oasis eluted with a gradient of a solution A: H2O/CH3CN/NH₄HCO₃ (1960 ml/40/3.16 g) to a solution B: CHjCN/HzO/NHqHCO? (1600 ml/400 ml/3.16 g) to afford the title compound compound (1 Img; 0.008 mmol).

Step 4: (2R)-2-[5-[3-chloro-4-[2-[4-[[4-[](2S)-2-]](2S)-2-]3-]2-(2,5-dioxopyrrol-lyl)ethoxy]propanoylamino]-3-methyl-butanoyl] amino]-3-methyl-5-ureido-pentanoyl] amino]-2sidfo-phenyl] methyl] -4-methyl-piperazin-4-ium-l -yl] ethoxy]-2-methyl-phenyl]-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-]]2-(2-methoxyphenyl)pyrimidin-4yl]methoxy]phenyl]propanoic acid L27-P1

To a solution of(2R)-2-[5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyl]amino]-5-ureido-pentanoyl]amino]-2-sulfo-phenyI]methyl]-4-methyl-piperazin-4-iuml-yl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (10.5 mg; 0.007 mmol) in DMF (0.4 mL), was added (2,5-dioxopynOlidin-l-yl) 3-[2-(2,5-dioxopyrrol-lyl)ethoxy]propanoate (5.7 mg; 0.018 mmol) and the solution was stirred at room température for 4 hours.The solution was purified by X-Bridge column Qs by direct deposit of the reaction mixture on the column and in using the TFA method to afford L27-P1 (10 mg; 0.006 mmol). HR-ESI+: [M+H]+ 1511.5018 / 1511.5002 [measured/theoretical]

Préparation of L28-P1: (2R)-2-[5-[3-chloro-4-[2-[4-|[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrroi-lyl)ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoyl]amino]-2-sulfophenyl|methyl]-4“methyl-piperazin-4-ium-l-yI]ethoxy]-2-methyl-phenyl]-6-(4fluorophcny l)thieno [ 2,3-d| pyrimidin-4-yl | oxy-3- [ 2- [ 12-(2-methoxypheny I)py rimidin-4yl]methoxy]phenyl]propanoic acid;2,2,2-trifluoroacetate

411

Step 1: 9H-fluoren-9-ylmethyl N-f(IS)-l-[[(lS)-2-[4-(chÎoromethyl)-3-methyl-aniHno]-l-methyl2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate

To a solution of 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]ammo]-2-(hydroxymethyl)benzenesulfonate (504.1 mg; 0.816 mmol) in NMP (5 mL), were added 6 times over a 75 minutes period, a solution of SOCb (60 pL; 0.816 mmol) in NMP (500pL). The reaction mixture was stirred at room température for 15 minutes. The crude product was purified by direct deposit of the reaction mixture on an Oasis column in using the TFA method to afford (337 mg) as a a mixture of 70% the title compound (384 mmol) and 30% of the starting material (170 mmol) as a white powder. IR (cm¹): 3600 to 2400, 1688+1648, 1599, 1518, 1022. UPLC-MS: MS (ESI) m/z [M+H]+= 614.17+616.18 (Cl) Step 2: (2R)-2-[5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyl] amino]propanoyl] amino]-2-methyl-phenyl] methyl]-4-methyl-piperazin-4-ium-l yl] ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl] oxy-3-[2[[2-(2-methoxyphenyl)pyrimîdin-4-yl] methoxy]phenyl]propanoîc acid;2,2,2-trifluoroacetate

To a solution of methyl (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-lyl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yI]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate (152 mg; 0.171 mmol) in NMP (4.5 ml), were successively added 9H-fluoren-9-yIm ethyl N-[(lS)-l-[[(lS)-2-[4-(chloromethyl)-3methyl-anilino]-l-methyl-2-oxo-ethyl] carbamoyl]-2-methyl-propyl] carbamate (150 mg; 0.171 mmol), DIPEA (238 pL; 1.37 mmol) and TBAI (76 mg; 0.205mmol). The reaction mixture was stirred at 80°C for 28 hours. The reaction mixture is cooled down to room température. A solution of L1OH.H2O (13.7 mg, 0.342 mmol) in water (500 pL) is then added. The reaction mixture was stirred at room température for 48 hours. The crude product was purified by Cis reverse phase prep-HPLC by direct deposit of the reaction mixture on the X-Bridge column and

412 using the TFA method to afford the title compound (40 mg; 0.0325 mmol) as a white powder. UPLC-MS: MS (ESI) m/z [M]+= 1230.61 + 1232.6I (Cl)

Step 3: (2R)-2-[5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[3-[2-(2,5-dioxopyrrol-lyl)ethoxy]propanoylamino]-3-methyl-butanoyl] amino]propanoyl] amino]-2-sulfophenyl] methyl] -4-methyl~piperazin-4-ium-l-yl] ethoxy] -2-methyl-phenyl] -6-(4fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[]2-(2-methoxyphenyl)pyrimidin-4yl]methoxy]phenyl]propanoic acid;2,2,2-trifluoroacetate L28-P1

To a solution of (2R)-2-[5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyl]amino]propanoyl]amino]-2-methyl-phenyl]methyl]-4-methyl-piperazin-4-ium-lyl]ethoxy]-3-chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2[[2-(2-methoxyphenyl)pyrimidin-4-yI]methoxy]phenyl]propanoic acid;2,2,2-trifluoroacetate (6.0 mg; 0.0049 mmol) in solution in DMF (l 80 pL), were successively added (2,5-dioxopyrrolidinl-yl) 3-[2-[2-(2,5-dioxopyrrol-l-yl)ethoxy]ethylcarbamoyl]oxetane-3-carboxylate (2.3 mg;

0.0073 mmol) and DIPEA (3.0 pL; 0.017 mmol). The reaction mixture was stirred at room température for l .5 hours and was monitored by UPLC-MS. The crude product was purified by direct deposit of the reaction mixture on the X-Bridge column in using the TFA method to afford L28-P1 (2.9 mg; 0.0020 mmol) as a white powder. HR-ES1+: m/z [M+H]+ = 1425.4534/ 1425.4527 [measured/theoreti cal]

Préparation of L29-C3: (2R)-2-[5-|4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[2-(2-azidoethoxy)acetyl]amino]-3-methylbutanoyI]amino]propanoyl|amino|-2-sulfo-phenyl]methoxycarbonyl]piperazin-lyl| ethoxy ]-3-chloro-2-methyl·phenyl]-6-(4-Πuorophenyl)thieπo[2,3“d]pyrimidin-4-yl]oxy-3[ 2-[|2-(2-methoxyphenyl) pyriniidin-4-yI] methoxy] phenyl] propanoic acid

N' H

413

Step 1: sodium; 5-[](2S)-2-(teri-butoxycarbonylamino)propanoyl]amino]-2(hydroxy methyl)benzenesulfonate

To a solution of Boc-L-Ala-OH (588 mg; 3.11 mmol) in DMF (38.6 mL), were successîvely added HATU (1.77 g; 4.67 mmol), sodium 5-amino-2(hydroxymethyl)benzenesulfonate (771 mg; 3.42 mmol) and DIPEA (1.29 mL; 7.78 mmol). The reaction mixture was stirred for 16 hours at room température then concentrated to dryness and co-evaporated with water to afford the crude reaction mixture. The resulting residue was purified by column chromatography on silica gel using ethyl acetate/methanol 95:5 to 80:20 as eluent to afford the title compound (1.17 g; 2.95 mmol) as a white solid. 'FI NMR (DMSO): δ 1.24 (s, 9H), 1.38 (m, 3H), 4.05-1.44 {m, IH), 4.73 (d, 2H, J= 4.8 Hz), 5.04 (t, IH, J = 5.6 Hz); 6.977.02 (m, IH), 7.33 {d, 1 H, J =8 Hz), 7.65-7.70 (m, IH), 7.83 (s, IH), 9.91 (s, IH).

Step 2: 5-[[(2S)-2-aminopropanoyl] amino]-2-(hydroxymethyl)benzenesulfonate, hydrochloride

Sodium 5-[[(2S)-2-(tert-butoxycarbonylamino)propanoyl]amino]-2-(hydroxymethyl) benzenesulfonate (1.17 g; 2.95 mmol; 1 eq.) was suspended in a solution of HCl 4N in dîoxane (10 mL). The mixture was stirred at room température for 2 hours then concentrated to dryness to afford the crude mixture (982 mg; 2.95 mmol) as a white solid. 'H NMR (DMSO): δ 1.45 (d, 3H, J = 5.6 Hz), 3.91-4.0 (m, IH), 4.76 G, 2H), 7.41 (d, IH, J = 7.6 Hz), 7.66 {d, IH, J= 7.6 Hz), 7.85 (5, IH), 8.17 A 2H), 10.44 (s, IH).

Step 3: 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]-2-(hydroxymethyl)benzenesulfonate

To a solution of 5-[[(2S)-2-aminopropanoyl]ammo]-2-(hydroxymethyl)benzenesulfonate, hydrochloride (981 mg; 2.95 mmol) in DMF (34.5 mL) were added Fmoc-L-Val-Osu (1.29 g; 2.95 mmol; 1 eq.) and DIPEA (975 pL; 5.9 mmol). The mixture was stirred ovemight at room température then concentrated to dryness and co evaporated with water to afford the crude mixture. The resulting residue was purified by column chromatography on silica gel using ethyl acetate /methanol 95:5 to 80:20 as eluent to afford the title compound (1.28 g; 2.072 mmol) as a coiorless oil. ^!H NMR (DMSO): δ 0.80-0.92 {m, 6H), 1.30 (d, 3H, J = 6.4 Hz), 2.02-2.10 (m, IH), 4.17-4.31 (m, 3H), 4.37-4.44 (m, 1 H), 4.73 (d, 2H, J= 5.6 Hz), 5.04(/, IH, J =6.4 Hz), 7.28-7.36 (m, 3H), 7.37-7.47 (m, 3H), 7.66 (d, 1 H, 7= 8.4 Hz), 7.71-7.77 (m, 2H), 7.83-7.85 (m, IH), 7.88 (d, 2H, 7= 7.6 Hz), 8.14 (d, 1H,7= 6.4 Hz), 9.99 (s, IH).

414

Step 4: 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-melhylbutanoyl] amino]propanoyl] amino]-2-[(4-nitrophenoxy)carbonyloxymethyl]benzenesulfonic acid To a suspension of5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3m ethyl-butanoyl] amino] propanoyl] amino]-2-(hydroxymethyl)benzenesulfbnate (1.28 g; 2.07 mmol) in THF (65 mL), were added pyridine (875 pL; 10.8 mmol), followed by 4-nitrophenyl chloroformate (1.09 g; 5.41 mmol). The mixture was stirred ovemight at room température. Then additional 4-nitrophenyl chloroformate (1.09 g; 5.41 mmol; 2.5 eq.) was added. After 5 hours stirring at room température, the mixture was concentrated to dryness then purified by column chromatography on Cie using water/acetonitrile 90/10 to 0/100 as eluent in 30 minutes. Acetonitrîle of the combined tubes was removed, and the rest was lyophilized to afford the title compound (650 mg; 0.83 mmol) as a white solid. ’H NMR (DMSO): δ 0.88 (m, 6H), 1.31 (d, 3H, J=4.8 Hz), 1.97-2.03 (m, 1H),3.92 (/, IH, .7=6.8 Hz), 4.23 (s, 2H), 4.24-4.34 (m, IH), 4.42 (ί, IH J= 5.6 Hz), 5.69 (ί, 2H), 7.30-7.48 (m, 6H), 7.62 (d, 2H, J = 8 Hz), 7.72-7.76 (m, 3H), 7.89 (d, 2H, J = 6.4 Hz), 7.94 (s, IH), 8.18 (d, IH, J = 5.6 Hz), 8.33 (d, 2H, J = 7.6 Hz), 10.11 (s, IH). ^I3C NMR (DMSO): δ 18.01, 18.26, 19.21,30.4,46.66,49.05, 59.91,65.67, 67.82, 117.7, 119.1, 120.06, 122.66, 125.37, 126.33, 127.05, 127.62, 128.0, 138.06, 140.67, 143.77, 143.86, 145.1, 146.23, 151.96, 155.47, 156.12, 171.0, 171.15. LCMS (2-100 ACN/H₂0+0.05% TFA); 90.41 % Tr = 12.7 min. Positiv mode 578.41 detected. Negativ mode 759.17 detected

Step 5: (2S)-2-[[5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fliioren-9ylmethoxycarbonylamino)-3-methyl-biitanoyl] amino]propanoyl] amino] -2-sidfophenyl] methoxycarbonyl]piperazin-l -yl] ethoxy] -2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-4-yl] methyl]-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl] methoxy]phenyl]propanoic acid

To a suspension of (2S)-2-[[5-[3-chIoro-2-methyl-4-(2-piperazin-l-ylethoxy)phenyl]-6(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]methyI]-3-[2-[[2-(2-methoxyphenyl)pyriinidin-4yl]m ethoxy] phenyl] propanoic acid;2,2,2-trifluoroacetic acid (178.4 mg; 0.183 mmol) in DMF (1.5 mL), were successively added 5-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)3-methyl-butanoyl ]amino]propanoyl] amino]-2-[(4nitrophenoxy)carbonyloxymethyl]benzenesulfonic acid (150 mg; 0.192 mmol) and DIPEA (91 pL; 0.549 mmol), The mixture was stirred ovemight at room température for 15 minutes. The crude product was purified by direct deposit of the reaction mixture on the X-Bridge column in

415 using the NH4HCO3 method to afford the title compound (176 mg, 0.11S mmol) as a white solid. UPLC-MS: MS (ESI) m/z [M+H]+ = 1482.15+1484.56(C1)

Step 6: (2S)-2-[[5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyl] amino]propanoyl] amino]-2-sulfo-phenyl]methoxycarbonyl]piperazin-l-yl] ethoxy]-3chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl] methyl]-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl] methoxy]phenyl]propanoic acid

To a solution of (2S)-2-[[5-[3-chloro-4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-(9H-fluoren-9ylmethoxycarbonylamîno)-3-methyl-butanoyl]amino]propanoyl]amino]-2-sulfophenyl]methoxycarbonyl]piperazin-l-yl]ethoxy]-2-methyl-phenyl]-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]methyl]-3-[2-[[2-(2-methoxyphenyl)pyriniidin-4yl]inethoxy]phenyl] propanoic acid (176 mg; 0.118 mmol) in DMF (1.0 mL), was added piperidine (24.17 pL; 0.237 mmol). The reaction mixture was stirred at room température for 18 hours. The crude product was purified by direct deposit of the réaction mixture on a X-Bridge column in using the NH4HCO3 method to afford the title compound (102 mg; 0.0809 mmol) as a white powder. IR (cm^-1): 3620-2680, 1683, 1235. UPLC-MS: MS (ESI) m/z [M+H]+ = 1260.37+1262.37(C1). Ή NMR (400 MHz, dmso-d6)Ôppm 10.20 (m, NH), 8.90 (m, 2H), 8.90 (m, IH), 8.60 (m, NH), 8.60 (s, IH), 7.90 (m, IH), 7.78 (m, IH), 7.70 (d, IH), 7.55 (d, IH), 7.48 (t, IH), 7.45 (d, IH), 7.3/7.2 (m, 4H), 7.20 (m, IH), 7.20 (d, IH), 7.15 (t, IH), 7.15 (d, IH), 7.05 (t, IH), 7.00 (d, IH), 6.7 (t, IH), 6.2 (d, IH), 5.48 (s, 2H), 5.50 (m, IH), 5.23 (s, 2H), 4.50 (m, IH), 4.25 (m, 2H), 3.75 (s, 3H), 3.4 (m, 4H), 3.40 (m, IH), 3.35 (m, IH) 2.80 (m, 2H), 2.5 (m, 4H), 2.50 (m, IH), 2.05 (m, IH), 1.80 (s, 3H), 1.30 (d, 3H), 0.90 (dd, 6H)

Step 7: (2,3,4,5,6-pentafluorophenyl) 2-[2-[2-(2-azidoethoxy)ethoxy] ethoxy] acetate

To a solution of 2-[2-[2-(2-azidoethoxy)ethoxy]ethoxy]acetic acid (74 mg; 0.317 mmol) in THF (500 pL) were succesively added 2,3,4,5,6-pentafluorophenol (70 mg; 0.380 mmol) in solution in THF (500 pL) and DCC (78.5 mg; 0.380 mmol) m solution in THF (500 pL). The reaction mixture was stirred at room température for 18 hours. The crude suspension is filtred through a coton pad in a pasteur pipette and the solution is used without further work-up in step 8.

Step 8: (2R)-2-[5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-[[2-(2-azidoethoxy)acetyl]amino]-3-methylbutanoyl]amino]propanoyl] amino] -2-sulfo-phenyl] methoxycarbonyl]piperazin-1 -yl] ethoxy]-3

416 chloro-2-methyl-phenyl]-6-(4-fliiorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl] methoxy]phenyl]propanoic acid L29-C3

To a solution of (2S)-2-[[5-[4-[2-[4-[[4-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyl]amino]propanoyl]amîno]-2-sulfo-phenyl]methoxycarbonyl]piperazin-l-yl]ethoxy]-3chloro-2-methyl-phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]methyl]-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (100 mg; 0.0793 mmol) in DMF (500 pL), were succesively added the solution of (2,3,4,5,6-pentafluorophenyl) 2-(2-(2-(2azidoethoxy)ethoxy]ethoxy]acetate in THF from step 7 (0.317 mmol) and DIPEA (39.3 pL; 0.238 mmol). The reaction mixture was stirred at room température for 15 minutes. The crude product was purified by direct deposit of the reaction mixture on a X-Bridge column in using the NH4HCO3 method to afford L29-C3 (43 mg, 0.0291 mmol) as a white powder. IR (cm’¹): 3257, 2102, 1663, 1236/1082, 834/756. Ή NMR (400 MHz, dmso-d6) δ ppm 10.03 (s, NH), 8.87 (d, IH), 8.59 (sNC, 1 H), 8.35 (d, NH), 7.89 (df, lH),7.69(dd, IH), 7.66 (m, 1H),7.53 (d, IH), 7.45 (t, IH), 7.36 (d, IH), 7.29 (dd, 2H), 7.21 (d, IH), 7.20 (t, 2H),7.19(d, IH), 7.14 (d, IH), 7.13 (m, IH), 7.09 (m, NH), 7.03 (t, IH), 7.00 (d, IH), 6.72 (t, IH), 6.24 (d, IH), 5.48 (dNC, IH), 5.44 (s, 2H), 5.23(AB, 2H), 4.40 ( t, IH), 4.30 (dd, IH), 4.24 (m, 2H), 3.94 (s, 2H), 3.75 (s, 3H), 3.58 (m, 10H), 3.38 (m, 4H), 3.36 (t, 2H), 3.30 (NC, IH), 2.75 (t, 2H), 2.50 (m, 4H), 2.50 (m, 1 H), 2.00 (m, IH), 1.51 (s, 3H), 1.30 (d, 3H), 0.88/0.82 (2d, 6H). ^I3C NMR (100/126 MHz, dmso-d6) δ ppm 158.2, 131.6, 131.1, 130.9, 130.9, 130.9, 130.9, 128.3, 126.7, 120.7, 120.4, 119.3, 117.9, 116.2, 112.5, 112.1, 110.8,70.2, 70.2; 69.6, 67.7, 64.0, 56.7, 56.5,55.8,53.2, 50.4, 49.2, 43.8, 31.7, 19.7, 18.7, 18.4, 17.8. ^,9F NMR (376/470 MHz, dmso-d6) δ ppm-112.0. HRESI+: m/z [M+H]+ = 1475.4643/1475.4638; [2M+H+Ca]3+ = 996.6289/996.6273; [M+2H]2+ = 738.2378/738.2355; [M+H+Na]2+ = 749.2255/749.2265.

Example 2. Synthesis and Characterization of Additional Linkers, Linker-Payloads, and Precursors thereof.

Exemplary linkers, linker-payloads, and precursors thereof were synthesized using exemplary methods described in this example.

Synthesis of 2-(bromomethyl)-4-nitrobenzoic acid

417

Br

To a stirred solution of 2-methyl-4-nitrobenzoic acid (300 g, 1.5371 mol) in CCU (3000 mL) was added NBS (300,93 g, l .6908 mol) and AIBN (37.86 g, 0.2305 mol) at room température. The reaction mixture was stirred at 80°C for 16 h. Reaction mixture was monitored by TLC analysis. The reaction mixture was diluted with sat. NaHCO₃ solution (2 L) and extracted with ethyl acetate (2x2 L). The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude compound was purified by column chromatography on silica gel using 2-3% of ethyl acetate in petroleum-ether as an eluent and 2-(bromomethyl)-4-nitrobenzoic acid was obtained. 'H NMR (400 MHz, CDCI3): Ô 8.35 (d, J=2.0 Hz, IH), 8.20 (q, J=8.8, 2.4 Hz, IH), 8.12 (d, J=8.8 Hz, lH),4.97(s, 2H), 4.00 (s, 3H).

Synthesis of 4-nitro-2-((prop-2-yn-l-yIoxy)methyl)benzoic acid NO_Z

To the mixture of 2-(bromomethyl)-4-nftrobenzoic acid (250 g, 0.9122 mol) in ACN (5000 mL) was added prop-2-yn-l-ol (255.68 g, 265.50 mL, 4.5609 mol, d=0.963 g/mL) and CS2CO3 (743.03 g, 2.2805 mol) at room température. The resulting mixture was heated to 80°C for 16 h. The reaction mixture was fîltered through celite pad washed with ethyl acetate (2 L). The filtrate was concentrated under reduced pressure. The obtained crude compound was added sat. NaHCOj solution (1 L) and the aq layer was acidified to PH 2 by using 2N HCI (2 L). After filteration vacuum drying 4-nitro-2-((prop-2-yn-l-yloxy)methyl)benzoic acid was obtained. 'H NMR (400 MHz, DMSO): δ 13.61 (brs, IH), 8.37 (d, J=2.4 Hz, IH), 8.23 (dd, J=2.4, 8.4 Hz, IH), 8.10 (d, J=8.8 Hz, IH), 4.95 (s, 2H), 4.37 (d, J=2.4 Hz, 2H), 3.52 (t, J=2.4 Hz, IH)

Synthesis of methyl 4-nitro-2-((prop-2-yn-l-yloxy)methyl)benzoate

To a stirred solution of 4-nitro-2-((prop-2-yn-l-yloxy)methyl)benzoic acid (130 g, 0.5527 mol) in MeOH (1300 mL) was added SOC1₂ (526.08 g, 320.78 mL, 4.4219 mol, d=1.64 g/mL) slowly at 0°C. The reaction stirred at 70°C for 4 h. The reaction solvent was evaporated under reduced pressure. The obtained residue was dissolved in ethyl acetate (1000 mL) and washed with sat. NaHCO₃ (600 mL), water (500 mL) and brine solution (500 mL). The separated organic

418 layer was dried over sodium sulphate, filtered and evaporated under reduced pressure to yield methyl 4-nitro-2-((prop-2-yn-1-yloxy)methyl)benzoate. ’H NMR (400 MHz, CDCh): δ 8.56 (t, J=0.8 Hz, l H), 8.18 - 8.09 (m, 2H), 5.03 (s, 2H), 4.35 (d, J=2.4 Hz, 2H), 3.96 (s, 3H), 2.49 (t, J=2.4 Hz, IH).

Synthesis of methyl 4-amino-2~((prop-2-yn-l-yloxy)methyl)bcnzoate

To a solution of methyl 4-nitro-2-((prop-2-yn-l-yloxy)methyl)benzoate (l 10 g, 0.4414 mol) in a mixture of EtOH (] I00 mL) and H₂O (550 mL) was added Fe Powder (197.21 g, 3.5310 mol) and NH₄C1 (188.88 g, 3.5310 mol) at room température. The resulting mixture was heated at 80°C for 16 h. The reaction mixture was cooled to room température and filtered through celite® and washed with ethyl acetate (2 L). The filtrate was concentrated under reduced pressure up to half of the volume. To the residue, ethyl acetate (1.5 L) was added and separated the two layers and the aqueous layer was extracted with ethyl acetate (2 L). The combined organic layer was dried over anhydrous sodium sulphate and concentrated under reduced pressure to obtain crude product. Purification by SiO₂ column chromatography (15-20% of ethyl acetate in petroleum-ether) yielded methyl 4-amino-2-((prop-2-yn-1 -yloxy)methyl)benzoate. ’ H NMR (400 MHz, CDCh): δ 7.67 (d, J=8.8 Hz, IH), 6.78 (t, J=1.6 Hz, IH), 6.48 (q, J=8.4, 2.4 Hz, IH), 4.79 (s, 2H), 4.25 (d, J=2.4 Hz, 2H), 3.70 (d, .1=4.0 Hz, 3H), 3.42 (t, J=2.4 Hz, IH).

Synthesis of (4-amino-2-((prop-2-yn-l-yloxy)niethyl)phenyl)methanol HO

To a stirred solution ofTHF (1000 mL) was added LÎA1H4 (1 M in THF) (21.23 g, 798.2 mmol, 798.2 mL) slowly at 0°C. A solution of methyl 4-amino-2-((prop-2-yn-lyloxy)methyl)benzoate (70 g, 3 19.3 mmol) in THF (800 mL) was added slowly at 0°C. The reaction was stirred at room température for 4 h. The reaction mixture was cooled to 0°C, then was added water (22 mL) very slowly and followed by the addition of 20% NaOH (22 mL) and water (66 mL). The reaction mixture was stirred at 0°C for 30 min. Anhydrous sodium sulphate was added to absorb excess of water. The mixture was filtered through celite®. The fi lier cake was washed with ethylacetate (1000 mL) and 10% MeOH/DCM (500 mL). The filtrate was concentrated under reduced pressure. The resulting crude compound was purified by SiO₂

419 column chromatography (35-40% of ethylacetate in petroleum-ether as an eluent) to give yield (4-amino-2-((prop-2-yn-I-yloxy)methyI)phenyl)methanol. ‘H NMR (400 MHz, CDCh): ô 6.98 (d, J=8.0 Hz, IH), 6.56 (d, J=2.4 Hz, IH), 6.43 (dd, J=2.4, 8.0 Hz, IH), 4.98 (s, 2H), 4.64 (t, J=5.2 Hz, IH), 4.47 (s, 2H), 4.34 (d, J=5.6 Hz, 2H), 4.15 (d, J=2.4 Hz, 2H), 3.46 (t, J=2.4 Hz, IH).

Synthesis of (9H-fluoren-9-yl)methyl (S)-(l-((4-(hydroxymethyl)-3-((prop-2-yn-lyloxy)methyl)phenyl)amino)-l-oxo-5-ureidopentan-2-yI)carbamate

To a solution of (4-amino-2-((prop-2-yn-l-yloxy)methyl)phenyl)methanol (1.92 g, 10.04 mmoles, 1.0 equiv.), (9H-fluoren-9-yl)methyl (S)-(l-amino-1 -oxo-5-ureidopentan-2yl)carbamate (3.99 g, 10.04 mmoles, 1.0 equiv.), and (l-[bis(dîmethylamino)methylene]-lHl,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (4.20 g, 11.04 mmoles, 1.1 equiv.) in DMF (10 mL) was added Ν,Ν-diîsopropylethylamine (2.62 mL, 15.06 mmoles, 1.5 equiv.). After stîrring at ambient température for I h, the mixture was poured into water (200 mL). The resulting solids were filtered, rinsed with water, and dried under vacuum, and (9H-fluoren-9yl)methyl (S)-(l-((4-(hydroxymethyl)-3-((prop-2-yn-l-yloxy)methyl)phenyI)amino)-l-oxo-5ureidopentan-2-yl)carbamate was obtained . LCMS; MH+=57L5; Rt=0.93 min (2 min acidic method).

Synthesis of (S)-2-amino-N-(4-(hydroxymethyl)-3-((prop-2-yn-l-yloxy)methyl)phenyl)-5ureidopen tan amide

To (9H-fluoren-9-yl)methyl (S)-(l-((4-(hydroxymethyl)-3-((prop-2-yn-lyloxy)methyi)phenyl)amino)-l-oxo-5-ureidopentan-2-yl)carbamate (6.08 g, 10.65 mmoles, 1.0 equiv.) was added dimethylamine (2 M in THF, 21.31 mL, 42.62 mmoles, 4 equiv.). After

420 stirring at ambient température for t.5 hours, the supematant solution was decanted from the gum-like residue that had formed. The residue was triturated with ether (3x50 mL) and the resulting solids were filtered, washed with ether, and dried under vacuum. (S)-2-amino-N-(4(hydroxymethyl)-3-((prop-2-yn-1 -yloxy)methyI)phenyl)-5-ureidopentananiide was obtained. LCMS: MH+ 349.3; Rt=0.42 min (2 min acidic method).

Synthesis of tert-butyl ((S)-l-(((S)-l-((4-(hydroxymethyl)-3-((prop-2-yn-lyloxy)methyl)phenyl)amino)-l-oxo-5-ureîdopentan-2-yl)amino)-3-niethyl-i-oxobutan-2yl)carbamate

To a solution of(S)-2-amino-N-(4-(hydroxymethyl)-3-((prop-2-yn-lyloxy)methyl)phenyl)-5-ureidopentanamide (3.50 g, 10.04 mmoles, LO equiv.), (tertbutoxycarbonyl)-L-valine (2.62 g, 12.05 mmol, 1.2 equiv.), and (1[bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridmium 3-oxid hexafluorophosphate (4.58 g, 12.05 mmoles, 1.2 equiv.) in DMF (10 mL) was added N,N-diisopropylethylamine (3.50 mL, 20.08 mmoles, 2.0 equiv). After stirring at ambient température for 2 h, the mixture was poured into water (200 mL) and the resulting suspension was extracted with EtOAc (3x100 mL). The combined organic layers were dried over sodium sulfate and concentrated under vacuum. After purification b y ISCO S1O2 chromatography (0-20% methanol / dichloromethane), tertbutyl ((S)-l-(((S)-l-((4-(hydroxymethyl)-3-((prop-2-yn-l-yloxy)methyl)phenyl)amino)-l-oxo-5ureidopentan-2-yl)amino)-3-methyl-l-oxobutan-2-yl)carbamate was obtained. ^!H NMR (400 MHz, DMSO-d6) 5 10.00 (s, 1 H), 7.96 (d, J = 7.7 Hz, 1 H), 7.55 (dq, J = 4.9, 2.2 Hz, 2H, aryl), 7.32 (d, J = 8.9 Hz, IH, aryl), 6.76 (d, J = 8.9 Hz, IH), 5.95 (t, J = 5.8 Hz, IH), 5.38 (s, 2H), 5.01 (t, J = 5.5 Hz, IH), 4.54 (s, 2H), 4.45 (dd, J = 25.2, 5.3 Hz, 3H), 4.20 (d, J = 2.4 Hz, 2H), 3.83 (dd, J = 8.9, 6.7 Hz, IH), 3.49 (t, J = 2.4 Hz, IH), 2.97 (dh, J = 26.0, 6.5 Hz, 2H), 1.96 (h, J = 6.6 Hz, IH), 1.74- 1.50 (m, 2H), 1.39 (m, 1 IH), 0.84 (dd, J = 16.2, 6.7 Hz, 6H). LCMS: Mna+ 570.5; Rt=0.79 min (2 min acidic method).

Synthesis of tert-butyl ((S)-l-(((S)-l-((4-(chloroniethvl)-3-((prop-2-vn-lyloxy)methyl)phenyl)amino)-l-oxo-5-ureidopentan-2-yl)amino)-3-methyl-l-oxobutan-2yl)carbamate 421

To a solution of tert-butyl ((S)-l-(((S)-l-((4-(hydroxymethyl)-3-((prop-2-yn-lyloxy)methyl)phenyl)amino)-1 -oxo-5-ureîdopentan-2-yl)amino)-3-methyl-1 -oxobutan-2yl)carbamate (2.00 grams, 3.65 mmol, l .0 equiv.) in acetonitrile (13.3 mL) at 0 °C was added thionyl chloride (0.53 mL, 7.30 mmol, 2.0 equiv). After stirring in the ice bath for one hour the solution was diluted with water (40 mL) and the resulting white precipitate was collected by filtration, air drying and drying under high vacuum to yield tert-butyl ((S)-1 -(((S)-l-((4(chloromethyl)-3-((prop-2-yn-l-yloxy)methyl)phenyl)amino)-l -oxo-5-ureidopentan-2yl)amino)-3-methyl-l-oxobutan-2-yl)carbamate. LCMS: Mna+ 588.5; Rt=2.17 min (5 min acidic method).

Synthesis of 2-(((tert-butyldiphenylsilyl)oxy)methyI)-5-nitrobenzoic acid

TBDPSO^JCÿJ

HO^O

To a solution of 6-nitroisobenzofuran-l(3H)-one (90 g, 502.43 mmol, l.OO eq) in MeOH (1000 mL) and KO H (28.19 g, 502.43 mmol, 1.00 eq) in H₂O (150 mL) was added. The brown mixture was stirred at 25°C for 1.5 h. The brown mixture was concentrated under reduced pressure to give a residue and dissolved in DCM (2000 mL). The mixture was added TBDPSC1 (296.91 g, 1.08 mol, 277.49 mL, 2.15 eq) and IMIDAZOLE (171.03 g, 2.51 mol, 5.00 eq) stirred at 25°C for 12 h. The mixture was concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (Petroleum ether/Ethyl acetate=l/0, 1/1) and 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzoic acid was obtained as a white solid. ’H NMR (400 MHz, METHANOL-d4) δ ppm 1.13 (s, 9 H) 5.26 (s, 2 H) 7.34 - 7.48 (m, 6 H) 7.68 (br d, J=8 Hz, 4 H) 8.24 (br d, J=8 Hz, 1 H) 8.46 (br d, J=8 Hz, 1 H) 8.74 (s, 1 H).

Synthesis of (2-(((tert-butyldiphenylsilyl)oxy)methyI)-5-nitrophenyl)methanol

422

To a mixture of 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzoic acid (41 g, 94.14 mmol, 1 eq) in THF (205 mL) was added BH3. THF (1 M, 470.68 mL, 5 eq). The yellow mixture was stirred at 60°C for 2h. The mixture was added MeOH (400mL), and concentrated under reduced pressure to give a residue. Then addition of H₂O (200mL) and DCM(300mL), extracted with DCM (3 *200 mL), washed with brine (300mL), dried over anhydrous MgSO₄, fîltered, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (Petroleum ether/Ethyl acetate=l/0, 1/1). (2-(((tertbutyldiphenylsilyl)oxy)methyl)-5-nitrophenyl)methanol was obtained as a white solid. ¹H NMR (400 MHz, METHANOL-d4) δ ppm 1.10 (s, 9 H) 4.58 (s, 2 H) 4.89 (s, 2 H) 7.32-7.51 (m, 6 H) 7.68 (dd, J=8, 1.38 Hz, 4 H) 7.76 (d, J=8 Hz, 1 H) 8.15 (dd, J=8 2.26 Hz, 1 H) 8.30 (d, J=2 Hz, 1 H).

Synthesis of 2-(((tert-butyldiphenylsilyl)oxy)methyi)-5-nitrobenzaldehyde

To a solution of (2-(((tert-butyldîphenylsilyl)oxy)methyl)-5-nitrophenyl)methanol (34 g, 80.65 mmol, 1 eq) in DCM (450 mL) was added MnO₂ (56.09 g, 645.22 mmol, 8 eq). The black mixture was stirred at 25°C for 36 h. The mixture was added MeOH (400mL), and concentrated under reduced pressure to give a residue. Then addition of H₂O (200mL) and DCM (300mL), extracted with DCM (3 *200 mL), washed with brine (300mL), dried over anhydrous MgSO₄, fîltered, and concentrated under reduced pressure to give a residue. The residue was purified by silica gel chromatography (CH₂Cl₂=100%). 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5nitrobenzaldehyde was obtained as a white solid. 'H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.14 (s, 9 H) 5.26 (s, 2 H) 7.34 - 7.53 (m, 6 H) 7.60 - 7.73 (m, 4 H) 8.13 (d, J=8Hz, 1 H) 8.48 (dd, J=8, 2.51 Hz, 1 H) 8.67 (d, J=2 Hz, 1 H) 10.16 (s, 1 H).

Synthesis of N-(2-(((tert-butyldiphenylsilyl)oxy)methyi)-5-nitrobenzyl)prop-2-yn-l-amine

To a solution of 2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzaldehyde (12.6 g, 30.03 mmol, 1 eq) in DCM (130 mL) was added prop-2-yn-l -amine (4.14 g, 75.08 mmol, 4.81 mL, 2.5 eq) and MgSO₄ (36.15 g, 300.33 mmol, 10 eq) then the suspension mixture was stirred 423 at 25°C for 24hr. Taking a little reaction solution and treating with NaBH4, the TLC showed one new point was formed. The reaction mixture was fîltered and concentrated under reduced pressure to give a residue. (E)-N-[[2-[[tert-butyl(diphenyl)silyl]oxymethyl]-5-nitrophenyl]methyl]prop-2-yn-l-imine was obtained as a yellow solid. *H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.11 (s, 9 H) 2.48 (t, J=2.38 Hz, 1 H) 4.52 (t, J=2.13 Hz, 2 H) 5.09 (s, 2 H) 7.35 - 7.49 (m, 6 H) 7.63 - 7.72 (m, 4 H) 7.79 (d, J=8.53 Hz, 1 H) S.25 (dd, J=8.53, 2.51 Hz, 1 H) 8.68 (d, J=2.26 Hz, 1 H) 8.S4 (t, J=1.88 Hz, 1 H).

(E)-N-[[2-[[tert-butyl(diphenyl)silyl]oxymethyl]-5-nitro-phenyl]methyl]prop-2-yn-limine (12 g, 26.28 mmol, 1 eq) was dissolved in MeOH (100 mL) and THF (50 mL), then NaBH4 (1.49 g, 39.42 mmol, 1.5 eq) was added and the yellow mixture was stirred at -20°C for 2hr. LCMS showed desired compound was detected. The reaction mixture was quenched by addition MeOH (200 mL) at -20 °C, and then concentrated under reduced pressure to give a residue. The residue was dissolved with EtOAc (500 mL) washed with brine (150 mL), dried over anhydrous Na₂SO4, fîltered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography ( Eluent of 0-10% Ethyl acetate/Petroleum ether gradient). N-(2-(((tert-butyldiphenylsîlyl)oxy)methyl)-5nitrobenzyl)prop-2-yn-l-amine was obtained as a pale yellow oil. NMR (400 MHz, CHLOROFORM-d) δ ppm 1.12 (s, 9 H) 2.13 (t, J=2.38 Hz, 1 H) 3.33 (d, J=2.51 Hz, 2 H) 3.80 (s, 2 H) 4.93 (s, 2 H) 7.36-7.49 (m, 6 H) 7.69 (dd, J=7.9l, 1.38 Hz, 4 H) 7.77 (d, J=8.53 Hz, 1 H) 8.16 (dd, J=8.41, 2.38 Hz, 1 H) 8.24 (d, J=2.26 Hz, 1 H).

Synthesis of (9 H-fluoren-9-yl) methyl (2-(((tert-butyldiphenylsilyI)oxy)methyl)-5nitrobenzyl)(prop-2-yn-l-yl)carbamate

To a solution of N-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)prop-2-yn-lamine (9 g, 19.62 mmol, 1 eq) and FMOC-OSU (7.28 g, 21.59 mmol, 1.1 eq) in dîoxane (90 mL) was added sat. NaHCOj (90 mL) and the white suspension was stirred at 20°C for 12 h. The reaction mixture was diluted with H₂O (150 mL) and extracted with EtOAc (150 mL x 2). The combined organic layers were washed with brine (200 mL), dried over anhydrous Na₂SO4, fîltered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (Eluent of 0-30% Ethyl acetate/Petroleum ether). (9H-fluoren-9yl)methyl (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(prop-2-yn-l-yl)carbamate 424 (7.7 g, 11.08 mmol, 56.48% yield, 98% purity) was obtained as a white solid. 'H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.12 (s, 9 H) 2.17 (br d, J=14.31 Hz, 1 H) 3.87-4.97 (m, 9 H) 6.98 - 8.28 (m, 21 H).

Synthesis of (9 H-fluoren-9-yl) methyl (5-amino-2-(((tertbutyldiphenylsiIyl)oxy)methyl)benzyl)(prop-2-yn-l-yl)carbamate

To an ice bath cooled solution of (9H-fluoren-9-yl)methyl (2-(((tertbutyldiphenylsilyl)oxy)methyl)-5-nitrobenzyl)(prop-2-yn-l-yl)carbamate (5.0 g, 7.34 mmoles, 1.0 equiv.) in 10% AcOH/CH₂C12 (100 mL) was added Zn (7.20 g, 110 mmoles, 15 equiv.). The ice bath was removed, and the resulting mixture stirred for 2 hours at which time it was filtered through a pad of celite®. The volatiles were removed in vacuo and the residue was dissolved in EtOAc, was washed with NaHCO3(sat.), NaCl(sat.), dried over MgSÛ4, filtered, concentrated and after 1SCO SiO₂ chromatography (0-75% EtOAc/Heptanes) (9H-fluoren-9-yl)methyl (5amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-l-yl)carbamate was obtained. LCMS: MH+=651.6; Rt=3.77 min (5 min acidic method).

Synthesis of (9H-fhioren-9-yI)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-(((tertbutyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-l-yi)carbamate

To (9H-fluoren-9-yl)methyl (5-amino-2-(((tertbutyldiphenylsiIyl)oxy)methyl)benzyl)(prop-2-yn-l-yl)carbamate (2.99 g, 4.59 mmoles, LO equiv) and (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanoic acid (1.72 g, 4.59 mmoles, 1.0 equiv.) in CH₂C1₂ (40 mL) was added ethyl 2-ethoxyquinolinel(2H)-carboxylate (2.27 g, 9.18 mmoles, 2.0 equiv.). After stirring for 10 min, MeOH (1 mL) was added and the solution became homogeneous. The reaction was stirred for 16 h, the

425 volatiles were removed in vacuo and after purification by ISCO SÎO₂ chromatography (0-15% MeOH/CH2Q2) (9H-fluoren-9-yl)m ethyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amîno)-3methylbutanamido)-5-ureidopentanamîdo)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop2-yn-l-yI)carbamate was obtaîned. LCMS: MH+=l 008.8; Rt=3.77 min (5 min acidic method).

Synthesis of prop-2-yn-l-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3niethylbutanamido)-5-ureidopentananndo)-2-(((tertbutyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-l-yl)carbamate

To (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldîphenylsilyl)oxy)methyl)benzyl)(prop2-yn-l-yl)carbamate (1.60 g, 1.588 mmoles, 1.0 equiv.) was added 2M dimethylamine in MeOH (30 mL, 60 mmol, 37 equiv.) and THF (10 mL). After standing for 3 h, the volatiles were removed in vacuo and the residue was triturated with Et₂O to remove FMOC deprotection byproducts. To the resulting solid was added CH₂C1₂ (16 mL) and pyridine (4 mL) and to the heterogeneous solution was added propargyl chloroformate (155 uL, 1.588 mmole, 1.0 equiv.). After stîrring for 30 minutes additional propargyl chloroformate (155 uL, 1.588 mmole, LO equiv.) was added. After stîrring for an additional 20 min, MeOH (1 mL) was added to quench remaining chloroformate and the volatiles were removed in vacuo. Upon purification by ISCO SiO₂ chromatography (0-15% MeOH/CH₂Cl₂) prop-2-yn-1-yl (5-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamîdo)-5-ureidopentanamido)-2-(((tertbutyldiphenylsilyl)oxy)methyl)benzyl)(prop-2-yn-l-yI)carbamate was obtaîned. LCMS: MH+=867.8; Rt=3.40 min (5 min acidic method).

Synthesis of prop-2-yn-l-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-(liydroxymethyl)benzyl)(prop-2-yn-lyl)carbaniate

426

To a solution of prop-2-yn-l-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(prop2-yn-l-yl)carbamate (984 mg, 1.135 mmoles, 1.0 equiv.) in THF (7.5 mL) was added 1.0 M tetrabutylammoniumn fluoride in THF (2.27 mL, 2.27 mmoles, 2.0 equiv.). After standing for 6 h, the volatiles were removed in vacuo, the residue was purified by ISCO S1O2 chromatography (0-40% MeOH/CH2C12) and prop-2-yn-I-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(prop-2-yn-l-yl)carbamate was obtained. LCMS: MH+=629.6; Rt=L74min (5 min acidic method).

Synthesis of prop-2-yn-l-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureîdopentanamido)-2-(chIoromethyl)benzyl)(prop-2-yn-lyl)carbamate

γ°· ο

Το prop-2-yn-l-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)5-ureîdopentanamido)-2-(hydroxymethyl)benzyl)(prop-2-yn-l-yl)carbamate (205 mg, 0.326 mmoles, l.O equiv.) in CH2CI2 (10 mL) was added pyridine (158 uL, 1.96 mmoles, 5 equiv.). The heterogeneous mixture was cooled in a 0 °C ice bath and thionyl chloride (71 uL, 0.98 mmoles, 3 equiv.). After stirring in the ice bath for 3 hours the reaction was directly purified by ISCO S1O2 chromatography (0-30% MeOH/CHsCh) and prop-2-yn-1 -yl (5-((S)-2-((S)-2-((tertb ut oxycarbonyl) amino)-3-methyl butanami do )-5-ureidopentanamido)-2(chloromethyl)benzyl)(prop-2-yn-l-yl)carbamate was obtained LCMS: MH+=647.6; Rt=2.54 min (5 min acîdic method).

Synthesis of 2-(hydroxyniethyi)-N-methyl-5-nitrobenzamide

427

I

To a stirred suspension of 6-nitroisobenzofuran-l(3H)-one (500 g, 2.79 mol) in MeOH (1500 mL) was added MeNH₂ (3.00 kg, 29.94 mol, 600 mL, 31.0% purity) at 25 °C and stirred for 1 h. The solid was filtered and washed with water twice (600 mL) and dried under high vacuum to get a residue. The product 2-(hydroxymethyl)-N-methyl-5-nitrobenzamide was obtained as white solid. LCMS: Rt = 0.537 min, MS m/z = 193.2. IH NMR: 400 MHz DMSO δ 8.57 (br d, J = 4.4 Hz, IH), 8.31 (dd, J = 2.4, 8.6 Hz, 1 H), 8.21 (d, J = 2.4 Hz, IH), 7.86 (d, J = 8.8 Hz, IH), 5.54 (t, J = 5.6 Hz, IH), 4.72 (d, J = 5.5 Hz, 2H), 2.78 (d, J = 4.4 Hz, 3H).

Synthesis of (2-((methylammo)methyl)-4-nitrophenyl)methanol ^^,no₂Il Ί ^hoA/ 'NH I

To a solution of 2-(hydroxymethyl)-N-methyl-5-nitrobenzamide (560 g, 2.66 mol) in THF (5000 mL) was cooled to 0 °C, then added BH3-Me2S (506 g, 6.66 mol) (2.0 M in THF) drop wise for 60 min and heated to 70 °C for 5 h. LCMS showed the starting material was consumed. After completion, 4M HCl (1200 mL) in Methanol was added to reaction mixture at 0 °C and heated at 65 °C for 8 h. The reaction mixture was cooled to 0 °C, the solid was filtered and concentrated in reduce pressure. The product (2-((methylamino)methyI)-4nitrophenyl)methanol (520 g) was obtained as a white solid. LCMS: Rt = 0.742 min, MS m/z = 197.1 [M+H]+. ‘H NMR: 400 MHz DMSO δ 9.25 (br s, 2H), 8.37 (d, J = 2.4 Hz, IH), 8.14 (dd, J = 2.4, 8.5 Hz, IH), 7.63 (d, J = 8.4 Hz, IH), 5.72 (br s, 1 H), 4.65 (s, 2H), 4.15 (br s, 2H), 2.55 2.45 (m, 3H)

Synthesis of l-(2-(((tert-butyldiphenylsilyl)oxy)methyl)-5“nitrophenyl)-Nmethylmethanamine ^<^no₂tbdpso^JA-NH I

To a solution of (2-((methylamino)methyl)-4-nitrophenyl)methanol (520 g, 2.65 mol) and imidazole (721 g, 10.6 mol) in DCM (2600 mL) was cooled to 0°C was added TBDPS-CL (1.09 kg, 3.98 mol, 1.02 L) drop wise and stirred for 2 h. The mixture was poured in ice cold water 428 ( 1000 mL) and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na₂SÛ4, fîltered and evaporated under vacuum to give crude product. The crude product was purified by chromatography on a silica gel eluted with Ethyl acetate: Petroleum ether (from 10/1 to 1) to give a residue. The product l-(2-(((tertbutyldiphenylsiIyl)oxy)methyl)-5-nitrophenyl)-N-methylmethanamine was obtained as yellow liquid. LCMS: product: Rt = 0.910 min, MS m/z = 435.2 [M+H]+. ‘H NMR: 400 MHz CDC13 δ 8.23 (d, J=2.4 Hz, IH), 8.15 (dd, J=2.4, 8.4 Hz, IH), 7.76 (d, J=8.4 Hz, IH), 7.71-7.66 (m, 4H), 7.50 - 7.37 (m, 6H), 4.88 (s, 2H), 3.65 (s, 2H), 2.39 (s, 3H), i. 12 (s, 9H)

Synthesis of (9H-fiuoren-9-yl)methyl (2-(((tert-butyidiphenylsilyI)oxy)methy])-5“ nitrobenzyl)(methyl)carbamate

TBDPSO^JAJ

L. , Fmoc N I

To a solution of l-(2-(((tert-butyldiphenylsilyI)oxy)methyI)-5-nitrophenyl)-Nmethylmethanamine (400 g, 920.3 mmol) in THF (4000 mL) was added FMOC-OSU (341.5 g, 1.01 mol) and Et₃N (186.2 g, 1.84 mol, 256.2 mL), the mixture was stirred at 25 °C for 1 h. The mixture was poured into water (1600 mL) and extracted with ethyl acetate (1000 mL x 2). The combined organic layers were washed with brine, dried over Na₂SO4, fîltered and evaporated under vacuum to give crude product. The crude product was purified by chromatography on a silica gel eluted with petroleum ether: ethyl acetate (from 1/0 to 1/1) to give (9H-fluoren-9yl)methyl (2-(((tert-butyldiphenylsîlyl)oxy)methyl)-5-nitrobenzyl)(methyl)carbamate as white solid. LCMS: Rt = 0.931 min, MS m/z = 657.2 [M+H]+. Ή NMR: EW16000-26-P1 A, 400 MHz CDC13 δ 8.21 - 7.96 (m, 1 H), 7.87 - 7.68 (m, 3EI), 7.68 - 7.62 (m, 4H), 7.62 - 7.47 (m, 2H), 7.47 - 7.28 (m, 9H), 7.26 - 7.05 (m, 2H), 4.81 (br s, IH), 4.62 - 4.37 (m, 4H), 4.31-4.19 (m, 1 H), 4.08 - 3.95 (m, IH), 2.87 (br d, J = 5.2 Hz, 3H), 1.12 (s, 9H).

Synthesis of (9H-fluoren-9-yl)methyI (5-amino-2-(((tertbutyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate ζ₂·/^ΝΗ2tbdpso^JLA k,,,Fmoc N I

A solution of (9H-fluoren-9-yI)methyI (2-(((tert-butyldiphenylsilyl)oxy)methyl)-5nitrobenzyl)(methyl)carbamate (3.0 g, 4.57 mmole, 1.0 equiv.) in MeOH (90 mL) and EtOAc (30 mL) was degassed and purged to a balloon of N₂ via three way stopcock. After repeating 429 degas/N₂ purge 2x, 10% Pd/C deGussa type (0.486 g, 0.457 mmoles, O.l equiv.) was added. The resulting mixture was degassed and purged to a balloon of 2 H₂ via three-way stopcock. After repeating degas/H₂ purge 2x, the reaction stirred under the balloon pressure of H₂ for 4 hours. The reaction was degassed and purged to N2, filtered through a pad of celite eluting further with MeOH. After removal of the volatiles in vacuo and pumping on high vac (9Hfluoren-9-yl)methyl (5-amino-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate was obtained. LCMS: MH+=627.7; Rt=l .59 min (2 min acidic method).

Synthesis of (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-(((tertbutyldiphenylsilyl)oxy)methyl)benzyl)(methyl) car ba mate nh₂

I

Fmoc

To (9H-fluoren-9-yl)methyl (5-amino-2-(((tertbutyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (2.86 g, 4.56 mmoles, LO equiv) and (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanoic acid ( l .71 g, 4.56 mmoles, l .0 equiv.) in 2:1 CH₂Cl₂/MeOH (60 mL) was added ethyl 2-ethoxyquinolînel(2H)-carboxylate (2.256 g, 9.12 mmoles, 2.0 equiv.). The homogeneous solution was stirred for 16 hours at which time additîonal (S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanoic acid (0.340 g, 0.2 equiv.) and ethyl 2-ethoxyquinolinel(2H)-carboxylate (0.452 g, 0.4 equiv.) were addd to drive the reaction to completion. After stirring for an adiditonal 5 hours the volatiles were removed in vacuo and after purification by ISCO SÎO₂ chromatography (0-5% MeOH/CH₂Cl₂) (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((tertbutyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate was obtained. LCMS: MH+=984.1; Rt=1.54 min (2 min acidic method).

Synthesis of prop-2-yn-l-yi (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-(((tertbutyldiphenylsilyi)oxy)methyl)benzyl)(methyl)carbamate

430

TSDPSO

To (9H-ftuoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamîdo)-5-ureidopentanamido)-2-(((tertbutyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (2.05 g, 2.085 mmol, 1.0 equiv) in THF (10 mL) was added 2.0 M dimethyl amine in MeOH (10.42 mL, 20.85 mmol, 10 equiv.). After stirrîng for 16 hours the volatiles were removed in vacuo. The residue was dissolved in CH2CI2 (20 mL) and D1EA (0.533 mL, 4.17 mmol, 2 equiv.) and propargyl chloroformate (0.264 mL, 2.71 mmol, 1.3 equiv.) were added. After stirring at rt for 16 hours the reaction was diluted with CH2CI2 (20 mL), was washed withNaHCO₃ (sat.), NaCl(sat), dried over MgSO₄, filtered, concentrated and purified by ISCO S1O2 chromatography (0-15% MeOH/CH₂Cb) to yield prop2-yn-l-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5ureidopentanaimdo)-2-(((tert-butyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate. LCMS: MH+=843.8; Rt=1.35 min (2 min acidîc method).

Synthesis of prop-2-yn-l-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-(hydroxyniethyl)benzyl)(methyl)carbaniate

To a 0 °C solution of prop-2-yn-l-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-(((tertbutyldiphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (1.6 g, 1.90 mmoles, 1.0 equiv.) în THF (10.0 mL) was added 1.0 M tetrabutylammoniumn fluoride in THF (3.80 mL, 3.80 mmoles, 2.0 equiv.). After warming to room température and stirring for 16 h the volatiles were removed in vacuo, the residue was dissolved in EtOAc, was washed with NaHCO₃(sat.), with NaCl(sat.), dried over MgSO₄, filtered, concentrated and the residue was purified by ISCO S1O2 chromatography (0-30% MeOH/CH2C12) to yield prop-2-yn-l-yl (5-((S)-2-((S)-2-((tert

431 butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2(hydroxymethyl)benzyl)(methyl)carbamate. LCMS: MH+=605.7; Rt=0.8I min (2 min acidic method).

