Camptothecin derivative

Abstract

Disclosed herein are novel cytotoxic compounds, and cytotoxic conjugates comprising these cytotoxic compounds and cell binding agents. More specifically, the present disclosure relates to novel camptothecin derivatives thereof, intermediates thereof, conjugates thereof, and pharmaceutically acceptable salts thereof, which are useful as medicaments, particularly as antiproliferative agents (anticancer agents).

Description

[0001] This application is a divisional application of the patent application with application number 202080031665.2, application date April 10, 2020, and name “Camptothecin Derivatives”.

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 839,440, filed April 26, 2019, U.S. Provisional Patent Application No. 62 / 875,169, filed July 17, 2019, and U.S. Provisional Patent Application No. 62 / 978,159, filed February 18, 2020. Each of the foregoing applications is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on March 27, 2020, is named 000219-0002-WO1_-_Sequence_Listing.txt, and is 61,926 bytes in size. Technical Field

[0005] Disclosed herein are novel compounds, and conjugates thereof. More specifically, the present disclosure relates to novel camptothecin derivatives, their intermediates, metabolites and conjugates, and pharmaceutically acceptable salts thereof, which are useful as pharmaceutical agents, particularly as antiproliferative agents (anticancer agents). Background Art

[0006] Cell-binding agent-drug conjugates, including antibody-drug conjugates (ADCs), have emerged as a powerful class of pharmaceutical agents with efficacy in a variety of abnormal cell growth or proliferative diseases, such as cancer. Cell-binding agent-drug conjugates, such as ADCs, are typically composed of three distinct elements: a cell-binding agent (e.g., an antibody); a linker; and a cytotoxic moiety.

[0007] Camptothecin (CPT) is a pentacyclic alkaloid isolated from the bark and trunk of Camptotheca acuminata (Camptotheca / Happy tree), a tree native to China. Camptothecin inhibits topoisomerase I, which causes cell death. Due to the cytotoxic mechanism and broad-spectrum anti-tumor activity of camptothecin, a lot of efforts have been made to develop clinical analogs of camptothecin. However, poor solubility and inactivity under physiological conditions have limited the clinical development of suitable camptothecin analogs. Camptothecin and most of its derivatives are insoluble in aqueous buffers. In addition, camptothecin is in equilibrium in the active lactone form and the inactive hydrolyzed carboxylate form, thus limiting its therapeutic efficacy.

[0008] There is a need for therapeutically effective camptothecin derivatives with increased solubility, potency, lactone stability, and bioavailability. Summary of the Invention

[0009] In one aspect, the present invention provides a compound of formula I or a pharmaceutically acceptable salt thereof:

[0010] Z-L 1 —D (Formula I)

[0011] in:

[0012] D is represented by the following structural formula:

[0013]

[0014] R 1 is -F, -CH3 or -CF3;

[0015] R 2 Yes -H, -F, -OR 3 、-SR 3 、-S(O)R 4 、-S(O)2R 4 , C1-C6 alkyl or C1-C6 fluoroalkyl; or R 1 and R 2 The carbon atoms to which they are attached together form a methylenedioxy or difluoromethylenedioxy ring;

[0016] R 3 is H or C1-C6 alkyl;

[0017] R 4 is a C1-C6 alkyl group;

[0018] L 1 does not exist, is -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, -X 1' -(C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; wherein * is the site of covalent attachment to Z;

[0019] X 1 Is -O-, -S-, -S(O)-, -S(O)2-, -C(=O)-, -NR 5 -、-NR 5 C(=O)- or -C(=O)NR 5 -;

[0020] X 1' is -O-, -S-, -S(O)- or -S(O)2-;

[0021] L 2 is phenylene;

[0022] Each R 5 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0023] Z is -H or -X 2 ;

[0024] X 2 Yes-OR 6 、-SR 6 、-S(O)R 6 、-S(O)2R 6 、-SSR 6 or -N(R 6 )2;

[0025] Each R 6 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0026] L 1 and L 2 Each independently optionally substituted by 1-4 selected from halogen, -CN, -OR 7 、-SR 7 、-N(R 7 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 is substituted with a substituent of heterocycloalkyl, aryl, or heteroaryl; and

[0027] Each R 7 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0028] The condition is that if R 1 If it is F, then L 1 -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, -X 1' -(C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; wherein * is the site of covalent attachment to Z; and Z is -X 2 ;and

[0029] The condition is that if R 1 is F and R 2 is -OMe, then -L1 -Z cannot be -NH2.

[0030] In some embodiments, the compounds of Formula I have the further proviso that if R 1 is F and R 2 If it is -Me, then -L1-Z cannot be -CH2OH.

[0031] In some embodiments, R 1 Is -H or -F. In some embodiments, R 1 In some embodiments, R 2 is -H, -F, -OCF3, -CF3, -OMe, -OEt, -SMe, -S(O)Me, -S(O)2Me, -SEt, -S(O)Et, -S(O2)Et, methyl or ethyl. In some embodiments, R 2 In some embodiments, R 2 is -OMe, -SMe, -S(O)Me or methyl. In some embodiments, R 2 In some embodiments, R 1 Yes -F and R 2 In some embodiments, R 1 is methyl and R 2 In some embodiments, R 1 Yes -F and R 2 It is -methyl.

[0032] In some embodiments, -L 1 -Z is -H. In some embodiments, -L 1 -Z is -(C1-C6 alkylene)-H or -(C1-C6 alkylene)-X 2 In some embodiments, -L 1 -Z is -(C1-C6 alkylene)-H. In some embodiments, -L 1 -Z is -(C1-C6 alkylene)-X 2 In some embodiments, -L 1 -Z is -(C1-C6 alkylene)-X 2 In some embodiments, -L 1 -Z is methyl, ethyl, propyl or butyl.

[0033] In some embodiments, -L 1 -Z is -(C1-C4 alkylene)-OR 6 、-(C1-C4 alkylene)-SR 6 or -(C1-C4 alkylene)-N(R 6)2. In some embodiments, -L 1 -Z is -(C1-C4 alkylene)-OR 6 In some embodiments, -L 1 -Z is -(C1-C4 alkylene)-SR 6 In some embodiments, -L 1 -Z is -(C1-C4 alkylene)-N(R 6 )2.

[0034] In some embodiments, -L 1 -Z is -CH2OH, -(CH2)2OH, -(CH2)3OH, -(CH2)4OH, -CH2OMe, -(CH2)2OMe, -(CH2)3OMe, -(CH2)4OMe, -CH2SH, -(CH2)2SH, -(CH2)3SH, -(CH2)4SH, -CH2SMe, -(CH2)2SMe, -(CH2)3SMe, -(CH2)4SMe, -CH2NH2, -(CH2)2NH2, -(CH2)3NH2 or -(CH2)4NH2.

[0035] In some embodiments, -L 1 -Z is -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-OR 6 、-(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-SR 6 , -(C1-C5 alkylene)-S-(C1-C5 alkylene)-SR 6 or -(C1-C5 alkylene)-S-(C1-C5 alkylene)-SSR 6 In some embodiments, -L 1 -Z is -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-OR 6 In some embodiments, -L 1 -Z is -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-SR 6 In some embodiments, -L 1 -Z is -(C1-C5 alkylene)-S-(C1-C5 alkylene)-SR 6 In some embodiments, -L 1 -Z is -(C1-C5 alkylene)-S-(C1-C5 alkylene)-SSR 6 .

[0036] In some embodiments, -L 1 -Z is -CH2NHC(=O)CH2OH, -CH2NHC(=O)(CH2)2OH, -CH2NHC(=O)(CH2)3OH, -CH2NHC(=O)(CH2)4OH, -CH2 NHC(=O)(CH2)5OH, -CH2NHC(=O)CH2OMe, -CH2NHC(=O)(CH2)2OMe, -CH2NHC(=O)(CH2)3OMe, -CH2NHC(= O)(CH2)4OMe, -CH2NHC(=O)(CH2)5OMe, -CH2NHC(=O)CH2SH, -CH2NHC(=O)(CH2)2SH, -CH2NHC(=O)(CH2 )3SH, -CH2NHC(=O)(CH2)4SH, -CH2NHC(=O)(CH2)5SH, -CH2NHC(=O)CH2SMe, -CH2NHC(=O)(CH2)2SMe, - CH2NHC(=O)(CH2)3SMe, -CH2NHC(=O)(CH2)4SMe, -CH2NHC(=O)(CH2)5SMe, -CH2SCH2OH, -CH2S(CH2)2O H, -CH2S(CH2)3OH, -CH2S(CH2)4OH, -CH2S(CH2)5OH, -CH2SCH2OMe, -CH2S(CH2)2OMe, -CH2S(CH2)3OMe , -CH2S(CH2)4OMe, -CH2S(CH2)5OMe, -CH2SCH2SH, -CH2S(CH2)2SH, -CH2S(CH2)3SH, -CH2S(CH2)4SH, -CH2S(CH2)5SH, -CH2SCH2SMe, -CH2S(CH2)2SMe, -CH2S(CH2)3SMe, -CH2S(CH2)4SMe or -CH2S(CH2)5SMe.

[0037] In some embodiments, each R 5 is independently -H, methyl or benzyl. In some embodiments, each R 5 is independently -H. In some embodiments, each R 5 In some embodiments, each R 5 It's benzyl.

[0038] In some embodiments, each R 6 is independently -H, methyl or benzyl. In some embodiments, each R 6 is independently -H. In some embodiments, each R 6In some embodiments, each R 6 It's benzyl.

[0039] In some embodiments, -L 1 -Z is -X 1' -(C1-C4 alkylene)-X 2 In some embodiments, -L 1 -Z is -OCH2OH, -O(CH2)2OH, -O(CH2)3OH, -O(CH2)4OH, -SCH2OH, -S(CH2)2OH, -S(CH2)3OH, -S(CH2)4OH, -S(O)CH2OH, -S(O )(CH2)2OH, -S(O)(CH2)3OH, -S(O)(CH2)4OH, -S(O)2CH2OH, -S(O)2(CH2)2OH, -S(O)2(CH2)3OH, -S(O)2(CH2)4OH, -OCH2S Me, -O(CH2)2SMe, -O(CH2)3SMe, -O(CH2)4SMe, -SCH2SMe, -S(CH2)2SMe, -S(CH2)3SMe, -S(CH2)4SMe, -S(O)CH2SMe, -S(O) (CH2)2SMe, -S(O)(CH2)3SMe, -S(O)(CH2)4SMe, -S(O)2CH2SMe, -S(O)2(CH2)2SMe, -S(O)2(CH2)3SMe or -S(O)2(CH2)4SMe.

[0040] In some embodiments, -L 1 -Z is -(C1-C6 alkylene)-X 1 -L 2 -X 2 In some embodiments, -L 1 -Z is In some embodiments, -L 1 -Z is In some embodiments, -L 1 -Z is

[0041] In some embodiments, the compound is any one of the following compounds:

[0042]

[0043]

[0044] In some embodiments, the compound is any one of the following compounds:

[0045]

[0046] In some embodiments, the compound is any one of the compounds selected from Table 1B. In another aspect, the present invention provides a compound of Formula II or a pharmaceutically acceptable salt thereof:

[0047] E—A—Z'—L 1 —D (Formula II)

[0048] in:

[0049] D is represented by the following structural formula:

[0050]

[0051] R 1 is -H, -F, -CH3 or -CF3;

[0052] R 2 Yes -H, -F, -OR 3 、-SR 3 、-S(O)R 4 、-S(O)2R 4 , C1-C6 alkyl or C1-C6 fluoroalkyl; or R 1 and R 2 The carbon atoms to which they are attached together form a methylenedioxy or difluoromethylenedioxy ring; provided that R 1 and R 2 Cannot be -H at the same time;

[0053] R 3 is H or C1-C6 alkyl;

[0054] R 4 is a C1-C6 alkyl group;

[0055] L 1 does not exist, is -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, X 1' -(C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; wherein * is the site of covalent attachment to Z';

[0056] X 1 Is -O-, -S-, -S(O)-, -S(O)2-, -C(=O)-, -NR 5 -、-NR 5 C(=O)- or -C(=O)NR 5 -;

[0057] X1' is -O-, -S-, -S(O)- or -S(O)2-;

[0058] L 2 is phenylene;

[0059] Each R 5 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0060] Z' is -O-CH2-NR 8 -*、-S-CH2-NR 8 -*, -NR 8 -*; wherein * is the site of covalent attachment to A;

[0061] Each R 8 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0062] L 1 and L 2 Each independently optionally substituted by 1-4 selected from halogen, -CN, -OR 7 、-SR 7 、-N(R 7 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 is substituted with a substituent of heterocycloalkyl, aryl, or heteroaryl; and

[0063] Each R 7 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0064] A is a peptide comprising 2 to 10 amino acids; wherein A is optionally substituted with one or more polyols; and

[0065] E is -C(=O)-L 3 -X 3 ;

[0066] L 3 Yes-(C1-C 10 Alkylene)-or-Y 1 -(C1-C 10 Alkylene)-X 4 -Y 2 -(C1-C 10 Alkylene)-*; wherein * is covalently linked to X 3 The site of

[0067] Y 1Does not exist, is-(CR a R b O) n -or-(CR a R b CR a' R b' O) m -;

[0068] X 4 Yes-NR 9 C(=O)- or -C(=O)NR 9 -;

[0069] Y 2 Does not exist, is-(CR c R d O) o -or--(CR c R d CR c' R d' O) p -;

[0070] n, m, o and p are each independently 1-10;

[0071] Each R a 、R b 、R a' 、R b' 、R c 、R d 、R c' and R d' is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0072] Among them L 3 Optionally substituted by 0-4 selected from halogen, -CN, -OR 11 、-SR 11 、-N(R 11 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 Substitution of heterocycloalkyl, aryl, heteroaryl, and polyol substituents;

[0073] Each R 11 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0074] X 3 yes

[0075] -C(=O)-CR bb R cc -W', -NR ee -C(=O)-CR bb R cc -W' or -SR 10 ;

[0076] Each W' is independently -H, -N(R gg 2. C1-C 10 Alkyl, C1-C 10 Alkenyl, C1-C 10 Alkynyl, C3-C6 cycloalkyl, aryl, heteroaryl or -(CH2CH2O) q -R ff ;

[0077] q is 1 to 24;

[0078] Each R aa 、R bb 、R cc 、R ee and R ff is independently -H or optionally substituted C1-C6 alkyl;

[0079] Each R YY and R XX are independently -H or C1-C6 alkyl;

[0080] R gg are each independently -H or C1-C6 alkyl; and

[0081] R 9 and R 10 Each is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl.

[0082] In some embodiments, R 1 Is -H or -F. In some embodiments, R 1 In some embodiments, R 2 is -H, -F, -OCF3, -CF3, -OMe, -OEt, -SMe, -S(O)Me, -S(O)2Me, -SEt, -S(O)Et, -S(O2)Et, methyl or ethyl. In some embodiments, R 2 In some embodiments, R 2 is -OMe, -SMe, -S(O)Me or methyl. In some embodiments, R 2 In some embodiments, R 1 Yes -F and R 2In some embodiments, R 1 is methyl and R 2 In some embodiments, R 1 Yes -F and R 2 It is -methyl.

[0083] In some embodiments, -L 1 -Z'-* is -(C1-C4 alkylene)-O-CH2-NR 8 -*, -(C1-C4 alkylene)-S-CH2-NR 8 -* or -(C1-C4 alkylene)-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C4 alkylene)-O-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C4 alkylene)-S-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C4 alkylene)-NR 8 -*.

[0084] In some embodiments, -L 1 -Z'-* is -CH2O-CH2NH-*, -(CH2)2O-CH2NH-*, -(CH2)3O-CH2NH-*, -(CH2)4O-CH2NH-*, -CH2S-CH2NH-*, -(CH2 )2S-CH2NH-*, -(CH2)3S-CH2NH-*, -(CH2)4S-CH2NH-*, -CH2NH-*, -(CH2)2NH-*, -(CH2)3NH-* or -(CH2)4NH-*.

[0085] In some embodiments, -L 1 -Z'-* is -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-O-CH2-NR 8 -*, -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-S-CH2-NR 8 -*, -(C1-C5 alkylene)-S-(C1-C5 alkylene)-S-CH2-NR 8 -* or -(C1-C5 alkylene)-S-(C1-C5 alkylene)-SS-CH2-NR 8 -*. In some embodiments, -L 1-Z'-* is -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-O-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-S-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C5 alkylene)-S-(C1-C5 alkylene)-S-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C5 alkylene)-S-(C1-C5 alkylene)-SS-CH2-NR 8 -*.

[0086] In some embodiments, -L 1 -Z'-* is -CH2NHC(=O)CH2O-CH2-NH-*, -CH2NH C(=O)(CH2)2O-CH2-NH-*, -CH2NHC(=O)(CH2)3O-CH2-NH-*, -CH2NHC(=O)(CH2)4O-CH2-NH-*, -CH2NHC(=O)(CH2)5O-CH2-NH-*, -CH2NHC( =O)CH2S-CH2-NH-*, -CH2NHC(=O)(CH2)2S-CH2-NH-*, -CH2NHC(=O)(CH2)3S-CH2-NH-*, -CH2NHC(=O)(CH2)4S-CH2-NH-*, -CH2NHC(=O)(C H2)5S-CH2-NH-*, -CH2SCH2O-CH2-NH-*, -CH2S(CH2)2O-CH2-NH-*, -CH2S(CH2)3O-CH2-NH-*, -CH2S(CH2)4O-CH2-NH-*, -CH2S(CH2)5O-C H2-NH-*, -CH2SCH2S-CH2-NH-*, -CH2S(CH2)2S-CH2-NH-*, -CH2S(CH2)3S-CH2-NH-*, -CH2S(CH2)4S-CH2-NH-* or -CH2S(CH2)5S-CH2-NH-*.

[0087] In some embodiments, each R 5 is independently -H, methyl or benzyl. In some embodiments, each R 5 is independently -H. In some embodiments, each R 5In some embodiments, each R 5 In some embodiments, each R 8 is independently -H, methyl or benzyl. In some embodiments, each R 8 is independently -H. In some embodiments, each R 8 In some embodiments, each R 8 It's benzyl.

[0088] In some embodiments, -L 1 -Z'-* is -X 1' -(C1-C4 alkylene)-O-CH2-NR 8 -*, -X 1' -(C1-C4 alkylene)-S-CH2-NR 8 -* or -X 1' -(C1-C4 alkylene)-NR 8 -*. In some embodiments, -L 1 -Z'-* is -X 1' -(C1-C4 alkylene)-O-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -X 1' -(C1-C4 alkylene)-S-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -X 1' -(C1-C4 alkylene)-NR 8 -*.

[0089] In some embodiments, -L 1-Z'-* is -OCH2O-CH2-NH-*, -O(CH2)2O-CH2-NH-*, -O(CH2)3O-CH2-NH-*, -O(CH2)4O-CH2-NH-*, -SCH2O-CH2-NH-*, -S(CH2)2O-CH2-N H-*, -S(CH2)3O-CH2-NH-*, -S(CH2)4O-CH2-NH-*, -S(O)CH2O-CH2-NH-*, -S(O)(CH2)2O-CH2-NH-*, -S(O)(CH2)3O-CH2-NH-*, -S(O)( CH2)4O-CH2-NH-*, -S(O)2CH2O-CH2-NH-*, -S(O)2(CH2)2O-CH2-NH-*, -S(O)2(CH2)3O-CH2-NH-*, -S(O)2(CH2)4O-CH2-NH-*, -OCH2S -CH2-NH-*, -O(CH2)2S-CH2-NH-*, -O(CH2)3S-CH2-NH-*, -O(CH2)4S-CH2-NH-*, -SCH2S-CH2-NH-*, -S(CH2)2S-CH2-NH-*, -S(CH2)3S -CH2-NH-*, -S(CH2)4S-CH2-NH-*, -S(O)CH2S-CH2-NH-*, -S(O)(CH2)2S-CH2-NH-*, -S(O)(CH2)3S-CH2-NH-*, -S(O)(CH2)4S-CH2-NH -*, -S(O)2CH2S-CH2-NH-*, -S(O)2(CH2)2S-CH2-NH-*, -S(O)2(CH2)3S-CH2-NH-*, -S(O)2(CH2)4S-CH2-NH-*, -OCH2-NH-*, -O(CH2)2 -NH-*, -O(CH2)3-NH-*, -O(CH2)4S-NH-*, -SCH2-NH-*, -S(CH2)2-NH-*, -S(CH2)3-NH-*, -S(CH2)4-NH-*, -S(O)CH2-NH-*, -S(O)(CH2 )2-NH-*, -S(O)(CH2)3-NH-*, -S(O)(CH2)4-NH-*, -S(O)2CH2-NH-*, -S(O)2(CH2)2-NH-*, -S(O)2(CH2)3-NH-* or -S(O)2(CH2)4-NH-*.

[0090] In some embodiments, -L 1 -Z'-* is -(C1-C6 alkylene)-X 1 -L 2 -Z'-*. In some embodiments, -L1 -Z'-*Yes In some embodiments, -L 1 -Z'-*Yes In some embodiments, -L 1 -Z'-*Yes

[0091] Disclosed herein -L 1 In various embodiments of -Z'-*, * is the site of covalent attachment to A.

[0092] In some embodiments, A is a peptide comprising 2 to 8 amino acids. In some embodiments, A is a peptide comprising 2 to 4 amino acids. In some embodiments, at least one amino acid in the peptide is an L amino acid. In some embodiments, each amino acid in the peptide is an L amino acid. In some embodiments, at least one amino acid in the peptide is a D amino acid.

[0093] In some embodiments, A is -(AA 1 )-(AA 2 ) a1 -*, where * is the site of covalent attachment to E; AA 1 and AA 2 are each independently an amino acid residue; and a1 is an integer from 1 to 9.

[0094] In some embodiments, -AA 1 -(AA 2 ) a1 -*is -Gly-Gly-Gly-*, -Ala-Val-*, -Val-Ala-*, -Val-Cit-*, -Val-Lys-*, -Lys-Val-*, -Phe-Lys-*, -Lys-Phe-*, -Lys-Ly s-*, -Ala-Lys-*, -Lys-Ala-*, -Phe-Cit-*, -Cit-Phe-*, -Leu-Cit-*, -Cit-Leu-*-Ile-Cit-*, -Phe-Ala-*, -Ala-Phe-*, -Phe-N 9 -Tosyl-Arg-*、-N 9 -Tosyl-Arg-Phe-*, -Phe-N 9 -Nitro-Arg-*, -N 9-Nitro-Arg-Phe*, -Phe-Phe-Lys-*, -Lys-Phe-Phe-*, -Gly-Phe-Lys-*, Lys-Phe-Gly-*, -Leu-Ala-Leu-*, -Ile-Ala-Leu-*, -Leu-Ala-Ile-*, - Val-Ala-Val-*, -Ala-Leu-Ala-Leu-*, -Leu-Ala-Leu-Ala-*, -β-Ala-Leu-Ala-Leu-*, -Gly-Phe-Leu-Gly-*, -Gly-Leu-Phe-Gly-*, -Val-Ar g-*, -Arg-Val-*, -Arg-Arg-*, -Ala-Ala-*, -Ala-Met-*, -Met-Ala-*, -Thr-Th r-*, -Thr-Met-*, -Met-Thr-*, -Leu-Ala-*, -Ala-Leu-*, -Cit-Val-*, -Gln-Val-*, -Val-Gln-*, -Ser-Val-*, -Val-Ser-*, -Ser-Ala-*, -Ser-Gly-*, -Ala-Ser-*, -Gl y-Ser-*, -Leu-Gln-*, -Gln-Leu-*, -Phe-Arg-*, -Arg-Phe-*, -Tyr-Arg-*, -Arg-Ty r-*, -Phe-Gln-*, -Gln-Phe-*, -Val-Thr-*, -Thr-Val-*, -Met-Tyr-* and -Tyr-Met-*.

[0095] In some embodiments, -AA 1 -(AA 2 ) a1 -*is -Val-D-Lys-*, -Val-D-Arg-*, -L-Val-Cit-*, -L-Val-Lys-*, -L-Val-Arg-*, -L-Val-D-Cit-*, -L-Phe-Phe-Lys-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Arg- D-Arg-*, -L-Ala-Ala-*, -L-Ala-D-Ala-*, -Ala-D-Ala-*, -Val-D-Cit-*, -L-Ala-L-Ala-*, -L-Ala-L-Val-*, -L-Gln-L-Val-*, -L-Gln-L-Leu-* or -L-Ser-L-Val-*.

[0096] In some embodiments, -AA 1 -(AA2 ) a1 -* is: -Ala-Ala-*, -Ala-Val-*, -Val-Ala-*, -Gln-Leu-*, -Leu-Gln-*, -Ala-Ala-Ala-*, -Ala-Ala-Ala-Ala-*, -Gly-Ala-Gly-Gly-*, -Gly-Gly-Ala-Gly-*, -Gly-Val-Gly-Gly-*, -Gly-Gly-Val-Gly-*, -Gly-Phe-Gly-Gly-* or -Gly-Gly-Phe-Gly-*.

[0097] In some embodiments, -AA 1 -(AA 2 ) a1 -* is: -L-Ala-L-Ala-*, -L-Ala-D-Ala-*, -L-Ala-L-Val-*, -L-Ala-D-Val-*, -L-Val-L-Ala-*, -L-Val-D-Ala-*, -L-Gln-L-Leu-*, -L-Gln-D-Leu-*, -L-Leu-L-Gln-*, -L-Leu-D-Gln-*, -L-Ala-L-Ala-L-Ala-*, -L-Ala-D-Ala-L-Ala-*, -L-Ala-L-Ala-D-Ala-*, -L-Ala-L-Ala-L-Ala-L-Ala-*, -L-Ala-D-Ala-L-Ala-L-Ala-*, -L-Ala-L-Ala-D-Ala-L-Ala-*, -L-Ala-L-Ala-L-Ala-D-Ala-*, -Gly-L-Ala-Gly-Gly-*, -Gly-Gly-L-Ala-Gly-*, -Gly-D-Ala-Gly-Gly-*, Gly-Gly-D-Ala-Gly-*, -Gly-L-Val-Gly-Gly-*, Gly-Gly-L-Val-Gly-*, -Gly-D-Val-Gly-Gly-*, Gly-Gly-D-Val-Gly-*, -Gly-L-Phe-Gly-Gly-* or Gly-Gly-L-Phe-Gly-*.

[0098] In some embodiments, -AA 1 -(AA 2 ) a1-* is: -L-Ala-L-Ala-*, -L-Ala-D-Ala-LAla-*, -L-Ala-L-Ala-L-Ala-* or -L-Ala-L-Ala-L-Ala-L-Ala-*.

[0099] Disclosed herein -AA 1 -(AA 2 ) a1 In various embodiments of -*, * is a site of covalent attachment to E.

[0100] In some embodiments, A is substituted with one or more polyols. In some embodiments, E is substituted with one or more polyols. In some embodiments, the polyol is -(C1-C6 alkylene)-X 5 -Y 3 ; Among them: X 5 Yes-NR 12 C(=O)- or -C(=O)NR 12 -;Y 3 Yes-C1-C 10 Alkyl, where Y 3 is substituted with 0-10 OH groups; and R 12 It is -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl.

[0101] In some embodiments, wherein the polyol is where R 12 It is H or methyl.

[0102] In some embodiments, E is -C(=O)-(C1-C 10 Alkylene)-X 3 In some embodiments, E is

[0103]

[0104] In some embodiments, E is -C(=O)-Y 1 -(C1-C 10 Alkylene)-X 4 -(C1-C 10 Alkylene)-X 3 ;

[0105] Y 1 Yes-(CR a R b O) n -or-(CR a R b CR a' R b' O)m -;

[0106] X 4 Yes-NR 9 C(=O)-; and

[0107] X 3 yes

[0108] -C(=O)-CR bb R cc -W',NR ee -C(=O)-CR bb R cc -W' or -SR 10 .

