CN116847844A

CN116847844A - Camptothecine antibody-drug conjugates and methods of use thereof

Info

Publication number: CN116847844A
Application number: CN202280011264.XA
Authority: CN
Inventors: S·丘普拉科夫; A·O·奥贡科亚; P·M·德雷克
Original assignee: RP Scherer Technologies LLC
Current assignee: RP Scherer Technologies LLC
Priority date: 2021-01-15
Filing date: 2022-01-13
Publication date: 2023-10-03

Abstract

The present disclosure provides antibody-drug conjugate (ADC) structures, wherein the antibody-drug conjugate comprises a camptothecin or camptothecin derivative linked to a polypeptide (e.g., an antibody) by a linker. In addition, the present disclosure also encompasses compounds and methods for producing such conjugates, as well as methods of using the conjugates.

Description

Camptothecine antibody-drug conjugates and methods of use thereof

Cross Reference to Related Applications

The disclosure of the present application claims the benefit of U.S. provisional application No. 63/237,355 filed on month 8, 26 of 2021, U.S. provisional application No. 63/214,525 filed on month 6, 24 of 2021, U.S. provisional application No. 63/186,489 filed on month 5, 10 of 2021, and U.S. provisional application No. 63/138,182 filed on month 1, 15 of 2021 is incorporated herein by reference.

Introduction to the application

The field of protein-small molecule therapeutic conjugates has made tremendous progress, providing many clinically beneficial agents, and hopefully providing more such agents in the next few years. Protein-conjugate therapeutic drugs may offer several advantages, resulting in reduced side effects, due to, for example, specificity, functional diversity, and relatively low off-target activity.

Disclosure of Invention

Camptothecins are a class of antineoplastic agents that have a common structural core i (figure 1, panel a). Because of their ability to inhibit the activity of DNA topoisomerase I, an intracellular enzyme critical for cell replication, several synthetic and semisynthetic camptothecins have been used as small molecule cancer chemotherapies (e.g., topotecan and irinotecan). In contrast, more potent analogs, such as SN-38 (1) and exenatide (3) (fig. 1, panel B), showed significant off-target toxicity, preventing their direct use in the treatment of cancer. Targeting delivery of highly potent camptothecins to tumor tissue can avoid toxicity issues and provide a more tolerated therapy. The present disclosure describes the preparation of antibody-drug conjugates (ADCs) containing camptothecins of general structure i (fig. 1, panel a). In each conjugate, the selected cytotoxin was linked to an antibody (e.g., mAb) either through a cleavable linker attached to a common C20 alcohol or through various chemical handles (chemical handles) specific for a particular camptothecin analog (fig. 1).

The present disclosure provides antibody-drug conjugate (ADC) structures, wherein the antibody-drug conjugate comprises a camptothecin or camptothecin derivative linked to a polypeptide (e.g., an antibody) by a linker. In addition, the present disclosure also encompasses compounds and methods for producing such conjugates, as well as methods of using these conjugates.

Aspects of the disclosure include conjugates of formula (I):

wherein:

z is CR ¹⁰ Or N, or a combination of two,

R ⁷ 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 ⁸ and R is ⁹ 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 ⁸ And R is ⁹ Optionally cyclic linked to form a 5-or 6-membered heterocyclyl;

each R is ¹⁰ 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, and substituted aryl Substituted cycloalkyl, heterocyclyl and substituted heterocyclyl;

w is a polypeptide;

l is R ¹ 、R ² 、R ³ 、R ⁴ 、R ⁵ Or R is ⁶ A linker attached to a compound of formula (II):

R ¹ and R is ² Each independently selected from hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, or R ¹ And R is ² Optionally cyclic linked to form a 5-or 6-membered cycloalkyl or heterocyclyl ring;

R ³ and R is ⁴ Each independently selected from hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, or R ³ And R is ⁴ Optionally cyclic linked to form a 5-or 6-membered cycloalkyl or heterocyclyl ring;

R ⁵ selected from the group consisting of hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

R ⁶ Selected from OH or OC (O) R ¹¹ The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

R ¹¹ Selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substitutedHeteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl,

wherein at least one R ¹⁰ Optionally to a second compound of formula (II).

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

wherein R is ³ OH and L is attached to R ⁶ A place; or L is attached to R ³ At and R is ⁶ OH; or wherein the compound of formula (II) has the structure of formula (IIb):

wherein R is ^1a Selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl, and L is attached to R ⁶ A place; or L is attached to R ^1a At and R is ⁶ OH; or (b)

Wherein the compound of formula (II) has the structure of formula (IIc):

wherein R is ^1b Selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl, and L is attached to R ⁶ A place; or L is attached to R ^1b At and R is ⁶ OH; or (b)

Wherein the compound of formula (II) has the structure of formula (IId):

wherein R is ^2a And R is ^2b Each independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl, and L is attached to R ⁶ A place; or L is attached to R ^2a At and R is ^2b And R is ⁶ OH; or wherein the compound of formula (II) has the structure of formula (IIe):

wherein R is ^2c 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, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl, and attachment to L is indicated by wavy lines.

In some embodiments, L comprises:

-(T ¹ -V ¹ ) _a -(T ² -V ² ) _b -(T ³ -V ³ ) _c -(T ⁴ -V ⁴ ) _d -(T ⁵ -V ⁵ ) _e -(T ⁶ -V ⁶ ) _f -，

wherein the method comprises the steps of

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

T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ and T ⁶ 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), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), 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 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;

V ¹ 、V ² 、V ³ 、V ⁴ 、V ⁵ and V ⁶ 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;

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

Each R is 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 is a linker, wherein:

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

T ² 、T ³ 、T ⁴ 、T ⁵ and T ⁶ 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, heterocyclylAnd substituted heterocyclyl, (EDA) _w 、(PEG) _n 、(AA) _p 、-(CR ¹³ OH) _m -, 4-amino-piperidine (4 AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal groups, hydrazine and esters; and is also provided with

V ¹ 、V ² 、V ³ 、V ⁴ 、V ⁵ And V ⁶ 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) asAnd is also provided with

Each R is ¹² 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 linked cyclic to form a piperazinyl ring.

In some embodiments, T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ And T ⁶ Each optionally substituted with a glycoside. In some embodiments, each of MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP is optionally substituted with a glycoside. In some embodiments, the glycoside is selected from the group consisting of glucuronide, galactoside, glucoside, Mannosides, fucosides, O-GlcNAc and O-GalNAc.

In some embodiments, L is a linker, 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 ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of; and is also provided with

T ⁶ Is EDA and V ⁶ is-CO-; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is absent and V ⁵ is-NR ¹⁵ (C ₆ H ₄ ) -; and is also provided with

T ⁶ Is absent and V ⁶ is-CO-; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ is-NR ¹⁵ -; and is also provided with

T ⁶ Is (C) ₁ -C ₁₂ ) Alkyl and V ⁶ is-CO-; or wherein:

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

T ² is AA and V ² Absence of;

T ³ is PABC and V ³ Absence of;

T ⁴ is EDA and V ⁴ is-CO-; and is also provided with

e and f are each 0; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of; and is also provided with

f is 0; or (b)

Wherein:

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

T ² is AA and V ² Absence of;

T ³ is PABC and V ³ Absence of; and is also provided with

d. e and f are each 0; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABA and V ⁵ is-CO-; and is also provided with

T ⁶ Is (C) ₁ -C ₁₂ ) Alkyl andV ⁶ is-SO ₂ -; or wherein:

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

T ² for (PEG) _n And V is ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

e and f are each 0; or (b)

Wherein:

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

T ² is substituted (C) ₁ -C ₁₂ ) Alkyl and V ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

e and f are each 0; or (b)

Wherein:

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

T ² for (PEG) _n And V is ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

T ⁵ is (C) ₁ -C ₁₂ ) Alkyl and V ⁵ Absence of;

f is 0; or (b)

Wherein:

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

T ² 4AP and V ² is-CO-;

T ³ is (C) ₁ -C ₁₂ ) Alkyl and V ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of;

f is 0; or (b)

Wherein:

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

T ² 4AP and V ² is-CO-;

T ³ is (C) ₁ -C ₁₂ ) Alkyl and V ³ is-O-;

T ⁴ is (C) ₁ -C ₁₂ ) Alkyl and V ⁴ is-CO-;

T ⁵ is AA and V ⁵ Absence of;

T ⁶ is PABC and V ⁶ Absence of; or (b)

Wherein:

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

T ² is an amino acid analog and V ² Absence of;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

e and f are each 0; or (b)

Wherein:

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

T ² for (PEG) _n And V is ² is-CONH-;

T ³ is substituted (C) ₁ -C ₁₂ ) Alkyl and V ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of;

f is 0; or (b)

Wherein:

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

T ² is AA and V ² is-NH-;

T ³ for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of;

f is 0; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABO and V ⁵ Absence of; and is also provided with

f is 0; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PAP and V ⁵ is-COO-; and is also provided with

f is 0; or (b)

Wherein:

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

T ² for (PEG) _n And V is ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PAP and V ⁴ is-COO-; and is also provided with

e and f are each 0.

In some embodiments, one R ¹⁰ Via a second joint L ^B Is linked to a second compound of formula (II).

In some embodiments, L ^B Comprising:

-(T ⁷ -V ⁷ ) _g -(T ⁸ -V ⁸ ) _h -(T ⁹ -V ⁹ ) _i -(T ¹⁰ -V ¹⁰ ) _j -(T ¹¹ -V ¹¹ ) _k -(T ¹² -V ¹² ) _l -, wherein

g. h, i, j, k and 1 are each independently 0 or 1;

T ⁷ 、T ⁸ 、T ⁹ 、T ¹⁰ 、T ¹¹ and T ¹² 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), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), 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 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;

V ⁷ 、V ⁸ 、V ⁹ 、V ¹⁰ 、V ¹¹ and V ¹² 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, wherein T ⁷ 、T ⁸ 、T ⁹ 、T ¹⁰ 、T ¹¹ And T ¹² Each optionally substituted with a glycoside. In some embodiments, each of MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP is optionally substituted with a glycoside. In some embodiments, the glycoside is selected from the group consisting of glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

In some embodiments, L ^B Is a joint, wherein:

T ⁷ is absent and V ⁷ is-NR ¹⁵ CO-；

T ⁸ Is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

T ¹¹ is EDA and V ¹¹ is-CO-; and is also provided with

l is 0; or (b)

Wherein:

T ⁷ is absent and V ⁷ is-NR ¹⁵ CO-；

T ⁸ Is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;and is also provided with

k and l are each 0; or (b)

Wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

T ⁸ is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is an amino acid analog and V ⁹ is-NH-;

T ¹⁰ for (PEG) _n And V is ¹⁰ is-CO-;

T ¹¹ is AA and V ¹¹ Absence of; and is also provided with

T ¹² Is PABC and V ¹² Absence of; or (b)

Wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

T ⁸ is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CONH-;

T ⁹ for (PEG) _n And V is ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of; and is also provided with

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ is substituted (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

k and l are each 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ for (PEG) _n And V is ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

T ¹¹ is (C) ₁ -C ₁₂ ) Alkyl and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ 4AP and V ⁸ is-CO-;

T ⁹ is (C) ₁ -C ₁₂ ) Alkyl and V ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ 4AP and V ⁸ is-CO-;

T ⁹ is (C) ₁ -C ₁₂ ) Alkyl and V ⁹ is-O-;

T ¹⁰ is (C) ₁ -C ₁₂ ) Alkyl and V ¹⁰ is-CO-;

T ¹¹ is AA and V ¹¹ Absence of;

T ¹² is PABC and V ¹² Absence of; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ is an amino acid analog and V ⁸ Absence of;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

k and l are each 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ for (PEG) _n And V is ⁸ is-CONH-;

T ⁹ is substituted (C) ₁ -C ₁₂ ) Alkyl and V ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ is AA and V ⁸ is-NH-;

T ⁹ for (PEG) _n And V is ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ for (PEG) _n And V is ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PAP and V ¹⁰ is-COO-; and is also provided with

k and l are each 0.

In some embodiments, the conjugate is selected from:

/>

aspects of the disclosure include compounds of formula (III):

wherein:

z is CR ¹⁰ Or N, or a combination of two,

each R is ¹⁰ 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;

R ¹¹ 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,

wherein at least one R ¹⁰ Optionally to a second compound of formula (II).

Wherein the compound of formula (II) has the structure of formula (IIc):

Wherein the compound of formula (II) has the structure of formula (IId):

In some embodiments, L comprises:

wherein the method comprises the steps of

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

T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ and T ⁶ 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), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), 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 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 30An integer of 1 to 20, and each m is an integer of 1 to 12;

In some embodiments, L is a linker, wherein:

T ² 、T ³ 、T ⁴ 、T ⁵ and T ⁶ 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), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal groups, hydrazine and esters; and is also provided with

wherein:

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

4-amino-piperidine (4 AP) asAnd is also provided with

In some embodiments, T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ And T ⁶ Each optionally substituted with a glycoside. In some embodiments, each of MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP is optionally substituted with a glycoside. In some embodiments, the glycoside is selected from the group consisting of glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

In some embodiments, L is a linker, 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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of; and is also provided with

T ⁶ Is EDA and V ⁶ is-CO-; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁶ Is absent and V ⁶ is-CO-; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ is-NR ¹⁵ -; and is also provided with

T ⁶ Is (C) ₁ -C ₁₂ ) Alkyl and V ⁶ is-CO-; or wherein:

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

T ² Is AA and V ² Absence of;

T ³ is PABC and V ³ Absence of;

T ⁴ is EDA and V ⁴ is-CO-; and is also provided with

e and f are each 0; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of; and is also provided with

f is 0; or (b)

Wherein:

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

T ² is AA and V ² Absence of;

T ³ is PABC and V ³ Absence of; and is also provided with

d. e and f are each 0; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABA and V ⁵ is-CO-; and is also provided with

T ⁶ Is (C) ₁ -C ₁₂ ) Alkyl and V ⁶ is-SO ₂ -; or wherein:

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

T ² for (PEG) _n And V is ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of; and is also provided with

e and f are each 0; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABA and V ⁵ is-CO-; and is also provided with

T ⁶ Is (C) ₁ -C ₁₂ ) Alkyl and V ⁶ is-SO ₂ -; or wherein:

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

T ² for (PEG) _n And V is ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

e and f are each 0; or (b)

Wherein:

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

T ² is substituted (C) ₁ -C ₁₂ ) Alkyl and V ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

e and f are each 0; or (b)

Wherein:

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

T ² for (PEG) _n And V is ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

T ⁵ is (C) ₁ -C ₁₂ ) Alkyl and V ⁵ Absence of;

f is 0; or (b)

Wherein:

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

T ² 4AP and V ² is-CO-;

T ³ is (C) ₁ -C ₁₂ ) Alkyl and V ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of;

f is 0; or (b)

Wherein:

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

T ² 4AP and V ² is-CO-;

T ³ is (C) ₁ -C ₁₂ ) Alkyl and V ³ is-O-;

T ⁴ is (C) ₁ -C ₁₂ ) Alkyl and V ⁴ is-CO-;

T ⁵ is AA and V ⁵ Absence of;

T ⁶ is PABC and V ⁶ Absence of; or (b)

Wherein:

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

T ² is an amino acid analog and V ² Absence of;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

e and f are each 0; or (b)

Wherein:

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

T ² for (PEG) _n And V is ² is-CONH-;

T ³ Is substituted (C) ₁ -C ₁₂ ) Alkyl and V ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of;

f is 0; or (b)

Wherein:

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

T ² is AA and V ² is-NH-;

T ³ for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of;

f is 0; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABO and V ⁵ Absence of; and is also provided with

f is 0; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PAP and V ⁵ is-COO-; and is also provided with

f is 0; or (b)

Wherein:

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

T ² for (PEG) _n And V is ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PAP and V ⁴ is-COO-; and is also provided with

e and f are each 0.

In some embodiments, L ^B Comprising:

-(T ⁷ -V ⁷ ) _g -(T ⁸ -V ⁸ ) _h -(T ⁹ -V ⁹ ) _i -(T ¹⁰ -V ¹⁰ ) _j -(T ¹¹ -V ¹¹ ) _k -(T ¹² -V ¹² ) _l -，

wherein the method comprises the steps of

g. h, i, j, k and 1 are each independently 0 or 1;

T ⁷ 、T ⁸ 、T ⁹ 、T ¹⁰ 、T ¹¹ and T ¹² 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), m-amino-benzyloxy (MABO), m-amino-benzyloxycarbonyl (MABC), p-amino-benzyloxy (PABO), p-amino-benzyloxycarbonylA group (PABC), p-aminobenzyl (PAB), p-amino-benzylamino (PABA), p-amino-phenyl (PAP), p-hydroxy-phenyl (PHP), 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;

In some embodiments, T ⁷ 、T ⁸ 、T ⁹ 、T ¹⁰ 、T ¹¹ And T ¹² Each optionally substituted with a glycoside. In some embodiments, each of MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP is optionally substituted with a glycoside. In some embodiments, the glycoside is selected from the group consisting of glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

In some embodiments, L ^B Is a joint, wherein:

T ⁷ is absent and V ⁷ is-NR ¹⁵ CO-；

T ⁸ Is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

T ¹¹ is EDA and V ¹¹ is-CO-; and is also provided with

l is 0; or (b)

Wherein:

T ⁷ is absent and V ⁷ is-NR ¹⁵ CO-；

T ⁸ Is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of; and is also provided with

k and l are each 0; or (b)

Wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

T ⁸ is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is an amino acid analog and V ⁹ is-NH-;

T ¹⁰ for (PEG) _n And V is ¹⁰ is-CO-;

T ¹¹ is AA and V ¹¹ Absence of; and is also provided with

T ¹² Is PABC and V ¹² Absence of; or (b)

Wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

T ⁸ is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CONH-;

T ⁹ for (PEG) _n And V is ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of; and is also provided with

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ is substituted (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

k and l are each 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ for (PEG) _n And V is ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

T ¹¹ is (C) ₁ -C ₁₂ ) Alkyl and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ 4AP and V ⁸ is-CO-;

T ⁹ is (C) ₁ -C ₁₂ ) Alkyl and V ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ 4AP and V ⁸ is-CO-;

T ⁹ is (C) ₁ -C ₁₂ ) Alkyl and V ⁹ is-O-;

T ¹⁰ is (C) ₁ -C ₁₂ ) Alkyl and V ¹⁰ is-CO-;

T ¹¹ is AA and V ¹¹ Absence of;

T ¹² is PABC and V ¹² Absence of; or wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ is an amino acid analog and V ⁸ Absence of;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

k and l are each 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ for (PEG) _n And V is ⁸ is-CONH-;

T ⁹ is substituted (C) ₁ -C ₁₂ ) Alkyl and V ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ is AA and V ⁸ is-NH-;

T ⁹ for (PEG) _n And V is ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ for (PEG) _n And V is ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PAP and V ¹⁰ is-COO-; and is also provided with

k and l are each 0.

In some embodiments, the compound is selected from:

/>

aspects of the present disclosure include a pharmaceutical composition comprising a conjugate according to the present disclosure and a pharmaceutically acceptable excipient.

Aspects of the disclosure include a method comprising administering to a subject an effective amount of a conjugate according to the disclosure.

Aspects of the present disclosure include a method of treating cancer in a subject by administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a conjugate according to the present disclosure, wherein administration is effective to treat the cancer in the subject.

Drawings

Figure 1 shows the chemical structure of the camptothecin family of topoisomerase I inhibitors. (FIG. 1, panel A) general structure of camptothecins. (fig. 1, panel B) representative examples of camptothecins used as antibody-drug conjugate payloads (arrows indicate modification sites for linker attachment).

Fig. 2 shows a schematic diagram of a synthesized HIPS connection for ADC. Antibodies carrying aldehyde moieties are reacted with hydro-iso-Pictet-Spengler (HIPS) linkers and payloads to generate site-specific conjugated ADCs with stable aza-carboline linkages.

Figure 3 shows a polymer reverse phase column (PLRP) trace of compound 12 conjugate 2941, which contains two tag sites and produces a DAR of 2.75, 2.2% HMW.

Figure 4 shows analytical Size Exclusion Chromatography (SEC) traces of compound 12 conjugate 2941, which contained two tag sites and produced a DAR of 2.75, 2.2% HMW.

Figure 5 shows PLRP trace of compound 12 conjugate 2944, which contains two tag sites and produces a DAR of 3.12, 4.5% HMW.

Figure 6 shows SEC trace of compound 12 conjugate 2944, which contains two tag sites and produces DAR of 3.12, 4.5% HMW.

Figure 7 shows PLRP trace of compound 12 conjugate 2746, which contains two tag sites and produces 2.78 DAR, 3.4% HMW.

Figure 8 shows a Hydrophobic Interaction Column (HIC) trace of compound 12 conjugate 2746, which contains two tag sites and produces 2.78 DAR, 3.4% HMW.

Figure 9 shows SEC trace of compound 12 conjugate 2746, which contains two tag sites and produces 2.78 DAR, 3.4% HMW.

Figure 10 shows PLRP trace of compound 12 conjugate 2749, which contains two tag sites and produces a DAR of 3.04, 0.9% HMW.

Figure 11 shows SEC trace of compound 12 conjugate 2749, which contains two tag sites and produces 3.04 DAR, 0.9% HMW.

Figure 12 shows PLRP trace of compound 12 conjugate 2752, which contains two tag sites and produces a DAR of 3.01, 4.3% HMW.

Fig. 13 shows the HIC trace of compound 12 conjugate 2752, which contains two tag sites and produces a DAR of 3.01, 4.3% HMW.

Figure 14 shows SEC trace of compound 12 conjugate 2752, which contains two tag sites and produces 3.01 DAR, 4.3% HMW.

Figure 15 shows PLRP trace of compound 12 conjugate 2755, which contains two tag sites and produces a DAR of 2.72, 3.2% HMW.

Fig. 16 shows the HIC trace of compound 12 conjugate 2755, which contains two tag sites and produces a DAR of 2.72, 3.2% HMW.

Figure 17 shows SEC trace of compound 12 conjugate 2755, which contains two tag sites and produces a DAR of 2.72, 3.2% HMW.

Fig. 18 shows the HIC trace of compound 12 conjugate 2758, which contains one tag site and produces 1.54 DAR, 1.6% HMW.

Figure 19 shows SEC trace of compound 12 conjugate 2758, which contains one tag site and produces DAR of 1.54, 1.6% HMW.

Figure 20 shows PLRP trace of compound 12 conjugate 2762, which contains two tag sites and produces 2.95 DAR, 3.6% HMW.

Figure 21 shows SEC trace of compound 20 conjugate 2762, which contains two tag sites and produces 2.95 DAR, 3.6% HMW.

Figure 22 shows PLRP trace of compound 20 conjugate 2765, which contains two tag sites and produces 2.97 DAR, 1.2% HMW.

Figure 23 shows SEC trace of compound 20 conjugate 2765, which contains two tag sites and produces 2.97 DAR, 1.2% HMW.

Figure 24 shows a PLRP trace of compound 20 conjugate 2768, which contains two tag sites and produces a DAR of 2.92, 5.3% HMW.

Figure 25 shows SEC trace of compound 20 conjugate 2768, which contains two tag sites and produces a DAR of 2.92, 5.3% HMW.

Figure 26 shows PLRP trace of compound 20 conjugate 2771, which contains two tag sites and produces DAR of 2.89, 4.3% HMW.

Figure 27 shows SEC trace of compound 20 conjugate 2771, which contains two tag sites and produces DAR of 2.89, 4.3% HMW.

Fig. 28 shows the HIC trace of compound 20 conjugate 2774, which contains one tag site and produces 1.22 DAR, 1.8% HMW.

Figure 29 shows SEC trace of compound 20 conjugate 2774, which contains one tag site and produces 1.22 DAR, 1.8% HMW.

Figure 30 shows a PLRP trace of compound 27 conjugate 2763, which contains two tag sites and produces a DAR of 2.43, 6.0% HMW.

Figure 31 shows SEC trace of compound 27 conjugate 2763, which contains two tag sites and produces a DAR of 2.43, 6.0% HMW.

Figure 32 shows a PLRP trace of compound 27 conjugate 2766, which contains two tag sites and produces a DAR of 2.43, 2.2% HMW.

Figure 33 shows SEC trace of compound 27 conjugate 2766, which contains two tag sites and produces a DAR of 2.43, 2.2% HMW.

Figure 34 shows a PLRP trace of compound 27 conjugate 2769, which contains two tag sites and produces a DAR of 2.42, 8.2% HMW.

Figure 35 shows SEC trace of compound 27 conjugate 2769, which contains two tag sites and produces a DAR of 2.42, 8.2% HMW.

Figure 36 shows PLRP trace of compound 27 conjugate 2772, which contains two tag sites and produces 2.47 DAR, 6.0% HMW.

Figure 37 shows SEC trace of compound 27 conjugate 2772, which contains two tag sites and produces DAR of 2.47, 6.0% HMW.

Fig. 38 shows the HIC trace of compound 27 conjugate 2775, which contains one tag site and produces a DAR of 0.45, 1.4% HMW.

Figure 39 shows the SEC trace of compound 27 conjugate 2775, which contains one tag site and produces a DAR of 0.45, 1.4% HMW.

Figure 40 shows the PLRP trace of compound 34 conjugate 2942, which contains two tag sites and produces a DAR of 2.78, 11.2% HMW.

Figure 41 shows the SEC trace of compound 34 conjugate 2942, which contains two tag sites and produces a DAR of 2.78, 11.2% HMW.

Figure 42 shows PLRP trace of compound 34 conjugate 2945, which contains two tag sites and produces a DAR of 3.03, 5.2% HMW.

Figure 43 shows SEC trace of compound 34 conjugate 2945, which contains two tag sites and produces a DAR of 3.03, 5.2% HMW.

Figure 44 shows PLRP trace of compound 42 conjugate 2943, which contains two tag sites and produces a DAR of 5.44, 21.3% HMW.

Figure 45 shows SEC trace of compound 42 conjugate 2943, which contains two tag sites and produces a DAR of 5.44, 21.3% HMW.

Figure 46 shows PLRP trace of compound 42 conjugate 2946, which contains two tag sites and produces a DAR of 5.07, 15.3% HMW.

Figure 47 shows SEC trace of compound 42 conjugate 2946, which contains two tag sites and produces a DAR of 5.07, 15.3% HMW.

Fig. 48 shows PLRP trace of compound 45 conjugate 2748, which contains two tag sites and produces a DAR of 3.51, 3.6% HMW.

Fig. 49 shows the HIC trace of compound 45 conjugate 2748, which contains two tag sites and produces a DAR of 3.51, 3.6% HMW.

Figure 50 shows SEC trace of compound 45 conjugate 2748, which contains two tag sites and produces a DAR of 3.51, 3.6% HMW.

Figure 51 shows the PLRP trace of compound 45 conjugate 2751, which contains two tag sites and produces a DAR of 2.98, 2.1% HMW.

Figure 52 shows the SEC trace of compound 45 conjugate 2751, which contains two tag sites and produces a DAR of 2.98, 2.1% HMW.

Figure 53 shows the PLRP trace of compound 45 conjugate 2754, which contains two tag sites and produces a DAR of 3.5, 2.3% HMW.

Fig. 54 shows the HIC trace of compound 45 conjugate 2754, which contains two tag sites and produces 3.5 DAR, 2.3% HMW.

Figure 55 shows SEC trace of compound 45 conjugate 2754, which contains two tag sites and produces 3.5 DAR, 2.3% HMW.

Figure 56 shows PLRP trace of compound 45 conjugate 2757, which contains two tag sites and produces a DAR of 3.69, 2.0% HMW.

Figure 57 shows SEC trace of compound 45 conjugate 2757, which contains two tag sites and produces DAR of 3.69, 2.0% HMW.

Fig. 58 shows the HIC trace of compound 45 conjugate 2760, which contains one tag site and produces 1.65 DAR, 0.8% HMW.

Figure 59 shows the SEC trace of compound 45 conjugate 2760, which contains one tag site and produces 1.65 DAR, 0.8% HMW.

Figure 60 shows the PLRP trace of compound 47 conjugate 3065, which contains two tag sites and produces a DAR of 6.82, 1.8% HMW.

Figure 61 shows the SEC trace of compound 47 conjugate 3065, which contains two tag sites and produces a DAR of 6.82, 1.8% HMW.

Fig. 62 shows PLRP trace of compound 47 conjugate 3066, which contains two tag sites and produces a DAR of 4.48.

Fig. 63 shows the HIC trace of compound 47 conjugate 3067, which contains one tag site and produces a DAR of 3.63, 4.4% HMW.

Figure 64 shows the SEC trace of compound 47 conjugate 3067, which contains one tag site and produces 3.63 DAR, 4.4% HMW.

Fig. 65 shows the HIC trace of compound 47 conjugate 3068, which contains one tag site and produces a DAR of 3.52, 0.8% HMW.

Fig. 66 shows SEC trace of compound 47 conjugate 3068, which contains one tag site and produces DAR of 3.52, 0.8% HMW.

Fig. 67 shows PLRP trace of compound 56 conjugate 3063, which contains two tag sites and produces a DAR of 3.35, 3.3% HMW.

Figure 68 shows the SEC trace of compound 56 conjugate 3063, which contains two tag sites and produces a DAR of 3.35, 3.3% HMW.

Fig. 69 shows PLRP trace of compound 56 conjugate 3064, which contains two tag sites and produces a DAR of 2.8.

FIG. 70 shows a graph of an in vitro cytotoxicity assay of a free topoisomerase inhibitor in NCI-N87 gastric cancer cells.

FIG. 71 shows a graph of an in vitro cytotoxicity assay of a free topoisomerase inhibitor in Sk-Br-3 breast cancer cells.

Figure 72 shows a graph of in vitro cytotoxicity assays of free topoisomerase inhibitors in Granta NHL cells.

FIG. 73 shows a graph of an in vitro cytotoxicity assay of a free topoisomerase inhibitor in MDA-MB-468 breast cancer cells.

FIG. 74 shows a graph of an in vitro cytotoxicity assay of a free topoisomerase inhibitor in MDA-PCa-2b prostate cancer cells.

FIG. 75 shows a graph of an in vitro cytotoxicity assay of TROP-2-targeting ADCs prepared using compound 61 in MDA-MB-468 breast cancer cells, according to an embodiment of the disclosure.

Figure 76 shows a graph of an in vitro cytotoxicity assay of HER 2-targeting ADCs prepared using compound 61 in NCI-N87 gastric cancer cells, according to an embodiment of the disclosure.

FIG. 77 shows a graph of an in vitro cytotoxicity assay of CD 25-targeted ADCs prepared using compound 61 in SU-DHL-1ALCL cells, according to an embodiment of the present disclosure.

FIG. 78 shows a graph of an in vitro cytotoxicity assay of TROP-2-targeting ADCs prepared using compound 61 in BxPC-3 pancreatic cancer cells according to an embodiment of the disclosure.

Figure 79 shows a graph of an in vitro cytotoxicity assay of HER 2-targeting ADCs prepared using compound 65 in NCI-N87 gastric cancer cells, according to an embodiment of the disclosure.

Figure 80 shows a graph of an in vitro cytotoxicity assay of HER 2-targeting ADCs prepared using compound 65 in Sk-Br-3 breast cancer cells, according to an embodiment of the disclosure.

FIG. 81 shows a schematic of ELISA assays for determining total antibody and ADC concentrations for Pharmacokinetic (PK) sample analysis.

FIG. 82 shows a graph of concentration (μg/mL) versus days post-administration after administration of trastuzumab antibody at a dose of 0.9 mg/kg.

Figure 83 shows a graph of concentration (μg/mL) versus days post-administration after use of a conventional HER2 topoisomerase inhibitor conjugated ADC with a protease cleavable linker at a dose of 0.9 mg/kg.

FIG. 84 shows a graph of concentration (μg/mL) versus days post-dosing after using a dose of 0.9mg/kg of CH 1-3/CT-labeled trastuzumab conjugated to construct 61 according to an embodiment of the present disclosure.

FIG. 85 shows a graph of concentration (μg/mL) versus days post-dosing after using a dose of 0.9mg/kg of CH 1-3/CT-labeled trastuzumab conjugated to construct 65 according to an embodiment of the present disclosure.

Figure 86 shows average tumor volume (mm ³ ) A graph of relationship to days indicating in vivo efficacy of TROP-2 targeted ADC carrying a topoisomerase inhibitor payload against NCI-H292 xenografts. n=8 mice/group; administration is indicated by arrows.

FIG. 87 shows average tumor volume (mm ³ ) Graph of the relationship with days, which indicates carrying topoisomeraseIn vivo efficacy of TROP-2 targeted ADC of enzyme inhibitor payload against NCI-H292 xenografts. n=7 mice/group. A single intravenous dose was delivered on day 0.

Fig. 88 shows average tumor volume (mm ³ ) A graph of relationship to days indicating in vivo efficacy of a topoisomerase inhibitor payload-targeted connexin-4 ADC against NCI-H1781 xenografts. n=5 mice/group. Doses of 5mg/kg were delivered intravenously on day 0 and day 7.

FIG. 89 shows a graph of in vitro potency of TROP-2-targeted or isotype control ADCs carrying (165), (65), (175) against MBA-MB-468 cells compared to (1) or (2).

FIG. 90 shows a graph of in vitro potency of TROP-2-targeted or isotype control ADCs carrying (165), (65), (175) against BxPC-3 cells compared to (1) or (2).

Figure 91 shows a graph of in vitro potency of HER 2-targeted or isotype control ADC carrying (65), (67) or (73) against SK-BR-3 cells compared to (2).

Figure 92 shows a graph of in vitro potency of HER 2-targeted or isotype control ADC carrying (65), (67) or (73) against NCI-N87 cells compared to (2).

FIG. 93 shows a graph of in vitro potency of TROP-2-targeted or isotype control ADCs carrying (65) or (80) against MDA-MB-468 cells as compared to (2).

FIG. 94 shows a graph of in vitro potency of TROP-2 targeted ADC or isotype control ADC carrying (65) or (80) against BxPC-3 cells compared to (2).

Figure 95 shows a graph of in vitro potency of HER 2-targeted or isotype control ADC carrying (99), (103) or (110) against NCI-N87 cells compared to (2).

Figure 96 shows a graph of in vitro potency of HER 2-targeted or isotype control ADC carrying (65), (86) or (92) against SK-BR-3 cells compared to (2).

FIG. 97 shows a graph of in vitro potency of a TROP-2-targeted ADC or isotype control ADC, trodelvy or CL2A-SN38 isotype control conjugate carrying (65) against SK-BR-3 cells compared to (1) or (2).

FIG. 98 shows a graph of in vitro potency of HER 2-targeted ADC or isotype control ADC, enhertu or MC-GGFG-Dxd isotype control conjugate carrying (65) against NCI-N87 cells compared to (2).

FIG. 99 shows a graph of in vitro potency of TROP-2-targeted or isotype control ADCs carrying (65) or (113) against MDA-MB-468 cells as compared to (2).

Graph 100 shows a graph of in vitro potency of HER 2-targeted or isotype control ADC carrying (65), (136) or (142) against NCI-N87 cells compared to (2).

FIG. 101 shows a graph of in vitro potency of TROP-2-targeted or isotype control ADCs carrying (65), (127) or (131) against MDA-MB-468 cells as compared to (2).

Figure 102 shows a graph of in vitro potency of HER 2-targeted or isotype control ADC carrying (65), (127) or (131) against NCI-N87 cells compared to (2).

FIG. 103 shows a graph of in vitro potency of TROP-2 targeted ADC or isotype control ADC carrying (151), (147) or (131) against NCI-N87 cells compared to (2), (148) or (144).

Figure 104 shows a graph of in vitro potency of HER 2-targeted or isotype control ADC carrying (151), (147) or (131) against SK-BR-3 cells compared to (2), (148) or (144).

FIG. 105 shows a graph of in vitro potency of TROP-2 targeted or isotype control ADCs carrying (155) or CL2A-SN38 against MDA-MB-468 cells as compared to (1).

FIG. 106 shows a graph of compound 127CH 1-3/CT-labeled trastuzumab conjugate that produced a DAR of 7.15 (as determined by PLRP).

FIG. 107 shows a graph of compound 131CH 1-3/CT-labeled trastuzumab conjugate that resulted in a DAR of 6.80 (as determined by PLRP).

FIG. 108 shows a graph of compound 127CH 1-3/CT-labeled trastuzumab conjugate, which is 94.4% monomer (as determined by analytical SEC).

FIG. 109 shows a graph of compound 131CH 1-3/CT-labeled trastuzumab conjugate, which is 93.6% monomer (as determined by analytical SEC).

FIG. 110 shows a graph of a compound 165CH 1-3/CT-labeled golian Sha Tuozhu mab conjugate that resulted in a DAR of 3.41 (as determined by HIC).

Figure 111 shows a graph of a compound 165CH 1-3/CT-labelled gor Sha Tuozhu mab conjugate that is 98.2% monomer (as determined by analytical SEC).

FIG. 112 shows a graph of compound 165CH 1-3/CT-labeled poloxamer conjugate that resulted in a DAR of 3.67 (as determined by HIC).

FIG. 113 shows a graph of compound 165CH 1-3/CT-labeled poloxamer conjugate, which is 97.4% monomer (as determined by analytical SEC).

FIG. 114 shows a graph of compound 80CH 1-3/CT-labeled trastuzumab conjugate that resulted in a DAR of 5.86 (as determined by PLRP).

FIG. 115 shows a graph of compound 80CH 1-3/CT-labeled trastuzumab conjugate, which is 97.4% monomer (as determined by analytical SEC).

FIG. 116 shows a graph of a compound 80CH 1-3/CT-labeled golian Sha Tuozhu mab conjugate that resulted in a DAR of 6.19 (as determined by PLRP).

FIG. 117 shows a graph of a compound 80CH 1-3/CT-labeled golian Sha Tuozhu mab conjugate that is 97.1% monomer (as determined by analytical SEC).

FIG. 118 shows a plot of compound 86CH 1-3/CT-labeled anti-FITC conjugate that resulted in a DAR of 5.46 (as determined by PLRP).

FIG. 119 shows a graph of compound 86CH 1-3/CT-labeled anti-FITC conjugate, which is 98.0% monomer (as determined by analytical SEC).

FIG. 120 shows a graph of compound 92CH 1-3/CT-labeled anti-FITC conjugate that resulted in a DAR of 1.58 (as determined by PLRP).

FIG. 121 shows a graph of compound 92CH 1-3/CT-labeled anti-FITC conjugate, which is 96.1% monomer (as determined by analytical SEC).

FIG. 122 shows a graph of compound 99CH 1-3/CT-labeled anti-FITC conjugate that produced 3.07 DAR (as determined by PLRP).

FIG. 123 shows a graph of a compound 99CH 1-3/CT-labeled anti-FITC conjugate that is 97.9% monomer (as determined by analytical SEC).

FIG. 124 shows a graph of compound 103CH 1-3/CT-labeled trastuzumab conjugate that resulted in a DAR of 6.56 (as determined by PLRP).

FIG. 125 shows a graph of compound 103CH 1-3/CT-labeled trastuzumab conjugate, which is 97.3% monomer (as determined by analytical SEC).

FIG. 126 shows a plot of compound 110CH 1-3/CT-labeled anti-FITC conjugate that resulted in a DAR of 5.66 (as determined by PLRP).

FIG. 127 shows a graph of compound 110CH 1-3/CT-labeled anti-FITC conjugate, which is 98.5% monomer (as determined by analytical SEC).

FIG. 128 shows a graph of compound 113CH 1-3/CT-labeled golian Sha Tuozhu mab conjugate that resulted in a DAR of 6.41 (as determined by PLRP).

FIG. 129 shows a graph of a compound 113CH 1-3/CT-labeled golian Sha Tuozhu mab conjugate that is 97.4% monomer (as determined by analytical SEC).

FIG. 130 shows a graph of compound 123CH 1-3/CT-labeled anti-FITC conjugate that resulted in a DAR of 5.56 (as determined by PLRP).

FIG. 131 shows a graph of compound 123CH 1-3/CT-labeled anti-FITC conjugate, which is 95.5% monomer (as determined by analytical SEC).

FIG. 132 shows a graph of compound 151CH 1-3/CT-labeled anti-FITC conjugate that resulted in a DAR of 5.67 (as determined by PLRP).

FIG. 133 shows a graph of compound 151CH 1-3/CT-labeled anti-FITC conjugate, which was 97.8% monomer (as determined by analytical SEC).

FIG. 134 shows a graph of compound 147CH 1-3/CT-labeled anti-FITC conjugate that resulted in a DAR of 6.47 (as determined by PLRP).

FIG. 135 shows a graph of compound 147CH 1-3/CT-labeled anti-FITC conjugate, 96.4% monomer (as determined by analytical SEC).

FIG. 136 shows a graph of compound 73CH 1-3/CT-labeled trastuzumab conjugate that resulted in a DAR of 5.41 (as determined by PLRP).