Synthesis of prop-2-yn-1-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-(chloromethyl)benzyl)(methyi)carbamate

0<^nh₂

To prop-2-yn-l-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (350 mg, 0.579 mmoles, 1.0 equiv.) in CH₂C1₂ (10 mL) was added pyridine (0.278 mL, 3.47 mmoles, 6 equiv.). The heterogeneous mixture was cooled in a 0 °C ice bath and thionyl chloride (0.126 mL, 1.73 mmoles, 3 equiv,). After stirring in the ice bath for 3 h, the reaction was purified by 1SCO SÎO₂chromatography (0-30% MeOH/CH₂CI₂) and prop-2-yn-l-yl (5-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentananiido)-2(chloromethyl)benzyl)(prop-2-yn-l-yl)carbamate was obtained. LCMS: MH+=623.7; Rt=2.19 min (5 min acidic method).

Synthesis of (9H-fluoren-9-yi) methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyI)amino)-3methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate o^,nh₂

To (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-(((tertbutyldîphenylsilyl)oxy)methyl)benzyl)(methyl)carbamate (2.6 g, 2.64 mmol, 1.0 equiv.) dissolved in THF (20 mL) was added acetic acid (0.757 mL, 13.22 mmol, 5.0 equiv.) and 1.0 M TBAF in THF (2.91 mL, 2.91 mmol, 1.1 equiv.). The solution was stirred for 72 hours at which tîme the volatiles were removed in vacuo. After purification by ISCO SiO₂ chromatography (0432

30% MeOH/CHzCh) (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate was obtained. LCMS: MH+=745.5; Rt=l.07 min (2 min acidic method).

Synthesis of (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyi)ammo)3-methylbutanamido)-5-ureidopentanamido)-2-(chloroniethyI)benzyl)(methyl)carbamate

I

To (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (200 mg, 0.269 mmoles, 1.0 equiv.) in CH2CI2 (10 mL) was added pyridine (0.130 mL, 1.61 mmoles, 6 equiv,). The heterogeneous mixture was cooled in a 0 °C ice bath and thionyl chloride (0.059 mL, 0.806 mmoles, 3 equiv.). After stirrîng in the ice bath briefly the reaction was stirred as it wanned up to room température for 2 hours. The reaction was purified b y ISCO SiO₂chromatography (0-30% MeOH/CH₂Cl₂) and (9H-fluoren-9-yl)m ethyl (5-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2(chloromethyl)benzyl)(methyl)carbamate was obtained. LCMS: MH+=763.2; Rt=l. 18 min (2 min acidic method).

GENERAL PROCEDURE 1

Synthesis of l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5ureidopentanamido)-2-((prop-2-yn-l-yl((prop-2-yn-lyloxy)carbonyl)amino)methyl)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium

433

To (R)-2-((5-(3-chloro-2-methyl-4-(2-(4-methylpiperazin-l-yl)ethoxy)phenyl)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-4-yI)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)propanoic acid hydrochloride (73.8 mg, 0.81 mmoles, l.O equiv.) and prop2-yn-1 -yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5ureidopentanamido)-2-(chloromethyl)benzyl)(prop-2-yn-l-yl)carbamate (78 mg, 0.122 mmoles, 1.5 equiv.) dissolved in DMF (0.5 mL) was added DIEA (70 uL, 0.405 mmoles, 5.0 equiv.) followed by tetrabutylammonium iodide (25.4 mg, 0.069 mmoles, 0.85 equiv.). After stirring for 5 h, the reaction was diluted with DMSO (3 mL) and was purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H2O, 0.1% NH4OH modifier). Upon lyophilization, l-(4((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2((prop-2-yn-l-yl((prop-2-yn-l-yloxy)carbonyl)amino)methyl)benzyl)-4-(2-(4-(4-((R)-l-carboxy2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l -methylpiperazin1-ium was obtained. LCMS: M+= 1486.3; Rt=2.70 min (5 min basic method).

GENERAL PROCEDURE 2

Synthesis of l-(2-(((((l-(2,5,8,ll,14,17,20,23-octaoxapentacosan-25-yl)-lH-l,2,3-triazol-4yl)methoxy)carbonyl)(( 1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-lH-l,2,3-triazol-4yl)methyI)amino)niethyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyI)pyrÎmidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium

434

Το l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)ammo)-3-methyIbutanamido)-5ureidopentanamido)-2-((prop-2-yn-l-yl((prop-2-yn-l-yloxy)carbonyl)amino)methyl)benzyl)-4(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyI)ethoxy)-6(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyI)-lmethylpiperazin-l-ium (24 mg, O.Ql 6 mmoles, l.O equiv) and 25-azido-2,5,8,11,14,17,20,23octaoxapentacosane (26.5 mg, 0.065 mmoles, 4 equiv.) was added t-BuOH (1 mL). The mixture was degassed via house vacuum and purged to a balloon of N₂ via a 3-way stopcock. Degas/purge was repeated 3 times. A 16 mg/mL aqueous solution of sodium ascorbate (297 uL, 0.024 mmoles, 1.5 equiv.) was added and the solution was degassed and purged to N₂ three times. A 4 mg/mL aqueous solution of copper sulfate (298 uL, 0.0048 mmoles, 0.3 equiv.) was added and the solution was degassed and purged to N₂ three times. After stîrring under N₂ for 3 h the réaction was diluted with DMSO (3 mL) and was purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization 1-(2-(((((1(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-l H-l,2,3-triazol-4-yl)methoxy)carbonyl)((l(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-l H-1,2,3-triazol-4-yl)methyl)amino)methyl)-4((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)4-(2-(4-(4-( (R)-l-carboxy-2-(2-((2-(2-methoxyph en yl)pyrimidin-4-yl)methoxy)phenyl) ethoxy)6-(4-fluorophenyl)thîeno[2,3-d]pyrimîdin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l methylpiperazin-1-ium was obtaîned. HRMS M+=2307.0730, Rt=2.69 min (5 min acidic method).

GENERAL PROCEDURE 3

435

Synthesis of 1-(2-(((((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-lH-l,2,3-triazol-4yl)methoxy)carbonyl)((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-y 1)-1 H-l,2,3-triazol-4yl)methyl)ainino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-IH-pyiTol-lyl)ethoxy)propanamÎdo)-3-methyIbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-dJpyrimidin-5-yl)-2-chIoro-3-methylphenoxy)ethyl)-lmethylpiperazin-l-ium (Ll-Pl)

Το 1 -(2-((((( 1 -(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-l H-1,2,3-triazol-4yl)methoxy)carbonyl )((1-(2,5,8,1 l,14,17,20,23-octaoxapentacosan-25-yl)-lH-l,2,3-triazol-4yl)methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thîeno[2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-methylpiperazin-I-ium (19 mg, 0.0082 mmoles, 1.0 equiv.) was added 25% TFA/CH2CI2 (2 mL). After standing for 45 min, the volatiles were removed in vacuo, CH2CI2 was added and the volatiles were removed in vacuo and pumped on. The residue was dissolved in DMF (1 mL) and DIEA (22 uL, 0.124 mmoles, 15 equiv.) and 2,5-dioxopyrrolidin1-yl 3-(2-(2,5-dioxo-2,5-dihydro-lH-pynOl-l-yl)ethoxy)propanoate (5.1 mg, 0.016 mmoles, 2 equiv. ) was added. After standing for 18 h, the solution was diluted with DMSO (3 mL) and was purified by RP-ISCO gold chromatography. LJpon lyophilization, 1-(2-(((((1(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-1 H-1,2,3-triazol-4-yl)methoxy)carbonyl)((l (2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-l H-1,2,3-triazoI-4-yl)methyl)amino)methyl)-4((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethoxy)propanamido)-3methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium (Ll-Pl) was

436 obtained. HRMS: M+=2399.0797, Rt=2.43 min (5 min acidic run). 'H NMR (400 MHz, DMSO4) δ ppm 0.83 (dd, 7=13.82, 6.72 Hz, 6 H) 1.30- 1.52 (m, 2 H) 1.55-1.76 (m, 2 H) 1.83 (s, 3 H) 1.88-2.08 (m, 1 H) 2.28-2.46 (m, 7 H) 2.73 - 2.84 (m, 4 H) 2.84 - 3.08 (m, 8 H) 3.15-3.27 (m, 3 H) 3.43 -3.66 (m, 68 H) 3.73 -3.83 (m, 7 H) 4.16-4.30 (m, 3 H) 4.32-4.43 (m, 2 H) 4.47 (br s, 6 H) 4.60 (br s, 3 H) 5.16 - 5.30 (m, 3 H) 5.40 (br s, 2 H) 5.44 - 5.52 (m, 1 H) 5.99 (br t, 7=5.07 Hz, 1 H) 6.21 (d, 7=6.48 Hz, 1 H) 6.71 (t, 7=7.40 Hz, 1 H) 6.97 - 7.04 (m, 2 H), 7.00 (s, 2H) 7.12-7.23 (m, 5 H) 7.27 - 7.54 (m, 8 H) 7.63 (d, 7=5.14 Hz, 1 H) 7.78-7.94 (m, 3 H) 7.99 (br s, 1 H) 8.05 - 8.23 (m, 2 H) 8.60 (s, 1 H) 8.88 (d, 7=5.13 Hz, 1 H) 10.24 (br s, IH).

Synthesis of l-(4-((S)-2-((S)-2-((tert-butoxycarbonyI)amino)-3-methylbutanamido)-5ureidopentanamido)-2-(((((l-(26-carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl)-lH-l,2,3triazol-4-yl)methoxy)carbonyl)((l-(26-carboxy-3,6,9,12,15,18,2 l,24-octaoxahexacosyl)-l H1,2,3-triazol-4-yl) methyl) amino) methyl)benzyI)-4-(2-(4-(4-((R)-l-car b oxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyi)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methyipiperazin-l-ium /—\ o—\

Following GENERAL PROCEDURE 2 with 1 -(4-((S)-2-((S)-2-((tertbutoxycarbonyI)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-l-yl((prop2-yn-1 -yloxy)carbonyl)amino)methyl)benzyl)-4-(2-(4-(4-((R)-1 -carboxy-2-(2-((2-(2m ethoxyphenyl)pyrimidin-4-yl)methoxy)ph en yl)ethoxy)-6-(4-fl uoro phenyl )thieno [2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-niethylpiperazin-l-ium (30 mg, 0.020 mmoles, 1.0 equiv) and l-azido-3,6,9,12,15,18,2L24-octaoxaheptacosan-27-oic acid (28.3 mg, 0.061 mmoles, 3 equiv.), l-(4-((S)-2-((S)-2-((tert-butoxycarbonyi)amino)-3-methyIbutanamido)5-ureidopentanamido)-2-((((( 1 -(26-carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl)-1 H-1,2,3triazol-4-yl)methoxy)carbonyl)((l-(26-carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl)-lH437

1,2,3-triazol-4-yl)methyl)amino)methyl)benzy 1)-4-(2-(4-(4-(( R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluoropheny1)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l -methylpipcrazin-1 -ium was obtained.

HRMS: M+=2420.0867, Rt=2.57 min (5 min acidic method).

EXAMPLE 2: Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyriniidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fhiorophenyI)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(2-(((((l-(26-carboxy3,6,9,12,15,18,21,24-octaoxahexacosyi)-lH-l,2,3-triazol-4-yl)methoxy)carbonyl)((l-(26carboxy-3,6,9,12,15,18,2l,24-oetaoxahexacosyI)-lH-l,2,3-triazol-4yl)methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyi)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-lmethylpiperazin-l-ium (L10-P1)

O NHj

Following GENERAL PROCEDURE 3 with l-(4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((((l-(26-carboxy3,6,9,12,15,18,21,24-octaoxahexacosyl)-lH-l,2,3-triazol-4-yl)methoxy)carbonyl)((l-(26carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl)-1 H-l ,2,3-triazol-4yl)methyl)amino)methyl)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3djpyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin-1-ium (30 mg, 0.012 mmoles, 1.0 equiv.), 4-(2-(4-(4-((R)-l-carboxy^-^-i^-P-methoxyphenyljpyrimidinAyljmeihoxyjphenyljethoxyj-ô-^-fluorophenyOthienolZJ-d^yrimidinA-yl^-chloro^methylphenoxy)ethyl)-1-(2-((((( l-(26-carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl)-1H1,2,3-triazol-4-yl)methoxy)carbonyl)((l -(26-carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl)1 H-1,2,3-triazol-4-yl)methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1H

438 pyrroLl-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-lmethylpiperazin-l-ium (L10-P1) ) was obtained. HR.MS: M+=25l 5.0879, Rt=2.43 min (5 min acidic method). Ή NMR (400 MHz, DMSO-^) Ô ppm 0.83 (dd, J=\3.82, 6.72 Hz, 6 H) 1.30 1.52 (m, 2 H) 1,55- 1.76 (m, 2 H) 1.83 (s, 3 H) 1.88-2.08 (m, 1 H) 2.28-2.46 (m, 7 H) 2.73 2.84 (m, 4 H) 2.84 - 3.08 (m, 8 H) 3.15 - 3.27 (m, 3 H) 3.43 - 3.66 (m, 68 H) 3.73 - 3.83 (m, 7 H) 4.16-4.30 (m, 3 H) 4.32-4.43 (m, 2 H) 4.47 (br s, 6 H) 4.60 (br s, 3 H) 5.16 - 5.30 (m, 3 H) 5.40 (brs, 2 H) 5.44-5.52 (m, 1 H) 5.99 (brt, >5.07 Hz, 1 H) 6.21 (d,>6.48 Hz, 1 H) 6.71 (t, >7.40 Hz, 1 H) 6.97 - 7.04 (m, 2 H), 7.00 (s, 2H) 7.12 - 7.23 (m, 5 H) 7.27 - 7.54 (m, 8 H) 7.63 (d, >5.14 Hz, 1 H) 7.78 - 7.94 (m, 3 H) 7.99 (br s, 1 H) 8.05 - 8.23 (m, 2 H) 8.60 (s, 1 H) 8.88 (d, >5.13 Hz, 1 H) 10.24 (brs, 1 H).

Synthesis of l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5ureidopentananiido)-2-((methyl((prop-2-yn-l-yloxy)carbonyl)amino)methyl)benzyl)-4-(2(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyI)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-inethylphenoxy)ethyl)-lmethylpiperazin-l-ium o nh₂

Following GENERAL PROCEDURE 1 with (R)-2-((5-(3-chloro-2-methyL4-(2-(4methylpiperazin-l-yl)ethoxy)phenyl)-6-(4-fIuorophenyl)Îhieno[2,3-d]pyrimidin-4-yI)oxy)-3-(2((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)propanoic acid hydrochloride (300 mg, 0.329 mmol, 1.0 equiv.) and prop-2-yn-l-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-(chloromethyl)benzyl)(methyl)carbamate (246 mg, 0.395 mmol, 1.2 equiv.), l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)5-ureidopentanamido)-2-((methyl((prop-2-yn-l-yloxy)carbonyl)amino)methyl)benzyI)-4-(2-(4(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l -methylpiperazin1-ium was obtained . H RM S: M+= 1461.5 800, Rt=2.53 min (5 min acidic method).

439

Synthesis 1-(2-(((((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-IH-l,2,3-triazol-4 yi)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)3-methyIbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazm-l-ium

Following GENERAL PROCEDURE 2 with l-(4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2-yn-Iyloxy)carbonyl)amino)methyl)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyI)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium (20 mg, 0.14 mmol, 1.0 equiv.) and 25-azido-2,5,8,l 1,14,17,20,23-octaoxapentacosane (16.8 mg, 0.041 mmol, 3.0 equiv.), 1-(2-(((((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-l H-1,2,3-triazol-4yl )methoxy)carbonyl)(m ethyl )amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium was obtained.

HRMS: M+=l 872.8359, Rt=2.56 min (5 min acidic method).

Synthesis of 1-(2-(((((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-lH-l,2,3-triazol-4yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydrolH-pyrrol-l-yl)ethoxy)propanamido)-3-nietliyibutanamido)-5-ureidopentanamido)benzyl)4-(2-(4-(4-((R)-l-carboxy-2“(2-((2“(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d|pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-methylpiperazin-l-ium (L4-P1)

440

Following GENERAL PROCEDURE 3 with 1 -(2-((((( 1 -(2,5,8,11,14,17,20,23octaoxapentacosan-25-yl)-l H-l,2,3-triazol-4-yI)methoxy)carbonyl)(methyl)amino)methyl)-4((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)6-(4-fluorophenyl)thieno[2,3-d]pyriinidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-lmethylpiperazin-l-ium (16.9 mg, 0.009 mmol, 1.0 equiv.), 1-(2-(((((1-(2,5,8,11,14,17,20,23octaoxapentacosan-25-yl)-lH-l,2,3-triazol-4-yl)methoxy)carbonyl)(methyl)amino)methyl)-4((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrroJ-l-yl)ethoxy)propanamido)-3methylbutanamido)-5-ureîdopentanamido)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin~4-yl)Tnethoxy)phenyl)eihoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimîdin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium (L4-P1) was obtained . HRMS: M+= 1967.8375, Rt=2.46 min (5 min acidic method).

Synthesis of l-(4-((S)-2-((S)-2-((tert-butoxycarbonyI)amino)-3-methylbntanamido)-5ureidopentanamido)-2-(((((l-(26-carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl)-lH-l,2,3triazoi-4-yl)methoxy)carbonyl)(methyl)amino)methyl)benzyi)-4-(2-(4-(4-((R)-l-carboxy-2(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy )-6-(4fluorophenyI)thieno[2,3-d]pynnndin-5-yl)-2-cliloro-3-methylphenoxy)ethyl)-l-

441

Following GENERAL PROCEDURE 2 with l-(4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2-yn-lyloxy)carbonyl)amino)methyl)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2m ethoxyphenyl)pyr imidin-4-yl )methoxy)phen yl)ethoxy)-6-(4-fluoro phenyl )thieno [2,3d]pyrimidin-5-yi)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium (12 mg, 0.0082 mmol, 1.0 equiv) and l-azido-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-oic acid (11.5 mg, 0.025 mmol, 3.0 equiv.), I-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)5-ureidopentanamido)-2-(((((l -(26-carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl)-1 H-1,2,3triazol-4-yl)methoxy)carbonyl)(methyl)amino)methyl)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2((2-(2-methoxyphenyl)pyrimidin-4-yi)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyiïmidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium was obtained . HRMS: M+= 1928.8459, Rt=2.52 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yI)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yi)-2-chloro-3niethylphenoxy)ethyl)-l-(2-(((((1-(26-carboxy-3,6,9,12,l5,l8,2l,24-octaoxahexacosyl)-lHl,2,3-triazoI-4-y])methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo2,5-dihydro-lH-pyrrol-l-yl)ethoxy)propanamido)-3-methylbutanamido)-5ureidopentananiido)benzyl)-l-niethylpiperazin-l-ium (L3-P1) o nh.

Following general procedure 3 with l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureîdopentanamido)-2-((((( l-(26-carboxy-3,6,9,12,15,18,21,24octaoxahexacosyl)-! H-l,2,3-triazol-4-yl)methoxy)carbonyl)(methyl)amino)methyl)benzyl)-4-(2(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy )-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin1-ium (12 mg, 0.006 mmol,1.00 equiv.), 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrîmi din-4-yl)methoxy)phenyl) ethoxy)-6-(4- fluorophenyl)thieno [2,3

442

d]pyrîmidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-1 -(2-((((( l -(26-carboxy3,6,9,12,15,18,21,24-octaoxahexacosyl)-lH-l ,2,3-triazol-4yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-l Hpyrrol-l-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-lmethylpiperazin-1-ium (L3-P1) was obtained . HRMS: M+=2024.85l6, Rt=2.42 min (5 min acidic method),

Synthesis of l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)aniino)-3-methylbutanamido)-5ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyriniidin-4yl)methoxy)phenyi)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3methylphenoxy)ethyl)-l-methylpiperazin-l-ium

N HBOC

To 4-methoxybenzyl (R)-2-((5-(3-chloro-2-meÎhyl-4-(2-(4-methylpiperazin-1 yl)ethoxy)plienyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyI)propanoate ( 160 mg, 0.161 mmol, 1,0 equiv.) and (9H-fluoren-9-yl)methyl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-(chloromethyl)benzyl)(methyl)carbamate (153 mg, 0.201 mmoles, 1.25 equiv.) dissolved in DMF (2 mL) was added DIEA (0.056 mL, 0.321 mmoles, 2.0 equiv.) followed by tetrabutylammonium iodide (65.3 mg, 0.177 mmoles, 1.1 equiv.). After standing for 16 h, 2.0 dimethylamine in THF (0.804 mL, 1.67 mmol, 10 equiv.) was added. After standing for 2 h, the volatiles were removed in vacuo, DM S O (6 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization, l-(4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methyl butanamido)-5-ureidopentanamido)-2((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4methoxybenzyl )oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan

443

2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l -methylpiperazin-1 -ium was obtained. HRMS: M+=l499.3700; Rt=2.59 min (5 min acidic method).

Synthesis of l-(4-((R)-2-((R)-2-((tert-butoxycarbonyl)amino)-3-niethylbutanamido)-5ureidopentanamido)-2-(2-methyl-3-oxo-4,7,10,13,16,19,22,25,28-nonaoxa-2azanonacosyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyI)pyrîmidin-4-yl)inethoxy)phenyl)-loxopropan-2-yl)oxy)thieno[2,3-d]pyrîmîdin-5-yl)-3-methylphenoxy)ethyl)-l-

To l-(4-((S)-2-((S)-2-((tert-butoxycarbony[)amino)-3-methylbutanamido)-5ureidopentanamido )-2-( (methyl amino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4(((R)-1 -((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1 oxopropan-2-yI)oxy)thieno[2_J3-d]pyi*imidin-5-yi)-3-methylphenoxy)ethyl)-1 -methylpiperazin-1 ium (40 mg, 0.027 mmol, l.O equiv.) and 2,5-dioxopyrrolidin-l-yl (2,5,8,11,14,17,20,23octaoxapentacosan-25-yl) carbonate (30.8 mg, 0.059 mmoles, 2.2 equiv.) dissolved in DMF (1.5 mL) was added DIEA (0.009 mL, 0.053 mmoles, 2.0 equiv.). After standing for 1 hour, DMSO (3 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (ΙΟΙ 00% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization l-(4-((R)-2-((R)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(2-methyl-3-oxo4,7,10,13,16,19,22,25,28-nonaoxa-2-azanonacosyl)benzyl)-4-(2-(2-chloro-4-(6-(4fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)-]-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidm-5-yl)-3methylphenoxy)ethyl)-l-methylpiperazin-l-ium was obtained. HRMS: M+=1909.3800; Rt=2.92 min (5 min acidic method).

444

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yI)niethoxy)phcnyI)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-(4-((R)-2-((R)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(2-niethyl-3-oxo4,7,10,13,16,19,22,25,28-nonaoxa-2-azanonacosyl)benzyl)-l-methylpiperazin-l-ium (L2-P1)

O nh.

Following GENERAL PROCEDURE 3 with l-(4-((R)-2-((R)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamîdo)-5-ureidopentanamido)-2-(2-methyl-3-oxo4,7,10,l 3,16,19,22,25,28-nonaoxa-2-azanonacosyl)benzyl)-4-(2-(2-chloro-4-(6-(4fiuorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidîn-4yl)methoxy)phenyl)~l-oxopropan-2-yi)oxy)thieno[2,3-d]pyrimidin-5-yl)-3methylphenoxy)ethyl)-l-methylpiperazin-l-inm (25.3 mg, 0.013 mmol, 1.0 equiv.), 4-(2-(4-(4((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(4-((R)-2-((R)-2(3-(2-(2,5-dîoxo-2,5-dihydro-lH-pyrrol-l-yl)ethoxy)propanamido)-3-methylbutanamido)-5ureidopentanamido)-2-(2-methyl-3-oxo-4,7,10,13_s16,19,22,25,28-nonaoxa-2azanonacosyl)benzyl)-l-methylpiperazîn-l-ium (L2-P1) wasobtaîned . HRMS: M+= 1884.7900, Rt=2.50 mîn (5 min acidic method).

Synthesis of l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5ureidopentanamido)-2-(80-carboxy-2-methyl-3-oxo6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78-pentacosaoxa-2azaoctacontyl)benzyI)-4-(2-(2-chloro-4-(6-(4-fluoropheny!)-4-(((R)-l-((4mcthoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyI)-loxopropan-2-yl)oxy)thieno[2,3-d]pyrimîdin-5-yl)-3-methylphenoxy)ethyl)-lmethylpiperazin-l-ium

445

To l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5ureidopentanamido)-2-((methylamino)m ethyl )benzyl )-4-(2-(2-chloro-4-(6-(4-fluorophenyl )-4(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l5 oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-lium (150 mg, 0.093 mmol, l.O equiv.) and 79-((2,5-dioxopyrrolidin-l-yl)oxy)-79-oxo4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52.55,58,61,64,67,70,73,76pentacosaoxanonaheptacontanoic acid (134 mg, 0.102 mmoles, 1.1 equiv.) dissolved in DMF (2 mL) was added DIEA (0.081 mL, 0.464 mmoles, 5.0 equiv.). After standing for 18 h, DMSO (6 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (ΙΟ-

Ι 00% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization l-(4-((S)-2-((S)-2-((tertbutoxycarbonyl) amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(8 0-carboxy-2-methy 1-3oxo-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78-pentacosaoxa-2azaoctacontyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)15 3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3d]pyrimidin-5-yl)-3-methylphenoxy)ethyI)-l-methyl piperazin-1-ium was obtained. H RM S: M+=2700.8701; Rt=2.83 min (5 min acidic method).

446

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-(2-(80-carboxy-2-methyl-3-oxo6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78-pentacosaoxa-2azaoctacontyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-lmcthylpiperazin-l-ium (Lll-Pl)

Following GENERAL PROCEDURE 3 with l -(4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(80-carboxy-2-methyl-3oxo-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78-pentacosaoxa-2azaoctacontyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluoiOphenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1 -methylpiperazin-1 -ium, 4-(2-(4-(4-((R)-l -carboxy2-(2-((2-(2-methoxyphenyI)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(2-(80-carboxy2-methyl-3-oxo-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78pentacosaoxa-2-azaoctacontyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrroI-lyl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-lmethylpiperazin-l-ium (Lll-Pl) was obtained . HRMS: M+= 2674.8201, Rt=2.44 min (5 min acidic method).

447

Synthesis of l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-niethylbutananiido)-5ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyI)pyrimidin-4-y])methoxy)phenyi)ethoxy)-6-(4-nuorophenyl)thieno[2,3d|pyrimidin-5-yl)-2-chIoro-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium

NHBcc

Me Me

Following GENERAL PROCEDURE l with (R)-2-((5-(3-chloro-2-methyl-4-(2-(4methylpiperazin-l-yl)ethoxy)phenyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl)oxy)-3-(2((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)ph en yl)propanoic acid (50 mg, 0.057 mmol, l .0 equiv.) and tert-butyl ((S)-l-(((S)-l-((4-(chloromethyl)phenyl)amino)-l-oxo-5-ureidopentan-2yl)amino)-3-methyl-l-oxobutan-2-yl)carbamate(34.l mg, 0.069 mmol, 1.2 equiv.), l-(4-((S)-2((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyI)-4-(2(4-(4-((R)-I -carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyi)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazinl-ium was obtained. LCMS: M+=1337.2, Rt=l.l 1 min (2 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyI)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidm-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopcntanamido)benzyl)-lmethylpiperazin-l-ium or (2R)-2-[(5S_a)-5-[3-chloro-4-ï2-[4-[[4-|((2S)-2-[[(2S)-2-[3-[2-(2,5dioxopyrrol-l-yOethoxylpropanoylaminopS-methyl-butanoyllaminol-S-ureidopentanoyl]aniino]phenyl]methyI]-4-niethyl-piperazm-4-ium-l-y]]ethoxy]-2-methyl-phenyl]6-(4-fluorophenyl)thieno[2,3-d|pyrimidin-4-yl]Qxy-3-[2-|[2-(2-methoxyphenyl)pynmidin-4yl]methoxy]phenyl]propanoic acid (L9-P1)

448

Following GENERAL PROCEDURE 3 with l-(4-((S)-2-((S)-2-((tertbutoxycarbonyi)amino)-3-inethylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-lcarboxy-2-(2-((2-(2-methoxyph en yl)pyrimidin-4-yl)methoxy)ph en yl)ethoxy )-6-(4fluorophenyI)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l -methylpiperazinl-ium (55 mg, 0.041 mmol, LO equiv.), 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pynmidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5dihydro-lH-pyrrol-l-yl)ethoxy)propanamido)-3-methylbutanamido)-5ureidopentanamido)benzyl)-l-methylpiperazin-l-ium (L9-P1) was obtained. HRMS: M+=l431.5400, Rt=2.50 min (5 min acidic method).

Synthesis of l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methyIbutanamitio)-5ureidopentanamido)-2-((prop-2-yn-l-yloxy)methyI)benzyl)-4-(2-(2-chIoro-4-(6-(4fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidm-4 yl)methoxy)phenyl)-l-oxopropan-2-yI)oxy)thieno[2,3-d]pyrimidin-5-yl)-3niethylphenoxy)ethyl)-l-methylpiperazin-l-ium

Following GENERAL PROCEDURE 1 with 4-methoxybenzyl (R)-2-((5-(3-chloro-2methyl-4-(2-(4-methylpiperazin-l-yl)ethoxy)phenyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin4-yl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)propanoate (85 mg, 0.085 mmol, 1.0 equiv.) and tert-butyi ((S)-l-(((S)-l-((4-(chloromethyl)-3-((prop-2-yn-lyloxy)methyl)phenyl)amîno)-l -oxo-5-ureidopentan-2-yl)amino)-3 -methyl-1 -oxobutan-2

449 yl)carbamate (58 mg, 0.102 mmol, 1.2 equiv.), I -(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-l-yloxy)methyl)benzyl)-4-(2-(2chloiO-4-(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin5-yl)-3-meth y! phenoxy)ethyl)-l-methylpiperazin-!-ium was obtained. HRMS: M+=1524.6200, Rt=2.95 min (5 min acidic method).

Synthesis of 1-(2-(((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-lH-l,2,3-triazol-4yl)methoxy)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5ureidopentanamîdo)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyI)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3dlpyrimîdin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium

N ''N

Following GENERAL PROCEDURE 2 with 1 -(4-(( S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-lyloxy)methyl)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)niethoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-methylpiperazin-1-ium (20 mg, 0.014 mmoles, 1.0 equiv) and 25-azido2,5,8,11,14,17,20,23-octaoxapentacosane (5.8 mg, 0.014 mmoles, 1 equiv.), 1-(2-(((1(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-l H-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin1-ium was obtained. LCMS: [(M+)+H+]+2/2=908.5, Rt=l. 15 min (2 min acidic method).

450

Synthesis of l-(2-(((l-(2,5,8,ll,14,17,20,23-octaoxapcntaeosan-25-yI)-lH-l,2,3-triazol-4y l) methoxy) methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-diox o-2,5-dihydr ο-1 H-pyr roi-1 yl)ethoxy)propanamido)-3-methylbutananudo)-5-ureidopentanamido)benzyl)-4-(2-(4-(4((R)-l-carboxy-2-(2-((2-(2-methoxyphenyI)pyirimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fIuorophenyl)thieno[2,3-d]pyrimidin-5-yH)-2-chloro-3-methylphenoxy)ethyl)-lmethylpiperazin-1-ium (L8-P1)

Following GENERAL PROCEDURE 3 with 1 -(2-(((1-(2,5,8,11,14,17,20,23octaoxapentacosan-25-yl)-lH-I,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-lcarboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyi)thieno[2,3-d]pyrimidin-5-yl)-2-chlo[O-3-methylphenoxy)ethyl)-I-methylpiperazin1-ium (20 mg, 0.010 mmol, 1.0 equiv.), l-(2-(((l-(2,5,8,ll,l4,17,20,23-octaoxapentacosan-25yl)-lH-l,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-IHpyrrol-l-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloiO-3-methylphenoxy)ethyl)-l -methylpiperazin1-ium (L8-P1) was obtained . HRMS; M+= 1908.8097, Rt=2.37 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-1-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy )-6-(4-fluorophenyI)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-IH-pyrrol-Iyl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-lyloxy)methyl)benzyl)-l-methylpiperazin-1-ium

451

Following GENERAL PROCEDURE 3 with l-(4-((S)-2-((S)-2-((tertbutoxycarbonyl)ainino)-3-methyibutanamido)-5-ureidopentanamido)-2-((prop-2-yn-lyloxy)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fhioiOphenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan-2-yl)0xy)thieno[2,3d]pyrimidin-5-y!)-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium (123.1 mg, 0.075 mmol, 1.0 equiv.), 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yI)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-l H-pyrrol-1yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-lyloxy)methyl)benzyl)-l-methylpiperazin-I-ium was obtained. HRMS: M+= 1499.5601, Rt=2.50 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yI)methoxy)phenyl)ethoxy)-6-(4-fIuorophenyl)thieno(2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyI)-l-(2-(((l-(26-carboxy-3,6,9,12,15,18,21,24-octaoxahexacosyl)-lHl,2,3-triazoI-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrroll-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-l-

Following GENERAL PROCEDURE 2 with 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5

452 dihydro-lH-pyrrol-l-yl)ethoxy)propanainido)-3-methylbutanamido)-5-ureidopentanamido)-2((prop-2-yn-l-yloxy)methyl)benzyl)-l-methylpiperazin-l-ium (40 mg, 0.027 mmoles, l.O equiv) and l-azido-3,6,9,I2,l5,l8,2l,24-octaoxaheptacosan-27-oic acid (37.4 mg, 0.080 mmoles, 3.0 equiv.), 4-(2-(4-(4-((R)-1 -carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidm-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-(2-(((l-(26-carboxy-3,6,9,l2,15,l8,2l,24-octaoxahexacosyl)-lH-l,2,3triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pynOl-lyl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-lmethylpiperazin-l-ium (L7-P1) was obtained. HRMS: M+=l965.560l, Rt=2.35 min (5 min acidic method).

Synthesis ofl-(2-(((l-(2,5,8,ll,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71tetracosaoxatriheptacontan-73-yl)-lH-l,2,3-triazol-4-yl)methoxy)methyi)-4-((S)-2-((S)-2-(3(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethoxy)propanamido)-3-methylbutanamido)-5ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidîn-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-inethylphenoxy)ethyl)-l-methyIpiperazin-l-ium (L5-P1)

N Ή

Following GENERAL PROCEDURE 2 with 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d] pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl )-1-(4-(( S )-2-((S )-2-(3-(2-(2,5-dioxo-2,5dihydro-lH-pynOl-l-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2((prop-2-yn-l-yloxy)methyI)benzyl)-l-methylpiperazin-l-ium (20.4 mg, 0.014 mmoles, 1.0 equiv) and 73-azido-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71

453 tetracosaoxatriheptacontane (28.1 mg, 0.025 mmoles, 1.5 equiv.), 1-(2-(((1(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71tetracosaoxatriheptacontan-73-yl)-lH-l,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2(2,5-dioxo-2,5-dîhydro-lH-pyrrol-l -yl)ethoxy)propanamido)-3-methylbutanamido)-5ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidm-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-methylpiperazin-l-ium (L5-P1) was obtained. HRMS: M+=2613.2100, Rt=2.44 min (5 min acidic method).

Synthesis of prop-2-yn-l-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-((((4nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate

A solution of prop-2-yn-l-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-(hydroxymethyl)benzyl)(methyl)carbamate (249 mg, 0.412 mmoles) and 4-nitrophenyl (4-nitrosophenyl) carbonate (356 mg, 1.24 mmoles, 3.0 equiv.) in DMF (2 mL) was swirled until homogeneous and sat for 16 hours. The solution was diluted with DMSO (6 mL) and was purified by RP-HPLC ISCO gold chromatography ( 1 ΟΙ 00% MeCN/H2O, no modifier). Upon lyophilization, prop-2-yn-l-yl (5-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanainido)-2-((((4nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate was obtained. LC/MS MH+=770.7, Rt=2.45 min (5 min acidic method).

Synthesis of prop-2-yn-l-yl (5-((R)-2-((R)-2-((tert-butoxycarbonyI)amino)-3methylbutanamido)-5-ureidopentanamido)-2-(((methyl(2(methylamino)ethyl) car bamoyi)oxy)methyl)benzyl)(niethyl)carba mate

454

To a solution of prop-2-yn-l-yl (5-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-((((4nitrophenoxy)carbonyl)oxy)methyl)benzyl)(methyl)carbamate (lOOmg, 0.130 mmol) in DMF (l ml) was added N,N’-Dimethyl-ethy1 en edi amine (22.90 mg, 0.260 mmol), followed by the addition of DIPEA (0.113 ml, 0.650 mmol) at room température. The resulting solution was stirred at room température overnight. The reaction was diluted with DMSO was purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H₂O, 0.1 % NH4OH modifier). Upon lyophilization prop-2-yn-1 -yl (5-((R)-2-((R)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-(((methyl(2(methylamino)ethyl)carbamoyl)oxy)methyl)benzyl)(methyl)carbamate was obtaîned. LCMS: MH+=719.9, Rt=0.73 min (2 min acidic method).

Synthesis of (R)-4-(2-(2-chloro-4-(6-(4-iluoro-3-hydroxyphenyl)-4-((l-methoxy-3-(2-((2-(2methoxypheny l)pyrimi din-4-yl)methoxy) phenyl)-l-oxopropan-2-y l)oxy)thieno [2,3d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methyl-l-(3-sulfopropyl)piperazin-l-ium

To a solution of (R)-4-(2-(4-(4-(l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluoro-3-hydroxyphenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro3-methylphenoxy)ethyl)-1-methyl-1-(3-sulfopropyl)piperazin-l-ium or (2Æ)-2-[(5S«)-5-[3chloro-2-methyl-4-[2-[4-methyl-4-(3-suIfopropyl)pîperazîn-4-ium-l-yl]ethoxy]phenyl]-6-(4fluoro-3-hydroxy-phenyl)thieno[2,3-</]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (100 mg, 0.099 mmoles) in

455

MeOH (1.5 mL) was added a few drops of HsSOAconc.). After stirring ovemight, the MeOH was removed in vacuo, the residue was dissolved in DMSO was purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization, (R)-4(2-(2-chIoro-4-(6-(4-fluoro-3-hydroxyphenyI)-4-((l-methoxy-3-(2-((2-(2methoxyphenyi)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin5-yl)-3-methylphenoxy)ethyl)-l-methyl-l-(3-sulfopropyl)piperazm-l-ium was obtained. HRMS M+= 1027.2900, Rt=2.31 min (5 min acidic method).

Synthesis of 4-(2-(4-(6-(3-(((2-((( (4-((S)-2-((S)-2-((tert-butoxycarbonyI)ammo)-3methylbutanamido)-5-ureÎdopentanamido)-2-((niethyl((prop-2-yn-lyloxy)carbonyl)amino)niethyl)benzyl)oxy)carbonyl)(methyl)aniino)ethyl)(methyl)carbamo yl)oxy)-4-fluorophenyl)-4-(((R)-l-methoxy-3-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)-l-oxopropan-2-yi)oxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3 methylphenoxy)ethyl)-l-niethyl-l-(3-sulfopropyl)piperazin-l-ium

To a solution of (R)-4-(2-(2-chloro-4-(6~(4-fluoro-3-hydiOxyphenyl)-4-((l-methoxy-3(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyI)-l-oxopropan-2-yl)oxy)thieno[2,3d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methyl-l-(3-sulfopropyl)piperazin-l-ium (50 mg, 0.049 mmoles, 1.0 equiv) in CH2C12 (1 mL) at 0 °C was added TEA (34 uL, 0.243 mmoles, 5.0 equiv.) followed by 4-Nitrophenyl chloroformate (10.8 mg, 0.054 mmoles, 1.1 equiv.). After stirring for 15 min, a solution ofprop-2-yn-l-yl (5-((R)-2-((R)-2-((tert-butoxycarbonyl)amino)3-methylbuianamido)-5-ureidopentanamido)-2-(((methyl(2(methylamino)ethyl)carbamoyl)oxy)methyl)benzyl)(methyl) carbamate (66.3 mg, 0.092 mmoles, 2.0 equiv) in DMF (1 mL) was added followed by DIEA (40 uL, 0.231 mmoles, 5.0 equiv.). After stirring for 2 h, the volatiles were removed in vacuo, the solution was diluted with DMSO (3 ml) and was purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization 4-(2-(4-(6-(3-(((2-((((4-((S)-2-((S)-2-((tert

456 butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2-yn-l yloxy)carbonyl)amîno)methyl)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)ox y)-4-fl uoropheny 1)-4-(( (R)-l-rnethoxy-3-(2-((2-(2-inethoxyphenyl)pyrimidin-4yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloiO-3methylphenoxy)ethyl)-l-methyl-l-(3-sulfopropyl)piperazin-I-ium was obtained. HRMS

M+=l 771.6700, Rt=2.57 min (5 min acidic method).

Synthesis of 4-(2-(4-(6-(3-(((2-((((4-((S)-2-((S)-2-((tert-butoxycarhonyl)amino)-3methylbutananiido)~5-ureidopentanamido)-2-((methyl((prop-2-yn-lyloxy)carbonyi)amino)methyi)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyi)carbamo yl)oxy)-4-fluorophenyI)-4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)thieno|2,3-d]pyriniidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)l-methyl-l-(3-sulfopropyl)piperazin-l-ium

NHBoc

O N H,

To a solution of 4-(2-(4-(6-(3-(((2-((((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methyIbutanamido)-5-ureidopentanamido)-2-((methyI((prop-2-yn-lyloxy)carbonyl)amino)m ethyl )benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)ox y)-4-fluorophenyl)-4-(((R)-l-methoxy-3-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-methyl-l-(3-sulfopiOpyl)piperazin-l-ium (23 mg, 0.013 mmoles, 1.0 equiv) in THF (1 mL) was added 2N LÎOH (0.032 mL, 0.065 mmoles, 5 equiv). After stimng for 2 h, the solution was neutralized with AcOH and purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H2O, 0.1% TFA modifier). Upon lyophîlization, 4-(2-(4-(6(3-(((2-((((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5ureidopentanamido)-2-((methyl((prop-2-yn-l yloxy)carbonyl)amino)methyl)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)ox y)-4-fluorophenyl)-4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4

457 yl)methoxy)phenyl)ethoxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-lmethyl-l-(3-sulfopropyi)piperazin-l-ium was obtained. HRMS M+=l 757.6200, Rt=2.46 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(3-(((2-((((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lHpyrrol-l-yl)ethoxy)propanamido)-3-niethylbiitanamido)-5-ureidopentanamido)-2((methyl((prop-2-yn-lyloxy)carbonyl)amino)niethyl)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbanio yl)oxy)-4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chIoro-3-methylphenoxy)ethyl)-lmethyl-l-(3-sulfopropyI)piperazin-l-ium o nh₂

Following GENERAL PROCEDURE 3 with 4-(2-(4-(6-(3-(((2-((((4-((S)-2-((S)-2((tert-butoxycarbonyl)ammo)-3-methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2yn-lyloxy)carbonyl) ami no)methyl)benzyl)oxy)carbonyl) (methyl )amino)ethyl)(methyl)carbamoyl)ox y)-4-fluoiOphenyl)-4-((R)-l-carboxy-2-(2-((2-(2-inethoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-lmethyl-l-(3-sulfopropyl)piperazîn-l-ium (17 mg, 0.0097 mmol, LO equiv.), 4-(2-(4-(4-((R)-lcarboxy-2-(2-((2-(2-methoxyphenyI)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(3-(((2-((((4((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethoxy)propanamido)-3methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2-yn-lyloxy)carbonyl)amino)meth yl)benzyl)oxy)carbonyl)(m ethyl) amino)ethyl )(m ethyl )carbamoyl)ox y)-4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methyl-l(3-sulfopropyl)piperazin-l-ium was obtained. HRMS: M+= 1852,5200, Rt=2.29 min (5 min acidic method).

458

Synthesis of 4-(2-(4-(6-(3-(((2-((((2-(((((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)1 H-l ,2,3-triazol-4-y])nicthoxy)carbonyl)(inethyl)amino)methyI)-4-((S)-2-((S)-2-(3-(2-(2,5dioxo-2,5-dihydro-1 H-pyrrol- l-yl)ethoxy)pr opan amido)-3- methy Ibu tan a mido)-5ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(niethyl)carbanioyl)oxy)-4fluorophenyl)-4-((R)-l-carboxy-2-(2-((2-(2-niethoxyphenyl)pyriniidin-4yl)niethoxy)phenyl)ethoxy)thieno|2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)l-niethyl-l-(3-sulfopropyl)piperazin-l-iiim (L12-P2)

Following GENERAL PROCEDURE 2 with 4-(2-(4-(4-((R)-l -carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(3-(((2-((((4-((S)-2-((S)-2-(3-(2-(2,5dioxo-2,5-dihydro-l H-pyrrol-l-yl)ethoxy)propanamido)-3-methylbutanamido)-5ureidopentanamido)-2-((methyl((prop-2-yn-1 yloxy)carbonyl)ainino)methyl)benzyl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)ox y)-4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methyl-l(3-sulfopropyl)piperazin-l-ium (10 mg, 0.0054 mmoles, 1.0 equiv.) and 25-azîdo2,5,8,11,14,17,20,23-octaoxapentacosane (4.4 mg, 0.011 mmoles, 2 equiv.), 4-(2-(4-(6-(3-(((2((((2-((((( 1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yI)-l H-1,2,3-triazol-4yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dîhydro-lHpyrrol-l-yl)ethoxy)propanainido)-3-methylbutanamido)-5urei dopentanami do )benzyl)oxy)carbonyl)(m ethyl )amino)ethyl)(m ethyl )carbamoyl)oxy)-4fluorophenyl)-4-((R)-I-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-lmethyl-l-(3-sulfopropyl)piperazin-l-îum (L12-P2 ) was obtained . HRMS: M+=2261.8601, Rt=2.24 min (5 min acidic method).

459

Synthesis of 4-(2-(4-(6-(3-((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopcntanamido)-2-((niethyl((prop-2-yn-lyloxy)carbonyl)amino)mcthyl)benzyl)oxy)-4-fluorophenyl)-4-((R)-l-carboxy-2-(2-((2-{2niethoxyphenyl)pyrimidin-4-yI)methoxy)phenyi)ethoxy)thieno]2,3-d]pyrimidin-5-yl)-2chloro-3-methyIphenoxy)ethyl)-l-methyl-l-(3-suIfopropyl)piperazin-l-ium

GENERAL PROCEDURE l was followed using (R)-4-(2-(2-chloro-4-(6-(4-fluoro-3hydroxyphenyl)-4-((l-methoxy-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-loxopropan-2-yl)oxy)thieno[2,3-d]pyriinidin-5-yl)-3-methylphenoxy)ethyl)-1 -methyl-1 -(3sulfopropyl)piperazin-l-ium (40 mg, 0.039 mmol, l.O equiv.) and prop-2-yn-l-yl (5-((R)-2-((R)2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2(chloromethyl)benzyl)(methyl)carbamate (36.4 mg, 0.058 mmol, l .5 equiv.), with the modification of after the alkylation was complété adding 2N LiOH (0.097 mL, 0.195 mmoles, 5.0 equiv.) and stirring for 2 h prior to neutralizing and purifying by RP-HPLC. Upon lyophilization, 4-(2-(4-(6-(3-((4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-((melhyl((prop-2-yn-lyloxy)carbonyl)amino)methyl)benzyl)oxy)-4-fluoiOphenyl)-4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloro3-methylphenoxy)ethyl)-l-methyl-l-(3-sulfopropyl)piperazin-l-ium was obtained. HRMS: M+=1599.5856, Rt=l.34 min (2 min acidic method).

460

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-niethoxyphcnyl)pyrimidm-4yl)methoxy)phenyl)ethoxy)-6-(3-((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)ethoxy)propanamido)-3-methylbutanamido)-5-ureîdopentanamido)-2-((methyl((prop-2yn-l-yloxy)carbonyI)amino)methyl)benzyl)oxy)-4-fluorophenyl)thieno[2,3-d]pyrimidin-5yl)-2-chloro-3-methyiphenoxy)ethyl)-l-methyl-l-(3-sulfopropyI)piperazin-l-ium so₃h

Foilowing GENERAL PROCEDURE 3 with 4-(2-(4-(6-(3-((4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamîdo)-5-ureidopentanamido)-2-((methyl((prop-2-yn-lyloxy)carbonyl)amino)methyl)benzyl)oxy)-4-fluorophenyl)-4-((R)-l-carboxy-2-(2-((2-(2niethoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloro3-methylphenoxy)ethyl)-1 -methyl-l-(3-sulfopropyl)piperazin-l -ium (46 mg, 0.029 mmol, 1.0 equiv.), 4-(2-(4-(4-((R)-1 -carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(3-((4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyirol-lyl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((methyl((prop-2-yn-lyloxy)carbonyl)amino)methyl)benzyl)oxy)-4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2chloro-3-methylphenoxy)ethyI)-l-methyl-l-(3-sulfopropyl)pîperazin-l-ium was obtained.

HRMS: M+= 1694.5699, Rt=2.55 min (5 min acidic method).

Synthesis of 4-(2-(4-(6-(3-((2-(((((1-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)-lH-l,2,3trÎazol-4-yi)methoxy)car b onyl)(methyl)amino) methyI)-4-((S)-2-((S )-2-(3-(2-(2,5-dioxo-2,5dihydro-lH-pyrroI-l-yl)ethoxy)propanamido)-3-methylbutanamido)-5ureidopentanamido)benzyl)oxy)-4-flnorophenyl)-4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)thieno[2,3-d]pyrimidin-5-yl)-2chloro-3-methylphenoxy)ethyl)-l-methyl-l-(3-sulfopropyl)piperazin-l-ium (L4-P2)

461

Following GENERAL PROCEDURE 2 with 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(3-((4-((S )-2-(( S )-2-(3-(2-(2,5-dioxo2,5-dihydro-IH-pyrrol-l-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)2-((methyl((prop-2-yn-l-yloxy)carbonyl)amino)methyl)benzyl)oxy)-4-fluorophenyl)thieno[2,3d]pyriinidin-5-yI)-2-chloro-3-methylphenoxy)ethyl)-1 -methyl-1 -(3-sulfopropyl)piperazin-l -ium (44 mg, 0.026 mmoles, l .0 equiv.) and 25-azido-2,5,8,11,14,17,20,23-octaoxapentacosane (21.2 mg, 0.052 mmoles, 2 equiv.), 4-(2-(4-(6-(3-((2-(((((1-(2,5,8,11,14,17,20,23-octaoxapentacosan25-yI)-lH-l,2,3-triazol-4-yl)methoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2(2,5-dioxo-2,5-dihydro-l H-pyrrol-l -yl)ethoxy)propanamido)-3-methylbutanamido)-5ureidopentanamido)benzyl)oxy)-4-fluorophenyl)-4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)thieno[2,3-d]pyrimidin-5-yl)-2-chloro3-methylphenoxy)ethyl)-l-methyl-l-(3-sulfopropyl)piperazin-l-ium (L4-P2) was obtained. HRMS: M+=2103.8000, Rt=2.47 min (5 min acidic method).

GENERAL PROCEDURE 4:

Synthesis of l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5ureidopentanamÎdo)-2-(39-methyl-38-oxo-2,5,8,ll,14,17,20,23,26,29,32,35-dodccaoxa-39azatetracontan-40-yl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-I-((4methoxy benzyl)oxy )-3-(2-( (2-(2-methoxyphenyl)pyrimidin-4-yI) methoxy) phenyl)-1oxopropan-2-yI)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-lmethylpiperazin-l-ium

462

To l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5ureidopentanamido)-2-((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)inethoxy)phenyl)-loxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-1ium (60 mg, 0.040 mmol, l.Ü equiv.) and 2,5-dioxopyrrolidin-l-yl

2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oate (35.7 mg, 0.052 mmoles, 1.3 equiv.) dissolved in DMF (1 mL) was added DIPEA (0.035 mL, 0.200 mmoles, 5.0 equiv.).

After standing for 18 h, DMSO (2 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-70% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization, 1(4-((S)-2-((S)-2-((tert-buioxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2(39-methyl-38-oxo-2,5,8,l l,14,17,20,23,26,29,32,35-dodecaoxa-39-azatetracontan-40yl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin5-yl)-3-methylphenoxy)ethyl)-l -methylpiperazin- 1-ium was obtained. HRMS: [M+N a]+=2092.9399; Rt=2.88 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphcnyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyI)-1-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)ethoxy)propanamido)-3-methylbutananiido)-5-ureidopentanamido)-2-(39-methyl-38oxo-2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxa-39-azatetracontan-40-yI)benzyl)-lmethylpiperazin-1-ium (L32-P1)

463

Following GENERAL PROCEDURE 3 with 1 -(4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(39-methyl-38-oxo2,5,8,1 l,14,I7,20,23,26,29,32,35-dodecaoxa-39-azatetracontan-40-yl)benzyI)-4-(2-(2-chloro-4(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3methylphenoxy)ethyl)-l-methylpiperazin-l-ium (43.4 mg, 0.021 mmol, 1 eq), 4-(2-(4-(4-((R)-lcarboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluoiOphenyl)thieno[2,3-d]pyrimidin-5-yi)-2-chloiO-3-inethylphenoxy)ethyl)-l-(4-((S)-2-((S)-2(3-(2-(2,5-dioxo-2,5-dihydro-lH-pynOl-l-yi)ethoxy)propanamido)-3-mcthylbutanamido)-5ureidopentanamido)-2-(39-methyl-38-oxo-2,5,8,l 1,14,17,20,23,26,29,32,35-dodecaoxa-3 9azatetracontan-40-yl)ben2yl)-l-methylpiperazin-l-ium was obtained. HRMS: [M+Na]+= 2066.8799; Rt=2.44 min (5 min acidic method).

Synthesis of l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5ureidopentanamido)-2-(51-methyl-50-oxo-2,5,8,ll,14,17,20,23,26,29,32,35,38,41,44,47hexadecaoxa-51-azadopentacontan-52-yI)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thicno[2,3-d]pyrimidin-5-yl)-3methylphenoxy)ethyl)-1 -methylpiper azin- 1-iu m

464

Following GENERAL PROCEDURE 4 with 2,5-dioxopyrrolidin-l-yl

2,5,8,11,14,17,20,23,26,29,32,3 5,38,41,44,47-hexadecaoxapentacontan-50-oate (44.8 mg, 0.021 mmol, 1 eq), 1-(4-( (S )-2-(( S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-55 ureidopentanamîdo)-2-(5 l-methyl-50-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47hexadecaoxa-51-azadopentacontan-52-yl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)1 -((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1 oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yI)-3-methylphenoxy)ethyl)-l-methylpiperazin-lium was obtaîned. HRMS: M+= 2246.0400; Rt=2.88 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(51-methyl-5015 oxo-2,5,8,1 l,14,17,20,23,26,29,32,35,38,41,44,47-hexadecaoxa-51-azadopentacontan-52yl)benzyl)-l-methylpiperazin-l-ium (L31-P1)

465

Following GENERAL PROCEDURE 3 with l-(4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(5l-methyl-50-oxo2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47-hexadecaoxa-5l-azadopentacontan-52-yl)benzyI)4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2methoxyphenyi)pyrimidin-4-yI)methoxy)phenyl)- l-oxopropan-2-yI)oxy)thieno[2,3-d]pyrimidin5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium (47,5 mg, 0.021 mmol, I eq), 4-(2-(4-(4((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(4-((S)-2-((S)-2(3-(2-(2,5-dioxo-2,5-dihydro-l H-pyrrol-l-yl)ethoxy)propanamido)-3-methylbutanamido)-5ureidopentanamido)-2-(51 -methyl-50-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47hexadecaoxa-51 -azadopentacontan-52-yl)benzyl)-1 -methylpiperazin-1 -ium was obtained.

HRMS: M+= 2221.0000; Rt=2.45 min (5 min acidic method).