[0109] In some embodiments, E is -C(=O)-Y 1 -(CH2)2-X 4 -(CH2)2-X 3 ;

[0110] Y 1 Yes -(CH2O) n -or-(CH2CH2O) m -;

[0111] X 4 is -NHC(=O)-;

[0112] n is 2; m is 2 to 6;

[0113] X 3 yes

[0114] -C(=O)-CR bb R cc -W',NR ee -C(=O)-CR bb R cc -W' or -SR 10 .

[0115] In some embodiments, the compound is any one of the compounds selected from Table 2.

[0116] In another aspect, the present invention provides a compound of formula III or a pharmaceutically acceptable salt thereof:

[0117] CBA—E'—A—Z'—L 1 —D (Formula III)

[0118] in:

[0119] D is represented by the following structural formula:

[0120]

[0121] R 1 is -H, -F, -CH3 or -CF3;

[0122] R 2 Yes -H, -F, -OR 3 、-SR 3 、-S(O)R 4 、-S(O)2R 4 , C1-C6 alkyl or C1-C6 fluoroalkyl; or R 1 and R 2 The carbon atoms to which they are attached together form a methylenedioxy or difluoromethylenedioxy ring; provided that R 1 and R 2 Cannot be -H at the same time;

[0123] R 3 is H or C1-C6 alkyl;

[0124] R 4 is a C1-C6 alkyl group;

[0125] L 1 does not exist, is -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, X 1' -(C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; wherein * is the site of covalent attachment to Z';

[0126] X 1 Is -O-, -S-, -S(O)-, -S(O)2-, -C(=O)-, -NR 5 -、-NR 5 C(=O)- or -C(=O)NR 5 -;

[0127] X 1' is -O-, -S-, -S(O)- or -S(O)2-;

[0128] L 2 is phenylene;

[0129] Each R 5 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0130] Z' is -O-CH2-NR 8 -*、-S-CH2-NR 8 -*, -NR 8 -*; wherein * is the site of covalent attachment to A;

[0131] Each R 8 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0132] L 1 and L 2 Each independently optionally substituted by 1-4 selected from halogen, -CN, -OR 7 、-SR 7 、-N(R 7 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 is substituted with a substituent of heterocycloalkyl, aryl, or heteroaryl; and

[0133] Each R 7 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0134] A is a peptide comprising 2 to 10 amino acids; wherein A is optionally substituted with one or more polyols;

[0135] E' is -C(=O)-L 3 -X 6 -*; wherein * is the site of covalent attachment to CBA;

[0136] L 3 Yes-(C1-C 10 Alkylene)-or-Y 1 -(C1-C 10 Alkylene)-X 4 -Y 2 -(C1-C 10 Alkylene)-*; wherein * is covalently linked to X 6 The site of

[0137] Y 1 Does not exist, is-(CR a R b O) n -or-(CR a R b CR a' R b' O) m -;

[0138] X4 Yes-NR 9 C(=O)- or -C(=O)NR 9 -;

[0139] Y 2 Does not exist, is-(CR c R d O) o -or-(CR c R d CR c' R d' O) p -;

[0140] n, m, o and p are each independently 1-10;

[0141] Each R a 、R b 、R a' 、R b' 、R c 、R d 、R c' and R d' is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0142] Among them L 3 Optionally substituted by 0-4 selected from halogen, -CN, -OR 11 、-SR 11 、-N(R 11 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 Substitution of heterocycloalkyl, aryl, heteroaryl, and polyol substituents;

[0143] Each R 11 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0144] X 6 yes

[0145] -C(=O)-CR bb R cc -* or -NR ee -C(=O)-CR bb R cc -*; wherein * is the site of covalent attachment to CBA;

[0146] Each R aa 、Rbb 、R cc and R ee is independently -H or optionally substituted C1-C6 alkyl;

[0147] Each R YY and R XX are independently -H or C1-C6 alkyl;

[0148] R 9 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl; and

[0149] CBA is a cell binding agent.

[0150] In some embodiments, R 1 Is -H or -F. In some embodiments, R 1 In some embodiments, R 2 is -H, -F, -OCF3, -CF3, -OMe, -OEt, -SMe, -S(O)Me, -S(O)2Me, -SEt, -S(O)Et, -S(O2)Et, methyl or ethyl. In some embodiments, R 2 In some embodiments, R 2 is -OMe, -SMe, -S(O)Me or methyl. In some embodiments, R 2 In some embodiments, R 1 Yes -F and R 2 In some embodiments, R 1 is methyl and R 2 In some embodiments, R 1 Yes -F and R 2 It is -methyl.

[0151] In some embodiments, -L 1 -Z'-* is -(C1-C4 alkylene)-O-CH2-NR 8 -*, -(C1-C4 alkylene)-S-CH2-NR 8 -* or -(C1-C4 alkylene)-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C4 alkylene)-O-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C4 alkylene)-S-CH2-NR 8 -*. In some embodiments, -L 1-Z'-* is -(C1-C4 alkylene)-NR 8 -*.

[0152] In some embodiments, -L 1 -Z'-* is -CH2O-CH2NH-*, -(CH2)2O-CH2NH-*, -(CH2)3O-CH2NH-*, -(CH2)4O-CH2NH-*, -CH2S-CH2NH-*, -(CH2 )2S-CH2NH-*, -(CH2)3S-CH2NH-*, -(CH2)4S-CH2NH-*, -CH2NH-*, -(CH2)2NH-*, -(CH2)3NH-* or -(CH2)4NH-.

[0153] In some embodiments, -L 1 -Z'-* is -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-O-CH2-NR 8 -*, -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-S-CH2-NR 8 -*, -(C1-C5 alkylene)-S-(C1-C5 alkylene)-S-CH2-NR 8 -* or -(C1-C5 alkylene)-S-(C1-C5 alkylene)-SS-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-O-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-S-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C5 alkylene)-S-(C1-C5 alkylene)-S-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C5 alkylene)-S-(C1-C5 alkylene)-SS-CH2-NR 8 -*.

[0154] In some embodiments, -L 1-Z'-* is -CH2NHC(=O)CH2O-CH2-NH-*, -CH2NHC(=O)(CH2)2O-CH2-NH-*, -CH2NHC(=O)(CH2)3O-CH2-NH-*, -CH2NHC(=O)(CH2)4O-CH2-NH-*, -CH2NHC( =O)(CH2)5O-CH2-NH-*, -CH2NHC(=O)CH2S-CH2-NH-*, -CH2NHC(=O)(CH2)2S-CH2-NH-*, -CH2NHC(=O)(CH2)3S-CH2-NH-*, -CH2NHC(=O)(CH2)4S-CH2- NH-*, -CH2NHC(=O)(CH2)5S-CH2-NH-*, -CH2SCH2O-CH2-NH-*, -CH2S(CH2)2O-CH2-NH-*, -CH2S(CH2)3O-CH2-NH-*, -CH2S(CH2)4O-CH2-NH-*, -CH2S (CH2)5O-CH2-NH-*, -CH2SCH2S-CH2-NH-*, -CH2S(CH2)2S-CH2-NH-*, -CH2S(CH2)3S-CH2-NH-*, -CH2S(CH2)4S-CH2-NH-* or -CH2S(CH2)5S-CH2-NH-*.

[0155] In some embodiments, each R 5 is independently -H, methyl or benzyl. In some embodiments, each R 5 is independently -H. In some embodiments, each R 5 In some embodiments, each R 5 In some embodiments, each R 8 is independently -H, methyl or benzyl. In some embodiments, each R 8 is independently -H. In some embodiments, each R 8 In some embodiments, each R 8 It's benzyl.

[0156] In some embodiments, -L 1 -Z'-* is -X 1' -(C1-C4 alkylene)-O-CH2-NR 8 -*, -X 1' -(C1-C4 alkylene)-S-CH2-NR 8 -* or -X 1' -(C1-C4 alkylene)-NR 8 -*. In some embodiments, -L 1-Z'-* is -X 1' -(C1-C4 alkylene)-O-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -X 1' -(C1-C4 alkylene)-S-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -X 1' -(C1-C4 alkylene)-NR 8 -*.

[0157] In some embodiments, -L 1-Z'-* is -OCH2O-CH2-NH-*, -O(CH2)2O-CH2-NH-*, -O(CH2)3O-CH2-NH-*, -O(CH2)4O-CH2-NH-*, -SCH2O-CH2-NH-*, -S(CH2)2O-CH2-N H-*, -S(CH2)3O-CH2-NH-*, -S(CH2)4O-CH2-NH-*, -S(O)CH2O-CH2-NH-*, -S(O)(CH2)2O-CH2-NH-*, -S(O)(CH2)3O-CH2-NH-*, -S(O)( CH2)4O-CH2-NH-*, -S(O)2CH2O-CH2-NH-*, -S(O)2(CH2)2O-CH2-NH-*, -S(O)2(CH2)3O-CH2-NH-*, -S(O)2(CH2)4O-CH2-NH-*, -OCH2S -CH2-NH-*, -O(CH2)2S-CH2-NH-*, -O(CH2)3S-CH2-NH-*, -O(CH2)4S-CH2-NH-*, -SCH2S-CH2-NH-*, -S(CH2)2S-CH2-NH-*, -S(CH2)3S -CH2-NH-*, -S(CH2)4S-CH2-NH-*, -S(O)CH2S-CH2-NH-*, -S(O)(CH2)2S-CH2-NH-*, -S(O)(CH2)3S-CH2-NH-*, -S(O)(CH2)4S-CH2-NH -*, -S(O)2CH2S-CH2-NH-*, -S(O)2(CH2)2S-CH2-NH-*, -S(O)2(CH2)3S-CH2-NH-*, -S(O)2(CH2)4S-CH2-NH-*, -OCH2-NH-*, -O(CH2)2 -NH-*, -O(CH2)3-NH-*, -O(CH2)4S-NH-*, -SCH2-NH-*, -S(CH2)2-NH-*, -S(CH2)3-NH-*, -S(CH2)4-NH-*, -S(O)CH2-NH-*, -S(O)(CH2 )2-NH-*, -S(O)(CH2)3-NH-*, -S(O)(CH2)4-NH-*, -S(O)2CH2-NH-*, -S(O)2(CH2)2-NH-*, -S(O)2(CH2)3-NH-* or -S(O)2(CH2)4-NH-*.

[0158] In some embodiments, -L 1 -Z'-* is -(C1-C6 alkylene)-X 1 -L 2 -Z'-*. In some embodiments, -L1 -Z'-*Yes In some embodiments, -L 1 -Z'-*Yes In some embodiments, -L 1 -Z'-*Yes

[0159] Disclosed herein -L 1 In various embodiments of -Z'-*, * is the site of covalent attachment to A.

[0160] In some embodiments, A is a peptide comprising 2 to 8 amino acids. In some embodiments, A is a peptide comprising 2 to 4 amino acids. In some embodiments, at least one amino acid in the peptide is an L amino acid. In some embodiments, each amino acid in the peptide is an L amino acid. In some embodiments, at least one amino acid in the peptide is a D amino acid.

[0161] In some embodiments, A is -(AA 1 )-(AA 2 ) a1 -*, where * is the point of connection with E', AA 1 and AA 2 are each independently an amino acid residue; and a1 is an integer from 1 to 9.

[0162] In some embodiments, -AA 1 -(AA 2 ) a1 -*is -Gly-Gly-Gly-*, -Ala-Val-*, -Val-Ala-*, -Val-Cit-*, -Val-Lys-*, -Lys-Val-*, -Phe-Lys-*, -Lys-Phe-*, -Lys-Ly s-*, -Ala-Lys-*, -Lys-Ala-*, -Phe-Cit-*, -Cit-Phe-*, -Leu-Cit-*, -Cit-Leu-*-Ile-Cit-*, -Phe-Ala-*, -Ala-Phe-*, -Phe-N 9 -Tosyl-Arg-*、-N 9 -Tosyl-Arg-Phe-*, -Phe-N 9 -Nitro-Arg-*, -N 9-Nitro-Arg-Phe*, -Phe-Phe-Lys-*, -Lys-Phe-Phe-*, -Gly-Phe-Lys-*, Lys-Phe-Gly-*, -Leu-Ala-Leu-*, -Ile-Ala-Leu-*, -Leu-Ala-Ile-*, - Val-Ala-Val-*, -Ala-Leu-Ala-Leu-*, -Leu-Ala-Leu-Ala-*, -β-Ala-Leu-Ala-Leu-*, -Gly-Phe-Leu-Gly-*, -Gly-Leu-Phe-Gly-*, -Val-Ar g-*, -Arg-Val-*, -Arg-Arg-*, -Ala-Ala-*, -Ala-Met-*, -Met-Ala-*, -Thr-Th r-*, -Thr-Met-*, -Met-Thr-*, -Leu-Ala-*, -Ala-Leu-*, -Cit-Val-*, -Gln-Val-*, -Val-Gln-*, -Ser-Val-*, -Val-Ser-*, -Ser-Ala-*, -Ser-Gly-*, -Ala-Ser-*, -Gl y-Ser-*, -Leu-Gln-*, -Gln-Leu-*, -Phe-Arg-*, -Arg-Phe-*, -Tyr-Arg-*, -Arg-Ty r-*, -Phe-Gln-*, -Gln-Phe-*, -Val-Thr-*, -Thr-Val-*, -Met-Tyr-* and -Tyr-Met-*.

[0163] In some embodiments, -AA 1 -(AA 2 ) a1 -*is -Val-D-Lys-*, -Val-D-Arg-*, -L-Val-Cit-*, -L-Val-Lys-*, -L-Val-Arg-*, -L-Val-D-Cit-*, -L-Phe-Phe-Lys-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Arg- D-Arg-*, -L-Ala-Ala-*, -L-Ala-D-Ala-*, -Ala-D-Ala-*, -Val-D-Cit-*, -L-Ala-L-Ala-*, -L-Ala-L-Val-*, -L-Gln-L-Val-*, -L-Gln-L-Leu-* or -L-Ser-L-Val-*.

[0164] In some embodiments, -AA 1 -(AA2 ) a1 -* is: -Ala-Ala-*, -Ala-Val-*, -Val-Ala-*-Gln-Leu-*, -Leu-Gln-*, -Ala-Ala-Ala-*, -Ala-Ala-Ala-Ala-*, -Gly-Ala-Gly-Gly-*, -Gly-Gly-Ala-Gly-*, -Gly-Val-Gly-Gly-*, -Gly-Gly-Val-Gly-*, -Gly-Phe-Gly-Gly-* or -Gly-Gly-Phe-Gly-*.

[0165] In some embodiments, -AA 1 -(AA 2 ) a1 -* is: -L-Ala-L-Ala-*, -L-Ala-D-Ala-*, -L-Ala-L-Val-*, -L-Ala-D-Val-*, -L-Val-L-Ala-*, -L-Val-D-Ala-*, -L-Gln-L-Leu-*, -L-Gln-D-Leu-*, -L-Leu-L-Gln-*, -L-Leu-D-Gln-*, -L-Ala-L-Ala-L-Ala-*, -L-Ala-D-Ala-L-Ala-*, -L-Ala-L-Ala-D-Ala-*, -L-Ala-L-Ala-L-Ala-L-Ala-*, -L-Ala-D-Ala-L-Ala-L-Ala-*, -L-Ala-L-Ala-D-Ala-L-Ala-*, -L-Ala-L-Ala-L-Ala-D-Ala-*, -Gly-L-Ala-Gly-Gly-*, -Gly-Gly-L-Ala-Gly-*, -Gly-D-Ala-Gly-Gly-*, Gly-Gly-D-Ala-Gly-*, -Gly-L-Val-Gly-Gly-*, Gly-Gly-L-Val-Gly-*, -Gly-D-Val-Gly-Gly-*, Gly-Gly-D-Val-Gly-*, -Gly-L-Phe-Gly-Gly-* or Gly-Gly-L-Phe-Gly-*.

[0166] In some embodiments, -AA 1 -(AA 2 ) a1-* is: -L-Ala-L-Ala-*, -L-Ala-D-Ala-L-Ala-*, -L-Ala-L-Ala-L-Ala-*, or -L-Ala-L-Ala-L-Ala-L-Ala-*.

[0167] Disclosed herein -AA 1 -(AA 2 ) a1 In various embodiments of -*, * is a site of covalent attachment to E'.

[0168] In some embodiments, A is substituted with one or more polyols. In some embodiments, E' is substituted with one or more polyols. In some embodiments, the polyol is -(C1-C6 alkylene)-X 5 -Y 3 ; Among them: X 5 Yes-NR 12 C(=O)- or -C(=O)NR 12 -;Y 3 Yes-C1-C 10 Alkyl, where Y 3 is substituted with 0-10 OH groups; and R 12 It is -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl.

[0169] In some embodiments, the polyol is where R 12 It is H or methyl.

[0170] In some embodiments, E' is -C(=O)-(C1-C 10 Alkylene)-X 6 -*. In some embodiments, E' is

[0171]

[0172]

[0173] -C(=O)CH2CH2-C(=O)-CR bb R cc -* or -C(=O)CH2CH2-NR ee -C(=O)-CR bb R cc -*; where * is the site of covalent attachment to CBA.

[0174] In some embodiments, E' is -C(=O)-Y 1 -(C1-C 10 Alkylene)-X4 -(C1-C 10 Alkylene)-X 6 -*;

[0175] Y 1 Yes-(CR a R b O) n -or-(CR a R b CR a' R b' O) m -;

[0176] X 4 Yes-NR 9 C(=O)-; and

[0177] X 6 yes

[0178] -C(=O)-CR bb R cc -* or -NR ee -C(=O)-CR bb R cc -*; where * is the site of covalent attachment to CBA.

[0179] In some embodiments, E' is -C(=O)-Y 1 -(CH2)2-X 4 -(CH2)2-X 6 -*;

[0180] Y 1 Yes -(CH2O) n -or-(CH2CH2O) m -;

[0181] X 4 is -NHC(=O)-;

[0182] n is 2; m is 2 to 6;

[0183] X 6 yes

[0184] -C(=O)-CR bb R cc -* or -NR ee -C(=O)-CR bb R cc -*; where * is the site of covalent attachment to CBA.

[0185] In some embodiments, the CBA comprises a -SH group covalently linked to E' to provide

[0186] -C(=O)-CR bb R cc -S-CBA or -NR ee -C(=O)-CR bb R cc -S-CBA.

[0187] In some embodiments, the CBA is an antibody and -E'-A-Z'-L 1 —D is the drug-linker structure, and the average number of drug-linker structures conjugated to each antibody ranges from 2 to 10.

[0188] In some embodiments, the average number of drug-linker structures conjugated per antibody is in the range of 2 to 10. In some embodiments, the average number of drug-linker structures conjugated per antibody is in the range of 6 to 8. In some embodiments, the average number of drug-linker structures conjugated per antibody is 8.

[0189] In some embodiments, the CBA is an antibody, a single-chain antibody, an antibody fragment that specifically binds to a target cell, a monoclonal antibody, a single-chain monoclonal antibody or a monoclonal antibody fragment that specifically binds to a target cell, a chimeric antibody, a chimeric antibody fragment that specifically binds to a target cell, a domain antibody, a domain antibody fragment that specifically binds to a target cell, a probody, a nanobody, a hexabody, a lymphokine, a hormone, a vitamin, a growth factor, a colony-stimulating factor, or a nutrient transport molecule.

[0190] In some embodiments, the CBA binds to a target cell selected from the group consisting of tumor cells, virus-infected cells, microbial-infected cells, parasite-infected cells, autoimmune cells, activated cells, myeloid cells, activated T cells, B cells, or melanocytes; cells expressing 5T4, ADAM-9, ALK, AMHRII, ASCT2, Axl, B7-H3, BCMA, C4.4a, CA6, CA9, CanAg, CD123, CD138, CD142, CD166, CD184, CD19, CD20, CD205, CD22, CD248, CD25, CD3, CD30, CD33, CD352, CD37, CD38, CD40L, CD44v 6. CD45, CD46, CD48, CD51, CD56, CD7, CD70, CD71, CD74, CD79b, CDH6, CEACAM5, CEACAM6, cKIT, CLDN18.2. CLDN6, CLL-1, c-MET, Cripto, CSP-1, CXCR5, DLK-1, DLL3, DPEP3, Dysadherin, EFNA4, EGFR, EGFRviii, ENPP3, EpCAM, EphA2, EphA3, ETBR, FGFR2, FGFR3, FLT3, FOLR-α, FSH, GCC, GD2, GD3, Globo H, GPC-1, GPC3, gpNMB, HER-2, HER-3, HLA-DR, HSP90, IGF-1R, IL-13R, IL1RAP, IL7R, interleukin-4 receptor (IL4R), KAAG-1, LAMP-1, Lewis Y antigen, LGALS3BP, LGR5, LH / hCG, LHRH, LIV-1, LRP-1, LRRC15, Ly6E, MAGE, mesothelin (MSLN), MET, MHC Cells expressing any one or more of class I chain-related proteins A and B (MICA and MICB), MT1-MMP, MTX3, MTX5, MUC1, MUC16, NaPi2b, Nectin-4, NOTCH3, OAcGD2, OX001L, p-cadherin, PD-L1, phosphatidylserine (PS), polymorphic epithelial mucin (PEM), prolactin receptor (PRLR), PSMA, PTK7, RNF43, ROR1, ROR2, SAIL, SLAMF7, SLC44A4, SLITRK6, SSTR2, STEAP-1, STING, STn, TIM-1, TM4SF1, TNF-α, TRA, TROP-2, tumor-associated glycoprotein 72 (TAG-72), tumor-specific epitope of mucin-1 (TA-MUC1), CD5, TIM-3, UPK2, or UPK1b antigens.

[0191] In some embodiments, the cell-binding agent is an anti-folate receptor antibody or an antibody fragment thereof, an anti-EGFR antibody or an antibody fragment thereof, an anti-CD33 antibody or an antibody fragment thereof, an anti-EpCAM antibody or an antibody fragment thereof, an anti-CD19 antibody or an antibody fragment thereof, an anti-Muc1 antibody or an antibody fragment thereof, or an anti-CD37 antibody or an antibody fragment thereof.

[0192] The present invention also includes a composition (e.g., a pharmaceutical composition) comprising a cytotoxic compound or conjugate of the present invention as described herein and a carrier (pharmaceutically acceptable carrier). The compound, conjugate, or composition of the present invention is suitable for inhibiting abnormal cell growth or treating a proliferative disorder (e.g., cancer), autoimmune disorder, destructive bone disorder, infectious disease, viral disease, fibrotic disease, neurodegenerative disorder, pancreatitis, or kidney disease in a mammal (e.g., a human).

[0193] The compounds, conjugates or compositions of the present invention are useful for treating cancer in a subject in need thereof. In some embodiments, the cancer is lymphoma or leukemia. In some embodiments, the cancer is acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), acute B-lymphoblastic leukemia or B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), acute promyelocytic leukemia (APL), B-cell chronic lymphoproliferative disorder (B-CLPD), atypical chronic lymphocytic leukemia, diffuse large B-cell lymphoma (DLBCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin lymphoma (NHL), mantle cell leukemia (MCL), small lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis, and Burkitt's lymphoma. In some embodiments, the cancer is endometrial cancer, lung cancer, colorectal cancer, bladder cancer, gastric cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, esophageal cancer, breast cancer, head and neck cancer, uterine cancer, ovarian cancer, liver cancer, cervical cancer, thyroid cancer, testicular cancer, bone marrow cancer, melanoma and lymphoma. In some embodiments, the lung cancer is non-small cell lung cancer or small cell lung cancer.

[0194] The present invention also includes the use of a cytotoxic compound, conjugate or composition of the present invention for the manufacture of a medicament for inhibiting abnormal cell growth or treating a proliferative disorder (e.g., cancer), an autoimmune disorder, a destructive bone disorder, an infectious disease, a viral disease, a fibrotic disease, a neurodegenerative disorder, pancreatitis or a renal disease in a mammal (e.g., a human).

[0195] The compounds, conjugates or compositions of the present invention are suitable for the manufacture of a medicament for treating cancer in a subject in need thereof. In some embodiments, the cancer is lymphoma or leukemia. In some embodiments, the cancer is acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), acute B-lymphoblastic leukemia or B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), acute promyelocytic leukemia (APL), B-cell chronic lymphoproliferative disorder (B-CLPD), atypical chronic lymphocytic leukemia, diffuse large B-cell lymphoma (DLBCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin lymphoma (NHL), mantle cell leukemia (MCL), small lymphocytic lymphoma (SLL), Hodgkin lymphoma, systemic mastocytosis, and Burkitt lymphoma. In some embodiments, the cancer is endometrial cancer, lung cancer, colorectal cancer, bladder cancer, gastric cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, esophageal cancer, breast cancer, head and neck cancer, uterine cancer, ovarian cancer, liver cancer, cervical cancer, thyroid cancer, testicular cancer, bone marrow cancer, melanoma and lymphoma. In some embodiments, the lung cancer is non-small cell lung cancer or small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0196] Figure 1 The first part of the synthesis of the camptothecin building block is depicted.

[0197] Figure 2 The second part of the synthesis of the camptothecin building block is depicted.

[0198] Figure 3 The first part of the synthesis of the side chain is depicted.

[0199] Figure 4 The second part of the synthesis of the side chain is depicted.

[0200] Figure 5 The coupling of the camptothecin building block to the first part of the side chain is depicted.

[0201] Figure 6 The coupling of the camptothecin building block to the second part of the side chain is depicted.

[0202] Figure 7 The third part of the camptothecin building block coupled to the side chain is depicted.

[0203] Figure 8 The synthesis of additional camptothecin metabolites is depicted.

[0204] Figure 9The coupling of the camptothecin building block to the side chain is depicted.

[0205] Figure 10 The synthesis of additional camptothecin compounds is depicted.

[0206] Figure 11 Depicted are compounds used for comparison, which include the generic Ab-999 structure of an ADC with the 999 moiety linked via the reduced interchain disulfide of the antibody.

[0207] Figure 12 The cytotoxicity of sulfide-bearing compound 8c and its sulfoxide 34a and sulfone 34b are depicted.

[0208] Figure 13 Depicts the pharmacokinetics of ML66-999 in mice. The top graph depicts the concentration (μg / mL) of the mAb component (mean) and the payload component versus time at 2 min, 1 day, and 3 days after administration in mice. The bottom graph depicts the concentration (μg / mL) of the mAb component (mean) and the concentration (μg / mL) that maintains bioactivity (pooled samples) versus time at 2 min, 1 day, and 3 days after administration in mice.

[0209] Figure 14 Depicts the pharmacokinetics of ML66-22a in mice. The top graph depicts the concentration (μg / mL) of the mAb component (mean) and the payload component versus time at 2 min, 1 day, and 3 days after administration in mice. The bottom graph depicts the concentration (μg / mL) of the mAb component (mean) and the concentration (μg / mL) of the ADC that maintains bioactivity (pooled samples) versus time at 2 min, 1 day, and 3 days after administration in mice.

[0210] Figure 15 In vitro cytotoxicity of ADCs against Ag+ and Ag- cells is depicted. ADC standards in formulations (standards) or serum (pooled) containing ADCs administered 2 min, 1 day, or 3 days after ML66-999 administration to mice.

[0211] Figure 16 In vitro cytotoxicity of ADCs against Ag+ and Ag- cells is depicted. ADC standards in formulations (standards) or serum (pooled) containing ADCs administered 2 min, 1 day, or 3 days after ML66-22a administration to mice.

[0212] Figure 17 The efficacy of the ADC in the HSC-2 xenograft model is depicted. Dosing was based on payload (75 μg / kg and 250 μg / kg based on approximately 3 mg / kg and approximately 10 mg / kg of antibody).

