CN116783208A

CN116783208A - Glycoside double cleavage linker for antibody-drug conjugates

Info

Publication number: CN116783208A
Application number: CN202180087979.9A
Authority: CN
Inventors: S·丘普拉科夫; A·O·奥贡科亚
Original assignee: RP Scherer Technologies LLC
Current assignee: RP Scherer Technologies LLC
Priority date: 2020-11-20
Filing date: 2021-11-19
Publication date: 2023-09-19

Abstract

The present disclosure provides antibody-drug conjugate structures comprising a cleavable linker connecting the antibody to the drug and having a first enzymatically cleavable moiety and a second enzymatically cleavable moiety, the second enzymatically cleavable moiety comprising a glycoside selected from the group consisting of galactosides, glucosides, mannosides, fucosides, O-GlcNAc, and O-GalNAc. The disclosure also encompasses compounds and methods for producing such conjugates, as well as methods of using the conjugates.

Description

Glycoside double cleavage linker for antibody-drug conjugates

Cross Reference to Related Applications

The present application claims the benefit of U.S. provisional application No. 63/116,632, filed 11/20 in 2020, and U.S. provisional application No. 63/139,685, filed 1/20 2021, the disclosures of which are incorporated herein by reference.

Background

The field of protein-small molecule therapeutic conjugates has made great progress, providing a number of clinically beneficial agents, hopefully more in the next few years. Protein conjugate therapeutics may provide several advantages due to, for example, specificity, functional diversity, and relatively low off-target activity, and thus fewer side effects. Chemical modification of proteins can make proteins more efficient, stable or multimodal to extend these advantages.

Disclosure of Invention

The present disclosure provides antibody-drug conjugate structures comprising a cleavable linker connecting an antibody to a drug and having a first enzymatically cleavable moiety and a second enzymatically cleavable moiety, the second enzymatically cleavable moiety comprising a glycoside selected from the group consisting of galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc. The disclosure also encompasses compounds and methods for producing such conjugates, as well as methods of using the conjugates.

Aspects of the disclosure include a conjugate comprising an antibody, a drug, and a cleavable linker connecting the antibody to the drug and having a first enzymatically cleavable moiety and a second enzymatically cleavable moiety, the second enzymatically cleavable moiety comprising a glycoside selected from the group consisting of galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

In some embodiments, the conjugate has formula (I):

wherein the method comprises the steps of

Z is CR ⁴ Or N;

x is O or NR ⁴ ；

R ¹ Selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl,Substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl;

R ² And R is ³ Each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or R ² And R is ³ Optionally linked in an cyclic manner to form a 5-or 6-membered heterocyclyl;

each R ⁴ Independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

each R ⁵ Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl;

Each R ⁶ Independently selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

k is an integer from 1 to 10;

R ⁷ comprising a second enzymatically cleavable moiety;

L ¹ is a first joint;

L ² is a second joint;

W ¹ is a drug; and is also provided with

W ² Is the antibody.

In some embodiments, k is 2; and the conjugate has the formula (Ia):

in some embodiments, the second enzymatically cleavable moiety comprises a galactoside. In some embodiments, the second enzymatically cleavable moiety comprises a glucoside. In some embodiments, the second enzymatically cleavable moiety comprises a mannoside. In some embodiments, the second enzymatically cleavable moiety comprises a fucoside. In some embodiments, the second enzymatically cleavable moiety comprises O-GlcNAc. In some embodiments, the second enzymatically cleavable moiety comprises O-GalNAc.

In some embodiments, the conjugate has formula (Ib):

in some embodiments, the conjugate has formula (Ic):

in some embodiments, the conjugate has formula (Id):

in some embodiments, the conjugate has formula (Ie):

In some embodiments, the conjugate has formula (If):

in some embodiments, the conjugate has the formula (Ig):

in some embodiments, L1 comprises:

-(T ¹ -V ¹ ) _a -(T ² -V ² ) _b -(T ³ -V ³ ) _c -(T ⁴ -V ⁴ ) _d -，

wherein the method comprises the steps of

a. b, c and d are each independently 0 or 1;

T ¹ 、T ² 、T ³ and T ⁴ Each independently selected from the group consisting of covalent bonds, (C) ₁ -C ₁₂ ) Alkyl, substituted (C) ₁ -C ₁₂ ) Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) _w 、(PEG) _n 、(AA) _p 、(CR ¹³ OH) _m -, 4-amino-piperidine (4 AP), acetal groups, hydrazines, disulfides and esters, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue or amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;

V ¹ 、V ² 、V ³ and V ⁴ Each independently selected from the group consisting of: covalent bond, -CO-, -NR ¹⁵ -、-NR ¹⁵ (CH ₂ ) _q -、-NR ¹⁵ (C ₆ H ₄ )-、-CONR ¹⁵ -、-NR ¹⁵ CO-、-C(O)O-、-OC(O)-、-O-、-S-、-S(O)-、-SO ₂ -、-SO ₂ NR ¹⁵ -、-NR ¹⁵ SO ₂ -and-P (O) OH-, whereinEach q is an integer from 1 to 6;

each R ¹³ Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl; and is also provided with

Each R ¹⁵ Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

In some embodiments, L ² Comprising:

-(T ⁵ -V ⁵ ) _e -(T ⁶ -V ⁶ ) _f -(T ⁷ -V ⁷ ) _g -(T ⁸ -V ⁸ ) _h -，

wherein the method comprises the steps of

e. f, g and h are each independently 0 or 1;

T ⁵ 、T ⁶ 、T ⁷ and T ⁸ Each independently selected from the group consisting of covalent bonds, (C) ₁ -C ₁₂ ) Alkyl, substituted (C) ₁ -C ₁₂ ) Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) _w 、(PEG) _n 、(AA) _p 、(CR ¹³ OH) _m -, 4-amino-piperidine (4 AP), acetal groups, hydrazines, disulfides and esters, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue or amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;

V ⁵ 、V ⁶ 、V ⁷ and V ⁸ Each independently selected from the group consisting of: covalent bond, -CO-, -NR ¹⁵ -、-NR ¹⁵ (CH ₂ ) _q -、-NR ¹⁵ (C ₆ H ₄ )-、-CONR ¹⁵ -、-NR ¹⁵ CO-、-C(O)O-、-OC(O)-、-O-、-S-、-S(O)-、-SO ₂ -、-SO ₂ NR ¹⁵ -、-NR ¹⁵ SO ₂ -and-P (O) OH-, wherein each q is an integer from 1 to 6;

In some embodiments, L ¹ As described herein, wherein:

T ¹ selected from (C) ₁ -C ₁₂ ) Alkyl and substituted (C) ₁ -C ₁₂ ) An alkyl group;

T ² 、T ³ and T ⁴ Each independently selected from aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) _w 、(PEG) _n 、(C ₁ -C ₁₂ ) Alkyl, substituted (C) ₁ -C ₁₂ ) Alkyl, (AA) _p 、-(CR ¹³ OH) _m -, 4-amino-piperidine (4 AP), acetal groups, hydrazines, and esters; and is also provided with

V ¹ 、V ² 、V ³ And V ⁴ Each independently selected from the group consisting of: covalent bond, -CO-, -NR ¹⁵ -、-NR ¹⁵ (CH ₂ ) _q -、-NR ¹⁵ (C ₆ H ₄ )-、-CONR ¹⁵ -、-NR ¹⁵ CO-、-C(O)O-、-OC(O)-、-O-、-S-、-S(O)-、-SO ₂ -、-SO ₂ NR ¹⁵ -、-NR ¹⁵ SO ₂ -and-P (O) OH-;

wherein:

(PEG) _n is thatWherein n is an integer from 1 to 30;

EDA is an ethylenediamine moiety having the structure:wherein y is an integer from 1 to 6 and r is 0 or 1;

4-amino-piperidine (4 AP) as

Each R ¹² And R is ¹⁵ Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, polyethylene glycol moieties, aryl, and substituted aryl, wherein any two adjacent R ¹² The groups may be connected in a cyclic manner to form a piperazinyl ring; and is also provided with

R ¹³ Selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl.

In some embodiments, L ¹ As described herein, wherein:

T ¹ is (C) ₁ -C ₁₂ ) Alkyl, and V ¹ is-CO-;

T ² is an amino acid analog, and V ² is-NH-;

T ³ is (PEG) _n And V is ³ is-CO-; and is also provided with

d is 0.

In some embodiments, L ² As described herein, wherein:

T ⁵ is a covalent bond, and V ⁵ is-CO-; and is also provided with

f. g and h are 0.

In some embodiments, L ² As described herein, wherein:

T ⁵ is a covalent bond, and V ⁵ is-CONR ¹⁵ -；

T ⁶ Is (C) ₁ -C ₁₂ ) Alkyl, and V ⁶ is-CO-; and is also provided with

g and h are 0.

In some embodiments, the drug is selected from the group consisting of cytotoxins, kinase inhibitors, immunostimulants, toll-like receptor (TLR) agonists, oligonucleotides, aptamers, cytokines, steroids, and peptides. In some embodiments, the drug is selected from the group consisting of auristatin (auristatin), maytansine (maytansine), and duocarmycin (duocarmycin). In some embodiments, the drug is selected from the group consisting of tepirtine M (Tubulysin M), calicheamicin (Calicheamicin), SN-38, irinotecan (Exatecan), STAT3 inhibitor, alpha-amanitine, aurora kinase inhibitor, belotecan (belotecan), 9-aminocamptothecin (9-AC), and anthracycline (anthracycline).

Aspects of the disclosure include a compound comprising a cleavable linker for linking an antibody to a drug, wherein the cleavable linker comprises a first enzymatically cleavable moiety and a second enzymatically cleavable moiety, the second enzymatically cleavable moiety having a glycoside selected from the group consisting of galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

In some embodiments, the compound has formula (II):

wherein the method comprises the steps of

Z is CR ⁴ Or N;

x is O or NR ⁴ ；

each R ⁴ Independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, acylaminoA group, an aminoacyl group, an alkylamide, a substituted alkylamide, a sulfonyl group, a thioalkoxy group, a substituted thioalkoxy group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, a cycloalkyl group, a substituted cycloalkyl group, a heterocyclic group, and a substituted heterocyclic group;

Each R ⁵ Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

k is an integer from 1 to 10;

R ⁷ comprising a second enzymatically cleavable moiety;

L ¹ is a first joint;

L ² is a second joint; and is also provided with

W ¹ Is a medicine.

In some embodiments, k is 2; and the compound has the formula (IIa):

in some embodiments, the second cleavable moiety comprises a galactoside. In some embodiments, the second cleavable moiety comprises a glucoside. In some embodiments, the second cleavable moiety comprises a mannoside. In some embodiments, the second enzymatically cleavable moiety comprises a fucoside. In some embodiments, the second enzymatically cleavable moiety comprises O-GlcNAc. In some embodiments, the second enzymatically cleavable moiety comprises O-GalNAc.

In some embodiments, the compound has formula (IIb):

in some embodiments, the compound has formula (IIc):

in some embodiments, the compound has formula (IId):

in some embodiments, the compound has formula (IIe):

in some embodiments, the compound has formula (IIf):

in some embodiments, the compound has formula (IIg):

in some embodiments, L ¹ Comprising:

-(T ¹ -V ¹ ) _a -(T ² -V ² ) _b -(T ³ -V ³ ) _c -(T ⁴ -V ⁴ ) _d -，

wherein the method comprises the steps of

a. b, c and d are each independently 0 or 1;

V ¹ 、V ² 、V ³ and V ⁴ Each independently selected from the group consisting of: covalent bond, -CO-, -NR ¹⁵ -、-NR ¹⁵ (CH ₂ ) _q -、-NR ¹⁵ (C ₆ H ₄ )-、-CONR ¹⁵ -、-NR ¹⁵ CO-、-C(O)O-、-OC(O)-、-O-、-S-、-S(O)-、-SO ₂ -、-SO ₂ NR ¹⁵ -、-NR ¹⁵ SO ₂ -and-P (O) OH-, wherein each q is an integer from 1 to 6;

In some embodiments, L ² Comprising:

wherein the method comprises the steps of

e. f, g and h are each independently 0 or 1;

T ⁵ 、T ⁶ 、T ⁷ and T ⁸ Each independently selected fromCovalent bond (C) ₁ -C ₁₂ ) Alkyl, substituted (C) ₁ -C ₁₂ ) Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) _w 、(PEG) _n 、(AA) _p 、(CR ¹³ OH) _m -, 4-amino-piperidine (4 AP), acetal groups, hydrazines, disulfides and esters, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue or amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;

In some embodiments, L ¹ As described herein, wherein:

wherein:

(PEG) _n is thatWherein n is an integer from 1 to 30;

4-amino-piperidine (4 AP) as

In some embodiments, L ¹ As described herein, wherein:

T ¹ is (C) ₁ -C ₁₂ ) Alkyl, and V ¹ is-CO-;

T ² is amino acid-likeObject, and V ² is-NH-;

T ³ is (PEG) _n And V is ³ is-CO-; and is also provided with

d is 0.

In some embodiments, L ² As described herein, wherein:

T ⁵ is a covalent bond, and V ⁵ is-CO-; and is also provided with

f. g and h are 0.

In some embodiments, L ² As described herein, wherein:

T ⁵ is a covalent bond, and V ⁵ is-CONR ¹⁵ -；

T ⁶ Is (C) ₁ -C ₁₂ ) Alkyl, and V ⁶ is-CO-; and is also provided with

g and h are 0.

In some embodiments, the drug is selected from the group consisting of cytotoxins, kinase inhibitors, immunostimulants, toll-like receptor (TLR) agonists, oligonucleotides, aptamers, cytokines, steroids, and peptides. In some embodiments, the drug is selected from the group consisting of auristatin, maytansine, and a sesquialter. In some embodiments, the drug is selected from the group consisting of topiramate M, calicheamicin, SN-38, irinotecan, STAT3 inhibitor, alpha-amanitine, aurora kinase inhibitor, belotecan, 9-aminocamptothecin (9-AC), and anthracycline.

Aspects of the disclosure include a pharmaceutical composition comprising a conjugate as described herein and a pharmaceutically acceptable excipient.

Aspects of the disclosure include a method for administering a conjugate to a subject, wherein the method comprises administering the conjugate as described herein to the subject.

Aspects of the disclosure include a method for treating cancer in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a conjugate as described herein, wherein the administration is effective to treat the cancer in the subject.

Drawings

Figure 1 shows cytotoxins commonly used to produce therapeutic antibody-drug conjugates. The arrow indicates the base labile group.

FIG. 2 shows HIPS ligation for the synthesis of antibody-drug conjugates (ADCs). Antibodies carrying aldehyde moieties are reacted with Hydrazino-iso-pick-Spengler (HIPS) linkers and payloads to generate site-specific conjugated ADCs with stable aza carboline bonds.

Fig. 3 shows Hydrophobic Interaction Column (HIC) traces of construct 30 trastuzumab conjugate, which resulted in a DAR of 1.7 as determined by HIC.

Figure 4 shows an analytical Size Exclusion Chromatography (SEC) trace of construct 30 trastuzumab conjugate, which indicates that the conjugate is 97.1% monomeric as determined by analytical SEC.

Fig. 5 shows the HIC trace of construct 33 trastuzumab conjugate, which resulted in a DAR of 1.66 as determined by HIC.

Fig. 6 shows an analytical SEC trace of construct 33 trastuzumab conjugate, which indicates that the conjugate is 97.6% monomeric as determined by analytical SEC.

Fig. 7 shows HIC trace of construct 30 polotouzumab (polatuzumab) conjugate, which resulted in DAR of 1.78 as determined by HIC.

Fig. 8 shows an analytical SEC trace of the construct 30 polotouzumab conjugate, which indicates that the conjugate is 95.4% monomeric as determined by analytical SEC.

Fig. 9 shows HIC trace of construct 33 poisotouzumab conjugate, which resulted in DAR of 1.6 as determined by HIC.

Fig. 10 shows an analytical SEC trace of the construct 33 polotouzumab conjugate, which indicates that the conjugate is 96.8% monomeric as determined by analytical SEC.

FIG. 11 shows the structure of the comparison molecules 35 (single cleavage maytansine construct) and 34 (glucuronide double cleavage MMAE construct).

FIG. 12 shows a graph of in vitro potency of maytansine conjugated ADCs carrying either single cleavage (35) or a galactose-modified double cleavage linker (30) against Granta-519 cells.

Figure 13 shows a graph of in vitro potency of maytansinoid conjugated ADCs carrying single-split (35) or galactose-modified double-split linker (30) against Ramos-RA cells.

Figure 14 shows a graph of in vitro potency of maytansinoid-conjugated ADCs carrying single-split (35) or galactose-modified double-split linkers (30) against NCI-N87 cells.

Figure 15 shows a graph of the in vitro potency of maytansinoid-conjugated ADCs carrying either single-split (35) or galactose-modified double-split linker (30) against Sk-Br-3 cells.

Figure 16 shows a graph of in vitro potency of MMAE conjugated ADCs carrying glucuronide modified double cleavage linker (34) or galactoside modified double cleavage linker (33) against Ramos-RA cells.

Figure 17 shows a graph of in vitro potency of MMAE conjugated ADCs carrying glucuronide modified double cleavage linker (34) or galactoside modified double cleavage linker (33) against NCI-N87 cells.

Figure 18 shows a graph of in vitro potency of MMAE conjugated ADCs carrying glucuronide modified double cleavage linker (34) or galactoside modified double cleavage linker (33) against Sk-Br-3 cells.

Fig. 19 shows PLRP traces of construct 46 against FITC conjugate, which resulted in DAR of 1.70 as determined by PLRP.

Figure 20 shows an analytical SEC trace of construct 46 against FITC conjugate, which was 95.9% monomeric as determined by analytical SEC.

Fig. 21 shows PLRP traces of construct 46 trastuzumab conjugate, which resulted in DAR of 1.79 as determined by PLRP.

Figure 22 shows the analytical SEC trace of the construct 46 trastuzumab conjugate, which was 96.2% monomeric as determined by analytical SEC.

Fig. 23 shows PLRP traces of construct 46 Sha Xituo bead mab (sacituzumab) conjugate, which resulted in DAR of 1.15 as determined by PLRP.

Figure 24 shows the analytical SEC trace of construct 46 Sha Xituo bead mab conjugate, which was 94.4% monomeric as determined by analytical SEC.

Fig. 25 shows PLRP traces of construct 44 against FITC conjugate, which resulted in DAR of 1.68 as determined by PLRP.

Figure 26 shows an analytical SEC trace of construct 44 against FITC conjugate, which was 96.0% monomeric as determined by analytical SEC.

Fig. 27 shows PLRP traces of construct 44 trastuzumab conjugate, which resulted in DAR of 1.78 as determined by PLRP.

Fig. 28 shows an analytical SEC trace of the construct 44 trastuzumab conjugate, which was 96.0% monomeric as determined by analytical SEC.

Fig. 29 shows PLRP traces of construct 44 Sha Xituo bead mab (sacituzumab) conjugates, which produced a DAR of 1.16 as determined by PLRP.

Figure 30 shows an analytical SEC trace of construct 44 Sha Xituo bead mab conjugate, which was 94.5% monomeric as determined by analytical SEC.

Figure 31 shows a graph of in vitro potency of maytansinoid-conjugated ADCs carrying either a glucoside modification (44) or a galactose-modified double cleavage linker (30) against MDA-MB-468 cells.

Figure 32 shows a graph of in vitro potency of maytansine conjugated ADCs carrying either glucoside modification (44) or galactose glycoside modification double cleavage linker (30) against BxPC3 cells.

Figure 33 shows a graph of in vitro potency of maytansinoid-conjugated ADCs carrying either a glucoside modification (44) or a galactose-modified double cleavage linker (30) against SKBR3 cells.

Figure 34 shows a graph of in vitro potency of maytansinoid-conjugated ADCs carrying either a glucoside modification (44) or a galactose-modified double-cleavage linker (30) against NCI-N87 cells.

FIG. 35 shows a graph of in vitro potency of MMAE-conjugated ADCs carrying glucuronide modification (34) or a glucoside modified double cleavage linker (46) against MDA-MB-468 cells.

Figure 36 shows a graph of in vitro potency of MMAE conjugated ADCs carrying glucuronide modified (34) or glucoside modified double cleavage linker (46) against BxPC3 cells.

Definition of the definition

Unless otherwise indicated, the following terms have the following meanings. Any undefined term has its technically accepted meaning.

"alkyl" refers to a monovalent saturated aliphatic hydrocarbyl group having 1 to 10 carbon atoms, for example 1 to 6 carbon atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms. The term includes, for example, straight and branched chain hydrocarbyl groups, such as methyl (CH) ₃ (-), ethyl (CH) ₃ CH ₂ (-), n-propyl (CH) ₃ CH ₂ CH ₂ (-), isopropyl ((CH) ₃ ) ₂ CH-), n-butyl (CH) ₃ CH ₂ CH ₂ CH ₂ (-), isobutyl ((CH) ₃ ) ₂ CHCH ₂ (-), sec-butyl ((CH) ₃ )(CH ₃ CH ₂ ) CH-), tert-butyl group ((CH) ₃ ) ₃ C-), n-pentyl (CH) ₃ CH ₂ CH ₂ CH ₂ CH ₂ (-) and neopentyl ((CH) ₃ ) ₃ CCH ₂ -)。

The term "substituted alkyl" refers to an alkyl group as defined herein wherein one or more carbon atoms (C ₁ Excluding carbon atoms) have optionally been interrupted by heteroatoms such as-O-, -N-, -S (O) _n - (wherein n is 0 to 2), -NR- (wherein R is hydrogen or alkyl) and has 1 to 5 substituents selected from the group consisting of: alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amido, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -aryl, -SO ₂ -heteroaryl and-NR ^a R ^b Wherein R 'and R' may be the same or different and are selected from hydrogen,Optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, and heterocyclyl.

"alkylene" means a divalent aliphatic hydrocarbon group preferably having 1 to 6, and more preferably 1 to 3 carbon atoms, which is straight or branched and which is optionally interrupted by one or more groups selected from-O-, -NR ¹⁰ -、-NR ¹⁰ C(O)-、-C(O)NR ¹⁰ -groups of the like. The term includes, for example, methylene (-CH) ₂ (-), ethylene (-CH) ₂ CH ₂ (-), n-propylene (-CH) ₂ CH ₂ CH ₂ (-), isopropylidene (-CH) ₂ CH(CH ₃ )-)、(-C(CH ₃ ) ₂ CH ₂ CH ₂ -)、(-C(CH ₃ ) ₂ CH ₂ C(O)-)、(-C(CH ₃ ) ₂ CH ₂ C(O)NH-)、(-CH(CH ₃ )CH ₂ (-), etc.

"substituted alkylene" refers to an alkylene group having 1 to 3 hydrogens replaced with a substituent group, as described below for carbon in the definition of "substituted".

The term "alkane" refers to an alkyl group and an alkylene group as defined herein.

The terms "alkylaminoalkyl", "alkylaminoalkenyl" and "alkylaminoalkynyl" refer to the groups R 'NHR "-, where R' is an alkyl group as defined herein, and R" is an alkylene, alkenylene or alkynylene group as defined herein.

The term "alkylaryl" or "aralkyl" refers to the groups-alkylene-aryl and-substituted alkylene-aryl, wherein alkylene, substituted alkylene, and aryl are as defined herein.

"alkoxy" refers to the group-O-alkyl, wherein alkyl is as defined herein. Alkoxy groups include, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, and the like. The term "alkoxy" also refers to the groups alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, wherein alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

The term "substituted alkoxy" refers to the group substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O-, wherein substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

The term "alkoxyamino" refers to the group-NH-alkoxy, wherein alkoxy is as defined herein.

The term "haloalkoxy" refers to the group alkyl-O-, wherein one or more hydrogen atoms on the alkyl group have been replaced with a halogen group, and includes groups such as trifluoromethoxy.

The term "haloalkyl" refers to a substituted alkyl group as described above wherein one or more hydrogen atoms on the alkyl group have been replaced with a halogen group. Examples of such groups include, but are not limited to, fluoroalkyl groups such as trifluoromethyl, difluoromethyl, trifluoroethyl, and the like.

The term "alkylalkoxy" refers to the groups-alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl and substituted alkylene-O-substituted alkyl, wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.

The term "alkylthio-alkoxy" refers to the groups-alkylene-S-alkyl, alkylene-S-substituted alkyl, substituted alkylene-S-alkyl and substituted alkylene-S-substituted alkyl, wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.

"alkenyl" refers to a straight or branched chain hydrocarbyl group having 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and having at least 1 and preferably 1 to 2 sites of double bond unsaturation. The term includes, for example, divinyl, allyl and but-3-en-1-yl. The term includes both cis and trans isomers or mixtures of these isomers.

The term "substituted alkenyl" refers to alkenyl groups as defined herein having 1 to 5 substituents or 1 to 3 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amido, acyloxy Amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl and-SO ₂ -heteroaryl.

"alkynyl" refers to a straight or branched monovalent hydrocarbon group having 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms and having at least 1 and preferably 1 to 2 sites of triple bond unsaturation. Examples of such alkynyl groups include ethynyl (-C≡CH) and propargyl (-CH) ₂ C≡CH)。

The term "substituted alkynyl" refers to an alkynyl group as defined herein having 1 to 5 substituents or 1 to 3 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amido, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl and-SO ₂ -heteroaryl.

"alkynyloxy" refers to the group-O-alkynyl, wherein alkynyl is as defined herein. Alkynyloxy includes, for example, ethynyloxy, propynyloxy, and the like.

"acyl" refers to the group H-C (O) -, alkyl-C (O) -, substituted alkyl-C (O) -, and alkenyl-C (O) -, substituted alkenyl-C (O) -, alkynyl-C (O)-, substituted alkynyl-C (O) -, cycloalkyl-C (O) -, substituted cycloalkyl-C (O) -, cycloalkenyl-C (O) -, substituted cycloalkenyl-C (O) -, aryl-C (O) -, substituted aryl-C (O) -, heteroaryl-C (O) -, substituted heteroaryl-C (O) -, heterocyclyl-C (O) -, and substituted heterocyclyl-C (O) -, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein. For example, the acyl group includes an "acetyl" group CH ₃ C(O)-。

"amido" means a group-NR ²⁰ C (O) alkyl, -NR ²⁰ C (O) -substituted alkyl, -NR ²⁰ C (O) cycloalkyl, -NR ²⁰ C (O) -substituted cycloalkyl, -NR ²⁰ C (O) cycloalkenyl, -NR ²⁰ C (O) -substituted cycloalkenyl, -NR ²⁰ C (O) alkenyl, -NR ²⁰ C (O) -substituted alkenyl, -NR ²⁰ C (O) alkynyl, -NR ²⁰ C (O) -substituted alkynyl, -NR ²⁰ C (O) aryl, -NR ²⁰ C (O) -substituted aryl, -NR ²⁰ C (O) heteroaryl, -NR ²⁰ C (O) -substituted heteroaryl, -NR ²⁰ C (O) heterocycle and-NR ²⁰ C (O) substituted heterocycles, wherein R ²⁰ Is hydrogen or alkyl, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein.

"aminocarbonyl" or the term "aminoacyl" refers to the group-C (O) NR ²¹ R ²² Wherein R is ²¹ And R is ²² Independently selected from the group consisting of: hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclyl and substituted heterocyclyl, and wherein R is as defined in the claims ²¹ And R is ²² Optionally together with the nitrogen to which it is bonded to form a heterocyclic ringA group or a substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

"aminocarbonylamino" refers to the group-NR ²¹ C(O)NR ²² R ²³ Wherein R is ²¹ 、R ²² And R is ²³ Independently selected from hydrogen, alkyl, aryl, or cycloalkyl, or wherein two R groups are joined to form a heterocyclyl group.

The term "alkoxycarbonylamino" refers to the group-NRC (O) OR, wherein each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, OR heterocyclyl, wherein alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

The term "acyloxy" refers to the groups alkyl-C (O) O-, substituted alkyl-C (O) O-, cycloalkyl-C (O) O-, substituted cycloalkyl-C (O) O-, aryl-C (O) O-, heteroaryl-C (O) O-, and heterocyclyl-C (O) O-, wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

"sulfamoyl" refers to the group-SO ₂ NR ²¹ R ²² Wherein R is ²¹ And R is ²² Independently selected from the group consisting of: hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl, and wherein R is as defined in claim 1 ²¹ And R is ²² Optionally together with the nitrogen to which it is bonded, form a heterocyclic group or a substituted heterocyclic group, and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

"Sulfonylamino" refers to the group-NR ²¹ SO ₂ R ²² Wherein R is ²¹ And R is ²² Independently selected from the group consisting of: hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclyl and substituted heterocyclyl, and wherein R is as defined in the claims ²¹ And R is ²² Optionally together with the atoms to which they are bonded form a heterocyclic group or a substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic are as defined herein.

"Aryl" or "Aryl (Ar)" refers to a monovalent aromatic carbocyclic group of 6 to 18 carbon atoms having a single ring (e.g., present in phenyl) or having multiple condensed rings (examples of such aromatic ring systems include naphthyl, anthracenyl, and indanyl groups), which may or may not be aromatic, provided that the point of attachment is through an atom of the aromatic ring. The term includes, for example, phenyl and naphthyl. Unless otherwise limited by the definition of aryl substituents, such aryl groups may be optionally substituted with 1 to 5 substituents or 1 to 3 substituents selected from the group consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkylaryl, aryl, aryloxy, azido, carboxy, carboxyalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyamido, thioalkoxy, substituted thioalkoxy, thioaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl, -SO ₂ -impuritiesAryl and trihalomethyl.

"aryloxy" refers to a group-O-aryl, wherein aryl is as defined herein, including, for example, phenoxy, naphthoxy, and the like, including optionally substituted aryl groups also as defined herein.

"amino" means a radical-NH ₂ 。

The term "substituted amino" refers to the group-NRR, wherein each R is independently selected from the group consisting of: hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl, provided that at least one R is not hydrogen.

The term "azido" refers to the group-N ₃ 。

"carboxyl", "carboxyl" or "carboxylate" refer to-CO ₂ H or a salt thereof.

"carboxyester" or the term "carboxyalkyl" or "carboxyalkyl" refers to the group-C (O) O-alkyl-C (O) O-substituted alkyl, -C (O) O-alkenyl, -C (O) O-substituted alkenyl, -C (O) O-alkynyl, -C (O) O-substituted alkynyl-C (O) O-aryl, -C (O) O-substituted aryl, -C (O) O-cycloalkyl, -C (O) O-substituted cycloalkyl, -C (O) O-cycloalkenyl, -C (O) O-substituted cycloalkenyl, -C (O) O-heteroaryl, -C (O) O-substituted heteroaryl, -C (O) O-heterocyclyl and-C (O) O-substituted heterocyclyl, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein.

"(carboxylester) oxy" or "carbonate" refers to the group-O-C (O) O-alkyl, -O-C (O) O-substituted alkyl, -O-C (O) O-alkenyl-O-C (O) O-substituted alkenyl, -O-C (O) O-alkynyl, -O-C (O) O-substituted alkynyl, -O-C (O) O-aryl, -O-C (O) O-substituted aryl-O-C (O) O-cycloalkyl, -O-C (O) O-substituted cycloalkyl, -O-C (O) O-cycloalkenyl, -O-C (O) O-substituted cycloalkenyl, -O-C (O) O-heteroaryl, -O-C (O) O-substituted heteroaryl, -O-C (O) O-heterocyclyl, -O-C (O) O-substituted heterocyclyl, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, and substituted heterocyclyl are as defined herein.

"cyano" or "nitrile" refers to the group-CN.

"cycloalkyl" refers to cycloalkyl groups of 3 to 10 carbon atoms having a single or multiple rings (including fused, bridged and spiro ring systems). Examples of suitable cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like. Such cycloalkyl groups include, for example, monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or polycyclic structures such as adamantyl and the like.