Syntheis of l-(4-((S)-2-((S)-2-((tert-butoxycarbonyI)amino)-3-methylbutanamido)-5ureidopentanamido)-2-(75-methyl-74-oxo2,5,8,U,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75azahexaheptacontan-76-yl)benzyl)-4-(2-(2-chIoro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4meth oxybenzyl)oxy)-3-(2-((2-(2-methoxy phenyl) pyrimidin-4-yl)methoxy)phenyl)-loxopropan-2-yl)oxy)thieno[2,3-d]pyrimidm-5-yl)-3-methylphenoxy)ethyl)-lmethylpiperazin-l-iuni

466

Following GENERAL PROCEDURE 4 with 2,5-dîoxopyrrolidin-l-yl

2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71tetracosaoxatetraheptacontan-74-oate (52.6 mg, 0.043 mmol, 1.3 eq), l-(4-((S)-2-((S)-2-((tert5 butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(75-methyl-74-oxo2,5,8,ll,14,l7,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75azahexaheptacontan-76-yl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium was obtained. HRMS: M+= 2598.2500; Rt=2.88 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno|2,3-d]pyrimidin-5-yI)-2-chloro-3methylphenoxy)ethyl)-l-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)ethoxy)propananiido)-3-niethylbutanamido)-5-ureidopentanamîdo)-2-(75-methyl-74oxo-2,5,8,1 l,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75

azahexahept acontan-76yl) benzyl)-1methylpiper azin-l-iuni (L30-PI)

Following GENERAL PROCEDURE 3 with l-(4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(75-methyl-74-oxo2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71-tetracosaoxa-75azahexaheptacontan-76-yl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l -oxopropan2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium (38,8 mg, 0.014 mmol, 1 eq), 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimîdin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(75-methyl-74-oxo2,5,8,ll,14,17,20,23,26,29,32,35,38,41,44,47,50,53_f56,59,62,65,68,71-tetracosaoxa-75azahexaheptacontan-76-yl)benzyl)-l-methylpiperazin-l-ium was obtained. HRMS: M+= 2573.2000; Rt=2.47 min (5 min acidic method).

Synthesis of l-(2-(((S)-2-((((9II-fluoren-9-yI)methoxy)carbonyl)amino)-5-(tertbutoxy)-N-methyl-5-oxopentanamido)methyl)-4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methyIbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2ehloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyI)-l-oxopropan-2-yI)oxy)thieno[2,3d]pyrimidin-5-yl)-3-methyIphenoxy)ethyl)-l-methylpiperazin-l-ium

468

Following GENERAL PROCEDURE 4 with 5-(tert-butyl) l-(2,5-dioxopyrroIidin-l-yI) (((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamate (39.0 mg, 0.075 mmol, 1.1 equiv.), l-(2(((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-N-methyl-5oxopentanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methyIbutanamido)-5ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyi)-l-oxopropan2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazm-l-ium was obtained. HRMS: M+= 1906.8101; Rt=3.03 min (5 min acidic method).

Synthesis of l-(2-(((S)-2-amino-5-(tert-butoxy)-N-methyl-5-oxopentanamido)methyI)-4((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5ureidopentananiido)benzyl)-4-(2-(2-chIoro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyi)pyrimidin-4-yl)methoxy)phenyl)-loxopropan-2-yI)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-

To 11 -(2-(((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-Nmethyl-5-oxopentanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanami do)-5-ureidopentanamido)benzyi)-4-(2-(2-chloro-4-(6-(4-fluoroph en yl)-4-(( (R)l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyriinidin-4-yl)methoxy)phenyl)-loxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methy!piperazin-lium (38.8 mg, 0.026 mmoles, 1.0 equiv.) dissolved in DMSO (2 mL) was added dimethylamine (0.192 mL, 0.384 mmoles, 20 equiv.). After standing for 4 hr, DMSO (2 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-70% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization, l-(2-(((S)-2-amino-5-(tert-butoxy)-N-methy1-5

469 oxopentanamido)methyI)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methyIbutanamido)-5ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxoprûpan2-y[)oxy)thieno[2,3-d]pyriinidin-5-yi)-3-methylphenoxy)ethyl)-l-methylpiperazin-l -ium was obtained. HRMS: M+=l684.4000; Rt=2.64 min (5 min acidic method).

GENERAL PROCEDURE 5

Synthesis of l-(2-(((R)-2-((((9H-fluoren-9-yl)methoxy)carbonyI)amino)-N-methyI-3sulfopropanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyI)amino)-3methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4y i) methoxy) ph enyl)-1 -oxopr opan-2-yl)oxy)thieno [ 2,3-d ] pyrimidin-5-yl)-3methylphenoxy)ethyi)-l-methylpiperazin-l-ium

NHFmoc

NHBoc

To (((9H-fluoren-9-yl)methoxy)carbonyl)(suIfo)-D-alanine (55.2 mg, 0.141 mmol, 1.3 eq) and 2-(3H-[ 1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouronium hexafluorophosphate(V) (41.3 mg, 0.109 mmoles, 1.0 equiv.) dissolved în DMF (2 mL) was added DIPEA (0.024 mL, 0.138 mmoles, 8.0 equiv.). After standing for 10 min, I-(4-((S)-2((S)-2-((tert-butoxycarbonyI)ainino)-3-methylbutanamido)-5-ureidopentanamido)-2((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyph en yl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium (200 mg, 0.109 mmol, 1.0 eq) was added. After standing for 2.5 hr, DMSO (4 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-70% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization, l-(2-(((R)-2-((((9H-fluoren-9470 yI)methoxy)carbonyl)amino)-N-methyl-3-sulfopropanamido)methyl)-4-((S)-2-((S)-2-((tertbutoxycarbonyl)aminû)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimîdin-5-yl)-3methylphcnoxy)eihyl)-l-methylpiperazin-l-ium was obtaîned. HRMS: M+=l872.7000; Rt=3.09 min (5 min acidic method).

Synthesis of l-(2-(((R)-2-amino-N-methyl-3-sulfopropanamido)methyl)-4-((S)-2-((S)2-((tert-butoxycarbonyl)aniino)-3-methyibutanamido)-5-ureÎdopentanamido)benzyI)-4-(2(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2methoxyphenyl)pyriniidin-4-yl)methoxy)phenyi)-l-oxopropan-2-yl)oxy)thieno[2,3d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium

N 'N

NHBoc

To l-(2-(((R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-N-methyl-3sulfopropanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)5-ureidopentanamido)benzyl)-4-(2-(2-chIoro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyI)pyrimidîn-4-yl)methoxy)phenyl)-l-oxopropan2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l -methylpiperazin-1 -ium ( 173 mg, 0.087 mmoles, 1.0 equiv.) dissolved in THF (2 mL) was added dimethylamine (0.870 mL, 1.740 mmoles, 20 equiv.). After standing for 5 hr, ail volatiles were removed in-vacuo. The solid was triturated with diethyl ether. DMSO (2 mL) was added and the solution was purified by RP-HPLC ISCO goto chromatography ( 10-100% MeCN/H2O, 0. i % TFA modifier). Upon lyophilization, l-(2-(((R)-2-amino-N-methyl-3-sulfopropanamîdo)methyl)-4-((S)-2-((S)-2-((tertbutoxycarbonyI)amîno)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3471 methylphenoxy)ethyl)-l-methylpiperazin-l-ium was obtained. HRMS: M+=l 650.5800;

Rt=2.7l min (5 min acidic method).

Synthesis of l-(2-(((R)-2-amino-N-methyl-3-suIfopropanamido)methyl)-4-((S)-2-((S)2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethoxy)propanamido)-3-mcthylbutanamido)5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2m ethoxy phenyl) pyrimidin-4-yl)methoxy)phenyI)ethoxy )-6-(4-fluor ophenyl)thieno [2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium (L70-PI)

N N

Following GENERAL PROCEDURE 3 with l-(2-(((S)-2-amino-5-(tert-butoxy)-NmeÎhyl-5-oxopentanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)benzyl )-4-(2-(2-chloro-4-(6-(4-fluoroph en yl)-4-(((R)l-((4-methoxybenzyI)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-loxopropan-2-yI)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-lium (145 mg, 0.088 mmol, l eq), l-(2-(((R)-2-amino-N-methyl-3-sulfopropanamido)methyl)-4((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yI)ethoxy)propanamido)-3methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyriinidin-4-yl)rnethoxy)phenyl)ethoxy)-6-(4-fluorOphenyl)thicno[2,3d]pyrimidin-5-yl)-2-chIoro-3-methylphenoxy)ethyl)-l -methylpiperazin-l-ium was obtained. HRMS: M+= 1625.5601; Rt=2.32 min (5 min acidic method).

GENERAL PROCEDURE 6

Synthesis of l-(2-(((S)-5-(tert-butoxy)-N-methyL5-oxo-2-(2sulfoacetamido)pentananiido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methy lbutanamido)-5-ureidopentanamido)benzy 1)-4-(2-(2-diloro-4-(6-(4-fluorophenyl)-4472 (((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno{2,3-d]pyriniidin-5-yl)-3methylphenoxy)ethyl)-l-methylpiperazin-1-ium

To 2-sulfoacetic acid (4.83 mg, 0.035 mmol, 2.0 equiv.) and 2-(3H-[I,2,3]triazolo[4,5b]pyridin-3-yl)-l,l,3,3-tetramethylisouronium hexafluorophosphate(V) (9.85 mg, 0.026 mmoies, 1.5 equiv.) dissolved in DMF (1 mL) was added DIPEA (0.024 mL, 0.138 mmoles, 8.0 equiv,). After standing for 10 min, l-(2-(((S)-2-amino-5-(tert-butoxy)-N-methyl-5oxopentanamido)meihyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyI)pyrimidin-4-yl)methoxy)phenyl)-i-oxopiOpan2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l -methylpiperazin-1 -ium (29.1 mg, 0.017 mmoles, 1.0) was added. After standing for 45 min, DMSO (2 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-70% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization, l-(2-(((S)-5-(tert-butoxy)-N-methyl-5-oxo-2-(2sulfoacetamido)pentanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyI)-loxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-1ium was obtained. HRMS: M+=l 806.7000; Rt=3.10 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yI)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methyIphenoxy)ethyl)-l-(2-(((S)-4-carboxy-N-methyi-2-(2473 sulfoacetamido)butanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dîoxo-2,5-dihydro-lHpyrrol-l-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-l-

Following GENERAL PROCEDURE 3 with l-(2-(((S)-5-(tert-butoxy)-N-methyl-5oxo-2-(2-sulfoacetamido)pentanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutatiamido)-5-ureidopentanamîdo)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-loxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-lium (23.7 mg, 0.01 î mmol, l eq), 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphen yl)pyrimidi n-4-yl)m ethoxy )phenyl)ethox y)-6-(4-fluorophenyl)thîeno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(2-(((S)-4-carboxy-N-methyi-2-(2sulfoacetamido)butanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-diüxo-2,5-dihydro-1 H-pyrrol-1 yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-l methylpiperazin-l-ium was obtained. FIRMS: M+= 1725.5900; Rt=2.39 min (5 min acidic method).

Synthesis of l-(2-((S)-40-(3-(tert-butoxy)-3-oxopropyI)-42-methyl-38,41-dioxo2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39,42-diazatritetracontan-43-yl)-4-((S)-2-((S)-2((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2chloro-4-(6-(4-fluorophenyI)-4-(((R)-l-((4-methoxybenzyl)oxy )-3-(2-((2-(2methoxyphenyl)pyrinûdin-4-yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno|2,3d]pyrimÎdin-5-yi)-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium

474

Foilowing GENERAL PROCEDURE 4 with l-(2-(((S)-2-amino-5-(tert-butoxy)-Nmethyl-5-oxopentanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methyl butanami do )- 5 -ureido pentanamido)benzyl) -4~(2 -(2-chloro -4-(6-(4-fluoro phenyl )-4-(( (R)l-((4-mcthoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidm-4-yl)methoxy)phenyl)-loxopropan-2-yl)oxy)thieno[2,3-d]pyrîmidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-!ium (30 mg, 0.018 mmol, LO eq) and Mal-PEG12-NHS Ester (18.3 1 mg, 0.027 mmol, 1.5 eq), l-(2-((S)-40-(3-(tert-butoxy)-3-oxopropyl)-42~methyl-38,41-dioxo2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxa-39,42-diazatritetracontan-43-yl)-4-((S)-2-((S)-2((tert-butoxycarbonyl)ami no)-3-methylbutanamido)-5-ureidopentanamido)benzyl )-4-(2-(2chioro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin5-yl)-3-methylphenoxy)ethyl)-I-methylpiperazin- 1-ium was obtained. HRMS: M+= 2255.0400; Rt=2.97 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-niethoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy )-6-(4-fluorophenyi)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyI)-l-(2-((S)-40-(2-carboxyethyi)-42-methyl-38,41-dioxo2,5,8,ll,14,17,20,23,26,29,32,35-dodccaoxa-39,42-diazatritetracontan-43-yI)-4-((S)-2-((S)-2(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethoxy)propananiido)-3-methylbutanainido)-5ureidopentanamido)benzyl)-l-methylpiperazin-l-ium (L72-P1)

475

O,.OH

Following GENERAL PROCEDURE 3 with l-(2-((S)-40-(3-(tert-butoxy)-3oxopropyl)-42-methyl-3 8,4 l-dioxo-2,5,8,1 l,l4,l7,20,23,26,29,32,35-dodecaoxa-39,42diazatritetracontan-43-yl)-4-((S)-2-((S)-2-((tert-buÎoxycarbonyl)amino)-3-methylbutanamido)-5ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4methoxybenzyl)oxy)-3-(2-((2-(2-inethoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1 -methylpiperazin-1 -ium (20.1 mg, 8.91 pmmol, l eq), 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-(2-((S)-40-(2-carboxyethyl)-42-methyl-38,41-dioxo2,5,8,11,14,17,20,23,26,29,32,35~dodecaoxa-39,42-diazatritetracontan-43-yl)-4-((S)-2-((S)-2-(3(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethoxy)propanamido)-3-methylbutanamido)-5ureidopentanamido)benzyl)-l-methylpiperazin-1-ium was obtained. HRMS: [M]+= 2173.9199; Rt=2.40 min (5 min acidic method).

Synthesis of l-(2-((2-(bis(2-(tert-butoxy)-2-oxoethyl)amino)-N methylacetamido)methyI)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentananiido)benzyl)-4-(2-(2-chloiO-4-(6-(4-fluorophenyl)-4(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyi)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3methylphenoxy)ethyl)-l-methylpiperazin-1-ium

476

Following GENERAL PROCEDURE 5 with l-(4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methyibutanamido)~5-ureidopentanamido)-2((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-iluorophenyl)-4-(((R)-1-((45 methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4~yl)methoxy)phenyl)-l-oxopropan2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1 -methylpiperazin-l -ium ( 17.0 mg, 0.011 mmol, 1.0 eq) and bis(2-(tert-butoxy)-2-oxoethyl) glycine (10.97 mg, 0.017 mmol, 1.5 eq), l-(2-((2-(bis(2-(tert-butoxy)-2-oxoethyl)amino)-N-methylacetamido)methyl)-4-((S)-2-((S)2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamidû)benzyl)-4-(2-(210 chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2“((2-(2· methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium was obtained. HRMS: M+= 1784.8000; Rt=3.31 min (5 min acidic method).

Synthesis of l-(2-((2-(bis(carboxymethyl)amino)-N-methylacetamido)methyl)-4-((S)2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethoxy)propanamido)-3methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2niethoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyriniidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium (L67-P1)

477

Following GENERAL PROCEDURE 3 with l-(2-((2-(bis(2-(tert-butoxy)-2oxoethyl)amino)-N-m ethyl acetamido)m ethyl )-4-(( S )-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-niethoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-loxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methyiphenoxy)ethyl)-l-methylpiperazin-lium (9.2 mg, 5.12 pmmol, 1 eq), l-(2-((2-(bis(carboxymethyl)amino)-Nmethylacetamido)methyl )-4-( (S )-2-(( S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-l H-pyrrol-lyl)ethoxy)propanamido)-3-methylbutananiido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-lcarboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl (methoxy )phenyl)ethoxy)-6-(4fluorophenyl )thieno [ 2,3 -d]pyrimidin-5-yl )-2-chloro-3 -m ethyl pheno xy)eîhyl )-1 -methylpip erazîn1-ium was obtained. HRMS: M+= 1647.6100; Rt=2.2S min (5 min acidîc method).

General Procedure 7

Synthesis of l-(2-(((S)-3-amino-4-(tert-butoxy)-N-methyl-4-oxobutanamido)methyl)4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-niethylbutanamido)-5ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4niethoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-loxopropan-2-y l)oxy)thieno 12,3-d] pyrimidin-5-y l)-3-methy lphenoxy)ethyl)-1 methylpiperazin-l-ium

478

PMBO

NHBoc

Reagents were used as DMF stock solution. To (S)-3-((((9H-fluoren-9yl)methoxy)carbonyl)amino)-4-(tert-butoxy)-4-oxobutanoic acid (8.04 mg, 161 pL, 0.020 mmol, 1.2 equiv.) and 2-(3H-[l,2,3]triazoIo[4,5-b]pyridin-3-yl)-l,l,3,3-tetramethylisouromum hexafluorophosphate(V) (6.81 mg, 681 pL, 0.018 mmoles, 1.1 equiv.) was added DIPEA (22.68 pL, 0.130 mmoles, 8.0 equiv.). Aller standing for 10 min, l-(4-((S)-2-((S)-2-((lertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyI)-4-(((R)-1-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-I-ium (30 mg, 353 pL, 0.016 mmoles, 1.0 equiv.) was added. Aller standing for 45 min, dimethyl amine (163 pl, 0.326 mmol) was added. After standing for 16 hours, DMSO (2 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H2O, 0.1% TFA modifier). Upon lyophîlization, l-(2-(((S)-3-amino-4-(tert-butoxy)-N-methyl-4oxobutanamido)methyI)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium was obtained. HRMS: M+=1670.2300; Rt=2.69 min (5 min acidic method).

Synthesis of l-(2-(((S)-4-(tert-butoxy)-N-methyl-4-oxo-3-(2sulfoacetamido)butanamido)methyl)-4-((S)-2-((S )-2-(( tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chIoro-4-(6-(4-fluorophenyl)-4(((R)-1-((4-methoxybenzyl)oxy )-3-(2-((2-(2-methoxy phenyl)py rÎniidin-4yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yI)-3methylphenoxy)ethyl)-l-methylpiperazin-l-ium 479

Following GENERAL PROCEDURE 6 with l-(2-(((S)-3-amino-4-(tert-butoxy)-Nmethy l-4-oxobutanami do )methyl)-4-((S )-2-( (S)-2-((tert-butoxycarbonyl )amino)-3methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)l -((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phcnyl)-1 oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-I-methylpiperazin-lium (20.6 mg, 10.23 μτηοΐ, l eq), l-(2-(((S)-4-(tert-butoxy)-N-methyl-4-oxo-3-(2sulfoacetamido)butanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloiO-4-(6-(4-fluorophenyl)-4-(((R)l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyI)-loxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-lium was obtained. HRMS: M+= 1792.6899; Rt=3.17 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-(2-(((S)-3-carboxy-N-methyl-3-(2suIfoacetamido)propanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lHpyrrol-l-yI)ethoxy)propanamido)-3-methylbutanamido)-5-ureÎdopentananiido)benzyl)-lmethylpiperazin-H-îum (L79-P1)

480

Following GENERAL PROCEDURE 3 with l-(2-(((S)-4-(tert-butoxy)-N-methyl-4oxo-3-(2-sulfoacetamido)butanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanami do)-5-urei do pentanamido)benzyl )-4-(2-(2-chloro-4-(6-(4-11 uoroph en yl)-4-(((R)5 l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-loxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl )-3-methyl phenoxy)ethyl)-l-methylpiperazin-lium (25.3 mg, 0.012 mmol, 1 eq), 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fliiorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(2-(((S)-3-carboxy-N-methyl-3-(210 sulfoacetamido)propanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)“lmethylpiperazin-l-ium was obtained. HRMS: M+= 1711.5699; Rt=2.48 min (5 min acidic method).

Synthesis of4-benzyl l-(tert-butyl) ((3-(tert-butoxy)-3-oxopropoxy)carbonyl)-L-

481

To tert-butyl 3-hydroxypropanoate (111 mg, 0.760 mmol, l equiv.) and bis(4nitrophenyl) carbonate (289 mg, 0.950 mmol, l .2 equiv.) dissolved in DMF (2 mL) was added DIPEA (0.221 mL, 1.267 mmoles, 2.0 equiv.). After standing for I hr, 4-benzyl l-(tert-butyl) Laspartate (200 mg, 0.633 mmol) was added. After standing for 16 hr, DMSO (4 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization, 4-benzyl 1-(tert-butyl) ((3-(tert-butoxy)-3oxopropoxy)carbonyl)-L-aspartate was obtaîned. HRMS: [M+H]+=452.4; Rt=2.68 min (5 min acidic method).

Synthesis of ((S)-4-(tert-butoxy)-3-(((3-(tert-butoxy)-3oxopropoxy)carbonyl)aniino)-4-oxobutanoic acid — O O

M Y^0H

To 4-benzyl 1-(tert-butyl) ((3-(tert-butoxy)-3-oxopropoxy)carbonyl)-L-aspartate (52.7 mg, 0.117 mmol) dissolved in MeOH (2 mL) was added Palladium hydroxide (8.20 mg, 0.012 mmol, 0.1 equiv.). The reaction atmosphère was swtiched to hygrogen. After stîrring for 16 hr, the reaction mixture was fïltered through a celite pad. The filtrate was removed in-vacuo to obtain, 4-benzyl 1-(tert-butyl) ((3-(tert-butoxy)-3-oxopropoxy)carbonyl)-L-aspartate.

Synthesis of l-(2-((S)-5-(tert-butoxycarbonyl)-2,13,13-trimethyI-3,7,ll-trioxo-8,I2dioxa-2,6-diazatetradecyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)“3methylbutanamido)-5-ureidopentanamîdo)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyI)-4(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyriniidin-5-yl)-3methylphenoxy)ethyl)-1-methylpïperazin-l-ium

482

NHBoC

PMBO

Following GENERAL PROCEDURE 7 with l -(4-((S)-2-((S)-2-((tertbutoxycarbonyI)amino)-3-methylbutanamido)-5-ureidopentanamido)-2«methyl amino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluoropheny 1)-4-((( R)-1-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopiOpan2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-1-ium (20 mg, 235 pL, 0.011 mmol, 1 eq) and 4-benzyl 1-(tert-butyl) «3-(tert-butoxy)-3oxopropoxy)carbonyl)-L-aspartate (7.84 mg, 204 pL, 0.022 mmol, 2 equiv.), l-(2-((S)-5-(tertbutoxycarbonyl)-2,13,13-trimethyl-3,7,11 -trioxo-8,12-dioxa-2,6-diazatetradecyl)-4-((S)-2-((S)2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3 -(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-1-ium was obtained. HRMS: M+= 1842.8000; Rt=3.23 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyI)pyriniidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pynmidin-5-yl)-2-chioro-3methylphenoxy)ethyl)-l-(2-(«S)-3-carboxy-3-(((2-carboxyethoxy)carbonyl)amino)-Nmethylpropanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)ethoxy)propanamido)-3-niethylbutanamido)-5“Ureidopentanamido)benzyi)-lmethylpiperazin-l-ium (L102-P1)

483

Following GENERAL PROCEDURE 3 with l-(2-((S)-5-(tert-butoxycarbonyl)-2,13,13trimethyl-3,7,1 l-trioxo-8,12-dioxa-2,6-diazatetradecyl)-4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanami do )benzyl )-4-(2-(2-chloro-4(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3methylphenoxy)ethyl)-I-methylpiperazin-l-ium (17.6 mg, 0.008 mmol, 1 eq), 4-(2-(4-(4-((R)-lcarboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyi‘imidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(2-(((S)-3carboxy-3-(((2-carboxyethoxy)carbonyl)amino)-N-methylpropanamido)methyl)-4-((S)-2-((S)-2(3-(2-(2,5-dioxo-2,5-dihydro-lH-pynOl-l-yl)ethoxy)propanamido)-3-methylbutanamido)-5ureidopentanamido)benzyl)-l-methyl piperazîn-l-îum was obtained. HRMS: M+= 1705.6200; Rt=2.41 min (5 min acidic method).

Synthesis of tert-butyl N-(((9H-tluoren-9-yl)methoxy)carbonyl)-0-((4nitrophenoxy)carbonyi)-L-serinate

NHFmoc

To tert-butyl (((9H-fluoren-9-yI)methoxy)carbonyl)-L-serinate (300 mg, 0.782 mmol, 1 equiv.) and bis(4-nitrophenyl) carbonate (357 mg, 1.174 mmol, 1.5 equiv.) dissolved in DMF (2 mL) was added DIPEA (0.136 mL, 0.782 mmoles, 1.0 equiv.). After standing for 16 hr, DMSO (4 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (ΙΟΙ 00% MeCN/H2O, 0.1% TFA modifier). Upon lyophilizatîon, tert-butyl N-(((9H-fluoren-9

484 yI)methoxy)carbonyl)-O-((4-nitrophenoxy)carbûnyl)-L-serinate was obtaîned. HRMS: M+= 566.4; Rt=3.01 min (5 min acidic method).

Synthesis of l-(2-(((((S)-2-amino-3-(tert-butoxy)-3 oxopropoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)3-methyIbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3methylphenoxy)ethyl)-l-methylpiperazin-l-iiini

PMBO^Yo

To tert-butyl N-(((9H-fluoren-9-yl)methoxy)carbonyl)-0-((4-nitrophenoxy)carbonyl)-Lserînate (7.14 mg, 143 pL, 0.0l3mmol, 1.2 equiv.) and l-(4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2((methyIamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium (20 mg, 235 pL, 0.011 mmol, 1.0 equiv.) was added DIPEA (15.1 pL, 0.087 mmoles, 8.0 equiv.). After standing for 16 hr, Dimethyl amine (109 pl, 0.217 mmol, 20 equiv.) was added. The reaction was stirred at RT for 1 hr. DMSO (4 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization, 1 -(2-(((((S)-2-amîno-3-(tert-butoxy)-3oxopropoxy)carbonyl)(m ethyl )amino)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl) amino )-3methylbutanamido)-5-urcidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fl uorophenyl)-4-(((R)l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l485 oxopropan-2-yl )oxy)thieno [2,3-d]pyrimidin-5-yl )-3-melhylphenoxy)cthyl )-1-methylpiperazin-lium was obtained. HRMS: M+= 1686.7200; Rt=2.71 min (5 min acidic method).

Synthesis l-(2-(((((S)-3-(tert-butoxy)-3-oxo-2-(2 sulfoaceta mido)propoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S )-2-(( tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2ch loro-4-(6-(4-fl no rophenyt)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno|2,3d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium

O NH₂

Following GENERAL PROCEDURE 6 with l-(2-(((((S)-2-amino-3-(tert-butoxy)-3oxopropoxy)carboDyl)(methyl)amino)methyl )-4-(( S )-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)benzyI)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)1 -((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1 oxopropan-2-y l)oxy)thieno[ 2,3-d]pyrimidin-5-yi)-3-methylphenoxy)ethyl)-I-methylpiperazin-lium (22.9 mg, 0.011 mmol, 1 eq), l-(2-(((((S)-3-(tert-butoxy)-3-oxo-2-(2suIfoacetamido)propoxy)carbonyI)(methyl)amino)methyl)-4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4(6-(4-fluorophenyl)-4-(((R)-l-((4-inethoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)-l-oxopropan-2-yl)üxy)thieno|2,3-d]pyrimidin-5-yl)-3methylphenoxy)ethyl)-l-methylpiperazin-l-ium was obtained. HRMS: M+= 1808.6899; Rt=3.18 min (5 min acidic method).

486

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyI)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyI)thieno|2,3-d|pyrimidin-5-yI)-2-chloro-3methylphenoxy)ethyl)-l-(2-(((((S)-2-carboxy-2-(2sulfoacetamido)ethoxy)carbonyI)(methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo2,5-dihydro-lH-pyrrol-l-yl)ethoxy)pro]pananiido)-3-methylbutananiido)-5ureidopentanamido)benzyl)-l-methylpiperazin-l-ium (L77-P1)

Following GENERAL PROCEDURE 3 with l-(2-(((((S)-2-amino-3-(tert-butoxy)-3oxopropoxy)carbonyl)(m ethyl )amino)methyl)-4-(( 5)-2-(( S )-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-inethoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-loxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1 -methylpiperazin-1 ium (15.3 mg, 7.11 pmol, 1 eq), 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(2-(((((S)-2-carboxy-2-(2sulfoacetamido)ethoxy)carbonyl)(methyl)amino)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5dihydro-1 H-pyrrol-l-yl)ethoxy)propanamido)-3-methylbutanamido)-5ureidopentanamido)benzyl)-l-methylpiperazin-l-ium was obtained. HRMS: M+= 1727.5800; Rt=2.47 min (5 min acidic method).

Synthesis of 4-benzyl l-(tert-butyl) (4-(diethoxyphosphoryl)butanoyl)-L-aspartate

487

To 4-benzyl l-(tert-butyl) L-aspartate (200 mg, 0.633 mmol, 1.0 equiv.) and 4(dîethoxyphosphoryl)butanoic acid (213 mg, 0.950 mmol, 1.5 equiv.) dissolved in DMF (2 mL) was added dicyclohexylmethanediimine (157 mg, 0.760 mmol, 1.2 equiv.), 1H[l,2,3]tnazolo[4,5-b]pyridin-l-ol hydrate (146 mg, 0.950 mmol, 1.5 equiv.) and DIPEA (0.110 mL, 0.633 mmol, 1.0 equiv.). After standing for 16 hr, DMSO (4 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization, 4-benzyl 1 -(tert-butyl) (4-(diethoxyphosphoryl)butanoyl)L-aspartate was obtained. HRMS: [M+H]+=486.4; Rt=2.15 min (5 min acidic method).

Synthesis of (S)-4-(tert-butoxy)-3-(4-(diethoxyphosphoryl)butanamido)-4oxobntanoic acid

To 4-benzyl 1-(tert-butyl) (4-(diethoxyphosphoryl)butanoyl)-L-aspartate (YUBI5-040EXP082-001 (100 mg, 0.206 mmol, 1.0 equiv.) dissolved in MeOH (2 mL) was added Palladium hydroxide (14,46 mg, 0.021 mmol, 0.1 equiv.). The reaction atmosphère was switched to hydrogen. After stirring for 16 hr, the reaction mixture was filtered through a celite pad. The filtrate was removed in-vacuo to obtain (S)-4-(tert-butoxy)-3-(4(diethoxyphosphoryl)butanamido)-4-oxobutanoîc acid. HRMS: [M+H]+= 396.3; Rt=1.35 min (5 min acidic method).

488

Synthesis of l-(2-(((S)-4-(tert-butoxy)-3-(4-(diethoxyphosphoryI)butanamido)-Nmethyl-4-oxobutanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3niethylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyi)pyrimidin-4yl)niethoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d|pyrimidin-5-yl)-3methylphenoxy)ethyl)-l-methylpiperazin-l-ium

Following GENERAL PROCEDURE 7 with l -(4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2((methylammo)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l -oxopropan2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-i-methylpiperazin-l-ium (30 mg, 353 pL, 0.016 mmol, 1 eq) and (S)-4-(tert-butoxy)-3-(4-(diethoxyphosphoryl)butanamido)4-oxobutanoic acid (12.87 mg, 161 μΐ, 0.033 mmol, 2 equiv.), l-(2-(((S)-4-(teri-butoxy)-3-(4(diethoxyphosphoryl)butanamido)-N-methyl-4-oxobutanamido)methyi)-4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopenîanamido)benzyl)-4-(2-(2-ch[oro-4(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3methylphenoxy)ethyl)-l-methylpiperazin-l-ium was obtained. MS: M/2+= 940.3; Rt=2.60 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yI)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methyIphenoxy)ethyl)-l-(2-(((S)-3-carboxy-3-(4-(diethoxyphosphoryl)butanamido)-Nmethylpropanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l489 yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-lmethylpiperazin-l-ium (L103-P1)

Following GENERAL PROCEDURE 3 with l-(2-(((S)-4-(tert-butoxy)-3-(4(diethoxyphosphoryl)buianamido)-N-methyl-4-oxobutanamido)in ethyl )-4-( (S )-2-(( S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrîmidin-5-yl)-3methylphenoxy)ethyl)-l -methylpiperazin-l-ium (20 mg, 0.009 mmol, l eq), 4-(2-(4-(4-((R)-lcarboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluoiOphenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(2-(((S)-3carboxy-3-(4-(diethoxyphosphoryl)butanamido)-N-methylpropanamido)methyl)-4-((S)-2-((S)-2(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethoxy)propanamido)-3-methylbutanamido)-5ureidopentanamido)benzyl)-l-methylpiperazin-l-ium was obtained. HRMS: M+= 1795.6700; Rt=2.44 min (5 min acidic method).

Synthesis of l-(4-((S)-2-((S)-2-((tert“butoxycarbonyI)anuno)-3-methylbutanamido)5-ureidopentanamido)-2-(((S)-2,6-diamino-N-metliylhexanamido)methyl)benzyI)-4-(2-(2chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2methoxyphenyl)pyrimidin-4-yI)methoxy)phenyI)-l-oxopropan-2-yl)oxy)thieno[2,3d]pynmidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium

490

NHBoc

PMBO

Following GENERAL PROCEDURE 7 with l-(4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamîdo)-5-ureidopentanamido)-2((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methyIphenoxy)ethyl)-l-methylpiperazin-l-ium (20 mg, 0.011 mmol, l eq) and (S)-2,5-bis((((9H-fluoren-9-yl)methoxy)carbonyl)amino)pentanoic acid (7.51 mg, 0.013 mmol, 1.2 eq), l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-(((S)-2,6-diamino-Nmethylhexanamido)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-flnorophenyl)-4-(((R)-1-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)metho^xy)phenyl)-l-oxopropan2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methyIpiperazin-l-ium was obtained. HRMS: M+= 1627.4000; Rt=2.48 min (5 min acidic method).

Synthesis of l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-mcthylbutanamido)5-ureidopentanamido)-2-(((S)-N-methyl-2,6-bis(2sulfoacetamido)hexananHdo)methyl)benzyl)-4-i(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l((4-methoxybenzyi)oxy)-3-(2-((2-(2-methoxyphenyl)pyriniidin-4-yl)methoxy)phenyI)-loxopropan-2-yi)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyI)-lmethylpipcrazin-1 -ium

491

Following GENERAL PROCEDURE 6 with l-(4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((S)-2,6-diamino-Nmethylhexanamido)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((45 meth oxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan2-yl)oxy)thieno[2,3-d]pyiïmidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpîperazin-l-ium (9.7 mg, 0.0049 mmol, l eq), l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)ammo)-3-methylbutanamido)5-ureidopentanamido)-2-(((S)-N-methyl-2,6-bis(2-sulfoacetamido)hexanamido)methyl)benzyl)4-(2-(2-chloro-4-(6-(4-fl uorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(210 methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1 -oxopropan-2-yI)oxy)thieno[2,3-d]pyrimidin5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-1-ium was obtained. HRMS: M+= 1871.6700; Rt=3.02 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-I-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-415 yl)methoxy)phenyI)ethoxy)-6-(4-fluorophenyl)thieno[2,3'd]pyrimidin-5-yI)-2-chloro-3methylphenoxy)ethyl)-l-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrroI-lyl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(((S)-N-methyl2,6-bis(2-sulfoacetamido)hcxanamido)methyl)benzyI)-l-methylpiperazin-l-îum (L78-P1)

492

Following GENERAL PROCEDURE 3 with-(4-((S)-2-((S)-2-((tertbutoxycarbûiiyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(((S)-N-methyl-2,6bis(2-sulfoacetamido)hexanamido)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)5 1 -((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-1 oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yi)-3-methylphenoxy)ethyl)-]-methylpiperazin-lium (15 mg, 7.55 pmol, 1 eq), 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin4-yl)methoxy)phenyI)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l10 yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(((S)-N-methyl-2,6bis(2-sulfoacetamido)hexanamido)methyl)benzyl)-1 -methylpiperazin-1 -ium was obtained.

HRMS: M+= 1846.6000; Rt=2.49 min (5 min acidic method).

Synthesis of 4-benzyI 1-(tert-butyl) ((2S,3S,4S,5R,6S)-3,4,5,615 tetraacetoxytetrahydro-2H-pyran-2-carbonyl)-L-aspartate

To (2S,3S,4S,5R,6S)-3,4,5,6-tetraacetoxytetrahydro-2H-pyran-2-carboxylic acid (344 mg, 0.950 mmol) and 4-benzyl 1-(tert-butyl) L-aspartate (300 mg, 0.950 mmol) dissolved in 493

DMF (3.2 mL) was added DIPEA (O.l 65 mL, 0.950 mmol, l.O equiv.), I H-[l,2,3]triazolo[4,5b]pyridin-1 -ol hydrate (154 mg, 0.997 mmol, 1.05 equiv.) and 3(((ethylimino)methylene)amino)-N,N-dimethylpropan-l-amine hydrochloride (191 mg, 0.997 mmol, 1.05 equiv.) were added. After standing for 16 hr, DMSO (4 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-70% MeCN/H2O, 0.1 % TFA modifier). Upon lyophilization, 4-benzyl 1-(tert-butyl) ((2S,3S,4S,5R,6S)-3,4,5,6tetraacetoxytetrahydro-2H-pyran-2-carbonyl)-L-aspartate was obtained. HRMS: M+=641.5; Rt=2.55 min (5 min acidic method).

Synthesis of (S)-4-(tert-butoxy)-4-oxo-3-((2S,3S,4S,5R,6S)-3,4,5,6tetraacetoxytetrahydro-2H-pyran-2-carboxamido)butanoic acid

To 4-benzyl 1-(tert-butyl) ((2S,3S,4S,5R_}6S)-3,4,5,6-tetraacetoxytetrahydro-2H-pyran-2carbonyl)-L-aspartate (100 mg, 0.160 mmol, 1.0 eq) dissolved in MeOH (2 mL) was added Palladium hydroxide (11.26 mg, 0.016 mmol, 0.1 equiv.). The reaction atmosphère was swtiched to hygrogen. After stirring for 16 hr, the reaction mixture was filtered through a celite pad. The filtrate was removed in-vacuo to obtain (S)-4-(tert-butoxy)-4-oxo-3-((2S,3S,4S,5R,6S)-3,4,5,6tetraacetoxytetrahydro-2H-pyran-2-carboxamido)butanoic acid. HRMS: [M-H]-= 532.3, Rt=l .71 min (5 min acidic method).

Synthesis of l-(2-(((S)-4-(tert-butoxy)-N-methyl-4-oxo-3-((2R,3R,4R,5S,6R)-3,4,5,6tetraacetoxytetrahydro-2H-pyran-2-carboxamido)butanamido)methyl)-4-((S)-2-((S)-2((tert-butoxycarbonyl)amino)-3-methylbutanainido)-5-ureidopentanamido)benzyl)-4-(2-(2chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyI)oxy)-3-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3494

d]pyrÎmîdin-5-yl)-3-methylphenoxy)ethyl)-l-niethylpiperazin-lI OAc

AcO,

H Y .

NHSoc

Foilowing GENERAL PROCEDURE 7 with l-(4-((S)-2-((S)-2-((tertbutoxycarbonyI)amino)-3-methylbutanamido)-5-ureidopentanamido)-2((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l -methylpiperazin- i -ium (30 mg, 0.016 mmol, 1.0 eq) and (S)-4-(tert-butoxy)-4-oxo-3-((2S,3S,4S,5R,6S)-3,4,5,6tetraacetoxytetrahydro-2H-pyran-2-carboxamido)butanoic acid (12.2 mg, 968 pL, 0.023 mmol, 1.4 eq), l-(2-(((S)-4-(tert-butoxy)-N-methyl-4-oxo-3-((2R,3R,4R,5S,6R)-3,4,5,6tetraacetoxytetrahydro-2H-pyran-2-carboxamido)butanamido)methyl)-4-((S)-2-((S)-2-((tertb utoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanami do )benzyl )-4-(2-(2-chloro-4(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyI)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3methylphenoxy)ethyl)-l-methylpiperazin-1-ium was obtained. HRMS: M+=2014.7800; Rt=3.21 min (5 min acidic method).

Synthesis of l-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)2-(((S)-3-carboxy-N-methyl-3-((2R,3R,4R,5S,6R)-3,4,5,6-tetraacetoxytetrahydro-2H-pyran2-earboxamido)propanamido)methyl)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2meth oxy phenyl) pyrimidin-4-yI) methoxy) phenyl)ethoxy)-6-(4-fl u orop henyl)thieno[ 2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium

495

To l-(2-(((S)-4-(tert-butoxy)-N-methyl-4-oxo-3-((2R,3R,4R,5S,6R)-3,4,5,6tetraacetoxytetrahydro-2H-pyran-2-carboxamido)butanamido)methyl)-4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyi)pyrimidin-4yl)methoxy)phenyl)-]-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3methylphenoxy)ethyl)-l-methylpiperazin-l-ium (22.7 mg, 0.009 mmoles, 1.0 equiv.) dissolved in DCM (32mL) was added TFA (0.67 mL). After stirring for 45 min, the solvent was removed in-vacuo to obtain l-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentananiido)-2(((S)-3-carboxy-N-methyl-3-((2R,3R,4R,5S,6R)-3,4,5,6-tetraacetoxytetrahydro-2H-pyran-2carboxamido)propanamido)methyl)benzyl)-4-(2-(4-(4-( (R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium. HRMS:

M+=l738.6200; Rt=2.30 min (5 min acidic method).

Synthesis of l-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)2-(((S)-3-carboxy-N-methyl-3-((2R,3R,4R,5S,6S)-3,4,5,6-tetrahydroxytetrahydro-2Hpyran-2-carboxamido)propananiido)methyl)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyriniidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chIoro-3-niethylphenoxy)ethyl)-l-niethylpiperazin-l-ium

496

OH

Το l -(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(((S)-3carboxy-N-methyl-3-((2R,3R,4R,5S,6R)-3,4,5,6-tetraacetoxytetrahydro-2H-pyran-2carboxamido)propanamido)methyl)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yI)methoxy)pbenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium (22.7 mg, 0.009 mmoles, l.O equiv.) dissolved in THF (1 mL) and MeOH (1 mL) was added lithium hydroxide (5.04 mg, 0.120 mmol, 10 equiv.). After stirring for 2 hour, the solvent was removed in-vacuo. Water (1 mL), TFA (0.2 mL), MeCN (1 mL) and DMSO (4 mL) were added and the solution was purified by RP-HPLC ISCO gold chromatography (10-70% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization, l-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5ureidopentanamido)-2-(((S)-3-carboxy-N-methyl-3-((2R,3R,4R,5S,6S)-3,4,5,6tetrahydroxytetrahydro-2H-pyran-2-carboxamido)propanami do )m ethyl )benzyl)-4-(2-(4-(4-((R)l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloiO-3-methylphenoxy)ethyl)-l-methylpiperazin1-ium was obtained. HRMS: M+= 1570.5900; Rt=2.02 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxy phenyl) pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-(2-(((S)-3-carboxy-N-methyl-3-((2R,3R,4R,5S,6S)-3,4,5,6tetrahydroxytetrahydro-2H-pyran-2-carboxamido)propanamido)methyl)-4-((S)-2-((S)-2(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethoxy)propanamido)-3-niethylbutananiido)-5ureïdopentanamido)benzyl)-l-methylpiperazin-l-inm (L68-P1)

497

To l-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(((S)-3carboxy-N-methyl-3-((2R,3R,4R,5S,6S)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2carboxamido)propanamido)methyl)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluoropheny])thieno[2,3d]pyriniidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium (10.8 mg, 0.0056 mmoles, 1.0 equiv.) and 2,5-dioxopyrrolidin-l-yI 3-(2-(2,5-dioxo-2,5-dihydro-IH-pyrrol-lyl)ethoxy)propanoate (5.25 mg, 0.017 mmol, 3.5 equiv.) dissolved in DMF (l mL) was added DIPEA (7.86 pL, 0.045 mmol, 8 equiv.). After standing for 1.5 hour, DMSO (2mL) were added and the solution was purified by RP-HPLC ISCO gold chromatography (10-70% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization, 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyI)ethoxy)-6~(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(2-(((S)-3-carboxy-N-methyl-3((2R,3R,4R,5S,6S)-3,4,5,6-tetrahydroxytetrahydro-2H-pyran-2carboxamido)propanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamîdo)benzyl)-lmethylpiperazin-l-ium was obtaîned. HRMS: M+=1765.6500; Rt=2.31 min (5 min acidic method).

Synthesis of 4-benzyl 1-(tert-butyl) ((((3R,4S,5S,6S)-3,4,5-triacetoxy-6(methoxycarbonyI)tetrahydro-2H-pyran-2-yl)oxy)carbonyl)-L-aspartate

498

To ((3R,4S,5S,6S)-2-hydroxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyI triacetate (254 mg, 0.760 mmol, 1.2 equiv.) and bis(4-nitrophenyl) carbonate (289 mg, 0.950 mmol, 1.5 equiv.) dissolved in DMF (2 mL) was added DIPEA (0.221 mL, 1.267 mmol, 2.0 equiv.). After standing for I hr, 4-benzyl l-(tert-butyl) L-aspartate (200 mg, 0.633 mmol, 1.0 equiv.) was added. After standing for 16 hr, DMSO (6 mL) was added and the solution was purified by RP-HPLC ISCO gold chromatography (10-70% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization, 4-benzyl 1-(tert-butyl) ((((3R,4S,5S,6S)-3,4,5-triacetoxy-6(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)carbonyl)-L-aspartate was obtained. HRMS: [M-H]-= 638.4; Rt=2.61 min (5 min acidic method).

Synthesis of (3S)-4-(tert-butoxy)-4-oxo-3-(((((3R,4S,5S,6S)-3,4,5-triacetoxy6-(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)carbonyl)amino)butanoic acid

To 4-benzyl 1-(tert-butyl) ((((3R,4S,5S,6S)-3,4,5-triacetoxy-6(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)carbonyl)-L-aspartate (50 mg, 0.078 mmol, 1.0 eq) dissolved in MeOH (2 mL) was added Palladium hydroxide (5.49 mg, 7.82 μιηοΐ, 0.1 equiv.). The reaction atmosphère was swtiched to hygrogen. After stirring for 16 hr, the reaction mixture was filtered through a celite pad. The filtrate was removed in-vacuo to obtain (3S)-4(tert-butoxy)-4-oxo-3-(((((3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2Hpyran-2-yl)oxy)carbonyl)amino)butanoic acid. HRMS: [M-H]-: 548.4, Rt=1.79 min (5 min acidic method).

499

Synthesis of l-(2-(((3S)-4-(tert-butoxy)-N-methyl-4-oxo-3-(((((3R,4S,5S,6S)-3,4,5triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2yl)oxy)carbonyt)amino)butanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluoropheny])4-(((R)-l-((4-niethoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)-l-oxopropan-2-yI)oxy)thieno[2,3-dJpyrimidin-5-yl)-3methy I phen oxy) ethyl)- 1-methyipiper azin-1 -iu m

NHBoc

Following GENERAL PROCEDURE 7 with 1 -(4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2((methylamino)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4methoxybenzyl)oxy)-3-(2-((2-(2-m ethox yphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan2-yl)oxy)thieno[2,3-d]pyrimîdin-5-yl)-3-methylphenoxy)ethyl)-l -methylpiperazin-l-ium (30 mg, 0.016 mmol, LO eq) and (3S)-4-(tert-butoxy)-4-oxo-3-(((((3R,4S,5S,6S)-3,4,5-triacetoxy-6(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)carbonyl)amino)butanoic acid (12.5 mg, 0.250 mL, 0.023 mmol ,1.4 eq), l-(2-(((3S)-4-(tert-butoxy)-N-methyl-4-oxo-3-(((((3R,4S,5S,6S)-3,4,5triacetoxy-6-(methoxycarbonyl)tetrahydro-2H-pyran-2yl)oxy)carbonyl)amino)butanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanami do)-5-ureidopentanamido)benzyl )-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-loxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-lium was obtained. HRMS: M+=2030.7900; Rt=3.l9 min (5 min acidic method).

500

Synthesis of l-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)2-(((3S)-3-carboxy-N-methyl-3-(((((3R,4S,5S,6S)-3,4,5-triacetoxy-6(methoxycarbonyl)tetrahydro-2H-pyran-2yl)oxy)carbonyl)annno)propanamido)methyI)benzyl)-4-(2-(4-(4-((R)“1-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yI)methoxy)phenyl)ethoxy)-6“(4-fluorophenyl)thieno[2,3dJpyrimidin-5-yl)-2-chloro-3-methylphcnoxy)ethyl)-l-niethyipiperazin-l-ium

To I-(2-(((3S)-4-(tert-butoxy)-N-methyl-4-oxo-3-(((((3R,4S,5S,6S)-3,4,5-triacetoxy-6(methoxycarbonyl)tetrahydro-2H-pyran-2-yl)oxy)carbonyl)amino)butanamido)methyl)-4-((S)-2((S)-2-((tert-butoxycarbonyl)amino)-3-methyIbutanamido)-5-ureidopentanamido)benzyI)-4-(2(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyI)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium (25,8 mg, 10.86 pmol, 1.0 equiv.) dissolved in DCM (2 mL) was added TFA (0.67 mL). After stirring for 2 hrs, the solvent was removed in-vacuo to obtain l-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5ureidopentanamido)-2-(((3S)-3~carboxy-N-methyl-3-(((((3R,4S,5S,6S)-3,4,5-triacetoxy-6(methoxycarbonyï)tetrahydro-2H-pyran-2-yl)oxy)carbonyl)amino)propanamido)methyl)benzyl)4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimÎdin-4-yl)methoxy)phenyl)ethoxy)6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methyl phenoxy)ethyl)-l methylpiperazin-l-ium. HRMS: M+=l 754.6200; Rt=2.31 min (5 min acidîc method).

Synthesis of l-(4-((S)-2-((S)-2-amino-3-methyIbutanamido)-5-ureidopentanamido)2-(((3S)-3-carboxy-3-(((((3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2501 yI)oxy)carbonyl)amino)-N-methyIpropanamido)niethyl)benzyl)-4-(2-(4-(4-((R)-l-carboxy2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yI)methoxy)phenyI)ethoxy)-6-(4fluorophenyI)thieno[2,3-d]pyrimidin-5-yI)-2-chloro-3-methylphenoxy)ethyl)”lmethylpiperazin-l-ium

To l-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(((3S)-3carboxy-N-methyl-3-(((((3R,4S,5S,6S)-3,4,5-triacetoxy-6-(methoxycarbonyl)tetrahydro-2Hpyran-2-yl)oxy)carbonyl)amino)propanamido)methyl)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fiuorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy) ethyl)-1-methylpiperazin-l-ium (26 mg, 0.012 mmol, 1.0 equiv.) dissolved in THF (1 mL) and MeOH (1 mL) was added lithium hydroxide (5.20 mg, 0.124 mmol, 10 equiv.). After stirring for 2 hour, the solvent was removed in-vacuo. Water (1 mL), TFA (0.2 mL), MeCN (1 mL) and DMSO (4 mL) were added and the solution was purified by RP-HPLC ISCO gold chromatography (10-70% MeCN/H2O, 0.1% TFA modifier), Upon lyophilization, l-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5ureidopentanamido)-2-(((3S)-3-carboxy-3-(((((3R,4S,5S,6S)-6-carboxy-3,4,5trihydroxytetrahydro-2H-pyran-2-yi)oxy)carbonyl)amino)-Nmethylpropanamîdo)methyl)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorQphenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methyiphenoxy)ethyl)-1 -methylpiperazin-l-ium was obtained. HRMS: M+=l 614.5800; Rt=2.04 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-(( R)-l-carboxy-2-(2-((2-(2-methoxy phenyl) pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyI)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-(2-(((3S)-3-carboxy-3-(((((3R,4S,5S,6S)-6-carboxy-3,4,5502 trihydroxytetrahydro-2H-pyran-2-yI)oxy)carbonyl)amino)-Nmethylpropanamido)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-!H-pyrrol-lyl)ethoxy)propanamîdo)-3-methyIbutanamido)-5-ureidopentanamÎdo)benzyl)-lmethylpiperazin-l-ium (L69-P1)

O /v

HO γ OH

ÔH

To l-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)-2-(((3S)-3carboxy-3-(((((3R,4S,5S,6S)-6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2yl)oxy)carbonyl)amino)-N-methy]propanamido)methyl)benzyI)-4-(2-(4-(4-((R)-l-carboxy-2-(2((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2_}3d]pyrimidin-5-yl)-2-chIoro-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium (lOmg, 5.11 μιηοΐ, 1.0 equiv.) and 2,5-dîoxopyrrolidin-l-yl 3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)ethoxy)propanoate (4.75 mg, 0.015 mmol, 3.5 equiv.) dissolved in DMF (1 mL) was added D1PEA (7.12 μΐ, 0.041 mmol, 8 equiv.). After standing for 1.5 hour, DMSO (2mL) were added and the solution was purified by RP-HPLC ISCO gold chromatography (10-70% MeCN/H2O, 0.1 % TFA modifier). Upon lyophilîzation, 4-(2-(4-(4-((R)-1 -carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(2-(((3S)-3-carboxy-3-(((((3R,4S,5S,6S)6-carboxy-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)oxy)carbonyl)amino)-Nm ethyl propan amido)m ethyl )-4-(( S)-2-((S )-2-(3-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-lmethylpiperazin-l-ium was obtained. HRMS: M+= 1809.6300; Rt=2.32 min (5 min acidic method).

503

Synthesis of l-(2-((3-(2-(2-aminoethoxy)ethoxy)-N-niethylpropanamido)methyl)-4-((S)-2((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)“5-ureidopentanamido)benzyl)4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3d]pyriniidin-5-yl)-3-methylphenoxy)ethyl)-l-niethylpiperazin-l-

A mixture of l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5ureidopentanamido)-2-((m ethyl amino)methyl)benzyl) -4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-loxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-lium (45 mg, 0.026 mmol), l-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azatridecan-l3-oic acid (12 mg, 0.030 mmol), HBTU (12 mg, 0.032 mmol), and DIPEA (0.023 mL, 0.13 mmol) in DMF (1 mL) was stirred at RT for 30 min. Me₂NH (2M in THF, 0.065 mL, 0.13 mmol) was added, and the mixture was stirred at RT for 1 h. Additional amount of Me₂NH (2M in THF, 0.1 mL, 0.2 mmol) was added. The mixture was continued to be stirred at RT for 1 h, diluted with DMSO (3 mL), and the solution was purified b y RP-HPLC ISCO gold chromatography (MeCN/H₂O, 0.1% TFA modifier). Upon lyophilization, l-(2-((3-(2-(2-aminoethoxy)ethoxy)-Nmethylpropanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fiuorophenyl)-4-(((R)l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yI)methoxy)phenyl)-loxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-inethylphenoxy)ethyl)~l-methylpiperazin-1ium was obtained. HRMS: M+= 1658.7200, Rt=2.57 min (5 min acidic method).