FIG. 137 shows a graph of compound 67CH 1-3/CT-labeled trastuzumab conjugate that produced a DAR of 4.02 (as determined by PLRP).

FIG. 138 shows a graph of compound 136CH 1-3/CT-labeled trastuzumab conjugate that resulted in a DAR of 7.26 (as determined by PLRP).

FIG. 139 shows a graph of compound 136CH 1-3/CT-labeled trastuzumab conjugate, which is 98.9% monomer (as determined by analytical SEC).

FIG. 140 shows a graph of compound 142CH 1-3/CT-labeled trastuzumab conjugate that resulted in a DAR of 6.9 (as determined by PLRP).

FIG. 141 shows a graph of compound 175CH 1-3/CT-labeled anti-FITC conjugate that produced a DAR of 5.08 (as determined by PLRP).

FIG. 142 shows a graph of compound 175CH 1-3/CT-labeled anti-FITC conjugate, which is 93.0% monomer (as determined by analytical SEC).

FIG. 143 shows a graph of compound 155CH 1-3/CT-labeled trastuzumab conjugate that resulted in a DAR of 2.82 (as determined by HIC).

FIG. 144 shows a graph of compound 155CH 1-3/CT-labeled anti-FITC conjugate, which is 89.7% monomer (as determined by analytical SEC).

Definition of the definition

Unless otherwise indicated, the following terms have the following meanings. Any undefined term has its art-recognized meaning.

"alkyl" means having 1 to 10 carbon atoms and such as 1 to 6 carbon atomsOr a monovalent saturated aliphatic hydrocarbon group of 1 to 5, or 1 to 4, or 1 to 3 carbon atoms. The term includes, by way of 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 (except C ₁ Outside the carbon atom) optionally substituted with heteroatoms such as-O-, -N-, -S (O) _n - (wherein n is 0 to 2), -NR- (wherein R is hydrogen or alkyl) substituted and having 1 to 5 substituents selected from the group consisting of: alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioonyl, 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 heterocycle.

"alkylene" means a divalent aliphatic hydrocarbon group, preferably having 1 to 6, and more preferably 1 to 3 carbon atoms, which is straight-chain orBranched and optionally selected from-O-, -NR ¹⁰ -、-NR ¹⁰ C(O)-、-C(O)NR ¹⁰ -one or more groups of the same are interrupted. The term includes, by way of 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 hydrogen atoms, which is replaced with a substituent as described for carbon atoms in the definition of "substituted" below.

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 "-wherein R' is an alkyl group as defined herein and R" is alkylene, alkenylene or alkynylene 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 halo 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 halo 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 hydrocarbon group having 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms, and having at least 1 and preferably 1 to 2 sites of double bond unsaturation. The term includes, by way of example, di-vinyl, 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, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocycle Oxy, 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, 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 alkynyl 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, acylamino, 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) -, alkenyl-C (O) -, substituted alkenyl-C (O) -, alkynyl-C (O) -, substituted alkynyl-C (O) -, cycloalkyl-C (O) -, and 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, heterocycle, and substituted heterocycle are as defined herein. For example, the acyl group includes an "acetyl" group CH ₃ C(O)-

"Acylamino" refers to the 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, heterocycle, and substituted heterocycle 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 bound to 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, heterocyclyl and substituted heterocyclyl 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, heterocycle, and substituted heterocycle, and wherein R is as defined in the claims ²¹ And R is ²² Optionally together with the nitrogen bound thereto to 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, alkeneA group, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocycle, and substituted heterocycle, and wherein R is a integer ²¹ And R is ²² Optionally together with the atoms to which they are bound to 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 "Ar" refers to a monovalent aromatic carbocyclic group of 6 to 18 carbon atoms having a single ring (such as found in phenyl) or a ring system having multiple fused rings (examples of such aromatic ring systems include naphthyl, anthracenyl and indanyl), where the fused rings may or may not be aromatic, so long as the attachment point 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, thioaheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl, -SO ₂ Heteroaryl 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 as also 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" or "carboxylate" refers to-CO ₂ H or a salt thereof.

"carboxyester" or "carboxylate" 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-heterocycle, and-C (O) O-substituted heterocycle, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, and substituted heterocycle 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-heterocycle and-O-C (O) O-substituted heterocycle, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, and substituted heterocycle are as defined herein.

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

"cycloalkyl" refers to a cyclic alkyl group having 3 to 10 carbon atoms, having 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 cycloalkyl groups 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, acylamino, 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 cyclic alkyl group having 3 to 10 carbon atoms, which has a single ring or multiple rings and has at least one double bond, preferably 1 to 2 double bonds.

The term "substituted cycloalkenyl" refers to cycloalkenyl groups 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, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido Nitrogen, cyano, halogen, hydroxy, keto, thioketone, carboxyl, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocycloxy, 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.

"hydroxyl" refers to the group-OH.

"heteroaryl" refers to an aromatic group having 1 to 15 carbon atoms in the ring, such as 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. Such heteroaryl groups may have multiple condensed rings in a single ring (such as pyridyl, imidazolyl, or furyl) or a ring system (e.g., in a group 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, such as 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, indolyl, thienyl and furyl. Unless otherwise limited by the definition of heteroaryl substituents, 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, carboxyl, carboxyalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyalkylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO ₂ -alkyl, -SO ₂ -substituted alkyl, -SO ₂ -aryl and-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", "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 3 to 20 ring atoms, including 1 to 10 heteroatoms. These ring atoms are selected from nitrogen, sulfur or oxygen, wherein in the fused ring system one or more 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 substituents, such as an alkyl group 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, indolizines, isoindoles, 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, morpholines, thiomorpholines (also known as thiomorpholinyl), 1-dioxothiomorpholinyl, piperidinyl, pyrrolidines, 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, acylamino, 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 heterocycle-S-.

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

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

"nitro" means a group-NO ₂ 。

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

"Sulfonyl" means-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 -groups of 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, heterocycle and substituted heterocycle are as defined herein. Sulfonyl groups include, by way of example, methyl-SO ₂ -, phenyl-SO ₂ -and 4-methylphenyl-SO ₂ -。

"Sulfonyloxy" means-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 -groups of 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, heterocycle and substituted heterocycle are as defined herein.

"sulfate" or "sulfate" 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-heterocycles and-O-SO ₂ -O-substituted heterocycles in which the alkaneThe groups, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, and substituted heterocycle 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 heterocycle, wherein alkyl, substituted alkyl, aryl, heteroaryl, and heterocycle are as defined herein.

"thiol" refers to the group-SH.

"thio" or the term "thione" 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 as also defined herein.

In addition to the disclosure herein, the term "substituted" when used to modify a particular group or radical may also mean that one or more hydrogen atoms of the particular group or radical are each independently replaced by the same or different substituents as defined below.

In addition to the groups disclosed with respect to the various terms herein, unless otherwise indicated, are used to replace saturated carbon in a given group or radicalOne or more hydrogens on the atom (any two hydrogens on a single carbon may be taken to be =o, =nr ⁷⁰ 、＝N-OR ⁷⁰ 、＝N ₂ Or = S substituted) is-R ⁶⁰ Halo, =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 is ⁸⁰ Independently is R ⁷⁰ Or alternatively, two R ⁸⁰ Together with the nitrogen atom to which it is bonded, form a 5-, 6-or 7-membered heterocycloalkyl group, which optionally includes 1 to 4 identical or different additional heteroatoms selected from the group consisting of: o, N and S, which Wherein N may have-H or C ₁ -C ₃ Alkyl substitution; and each M ⁺ Is a counter ion with a single net positive charge. Each M ⁺ May independently be, for example, an alkali metal ion such as K ⁺ 、Na ⁺ 、Li ⁺ The method comprises the steps of carrying out a first treatment on the surface of the Ammonium ions, such as ⁺ N(R ⁶⁰ ) ₄ The method comprises the steps of carrying out a first treatment on the surface of the Or alkaline earth ions, such as [ Ca ] ²⁺ ] _0.5 、[Mg ²⁺ ] _0.5 Or [ Ba ] ²⁺ ] _0.5 (subscript 0.5 indicates that one of the counter ions of such divalent alkaline earth ions may be the ionized form of the compound of the invention, while the other may be a typical counter ion such as chloride, or that two of the ionizing compounds disclosed herein may be used as counter ions of such divalent alkaline earth ions, or that the dual ionizing compounds of the invention may be used as counter ions of such divalent alkaline earth 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.

In addition to the disclosure herein, unless otherwise indicated, the substituent of a hydrogen on an unsaturated carbon atom in "substituted" alkene, alkyne, aryl and heteroaryl groups is-R ⁶⁰ Halo, -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 、-OS O 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 ⁺ With the proviso that in the case of substituted alkenes or alkynes, the substituents are not-O ^- M ⁺ 、-OR ⁷⁰ 、-SR ⁷⁰ or-S ^– M ⁺ 。

The substituents of hydrogen on nitrogen atoms in "substituted" heteroalkyl and cycloheteroalkyl groups are-R, except where disclosed with respect to each term herein, unless otherwise indicated 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 defined above.

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 substituted groups defined hereinabove, polymers obtained by defining substituents as having themselves further substituents (e.g., substituted aryl groups having substituted aryl groups as substituents, the substituents themselves being substituted with substituted aryl groups, the substituted aryl groups being further substituted with substituted aryl groups, etc.) are not intended to be encompassed herein. In such cases, the maximum number of such substitutions is three. For example, the sequential substitution of substituted aryl groups specifically contemplated herein is limited to substituted aryl- (substituted aryl) -substituted aryl.

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

For any of the groups disclosed herein that contain one or more substituents, it should of course be understood that these groups do not contain any substituents or substitution patterns that are sterically impractical and/or synthetically infeasible. Furthermore, the subject compounds include all stereochemical isomers arising from substituents of these compounds.

The term "pharmaceutically acceptable salt" means a salt that is acceptable for administration to a patient, such as a mammal (a counterion-containing salt that has acceptable mammalian safety for a given dosage regimen). Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and from 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 counterions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and at this time, the molecule contains a basic functional group, a salt of an organic or inorganic acid, such as hydrochloride, hydrobromide, formate, tartrate, benzenesulfonate, methanesulfonate, acetate, maleate, oxalate, and the like.

The term "salt thereof" means a compound formed when the proton of an acid is replaced by a cation such as a metal cation or an organic cation, etc. 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. By way of example, salts of the compounds of the present invention include salts wherein the compound is protonated by an inorganic or organic acid to form a cation and a conjugate base of the inorganic or organic acid is used as the anionic component of the salt.

"solvate" refers to a complex formed by the combination of a solvent molecule with 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, dimethyl sulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.

"stereoisomers" refers to compounds having identical atomic connectivity but differing in the arrangement of atoms in space. Stereoisomers include cis, trans, E and Z isomers, enantiomers and diastereomers.

"tautomer" refers to alternative forms of the molecule that differ only in the electron bonding and/or proton positions of the atoms, 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, tetrazole. One of ordinary skill in the art will recognize that other tautomeric ring atom arrangements are 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 effective amount or therapeutically effective amount includes an amount sufficient to cause, among other things, tumor shrinkage or decrease the rate of tumor growth.

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

The term "treatment" as used herein means the treatment of a disease or medical condition of a patient, such as a mammal (particularly a human), including: (a) Preventing the occurrence of a disease or medical condition, such as prophylactic treatment of a subject; (b) Improving a disease or medical condition, such as eliminating or causing regression of the 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; (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 amino acids of any length in polymerized form. Unless explicitly indicated otherwise, "polypeptide", "peptide" and "protein" may include genetically encoded and non-encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including but not limited to fusion proteins having heterologous amino acid sequences, fusion proteins having heterologous and homologous leader sequences, proteins containing at least one N-terminal methionine residue (e.g., to facilitate production in recombinant host cells); an immunologically labeled protein, and the like. In certain embodiments, the polypeptide is an antibody.

"native amino acid sequence" or "parent amino acid sequence" is used interchangeably herein to refer to an amino acid sequence of a polypeptide prior to modification to include at least one modified amino acid residue.

The terms "amino acid analog," "unnatural amino acid," and the like, are used interchangeably, and include amino acid compounds that are similar in structure and/or overall shape to one or more amino acids common in naturally occurring proteins (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). 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 modified groups in the molecule. Such modifications may include, but are not limited to, substitution of one atom (such as N) for one related atom (such as O or S), addition of groups (such as methyl or hydroxy, etc.) or atoms (such as Cl or Br, etc.), deletion of groups, substitution of covalent bonds (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. Examples of amino acid analogs include, but are not limited to, cysteic acid, 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 herein in the context of modified amino acids and/or conjugates.

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 (by any convenient chemical conversion to another group), or absent (e.g., eliminated or replaced by H). A wide variety of carbohydrates and carbohydrate derivatives are available and may be suitable for use with the subject compounds and conjugates.

The term "glycoside" or "glycosyl" refers to a sugar molecule or group that is bound to a moiety via a glycosidic bond. For example, the moiety to which the glycoside binds may be a cleavable linker as described herein. The glycosidic bond may connect 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 (sugar amine), an S-glycosidic bond (thio glycoside) 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, such as 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., convers, p., walport, m., shomchik (2001) immune Biology, 5 th edition, garland Publishing, new York). The target antigen may have one or more binding sites (also referred to as epitopes) that are 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 formed 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 amino acids from the corresponding positions of 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. Nos. 5,225,539, 5,530,101 and 5,585,089), veneering or surface reshaping (EP 592,106;EP 519,596;Padlan,Molecular Immunology 28 (4/5): 489-498 (1991); studnicka et al Protein Engineering (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 by sequence comparison to identify aberrant framework residues at specific positions (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; U.S. Pat. No. 4,816,567; PCT publications WO 98/45331 and WO 98/45332. In particular embodiments, subject rabbit antibodies can be humanized according to the methods set forth in US20040086979 and US 20050033031. Thus, the antibodies described above can 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 segments of genes from mouse monoclonal antibodies can be linked to human constant segments, such as γ1 and γ3. An example of a therapeutic chimeric antibody is a hybrid protein 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 also be used.

Immunoglobulin polypeptide immunoglobulin light or heavy chain variable regions are composed of Framework Regions (FR) (also known as "complementarity determining regions" or "CDRs") interrupted by three hypervariable regions. The framework regions and CDR ranges have been established (see "Sequences of Proteins of Immunological Interest", E.Kabat et al, U.S. department of health and public service, 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 mainly responsible for binding to epitopes of antigens.

A "parent Ig polypeptide" is a polypeptide comprising an amino acid sequence that lacks an aldehyde-tagged constant region as 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, such as 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. "isolated" is intended to include compounds in a sample that are substantially enriched in the 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 more than 98% free of other components with which it is naturally associated.

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

By "reactive partner" is meant a molecule or molecular moiety 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 a reaction product containing a converted aldehyde tag of formylglycine (fgy) in place of cysteine or serine in the motif. Other exemplary reaction partners include aldehydes (e.g., reactive aldehyde groups) and "aldehyde-reactive partners" of converted aldehyde-tagged fGly residues, which 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 the modified polypeptides via the fGly residues.

"N-terminal" refers to the terminal amino acid residue of a polypeptide having a free amine group, the amine group in which is not the 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 the amino acid sequence of a polypeptide means a region of the polypeptide that is not at the N-terminus or C-terminus.

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 sub-combination. All combinations of embodiments related to the invention are specifically contemplated by the invention and disclosed herein as if each combination were individually and specifically disclosed, such combinations encompass, to some extent, the inventive substance (i.e., a compound that can be prepared, isolated, characterized, and tested for biological activity) as a compound that stabilizes the compound. In addition, the invention specifically encompasses all subcombinations of the various embodiments and elements thereof (e.g., elements of the chemical groups listed in the embodiments describing such variables) and is disclosed herein as if each subcombination and each such subcombination were 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 is further 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, "or use of negative" limitation in connection with recitation of claim elements.

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 sub-combination.

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 publication by virtue of prior application. In addition, 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 (ADC) structures comprising camptothecins or camptothecine derivatives linked to polypeptides (e.g., antibodies) through linkers. The disclosure also encompasses compounds and methods for producing such conjugates, as well as methods of using these conjugates.

Antibody-drug conjugates

The present disclosure provides a conjugate, such as an antibody-drug conjugate (ADC). By "conjugate" is meant that the polypeptide (e.g., antibody) is covalently attached to one or more other moieties (e.g., drug or active agent). For example, an antibody-drug conjugate according to the present disclosure includes one or more drugs or active agents covalently attached to an antibody. In certain embodiments, a polypeptide (e.g., an antibody) and one or more drugs or active agents are bound to each other by one or more functional groups and covalent bonds. For example, the one or more functional groups and covalent bonds may include a cleavable linker as described herein.

In certain embodiments, the conjugate is a polypeptide conjugate that includes a polypeptide (e.g., an antibody) conjugated to one or more other moieties. In certain embodiments, one or more moieties conjugated to the polypeptide may each independently 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, thus providing an antibody-drug conjugate. For example, the conjugate may be a drug conjugate in which the polypeptide is conjugated to one or more drugs or active agents. In certain embodiments, the drug or active agent is camptothecin or a camptothecin derivative. Various types of camptothecins or camptothecine derivatives may be used in the conjugates and are described in more detail below.

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

In certain embodiments, the conjugates of the present disclosure include one or more drugs or active agents 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 and attached to one or more drugs or active agents (e.g., through 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 and attached to one or more drugs or active agents (e.g., attached to one or more drugs or active agents through a linker as described herein).

Embodiments of the present disclosure include conjugates in which a polypeptide is conjugated to one or more moieties, such as 2 moieties, 3 moieties, 4 moieties, 5 moieties, 6 moieties, 7 moieties, 8 moieties, 9 moieties, or 10 or more moieties. These moieties 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 the polypeptide and the second moiety is conjugated to a second amino acid residue of the polypeptide. Combinations of the above are also possible, for example wherein 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 portions at a first amino acid residue and to the third and fourth portions at a second amino acid residue, and so forth. In some cases, two or more amino acid residues in a polypeptide are each conjugated to a pair of moieties (i.e., two moieties), wherein each pair of moieties is conjugated to the polypeptide through a branched linker as described herein. In some cases, 1 amino acid residue in a polypeptide is conjugated to a pair of moieties through a branched linker as described herein.

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 one or more drugs or active agents conjugated to naturally occurring amino acid residues of a polypeptide. In other cases, the conjugate may include one or more drugs or active agents conjugated to an unnatural amino acid residue of the polypeptide. As described above, one or more drugs or active agents may be conjugated to a 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 as 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 conjugated to the moiety of interest.

In certain embodiments, the polypeptide (e.g., antibody) and the moiety of interest (e.g., drug or active agent) are conjugated via a conjugate moiety. For example, the polypeptide and the moiety of interest may each be bound (e.g., covalently bound) to a conjugate moiety, such that the polypeptide and the moiety of interest are indirectly bound together by the conjugate moiety. In some cases, the conjugate 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 conjugate moiety is shown in the general reaction scheme below. The hydrazino-indolyl and hydrazino-pyrrolo-pyridinyl conjugate moieties are also referred to herein as hydrazino-iso-Pictet-Spengler (HIPS) conjugate moieties and aza-hydrazino-iso-Pictet-Spengler (azaHIPS) conjugate 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 (fgy) is reacted with a conjugate moiety modified to include a conjugate moiety (e.g., a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety) to produce a polypeptide conjugate attached to the conjugate moiety, whereby the drug or active agent is attached to the polypeptide through the conjugate 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 can 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, a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl coupling moiety may be attached (e.g., covalently attached) to the linker. Thus, embodiments of the present disclosure include hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moieties attached to a drug or active agent via a linker. Various embodiments of linkers that can couple hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moieties to a drug or active agent are described in detail herein. For example, in some cases, the linker is a cleavable linker, such as the cleavable linkers described herein.

In some cases, the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety may be attached (e.g., covalently attached) to two or more linkers. Thus, embodiments of the present disclosure include hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moieties each attached to two or more drugs or active agents via a corresponding linker. Thus, conjugates of the present disclosure may include two or more linkers, wherein each linker attaches a respective drug or active agent to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety. Thus, a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety and two or more linkers may be considered as "branched linkers" in general, wherein the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety is attached to two or more "branches", wherein each branch comprises a linker attached to a drug or active agent.

In certain embodiments, a polypeptide may be conjugated to one or more moieties of interest, wherein one or more amino acid residues of the polypeptide are modified prior to conjugation to the moiety of interest. Modification of one or more amino acid residues of the polypeptide may result in a polypeptide containing one or more reactive groups suitable for conjugation to a moiety of interest. In some cases, the polypeptide may include one or more modified amino acid residues to provide one or more reactive groups suitable for conjugation to a moiety of interest (e.g., one or more moieties including a conjugation moiety, such as a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugation moiety as described above). For example, the amino acid of the polypeptide can be modified to include a reactive aldehyde group (e.g., a reactive aldehyde). Reactive aldehydes may be included in "aldehyde tags" or "ald-tags," which as used herein, refers to amino acid sequences derived from a sulfatase motif (e.g., L (C/S) TPSR) that has been converted to contain a 2-formylglycine residue (referred to herein as "fggly") by the action of a Formylglycine Generating Enzyme (FGE). The FGE-derived fcy residue 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 fggly by the action of FGE, e.g., L (fggly) TPSR). The converted sulfatase motif may be produced from an amino acid sequence that includes an "unconverted" sulfatase motif (i.e., a sulfatase motif in which a cysteine or serine residue is not converted to fGly by FGE but is capable of being converted, e.g., an unconverted sulfatase motif having the sequence L (C/S) TPSR). "conversion" 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 (fgy) residue (e.g., conversion of Cys to fgy, or conversion of Ser to fgy). 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, to produce a conjugate, a polypeptide containing a fGly residue can be conjugated to the moiety of interest by reacting fGly with a compound (e.g., a compound containing a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety, as described above). For example, a polypeptide comprising fGly may be contacted with a drug comprising a reactive partner under conditions suitable to provide conjugation of the drug to the polypeptide. In some cases, the drug containing the reactive partner may include a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety as described above. For example, the drug or active agent may be modified to include a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety. In some cases, the drug or active agent is attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl group, such as covalently attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl group through a linker (such as the linker detailed herein).

In certain embodiments, conjugates of the present disclosure include polypeptides (e.g., antibodies) having at least one amino acid residue attached to one or more moieties of interest (e.g., a drug or active agent). To prepare the conjugates, the amino acid residues of the polypeptides may be modified and then coupled to one or more drugs or active agents attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety as described above. In certain embodiments, the amino acid residue of a polypeptide (e.g., an antibody) is a cysteine or serine residue modified to a fGly residue, as described above. In certain embodiments, modified amino acid residues (e.g., fGly residues) are conjugated to a drug or active agent containing a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety as described above to provide a conjugate of the present disclosure, wherein one or more drugs or active agents are conjugated to the polypeptide through the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety. As used herein, the term fgy' refers to modified amino acid residues of a polypeptide (e.g., an antibody) coupled to a moiety of interest (e.g., a drug or active agent).

In certain embodiments, conjugates include a polypeptide (e.g., an antibody) having at least one amino acid residue attached to a linker as described herein, which in turn is attached to one or more drugs or active agents. For example, a conjugate may include a polypeptide (e.g., an antibody) having at least one amino acid residue (fGly') conjugated to one or more moieties of interest (e.g., one or more drugs or active agents) as described above.

Aspects of the disclosure include conjugates of formula (I):

wherein the method comprises the steps of

Z is CR ¹⁰ Or N, or a combination of two,

w is a polypeptide;

R ³ and R is ⁴ Each independently of the otherSelected from hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, or R ³ And R is ⁴ Optionally cyclic linked to form a 5-or 6-membered cycloalkyl or heterocyclyl ring;

wherein at least one R ¹⁰ Optionally to a second compound of formula (II).

Substituents associated with the conjugates of formula (I) are described in more detail below.

In certain embodiments, Z is CR ¹⁰ Or N. In certain embodiments, Z is CR ¹⁰ . In certain embodiments, Z is N.

In certain embodiments, R ⁷ 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 ⁷ Is hydrogen. In certain embodiments, R ⁷ Is alkyl or substituted alkyl, such as 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 ⁷ Is methyl. In certain embodiments, R ⁷ Alkenyl or substituted alkenyl, such as 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 ⁷ Alkynyl or substituted alkynyl, such as 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 ⁷ Is aryl or substituted aryl, such as C _5-8 Aryl or C _5-8 Substituted aryl groups, such as C ₅ Aryl or C ₅ Substituted aryl, or C ₆ Aryl or C ₆ Substituted aryl. In certain embodiments, R ⁷ Is heteroaryl or substituted heteroaryl, such as C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, such as C ₅ Heteroaryl or C ₅ Substituted heteroaryl, or C ₆ Heteroaryl or C ₆ Substituted heteroaryl groups. In certain embodiments, R ⁷ Cycloalkyl or substituted cycloalkyl, such as C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl groups, such as 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 ⁷ Is a heterocyclic group or a substituted heterocyclic group, such as C _3-8 Heterocyclyl or C _3-8 Substituted heterocyclyl groups, such as 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 ⁸ And R is ⁹ Each 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 thioSubstituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, or R ⁸ And R is ⁹ Optionally cyclic to form a 5-or 6-membered heterocyclyl.

In certain embodiments, R ⁸ 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 ⁸ Is hydrogen. In certain embodiments, R ⁸ Is alkyl or substituted alkyl, such as 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 ⁸ Is methyl. In certain embodiments, R ⁸ Alkenyl or substituted alkenyl, such as 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 ⁸ Is alkynyl or substituted alkynyl. In certain embodiments, R ⁸ Is an alkoxy group or a substituted alkoxy group. In certain embodiments, R ⁸ Is amino or substituted amino. In certain embodiments, R ⁸ Is carboxyl or carboxyl ester. In certain embodiments, R ⁸ Is acyl or acyloxy. In certain embodiments, R ⁸ Is an acylamino group or an aminoacyl group. In certain embodiments, R ⁸ Is an alkylamide or a substituted alkylamide. In certain embodiments, R ⁸ Is sulfonyl. In certain embodiments, R ⁸ Is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ⁸ Is aryl or substituted aryl, such as C _5-8 Aryl or C _5-8 Substituted aryl groups, such as C ₅ Aryl or C ₅ Substituted aryl, or C ₆ Aryl or C ₆ Substituted aryl. In certain embodiments, R ⁸ Is heteroaryl or substituted heteroaryl, such as C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, such as C ₅ Heteroaryl or C ₅ Substituted heteroaryl, or C ₆ Heteroaryl or C ₆ Substituted heteroaryl groups. In certain embodiments, R ⁸ Cycloalkyl or substituted cycloalkyl, such as C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl groups, such as 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 ⁸ Is a heterocyclic group or a substituted heterocyclic group, such as 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 ⁹ 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 ⁹ Is hydrogen. In certain embodiments, R ⁹ Is alkyl or substituted alkyl, such as 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 ⁹ Is methyl. In certain embodiments, R ⁹ Alkenyl or substituted alkenyl, such as 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 some embodiments of the present invention, in some embodiments,R ⁹ is alkynyl or substituted alkynyl. In certain embodiments, R ⁹ Is an alkoxy group or a substituted alkoxy group. In certain embodiments, R ⁹ Is amino or substituted amino. In certain embodiments, R ⁹ Is carboxyl or carboxyl ester. In certain embodiments, R ⁹ Is acyl or acyloxy. In certain embodiments, R ⁹ Is an acylamino group or an aminoacyl group. In certain embodiments, R ⁹ Is an alkylamide or a substituted alkylamide. In certain embodiments, R ⁹ Is sulfonyl. In certain embodiments, R ⁹ Is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ⁹ Is aryl or substituted aryl, such as C _5-8 Aryl or C _5-8 Substituted aryl groups, such as C ₅ Aryl or C ₅ Substituted aryl, or C ₆ Aryl or C ₆ Substituted aryl. In certain embodiments, R ⁹ Is heteroaryl or substituted heteroaryl, such as C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, such as C ₅ Heteroaryl or C ₅ Substituted heteroaryl, or C ₆ Heteroaryl or C ₆ Substituted heteroaryl groups. In certain embodiments, R ⁹ Cycloalkyl or substituted cycloalkyl, such as C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl groups, such as 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 ⁹ Is a heterocyclic group or a substituted heterocyclic group, such as C _3-8 Heterocyclyl or C _3-8 Substituted heterocyclyl groups, such as 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 ⁸ And R is ⁹ Optionally cyclic to form a 5-or 6-membered heterocyclyl. In certain embodiments, R ⁸ And R is ⁹ Cyclic linkages to form a 5-or 6-membered heterocyclyl. In certain embodiments, R ⁸ And R is ⁹ Cyclic linkages to form a 5 membered heterocyclyl. In certain embodiments, R ⁸ And R is ⁹ Annular linkFollowed by formation of a 6 membered heterocyclic group.

In certain embodiments, each R ¹⁰ 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 is described in more detail below ¹⁰ Is a combination of the above-mentioned various possibilities. In certain embodiments, R ¹⁰ Is hydrogen. In certain embodiments, each R ¹⁰ Is hydrogen. In certain embodiments, R ¹⁰ Halogen such as F, cl, br or I. In certain embodiments, R ¹⁰ F. In certain embodiments, R ¹⁰ Is Cl. In certain embodiments, R ¹⁰ Is Br. In certain embodiments, R ¹⁰ Is I. In certain embodiments, R ¹⁰ Is alkyl or substituted alkyl, such as 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 ¹⁰ Is methyl. In certain embodiments, R ¹⁰ Alkenyl or substituted alkenyl, such as 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 ¹⁰ Is alkynyl or substituted alkynyl. In certain embodiments, R ¹⁰ Is an alkoxy group or a substituted alkoxy group. In certain embodiments, R ¹⁰ Is amino or substituted amino. In certain embodiments, R ¹⁰ Is carboxyl or carboxyl ester. In certain embodiments, R ¹⁰ Is acyl or acyloxy. In certain embodiments, R ¹⁰ Is an acylamino group or an aminoacyl group. In certain embodiments, R ¹⁰ Is an alkylamide or a substituted alkaneAnd (3) a alkylamide. In certain embodiments, R ¹⁰ Is sulfonyl. In certain embodiments, R ¹⁰ Is thioalkoxy or substituted thioalkoxy. In certain embodiments, R ¹⁰ Is aryl or substituted aryl, such as C _5-8 Aryl or C _5-8 Substituted aryl groups, such as 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 ¹⁰ Is heteroaryl or substituted heteroaryl, such as C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, such as C ₅ Heteroaryl or C ₅ Substituted heteroaryl, or C ₆ Heteroaryl or C ₆ Substituted heteroaryl groups. In certain embodiments, R ¹⁰ Cycloalkyl or substituted cycloalkyl, such as C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl groups, such as 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 ¹⁰ Is a heterocyclic group or a substituted heterocyclic group, such as C _3-8 Heterocyclyl or C _3-8 Substituted heterocyclyl groups, such as 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, W is a polypeptide. For example, W may be an antibody. In certain embodiments, W comprises one or more fGly' residues as described herein. In certain embodiments, the polypeptide is attached to the remainder of the conjugate by an fGly' residue as described herein. Further description of polypeptides and antibodies used in the subject conjugates are found in the disclosure herein.

In certain embodiments, L is R ¹ 、R ² 、R ³ 、R ⁴ 、R ⁵ Or R is ⁶ A linker attached to a compound of formula (II). A suitable linker for L is described in more detail below.

In certain embodiments, the conjugate of formula (I) comprises a linker L. The linker can be used to bind one or more moieties of interest (e.g., a drug or active agent) to one or more polypeptides via a conjugate moiety. The linker can be bound (e.g., covalently bonded) to the conjugate moiety at any convenient location (e.g., as described herein). For example, the linker may attach the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety to a drug (e.g., camptothecin or a camptothecin derivative). The hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety may be used to conjugate a linker (and thus a drug) to a polypeptide such as an antibody. For example, the conjugate 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, as described herein.

For example, as shown in formula (I) above, L is attached to W through the conjugate moiety, and thus W is indirectly bonded to linker L through the conjugate moiety. As described above, W is a polypeptide (e.g., an antibody), and thus L is attached to the polypeptide (antibody) through a conjugate moiety, e.g., linker L is indirectly bound to the polypeptide (antibody) through a conjugate moiety (e.g., through a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety as described herein).

Any convenient linker may be used for the linker L in the subject conjugates and compounds. In certain embodiments, the linker L may comprise 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 linker L may comprise an alkyl or substituted alkyl group. In certain embodiments, the linker L may comprise an alkenyl or substituted alkenyl group. In certain embodiments, linker L may include an alkynyl or substituted alkynyl group. In certain embodiments, the linker L may include an alkoxy or substituted alkoxy group. In certain embodiments, the linker L may include an amino group or a substituted amino group. In certain embodiments, the linker L may include a carboxyl or carboxyl ester group. In certain embodiments, the linker L can include an acylamino group. In certain embodiments, the linker L may comprise an alkylamide or a substituted alkylamide group. In certain embodiments, the linker L may comprise an aryl group or a substituted aryl group. In certain embodiments, linker L may comprise a heteroaryl or substituted heteroaryl. In certain embodiments, linker L may comprise cycloalkyl or substituted cycloalkyl. In certain embodiments, linker L may comprise a heterocyclyl or substituted heterocyclyl group.

In certain embodiments, the linker L 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, copolymers of ethylene glycol and propylene glycol (e.g., wherein the homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ether, polyvinyl pyrrolidone, 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 is a linker (e.g., a first linker) described by the formula:

-(L ¹ ) _a -(L ² ) _b -(L ³ ) _c -(L ⁴ ) _d -(L ⁵ ) _e -(L ⁶ ) _f -，

wherein L is ¹ 、L ² 、L ³ 、L ⁴ 、L ⁵ And L ⁶ Each independently is a linker subunit, and a, b, c, d, e and f are each independently 0 or 1, wherein the sum of a, b, c, d, e and f is 1 to 6.

In certain embodiments, the sum of a, b, c, d, e and f is 1. In certain embodiments, the sum of a, b, c, d, e and f is 2. In certain embodiments, the sum of a, b, c, d, e and f is 3. In certain embodiments, the sum of a, b, c, d, e and f is 4. In certain embodiments, the sum of a, b, c, d, e and f is 5. In certain embodiments, the sum of a, b, c, d, e and f is 6. In certain embodiments, a, b, c, d, e and f are each 1. In certain embodiments, a, b, c, d and e are each 1 and f is 0. In certain embodiments, a, b, c, and d are each 1 and e and f are each 0. In certain embodiments, a, b, and c are each 1 and d, e, and f are each 0. In certain embodiments, a and b are each 1 and c, d, e, and f are each 0. In certain embodiments, a is 1 and b, c, d, e and f are each 0.

In certain embodiments, the linker subunit L ¹ Attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety (e.g., as shown in formula (I) above). In certain embodiments, the linker subunit L ² Attached to camptothecin or camptothecin derivatives when present. In certain embodiments, the linker subunit L ³ Attached to camptothecin or camptothecin derivatives when present. In certain embodiments, the linker subunit L ⁴ Attached to camptothecin or camptothecin derivatives when present. In certain embodiments, the linker subunit L ⁵ Attached to camptothecin or camptothecin derivatives when present. In certain embodiments, the linker subunit L ⁶ Attached to camptothecin or camptothecin derivatives when present.

Any convenient linker subunit may be used in linker L. The linker subunits of interest include, but are not limited to: units of polymers such as polyethylene glycol, polyethylene and polyacrylate; amino acid residues; a carbohydrate-based polymer or carbohydrate residues and derivatives thereof; a polynucleotide; an alkyl group; an aryl group; a heterocyclic group; combinations thereof; and substituted versions thereof. In some embodiments, L ¹ 、L ² 、L ³ 、L ⁴ 、L ⁵ And L ⁶ Each (if present) comprising 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., tethering groups including alkylene diamines).

In some embodiments, L ¹ 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 ¹ Comprises polyethylene glycol. In some embodiments, L ¹ Comprises modified polyethylene glycol. In some embodiments, L ¹ Comprising amino acid residues. In some embodiments, L ¹ Comprising an alkyl group or a substituted alkyl group. In some embodiments, L ¹ Comprising an aryl group or a substituted aryl group. In some embodiments, L ¹ Comprising a diamine (e.g., comprising a linking group of an alkylene diamine).

In some embodiments, L ² 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 ² Comprises polyethylene glycol. In some embodiments, L ² Comprises modified polyethylene glycol. In some embodiments, L ² Comprising amino acid residues. In some embodiments, L ² Comprising an alkyl group or a substituted alkyl group. In some embodiments, L ² Comprising an aryl group or a substituted aryl group. In some embodiments, L ² Comprising a diamine (e.g., comprising a linking group of an alkylene diamine).

In some embodiments, L ³ 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 ³ Comprises polyethylene glycol. In some embodiments, L ³ Comprises modified polyethylene glycol. In some embodiments, L ³ Comprising amino acid residues. In some embodiments, L ³ Comprising an alkyl group or a substituted alkyl group. In some embodiments, L ³ Comprising an aryl group or a substituted aryl group. In some embodiments, L ³ Comprising a diamine (e.g., comprising a linking group of an alkylene diamine).

In some embodiments, L ⁴ 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 ⁴ Comprises polyethylene glycol. In some embodiments, L ⁴ IncludedModified polyethylene glycol. In some embodiments, L ⁴ Comprising amino acid residues. In some embodiments, L ⁴ Comprising an alkyl group or a substituted alkyl group. In some embodiments, L ⁴ Comprising an aryl group or a substituted aryl group. In some embodiments, L ⁴ Comprising a diamine (e.g., comprising a linking group of an alkylene diamine).

In some embodiments, L ⁵ 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 ⁵ Comprises polyethylene glycol. In some embodiments, L ⁵ Comprises modified polyethylene glycol. In some embodiments, L ⁵ Comprising amino acid residues. In some embodiments, L ⁵ Comprising an alkyl group or a substituted alkyl group. In some embodiments, L ⁵ Comprising an aryl group or a substituted aryl group. In some embodiments, L ⁵ Comprising a diamine (e.g., comprising a linking group of an alkylene diamine).

In some embodiments, L ⁶ 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 ⁶ Comprises polyethylene glycol. In some embodiments, L ⁶ Comprises modified polyethylene glycol. In some embodiments, L ⁶ Comprising amino acid residues. In some embodiments, L ⁶ Comprising an alkyl group or a substituted alkyl group. In some embodiments, L ⁶ Comprising an aryl group or a substituted aryl group. In some embodiments, L ⁶ Comprising a diamine (e.g., comprising a linking group of an alkylene diamine).

In some embodiments, L is a polypeptide comprising- (L) ¹ ) _a -(L ² ) _b -(L ³ ) _c -(L ⁴ ) _d -(L ⁵ ) _e -(L ⁶ ) _f -a linker, wherein:

-(L ¹ ) _a -is- (T) ¹ -V ¹ ) _a -；

-(L ² ) _b -is- (T) ² -V ² ) _b -；

-(L ³ ) _c -is- (T) ³ -V ³ ) _c -；

-(L ⁴ ) _d -is- (T) ⁴ -V ⁴ ) _d -；

-(L ⁵ ) _e -is- (T) ⁵ -V ⁵ ) _e -; and is also provided with

-(L ⁶ ) _f -is- (T) ⁶ -V ⁶ ) _f -，

Wherein T is ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ And T ⁶ A tethering group when present;

V ¹ 、V ² 、V ³ 、V ⁴ 、V ⁵ and V ⁶ Covalent bonds or linking functional groups when present; and is also provided with

a. b, c, d, e and f are each independently 0 or 1, wherein the sum of a, b, c, d, e and f is 1 to 6.

As described above, in certain embodiments, L ¹ Attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety (e.g., as shown in formula (I) above). Thus, in certain embodiments, T ¹ Attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety (e.g., as shown in formula (I) above). In certain embodiments, V ¹ Attached to camptothecin or camptothecin derivatives. In certain embodiments, L ² Attached to camptothecin or camptothecin derivatives when present. Thus, in certain embodiments, T ² Attached to camptothecin or camptothecin derivatives, or V when present ² Attached to camptothecin or camptothecin derivatives when present. In certain embodiments, L ³ Attached to camptothecin or camptothecin derivatives when present. Thus, in certain embodiments, T ³ Attached to camptothecin or camptothecin derivatives, or V when present ³ Attached to camptothecin or camptothecin derivatives when present. In certain embodiments, L ⁴ Attached to camptothecins or camptothecins when presentA base derivative. Thus, in certain embodiments, T ⁴ Attached to camptothecin or camptothecin derivatives, or V when present ⁴ Attached to camptothecin or camptothecin derivatives when present. In certain embodiments, L ⁵ Attached to camptothecin or camptothecin derivatives when present. Thus, in certain embodiments, T ⁵ Attached to camptothecin or camptothecin derivatives, or V when present ⁵ Attached to camptothecin or camptothecin derivatives when present. In certain embodiments, L ⁶ Attached to camptothecin or camptothecin derivatives when present. Thus, in certain embodiments, T ⁶ Attached to camptothecin or camptothecin derivatives, or V when present ⁶ Attached to camptothecin or camptothecin derivatives when present.

