Tandem cleavage linker and its use for antibody-drug conjugates

Abstract

This disclosure provides a cleavable linker for antibody-drug conjugates (ADCs), comprising a tandem cleavage portion. This disclosure also provides an ADC comprising a cleavable linker having a tandem cleavage portion. In addition, this disclosure encompasses compounds and methods for producing such ADCs, as well as methods for using the ADCs.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of the earlier filing dates of U.S. Provisional Application No. 63 / 538,972, filed on 18 September 2023, U.S. Provisional Application No. 63 / 544,310, filed on 16 October 2023, and U.S. Provisional Application No. 63 / 605,310, filed on 1 December 2023, the disclosures of each of these are incorporated herein by reference.

[0002] Introduction Antibody-drug conjugates (ADCs) are emerging as a promising therapeutic format that combines the highly specific affinity of an antibody (targeting element) for a cell surface antigen with the potency of a small molecule drug (payload). The latter is attached to a protein via various chemical linkers using one of the available conjugation methods. While the concept of an ADC can be represented in a simple diagram (Figure 1), the details of ADC composition and how it relates to function (including efficacy and tolerability) are far more complex. Furthermore, practical concerns, including both the translatability from preclinical to clinical outcomes and the manufacturability / stability of the compounds, influence which concepts can be successfully incorporated into therapeutics. Indeed, the field's understanding of the best ways to construct, generate, and use ADCs is still evolving. [Overview of the project]

[0003] This disclosure provides a cleavable linker for antibody-drug conjugates (ADCs) in which the linker includes a tandem cleavage portion. In addition, this disclosure also encompasses compounds and methods for producing such ADCs, as well as methods for using ADCs.

[0004] A part of this disclosure is a conjugate of formula (I), [ka] During the ceremony, Each R 1 is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R 2 is independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, k1 is an integer of 1 to 10, R 3 comprises a cleavable moiety, L A is a first linker, W 1 is a drug, W 2 is an antibody, X is a conjugation moiety that binds to the antibody at a natural amino acid residue of the antibody, and the conjugate is included.

[0005] In some embodiments, R 1 is H, and R 2 is alkyl or substituted alkyl.

[0006] In some embodiments, k1 is 2.

[0007] In some embodiments, R 3 is a chemically cleavable moiety.

[0008] In some embodiments, R 3 is an enzymatically cleavable moiety.

[0009] In some embodiments, R 3 is a glycoside or a glycoside derivative.

[0010] In some embodiments, the glycoside or glycoside derivative is selected from glucuronides, galactosides, glucosides, mannosides, fucosides, O-GlcNAc, and O-GalNAc.

[0011] In some embodiments, X includes a conjugation moiety configured to bind to a cysteine ​​residue or a lysine residue of the antibody.

[0012] In some embodiments, X comprises maleimide, and the native amino acid residue of the antibody comprises a cysteine ​​residue.

[0013] In some embodiments, the conjugate is the conjugate of formula (II), [ka] During the ceremony, Each R 1 These are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl. Each R 2 These are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k1 is an integer between 1 and 10. R 3 This includes a severable portion, L A This is the first linker, W 1 It is a drug, W 2 It is an antibody.

[0014] In a particular embodiment of formula (I), the conjugate is the conjugate of formula (IIa), [ka] During the ceremony, Each R 1 These are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl. Each R 2 These are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k1 is an integer between 1 and 10. R 3 This includes a severable portion, L A This is the first linker, W 1 It is a drug, W 2 It is an antibody.

[0015] In some embodiments, L A teeth, -(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 - Includes, During the ceremony, a, b, c, d, e, and f are each independently either 0 or 1. T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 Each is independent, and covalently bonded, (C1~C 12 ) alkyl, substituted (C1~C12 ) alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl, (EDA) w (PEG) n , (AA) p ,-(CR 13 OH) m - Selected from 4-amino-piperidine (4AP), acetal group, hydrazine, disulfide, and ester, EDA is the ethylenediamine moiety, PEG is polyethylene glycol, AA is an amino acid residue or amino acid analog, 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 These are, independently, covalent, -CO-, and -NR bonds. 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 Selected from the group consisting of SO2- and -P(O)OH-, where each q is an integer from 1 to 6. Each R 13 These are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. Each R 15 These are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0016] In some embodiments, L A comprises: T 1 is selected from (C1-C 12 )alkyl and substituted (C1-C 12 )alkyl, T 2 , T 3 , T 4 , T 5 , and T 6 are each independently selected from a covalent bond, (C1-C 12 )alkyl, substituted (C1-C 12 )alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, (EDA) w , (PEG) n , (AA) p , -(CR 13 OH) m -, 4-amino-piperidine (4AP), acetal group, hydrazine, and ester, V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 are each independently selected from the group consisting of a covalent bond, -CO-, -NR 15 -, -NR 15 (CH₂) q -, -NR 15 (C₆H₄)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO₂-, -SO₂NR 15 -, -NR 15 SO₂-, and -P(O)OH-, (PEG) n is

Chemical Formula

Chemical Formula

[0017] In some embodiments, the conjugate includes: T 1 (C1~C 12 ) is alkyl, V 1 It is -CO-, b, c, d, e, and f are each 0 or T 1 (C1~C 12 ) is alkyl, V 1 It is -CO-, T 2 It is an amino acid analog, V 2 It is -NH-, T 3 (PEG) n V 3 It is -CO-, d, e, and f are each 0, or T 1 (C1~C 12 ) is alkyl, V 1 is -CONH-, T 2 This is a substitution (C1~C 12 ) is alkyl, V 2 It is -CO-, c, d, e, and f are all 0.

[0018] In some embodiments, T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , V 1 , V 2 , V 3 , V 4 , V 5 , or V 6 One of them is a branching element.

[0019] In some embodiments, the branching group is -CONR 15 - and selected from 4AP.

[0020] In some embodiments, the branching base is a second linker L B It is connected.

[0021] In some embodiments, the second linker L B teeth, -(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 - Includes, During the ceremony, g, h, i, j, k, and l are each independently either 0 or 1. T 7 , T 8 , T 9 , T 10 , T 11 , and T 12 Each is independent, and covalently bonded, (C1~C 12 ) alkyl, substituted (C1~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 - Selected from 4-amino-piperidine (4AP), acetal group, hydrazine, disulfide, and ester, EDA is the ethylenediamine moiety, PEG is polyethylene glycol, AA is an amino acid residue or amino acid analog, 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 These are, independently, covalent, -CO-, and -NR bonds. 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 Selected from the group consisting of SO2- and -P(O)OH-, where each q is an integer from 1 to 6. Each R 13 These are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. Each R 15 These are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0022] In some embodiments, LB It is bonded to the compound of formula (III), [ka] During the ceremony, Each R 4 These are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl. Each R 5 These are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k2 is an integer between 1 and 10. R 6 This includes a severable portion, W 1a It is a drug.

[0023] In some embodiments, R 4 H is R 5 It is an alkyl or substituted alkyl group.

[0024] In some embodiments, k2 is 2.

[0025] In some embodiments, R 6 This is a part that can be chemically cleaved.

[0026] In some embodiments, R 6 This is the portion that can be enzymatically cleaved.

[0027] In some embodiments, R 6 It is a glycoside or a glycoside derivative.

[0028] In some embodiments, the glycoside or glycoside derivative is selected from glucuronides, galactosides, glucosides, mannosides, fucosides, O-GlcNAc, and O-GalNAc.

[0029] In some embodiments, the conjugate includes: T 1 (C1~C 12 ) is alkyl, V 1 is a branching group (-CONR 15 )- and, T 2 (C1~C 12 ) is alkyl, V 2 is -CONH-, T 3 This is a substitution (C1~C 12 ) is alkyl, V 3 It is -CO-, d, e, and f are each 0, T 7 (C1~C 12 ) is alkyl, V 7 is -CONH-, T 8 This is a substitution (C1~C 12 ) is alkyl, V 8 It is -CO-, i, j, k, and l are each 0 or T 1 (C1~C 12 ) is alkyl, V 1 It is -CO-, T 2 It is a branching group (4AP), and V 2 It is -CO-, T 3 (PEG) n V 3 is -CONH-, T 4 This is a substitution (C1~C 12 ) is alkyl, V 4 It is -CO-, e and f are both 0, T 7 (PEG) n V 7 is -CONH-, T 8 This is a substitution (C1~C 12) is alkyl, V 8 It is -CO-, i, j, k, and l are each 0 or T 1 (C1~C 12 ) is alkyl, V 1 It is -CO-, T 2 It is a branching group (4AP), and V 2 It is -CO-, T 3 (C1~C 12 ) is alkyl, V 3 It does not exist, T 4 It is a heteroaryl (triazole), and V 4 It does not exist, T 5 (C1~C 12 ) is alkyl, V 5 is -CONH-, T 6 This is a substitution (C1~C 12 ) is alkyl, V 6 It is -CO-, T 7 (PEG) n V 7 is -CONH-, T 8 This is a substitution (C1~C 12 ) is alkyl, V 8 It is -CO-, i, j, k, and l are all 0.

[0030] In some embodiments, W 1 and W 1a These are the same drug.

[0031] In some embodiments, W 1 and W 1a These are different drugs.

[0032] In some embodiments, the conjugate is selected from the following: [ka] [ka] [ka]

[0033] Aspects of this disclosure are compounds of formula (IV), [ka] During the ceremony, Each R 1 These are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl. Each R 2 These are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k1 is an integer between 1 and 10. R 3 This includes a severable portion, L A This is the first linker, W 1 It is a drug, X comprises a compound that is a conjugation moiety configured to bind to the antibody at a native amino acid residue of the antibody.

[0034] In some embodiments, R 1 H is R 2 It is an alkyl or substituted alkyl group.

[0035] In some embodiments, k1 is 2.

[0036] In some embodiments, R 3 This is a part that can be chemically cleaved.

[0037] In some embodiments, R 3 This is the portion that can be enzymatically cleaved.

[0038] In some embodiments, R 3 It is a glycoside or a glycoside derivative.

[0039] In some embodiments, the glycoside or glycoside derivative is selected from glucuronides, galactosides, glucosides, mannosides, fucosides, O-GlcNAc, and O-GalNAc.

[0040] In some embodiments, X includes a conjugation moiety configured to bind to a cysteine ​​residue or a lysine residue of the antibody.

[0041] In some embodiments, X comprises maleimide, and the native amino acid residue of the antibody comprises a cysteine ​​residue.

[0042] In some embodiments, the compound is a compound of formula (V), [ka] During the ceremony, Each R 1 These are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl. Each R 2 These are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k1 is an integer between 1 and 10. R 3 This includes a severable portion, L A This is the first linker, W 1 It is a drug.

[0043] In some embodiments of the compound described in formula (IV), the compound is the compound of formula (Va), [ka] During the ceremony, Each R 1 These are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl. Each R 2 These are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k1 is an integer between 1 and 10. R 3 This includes a severable portion, L A This is the first linker, W 1 It is a drug.

[0044] In some embodiments, L A teeth, -(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 - Includes, During the ceremony, a, b, c, d, e, and f are each independently either 0 or 1. T 1 , T 2 , T 3 , T4 , T 5 , and T 6 Each is independent, and covalently bonded, (C1~C 12 ) alkyl, substituted (C1~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 - Selected from 4-amino-piperidine (4AP), acetal group, hydrazine, disulfide, and ester, EDA is the ethylenediamine moiety, PEG is polyethylene glycol, AA is an amino acid residue or amino acid analog, 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 These are, independently, covalent, -CO-, and -NR bonds. 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 Selected from the group consisting of SO2- and -P(O)OH-, where each q is an integer from 1 to 6. Each R 13 These are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. Each R 15These are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0045] In some embodiments, L A This includes: T 1 (C1~C 12 ) alkyl and substituted (C1~C 12 ) Selected from alkyl groups, T 2 , T 3 , T 4 , T 5 , and T 6 Each is independent, and covalently bonded, (C1~C 12 ) alkyl, substituted (C1~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 - Selected from 4-amino-piperidine (4AP), acetal group, hydrazine, and ester, V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 These are, independently, covalent, -CO-, and -NR bonds. 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15Selected from the group consisting of SO2- and -P(O)OH-, (PEG) n teeth, [ka] In the formula, n is an integer between 1 and 30. The EDA has the following structure: [ka] The ethylenediamine moiety has the following characteristics, where y is an integer from 1 to 6, and r is 0 or 1. 4-amino-piperidine (4AP) is, [ka] And, Each R 12 These are independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, and any two adjacent R 12 The groups can be linked in a cyclic manner to form a piperazinyl ring.

[0046] In some embodiments, the compound includes: T 1 (C1~C 12 ) is alkyl, V 1 It is -CO-, b, c, d, e, and f are each 0 or T 1 (C1~C 12 ) is alkyl, V 1 It is -CO-, T 2 It is an amino acid analog, V 2 It is -NH-, T 3 (PEG) n V 3 It is -CO-, d, e, and f are each 0, or T 1 (C1~C12 ) is alkyl, V 1 is -CONH-, T 2 This is a substitution (C1~C 12 ) is alkyl, V 2 It is -CO-, c, d, e, and f are all 0.

[0047] In some embodiments, T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , V 1 , V 2 , V 3 , V 4 , V 5 , or V 6 One of them is a branching element.

[0048] In some embodiments, the branching group is -CONR 15 - and selected from 4AP.

[0049] In some embodiments, the branching base is a second linker L B Includes.

[0050] In some embodiments, the second linker L B teeth, -(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 - Includes, During the ceremony, g, h, i, j, k, and l are each independently either 0 or 1. T 7 , T 8 , T 9 , T 10 , T 11 , and T 12 Each is independent, and covalently bonded, (C1~C 12 ) alkyl, substituted (C1~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 - Selected from 4-amino-piperidine (4AP), acetal group, hydrazine, disulfide, and ester, EDA is the ethylenediamine moiety, PEG is polyethylene glycol, AA is an amino acid residue or amino acid analog, 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 These are, independently, covalent, -CO-, and -NR bonds. 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 Selected from the group consisting of SO2- and -P(O)OH-, where each q is an integer from 1 to 6. Each R 13 These are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. Each R 15These are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0051] In some embodiments, L B It is bonded to the compound of formula (VI), [ka] During the ceremony, Each R 4 These are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl. Each R 5 These are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k2 is an integer between 1 and 10. R 6 This includes a severable portion, W 1a It is a drug.

[0052] In some embodiments, R 4 H is R 5 It is an alkyl or substituted alkyl group.

[0053] In some embodiments, k2 is 2.

[0054] In some embodiments, R 6 This is a part that can be chemically cleaved.

[0055] In some embodiments, R 6 This is the portion that can be enzymatically cleaved.

[0056] In some embodiments, R 6 It is a glycoside or a glycoside derivative.

[0057] In some embodiments, the glycoside or glycoside derivative is selected from glucuronides, galactosides, glucosides, mannosides, fucosides, O-GlcNAc, and O-GalNAc.

[0058] In some embodiments, the compound includes: T 1 (C1~C 12 ) is alkyl, V 1 is a branching group (-CONR 15 )- and, T 2 (C1~C 12 ) is alkyl, V 2 is -CONH-, T 3 This is a substitution (C1~C 12 ) is alkyl, V 3 It is -CO-, d, e, and f are each 0, T 7 (C1~C 12 ) is alkyl, V 7 is -CONH-, T 8 This is a substitution (C1~C 12 ) is alkyl, V 8 It is -CO-, i, j, k, and l are each 0 or T 1 (C1~C 12 ) is alkyl, V 1 It is -CO-, T 2 It is a branching group (4AP), and V 2 It is -CO-, T 3 (PEG) n V 3 is -CONH-, T 4 This is a substitution (C1~C12 ) is alkyl, V 4 It is -CO-, e and f are both 0, T 7 (PEG) n V 7 is -CONH-, T 8 This is a substitution (C1~C 12 ) is alkyl, V 8 It is -CO-, i, j, k, and l are each 0 or T 1 (C1~C 12 ) is alkyl, V 1 It is -CO-, T 2 It is a branching group (4AP), and V 2 It is -CO-, T 3 (C1~C 12 ) is alkyl, V 3 It does not exist, T 4 It is a heteroaryl (triazole), and V 4 It does not exist, T 5 (C1~C 12 ) is alkyl, V 5 is -CONH-, T 6 This is a substitution (C1~C 12 ) is alkyl, V 6 It is -CO-, T 7 (PEG) n V 7 is -CONH-, T 8 This is a substitution (C1~C 12 ) is alkyl, V 8 It is -CO-, i, j, k, and l are all 0.

[0059] In some embodiments, W 1 and W 1aThese are the same drug.

[0060] In some embodiments, W 1 and W 1a These are different drugs.

[0061] In some embodiments, the conjugate is selected from the following: [ka] [ka] [ka]

[0062] Aspects of this disclosure include a pharmaceutical composition comprising the conjugate provided for in this disclosure and a pharmaceutically acceptable excipient.

[0063] A part of this disclosure includes a method that involves administering the conjugate provided for this disclosure to a subject.

[0064] Aspects of the present disclosure include a method for treating cancer in a subject, wherein the subject is administered a therapeutically effective amount of a pharmaceutical composition comprising the conjugate of the present disclosure, the administration being effective in treating cancer in the subject. [Brief explanation of the drawing]

[0065] [Figure 1] A diagram illustrates how ADCs contain antibodies conjugated to a linker and payload via a specific conjugation approach. The chemistry involved in conjugation can be diverse and contribute to the biophysical and functional properties of the molecule. [Figure 2] A schematic diagram illustrates how NHS esters and maleimide conjugation chemistry react with naturally occurring nucleophilic amino acid residues in proteins. [Figure 3]A schematic diagram illustrating a maleimide conjugate that is reversible in plasma and can transfer the linker-payload to other thiol-containing molecules such as albumin is shown. [Figure 4] This diagram illustrates how ADC payloads released early in circulation drive systemic toxicity. [Figure 5] The diagram illustrates how a tandem cleavage linker requires two consecutive enzymatic steps to occur for drug release after ADC internalization. [Figure 6] This diagram illustrates a tandem cleavage linker that prevents the release of a circulating cytotoxic payload in the case of retro-Michael deconjugation of a maleimide-thioether bond. [Figure 7-1] This example demonstrates a branched maleimide linker design that allows for the conjugation of two or more payload molecules in a single step. [Figure 7-2] This example demonstrates a branched maleimide linker design that allows for the conjugation of two or more payload molecules in a single step. [Figure 8] This demonstrates an orthogonal substitution branch linker that allows conjugation of two different payloads in a single step (showing MMAE / Belotecan conjugation). [Figure 9] The HIC evaluation of the trastuzumab antibody conjugated to compound 38 is shown. [Figure 10] The HIC evaluation of the trastuzumab antibody conjugated to compound 14 is shown. [Figure 11] The HIC evaluation of the trastuzumab antibody conjugated to compound 18 is shown. [Figure 12] This shows the HIC evaluation of a trastuzumab antibody with a CH1 / CT tag conjugated to compound 24. [Figure 13] The SEC evaluation of the trastuzumab antibody conjugated to compound 38 is shown. [Figure 14] When the trastuzumab antibody conjugated to compound 14 was measured by SEC, it was found that 97.3% of it was monomer. [Figure 15] When the trastuzumab antibody conjugated to compound 18 was measured by SEC, it was found that 97.5% of it was monomer. [Figure 16] This study shows that trastuzumab antibody conjugated to compound 38 yields a DAR of 3.80, as measured by PLRP. [Figure 17] This study shows that trastuzumab antibody conjugated to compound 14 yields a DAR of 3.80, as measured by PLRP. [Figure 18] The trastuzumab antibody conjugated to compound 18 yielded a DAR of 3.85, as measured by PLRP. [Figure 19] The HIC evaluation of the trastuzumab antibody conjugated to compound 36 is shown. [Figure 20] The HIC evaluation of the trastuzumab antibody conjugated to compound 34 is shown. [Figure 21] This shows the HIC (High Intensity Compensation) evaluation of Enhertu (trastuzumab deruxtecan). [Figure 22] When the trastuzumab antibody conjugated to compound 36 was measured by SEC, it was found that 97% of it was monomer. [Figure 23] This study shows that trastuzumab antibody conjugated to compound 36 yields a DAR of 7.61, as measured by PLRP. [Figure 24] This study demonstrates the in vitro efficacy of trastuzumab-based ADCs conjugated with MMAE in NCI-N87 cells. MMAE alone (compound 11) is included as a reference. [Figure 25] This study demonstrates the in vitro efficacy of trastuzumab-based ADCs conjugated with a topoisomerase I inhibitor against NCI-N87 cells. [Figure 26] This demonstrates the in vitro stability of trastuzumab MMAE conjugate incubated in rat serum at 37°C for up to 7 days. [Figure 27]This demonstrates the in vitro stability of trastuzumab MMAE conjugate incubated in rat serum at 37°C for up to 7 days. [Figure 28] This demonstrates the in vitro stability of trastuzumab topoisomerase I inhibitor conjugates incubated in rat serum at 37°C for up to 7 days. [Figure 29] This demonstrates the in vitro stability of trastuzumab topoisomerase I inhibitor conjugates incubated in cynomolgus monkey serum at 37°C for up to 7 days. [Figure 30] This study shows that trastuzumab antibody conjugated to compound 37 yields a DAR of 7.55, as measured by PLRP. [Figure 31] The HIC evaluation of the trastuzumab antibody conjugated to compound 37 is shown. [Figure 32] When the trastuzumab antibody conjugated to compound 37 was measured by SEC, it was found that 94.9% of it was monomer. [Figure 33] This invention provides a scheme illustrating cysteine-maleimide conjugation according to embodiments of this disclosure. [Figure 34] This disclosure presents a scheme illustrating various conjugation chemistry methods that can be used for binding to cysteine ​​residues according to embodiments of this disclosure. [Figure 35] This invention presents a scheme illustrating the conjugation of a P5 phosphoramide to a cysteine ​​residue according to embodiments of this disclosure. [Figure 36] This invention illustrates a scheme for explaining thiobridge conjugation to a cysteine ​​residue according to embodiments of this disclosure. [Figure 37] Examples of NHS-ester conjugation moieties that can be used for binding to lysine residues according to embodiments of this disclosure are shown. [Figure 38]Embodiments of this disclosure illustrate a scheme illustrating that an N-terminal amine may be oxidized with an N-methylpyridinium-4-carboxyaldehyde benzenesulfonate salt (Rapoport's salt, RS) to yield an aldehyde or ketone-tagged antibody, which may then be conjugated using an oxime or hydrazone linkage. [Figure 39] This disclosure presents a scheme illustrating different click chemical reactions that can be used to produce the linker. Examples of click chemical reactions include the azide-alkyne reaction, the alkyne-DBCO (dibenzocyclooctin) reaction, and the TCO-Tz (trans-cycloctentetrazine) reaction. [Figure 40] The graph illustrates the efficacy of trastuzumab MMAE conjugate to a HER2+NCI-N87 gastric tumor xenograft model according to embodiments of this disclosure. [Figure 41] This is a schematic diagram showing an ELISA assay configured for the analysis of total antibody (left) and total ADC concentrations (right) in rat plasma according to embodiments of the present disclosure. [Figure 42] The graph shows rat pharmacokinetic data, indicating the total antibody and total ADC concentrations measured by ELISA in rat-derived plasma samples sampled at the indicated time after administration of the indicated compound, according to embodiments of the present disclosure. [Figure 43] The graph shows clinical observations (average total per animal) from rat toxicity studies comparing enfortumab conjugates carrying different linker-payloads according to embodiments of the present disclosure. The vedotin conjugate (compound 38 conjugate) induced clinical observations (primarily fur and skin-related) in all administered animals, reaching a maximum of 2.6 average clinical observations / animal on day 17, when the animals died. In contrast, no clinical observations were observed in animals administered enfortumab conjugated with either compound 14 or compound 18. [Figure 44]The graph shows the number of red blood cells (RBCs) in rats administered with enfortumab conjugate or vehicle alone, according to embodiments of the present disclosure. [Figure 45] The graph shows the number of neutrophils in rats administered with enfortumab conjugate or vehicle alone, according to embodiments of the present disclosure. [Figure 46] The graph shows alanine transaminase levels in rats administered with enfortumab conjugate or vehicle alone, according to embodiments of the present disclosure. [Figure 47] The graph shows the reticulocyte count in rats administered with enfortumab conjugate or vehicle alone, according to embodiments of the present disclosure. [Figure 48] The graph illustrates the efficacy of a trastuzumab topoisomerase I inhibitor conjugate to a HER2+NCI-N87 gastric tumor xenograft model according to embodiments of this disclosure. [Figure 49] The graph illustrates rat pharmacokinetic data showing total antibody and total ADC concentrations measured by ELISA in rat-derived plasma samples sampled at indicated times after administration of the indicated compounds, according to embodiments of this disclosure. [Figure 50] The graph illustrates rat pharmacokinetic data showing total antibody and total ADC concentrations measured by ELISA in rat-derived plasma samples sampled at indicated times after administration of the indicated compounds, according to embodiments of this disclosure. [Figure 51] The graph illustrates rat pharmacokinetic data showing total antibody and total ADC concentrations measured by ELISA in rat-derived plasma samples sampled at indicated times after administration of the indicated compounds, according to embodiments of this disclosure. [Figure 52] The graph illustrates rat pharmacokinetic data showing total antibody and total ADC concentrations measured by ELISA in rat-derived plasma samples sampled at indicated times after administration of the indicated compounds, according to embodiments of this disclosure. [Figure 53]A graph illustrating the efficacy of the trastuzumab conjugate according to the embodiments of this disclosure to a HER2-moderate JIMT-1 breast tumor xenograft model according to the embodiments of this disclosure is shown. [Figure 54] A graph illustrating the in vitro efficacy of the enfortumab conjugate according to the embodiments of this disclosure against HEK-293 cells overexpressing rat nectin-4 is shown. [Figure 55] A graph illustrating the in vitro efficacy of the enfortumab conjugate according to the embodiments of this disclosure against HEK-293 cells overexpressing human nectin-4 is shown. [Figure 56] A graph illustrating the in vitro efficacy of trastuzumab conjugate according to the embodiments of this disclosure against the HER2-positive cell line NCI-N87 is shown. [Figure 57] A graph illustrating the in vitro efficacy of the trastuzumab conjugate according to the embodiments of this disclosure against the HER2-positive cell line SK-BR-3 is shown. [Figure 58] A graph illustrating the in vitro efficacy of trastuzumab conjugate according to the embodiments of this disclosure against the HER2-positive cell line NCI-N87 is shown. [Figure 59] A graph illustrating the in vitro efficacy of trastuzumab conjugate according to the embodiments of this disclosure against the HER2-positive cell line NCI-N87 is shown. [Figure 60] A graph illustrating the in vitro efficacy of trastuzumab conjugate according to the embodiments of this disclosure against the HER2-positive cell line NCI-N87 is shown. [Figure 61] A graph illustrating the in vitro efficacy of trastuzumab conjugate according to the embodiments of this disclosure against the HER2-positive cell line NCI-N87 is shown. [Figure 62] The HPLC trace demonstrates that compound 14 conjugated to enfortumab yields a DAR of 3.7 when measured by reduced PLRP. [Figure 63]Compound 14, conjugated to enfortumab, showed HPLC traces demonstrating that 97.1% of it was the monomer, as measured by analytical SEC. [Figure 64] The HPLC trace demonstrates that compound 18, conjugated to enfortumab, yields a DAR of 3.7 when measured by reduced PLRP. [Figure 65] Compound 18, conjugated to enfortumab, showed HPLC traces demonstrating that 98.0% of it was monomer, as measured by analytical SEC. [Figure 66] The HPLC trace demonstrates that compound 36, conjugated to trastuzumab, yields a DAR of 7.84 when measured by reduced PLRP. [Figure 67] Compound 36, conjugated to trastuzumab, showed HPLC traces demonstrating that 96.9% of it was monomer, as measured by analytical SEC. [Figure 68] The HPLC trace demonstrates that compound 37, conjugated to trastuzumab, yields a DAR of 7.81 when measured by reduced PLRP. [Figure 69] Compound 37, conjugated to trastuzumab, showed HPLC traces demonstrating that 95.5% of it was monomer, as measured by analytical SEC. [Figure 70] The HPLC trace demonstrates that compound 37 conjugated to ivalizumab yields a DAR of 7.1 when measured by reduced PLRP. [Figure 71] Compound 37, conjugated to ivalizumab, showed HPLC traces demonstrating that 98.0% of it was the monomer, as measured by analytical SEC. [Figure 72] The HPLC trace demonstrates that compound 42, conjugated with anti-FITC, yields a DAR of 7.5 when measured by reducing PLRP. [Figure 73]Compound 42, conjugated with anti-FITC, showed HPLC traces demonstrating that 96.6% of it was monomer, as measured by analytical SEC. [Figure 74] The HPLC trace demonstrates that compound 46, conjugated to trastuzumab, yields a DAR of 7.9 when measured by reduced PLRP. [Figure 75] Compound 46, conjugated to trastuzumab, showed HPLC traces demonstrating that 94.9% of it was monomer, as measured by analytical SEC. [Figure 76] The HPLC trace demonstrates that compound 46, conjugated with anti-FITC, yields a DAR of 7.7 when measured by reducing PLRP. [Figure 77] Compound 46, conjugated with anti-FITC, showed HPLC traces demonstrating that 97.2% of it was monomer, as measured by analytical SEC. [Figure 78] The HPLC trace demonstrates that compound 50, conjugated to trastuzumab, yields a DAR of 7.75 when measured by reduced PLRP. [Figure 79] Compound 50, conjugated to trastuzumab, showed HPLC traces demonstrating that 96.0% of it was monomer, as measured by analytical SEC. [Figure 80] The HPLC trace demonstrates that compound 50, conjugated with anti-FITC, yields a DAR of 7.82 when measured by reducing PLRP. [Figure 81] Compound 50, conjugated with anti-FITC, showed HPLC traces demonstrating that 97.8% of it was monomer, as measured by analytical SEC. [Figure 82] The HPLC trace demonstrates that compound 57, conjugated to trastuzumab, yields a DAR of 7.61 when measured by reduced PLRP. [Figure 83]Compound 57, conjugated to trastuzumab, showed HPLC traces demonstrating that 95.9% of it was monomer, as measured by analytical SEC. [Figure 84] The HPLC trace demonstrates that compound 57, conjugated with anti-FITC, yields a DAR of 7.72 when measured by reducing PLRP. [Figure 85] Compound 57, conjugated with anti-FITC, showed HPLC traces demonstrating that 97.3% of it was monomer, as measured by analytical SEC. [Figure 86] The HPLC trace demonstrates that compound 60, conjugated to trastuzumab, yields a DAR of 7.8 when measured by reduced PLRP. [Figure 87] Compound 60, conjugated to trastuzumab, showed HPLC traces demonstrating that 96.2% of it was monomer, as measured by analytical SEC. [Figure 88] The HPLC trace demonstrates that compound 60 conjugated with anti-FITC yields a DAR of 7.7 when measured by reducing PLRP. [Figure 89] Compound 60, conjugated with anti-FITC, showed HPLC traces demonstrating that 97.4% of it was monomer, as measured by analytical SEC. [Figure 90] The HPLC trace demonstrates that compound 61, conjugated to trastuzumab, yields a DAR of 3.9 when measured by reduced PLRP. [Figure 91] Compound 61, conjugated to trastuzumab, showed HPLC traces demonstrating that 97.1% of it was monomer, as measured by analytical SEC. [Figure 92] The HPLC trace demonstrates that compound 61, conjugated with anti-FITC, yields a DAR of 3.9 when measured by reducing PLRP. [Figure 93]Compound 61, conjugated with anti-FITC, showed HPLC traces demonstrating that 98.6% of it was monomer, as measured by analytical SEC. [Figure 94] The HPLC trace demonstrates that compound 65, conjugated to trastuzumab, yields a DAR of 6.51 when measured by reduced PLRP. [Figure 95] Compound 65, conjugated to trastuzumab, showed HPLC traces demonstrating that 95.3% of it was monomer, as measured by analytical SEC. [Figure 96] The HPLC trace demonstrates that compound 65, conjugated to trastuzumab, yields a DAR of 6.9 when measured by reduced PLRP. [Figure 97] Compound 65, conjugated to trastuzumab, exhibits HPLC traces demonstrating that 97.9% of it is monomer, as measured by analytical SEC. [Figure 98] The HPLC trace demonstrates that compound 62, conjugated with anti-FITC, yields a DAR of 3.7 when measured by reducing PLRP. [Figure 99] Compound 62, conjugated with anti-FITC, exhibits HPLC traces demonstrating that 98.7% of it is monomer, as measured by analytical SEC. [Figure 100] The HPLC trace demonstrates that compound 62, conjugated to trastuzumab, yields a DAR of 3.8 when measured by reduced PLRP. [Figure 101] Compound 62, conjugated to trastuzumab, showed HPLC traces demonstrating that 96.9% of it was monomer, as measured by analytical SEC. [Figure 102] The HPLC trace demonstrates that compound 66, conjugated to trastuzumab, yields a DAR of 3.0 when measured by reduced PLRP. [Figure 103]Compound 66, conjugated to trastuzumab, exhibits HPLC traces demonstrating that 95.4% of it is monomer, as measured by analytical SEC. [Figure 104] The HPLC trace demonstrates that compound 66 conjugated with anti-FITC yields a DAR of 3.0 when measured by reducing PLRP. [Figure 105] Compound 66, conjugated with anti-FITC, exhibits HPLC traces demonstrating that 97.0% of it is monomer, as measured by analytical SEC. [Figure 106] The HPLC trace demonstrates that compound 59, conjugated with anti-FITC, yields a DAR of 5.72 when measured by reducing PLRP. [Figure 107] Compound 59, conjugated with anti-FITC, showed HPLC traces demonstrating that 97.2% of it was monomer, as measured by analytical SEC. [Figure 108] The HPLC trace demonstrates that compound 59, conjugated to trastuzumab, yields a DAR of 7.06 when measured by reduced PLRP. [Figure 109] Compound 59, conjugated to trastuzumab, showed HPLC traces demonstrating that 95.8% of it was monomer, as measured by analytical SEC.