Synthesis of l-(4-((S)-2-((S)-2-((tert-butoxycarbonyÎ)amino)-3-methylbutanamido)-5ureidopentanamido)-2-(2-methyl-3,13-dioxo-15-phosphono-6,9-dÎoxa-2,12diazapentadecyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4niethoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyI)pyrimidin-4-yl)methoxy)phenyl)-l504 oxopropan-2-yl)oxy)thieno[2,3-d|pyrimidiii-5-yl)-3-methylphenoxy)ethyl)-lmethylpiperazin-l-ium

ΗΟ-γ

OH

A mixture of 3-phosphopropionic acid (11 mg, 0.071 mmol), HBTU (27 mg, 0.071 mmol), and DIPEA (0.060 mL, Û.34 mmol) in DMF (0.5 mL) was stirred at RT for 10 min. This mixture was added to a solution ofl-(2-((3-(2-(2-aminoethoxy)ethoxy)-Ninethylpropanamido)methyl)-4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)“5’Ureidopentanamido)benzyl)-4-(2-(2-chloro-4-(6-(4-fluoiOphenyl)-4-(((R)l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l10 oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-lium (40 mg, 0.021 mmol) and DIPEA (0.010 mL, 0.057 mmol) in DMF (0.5 mL). The mixture was stirred at RT for 2 days. The mixture was diluted with DMSO (3 mL), and the solution was purified by RP-HPLC ISCO gold chromatography (MeCN/H?O, 0.1% TFA modifier). Upon lyophilization, I-(4-((S)-2-((S)-2-((tert-butoxycarbonyI)amino)-3-methylbutanamido)-515 ureidopentanamido)-2-(2-methyl-3,13-dioxo-l 5-phosphono-6,9-dioxa-2,12diazapentadecyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium was obtained. HRMS: M+= 1794.7100, Rt=2.78 min (5 min acidic method).

505

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyI)ethoxy)-6-(4-fluorophenyI)thieno[2,3-d]pyrimidin-5-yI)-2-chloro-3methylphenoxy)ethyl)-1 -(4- ((S )-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 yI)ethoxy)propanamido)-3-methyIbutanamido)-5-ureidopentanamido)-2-(2-methyl-3,13dioxo-15-phosphono-6,9-dioxa-2,12-diazapentadecyl)benzyl)-l-methyIpiperazin-l-ium (L41-P1)

HO-P^

OH

Following GENERAL PROCEDURE 3 (except that the product after the first step with TFA/CH2CI2 was purified by RP-HPLC ISCO gold chromatography [MeCN/HzO, 0.1% NH4OH modifier]) with 1 -(4-((S)-2-((S)-2-((tert-butoxyearbonyl)amino)-3-methylbutanamido)-5ureidopentanamido)-2-(2-methyI-3,13-dioxo-15-phosphono-6,9-dioxa-2,12diazapentadecyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yI)methoxy)phenyl)-l-oxopropan2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium, 4-(2(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2₎3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l -(4-((S)-2-((S)-2(3-(2-(2,5-dioxo-2,5-dihydro-l H-pyrrol- l-yl)ethoxy)propanamido)-3-methylbutanamido)-5ureidopentanamido)-2-(2-methyl-3,13-dioxo-l 5-phosphono-6,9-dioxa-2,12diazapcntadecyl)benzyl)-l-methylpiperazin-l-ium was obtained. HRMS: M+= 1769.4500, Rt=2.33 min (5 min acidic method).

Synthesis of l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5ureidopentanamido)-2-(2,5,8,ll,14,17,20,23,26,29,32,35,38,44,44-pentadecamethyl3,6,9,12,15,18,21,24,27,30,33,36,39,42-tetradecaoxo-43-oxa2,5,8,11,14,17,20,23,26,29,32,35,38-tridecaazapentatetracontyl)benzyI)-4-(2-(2-chloro-4-(6506 (4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-niethoxyphenyI)pyrimidin-4yI)mcthoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3methylphen oxy)ethy l)-1 - methy lp iper azin-1 -i u m

To a stirred solution of 1 -(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylb utanamido)-5-ureidopentanamido)-2-((mcthyl amino )m ethyl )benzyl )-4-( 2-(2-chloro-4(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3methylphenoxy)ethyl)-l-methylpiperazin-l-ium (43.6 mg, 0.025 mmol, 1.0 equiv.),

3,6,9,12,15,18,21,24,27,30,33,36,42,42-tetradecamethyl-4,7,l 0,13,16,19,22,25,28,31,34,37,40tridecaoxo-41-oxa-3,6,9,12,15,18,21,24,27,30,33,36-dodecaazatritetracontanoic acid (25.9 mg, 0.025 mmol, 1.0 equiv.), and HATU (10.5 mg, 0.028 mmol, 1.1 equiv.) in DMF (0.25 mL) was added DIPEA (22 pL, 0.126 mmol, 5.0 equiv.). The resulting solution was stirred at ambient température for 1 hour. The reaction was diluted with 1 mL DMSO and purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2(2,5,8,11,14,17,20,23,26,29,32,35,38,44,44-pentadecamethyl3,6,9,12,15,18,21,24,27,30,33,36,39,42-tetradecaoxo-43-oxa2,5,8,1 l,14,17,20,23,26,29,32,35,38-tridecaazapentatetracontyl)benzyl)-4-(2-(2-chloro-4-(6-(4fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3methylphenoxy)ethyl)-l-methylpiperazin-l-ium was obtained. HRMS M+ 2508.1499 Rt=2.78 min (5 min acidic method).

507

Synthesis of l-(2-(41-carboxy-2,5,8,ll,14,17,20,23,26,29,32,35,38-tridecamethyl3,6,9,12,15,18,21,24,27,30,33,36,39-tndecaoxo-2,5,8,ll,14,17,20,23,26,29,32,35,38tridecaazahentetracontyI)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrroi-lyl)ethoxy)propanamido)-3-methylbutananudo)-5-ureidopentananiÎdo)benzyl)-4-(2-(4-(4((R)-l-carboxy-2-(2-((2-(2-niethoxyphenyl)pyrimidin-4-yl)methoxy)phenyI)ethoxy)“6-(4fluorophenyl)thieno[2,3-d]pyrimidïn-5-yl)-2-chloro-3-methylphenoxy)ethyI)-lmethylpiperazin-l-ium (L35-PI)

Following GENERAL PROCEDURE 3 with 1 -(4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methyIbutanamido)-5-ureidopentanamido)-2(2,5,8,11,14,17,20,23,26,29,32,35,38,44,44-pentadecamethyl3,6,9,12,15,18,21,24,27,30,33,36,39,42-tetradecaoxo-43-oxa2,5,8,1 l,14,17,20,23,26,29,32,35,38-tridecaazapentatetracontyl)benzyl)-4-(2-(2-chloro-4-(6-(4fluorophenyl)-4-(((R)-l-((4-methoxybenzyï)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3methylphenoxy)ethyl)-I-methylpiperazin-l-ium (50.4 mg, 0.019 mmol, 1.0 equiv.), 1-(2-(41carboxy-2,5,8,11,14,17,20,23,26,29,32,35,38-tridecamethyl3,6,9,12,15,18,21,24,27,30,33,36,39-tridecaoxo-2,5,8,11,14,17,20,23,26,29,32,35,38tridecaazahentetracontyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dih ydro-lH-pyrrol-1 yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-lcarboxy-2-(2-((2-(2-methoxyphenyI)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4508 fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methyÎphenoxy)ethyl)-l-methylpiperazinl-ium was obtained. HRMS: M+ 2427.0400, rt = 2.30 min. (5 min acidic method).

Synthesis of l-(4~((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5ureidopentanamido)-2-(2,5,8,U,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,62,625 henicosamethyl-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60-icosaoxo-61-oxa2,5,8,ll,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56-nonadecaazatrihexacontyi)benzyl)-4(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3d]pyrimidin-5-yi)-3-methylphenoxy)ethyl)-l-methylpiperazin- 1-ium

OMe

To a stirred solution of 1 -(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanamido)-5-ureidopentanamido)-2-((methylammo)methyl)benzyl)-4-(2-(2-chloro-4(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-niethoxyphenyl)pyrimidin-4yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-315 methylphenoxy)ethyl)-l-methylpiperazin-1-ium (31.9 mg, 0.018 mmol, 1.0 equiv.),

3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,60,60-icosamethyl4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58-nonadecaoxo-59-oxa3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54-octadecaazahenhexacontanoic acid (26.8

509 mg, 0.018 mmol, 1.0 equiv.), and HATU (7.7 mg, 0.020 mmol, 1.1 equiv.) in DMF (0.25 mL) was added DIPEA (16 pL, 0.092 mmol, 5.0 equiv.). The resulting solution was stirred at ambient température for 1 hour. The reaction was diluted with 1 mL DMSO and purified by RPHPLC ISCO gold chromatography (10-100% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5ureidopentanamido)-2-(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,62,62henicosamethyI-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60-icosaoxo-61 -oxa2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56-nonadecaazatrihexacontyl)benzyI)-4-(2(2-chloro-4-(6-(4-fIuorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2methoxyphenyl)pyrimidin-4-yI)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin5-yl)-3-methylphenoxy)ethyl)-l -methylpiperazin- 1-ium was obtained. HRMS M+ 2934.3601 Rt=2.73 min (5 min acidic method).

Synthesis of l-(2-(59-carboxy-2,5,8,ll,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56nonadecamethyl-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57-nonadecaoxo2,5,8,ll,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56-nonadecaazanonapentacontyl)-4-((S)2“((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH“pyrrol-l-yl)ethoxy)propanamido)-3methylbutanamido)-5-ureidopentanamido)beinzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2“ methoxyphenyl)pyrinndm-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin-l-iuin (L36-P1)

510

Following GENERAL PROCEDURE 3 with l-(4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2(2,5,8,11,14,17,20,23,26,29,32,3 5,3 8,41,44,47,50,53,56,62,62-henicosamethyl5 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60-icosaoxo-61-oxa2,5,8,1 l,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56-nonadecaazatrihexacontyl)benzyl)-4-(2(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin5-yl)-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium (45.5 mg, 0.015 mmol, 1.0 equiv.), l-(210 (59-carboxy-2,5,8,l 1,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56-nonadecamethyl3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57-nonadecaoxo2,5,8,ll,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56-nonadecaazanonapentacontyl)-4-((S)-2((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethoxy)piOpanamido)-3-methylbutanamido)5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin15 4-yI)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chIoiO-3methylphenoxy)ethyl)-1 -methylpiperazin-l-ium was obtained. HRMS: M+ 2853.2766, rt = 2.20 min. (5 min acidic method).

511

Synthesis of l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)aniÎno)-3-mcthylbutanamido)-5ureidopentanamido)-2(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,80,80heptacosamethyl-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78hexacosaoxo-79-oxa-2,5,8,ll,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74pentaeosaazahenoctacontyl)benzyl)-4~(2-(2-chIoro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4methoxybenzyl)oxy )-3-(2-((2-(2-methoxyphenyI)pyrimidin-4-yl)methoxy)phenyl)-loxopropan-2-yI)oxy)thieno[2,3-d]pyrimidm-5-yl)-3-methylphenoxy)ethyl)-lmethylpiperazin-l-ium

To a stirred solution ofl-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3methylbutanami do)-5-urei dopentanamido)-2-((methyl amino)m ethyl )benzyl )-4-(2-(2-chloro-4(6-(4-fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl )pyrimidin-4yl)methoxy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yI)-3mcthylphenoxy)ethyl)-l-methylpiperazin-l-ium (27.8 mg, 0.016 mmol, 1.0 equiv.), 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,78,78-hexacosamethyl4,7,I0,13,16,I9,22,25,28,31,34,37,40,43_}46,49,52,55,58,61,64,67,70,73,76-pentacosaoxo-77oxa-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72tetracosaazanonaheptacontanoic acid (30.2 mg, 0.016 mmol, 1.0 equiv.), and HATU (6.7 mg, 0.018 mmol, 1.1 equiv.) in DMF (0.25 mL) was added DIPEA (14 pL, 0.080 mmol, 5.0 equiv.).

512

The resulting solution was stirred at ambient température for l hour. The reaction was diluted with 1 mL DMSO and purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization l-(4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,80,80heptacosamethyl-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78hexacosaoxo-79-oxa-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74pentacosaazahenoctacontyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-1-((4meihoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)-]-oxopropan2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1 -methylpiperazin-1 -ium was obtained. HRMS M+ 3360.5798 Rt=2.68 min (5 min acidic method).

Synthesis of l-(2-(77-carboxy2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74-pentacosamethyl3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75-pentacosaoxo2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74pentacosaazaheptaheptacontyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4((R)-l-carboxy-2-(2-((2-(2-mcthoxyphenyl)pyirimidin-4-yl)methoxy)phenyI)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yll)-2-chloro-3-methylphenoxy)ethyl)-lmethylpiperazin-l-ium (L37-P1)

513

Following GENERAL PROCEDURE 3 with l-(4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2(2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,80,80heptacosamethyl-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75,78hexacosaoxo-79-oxa-2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74pentacosaazahenoctacontyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluoroph en yI)-4-(((R)-1-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidm-4-yl)methoxy)phenyl)-l-oxopropan2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-1 -methylpiperazin-1 -ium (41.3 mg, 0.012 mmol, 1.0 equiv.), l-(2-(77-carboxy2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74-pentacosamethyl3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75-pentacosaoxo2,5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74pentacosaazaheptaheptacontyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dîoxo-2,5-dihydro-lH-pyrrol-lyl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-l carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)m ethoxy )phenyl)ethoxy )-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l -methylpiperazin1-ium was obtained. HRMS: M+ 3279.4678, rt = 2.21 min. (5 min acidic method).

Synthesis of Tert-butyl l-((N-i(2-azidoethoxv)carbonyI)sulfamoyl)amino)-3,6,9,l 2,15,18hcxaoxahenicosan-21-oate

514

To 2-azîdoethan-1-ol (105 mg, l.2l mmol) in CH2CI2 (15 ml) was added sulfurisocyanatidic chloride (0.105 ml, 1.21 mmol) at 0°C. The mixture was stirred at 0°C for 30 min. then TEA (0.336 ml, 2.41 mmol) and tert-butyl l-amino-3,6,9,12,15,18-hexaoxahenicosan21-oate (518 mg, 1.27 mmol) in CH2CI2 (1 mL) were added. The mixture was stirred at 0°C for Ih and rt for 2 h, then was quenched with Satd NH4C1, and 1 N HCl (2.4 mL). The aqueous was extracted with CH2C12 (5X). The organic layers were dried over anh, NasSCU, fdtered and concentrated via rotary évaporation to give a clear oil. Purification via flash chromatography (ΟΙ 5% MeOH in CH2CI2, ELSD détection) provided tert-butyl l-((N-((2azîdoethoxy)carbonyl)sulfamoyl)amino)-3,6,9,12,15,I8-hexaoxahenicosan-21-oate as a clear oil (474 mg, 0.788 mmol): LCMS: M+NH4+=619.5, Rt=0.94 min (acidic, 2 min). IH NMR (400 MHz, DMSO-d6) δ 11.33 (s, IH), 7.76 (s, IH), 4.28 - 4.20 (m, 2H), 3.60 (td, J = 5.6, 5.0, 2.9 Hz, 4H), 3.55 - 3.45 (m, 22H), 3.16 - 3.04 (m, 2H), 2.46 - 2.38 (m, 2H), 1.41 (s, 9H).

Synthesis of l-((N-((2-azidoethoxy)carbonyl)sulfamoyl)amino)-3,6,9,12,15,18hexaoxahenicosan-21-oic acid

OH _n O

H

To tert-butyl 1 -((N-((2-azidoethoxy)carbonyl)sulfamoyl)amino)-3,6,9,12,15,18hexaoxahenicosan-2I-oate (145 mg, 0.241 mmol) in CH2CI2 (1 mL) at 0°C was added TFA (1 mL, 12.98 mmol). The mixture was stirred at rt for 1.45 h, then concentrated via rotary évaporation at 25°C water bath and dried in high vac for 30 min. The residue was azeotropically dried with anh toluene (3X2 mL), and dried in vacuum overnight to provide l-((N-((2-azidoethoxy)carbonyl)sulfamoyl)amino)-3,6,9,12,15,18-hexaoxahenicosan21-oic acid as a clear oil (147 mg, 89% by weight based on theoretical yield): LCMS: M+=546.3, 0.65 min (acidic, 2 min, ELSD); 1HNMR (400 MHz, DMSO-d6) δ 11.30 (s, IH), 7.77 - 7.71 (m, IH), 4.25 - 4.19 (m, 2H), 3.63 - 3.55 (m, 4H), 3.54 - 3.45 (m, 22H), 3.07 (q, J = 6.0 Hz, 2H), 2.47 - 2.40 (m, 2H).

Synthesis of l-(4-((S)-2-((S)-2-amino-3-niethylbutanamido)-5-ureidopentanamido)-2((prop-2-yn-l-y!oxy)methyl)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2515 methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyI)thieno[2,3“ d]pyrimidin-5-yl)-2-chloro-3-methyIphenoxy)ethyl)-l-methyIpiperazin-l-ium trifluoroacetate

To l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)-5ureidopentanamido)-2-((prop-2-yn-l-yloxy)methyl)benzyl)-4-(2-(2-chloro-4-(6-(4fluorophenyl)-4-(((R)-l-((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyi)pyriinidin-4yl)metho^xy)phenyl)-l-oxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3methylphenoxy)ethyl)-l -methylpiperazin-l-ium trifluoroacetate (321 mg, 0.210 mmol) at 0°C was added 25% TFA in CH2C12 (12.3 mL, 40.0 mmol). The reaction mixture was raised to Rt, stirred for Ih, then concentrated under high vacuum at RT water bath. The crude was diluted with DMSO (3 mL) and was purified by RP-HPLC 1SCO gold chromatography (l 50 g, 10-80% MeCN/H2O, 0.1% TFA modifier). Upon lyophîlization l-(4-((S)-2-((S)-2-amino-3methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-1-yloxy)methyl )benzyl)-4-(2-(4-(4((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidm-5-yl)-2-chloro-3-methylphenoxy)ethyl)-i-methylpiperazin1-ium trifluoroacetate was obtained as a white powder (224 mg, 0.158 mmol): HRMS: M+=l 304.5100, Rt=2.15 min (5 min acidic)

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yI)-2-chloro-3methyIphenoxy)ethyl)-l-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-lyloxy ) me thyl)benzyl)-1 -methylpiperazin- 1-ium trîflu oroacetate

516

Following GENERAL PROCEDURE 3 with l-(4-((S)-2-((S)-2-amino-3methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-l-yloxy)methyl)benzyl)-4-(2-(4-(4((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyI)-1 -methylpiperazinl-ium trifluoroacetate (164 mg, 0.115 mmol) and Mal-Pegl -NHS ester (72 mg, 0.23 mmol), 4(2-(4-(4-((R)-l-carboxy-2“(2-((2-(2-methoxyphenyI)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2~chloro-3-methylphenoxy)ethyl)-l-(4-((S)-2-((S)2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethoxy)propanamido)-3-methylbutanamido)-5ureidopentanamîdo)-2-((prop-2-yn-1 -yloxy)methyl)benzyl)-l -methylpiperazin-1 -ium trifluoroacetatewas obtained as a white powder (170 mg, 0.105 mmol), HRMS: M+ =1499.5699, Rt=2.45 min (5 min acidic); 1HNMR (400 MHz, DMSO-d6) δ 10.16 (s, IH), 8.81 (d, J = 5.1 Hz, IH), 8.54 (s, IH), 8.07 (d, J = 7.2 Hz, IH), 7.75 (d, J = 8.5 Hz, IH), 7.69 (d, J = 6.9 Hz, 2H), 7.56 (d, J = 5.1 Hz, IH), 7.45 (dd, J = 7.5, 1.8 Hz, IH), 7.42-7.29 (m, 3H), 7.24 (dd, J = 8.9, 5.4 Hz, 2H), 7.18 - 7.05 (m, 5H), 6.99 - 6.91 (m, 4H), 6.65 (t, J = 7.4 Hz, IH), 6.15 (d, J = 7.0 Hz, IH), 5.91 (s, 1 H), 5.43 (dd, J = 9.8, 3.5 Hz, IH), 5.23 - 5.12 (m, 2H), 4.54 (d, J= 11.2 Hz, 4H), 4.33 - 4.09 (m, 7H), 3.69 (s, 3H), 3.42 - 2.78 (m, 32H, overlapping with DMSO), 2.39 - 2.20 (m,2H), 1.91 - 1.81 (m, IH), 1.77 (s, 3H), 1.46 (dd, J = 93.8, 31.3 Hz, 3H), 0.76 (dd, J= 13.8, 6.7 Hz, 6H); 19F NMR (376 MHz, DMSO-d6): -112.18 ppm

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyI)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d|pyriniidin-5-yl)-2-£hloro-3methylphenoxy)ethyl)-l-(2-(((l-(2-(((N-(20-carboxy-3,6,9,12,15,18hexaoxaicosyl)siilfamoyl)carbamoyl)oxy)ethyl)-lH-l,2,3-tnazol-4-yl)methoxy)methyl)-4((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethoxy)propanamido)-3517 methylbutanamido)-5-ureidopentanamido)benzyl)-l-inethylpiperazin-l-ium trifluoroacetate (L59-P1)

Following GENERAL PROCEDURE 2 with 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidm-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l -(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5dihydiO-lH-pynOl-l-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2((prop-2-yn-l-yloxy)methyl)benzyl)-l-methy]piperazîn-l-ium trifluoroacetate (32 mg, 0.021 mmol) and l “((N~((2-azidoethoxy)carbonyl)sulfamoyl)amino)-3,6,9,12,15,18-hexaoxahenicosan2i-oic acid (26.2 mg, 0.043 mmol), 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(2-(((1-(2-(((N-(20-carboxy3,6,9,12,15,18-hexaoxaicosyl)sulfamoyl)carbamoyl)oxy)ethyl)-l H-1,2,3-triazol-4yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-l H-pyrrol-1yl)ethoxy)propanamido)-3-methyIbutanamido)-5-ureidopentanamîdo)benzyl)-Imethylpiperazin-1-ium trifluoroacetate was obtained as a white powder: HRMS: M+ =2044.7700, Rt=2.36 min (5 min acidic).

Synthesis of 3-(2-(2-(2-((N-((2azidoethoxy)carbonyl)sulfamoyl)amino)ethoxy)ethoxy)ethoxy)propanoic acid 518

H

To 2-azidoethan-l-ol ( 105 mg, l ,21 mmol) in CH2CI2 (15 ml) was added sulfurisocyanatidic chloride (0.105 ml, l.2l mmol) at 0°C. The mixture was stirred at 0°C for 30 min. At 0°C TEA (0.336 ml, 2.41 mmol), and tert-butyl 3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy) propanoate (401 mg, >80% technical purity, 1.447 mmol) in CH2CI2 (1 mL) were added. After being stirred at 0°C for 1 h, then rt for 1 h, the mixture was quenched with satd. NH4CI, and 1 N HCl (2.4 mL). The aqueous was extracted with CH2CI2 (5X). The organic layers were dried over anh, Na2SO₄, filtered and concentrated via rotary évaporation to provide a clear oil. Following purification by flash chromatography (0-15% MeOH in CH2CI2, ELSD détection) tert-butyl 3-(2-(2-(2-((N-((2azidoethoxy)carbonyl)sulfamoyl)amino)ethoxy)ethoxy)ethoxy)propanoate was obtained as thick clear oil (356 mg, 0.910 mmol): LCMS: MS+NH4+=487.4, Rt=0.90 min (acidic, 2 min, ELSD); 1H NMR (400 MHz, DMSO-d6) δ 11.31 (s, 1 H), 7.78 - 7.70 (m, 1 H), 4.26-4.19 (m, 2H), 3.58 (td, J = 5.6, 5.0, 3.7 Hz, 4H), 3.53 -3.39 (m, 10H), 3.10 - 3.03 (m, 2H), 2.44 - 2.39 (m, 2H), 1.40 (s, 9H).

To tert-butyl 3-(2-(2-(2-((N-((2azidoethoxy)carbonyl)sulfamoyl)amino)ethoxy)ethoxy)ethoxy)propanoate (162 mg, 0.345 mmol) in CH2CI2 (1 mL) at 0°C was added TFA (1 mL, 13.0 mmol). After being stirred at rt for 2 h, the mixture was concentrated via rotary évaporation with a water bath at 25 °C. The residue was dried in high vac for 30 min, by azeotropic distillation with anh. Toluene (3X 2 mL), and in vacuo ovemight to provide 3-(2-(2-(2-((N-((2azidoethoxy)carbonyl)sulfamoyl)amino)ethoxy)ethoxy)ethoxy)propanoic acid as thick oil (139 mg, 0.335 mmol): LCMS: MS+NH4+=431.4, Rt=0.62 min (acidic, 2 min, ELSD): 1HNMR (400 MHz, DMSO-d6) δ 11.32 (d, J = 15.4 Hz, IH), 7.78 - 7.69 (m, l H), 4.27 - 4.15 (m, 2H), 3.67 - 3.54 (m, 8H), 3.49 - 3.42 (m, 4H), 3.07 (q, J = 6.0 Hz, 2H), 2.47 - 2.39 (m, 4H).

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidm-4yl)methoxy)phenyl)ethoxy )-6-(4-fluorophenyl)thieno [2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-(2-(((l-(2-(((N-(2-(2-(2-(2519 carboxyethoxy)ethoxy)ethoxy)ethyl)sulfamoyl)carbamoyl)oxy)ethyl)-lH-l,2,3-tnazol-4yl)methoxy)metliyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dîoxo-2,5-dihydro-lH-pyrrol-lyl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-lmethylpiperazin-l-iuin trifluoroacetate (L60-P1)

Foilowing GENERAL PROCEDURE 2 with 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyI)pyrimidm-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5dihydro-1 H-pyrrol-1 -yl)ethoxy)propanamido)-3-methylbuÎanamido)-5-ureidopentanamido)-2((prop-2-yn-I-yloxy)methyl)benzyI)-l-methylpiperazin-l-ium trifluoroacetate (15 mg, 10 μτηοΐ) and 3-(2-(2-(2-((N-((2-azidoethoxy)carbonyl)sulfamoyl)amino)ethoxy)ethoxy)ethoxy)propanoic acid (12 mg, 0.029 mmol), 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-nuorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methyîphenoxy)ethyl)-l-(2-(((l-(2-(((N-(2-(2-(2-(2carboxyethoxy)ethoxy)ethoxy)ethyl)sulfamoyl)carbamoyl)oxy)ethyl)-lH-l,2,3-triazol-4yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-l H-pyrrol-1yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-lmethylpiperazin-1-ium trifluoroacetate was obtained as a white powder: HRMS: MS+=Ï 912.7000, Rt=2.38 min (5 min acidic).

520

Synthesis of (2R,3R,4S,5R,6R)-2-(acctoxymethyl)-6-(2-(2-((tertbutoxycarbonyl)amino)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-trïyi triacetate

OAc | (WN ° .WA AcO OAc

OAc

A mixture of tert-butyl (2-(2-hydroxyethoxy)ethyl)carbamate (1535 mg, 7.48 mmol), Ag2CO3 (8248 mg, 14.96 mmol) and a piece of crystalline iodine in CH2CI2 (6 mL) was stirred with powdered 4A molécule sieve (1400 mg) for 15 min. To the mixture was added (2R,3R,4S,5R, 6R)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl triacetate (2050 mg, 4.99 mmol) in CH2CI2 (6.00 ml), also stirred with powdered 4A molécule sieve (1400 mg) for 15 min prior to addition. The resulting mixture was covered with aluminum foil and stirred at rt for 60 h, and then filtered through celite with EtOAc washîng. The filtrate was concentrated to give a clear oil that was purified by flash chromatography (0-10% MeOH in CH2CI2, ELSD détection) to provide, after concentration of appropriate fractions, a thick oil (640 mg) as a 47/53% mixture of the desired (2R,3R,4S,5R,6R)-2-(a.cetoxymethyl)-6-(2-(2-((tertbutoxycarbonyl)amino)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate and (2S,3aR,5R,6R, 7S, 7aR)-5-(acetoxymethyl)-2-(2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethoxy)2-methyltetrahydro-5H-[l,3]dioxolo[4,5-b]pyran-6,7-diyl diacetate: LCMS: MS+=536.4, Rt=0.96 min (2 min, acid, ELSD); IH NMR (400 MHz, DMSO-d6) δ 6.77 (s, 2H), 5.78 (s, 2H), 5.76 (s, IH), 5.28 (t, J = 9.5 Hz, IH), 5.04 (t, J = 3.1 Hz, IH), 4.91 (t, J = 9.7 Hz, IH), 4.87 4.74 (m, 3H), 4.38 (ddd, J = 5.2, 3.1, 0.9 Hz, IH), 4.24 - 4.10 (m, 3H), 4.07 - 3.96 (m, 2H), 3.92 (dt, J = 8.8, 4.1 Hz, IH), 3.80 (dt, J = 11.2, 4.4 Hz, IH), 3.68 - 3.60 (m, IH), 3.57 - 3.45 (m, 7H), 3.38 (td, J =6.2, 1.5 Hz, 7H), 3.07 (dt, J = 6.1, 3.0 Hz, 4H), 2.08 (s, 3H), 2.06 (s, 3H), 2.04 (s, 6H), 2.01 (s, 3H), 2.00 (s, 3H), 1.95 (s, 3H), 1.65 (s, 3H), 1.39 (s, 19H).

Synthesis of (2R,3R,4S,5R,6S)-2-(acetoxymethyl)-6-(2-(2-aminoethoxy)ethoxy)tetrahydro2H-pyran-3,4,5-triyl triacetate

OAc

To a 47/53% mixture of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(2-((tertbutoxycarbon yl)amino)ethoxy)ethoxy)tetrah ydro-2 H -pyran-3,4,5 -tri yl triacetate and (2S,3aR,5R,6R,7S,7aR)-5-(acetoxymethyl)-2-(2-(2-((tert521 butoxycarbonyl)amino)ethoxy)ethoxy)-2-methyltetrahydro-5H-[l,3]dioxolo[4,5-b]pyran-6,7-diyl diacetate (622 mg, i .161 mmol) in CH2CI2 (16 mL) at 0°C was added TFA (4.0 mL, 52 mmol). The reaction mixture was raised to Rt and stirred for 1 h. The mixture was concentrated and the residue was dried under vacuo for 60 min to give a light yellow oil. The crude product was diluted with DMSO (4 mL) and was purified by RP-HPLC ISCO gold chromatography (5-60% MeCN/H2O, 0.1% TFA modifier, ELSD détection). Upon lyophilization of appropriate fractions, (2R,3R,4S,5R,6S)-2-(acetoxymethyl)-6-(2-(2-aminoethoxy)ethoxy)tetrahydro-2Hpyran-3,4,5-triyl triacetate, as a TFA sait, was obtained as a white powder (195 mg, 0.355 mmol): LCMS: MS+=436.4, Rt=0.58 min (acidic, 2 min); 1H NMR (400 MHz, DMSO-d6) δ 7.72 (s, 3H), 5.27 (t, J = 9.4 Hz, IH), 4.92 (t, J = 9.6 Hz, IH), 4.86 - 4.75 (m, 2H), 4.22 - 4.15 (m, IH), 4.07-3.94 (m, 3H), 3.89 - 3.53 (m, 14H,mixed with DMSO), 3.03 - 2.91 (m, 2H), 2.03 (s, 3H), 2.01 (s, 3H), 1.99 (s, 3H), 1.94 (s, 3H).

Synthesis of 2-azidoethyl (N-(2-(2-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6(hydroxymethyI)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)sulfamoyl) carbamate

To 2-azidoethan-l-ol (16 mg, 0.18 mmol) in CH₂C12 (2.5 ml) was added sulfurisocyanatidic chloride (0.016 ml, 0.184 mmol) at 0°C. The mixture was stirred at 0 C for 1 h, then TEA (0.128 ml, 0.919 mmol) and (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(2aminoethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate, as a TFA sait (116 mg, 0.211 mmol) in CH2CI2 (1.5 mL) were added. After being stirred at 0°C for 1 h, then rt for 1 h, the mixture was quenched with satd NH4CI, and 1 N HCl (0.919 mL, 0.919 mmol). The aqueous was extracted with CH2CI2 (5X). The organic layer was over anh, Na₂SO4, fîltered and concentrated via rotary évaporation to afford (2Æ,3£,45,5£,6Â7-2-(acetoxymethyl)-6-(2-(2-((N((2-azidoethoxy)carbonyl)sulfamoyI)amino)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate as a clear oil (121 mg): LCMS: MS+=628, Rt=0.85 min (acidic, 2min, ELSD); IH NMR (400 MHz, DMSO-d6) δ 11.32 (s, IH), 7.79 - 7.71 (m, IH), 5.26 (t, J = 9.5 Hz, IH), 4.90 (t, J = 9.7 Hz, IH), 4.85 - 4.73 (m, 2H), 4.25 - 4.14 (m, 3H), 4.06 - 3.94 (m, 2H), 3.79 (ddd, J = 11.3, 5.3, 3.8 Hz, IH), 3.68-3.40 (m, 8H), 3.16-3.00 (m, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.98 (s, 3H), 1.94 (s, 3H).

522

The product above was dissolved in dioxane (3 mi) and cooled at 0°C. L1OH.H2O (0.5 M in water, 2.94 ml, l .47 mmol) was added. The resulting clear solution was stirred at rt for l h and then quenched at 0°C with HCl (5N, 0.147 mL, 0.735 mmol). The mixture was concentrated via rotary évaporation at 20 °C water bath to remove most of dioxane. The residual solution (ca. 3 mL) was purified by prep HPLC (Sunfire 5pm 30x50 mm column, 2-12% of Acetonitrile with 0.1% FA in Water. Flow Rate: 75 mL/min., MS 459.3, 476.3 détection) to provide, after removal of solvent of appropriate fractions via lyophilization, 2-azidoethyl (N-(2-(2-(((2R,3R,4S,5S,6R)3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2yl)oxy)ethoxy)ethyl)sulfamoyl)carbamate as a semi-solîd (64 mg, 0.14 mmol): LCMS: M+NH4+=477.3, MS-=458 (acidic, 2min, ELSD); IH NMR (400 MHz, DMSO-d6) δ 11.32 (s, IH), 7.71 (s, IH), 4.96 (d, J = 4.9 Hz, IH), 4.89 (dd, J = 13.7, 4.8 Hz, 2H), 4.47 (t, J = 5.9 Hz, IH), 4.22 (t, J = 5.0 Hz, 2H), 4.15 (d, J = 7.8 Hz, IH), 3.90-3.81 (m, IH), 3.67 (ddd, J = 12.0, 5.7,2.0 Hz, IH), 3.62-3.39 (m, 8H), 3.19-2.90 (m, 6H).

Syntheis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyI)pyrimidni-4yI)methoxy)phenyl)etlioxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5“yI)-2-chloro-3methylphenoxy)ethyi)-l-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentananiido)-2-(((1-(2-(( (N-(2-(2(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyI)tetrahydro-2H-pyran-2yl)oxy)ethoxy)ethyl)s ulfamoy I) ca r ba moyl)oxy)ethy 1)-1 H-1,2,3-triazol-4yl)methoxy)methyl)benzyl)-l-methylpiperazin-l-ium trifluoroacetate (L63-P1)

523

Following GENERAL PROCEDURE 2 with 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5dihydro-lH-pyrrol-l-yI)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2((prop-2-yn-l-yloxy)methyl)benzyl)-l-methylpiperazin-l-ium trifluoroacetate (36 mg, 0.022 mmol) and 2-azidoethyl (N-(2-(2-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6(hydroxymethyI)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)sulfamoyl)carbamate (22 mg, 0.048 mmol), 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-IH-pyrrol-lyl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(((l-(2-(((N-(2-(2(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2yl)oxy)ethoxy)ethyl)sulfamoyl)carbamoyl)oxy)ethyl)-lH-l,2,3-triazol-4yl)methoxy)methyl)benzyl)-l-methylpiperazin-l-ium trifluoroacetate was obtained as a white powder: HRMS: MS+=1958.6899, Rt=2.31 min (5 min acidic); IH NMR (400 MHz, DMSO-d6) δ 11.17 (s, IH), 10.16 (s, IH), 8.81 (d, J = 5.2 Hz, 1 H), 8.54 (s, IH), 8.07 (d, J = 13.5 Hz, 2H), 7.74 (d, J = 8.6 Hz, IH), 7.68 (q, J = 4.3, 2.7 Hz, 3H), 7.56 (d, J = 5.1 Hz, IH), 7.45 (dd, J = 7.6, 1.8 Hz, IH), 7.42-7.28 (m, 3H), 7.23 (ddd, J = 8.5, 5.4, 2.6 Hz, 2H), 7.17-7.04 (m, 5H), 6.99 -6.91 (m, 4H), 6.65 (t, J = 7.4 Hz, lH),6.14(dd, J = 7.6, 1.8 Hz, IH), 5.93 (s, IH), 5.43 (dd, J = 9.8,3.6 Hz, IH), 5.23-5.11 (m, 2H), 4.59 (dd, J = 11.3, 7.4 Hz, 8H), 4.42 - 4.06 (m, 12H),3.53 -3.17 (m, 27H, overlappîng with DMSO), 3.09 - 2.80 (m, 17H, overlapping with DMSO), 2.39 -2.20 (m, 3H), 1.86 (h, J = 6.9 Hz, IH), 1.77 (s, 3H), 1.68-1.21 (m, 5H), 0.76 (dd, J = 13.8, 6.8 Hz, 6H); 19F NMR (376 MHz, DMSO-d6): -112.18 ppm.

Synthesis of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-((2,2-dimethyl-4-oxo-3,8,l l-trioxa-5azatridecan-13-yI)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate

A mixture of tert-butyl (2-(2-(2-hydroxyethoxy)ethoxy)ethyl)carbamate ( 1046 mg, 4.20 mmol), AgzCOs (4627 mg, 8.39 mmol) and a piece of crystalline iodine in CH2CI2 (3 mL) was stirred with powdered 4A molécule sieve (700 mg) for 15 min. To the mixture was added (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl triacetate (1150

524 mg, 2.80 mmol) in CH2CÎ2 (3.00 mL) (also stirred with powdered 4A molécule sîeve (700 mg) for 15 min). The resulting mixture was covered with aluminum foil and stirred at rt for 60 h, then fïltered through celite with EtOAc washing. The filtrate was concentrated to give a clear oil. Purification via flash chromatography (20-100% EtOAc in heptane, ELSD détection) provided a 28/72% mixture of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-((2,2-dimethyl-4-oxo-3,8,l 1trioxa-5-azatridecan-l 3-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate and (2S,3aR,5R,6R,7S, 7aR)-5-(acetoxymethyl)-2-((2,2-dimethyl-4-oxo-3,8,l l-trioxa-5-azatrîdecan13-yl)oxy)~2-methyltetrahydro-5H-[l,3]dioxolo[4.5-b]pyran-6,7-diyl diacetate as a thick clear oil (307 mg, 0.529 mmol): LCMS: MS+=580.4, Rt=0.96 min (acid, 2 min, ELSD only); IH NMR (400 MHz, DMSO-d6) 6 6.74 (s, 3H), 5.76 (s, 8H), 5.26 (t, J = 9.5 Hz, IH), 5.02 (t, J = 3.1 Hz, 3H), 4.90 (t, J = 9.7 Hz, I H), 4.84 - 4.72 (m, 5H), 4.37 (ddd, J = 5.2, 3.1, 0.9 Hz, 3H), 4.18 (dd, J = 12.2, 5.0 Hz, IH), 4.11 (d, J = 4.5 Hz, 5H), 4.08 - 3.94 (m, 4H), 3.91 (dt, J = 8.6, 4.1 Hz, 3H), 3.85 -3.75 (m, IH), 3.66-3.58 (m, IH), 3.58-3.51 (m, 7H), 3.42-3.31 (m, 14H), 3.06 (q, J = 5.9 Hz, 8H), 2.07 (s, 7H), 2.05 (s, 7H), 2.02 (s, 10H), 1.99 (d, J = 1.0 Hz, 6H), 1.99 (s, 3H), 1.94 (s, 3H), 1.63 (s, 7H), L38 (s, 36H), 1.18 (t, J = 7.1 Hz, 4H).

Synthesis of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(2-(2aminoethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate

OAc

To a mixture of (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-((2,2-dimethyl-4-oxo-3,8,11trioxa-5-azatridecan-13-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate and (2S,3aR,5R,6R, 7S, 7aR)-5-(acetoxymethyl)-2-((2,2-dimethyl-4-oxo-3,8,1 l-trioxa-5-azatridecan13-yl)oxy)-2-methyltetrahydro-5H-[l,3]dioxolo[4,5-b]pyran-6,7-diyl diacetate (323 mg, 0.557 mmol) in CH2CI2 (8 mL) at 0°C was added TFA (1.9 mL, 25 mmol). After being stirred at rt for for 45 min, the mixture was concentrated and the residue was dried under vacuum for 60 min to give a light yellow oil. Purification via flash chromatography (0-20% MeOH in CH2CI2, with 0.2% NH4OH modifier in MeOH, ELSD détection) afforded (2R,3R,4S,5R,6R)-2(acetoxymethyl)-6-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate as a clear oil (82 mg, 0.171 mmol). LCMS: MS+=480.4, Rt=0.6l min (acidic, 2 min, ELSD); 1H NMR (400 MHz, DMSO-d6) δ 7.76 (s, 2H), 5.28 (t, J = 9.4 Hz, IH), 4.93 (t, J = 9.7 Hz, IH), 4.86 - 4.75 (m, 2H), 4.20 (dd, J = 12.2, 5.0 Hz, 1 H), 4.08 - 3.95 (m, 2H), 3.88 - 3.79 525 (m, IH), 3.67 - 3.53 (m, i IH, overlapping with DMSO), 3.00 (q, J = 5.5 Hz, 2H), 2.04 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.96 (s, 3H).

Synthesis of 2-azidoethyl (N-(2-(2-(2-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyI)sulfamoyl)carbamate

OH

To 2-azidoethan-l -ol (12.5 mg, 0.144 mmol) in CH2CI2 (2 mL) was added sulfürisocyanatidic chloride (0.012 ml, 0.14 mmol) at 0°C. The mixture was stirred at 0°C for 45 min, then TEA (0.040 ml, 0.29 mmol) and (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(2-(2aminoethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (77 mg, 0.16 mmol) in CH2CI2 (1 mL) were added. After being stirred at 0°C for Ih, then at rt for 1 h, the mixture was quenched with satd. NH4CI, and 1 N HCl (0.29 mL). The aqueous was extracted with CH2CI2 (5X). The organic layers were dried over anh. NasSCE, filtered and concentrated via rotary évaporation to give (2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(2-(2-((N-((2azidoethoxy)carbonyl)sulfamoyl)amino)ethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate as a clear oil (75 mg): LCMS: 0.97 min, MS+=672.4, 96, Rt-0.87 min (acidic, 2min, ELSD).

To the product above in dioxane (4 mL) at 0°C was added LÎOH.H2O (0.5 M in water, 3.45 ml, 1.72 mmol). The resulting clear solution was stirred at rt for 1 h and then was concentrated via rotary évaporation with 20°C water bath. The residue was purified by prep H P LC (Sunfire 5μηι 30x50 mm column, 2-12% of Acetonitrile with 0.1% FA in Water. Flow Rate: 75 mL/min., MS 503.5, 520.3 détection) to provide, after lyophilization, 2-azidoethyl (N(2-(2-(2-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2yl)oxy)ethoxy)ethoxy)ethyl)sulfamoyl)carbamate as a clear thin film (22 mg, 0.044 mmol): LCMS: MS+=504.3, Rt=0.52 min (acidic, 2min, ELSD); IH NMR (400 MHz, DMSO-d6) δ 11.31 (s, IH), 7.74 (s, 1 H), 4.96 (d, J = 4.9 Hz, IH), 4.89 (dd, J = 12.5,4.8 Hz, 2FI), 4.47 (t, J = 5.9 Hz, 1 H), 4.22 (t, J = 5.0 Hz, 2H), 4.15 (d, J = 7.8 Hz, 1 H), 3.93 - 3.82 (m, I H), 3.67 (dd, J = 11.2, 5.8 Hz, IH), 3.62-3.40 (m, 12H), 3.17 - 2.90 (m, 6H).

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrîmidin-5-yl)-2-chloro-3526 methylphenoxy)ethyl)-l-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(((l-(2-(((N-(2-(2(2-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2yl)oxy) ethoxy)eth oxy)ethyl)s ulfamoyl)car bamoyl)oxy )ethy l)-1 H-1,2,3-triazol-45 yl)methoxy)methyl)benzyl)-l-methylpiperazin-l-ium trifluoroacetate (L62-P1)

Following GENERAL PROCEDURE 2 with 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(210 methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5dihydro-1 H-pyrrol-l-yl)ethoxy)propanamido)-3-methyIbutanamido)-5-ureidopentanamido)-2((prop-2-yii-Lyloxy)methyl)benzyl)-l-methylpiperazin-l-ium trifluoroacetate (32 mg, 0.020 mmol) and 2-azidoethyl (N-(2-(2-(2-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-615 (hydroxymethyl)tetrahydro-2H-pyran-2=yl)oxy)ethoxy)ethoxy)ethyl)sulfamoyl)carbamate (21 mg, 0.042 mmol), 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-l H-pyrrol-1yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(((l-(2-(((N-(2-(2-(2527 (((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2yl)oxy)ethoxy)ethoxy)ethyl)sulfamoyl)carbamoyl)oxy)ethyl)-1 H-1,2,3-triazol-4yl)methoxy)methyl)benzyl)-l-methylpiperazin-l-ium trifluoroacetate was obtained as a white powder: HRMS: MS+=2002.7l00, Rt=2.37 min (5 min acidic).

Synthesis of Di-tert-butyl 3,3’-((3-((((9H-fluoren-9yl)mcthoxy)carbonyl)amino)propanoyl)azanediyl)dipropionate

To a mixture of dî-tert-butyl 3,3’-azanediyldipropionate (403 mg, 1.47 mmol), 3-((((9Hfluoren-9-yl)methoxy)carbonyl)amino)propanoic acid (505 mg, 1.62 mmol) and DIPEA (0.309 mL, 1.77 mmol) in CH2CI2 (3 mL) was added ÿV-(Dimethylaminopropyl)-M-ethyl-carbodiimide hydrochloride.HCl (367 mg, 1.92 mmol). After being stirred at ri for 2h, the mixture was quenched with satd. NH4CI, and extracted with CH2CI2 (3X). The combined organic phase was washed with brine, dried over anh. NasSCU, filtered and concentrated. The resulting crude product was purified by flash chromatography (0-50% EtOAc in heptane) to provide di-tert-butyl 3,3’-((3-((((9H-fluoren-9yl)methoxy)carbonyl)amino)propanoyl)azanediyl)dipropionate as a white foam (290 mg, 0.511 mmol): LCMS: MS+=567.5, Rt= 1.32 min (acid, 2 min); PMR: IH NMR (400 MHz, DMSO-d6) δ 7.94 - 7.88 (m, 2H), 7.70 (d, J = 7.5 Hz, 2H), 7.47 - 7.40 (m, 2H), 7.40 - 7.31 (m, 2H), 7.22 (t, J = 5.7 Hz, 1 H), 4.31 (d, J = 6.8 Hz, 2H), 4.26 - 4.18 (m, IH), 3.56-3.03 (m, 8H), 2.50 - 2.38 (m, 4H), 1.41 (s, 9H), 1.40 (s, 9H).

Synthesis of Di-tert-butyl 3,3’-((3-aminopropanoyl)azanediyl)dipropionate, l-(9H-fluoren9-yl)-N,N-dimethylmethanamme

I O O

N NH,

To di-tert-butyl 3,3’-((3-((((9H-fluoren-9yl)methoxy)carbonyl)amino)propanoyl)azanediyl)dipropionate (288 mg, 0.508 mmol) in CH2CI2

528 (3 mL) was added dimethylamine (2 N in THF, l mL, 2 mmol). The mixture was stirred at rt for l h. More dîmethylamine (2 N in THF, 1 mL, 2 mmol) was added. After being stirred for additional 4 h, the mixture was concentrated and the residue was purified by flash chromatography (0-25% MeOH in CH2CI2, 37 min, 0.2% NH4OH modifier was in MeOH, ELSD détection) to provide di-tert-butyl 3,3’-((3aminopropanoyl)azanediyl)dipropionate, l-(9H-fluoren-9-yl)-N,N-dimethylmethanamine as a light brown oil (62 mg, 0.472 mmol): LCMS: MS+=345.4, Rt=0.76 min (acide, 2 min, ELSD); IH NMR (400 MHz, DMSO-76) δ 4.05 (s, 2H), 3.51 (t, J = 7.2 Hz, 2H), 3.43 (i, J = 7.3 Hz, 2H), 3.31 (s, 2H), 2.79 (t, J= 6.4 Hz, 2H), 2.55 - 2.45 (m,17H, overlapping with DMSO), 2.41 (t,7=7.3 Hz, 2H), 1.41 (s, 9H), 1.40 (s, 9H).

Synthesis of 3,3’-((3-((N-((2azidoethoxy)carbonyl)sulfamoyl)aniino)propanoyl)azanediyl)dipropiomc acid

H H -O IJ

HO ' N ' NH N O ï ^HO^OH

To 2-azidoethan-l-ol (16 mg, 0.18 mmol) in CH2CI2 (2.5 ml) was added sulfürisocyanatidic chloride (0.016 ml, 0. IS mmol) at 0°C. The mixture was stirring at 0°C for 30 min, then TE A (0.051 ml, 0.37 mmol) and di-tert-butyl 3,3 ’-((3aminopropanoyl)azanediyl)dipropionate (73 mg, 0.21 mmol) in CH2CI2 (1 mL). After being stirred at 0°C for Ih and then rt for 1 h, the mixture was quenched with Satd NH4CI, and 1 N HCl (0.37 mL). The aqueous was extracted with CH2CI2 (5X). The organic layers were dried over anh. Na₂SO₄, filtered and concentrated via rotary évaporation. The resulting residue was purified by flash chromatography (0-10% MeOH in CH2CI2 , ELSD and UV214 détection) to provide di-tert-butyl 3,3’-((3-((N-((2azidoethoxy)carbonyl)sulfamoyl)amino)propanoyl)azanediyl)dipropionate, as a thick clear oil (70 mg, 0.13 mmol): LCMS MS+= 537.4 , Rt=1.08 min (acidic, 2 min, ELSD); IH NMR (400 MHz, DMSO-d6) δ 11.39 (s, IH), 7.65 - 7.59 (m, IH), 4.28 - 4.23 (m, 2H), 3.63 - 3.59 (m, 2H), 3.54 _ 3.47 (m, 2H), 3.47 - 3.38 (m, 2H), 3.18 - 3.09 (m, 2H), 2.61 - 2.54 (m, 4H), 2.43 (dd, J = 8.5, 6.0 Hz, 2H), i .43 (s, 9H), 1.42 (s, 9H).

To the product above in CH2CI2 (2 ml) at 0°C was added TFA (2 ml). After being stirred at rt for 1.5 h, the mixture was concentrated via rotary évaporation at 25 °C water bath. The residue was dried in high vac for 30 min, then by azeotropic distillation with anh. Toluene

529 (3X 3 mL), and further dried in high vac ovemight to provide 3,3’-((3-((N-((2azidoethoxy)carbonyl)sulfamoyl)amino)propanoyl)azanediyl)dipropionic acid as a white solid (72 mg, 77% by weight based on theoretical yield. It was used directly in the next step): LCMS MS+=425.3 , Rt=0.52 min (acidic, 2 min, ELSD); IH NMR (400 MHz, DMSO-d6) δ 12.28 (s, IH), 11.35 (s, IH), 7.58 (t, J = 5.8 Hz, IH), 4.26 - 4.20 (m, 2H),3.6l -3.55 (m, 2H), 3.50 (t, J = 7.4 Hz, 2H), 3.42 (t, J = 7.4 Hz, 2H), 3.12 (q, J = 6.8 Hz, 2H), 2.56 (dd, J = 15.1, 7.4 Hz, 4H), 2.43 (t, J = 7.4 Hz, 2H), 2.08 (s, IH).

Synthesis of l-(2-(((l-(2-(((N-(3-(bis(2-carboxyethyl)amino)-3oxopropyl)sulfamoyl)carbamoyl)oxy)ethyl)-lH-1,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2 ((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethoxy)propanamido)-3 methyIbutanamido)-5-ureidopentanamido)benzyI)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2inethoxyphenyl)pyriniidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyriniidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium trifluoroacetate (L61-P1)

Following GENERAL PROCEDURE 2 with 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l -(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5dihydro-lH-pynOl-l-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2((prop-2-yn-l-yloxy)methyl)benzyl)-l-methylpiperazin-l-ium trifluoroacetate (20 mg, 0.012

530 mmol) and 3,3’-((3-((N-((2azidoethoxy)carbonyl)sulfamoyl)amino)propanoyl)azanediyl)dipropionic acid (15 mg, 0.027 mmol), l -(2-((( l -(2-(((N-(3-(bis(2-carboxyethyl)amino)-3oxopropyl)sulfamoyl)carbamoyl)oxy)ethyl)-lH-l,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-l H-pyrrol-l-yl)ethoxy)propanamido)-3-methylbutanamido)5-ureidopentanainido)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-methylpiperazin-l-ium trifluoroacetate was obtained as a white powder HRMS: MS+= 1923.6500 Rt=2.34 min (5 min acidic); IH NMR (400 MHz, DMSO-d6) δ 11.21 (s, IH), 10.16 (s, IH), 8.81 (d, J = 5.1 Hz, IH), 8.54 (s, IH), 8.08 (d, J= 13.7 Hz, 2H), 7.77 7.50 (m, 5H), 7.47 - 7.20 (m, 6H), 7.18- 7.04 (m, 5H), 6.99 - 6.90 (m, 4H), 6.65 (t, J = 7.4 Hz, IH), 6.14 (dd, J = 7.6, 1.7 Hz, IH), 5.92 (s, IH), 5.43 (dd, J = 9.8, 3.5 Hz, IH), 5.24 - 5.11 (m, 3H), 4.65-4.51 (m, 9H), 4.45 - 3.81 (m, 52H, overlapping with DMSO), 3.68 (s, 3H), 3.542.77 (m, 3 IH), 2.39 - 2.20 (m, 5H), 1.86 (q, J = 6.8 Hz, 1 H), 1.77 (s, 3H), 1.69- 1.00 (m, 6H), 0.76 (dd, J = 13.9, 6.8 Hz, 6H); 19F NMR (376 MHz, DMSO-d6): -112.16 ppm.

Synthesis of 1-(2-(((1-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)lH-l,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-amino-3-methyIbutanainido)-5 ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2 inethoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-nuorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin-l-ium

531

Following GENERAL PROCEDURE 2 with l-(4-((S)-2-((S)-2-amino-3methyIbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-l-yloxy)methyl)benzyl)-4-(2-(4-(4((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimîdin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazinl-ium (87.5 mg, 0.063 mmoles, 1.0 equiv) and mPEG12-Azide (73.3 mg, 0.125 mmol, 2 equiv ), 1-(2-(((1-(2,5,8,1 l,14,17,20,23,26₎29,32,35-dodecaoxaheptatriacontan-37-yl)-lH-l,2,3-triazol-4yi)methoxy)methyl)-4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chioro-3-methylphenoxy)ethyl)-l methylpiperazin-l-ium was obtained. HRMS: M+= 1889.8544, Rt=2.19 min (5 min acidic method).