With respect to the tethering group T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ And T ⁶ Any convenient tethering group may be used in the subject linker. In some embodiments, T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ And T ⁶ Each comprising one or more groups independently selected from: covalent 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), meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), acetal groups, hydrazine, disulfides and esters, 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 certain embodiments, the tether group (e.g., T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ And/or T ⁶ ) Comprises (C) ₁ -C ₁₂ ) Alkyl (C)Of radicals 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, such as 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 ) Alkyl may be alkyl or substituted alkyl, such as 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 such as 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 ₃ Alkylene (e.g., CH ₂ 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, such as 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, such as a 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 being substituted C ₂ -an alkyl group. For example, substituted (C ₁ -C ₁₂ ) The alkyl group may be a substituted alkylene group, such as 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 being substituted C ₂ -an alkylene group. In some cases, substituted (C ₁ -C ₁₂ ) Alkyl being substituted C ₃ -an alkylene group. For example, substituted (C ₁ -C ₁₂ ) Alkyl groups may include those described herein (PEG) _n Radicals (e.g. -CONH (PEG) ₃ or-NHCO (PEG) ₇ ) Substituted C ₁ -C ₁₂ Alkylene (e.g., C ₃ Alkylene or C ₅ -alkylene) or may comprise a per-CONHCH ₂ CH ₂ SO ₃ C substituted by H groups ₁ -C ₁₂ Alkylene (e.g., C ₃ -alkylene) or may comprise a quilt-NHCOCH ₂ SO ₃ C substituted by H groups ₁ -C ₁₂ Alkylene (e.g., C ₅ -an alkylene group).

In certain embodiments, the tether group (e.g., T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ And/or T ⁶ ) 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 、T ⁴ 、T ⁵ And T ⁶ ) Including aryl or substituted aryl. For example, aryl may be phenyl. In some cases, the substituted aryl is a substituted phenyl. The substituted phenyl group may be selected from (C ₁ -C ₁₂ ) Alkyl, substituted (C) ₁ -C ₁₂ ) Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and one or more substituents of the substituted heterocyclyl. In some cases, a substituted aryl is a substituted phenyl, wherein the substituents include cleavable moieties (e.g., enzymatically cleavable moieties such as glycosides or glycoside derivatives) as described herein.

In some cases, the tether group (e.g., T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ And/or T ⁶ ) Including heteroaryl or substituted heteroaryl. In some cases, the tether group (e.g., T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ And T ⁶ ) Including cycloalkyl or substituted cycloalkyl. In some cases, the tether group (e.g., T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ And T ⁶ ) Including heterocyclyl or substituted heterocyclyl. In some cases, the substituents on the substituted heteroaryl, substituted cycloalkyl, or substituted heterocyclyl include cleavable moieties (e.g., enzymatically cleavable moieties such as glycosides or glycoside derivatives) as described herein.

In certain embodiments, the tether group (e.g., T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ And/or T ⁶ ) Including Ethylenediamine (EDA) moieties, e.g., EDA-containing tethering groups. In certain embodiments, (EDA) _w Including one or more EDA moieties such as where w 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 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, or 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, and substituted arylSubstituted 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 linked cyclic, for example to form 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 、T ⁴ 、T ⁵ And/or T ⁶ ) 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, amino, aminoacyl, thioamide, thioalkoxy, aryl, and substituted heteroaryl,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:

where 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, OR, COOH or COOR, wherein R is selected from 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 ¹⁷ COOH. In certain embodiments, R ¹⁷ Is OH. In certain embodiments, R ¹⁷ Is OR, such as OCH ₃ 。

In certain embodiments, the tether group (e.g., T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ And/or T ⁶ ) Comprises (PEG) _n Wherein (PEG) _n Is polyethylene glycol or modified polyethylene glycol connecting 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 、T ⁴ 、T ⁵ And/or T ⁶ ) Comprises (AA) _p Wherein AA is an amino acid residue. Any convenient amino acid may be utilized. Amino acids of interest include, but are not limited to, L-amino acids and D-amino acids, naturally occurring amino acids (such as any of the 20 major alpha-amino acids and beta-alanine), non-naturally occurring amino acids (e.g., amino acid analogs such as non-naturally occurring alpha-amino acids or non-naturally occurring beta-amino acids, etc.). 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 、T ⁴ 、T ⁵ And/or T ⁶ ) Including amino acid analogs. Amino acid analogs include compounds that are similar in structure and/or overall shape to one or more amino acids commonly found in naturally occurring proteins (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). 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 modified groups in the molecule. Such modifications may include, but are not limited to, substitution of one atom (such as N) for a related atom (such as O or S), addition of groups (such as methyl or hydroxy, etc.), or atoms (such as Cl or Br, etc.), deletion of groups, substitutionCovalent bonds (single bonds instead of double bonds, etc.) or combinations thereof. For example, amino acid analogs can include alpha-hydroxy acids, alpha-amino acids, and the like. Examples of amino acid analogs include, but are not limited to, cysteic acid, and the like.

In certain embodiments, the tether group (e.g., T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ And/or T ⁶ ) Including those made of- (CR) ¹³ OH) _m Part of the description, where m 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, m is 1. In certain embodiments, m 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, such as 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 Alkenyl or substituted alkenyl, such as 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 amino or substituted amino. 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 acylAn amino 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, such as C _5-8 Aryl or C _5-8 Substituted aryl groups, such as 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, such as C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, such as 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, such as C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl groups, such as 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, such as C _3-8 Heterocyclyl or C _3-8 Substituted heterocyclyl groups, such as 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 ¹³ Said method.

In certain embodiments, the tether group (e.g., T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ And/or T ⁶ ) Including meta-amino-benzyloxy (MABO), meta-amino-benzyloxycarbonyl (MABC), para-amino-benzyloxy (PABO), para-amino-benzyloxycarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP) or para-hydroxy-phenyl (PHP).

In some embodiments, the tether comprises a MABO group described by the following structure:

in some embodiments, the tether comprises a MABC group described by the following structure:

in some embodiments, the tether includes a PABO group described by the following structure:

in some embodiments, the tether comprises a PABC group described by the following structure:

In some embodiments, the tether includes a PAB group described by the following structure:

in some embodiments, the tether comprises a PABA group described by the following structure:

in some embodiments, the tether comprises a PAP group described by the following structure:

in some embodiments, the tether comprises a PHP group described by the following structure:

in certain embodiments, each R ¹⁴ 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, R ¹⁴ Is hydrogen. In certain embodiments, each R ¹⁴ Is hydrogen. In certain embodiments, R ¹⁴ Is alkyl or substituted alkyl, such as 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 ¹⁴ Alkenyl or substituted alkenyl, such as 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 ¹⁴ Is alkynyl or substituted alkynyl. In certain embodiments, R ¹⁴ Is an alkoxy group or a substituted alkoxy group. In certain embodiments, R ¹⁴ Is amino or substituted amino. In certain embodiments, R ¹⁴ Is carboxyl or carboxyl ester. In certain embodiments, R ¹⁴ Is acyl or acyloxy. In certain embodiments, R ¹⁴ Is an acylamino group or an aminoacyl group. In certain embodiments, R ¹⁴ Is an alkylamide or a substituted alkylamide. In certain embodiments, R ¹⁴ Is sulfonyl. In certain embodiments, R ¹⁴ Is thioalkoxy orSubstituted thioalkoxy groups. In certain embodiments, R ¹⁴ Is aryl or substituted aryl, such as C _5-8 Aryl or C _5-8 Substituted aryl groups, such as C ₅ Aryl or C ₅ Substituted aryl, or C ₆ Aryl or C ₆ Substituted aryl. In certain embodiments, R ¹⁴ Is heteroaryl or substituted heteroaryl, such as C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, such as C ₅ Heteroaryl or C ₅ Substituted heteroaryl, or C ₆ Heteroaryl or C ₆ Substituted heteroaryl groups. In certain embodiments, R ¹⁴ Cycloalkyl or substituted cycloalkyl, such as C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl groups, such as 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 ¹⁴ Is a heterocyclic group or a substituted heterocyclic group, such as C _3-8 Heterocyclyl or C _3-8 Substituted heterocyclyl groups, such as 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 of MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP tether structures shown above, the benzene ring may be substituted with one or more additional groups selected from: 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.

In certain embodiments of linker L, the tethering group T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ Or T ⁶ Each optionally substituted with a glycoside or glycoside derivative. In certain embodiments, the glycoside or glycoside derivative is selected from Glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc and O-GalNAc.

In certain embodiments, the MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP tether structures shown above may be substituted with one or more additional groups selected from glycoside and glycoside derivatives. For example, in some embodiments of MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP tether structures shown above, the benzene ring may be substituted with one or more additional groups selected from glycoside and glycoside derivatives. In certain embodiments, the glycoside or glycoside derivative is selected from the group consisting of glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

For example, in some embodiments, the glycoside or glycoside derivative may be selected from the following structures:

with respect to the linking functional group V ¹ 、V ² 、V ³ 、V ⁴ 、V ⁵ And V ⁶ Any convenient linking functionality may be used in linker L. The linking functional groups of interest include, but are not limited to, amino, carbonyl, amido, oxycarbonyl, carboxyl, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phosphoryl, phosphoramidite, thiophosphorous amide, and the like. In some embodiments, V ¹ 、V ² 、V ³ 、V ⁴ 、V ⁵ And V ⁶ Each independently selected from: 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 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 certain embodiments, q is 3. At a certain positionIn some embodiments, q is 4. In certain embodiments, q is 5. In certain embodiments, q is 6.

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.

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, such as 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 Alkenyl or substituted alkenyl, such as 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 amino or substituted amino. 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 certain embodiments, R ¹⁵ Is aryl or substituted aryl, such as C _5-8 Aryl or C _5-8 Substituted aryl groups, such as 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, such as C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, such as 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, such as C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl groups, such as 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, such as C _3-8 Heterocyclyl or C _3-8 Substituted heterocyclyl groups, such as 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, 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 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 hydrazine. In some embodiments, the tether group comprises a disulfide. In some embodiments, the tether group comprises an ester.

As described above, in some embodiments, L is a compound comprising (T ¹ -V ¹ ) _a -(T ² -V ² ) _b -(T ³ -V ³ ) _c -(T ⁴ -V ⁴ ) _d -(T ⁵ -V ⁵ ) _e -(T ⁶ -V ⁶ ) _f -a linker wherein a, b, c, d, e and f are each independently 0 or 1, wherein the sum of a, b, c, d, e and f is 1 to 6.

In some embodiments, in the joint L:

T ² 、T ³ 、T ⁴ 、T ⁵ and T ⁶ 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) _m -, 4-amino-piperidine (4 AP), MABO, MABC, PABO, PABC, PAB, PABA, PAP, PHP, acetal groups, disulfides, hydrazines, and esters; and is also provided with

V ¹ 、V ² 、V ³ 、V ⁴ 、V ⁵ And V ⁶ Each independently selected from: 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 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 is ¹² 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 linked cyclic to form a piperazinyl ring;

In certain embodiments, T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ And T ⁶ V (V) ¹ 、V ² 、V ³ 、V ⁴ 、V ⁵ And V ⁶ Selected from the following:

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of; and is also provided with

T ⁶ Is EDA and V ⁶ is-CO-; or (b)

Wherein:

T ¹ is (C) ₁ -C ₁₂ ) Alkyl (C)And V is a radical ¹ is-CO-;

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

T ³ for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁶ Is absent and V ⁶ is-CO-; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ is-NR ¹⁵ -; and is also provided with

T ⁶ Is (C) ₁ -C ₁₂ ) Alkyl and V ⁶ is-CO-; or wherein:

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

T ² is AA and V ² Absence of;

T ³ is PABC and V ³ Absence of;

T ⁴ is EDA and V ⁴ is-CO-; and is also provided with

e and f are each 0; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of; and is also provided with

f is 0; or (b)

Wherein:

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

T ² is AA and V ² Absence of;

T ³ is PABC and V ³ Absence of; and is also provided with

d. e and f are each 0; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABA and V ⁵ is-CO-; and is also provided with

T ⁶ Is (C) ₁ -C ₁₂ ) Alkyl and V ⁶ is-SO ₂ -; or wherein:

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

T ² for (PEG) _n And V is ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

e and f are each 0; or (b)

Wherein:

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

T ² is substituted (C) ₁ -C ₁₂ ) Alkyl and V ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

e and f are each 0; or (b)

Wherein:

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

T ² for (PEG) _n And V is ² is-CO-;

T ³ Is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

T ⁵ is (C) ₁ -C ₁₂ ) Alkyl and V ⁵ Absence of;

f is 0; or (b)

Wherein:

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

T ² 4AP and V ² is-CO-;

T ³ is (C) ₁ -C ₁₂ ) Alkyl and V ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of;

f is 0; or (b)

Wherein:

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

T ² 4AP and V ² is-CO-;

T ³ is (C) ₁ -C ₁₂ ) Alkyl and V ³ is-O-;

T ⁴ is (C) ₁ -C ₁₂ ) Alkyl and V ⁴ is-CO-;

T ⁵ is AA and V ⁵ Absence of;

T ⁶ is PABC and V ⁶ Absence of; or (b)

Wherein:

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

T ² is an amino acid analog and V ² Absence of;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

e and f are each 0; or (b)

Wherein:

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

T ² for (PEG) _n And V is ² is-CONH-;

T ³ is substituted (C) ₁ -C ₁₂ ) Alkyl and V ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of;

f is 0; or (b)

Wherein:

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

T ² is AA and V ² is-NH-;

T ³ for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of;

f is 0; or (b)

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 For (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and isAnd V is ⁴ Absence of;

T ⁵ is PABO and V ⁵ Absence of; and is also provided with

f is 0; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PAP and V ⁵ is-COO-; and is also provided with

f is 0; or (b)

Wherein:

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

T ² for (PEG) _n And V is ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PAP and V ⁴ is-COO-; and is also provided with

e and f are each 0.

In certain embodiments, the left side of the linker structure is attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety, and the right side of the linker structure is attached to a camptothecin or camptothecin derivative.

In certain embodiments of the conjugates of formula (I), the linker L is attached to a camptothecin or a camptothecin derivative. In some cases, linker L is at R ¹ 、R ² 、R ³ 、R ⁴ 、R ⁵ Or R is ⁶ Attached to a compound of formula (II):

wherein:

R ¹ and R is ² Each of which is a single pieceIndependently selected from hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, or R ¹ And R is ² Optionally cyclic linked to form a 5-or 6-membered cycloalkyl or heterocyclyl ring;

R ³ and R is ⁴ Each independently selected from hydrogen, halo, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, or R ³ And R is ⁴ Optionally cyclic linked to form a 5-or 6-membered cycloalkyl or heterocyclyl ring;

wherein at least one R ¹⁰ Optionally to a second compound of formula (II).

In certain embodiments, R ¹ And R is ² Each independently selected from the group consisting of hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl,Substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, or R ¹ And R is ² Optionally cyclic to form a 5-or 6-membered cycloalkyl or heterocyclyl ring.

In certain embodiments, R ¹ Selected from the group consisting of hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, 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 Halogen (e.g., F, cl, br, I). In certain embodiments, R ¹ Is hydroxyl. In certain embodiments, R ¹ Is amino or substituted amino. In certain embodiments, R ¹ Is alkyl or substituted alkyl, such as 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 Alkenyl or substituted alkenyl, such as 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 alkynyl or substituted alkynyl. In certain embodiments, R ¹ Is an alkoxy group or a substituted alkoxy group. In certain embodiments, R ¹ Is aryl or substituted aryl, such as C _5-8 Aryl or C _5-8 Substituted aryl groups, such as 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, such as C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, such 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, such as C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl groups, such as 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, such as 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 Selected from the group consisting of hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, 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 Halogen (e.g., F, cl, br, I). In certain embodiments, R ² Is hydroxyl. In certain embodiments, R ² Is amino or substituted amino. In certain embodiments, R ² Is alkyl or substituted alkyl, such as 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 Alkenyl or substituted alkenyl, such as 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 aryl or substituted aryl, such as C _5-8 Aryl or C _5-8 Substituted aryl groups, such as 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, such as C _5-8 Heteroaryl groupOr C _5-8 Substituted heteroaryl groups, such as 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, such as C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl groups, such as 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, such as 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 ¹ And R is ² Optionally cyclic to form a 5-or 6-membered cycloalkyl or heterocyclyl ring. In certain embodiments, R ¹ And R is ² Cyclic linkages to form 5-or 6-membered cycloalkyl groups. In certain embodiments, R ¹ And R is ² Cyclic linkages to form a 5-or 6-membered heterocyclyl. In certain embodiments, R ¹ And R is ² Cyclic linkage to form a 5 membered cycloalkyl. In certain embodiments, R ¹ And R is ² Cyclic linkage to form a 6 membered cycloalkyl. In certain embodiments, R ¹ And R is ² Cyclic linkages to form a 5 membered heterocyclyl. In certain embodiments, R ¹ And R is ² Cyclic linkages to form a 6 membered heterocyclyl.

In certain embodiments, R ³ And R is ⁴ Each independently selected from hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl, or R ³ And R is ⁴ Optionally cyclic to form a 5-or 6-membered cycloalkyl or heterocyclyl ring.

In certain embodiments, R ³ Selected from the group consisting of hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl,Alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, 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 Halogen (e.g., F, cl, br, I). In certain embodiments, R ³ Is hydroxyl. In certain embodiments, R ³ Is amino or substituted amino. In certain embodiments, R ³ Is alkyl or substituted alkyl, such as 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 Alkenyl or substituted alkenyl, such as 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 aryl or substituted aryl, such as C _5-8 Aryl or C _5-8 Substituted aryl groups, such as 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, such as C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, such as 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, such as C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl groups, such as 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, such as C _3-6 Heterocyclyl or C _3-6 Substituted heterocyclyl, orC _3-5 Heterocyclyl or C _3-5 Substituted heterocyclyl groups.

In certain embodiments, R ⁴ Selected from the group consisting of hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R ⁴ Is hydrogen. In certain embodiments, R ⁴ Halogen (e.g., F, cl, br, I). In certain embodiments, R ⁴ Is hydroxyl. In certain embodiments, R ⁴ Is amino or substituted amino. In certain embodiments, R ⁴ Is alkyl or substituted alkyl, such as 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 Alkenyl or substituted alkenyl, such as 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 aryl or substituted aryl, such as C _5-8 Aryl or C _5-8 Substituted aryl groups, such as C ₅ Aryl or C ₅ Substituted aryl, or C ₆ Aryl or C ₆ Substituted aryl. In certain embodiments, R ⁴ Is heteroaryl or substituted heteroaryl, such as C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, such as 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, such as C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl groups, such as C _3-6 Cycloalkyl orC _3-6 Substituted cycloalkyl, or C _3-5 Cycloalkyl or C _3-5 Substituted cycloalkyl groups. In certain embodiments, R ⁴ Is a heterocyclic group or a substituted heterocyclic group, such as 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 ³ And R is ⁴ Optionally cyclic to form a 5-or 6-membered cycloalkyl or heterocyclyl ring. In certain embodiments, R ³ And R is ⁴ Cyclic linkages to form 5-or 6-membered cycloalkyl groups. In certain embodiments, R ³ And R is ⁴ Cyclic linkages to form a 5-or 6-membered heterocyclyl. In certain embodiments, R ³ And R is ⁴ Cyclic linkage to form a 5 membered cycloalkyl. In certain embodiments, R ³ And R is ⁴ Cyclic linkage to form a 6 membered cycloalkyl. In certain embodiments, R ³ And R is ⁴ Cyclic linkages to form a 5 membered heterocyclyl. In certain embodiments, R ³ And R is ⁴ Cyclic linkages to form a 6 membered heterocyclyl.

In certain embodiments, R ⁵ Selected from the group consisting of hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R ⁵ Is hydrogen. In certain embodiments, R ⁵ Halogen (e.g., F, cl, br, I). In certain embodiments, R ⁵ Is hydroxyl. In certain embodiments, R ⁵ Is amino or substituted amino. In certain embodiments, R ⁵ Is alkyl or substituted alkyl, such as 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 Alkenyl or substituted alkenyl, such as C _2-6 Alkenyl or C _2-6 SubstitutedAlkenyl, 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, R ⁵ Is an alkoxy group or a substituted alkoxy group. In certain embodiments, R ⁵ Is aryl or substituted aryl, such as C _5-8 Aryl or C _5-8 Substituted aryl groups, such as C ₅ Aryl or C ₅ Substituted aryl, or C ₆ Aryl or C ₆ Substituted aryl. In certain embodiments, R ⁵ Is heteroaryl or substituted heteroaryl, such as C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, such as C ₅ Heteroaryl or C ₅ Substituted heteroaryl, or C ₆ Heteroaryl or C ₆ Substituted heteroaryl groups. In certain embodiments, R ⁵ Cycloalkyl or substituted cycloalkyl, such as C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl groups, such as 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 ⁵ Is a heterocyclic group or a substituted heterocyclic group, such as 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 Selected from OH or OC (O) R ¹¹ . In certain embodiments, R ⁶ Is OH. In certain embodiments, R ⁶ Is OC (O) R ¹¹ 。

In certain embodiments, R ¹¹ 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 ¹¹ Is hydrogen. In certain embodiments, R ¹¹ Is alkyl or substituted alkyl, such as 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 ¹¹ Alkenyl or substituted alkenyl, such as 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 ¹¹ Is alkynyl or substituted alkynyl. In certain embodiments, R ¹¹ Is aryl or substituted aryl, such as C _5-8 Aryl or C _5-8 Substituted aryl groups, such as C ₅ Aryl or C ₅ Substituted aryl, or C ₆ Aryl or C ₆ Substituted aryl. In certain embodiments, R ¹¹ Is heteroaryl or substituted heteroaryl, such as C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, such as C ₅ Heteroaryl or C ₅ Substituted heteroaryl, or C ₆ Heteroaryl or C ₆ Substituted heteroaryl groups. In certain embodiments, R ¹¹ Cycloalkyl or substituted cycloalkyl, such as C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl groups, such as 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 ¹¹ Is a heterocyclic group or a substituted heterocyclic group, such as 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, the compound of formula (II) has the structure of formula (IIa):

in certain embodiments of the compounds of formula (IIa), R ³ As described above.

In certain embodiments of the compounds of formula (IIa), R ⁶ As described above.

In certain embodiments of the compounds of formula (IIa), R ³ OH, and L is attached to R ⁶ Where it is located. In certain embodiments of the compounds of formula (IIa), L is attached to R ³ At (a), and R ⁶ Is OH.

In certain embodiments, the compound of formula (II) has the structure of formula (IIb):

in certain embodiments of the compounds of formula (IIb), R ^1a Selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl. In certain embodiments, R ^1a Is hydrogen. In certain embodiments, R ^1a Is alkyl or substituted alkyl, such as 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 ^1a Is aryl or substituted aryl, such as C _5-8 Aryl or C _5-8 Substituted aryl groups, such as C ₅ Aryl or C ₅ Substituted aryl, or C ₆ Aryl or C ₆ Substituted aryl. In certain embodiments, R ^1a Is heteroaryl or substituted heteroaryl, such as C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, such as C ₅ Heteroaryl or C ₅ Substituted heteroaryl, or C ₆ Heteroaryl or C ₆ Substituted heteroaryl groups. In certain embodiments, R ^1a Cycloalkyl or substituted cycloalkyl, such as C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl groups, such as 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 ^1a Is a heterocyclic group or a substituted heterocyclic group, such as 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 ^1a Is carboxyl. In certain embodiments, R ^1a Is a carboxyl ester. In some casesIn embodiments, R ^1a Is acyl. In certain embodiments, R ^1a Is sulfonyl.

In certain embodiments of the compounds of formula (IIb), R ⁶ As described above.

In certain embodiments of the compounds of formula (IIb), R ^1a Selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl, and L is attached to R ⁶ Where it is located. In certain embodiments of the compounds of formula (IIb), L is attached to R ^1a At (a), and R ⁶ Is OH.

In certain embodiments, the compound of formula (II) has the structure of formula (IIc):

in certain embodiments of the compounds of formula (IIc), R ^1b Selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl. In certain embodiments, R ^1b Is hydrogen. In certain embodiments, R ^1b Is alkyl or substituted alkyl, such as 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 ^1b Is aryl or substituted aryl, such as C _5-8 Aryl or C _5-8 Substituted aryl groups, such as C ₅ Aryl or C ₅ Substituted aryl, or C ₆ Aryl or C ₆ Substituted aryl. In certain embodiments, R ^1b Is heteroaryl or substituted heteroaryl, such as C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, such as C ₅ Heteroaryl or C ₅ Substituted heteroaryl, or C ₆ Heteroaryl or C ₆ Substituted heteroaryl groups. At a certain positionIn some embodiments, R ^1b Cycloalkyl or substituted cycloalkyl, such as C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl groups, such as 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 ^1b Is a heterocyclic group or a substituted heterocyclic group, such as 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 ^1b Is carboxyl. In certain embodiments, R ^1b Is a carboxyl ester. In certain embodiments, R ^1b Is acyl. In certain embodiments, R ^1b Is sulfonyl.

In certain embodiments of the compounds of formula (IIc), R ⁶ As described above.

In certain embodiments of the compounds of formula (IIc), R ^1b Selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl, and L is attached to R ⁶ Where it is located. In certain embodiments of the compounds of formula (IIc), L is attached to R ^1b At (a), and R ⁶ Is OH.

In certain embodiments, the compound of formula (II) has the structure of formula (IId):

in certain embodiments of the compounds of formula (IId), R ^2a And R is ^2b Each independently selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl.

In certain embodiments of the compounds of formula (IId), R ^2a Selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkaneA group, a substituted cycloalkyl group, a heterocyclic group, a substituted heterocyclic group, a carboxyl ester, an acyl group, and a sulfonyl group. In certain embodiments, R ^2a Is hydrogen. In certain embodiments, R ^2a Is alkyl or substituted alkyl, such as 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 ^2a Is aryl or substituted aryl, such as C _5-8 Aryl or C _5-8 Substituted aryl groups, such as C ₅ Aryl or C ₅ Substituted aryl, or C ₆ Aryl or C ₆ Substituted aryl. In certain embodiments, R ^2a Is heteroaryl or substituted heteroaryl, such as C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, such as C ₅ Heteroaryl or C ₅ Substituted heteroaryl, or C ₆ Heteroaryl or C ₆ Substituted heteroaryl groups. In certain embodiments, R ^2a Cycloalkyl or substituted cycloalkyl, such as C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl groups, such as 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 ^2a Is a heterocyclic group or a substituted heterocyclic group, such as 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 ^2a Is carboxyl. In certain embodiments, R ^2a Is a carboxyl ester. In certain embodiments, R ^2a Is acyl. In certain embodiments, R ^2a Is sulfonyl.

In certain embodiments of the compounds of formula (IId), R ^2b Selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl. In certain embodiments, R ^2b Is hydrogen. In certain embodiments, R ^2b Is alkyl or substituted alkyl, such as C _1-6 Alkyl groupOr 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 ^2b Is aryl or substituted aryl, such as C _5-8 Aryl or C _5-8 Substituted aryl groups, such as C ₅ Aryl or C ₅ Substituted aryl, or C ₆ Aryl or C ₆ Substituted aryl. In certain embodiments, R ^2b Is heteroaryl or substituted heteroaryl, such as C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, such as C ₅ Heteroaryl or C ₅ Substituted heteroaryl, or C ₆ Heteroaryl or C ₆ Substituted heteroaryl groups. In certain embodiments, R ^2b Cycloalkyl or substituted cycloalkyl, such as C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl groups, such as 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 ^2b Is a heterocyclic group or a substituted heterocyclic group, such as 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 ^2b Is carboxyl. In certain embodiments, R ^2b Is a carboxyl ester. In certain embodiments, R ^2b Is acyl. In certain embodiments, R ^2b Is sulfonyl.

In certain embodiments of the compounds of formula (IId), R ⁶ As described above.

In certain embodiments of the compounds of formula (IId), R ^2a And R is ^2b Each independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl, and L is attached to R ⁶ Where it is located. In certain embodiments of the compounds of formula (IId), L is attached to R ^2a Or R is ^2b At (a), and R ⁶ Is OH. In certain embodiments of the compounds of formula (IId), L is attached to R ^2a At (a), and R ⁶ Is OH. In certain embodiments of the compounds of formula (IId), L is attached to R ^2b At (a), and R ⁶ Is OH.

In certain embodiments, the compound of formula (II) has the structure of formula (IIe):

in certain embodiments of the compounds of formula (IIe), R ^2c 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, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl, and attachment to L is indicated by wavy lines.

In certain embodiments, R ^2c Is alkyl or substituted alkyl, such as 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 ^2c Alkenyl or substituted alkenyl, such as 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 ^2c Alkynyl or substituted alkynyl, such as C _2-6 Alkynyl or C _2-6 Substituted alkynyl, or C _2-4 Alkynyl or C _2-4 Substituted alkynyl, or C _2-3 Alkynyl or C _2-3 Substituted alkynyl. In certain embodiments, R ^2c Is aryl or substituted aryl, such as C _5-8 Aryl or C _5-8 Substituted aryl groups, such as C ₅ Aryl or C ₅ Substituted aryl, or C ₆ Aryl or C ₆ Substituted aryl. In certain embodiments, R ^2c Is heteroaryl or substituted heteroaryl, such as C _5-8 Heteroaryl or C _5-8 Substituted heteroaryl groups, such as C ₅ Heteroaryl or C ₅ Substituted heteroaryl, or C ₆ Heteroaryl or C ₆ Substituted heteroaryl groups. In certain embodiments, R ^2c Cycloalkyl or substituted cycloalkyl, such as C _3-8 Cycloalkyl or C _3-8 Substituted cycloalkyl groups, such as 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 ^2c Is a heterocyclic group or a substituted heterocyclic group, such as 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 ^2c Is carboxyl. In certain embodiments, R ^2c Is a carboxyl ester. In certain embodiments, R ^2c Is acyl. In certain embodiments, R ^2c Is sulfonyl.

In certain embodiments of the compounds of formula (IIe), the attachment to L is indicated by wavy lines. In other words, the bond with the wavy line indicates the bond attaching the compound of formula (IIe) to the linker.

In certain embodiments of the conjugates of formula (I), the polypeptide (e.g., antibody) may be linked to a drug or active agent through a conjugate moiety. In some cases, a polypeptide (e.g., an antibody) may be linked to more than one drug or active agent through a conjugate moiety. For example, the conjugate moiety may be linked to two or more drugs or active agents. Each drug or active agent may be linked via a respective linker to the same conjugate moiety, which in turn may be attached to a polypeptide (e.g., an antibody) as described herein, thereby attaching the polypeptide (e.g., an antibody) to two or more drugs or active agents.

For example, in certain embodiments of the conjugates of formula (I), one or more R ¹⁰ Optionally to a second compound of formula (II). In some cases, one or more R ¹⁰ Is linked to a second compound of formula (II). In other cases, R ¹⁰ Is not linked to the second compound of formula (II). For example, at least one R ¹⁰ Optionally to a second compound of formula (II). In some cases, one R ¹⁰ Is linked to a second compound of formula (II).

In some embodiments, one ofR is a number of ¹⁰ Via a second joint L ² Is linked to a second compound of formula (II). In certain embodiments, the second joint L ² For a linker (e.g., a second linker) described by the formula:

-(L ⁷ ) _g -(L ⁸ ) _h -(L ⁹ ) _i -(L ¹⁰ ) _j -(L ¹¹ ) _k -(L ¹² ) _l -，

wherein L is ⁷ 、L ⁸ 、L ⁹ 、L ¹⁰ 、L ¹¹ And L ¹² Each independently is a linker subunit, g, h, i, j, k and l are each independently 0 or 1, wherein g, h, i, j, k and l are 1 to 6.

In certain embodiments, the sum of g, h, i, j, k and l is 1. In certain embodiments, the sum of g, h, i, j, k and l is 2. In certain embodiments, the sum of g, h, i, j, k and l is 3. In certain embodiments, the sum of g, h, i, j, k and l is 4. In certain embodiments, the sum of g, h, i, j, k and l is 5. In certain embodiments, the sum of g, h, i, j, k and l is 6. In certain embodiments, g, h, i, j, k and l are each 1. In certain embodiments, g, h, i, j and k are each 1 and l is 0. In certain embodiments, g, h, i, and j are each 1 and k and l are each 0. In certain embodiments, g, h, and i are each 1 and j, k, and l are each 0. In certain embodiments, g and h are each 1 and i, j, k, and l are each 0. In certain embodiments, g is 1 and h, i, j, k and l are each 0.

In certain embodiments, the linker subunit L ⁷ Attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety (e.g., as shown in formula (I) above). In certain embodiments, the linker subunit L ⁸ Attached to camptothecin or camptothecin derivatives when present. In certain embodiments, the linker subunit L ⁹ Attached to camptothecin or camptothecin derivatives when present. In certain embodiments, the linker subunit L ¹⁰ Attached to camptothecin or camptothecin derivatives when present. In certain embodiments, the linker subunit L ¹¹ Attached to camptothecin or camptothecin derivatives when present. In some implementationsIn examples, linker subunit L ¹² Attached to camptothecin or camptothecin derivatives when present.

Any convenient linker subunit can be used for the second linker L ^B Is a kind of medium. For example, the above is for L ¹ 、L ² 、L ³ 、L ⁴ 、L ⁵ And L ⁶ Any of the described linker subunits may be used for linker subunit L ⁷ 、L ⁸ 、L ⁹ 、L ¹⁰ 、L ¹¹ And L ¹² 。

In certain embodiments, the second joint L ^B Is a linker- (L) comprising ⁷ ) _g -(L ⁸ ) _h -(L ⁹ ) _i -(L ¹⁰ ) _j -(L ¹¹ ) _k -(L ¹² ) _l -, wherein:

-(L ⁷ ) _g -is- (T) ⁷ -V ⁷ ) _g -；

-(L ⁸ ) _h -is- (T) ⁸ -V ⁸ ) _h -；

-(L ⁹ ) _i -is- (T) ⁹ -V ⁹ ) _i -；

-(L ¹⁰ ) _j -is- (T) ¹⁰ -V ¹⁰ ) _j -；

-(L ¹¹ ) _k -is- (T) ¹¹ -V ¹¹ ) _k -; and is also provided with

-(L ¹² ) _l -is- (T) ¹² -V ¹² ) _l -，

Wherein T is ⁷ 、T ⁸ 、T ⁹ 、T ¹⁰ 、T ¹¹ And T ¹² A tethering group when present;

V ⁷ 、V ⁸ 、V ⁹ 、V ¹⁰ 、V ¹¹ and V ¹² Covalent bonds or linking functional groups when present; and is also provided with

g. h, i, j, k and l are each independently 0 or 1, wherein the sum of g, h, i, j, k and l is 1 to 6.

Thus, in certain embodiments, the second joint L ^B Comprising:

wherein the method comprises the steps of

g. h, i, j, k and 1 are each independently 0 or 1;

each R is ¹³ Independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted Aryl of (a); and is also provided with

Any convenient tethering group may be used for T ⁷ 、T ⁸ 、T ⁹ 、T ¹⁰ 、T ¹¹ And T ¹² . For example, the above is about T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ And T ⁶ Any of the tethering groups described may be used for tethering group T ⁷ 、T ⁸ 、T ⁹ 、T ¹⁰ 、T ¹¹ And T ¹² 。

Any convenient linking functionality can be used for V ⁷ 、V ⁸ 、V ⁹ 、V ¹⁰ 、V ¹¹ And V ¹² . For example, the above is about V ¹ 、V ² 、V ³ 、V ⁴ 、V ⁵ And V ⁶ Any of the described linking functionalities may be used for linking functionality V ⁷ 、V ⁸ 、V ⁹ 、V ¹⁰ 、V ¹¹ And V ¹² 。

In certain embodiments, each R ¹³ Independently 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 ¹³ Said method.

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, 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 are as described above for R ¹⁵ Said method. In these embodiments, the various possible substituents are as above for R ¹⁵ Said method.

At the second joint L ^B In certain embodiments of (2), the tethering group T ⁷ 、T ⁸ 、T ⁹ 、T ¹⁰ 、T ¹¹ And T ¹² Each optionally substituted with a glycoside or glycoside derivative. In certain embodiments, the glycoside or glycoside derivative is selected from the group consisting of glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

At the second joint L ^B In certain embodiments of MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP tether structures as shown above may be substituted with one or more additional groups selected from glycoside and glycoside derivatives. For example, in some embodiments of MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP tether structures shown above, the benzene ring may be substituted with one or more additional groups selected from glycoside and glycoside derivatives. In certain embodiments, the glycoside or glycoside derivative is selected from the group consisting of glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

In certain embodiments, T ⁷ 、T ⁸ 、T ⁹ 、T ¹⁰ 、T ¹¹ And T ¹² V (V) ⁷ 、V ⁸ 、V ⁹ 、V ¹⁰ 、V ¹¹ And V ¹² Selected from the following:

Wherein:

T ⁷ is absent and V ⁷ is-NR ¹⁵ CO-；

T ⁸ Is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

T ¹¹ is EDA and V ¹¹ is-CO-; and is also provided with

l is 0; or (b)

Wherein:

T ⁷ is absent and V ⁷ is-NR ¹⁵ CO-；

T ⁸ Is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of; and is also provided with

k and l are each 0; or (b)

Wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

T ⁸ is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is an amino acid analog and V ⁹ is-NH-;

T ¹⁰ for (PEG) _n And V is ¹⁰ is-CO-;

T ¹¹ is AA and V ¹¹ Absence of; and is also provided with

T ¹² Is PABC and V ¹² Absence of; or (b)

Wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

T ⁸ is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CONH-;

T ⁹ for (PEG) _n And V is ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of; and is also provided with

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ is substituted (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

k and l are each 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ for (PEG) _n And V is ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

T ¹¹ is (C) ₁ -C ₁₂ ) Alkyl and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ Is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ 4AP and V ⁸ is-CO-;

T ⁹ is (C) ₁ -C ₁₂ ) Alkyl and V ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ 4AP and V ⁸ is-CO-;

T ⁹ is (C) ₁ -C ₁₂ ) Alkyl and V ⁹ is-O-;

T ¹⁰ is (C) ₁ -C ₁₂ ) Alkyl and V ¹⁰ is-CO-;

T ¹¹ is AA and V ¹¹ Absence of;

T ¹² is PABC and V ¹² Absence of; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ is an amino acid analog and V ⁸ Absence of;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

k and l are each 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ for (PEG) _n And V is ⁸ is-CONH-;

T ⁹ is substituted (C) ₁ -C ₁₂ ) Alkyl and V ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ is AA and V ⁸ is-NH-;

T ⁹ for (PEG) _n And V is ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ for (PEG) _n And V is ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PAP andV ¹⁰ is-COO-; and is also provided with

k and l are each 0.

At the second joint L ^B In some embodiments of (2), the left side of the linker structure is attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety, and the right side of the linker structure is attached to a camptothecin or camptothecin derivative. At the second joint L ^B In some embodiments of (2) the left side of the linker structure is attached to the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety through a linking functional group such as-NHCO-.

In certain embodiments, the conjugate is an antibody-drug conjugate, wherein the antibody and drug are conjugated through a linker (e.g., L or L ^B ) Are connected together as described above. In some cases, the joint is a cleavable joint. 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, the cleavable moiety may comprise one or more covalent bonds that may dissociate or break under certain conditions to separate the cleavable linker into two or more moieties. Thus, a cleavable linker 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 site of action target of the drug.

In some cases, the cleavable joint includes two cleavable portions, such as a first cleavable portion and a second cleavable portion. The cleavable moiety may be configured such that cleavage of both cleavable moieties is required in order to separate or release the drug from the antibody at the desired site of action target of the drug. For example, the cutting of the cleavable joint may be achieved by first cutting one of the two cleavable portions, and then cutting the other of the two cleavable portions. In certain embodiments, the cleavable joint comprises a first cleavable portion and a second cleavable portion that impedes the cleavage of the first cleavable portion. By "hindering cutting" is meant that the presence of an uncut second cleavable portion reduces the likelihood of or substantially inhibits the cleavage of the first cleavable portion, thereby significantly reducing the amount of or preventing the cleavage of the cleavable linker. For example, the presence of an uncut second cuttable portion may prevent the first cuttable portion from cutting. The presence of the second cleavable moiety prevents cleavage of the first cleavable moiety from continuing to significantly reduce the amount of drug released from the antibody or prevent release of drug from the antibody. For example, premature release of the drug from the antibody can be significantly reduced or prevented until the antibody-drug conjugate is at or near the desired site of action target of the drug.