[0213] Figure 18 The efficacy of the ADC in the FaDu xenograft model is depicted. Dosing was based on payload (75 μg / kg and 250 μg / kg based on approximately 3 mg / kg and approximately 10 mg / kg of antibody).

[0214] Figure 19 Tolerability of mice to ML66-999, ML66-22a, and ML66-28a ADCs is depicted.

[0215] Figure 20 The anti-tumor activity of the ADC in the H1703 mouse xenograft model is depicted. Dosing was based on payload (75 μg / kg and 250 μg / kg based on approximately 3 mg / kg and approximately 10 mg / kg of antibody).

[0216] Figure 21 Depicted is the mouse tolerability of a non-cross-reactive ADC at a 5000 μg / kg payload dose (approximately 200 mg / kg based on the Ab component). F =Humanized anti-folate receptor antibody. DETAILED DESCRIPTION

[0217] In order to fully understand the invention described herein, the following specific embodiments are set forth. Although the present invention will be described in conjunction with the enumerated embodiments, it should be understood that it is not intended to limit the invention to those embodiments. On the contrary, the present invention is intended to encompass all alternatives, modifications, and equivalents that may be included within the scope of the invention as defined in the claims. Those skilled in the art will recognize that a variety of methods and materials similar or equivalent to those described herein may be used in the practice of the present invention.

[0218] The term "herein" refers to the entire application.

[0219] Unless otherwise defined herein, the scientific and technical terms used herein shall have the meanings commonly understood by those of ordinary skill in the art to which the invention belongs. Generally speaking, the nomenclature used in conjunction with the compounds, compositions and methods described herein are those well known and commonly used in the art.

[0220] It should be understood that any embodiment described herein, including those described under different aspects of the invention and different parts of this specification (including embodiments described only in the examples), can be combined with one or more other embodiments of the invention unless expressly denied or inappropriate. The combination of embodiments is not limited to those specific combinations claimed via multiple dependent claims.

[0221] Chemical terms used herein are used according to conventional usage in the art as exemplified by "The McGraw-Hill Dictionary of Chemical Terms", Parker S. ed., McGraw-Hill, San Francisco, CA (1985).

[0222] All of the above and any other publications, patents, and published patent applications mentioned in this application are specifically incorporated herein by reference. Any information in any material incorporated herein by reference is incorporated by reference only to the extent that there is no conflict between that information and the other statements and drawings set forth herein. If such a conflict exists, any such conflicting information in such incorporated by reference materials is not specifically incorporated herein by reference, and the present specification (including its specific definitions) will control.

[0223] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer (or components) or group of integers (or components) but not the exclusion of any other integer (or component) or group of integers (or components).

[0224] Throughout this specification, where a composition is described as having, including or comprising (or variations thereof) a particular component, it is contemplated that the composition may also consist essentially of, or be composed of, the components. Similarly, where a method or process is described as having, including or comprising a specific process step, the process may also consist essentially of, or be composed of, the process steps. Furthermore, it will be understood that the order of steps or the order in which certain actions are performed is not important, as long as the compositions and methods described herein remain operable. Furthermore, two or more steps or actions may be performed simultaneously.

[0225] The term "including" is used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0226] As used herein, "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system.

[0227] The use of the terms “a” and “an” and “said” and similar referents in the context of describing elements (especially in the context of the following claims) are intended to be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by context.

[0228] As used herein, the term "or" should be understood to mean "and / or" unless the context clearly indicates otherwise.

[0229] Unless otherwise indicated herein, the value ranges set forth herein are only intended to serve as a shorthand method for individually referring to each individual value belonging to the scope, and each individual value is incorporated into this specification as if it were individually narrated herein. Unless otherwise indicated herein or otherwise clearly contradicted by this article, all methods described herein can be performed in any suitable order. Unless otherwise specified, any and all embodiments used, or the exemplary language provided herein (e.g., "such as") are only intended to better illustrate the embodiments and are not intended to limit the scope of the claims. The language in this specification should not be interpreted as indicating that any unclaimed element is necessary.

[0230] definition

[0231] As used herein, the term "alkyl" or "straight or branched chain alkyl" refers to a saturated straight or branched chain monovalent hydrocarbon radical. In preferred embodiments, the straight or branched chain alkyl group has thirty or fewer carbon atoms (e.g., C1-C2 for straight chain alkyl). 30 And the branched alkyl group is C3-C 30 ), and more preferably twenty or fewer carbon atoms. Even more preferably, the straight or branched chain alkyl group has ten or fewer carbon atoms (i.e., C1-C 10 And the branched alkyl group is C3-C 10 In other embodiments, the straight chain or branched chain alkyl group has six or fewer carbon atoms (i.e., C1-C 6- and C3-C6 with respect to branched alkyl groups. Examples of alkyl groups include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, -CH2CH(CH3)2, 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl), 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and the like. Furthermore, the term "alkyl" as used throughout the specification, examples, and claims is intended to include "unsubstituted alkyl" and "substituted alkyl," wherein the latter refers to an alkyl moiety having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. As used herein, (C x -C xx ) alkyl or C x-xxThe alkyl group refers to a straight-chain or branched-chain alkyl group having x to xx number of carbon atoms.

[0232] As used herein, the term "alkylene" refers to a saturated straight or branched chain divalent hydrocarbon radical. In preferred embodiments, a straight or branched chain alkylene radical has thirty or fewer carbon atoms (e.g., C1-C2 for straight chain alkylene). 30 And the branched alkylene is C3-C 30 ), and more preferably twenty or fewer carbon atoms. Even more preferably, the straight or branched chain alkylene group has ten or fewer carbon atoms (i.e., C1-C 10 And the branched alkylene is C3-C 10 In other embodiments, a straight chain or branched chain alkylene group has six or fewer carbon atoms (i.e., C1-C6 for straight chain alkylene groups and C3-C6 for branched chain alkylene groups). As used herein, (C x -C xx ) alkylene or C x-xx The alkylene group refers to a straight or branched chain alkylene group having x to xx number of carbon atoms.

[0233] The term "alkenyl" or "straight or branched alkenyl" refers to a straight or branched monovalent hydrocarbon radical having from two to twenty carbon atoms and having at least one site of unsaturation (i.e., a carbon-carbon double bond), wherein alkenyl includes groups having "cis" and "trans" orientations or alternatively "E" and "Z" orientations. Examples include, but are not limited to, vinyl (-CH=CH2), allyl (-CH2CH=CH2), and the like. Preferably, alkenyl has from two to ten carbon atoms. More preferably, alkyl has from two to four carbon atoms.

[0234] The term "alkynyl" or "straight or branched alkynyl" refers to a straight or branched monovalent hydrocarbon radical having from two to twenty carbon atoms and having at least one site of unsaturation (i.e., a carbon-carbon triple bond). Examples include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, hexynyl, and the like. Preferably, the alkynyl radical has from two to ten carbon atoms. More preferably, the alkynyl radical has from two to four carbon atoms.

[0235] The terms "cyclic alkyl" and "cycloalkyl" are used interchangeably. As used herein, the terms refer to a group of a saturated carbocyclic ring. In a preferred embodiment, the cycloalkyl has 3 to 10 carbon atoms in its ring structure, and more preferably has 5 to 7 carbon atoms in the ring structure. In some embodiments, the two rings may have two or more atoms in common, for example, the ring is a "fused ring". Suitable cycloalkyls include, but are not limited to, cycloheptyl, cyclohexyl, cyclopentyl, cyclobutyl, and cyclopropyl. In some embodiments, the cycloalkyl is a monocyclic group. In some embodiments, the cycloalkyl is a bicyclic group. In some embodiments, the cycloalkyl is a tricyclic group.

[0236] The term "cycloalkylalkyl" refers to an alkyl group as defined above substituted by a cycloalkyl group.

[0237] The term "cycloalkenyl" refers to a carbocyclic group having at least one double bond in the ring structure.

[0238] The term "cyclic alkynyl" refers to a carbocyclic group having at least one triple bond in the ring structure.

[0239] As used herein, the term "aryl" or "aromatic ring" includes substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon. Preferably, the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. Aryl groups include, but are not limited to, phenyl, phenol, aniline, and the like. The term "aryl" also includes "polycyclic," "polycyclic," and "polycyclic" ring systems having two or more rings, in which two or more atoms are shared by two adjacent rings, for example, the rings are "fused rings," in which at least one of the rings is aromatic, for example, the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, or aromatic rings. In some preferred embodiments, the polycyclic ring has 2-3 rings. In certain preferred embodiments, the polycyclic ring system has two rings, both of which are aromatic. Each ring of the polycyclic ring may be substituted or unsubstituted. In certain embodiments, each ring of the polycyclic ring contains 3 to 10 carbon atoms, preferably 5 to 7. For example, aryl includes, but is not limited to, phenyl, tolyl, anthracenyl, fluorenyl, indenyl, azulenyl, and naphthyl, as well as benzo-fused carbocyclic moieties such as 5,6,7,8-tetrahydronaphthyl. In some embodiments, aryl is a monocyclic aromatic group. In some embodiments, aryl is a bicyclic aromatic group. In some embodiments, aryl is a tricyclic aromatic group.

[0240] The term "heteroalkyl" refers to an alkyl group wherein one or more of the backbone atoms of the alkyl group is selected from atoms other than carbon, such as O, S, N (e.g., -NH, -N(alkyl)-), or a combination thereof. The heteroalkyl group is attached to the remainder of the molecule at a carbon atom of the heteroalkyl group. In one aspect, the heteroalkyl group is a C1-C 30 Heteroalkyl. C1-C 30Heteroalkyl refers to an alkyl group having 1 to 30 carbon atoms and 1 to 15 heteroatoms. 30 Examples of heteroalkyl groups include, but are not limited to, ethers (e.g., -CH2-O-CH3, -(CH2)2-O-CH3, -(CH2)3-O-(CH2)2-O-CH3, -(CH2)2-O-(CH2)3CH3, CH2-O-CH2-O-CH3, -CH2-O-(CH2)3-O-CH3), polyethylene glycol (PEG) derivatives (e.g., -[(CH2)2O] 10 CH2CH3), thioethers (e.g., -CH2-S-CH3, -(CH2)2-S-CH3, -(CH2)3-S-(CH2)2CH3, -((CH2)2S) 10 -CH2CH3), -CH2-SS-CH2, -(CH2)2-S-(CH2)3CH3, CH2-S-CH2-S-CH3, -CH2-S-(CH2)3-S-CH3), amines (e.g., -CH2-NH-CH3, -(CH2)2-N(alkyl)-CH3, -(CH2)3-NH-(CH2)2CH3, -(CH2)2-N(alkyl)-(CH2)3CH3, CH2-NH-CH2-NH-CH3, -CH2-NH-(CH2)3-NH-CH3), or combinations thereof. The present disclosure also encompasses C1-C 30 Heteroalkyl groups wherein one of 1 to 15 heteroatoms occupies the terminal position of the alkyl group, for example, creating an alcohol (ie, OH), a thiol (ie, SH), or an amine (eg, -NH2) in the terminal position of the moiety.

[0241] The term "heteroalkenyl" refers to an alkenyl group as defined herein in which one or more carbon atoms have been replaced by a heteroatom such as O, S, N (e.g., -NH, -N(alkyl)-). The heteroalkenyl group is attached to the remainder of the molecule at a carbon atom of the heteroalkenyl group. In one aspect, the heteroalkenyl group is a C1-C 30 Heteroalkenyl. C1-C 30 Heteroalkenyl refers to an alkenyl group having 1 to 30 carbon atoms and 1 to 15 heteroatoms (e.g., 1 to 10 heteroatoms or 1 to 5 heteroatoms). 30 Heteroalkenyl groups wherein one of 1 to 15 heteroatoms occupies the terminal position of the alkenyl group, for example, creating an alcohol (i.e., OH), a thiol (i.e., SH), an amine (e.g., -NH2), or an imine (-C=N) in the terminal position of the moiety.

[0242] The term "heteroalkynyl" refers to an alkenyl group as defined herein in which one or more carbon atoms have been replaced by a heteroatom such as O, S, N (e.g., -NH, -N(alkyl)-). The heteroalkynyl group is attached to the remainder of the molecule at a carbon atom of the heteroalkynyl group. In one aspect, the heteroalkynyl group is a C1-C 30 Heteroalkynyl. C1-C 30 Heteroalkynyl refers to an alkynyl group having 1 to 30 carbon atoms and 1 to 15 heteroatoms (e.g., 1 to 10 heteroatoms or 1 to 5 heteroatoms). 30 Heteroalkynyl groups, wherein one of 1 to 15 heteroatoms occupies the terminal position of the alkynyl group, for example, creating an alcohol (ie, OH), a thiol (ie, SH), an amine (eg, -NH2), or a nitrile (-C≡N) in the terminal position of the moiety.

[0243] As used herein, the terms "heterocycle", "heterocyclyl" and "heterocyclic ring" refer to a 3- to 18-membered ring, preferably a 3- to 10-membered ring, more preferably a 3- to 7-membered ring, which is a substituted or unsubstituted non-aromatic ring structure, the ring structure of which includes at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. In certain embodiments, the ring structure may have two rings. In some embodiments, the two rings may have two or more atoms in common, for example, the ring is a "fused ring". Heterocyclyl includes, for example, piperidine, piperazine, pyrrolidine, morpholine, lactone, lactam, etc. Heterocycles are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950 to present), particularly Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. Examples of heterocycles include, but are not limited to, tetrahydrofuran, dihydrofuran, tetrahydrothiophene, tetrahydropyran, dihydropyran, tetrahydrothiopyran, thiomorpholine, thioxane, homopiperazine, azetidine, oxetane, thietane, homopiperidine, piperidine, piperazine, pyrrolidine, morpholine, oxepane, thieptan, oxazepine, diazepine, thiazepine, 2-pyrroline, 3-pyrroline, indoline, 2H-pyran, 4H-pyran, dioxane, 1,3-dioxolane, pyrazoline, dithiane, dithiolane, dihydropyran, dihydrothiophene, dihydrofuran, pyrazolidinimidazoline, imidazolidine, 3-azabicyclo[3.1.0]hexane, 3-azabicyclo[4.1.0]heptane, and azabicyclo[2.2.2]hexane. Spiro moieties are also included within the scope of this definition.Examples of heterocyclic groups wherein a ring atom is substituted with an oxo (=0) moiety are pyrimidone and 1,1-dioxo-thiomorpholine.

[0244] As used herein, the term "heteroaryl" or "heteroaromatic ring" refers to a substituted or unsubstituted aromatic monocyclic ring structure, preferably a 6- to 18-membered ring, preferably a 5- to 7-membered ring, more preferably a 5- to 6-membered ring, whose ring structure includes at least one heteroatom (e.g., O, N, or S), preferably one to four or one to three heteroatoms, more preferably one or two heteroatoms. When two or more heteroatoms are present in a heteroaryl ring, they may be the same or different. The term "heteroaryl" also includes "polycyclic," "polycyclic," and "polycyclic" ring systems having two or more rings, wherein two or more ring atoms are shared by two adjacent rings, for example, the ring is a "fused ring," wherein at least one of the rings is heteroaromatic, for example, the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaromatic, and / or heterocyclyl. In some preferred embodiments, the polycyclic heteroaryl has 2-3 rings. In certain embodiments, preferred polycyclic heteroaryl has two rings, wherein both rings are aromatic.In certain embodiments, each ring of polycyclic contains 3 to 10 atoms in the ring, preferably contains 5 to 7 atoms in the ring.For example, heteroaryl includes but is not limited to pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, quinoline, pyrimidine, indolizine, indole, indazole, benzimidazole, benzothiazole, benzofuran, benzothiophene, cinnoline, phthalazine, quinazoline, carbazole, phenoxazine, quinoline, purine etc.In some embodiments, heteroaryl is a monocyclic aromatic group.In some embodiments, heteroaryl is a bicyclic aromatic group.In some embodiments, heteroaryl is a tricyclic aromatic group.

[0245] Where possible, the heterocycle or heteroaryl group may be carbon-linked (carbon-linked) or nitrogen-linked (nitrogen-linked). By way of example and not limitation, a carbon-linked heterocycle or heteroaryl group is bonded at the 2, 3, 4, 5, or 6 position of a pyridine, the 3, 4, 5, or 6 position of a pyridazine, the 2, 4, 5, or 6 position of a pyrimidine, the 2, 3, 5, or 6 position of a pyrazine, the 2, 3, 4, or 5 position of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole, the 2, 4, or 5 position of an oxazole, imidazole, or thiazole, the 3, 4, or 5 position of an isoxazole, pyrazole, or isothiazole, the 2 or 3 position of an aziridine, the 2, 3, or 4 position of an azetidine, the 2, 3, 4, 5, 6, 7, or 8 position of a quinoline, or the 1, 3, 4, 5, 6, 7, or 8 position of an isoquinoline.

[0246] By way of example and not limitation, nitrogen-bonded heterocyclic or heteroaryl groups are bonded to the 1-position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, the 2-position of isoindole or isoindoline, the 4-position of morpholine, and the 9-position of carbazole or O-carboline.

[0247] Heteroatoms present in a heteroaryl or heterocyclyl group include oxidized forms such as NO, SO and SO2.

[0248] In some embodiments, the heteroaryl ring is a 5- to 18-membered ring.

[0249] The term "halo" or "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I). In some embodiments, the halogen is fluorine. In some embodiments, the halogen is chlorine. In some embodiments, the halogen is bromine. In some embodiments, the halogen is iodine. As used herein, the term "haloalkyl" refers to an alkyl group as defined herein, which is substituted by one or more halogen groups as defined herein. The haloalkyl group may be a monohaloalkyl group, a dihaloalkyl group or a polyhaloalkyl group. A monohaloalkyl group may have one fluorine, chlorine, bromine or iodine substituent. A dihaloalkyl group or a polyhaloalkyl group may be substituted by two or more of the same halogen atoms or a combination of different halogen groups. Examples of haloalkyl groups include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl (diflosoropropyl), dichloroethyl and dichloropropyl.

[0250] The term "alkoxy" as used herein refers to alkyl-O-, wherein alkyl is as defined above. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.

[0251] The alkyl, haloalkyl, alkoxy, alkenyl, alkynyl, cyclic alkyl, cyclic alkenyl, cyclic alkynyl, carbocyclyl, aryl, heterocyclyl and heteroaryl groups described above may be optionally substituted with one or more (e.g., 2, 3, 4, 5, 6 or more) substituents.

[0252] Unless explicitly stated as "unsubstituted", references to chemical moieties herein are understood to also include substituted variants. For example, reference to "alkyl" or a moiety implicitly includes both substituted and unsubstituted variants. Examples of substituents on a chemical moiety include, but are not limited to, halogen, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (e.g., thioester, thioacetate or thioformate), alkoxy, alkylthio, acyloxy, phosphoryl, phosphate, phosphonate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aryl or heteroaryl moieties.

[0253] "Optional" or "optionally" means that the subsequently described circumstance may or may not occur, such that the disclosure includes instances where said circumstance occurs and instances where said circumstance does not occur. For example, the phrase "optionally substituted" means that a non-hydrogen substituent may or may not be present on a given atom, and thus, the disclosure includes structures where the non-hydrogen substituent is present and structures where the non-hydrogen substituent is not present.

[0254] The term "substituted" refers to a moiety having a substituent that replaces the hydrogen on one or more carbon, nitrogen, oxygen or sulfur atoms. It should be understood that "replacement" or "substituted" includes implicit restrictions, i.e., the substitution is consistent with the allowed valence of the substituted atom and the substituent, and the substitution produces a stable compound, such as the compound will not spontaneously undergo a transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is intended to include all permissible substituents of an organic compound. In a broad aspect, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of an organic compound. Permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of the present invention, for example, a heteroatom of nitrogen can have a hydrogen substituent and / or any permissible substituent that satisfies the heteroatom valence of an organic compound as described herein. Substituents may include any substituent described herein, such as halogen, hydroxy, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxy, alkylthio, acyloxy, phosphoryl, phosphate, phosphonate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. For illustration, a monofluoroalkyl group is an alkyl group substituted with one fluorine substituent, and a difluoroalkyl group is an alkyl group substituted with two fluorine substituents. It should be understood that if more than one substitution is present on a substituent, each non-hydrogen substituent may be the same or different (unless otherwise specified).

[0255] If a carbon of a substituent is described as being optionally substituted with one or more of the substituents listed, then one or more hydrogens on that carbon (to the extent that some are present) may be replaced, independently and / or collectively, with independently selected optional substituents. If a nitrogen of a substituent is described as being optionally substituted with one or more of the substituents listed, then one or more hydrogens on that nitrogen (to the extent that some are present) may each be replaced with an independently selected optional substituent. An exemplary substituent may be depicted as -NR'R", wherein R' and R" together with the nitrogen atom to which they are attached may form a heterocyclic ring. The heterocyclic ring formed by R' and R" together with the nitrogen atom to which they are attached may be partially or fully saturated. In some embodiments, the heterocyclic ring consists of 3 to 7 atoms. In other embodiments, the heterocyclic ring is selected from pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, pyridinyl, and thiazolyl.

[0256]

[0026] This specification uses the terms "substituent," "radical," and "group" interchangeably.

[0257] If a group of substituents is collectively described as being optionally substituted with one or more of the listed substituents, the group may include: (1) unsubstituted substituents, (2) substitutable substituents that are not substituted with the optional substituents, and / or (3) substitutable substituents that are substituted with one or more of the optional substituents.

[0258] If a substituent is described as being optionally substituted with up to a specified number of non-hydrogen substituents, then the substituent may be (1) unsubstituted; or (2) substituted with the specified number of non-hydrogen substituents or with up to the maximum number of substitutable positions on the substituent, whichever is less. Thus, for example, if a substituent is described as being a heteroaryl optionally substituted with up to 3 non-hydrogen substituents, then any heteroaryl having fewer than 3 substitutable positions will be optionally substituted with up to as many non-hydrogen substituents as the heteroaryl has substitutable positions. In non-limiting examples, such substituents may be selected from a linear, branched, or cyclic alkyl, alkenyl, or alkynyl group having from 1 to 10 carbon atoms, an aryl, a heteroaryl, a heterocyclyl, a halogen, a guanidinium salt [-NH(C=NH)NH2], a -OR 100 NR 101 R 102 、-NO2、-NR 101 COR 102 、-SR 100 , by-SOR 101 Sulfoxide represented by -SO2R 101 Sulfone, sulfonate -SO3M, sulfate -OSO3M, -SO2NR 101 R 102Sulfonamide, cyano, azide, -COR 101 、-OCOR 101 、-OCONR 101 R 102 and polyethylene glycol units (-OCH2CH2) n R 101 , where M is H or a cation (e.g. Na + or K + );R 101 、R 102 and R 103 Each independently selected from H, a linear, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms, a polyethylene glycol unit (-OCH2CH2) n -R 104 (wherein n is an integer from 1 to 24), an aryl group having from 6 to 10 carbon atoms, a heterocyclic ring having from 3 to 10 carbon atoms, and a heteroaryl group having from 5 to 10 carbon atoms; and R 104 is H or a linear or branched alkyl group having 1 to 4 carbon atoms, wherein the group is represented by R 100 、R 101 、R 102 、R 103 and R 104 The alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclic radicals represented by the present invention are optionally substituted by one or more (e.g., 2, 3, 4, 5, 6 or more) substituents independently selected from halogen, -OH, -CN, -NO2 and unsubstituted straight or branched chain alkyl groups having 1 to 4 carbon atoms. Preferably, the substituents for the optionally substituted alkyl, alkenyl, alkynyl, cyclic alkyl, cyclic alkenyl, cyclic alkynyl, carbocyclic radical, aryl, heterocyclic radical and heteroaryl described above include halogen, -CN, -NR 102 R 103 、-CF3、-OR 101 , aryl, heteroaryl, heterocyclic, -SR 101 、-SOR 101 、-SO2R 101 and -SO3M.

[0259] With respect to -SOR as indicated in the preceding paragraph 101 The sulfoxide represented by encompasses two optical isomers (R and S configurations at the sulfur atom of the sulfoxide group).

[0260] The number of carbon atoms in a group may be determined herein by the prefix "C x-xx ” or “C x -C xx " specifies, where x and xx are integers. For example, "C 1-4"Alkyl" or "C1-C4 alkyl" is an alkyl group having 1 to 4 carbon atoms.

[0261] The terms "compound" or "cytotoxic compound", "cytotoxic dimer" and "cytotoxic dimer compound" are used interchangeably. They are intended to include the following compounds, whose structures or formulas or any derivatives have been disclosed in the present invention or whose structures or formulas or any derivatives have been incorporated by reference. The term also includes stereoisomers, geometric isomers, tautomers, solvates, metabolites, salts (e.g., pharmaceutically acceptable salts) and prodrugs and prodrug salts of compounds of all formulas disclosed in the present invention. The term also includes any solvates, hydrates and polymorphs of any of the foregoing. In certain aspects of the invention described in this application, the specific description of "stereoisomers", "geometric isomers", "tautomers", "solvates", "metabolites", "salts", "prodrugs", "prodrug salts", "conjugates", "conjugate salts", "solvates", "hydrates" or "polymorphs" should not be interpreted as other aspects of the invention in which the term "compound" is used without describing these other forms. These forms are omitted.

[0262] As used herein, the term "conjugate" refers to a compound described herein, or a derivative thereof, linked to a cell-binding agent.

[0263] The term "chiral" refers to molecules that have the property of non-superimposability on their mirror image partner, whereas the term "achiral" refers to molecules that are superimposable on their mirror image partner.

[0264] The term "stereoisomers" refers to compounds that have identical chemical constitution and connectivity but different orientations of their atoms in space that are not interconvertible by rotation about single bonds.

[0265] The term "diastereomer" refers to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of one another. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivity. Mixtures of diastereomers can be separated by high-resolution analytical procedures such as crystallization, electrophoresis, and chromatography.

[0266] The term "enantiomers" refers to two stereoisomers of a compound that are non-superimposable mirror images of one another.

[0267] The stereochemical definitions and conventions used herein generally follow those in SP Parker, ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds," John Wiley & Sons, Inc., New York, 1994. The compounds of the present invention may contain asymmetric or chiral centers and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the present invention form part of the present invention, including but not limited to diastereomers, enantiomers and hindered isomers, as well as mixtures thereof, such as racemic mixtures. Many organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of plane-polarized light. When describing an optically active compound, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule about its chiral center. The prefixes d and l or (+) and (-) are used to indicate that the compound rotates plane-polarized light, where (-) or l means that the compound is left-handed. Compounds prefixed with (+) or d are right-handed. With respect to a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Specific stereoisomers may also be referred to as enantiomers, and mixtures of such isomers are often referred to as enantiomeric mixtures. A 50:50 mixture of enantiomers is referred to as a racemic mixture or racemate, which can exist in a chemical reaction or process without stereoselectivity or stereospecificity. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric substances that lack optical activity.

[0268] The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions by reorganization of some of the bonding electrons.

[0269] As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound of the invention. Exemplary salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, gentisates, fumarates, gluconates, glucuronates, saccharates, formates, benzoates, glutamates, methanesulfonates "mesylate," ethanesulfonates, benzenesulfonates, p-toluenesulfonates, pamoates (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)), alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Additionally, a pharmaceutically acceptable salt may have more than one charged atom in its structure. In cases where multiple charged atoms are part of a pharmaceutically acceptable salt, there may be multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0270] If the compound of the present invention is a base, the desired pharmaceutically acceptable salt can be prepared by any suitable method available in the art, for example, by treating the free base with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, methanesulfonic acid, phosphoric acid, or the like, or with an organic acid such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosonic acid (e.g., glucuronic acid or galacturonic acid), an alpha hydroxy acid (e.g., citric acid or tartaric acid), an amino acid (e.g., aspartic acid or glutamic acid), an aromatic acid (e.g., benzoic acid or cinnamic acid), a sulfonic acid (e.g., p-toluenesulfonic acid or ethanesulfonic acid), or the like.