Synthesis of l-(2-(((l-(2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)lH-l,2,3-triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lHpyrrol-l-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamîdo)benzyl)-4(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyI)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methy Iphenoxy)ethyl)-1 -methylpiperazin- 1-ium (L 104-P1 )

Following GENERAL PROCEDURE 3 with 1-(2-(((1(2,5,8,1 l,I4,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-lH-l,2,3-triazol-4yl)methoxy)methyl)-4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)532

6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-lmethylpiperazin-1-ium (22 mg, 0.011 mmol, 1.0 equiv.), 1-(2-(((1(2,5,8,1 l,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontan-37-yl)-lH-l,2,3-triazol-4yl)m ethoxy )methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-l carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin1-ium was obtained. HRMS: M+= 2084.9099, Rt=2.45 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yi)methoxy)phenyl)ethoxy)-6-(4-fluorophenyI)thieno[2,3-d|pyrimidm-5-yi)-2-chloro-3methylphenoxy)ethyl)-l-(4-((S)-2-((S)-2-(3“(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-lyloxy)niethyl)benzyl)-l-methylpiperazin“l-ium h γ ;

Me Me

Foilowing GENERAL PROCEDURE 3 with l-(4-((S)-2-((S)-2-amino-3methyIbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-l-yloxy)methyI)benzyl)-4-(2-(4-(4((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin1-ium (155 mg, 0.119 mmol, 1.0 equiv.), 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxypheny!)pynmidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chIoro-3-methylphenoxy)ethyl)-l-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5dihydro-1 H-pyrrol-1 -yl)ethoxy)propanamido )-3-methylbutanamido)-5 -ureidopentanamido)-2((prop-2-yn-l-yloxy)methyl)benzyl)-l-methylpiperazin-l-ium was obtained. HRMS: M+= 1499.5699, Rt=2.39 min (5 min acidic method).

533

Synthesis ofl-(2-(((l-(2,5,8,1l,l4,l7,20,23,26,29,32,35,38,4l,44,47hexadccaoxanonatctracontan-49-yl)-lH-l,2,3-triazol-4-yl)methoxy)niethyl)-4-((S)-2-((S)-2(3-(2-(2,5-dîoxo-2,5-dihydro-lH-pyrrol-l-yl)ethoxy)propananiido)-3-methylbutananiido)-5ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyriinidin-4-yI)methoxy)phenyI)ethoxy)-6-(4-fluorophenyl)thieno|2,3d]pyrimidin-5-yI)-2-chloro-3-methylphenoxy)ethyl)-l-methyIpiperazin-l-ium (L34-PI)

Following GENERAL PROCEDURE 2 with 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyI)thieno[2,3d]pyriinidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5dihydro-lH-pyrrol-l-yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2((prop-2-yn-l-yloxy)methyl)benzyl)-l-methylpiperazin-l-ium (50 mg, 0.033 mmoles, 1.0 equiv) and m-PEG16-azide (from Broadpharm BP-23558) (50.8 mg, 0.067 mmol, 2 equiv), 1-(2-(((1(2,5,8,1 l,14,17,20,23,26,29,32,35,38,41,44,47-hexadecaoxanonatetracontan-49-yl)-lH-I,2,3triazol-4-yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pynOl-lyl)ethoxy)propanamido)-3-methyIbutanamido)-5-ureidopentanamido)benzyl)-4-(2-(4-(4-((R)-lcarboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazinl-ium was obtained. HRMS: M+=2261.0196, Rt=2.28 min (5 min acidic method).

534

Synthesis of l-(4-((S)-2-((S)-2-aniino-3-niethyibutanainido)-5-ureidopentanamido)-2-(((l((2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyI)-5-(((2S,3R,4S,5R,6R)-3,4,5triliydroxy-6-(hydroxyniethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)lH-l,2,3-tiïazol-4-yl)methoxy)methyl)benzyI)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(25 methoxyphenyl)pyrimidm-4-yI)methoxy)phenyl)ethoxy)-6-(4-flnorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methyipiperazin-1-ium

Following GENERAL PROCEDURE 2 with l -(4-((S)-2-((S)-2-amino-3methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-l-yloxy)methyl)benzyl)-4-(2-(4-(410 ((R)-1 -carboxy-2-(2-((2-(2-methoxyphenyI)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-I-methylpiperazin1-ium (87.5 mg, 0.063 mmoles, 1.0 equiv) and 1-azido-l-deoxy-beta-D-lactopyranoside (22.99 mg, 0.063 mmol, 1 equiv), l-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5ureidopentanamido)-2-(((l-((2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-515 (((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2yl)oxy)tetrahydiO-2H-pyran-2-yl)-lH-l,2,3-triazol-4-yl)methoxy)methyl)benzyl)-4-(2-(4-(4((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin1-ium was obtaîned. HRMS: M+= 1671.6400, Rt=1.95 min (5 min acidic method).

535

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyi)ethoxy)-6-(4-fluorophenyl)thicno[2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-(2-(((l-((2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5(((2S JR.4S.5R.6R)-3.4.5-trihydroxy-6-(hvdroxyrnethvl)(etraliydro-2H-pyran-2yl)oxy)tetrahydro-2H-pyran-2-yI)-lH-l,2,3-triazol-4-yl)methoxy)methyi)-4-((S)-2-((S)-2-(3(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yI)ethoxy)propanamido)-3-methylbutanamido)-5ureidopentanamido)benzyl)-l-methylpÎperazm-l-ium (L46-P1)

Following GENERAL PROCEDURE 3 with l-(4-((S)-2-((S)-2-amino-3methylbutanamido)-5-ureidopentanamido)-2-(((l-((2R,3R,4R,5S,6R)-3,4-dihydroxy-6(hydroxymethyl)-5-(((2S₎3R,4S,5R,6R)-3_}4₎5-trihydroxy-6-(hydroxymethyl)tetrahydro-2Hpyran-2-yl)oxy)tetrahydro-2H-pyran~2-yl)-lH-l,2,3-triazol-4-yl)methoxy)methyl)benzyl)-4-(2(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidm-4-y])methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-:methylphenoxy)ethyl)-l -methylpiperazin1-ium (35 mg, 0.020 mmol, 1.0 equiv.), 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(2-(((l-((2R,3R,4R,5S,6R)-3,4dihydroxy-6-(hydroxymethyl)-5-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)-l H-1,2,3-triazol-4yl)methoxy)methyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-lyl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-l536 methylpiperazin-l-ium was obtained. HRMS: M+= 1866.6899, Rt=2.28 min (5 min acidic method).

Synthesis 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimîdin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophcnyl)thieno[2,3-d]pyrimidin-5-yl)-2-chIoro-3niethylphenoxy)ethyl)-l-(4-((6S,9S,12S)-9-isopropyl-2,2-dimethyl-4,7,10-trioxo-6-(prop-2yn-l-yl)-12-(3-ureidopropyI)-3-oxa-5,8,ll-triazatridecan-13-amido)-2-((prop-2-yn-lyloxy)methyl)benzyl)-l-methylpiperazin-l-iuni

N ''N

NHBoc

To the mixture of Boc-Propargyl-Gly-OH (40 mg, 0.188 mmol, 1 equiv ) and HATU (71.3 mg, 0.188 mmol, 1 equiv) in DMF (0.5 ml) was added DIPEA (65.5 μΐ, 0.375 mmol, 2 equiv). The mixture was stirred at RT for 30 min. Then a solution of I-(4-((S)-2-((S)-2-amîno-3methylbutanamîdo)-5-ureidopentanamido)-2-((prop-2-yn-l-yloxy)methyl)benzyl)-4-(2-(4-(4((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazm1-ium (245 mg, 0.188 mmol, 1 equiv ) in DMF (1 ml) was added into the reaction mixture. The reaction mixture was stirred at RT for 30min. The crude mixture was separated with C18 column ( 100 cartridge, MeCN/Water with 0.1 % Formîc Acid, 0-100% over 15CV) to obtain 4(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyriniidin-4-yI)methoxy)phenyl)ethoxy)-6(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(4((6S,9S,12S)-9-isopropyl-2,2-dimethyl-4,7,10-trioxo-6-(prop-2-yn-l-yl)-12-(3-ureidopropyl)-3oxa-5,8,11-triazatridecan-13-amido)-2-((prop-2-yn-l-yloxy)methyl)benzyI)-l-niethylpiperazm1-ium. HRMS: M+= 1499.6000 , Rt= 2.91 min (5 min acidic method).

537

Synthesis l-(4-((S)-2-((S)-2-((S)-2-aminopent-4-ynamido)-3-methylbutanamido)-5ureidopentanamido)-2-((prop-2-yn-l-yloxy)methyl)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy )-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-cliloro-3-methylplienoxy)ethyi)-lmethylpiperazin-l-ium

At 0°C i ce-water bath, to 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(4-((6S,9S,12S)-9-isopropyl-2,2dimethyÎ-4,7,I0-trioxo-6-(prop-2-yn-l-yl)-l2-(3-ureidopropyl)-3-oxa-5,8,l l-triazatridecan-13amido)-2-((prop-2-yn-l-yloxy)methyl)benzyl)-l-methylpiperazin-l-ium (56 mg, 0.037 mmol) was added TFA (25% in DCM) 2 mL, Then the reaction mixture was raised to RT and stirred for Ih. The crude mixture was concentrated under high vacuum . Then the mixture was disolved in MeOH, and was purified by C-l 8 column(50 g cartridge, MeCN/Water with 0.1% Formic Acid 0-100% over 16 CV) to obtain l-(4-((S)-2-((S)-2-((S)-2-aminopent-4-ynamido)-3m ethylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn- 1-yl oxy)m ethyl )benzyl) -4-(2-(4-(4((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-methylpiperazin1-ium. HRMS: M+= 1399.5400 , Rt= 2.17 min (5 min acidic method).

538

Synthesis ofl-(4-((S)-2-((S)-2-((S)-2-amino-3-(l-((2R,3R,4R,5S,6R)-3,4-dihydroxy-6(hydroxymethyI)-5-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2Hpyran-2-yI)oxy)tetrahydro-2H-pyran-2-yI)-lH-l,2,3-triazol-4-yl)propanamido)-3methylbutanamido)-5-ureidopentanamido)-2-(((l-((2S,3S,4S,5R,6S)-3,4-dihydroxy-6(hydroxy methyl)-5-(((2 R,3 S,4R,5 S,6S)-3,4,5-trihydroxy-6-(hydr oxy methy l)tetr ahy dro-2 Hpyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)-lH-l,2,3-tnazol-4-yl)methoxy)methyl)benzyl)-4(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy )-6-(4-0 uorophenyl)thieno [2,3-d]pyrimidin-5-yI)-2-chloro-3methylphenoxy)ethyl)-l-methylpiperazin-l-ium

Following GENERAL PROCEDURE 2 with l-(4-((S)-2-((S)-2-((S)-2-aminopent-4ynamido)-3-methylbutanamido)-5-ureidopentanamido)-2-((prop-2-yn-l-yloxy)methyl)benzyl)-4(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyI)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-lmethylpiperazin-î-ium (44 mg, 0.031 mmol, LO equiv) and l-azîdo-l-deoxy-beta-Dlactopyranoside (69.2 mg, 0.188 mmol, 6 eq), l-(4-((S)-2-((S)-2-((S)-2-amino-3-(l((2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)-l H-1,2,3-triazol-4yI)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(((l-((2S,3S,4S,5R,6S)-3,4dihydroxy-6-(hydroxymethyl)-5-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6(hydroxymethyI)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)-1 H-l ,2,3-triazol-4yl)methoxy)methyl)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4

539 yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrîmidin-5-yl)-2-chloro-3methylphenoxy)ethyl)-I-methylpiperazin-l-ium was obtained. HRMS: M+= 2133.7800 , Rt= 1.95 min (5 min acidic method).

Synthesis of 4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-(2-(((l-((2S,3S,4S,5R,6S)-3,4-dihydroxy-6-(hydroxymethyl)-5(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymcthyl)tetrahydro-2H-pyran-2yl)oxy)tetrahydro-2H-pyran-2-yl)-lH-l,2,3-triazol-4-yl)methoxy)methyl)-4-((2S,5S,8S)-8((l-((2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-(((2S,3R,4S,5R,6R)-3,4,5tr ihydroxy-6-(hydroxy methyl) te trahyd ro-2 H-pyran-2-yI)oxy)tetr ahydro-2H-py r an-2 -y 1)lH-l,2,3-triazol-4-yl)methyl)-15-(2,5-dioxo-2,5-dihydro-l H-pyrrol-l-yI)-5-isopropyl-4,7,10trioxo-2-(3-ureidopropyl)-13-oxa-3,6,9-triazapentadecanamido)benzyl)-l-methylpîperazin1-ium (L47-P1)

Following GENERAL PROCEDURE 3 with l-(4-((S)-2-((S)-2-((S)-2-amino-3-(l((2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)-5-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)-1 H-l,2,3-triazol-4yl)propanamido)-3-methylbutanamido)-5-ureidopentanamido)-2-(((l-((2S_J3S_J4S,5R,6S)-3,4dihydroxy-6-(hydroxymethyl)-5-(((2R,3S_f4R,5S,6S)-3_J4,5-trihydroxy-6(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)-1 H-l ,2,3-triazol-4yl)methoxy)methyl)benzyl)-4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3

540 methylphenoxy)ethyl)-l-methylpiperazin-l-ium (19 mg, 0.009 mmol, LO equiv.), 4-(2-(4-(4((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidîn-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(2-(((l((2S,3S,4S,5R,6S)-3,4-dihydroxy-6-(hydroxymethyl)-5-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)-1 H-l,2,3-triazol-4yl)methoxy)methyl)-4-((2S,5S,8S)-8-((l-((2R,3R,4R,5S,6R)-3,4-dihydroxy-6-(hydroxymethyl)5-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2y!)oxy)tetrahydro-2H-pyran-2-yl)-lH-l,2,3-triazol-4-yl)methyl)-15-(2,5-dioxo-2,5-dihydro-lHpyrrol-l-yl)-5-îsopropyl-4,7,l0-trioxo-2-(3-ureidopropyl)-l3-oxa-3,6,9triazapentadecanamido)benzyl)-l-methylpiperazin-1-ium was obtained. HRMS: M+= 2328.8301, Rt=2.15 min (5 min acidic method).

l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amîno)-3-methylbutanamido)-5 ureidopentanamido)-2-(78-carboxy-2-niethyl-3-oxo7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76-tetracosaoxa-2,4diazaoctaheptacontyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)metlioxy)phenyl)-loxopropan-2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-lmethylpiperazin-l-ium

A mixture of 1 -amino3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72tetracosaoxapentaheptacontan-75-oic acid (50 mg, 0.044 mmol), bis(4-nitrophenyl) carbonate (13 mg, 0.043 mmol), and DIPEA (20 pL, 0.12 mmol) in THF (2 mL) was stirred at RT for 2 h. The mixture was concentrated by blowing nitrogen gas to it. The resulting solid residue was

541 taken up in DMF (1 mL). l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)amino)-3-methylbutanamido)5-ureidopentanamido)-2-((methyl amino )methyl)benzyI)-4-(2-(2-chlorO“4-(6-(4-fluorophen y l)-4(((R)-1 -((4-methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)inethoxy)phenyl)-1 oxopropan-2-yI)oxy)thienû[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyI)-l-methy]piperazin-lium (50 mg, 0.029 mmol) and DIPEA (100 gL, 0.573 mmol) were added. The mixture was stirred at RT for 5 min. The mixture was diluted with DMSO (2 mL), and the solution was purified by RP-HPLC ISCO gold chromatography (MeCN/HsO, 0.1 % TFA modifier). Upon lyophilization, l-(4-((S)-2-((S)-2-((tert-butoxycarbonyl)^am’ⁿo)-3-methylbutanamido)-5ureidopentanamido)-2-(78-carboxy-2-methyl-3-oxo7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76-tetracosaoxa-2,4diazaoctaheptacontyl)benzyl)-4-(2-(2-chloro-4-(6-(4-fluorophenyl)-4-(((R)-l-((4methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yI)methoxy)phenyl)-l-oxopropan2-yl)oxy)thieno[2_f3-d]pyriinidin-5-yl)-3-methyIphenoxy)ethyl)-l-methylpiperazîn-l-ium was obtained. HRMS: M+= 2671.2700, Rt=2.88 min (5 min acidic method).

4-(2-(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4yl)methoxy)phenyl)ethoxy)-6-(4-fluorophenyl)thieno[2,3-d]pyrimîdin-5-yl)-2-chloro-3methylphenoxy)ethyl)-l-(2-(78-carboxy-2-methyl-3-oxo7,10,13,l6,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,7Û,73,76-tetracosaoxa-2,4diazaoctaheptacontyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l“ yi)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)-lmethylpiperazin-l-ium (L42-P1)

542

Following GENERAL PROCEDURE 3 withl-(4-((S)-2-((S)-2-((tertbutoxycarbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)-2-(78-carboxy-2-methyI-3oxo-7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76-tetracosaoxa-2,4dîazaoctaheptacontyl)benzyI)-4-(2-(2-chIoro-4-(6-(4-fluorophenyl)-4-(((R)-1-((45 methoxybenzyl)oxy)-3-(2-((2-(2-methoxyphenyl)pyriinidin-4-yl)methoxy)phenyi)-l-oxopropan2-yl)oxy)thieno[2,3-d]pyrimidin-5-yl)-3-methylphenoxy)ethyl)-l -methylpiperazin-l-ium, 4-(2(4-(4-((R)-l-carboxy-2-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)ethoxy)-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-5-yl)-2-chloro-3-methylphenoxy)ethyl)-l-(2-(78-carboxy2-methyl-3~oxo-7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,7610 tetracosaoxa-2,4-diazaoctaheptacontyl)-4-((S)-2-((S)-2-(3-(2-(2,5-dioxo-2,5-dîhydro-lH-pyrrol1 -yl)ethoxy)propanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyI)-1 methylpiperazin-l-ium (L42-P1) was obtained. HRMS: M+= 2646.7700, Rt=2.38 min (5 min acidic method).

The following compounds were prepared using procedures similar to those described above.

Code	Linker Payload Structure	Synthetic Methods	Characterizations
L33- P1	.A,· h = il H P αΑΑΑ'Α'Ο) rLa L O ~ 0 J	General Procedure 2	HRMS: M+ 2142.9099, rt = 2.39 min. (5 min acidic method).

543

L38- P1	I fi I B I H γ n γ n γ n γ O ¹ O ¹ 0 ¹ Ο I o I 'N'^O yy . AzFxL· JL j y- Y^N^- Y N'Y ^οΐΑ O O Ïi ? r^N’Me QAY)A » „“v“o Y aFci TA_n Αγ γΎ A_^._o^^-n A ^hoyS va ^h J â ^h o ⁰ N \|Ty A-F H N '^J A ^s hjAo	Similar to synthesis of L35-P1 and using General Procedure 3	HRMS: M+ 2369.0400, rt = 2.11 min. (5 min acidic method).
L39- Pl	H ¹ oy o .A „ Ί । 1 1 n 1? 1 ? A λ N A Λ N A N. A. A A .O o^; n a ^Ny - N y - N y ^ rr y 1 0 1 O È O 1 O 1 □ 1 Ο I N^O jy Vy °<Α ο ¹ ο A Υ^-'^'Α-Α /'Ά’χ.,Α ο Me Me ^Οχ_.. Ll V^{X j}U A ? h A ° A. Άν /A⁰¹ ΑΑνγγ._Λγ^^^ -y_o Ας ^Η J A & ⁰ Ν ΑτΑγΑρ HN·^ k'AsA=/ _η=νλ_ο	Similar to synthesis ofL35-Pl and using General Procedure 3	HRMS: M+ 2795.2537, rt = 2.07 min. (5 min acidic method).
L40- Pï	B i ? 1 H 1 fl 1 ΝΑγ_ΝΑγ_ΝΑγ_Νγγ o ¹ o ¹ o ¹ o F ¹ 11 ¹ 11 1 B 1 ? iL A A .--w ,bk ,A A. ^ONA ^N T ^N A ^N Y n γ l o । o । o । o । .--A o । o । ^χνΆ riAl -w. A A Λ. A^kA AJ 1 TA T ''o'A^ °<A ° ⁰ p ï A^Me _a;<. °.Yw A ,aA- Î Me Me L I ?^^NxA ΎΑ h h ^Ύ b A - Ί aAci ΧΑ^νΑνΑ-ο^υ ^ΗΟγΑ_ο AT ^H J ο ^H o ° Fy?—tt~f ^ha N ^{S —} H_;nA	Similar to synthesis ofL35-Pl and using General Procedure 3	HRMS: M+ 3221.4653, rt = 2.05 min. (5 min acidic method).

544

L43- P1	s Q HR ? ° J ^Ζ“Ά O O O O ( V Q-YH ) O 0 O O 2 PA 0 Ό _VH A H ° P? M ° / ° ) Z ZZE '--' ) O ~ 0 IZ \ r ? O G	Similar to synthesis ofL35-Pl and using General Procedure 3	HRMS: M+ 2673.7827, rt = 2.46 mîn. (5 min acidic method).
L44- Pl	-o^° O ^-=0.--=^0 ^=_Q^=^, ο -..-=0--=-^⁰ 'ν'-'Ό Y 7- A Α-γΑ o _H c A _ oJGp,^ RAvA^A' CK Ha r ° ^H ° ho~/o __ nh T L Y Y w A ° n Y -yL/ ° ^nh3	Similar to synthesis ofL35-Pl and using General Procedure 3	HRMS: M+ 2673.7832, rt = 2.39 min. (5 min acidic method).
L45PI	⁵ 'Q hb Hh Ga-q <o> t Q iA > o o c o ) σΑ A v A ' p 2 ¹ ^ΊΤ \—Z \ O ^J Y-./ U A o ? o ^ο=Αγ>	Similar to synthesis ofL35-Pl and using General Procedure 3	HRMS: M+ 2644.2253, rt = 2.50 min. (5 min acidic method).

545

L95- Pl	Yyo o o . 4. 3 „ ^xz _x T 7 O ZZ zY —; 0=^ W o '--V / zz À I ï lO φχχχ /° / aY / O y^Z Z-- \=i 7=0 w °	Similar to synthesis ofL71-PI	HRMS: M+ 1725.6000, rt = 2.32 min. (5 min acidic method)
LlOlPl	HO ΑΧΌΗ Η0ΑΊ. \ _zo '7 o ^HNA 0 Lo 0. L.o A γΑθ aA yS Αφ xX cV a ° mY	Similar to synthesis ofL62-Pl	HRMS: M+ l962.7100,rt = 2.50 min. (5 min acidic method).
L105- Pl	,0H Ht hoAa ÔH o HN-q _t£'NH o. Yj] A AA. A OC U ^H J o ^M 0 ίΎ^ΰ C ^hcAo AA ^NH ° Vax °^ΛνΗϊ SiA —⁷	Similar to synthesis ofL62-Pl	HRMS: M+ 1962.7100,11 = 2.50 min. (5 min acidic method).

546

The synthetic methods for preparing the polyethylene glycols in L43-P1, L44-P1 and L45-P1 are described below.

Synthesis of 2-oxo-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75tetracosaoxa-3-azaoctaheptacontan-78-oic acid

To a stirred solution ofl-amino3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72tetracosaoxapentaheptacontan-75-oic acid (100 mg, 0.087 mmol, 1.0 equiv.) and DIPEA (24.8 mg, 34 pL, 0.192 mmol, 2.2 equiv.) in dichloromethane (0.5 mL) was added acetic anhydride (8.9 mg, 8.25 pL, 1.0 equiv.). The resulting mixture was stirred at ambient température for 1.5 hours. The solvent was removed under reduced pressure. The resulting residue was taken up in DMSO (1 mL) and purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization 2-oxo6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72,75-tetracosaoxa-3azaoctaheptacontan-78-oic acid (62.3 mg, 0.052 mmol, 60% yield) was obtaîned. LC/MS [ΜΗ]- 1187.3 Rt=0.75 min. (2 min acidic method). 1H NMR (400 MHz, DMSO-d6) Ô 7.86 (s, 1 H), 3.60 (t, J = 6.4 Hz, 3H), 3.50 (d, J = 4.9 Hz, 91 H), 3.40 (t, J = 5.9 Hz, 2H), 3.18 (q, J = 5.8 Hz, 2H), 2.44 (t, J = 6.4 Hz, 2H), 1.80 (s, 3H).

Synthesis of 4-oxo-3,8,ll,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77pentacosaoxa-5-azaoctacontan-80-oic acid

To a stirred solution of 1-amino3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72

547 tetracosaoxapentaheptacontan-75-oic acid (100 mg, 0.087 mmol, l.O equiv.) and DIPEA (24.8 mg, 34 pL, 0.192 mmol, 2.2 equiv.) in dichloromethane (0.5 mL) was added ethyl chloroformate (9.5 mg, 8.34 pL, 1.0 equiv.). The resulting mixture was stirred at ambient température for 1.5 hours. The solvent was removed under reduced pressure. The resulting residue was taken up in DMSO (1 mL) and purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization 4-oxo3,8,11,14,17,20,23,26,29,32,3 5,38,41,44,47,50,53,56,59,62,65,68,71,74,77-pentacosaoxa-5azaoctacontan-80-oic acid (75 mg, 0.062 mmol, 71 % yield) was obtained. LC/MS [M-H]1217.3 Rt=0.81 min. (2 min acidic method). IH NMR (400 MHz, DMSO-d6) Ô 7.03 (s, IH), 3.97 (q, J = 7.1 Hz, 2H), 3.60 (t, J = 6.4 Hz, 2H), 3.50 (d, J = 5.0 Hz, 92H), 3.40 (t, J = 6.1 Hz, 2H), 3.11 (q, J = 5.9 Hz, 2H), 2.45 (q, J = 6.5 Hz, 2H), 1.15 (t, J = 7.1 Hz, 3H,).

Synthesis of 4-oxo-2,8,11,l 4,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77pentacosaoxa-5-azaoctacontan-80-oîc acid

OMe

To a stirred solution of 1-amino3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72tetracosaoxapentaheptacontan-75-oic acid (100 mg, 0.087 mmol, 1.0 equiv.) and DIPEA (24.8 mg, 34 pL, 0.192 mmol, 2.2 equiv.) in dichloromethane (0.5 mL) was added methoxyacetyl chloride (11,36 mg, 9.57 pL, 1.2 equiv.). The resulting mixture was stirred at ambient température for l .5 hours. The solvent was removed under reduced pressure. The resulting residue was taken up in DMSO (I mL) and purified by RP-HPLC ISCO gold chromatography (10-100% MeCN/H2O, 0.1% TFA modifier). Upon lyophilization 4-oxo2,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50,53,56,59,62,65,68,71,74,77-pentacosaoxa-5azaoctacontan-80-oic acid (69 mg, 0.057 mmol, 65% yield) was obtained. LC/MS [M-H]1217.4 Rt=0.75 min. (2 min acidic method). IH NMR (400 MHz, DMSO-d6) δ 7.68 (s, IH), 3.79 (s, 2H), 3.60 (t, J = 6.4 Hz, 2H), 3.51 (s, 92H), 3.43 (t, J = 6.0 Hz, 2H), 3.30 (s, 3H), 3.26 (q, J = 6.0 Hz, 2H), 2.44 (t, J = 6.4 Hz, 2H).

548

Example 3. Synthesis and Characterization of Mcl-l Payloads

Exemplary payloads were synthesized using excmplary methods described in this example.

Préparation of Cl:

(2/?)-2-{[(55^)-5-{3-chloro-2-methyl-4-(2-(4-methylpiperazm-l-yl)ethoxy] phenyl}-6-(4fluorophenyI)thieno[2,3-rfJpyrimidin-4-yl]oxy}-3-(2-{[2-(2-methoxyphenyl)pyriniidin-4 yl]methoxy}phenyl)propanoic acid

Cl was prepared according to Example 30 in WO 2015/097123.

Préparation of C2:

(2J?)“2-[(5S_a)-5-[3-chloro-2-methyl-4-[2-[4-methyl-4-(3-sulfopropyl) piperazin-4-ium-l-yl]ethoxy]phenyl]-6-(4'fluoro-3-hydroxy-phenyl)thieno[2,3-i/] pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxypheiiyl)pyrimidm-4-yl]methoxy]phenyl]propanoic acid

Step A : 5-bramo-4-chloro-6-(4-fluoro-3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3d]pyrimidine

549

4.49 g 5-bromo-4-chloro-6-iodo-thieno[2,3-d]pyrimidine (l 1.96 mmol; obtained according to WO 2015/097123, Préparation la) and 4.31 g (4-fluoro-3-tetrahydropyran-2-yloxyphenyl)boronic acid (17.94 mmol) were dissolved in 60 mL THF, then 134 mg Pd(Oac)2 (0.60 mmol), 508 mg tBuXPhos (1.20 mmol), 11.69 g Cs2CO3 (35.88 mmol) and 60 mL water were added and the mixture was stirred at 70°C under N 2 atmosphère until no further conversion was observed. Then it was diluted with water, neutralized with 2 M aqueous HCl solution, and extracted with DCM. The combined organic layer was dried over Na₂SO₄, filtered and the fîltrate was concentrated under reduced pressure. The crude product product was purified via flash chromatography using heptane and EtOAc as eluents to give 5-bromo-4-chforo-6-(4-fluoro-3tetrahydropyran-2-yloxy-phenyl)thieno[2,3-d]pyrimidine. *H NMR (500 MHz, DMSO-dô) Ô: 9.02 (s, IH), 7.64 (dd, J= 7.9, 2.1 Hz, IH), 7.47 (dd, J = 11.0,8.5 Hz, IH), 7.36 (m, IH), 5.63 (m, 1 H), 3.81 (m, 1 H), 3.61 (m, IH), 1.94-1.78 (m, 3H), 1.69-1.50 (m, 3H). ^I3CNMR(125 MHz, DMSO-dô) δ: 166.6, 153.9, 153.1, 152.7, 144.3, 139.2, 127.7, 126.6, 124.2, 119.9, 117.1, 100.7, 97.2, 61.6, 29.5, 24.5, 18.2. HRMS calculated for CnHuWASBrClF: 441.9554; found 442.9624 (M+H).

Step B: ethyl (2R)-2-[5-bromo-6-(4-fluoro-3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3d]pyrimidin-4-yl] oxy-3-[2-[[2-(2-methoxyphenyl)pyi'lmldin-4yl] methoxy]phenyl]propanoate

3.09 g 5-bromo-4-chloro-6-(4-fluoro-3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3d]pyrimidine (6.97 mmol), 3.28 g ethyl (2R)-2-hydroxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4yl] methoxy] ph en yl]propanoate (8.02 mmol; obtained according to WO 2015/097123, Préparation 3bs) were dissolved in 70 mL /eri-butanol, then 6.82 g Cs₂CO3 (20.9 mmol) was added and the mixture was stirred under N₂ atmosphère at 70°C until no further conversion was observed. Then it was diluted with water, neutralized with 2 M aqueous HCl solution, and extracted with DCM. The combined organic layer was dried over Na₂SO₄, filtered and the fîltrate was concentrated under reduced pressure. The crude product was purified via flash chromatography using heptane and EtOAc as eluents to give ethyl (2R)-2-[5-bromo-6-(4-fluoro3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate as a mixture of diastereoisomers. HRMS calculated for C₄oH₃₆N₄07SBrF: 814.1472; found 815.1539 (M+H).

550

Step C: ethyl (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl]-6(4-fluoro-3-tetrahydropyran-2-yloxy~phenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2][2-(2-methoxyphenyl)pyrimidin-4-yl] methoxy]phenyl]propanoate

3.65 g ethyl (2R)-2-[5-bromo-6-(4-fluoro-3-tetrahydropyran-2-yloxy-pheny!)thieno[2,3d]pyrimidm-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]niethoxy]phenyl]propanoate (4.47 mmol) and 2.12 g l-[2-[2-chloro-3-methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2yl)phenoxy]ethyl]-4-methyl-piperazine (5.36 mmol; obtained according to WO 2015/097123, Préparation 5b) were dissolved in 22 mL dioxane, then 315 mg PdCb^xAtaPhos (0.45 mmol), 4.37 g CS2CO3 (13.41 mmol) and 22 mL water were added and the mixture was stirred at 70°C under N2 atmosphère until complété conversion. Then it was diluted with water, neutralized with 2 M aqueous HCl solution, and extracted with EtOAc. The combined organic layer was dried over Na₂SO₄, fîltered and the filtrate was concentrated under reduced pressure. The crude product product was purified via flash chromatography using EtOAc and MeOH, then DCM and MeOH as eluents to give ethyl (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-lyl)ethoxy]phenyl]-6-(4-fluoro-3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3-d]pyrimîdin-4yi]oxy-3-[2-[[2-(2-inethoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate as a mixture of two diastereoisomer pairs. HRMS calculated for C54H56N6O8SCIF: 1002.3553; found 1003.3614 and 1003.3622 (M+H).

Step D: (2R)-2-[(5Sa)-5-[3-chloro-2-methyl~4-f2-(4-meihylpiperazin-I-yl)ethoxy]phenyl]-6(4-Jluoro-3-teirahydropyran-2-yloxy-phenyl)thieno[2,3-d]pyrimidîn-4-yl]oxy-3-[2[[2-(2-methoxyphenyl)pyrimidin-4-yl] methoxy]phenyl]propanoic acid

3.47 g ethyl (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-1 -yl)ethoxy]phenyl]-6-(4' fluoro-3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate (3.46 mmol) was dissolved in 35 mL dioxane, then 1.45 g LiOH^xH₂O (34.6 mmol) and 35 mL water were added. The mixture was stirred at room température until complété hydrolysis. Then it was diluted with water, acîdified to pH 4 with 2 M aqueous HCl solution, and extracted with DCM. The combined organic phase was dried over Na₂SO₄, fîltered and the filtrate was concentrated under reduced pressure. The atropisomers were purified and separated via préparative reverse phase chromatography using 25 mM aqueous NH4HCO3 solution and MeCN as eluents. The atropisomer pair eluting later was isolated as (2R)-2-[(5Sa)-5-[3-chloro-2-methyl-4-[2-(4methylpiperazin-l-yl)ethoxy]phenyl]-6-(4-fluoro-3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3~

551

d]pyrimidin-4-yl]oxy~3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl] methoxy] phenyl] propanoic acid HRMS calculated for C52H52N6O8SCIF: 974.3240; found 975.3303 (M+H).

Step E: (4-methoxyphenyl)methyl (2R)-2-[(5Sa)-5-[3-chloro-2-methyl-4-[2-(4methylpiperazin-1 -yl)ethoxy]phenyl] -6-(4-jluoro-3-tetrahydropyran-2-yloxyphenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4yl] met h oxy]phenyl]propanoate

2.39 g (2R)-2-[(5Sa)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl]6-(4-fIuoro-3-tetrahydropyran-2-yloxy-phenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid (2.45 mmol), 1.13 g DTBAD (4.91 mmol) and 1.29gPPh3 (4.91 mmol) were dissolved in 49 mL toluene, then 0.61 mL PMBOH (4,91 mmol) was added and the reaction mixture was stirred at 50°C until complété conversion. Then the mixture was diluted with DCM and then concentrated under reduced pressure and then purified via flash chromatography, using heptane and EtOAc as eluents to give (4-methoxyphenyl)methyl (2R)-2-[(5Sa)-5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-lyl)ethoxy]phenyl]-6“(4-fluoro-3-tetrahydiOpyran-2-yloxy-phenyl)thieno[2,3-d]pyrimidin-4yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate as a mixture of diastereoisomers. HRMS calculated for CôoHôoNôOqSCIF: 1094.3815; found 1095.3880 (M+H).

Step F: (2'R)-2-[(5‘&d-5-[3-chloro-2-meihyl-4-l2-[4-methyl-4-(3-sidfopropyl) piperazin-4-ium-1-yl] ethoxy]phenyl]-6-(4-fluoro-3-hydroxy-phenyl)thieno[2,3-(3] pyfimidin-4-yl] oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4yl] methoxy]phenyl]propanoic acid (C2)

600 mg (4-methoxyphenyl)methyl (2R)-2-[(5Sa)-5-[3-chIoro-2-methyl-4-[2-(4methylpiperazin-l-yl)ethoxy]phenyl]-6-(4-fluoro-3-tetrahydropyran-2-yloxy-phenyl)thîeno[2,3d]pyrimidin-4-yl] oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidm-4-yl]methoxy] phenyl] propanoate (0.548 mmol) was dissolved in 11 mL MeCN, then 0.48 mL oxathiolane 2,2-dioxide (5.48 mmol) was added, and the mixture was stirred under N2 atmosphère at 60°C until complété conversion. Then it was concentrated under reduced pressure, dissolved in 8 mL DCM, then 2.2 mL TFA was added and mixture was stirred at room température until complété cleavage of ΤΗΡ and PMB. Then it was concentrated (heating bath removed). It was dissolved in 10 mL THF, and concentrated again under reduced pressure in 30°C bath. The crude product was purified via préparative reverse phase chromatography using 5 mM aqueous NH4HCO3 solution and MeCN as eluents to obtain give C2. ^lH NMR (400 MHz, DMSO-dô) δ: 13.19 (br s, IH), 10.16 (br s,

552

IH), 8.89 (d,J = 5.2 Hz, IH), 8.58 (s, IH), 7.68 (br s, IH), 7.52 (dd, J=7.5, 1.8 Hz, IH), 7.46 (m, IH), 7.33 (d, J= 8.3 Hz, IH), 7.22-7.09 (m, 4H), 7.06-7.00 (m, 2H), 6.86 (dd, J = 8.3, 2.0 Hz, 1 H), 6.74 (t, J =7.4 Hz, IH), 6.66 (m, IH), 6.23 (d, J =6.7 Hz, IH), 5.46 (dd, J =9.8, 3.3 Hz, IH), 5.27 (d, J= 15.2 Hz, IH), 5.22 (d, J= 15.2 Hz, IH), 4.23 (m, 2H), 3.76 (s, 3H), 3.46 (m, 2H), 3.41-3.23 (m, 5H), 2.97 (s, 3H), 2.94-2.77 (m, 6H), 2.48 (m, IH), 2.45 (t, J= 7.0 Hz, 2H), 2.00-1.90 (m, 2H), 1.86 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ: 170.8, 166.2, 165.9, 164.7, 157.7, 157.2, 155.4, 153.6, 152.7, 152.3, 149.9, 145.1, 137.0, 135.9, 131.0, 130.8, 130.3, 129.2, 128.34, 128.32, 128.2, 128.0, 122.0, 120.5, 120.1, 118.8, 118.2, 116.6, 115.6, 112.2, 111.9, 110.6, 73.3, 69.0, 67.3, 59.2, 59.1, 55.71, 55.68,47.6, 46.1,31.8, 18.1, 17.6. HRMS calculated for C50H50N6O10S2CIF: 1012.2703; found 1013.2775 (M+H).

Préparation of C3:

(2R)-2-{[(5Sa)-5-{3-chloro-2-methyl-4-[2-(piperazin-l-yl)ethoxy]phenyl}-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2-(2-methoxyphenyl)pyrîmidîn-4 yl]methoxy}phenyl)propanoic acid

C3 was prepared according to Example 744 in WO 2015/097123.

Préparation of C4:

(2R)-2-{[(5Sa)-5-{3-chloro-2-niethyI-4-[2-(piperazin-l-yl)ethoxy]phenyl}-6-(4f]uorophenyl)thieno(2,3-d|pyrimidin-4-yl]oxy}-3-(2-{[2-(2-methoxyphenyl)pyrimîdin-4 yl]methoxy}phenyl)propanoic acid

553

Step 1: ethyl (2R)-2-[5-bromo-6-(4-fhiorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-])(2chloropyrimidin-4-yl)methoxy]phenyl]propanoate

To a solution of ethyl (2R)-3-[2-[(2-chloropyrimidin-4-yl)methoxy]phenyl]-2-hydroxypropanoate (25 g, 74,2 mmol) in THF (38 mL) were successively added 5-bromo-6-(4fluorophenyl)-4-iodo-thieno[2,3-d]pyrimidine (23 g, 67.5mmol) and césium carbonate (67 g, 203 mmol). The reaction was heated at reflux ovemight, and the volatiles were evaporated. The residue was diluted with ethyl acetate and water (respectively 500 and 400 mL) and the solution is filtered. The organic layer was separated, washed with brine, dried over magnésium sulfate and concentrated under vacuum. The residue was purified by silica gel chromatography (gradient of ethyl acetate in petroleum ether to afford ethyl (2R)-2-[5-bromo-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-4-yl]oxy-3-[2-[(2-chloropyrimidin-4-yl)methoxy]phenyl]propanoate as a slightly orange solid. Ή NMR (400 MHz, dmso-d6): δ 8.83 (d, IH), 8.65 (s, IH), 7.75 (m, 2H), 7.71 (d, IH), 7.48 (d, IH), 7.45 (m, 2H), 7.25 (t, IH), 7.06 (d, IH), 6.95 (t, IH), 5.75 (dd, IH), 5.28 (2*d, 2H), 4.18 (q, 2H), 3.6/3.3 (2*dd, 2H), 1.12 (t, 3H). IR Wavelenght (cm’¹): 1749.

Step 2: (4-bronw-2-chloro-3-methyl-phenoxy)-triisopropyl-silane

To a solution of 4-bromo-2-chloro-3-methyl-phénol (100 g, 482 mmol) in dichloromethane (1.5 L) were added imidazole (82 g, 1.2 mol) and dropwise over 1 h chloro(triisopropyl)silane (102 mL, 482 mmol). The reaction was stirred at room température for 1 h and water was added (500 mL). The organic layers were washed with brine (200 mL), dried over Magnésium sulfate and concentrated. The residue was used without further purification. ’H NMR (400 MHz, CDCh): δ 7.48 (s, IH), 7.2 (dd, IH), 6.7 (d, IH), 1.3 (m, 3H), 1.1 (2s, 18H).

Step 3: tert-butyl-[2-chloro-3-methyl-4-(4,4,5,5-tetramethyl-l,3,2~dioxaborolan-2yl)phenoxy]-dimelhyl-silane

To a solution of (4-bromo-2-chloro-3-methyl-phenoxy)-triisopropyl-silane (27.2 g, 71.9 mmol) in THF (350 mL) at -78°C under Argon was added dropwise over 30 min a solution of nbutyl lithium 1.6 M tn THF (49.5 mL, 79.9 mmol). The reaction was stirred at -78 °C for 2 h and a solution of 2-Isopropoxy-4,4,5,5-tetramethyl-L3,2-dioxaborolane (16.1 g, 86.4 mmol) in THF (50 mL) was added dropwise over 30 min. After 2h stirring at -78°C, the reaction mixture was quenched by a slow addition of water (20 mL) and warmed to room température, diluted with water (200 mL) and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Magnésium sulfate and concentrated under vacuum. The residue was used

554 without further purification. ^lH NMR (400 MHz, dmso-d6): δ 7.5 (d, IH), 6.82 (d, IH), 2.52 (s, 3H), 1.32 (m, 3H), 1.3 (s, 12H), 1.08 (s, 18H).

Step 4: 2-chloro-3-methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)phenol

To a solution of tert-butyl-[2-chloro-3-methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan2-yl)phenoxy]-dimethyl-si!ane (25.4 g, 59.8 mmol) in THF (750 mL) was added dropwise at room température a solution of Tetrabutylammonium Fluoride 1 M in THF (90 mL, 90 mmol). The reaction mixture was stirred for 2 h, concentrated, diluted with ethyl acetate, partitioned with water and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Magnésium sulfate and concentrated under vacuum. The residue was purified by silica gel chromatography (gradient of éthanol in dîchloromethane) to afford 2-chloro-3-methyl 4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)phenol. ^lH NMR (400 MHz, dmso-d6): δ 10.4 (m, IH), 7.4 (d, IH), 6.8 (d, IH), 2.5 (s, 3H), 1.3 (s, 12H).IR Wavelenght (cm·¹): 3580-3185, 1591,857, 827.

Step 5: ethyl (2R)-2-![(5S_a)-5-(3-chloro-4-hydroxy-2-methylphenyl)-6-(4fluorophenyl)lhieno[2,3-d]pyrimidin-4-yl]oxy}-3-{2-[(2-chloropyrimidin-4yl)methoxy]phenyl}propanoate

To a solution of ethyl (2R)-2-[5-bromo-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4yl]oxy-3-[2-[(2-chIoropyrimidin-4-yl)methoxy]phenyl]propanoate (43.8 g, 61.2 mmol) and 2chloro-3-methyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)phenol (19.7 g, 73.5 mmol) in a mixture of THF/H₂O 1/1 (800 mL) was added césium carbonate (40 g, 122 mmol). The reaction was degased by bubbling argon through the solution for 20 min and Bis(di-tert-butyl(4dimethylaminophenyl)phosphine)dichloropalladium(II) (4.35g, 6.1 mmol) was added . The reaction mixture was heated at 80°C under argon overnight. The reaction was diluted with water, partitioned with ethyl acetate and extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Magnésium sulfate and concentrated under vacuum. The residue was purified by silica gel chromatography (gradient of methanol in dîchloromethane) to afford ethyl (2R)-2-[5-(3-chloro-4-hydroxy-2-methyl-phenyl)-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-4-yl]oxy-3-[2-[(2-chloropyrimidin-4-yl)methoxy]phenyl]piOpanoate as a mixture of diastereoisomers 85/15 (aS/aR or S_a/R_a).Optically pure aS (or S_a) was obtaîned by Préparative SFC purification.

555

Step 6: tert-butyl 4-(2-{2-chloro-4-[4-{[(2R)-3-{2-[(2-chloropyrimidin-4-yl)methoxy]phenyl}l-ethoxy-l-oxopropan-2-yl]oxy}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-3methylphenoxy}ethyl)piperazine-I-carboxylate

To a solution of triphenylphosphîne (2.66 g, 10 mmol) in THF was added at room température under argon Diisopropyl azodicarboxylate (2.33 g, 10 mmol). After 15 min of stirring, was added a solution of tert-butyl 4-(2-hydroxyethyl)piperazine-l-carboxylate (2.33g, 10 mmol) in THF (8 mL). The reaction was stirred at room température for i h, then was added dropwise a solution of and (2R)-2-[(5S_a)-5-(3-chloro-4-hydroxy-2-methyl-phenyl )-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[(2-chloropyrimidin-4yl)methoxy]phenyl]propanoic acid (3.57 g, 5 mmol) in THF (8 mL). The reaction was stirred at room température for 96 h and concentrated. The residue was purified by silica gel chromatography (gradient of methanol (containing 7M ammonia) in dichloromethane) to afford tert-butyl 4-(2-{2-chloro-4-[4-{[(2R)-3-{2-[(2-chloropyrimidin-4-yl)methoxy]phenyl}-l-ethoxyl-oxopropan-2-yl]oxy}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-3methylphenoxy}ethyl)piperazine-l-carboxylate.^lH NMR (400 MHz, CDCfi): Ô 8.95 (d, IH), 8.58 (s, IH), 8.32 (d, 2H), 7.58 (d, IH), 7.41 (dd, 2H), 7.32 (d, IH), 7.29 (dd, 2H), 7.22 (t, 2H), 7.21 (d, IH), 7.19 (t, IH), 7.05 (d, IH), 6.75 (t, IH), 6.31 (dd, IH), 5.53 (dd, IH), 5.29 (dd, 2H), 4.2 (m, 2H), 4.05 (q, 2H), 3.97 (m, 4H), 3.3 (m, 2H), 3.2 (t, 4H), 3.19/2.59 (m, 2H), 2.72 (t, 2H), 2.4 (t, 4H), 1.87 (s,3H), 1.37 (s, 9H), 1.18 (t, 6H), 1.05 (t, 3H). ¹³CNMR(125 MHz, CDCh): δ 158, 152, 131, 131, 130, 130, 128, 127, 120.5, 116, 116, 112, 110, 73, 68.5, 67, 62,61,56, 52, 43,32, 32, 28, 17, 16, 14.

Step 7: tert-butyl 4-(2-{2-chloro-4-[4-{[(2R)-3-{2-[(2-{4[(dlethoxyphosphoryl)methy l]phenyl}pyrimidin-4 -y l)methoxy]phenyl}-!-ethoxy-1 oxopropan-2-yl] oxy}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-3methylphenoxy}ethyl)piperazine-l-carboxylate

To a solution of tert-butyl 4-(2-{2-chloro-4-[4-{[(2R)-3-{2-[(2-chloropyrimidin-4yl)methoxy]phenyl}-l-ethoxy-l-oxopropan-2-yl]oxy}-6-(4-fluorophenyl)thieno[2,3d]pyrimidin-5-yl]-3-methylphenoxy}ethyl)piperazine-l-carboxylate (337 mg, 0.367 mmol) and [4-(diethoxyphosphorylmethyl)phenyl]boronic acid (200mg, 0.735 mmol) in dioxane (2.5 mL), were added water (2.5 mL) and césium carbonate (241 mg, 0.735 mmol). The réaction mixture was degased by bubbling argon through the solution for 30 min, Bis(triphenylphosphine)palladium(II) dichloride (2.5 mg, 3.6 μιτιοί) was added and the reaction mixture was heated b y micro wave irradiation in a sealed vessel at 90 °C for 3 h. The reaction 556 was diluted with ethyl acetate and water. The aqueous layer was extracted with ethyl acetate and the combined organic layers were washed with brine, dried over Magnésium sulfate and concentrated under vacuum. The residue was purified by silica gel chromatography (gradient of methanol in dîchloromethane) to afford tert-butyl 4-(2-{2-chloro-4-[4-{[(2R)-3-{2-[(2-{4[(diethoxyphosphoryl)methyl]phenyl}pyrimidin-4-yl)methoxy]phenyl}-l-ethoxy-l-oxopropan2-yl]oxy}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-3-methylphenoxy}ethyl)piperazine-lcarboxylate.'H NMR (500 MHz, dmso-d6): δ 8.95 (d, IH), 8.58 (s, IH), 8.32 (d, 2H), 7.58 (d, IH), 7.41 (dd, 2H), 7.32 (d, IH), 7.29 (dd, 2H), 7.22 (t, 2H), 7.21 (d, IH), 7.19 (t, IH), 7.05 (d, IH), 6.75 (t, IH), 6.31 (dd, IH), 5.53 (dd, IH), 5.29 (2*d, 2H), 4.2 (m, 2H), 4.05 (q, 2H), 3.97 (m, 4H), 3.3 (m, 2H), 3.2 (t, 4H), 3.19/2.59 (2*dd, 2H), 2.72 (t, 2H), 2.4 (t, 4H), 1.87 (s, 3H), L37 (s, 9H), I.18 (t,6H), 1.05 (t, 3H). ¹³CNMR(125 MHz, dmso-d6) δ 158, 152, 131, 131, 130, 130, 128, 127, 120.5, 116, 116, 112, 110, 73,68.5,67, 62,61,56, 52,43, 32,32, 28, 17, 16, 14

Step 8: Synthesis of (2R)-2-[(5S_u)-5-[3-chloro-2-methyl-4-(2-piperazin-l-ylethoxy)phenyl]-6(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-[4(phosphonomethyl)phenyl]pyrimidin-4-yl] methoxy]phenyl]propanoic acid

To a solution of tert-butyl 4-(2-{2-chloro-4-[4-{[(2R)-3-{2-[(2-{4[(diethoxyphosphoryl)methyI]phenyl}pyrimidiu-4-yl)methoxy]phenyl}-l -ethoxy-l-oxopropan2-yl]oxy}-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-5-yl]-3-methylphenoxy}ethyl)piperazine-lcarboxylate (540 mg, 0.486 mmol) în dîchloromethane (5 mL) was added bromotrimethylsilane ( 186 pL, 1.46 mmol). The reaction mixture was heated to reflux ovemight. Another portion of bromotrimethylsilane was added at room température ( 186 pL, 1.46mmol) and the reaction was heated at reflux for 20h and concentrated to dryness. The residue was taken up in methanol, stirred at room température for 3h and concentrated to afford a brown viscous oil which was diluted with dîoxane (4 mL) and water (4 mL). Lithium hydroxide monohydrate (100 mg, 24 mmol) was added by portions and the reaction mixture was stirred at room température for 1 h, heated at 45°C for 3 h and concentrated. The residue was diluted with water (5 mL), acidified to pH2 by dropwise addition of an aqueous 2 M HCl solution. The precipitate was fdtered, washed with THF and purified by C18 reverse phase prep-HPLC by direct deposit of the reaction mixture on the Xbridge column and using the NH4HCO3 method to afford C4. *H NMR (500 MHz, dmso-d6): δ 8.88 (br d, 1 H), 8.25 (d, 2 H), 7.75 (t, l H), 7.59 (s, 1 H), 7.52 (d, 1 H), 7.35 (d, 2 H), 7.23 (dd, 2 H), 7.18 (d, 1 H), 7.15 (t, 2 H), 7.11 (t, 1 H), 7.02 (d, 1 H), 6.82 (d, 1 H), 6.64 (m, 1 H), 5.51 (d, 1 H), 5.28/5.07 (m, 2 H), 3.82/3.55 (2m, 2 H), 3.35/2.55 (br s, 2 H), 2.S1 (d, 2 H), 2.55 (m, 4 H), 2.4/2.27 (2m, 2 H), 2.21 (m, 4 H), 1.65 (br s, 3 H). ^I3CNMR(125 MHz, 557 dmso-d6): δ 131.5, 130.2, 129.7, 127.4, 127.2, 120.3, 115.9, 115.3, 111.9, 110.3, 75.1, 69.2, 67.3, 56.4,49.9, 42.4, 40, 38.9, 18.1.³¹P NMR (200 MHz, dmso-d6): δ 15 HR-ESI+ : m/z [M+H]+ = 925.2356/925.2346 (measured/theoretical)

Préparation of C5:

(2R)-2-{ [(5Sa)-5-{3-chloro-2-methyl-4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl}-6-(4fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy}-3-(2-{[2-(4-hydroxyphenyl)pyrimidin-4yl] methoxy} ph en yl)propanoic acid

C5 was prepared according to Example 3 in WO 2016/207216.

Préparation of C6:

(2//)-2-[ [(5^)-5-{3-chloro-2-methy 1-4-(2-(4- methyl piper azin-l-yl)eth oxy] phenyl }-6-(4 fluorophenyI)thieno[2,3-d]pyrimidin-4-yf|oxy}-3-[2-({2-[2(hydroxymethyl)phenyl]pyrimidin-4-yl}mcthoxy)phenyl] propanoic acid

C6 was prepared according to Example 728 in WO 2015/097123.

Préparation of C7:

558 (2R)-2-|(5Sa)-5-[3-chloro-2-ethyl-4-|2-(4-methylpiperazin-1 -yl)ethoxy]phenyl]-6-prop-1ynyl-thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4 yl] methoxy] phenyl] propanoic acid

Step A: ethyl (2R)-2-(5-iodo-6-prop-l-ynyl-thieno[2,3-d]pyrimidin-4-yl)oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl] methoxy]phenyl]propanoate

5.0 g 4-chloro-5-iodo-6-prop-l-ynyl-thieno[2,3-d]pyrimidine (15.0 mmol; obtained according to WO 2015/097123, Préparation 2f) and 6.10 g ethyl (2R)-2-hydroxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate (15.0 mmol; obtained according to WO 2015/097123, Préparation 3bs) were dissolved in 150 mL ierHbutanol, then 14.7 g CS2CO3 (45.0 mmol) was added and the mixture was stirred under N₂ atmosphère at 50°C until no further conversion was observed. Then water and brine was added and the mixture was extracted with EtOAc. The combined organic layer was dried over Na₂SO₄, filtered and the filtrate was concentrated under reduced pressure. The crude product was purified via flash chromatography using heptane and EtOAc as eluents to give ethyl (2R)-2-(5-iodo-6-prop-l-ynyl-thieno[2,3d]pyrimidin-4-yl)oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate. ^lH NMR (400 MHz, DMSO-d₆) Ô: 8.89 (d, J = 5.1 Hz, IH), 8.59 (s, IH), 7.62 (d, J= 5.2 Hz, IH), 7.55 (dd, J=7.5, 1.6 Hz, IH), 7.51 (dd, J = 7.5, 1.6 Hz, IH), 7.43 (m, IH), 7.26 (m, IH), 7.11 (m, 2H), 7.02 (td, J = 7.5, 0.9 Hz, 1 H), 6.94 (td, J = 7.4, 0.8 Hz, 1 H), 5.79 (dd, J = 9.1,4.6 Hz, IH), 5.31 (d, J= 14.9 Hz, IH), 5.26 (d,J= 14.9 Hz, 1H),4.13 (m, 2H),3.76 (s, 3H), 3.60 (dd, J = 13.8,4.5 Hz, IPI), 3.33 (m, IH), 2.21 (s, 3H), 1.10 (t, J = 7.1 Hz, 3H). ^I3C NMR (100 MHz, DMSO-ds) Ô: 169.4, 166.5, 165.7, 164.8, 161.3, 157.7, 155.8, 153.6, 132.2, 131.0, 130.8, 128.3, 124.0, 120.9, 120.1, 115.5, ] 12.2, 112.0, 110.5, 98.9,79.5, 74.4, 74.3, 69.1,61.1, 55.7, 13.9, 4.6.