In some cases, the cutting of the cleavable joint may be achieved by first cutting the second cleavable portion and then cutting the first cleavable portion, as the second cleavable portion hinders the first cleavable portion from cutting. Cutting of the second cuttable portion may reduce or eliminate obstruction of cutting of the first cuttable portion, thereby allowing the first cuttable portion to be cut. Cleavage of the first cleavable moiety may result in dissociation 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, substantially no cutting of the first cleavable portion occurs in the presence of the second cleavable portion that is not cut. By "substantially" is meant that about 10% or less of the first cleavable moiety is cleaved in the presence of the second cleavable moiety that is not cleaved, such as 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 is cleaved in the presence of the second cleavable moiety that is not cleaved.

In other words, the second cuttable portion may protect the first cuttable portion from cutting. For example, the presence of an uncleaved second cleavable moiety can protect the first cleavable moiety from cleavage, thereby significantly reducing or preventing 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, which in turn, as described above, separates or releases the drug from the antibody at the desired site of action target of the drug. In some cases, the cutting of the second cuttable portion exposes the first cuttable portion to a subsequent cut, but the cutting of the second cuttable portion does not itself result in the cutting of the cuttable joint (i.e., the first cuttable portion still needs to be cut in order to cut the cuttable joint).

The cleavable moieties contained 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 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, such as 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 substantial amounts in other areas (such as in 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, while cleavage does not occur in substantial amounts 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, such as for delivering a cancer therapeutic drug to a desired site of action where cancer cells are present. In some cases, an enzyme such as protease cathepsin B may be a biomarker for cancer that is overexpressed in cancer cells. Overexpression of certain enzymes in cancer and localization thereby 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., cancer (and overexpressed enzymes) site). Thus, in some embodiments, the enzymatically cleavable moiety is a cleavable moiety (e.g., a peptide) 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) 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 that the second cleavable moiety protects from premature cleavage) 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), thereby significantly reducing or preventing 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 linked 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 or glycoside derivative suitable for use in a cleavable linker and cleavable 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 or glycoside derivative selected from the group consisting of: glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc and O-GalNAc. In some cases, the second cleavable moiety is a glucuronide. 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 attached (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 (sugar amine), an S-glycosidic bond (thio glycoside) 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 effecting cleavage (hydrolysis) of 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 glucuronidase, glycosidases (such as galactosidase, glucosidase, mannosidase, fucosidase, etc.). Other suitable enzymes may also be used to mediate cleavage (hydrolysis) of the glycosidic bond attaching the glycoside to the cleavable linker. In some cases, the enzyme used to mediate cleavage (hydrolysis) of the glycosidic bond attaching the glycoside to the cleavable linker is present 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, that 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 present at or near a target site at which an enzyme is present that mediates cleavage of the first cleavable moiety.

In certain embodiments, the conjugate of formula (I) has a structure selected from the group consisting of:

/>

any of the chemical entities, linkers, and coupling moieties shown in the structures described above may be suitable for use in the subject compounds and conjugates.

Additional disclosures relating to hydrazino-indolyl and hydrazino-pyrrolo-pyridinyl compounds and methods for producing 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. Additional disclosure regarding cleavable linkers is found in U.S. provisional application No. 63/116,632, filed 11/20 in 2020, the disclosure of which is incorporated herein by reference.

Compounds useful for the production of conjugates

The present disclosure provides hydrazino-indolyl and hydrazino-pyrrolo-pyridinyl compounds useful for the production of conjugates described herein. In certain embodiments, the hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl compounds may be conjugated moieties for conjugation of polypeptides (e.g., antibodies) and drugs or active agents (e.g., camptothecins or camptothecine derivatives). For example, a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl compound may bind to a polypeptide (antibody) and also to a drug or active agent, thereby indirectly binding the polypeptide (antibody) to the drug.

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

wherein the method comprises the steps of

Z is CR ⁴ Or N;

R ⁵ selected from the group consisting of hydrogen, halo, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

wherein at least one R ¹⁰ Optionally to a second compound of formula (II).

In some cases, the compound of formula (II) has the structure of formula (IIa):

wherein R is ³ OH and L is attached to R ⁶ A place; or L is attached to R ³ At and R is ⁶ Is OH

In some cases, the compound of formula (II) has the structure of formula (IIb):

wherein R is ^1a Selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl, and L is attached to R ⁶ A place; or L is attached to R ^1a At and R is ⁶ Is OH.

In some cases, the compound of formula (II) has the structure of formula (IIc):

wherein R is ^1b Selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl, and L is attached to R ⁶ A place; or L is attached to R ^1b At and R is ⁶ Is OH.

In some cases, the compound of formula (II) has the structure of formula (IId):

wherein R is ^2a And R is ^2b Each independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl, and L is attached to R ⁶ A place; or L is attached to R ^2a At and R is ^2b And R is ⁶ Is OH.

In some cases, the compound of formula (II) has the structure of formula (IIe):

wherein R is ^2c Selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl,Alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl, and attachment to L is indicated by wavy lines.

Substituents associated with the compounds of formulae (II), (IIa), (IIb), (IIc), (IId) and (IIe) are as described above. The reference to formula (II) is intended to also encompass formulae (IIa), (IIb), (IIc), (IId) and (IIe).

Regarding the compound of formula (III), substituent Z, R ⁸ 、R ⁹ 、R ¹⁰ 、L、L ^B And W is as described above for the conjugate of formula (I). Similarly, with respect to the first linker L and the second linker L of formula (III) ^B ，T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ 、T ⁶ 、V ¹ 、V ² 、V ³ 、V ⁴ 、V ⁵ And V ⁶ T is as follows ⁷ 、T ⁸ 、T ⁹ 、T ¹⁰ 、T ¹¹ 、T ¹² 、V ⁷ 、V ⁸ 、V ⁹ 、V ¹⁰ 、V ¹¹ And V ¹² The substituents are as described above for the conjugates of formula (I).

For example, in some cases T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ And T ⁶ V (V) ¹ 、V ² 、V ³ 、V ⁴ 、V ⁵ And V ⁶ Selected from the following:

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of; and is also provided with

T ⁶ Is EDA and V ⁶ is-CO-; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁶ Is absent and V ⁶ is-CO-; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ is-NR ¹⁵ -; and is also provided with

T ⁶ Is (C) ₁ -C ₁₂ ) Alkyl and V ⁶ is-CO-; or (b)

Wherein:

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

T ² is AA and V ² Absence of;

T ³ is PABC and V ³ Absence of;

T ⁴ is EDA and V ⁴ is-CO-; and is also provided with

e and f are each 0; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of; and is also provided with

f is 0; or (b)

Wherein:

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

T ² is AA and V ² Absence of;

T ³ is PABC and V ³ Absence of; and is also provided with

d. e and f are each 0; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABA and V ⁵ is-CO-; and is also provided with

T ⁶ Is (C) ₁ -C ₁₂ ) Alkyl and V ⁶ is-SO ₂ -; or (b)

Wherein:

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

T ² for (PEG) _n And V is ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

e and f are each 0; or (b)

Wherein:

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

T ² is substituted (C) ₁ -C ₁₂ ) Alkyl and V ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

e and f are each 0; or (b)

Wherein:

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

T ² for (PEG) _n And V is ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

T ⁵ is (C) ₁ -C ₁₂ ) Alkyl and V ⁵ Absence of;

f is 0; or (b)

Wherein:

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

T ² 4AP and V ² is-CO-;

T ³ is (C) ₁ -C ₁₂ ) Alkyl and V ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of;

f is 0; or (b)

Wherein:

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

T ² 4AP and V ² is-CO-;

T ³ is (C) ₁ -C ₁₂ ) Alkyl and V ³ is-O-;

T ⁴ is (C) ₁ -C ₁₂ ) Alkyl and V ⁴ is-CO-;

T ⁵ is AA and V ⁵ Absence of;

T ⁶ is PABC and V ⁶ Absence of; or (b)

Wherein:

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

T ² is an amino acid analog and V ² Absence of;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

e and f are each 0; or (b)

Wherein:

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

T ² for (PEG) _n And V is ² is-CONH-;

T ³ is substituted (C) ₁ -C ₁₂ ) Alkyl and V ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of;

f is 0; or (b)

Wherein:

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

T ² is AA and V ² is-NH-;

T ³ for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of;

f is 0; or (b)

Wherein:

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

T ² is amino acidAnalogue and V ² is-NH-;

T ³ for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABO and V ⁵ Absence of; and is also provided with

f is 0; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PAP and V ⁵ is-COO-; and is also provided with

f is 0; or (b)

Wherein:

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

T ² for (PEG) _n And V is ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PAP and V ⁴ is-COO-; and is also provided with

e and f are each 0.

For example, in some cases T ⁷ 、T ⁸ 、T ⁹ 、T ¹⁰ 、T ¹¹ And T ¹² V (V) ⁷ 、V ⁸ 、V ⁹ 、V ¹⁰ 、V ¹¹ And V ¹² Selected from the following:

wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

T ⁸ is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

T ¹¹ is EDA and V ¹¹ is-CO-; and is also provided with

l is 0; or (b)

Wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

T ⁸ is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of; and is also provided with

k and l are each 0; or (b)

Wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

T ⁸ is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is an amino acid analog and V ⁹ is-NH-;

T ¹⁰ for (PEG) _n And V is ¹⁰ is-CO-;

T ¹¹ is AA and V ¹¹ Absence of; and is also provided with

T ¹² Is PABC and V ¹² Absence of; or (b)

Wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

T ⁸ is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CONH-;

T ⁹ for (PEG) _n And V is ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of; and is also provided with

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ is substituted (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ Is PABC and V ¹⁰ Absence of;

k and l are each 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ for (PEG) _n And V is ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

T ¹¹ is (C) ₁ -C ₁₂ ) Alkyl and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ 4AP and V ⁸ is-CO-;

T ⁹ is (C) ₁ -C ₁₂ ) Alkyl and V ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ 4AP and V ⁸ is-CO-;

T ⁹ is (C) ₁ -C ₁₂ ) Alkyl and V ⁹ is-O-;

T ¹⁰ is (C) ₁ -C ₁₂ ) Alkyl andV ¹⁰ is-CO-;

T ¹¹ is AA and V ¹¹ Absence of;

T ¹² is PABC and V ¹² Absence of; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ is an amino acid analog and V ⁸ Absence of;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

k and l are each 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ for (PEG) _n And V is ⁸ is-CONH-;

T ⁹ is substituted (C) ₁ -C ₁₂ ) Alkyl and V ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ is AA and V ⁸ is-NH-;

T ⁹ for (PEG) _n And V is ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ Is PABC and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ for (PEG) _n And V is ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PAP and V ¹⁰ is-COO-; and is also provided with

k and l are each 0.

The compounds of formula (III) may be used in the conjugation reactions described herein, wherein a drug or active agent attached to a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety is conjugated to a polypeptide (e.g., an antibody) to form an antibody-drug conjugate.

In certain embodiments, the compound of formula (III) has the following structure:

/>

any of the chemical entities, linkers, and conjugate moieties shown in the structures described above may be suitable for use with the subject compounds and conjugates.

Polypeptides and antibodies

As described above, the subject conjugates can comprise a polypeptide (e.g., an antibody) as substituent W. The amino acid sequence of a polypeptide (antibody) has been modified to include 2-formylglycine (fGly) residues. As used herein, amino acids may be referred to by their standard names, their standard three-letter abbreviations, and/or their standard single-letter abbreviations, such as: 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 polypeptide (antibody) is modified to include a sulfatase motif comprising serine or cysteine residues, which sulfatase motif is capable of converting (oxidizing) to 2-formylglycine (fggly) residues by the action of Formylglycine Generating Enzymes (FGEs) in vivo (e.g., when translating aldehyde-tagged containing proteins in cells) or in vitro (e.g., by contacting aldehyde-tagged containing proteins with FGEs in a cell-free system). Such sulfatase motifs may also be referred to herein as FGE modification sites.

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. 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 so as to define sulfatase motifs less than 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 amino acid residues in length.

In certain embodiments, the polypeptide of interest includes those 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) with respect to the native amino acid sequence to provide a sequence of the sulfatase motif in the polypeptide. In certain embodiments, the 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. Where 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 residue modifications may be reduced, e.g., by site-specific modification (insertion, addition, deletion, substitution/substitution) of the 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 to minimize the number of amino acid residues inserted, deleted, substituted (substituted), or added (e.g., added to the N-terminus or C-terminus). Minimizing the extent of amino acid sequence modifications of a target antibody may minimize the effects such modifications may have on antibody function and/or structure.

It should be noted that while aldehyde tags of particular interest are those that comprise at least a minimal sulfatase motif (also referred to as a "consensus sulfatase motif"), it is readily understood that longer aldehyde tags are 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 that may flank additional amino acid residues at the N-terminal and/or C-terminal end of the motif. 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. The aldehyde tag may be present in the CH1 domain of the Ig heavy chain. 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, for example, in 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 (also denoted by (C/S));

Z ²⁰ proline or alanine residues (which may also be denoted (P/a));

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

The amino acid sequence of the antibody heavy and/or light chain may be modified to provide at least 5 of formula X ¹ Z ¹⁰ X ² Z ²⁰ X ³ Z ³⁰ Wherein

Z ¹⁰ Is cysteine or serine;

Z ²⁰ is a proline or alanine residue;

Z ³⁰ is aliphatic amino acid or 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 in which the aldehyde-tagged polypeptide is expressed or FGE that will be contacted with the aldehyde-tagged polypeptide in a cell-free in vitro method).

For example, where FGE is 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., other than an aromatic amino acid or a charged amino acid), such as L, M, S or V, provided that when the sulfatase motif is N-terminal to the target polypeptide, X is present ¹ ；

X ² And X ³ May independently be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., other than 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), such as lysine) or an aliphatic amino acid (alanine (a), glycine (G), leucine (L), valine (V), isoleucine (I) or proline (P), such as 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: /).

fGly-containing sequences

After FGE acts on the antibody heavy and/or light chain, serine or cysteine in the sulfatase motif is modified to fcy. Thus, a sulfatase motif containing fGly 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 formylglycine residue;

Z ²⁰ Proline or alanine residues (which may also be denoted (P/a));

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., other than 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 May independently be any amino acid, such as an aliphatic amino acid, a sulfur-containing amino acid, or a polar uncharged amino acid (i.e., other than 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, to produce a conjugate, a polypeptide containing a fGly residue may be conjugated to a drug or active agent by reacting the fGly with a reactive moiety of a linker (e.g., a hydrazino-indolyl or hydrazino-pyrrolo-pyridinyl conjugate moiety, as described above) that is attached to the drug or active agent to produce a sulfatase motif containing fGly'. As used herein, the term "fGly'" refers to an amino acid residue coupled to the sulfatase motif of a drug or active agent through a linker, as described herein. Thus, a sulfatase motif containing fGly' 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 an amino acid residue coupled to a drug or active agent through a linker as described herein;

Z ²⁰ proline or alanine residues (which may also be denoted (P/a));

Modification site

As described above, the amino acid sequence of a polypeptide (antibody) is modified to include a sulfatase motif comprising serine or cysteine residues, which sulfatase motif is capable of being converted (oxidized) to a fGly residue by the action of FGE, either in vivo (e.g., when an aldehyde-tagged-containing protein is translated in a cell) or in vitro (e.g., by contacting an aldehyde-tagged-containing 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 the antibody into which the sulfatase motif is introduced 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 to minimize the number of amino acid residues inserted, deleted, substituted (substituted), and/or added (e.g., added to the N-terminus or C-terminus). Minimizing the extent of amino acid sequence modifications of a target antibody may minimize the effects such modifications may have on antibody function and/or structure.

Antibody heavy chain constant regions can 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 amino acid sequence can be modified to include an aldehyde tag, wherein the aldehyde tag is present in or near the solvent accessible loop region of the Ig constant region. The Ig constant region amino acid sequence 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 sequence of the sulfatase motif 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 IgA, igM, igD, igE, igG, igG2, igG3 or IgG4 isotype heavy chain or any allotypic variant 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, or the like, which heavy chain constant region sequence has been modified to include at least one sulfatase motif that can be modified by FGE to produce a fgy modified Ig polypeptide. 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. Aldehyde-labeled Ig light chain constant regions can include kappa light chains, lambda light chain constant region sequences (e.g., human kappa or lambda light chain constant regions), hybrid light chain constant regions, synthetic light chain constant regions, or consensus light chain constant region sequences, and the like, which include at least one sulfatase motif that can be modified by FGE to produce an fgy modified antibody. Exemplary constant regions include human γ1 and γ3 regions. The modified constant region may have a wild-type amino acid sequence in addition to the thioesterase motif, or it may 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 or in addition to the C-terminus of the Ig polypeptide heavy chain. As described above, the isolated aldehyde-labeled antibody may comprise a heavy chain constant region amino acid sequence modified to include a sulfatase motif as described above, wherein the sulfatase motif is located at or adjacent to a surface accessible loop region of the heavy chain constant region of the antibody.

Sulfatase motifs may be provided within or near one or more of these amino acid sequences of such modification sites of Ig heavy chains. For example, an Ig heavy chain polypeptide amino acid sequence can be modified (e.g., wherein the modification includes one or more amino acid residue insertions, deletions, and/or substitutions) at one or more of these amino acid sequences 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 amino acid sequence can be modified (e.g., wherein the modification includes one or more amino acid residue insertions, deletions, and/or substitutions) at one or more of these amino acid sequences to provide a sulfatase motif between any two residues of the Ig heavy chain modification site. In some embodiments, the Ig heavy chain polypeptide amino acid sequence 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, in cases where the native amino acid sequence provides one or more amino acid residues of the sulfatase motif sequence, selected amino acid residues of the modification site(s) of the Ig heavy chain polypeptide amino acid sequence may be modified (e.g., where the modification includes one or more amino acid residue insertions, deletions, and/or substitutions) so as to provide the sulfatase motif at the modification site(s).

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, for example: an antigen present on a cancer cell; 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); antigens present on diseased cells, and the like. 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 are capable of binding with suitable binding affinity (e.g., 5x10 ^-6 M to 10 ^-7 M、10 ^-7 M to 5x10 ^-7 M、5x 10 ^-7 M to 10 ^-8 M、10 ^-8 M to 5x10 ^-8 M、5x 10 ^-8 M to 10 ^-9 M) or greater than 10 ^-9 The binding affinity of M binds to the antigen.

As a non-limiting example, the subject antibody conjugates 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 conjugated 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 conjugated 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., wherein the antigen is encoded by a virus; wherein the antigen is expressed on a virus-infected cell type, etc.), and the conjugated moiety can be a drug, such as a viral fusion inhibitor. For example, the subject antibody conjugates can bind to an antigen present on a virus-infected cell, and the conjugated moiety can 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). Drugs suitable for use as or that can be modified to render them suitable for use as reactive partners conjugated to polypeptides (e.g., antibodies) as described herein include camptothecins or camptothecine derivatives. For example, suitable camptothecins and camptothecine derivatives for use in the conjugates and compounds described herein include, but are not limited to, compounds of formulas (II), (IIa), (IIb), (IIc), and (IId), as described above.

Embodiments of the present disclosure include conjugates in which a polypeptide (e.g., an antibody) is conjugated to one or more drug moieties, such as 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. As described herein, a drug moiety may be conjugated to an antibody at one or more sites of the polypeptide (antibody). 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 conjugate has an average DAR of 1 to 3 (such as 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3), and in certain embodiments, the conjugate has an average DAR of 1 to 2, in certain embodiments, the conjugate has an average DAR of 2 to 3.

Formulations

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

In some embodiments, a pharmaceutical composition is provided that includes any of the conjugates of the present disclosure and a pharmaceutically acceptable excipient.

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

The method of formulating the conjugate can be adapted from those 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 amenable to treatment by administration of a parent drug (i.e., camptothecin or a camptothecin derivative prior to conjugation to an antibody).

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

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 administration is effective to release a therapeutically effective amount of the drug (e.g., camptothecin or a camptothecin derivative) 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, such as 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 moiety that protects the first cleavable moiety from cleavage may be cleaved so as 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 facilitates 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 facilitates 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 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 desired site of action further includes the presence of a first enzyme. In some cases, the first enzyme is present in excess at the desired site of action compared to other parts of the subject's body to be treated. For example, the first enzyme may be overexpressed at a desired site of action compared to other parts of the subject's body to be treated. In some cases, the first enzyme is present in excess at the desired site of action because the first enzyme is located at a particular site or position. For example, a first enzyme may be associated with a structure (such as a lysosome) within a desired site of action. In some cases, the first enzyme is present in excess in the lysosome as compared to other parts of the subject's body. In some embodiments, the lysosome comprising the first enzyme is present at a desired site of action of the drug of the antibody-drug conjugate, such as at 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 the desired site of action further includes the presence of a second enzyme. In some cases, the second enzyme is present in excess at the desired site of action compared to other parts of the subject's body to be treated. For example, the second enzyme may be overexpressed at a desired site of action compared to other parts of the subject's body to be treated. In some cases, the second enzyme is present in excess at the desired site of action because the second enzyme is located at a particular site or position. For example, the second enzyme may be associated with a structure (such as a lysosome) within the desired site of action. In some cases, the second enzyme is present in excess in the lysosome as compared to other parts of the subject's body. In some embodiments, the lysosome comprising the second enzyme is present at a desired site of action of the drug of the antibody-drug conjugate, such as at a site of a cancer or tumor to be treated with the drug. In certain embodiments, the second enzyme is a glycosidase, such as a galactosidase, glucosidase, or mannosidase.

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, rabbit, 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 substantial 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 substantial 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 (such as 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) of the antibody-drug conjugate is cleaved in the absence of the first enzyme and/or the second enzyme. In some cases, the antibody-drug conjugate is substantially stable such that the cleavable linker of the antibody-drug conjugate does not undergo substantial cleavage in the absence of the first enzyme and/or the second enzyme, but is capable of being cleaved in the presence of the first enzyme and/or 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 may be cleaved from the cleavable linker, thereby exposing the first cleavable moiety to subsequent cleavage by the first enzyme, which in turn releases the drug at the desired site of action. In certain embodiments, the antibody-drug conjugate remains stable for an extended period of time (such as 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 more, or 24 hours (1 day) or more, or 2 days or more, or 3 days or more, or 4 days or more, or 5 days or more, or 6 days or more, or 7 days (1 week) or more) in the absence of the first enzyme and/or the second enzyme after administration to the subject. In certain embodiments, the antibody-drug conjugate remains stable for an extended period of time in the absence of the first enzyme and/or the second enzyme at a range of pH values, such as in a pH range of 2 to 10, or 3 to 9, or 4 to 8, or 5 to 8, or 6 to 8, or 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 that is amenable to treatment by administration of a parent drug. By "treatment" is meant that at least an improvement in symptoms associated with the condition afflicting the host is achieved, wherein the improvement is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, such as a symptom associated with the condition being treated. Thus, treatment also includes situations in which the pathological condition, or at least symptoms associated therewith, are completely inhibited (e.g., prevented from occurring) or stopped (e.g., terminated) such that the host is no longer suffering from the condition, or at least symptoms that characterize the condition. The treatment thus comprises: (i) Prevention, i.e., reducing the risk of developing clinical symptoms, including preventing the clinical symptoms from developing, e.g., preventing disease from progressing 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 suitable for treatment with the antibody-drug conjugates disclosed herein. Generally, such subjects are "mammals," in which the human is 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, e.g., in animal models of disease), and non-human primates (e.g., chimpanzees and monkeys).

The amount of antibody-drug conjugate administered can be initially determined based on the dosage of the parent drug and/or the guidance of the dosing regimen. In general, the antibody-drug conjugate can provide targeted delivery of the conjugated drug and/or an extended serum half-life, providing at least one of a reduced dose or reduced administration of the drug in a dosing regimen. Thus, the antibody-drug conjugate may provide a reduced dose and/or a reduced number of administrations 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, since the antibody-drug conjugate can provide a controlled stoichiometry of drug delivery, the dosage of the antibody-drug conjugate can be calculated based on the number of drug molecules provided per antibody-drug conjugate.

In some embodiments, multiple doses of the antibody-drug conjugate are administered. The frequency of administration of the antibody-drug conjugate can vary based 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 frequency of administration of the antibody-drug conjugate is 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/od), 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. These methods can be used to treat a variety of cancers, including but not limited to breast, ovarian, colon, lung, gastric and pancreatic. 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 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, colon cancer, colorectal cancer, gastric cancer, lung cancer (including lung small cell cancer and non-small cell cancer), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or cholangiocarcinoma, choriocarcinoma, seminoma, embryonic carcinoma, cervical cancer, uterine cancer, testicular cancer, epithelial cancer, and the like.

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 present 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. By "average" is meant an arithmetic average. Standard abbreviations may be used, for example, bp: base pairing; kb: kilobases; pl: lifting the skin; s or sec: second, wherein the second is; min: minutes; h or hr: hours; aa: amino acids; kb: kilobases; the method comprises the following steps: base pairing; nt, nucleotide; i.m.: intramuscular (earth); i.p.: intraperitoneal (ground); s.c.: subcutaneous (ground); etc.

General Synthesis procedure

Many general references are available that provide well-known chemical synthesis schemes and conditions for synthesizing the disclosed compounds (see, e.g., smith and March, march's Advanced Organic Chemistry: reactions, mechanisms, and structures, fifth edition, wiley journal, 2001; or Vogel, textbooks on practical organic chemistry, including qualitative organic analysis (A Textbook of Practical Organic Chemistry, including Qualitative Organic Analysis), fourth edition, new York: lantern publishing, 1978).

The compounds as described herein may be purified by any purification scheme known in the art, including chromatographic methods such as HPLC, preparative thin layer chromatography, flash column chromatography, and ion exchange chromatography. Any suitable stationary phase including normal and reverse phases and ionic resins may be used. In certain embodiments, the disclosed compounds are purified via silica gel and/or alumina chromatography. See, e.g., modern liquid chromatography guide (Introduction to Modern Liquid Chromatography), 2 nd edition, l.r. snyder and j.j.kirkland editions, john wili father-son press (John Wiley and Sons), 1979; and thin layer chromatography (Thin Layer Chromatography), edited by e.stahl, schpringer publishing (Springer-Verlag), new york, 1969.

During any process for 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 described in standard literature, such as J.F.W.McOmie, protecting groups in organic chemistry (Protective Groups in Organic Chemistry), plenum Press, london and New York, 1973; T.W.Greene and P.G.M.Wuts, protecting group in organic Synthesis (Protective Groups in Organic Synthesis), third edition, wili Press, new York, 1999; peptides (The Peptides), volume 3 (e.gross and j.meienhofer, editors), academic Press (Academic Press), london and new york, 1981; organic chemistry methods (Methoden der organischen Chemie), haobhen-Wei Er (Houben-Weyl), 4 th edition, volume 15/l, georg Thieme Verlag, stuttgart, 1974; H. jakuske and H.Jescheit, amino acids, peptides, proteins, chemical publishers (Verlag Chemie), weinheim, deerfield beacons and Basel,1982; and/or Jochen Lehmann, hydrogenation chemistry: monosaccharides and derivatives (Chemie der Kohlenhydrate: monosaccharide and Derivate), georg Thieme 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 via a variety of different synthetic routes using commercially available starting materials and/or starting materials prepared by conventional synthetic methods. Examples of the various synthetic routes that can be used to synthesize the compounds disclosed herein are described in the schemes below.

Example 1

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. Compounds 7, 13, 21, 28, 74 and 152 and HIPS linkers 10, 18 and 36 were purchased from Shanghai Meidixi and used without purification. Compounds 156, 157, 169, and 171 were purchased from other commercial sources; the synthesis of compounds 87 and 93 was previously reported. In all cases, buchi Rotovapor R-114, equipped with a Buchi V-700 vacuum pump, was used to remove the solvent under reduced pressure. 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 150x 21.2mm 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, a Agilent Poroshell SB C18 column (4.6 mm x 50 mm) using a gradient of 10% to 100% water and acetonitrile containing 0.1% formic acid at room temperature. HPLC was monitored at 254nm or 205 nm.

Synthesis of HIPS constructs with camptothecins

Scheme 1. Synthesis of intermediate 6.

Preparation of (S) -tert-butyl (4, 11-diethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-9-yl) carboxylate (5)

SN-38 1 (158 mg,0.402 mmol) and Boc at 0deg.C ₂ To a solution of O (114 mg,1.3 mmol) in dichloromethane (16 mL) was added pyridine (0.480 mL,12.2 mmol). After one hour, the solution was warmed to room temperature and stirred for two hours. The reaction mixture was then concentrated under vacuum and the residue was purified on silica gel (hexane/EtOAc, 100:0 to 0:100 v/v) to give 5 (160 mg, 80%) as an off-white solid. LRMS (ESI): m/z 493.2[ M+H ]] ⁺ ，C ₂₇ H ₂₉ N ₂ O ₇ m/z calculated 493.2.

Preparation of (S) -tert-butyl (4, 11-diethyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-4, 9-diyl) (4-nitrophenyl) bis (carbonate) (6)

To a solution of 5 (15 mg,0.030 mmol) in dichloromethane (1 mL) was added nitrophenyl chloroformate (6 mg,0.030 mmol) and DIPEA (11 mg,0.060 mmol) at 0deg.C. The solution was warmed to room temperature and stirred for 3 hours. The reaction mixture was then concentrated and used in the next step without further purification. LRMS (ESI): m/z 658.2[ M+H ] ] ⁺ ，C ₃₄ H ₃₂ N ₃ O ₁₁ m/z calculated 652.2.

Scheme 2. Synthesis of construct 12.

Preparation of 4- ((S) -2- (((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -3-methylbutanamide) propanamido) benzyl ((S) -9- ((tert-butoxycarbonyl) oxy) -4, 11-diethyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) ethane-1, 2-diylbis (methylcarbamate) (8)

To PNP carbonate 6 (20 mg,0.030 mmol) and amine 7 (46 mg,0.076 mmol) in DMF (1 mL)DIPEA (26 uL,0.15 mmol) was added to the solution. The reaction mixture was stirred at room temperature overnight and used further in the synthesis without purification. LRMS (ESI): m/z 1148.4[ M+H ]] ⁺ ，C ₆₃ H ₇₀ N ₇ O ₁₄ m/z calculated 1148.5.4- ((S) -2- ((S) -2-amino-3-methylbutanamide) propanamido) benzyl ((S) -4, 11-diethyl-9-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6, 7)]Indolazino [1,2-b ]]Preparation of quinolin-4-yl) ethane-1, 2-diylbis (methylcarbamate) (9)

To a solution of compound 8 (35 mg, 30. Mu. Mol) in DMF (1 mL) was added piperidine (100. Mu.L) at room temperature. After 30 min, the mixture was purified by reverse phase chromatography on a C18 column (H with 0.05% TFA) ₂ O/CH ₃ CN,9:1 to 35:65 v/v). Fractions containing the desired compound were combined and concentrated in vacuo to afford compound 9 (2.0 mg, 8% yield) as an off-white solid. LRMS (ESI): m/z 826.3[ M+H ] ] ⁺ ，C ₄₃ H ₅₂ N ₇ O ₁₀ m/z calculated 826.4.

Preparation of (2S, 5S, 36R) -36- (3- (2- ((2- (((9H-fluoren-9-yl) methoxy) carbonyl) -1, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2,3-b ] pyridin-1-yl) propanamido) -1- ((4- (((((S) -4, 11-diethyl-9-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) oxy) carbonyl) phenyl) amino) -5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazatridecane-37-sulfonic acid (11)

To a solution of amine 9 (2 mg, 2.4. Mu. Mol) and carboxylic acid 10 (2 mg, 2.5. Mu. Mol) in DMF (0.5 mL) was added HATU (1.3 mg, 3.3. Mu. Mol) and DIPEA (1.3. Mu.L, 7. Mu. Mol). The reaction was completed after 30 minutes and the crude solution of 11 was used in the next step without further purification. LRMS (ESI): m/z 1603.2[ M+H ]] ⁺ ，C ₈₁ H ₉₆ N ₁₃ O ₂₀ Sm/z calculated 1602.7.

Preparation of (2S, 5S, 36R) -1- ((4- ((((S) -4, 11-diethyl-9-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) oxy) carbonyl) phenyl) amino) -36- (3- (2- ((1, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2,3-b ] pyridin-1-yl) propanamido) -5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triaza-heptadecane-37-sulfonic acid (12)

To a crude solution of 11 (about 2.4. Mu. Mol) in DMF (0.5 mL) was added piperidine (50. Mu.L). After stirring at room temperature for 15 min, the reaction mixture was directly subjected to reverse phase HPLC (H with 0.05% TFA) using a C18 column ₂ O/CH ₃ CN,100:0 to 40:60 v/v). Product 12 (0.8 mg, 20% yield) was obtained as a white solid. LRMS (ESI): m/z 690.9[ M+2H ]] ⁺⁺ ，C ₆₆ H ₈₇ N ₁₃ O ₁₈ S m/z calculated 690.6.

Scheme 3. Synthesis of sn-38 construct 20.

Preparation of 4- ((S) -2- ((S) -2- (((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -3-methylbutanamido) propanamido) benzoic acid (15)

DIPEA (32 uL, 196. Mu. Mol) was added to a solution of Fmoc-Val-Ala-OH 13 (38 mg, 93. Mu. Mol) and HATU (35 mg, 93. Mu. Mol) in DMF (1 mL). The solution was stirred at room temperature for 30 minutes, then 4-aminobenzoic acid 14 (51 mg, 372. Mu. Mol) was added. After stirring for 15 min, the reaction mixture was purified by reverse phase chromatography on a C18 column (H with 0.05% TFA) ₂ O/CH ₃ CN,90:10 to 30:70 v/v) to remove excess 4-aminobenzoic acid and used in the next step without further purification. LRMS (ESI): m/z 530.2[ M+H ]] ⁺ ，C ₃₀ H ₃₂ N ₃ O ₆ m/z calculated 530.2.

Preparation of (S) -9- ((tert-Butoxycarbonyl) oxy) -4, 11-diethyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl 4- ((S) -2- ((S) -2- (((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -3-methylbutanamido) propanamido) benzoate (16)

Boc-protected SN-38 5 (10 mg, 20. Mu. Mol) was added to dichloromethane (2 mL) and DMF (0.5 mL) containing crude carboxylic acid 15 at 0deg.C, thenDCC (38 mg, 180. Mu. Mol) and DMAP (16 mg, 130. Mu. Mol) were added. After 1h, the reaction was warmed to room temperature and stirred overnight. The mixture was concentrated in vacuo and used without further purification. LRMS (ESI): m/z 1004.5[ M+H ]] ⁺ ，C ₅₇ H ₅₈ N ₅ O ₁₂ m/z calculated 1004.4.

(S) -4, 11-diethyl-9-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl 4- ((S) -2- ((S) -2-amino-3-methylbutanamido) propanamido) benzoate (2S, 5S, 36R) -36- (3- (2- ((2- (((9H-fluoren-9-yl) methoxy) carbonyl) -1, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2,3-b ] pyridin-1-yl) propanamido) -1- ((4- (((((S) -4, 11-diethyl-9-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3', preparation of 4':6,7] indolizino [1,2-b ] quinolin-4-yl) oxy) carbonyl) phenyl) -amino) -5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazatridecane-37-sulfonate (R) -1- (2- ((2- (((9H-fluoren-9-yl) methoxy) carbonyl) -1, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2,3-b ] pyridin-1-yl) -3, 6-dioxo-5- (sulfomethyl) -10,13,16,19,22,25,28,31-octaoxa-4, 7-diaza-triodec-34-carboxylate (17)

DMF (1 mL) was added to crude compound 16 at room temperature followed by piperidine (50. Mu.L). The mixture was stirred for 15 min and directly by reverse phase HPLC using a C18 column (H with 0.05% TFA ₂ O/CH ₃ CN,100:0 to 30:70 v/v). Fractions containing the desired compound were combined and concentrated in vacuo to give 17 (3 mg, 27% yield over 2 steps) as an off-white solid. LRMS (ESI): m/z 682.3[ M+H ]] ⁺ ，C ₃₇ H ₄₀ N ₅ O ₈ m/z calculated 682.3.

(S) -4, 11-diethyl-9-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl 4- ((S) -2- ((S) -2-amino-3-methylbutanamido) propanamido) benzoate (2S, 5S, 36R) -36- (3- (2- ((2- (((9H-fluoren-9-yl) methoxy) carbonyl) -1, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2,3-b ] pyridin-1-yl) propanamido) -1- ((4- (((((S) -4, 11-diethyl-9-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3', preparation of 4':6,7] indolizino [1,2-b ] quinolin-4-yl) oxy) carbonyl) phenyl) amino) -5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazatridecane-37-sulfonate (R) -1- (2- ((2- (((9H-fluoren-9-yl) methoxy) carbonyl) -1, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2,3-b ] pyridin-1-yl) -3, 6-dioxo-5- (sulfomethyl) -10,13,16,19,22,25,28,31-octaoxa-4, 7-diaza-triodec-34-carboxylate (19)

To a solution of amine 17 (3 mg, 4.4. Mu. Mol) and carboxylic acid 18 (7 mg, 6.6. Mu. Mol) in DMF (0.5 mL) was added HATU (2.5 mg, 6.6. Mu. Mol) and DIPEA (3.4. Mu.L, 20. Mu. Mol). The reaction was completed after 30 minutes and product 19 was used as crude product in the next step without purification. LRMS (ESI): m/z 1722.5[ M+H ]] ⁺ ，C ₈₇ H ₁₀₈ N ₁₁ O ₂₄ S m/z calculated 1722.7.

Preparation of (2S, 5S,36 r) -36- (3- (2- ((2- (((9H-fluoren-9-yl) methoxy) carbonyl) -1, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2,3-b ] pyridin-1-yl) propanamido) -1- ((4- ((((4- (((S) -4, 11-diethyl-9-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) oxy) -4-oxobutyl) (methyl) carbamoyl) oxy) methyl) phenyl) amino) -5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptadecane-37-sulfonic acid (20)

To a crude solution of 19 (4.4. Mu. Mol) in DMF (0.5 mL) was added piperidine (50. Mu.L). After stirring for 15 min, the solution was purified by reverse phase chromatography using a C18 column (H with 0.05% TFA) ₂ O/CH ₃ CN,100:0 to 40:60 v/v). Product 20 (1.5 mg, 21% yield) was obtained as a white solid. LRMS (ESI): m/z 1722.5[ M+2H] ⁺⁺ ，C ₇₂ H ₉₉ N ₁₁ O ₂₂ S m/z calculated 750.8.

Scheme 3. Synthesis of SN-38 construct 27.

Preparation of 4- ((((4- ((S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -3-methylbutanamido) propanamido) benzyl) oxy) carbonyl) (methyl) amino) butanoic acid (23)

To a solution of PNP carbonate 21 (100 mg,0.15 mmol) and 4- (methylamino) butyric acid 22 (27 mg,0.18 mmol) in DCM (1 mL) was added DIPEA (72 uL,0.36 mmol). The reaction was stirred overnight and then purified by reverse phase chromatography on a C18 column (H with 0.05% TFA ₂ O/CH ₃ CN,100:0 to 0:100 v/v). Product 23 was obtained as a white solid (60 mg, 63% yield). LRMS (ESI): m/z 681.3[ M+Na ]] ⁺ ，C ₃₆ H ₄₂ N _4Na O ₈ m/z calculated 681.3.

Preparation of (S) -9- ((tert-Butoxycarbonyl) oxy) -4, 11-diethyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl 4- ((((4- ((S) -2- ((S) -2- (((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -3-methylbutanamid yl) propanamido) benzyl) oxy) carbonyl) (methyl) amino) butanoate (24)

To a solution of carboxylic acid 23 (60 mg, 92. Mu. Mol) in dichloromethane (1 mL) and DMF (0.25 mL) was added Boc-protected SN-38 5 (15 mg, 30. Mu. Mol) followed by DCC (21 mg, 90. Mu. Mol) and DMAP (12 mg, 99. Mu. Mol) at 0deg.C. After 1h, the reaction was warmed to room temperature and stirred overnight. The reaction mixture was concentrated under vacuum and the crude product 24 was used without further purification. LRMS (ESI): m/z 1133.4[ M+H ] ] ⁺ ，C ₆₃ H ₆₉ N ₆ O ₁₄ m/z calculated 1133.5.