[0066] definition The following terms have the meanings set forth below unless otherwise indicated. Any undefined terms have the meanings recognized in the relevant technical field.

[0067] "Alkyl" refers to monovalent saturated aliphatic hydrocarbyl groups having 1 to 10 carbon atoms, such as 1 to 6 carbon atoms, 1 to 5 carbon atoms, 1 to 4 carbon atoms, or 1 to 3 carbon atoms. This term includes linear and branched hydrocarbyl groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0068] The term "substituted alkyl" is an alkyl group as defined herein, wherein one or more carbon atoms (excluding the C1 carbon atom) in the alkyl chain are -O-, N-, S-, -S(O) n -(n is 0-2), -NR-(R is hydrogen or alkyl), and are optionally replaced by heteroatoms such as alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NR a R b (R ’ and R ”The alkyl group has 1 to 5 substituents selected from the group consisting of hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, and heterocyclic rings, and may be the same or different.

[0069] "Alkylene" is preferably linear or branched, having 1 to 6 carbon atoms, more preferably 1 to 3 carbon atoms, and optionally -O-, -NR 10 -, -NR 10 C(O)-, -C(O)NR 10 This term refers to a divalent aliphatic hydrocarbyl group that is interrupted by one or more groups selected from the following. Examples of this term include methylene (-CH2-), ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), isopropylene (-CH2CH(CH3)-), (-C(CH3)2CH2CH2-), (-C(CH3)2CH2C(O)-), (-C(CH3)2CH2C(O)NH-), (-CH(CH3)CH2-), etc.

[0070] "Substitutive alkylene" refers to an alkylene group having 1 to 3 hydrogen atoms replaced by substituents, as described in the definition of "substitution" below for carbon.

[0071] The term "alkane" refers to alkyl and alkylene groups as defined herein.

[0072] The terms “alkylaminoalkyl”, “alkylaminoalkenyl”, and “alkylaminoalkynyl” refer to an R'NHR”-group (where R' is an alkyl group as defined herein, and R” is an alkylene group, alkenylene group, or alkynylene group as defined herein).

[0073] The terms "alkaryl" or "aralkyl" refer to alkylene-aryl and substituted alkylene-aryl groups, where alkylene, substituted alkylene, and aryl are defined herein.

[0074] "Alkoxy" refers to an -O-alkyl group, where alkyl is as defined herein. Examples of alkoxys include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. The term "alkoxy" also refers to alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O- groups, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0075] The term "substituted alkoxy" refers to substituted alkyl-O-groups, substituted alkenyl-O-groups, substituted cycloalkyl-O-groups, substituted cycloalkenyl-O-groups, and substituted alkynyl-O-groups, where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0076] The term "alkoxyamino" refers to an -NH-alkoxy group, where alkoxy is defined herein.

[0077] The term "haloalkoxy" refers to an alkyl-O- group in which one or more hydrogen atoms on the alkyl group are substituted with a halo group. Examples of such groups include trifluoromethoxy.

[0078] The term "haloalkyl" refers to the substituted alkyl groups described above, in which one or more hydrogen atoms on the alkyl group are substituted with a halo group. Examples of such groups include, but are not limited to, fluoroalkyl groups such as trifluoromethyl, difluoromethyl, and trifluoroethyl.

[0079] The term "alkylalkoxy" refers to alkylene-O-alkyl groups, alkylene-O-substituted alkyl groups, substituted alkylene-O-alkyl groups, and substituted alkylene-O-substituted alkyl groups, where alkyl, substituted alkyl, alkyl, alkylene, and substituted alkylene are as defined herein.

[0080] The term "alkylthioalkoxy" refers to alkylene-S-alkyl, alkylene-S-substituted alkyl, substituted alkylene-S-alkyl, and substituted alkylene-S-substituted alkyl, where alkyl, substituted alkyl, alkyl, alkylene, and substituted alkylene are as defined herein.

[0081] "Alkenyl" refers to a linear or branched hydrocarbyl group having 2 to 6 carbon atoms, preferably 2 to 4 carbon atoms, and at least 1, preferably 1 to 2 double bond unsaturated sites. This term includes, as examples, be-vinyl, allyl, and buta-3-en-1-yl. This term includes cis isomers and trans isomers, or mixtures thereof.

[0082] The term "substituted alkenyl" refers to an alkenyl group as defined herein having 1 to 5 substituents or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0083] "Alkynyl" refers to a linear or branched monovalent hydrocarbyl group having 2 to 6 carbon atoms, preferably 2 to 3 carbon atoms, and at least 1, preferably 1 to 2 triple bond unsaturated moieties. Examples of such alkynyl groups include acetylenyl (-C≡CH) and propargyl (-CH2C≡CH).

[0084] The term "substituted alkynyl" refers to an alkynyl group as defined herein having 1 to 5 substituents or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0085] "Alkynyloxy" refers to an -O-alkynyl group, and alkynyl is as defined herein. Examples of alkynyloxy include ethynyloxy and propynyloxy.

[0086] "Acyl" includes HC(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)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, and substituted This refers to heteroaryl-C(O)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)- groups, while 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. For example, acyl contains the "acetyl" group CH3C(O)-.

[0087] "Acylamino" is -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) heterocyclic equation, and -NR 20 This refers to a C(O) substituted heterocyclic group, where R is located. 20is hydrogen or alkyl, 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.

[0088] The terms "aminocarbonyl" or "aminoacyl" are derived from -C(O)NR 21 R 22 It refers to the base, R 21 and R 22 R is 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. 21 and R 22 These are optionally linked together with the nitrogen atoms to which they are bonded to form heterocyclic or substituted heterocyclic groups, where 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 groups are as defined herein.

[0089] "Aminocarbonylamino" is -NR 21 C(O)NR 22 R 23 It refers to the base, and in the formula, R 21 , R 22 , and R 23 These are independently selected from hydrogen, alkyl, aryl, or cycloalkyl, or two R groups are bonded together to form a heterocycline group.

[0090] The term "alkoxycarbonylamino" refers to the -NRC(O)OR group, where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl, which are defined herein.

[0091] The term "acyloxy" refers to alkyl-C(O)O-groups, substituted alkyl-C(O)O-groups, cycloalkyl-C(O)O-groups, substituted cycloalkyl-C(O)O-groups, aryl-C(O)O-groups, heteroaryl-C(O)O-groups, and heterocyclyl-C(O)O-groups, where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0092] "Aminosulfonyl" is -SO2NR 21 R 22 It refers to the base, and in the formula, R 21 and R 22 R is 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, heterocyclic, and substituted heterocyclic. 21 and R 22 These groups may optionally bond with the nitrogen atoms attached to them to form heterocyclic or substituted heterocyclic groups, where 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 groups are as defined herein.

[0093] "Sulfonylamino" is -NR 21 SO2R 22 It refers to the base, and in the formula, R 21 and R 22R is 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, heterocyclic, and substituted heterocyclic. 21 and R 22 These can optionally bond with atoms attached to them to form heterocyclic or substituted heterocyclic groups, where 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 groups are as defined herein.

[0094] "Aryl" or "Ar" refers to a monovalent aromatic carbocyclic group of 6 to 18 carbon atoms that has a single ring (e.g., those present in a phenyl group) or a ring system with multiple fused rings (examples of such aromatic ring systems include naphthyl, anthryl, and indanyl) where the fused ring may or may not be aromatic, provided that the bond site passes through an atom of the aromatic ring. This term includes phenyl and naphthyl as examples. Unless otherwise restricted by the definition of aryl substituents, such aryl groups may be optionally substituted with 1 to 5 substituents selected from 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, alkaryl, aryloxy, azide, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl.

[0095] "Aryloxy" refers to an -O-aryl group in which aryl is as defined herein, and includes, for example, phenoxy, naphthoxy, and other optionally substituted aryl groups as also defined herein.

[0096] "Amino" refers to the -NH2 group.

[0097] The term "substituted amino" refers to the -NRR group, where 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.

[0098] The term "azide" refers to the -N3 group.

[0099] "Carboxyl," "carboxy," or "carboxylate" refers to -CO2H or a salt thereof.

[0100] The terms "carboxyl ester" or "carboxyester," or "carboxyalkyl" or "carboxylalkyl" are defined as -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-heterocyclic, and -C(O)O-substituted heterocyclic groups are defined herein as follows: 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 groups.

[0101] "(carboxyl ester)oxy" or "carbonate" refers to -OC(O)O-alkyl, -OC(O)O-substituted alkyl, -OC(O)O-alkenyl, -OC(O)O-substituted alkenyl, -OC(O)O-alkynyl, -OC(O)O-substituted alkynyl, -OC(O)O-aryl, -OC(O)O-substituted aryl, -OC(O)O-cycloalkyl, -OC(O)O-substituted cycloalkyl, -OC(O)O-cycloalkenyl, -OC(O)O-substituted cycloalkenyl Nyl, -OC(O)O-heteroaryl, -OC(O)O-substituted heteroaryl, -OC(O)O-heterocyclic, and -OC(O)O-substituted heterocyclic groups are defined herein, while 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 groups are defined herein.

[0102] "Cyano" or "nitrile" refers to the -CN group.

[0103] "Cycloalkyl" refers to a cyclic alkyl group of 3 to 10 carbon atoms having one or more cyclic rings, including condensation, crosslinking, and spiro-ring systems. Examples of suitable cycloalkyl groups include adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclooctyl. Such cycloalkyl groups include monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, and cyclooctyl, or polycyclic structures such as adamantanyl.

[0104] The term "substituted cycloalkyl" refers to a cycloalkyl group having 1 to 5 substituents or 1 to 3 substituents selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0105] "Cycloalkenyl" refers to a non-aromatic cyclic alkyl group of 3 to 10 carbon atoms having one or more rings and at least one double bond, preferably 1 to 2 double bonds.

[0106] The term "substituted cycloalkenyl" refers to a cycloalkenyl group having 1 to 5 substituents or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0107] "Cycloalkynyl" refers to a non-aromatic cycloalkyl group consisting of 5 to 10 carbon atoms, having one or more rings and at least one triple bond.

[0108] "Cycloalkoxy" refers to -O-cycloalkyl.

[0109] "Cycloalkenyloxy" refers to -O-cycloalkenyl.

[0110] "Halo" or "halogen" refers to fluoro, chloro, bromo, and iodine compounds.

[0111] "Hydroxy" or "hydroxyl" refers to the -OH group.

[0112] A "heteroaryl" refers to an aromatic group having 1 to 15 carbon atoms, such as 1 to 10 carbon atoms, within a ring, and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. Such a heteroaryl group may have multiple fused rings within a single ring (e.g., pyridinyl, imidazolyl, or furyl) or a ring system (e.g., groups such as indolidinyl, quinolinyl, benzofuran, benzimidazolyl, or benzothienyl), with at least one ring in the ring system being aromatic. To satisfy the valence requirement, any heteroatom within such a heteroaryl ring may or may not be bonded to H or a substituent, such as an alkyl group or other substituents described herein. In certain embodiments, the nitrogen and / or sulfur ring atoms(s) of the heteroaryl group may optionally be oxidized to provide an N-oxide (N→O) moiety, a sulfinyl moiety, or a sulfonyl moiety. The term includes, by example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise restricted 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 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, alkaryl, aryl, aryloxy, azide, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl, as well as trihalomethyl.

[0113] The term "heteroaralkyl" refers to an alkylene-heteroaryl group, where alkylene and heteroaryl are defined herein. This term includes, by example, pyridylmethyl, pyridylethyl, indolylmethyl, and others.

[0114] "Heteroaryloxy" refers to -O-heteroaryl.

[0115] "Heterocyclic," "heterocyclic," "heterocycloalkyl," and "heterocyclyl" refer to saturated or unsaturated groups having a single or multiple fused rings, including condensation, crosslinking, and spiro-ring systems, and having 3 to 20 ring atoms containing 1 to 10 heteroatoms. These ring atoms are selected from nitrogen, sulfur, or oxygen, and in the fused ring system, one or more of the rings may be cycloalkyl, aryl, or heteroaryl, provided that the bonding site passes through a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atoms(s) of the heterocyclic group are optionally oxidized to provide an N-oxide, -S(O)-, or -SO2- moiety. To satisfy valence requirements, any heteroatom in such a heterocyclic ring may or may not be bonded to one or more H atoms or one or more substituents, e.g., alkyl groups or other substituents described herein.

[0116] Examples of heterocyclic and heteroaryl compounds include azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indidine, isoindole, indole, dihydroindole, indazole, purine, quinoridine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carborin, phenantholidine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, and This includes, but is not limited to, enoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinil, thiomorpholinil (also called thiamorpholinil), 1,1-dioxothiomorpholinil, piperidinil, pyrrolidine, tetrahydrofuranil, etc.

[0117] Unless otherwise restricted by the definition of heterocyclic substituents, such heterocyclic groups may be optionally substituted with 1 to 5 or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and condensed heterocycles.

[0118] "Heterocyclyloxy" refers to the -O-heterocyclyl group.

[0119] The term "heterocyclilthio" refers to a heterocyclic -S- group.

[0120] The term "heterocyclene" refers to a diradical group formed from a heterocycle, as defined herein.

[0121] The term "hydroxyamino" refers to the -NHOH group.

[0122] "Nitro" refers to the -NO2 group.

[0123] "Oxo" refers to an atom (=O).

[0124] "Sulfonyl" refers to -SO2-alkyl groups, -SO2-substituted alkyl groups, -SO2-alkenyl groups, -SO2-substituted alkenyl groups, -SO2-cycloalkyl groups, -SO2-substituted silcoalkyl groups, -SO2-cycloalkenyl groups, -SO2-substituted silcoalkenyl groups, -SO2-aryl groups, -SO2-substituted aryl groups, -SO2-heteroaryl groups, -SO2-substituted heteroaryl groups, -SO2-heterocyclic groups, and -SO2-substituted heterocyclic groups, where 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. Examples of sulfonyls include methyl-SO2-, phenyl-SO2-, and 4-methylphenyl-SO2-.

[0125] "Sulfonyloxy" refers to -OSO2-alkyl, -OSO2-substituted alkyl, -OSO2-alkenyl, -OSO2-substituted alkenyl, -OSO2-cycloalkyl, -OSO2-substituted sylcoalkyl, -OSO2-cycloalkenyl, -OSO2-substituted sylcoalkenyl, -OSO2-aryl, -OSO2-substituted aryl, -OSO2-heteroaryl, -OSO2-substituted heteroaryl, -OSO2-heterocyclic, and -OSO2-substituted heterocyclic, where 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.

[0126] "Sulfate" or "sulfate ester" refers to -O-SO2-OH, -O-SO2-O-alkyl, -O-SO2-O-substituted alkyl, -O-SO2-O-alkenyl, -O-SO2-O-substituted alkenyl, -O-SO2-O-cycloalkyl, -O-SO2-O-substituted cycloalkyl, -O-SO2-O-cycloalkenyl, -O-SO2-O-substituted cycloalkenyl, -O-SO2-O-aryl, -O-SO2-O-substituted aryl, -O-SO2-O- Telloaryl, -O-SO2-O-substituted heteroaryl, -O-SO2-O-heterocyclic, and -O-SO2-O-substituted heterocyclic groups refer to 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.

[0127] The term "aminocarbonyloxy" refers to the -OC(O)NRR group, where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic, and alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclic are as defined herein.

[0128] "Thiol" refers to the -SH group.

[0129] The terms "thioxo" or "thioketo" refer to an atom (=S).

[0130] The terms "alkylthio" or "thioalkoxy" refer to an -S-alkyl group, where alkyl is as defined herein. In certain embodiments, sulfur may be oxidized to -S(O)-. The sulfoxide may exist as one or more stereoisomers.

[0131] The term "substituted thioalkoxy" refers to an -S-substituted alkyl group.

[0132] The term "thioaryloxy" refers to an aryl-S-group in which the aryl group is as defined herein and includes an aryl group optionally substituted as also defined herein.

[0133] The term "thioheteroaryloxy" refers to a heteroaryl-S-group in which the heteroaryl group is as defined herein and includes an optionally substituted aryl group as also defined herein.

[0134] The term "thioheterocyclooxy" refers to a heterocyclyl-S-group, wherein the heterocyclyl group is as defined herein and includes a heterocyclyl group optionally substituted as also defined herein.

[0135] In addition to the disclosures herein, the term “substituted” may also mean, when used to modify a particular group or radical, that one or more hydrogen atoms of that particular group or radical are each independently replaced by the same or different substituents as defined below.

[0136] In addition to the groups disclosed with respect to the individual terms herein, one or more hydrogen atoms on a saturated carbon atom of a specified group or radical (any two hydrogen atoms on a single carbon atom = O, = NR) 70 、=N-OR 70 The substituent to replace (which can be replaced by =N2 or =S) is -R unless otherwise specified. 60 , halo, =O, -OR 70 , -SR 70 , -NR 80 R 80 , trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R 70 , -SO2O - M + , -SO2OR 70 , -OSO2R 70 , -OSO2O - M + , -OSO2OR 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 CO2 -M + , -NR 70 CO2R 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 And in the formula, R 60 The group consists of alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl, and each R 70 These are, independently, hydrogen or R 60 And each R 80 R is independent of R 70 Alternatively, two R 80 However, together with the nitrogen atom to which they are bonded, they form a 5-membered, 6-membered, or 7-membered heterocycloalkyl, which may optionally contain 1 to 4 additional heteroatoms of the same or different type selected from the group consisting of O, N, and S, wherein the N may have a -H or C1-C3 alkyl substitution, and each M + Each M is a counterion with a single net positive charge. + These are independent, for example, alkaline ions, for example, K + na + Li + ; Ammonium ions, for example, + N(R 60 )4; or alkaline earth ions, for example, [Ca 2+ ] 0.5 [Mg 2+ ] 0.5 , or [Ba 2+ ] 0.5(The subscript 0.5 means that one of the counterions of such divalent alkaline earth ions may be the ionized form of the compound of the present invention, and the other may be a typical counterion such as a chloride, or that two ionized compounds disclosed herein may function as counterions of such divalent alkaline earth ions, or that the biionized compound of the present invention may function as a counterion of such divalent alkaline earth ions.) As a specific example, -NR 80 R 80 It is intended to contain -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methylpiperazin-1-yl, and N-morpholinyl.

[0137] In addition to the disclosures herein, the substituents of hydrogen on unsaturated carbon atoms in "substituted" alkenes, alkynes, aryls, and heteroaryl groups are -R unless otherwise specified. 60 Hello, -O - M + , -OR 70 , -SR 70 , -SM + , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R 70 , -SO3 - M + , -SO3R 70 , -OSO2R 70 , -OSO3 - M + , -OSO3R 70 , -PO3 -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 , -CO2 - M + , -CO2R 70 , -C(S)OR70 -C(O)NR 80 R 80 -C(NR 70 )NR 80 R 80 -OC(O)R 70 ,-OC(S)R 70 , -OCO2 - M + , -OCO2R 70 , -OC(S)OR 70 , -NR 70 C(O)R 70 , -NR 70 C(S)R 70 , -NR 70 CO2 - M + , -NR 70 CO2R 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 And in the formula, R 60 , R 70 , R 80 , and M + This is as previously defined, except in the case of substituted alkenes or alkynes, the substituent is -O - M + , -OR 70 , -SR 70 , or -S - M + This is conditional on the fact that it is not the case.

[0138] In addition to the groups disclosed with respect to the individual terms herein, the substituents of hydrogen on the nitrogen atom in "substituted" heteroalkyl and cycloheteralkyl groups are, unless otherwise specified, -R 60 , -O - M + , -OR 70 , -SR 70 , -S - M+ , -NR 80 R 80 , trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R 70 , -S(O)2O - M + , -S(O)2OR 70 , -OS(O)2R 70 , -OS(O)2O - M + , -OS(O)2OR 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 , and in the formula, R 60 , R 70 , R 80, and M + This is as previously defined.

[0139] In addition to the disclosures herein, in certain embodiments, the substituted group has one, two, three, or four substituents, one, two, or three substituents, one or two substituents, or one substituent.

[0140] It is understood that polymers obtained by defining substituents having further substituents on themselves in all the substituted groups defined above (for example, a substituted aryl having a substituted aryl group as a substituent, which itself is substituted with a substituted aryl group and is further substituted with a substituted aryl group, etc.) are not intended to be included herein. In such cases, the maximum number of such substitutions is 3. For example, the sequential substitution of substituted aryl groups specifically intended herein is limited to substituted aryl-(substituted aryl)-substituted aryl.

[0141] Unless otherwise indicated, substituents not explicitly defined herein are named by naming the terminal portion of the functional group, followed by the functional groups adjacent to the bond site. For example, the substituent "arylalkyloxycarbonyl" refers to the (aryl)-(alkyl)-OC(O)- group.

[0142] With respect to any of the groups disclosed herein that include one or more substituents, it is understood that such groups do not include any substitutions or substitution patterns that are sterically unrealistic and / or synthetically impossible. In addition, the compounds of interest include all stereochemical isomers resulting from the substitutions of these compounds.

[0143] The term “pharmaceutically acceptable salt” means a salt that is acceptable for administration to patients such as mammals (a salt having a counterion that has mammalian safety acceptable for a given drug regimen). Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids. “pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts of compounds derived from various organic and inorganic counterions known in the art, including but not limited to sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium, and, if the molecule contains a basic functional group, salts of organic or inorganic acids such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, and oxalate.

[0144] The term "salt" refers to a compound formed when a proton of an acid is replaced by a cation, such as a metal cation or an organic cation. Where applicable, the salt is pharmaceutically acceptable, which is not required for salts of intermediate compounds not intended for administration to a patient. As an example, salts of this compound include those in which the compound is protonated by an inorganic or organic acid to form a cation, and the conjugate base of the inorganic or organic acid becomes the anionic component of the salt.

[0145] A "solvate" refers to a complex formed by a combination of solvent molecules and solute molecules or ions. The solvent can be an organic compound, an inorganic compound, or a mixture of both. Some examples of solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, and water. When the solvent is water, the solvate formed is a hydrate.

[0146] "Stereoisomers" refer to compounds that have the same atomic bonding but different atomic arrangements in space. Stereoiomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.

[0147] "Tautomers" refers to alternative forms of molecules that differ only in the electron bonding of atoms and / or the position of protons, e.g., enol-keto and imine-enamine tautomers, or tautomers of heteroaryl groups containing the -N=C(H)-NH- ring configuration, e.g., pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles. Those skilled in the art will recognize that other tautomer ring configurations are possible.

[0148] It should be understood that the term "its salts, solvates, or stereoisomers" is intended to include all permutations of salts, solvates, and stereoisomers, such as solvates of pharmaceutically acceptable salts of the stereoisomers of the compound in question.

[0149] "Pharmacologically effective dose" and "therapeutic effective dose" refer to the amount of a compound sufficient to treat one or more of a particular disorder or disease or its symptoms, and / or to prevent the onset of that disease or disorder. With respect to tumorigenic proliferative disorders, the pharmacochemically effective dose or therapeutic effective dose includes, in particular, an amount sufficient to shrink the tumor or slow its growth rate.

[0150] "Patient" refers to both human and non-human subjects, particularly mammals.

[0151] As used herein, the terms “to treat” or “to cure” mean treating or curing a disease or medical condition of a patient, such as a mammal (especially a human), and include (a) preventing the onset of a disease or medical condition, such as by prophylactic treatment of the subject; (b) improving a disease or medical condition, such as eliminating or regressing the disease or medical condition of the patient; (c) suppressing a disease or medical condition, such as by slowing or stopping the progression of the disease or medical condition of the patient; or (d) alleviating the symptoms of a disease or medical condition of the patient.

[0152] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymeric forms of amino acids of any length. Unless otherwise specifically indicated, “polypeptide,” “peptide,” and “protein” may include genetically encoded and unencoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backbone. The term includes, but is not limited to, fusion proteins having heterologous amino acid sequences, fusions having heterologous and homologous leader sequences, proteins containing at least one N-terminal methionine residue (e.g., to facilitate production in recombinant host cells), immunotagged proteins, and the like. In certain embodiments, the polypeptide is an antibody.

[0153] "Natural amino acid sequence" or "parent amino acid sequence" are used interchangeably herein and refer to the amino acid sequence of the unmodified polypeptide that contains at least one modified amino acid residue.

[0154] "Natural amino acid" or "natural amino acid residue" is used interchangeably herein to refer to natural 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). Natural amino acids may be located at their naturally occurring positions within a natural amino acid sequence.

[0155] The terms "amino acid analog" and "non-natural amino acid" are used interchangeably and may include amino acid-like compounds that are structurally and / or in shape similar 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 with modified side chains or backbone. Amino acid analogs also include amino acid analogs that have the same stereochemistry as the naturally occurring D form, and L forms of amino acid analogs. In some cases, amino acid analogs share the backbone and / or side-chain structures of one or more natural amino acids, and the difference(s) is one or more modifying groups in the molecule. Such modifications may include, but are not limited to, substitution of a relevant atom (such as S) with another atom (such as N), addition of a group (such as methyl or hydroxyl) or an atom (such as Cl or Br), deletion of a group, substitution of a covalent bond (substitution of a double bond with a single bond), or combinations thereof. For example, amino acid analogs may include α-hydroxy acids and α-amino acids. Examples of amino acid analogs include, but are not limited to, sulfoalanine.

[0156] Terms such as “amino acid side chain” or “side chain of amino acids” may be used to refer to substituents attached to the α-carbon of an amino acid residue, including natural amino acids, non-natural amino acids, and amino acid analogs. Amino acid side chains may also include amino acid side chains described herein in the context of modified amino acids and / or conjugates.

[0157] The term "carbohydrate" 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 and disaccharides. The term "carbohydrate derivative" includes compounds in which one or more functional groups of the carbohydrate of interest are substituted (replaced by any convenient substituent), modified (converted to another group using any convenient chemical method), or absent (e.g., excluded or replaced by H). A variety of carbohydrates and carbohydrate derivatives are available and may be adapted for use in the subject compound and conjugate.

[0158] The terms “glycoside” or “glycosyl” refer to a sugar molecule or group linked to a part via a glycosidic bond. For example, the part to which a glycoside is linked may be a cleavable linker as described herein. Glycosidic bonds can link a glycoside to another part via various types of bonds, such as, but not limited to, O-glycosidic bonds (O-glycosides), N-glycosidic bonds (glycosylamines), S-glycosidic bonds (thioglycosides), or C-glycosidic bonds (C-glycosides or C-glycosyls). In some cases, glycosides may be cleaved from the part to which they are linked by, for example, chemically mediated hydrolysis or enzymatically mediated hydrolysis.

[0159] 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, and antibody fragments (e.g., Fab fragments). Antibodies can bind to target antigens. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). Target antigens may have one or more binding sites, also called epitopes, that are recognized by complementarity-determining regions (CDRs) formed by one or more variable regions of the antibody.

[0160] The term "natural antibody" refers to an antibody in which the heavy and light chains have been produced and paired by the immune system of a multicellular organism. The spleen, lymph nodes, bone marrow, and serum are examples of tissues that produce natural antibodies. For example, antibodies produced by antibody-producing cells isolated from a first animal immunized with an antigen are natural antibodies.