559

Step B: 2-chloro-3-ethyl-4-(4,4,5,5-tetramethyl-l ,3,2-dioxaborolan-2-yl)phenol

33.7 g [2-chloro-3-ethyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)phenoxy]triisopropyl-silane (76.9 mmol; obtained according to WO 2015/097123, Préparation 5e) was dissolved in 600 mL THF and was cooled to 0°C, then 92.3 mL TBAF (92.3 mmol, IM solution in THF) was added dropwise and the mixture was stirred until complété conversion. Then it was diluted with brine, acidified with citric acid then extracted with DCM. The combined organic layer was dried over Na₂SO₄, filtered and the filtrate was concentrated under reduced pressure. The crude product was purified via flash chromatography using heptane and EtOAc as eluents to give 2-chloro-3-ethyl-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)phen01. ^lH NMR (400 MHz, CDCh)Ô: 7.63 (d, J = 8.2 Hz, 1H),6.86 (d,./=8.2 Hz, IH), 5.87 (s, IH), 3.09 (q, J = 7.44 Hz, 2H), 1.33(s, 12H), 1.15 (t,J= 7.44 Hz, 3H).

Step C: ethyl (2R)-2-[5~(3~chloro-2-ethyl-4~hydroxy-phenyl)~6-prop-l-ynyl-thieno[2,3d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4yl] methoxy]phenyl]propanoate

353 mg ethyl (2R)-2-(5-iodo~6-prop-l-ynyl-thieno[2,3~d]pyrimidm-4-yl)oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate (0.50 mmol) and 282 mg 2-chloro3-ethyl-4-(4,4,5,5-tetramethyLl,3,2-dioxaborolan-2-yl)phenol (0.55 mmol) were dissolved in 2 mL dioxane, then 35 mg PdCfixAtaPhos (0.05 mmol), 326 mg Cs₂CO₃ (1.00 mmol) and 1 mL water were added and the mixture was stirred in a microwave reactor at !00°C under N₂atmosphère for 20 minutes. Then it was diluted with brine, acidified to pH 5 with 1 M aqueous HCl solution and extracted with DCM. The combined organic layer was dried over Na₂SO₄, filtered and the filtrate was concentrated under reduced pressure. The crude product was purified via flash chromatography using heptane and EtOAc as eluents. Then it was further purified via préparative reverse phase chromatography using 5 mM aqueous NH₄HCO₃ solution and MeCN as eluents to give ethyl (2R)-2-[5-(3-chloro-2-ethyl-4-hydroxy-phenyl)-6-prop-l-ynylthieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimidin-4yl]methoxy]phenyl]propanoate as a 2:1 mixture of atropisomers. 'H NMR (500 MHz, DMSO-dô) Ô: 10.35/10.29 (s, IH), 8.93 (d, J=5.1 Hz, IH), 8.59/8.57 (s, 1 H), 7.63/7.60 (d, J = 5.1 Hz, IH), 7.54-6.93 (m, 8 H), 6.84/6.74 (t, J = 7.5 Hz, IH), 6.43/6.18 (dd, J = 7.5, 1.4 Hz, IH), 5.51/5.40 (m, IH), 5.30-5.16 (m, 2H), 4.17-3.99 (m, 2H), 3.76/3.75 (s, 3H), 3.34-3.14 (m, IH), 2.93-2.35 (m, 3H), 2.02/1.98 (s, 3H), 1.08/1.04 (t, J= 7.0 Hz, 3H), 0.94/0.76 (t, J= 7.5 Hz, 3H).

560

Step D: (2R)-2-f(5Sa)-5-[3-chloro-2-ethyl-4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl]-6prop-l-ynyl-thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2-methoxyphenyl)pyrimîdin4-yl] methoxy]phenyl]propanoic acid, C7

322 mg ethyl (2R)-2-[5-(3-chloro-2-ethyl-4-hydroxy-phenyI)-6-prop-l-ynyI-thieno[2,3d] pyrimidin-4-yl] oxy-3 -[ 2-[[ 2-(2-methoxyph en yl) pyrimidin-4-yi] methoxy ] phenyl] propanoate (0.44 mmol), 190 mg 2-(4-methylpîperazin-l-yl)ethanol (I.32 mmol) and 346 mg PPh₃ (1.32 mmol) were dissolved in 10 mL dry toluene, then 304 mg DTBAD (1.32 mmol) was added and the mixture was stirred at 50°C under N₂ atmosphère until no further conversion was observed. Then the mixture was concentrated under reduced pressure and the residue was purified via flash chromatography using heptane, EtOAc and MeOH as eluents, then further purified via préparative reverse phase chromatography using 5 mM aqueous NH4HCO3 solution and MeCN as eluents to obtain the ester intermediate. It was dissolved in 2 mL dioxane, then 84 mg LiOHxH₂O (2.00 mmol) and 1 mL water were added. The mixture was stirred at 50°C until complété hydrolysis. Then it was diluted with brine, neutralized with 2 M aqueous HCl solution, and extracted with DCM. The combined organic phase was dried over Na₂SO4, filtered and the fîltrate was concentrated under reduced pressure. The atropoisomers were purified and separated via préparative reverse phase chromatography using 5 mM aqueous NH4HCO3 solution and MeCN as eluents. The atropoisomer eluting later was isolated as C7. 'H NMR (400 MHz, DMSO-dô) δ: 8.88 (d, J= 5.2 Hz, IH), 8.60 (s, IH), 7.76 (d,J=5.0 Hz, IH), 7.54 (dd, J= 7.6, 1.8 Hz, IH), 7.46 (m, IH), 7.26 (d, A 8.5 Hz, IH), 7.20-7.13 (m, 3H), 7.04 (td, J =1.5, 0.9 Hz, IH), 7.00 (d, 8.0 Hz, IH), 6.78 (t, J =1.5 Hz, IH), 6.32 (dd, J = 7.4, 1.5 Hz, IH), 5.48 (dd, J = 9.7, 2.9 Hz, IH), 5.27 (d, J= 15.0 Hz, IH), 5.19 (d, J = 15.0 Hz, 1 H), 4.23 (m, 2H), 3.76 (s, 3H), 3.31 (m, IH), 2.75 (m, 2H), 2.47 (m, IH), 2.64-2.36 (m, 10H), 2.22 (s, 3H), 2.01 (s, 3H), 0.74 (t,J= 7.5 Hz, 3H). ¹³CNMR(100 MHz, DMSO-dû) δ: 165.95, 165.89, 164.7, 157.8, 157.2, 155.3, 154.0, 141.6, 135.6, 131.0, 130.8, 130.1, 128.4, 128.0, 127.2, 121.4, 120.1, 117.8, 112.2, 111.7, 97.2, 74.9, 68.9, 67.1, 56.1,55.7, 54.0, 32.7, 24.4, 13.1, 4.4. HRMS calculated for C45H45N6O6SCI: 832.2810; found 833.2878 (M+H).

Préparation of C8: (2R)-2-[(5Sa)-5-[3-chloro-2-methyl-4-[2-(4-methyIpÎperazin-l-yI)ethoxy|phenyl]-6-(4fluoroplienyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[|2-(3-sulfooxyphenyl)pyrinudin-4yl]methoxy]phenyl]propanoic acid

561

2I0 mg ethyï (2R)-2-[5-[3-chloro-2-methyI-4-[2-(4-methylpiperazin-lyl)ethoxy]phenyl]-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(3hydroxyphenyl)pyrimidin-4-yl]methoxy]phenyl]piOpanoate (0.24 mmol; obtained accordîng to WO 2016/207216, Préparation 2) was dissolved in 9.3 mL pyridine, than 0.38 mL SOj^pyridine (2.36 mmol) was added and the mixture was stirred at 70°C until complété conversion. Then the mixture was concentrated under reduced pressure and the residue was dissolved in 2 mL dioxane, then 200 mg KOH (3.56 mmol) and 1 mL water were added. The mixture was stirred at 70°C until complété hydrolysis of the Et ester. Then it was neutralîzed with 5 M aqueous HCl solution, and purified via préparative reverse phase chromatography (direct injection) using 25 mM aqueous NH4HCO3 solution and MeCN as eluents to give C8. ^lH NMR (500 MHz, DMSOd6) δ: 8.93 (d, 7=5.1 Hz, IH), 8.63 (s, IH), 8.27 (m, IH), 8.13 (m, IH), 7.61 (d,7=5.1 Hz, IH), 7.43 (t,7=7.8 Hz, IH), 7.33 (d,7=7.8 Hz, IH), 7.32-7.27 (m, 3H), 7.23-7.13 (m, 4H), 7.05 (d, 7=7.8 Hz, IH), 6.73 (t,7=7.5 Hz, IH), 6.31 (dd,7=7.5, 1.3 Hz, IH), 5.51 (dd,7=9.8, 3.5 Hz, IH), 5.34 (d, 7= 15.2 Hz, IH), 5.27 (d,7 = 15.2 Hz, IH), 4.24-4.08 (m, 2H), 3.26 (dd,7 = 14.3, 3.4 Hz, IH), 3.10-2.52 (m, 14H), 1.82 (s, 3H). ^I3CNMR(125 MHz, DMSO-d6) δ: 170.8, 166.5, 166.4, 162.8, 162.7, 158.3, 155.3, 154.0, 153.6, 152.9, 137.8, 136.8, 135.9, 131.12, 131.05, 130.4, 130.3, 129.1, 128.5, 128.2, 127.8, 124.4, 123.5, 122.7, 121.9, 120.5, 120.2, 118.8, 116.0, 115.9, 112.0, 110.5, 73.5,69.1,67.2, 55.4,43.1, 31.8, 17.5. HRMS calculated for C46H42N6O9S2CIF: 940.2127; found 941.2191 (M+H).

Préparation of C9:

2Æ)-2-{[5-{3-chloro-2-methyl-4-[2-(4-methylpîperazin-l-yl)ethoxy]phenyl}-6-(4fluorophenyl)thieno[2,3-rf]pyrimidin-4-yl]oxy}-3-(2-{[2-(3-sulfophenyI)pyrimidîn-4yl]methoxy}phenyi)propanoic acid

562

750 mg ethyl (2R)-2-[(5Sa)-5-[3-chloro-2-methyl-4-[2-(4-methy1piperazin-1yl)ethoxy]phenyl]-6-(4-f]uorophenyl)thieno[2,3-d]pyrimidin-4-yI]oxy-3-[2-[(2methylsulfanylpyrimidin-4-yl)methoxy]phenyljpropanoate (0.89 mmol; obtained according to WO 2015/097123, Préparation 10a) was dissolved in 9 mL THF, then 726 mg [3-(2,2dimethylpropoxysulfonyl)phenyl]boronic acid (2.67 mmol), 62 mg Pd(PPh₃)₄ (0.05 mmol ) and 509 mg thiophene-2-carbonyloxycopper (2.67 mmol) were added and the mixture was stirred at 75°C until complété conversion. Then it was concentrated under reduced pressure and was purified via flash chromatography using heptane, EtOAc and 0.7 M NH₃ solution in MeOH as eluents. Then it was dissolved in 20 mL l,l,l,3,3,3-hexafluoropropan-2-oI, 4.5 mL TFA was added and the mixture was stirred at 80°C for until complété hydrolysis of the sulfonîc ester. The mixture was concentrated under reduced pressure and then dissolved in 5 mL dioxane, then 210 mg LiOHxH₂O (5.00 mmol) and 2 mL water were added. The mixture was stirred at room température until complété hydrolysis. Then it was diluted with brine, neutralized with 2 M aqueous HCl solution, and extracted with DCM. The combined organic phase was dried over Na₂SO₄, filtered and the filtrate was concentrated under reduced pressure. The formed atropisomers were purified and separated via préparative reverse phase chromatography using 25 mM aqueous NH₄HCOj solution and MeCN as eluents, then further purified using 0.1% aqueous TFA solution and MeCN as eluents to give C9. *H NMR (500 MHz, DMSO-dô) δ: 13.19 (br s, IH), 9.48 (brs, IH), 8.94 (d, J= 5.1 Hz, IH), 8.74 (t, J= 1.6 Hz, IH), 8.65 (s, IH), 8.37 (dt, J = 7.8, 2.9 Hz, 1 H), 7.78 (dt, J = 7.6, 1.5 Hz, 1 H), 7.60 (d, J = 5.1 Hz, 1 H), 7.51 (t, J = 7.7 Hz, 1 H), 7.29 (m, 3H), 7.22-7.14 (m, 3H), 7.13-7.05 (m, 2H), 6.74 (t, J= 7.5 Hz, IH), 6.38 (d, J = 7.6 Hz, IH), 5.53 (dd, 7=9.6, 3.6 Hz, IH), 5.37 (d, J= 15.3 Hz, IH), 5.31 (d, J= 15.3 Hz, IH), 4.2 (m, 2H), 3.50-2.88 (m, 11H),2.76 (s, 3H), 2.61 (dd, J= 14.2, 9.7 Hz, IH), 1.79 (s, 3H). ⁿCNMR (125 MHz, DMSO-dû) δ: 170.8, 166.5, 163.0, 162.7, 161.1, 158.4, 155.4, 153.3, 152.9, 148.6, 136.6, 136.0, 131.1, 130.1, 129.0, 128.5, 128.3, 128.1, 127.9, 125.3, 124.4, 121.9, 120.5, 118.8,

563

116.2, 116.0, 115.9, 1 12.1, 110.5, 73.2,69.1, 66.5, 55.0, 51.7,49.7, 42.1, 31.5, 17.5. HRMS calculated for C46H42N6O8S2CIF: 924.2178; found 925.2274 (M+H).

Préparation of CIO:

(2R)-2-[(5Sa)-5-|3-chloro-2-methyl-4-[2-(4-inetliylpiperazin-l-yl)ethoxy]plienyl]-6-[4fluoro-3-(2,2,2-trifluorocthoxy)phenyl]thieno[2,3-dJpyrimidin-4-yI|oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

Ν'' N

250 mg ethyl (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-lyI)ethoxy]phenyl]-6-iodo-thieno[2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2methoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoate (0.27 mmol; obtaîned according to WO 2015/097123, Préparation 30) and 79 mg [4-fluoro-3-(2,2,2-trifluoroethoxy)phenyl]boronic acid (0.40 mmol) were dissolved in 1 mL THF, then 3.0 mg PdOAc₂ (0.013 mmol), 5.7 mg tBuXPhos (0.013 mmol), 174 mg Cs₂CO3 (0.53 mmol) and 0.27 mL water were added and the mixture was stirred at 70°C under N₂ atmosphère for 2 hours. Then it was diluted with brine, and extracted with 2-MeTHF. The combined organic layer was dried over Na₂SO₄, fïltered and the filtrate was concentrated under reduced pressure. The crude ester product was purified via flash chromatography using heptane, EtOAc and 0.7 M NH₃ solution in MeOH as eluents. Then it was dissolved in 5.3 mL dioxane, then 64 mg LiOHxH₂O (1.52 mmol) and 1.3 mL water were added. The mixture was stirred at room température until complété hydrolysis. Then it was diluted with brine, neutralized with 2 M aqueous HCl solution, and extracted with DCM. The combined organic phase was dried over Na₂SO4, fïltered and the filtrate was concentrated under reduced pressure. The atropisomers were purified and separated via préparative reverse phase chromatography using 25 mM aqueous NH4HCO3 solution and MeCN as eluents. The atropisomer eluting later was isolated as CIO. 'H NMR (500 MHz, DMSO-d_d) δ: 8.91 (d, J = 5.2 Hz, IH), 8.57 (s, IH), 7.82 (d, J = 5.1 Hz, IH), 7.53 (dd, J= 7.5, 1.7 Hz, IH), 7.45 (m, 2H),

564

7.27 (dd, J= ll.O, 8.6 Hz, IH), 7.22 (d, J =8.6 Hz, IH), 7.16-7.09 (m, 3H), 7.03 (t, J =1.5 Hz, 1H), 6.98 (d,J= 8.3 Hz, IH), 6.92 (m, 1H),6.69 (t, J = 7.4 Hz, IH), 6.16 (d, 7.2 Hz, IH),

5.47 (dd, .7= 10.3, 2.6 Hz, IH), 5.25 (d, J = 15.1 Hz, IH), 5.19 (d,J= 15.1 Hz, IH), 4.75-4.53 (m, 2H), 4.21 (t, J= 5.5 Hz, 2H), 3.75 (s, 3H), 3.41 (d, J = 12.0 Hz, IH), 2.77-2.30 (m, 12H),

2.24 (s, 3H), 1.81 (s, 3H). ^I3CNMR(125 MHz, DMSO-d₆) δ: 171.3, 166.2, 166.0, 164.6, 163.5,

157.9, 157.2, 155.3, 153.7, 153.1, 152.1, 150.5, 144.6, 135.8, 131.0, .130.8, 130.7, 129.6, 129.1, 128.4, 128.1, 127.8, 125.4, 123.7, 122.0, 120.4, 120.1, 118.8, 117.0, 116.7, 115.6, 112.2, 111.7, i 10.5, 74.8, 68.9, 67.2, 65.6, 56.0, 55.7, 53.8, 52.0, 44.6, 32.7, 17.5. HRMS calculated for C49H45N6O7SCIF4: 972.2695; found 973.2761 (M+H).

Préparation of Cl 1 :

(2R)-2“{[(5Sa)-5-{3-chIoro-2-mcthyl·4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl}-6-(4fluorophenyI)thienol2,3-d]pyrimidin-4-ylJoxy}-3-(2-{[2-(4-methoxyphenyl)pyrimidin-4yl]methoxy}phenyl)propanoic acid

Cl 1 was prepared according to Example 107 in WO 2015/097123.

Préparation of C12 (2/f)-2-{[(5S'_e)-5-{3-chloro-2-methyi-4-[2-(4-methylpiperazin-l-yl)ethoxy]phenyl}-6-(4fluorophenyl)thieno[2,3-</]pyrimidin-4-yHoxy}-3-(2-{[l-(2,2,2-trifIuoroethyl)-lH-pyrazol-5yl| methoxy} phenyl)propan oie acid

565

Cl2 was prepared according to Example 77 in WO 2015/097123.

Préparation of C13 (2R)-2-[(5S_a)-6-(3-amino-4,5-difluoro-phenyl)-5-[3-chloro-2-methyl-4-[2-(45 methylpiperazin-l-yl)ethoxy]phenyl]thieno|2,3-d]pyrimidin-4-yl]oxy-3-[2-[[2-(2niethoxyphenyl)pyrimidin-4-yl]methoxy]phenyl]propanoic acid

250 mg ethyl (2R)-2-[5-[3-chloro-2-methyI-4-[2-(4-methylpiperazin-lyl)ethoxy]phenyl]-6-iodo-thîeno[2,3-d]pyrimidîn-4-yl]oxy-3-[2-[[2-(2- methoxyphenyl)pyrimidin-4-yl] methoxy] phenyl] propanoate (0.27 mmol; obtained according to

WO 2015/097123, Préparation 30) and 102 mg 2,3-difluoro-5-(4,4,5,5-tetramethyl-1,3,2dîoxaborolan-2-yl)aniline (0.41 mmol) were dissolved in 1 mL THF, then 3.0 mg PdOAc₂ (0.013 mmol), 5.7 mg tBuXPhos (0.013 mmol), 174 mg CS2CO3 (0.53 mmol) and 0.27 mL water were added and the mixture was stirred at 70°C under N2 atmosphère for 2 hours. Then it was diluted

5 with brine, and extracted with 2-MeTHF. The combined organic layer was dried over Na₂SÛ4, fïltered and the fdtrate was concentrated under reduced pressure. The crude ester product was purified via flash chromatography using heptane, EtOAc and 0.7 Μ NH₃ solution in MeOH as eluents. Then it was dissolved in 0.7 mL dioxane, then 60 mg LiOHxH₂O (1.43 mmol) and 0.18 566 mL water were added. The mixture was stirred at room température until complété hydrolysis. Then it was diluted with brine, neutralîzed with 2 M aqueous HCl solution, and extracted with DCM. The combined organic phase was dried over Na₂SO₄, filtered and the filtrate was concentrated under reduced pressure. The atropoisomers were purified and separated via préparative reverse phase chromatography usîng 25 mM aqueous NH4HCO3 solution and MeCN as eluents. The atropisomer eluting later was isolated as C13. 'H NMR (500 MHz, DMSO-dô) δ:

8.89 (d, 7=5.2 Hz, IH), 8.56 (s, IH), 7.76 (d, 7= 5.0 Hz, IH), 7.53 (dd,7= 7.6, 1.8 Hz, IH), 7.45 (m, IH), 7.36 (d, J= 8.6 Hz, IH), 7.20 (d, 7= 8.7 Hz, IH), 7.13 (m, 2H), 7.03 (td,7=7.5, 1.0 Hz, IH), 6.99 (d, 7= 8.1 Hz, IH), 6.71 (t,7=7.3 Hz, IH), 6.62 (m, IH), 6.21 (d,7=7.5, 1.3 Hz, IH), 6.12 (m, IH), 5.73 (s, 2H), 5.46 (dd,7 = 10.1, 3.1 Hz, IH), 5.25 (d,7= 15.1 Hz, IH), 5.19 (d,7 = 15.2 Hz, 1H),4.22 (m, 2H), 3.75 (s, 3H), 3.35 (m, 1 H), 2.73 (m, 2H), 2.65-2.35 (m, 9H), 2.22 (s, 3H), 1.85 (s, 3H). ^1JC NMR (125 MHz, DMSO-d₆) δ: 171.1, 166.0, 165.3, 163.3, 157.8, 157.2, 155.3, 153.7, 152.9, 149.0, 138.9, 137.1, 135.8, 131.0, 130.8, 130.4, 128.7, 128.4, 128.1, 127.8, 125.2, 122.0, 120.4, 120.1, 118.9, 115.6, 112.2, 111.9, 110.6, 103.2, 74.5, 68.9, 67.1, 56.0, 55.7, 53.9, 52.1,44.7, 32.6, 17.6. HRMS calculated for C47H44N7O6SCÏF2: 907.2730; found 908.2803 (M+H).

Préparation of C14 (2Æ)-2-{[(5Sa)-5-{3-chloro-2-niethyl-4-[2-(4-methyIpiperazin-l-yl)ethoxy]phenyl}-6-(4fluorophenyl)thieno[2,3-7|pyrimidin-4-yl]oxy}-3-(2-{[2-(3-hydroxy-2methoxyphenyl)pyrimidin-4-yl]methoxy}phcnyl)propanoic acid

210 mg ethyl (2R)-2-[5-[3-chloro-2-methyl-4-[2-(4-methylpiperazin-lyl)ethoxy]phenyl]-6-(4-fIuorophenyl)thieno[2,3-d]pyrimidm-4-yl]oxy-3-[2-[(2-chloropyrimidm4-yl)methoxy]phenyl]propanoate (0.25 mmol, WO2016/207216 Préparation I) and 84 mg (3hydroxy-2-methoxy-phenyl)boronic acid (0.50 mmol) were dissolved in 3.8 mL 1,4-dioxane, then 18 mg Pd(PPh3)₂Cl₂ (0.025 mmol), 240 mg Cs₂CO₃ (0.75 mmol) and 3.8 mL water were added and the mixture was stirred under N2 atmosphère at 70°C until complété conversion. Then

567 it was diluted with water, neutralized with 2 M aqueous HCl solution, and extracted with DCM. The combined organic phase was dried over Na₂SO4, filtered and the filtrate was concentrated under reduced pressure. The crude ester was purified via flash chromatography using heptane, EtOAc and 0.7 M NHj solution in MeOH as eluents to obtain a mixture of diastereoisomers. It was in dissolved in 2 mL dioxane, then 245 mg LiOH*H₂O (5.85 mmol) and 1 mL water were added. The mixture was stirred at rt until complété hydrolysis. Then it was neutralized with 2 M aqueous HCl solution, and directly injected on prep-RP-HPLC, using 0.1 % aqueous TFA solution and MeCN as eluents. The diastereoisomer eluting later was collected as C14. 'H NMR (500 MHz, DMSO-dô) δ: 9.53 (brs, IH), 8.91 (d, IH), 8.56 (s, IH), 7.79 (d, IH), 7.42 (d, IH), 7.26 (m, 2H), 7.19 (d, IH), 7.18 (m, 2H),7.12(t, JH), 7.06 (dd, 1 H), 6.98 (m, lH),6.98(m, IH), 6.97 (d, IH), 6.68 (t, IH), 6.16 (d, IH), 5.47 (m, IH), 5.27/5.20 (d+d, 2H), 4.26/4.19 (m+m, 2H), 3.76 (s, 3H), 3.38/2.42 (dd+dd, 2H), 2.74 (m, 2H), 2.55 (br., 4H), 2.47 (br., 4H), 2.25 (s, 3H), 1.80 (s,3 H). HRMS calculated for C47H44N6O7SCIF: 890.2665; found 891.2721 (M+H).

Préparation of P15 ( 11 R,20R)-23,26-dichloro-3-(4-fluorophenyl)-14-[[2-(2-methoxyphenyl)pyrimîdin-4yl]methoxy]-24,25-dimethyl-20-[(4-methylpiperazin-l-yI)methyl]-10,18,21-trioxa-4-thia“ 6,8-diazapentacyclo[20.2.2.12,5.113,17.09,28]octacosa1(25),2,5(28),6,8,13,15,17(27),22(26),23-decaene-ll-carboxylic acid

HOOC _R O

P15 was prepared according to Example 116 in WO 2019/035914.

Préparation of Pl 6 (llR,20R)-23,26-dîchloro-14-[[2-[4-[[(2S)-l,4-dioxan-2-yl]methoxymethyI]-4-fluorocyclohexyl]pyrimidin-4-yl]methoxy]-3-(4-fluorophenyl)-24,25-dimethyl-20-[(4methy Ipiper azin-1 -yl) methyl] -10,18,2 l-trioxa-4-thia-6,8568 diazapentacyclo[20^.2.12,5.113,17.09^8|octacosa-l(24),2,5(28),6,8,13,15,17(27),22,25decaene-ll-carboxylic acid

P16 was prepared according to Example 28 in WO 2019/035911.

Préparation of P17 (Il R,20R)-23,26-dichloro-14-[[2-[4-[[(2S)-l,4-dioxan-2-yl]mcthoxy] cyclohexyl] pyrimidin-4yl]mcthoxy]-3-(4-fluorophenyl)-24,25-dimethyl-20-[(4-methylpiperazin-l-yl)methyl]10,18,21-trioxa-4-thia-6,8-diazapentacyclo[20.2.2.12,5.113,17.09,28]octacosa10 1(24),2,5(28),6,8,13,15,17(27),22,25-decaene-ll-carboxylic acid

P17 was prepared according to Example 44 in WO 2019/035911.

Exaniple 4: Conjugation and analytical characterization of anti-CD74 ADCs

5 Exemplary antibody-drug conjugates (ADCs) were synthesized using the exemplary methods described below. Antibodies anti-CD74 (Milatuzumab) used for the préparation of the exemplary ADCs is deftned by the abbreviation Ab M, CD74, or CD74_CysmAb (Table 6). The tenu “CysmAb” refers to cysteine mutations in the heavy chain of the antibody that are used to 569 conjugate linker-payloads to the antibody via maleimide group. Ail full length, conjugated CD74 Abs used herein contain the engineered cysteine mutations E152C and S375C (numbered according to the EU system).

Table 6. Antibodies used for the synthesis of the exempiified ADCs

Antibody abbreviation	Antibody	Fc Mutation	Sequence (Heavy Chain/Light Chain)
Ab M or CD74	mîlatuzumab	Wild Type-	SEQ ID Nos 12,25
Anti-CD74CysmAb Fc silent or CD74 Fc silent	mîlatuzumab	DANAPA Fc silencing	SEQ ID Nos 15, 25

Conjugation

Method Cl: The antibody was bound on rmp Protein A resin (GE Healthcare) at a ratio of 10 mg Ab to l ml resin in PBS for 30 minutes by mixing in Biorad sized disposable column. To deblock the reactive cysteines, cysteine hydrochloride monohydrate was added to a final concentration of 20 mM. The mixture was agitated for 30 minutes at room température followed by washing 5 times the resin with 50 column volumes PBS on a vacuum manifold. The resin was then resuspended in an equal volume PBS containing 250 nM CuCb and incubated for l hour and 30 min. The re-oxidized antibody was washed 5 times with 50 column volumes PBS on a vacuum manifold and resuspended in an equal volume PBS. To the mixture were added 10 fold-molar excess of 20mM linker-payload solution and equal volume of DMSO. The reaction was incubated at room température for 2 hours. To monitor the conjugation 20μ 1 of resin slurry were removed, centrifuged, supematant removed, and then eluted with 40 μΐ Antibody elution buffer (Thermo Fisher Scientific). The supematant was analyzed by reverse phase chromatography using an Agilent PLRP-S 4000A 5 um, 4.6 x 50 mm column (Buffer A is water, Ü.1% TFA, Buffer B Acetonitrile, 0.1% TFA, column held at 80°C, Flowrate 1.5 ml/min). After élimination the excess of linker-payload by washing the resin 5 times with 50 column volumes PBS on a vacuum manifold, the ADC was eluted from protein A with antibody elution buffer and neutralized with 1/10 volume 1 M Tris pH 9.0. Ail exempiified ADCs were buffer exchange

570 by dialysis in PBS IX pH 7.4 (Sigma Life Science, P3813, 10PAK), concentrated using Vivaspin 20, 50K.D, PES (Sartorius Stedim, VS2031), filtered sterilely through 0.2pm stérile PES Filter, 25mm (Whatmann, G896-2502) and stored at 4°C. The conjugations were performed în a range of 5mg of antibody. Ail exemplified ADC were characterized by analyti cal size exclusion chromatography Superdex 200 Increase 5/150 GL (GE Healthcare, 28990945) to déterminé monomer percentage and LC-MS for DAR détermination (Tablel6).

Method C2. Antibody was incubated with RMP Protein A resin (GE) at a ratio of 10 mg Ab to 1 ml resin in PBS for 15 minutes with mixing in an appropriately sized disposable column. Cysteine HCl was added to a final concentration of 20 mM and incubated with agitation for 30 min at room température to allow the reactive cysteines to be deblocked. The resin was quickly washed with 50 column volumes PBS on a vacuum manîfold. The resin was then resuspeneded in an equal volume PBS containing 250 nM CuCb. Reformation of antibody interchain disulfides was monitored by taking time points. At each time point, 25 pL of resin slurry was removed, 1 pL of 20 mM MC-valcit-MMAE was added, and the tube flicked several times. The resin was spun down, supematant removed, and then eluted with 50 pL Antibody elution buffer (Thermo). The resin was pelleted and the supernatant analyzed by reverse phase chromatography using an Agilent PLRP-S 4000A 5um, 4.6x50mm column (Buffer A is water, 0.1 % TFA, Buffer B Acetonitrile, 0.1 % TFA, column held at 80 °C, Flowrate 1.5 ml/min). Once it was determined that the antibody has reformed its interchain disulfïde bonds, the resin was washed with 10 column volumes PBS and the resin was resuspended in an equal volume PBS and 8-12 équivalents of linker-payload in DMSO was added, with a final concentration of 10% DM S O in reaction and then incubated at room température for 3 hours. The resin was then washed with 50 column volumes PBS. The ADC was eluted from the protein A resin with Antibody elution buffer. The ADC was then buffer exchanged into PBS or other suitable buffer and préparative size exclusion chromatography to remove aggregates was performed (S200 Increase; GE), if required to remove aggregates. The foilowing analyses were performed analytical SEC to détermine percent monomer, mass spectroscopy to détermine DAR, LAL test to déterminé endotoxin load and protein concentration was determined by A280 utilizing extinction coefficient and molecular weight of antibody.

Method C3 (Drug Substance Intermediate (DSi) Préparation). 200 mg of antibody (1.36 _M) at 10 mg/ml was incubated with 20ml of settled RMP Protein A resin (GE Lifesciences, 17513803) and agîtated for 15 minutes. Cysteine HCl monohydrate was added to a final concentration of 20 mM and incubated with agitation for 30 min at room température to

571 allow the reactive cysteines to be deblocked. The resin was quickly washed with 50 column volumes PBS on a vacuum manifold. The resin was then resuspeneded in an equal volume PBS containing 250 nM CuCh- Reformation of antibody interchain disulfides was monitored by taking time points. At each time point, 25 pL of resin siurry was removed, 1 pL of 20 mM MCvalcit-MMAE was added, and the tube flicked several times. The resin was spun down, supematant removed, and then eluted with 50 μL Antibody elution buffer (Thermo). The resin was pelleted and the supematant analyzed by reverse phase chromatography using an Agilent PLRP-S 4000A 5um, 4.6x50mm column (Buffer A is water, 0.1% TFA, Buffer B Acetonitrile, 0.1% TFA, column held at 80 C, Flowrate 1.5 ml/min; Gradient 0 minutes - 30%B, 5 minutes 45%B, 6.5 min - 100%B, 8 minutes - 100%B, 10 minutes - 30%). At 60 minutes after addition of CuC12, was removed by washing with 50 column volumes ofPBS on a vacuum manifold and then20 ml ofPBS was added to resuspend and drained by gravity. The antibody was eluted with 100 ml antibody elution buffer (Thermo Scientifïc, 21004) and then buffer exchanged into IX PBS pH 7.2. The material was then concentrated using a centrifugal concentrator using an Amicon Ultra-15, 50KDa, regenerated cellulose (Millipore, UFC0905024), to 6.6 mg/ml aliquoted into 5 mg aliquots and flash frozen in liquid nitrogen and stored at -80 C until used (CD74-L5-P1): 120 mg of antibody (0.816 pmoles, 1.0 equiv.) was incubated with 12 ml of settled RMP Protein A resin (GE Lifesciences, 17513803) and agîtated for 15 minutes. Cysteine HCl monohydrate was added to a final concentration of 20 mM and incubated with agitation for 30 min at room température to allow the reactive cysteines to be deblocked. The resin was quickly washed with 50 column volumes PBS on a vacuum manifold. The resin was then resuspeneded in an equal volume PBS containing 250 nM CuCh. Reformation of antibody interchain disulfides was monitored by taking time points. At each time point, 25 pL of resin siurry was removed, 1 pL of 20 mM MC-valcit-MMAE was added, and the tube flicked several times. The resin was spun down, supematant removed, and then eluted with 50 pL Antibody elution buffer (Thermo, 24001 ). The resin was pelleted and the supematant analyzed b y reverse phase chromatography using an Agilent PLRP-S 4000A 5uni, 4.6x50mm column (Buffer A is water, 0.1% TFA, Buffer B Acetonitrile, 0.1% TFA, column held at 80 C, Flowrate 1.5 ml/min; Gradient 0 minutes - 30%B, 5 minutes - 45 %B, 6.5 min — 100%B, 8 minutes - 100%B, 10 minutes - 30%). At 60 minutes after addition of CuC12, was removed by washing with 50 column volumes ofPBS on a vacuum manifold and then 12 ml ofPBS was added to resuspend. To this siurry of resin and antibody. L5-P1 (365 pl of a 20 mM solution in DMSO, 6.528 pmoles, 8 equiv.) was added. The resulting mixture was then incubated at ambient température

572 for 3 hours. The resin was then washed with 50 column volumes PBS. The ADC was eluted from the resin with antibody elution buffer (Thermo Scientific, 21004). The ADC was then buffer exchanged into IX PBS (20X PBS, TeknovaP0191) by dialysis and préparative size exclusion chromatography eluted in Dulbecco’s PBS pH 7.2 (Hyclone SH30028.03) was done to remove aggregates was performed with a HiLoad 26/600 Superdex 200 pg (GE Healthcare, 28989336). The material was then concentrated using a centrifugal concentrator using an Amîcon Ultra-15, 50KDa, regenerated cellulose (Millîpore, UFCÛ905024), to 8 mg/ml and filtered sterilely through 0.22 pm stérile PVDF Filter, 25mm (Millapore, SLGV013SL) and stored at 4°C. The final yeild was 74.1 mg (0.504 . mol) The following analyses were performed: analytical size-exclusion chromatography (SEC) to déterminé percent monomer, mass spectroscopy (MS) to détermine DAR, LAL test to détermine en do fox in load and protein concentration determîned by A280 utilizing extinction coefficient and molecular weight of antibody. HRMS data (protein method) indicated a dominant mass of the heavy chain was 54294 da, giving a DAR of 4.4 as calculated by comparing MS intensifies of peaks for DAR1 DAR2 and DAR3 species. SEC indicated </= 1% aggregation, as determîned by comparison of the area of the high-molecular-weight peak absorbance at 210 and 280 nm with the area of the peak absorbance for monomeric ADC.

(CD74-L7-P1): 20 mg of antibody (0.136 pmoles, 1.0 equiv.) was incubated with 2 ml of settled RMP Protein A resin (GE Lifesciences, 17513803) and agitated for 15 minutes. Cysteine HCl monohydrate was added to a final concentration of 20 mM and incubated with agitation for 30 min at room température to allow the réactivé cysteines to be deblocked. The resin was quickly washed wîth 50 column volumes PBS on a vacuum manifold. The resin was then resuspeneded in an equal volume PBS containîng 250 nM CuCh. Reformation of antibody interchain disulfides was monitored by taking time points. At each time point, 25 pL of resin slurry was removed, 1 pL of 20 mM MC-valcît-MMAE was added, and the tube flicked several times. The resin was spun down, supernatant removed, and then eluted with 50 pL Antibody elution buffer (Thermo). The resin was pelleted and the supernatant analyzed by reverse phase chromatography using an Agilent PLRP-S 4000A 5um, 4.6x50mm column (Buffer A is water, 0.1% TFA, Buffer B Acetonitrîle, 0.1% TFA, column held at 80 C, Flowrate 1.5 ml/min; Gradient 0 minutes - 30%B, 5 minutes - 45%B, 6.5 min - 100%B, 8 minutes - 100%B, 10 minutes - 30%). At 60 minutes after addition of CuC12, was removed by washing with 50 column volumes of PBS on a vacuum manifold and then 2 ml of PBS was added to resuspend. To this slurry of resin and antibody. L7-P1 (55 pl of a 20 mM solution in DMSO, 1.088

573 pmoles, 8 equiv.) was added. The resulting mixture was then incubated at ambient température for 3 hours. The resin was then washed with 50 column volumes PBS. The ADC was eluted from the resin with Antibody elution buffer (Thermo Scîentifîc, 21004). The ADC was then buffer exchanged into IX PBS (20X PBS, TeknovaP0191) by dialysis and préparative sîze exclusion chromatography eluted in Dulbecco’s PBS pH 7.2 (Hyclone SH30028.03) was done to remove aggregates was performed with a HiLoad 16/600 Superdex 200 pg (GE Healthcare, 28989335). The material was then concentrated using a centrifugal concentrator using an Amicon Ultra-15, 50KDa, regenerated cellulose (Millipore, UFC0905024), to 4.5 mg/ml and fïltered sterîlely through 0.22 pm stérile PVDF Filter, 25mm (Millapore, SLGV013SL) and stored at 4°C. The final yeild was 1.6 mg (0.00681 J mol) The following analyses were performed: analytical size-exclusion chromatography (SEC) to détermine percent monomer, mass spectroscopy (MS) to détermine DAR, LAL test to détermine endotoxin load and protein concentration determined by A280 utiïizing extinction coefficient and molecular weight of antibody. HRMS data (protein method) indicated a dominant mass of the heavy chain was 54562 da, giving a DAR of 3.8 was calculated by comparîng MS intensities of peaks for DAR1 DAR2 and DAR3 species. SEC indicated 1.8% aggregation, as determined by comparison ofthe area of the high-molecular-weight peak absorbance at 210 and 280 nm with the area of the peak absorbance for monomeric ADC.

Analytical Methods

Liquid Chromatography-Mass Spectrometry (LC/MS) I

General Methodology: Drug-to-antibody ratio (DAR) of exemplary ADCs was determined by liquid chromatography-mass spectrometry (LC/MS) according to one of the following methods (i.e., LC-I, LCII and LC-TII). For ail LC methods, mobile phase A was purified MS grade water (Biosolve, Dîeuzc, France, 00232141B IBS), mobile phase B was MS grade acetonitrile (Biosolve, Dîeuze, France, 0001204101BS), and mobile phase D was purified MS grade water supplemented with 1 % of formic acid (FA) (Honeywell/Fluka, Bucharest, Romania, 56302). Mobile phase D was fixed at 10 % in order to maintain a 0.1% FA mobile phase composition and column température was set at 80°C. A general MS method was optimized for ail ADCs synthesîzed, except for one ADC where native MS was used în order to détermine average DAR (see MS-I and Table 16 below).

General Methodology (1): Drug-to-antibody ratio (DAR) of exemplary ADCs was determined by liquid chromatography-mass spectrometry (LC/MS) according to the following

574 method. For ah LC methods, mobile phase A was purified MS grade water (Honeywell, LC0151), mobile phase B was MS grade S0% Isopropanol (Honeywell LC323-1): 20% acetonitrile (Honeywell, LC015-1), LC323-1), supplemented with 1 % of formic acid (FA) (Thermo Scientifie, 85178). The column température was set at 80°C. A general MS method was optimized for ail ADCs synthesized. The column used for analysis was an Agilent PLRP-S 4000 A; 2.1x150mm, 8um (Agilent, PL1912-3803). Flowrate used was 0.3 ml/mîn. The gradient used was 0-0.75 minute 95%A, 0.76 -1.9 minute 75%A, 1.91-1 LO minute 50%A, 11.01-11.50 10%A, 11.51-13.50 minute 95%A, 13.51 -18 minute 95%A ona Acquîtty Bio H-Class Quaremary UPLC (Waters). MS system was Xevo G2-XS QToF ESI mass spectrometer (Waters) and data acquired from 1.5-11 minutes and masses were analyzed between 15000-80000 dallons. DAR was determined from the deconvoluted spectra or UV chromatogram by summing the integrated MS (total ion current) or UV (280 nm) peak area of unconjugated and conjugated given species (mAb or associaied fragment), weighted by multiplying each area by the number of drug attached. The summed, weighted areas were divided b y the sum of total area and the results produced a final average DAR value for the full ADC.

LC-I: ADC was loaded onto a MassPREP Micro desalting column (2.1 x 5.0 mm, Waters, Saint- Quentin-en-Yvelines, France, 186004032). For intact mass analysis, a desalting step was perfonned for 0.5 min at 5% mobile phase B with a flow rate of 0.5 ml/min. Elution step was performed with a gradient from 0.51 min at 5% B to 2.0 min at 90 % B with a flow rate of 0.2 ml/min. Two wash steps were set from 2.1 min to 2.7 min and from 2.8 min to 3.4 min at 5% B to 90% B with a flow rate of 0.5 ml/min. Finally, a conditionîng step was used at 3.5 min for 0.5 min at 5 % B (0.5 ml/min).

For ADC analysis in reduced conditions, a desalting step was perfonned for 0.5 min at 5% B with a flow rate of 0.2 ml/min. Then, the elution step started with a gradient from 0.51 min at 10% B to 7.61 min at 80 % B with a flow rate of 0.2 ml/min. Two washing steps were set from 8.1 min to 8.6 min and from 8.7 min to 9.2 min from 5% B to 90% B (0.5 ml/min). Finally, a conditionîng step was perfonned at 9.3 min for 0.5 min at 5 % B with a flow rate of 0.5 ml/min.

LC-IL ADC was loaded onto a MabPac RP column (2,1 x 100 mm, 4 pm, Thermo Fisher Scientific, Rockford, IL, 088647). For analysis in both intact and reduced conditions, a desalting step was performed for 1.4 min at 20% of B with a flow rate of 0.4 mL/min. Then, the ehition step was performed with a gradient from 1.5 min at 20% B to 11.5 min at 70 % B with a flow rate of 0.3 ml/min. A wash step was set from 11.75 min to 13.75 min at 90% B with a flow

575 rate of 0.5 ml/min. Finally, a conditioning step was used at 14.0 min for l.O min at 20 % B with a flow rate of 0.4 ml/min.

LC-ΠΙ: ADC was loaded onto a Bioresolve RP mAb Polyphenyl, 450A, 2.7 pm, 2.1 x 150 mm (Waters, Saint- Quentin-en-Yvelines, France, 186008946). For analysis in both intact and reduced conditions, a desalting step was performed for 1.4 min at 20% of B with a flow rate of 0.4 ml/min. Elution step was perfonned with a gradient from 1.5 min at 20% B to 11.5 min at 70 % B with a flow rate of 0.3 ml/min. A wash step was set from 11.75 min to 13.75 min at 90% B with a flow rate of 0.5 ml/min. Finally, a conditioning step was used at 14.0 min for 1.0 min at 20 % B with a flow rate of 0.4 ml/min.

LC-IV: ADC was loaded onto a Bioresolve RP mAb Polyphenyl, 450A, 2.7pm, 2.1 x 150mm (Waters, Saint- Quentin-en-Yvelines, France, 186008946). For analysis in both intact and reduced conditions, a desalting step was performed for 1.5 min at 20% of B with a flow rate of 0.6 mL/min. Elution step was performed with a gradient from 1.5 min at 20% B to 16.5 min at 50 % B with a flow rate of 0.6 mL/min. A wash step was set from 16.8 min to 18.8 min at 90% B with a flow rate of 0.6 mL/min. Finally, a conditioning step was used at 19.1 min for 1.9 min at 20 % B with a flow rate of 0.6 mL/min (Total run time = 21 minutes).

MS-I: ADC was buffer exchanged with a MS compatible buffer and infused directly into a hybrid Q-TOF MS instrument (Synapt G2-S HDMS, Waters, Manchester, UK) equipped with an automated chip-based nanoESI source (Triversa Nanomate, Advion Biosciences, Ithaca, NY, USA) operating in positive ion mode. Instrumental MS parameters were tuned to preserve native 3D structure in the gas phase and ensure efficient ion desolvatîon and transmission.

LC-MS analysis was performed using a Waters UPLC H-Class Bio chromatography system hyphenated with a Xevo G2 XS Q-TOF ESI mass spectrometer (Waters, Manchester, UK). The ADC was either analyzed in intact condition (no prelimînary treatment) or following réduction with 5 mM (final concentration) of DTT (Thermo Scientific, Rockford, IL, 20291). Subsequently, treated ADC was analyzed using the aforementioned LC-IV (Table 16). Electrospray-ionization time-of-flight mass spectra of the analytes were acquired using MassLynx™ acquisition software (Waters, Manchester, UK). Then, the extracted intensity vs. m/z spectrum was deconvoluted using Maximum Entropy (MaxEnt) method of MassLynx™ software in order to détermine the mass of each intact antibody species or each reduced antibody fragment depending on the treatment used. Finally, DAR was determined from the deconvoluted spectra or UV chromatogram by summing the integrated MS (total ion current) or UV (280 nm) peak area of unconjugated and conjugated given species (mAb or associated fragment). For the

576

DAR détermination by UV chromatogram, relative area percentage of each species was multiplied by the number of drug attached. The summed, weighted areas of each species were divided by the sum of total relative area percentage and the results produced an estimation of the final average DAR value for the full ADC. For the DAR détermination by deconvoluted spectra, 5 the percentage of each species îdentified was calculated by intensîty peak value from deconvoluted spectra. The percentage obtained, was multiplied by the number of drug attached. The summed results produced an estimation of the final average DAR value for the full ADC.

Liquid Chromatography-Mass Spectrometry (LC/MS) II

LC/MS data were acquired using an instrument with the following parameters (Table 7):

Table 7. Parameters

Pump	Waters AcQuity UPLC Binary Solvent Manager
Sample Manager Column Compartment Detector ELSD Mass Spec Columns Eluent Al Eluent B1 Eluent A2 Eluent B2	Waters AcQuity UPLC Sample Manager Waters AcQuity UPLC Column Manager Waters AcQuity UPLC PDA Shimadzu ELSD-LTII Waters SQD AcQuity UPLC BEH C18 L7pm 2.1x50mm 0.1% Fonnic Acid in Water 0.1 % Fonnic Acid in Acetonitrile 5mM Ammonium Hydroxide in Water 5mM Ammonium Hydroxide in Acetonitrile

The methods used to generate LC/MS data were as follows (Tables 8-10):

Table 8. 2 minute acidic method min acidic method

Eluent Al	0.1% Fonnic Acid in Water
Eluent B1	0.1% Fonnic Acid in Acetonitrile
Flow	LO mL/min
Stop Time	3.00 min
pH	2.6
Gradient	Time %A (Eluent %B (Eluent Bl)

577

Al)
0.00	95	5
0.20	95	5
2.00	5	95
2.50	5	95
2.60	95	5
3.00	95	5

Column	AcQuity UPLC BEH Cl8 l.7pm 2.lx50mm
Column Température	50^ûC
TAC	210-400 nm
Mass Range	120-1500 Da
Scan Time	0.3 sec

Table 9. 2 minute basic method

2 min basic method
Eluent A2	5mM Ammonium Hydroxide in Water
Eluent B 2	5mM Ammonium Hydroxide in Acetonitrîle
Flow	1.0 mL/min
Stop Time	3.00 min
pH	10.2
Gradient	Time %A (Eluent A2)	%B (Eluent B2)
	0.00	95	5
	0.20	95	5
	2.00	5	95
	2.50	5	95
	2.60	95	5

578

	3.00 95	5
Column	AcQuity UPLC B EH Cl 8 1.7pm 2.1x50mm
Column	50°C
T emperature
TAC	210-400 nm
Mass Range	120-1500 Da
Scan Time	0.3 sec

Table 10. 5 minute acidic method

5 min acidic method
Flow	1.0 mL/min
Stop Time	5.20 min
pH	2.6
Gradient	Time %A (Eluent	%B (Eluent
	Al)	Bl)
	0.00 98	2
	4.40 2	98
	5.15 2	98
	5.19 98	2
Column	AcQuity UPLC BEH Cl 8 1.7pm 2.1x50mm
Column Température	50^ûC
TAC	210-400 nm
Mass Range	120-1500 Da
Scan Time	0.3 sec

579

High Resolution Mass Spectrometry (HRMS)

HRMS data were acquired using an instrument with the following parameters (Table 11):

Table 11, Parameters

Pump	Waters AcQuity UPLC Binary Solvent Manager
Sample Manager	Waters AcQuity UPLC Sample Manager
Column	Waters AcQuity UPLC Column Manager
Compartment Detector	Waters AcQuity UPLC PDA
ELSD	n/a
Mass Spec	Waters Xevo G2 Qtof AcQuity UPLC PrSTC4 300Â L7pm
Columns	2,1x100mm AcQuity UPLC CSH Cl 8 1.7 um 2.1x50mm ProSwift RP-3U 4.6x50mm SS

Eluent Al	0.1% Formic Acid in Water
Eluent B1	0.1% Formic Acid in Acetonitrile
Eluent A2	0.05% Trifluoroacetic Acid in Water
Eluent B2	0.05% Trifluoroacetic Acid in Acetonitrile

580

The methods used to generate HRMS data for linker/payloads and synthetic intermediates were as follows (Tables 7 and 8):

Table 12. 5 minute acidic method

5 min acidic method:
Flow Stop Time pH Gradient	1 .0 mL/min 5.2 min 2.6 Time %A (Eluent 0.00 98 4.40 2 5.15 2 5.19 98	%B (Eluent 2 98 98 2
Column Column TAC Mass Range Processing Scan Time	AcQuity UPLC BEHC18 1.7pm 2.1x50mm 50°C 210-400 nm 300-4000 Da n/a 0.5 sec

Table 13. 2 minute acidic method

2 min acidic method:	1.0 mL/min
Flow Stop Time pH Gradient	2.20 min 2.6 Time %A (Eluent	%B (Eluent
Column	A2) B2) 0.00 98 2 0.06 98 2 1.76 2 98 2.00 2 98 2.16 98 2 AcQuity UPLC CSH C18 1.7μιη 2.1x50mm

581

Column Température	50°C
UV	210-400 nm
Mass Range	100-2050 Da
Scan Time	0.2 sec

The method used to generate HRMS data for the ADCs was as follows (Table 14):

Table 14. Protein method

Protein method:
Flow Stop Time pH Gradient	1.0 mL/min 3.30 min 2.6 Time %A	%B
Column	0.00 98 2 0.70 98 2 2.00 2 98 2.10 2 98 2.30 98 2 3.30 98 2 ProSwift RP-3U 4.6x50mm SS
Column TAC Mass Range Processing Scan Time	50°C 210-400 nm 600-3900 Da 14000-170000 Da 1.5 sec

Size Exclusion Chromatography (SEC) I

Size exclusion chromatography (SEC) was performed to détermine the quality of the ADCs and aggregation percentage (%) after purification. The analysis was performed on analytical column Superdex 200 Increase 5/150 GL (GE Healthcare, 28990945) in isocratic conditions 100% PBS pH 7.4 (Sigma, P3813, 10PAK), flow 0.45 ml/min for 12 minutes. The % 10 aggregate fraction of the ADC sample was quantified based on the peak area absorbance at 280 nm. Calculation was based on the ratio between the high molecular weight eluent at 280 nm divîded by the sum of peak area absorbance at the same wavelength of the high molecular weight and monomeric eluents multiplied by 100%.

582

Size exclusion chromatography (SEC) (1) was performed to déterminé the quality of the ADCs and aggregation percentage (%) after purification. The analysis was performed on analytical column Superdex 200 Increase 5/150 GL (GE Healthcare, 28990945) in isocratic conditions PBS pH 7.2 ((Hyclone SH30028.03)), supplemented with 150 mM NaCl and 1 mM EDTA, flow 0.45 5 ml/min for 8 minutes. The % aggregate fraction of the ADC sample was quantified based on the peak area absorbance at 280 nm. Calculation was based on the ratio between the high molecular weight eluent at 280 nm dîvided by the sum of peak area absorbance at the same wavelength of the high molecular weight and monomeric eluents multiplied by 100%. Data was aquired on an Agilent Bio-Inert 1260 HPLC outfitted with a Wyatt miniDAWN light scattering and Treos refractive index detectors (Wyatt Technologies, Santa Barbara, CA).

Size Exclusion Chromatography (SEC) Il

SEC data were acquired using an instrument with the following parameters (Table 15) and a run length of 12 minutes:

Table 15. Parameters

Pump	Waters bioAcQuity UPLC Quatemary Solvent Manager
Sample Manager	Waters bioAcQuity UPLC Sample Manager FTN
Column	Waters AcQuity UPLC 30cm Column Heater
Compartiment Detector	Waters AcQuity UPLC PDA
ELSD	n/a
Mass Spec	n/a
Columns	Superdex 200 Increase 5/150 GL
Eluent Al	IX PBS (Phosphate Buffered Saline) + 0.1M
Eluent B1	NaCl + 5% Isopropanol n/a

583

Results

Characterization of exemplary ADCs is summarized in Table 16, 16a and 16b (coupling and LC/MS method, aggregation status, and DAR). The average DAR values were determined using the above LC/MS methods (LC/MS I) and percentage aggregation was determined using 5 the above SEC methods (SEC I).