Preparation of (S) -4, 11-diethyl-9-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl 4- ((((4- ((S) -2- ((S) -2-amino-3-methylbutanamido) propanamido) benzyl) oxy) carbonyl) (methyl) amino) butanoate (25)

DMF (1 mL) was added to crude compound 24 followed by piperidine (50. Mu.L). The mixture was stirred for 15 min and purified by reverse phase HPLC using a C18 column (H with 0.05% TFA) ₂ O/CH ₃ CN,100:0 to 40:60 v/v). Fractions containing the desired compound were combined and concentrated in vacuo to give 25 (14 mg, 57% 2 steps yield) as ashWhite solid. LRMS (ESI): m/z 811.3[ M+H ]] ⁺ ，C ₄₃ H ₅₁ N ₆ O ₁₀ m/z calculated 811.4. (2 s,5s,36 r) -36- (3- (2- ((2- (((9H-fluoren-9-yl) methoxy) carbonyl) -1, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2, 3-b)]Pyridin-1-yl) propionamido) -1- ((4- ((((S) -4, 11-diethyl-9-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6, 7)]Indolazino [1,2-b ]]Quinolin-4-yl) oxy) carbonyl) phenyl) -5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triaza-heptadecane-37-sulfonic acid (26)

To a mixture of amine 25 (4 mg, 4.9. Mu. Mol) and carboxylic acid 18 (7.8 mg, 7.4. Mu. Mol) in DMF (0.5 mL) was added HATU (2.8 mg, 7.4. Mu. Mol) and DIPEA (3.8. Mu.L, 22. Mu. Mol) at room temperature. After 30 minutes, the reaction mixture was concentrated in vacuo and crude compound 26 was used in the next step without further purification. LRMS (ESI): m/z 926.6[ M+2H] ⁺⁺ ，C ₉₃ H ₁₂₀ N ₁₂ O ₂₆ S m/z calculated 926.9.

Preparation of (2S, 5S,36 r) -36- (3- (2- ((2- (((9H-fluoren-9-yl) methoxy) carbonyl) -1, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2,3-b ] pyridin-1-yl) propanamido) -1- ((4- ((((4- (((S) -4, 11-diethyl-9-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) oxy) -4-oxobutyl) (methyl) carbamoyl) oxy) methyl) phenyl) amino) -5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptadecane-37-sulfonic acid (27)

To a crude solution of 26 (4.9. Mu. Mol) in DMF (0.5 mL) was added piperidine (45. Mu.L). After stirring at room temperature for 15 min, the reaction mixture was directly applied to a C18 column (H with 0.05% TFA) by reverse phase HPLC ₂ O/CH ₃ CN,100:0 to 40:60 v/v). Product 27 was obtained as a white solid (3.4 mg, 42% yield). LRMS (ESI): m/z 815.5[ M+2H ] ⁺⁺ ，C ₇₈ H ₁₀₉ N ₁₂ O ₂₄ S m/z calculated 814.9.

Scheme 4. Synthesis of sn-38 construct 34.

Preparation of (2S, 3R,4S,5S, 6S) -2- (2- ((S) -2- ((S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -3-methylbutanamido) propanamido) -5- (((methyl (2- (methylamino) ethyl) carbamoyl) oxy) methyl) phenoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-trisyl triacetate (29)

PNP carbonate 28 (20 mg, 0.020mmol), N at room temperature ¹ ,N ² To a mixture of dimethylethane-1, 2-diamine (3.3 mg,0.038 mmol) and HOAT (2.7 mg, 0.020mmol) in DMF (1 mL) was added DIPEA (6.9 uL,0.040 mmol). After stirring the resulting mixture for 2 hours, the solution was applied to a C18 column (H with 0.05% TFA) by reverse phase HPLC ₂ O/CH ₃ CN,9:1 to 25:75 v/v) and semi-pure product 29 was used in the next step. LRMS (ESI): m/z 962.3[ M+H ]] ⁺ ，C ₄₈ H ₆₀ N ₅ O ₁₆ m/z calculated 962.4.

Preparation of (2S, 3R,4S,5S, 6S) -2- (2- ((S) -2- ((S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -3-methylbutanamide) propanamido) -5- ((((2- (((((S) -9- ((tert-butoxycarbonyl) oxy) -4, 11-diethyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) oxy) carbonyl) (methyl) amino) ethyl) (methyl) oxy) methyl) phenoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-trisyltriacetate (30)

To a mixture of PNP carbonate 6 (13 mg, 0.020mmol), crude compound 29 and HOAT (5.4 mg,0.040 mmol) in DMF (1 mL) was added DIPEA (6.9 uL,0.040 mmol). The reaction was stirred for 1 hour and used without further purification. LRMS (ESI): m/z 1480.5[ M+H ]] ⁺ ，C ₇₆ H ₈₆ N ₇ O ₂₄ m/z calculated 1480.6. (2S, 3R,4S,5S, 6S) -2- (2- ((S) -2- ((S) -2-amino-3-methylbutanamide) propanamido) -5- (((2- ((((((S) -4, 11-diethyl-9-hydroxy-3, 14-dioxo-3,4,12,14) -tetrahydro-1H-pyrano [3',4':6, 7)]Indolazino [1,2-b ]]Quinolin-4-yl) oxy) carbonyl) (methyl) amino) ethyl (methyl) carbamoyl) oxy) methyl) Preparation of phenoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate (31)

Piperidine (30. Mu.L) was added to a crude solution of 30 in DMF (0.5 mL) at 0deg.C. After stirring for 2 hours, the reaction mixture was directly applied to a C18 column (H with 0.05% TFA) by reverse phase HPLC ₂ O/CH ₃ CN,100:0 to 40:60 v/v). Product 31 was obtained as a white solid (5 mg, 22% yield over 3 steps). LRMS (ESI): m/z1158.4[ M+H ]] ⁺ ，C ₅₆ H ₆₈ N ₇ O ₂₀ m/z calculated 1158.5.

Preparation of (2S, 5S, 18R) -18- (3- (2- ((2- (((9H-fluoren-9-yl) methoxy) carbonyl) -1, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2,3-b ] pyridin-1-yl) propanamido) -1- ((4- ((((2- ((((S) -4, 11-diethyl-9-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) oxy) carbonyl) (methyl) amino) ethyl) (methyl) carbamoyl) oxy) methyl) -2- (((2S, 3R,4S,5S, 6S) -3,4, 5-triacetoxy-6- (methoxycarbonyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl) amino) -5-isopropyl-2-methyl-1,4,7,17-tetraoxo-10, 13-dioxa-3,6,16-trioxazine (33-sulfonic acid)

To a solution of amine 31 (2 mg, 2.6. Mu. Mol) and carboxylic acid 10 (2 mg, 2.6. Mu. Mol) in DMF (0.5 mL) was added HATU (1 mg, 2.6. Mu. Mol) and DIPEA (1.3. Mu.L, 7.8. Mu. Mol). The reaction was completed after 30 minutes and crude compound 33 was used without further purification. LRMS (ESI): m/z 1017.1[ M+2H] ⁺⁺ ，C ₉₄ H ₁₁₁ N ₁₃ O ₃₀ S m/z calculated 967.9.

Preparation of (2S, 3S,4S,5R, 6S) -6- (5- (((2- ((((S) -4, 11-diethyl-9-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) oxy) carbonyl) (methyl) amino) ethyl) (methyl) carbamoyl) oxy) methyl) -2- ((2S, 5S, 18R) -22- (2- ((1, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2,3-b ] pyridin-1-yl) -5-isopropyl-2-methyl-4,7,17,20-tetraoxo-18- (sulfomethyl) -10, 13-dioxa-3,6,16,19-tetraazabehenyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (34)

Crude chemical combination to 0 ℃To a solution of material 33 in MeOH (0.6 mL) was added a solution of LiOH (22 mg) in water (0.9 mL). After stirring the reaction mixture for 2H, the mixture was purified by reverse phase HPLC on a C18 column (H with 0.05% tfa) ₂ O/CH ₃ CN,100:0 to 35:65 v/v). Fractions containing the desired compound were combined and concentrated in vacuo to afford compound 34 (0.6 mg, 20% yield over 2 steps) as a white solid. LRMS (ESI): m/z 1572.5[ M+H ] ] ⁺ ，C ₇₂ H ₉₄ N ₁₃ O ₂₅ S m/z calculated 1572.6.

Scheme 5. Synthesis of intermediate 40.

Preparation of (9H-fluoren-9-yl) methyl 2- ((5-amino-1- (3- (tert-butoxy) -3-oxopropyl) -1H-indol-2-yl) methyl) -1, 2-dimethylhydrazine-1-carboxylate (36)

Nitro compound 35 (116 mg,0.20 mmol) was dissolved in 1mL THF and combined with a solution of ammonium chloride (85 mg,1.6 mmol) in 0.5mL water and 1mL methanol. The resulting mixture was stirred vigorously at room temperature and treated with a small amount of zinc powder (104 mg,1.6 mmol) over 5 minutes. The reaction mixture was stirred for 2 hours, the solid was filtered off, and the filtrate was diluted with 20mL of saturated aqueous ammonium chloride and extracted with ethyl acetate (2×25 mL). The organic extract was dried over sodium sulfate and the solvent removed under vacuum to give crude product 36, which was used in the next step without further purification. LRMS (ESI): m/z 555.3[ M+H ]] ⁺ ，C ₃₃ H ₃₈ N ₄ O ₄ m/z calculated 555.3.

Preparation of (9H-fluoren-9-yl) methyl 2- ((1- (3- (tert-butoxy) -3-oxopropyl) -5- (4- (tert-butoxy) -4-oxobutanamide) -1H-indol-2-yl) methyl) -1, 2-dimethylhydrazine-1-carboxylate (38)

Crude compound 36 (about 0.20 mmol) was combined with 4- (tert-butoxy) -4-oxobutanoic acid 37 (40 mg,0.23 mmol) in 2mL DMF. DIPEA (0.12 mL,0.6 mmol) was added to the mixture at room temperature, followed by a portion of PyAOP (110 mg,0.21 mmol). After 30 minutes, the mixture is poured into saturated ammonium chloride water solution The reaction was quenched in solution, extracted with ethyl acetate, washed with brine and dried over sodium sulfate. The solvent was removed under vacuum to give 120mg (0.17 mmol, 85% yield over 2 steps) of product 38 as a dark oil which was used further without additional purification. LRMS (ESI): m/z 733.4[ M+Na] ⁺ ，C ₄₁ H ₅₀ N ₄ O ₇ m/z calculated 733.4.

Preparation of 4- ((2- ((2- (((9H-fluoren-9-yl) methoxy) carbonyl) -1, 2-dimethylhydrazino) methyl) -1- (2-carboxyethyl) -1H-indol-5-yl) amino) -4-oxobutanoic acid (39)

Bis-tert-butyl ester compound 38 (120 mg,0.17 mmol) was dissolved in a mixture of 2mL anhydrous DCM, 2mL TFA and 0.5mL triisopropylsilane. The resulting mixture was allowed to stand at room temperature for 4 hours. The solvent was removed under vacuum and the residue was purified by reverse phase chromatography (C18 column, gradient 0% to 70% v/v CH with 0.05% TFA ₃ CN/H ₂ O) to obtain 53mg (0.09 mmol, 53% yield) of diacid product 39.LRMS (ESI): m/z 599.3[ M+H ]] ⁺ ，C ₃₃ H ₃₄ N ₄ O ₇ m/z calculated 599.2.

Preparation of (9H-fluoren-9-yl) methyl 1, 2-dimethyl-2- ((1- (3-oxo-3- (perfluorophenoxy) propyl) -5- (4-oxo-4- (perfluorophenoxy) butyramide) -1H-indol-2-yl) methyl) hydrazine-1-carboxylate (40)

To a mixture of diacid 39 (50 mg,0.084 mmol) and pentafluorophenol (46 mg,0.25 mmol) in 2mL dry THF was added one portion of DCC (51 mg,0.25 mmol) at room temperature. The resulting mixture was stirred for 16 hours, the solid was filtered off, the filtrate was concentrated and purified by reverse phase chromatography (C18 column, 0% to 100% v/v gradient CH with 0.05% TFA ₃ CN/H ₂ O) purifying. The product containing fractions were concentrated to about 20mL, poured into 50mL of 10% aqueous citric acid and extracted with ethyl acetate (2 x20 mL), dried over sodium sulfate. The solvent was removed under vacuum to give 67mg (0.072 mmol, 86% yield) of bis-pfp ester product 40 as a dark viscous oil. LRMS (ESI): m/z 953.1[ M+Na] ⁺ ，C ₄₅ H ₃₂ F ₁₀ N ₄ O ₇ m/z calculated 953.2.

Scheme 6. Synthesis of SN-38 construct 42.

(2S, 3R,4S,5S, 6S) -2- (2- ((S) -2- ((2- ((2- (((9H-fluoren-9-yl) methoxy) carbonyl) -1, 2-dimethylhydrazino) methyl) -1- (3- (((S) -1- (((S) -1- ((4- ((((S) -4, 11-diethyl-9-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) oxy) carbonyl) (methyl) amino) ethyl) (methyl) carbamoyl) oxy) methyl) -2- (((2S, 3R,4S,5S, 6S) -3, 4-triacetoxy-6- (methoxycarbonyl) tetrahydro-2H-pyran-2-yl) phenyl) amino) -1-oxopropan-2-yl) -3-oxo-1H-pyran-2-oxo-2-oxo-butan-3-yl) -amino) -butan-3-yl-3-oxo-butan-3-yl-amino) -2- (-2S, 3-triacetoxy-methyl) butan-2-methyl-2-butan-3-oxo-butan-3-amino Preparation of methylbutanamido) propanamido) -5- (((2- (((((S) -4, 11-diethyl-9-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) oxy) carbonyl) (methyl) amino) ethyl) (methyl) carbamoyl) oxy) methyl) phenoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-trisyl triacetate (41)

To a mixture of bis-PFP ester 40 (1.2 mg, 1.3. Mu. Mol), amine 31 (3 mg, 2.6. Mu. Mol) and HOAT (0.45 mg, 3.3. Mu. Mol) in DMF (0.5 mL) was added DIPEA (1.1 uL, 6.5. Mu. Mol). The reaction was stirred at room temperature for 1 hour. After removal of the solvent under vacuum, crude intermediate 41 was used in the next step without further purification. LRMS (ESI): m/z 1439.9[ M+2H] ⁺⁺ ，C ₁₄₅ H ₁₆₆ N ₁₈ O ₄₅ m/z calculated 1439.6.

(2S, 3S,4S,5R, 6S) -6- (2- ((S) -2- ((S) -2- (4- ((1- (3- (((S) -1- (((S) -1- ((2- (((2S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) o) -4- (((2- (((((S) -4, 11-diethyl-9-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3', 4'): 6,7] Indolazino [1,2-b ] quinolin-4-yl) oxy) carbonyl) (methyl) amino) ethyl) (methyl) carbamoyl) oxy) methyl) phenyl) -1-oxopropan-2-yl) amino) -3-methyl-1-oxo butan-2-yl) amino) -3-oxopropyl) -2- ((1, 2-dimethylhydrazino) methyl) -1H-indol-5-yl amino) -4-oxobutyrylamido) -3-methylbutanamido) propanamido) -5- (((2- ((((S)) S) Preparation of (E) -4, 11-diethyl-9-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) oxy) carbonyl) (methyl) amino) ethyl) (methyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (42)

To a solution of crude compound 41 in MeOH (0.6 mL) at 0 ℃ was added a solution of LiOH (22 mg) in water (0.9 mL). After stirring for 2H, the mixture was purified by reverse phase C18 column chromatography (H with 0.05% TFA) ₂ O/CH ₃ CN,100:0 to 35:65 v/v). Fractions containing the desired compound were combined and concentrated in vacuo to give 42 (0.3 mg, 8% yield over 2 steps) as a white solid. LRMS (ESI): m/z 1188.6[ M+2H ]] ⁺⁺ ，C ₁₁₆ H ₁₄₀ N ₁₈ O ₃₇ m/z calculated 1188.5

Scheme 7 Synthesis of belotecan (belotecan) construct 45

Preparation of 4- ((S) -2- ((S) -2-amino-3-methylbutanamino) propanamido) benzyl (43) carbamate of (2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) amino-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) propanamido) ethyl (3-methyl-5-yl) pyran-o-1H-pyrano-1-yl

To a mixture of belloteprednol hydrochloride (9.4 mg, 20. Mu. Mol), HOAT (2.8 mg, 20. Mu. Mol) and 7. Mu.L DIPEA (40. Mu. Mol) in 1mL DMF was added a portion of PNP carbonate 18 (13.6 mg, 20. Mu. Mol) at room temperature. After one hour, 40. Mu.L (0.40 mmol) of piperidine was added directly to the reaction mixture. After 20 min, the reaction mixture was subjected to reverse phase HPLC (C18 column, gradient of 0% to 50% v/v CH with 0.05% TFA) ₃ CN/H ₂ O) to give 10.8mg (14.3 μmol, 72% yield) of compound 43 as a yellow solid. LRMS (ESI): m/z 753.4[ M+H ] ] ⁺ ，C ₄₁ H ₄₈ N ₆ O ₈ m/z calculated 753.4.

Preparation of (2S, 5S,36 r) -36- (3- (2- ((1- (((9H-fluoren-9-yl) methoxy) carbonyl) -2, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2,3-b ] pyridin-1-yl) propanamido) -1- ((4- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenyl) amino) -5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptadecane-37-sulfonic acid (44)

To a mixture of compound 43 (10.8 mg, 14.3. Mu. Mol) and carboxylic acid 18 (15.2 mg, 14.3. Mu. Mol) in 2mL DMF was added 8. Mu.L DIPEA (46. Mu. Mol) followed by PyAOP (7.5 mg, 14.3. Mu. Mol). After 30 min at room temperature, the reaction mixture was directly subjected to reverse phase HPLC (C18 column, 5% to 95% v/v gradient CH with 0.05% TFA) ₃ CN/H ₂ O) purifying. The fractions were lyophilized to give 16.5mg (9.2. Mu. Mol, 64% yield) of compound 44 as a yellowish powder. LRMS (ESI): m/z 1794.8[ M+H ]] ⁺ ，C ₉₁ H ₁₁₆ N ₁₂ O ₂₄ S m/z calculated 1794.8.

Preparation of (2S, 5S, 36R) -36- (3- (2- ((1, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2,3-b ] pyridin-1-yl) propanamido) -1- ((4- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenyl) amino) -5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptadecane-37-sulfonic acid (45)

To a solution of compound 44 (16.5 mg, 9.2. Mu. Mol) in 2mL of DMA was added piperidine (18. Mu.L, 0.18 mmol) at room temperature. After 30 min, the reaction mixture was purified on reverse phase HPLC (C18 column, 0% to 50% v/v gradient CH with 0.05% TFA ₃ CN/H ₂ O) purifying. The pure fraction was lyophilized to give 11.2mg (7.1. Mu. Mol, 77% yield) of compound 45 as a yellow powder. LRMS (ESI): m/z 1572.7[ M+H ]] ⁺ ，C ₇₆ H ₁₀₆ N ₁₂ O ₂₂ S m/z calculated 1572.8.

Scheme 8. Synthesis of branched belloteprednol etabonate construct 47

Preparation of (2S, 3S,4S,5R, 6S) -6- (2- ((S) -2- ((S) -2-amino-3-methylbutanamide) propanamido) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (46)

To a solution of belloteprednol hydrochloride (20 mg, 43. Mu. Mol) in 2mL DMF was added 15uL DIPEA (86. Mu. Mol) and 6mg HOAt (43. Mu. Mol). The resulting mixture was combined with PNP carbonate 28 (43 mg,43 μmol) at room temperature and stirred for one hour, then DMF was removed under vacuum. The residue was dissolved in 1mL MeOH and treated with 1mL of 1m aqueous LiOH. After 10 minutes, 1mL of 1M aqueous HCl was added to the mixture of shocking ml and then 1mL of a 0.5M mixture of acetate at pH 4.7. The resulting mixture was stirred at room temperature for 30 min and passed directly through reverse phase HPLC (C18 column, 0% to 50% v/v gradient CH with 0.05% TFA) ₃ CN/H ₂ O) purifying. The solvent was removed under vacuum to give 17mg (18. Mu. Mol, 43% yield) of compound 46 as a glassy yellow solid. LRMS (ESI): m/z 945.4[ M+H ]] ⁺ ，C ₄₇ H ₅₆ N ₆ O ₁₅ m/z calculated 945.4.

(2S, 3S,4S,5R, 6S) -6- (2- ((S) -2- ((S) -2- (4- ((1- (3- (((S) -1- (((S) -1- ((2- (((2S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) 4- (((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] Indolazino [1,2-b ] quinolin-11-yl) ethyl (isopropyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl amino) -3-methyl-1-oxobutan-2-yl amino) -3-oxopropyl) -2-1, 2-dimethylhydrazino methyl) -1H-indol-5-yl) -4-oxobutanamido) -3-methylbutanamido) propanamido) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14- Preparation of tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (47)

Compound 46 (17 mg, 18. Mu. Mol) was combined with bis-PFP ester 40 (8.3 mg, 9. Mu. Mol), 2.5mg HOAt (18. Mu. Mol) and 10. Mu.L DIPEA (54. Mu. Mol) in 2mL DMA at room temperature. After one hour piperidine (35 μl,0.36 mmol) was added to the reaction mixture. After 30 min, the reaction mixture was directly subjected to reverse phase HPLC (C18 column, gradient 0% to 50% v/v CH with 0.05% TFA) ₃ CN/H ₂ O) purifying. The pure fraction was lyophilized to give 5.0mg of compound 47 (2.2. Mu. Mol, 24% yield) as a yellowish powder. LRMS (ESI): m/z 1116.1[ M+2H] ⁺⁺ ，C ₁₁₂ H ₁₃₂ N ₁₆ O ₃₃ m/z calculated 1116.0.

Scheme 8. Synthesis of intermediate 50

Preparation of (9H-fluoren-9-yl) methyl ((S) -1- (((S) -1- ((4- (((tert-butoxycarbonyl) amino) methyl) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) carbamate (49)

To a stirred mixture of Fmoc-Val-Ala-OH 13 (410 mg,1.0 mmol) and tert-butyl (4-aminobenzyl) carbamate 48 (267 mg,1.2 mmol) in 5mL DCM and 0.5mL MeOH was added one portion of EEDQ (495mg, 2.0 mmol) at room temperature. The resulting mixture was stirred overnight in the dark, the precipitate was collected by filtration, washed with MTBE and dried in air to give 520mg of product 49 (0.85 mmol, 85% yield) as a tan powder. LRMS (ESI): m/z 637.3[ M+Na] ⁺ ，C ₃₅ H ₄₂ N ₄ O ₆ m/z calculated 637.3.

Preparation of (9H-fluoren-9-yl) methyl ((S) -1- (((S) -1- ((4- (aminomethyl) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) carbamate 2, 2-trifluoroacetate (50)

Boc-protected compound 49 (520 mg,0.85 mmol) was dissolved in 5mL DCM-TFA mixture (1:1) at room temperature. The reaction mixture was stirred for 15 minutes and then under vacuum The solvent was removed. The residue was triturated with 20mL of MTBE and the resulting precipitate collected by filtration, washed with MTBE and dried in air to give 525mg of product 50 (0.84 mmol, 99% yield) as a tan powder (TFA salt). LRMS (ESI): m/z 515.3[ M+H ]] ⁺ ，C ₃₀ H ₃₄ N ₄ O ₄ m/z calculated 515.3.

Scheme 9. Synthesis of irinotecan construct 56

Preparation of methyl 3- (N- ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) sulfamoyl) propanoate (52)

To a solution of irinotecan mesylate (10 mg, 19. Mu. Mol) in 1mL of anhydrous DMF was added 10. Mu. LDIPEA (56. Mu. Mol) followed by 4. Mu.L of sulfonyl chloride 51 (21. Mu. Mol) at room temperature. After one hour, the reaction mixture was purified directly on reverse phase HPLC (C18 column, 0% to 75% v/v gradient CH with 0.05% TFA ₃ CN/H ₂ O) to give 5mg (9 μmol, 47% yield) of the product as a yellow solid. LRMS (ESI): m/z 586.2[ M+H ]] ⁺ ，C ₂₈ H ₂₈ FN ₃ O ₈ S m/z calculated 586.2.

Preparation of 3- (N- ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) sulfamoyl) propionic acid (53)

Methyl ester 52 (5 mg, 8.5. Mu. Mol) was dissolved in 2mL of methanol at room temperature and treated with 1mL of 1M aqueous LiOH. The resulting mixture was stirred for one hour, quenched with 1mL of 1M aqueous HCl followed by 1mL of 0.5M acetate buffer at pH 4.7. After 10 min, the reaction mixture was concentrated in vacuo and purified by reverse phase HPLC (C18 column, 0% to 75% v/v gradient CH with 0.05% TFA ₃ CN/H ₂ O) to give 5.0mg of carboxylic acid product 53 (8.7. Mu. Mol, quantitative) as a bright yellow solid. LRMS (ESI): m is m/z 572.2[M+H] ⁺ ，C ₂₈ H ₂₈ FN ₃ O ₈ S m/z calculated 572.6

Preparation of (S) -2-amino-N- ((S) -1- ((4- ((3- (N- ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) sulfamoyl) propanamido) methyl) phenyl) amino) -1-oxopropan-2-yl) -3-methylbutanamide (54)

DIPEA (4.6. Mu.L, 26. Mu. Mol) was added to a mixture of carboxylic acid 53 (5.0 mg, 8.7. Mu. Mol) and amine 50 (5.5 mg, 8.7. Mu. Mol) in 1mL DMF at room temperature followed by a portion of PyAOP (4.6 mg, 8.7. Mu. Mol). After 30 min, the reaction mixture was treated with piperidine (17 μl,0.17 mmol), stirred at room temperature for 15 min, and purified by reverse phase HPLC (C18 column, 0% to 50% v/v gradient of CH with 0.05% TFA) ₃ CN/H ₂ O) purifying. The solvent was removed under vacuum to give 5.0mg (5.7. Mu. Mol, 66% yield) of compound 54 as a deep yellow oil. LRMS (ESI): m/z846.4[ M+H ]] ⁺ ，C ₄₂ H ₄₈ FN ₇ O ₉ S m/z calculated 846.3.

Preparation of (2 s,5s,36 r) -36- (3- (2- ((2- (((9H-fluoren-9-yl) methoxy) carbonyl) -1, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2,3-b ] pyridin-1-yl) propanamido) -1- ((4- ((3- (N- ((1 s,9 s) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) sulfamoyl) propanamido) methyl) phenyl) amino) -5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptadecane-37-sulfonic acid (55)

To a mixture of compound 54 (5.0 mg, 4.7. Mu. Mol) and carboxylic acid 18 (6.3 mg, 4.7. Mu. Mol) in 1mL of DMF was added DIPEA (3. Mu.L, 14. Mu. Mol) followed by a portion of PyAOP (3.1 mg, 4.7. Mu. Mol) at room temperature. After 30 min, the reaction mixture was directly subjected to reverse phase HPLC (C18 column, gradient 0% to 75% v/v CH with 0.05% TFA) ₃ CN/H ₂ O) was directly purified to give 6.0mg (2.6 μmol, yield 55%) of compound 55 as a yellow powder. LRMS (ESI): m/z 1887.8[ M+H ]] ⁺ ，C ₉₂ H ₁₁₆ FN ₁₃ O ₂₅ S ₂ m/z calculated 1886.8.

Preparation of (2S, 5S, 36R) -36- (3- (2- ((1, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2,3-b ] pyridin-1-yl) propanamido) -1- ((4- ((3- (N- ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) sulfamoyl) propanamido) methyl) phenyl) amino) -5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptadecane-37-sulfonic acid (56)

To a solution of compound 55 (6.0 mg, 3.2. Mu. Mol) in 1mL of DMA was added piperidine (6.3. Mu.L, 63. Mu. Mol) at room temperature. After 30 min, the reaction mixture was directly subjected to reverse phase HPLC (C18 column, gradient 0% to 50% v/v CH with 0.05% TFA) ₃ CN/H ₂ O) purifying. The product-containing fraction was lyophilized to obtain 3.0mg of compound 56 (1.8. Mu. Mol, yield 56%) as a yellow powder. LRMS (ESI): m/z 1665.7[ M+H ]] ⁺ ，C ₇₇ H ₁₀₆ FN ₁₃ O ₂₃ S ₂ m/z calculated 1664.7.

Scheme 10 Synthesis of branched belatinecan construct 61

Preparation of (2R, 3S,4S,5R, 6S) -2- (Acetoxymethyl) -6- (2- ((S) -2- ((S) -2-amino-3-methylbutanamide) propanamido) -5- (((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) tetrahydro-2H-pyran-3, 4, 5-trisyl triacetate (58)

To a dried 20mL scintillation vial was added beloxsulam hydrochloride (2,48 mg, 102. Mu. Mol) and 1.6mL anhydrous DMF followed by 47. Mu.L DIPEA (269. Mu. Mol) and 13mg HOAt (96. Mu. Mol). The resulting mixture was treated with one portion of PNP carbonate 57 (104 mg, 101. Mu. Mmol) as a solid at room temperature and stirred overnight. After consumption of the starting material, 200. Mu.L of piperidine (2 mmol) was added. Stirring the mixture30 minutes and monitored by LC-MS. Passing the reaction mixture through reverse phase(C18, 0% to 100% v/v CH with 0.05% TFA) ₃ CN-H ₂ O) purifying. The pure fractions were lyophilized to give 100mg of compound 58 (91. Mu. Mol, 90% yield) as a yellow powder. LRMS (ESI): m/z 1099.4[ M+H ]] ⁺ ，C ₅₅ H ₆₇ N ₆ O ₁₈ m/z calculated 1099.5.

Preparation of (2S, 5S, 18R) -18-amino-1- ((4- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) -2- (((2S, 3R,4S,5R, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl) amino) -5-isopropyl-2-methyl-1,4,7,17-tetraoxo-10, 13-dioxa-3,6,16-triazanona-ne-19-sulfonic acid (60)

To a dried 20mL scintillation vial was added amine (58, 50mg, 46. Mu. Mol), a cysteic acid linker (59, 27mg, 49. Mu. Mol) and 0.5mL anhydrous DMF followed by 24. Mu.L DIPEA (138. Mu. Mol) and 18mg HATU (46. Mu. Mol). The resulting mixture was stirred at room temperature and monitored by LCMS. After the starting material was consumed, the solution was concentrated under vacuum to remove DMF. The residue was dissolved in 1mL of methanol and slowly treated with 1.5mL of 1m aqueous LiOH at 0 ℃. After stirring the reaction mixture for 15 minutes, it was then warmed to room temperature and stirring was continued for 1 hour until the hydrolysis was judged complete by LCMS analysis. 1mL of 1M HCl was added, followed by 1mL of 0.5M pH 4.7 acetate buffer, the reaction mixture quenched, concentrated in vacuo and purified by reverse phase HPLC (C18, 0% to 75% v/v CH with 0.05% TFA) ₃ CN-H ₂ O) purifying. The pure fractions were lyophilized to give 39mg of compound 60 (31. Mu. Mol, 68% yield) as a yellow powder. LRMS (ESI): m/z 1241.5[ M+H ]] ⁺ ，C ₅₇ H ₇₇ N ₈ O ₂₁ S m/z calculated 1241.5.

(2S, 5S,18 r) -18- (4- ((2- ((1, 2-dimethylhydrazino) methyl) -1- ((2S, 5S,18 r) -1- ((4- (((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4 '): 6,7] Indolazino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) -2- (((2S, 3R,4S,5R, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl) amino) -5-isopropyl-2-methyl-1,4,7,17,20-pentoxy-18- (sulfomethyl) -10, 13-dioxa-3,6,16,19-tetraazabehen-22-yl) -1H-indol-5-yl) amino) -4-oxobutanamide) -1- ((4- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3', preparation of 4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) -2- (((2S, 3R,4S,5R, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl) amino) -5-isopropyl-2-methyl-1,4,7,17-tetraoxo-10, 13-dioxa-3,6,16-triazanona-ne-19-sulfonic acid (61)

Compound 60 (39 mg, 31. Mu. Mol) was combined with bis-PFP ester 40 (14.7 mg, 15.5. Mu. Mol), 5mg HOAt (31. Mu. Mol) and 17. Mu.L DIPEA (93. Mu. Mol) in 1mL DMF at room temperature. After one hour, 61. Mu.L (0.62 mmol) of piperidine was added to the reaction mixture. After 30 min, the reaction mixture was directly subjected to reverse phase HPLC (C18 column, gradient 0% to 75% v/v CH with 0.05% TFA) ₃ CN/H ₂ O) purifying. The pure fractions were lyophilized to give 29mg of compound 61 (10.3. Mu. Mol, 66% yield) as a yellowish powder. LRMS (ESI): m/z 1412.1[ M+2H] ⁺⁺ ，C ₁₃₂ H ₁₇₄ N ₂₀ O ₄₅ S ₂ m/z calculated 1412.1.

Scheme 11 synthesis of branched belatinecan construct 65

Preparation of 1- (9H-fluoren-9-yl) -3-oxo-2,7,10,13,16-pentaoxa-4-aza-nineteen-19-acid perfluorophenyl ester (63)

In a dried scintillation vial, 1- (9H-fluoren-9-yl) -3-oxo-2,7,10,13,16-pentaoxa-4-azanonadecan-19-oic acid (62, 487mg,1 mmol) and pentafluorophenol (365 mg,2 mmol) were combined in 5mL anhydrous THF. The resulting mixture was treated with one portion of DCC (247 mg,1.2 mmol) at room temperature andthe reaction mixture was stirred overnight. The precipitated solid was filtered off, the solvent was removed under vacuum, and the residue was purified by reverse phase chromatography (C18 column, 10% to 100% v/v gradient CH with 0.05% TFA ₃ CN/H ₂ O) to give 670mg of PFP ester 63 (570 mg,0.87mmol, 87% yield) as a colorless oil. LRMS (ESI): m/z 654.2[ M+H ]] ⁺ ，C ₃₂ H ₃₂ F ₅ NO ₈ m/z calculated 654.2.

Preparation of (2S, 3S,4S,5R, 6S) -6- (2- ((17S, 20S) -1-amino-17-isopropyl-20-methyl-15, 18-dioxo-3, 6,9, 12-tetraoxa-16, 19-diaza-eicosane-21-amido) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (64)

Compound 46 (262 mg,0.22 mmol) was dissolved in 4mL DMF. To this solution was added DIPEA (105. Mu.L, 0.66 mmol) and PFP ester 63 (181 mg,0.22 mmol) in 0.5mL DMF followed by HOAt (38 mg,0.22 mmol). The resulting mixture was allowed to stand at room temperature for one hour, then treated directly with 4mL of triethylamine. The reaction mixture was stirred for 5 hours until Fmoc-deprotection was judged complete by LCMS analysis. The reaction mixture was concentrated in vacuo and purified by reverse phase chromatography (C18 column, 0% to 50% v/v gradient CH with 0.05% TFA ₃ CN/H ₂ O) to give 185mg (0.16 mmol, 73% yield) of compound 64 as a yellow powder. LRMS (ESI): m/z 1192.5[ M+H ]] ⁺ ，C ₅₈ H ₇₇ N ₇ O ₂ m/z calculated 1192.5.

(2S, 3S,4S,5R, 6S) -6- (2- ((2S, 5S) -25- (5- ((2S, 5S) -1- ((2- (((2S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) 4- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4 '): 6,7] Indolazino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenyl) -5-isopropyl-2-methyl-1,4,7,23-tetraoxo-10,13,16,19-tetraoxa-3,6,22-triazahexa-ne-26-amido) -2- ((1, 2-dimethylhydrazino) methyl) -1H-indol-1-yl) -5-isopropyl-2-methyl-4,7,23-trioxo-10,13,16,19-tetraoxa-3,6,22-triazaeicosamido) -5- (((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3', preparation of 4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (65)

Compound 64 (23 mg, 19. Mu. Mol) was dissolved in 2mL anhydrous DMA. To this solution was added a portion of DIPEA (10. Mu.L, 57. Mu. Mol) and bis-PFP ester 40 (8 mg, 8.6. Mu. Mol) as a solid at room temperature, followed by HOAt (2.6 mg, 19. Mu. Mol). The resulting mixture was left to stand at room temperature for one hour, and then treated directly with 17. Mu.L of piperidine (172. Mu. Mol). After 20 min, the reaction mixture was purified by reverse phase chromatography HPLC (C18 column, gradient 0% to 50% v/v CH with 0.05% TFA) ₃ CN/H ₂ O) purifying. The pure fraction was lyophilized to give 5.8mg (2.1. Mu. Mol, yield 24%) of compound 65 as a yellow powder. LRMS (ESI): m/z 1363.1[ M+2H] ⁺⁺ ，C ₁₃₄ H ₁₇₄ N ₁₈ O ₄₃ m/z calculated 1362.6.

Scheme 12. Synthesis of branched belatinecan construct 67

Preparation of 4- ((17S, 20S) -1-amino-17-isopropyl-20-methyl-15, 18-dioxo-3, 6,9, 12-tetraoxa-16, 19-diaza-eicosan-21-amido) -3- (((2S, 3R,4S,5R, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) benzyl (2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyran [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamate (66)

To a mixture of compounds 58 (30 mg, 27. Mu. Mol) and 62 (17 mg, 35. Mu. Mol) in DMF (0.5 mL) was added HATU (12 mg, 32. Mu. Mol) followed by DIPEA (14. Mu.L, 82. Mu. Mol) and the resulting solution was stirred for 1h. The solvent was removed under reduced pressure, and the residue was dissolved in MeOH (1 mL). Then 1M aqueous LiOH (1 mL) was added to the solution at 0deg.C, and the reaction mixture was slowed down Slowly warm to room temperature. After completion of hydrolysis as judged by LCMS analysis, the reaction mixture was quenched with pH 4.7 acetate buffer (1 mL). The solid was filtered off and the filtrate was purified by reverse phase prep HPLC (C18 column, 0% to 75% acetonitrile-water with 0.05% TFA). The pure fractions were collected and lyophilized to give product 66 as a yellow solid (19 mg,16 μmol, 59% yield). LRMS (ESI): m/z 1178.5[ M+H ]] ⁺ ，C ₅₈ H ₇₉ N ₇ O ₁₉ m/z calculated: 1178.5.

(2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamic acid 4- ((2S, 5S) -25- (2- ((1, 2-dimethylhydrazino) methyl) -5- ((2S, 5S) -1- ((4- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] Indolazino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) -2- (((2S, 3R,4S,5R, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl) amino) -5-isopropyl-2-methyl-1,4,7,23-tetraoxo-10,13,16,19-tetraoxa-3,6,22-triazahexa-ne-26-amido) -1H-indol-1-yl) -5-isopropyl-2-methyl-4,7,23-trioxo-10,13,16,19-tetraoxa-3,6,22-triazaeicosanamido ) Preparation of (2S, 3R,4S,5R, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) benzyl ester (67)

DIPEA (9. Mu.L, 48. Mu. Mol and HOAt (3 mg, 21. Mu. Mol) were added to a solution of compound 66 (19 mg, 16. Mu. Mol) in DMF (0.5 mL) at room temperature, then bis-PFP ester 40 (7.4 mg, 8. Mu. Mol) was added the resulting mixture was stirred for 1h until judged to be coupled to completion by LCMS analysis, then piperidine (32. Mu.L, 0.32 mmol) was added directly to the reaction mixture at room temperature and stirring continued for 15 min, then the reaction mixture was purified by reverse phase preparative HPLC (C18 column, 0% to 70% acetonitrile-water containing 0.05% TFA), pure fractions were collected and lyophilized to give product 67 as a yellow solid (13 mg, 4.8. Mu. Mol, 60% yield.) LRMS (ESI): m/z 1349.0[ M+2H] ²⁺ ，C ₁₃ 4H ₁₇₈ N ₁₈ O ₄₁ m/z calculated: 1349.1.

scheme 13 Synthesis of branched belatinecan construct 73

Preparation of (S) -14- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -1-hydroxy-13-oxo-3, 6, 9-trioxa-12-aza-heptadecane-17-oic acid tert-butyl ester (70)

To a mixture of Fmoc-Glu (OtBu) -OH (68, 42mg,0.1 mmol) and amino-PEG 4-OH (69, 19mg,0.1 mmol) in DMF (1 mL) was added HATU (38 mg,0.1 mmol) and DIPEA (52. Mu.L, 0.3 mmol) at room temperature. The reaction mixture was stirred for 1h and purified directly by reverse phase chromatography (C18 column, 0% to 70% acetonitrile-water with 0.05% TFA). The pure fractions were lyophilized to give compound 70 as a white solid (50 mg,0.83mmol, 83% yield). LRMS (ESI): m/z 601.3[ M+H ] ] ⁺ ，C ₃₂ H ₄₄ N ₂ O ₉ m/z calculated: 601.3. preparation of (S) -14- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -1-hydroxy-13-oxo-3, 6, 9-trioxa-12-aza-heptadecane-17-acid (71)

Compound 70 (50 mg,83 μmol) was dissolved in TFA (2 mL) and stirred at room temperature for 1 min. The solvent was removed under reduced pressure and the residue was purified by reverse phase chromatography (C18 column, 0% to 75% acetonitrile-water with 0.05% TFA). The pure fractions were collected and lyophilized to give compound 71 as a white solid (35 mg,83 μmol, 77% yield). LRMS (ESI): m/z 545.3[ M+H ]] ⁺ ，C ₂₈ H ₃₆ N ₂ O ₉ m/z calculated: 545.2.

preparation of 4- ((14S, 19S, 22S) -14-amino-1-hydroxy-19-isopropyl-22-methyl-13,17,20-trioxo-3, 6, 9-trioxa-12,18,21-triazatricosan-23-amido) -3- (((2S, 3R,4S,5R, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) benzyl (2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyran-3 ',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamate (72)

HATU (10 mg, 27. Mu. Mol) was added to a mixture of amine 58 (30 mg, 27. Mu. Mol) and carboxylic acid 71 (15 mg, 28. Mu. Mol) in DMF (0.5 mL) at room temperatureDIPEA (14. Mu.L, 82. Mu. Mol) was then added. The reaction mixture was stirred for 1h until the coupling was found to be complete by LCMS analysis. Removing the solvent under reduced pressure; the residue was dissolved in MeOH (1 mL) and treated with 1M aqueous LiOH solution (1 mL) at 0 ℃. The reaction mixture was slowly warmed to room temperature, stirred for an additional 1h, and quenched with pH 4.7 acetate buffer (1 mL). The solid was filtered off and the clear filtrate was purified by reverse phase prep HPLC (C18 column, 0% to 75% acetonitrile-water with 0.05% TFA). The pure fractions were combined and lyophilized to give 30mg (24 μmol, 89% yield) of compound 72 as a yellow solid. LRMS (ESI): m/z 1235.5[ M+H ] ] ⁺ ，C ₆₀ H ₈₂ N ₈ O ₂ m/z calculated: 1235.6.