[0161] The terms "humanized antibody" or "humanized immunoglobulin" refer to non-human (e.g., mouse or rabbit) antibodies that contain one or more amino acids (e.g., in the framework region, constant region, or CDR) that are substituted with correspondingly positioned amino acids derived from human antibodies. Generally, humanized antibodies produce a reduced immune response in the human host compared to the non-humanized version of the same antibody. Antibodies can be humanized using various techniques known in the art, including, for example, CDR grafting (EP239,400, PCT Publication No. WO91 / 09967, U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP592,106, EP519,596, Padlan, Molecular Immunology 28(4 / 5):489-498 (1991), Studnicka et al., Protein Engineering 7(6):805-814 (1994), Roguska et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Patent No. 5,565,332). In certain embodiments, framework substitutions are identified by CDR and interaction modeling to identify framework residues important for antigen binding, as well as by sequence comparison to identify abnormal framework residues at specific locations (see, for example, U.S. Patent No. 5,585,089, Riechmann et al., Nature 332:323 (1988)). Additional methods for humanizing antibodies intended for use in the present invention are described in U.S. Patents No. 5,750,078, No. 5,502,167, No. 5,705,154, No. 5,770,403, No. 5,698,417, No. 5,693,493, No. 5,558,864, No. 4,935,496, and No. 4,816,567, as well as PCT Publications WO98 / 45331 and WO98 / 45332. In certain embodiments, the subject rabbit antibody may be humanized according to the methods described in US2004 / 0086979 and US2005 / 0033031.Therefore, the antibodies described above can be humanized using methods well known in the art.

[0162] The term "chimeric antibody" refers to an antibody in which the light and heavy chain genes are typically constructed through genetic engineering from antibody variable and constant region genes belonging to different species. For example, the variable segment of a gene derived from a mouse monoclonal antibody may bind to human constant segments such as gamma 1 and gamma 3. An example of a therapeutic chimeric antibody is a hybrid protein composed of a variable domain or antigen-binding domain derived from a mouse antibody and a constant domain or effector domain derived from a human antibody, although domains from other mammalian species may also be used.

[0163] The immunoglobulin light or heavy chain variable region of an immunoglobulin polypeptide consists of a framework region (FR) interrupted by three hypervariable regions, also known as "complementarity-determining regions" or "CDRs." The extent of the framework region and CDRs is defined (see "Sequences of Proteins of Immunological Interest," E. Kabat et al., USD Department of Health and Human Services, 1991). The framework region of an antibody, which is the framework region into which the constituent light and heavy chains combine, helps to locate and align the CDRs. The CDRs are primarily involved in the binding of antigens to epitopes.

[0164] A "parent Ig polypeptide" is a polypeptide comprising an amino acid sequence lacking the aldehyde-tagged constant region described herein. The parent polypeptide may include a natural sequence constant region or a constant region having existing amino acid sequence modifications (such as additions, deletions, and / or substitutions).

[0165] As used herein, the term “isolated” is intended to describe a compound of interest that is in an environment different from the environment in which the compound naturally occurs. “Isolated” is intended to include compounds in samples in which the compound of interest is substantially concentrated and / or in samples in which the compound of interest is partially or substantially purified.

[0166] As used herein, the term “substantially purified” means a compound that has been removed from the natural environment and contains at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more than 98% of other components that naturally associate with it.

[0167] The term "physiological conditions" is intended to encompass conditions that are compatible with living cells, such as temperature, pH, salinity, and other primarily aqueous conditions that are compatible with living cells.

[0168] A "reactive partner" refers to a molecule or molecular part that specifically reacts with another reactive partner to produce a reaction product. Exemplary reactive partners include cysteine ​​residues or lysine residues that can react with a cysteine-reactive moiety or a lysine-reactive moiety to form a reaction product in which the desired moiety binds to a cysteine ​​residue or a lysine residue. For example, a maleimide group may be a reactive partner of a cysteine ​​residue, or an N-hydroxysuccinimide ester (NHS ester) may be a reactive partner of a lysine residue.

[0169] The "N-terminus" refers to the terminal amino acid residue of a polypeptide that has a free amine group, and the amine group in the non-N-terminal amino acid residue usually forms part of the covalent backbone of the polypeptide.

[0170] The "C-terminus" refers to the terminal amino acid residue of a polypeptide that has a free carboxyl group, while carboxyl groups in non-C-terminal amino acid residues typically form part of the polypeptide's covalent backbone.

[0171] When used in reference to polypeptides or the amino acid sequences of polypeptides, the term "internal region" refers to a region of the polypeptide that is not located at the N-terminus or C-terminus.

[0172] Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described, and that the specific embodiments described may, of course, be modified. Furthermore, it should be understood that the scope of the present invention is limited only by the appended claims, and that the terminology used herein is intended solely to describe specific embodiments and not to limit them.

[0173] Where a range of values ​​is provided, unless otherwise explicitly indicated in the context, it is understood that each intermediate value up to one-tenth of the lower limit between the upper and lower limits of that range, and any other stated values ​​or intermediate values ​​within that stated range, are included in the present invention. The upper and lower limits of these smaller ranges may independently be included within the smaller range, and may also be included in the present invention according to any specifically excluded limit values ​​within the stated range. Where a stated range includes one or both limit values, a range excluding one or both of those included limit values ​​is also included in the present invention.

[0174] For clarity, it is understood that certain features of the Invention described in relation to separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the Invention described in relation to a single embodiment for brevity may also be provided separately or in any preferred partial combination. All combinations of embodiments relating to the Invention are specifically encompassed by the Invention and are disclosed herein as if every possible combination were disclosed individually and expressly to the extent that such combinations encompass a subject, e.g., a stable compound (i.e., a compound that can be prepared, isolated, characterized, and tested for biological activity). In addition, all partial combinations of various embodiments and their elements (e.g., elements of chemical groups listed in embodiments illustrating such variables) are also specifically encompassed by the Invention and are disclosed herein as if every possible combination were disclosed individually and expressly.

[0175] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which the invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the invention, but preferred methods and materials are described herein. All publications referenced herein are incorporated herein by reference for disclosing and explaining in relation to the methods and / or materials cited herein.

[0176] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless otherwise explicitly indicated by the context. It should also be noted that the claims may be written to exclude any optional elements. Therefore, this statement is intended to serve as a basis for prior art for the use of exclusive terms such as “solely,” “only,” or “negative” limitations in relation to the enumeration of elements of the claims.

[0177] For clarity, it is understood that certain features of the present invention described in relation to separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the present invention described in relation to a single embodiment may also be provided separately or in any preferred partial combination.

[0178] The publications discussed herein are presented solely to indicate that they were published prior to the filing date of this application. Nothing herein should be construed as acknowledging that the present invention does not have prior rights to such publications by prior art. Furthermore, the dates of the presented publications may differ from the actual publication dates that may need to be independently verified. [Modes for carrying out the invention]

[0179] This disclosure provides a cleavable linker for antibody-drug conjugates (ADCs) in which the linker includes a tandem cleavage portion. In addition, this disclosure also encompasses compounds and methods for producing such ADCs, as well as methods for using ADCs.

[0180] Antibody-drug conjugates This disclosure provides conjugates, such as antibody-drug conjugates (ADCs). “Conjugate” means a polypeptide (e.g., an antibody) covalently bound to one or more other parts (e.g., a drug or active agent). For example, an antibody-drug conjugate according to this disclosure comprises one or more drugs or active agents covalently bound to an antibody. In certain embodiments, the polypeptide (e.g., an antibody) and one or more drugs or active agents are linked to one or more functional groups and covalent bonds. For example, one or more functional groups and covalent bonds may include cleavable linkers as described herein (e.g., tandem cleavage linkers as described herein).

[0181] In certain embodiments, the conjugate is a polypeptide conjugate comprising a polypeptide (e.g., an antibody) conjugated to one or more other parts. In certain embodiments, the one or more parts conjugated to the polypeptide may, independently and without limitation, be any of a variety of parts for various purposes, such as a drug, an active agent, a detectable label, a water-soluble polymer, or a part for immobilizing the polypeptide onto a membrane or surface. In certain embodiments, the conjugate is a drug conjugate, where the polypeptide is an antibody, and therefore provides 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.

[0182] One or more drugs or active agents can be conjugated to a polypeptide (e.g., an antibody) at any desired site of the polypeptide. Therefore, this disclosure provides polypeptides having a drug or active agent conjugated, for example, at or near the C-terminus of the polypeptide. Other examples include polypeptides having a drug or active agent conjugated at or near the N-terminus of the polypeptide. Also, examples include polypeptides having a drug or active agent conjugated between the C-terminus and N-terminus of the polypeptide (e.g., at an internal site of the polypeptide). The above combinations are also possible when the polypeptide is conjugated with two or more drugs or active agents.

[0183] In some embodiments, one or more drugs or active agents are conjugated to a polypeptide (e.g., an antibody) at one or more native amino acid residues of the polypeptide. For example, maleimide-based conjugations can be used to conjugate one or more drugs or active agents to a polypeptide (e.g., an antibody) at one or more native cysteine ​​residues of the polypeptide (e.g., an antibody). In other cases, N-hydroxysuccinimide (NHS ester)-based conjugations can be used to conjugate one or more drugs or active agents to a polypeptide (e.g., an antibody) at one or more native lysine residues of the polypeptide (e.g., an antibody).

[0184] In certain embodiments, the conjugates of the Disclosure comprise one or more drugs or active agents conjugated to amino acid residues of a polypeptide at the α-carbon of an amino acid residue. In other words, the conjugate comprises a polypeptide in which the side chains of one or more amino acid residues in the polypeptide are modified to bind to one or more drugs or active agents (e.g., to one or more drugs or active agents via linkers as described herein). For example, the conjugate comprises a polypeptide in which the α-carbon of one or more amino acid residues in the polypeptide is modified to bind to one or more drugs or active agents (e.g., to one or more drugs or active agents via linkers as described herein).

[0185] Embodiments of the present disclosure include conjugates in which a polypeptide is conjugated to one or more parts, for example, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more parts. A part can be conjugated to a polypeptide at one or more sites in the polypeptide. For example, one or more parts can be conjugated to a single amino acid residue of the polypeptide. In some cases, one part is conjugated to an amino acid residue of the polypeptide. In other embodiments, two parts can be conjugated to the same amino acid residue of the polypeptide. In other embodiments, a first part is conjugated to a first amino acid residue of the polypeptide, and a second part is conjugated to a second amino acid residue of the polypeptide. For example, the above combination is also possible, where the polypeptide is conjugated to a first part at a first amino acid residue and to two other parts at a second amino acid residue. Other combinations are also possible, for example, polypeptides in which the first and second portions are conjugated to a first amino acid residue, and the third and fourth portions are conjugated to a second amino acid residue. In some cases, two or more amino acid residues in the polypeptide are each conjugated to a pair of portions (i.e., two portions), and each pair of portions is conjugated to the polypeptide via a branched linker as described herein. In some cases, one amino acid residue in the polypeptide is conjugated to a pair of portions via a branched linker as described herein. In some cases, two amino acid residues in the polypeptide are each conjugated to a pair of portions via a branched linker as described herein. In some cases, three amino acid residues in the polypeptide are each conjugated to a pair of portions via a branched linker as described herein. In some cases, four amino acid residues in the polypeptide are each conjugated to a pair of portions via a branched linker as described herein. In some cases, five amino acid residues in the polypeptide are each conjugated to a pair of portions via a branched linker as described herein.In some cases, six amino acid residues in the polypeptide are each conjugated to a pair of parts via a branched linker as described herein. In other cases, seven amino acid residues in the polypeptide are each conjugated to a pair of parts via a branched linker as described herein. In other cases, eight amino acid residues in the polypeptide are each conjugated to a pair of parts via a branched linker as described herein.

[0186] One or more amino acid residues of a polypeptide that are conjugated to one or more parts may be naturally occurring amino acids, unnatural amino acids, or a combination thereof. For example, a conjugate may comprise one or more drugs or active agents conjugated to one or more naturally occurring amino acid residues (e.g., natural amino acid residues) of a polypeptide. In other cases, a conjugate may comprise one or more drugs or active agents conjugated to one or more unnatural amino acid residues of a polypeptide. One or more drugs or active agents may be conjugated to a single natural (e.g., native) or unnatural amino acid residue of a polypeptide, as described above. One or more natural (e.g., native) or unnatural amino acid residues in a polypeptide may be conjugated to one or more parts as described herein. For example, two or more amino acid residues (e.g., natural (e.g., native) or unnatural amino acid residues) in a polypeptide may each be conjugated to one or two parts such that multiple sites in the polypeptide are conjugated to the desired parts.

[0187] In certain embodiments, a polypeptide (e.g., an antibody) and a moiety of interest (e.g., a drug or active agent) are conjugated via a conjugation moiety. For example, the polypeptide and the moiety of interest are each bound (e.g., covalently) to the conjugation moiety, and thus the polypeptide and the moiety of interest can be indirectly linked together via the conjugation moiety.

[0188] In some cases, the conjugation moiety includes maleimide or a derivative thereof. For example, the maleimide conjugation moiety may be conjugated to a cysteine ​​residue of a polypeptide (e.g., an antibody), such as a native cysteine ​​residue of the polypeptide (e.g., an antibody), thereby indirectly linking the polypeptide (e.g., an antibody) and the moiety of interest (e.g., a drug or active agent) together via the maleimide conjugation moiety.

[0189] In some cases, the conjugation moiety comprises an N-hydroxysuccinimide ester (NHS ester) or a derivative thereof. For example, the NHS-ester conjugation moiety may be conjugated to a lysine residue of a polypeptide (e.g., an antibody), such as a native lysine residue of the polypeptide (e.g., an antibody), thereby indirectly linking the polypeptide (e.g., an antibody) and the moiety of interest (e.g., a drug or active agent) together via the NHS-ester conjugation moiety.

[0190] In some cases, a drug or active agent can be modified to include a conjugation moiety, which is then conjugated to a polypeptide (e.g., an antibody) to produce a polypeptide (e.g., antibody) conjugate, thereby linking the drug or active agent to the polypeptide (e.g., an antibody) via the conjugation moiety.

[0191] In certain embodiments, the conjugation portion may be coupled (e.g., covalently) to a linker. Therefore, embodiments of the present disclosure include a conjugation portion coupled to a drug or active agent via a linker. Various embodiments of linkers that may be used to couple the conjugation portion to a drug or active agent are described in detail herein. For example, in some cases, the linker is a cleavable linker, e.g., a cleavable linker as described herein.

[0192] In certain embodiments, the linker is a branched linker, for example, a branched linker as described herein. In some cases, the conjugation portion may be coupled (e.g., covalently) to two or more linkers. Thus, embodiments of the present disclosure include a conjugation portion coupled to two or more drugs or active agents via corresponding linkers. Thus, the conjugate of the present disclosure may include two or more linkers, each linker coupling a corresponding drug or active agent to the conjugation portion. Thus, the conjugation portion and the two or more linkers together can be considered a “branched linker,” where the conjugation portion is coupled to two or more “branches,” and each branch includes a linker coupled to a drug or active agent.

[0193] In certain embodiments, the polypeptide may be conjugated to one or more moieties of interest, and one or more amino acid residues of the polypeptide are native amino acid residues of the polypeptide. For example, the polypeptide may be conjugated to one or more moieties of interest, and one or more amino acid residues of the polypeptide are not modified before conjugation to the moieties of interest. Examples of native amino acid residues that can be conjugated to one or more moieties of interest include, but are not limited to, cysteine ​​and lysine. In some cases, the amino acid residues conjugated to one or more moieties of interest are native amino acid residues that do not have an aldehyde group, and the conjugation moiety is not a hydrazinyl-indolyl or hydrazinyl-pyrrolo-pyridinyl conjugation moiety.

[0194] In some cases, to generate a conjugate, a polypeptide may be conjugated to a desired portion by a reaction between amino acid residues of the polypeptide and a compound (e.g., a compound containing a conjugation moiety as described herein). For example, a polypeptide may be contacted with a drug containing a reactive partner under conditions suitable for providing a drug conjugation to the polypeptide. In some cases, the drug containing a reactive partner may contain the conjugation moiety described above. For example, a drug or active agent may be modified to contain a conjugation moiety. In some cases, the drug or active agent is bound to the conjugation moiety, such as by covalent bonding via a linker, such as a linker as described in detail herein.

[0195] In certain embodiments, the conjugate of the present disclosure comprises a polypeptide (e.g., an antibody) having at least one amino acid residue conjugated to one or more target moieties (e.g., a drug or active agent). To construct a conjugate, an amino acid residue of the polypeptide (e.g., a native amino acid residue) can be coupled to one or more drugs or active agents conjugated to the conjugation moiety described above. In certain embodiments, the native amino acid residue of the polypeptide (e.g., an antibody) is a cysteine ​​or lysine residue. In certain embodiments, the amino acid residue is conjugated to a drug or active agent containing the conjugation moiety described above to provide the conjugate of the present disclosure in which one or more drugs or active agents are conjugated to the polypeptide via the conjugation moiety.

[0196] In certain embodiments, the conjugate comprises a polypeptide (e.g., an antibody) having at least one native amino acid residue conjugated to a linker described herein, which in turn is conjugated to one or more drugs or active agents. For example, the conjugate may comprise a polypeptide (e.g., an antibody) having at least one native amino acid residue conjugated to one or more target portions (e.g., one or more drugs or active agents) described above.

[0197] A part of this disclosure is a conjugate of formula (I), [ka] During the ceremony, Each R 1 However, they are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R 2 However, they are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k1 is an integer between 1 and 10. R 3 However, it includes parts that can be cut, L A However, it was the first linker, W 1 However, it is a drug, W 2 However, it is an antibody, X contains a conjugate, which is a conjugation portion that binds to the antibody at a native amino acid residue of the antibody.

[0198] The substituents associated with the conjugate of formula (I) are described in more detail below.

[0199] In a particular embodiment, each R 1R is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl. In certain embodiments, R 1 is hydrogen. In a particular embodiment, R 1 C 1~6 Alkyl or C 1~6 Substituting alkyl, or C 1~4 Alkyl or C 1~4 Substituting alkyl, or C 1~3 Alkyl or C 1~3 It is an alkyl or substituted alkyl, such as a substituted alkyl. In a particular embodiment, R 1 C 2~6 Alkenil or C 2~6 Substituting alkenyl, or C 2~4 Alkenil or C 2~4 Substituting alkenyl, or C 2~3 Alkenil or C 2~3 It is an alkenyl or substituted alkenyl, such as a substituted alkenyl. In a particular embodiment, R 1 This is an alkynyl or substituted alkynyl.

[0200] In a particular embodiment, each R 2 R is independently 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 2 is hydrogen. In a particular embodiment, R 2 C 1~6 Alkyl or C 1~6 Substituting alkyl, or C 1~4 Alkyl or C 1~4 Substituting alkyl, or C 1~3 Alkyl or C 1~3 It is an alkyl or substituted alkyl, such as a substituted alkyl. In a particular embodiment, R 2 is methyl. In a particular embodiment, R 2is isopropyl. In certain embodiments, R 2 C 2~6 Alkenil or C 2~6 Substituting alkenyl, or C 2~4 Alkenil or C 2~4 Substituting alkenyl, or C 2~3 Alkenil or C 2~3 It is an alkenyl or substituted alkenyl, such as a substituted alkenyl. In a particular embodiment, R 2 is an alkynyl or substituted alkynyl. In certain embodiments, R 2 C 5~8 Aryl or C 5~8 Substitutive aryls are aryls or substitution aryls such as C5 aryl or C5 substitution aryl, or C6 aryl or C6 substitution aryl. In certain embodiments, R 2 C 5~8 heteroaryl or C 5~8 A substituted heteroaryl, for example, a C5 heteroaryl or C5-substituted heteroaryl, or a C6 heteroaryl or C6-substituted heteroaryl, or any other heteroaryl or substituted heteroaryl. In a particular embodiment, R 2 C 3~8 Cycloalkyl or C 3~8 Substitutive cycloalkyls, for example, C 3~6 Cycloalkyl or C 3~6 Substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 It is a cycloalkyl or substituted cycloalkyl, such as a substituted cycloalkyl. In a particular embodiment, R 2 C 3~6 Heterocycline or C 3~6 Substituted heterocyclyl, or C 3~5 Heterocycline or C 3~5 It is a heterocyclyl, such as a substituted heterocyclyl, or a substituted heterocyclyl.

[0201] In a particular embodiment, R 1 H is R 2is an alkyl or substituted alkyl. For example, in some cases, R 1 H is R 2 Is it methyl, or R 1 H is R 2 It is isopropyl.

[0202] In a particular embodiment, k1 is an integer between 1 and 10. In a particular embodiment, k1 is 1. In a particular embodiment, k1 is 2. In a particular embodiment, k1 is 3. In a particular embodiment, k1 is 4. In a particular embodiment, k1 is 5. In a particular embodiment, k1 is 6. In a particular embodiment, k1 is 7. In a particular embodiment, k1 is 8. In a particular embodiment, k1 is 9. In a particular embodiment, k1 is 10.

[0203] In a particular embodiment, k1 is 2, and each R 1 H is H, and each R 2 is an alkyl or substituted alkyl. For example, in some cases k1 is 2, and each R 1 H is H, while R is H. 2 R is methyl, and the other R is methyl 2 It is isopropyl.

[0204] In a particular embodiment, R 3 This includes severable portions, as described in detail herein. For example, in some cases, R 3 This may be a chemically cleavable portion, as described in detail herein. In some cases, R 3 This may be an enzymatically cleavable portion (e.g., a glycoside or a glycoside derivative), as described in detail herein.

[0205] In a particular embodiment, W 1 is a drug (or active agent). Further descriptions of drugs (or active agents) used in the conjugate of this subject are used in the disclosure herein.

[0206] In a particular embodiment, W 2 It is a polypeptide. For example, W 2 This may be an antibody. In a particular embodiment, W 2 As described herein, via linker L, drug W 1 It comprises one or more amino acid residues (e.g., native amino acid residues) that bind to it. In certain embodiments, a polypeptide (e.g., an antibody) binds to the rest of the conjugate via the native amino acid residues, as described herein. Further descriptions of polypeptides and antibodies used in the conjugates of this subject can be found in the disclosure herein.

[0207] In a particular embodiment, L A This is a linker (for example, the first linker). Linker L A At one end of the linker, the drug W 1 It may also bind to the antibody W at the other end of the linker. 2 It may be joined to L. A Suitable linkers are described in more detail below.

[0208] In certain embodiments, X is a conjugation moiety that binds to the antibody at a native amino acid residue of the antibody. In certain embodiments, the antibody and the drug are conjugated via the conjugation moiety X. For example, the antibody and the drug may each bind (e.g., covalently) to the conjugation moiety X, and thus the antibody and the drug are indirectly bound together via the conjugation moiety X.

[0209] In some cases, the conjugation moiety X includes maleimide or a derivative thereof. For example, the maleimide conjugation moiety may react with a cysteine ​​residue of the antibody, e.g., a native cysteine ​​residue of the antibody, and thus bind to it (e.g., covalently), thereby indirectly binding the antibody and the drug together via the maleimide conjugation moiety. An example of a maleimide conjugation moiety is illustrated in Figure 33 of this specification.

[0210] Other conjugation groups that may be used to bind to antibody cysteine ​​residues are illustrated in Figure 34 of this specification. Other conjugation groups that may be used to bind to antibody cysteine ​​residues include P5 phosphoramidates or thiobridge conjugation moieties, as shown in Figures 35 and 36, respectively.

[0211] In some cases, the conjugation moiety X comprises N-hydroxysuccinimide (NHS) or a derivative thereof. For example, the NHS-ester conjugation moiety may react with a lysine residue of the antibody, e.g., a native lysine residue of the antibody, and thus bind to it (e.g., covalently), thereby indirectly binding the antibody and the drug together via the NHS-ester conjugation moiety. An example of an NHS-ester conjugation moiety is illustrated in Figure 37 of this specification.

[0212] Other conjugation groups that may be used for conjugation include conjugation moieties for binding to the N-terminal amine of an antibody. For example, the N-terminal amine can be oxidized with N-methylpyridinium-4-carboxyaldehyde benzenesulfonate salt (Lapoport salt, RS) to yield an aldehyde or ketone-tagged antibody, which can then be conjugated using an oxime or hydrazone linkage. An example of conjugation to an N-terminal amine is illustrated in Figure 38 of this specification.

[0213] In a particular embodiment, the conjugate of formula (I) is the linker LA The linker may be used to conjugate one or more desired moieties (e.g., a drug or active agent) to one or more polypeptides via a conjugation moiety X. The linker may be conjugated (e.g., covalently) to the conjugation moiety at any convenient position (e.g., as described herein). For example, the linker may be conjugated to a drug. The conjugation moiety may be used to conjugate the linker (and therefore the drug) to a polypeptide, e.g., an antibody. For example, the conjugation moiety may be used to conjugate the linker (and therefore the drug) to a native amino acid residue of an antibody, as described herein.

[0214] In some cases, as shown in equation (I) above, L A W via the conjugation portion X 2 It joins to W, and therefore W 2 Linker L via the conjugation portion A It is indirectly connected to W. 2 It is an antibody, and therefore, L A It binds to the antibody via the conjugation portion X, for example, the linker L A It indirectly binds to the antibody via the conjugation portion X.

[0215] Any suitable linker, linker L in the conjugate and compound of this subject. A It can be used for the following purposes. In a particular embodiment, linker L A The group may include a group selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, 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 AThis may include alkyl or substituted alkyl groups. In certain embodiments, linker L A This may include an alkenyl or substituted alkenyl group. In certain embodiments, the linker L A This may include an alkynyl or substituted alkynyl group. In certain embodiments, the linker L A It may contain an alkoxy or substituted alkoxy group. In certain embodiments, linker L A It may contain an amino or substituted amino group. In certain embodiments, linker L A It may contain a carboxyl or carboxyl ester group. In certain embodiments, linker L A It may contain an acylamino group. In certain embodiments, linker L A This may include an alkylamide or a substituted alkylamide group. In certain embodiments, the linker L A This may include an aryl or substituted aryl group. In certain embodiments, the linker L A This may include a heteroaryl or substituted heteroaryl group. In certain embodiments, the linker L A This may include a cycloalkyl or substituted cycloalkyl group. In certain embodiments, the linker L A This may include a heterocyclyl or a substituted heterocyclyl group.

[0216] In a particular embodiment, linker L AThe polymer may include polymers. For example, the polymer may include polyalkylene glycols and their derivatives, including polyethylene glycol, methoxypolyethylene glycol, polyethylene glycol homopolymer, polypropylene glycol homopolymer, copolymers of ethylene glycol and propylene glycol (e.g., homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group), polyvinyl alcohol, polyvinyl ethyl ether, polyvinylpyrrolidone, and combinations thereof. In certain embodiments, the polymer is 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.

[0217] In some embodiments, L A This is a linker (for example, the first linker) described by the following formula: -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e -(L 6 ) f -, In the formula, L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 Each of these is an independent linker subunit, and a, b, c, d, e, and f are each independently 0 or 1, and the sum of a, b, c, d, e, and f is between 1 and 6.

[0218] 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.

[0219] In certain embodiments, linker subunit L 1 binds to conjugation moiety X (e.g., as shown in Formula (I) above). In certain embodiments, linker subunit L 2 , when present, binds to the -NR 1 - group (e.g., as shown in Formula (I) above). In certain embodiments, linker subunit L 3 , when present, binds to the -NR 1 - group (e.g., as shown in Formula (I) above). In certain embodiments, linker subunit L 4 , when present, binds to the -NR 1 - group (e.g., as shown in Formula (I) above). In certain embodiments, linker subunit L 5 , when present, binds to the -NR 1 - group (e.g., as shown in Formula (I) above). In certain embodiments, linker subunit L 6 , when present, binds to the -NR 1- group (for example, as shown in Formula (I) above).

[0220] Any convenient linker subunit may be used for linker L A . Suitable linker subunits of interest include, but are not limited to, units of polymers such as polyethylene glycol, polyethylene and polyacrylate, amino acid residue(s), carbohydrate-based polymers or carbohydrate residues and derivatives thereof, polynucleotides, alkyl groups, aryl groups, heterocyclic groups, combinations thereof, and substituted versions thereof. In some embodiments, L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 (if present) each independently comprise one or more groups selected from polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl, aryl groups, substituted aryl groups, and diamines (e.g., linking groups including alkylene diamines).

[0221] In some embodiments, L 1 (if present) comprises polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 1 comprises polyethylene glycol. In some embodiments, L 1 comprises modified polyethylene glycol. In some embodiments, L 1 comprises an amino acid residue. In some embodiments, L 1 comprises an alkyl group or substituted alkyl. In some embodiments, L 1 comprises an aryl group or substituted aryl group. In some embodiments, L 1 comprises a diamine (e.g., a linking group comprising an alkylene diamine).

[0222] In some embodiments, L 2(If present) includes polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 2 It contains polyethylene glycol. In some embodiments, L 2 It contains modified polyethylene glycol. In some embodiments, L 2 It contains an amino acid residue. In some embodiments, L 2 This includes an alkyl group or a substituted alkyl group. In some embodiments, L 2 This includes an aryl group or a substituted aryl group. In some embodiments, L 2 It contains a diamine (for example, a linking group including alkylenediamine).

[0223] In some embodiments, L 3 (If present) includes polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 3 It contains polyethylene glycol. In some embodiments, L 3 It contains modified polyethylene glycol. In some embodiments, L 3 It contains an amino acid residue. In some embodiments, L 3 This includes an alkyl group or a substituted alkyl group. In some embodiments, L 3 This includes an aryl group or a substituted aryl group. In some embodiments, L 3 It contains a diamine (for example, a linking group including alkylenediamine).

[0224] In some embodiments, L 4 (If present) includes polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 4 It contains polyethylene glycol. In some embodiments, L 4It contains modified polyethylene glycol. In some embodiments, L 4 It contains an amino acid residue. In some embodiments, L 4 This includes an alkyl group or a substituted alkyl group. In some embodiments, L 4 This includes an aryl group or a substituted aryl group. In some embodiments, L 4 It contains a diamine (for example, a linking group including alkylenediamine).

[0225] In some embodiments, L 5 (If present) includes polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 5 It contains polyethylene glycol. In some embodiments, L 5 It contains modified polyethylene glycol. In some embodiments, L 5 It contains an amino acid residue. In some embodiments, L 5 This includes an alkyl group or a substituted alkyl group. In some embodiments, L 5 This includes an aryl group or a substituted aryl group. In some embodiments, L 5 It contains a diamine (for example, a linking group including alkylenediamine).

[0226] In some embodiments, L 6 (If present) includes polyethylene glycol, modified polyethylene glycol, amino acid residues, alkyl groups, substituted alkyl groups, aryl groups, substituted aryl groups, or diamines. In some embodiments, L 6 It contains polyethylene glycol. In some embodiments, L 6 It contains modified polyethylene glycol. In some embodiments, L 6 It contains an amino acid residue. In some embodiments, L 6 This includes an alkyl group or a substituted alkyl group. In some embodiments, L 6comprises an aryl group or a substituted aryl group. In some embodiments, L 6 comprises a diamine (e.g., a linking group including an alkylenediamine).