The term "substituted cycloalkyl" refers to a cycloalkyl group having 1 to 5 substituents or 1 to 3 substituents selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amido, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl and-SO ₂ -heteroaryl.

"cycloalkenyl" refers to a non-aromatic cycloalkyl group of 3 to 10 carbon atoms having a single ring or multiple rings and having at least one double bond, and preferably 1 to 2 double bonds.

The term "substituted cycloalkenyl" refers to compounds having 1 to 5 substituents or 1 to 3Cycloalkenyl groups of substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amido, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, keto, thioketo, carboxyl, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl and-SO ₂ -heteroaryl.

"cycloalkynyl" refers to a non-aromatic cycloalkyl group of 5 to 10 carbon atoms having a single ring or multiple rings and at least one triple bond.

"Cycloalkoxy" refers to-O-cycloalkyl.

"cycloalkenyloxy" refers to an-O-cycloalkenyl group.

"halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

"hydroxy" or "hydroxy" refers to the group-OH.

"heteroaryl" refers to an aromatic group having 1 to 15 carbon atoms, such as 1 to 10 carbon atoms, and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur, within the ring. Such heteroaryl groups may have a single ring (e.g., pyridinyl, imidazolyl, or furanyl) or multiple condensed rings in the ring system (e.g., in groups such as indolizinyl, quinolinyl, benzofuran, benzimidazolyl, or benzothienyl), wherein at least one ring in the ring system is aromatic. To meet valence requirements, any heteroatom in such heteroaryl rings may or may not be bonded to H or a substituent group (e.g., an alkyl group or other substituent as described herein). In certain embodiments, the nitrogen and/or sulfur ring atoms of the heteroaryl group are optionally oxidized to provide an N-oxide (n→o), sulfinyl, or sulfonyl moiety. The term includes, for example, pyridyl, pyrrolyl, and indolyl Indolyl, thienyl and furyl. Unless otherwise limited by the definition of heteroaryl substituent, such heteroaryl groups may be optionally substituted with 1 to 5 substituents or 1 to 3 substituents selected from the group consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkylaryl, aryl, aryloxy, azido, carboxy, carboxyalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyamido, thioalkoxy, substituted thioalkoxy, thioaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl, -SO ₂ Heteroaryl and trihalomethyl.

The term "heteroarylalkyl" refers to the group-alkylene-heteroaryl, wherein alkylene and heteroaryl are as defined herein. The term includes, for example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

"heteroaryloxy" refers to an-O-heteroaryl group.

"heterocycle", "heterocyclyl", "heterocycloalkyl" and "heterocyclyl" refer to saturated or unsaturated groups having a single ring or multiple condensed rings (including fused bridged and spiro ring systems) and having from 3 to 20 ring atoms (including from 1 to 10 heteroatoms). These ring atoms are selected from nitrogen, sulfur or oxygen, wherein in the fused ring system one or more of the rings may be cycloalkyl, aryl or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In certain embodiments, the nitrogen and/or sulfur atoms of the heterocyclic group are optionally oxidized to provide an N-oxide, -S (O) -or-SO ₂ -a portion. To meet valence requirements, any heteroatom in such a heterocycle may or may not be bonded to one or more H or one or more substituent groups, such as alkyl groups or other substituents as described herein.

Examples of heterocycles and heteroaryls include, but are not limited to, azetidines, pyrroles, imidazoles, pyrazoles, pyridines, pyrazines, pyrimidines, pyridazines, indolines, indoles, indolines, indazoles, purines, quinolizines, isoquinolines, quinolines, phthalazines, naphthyridines, quinoxalines, quinazolines, cinnolines, pteridines, carbazoles, carbolines, phenanthridines, acridines, phenanthrolines, isothiazoles, phenazines, isoxazoles, phenoxazines, phenothiazines, imidazolidines, imidazolines, piperidines, piperazines, indolines, phthalimides, 1,2,3, 4-tetrahydroisoquinolines, 4,5,6, 7-tetrahydrobenzo [ b ] thiophenes, thiazoles, thiazolidines, thiophenes, benzo [ b ] thiophenes, morpholinyl, thiomorpholinyl (also known as thiomorpholinyl), 1-dioxothiomorpholinyl, piperidinyl, pyrrolidinyl, tetrahydrofuranyl, and the like.

Unless otherwise limited by the definition of heterocyclic substituents, such heterocyclic groups may be optionally substituted with 1 to 5 or 1 to 3 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amido, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl, -SO ₂ Heteroaryl and fused heterocycles.

"Heterocyclyloxy" refers to the group-O-heterocyclyl.

The term "heterocyclylthio" refers to the group heterocyclyl-S-.

The term "heterocyclylene" refers to a diradical formed from a heterocycle as defined herein.

The term "hydroxyamino" refers to the group-NHOH.

"nitro" isFinger group-NO ₂ 。

"oxo" refers to an atom (=o).

"Sulfonyl" refers to the group-SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -alkenyl, -SO ₂ -substituted alkenyl, -SO ₂ -cycloalkyl, -SO ₂ -substituted cycloalkyl, -SO ₂ -cycloalkenyl, -SO ₂ -substituted cycloalkenyl, -SO ₂ -aryl, -SO ₂ -substituted aryl, -SO ₂ -heteroaryl, -SO ₂ -substituted heteroaryl, -SO ₂ -heterocyclyl and-SO ₂ -a substituted heterocyclyl group, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl and substituted heterocyclyl are as defined herein. Sulfonyl groups include, for example, methyl-SO ₂ -, phenyl-SO ₂ -and 4-methylphenyl-SO ₂ -。

"Sulfonyloxy" refers to the group-OSO ₂ -alkyl, -OSO ₂ -substituted alkyl, -OSO ₂ -alkenyl, -OSO ₂ -substituted alkenyl, -OSO ₂ -cycloalkyl, -OSO ₂ -substituted cycloalkyl, -OSO ₂ -cycloalkenyl, -OSO ₂ -substituted cycloalkenyl, -OSO ₂ -aryl, -OSO ₂ -substituted aryl, -OSO ₂ -heteroaryl, -OSO ₂ -substituted heteroaryl, -OSO ₂ -heterocyclyl and-OSO ₂ -a substituted heterocyclyl group, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl and substituted heterocyclyl are as defined herein.

"sulfate" or "sulfate ester" refers to the group-O-SO ₂ -OH、-O-SO ₂ -O-alkyl, -O-SO ₂ -O-substituted alkyl, -O-SO ₂ -O-alkenyl, -O-SO ₂ -O-substituted alkenyl, -O-SO ₂ -O-cycloalkyl, -O-SO ₂ -O-substituted cycloalkyl, -O-SO ₂ -O-cycloalkenyl, -O-SO ₂ -O-substituted cycloalkenyl, -O-SO ₂ -O-aryl, -O-SO ₂ -O-substituted aryl, -O-SO ₂ -O-heteroaryl, -O-SO ₂ -O-substituted heteroaryl, -O-SO ₂ -O-heterocyclyl and-O-SO ₂ -O-substituted heterocyclyl wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl and substituted heterocyclyl are as defined herein.

The term "aminocarbonyloxy" refers to the group-OC (O) NRR, wherein each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl, wherein alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

"thiol" refers to the group-SH.

"thio" or the term "thioketone group" refers to an atom (=s).

"alkylthio" or the term "thioalkoxy" refers to the group-S-alkyl, wherein alkyl is as defined herein. In certain embodiments, sulfur may be oxidized to-S (O) -. Sulfoxides may exist as one or more stereoisomers.

The term "substituted thioalkoxy" refers to the group-S-substituted alkyl.

The term "thioaryloxy" refers to the group aryl-S-, wherein aryl groups are as defined herein, including optionally substituted aryl groups as also defined herein.

The term "thioheteroaryloxy" refers to the group heteroaryl-S-, wherein heteroaryl groups are as defined herein, including optionally substituted aryl groups as also defined herein.

The term "thioheterocyclyloxy" refers to the group heterocyclyl-S-, wherein heterocyclyl groups are as defined herein, including optionally substituted heterocyclyl groups also as defined herein.

In addition to the disclosure herein, the term "substituted" when used to modify a specified group or radical may also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, substituted with the same or different substituent groups as defined below.

Except for the groups disclosed in relation to the various terms herein, unless otherwise indicated, are used to replace one or more hydrogens on the saturated carbon atom in the indicated group or group (any two hydrogens on a single carbon may be taken to be =o, =nr ⁷⁰ 、＝N-OR ⁷⁰ 、＝N ₂ Or = substituted) substituent group is-R ⁶⁰ Halogen, =o, -OR ⁷⁰ 、-SR ⁷⁰ 、-NR ⁸⁰ R ⁸⁰ Trihalomethyl, -CN, -OCN, -SCN, -NO ₂ 、＝N ₂ 、-N ₃ 、-SO ₂ R ⁷⁰ 、-SO ₂ O ^- M ⁺ 、-SO ₂ OR ⁷⁰ 、-OSO ₂ R ⁷⁰ 、-OSO ₂ O ^- M ⁺ 、-OSO ₂ OR ⁷⁰ 、-P(O)(O ^- ) ₂ (M ⁺ ) ₂ 、-P(O)(OR ⁷⁰ )O ^- M ⁺ 、-P(O)(OR ⁷⁰ ) ₂ 、-C(O)R ⁷⁰ 、-C(S)R ⁷⁰ 、-C(NR ⁷⁰ )R ⁷⁰ 、-C(O)O ^- M ⁺ 、-C(O)OR ⁷⁰ 、-C(S)OR ⁷⁰ 、-C(O)NR ⁸⁰ R ⁸⁰ 、-C(NR ⁷⁰ )NR ⁸⁰ R ⁸⁰ 、-OC(O)R ⁷⁰ 、-OC(S)R ⁷⁰ 、-OC(O)O ^- M ⁺ 、-OC(O)OR ⁷⁰ 、-OC(S)OR ⁷⁰ 、-NR ⁷⁰ C(O)R ⁷⁰ 、-NR ⁷⁰ C(S)R ⁷⁰ 、-NR ⁷⁰ CO ₂ ^- M ⁺ 、-NR ⁷⁰ CO ₂ R ⁷⁰ 、-NR ⁷⁰ C(S)OR ⁷⁰ 、-NR ⁷⁰ C(O)NR ⁸⁰ R ⁸⁰ 、-NR ⁷⁰ C(NR ⁷⁰ )R ⁷⁰ and-NR ⁷⁰ C(NR ⁷⁰ )NR ⁸⁰ R ⁸⁰ Wherein R is ⁶⁰ Selected from the group consisting of: optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R ⁷⁰ Independently is hydrogen or R ⁶⁰ The method comprises the steps of carrying out a first treatment on the surface of the Each R ⁸⁰ Independently is R ⁷⁰ Or alternatively, two R ⁸⁰ Together with the nitrogen atom to which they are bound form a 5-, 6-or 7-membered heterocycloalkyl which may optionally include 1 to 4 identical or different further heteroatoms selected from the group consisting of O, N and S, where N may have-H or C ₁ -C ₃ An alkyl substituent; and each M ⁺ Is a counterion having a net single positive charge. Each M ⁺ Can be independently, for example, a basic ion, for example, K ⁺ 、Na ⁺ 、Li ⁺ The method comprises the steps of carrying out a first treatment on the surface of the Ammonium ions, e.g. ⁺ N(R ⁶⁰ ) ₄ The method comprises the steps of carrying out a first treatment on the surface of the Or alkaline earth metal ions, e.g. [ Ca ] ²⁺ ] _0.5 、[Mg ²⁺ ] _0.5 Or [ Ba ] ²⁺ ] _0.5 ("subscript 0.5" means that one of the counter ions of such divalent alkaline earth metal ions may be the ionized form of the compound of the invention, the other is a typical counter ion, such as chloride, or that the two ionic compounds disclosed herein may act as counter ions for such divalent alkaline earth metal ions, or that the zwitterionic compounds of the invention may act as counter ions for such divalent alkaline earth metal ions). As a specific example, -NR ⁸⁰ R ⁸⁰ Is intended to include-NH ₂ -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl and N-morpholinyl.

Unless otherwise indicated, the substituent groups for hydrogen on unsaturated carbon atoms in "substituted" alkene, alkyne, aryl and heteroaryl groups are-R, in addition to the disclosure herein ⁶⁰ Halogen, -O ^- M ⁺ 、-OR ⁷⁰ 、-SR ⁷⁰ 、-S-M ⁺ 、-NR ⁸⁰ R ⁸⁰ Trihalomethyl, -CF ₃ 、-CN、-OCN、-SCN、-NO、-NO ₂ 、-N ₃ 、-SO ₂ R ⁷⁰ 、-SO ₃ ^- M ⁺ 、-SO ₃ R ⁷⁰ 、-OSO ₂ R ⁷⁰ 、-OSO ₃ -M ⁺ 、-OSO ₃ R ⁷⁰ 、-PO ₃ ^-2 (M ⁺ ) ₂ 、-P(O)(OR ⁷⁰ )O ^- M ⁺ 、-P(O)(OR ⁷⁰ ) ₂ 、-C(O)R ⁷⁰ 、-C(S)R ⁷⁰ 、-C(NR ⁷⁰ )R ⁷⁰ 、-CO ₂ ^- M ⁺ 、-CO ₂ R ⁷⁰ 、-C(S)OR ⁷⁰ 、-C(O)NR ⁸⁰ R ⁸⁰ 、-C(NR ⁷⁰ )NR ⁸⁰ R ⁸⁰ 、-OC(O)R ⁷⁰ 、-OC(S)R ⁷⁰ 、-OCO ₂ ^- M ⁺ 、-OCO ₂ R ⁷⁰ 、-OC(S)OR ⁷⁰ 、-NR ⁷⁰ C(O)R ⁷⁰ 、-NR ⁷⁰ C(S)R ⁷⁰ 、-NR ⁷⁰ CO ₂ ^- M ⁺ 、-NR ⁷⁰ CO ₂ R ⁷⁰ 、-NR ⁷⁰ C(S)OR ⁷⁰ 、-NR ⁷⁰ C(O)NR ⁸⁰ R ⁸⁰ 、-NR ⁷⁰ C(NR ⁷⁰ )R ⁷⁰ and-NR ⁷⁰ C(NR ⁷⁰ )NR ⁸⁰ R ⁸⁰ Wherein R is ⁶⁰ 、R ⁷⁰ 、R ⁸⁰ And M ⁺ As previously defined, provided that in the case of a substituted alkene or alkyne, the substituent is not-O ^- M ⁺ 、-OR ⁷⁰ 、-SR ⁷⁰ or-S ^- M ⁺ 。

The substituent groups of hydrogen on the nitrogen atom in "substituted" heteroalkyl and cycloheteroalkyl groups are-R, except where the terms are disclosed with respect to ⁶⁰ 、-O ^- M ⁺ 、-OR ⁷⁰ 、-SR ⁷⁰ 、-S ^- M ⁺ 、-NR ⁸⁰ R ⁸⁰ Trihalomethyl, -CF ₃ 、-CN、-NO、-NO ₂ 、-S(O) ₂ R ⁷⁰ 、-S(O) ₂ O ^- M ⁺ 、-S(O) ₂ OR ⁷⁰ 、-OS(O) ₂ R ⁷⁰ 、-OS(O) ₂ O ^- M ⁺ 、-OS(O) ₂ OR ⁷⁰ 、-P(O)(O ^- ) ₂ (M ⁺ ) ₂ 、-P(O)(OR ⁷⁰ )O ^- M ⁺ 、-P(O)(OR ⁷⁰ )(OR ⁷⁰ )、-C(O)R ⁷⁰ 、-C(S)R ⁷⁰ 、-C(NR ⁷⁰ )R ⁷⁰ 、-C(O)OR ⁷⁰ 、-C(S)OR ⁷⁰ 、-C(O)NR ⁸⁰ R ⁸⁰ 、-C(NR ⁷⁰ )NR ⁸⁰ R ⁸⁰ 、-OC(O)R ⁷⁰ 、-OC(S)R ⁷⁰ 、-OC(O)OR ⁷⁰ 、-OC(S)OR ⁷⁰ 、-NR ⁷⁰ C(O)R ⁷⁰ 、-NR ⁷⁰ C(S)R ⁷⁰ 、-NR ⁷⁰ C(O)OR ⁷⁰ 、-NR ⁷⁰ C(S)OR ⁷⁰ 、-NR ⁷⁰ C(O)NR ⁸⁰ R ⁸⁰ 、-NR ⁷⁰ C(NR ⁷⁰ )R ⁷⁰ and-NR ⁷⁰ C(NR ⁷⁰ )NR ⁸⁰ R ⁸⁰ Wherein R is ⁶⁰ 、R ⁷⁰ 、R ⁸⁰ And M ⁺ As previously defined.

In addition to the disclosure herein, in certain embodiments, a substituted group has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

It is to be understood that, of all the substituted groups defined above, polymers obtained by defining substituents with further substituents themselves (e.g., substituted aryl groups having substituted aryl groups as substituents, themselves substituted with substituted aryl groups, substituted aryl groups further substituted with substituted aryl groups, etc.) are not intended to be included herein. In this case, the maximum number of such substitutions is 3. For example, the sequential substitution of substituted aryl groups specifically contemplated herein is limited to substituted aryl- (substituted aryl) -substituted aryl.

Unless otherwise indicated, naming of substituents not explicitly defined herein is accomplished by naming the terminal portion of a functional group, followed by naming of the adjacent functional group toward the point of attachment. For example, the substituent "arylalkoxycarbonyl" refers to the group (aryl) - (alkyl) -O-C (O) -.

With respect to any of the groups disclosed herein that contain one or more substituents, it is of course understood that such groups do not contain any substitution or pattern of substitution that is sterically impractical and/or synthetically infeasible. Furthermore, the subject compounds include all stereochemical isomers resulting from the substitution of these compounds.

The term "pharmaceutically acceptable salt" means a salt (salt with a counter ion that has acceptable mammalian safety for a given dosage regimen) that is acceptable for administration to a patient (e.g., a mammal). Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. "pharmaceutically acceptable salts" refers to pharmaceutically acceptable salts of the compounds which are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; when the molecule contains basic functional groups, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, benzenesulfonate, methanesulfonate, acetate, maleate, oxalate, and the like.

The term "salt thereof" refers to a compound formed when the proton of an acid is replaced by a cation, such as a metal cation or an organic cation, or the like. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not necessary for salts of intermediate compounds that are not intended for administration to a patient. For example, salts of the compounds of the invention include those in which the compound is protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.

"solvate" refers to a complex formed by the combination of a solvent molecule and a molecule or ion of a solute. The solvent may be an organic compound, an inorganic compound, or a mixture of both. Some examples of solvents include, but are not limited to, methanol, N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.

"stereoisomers" refers to compounds having the same atomic connectivity but different atomic arrangements in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers and diastereomers.

"tautomer" refers to alternative forms of the molecule that differ only in the position of the electron bond and/or proton of the atom, such as enol-ketone and imine-enamine tautomers, or tautomeric forms of heteroaryl groups containing an array of-n=c (H) -NH-ring atoms, such as pyrazole, imidazole, benzimidazole, triazole and tetrazole. One of ordinary skill in the art will recognize that other tautomeric ring atom arrangements are also possible.

It is to be understood that the term "or a salt or solvate or stereoisomer thereof" is intended to include all permutations of salts, solvates and stereoisomers, such as solvates of pharmaceutically acceptable salts of stereoisomers of the subject compounds.

"pharmaceutically effective amount" and "therapeutically effective amount" refer to an amount of a compound sufficient to treat a particular disorder or disease or one or more symptoms thereof and/or prevent the occurrence of a disease or disorder. With respect to tumorigenic proliferative disorders, a pharmaceutically or therapeutically effective amount includes an amount sufficient to cause a tumor to shrink or reduce the rate of tumor growth.

"patient" refers to both human and non-human subjects, particularly mammalian subjects.

The term "treatment" or "treatment" as used herein refers to treating a disease or medical condition of a patient, such as a mammal (particularly a human), including: (A) Preventing the occurrence of the disease or medical condition, e.g., prophylactic treatment of a subject; (b) Improving a disease or medical condition, e.g., eliminating or causing regression of a disease or medical condition in a patient; (c) Inhibiting the disease or medical condition, for example, by slowing or arresting the progression of the disease or medical condition in the patient; or (d) alleviating a symptom of a disease or medical condition in the patient.

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymeric form of amino acids of any length. Unless otherwise specifically indicated, "polypeptide," "peptide" and "protein" may include genetically encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backbone. The term includes fusion proteins, including but not limited to fusion proteins having heterologous amino acid sequences, fusions having heterologous and homologous leader sequences, proteins containing at least one N-terminal methionine residue (e.g., to facilitate production in a recombinant host cell); an immunolabeling protein; etc.

"native amino acid sequence" or "parent amino acid sequence" is used interchangeably herein to refer to the amino acid sequence of a polypeptide prior to modification to include modified amino acid residues.

The terms "amino acid analog," "unnatural amino acid," and the like, are used interchangeably and include amino acid-like compounds (e.g., ala or A, cys or C, asp or D, glu or E, phe or F, gly or G, his or H, ile or I, lys or K, leu or L, met or M, asn or N, pro or P, gln or Q, arg or R, ser or S, thr or T, val or V, trp or W, tyr or Y) that are similar in structure and/or overall shape to one or more amino acids commonly found in naturally occurring proteins. Amino acid analogs also include natural amino acids having modified side chains or backbones. Amino acid analogs also include amino acid analogs that have the same stereochemistry as naturally occurring D-type and L-type amino acid analogs. In some cases, amino acid analogs share one or more backbone structures and/or side chain structures of the natural amino acid, differing in one or more modifying groups in the molecule. Such modifications may include, but are not limited to, substitution of an atom (e.g., N) for a related atom (e.g., S), addition of a group (e.g., methyl or hydroxyl, etc.) or an atom (e.g., cl or Br, etc.), deletion of a group, substitution of a covalent bond (single bond for double bond, etc.), or combinations thereof. For example, amino acid analogs can include alpha-hydroxy acids, alpha-amino acids, and the like.

The terms "amino acid side chain" or "side chain of an amino acid" and the like may be used to refer to substituents attached to the alpha-carbon of an amino acid residue (including natural amino acids, unnatural amino acids, and amino acid analogs). The amino acid side chains may also include amino acid side chains as described in the context of modified amino acids and/or conjugates as described herein.

The term "carbohydrate" and the like may be used to refer to monomeric units and/or polymers of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The term sugar may be used to refer to smaller carbohydrates, such as monosaccharides, disaccharides. The term "carbohydrate derivative" includes compounds in which one or more functional groups of the carbohydrate of interest are substituted (by any convenient substituent), modified (converted to another group using any convenient chemical method), or absent (e.g., eliminated or replaced by H). A wide variety of carbohydrates and carbohydrate derivatives are available and are suitable for use in the subject compounds and conjugates.

The term "glycoside" or "glycosyl" refers to a sugar molecule or group that is bound to a moiety through a glycosidic bond. For example, the moiety to which the glycoside binds may be a cleavable linker as described herein. The glycosidic bond may link the glycoside to other moieties by various types of bonds, such as, but not limited to, an O-glycosidic bond (O-glycoside), an N-glycosidic bond (glycosylamine), an S-glycosidic bond (thioglycoside), or a C-glycosidic bond (C-glycoside or C-glycosyl). In some cases, the glycosides may be cleaved from the moiety to which they are attached, for example by chemical-mediated hydrolysis or enzyme-mediated hydrolysis.

The term "antibody" is used in its broadest sense and includes monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, single chain antibodies, chimeric antibodies, antibody fragments (e.g., fab fragments), and the like. Antibodies are capable of binding to target antigens. (Janeway, C, transitions, P., walport, M., shomchik (2001) & immunobiology, 5 th edition, galand Press (Garland Publishing), new York). The target antigen may have one or more binding sites, also known as epitopes, recognized by Complementarity Determining Regions (CDRs) formed by one or more variable regions of the antibody.

The term "natural antibody" refers to an antibody in which the heavy and light chains of the antibody have been produced and paired by the immune system of a multicellular organism. Spleen, lymph nodes, bone marrow and serum are examples of tissues that produce natural antibodies. For example, the antibody produced by the antibody-producing cell isolated from the first animal immunized with the antigen is a natural antibody.

The term "humanized antibody" or "humanized immunoglobulin" refers to a non-human (e.g., mouse or rabbit) antibody that contains one or more amino acids (e.g., in the framework regions, constant regions, or CDRs) that have been substituted with correspondingly positioned amino acids from a human antibody. In general, humanized antibodies produce a reduced immune response in a human host compared to non-humanized versions of the same antibody. Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. No. 5,225,539; U.S. Pat. No. 5,530,101; and U.S. Pat. No. 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; padlan, molecular immunology (Molecular Immunology) 28 (4/5): 489-498 (1991); studnicka et al, protein engineering (Protein Engineering) 7 (6): 805-814 (1994); roguska et al, PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). In certain embodiments, framework substitutions are identified by modeling the interactions of CDRs and framework residues to identify framework residues important for antigen binding, and unusual framework residues at specific positions are identified by sequence comparison (see, e.g., U.S. Pat. No. 5,585,089; riechmann et al, nature, 332:323 (1988)). Other methods of humanizing antibodies contemplated for use in the present invention are described in U.S. patent No. 5,750,078; 5,502,167; 5,705,154; 5,770,403; 5,698,417; 5,693,493; no. 5,558,864; 4,935,496; and No. 4,816,567, PCT publications WO 98/45331 and WO 98/45332. In particular embodiments, the subject rabbit antibodies may be humanized according to the methods described in US20040086979 and US 20050033031. Thus, the antibodies described above may be humanized using methods well known in the art.

The term "chimeric antibody" refers to an antibody whose light and heavy chain genes are constructed, typically by genetic engineering, from antibody variable and constant region genes belonging to different species. For example, variable fragments of genes from mouse monoclonal antibodies can be ligated to human constant fragments, such as γ1 and γ3. Examples of therapeutic chimeric antibodies are hybrid proteins consisting of a variable or antigen binding domain from a mouse antibody and a constant or effector domain from a human antibody, although domains from other mammalian species may be used.

Immunoglobulin polypeptide immunoglobulin light or heavy chain variable regions consist of Framework Regions (FR) interrupted by three hypervariable regions (also known as "complementarity determining regions" or "CDRs"). The framework regions and CDR ranges have been defined (see, "immunological protein sequences of interest (Sequences of Proteins of Immunological Interest)", "E.Kabat et al, U.S. department of health and human services (U.S. part of Health and Human Services), 1991). The framework regions of antibodies, i.e., the combined framework regions that make up the light and heavy chains, are used to position and align the CDRs. CDRs are primarily responsible for binding to epitopes of the antigen.

A "parent Ig polypeptide" is a polypeptide comprising an amino acid sequence that lacks the aldehyde-tagged constant regions described herein. The parent polypeptide may comprise a native sequence constant region, or may comprise a constant region with pre-existing amino acid sequence modifications (e.g., additions, deletions, and/or substitutions).

As used herein, the term "isolated" is intended to describe a compound of interest in an environment different from the environment in which the compound naturally occurs. By "isolated" is meant a compound included in a sample that is substantially enriched in a compound of interest and/or wherein the compound of interest is partially or substantially purified.

As used herein, the term "substantially purified" refers to a compound that is removed from its natural environment and that is at least 60% free, at least 75% free, at least 80% free, at least 85% free, at least 90% free, at least 95% free, at least 98% free, or greater than 98% free of other components with which it is naturally associated.

The term "physiological conditions" is intended to encompass those conditions that are compatible with living cells, such as temperature, pH, salinity, etc., that are primarily aqueous conditions that are compatible with living cells.

"reactive partner" refers to a molecule or portion of a molecule that specifically reacts with another reactive partner to produce a reaction product. Exemplary reactive partners include cysteine or serine of the sulfatase motif and Formylglycine Generating Enzymes (FGEs) that react to form reaction products containing converted aldehyde tags that replace the cysteine or serine in the motif, formylglycine (FGly). Other exemplary reactive partners include aldehydes (e.g., reactive aldehyde groups) of converted aldehyde-tagged fGly residues and "aldehyde-reactive partners" that comprise aldehyde-reactive groups and moieties of interest and which react to form reaction products of modified aldehyde-tagged polypeptides having moieties of interest conjugated to modified polypeptides through modified fGly residues.

"N-terminal" refers to the terminal amino acid residue of a polypeptide having a free amine group, the amine group in the non-N-terminal amino acid residue typically forming part of the covalent backbone of the polypeptide.

"C-terminal" refers to the terminal amino acid residue of a polypeptide having a free carboxyl group, the carboxyl group in a non-C-terminal amino acid residue typically forming part of the covalent backbone of the polypeptide.

"internal site" as used with reference to a polypeptide or an amino acid sequence of a polypeptide refers to a region of the polypeptide that is not N-terminal or C-terminal.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of embodiments relating to the present invention are specifically included by the present invention and disclosed herein as if each combination were individually and specifically disclosed to the extent that such combinations include the subject matter of a compound (i.e., a compound that can be prepared, isolated, characterized, and tested for biological activity) that is, for example, a stable compound. Moreover, all subcombinations of the various embodiments and elements thereof (e.g., elements of the chemical groups listed in the embodiments describing such variables) are also specifically contemplated by the present invention and disclosed herein as if each such subcombination was individually and explicitly disclosed herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as antecedent basis for use of exclusive terminology such as "solely," "only" and the like in connection with recitation of claim elements, or use of "negative" limitations.

It is appreciated that certain features of the application, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the application, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the application is not entitled to antedate such disclosure by virtue of prior application. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Detailed Description

The present disclosure provides antibody-drug conjugate structures that include cleavable linkers that link an antibody to a drug. The cleavable linker comprises a first enzymatically cleavable moiety and a second enzymatically cleavable moiety, the second enzymatically cleavable moiety comprising a glycoside selected from the group consisting of galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc. The disclosure also encompasses methods of making such conjugates and methods of using the conjugates.

Antibody-drug conjugates

The present disclosure provides a conjugate, such as an antibody-drug conjugate (ADC). By "conjugate" is meant that a first moiety (e.g., an antibody) is stably bound to a second moiety (e.g., a drug or active agent). For example, an antibody-drug conjugate includes a drug or active agent stably bound to another moiety (e.g., an antibody). "stable binding" refers to the binding of one moiety to another moiety or structure under standard conditions. In certain embodiments, the first moiety and the second moiety are bound to each other by one or more functional groups and covalent bonds. For example, one or more functional groups and covalent bonds may include cleavable linkers as described herein.

In certain embodiments, the conjugate is a polypeptide conjugate that includes a polypeptide (e.g., an antibody) conjugated to a second moiety. In certain embodiments, the moiety conjugated to the polypeptide may be any of a variety of moieties of interest, such as, but not limited to, a drug, an active agent, a detectable label, a water-soluble polymer, or a moiety for immobilizing the polypeptide to a membrane or surface. In certain embodiments, the conjugate is a drug conjugate, wherein the polypeptide is an antibody, thereby providing an antibody-drug conjugate. For example, the conjugate may be a drug conjugate, wherein the polypeptide is conjugated to a drug or active agent. Various types of drugs or active agents may be used in the conjugates, described in more detail below.

In certain embodiments, the conjugate is an antibody-drug conjugate, wherein the antibody and the drug are linked together by a linker. In some cases, the linker is a cleavable linker. A cleavable linker is a linker comprising one or more cleavable moieties, wherein the cleavable moiety comprises one or more bonds that are cleavable under certain conditions, thereby separating the cleavable linker into two or more cleavable moieties. For example, a cleavable moiety may comprise one or more covalent bonds that can dissociate or cleave under certain conditions to separate the cleavable linker into two or more moieties. Thus, such cleavable linkers may be included in an antibody-drug conjugate such that under appropriate conditions, the cleavable linker is cleaved to separate or release the drug from the antibody at the desired target site of drug action.