(2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamic acid 4- ((14S, 19S, 22S) -14- (4- ((2- ((1, 2-dimethylhydrazino) methyl) -1- ((S) -14- (3- (((S) -1- (((S) -1- ((4- (((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 '), 4':6,7] Indolazino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) -2- (((2S, 3R,4S,5R, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -3-oxopropyl) -1-hydroxy-13, 16-dioxo-3, 6,9 preparation of (2S, 3R,4S,5R, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) benzyl (73) -trioxa-12, 15-diazaoctadelan-18-yl) -1H-indol-5-yl) amino) -4-oxobutanamido) -1-hydroxy-19-isopropyl-22-methyl-13,17,20-trioxo-3, 6, 9-trioxa-12,18,21-triaza-23-amido) -3- (((2S, 3R,4S,5R, 6R) -3,4, 5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy) benzyl ester

To a solution of compound 72 (30 mg, 24. Mu. Mol) in DMF (1.0 mL) was added DIPEA (13. Mu.L, 73. Mu. Mol) and HOAt (4.2 mg, 32. Mu. Mol) at room temperature, followed by a portion of bis-PFP ester 40 (11 mg, 12. Mu. Mol). The reaction mixture was left to stand for 1h until the reaction was judged complete by LCMS analysis and treated with piperidine (49 μl,0.49 mmol) at room temperature. The reaction mixture was purified directly by reverse phase prep HPLC (C18 column, 0% to 70% acetonitrile-water with 0.05% TFA). Collect and The pure fractions were lyophilized to give 24mg of compound 73 as a yellow solid (8.5. Mu. Mol, 70% yield). LRMS (ESI): m/z 1406.3[ M+2H ]] ²⁺ ，C ₁₃₈ H ₁₈₄ N ₂₀ O ₄₃ m/z calculated: 1406.2. scheme 14. Synthesis of branched belatinecan construct 80

Preparation of (2S, 3R,4S,5S, 6S) -2- (2- ((S) -2- ((S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -3-methylbutanamido) propanamido) -5-formylphenoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-trisyl triacetate (75)

To a round bottom flask containing a stir bar was added alcohol 74 (0.075 g,0.088 mmol) and anhydrous DCM (15 mL) and then a portion of MnO was added at ambient temperature ₂ (0.400 g,4.6 mmol) activated by heating overnight in an oven at 130 ℃. The reaction mixture was stirred for 90 minutes until the starting material was judged to have been completely consumed by TLC analysis. The reaction mixture was filtered through a celite pad eluting with DCM. The combined filtrates were concentrated and purified by silica gel chromatography (0% to 50% gradient of EtOAc-hexanes) to give aldehyde 75 as a white solid (0.057 g,0.068mmol, 77% yield). LRMS (ESI): m/z 846.5[ M+H ]] ⁺ ，C ₄₃ H ₄₇ N ₃ O ₁₅ m/z calculated: 846.3.

preparation of (2S, 3R,4S,5S, 6S) -2- (2- ((S) -2- ((S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -3-methylbutanamido) propanamido) -5- (aminomethyl) phenoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-trisyl triacetate (76)

To a dried vial containing a stir bar was added aldehyde 75 (0.100 g;0.118 mmol) and anhydrous MeOH (10 mL), followed by the addition of dried 4A molecular sieve (about 1 g). The resulting mixture was stirred at room temperature for 10min. Anhydrous ammonium acetate (0.911 g;11.8 mmol) is then added to the mixture and continued for 1h, followed by the addition of a portion of sodium cyanoborohydride (0.038 g;0.591 mmol) at room temperature. After stirring for a further 1h, the reaction mixture was filtered and concentrated under reduced pressureAnd purified by silica gel chromatography (gradient of DCM containing 0% to 10% MeOH) to give 0.043g of amine product 76 (0.051 mmol, 43% yield). LRMS (ESI): m/z 847.4[ M+H ]] ⁺ ，C ₄₃ H ₅₀ N ₄ O ₁₄ m/z calculated: 847.3.

preparation of (S) -N- (2- (4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) -N-isopropylglycine (77)

Belotekang hydrochloride (2, 0.025g;0.057 mmol) was dissolved in DMF (0.25 mL) and diluted with MeOH (3.0 mL). The resulting solution was combined with glyoxylate (0.0111 g;0.115 mmol) and sodium acetate (0.033 g;0.40 mmol) and stirred at room temperature for 1h. The reaction mixture was then treated with sodium cyanoborohydride (0.025 g;0.40 mmol), stirred at room temperature overnight, and quenched with 1mL of 0.05% TFA in water. The solvent was removed in vacuo to leave a crude oil which was purified by reverse phase prep HPLC (C18 column, 5% to 55% acetonitrile-water/0.05% TFA). Fractions containing the desired product were collected and lyophilized to give 0.027g (0.055 mmol, 96% yield) of compound 77 as a pale yellow solid. LRMS (ESI): m/z 492.2[ M+H ] ] ⁺ ，C ₂₇ H ₂₉ N ₃ O ₆ m/z calculated: 492.2.

preparation of (2S, 3S,4S,5R, 6S) -6- (2- ((S) -2- ((S) -2-amino-3-methylbutanamide) propanamido) -5- ((2- ((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) amino) acetamido) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (78)

To a dried scintillation vial containing a stir bar was added carboxylic acid 77 (0.018 g;0.037 mmol) and anhydrous DMF (2 mL) at room temperature followed by HATU (0.013 g;0.034 mmol) and DIPEA (30. Mu.L). The mixture was stirred for 45min and then combined with amine 76 (0.026 g;0.030 mmol) and DIPEA (30. Mu.L) in 2mL DMF. The reaction mixture was stirred for 1h, quenched by addition of 1% aqueous TFA (15 mL), transferred to a separatory funnel and extracted with EtOAc. The organic layer was washed with water and brine, and was purified by Na ₂ SO ₄ And (5) drying. In true senseThe solvent was removed under air to give a crude yellow oily solid (0.048 g) which was dissolved in 5mL THF. The solution was cooled to 0 ℃ in an ice bath and slowly treated with chilled aqueous LiOH (1 m,2.0 ml). The reaction mixture was stirred at 0 ℃ for 1h, warmed slowly to room temperature and quenched by addition of aqueous HCl (1.0M) to pH 4. The mixture was purified by reverse phase prep HPLC (C18 column, 0% to 50% acetonitrile-water/0.05% TFA) to give 0.020g of compound 78 (0.021 mmol, 70% yield) as an off-white solid. LRMS (ESI): m/z 959.1[ M+H ] ] ⁺ ，C ₄₈ H ₅₉ N ₇ O ₁₄ m/z calculated: 958.4

(2S, 3S,4S,5R, 6S) -6- (2- ((17S, 20S) -1-amino-17-isopropyl-20-methyl-15, 18-dioxo-3, 6,9, 12-tetraoxa-16, 19-diaza-eicosane-21-amido) -5- ((2- ((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) amino) acetamido) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (79)

To a solution of amine 78 (0.020g, 0.021 mmol) in anhydrous DMF (2 mL) was added PFP ester 63 (0.020g, 0.031 mmol), HOAt (0.04 g;0.031 mmol) and DIPEA (11. Mu.L) at room temperature. The reaction mixture was stirred for 45min, then treated with piperidine (50 μl) and stirred for an additional 20min. The mixture was purified by reverse phase prep HPLC (C18 column, 0% to 50% acetonitrile-water/0.05% TFA). The pure fractions were combined and lyophilized to give amine product 79 as a pale yellow solid (0.015 g,0.012mmol, 57% yield). LRMS (ESI): m/z 1205.5[ M+H ]] ⁺ ，C ₅₉ H ₈₀ N ₈ O ₁₉ m/z calculated: 1205.6.

(2S, 3S,4S,5R, 6S) -6- (2- ((2S, 5S) -25- (5- ((2S, 5S) -1- ((2- (((2S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxy tetrahydro-2H-pyran-2-yl) oxy) -4- ((2- ((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] Indolazino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) amino) acetamido) methyl) phenyl) -5-isopropyl-2-methyl-1,4,7,23-tetraoxo-10,13,16,19-tetraoxa-3,6,22-triazahexa-2- ((1, 2-dimethylhydrazino) methyl) -1H-indol-1-yl) -5-isopropyl-2-methyl-4,7,23-trioxo-10,13,16,19-tetraoxa-3,6,22-triazaeicosamido) -5- ((2- ((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',3', 4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) amino) acetamido) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (80)

To a solution of amine 79 (15 mg; 12. Mu. Mol) in anhydrous DMF (2 mL) was added bis-PFP ester 40 (5.5 mg; 6. Mu. Mol) followed by HOAt (3.4 mg; 2.5. Mu. Mol) and DIPEA (22. Mu.L) at room temperature. The resulting mixture was stirred for 30min, then 50 μl piperidine was added and stirring was continued for 20min. The reaction mixture was diluted with 0.05% TFA (1 mL) and purified by reverse phase preparative HPLC (C18 column, 0% to 50% acetonitrile-water/0.05% TFA). The pure fractions were collected and immediately subjected to lyophilization to give 5.2mg of compound 80 as a yellow solid (1.9 μmol, 32% yield). LRMS (ESI): m/z 1376.2[ M+2H] ²⁺ ，C ₁₃₆ H ₁₈₀ N ₂₀ O ₄₁ m/z calculated: 1376.1.

scheme 15. Synthesis of branched belatinecan construct 86

Preparation of (S) -22- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -21-oxo-2, 5,8,11,14, 17-hexaoxa-20-azaeicosane-25-oic acid tert-butyl ester (82)

Fmoc-Glu (OtBu) -OH 68 (0.319 g; 0.319 mmol) and DMF (15 mL) were added to a round bottom flask containing a stir bar at room temperature followed by HATU (0.215 g; 0.5538 mmol) and DIPEA (440. Mu.L; 2.54 mmol). The resulting mixture was stirred for 30min and combined with mPEG 6-amine 81 (0.150 g;0.507 mmol). After 1h, the reaction mixture was transferred to a separatory funnel, diluted with water (30 mL), and extracted with EtOAc (2×30 mL). The organic layer was washed with water and brine, dried over sodium sulfate. The solvent was removed in vacuo to give 0.50g of crude product 82 as a colourless oil, which was further used without purification. LRMS (ESI): m/z 703.4[ M+H ] ] ⁺ ，C ₃₇ H ₅₄ N ₂ O ₁₁ m/z calculated: 703.4.

preparation of (S) -22- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -21-oxo-2, 5,8,11,14, 17-hexaoxa-20-azaeicosane-25-oic acid (83)

The crude compound 82 (0.50 g) was dissolved in anhydrous DCM (5 mL) and treated with TFA (2 mL) at room temperature. The reaction mixture was stirred for 2h, then the solvent was removed under reduced pressure, and the residue was dried under high vacuum overnight to give 0.50g of crude carboxylic acid 83 as a colorless oil, which was used further without purification. LRMS (ESI): m/z 647.7[ M+H ]] ⁺ ，C ₃₃ H ₄₆ N ₂ O ₁₁ m/z calculated: 647.3.

preparation of (S) -22- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -21-oxo-2, 5,8,11,14, 17-hexaoxa-20-azaeicosane-25-oic acid perfluorophenyl ester (84)

To a stirred solution of crude carboxylic acid 83 (0.50 g) in anhydrous THF (20 mL) was added pentafluorophenol (1.42 g;7.73 mmol) at room temperature followed by a portion of DCC (0.32 g;1.55 mmol). The reaction mixture was stirred at room temperature overnight, filtered and concentrated in vacuo. The residue was then purified by silica gel chromatography (DCM gradient with 0% to 10% MeOH) to give PFP-ester 84 as a colourless solid (0.43 g,0.53mmol, 87% yield in 3 steps). LRMS (ESI): m/z 813.7[ M+H ] ] ⁺ ，C ₃₉ H ₄₅ F ₅ N ₂ O ₁₁ m/z calculated: 813.3.

preparation of (2S, 3S,4S,5R, 6S) -6- (2- ((22S, 27S, 30S) -22-amino-27-isopropyl-30-methyl-21,25,28-trioxo-2, 5,8,11,14, 17-hexaoxa-20,26,29-triazatrioundecane-31-amido) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (85)

To a solution of compound 46 (30 mg, 31. Mu. Mol) in anhydrous DMF (3 mL) was added PFP-ester 84 (31 mg, 38. Mu. Mol) at room temperature followed by HOAt (1.5 mg, 47. Mu. Mol) and DIPEA (10. Mu.L). The reaction mixture was stirred for 45min, thenTreatment with piperidine (50 μl) was performed directly. After 30min, the reaction mixture was quenched with 0.05% aqueous TFA (1 mL) and purified by reverse phase preparative HPLC (C18 column, 0% to 50% acetonitrile-water/0.05% TFA). Fractions containing the desired product were combined and lyophilized to give 38mg of amine 85 as a pale yellow solid (28 μmol, 90% yield). LRMS (ESI): m/z 1351.6[ M+H ]] ⁺ ，C ₆₅ H ₉₀ N ₈ O ₂₃ m/z calculated: 1351.6.

(2S, 3S,4S,5R, 6S) -6- (2- ((22S, 27S, 30S) -22- (3- (5- ((S) -22- (3- (((S) -1- (((S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxy tetrahydro-2H-pyran-2-yl) oxy) -4- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) -amino) -3-methyl-1-oxo-butan-2-yl) -3-oxo-butan-2, 24-dioxo-2, 5,8,11,14, 17-dioxa-20-diaza-2, 23-diaza-27-oxa-2, 35-oxa-3, 24-dioxa-2, 5,8, 17-dioxa-2, 17-dioxa-3, 17-oxa-yl) propan-2-yl) amino) -3-oxo-3-oxo-oxa-2-oxamide Alkyl-31-amido) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (86)

To a stirred solution of amine 85 (20 mg; 15. Mu. Mol) in 3mL of anhydrous DMF was added bis-PFP ester 40 (6.8 mg; 7.3. Mu. Mol) followed by HOAt (2.5 mg; 18. Mu. Mol) and DIPEA (13. Mu.L) at room temperature. The reaction mixture was stirred for 30min and then treated directly with piperidine (50 μl). After 20 min, the reaction mixture was purified by reverse phase prep HPLC (C18 column, 0% to 50% acetonitrile-water/0.05% TFA). The pure fractions containing the product were combined and lyophilized to give 15mg of compound 86 (5 μmol, 69% yield) as a pale yellow solid. LRMS (ESI): m/z 1522.2[ M+2H] ²⁺ ，C ₁₄₈ H ₂₀₀ N ₂₀ O ₄₉ m/z calculated: 1522.2.

scheme 16. Synthesis of branched belatinecan construct 92

Preparation of 13- (1- (((9H-fluoren-9-yl) methoxy) carbonyl) piperidin-4-yl) -2, 2-dimethyl-4, 14-dioxo-3, 7, 10-trioxa-13-aza-heptadecane-17-acid (88)

Succinic anhydride (93 mg, 928. Mu. Mol) and triethylamine (129. Mu.L, 928. Mu. Mol) were added to a stirred solution of compound 87 (100 mg, 186. Mu. Mol) in MeCN (2 mL) at ambient temperature. The reaction mixture was stirred for 10min and then purified directly by reverse phase chromatography (C18 column, 0% to 50% acetonitrile-water/0.05% TFA). The pure fractions were collected and lyophilized to give compound 88 as a colorless oil (90 mg,141 μmol, 76% yield).

Preparation of (9H-fluoren-9-yl) methyl 4- (N- (2- (2- (3- (tert-butoxy) -3-oxopropoxy) ethoxy) ethyl) -4-oxo-4- (perfluorophenoxy) butyramide) piperidine-1-carboxylate (89)

DCC (101 mg, 493. Mu. Mol) was added to a mixture of carboxylic acid 88 (90 mg, 141. Mu. Mol) and pentafluorophenol (91 mg, 493. Mu. Mol) in 2mL of anhydrous THF at room temperature. The reaction mixture was stirred overnight, the solid was filtered off, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (EtOAc-hexanes, gradient 0% to 50%) to give 42mg PFP-ester 89 (52 μmol, 37% yield) as an off-white solid.

Preparation of (2S, 3S,4S,5R, 6S) -6- (2- ((2S, 5S) -11- (1- (((9H-fluoren-9-yl) methoxy) carbonyl) piperidin-4-yl) -5-isopropyl-2,22,22-trimethyl-4,7,10,20-tetraoxo-14,17,21-trioxa-3,6,11-triazatricosamide-yl) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (90)

To a solution of compound 46 (25 mg, 26. Mu. Mol) in DMF (1.0 mL) was added DIPEA (14. Mu.L, 73. Mu. Mol) and HOAt (5 mg, 35. Mu. Mol) at room temperature followed by PFP-ester 89 (21 mg, 26. Mu. Mol). The reaction mixture was stirred for 30min and then was allowed to stand Followed by reverse phase chromatography (C18, 0% to 100% v/vMeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions were lyophilized to give 38mg of compound 90 (24. Mu. Mol, 92% yield) as a yellow powder. LRMS (ESI): m/z 1565.7[ M+H ]] ⁺ ，C ₈₂ H ₁₀₀ N ₈ O ₂₃ m/z calculated: 1565.7.

preparation of (2S, 5S) -1- ((2- (((2S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxy tetrahydro-2H-pyran-2-yl) oxy) -4- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenyl) amino) -5-isopropyl-2-methyl-1, 4,7, 10-tetraoxo-11- (piperidin-4-yl) -14, 17-dioxa-3,6,11-triazaeicosane-20-oic acid (91)

A solution of compound 90 (38 mg, 24. Mu. Mol) in TFA (2 mL) was stirred for one minute, then diluted with 2mL of a water-acetonitrile mixture (1:1 v/v) and lyophilized to give a white solid. The solid was dissolved in DMF (1 mL) and treated with piperidine (49. Mu.L, 0.49 mmol) at room temperature. After 20 min, the reaction mixture was purified by reverse phase prep HPLC (C18, 0% to 70% v/v MeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions were lyophilized to give 10mg of compound 91 (7. Mu. Mol, 34% yield) as a yellow powder. LRMS (ESI): m/z 1287.5[ M+H ] ] ⁺ ，C ₆₃ H ₈₂ N ₈ O ₂₁ m/z calculated 1287.6.

(2S, 3S,4S,5R, 6S) -6- (2- ((15S, 18S) -1-carboxy-9- (1- (3- (5- (4- (4- (((S) -1- (((S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxy tetrahydro-2H-pyran-2-yl) oxy) -4- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) -3-methyl-1-oxobutan-2-yl-2-oxo-2-yl) amino) -N- (2- (2- (2-carboxyethoxy) ethyl) -4-oxobutan-1-oxo-amino) -2-butan-yl) -1-oxo-butan-1-yl) -2-methyl-butanyl) -1-2-yl) propan-yl-1-yl) butanyl-hydrazo-1-yl-1-methyl) butanyl-1-hydrazo-yl Preparation of (15-isopropyl-18-methyl-10, 13, 16-trioxo-3, 6-dioxa-9,14,17-triazanineteen-19-amido) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (92)

To a solution of amine 91 (10 mg, 8. Mu. Mol) in anhydrous DMF (0.5 mL) was added DIPEA (2. Mu.L, 12. Mu. Mol) and HOAt (0.7 mg, 5. Mu. Mol) at room temperature, followed by one portion of compound 40 (3.5 mg, 4. Mu. Mol). The reaction mixture was stirred for 1h and then treated directly with piperidine (8 μl,160 μmol) at room temperature. After 20 min, the reaction mixture was purified by reverse phase prep HPLC (C18, 0% to 70% v/v MeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions were lyophilized to give 2.8mg of compound 92 (1. Mu. Mol, 26% yield) as a yellow powder. LRMS (ESI): m/z1458.2[ M+2H ]] ²⁺ ，C ₁₄₄ H ₁₈₄ N ₂₀ O ₄₅ m/z calculated 1458.1.

Scheme 17 Synthesis of branched belatinecan construct 99

Preparation of (9H-fluoren-9-yl) methyl 4- ((2, 5,8, 11-tetraoxatridecan-13-yl) amino) piperidine-1-carboxylate (95)

A mixture of N-Fmoc-piperidone (93, 640 mg,2 mmol) and mPEG 4-amine (94, 414mg,2 mmol) in DCE (10 mL) was stirred at room temperature for 30min and then treated with a small amount of STAB (840 mg,4 mmol). The resulting mixture was stirred for 2h, quenched with saturated sodium bicarbonate solution (5 mL), and extracted with EtOAc (3×15 mL). The combined organic layers were washed with brine and dried over sodium sulfate. The solvent was removed in vacuo to give crude 95 as a colorless oil (900 mg) which was used further without purification.

Preparation of 14- (1- (((9H-fluoren-9-yl) methoxy) carbonyl) piperidin-4-yl) -15-oxo-2,5,8,11,17-pentaoxa-14-aza-nineteen-19-oic acid (96)

To a solution of crude compound 95 (900 mg) in anhydrous MeCN (10 mL) was added 1, 4-dioxane-2, 6-dione (1.0 g,0.93 mmol) and triethylamine (0.85 mL,0.93 mmol) at room temperature. Will beThe reaction mixture was stirred for 30min and then purified directly by reverse phase chromatography (C18, 0% to 70% v/v MeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions were collected and lyophilized to give compound 96 as a colorless oil (260 mg,0.45mmol, 23% yield over 2 steps). LRMS (ESI): m/z 629.3[ M+H ]] ⁺ ，C ₃₃ H ₄₄ N ₂ O ₁₀ m/z calculated 629.3.

Preparation of (9H-fluoren-9-yl) methyl 4- (2- (2-oxo-2- (perfluorophenoxy) ethoxy) -N- (2, 5,8, 11-tetraoxatridecan-13-yl) acetamido) piperidine-1-carboxylate (97)

DCC (255 mg,1.23 mmol) was added to a mixture of acid 96 (260 mg,0.41 mmol) and pentafluorophenol (264 mg,1.23 mmol) in 2mL dry THF at room temperature. The reaction mixture was stirred overnight, the solid was filtered off, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (EtOAc-hexanes, 0% to 50% v/v gradient) to give 163mg PFP-ester 97 (0.20 mmol, 50% yield) as a colorless oil. LRMS (ESI): m/z 795.3[ M+H ]] ⁺ ，C ₃₉ H ₄₃ F ₅ N ₂ O ₁₀ m/z calculated 795.3.

Preparation of (2S, 3S,4S,5R, 6S) -6- (5- (((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) -2- ((21S, 24S) -21-isopropyl-24-methyl-15,19,22-trioxo-14- (piperidin-4-yl) -2,5,8,11,17-pentaoxa-14,20,23-triazaeicosan-25-ylamino) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (98)

DIPEA (14. Mu.L, 73. Mu. Mol) and HOAt (4.6 mg, 34. Mu. Mol) were added to a solution of compound 46 (25 mg, 26. Mu. Mol) in anhydrous DMF (1.0 mL) at room temperature, followed by PFP-ester 97 (21 mg, 26. Mu. Mol). The reaction mixture was stirred for 30min, then piperidine (52 ul,0.52 mmol) was added and stirring was continued for 20 min. The reaction mixture was directly subjected to reverse phase chromatography (C18, 0% to 100% v/v MeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions were lyophilized to give 22mg of compound 98 (16. Mu. Mol, 62% yield) as a yellow powder. LRMS (ESI): m/z 1333.6[ M+H ]]+，C ₆₅ H ₈₈ N ₈ O ₂₂ m/z calculated 1333.6.

(2S, 3S,4S,5R, 6S) -6- (2- ((21S, 24S) -14- (1- (4- (2- (((S) -1- (((S) -1- ((2- (((2S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxy tetrahydro-2H-pyran-2-yl) oxy) -4- (((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3', 4'); 6,7] Indolazino [1,2-b ] quinolin-11-yl) ethyl (isopropyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl amino) -2-oxoethoxy) -N- (2, 5,8, 11-tetraoxatridecan-13-yl) acetamido) piperidin-1-yl) -3-oxopropyl) -2- ((1, 2-dimethylhydrazino) methyl) -1H-indol-5-yl) amino) -4-oxobutan oyl) piperidin-4-yl ) Preparation of (21-isopropyl-24-methyl-15,19,22-trioxo-2,5,8,11,17-pentaoxa-14,20,23-triazaeicosane-25-amido) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (99)

DIPEA (4.3. Mu.L, 24. Mu. Mol) and HOAt (1.4 mg, 11. Mu. Mol) were added to a solution of compound 98 (22 mg, 16. Mu. Mol) in anhydrous DMF (1.0 mL) at room temperature, followed by compound 40 (7 mg, 8. Mu. Mol). After 30 minutes piperidine (16 μl,0.16 mmol) was added in one portion at room temperature. The reaction mixture was stirred for 15 min and then directly subjected to reverse phase prep HPLC (C18, 0% to 70% v/v MeCN-H with 0.05% TFA) ₂ O) purifying. The pure fractions were lyophilized to give 15mg 99 (5. Mu. Mol, 63% yield) as a yellow powder. LRMS (ESI): m/z 1504.2[ M+2H] ²⁺ ，C ₁₄₈ H ₁₉₆ N ₂₀ O ₄₇ m/z calculated 1504.2.

Scheme 18. Synthesis of branched belloteprednol etabonate construct 103

Preparation of (R) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -3-oxo-3- (perfluorophenoxy) propane-1-sulfonic acid (101)

To a stirred mixture of Fmoc-L-cysteic acid 100 (100 mg,0.26 mmol) and pentafluorophenol (94 mg,0.51 mmol) in 2mL anhydrous DMF was added one portion of EDCI-HCl (98 mg,0.51 mmol) at room temperature. The resulting mixture was stirred overnight and then directly subjected to reverse phase chromatography (C18, 0% to 100% v/v MeCN-H with 0.05% TFA ₂ O) purifying. The pure fraction was concentrated under reduced pressure until the solution became cloudy, and it was lyophilized to give 122mg of PFP-ester 101 (0.22 mmol, yield 85%) as an off-white solid. LRMS (ESI-): m/z556.2[ M-H ] ] ^- ，C ₂₄ H ₁₆ F ₅ NO ₇ S m/z calculated 556.1.

Preparation of (2S, 3S,4S,5R, 6S) -6- (2- ((S) -2- ((S) -2- ((R) -2-amino-3-sulfopropionamido) -3-methylbutanamido) propanamido) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (102)

To a mixture of compound 46 (30 mg, 32. Mu. Mol) and DIPEA (11. Mu.L, 64. Mu. Mol) in 2mL anhydrous DMF was added PFP-ester 101 (18 mg, 32. Mu. Mol) followed by HOAt (4.5 mg, 32. Mu. Mol) at room temperature. The resulting mixture was left to stand at room temperature for 1h, and then treated with piperidine (63. Mu.L, 0.63 mmol). After 20 min, the reaction mixture was purified by reverse phase prep HPLC (C18, 0% to 50% v/v MeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions containing the product were combined and lyophilized to give 12mg of compound 102 (11 μmol, 34% yield) as a yellow solid.

(2S, 3S,4S,5R, 6S) -6- (2- ((S) -2- ((S) -2- ((R) -2- (4- ((1- (3- (((S) -1- (((2S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) -4- (((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [ 3'), preparation of (2) tetrahydro-2H-pyrano [3',4':6,7] indolizino [1, 2-dimethylhydrazino ] quinolin-11-yl) amino) -1H-indol-5-yl) amino) -4-oxobutyrylamino) -3-sulfopropionamido) -3-methylbutanamido-propanamido) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy-2H-pyran-2-carboxylic acid

To a mixture of compound 102 (12 mg, 11. Mu. Mol) and DIPEA (4. Mu.L, 22. Mu. Mol) in 2mL anhydrous DMF was added bis-PFP-ester 40 (4.5 mg, 5. Mu. Mol) followed by HOAt (1.5 mg, 11. Mu. Mol) at room temperature. The resulting mixture was left to stand at room temperature for 1h, then treated with piperidine (22. Mu.L, 0.22 mmol). After 20 min, the reaction mixture was purified by reverse phase prep HPLC (C18, 0% to 50% v/v MeCN-H ₂ O/10mM ammonium formate). The pure fractions containing the product were combined and lyophilized to give 7mg of compound 103 (2.8 μmol, 56% yield) as a tan powder. LRMS (ESI): m/z 1266.5[ M+2H] ²⁺ ，C ₁₁₈ H ₁₄₂ N ₁₈ O ₄₁ S ₂ m/z calculated 1266.5. Scheme 19. Synthesis of branched belloteprednol etabonate construct 110

Preparation of (S) -16-amino-15-oxo-2, 5,8, 11-tetraoxa-14-aza-nineteen-19-acid tert-butyl ester (104)

To a solution of Fmoc-Glu-OtBu 68 (0.49 g,1.2 mmol) in DMF (15 mL) was added HATU (0.42 g,1.1 mmol) and DIPEA (1 mL) at room temperature. The resulting mixture was stirred for 45min, then combined with mPEG 4-amine 94 (0.20 g,0.96 mmol) and stirred at room temperature for 30min. Water (30 mL) containing 0.05% TFA was added to quench the reaction, and extracted with EtOAc (2X 30 mL). The organic layer was washed with water and brine, dried over Na ₂ SO ₄ Dried, filtered and concentrated in vacuo to give a crude oil. The crude product was resuspended in acetonitrile (20 mL) and treated with piperidine (1.0 mL,1 mmol) at room temperature. After 45min, the solvent was removed in vacuo to give a crude oil, which was washed once with hexane (10 mL) and purified by reverse phase chromatography (C18, 0% to 50% with 0.05% TFA v/v MeCN-H ₂ O) purifying. The pure fractions were combined and concentrated, then lyophilized to give amine 104 (0.23 g,0.57mmol, 58% yield) as an oily solid. LRMS (ESI): m/z 393.3[ M+H ]] ⁺ ，C ₁₈ H ₃₆ N ₂ O ₇ m/z calculated 393.3.

Preparation of (S) -16- (1- (9H-fluoren-9-yl) -3-oxo-2,7,10-trioxa-4-azatridecan-13-ylamino) -15-oxo-2, 5,8, 11-tetraoxa-14-azanona-19-oic acid tert-butyl ester (106)

To a solution of amine 104 (0.23 g;0.57 mmol) in DMF (10 mL) was added carboxylic acid 105 (0.29 g;0.72 mmol), HATU (0.27 g;0.69 mmol) and DIPEA (0.50 mL,2.9 mmol) at room temperature. The reaction mixture was stirred for 2h, then poured into 0.05% aqueous TFA (15 mL) and extracted with EtOAc (2×25 mL). The organic layer was washed with water and brine, and dried over sodium sulfate. The solvent was removed under vacuum to give crude compound 106 as an oil (0.50 g) which was used further without purification. LRMS (ESI): m/z 774.9[ M+H ]] ⁺ ，C ₄₀ H ₅₉ N ₃ O ₁₂ m/z calculated 774.4.

Preparation of (S) -16- (1- (9H-fluoren-9-yl) -3-oxo-2,7,10-trioxa-4-azatridecan-13-ylamino) -15-oxo-2, 5,8, 11-tetraoxa-14-azanineteen-19-oic acid (107)

To a solution of crude ester 106 (0.25 g,0.32 mmol) in DCM (10 mL) was added TFA (4.0 mL) and the resulting solution was stirred at room temperature for 6h. The solvent was removed in vacuo to give 0.23g (0.32 mmol, quantitative yield) of crude compound 107 as an oil. LRMS (ESI): m/z 718.4[ M+H ] ] ⁺ ，C ₃₆ H ₅₁ N ₃ O ₁₂ m/z calculated 718.4.

Preparation of (S) -16- (1- (9H-fluoren-9-yl) -3-oxo-2,7,10-trioxa-4-azatridecan-13-amido) -15-oxo-2, 5,8, 11-tetraoxa-14-aza-ninety-19-acid perfluorophenyl ester (108)

To a solution of crude acid 107 (0.23 g;0.32 mmol) in anhydrous THF (10 mL) was added DCC (0.33 g;1.57 mmol) and pentafluorophenol (0.29 g;1.57 mmol) at room temperature. The reaction mixture was stirred at room temperature overnight, then filtered and concentrated under vacuumAnd (5) shrinking. The residue was purified by chromatography on silica gel using a gradient of DCM containing 0% to 10% MeOH to give 0.23g PFP-ester 106 as a colourless oil (0.23 g,0.26mmol, 81% yield). LRMS (ESI): m/z 884.9[ M+H ]] ⁺ ，C ₄₂ H ₅₀ F ₅ N ₃ O ₁₂ m/z calculated 884.3.

Preparation of (2S, 3S,4S,5R, 6S) -6- (2- ((16R, 21S, 24S) -16- (3- (2- (2-aminoethoxy) ethoxy) propionamido) -21-isopropyl-24-methyl-15,19,22-trioxo-2, 5,8, 11-tetraoxa-14,20,23-triazaeicosane-25-amido) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (109)

To a solution of amine 46 (10 mg; 10. Mu. Mol) in anhydrous DMF (2 mL) was added PFP-ester 106 (11.5 mg; 13. Mu. Mol) followed by HOAt (3 mg, 22. Mu. Mol) and DIPEA (10. Mu.L) at room temperature. The reaction mixture was stirred for 1h, then piperidine (50 μl) was added directly to the mixture and stirring was continued for 30min. The reaction mixture was quenched by addition of 2mL of 0.05% aqueous TFA and purified by reverse phase prep HPLC (C18, 0% to 50% v/v MeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions were lyophilized to give 13mg of compound 109 (9 μmol, 90% yield) as a pale yellow solid. LRMS (ESI): m/z 1422.6[ M+H ]] ⁺ ，C ₆₈ H ₉₅ N ₉ O ₂₄ m/z calculated 1422.7.

(2S, 3S,4S,5R, 6S) -6- (2- ((16S, 21S, 24S) -16- (3- (2- (2- (3- ((R) -16- (3- (((S) -1- (((S) -2- (((2S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxy tetrahydro-2H-pyran-2-yl) oxy) -4- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) -3-methyl-1-oxo-1-butan-2-yl) -3-oxo-amino) -3-oxopropyl) -15,18,28-trioxo-2,5,8,11,21,24-trioxa-14,17,27-triazol-undec-2-yl) -propan-2-yl) oxy-2- (-methyl) propan-1-yl) 2-yl) propan-1-yl) 2-ethoxy) propan-1-yl-1-2-yl-ethoxy-oxamide Preparation of (2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (110)

To a solution of amine 110 (13 mg, 9. Mu. Mol) in 2.5mL of anhydrous DMF was added bis-PFP-ester 40 (4.2 mg, 4.5. Mu. Mol), followed by HOAt (2.4 mg, 18. Mu. Mol) and DIPEA (5. Mu.L). The reaction mixture was stirred for 30mins, then piperidine (50 μl) was added directly to the mixture and stirring was continued for 30min. The reaction mixture was purified by reverse phase prep HPLC (C18, 0% to 50% v/v MeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions were lyophilized to give 5mg of compound 110 (1.6. Mu. Mol, 36% yield) as a pale yellow solid. LRMS (ESI): m/z 1593.3[ M+2H] ²⁺ ，C ₁₅₄ H ₂₁₀ N ₂₂ O ₅₁ m/z calculated 1593.2.

Scheme 20. Synthesis of branched belloteprednol etabonate construct 113

Preparation of (R) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -3-oxo-3- ((2- (2- (3-oxo-3- (perfluorophenoxy) propoxy) ethoxy) ethyl) amino) propane-1-sulfonic acid (111)

DCC (155 mg,0.75 mmol) was added to a mixture of compound 59 (136 mg,0.25 mmol) and pentafluorophenol (136 mg,0.75 mmol) in 2mL dry THF at room temperature. The reaction mixture was stirred overnight, the solid was filtered off, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (EtOAc-hexanes, 0% to 50% v/v gradient) to give PFP-ester 111 (27 mg,38 μmol, 15% yield) as a colorless oil. LRMS (ESI): m/z 717.2[ M+H ] ] ⁺ ，C ₃₁ H ₂₉ F ₅ N ₂ O ₁₀ S m/z calculated 717.2.

Preparation of (2S, 3S,4S,5R, 6S) -6- (2- ((2S, 5S, 18R) -18-amino-5-isopropyl-2-methyl-4,7,17-trioxo-19-sulfo-10, 13-dioxa-3,6,16-triazanonamido) -5- (((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (112)

To a stirred solution of compound 46 (25 mg, 26. Mu. Mol) in anhydrous DMF (1.0 mL) was added DIPEA (14. Mu.L, 73. Mu. Mol) and HOAt (5 mg, 35. Mu. Mol) at room temperature, followed by a portion of PFP-ester 111 (19 mg, 27. Mu. Mol). The reaction mixture was stirred for 1H and then purified by reverse phase chromatography (C18, 0% to 100% v/v MeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions were lyophilized to give 9mg of compound 112 (7. Mu. Mol, 26% yield) as a yellow powder. LRMS (ESI): m/z 1255.4[ M+H ]]+，C ₅₇ H ₇₄ N ₈ O ₂₂ S m/z calculated 1255.5.

(2S, 3S,4S,5R, 6S) -6- (2- ((2S, 5S, 18R) -23- ((1- ((2S, 5S, 18R) -1- ((2- (((2S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxy tetrahydro-2H-pyran-2-yl) oxy) 4- (((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] Indolazino [1,2-b ] quinolin-11-yl) ethyl (isopropyl) carbamoyl) oxy) methyl) phenyl) -5-isopropyl-2-methyl-1,4,7,17,20-pentoxy-18- (sulfomethyl) -10, 13-dioxa-3,6,16,19-tetrazabehenn-22-yl) -2- ((1, 2-dimethylhydrazino) methyl) -1H-indol-5-yl) amino) -5-isopropyl-2-methyl-4,7,17,20,23-pentoxy-18- (sulfomethyl) -10, 13-dioxa-3,6,16,19-tetraazatricosamido) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3', preparation of 4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (113)

DIPEA (2. Mu.L, 10. Mu. Mol) and HOAt (1.4 mg, 10. Mu. Mol) were added to a solution of compound 112 (9 mg, 7. Mu. Mol) in anhydrous DMF (1.0 mL) at room temperature, followed by a portion of bis-PFP ester 40 (3.0 mg, 3.5. Mu. Mol). The reaction mixture was stirred for 30min, then treated with piperidine (7. Mu.L, 70. Mu. Mol), stirred for 15 min, then directlyBy reverse phase preparative HPLC (C18, 0% to 70% v/v MeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions were lyophilized to give 6mg of compound 113 (2.0. Mu. Mol, 57% yield) as a yellow powder. LRMS (ESI): m/z1426.1[ M+2H ]] ²⁺ ，C ₁₃₂ H ₁₆₈ N ₂₀ O ₄₇ S ₂ m/z calculated 1426.0.