[0227] In some embodiments, L A is a linker comprising -(L 1 ) a -(L 2 ) b -(L 3 ) c -(L 4 ) d -(L 5 ) e -(L 6 ) f -, 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 -, -(L 6 ) f - is -(T 6 -V 6 ) f -, T 1 , T 2 , T 3 , T 4 , T 5, and T 6 If present, it is a tether group. V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 If present, it is a covalent bond or linking functional group. a, b, c, d, e, and f are each independently either 0 or 1, and the sum of a, b, c, d, e, and f is between 1 and 6.

[0228] As described above, in a particular embodiment, L 1 T is coupled to the conjugation portion X (for example, as shown in equation (I) above). Therefore, in a particular embodiment, T 1 It is joined to the conjugation portion X (for example, as shown in equation (I) above). In a particular embodiment, V 1 -NR 1 - Bonds to the base (for example, as shown in formula (I) above). In a particular embodiment, L 2 If present, -NR 1 - Bonds to the base (for example, as shown in formula (I) above). Therefore, in a particular embodiment, T 2 If present, -NR 1 - Bonds to a base (for example, as shown in formula (I) above), or V 2 If present, -NR 1 - Bonds to the base (for example, as shown in formula (I) above). In a particular embodiment, L 3 If present, -NR 1 - Bonds to the base (for example, as shown in formula (I) above). Therefore, in a particular embodiment, T 3 If present, -NR 1 - Bonds to a base (for example, as shown in formula (I) above), or V 3 If present, -NR 1 - Bonds to the base (for example, as shown in formula (I) above). In a particular embodiment, L 4 If present, -NR1 - Bonds to the base (for example, as shown in formula (I) above). Therefore, in a particular embodiment, T 4 If present, -NR 1 - Bonds to a base (for example, as shown in formula (I) above), or V 4 If present, -NR 1 - Bonds to the base (for example, as shown in formula (I) above). In a particular embodiment, L 5 If present, -NR 1 - Bonds to the base (for example, as shown in formula (I) above). Therefore, in a particular embodiment, T 5 If present, -NR 1 - Bonds to a base (for example, as shown in formula (I) above), or V 5 If present, -NR 1 - Bonds to the base (for example, as shown in formula (I) above). In a particular embodiment, L 6 If present, -NR 1 - Bonds to the base (for example, as shown in formula (I) above). Therefore, in a particular embodiment, T 6 If present, -NR 1 - Bonds to a base (for example, as shown in formula (I) above), or V 6 If present, -NR 1 - It binds to the base (for example, as shown in equation (I) above).

[0229] Tether group T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 In relation to this, any convenient tether group can be used in the linker of this subject. In some embodiments, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 Each of these is a covalent bond, (C1~C 12 ) alkyl, substituted (C1~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 - comprises one or more groups independently selected from 4-amino-piperidine (4AP), acetal groups, hydrazines, disulfides, and esters, where 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.

[0230] In certain embodiments, a tether group (e.g., T) is used. 1 , T 2 , T 3 , T 4 , T 5 , and / or T 6 ) is (C1~C 12 ) alkyl or substituted (C1~C 12 ) Contains alkyl. In certain embodiments, (C1~C 12 Alkyl is a linear or branched alkyl group containing 1 to 12 carbon atoms, for example, 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some cases, (C1-C 12 ) Alkyl is C1~C 12 Alkyl, or C1-C 10 It may be an alkyl or substituted alkyl such as an alkyl, C1-C6 alkyl, or C1-C3 alkyl. In some cases, (C1-C 12 ) Alkyl is C2-alkyl. For example, (C1~C 12 ) Alkyl is C1~C 12 Alkylene, or C1-C 10 It may be an alkylene, or an alkylene or substituted alkylene such as a C1-C6 alkylene, or a C1-C3 alkylene. In some cases, (C1-C 12Alkyl is C2-alkylene (e.g., CH2CH2). In some cases, (C1~C 12 Alkyl is a C3-alkylene (e.g., CH2CH2CH2).

[0231] In a particular embodiment, substitution (C1~C 12 Alkyl is a linear or branched substituted alkyl group containing 1 to 12 carbon atoms, for example, 1 to 10 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms, or 1 to 5 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms. In some cases, substitution (C1-C 12 ) Alkyl is a substitution of C1~C 12 Alkyl or substituted C1-C 10 Alkyl, or substituted C1-C6 alkyl, or substituted C1-C3 alkyl, etc. may be substituted alkyl. In some cases, substituted (C1-C 12 )alkyl is a substituted C2-alkyl. For example, substituted (C1~C 12 ) Alkyl is a substitution of C1~C 12 Alkylene, or substituted C1-C 10 It may be an alkylene, or a substituted alkylene such as a substituted C1-C6 alkylene, or a substituted C1-C3 alkylene. In some cases, substitution (C1-C 12 )alkyl is a substituted C2 alkylene. In some cases, substituted (C1~C 12 )alkyl is a substituted C3 alkylene. For example, substituted (C1~C 12 ) Alkyl is (PEG) as described herein. n C1-C substituted with a group 12 It may contain alkylenes (e.g., C3-alkylene or C5-alkylene) (e.g., -CONH(PEG)3 or -NHCO(PEG)7), or C1-C substituted with -CONHCH2CH2SO3H groups. 12 It may contain alkylene (e.g., C3-alkylene), or C1-C substituted with -NHCOCH2SO3H groups. 12 It may contain alkylenes (e.g., C5-alkylenes).

[0232] In certain embodiments, a tether group (e.g., T) is used. 1 , T 2 , T 3 , T 4 , T 5 , and / or T 6 ) includes aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl. In some cases, a tether group (e.g., T) is included. 1 , T 2 , T 3 , T 4 , T 5 , and T 6 ) includes aryl or substituted aryl. For example, aryl can be phenyl. In some cases, substituted aryl is substituted phenyl. Substituted phenyl is (C1~C 12 ) alkyl, substituted (C1~C 12 The substituents may be substituted with one or more substituents selected from alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In some cases, the substituted aryl is a substituted phenyl, and the substituents include cleavable moieties as described herein (e.g., enzymatically cleavable moieties such as glycosides or glycoside derivatives).

[0233] In some cases, the tether group (for example, T 1 , T 2 , T 3 , T 4 , T 5 , and / or T 6 ) includes heteroaryl or substituted heteroaryl groups. In some cases, a tether group (e.g., T) is used. 1 , T 2 , T 3 , T 4 , T 5 , and T 6 ) includes cycloalkyl or substituted cycloalkyl. In some cases, a tether group (e.g., T1 , T 2 , T 3 , T 4 , T 5 , and T 6 ) comprises a heterocyclyl or a substituted heterocyclyl. In some cases, substituents on a substituted heteroaryl, substituted cycloalkyl, or substituted heterocyclyl include cleavable moieties as described herein (e.g., enzymatically cleavable moieties such as glycosides or glycoside derivatives).

[0234] In certain embodiments, a tether group (e.g., T) is used. 1 , T 2 , T 3 , T 4 , T 5 , and / or T 6 The group includes aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl, which are produced from reactions such as click reactions. For example, click reactions can be used to join two parts of a linker together during the synthesis of a linker. Examples of click reactions that can be used to produce aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl tether groups include, but are not limited to, azide-alkyne reactions, alkyne-DBCO (dibenzocyclooctin) reactions, and TCO-Tz (transcycloctentetrazine) reactions (see, for example, Figure 39).

[0235] In certain embodiments, a tether group (e.g., T) is used. 1 , T 2 , T 3 , T 4 , T 5 , and / or T 6) comprises an ethylenediamine (EDA) moiety, for example, an EDA-containing tether group. In certain embodiments, (EDA) w The EDA moiety comprises one or more EDA moieties, for example, w is an integer from 1 to 50, e.g., 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, e.g., 1, 2, 3, 4, 5, or 6). The linked ethylenediamine (EDA) moieties may optionally be substituted at one or more favorable positions with any favorable substituent, e.g., alkyl, substituted alkyl, acyl, substituted acyl, aryl, or substituted aryl. In a particular embodiment, the EDA moiety is described by the following structure: [ka] In the formula, y is an integer from 1 to 6, or 0 or 1, and each R 12 R is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, y is 1, 2, 3, 4, 5, or 6. In certain embodiments, y is 1 and r is 0. In certain embodiments, y is 1 and r is 1. In certain embodiments, y is 2 and r is 0. In certain embodiments, each R 12 These are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In a particular embodiment, any two adjacent R of EDA 12 The groups can be linked in a ring, for example, to form a piperazinyl ring. In a particular embodiment, y is 1 and two adjacent R 12The group is an alkyl group linked cyclically 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).

[0236] In certain embodiments, a tether group (e.g., T) is used. 1 , T 2 , T 3 , T 4 , T 5 , and / or T 6 ) comprises a 4-amino-piperidine (4AP) moiety (also referred to herein as piperidine-4-amino, P4A). The 4AP moiety may optionally be substituted at one or more favorable positions with any favorable substituent, such as alkyl, substituted alkyl, polyethylene glycol moiety, acyl, substituted acyl, aryl, or substituted aryl. In certain embodiments, the 4AP moiety is described by the following structure: [ka] R 12 R is selected from 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, carboxyl ester, acyl, acyloxy, acylamino, aminoacyl, alkylamide, substituted alkylamide, sulfonyl, thioalkoxy, substituted thioalkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R 12 This is the polyethylene glycol portion. In a particular embodiment, R 12 This is carboxy-modified polyethylene glycol.

[0237] In a particular embodiment, R12 The formula is (PEG) k It contains a polyethylene glycol portion as described by, which may be represented by the following structure: [ka] In the formula, k is an integer between 1 and 20, for example, 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, for example, 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 a particular embodiment, R 17 R is selected from OH, OR, COOH, or COOR, where R is selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. In certain embodiments, R 17 is COOH. In a particular embodiment, R 17 is OH. In a particular embodiment, R 17 This is an OR, for example, OCH3.

[0238] In certain embodiments, a tether group (e.g., T) is used. 1 , T 2 , T 3 , T 4 , T 5 , and / or T 6 ) is (PEG) n (PEG) n (PEG) is a polyethylene glycol or modified polyethylene glycol linkage unit. In certain embodiments, (PEG) n It is described by the following structure: [ka] In the formula, n is an integer between 1 and 50, for example, 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, for example, 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.

[0239] In certain embodiments, a tether group (e.g., T) is used. 1 , T 2 , T 3 , T 4 , T 5 , and / or T 6 ) is (AA) p The formula includes, where AA is an amino acid residue. Any convenient amino acid may be used. The amino acid of interest may include, but is not limited to, L-amino acids and D-amino acids, naturally occurring amino acids, e.g., any of the 20 main alpha-amino acids and beta-alanine, or amino acids that do not exist naturally (e.g., amino acid analogs), e.g., naturally occurring alpha-amino acids or naturally occurring beta-amino acids. In certain embodiments, p is an integer from 1 to 50, e.g., 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, e.g., 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.

[0240] In certain embodiments, a tether group (e.g., T) is used. 1 , T 2 , T 3 , T 4 , T 5 , and / or T 6) includes amino acid analogs. Amino acid analogs include compounds that are structurally and / or in shape similar 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 that have modified side chains or backbone. Amino acid analogs also include amino acid analogs that have the same stereochemistry as the naturally occurring D form, and L forms of amino acid analogs. In some cases, amino acid analogs share the backbone structure and / or side chain structure of one or more natural amino acids, and the difference(s) is one or more modifying groups in the molecule. Such modifications may include, but are not limited to, substitution of a relevant atom (such as S) with another atom (such as N), addition of a group (such as methyl or hydroxyl) or an atom (such as Cl or Br), deletion of a group, substitution of a covalent bond (substitution of a double bond with a single bond), or combinations thereof. For example, amino acid analogs may include α-hydroxy acids and α-amino acids. Examples of amino acid analogs include, but are not limited to, sulfoalanine.

[0241] In certain embodiments, a tether group (e.g., T) is used. 1 , T 2 , T 3 , T 4 , T 5 , and / or T 6 ) is formula - (CR 13 OH) m-Includes the portion described by, where in the formula, m is 0 or n is an integer such as 1 to 50, e.g., 1 to 40, 1 to 30, 1 to 20, 1 to 12, or 1 to 6, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In a particular embodiment, m is 1. In a particular embodiment, m is 2. In a particular embodiment, each R 13 is selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, 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 a particular embodiment, R 13 C 1~6 Alkyl or C 1~6 Substituting alkyl, or C 1~4 Alkyl or C 1~4 Substituting alkyl, or C 1~3 Alkyl or C 1~3 It is an alkyl or substituted alkyl, such as a substituted alkyl. In a particular embodiment, R 13 C 2~6 Alkenil or C 2~6 Substituting alkenyl, or C 2~4 Alkenil or C 2~4 Substituting alkenyl, or C 2~3 Alkenil or C 2~3 It is an alkenyl or substituted alkenyl, such as a substituted alkenyl. In a particular embodiment, R 13 is an alkynyl or substituted alkynyl. In certain embodiments, R 13 is an alkoxy or substituted alkoxy. In certain embodiments, R 13 is an amino acid or a substituted amino acid. In certain embodiments, R 13is a carboxyl or carboxyl ester. In certain embodiments, R 13 is an acyl or acyloxy. In a particular embodiment, R 13 is acylamino or aminoacyl. In certain embodiments, R 13 is an alkylamide or a substituted alkylamide. In certain embodiments, R 13 is a sulfonyl. In certain embodiments, R 13 is a thioalkoxy or a substituted thioalkoxy. In certain embodiments, R 13 C 5~8 Aryl or C 5~8 Substitutive aryls are aryls or substitution aryls such as C5 aryl or C5 substitution aryl, or C6 aryl or C6 substitution aryl. In certain embodiments, R 13 C 5~8 heteroaryl or C 5~8 A substituted heteroaryl, for example, a C5 heteroaryl or C5-substituted heteroaryl, or a C6 heteroaryl or C6-substituted heteroaryl, or any other heteroaryl or substituted heteroaryl. In a particular embodiment, R 13 C 3~8 Cycloalkyl or C 3~8 Substitutive cycloalkyls, for example, C 3~6 Cycloalkyl or C 3~6 Substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 It is a cycloalkyl or substituted cycloalkyl, such as a substituted cycloalkyl. In a particular embodiment, R 13 C 3~8 Heterocycline or C 3~8 Substituting heterocyclyls, e.g., C 3~6 Heterocycline or C 3~6 Substituted heterocyclyl, or C 3~5 Heterocycline or C 3~5 It is a heterocyclyl, such as a substituted heterocyclyl, or a substituted heterocyclyl.

[0242] In a particular embodiment, R 13 R is selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are R 13 The details are as described above.

[0243] Linking functional group, V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 In relation to this, any convenient linking functional group can be used in the linker. Examples of desired linking functional groups, but not limited to, include amino, carbonyl, amide, oxycarbonyl, carboxy, sulfonyl, sulfoxide, sulfonylamino, aminosulfonyl, thio, oxy, phospho, phosphoramidate, and thiophosphoride. In some embodiments, V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 These are, independently, covalent, -CO-, and -NR bonds. 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 Selected from SO2- and -P(O)OH-, where 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. In certain embodiments, q is 4. In certain embodiments, q is 5. In certain embodiments, q is 6.

[0244] Several embodiments, each R15 These are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, 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.

[0245] In a particular embodiment, R 15 is hydrogen. In a particular embodiment, each R 15 is hydrogen. In a particular embodiment, R 15 C 1~6 Alkyl or C 1~6 Substituting alkyl, or C 1~4 Alkyl or C 1~4 Substituting alkyl, or C 1~3 Alkyl or C 1~3 It is an alkyl or substituted alkyl, such as a substituted alkyl. In a particular embodiment, R 15 C 2~6 Alkenil or C 2~6 Substituting alkenyl, or C 2~4 Alkenil or C 2~4 Substituting alkenyl, or C 2~3 Alkenil or C 2~3 It is an alkenyl or substituted alkenyl, such as a substituted alkenyl. In a particular embodiment, R 15 is an alkynyl or substituted alkynyl. In certain embodiments, R 15 is an alkoxy or substituted alkoxy. In certain embodiments, R 15 is an amino acid or a substituted amino acid. In certain embodiments, R 15 is a carboxyl or carboxyl ester. In certain embodiments, R 15 is an acyl or acyloxy. In a particular embodiment, R 15is acylamino or aminoacyl. In certain embodiments, R 15 is an alkylamide or a substituted alkylamide. In certain embodiments, R 15 is a sulfonyl. In certain embodiments, R 15 is a thioalkoxy or a substituted thioalkoxy. In certain embodiments, R 15 C 5~8 Aryl or C 5~8 Substitutive aryls are aryls or substitution aryls such as C5 aryl or C5 substitution aryl, or C6 aryl or C6 substitution aryl. In certain embodiments, R 15 C 5~8 heteroaryl or C 5~8 A substituted heteroaryl, for example, a C5 heteroaryl or C5-substituted heteroaryl, or a C6 heteroaryl or C6-substituted heteroaryl, or any other heteroaryl or substituted heteroaryl. In a particular embodiment, R 15 C 3~8 Cycloalkyl or C 3~8 Substitutive cycloalkyls, for example, C 3~6 Cycloalkyl or C 3~6 Substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 It is a cycloalkyl or substituted cycloalkyl, such as a substituted cycloalkyl. In a particular embodiment, R 15 C 3~8 Heterocycline or C 3~8 Substituting heterocyclyls, e.g., C 3~6 Heterocycline or C 3~6 Substituted heterocyclyl, or C 3~5 Heterocycline or C 3~5 It is a heterocyclyl, such as a substituted heterocyclyl, or a substituted heterocyclyl.

[0246] In a particular embodiment, each R 15R is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, 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, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl are R 15 The details are as described above.

[0247] In certain embodiments, the tether group includes an acetal group, a disulfide, a hydrazine, or an ester. In some embodiments, the tether group includes an acetal group. In some embodiments, the tether group includes a hydrazine. In some embodiments, the tether group includes a disulfide. In some embodiments, the tether group includes an ester.

[0248] As described above, in some embodiments, L A is, -(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 containing -, where a, b, c, d, e, and f are each independently either 0 or 1, and the sum of a, b, c, d, e, and f is between 1 and 6.

[0249] In some embodiments, linker LA In, T 1 (C1~C 12 ) alkyl and substituted (C1~C 12 ) Selected from alkyl groups, T 2 , T 3 , T 4 , T 5 , and T 6 However, each operates independently, (C1~C 12 ) alkyl, substituted (C1~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 - Selected from 4-amino-piperidine (4AP), acetal group, disulfide, hydrazine, and ester, V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 These are, independently, covalent, -CO-, and -NR bonds. 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 Selected from SO2- and -P(O)OH-, where q is an integer from 1 to 6. (PEG) n teeth, [ka] In the formula, n is an integer between 1 and 30. The EDA has the following structure: [ka] The ethylenediamine moiety has the following characteristics, where y is an integer from 1 to 6, and r is 0 or 1. 4-amino-piperidine (4AP) is, [ka] And, AA represents an amino acid residue, and p is an integer from 1 to 20. Each R 12 These are independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, and any two adjacent R 12 The groups can be linked in a ring to form a piperazinyl ring. Each R 13 These are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. Each R 15 These are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0250] In a particular embodiment, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 , and V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 The following can be selected: T 1 (C1~C 12 ) is alkyl, V 1 It is -CO-, b, c, d, e, and f are each 0 or T1 (C1~C 12 ) is alkyl, V 1 It is -CO-, T 2 It is an amino acid analog, V 2 It is -NH-, T 3 (PEG) n V 3 It is -CO-, d, e, and f are each 0, or T 1 (C1~C 12 ) is alkyl, V 1 is -CONH-, T 2 This is a substitution (C1~C 12 ) is alkyl, V 2 It is -CO-, c, d, e, and f are all 0.

[0251] As described above, in some cases, the conjugation moiety X includes maleimide or a derivative thereof. For example, the maleimide conjugation moiety may bind to a cysteine ​​residue of the antibody, for example, a native cysteine ​​residue of the antibody. In these cases, the conjugate may be the conjugate of formula (II), [ka] During the ceremony, Each R 1 However, they are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R 2 These are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k1 is an integer between 1 and 10. R 3 This includes a severable portion, LA However, it was the first linker, W 1 However, it is a drug, W 2 However, it is an antibody.

[0252] In some cases, the conjugation moiety X includes acetamide or a derivative thereof. For example, the acetamide conjugation moiety may bind to a cysteine ​​residue of the antibody, e.g., a native cysteine ​​residue of the antibody. In these cases, the conjugate may be a conjugate of formula (IIa), [ka] During the ceremony, Each R 1 However, they are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R 2 These are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k1 is an integer between 1 and 10. R 3 This includes a severable portion, L A However, it was the first linker, W 1 However, it is a drug, W 2 However, it is an antibody.

[0253] In embodiments of formulas (II) and (IIa), R 1 , R 2 , k1, R 3 , L A , W 1 , and W 2 This is as described herein (for example, as with the description of formula (I) herein).

[0254] In certain embodiments of the conjugate of formula (I), an antibody may be conjugated to one drug or active agent via a conjugation moiety and a linker. In some cases, an antibody may be conjugated to two or more drugs or active agents via a conjugation moiety and a linker. For example, a conjugation moiety may be conjugated to two or more drugs or active agents. Each drug or active agent may be conjugated to the same conjugation moiety via a corresponding linker, which can then be bound to the antibody as described herein, thus conjugating the antibody to two or more drugs or active agents.

[0255] For example, in a particular embodiment of the conjugate of formula (I), the linker, L A This is a branched linker. In embodiments having a branched linker, T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , V 1 , V 2 , V 3 , V 4 , V 5 , or V 6 One of these is a branching group. In some embodiments, the branching group is -CONR 15 - and 4AP are selected. In some embodiments, the branching group is -CONR 15 - In some embodiments, the branching base is 4AP. In certain embodiments, the branching base is the second linker L B It connects to the first linker L. A The branching group is the first drug W, as shown in equation (I) above. 1 It can be coupled to the second linker L B Through, the second drug W 1a It can be combined with (for example, as shown in equation (III) below).

[0256] In a particular embodiment, the second linker L B teeth, -(L7 ) g -(L 8 ) h -(L 9 ) i -(L 10 ) j -(L 11 ) k -(L 12 ) l - It is a linker described by In the formula, L 7 , L 8 , L 9 , L 10 , L 11 , and L 12 Each of these is an independent linker subunit, and each of g, h, i, j, k, and l is independently 0 or 1, and the sum of g, h, i, j, k, and l is between 1 and 6.

[0257] In a particular embodiment, the sum of g, h, i, j, k, and l is 1. In a particular embodiment, the sum of g, h, i, j, k, and l is 2. In a particular embodiment, the sum of g, h, i, j, k, and l is 3. In a particular embodiment, the sum of g, h, i, j, k, and l is 4. In a particular embodiment, the sum of g, h, i, j, k, and l is 5. In a particular embodiment, the sum of g, h, i, j, k, and l is 6. In a particular embodiment, g, h, i, j, k, and l are each 1. In a particular embodiment, g, h, i, j, and k are each 1, and l is 0. In a particular embodiment, g, h, i, and j are each 1, and k and l are each 0. In a particular embodiment, g, h, and i are each 1, and j, k, and l are each 0. In a particular embodiment, g and h are each 1, and i, j, k, and l are each 0.

[0258] In a particular embodiment, linker subunit L 7It is bonded to the branching base. In a particular embodiment, the linker subunit L 8 If present, the second drug W 1a It is coupled to the linker subunit L. In a particular embodiment, 9 If present, the second drug W 1a It is coupled to the linker subunit L. In a particular embodiment, 10 If present, the second drug W 1a It is coupled to the linker subunit L. In a particular embodiment, 11 If present, the second drug W 1a It is coupled to the linker subunit L. In a particular embodiment, 12 If present, the second drug W 1a It is connected.

[0259] Any convenient linker subunit, second linker L B It can be used for this purpose. For example, L 1 , L 2 , L 3 , L 4 , L 5 , and L 6 In relation to this, any of the linker subunits described above, linker subunit L 7 , L 8 , L 9 , L 10 , L 11 , and L 12 It may be used for this purpose.

[0260] In a particular embodiment, the second linker L B teeth, -(L 7 ) g -(L 8 ) h -(L 9 ) i -(L 10 ) j -(L 11 ) k -(L 12 ) l - is a linker that includes, in the formula, -(L7 ) g - is, - (T 7 -V 7 ) g -and, -(L 8 ) h - is, - (T 8 -V 8 ) h -and, -(L 9 ) i - is, - (T 9 -V 9 ) i -and, -(L 10 ) j - is, - (T 10 -V 10 ) j -and, -(L 11 ) k - is, - (T 11 -V 11 ) k -and, -(L 12 ) l - is, - (T 12 -V 12 ) l -and, T 7 , T 8 , T 9 , T 10 , T 11 , and T 12 If present, it is a tether group. V 7 , V 8 , V 9 , V 10 , V 11 , and V 12 If present, it is a covalent bond or linking functional group. g, h, i, j, k, and l are each independently either 0 or 1, and the sum of g, h, i, j, k, and l is between 1 and 6.

[0261] Therefore, in a particular embodiment, the second linker L B teeth, -(T 7 -V7 ) 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 - Includes, During the ceremony, g, h, i, j, k, and l are each independently either 0 or 1. T 7 , T 8 , T 9 , T 10 , T 11 , and T 12 Each is independent, and covalently bonded, (C1~C 12 ) alkyl, substituted (C1~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 - Selected from 4-amino-piperidine (4AP), acetal group, hydrazine, disulfide, and ester, where EDA is the ethylenediamine moiety, PEG is polyethylene glycol, AA is an amino acid residue or amino acid analog, 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 These are, independently, covalent, -CO-, and -NR bonds. 15 -, -NR 15 (CH2)q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 Selected from the group consisting of SO2- and -P(O)OH-, where each q is an integer from 1 to 6. Each R 13 These are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. Each R 15 These are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

[0262] Any convenient tether group, T 7 , T 8 , T 9 , T 10 , T 11 , and T 12 It can be used for the following purposes. For example, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 In relation to this, any of the tether groups described above, tether group T 7 , T 8 , T 9 , T 10 , T 11 , and T 12 It may be used for this purpose.

[0263] Any convenient linking functional group, V 7 , V 8 , V 9 , V 10 , V 11 , and V 12 It can be used for V. 1 , V2 , V 3 , V 4 , V 5 , and V 6 In relation to the above, any of the linked functional groups, linked functional group V 7 , V 8 , V 9 , V 10 , V 11 , and V 12 It may be used for this purpose.

[0264] In a particular embodiment, each R 13 R is independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl. In these embodiments, alkyl, substituted alkyl, aryl, and substituted aryl are R 13 The details are as described above.

[0265] In a particular embodiment, each R 15 R is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, 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, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl are R 15 As described above, in these embodiments, various possible substituents are R 15 The details are as described above.

[0266] As described above, in a particular embodiment, the branching base is the second linker L B It is coupled to the second linker L. B As shown in equation (III), the second drug W 1a It can be joined, [ka] During the ceremony, Each R 4 These are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl. Each R 5 These are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k2 is an integer between 1 and 10. R 6 This includes a severable portion, W 1a It is a drug.

[0267] Therefore, L B It can bind to the compound of formula (III). For example, L B It can bond to the compound of formula (III) by the bond shown by the wavy line in formula (III).

[0268] The substituents associated with the conjugate of formula (III) are described in more detail below.

[0269] In a particular embodiment, each R 4 R is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl. In certain embodiments, R 4 is hydrogen. In a particular embodiment, R 4 C 1~6 Alkyl or C 1~6 Substituting alkyl, or C 1~4 Alkyl or C 1~4 Substituting alkyl, or C 1~3 Alkyl or C 1~3 It is an alkyl or substituted alkyl, such as a substituted alkyl. In a particular embodiment, R 4 C 2~6Alkenil or C 2~6 Substituting alkenyl, or C 2~4 Alkenil or C 2~4 Substituting alkenyl, or C 2~3 Alkenil or C 2~3 It is an alkenyl or substituted alkenyl, such as a substituted alkenyl. In a particular embodiment, R 4 This is an alkynyl or substituted alkynyl.

[0270] In a particular embodiment, each R 5 R is independently 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 5 is hydrogen. In a particular embodiment, R 5 C 1~6 Alkyl or C 1~6 Substituting alkyl, or C 1~4 Alkyl or C 1~4 Substituting alkyl, or C 1~3 Alkyl or C 1~3 It is an alkyl or substituted alkyl, such as a substituted alkyl. In a particular embodiment, R 5 is methyl. In a particular embodiment, R 5 is isopropyl. In certain embodiments, R 5 C 2~6 Alkenil or C 2~6 Substituting alkenyl, or C 2~4 Alkenil or C 2~4 Substituting alkenyl, or C 2~3 Alkenil or C 2~3 It is an alkenyl or substituted alkenyl, such as a substituted alkenyl. In a particular embodiment, R 5 is an alkynyl or substituted alkynyl. In certain embodiments, R 5 C 5~8 Aryl or C 5~8Substitutive aryls are aryls or substitution aryls such as C5 aryl or C5 substitution aryl, or C6 aryl or C6 substitution aryl. In certain embodiments, R 5 C 5~8 heteroaryl or C 5~8 A substituted heteroaryl, for example, a C5 heteroaryl or C5-substituted heteroaryl, or a C6 heteroaryl or C6-substituted heteroaryl, or any other heteroaryl or substituted heteroaryl. In a particular embodiment, R 5 C 3~8 Cycloalkyl or C 3~8 Substitutive cycloalkyls, for example, C 3~6 Cycloalkyl or C 3~6 Substituted cycloalkyl, or C 3~5 Cycloalkyl or C 3~5 It is a cycloalkyl or substituted cycloalkyl, such as a substituted cycloalkyl. In a particular embodiment, R 5 C 3~6 Heterocycline or C 3~6 Substituted heterocyclyl, or C 3~5 Heterocycline or C 3~5 It is a heterocyclyl, such as a substituted heterocyclyl, or a substituted heterocyclyl.

[0271] In a particular embodiment, R 4 H is R 5 is an alkyl or substituted alkyl. For example, in some cases, R 4 H is R 5 Is it methyl, or R 4 H is R 5 It is isopropyl.

[0272] In a particular embodiment, k2 is an integer between 1 and 10. In a particular embodiment, k2 is 1. In a particular embodiment, k2 is 2. In a particular embodiment, k2 is 3. In a particular embodiment, k2 is 4. In a particular embodiment, k2 is 5. In a particular embodiment, k2 is 6. In a particular embodiment, k2 is 7. In a particular embodiment, k2 is 8. In a particular embodiment, k2 is 9. In a particular embodiment, k2 is 10.

[0273] In a particular embodiment, k2 is 2, and each R 4 H is H, and each R 5 is an alkyl or substituted alkyl. For example, in some cases k2 is 2, and each R 4 H is H, while R is H. 5 R is methyl, and the other R is methyl 5 It is isopropyl.