Table 16. ADC analytîcal characterization and conjugation methodology

ADS	Conjugation Method	LC-MS method	DAR	% Agg. (by SEC I)
AbM-L9-P16	Cl	LC-IV	2.8	0.2
AbM-L9-P17	Cl	LC-IV	3.2	0.2
AbM-L9-P15	Cl	LC-IV	2.7	0.2
AbM-L9-C1	Cl	LC-IV	2.7	3.8

Table 16a. ADC analytîcal characterization and conjugation methodology

ADC	Conjugation Method	LC-MS Method I	DAR (by LC-MS D	% Agg. (by SEC I)
CD74_Fc Silenced-L5P1	C2	1	4.4	0.9
CD74_Fc Silenced-L7P1	C2	1	3.8	1.8

Table 16b. ADC analytîcal characterization and conjugation methodology

ADC	Conjugation Method	HRMS Method	DAR (by HRMS)	% Agg. (by SEC II)
CD74-L5-P1	C3	Protein	3.9	6.0
CD74-L30-P1	C3	Protein	3.8	5.7
CD74-L31-P1	C3	Protein	3.7	5.3
CD74-L32-P1	C3	Protein	3.6	10.3
CD74-L33-P1	C3	Protein	3.5	10.7
CD74-L34-PI	C3	Protein	3.9	10.1
CD74-L35-P1	C3	Protein	3.8	10.0

584

ADC	Conjugation Method	HRMS Method	DAR (by HRMS)	% Agg. (by SEC H)
CD74-L36-P1	C3	Protein	3.8	8.0
CD74-L37-P1	C3	Protein	3.8	11.0
CD74-L60-P1	C3	Protein	3.7	8.0
CD74-L61-P1	C3	Protein	3.8	3.4
CD74-L62-P1	C3	Protein	3.8	5.5
CD74-L63-P1	C3	Protein	3.7	12.0
CD74-L67-P1	C3	Protein	3.7	9.1
CD74-L68-P1	C3	Protein	3.8	17
CD74-L69-P1	C3	Protein	3.8	5.7
CD74-L71-P1	C3	Protein	3.8	12.3
CD74-L72-P1	C3	Protein	3.5	6.0
CD74-L77-P1	C3	Protein	3.5	11.0
CD74-L78-PI	C3	Protein	3.6	18.0
CD74-L79-PI	C3	Protein	3.7	21.0
CD74-L86-PI	C3	Protein	3.7	9.0

Example 5: In Vitro Assessment I

Inhibition of Mcl-l by Fluorescence Polarization fFP)

The relative binding potency of each compound was determined via Fluorescence

Polarization (FP). The method utilized a fluorescein-labelled ligand (Fluorescein-pAla-Ahx-AREIGAQLRRMADDLNAQY-OH (SEQ ID NO:72); MW 2,765) which binds to Mcl-l protein (UniProt Reference Sequence: Q07820; SEQ ID NO:48), leading to an increased anîsotropy measured in milli-polarization (mP) units using a reader. The addition of a compound which binds competitively to the same site as the ligand results in a greater proportion of unbound ligand in the System, as indicated by a decrease in mP units.

An 1 l-point serial dilution of each compound was prepared in DMSO and 2 μΐ transferred into fiat bottomed, low binding, 384-well plate (final DMSO concentration 5 %). 38 μΐ of buffer (10 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid [HEPES], 150 mM 585

NaCl, 0.05 % Tween 20, pH 7.4), containing the fluorescein-labelled ligand (final concentration l nM) and Mcl-l protein (final concentration 5 nM) was then added.

Assay plates were incubated -2 hours at room température before FP was measured on a Biomek Synergy2 reader (Ex. 528 nm, Em. 640 nm, Cut off 510 nm) and mP units calculated. The binding of increasing doses of test compound was expressed as a percentage réduction in mP compared to a window established between ‘5 % DMSO only’ and ‘100 % inhibition’ Controls. 11-point dose response curves were plotted with XL-Fit software using a 4-Parameter Logistic Model (Sîgmoidal Dose-Response Model) and the inhibitory concentrations that gave a 50 % réduction in mP (ICso) were determined. The results are presented in Table 17 below. The results suggest that the tested compounds inhibit interaction between the Mcl-l protein and the fluorescein-labelled peptide.

In Vitro Cvtotoxicity

Cytotoxicity studies were carried out on the NCI-H929 multiple myeloma cell line. The cells were distributed onto microplates and exposed to the test compounds for 48 hours. The cell viability was then quantified by a colorimétrie assay, the Microculture Tétrazolium Assay (Carmichael et al. (1987) Cancer Res. 47:936-42).

The results are expressed in IC50 (the concentration of compound that inhibits cell viability by 50%) and are presented in Table 17 below. The results show that the tested compounds are cytotoxic in NCJ-H929 cells.

Table 17. ICso of Mcl-l inhibition and cytotoxicity in NCI-H929 cells

Payloa	IC50 (M) Mcl-l FP	IC50 (Μ) MTT H929	Payloa	IC50 (M) Mcl-l FP	IC50 (M) MTT H929
Cl	2.8E-09	3.0E-09	C8	3.0E-09	5.74E-07
C2	2.6E-09	8.51E-06	C9	2.5E-09	2.27E-06
C3	9.48E-10	4.2E-08	CIO	3.4E-09	1.35E-08
C4	2.6E-09	3.21 E-06	Cil	5.3E-09	7.0E-09
C5	4.05E-09	4.08E-09	C12	5.9E-09	4.2E-08
C6	1.18E-09	3.0E-09	C13	2.2E-09	3.76E-09
C7	2.6E-09	1.88E-09	C14	SAE-10	2.61E-09

586

Example 6: In Vitro Assessment II - In vitro activity of CD74-L7-P1 and PI payload in

DLBCL cell lines (CTG 72h)

DLBCL cells lines (Karpas422, SUDHL4, NUDHL1, SUDHL6, SUDHLIO, DOHHL2 and SUDHL5) were cultivated in RPMI supplemented with 10% heat inactivated fêtai bovine sérum, penîcillin (100 lU/ml), streptomycin (100 pg/ml) and L-glutamine (2 mM). Cells were cultured at 37°C in a humidified atmosphère containing 5% CO₂. Cells were seeded in 96 microwell plates and exposed to the ADCs or the corresponding payload for 72h (serially diluted; 9 concentrations each, triplicates). Effects of ADCs or payloads on cell viability were assessed after 3 days of incubation at 37°C/5% CO₂ by quantification of cellular ATP levels using CellTiterGlo ai 75pL reagent/well. Ail the conditions were tested in triplicates. Luminescence was quantified on a multipurpose plate reader. IC50S were calculated using standard fourparametric curve fitting. ICso is defined as the compound concentration at which the CTG signal is reduced to 50% of that measured for the control. Each experiment was performed at least twice, with results being reproducible. As shown in Table 18 and FIGs. IA and IB, the payload PI and the ADC CD74-L7-P1 induced a dose dépendent decrease in the viability of DLBCL cells in CTG assay.Table 18: ICso of cytotoxicity of ADCs and payload in varions cell lines

IC50 M (CTG 72h)	Karpas42 2	SUDHL 4	NUDHL 1	SUDHL6	SUDHL 10	DOHHL 2	SUDHL5
lgG-L7- P1	Not tested	Not tested	Not tested	Not tested	>3.0e007	>3.0e007	>3.0e-007
CD74-L7- P1	1.054e-009	4.793 e010	1.033e- 008	1.453e- 009	9.4 He- Oll	1.195e- 009	3.139e- Üil
PI	1.921e-009	9.373e010	1.849e- oos	3.087e009	2.164e010	1.824e- 009	2.164e010

Example 7: In Vitro Assessment III - In vitro activity of CD74-Mcl-1 ADCs and payloads in Monomacl AML cell line (CTG 72h)Monomacl cells were cultivated in RPMI supplemented with 10% heat inactivated fêtai bovine sérum, penicillin (100 lU/ml), streptomycin (100 pg/ml) and L-glutamine (2 mM). Cell Unes were cultured at 37°C in a humidified atmosphère containing 5% CO₂. Cells were seeded in 96 microwell plates and exposed to the payloads or ADCs for 72h (serially diluted; 9 concentrations each, triplicates). Effects of payloads or ADCs on cell viability were assessed after 3 days of incubation at 37°C/5% CO₂ by 587 quantification of cellular ATP levels using CellTîterGlo at 75pL reagent/well. AH the conditions were tested in triplicates. Luminescence was quantified on a multipurpose plate reader. IC50S were calculated using standard four-parametric curve fittîng. IC50 is defined as the compound concentration at which the CTG signal is reduced to 50% of that measured for the control. Each experiment was perfonned at least twice, with results being reproducible. As shown in Table 19 and FIG. 2, the payloads and the anti-CD74-Mcl-l ADCs induced a dose dépendent decrease in the viability of Monomacl cells in CTG assay.

Table 19. IC50 of cytotoxicity of of ADCs and payload in Monomac-1 cell

ADCs or payloads	IC50 M (CTG 72h) Monomacl
CD74-L7-P1	9.217e-011
PI	1.294e-009
CD74-L9-P1	4.815e-010
PI	1.322e-009
CD74-L9-P17	2.367e-01I
P17	6.20le-010
CD74-L9-P16	2.064e-011
P16	2.812e-010
CD74-L9-P15	3.996e-011
P15	3.86e-010

Example 8: In Vitro Assessment IV - In vitro activity of CD74 ADCs in varions cancer cell lines

Cell Lines

The CD74 MCL-1 antibody drug conjugate CD74-L7-P1, Isotype IgG-L7-Pl ADC, and MCL-1 free payload PI were tested against six endogenous cancer cell lines. KMS-21BM (JCRB No. JCRB1185 cultured in RPMI-1640 + 10% FBS), KMS-20 (JCRB No. JCRB1196 cultured in RPMI-1640+ 10% FBS), MONO-MAC-1 (DSMZ No. ACC-252 cultured in RPMI1640 + 10% FBS + Ix non-essential amino acids + 1 mM sodium pyruvate), NOMO-1 (DSMZ No. ACC-542 cultured in RPMI-1640 + 10% FBS), Kasumi-6 (ATCC No. CRL-2775 cultured in RPMI-1640 + 20% FBS + 1.5 g/L sodium bicarbonate + 4.5 g/L glucose +10 mM HEPES, + 1

588 mM sodium pyruvate supplemented with 2 ng/ml human recombinant granulocyte macrophage colony stimulating factor (GM-CSF) and EOL-1 (DSMZ No. ACC-386 cultured in RPMI-1640 + 10%FBS)

Inhibition of cell prolifération and survival

The ability of the CD74 MCL-1 antibody drug conjugale (CD74-L7-P1), Isotype IgG-L7P1 ADC, and MCL-1 free payload PI to inhibit cell prolifération and survival was assessed using the Promega CellTiter-Glo® prolifération assay. Cell Unes were cultured in media that is optimal for their growth at 5% CO2, 37°C in a tissue culture incubator. Prior to seeding for the prolifération assay, the cells were split at least 2 days before the assay to ensure optimal growth density. On the day of seeding, suspension cells were harvested. Cell viability and cell density were detennined using a cell counter (Vi-Cell XR Cell Viability Analyzer, Beckman Coulter). Cells with higher than 85% viability were seeded in white clear bottom 384-well TC treated plates (Corning cat. # 3765). Cells were seeded at a density of Ι,θθθ cells per well in 45 pL of standard growth media. Plates were incubated at 5% CO2, 37°C overnight in a tissue culture incubator. The next day, free MCL-1 payload (PI), targeting MCL-1 ADC, and non-targeting isotype ADCs were prepared at 10X in standard growth media. The prepared drug treatments were then added to the cells resulting in final concentrations of 0.0005 - 500 nM and a final volume of 50 pL per well. Each drug concentration was tested in quadruplets. Plates were incubated at 5% CO₂, 37°C for 5 days in a tissue culture incubator, after which cell viability was assessed through the addition of 25 pL of CellTiter Glo® (Promega, cat# G7573), a reagent which lyses cells and measures total adenosine triphosphate (ATP) content. Plates were incubated at room température for 10 minutes to stabilize luminescent signais prior to reading using a luminescence reader (EnVisîon Multiiabel Plate Reader, PerkinElmer). To evaluate the effect of the drug treatments, luminescent counts from wells containing untreated cells (100% viability) were used to normalize treated samples. A variable slope model was applied to fît a nonlinear régression curve to the data in GraphPad PRISM version 7.02 software. IC50 and Amax values were extrapolated from the résultant curves.

Resuit

The dose response curves of représentative cancer cell Unes expressing the CD74 target of interest are shown in FIG. 3 (square = CD74-L7-P1, circle IgG-L7-Pl, triangle = free MCL1 payload P1 ). The concentrations of treatment required to inhibit 50% of cell growth or survival

589 (IC50) were calculated with représentative IC50 values of the cell lines tested summarized in

Table 20.

Table 20: CD74 MCL1 ADCs

	CD74-L7-P1	IgG-L7-Pl	Pl
	IC50 (nM)	Amax	IC50 (nM)	Amax	IC50 (nM)	Amax
KMS-21BM	0.948	98.75	>200	—	0.941	85.27
KMS-20	0.814	101.3	>200	—	4.946	94.34
MONO-MAC-1	**	t*	>200	—	1.151	82.41
Kasumi-6	2.947	127.8	>200	—	9.266	88.12
NOMO-1	1.433	95.88	>200	—	5.015	110.9
EOL-1	0.118	124.0	>200		1.095	67.97

** tested but curve did not converge

Example 9: In Vitro Assessment V - In vitro activity of CD74 ADCs as single agent and in combination with Bcl-2 inhbitors

Cell Lines

The CD74 MCL-1 antibody drug conjugales were tested against three endogenous cancer cell lines. MONO-MAC-1 (DSMZ No. ACC-252 cultured in RPMI-1640 + 10% FBS + Ix nonessentîal amino acids + 1 mM sodium pyruvate), NOMO-1 (DSMZ No. ACC-542 cultured in RPMI-1640 + 10% FBS) and EOL-1 (DSMZ No. ACC-386 cultured in RPMI-1640 + 10% FBS)

Inhibition qfcellprolifération and sinxival

The ability of the CD74 MCL-1 antibody drug conjugates to inhibit cell prolifération in combination with BCL-2 inhibitor and survival was assessed using the Promega CellTiter-Glo® prolifération assay. Cell lines were cultured in media that is optimal for their growth at 5% CO₂, 37°C in a tissue culture încubator. Prior to seeding for the prolifération assay, the cells were split ai least 2 days befbre the assay to ensure optimal growth density. On the day of seeding, suspension cells were harvested. Cell viability and cell density were determîned using a cell counter (Vi-Cell XR Cell Viability Analyzer, Beckman Coulter). Cells with higher than 85% viability were seeded in white clear bottom 384-well TC treated plates (Corning cat. # 3765). Cells were seeded at a density of 1,000 cells per well in 45 pL of standard growth media. Plates were incubated at 5% CO₂, 37°C ovemight in a tissue culture incubator. The next day, free MCL-1 payload (Pl), targeting MCL-1 ADCs and non-targeting isotype ADCs were prepared at

590

10X in standard growth media. The prepared drug treatments were then added to the cells resulting in final concentrations of 0.0005 - 300 nM and a final volume of 50 pL per well. Each drug concentration was tested in quadruplets. Three conditions were evaluated for each compound. The compounds were evaluated as single agents and in combination with Venetoclax and in combination with Compound Al. The Venetoclax and Compound Al were dosed at either 5 nM or 50 nM final concentration. Plates were incubated at 5% CO₂, 37°C for 5 days in a tissue culture incubator, after which cell viabilîty was assessed through the addition of 25 pL of CellTiter Glo® (Promega, cat# G7573), a reagent which lyses cells and measures total adenosine triphosphate (ATP) content. Plates were incubated at room température for 10 minutes to stabilize luminescent signais prier to reading using a luminescence reader (EnVision Multilabel Plate Reader, PerkinElmer). To evaiuate the effect of the drug treatments, luminescent counts from wells containing untreated cells (100% viabilîty) were used to normalize treated samples. A variable slope model was applied to fit a nonlinear régression curve to the data in GraphPad PR1SM version 7.02 software. IC50 and Amax values were extrapolated from the résultant curves.

Resuit

The dose response curves of représentative cancer cell Unes ex pressing the CD 74 targets of interest are shown în FIG. 4 and FIG. 5. The concentrations of treatment required to inhibit 50% of cell growth or survival (1C50) were calculated with représentative IC50 values of the cell lines tested summarized in Table 21.

Table 21: CD74-MCL-1 ADCs in Combination with Venetoclax and Compound Al

	NOMO-1	MONO-MAC-1	EOL-1
	IC50 (nM)	Amax (Span)	IC50 (nM)	Amax (Span)	IC50 (nM)	Amax (Span)
CD74-L5-P1	3.70	74.45	7.95	84.9	1.419	91.25
CD74-L5-P1 +5 nM Venetoclax	1.03	89.68	1.31	115.5	0.164	113.4
CD74-L5-P1 +50 nM Venetoclax	0.278	92.24	0.409	88.73	n/a	n/a
CD74-L5-P1 + 5 nM Compound Al	0.457	91.5	0.636	113.5	0.1364	99.58
CD74-L5-P1 + 50 nM Compound Al	0.138	100.6	0.156	115.4	n/a	n/a

591

PI	1.45	94.01	0.800	94.52	1.069	94.92
PI + 5 nM Venetoclax	1.59	70.4	0.183	110.2	0.365	87.42
PI + 50 nM Venetoclax	0.567	69.95	0.122	84.64	n/a	n/a
P1 + 5 nM Compound A1	0.640	60.65	0.100	121.5	0.2294	89.37
PI + 50 nM Compound Al	0.222	97.61	0.052	102.9	n/a	n/a

IgGl-L5-Pl	>50	—	107.9	73.83	>50	—
IgGLL5-Pl + 5 nM Venetoclax	>50	—	>50	107.1	>50	—
lgGI-L5-Pl + 50 nM Venetoclax	>50	--	47.57	108.7	n/a	n/a
IgGl-L5-Pl + 5 nM Compound Al	137.6	123.2	57.93	122.4	92.33	122.1
IgGl-L5-Pl + 50 nM Compound Al	>50	—	30.8	114.8	n/a	n/a

Example 10: In Vitro Assessment VI — In vitro activity of CD74 MCL-1 ADCs

Cell Lines

The CD74 MCL-i antibody drug conjugates were tested against three endogenous cancer cell lines. MONO-MAC-I (DSMZ No. ACC-252 cultured in RPMI-1640 + 10% FBS + Ix nonessential amino acids + 1 mM sodium pyruvate), NOMO-1 (DSMZ No. ACC-542 cultured in RPMI-1640 + 10% FBS) and EOL-1 (DSMZ No. ACC-386 cultured in RPMI-1640 + 10% FBS).

Inhibition of cellprolifération and survival

The abiiity of the CD74 MCL-1 antibody drug conjugates to inhibit cell prolifération and survival was assessed using the Promega CellTiter-Glo® prolifération assay. Cell lines were cultured in media that is optimal for their growth at 5% CCh, 37°C in a tissue culture incubator. Prior to seeding for the prolifération assay, the cells were split at least 2 days before the assay to ensure optimal growth density. On the day of seeding, suspension cells were harvested. Cell viability and cell density were determined using a cell counter (Vi-Cell XR Cell Viability Analyzer, Beckman Coulter). Cells with higher than 85% viability were seeded in white clear bottom 384-weil TC treated plates (Corning cat. # 3765). Cells were seeded at a density of 1,000 cells per well in 45 pL of standard growth media. Plates were incubated at 5% CO2, 37°C

592 overnight in a tissue culture incubator. The next day targeting MCL-l ADCs were prepared at 10X in standard growth media. The prepared drug treatments were then added to the cells resulting in final concentrations of 0.01 — 200 nM and a final volume of 50 pL per well. Each drug concentration was tested in quadruplets. Plates were incubated at 5% CCh, 37°C for 5 days in a tissue culture incubator, after which cell viability was assessed through the addition of 25 pL of CellTiter Glo® (Promega, cat# G7573), a reagent which lyses cells and measures total adenosine triphosphate (ATP) content. Plates were incubated at room température for 10 minutes to stabilize luminescent signais prior to reading using a luminescence reader (EnVision Multilabel Plate Reader, PerkinElmer). To evaluate the effect of the drug treatments, luminescent 10 counts from wells containing untreated cells ( 100% viability) were used to nonnalize treated samples. A variable slope model was applied to fit a nonlinear régression curve to the data in GraphPad P RI SM version 7.02 software. IC 50 and Amax values were extrapolai ed from the résultant curves and shown in Table 22.

Table 22: ICso values

LinkerPayload in ADC	Liberate d Payload	MONO- MAC-1 IC50 (nM)	MONOMAC-1 Amax	ΝΟΜΟ -1 IC50 (nM)	ΝΟΜΟ -1 Amax	EOL-1 IC50 (nM)	EOL1 Amax
L5-P1	Pl	7.95	84.9	2.221	55.58	1.018	75.06
L30-P1	Pl	5.451	86.78	1.617	78.95	0.2508	197.6
L31-P1	Pl	3.959	110.3	1.195	73.4	0.466	83.98
L32-P1	Pl	6.721	97.57	1.504	62.58	0.1604	125
L33-P1	Pl	6.716	89.18	2.584	58.95	0.6202	66.96
L34-P1	Pl	6.275	104.4	2.961	66.95	0.736	70.92
L35-P1	Pl		**	6.904	62.73	0.8938	75.56
L36-P1	Pl	0.519	66.3	4.578	63.34	0.6856	100.8
L37-P1	Pl	**	**	5.141	83.05	0.0425	243
L60-P1	Pl	**		2.625	76.94	0.5348	79.04
L61-P1	Pl	**	n	0.5457	45.63	0.03283	63.76
L62-P1	Pl	**	**	3.411	65.18	0.7708	83.03
L63-PI	Pl		**	0.7138	61.25	**	**
L67-P1	Pl		**	2.493	79.39	0.2614	119.3
L68-P1	PI		**	6.912	91.25	0.788	127.3
L69-P1	Pl	K	**	14.75	95.03	0.7043	159.6
L71-Pl	Pl	*4=	**	9.825	53.62	0.3409	93.09
L72-P1	Pl	**	**		57.68	0.3857	109.8
L77-P1	Pl	0.9865	108.2	3.185	48.53	0.3832	102.6
L78-P1	Pl	**	O	3.735	53.4	0.4304	87.22
L79-P1	Pl	**	* t	3.136	56.07	0.4916	91.3

593

L86-P1

PI

**

7.475

74.52

0.7212

105.5

** tested but curve did not converge

Example 11. In Vivo Assessment I - In vivo activity of CD74 ADCs in Nomol acute myeloid leukemia model after intraveneous (IV) administration

The in vivo therapeutic effect of a CD74-targeting ADC formulated in PhosphateBuffered Saline (PBS) is determined in Nomol acute myeloid leukemia model after intravenous (IV) administration, The follow compounds were tested: anti-CD74_CysmAb Fc silent_L5-Pl, anti-CD74_CysmAb Fc silent and IgGl-Linker-Payload Fc silent.

Materials and methods

Nomol cells, obtained from DSMZ, were cultured in RPMI supplemented with 10% FBS. 0.1ml containing 5x10⁶ cells were subcutaneously inoculated into the right flank of female SCID mice, provided by Charles River. When tumors reached the appropriate volume, mice were randomized, 6 animais per group, using Easy stat software. IgGl-Linker-Payload Fc silent, anti-CD74_CysmAb Fc silent and anti-CD74_CysmAb Fc silent_L5-Pl (30 mg/kg) were injected once IV in PBS. Venetoclax (synthetized at the Servier Research Institute - IdRS) was formulated in PEG/EtOH/Phosaî (Polyethylene g/yco Z/ethanol/phosal, 30/10/60) and adminîstered per os (PO) daily on weekdays (50mpk, 4ON/3OFF/4ON). Mice body weight was monitored three times a week and tumor size measured using electronic calipers. Tumor volume was estimated by measuring the minimum and maximum tumor diameters using the fonnula: (minimum diameter)²(maximum diameter)/2. The last day with at least half of animais of ail groups still présent in the study (d9), tumor growth inhibition was calculated using the formula:

(, Médian (DTV at Dx in treated group) ) , „„ 1---------------------------— x 100

Médian (DTV at Dx in Control group) J

With DTV (Delta Tumor Volume) at Dx, calculated being TV at Dx - TV at Randomizatîon. Mice were sacrificed at the first measurement for which tumor volume exceeded 2000 mm³ or at the first sîgns of animal health détérioration. Ail experiments were conducted in accordance with the French régulations in force in 2018 after approval by IdRS Ethical Committee. SCID mice were maintained according to institutional guidelines.

594

Results

The efficacy of anti-CD 74_CysmAb Fc silent_L5-Pl on Nomol xenografts is illustrated in FIG. 3. Treatment was started 10 days post tumor cells inoculation (médian size: I9l .6 mm³). JgGl-Linker-Payload Fc silent (non-targeting ADC FS), anti-CD74_CysmAb Fc silent (naked antibody FS) and anti-CD74_CysmAb Fc silent_L5-Pl (CD74-targeting ADC FS) were administered once IV at 30mg/kg. The non-targeting ADC FS and the CD74-targeting ADC FS were also combined to venetoclax, which was dosed PO 4ON/3OFF/4ON at 50mg/kg. On day 9, no or very slight Tumor Growth Inhibition (%TGI) was induced by the naked antibody FS, the CD74-targeting ADC FS or the correspondent non-targeting ADC FS at 30mg/kg (TGI= 32.7%, -5.6% and -12.2 respectively), as depicted in FIG. 6 and Table 23. However, în combination with venetoclax, the anti-tumor activity induced by the CD74-targeting ADC FS was much greater than the correspondent non-targeting ADC FS (TGI= 98.6% vs 5.5% respectively, p<0.Ü01). Of note, venetoclax alone had barely any effect on tumor growth (TGI= 26.2%). No clinically relevant body weight loss or other clinical signs due to the treatment were observed (FIG. 7).

Table 23: Nomol tumor growth inhibition

Treatment	Dose/Schedule	%TGI (d9)
IgGl-Linker-Payload Fc silent	30MK QD, IV	-12.2
anti-CD74_CysmAb Fc silent	30MK QD, IV	32.7
anti-CD74_CysmAb Fc silent_L5- P1	30MK QD, IV	-5.6
Venetoclax	50MK 4ON/3OFF/4ON, PO	26.2
IgGl-Linker-Payload Fc silent+ venetoclax	30MK QD, IV+ 50MK 4ON/3OFF/4ON, PO	5.5
anti-CD74_CysmAb Fc silent_L5- P1 + venetoclax	30MK QD, IV+ 50MK 4ON/3OFF/4ON, PO	98.6***

*** p <= 0.001 compared to control group.

Example 12. In Vivo Assessment II - In vivo activity of CD74 ADCs in Karpas422 diffuse large B-cell lymphoma model after intraveneous (IV) administration

595

The in vivo therapeutîc effect of a CD74-targeting ADC formulated in PhosphateBuffered Saline (PBS) was determined in Karpas422 diffuse large B-cell lymphoma model after întravenous (IV) administration. The following compounds were tested: CD74-targeting ADC is anti-CD74_CysmAb Fc silent_L5-Pl, IgGl-Linker-Payload Fc silent and anti-CD74_CysmAb Fc silent.

Materials and methods

Karpas422 cells, obtained from ATCC, were cultured in RPMI supplemented with 20% FBS. Cells were resuspended in 100% Matrigei (BD Biosciences) and 0.1ml containing 1 IxIO⁶cells were subcutaneously inoculâted into the right flank of female NSG mice, provided by Charles River. When tumors reached the appropriate volume, mice were randomized, 6 animais per group, using Easy stat software. IgG 1-Linker-Payload Fc silent, anti-CD74_CysmAb Fc silent and anti-CD74_CystnAb Fc silent_L5-Pl (30 mg/kg) were injected once IV în PBS. Venetoclax (synthetized at the Servier Research Institute - IdRS) was formulated in PEG/EtOH/Phosal (Polyethylene g/ycoZ/ethanol/phosal, 30/10/60) and administered per os (PO) daily on weekdays (50mpk, 4ON/3OFF/4ON). Mice body weight was monîtored three tîmes a week and tumor size measured using electronic calipers. Tumor volume was estimated by measuring the minimum and maximum tumor diameters using the formula: (minimum diameter)²(maximum diameter)/2. The last day with at least half of control animais still présent in the study (d23), tumor growth inhibition was calculated using the formula:

(, Médian (DTV atDx intreated group) ) 1--¹--------------------²---— x 100

Médian (DTV at Dx in Control group) J

With DTV (Delta Tumor Volume) at Dx, calculated being TV at Dx - TV at Randomization. Response was evaluated as it follows: CR (Complété Response) îf tumor size was <25mm³ for at least three measurements, PR (Partial Response) if tumor sîze was comprised between 25mm³and 168.3 mm³ (half of the starting size) for at least three measurements. Mice were sacrifïced at the first measurement for which tumor volume exceeded 2000 mm³ or at the first signs of animal health détérioration. Ail experîments were conducted in accordance with the French régulations in force in 2018 after approval by IdRS Ethical Committee. SCID mice were maintained according to institutîonal guidelines.

Residts

596

The efficacy of anti-CD74_CysmAb Fc silent_L5-Pl on Karpas422 xenografts is illustrated in FIG. 8. Treatment was started 17 days post tumor cells inoculation (médian size: 336.6 mm3). IgGl-Linker-Payload Fc silent (non-targeting ADC FS), anti-CD74_CysmAb Fc sîlent (naked antibody FS) and anti-CD74_CysmAb Fc silent_L5-Pl (CD74-targeting ADC FS) were administered once IV at 30mg/kg. The non-targeting ADC FS and the CD74-targeting ADC FS were also combined to venetoclax, which was dosed PO 4ON/3OFF/4ON at 50mg/kg. On day 23, no Tumor Growth Inhibition (%TGI) was induced by the naked antibody FS, the CD74-targeting ADC FS or the correspondent non-targeting ADC FS at 30mg/kg (TGI= -l%, 61.8% and -76.2% respectively), as depicted in FIG. 8 and Table 24. However, in combination with venetoclax, the anti-tumor activity induced by the CD74-targeting ADC FS was much greater than the correspondent non-targeting ADC FS (TG1= 154.9% vs -9% respectively, p<0.001), and complété régression (CR) was exclusively achieved in this treatment group (Table 24). Of note, venetoclax alone had barely any effect on tumor growth (TGI= 31.1%). No clinical signs due to the treatment were observed, apart from a moderate body weight ioss (still not clinically relevant) in the groups dosed with venetoclax (FIG. 9).

Table 24:: Karpas422 tumor growth inhibition

Treatment	Dose/Schedule	%TGI (d23)	%CR	%PR
IgGl-Linker-Payload Fc silent	30MK QD, IV	-76.2	0	0
anti-CD74_CysmAb Fc silent	30MK QD, IV	-1	0	0
anti-CD74_CysmAb Fc silent_L5- P1	30MK QD, IV	-61.8	0	0
Venetoclax	50MK 4ON/3OFF/4ON, PO	31.1	0	0
IgGl-Linker-Payload Fc silent+ venetoclax	30MK QD, IV+ 50MK 4ON/3OFF/4ON, PO	-9	0	0
anti-CD74_CysmAb Fc silent_L5PI+ venetoclax	30MK QD, IV+ 50MK 4ON/3OFF/4ON, PO	154 9***	33	67

*** p <= o.oûl compared to control group

Example 13. In Vivo Assessment III - In vivo activity of CD74 ADCs in Monomacl acute myeloid leukemia model after intraveneous (IV) administration

597

The in vivo therapeutic effect of a CD74-targeting ADC formulated in PhosphateBuffered Saline (PBS) was determined in Monomacl acute myeloid leukemia model after intravenous (IV) administration. The following compounds were tested: CD74-targeting ADC is anti-CD74_CysmAb Fc silent_L5-Pl, IgGl-Linker-Payload Fc silent, and anti-CD74_CysmAb Fc silent.

Materials and methods

Monomacl cells, obtained from DSMZ, were cultured in RPMI supplemented with 10% FBS. 0.1ml containing 5x10⁶ cells were subcutaneously inoculated into the right flank of female SCID mice, provided by Charles River. When tumors reached the appropriate volume, mice were randomized, 6 animais per group, using Easy stat software. IgGl-Linker-Payload Fc silent, anti-CD74_CysmAb Fc silent and anti-CD74_CysmAb Fc silent_L5-Pl (30 mg/kg) were injected once IV in PBS. Venetoclax (synthetized at the Servier Research Institute - IdRS) was formulated in PEG/EtOH/Phosal (Polyethylene g/ycoZ/ethanol/phosal, 30/10/60) and administered per os (PO) daily on weekdays (50mpk, 2ON/2OFF/5ON/2OFF/4ON). Mice body weight was monitored three times a week and tumor size measured using electronic calîpers. Tumor volume was estimated by measuring the minimum and maximum tumor diameters using the formula: (minimum diameter)²(maximum diameter)/2. The last day with at least half of control animais still présent in the study (dl6), tumor growth inhibition was calculated using the formula:

Médian (DTV at Dx in treated group) ] , „„ Médian (DTV at Dx in Control group) )

With DTV (Delta Tumor Volume) at Dx, calculated being TV at Dx - TV at Randomization. Response was evaluated as ît follows: CR (Complété Response) if tumor size was <25mm³ for at least three measurements, PR (Partial Response) if tumor size was comprised between 25mm³and 60.1 mm³ (half of the starting size) for at least three measurements. Mice were sacrificed at the first measurement for which tumor volume exceeded 2000 mm³ or at the first signs of animal health détérioration. Ail experiments were conducted in accordance with the French régulations in force in 2018 after approval by IdRS Ethical Committee. SCID mice were maintained according to institutional guidelînes.

Results

598

The efficacy of anti-CD74_CysmAb Fc silent_L5-Pl on Monomacl xenografts is illustrated in FIG. 10. Treatment was started 7 days post tumor ceils inoculation (médian size: 120.2 mm³). IgGl-Linker-Payload Fc silent (non-targeting ADC FS), anti-CD74_CysmAb Fc silent (naked antibody FS) and anti-CD74_CysmAb Fc silent_L5-Pl (CD74-targeting ADC FS) were administered once IV at 30mg/kg. The non-targeting ADC FS and the CD74-targeting ADC FS were also combined to venetoclax, which was dosed PO 2ON/2OFF/5ON/2OFF/4ON at 50mg/kg. On day 16, the Tumor Growth Inhibition (%TGI) induced by the CD74-targeting ADC FS at 30mg/kg was greater than the naked antibody FS (TGI= 33.8% vs -23% respectively, p<0.05) and the correspondent non-targeting ADC FS (TGI= -14.6%), as depicted in FIG. 10 and Table 25. When combined to venetoclax, the non-targeting ADC FS displayed some antitumor activity (TGI= 34.4%, p<0.05), but a complété and long-1asting tumor régression was observed only in combination with the CD74-targeting ADC FS (TG1= 108.5%, p<0.001). No clinically relevant body weight loss or other clinical signs due to the treatment were observed (FIG. 11).

Table 25: Monomacl tumor growth inhibition

T reatment	Dose/Schedule	%TGI (dl6)	%CR	%PR
IgGl-Linker-Payload Fc silent	30MK QD, IV	-14.6	0	0
anti-CD74_CysmAb Fc silent	30MK QD, IV	-23	0	0
anti-CD74_CysmAb Fc silent_L5-P 1	30MK QD, IV	33.8*	0	0
Venetoclax	50MK 2ON/2OFF/5ON/2OFF/4ON, PO	4.5	0	0
IgGl-Linker-Payload Fc silent+ venetoclax	30MK QD, IV+50MK. 2ON/2OFF/5ON/2OFF/4ON, PO	34.4*	0	0
anti-CD74_CysmAb Fc silent_L5-Pl + venetoclax	30MK QD, IV+50MK 2ON/2OFF/5ON/2OFF/4ON, PO	108.5***	100	0

* p <= 0.05, ** p <= 0.01, *** p <= 0.001 compared to control group

Example 14: In vivo efficacy of CD74-L5-P1 Fc silent against the EOL1 human acute myeloid (éosinophilie) leukemia xenograft model in mice

As the in vitro studies had shown target-dependent and potent inhibition of cell growth in the EOL1 cell line by combination of CD74-L5-P1 Fc silent with venetoclax, the antitumor activity of this combination treatment was evaluated in vivo in this AML model.

599

Methods

EOL1 lucîferase transfected cells were cultured at 37 °C in an atmosphère of 5 % CO₂ in air in RPMH640 (BioConcept Ltd. Amimed) supplemented with 10 % FCS (BioConcept Ltd. Amimed, # 2-01F30-I), 2 mM L-glutamine (BioConcept Ltd. Amimed, #5-10K00-H) and ImM sodium pyruvate (BioConcept Ltd. Amimed, #5-60F00~H), 10 mM HEPES (Gibco #11560496). To establish EOL1 xenografts cells were harvested and re-suspended in HBSS (Gibco, #14175) before injecting 100 pL containing 5 x 10⁶ cells subcutaneously in the flanks of female SCID beige mice (Charles River, Germany). Tumor growth was momtored regularly post cell inoculation and animais were randomized into treatment groups (n = 6) with a mean tumor volume of about 130 mm³. Control and various treatment groups were dosed as indîcated in FIG. 12. CD74-L5-P1 Fc silent, isotype IgGl ADC Fc silent and CD74 Fc silent antibody were administered intravenously (iv) once at the start of treatment at the doses indîcated in FIG. 12 either alone or in combination with venetoclax which was administered orally (po) daily (qd) at 50 mg/kg. Doses were adjusted to individual mouse body weights. The iv dose volume was 10 ml/kg and each ADC was dissolved in 0.9% (w/v) NaCl in water. Tumor volume data on day 10 post treatment were analyzed statistically using Kruskal-Wallis rank sum test (Indigo Software). When applicable, results are presented as mean ± SEM. As a mcasure of efficacy the %T/C value is calculated on day 10 according to:

(Atumor volume^treated/Atumor volume^control)*lÜ0 Tumor régression was calculated according to:

-(Atumor volume^tr£ated/tumor volume^{11 ealedal SÎart})*100

Where Atumor volumes represent the mean tumor volume on the évaluation day minus the mean tumor volume at the start of the experiment.

Results: Efficacy and Tolerability

The antitumor effects of CD74-L5-P1 Fc silent in combination with venetoclax along with various control groups are summarized in FIG. 12 and Table 26. Limited efficacy was observed with venetoclax (50 mg/kg po qd) alone and the combination of CD74 Fc silent antibody plus venetoclax. A slight tumor growth delay (TGD) was observed with combination of isotype IgGI ADC (30 mg/kg) plus venetoclax (50 mg/kg) and also with CD74-L5-P1 Fc silent alone (30 mg/kg) but neither were statistically different from the vehicle control group. In contrast complété régressions (CRs) were observed with CD74-L5-P1 Fc silent dosed at 10 or 30 mg/kg iv once in combination with venetoclax 50 mg/kg po qdx21 (p<0.05 from vehicle group

600 and isotype IgGl ADC plus venetoclax group). ΑΠ treatments were well tolérâted based on body weight changes (FIG. 13).

Table 26: Antitumor effects of CD74-L5-P1 Fc silent in combination with venetoclax

Test agent, dose schedule and route	Day	ATumor volume (mm³)	T/C (%)	Régression (%)
Vehicle p.o. + vehicle i.v.	10	1082	100
Vehicle po + CD74-L5-P1 Fc silent 30 mg/kg i.v.	10	817	76
Venetoclax 50 mg/kg p.o.qd + vehicle for ADC	10	977	90
Venetoclax 50 mg/kg p.o. qd + CD74L5-P1 Fc silent 30 mg/kg i.v.	10	-128		-100*^s
Venetoclax 50 mg/kg p.o. qd + CD74L5-P1 Fc silent 10 mg/kg i.v.	10	-127		-I00*^3,
Venetoclax 50 mg/kg p.o. qd + isotypeIgGl ADC Fc silent 30 mg/kg i.v.	10	963	88.99
Venetoclax 50 mg/kg p.o. qd + CD74 Fc silent 30 mg/kg i.v.	10	1028	95.03

Delta tumor volumes and T/C% values were calculated for day 10 and are presented as means. * Indicates statistical différence (p<0.05) compared to vehicle control and $ indicates statistical différence (<0.05) compared to isotype IgGl ADC + venetoclax on day 10 (Kruskal-Wallis rank sum test; Indigo Software).

Examplc 15:7« vivo efficacy of CD74-ADCs Fc silent with different linker payloads against the EOL1 human acute myeloid (éosinophilie) leukemia xenograft model in mice

To optîmize the linker payload, efficacy of 6 CD74-ADCs with different linker payloads (L5-P1, L30-P1, L31-P1, L32-P1, L33-P1 and L34-P1) was evaluated in the EOL1 mode) in vivo.

Methods

EOL1 luciferase transfected cells were cultured at 37 °C in an atmosphère of 5 % CCh in air in R.PMI1640 (BioConcept Ltd. Amimed) supplemented with 10 % FCS (BioConcept Ltd. Amimed, # 2-01F30-1), 2 mM L-glutamine (BioConcept Ltd. Amimed, #5-10K00-H) and ImM sodium pyruvate (BioConcept Ltd. Amimed, #5-60F00-H), 10 mM HEPES (Gibco #11560496). To establish EOL1 xenografts cells were harvested and re-suspended in HBSS (Gibco, #14175) before înjecting 100 pL containing 5 x 106 cells subcutaneously in the flanks of female SCID beige mice (Charles River, Germany). Tumor growth was monitored regularly post cell

601 inoculation and animais were randomized into treatment groups (n = 6) with a mean tumor volume of about 130 mm³. Control and various CD74-ADCs fc silent (L5-P1, L30-P1, L31-P1, L32-P1, L33-P1 and L34-P1) were dosed as indicated in FIG. 14. The ADCs were administered intravenously (iv) once at the start of treatment at 2.5 mg/kg or at 3.3 mg/kg in combination with venetoclax which was administered orally (po) daily (qd) at 50 mg/kg. Doses were adjusted to individual mouse body weights. The iv dose volume was 10 ml/kg and each ADC was dissolved in 0.9% (w/v) NaCl in water. Tumor volume data on day 10 post treatment were analyzed statistically using one way ANOVA post hoc Tukey's multiple comparîsons test (Indigo Software). Results are presented as mean ± SEM. As a measure of efficacy the %T/C value is calculated on day 10 according to:

(Atumor volume^treated/Atumor volume^conl,Ol)*100

Tumor régression was calculated according to:

-(Atumor vohime'^reatetl/tumor volume^{treated at start})* 100

Results: Efficacy and Tolerability

Ail ADCs, except CD74-L5-P1 Fc silent, signîficantly (p<0.05) reduced the growth of EOL1 tumors after administering a low dose of 2.5 mg/kg in combination with venetoclax compared with the vehicle control group on day 10 (FIG. 14 and Table 27). CD74-L34-PI Fc silent exhibited a dose-related response between 2.5 and 3.3 mg/kg. Ail treatment schedules were well tolerated based on body weight changes (FIG. 15).

Table 27: Antitumor effects of CD74-ADCs in combination with venetoclax

Test agent, dose schedule and route	Day	ATumor volume (mm³)	T/C (%)
Vehicle p.o. + vehicle i.v.	10	1082	100
Venetoclax 50 mg/kg p.o.qd + CD74-L5-P1 2.5 mg/kg i.v.	10	702	70
Venetoclax 50 mg/kg p.o. qd + CD74-L30-P1 2.5 mg/kg i.v.	10	47	5*^s
Venetoclax 50 mg/kg p.o. qd + CD74-L31-P1 2.5 mg/kg i.v.	10	144
Venetoclax 50 mg/kg p.o. qd CD74-L32-P1 2.5 mg/kg i.v.	10	38	4*$
Venetoclax 50 mg/kg p.o. qd + CD74-L33-P1 2.5 mg/kg i.v.	10	228	2₃*s
Venetoclax 50 mg/kg p.o. qd + CD74-L34-P1 2.5 mg/kg i.v.	10	401	40*

602

Venetoclax 50 mg/kg p.o. qd + CD74-L34-P1 3.3 mg/kg i.v.

10

170

17*^s

Delta tumor volumes and T/C% values were calculated for day 10 and are presented as means. * Indicates statistical différence (p<0.05) compared to vehicle control and ^s indicates statistical différence (<0.05) compared to CD74-L5-P1 + venetoclax on day 10 (one way ANOVA post hoc Tukey's multiple comparisons test; Indigo Software).

Example 16 - expression of humanized LL1 variants

Expression vectors containing different humanized LL1 heavy chains and humanized

LL1 light chains were cotransfected into Expi293 cells (Invitrogen, Carlsbad, CA) using expifectamine and cultured for 7 days. Culture supernatants were harvested b y centrifugation, 10 fdtered, and antibodies purified b y Protein A affinity chromatography. Antibody purity after affïnity chromatography was tested by analytical size exclusion chromatography.

The first round of transfections (Table 28) focused on evaluating the different humanized heavy and light chains, along with PTM removal mutations (see Example 17).

Table 28: Expression of humanized LL1 variants, first round

Heavy Chain	Light chain	Final yield (mg)	Purity (% monomer)
hLLl	hLLl	2.58	99.73
hLLl	hzLLl-Vklb	2.57	99.75
hLLl	hzLLl-Vkla	2.28	99.79
hLLl	hLLl (NQ)	1.34	99.78
hLLl	hLLl (QG)	1.05	99.78
hLLl	hLLl (TG)	1.89	99.7
hLLl	hLLl (SG)	2.06	99.67
hLLl	hLLl (NV)	0.53	99.53
hzLLl-VH2	hzLLl-Vklb	2.98	99.74
hzLLl-VHlb	hzLLl-Vklb	2.60	99.5
hzLLl-VHla	hzLLl-Vklb	2.60	99.62
hzLLl-VH3	hLLl	3.49	99.69
hzLLl-VH2	hLLl	2.10	99.81
hzLLl-VH3	hzLLl-Vkla	2.71	99.66
hzLLl-VHlb	hLLl	2.96	99.62
hzLLl-VH2	hzLLl-Vkla	2.77	99.77
hzLLl-VHla	hLLl	2.80	99.67
hzLLl-VHlb	hzLLl-Vkla	3.00	99.55

603

hzLLl-VH3	hzLLl-Vklb	3.07	99.62
hzLLl-VH la	hzLLl-Vkla	2.64	99.61

Overail expression and purity was comparable between most of the humanized variants and the original humanized version (hLLl). A réduction in overail yield was seen for most of the PTM removal variants compared to tire other humanized variants,

The NQ mutation set was incorporated into the hzLLl -Vklb and hzLLl-Vkla light chains, and a second round of variants were expressed using transient transfection into Expi293 cells (Table 29):

Table 29: Expression of humanized LL1 variants, second round

Heavy Chain	Light chain	Final yield (mg)	Purity (% monomer)
hLLl	hzLLl-Vkla (NQ)	2.8	96.0
hLLl	hzLLl-Vklb (NQ)	0.5	98.0
hzLLl-VHla Y95F	hzLLl-Vkla (NQ)	2.75	98.0
hzLLl-VHla Y95F	hzLLl-Vklb (NQ)	0.8	99.0
hzLLl-VHla V5Q Y95F	hzLLl-Vkla (NQ)	1.8	98.0
hzLLl-VHla V5Q Y95F	hzLLl-Vklb (NQ)	0.8	99.0

Overail expression of these variants seemed to correlate with the sélection of light chain - variants with the hzLLl-Vkla (NQ) light chain expressed at a higher level than variants with the hzLLl-Vklb (NQ) light chain.

Humanization variant antibodies were tested using FACS and SPR for binding to human CD74 protein. Variant antibodies with binding characterîstics similar to milatuzumab were selected.

Example 17: humanization and PTM removal from LL1 VH and VL sequences

Variable domains for the murine anti-CD74 antibody LL1 were described in US7931903 in the context of chimeric antibody sequences (cLLl):

LLl VH

QVQLQQSGPELKKPGETVKVTCKTSGYTFTNYGVNW1KQTPGEGLQWMGWINP NTGEPTFDDDFKGRFAFSLESSASTAFLQISNLKNEDMGTYFCSRSRGKNEAWFDYWGQ GTLVTVSS (SEQ ID NO:49)

604

LLl VL

DIQLTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIYTV SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYFCSQSSHVPPTFGAGTKLEIK (SEQ ID NO:50)

These were disclosed at the same time as sequences for humanized VH and VL domains, hLLl VH and hLLl VL, respectively:

hLLl VH

QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWIKQAPGQGLQWMGWINPNTGE PTFDDDFKGRFAFSLDTSVSTAYLQISSLKADDTAVYFCSRSRGKNEAWFAYWGQGTL VTVSS (SEQ IDNO:lO) hLLl VL

DIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWFQQRPGQSPRLLIYTVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHVPPTFGAGTRLEIK (SEQ ID NO:23)

Humanization of these sequences was described in example 5 of US7931903. This humanization process describes humanizing both variable domains using non-germline antibody sequences of humanized antibodies (RT-TS3, HF-21/28) as a template, which introduces a number of murine framework residues into the final antibody sequences. Also, a problematic site (Asn-Gly, or N G) for a common protein post-translational modification (deamidation) was left intact in the CDR L1 région of the humanized light chain. These properties are undesirable for an antibody therapeutic, as they could increase immunogenicity or decrease the binding affinity of the antibody upon storage.

Two separate efforts were undertaken to résolve these complications. First, both the heavy and light chains of LLl were rehumanized using human germline frameworks as a template. Second, several mutations were deliberately introduced into the Asn-Gly sequence in the CDR L1 région of the LLl light chain to disrupt the post-translational modification site. Humanized constructs with reasonable expression and binding to CD74 were combined with PTM removal mutations to produce final lead candidates.

Humanization of LLl

Initially, murine LLl VH and VL sequences were compared against mouse germline frameworks. For the VH, the closest V-gene match (79/98 residues) appears to be murine IGHV9-1 *02 (IMGT nomenclature), with mutations from germline scattered between CDR and

605

Framework régions. For the VL, the closest V-gene match (93/100 residues) appears to be murine IGKVl-100*01, with mutations divided between CDR régions and the N terminus.

Alîgnment of the murine LL1 VH région to human germlines revealed a reasonable match to three human germlines: IGHV7-4-l*02 (closest match), 1GHV1-3*O2, and IGHV551 *02. Several murine residues in framework régions maintained in the prior heavy chain humanization (hLLl VH) were also flagged as being potentialiy important to binding. These three human germlines and murine residues were used to préparé 6 variant heavy chaîns:

1. humanization to IGHV7-4-l*02 (hzLLl-VHIa)

QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGVNWVRQAPGQGLEWMGWIN PNTGEPTFDDDFKGRFVFSLDSSVSTAYLQISSLKAEDTAVYYCSRSRGKNEAWFDYWG QGTLVTVSS (SEQ ID NO:51)

2. Humanization to IGHV7-4-l*02 with rétention of V37I mutation (hzLLl-VHlb) QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGVNWIRQAPGQGLEWMGWINPNTGE PTFDDDFKGRFVFSLDSSVSTAYLQISSLKAEDTAVYYCSRSRGKNEAWFDYWGQGTL VTVSS (SEQ IDNO:52)

3. Humanization to IGHV7-4-l*02 with rétention ofY95F mutation (hzLLl-VHIa Y95F)

QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGVNWVRQAPGQGLEWMGWINPNTG EPTFDDDFKGRFVFSLDSSVSTAYLQISSLKAEDTAVYFCSRSRGKNEAWFDYWGQGTL VTVSS (SEQ ID NO:53)

4. Humanization to IGHV7-4-l*02 with rétention of V5Q Y95F mutations (hzLLl -VH la V5Q Y95F)

QVQLQQSGSELKKPGASVKVSCKASGYTFTNYGVNWVRQAPGQGLEWMGWINPNTG EPTFDDDFKGRFVFSLDSSVSTAYLQISSLKAEDTAVYFCSRSRGKNEAWFDYWGQGTL VTVSS (SEQ ID NO:54)

5. Humanization to IGHVl-3*02 (hzLLl-VH2)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGVNWVRQAPGQRLEWMGWINPNTG EPTFDDDFKGRVTITLDSSASTAYMELSSLRSEDMAVYYCSRSRGKNEAWFDYWGQGT LVTVSS (SEQ ID NO:55)

6. Humanization to IGHV5-51*O1 (hzLLl-VH3)

EVQLVQSGAEVKKPGESLK1SCKGSGYTFTNYGVNWVRQMPGKGLEWMGWINPNTGE PTFDDDFKGQVTISLDSSISTAYLQWSSLKASDTAMYYCSRSRGKNEAWFDYWGQGTL VTVSS (SEQ IDNO:56)

606

For the light chain, alignaient of the murine LL1 light chain to human germlines revealed reasonable matches with two human germlines - 1GKV2-4O*O1 and IGKV2-3Û*02. These sequences were used to préparé two variant humanized light chai ns:

1. Humanization to IGKV2-40*01 (hzLLl-Vkia)

DIVMTQTPLSLPVTPGEPASISCRSSQSLVHRNGNTYLHWYLQKPGQSPQLLIYTVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHVPPTFGQGTKLEIK (SEQ ID NO:27)

2. Humanizationto 1GKV2-3O*O2 (hzLLl-Vklb)

DVVMTQSPLSLPVTLGQPASISCRSSQSLVHRNGNTYLHWYQQRPGQSPRLLIYTVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHVPPTFGQGTKLEIK (SEQ ID

NO:31)

Humanized heavy chains and light chains were synthesized and used for recombinant protein expression (see Example 16).

PTM Removed from LL1 light chain

To remove the deamidation site (Asn-Gly, N G) from the CDR L1 région of the light chain, sequences containing the foilowing 5 altemate residues (QG, SG, TG, NQ, NV) were constructed in the hLLl VL sequence. This was used to make the foilowing 5 variant light chains:

hLLl VL(QG)

DIQLTQSPLSLPVTLGQPASISCRSSQSLVHRQGNTYLHWFQQRPGQSPRLLIYTVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHVPPTFGAGTRLEIK (SEQ ID NO:57) hLLl VL (SG)

DIQLTQSPLSLPVTLGQPASISCRSSQSLVHRSGNTYLHWFQQRPGQSPRLLIYTVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHVPPTFGAGTRLEIK (SEQ ID NO:58) hLLl VL (TG)

DIQLTQSPLSLPVTLGQPASISCRSSQSLVHRTGNTYLHWFQQRPGQSPRLLIYTVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHVPPTFGAGTRLEIK (SEQ ID NO:59) hLLl VL(NQ)

607

DIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNQNTYLHWFQQRPGQSPRLLIYTVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHVPPTFGAGTRLEIK (SEQ ID NO:36) hLLl VL (NV)

DIQLTQSPLSLPVTLGQPASISCRSSQSLVHRNVNTYLHWFQQRPGQSPRLLIYTVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSHVPPTFGAGTRLEIK (SEQ ID NO:60)

Mutant humanized light chains were synthesized and used for recombinant protein expression.

Example 18 Biacore Kd Détermination

Binding to human and cyno CD74 for the WT IgG and humanized variant antibodies were assessed for affinity déterminations using Biacore. Kinetic rate constants were performed via SPR using the Biacore 8K instrument (Cytiva, formerly GE Healthcare Lifescîences) as described below. The anti-IgG Fc capture method was utilized in order to déterminé kinetics for the antibodies. A commercial!y available anti-IgG Fc capture kit was used (Cytiva, formerly GE Healthcare Lifescîences). The provided IgG was immobilized on the chip surface using the provided amine couplîng protocol from GE. This immobilized antibody captured the WT and humanized variants, and the soluble human and cyno CD74 flowed over as the analytes. The CD74 concentration started at 100 nM and was serially diluted at one part to one part of running buffer for seven concentrations. Régénération was performed at the end of each concentration using the specified conditions in the kit.

Double reference subtraction was completed to generate the final data. The raw data was fitted to a L1 binding mode!, with parameter(s) Refractîve Index set to local. Due to the nature of the antigen, the following table lists the apparent kinetic results for the WT and humanized IgG variants.