Scheme 21. Synthesis of branched belloteprednol etabonate construct 118

Preparation of (S) -2- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -5- (tert-butoxy) -5-oxopentanoylamino) ethane-1-sulfonic acid (114)

Fmoc-Glu (OtBu) -OH 68 (0.750 g,1.77 mmol) and anhydrous DMF (20 mL) were added to a 100mL round bottom flask at room temperature followed by HATU (1.02 g,2.64 mmol), HOAt (0.250 g,2.12 mmol) and DIPEA (500. Mu.L). The resulting mixture was stirred for 45min, then taurine (0.4475 g,3.53 mmol) was added and the mixture was stirred overnight. The reaction mixture was poured into water and extracted with DCM. The organic layer was washed with water, brine, and dried over Na ₂ SO ₄ And (5) drying. The solvent was removed in vacuo to give crude compound 114 (1.4 g) as a white solid. LRMS (ESI-): m/z 531.2[ M-H ]] ^- ，C ₂₆ H ₃₂ N ₂ O ₈ S m/z calculated 531.2.

Preparation of (S) -4- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -5-oxo-5- ((2-sulfoethyl) amino) pentanoic acid (115)

To a solution of crude compound 114 (1.4 g) in DCM (10 mL) was added TFA (5 mL) at room temperature. The reaction mixture was stirred overnight, then the solvent was removed in vacuo, and the residue was purified by reverse phase chromatography (C18 column, 0% to 50% v/v MeCN-H with 0.05% TFA ₂ O) to give 0.74g of product 115 as a white solid (1.6 mmol, 88% yield over 2 steps). LRMS (ESI-): m/z 475.1[ M-H ]] ^- ，C ₂₂ H ₂₄ N ₂ O ₈ S m/z calculated 475.1.

Preparation of (S) -2- (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -5-oxo-5- (perfluorophenoxy) pentanamido) ethane-1-sulfonic acid (116)

To a 100mL round bottom flask containing anhydrous THF (25 mL) was added carboxylic acid 115 (0.25 g;0.53 mmol) and pentafluorophenol (0.49 g;2.6 mmol) followed by DCC (0.83 g;3.9 mmol) at room temperature. The resulting mixture was stirred at room temperature overnight, then filtered, concentrated in vacuo and purified by silica gel chromatography (DCM gradient containing 0% to 10% MeOH) to give 0.18g PFP-ester 116 as a white solid (0.28 mmol, 53% yield). LRMS (ESI-): m/z 641.1[ M-H ] ] ^- ，C ₂₈ H ₂₃ F ₅ N ₂ O ₈ S m/z calculated 641.1.

Preparation of (2S, 3S,4S,5R, 6S) -6- (2- ((S) -2- ((S) -2- ((R) -4-amino-5-oxo-5- ((2-sulfoethyl) amino) pentanamido) -3-methylbutanamide) propanamido) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (117)

To a solution of PFP-ester 116 (30 mg, 48. Mu. Mol) in anhydrous DMF (3 mL) was added amine 46 (29 mg, 32. Mu. Mol) at room temperature followed by HOAt (7.4 mg, 54. Mu. Mol) and DIPEA (30. Mu.L). The resulting mixture was stirred for 45min, then piperidine (50 μl) was added to the mixture and stirring was continued for 30min. The reaction mixture was directly subjected to reverse phase prep HPLC (C18 column, 0% to 50% v/v MeCN-H with 0.05% TFA ₂ O) purifying. The product-containing fractions were concentrated and lyophilized to give 28mg (24 μmol, 75% yield) of compound 117 as a bright yellow solid. LRMS (ESI-): m/z 1179.4[ M-H ]] ^- ，C ₅₄ H ₆₈ N ₈ O ₂₀ S m/z calculated 1179.4.

(2S, 3S,4S,5R, 6S) -6- (2- ((S) -2- ((S) -2- ((S) -4- (4- ((1- (3- (((S) -5- (((S) -1- (((2S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) -4- (((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [ 3'), 4':6,7] Indolazino [1,2-b ] quinolin-11-yl) ethyl (isopropyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -1, 5-dioxo-1- ((2-sulfoethyl) amino) pentan-2-yl) amino) -3-oxopropyl) -2- ((1, 2-dimethylhydrazino) methyl) -1H-indol-5-yl) amino) -4-oxobutanamido) -5-oxo-5- ((2-sulfoethyl) amino) pentanamido) -3-methylbutanamino) -5- (((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indol-1, 2-b ] quinolin-11-yl) ethyl) (isopropyl) amino) pentan-yl) -3-methyl) phenoxy) -3-5-oxobutan-yl) propan-5- (((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyran [3',4':6,7] indol-11-yl) ethyl) amino) pentan-yl) oxy) methyl) 3-hydroxy-5-butan-yl) 6-hydroxy-5-butan-yl

To a solution of compound 117 (28 mg, 24. Mu. Mol) in 2mL of anhydrous DMF was added bis-PFP-ester 40 (11 mg, 11.6. Mu. Mol), followed by HOAt (39 mg, 28. Mu. Mol) and DIPEA (21. Mu.L). The resulting mixture was stirred for 45min, then treated with piperidine (50 μl), stirred for another 30min, and purified by reverse phase prep HPLC (C18 column, 0% to 50% v/v MeCN-H with 0.05% TFA) ₂ O) purifying. Fractions containing the desired product were collected and lyophilized to give 15mg of compound 118 as a pale yellow solid (5.5 μmol, 47% yield). LRMS (ESI-): m/z 1350.0[ M-2H] ^2- ，C ₁₂₆ H ₁₅₆ N ₂₀ O ₄₃ S ₂ m/z calculated 1350.0.

Scheme 22. Synthesis of branched belatinecan construct 123

N ⁶ - (((9H-fluoren-9-yl) methoxy) carbonyl) -N ² Preparation of- (2-sulfoacetyl) -L-lysine (120)

To a solution of 2-sulfoacetic acid (280 mg,2.0 mmol) in DMF (3 mL) was added HATU (760 mg,2.0 mmol) and DIPEA (695. Mu.L, 4.0 mmol) at room temperature. After stirring the mixture for 30 minutes, amino acid 119 (330 mg,0.90 mmol) was added and stirring was continued for one hour. The reaction mixture was directly subjected to reverse phase HPLC (H with 0.05% TFA) using a C18 column ₂ O/CH ₃ CN,90:10 to 0:100 v/v). Fractions containing the desired compound were separatedAnd lyophilized to give compound 120 (280 mg,0.57mmol, 63% yield). LRMS (ESI): m/z 491.2[ M+H ] ] ⁺ ，C ₂₃ H ₂₆ N ₂ O ₈ Sm/z calculated 491.1.

Preparation of (S) -2- ((6- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -1-oxo-1- (perfluorophenoxy) hexan-2-yl) amino) -2-oxoethane-1-sulfonic acid (121)

To a stirred mixture of carboxylic acid 120 (280 mg,0.76 mmol) and pentafluorophenol (315 mg,1.7 mmol) in DCM (5 mL) was added DCC (35 mg,1.7 mmol) at room temperature. After stirring for one hour, the reaction mixture was filtered, concentrated, and purified by reverse phase chromatography on a C18 column (H with 0.05% TFA ₂ O/CH ₃ CN,90:10 to 0:100 v/v) to give compound 121 as a white solid (80 mg,0.12mmol, 16% yield). LRMS (ESI): m/z 657.1[ M+H ]] ⁺ ，C ₂₉ H ₂₅ F ₅ N ₂ O ₈ Sm/z calculated 657.1.

Preparation of (2S, 3S,4S,5R, 6S) -6- (2- ((S) -2- ((S) -2- ((S) -6-amino-2- (2-sulfoacetamido) hexanamido) -3-methylbutanamido) propanamido) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (122)

To a mixture of amine 46 (10 mg, 11. Mu. Mol) and PFP-ester 121 (15 mg, 23. Mu. Mol) in anhydrous DMF (0.5 mL) was added DIPEA (4.4. Mu.L, 26. Mu. Mol) at room temperature. After stirring overnight, and piperidine (50 μl) was added to the reaction mixture. After stirring at room temperature for 15 min, the reaction mixture was directly subjected to reverse phase prep HPLC (H with 0.05% TFA) using a C18 column ₂ O/CH ₃ CN,90:10 to 45:55 v/v). Fractions containing the desired compound were combined and lyophilized to give compound 122 (7 mg,5.9 μmol, 54% yield). LRMS (ESI): m/z 1195.5[ M+H ]] ⁺ ，C ₅₅ H ₇₀ N ₈ O ₂₀ Sm/z calculated 1195.4.

(2S, 3S,4S,5R, 6S) -6- (2- ((S) -2- ((S) -2- ((S) -6- (4- ((1- (3- (((S) -6- (((S) -1- (((2S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) -4- (((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3', preparation of 4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -6-oxo-5- (2-sulfoacetamido) hexyl) amino) -3-oxopropyl) -2- ((1, 2-dimethylhydrazino) methyl) -1H-indol-5-yl) amino) -4-oxobutanamido) -2- (2-sulfoacetamido) hexanamido) -3-methylbutanamido) propanamido) -5- (((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4 '. 6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) oxy) phenoxy) 3, 5-hydroxy) phenoxy) -3, 5-trimethylpyran-2-yl) 3, 123-tetrahydro-2-hydroxy-pyran-2-carboxylic acid

DIPEA (5. Mu.L, 26. Mu. Mol) was added to a stirred mixture of amine 122 (7 mg, 6. Mu. Mol) and bis-PFP-ester 40 (2.6 mg, 2.8. Mu. Mol) in DMF (0.5 mL) at room temperature. The resulting mixture was stirred for 2 hours, and then piperidine (50 μl) was added to the mixture. After stirring at room temperature for 15 min, the partial reaction mixture was directly subjected to reverse phase prep HPLC (H with 0.05% TFA) using a C18 column ₂ O/CH ₃ CN,90:10 to 45:55 v/v). Fractions containing the desired compound were combined and lyophilized to give compound 123 (3 mg,1 μmol, 36% yield). LRMS (ESI): m/z 1365.5[ M+H ]] ²⁺ ，C ₁₂₈ H ₁₆₀ N ₂₀ O ₄₃ S ₂ m/z calculated 1365.5.

Scheme 23 Synthesis of branched belatinecan construct 127

Preparation of 1- (9H-fluoren-9-yl) -3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-aza-trioundecane-31-oic acid perfluorophenyl ester (125)

To a stirred mixture of carboxylic acid 124 (100 mg,0.15 mmol) and pentafluorophenol (140 mg,0.75 mmol) in dry THF (2 mL) was added one portion of DCC (37 mg,0.18 mmol) at room temperature. Mixing the obtained mixtureThe mixture was stirred overnight, filtered and concentrated in vacuo. The residue was purified by reverse phase chromatography (C18 column, 0% to 70% v/v MeCN-H with 0.05% TFA ₂ O) to give 120mg of compound 125 (0.14 mmol, 93% yield) as a clear colorless oil. LRMS (ESI): m/z 830.3[ M+H ] ] ⁺ ，C ₄₀ H ₄₈ F ₅ NO ₁₂ m/z calculated 830.3.

Preparation of (2S, 3S,4S,5R, 6S) -6- (2- ((29S, 32S) -1-amino-29-isopropyl-32-methyl-27, 30-dioxo-3,6,9,12,15,18,21,24-octaoxa-28, 31-diazatridecan-33-amido) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (126)

A solution of amine 46 (55 mg, 58. Mu. Mol) in 2mL of anhydrous DMF was treated with DIPEA (20. Mu.L, 0.12 mmol) and HOAt (8 mg, 58. Mu. Mol) and then combined at room temperature with DMF (1 mL) containing PFP-ester 125 (48 mg, 58. Mu. Mol). The resulting mixture was stirred for 30 minutes, and then piperidine (115. Mu.L, 115. Mu. Mol) was added to the mixture. After 20 min, the reaction mixture was subjected to reverse phase prep HPLC (C18 column, 0% to 50% v/vMeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions containing the product were combined and lyophilized to give 49mg of compound 126 as a yellowish solid (36 μmol, 62% yield). LRMS (ESI): m/z 1368.6[ M+H ]] ⁺ ，C ₆₆ H ₉₃ N ₇ O ₂₄ m/z calculated 1368.6.

(2S, 3S,4S,5R, 6S) -6- (2- ((2S, 5S) -38- ((1- ((2S, 5S) -1- ((2- (((2S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxy tetrahydro-2H-pyran-2-yl) oxy) -4- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenyl) -5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxo-3,6,34-triazatridecan-37-yl) -2- ((1, 2-dimethylhydrazino) methyl) -1H-indol-5-amino) -5-isopropyl-2-methyl-96-tetraoxo-92-oxa-3 ' -oxa-35-2-oxo-3, 3,4,12,14-oxo-3-octa-2-oxo-3- (((3, 2-b) ethyl) amino) -5-oxo-3- (-2-oxo-3, 35-oxo-2H-yl) amino) -5-oxo-6-1- ((2H-oxo-6-5H-pyran-yl) amino) -6-, preparation of 4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (127)

To a mixture of compound 126 (49 mg, 36. Mu. Mol) and DIPEA (13. Mu.L, 72. Mu. Mol) in 2mL of DMA was added a portion of bis-PFP-ester 40 (14.6 mg, 16. Mu. Mol) followed by HOAt (5 mg, 36. Mu. Mol) at room temperature. The resulting mixture was stirred at room temperature for 30 minutes, then piperidine (21 μl) was added, and stirring was continued for 20 minutes. The reaction mixture was directly subjected to reverse phase prep HPLC (C18 column, 0% to 50% v/v MeCN-H with 0.05% TFA ₂ O) purifying. The fractions were lyophilized to give 32mg of compound 127 (10. Mu. Mol, 63% yield) as a yellow powder. LRMS (ESI): m/z 1539.3[ M+H ]] ²⁺ ，C ₁₅₀ H ₂₀₆ N ₁₈ O ₅ m/z calculated 1538.7.

Scheme 24. Synthesis of branched belatinecan construct 131

N ⁶ - (((9H-fluoren-9-yl) methoxy) carbonyl) -N ² Preparation of- (3- (2- (2-methoxyethoxy) ethoxy) propionyl) -L-lysine (129)

To a solution of mPEG 8-acid 128 (100 mg,0.24 mmol) in 2mL anhydrous DMF was added DIPEA (0.13 mL,0.72 mmol) and HATU (93 mg,0.24 mmol) at room temperature. The resulting mixture was stirred for one hour, then Lys (Fmoc) -OH 119 (89 mg,0.24 mmol) was added to the mixture and stirring was continued for one hour. The reaction mixture was directly subjected to reverse phase chromatography HPLC (C18, 0% to 70% v/v MeCN-H with 0.05% TFA ₂ O) to give 120mg of compound 129 (0.16 mmol, 67% yield) as a colorless oil. LRMS (ESI): m/z 763.4[ M+H ] ] ⁺ ，C ₃₉ H ₅₈ N ₂ O ₁₃ m/z calculated 763.4.

Preparation of (2S, 3S,4S,5R, 6S) -6- (2- ((28S, 31S, 34S) -28- (4-aminobutyl) -31-isopropyl-34-methyl-26,29,32-trioxo-2,5,8,11,14,17,20,23-octaoxa-27,30,33-triazatripentadec-ne-35-amido) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (130)

DIPEA (21. Mu.L, 120. Mu. Mol) and HATU (22 mg, 59. Mu. Mol) were added to a solution of carboxylic acid 129 (45 mg, 59. Mu. Mol) in 3mL anhydrous DMF at room temperature. The resulting mixture was stirred for 20 min and combined with amine 46 (55 mg, 58. Mu. Mol) in 1mL DMF. The reaction mixture was stirred for 30 min, then piperidine (115 μl,1.2 mmol) was added to the mixture at room temperature. After 20 min, the reaction mixture was directly subjected to reverse phase prep HPLC (C18, 0% to 50% v/v MeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions were lyophilized to give 34mg (23. Mu. Mol, 40% yield) of compound 130 as a yellow powder. LRMS (ESI): m/z 1467.7[ M+H ]] ⁺ ，C ₇₁ H ₁₀₂ N ₈ O ₂₅ m/z calculated 1467.7.

(2S, 3S,4S,5R, 6S) -6- (2- ((28S, 31S, 34S) -28- (4- (3- (5- ((S) -28- (((S) -1- (((2S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxy tetrahydro-2H-pyran-2-yl) oxy) -4- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) methyl) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxo-2-butan-2-yl) -amino) -26, 34-dioxo-2,5,8,11,14,17,20,23-octaoxa-27, 33-diazaheptadecan-3-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indol-11-yl) ethyl) (isopropyl) methyl) oxo) phenyl) amino) -3-oxopropan-2-yl) -3-oxo-2-yl ] amino) -3-oxo-6, 33-diaza-2H-7-oxa-methyl-2-1-to 35-to the 3-oxo-to the 3-base of the 3-base Preparation of (2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (131)

Compound 130 (34 mg, 23. Mu. Mol) and DIPEA (8. Mu.L, 46. Mu. Mol) were added at room temperature in 2mLTo the mixture in DMA was added bis-PFP ester 40 (9.4 mg, 10.5. Mu. Mol) followed by HOAt (3 mg, 23. Mu. Mol). The resulting mixture was allowed to stand at room temperature for 30 minutes, and then piperidine (21 μl,0.21 mmol) was added to the mixture at room temperature. After 20 min, the reaction mixture was directly subjected to reverse phase prep HPLC (C18, 0% to 50% v/vMeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions were combined and lyophilized to give compound 131 as a yellow solid (23 mg,7 μmol, 67% yield). LRMS (ESI): m/z 1638.3[ M+H ]] ²⁺ ，C ₁₆₀ H ₂₂₄ N ₂₀ O ₅₃ m/z calculated 1638.8. Scheme 25 Synthesis of branched belatinecan construct 136

Preparation of (R) -21- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -2, 2-dimethyl-4, 20-dioxo-3,7,10,13,16-pentaoxa-19-azabehene-22-sulfonic acid (133)

To a mixture of Fmoc-L-cysteic acid 100 (399mg, 1.0 mmol) and amine 132 (321 mg,1.0 mmol) in anhydrous DMF (2 mL) was added HATU (400 mg,1.05 mmol) and DIPEA (0.52 mL,3 mmol). The reaction mixture was stirred for one hour and then purified directly by reverse phase chromatography (C18, 0% to 50% v/v MeCN-H with 0.05% TFA ₂ O) to give compound 133 as a colourless oil (500 mg,0.72mmol, 72% yield). LRMS (ESI-): m/z 693.3[ M-H ] ] ^- ，C ₃₃ H ₄₆ N ₂ O ₁₂ Sm/z calculated 693.3.

Preparation of (R) -1- (9H-fluoren-9-yl) -3, 6-dioxo-5- (sulfomethyl) -2,10,13,16,19-pentaoxa-4, 7-diaza-behene-22-oic acid (134)

To a solution of compound 133 (100 mg,0.14 mmol) in DCM (2 mL) was added TFA (2 mL) at ambient temperature. The reaction mixture was stirred for 10 min, then the solvent was removed under vacuum, and the residue was purified by reverse phase chromatography (C18, 0% to 75% v/v MeCN-H with 0.05% TFA ₂ O) to give compound 134 as a colourless oil (80 mg,0.12mmol, 86% yield). LRMS (E)SI-)：m/z 637.2[M-H] ^- ，C ₂₉ H ₃₈ N ₂ O ₁₂ Sm/z calculated 637.2.

Preparation of (2S, 3S,4S,5R, 6S) -6- (2- ((2S, 5S, 24R) -24-amino-5-isopropyl-2-methyl-4,7,23-trioxo-25-sulfo-10,13,16,19-tetraoxa-3,6,22-triazaeicosamido) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (135)

To a solution of compound 134 (9 mg, 14. Mu. Mol) in anhydrous DMF (1.0 mL) was added DIPEA (7.4. Mu.L, 42. Mu. Mol) and HATU (5 mg, 13. Mu. Mol) at room temperature. The resulting mixture was stirred for 30 minutes and then combined with compound 46 (14 mg, 15. Mu. Mol) at room temperature. After one hour piperidine (30 μl) was added to the reaction mixture and stirring was continued for 20 minutes. The reaction mixture was purified by reverse phase prep HPLC (C18, 0% to 70% v/vMeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions were lyophilized to give 13mg of compound 135 (10. Mu. Mol, 68% yield) as a yellow powder. LRMS (ESI): m/z 1343.5[ M+H ]] ⁺ ，C ₆₁ H ₈₂ N ₈ O ₂₄ S m/z calculated 1343.5.

(2S, 3S,4S,5R, 6S) -6- (2- ((2S, 5S, 24R) -28- (5- ((2S, 5S, 24R) -1- ((2- (((2S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxy tetrahydro-2H-pyran-2-yl) oxy) 4- (((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] Indolazino [1,2-b ] quinolin-11-yl) ethyl (isopropyl) carbamoyl) oxy) methyl) phenyl) -5-isopropyl-2-methyl-1,4,7,23,26-pentoxy-24- (sulfomethyl) -10,13,16,19-tetraoxa-3,6,22,25-tetraazaicosane-29-amido) -2- ((1, 2-dimethylhydrazino) methyl) -1H-indol-1-yl) -5-isopropyl-2-methyl-4,7,23,26-tetraoxo-24- (sulfomethyl) -10,13,16,19-tetraoxa-3,6,22,25-tetraazaocta-amido) -5- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3', preparation of 4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (136)

To a solution of compound 135 (13 mg, 10. Mu. Mol) in anhydrous DMF (0.5 mL) was added DIPEA (5. Mu.L, 15. Mu. Mol) and HOAt (2 mg, 15. Mu. Mol) at room temperature followed by bis-PFP ester 40 (4.3 mg, 5. Mu. Mol). After 30 min, the reaction was judged complete by LCMS analysis, and piperidine (10 μl,97 μmol) was added directly to the mixture at room temperature in one portion. After 15 min, the reaction mixture was purified by reverse phase prep HPLC (C18, 0% to 70% v/v MeCN-H with 0.05% TFA ₂ O) purifying. The pure fraction was lyophilized to give 7.4mg of compound 136 (2.4. Mu. Mol, 57% yield) as a yellow powder. LRMS (ESI): m/z 1514.2.1[ M+2H] ²⁺ ，C ₁₄₀ H ₁₈₄ N ₂₀ O ₅₁ S ₂ m/z calculated 1514.1.

Scheme 26 Synthesis of branched belatinecan construct 142

Preparation of (S) -5- ((9H-fluoren-9-yl) methoxy) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -5-oxopentanoic acid (138)

To a mixture of Fmoc-Glu-OtBu 137 (426 mg,1 mmol) and (9H-fluoren-9-yl) methanol (216 mg,1.1 mmol) in 5mL anhydrous THF was added one portion of DCC (247 mg,1.2 mmol) at room temperature. The resulting mixture was stirred overnight, filtered and concentrated under vacuum. The residue was dissolved in DCM-TFA mixture (1:1 v/v,6 mL) and left to stand at room temperature for 30 min. The solvent was removed in vacuo, the residue was dissolved in 40mL EtOAc, washed with saturated ammonium chloride, water and brine, dried over sodium sulfate, and purified by silica gel chromatography (0% to 25% v/v EtOAc-hexanes) to give 230mg Fmoc-Glu (OFm) -OH 138 as a colorless solid (0.42 mmol, 42% yield). LRMS (ESI): m/z 548.2[ M+H ]] ⁺ ，C ₃₄ H ₂₉ NO ₆ m/z calculated 548.2.

Preparation of (S) -5- (3- ((9H-fluoren-9-yl) methoxy) -3-oxopropyl) -1- (9H-fluoren-9-yl) -3, 6-dioxo-2,10,13,16,19-pentaoxa-4, 7-diaza-behene-22-oic acid (139)

Fmoc at room temperatureGlu (OFm) -OH 138 (230 mg,0.42 mmol) and amino-PEG 4-OtBu 132 (162 mg,0.46 mmol) in 2mL DMF was added DIPEA (0.22 mL,1.26 mmol) followed by PyAOP (240 mg,0.42 mmol). The reaction mixture was stirred for 30 min, then poured into saturated ammonium chloride solution and extracted with EtOAc. The organic layer was washed with brine and dried over sodium sulfate. After removal of the solvent in vacuo, the residue was redissolved in a DCM-TFA mixture (1:1 v/v,4 mL) at room temperature and stirred for 15 min, then the solvent was removed in vacuo and the residue was purified by reverse phase chromatography (C18, 0% to 70% v/vMeCN-H with 0.05% TFA ₂ O) to give 306mg of compound 139 as a clear colorless oil (0.39 mmol, 92% yield). LRMS (ESI): m/z 795.3[ M+H ]] ⁺ ，C ₄₅ H ₅₀ N ₂ O ₁₁ m/z calculated 795.3.

Preparation of (S) -18- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -17-oxo-4, 7,10, 13-tetraoxa-16-aza-di-undecanedioic acid 21- ((9H-fluoren-9-yl) methyl) 1- (2, 3,5, 6-tetrafluorophenyl) ester (140)

To a mixture of compound 139 (145 mg,0.18 mmol) and 2,3,5,6, -tetrafluorophenol (61 mg,0.36 mmol) in 2mL THF was added one portion of DCC (45 mg,0.36 mmol) at room temperature. The resulting mixture was stirred overnight, filtered, concentrated in vacuo, and purified by reverse phase chromatography (C18, 0% to 80% v/v MeCN-H with 0.05% TFA ₂ O) to give 84mg TFP-ester 140 as a colourless oil (0.09 mmol, 50% yield). LRMS (ESI): m/z 965.3[ M+Na] ⁺ ，C ₅₁ H ₅₀ F ₄ N ₂ O ₁₁ m/z calculated 965.3.

Preparation of (2S, 5S, 24S) -24-amino-1- ((2- (((2S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxy tetrahydro-2H-pyran-2-yl) oxy) -4- ((((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenyl) amino) -5-isopropyl-2-methyl-1,4,7,23-tetraoxo-10,13,16,19-tetraoxa-3,6,22-triazadihexadecane-27-oic acid (141)

A solution of Compound 46 (19 mg, 20. Mu. Mol) in 3mL anhydrous DMF was added at room temperatureDIPEA (9. Mu.L, 60. Mu. Mol) and HOAt (2.7 mg, 20. Mu. Mol) were added thereto, followed by a portion of TFP ester 140 (19 mg, 20. Mu. Mol). The reaction mixture was stirred for 30 min and monitored by LCMS analysis. After the completion of the reaction was judged to be complete, piperidine (40. Mu.L) was added to the mixture and stirring was continued for 20 minutes. The reaction mixture was then purified by reverse phase prep HPLC (C18, 0% to 50% v/v MeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions were lyophilized to give 14.6mg of compound 141 as a yellow powder (11 μmol, 55% yield). LRMS (ESI): m/z 1321.5[ M+H ] ] ⁺ ，C ₆₃ H ₈₄ N ₈ O ₂₃ m/z calculated 1321.6.

(2S, 3S,4S,5R, 6S) -6- (2- ((2S, 5S, 24S) -28- (5- ((2S, 5S, 24S) -1- ((2- (((2S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxy tetrahydro-2H-pyran-2-yl) oxy) -4- (((2- ((S) -4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenyl) -24- (2-carboxyethyl) -5-isopropyl-2-methyl-1,4,7,23,26-pentaoxo-10,13,16,19-tetraoxa-3,6,22,25-tetraazadi-nona-29-amido) -2- ((1, 2-dimethyl) methyl) -1H-indol-1-yl) -24- (6, 7] indol-1-yl) -2- (3, 2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) methyl) -24- (2-oxo-1,4,7,23,26-pentaoxo-10,13,16,19-tetraoxa-29-amido) -2- ((1, 2-dimethyl) methyl) -1H-2-oxo-1H-2- (((3S, 4' - [ 6,7] indolizino [1,2-b ] quinolin-11-yl ] ethyl) -11-yl) -ethyl) -96-yl-5- ((2-yl) -amino-5-yl-amino. Preparation of 4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (142)

To a solution of compound 141 (14.6 mg, 11. Mu. Mol) in 2mL of DMA was added DIPEA (6. Mu.L, 33. Mu. Mol) and HOAt (1.5 mg, 11. Mu. Mol) at room temperature, followed by a portion of bis-PFP ester 40 (4.5 mg, 5. Mu. Mol). The resulting mixture was allowed to stand at room temperature for 30 minutes, and then piperidine (10. Mu.L) was directly added to the mixture. After 20 min, the reaction mixture was purified by reverse phase prep HPLC (C18, 0% to 50% v/v MeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions were combined and lyophilized to give 6mg (2 μmol, 40% yield) of compound 142 as a yellow solid. LRMS (ESI): m/z 1491.2[ M+2H] ²⁺ ，C ₁₄₄ H ₁₈₈ N ₂₀ O ₄₉ m/z calculated 1491.6.

Scheme 27 Synthesis of branched des-Me-topotecan construct 147

Preparation of (S) -4-ethyl-4, 9-dihydroxy-10- ((methylamino) methyl) -1, 12-dihydro-14H-pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-3, 14 (4H) -dione (144)

To a solution of 10-hydroxycamptothecin 143 (500 mg,1.37 mmol) in acetic acid (30 mL) and EtOH (15 mL) was added formaldehyde (1 mL,37wt%, at H) ₂ O) and MeNH ₂ (1 mL,40% w/w aqueous solution). The reaction mixture was stirred at room temperature overnight and then concentrated under reduced pressure. The residue was purified by reverse phase chromatography (C18, 0% to 70% v/v MeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions were collected and lyophilized to give des-Me-topotecan 144 as a pale yellow solid (150 mg,0.46mmol, 27% yield). LRMS (ESI): m/z 408.2[ M+H ]] ⁺ ，C ₂₂ H ₂₁ N ₃ O ₅ m/z calculated 408.2.

Preparation of (2S, 3S,4S,5R, 6S) -6- (2- ((S) -2- ((S) -2-amino-3-methylbutanamide) propanamido) -5- (((((((S) -4-ethyl-4, 9-dihydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-10-yl) methyl) (carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (145)

To a stirred solution of des-Me-topotecan 144 (25 mg, 61. Mu. Mol) in DMF (1.5 mL) was added HOAt (8.5 mg, 62. Mu. Mol) and DIPEA (30. Mu.L, 184. Mu. Mol) at room temperature. The resulting mixture was then purified at room temperature with a portion of PNP-carbonate 28 (58 mg, 62. Mu. Mol). The reaction mixture was stirred overnight until all starting material was consumed as judged by LCMS analysis. The reaction mixture was poured into 10mL of water, and the resulting precipitate was collected and dissolved in THF (2 mL). The THF solution was then slowly treated with aqueous LiOH (1 ml,1 m) at 0 ℃ and stirred for 30min. The reaction mixture was slowly warmedTo room temperature and stirred for an additional hour, quenched by addition of 1M HCl aqueous solution to pH about 4, filtered, and purified by reverse phase preparative HPLC (C18, 0% to 70% v/v MeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions were collected and lyophilized to give compound 145 as a yellow solid (25 mg,27 μmol, 44% yield). LRMS (ESI): m/z 919.3[ M+H ]] ⁺ ，C ₄₄ H ₅₀ N ₆ O ₁₆ m/z calculated 919.3.

Preparation of (2S, 3S,4S,5R, 6S) -6- (2- ((28S, 31S, 34S) -28- (4-aminobutyl) -31-isopropyl-34-methyl-26,29,32-trioxo-2,5,8,11,14,17,20,23-octaoxa-27,30,33-triazatripentadec-ne-35-amido) -5- ((((((S) -4-ethyl-4, 9-dihydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-10-yl) methyl) (carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (146)

To a solution of compound 128 (21 mg, 27.5. Mu. Mol) in DMF (2 mL) was added HATU (10 mg, 31. Mu. Mol) and DIPEA (14. Mu.L, 82. Mu. Mol) at room temperature. The resulting mixture was stirred for one hour, then compound 145 (25 mg,27 μmol) was added to the mixture and stirring was continued for 1h until the coupling was judged complete by LCMS analysis. Next, the reaction mixture was treated with triethylamine (0.4 mL) and stirred at room temperature for 5h. The reaction mixture was purified by reverse phase prep HPLC (C18, 0% to 70% v/v MeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions were collected and lyophilized to give compound 146 as a yellow solid (26 mg,18 μmol, 67% yield). LRMS (ESI): m/z 1441.6[ M+H ]]+，C ₆₈ H ₉₆ N ₈ O ₂₆ m/z calculated 1441.6.

(2S, 3S,4S,5R, 6S) -28- (4- (3- (5- ((S), 31S, 34S) -28- (((S) -1- (((S) -1- ((2- (((2S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) 4- ((((((S) -4-ethyl-4, 9-dihydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3', 4') for use) a) hydroxy group of 4- (2S, 3S,4S,5R, 6S) oxy group; 6,7] Indolazino [1,2-b ] quinolin-10-yl) methyl (methyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl carbamoyl) -26, 34-dioxo-2,5,8,11,14,17,20,23-octaoxa-27, 33-diazatrioctadecyl-37-amido) -2, 1, 2-dimethylhydrazino) methyl) -1H-indol-1-yl) propanamido) butyl) -31-isopropyl-34-methyl-26,29,32-trioxo-2,5,8,11,14,17,20,23-octaoxa-27,30,33-triazatripentadecyl-35-acyl Preparation of amino) -5- ((((((S) -4-ethyl-4, 9-dihydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-10-yl) methyl) (methyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (147)

To a solution of compound 146 (26 mg, 18. Mu. Mol) in DMF (1.5 mL) was added DIPEA (10. Mu.L, 55. Mu. Mol) and HOAt (7 mg, 23. Mu. Mol) at room temperature followed by a portion of bis-PFP ester 40 (8.4 mg, 9. Mu. Mol). The reaction mixture was stirred for 30 min until the coupling was judged complete by LCMS analysis, then diethylamine (37 μl,0.36 mmol) was added to the mixture and stirring was continued for 2 h. The reaction mixture was purified by reverse phase prep HPLC (C18, 0% to 70% v/v MeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions were collected and lyophilized to give compound 147 as a yellow solid (18 mg,6 μmol, 67% yield). LRMS (ESI): m/z 1612.2[ M+2H] ²⁺ ，C ₁₅₄ H ₂₁₂ N ₂₀ O ₅₅ m/z calculated 1612.2.

Scheme 28 Synthesis of branched construct 151

Preparation of (S) -4-ethyl-4, 9-dihydroxy-10- ((isopropylamino) methyl) -1, 12-dihydro-14H-pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-3, 14 (4H) -dione (148)

To a solution of 10-hydroxycamptothecin (500 mg,1.37 mmol) in HOAc (30 mL) and EtOH (15 mL) at room temperature was added formaldehyde (1 mL,37wt%, at H) ₂ O) and i-PrNH ₂ (150. Mu.L, 1.83 mmol). The reaction mixture was stirred overnight and then concentrated in vacuo. The residue was purified by reverse phase chromatography (C18, 0% to 70% v/v MeCN-H with 0.05% TFA ₂ O) purifying. Collecting and freeze-drying the pure fractions to obtainCompound 148 was obtained as an orange solid (200 mg,0.46mmol, 36% yield). LRMS (ESI): m/z 436.2[ M+H ]] ⁺ ，C ₂₄ H ₂₅ N ₃ O ₅ m/z calculated 436.2.

Preparation of (2S, 3S,4S,5R, 6S) -6- (2- ((S) -2- ((S) -2-amino-3-methylbutanamide) propanamido) -5- ((((((S) -4-ethyl-4, 9-dihydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-10-yl) methyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (149)

To a solution of compound 148 (50 mg, 115. Mu. Mol) in DMF (3 mL) was added HOAt (16 mg, 115. Mu. Mol) and DIPEA (60. Mu.L, 344. Mu. Mol) at room temperature. The resulting mixture was treated with PNP-carbonate 28 (116 mg,115 μmol) and stirred at room temperature overnight until all starting material was consumed as judged by HPLC analysis. The reaction mixture was then diluted with water (10 mL), the resulting precipitate was collected, and dissolved in THF (3 mL). The THF solution was then treated with LiOH (1 mL, 1M) at 0deg.C, stirred for 30min, warmed to room temperature and stirred for 1h. The reaction mixture was purified by reverse phase prep HPLC (C18, 0% to 70% v/v MeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions were combined and lyophilized to give compound 149 as a yellow solid (31 mg,33 μmol, 29% yield). LRMS (ESI): m/z 947.4[ M+H ]] ⁺ ，C ₄₆ H ₅₄ N ₆ O ₁₆ m/z calculated 947.4.

Preparation of (2S, 3S,4S,5R, 6S) -6- (2- ((28S, 31S, 34S) -28- (4-aminobutyl) -31-isopropyl-34-methyl-26,29,32-trioxo-2,5,8,11,14,17,20,23-octaoxa-27,30,33-triazatripentadec-ne-35-amido) -5- ((((((S) -4-ethyl-4, 9-dihydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-10-yl) methyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (150)

To a stirred solution of carboxylic acid 129 (31 mg, 41. Mu. Mol) in anhydrous DMF (2 mL) was added HATU (15 mg, 36. Mu. Mol) and DIPEA (17. Mu.L, 94. Mu. Mol) at room temperature. The resulting mixture was stirred for 1h, then compound 149 (31 mg, 33. Mu. Mol) was added to the mixtureThe mixture was stirred for 1h. Next, the reaction mixture was treated directly with piperidine (62. Mu.L, 0.63 mmol) at room temperature, stirred for 20 min, and purified by reverse phase preparative HPLC (C18, 0% to 70% v/v MeCN-H with 0.05% TFA) ₂ O) purifying. The pure fractions were collected and lyophilized to give compound 150 as a yellow solid (26 mg,18 μmol, 55% yield). LRMS (ESI): m/z 1469.7[ M+H ] ]+，C ₇₀ H ₁₀₀ N ₈ O ₂₆ m/z calculated 1469.7.

(2S, 3S,4S,5R, 6S) -6- (2- ((28S, 31S, 34S) -28- (4- (3- (5- ((S) -28- (((S) -1- (((S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxy tetrahydro-2H-pyran-2-yl) oxy) -4- (((((((S) -4-ethyl-4, 9-dihydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolo [1,2-b ] quinolin-10-yl) methyl) (isopropyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) -3-methyl-1-oxobutan-2-yl) -3-oxo-2-yl-carbamoyl) -26, 34-dioxo-2,5,8,11,14,17,20,23-octaoxa-27, 33-diaza-heptadecan-2-yl) -2, 33-diaza-triazol-37-oxa-2-yl) -2, 34-oxobutan-3-yl) n-yl-3-oxobutan-2-yl) amino) -1-oxopropan-2-oxo-2-yl) amino) -35-pentadecan-yl-3-oxobutan-35-3-yl-oxa-yl-3-methyl-2-oxo-6-methyl-oxa-6-1-oxo-6-hydroxy-6H-6-7-1-7-hydroxy-methyl-7-1-hydroxy-7-methyl-carboxylate Preparation of amido) -5- ((((((S) -4-ethyl-4, 9-dihydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-10-yl) methyl) (isopropyl) carbamoyl) oxy) methyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (151)

To a solution of compound 150 (27 mg, 18. Mu. Mol) in DMF (1.5 mL) was added DIPEA (10. Mu.L, 55. Mu. Mol) and HOAt (8 mg, 24. Mu. Mol) at room temperature followed by a portion of bis-PFP ester 40 (8 mg, 9. Mu. Mol). The resulting mixture was stirred for 30 minutes, then piperidine (36 μl,0.36 mmol) was added to the mixture at room temperature. After 20 min, the reaction mixture was purified by reverse phase prep HPLC (C18, 0% to 70% v/vMeCN-H with 0.05% TFA ₂ O) purifying. The pure fractions were collected and lyophilized to give compound 151 as a yellow solid (19 mg,5.8 μmol, 64% yield). LRMS (ESI): m/z 1640.4[ M+2H] ²⁺ ，C ₁₅₈ H ₂₂₀ N ₂₀ O ₅₅ m/z calculated: 1640.3。

scheme 29 synthesis of SN-38 construct 154

Preparation of (S) -2-amino-N- ((S) -1- ((4- ((((S) -4, 11-diethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-9-yl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) -3-methylbutanamide (154)

To a solution of Fmoc-Val-Ala-OH 152 (30 mg, 59. Mu. Mol) in DMF (1 mL) was added MsCl (13.5 mg, 118. Mu. Mol) and DIPEA (20. Mu.L, 118. Mu. Mol) at room temperature. After one hour, the reaction mixture was concentrated and partitioned between ethyl acetate and saturated sodium bicarbonate. The organic layer was washed with brine and was purified by Na ₂ SO ₄ And (5) drying. The solvent was removed under vacuum to give crude chloride 153, which was dissolved in DMF (1 mL). To this solution, SN38 (1, 23mg, 59. Mu. Mol) and K were added ₂ CO ₃ (24 mg,0.18 mmol) and the reaction mixture was stirred vigorously at 45℃overnight. The reaction mixture was directly subjected to reverse phase HPLC (H with 0.05% TFA) using a C18 column ₂ O/CH ₃ CN,90:10 to 45:55 v/v). Fractions containing the desired compound were combined and lyophilized to give compound 154 (2.7 mg, 16% yield).