[0271] If the compound of the invention is an acid, the desired pharmaceutically acceptable salt may be prepared by any suitable method, for example, by treating the free acid with an inorganic or organic base such as an amine (primary, secondary or tertiary), an alkali metal hydroxide or an alkaline earth metal hydroxide, etc. Illustrative examples of suitable salts include, but are not limited to, organic salts derived from amino acids such as glycine and arginine, ammonia, primary, secondary and tertiary amines, and cyclic amines such as piperidine, morpholine and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

[0272] As used herein, the term "solvate" refers to a compound that further includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces, such as water, isopropanol, acetone, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine, dichloromethane, 2-propanol, etc. Solvates or hydrates of the compounds are readily prepared by adding at least one molar equivalent of a hydroxylic solvent (e.g., methanol, ethanol, 1-propanol, 2-propanol, or water) to the compound to produce solvation or hydration of the imine moiety.

[0273] The phrase "pharmaceutically acceptable" indicates that the substance or composition must be compatible chemically and / or toxicologically with the other ingredients making up the formulation and / or the mammal to be treated therewith.

[0274] The term "leaving group" refers to a group of a charged or uncharged moiety that leaves during substitution or displacement. Such leaving groups are well known in the art and include, but are not limited to, halogens, esters, alkoxy groups, hydroxy groups, tosylates, trifluoromethanesulfonates, mesylate groups, nitriles, azides, carbamates, disulfides, thioesters, thioethers, and diazo compounds.

[0275] The term "reactive ester" refers to an ester having a readily displaceable leaving group that can readily react with an amine group to form an amide bond. Examples of reactive esters include, but are not limited to, N-hydroxysuccinimide esters, N-hydroxysulfosuccinimide esters, nitrophenyl (e.g., 2 or 4-nitrophenyl) esters, dinitrophenyl (e.g., 2,4-dinitrophenyl) esters, sulfo-tetrafluorophenyl (e.g., 4-sulfo-2,3,5,6-tetrafluorophenyl) esters, or pentafluorophenyl esters.

[0276] The term "reactive group" refers to a group that can react with a moiety on another molecule (e.g., a cell-binding agent or cytotoxic compound) to form a covalent bond. Reactive groups include, but are not limited to, amine-reactive groups and thiol-reactive groups.

[0277] The term "amine-reactive group" refers to a group that can react with an amine group to form a covalent bond. Exemplary amine-reactive groups include, but are not limited to, reactive ester groups, acyl halides, sulfonyl halides, imidoesters, or reactive thioester groups. In certain embodiments, the amine-reactive group is a reactive ester group. In one embodiment, the amine-reactive group is an N-hydroxysuccinimide ester or an N-hydroxysulfo-succinimide ester.

[0278] The term "thiol-reactive group" refers to a group that can react with a thiol (-SH) group to form a covalent bond. Exemplary thiol-reactive groups include, but are not limited to, maleimide, haloacetyl, haloacetamide, vinyl sulfone, vinyl sulfonamide, or vinyl pyridine. In one embodiment, the thiol-reactive group is maleimide.

[0279] The term "bifunctional cross-linker", "bifunctional linker" or "cross-linking agent" refers to a modifier having two reactive groups; one of the reactive groups is capable of reacting with a cell binding agent, while the other reacts with a cytotoxic compound to link the two moieties together. The bifunctional cross-linking agents are well known in the art (see, for example, Isalm and Dent Bioconjugation Chapter 5, pages 218-363, Groves Dictionaries Inc. New York, 1999). For example, bifunctional cross-linking agents that enable connection via thioether bonds include N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) to introduce maleimide groups, or N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB) to introduce iodoacetyl groups.Other bifunctional cross-linking agents that introduce maleimide or haloacetyl groups into cell binding agents are well known in the art (see U.S. Patent Applications 2008 / 0050310, 20050169933, available from Pierce Biotechnology Inc. PO Box 117, Rockland, IL). 61105, USA) and include but are not limited to bismaleimido polyethylene glycol (BMPEO), BM(PEO)2, BM(PEO)3, N-(β-maleimidopropoxy) succinimide ester (BMPS), γ-maleimidobutyric acid N-succinimide ester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), 5-maleimidovaleric acid NHS, HBVS, N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidohexanoate) (which is a "long-chain" analog of SMCC (LC-SMCC)), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)-butyric acid hydrazide or HCl salt (MPBH), N-succinimidyl 3-(bromoacetamido) propionate (SBAP), N-succinimidyl iodoacetate (SIA), kappa-maleimidoundecanoic acid N-succinimidyl ester (KMUA), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), succinimidyl-6-(β-aminobenzoyl)propionate (SBAP), N-succinimidyl iodoacetate (SIA), kappa-maleimidoundecanoic acid N-succinimidyl ester (KMUA), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), succinimidyl-6-(β-aminobenzoyl)propionate (SBAP), N-succinimidyl iodoacetate (SIA), kappa-maleimidoundecanoic acid N-succinimidyl ester (KMUA), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), -maleimidopropionylamino)hexanoate (SMPH), succinimidyl-(4-vinylsulfonyl)benzoate (SVSB), dithiobismaleimidoethane (DTME), 1,4-bismaleimidobutane (BMB), 1,4-bismaleimido-2,3-dihydroxybutane (BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), sulfosuccinimide The present invention also provides the following compounds: 1, 2-aminobenzoyl (4-iodoacetyl) aminobenzoate (sulfo-SIAB), 2, 3-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS), 3, 4-(maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBS), 4, 5-(maleimidohexanoyloxy)sulfosuccinimide ester (sulfo-EMCS), 5, 6-(maleimidoundecanoyloxy)sulfosuccinimide ester (sulfo-KMUS), 6, 7-(4-iodoacetyl) aminobenzoate (sulfo-SIAB), 3, 4-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS), 3, 5-(maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBS), 3, 6-(maleimidohexanoyloxy)sulfosuccinimide ester (sulfo-EMCS), 3, 7-(maleimidoundecanoyloxy)sulfosuccinimide ester (sulfo-KMUS), and 3, 5-(4-maleimidophenyl)butyrate (sulfo-SMPB).

[0280] Heterobifunctional cross-linking agents are bifunctional cross-linking agents with two different reactive groups. Heterobifunctional cross-linking agents containing both amine-reactive N-hydroxysuccinimide groups (NHS groups) and carbonyl-reactive hydrazine groups can also be used to connect the cytotoxic compounds described herein to cell-binding agents (e.g., antibodies). Examples of such commercially available heterobifunctional cross-linking agents include succinimidyl 6-hydrazinonicotinamide acetone hydrazone (SANH), succinimidyl 4-hydrazino terephthalate hydrochloride (SHTH), and succinimidyl hydrazine nicotinate hydrochloride (SHNH). Conjugates with acid-labile bonds can also be prepared using benzodiazepine derivatives with hydrazines of the present invention. Examples of usable bifunctional cross-linking agents include succinimidyl-p-formyl benzoate (SFB) and succinimidyl-p-formyl phenoxyacetate (SFPA).

[0281] Bifunctional cross-linking agents that enable attachment of cell-binding agents to cytotoxic compounds via disulfide bonds are known in the art and include N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio) pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB), N-succinimidyl-4-(2-pyridyldithio) 2-sulfobutyrate (sulfo-SPDB) to introduce dithiopyridyl groups. Other bifunctional cross-linking agents that can be used to introduce disulfide groups are known in the art and are disclosed in U.S. Patents 6,913,748, 6,716,821, and U.S. Patent Publications 20090274713 and 20100129314, all of which are incorporated herein by reference. Alternatively, a cross-linking agent that introduces a thiol group, such as 2-iminothiolane, homocysteine ​​thiolactone, or S-acetylsuccinic anhydride, may also be used.

[0282] As defined herein, the terms "linker," "linker moiety," or "linking group" refer to a moiety that connects two groups, such as a cell-binding agent and a cytotoxic compound. Typically, the linker is substantially inert under the conditions under which the two groups to which it is connected are connected. A bifunctional cross-linking agent may comprise two reactive groups, one at each end of the linker moiety, such that one reactive group can first react with the cytotoxic compound to provide a compound bearing the linker moiety and a second reactive group, which can then react with the cell-binding agent. Alternatively, one end of the bifunctional cross-linking agent can first react with the cell-binding agent to provide a cell-binding agent bearing the linker moiety and the second reactive group, which can then react with the cytotoxic compound. The linking moiety can contain a chemical bond that allows release of the cytotoxic moiety at a specific site. Suitable chemical bonds are well known in the art and include disulfide bonds, thioether bonds, acid-labile bonds, photolabile bonds, peptidase-labile bonds, and esterase-labile bonds (see, e.g., U.S. Patents 5,208,020; 5,475,092; 6,441,163; 6,716,821; 6,913,748; 7,276,497; 7,276,499; 7,368,565; 7,388,026 and 7,414,073). Disulfide bonds, thioether bonds, and peptidase-labile bonds are preferred. Other linkers useful in the present invention include non-cleavable linkers, such as those described in detail in U.S. Publication No. 20050169933, or the charged linkers or hydrophilic linkers described in US 2009 / 0274713, US 2010 / 01293140, and WO 2009 / 134976, each of which is expressly incorporated herein by reference.

[0283] The term "self-degrading joint" refers to a joint that allows the release of cytotoxic compounds when a remote site is activated. In certain embodiments, the joint includes a p-aminobenzyl unit. In some such embodiments, p-aminobenzyl alcohol is connected to an amino acid unit via an amide bond, and carbamate, methylcarbamate or carbonate (Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15: 1087-1103) are produced between benzyl alcohol and the drug. In some embodiments, the joint includes p-aminobenzyloxycarbonyl (PAB). Other examples of self-degrading joints include, but are not limited to, aromatic compounds electronically similar to PAB groups, such as 2-aminoimidazole-5-methanol derivatives (U.S. Patent No. 7,375,078; Hay et al. (1999) Bioorg. Med. Chem. Lett. 9: 2237) and o-aminobenzyl acetal or p-aminobenzyl acetal. In some embodiments, spacers that undergo cyclization upon amide bond hydrolysis may be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al. (1995) Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al. (1972) J. Amer. Chem. Soc. 94:5815), and 2-aminophenylpropionic acid amide (Amsberry et al. (1990) J. Org. Chem. 55:5867). Attachment of the drug to the α-carbon of a glycine residue is another example of a self-immolative linker suitable for use in ADCs (Kingsbury et al. (1984) J. Med. Chem. 27:1447).

[0284] The term "amino acid" refers to naturally occurring amino acids or non-naturally occurring amino acids. In some embodiments, the amino acid is composed of NH2-C(R aa' R aa )-C(=O)OH, wherein R aa and R aa' are each independently H, an optionally substituted linear, branched or cyclic alkyl, alkenyl or alkynyl group having 1 to 10 carbon atoms, an aryl group, a heteroaryl group or a heterocyclic group, or R aa The N-terminal nitrogen atom and the N-terminal nitrogen atom may be combined to form a heterocyclic ring (e.g., as in proline). The term "amino acid residue" refers to the corresponding residue when one hydrogen atom is removed from the amine and / or carboxyl terminus of an amino acid, e.g., -NH-C(R aa' R aa )-C(=O)-.

[0285] The term "peptide" refers to a short chain of amino acid monomers connected by peptide (amide) bonds. In some embodiments, the peptide contains 2 to 20 amino acid residues. In other embodiments, the peptide contains 2 to 10 or 2 to 8 amino acid residues. In other embodiments, the peptide contains 2 to 5 amino acid residues. As used herein, when a peptide is a portion represented by a specific sequence of amino acids in a cytotoxic agent or a linker as described herein, the peptide can be connected to the remainder of the cytotoxic agent or the linker in two directions.

[0286] The term "cation" refers to an ion having a positive charge. The cation may be monovalent (e.g., Na + , K + etc.), divalent (e.g., Ca 2+ Mg 2+ etc.) or multivalent (e.g., Al 3+ Preferably, the cation is monovalent.

[0287] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing, via at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses complete polyclonal antibodies, complete monoclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) mutants, multispecific antibodies (e.g., bispecific antibodies, biparatopic antibodies, etc.), multivalent antibodies (e.g., trivalent, tetravalent, etc. antibodies having three, four, or more antigen binding sites), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigenic determinant of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site, as long as the antibody exhibits the desired biological activity. Antibodies can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains (referred to as α, δ, ε, γ, and μ, respectively). The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules, such as toxins, radioisotopes, and the like. As used herein, "antibody" also includes activatable antibodies (e.g., probodies). Activatable means that when the activatable antibody is in an inhibited, masked, intact, or uncleaved state (i.e., a first conformation), the activatable antibody exhibits a first level of binding to the target, and exhibits a second level of binding to the target in an uninhibited, unmasked, and / or cleaved state (i.e., a second conformation), wherein the second level of target binding is greater than the first level of binding.

[0288] In some embodiments, the antibody is a non-naturally occurring antibody. In some embodiments, the antibody is purified from natural components. In some embodiments, the antibody is recombinantly produced. In some embodiments, the antibody is produced by a hybridoma.

[0289] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and F v Fragments, linear antibodies, single-chain antibodies, and multispecific antibodies (e.g., bispecific, biparatopic) formed from antibody fragments. The term "antigen-binding fragment" of an antibody includes one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by certain fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) Fab fragments, i.e., fragments consisting of V L 、V H 、C L and C H1 (ii) single-chain Fab (scFab), i.e., a monovalent fragment consisting of V L 、V H 、C L and C H1 The fragment consists of a domain, in which C L and V H The domains are connected via a linker peptide; (iii) F(ab')2 fragments, i.e., bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region (e.g., pepsin digestion of an antibody produces two fragments: a bivalent antigen-binding F(ab')2 fragment and a pFc' fragment that does not bind antigen) and their related F(ab') monovalent units; (iv) F d Fragment, which consists of V H and C H1 domain (i.e., the portion of the heavy chain included in the Fab); (v) V domains of a single antibody arm L and V H F domain v Fragment, and related disulfide-linked F v (vi) dAb (domain antibody) or sdAb (single domain antibody) fragments (Ward et al., Nature 341:544-546, 1989), which are composed of V H domains; (vii) isolated complementarity determining regions (CDRs); (viii) single chain variable fragments (scFvs), which consist of VH and V L domain-comprising fragments; and (ix) tetravalent antibodies, which may comprise multiple forms (structures) whereby the antibody comprises four antigen-binding sites.

[0290] The term "monoclonal antibody" refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically include different antibodies directed against different antigenic determinants. The term "monoclonal antibody" encompasses intact and full-length monoclonal antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2, F v ), single chain (scFv) mutants, fusion proteins comprising antibody portions, and any other modified immunoglobulin molecules comprising antigen recognition sites. In addition, "monoclonal antibody" refers to such antibodies produced in a variety of ways, including but not limited to hybridomas, phage selection, recombinant expression, and transgenic animals.

[0291] The term "humanized antibody" refers to a form of non-human (e.g., murine) antibodies that is a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof containing minimal non-human (e.g., murine) sequence. Typically, humanized antibodies are human immunoglobulins in which residues from the complementary determining regions (CDRs) are replaced by residues from CDRs of non-human species (e.g., mouse, rat, rabbit, hamster) with the desired specificity, affinity, and capacity (Jones et al., Nature 321:522-525, 1986; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988).

[0292] In some cases, the F v Framework region (FR) residues are replaced with corresponding residues in antibodies from non-human species having the desired specificity, affinity, and capacity. v The substitution of additional residues in the framework regions and / or in the replaced non-human residues is further modified to refine and optimize antibody specificity, affinity and / or ability. In general, a humanized antibody will comprise substantially all of at least one and typically two or three variable domains containing all or substantially all of the CDR regions corresponding to non-human immunoglobulins, while all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. A humanized antibody may also comprise an immunoglobulin constant region or domain (F c) (typically, a constant region or domain of a human immunoglobulin). Examples of methods for producing humanized antibodies are described in U.S. Patents 5,225,539 and 5,639,641, Roguska et al., Proc. Natl. Acad. Sci. USA 91(3):969-973, 1994; and Roguska et al., Protein Eng. 9(10):895-904, 1996 (each incorporated herein by reference). In some embodiments, a "humanized antibody" is a surface reshaped antibody. In some embodiments, a "humanized antibody" is a CDR-grafted antibody.

[0293] The term "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, alone or in combination. The variable regions of the heavy and light chains are each composed of four framework regions (FRs) connected by three complementary determining regions (CDRs) (also called hypervariable regions). The CDRs in each chain are tightly held together by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antibody's antigen binding site. There are at least two techniques for determining CDRs: (1) a method based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, 1991, National Institutes of Health, Bethesda Md.); and (2) a method based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., J. Molec. Biol. 273:927-948, 1997). In addition, a combination of these two methods is sometimes used in the art to determine CDRs.

[0294] The Kabat numbering system (e.g., Kabat et al., Sequences of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) is generally used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain).

[0295] As in Kabat, amino acid position numbering refers to the numbering system of the heavy chain variable domain or light chain variable domain for the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991) (incorporated herein by reference). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening of the FR or CDR of the variable domain or insertions therein. For example, the heavy chain variable domain may include a single amino acid insertion (according to Kabat's residue 52a) after residue 52 of H2 and a residue inserted after heavy chain FR residue 82 (e.g., according to Kabat's residues 82a, 82b, and 82c, etc.). The Kabat numbering of a residue can be determined for a given antibody by comparing it to a "standard" Kabat numbering sequence at the homology region of the sequence of the antibody. Chothia instead refers to the positions of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987). When numbered using the Kabat numbering convention, the ends of the Chothia CDR-H1 loop vary between H32 and H34, depending on the length of the loop. This is because the Kabat numbering scheme places the insertion at H35A and H35B—if both 35A and 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34. The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.

[0296]

[0297] The EU index, or EU index as in Kabat or EU numbering scheme, refers to the numbering system for human IgG1 Eu antibodies based on Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, incorporated herein by reference.

[0298] The term "human antibody" refers to an antibody produced by a human, or an antibody having an amino acid sequence corresponding to an antibody produced by a human, made using any technique known in the art. In certain embodiments, the human antibody does not have non-human sequences. This definition of a human antibody includes intact or full-length antibodies, or antigen-binding fragments thereof.

[0299] The term "chimeric antibody" refers to an antibody in which the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to the variable regions of antibodies derived from a mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in antibodies derived from another species (usually human) to avoid or reduce the probability of eliciting an immune response in that species (e.g., human). In certain embodiments, a chimeric antibody may include an antibody or antigen-binding fragment thereof comprising at least one human heavy and / or light chain polypeptide, such as an antibody comprising a murine light chain and a human heavy chain polypeptide.

[0300] The terms "epitope" or "antigenic determinant" are used interchangeably herein and refer to the portion of an antigen that can be recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, an epitope can be formed by contiguous amino acids and non-contiguous amino acids juxtaposed by triple folding of the protein. Epitopes formed by contiguous amino acids are generally retained upon protein denaturation, while epitopes formed by triple folding are generally lost upon protein denaturation. An epitope typically includes at least 3, and more typically at least 5 or 8-10, amino acids in a unique spatial configuration.

[0301] "Binding affinity" generally refers to the aggregate strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K d ) or half-maximal effective concentration (EC 50 ) represents. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate quickly, while high-affinity antibodies generally bind antigen faster and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention. Specific illustrative embodiments are described herein.

[0302] "Specifically binds" generally means that an antibody binds to an epitope via its antigen binding domain, and the binding requires some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope more quickly via its antigen binding domain than it would to a random, unrelated epitope. The term "specificity" is used herein to define the relative affinity of a particular antibody when it binds to a particular epitope. For example, antibody "A" can be considered to have a higher specificity for a given epitope than antibody "B", or antibody "A" can be said to bind to epitope "C" with a higher specificity than the specificity of antibody "A" for a related epitope "D".

[0303] As used herein, the term "immunoconjugate," "conjugate," or "ADC" refers to a compound or derivative thereof linked to a cell-binding agent, such as an antibody or antigen-binding fragment thereof.

[0304] The term "cysteine ​​engineered antibody" includes antibodies having at least one Cys that is not typically present at a given residue of an antibody light chain or heavy chain. The Cys may also be referred to as an "engineered Cys" and may be engineered using any conventional molecular biology or recombinant DNA technology (e.g., by replacing the coding sequence of a non-Cys residue at a target residue with the coding sequence of a Cys). For example, if the original residue is a Ser with a coding sequence of 5'-UCU-3', the coding sequence may be mutated (e.g., induced by site-directed mutagenesis) to 5'-UGU-3', which encodes a Cys. In certain embodiments, the Cys engineered antibodies of the present invention have an engineered Cys in the heavy chain. In certain embodiments, the engineered Cys is in or near the CH3 domains of the heavy chain. In certain embodiments, the engineered Cys is at residue 442 (EU / OU numbering) of the heavy chain. The C442 residue can be conjugated to a cytotoxic drug / agent via the free thiol group of the C442 residue, for example, via reaction with a thiol-reactive agent (eg, a maleimide group) of the cytotoxic drug.

[0305] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells is characterized by unregulated cell growth. "Tumor" and "neoplasm" refer to a growth or proliferation of one or more cells, either benign (non-cancerous) or malignant (cancerous), including precancerous lesions.

[0306] Examples of cancer include endometrial cancer, lung cancer (e.g., non-small cell lung cancer), colorectal cancer, bladder cancer, gastric cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, esophageal cancer, breast cancer, head and neck cancer, uterine cancer, ovarian cancer, liver cancer, cervical cancer, thyroid cancer, testicular cancer, bone marrow cancer, melanoma, and lymphoma. In certain embodiments, the cancer is non-small cell lung cancer, colorectal cancer, gastric cancer, or pancreatic cancer. In certain embodiments, the cancer is non-small cell lung cancer (squamous cell, non-squamous cell, adenocarcinoma, or large cell undifferentiated carcinoma), colorectal cancer (adenocarcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, primary colorectal lymphoma, leiomyosarcoma, or squamous cell carcinoma), or breast cancer (e.g., triple negative breast cancer (TNBC)). In certain embodiments, cancer is lymphoma and leukemia. In certain embodiments, examples of cancer include AML, CML, ALL (e.g., B-ALL), CLL, myelodysplastic syndrome, blastic plasmacytoid DC neoplasm (BPDCN) leukemia, B-cell lymphomas (including non-Hodgkin lymphoma (NHL)), precursor B-cell lymphoblastic leukemia / lymphoma and mature B-cell neoplasms (e.g., B-cell chronic lymphocytic leukemia (B-CLL) / small lymphocytic lymphoma (SLL)), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), and myelodysplastic syndrome. In some embodiments, the cancer is BPDCN leukemia. In some embodiments, the cancer is ALL. In other embodiments, the cancer is AML.

[0307] The term "subject" refers to any animal (e.g., mammal) that is to be the recipient of a particular treatment, including but not limited to humans, non-human primates, rodents, etc. Generally, the terms "subject," "patient," and "individual" are used interchangeably herein with respect to human subjects.

[0308] The term "pharmaceutical composition" refers to a preparation that is in a form that permits the biological activity of the active ingredient to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the composition would be administered. Such compositions may be sterile.

[0309] As used herein, a "therapeutically effective amount" is an amount of a compound or composition sufficient to carry out a specific stated purpose. Full therapeutic effect may not occur after administration of a single dose, but may occur only after administration of a series of doses. The specific "therapeutically effective amount" will depend on, for example, the age, weight, and medical condition of the subject, as well as the method of administration and the therapeutic agent or combination of therapeutic agents selected for administration. A "therapeutically effective amount" can be determined empirically and in a routine manner for the stated purpose.

[0310] As used herein, the terms "treating," "treat," or "treatment" include reversing, reducing, or preventing the symptoms, clinical signs, or underlying pathology of a disorder in a manner that improves or stabilizes the disorder in a subject. As used herein, and as is well understood in the art, "treatment" is a method for obtaining beneficial or desired results, including clinical results. Beneficial or desired results include, but are not limited to, prevention, alleviation, improvement, or slowing of progression of one or more symptoms or disorders associated with a disorder, weakening of the extent of the disease, a stabilized state of the disease, delaying or slowing of disease progression, improvement or alleviation of the disease state, and relief (partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to the expected survival if not receiving treatment.

[0311] Compound

[0312] In one aspect, the present invention provides a compound of formula I or a pharmaceutically acceptable salt thereof:

[0313] Z-L 1 —D (Formula I)

[0314] in:

[0315] D is represented by the following structural formula:

[0316]

[0317] R 1 is -F, -CH3 or -CF3;

[0318] R 2 Yes -H, -F, -OR 3 、-SR 3 、-S(O)R 4 、-S(O)2R 4 , C1-C6 alkyl or C1-C6 fluoroalkyl; or R 1 and R 2 The carbon atoms to which they are attached together form a methylenedioxy or difluoromethylenedioxy ring;

[0319] R 3 is H or C1-C6 alkyl;

[0320] R 4 is a C1-C6 alkyl group;

[0321] L 1 does not exist, is -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, -X 1' -(C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; wherein * is the site of covalent attachment to Z;

[0322] X 1 Is -O-, -S-, -S(O)-, -S(O)2-, -C(=O)-, -NR 5 -、-NR 5 C(=O)- or -C(=O)NR 5 -;

[0323] X 1' is -O-, -S-, -S(O)- or -S(O)2-;

[0324] L 2 is phenylene;

[0325] Each R 5 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0326] Z is -H or -X 2 ;

[0327] X 2 Yes-OR 6 、-SR 6 、-S(O)R 6 、-S(O)2R 6 、-SSR 6 or -N(R 6 )2;

[0328] Each R 6 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0329] L 1 and L 2 Each independently optionally substituted by 1-4 selected from halogen, -CN, -OR 7 、-SR 7 、-N(R 7)2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 is substituted with a substituent of heterocycloalkyl, aryl, or heteroaryl; and

[0330] Each R 7 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0331] The condition is that if R 1 If it is F, then L 1 -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, -X 1' -(C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; wherein * is the site of covalent attachment to Z; and Z is -X 2 ;and

[0332] The condition is that if R 1 is F and R 2 is -OMe, then -L 1 -Z cannot be -NH2.

[0333] In some embodiments, the present invention provides a compound of Formula I or a pharmaceutically acceptable salt thereof:

[0334] Z-L 1 —D (Formula I)

[0335] in:

[0336] D is represented by the following structural formula:

[0337]

[0338] R 1 is -F, -CH3 or -CF3;

[0339] R 2 Yes -H, -F, -OR 3 、-SR 3 、-S(O)R 4 、-S(O)2R 4 , C1-C6 alkyl or C1-C6 fluoroalkyl; or R 1 and R 2 The carbon atoms to which they are attached together form a methylenedioxy or difluoromethylenedioxy ring;

[0340] R 3 is H or C1-C6 alkyl;

[0341] R 4 is a C1-C6 alkyl group;

[0342] L 1 does not exist, is -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, -X 1' -(C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; wherein * is the site of covalent attachment to Z;

[0343] X 1 Is -O-, -S-, -S(O)-, -S(O)2-, -C(=O)-, -NR 5 -、-NR 5 C(=O)- or -C(=O)NR 5 -;

[0344] X 1' is -O-, -S-, -S(O)- or -S(O)2-;

[0345] L 2 is phenylene;

[0346] Each R 5 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0347] Z is -H or -X 2 ;

[0348] X 2 Yes-OR 6 、-SR 6 、-S(O)R 6 、-S(O)2R 6 、-SSR 6 or -N(R 6 )2;

[0349] Each R 6 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0350] L 1 and L 2 Each independently optionally substituted by 1-4 selected from halogen, -CN, -OR 7 、-SR 7 、-N(R 7)2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 is substituted with a substituent of heterocycloalkyl, aryl, or heteroaryl; and

[0351] Each R 7 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0352] The condition is that if R 1 If it is F, then L 1 -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, -X 1' (C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; wherein * is the site of covalent attachment to Z; and Z is -X 2 ;

[0353] The condition is that if R 1 is F and R 2 is -OMe, then -L 1 -Z cannot be -NH2; and

[0354] The condition is that if R 1 is F and R 2 If it is -Me, then -L1-Z cannot be -CH2OH.