[0274] In a particular embodiment, R 6 This includes severable portions, as described in detail herein. For example, in some cases, R 6 This may be a chemically cleavable portion, as described in detail herein. In some cases, R 6 This may be an enzymatically cleavable portion (e.g., a glycoside or a glycoside derivative), as described in detail herein.

[0275] In a particular embodiment, W 1a This is a drug (or active agent). Second linker L B In a particular embodiment, as shown in formula (III), the left side of the linker structure is bonded to a branching group, and the right side of the linker structure is bonded to a second drug W 1a It binds to. Further description of the drugs (or active agents) used in the conjugate of this subject can be found in the disclosure herein.

[0276] In a particular embodiment of the branch linker, T 1 , T2 , T 3 , T 4 , T 5 and T 6 and V 1 , V 2 , V 3 , V 4 , V 5 and V 6 , and T 7 , T 8 , T 9 , T 10 , T 11 and T 12 and V 7 , V 8 , V 9 , V 10 , V 11 and V 12 The following can be selected: T 1 (C1~C 12 ) is alkyl, V 1 is a branching group (-CONR 15 )- and, T 2 (C1~C 12 ) is alkyl, V 2 is -CONH-, T 3 This is a substitution (C1~C 12 ) is alkyl, V 3 It is -CO-, d, e, and f are each 0, T 7 (C1~C 12 ) is alkyl, V 7 is -CONH-, T 8 This is a substitution (C1~C 12 ) is alkyl, V 8 It is -CO-, i, j, k, and l are each 0 or T 1 (C1~C 12 ) is alkyl, V 1 It is -CO-, T 2 It is a branching group (4AP), and V 2It is -CO-, T 3 (PEG) n V 3 is -CONH-, T 4 This is a substitution (C1~C 12 ) is alkyl, V 4 However, it is -CO-, e and f are both 0, T 7 (PEG) n V 7 is -CONH-, T 8 This is a substitution (C1~C 12 ) is alkyl, V 8 It is -CO-, i, j, k, and l are each 0 or T 1 (C1~C 12 ) is alkyl, V 1 It is -CO-, T 2 It is a branching group (4AP), and V 2 It is -CO-, T 3 (C1~C 12 ) is alkyl, V 3 It does not exist, T 4 It is a heteroaryl (triazole), and V 4 It does not exist, T 5 (C1~C 12 ) is alkyl, V 5 is -CONH-, T 6 This is a substitution (C1~C 12 ) is alkyl, V 6 It is -CO-, T 7 (PEG) n V 7 is -CONH-, T 8 This is a substitution (C1~C 12 ) is alkyl, V 8It is -CO-, i, j, k, and l are all 0.

[0277] A combination of the same or different payloads (e.g., drugs or active agents) may be conjugated to an antibody via a branched linker. In certain embodiments, the two payloads (e.g., drugs or active agents) bound to the branched linker are the same payload (e.g., drugs or active agents). For example, a first branch of the branched linker may bind to a payload (e.g., drugs or active agents), and a second branch of the branched linker may bind to the same payload (e.g., drugs or active agents) as the first branch. In other embodiments, the two payloads (e.g., drugs or active agents) bound to the branched linker are different payloads (e.g., drugs or active agents). For example, a first branch of the branched linker may bind to a first payload (e.g., drugs or active agents), and a second branch of the branched linker may bind to a second payload (e.g., drugs or active agents) different from the first payload (e.g., drugs or active agents) bound to the first branch.

[0278] For example, in a particular embodiment, the branched linker is a first drug W 1 and the second drug W 1a It joins with W. 1 and W 1a These are the same drug. In other cases, W 1 and W 1a These are different drugs.

[0279] In some embodiments, when two different drugs or active agents are bound to a branched linker, the drugs or active agents may be selected from drugs and active agents that have a synergistic therapeutic effect. "Synergistic," "synergistic effect," or "synergistic effect" means a therapeutic effect greater than the sum of the effects of using the drugs or active agents individually. For example, in some cases, the use of two different drugs or active agents bound to a branched linker may provide a low therapeutically effective concentration in which both payloads act, thereby increasing the overall efficacy of the ADC.

[0280] In some embodiments, when two different drugs or active agents are bound to a branched linker, the drugs or active agents may be selected from drugs and active agents that provide enhanced therapeutic benefits compared to when the drugs or active agents are used separately. For example, the drugs or active agents may provide an increased effect on drug delivery by the ADC (for example, some payloads, such as iRGD peptides, may increase extravasation into tissues and enhance penetration into tumors).

[0281] In some embodiments, when two different drugs or active agents are bound to a branched linker, the drugs or active agents may be selected from drugs and active agents that use different mechanisms of action. In some cases, this can result in a reduction of tumor drug resistance by targeting multiple pathways. Examples of payload combinations include, but are not limited to, cytotoxic drugs, immunomodulatory molecules for activating or inhibiting immune cell populations, cytokines, hormones, and radioisotopes-filled chelating agents.

[0282] In some embodiments, when two different payloads are coupled to the branched linker, the payloads may be selected from a combination of a drug or active agent and a detectable label. For example, the first payload may be a detectable label used as a contrast agent or tracer to detect the location of the ADC in vivo, while the second payload may be a drug or active agent that provides therapeutic activity.

[0283] Various embodiments of linkers capable of coupling a conjugation portion to a drug or active agent are described in detail herein. For example, in some cases, the linker is a cleavable linker, e.g., a cleavable linker as described herein.

[0284] In a particular embodiment, the conjugate is such that the antibody and drug are linked to a linker (e.g., L) as described above. A and / or L B An antibody-drug conjugate is linked together by a linker. In some cases, the linker is a cleavable linker. A cleavable linker is a linker containing one or more cleavable moieties, each containing one or more bonds that can dissociate under certain conditions, and thus separate the cleavable linker into two or more separable parts. For example, a cleavable moiety may contain one or more covalent bonds that can dissociate or decompose under certain conditions, separating the cleavable linker into two or more parts. Thus, a cleavable linker may be included in an antibody-drug conjugate such that, under suitable conditions, the cleavable linker is cleaved to separate or release the drug from the antibody at the desired target site of action of the drug.

[0285] In some cases, a cleavable linker includes two cleavable parts, such as a first cleavable part and a second cleavable part. The cleavable parts may be configured such that cleavage of both cleavable parts is required to separate or release the drug from the antibody at a desired target site of action of the drug. For example, cleavage of a cleavable linker can be achieved by first cleaving one of the two cleavable parts and then cleaving the other of the two cleavable parts. In certain embodiments, a cleavable linker includes a first cleavable part and a second cleavable part that interferes with the cleavage of the first cleavable part. "Interfering with cleavage" means that the presence of the uncleaved second cleavable part reduces the likelihood of cleavage of the first cleavable part, or substantially inhibits cleavage, and therefore substantially reduces or prevents cleavage of the cleavable linker. For example, the presence of the uncleaved second cleavable part can interfere with the cleavage of the first cleavable part. The presence of a second cleavable region interferes with the cleavage of the first cleavable region, thereby substantially reducing or preventing the release of drug from the antibody. For example, premature release of drug from the antibody can be substantially reduced or prevented until the antibody-drug conjugate is at or near the desired target site of action of the drug.

[0286] In some cases, the second cleavable portion interferes with the cleavage of the first cleavable portion, so cleavage of the cleavable linker can be achieved by first cleaving the second cleavable portion and then cleaving the first cleavable portion. Cleavage of the second cleavable portion can reduce or eliminate interference with the cleavage of the first cleavable portion by exposing the first cleavable portion, and thus allow the first cleavable portion to be cleaved. Cleavage of the first cleavable portion can release the drug from the antibody-drug conjugate by dissociating or separating the cleavable linker into two or more parts as described above. In some cases, cleavage of the first cleavable portion does not substantially occur in the presence of an uncleaved second cleavable portion. Substantially, this means that in the presence of a second, uncuttable portion, the cut of the first cuttable portion is less than or equal to about 10%; for example, in the presence of a second, uncuttable portion, the cut of the first cuttable portion is less than or equal to about 9%, or less than or equal to about 8%, or less than or equal to about 7%, or less than or equal to about 6%, or less than or equal to about 5%, or less than or equal to about 4%, or less than or equal to about 3%, or less than or equal to about 2%, or less than or equal to about 1%, or less than or equal to about 0.5%, or less than or equal to about 0.1%.

[0287] In other words, the second cleavable portion can protect the first cleavable portion from cleavage. For example, the presence of an uncleaved second cleavable portion protects the first cleavable portion from cleavage, and thus substantially reduces or prevents premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired target site of action of the drug. Thus, cleavage of the second cleavable portion exposes the first cleavable portion (e.g., deprotects the first cleavable portion), and thus allows the first cleavable portion to be cleaved, which results in cleavage of the cleavable linker, and consequently separates or releases the drug from the antibody at the desired target site of action of the drug, as described above. In some cases, cleavage of the second cleavable portion exposes the first cleavable portion to subsequent cleavage, but cleavage of the second cleavable portion itself does not result in cleavage of the cleavable linker (i.e., cleavage of the first cleavable portion is still necessary to cleave the cleavable linker).

[0288] Each cleavable portion within a cleavable linker may be an enzymatically cleavable portion. For example, the first cleavable portion may be the first enzymatically cleavable portion, and the second cleavable portion may be the second enzymatically cleavable portion. An enzymatically cleavable portion is a cleavable portion that can be separated into two or more parts as described above through the enzymatic action of an enzyme. An enzymatically cleavable portion may be any cleavable portion that can be cleaved through the enzymatic action of an enzyme, such as a peptide or glycoside, but is not limited to these. In some cases, the enzyme that cleaves the enzymatically cleavable portion is present at the desired target site of action, such as the desired target site of action of a drug released from an antibody-drug conjugate. In some cases, the enzyme that cleaves the enzymatically cleavable portion is not present in significant amounts in other areas, such as whole blood, plasma, or serum. Therefore, while the cleavage of the enzymatically cleavable portion can be controlled so that substantial cleavage occurs at the desired site of action, the cleavage may not significantly occur in other areas (e.g., systemically) or before the antibody-drug conjugate reaches the desired site of action.

[0289] For example, as described herein, the antibody-drug conjugates of this disclosure can be used for the treatment of cancer, such as for the delivery of cancer therapy drugs to desired sites of action where cancer cells are located. In some cases, enzymes such as the protease enzyme cathepsin B may be cancer biomarkers that are overexpressed in cancer cells. The overexpression, and therefore localization, of a specific enzyme in cancer can be used in the context of an enzymatically cleavable moiety contained in the cleavable linker of the antibody-drug conjugate of this disclosure to specifically release a drug at a desired site of action (i.e., the site of cancer (and the overexpressed enzyme)). Thus, in some embodiments, the enzymatically cleavable moiety is a cleavable moiety (e.g., a peptide) that can be cleaved by an enzyme overexpressed in cancer cells. For example, the enzyme may be the protease enzyme cathepsin B. Thus, in some cases, the enzymatically cleavable moiety is a cleavable moiety (e.g., a peptide) that can be cleaved by a protease enzyme such as cathepsin B.

[0290] In certain embodiments, the enzymatically cleavable portion is a peptide. The peptide can be any peptide that is suitable for use in a cleavable linker and can be cleaved through the enzymatic action of an enzyme. Non-limiting examples of peptides that can be used as an enzymatically cleavable portion include, for example, Val-Ala and Phe-Lys. For example, the first cleavable portion (i.e., the cleavable portion protected from premature cleavage by the second cleavable portion) may include a peptide. The presence of the uncleaved second cleavable portion protects the first cleavable portion (peptide) from cleavage by a protease enzyme (e.g., cathepsin B), and thus substantially reduces or prevents premature release of the drug from the antibody until the antibody-drug conjugate is at or near the desired target site of action of the drug. In some cases, one of the amino acid residues of the peptide containing the first cleavable portion is linked to or contains a substituent, the substituent contains the second cleavable portion. In some cases, the second cleavable portion contains a glycoside.

[0291] For example, in the context of the formulas described herein, the cleavable portion is a peptide, for example, a portion included in the formulas described herein (for example, a portion included in formulas (I), (II), (III), (IV), (V), or (VI) described herein): [ka] It may be a peptide described by [the relevant authority].

[0292] In some embodiments, the enzymatically cleavable portion is a sugar moiety such as a glycoside (or glycosyl). In some cases, the glycoside can promote increased hydrophilicity of the cleavable linker compared to a cleavable linker that does not contain a glycoside. The glycoside can be any glycoside or glycoside derivative that is suitable for use in the cleavable linker and can be cleaved by the enzymatic action of an enzyme. For example, the second cleavable portion (i.e., a cleavable portion that protects the first cleavable portion from premature cleavage) can be a glycoside or a glycoside derivative. For example, in some embodiments, the first cleavable portion includes a peptide, and the second cleavable portion includes a glycoside or a glycoside derivative. In certain embodiments, the second cleavable portion is a glycoside or glycoside derivative selected from glucuronides, galactosides, glucosides, mannosides, fucosides, O-GlcNAc, and O-GalNAc. In some cases, the second cleavable part is a glucuronide. In some cases, the second cleavable part is a galactoside. In some cases, the second cleavable part is a glucoside. In some cases, the second cleavable part is a mannoside. In some cases, the second cleavable part is a fucoside. In some cases, the second cleavable part is O-GlcNAc. In some cases, the second cleavable part is O-GalNAc.

[0293] For example, in the context of the formulas described herein, the cleavable portion may be a glycoside or a glycoside derivative, for example, R 3 or R 6 is a glycoside or a glycoside derivative (for example, as shown in formulas (I), (II), (III), (IV), (V), or (VI) described herein).

[0294] Glycosides or glycoside derivatives can be covalently bonded to cleavable linkers via glycosidic bonds. Glycosidic bonds can link glycosides or glycoside derivatives to cleavable linkers via various types of bonds, including, but not limited to, O-glycosidic bonds (O-glycosids), N-glycosidic bonds (glycosylamines), S-glycosidic bonds (thioglycosids), or C-glycosidic bonds (C-glycosids or C-glycosyls). In some cases, the glycosidic bond is an O-glycosidic bond (O-glycosid). In some cases, glycosides or glycoside derivatives can be enzymatically cleaved away from the cleavable linker they bond to (e.g., via enzymatic hydrolysis of the glycosidic bond). A glycoside or glycoside derivative can be removed from or cleaved from a cleavable linker by any convenient enzyme capable of performing cleavage (hydrolysis) of the glycosidic bond that binds the glycoside or glycoside derivative to the cleavable linker. Examples of enzymes that can be used to mediate the cleavage (hydrolysis) of the glycosidic bond that binds the glycoside or glycoside derivative to the cleavable linker include glucuronidases, glycosidases, e.g., galactosidases, glucosidases, mannosidases, and fucosidases. Other suitable enzymes may also be used to mediate the cleavage (hydrolysis) of the glycosidic bond that binds the glycoside or glycoside derivative to the cleavable linker. In some cases, the enzyme used to mediate the cleavage (hydrolysis) of the glycosidic bond that binds the glycoside or glycoside derivative to the cleavable linker is found at or near the desired site of action for the drug in the antibody-drug conjugate. For example, the enzyme may be a lysosomal enzyme, such as a lysosomal glycosidase, found in cells at or near the desired site of action of the drug in an antibody-drug conjugate. In some cases, the enzyme is an enzyme found at or near the target site where an enzyme mediating the cleavage of the first cleavable portion is found.

[0295] Embodiments including a linker having the two severable portions described above may also be referred to as having a “tandem-cut” linker.

[0296] In certain embodiments, the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc. For example, in some embodiments, the glycoside or glycoside derivative has the following structure: [ka] [ka] It can be selected from the following.

[0297] In a particular embodiment, the conjugate of formula (I) is as follows: [ka] [ka] [ka] [ka] [ka] [ka]

[0298] Any of the chemical entities, linkers, conjugation portions, or cleavable portions described above may be adapted for use in the compounds and conjugates of this subject.

[0299] Further disclosures relating to severable linkers can be found in U.S. Application No. 17 / 423,796, filed on 22 January 2020, and U.S. Application No. 17 / 531,343, filed on 19 November 2021, the respective disclosures of which are incorporated herein by reference.

[0300] Compounds useful for generating conjugates This disclosure provides compounds useful for generating the conjugates described herein. In certain embodiments, the compounds may comprise a conjugation moiety useful for conjugating a polypeptide (e.g., an antibody) with a drug or active agent. For example, the compound may be conjugated to a polypeptide (antibody) and also to a drug or active agent, and thus indirectly conjugate the polypeptide (antibody) and the drug together.

[0301] In a particular embodiment, the compound is a compound of formula (IV), [ka] During the ceremony, Each R 1 However, they are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R 2 These are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k1 is an integer between 1 and 10. R 3 This includes a severable portion, L A However, it was the first linker, W 1 However, it is a drug, X is a conjugation region configured to bind to the antibody at a native amino acid residue of the antibody.

[0302] Regarding the compound of formula (IV), substituent R 1 , R 2 , R 3 , R 4 , R 5 , R 6 k1, k2, L A , L B , W 1 , W 1a , and X are as described above with respect to the conjugate of equation (I). Similarly, the first linker L A and the second linker L B Regarding 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 , and T 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 with respect to the conjugate of formula (I).

[0303] As described herein, in some cases, the conjugation moiety X comprises maleimide or a derivative thereof. For example, the maleimide conjugation moiety may bind to a cysteine ​​residue of the antibody, e.g., a native cysteine ​​residue of the antibody. In these cases, the compound may be a compound of formula (V), [ka] During the ceremony, Each R 1However, they are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R 2 These are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k1 is an integer between 1 and 10. R 3 This includes a severable portion, L A This is the first linker, W 1 It is a drug.

[0304] In certain embodiments disclosed herein, the conjugation portion X comprises an alpha-haloacetamide, such as alpha-bromoacetamide. In such embodiments, a cysteine ​​residue of the antibody, such as a natural cysteine ​​residue, can be replaced by a halogen to achieve conjugation. In such one embodiment where X comprises an alpha-haloacetamide, the compound is of formula (Va): [ka] It has, in the formula, Each R 1 However, they are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R 2 These are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k1 is an integer between 1 and 10. R 3 This includes a severable portion, L A This is the first linker, W 1 It is a drug.

[0305] In the embodiment of formula (V), R 1 , R 2 , R 3 k1, L A , and W 1 This is as described herein (for example, as with the descriptions of formulas (I) and (IV) herein).

[0306] In a particular embodiment, T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 , and V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 The following can be selected: T 1 (C1~C 12 ) is alkyl, V 1 It is -CO-, b, c, d, e, and f are each 0 or T 1 (C1~C 12 ) is alkyl, V 1 It is -CO-, T 2 It is an amino acid analog, V 2 It is -NH-, T 3 (PEG) n V 3 It is -CO-, d, e, and f are each 0, or T 1 (C1~C 12 ) is alkyl, V 1 is -CONH-, T 2 This is a substitution (C1~C 12 ) is alkyl, V 2It is -CO-, c, d, e, and f are all 0.

[0307] As described herein, in certain embodiments, the branching group is the second linker L B It is coupled to the second linker L. B As shown in equation (VI), the second drug W 1a It can be joined, [ka] During the ceremony, Each R 4 However, they are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R 5 These are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k2 is an integer between 1 and 10. R 6 This includes a severable portion, W 1a It is a drug.

[0308] Therefore, L B L can bind to the compound of formula (VI). For example, L B It can bond to the compound of formula (VI) by the bond shown by the wavy line in formula (VI).

[0309] In the embodiment of formula (VI), R 4 , R 5 , R 6 , k2, and W 1a This is as described herein (for example, as with respect to formula (III) herein).

[0310] In a particular embodiment of the branch linker, T 1 , T 2, T 3 , T 4 , T 5 and T 6 and V 1 , V 2 , V 3 , V 4 , V 5 and V 6 , and T 7 , T 8 , T 9 , T 10 , T 11 and T 12 and V 7 , V 8 , V 9 , V 10 , V 11 and V 12 The following can be selected: T 1 (C1~C 12 ) is alkyl, V 1 is a branching group (-CONR 15 )- and, T 2 (C1~C 12 ) is alkyl, V 2 is -CONH-, T 3 This is a substitution (C1~C 12 ) is alkyl, V 3 It is -CO-, d, e, and f are each 0, T 7 (C1~C 12 ) is alkyl, V 7 is -CONH-, T 8 This is a substitution (C1~C 12 ) is alkyl, V 8 It is -CO-, i, j, k, and l are each 0 or T 1 (C1~C 12 ) is alkyl, V 1 It is -CO-, T 2 It is a branching group (4AP), and V 2 It is -CO-, T 3 (PEG) n V 3 is -CONH-, T 4 This is a substitution (C1~C 12 ) is alkyl, V 4 It is -CO-, e and f are both 0, T 7 (PEG) n V 7 is -CONH-, T 8 This is a substitution (C1~C 12 ) is alkyl, V 8 It is -CO-, i, j, k, and l are each 0 or T 1 (C1~C 12 ) is alkyl, V 1 It is -CO-, T 2 It is a branching group (4AP), and V 2 It is -CO-, T 3 (C1~C 12 ) is alkyl, V 3 It does not exist, T 4 It is a heteroaryl (triazole), and V 4 It does not exist, T 5 (C1~C 12 ) is alkyl, V 5 is -CONH-, T 6 This is a substitution (C1~C 12 ) is alkyl, V 6 It is -CO-, T 7 (PEG) n V 7 is -CONH-, T 8 This is a substitution (C1~C 12 ) is alkyl, V 8 It is -CO-, i, j, k, and l are all 0.

[0311] The compound of formula (VI) can be used in the conjugation reactions described herein, in which a drug or active agent bound to the conjugation moiety is conjugated to a polypeptide (e.g., an antibody) to form an antibody-drug conjugate. In a particular embodiment, the compound of formula (VI) has the following structure [ka] [ka] [ka] [ka] [ka] [ka] It holds.

[0312] Any of the chemical entities, linkers, conjugation portions, and cleavable portions described above may be adapted for use in the compounds and conjugates of this subject.

[0313] Polypeptides and antibodies As shown above, the subject conjugate is substituent W 2This may include polypeptides (e.g., antibodies). As used herein, amino acids may be referred to by their standard names, their standard three-letter abbreviations, and / or their standard one-letter abbreviations, for example, alanine or Ala or A, cysteine ​​or Cys or C, aspartic acid or Asp or D, glutamic acid or Glu or E, phenylalanine or Phe or F, glycine or Gly or G, histidine or His or H, isoleucine or Ile or I, lysine or Lys or K, leucine or Leu or L, methionine or Met or M, asparagine or Asn or N, proline or Pro or P, glutamine or Gln 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.

[0314] In certain embodiments, the amino acid sequence of the polypeptide (antibody) is the native amino acid sequence of the polypeptide (antibody). In these embodiments, the conjugation moiety can be conjugated to the antibody at the native amino acid residues of the antibody. For example, a maleimide-based conjugation can be used to conjugate one or more drugs or active agents to the polypeptide (e.g., antibody) at one or more native cysteine ​​residues of the polypeptide (e.g., antibody). The native cysteine ​​residues of the antibody may typically originate from cysteine ​​residues involved in disulfide bonding, such as the eight hinged cysteine ​​residues that form the intrachain disulfide bond of the antibody. In some cases, the disulfide bond can be reduced to provide cysteine ​​residues whose sulfhydryl groups react with and become available for conjugation (e.g., covalent bonding) to the conjugation moiety (e.g., maleimide conjugation moiety) described herein.

[0315] In other instances, N-hydroxysuccinimide (NHS ester)-based conjugations can be used to conjugate one or more drugs or active agents to a polypeptide (e.g., an antibody) at one or more native lysine residues of the polypeptide (e.g., an antibody). The native lysine residues of the antibody may contain amine groups that are available to react with and bind (e.g., covalently) to the conjugation moiety (e.g., the NHS-ester conjugation moiety) described herein.

[0316] The antibodies used in the antibody-drug conjugates of this disclosure are, but are not limited to, antigens present on cancer cells, antigens present on autoimmune cells, antigens present on pathogenic microorganisms, antigens present on virus-infected cells (e.g., human immunodeficiency virus-infected cells), and antigens present on disease cells, and may have any of the following antigen-binding specificities. For example, the antibody conjugate may bind to an antigen, and the antigen may be present on the surface of a cell. The antibody conjugates of this disclosure have a suitable binding affinity, for example, 5 × 10⁻⁶ -6 M~10 -7 M, 10 -7 M~5×10 -7 M, 5×10 -7 M~10 -8 M, 10 -8 M~5×10 -8 M, 5×10 -8 M~10 -9 M, or 10 -9 It can bind to antigens with a binding affinity greater than M.

[0317] As a non-limiting example, the antibody conjugate of the subject may bind to an antigen present on cancer cells (e.g., tumor-specific antigen, antigen overexpressed on cancer cells, etc.), and the conjugated portion may be a drug, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.). For example, the antibody conjugate of the subject may be specific to an antigen on cancer cells, and the conjugated portion may be a drug, such as a cytotoxic compound (e.g., a cytotoxic small molecule, a cytotoxic synthetic peptide, etc.).

[0318] As a further non-limiting example, the antibody conjugate of the subject may bind to an antigen present on a virus-infected cell (for example, if the antigen is encoded by a virus, or if the antigen is expressed on a cell type that is infected by the virus), and the conjugated portion may be a drug, such as a viral fusion inhibitor.

[0319] Drugs for conjugation into polypeptides As described above, the conjugate or compound of this disclosure is substituent W 1 and W 1a This may include drugs or active agents. Some of these drugs are suitable for use as a reactive partner to conjugate to an antibody, or can be modified to be suitable for use. Examples of drugs include small molecule drugs and peptide drugs.

[0320] As used herein, “small molecule drug” refers to a compound, such as an organic compound, which exhibits the desired pharmaceutically active substance and has a molecular weight of generally about 800 Da or less, or 2000 Da or less, but can include molecules up to 5 kDa and may be as large as 10 kDa. Small inorganic molecules refer to molecules that do not contain carbon atoms, while small organic molecules refer to compounds that contain at least one carbon atom.

[0321] For example, the drug or active agent may be a topoisomerase inhibitor (e.g., a topoisomerase I inhibitor), such as camptothecin, or its analogues or derivatives, or a pharmaceutically active camptothecin moiety and / or a portion thereof. The topoisomerase inhibitor conjugated to the polypeptide (e.g., camptothecin, or its analogues or derivatives) may be any of a variety of topoisomerase inhibitors, including, but are not limited to, camptothecin or camptothecin moieties, as well as their analogues and derivatives, as described herein. Examples of drugs used in the conjugates and compounds described herein include, but are not limited to, topoisomerase inhibitors such as SN-38, berotecan, exatecan, 9-aminocamptothecin (9-AC), topotecan, des-Me-topotecan, and their derivatives, as well as camptothecin or camptothecin derivatives. Additional examples of topoisomerase inhibitors used in this disclosure are described in PCT / US2022 / 012325, which is incorporated herein by reference.

[0322] In other embodiments, the drug or active agent may be mytansine. "Meitansine," "meitansine moiety," "meitansine active agent moiety," and "meitansinoid" refer to meitansine and its analogues and derivatives, as well as pharmaceutically active meitansine moieties and / or parts thereof. Meitansine conjugated to a polypeptide may be any of the various meitansinoid moieties described herein, including, but not limited to, meitansine and its analogues and derivatives (e.g., deacylmeitansine).

[0323] In other instances, the drug or active agent may be auristatin, its analogues or derivatives, or a pharmaceutically active auristatin moiety and / or its moiety. Auristatin conjugated to a polypeptide may be any of the various auristatin moieties, including but not limited to auristatin and its analogues and derivatives, as described herein. Examples of drugs used in the conjugates and compounds described herein include, but are not limited to, auristatin or auristatin derivatives such as monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and their derivatives.

[0324] In other cases, the drug or active agent may be duocalmycin, its analogues or derivatives, or a pharmaceutically active duocalmycin moiety and / or moiety thereof. Duocalmycin conjugated to a polypeptide may be any of the various duocalmycin moieties, including duocalmycin and its analogues and derivatives, as described herein, but are not limited. Examples of drugs used in the conjugates and compounds described herein include, but are not limited to, duocalmycin, or duocalmycin derivatives such as duocalmycin A, duocalmycin B1, duocalmycin B2, duocalmycin C1, duocalmycin C2, duocalmycin D, duocalmycin SA, and CC-1065, and their derivatives. In some embodiments, duocalmycin is a duocalmycin analog, such as adzeresin, bizeresin, or karzeresin, but are not limited to.

[0325] In certain embodiments, the drug is selected from cytotoxins, kinase inhibitors, selective estrogen receptor modulators, immunostimulants, Toll-like receptor (TLR) agonists, oligonucleotides, aptamers, cytokines, steroids, and peptides.

[0326] For example, cytotoxins can include any compound that leads to cell death (e.g., necrosis or apoptosis) or reduced cell survival.

[0327] Kinase inhibitors include, but are not limited to, adavocertib, afatinib, axitinib, bosutinib, cetuximab, cobimetinib, crizotinib, cabozantinib, dacomitinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mbritinib, nilotinib, pazopanib, pegaptanib, ruxolitinib, sorafenib, sunitinib, tucatinib, vandetanib, and vemurafenib.

[0328] For example, selective estrogen receptor modulators include, but are not limited to, endoxifen, tamoxifen, afimoxifen, and toremifene.

[0329] Immunostimulants include, but are not limited to, vaccines (e.g., bacterial or viral vaccines), colony-stimulating factors, interferons, and interleukins. TLR agonists include, but are not limited to, imiquimod and reximod.

[0330] Oligonucleotide drugs include, but are not limited to, fomivirsen, pegaptanib, mipomersen, eteplirsen, defibrotide, nusinersen, golozyrcene, viltolarsen, boranesolsen, inotercene, tofersen, and tominersen.

[0331] Aptamer drugs include, but are not limited to, pegaptanib, AS1411, REG1, ARC1779, NU172, ARC1905, E10030, NOX-A12, and NOX-E36.

[0332] Cytokines include Albuinterferon alpha-2B, Aldesleukin, ALT-801, Anakinra, Ancestim, Avotermin, Valgrastim, Benpegaldesleukin, Vinetraquin, Syntredekin Besdotox, CTCE-0214, Darbepoetin alpha, Denileukin Difutox, Duranermin, Edodekin alpha, Emfilermin, Epoetin Delta, Erythropoietin, Human Interleukin-2, Interferon alpha, Interferon alpha-2c, Interferon alpha-n1, Interferon alpha-n3, Interferon alpha-1, Interferon beta-1a, Interferon beta-1b, Interferon gamma-1b, Interferon Ca This includes, but is not limited to, ppa, interleukin-1 alpha, interleukin-10, interleukin-7, lenograstim, religistim, repegfilgrastim, lorcafusp alpha, Maxy-G34, methoxypolyethylene glycol epoetin beta, moglamostim, muprestim, nagrestipen, oprelbequin, pegfilgrastim, pegrodecakine, peginterferon alpha-2a, peginterferon alpha-2b, peginterferon beta-1a, peginterferon lambda-1a, recombinant CD40 ligand, reglamostim, romiplostim, salglamostim, thrombopoietin, tucozotzumab cermoloukin, viral macrophage inflammatory proteins, etc.