In some cases, the cleavable linker comprises two cleavable moieties, e.g., a first cleavable moiety and a second cleavable moiety. The cleavable moiety may be configured such that cleavage of both cleavable moieties is required to separate or release the drug from the antibody at the desired site of action target of the drug. For example, cleavage of a cleavable linker may be achieved by first cleaving one of the two cleavable moieties and then cleaving the other of the two cleavable moieties. In certain embodiments, the cleavable linker comprises a first cleavable moiety and a second cleavable moiety that blocks cleavage of the first cleavable moiety. By "hindering cleavage" is meant that the presence of an uncleaved second cleavable moiety reduces the likelihood of cleavage of the first cleavable moiety or substantially inhibits cleavage of the first cleavable moiety, thus substantially reducing the amount of cleavable linker or preventing cleavage of cleavable linker. For example, the presence of an uncleaved second cleavable moiety may hinder cleavage of the first cleavable moiety. Cleavage of the first cleavable moiety is hindered by the presence of the second cleavable moiety, thereby significantly reducing the amount of drug or preventing release of drug from the antibody. For example, premature release of the drug from the antibody may be substantially reduced or prevented until the antibody-drug conjugate is at or near the desired site of action target of the drug.

In some cases, cleavage of the cleavable linker may be achieved by first cleaving the second cleavable moiety and then cleaving the first cleavable moiety, as the second cleavable moiety blocks cleavage of the first cleavable moiety. Cleavage of the second cleavable moiety may reduce or eliminate the obstruction of cleavage of the first cleavable moiety, thereby allowing the first cleavable moiety to be cleaved. Cleavage of the first cleavable moiety may result in cleavage or separation of the cleavable linker into two or more moieties as described above to release the drug from the antibody-drug conjugate. In some cases, cleavage of the first cleavable moiety does not substantially occur in the presence of the uncleaved second cleavable moiety. By "substantially" is meant that cleavage of about 10% or less of the first cleavable moiety occurs in the presence of the uncleaved second cleavable moiety, e.g., cleavage of about 9% or less, or about 8% or less, or about 7% or less, or about 6% or less, or about 5% or less, or about 4% or less, or about 3% or less, or about 2% or less, or about 1% or less, or about 0.5% or less, or about 0.1% or less of the first cleavable moiety occurs in the presence of the uncleaved second cleavable moiety.

In other words, the second cleavable moiety may protect the first cleavable moiety from cleavage. For example, the presence of an uncleaved second cleavable moiety can protect the first cleavable moiety from cleavage and thus substantially reduce or prevent premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired site of action target of the drug. Thus, cleavage of the second cleavable moiety exposes (e.g., deprotects) the first cleavable moiety, allowing the first cleavable moiety to be cleaved, which results in cleavage of the cleavable linker, thereby separating or releasing the drug from the antibody at the desired site of action target of the drug as described above. In some cases, cleavage of the second cleavable moiety exposes the first cleavable moiety to subsequent cleavage, but cleavage of the second cleavable moiety does not itself result in cleavage of the cleavable linker (i.e., cleavage of the first cleavable moiety is still required to cleave the cleavable linker).

The cleavable moieties comprised in the cleavable linker may each be an enzymatically cleavable moiety. For example, the first cleavable moiety may be a first enzymatically cleavable moiety and the second cleavable moiety may be a second enzymatically cleavable moiety. An enzymatically cleavable moiety is a cleavable moiety that can be separated into two or more moieties as described above by enzymatic action of an enzyme. The enzymatically cleavable moiety may be any cleavable moiety cleavable by the enzymatic action of an enzyme, such as, but not limited to, peptides, glycosides, and the like. In some cases, the enzyme that cleaves the enzymatically cleavable moiety is present at a desired action target site, e.g., a desired action target site of a drug to be released from an antibody-drug conjugate. In some cases, the enzyme that cleaves the enzymatically cleavable moiety is not present in a significant amount in other areas, such as whole blood, plasma, or serum. Thus, cleavage of the enzymatically cleavable moiety may be controlled such that substantial cleavage occurs at the desired site of action without significant cleavage occurring in other regions or before the antibody-drug conjugate reaches the desired site of action.

For example, as described herein, the antibody-drug conjugates of the present disclosure can be used to treat cancer, e.g., for delivering a cancer therapeutic drug to a desired site of action in the presence of cancer cells. In some cases, the enzyme, e.g., protease cathepsin B, may be a biomarker for cancer that is overexpressed in cancer cells. Overexpression and thus localization of certain enzymes in cancer can be used in the context of enzymatically cleavable moieties included in the cleavable linkers of the antibody-drug conjugates of the present disclosure to specifically release the drug at the desired site of action (i.e., the site of cancer (and overexpressed enzymes). Thus, in some embodiments, the enzymatically cleavable moiety is a cleavable moiety (e.g., a peptide) that is cleavable by an enzyme that is overexpressed in a cancer cell. For example, the enzyme may be protease cathepsin B. Thus, in some cases, the enzymatically cleavable moiety is a cleavable moiety (e.g., a peptide) that is cleavable by a protease such as cathepsin B.

In certain embodiments, the enzymatically cleavable moiety is a peptide. The peptide may be any peptide suitable for use in a cleavable linker and cleavable by enzymatic action of an enzyme. Non-limiting examples of peptides that can be used as enzymatically cleavable moieties include, for example, val-Ala, phe-Lys, and the like. For example, the first cleavable moiety described above (i.e., the cleavable moiety protected from premature cleavage by the second cleavable moiety) can comprise a peptide. The presence of the uncleaved second cleavable moiety can protect the first cleavable moiety (peptide) from cleavage by a protease (e.g., cathepsin B) and thus substantially reduce or prevent premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired site of action target of the drug. In some cases, one of the amino acid residues of the peptide comprising the first cleavable moiety is attached to or comprises a substituent, wherein the substituent comprises the second cleavable moiety. In some cases, the second cleavable moiety comprises a glycoside.

In some embodiments, the enzymatically cleavable moiety is a sugar moiety, such as a glycoside (or glycosyl). In some cases, the glycoside may promote increased hydrophilicity of the cleavable linker as compared to a cleavable linker that does not include the glycoside. The glycoside may be any glycoside which is suitable for use in a cleavable linker and which can be cleaved by enzymatic action of an enzyme. For example, the second cleavable moiety (i.e., the cleavable moiety that protects the first cleavable moiety from premature cleavage) can be a glycoside. For example, in some embodiments, the first cleavable moiety comprises a peptide and the second cleavable moiety comprises a glycoside. In certain embodiments, the second cleavable moiety is a glycoside selected from the group consisting of galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc. In some cases, the second cleavable moiety is a galactoside. In some cases, the second cleavable moiety is a glucoside. In some cases, the second cleavable moiety is a mannoside. In some cases, the second cleavable moiety is a fucoside. In some cases, the second cleavable moiety is O-GlcNAc. In some cases, the second cleavable moiety is O-GalNAc.

The glycoside may be linked (covalently bonded) to the cleavable linker by a glycosidic bond. The glycosidic bond may connect the glycoside to the cleavable linker by various types of bonds such as, but not limited to, an O-glycosidic bond (O-glycoside), an N-glycosidic bond (glucosamine), an S-glycosidic bond (thioglycoside) or a C-glycosidic bond (C-glycoside or C-glycosyl). In some cases, the glycosidic bond is an O-glycosidic bond (O-glycoside). In some cases, the glycoside may be cleaved from the cleavable linker to which it is attached by an enzyme (e.g., by enzyme-mediated hydrolysis of the glycosidic bond). The glycoside may be removed or cleaved from the cleavable linker by any convenient enzyme capable of cleaving (hydrolyzing) the glycosidic bond linking the glycoside to the cleavable linker. Examples of enzymes that can be used to mediate cleavage (hydrolysis) of the glycosidic bond connecting the glycoside to the cleavable linker are glycosidases, such as galactosidase, glucosidase, mannosidase, fucosidase and the like. Other suitable enzymes may also be used to mediate cleavage (hydrolysis) of the glycosidic bond connecting the glycoside to the cleavable linker. In some cases, the enzyme used to mediate cleavage (hydrolysis) of the glycosidic bond connecting the glycoside to the cleavable linker is found at or near the desired site of action of the drug of the antibody-drug conjugate. For example, the enzyme may be a lysosomal enzyme, such as a lysosomal glycosidase, which is present in the cell at or near the desired site of action of the drug of the antibody-drug conjugate. In some cases, the enzyme is an enzyme found at or near the enzyme target site found to mediate cleavage of the first cleavable moiety.

The moiety of interest (e.g., a drug or active agent) can be conjugated to the polypeptide (e.g., an antibody) at any desired site of the polypeptide. Thus, the present disclosure provides modified polypeptides, for example, having a moiety conjugated at or near the C-terminus of the polypeptide. Other examples include modified polypeptides having a moiety conjugated at or near the N-terminus of the polypeptide. Examples also include modified polypeptides having a moiety conjugated at a position between the C-terminus and the N-terminus of the polypeptide (e.g., at an internal site of the polypeptide). Combinations of the above are also possible when the modified polypeptide is conjugated to two or more moieties.

In certain embodiments, the conjugates of the present disclosure include a drug or active agent conjugated to an amino acid residue of a polypeptide at the α -carbon of the amino acid residue. In other words, conjugates include polypeptides in which the side chains of one or more amino acid residues in the polypeptide have been modified to be attached to a drug or active agent (e.g., by a linker as described herein). For example, conjugates include polypeptides in which the alpha-carbon of one or more amino acid residues in the polypeptide has been modified to be linked to a drug or active agent (e.g., by a linker as described herein).

Embodiments of the present disclosure include conjugates in which a polypeptide is conjugated to one or more moieties, e.g., 2 moieties, 3 moieties, 4 moieties, 5 moieties, 6 moieties, 7 moieties, 8 moieties, 9 moieties, or 10 or more moieties. The moiety may be conjugated to the polypeptide at one or more sites in the polypeptide. For example, one or more moieties may be conjugated to a single amino acid residue of a polypeptide. In some cases, one moiety is conjugated to an amino acid residue of a polypeptide. In other embodiments, both moieties may be conjugated to the same amino acid residue of the polypeptide. In other embodiments, the first moiety is conjugated to a first amino acid residue of a polypeptide and the second moiety is conjugated to a second amino acid residue of a polypeptide. Combinations of the above are also possible, for example, when the polypeptide is conjugated to a first moiety at a first amino acid residue and to two other moieties at a second amino acid residue. Other combinations are also possible, such as, but not limited to, polypeptides conjugated to the first and second moieties at a first amino acid residue and to the third and fourth moieties at a second amino acid residue, and the like.

The one or more amino acid residues of the polypeptide conjugated to one or more moieties may be naturally occurring amino acids, non-natural amino acids, or a combination thereof. For example, a conjugate may include a moiety conjugated to a naturally occurring amino acid residue of a polypeptide. In other cases, the conjugate may include a moiety conjugated to an unnatural amino acid residue of the polypeptide. One or more moieties may be conjugated to the polypeptide at a single natural or unnatural amino acid residue. One or more natural or unnatural amino acid residues in a polypeptide can be conjugated to one or more moieties described herein. For example, two (or more) amino acid residues (e.g., natural or unnatural amino acid residues) in a polypeptide can each be conjugated to one or both moieties, such that multiple sites in the polypeptide are modified.

The polypeptide may be conjugated to one or more moieties, as described herein. In certain embodiments, the moiety of interest is a chemical entity, such as a drug, an active agent, or a detectable label. For example, the drug (or active agent) may be conjugated to the polypeptide, or in other embodiments, the detectable label may be conjugated to the polypeptide. Thus, for example, embodiments of the present disclosure include, but are not limited to, the following: conjugates of polypeptides and drugs; conjugates of a polypeptide and an active agent; a conjugate of a polypeptide and a detectable label; conjugates of two or more drugs and polypeptides; conjugates of two or more detectable labels and a polypeptide; etc.

In certain embodiments, the polypeptide (e.g., antibody) and the moiety of interest (e.g., drug or active agent) are conjugated via a coupling moiety. For example, the polypeptide and the moiety of interest may each be bound (e.g., covalently bound) to a coupling moiety, thereby indirectly binding the polypeptide and the moiety of interest together via the coupling moiety. In some cases, the coupling moiety comprises a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl compound, or a derivative of a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl compound. For example, the general scheme for coupling a moiety of interest to a polypeptide via a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety is shown in the general reaction scheme below. The hydrazino-indolyl and hydrazino-pyrrolo-pyridinyl coupling moieties are also referred to herein as hydrazino-iso-picket-spengler (HIPS) coupling moieties and aza-hydrazino-iso-picket-spengler (aza HIPS) coupling moieties, respectively.

In the above reaction schemes, R comprises a moiety of interest (e.g., a drug or active agent) conjugated to a polypeptide (e.g., conjugated to a polypeptide via a cleavable linker as described herein). As shown in the above reaction schemes, a polypeptide comprising a 2-formylglycine residue (fGly) is reacted with a drug or active agent that has been modified to include a coupling moiety (e.g., a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety) to produce a polypeptide conjugate linked to the coupling moiety, whereby the drug or active agent is linked to the polypeptide through the coupling moiety.

As described herein, the moiety may be any of a variety of moieties, such as, but not limited to, a chemical entity, such as a detectable label, or a drug or active agent. R 'and R' may each independently be any desired substituent such as, but not limited to, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. Z may be CR ²¹ 、NR ²² N, O or S, wherein R ²¹ And R is ²² Each independently selected from any of the substituents described above for R' and R ".

Other hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moieties are also possible, as shown in the conjugates and compounds described herein. For example, the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety may be modified to be linked (e.g., covalently linked) to a linker. Thus, embodiments of the present disclosure include hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moieties linked to a drug or active agent through a linker. Various embodiments of linkers that can couple hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moieties to drugs or active agents are described in detail herein. For example, in some cases, the linker is a cleavable linker, such as described herein.

In certain embodiments, the polypeptide may be conjugated to a moiety of interest, wherein the polypeptide is modified prior to conjugation to the moiety of interest. Modification of the polypeptide may result in a modified polypeptide containing one or more reactive groups suitable for conjugation to a moiety of interest. In some cases, the polypeptide may be modified at one or more amino acid residues to provide one or more reactive groups suitable for conjugation to a moiety of interest (e.g., a moiety comprising a coupling moiety, such as the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety described above). For example, the polypeptide may be modified to include a reactive aldehyde group (e.g., a reactive aldehyde). Reactive aldehydes may be included in "aldehyde tags" or "ald-tags," as used herein, refers to amino acid sequences derived from sulfatase motifs (e.g., L (C/S) TPSR) that have been converted to contain 2-formylglycine residues (referred to herein as "FGly") by the action of Formylglycine Generating Enzymes (FGEs). The FGly residue generated by FGE may also be referred to as "formylglycine". In other words, the term "aldehyde tag" is used herein to refer to an amino acid sequence that includes a "converted" sulfatase motif (i.e., a sulfatase motif in which a cysteine or serine residue has been converted to FGly by the action of FGE, e.g., L (FGly) TPSR). The converted sulfatase motif may be derived from an amino acid sequence comprising an "unconverted" sulfatase motif (i.e., wherein the cysteine or serine residue is not converted to FGly by FGE, but is capable of being converted to a sulfatase motif, e.g., an unconverted sulfatase motif having the sequence L (C/S) TPSR). "transformation" as used in the context of the action of Formylglycine Generating Enzyme (FGE) on a sulfatase motif refers to the biochemical modification of a cysteine or serine residue in the sulfatase motif to a formylglycine (FGly) residue (e.g., cys to FGly, or Ser to FGly). Other aspects of aldehyde tags and their use in site-specific protein modification are described in U.S. patent No. 7,985,783 and U.S. patent No. 8,729,232, the disclosures of each of which are incorporated herein by reference.

In some cases, modified polypeptides containing FGly residues can be conjugated to a moiety of interest by reaction of FGly with a compound (e.g., a compound containing a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety, as described above). For example, a FGly-containing polypeptide may be contacted with a drug containing a reactive partner under conditions suitable to conjugate the drug to the polypeptide. In some cases, the drug containing the reactive partner may include a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety as described above. For example, the drug or active agent may be modified to include a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety. In some cases, the drug or active agent is linked to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl group, for example, covalently linked to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl group through a linker, for example, a cleavable linker as described in detail herein.

In certain embodiments, conjugates of the present disclosure include polypeptides (e.g., antibodies) having at least one modified amino acid residue. The modified amino acid residues of the polypeptides may be conjugated to a drug or active agent comprising a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety as described above. In certain embodiments, modified amino acid residues of a polypeptide (e.g., an antibody) may be derived from cysteine or serine residues that have been converted to FGly residues as described above. In certain embodiments, the FGly residue is conjugated to a drug or active agent containing a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety as described above, to provide a conjugate of the present disclosure, wherein the drug or active agent is conjugated to the polypeptide through the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety. As used herein, the term FGly' refers to a modified amino acid residue of a polypeptide (e.g., an antibody) coupled to a moiety of interest (e.g., a drug or active agent).

In certain embodiments, the conjugate comprises at least one modified amino acid residue as described herein, wherein the modified amino acid residue is attached to a linker (cleavable linker) as described herein, which in turn is attached to a drug or active agent. For example, the conjugate may comprise at least one modified amino acid residue (FGly') as described above. In some embodiments, the conjugate has formula (I):

wherein the method comprises the steps of

Z is CR ⁴ Or N;

x is O or NR ⁴ ；

R ¹ Selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedAryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl;

k is an integer from 1 to 10;

R ⁷ comprising a second enzymatically cleavable moiety;

L ¹ is a first joint;

L ² is a second joint;

W ¹ is a drug; and is also provided with

W ² Is the antibody.

In certain embodiments of formula (I), k is 2, and the conjugate has formula (Ia):

In certain embodiments of formula (I), the conjugate has formula (Ib):

in certain embodiments of formula (I), the conjugate has formula (Ic):

in certain embodiments of formula (I), the conjugate has formula (Id):

in certain embodiments of formula (I), the conjugate has formula (Ie):

in certain embodiments of formula (I), the conjugate has formula (If):

/>

in certain embodiments of formula (I), the conjugate has formula (Ig):

substituents associated with the conjugates of formula (I) are described in more detail below. References to formula (I) also encompass formulas (Ia), (Ib), (Ic), (Id), (Ie), (If) and (Ig).

In certain embodiments, Z is CR ⁴ Or N. In certain embodiments, Z is CR ⁴ . In certain embodiments, Z is N.

In certain embodiments, X is O or NR ⁴ . In some cases, X is O. In some cases, X is NR ⁴ . In some cases, X is NH.

In certain embodiments, R ¹ Selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R ¹ Is hydrogen. In certain embodiments, R ¹ Is alkyl or substituted alkyl, e.g. C _1-6 Alkyl or C _1-6 Substituted alkyl, or C _1-4 Alkyl or C _1-4 Substituted alkyl or C _1-3 Alkyl or C _1-3 Substituted alkyl. In certain embodiments, R ¹ Is methyl. In certain embodiments, R ¹ Is alkenyl or substituted alkenyl, e.g. C _2-6 Alkenyl or C _2-6 Substituted alkenyl, or C _2-4 Alkenyl or C _2-4 Substituted alkenyl, or C _2-3 Alkenyl or C _2-3 Substituted alkenyl groups. In certain embodiments, R ¹ Alkynyl or substituted alkynyl, e.g. C _2-6 Alkenyl or C _2-6 Substituted alkenyl, or C _2-4 Alkenyl or C _2-4 Substituted alkenyl, or C _2-3 Alkenyl or C _2-3 Substituted alkenyl groups. In certain embodiments, R ¹ Is aryl or substituted aryl, e.g. C _5-8 Aryl or C _5-8 Substituted aryl groups, e.g. C ₅ Aryl or C ₅ Substituted aryl, or C ₆ Aryl or C ₆ Substituted aryl. In certain embodiments, R ¹ Is heteroaryl or substituted heteroaryl, e.g. C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, e.gSuch as C ₅ Heteroaryl or C ₅ Substituted heteroaryl, or C ₆ Heteroaryl or C ₆ Substituted heteroaryl groups. In certain embodiments, R ¹ Cycloalkyl or substituted cycloalkyl, e.g. C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl radicals, e.g. C _3-6 Cycloalkyl or C _3-6 Substituted cycloalkyl, or C _3-5 Cycloalkyl or C _3-5 Substituted cycloalkyl groups. In certain embodiments, R ¹ Is a heterocyclic group or a substituted heterocyclic group, e.g. C _3-8 Heterocyclyl or C _3-8 Substituted heterocyclic groups, e.g. C _3-6 Heterocyclyl or C _3-6 Substituted heterocyclyl, or C _3-5 Heterocyclyl or C _3-5 Substituted heterocyclyl groups.

In certain embodiments, R ² And R is ³ Each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, or R ² And R is ³ Optionally linked in an cyclic manner to form a 5-or 6-membered heterocyclic group.

In certain embodiments, R ² Selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R ² Is hydrogen. In certain embodiments, R ² Is alkyl or substituted alkyl, e.g. C _1-6 Alkyl or C _1-6 Substituted alkyl, or C _1-4 Alkyl or C _1-4 Substituted alkyl or C _1-3 Alkyl or C _1-3 Substituted alkyl. In certain embodiments, R ² Is methyl. In certain embodiments, R ² Is alkenyl or substituted alkenyl, e.g. C _2-6 Alkenyl or C _2-6 Substituted alkenyl, or C _2-4 Alkenyl or C _2-4 Substituted alkenyl, or C _2-3 Alkenyl or C _2-3 Substituted alkenyl groups. In certain embodiments, R ² Is alkynyl or substituted alkynyl. In certain embodiments, R ² Is an alkoxy group or a substituted alkoxy group. In certain embodiments, R ² Is an amino group or a substituted amino group. In certain embodiments, R ² Is carboxyl or carboxyl ester. In certain embodiments, R ² Is acyl or acyloxy. In certain embodiments, R ² Is an acylamino group or an aminoacyl group. In certain embodiments, R ² Is an alkylamide or a substituted alkylamide. In certain embodiments, R ² Is sulfonyl. In certain embodiments, R ² Is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ² Is aryl or substituted aryl, e.g. C _5-8 Aryl or C _5-8 Substituted aryl groups, e.g. C ₅ Aryl or C ₅ Substituted aryl, or C ₆ Aryl or C ₆ Substituted aryl. In certain embodiments, R ² Is heteroaryl or substituted heteroaryl, e.g. C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, e.g. C ₅ Heteroaryl or C ₅ Substituted heteroaryl, or C ₆ Heteroaryl or C ₆ Substituted heteroaryl groups. In certain embodiments, R ² Cycloalkyl or substituted cycloalkyl, e.g. C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl radicals, e.g. C _3-6 Cycloalkyl or C _3-6 Substituted cycloalkyl, or C _3-5 Cycloalkyl or C _3-5 Substituted cycloalkyl groups. In certain embodiments, R ² Is a heterocyclic group or a substituted heterocyclic group, e.g. C _3-6 Heterocyclyl or C _3-6 Substituted heterocyclyl, or C _3-5 Heterocyclyl or C _3-5 Substituted heterocyclyl groups.

In some embodiments，R ³ Selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R ³ Is hydrogen. In certain embodiments, R ³ Is alkyl or substituted alkyl, e.g. C _1-6 Alkyl or C _1-6 Substituted alkyl, or C _1-4 Alkyl or C _1-4 Substituted alkyl or C _1-3 Alkyl or C _1-3 Substituted alkyl. In certain embodiments, R ³ Is methyl. In certain embodiments, R ³ Is alkenyl or substituted alkenyl, e.g. C _2-6 Alkenyl or C _2-6 Substituted alkenyl, or C _2-4 Alkenyl or C _2-4 Substituted alkenyl, or C _2-3 Alkenyl or C _2-3 Substituted alkenyl groups. In certain embodiments, R ³ Is alkynyl or substituted alkynyl. In certain embodiments, R ³ Is an alkoxy group or a substituted alkoxy group. In certain embodiments, R ³ Is an amino group or a substituted amino group. In certain embodiments, R ³ Is carboxyl or carboxyl ester. In certain embodiments, R ³ Is acyl or acyloxy. In certain embodiments, R ³ Is an acylamino group or an aminoacyl group. In certain embodiments, R ³ Is an alkylamide or a substituted alkylamide. In certain embodiments, R ³ Is sulfonyl. In certain embodiments, R ³ Is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ³ Is aryl or substituted aryl, e.g. C _5-8 Aryl or C _5-8 Substituted aryl groups, e.g. C ₅ Aryl or C ₅ Substituted aryl, or C ₆ Aryl or C ₆ Substituted aryl. In certain embodiments, R ³ Is heteroaryl or substituted heteroaryl, e.g. C _5-8 Heteroaryl or C _5-8 Substituted heterogeniesAryl radicals, e.g. C ₅ Heteroaryl or C ₅ Substituted heteroaryl, or C ₆ Heteroaryl or C ₆ Substituted heteroaryl groups. In certain embodiments, R ³ Cycloalkyl or substituted cycloalkyl, e.g. C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl radicals, e.g. C _3-6 Cycloalkyl or C _3-6 Substituted cycloalkyl, or C _3-5 Cycloalkyl or C _3-5 Substituted cycloalkyl groups. In certain embodiments, R ³ Is a heterocyclic group or a substituted heterocyclic group, e.g. C _3-8 Heterocyclyl or C _3-8 Substituted heterocyclic groups, e.g. C _3-6 Heterocyclyl or C _3-6 Substituted heterocyclyl, or C _3-5 Heterocyclyl or C _3-5 Substituted heterocyclyl groups.

In certain embodiments, R ² And R is ³ Optionally linked in an cyclic manner to form a 5-or 6-membered heterocyclic group. In certain embodiments, R ² And R is ³ Cyclic linkages to form 5 or 6 membered heterocyclyl. In certain embodiments, R ² And R is ³ Cyclic linkages to form a 5 membered heterocyclyl. In certain embodiments, R ² And R is ³ Cyclic linkages to form a 6 membered heterocyclyl.

In certain embodiments, each R ⁴ Independently selected from the group consisting of hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

Each R ⁴ The various possibilities of which are described in more detail below. In certain embodiments, R ⁴ Is hydrogen. In certain embodiments, each R ⁴ Is hydrogen. In certain embodiments, R ⁴ Halogen, such as F, cl, br or I. In certain embodiments, R ⁴ Is F. In certain embodiments, R ⁴ Is Cl. In certain embodiments, R ⁴ Is Br. In certain embodiments, R ⁴ Is I. In certain embodiments, R ⁴ Is alkyl or substituted alkyl, e.g. C _1-6 Alkyl or C _1-6 Substituted alkyl, or C _1-4 Alkyl or C _1-4 Substituted alkyl or C _1-3 Alkyl or C _1-3 Substituted alkyl. In certain embodiments, R ⁴ Is methyl. In certain embodiments, R ⁴ Is alkenyl or substituted alkenyl, e.g. C _2-6 Alkenyl or C _2-6 Substituted alkenyl, or C _2-4 Alkenyl or C _2-4 Substituted alkenyl, or C _2-3 Alkenyl or C _2-3 Substituted alkenyl groups. In certain embodiments, R ⁴ Is alkynyl or substituted alkynyl. In certain embodiments, R ⁴ Is an alkoxy group or a substituted alkoxy group. In certain embodiments, R ⁴ Is an amino group or a substituted amino group. In certain embodiments, R ⁴ Is carboxyl or carboxyl ester. In certain embodiments, R ⁴ Is acyl or acyloxy. In certain embodiments, R ⁴ Is an acylamino group or an aminoacyl group. In certain embodiments, R ⁴ Is an alkylamide or a substituted alkylamide. In certain embodiments, R ⁴ Is sulfonyl. In certain embodiments, R ⁴ Is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ⁴ Is aryl or substituted aryl, e.g. C _5-8 Aryl or C _5-8 Substituted aryl groups, e.g. C ₅ Aryl or C ₅ Substituted aryl, or C ₆ Aryl or C ₆ Substituted aryl (e.g., phenyl or substituted phenyl). In certain embodiments, R ⁴ Is heteroaryl or substituted heteroaryl, e.g. C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, e.g. C ₅ Heteroaryl or C ₅ Substituted heteroaryl, or C ₆ Heteroaryl or C ₆ Substituted heteroaryl groups. In certain embodiments, R ⁴ Cycloalkyl or substituted cycloalkyl, e.g. C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl radicals, e.g. C _3-6 Cycloalkyl or C _3-6 Substituted cycloalkyl, or C _3-5 Cycloalkyl or C _3-5 Substituted cycloalkyl groups. In some casesIn embodiments, R ⁴ Is a heterocyclic group or a substituted heterocyclic group, e.g. C _3-8 Heterocyclyl or C _3-8 Substituted heterocyclic groups, e.g. C _3-6 Heterocyclyl or C _3-6 Substituted heterocyclyl, or C _3-5 Heterocyclyl or C _3-5 Substituted heterocyclyl groups.

In certain embodiments, each R ⁵ Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl. In certain embodiments, R ⁵ Is hydrogen. In certain embodiments, R ⁵ Is alkyl or substituted alkyl, e.g. C _1-6 Alkyl or C _1-6 Substituted alkyl, or C _1-4 Alkyl or C _1-4 Substituted alkyl or C _1-3 Alkyl or C _1-3 Substituted alkyl. In certain embodiments, R ⁵ Is methyl. In certain embodiments, R ⁵ Is ethyl. In certain embodiments, R ⁵ Is propyl (e.g., n-propyl or isopropyl). In certain embodiments, R ⁵ Is butyl (e.g., n-butyl, isobutyl, sec-butyl, or tert-butyl). In certain embodiments, R ⁵ Is a pentyl group (e.g., n-pentyl or neopentyl, etc.). In certain embodiments, R ⁵ Is neopentyl. In certain embodiments, R ⁵ Is alkenyl or substituted alkenyl, e.g. C _2-6 Alkenyl or C _2-6 Substituted alkenyl, or C _2-4 Alkenyl or C _2-4 Substituted alkenyl, or C _2-3 Alkenyl or C _2-3 Substituted alkenyl groups. In certain embodiments, R ⁵ Is alkynyl or substituted alkynyl.

In certain embodiments, each R ⁶ Independently selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; in certain embodiments, R ⁶ Is hydrogen. In certain embodiments, R ⁶ Is alkyl or substituted alkyl, e.g. C _1-6 Alkyl or C _1-6 Substituted alkyl, or C _1-4 Alkyl or C _1-4 Substituted alkyl or C _1-3 Alkyl or C _1-3 Substituted alkyl. In certain embodiments, R ⁶ Is methyl. In certain embodiments, R ⁶ Is ethyl. In certain embodiments, R ⁶ Is propyl (e.g., n-propyl or isopropyl). In certain embodiments, R ⁶ Is butyl (e.g., n-butyl, isobutyl, sec-butyl, or tert-butyl). In certain embodiments, R ⁶ Is a pentyl group (e.g., n-pentyl or neopentyl, etc.). In certain embodiments, R ⁶ Is neopentyl. In certain embodiments, R ⁶ Is alkenyl or substituted alkenyl, e.g. C _2-6 Alkenyl or C _2-6 Substituted alkenyl, or C _2-4 Alkenyl or C _2-4 Substituted alkenyl, or C _2-3 Alkenyl or C _2-3 Substituted alkenyl groups. In certain embodiments, R ⁶ Is alkynyl or substituted alkynyl.

In certain embodiments, R ⁶ Is aryl or substituted aryl, e.g. C _5-8 Aryl or C _5-8 Substituted aryl groups, e.g. C ₅ Aryl or C ₅ Substituted aryl, or C ₆ Aryl or C ₆ Substituted aryl (e.g., phenyl or substituted phenyl). In certain embodiments, R ⁶ Is heteroaryl or substituted heteroaryl, e.g. C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, e.g. C ₅ Heteroaryl or C ₅ Substituted heteroaryl, or C ₆ Heteroaryl or C ₆ Substituted heteroaryl groups. In certain embodiments, R ⁶ Cycloalkyl or substituted cycloalkyl, e.g. C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl radicals, e.g. C _3-6 Cycloalkyl or C _3-6 Substituted cycloalkyl, or C _3-5 Cycloalkyl or C _3-5 Substituted cycloalkyl groups. In certain embodiments, R ⁶ Is a heterocyclic group or a substituted heterocyclic group, e.g. C _3-8 Heterocyclyl or C _3-8 Substituted heterocyclic groups, e.g. C _3-6 Heterocyclyl or C _3-6 Substituted heterocyclyl, or C _3-5 Heterocyclyl or C _3-5 Substituted heterocyclyl groups.