[0355] In some embodiments, R 1 It is F and -L 1 -Z is -(C1-C6 alkylene)-X 2 、-(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-X 2 、-X 1' -(C1-C6 alkylene)-X 2 or -(C1-C6 alkylene)-X 1 -L 2 -X 2 In some embodiments, R 1 It is F and -L 1 -Z is -(C1-C6 alkylene)-OR 6 、-(C1-C6 alkylene)-SR 6 、-(C1-C6 alkylene)-S(O)R 6 、-(C1-C6 alkylene)-S(O)2R 6 、-(C1-C6 alkylene)-SSR6 or -(C1-C6 alkylene)-N(R 6 )2. In some embodiments, R 1 It is F and -L 1 -Z is -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-OR 6 、-(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-SR 6 、-(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-S(O)R 6 、-(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-S(O)2R 6 、-(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-SSR 6 or -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-N(R 6 )2. In some embodiments, R 1 It is F and -L 1 -Z is -X 1' -(C1-C6 alkylene)-OR 6 、-X 1' -(C1-C6 alkylene)-SR 6 、-X 1' -(C1-C6 alkylene)-S(O)R 6 、-X 1' -(C1-C6 alkylene)-S(O)2R 6 、-X 1' -(C1-C6 alkylene)-SSR 6 or -X 1' -(C1-C6 alkylene)-N(R 6 )2. In some embodiments, R 1 It is F and -L 1 -Z is -(C1-C6 alkylene)-X 1 -L 2 -OR 6 、-(C1-C6 alkylene)-X 1 -L 2 -SR 6 、-(C1-C6 alkylene)-X 1 -L 2 -S(O)R 6 、-(C1-C6 alkylene)-X 1 -L 2-S(O)2R 6 、-(C1-C6 alkylene)-X 1 -L 2 -SSR 6 or -(C1-C6 alkylene)-X 1 -L 2 -N(R 6 )2.

[0356] In some embodiments, R 1 Is -H or -F. In some embodiments, R 1 In some embodiments, R 2 is -H, -F, -OCF3, -CF3, -OMe, -OEt, -SMe, -S(O)Me, -S(O)2Me, -SEt, -S(O)Et, -S(O2)Et, methyl or ethyl. In some embodiments, R 2 In some embodiments, R 2 is -OMe, -SMe, -S(O)Me or methyl. In some embodiments, R 2 In some embodiments, R 1 Yes -F and R 2 In some embodiments, R 1 is methyl and R 2 In some embodiments, R 1 Yes -F and R 2 It is -methyl.

[0357] In some embodiments, -L 1 -Z is -H. In some embodiments, -L 1 -Z is -(C1-C6 alkylene)-H or -(C1-C6 alkylene)-X 2 In some embodiments, -L 1 -Z is -(C1-C6 alkylene)-H. In some embodiments, -L 1 -Z is -(C1-C6 alkylene)-X 2 In some embodiments, -L 1 -Z is -(C1-C6 alkylene)-X 2 In some embodiments, -L 1 -Z is methyl, ethyl, propyl or butyl.

[0358] In some embodiments, -L 1 -Z is -(C1-C4 alkylene)-OR 6 、-(C1-C4 alkylene)-SR 6 or -(C1-C4 alkylene)-N(R6 )2. In some embodiments, -L 1 -Z is -(C1-C4 alkylene)-OR 6 In some embodiments, -L 1 -Z is -(C1-C4 alkylene)-SR 6 In some embodiments, -L 1 -Z is -(C1-C4 alkylene)-N(R 6 )2.

[0359] In some embodiments, -L 1 -Z is -CH2OH, -(CH2)2OH, -(CH2)3OH, -(CH2)4OH, -CH2OMe, -(CH2)2OMe, -(CH2)3OMe, -(CH2)4OMe, -CH2SH, -(CH2)2SH, -( CH2)3SH, -(CH2)4SH, -CH2SMe, -(CH2)2SMe, -(CH2)3SMe, -(CH2)4SMe, -CH2NH2, -(CH2)2NH2, -(CH2)3NH2, -(CH2)4NH2.

[0360] In some embodiments, -L 1 -Z is -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-OR 6 、-(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-SR 6 , -(C1-C5 alkylene)-S-(C1-C5 alkylene)-SR 6 or -(C1-C5 alkylene)-S-(C1-C5 alkylene)-SSR 6 In some embodiments, -L 1 -Z is -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-OR 6 In some embodiments, -L 1 -Z is -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-SR 6 In some embodiments, -L 1 -Z is -(C1-C5 alkylene)-S-(C1-C5 alkylene)-SR 6 In some embodiments, -L 1 -Z is -(C1-C5 alkylene)-S-(C1-C5 alkylene)-SSR 6 .

[0361] In some embodiments, -L 1 -Z is -CH2NHC(=O)CH2OH, -CH2NHC(=O)(CH2)2OH, -CH2NHC(=O)(CH2)3OH, -CH2NHC(=O)(CH2)4OH, -CH2 NHC(=O)(CH2)5OH, -CH2NHC(=O)CH2OMe, -CH2NHC(=O)(CH2)2OMe, -CH2NHC(=O)(CH2)3OMe, -CH2NHC(= O)(CH2)4OMe, -CH2NHC(=O)(CH2)5OMe, -CH2NHC(=O)CH2SH, -CH2NHC(=O)(CH2)2SH, -CH2NHC(=O)(CH2 )3SH, -CH2NHC(=O)(CH2)4SH, -CH2NHC(=O)(CH2)5SH, -CH2NHC(=O)CH2SMe, -CH2NHC(=O)(CH2)2SMe, - CH2NHC(=O)(CH2)3SMe, -CH2NHC(=O)(CH2)4SMe, -CH2NHC(=O)(CH2)5SMe, -CH2SCH2OH, -CH2S(CH2)2O H, -CH2S(CH2)3OH, -CH2S(CH2)4OH, -CH2S(CH2)5OH, -CH2SCH2OMe, -CH2S(CH2)2OMe, -CH2S(CH2)3OMe , -CH2S(CH2)4OMe, -CH2S(CH2)5OMe, -CH2SCH2SH, -CH2S(CH2)2SH, -CH2S(CH2)3SH, -CH2S(CH2)4SH, -CH2S(CH2)5SH, -CH2SCH2SMe, -CH2S(CH2)2SMe, -CH2S(CH2)3SMe, -CH2S(CH2)4SMe or -CH2S(CH2)5SMe.

[0362] In some embodiments, each R 5 is independently -H, methyl or benzyl. In some embodiments, each R 5 is independently -H. In some embodiments, each R 5 In some embodiments, each R 5 It's benzyl.

[0363] In some embodiments, each R 6 is independently -H, methyl or benzyl. In some embodiments, each R 6 is independently -H. In some embodiments, each R 6In some embodiments, each R 6 It's benzyl.

[0364] In some embodiments, -L 1 -Z is -X 1' -(C1-C4 alkylene)-X 2 In some embodiments, -L 1 -Z is -OCH2OH, -O(CH2)2OH, -O(CH2)3OH, -O(CH2)4OH, -SCH2OH, -S(CH2)2OH, -S(CH2)3OH, -S(CH2)4OH, -S(O)CH2OH, -S(O )(CH2)2OH, -S(O)(CH2)3OH, -S(O)(CH2)4OH, -S(O)2CH2OH, -S(O)2(CH2)2OH, -S(O)2(CH2)3OH, -S(O)2(CH2)4OH, -OCH2S Me, -O(CH2)2SMe, -O(CH2)3SMe, -O(CH2)4SMe, -SCH2SMe, -S(CH2)2SMe, -S(CH2)3SMe, -S(CH2)4SMe, -S(O)CH2SMe, -S(O) (CH2)2SMe, -S(O)(CH2)3SMe, -S(O)(CH2)4SMe, -S(O)2CH2SMe, -S(O)2(CH2)2SMe, -S(O)2(CH2)3SMe or -S(O)2(CH2)4SMe.

[0365] In some embodiments, -L 1 -Z is -(C1-C6 alkylene)-X 1 -L 2 -X 2 In some embodiments, -L 1 -Z is In some embodiments, -L 1 -Z is In some embodiments, -L 1 -Z is

[0366] In another aspect, the present invention provides a compound of formula II or a pharmaceutically acceptable salt thereof:

[0367] E—A—Z'—L 1 —D (Formula II)

[0368] in:

[0369] D is represented by the following structural formula:

[0370]

[0371] R 1 is -H, -F, -CH3 or -CF3;

[0372] R 2 Yes -H, -F, -OR 3 、-SR 3 、-S(O)R 4 、-S(O)2R 4 , C1-C6 alkyl or C1-C6 fluoroalkyl; or R 1 and R 2 The carbon atoms to which they are attached together form a methylenedioxy or difluoromethylenedioxy ring; provided that R 1 and R 2 Cannot be -H at the same time;

[0373] R 3 is H or C1-C6 alkyl;

[0374] R 4 is a C1-C6 alkyl group;

[0375] L 1 does not exist, is -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, X 1' -(C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; wherein * is the site of covalent attachment to Z';

[0376] X 1 Is -O-, -S-, -S(O)-, -S(O)2-, -C(=O)-, -NR 5 -、-NR 5 C(=O)- or -C(=O)NR 5 -;

[0377] X 1' is -O-, -S-, -S(O)- or -S(O)2-;

[0378] L 2 is phenylene;

[0379] Each R 5 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0380] Z' is -O-CH2-NR 8 -*、-S-CH2-NR 8 -*, -NR8 -*; wherein * is the site of covalent attachment to A;

[0381] Each R 8 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0382] L 1 and L 2 Each independently optionally substituted by 1-4 selected from halogen, -CN, -OR 7 、-SR 7 、-N(R 7 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 is substituted with a substituent of heterocycloalkyl, aryl, or heteroaryl; and

[0383] Each R 7 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0384] A is a peptide comprising 2 to 10 amino acids; wherein A is optionally substituted with one or more polyols; and

[0385] E is -C(=O)-L 3 -X 3 ;

[0386] L 3 Yes-(C1-C 10 Alkylene)-or-Y 1 -(C1-C 10 Alkylene)-X 4 -Y 2 -(C1-C 10 alkylene)-*; wherein * is covalently linked to X 3 The site of

[0387] Y 1 Does not exist, is-(CR a R b O) n -or-(CR a R b CR a' R b' O) m -;

[0388] X 4 Yes-NR 9 C(=O)- or -C(=O)NR 9 -;

[0389] Y2 Does not exist, is-(CR c R d O) o -or-(CR c R d CR c' R d' O) p -;

[0390] n, m, o and p are each independently 1-10;

[0391] Each R a 、R b 、R a' 、R b' 、R c 、R d 、R c' and R d' is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0392] Among them L 3 Optionally substituted by 0-4 selected from halogen, -CN, -OR 11 、-SR 11 、-N(R 11 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 Substitution of heterocycloalkyl, aryl, heteroaryl, and polyol substituents;

[0393] Each R 11 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0394] X 3 yes -C(=O)-CR bb R cc -W', -NR ee -C(=O)-CR bb R cc -W' or -SR 10 ;

[0395] Each W' is independently -H, -N(R gg 2. C1-C 10 Alkyl, C1-C 10 Alkenyl, C1-C 10 Alkynyl, C3-C6 cycloalkyl, aryl, heteroaryl or -(CH2CH2O) q -Rff ;

[0396] q is 1 to 24;

[0397] Each R aa 、R bb 、R cc 、R ee and R ff is independently -H or optionally substituted C1-C6 alkyl;

[0398] Each R YY and R XX are independently -H or C1-C6 alkyl;

[0399] R gg are each independently -H or C1-C6 alkyl; and

[0400] R 9 and R 10 Each is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl.

[0401] In some embodiments, R 1 Is -H or -F. In some embodiments, R 1 In some embodiments, R 2 is -H, -F, -OCF3, -CF3, -OMe, -OEt, -SMe, -S(O)Me, -S(O)2Me, -SEt, -S(O)Et, -S(O2)Et, methyl or ethyl. In some embodiments, R 2 In some embodiments, R 2 is -OMe, -SMe, -S(O)Me or methyl. In some embodiments, R 2 In some embodiments, R 1 Yes -F and R 2 In some embodiments, R 1 is methyl and R 2 In some embodiments, R 1 Yes -F and R 2 It is -methyl.

[0402] In some embodiments, -L 1 -Z'-* is -(C1-C4 alkylene)-O-CH2-NR 8 -*, -(C1-C4 alkylene)-S-CH2-NR 8 -* or -(C1-C4 alkylene)-NR 8 -*. In some embodiments, -L1 -Z'-* is -(C1-C4 alkylene)-O-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C4 alkylene)-S-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C4 alkylene)-NR 8 -*.

[0403] In some embodiments, -L 1 -Z'-* is -CH2O-CH2NH-*, -(CH2)2O-CH2NH-*, -(CH2)3O-CH2NH-*, -(CH2)4O-CH2NH-*, -CH2S-CH2NH-*, -(CH2 )2S-CH2NH-*, -(CH2)3S-CH2NH-*, -(CH2)4S-CH2NH-*, -CH2NH-*, -(CH2)2NH-*, -(CH2)3NH-* or -(CH2)4NH-*.

[0404] In some embodiments, -L 1 -Z'-* is -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-O-CH2-NR 8 -*, -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-S-CH2-NR 8 -*, -(C1-C5 alkylene)-S-(C1-C5 alkylene)-S-CH2-NR 8 -* or -(C1-C5 alkylene)-S-(C1-C5 alkylene)-SS-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-O-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-S-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C5 alkylene)-S-(C1-C5 alkylene)-S-CH2-NR 8 -*. In some embodiments, -L 1-Z'-* is -(C1-C5 alkylene)-S-(C1-C5 alkylene)-SS-CH2-NR 8 -*.

[0405] In some embodiments, -L 1 -Z'-* is -CH2NHC(=O)CH2O-CH2-NH-*, -CH2NHC(=O)(CH2)2O-CH2-NH-*, -CH2NHC(=O)(CH2)3O-CH2-NH-*, -CH2NHC(=O)(CH2)4O-CH2-NH-*, -CH2NHC( =O)(CH2)5O-CH2-NH-*, -CH2NHC(=O)CH2S-CH2-NH-*, -CH2NHC(=O)(CH2)2S-CH2-NH-*, -CH2NHC(=O)(CH2)3S-CH2-NH-*, -CH2NHC(=O)(CH2)4S-CH2- NH-*, -CH2NHC(=O)(CH2)5S-CH2-NH-*, -CH2SCH2O-CH2-NH-*, -CH2S(CH2)2O-CH2-NH-*, -CH2S(CH2)3O-CH2-NH-*, -CH2S(CH2)4O-CH2-NH-*, -CH2S (CH2)5O-CH2-NH-*, -CH2SCH2S-CH2-NH-*, -CH2S(CH2)2S-CH2-NH-*, -CH2S(CH2)3S-CH2-NH-*, -CH2S(CH2)4S-CH2-NH-* or -CH2S(CH2)5S-CH2-NH-*.

[0406] In some embodiments, each R 5 is independently -H, methyl or benzyl. In some embodiments, each R 5 is independently -H. In some embodiments, each R 5 In some embodiments, each R 5 In some embodiments, each R 8 is independently -H, methyl or benzyl. In some embodiments, each R 8 is independently -H. In some embodiments, each R 8 In some embodiments, each R 8 It's benzyl.

[0407] In some embodiments, -L 1 -Z'-* is -X 1' -(C1-C4 alkylene)-O-CH2-NR 8 -*, -X1' -(C1-C4 alkylene)-S-CH2-NR 8 -* or -X 1' -(C1-C4 alkylene)-NR 8 -*. In some embodiments, -L 1 -Z'-* is -X 1' -(C1-C4 alkylene)-O-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -X 1' -(C1-C4 alkylene)-S-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -X 1' -(C1-C4 alkylene)-NR 8 -*.

[0408] In some embodiments, -L 1-Z'-* is -OCH2O-CH2-NH-*, -O(CH2)2O-CH2-NH-*, -O(CH2)3O-CH2-NH-*, -O(CH2)4O-CH2-NH-*, -SCH2O-CH2-NH-*, -S(CH2)2O-CH2-N H-*, -S(CH2)3O-CH2-NH-*, -S(CH2)4O-CH2-NH-*, -S(O)CH2O-CH2-NH-*, -S(O)(CH2)2O-CH2-NH-*, -S(O)(CH2)3O-CH2-NH-*, -S(O)( CH2)4O-CH2-NH-*, -S(O)2CH2O-CH2-NH-*, -S(O)2(CH2)2O-CH2-NH-*, -S(O)2(CH2)3O-CH2-NH-*, -S(O)2(CH2)4O-CH2-NH-*, -OCH2S -CH2-NH-*, -O(CH2)2S-CH2-NH-*, -O(CH2)3S-CH2-NH-*, -O(CH2)4S-CH2-NH-*, -SCH2S-CH2-NH-*, -S(CH2)2S-CH2-NH-*, -S(CH2)3S -CH2-NH-*, -S(CH2)4S-CH2-NH-*, -S(O)CH2S-CH2-NH-*, -S(O)(CH2)2S-CH2-NH-*, -S(O)(CH2)3S-CH2-NH-*, -S(O)(CH2)4S-CH2-NH -*, -S(O)2CH2S-CH2-NH-*, -S(O)2(CH2)2S-CH2-NH-*, -S(O)2(CH2)3S-CH2-NH-*, -S(O)2(CH2)4S-CH2-NH-*, -OCH2-NH-*, -O(CH2)2 -NH-*, -O(CH2)3-NH-*, -O(CH2)4S-NH-*, -SCH2-NH-*, -S(CH2)2-NH-*, -S(CH2)3-NH-*, -S(CH2)4-NH-*, -S(O)CH2-NH-*, -S(O)(CH2 )2-NH-*, -S(O)(CH2)3-NH-*, -S(O)(CH2)4-NH-*, -S(O)2CH2-NH-*, -S(O)2(CH2)2-NH-*, -S(O)2(CH2)3-NH-* or -S(O)2(CH2)4-NH-*.

[0409] In some embodiments, -L 1 -Z'-* is -(C1-C6 alkylene)-X 1 -L 2 -Z'-*. In some embodiments, -L1 -Z'-*Yes In some embodiments, -L 1 -Z'-*Yes In some embodiments, -L 1 -Z'-*Yes

[0410] Disclosed herein -L 1 In various embodiments of -Z'-*, * is the site of covalent attachment to A.

[0411] In some embodiments, A is a peptide comprising 2 to 8 amino acids. In some embodiments, A is a peptide comprising 2 to 4 amino acids. In some embodiments, at least one amino acid in the peptide is an L amino acid. In some embodiments, each amino acid in the peptide is an L amino acid. In some embodiments, at least one amino acid in the peptide is a D amino acid.

[0412] In some embodiments, A is -(AA 1 )-(AA 2 ) a1 -*, where * is the site of covalent attachment to E; AA 1 and AA 2 are each independently an amino acid residue; and a1 is an integer from 1 to 9.

[0413] In some embodiments, -AA 1 -(AA 2 ) a1 -*is -Gly-Gly-Gly-*, -Ala-Val-*, -Val-Ala-*, -Val-Cit-*, -Val-Lys-*, -Lys-Val-*, -Phe-Lys-*, -Lys-Phe-*, -Lys-Ly s-*, -Ala-Lys-*, -Lys-Ala-*, -Phe-Cit-*, -Cit-Phe-*, -Leu-Cit-*, -Cit-Leu-*-Ile-Cit-*, -Phe-Ala-*, -Ala-Phe-*, -Phe-N 9 -Tosyl-Arg-*、-N 9 -Tosyl-Arg-Phe-*, -Phe-N 9 -Nitro-Arg-*, -N 9-Nitro-Arg-Phe*, -Phe-Phe-Lys-*, -Lys-Phe-Phe-*, -Gly-Phe-Lys-*, Lys-Phe-Gly-*, -Leu-Ala-Leu-*, -Ile-Ala-Leu-*, -Leu-Ala-Ile-*, - Val-Ala-Val-*, -Ala-Leu-Ala-Leu-*, -Leu-Ala-Leu-Ala-*, -β-Ala-Leu-Ala-Leu-*, -Gly-Phe-Leu-Gly-*, -Gly-Leu-Phe-Gly-*, -Val-Ar g-*, -Arg-Val-*, -Arg-Arg-*, -Ala-Ala-*, -Ala-Met-*, -Met-Ala-*, -Thr-Thr-*, -Thr-Met-*, -Met- Thr-*, -Leu-Ala-*, -Ala-Leu-*, -Cit-Val-*, -Gln-Val-*, -Val-Gln-*, -Ser-Val-*, -Val-Ser-*, -Se r-Ala-*, -Ser-Gly-*, -Ala-Ser-*, -Gly-Ser-*, -Leu-Gln-*, -Gln-Leu-*, -Phe-Arg-*, -Arg-Phe-*, - Tyr-Arg-*, -Arg-Tyr-*, -Phe-Gln-*, -Gln-Phe-*, -Val-Thr-*, -Thr-Val-*, -Met-Tyr-* and -Tyr-Met-*.

[0414] In some embodiments, -AA 1 -(AA 2 ) a1 -*is -Val-D-Lys-*, -Val-D-Arg-*, -L-Val-Cit-*, -L-Val-Lys-*, -L-Val-Arg-*, -L-Val-D-Cit-*, -L-Phe-Phe-Lys-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Arg- D-Arg-*, -L-Ala-Ala-*, -L-Ala-D-Ala-*, -Ala-D-Ala-*, -Val-D-Cit-*, -L-Ala-L-Ala-*, -L-Ala-L-Val-*, -L-Gln-L-Val-*, -L-Gln-L-Leu-* or -L-Ser-L-Val-*.

[0415] In some embodiments, -AA 1 -(AA2 ) a1 -* is: -Ala-Ala-*, -Ala-Val-*, -Val-Ala-*, -Gln-Leu-*, -Leu-Gln-*, -Ala-Ala-Ala-*, -Ala-Ala-Ala-Ala-*, -Gly-Ala-Gly-Gly-*, -Gly-Gly-Ala-Gly-*, -Gly-Val-Gly-Gly-*, -Gly-Gly-Val-Gly-*, -Gly-Phe-Gly-Gly-* or -Gly-Gly-Phe-Gly-*.

[0416] In some embodiments, -AA 1 -(AA 2 ) a1 -* is: -L-Ala-L-Ala-*, -L-Ala-D-Ala-*, -L-Ala-L-Val-*, -L-Ala-D-Val-*, -L-Val-L-Ala-*, -L-Val-D-Ala-*, -L-Gln-L-Leu-*, -L-Gln-D-Leu-*, -L-Leu-L-Gln-*, -L-Leu-D-Gln-*, -L-Ala-L-Ala-L-Ala-*, -L-Ala-D-Ala-L-Ala-*, -L-Ala-L-Ala-D-Ala-*, -L-Ala-L-Ala-L-Ala-L-Ala-*, -L-Ala-D-Ala-L-Ala-L-Ala-*, -L-Ala-L-Ala-D-Ala-L-Ala-*, -L-Ala-L-Ala-L-Ala-D-Ala-*, -Gly-L-Ala-Gly-Gly-*, -Gly-Gly-L-Ala-Gly-*, -Gly-D-Ala-Gly-Gly-*, Gly-Gly-D-Ala-Gly-*, -Gly-L-Val-Gly-Gly-*, Gly-Gly-L-Val-Gly-*, -Gly-D-Val-Gly-Gly-*, Gly-Gly-D-Val-Gly-*, -Gly-L-Phe-Gly-Gly-* or Gly-Gly-L-Phe-Gly-*.

[0417] In some embodiments, -AA 1 -(AA 2 ) a1-* is: -L-Ala-L-Ala-*, -L-Ala-D-Ala-LAla-*, -L-Ala-L-Ala-L-Ala-* or -L-Ala-L-Ala-L-Ala-L-Ala-*.

[0418] Disclosed herein -AA 1 -(AA 2 ) a1 In various embodiments of -*, * is a site of covalent attachment to E.

[0419] In some embodiments, A is substituted with one or more polyols. In some embodiments, E is substituted with one or more polyols. In some embodiments, the polyol is -(C1-C6 alkylene)-X 5 -Y 3 ; Among them: X 5 Yes-NR 12 C(=O)- or -C(=O)NR 12 -;Y 3 Yes-C1-C 10 Alkyl, where Y 3 is substituted with 0-10 OH groups; and R 12 It is -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl.

[0420] In some embodiments, wherein the polyol is where R 12 It is H or methyl.

[0421] In some embodiments, wherein the polyol is where R 12 It is H or methyl.

[0422] In some embodiments, E is -C(=O)-(C1-C 10 Alkylene)-X 3 In some embodiments, E is

[0423]

[0424] In some embodiments, E is -C(=O)-Y 1 -(C1-C 10 Alkylene)-X 4 -(C1-C 10 Alkylene)-X 3 ;

[0425] Y 1 Yes-(CR a R b O)n -or-(CR a R b CR a' R b' O) m -;

[0426] X 4 Yes-NR 9 C(=O)-; and

[0427] X 3 yes -C(=O)-CR bb R cc -W',NR ee -C(=O)-CR bb R cc -W' or -SR 10 .

[0428] In some embodiments, E is -C(=O)-Y 1 -(CH2)2-X 4 -(CH2)2-X 3 ;

[0429] Y 1 Yes -(CH2O) n -or-(CH2CH2O) m -;

[0430] X 4 is -NHC(=O)-;

[0431] n is 2; m is 2 to 6;

[0432] X 3 yes -C(=O)-CR bb R cc -W',NR ee -C(=O)-CR bb R cc -W' or -SR 10 .

[0433] In another aspect, the present invention provides a compound of formula III or a pharmaceutically acceptable salt thereof:

[0434] CBA—E'—A—Z'—L 1 —D (Formula III)

[0435] in:

[0436] D is represented by the following structural formula:

[0437]

[0438] R 1 is -H, -F, -CH3 or -CF3;

[0439] R 2 Yes -H, -F, -OR 3 、-SR 3 、-S(O)R 4 、-S(O)2R 4 , C1-C6 alkyl or C1-C6 fluoroalkyl; or R 1 and R 2 The carbon atoms to which they are attached together form a methylenedioxy or difluoromethylenedioxy ring; provided that R 1 and R 2 Cannot be -H at the same time;

[0440] R 3 is H or C1-C6 alkyl;

[0441] R 4 is a C1-C6 alkyl group;

[0442] L 1 does not exist, is -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, X 1' -(C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; wherein * is the site of covalent attachment to Z';

[0443] X 1 Is -O-, -S-, -S(O)-, -S(O)2-, -C(=O)-, -NR 5 -、-NR 5 C(=O)- or -C(=O)NR 5 -;

[0444] X 1' is -O-, -S-, -S(O)- or -S(O)2-;

[0445] L 2 is phenylene;

[0446] Each R 5 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0447] Z' is -O-CH2-NR 8-*、-S-CH2-NR 8 -*, -NR 8 -*; wherein * is the site of covalent attachment to A;

[0448] Each R 8 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0449] L 1 and L 2 Each independently optionally substituted by 1-4 selected from halogen, -CN, -OR 7 、-SR 7 、-N(R 7 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 is substituted with a substituent of heterocycloalkyl, aryl, or heteroaryl; and

[0450] Each R 7 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0451] A is a peptide comprising 2 to 10 amino acids; wherein A is optionally substituted with one or more polyols;

[0452] E' is -C(=O)-L 3 -X 6 -*; wherein * is the site of covalent attachment to CBA;

[0453] L 3 Yes-(C1-C 10 Alkylene)-or-Y 1 -(C1-C 10 Alkylene)-X 4 -Y 2 -(C1-C 10 alkylene)-*; wherein * is covalently linked to X 6 The site of

[0454] Y 1 Does not exist, is-(CR a R b O) n -or-(CR a R b CR a' R b' O) m -;

[0455] X 4 Yes-NR 9C(=O)- or -C(=O)NR 9 -;

[0456] Y 2 Does not exist, is-(CR c R d O) o -or-(CR c R d CR c' R d' O) p -;

[0457] n, m, o and p are each independently 1-10;

[0458] Each R a 、R b 、R a' 、R b' 、R c 、R d 、R c' and R d' is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0459] Among them L 3 Optionally substituted by 0-4 selected from halogen, -CN, -OR 11 、-SR 11 、-N(R 11 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 Substitution of heterocycloalkyl, aryl, heteroaryl, and polyol substituents;

[0460] Each R 11 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0461] X 6 yes -C(=O)-CR bb R cc -* or -NR ee -C(=O)-CR bb R cc -*; wherein * is the site of covalent attachment to CBA;

[0462] Each R aa 、R bb 、R cc and R eeis independently -H or optionally substituted C1-C6 alkyl;

[0463] Each R YY and R XX are independently -H or C1-C6 alkyl;

[0464] R 9 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl; and

[0465] CBA is a cell binding agent.