[0333] Steroid drugs include, but are not limited to, prednisolone, betamethasone, dexamethasone, hydrocortisone, methylprednisolone, and deflazacort.

[0334] As used herein, “peptide drugs” refers to amino acid-containing polymer compounds and is intended to encompass naturally occurring and non-naturally occurring peptides, oligopeptides, cyclic peptides, polypeptides, as well as proteins and peptide mimes. Peptide drugs may be obtained by chemical synthesis or produced from genetically encoded sources (e.g., recombinant sources). Peptide drugs may have a molecular weight range of 200 Da to 10 kDa or greater. Suitable peptides include, but are not limited to, cytotoxic peptides, angiogenic peptides, anti-angiogenic peptides, B cell-activating peptides, T cell-activating peptides, antiviral peptides, peptides that inhibit viral fusion, peptides that increase the production of one or more lymphocyte populations, antimicrobial peptides, growth factors, growth hormone-releasing factors, vasoactive peptides, anti-inflammatory peptides, peptides that regulate glucose metabolism, antithrombotic peptides, anti-nociceptive peptides, vasodilatory peptides, platelet aggregation inhibitors, analgesics, and the like.

[0335] Additional examples of drugs used in the conjugates and compounds described herein include, but are not limited to, tubulisin M, calicheamycin, STAT3 inhibitors, alpha-amanitin, aurora kinase inhibitors, berotecan, and anthracyclines.

[0336] Other examples of drugs include small molecule drugs such as cancer chemotherapeutic agents. For example, if the polypeptide is an antibody (or fragment thereof) having specificity against tumor cells, the antibody may be produced to include modified amino acids, as described herein, and then conjugate to a cancer chemotherapeutic agent. Cancer chemotherapeutic agents include non-peptidic (i.e., non-proteinic) compounds that reduce the proliferation of cancer cells and encompass cytotoxic agents and cell proliferation inhibitors. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones. Peptidic compounds may also be used.

[0337] Suitable cancer chemotherapeutic agents include drastatin and its active analogs and derivatives, and auristatin and its active analogs and derivatives (e.g., monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), etc.). See, for example, WO96 / 33212, WO96 / 14856, and US6,323,315. For example, drastatin 10 or auristatin PE may be included in the antibody-drug conjugate of this disclosure. Suitable cancer chemotherapeutic agents also include mytansinoids and their active analogues and derivatives (see, for example, EP1391213 and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), duocalmycin and its active analogues and derivatives (including, for example, synthetic analogues, KW-2189 and CB 1-TM1), and benzodiazepines and their active analogues and derivatives (for example, pyrrolobenzodiazepine (PBD)).

[0338] Drugs that act to reduce cell proliferation are known and widely used in the art. Such drugs include alkylating agents such as nitrogen mustard, nitrosourea, ethyleneimine derivatives, alkyl sulfonates, and triazenes, and include, but are not limited to, mechloretamine, cyclophosphamide (Cytoxan®), melphalan (L-sarcolicin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozotocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobromane, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.

[0339] Antimetabolites include folate analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, and include, but are not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolate (DDATHF), leucovorin, fludarabine phosphate, pentostatin, and gemcitabine.

[0340] Suitable natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins) include Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinal; alkaloids, e.g., vincristine, vinblastine, vinorelbine, vindesine; podophyllotoxins, e.g., etoposide, teniposide; antibiotics, e.g., anthracyclines, daunorubicin hydrochloride. Examples of these include (daunomycin, rubidomycin, serubicin), idarubicin, doxorubicin, epirubicin, and morpholino derivatives; phenoxyzombine cyclopeptides, e.g., dactinomycin; basic glycopeptides, e.g., bleomycin; anthraquinone glycosides, e.g., plicamycin (mitramycin); anthracendions, e.g., mitoxantrone; azilinopyrroindolediones, e.g., mitomycin; macrocyclic immunosuppressants, e.g., cyclosporine, FK-506 (tacrolimus, Prograf), rapamycin, etc., but are not limited to these.

[0341] Other antiproliferative cytotoxic agents include navelbine, CPT-11, anastrozole, letrozole, capecitabine, reloxafene, cyclophosphamide, ifosfamide, and droloxifene.

[0342] Microtubule agonists with antiproliferative activity are also suitable for use, including alocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), meitansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), and thiocolchicine (NSC This includes, but is not limited to, natural and synthetic epotilones, estramustine, nocodazole, etc., as well as tritylcysteine, vinblastine sulfate, vincristine sulfate, epotilone A, epotilone B, and discodermold.

[0343] Suitable hormone regulators and steroids (including synthetic analogs) for use include, but are not limited to, corticosteroids such as prednisone and dexamethasone; estrogens and pregestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, and tamoxifen; and corticosteroids such as aminoglutethimide, 17α-ethinylestradiol, diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testactone, methylprednisolone, methyl-testosterone, prednisolone, triamcinolone, chlorotrianicene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide (Drogenil), toremifene (Fareston), and Zoladex®. Estrogen stimulates proliferation and differentiation; therefore, compounds that bind to estrogen receptors are used to inhibit this activity. Corticosteroids can inhibit T cell proliferation.

[0344] Other suitable chemotherapeutic agents include metal complexes, e.g., cisplatin (cis-DDP), carboplatin; urea, e.g., hydroxyurea; and hydrazine, e.g., N-methylhydrazine; epidophyllotoxin; topoisomerase inhibitors; procarbazine; mitoxantrone; leucovorin; tegafur, etc. Other antiproliferative agents of interest include immunosuppressants, e.g., mycophenolic acid, thalidomide, desoxysperguarin, azasporin, leflunomide, mizoribine, azaspiran (SKF105685); and Iressa (registered trademark) (ZD1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline).

[0345] Taxanes are preferred for use. “Taxanes” include paclitaxel and any active taxane derivatives or prodrugs. “Paclitaxel” (which should be understood herein to include analogues, formulations, and derivatives such as, for example, docetaxel, TAXOL®, TAXOTERE® (a formulation of docetaxel), 10-desacetyl analogues of paclitaxel, and 3'N-desbenzoyl-3'Nt-butoxycarbonyl analogues of paclitaxel) is readily prepared using techniques known to those skilled in the art (WO94 / 078). See also U.S. Patent Nos. 82, WO94 / 07881, WO94 / 07880, WO94 / 07876, WO93 / 23555, WO93 / 10076, U.S. Patents Nos. 5,294,637, 5,283,253, 5,279,949, 5,274,137, 5,202,448, 5,200,534, 5,229,529, and EP590,267) or it may be available from various commercial sources, including, for example, Sigma Chemical Co. (St. Louis, Mo.) (T7402 derived from Taxus brevifolia, or T-1912 derived from Taxus yannanensis).

[0346] It should be understood that paclitaxel refers not only to the commonly available chemical forms of paclitaxel, but also to its analogues and derivatives (e.g., Taxotere® docetaxel as described above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

[0347] Furthermore, the term "taxane" also includes a variety of known derivatives, including both hydrophilic and hydrophobic derivatives. Taxane derivatives include, but are not limited to, the galactose and mannose derivatives described in International Patent Application No. 99 / 18113; piperazino and other derivatives described in WO99 / 14209; taxane derivatives described in WO99 / 09021, WO98 / 22451, and U.S. Patent No. 5,869,680; 6-thio derivatives described in WO98 / 28288; sulfenamide derivatives described in U.S. Patent No. 5,821,263; and taxol derivatives described in U.S. Patent No. 5,415,869. It further includes, but is not limited to, prodrugs of paclitaxel described in WO98 / 58927, WO98 / 13059, and U.S. Patent No. 5,824,701.

[0348] Suitable biological reaction modifiers for use include, but are not limited to, (1) inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors of serine / threonine kinase activity; (3) tumor-associated antigen antagonists such as antibodies that specifically bind to tumor antigens; (4) apoptosis receptor agonists; (5) interleukin-2; (6) IFN-α; (7) IFN-γ; (8) colony-stimulating factors; and (9) inhibitors of angiogenesis.

[0349] Examples of drugs include small molecule drugs such as cancer chemotherapy agents. For example, if the polypeptide is an antibody (or fragment thereof) having specificity for tumor cells, the antibody may be generated to include modified amino acids as described herein and then conjugate to cancer chemotherapy agents such as microtubule agonists. In certain embodiments, the drug is a microtubule agonist having antiproliferative activity, such as a meitansinoid.

[0350] Embodiments of the present disclosure include a conjugate in which an antibody is conjugated to one or more drug moieties, for example, two or more drug moieties, for example, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty or more drug moieties. The drug moieties may be conjugated to the antibody at one or more sites in the antibody as described herein. In certain embodiments, the conjugate has an average drug-to-antibody ratio (DAR) (molar ratio) in the range of 0.1 to 20, or 0.5 to 20, or 1 to 20, for example, 1 to 19, or 1 to 18, or 1 to 17, or 1 to 16, or 1 to 15, or 1 to 14, or 1 to 13, or 1 to 12, or 1 to 11, or 1 to 10, or 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 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the conjugate has an average DAR of 10 to 20, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, the conjugate has an average DAR of 1 to 10. In certain embodiments, the conjugate has an average DAR of 1 to 5 (e.g., 4). In certain embodiments, the conjugate has an average DAR of 5 to 10 (e.g., 8). In certain embodiments, the conjugate has an average DAR of 8 to 12 (e.g., 10). In certain embodiments, the conjugate has an average DAR of 10 to 15 (e.g., 12). In certain embodiments, the conjugate has an average DAR of 15 to 20 (e.g., 16). Average means arithmetic mean.

[0351] As described herein, in some cases the antibody-drug conjugate may include a branched linker, and two drugs or active agents are bound to the branched linker. In certain embodiments, the two drugs or active agents bound to the branched linker are the same drug or active agent. For example, the first branch of the branched linker may be bound to a drug or active agent, and the second branch of the branched linker may be bound to the same drug or active agent as the first branch. In other embodiments, the two drugs or active agents bound to the branched linker are different drugs or active agents. For example, the first branch of the branched linker may be bound to a first drug or first active agent, and the second branch of the branched linker may be bound to a second drug or second active agent different from the first drug or first active agent bound to the first branch.

[0352] In some embodiments, when two different drugs or active agents are bound to a branched linker, the drugs or active agents may be selected from drugs and active agents that have a synergistic therapeutic effect. For example, in some cases, the use of two different drugs or active agents bound to a branched linker may provide a low therapeutically effective concentration in which both payloads act, thereby increasing the overall efficacy of the ADC.

[0353] In some embodiments, when two different drugs or active agents are bound to a branched linker, the drugs or active agents may be selected from drugs and active agents that provide enhanced therapeutic benefits compared to when the drugs or active agents are used separately. For example, the drugs or active agents may provide an increased effect on drug delivery by the ADC (for example, some payloads, such as iRGD peptides, may increase extravasation into tissues and enhance penetration into tumors).

[0354] In some embodiments, when two different drugs or active agents are bound to a branched linker, the drugs or active agents may be selected from drugs and active agents that use different mechanisms of action. In some cases, this can result in a reduction of tumor drug resistance by targeting multiple pathways. Examples of payload combinations include, but are not limited to, cytotoxic drugs, immunomodulatory molecules for activating or inhibiting immune cell populations, cytokines, hormones, and radioisotopes-filled chelating agents.

[0355] In some embodiments, when two different drugs or active agents are bound to a branched linker, the two different drugs or active agents are a topoisomerase inhibitor described herein (e.g., belotecan) and auristatin described herein (e.g., MMAE). In some embodiments, when two different drugs or active agents are bound to a branched linker, the two different drugs or active agents are a topoisomerase inhibitor described herein (e.g., belotecan) and an iRGD peptide described herein. In some embodiments, when two different drugs or active agents are bound to a branched linker, the two different drugs or active agents are auristatin described herein (e.g., MMAE) and an iRGD peptide described herein. In some embodiments, when two different drugs or active agents are bound to a branched linker, the two different drugs or active agents are auristatin described herein (e.g., MMAE) and a kinase inhibitor described herein (e.g., sorafenib, lapatinib, gefitinib, etc.). In some embodiments, when two different drugs or active agents are bound to a branched linker, the two different drugs or active agents are a topoisomerase inhibitor (e.g., berotecan) and a kinase inhibitor (e.g., sorafenib, lapatinib, gefitinib, etc.) as described herein. In some embodiments, when two different drugs or active agents are bound to a branched linker, the two different drugs or active agents are auristatin (e.g., MMAE) and a selective estrogen receptor modulator (e.g., endoxifen) as described herein. In some embodiments, when two different drugs or active agents are bound to a branched linker, the two different drugs or active agents are a topoisomerase inhibitor (e.g., berotecan) and a selective estrogen receptor modulator (e.g., endoxifen) as described herein.

[0356] Drugs conjugated to polypeptides can be modified to incorporate a reactive partner for reaction with the polypeptide. If the drug is a peptide drug, the reactive moiety (e.g., aminooxy or hydrazide) can be located in the N-terminal region, the N-terminus, the C-terminal region, the C-terminus, or within the peptide. For example, one method involves synthesizing a peptide drug having an aminooxy group. In this example, the peptide is synthesized from a Boc-protected precursor. The amino group of the peptide can react with compounds containing a carboxylic acid group and an oxy-N-Boc group. As an example, the amino group of the peptide reacts with 3-(2,5-dioxopyrrolidine-1-yloxy)propanoic acid. Other variations on compounds containing a carboxylic acid group and an oxy-N-protecting group can include different numbers of carbons in the alkylene linker and substituents on the alkylene linker. The reaction between the amino group of the peptide and compounds containing a carboxylic acid group and an oxy-N-protecting group occurs through standard peptide coupling chemistry.Examples of peptide coupling reagents that can be used include DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), di-p-toluoylcarbodiimide, BDP (1-benzotriazolediethylphosphate-1-cyclohexyl-3-(2-morpholinylethyl)carbodiimide), EDC (1-(3-dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), cyanuryl fluoride, cyanuryl chloride, and TFFH (tetramethylfluoride). (O-formamidinium hexafluorophosphate), DPPA (diphenylphosphoradidate), BOP (benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate), HBTU (O-benzotriazole-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate), TBTU (O-benzotriazole-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate), TSTU (O-( N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate), HATU (N-[(dimethylamino)-1-H-1,2,3-triazolo[4,5,6]-pyridine-1-ylmethylene]--N-methylmethaneaminium hexafluorophosphate N-oxide), BOP-Cl (bis(2-oxo-3-oxazolidinyl)phosphinate chloride), PyBOP ((1-H-1,2,3-benzotriazole-1-yloxy)-tris(pyro Examples include, but are not limited to, lysino(phosphonium tetrafluorophosphate), BrOP (bromotris(dimethylamino)phosphonium hexafluorophosphate), DEPBT (3-(diethoxyphosphoryloxy)-1,2,3-benzotriazine-4(3H)-one), and PyBrOP (bromotris(pyrrolidino)phosphonium hexafluorophosphate). Non-limiting examples include HOBt and DIC, which can be used as peptide coupling reagents.

[0357] Deprotection to expose aminooxy functionalities is carried out in peptides containing N-protecting groups. Deprotection of N-oxysuccinimide groups occurs, for example, according to standard deprotection conditions for cyclic amide groups. Deprotection conditions can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3rd Ed., 1999, John Wiley & Sons, NY and Harrison et al. Certain deprotection conditions involve hydrazine reagents, amino reagents, or sodium borohydride. Deprotection of Boc protecting groups occurs using TFA. Other reagents for deprotection include, but are not limited to, hydrazine, methylhydrazine, phenylhydrazine, sodium borohydride, and methylamine. Products and intermediates can be purified by conventional means such as HPLC purification.

[0358] Those skilled in the art will understand that factors such as pH and steric hindrance (i.e., the possibility of contact between amino acid residues for reaction with the desired reactive partner) are important, and modification reaction conditions to provide optimal conjugate conditions are well-established within the art and are customary in the art. When conjugation is carried out using polypeptides present in or on living cells, the conditions are selected to be physiologically compatible. For example, the pH may be temporarily reduced for a period sufficient for the reaction to occur but within a period tolerable by the cells (e.g., about 30 minutes to 1 hour). Physiological conditions for modifying polypeptides on the cell surface may be similar to those used in ketone-azide reactions in the modification of cells with cell surface azides (see, e.g., US6,570,040).

[0359] Small molecule compounds containing or modified to contain an α-nucleophile that function as a reactive partner with the compounds or conjugates disclosed herein are also intended for use as drugs in the polypeptide drug conjugates of this disclosure. General methods are known in the art for useful chemical synthesis schemes and conditions for synthesizing the compounds of interest (see, for example, Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001, or Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978).

[0360] formulation The conjugates of this disclosure can be formulated in a variety of different ways. Generally, if the conjugate is an antibody-drug conjugate, the conjugate is formulated in a manner that is appropriate to the drug, antibody, the condition being treated, and the route of administration used.

[0361] In some embodiments, a pharmaceutical composition is provided comprising one of the conjugates of the present disclosure and a pharmaceutically acceptable excipient.

[0362] Conjugates (e.g., antibody-drug conjugates) can be provided in any preferred form, for example, in the form of a pharmaceutically acceptable salt, and can be formulated for any preferred route of administration, e.g., oral, topical, or parenteral administration. If the conjugates are provided as injectable liquids (e.g., embodiments in which they are administered intravenously or directly to tissues), the conjugates can be provided as ready-to-use dosage forms or as reconstituted, storage-stable powders or liquids composed of pharmaceutically acceptable carriers and excipients.

[0363] Methods for formulating the conjugate can be adapted from readily available ones. 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., physiological saline). The pharmaceutical composition may optionally contain other additives (e.g., buffers, stabilizers, preservatives, etc.). In some embodiments, the formulation is suitable for administration to mammals, for example, to humans.

[0364] Treatment method The antibody-drug conjugates of this disclosure are useful for treating conditions or diseases in subjects who are able to receive treatment by administering the parent drug (i.e., the drug before conjugation to the antibody).

[0365] In some embodiments, methods are provided that involve administering a certain amount (e.g., a therapeutically effective dose) of any of the conjugates of this disclosure to a target.

[0366] In certain embodiments, a method is provided for delivering a drug to a target site in a subject, wherein the method comprises administering a pharmaceutical composition comprising one of the conjugates of the present disclosure to the subject, the administration being effective in releasing a therapeutically effective amount of the drug from the conjugate to a target site in the subject. For example, as described above, the antibody-drug conjugate of the present disclosure may comprise a cleavable linker, such as an enzymatically cleavable linker comprising a first enzymatically cleavable portion and a second enzymatically cleavable portion. In some cases, the cleavable linker is cleaved under appropriate conditions to separate or release the drug from the antibody at a desired site of action for that drug. For example, a second cleavable portion protecting the first cleavable portion from cleavage may be cleaved to enable cleavage of the first cleavable portion, thereby cleaving the cleavable linker into two or more portions, and thus releasing the drug from the antibody-drug conjugate at a desired site of action.

[0367] In certain embodiments, the first cleavable portion may be an enzymatically cleavable portion. In some cases, the enzyme that facilitates the cleavage of the first cleavable portion is an enzyme administered to the subject being treated (i.e., an exogenous enzyme to the subject being treated). For example, the first enzyme may be administered before, concurrently with, or after the administration of the antibody-drug conjugate described herein.

[0368] In certain embodiments, the second cleavable portion may be an enzymatically cleavable portion. In some cases, the enzyme that facilitates the cleavage of the second cleavable portion is an enzyme administered to the subject being treated (i.e., an exogenous enzyme to the subject being treated). For example, the second enzyme may be administered before, concurrently with, or after the administration of the antibody-drug conjugate described herein. In certain embodiments, the first and second enzymes are different enzymes.

[0369] In other cases, the first enzyme that facilitates the cleavage of the first cleavable portion is an enzyme present in the target being treated (i.e., endogenous to the target being treated). For example, the first enzyme may be present at the desired site of action for the drug in an antibody-drug conjugate. The antibody in the antibody-drug conjugate may be specifically targeted to the desired site of action (e.g., specifically bound to an antigen present at the desired site of action), and the desired site of action also includes the presence of the first enzyme. In some cases, the first enzyme is present in excess at the desired site of action compared to other areas of the target body being treated. For example, the first enzyme may be overexpressed at the desired site of action compared to other areas of the target body being treated. In some cases, the first enzyme is present in excess at the desired site of action due to localization of the first enzyme in a particular region or location. For example, the first enzyme may be associated with a particular structure within the desired site of action, such as a lysosome. In some cases, the first enzyme is present in excess in lysosomes compared to other areas of the target body. In some embodiments, the lysosome containing the first enzyme is located at the desired site of action for the drug in the antibody-drug conjugate, for example, at the site of cancer or tumor to be treated with the drug. In certain embodiments, the first enzyme is a protease, such as a human protease enzyme (e.g., cathepsin B).

[0370] In certain embodiments, the second enzyme that facilitates the cleavage of the second cleavable portion is an enzyme present in the target being treated (i.e., endogenous to the target being treated). For example, the second enzyme may be present at the desired site of action for the drug in an antibody-drug conjugate. The antibody in the antibody-drug conjugate may be specifically targeted to the desired site of action (e.g., specifically bound to an antigen present at the desired site of action), and the desired site of action also includes the presence of the second enzyme. In some cases, the second enzyme is present in excess at the desired site of action compared to other areas of the body of the target being treated. For example, the second enzyme may be overexpressed at the desired site of action compared to other areas of the body of the target being treated. In some cases, the second enzyme is present in excess at the desired site of action due to localization of the second enzyme in a particular region or location. For example, the second enzyme may be associated with a particular structure within the desired site of action, such as a lysosome. In some cases, the second enzyme is present in excess in lysosomes compared to other areas of the body of the target. In some embodiments, lysosomes containing the second enzyme are located at the desired site of action for the drug in the antibody-drug conjugate, for example, at the site of cancer or tumor to be treated with the drug. In certain embodiments, the second enzyme is a glucuronidase, glycosidase, such as galactosidase, glucosidase, mannosidase, or fucosidase.

[0371] Any suitable enzyme can be used to cleave the first and second cleavable portions of the antibody-drug conjugate described herein. Other enzymes, for example, but not limited to, those derived from other vertebrates (e.g., primates, mice, rats, cats, pigs, quail, goats, dogs, rabbits, etc.), may also be suitable for cleaving the first and second cleavable portions of the antibody-drug conjugate described herein.

[0372] In certain embodiments, the antibody-drug conjugate is substantially stable under standard conditions. Substantially stable means that the cleavable linker of the antibody-drug conjugate does not undergo a significant amount of cleavage in the absence of the first and second enzymes described above. For example, as described above, the second cleavable portion can be protected from cleavage of the first cleavable portion, and therefore the cleavable linker of the antibody-drug conjugate does not undergo a significant amount of cleavage in the absence of the second enzyme described above. For example, the cleavable linker of the antibody-drug conjugate may be substantially stable such that 25% or less of the antibody-drug conjugate, e.g., 20%, or 15%, or 10%, or 5%, or 4%, or 3%, or 2%, or 1%, is cleaved in the absence of the first and / or second enzymes. In some cases, an antibody-drug conjugate is substantially stable such that its cleavable linker does not undergo significant cleavage in the absence of a first enzyme and / or a second enzyme, but can be cleaved in the presence of both the first and second enzymes. For example, an 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 in the presence of a second enzyme at a desired site of action, the second cleavable portion is cleaved from the cleavable linker, thus exposing the first cleavable portion to subsequent cleavage by the first enzyme, and subsequently releasing the drug at its desired site of action. In certain embodiments, after administration to a subject, the antibody-drug conjugate remains stable for a long period of time, for example, 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.In certain embodiments, the antibody-drug conjugate is stable for extended periods in a certain pH range, for example, in the range of 2-10, or 3-9, or 4-8, or 5-8, or 6-8, or 7-8, in the absence of the first and / or second enzymes.

[0373] As described above, the antibody-drug conjugates of this disclosure are used to treat a condition or disease of a subject treatable by the administration of the parent drug. "Treatment" means that at least improvement of the condition and associated symptoms of the host is achieved, and improvement is used broadly to mean at least reduction of parameters associated with the condition being treated, such as the severity of the symptoms. Thus, treatment also includes situations in which a pathological condition, or at least symptoms associated therewith, is completely inhibited, e.g., prevented from occurring or stopped, e.g., terminated, such that the host no longer suffers from that condition, or at least the symptoms that characterize that condition. Thus, treatment includes (i) prevention, i.e., reducing the risk of developing clinical symptoms, including preventing the development of clinical symptoms, such as preventing disease progression to an adverse condition; (ii) inhibition, i.e., preventing the development or further development of clinical symptoms, such as mitigating or completely inhibiting active disease; and / or (iii) mitigation, i.e., regressing clinical symptoms.

[0374] The subjects to be treated may be those in need of therapy, and the subjects to be treated are those 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,” with humans being the subject of interest. Other subjects may include domestic pets (e.g., dogs and cats), livestock (e.g., cattle, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs, and rats, e.g., animal models of diseases), and non-human primates (e.g., chimpanzees and monkeys).

[0375] The amount of antibody-drug conjugate administered may be initially determined based on the dose of the parent drug and / or guidance of the dosing regimen. Generally, the administered antibody-drug conjugate may provide targeted delivery of the conjugated drug and / or an enhanced serum half-life, thus providing at least one of a reduced dose or a reduction in the number of doses of the dosing regimen. Therefore, an antibody-drug conjugate may provide a reduced dose and / or a reduction in the number of doses of the dosing regimen to the parent drug before being conjugated into the antibody-drug conjugate of this disclosure.

[0376] Furthermore, as described above, antibody-drug conjugates can provide a controlled stoichiometry of drug delivery; therefore, the dosage of an antibody-drug conjugate can be calculated based on the number of drug molecules delivered per antibody-drug conjugate.

[0377] In some embodiments, multiple doses of the antibody-drug conjugate may be administered. The frequency of administration of the antibody-drug conjugate may vary depending on various factors, such as the severity of symptoms or the condition of the subject. For example, in some embodiments, the antibody-drug conjugate may be administered once a month, twice a month, three times a month, every other week, once a week (qwk), twice a week, three times a week, four times a week, five times a week, six times a week, every other day, daily (qd / od), twice a day (bds / bid), or three times a day (tds / tid).

[0378] Methods of treating cancer This disclosure provides a method comprising delivering the conjugate of this disclosure to an individual having cancer. For example, this method may comprise administering a pharmaceutical composition containing a therapeutically effective amount of the conjugate of this disclosure to a subject, the administration of which is effective in treating cancer in the subject. This method is useful for treating a wide variety of cancers, including but not limited to breast cancer, ovarian cancer, colon cancer, lung cancer, gastric cancer, and pancreatic cancer. In the context of cancer, the term “treat” includes, for example, reducing the growth of a solid tumor, inhibiting the replication of cancer cells, reducing the overall tumor burden, and improving one or more symptoms associated with cancer (each of these).

[0379] Cancers that can be treated using this method include, but are not limited to, colon cancer, colorectal cancer, gastric cancer, lung cancer (including small cell and non-small cell lung cancer), pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or cholangiocarcinoma, choriocarcinoma, seminoma, fetal cancer, cervical cancer, uterine cancer, testicular cancer, and epithelial cancer.

[0380] In certain embodiments, a method is provided for treating cancer in a subject, such method comprising administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of any of the conjugates of the present disclosure, the administration being effective in treating cancer in the subject.