In certain embodiments, R ⁶ Representing the side chain of an amino acid. For example, R ⁶ Can represent the amino acid residue [ ], the amino acid residue ]Including natural amino acids, unnatural amino acids, and amino acid analogs). In some cases, R ⁶ Represents the side chains of amino acids found in naturally occurring proteins (e.g., side chains of Ala or A, cys or C, asp or D, glu or E, phe or F, gly or G, his or H, ile or I, lys or K, leu or L, met or M, asn or N, pro or P, gln or Q, arg or R, ser or S, thr or T, val or V, trp or W, tyr or Y). In certain embodiments, R ⁶ A side chain representing valine (Val); namely, R ⁶ Is isopropyl. In certain embodiments, R ⁶ Represents the side chain of alanine (Ala); namely, R ⁶ Is methyl. In certain embodiments, R ⁶ Represents the side chain of phenylalanine (Phe); namely, R ⁶ Is benzyl. In certain embodiments, R ⁶ A side chain representing lysine (Lys); namely, R ⁶ Is 4-amino-butyl.

In certain embodiments, k is an integer from 1 to 10. In certain embodiments, k is 1. In certain embodiments, k is 2. In certain embodiments, k is 3. In certain embodiments, k is 4. In certain embodiments, k is 5. In certain embodiments, k is 6. In certain embodiments, k is 7. In certain embodiments, k is 8. In certain embodiments, k is 9. In certain embodiments, k is 10.

In certain embodiments, the portion of formula (I) enclosed by brackets-subscript k represents one or more amino acids (e.g., a peptide). For example, as described above, a conjugate of the present disclosure can include a first enzymatically cleavable moiety, wherein the first enzymatically cleavable moiety is a peptide. As shown in formula (I), one or more of the amino acids may be a peptide comprising a first enzymatically cleavable moiety.

In certain embodiments, R ⁷ Is a second enzymatically cleavable moiety as described herein. For example, R ⁷ May comprise a glycoside selected from the group consisting of galactoside, glucoside, mannoside, fucoside, O-GlcNAc and O-GalNAc. In some cases, R ⁷ Comprises a galactoside. In some cases, R ⁷ Comprises a glucoside. In some cases, R ⁷ Comprises mannosides. In some cases, R ⁷ Comprises fucoside. In some cases, R ⁷ Comprising O-GlcNAc. In some cases, R ⁷ Comprising O-GalNAc.

In certain embodiments, L ¹ Is a first joint. Is suitable for L ¹ Is described in more detail below.

In certain embodiments, L ² Is a second joint. Is suitable for L ² Is described in more detail below.

In some embodiments, W ¹ Is a drug (or active agent). Drugs and active agents suitable for use in the conjugates and compounds described herein are described further in more detail below.

In some embodiments, W ² Is an antibody. Further description of antibodies useful in the conjugates of the invention are provided herein.

In certain embodiments, the conjugate of formula (I) comprises one or more linkers. The linker may be used to bind the coupling moiety to one or more moieties of interest and/or one or more polypeptides. In some embodiments, the linker binds the coupling moiety to a polypeptide or chemical entity, such as a drug. The linker can be bound (e.g., covalently bound) to the coupling moiety at any convenient location (e.g., as described herein). For example, the linker may link the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety to a drug (e.g., maytansine or auristatin). The hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moieties may be used to conjugate a linker (and thus a drug) to a polypeptide, such as an antibody. For example, the coupling moiety may be used to conjugate a linker (and thus a drug) to a modified amino acid residue of a polypeptide, such as the FGly residue of an antibody.

In certain embodiments, the linker comprises one or more linkers, such as a first linker L ¹ And a second joint L ² . Further, the linker can include one or more cleavable moieties (e.g., a first cleavable moiety and a second cleavable moiety) as described herein. In some cases, the linker comprises one or more linkers, e.g., a first linker L ¹ And a second joint L ² . For example, the linker may comprise a first cleavableA first linker (L) partially linked to a coupling moiety (e.g., a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety as described herein) ¹ ) And a second linker (L) connecting the first cleavable moiety to a chemical entity (a drug or active agent as described herein) ² ). Thus, the linker can comprise a first linker (L) that connects the first cleavable moiety to the antibody (e.g., via a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety as described herein) ¹ ) And a second linker (L) connecting the first cleavable moiety to a chemical entity (a drug or active agent as described herein) ² )。

For example, as shown in the above formula (I), L ¹ Attachment to W through a coupling moiety ² And thus W ² Indirectly bonded to the first linker L through a coupling moiety ¹ . As described above, W ² Is an antibody, thus L ¹ Attachment to an antibody via a coupling moiety, e.g. first linker L ¹ Indirectly binds to the antibody through a coupling moiety (e.g., through a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety as described herein). Furthermore, as shown in the above formula (I), L ¹ (indirectly) connected to L ² And L is ² To W ¹ . As described above, W ¹ Is a drug, and thus the second linker L ² Through the first joint L ¹ And a coupling moiety (e.g., a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety as described herein) to attach the drug to antibody W ² 。

Any convenient linker may be used for the first linker (L ¹ ) And a second joint (L) ² ). In certain embodiments, the first joint (L ¹ ) And a second joint (L) ² ) Each independently may include a group selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acylamino, alkylamide, substituted alkylamide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl . In certain embodiments, the first joint (L ¹ ) And a second joint (L) ² ) Each independently may include an alkyl group or a substituted alkyl group. In certain embodiments, the first joint (L ¹ ) And a second joint (L) ² ) Each independently may include an alkenyl or substituted alkenyl group. In certain embodiments, the first joint (L ¹ ) And a second joint (L) ² ) Each independently may include an alkynyl group or a substituted alkynyl group. In certain embodiments, the first joint (L ¹ ) And a second joint (L) ² ) Each independently may include an alkoxy group or a substituted alkoxy group. In certain embodiments, the first joint (L ¹ ) And a second joint (L) ² ) Each independently may include an amino group or a substituted amino group. In certain embodiments, the first joint (L ¹ ) And a second joint (L) ² ) Each independently may include a carboxyl or carboxyl ester group. In certain embodiments, the first joint (L ¹ ) And a second joint (L) ² ) Each independently may comprise an acylamino group. In certain embodiments, the first joint (L ¹ ) And a second joint (L) ² ) Each independently may comprise an alkylamide or a substituted alkylamide group. In certain embodiments, the first joint (L ¹ ) And a second joint (L) ² ) Each independently may comprise an aryl or substituted aryl group. In certain embodiments, the first joint (L ¹ ) And a second joint (L) ² ) Each independently may comprise a heteroaryl or substituted heteroaryl group. In certain embodiments, the first joint (L ¹ ) And a second joint (L) ² ) Each independently may include cycloalkyl or substituted cycloalkyl groups. In certain embodiments, the first joint (L ¹ ) And a second joint (L) ² ) Each independently may include a heterocyclyl or substituted heterocyclyl group.

In certain embodiments, the first joint (L ¹ ) And a second joint (L) ² ) Each independently may comprise a polymer. For example, the polymer may include polyalkylene glycols and derivatives thereof, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymers, polypropylene glycol homopolymers, ethylene glycol and propylene glycolCopolymers (e.g., wherein the homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohols, polyvinyl ethyl ethers, polyvinyl pyrrolidones, combinations thereof, and the like. In certain embodiments, the polymer is a polyalkylene glycol. In certain embodiments, the polymer is polyethylene glycol. Other linkers are also possible, as shown in the conjugates and compounds described in more detail below.

In some embodiments, L ¹ Is formed by- (L) ¹¹ ) _a -(L ¹² ) _b -(L ¹³ ) _c -(L ¹⁴ ) _d -a first linker as described, wherein L ¹¹ 、L ¹² 、L ¹³ And L ¹⁴ Each independently is a first linker subunit, and a, b, c, and d are each independently 0 or 1, wherein the sum of a, b, c, and d is 1 to 4.

In certain embodiments, the sum of a, b, c, and d is 1. In certain embodiments, the sum of a, b, c, and d is 2. In certain embodiments, the sum of a, b, c, and d is 3. In certain embodiments, the sum of a, b, c, and d is 4. In certain embodiments, a, b, c, and d are each 1. In certain embodiments, a, b, and c are each 1, and d is 0. In certain embodiments, a and b are each 1, and c and d are each 0. In certain embodiments, a is 1 and b, c, and d are each 0.

In certain embodiments, L ¹¹ To a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety (e.g., as shown in formula (I) above). In certain embodiments, L ¹² If present, to the first cleavable moiety. In certain embodiments, L ¹³ If present, to the first cleavable moiety. In certain embodiments, L ¹⁴ If present, to the first cleavable moiety.

At the first joint L ¹ Any convenient linker subunit may be used. Linker subunits of interest include, but are not limited to, polymeric units (e.g., polyethylene glycol, polyethylene, and polyacrylate), amino acid residues, carbohydrate-based polymers or carbohydrate residues and derivatives thereof, polynucleotides, alkyl groups, aryl groups, Heterocyclic groups, combinations thereof, and substituted forms thereof. In some embodiments, L ¹¹ 、L ¹² 、L ¹³ And L ¹⁴ Each (if present) comprises one or more groups independently selected from polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, and diamines (e.g., linker groups including alkylene diamines).

In some embodiments, L ¹¹ Comprising polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups or diamines, if present. In some embodiments, L ¹¹ Comprises polyethylene glycol. In some embodiments, L ¹¹ Comprising modified polyethylene glycol. In some embodiments, L ¹¹ Comprising amino acid residues. In some embodiments, L ¹¹ Comprising an alkyl group or a substituted alkyl group. In some embodiments, L ¹¹ Comprising an aryl group or a substituted aryl group. In some embodiments, L ¹¹ Comprising a diamine (e.g., a linker group comprising an alkylene diamine).

In some embodiments, L ¹² Comprising polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups or diamines, if present. In some embodiments, L ¹² Comprises polyethylene glycol. In some embodiments, L ¹² Comprising modified polyethylene glycol. In some embodiments, L ¹² Comprising amino acid residues. In some embodiments, L ¹² Comprising an alkyl group or a substituted alkyl group. In some embodiments, L ¹² Comprising an aryl group or a substituted aryl group. In some embodiments, L ¹² Comprising a diamine (e.g., a linker group comprising an alkylene diamine).

In some embodiments, L ¹³ Comprising polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups or diamines, if present. In some embodiments, L ¹³ Comprises polyethylene glycol. In some implementationsIn embodiments, L ¹³ Comprising modified polyethylene glycol. In some embodiments, L ¹³ Comprising amino acid residues. In some embodiments, L ¹³ Comprising an alkyl group or a substituted alkyl group. In some embodiments, L ¹³ Comprising an aryl group or a substituted aryl group. In some embodiments, L ¹³ Comprising a diamine (e.g., a linker group comprising an alkylene diamine).

In some embodiments, L ¹⁴ Comprising polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups or diamines, if present. In some embodiments, L ¹⁴ Comprises polyethylene glycol. In some embodiments, L ¹⁴ Comprising modified polyethylene glycol. In some embodiments, L ¹⁴ Comprising amino acid residues. In some embodiments, L ¹⁴ Comprising an alkyl group or a substituted alkyl group. In some embodiments, L ¹⁴ Comprising aryl or substituted aryl groups. In some embodiments, L ¹⁴ Comprising a diamine (e.g., a linker group comprising an alkylene diamine).

In some embodiments, L ¹ Is comprised of (L) ¹¹ ) _a -(L ¹² ) _b -(L ¹³ ) _c -(L ¹⁴ ) _d -a first linker, wherein:

-(L ¹¹ ) _a -is- (T) ¹ -V ¹ ) _a -；

-(L ¹² ) _b -is- (T) ² -V ² ) _b -；

-(L ¹³ ) _c -is- (T) ³ -V ³ ) _c -; and is also provided with

-(L ¹⁴ ) _d -is- (T) ⁴ -V ⁴ ) _d -，

Wherein T is ¹ 、T ² 、T ³ And T ⁴ (if present) is a tethering group;

V ¹ 、V ² 、V ³ and V ⁴ Covalent bonds or linking functional groups, if present; and is also provided with

a. b, c and d are each independently 0 or 1, wherein the sum of a, b, c and d is 1 to 4.

As described above, in certain embodiments, L ¹¹ To a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety (e.g., as shown in formula (I) above). Thus, in certain embodiments, T ¹ To a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety (e.g., as shown in formula (I) above). In certain embodiments, V ¹ Is attached to the first cleavable moiety. In certain embodiments, L ¹² If present, to the first cleavable moiety. Thus, in certain embodiments, T ² Attached to the first cleavable moiety, if present, or V ² If present, to the first cleavable moiety. In certain embodiments, L ¹³ If present, to the first cleavable moiety. Thus, in certain embodiments, T ³ Attached to the first cleavable moiety, if present, or V ³ If present, to the first cleavable moiety. In certain embodiments, L ¹⁴ If present, to the first cleavable moiety. Thus, in certain embodiments, T ⁴ Attached to the first cleavable moiety, if present, or V ⁴ If present, to the first cleavable moiety.

With respect to the linker group T ¹ 、T ² 、T ³ And T ⁴ Any convenient linker group may be used in the subject linker. In some embodiments, T ¹ 、T ² 、T ³ And T ⁴ Each comprising one or more groups independently selected from (C) ₁ -C ₁₂ ) Alkyl, substituted (C) ₁ -C ₁₂ ) Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) _w 、(PEG) _n 、(AA) _p 、-(CR ¹³ OH) _h -, 4-amino-piperidine (4 AP), acetal groups, disulfides, hydrazines and esters, wherein w is an integer from 1 to 20, n is an integer from 1 to 30, p is an integer from 1 to 20, and h is an integer from 1 to 12.

In certain embodiments, the tether group (e.g., T ¹ 、T ² 、T ³ And/or T ⁴ ) Comprises (C) ₁ -C ₁₂ ) Alkyl or substituted (C) ₁ -C ₁₂ ) An alkyl group. In certain embodiments, (C) ₁ -C ₁₂ ) Alkyl is a straight or branched alkyl group comprising 1 to 12 carbon atoms, for example 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some cases, (C) ₁ -C ₁₂ ) The alkyl group may be an alkyl group or a substituted alkyl group, for example C ₁ -C ₁₂ Alkyl, or C ₁ -C ₁₀ Alkyl, or C ₁ -C ₆ Alkyl, or C ₁ -C ₃ An alkyl group. In some cases, (C) ₁ -C ₁₂ ) Alkyl is C ₂ -an alkyl group. For example, (C) ₁ -C ₁₂ ) The alkyl group may be an alkylene group or a substituted alkylene group, for example C ₁ -C ₁₂ Alkylene, or C ₁ -C ₁₀ Alkylene, or C ₁ -C ₆ Alkylene, or C ₁ -C ₃ An alkylene group. In some cases, (C) ₁ -C ₁₂ ) Alkyl is C ₂ Alkylene (e.g., CH ₂ CH ₂ )。

In certain embodiments, substituted (C ₁ -C ₁₂ ) Alkyl is a straight or branched substituted alkyl group comprising 1 to 12 carbon atoms, for example 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some cases, substituted (C ₁ -C ₁₂ ) The alkyl group may be a substituted alkyl group, for example substituted C ₁ -C ₁₂ Alkyl, or substituted C ₁ -C ₁₀ Alkyl, or substituted C ₁ -C ₆ Alkyl, or substituted C ₁ -C ₃ An alkyl group. In some cases, substituted (C _l -C ₁₂ ) Alkyl is substituted C ₂ -an alkyl group. For example, substituted (C ₁ -C ₁₂ ) The alkyl group may be a substituted alkylene group, e.g. a substituted C ₁ -C ₁₂ Alkylene, or substituted C ₁ -C ₁₀ Alkylene, or substituted C ₁ -C ₆ Alkylene, or substituted C ₁ -C ₃ An alkylene group. In some cases, substituted (C ₁ -C ₁₂ ) Alkyl is substituted C ₂ -an alkylene group.

In certain embodiments, the tether group (e.g., T ¹ 、T ² 、T ³ And/or T ⁴ ) Including aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl. In some cases, the tether group (e.g., T ¹ 、T ² 、T ³ And/or T ⁴ ) Including aryl or substituted aryl. For example, the aryl group may be phenyl. In some cases, the substituted aryl is a substituted phenyl. The substituted phenyl groups may be substituted with one or more groups selected from (C ₁ -C ₁₂ ) Alkyl, substituted (C) ₁ -C ₁₂ ) Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl substituents. In some cases, the substituted aryl is a substituted phenyl, wherein the substituents include a second cleavable moiety (e.g., an enzymatically cleavable moiety, such as a glycoside) as described herein.

In some cases, the tether group (e.g., T ¹ 、T ² 、T ³ And/or T ⁴ ) Including heteroaryl or substituted heteroaryl. In some cases, the tether group (e.g., T ¹ 、T ² 、T ³ And/or T ⁴ ) Including cycloalkyl or substituted cycloalkyl. In some cases, the tether group (e.g., T ¹ 、T ² 、T ³ And/or T ⁴ ) Including heterocyclyl or substituted heterocyclyl. In some cases, the substituents on the substituted heteroaryl, substituted cycloalkyl, or substituted heterocyclyl include a second cleavable moiety (e.g., an enzymatically cleavable moiety, such as a glycoside) as described herein.

In certain embodiments, the tether group (e.g., T ¹ 、T ² 、T ³ And/or T ⁴ ) Comprising Ethylenediamine (EDA) moieties, e.gSuch as EDA-containing tethers. In certain embodiments, (EDA) _w Including one or more EDA moieties, for example where w is an integer from 1 to 50, for example from 1 to 40, 1 to 30, 1 to 20, 1 to 12 or 1 to 6, for example 1, 2, 3, 4, 5 or 6. The attached Ethylenediamine (EDA) moiety may optionally be substituted at one or more convenient positions with any convenient substituent, for example with an alkyl group, a substituted alkyl group, an acyl group, a substituted acyl group, an aryl group or a substituted aryl group. In certain embodiments, the EDA part is described by the following structure:

Wherein y is an integer from 1 to 6, alternatively 0 or 1, and each R ¹² Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, y is 1, 2, 3, 4, 5, or 6. In certain embodiments, y is 1 and r is 0. In certain embodiments, y is 1 and r is 1. In certain embodiments, y is 2 and r is 0. In certain embodiments, y is 2 and r is 1. In certain embodiments, each R ¹² Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In certain embodiments, any two adjacent R's of EDA ¹² The groups may be cyclic, for example forming a piperazinyl ring. In certain embodiments, y is 1 and two adjacent R ¹² The groups are alkyl groups which are cyclic linked to form a piperazinyl ring. In certain embodiments, y is 1 and adjacent R ¹² The group is selected from hydrogen, alkyl (e.g., methyl) and substituted alkyl (e.g., lower alkyl-OH, such as ethyl-OH or propyl-OH).

In certain embodiments, the tether group (e.g., T ¹ 、T ² 、T ³ And/or T ⁴ ) Comprising a 4-amino-piperidine (4 AP) moiety (also referred to herein as piperidine-4-amino, P4A). The 4AP moiety may be optionally substituted at one or more convenient positions with any convenient substituent, for example with an alkyl group, a substituted alkyl group, a polyethylene glycol moiety, an acyl group, a substituted acyl group, an aryl group or a substituted aryl group. In some embodiments, the 4AP portion is described by the following structure:

wherein R is ¹² Selected from the group consisting of hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety (e.g., polyethylene glycol or modified polyethylene glycol), alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R ¹² Is a polyethylene glycol moiety. In certain embodiments, R ¹² Is carboxyl modified polyethylene glycol.

In certain embodiments, R ¹² Comprising a polyethylene glycol moiety described by the formula: (PEG) _k It can be represented by the following structure:

wherein k is an integer from 1 to 20, such as 1 to 18, or 1 to 16, or 1 to 14, or 1 to 12, or 1 to 10, or 1 to 8, or 1 to 6, or 1 to 4, or 1 or 2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, k is 2. In certain embodiments, R ¹⁷ Selected from OH, COOH, or COOR, wherein R is selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R ¹⁷ Is COOH.

In certain embodiments, the tether group (e.g., T ¹ 、T ² 、T ³ And/or T ⁴ ) Comprises (PEG) _n Wherein (PEG) _n Is polyethylene glycol or modified polyethylene glycol linking unit. In certain embodiments, (PEG) _n Described by the following structure:

wherein n is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, n is 2. In some cases, n is 3. In some cases, n is 6. In some cases, n is 12.

In certain embodiments, the tether group (e.g., T ¹ 、T ² 、T ³ And/or T ⁴ ) Comprises (AA) _p Wherein AA is an amino acid residue. Any convenient amino acid may be used. Amino acids of interest include, but are not limited to, L-and D-amino acids, naturally occurring amino acids (e.g., any of 20 primary alpha-amino acids and beta-alanine), non-naturally occurring amino acids (e.g., amino acid analogs), e.g., non-naturally occurring alpha-amino acids or non-naturally occurring beta-amino acids, and the like. In certain embodiments, p is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, p is 1. In certain embodiments, p is 2.

In certain embodiments, the tether group (e.g., T ¹ 、T ² 、T ³ And/or T ⁴ ) Including those made of- (CR) ¹³ OH) _h -a moiety as described wherein h is 0, or n is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12 or 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In certain embodiments, h is 1. In certain embodiments, h is 2. In certain embodiments, R ¹³ Selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R ¹³ Is hydrogen. In certain embodiments, R ¹³ Is alkyl or substituted alkyl, e.g. C _1-6 Alkyl or C _1-6 Substituted alkyl, or C _1-4 Alkyl or C _1-4 Substituted alkyl or C _1-3 Alkyl or C _1-3 Substituted alkyl. In certain embodiments, R ¹³ Is alkenyl or substituted alkenyl, e.g. C _2-6 Alkenyl or C _2-6 Substituted alkenyl, or C _2-4 Alkenyl or C _2-4 Substituted alkenyl, or C _2-3 Alkenyl or C _2-3 Substituted alkenyl groups. In certain embodiments, R ¹³ Is alkynyl or substituted alkynyl. In certain embodiments, R ¹³ Is an alkoxy group or a substituted alkoxy group. In certain embodiments, R ¹³ Is an amino group or a substituted amino group. In certain embodiments, R ¹³ Is carboxyl or carboxyl ester. In certain embodiments, R ¹³ Is acyl or acyloxy. In certain embodiments, R ¹³ Is an acylamino group or an aminoacyl group. In certain embodiments, R ¹³ Is an alkylamide or a substituted alkylamide. In certain embodiments, R ¹³ Is sulfonyl. In certain embodiments, R ¹³ Is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ¹³ Is aryl or substituted aryl, e.g. C _5-8 Aryl or C _5-8 Substituted aryl groups, e.g. C ₅ Aryl or C ₅ Substituted aryl, or C ₆ Aryl or C ₆ Substituted aryl. In certain embodiments, R ¹³ Is heteroaryl or substituted heteroaryl, e.g. C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, e.g. C ₅ Heteroaryl or C ₅ Substituted heteroaryl, or C ₆ Heteroaryl or C ₆ Substituted heteroaryl groups. In certain embodiments, R ¹³ Cycloalkyl or substituted cycloalkyl, e.g. C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl radicals, e.g. C _3-6 Cycloalkyl or C _3-6 Substituted cycloalkyl, or C _3-5 Cycloalkyl or C _3-5 Substituted cycloalkyl groups. In certain embodiments, R ¹³ Is a heterocyclic group or a substituted heterocyclic group, e.g. C _3-8 Heterocyclyl or C _3-8 Substituted heterocyclic groups, e.g. C _3-6 Heterocyclyl or C _3-6 Substituted heterocyclyl, or C _3-5 Heterocyclyl or C _3-5 Substituted heterocyclyl groups.

In certain embodiments, R ¹³ Selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are as described above for R ¹⁵ Said method.

With respect to the linking functional group V ¹ 、V ² 、V ³ And V ⁴ At the first joint L ¹ Any convenient linking functionality may be used. Linking functionalities of interest include, but are not limited to, amino, carbonyl, amido, oxycarbonyl, carboxyl, sulfonyl, sulfoxide, sulfonylamino, sulfamoyl, thio, oxy, phosphate, phosphoramidate, phosphorothioate, and the like. In some embodiments, V ¹ 、V ² 、V ³ And V ⁴ Each independently selected from covalent bonds, -CO-, -NR ¹⁵ -、-NR ¹⁵ (CH ₂ ) _q -、-NR ¹⁵ (C ₆ H ₄ )-、-CONR ¹⁵ -、-NR ¹⁵ CO-、-C(O)O-、-OC(O)-、-O-、-S-、-S(O)-、-SO ₂ -、-SO ₂ NR ¹⁵ -、-NR ¹⁵ SO ₂ -sum of-P (O) OH-, wherein q is an integer from 1 to 6. In certain embodiments, q is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6). In certain embodiments, q is 1. In certain embodiments, q is 2.

In some embodiments, each R ¹⁵ Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

Each R ¹⁵ The various possibilities of which are described in more detail below. In certain embodiments, R ¹⁵ Is hydrogen. In certain embodiments, each R ¹⁵ Is hydrogen. In certain embodiments, R ¹⁵ Is alkyl or substituted alkyl, e.g. C _1-6 Alkyl or C _1-6 Substituted alkyl, or C _1-4 Alkyl or C _1-4 Substituted alkyl or C _1-3 Alkyl or C _1-3 Substituted alkyl. In certain embodiments, R ¹⁵ Is alkenyl or substituted alkenyl, e.g. C _2-6 Alkenyl or C _2-6 Substituted alkenyl, or C _2-4 Alkenyl or C _2-4 Substituted alkenyl, or C _2-3 Alkenyl or C _2-3 Substituted alkenyl groups. In certain embodiments, R ¹⁵ Is alkynyl or substituted alkynyl. In certain embodiments, R ¹⁵ Is an alkoxy group or a substituted alkoxy group. In certain embodiments, R ¹⁵ Is an amino group or a substituted amino group. In certain embodiments, R ¹⁵ Is carboxyl or carboxyl ester. In certain embodiments, R ¹⁵ Is acyl or acyloxy. In certain embodiments, R ¹⁵ Is an acylamino group or an aminoacyl group. In certain embodiments, R ¹⁵ Is an alkylamide or a substituted alkylamide. In certain embodiments, R ¹⁵ Is sulfonyl. In certain embodiments, R ¹⁵ Is thioalkoxy or substituted thioalkoxy. In some casesIn embodiments, R ¹⁵ Is aryl or substituted aryl, e.g. C _5-8 Aryl or C _5-8 Substituted aryl groups, e.g. C ₅ Aryl or C ₅ Substituted aryl, or C ₆ Aryl or C ₆ Substituted aryl. In certain embodiments, R ¹⁵ Is heteroaryl or substituted heteroaryl, e.g. C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, e.g. C ₅ Heteroaryl or C ₅ Substituted heteroaryl, or C ₆ Heteroaryl or C ₆ Substituted heteroaryl groups. In certain embodiments, R ¹⁵ Cycloalkyl or substituted cycloalkyl, e.g. C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl radicals, e.g. C _3-6 Cycloalkyl or C _3-6 Substituted cycloalkyl, or C _3-5 Cycloalkyl or C _3-5 Substituted cycloalkyl groups. In certain embodiments, R ¹⁵ Is a heterocyclic group or a substituted heterocyclic group, e.g. C _3-8 Heterocyclyl or C _3-8 Substituted heterocyclic groups, e.g. C _3-6 Heterocyclyl or C _3-6 Substituted heterocyclyl, or C _3-5 Heterocyclyl or C _3-5 Substituted heterocyclyl groups.

In certain embodiments, each R ¹⁵ Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In these embodiments, hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl substituents are as described above for R ¹⁵ Said method.

In certain embodiments, the tether group comprises an acetal group, a disulfide, hydrazine, or an ester. In some embodiments, the tether group comprises an acetal group. In some embodiments, the tether group comprises a disulfide. In some embodiments, the tether group comprises hydrazine. In some embodiments, the tether group comprises an ester.

As described above, in some embodiments, L ¹ Is comprised of (T) ¹ -V ¹ ) _a -(T ² -V ² ) _b -(T ³ -V ³ ) _c -(T ⁴ -V ⁴ ) _d -a first linker wherein a, b, c and d are each independently 0 or 1, wherein the sum of a, b, c and d is 1 to 4.

In some embodiments, at the first joint L ¹ In (a):

T ² 、T ³ and T ⁴ Each independently selected from (C) ₁ -C ₁₂ ) Alkyl, substituted (C) ₁ -C ₁₂ ) Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) _w 、(PEG) _n 、(AA) _p 、-(CR ¹³ OH) _h -, 4-amino-piperidine (4 AP), acetal groups, disulfides, hydrazines and esters; and is also provided with

V ¹ 、V ² 、V ³ And V ⁴ Each independently selected from covalent bonds, -CO-, -NR ¹⁵ -、-NR ¹⁵ (CH ₂ ) _q -、-NR ¹⁵ (C ₆ H ₄ )-、-CONR ¹⁵ -、-NR ¹⁵ CO-、-C(O)O-、-OC(O)-、-O-、-S-、-S(O)-、-SO ₂ -、-SO ₂ NR ¹⁵ -、-NR ¹⁵ SO ₂ -and-P (O) OH-, wherein q is an integer from 1 to 6;

wherein:

(PEG) _n is thatWherein n is an integer from 1 to 30;

EDA is an ethylenediamine moiety having the structure:

wherein y is an integer from 1 to 6 and r is 0 or 1;

4-amino-piperidine (4 AP) as

AA is an amino acid residue, wherein p is an integer from 1 to 20; and is also provided with

Each R ¹⁵ And R is ¹² Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl, wherein any two adjacent R ¹² The groups may be connected in a cyclic manner to form a piperazinyl ring; and is also provided with

In certain embodiments, T ¹ 、T ² 、T ³ And T ⁴ V (V) ¹ 、V ² 、V ³ And V ⁴ Selected from the following:

T ¹ is (C) ₁ -C ₁₂ ) Alkyl, and V ¹ is-CO-;

T ² is an amino acid analog, and V ² is-NH-;

T ³ is (PEG) _n And V is ³ is-CO-; and is also provided with

d is 0 (i.e., T ⁴ And V ⁴ Absence).

In certain embodiments, the left hand side of the linker structure is attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety, and the right hand side of the linker structure is attached to a first cleavable moiety. For example, where the first cleavable moiety comprises a peptide, the right hand side of the above-described linker structure may be attached to an amino acid of the peptide comprising the first cleavable moiety. In some cases, the carbonyl group on the right hand side of the above linker structure may form an amide bond with an amino acid of the peptide comprising the first cleavable moiety.

In some embodiments, L ² Is formed by- (L) ²¹ ) _e -(L ²² ) _f -(L ²³ ) _g -(L ²⁴ ) _h -a second linker as described, wherein L ²¹ 、L ²² 、L ²³ And L ²⁴ Each independently is a second linker subunit, and e, f, g and h are each independently 0 or 1, wherein the sum of e, f, g and h is 0 to 4.

In certain embodiments, the sum of e, f, g, and h is 0. In these cases, the second linker L is absent ² . In other words, when the sum of e, f, g and h is 0, then the second linker L ² Is a covalent bond. In certain embodiments, the sum of e, f, g, and h is 1. In certain embodiments, the sum of e, f, g, and h is 2. In certain embodiments, the sum of e, f, g, and h is 3. In certain embodiments, the sum of e, f, g, and h is 4. In certain embodiments, e, f, g, and h are each 1. In certain embodiments, e, f, and g are each 1, and h is 0. In certain embodiments, e and f are each 1, and g and h are each 0. In certain embodiments, e is 1 and f, g, and h are each 0.