[0466] In some embodiments, R 1 Is -H or -F. In some embodiments, R 1 In some embodiments, R 2 is -H, -F, -OCF3, -CF3, -OMe, -OEt, -SMe, -S(O)Me, -S(O)2Me, -SEt, -S(O)Et, -S(O2)Et, methyl or ethyl. In some embodiments, R 2 In some embodiments, R 2 is -OMe, -SMe, -S(O)Me or methyl. In some embodiments, R 2 In some embodiments, R 1 Yes -F and R 2 In some embodiments, R 1 is methyl and R 2 In some embodiments, R 1 Yes -F and R 2 It is -methyl.

[0467] In some embodiments, -L 1 -Z'-* is -(C1-C4 alkylene)-O-CH2-NR 8 -*, -(C1-C4 alkylene)-S-CH2-NR 8 -* or -(C1-C4 alkylene)-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C4 alkylene)-O-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C4 alkylene)-S-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C4 alkylene)-NR 8 -*.

[0468] In some embodiments, -L 1 -Z'-* is -CH2O-CH2NH-*, -(CH2)2O-CH2NH-*, -(CH2)3O-CH2NH-*, -(CH2)4O-CH2NH-*, -CH2S-CH2NH-*, -(CH2 )2S-CH2NH-*, -(CH2)3S-CH2NH-*, -(CH2)4S-CH2NH-*, -CH2NH-*, -(CH2)2NH-*, -(CH2)3NH-* or -(CH2)4NH-.

[0469] In some embodiments, -L 1 -Z'-* is -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-O-CH2-NR 8 -*, -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-S-CH2-NR 8 -*, -(C1-C5 alkylene)-S-(C1-C5 alkylene)-S-CH2-NR 8 -* or -(C1-C5 alkylene)-S-(C1-C5 alkylene)-SS-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-O-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C5 alkylene)-NR 5 C(=O)-(C1-C5 alkylene)-S-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C5 alkylene)-S-(C1-C5 alkylene)-S-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -(C1-C5 alkylene)-S-(C1-C5 alkylene)-SS-CH2-NR 8 -*.

[0470] In some embodiments, -L 1-Z'-* is -CH2NHC(=O)CH2O-CH2-NH-*, -CH2NHC(=O)(CH2)2O-CH2-NH-*, -CH2NHC(=O)(CH2)3O-CH2-NH-*, -CH2NHC(=O)(CH2)4O-CH2-NH-*, -CH2NHC( =O)(CH2)5O-CH2-NH-*, -CH2NHC(=O)CH2S-CH2-NH-*, -CH2NHC(=O)(CH2)2S-CH2-NH-*, -CH2NHC(=O)(CH2)3S-CH2-NH-*, -CH2NHC(=O)(CH2)4S-CH2- NH-*, -CH2NHC(=O)(CH2)5S-CH2-NH-*, -CH2SCH2O-CH2-NH-*, -CH2S(CH2)2O-CH2-NH-*, -CH2S(CH2)3O-CH2-NH-*, -CH2S(CH2)4O-CH2-NH-*, -CH2S (CH2)5O-CH2-NH-*, -CH2SCH2S-CH2-NH-*, -CH2S(CH2)2S-CH2-NH-*, -CH2S(CH2)3S-CH2-NH-*, -CH2S(CH2)4S-CH2-NH-* or -CH2S(CH2)5S-CH2-NH-*.

[0471] In some embodiments, each R 5 is independently -H, methyl or benzyl. In some embodiments, each R 5 is independently -H. In some embodiments, each R 5 In some embodiments, each R 5 In some embodiments, each R 8 is independently -H, methyl or benzyl. In some embodiments, each R 8 is independently -H. In some embodiments, each R 8 In some embodiments, each R 8 It's benzyl.

[0472] In some embodiments, -L 1 -Z'-* is -X 1' -(C1-C4 alkylene)-O-CH2-NR 8 -*, -X 1' -(C1-C4 alkylene)-S-CH2-NR 8 -* or -X 1' -(C1-C4 alkylene)-NR 8 -*. In some embodiments, -L 1-Z'-* is -X 1' -(C1-C4 alkylene)-O-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -X 1' -(C1-C4 alkylene)-S-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -X 1' -(C1-C4 alkylene)-NR 8 -*.

[0473] In some embodiments, -L 1-Z'-* is -OCH2O-CH2-NH-*, -O(CH2)2O-CH2-NH-*, -O(CH2)3O-CH2-NH-*, -O(CH2)4O-CH2-NH-*, -SCH2O-CH2-NH-*, -S(CH2)2O-CH2-N H-*, -S(CH2)3O-CH2-NH-*, -S(CH2)4O-CH2-NH-*, -S(O)CH2O-CH2-NH-*, -S(O)(CH2)2O-CH2-NH-*, -S(O)(CH2)3O-CH2-NH-*, -S(O)( CH2)4O-CH2-NH-*, -S(O)2CH2O-CH2-NH-*, -S(O)2(CH2)2O-CH2-NH-*, -S(O)2(CH2)3O-CH2-NH-*, -S(O)2(CH2)4O-CH2-NH-*, -OCH2S -CH2-NH-*, -O(CH2)2S-CH2-NH-*, -O(CH2)3S-CH2-NH-*, -O(CH2)4S-CH2-NH-*, -SCH2S-CH2-NH-*, -S(CH2)2S-CH2-NH-*, -S(CH2)3S -CH2-NH-*, -S(CH2)4S-CH2-NH-*, -S(O)CH2S-CH2-NH-*, -S(O)(CH2)2S-CH2-NH-*, -S(O)(CH2)3S-CH2-NH-*, -S(O)(CH2)4S-CH2-NH -*, -S(O)2CH2S-CH2-NH-*, -S(O)2(CH2)2S-CH2-NH-*, -S(O)2(CH2)3S-CH2-NH-*, -S(O)2(CH2)4S-CH2-NH-*, -OCH2-NH-*, -O(CH2)2 -NH-*, -O(CH2)3-NH-*, -O(CH2)4S-NH-*, -SCH2-NH-*, -S(CH2)2-NH-*, -S(CH2)3-NH-*, -S(CH2)4-NH-*, -S(O)CH2-NH-*, -S(O)(CH2 )2-NH-*, -S(O)(CH2)3-NH-*, -S(O)(CH2)4-NH-*, -S(O)2CH2-NH-*, -S(O)2(CH2)2-NH-*, -S(O)2(CH2)3-NH-* or -S(O)2(CH2)4-NH-*.

[0474] In some embodiments, -L 1 -Z'-* is -(C1-C6 alkylene)-X 1 -L 2 -Z'-*. In some embodiments, -L1 -Z'-*Yes In some embodiments, -L 1 -Z'-*Yes In some embodiments, -L 1 -Z'-*Yes

[0475] Disclosed herein -L 1 In various embodiments of -Z'-*, * is the site of covalent attachment to A.

[0476] In some embodiments, A is a peptide comprising 2 to 8 amino acids. In some embodiments, A is a peptide comprising 2 to 4 amino acids. In some embodiments, at least one amino acid in the peptide is an L amino acid. In some embodiments, each amino acid in the peptide is an L amino acid. In some embodiments, at least one amino acid in the peptide is a D amino acid.

[0477] In some embodiments, A is -(AA 1 )-(AA 2 ) a1 -*, where * is the point of connection with E', AA 1 and AA 2 are each independently an amino acid residue; and a1 is an integer from 1 to 9.

[0478] In some embodiments, -AA 1 -(AA 2 ) a1 -*is -Gly-Gly-Gly-*, -Ala-Val-*, -Val-Ala-*, -Val-Cit-*, -Val-Lys-*, -Lys-Val-*, -Phe-Lys-*, -Lys-Phe-*, -Lys-Ly s-*, -Ala-Lys-*, -Lys-Ala-*, -Phe-Cit-*, -Cit-Phe-*, -Leu-Cit-*, -Cit-Leu-*-Ile-Cit-*, -Phe-Ala-*, -Ala-Phe-*, -Phe-N 9 -Tosyl-Arg-*、-N 9 -Tosyl-Arg-Phe-*, -Phe-N 9 -Nitro-Arg-*, -N 9-Nitro-Arg-Phe*, -Phe-Phe-Lys-*, -Lys-Phe-Phe-*, -Gly-Phe-Lys-*, Lys-Phe-Gly-*, -Leu-Ala-Leu-*, -Ile-Ala-Leu-*, -Leu-Ala-Ile-*, - Val-Ala-Val-*, -Ala-Leu-Ala-Leu-*, -Leu-Ala-Leu-Ala-*, -β-Ala-Leu-Ala-Leu-*, -Gly-Phe-Leu-Gly-*, -Gly-Leu-Phe-Gly-*, -Val-Ar g-*, -Arg-Val-*, -Arg-Arg-*, -Ala-Ala-*, -Ala-Met-*, -Met-Ala-*, -Thr-Th r-*, -Thr-Met-*, -Met-Thr-*, -Leu-Ala-*, -Ala-Leu-*, -Cit-Val-*, -Gln-Val-*, -Val-Gln-*, -Ser-Val-*, -Val-Ser-*, -Ser-Ala-*, -Ser-Gly-*, -Ala-Ser-*, -Gl y-Ser-*, -Leu-Gln-*, -Gln-Leu-*, -Phe-Arg-*, -Arg-Phe-*, -Tyr-Arg-*, -Arg-Ty r-*, -Phe-Gln-*, -Gln-Phe-*, -Val-Thr-*, -Thr-Val-*, -Met-Tyr-* and -Tyr-Met-*.

[0479] In some embodiments, -AA 1 -(AA 2 ) a1 -*is -Val-D-Lys-*, -Val-D-Arg-*, -L-Val-Cit-*, -L-Val-Lys-*, -L-Val-Arg-*, -L-Val-D-Cit-*, -L-Phe-Phe-Lys-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Arg- D-Arg-*, -L-Ala-Ala-*, -L-Ala-D-Ala-*, -Ala-D-Ala-*, -Val-D-Cit-*, -L-Ala-L-Ala-*, -L-Ala-L-Val-*, -L-Gln-L-Val-*, -L-Gln-L-Leu-* or -L-Ser-L-Val-*.

[0480] In some embodiments, -AA 1 -(AA2 ) a1 -* is: -Ala-Ala-*, -Ala-Val-*, -Val-Ala-*-Gln-Leu-*, -Leu-Gln-*, -Ala-Ala-Ala-*, -Ala-Ala-Ala-Ala-*, -Gly-Ala-Gly-Gly-*, -Gly-Gly-Ala-Gly-*, -Gly-Val-Gly-Gly-*, -Gly-Gly-Val-Gly-*, -Gly-Phe-Gly-Gly-* or -Gly-Gly-Phe-Gly-*.

[0481] In some embodiments, -AA 1 -(AA 2 ) a1 -* is: -L-Ala-L-Ala-*, -L-Ala-D-Ala-*, -L-Ala-L-Val-*, -L-Ala-D-Val-*, -L-Val-L-Ala-*, -L-Val-D-Ala-*, -L-Gln-L-Leu-*, -L-Gln-D-Leu-*, -L-Leu-L-Gln-*, -L-Leu-D-Gln-*, -L-Ala-L-Ala-L-Ala-*, -L-Ala-D-Ala-L-Ala-*, -L-Ala-L-Ala-D-Ala-*, -L-Ala-L-Ala-L-Ala-L-Ala-*, -L-Ala-D-Ala-L-Ala-L-Ala-*, -L-Ala-L-Ala-D-Ala-L-Ala-*, -L-Ala-L-Ala-L-Ala-D-Ala-*, -Gly-L-Ala-Gly-Gly-*, -Gly-Gly-L-Ala-Gly-*, -Gly-D-Ala-Gly-Gly-*, Gly-Gly-D-Ala-Gly-*, -Gly-L-Val-Gly-Gly-*, Gly-Gly-L-Val-Gly-*, -Gly-D-Val-Gly-Gly-*, Gly-Gly-D-Val-Gly-*, -Gly-L-Phe-Gly-Gly-* or Gly-Gly-L-Phe-Gly-*.

[0482] In some embodiments, -AA 1 -(AA 2 ) a1-* is: -L-Ala-L-Ala-*, -L-Ala-D-Ala-L-Ala-*, -L-Ala-L-Ala-L-Ala-*, or -L-Ala-L-Ala-L-Ala-L-Ala-*.

[0483] Disclosed herein -AA 1 -(AA 2 ) a1 In various embodiments of -*, * is a site of covalent attachment to E'.

[0484] In some embodiments, A is substituted with one or more polyols. In some embodiments, E' is substituted with one or more polyols. In some embodiments, the polyol is -(C1-C6 alkylene)-X 5 -Y 3 ; Among them: X 5 Yes-NR 12 C(=O)- or -C(=O)NR 12 -;Y 3 Yes-C1-C 10 Alkyl, where Y 3 is substituted with 0-10 OH groups; and R 12 It is -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl.

[0485] In some embodiments, the polyol is where R 12 It is H or methyl.

[0486] In some embodiments, wherein the polyol is where R 12 It is H or methyl.

[0487] In some embodiments, E' is -C(=O)-(C1-C 10 Alkylene)-X 6 -*. In some embodiments, E' is

[0488]

[0489]

[0490] -C(=O)CH2CH2-C(=O)-CR bb R cc -* or -C(=O)CH2CH2-NR ee -C(=O)-CR bb R cc -*; wherein * is a site of covalent attachment to CBA. In some embodiments, RYY 、R XX 、R aa 、R bb Each is independently -H or C1-C6 alkyl.

[0491] In some embodiments, E' is -C(=O)-Y 1 -(C1-C 10 Alkylene)-X 4 -(C1-C 10 Alkylene)-X 6 -*;

[0492] Y 1 Yes-(CR a R b O) n -or-(CR a R b CR a' R b' O) m -;

[0493] X 4 Yes-NR 9 C(=O)-; and

[0494] X 6 yes

[0495] -C(=O)-CR bb R cc -* or -NR ee -C(=O)-CR bb R cc -*; where * is the site of covalent attachment to CBA.

[0496] In some embodiments, E' is -C(=O)-Y 1 -(CH2)2-X 4 -(CH2)2-X 6 -*;

[0497] Y 1 Yes -(CH2O) n -or-(CH2CH2O) m -;

[0498] X 4 is -NHC(=O)-;

[0499] n is 2; m is 2 to 6;

[0500] X 6 yes

[0501] -C(=O)-CR bb R cc -* or -NR ee -C(=O)-CR bb R cc -*; where * is the site of covalent attachment to CBA.

[0502] In some embodiments, the CBA comprises a -SH group covalently linked to E' to provide

[0503] -C(=O)-CR bb R cc -S-CBA or -NR ee -C(=O)-CR bb R cc -S-CBA.

[0504] In another aspect, the present invention provides a compound of formula IV or a pharmaceutically acceptable salt thereof:

[0505] E—A—Z'—L 1 —D (Formula IV)

[0506] in:

[0507] D is represented by the following structural formula:

[0508]

[0509] R 1 is -H, -F, -CH3 or -CF3;

[0510] R 2 Yes -H, -F, -OR 3 、-SR 3 、-S(O)R 4 、-S(O)2R 4 , C1-C6 alkyl or C1-C6 fluoroalkyl; or R 1 and R 2 The carbon atoms to which they are attached together form a methylenedioxy or difluoromethylenedioxy ring; provided that R 1 and R 2 Cannot be -H at the same time;

[0511] R 3 is H or C1-C6 alkyl;

[0512] R 4 is a C1-C6 alkyl group;

[0513] L 1 does not exist, is -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, X 1' -(C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; wherein * is the site of covalent attachment to Z';

[0514] X 1 Is -O-, -S-, -S(O)-, -S(O)2-, -C(=O)-, -NR 5 -、-NR 5 C(=O)- or -C(=O)NR 5 -;

[0515] X 1' is -O-, -S-, -S(O)- or -S(O)2-;

[0516] L 2 is phenylene;

[0517] Each R 5 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0518] Z' is -O-CH2-NR 8 -*、-S-CH2-NR 8 -*, -NR 8 -*; wherein * is the site of covalent attachment to A;

[0519] Each R 8 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0520] L 1 and L 2 Each independently optionally substituted by 1-4 selected from halogen, -CN, -OR 7 、-SR 7 、-N(R 7 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 Substitution of heterocycloalkyl, aryl or heteroaryl groups;

[0521] Each R 7 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0522] A is a peptide comprising 2 to 10 amino acids; wherein A is optionally substituted with one or more polyols;

[0523] E is -C(=O)OL 3 -X 3 ;

[0524] L 3 Yes-(C1-C 10 alkylene)-;

[0525] Among them L 3 Optionally substituted by 0-4 selected from halogen, -CN, -OR 9 、-SR 9 、-N(R 9 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 Substitution of heterocycloalkyl, aryl, heteroaryl, and polyol substituents;

[0526] Each R 9 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl; and

[0527] X 3 yes

[0528] In some embodiments, E is -C(=O)OL 3 -X 3 And L 3 is -(C1 alkylene)-. In some embodiments, L 3 It is -CH2-.

[0529] In another aspect, the present invention provides a compound of formula V or a pharmaceutically acceptable salt thereof:

[0530] A—Z'—L 1 —D(Formula V)

[0531] in:

[0532] D is represented by the following structural formula:

[0533]

[0534] R 1 is -H, -F, -CH3 or -CF3;

[0535] R 2 Yes -H, -F, -OR 3 、-SR 3 、-S(O)R 4、-S(O)2R 4 , C1-C6 alkyl or C1-C6 fluoroalkyl; or R 1 and R 2 The carbon atoms to which they are attached together form a methylenedioxy or difluoromethylenedioxy ring; provided that R 1 and R 2 Cannot be -H at the same time;

[0536] R 3 is H or C1-C6 alkyl;

[0537] R 4 is a C1-C6 alkyl group;

[0538] L 1 does not exist, is -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, X 1' -(C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; wherein * is the site of covalent attachment to Z';

[0539] X 1 Is -O-, -S-, -S(O)-, -S(O)2-, -C(=O)-, -NR 5 -、-NR 5 C(=O)- or -C(=O)NR 5 -;

[0540] X 1' is -O-, -S-, -S(O)- or -S(O)2-;

[0541] L 2 is phenylene;

[0542] Each R 5 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0543] Z' is -O-CH2-NR 8 -*、-S-CH2-NR 8 -*, -NR 8 -*; wherein * is the site of covalent attachment to A;

[0544] Each R 8 is independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl;

[0545] L 1 and L2 Each independently optionally substituted by 1-4 selected from halogen, -CN, -OR 7 、-SR 7 、-N(R 7 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 Substitution of heterocycloalkyl, aryl or heteroaryl groups;

[0546] Each R 7 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl or benzyl; and

[0547] A is a peptide comprising 2 to 10 amino acids; wherein A is optionally substituted with one or more polyols.

[0548] In some embodiments, the compound is a compound of Formula I and is any one of the following compounds from the following table:

[0549] Table 1A.

[0550]

[0551]

[0552] In some embodiments, the compound is a compound of Formula I and is any one of the following from the following table:

[0553] Table 1B

[0554]

[0555]

[0556]

[0557]

[0558] In some embodiments, the compound is a compound of Formula II and is any one of the following from the following table:

[0559] Table 2

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566]

[0567] In some embodiments, wherein the compound comprises a sulfoxide and the sulfoxide is in the R or S configuration. In some embodiments, the compound is any of the following:

[0568]

[0569] In some embodiments, the compound is any of the following:

[0570]

[0571]

[0572]

[0573] Synthesis of Compounds The compounds described herein are prepared by the general synthetic routes described in the following schemes. In some embodiments, the compounds described herein are prepared as outlined in Scheme 1. Scheme 1. General synthetic scheme for preparing RNH2 compound (A-6)

[0574]

[0575] In some embodiments, the compounds described herein are prepared as outlined in Scheme 2. Scheme 2. General synthetic scheme for the preparation of ROH compounds (B-2)

[0576]

[0577] In some embodiments, the compounds described herein are prepared as outlined in Scheme 3. Scheme 3. For the preparation of RNHR C1 General synthetic scheme of compound (C-4)

[0578]

[0579] In some embodiments, the compounds described herein are prepared as outlined in Scheme 4.

[0580] Scheme 4. For the preparation of RS(CH2) n General synthetic scheme of OH compound (D-2)

[0581]

[0582] In some embodiments, the compounds described herein are prepared as outlined in Scheme 5.

[0583] Scheme 5. General method for the preparation of thiol-bearing camptothecin derivatives (E-4)

[0584]

[0585] In some embodiments, v is 1 to 4. In some embodiments, R is alkyl, aryl, or heteroaryl. In some embodiments, R is methyl, ethyl, phenyl, or pyridyl.

[0586] In some embodiments, the compounds described herein are prepared as outlined in Scheme 6.

[0587] Scheme 6. General method for preparing thioamine-peptide bonds that can be further derivatized to incorporate thiols or maleimides

[0588]

[0589] In some embodiments, v is 1 to 4. In some embodiments, v2 is 1 to 4. In some embodiments, R is alkyl, aryl, or heteroaryl. In some embodiments, R is methyl, ethyl, phenyl, or pyridyl.

[0590] In some embodiments, the compounds described herein are prepared as outlined in Scheme 7.

[0591] Plan 7.

[0592]

[0593] In some embodiments, n is 1 to 10. In some embodiments, n is 1 to 5. In some embodiments, v is 1 to 10. In some embodiments, v is 1 to 5.

[0594] In some embodiments, the compounds described herein are prepared as outlined in Scheme 8.

[0595] Plan 8.

[0596]

[0597] In some embodiments, v is 1 to 10. In some embodiments, v is 1 to 5.

[0598] Cell binding agents

[0599] The cell-binding agent in the immunoconjugate of the present invention can be of any type currently known or becoming known, including peptides and non-peptides that bind to cells or cellular components (e.g., receptors, proteins, DNA, RNA, etc.). Generally speaking, these cell-binding agents can be antibodies (e.g., polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies) or fragments thereof, lymphokines, hormones, growth factors, vitamins (e.g., folate, etc., which can bind to their cell surface receptors, such as folate receptors), nutrient transport molecules (e.g., transferrin), probodies, nanobodies, or any other cell-binding molecules or substances.

[0600] In certain embodiments, the cell-binding agent is an antibody, a single chain antibody, an antibody fragment that specifically binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody, a monoclonal antibody fragment (or "antigen-binding portion" or "antigen-binding fragment") that specifically binds to a target cell, a chimeric antibody, a chimeric antibody fragment (or "antigen-binding portion" or "antigen-binding fragment") that specifically binds to a target cell, a domain antibody (e.g., sdAb), or a domain antibody fragment that specifically binds to a target cell.

[0601] In certain embodiments, the cell-binding agent is a humanized antibody, a humanized single chain antibody, or a humanized antibody fragment (or "antigen-binding portion" or "antigen-binding fragment").

[0602] In certain embodiments, the cell-binding agent is a resurfaced antibody, a resurfaced single chain antibody, or a resurfaced antibody fragment (or "antigen-binding portion" or "antigen-binding fragment").

[0603] In certain embodiments, the cell-binding agent is an antibody or antigen-binding portion thereof (including antibody derivatives), and the CBA can bind to a ligand on a target cell, such as a cell-surface ligand, including a cell-surface receptor.

[0604] In certain embodiments, the cell binding agent (CBA) binds to a target cell selected from a tumor cell, a virally infected cell, a microbial infected cell, a parasite infected cell, an autoimmune cell, an activated cell, a myeloid cell, an activated T cell, a B cell, or a melanocyte.In some embodiments, the CBA binds to a protein expressing 5T4, ADAM-9, ALK, AMHRII, ASCT2, Axl, B7-H3, BCMA, C4.4a, CA6, CA9, CanAg, CD123, CD138, CD142, CD166, CD184, CD19, CD20, CD205, CD22, CD248, CD25, CD3, CD30, CD33, CD352, CD37, CD38, CD40L, CD44v6, CD45, CD46, CD48, CD51, CD56, CD7, CD70, CD71, CD74, CD79b, CDH6, CEACAM5, CEACAM6, cKIT, CLDN18.2, CLDN6, CLL-1, c-MET, Cripto, CSP-1, CXCR5, DLK-1, DLL3, DPEP3, antiadhesin, EFNA4, EGFR, EGFRviii, ENPP3, EpCAM, EphA2, EphA3, ETBR, FGFR2, FGFR3, FLT3, FOLR-α, FSH, GCC, GD2, GD3, Globo H, GPC-1, GPC3, gpNMB, HER-2, HER-3, HLA-DR, HSP90, IGF-1R, IL-13R, IL1RAP, IL7R, interleukin-4 receptor (IL4R), KAAG-1, LAMP-1, Lewis Y antigen, LGALS3BP, LGR5, LH / hCG, LHRH, LIV-1, LRP-1, LRRC15, Ly6E, MAGE, mesothelin (MSLN), MET, MHC cells harboring any one or more of class I chain-related proteins A and B (MICA and MICB), MT1-MMP, MTX3, MTX5, MUC1, MUC16, NaPi2b, Nectin-4, NOTCH3, OAcGD2, OX001L, p-cadherin, PD-L1, phosphatidylserine (PS), polymorphic epithelial mucin (PEM), prolactin receptor (PRLR), PSMA, PTK7, RNF43, ROR1, ROR2, SAIL, SLAMF7, SLC44A4, SLITRK6, SSTR2, STEAP-1, STING, STn, TIM-1, TM4SF1, TNF-α, TRA, TROP-2, tumor-associated glycoprotein 72 (TAG-72), a tumor-specific epitope of mucin-1 (TA-MUC1), CD5, TIM-3, UPK2, or UPK1b antigens.