[0381] A certain embodiment This disclosure intends, in particular, to include the following numbered embodiments. 1. The conjugate of equation (I), [ka] During the ceremony, Each R 1 However, they are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R 2However, they are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k1 is an integer between 1 and 10. R 3 However, it includes parts that can be cut, L A However, it was the first linker, W 1 However, it is a drug, W 2 However, it is an antibody, A conjugate in which X is a conjugation portion that binds to the antibody at a natural amino acid residue of the antibody. 2.R 1 However, H is R 2 The conjugate according to Embodiment 1, wherein the conjugate is alkyl or substituted alkyl. 3. The conjugate according to Embodiment 1 or 2, wherein k1 is 2. 4.R 3 However, the conjugate according to any one of embodiments 1 to 3 is a portion that can be chemically cleaved. 5.R 3 However, the conjugate according to any one of Embodiments 1 to 3 is a portion that can be enzymatically cleaved. 6.R 3 The conjugate according to Embodiment 5, wherein the conjugate is a glycoside or a glycoside derivative. 7. The conjugate according to Embodiment 6, wherein the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc. 8. The conjugate according to any one of embodiments 1 to 7, wherein X comprises a conjugation moiety configured to bind to a cysteine ​​residue or lysine residue of the antibody. 9. The conjugate according to any one of Embodiments 1 to 7, wherein X comprises maleimide, and the natural amino acid residue of the antibody comprises a cysteine ​​residue. 10. The conjugate is the conjugate of formula (II), [ka] During the ceremony, Each R 1 However, they are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R 2 However, they are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k1 is an integer between 1 and 10. R 3 However, it includes parts that can be cut, L A However, it was the first linker, W 1 However, it is a drug, W 2 However, the conjugate described in Embodiment 8 is an antibody. 11. The first linker L A but, -(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 - Includes, During the ceremony, a, b, c, d, e, and f are each independently either 0 or 1. T 1 , T 2 , T 3 , T4 , T 5 , and T 6 However, each is independent, and covalent, (C1~C 12 ) alkyl, substituted (C1~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 - Selected from 4-amino-piperidine (4AP), acetal group, hydrazine, disulfide, and ester, where EDA is the ethylenediamine moiety, PEG is polyethylene glycol, AA is an amino acid residue or amino acid analog, 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 However, each operates independently as a covalent bond, -CO-, -NR. 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 Selected from the group consisting of SO2- and -P(O)OH-, where each q is an integer from 1 to 6. Each R 13 However, they are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl, Each R 15The conjugate according to any one of Embodiments 1 to 10, independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 12. T 1 However, (C1~C 12 ) alkyl and substituted (C1~C 12 ) Selected from alkyl groups, T 2 , T 3 , T 4 , T 5 , and T 6 However, each is independent, and covalent, (C1~C 12 ) alkyl, substituted (C1~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 - Selected from 4-amino-piperidine (4AP), acetal group, hydrazine, and ester, V 1 , V 2 , V 3 , V 4 , V 5 , and V 6 However, each operates independently as a covalent bond, -CO-, -NR. 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 Selected from the group consisting of SO2- and -P(O)OH-, (PEG)n but, [ka] In the formula, n is an integer between 1 and 30. The EDA has the following structure: [ka] The ethylenediamine moiety has the following characteristics, where y is an integer from 1 to 6, and r is 0 or 1. 4-amino-piperidine (4AP) [ka] And, Each R 12 However, independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, any two adjacent R 12 The conjugate according to embodiment 11, wherein the groups can be linked in a cyclic manner to form a piperazinyl ring. 13. T 1 However, (C1~C 12 ) is alkyl, V 1 However, it is -CO-, b, c, d, e, and f are each 0, or T 1 However, (C1~C 12 ) is alkyl, V 1 However, it is -CO-, T 2 However, it is an amino acid analog, V 2 However, it is -NH-, T 3 However, (PEG) n V 3 However, it is -CO-, d, e, and f are each 0, or T 1 However, (C1~C 12 ) is alkyl, V 1 However, it is -CONH-, T 2However, substitution (C1~C 12 ) is alkyl, V 2 However, it is -CO-, The conjugate according to embodiment 11 or 12, wherein c, d, e, and f are each 0. 14.T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , V 1 , V 2 , V 3 , V 4 , V 5 , or V 6 The conjugate according to embodiment 11 or 12, wherein one of the elements is a branching element. 15. The branching base is -CONR 15 - and a conjugate selected from 4AP, as described in Embodiment 14. 16. The branching base is the second linker L B A conjugate according to embodiment 14 or 15, which is coupled to the 17. The second linker L mentioned above B but, -(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 - Includes, During the ceremony, g, h, i, j, k, and l are each independently either 0 or 1. T 7 , T 8 , T 9 , T 10 , T 11 , and T 12However, each is independent, and covalent, (C1~C 12 ) alkyl, substituted (C1~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 - Selected from 4-amino-piperidine (4AP), acetal group, hydrazine, disulfide, and ester, where EDA is the ethylenediamine moiety, PEG is polyethylene glycol, AA is an amino acid residue or amino acid analog, 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 However, each operates independently as a covalent bond, -CO-, -NR. 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 Selected from the group consisting of SO2- and -P(O)OH-, where each q is an integer from 1 to 6. Each R 13 However, they are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl, Each R 15The conjugate according to Embodiment 16, independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 18.L B However, it is bonded to the compound of formula (III), [ka] During the ceremony, Each R 4 However, they are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R 5 However, they are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k2 is an integer between 1 and 10. R 6 However, it includes parts that can be cut, W 1a However, the conjugate according to embodiment 16 or 17 is a drug. 19.R 4 However, H is R 5 The conjugate according to Embodiment 18, wherein the conjugate is alkyl or substituted alkyl. 20. The conjugate according to Embodiment 18 or 19, wherein k2 is 2. 21.R 6 However, the conjugate according to any of embodiments 18 to 20 is a portion that can be chemically cleaved. 22.R 6 However, the conjugate according to any of embodiments 18 to 20 is a portion that can be enzymatically cleaved. 23.R 6The conjugate according to Embodiment 22, wherein the conjugate is a glycoside or a glycoside derivative. 24. The conjugate according to Embodiment 23, wherein the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc. twenty five. T 1 However, (C1~C 12 ) is alkyl, V 1 However, branching base (-CONR 15 )- and, T 2 However, (C1~C 12 ) is alkyl, V 2 However, it is -CONH-, T 3 However, substitution (C1~C 12 ) is alkyl, V 3 However, it is -CO-, d, e, and f are each 0, T 7 However, (C1~C 12 ) is alkyl, V 7 However, it is -CONH-, T 8 However, substitution (C1~C 12 ) is alkyl, V 8 However, it is -CO-, i, j, k, and l are each 0, or T 1 However, (C1~C 12 ) is alkyl, V 1 However, it is -CO-, T 2 However, it is a branching group (4AP), V 2 However, it is -CO-, T 3 However, (PEG) n V 3 However, it is -CONH-, T 4 However, substitution (C1~C 12 ) is alkyl, V 4 However, it is -CO-, e and f are both 0, T 7 However, (PEG) n V 7 However, it is -CONH-, T 8 However, substitution (C1~C 12 ) is alkyl, V 8 However, it is -CO-, i, j, k, and l are each 0, or T 1 However, (C1~C 12 ) is alkyl, V 1 However, it is -CO-, T 2 However, it is a branching group (4AP), V 2 However, it is -CO-, T 3 However, (C1~C 12 ) is alkyl, V 3 However, it does not exist. T 4 However, it is a heteroaryl (triazole), V 4 However, it does not exist. T 5 However, (C1~C 12 ) is alkyl, V 5 However, it is -CONH-, T 6 However, substitution (C1~C 12 ) is alkyl, V 6 However, it is -CO-, T 7 However, (PEG) n V 7 However, it is -CONH-, T 8 However, substitution (C1~C 12 ) is alkyl, V 8 However, it is -CO-, A conjugate according to any one of embodiments 11 to 24, wherein i, j, k, and l are each 0. 26.W 1 and W 1a However, the conjugate described in any of Embodiments 14 to 25 is the same drug. 27.W 1 and W 1aHowever, the conjugate described in any of embodiments 14 to 25 is a different drug. 28. The aforementioned conjugate, [ka] [ka] and [ka] A conjugate according to any of embodiments 1 to 27, selected from the above. 29. A compound of formula (IV), [ka] During the ceremony, Each R 1 However, they are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R 2 However, they are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k1 is an integer between 1 and 10. R 3 However, it includes parts that can be cut, L A However, it was the first linker, W 1 However, it is a drug, A compound in which X is a conjugation moiety configured to bind to the antibody at a natural amino acid residue of the antibody. 30.R 1 However, H is R 2 The compound according to Embodiment 29, wherein the compound is alkyl or substituted alkyl. 31. The compound according to Embodiment 29 or 30, wherein k1 is 2. 32.R 3However, the compound according to any one of embodiments 29 to 31 is a chemically cleavable portion. 33.R 3 However, the compound according to any of embodiments 29 to 31 is an enzymatically cleavable portion. 34.R 3 The compound according to Embodiment 33, which is a glycoside or a glycoside derivative. 35. The compound according to Embodiment 34, wherein the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc. The compound according to any one of embodiments 29 to 35, wherein 36.X comprises a conjugation moiety configured to bind to a cysteine ​​residue or lysine residue of the antibody. 37. A compound according to any one of Embodiments 29 to 35, wherein X contains maleimide, and the natural amino acid residue of the antibody contains a cysteine ​​residue. 38. The compound is a compound of formula (V), [ka] During the ceremony, Each R 1 However, they are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R 2 However, they are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k1 is an integer between 1 and 10. R 3 However, it includes parts that can be cut, L A However, it was the first linker, W 1 However, the compound described in Embodiment 36 is a drug. 39. The first linker L A but, -(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 - Includes, During the ceremony, a, b, c, d, e, and f are each independently either 0 or 1. T 1 , T 2 , T 3 , T 4 , T 5 , and T 6 However, each is independent, and covalent, (C1~C 12 ) alkyl, substituted (C1~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 - Selected from 4-amino-piperidine (4AP), acetal group, hydrazine, disulfide, and ester, where EDA is the ethylenediamine moiety, PEG is polyethylene glycol, AA is an amino acid residue or amino acid analog, 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 However, each operates independently as a covalent bond, -CO-, -NR.15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 Selected from the group consisting of SO2- and -P(O)OH-, where each q is an integer from 1 to 6. Each R 13 However, they are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl, Each R 15 The compounds according to any one of Embodiments 29 to 38, which are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 40. T 1 However, (C1~C 12 ) alkyl and substituted (C1~C 12 ) Selected from alkyl groups, T 2 , T 3 , T 4 , T 5 , and T 6 However, each is independent, and covalent, (C1~C 12 ) alkyl, substituted (C1~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 - Selected from 4-amino-piperidine (4AP), acetal group, hydrazine, and ester, V 1 , V 2, V 3 , V 4 , V 5 , and V 6 However, each operates independently as a covalent bond, -CO-, -NR. 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR 15 Selected from the group consisting of SO2- and -P(O)OH-, (PEG) n but, [ka] In the formula, n is an integer between 1 and 30. The EDA has the following structure: [ka] The ethylenediamine moiety has the following characteristics, where y is an integer from 1 to 6, and r is 0 or 1. 4-amino-piperidine (4AP) [ka] And, Each R 12 However, independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, any two adjacent R 12 The compound according to embodiment 39, wherein the groups can be linked cyclically to form a piperazinyl ring. 41. T 1 However, (C1~C 12 ) is alkyl, V 1 However, it is -CO-, b, c, d, e, and f are each 0, or T 1 However, (C1~C 12) is alkyl, V 1 However, it is -CO-, T 2 However, it is an amino acid analog, V 2 However, it is -NH-, T 3 However, (PEG) n V 3 However, it is -CO-, d, e, and f are each 0, or T 1 However, (C1~C 12 ) is alkyl, V 1 However, it is -CONH-, T 2 However, substitution (C1~C 12 ) is alkyl, V 2 However, it is -CO-, The compound according to Embodiment 39 or 40, wherein c, d, e, and f are each 0. 42.T 1 , T 2 , T 3 , T 4 , T 5 , T 6 , V 1 , V 2 , V 3 , V 4 , V 5 , or V 6 The compound according to embodiment 39 or 40, wherein one of the groups is a branched group. 43. The branching group is -CONR 15 - and the compound according to Embodiment 42, selected from 4AP. 44. The branching base is the second linker L B A compound according to embodiment 42 or 43, including the compound described in embodiment 42 or 43. 45. The second linker L B but, -(T 7 -V 7 ) g -(T 8 -V 8 ) h -(T 9 -V 9 ) i -(T 10 -V10 ) j -(T 11 -V 11 ) k -(T 12 -V 12 ) l - Includes, During the ceremony, g, h, i, j, k, and l are each independently either 0 or 1. T 7 , T 8 , T 9 , T 10 , T 11 , and T 12 However, each is independent, and covalent, (C1~C 12 ) alkyl, substituted (C1~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 - Selected from 4-amino-piperidine (4AP), acetal group, hydrazine, disulfide, and ester, where EDA is the ethylenediamine moiety, PEG is polyethylene glycol, AA is an amino acid residue or amino acid analog, 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 However, each operates independently as a covalent bond, -CO-, -NR. 15 -, -NR 15 (CH2) q -, -NR 15 (C6H4)-, -CONR 15 -, -NR 15 CO-, -C(O)O-, -OC(O)-, -O-, -S-, -S(O)-, -SO2-, -SO2NR 15 -, -NR15 Selected from the group consisting of SO2- and -P(O)OH-, where each q is an integer from 1 to 6. Each R 13 However, they are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl, Each R 15 The compounds according to Embodiment 44, which are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 46.L B However, it is bonded to the compound of formula (VI), [ka] During the ceremony, Each R 4 However, they are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R 5 However, they are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k2 is an integer between 1 and 10. R 6 However, it includes parts that can be cut, W 1a However, the compound described in Embodiment 44 or 45 is a drug. 47.R 4 However, H is R 5 The compound according to Embodiment 46, wherein the compound is alkyl or substituted alkyl. 48. The compound according to embodiment 46 or 47, wherein k2 is 2. 49.R 6 However, the compound according to any of embodiments 46 to 48 is a chemically cleavable portion. 50.R 6 However, the compound according to any one of embodiments 46 to 48, which is an enzymatically cleavable portion. 51.R 6 The compound according to Embodiment 50, which is a glycoside or a glycoside derivative. 52. The compound according to Embodiment 51, wherein the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc. 53. T 1 However, (C1~C 12 ) is alkyl, V 1 However, branching base (-CONR 15 )- and, T 2 However, (C1~C 12 ) is alkyl, V 2 However, it is -CONH-, T 3 However, substitution (C1~C 12 ) is alkyl, V 3 However, it is -CO-, d, e, and f are each 0, T 7 However, (C1~C 12 ) is alkyl, V 7 However, it is -CONH-, T 8 However, substitution (C1~C 12 ) is alkyl, V 8 However, it is -CO-, i, j, k, and l are each 0, or T 1 However, (C1~C 12 ) is alkyl, V 1 However, it is -CO-, T 2 However, it is a branching group (4AP), V 2 However, it is -CO-, T 3 However, (PEG) n V 3 However, it is -CONH-, T 4 However, substitution (C1~C12 ) is alkyl, V 4 However, it is -CO-, e and f are both 0, T 7 However, (PEG) n V 7 However, it is -CONH-, T 8 However, substitution (C1~C 12 ) is alkyl, V 8 However, it is -CO-, i, j, k, and l are each 0, or T 1 However, (C1~C 12 ) is alkyl, V 1 However, it is -CO-, T 2 However, it is a branching group (4AP), V 2 However, it is -CO-, T 3 However, (C1~C 12 ) is alkyl, V 3 However, it does not exist. T 4 However, it is a heteroaryl (triazole), V 4 However, it does not exist. T 5 However, (C1~C 12 ) is alkyl, V 5 However, it is -CONH-, T 6 However, substitution (C1~C 12 ) is alkyl, V 6 However, it is -CO-, T 7 However, (PEG) n V 7 However, it is -CONH-, T 8 However, substitution (C1~C 12 ) is alkyl, V 8 However, it is -CO-, A compound according to any one of embodiments 39 to 52, wherein i, j, k, and l are each 0. 54.W 1 and W 1aHowever, the conjugate described in any of embodiments 42 to 53 is the same drug. 55.W 1 and W 1a However, the conjugate described in any of embodiments 42 to 53 is a different drug. 56. The aforementioned conjugate, [ka] [ka] [ka] A conjugate according to any of embodiments 29 to 55, selected from the above. 57. A pharmaceutical composition, A conjugate described in any one of Embodiments 1 to 28, A pharmaceutical composition comprising a pharmaceutically acceptable excipient. 58. A method, A method comprising administering a conjugate described in any of Embodiments 1 to 28 to a subject. 59. A method for treating cancer in a subject, A method comprising administering to the subject a pharmaceutical composition containing a conjugate according to any one of Embodiments 1 to 28 in a therapeutically effective amount, wherein the administration is effective in treating the cancer of the subject. [Examples]

[0382] The following examples are provided to those skilled in the art to provide a complete disclosure and description of the methods of preparation and use of the present invention and are not intended to limit the scope of what the inventors consider to be the invention, nor are they intended to represent all or only experiments that have been conducted. While efforts have been made to ensure accuracy of the figures used (e.g., quantities, temperatures, etc.), some degree of experimental error and deviation should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is Celsius, and pressure is atmospheric pressure or close to it. "Average" means arithmetic mean. Standard abbreviations may also be used, such as bp: base pair(s), kb: kilobase(s), pl: picoliters(s), s or sec: seconds(s), min: minutes(s), h or hr: hours(s), aa: amino acids(s), kb: kilobase(s), bp: base pair(s), nt: nucleotide(s), im: intramuscular, ip: intraperitoneal, sc: subcutaneous, etc.

[0383] Example 1 overview Historically, first-generation ADCs relied on probabilistic conjugation chemistry utilizing naturally occurring nucleophilic amino acid residues, primarily cysteine ​​and lysine side chains (Figure 2). For example, maleimide and NHS-ester-based conjugation methods were used due to their simplicity.

[0384] However, despite the initial optimism that saw a surge in heterogeneously conjugated ADCs moving into clinical practice, it was eventually recognized that these common conjugation approaches were not the optimal way to construct complex molecular systems designed for use as therapeutic agents. Heterogeneous preparations were not only difficult to produce reproducible batch-to-batch results, but were also extremely difficult to characterize due to their complexity. Clinically, poor tolerability and mediocre efficacy were obstacles that needed to be overcome to make clinical trials successful. Multiple efforts were made to redesign, improve, and stabilize the linker-payload design (Jain et al. Pharm Res, 32, 3526-3540 (2015), Su and Zhang Frontiers in Pharmacology Vol. 12, 2021).

[0385] In addition to these, various approaches have been introduced to create more homogeneous conjugates ligated with intra-antibody disulfide, using thiol crosslinking reagents (modified maleimide reagents) to form crosslinks between adjacent cysteine ​​molecules, resulting in more homogeneously constructed DAR4 molecules (Behrens et al. Mol Pharm 12,3986-3998 (2015), Badescu et al. Bioconjugate Chemistry 25,1124-1136 (2014), Schumacher et al. Org Biomol Chem 12,7261-7269 (2014)). As the causes and effects of ADC design have been better understood, the excessive toxicity and insufficient efficacy observed in patients treated with early ADCs have been linked to two characteristics related to conjugation and linker chemistry (Drake and Rabuka, Curr Opin Chemical Biology, 28, 174-180 (2015), Su and Zhang, Frontiers in Pharmacology Vol. 12, 2021). Specifically, these were 1) heterogeneous conjugate mixtures and 2) linker instability.

[0386] Heterogeneous conjugations resulted from (random) incomplete ligation to available reactive groups on the protein. For example, most maleimide and NHS-ester conjugates targeted a drug-to-antibody ratio (DAR) of 4, which was below the maximum number of conjugateable sites. ADC linker instability is evident in two locations: the conjugation chemistry and the cleavable linker element, which can be a disulfide, an acid-sensitive group, or a peptide.

[0387] The heterogeneity of first-generation ADCs was characterized by both under-conjugate and over-conjugate species within the conjugate mixture. In particular, over-conjugate species tended to be hydrophobic and aggregated and were often rapidly cleared by the liver upon injection into the body (Beck et al. Nature Reviews Drug Discovery, 16, 315-337 (2017), Sun et al. Bioconjugate Chemistry, 28, 1371-1381 (2017), Lyon et al. Nature Biotechnology, 33, 733-735 (2015)). Consequently, hepatotoxicity was observed as a common side effect of ADC administration among these first-generation heterogeneous constructs.

[0388] One common method for characterizing ADCs in terms of hydrophobicity is to use hydrophobic interaction chromatography, i.e., HIC. Conjugated antibodies have longer retention times on HIC columns compared to unconjugated antibodies. For this reason, HIC is often used as a tool to evaluate DAR. It can also be used as a tool to compare the relative hydrophobicity of different conjugates or antibodies during optimization or lead selection (Lyon et al. Nature Biotechnology, 33, 733-735 (2015)). (Similar information can be obtained from analyzing reduced ADCs on PLRP columns, which also retain more hydrophobic compounds longer than hydrophilic compounds.) Generally, the more hydrophilic the conjugate (the shorter the HIC retention time [RT]), the better the in vivo outcomes in terms of pharmacokinetics and efficacy. More hydrophilic molecules typically enjoy longer in vivo exposure after administration and, in turn, show stronger in vivo antitumor efficacy, all other conditions equal. The rationale for this outcome is that ADCs that remain in circulation longer (have longer exposure) can deliver more payload to the tumor over time and therefore exhibit higher efficacy. This reasoning assumes that the conjugate is stable and that the payload remains on the antibody and is available for delivery to the tumor.

[0389] To address the problems of conjugate heterogeneity and apparent hydrophobicity, site-specific conjugation methods have been developed. One of the most prominent and commonly used site-specific conjugation approaches involved genetically engineering an exogenous cysteine ​​residue within the antibody constant region and conjugating a linker-payload to that cysteine ​​using standard maleimide chemistry. This approach has been referred to as ThioMab (Junutula et al. Nature Biotechnology 26, 925-932 (2008), Junutula et al. Clinical Cancer Research 16, 4769-4778 (2010)). The ThioMab approach has generally been used to produce ADCs with lower overall drug-to-antibody ratios (e.g., DAR2) than those typically generated using probabilistic conjugation methods (e.g., DAR4). The resulting site-specific DAR2 conjugates have been shown to possess improved biophysical and functional properties compared to first-generation probabilistically induced conjugates. Other methods have been developed to produce site-specific conjugated ADCs, and various methods have been used to introduce bioorthogonal reactive chemical handles at defined locations on the antibody.These include a) the incorporation of functionalized non-natural amino acids at specific positions within a sequence (Xu et al. Org Process Res Dev 20,1034-1043 (2016) and Tian et al. PNAS 111,1766-1771 (2014), b) the derivatization of manipulated consensus peptide sequences and the introduction of reactive functional groups using enzyme specificity (Lee et al. Angewandte Chemie 54,12020-12024 (2015), Strop et al. Chemistry & Biology 20,161-167 (2013), and Drake et al. Bioconjugate Chemistry 25,1331-1341 (2014), and c) the modification of natural N-linked glycans to incorporate reactive handles via non-natural monosaccharide groups (van Geel et al. Bioconjugate Chemistry This includes 26,2233-2242 (2015). In addition to these, various approaches have been introduced to create more homogeneous conjugates ligated with intra-antibody disulfide, using thiol crosslinking reagents (modified maleimide reagents) to form crosslinks between adjacent cysteine ​​molecules, resulting in more homogeneously constructed DAR4 molecules (Behrens et al. Mol Pharm 12,3986-3998 (2015), Badescu et al. Bioconjugate Chemistry 25,1124-1136 (2014), Schumacher et al. Org Biomol Chem 12,7261-7269 (2014)).

[0390] Among the advantages of these various site-specific conjugation methods is the predictability of structural outcomes (conjugate homogeneity). In addition, several site-specific approaches, such as engineered cysteine ​​and aldehyde tags when used in combination with HIPS conjugation chemistry, allow for variations in linker-payload configuration on antibodies. This flexibility in linker-payload configuration is useful for structure-activity relationship mapping. Studies across conjugation platforms have shown that the location of the linker-payload configuration on the antibody influences the biophysical properties of ADCs and the in vivo functional outcomes of the molecule (e.g., efficacy, pharmacokinetics, and tolerability) (Strop et al. Chemistry & Biology 20, 161-167 (2013), Drake et al. Bioconjugate Chemistry 25, 1331-1341 (2014), Shen et al. Nature Biotechnology 30, 184-189 (2012), Su and Zhang Frontiers in Pharmacology Vol. 12, 2021).

[0391] Secondly, because the majority of clinically tested ADC linkers are peptide-based, they are susceptible to cleavage by incidental plasma proteases or other enzymes, releasing a very potent ADC payload into circulation. This resulted in systemic toxicity and poor tolerability of the ADC. Site-directed conjugation methods (and the resulting improvement in product homogeneity) do not, in themselves, address the second major design problem identified in first-generation conjugates: the element of conjugate instability driven by the linker itself. Most cleavable linkers are unstable in circulation, and the premature release of the cytotoxic payload into the patient's bloodstream represents a significant cause of systemic toxicity and poor tolerability of the ADC (Figure 4).

[0392] To address these issues, two points of instability had to be considered: the conjugation chemistry used to conjugate protein and small molecule components, as well as the composition of the linker connecting the conjugation handle to the payload. With respect to conjugation chemistry, the commonly used maleimide-cysteine ​​conjugation chemistry has inherent weaknesses. The conjugation product can undergo a retro-Michael reaction, resulting in the separation of the antibody from the drug-linker (Alley et al. Bioconjugate Chemistry 19, 759-765 (2008)), the latter of which can further react with free plasma thiols (such as those found in albumin (e.g., Wei et al. Analytical Chemistry 88, 4979-4986 (2016))) and continue to circulate as a secondary cytotoxic conjugate (Figure 3).

[0393] Historically, designing cleavable linkers that remain stable while ADCs circulate has been extremely challenging. Typical release mechanisms used in cleavable linkers include acid-mediated hydrolysis (Laguzza et al. J Med Chem 32, 548-555 (1989)), reduction of disulfide bonds (Saito et al. Advanced Drug Delivery Reviews 55, 199-215 (2003), Kellogg et al. Bioconjugate Chemistry 22, 717-727 (2011)), and proteolytic cleavage of peptide motifs. The latter is most notably exemplified by the Val-Cit-PABC linker with an MMAE payload, also known as vedotin (Doronina et al. Nature Biotechnology 21, 778-784 (2003)).

[0394] Regardless of the target antigen, common side effects of vcMMAE-conjugated ADCs include myelosuppression, including neutropenia, thrombocytopenia, and anemia. This off-target toxicity is at least partially caused by premature cleavage of the ValCit dipeptide by extracellular enzymes encountered in circulation (Masters et al. Investigational New Drugs 36, 121-135 (2018)).

[0395] A highly successful approach for stabilizing cleavable peptide-based linkers is a tandem cleavage linker system, as disclosed herein, which requires two consecutive enzymatic steps to occur for payload release (Figure 5). The monosaccharide unit bound to the phenol auto-leaving moiety (PABA) acts as a protective element and must first be removed by the corresponding lysosomal glycosidase, and then the peptide portion of the linker, which is readily cleaved by a protease to release the payload, must be exposed. Glucuronidase is only activated in the low-pH environment of the lysosomal compartment and therefore generally remains unactivated when found in serum. Thus, for tandem cleavage linkers to be enzymatically degraded, they must first be internalized into the cell and transported to lysosomes. This requirement significantly reduces the release of linkers during ADC circulation.

[0396] Most site-specific conjugation methods require extensive protein engineering of either the protein sequence itself or the conjugated carbohydrate group, which can significantly extend development time and / or involve complex manufacturing approaches with low yields. Furthermore, most site-specific approaches are incompatible with wild-type antibody formats and therefore cannot be applied to existing antibodies (often representing a significant investment in both time and cost). In addition, some site-specific methods appear incompatible for use with many desirable payloads.

[0397] Example 2 Improved ADC including maleimide tandem cut conjugate Surprisingly, it has now been discovered that the tandem cleavage element itself is sufficient to overcome many of the well-known hurdles to constructing stable and well-tolerated ADCs. In one embodiment, it has been shown that by adding the tandem cleavage element to a linker equipped with a maleimide handle (and introduced by various types of payloads), it is possible to produce safe and effective ADCs that exhibit quality equal to or better than those produced using common maleimide peptide-based linkers or using site-specific conjugation approaches with aldehyde tag / HIPS-functionalized linkers. For example, with respect to elements typically evaluated after conjugation, including yield, conjugate retention time, DAR, aggregate % (high molecular weight species, HMW), and evidence of precipitation in the conjugation mixture, maleimide tandem cleavage functionalized conjugates performed better than conjugates prepared with a standard vedotin linker (maleimide-Val-Cit-PABC-MMAE, compound 38), which also contained maleimide and MMAE but lacked the tandem cleavage element (see Table 1 and Figures 9-23, 30-32). The tandem cleavage-containing ADCs showed evidence of better reaction yield, shorter PLRP retention time, and reduced precipitation, all of which indicate improved hydrophilicity of the constructs, resulting in improved overall biophysical properties. The improved outcomes observed in linkers incorporating tandem cleavage elements may be due to the effect of the tandem cleavage elements on the overall conjugate hydrophilicity, resistance to protease degradation (via steric hindrance / shielding of protease cleavage sites), improved stability of the maleimide conjugation by influencing the exchange rate with surrounding thiols, and reduced tendency to form aggregates, self-associate, or interact with charged species. [Table 1]

[0398] Example 3 Improved hydrophilicity of maleimide tandem cleavage conjugates When comparing the HIC retention time of maleimide tandem cleavage conjugates with that of site-specific (aldehyde tag / HIPS) conjugates using tandem cleavage elements, similar, but even more dramatic, effects were observed. As shown in Table 2, DAR8 topoisomerase I inhibitor conjugates prepared using maleimide tandem cleavage eluted from the HIC column almost 2 minutes faster than site-specific HIPS tandem cleavage conjugates using the same payload. This clear improvement in hydrophilicity of maleimide tandem cleavage conjugates compared to optimized site-specific conjugates was unexpected and, furthermore, a welcome surprise considering the potential benefits obtained by conjugation to native cysteine ​​rather than specific modification of antibodies for a unique conjugation approach. [Table 2]

[0399] Example 4 Improved hydrophilicity of maleimide tandem cleavage conjugates Simultaneously, maleimide topoisomerase inhibitor conjugates with tandem cleavage linkers (compounds 36 and 37) showed improved (more hydrophilic) HIC retention time compared to Enhertu (an ADC prepared using trastuzumab conjugated to a maleimide-based deruxtecan linker without a tandem cleavage element) (Table 2). The latter observation again points to the improvements widely offered when using tandem cleavage elements in linkers, regardless of the context of conjugation chemistry or payload.

[0400] In the context of maleimide conjugates, tandem cleavage elements can even confer an additional layer of stability and tolerability to linkers that are ligated to albumin (or other serum thiols) by deconjugating from the antibody and functioning as a “prodrug” inactive form (Figure 6). [Table 3]

[0401] Example 5 Tandem-cleaved maleimide conjugates show no impact on HER2+ in vitro efficacy or ability to kill gastric cell lines. Regarding efficacy and the ability to kill target cells, trastuzumab ADCs conjugated with tandem-cleaved maleimide were equivalent in efficacy and ability to kill the HER2+ gastric cell line NCI-N87 compared to ADCs prepared with a conventional maleimide linker (without tandem cleavage) (Figures 24 and 25). In other words, the improved stability provided by the tandem-cleaved element did not reduce in vitro efficacy or adversely affect the ADC's ability to kill target antigen-expressing cells.

[0402] Example 6 Tandem-cleaved maleimide conjugates exhibit improved serum stability, improved in vivo efficacy, and better tolerability. In several embodiments, tandem-cleaved maleimide linkers have been shown to produce conjugates with improved in vitro serum stability compared to conventional maleimide linkers (e.g., vedotin) (see Figures 26–29). These linkers also appeared to exhibit improved resistance to glutathione substitution when incubated in vitro at 37°C for up to 6 days (Table 4). [Table 4]

[0403] This observation indicates that the tandem cleavage-containing linker affects the stability of the maleimide bond site itself, possibly by direct stabilization of the thioether bond, and possibly by influencing ring-opening of the maleimide ring. These unexpected observations further suggest that conjugates containing tandem cleavage-containing linkers exhibit improved in vivo efficacy and tolerability compared to ADCs containing non-tandem cleavage. These anticipated benefits may be observed for tandem cleavage-containing ADCs conjugated with maleimide, or with other conjugation chemistry such as click reactions or NHS esters.

[0404] The improved outcomes observed in linkers incorporating tandem cleavage elements may be due to the effect of the tandem cleavage elements on the overall conjugate hydrophilicity, resistance to protease degradation (via steric hindrance / shielding of protease cleavage sites), improved stability of the maleimide conjugation by influencing the exchange rate with surrounding thiols, and reduced tendency to form aggregates, self-associate, or interact with charged species.

[0405] Another advantage of maleimide-functionalized tandem cleavage linkers is that, unlike ADCs formed based on HIPS conjugation chemistry, they can be used in combination with a wide variety of acid-sensitive payloads (such as anthracyclines), thus broadening the range of applications for tandem cleavage elements.

[0406] Example 7 Conjugate with maleimide-functionalized tandem cleavage linker carrying multiple payloads In one embodiment, the modularity of the disclosed method has been demonstrated to enable the creation of conjugates with a high drug-to-antibody ratio (DAR) through the use of branched linkers carrying two or more payloads per conjugation unit. An example of such a linker is shown in Figure 7. Dicarboxylic acid 1 or 2 is further chemically modified to include a conjugation element (e.g., maleimide), and then the carboxylic acid functions of 3 and 4 serve as chemical handles for linking two linker-payload systems selected to construct compounds such as 5 and 6, the payloads being selected from topoisomerase I, kinases, or PARP inhibitors, or several other biologically active compounds.