In certain embodiments, L ²¹ To a drug (e.g., W as shown in formula (I) above ¹ ). In certain embodiments, L ²² And (if present) to the drug. In certain embodiments, L ²³ And (if present) to the drug. In certain embodiments, L ²⁴ And (if present) to the drug.

At the second joint L ² Any convenient linker subunit may be used. Linker subunits of interest include, but are not limited to, polymeric units (e.g., polyethylene glycol, polyethylene, and polyacrylate), amino acid residues, carbohydrate-based polymers or carbohydrate residues and derivatives thereof, polynucleotides, alkyl groups, aryl groups, heterocyclic groups, combinations thereof, and substituted versions thereof. In some embodiments, L ²¹ 、L ²² 、L ²³ And L ²⁴ Each (if present) comprises one or more groups independently selected from polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, and diamines (e.g., including the linker groups of alkylene diamines).

In some embodiments, L ²¹ Comprising polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups or diamines, if present. In some embodiments, L ²¹ Comprises polyethylene glycol. In some embodiments, L ²¹ Comprising modified polyethylene glycol. In some embodiments, L ²¹ Comprising amino acid residues. In some embodiments, L ²¹ Comprising an alkyl group or a substituted alkyl group. In some embodiments, L ²¹ Comprising an aryl group or a substituted aryl group. In some embodiments, L ²¹ Comprising a diamine (e.g., a linker group comprising an alkylene diamine).

In some embodiments, L ²² Comprising polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups or diamines, if present. In some embodiments, L ²² Comprises polyethylene glycol. In some embodiments, L ²² Comprising modified polyethylene glycol. In some embodiments, L ²² Comprising amino acid residues. In some embodiments, L ²² Comprising an alkyl group or a substituted alkyl group. In some embodiments, L ²² Comprising an aryl group or a substituted aryl group. In some embodiments, L ²² Comprising a diamine (e.g., a linker group comprising an alkylene diamine).

In some embodiments, L ²³ Comprising polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups or diamines, if present. In some embodiments, L ²³ Comprises polyethylene glycol. In some embodiments, L ²³ Comprising modified polyethylene glycol. In some embodiments, L ²³ Comprising amino acid residues. In some embodiments, L ²³ Comprising an alkyl group or a substituted alkyl group. In some embodiments, L ²³ Comprising an aryl group or a substituted aryl group. In some embodiments, L ²³ Comprising a diamine (e.g., a linker group comprising an alkylene diamine).

In some embodiments, L ²⁴ (if it isPresent) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups or diamines. In some embodiments, L ²⁴ Comprises polyethylene glycol. In some embodiments, L ²⁴ Comprising modified polyethylene glycol. In some embodiments, L ²⁴ Comprising amino acid residues. In some embodiments, L ²⁴ Comprising an alkyl group or a substituted alkyl group. In some embodiments, L ²⁴ Comprising an aryl group or a substituted aryl group. In some embodiments, L ²⁴ Comprising a diamine (e.g., a linker group comprising an alkylene diamine).

In some embodiments, L ² Is comprised of (L) ²¹ ) _e -(L ²² ) _f -(L ²³ ) _g -(L ²⁴ ) _h -a second linker, wherein:

-(L ²¹ ) _e -is- (T) ⁵ -V ⁵ ) _e -；

-(L ²² ) _f -is- (T) ⁶ -V ⁶ ) _f -；

-(L ²³ ) _g -is- (T) ⁷ -V ⁷ ) _g -; and is also provided with

-(L ²⁴ ) _h -is- (T) ⁸ -V ⁸ ) _h -，

Wherein T is ⁵ 、T ⁶ 、T ⁷ And T ⁸ (if present) is a tethering group;

V ⁵ 、V ⁶ 、V ⁷ and V ⁸ Covalent bonds or linking functional groups, if present; and is also provided with

e. f, g and h are each independently 0 or 1, wherein the sum of e, f, g and h is 0 to 4.

In certain embodiments, L ²¹ Is attached to the first cleavable moiety. Thus, in certain embodiments, T ⁵ Is attached to the first cleavable moiety. In certain embodiments, V ⁵ To a drug (e.g., W as shown in formula (I) above ¹ ). In certain embodiments, L ²² And (if present) to the drug. Thus (2)In certain embodiments, T6 (if present) is linked to a drug, or V ⁶ And (if present) to the drug. In certain embodiments, L ²³ And (if present) to the drug. Thus, in certain embodiments, T ⁷ Attached to the drug, if present, or V ⁷ And (if present) to the drug. In certain embodiments, L ²⁴ And (if present) to the drug. Thus, in certain embodiments, T ⁸ Attached to the drug, if present, or V ⁸ And (if present) to the drug.

With respect to the linker group T ⁵ 、T ⁶ 、T ⁷ And T ⁸ Any convenient linker group may be used in the subject linker. In some embodiments, T ⁵ 、T ⁶ 、T ⁷ And T ⁸ Each comprising one or more groups independently selected from (C) ₁ -C ₁₂ ) Alkyl, substituted (C) ₁ -C ₁₂ ) Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) _w 、(PEG) _n 、(AA) _p 、-(CR ¹³ OH) _h -, 4-amino-piperidine (4 AP), acetal groups, disulfides, hydrazines and esters, wherein w is an integer from 1 to 20, n is an integer from 1 to 30, p is an integer from 1 to 20, and h is an integer from 1 to 12.

In certain embodiments, the tether group (e.g., T ⁵ 、T ⁶ 、T ⁷ And/or T ⁸ ) Comprises (C) ₁ -C ₁₂ ) Alkyl or substituted (C) ₁ -C ₁₂ ) An alkyl group. In certain embodiments, (C) ₁ -C ₁₂ ) Alkyl is a straight or branched alkyl group comprising 1 to 12 carbon atoms, for example 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some cases, (C) ₁ -C ₁₂ ) The alkyl group may be an alkyl group or a substituted alkyl group, for example C ₁ -C ₁₂ Alkyl, or C ₁ -C ₁₀ Alkyl, or C ₁ -C ₆ Alkyl, or C ₁ -C ₃ An alkyl group. At the position ofIn some cases, (C) ₁ -C ₁₂ ) Alkyl is C ₂ -an alkyl group. For example, (C) ₁ -C ₁₂ ) The alkyl group may be an alkylene group or a substituted alkylene group, for example C ₁ -C ₁₂ Alkylene, or C ₁ -C ₁₀ Alkylene, or C ₁ -C ₆ Alkylene, or C ₁ -C ₃ An alkylene group. In some cases, (C) ₁ -C ₁₂ ) Alkyl is C ₂ Alkylene (e.g., CH ₂ CH ₂ ). In some cases, (C) ₁ -C ₁₂ ) Alkyl is C ₁ Alkylene (e.g., CH ₂ )。

In certain embodiments, substituted (C ₁ -C ₁₂ ) Alkyl is a straight or branched substituted alkyl group comprising 1 to 12 carbon atoms, for example 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some cases, substituted (C ₁ -C ₁₂ ) The alkyl group may be a substituted alkyl group, for example substituted C ₁ -C ₁₂ Alkyl, or substituted C ₁ -C ₁₀ Alkyl, or substituted C ₁ -C ₆ Alkyl, or substituted C ₁ -C ₃ An alkyl group. In some cases, substituted (C ₁ -C ₁₂ ) Alkyl is substituted C ₂ -an alkyl group. For example, substituted (C ₁ -C ₁₂ ) The alkyl group may be a substituted alkylene group, e.g. a substituted C ₁ -C ₁₂ Alkylene, or substituted C ₁ -C ₁₀ Alkylene, or substituted C ₁ -C ₆ Alkylene, or substituted C ₁ -C ₃ An alkylene group. In some cases, substituted (C ₁ -C ₁₂ ) Alkyl is substituted C ₂ -an alkylene group. In some cases, substituted (C ₁ -C ₁₂ ) Alkyl is substituted C ₁ -an alkylene group.

In certain embodiments, the tether group (e.g., T ⁵ 、T ⁶ 、T ⁷ And/or T ⁸ ) Comprising aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl or substitutedHeterocyclic groups of (a). In some cases, the tether group includes an aryl group or a substituted aryl group. For example, aryl may be phenyl or substituted phenyl. In some cases, the tether group (e.g., T ⁵ 、T ⁶ 、T ⁷ And/or T ⁸ ) Including heteroaryl or substituted heteroaryl. In some cases, the tether group (e.g., T ⁵ 、T ⁶ 、T ⁷ And/or T ⁸ ) Including cycloalkyl or substituted cycloalkyl. In some cases, the tether group (e.g., T ⁵ 、T ⁶ 、T ⁷ And/or T ⁸ ) Including heterocyclyl or substituted heterocyclyl.

In certain embodiments, the tether group (e.g., T ⁵ 、T ⁶ 、T ⁷ And/or T ⁸ ) Including Ethylenediamine (EDA) moieties, for example, as described above, such as those described by the following structure:

wherein y is an integer from 1 to 6, alternatively 0 or 1, and each R ¹² Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, y is 1, 2, 3, 4, 5, or 6. In certain embodiments, y is 1 and r is 0. In certain embodiments, y is 1 and r is 1. In certain embodiments, y is 2 and r is 0. In certain embodiments, y is 2 and r is 1. In certain embodiments, each R ¹² Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In certain embodiments, any two adjacent R's of EDA ¹² The groups may be cyclic, e.g. formingA piperazinyl ring. In certain embodiments, y is 1 and two adjacent R ¹² The groups are alkyl groups which are cyclic linked to form a piperazinyl ring. In certain embodiments, y is 1 and adjacent R ¹² The group is selected from hydrogen, alkyl (e.g., methyl) and substituted alkyl (e.g., lower alkyl-OH, such as ethyl-OH or propyl-OH).

In certain embodiments, the tether group (e.g., T ⁵ 、T ⁶ 、T ⁷ And/or T ⁸ ) Including a 4-amino-piperidine (4 AP) moiety as described above, such as the 4AP moiety described by the structure:

wherein k is an integer from 1 to 20, such as 1 to 18, or 1 to 16, or 1 to 14, or 1 to 12, or 1 to 10, or 1 to 8, or 1 to 6, or 1 to 4, or 1 or 2, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14. 15, 16, 17, 18, 19 or 20. In some cases, k is 2. In certain embodiments, R ¹⁷ Selected from OH, COOH, or COOR, wherein R is selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R ¹⁷ Is COOH.

In certain embodiments, the tether group (e.g., T ⁵ 、T ⁶ 、T ⁷ And/or T ⁸ ) Comprising a polyethylene glycol moiety (PEG) as described above _n For example, by the following structure (PEG) _n Part (c):

In certain embodiments, the tether group (e.g., T ⁵ 、T ⁶ 、T ⁷ And/or T ⁸ ) Comprises (AA) _p Wherein AA is an amino acid residue. Any convenient amino acid may be used. Amino acids of interest include, but are not limited to, L-and D-amino acids, naturally occurring amino acids (e.g., any of 20 primary alpha-amino acids and beta-alanine), non-naturally occurring amino acids (e.g., amino acid analogs), e.g., non-naturally occurring alpha-amino acids or non-naturally occurring beta-amino acids, and the like. In certain embodiments, p is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, p is 1. In certain embodiments, p is 2.

In certain embodiments, the tether group (e.g., T ⁵ 、T ⁶ 、T ⁷ And/or T ⁸ ) Including those made of- (CR) ¹³ OH) _h -a moiety as described wherein h is 0, or n is an integer from 1 to 50, such as 1 to 40, 1 to 30, 1 to 20, 1 to 12 or 1 to 6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In certain embodiments, h is 1. In certain embodiments, h is 2. In certain embodiments, R ¹³ Selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R ¹³ Is hydrogen. In certain embodiments, R ¹³ Is alkyl or substituted alkyl, e.g. C _1-6 Alkyl or C _1-6 Substituted alkyl, or C _1-4 Alkyl or C _1-4 Substituted alkyl or C _1-3 Alkyl or C _1-3 Substituted alkyl. In certain embodiments, R ¹³ Is alkenyl or substituted alkenyl, e.g. C _2-6 Alkenyl or C _2-6 Substituted alkenyl, or C _2-4 Alkenyl or C _2-4 Substituted alkenyl, or C _2-3 Alkenyl or C _2-3 Substituted alkenyl groups. In certain embodiments, R ¹³ Is alkynyl or substituted alkynyl. In certain embodiments, R ¹³ Is an alkoxy group or a substituted alkoxy group. In certain embodiments, R ¹³ Is an amino group or a substituted amino group. In certain embodiments, R ¹³ Is carboxyl or carboxyl ester. In certain embodiments, R ¹³ Is acyl or acyloxy. In certain embodiments, R ¹³ Is an acylamino group or an aminoacyl group. In certain embodiments, R ¹³ Is an alkylamide or a substituted alkylamide. In certain embodiments, R ¹³ Is sulfonyl. In certain embodiments, R ¹³ Is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ¹³ Is an aryl group or a substituted aryl group,for example C _5-8 Aryl or C _5-8 Substituted aryl groups, e.g. C ₅ Aryl or C ₅ Substituted aryl, or C ₆ Aryl or C ₆ Substituted aryl. In certain embodiments, R ¹³ Is heteroaryl or substituted heteroaryl, e.g. C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, e.g. C ₅ Heteroaryl or C ₅ Substituted heteroaryl, or C ₆ Heteroaryl or C ₆ Substituted heteroaryl groups. In certain embodiments, R ¹³ Cycloalkyl or substituted cycloalkyl, e.g. C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl radicals, e.g. C _3-6 Cycloalkyl or C _3-6 Substituted cycloalkyl, or C _3-5 Cycloalkyl or C _3-5 Substituted cycloalkyl groups. In certain embodiments, R ¹³ Is a heterocyclic group or a substituted heterocyclic group, e.g. C _3-8 Heterocyclyl or C _3-8 Substituted heterocyclic groups, e.g. C _3-6 Heterocyclyl or C _3-6 Substituted heterocyclyl, or C _3-5 Heterocyclyl or C _3-5 Substituted heterocyclyl groups.

With respect to the linking functional group V ⁵ 、V ⁶ 、V ⁷ And V ⁸ At the second joint L ² Any convenient linking functionality may be used. Linking functionalities of interest include, but are not limited to, amino, carbonyl, amido, oxycarbonyl, carboxyl, sulfonyl, sulfoxide, sulfonylamino, sulfamoyl, thio, oxy, phosphate, phosphoramidate, phosphorothioate, and the like. In some embodiments, V ⁵ 、V ⁶ 、V ⁷ And V ⁸ Each independently selected from covalent bonds, -CO-, -NR ¹⁵ -、-NR ¹⁵ (CH ₂ ) _q -、-NR ¹⁵ (C ₆ H ₄ )-、-CONR ¹⁵ -、-NR ¹⁵ CO-、-C(O)O-、-OC(O)-、-O-、-S-、-S(O)-、-SO ₂ -、-S0 ₂ NR ¹⁵ -、-NR ¹⁵ SO ₂ -and-P (O) OH-, wherein q is an integer from 1 to 6. In certain embodiments, q is an integer from 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6). In certain embodiments, q is 1. In certain embodiments, q is 2.

Each R ¹⁵ The various possibilities of which are described in more detail below. In certain embodiments, R ¹⁵ Is hydrogen. In certain embodiments, each R ¹⁵ Is hydrogen. In certain embodiments, R ¹⁵ Is alkyl or substituted alkyl, e.g. C _1-6 Alkyl or C _1-6 Substituted alkyl, or C _1-4 Alkyl or C _1-4 Substituted alkyl or C _1-3 Alkyl or C _1-3 Substituted alkyl. In certain embodiments, R ¹⁵ Is alkenyl or substituted alkenyl, e.g. C _2-6 Alkenyl or C _2-6 Substituted alkenyl, or C _2-4 Alkenyl or C _2-4 Substituted alkenyl, or C _2-3 Alkenyl or C _2-3 Substituted alkenyl groups. In certain embodiments, R ¹⁵ Is alkynyl or substituted alkynyl. In certain embodiments, R ¹⁵ Is an alkoxy group or a substituted alkoxy group. In certain embodiments, R ¹⁵ Is an amino group or a substituted amino group. In certain embodiments, R ¹⁵ Is carboxyl or carboxyl ester. In certain embodiments, R ¹⁵ Is acyl or acyloxy. In certain embodiments, R ¹⁵ Is an acylamino group or an aminoacyl group. In certain embodiments, R ¹⁵ Is an alkylamide or a substituted alkylamide. In certain embodiments, R ¹⁵ Is sulfonyl. In certain embodiments, R ¹⁵ Is thioalkoxyA group or a substituted thioalkoxy group. In certain embodiments, R ¹⁵ Is aryl or substituted aryl, e.g. C _5-8 Aryl or C _5-8 Substituted aryl groups, e.g. C ₅ Aryl or C ₅ Substituted aryl, or C ₆ Aryl or C ₆ Substituted aryl. In certain embodiments, R ¹⁵ Is heteroaryl or substituted heteroaryl, e.g. C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, e.g. C ₅ Heteroaryl or C ₅ Substituted heteroaryl, or C ₆ Heteroaryl or C ₆ Substituted heteroaryl groups. In certain embodiments, R ¹⁵ Cycloalkyl or substituted cycloalkyl, e.g. C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl radicals, e.g. C _3-6 Cycloalkyl or C _3-6 Substituted cycloalkyl, or C _3-5 Cycloalkyl or C _3-5 Substituted cycloalkyl groups. In certain embodiments, R ¹⁵ Is a heterocyclic group or a substituted heterocyclic group, e.g. C _3-8 Heterocyclyl or C _3-8 Substituted heterocyclic groups, e.g. C _3-6 Heterocyclyl or C _3-6 Substituted heterocyclyl, or C _3-5 Heterocyclyl or C _3-5 Substituted heterocyclyl groups.

As described above, in some embodiments, L ² Is comprised of (T) ⁵ -V ⁵ ) _e -(T ⁶ -V ⁶ ) _f -(T ⁷ -V ⁷ ) _g -(T ⁸ -V ⁸ ) _h -a second linker wherein e, f, g and h are each independently 0 or 1, wherein the sum of e, f, g and h is 0 to 4.

In some embodiments, at the second joint L ² In (a):

T ⁵ 、T ⁶ 、T ⁷ and T ⁸ Each independently selected from (C) ₁ -C ₁₂ ) Alkyl, substituted (C) ₁ -C ₁₂ ) Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl or substituted heterocyclyl, (EDA) _w 、(PEG) _n 、(AA) _p 、-(CR ¹³ OH) _h -, 4-amino-piperidine (4 AP), acetal groups, disulfides, hydrazines and esters; and is also provided with

V ⁵ 、V ⁶ 、V ⁷ And V ⁸ Each independently selected from covalent bonds, -CO-, -NR ¹⁵ -、-NR ¹⁵ (CH ₂ ) _q -、-NR ¹⁵ (C ₆ H ₄ )-、-CONR ¹⁵ -、-NR ¹⁵ CO-、-C(O)O-、-OC(O)-、-O-、-S-、-S(O)-、-SO ₂ -、-SO ₂ NR ¹⁵ -、-NR ¹⁵ SO ₂ -and-P (O) OH-, wherein q is an integer from 1 to 6;

wherein:

(PEG) _n is thatWherein n is an integer from 1 to 30;

EDA is an ethylenediamine moiety having the structure:

wherein y is an integer from 1 to 6 and r is 0 or 1;

4-amino-piperidine (4 AP) as

AA is an amino acid residue, wherein p is an integer from 1 to 20;

In certain embodiments, T ⁵ 、T ⁶ 、T ⁷ And T ⁸ V (V) ⁵ 、V ⁶ 、V ⁷ And V ⁸ Absent (i.e., the sum of e, f, g, and h is 0).

In certain embodiments, T ⁵ 、T ⁶ 、T ⁷ And T ⁸ V (V) ⁵ 、V ⁶ 、V ⁷ And V ⁸ Selected from the following:

T ⁵ is a covalent bond, and V ⁵ is-C (O) -;

f is 0 (i.e., T ⁶ And V ⁶ Absence of presence);

g is 0 (i.e., T ⁷ And V ⁷ Absence of presence); and is also provided with

h is 0 (i.e., T ⁸ And V ⁸ Absence).

T ⁵ is a covalent bond, and V ⁵ is-CONR ¹⁵ -；

T ⁶ Is alkyl, and V ⁶ is-CO-;

g is 0 (i.e., T ⁷ And V ⁷ Absence of presence); and is also provided with

h is 0 (i.e., T ⁸ And V ⁸ Absence).

In some embodiments, the left hand side of the linker structure is attached to the first cleavable moiety and the right hand side of the linker structure is attached to the drug.

Any of the chemical entities, linkers, and coupling moieties listed in the structures above may be suitable for use with the subject compounds and conjugates.

Additional disclosures relating to hydrazino-indolyl and hydrazino-pyrrolo-pyridinyl compounds and methods of making conjugates are found in U.S. patent No. 9,310,374 and U.S. patent No. 9,493,413, the disclosures of each of which are incorporated herein by reference.

Compounds for preparing conjugates

The present disclosure provides hydrazino-indolyl and hydrazino-pyrrolo-pyridinyl compounds useful for preparing conjugates described herein. In certain embodiments, the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl compounds may be a coupling moiety for conjugating an antibody to a drug or active agent. For example, the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl compounds may be conjugated to antibodies or to drugs, thereby indirectly binding the antibodies and drugs together.

In certain embodiments, the compound comprises a cleavable linker for linking the antibody to the drug, wherein the cleavable linker comprises a first enzymatically cleavable moiety and a second enzymatically cleavable moiety, the second enzymatically cleavable moiety comprising a glycoside selected from the group consisting of: galactoside, glucoside, mannoside, fucoside, O-GlcNAc and O-GalNAc.

In certain embodiments, the compound is a compound of formula (II):

wherein the method comprises the steps of

Z is CR ⁴ Or N;

x is O or NR ⁴ ；

k is an integer from 1 to 10;

R ⁷ is a second enzymatically cleavable moiety;

L ¹ is a first joint;

L ² is a second joint; and is also provided with

W ¹ Is a medicine.

In some cases, k is 2 and the compound is a compound of formula (IIa):

for example, the compound may be a compound of formula (IIb):

in some cases, the compound may be a compound of formula (IIc):

in some cases, the compound may be a compound of formula (IId):

in some cases, the compound may be a compound of formula (IIe):

in some cases, the compound may be a compound of formula (IIf):

in some cases, the compound may be a compound of formula (IIg):

substituents associated with conjugates of formula (II) are described herein. Mention of formula (II) is also intended to encompass formulae (IIa), (IIb), (IIc), (IId), (IIe), (IIf) and (IIg).

Regarding the compound of formula (II), substituent Z, R ² 、R ³ 、R ⁴ 、R ⁵ 、R ⁶ 、R ⁷ 、L ¹ 、L ² And W is ¹ As described above for the conjugates of formula (I). Similarly, with respect to the first linker L of formula (II) ¹ And a second joint L ² ，T ¹ 、T ² 、T ³ 、T ⁴ 、V ¹ 、V ² 、V ³ And V ⁴ And T ⁵ 、T ⁶ 、T ⁷ 、T ⁸ 、V ⁵ 、V ⁶ 、V ⁷ And V ⁸ The substituents are as described above for the conjugates of formula (I).

In certain embodiments, the compound has the following structure:

antibodies to

As described above, the subject conjugates can comprise an antibody as substituent W2, wherein the antibody has been modified to include a 2-formylglycine (FGly) residue. As used herein, amino acids may be referred to by their standard names, their standard three-letter abbreviations, and/or their standard one-letter abbreviations, for example: alanine or Ala or a; cysteine or Cys or C; aspartic acid or Asp or D; glutamic acid or Glu or E; phenylalanine or Phe or F; glycine or Gly or G; histidine or His or H; isoleucine or Ile or I; lysine or Lys or K; leucine or Leu or L; methionine or Met or M; asparagine or Asn or N; proline or Pro or P; glutamine or gin or Q; arginine or Arg or R; serine or Ser or S; threonine or Thr or T; valine or Val or V; tryptophan or Trp or W; and tyrosine or Tyr or Y.

In certain embodiments, the amino acid sequence of the antibody is modified to include a sulfatase motif comprising serine or cysteine residues capable of being converted (oxidized) to 2-formylglycine (FGly) residues by the action of Formylglycine Generating Enzymes (FGEs) in vivo (e.g., upon translation of an aldehyde-tagged protein in a cell) or in vitro (e.g., by contacting the aldehyde-tagged protein with FGEs in a cell-free system). Such sulfatase motif may also be referred to herein as FGE-modification site.

Sulfatase motif

The minimum sulfatase motif of an aldehyde tag is typically 5 or 6 amino acid residues in length, typically no more than 6 amino acid residues in length. Sulfatase motifs provided in Ig polypeptides are at least 5 or 6 amino acid residues in length, and may be, for example, 5 to 16, 6 to 16, 5 to 15, 6 to 15, 5 to 14, 6 to 14, 5 to 13, 6 to 13, 5 to 12, 6 to 12, 5 to 11, 6 to 11, 5 to 10, 6 to 10, 5 to 9, 6 to 9, 5 to 8, or 6 to 8 amino acid residues in length to define sulfatase motifs of less than 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 amino acid residues in length.

In certain embodiments, polypeptides of interest include polypeptides in which one or more amino acid residues, such as 2 or more, or 3 or more, or 4 or more, or 5 or more, or 6 or more, or 7 or more, or 8 or more, or 9 or more, or 10 or more, or 11 or more, or 12 or more, or 13 or more, or 14 or more, or 15 or more, or 16 or more, or 17 or more, or 18 or more, or 19 or more, or 20 or more amino acid residues, have been inserted, deleted, substituted (substituted) relative to the native amino acid sequence to provide a sequence of a sulfatase motif in the polypeptide. In certain embodiments, a polypeptide comprises modifications (insertions, additions, deletions, and/or substitutions/substitutions) of less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues of the amino acid sequence relative to the native amino acid sequence of the polypeptide. When the native amino acid sequence of a polypeptide (e.g., an antibody) contains one or more residues of a desired sulfatase motif, the total number of modifications of the residues may be reduced, e.g., by site-specific modification (insertion, addition, deletion, substitution/substitution) of amino acid residues flanking the native amino acid residue to provide the sequence of the desired sulfatase motif. In certain embodiments, the degree of modification of the native amino acid sequence of the target antibody is minimized in order to minimize the number of amino acid residues inserted, deleted, substituted (substituted) or added (e.g., to the N or C-terminus). Minimizing the extent of amino acid sequence modification of a target antibody may minimize the impact of such modification on antibody function and/or structure.

It should be noted that while aldehyde tags of particular interest are those comprising at least one minimal sulfatase motif (also referred to as a "consensus sulfatase motif"), it will be readily understood that longer aldehyde tags are also contemplated and encompassed by the present disclosure and are useful in the compositions and methods of the present disclosure. Thus, the aldehyde tag may comprise a minimal sulfatase motif of 5 or 6 residues, or may be longer and comprise a minimal sulfatase motif, which may be flanked on the N-and/or C-terminal side of the motif by additional amino acid residues. Aldehyde tags of, for example, 5 or 6 amino acid residues are contemplated, as well as longer amino acid sequences of more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues.

The aldehyde tag may be present at or near the C-terminus of the Ig heavy chain; for example, an aldehyde tag may be present within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of the C-terminal end of a naturally wild-type Ig heavy chain. Aldehyde tags may be present in the CH1 domain of Ig heavy chains. The aldehyde tag may be present in the CH2 domain of the Ig heavy chain. The aldehyde tag may be present in the CH3 domain of the Ig heavy chain. The aldehyde tag may be present in an Ig light chain constant region, such as a kappa light chain constant region or a lambda light chain constant region.

In certain embodiments, the sulfatase motif used may be described by the formula:

X ¹ Z ¹⁰ X ² Z ²⁰ X ³ Z ³⁰ (I′)

wherein the method comprises the steps of

Z ¹⁰ Is cysteine or serine (which may also be represented by (C/S));

Z ²⁰ is a proline or alanine residue (which may also be represented by (P/a));

Z ³⁰ is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), e.g., lysine), or an aliphatic amino acid (alanine (a), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I);

X ¹ is present or absent and, when present, can be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., not an aromatic amino acid or a charged amino acid), such as L, M, V, S or T, such as L, M, S or V, provided that X is present when the sulfatase motif is at the N-terminus of the target polypeptide ¹ The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

X ² And X ³ May independently be any amino acid, but is typically an aliphatic amino acid, a polar uncharged amino acid, or a sulfur-containing amino acid (i.e., other than an aromatic amino acid or a charged amino acid), such as S, T, A, V, G or C, e.g., S, T, A, V or G.

The amino acid sequence of the antibody heavy and/or light chain may be modified to provide formula X ¹ Z ¹⁰ X ² Z ²⁰ X ³ Z ³⁰ At least 5 amino acids in the sequence of (2), wherein

Z ¹⁰ Is cysteine or serine;

Z ²⁰ is a proline or alanine residue;

Z ³⁰ is an aliphatic amino acid or a basic amino acid;

X ¹ presence or absence, and when present is any amino acid, provided that when the heterologous sulfatase motif is at the N-terminus of the polypeptide, X is present ¹ ；

X ² And X ³ Each independently is any amino acid.

The sulfatase motif is typically selected so as to be able to be transformed by a selected FGE, e.g., FGE present in a host cell expressing the aldehyde-labeled polypeptide or FGE contacted with the aldehyde-labeled polypeptide in a cell-free in vitro method.

For example, when the FGE is a eukaryotic FGE (e.g., mammalian FGE, including human FGE), the sulfatase motif may have the formula:

X ¹ CX ² pX ³ Z ³⁰ (I″)

wherein the method comprises the steps of

X ¹ May be present or absent and, when present, may be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., not an aromatic amino acid or a charged amino acid), such as L, M, S or V, provided that when the sulfatase motif is at the N-terminus of the target polypeptide, X is present ¹ ；

X ² And X ³ Independently can be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., not an aromatic amino acid or a charged amino acid), such as S, T, A, V, G or C, such as S, T, A, V or G; and is also provided with

Z ³⁰ Is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), e.g., lysine), or an aliphatic amino acid (alanine (a), glycine (G), leucine (L), valine (V), isoleucine (I), or proline (P), e.g., A, G, L, V, or I).

Specific examples of sulfatase motifs include LCTPSR (SEQ ID NO: MCTPSR (SEQ ID NO: /), VCTPSR (SEQ ID NO: /), LCPSR (SEQ ID NO: /), LCAPSR (SEQ ID NO: /), LCVPSR (SEQ ID NO: /), LCGPSR (SEQ ID NO: /), ICTPAR (SEQ ID NO: /), LCTPSK (SEQ ID NO: /), MCTASK (SEQ ID NO: /), VCTPSK (SEQ ID NO: /), LCPSK (SEQ ID NO: /), LCAPSK (SEQ ID NO: /), LCVPSK (SEQ ID NO://), LCGPSK (SEQ ID NO://), LCTPSA (SEQ ID NO://), ICTPAA (SEQ ID NO://), LCPSA (SEQ ID NO: /), VCTPRD (SEQ ID NO: /), LCTPSA (SEQ ID NO: /), LCAPPSK (SEQ ID NO://), LCTPSA (SEQ ID NO: /), and LCGPSA (SEQ ID NO: /).