[0605] In certain embodiments, the cell-binding agent is a cysteine ​​engineered antibody or antigen-binding fragment thereof that specifically binds to a cell expressing 5T4, ADAM-9, ALK, AMHRII, ASCT2, Axl, B7-H3, BCMA, C4.4a, CA6, CA9, CanAg, CD123, CD138, CD142, CD166, CD184, CD19, CD20, CD205, CD22, CD248, CD25, CD3, CD30, CD33, CD352, CD37, CD38, CD40L, CD44v6, CD45, CD46, CD 48, CD51, CD56, CD7, CD70, CD71, CD74, CD79b, CDH6, CEACAM5, CEACAM6, cKIT, CLDN18.2, CLDN6, CLL-1, c-MET, Cripto, CSP-1, CXCR5, DLK-1, DLL3, DPEP3, antiadhesin, EFNA4, EGFR, EGFRviii, ENPP3, EpCAM, EphA2, EphA3, ETBR, FGFR2, FGFR3, FLT3, FOLR-α, FSH, GCC, GD2, GD3, Globo H, GPC-1, GPC3, gpNMB, HER-2, HER-3, HLA-DR, HSP90, IGF-1R, IL-13R, IL1RAP, IL7R, interleukin-4 receptor (IL4R), KAAG-1, LAMP-1, Lewis Y antigen, LGALS3BP, LGR5, LH / hCG, LHRH, LIV-1, LRP-1, LRRC15, Ly6E, MAGE, mesothelin (MSLN), MET, MHC cells harboring any one or more of class I chain-related proteins A and B (MICA and MICB), MT1-MMP, MTX3, MTX5, MUC1, MUC16, NaPi2b, Nectin-4, NOTCH3, OAcGD2, OX001L, p-cadherin, PD-L1, phosphatidylserine (PS), polymorphic epithelial mucin (PEM), prolactin receptor (PRLR), PSMA, PTK7, RNF43, ROR1, ROR2, SAIL, SLAMF7, SLC44A4, SLITRK6, SSTR2, STEAP-1, STING, STn, TIM-1, TM4SF1, TNF-α, TRA, TROP-2, tumor-associated glycoprotein 72 (TAG-72), a tumor-specific epitope of mucin-1 (TA-MUC1), CD5, TIM-3, UPK2, or UPK1b antigens.

[0606] In certain embodiments, the CBA is an antibody selected from the group consisting of an anti-CD37 antibody (e.g., as disclosed in USPN 8,765,917, the contents of which are incorporated herein by reference in their entirety), an anti-CD19 antibody (e.g., the huB4 antibody as disclosed in USPN 9,555,126, the contents of which are incorporated herein by reference in their entirety), and an anti-EGFR antibody (e.g., the huML66 antibody as disclosed in USPNs 9,238,690 8,790,649 and 9,125,896, the contents of which are incorporated herein by reference in their entirety).

[0607] In certain embodiments, the CBA is an anti-CD123 antibody or an antigen-binding fragment thereof, which may comprise: a) at least one light chain variable region or fragment thereof, each comprising three consecutive complementarity determining regions (CDRs); L 1. CDR L 2 and CDR L 3, including CDR L 1 has the amino acid sequence RASQDINSYLS (SEQ ID NO: 1), CDR L 2 has the amino acid sequence RVNRLVD (SEQ ID NO: 2), and CDR L 3 having the amino acid sequence LQYDAFPYT (SEQ ID NO: 3); and b) at least one heavy chain variable region or fragment thereof, each comprising three consecutive complementarity determining regions (CDRs) CDR H 1. CDR H 2 and CDR H 3, including CDR H 1 has the amino acid sequence SSIMH (SEQ ID NO: 4), CDR H 2 has the amino acid sequence YIKPYNDGTKYNEKFKG (SEQ ID NO: 5), and CDR H 3 has the amino acid sequence EGGNDYYDTMDY (SEQ ID NO: 6).

[0608] In certain embodiments, an anti-CD123 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V H ), which has the amino acid sequence

[0609]

[0610]

[0611] CDR H 1. CDR H 2 and CDR H 3 add double underline;

[0612] and light chain variable region (V L ), which has the amino acid sequence

[0613]

[0614] CDR L 1. CDR L 2 and CDR L 3 Add double underscore.

[0615] In certain embodiments, the anti-CD123 antibody has a heavy chain full-length sequence

[0616] CDR H 1. CDR H 2 and CDR H 3 add double underline;

[0617] and full-length light chain sequence

[0618]

[0619] CDR L 1. CDR L 2 and CDR L 3 Add double underscore.

[0620] In certain embodiments, anti-CD123 antibody or its Fab is provided as activatable antibody or activatable antibody binding antibody fragment as further described below.In certain other embodiments, described anti-CD123 activatable antibody or activatable CD123 antibody binding antibody fragment can be conjugated to formula I compound.

[0621] In certain embodiments, the CBA is an anti-CD123 antibody or antigen-binding fragment thereof as described in U.S. Patent Nos. 7,342,110 and 7,557,189, which are incorporated herein by reference.

[0622] In certain embodiments, the anti-CD33 antibody or antigen-binding fragment thereof may comprise: a) at least one light chain variable region or fragment thereof, each comprising three consecutive complementarity determining regions (CDRs); L 1. CDR L 2 and CDR L 3, including CDR L 1 has the amino acid sequence KSSQSVFFSSSQKNYLA (SEQ ID NO: 11), CDR L2 has the amino acid sequence WASTRES (SEQ ID NO: 12), and CDR L 3 having the amino acid sequence HQYLSSRT (SEQ ID NO: 13); and b) at least one heavy chain variable region or fragment thereof, each comprising three consecutive complementarity determining regions (CDRs) CDR H 1. CDR H 2 and CDR H 3, including CDR H 1 has the amino acid sequence SYYIH (SEQ ID NO: 14), CDR H 2 has the amino acid sequence VIYPGNDDISYNQKFQG (SEQ ID NO: 15), and CDR H 3 has the amino acid sequence EVRLRYFDV (SEQ ID NO: 16).

[0623] In certain embodiments, an anti-CD33 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V H ), which has the amino sequence

[0624]

[0625] CDR H 1. CDR H 2 and CDR H 3 add double underline;

[0626] and light chain variable region (V L ), which has the amino acid sequence

[0627]

[0628] CDR L 1. CDR L 2 and CDR L 3 Add double underscore.

[0629] In certain embodiments, the anti-CD33 antibody has a full-length heavy chain sequence

[0630]

[0631]

[0632] CDR H 1. CDR H 2 and CDR H 3 add double underline;

[0633] and full-length light chain sequence

[0634]

[0635] CDR L 1. CDR L 2 and CDR L 3 Add double underscore.

[0636] In certain embodiments, the anti-CD33 antibody is the huMy9-6 antibody.

[0637] In certain embodiments, the anti-CD33 antibody or antigen-binding fragment thereof is provided as an activatable antibody or activatable antibody-binding antibody fragment as further described below. In certain other embodiments, the anti-CD33 activatable antibody or activatable CD33 antibody-binding antibody fragment may be conjugated to a compound of Formula I.

[0638] In certain embodiments, the CBA is an anti-ADAM9 antibody or antigen-binding fragment thereof as described in WO2018 / 119196 and U.S. Provisional Application Nos. 62 / 690052 and 62 / 691342, each of which is incorporated herein by reference.

[0639] In certain embodiments, the anti-ADAM9 antibody or antigen-binding fragment thereof is a humanized anti-ADAM9 antibody or antigen-binding fragment thereof that specifically binds to human ADAM9 and cynomolgus monkey ADAM9.

[0640] In certain embodiments, the humanized anti-ADAM9 antibody or ADAM9-binding fragment thereof is optimized to have at least a 100-fold enhanced binding affinity for cynomolgus monkey ADAM9 while retaining high affinity binding to human ADAM9 compared to the chimeric or murine parent antibody.

[0641] In certain embodiments, an anti-ADAM9 antibody or antigen-binding fragment thereof (e.g., a humanized anti-ADAM9 antibody or antigen-binding fragment thereof) comprises: a) at least one light chain variable region or fragment thereof, each comprising three consecutive complementarity determining regions (CDRs); L 1. CDR L 2 and CDR L 3, including CDR L 1 has the amino acid sequence KASQSVDYSGDSYMN (SEQ ID NO: 21), CDR L 2 has the amino acid sequence AASDLES (SEQ ID NO: 22), and CDR L3 having the amino acid sequence QQSHEDPFT (SEQ ID NO: 23); and b) at least one heavy chain variable region or fragment thereof, each comprising three consecutive complementarity determining regions (CDRs) CDR H 1. CDR H 2 and CDR H 3, including CDR H 1 has the amino acid sequence SYWMH (SEQ ID NO: 24), CDR H 2 has the amino acid sequence EIIPIFGHTNYNEKFKS (SEQ ID NO: 25), and CDR H 3 has the amino acid sequence GGYYYYPRQGFLDY (SEQ ID NO: 26).

[0642] In certain embodiments, an anti-ADAM9 antibody or antigen-binding fragment thereof (e.g., a humanized anti-ADAM9 antibody or antigen-binding fragment thereof) comprises a heavy chain variable region (V H ), which has the amino sequence

[0643] CDR H 1. CDR H 2 and CDR H； 3 add double underline;

[0644] and light chain variable region (V L ), which has the amino acid sequence

[0645]

[0646] CDR L 1. CDR L 2 and CDR L 3 Add double underscore.

[0647] In certain embodiments, the anti-ADAM9 antibody has a heavy chain full-length sequence

[0648]

[0649] CDR H 1. CDR H 2 and CDR H 3 add double underline;

[0650] and full-length light chain sequence

[0651]

[0652] CDR L 1. CDR L 2 and CDR L 3 Add double underscore.

[0653] In certain embodiments, the anti-ADAM9 antibody or antigen-binding fragment thereof is provided as an activatable antibody or activatable antibody-binding antibody fragment as further described below. In certain other embodiments, the anti-ADAM9 activatable antibody or activatable ADAM9 antibody-binding antibody fragment can be conjugated to a compound of Formula I.

[0654] In certain embodiments, the CBA is an anti-folate receptor antibody (ie, FOLR1 or FRα) (eg, as described in US Patent No. 8,709,432, US Patent No. 8,557,966, and WO2011106528, each of which is incorporated herein by reference).

[0655] In certain embodiments, an anti-FRα antibody or antigen-binding fragment thereof may comprise: a) at least one light chain variable region or fragment thereof, each comprising three consecutive complementarity determining regions (CDRs); L 1. CDR L 2 and CDR L 3, including CDR L 1 has the amino acid sequence KASQSVSFAGTSLMH (SEQ ID NO: 31), CDR L 2 has the amino acid sequence RASNLEA (SEQ ID NO: 32), and CDR L 3 having the amino acid sequence QQSREYPYT (SEQ ID NO: 33); and b) at least one heavy chain variable region or fragment thereof, each comprising three consecutive complementarity determining regions (CDRs) CDR H 1. CDR H 2 and CDR H 3, including CDR H 1 has the amino acid sequence GYFMN (SEQ ID NO: 34) or GYTFTGYFMN (SEQ ID NO: 37), CDR H 2 has the amino acid sequence RIHPYDGDTFYNQKFQG (SEQ ID NO: 35) or RIHPYDGDTF (SEQ ID NO: 38), and CDR H 3 has the amino acid sequence YDGSRAMDY (SEQ ID NO: 36). In certain embodiments, the anti-FRα antibody or antigen-binding fragment thereof comprises a) a light chain variable region comprising a CDR having the amino acid sequence shown in SEQ ID NO: 31. L1. CDR having the amino acid sequence shown in SEQ ID NO: 32 L 2 and a CDR having the amino acid sequence shown in SEQ ID NO: 33 L 3; and b) a heavy chain variable region comprising a CDR having an amino acid sequence as shown in SEQ ID NO: 34 H 1. CDR having the amino acid sequence shown in SEQ ID NO: 35 H 2 and a CDR having the amino acid sequence shown in SEQ ID NO: 36 H 3. In certain embodiments, the anti-FRα antibody or antigen-binding fragment thereof comprises a) a light chain variable region comprising a CDR having the amino acid sequence shown in SEQ ID NO: 31. L 1. CDR having the amino acid sequence shown in SEQ ID NO: 32 L 2 and a CDR having the amino acid sequence shown in SEQ ID NO: 33 L 3; and b) a heavy chain variable region comprising a CDR having an amino acid sequence as shown in SEQ ID NO: 37 H 1. CDR having the amino acid sequence shown in SEQ ID NO: 38 H 2 and a CDR having the amino acid sequence shown in SEQ ID NO: 36 H 3.

[0656] In certain embodiments, an anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V H ), which has the amino sequence

[0657]

[0658] CDR H 1. CDR H 2 and CDR H 3 add double underline;

[0659] and light chain variable region (V L ), which has the amino acid sequence

[0660]

[0661] CDR L 1. CDR L 2 and CDR L 3 Add double underscore.

[0662] In certain embodiments, an anti-FRα antibody has a heavy chain full-length sequence

[0663]

[0664] CDR H 1. CDR H 2 and CDR H 3 add double underline;

[0665] and full-length light chain sequence

[0666]

[0667]

[0668] CDR L 1. CDR L 2 and CDR L 3 Add double underscore.

[0669] In certain embodiments, the anti-FRα antibody is huMov19 or M9346A antibody.

[0670] In certain embodiments, the anti-FRα antibody or antigen-binding fragment thereof is provided as an activatable antibody or activatable antibody-binding antibody fragment as further described below. In certain other embodiments, the anti-FRα activatable antibody or activatable FRα antibody-binding antibody fragment can be conjugated to a compound of Formula I.

[0671] In certain embodiments, the CBA is an anti-EpCAM antibody or an antigen-binding fragment thereof. In certain embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof may comprise: a) at least one light chain variable region or fragment thereof, each comprising three consecutive complementarity determining regions (CDRs); L 1. CDR L 2 and CDR L 3, including CDR L 1 has the amino acid sequence RSSRSLLHSDGFTYLY (SEQ ID NO: 45), CDR L 2 has the amino acid sequence QTSNLAS (SEQ ID NO: 46), and CDR L 3 having the amino acid sequence AQNLELPNT (SEQ ID NO: 47); and b) at least one heavy chain variable region or fragment thereof, each comprising three consecutive complementarity determining regions (CDRs) CDR H 1. CDR H 2 and CDR H 3, including CDR H 1 has the amino acid sequence NYYIH (SEQ ID NO: 48), CDR H2 has the amino acid sequence WIYPGNVYIQYNEKFKG (SEQ ID NO: 49), and CDR H 3 has the amino acid sequence DGPWFAY (SEQ ID NO: 50).

[0672] In certain embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region (V H ), which has the amino acid sequence

[0673]

[0674] CDR H 1. CDR H 2 and CDR H 3 add double underline;

[0675] and light chain variable region (V L ), which has the amino acid sequence

[0676]

[0677] CDR L 1. CDR L 2 and CDR L 3 Add double underscore.

[0678] In certain embodiments, the anti-EpCAM antibody has a heavy chain full-length sequence

[0679]

[0680] CDR H 1. CDR H 2 and CDR H 3 add double underline;

[0681] and full-length light chain sequence

[0682]

[0683] CDR L 1. CDR L 2 and CDR L 3 Add double underscore.

[0684] In certain embodiments, the CBA is an anti-EpCAM antibody or an antigen-binding fragment thereof, which may comprise: a) at least one light chain variable (VL) region or fragment thereof, each comprising three consecutive complementarity determining regions (CDRs); L1. CDR L 2 and CDR L 3, including CDR L 1 having an amino acid sequence selected from SEQ ID NOs: 78, 45, 79, 80, and 82; CDRs L 2 has the amino acid sequence SEQ ID NO: 46; and CDR L 3 having an amino acid sequence selected from the group consisting of SEQ ID NO: 47, 81, 83, 84, 85, 86 and 87, and b) at least one heavy chain variable (VH) region or fragment thereof, each comprising three consecutive complementarity determining regions (CDRs) CDRs H 1. CDR H 2 and CDR H 3, including CDR H 1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 57, 58, 60, 61, 62, 68, 69, 70, 77, and 88; CDRs H 2 has an amino acid sequence selected from SEQ ID NOs: 49, 56, 59, 63, and 64; and CDRs H 3 has an amino acid sequence selected from SEQ ID NO: 55, 65, 66, 67, 71, 72, 73, 74, 75 and 76.

[0685] In certain embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof is provided as an activatable antibody or activatable antibody-binding antibody fragment as further described below. In certain other embodiments, the anti-EpCAM activatable antibody or activatable EpCAM antibody-binding antibody fragment may be conjugated to a compound of Formula I.

[0686] The variants in the CDRs of the anti-EpCAM antibodies or antigen-binding fragments are summarized in Tables 3-6 below.

[0687] Table 3. huEpCAM-23 4.2 (CDRs and variable domains)

[0688]

[0689] Table 4. Humanized variants of huEpCAM-23 (full-length sequence)

[0690]

[0691] Table 5. EpCAM23 variant heavy chain CDR sequences

[0692]

[0693]

[0694]

[0695] Table 6. EpCAM23 variant light chain CDR sequences

[0696]

[0697]

[0698] In certain embodiments, the antibodies described herein are murine, non-human mammalian, chimeric, humanized, or human antibodies. For example, the humanized antibody may be a CDR-grafted antibody or a surface-reshaped antibody. In certain embodiments, the antibody is a full-length antibody. In certain embodiments, its antigen-binding fragment is Fab, Fab', F(ab')2, F d , single-chain Fab (scFab), single-chain Fv or scFv, disulfide-linked F v , V-NAR domain, IgNar, endosome, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2 or scFv-Fc.

[0699] In certain embodiments, the cell binding agent is an alternative protein scaffold, such as a peptide targeting the somatostatin receptor (see Barbieri F, Bajetto A, Pattarozzi A, Gatti M, Wurth R, Thellung S et al., Peptide receptor targeting in cancer: the somatostatin paradigm. Int J Pept 2013; 2013: 926295), an inhibitor of cystine knot (also known as ICK or Knottin; see Knottins: disulfide-bonded therapeutic and diagnostic peptides. Drug Discov Today Technol 2012; 9(1): e1-e70; Moore SJ, Cochran JR. Engineering knottins as novel binding agents. Methods Enzymol 2012; 503: 223-51), a diabody (see Labrijn AF, Meesters JI, Priem P, de Jong RN, van den Bremer ET, van Kampen MD et al., Controlled Fab-arm exchange for the generation of stable bispecific IgG1. Nat Protoc 2014;9(10):2450-63), hexabody (see de Jong RN, Beurskens FJ, Verploegen S, Strumane K, van Kampen MD, Voorhorst M et al., A Novel Platform for the Potentiation of TherapeuticAntibodies Based on Antigen-Dependent Formation of IgG Hexamers at the CellSurface.PLoS Biol2016;14(1):e1002344); single-chain Fab (scFab) fragment (see Koerber JT, Hornsby MJ, Wells JA. An improved single-chain Fab platform for efficient display and recombinant expression.J Mol Biol 2015; 427(2): 576-86; Hust M, Jostock T, Menzel C, Voedisch B, Mohr A, Brenneis M et al., Single chain Fab (scFab) fragment. BMC Biotechnol 2007; 7: 14), targets identified by drug affinity responsive target stability (DARTS) (see Pai MY, Lomenick B, Hwang H, Schiestl R, McBride W, Loo JA et al., Drugaffinity responsive target stability (DARTS) for small-molecule target identification. Methods in molecular biology 2015; 1263: 287-98), Centyrin (a protein scaffold based on the consensus sequence of fibronectin type III (FN3) repeats; see U.S. Patent Publication Nos. 2010 / 0255056, 2010 / 0216708, and 2011 / 0274623, incorporated herein by reference), ankyrin repeat proteins (e.g., designed ankyrin repeat proteins, termed DARPins; see U.S. Patent Publication Nos. 2004 / 0132028, 2009 / 0082274, 2011 / 0118146, and 2011 / 0224100, incorporated herein by reference, and see also C. Zahnd et al., Cancer Res. (2010) 70: 1595-1605; Zahnd et al., J. Biol. Chem. (2006) 281(46): 35167-35175; and Binz, HK, Amstutz, P. and Pluckthun, A., Nature Biotechnology (2005) 23: 1257-1268, incorporated herein by reference), ankyrin-like repeat proteins or synthetic peptides (see, e.g., U.S. Patent Publication No. 2007 / 0238667; U.S. Patent No. 7,101,675; WO 2007 / 147213; and WO 2007 / 062466, incorporated herein by reference), fibronectin (fibronectin domain scaffold protein; see U.S. Patent Publication Nos. 2007 / 0082365; 2008 / 0139791, incorporated herein by reference), knottin (small disulfide-rich protein characterized by disulfides linked by disulfide knots), bicyclic peptides (also known as bicyclic; see Heinis C, Rutherford T, Freund S, Winter G.Phage-encoded combinatorial chemical libraries based on bicyclicpeptides. Nat Chem Biol 2009; 5(7): 502-7; Teufel DP, Bennett G, Harrison H, van Rietschoten K, Pavan S, Stace C, et al., Stable and Long-Lasting, Novel BicyclicPeptide Plasma Kallikrein Inhibitors for the Treatment of Diabetic Macular Edema. J Med Chem 2018; 61(7): 2823-36), Avibodies (including bifunctional antibodies, trifunctional antibodies and tetrafunctional antibodies; see U.S. Publication Nos. 2008 / 0152586 and 2012 / 0171115), dual receptor retargeting (DART) molecules (PA Moore et al., Blood, 2011; 117(17): 4542-4551; Veri MC et al., Arthritis Rheum, 2010 Mar 30;62(7):1933-43; Johnson S et al., J Mol Biol, 2010 Apr 9;399(3):436-49) and cell-permeable supercharged proteins (Methods in Enzymol. 502, 293-319 (2012)).

[0700] Activated CBA

[0701] In additional embodiments, the CBA provided is an activatable antibody or activatable antigen-binding antibody fragment (collectively referred to as AA). In some embodiments, the activatable antibody or activatable antigen-binding antibody fragment comprises an antibody or antigen-binding antibody fragment (e.g., an antibody or antigen-binding antibody fragment described herein) that specifically binds to a ligand on a target cell (or "target"), which is coupled to a masking moiety (MM) such that the coupling of the MM reduces the ability of the antibody or antigen-binding antibody fragment to bind to the target. In some embodiments, the MM is coupled via a sequence comprising a substrate for a protease, such as a protease that is active in diseased tissue and / or a protease that is co-localized with the target at a treatment site in a subject. The activatable antibody is preferably stable in circulation, activated at the intended site of therapy and / or diagnosis but not in normal (e.g., healthy) tissue or other tissues not targeted for therapy and / or diagnosis, and when activated, exhibits binding to the target that is at least equivalent to that of the corresponding, unmodified antibody. In some embodiments, the AAs are those described in WO 2009 / 025846, WO 2010 / 081173, WO 2015 / 048329, WO 2015 / 116933, and WO 2016 / 118629, each of which is incorporated by reference in its entirety.

[0702] In some embodiments, the activatable antibody or antibody fragment comprises:

[0703] (a) a cleavable moiety (CM) coupled to the antibody or antibody fragment (collectively referred to as "AB"), wherein the CM is a polypeptide that serves as a protease substrate; and

[0704] (b) a masking moiety (MM) coupled to the antibody or antibody fragment, wherein the MM inhibits binding of the antibody or antibody fragment to a ligand when the activatable antibody is in an uncleaved state,

[0705] The activatable antibody in the uncleaved state has the following structural arrangement from N-terminus to C-terminus: (MM)-(CM)-(AB) or (AB)-(CM)-(MM).

[0706] In some embodiments, the masking moiety (or "mask") is an amino acid sequence that is coupled or otherwise attached to the antibody and positioned within the activatable antibody construct such that the masking moiety reduces the ability of the antibody to specifically bind to the target. Suitable masking moieties are identified using any of a variety of known techniques. For example, peptide masking moieties are identified using the methods described in WO 2009 / 025846, the contents of which are incorporated herein by reference in their entirety.

[0707] K of MM-modified AB against target d Comparable K of AB without MM modification or parental AB against target d at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 times or more, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-1,000,000, 10-10,000,000, 100-1,000,000, 100-1,000,000, 100-1,000,000, 100-1,000,000 , 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-10,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 100,000-1,000,000 or 100,000-10,000,000 times. Conversely, the binding affinity of an AB modified with an MM for a target may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 fold or more, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, or more, than the binding affinity of an AB not modified with an MM or a parental AB for a target. 10-10,000,000, 100-1,000, 100-10,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,000, 1,000-10,000, 1,000-10,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000, 100,000-1,000,000 or 100,000-10,000,000 times.

[0708] The dissociation constant (K) of MM against AB d ) is generally greater than the K of AB against the target dMM against AB's K d Comparable AB K against target d AB. MMs can have a binding affinity that is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times greater than the binding affinity of AB for the target. Conversely, the binding affinity of MMs for AB is generally lower than the binding affinity of AB for the target. The binding affinity of MMs for AB can be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000, or even 10,000,000 times less than the binding affinity of AB for the target.

[0709] When the AB is modified with an MM and in the presence of the target, the specific binding of the AB to its target may be reduced or inhibited compared to the specific binding of the AB not modified with the MM to the target or the specific binding of the parent AB to the target. When measured in vivo or in a target displacement in vitro immunosorbent assay as described in WO 2010 / 081173, the ability of the AB to bind to the target when modified with a MM may be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer when compared to the binding of the AB not modified with the MM or the binding of the parental AB to the target.

[0710] The MM can inhibit binding of the AB to the target. The MM can bind to the antigen binding domain of the AB and inhibit binding of the AB to its target. The MM can sterically inhibit binding of the AB to the target. The MM can allosterically inhibit binding of the AB to its target. In these embodiments, when the AB is modified with or conjugated to the MM and in the presence of the target, the AB does not bind or does not substantially bind to the target, or there is only .001%, .01%, .1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, %, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 50% combined for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer.

[0711] When an AB is conjugated to or modified by an MM, the MM may 'mask' or reduce or inhibit the specific binding of the AB to its target. When an AB is conjugated to or modified by an MM, the conjugation or modification may effect a structural change that reduces or inhibits the ability of the AB to specifically bind to its target.

[0712] AB coupled to or modified with MM can be represented by the following formulas (in order from the amino (N) terminal region to the carboxyl (C) terminal region):

[0713] (MM)-(AB)

[0714] (AB)-(MM)

[0715] (MM)-L-(AB)

[0716] (AB)-L-(MM)

[0717] wherein MM is a masking moiety, Ab is an antibody or target binding antigen fragment, and L is a linker. In various embodiments, it may be desirable to insert one or more linkers (eg, flexible linkers) into the composition to provide flexibility.

[0718] In certain embodiments, the MM is not a natural binding partner of the Ab. In some embodiments, the MM contains no or substantially no homology to any natural binding partner of the Ab. In some embodiments, the MM is only 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to any natural binding partner of the Ab. In some embodiments, the MM is only 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding partner of the Ab. In some embodiments, the MM is only 25% identical to any natural binding partner of the Ab. In some embodiments, the MM is only 50% identical to any natural binding partner of the Ab. In some embodiments, the MM is only 20% identical to any natural binding partner of Ab. In some embodiments, the MM is only 10% identical to any natural binding partner of Ab.

[0719] The activatable antibodies provided herein may also include a cleavable moiety (CM). Such AA exhibits activatable / convertible binding to the target of the AB. AA generally includes an antibody or antibody fragment (AB) that is modified or coupled to a masking moiety (MM) and a modifiable or cleavable moiety (CM) by a masking moiety (MM) and a modifiable or cleavable moiety (CM). In some embodiments, the CM contains an amino acid sequence that serves as a target protease substrate. In other embodiments, the CM provides a cysteine-cysteine ​​disulfide bond that can be cleaved by reduction. In other embodiments, the CM provides a photolytic substrate that can be activated by photolysis.

[0720] In some embodiments, the cleavable moiety (or "substrate") comprises an amino acid sequence that is a substrate for a protease, typically an extracellular protease. Suitable substrates are identified using any of a variety of known techniques. For example, peptide substrates are identified using methods described in U.S. Patents Nos. 7,666,817 and 8,563,269; and WO 2014 / 026136, the contents of each of which are incorporated herein by reference in their entirety. (See also Boulware et al., Biotechnol Bioeng. 106(3):339-346(2010)). Optionally, the CM comprises a cysteine-cysteine ​​pair capable of forming a disulfide bond that can be cleaved by the action of a reducing agent. The CM is positioned such that when the CM is cleaved by a cleavage agent in the presence of a target (e.g., a protease substrate of the CM is cleaved by a protease and / or a cysteine-cysteine ​​disulfide bond is disrupted by reduction via exposure to a reducing agent), resulting in a cleaved state, the Ab binds to the target, and in the uncleaved state, in the presence of the target, the binding of the Ab to the target is inhibited by the MM. It should be noted that the amino acid sequence of the CM may overlap with or be included within the MM, such that all or a portion of the CM contributes to "masking" of the Ab when the activatable antibody is in the uncleaved conformation.