[0407] Example 8 Dual-payload ADC in an ADC construct including maleimide-functionalized tandem cutting linker In further embodiments, branched linkers have been shown to serve as a platform for developing dual-payload ADCs, providing reliable access to cytotoxin combination modalities. This can be achieved by designing a branched linker with orthogonal connecting elements, as shown for compound 7 (Figure 8), where the carboxylic acid moiety is used to link a selected first payload, while the azide function is used to link a second payload by azide-alkyne ring addition, to access the dual-payload linker 8.

[0408] Example 9 material and method general The subject conjugates and compounds can be synthesized via a variety of different synthetic routes using commercially available starting materials and / or starting materials prepared by conventional synthetic methods. Various examples of synthetic routes that can be used to synthesize the compounds disclosed herein are shown in the following scheme.

[0409] 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 10 and 35 were commercially obtained from Shanghai Medicilon and used without purification. Compounds 58 and 63 were either commercially available or synthesized according to literature procedures. Payloads 11, 26, 39, and 67, as well as MC-VC-PAB-MMAE38 (vedotin linker), were purchased from commercial sources. In all cases, solvents were removed under reduced pressure using a Buchi Rotovapor R-114 equipped with a Buchi V-700 vacuum pump. Column chromatography was performed using a Biotage chromatography system. Preparative HPLC purification was performed using a Waters preparative HPLC unit equipped with a Phenomenex Kinetex 5μm EVO C18 150×21.2mm column. HPLC analysis was performed at room temperature using a 10–100% gradient of water and acetonitrile containing 0.05% trifluoroacetic acid on an Agilent 1100 series analytical HPLC system equipped with a Model G1322A degasser, Model G1311A quaternary pump, Model G1329A autosampler, Model G1314 tunable wavelength detector, and Agilent Poroshell 120 SB C18, 4.6 mm × 50 mm column. The HPLC was monitored at 254 or 205 nm. Low-resolution mass spectra (LRMS) were acquired at room temperature using a 10–100% gradient of water and acetonitrile containing 0.1% formic acid on an Agilent Technology 6120 quadrupole LC / MS system equipped with an Agilent 1260 Infinity HPLC system, G1314 tunable wavelength detector, and Agilent Poroshell 120 SB C18, 4.6 mm × 50 mm column.

[0410] Drug-linker synthesis Example 10 (2S,3S,4S,5R,6S)-6-(5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-5,8-diisopropyl-4 Preparation of 10-dimethyl-3,6,9-trioxo-2,13-dioxa-4,7,10-triazatetradecyl)-2-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide)-3-methylbutanamide)propanamide)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (14) [ka] In a 20 mL glass vial, monomethyl auristatin A11 (MMAE, 720 mg, 1.0 mmol), 5 mL of anhydrous DMF, and 0.35 mL of DIPEA (2.0 mmol) were combined at room temperature. The resulting mixture was stirred and treated in small amounts with PNP carbonate 10 (1014 mg, 1.0 mmol) as a solid, followed by the addition of HOAt (136 mg, 1.0 mmol) all at once at room temperature. The reaction mixture was stirred for 6 hours (HPLC) until the reaction was deemed complete. The reaction mixture was poured into 30 mL of water, the resulting white precipitate was collected by centrifugation, washed with 5 mL of water, and briefly dried under high vacuum to yield 1.87 g of crude coupling product as an off-white solid, which was proceeded to the next step without purification.

[0411] A solution of the crude intermediate (1.87 g) in 15 mL of THF was cooled to 0°C in an ice bath and slowly treated with 3 mL of 1 M lithium hydroxide aqueous solution. The reaction mixture was stirred at 0°C for 3 hours, then heated to ambient temperature, treated with 3 mL of 1 M lithium hydroxide aqueous solution, and diluted with 3 mL of methanol. The resulting mixture was stirred at room temperature for 3 hours (HPLC) until hydrolysis was complete, and then quenched by adding 1 M HCl aqueous solution to pH 7. The reaction mixture was then concentrated under reduced pressure and washed with 10 mL of MTBE. The aqueous layer was purified by reverse-phase chromatography (C18 column, 0-40% acetonitrile-water with 0.05% TFA). The pure product fractions were combined, concentrated under reduced pressure, and lyophilized to yield compound 12 as a white powder (735 mg, 0.60 mmol, 60% yield over two steps). LRMS (ESI): m / z 1229.7 [M+H] + , C 61 H 96 N8O 18 The calculated value for m / z is 1229.7.

[0412] In a 20 mL glass vial dried in an oven, compound 12 (50 mg, 40 μmol), HOAt (50 μmol, 50 μL of 1 M DMA), and DIPEA (21 μL, 150 μmol) were combined in 3 mL of DMF at room temperature. PFP-ester 13 was added to this mixture all at once. After 10 minutes, the reaction was determined to be complete by LC-MS analysis. The mixture was directly purified by reverse-phase preparative HPLC (C18, 0-75% acetonitrile-water, 0.05% TFA). The pure fractions were combined and lyophilized to obtain 46 mg of product 14 as a white powder (32 μmol, 80% yield). LRMS (ESI): m / z 1422.7 [M+H] + , C 71 H 107 N9O 21 The calculated value for m / z is 1422.8.

[0413] Synthesis of compound 13. To a mixture of 6-maleimidohexanoic acid (2.11 g, 10 mmol) and pentafluorophenol (2.2 g, 12 mmol) in 25 mL of anhydrous THF, DCC (2.06 g, 10 mmol) was added all at once at room temperature. The resulting mixture was stirred overnight, then the solid was filtered off and washed with THF. The combined filtrate was concentrated under vacuum and then purified by silica gel chromatography using (0-15% siRNA) to obtain 3.22 g of product 13 as a white solid (8.5 mmol, yield 85%). LRMS (ESI): m / z 400.1 [M+Na] + , C 16 H 12 The calculated value for F5NO4 is m / z 400.1.

[0414] Example 11 (2S,3S,4S,5R,6S)-6-(5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13 Preparation of -Dioxa-4,7,10-triazatetradecyl)-2-((2S,5S,18R)-22-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-5-isopropyl-2-methyl-4,7,17,20-tetraoxo-18-(sulfomethyl)-10,13-dioxa-3,6,16,19-tetraazadocosaneamide)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (18) Synthesis of Linker 15. [ka] To a mixture of Fmoc-L-cysteic acid 19 (0.39 g, 1 mmol) and amine 20 (233 mg, 1 mmol) in 4 mL of DMF, DIPEA (0.52 mL, 3 mmol) was added, followed by the one-time addition of HATU (380 mg, 1 mmol) at room temperature. After 30 minutes, the reaction was determined to be complete by LC-MS analysis. The reaction mixture was directly purified by reverse-phase chromatography (C18, 0-50% acetonitrile-water, 0.05% TFA). The pure fractions were combined, and the solvent was removed under vacuum to obtain 485 mg of product 21 as a white foaming solid (0.8 mmol, 80% yield). LRMS (ESI negative): m / z 605.2 [MH] - , C 29 H 38 N2O 10 The calculated value for S is m / z 605.2.

[0415] Compound 21 (485 mg, 0.8 mmol) was dissolved in a DCM-TFA mixture (1:1, 6 mL). The resulting solution was allowed to stand at room temperature for 10 minutes, then the solvent was removed under vacuum, and the residue was purified by reverse-phase chromatography (C18 column, 0-50% acetonitrile-water, 0.05% TFA). The pure fractions were combined, and the solvent was removed under vacuum to obtain 420 mg of Compound 22 as a white foaming solid (0.76 mmol, 95% yield). LRMS (ESI negative): m / z 549.1 [MH] - , C 25 H 30 N2O 10 The calculated value for S is m / z 549.2.

[0416] Carboxylic acid 22 (420 mg, 0.76 mmol) was combined with 421 mg (2.3 mmol) of pentafluorophenol in 5 mL of anhydrous DMF. This mixture was treated with EDCI-HCl (292 mg, 1.5 mmol) at room temperature and stirred for 48 hours until LC-MS analysis determined that 22 was completely consumed. The reaction mixture was directly purified by reverse-phase chromatography (C18, 0-70% acetonitrile-water, 0.05% TFA). The pure fractions were combined, concentrated under vacuum, and lyophilized to obtain 254 mg of PFP-ester 15 as a yellowish-brown solid (0.36 mmol, 47% yield). LRMS (ESI negative): m / z 715.1 [MH] - , C 31 H 29 F5N2O 10 The calculated value for S is m / z 715.2. [ka]

[0417] In a 20 mL glass vial dried in an oven, 60 mg of compound 12 (49 μmol), 26 μL of DIPEA (147 μmol), and 50 μL of HOAt solution (1 M in DMA) were mixed in 3 mL of DMF. The mixture was stirred and treated in one step at room temperature with PFP ester 15 (35 mg, 49 μmol). After 30 minutes, LC-MS analysis confirmed that the reaction was complete. The reaction mixture was directly treated at room temperature with 96 μL of piperidine (0.98 mmol). After 20 minutes, the reaction mixture was directly purified by reverse-phase preparative HPLC (C18, 0-50% acetonitrile-water, 0.05% TFA). The pure fractions were combined and lyophilized to obtain 47 mg of compound 16 (31 μmol, 63% yield) as a white solid. LRMS (ESI): m / z 1539.7 [M+H] + , C 71 H 114 N 10 O 25 The calculated value for S is m / z 1539.8.

[0418] In a glass vial, compound 16 (47 mg, 31 μmol), 2 mL of DMF, and 16 μL of DIPEA (93 μmol) were mixed. The resulting mixture was stirred and combined with 10 mg of PFP ester 17 (31 μmol) at room temperature. After 2 hours, the reaction mixture was directly purified by reverse-phase preparative HPLC (C18, 0-50% acetonitrile-water, 0.05% TFA). The pure fractions were combined and lyophilized to obtain 28 mg of compound 18 as a white solid (17 μmol, 55% yield). LRMS (ESI): m / z 1690.7 [M+H] + , C 78 H 119 N 11 O 28 The calculated value for S is m / z 1690.8.

[0419] Synthesis of compound 17. 3-Maleimidopropanoic acid (169 mg, 1 mmol) was combined with pentafluorophenol (368 mg, 2 mmol) in 4 mL of anhydrous THF. DCC (206 mg, 1 mmol) was added to this mixture all at once at room temperature. The resulting mixture was stirred overnight at room temperature. The solid was filtered and washed with THF. The combined filtrate was concentrated under vacuum and purified by silica gel chromatography (0-20% siRNA) to obtain 250 mg of compound 17 as an off-white solid. LRMS (ESI): m / z 358.1 [M+Na] + , C 13 The calculated value for H6F5NO4 is m / z 358.0.

[0420] Example 12 (2S,3S,4S,5R,6S)-6-(5-((5S,8S,11S,12R)-11-((S)-sec-butyl)-12-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropane-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-yl)-2-oxoethyl)-5,8-diisopropyl-4,10-dimethyl-3,6,9-trioxo-2,13-dioxa-4, Preparation of 7,10-Triazatetradecyl)-2-((2S,5S,18R)-22-(2-((1,2-dimethylhydrazinyl)methyl)-1H-pyrrolo[2,3-b]pyridine-1-yl)-5-isopropyl-2-methyl-4,7,17,20-tetraoxo-18-(sulfomethyl)-10,13-dioxa-3,6,16,19-tetraazadocosaneamide)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (24) [ka] To a stirred solution of compound 12 (735 mg, 0.60 mmol) in 3 mL of anhydrous DMA, DIPEA (0.21 mL, 1.2 mmol) and a solution of PFP-ester 23 (575 mg, 0.60 mmol) in 2 mL of DMA were added at room temperature, followed by the addition of HOAt (84 mg, 0.60 mmol). The resulting mixture was stirred for 30 minutes until coupling was deemed complete (HPLC analysis), and then directly treated with 1.2 mL of piperidine at room temperature. After 15 minutes, the reaction mixture was purified by reverse-phase chromatography (C18 column, 0-40% gradient of acetonitrile-water, no acid additives). The pure fractions were combined, concentrated under reduced pressure to a final volume of approximately 50 mL, and lyophilized to yield compound 24 (808 mg, 0.45 mmol, 75% yield) as a white, cottony powder. LRMS(ESI): m / z 1783.9[M+H] + , C 84 H 130 N 14 O 26 The calculated value for S is m / z 1783.9.

[0421] Synthesis of compound 23. [ka] Carboxylic acid 25 (1.33 g, 1.67 mmol) was combined with pentafluorophenol (1.23 g, 6.68 mmol) in 6.5 mL of anhydrous DMF. This mixture was treated in one step with EDCI-HCl (0.64 g, 3.34 mmol) at room temperature and stirred for 20 hours until 25 was completely consumed, as determined by HPLC analysis. The reaction mixture was placed on a C18 column and eluted with acetonitrile-water with 0.05% TFA added, under a 0-80% gradient. The pure fractions were combined, concentrated under reduced pressure until slightly turbid, and lyophilized to yield PFP-ester product 23 (1.40 g, 1.46 mmol, 87% yield) as a yellowish-brown powder. LRMS (ESI): m / z 961.2 [M+H] + , C 44 H 45 F5N6O 11 The calculated value for S is m / z 961.3.

[0422] Example 13 (2S,3S,4S,5R,6S)-6-(2-((28S,31S,34S)-28-(4-(3-(5-((S)...

Claims

1. The conjugate of equation (I), 【Chemistry 1】 During the ceremony, Each R １ However, they are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R ２ However, they are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k1 is an integer between 1 and 10. R ３ However, it includes parts that can be cut, L Ａ However, it was the first linker, W １ However, it is a drug, W ２ However, it is an antibody, X is a conjugate, which is a conjugation portion that binds to the antibody at a natural amino acid residue of the antibody.

2. R １ However, H is R ２ The conjugate according to claim 1, wherein the conjugate is an alkyl or substituted alkyl.

3. The conjugate according to claim 1 or 2, wherein k1 is 2.

4. R ３ The conjugate according to any one of claims 1 to 3, which is a chemically cleavable moiety.

5. R ３ The conjugate according to any one of claims 1 to 3, wherein the portion is enzymatically cleavable.

6. R ３ The conjugate according to claim 5, wherein the conjugate is a glycoside or a glycoside derivative.

7. The conjugate according to claim 6, wherein the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

8. The conjugate according to any one of claims 1 to 7, wherein X includes a conjugation portion configured to bind to a cysteine ​​residue or lysine residue of the antibody.

9. The conjugate according to any one of claims 1 to 7, wherein X contains maleimide, and the natural amino acid residue of the antibody contains a cysteine ​​residue.

10. The aforementioned conjugate is the conjugate of equation (II), 【Chemistry 2】 During the ceremony, Each R １ However, they are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R ２ However, they are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k1 is an integer between 1 and 10. R ３ However, it includes parts that can be cut, L Ａ However, it was the first linker, W １ However, it is a drug, W ２ The conjugate according to claim 8, wherein the conjugate is an antibody.

11. The aforementioned conjugate is the conjugate of equation (IIa), 【Transformation 3】 During the ceremony, Each R １ However, they are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R ２ However, they are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k1 is an integer between 1 and 10. R ３ However, it includes parts that can be cut, L Ａ However, it was the first linker, W １ However, it is a drug, W ２ The conjugate according to claim 8, wherein the conjugate is an antibody.

12. The first linker L Ａ but, -(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 - Includes, During the ceremony, a, b, c, d, e, and f are each independently either 0 or 1. T １ , T ２ , T ３ , T ４ , T ５ , and T ６ However, each is independent, and covalently bonded, (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 - Selected from 4-amino-piperidine (4AP), acetal group, hydrazine, disulfide, and ester, where EDA is the ethylenediamine moiety, PEG is polyethylene glycol, AA is an amino acid residue or amino acid analog, 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 However, each is independent of the 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 Selected from the group consisting of - and -P(O)OH-, where each q is an integer from 1 to 6, Each R １３ However, they are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl, Each R １５ The conjugate according to any one of claims 1 to 11, wherein the conjugate is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

13. T １ However, (C １ ~C １２ ) alkyl and substituted (C １ ~C １２ ) Selected from alkyl groups, T ２ , T ３ , T ４ , T ５ , and T ６ are each 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 (4AP), acetal group, hydrazine, and ester V １ , V ２ , V ３ , V ４ , V ５ , and V ６ However, each is independent of the 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 Selected from the group consisting of - and -P(O)OH-, (PEG) ｎ but, 【Chemistry 4】 In the formula, n is an integer between 1 and 30. EDA has the following structure: 【Transformation 5】 The ethylenediamine moiety has the following characteristics, where y is an integer from 1 to 6, and r is 0 or 1. 4-amino-piperidine (4AP) 【Transformation 6】 And, Each R １２ However, independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, any two adjacent R １２ The conjugate according to claim 12, wherein the groups can be linked in a ring to form a piperazinyl ring.

14. T １ However, (C １ ~C １２ ) is alkyl, V １ However, it is -CO-, b, c, d, e, and f are each 0, or T １ However, (C １ ~C １２ ) is alkyl, V １ However, it is -CO-, T ２ However, it is an amino acid analog, V ２ However, it is -NH-, T ３ However, (PEG) ｎ V ３ However, it is -CO-, d, e, and f are each 0, or T １ However, (C １ ~C １２ ) is alkyl, V １ However, it is -CONH-, T ２ However, substitution (C １ ~C １２ ) is alkyl, V ２ However, it is -CO-, The conjugate according to claim 12 or 13, wherein c, d, e, and f are each 0.

15. T １ , T ２ , T ３ , T ４ , T ５ , T ６ , V １ , V ２ , V ３ , V ４ , V ５ , or V ６ The conjugate according to claim 12 or 13, wherein one of the members is a branching group.

16. The aforementioned branching group is -CONR １５ - and 4AP selected, the conjugate according to claim 15.

17. The aforementioned branching base is the second linker L Ｂ The conjugate according to claim 15 or 16, which is coupled to the following.

18. The second linker L Ｂ but, -(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 - Includes, During the ceremony, g, h, i, j, k, and l are each independently either 0 or 1. T ７ , T ８ , T ９ , T １０ , T １１ , and T １２ However, each is independent, and covalently bonded, (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 - Selected from 4-amino-piperidine (4AP), acetal group, hydrazine, disulfide, and ester, where EDA is the ethylenediamine moiety, PEG is polyethylene glycol, AA is an amino acid residue or amino acid analog, 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 However, each is independent of the 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 Selected from the group consisting of - and -P(O)OH-, where each q is an integer from 1 to 6, Each R １３ However, they are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl, Each R １５ The conjugate according to claim 17, wherein the elements are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

19. L Ｂ However, it is bonded to the compound of formula (III), 【Transformation 7】 During the ceremony, Each R ４ However, they are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R ５ However, they are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k2 is an integer between 1 and 10. R ６ However, it includes parts that can be cut, W １ａ The conjugate according to claim 17 or 18, wherein the conjugate is a drug.

20. R ４ However, H is R ５ The conjugate according to claim 19, wherein the conjugate is an alkyl or substituted alkyl.

21. The conjugate according to claim 19 or 20, wherein k2 is 2.

22. R ６ The conjugate according to any one of claims 19 to 21, wherein the portion is chemically cleavable.

23. R ６ The conjugate according to any one of claims 19 to 21, wherein the portion is enzymatically cleavable.

24. R ６ The conjugate according to claim 23, wherein the conjugate is a glycoside or a glycoside derivative.

25. The conjugate according to claim 24, wherein the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

26. T １ However, (C １ ~C １２ ) is alkyl, V １ However, branching group (-CONR １５ ) - and T ２ However, (C １ ~C １２ ) is alkyl, V ２ However, it is -CONH-, T ３ However, substitution (C １ ~C １２ ) is alkyl, V ３ However, it is -CO-, d, e, and f are each 0, T ７ However, (C １ ~C １２ ) is alkyl, V ７ However, it is -CONH-, T ８ However, substitution (C １ ~C １２ ) is alkyl, V ８ However, it is -CO-, i, j, k, and l are each 0, or T １ However, (C １ ~C １２ ) is alkyl, V １ However, it is -CO-, T ２ However, it is a branching group (4AP), V ２ However, it is -CO-, T ３ However, (PEG) ｎ V ３ However, it is -CONH-, T ４ However, substitution (C １ ~C １２ ) is alkyl, V ４ However, it is -CO-, e and f are both 0, T ７ However, (PEG) ｎ V ７ However, it is -CONH-, T ８ However, substitution (C １ ~C １２ ) is alkyl, V ８ However, it is -CO-, i, j, k, and l are each 0, or T １ However, (C １ ~C １２ ) is alkyl, V １ However, it is -CO-, T ２ However, it is a branching group (4AP), V ２ However, it is -CO-, T ３ However, (C １ ~C １２ ) is alkyl, V ３ However, it does not exist. T ４ However, it is a heteroaryl (triazole), V ４ However, it does not exist. T ５ However, (C １ ~C １２ ) is alkyl, V ５ However, it is -CONH-, T ６ However, substitution (C １ ~C １２ ) is alkyl, V ６ However, it is -CO-, T ７ However, (PEG) ｎ V ７ However, it is -CONH-, T ８ However, substitution (C １ ~C １２ ) is alkyl, V ８ However, it is -CO-, The conjugate according to any one of claims 12 to 25, wherein i, j, k, and l are each 0.

27. W １ and W １ａ However, the conjugate according to any one of claims 15 to 26 is the same drug.

28. W １ and W １ａ However, the conjugate according to any one of claims 15 to 26 is a different drug.

29. The aforementioned conjugate, 【Chemistry 8-1】 【Chemistry 8-2】 【Chemistry 8-3】 【Chemistry 8-4】 【Chemistry 8-5】 【Chemistry 8-6】 A conjugate according to any one of claims 1 to 28, selected from the above.

30. A compound of formula (IV), 【Chemistry 9】 During the ceremony, Each R １ However, they are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R ２ However, they are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k1 is an integer between 1 and 10. R ３ However, it includes parts that can be cut, L Ａ However, it was the first linker, W １ However, it is a drug, A compound in which X is a conjugation moiety configured to bind to the antibody at a natural amino acid residue of the antibody.

31. R １ However, H is R ２ The compound according to claim 30, wherein the compound is alkyl or substituted alkyl.

32. The compound according to claim 30 or 31, wherein k1 is 2.

33. R ３ The compound according to any one of claims 30 to 32, wherein the portion is chemically cleavable.

34. R ３ The compound according to any one of claims 30 to 32, wherein the portion is enzymatically cleavable.

35. R ３ The compound according to claim 34, wherein the compound is a glycoside or a glycoside derivative.

36. The compound according to claim 35, wherein the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

37. The compound according to any one of claims 30 to 36, wherein X includes a conjugation moiety configured to bind to a cysteine ​​residue or lysine residue of the antibody.

38. The compound according to any one of claims 30 to 36, wherein X contains maleimide, and the natural amino acid residue of the antibody contains a cysteine ​​residue.

39. The aforementioned compound is a compound of formula (V), 【Chemistry 10】 During the ceremony, Each R １ However, they are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R ２ However, they are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k1 is an integer between 1 and 10. R ３ However, it includes parts that can be cut, L Ａ However, it was the first linker, W １ However, the compound according to claim 37 is a drug.

40. The aforementioned compound is a compound of formula (Va), 【Chemistry 11】 During the ceremony, Each R １ However, they are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R ２ However, they are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k1 is an integer between 1 and 10. R ３ However, it includes parts that can be cut, L Ａ However, it was the first linker, W １ However, the compound according to claim 37 is a drug.

41. The first linker L Ａ but, -(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 - Includes, During the ceremony, a, b, c, d, e, and f are each independently either 0 or 1. T １ , T ２ , T ３ , T ４ , T ５ , and T ６ However, each is independent, and covalently bonded, (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 - Selected from 4-amino-piperidine (4AP), acetal group, hydrazine, disulfide, and ester, where EDA is the ethylenediamine moiety, PEG is polyethylene glycol, AA is an amino acid residue or amino acid analog, 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 However, each is independent of the 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 Selected from the group consisting of - and -P(O)OH-, where each q is an integer from 1 to 6, Each R １３ However, they are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl, Each R １５ The compound according to any one of claims 30 to 40, which is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

42. T １ However, (C １ ~C １２ ) alkyl and substituted (C １ ~C １２ ) Selected from alkyl groups, T ２ , T ３ , T ４ , T ５ , and T ６ However, each is independent, and covalently bonded, (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 - Selected from 4-amino-piperidine (4AP), acetal group, hydrazine, and ester, V １ , V ２ , V ３ , V ４ , V ５ , and V ６ However, each is independent of the 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 Selected from the group consisting of - and -P(O)OH-, (PEG) ｎ but, 【Chemistry 12】 In the formula, n is an integer between 1 and 30. EDA has the following structure: 【Chemistry 13】 The ethylenediamine moiety has the following characteristics, where y is an integer from 1 to 6, and r is 0 or 1. 4-amino-piperidine (4AP) 【Chemistry 14】 And, Each R １２ However, independently selected from hydrogen, alkyl, substituted alkyl, polyethylene glycol moiety, aryl, and substituted aryl, any two adjacent R １２ The compound according to claim 41, wherein the groups can be linked in a cyclic manner to form a piperazinyl ring.

43. T １ However, (C １ ~C １２ ) is alkyl, V １ However, it is -CO-, b, c, d, e, and f are each 0, or T １ However, (C １ ~C １２ ) is alkyl, V １ However, it is -CO-, T ２ However, it is an amino acid analog, V ２ However, it is -NH-, T ３ However, (PEG) ｎ V ３ However, it is -CO-, d, e, and f are each 0, or T １ However, (C １ ~C １２ ) is alkyl, V １ However, it is -CONH-, T ２ However, substitution (C １ ~C １２ ) is alkyl, V ２ However, it is -CO-, The compound according to claim 41 or 42, wherein c, d, e, and f are each 0.

44. T １ , T ２ , T ３ , T ４ , T ５ , T ６ , V １ , V ２ , V ３ , V ４ , V ５ , or V ６ The compound according to claim 41 or 42, wherein one of the groups is a branched group.

45. The aforementioned branching group is -CONR １５ - and 4AP, the compound according to claim 44.

46. The aforementioned branching base is the second linker L Ｂ The compound according to claim 44 or 45, comprising:

47. The second linker L Ｂ but, -(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 - Includes, During the ceremony, g, h, i, j, k, and l are each independently either 0 or 1. T ７ , T ８ , T ９ , T １０ , T １１ , and T １２ However, each is independent, and covalently bonded, (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 - Selected from 4-amino-piperidine (4AP), acetal group, hydrazine, disulfide, and ester, where EDA is the ethylenediamine moiety, PEG is polyethylene glycol, AA is an amino acid residue or amino acid analog, 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 However, each is independent of the 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 Selected from the group consisting of - and -P(O)OH-, where each q is an integer from 1 to 6, Each R １３ However, they are independently selected from hydrogen, alkyl, substituted alkyl, aryl, and substituted aryl, Each R １５ The compound according to claim 46, wherein the compound is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, carboxyl, carboxyl ester, acyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl.

48. L Ｂ However, it is bonded to the compound of formula (VI), 【Chemistry 15】 During the ceremony, Each R ４ However, they are independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, and substituted alkynyl, Each R ５ However, they are independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. k2 is an integer between 1 and 10. R ６ However, it includes parts that can be cut, W １ａ The compound according to claim 46 or 47, which is a drug.

49. R ４ However, H is R ５ The compound according to claim 48, wherein the compound is alkyl or substituted alkyl.

50. The compound according to claim 48 or 49, wherein k2 is 2.

51. R ６ The compound according to any one of claims 48 to 50, wherein the portion is chemically cleavable.

52. R ６ The compound according to any one of claims 48 to 50, wherein the portion is enzymatically cleavable.

53. R ６ The compound according to claim 52, wherein the compound is a glycoside or a glycoside derivative.

54. The compound according to claim 53, wherein the glycoside or glycoside derivative is selected from glucuronide, galactoside, glucoside, mannoside, fucoside, O-GlcNAc, and O-GalNAc.

55. T １ However, (C １ ~C １２ ) is alkyl, V １ However, branching group (-CONR １５ ) - and T ２ However, (C １ ~C １２ ) is alkyl, V ２ However, it is -CONH-, T ３ However, substitution (C １ ~C １２ ) is alkyl, V ３ However, it is -CO-, d, e, and f are each 0, T ７ However, (C １ ~C １２ ) is alkyl, V ７ However, it is -CONH-, T ８ However, substitution (C １ ~C １２ ) is alkyl, V ８ However, it is -CO-, i, j, k, and l are each 0, or T １ However, (C １ ~C １２ ) is alkyl, V １ However, it is -CO-, T ２ However, it is a branching group (4AP), V ２ However, it is -CO-, T ３ However, (PEG) ｎ V ３ However, it is -CONH-, T ４ However, substitution (C １ ~C １２ ) is alkyl, V ４ However, it is -CO-, e and f are both 0, T ７ However, (PEG) ｎ V ７ However, it is -CONH-, T ８ However, substitution (C １ ~C １２ ) is alkyl, V ８ However, it is -CO-, i, j, k, and l are each 0, or T １ However, (C １ ~C １２ ) is alkyl, V １ However, it is -CO-, T ２ However, it is a branching group (4AP), V ２ However, it is -CO-, T ３ However, (C １ ~C １２ ) is alkyl, V ３ However, it does not exist. T ４ However, it is a heteroaryl (triazole), V ４ However, it does not exist. T ５ However, (C １ ~C １２ ) is alkyl, V ５ However, it is -CONH-, T ６ However, substitution (C １ ~C １２ ) is alkyl, V ６ However, it is -CO-, T ７ However, (PEG) ｎ V ７ However, it is -CONH-, T ８ However, substitution (C １ ~C １２ ) is alkyl, V ８ However, it is -CO-, The compound according to any one of claims 41 to 54, wherein i, j, k, and l are each 0.

56. W １ and W １ａ However, the compound described in any one of claims 44 to 55 is the same drug.

57. W １ and W １ａ However, the compound according to any one of claims 44 to 55 is a different drug.

58. The aforementioned compound, 【Chemistry 16-1】 【Chemistry 16-2】 【Chemistry 16-3】 【Chemistry 16-4】 【Chemistry 16-5】 【Chemistry 16-6】 A compound according to any one of claims 30 to 55, selected from the above.

59. A pharmaceutical composition, A conjugate according to any one of claims 1 to 29, A pharmaceutical composition comprising a pharmaceutically acceptable excipient.

60. It is a method, A method comprising administering a conjugate according to any one of claims 1 to 29 to a subject.

61. A method for treating cancer in a subject, A method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a conjugate according to any one of claims 1 to 29, wherein the administration is effective in treating the cancer of the subject.

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