Containing FGly sequences

Serine or cysteine in the sulfatase motif is modified to FGly when FGE is acted upon the modified antibody heavy and/or light chain. Thus, the FGly-containing sulfatase motif may have the formula:

X ¹ (FGly)X ² Z ²⁰ X ³ Z ³⁰ (I″′)

Wherein the method comprises the steps of

FGly is a formylglycine residue;

Z ³⁰ is a basic amino acid (e.g., arginine (R), and may be lysine (K) or histidine (H), typically lysine), or an aliphatic amino acid (alanine (a), glycine (G), leucine (L), valine (V), isoleucine (I) or proline (P), e.g., A, G, L, V, or I;

X ¹ may be present or absent and, when present, may be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., not an aromatic amino acid or a charged amino acid), such as L, M, V, S or T, such as L, M or V, provided that X is present when the sulfatase motif is at the N-terminus of the target polypeptide ¹ The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

X ² And X ³ Independently, any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., not an aromatic amino acid or a charged amino acid), such as S, T, A, V, G or C, such as S, T, A, V or G.

As described above, modified polypeptides containing FGly residues can be conjugated to a drug (e.g., maytansinoids) by reaction of FGly with the drug (e.g., a drug containing a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety, as described above) to produce a sulfatase motif containing FGly'. The term "FGly" as used herein refers to a modified amino acid residue of a sulfatase motif, such as maytansine or auristatin, coupled to a drug. Thus, the FGly' containing sulfatase motif may have the formula:

X ¹ (FGly′)X ² Z ²⁰ X ³ Z ³⁰ (II)

Wherein the method comprises the steps of

FGly' is a modified amino acid residue of formula (I);

Modification site

As described above, the amino acid sequence of the antibody is modified to include a sulfatase motif comprising serine or cysteine residues that are capable of being converted (oxidized) to FGly residues by the action of FGE, either in vivo (e.g., when the aldehyde-tagged protein is translated in a cell) or in vitro (e.g., by contacting the aldehyde-tagged protein with FGE in a cell-free system). Antibodies for use in producing conjugates of the present disclosure include at least an Ig constant region, such as an Ig heavy chain constant region (e.g., at least a CH1 domain, at least CH1 and CH2 domains, CH1, CH2 and CH3 domains, or CH1, CH2, CH3 and CH4 domains), or an Ig light chain constant region. Such Ig polypeptides are referred to herein as "target Ig polypeptides" or "target antibodies.

The site of introduction of the sulfatase motif in the antibody may be any convenient site. As described above, in some cases, the degree of modification of the native amino acid sequence of the target polypeptide is minimized in order to minimize the number of amino acid residues inserted, deleted, substituted (substituted), and/or added (e.g., to the N-or C-terminus). Minimizing the extent of amino acid sequence modification of a target antibody may minimize the impact of such modification on antibody function and/or structure.

Antibody heavy chain constant regions may include Ig constant regions of any heavy chain isotype, non-naturally occurring Ig heavy chain constant regions (including consensus Ig heavy chain constant regions). The Ig constant region can be modified to include an aldehyde tag, wherein the aldehyde tag is present in or adjacent to a solvent accessible loop region of the Ig constant region. Ig constant regions can be modified by insertion and/or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids or more than 16 amino acids to provide the amino acid sequences of sulfatase motifs as described above.

In some cases, the aldehyde-labeled antibody comprises an aldehyde-labeled Ig heavy chain constant region (e.g., at least a CH1 domain, at least CH1 and CH2 domains, CH1, CH2 and CH3 domains, or CH1, CH2, CH3 and CH4 domains). The aldehyde-labeled Ig heavy chain constant region may comprise a heavy chain constant region sequence of a heavy chain of the IgA, igM, igD, igE, igG, igG2, igG3 or IgG4 isotype, or any allotype thereof, such as a human heavy chain constant region sequence or a mouse heavy chain constant region sequence, a hybrid heavy chain constant region, a synthetic heavy chain constant region or a consensus heavy chain constant region sequence, etc., which is modified to include at least one sulfatase motif that may be modified by FGE to produce FGly modified Ig polypeptides. Allotypic variants of Ig heavy chains are known in the art. See, e.g., jefferis and Lefranc (2009) MAbs 1:4.

In some cases, the aldehyde-labeled antibody comprises an aldehyde-labeled Ig light chain constant region. The aldehyde-labeled Ig light chain constant region may include a kappa light chain, a lambda light chain constant region sequence, such as a human kappa or lambda light chain constant region, a hybrid light chain constant region, a synthetic light chain constant region, or a consensus light chain constant region sequence, or the like, modified to include at least one sulfatase motif that may be modified by FGE to produce FGly modified antibodies. Exemplary constant regions include human γ1 and γ3 regions. In addition to the sulfatase motif, the modified constant region can have a wild-type amino acid sequence, or it can have an amino acid sequence that is at least 70% identical (e.g., at least 80%, at least 90%, or at least 95% identical) to the wild-type amino acid sequence.

In some embodiments, the sulfatase motif is located at a position other than the C-terminus of the Ig polypeptide heavy chain. As described above, the isolated aldehyde-labeled antibody may comprise a heavy chain constant region modified to include a sulfatase motif as described above, wherein the sulfatase motif is in or adjacent to a surface accessible loop region of the heavy chain constant region of the antibody.

Sulfatase motifs may be provided within or adjacent to one or more of these amino acid sequences at such modification sites of an Ig heavy chain. For example, ig heavy chain polypeptides may be modified at one or more of these amino acid sequences (e.g., wherein the modification includes one or more amino acid residue insertions, deletions, and/or substitutions) to provide a sulfatase motif adjacent and N-terminal and/or adjacent and C-terminal to these modification sites. Alternatively or additionally, an Ig heavy chain polypeptide can be modified at one or more of these amino acid sequences (e.g., wherein the modification includes one or more amino acid residue insertions, deletions, and/or substitutions) to provide a sulfatase motif between any two residues of the Ig heavy chain modification site. In some embodiments, an Ig heavy chain polypeptide can be modified to include two motifs that can be adjacent to each other or can be separated by one, two, three, four, or more (e.g., about 1 to about 25, about 25 to about 50, or about 50 to about 100 or more) amino acids. Alternatively or additionally, when the natural amino acid sequence provides one or more amino acid residues of a sulfatase motif sequence, selected amino acid residues of the modification site of the Ig heavy chain polypeptide amino acid sequence may be modified (e.g., when the modification includes one or more amino acid residue insertions, deletions, and/or substitutions) so as to provide a sulfatase motif at the modification site.

Antibodies used in the antibody-drug conjugates of the present disclosure may have any of a variety of antigen binding specificities, including, but not limited to, antigens present on cancer cells, for example; an antigen present on an autoimmune cell; an antigen present on a pathogenic microorganism; an antigen present on a virus-infected cell (e.g., a human immunodeficiency virus-infected cell); an antigen present on the diseased cells; etc. For example, the antibody conjugate may bind to an antigen, wherein the antigen is present on the cell surface. The antibody conjugates of the present disclosure can bind antigen with suitable binding affinity, e.g., 5x10 ^-6 M to 10 ^-7 M、10 ^-7 M to 5x10 ^- ⁷ M、5x10 ^-7 M to 10 ^-8 M、10 ^-8 M to 5x10 ^-8 M、5x10 ^-8 M to 10 ^-9 M or greater than 10 ^-9 Binding affinity of M.

As a non-limiting example, the antibody conjugates of the invention can bind to an antigen present on a cancer cell (e.g., a tumor-specific antigen; an antigen overexpressed on a cancer cell; etc.), and the conjugate moiety can be a drug, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.). For example, the subject antibody conjugates can be specific for an antigen on a cancer cell, wherein the conjugate moiety is a drug, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.).

As a further non-limiting example, the subject antibody conjugates can bind to an antigen present on a virus-infected cell (e.g., where the antigen is encoded by a virus; where the antigen is expressed on a virus-infected cell type; etc.), and the conjugate moiety can be a drug, such as a viral fusion inhibitor. For example, the antibody conjugates of the invention may bind to an antigen present on a virus-infected cell, and the conjugate moiety may be a drug, such as a viral fusion inhibitor.

Medicaments for conjugation to polypeptides

The present disclosure provides drug-polypeptide conjugates (e.g., antibody-drug conjugates). Any of a number of drugs are suitable for use as or can be modified to be suitable for use as a reactive partner for conjugation with an antibody. Examples of the drug include small molecule drugs and peptide drugs.

As used herein, "small molecule drug" refers to a compound, such as an organic compound, that exhibits a pharmaceutical activity of interest and typically has a molecular weight of 800Da or less or 2000Da or less, but may encompass molecules up to 5kDa and may be as large as 10 kDa. Small inorganic molecules refer to molecules that do not contain carbon atoms, while small organic molecules refer to compounds that contain at least one carbon atom.

For example, the drug or active agent may be maytansine. "maytansine," "maytansine moiety," "maytansine active agent moiety," and "maytansinoids" refer to maytansine and analogs and derivatives thereof, as well as pharmaceutically active maytansine moieties and/or portions thereof. Maytansine conjugated to a polypeptide can be any of a variety of maytansinoid moieties, such as, but not limited to, maytansine and analogs and derivatives thereof (e.g., deacylated maytansine) described herein. In other cases, the drug or active agent may be an auristatin, or an analog or derivative thereof, or a pharmaceutically active auristatin moiety and/or portion thereof. The auristatin conjugated to the polypeptide may be any of a variety of auristatin moieties, such as, but not limited to, auristatins and analogs and derivatives thereof described herein. In other cases, the drug or active agent may be a sesquialter, or an analog or derivative thereof, or a pharmaceutically active sesquialter moiety and/or a portion thereof. The duocarmycin conjugated to the polypeptide may be any of a variety of duocarmycin moieties, such as, but not limited to, the duocarmycin and analogs and derivatives thereof described herein.

In certain embodiments, the drug is selected from the group consisting of cytotoxins, kinase inhibitors, immunostimulants, toll-like receptor (TLR) agonists, oligonucleotides, aptamers, cytokines, steroids, and peptides.

For example, a cytotoxin may include any compound that results in cell death (e.g., necrosis or apoptosis) or reduced cell viability.

Kinase inhibitors may include, but are not limited to, adalook (adavitertib), afatinib (Afatinib), axitinib (Axitinib), bosutinib (Bosutinib), cetuximab (Cetuximab), cobacitinib (cobimatinib), crizotinib (Crizotinib), caboztinib (Cabozantinib), dacatinib (Dacomitinib), dasatinib (Dasatinib), emtrictinib (Entrectinib), edafitinib (erdatatinib), erlotinib (Erlotinib), fotematinib (Gefitinib), ibrutinib (ibutinib), imatinib (Imatinib), lapatinib (lapatib), valatinib (levatinib), muzantinib (muxitinib), vanatinib (vanatinib), vandetatinib (vandetatinib), vandetatinib (Vandetanib), vandetatinib (vanatib), vandetatinib (vanab), vanatib (vanatib ) and the like.

Immunostimulants may include, but are not limited to, vaccines (e.g., bacterial or viral), colony stimulating factors, interferons, interleukins, etc. TLR agonists include, but are not limited to, imiquimod (imiquimod), resiquimod (resiquimod), and the like.

Oligonucleotide drugs include, but are not limited to, fomivirsen (fomivirsen), pegaptanib (pegaptanib), mi Bomei (mipomersen), etiqueleno (eteplirsen), defibrotide (defibrotide), norcin (nusinesen), golodirsen (golodirsen), viterbi (villearsen), wo Lannai (volanesorsen), inotersen (inotersen), tolfresen (tofersen), tol Mi Sen (tominersen), and the like.

Aptamer drugs include, but are not limited to, pie-taganib, AS1411, REG1, ARC1779, NU172, ARC1905, E10030, NOX-a12, NOX-E36, and the like.

Cytokines include, but are not limited to, interferon alpha-2B (Albinterferon Alfa-2B), aldileukins (Aldoleukin), ALT-801, anakinra, ancestim (Ancestim), A Fu Teming (Avotermin), balustrin (Balutwatch), BEMPEG (Bempegaldesleukin), binemakin-4 (Binetrakin), bei Shutuo-Xin Baijie (Cintredekin Besudotox), CTCE-0214 (CTCE-0214), dapoxetine alpha (Darbepoetin alfa), ditoretin-dilin (Denileukin diftitox), du Lale min (Dulanermin), edolein alpha (Edodekin alfa), enfehlamine (Emfildelta ermin), epoetin delta (Epoetin) Erythropoietin (Erythropoietin), human Interleukin-2 (Human Interleukin-2), interferon alpha (Interferon alfa), interferon alpha-2 c (Interferon alfa-2 c), interferon a-n1 (Interferon alfa-n 1), interferon alpha-n 3 (Interferon alfa-n 3), consensus Interferon (Interferon alfacon-1), interferon beta-1a (Interferon beta-1 a), interferon beta-1B (Interferon beta-1B), interferon gamma-1B (Interferon gamma-1B), interferon Kappa (Interferon Kappa), interleukin-1 alpha (Interferon alpha-1), interleukin-10 (Interferon kinase-10), interleukin-7 (Interferon kinase-7), interleukin-7, lenogastrin (Lenogastrim), lerisstim (Lerdistim), li Peifei grastim (Lipegfiggrastim), rockwell alpha (Lorukafusp alfa), maxy-G34, methoxypolyethylene glycol-epoetinbeta (Methoxy polyethylene glycol-epoetin beta), mo Gesi pavilion (Molgastrim), mo Laisi pavilion (Muplestim), narrotigotine (Nagrestipen), opril interleukin (Oprelvekin), pefegrid (Pegiigstim), pegilobiginet (Pegilocal interleukin), polyethylene glycol interferon alpha-2 a (Peginterferon alfa-2 a), polyethylene glycol interferon alpha-2 b (Peginterferon alfa-2 b), polyethylene glycol interferon beta-1a (Peginterferon beta-1 a), polyethylene glycol interferon lambda-1a (Peginterferon lambda-1 a), recombinant CD40 ligand (Realbona CD 40-ligands), remostimothin (Remostimum), hemangiostatin (Remostimigim), heme (26-angustorin), and the like, and so on (Lemongrastim), and so on.

Steroid drugs include, but are not limited to, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, deflazacort, and the like.

As used herein, "peptide drug" refers to polymeric compounds containing amino acids and is intended to encompass naturally occurring and non-naturally occurring peptides, oligopeptides, cyclic peptides, polypeptides and proteins, as well as peptidomimetics. Peptide drugs may be obtained by chemical synthesis or produced from genetically encoded sources (e.g., recombinant sources). The molecular weight of the peptide drug may range from 200Da to 10kDa or greater. Suitable peptides include, but are not limited to, cytotoxic peptides; angiogenic peptides; an anti-angiogenic peptide; peptides that activate B cells; peptides that activate T cells; an antiviral peptide; peptides that inhibit viral fusion; increasing the production of peptides by one or more lymphocyte populations; an antimicrobial peptide; a growth factor; growth hormone releasing factor; a vasoactive peptide; an anti-inflammatory peptide; peptides that regulate glucose metabolism; antithrombotic peptides; antinociception peptides; vasodilator peptides; platelet aggregation inhibitors; an analgesic; etc.

Examples of drugs that may be used in the conjugates and compounds described herein include, but are not limited to, topiramate M, calicheamicin, SN-38, irinotecan, STAT3 inhibitors, alpha-amanitine, aurora kinase inhibitors, belotecan, 9-aminocamptothecin (9-AC), and anthracyclines.

Other examples of drugs include small molecule drugs, such as cancer chemotherapeutic agents. For example, where the polypeptide is an antibody (or fragment thereof) specific for a tumor cell, the antibody may be modified as described herein to include modified amino acids, which may then be conjugated to a cancer chemotherapeutic agent. Cancer chemotherapeutic agents include non-peptide (i.e., non-protein) compounds that reduce proliferation of cancer cells, and encompass cytotoxic and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids and steroid hormones. Peptide compounds may also be used.

Suitable cancer chemotherapeutic agents include dolastatin (dolastatin) and active analogs and derivatives thereof; and auristatin and active analogs and derivatives thereof (e.g., monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), etc.). See, for example, WO 96/33212, WO 96/14856 and U.S.6,323,315. For example, dolastatin 10 or auristatin PE can be included in the antibody-drug conjugates of the present disclosure. Suitable cancer chemotherapeutic agents also include maytansinoids and active analogues and derivatives thereof (see, e.g., EP 1391213; and Liu et al (1996) Proc. Natl. Acad. Sci. USA) 93:8618-8623; the sesqui-carcinomycin and active analogues and derivatives thereof (including, for example, synthetic analogues, KW-2189 and CB 1-TM 1); and benzodiazepines and active analogs and derivatives thereof (e.g., pyrrolobenzodiazepine (PBD).

Agents for reducing cell proliferation are known in the art and are widely used. Such agents include alkylating agents, such as nitrogen mustards, nitrosoureas, ethyleneimine derivatives, alkyl sulfonates, and triazenes, including but not limited to nitrogen mustards, cyclophosphamide (Cytoxan) ^TM ) Melphalan (L-sarcolysin), carmustine (BCNU), lomustine (lomustine) (CCNU), semustine (semustine) (methyl-CCNU), streptozotocin, chlorourea, uracil nitrogen mustard, ifosfamide, chlorambucil, pipobroman (pipobroman), triethylamine, triethylenethiophosphamine, busulfan, dacarbazine (dacarbazine) and temozolomide (temozolomide).

Antimetabolites include folic acid analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytarabine (cytosine arabinoside), fluorouracil (5-FU), fluorouridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), penstatin (pentastatin), 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5, 8-diazepino (PDDF, CB 3717), 5, 8-diazatetrahydrofolic acid (DDATHF), folinic acid, fludarabine phosphate (fiudarabine phosphate), pentcstatine (pentastatin), and gemcitabine (gemcitabine).

Suitable natural products and derivatives thereof (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines and epipodophyllotoxins) include, but are not limited to, ara-C, paclitaxelDocetaxel->Deoxidizing syndiotactic-assisting type mycin, mitomycin-C, L-asparaginase and azathioprine; bucona; alkaloids, e.g. vincristine, vinblastine, vinorelbine,Vindesine, etc.; podophyllotoxins, such as etoposide, teniposide, and the like; antibiotics such as anthracyclines, daunorubicin (daunomycin), rubicin (rubicin), sirubicin (cerubidine), idarubicin (idarubicin), doxorubicin (doxorubicin), epirubicin (epirubicin), morpholino derivatives, and the like; phenoxy hydrazone bicyclic peptides such as dactinomycin; basic glycopeptides such as bleomycin (bleomycin); anthraquinone glycosides, for example plicamycin (plicamycin) (mithramycin); anthracenediones, such as mitoxantrone; aziridinopyrroloindolediones, such as mitomycin (mitomycin); macrocyclic immunosuppressants such as cyclosporin, FK-506 (tacrolimus), rapamycin (rapamycin), and the like; etc.

Other anti-proliferative cytotoxic agents are novelties (navlbene), CPT-11, anastrozole (anastrazole), letrozole (letrozole), capecitabine (capecitabine), raloxifene (reloxafine), cyclophosphamide, ifosfamide and droloxifene (droloxifene).

Microtubule-affecting agents with antiproliferative activity are also suitable for use, and include, but are not limited to, isochorisonine (NSC 406042), halichondrin B (Halichondrin B) (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolastatin 10 (dolastatin 10) (NSC 376128), maytansine (NSC 153858), rhizotoxin (rhizoxin) (NSC 332598), paclitaxelDerivatives, docetaxelThiocolchicine (NSC 361792), trityl cystine, vinblastine sulfate, vincristine sulfate, natural and synthetic epothilones, including but not limited to epothilone A, epothilone B, discodermolide; estramustine (estramustine), nocodazole (nocodazole), and the like.

Hormone modulators and steroids (including synthetic analogs) suitable for use include, but are not limited to, adrenocortical stimulationA pharmaceutical agent such as prednisone (prednisone), dexamethasone (dexamethasone) and the like; estrogens and progestogens, such as, for example, medroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen (tamoxifen); etc.; and adrenocortical inhibitors such as aminoglutethimide (amiglutethimide); 17 alpha-ethinyl estradiol; diethylstilbestrol, testosterone (testosterone), fluoxymesterone, droxidone propionate, testosterone, methylprednisolone, methyltestosterone (methyl-testosterone), prednisolone, triamcinolone (triamcinolone), chlorotrianisole, medroxyprogesterone, aminoglutethimide, estramustine (estramustine), medroxyprogesterone acetate, leuprorelin (leuprolide), flutamide (Flutamide) (droginil), toremifene (Toremifene) (farston) and Estrogens stimulate proliferation and differentiation; thus, compounds that bind to estrogen receptors are used to block this activity. Corticosteroids can inhibit T cell proliferation.

Other suitable chemotherapeutic agents include metal complexes such as cisplatin (cis-DDP), carboplatin, and the like; urea, such as hydroxyurea; and hydrazines, such as N-methyl hydrazine; epipodophyllotoxin; topoisomerase inhibitors; methyl benzyl hydrazine; mitoxantrone; folinic acid; tegafur (tegafur); etc. Other antiproliferative agents of interest include immunosuppressants such as mycophenolic acid (mycophenolic acid), thalidomide (thalidomide), deoxyspergualin (desoxyspergualin), azasporine (azasporine), leflunomide (leflunomide), mizoribine (mizoribine), azaspirane (azaspirane) (SKF 105685);(ZD 1839,4- (3-chloro-4-fluorophenylamino) -7-methoxy-6- (3- (4-morpholino) propoxy) quinazoline); etc.

Taxanes are suitable for use. "taxane" includes paclitaxel and any active taxane derivative or prodrug. "paclitaxel" (which is understood herein to include analogs, formulations and derivatives, such as docetaxel),TAXOL ^TM 、TAXOTERE ^TM (formulation of docetaxel), 10-deacetyl analogs of paclitaxel and 3 'n-debenzoyl-3' n-t-butoxycarbonyl analogs of paclitaxel can be readily prepared using techniques known to those skilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO 94/07876, WO 93/23555, WO 93/10076; U.S. patent No. 5,294,637; 5,283,253; 5,279,949; 5,274,137; no. 5,202,448; 5,200,534; 5,229,529; and EP 590,267), or from a variety of commercial sources including, for example, sigma Chemical co. Company of st louis, missouri (Sigma Chemical Co, st.louis, mo). (T7402 is derived from Taxus brevifolia (Taxus brevensis) or T-1912 is derived from Taxus yunnanensis (Taxus yannanensis)).

Paclitaxel is understood to mean not only the usual chemically usable forms of paclitaxel but also analogues and derivatives (e.g. Taxotere as described above ^TM Docetaxel) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

The term "taxane" also includes a variety of known derivatives including hydrophilic derivatives and hydrophobic derivatives. Taxane derivatives include, but are not limited to, galactose and mannose derivatives as described in International patent application WO 99/18113; piperazine and other derivatives described in WO 99/14209; taxane derivatives described in WO 99/09021, WO 98/22451 and U.S. Pat. No. 5,869,680; 6-thio derivatives as described in WO 98/28288; sulfinamide derivatives described in us patent No. 5,821,263; and paclitaxel derivatives described in U.S. patent No. 5,415,869. It also includes prodrugs of paclitaxel, including but not limited to WO 98/58927; WO 98/13059; and those described in U.S. patent No. 5,824,701.

Suitable biological response modifiers include, but are not limited to, (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine/threonine kinase activity; (3) Tumor-associated antigen antagonists, such as antibodies that specifically bind to tumor antigens; (4) an apoptosis receptor agonist; (5) interleukin-2; (6) IFN- α; (7) IFN-gamma; (8) colony stimulating factor; and (9) an angiogenesis inhibitor.

Examples of drugs include small molecule drugs, such as cancer chemotherapeutic agents. For example, where the polypeptide is an antibody (or fragment thereof) specific for a tumor cell, the antibody may be modified as described herein to include modified amino acids, which may be subsequently conjugated to a cancer chemotherapeutic agent, such as a microtubule-affecting agent. In certain embodiments, the drug is a microtubule-affecting agent having antiproliferative activity, e.g., a maytansinoid.

In certain embodiments, the drug is a maytansinoid having the following structure:

wherein the method comprises the steps ofRepresents maytansinoids and a second linker L in the conjugates and compounds described herein ² A connection point between them. "connection point" means>The symbols represent N and the second linker L of maytansinoids in the conjugates and compounds described herein ² A bond therebetween. For example, in formula (I), W ¹ Can be maytansinoids, such as maytansinoids of the above structure, wherein +.>Represents a maytansinoid and a second linker L ² A connection point between them. In some cases, maytansinoids of the above structure may be referred to as deacylated maytansinoids.

In certain embodiments, the agent is an anti-mitotic agent, such as an auristatin or an active auristatin analog or derivative thereof (e.g., monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), etc.). In certain embodiments, the drug is MMAE, having the following structure:

For example, MMAE active agents may be included in antibody-drug conjugates as follows:

wherein the method comprises the steps ofRepresents the auristatin and the second linker L in the conjugates and compounds described herein ² A connection point between them. For example, a->The symbols indicate the N of auristatin and the second linker L ² The bond between them, for example, is as shown in formula (I). For example, in formula (I), W ¹ Can be an auristatin, e.g. MMAE, wherein +.>Represents MMAE and second linker L ² A connection point between them.

In certain embodiments, the drug is a DNA alkylating agent, such as a sesquialter. Examples of the duocarmycin include, but are not limited to, duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, and CC-1065. In some embodiments, the duocarmycin is a duocarmycin analog such as, but not limited to, adozelesin, bizelesin, or carbozelesin.

In some cases, the duocarmycin is a compound having the structure:

for example, a milbemycin active agent may be included in an antibody-drug conjugate as follows:

wherein the method comprises the steps ofRepresents the binding of a sesquialter mycin to a second linker L in the conjugates and compounds described herein ² A connection point between them. For example, a->Symbolism of the sesquialter mycin and the second linker L ² The bond between them, for example, is as shown in formula (I). For example, in formula (I), W ¹ May be a sesquialter, such as the one shown above, wherein +.>Represents a sesquialter mycin and a second linker L ² A connection point between them. />

As described above, in certain embodiments, L ² Is formed by- (L) ²¹ ) _e -(L ²² ) _f -(L ²³ ) _g -(L ²⁴ ) _h -a second linker as described, wherein L ²¹ 、L ²² 、L ²³ And L ²⁴ Each independently is a second linker subunit. In certain embodiments, L ²¹ Is attached to the first cleavable moiety. In certain embodiments, L ²¹ Is also connected to W, if present ¹ (drug). In certain embodiments, L ²² Attached to W, if present ¹ (drug). In certain embodiments, L ²³ Attached to W, if present ¹ (drug). In certain embodiments, L ²⁴ Attached to W, if present ¹ (drug).

As described above, in certain embodiments, the second linker- (L) ²¹ ) _e -(L ²² ) _f -(L ²³ ) _g -(L ²⁴ ) _h -by- (T) ⁵ -V ⁵ ) _e -(T ⁶ -V ⁶ ) _f -(T ⁷ -V ⁷ ) _g -(T ⁸ -V ⁸ ) _h -description wherein e, f, g and h are each independently 0 or 1, wherein the sum of e, f, g and h is 0 to 4. In certain embodiments, L, as described above ²¹ Is attached to the first cleavable moiety. Thus, in certain embodiments, T ⁵ Is attached to the first cleavable moiety. In certain embodiments, V ⁵ To W ¹ (drug). In certain embodiments, L, as described above ²² Attached to W, if present ¹ (drug). Thus, in certain embodiments, T ⁶ Attached to W, if present ¹ (drug), or V ⁶ Attached to W, if present ¹ (drug). In certain embodiments, L, as described above ²³ Attached to W, if present ¹ (drug). Thus, in certain embodiments, T ⁷ Attached to W, if present ¹ (drug), or V ⁷ Attached to W, if present ¹ (drug). In certain embodiments, L, as described above ²⁴ Attached to W, if present ¹ (drug). Thus, in certain embodiments, T ⁸ Attached to W, if present ¹ (drug), or V ⁸ Attached to W, if present ¹ (drug).

Embodiments of the present disclosure include conjugates in which an antibody is conjugated to one or more drug moieties, e.g., 2 drug moieties, 3 drug moieties, 4 drug moieties, 5 drug moieties, 6 drug moieties, 7 drug moieties, 8 drug moieties, 9 drug moieties, or 10 or more drug moieties. The drug moiety may be conjugated to the antibody at one or more sites in the antibody, as described herein. In certain embodiments, the conjugate has an average drug to antibody ratio (DAR) (molar ratio) in the range of 0.1 to 10, or 0.5 to 10, or 1 to 10, such as 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2. In certain embodiments, the average DAR of the conjugate is 1 to 2, e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2. In certain embodiments, the conjugate has an average DAR of 1 to 1.5. In certain embodiments, the conjugate has an average DAR of 1.5 to 2. Mean refers to an arithmetic mean.

Drugs coupled to polypeptides may be modified to incorporate reactive partners that react with the polypeptide. When the drug is a peptide drug, the reactive moiety (e.g., an aminooxy or hydrazide) may be located at the N-terminal region, the N-terminal, the C-terminal region, the C-terminal, or at a position internal to the peptide. For example, examples of methods relate to the synthesis of peptide drugs having an aminooxy group. In this example, the peptide was synthesized from a Boc protected precursor. The amino group of the peptide may be reacted with a compound comprising a carboxylic acid group and an oxy-N-Boc group. For example, the amino group of the peptide is reacted with 3- (2, 5-dioxopyrrolidin-1-yloxy) propionic acid. Other variants of compounds comprising a carboxylic acid group and an oxy-N-protecting group may include a different number of carbons in the alkylene linker and substituents on the alkylene linker. The reaction between the amino group of the peptide and the compound comprising the carboxylic acid group and the oxy-N-protecting group occurs by standard peptide coupling chemistry. Examples of peptide coupling reagents that may be used include, but are not limited to DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), di-p-toluoyl carbodiimide, BDP (diethyl 1-benzotriazole phosphate-1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide), EDC (1- (3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride), cyanuric fluoride, cyanuric chloride, TFFH (tetramethyl fluoro formamidine hexafluorophosphate), DPPA (diphenyl phosphate), BOP (benzotriazole-1-oxo-tris (dimethylamino) hexafluorophosphate), HBTU (O-benzotriazole-1-yl-N, N, N ', N' -tetramethyluronium hexafluorophosphate), TBTU (O-benzotriazol-1-yl-N, N, N ', N' -tetramethyluronium tetrafluoroborate), TSTU (O- (N-succinimidyl) -N, N, N ', N' -tetramethyluronium tetrafluoroborate), HATU (N- [ (dimethylamino) -1-H-1,2, 3-triazolo [4,5,6] -pyridin-1-ylmethylene ] -N-methyl-ammonium hexafluorophosphate N-oxide), BOP-Cl (bis (2-oxo-3-oxazolidinyl) phosphinic acid chloride), and, pyBOP ((1-H-1, 2, 3-benzotriazol-1-yloxy) -tris (pyrrolidinyl) tetrafluoro-sphonium) BrOP (bromotris (dimethylamino) hexafluoro-phosphate), DEPBT (3- (diethoxyphosphoryloxy) -1,2, 3-benzotriazin-4 (3H) -one) PyBrOP (bromotris (tetrahydropyrrole) hexafluoro-phosphate). As non-limiting examples HOBt and DIC can be used as peptide coupling reagents.

The peptide containing the N-protecting group is deprotected to expose the amino-oxy functional group. Deprotection of the N-oxysuccinimide group occurs, for example, according to standard deprotection conditions for the cyclic amide group. Deprotection conditions can be found in Greene and Wuts, protecting groups in organic chemistry (Protective Groups in Organic Chemistry), 3 rd edition, 1999, john Wiley parent-child publishing company (John Wiley & Sons), NY and Harrison et al. Some deprotection conditions include hydrazine reagent, amino reagent or sodium borohydride. Deprotection of the Boc protecting group may be performed with TFA. Other reagents for deprotection include, but are not limited to, hydrazine, methyl hydrazine, phenyl hydrazine, sodium borohydride, and methylamine. The products and intermediates can be purified by conventional methods, such as HPLC purification.