Table 30 Kinetic Results

Sample	Antigen	ka (1/Ms)	kd (1/s)	KD (M)	Antigen	ka (1/Ms)	kd (1/s)	KD (M)
milatuzumab	Human CD74	1.0E+05	5.1E- 04	4.9E-09	Cyno CD74	5.6E+04	8.8E-04	1.6E- 08
Hcmil x LCmil_NQ	9.6E+04	3.3E04	3.4E-09	3.0E+04	7.7E-04	2.6E08
Mil-HC x hzVklb	2.3E+O5	1.7E04	7.2E-10	1. 1E+05	5.8E-04	5.1E09

608

Mil_HC x hzVkla		2.1E+05	1.8E- 04	8.5E-10		1.0E+05	5.SE-04	5.7E09
VHmil x VKlaNQ	2.0E+05	1.8E04	9.1E-I0	1.0E+05	6.2E-04	5.9E09
VHmil x VKlbNQ	2.2E+05	1.6E- 04	7.6E-10	1.1E+05	5.8E-04	5.3E09

609

Claims

( 1 ) the cancer cells in the sample express CD74;

( 1 ) the cancer expresses CD74;

(1) D comprises:

642 or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait of any of the foregoing;

1+

N' (iv) Rq3 comprises the formula: , wherein * is a bond to the linker;

(v) R09 is Cyo₂; preferably Cyo₂ is an optionally substituted aryl group;

(vi) Cyos comprises a heteroaryl group selected from a pyrazolyl group and a pyrimidinyl group; (vii) Cyos îs a pyrimidinyl group; and/or viii) the L is attached to D by a covalent bond from L to R03 of formula (I), (II), or (III); or the L is attached to D by a covalent bond from L to R09 of formula (I), (II), or (III).

(1) D comprises a compound of Formula (I):

631

wherein:

Ring Do is a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group,

Ring Eo is a furyl, thienyl or pyrrolyl ring,

Xoi, Xoj, Xo4 and Xos independently of one another are a carbon atom or a nitrogen atom,

Xo2 is a C-Ro26 group or a nitrogen atom, ( j means that the ring is aromatic,

Yo is a nitrogen atom or a C-Ro₃ group,

Zo is a nitrogen atom or a C-R04 group,

Roi îs a halogen atom, a linear or branched (Ci-Cô)alkyl group, a linear or branched (C₂-Cô)alkenyl group, a linear or branched (C₂-Cô)alkynyl group, a linear or branched (Ci-Côjhaloalkyl group, a hydroxy group, a hydroxy(Ci-Cô)alkyl group, a linear or branched (Ci-Côjalkoxy group, -S-(Ci-C&)alkyl group, a cyano group, a nîtro group, -Cyos, -(Co-C6)alkyl-NRonRon’, -0-(Ci-C6)alkyl-NRouRon’, -0-(Ci-C6)alky1-Roi2, C(0)-ORon, -0-C(0)-Ron, -C(0)-NRoi 1R01i’, -NRoh-C(0)-Roii’, -NRou-C(0)-ORoh’, -(Ci-C6)alkyl-NR_oli-C(0)-Rou’,-S0₂-NRoiiRoii’,or -SO₂-(Ci-C₆)alkyl,

Ro?, Ro3, Ro4 and Ros independently of one another are a hydrogen atom, a halogen atom, a linear or branched (Ci-Cé)alkyl group, a linear or branched (C₂Cé)alkenyl group, a linear or branched (C₂-C&)alkynyl group, a linear or branched (Ci632

Côjhaloalkyl, a hydroxy group, a hydroxy(Ci-C&)alkyï group, a linear or branched (CiCô)alkoxy group, a -S-(Ci-C&)alkyl group, a cyano group, a nitro group, -(Co-CôjalkylNRonRou’, -O-Cyoi, -(Co-C6)alkyl-Cyoi, -(C2-Cô)alkenyl-Cyoi, -(C2-C6)alkynyl-Cyoi, -0-(Ci-C6)alkyl-NRoi iRo11 -O-(Ci-C₆)alkyl-R_ü3i,-O-(Ci-C6)alkyl-R₀|_2î -C(0)-ORon, -0-C(0)-Roii, -C(O)NRonRoii’, -NRon-C(0)-Roii\ -NRon-C(0)-ORon’, -(Ci-C6)alkyl-NRon-C(0)-Ron’, -S0₂-NRoi iRo11’, or -SO₂-(Ci-C6)alkyl, or the pair (Roi, R02), (R02, Roa), (R03, R04), or (R04, R05) together with the carbon atoms to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contants l to 3 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted by l or 2 groups selected from halogen, linear or branched (Ci-Cô)alkyl, (Co-Côjalkyi-NRonRoii’, -NRoiîRoij’, -(CoCô)alkyl-Cyoi or oxo,

Roô and Ro? independently of one another are a hydrogen atom, a halogen atom, a linear or branched (Ci-Cô)alkyl group, a linear or branched (C₂-Cô)alkenyl group, a linear or branched (C₂-C&)alkynyl group, a linear or branched (Ci-C(j)haloalkyl, a hydroxy group, a linear or branched (Ci-C&)alkoxy group, a -S-(Ci-C6)alkyl group, a cyano group, a nitro group, -(Co-C&)alkyl-NRoi 1R01i’, -0-(Ci-C₆)alkyl-NRoiiRon’, -O-Cyoi, -(Co-C₆)alkyl-Cyoi, -(C₂-C₆)alkenyl-Cyoi, -(C₂-Cô)alkynyl-Cyoi, -O-(Ci-C₆)alkyl-R₍)i2, -C(0)-ORon, -O-C(O)-R₀h, -C(0)-NRoi iRo11’, -NRoirC(O)-R₀ii’, -NRoi i-C(0)-ORoj i’, -(Ci-C6)alkyl-NR(Hi-C(O)-R₀ii>^ -SO₂-(Ci-C₆)alkyl, or the pair (R00, Ro?), when fused with the two adjacent carbon atoms, together with the carbon atoms to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contai ns l to 3 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted by a linear or branched (Cj-Côjalkyl group, -NRo_l3Roi3’, -(Co-Cô)alkyl-Cyoi or an oxo,

Wo is a -CH₂- group, a -NH- group or an oxygen atom,

Rq₈ is a hydrogen atom, a linear or branched (Ci-CQalkyl group, a -CHRoaRob group, an aryl group, a heteroaryl group, an aryl(Cj-Cô)alkyl group, or a heteroaryl(CiCôjalkyl group,

R09 is a hydrogen atom, a linear or branched (Ci-Cô)alkyl group, a linear or

633 branched (C₂-C&)alkenyl group, a linear or branched (C₂-Cô)alkynyl group, -Cyoz, -(CiC&)alkyl-Cyo2, -(C2-C&)alkenyl-Cyo2, -(C₂-Cô)alkynyl-Cyo2, -Cyo2-Cyo₃, -(C2-Cô)alkynyl0-Cyo2, -Cyo2-(Co-C6)alkyI-0-(Co-C6)alkyl·Cy()î, a halogen atom, a cyano group, -C(O)Roi4, or -C(0)-NRoi₄Roi4’,

Roio is a hydrogen atom, a linear or branched (Ci-Cô)alkyl group, a linear or branched (C₂-Cô)alkenyl group, a linear or branched (C₂-C6)alkynyl group, an arylfCiCô)alkyl group, a (Ci-Cô)cycloalkylalkyl group, a linear or branched (Ci-Côjhaloalkyl, or -(Ci-C₆)alky1-0-Cyo4, or the pair (Ro% Roio), when fiised with the two adjacent carbon atoms, together with the carbon atoms to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ringmembers, which optionally contains 1 to 3 heteroatoms selected from O, S and N,

Roii and Ron’ independently of one another are a hydrogen atom, an optionally substituted linear or branched (Ci-Cô)alkyl group, or -(Co-Côjalkyl-Cyoi, or the pair (Ron, Roii’) together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains, in addition to the nitrogen atom, 1 to 3 heteroatoms selected from O, S, and N, wherein the N atom may be substituted by 1 or 2 groups selected from a linear or branched (Ci-Cô)alkyl group, and wherein one or more of the carbon atoms of the linear or branched (Ci-Cô)alkyl group is optionally deuterated,

R012 is -Cyos, -Cyo5-(Co-C6)alkyl-0-(Co-C6)alkyl-Cyo6, -Cyo5-(Co-Cô)alkyl-Cyo6, -Cy₀₅-(C₀-C6)alkyI-NR₀i i-(C₀-C₆)alkyi-Cy₀₆, -Cy₀₅-Cy₀₆-O-(C₀-C6)alkyl-Cy₀7, -Cyo₅-(C_oC6)alky]-0-(Co-C₆)alkyl-Cyo9, -Cyo₅-(Co-C₆)alkyl-Cyo9, -NH-C(0)-NH-Ron,-Cyo5-(Cü-Cô)alkyl-NRûii-(Co-C6)alkyl-Cyo9, -C(O)-NR_0J1R₀h’, -NRonRoii\ -ORoii, -NRqii-C(O)-Rqii’, -0-(Ci-Cô)alkyl-ORoi i, -SO2-R011, -C(O)-ORch 1,

Roi3, Roi3’, R014 and Roi4’ independently of one another are a hydrogen atom, or an optionally substituted linear or branched (Ci-Cô)alkyl group,

Roa îs a hydrogen atom or a linear or branched (Ci-Côjalkyl group,

Rob is a -0-C(0)-0-Roc group, a -0-C(0)-NRocRoe’ group, or a -0-P(0)(QRo_c)₂group,

Roc and Ro? independently of one another are a hydrogen atom, a linear or branched (Ci-Cs)alkyl group, a cycloalkyl group, a (Ci-C6)alkoxy(Ci-C&)alkyl group, or a (Ci-C6)alkoxycarbonyl(Ci-Cô)alkyl group,

634 or the pair (Roc, Ro?) together with the nitrogen atom to which they are attached form a non-aromatic ring composed of from 5 to 7 ring members, which may contain in addition to the nitrogen atom from 1 to 3 heteroatoms selected from oxygen and nitrogen, wherein the nitrogen is optionally substituted by a linear or branched (Ci-Côjalkyl group,

Cyoi, Cyo2, Cyo3, Cyo4, Cyos, Cyo&, Cyo7, Cyos and Cyoïo independently of one another, are a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group, each of which is optionally substituted,

or Cyo9 is a heteroaryl group which is substituted by a group selected from -OP(0)(ORo2o)2; -O-P(O)(O'M⁺)₂; -(CH₂)pO-0-(CHRo_ls-CHRoi9-0)_qo-R₀2o; hydroxy;

hydroxy(Ci-Cô)alkyl; -(CH₂)io-Uo-(CH₂)_so-heterocycloalkyl; and -Uo-(CH₂)qo-NRo2iRo2i\

Rois îs a hydrogen atom; a -(CH₂)po-0-(CHRoi8-CHRoi9-0)_qo-Ro2o group; a linear or branched (Ci-C6)alkoxy(Ci-C6)alkyl group; a -Uo-(CH₂)_qo-NRo2iRo2i’ group; or a -(CH₂)iQ-Uo-(CH₂)so-heterocycloalkyl group,

Rois îs a hydrogen atom; a hydroxy group; a hydroxy(Ci-C&)alkyl group; a (CHQ^-Uo-tCHQso-heterocycloalkyl group; a (CH₂)iî)-Uo-Vo-0-P(0)(ORo2o)₂ group; a -O-P(O)(O’M⁺)₂ group; a -O-S(O)₂ORq₂0 group; a -S(0)₂ORo₂o group; a -(CH₂)_po-0(CHRoi8-CHRo!9-0)_qo-Ro20 group; a -(CH₂)po-0-C(0)-NRo2₂Ro23 group; ora-Uo-(CH₂)_qoNR021R021’ group,

R017 is a hydrogen atom; a -(CH₂)po-0-(CHRoi8-CHRoi9-0)_qo-Ro₂o group; a -CH₂P(0)(ORo2o)₂ group, a -0-P(0)(ORo₂o)₂ group; a -O-P(O)(O'M⁺)₂ group; a hydroxy group; a hydroxy(Ci-C6)alkyl group; a -(CH₂)ro-Uo-(CH₂)_so-heterocycloalkyl group; a -Uo-(CH₂)qo-NRo₂]Ro2]’ group; or an aldonic acid,

M⁺ is a pharmaceutically acceptable monovalent cation,

Uo is a bond or an oxygen atom,

Vo is a -(CH₂)so- group or a -C(O)- group,

Roi8 is a hydrogen atom or a (Ci-CôjalkoxylCj-CQalkyl group,

R019 is a hydrogen atom or a hydroxy(C]-Cô)alkyl group,

Ro₂o is a hydrogen atom or a linear or branched (Ci-CQaïkyl group,

635

Ro2i and Ro₂i* independently of one are a hydrogen atom, a linear or branched (Ci-Cô)alkyl group, or a hydroxy(Ci-Cô)alkyl group, or the pair (R021, R021’) together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains, in addition to the nitrogen atom, 1 to 3 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted by a hydrogen atom or a linear or branched (Ci-Ce)alkyl group,

Rû22 is a (Ci-C6)alkoxy(Ci-Cô)alkyl group, a -(CH₂)_po-NRo₂4Ro₂4’ group, or a -(CH₂)_Po-0-(CHRo । s-CHRo 19-0)qo-R₂o group,

R023 is a hydrogen atom or a (Ci-C6)alkoxy(Ci-Cô)aIkyl group, or the pair (Ro₂₂, R023) together with the nitrogen atom to which they are attached fonn an aromatic or non-aromatic ring containing 5 to 18 ring members, which optionally contains, in addition to the nitrogen atom, 1 to 5 heteroatoms selected from O, S and N, wherein the resulting ring is optionally substituted by a hydrogen atom, a linear or branched (Ci-Cô)alkyl group or a heterocycloalkyl group,

Ro24 and R024’ independently of one another are a hydrogen atom or a linear or branched (Ci-Côjalkyl group, or the pair (R024, Ro₂₄’) together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring composed of from 5 to 7 ring members, which may contaîn in addition to the nitrogen atom from 1 to 3 heteroatoms selected from O, S and N, and wherein the resulting ring is optionally substituted by a hydrogen atom or a linear or branched (Ci-Ce)alkyl group,

R025 is a hydrogen atom, a hydroxy group, or a hydroxy(Ci-CQalkyl group,

Ro₂6 is a hydrogen atom, a halogen atom, a linear or branched (Ci-Cô)alkyl group, or a cyano group,

Ro₂7 is a hydrogen atom or a linear or branched (Ci-Cô)alkyl group,

Ro₂8 is a -O-P(O)(O)(O) group, a -0-P(0)(0 )(ORojo) group, a -0-P(0)(ORo3o)(ORo3o’) group, a -(CH₂)_P0-O-SO₂-O· group, a -(CH₂)_Po-S02-0’ group, a -(CH₂)_Po-0-S0₂-ORo30 group, -Cyoïo, a -(CH₂)_Po-S0₂-ORo30 group, a -0-C(0)-Ro₂₉group, a -O-C(O)-ORq₂₉ group or a -O-C(O)-NRq₂₉Ro₂9’ group;

R029 and Rom’ independently of one another are a hydrogen atom, a linear or branched (Ci-Cô)alkyl group or a linear or branched amino(Ci-Cô)alkyl group,

Roîo and Roîo’ independently of one another are a hydrogen atom, a linear or

636 branched (Ci-Cô)alkyl group or an aryl(Ci-C6)alkylgroup,

zwitterionic form or has a monovalent anionic counterion,

1 5 8. The antibody-drug conjugate of any one of claims 1 to 7, wherein -(L-D) comprises or is formed from a compound of formula:

ri ? V h v 0 ^H 0 < ^H i L N-N 0—Z 0) h₂no \—/ R is H, -CH₃ or -CH₂CH₂C(=O)OH; _ZO-R ioYîv'· , wherein:

623

A is a bond,-OC(=O)-*, OH , OH OH , OH

O O _z * < H II TA

YO-P-O-P-⁷

OH OH , -OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or

-OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH₃)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C₃-Cs cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-l inhibitor;

(1) the attachment group is formed by a réaction comprising at least one reactive group;

1. An antibody-drug conjugate of Formula ( 1 ): Ab-tL-Db (1) wherein:

Ab is an anti-CD74 antibody or an antigen-binding fragment thereof; p is an integer from 1 to 16; and

-(L-D) is of the formula (C):

-^R¹—Q—Lp— ^l3-r² (C), wherein:

R¹ îs an attachment group;

Li is a bridging spacer;

L_p is a peptide group comprising 1 to 6 amino acids;

D is an Mcl-l inhibitor;

G1-L2-A is a self-immolative spacer;

L2 is a bond, a methylene, a neopentylene or a C2-C3 alkenylene;

0 * . Il s γ-ο-ργA is a bond, -OC(=O)-*, OH , 0 0 , * n n 00* 0 . n n . n * -i-O-P-O-PA- -|-Ο-Ρ-Ογγ OH OH OH ï 5

-|-O-P-O-P^

OH OH , -OC(=O)N(CH₃)CH2CH2N(CH₃)C(=O)-* or

-OC(=O)N(CH₃)C(R^a)2C(R^a)₂N(CH₃)C(=O^ wherein each R^a is independently selected from H, Ci-C& alkyl, and C₃-Cs cycloalkyl and the * of A indicates the point of attachment to D;

L₃ is a spacer moiety; and

R² is a hydrophilic moiety.
(2) the cancer expresses CD74;

(2) the tumor is a breast cancer, gastric cancer, bladder cancer, brain cancer, cervical cancer, colorectal cancer, esophageal cancer, hepatocellular cancer, melanoma, oral cancer, ovarian cancer, non-small cell lung cancer, prostate cancer, small cell lung cancer, or spleen cancer, or the cancer cell population is from a tumor or a hematological cancer;

(2) the cancer is a tumor or a hematological cancer;

(2) D comprises:

(2) D comprises a compound of Formula (II):

637

wherein:

Zo is a nitrogen atom or a C-R04 group,

Roi is a halogen atom, a linear or branched (Ci-Cô)alkyl group, a linear or branched (C₂-Cô)alkenyl group, a linear or branched (C₂-Cô)alkynyl group, a linear or branched (Ci-Cô)haloalkyl group, a hydroxy group, a linear or branched (Ci-Cô)alkoxy group, a -S-(Ci-Cô)alkyl group, a cyano group, -Cyos, -NRohRoh’,

Ro2, R03 and R04 independently of one another are a hydrogen atom, a halogen atom, a linear or branched (Ci-CfOalkyl group, a linear or branched (C₂-Ce)alkenyl group, a linear or branched (C2-Cô)alkynyl group, a linear or branched (C i-Cô)haloalkyl, a hydroxy group, a linear or branched (Ci-Cô)alkoxy group, a -S-(CiCô)alkyl group, a cyano group, a nitro group, -(Co-Cô)alkyl-NRonRoir, -O-Cyoi, -(CoC₆)alkyl-Cyoi, -(C₂-C₆)alkenyl-Cyoi, -(C2-C6)alkynyl-Cyoi, -0-(Ci-C6)alkyI-NR(HiRoji’, -0-(Ci-Cô)alkyl-Ro3i, -C(0)-ORoii, -0-C(0)-Roii, -C(0)-NRohRoii’, -NRoii-C(0)-Ron’, -NRoii-C(0)-ORouk-(Ci-C6)alkyl-NRoii-C(0)-R_oli\-S02-NRm^

Cô)alkyl, or the pair (R02, Rm) or (R03, R04) together with the carbon atoms to which they are attached form an aromatic or non-aromatic ring containîng 5 to 7 ring members, which optionally contains 1 to 3 heteroatoms selected from O, S and N, wherein the ring is optionally substituted by a group selected from a linear or branched (Ci-Côjalkyl, -NRoiîRoiî’, -(Co-Cô)alkyl-Cyoi and oxo,

Roô and Rov independently of one another are a hydrogen atom, a halogen atom, a linear or branched (Ci-Cô)alkyl group, a linear or branched (C₂-Cô)alkenyl group, a linear or branched (C2-Cô)alkynyl group, a linear or branched

638 (Ci-Cô)haloalkyl, a hydroxy group, a linear or branched (Ci-Cô)alkoxy group, a -S-(Ci-C&)alkyl group, a cyano group, a nitro group, -(Co-Ce)alkyl-NRoi iRoii’, -O-Cyoi, -(Co-C₆)alkyl-Cyoi, -(C₂-C6)alkenyl-Cyoi, -(C2-C₆)alkynyl-Cy_Oi, -O-(Ci-C6)alkyl-R_0i2, -C(O)-OR_0Ii, -O-C(O)-R₀ii, -C(0)-NRoiiRoii-NRoii-C(O)-R₀ji -NRoii-C(0)-ORoii’,-(Ci-C6)alkyl-NRoii-C(0)-Roii’,-S02-NRoiiRoii’,or -SO2-(Ci-C₆)alkyl, or the pair (Roô, Ro?), when fused with two adjacent carbon atoms, together with the carbon atoms to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains l to 3 heteroatoms selected from O, S and N, and wherein the resulting ring is optionally substituted by a group selected from a linear or branched (Ci-Cô)alkyl group, -NR0J3R013’, -(Co-CQalkyl-Cyoi and an oxo,

Rus is a hydrogen atom, a linear or branched (Ci-Cs)alkyl group, an aryl group, a heteroaryl group, an aryl-(Ci-C6)alkylgroup, or a heteroaryltCi-CQalkyl group,

Roy is a linear or branched (Ci-Cô)alkyl group, a linear or branched (C2-Cb)alkenyl group, a linear or branched (C₂-Cô)alkynyl group, -Cyo2, -(Ci-C6)alkyl-Cyo2, -(C₂-Cô)alkenyl-Cyo2, -(C₂-C6)alkynyl-Cyo₂, -Cyo₂-Cyo3, -(C2-C6)alkynyl-0-Cyo2, -Cyo2-(Co-Cô)alkyl-0-(Co-C6)alkyl-Cyo3, a halogen atom, a cyano group, -C(0)-Roi₄, -C(0)-NRoi₄Roi4’,

Rom and Roii’ independently of one another are a hydrogen atom, an optionally substituted linear or branched (Cj-Cô)alkyl group, or -(Co-Cô)alkyl-Cyoi, or the pair (Roi i. Roi i’) together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains, in addition to the nitrogen atom, l to 3 heteroatoms selected from O, S and N, wherein the N atom is optionally substituted by a linear or branched (Ci-Côjalkyl group, and wherein one or more of the carbon atoms of the linear or branched (C । -Céjalkyl group is optionally deuterated,

R012 is -Cyos, -Cyo5-(Co-C6)alkyl-Cyo6, -Cyo5-(Co-C6)alkyl-0-(Co-C6)alkyl-Cyo6, -Cyo5-(Co-C₆)alkyl-NRoi !-(C_o-C₆)alkyl-Cyo6, -Cyo5<yo6-0~(Co-C6)alkyl-Cy₀7, -Cy_O5-(CoC₆)alkyl-Cy₀9, -NH-C(O)-NH-R₀ii, -C(0)-NR_0iiRoti’, -NRojiRoii’, -ORoii, -NR₀n-C(O)Ron’, -0-(Ci-C6)alkyl-ORon,-S0₂-Roii,or -C(O)-ORnn,

Roî3, R013’, R014 and Rom’ independently of one another are a hydrogen atom, or an optionally substituted linear or branched (Ci-C&)alkyl group,

639

Cyoi, Cyo2, Cyo₃, Cyoj, Cyoô, Cyo? and Cyos independently of one another, are a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group, each of which is optionally substituted,

Cy₀₉ is

0I5, Ry^, and R_0]7 are as defined for formula (I),

R<m is

2' , wherein:

each R independently is H, -CH₃ or -CH₂CH₂C(=O)OH;

A is a bond, -OC(=O)-*, O O , *

OH

OH OH

OH

OH OH , -OC(=O)N(CHî)CH₂CH2N(CHj)C(=O)-* or -OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH₃)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and Cî-Cs cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-l inhibitor.

(2) ^hîⁿ ° , wherein:

R is H, -CH₃ or -CH₂CH₂C(=O)OH;

O * O O * 0 , II s , Il I 1 ς II *

-ξ-ο-ρ-ο-ρφ- n-o-p-o<^

A is a bond,-OC(=O)-*, OH , OH OH , OH

O O * _t n n

(2) the peptide group comprises Val-Cit, Phe-Lys, Val-Ala, Val-Lys, Leu-Cit, sulfo-Ala-Val,

(2) the attachment group is formed by reacting:

a fïrst reactive group that is attached to the linker, and a second reactive group that is attached to the antibody or is an amino acid residue of the antibody;

(2) Li is ⁿ , and n is an integer from 1 to 12 or n is 1 or n is 12, wherein the * of Li indicates the point of direct or indirect attachment to Lp, and the ** of Li indicates the point of direct or indirect attachment to R¹;

611 (3) Li is

, and n is an integer from l to 12, wherein the * of Li indicates the point of direct or indirect attachment to Lp, and the ** of Li indicates the point of direct or indirect attachment to R¹;

OH OH

2. The antibody-drug conjugate of claim 1, or pharmaceutically acceptable sait thereof, wherein -(L-D) is of Formula (D):

610

wherein:

R¹ is an attachment group;

Li is a bridging spacer;

Lp is a peptide group comprising 1 to 6 amino acids;

O * O O * O

-bo-p-O-P-l· TO-P-O^

A is a bond, -OC(=O)-*, OH , OH OH , OH ° 0 * r μ H XL -bo-p-o-A

OH OH , -OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or

-OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH₃)C(=O)-*, wherein each R^a is independently selected from H, Ci-Ce alkyl, and C₃-Cs cycloalkyl and the * of A indicates the point of attachment to D;

L₃ is a spacer moiety; and

R² is a hydrophilic moiety.
(3) the cancer is a tumor or a hematologîcal cancer;

(3) the cancer cell population is from a breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer;

(3) the cancer is a breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or Β-ceII origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer; and/or (4) the antibody-drug conjugate, the composition or the pharmaceutical composition is used in combination with at least one additional therapeutic agent.

(3) D comprises:

or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or

(3) the attachment group is formed by reaction comprising at least one reactive group, wherein the at least one of the reactive groups comprises:

a thiol, a maleimide, a haloacetamide, an azide, an alkyne, a cyclcooctene,

615 a trîaryl phosphine, an oxanobomadiene, a cyclooctyne, a diaryl tetrazine, a monoaryl tetrazine, a norbornene, an aldéhyde, a hydroxylamine, a hydrazine, NH₂-NH-C(=O)-, a ketone, a vinyl sulfone, an aziridine, an amino acid residue,

-SSR⁴, -S(=O)₂(CH=CH₂), -(CH₂)₂S(=O)₂(CH=CH₂), -NHS(=O)₂(CH=CH₂), -

616

wherein:

each R³ is independently selected from H and Ci-Céalkyl;

each R⁴ is 2-pyridyl or 4-pyridyl;

each R⁵ îs independently selected from H, Ci-Côalkyl, F, Cl, and -OH;

each R⁶ is independently selected from H, Ci-Cf>alkyl, F, Cl, -NH₂, -OCH3,

-OCH2CH3, -N(CH₃)2, -CN, -NO2 and -OH;

each R⁷ is independently selected from H, Ci.6alkyl, fluoro, benzyloxy substituted with =C(=O)OH, benzyl substituted with -C(=O)OH, Ci-4alkoxy substituted with -C(=O)OH and Ci-4alkyl substituted with -C(=O)OH;

617 (4) the attachment group is formed by reacting: a first reactive group that is attached to the linker, and a second reactive group that is attached to the antibody or is an amino acid residue of the antibody, wherein the first reactive group and second reactive group comprise:

(3) the hydrophilîc moiety represented by R³ comprises:

(i) a polysarcosine with the following moiety:

O _; wherein n is an integer between 3 and 25; and R is H, -CH₃ or

-CH₂CH₂C(=O)OH; or

(ii) a polyethylene glycol of formula: ⁿ or ^m , wherein

R is H, -CH₃, CH₂CH₂NHC(=O)OR_a, -CH₂CH₂NHC(=O)R_a, or -CH₂CH₂C(=O)OR_a, R’ is OH, -OCH₃, -CH₂CH₂NHC(=O)OR₃, -CH₂CH₂NHC(=O)R_a, or -OCH₂CH₂C(=O)OR₃, in which R_a is H or Cm alkyl optionally substituted with either OH or Cm alkoxyl, and each of m and n is independently an integer between 2 and 25; or

3. The antibody-drug conjugate of claim 1 or 2, wherein (1) Li comprises:

*-CH(OH)CH(OH)CH(OH)CH(OH)-**, wherein each n is an integer from 1 to 12, wherein the * of Li indicates the point of direct or indirect attachment to Lp, and the ** of Li indicates the point of direct or indirect attachment to R¹ ;

O
(4) the cancer is a breast cancer, multiple myeloma, plasma cell myeloma, leukemia, lymphoma, gastric cancer, acute myeloid leukemia, bladder cancer, brain cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell o ri gin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small cell lung cancer, chronic lymphocytic leukemia, prostate cancer, small cell lung cancer, or spleen cancer; and/or (5) the sample is a tissue biopsy sample, a blood sample, or a bone marrow sample.

(4) the antibody-drug conjugate, composition, or pharmaceutical composition reduces or inhibits the growth of the tumor or reduces the cancer cell population or slows the expansion of the

654 cancer cell population by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99%; and/or (5) the antibody-drug conjugate, composition, or pharmaceutical composition is used in combination with at least one additional therapeutic agent.

(4) D comprises:

or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait of any of the foregoing;

4-o-P’O-p^

OH OH , -OC(=O)N(CH₃)CH2CH₂N(CH₃)C(=O)-* or

-OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and Cs-Cg cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-l inhibitor;

Xb is -CH2-, -OCH2-, -NHCH2- or -NRCH2- and each R independently is H, -CH₃ or CH₂CH2C(=O)OH;

O *

-|-o-p4~

A is a bond, -OC(=O)-*, OH

OH

O O „ / * <, Il II Th

-|-o-p-o-p^

OH OH _; -OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or

-OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH₃)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C₃-Cs cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-l inhibitor;

627

Il II T

-O-P-O-P—⁷

OH OH , -OC(=O)N(CH3)CH2CH₂N(CH3)C(=O)-* or

-OC(=O)N(CH₃)C(R^ahC^

4-0-p-r

A is a bond, -0C(=0)-*, OH

, -OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or

4-o-p-o-p—⁷

OH OH , -OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or

-OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH₃)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C₃-C₈ cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-1 inhibitor;

R is H, -CH₃ or -CH₂CH₂C(=O)OH;

624

Ο *

-|-o-p-h

A is a bond, -OC(=O)-*, OH

OH OH

OH

OH OH , -OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or

-OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C₃-Cs

(4) the hydrophilîc moiety represented by R² comprises OH

4-0-P-OH j-P-OH an oligosaccharide, a polypeptide, or Cz-Ce alkyl substituted with 1 to 3 OH or OH groups;

613

4. The antibody-drug conjugate of any one of daims 1 to 3, wherein: (1) R² is a hydrophilic moiety comprisîng polyethylene glycol, polyaikylene glycol, a polyol, a polysarcosine, a sugar,

0 0

(4) Li comprises OH OH , wherein the * of Li indicates the point of direct or indirect attachment to Lp, and the ** of Li indicates the point of direct or indirect attachment to R¹; or (5) Li is a bridging spacer comprising:

*-C(=O)(CH₂)_mO(CH2)_m-**; *-C(=O)((CH₂)_mO)_t(CH₂)_n-**; *-C(=O)(CH₂)_ni-**;

*-C(=O)NH((CH₂)_mO)_t(CH2)_n-**;

*-C(=O)O(CH₂)i_nSSC(R³)2(CH2)mC(=O)NR³(CH2)_1IlNR³C(=O)(CH₂)_m-**;

*-C(=O)O(CH2)_mC(=O)NH(CH2)_m-*^*-C(=O)(CH₂)_inNH(CH^ *-C(=O)(CH2)_mNH(CH₂)_nC(=O)-**; *-C(=O)(CH₂)_mXi(CH₂)_m-**;

*-C(=O)((CH2)mO)_t(CH₂)_nX₁(CH₂)n-**;*-C(=O)(CH₂)_mNHC(=O)(CH₂)n-**;

*-C(=O)((CH2)_mO)_t(CH₂)_nNHC(=O)(CH₂^ *-C(=O)(CH₂)_mNHC(=O)(CH₂)_nXi(Œ^ *-C(=O)((CH₂)mO)t(CH2)nNHC(=O)(CH₂)_riXi(CH₂)n-**;

*-C(=O)((CH2)mO)t(CH₂)nC(=O)^ *-C(=O)(CH₂)_mC(R³)2-** or *-C(=O)(CH₂)mC(=O)NH(CH₂)_m-**, wherein the * of Li indicates the point of direct or indirect attachment to Lp, and the ** of Li indicates the point of direct or indirect attachment to R¹;

ν' lp N^/z |] ^HOY«

Il _?/ ^N N^AqH Jl

Xi is Ά , , Ύ _or Λn and each m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

each n is independently selected from L 2, 3, 4, 5, 6, 7, 8, 9 and 10; and each t is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30;

and each R³ îs independently selected from H and Ci-C&alkyL

612
5 p is an integer from 1 to 16;

L is a linker; and

D is an MCL1 inhibitor compound.

5 diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait thereof.

5 or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait of any of the foregoing;

5 no îs an integer equal to 0 or l, po is an integer equal to 0, l, 2, or 3, qo is an integer equal to 1,2,3 or 4, ro and so are independently an integer equal to 0 or 1;

wherein, at most, one of the R03, Rœ, or R012 groups, if présent, is covalently attached to

5 cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-l inhibitor;

O O *

OH OH , -OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or

-OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH₃)C(=O)-*, wherein each R^a is independently selected from H, Ci-C& alkyl, and C₃-Cs cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-l inhibitor;

wherein:

630

A is a bond, -OC(=O)-*, OH _} OH OH , OH

OH OH , -OC(=O)N(CH₃)CH2CH2N(CHî)C(=O)-* or

-OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH₃)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C₃-Cs cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-l inhibitor; or (16)

5 cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-l inhibitor;

O O * . n n X,

-bo-P-O-P^

OH OH

-OC(=O)N(CH3)CH₂CH₂N(CH3)C(=O)-*or

5 wherein each R^a îs independently selected from H, Ci-Cô alkyi, and C₃-Cs cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-l inhibitor;

O O _z * i ^{11 11} A

-|-o-p-o-p^

OH OH , -OC(=O)N(CH3)CH₂CH2N(CH3)C(=O)-* or

-OC(=O)N(CH3)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyi, and C3-C8 cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-l inhibitor;

628

Ο *

-|-o-p-h

Α is a bond, -OC(=O)-*, OH

O O „ . Il Il / -bo-p-o-p-l·

OH OH

O Il *

OH

0 0 , Il II A

-f-o-p-o-p—' I I

OH OH , -OC(=O)N(CH3)CH₂CH₂N(CH3)C(=O)-* or

-OC(=O)N(CH3)C(R^a)2C(R^a)2N(CH3)C(=O)-*, wherein each R^a is independently selected from H, Cj-Cé alkyl, and C₃-Cs

5 -OC(=O)N(CH₃)C(R^a)2C(R^a)₂N(CH3)C(=O)>

wherein each R^a îs independently selected from H, Ci-Cô alkyl, and C₃-Cs cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-l inhibitor;

wherein:

5 cycloalkyl and the * of A indicates the point of attachment to D; and D is an Mcl-l inhibitor;

each R is independently selected from H, -CH₃, and -CH₂CH₂C(=O)OH;

A is a bond, -OC(=O)-*, OH _} OH OH , OH

O O * , Il II 7^

-i-o-p-o-p—⁷

5 each R⁷ is independently selected from H, Ci-6 alkyl, fluoro, benzyloxy substituted with

-C(=O)OH, benzyl substituted with -C(=O)OH, Cm alkoxy substituted with -C(=0)0H and Cm alkyl substituted with-C(=O)OH;

R³⁷ is independently selected from H, phenyl and pyridine;

q is 0,1, 2 or 3;

(5) the attachment group comprises a group selected from:

618

619

H

620

621

disulfide, wherein:

R³² is H, Ci-4 alkyl, phenyl, pyrimidine or pyridine;

R³⁵ is H, Ci-6 alkyl, phenyl or Cm alkyl substituted with 1 to 3 -OH groups;

5 a thiol and a maleimide, a thiol and a haloacetamide, a thiol and a vinyl sulfone, a thiol and an aziridine, an azide and an alkyne,

5. The antibody-drug conjugate of any one of daims 1 to 4, wherein:

(i) L₃ is a spacer moiety having the structure ~Y^{W x} , wherein:

W is -CH₂-, -CH₂O-, -CH₂N(R^b)C(=O)O-, -NHC(=O)C(R^b)2NHC(=O)O-, -NHC(=O)C(R^b)2NH-, -NHC(=O)C(R^b)₂NHC(=O)-, -CH₂N(X-R³)C(=O)O-, -C(=O)N(X-R³)-,

614

-CH₂N(X-R²)C(=O)-, -C(=O)NR^b-, -C(=O)NH-, -CH2N R^b C(=O)-, -CH2N R^b C(=O)NH-, -CH2NR^bC(=O)NR^b-, -NHC(=O)-, -NHC(=O)O-, -NHC(=O)NH-, -OC(=O)NH-, -S(O)2NH-, -NHS(O)₂-, -C(=O)-, -C(=O)O- or -NH-, wherein each R^b is independently selected from H, Cj-Côalkyl, and Cj-Cg cycloalkyl; and

X is a bond, triazolyl, or -CH₂-triazolyI-; or —| yy |— (ii) La is a spacer moiety having the structure ⁵ , wherein:

W is -CH₂-, -CH₂O-, -CH₂N(R^h)C(=O)O-, -NHC(=O)C(R^b)2NHC(=O)O-, -NHC(=O)C(R^b)2NH-, -NHC(=O)C(R^b)2NHC(=O)-, -CH2N(X-R²)C(=O)O-, -C(=O)N(X-R²)-, -CH2N(X-R²)C(=O)-, -C(=O)NR^b-, -C(=O)NH-, -CH2NR^bC(=O)-, -CH2NR^bC(=O)NH-, -CH2NR^bC(=O)NR^b-, -NHC(=O)-, -NHC(=O)O-, -NHC(=O)NH-, -OC(=O)NH-, -S(O)2NH-, -NHS(O)₂-, -C(=O)-, -C(=O)O- or -NH-, wherein each R^b îs independently selected from H, Ci-Csalkyl, and C3-C8 cycloalkyl; and

X is -CH₂-triazolyl-Ci-4 a!kylene-OC(O)NHS(O)₂NH-,

-C4-6 cycloaïkylene-OC(O)NHS(O)₂NH-, -(CH₂CH₂O)_n-C(O)NHS(O)₂NH-, -(CH₂CH₂O)_n-C(O)NHS(O)₂NH-(CH₂CH₂O)_n-, or

-CH₂-triazolyl-Ci-4 alkylene-OC(O)NHS(O)₂NH-(CH₂CH₂O)_n-, wherein each n independently is l,2, or 3.
(6) D comprises:

or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait of any of the foregoing;

6. The antibody-drug conjugale of any one of claims l to 5, wherein:
(7) D comprises:

or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait of any of the foregoing;

7. The antibody-drug conjugate of any one of claims l to 6, wherein:

(l) the peptide group represented by Lp comprises 1 to 4 amino acid residues, 1 to 3 amino acid 5 residues, or 1 to 2 amino acid residues, optionally wherein the amino acid residues are selected from L-glycine (Gly), L-valine (Val), L-citrulline (Cit), L-cysteic acid (sulfo-Ala), L-lysine (Lys), L-îsoleucine (Ile), L-phenylalanine (Phe), L-methionîne (Met), L-asparagine (Asn), Lproline (Pro), L-alanine (Ala), L-ieucine (Leu), L-tryptophan (Trp), and L-tyrosine (Tyr);
(8) D comprises:

644

or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait of any of the foregoing;
(9) D comprises:

or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or phannaceutically acceptable sait of any of the foregoing;

9. The antibody-drug conjugate of claim 8, wherein A is a bond and R is -CH₃.
10 NO:3; light chain CDR1 (LCDR1) consisting of SEQ ID NO: 16, light chain CDR2 (LCDR2) consisting of SEQ ID NO:70, and light chain CDR3 (LCDR3) consisting of SEQ ID NO: 18;

(ii) heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:4, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:2, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; light chain CDR1 (LCDR1) consisting of SEQ ID NO: 16, light chain CDR2 (LCDR2)

10 or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait of any of the foregoing;

10 or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or phannaceutically acceptable sait of any of the foregoing;

10 or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait of any of the foregoing;

(l l) D comprises:

645

or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait of any of the foregoing;

(10) D comprises:

10 pharmaceutically acceptable sait of any of the foregoing;

10 the linker, and wherein the valency of an atom is not exceeded by virtue of one or more substituents bonded thereto, or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait of any of the foregoing;

10. The antibody-drug conjugate of any one of daims 1 to 9, wherein:

10 -OC( =O)N(CH₃)C(R^ahC(R^ wherein each R^a is independently selected from H, Ci-Cô alkyl, and C₃-Cs cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-l inhibitor;

629

Ο *

H S

-|-ο-ρ-α

A is a bond, -OC(=O)-% OH

OH OH

0 n *

-o-p-o^v

OH

O O

L II H 7

-f-O-P-O-P—'

OH OH , -OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or

-OC(=O)N(CH₃)C(R^a)2C(R^a)₂N(CH₃)C(=O)-*, wherein each R^a is independently selected from H, Cj-C& alkyl, and C₃-Cs

10 Xa is -CH₂-, -OCH2-, -NHCH₂- or —NRCH₂- and each R independently is H, -CH₃ or

-CH₂CH₂C(=O)OH;

O * O O * O . Ils , Il II i S γ-°_ργ_ _|.Q_p_Q_pY -l-O-PA is a bond, -OC(=O)-*, OH , OH OH , OH

O O , , * , Il II

-I-O-P-O-P^ OH OH , -OC(=O)N(CH₃)CH₂CH₂N(CH₃)C(=O)-* or

-OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH₃)C(=O)-*,

626 wherein each R^a is independently selected from H, Ci-Cô alkyl, and Cj-Cs cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-l inhibitor;

R is H, -CH₃ or -CH2CH₂C(=O)OH;

O * O O * O

10 OH OH , -OC(=O)N(CH3)CH₂CH₂N(CH3)C(=O)-* or

-OC(=O)N(CH₃)C(R^a)2C(R^a)2N(CH₃)C(=O)-*, wherein each R^a is independently selected from H, Ci-Cô alkyl, and C₃-Cs cycloalkyl and the * of A indicates the point of attachment to D; and

D is an Mcl-l inhibitor;

625

each R is independently selected from H, -CH₃, and -CH₂CH₂C(=O)OH;

O * < Ils

10 and/or sulfo-Ala-Val-Ala; or (3) Lp is selected from:

10 R⁸ is H or methyl; and

622

R⁹ is H, -CH₃ or phenyl.

10 an azide and a cyclooctyne, an azide and a cyclooctene, an azide and a tri aryl phosphine, an azide and an oxanobornadiene, a diaryl tetrazine and a cyclooctene,
11. The antibody-drug conjugate of claim 10, wherein:

(i) Roi is methyl or ethyl;

(ii) R03 is -O-CH2-CH2-NR01 iRoi 1 ’ in which Rou and Roi 1’ form, together with the nitrogen atom carrying them, a piperazinyl group which may be substituted by a substituted by a group areing a hydrogen atom or a linear or branched (Ci-Cô)alkyl group);

^R027

ÇK₃ ^R028 (iii) R03 comprises the formula: ⁰ a hydrogen atom and R028 is a -(CH2)pO-S02-ORo30 group;

, wherein R027 is

ÇH₃

11 5 < Il II ς C II

-ξ-0-ΡA is a bond, -OC(=O)-*, OH , OH OH ₅ OH ⁰ O „ / * r II II vV
(12) D comprises:

or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or phannaceutically acceptable sait of any of the foregoing; or

12. The antibody-drug conjugate of any one of daims 1 to 11, wherein:
13. The antibody-drug conjugate of claim 1, wherein the -(L-D) comprises or is formed from the following compound:

648

L30-PI,

L37-P1,

649

(13) D comprises:
14. An anti-CD74 antibody or antigen binding fragment comprising (1) three heavy chain CDRs and three light chain CDRs as follows:

(i) heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:1, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:2, heavy chain CDR3 (HCDR3) consisting of SEQ ID

(14) D comprises:

646

or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait of any of the foregoing;

or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or phannaceutically acceptable sait of any of the foregoing;
15. A nucleic acid encoding the antibody of claim 14.

15 consisting of SEQ ID NO:70, and light chain CDR3 (LCDR3) consisting of SEQ IDN0:18;

650 (iii) heavy chain CDRl (HCDRl) consisting of SEQ ID NO:5, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:6, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; light chain CDRl (LCDRl) consisting of SEQ ID NO: 19, light chain CDR2 (LCDR2) consisting of SEQ ID NO:20, and light chain CDR3 (LCDR3) consisting of SEQ ID NO:21;

(iv) heavy chain CDRl (HCDRl) consisting of SEQ ID NO:7, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:S, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:9; light chain CDRl (LCDRl) consisting of SEQ ID NO:22, light chain CDR2 (LCDR2) consisting of SEQ ID NO:20, and light chain CDR3 (LCDR3) consisting of SEQ ID NO: 18;

(v) heavy chain CDRl (HCDRl) consisting of SEQ ID NO:1, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:2, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; light chain CDRl (LCDRl) consisting of SEQ ID NO:35, light chain CDR2 (LCDR2) consisting of SEQ ID NO:70, and light chain CDR3 (LCDR3) consisting of SEQ ID NO: 18;

(vi) heavy chain CDRl (HCDRl) consisting of SEQ ID NO:4, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:2, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; light chain CDRl (LCDRl) consisting of SEQ ID NO:35, light chain CDR2 (LCDR2) consisting of SEQ ID NO:70, and light chain CDR3 (LCDR3) consisting of SEQ ID NO: 18;

(vii) heavy chain CDRl(HCDRl) consisting of SEQ ID NO:4, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:2, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; light chain CDRl (LCDRl) consisting of SEQ ID NO:35, light chain CDR2 (LCDR2) consisting of SEQ ID NO:70, and light chain CDR3 (LCDR3) consisting of SEQ ID NO: 1 8.

(viiî) heavy chain CDRl (HCDRl) consisting of SEQ ID NO:5, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:6, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; light chain CDRl (LCDRl) consisting of SEQ ID NO:71, light chain CDR2 (LCDR2) consisting of SEQ ID NO:20, and light chain CDR3 (LCDR3) consisting of SEQ ID NO:21.or (ix) heavy chain CDRl (HCDRl) consisting of SEQ ID NO:7, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:8, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:9; light chain CDRl (LCDRl) consisting of SEQ ID NO: 17, light chain CDR2 (LCDR2) consisting of SEQ ID NO:20, and light chain CDR3 (LCDR3) consisting of SEQ ID NO: 18; (2) a heavy chain variable région and a light chain variable région as follows:

(i) a heavy chain variable région comprising the amino acîd sequence of SEQ ID NO: 10, and a light chain variable région comprising the amino acid sequence of SEQ ID NO:23;

(ii) a heavy chain variable région comprising the amino acid sequence of SEQ ID NO: 10, and a lîght chain variable région comprising the amino acîd sequence of SEQ ID NO:27;

651 (iii) a heavy chain variable région comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable région comprising the amino acid sequence of SEQ ID NO:31;

(iv) a heavy chain variable région comprising the amino acid sequence of SEQ ID

NO: 10, and a light chain variable région comprising the amino acid sequence of SEQ ID NO:36;

(y) a heavy chain variable région comprising the amino acid sequence of SEQ ID NO:lO, and a light chain variable région comprising the amino acid sequence of SEQ ID NO:40; or (vi) a heavy chain variable région comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable région comprising the amino acid sequence of SEQ ID NO:44; or (3) a heavy chain and light chain as follows:

(a) the heavy chain having amino acid sequence of SEQ ID NO: 12 and the light chain having amino acid sequence of SEQ ID NO:25;

(b) the heavy chain having amino acid sequence of SEQ ID NO: I4 and the light chain having amino acid sequence of SEQ ID NO:25;

(c) the heavy chain having amino acid sequence of SEQ ID NO: 15 and the light chain having amino acid sequence of SEQ ID NO:25;

(d) the heavy chain having amino acid sequence of SEQ ID NO:l2 and the light chain having amino acid sequence of SEQ ID NO:29;

(e) the heavy chain having amino acid sequence of SEQ ID NO:l4 and the light chain having amino acid sequence of SEQ ID NO:29;

(f) the heavy chain having amino acid sequence of SEQ ID NO:l5 and the light chain having amino acid sequence of SEQ ID NO:29;

(g) the heavy chain having amino acid sequence of SEQ ID NO: 12 and the light chain having amino acid sequence of SEQ ID NO:33;

(h) the heavy chain having amino acid sequence of SEQ ID NO: 14 and the light chain having amino acid sequence of SEQ ID NO:33;

(i) the heavy chain having amino acid sequence of SEQ ID NO: 15 and the light chain having amino acid sequence of SEQ ID NO:33;

(j) the heavy chain having amino acid sequence of SEQ ID NO: 12 and the light chain having amino acid sequence of SEQ ID NO:38;

(k) the heavy chain having amino acid sequence of SEQ ID NO: 14 and the light chain having amino acid sequence of SEQ ID NO:38;

652 (l) the heavy chain having amino acid sequence of SEQ ID NO:l 5 and the light chain having amino acid sequence of SEQ ID NO:38;

(rn ) the heavy chain having amino acid sequence of SEQ ID NO: 12 and the light chain having amino acid sequence of SEQ ID NO:42;

(n) the heavy chain having amino acid sequence of SEQ ID NO: 14 and the light chain having amino acid sequence of SEQ ID NO:42;

(o) the heavy chain having amino acid sequence of SEQ ID NO: 15 and the light chain having amino acid sequence of SEQ ID NO:42;

(p) the heavy chain having amino acid sequence of SEQ ID NO: 12 and the light chain having amino acid sequence of SEQ ID NO:46;

(q) the heavy chaîn having amino acid sequence of SEQ ID NO: 14 and the light chain having amino acid sequence of SEQ ID NO:46; or (r) the heavy chain having amino acid sequence of SEQ ID NO: 15 and the light chain having amino acid sequence of SEQ ID NO:46.

15 (5) D comprises:

643

or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait of any of the foregoing;

15 a monoaryl tetrazine and a nonboniene, an aldéhyde and a hydroxylamine, an aldéhyde and a hydrazine, an aldéhyde and NH₂-NH-C(=O)-, a ketone and a hydroxylamine,
16. A vector comprising the nucleic acid of claim 15.

(16) D comprises:
17. A cell line comprising the vector of claim 16 or the nucleic acid of claim 15.

(17) D comprises:

647

or an enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait of any of the foregoing; or (l 8) -(L-D) is formed from a compound selected from Table A or an enantiomer,
18. A method of making the antibody of claim 14 comprising growîng the cell line of claim 17 under suitable conditions to express the antibody, then purifying and îsolating the antibody.
19. The antibody drug conjugale of any one of claims 1 to 13, wherein the anti-CD74 antibody comprises the antibody of claim 14.
20. A composition comprising multiple copies of the antibody-drug conjugale of any one of claims 1 to 13 and 19, wherein the average p of the antibody-drug conjugales in the composition îs from about 2 to about 16, about 2 to about 8, or about 2 to about 4.

20 a ketone and a hydrazine, a ketone and NH₂-NH-C(=O)-> 0 Δ a hydroxylamine and 75 γ o. o_x /^s°³' ^Na+ _n n ^R γΎ γΥ ,γΧΧ YX an amine and 0 , θ , ^F , ^F , or CK 0 V iT M Cl , or
21. A pharmaceutical composition comprising the antibody-drug conjugale of any one of claims l to 13 and 19 or the composition of claim 20, and a pharmaceutically acceptable carrier.

653
22. An antibody-drug conjugate of any one of claims I to 13 and 19, a composition of claim 20, or a pharmaceutical composition of claim 21 for use in treating a subject having or suspected of having a cancer; wherein:
23. An antibody-drug conjugate of any one of claims 1 to 13 and 19, a composition of claim 20, or a pharmaceutical composition of claim 21 for use in reducing or înhibiting the growth of a tumor or reducing or slowing the expansion of a cancer cell population in a subject; wherein: ( 1 ) the tumor or the cancer cell population expresses CD74;

^023 . wherein and are as defmed for formula (I) wherein, at most, one of the Rot, Ro% or R012 groups, if present, is covalently attached to the linker, or an enantiomer, diastereoîsomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait of any of the foregoing; or (3) D comprises a compound of Fonnula (III):

640 wherein:

Roi is a linear or branched (Ci-Cô)alkyl group,

Ros is -0-(C]-C6)aIkyl-NRoiiRoii’,

Rq27

wherein Rou and Roi i’ independently of one another are a hydrogen atom, an optionally substituted linear or branched (Ci-C6)alkyl group, or -(Co-Cô)alkyl-Cyoi;

or the pair (Ron, Roi Γ) together with the nitrogen atom to which they are attached form an aromatic or non-aromatic ring containing 5 to 7 ring members, which optionally contains, in addition to the nitrogen atom, 1 to 3 heteroatoms selected from O, S and N, wherein the N atom may be substituted by 1 or 2 groups selected from a hydrogen atom or a linear or branched (Ci-Cft)alkyl group, and wherein R027 is a hydrogen atom and Ross is a -(CH₂)_po-0-S0₂-0' group or a -(CH2)po-S0₂-ORo30 group;

R09 is a linear or branched (C₂-Câ)alkynyl group or -Cyo₂,

Roi₂ is -Cyos, -Cyo5-(Co-Ci,)alkyl-Cyoô, or -Cyo5-(Co-C6)alkyl-Cyo9,

Cyoi, Cyo2, Cyos and Cyoô independently of one another, are a cycloalkyl group, a heterocycloalkyl group, an aryl group or a heteroaryl group, each of which is optionally substituted,

R015, Roté, and R017 are as defined for formula (1), wherein, at most, one of the R03, R09, or Roi 2 groups, if présent, is covalently attached to the linker, or the enantiomer, diastereoisomer, atropisomer, deuterated dérivative, and/or pharmaceutically acceptable sait of any of the foregoing.

641
24. The antibody-drug conjugate, composition or pharmaceutical composition for use of claim 22 or 23, wherein the additional therapeutic agent is (1) a Bcl-2 inhibitor, or (2) venetoclax, compound Al or compound A2.
25. An in vitro or ex vivo method of determining whether a subject having or suspected of having a cancer will be responsive to treatment with the antibody-drug conjugate of any one of daims 1 to 13 and 19, the composition of claim 20, or the pharmaceutical composition of claim 21, comprising providing a bîological sample from the subject; contacting the sample with the antibody-drug conjugate; and detecting binding of the antibody-drug conjugate to cancer cells in the sample, wherein:

25 a CoA or CoA analogue and a serine residue; or
26. A method of producing the antibody-drug conjugate of any one of daims 1 to 13 and 19, comprising reacting an antibody or antigen-binding fragment with a cleavable linker joined to an MCL1 inhibitor under conditions that allow conjugation.
27. An antibody-drug conjugate of Formula ( 1 ):

655

Ab-(L-D)_p (I) wherein:

Ab is an anti-CD74 antibody or an antigen-binding fragment thereof, optionally wherein the Ab is a Fc siient antibody;
28. An antibody-drug conjugate of claim 27 for use in treating a disease or disorder, wherein 10 the antibody-drug conjugate is used in combination with a Bcl-2 inhibitor in a subject in need thereof, and wherein the disease or disorder is mediated by CD74,