LRMS(ESI)：m/z 668.3[M+H] ⁺ ，C ₃₇ H ₄₁ N ₅ O ₇ m/z calculated 668.3.

Preparation of (2S, 5S, 18R) -1- ((4- ((((S) -4, 11-diethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-9-yl) oxy) methyl) phenyl) amino) -18- (3- (2- ((1, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2,3-b ] pyridin-1-yl) propanamido) -5-isopropyl-2-methyl-1,4,7,17-tetraoxo-10, 13-dioxa-3,6,16-triazanona-ne-19-sulfonic acid (155)

To a mixture of amine 154 (8 mg, 9. Mu. Mol) and PFP ester 111 (12 mg, 10. Mu. Mol) in DMF (0.5 mL) was added HOAT (1.2 mg, 9. Mu. Mol) and DIPEA (5. Mu.L, 27. Mu. Mol) at room temperature. Stirring the obtained mixtureAfter stirring for 1 hour, DMF (0.5 mL) and piperidine (50. Mu.L) were added to the mixture. After stirring at room temperature for 15 min, the reaction mixture was directly subjected to reverse phase HPLC (H with 0.05% TFA) using a C18 column ₂ O/CH ₃ CN,90:10 to 45:55 v/v). Fractions containing the desired compound were combined and lyophilized to give compound 155 (3.5 mg,2.9 μmol, 32% yield). LRMS (ESI): m/z 1222.5[ M+H ]] ⁺ ，C ₆₀ H ₇₅ N ₁₁ O ₁₅ Sm/z calculated 1222.5.

Scheme 30. Synthesis of SN-38 construct 165

/>

Preparation of (2S, 3R,4S,5S, 6S) -2- (2- (tert-butoxycarbonyl) -5-nitrophenoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-trisyl triacetate (158)

To a mixture of tert-butyl 2-hydroxy-4-nitrobenzoate 157 (1.57 g,6.6 mmol) and bromide 156 (2.37 g,6.0 mmol) in 25mL acetonitrile was added silver (I) oxide (1.53 g,6.6 mmol). The resulting mixture was stirred overnight in the dark, then filtered through a pad of silica gel, eluted with ethyl acetate and concentrated in vacuo. The residue was purified by silica gel chromatography (0% to 10% EtOAc-hexanes) to give 2.3g of compound 158 as a white solid (4.1 mmol, 68% yield). LRMS (ESI): m/z 578.2[ M+Na] ⁺ ，C ₂₄ H ₂₉ NO ₁₄ m/z calculated 578.2.

Preparation of 4-nitro-2- (((2S, 3R,4S,5S, 6S) -3,4, 5-triacetoxy-6- (methoxycarbonyl) tetrahydro-2H-pyran-2-yl) oxy) benzoic acid (159)

Compound 158 (180 mg,0.32 mmol) was dissolved in 4mL of DCM-TFA mixture (1:1 v/v) at room temperature. The resulting solution was left to stand for 30 min, then the solvent was removed under vacuum, and the residue was purified by silica gel chromatography (0% to 5% meoh-DCM) to give 160mg of carboxylic acid 159 (0.32 mmol, quantitative yield) as a pink foamy solid. LRMS (ESI): m/z 522.1[ M+Na ]] ⁺ ，C ₂₄ H ₂₉ NO ₁₄ m/z calculated 522.1.

Preparation of (2S, 3R,4S,5S, 6S) -2- (2- ((((S) -9- ((tert-butoxycarbonyl) oxy) -4, 11-diethyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) oxy) carbonyl) -5-nitrophenoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-trisyltriacetate (160)

To a solution of carboxylic acid 159 (18 mg, 36. Mu. Mol) in dichloromethane (1 mL) and DMF (0.5 mL) was added Boc-protected SN-38 5 (14 mg, 28. Mu. Mol) followed by DCC (6 mg, 29. Mu. Mol) and DMAP (3 mg, 25. Mu. Mol) at 0deg.C. After 1h, the reaction mixture was warmed to room temperature and stirring was continued for 2h. The reaction mixture was purified by reverse phase chromatography using a C18 column (H with 0.05% TFA ₂ O/CH ₃ CN,100:0 to 0:100 v/v) to give compound 160 (25 mg, 26. Mu. Mol, 93% yield) as a yellow solid. LRMS (ESI): m/z 974.3[ M+H ]] ⁺ ，C ₄₇ H ₄₇ N ₃ O ₂₀ m/z calculated 974.3.

Preparation of (2S, 3R,4S,5S, 6S) -2- (5-amino-2- ((((S) -9- ((tert-butoxycarbonyl) oxy) -4, 11-diethyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) oxy) carbonyl) phenoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-trisyl triacetate (161)

To a solution of compound 160 (35 mg, 36. Mu. Mol) in EtOAc (0.5 mL) were added Pd/C (10 wt%,2 mg) and triethylamine (2. Mu.L, 22. Mu. Mol). The flask was then evacuated and filled with hydrogen from the balloon and the cycle repeated three times. The reaction mixture was treated with attached H at room temperature ₂ The balloon was vigorously stirred for 48h. Filtration through a pad of silica gel to remove solids, the filtrate was concentrated and dried under vacuum to give 35mg of crude compound 161, which was used in the next step without further purification. LRMS (ESI): m/z 944.3[ M+H ] ] ⁺ ，C ₄₇ H ₄₉ N ₃ O ₁₈ m/z calculated 944.3.

Preparation of (2S, 3R,4S,5S, 6S) -2- (5- ((S) -2- ((S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -3-methylbutanamide) propanoyl) -2- (((((S) -9- ((tert-butoxycarbonyl) oxy) -4, 11-diethyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) oxy) carbonyl) phenoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyltriacetate (162)

To a mixture of crude amine 161 (35 mg) and Fmoc-Val-Ala-OH 13 (60 mg,0.15 mmol) in DMF (0.5 mL) was added HATU (56 mg,0.15 mmol) and DIPEA (51. Mu.L, 0.30 mmol) at room temperature. The reaction mixture was stirred overnight and applied to a C18 column (H with 0.05% TFA) by reverse phase chromatography ₂ O/CH ₃ CN,100:0 to 0:100 v/v) to give compound 162 (46 mg, 34. Mu. Mol, 94% in two steps) as a yellow solid. LRMS (ESI): m/z 1336.5[ M+H ]] ⁺ ，C ₇₀ H ₇₃ N ₅ O ₂₂ m/z calculated 1336.5.

Preparation of (2S, 3S,4S,5R, 6S) -6- (5- ((S) -2- ((S) -2-amino-3-methylbutanamide) propanamido) -2- ((((S) -4, 11-diethyl-9-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) oxy) carbonyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (163)

Compound 162 (20 mg, 15. Mu. Mol) was quenched with MeOH-H at room temperature ₂ Sc (OTf) was added to a solution in O mixture (4:1 v/v,1 mL) ₃ (180 mg,0.36 mmol). The resulting mixture was stirred for 2 days and concentrated under vacuum. The residue was redissolved in DMF-piperidine mixture (10:1 v/v,1.1 mL) and stirred at room temperature for 1 hour. The reaction mixture was purified by reverse phase chromatography on a C18 column (H with 0.05% TFA) ₂ O/CH ₃ CN,90:10 to 35:65 v/v) to give compound 163 (8 mg, 9. Mu. Mol, 60% yield). LRMS (ESI): m/z 874.3[ M+H ]] ⁺ ，C ₄₃ H ₄₇ N ₅ O ₁₅ m/z calculated 874.3.

Preparation of (R) -2- (3- (2- ((2- (((9H-fluoren-9-yl) methoxy) carbonyl) -1, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2,3-b ] pyridin-1-yl) propanamido) -3, 31-dioxo-31- (perfluorophenoxy) -7,10,13,16,19,22,25,28-octaoxa-4-aza-trioundecane-1-sulfonic acid (164)

DCC (68 mg,0.33 mmol) was added to a mixture of carboxylic acid 18 (180 mg,0.17 mmol) and pentafluorophenol (125 mg,0.68 mmol) in 4mL dry THF at room temperature. The obtained product is then processedThe mixture was stirred overnight, filtered through celite pad, concentrated in vacuo, and purified by reverse phase chromatography (C18, 0% to 80% acetonitrile-water/0.05% TFA) to give 100mg PFP ester 164 as a colorless oil (0.08 mmol, 47% yield). LRMS (ESI): m/z 1225.4[ M+H ] ] ⁺ ，C ₅₆ H ₆₉ F ₅ N ₆ O ₁₇ Sm/z calculated 1225.4.

Preparation of (2S, 3S,4S,5R, 6S) -6- (2- (((S) -4, 11-diethyl-9-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) oxy) carbonyl) -5- ((2S, 5S, 36R) -40- (2- ((1, 2-dimethylhydrazino) methyl) -1H-pyrrolo [2,3-b ] pyridin-1-yl) -5-isopropyl-2-methyl-4,7,35,38-tetraoxo-36- (sulfomethyl) -10,13,16,19,22,25,28,31-octaoxa-3,6,34,37-tetraazatetrazotetramido) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (165)

To a mixture of amine 163 (8 mg, 9. Mu. Mol) and PFP ester 164 (12 mg, 10. Mu. Mol) in DMF (0.5 mL) was added HOAt (1.2 mg, 9. Mu. Mol) and DIPEA (5. Mu.L, 27. Mu. Mol) at ambient temperature. The reaction mixture was stirred for one hour, then DMF (0.5 mL) was added to the mixture, followed by piperidine (50. Mu.L). After stirring at room temperature for 15 min, the reaction mixture was directly subjected to reverse phase HPLC (H with 0.05% TFA) using a C18 column ₂ O/CH ₃ CN,90:10 to 45:55 v/v). Fractions containing the desired compound were combined and lyophilized to give compound 165 (3.5 mg,2 μmol, 22% yield) as a yellow solid. LRMS (ESI): m/z 1692.7[ M+H ]] ⁺ ，C ₇₈ H ₁₀₅ N ₁₁ O ₂₉ S m/z calculated 1692.7.

Scheme 31 Synthesis of Belotecan branched construct 175

Preparation of tert-butyl (S) - (2- (4-ethyl-4-hydroxy-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-11-yl) ethyl) (isopropyl) carbamate (166)

Belote at room temperatureKang 2 (50 mg,0.11 mmol) and Boc ₂ To a mixture of O (12 mg,0.23 mmol) in dichloromethane (2 mL) was added DIPEA (40. Mu.L, 0.23 mmol). After stirring for 6 hours, the reaction mixture was directly purified by silica gel chromatography (DCM-MeOH, 100:0 to 95:5 v/v) to give compound 166 (44 mg,0.08mmol, 73% yield) as an off-white solid. LRMS (ESI): m/z 534.3[ M+H ]] ⁺ ，C ₃₀ H ₃₅ N ₃ O ₆ m/z calculated 534.3.

Preparation of (2S, 3R,4S,5S, 6S) -2- (5-amino-2- ((((S) -11- (2- ((tert-butoxycarbonyl) (isopropyl) amino) ethyl) -4-ethyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) oxy) carbonyl) phenoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-trisyl triacetate (168)

To a solution of carboxylic acid 159 (240 mg, 480. Mu. Mol) in dichloromethane (1 mL) and DMF (0.5 mL) was added Boc-protected belote 166 (100 mg, 190. Mu. Mol) followed by DCC (6 mg, 29. Mu. Mol) and DMAP (3 mg, 25. Mu. Mol) at 0deg.C. After 1h, the reaction mixture was warmed to room temperature and stirred overnight. The mixture was simply purified by silica gel pad (0% to 6% MeOH-DCM as eluent) to give crude compound 167, which was dissolved in EtOAc (2 mL) and combined with Pd/C (10 wt%,20 mg) and triethylamine (20 μl,220 μmol). The reaction flask was then evacuated and filled with hydrogen from the balloon and the cycle repeated three times. The reaction mixture was treated with attached H at room temperature ₂ The balloon was vigorously stirred for 48h and then filtered through a pad of celite. The filtrate was concentrated in vacuo and purified by silica gel chromatography (0% to 5% MeOH-DCM) to give compound 168 (60 mg,61 μmol, 33% yield) as a yellow solid. LRMS (ESI): m/z 985.4[ M+H ]] ⁺ ，C ₅₀ H ₅₆ N ₄ O ₁₇ m/z calculated 985.4. (2S, 3R,4S,5S, 6S) -2- (5- ((S) -2- ((S) -2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -3-methylbutanamido) propanamido) -2- ((((S) -11- (2- ((tert-butoxycarbonyl) (isopropyl) amino) ethyl) -4-ethyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6, 7)]Indolazino [1,2-b ]]Quinolin-4-yl) oxy) carbonyl) phenoxy) -6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyltriazinePreparation of acetate (172)

DIPEA (22. Mu.L, 120. Mu. Mol) was added to a mixture of amine 168 (60 mg, 61. Mu. Mol) and Fmoc-Ala-Cl 169 (20 mg, 61. Mu. Mol) in DMF (1 mL) at room temperature. The reaction mixture was stirred for 1h, then DMF (0.5 mL) and piperidine (50. Mu.L) were added to the mixture. After 30 min, the reaction was semi-purified by silica gel chromatography (gradient with DCM containing 0 to 5% MeOH) to give crude compound 170. Next, a solution of 170 in 1mL of acetonitrile was treated with Fmoc-Val-OPfp 171 (62 mg, 120. Mu. Mol) and DIPEA (22. Mu.L, 120. Mu. Mol) at room temperature. After stirring for 20 min, the reaction mixture was purified by silica gel chromatography (MeOH-DCM 0% to 5% gradient) to give compound 172 (70 mg,51 μmol, 83% yield) as a yellow solid.

LRMS(ESI)：m/z 1377.5[M+H] ⁺ ，C ₇₃ H ₈₀ N ₆ O ₂₁ m/z calculated 1377.5.

Preparation of (2S, 3S,4S,5R, 6S) -6- (5- ((S) -2- ((S) -2-amino-3-methylbutanamide) propanamido) -2- ((((S) -11- (2- ((tert-butoxycarbonyl) (isopropyl) amino) ethyl) -4-ethyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-4-yl) oxy) carbonyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (173)

Compound 172 (70 mg, 51. Mu. Mol) in MeOH-H at room temperature ₂ Sc (OTf) was added to a solution in O mixture (4:1 v/v,1 mL) ₃ (640 mg,1.3 mmol). The reaction mixture was stirred for two days, then concentrated and redissolved in DMF-piperidine mixture (10:1 v/v,1.1 mL). The reaction mixture was stirred for 1 hour and applied to a C18 column (H with 0.05% TFA) by reverse phase chromatography ₂ O/CH ₃ CN,90:10 to 20:80 v/v) purified as compound 173 (5 mg, 5. Mu. Mol, 10% yield). LRMS (ESI): m/z 1015.4[ M+H ]] ⁺ ，C ₅₁ H ₆₂ N ₆ O ₁₆ m/z calculated 1015.4. (2S, 3S,4S,5R, 6S) -6- (5- ((29S, 32S) -1-amino-29-isopropyl-32-methyl-27, 30-dioxo-3,6,9,12,15,18,21,24-octaoxa-28, 31-diazatridecan-33-amido) -2- ((((S) -11- (2- ((tert-butoxycarbonyl) (isopropyl) amino) ethyl) -4-ethyl-3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3 ')' ,4':6,7]Indolazino [1,2-b ]]Quinolin-4-yl) oxy) carbonyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (174)

DIPEA (5. Mu.L, 29. Mu. Mol) was added to a mixture of amine 174 (5 mg, 5. Mu. Mol) and PFP ester 125 (12 mg, 6. Mu. Mol) in DMF (0.5 mL) at room temperature. The reaction mixture was stirred for 1 hour, and then DMF (0.5 mL) and piperidine (50. Mu.L) were added to the mixture. After stirring at room temperature for 15 min, the partial reaction mixture was directly subjected to reverse phase prep HPLC (H with 0.05% TFA) using a C18 column ₂ O/CH ₃ CN,90:10 to 30:70 v/v). Fractions containing the desired compound were combined and lyophilized to give compound 174 (2 mg,1.4 μmol, 28% yield). LRMS (ESI): m/z 1438.7[ M+H ]] ⁺ ，C ₇₀ H ₉₉ N ₇ O ₂₅ m/z calculated 1438.7.

(2S, 3S,4S,5R, 6S) -6- (5- ((2S, 5S) -38- ((1- ((2S, 5S) -1- ((3- (((2S, 3R,4S,5S, 6S) -6-carboxy-3, 4, 5-trihydroxytetrahydro-2H-pyran-2-yl) oxy) -4- ((((S) -4-ethyl-11- (2- (isopropylamino) ethyl) -3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] Indolazino [1,2-b ] quinolin-4-yl) oxy) carbonyl) phenyl) -5-isopropyl-2-methyl-1,4,7,35-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazatridecan-37-yl) -2 (1, 2-dimethylhydrazino) methyl) -1H-indol-5-yl) amino) -5-isopropyl-2-methyl-4,7,35,38-tetraoxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazatriacontamido) -2- ((((S) -4-ethyl-11- (2- (isopropylamino) ethyl) -3, 14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3', preparation of 4':6,7] indolizino [1,2-b ] quinolin-4-yl) oxy) carbonyl) phenoxy) -3,4, 5-trihydroxy tetrahydro-2H-pyran-2-carboxylic acid (175)

To a stirred mixture of amine 174 (2 mg, 1.4. Mu. Mol) and bis-PFP-ester 40 (0.8 mg, 0.7. Mu. Mol) in DMF (1 mL) was added DIPEA (0.5. Mu.L, 2.8. Mu. Mol) at room temperature. After 2 hours, the reaction mixture was concentrated and redissolved in formic acid (1 mL) at room temperature. After 30 min, formic acid was removed in vacuo and the residue was redissolved in DMF (1 mL) and piperidine (50. Mu.L). After stirring at room temperature for 15 min, the reaction mixture was directly subjected to reverse phase HPLC (H with 0.05% TFA) using a C18 column ₂ O/CH ₃ CN,90:10 to 35:65 v/v). Fractions containing the desired compound were combined and lyophilized to give compound 175 (0.7 mg,0.2 μmol, 33% yield) as a yellow powder. LRMS (ESI): m/z 1509.2[ M+2H] ²⁺ ，C ₁₄₈ H ₂₀₂ N ₁₈ O ₄₉ m/z calculated 1508.7.

Example 2

Preparation of conjugates

Using HIPS ligation, all synthetic constructs containing camptothecins were conjugated to a set of aldehyde-labeled monoclonal antibodies (figure 2). Analytical characterization of the resulting conjugates is shown in figures 3 to 69 and figures 106 to 144.

Bioconjugation, purification and HPLC analysis

Aldehyde-labeled antibody (15 mg/mL) was conjugated to linker-payload (8 mol equivalent drug: antibody) at 37℃in 20mM sodium citrate, 50mM NaCl pH 5.5 (containing 0.85% DMA) for 72h. In some cases, additional DMA up to 10% vol/vol is added to improve the solubility of the linker-payload. After conjugation, free drug was removed by multiple rounds of dilution into 20mM sodium citrate, 50mM NaCl pH 5.5, and concentration using an Amicon 0.5mL 30kD MWCO centrifugal filter (Millipore Sigma #UFC5030 BK). To determine the DAR of the final product, the ADC was checked by analytical HIC or PLRP. HIC column (Tosoh # 14947) used 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. PLRP column (Agilent #PL 1912-1802) uses mobile phase A: H2O containing 0.1% trifluoroacetic acid and mobile phase B: the column was heated to 80 ℃ by running a CH3CN solution containing 0.1% trifluoroacetic acid. To determine aggregation, samples were analyzed using analytical size exclusion chromatography (SEC; tosoh # 08541) using a mobile phase of 300mM NaCl, 25mM sodium phosphate pH 6.8, 5% isopropyl alcohol.

The conjugation results of compounds 47, 61 and 65 with 10 different antibodies are shown in table 1 below. Table 1 shows the drug-to-antibody ratio (DAR) and the percent high molecular weight species (% HMW).

TABLE 1

* DAR, drug-to-antibody ratio

* HMW, high molecular weight species

Example 3

In vitro cytotoxicity assay

Cell lines were seeded into 96-well plates (Costar 3610) at a density of 5x 104 cells/well in 100 μl of growth medium. The following day, cells were treated with 20 μl of test compound serially diluted in culture. At 37℃and 5% CO ₂ After 5 days incubation under conditions, promega CellTiter was used according to manufacturer's recommendationsThe reagent measures survival. GI50 curves were calculated in GraphPad Prism, normalized to payload concentration. Cytotoxicity assays (% survival versus drug concentration (nM)) are shown in figures 70 to 80 and figures 89 to 105.

Example 4

Rat Pharmacokinetic (PK) studies

A single intravenous bolus of 0.9mg/kg of the test sample was administered to male Sprague-Dawley rats (3 per group). Plasma stabilized with K2EDTA was collected at 1, 8 and 24 hours and 2, 4, 6, 8, 10 and 14 days post-dose.

PK sample analysis

Total antibodies and total ADC concentrations were quantified by ELISA as in fig. 81 for total antibodies, conjugates were captured with anti-human IgG specific antibodies and detected with HRP conjugated anti-human Fc specific antibodies. For total ADC, the conjugate was captured with anti-human Fab specific antibody and detected with mouse anti-payload primary antibody followed by HRP conjugated anti-mouse IgG subclass 1 specific secondary antibody. Bound secondary antibodies were detected using Ultra TMB one-step ELISA substrate (Thermo Fisher). After quenching the reaction with sulfuric acid, the signal was read by obtaining absorbance at 450nm on a Molecular Devices Spectra Max M microplate reader equipped with SoftMax Pro software. Data were analyzed using GraphPad Prism and Microsoft Excel software.

The results of PK sample analysis are shown in figures 82 to 85. FIG. 82 shows a graph of concentration (μg/mL) versus days post-administration after administration of trastuzumab antibody at a dose of 0.9 mg/kg. Figure 83 shows a graph of concentration (μg/mL) versus days post-administration after use of a conventional HER2 topoisomerase inhibitor conjugated ADC with a protease cleavable linker at a dose of 0.9 mg/kg. FIG. 84 shows a plot of concentration (μg/mL) versus days post-administration after using CH 1-3/CT-labeled trastuzumab conjugated to construct 61 at a dose of 0.9 mg/kg. FIG. 85 shows a plot of concentration (μg/mL) versus days post-administration after using CH 1-3/CT-labeled trastuzumab conjugated to construct 65 at a dose of 0.9 mg/kg.

Example 5

Xenograft study

The method comprises the following steps:

NCI-H292 xenograft: female SCID Beige mice (7 or 8/group) were inoculated subcutaneously with 500 ten thousand NCI-H292 cells in PBS. When the average tumor reaches 121mm ³ Administration was started at (day 1). Intravenous administration of vehicle alone, trodel vy, DS-1062 or conjugate 3485 (a TROP-2-targeting ADC comprising two tag sites conjugated to compound 65 (DAR with 6.85)) to animals for study 1, ADC was administered at 10mg/kg on day 0, day 7 and day 21 (Trodel vy) or at 6mg/kg on day 0 and day 21 (DS-1062 and conjugate 3485.) intravenous administration of vehicle alone, DS-1062 or conjugate 3485, 3789 or 3790 (TROP-2-targeting ADC comprising two tag sites conjugated to compound 65, 127 or 131, respectively) to animals was monitored twice weekly for body weight and tumor size when tumors reached 2000mm ³ Or weight loss of more than 15%, animals were euthanized.

Results:

the results of study 1 are shown in FIG. 86, which shows the average tumor volume (mm ³ ) Graph of relationship to days and indicates in vivo targeting TROP-2 ADC carrying topoisomerase inhibitor payload against NCI-H292 xenograftsEfficacy. n=8 mice/group; administration is indicated by arrows.

The results of study 2 are shown in FIG. 87, which shows the average tumor volume (mm ³ ) Graph of days and indicates in vivo efficacy of TROP-2 targeted ADC carrying a topoisomerase inhibitor payload against NCI-H292 xenografts. n=7 mice/group. A single intravenous dose was delivered on day 0.

The method comprises the following steps:

NCI-H1781 xenograft: female BALB/c nude mice (5/group) were inoculated subcutaneously with 2000 ten thousand NCI-H1781 cells in PBS. When the average tumor size reaches 222mm ³ Administration was started at (day 1). Animals were given intravenous vehicle alone, a connexin-4 compound 65 conjugate with a DAR of 6.8, or a connexin-4 mc-GGFG-Dxd conjugate with a DAR of 3.7. On days 0 and 7, the ADC was dosed intravenously at 5 mg/kg. Body weight and tumor size were monitored twice weekly. When the tumor reaches 2000mm ³ Or weight loss of more than 15%, animals were euthanized.

Results:

the results of the NCI-H1781 study are shown in FIG. 88, where mean tumor volume (mm ³ ) Graph of days and indicates in vivo efficacy of topoisomerase inhibitor payload-carrying, connexin-4 targeted ADC against NCI-H1781 xenografts. n=5 mice/group. Doses of 5mg/kg were delivered intravenously on day 0 and day 7.

Although the invention has been described with reference to specific embodiments, 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 fall within the scope of the appended claims.

Claims

1. A conjugate of formula (I):

wherein:

z is CR ¹⁰ Or N, or a combination of two,

w is a polypeptide;

wherein at least one R ¹⁰ Optionally to a second compound of formula (II).

2. The conjugate according to claim 1,

wherein the compound of formula (II) has the structure of formula (IIa):

Wherein the compound of formula (II) has the structure of formula (IIc):

Wherein the compound of formula (II) has the structure of formula (IId):

wherein R is ^2a And R is ^2b Each independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroarylA group, cycloalkyl group, substituted cycloalkyl group, heterocyclic group, substituted heterocyclic group, carboxyl ester, acyl group and sulfonyl group, and L is attached to R ⁶ A place; or L is attached to R ^2a At and R is ^2b And R is ⁶ OH; or (b)

Wherein the compound of formula (II) has the structure of formula (IIe):

3. The conjugate according to any one of claims 1 to 2, wherein L comprises:

wherein the method comprises the steps of

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

T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ and T ⁶ 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), m-amino-benzyloxy (MABO), m-amino-benzyloxycarbonyl (MABC), p-amino-benzyloxy (PABO), p-amino-benzyloxyCarbonyl (PABC), para-aminobenzyl (PAB), para-amino-benzylamino (PABA), para-amino-phenyl (PAP), para-hydroxy-phenyl (PHP), 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;

4. A conjugate according to claim 3, wherein:

wherein:

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

4-amino-piperidine (4 AP) asAnd is also provided with

5. The conjugate of any one of claims 3 to 4, wherein T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ And T ⁶ Each optionally substituted with a glycoside.

6. The conjugate of any one of claims 3 to 4, wherein each of MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP is optionally substituted with a glycoside.

7. The conjugate of any one of claims 5 to 6, wherein the glycoside is selected from the group consisting of glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc and O-GalNAc.

8. The conjugate according to any one of claim 3 to 7,

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of; and is also provided with

T ⁶ Is EDA and V ⁶ is-CO-; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁶ Is absent and V ⁶ is-CO-; or (b)

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-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ is-NR ¹⁵ -; and is also provided with

T ⁶ Is (C) ₁ -C ₁₂ ) Alkyl and V ⁶ is-CO-; or wherein:

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

T ² is AA and V ² Absence of;

T ³ is PABC and V ³ Absence of;

T ⁴ is EDA and V ⁴ is-CO-; and is also provided with

e and f are each 0; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of; and is also provided with

f is 0; or (b)

Wherein:

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

T ² is AA and V ² Absence of;

T ³ is PABC and V ³ Absence of; and is also provided with

d. e and f are each 0; or (b)

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 (P)EG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABA and V ⁵ is-CO-; and is also provided with

T ⁶ Is (C) ₁ -C ₁₂ ) Alkyl and V ⁶ is-SO ₂ -; or wherein:

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

T ² for (PEG) _n And V is ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

e and f are each 0; or (b)

Wherein:

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

T ² is substituted (C) ₁ -C ₁₂ ) Alkyl and V ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

e and f are each 0; or (b)

Wherein:

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

T ² for (PEG) _n And V is ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

T ⁵ is (C) ₁ -C ₁₂ ) Alkyl and V ⁵ Absence of;

f is 0; or (b)

Wherein:

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

T ² 4AP and V ² is-CO-;

T ³ is (C) ₁ -C ₁₂ ) Alkyl and V ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of;

f is 0; or (b)

Wherein:

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

T ² 4AP and V ² is-CO-;

T ³ is (C) ₁ -C ₁₂ ) Alkyl and V ³ is-O-;

T ⁴ is (C) ₁ -C ₁₂ ) Alkyl and V ⁴ is-CO-;

T ⁵ is AA and V ⁵ Absence of;

T ⁶ is PABC and V ⁶ Absence of; or (b)

Wherein:

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

T ² is an amino acid analog and V ² Absence of;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

e and f are each 0; or (b)

Wherein:

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

T ² for (PEG) _n And V is ² is-CONH-;

T ³ is substituted (C) ₁ -C ₁₂ ) Alkyl and V ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC andand V is ⁵ Absence of;

f is 0; or (b)

Wherein:

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

T ² is AA and V ² is-NH-;

T ³ for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of;

f is 0; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABO and V ⁵ Absence of; and is also provided with

f is 0; or (b)

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 For (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PAP and V ⁵ is-COO-; and is also provided with

f is 0; or (b)

Wherein:

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

T ² for (PEG) _n And V is ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PAP and V ⁴ is-COO-; and is also provided with

e and f are each 0.

9. The conjugate according to any one of claims 1 to 8, wherein one R ¹⁰ Via a second joint L ^B To a compound of the second formula fII).

10. The conjugate of claim 9, wherein L ^B Comprising:

-(T ⁷ -V ⁷ ) _g -(T ⁸ -V ⁸ ) _h -(T ⁹ -V ⁹ ) _i -(T ¹⁰ -V ¹⁰ ) _j -(T ¹¹ -V ¹¹ ) _k -(T ¹² -V ¹² ) ₁ -，

wherein the method comprises the steps of

g. h, i, j, k and 1 are each independently 0 or 1;

V ⁸ 、V ⁸ 、V ⁹ 、V ¹⁰ 、V ¹¹ And V ¹² 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;

11. The conjugate of claim 10, wherein T ⁷ 、T ⁸ 、T ⁹ 、T ¹⁰ 、T ¹¹ And T ¹² Each optionally substituted with a glycoside.

12. The conjugate of claim 10, wherein MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP are each optionally substituted with a glycoside.

13. The conjugate of any one of claims 11 to 12, wherein the glycoside is selected from the group consisting of glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc and O-GalNAc.

14. The conjugate according to any one of claim 10 to 13,

wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

T ⁸ is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

T ¹¹ is EDA and V ¹¹ is-CO-; and is also provided with

1 is 0; or (b)

Wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

T ⁸ is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of; and is also provided with

k and l are each 0; or (b)

Wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

T ⁸ is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is an amino acid analog and V ⁹ is-NH-;

T ¹⁰ for (PEG) _n And V is ¹⁰ is-CO-;

T ¹¹ is AA and V ¹¹ Absence of; and is also provided with

T ¹² Is PABC and V ¹² Absence of; or (b)

Wherein:

T ⁸ is absent and V ⁷ is-NHCO-;

T ⁸ is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CONH-;

T ⁹ for (PEG) _n And V is ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of; and is also provided with

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ is substituted (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

k and l are each 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ for (PEG) _n And V is ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

T ¹¹ is (C) ₁ -C ₁₂ ) Alkyl and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ 4AP and V ⁸ is-CO-;

T ⁹ is (C) ₁ -C ₁₂ ) Alkyl and V ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ 4AP and V ⁸ is-CO-;

T ⁹ is (C) ₁ -C ₁₂ ) Alkyl and V ⁹ is-O-;

T ¹⁰ is (C) ₁ -C ₁₂ ) Alkyl radical andand V is ¹⁰ is-CO-;

T ¹¹ is AA and V ¹¹ Absence of;

T ¹² is PABC and V ¹² Absence of; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ is an amino acid analog and V ⁸ Absence of;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

k and l are each 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ for (PEG) _n And V is ⁸ is-CONH-;

T ⁹ is substituted (C) ₁ -C ₁₂ ) Alkyl and V ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ is AA and V ⁸ is-NH-;

T ⁹ for (PEG) _n And V is ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ is (PE)G) _n And V is ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PAP and V ¹⁰ is-COO-; and is also provided with

k and l are each 0.

15. The conjugate of any one of claims 1 to 14, wherein the conjugate is selected from the group consisting of:

/>

16. a compound of formula (III):

Wherein:

z is CR ¹⁰ Or N, or a combination of two,

L is R ¹ 、R ² 、R ³ 、R ⁴ 、R ⁵ Or R is ⁶ A linker attached to a compound of formula fhi):

R ⁵ selected from the group consisting of hydrogen, halogen, hydroxy, amino, substituted amino, alkyl, substituted alkyl, alkenyl,Substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl and substituted heterocyclyl;

wherein at least one R ¹⁰ Optionally to a second compound of formula (II).

17. A compound according to claim 16,

wherein the compound of formula (II) has the structure of formula (IIa):

Wherein the compound of formula (II) has the structure of formula (IIc):

Wherein the compound of formula (II) has the structure of formula (IId):

wherein R is ^2a And R is ^2b Each independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, carboxyl ester, acyl, and sulfonyl, and L is attached to R ⁶ A place; or L is attached to R ^2a At and R is ^2b And R is ⁶ OH; or (b)

Wherein the compound of formula (II) has the structure of formula (IIe):

18. The compound according to any one of claims 16 to 17, wherein L comprises:

wherein the method comprises the steps of

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

Each R is ¹⁵ Independently selected from 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.

19. A compound according to claim 18, wherein:

wherein:

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

4-amino-piperidine (4 AP) asAnd is also provided with

20. The compound according to any one of claims 18 to 19, wherein T ¹ 、T ² 、T ³ 、T ⁴ 、T ⁵ And T ⁶ Each optionally substituted with a glycoside.

21. The compound of any one of claims 18 to 19, wherein MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP are each optionally substituted with a glycoside.

22. The compound of any one of claims 20 to 21, wherein the glycoside is selected from the group consisting of glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

23. A compound according to any one of claims 18 to 22, 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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of; and is also provided with

T ⁶ Is EDA and V ⁶ is-CO-; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁶ Is absent and V ⁶ is-CO-; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ is-NR ¹⁵ -; and is also provided with

T ⁶ Is (C) ₁ -C ₁₂ ) Alkyl and V ⁶ is-CO-; or (b)

Wherein:

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

T ² is AA and V ² Absence of;

T ³ is PABC and V ³ Absence of;

T ⁴ is EDA and V ⁴ is-CO-; and is also provided with

e and f are each 0; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of; and is also provided with

f is 0; or (b)

Wherein:

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

T ² is AA and V ² Absence of;

T ³ is PABC and V ³ Absence of; and is also provided with

d. e and f are each 0; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABA and V ⁵ is-CO-; and is also provided with

T ⁶ Is (C) ₁ -C ₁₂ ) Alkyl and V ⁶ is-SO ₂ -; or (b)

Wherein:

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

T ² for (PEG) _n And V is ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

e and f are each 0; or (b)

Wherein:

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

T ² is substituted fC ₁ -C ₁₂ ) Alkyl and V ² is-CO-; t (T) ³ Is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

e and f are each 0; or (b)

Wherein:

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

T ² for (PEG) _n And V is ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and y ⁴ Absence of;

T ⁵ is (C) ₁ -C ₁₂ ) Alkyl and V ⁵ Absence of;

f is 0; or (b)

Wherein:

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

T ² 4AP and V ² is-CO-;

T ³ Is (C) ₁ -C ₁₂ ) Alkyl and V ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of;

f is 0; or (b)

Wherein:

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

T ² 4AP and V ² is-CO-;

T ³ is (C) ₁ -C ₁₂ ) Alkyl and V ³ is-O-;

T ⁴ is (C) ₁ -C ₁₂ ) Alkyl and V ⁴ is-CO-;

T ⁵ is AA and V ⁵ Absence of;

T ⁶ is PABC and V ⁶ Absence of; or (b)

Wherein:

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

T ² is an amino acid analog and V ² Absence of;

T ³ is AA and V ³ Absence of;

T ⁴ is PABC and V ⁴ Absence of;

_e and f are each 0; or (b)

Wherein:

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

T ² for (PEG) _n And V is ² is-CONH-;

T ³ is substituted (C) ₁ -C ₁₂ ) Alkyl and V ³ is-CO-; t (T) ⁴ Is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of;

f is 0; or (b)

Wherein:

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

T ² is AA and V ² is-NH-;

T ³ for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABC and V ⁵ Absence of;

f is 0; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PABO and V ⁵ Absence of; and is also provided with

f is 0; or (b)

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 for (PEG) _n And V is ³ is-CO-;

T ⁴ is AA and V ⁴ Absence of;

T ⁵ is PAP and V ⁵ is-COO-; and is also provided with

f is 0; or (b)

Wherein:

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

T ² for (PEG) _n And V is ² is-CO-;

T ³ is AA and V ³ Absence of;

T ⁴ is PAP and V ⁴ is-COO-; and is also provided with

e and f are each 0.

24. A compound according to any one of claims 16 to 23, wherein one R ¹⁰ Via a second joint L ^B Is linked to a second compound of formula (II).

25. The compound of claim 24, wherein L ^B Comprising:

wherein the method comprises the steps of

g. h, i, j, k and 1 are each independently 0 or 1;

26. The compound of claim 25, wherein T ⁷ 、T ⁸ 、T ⁹ 、T ¹⁰ 、T ¹¹ And T ¹² Each optionally substituted with a glycoside.

27. The compound of claim 25, wherein MABO, MABC, PABO, PABC, PAB, PABA, PAP and PHP are each optionally substituted with a glycoside.

28. The compound of any one of claims 26 to 27, wherein the glycoside is selected from the group consisting of glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

29. The compound according to any one of claim 25 to 28,

wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

T ⁸ is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

T ¹¹ is EDA and V ¹¹ is-CO-; and is also provided with

1 is 0; or (b)

Wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

T ⁸ is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of; and is also provided with

k and l are each 0; or (b)

Wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

T ⁸ is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is an amino acid analog and V ⁹ is-NH-;

T ¹⁰ for (PEG) _n And V is ¹⁰ is-CO-;

T ¹¹ is AA and V ¹¹ Absence of; and is also provided with

T ¹² Is PABC and V ¹² Absence of; or (b)

Wherein:

T ⁷ is absent and V ⁷ is-NHCO-;

T ⁸ is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CONH-;

T ⁹ for (PEG) _n And V is ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of; and is also provided with

1 is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ is substituted (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

k and 1 are each 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ for (PEG) _n And V is ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

T ¹¹ is (C) ₁ -C ₁₂ ) Alkyl and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ 4AP and V ⁸ is-CO-;

T ⁹ is (C) ₁ -C ₁₂ ) Alkyl and V ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ 4AP and V ⁸ is-CO-;

T ⁹ is (C) ₁ -C ₁₂ ) Alkyl and V ⁹ is-O-;

T ¹⁰ is (C) ₁ -C ₁₂ ) Alkyl and V ¹⁰ is-CO-;

T ¹¹ is AA and V ¹¹ Absence of;

T ¹² is PABC and V ¹² Absence of; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ is an amino acid analog and V ⁸ Absence of;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PABC and V ¹⁰ Absence of;

k and l are each 0; or (b)

Wherein:

T ⁸ is (C) ₁ -C ₁₂ ) Alkyl and V ⁸ is-CONH-;

T ⁸ for (PEG) _n And V is ⁸ is-CONH-;

T ⁹ is substituted (C) ₁ -C ₁₂ ) Alkyl and V ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CO-;

T ⁸ is AA and V ⁸ is-NH-;

T ⁹ for (PEG) _n And V is ⁹ is-CO-;

T ¹⁰ is AA and V ¹⁰ Absence of;

T ¹¹ is PABC and V ¹¹ Absence of;

l is 0; or (b)

Wherein:

T ⁷ is (C) ₁ -C ₁₂ ) Alkyl and V ⁷ is-CONH-;

T ⁸ for (PEG) _n And V is ⁸ is-CO-;

T ⁹ is AA and V ⁹ Absence of;

T ¹⁰ is PAP and V ¹⁰ is-COO-; and is also provided with

k and l are each 0.

30. A compound according to any one of claims 16 to 29, wherein the compound is selected from:

/>

31. a pharmaceutical composition comprising:

The conjugate of any one of claims 1 to 15; and

pharmaceutically acceptable excipients.

32. A method, comprising:

administering to a subject an effective amount of the conjugate of any one of claims 1 to 15.

33. A method of treating cancer in a subject, comprising:

administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising the conjugate of any one of claims 1 to 15, wherein the administration is effective to treat cancer in the subject.