One of ordinary skill will appreciate that factors such as pH and steric hindrance (i.e., the accessibility of the amino acid residue to react with the reactive partner of interest) are important. It is well within the skill of the ordinary artisan to modify the reaction conditions to provide optimal conjugation conditions and is routine in the art. When coupled to a polypeptide present in or on a living cell, physiologically compatible conditions are selected. For example, the pH may be temporarily lowered for a time sufficient to allow the reaction to occur, but within a period of time that the cells are tolerant (e.g., about 30 minutes to 1 hour). The physiological conditions used for polypeptide modification on the cell surface may be similar to those used in ketone-azide reactions when modifying cells carrying cell surface azides (see, e.g., U.S.6,570,040).

Small molecule compounds containing or modified to contain an alpha-nucleophilic group that serves as a reactive partner with the compounds or conjugates disclosed herein are also contemplated for use as medicaments in polypeptide-medicament conjugates of the disclosure. General methods for chemical synthesis schemes and conditions for synthesizing compounds of interest are known in the art (see, e.g., smith and March, march's Advanced Organic Chemistry. Advanced chemistry: reactions, mechanisms, and structures, fifth edition, wiley-Interscience publishers, 2001; or Vogel, textbooks on practical organic chemistry, including qualitative organic chemistry (A Textbookof practical organic chemistry, including qualitative organic analysis), fourth edition, new York (New York): langman, 1978).

Formulations

The conjugates of the present disclosure can be formulated in a number of different ways. Typically, when the conjugate is an antibody-drug conjugate, the conjugate is formulated in a manner compatible with the drug, the antibody, the disorder to be treated, and the route of administration to be used.

In some embodiments, pharmaceutical compositions comprising any of the conjugates of the present disclosure and a pharmaceutically acceptable excipient are provided.

The conjugate (e.g., antibody-drug conjugate) may be provided in any suitable form, e.g., in the form of a pharmaceutically acceptable salt, and may be formulated for any suitable route of administration, e.g., oral, topical, or parenteral administration. When the conjugate is provided in liquid injectable form (e.g., in those embodiments where it is administered intravenously or directly into tissue), the conjugate may be provided as a ready-to-use dosage form, or as a reconstitutable, storage-stable powder or liquid composed of a pharmaceutically acceptable carrier and excipient.

The method for formulating the conjugate may be modified according to those methods readily available. For example, the conjugate may be provided in a pharmaceutical composition comprising a therapeutically effective amount of the conjugate and a pharmaceutically acceptable carrier (e.g., saline). The pharmaceutical composition may optionally include other additives (e.g., buffers, stabilizers, preservatives, etc.). In some embodiments, the formulations are suitable for administration to mammals, such as those suitable for administration to humans.

Therapeutic method

The antibody-drug conjugates of the present disclosure are useful for treating a condition or disease in a subject that is suitable for treatment by administration of a parent drug (i.e., a drug prior to conjugation to an antibody).

In some embodiments, methods are provided that include administering to a subject an effective amount (e.g., a therapeutically effective amount) of any of the conjugates of the disclosure.

In certain aspects, methods of delivering a drug to a target site in a subject are provided, the methods comprising administering to the subject a pharmaceutical composition comprising any of the conjugates of the disclosure, wherein the administration is effective to release a therapeutically effective amount of the drug from the conjugate at the target site in the subject. For example, as described herein, an antibody-drug conjugate of the present disclosure can include a cleavable linker, e.g., an enzymatically cleavable linker that includes a first enzymatically cleavable moiety and a second enzymatically cleavable moiety. In some cases, the cleavable linker may be cleaved under appropriate conditions to separate or release the drug from the antibody at the desired site of action target of the drug. For example, a second cleavable linker that protects the first cleavable linker from cleavage may be cleaved to allow the first cleavable moiety to be cleaved, which results in cleavage of the cleavable linker into two or more moieties, thereby releasing the drug from the antibody-drug conjugate at the desired site of action.

In certain embodiments, the first cleavable moiety may be an enzymatically cleavable moiety. In some cases, the enzyme that promotes cleavage of the first cleavable moiety is an enzyme that is administered to the subject to be treated (i.e., exogenous to the subject to be treated). For example, the first enzyme may be administered prior to, concurrently with, or after administration of the antibody-drug conjugates described herein.

In certain embodiments, the second cleavable moiety may be an enzymatically cleavable moiety. In some cases, the enzyme that promotes cleavage of the second cleavable moiety is an enzyme that is administered to the subject to be treated (i.e., exogenous to the subject to be treated). For example, the second enzyme may be administered prior to, concurrently with, or after administration of the antibody-drug conjugates described herein. In certain embodiments, the first enzyme and the second enzyme are different enzymes.

In other cases, the first enzyme that facilitates cleavage of the first cleavable moiety is an enzyme that is present in the subject to be treated (i.e., endogenous to the subject to be treated). For example, the first enzyme may be present at a desired site of action of the drug of the antibody-drug conjugate. The antibody of the antibody-drug conjugate can specifically target a desired site of action (e.g., can specifically bind to an antigen present at the desired site of action), wherein the first enzyme is also present at the desired site of action. In some cases, the first enzyme is present in excess at the desired site of action compared to other regions in the subject to be treated. For example, the first enzyme may be overexpressed at the desired site of action compared to other regions in the subject to be treated. In some cases, the first enzyme is present in excess at the desired site of action due to its localization at a particular region or location. For example, the first enzyme may be associated with a structure within the desired site of action, such as lysosomes. In some cases, the first enzyme is present in excess in the lysosome compared to other regions within the subject. In some embodiments, lysosomes comprising the first enzyme are found at a desired site of action of a drug of an antibody-drug conjugate, e.g., a site of a cancer or tumor to be treated with the drug. In certain embodiments, the first enzyme is a protease, such as a human protease (e.g., cathepsin B).

In certain embodiments, the second enzyme that facilitates cleavage of the second cleavable moiety is an enzyme that is present in the subject to be treated (i.e., endogenous to the subject to be treated). For example, the second enzyme may be present at a desired site of action of the drug of the antibody-drug conjugate. The antibody of the antibody-drug conjugate can specifically target a desired site of action (e.g., can specifically bind to an antigen present at the desired site of action), wherein a second enzyme is also present at the desired site of action. In some cases, the second enzyme is present in excess at the desired site of action compared to other regions in the subject to be treated. For example, the second enzyme may be overexpressed at the desired site of action compared to other regions in the subject to be treated. In some cases, the second enzyme is present in excess at the desired site of action due to its localization at a particular region or location. For example, the second enzyme may be associated with a structure within the desired site of action, such as lysosomes. In some cases, the second enzyme is present in excess in the lysosome compared to other regions within the subject. In some embodiments, lysosomes comprising the second enzyme are found at a desired site of action of the drug of the antibody-drug conjugate, e.g., at a site of cancer or tumor to be treated with the drug. In certain embodiments, the second enzyme is a glycosidase, e.g., a galactosidase, glucosidase, mannosidase, fucosidase, and the like.

Any suitable enzyme may be used to cleave the first cleavable moiety and the second cleavable moiety of the antibody-drug conjugate described herein. Other enzymes may also be suitable for cleaving the first cleavable moiety and the second cleavable moiety of the antibody-drug conjugates described herein, such as, but not limited to, enzymes from other vertebrates (e.g., primate, mouse, rat, cat, pig, quail, goat, dog, etc.).

In certain embodiments, the antibody-drug conjugate is substantially stable under standard conditions. By substantially stable is meant that the cleavable linker of the antibody-drug conjugate does not undergo significant amounts of cleavage in the absence of the first and second enzymes as described above. For example, as described above, the second cleavable moiety may protect the first cleavable moiety from cleavage, and thus the cleavable linker of the antibody-drug conjugate does not undergo significant amounts of cleavage in the absence of the second enzyme as described above. For example, the cleavable linker of the antibody-drug conjugate may be substantially stable such that 25% or less of the antibody-drug conjugate is cleaved in the absence of the first enzyme and/or the second enzyme, e.g., 20% or less, or 15% or less, or 10% or less, or 5% or less, or 4% or less, or 3% or less, or 2% or less, or 1% or less. In some cases, the antibody-drug conjugate is substantially stable such that the cleavable linker of the antibody-drug conjugate does not undergo a significant amount of cleavage in the absence of the first enzyme and/or the second enzyme, but can be cleaved in the presence of the first enzyme and the second enzyme. For example, the antibody-drug conjugate may be substantially stable after administration to a subject. In some cases, the antibody-drug conjugate is substantially stable after administration to a subject, and then, when the antibody-drug conjugate is present at the desired site of action, the second cleavable moiety can be cleaved from the cleavable linker, such that the first cleavable moiety is subsequently cleaved by the first enzyme, which then releases the drug at the desired site of action. In certain embodiments, after administration to a subject, the antibody-drug conjugate is stable in the absence of the first enzyme and/or the second enzyme for an extended period of time, e.g., 1 hour or more, or 2 hours or more, or 3 hours or more, or 4 hours or more, or 5 hours or more, or 6 hours or more, or 7 hours or more, or 8 hours or more, or 9 hours or more, or 10 hours or more, or 15 hours or more. Or 20 hours or longer, or 24 hours (1 day) or longer, or 2 days or longer, or 3 days or longer, or 4 days or longer, or 5 days or longer, or 6 days or longer, or 7 days (1 week) or longer. In certain embodiments, the antibody-drug conjugate is stable over an extended period of time in the absence of the first enzyme and/or the second enzyme over a range of pH values, e.g., over a range of pH from 2 to 10, or from 3 to 9, or from 4 to 8, or from 5 to 8, or from 6 to 8, or from 7 to 8.

As described above, the antibody-drug conjugates of the present disclosure are useful for treating a condition or disease in a subject, which is suitable for treatment by administration of a parent drug. "treating" refers to effecting at least an improvement in a symptom associated with a condition afflicting a host, wherein in a broad sense, improvement refers to at least a reduction in the magnitude of a parameter (e.g., symptom) associated with the treated condition. Thus, treatment also includes situations in which a pathological condition, or at least a symptom associated therewith, is completely inhibited (e.g., prevented from occurring) or stopped (e.g., terminated) such that the host no longer has the condition, or at least a symptom that characterizes the condition. Whereby the treatment comprises: (i) Prevention, i.e., reducing the risk of developing clinical symptoms, including leaving the clinical symptoms non-developing, e.g., preventing disease progression to a deleterious state; (ii) Inhibition, i.e., arresting the development or further development of clinical symptoms, e.g., alleviating or completely inhibiting active disease; and/or (iii) relief, i.e., causing regression of clinical symptoms.

The subject to be treated may be a subject in need of treatment, wherein the subject to be treated is a subject suitable for treatment with the parent drug. Thus, a variety of subjects may be amenable to treatment with the antibody-drug conjugates disclosed herein. Typically, such subjects are "mammals" and humans are of interest. Other subjects may include domestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs, and rats, for example, as in animal models of disease), and non-human primates (e.g., chimpanzees and monkeys).

The amount of antibody-drug conjugate administered may initially be determined based on the dosage of the parent drug and/or the guidelines of the dosing regimen. In general, the antibody-drug conjugate can provide targeted delivery of the conjugated drug and/or enhanced serum half-life, thereby achieving at least one of reduced dose or reduced dosing in a dosage regimen. Thus, the antibody-drug conjugate may achieve reduced doses and/or reduced dosing in a dosing regimen relative to the parent drug prior to conjugation in the antibody-drug conjugate of the present disclosure.

Furthermore, as described above, because antibody-drug conjugates can achieve controlled stoichiometric drug delivery, the dosage of the antibody-drug conjugate can be calculated based on the number of drug molecules provided on a per antibody-drug conjugate basis.

In some embodiments, multiple doses of the antibody-drug conjugate are administered. The frequency of administration of the antibody-drug conjugate can vary depending on any of a variety of factors, such as the severity of the symptoms, the condition of the subject, and the like. For example, in some embodiments, the antibody-drug conjugate is administered once a month, twice a month, three times a month, every other week, once a week (qwk), twice a week, three times a week, four times a week, five times a week, six times a week, every other day, daily (qd/°c d), twice a day (bds/bid), or three times a day (tds/tid), etc.

Methods of treating cancer

The present disclosure provides methods comprising delivering a conjugate of the present disclosure to an individual having cancer. The methods are useful for treating a variety of cancers, including carcinoma, sarcoma, leukemia, and lymphoma. In the context of cancer, the term "treatment" includes one or more (e.g., each) of the following: reducing the growth of solid tumors, inhibiting the replication of cancer cells, reducing the overall tumor burden, and ameliorating one or more symptoms associated with cancer.

Cancers that may be treated using the subject methods include, but are not limited to, esophageal cancer, hepatocellular cancer, basal cell cancer (a form of skin cancer), phosphonium cell cancer (tissues), bladder cancer (including transitional cell cancer (malignancy of the bladder)), bronchial cancer, colon cancer, colorectal cancer, gastric cancer, lung cancer (including small cell and non-small cell cancers of the lung), adrenal cortex cancer, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary gland cancer, cyst gland cancer, myeloid cancer, renal cell carcinoma, ductal carcinoma in situ or cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, wilms' tumor, cervical cancer, uterine cancer, testicular cancer, osteogenic cancer, epithelial cancer, and nasopharyngeal cancer, among others.

Sarcomas that can be treated using the subject methods include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangio-endothelial sarcoma, synovial carcinoma, mesothelioma, ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

Other solid tumors that may be treated using the subject methods include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngeal tube tumor, ependymoma, pineal tumor, angioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

Leukemias that may be treated using the subject methods include, but are not limited to: a) Chronic myeloproliferative syndrome (neoplastic disorder of pluripotent hematopoietic stem cells); b) Acute myelogenous leukemia (neoplastic transformation of pluripotent hematopoietic stem cells or hematopoietic cells that limit lineage potential); c) Chronic lymphocytic leukemia (CLL; clonal proliferation of immunologically immature and functionally disabled small lymphocytes), including B-cell CLL, T-cell CLL prolymphocytic leukemia and hairy cell leukemia; and d) acute lymphoblastic leukemia (characterized by the accumulation of lymphoblastic cells). Lymphomas that can be treated using the methods of the invention include, but are not limited to, B cell lymphomas (e.g., burkitt's lymphomas); hodgkin lymphoma; non-hodgkin's B-cell lymphoma; etc.

In certain aspects, methods of treating cancer in a subject are provided, such methods comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising any of the conjugates of the disclosure, wherein administration is effective to treat cancer in the subject.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric pressure. "average" refers to an arithmetic mean. Standard abbreviations may be used, e.g., bp, base pairs; kb, kilobases; p1, picoliter; s or sec, seconds; min, min; h or hr, hr; aa, amino acids; kb, kilobases; bp, base pairs; nt, nucleotide; i.m, intramuscular; p, intraperitoneal; s.c, subcutaneously; etc.

General synthetic method

Many general references are available that provide generally known chemical synthesis schemes and conditions for synthesizing the disclosed compounds (see, e.g., smith and March, march's advanced chemistry: reactions, mechanisms and structures (March's Advanced Organic Chemistry), fifth edition, wiley-interscience Press, 2001; or Vogel, textbooks on practical organic chemistry, including qualitative organic chemistry (A Textbookof practical organic chemistry, including qualitative organic analysis), fourth edition, new York (New York): langman, 1978).

The compounds described herein may be purified by any purification scheme known in the art, including chromatography, such as HPLC, preparative thin layer chromatography, flash column chromatography, and ion exchange chromatography. Any suitable stationary phase may be used, including normal and reverse phases, as well as ion resins. In certain embodiments, the disclosed compounds are purified by silica gel and/or alumina chromatography. See, e.g., modern liquid chromatography guide (Introduction to Modern Liquid Chromatography), 2 nd edition, ed.l.r.snyder and j.j.kirkland, john wili parent-child publishing company (John Wiley & Sons), 1979; and thin layer chromatography (Thin Layer Chromatography), ed E.Stahl, springer-Verlag, new York, 1969.

In any method of preparing the subject compounds, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the relevant molecules. This can be achieved by conventional protecting groups as described in The standard works, for example J.F.W.McOmie, protecting group "Protective Groups in Organic Chemistry" in organic chemistry, plenum Press, london and New York 1973, protecting group (Protective Groups in Organic Synthesis) in organic synthesis, third edition, wiley Press, new York 1999, peptides (The Peptides); volume 3 (editions: E.Gross and J.Meienhofer), academic Press (Academic Press), london and New York 1981, in "histochemistry methods (Methoden der organischen Chemie), houben-Weyl, 4 th edition, volume 15/l, georg Thieme Verlag Press, stuttgart 1974, in H.—D.Jakuske and H.Jescheit, amino acids, peptides, proteins (amino acids, proteins), protein), germany chemical Press (Verlag Chemie), wei Enhai mu, dierfield beach and Basel 1982, and/or in Jochen Lehmann, carbohydrate chemistry: monosaccharides and derivatives (Chemie der Kohlenhydrate: monosaccharide and Derivate), georg Thieme Verlag Verlag, stuttgart 1974. The protecting groups may be removed at a convenient subsequent stage using methods known in the art.

The subject compounds may be synthesized using commercially available starting materials and/or starting materials prepared by conventional synthetic methods. Various examples of synthetic pathways that can be used to synthesize the compounds disclosed herein are described in the following schemes.

Actual pouring 1

Galactoside and glucoside double cleavage linker for antibody-drug conjugates

Introduction to the invention

The utility of glucuronide-dipeptide double cleavage linkers has been demonstrated, for example, in scheme 1 below, wherein MMAE construct 4 carries a Val-Ala-PABC linker with a glucuronide moiety attached to a self-cleaving unit, which when conjugated to an antibody provides an antibody-drug conjugate (ADC) that has superior stability compared to the universal dipeptide MMAE drug-linker but similar efficacy in vivo. As shown in scheme 1, the glucuronide-based MMAE construct was synthesized in 10 synthetic steps and good overall yield. The synthesis starts with the commercially available fully protected beta-glucuronic acid bromide. The desired overall deprotection of all acetate groups in the glucuronide moiety of intermediate 3 and cleavage of the methyl ester is accomplished by hydrolysis under strongly basic conditions (solution of LiOH in methanol).

Scheme 1.P1' -glucuronide double cleavage MMAE construct 4.

Although these strongly basic conditions are compatible with MMAE drugs (not particularly sensitive to strong bases), the need to conduct basic hydrolysis in the final step of synthesis significantly limits the use of double cleavage linkers based on glucuronide. Indeed, many cytotoxins often used to produce antibody-drug conjugates contain base labile functional groups (fig. 1). For example, the primary alkyl chloride and amide groups in the sesqui-carcinomycin DM 5 are highly susceptible to alkaline hydrolysis, as are the acetate groups in the terpirtine M6 and the ester functions in the calicheamicin 7. Maytansinoids 8 and 9 are particularly unstable under alkaline conditions because their structures contain esters and epoxides. Similarly, DNA topoisomerase I inhibitors such as SN-3810 and lactones in irinotecan 11 are known to hydrolyze even under mildly alkaline conditions.

Despite the above compatibility problems, we have previously shown that certain maytansinoids, such as 4 AP-maytansine 9 (fig. 1), can still pair with glucuronide-based linkers, however, synthesizing such constructs is laborious and inefficient (17 steps, 1.8% overall yield, scheme 2). In this synthetic route, the glucuronide linker 12 must be prepared separately and later entered into the synthetic sequence in a fully deprotected form to react with the separately synthesized maytansinoid derivative 13.

Scheme 2 synthesis of p1' -glucuronide 4 AP-maytansine construct 16.

Although this particular maytansinoid (9) is suitable for synthetic modification to some extent, the more attractive NMC 3-maytansinoids (8) cannot be used in a similar manner due to their structural units. If the synthesis strategy used in 9 (scheme 2) is used in 8, then the problem of selectivity in distinguishing the two carboxyl groups in glucuronic acid intermediate 18 (scheme 3) at the later stages of synthesis will inevitably be faced and an alternative and lengthy protection-deprotection method will be required. Scheme 3. Synthetic method of P1' -glucuronide NMC 3-maytansinoid construct 19 (not viable due to the presence of free carboxylic acid in the glucuronide moiety).

Results and discussion

In contrast to the examples described in the introduction, a greater range of cytotoxins can be successfully used in the synthesis when different biologically relevant glycosides are used in the double cleavage linker. Thus, the use of galactoside instead of glucuronide allows the use of NMC 3-maytansine (8, fig. 1) and the production of P1' -galactoside construct 30 in good overall yield (7.5%) in 11 synthetic steps (scheme 4). Due to the absence of carboxyl groups (as in compound 29, scheme 4), the galactoside moiety is in its fully unprotected form, does not cause chemoselectivity problems and can be successfully used with a large number of base sensitive ADC payloads. The same is expected for glucosides and mannosides, as their structures are not significantly different from that of galactosyls.

Scheme 4 synthesis of P1' -galactoside NMC 3-maytansinoid construct 30.

Indeed, similar to galactosides, other monosaccharide derivatives for producing an alkali sensitive payload of ADC can be prepared. Thus, the double-cleaved glucoside linker successfully paired with NMC 3-maytansinoid payload, providing the corresponding construct 44 in good overall yield (11 steps, 4.9%) as shown in scheme 5.

Scheme 5 synthesis of P1' -glucoside NMC 3-maytansinoid construct 44.

Furthermore, the use of galactosides or glucosides has been found to allow for consistently more efficient and high yield synthesis of drug-linkers compared to the glucuronide counterparts. Thus, the synthetic route of P1' -galactoside MMAE construct 33 shown in scheme 6 produced the desired compound in excellent overall yield (9.4% versus 4.2% for the glucuronide construct, table 1).

Scheme 6 synthesis of p1' -galactoside MMAE construct 33.

Similarly, when compared to compound 4 carrying glucuronide, the glucuronide homolog 46 was prepared in a more efficient manner starting from the corresponding monosaccharide derivative 41 according to an improved synthetic pathway (scheme 7) (7.6% yield versus 4.2% for the glucuronide construct, table 1).

Scheme 7. Synthesis of glucoside MMAE construct 46.

Furthermore, comparison of material costs shows that the desired galactose and glucose derivatives are on average 10 to 20 times cheaper than the corresponding glucuronic acid derivatives (see table 1).

Table 1. The synthesis efficiency and cost are compared according to the type of glycoside used.

* Cost of glucuronic acid, galactose and acetate protected bromide derivatives of glucose (AK Scientific catalogue, month 9 2020)

When incorporated into a linker adjacent to an enzymatically cleavable dipeptide, the glycoside can act as a temporary protecting group that prevents premature cleavage of the antibody-drug conjugate dipeptide linker in circulation. Once the ADC is transported into the lysosomal compartment of the cell, the glycoside moiety must be cleaved by the lysosomal glycosidase to expose the dipeptide linker, which in turn undergoes enzyme-mediated cleavage to release the payload. There are many human glycosidases with different substrate preferences and different cell and tissue expression patterns. Although we have previously shown that the glucuronide moiety can be effectively removed in a variety of cell types as a first step in achieving payload release, it is unclear whether the galactoside or the glucuronide moiety can be similarly processed. To address this problem, constructs 30 and 33 were conjugated to aldehyde-labeled anti-HER 2 and anti-CD 79b antibodies using HIPS ligation, and constructs 44 and 46 were conjugated to aldehyde-labeled anti-FITC, anti-HER 2, and anti-TROP-2 antibodies using HIPS ligation (fig. 2). Analytical characterizations of the resulting conjugates are shown in figures 3 to 10 and 19 to 30.

The resulting conjugates were tested for in vitro activity using HER2+ (NCI-N87, sk-Br-3), CD79b+ (Granta-519, ramos-RA) and TROP-2+ (MDA-MB-468, bxPC 3) cancer cell lines. In some studies, the efficacy of maytansine-conjugated ADCs carrying a galactose-modified double cleavage linker (30) was compared to the activity of the corresponding ADCs generated from single cleavage linker-payload 35 (construct 35 shown in fig. 11, in vitro efficacy data shown in fig. 12-15). In other studies, the efficacy of maytansine-conjugated ADCs carrying a galactose-modified double cleavage linker (30) was compared to the activity of the corresponding ADCs generated using a glucoside-modified double cleavage linker 44 (in vitro efficacy data are shown in figures 31 to 34). Regarding MMAE conjugated ADCs, the in vitro potency of either the galactose modified (33) or the glucoside modified (46) double-cleavage linker was compared to the in vitro potency of the glucuronide modified double-cleavage linker 34 (construct 34 shown in fig. 11, in vitro potency data shown in fig. 16-18, 35 and 36). In summary, the results show that ADCs prepared with mono-and di-lytic linkers (including glucuronide, galactoside and glucoside variants) provide similar levels of potency in cell lines representing solid tumors (gastric, pancreatic and breast cancers) and hematological tumor indications. The data indicate that although glycosidase expression pattern and substrate specificity are unknown, removal of the galactoside and glucoside moieties is robust and efficient, and overall ADC catabolism and payload release are comparable for all tested linkers.

Materials and methods

SUMMARY

Synthetic reagents were purchased from Sigma-Aldrich, acros, AK Scientific or other commercial sources and used without purification. Anhydrous solvents were obtained from commercial sources in sealed bottles. Acetyl bromide-a-D-galactose 21, acetyl bromide-a-D-glucose 36, MMAE 2 and maytansinoid 17 were purchased from commercial sources. HIPS linker compound 32 was commercially available from Shanghai Medicilon and was used without purification. Pentafluoro-ester 28 was synthesized from 32 using standard literature procedures. The synthesis of compounds 34 and 35 was as described previously. In all cases, the solvent was removed under reduced pressure using a Buchi rotary evaporator R-114 equipped with a Buchi V-700 vacuum pump. Column chromatography was performed using a Biotage Isolera chromatography system. Preparative HPLC purification was performed using a Waters preparative HPLC unit equipped with a Phenomenex Kinetex μm EVO C18 150x21.2mm column. HPLC analysis was performed on an Agilent 1100 series analytical HPLC equipped with a G1322A degasser, a G1311A quaternary pump, a G1329A autosampler, a G1314 variable wavelength detector, agilent Poroshell 120sb c18, 4.6mm x 50mm, using a gradient of 10-100% water and acetonitrile containing 0.1% formic acid at room temperature. HPLC was monitored at 254 nm.

Synthesis of constructs 30 and 33

Preparation of (2R, 3S,4S,5R, 6S) -2- (Acetoxymethyl) -6- (5-formyl-2-nitrophenoxy) tetrahydro-2H-pyran-3, 4, 5-trisyl triacetate (21 a)

A50 mL round bottom flask was charged with 204mg (1.22 mmol) of 3-hydroxy-4-nitrobenzaldehyde 20 and acetyl bromide-. Alpha. -D-galactose 21 (500 mg,1.22 mmol) followed by 20mL of anhydrous acetonitrile. The solution was treated with silver (I) oxide (986 mg,4.3 mmol) and the resulting mixture was stirred vigorously in the dark at room temperature for 24 hours. After concentrating the reaction mixture under vacuum, the residue was purified on silica gel using ethyl acetate-hexane mixture as eluent (0-100% gradient) to give 420mg (0.85 mmol,70% yield) of the product as pale yellow solid. LRMS (ESI): m/z 520.0[ M+Na] ⁺ For C ₂₁ H ₂₃ NO ₁₃ m/z 520.1 calculation.

Preparation of (2R, 3S,4S,5R, 6S) -2- (Acetoxymethyl) -6- (2-amino-5- (hydroxymethyl) phenoxy) tetrahydro-2H-pyran-3, 4, 5-trisyl triacetate (22)

To a solution of acetate protected galactoside intermediate 21a (350 mg,0.70 mmol) in 3mL ethyl acetate was added 30mg palladium on carbon (10 wt%) and 30. Mu.L triethylamine. After removing the air, the flask was fitted with a hydrogen balloon and the reaction mixture was stirred at room temperature for 48 hours. After filtration through celite pad, the solvent was removed under vacuum and the residue was dried under high vacuum to give 300mg of crude product 22 as a white solid which was further used for synthesis without further purification. LRMS (ESI): m/z 470.1[ M+H ] ] ⁺ For C ₂₁ H ₂₇ NO ₁₁ m/z 470.2 calculation.

Preparation of (2R, 3R,4S5S, 6S) -2- (2- ((S) -2- ((S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -3-methylbutanamidyl) propanamido) -5- (((((4-nitrophenoxy) carbonyl) oxy) methyl) phenoxy) -6- (acetoxymethyl) tetrahydro-2H-pyran-3, 4, 5-trisyl triacetate (26)

Boc-L-alanine (220 mg,1.11 mmol) and aniline derivative 22 (520 mg,1.16 mmol) were combined in 5mL dry dichloromethane and 0.50mL dry methanol. The resulting solution was treated with EEDQ (280 mg,1.13 mmol) at room temperature. The reaction mixture was stirred in the dark for 1 hour, then the solvent was removed in vacuo. The resulting crude compound 23 was dried under high vacuum for 1 hour and dissolved in 6mL of 1:1 TFA-DCM mixture at room temperature. The solution was allowed to stand for 1 hour, then the solvent was removed, and the resulting crude amine 24 was kept under high vacuum for several hours.

Fmoc-L-valine (375 mg,1.1 mmol) was mixed with HATU (420 mg,1.1 mmol) and DIPEA (375. Mu.L, 2.15 mmol) in 3mL anhydrous DMF in a separate 20mL scintillation vial. The mixture was stirred at room temperature for 30 min, combined with crude compound 24, and stirred overnight until the reaction was complete as judged by LCMS analysis. The reaction mixture was purified directly on a reverse phase C18 column (CH ₃ CN-H ₂ O/0.05% TFA, gradient 0-80%) to give compound 25 as a mixture with small amounts of impurities. The product was thoroughly dried under high vacuum and carried on to the next step without additional purification.

Compound 25 was dissolved in 6mL of anhydrous DCM and treated with DIPEA (375 μl,2.15 mmol) and then 686mg (2.26 mmol) of bis (4-nitrophenyl) carbonate was added in one portion at room temperature. The reaction mixture was stirred for 1 hour, then the solvent was removed in vacuo, and the residue was purified by silica gel column chromatography (EtOAc-hexanes, 10-100% gradient) to give p-nitrophenylcarbonate product 26 (640 mg,0.62mmol,56% yield, over 4 steps) as a pale yellow solid. LRMS (ESI): m/z 1049.3[ M+Na] ⁺ For C ₅₁ H ₅₄ N ₄ O ₁₉ m/z 1049.3 calculation.

Preparation of 4- ((((4- ((S) -2- ((S) -2-amino-3-methylbutanamide) propanamido) -3- (((2R, 3R,4S,5R, 6S) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) benzyl) oxy) carbonyl) (methyl) amino) butanoic acid (27)

To a 20mL scintillation vial was added 4- (methylamino) butyric acid (61 mg,0.52 mmol), 61. Mu.L (0.35 mmol) DIPEA and 1mL anhydrous DMF. The resulting solution was combined with 180mg (0.175 mmol) of PNP-carbonate 26 at room temperature. The reaction mixture was stirred for 1 hour until the PNP-carbonate was completely consumed as judged by LCMS analysis. The solvent was removed in vacuo and the residue was dissolved in 3mL of methanol. The resulting solution was cooled to 0 ℃ and treated with 3ml of 1m aqueous lithium hydroxide. The reaction mixture was stirred for 30 minutes, then warmed to room temperature and concentrated under vacuum. Direct purification of the residue on a reverse phase C18 column (CH ₃ CN-H ₂ O/0.05% TFA,0-50% gradient) to afford 90mg (0.146 mmol,83% yield over 2 steps) of compound 27 as a yellow oil. LRMS (ESI): m/z 615.3[ M+H ]] ⁺ For C ₂₇ H ₄₂ N ₄ O ₁₂ m/z 615.3 calculation.

Preparation of (2S, 5S, 18R) -1- ((4- (((S) -1- (((1)) ⁴ S，1 ⁶ S，3 ² R，3 ³ R,2R,4S,10E,12E, 14R) -86-chloro-1 ⁴ -hydroxy 8 ⁵ 14-dimethoxy-3 ³ 2,7, 10-tetramethyl-1 ² 6-dioxo-7-aza-1 (64) -oxazinan-3 (2, 3) -oxirane-8 (1, 3) -benzocyclotetradec-10, 12-dien-4-yl) oxy) -1-oxopropan-2-yl) (methyl) amino) -4-oxobutyl) (methyl) oxy) methyl) -2- (((2R, 3R,4S,5R, 6S) -3,4, 5-trihydroxy 6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl) amino) -18- (3- (2- ((1, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2,3-b]Pyridin-1-yl) propionylamino) -5-isopropyl-2-methyl-1, 4,7, 17-tetraoxo-10, 13-dioxa-3, 6, 16-triazanonane-19-sulfonic acid (30)

To a 20mL scintillation vial was added compound 27 (84 mg,0.14 mmol), 1mL anhydrous DMF and DIPEA (48. Mu.L, 0.28 mmol), followed by 130mg (0.14 mmol) of pentafluoro-ester 28. The resulting mixture was stirred for 30 min until judged complete coupling (LCMS). Maytansine 17 (89 mg,0.14 mmol) was directly used as a solution in 0.5mL DMF To the reaction mixture was added, followed by HATU (52 mg,0.14 mmol). After 30 min, the reaction mixture was purified on a reverse phase C18 column (CH ₃ CN-H ₂ O/0.05% TFA,10-95% gradient). After complete removal of the solvent, the resulting intermediate 29 was dissolved in 3mL DMF and treated with 150. Mu.L piperidine at room temperature. After 30 min, the reaction mixture was directly purified by reverse phase prep HPLC (C18 column, CH ₃ CN-H ₂ O/0.05% TFA,0-50% gradient). The pure fractions were lyophilized to give 58mg (0.032 mmol, 23% yield over 3 steps) of compound 30.LRMS (ESI): m/z 1801.8[ M+H ]] ⁺ For C ₈₂ H ₁₁₈ CIN ₁₃ O ₂₈ S m/z 1800.8.

Preparation of (2R, 3S,4S,5R, 6S) -2- (Acetoxymethyl) -6- (2- ((S) -2- ((S) -2-amino-3-methylbutanamido) propanamido) -5- ((5S, 8S,11S, 12R) -11- ((((S) -sec-butyl) -12- (2- ((S) -2- ((1R, 2R) -3- (((1S, 2R) -1-hydroxy-1-phenylpropan-2-yl) amino) -1-methoxy-2-methyl-3-oxopropyl) pyrrolidin-1-yl) -2-oxoethyl) -5, 8-diisopropyl-4, 10-dimethyl 3,6, 9-trioxo-2, 13-dioxa-4, 7, 10-triazatetradecyl) phenoxy) tetrahydro-2H-pyran-3, 4, 5-trioxytriacetate (31)

In a 4mL glass vial was mixed MMAE 2 (13.4 mg TFA salt, 0.016 mmol), DIPEA (3.8. Mu.L, 0.022 mmol) and 1mL anhydrous DMF. The resulting solution was treated with PNP-carbonate 26 (15 mg,0.015 mmol) and HOAt (1.0 mg,0.008 mmol). After stirring overnight at room temperature, piperidine (30 μl) was added directly to the reaction mixture. After 30 min, the reaction mixture was purified by reverse phase prep HPLC (C18 column, CH ₃ CN-H ₂ O/0.05% TFA,0-50% gradient). The pure fractions were lyophilized to give 8mg (0.006mmol, 40% yield over 2 steps) of compound 31 as an off-white solid. LRMS (ESI): m/z 1383.5[ M+H ]] ⁺ For C ₆₉ H ₁₀₆ N ₈ O ₂₁ m/z 1383.8 calculation.

Preparation of (2S, 5S, 18R) -1- ((4- ((5S, 8S,11S, 12R) -11- ((S) -sec-butyl) -12- (2- ((S) -2- ((1R, 2R) -3- (((1S, 2R) -1-hydroxy-1-phenylpropan-2-yl) amino) -1-methoxy-2-methyl-3-oxopropyl) pyrrolidin-1-yl) -2-oxoethyl) -5, 8-diisopropyl-4, 10-dimethyl-3, 6, 9-trioxo-2, 13-dioxa-4, 7, 10-triazatetradecyl) -2- (((2R, 3R,4S,5R, 6S) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl) amino) -18- (3- (2- ((1, 2-dimethylhydrazino) methyl) -IH-pyrrolo [2,3-b ] pyridin-1-yl) propionylamino) -5-isopropyl-2, 7, 17-dioxa-1, 7, 17-dioxa-3, 6-dioxa-13, 16-Triazanonane-19-sulfonic acid (33)

In a 4mL glass vial, compound 31 (8 mg, 0.006mmol), HIPS linker compound 32 (4.5 mg, 0.006mmol), DIPEA (1.5. Mu.L, 0.009 mmol) and 2.3mg (0.06 mmol) of HATU were combined in 1mL DMF. The reaction mixture was stirred at room temperature for 30 minutes, then DMF was removed in vacuo and the residue was dissolved in 1mL of methanol. To this solution was added 1mL of a 1M aqueous lithium hydroxide solution, and the resulting mixture was stirred at room temperature overnight. The reaction mixture was purified by reverse phase prep HPLC (C18 column, CH ₃ CN-H ₂ o/0.05% TFA, gradient 10-60%) and freeze-drying the pure fractions gave 6mg (0.0033 mmol,60% yield over 2 steps) of compound 33 as an off-white powder. LRMS (ESI): m/z 1770.9[ M+H ]] ⁺ For C ₈₄ H ₁₃₂ N ₁₄ O ₂₅ S m/z 1769.9.

Synthesis of constructs 44 and 46

Preparation of (2R, 3R,4S,5R, 6S) -2- (Acetoxymethyl) -6- (2-amino-5- (hydroxymethyl) phenoxy) tetrahydro-2H-pyran-3, 4, 5-trisyl triacetate (37).

To a mixture of compound 20 (5.0 g,29.9mmol,1.7 eq.) and compound 36 (7.23 g,17.6mmol,1 eq.) in anhydrous acetonitrile (100 mL) was added silver (I) oxide (15.6 g,87.9mmol,5 eq.). The mixture was stirred under nitrogen at 25 ℃ for 24 hours in the dark. The reaction mixture was diluted with EtOAc (100 mL), filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (0-30% hexanes-EtOAc). To a mixture of intermediate aldehyde (5.61 g,11.3 mmol) and triethylamine (2.5 mL) in EtOAc (80 mL) was added palladium on carbon (10 wt%, 800mg,0.75 mmol) in one portion. The reaction mixture was stirred at 25℃under H ₂ Stirring is carried out for 24 hours under an atmosphere. Filtering out the solid, and concentrating the obtained filtrate to obtain5.2g (11.1 mmol,98% yield) of product 37 as a white solid which is used in the next step without further purification.

Preparation of (2R, 3R,4S,5R, 6S) -2- (Acetoxymethyl) -6- (2- ((S) -2- ((tert-butoxycarbonyl) amino) propionylamino) -5- (hydroxymethyl) phenoxy) tetrahydro-2H-pyran-3, 4, 5-trisyl triacetate (38).

A mixture of compound 37 (5.20 g,11.1 mmol), boc-L-Ala-OH (1.75 g,9.25 mmol) and EEDQ (2.3 g,9.25 mmol) in anhydrous DCM (40 mL) and MeOH (4 mL) was stirred at room temperature in the dark for 1 hour. The reaction mixture was concentrated to give 5.5g of crude product 38 as a yellow solid. The crude product was used in the next step without further purification.

Preparation of (2R, 3R,4S,5R, 6S) -2- (Acetoxymethyl) -6- (2- ((S) -2-aminopropionamido) -5- (hydroxymethyl) phenoxy) tetrahydro-2H-pyran-3, 4, 5-trisyl triacetate (39).

The crude compound 38 (5.5 g,8.6 mmol) was dissolved in TFA (23 mL). The resulting solution was stirred at room temperature for 10 minutes and concentrated in vacuo. The residue was purified by reverse phase chromatography (C18 column, 0-75% acetonitrile-water containing 0.05% tfa). The pure fractions were combined and concentrated to give 4.0g of product amine 39 as a yellow oil (7.5 mmol,66% yield over 3 steps).

Preparation of (2S, 3R,4S,5R, 6R) -2- (2- ((S) -2- ((S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -3-methylbutanamidyl) propanamido) -5- (hydroxymethyl) phenoxy) -6- (acetoxymethyl) tetrahydro-2H-pyran-3, 4, 5-trisyl triacetate (40).

HATU (3.5 g,9.2 mmol) was added in one portion to a mixture of Fmoc-L-valine (3.1 g,9.2 mmol) and DIPEA (3.9 mL,22.2 mmol) in anhydrous DMF (20 mL) at room temperature. The resulting solution was stirred at room temperature for 30 minutes and then combined with amine 39 (4.0 g,7.5 mmol). The reaction mixture was stirred for 16 hours and concentrated in vacuo. The residue was purified by silica gel chromatography (hexanes: etOAc, 0-100%) to give compound 40 (4.5 g,5.2mmol,70% yield) as a white solid.

Preparation of (2S, 3R,4S,5R, 6R) -2- (2- ((S) -2- ((S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -3-methylbutanamidyl) propanamido) -5- (((((4-nitrophenoxy) carbonyl) oxy) methyl) phenoxy) -6- (acetoxymethyl) tetrahydro-2H-pyran-3, 4, 5-trisyl triacetate (41).

To a mixture of alcohol 40 (4.5 g,5.2 mmol) and DIPEA (4.5 mL,26.1mmol,5 eq.) in anhydrous THF (20 mL) was added bis (4-nitrophenyl) carbonate (7.9 g,26.1mmol,5 eq.). The resulting mixture was stirred at room temperature for 24 hours and concentrated under vacuum. The residue was purified by reverse phase chromatography (acetonitrile-water 0-70%, containing 0.05% tfa) to give 4-nitrophenyl carbonate product 41 as a white solid (3.9 g,73% yield). LRMS (ESI): m/z 1027.3[ M+H ] ] ⁺ For C ₅₁ H ₅₄ N ₄ O ₁₉ m/z 1027.3 calculation.

Preparation of 4- ((((4- ((S) -2-amino-3-methylbutanamide) propanamido) -3- (((2S, 3r,4S5r,6 r) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) benzyl) oxy) carbonyl) (methyl) amino) butanoic acid (42).

To a mixture of p-nitrophenyl carbonate 41 (380 mg,0.37m,1 eq.) and 4- (methylamino) butanoic acid (55 mg,0.47mmol,1.25 eq.) in anhydrous DMF (2 mL) was added DIPEA (190. Mu.L, 1.11mmol,3 eq.). The resulting mixture was stirred for 16 hours, concentrated in vacuo, and the residue was redissolved in THF (2 mL). To the solution in THF was added 2ml of 1m aqueous LiOH solution at room temperature and stirred for 2 hours until the hydrolysis was judged complete by HPLC analysis. The reaction mixture was quenched with 1M HCl (2 mL), diluted with DMF (1 mL) and purified by reverse phase chromatography (C18 column, acetonitrile-water with 0.05% tfa 0-75% mecn). The pure fractions were collected, concentrated under reduced pressure, and lyophilized to give compound 42 as a white solid (160 mg,0.26mmol,70% yield, over two steps). LRMS (ESI): m/z 615.3[ M+H ]] ⁺ For C ₂₇ H ₄₂ N ₄ O ₁₂ m/z 615.3 calculation.

Preparation of 4- ((((4- ((2 s,5s,18 r) -5-isopropyl-2-methyl-4, 7, 17, 20-tetraoxo-18- (sulfomethyl) -22- (2- ((1, 2-trimethylhydrazino) methyl) -1H-pyrrolo [2,3-b ] pyridin-1-yl) -10, 13-dioxa-3, 6, 16, 19-tetraazaeicosanamide) -3- (((2 s,3r,4s,5r,6 r) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) benzyl) oxy) carbonyl) (methyl) amino) butanoic acid (43).

To a solution of compound 32 (97 mg,0.12mmol,1 eq.) in DMF (2 mL) was added HATU (40 mg,0.1mmol,0.85 eq.) followed by DIPEA (63. Mu.L, 0.36mmol,3 eq.). The resulting mixture was stirred for 1 hour and then combined with a 0.1M solution of compound 42 in DMF (1.22 mL,0.12mmol,1 eq.). The reaction mixture was stirred for 30 minutes until all starting material was consumed as judged by HPLC analysis. The mixture was purified directly by reverse phase chromatography (C18 column, acetonitrile-water 0-75% mecn, containing 0.05% tfa). The pure fractions were collected and lyophilized to give product 43 as a white solid (107 mg,0.08mmol,67% yield). LRMS (ESI): m/z 1391.6[ M+H ]] ⁺ For C ₆₅ H ₈₆ N ₁₀ O ₂₂ S m/z 1391.6.

Preparation of (2S, 5S 18R) -1- ((4- (((S) -1- (((14S, 16S,32R,33R,2R,4S,10E,12E, 14R) -86-chloro-14-hydroxy-85, 14-dimethoxy 33,2,7, 10-tetramethyl-12, 6-dioxo-7-aza-1 (64) -oxazinan-3 (2, 3) -oxiran-8 (1, 3) -benzocyclotetradec-10, 12-dien-4-yl) oxy) -1-oxopropan-2-yl) (methyl) amino) -4-oxobutyl) (methyl) carbamoyl) oxy) methyl) -2- (((2S, 3R,4S,5R, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl) amino) -18- (3- (2- ((1, 2-dimethyl) methyl) -1H-pyrrolo [2,3-b ] pyridin-2-yl) amino) -3- (2- ((1, 2-dimethyl) pyrrol-2-yl) amino) -1-3-oxo-3, 3-oxo-propionyl-3, 17-hydrazo-yl, 6, 16-Triazanonane-19-sulfonic acid (44).

To a mixture of compound 43 (35 mg, 25. Mu. Mol) and maytansinoid 17 (16 mg, 25. Mu. Mol) in DMF (2 mL) was added HATU (19 mg, 51. Mu. Mol,2 eq.) followed by DIPEA (13. Mu.L, 76. Mu. Mol,3 eq.) at room temperature. The reaction mixture was stirred for 30 minutes until all starting material was consumed as judged by HPLC analysis. Piperidine (49 μl,0.5mmol,20 eq.) was then added directly to the solution at room temperature in one portion and the reaction mixture was stirred for 30 min. The mixture was purified by preparative HPLC (C18 column,acetonitrile-water containing 0.05% tfa 0-75% mecn). The pure fractions were combined and lyophilized to give compound 44 as a white solid (21 mg,12 μmol,48% yield). LRMS (ESI): m/z 1800.7[ M+H ]] ⁺ For C ₈₂ H ₁₁₈ CIN ₁₃ O ₂₈ 8.1800.8 calculations.

Preparation of tert-butyl 4- ((S) -2- ((S) -2-amino-3-methylbutanamidyl) propan-1-yl) -3- (((2S, 3R,4S,5S, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) benzyl ((S) -1- (((S) -1- (((3R, 4S, 5S) -1- ((S) -2- ((1R, 2R) -3- (((1S, 2R) -1-hydroxy-1-phenylpropan-2-yl) amino) -1-methoxy-2-methyl-3-oxopropyl) pyrrolidin-1-yl) -3-methoxy 5-methyl-1-oxohept-4-yl) (methyl) amino) -3-methyl-1-oxobutan-2-yl) (methyl) carbamate (45).

A solution of monomethyl auristatin A2 (50 mg, 70. Mu. Mol) in anhydrous DMF (2 mL) was treated with HOAt (9.5 mg, 70. Mu. Mol) and DIPEA (36. Mu.L, 209. Mu. Mol) at room temperature. To this mixture was added compound 41 (72 mg, 70. Mu. Mol,1 eq.) in solid form at room temperature in one portion. The reaction mixture was stirred for 4 hours until all starting material was consumed as judged by LCMS analysis. The solvent was removed in vacuo and the residue was redissolved in THF (2 mL). The THF solution was treated with 1M LiOH solution (2 mL) at room temperature and stirred for 2 hours until hydrolysis was complete. The reaction mixture was quenched with 1M HCl (2 mL), diluted with DMF (1 mL) and purified by reverse phase chromatography (C18 column, acetonitrile-water with 0.05% TFA 0-75% MeCN). The pure fractions were combined, concentrated under reduced pressure, and lyophilized to give product 45 as a white solid (68 mg,56 μmol,80% yield).

Preparation of (2S, 5S, 18R) -1- ((4- ((5S, 8S,11S, 12R) -11- ((S) -sec-butyl) -12- (2- ((S) -2- ((1R, 2R) -3- (((1S, 2R) -1-hydroxy-1-phenylpropan-2-yl) amino) -1-methoxy-2-methyl-3-oxopropyl) pyrrolidin-1-yl) -2-oxoethyl) -5, 8-diisopropyl-4, 10-dimethyl-3, 6, 9-trioxo-2, 13-dioxa-4, 7, 10-triazatetradecyl) -2- (((2S, 3R,4S,5S, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl) amino) -18- (3- (2- ((1, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2,3-b ] pyridin-1-yl) propionylamino) -5-isopropyl-1, 7, 17-dioxa-3, 6-oxa-3, 17, 6-dioxa-3, 17, 16-triazanonane-19-sulfonic acid (46).

To a solution of carboxylic acid 32 (50 mg, 63. Mu. Mol) in DMF (2 mL) was added HATU (24 mg, 63. Mu. Mol) followed by DIPEA (33. Mu.L, 189. Mu. Mol) at room temperature. The resulting mixture was stirred for 1 hour and combined with compound 45 (68 mg, 63. Mu. Mol). The reaction mixture was stirred for 5 hours until all starting material was consumed as judged by HPLC analysis. Piperidine (110 μl,20 eq) was then added to the solution at room temperature in one portion and the reaction mixture was stirred for 30 min, diluted with pH 4.70.5M acetate buffer until the solution became acidic and purified by preparative HPLC (C18 column, acetonitrile-water containing 0.05% tfa 0-75% mecn). The pure fractions were collected and lyophilized to give compound 46 as a white solid (47 mg,43% yield). LRMS (ESI): m/z 1769.9[ M+H ]] ⁺ For C ₈₄ H ₁₃₂ N ₁₄ O ₂₅ S1769.9 calculation.

Example 2

Bioconjugation, purification and HPLC analysis

Method

The C-terminal aldehyde-labeled antibody (15 mg/mL) was conjugated to linker-payloads 30, 33, 44, and 46 at 0.85mM (8 mol equivalent drug: antibody) in 20mM sodium citrate, 50mM sodium chloride pH 5.5 (20/50 buffer) containing 0.85% DMA at 37℃and reacted for 72 hours. The free drug was removed using a 30kD MWCO 0.5mL Amicon spin concentrator. The sample was added to a spin concentrator and centrifuged at 15,000Xg for 7 minutes, then diluted with 450. Mu.L of 20mM sodium citrate, 50mM sodium chloride pH 5.5 and centrifuged again. This process was repeated 10 times. To determine DAR of the final product, ADC was checked by analytical HIC (Tosoh # 14947), mobile phase a:1.5M ammonium sulfate, 25mM sodium phosphate pH 7.0 and mobile phase B:25% isopropyl alcohol, 18.75mM sodium phosphate pH 7.0. To determine aggregation, the samples were analyzed using analytical size exclusion chromatography (SEC; tosoh # 08541) with a mobile phase of 300mM sodium chloride, 25mM sodium phosphate pH 6.8.

Results

Alpha HER2 (trastuzumab) and alpha CD79b (polobutyzumab) antibodies modified to contain an aldehyde tag at the heavy chain C-terminus (CT) were conjugated to linker-payloads 30 and 33 carrying maytansine and MMAE, respectively. These reactions are high yield, with almost quantitative conjugation efficiency and > 95% overall yield. The resulting ADCs have a drug to antibody ratio (DAR) of 1.79 to 1.89 and are predominantly monomeric. Fig. 3-10 show representative ADCs for DAR as determined by HIC and monomer integrity as determined by SEC.

Alpha HER2 (trastuzumab), aTROP-2 (Sha Xituo bead mab) and anti-FITC antibodies modified to contain an aldehyde tag at the heavy chain C-terminus (CT) were conjugated to linker-payloads 44 and 46 carrying maytansine and MMAE, respectively. These reactions are generally in high yields. The resulting ADCs have a drug to antibody ratio (DAR) of 1.15 to 1.79 and are predominantly monomeric. Figures 19 to 30 record representative ADCs for DAR as determined by PLRP and monomer integrity as determined by SEC.

Example 3

In vitro cytotoxicity

Method

CD79B positive B cell lymphoma cell lines (Granta 519 and Ramos-RA), HER2+ gastric and breast cancer cell lines (NCI-N87 and SKBR 3) and TROP-2+ pancreatic and breast cancer cell lines (BxPC 3 and MDA-MB-468) were obtained from DSMZ and ATCC cell banks. Cells were maintained in growth medium as recommended by the supplier. Cells were passaged 24 hours prior to plating to ensure log phase growth. On the day of plating, 5000 cells/well were seeded into 100 μl of normal growth medium in 96-well plates. Cells were treated with 20 μl of diluted analyte at various concentrations and plates were incubated at 37 ℃ at 5% co ₂ Is incubated in an atmosphere of (2). After 5 days, 100. Mu.L/well of Cell Titer-Glo reagent (Promega) was added and luminescence was measured using a Molecular Devices SpectraMax M plate reader. Data analysis was performed using GraphPad Prism software.

Results

The galactoside and glucoside modified double-cleavage linker showed potent (sub-nanomolar) in vitro cytotoxicity against antigen-positive cell lines with activity comparable to that of the free payload and ADC carrying either single cleavage linker or glucuronide modified double cleavage linker (fig. 12 to 1 and 31 to 36).

While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

Claims

1. A conjugate, comprising:

an antibody;

a drug; and

a cleavable linker connecting the antibody to the drug and comprising a first enzymatically cleavable moiety and a second enzymatically cleavable moiety, said second enzymatically cleavable moiety comprising a glycoside selected from the group consisting of: galactoside, glucoside, mannoside, fucoside, O-GlcNAc and O-GalNAc.

2. The conjugate of claim 1, wherein the conjugate is of formula (I):

wherein the method comprises the steps of

Z is CR ⁴ Or N;

x is O or NR ⁴ ；

R ¹ Selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

R ² and R is ³ Each independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedAlkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, or R ² And R is ³ Optionally linked in an cyclic manner to form a 5-or 6-membered heterocyclyl;

k is an integer from 1 to 10;

R ⁷ comprising the second enzymatically cleavable moiety;

L ¹ is a first joint;

L ² is a second joint;

W ¹ is the medicament; and is also provided with

W ² Is the antibody.

3. The conjugate of claim 2, wherein:

k is 2; and is also provided with

The conjugate has the formula (Ia):

4. a conjugate according to any one of claims 1 to 3, wherein the second enzymatically cleavable moiety comprises a galactoside.

5. A conjugate according to any one of claims 1 to 3, wherein the second enzymatically cleavable moiety comprises a glucoside.

6. A conjugate according to any one of claims 1 to 3, wherein the second enzymatically cleavable moiety comprises a mannoside.

7. A conjugate according to any one of claims 1 to 3, wherein the second enzymatically cleavable moiety comprises a fucoside.

8. The conjugate of any one of claims 1 to 3, wherein the second enzymatically cleavable moiety comprises O-GlcNAc.

9. The conjugate of any one of claims 1 to 3, wherein the second enzymatically cleavable moiety comprises O-GalNAc.

10. A conjugate according to claim 3, wherein the conjugate has formula (Ib):

11. the conjugate of claim 3, wherein the conjugate is of formula (Ic):

12. the conjugate of claim 3, wherein the conjugate has the formula (Id):

13. a conjugate according to claim 3, wherein the conjugate has formula (Ie):

14. a conjugate according to claim 3, wherein the conjugate has formula (If):

15. the conjugate of claim 3, wherein the conjugate has the formula (Ig):

16. the conjugate of any one of claims 2 to 15, wherein L ¹ Comprising:

-(T ¹ -V ¹ ) _a -(T ² -V ² ) _b -(T ³ -V ³ ) _c -(T ⁴ -V ⁴ ) _d -，

wherein the method comprises the steps of

a. b, c and d are each independently 0 or 1;

17. The conjugate of any one of claims 2 to 16, wherein L ² Comprising:

wherein the method comprises the steps of

e. f, g and h are each independently 0 or 1;

18. The conjugate of claim 16, wherein:

wherein:

(PEG) _n is thatWherein n is an integer from 1 to 30;

4-amino-piperidine (4 AP) as

Each R ¹² And R is ¹⁵ Independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, polyethylene glycol moieties, aryl, and substituted aryl, wherein any two adjacent R ¹² The groups can be connected in a cyclic manner to form a piperazinyl ring; and is also provided with

19. The conjugate of any one of claims 16 to 18, wherein:

T ¹ is (C) ₁ -C ₁₂ ) Alkyl, and V ¹ is-CO-;

T ² is an amino acid analog, and V ² is-NH-;

T ³ is (PEG) _n And V is ³ is-CO-; and is also provided with

d is 0.

20. The conjugate of any one of claims 16 to 19, wherein:

T ⁵ is a covalent bond, and V ⁵ is-CO-; and is also provided with

f. g and h are 0.

21. The conjugate of any one of claims 16 to 19, wherein:

T ⁵ is a covalent bond, and V ⁵ is-CONR ¹⁵ -；

T ⁶ Is (C) ₁ -C ₁₂ ) Alkyl, and V ⁶ is-CO-; and is also provided with

g and h are 0.

22. The conjugate of any one of claims 1 to 21, wherein the drug is selected from the group consisting of: cytotoxins, kinase inhibitors, immunostimulants, toll-like receptor (TLR) agonists, oligonucleotides, aptamers, cytokines, steroids and peptides.

23. The conjugate of any one of claims 1 to 21, wherein the drug is selected from the group consisting of: australian statin (auristatin), maytansine (maytansine) and duocarmycin (duocarmycin).

24. The conjugate of any one of claims 1 to 21, wherein the drug is selected from the group consisting of: terpirtine M, calicheamicin, SN-38, irinotecan, STAT3 inhibitor, alpha-amanitine, aurora kinase inhibitor, belotecan, 9-aminocamptothecin (9-AC) and anthracyclines.

25. A compound, comprising:

a cleavable linker for linking an antibody to a drug, wherein the cleavable linker comprises a first enzymatically cleavable moiety and a second enzymatically cleavable moiety, the second enzymatically cleavable moiety comprising a glycoside selected from the group consisting of: galactoside, glucoside, mannoside, fucoside, O-GlcNAc and O-GalNAc.

26. The compound of claim 25, wherein the compound has formula (II):

wherein the method comprises the steps of

Z is CR ⁴ Or N;

x is O or NR ⁴ ；

each R ⁴ Independently selected from hydrogen, halogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substitutedAmino, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

k is an integer from 1 to 10;

R ⁷ comprising the second enzymatically cleavable moiety;

L ¹ is a first joint;

L ² is a second joint; and is also provided with

W1 is a drug.

27. A compound according to claim 26, wherein:

k is 2; and is also provided with

The compounds have the formula (IIa):

28. the compound of any one of claims 25-27, wherein the second cleavable moiety comprises a galactoside.

29. The compound of any one of claims 25 to 27, wherein the second cleavable moiety comprises a glucoside.

30. The compound of any one of claims 25 to 27, wherein the second cleavable moiety comprises a mannoside.

31. The compound of any one of claims 25 to 27, wherein the second cleavable moiety comprises a fucoside.

32. The compound of any one of claims 25 to 27, wherein the second cleavable moiety comprises O-GlcNAc.

33. The compound of any one of claims 25 to 27, wherein the second cleavable moiety comprises O-GalNAc.

34. The compound of claim 27, wherein the compound is of formula (IIb):

35. the compound of claim 27, wherein the compound is of formula (IIc):

36. the compound of claim 27, wherein the compound has formula (IId):

37. the compound of claim 27, wherein the compound is of formula (IIe):

38. the compound of claim 27, wherein the compound has formula (IIf):

39. the compound of claim 27, wherein the compound has formula (IIg):

40. the compound of any one of claims 26 to 39, wherein L ¹ Comprising:

-(T ¹ -V ¹ ) _a -(T ² -V ² ) _b -(T ³ -V ³ ) _c -(T ⁴ -V ⁴ ) _d -，

wherein the method comprises the steps of

a. b, c and d are each independently 0 or 1;

T ¹ 、T ² 、T ³ and T ⁴ Each independently selected from the group consisting of covalent bonds, (C) ₁ -C ₁₂ ) Alkyl, substituted (C) ₁ -C ₁₂ ) Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) _w 、(PEG) _n 、(AA) _p 、(CR ¹³ OH) _m -, 4-amino-piperidine (4 AP), acetal groups, hydrazines, disulfides and esters, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol, andand AA is an amino acid residue or amino acid analog, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;

41. The compound of any one of claims 26 to 40, wherein L ² Comprising:

wherein the method comprises the steps of

e. f, g and h are each independently 0 or 1;

T ⁵ 、T ⁶ 、T ⁷ And T ⁸ Each independently selected from the group consisting of covalent bonds, (C) ₁ -C ₁₂ ) Alkyl, substituted (C) ₁ -C ₁₂ ) Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, (EDA) _w 、(PEG) _n 、(AA) _p 、(CR ¹³ OH) _m -, 4-amino group-piperidine (4 AP), acetal groups, hydrazine, disulfides and esters, wherein EDA is an ethylenediamine moiety, PEG is polyethylene glycol, and AA is an amino acid residue or amino acid analogue, wherein each w is an integer from 1 to 20, each n is an integer from 1 to 30, each p is an integer from 1 to 20, and each m is an integer from 1 to 12;

42. The compound of claim 40, wherein:

wherein:

(PEG) _n is thatWherein n is an integer from 1 to 30;

4-amino-piperidine (4 AP) as

43. The compound of any one of claims 40 to 42, wherein:

T ¹ is (C) ₁ -C ₁₂ ) Alkyl, and V ¹ is-CO-;

T ² is an amino acid analog, and V ² is-NH-;

T ³ is (PEG) _n And V is ³ is-CO-; and is also provided with

d is 0.

44. The compound of any one of claims 40 to 43, wherein:

T ⁵ Is a covalent bond, and V ⁵ is-CO-; and is also provided with

f. g and h are 0.

45. The compound of any one of claims 40 to 43, wherein:

T ⁵ is a covalent bond, and V ⁵ is-CONR ¹⁵ -；

T ⁶ Is (C) ₁ -C ₁₂ ) Alkyl, and V ⁶ is-CO-; and is also provided with

g and h are 0.

46. The compound of any one of claims 25 to 45, wherein the drug is selected from the group consisting of: cytotoxins, kinase inhibitors, immunostimulants, toll-like receptor (TLR) agonists, oligonucleotides, aptamers, cytokines, steroids and peptides.

47. The compound of any one of claims 25 to 45, wherein the drug is selected from the group consisting of: auristatin, maytansine, and sesquialter.

48. The compound of any one of claims 25 to 45, wherein the drug is selected from the group consisting of: terpirtine M, calicheamicin, SN-38, irinotecan, STAT3 inhibitor, alpha-amanitine, aurora kinase inhibitor, belotecan, 9-aminocamptothecin (9-AC) and anthracyclines.

49. A pharmaceutical composition comprising:

the conjugate of any one of claims 1 to 24; and

pharmaceutically acceptable excipients.

50. A method for administering a conjugate to a subject, the method comprising:

administering the conjugate of any one of claims 1 to 24 to a subject.

51. A method for treating cancer in a subject, the method comprising:

administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising the conjugate of any one of claims 1 to 24, wherein the administration is effective to treat cancer in the subject.