[0721] In some embodiments, the CM can be selected based on a protease that colocalizes with the desired target of the activatable antibody in the tissue. A variety of different conditions are known under which a target of interest is colocalized with a protease, wherein the substrate of the protease is known in the art. For example, the target tissue can be cancerous tissue, particularly cancerous tissue of a solid tumor. The literature has repeatedly reported that proteases with known substrates have increased levels in various cancers (e.g., solid tumors). See, for example, LaRocca et al., (2004) British J. of Cancer 90(7):1414-1421.

[0722] Exemplary substrates can include, but are not limited to, substrates that can be cleaved by one or more of the following enzymes: MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, plasmin, PSA, PSMA, cathepsin D, cathepsin K, cathepsin S, ADAM10, ADAM12, ADAMTS, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, caspase-12, caspase-13, caspase-14, and TACE.

[0723] In some embodiments, the CM is a polypeptide up to 15 amino acids in length.

[0724] In some embodiments, the CM is a polypeptide comprising a first cleavable moiety (CM1) that is a substrate for at least one matrix metalloproteinase (MMP) and a second cleavable moiety (CM2) that is a substrate for at least one serine protease (SP). In some embodiments, each of the CM1 substrate sequence and the CM2 substrate sequence of the CM1-CM2 substrate is independently a polypeptide of up to 15 amino acids in length.

[0725] In some embodiments, the CM is a substrate for legumin, plasmin, TMPRSS-3 / 4, MMP-9, MT1-MMP, cathepsin, caspase, human neutrophil elastase, β-secretase, uPA, or PSA.

[0726] K of Ab modified with MM and CM against targets d Comparable K of Ab not modified with MM and CM or parent Ab against target dat least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 times or more, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-1,000,000, 10-10,000,000, 100-1,000,000, 100-1,000,000, 100-1,000,000, 100-1,000,000 , 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-10,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 100,000-1,000,000 or 100,000-10,000,000 times. In contrast, the binding affinity of an Ab modified with MM and CM for a target may be at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, or more, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, or more, than the binding affinity of an Ab not modified with MM and CM or a parent Ab for a target. 00, 10-10,000,000, 100-1,000, 100-10,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,000, 1,000-10,000,000, 1,000-10,000,000, 10,000-10,000,000, 10,000-10,000,000, 10,000-1,000,000, 100,000-1,000,000 or 100,000-10,000,000 times.

[0727] When an Ab is modified with MM and CM and in the presence of the target but in the absence of a modifying agent (e.g., an enzyme, a protease, a reducing agent, light), the specific binding of an Ab to its target can be reduced or inhibited, compared to the specific binding of an Ab not modified with MM and CM or a parent Ab to the target. The ability of the Ab to bind to its target when modified with MM and CM may be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer when compared to the binding of the parent Ab to its target or the binding of the Ab not modified with MM and CM to its target when measured in vivo or in a target shift in vitro immunosorbent assay as described in WO201008117 (incorporated herein by reference in its entirety).

[0728] As used herein, the term "cleaved state" refers to the state of an AA after protease modification of the CM and / or reduction and / or photoactivation of the cysteine-cysteine ​​disulfide bonds of the CM. As used herein, the term "uncleaved state" refers to the state of an AA in the absence of protease cleavage of the CM and / or reduction of the cysteine-cysteine ​​disulfide bonds of the CM and / or in the absence of light. As discussed above, the term "AA" is used herein to refer to an AA in its uncleaved (native) state as well as in its cleaved state. It should be apparent to one of ordinary skill that, in some embodiments, a cleaved AA may lack an MM due to protease cleavage of the CM resulting in the release of at least the MM (e.g., where the MM is not attached to the AA via a covalent bond (e.g., a disulfide bond between cysteine ​​residues)).

[0729] Activatable or convertible means that the AA exhibits a first level of binding to the target when in an inhibited, masked, or uncleaved state (i.e., a first conformation), and exhibits a second level of binding to the target in an uninhibited, unmasked, and / or cleaved state (i.e., a second conformation), wherein the second level of target binding is greater than the first level of binding. Generally, the target has greater access to the Ab of the AA in the presence of a cleavage agent capable of cleaving the CM than in the absence of such a cleavage agent. Thus, when the AA is in the uncleaved state, the Ab is inhibited from target binding and can be masked from target binding (i.e., the first conformation renders the Ab unable to bind the target), and in the cleaved state, the AB is not inhibited or masked from target binding.

[0730] The CM and AB of the AA can be selected such that the AB represents a binding moiety for the target of interest and the CM represents a substrate for a protease co-localized with the target at a treatment site in a subject. Alternatively or additionally, the CM is a cysteine-cysteine ​​disulfide bond that can be cleaved upon reduction of the disulfide bond. The AA contains at least one of a protease-cleavable CM or a cysteine-cysteine ​​disulfide bond, and in some embodiments, includes both types of CM. The AA may alternatively or further include a photolabile substrate that can be activated by a light source. The AAs disclosed herein find particular use where, for example, a protease capable of cleaving a site in the CM is present at relatively higher levels in target-containing tissue at the treatment site (e.g., diseased tissue; e.g., for therapeutic or diagnostic treatments) compared to tissue at a non-treatment site (e.g., healthy tissue). The AAs disclosed herein also find particular use where, for example, a reducing agent capable of reducing a site in the CM is present at relatively higher levels in target-containing tissue at the treatment or diagnostic site compared to tissue at a non-treatment, non-diagnostic site. The AAs disclosed herein also find particular use where, for example, a light source (eg, by means of a laser) capable of photolyzing sites in the CM is introduced to target-containing tissue at the site of treatment or diagnosis.

[0731] In some embodiments, the AAs may provide reduced toxicity and / or adverse side effects that might otherwise result from Ab binding at non-therapeutic sites if the Ab were not masked or otherwise inhibited from binding its target. In cases where the AAs contain a CM that is cleavable by a reducing agent that promotes disulfide bond reduction, ABs of such AAs may be selected to study activation of the Ab in the presence of the target of interest at a desired therapeutic site, characterized by elevated reducing agent levels such that the environment has a higher reducing potential than, for example, the environment at a non-therapeutic site.

[0732] In general, an AA can be designed by selecting an Ab of interest and constructing the remainder of the AA such that the MM provides masking of the Ab or reduced binding of the Ab to its target when conformationally constrained. Structural design criteria should consider providing this functional feature.

[0733] A Aa that exhibits a switchable phenotype for target binding in an inhibited conformation relative to an uninhibited conformation is provided. Dynamic range generally refers to the ratio of (a) the maximum detection level of a parameter under a first set of conditions to (b) the minimum detection value of the parameter under a second set of conditions. For example, in the case of AA, dynamic range refers to the ratio of (a) the maximum detection level of the target protein bound to the AA in the presence of a protease capable of cleaving the CM of the AA to (b) the minimum detection level of the target protein bound to the AA in the absence of the protease. The dynamic range of an AA can be calculated as the ratio of the equilibrium dissociation constant for treatment with an AA cleavage agent (e.g., an enzyme) to the equilibrium dissociation constant for treatment with the AA cleavage agent. The greater the dynamic range of the AA, the better the switchable phenotype of the AA. Aas with relatively high dynamic range values ​​(e.g., greater than 1) exhibit a more desirable switchable phenotype such that target protein binding of the AA occurs to a greater extent (e.g., predominantly) in the presence of a cleavage agent (e.g., an enzyme) capable of cleaving the CM of the AA compared to in the absence of the cleavage agent.

[0734] AA can be provided in a variety of structural configurations. Exemplary formulas for AA are provided below. It is specifically contemplated that the N-terminal to C-terminal order of AB, MM, and CM can be reversed within an AA. It is also specifically contemplated that the amino acid sequences of CM and MM can overlap, for example, such that the CM is contained within the MM.

[0735] For example, AA can be represented by the following formula (in order from the amino (N) terminal region to the carboxyl (C) terminal region):

[0736] (MM)-(CM)-(AB)

[0737] (AB)-(CM)-(MM), wherein MM is a masking moiety, CM is a cleavable moiety, and AB is an antibody or fragment thereof. It should be noted that although MM and CM are indicated as different components in the above formula, in all exemplary embodiments disclosed herein (including the various formulas), it is contemplated that the amino acid sequences of MM and CM may overlap, for example, such that CM is completely or partially contained within MM. In addition, the above formulas provide additional amino acid sequences that can be located at the N-terminus or C-terminus of the AA element.

[0738] In certain embodiments, it may be desirable to insert one or more linkers (e.g., flexible linkers) into the AA construct to provide flexibility at one or more of the MM-CM junction, the CM-AB junction, or both. For example, the AB, MM, and / or CM may not contain a sufficient number of residues (e.g., G1y, Ser, Asp, Asn, particularly G1y and Ser, and especially G1y) to provide the required flexibility. Thus, the switchable phenotype of such AA constructs may benefit from the introduction of one or more amino acids to provide a flexible linker. Additionally, as described below, where the AA is provided as a conformationally constrained construct, a flexible linker may be operatively inserted to facilitate the formation and maintenance of a ring structure in the uncleaved AA.

[0739] For example, in certain embodiments, AA comprises one of the following formulae (wherein the following formulae represent amino acid sequences in either an N-terminal to C-terminal orientation or a C-terminal to N-terminal orientation):

[0740] (MM)-L1-(CM)-(AB)

[0741] (MM)-(CM)-L1-(AB)

[0742] (MM)-L1-(CM)-L2-(AB)

[0743] Ring [L1-(MM)-L2-(CM)-L3-(AB)]

[0744] wherein MM, CM, and AB are as defined above; wherein L1, L2, and L3 are each independently and optionally present or absent, the same or different flexible linkers comprising at least one flexible amino acid (e.g., Gly); and wherein, when a ring is present, the AA is in the form of a cyclic structure due to the presence of a disulfide bond between a pair of cysteines in the AA. Additionally, the above formulas provide additional amino acid sequences that can be positioned at the N-terminus or C-terminus of the AA element. It will be understood that in the formula ring [L1-(MM)-L2-(CM)-L3-(AB)], the cysteines responsible for the disulfide bond can be positioned in the AA to allow for one or two tails, thereby creating a lasso or omega structure when the AA is in a disulfide-bonded structure (and thus conformationally constrained state). The amino acid sequence of the tail can provide additional AA characteristics, such as binding to a target receptor to facilitate localization of the AA, thereby increasing the serum half-life of the AA, and the like. A targeting moiety (e.g., a ligand for a receptor present on cells in the target tissue) and a serum half-life extending moiety (e.g., a polypeptide that binds a serum protein, such as an immunoglobulin (e.g., IgG) or serum albumin (e.g., human serum albumin (HSA))).

[0745] The linkers suitable for use with the AA described herein are generally linkers that provide flexibility to the modified AB or AA to promote inhibition of the binding of the AB to the target. Such linkers are generally referred to as flexible linkers. Suitable linkers can be easily selected and can have any suitable different lengths, such as 1 amino acid (e.g., 1 μg) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6 or 7 amino acids.

[0746] Exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers (including, for example, (GS) n 、(GSGGS) n and (GGGS) n , wherein n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and therefore may be able to serve as neutral tethers between components. Glycine approaches significantly more phi-psi spaces than even alanine and is less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers include, but are not limited to, Gly-Gly-Ser-Gly, Gly-Gly-Ser-Gly-Gly, Gly-Ser-Gly-Ser-Gly, Gly-Ser-Gly-Gly-Gly, Gly-Gly-Gly-Ser-Gly, Gly-Ser-Ser-Gly etc. One of ordinary skill will recognize that the design of an AA may include a fully or partially flexible joint, such that the joint may include a flexible joint and one or more portions that impart a less flexible structure to provide the desired AA structure.

[0747] In addition to the elements described above, the modified AB and AA may also contain additional elements, such as the N-terminus or C-terminus of the amino acid sequence of the AA. For example, the AA may include a targeting moiety to facilitate delivery to the target cell or tissue. Furthermore, the AA may be provided in the context of a scaffold protein to facilitate display of the AA on the cell surface.

[0748] In some embodiments, the activatable antibody further comprises a signal peptide. In some embodiments, the signal peptide is bound to the activatable antibody via a spacer. In some embodiments, the spacer is conjugated to the activatable antibody in the absence of a signal peptide. In some embodiments, the spacer is directly attached to the MM of the activatable antibody. In some embodiments, in a structural arrangement having spacer-MM-CM-AB from the N-terminus to the C-terminus, the spacer is directly attached to the MM of the activatable antibody.

[0749] Suitable spacers and spacer technology are known in the art and can be routinely used to incorporate spacers in some embodiments of the provided activatable antibodies. See, for example, WO 2016 / 179285 (e.g., at pages 52-53), the contents of which are incorporated herein by reference in their entirety.

[0750] In some embodiments, the activatable antibody is exposed to and cleaved by a protease such that, in the activated or cleaved state, the activatable antibody comprises a light chain sequence that includes at least a portion of the LP2 and / or CM sequence after the protease has cleaved the CM.

[0751] The CM is composed of at least one protease at a concentration of about 0.001-1500×10 4 M -1 S -1 or at least 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or 1500×10 4 M -1 S -1 In some embodiments, the CM is specifically cleaved at a rate of about 100,000 M -1 S -1 In some embodiments, the CM is specifically cleaved at a rate of about 1×10 2 to about 1×10 6 M -1 S -1 (i.e., approximately 1×10 2 to about 1×10 6 M -1 S -1) specifically cleaves at a rate of 100%. For specific cleavage achieved by an enzyme, contact is established between the enzyme and the CM. When an activatable antibody comprising an Ab (e.g., an antibody or EpCAM-binding antibody fragment) conjugated to both the MM and the CM is present in the presence of a target and sufficient enzymatic activity, the CM can be cleaved. Sufficient enzymatic activity can refer to the ability of the enzyme to contact the CM and achieve cleavage. It is readily conceivable that an enzyme could be in proximity to the CM but unable to cleave due to modification by other cellular factors or proteins.

[0752] Generation of cell-binding agent-drug conjugates

[0753] In order to link any of the cytotoxic compounds described herein or their derivatives to a cell-binding agent, the cytotoxic compound may comprise a linking moiety having a reactive group bonded thereto. These compounds may be directly linked to the cell-binding agent. Representative processes for linking a cytotoxic compound having a reactive group bonded thereto to a cell-binding agent to produce a cell-binding agent-cytotoxic agent conjugate are described in the Examples.

[0754] In some embodiments, a bifunctional cross-linking agent can first react with the cytotoxic compound to provide a compound with a linking moiety having one reactive group bonded thereto (i.e., a drug-linker compound), which can then react with a cell-binding agent. Alternatively, one end of the bifunctional cross-linking reagent can first react with a cell-binding agent to provide a cell-binding agent with a linking moiety having one reactive group bonded thereto, which can then react with the cytotoxic compound. The linking moiety can contain a chemical bond that allows release of the cytotoxic moiety at a specific site. Suitable chemical bonds are well known in the art and include disulfide bonds, thioether bonds, acid-labile bonds, photolabile bonds, peptidase-labile bonds, and esterase-labile bonds (see, e.g., U.S. Patents 5,208,020; 5,475,092; 6,441,163; 6,716,821; 6,913,748; 7,276,497; 7,276,499; 7,368,565; 7,388,026 and 7,414,073). Disulfide bonds, thioether bonds, and peptidase-labile bonds are preferred. Other linkers useful in the present invention include non-cleavable linkers, such as those described in detail in U.S. Publication No. 2005 / 0169933, or the charged linkers or hydrophilic linkers described in US 2009 / 0274713, US 2010 / 01293140, and WO 2009 / 134976, each of which is expressly incorporated herein by reference.

[0755] In some embodiments, a solution of a cell-binding agent (e.g., an antibody) in an aqueous buffer can be incubated with a molar excess of a bifunctional cross-linking agent such as N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB), or N-succinimidyl-4-(2-pyridyldithio)2-sulfobutyrate (sulfo-SPDB) to introduce dithiopyridyl groups. The modified cell-binding agent (e.g., a modified antibody) is then reacted with a thiol-containing cytotoxic compound described herein to produce a disulfide-linked cell-binding agent-cytotoxic agent conjugate of the invention.

[0756] In another embodiment, the thiol-containing cytotoxic compounds described herein can be reacted with a bifunctional cross-linking agent such as N-succinimidyl-4-(2-pyridyldithio) pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio) butyrate (SPDB), N-succinimidyl-4-(2-pyridyldithio) 2-sulfobutyrate (sulfo-SPDB) to form a cytotoxic agent-linker compound, which can then be reacted with a cell-binding agent to produce a disulfide-linked cell-binding agent-cytotoxic agent conjugate of the present invention. The cytotoxic agent-linker compound can be prepared in situ without purification and then reacted with the cell-binding agent. Alternatively, the cytotoxic agent-linker compound can be purified prior to reaction with the cell-binding agent.

[0757] The cell-binding agent-cytotoxic agent conjugate can be purified using any purification method known in the art, such as those described in U.S. Patent No. 7,811,572 and U.S. Publication No. 2006 / 0182750 (both incorporated herein by reference). For example, the cell-binding agent-cytotoxic agent conjugate can be purified using tangential flow filtration, adsorption chromatography, adsorption filtration, selective precipitation, non-absorptive filtration, or a combination thereof. Preferably, tangential flow filtration (TFF, also known as cross-flow filtration, ultrafiltration, and diafiltration) and / or adsorption chromatography resins are used for purification of the conjugate.

[0758] The number of cytotoxic molecules bound by each antibody molecule can be determined spectrophotometrically by measuring the ratio of absorbance at 280nm and 330nm. In some embodiments, an average of 1-10 cytotoxic compounds / antibody molecules can be connected by the above method. In some embodiments, the average number of cytotoxic compounds connected per antibody molecule (DAR) is 2-12. In some embodiments, the DAR value is 2-10. In some embodiments, the DAR value is 2-8. In some embodiments, the DAR value is 2.5-4.0. In some embodiments, the DAR value is 4-8. In some embodiments, the DAR value is 5-8. In some embodiments, the DAR value is 6-8. In some embodiments, the DAR value is 6.5-8. In some embodiments, the DAR value is 7-8. In some embodiments, the DAR value is 7.1-8. In some embodiments, the DAR value is 7.2-8. In some embodiments, the DAR value is 7.3-8. In some embodiments, the DAR value is 7.4-8. In some embodiments, the DAR value is 7.5-8. In some embodiments, the DAR value is 7.6-8. In some embodiments, the DAR value is 7.7-8. In some embodiments, the DAR value is 7.8-8. In some embodiments, the DAR value is 7.9-8. In some embodiments, the composition (e.g., pharmaceutical composition) comprising the conjugate of the present invention has a DAR value between 2 and 12, between 2 and 10, more specifically between 6 and 8. In some embodiments, the average number of cytotoxic compounds attached per antibody molecule (DAR) is 7-8, and more specifically 8.

[0759] In some embodiments, when the antibody is connected to a cytotoxic agent via a cysteine ​​thiol group, the conjugate has 1 cytotoxic compound per antibody molecule. In some embodiments, the conjugate has 1 or 2 cytotoxic compounds per antibody molecule. In some embodiments, the conjugate has 2 cytotoxic compounds per antibody molecule. In some embodiments, the average number of cytotoxic compounds connected per antibody molecule (DAR) is 1.5-2.5, more specifically 1.8-2.2. In some embodiments, the composition (e.g., pharmaceutical composition) comprising the conjugate of the present invention has a DAR value between 1.0 and 2.5, between 1.5 and 2.5, more specifically between 1.8 and 2.2 or between 1.9 and 2.1.

[0760] Representative processes for preparing the cell-binding agent-drug conjugates of the present invention are described in 8,765,740 and US Application Publication No. 2012 / 0238731. The entire teachings of these references are incorporated herein by reference.

[0761] Composition

[0762] The present invention includes compositions (eg, pharmaceutical compositions) comprising any one of the compounds described herein, derivatives thereof, or conjugates thereof (and / or solvates, hydrates, and / or salts thereof) and a carrier (a pharmaceutically acceptable carrier).

[0763] The pharmaceutical compositions described herein can be administered in a variety of ways for local or systemic treatment. Administration can be surface (e.g., to mucous membranes, including vaginal and rectal delivery), such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary (e.g., by inhalation or insufflation of a powder or aerosol, including by a nebulizer; intratracheal, intranasal, epidermal and transdermal); oral; or parenteral, including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial (e.g., intrathecal or intraventricular) administration. In some specific embodiments, the administration is intravenous. The pharmaceutical compositions described herein can also be used in vitro or ex vivo.

[0764] Suitable pharmaceutically acceptable carriers, diluents, and excipients are well known and can be determined by one skilled in the art based on the clinical situation. Examples of suitable carriers, diluents, and / or excipients include: (1) Dulbecco's phosphate buffered saline, approximately pH 7.4, with or without approximately 1 mg / mL to 25 mg / mL human serum albumin, (2) 0.9% saline (0.9% w / v NaCl), and (3) 5% (w / v) dextrose; and may also contain antioxidants such as tryptamine and stabilizers such as Tween 20.

[0765] How to use

[0766] The compositions of the present invention are useful for inhibiting abnormal cell growth or treating proliferative disorders in mammals (eg, humans).

[0767] The present invention includes a method of inhibiting abnormal cell growth or treating a proliferative disorder, autoimmune disorder, destructive bone disorder, infectious disease, viral disease, fibrotic disease, neurodegenerative disorder, pancreatitis or renal disease in a mammal (e.g., a human), comprising administering to the mammal a therapeutically effective amount of a cytotoxic compound, a derivative thereof, or a conjugate thereof (and / or a solvate and salt thereof), or a composition thereof, as described herein.

[0768] In certain embodiments, the proliferative disorder of a mammal is a cancer, including a hematological cancer, leukemia, or lymphoma. In certain embodiments, the proliferative disorder is a cancer of a lymphoid organ, or a hematological malignancy. In some embodiments, the cancer is a lymphoma or a leukemia.

[0769] For example, the cancer can be selected from the group consisting of acute myeloid leukemia (AML, including CD33 low AML, P-glycoprotein positive AML, relapsed AML or refractory AML), chronic myeloid leukemia (CML) (including blast crisis of CML and Abelson oncogene (Bcr-ABL translocation) associated with CML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) (including but not limited to acute B lymphoblastic leukemia or B cell acute lymphoblastic leukemia (B-ALL)), chronic lymphocytic leukemia (CLL) (including Richter syndrome or Richter CLL transformation), hairy cell leukemia (HCL), acute promyelocytic leukemia (APL), B-cell chronic lymphoproliferative disorder (B-CLPD), atypical chronic lymphocytic leukemia (preferably with expression of the marker CD11c), diffuse large B-cell lymphoma (DLBCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin lymphoma (NHL) (including mantle cell leukemia (MCL) and small lymphocytic lymphoma (SLL), Hodgkin lymphoma, systemic mastocytosis, and Burkitt lymphoma).

[0770] In some embodiments, the cancer is endometrial cancer, lung cancer, colorectal cancer, bladder cancer, gastric cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, esophageal cancer, breast cancer (e.g., triple-negative breast cancer (TNBC)), head and neck cancer, uterine cancer, ovarian cancer, liver cancer, cervical cancer, thyroid cancer, testicular cancer, bone marrow cancer, melanoma, and lymphoma. In some embodiments, the lung cancer is non-small cell lung cancer or small cell lung cancer. In other embodiments, the compounds of the present invention may be useful for treating non-small cell lung cancer (squamous cell, non-squamous cell, adenocarcinoma, or large cell undifferentiated carcinoma), colorectal cancer (adenocarcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, primary colorectal lymphoma, leiomyosarcoma, or squamous cell carcinoma), or breast cancer (e.g., triple-negative breast cancer (TNBC)).

[0771] The invention thus generally described will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the invention.

[0772] Example

[0773] The following abbreviations are used for the following terms:

[0774] DAR drug:antibody ratio;

[0775] DIPEA diisopropylethylamine;

[0776] DMF dimethylformamide;

[0777] DMSO dimethyl sulfoxide;

[0778] DMTMM 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride;

[0779] EDTA ethylenediaminetetraacetic acid;

[0780] EPPS 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid;

[0781] Fmoc 9-fluorenylmethoxycarbonyl;

[0782] HMPA hexamethylphosphoramide;

[0783] iv intravenous;

[0784] NMM N-methylmorpholine;

[0785] PPTS pyridinium p-toluenesulfonate;

[0786] SEC size exclusion chromatography;

[0787] SEM standard error of the mean;

[0788] TCEP 3,3′,3″-phosphinetriyltripropionate hydrochloride;

[0789] TEA triethylamine; and

[0790] TFA trifluoroacetic acid.

[0791] Figure 1 and Figure 2 The synthesis of camptothecin building blocks is depicted. Figure 3 、 Figure 4 and Figure 5 The synthesis of the side chain is depicted. Figure 6 and Figure 7 The coupling of the camptothecin building block to the side chain is depicted. Figure 8 The synthesis of additional camptothecin metabolites is depicted. Figure 9 The coupling of the camptothecin building block to the side chain is depicted. Figure 10 The synthesis of additional camptothecin compounds is depicted.

[0792] Figure 11Depicted are compounds used for comparison, including the generic Ab-999 structure of an ADC with a 999 moiety linked via a reduced interchain disulfide of the antibody. Compounds 998 and 999 were prepared as described in U.S. Patent 2016 / 0297890A1 and references therein.

[0793] Example 1. Synthesis of Compounds 2a-2e

[0794]

[0795] 2a: To a flask containing anhydrous 1,2-dichloroethane (80 mL) was added dichloromethane (16 mL, 16 mmol) containing 1 M boron trichloride, which was then cooled to 0 ° C with an ice-water bath. 1a (2.5 g, 20 mmol) was added in portions, followed by stirring at 0 ° C for 10 min, followed by addition of chloroacetonitrile (2.7 mL, 23.5 mmol), followed by addition of aluminum chloride (3.5 g, 26 mmol). The ice bath was removed and the reaction solution was gradually warmed to room temperature. After stirring at room temperature for 10 min, the reaction mixture was heated under reflux for 39 h. The reaction solution was cooled to room temperature and cold water (40 mL) was slowly added, followed by addition of 5% HCl aqueous solution. After 30 min, it was diluted with dichloromethane (80 mL). The organic layer was washed with water and brine, dried over anhydrous sodium sulfate and filtered. The filtrate was stripped under reduced pressure and the residue was purified by reverse phase HPLC (200 g C18 column, 80 mL / min, CH3CN / H2O, 25% CH3CN for 5 min, then 95% CH3CN over 22 min, then 95% CH3CN for 5 min) to afford compound 2a (1.52 g, 38% yield) as an off-white solid. MS (ESI): (M+H) + Calculated value 202.0, experimental value 202.2.

[0796]

[0797] 2b: AlCl3 (1.0 g, 7.7 mmol) and toluene (3.4 mL) were added to a 100 mL flask equipped with a magnetic stirrer. A solution of 1b (1.0 g, 6.4 mmol) in toluene (3.85 mL) was added to the flask under a nitrogen atmosphere, followed by the addition of toluene (3.85 mL, 7.6 mmol) containing 1 M BCl3. 2-Chloroacetonitrile (1.7 mL, 26.6 mmol) was added dropwise over approximately 2 min, and the flask was then equipped with a reflux condenser and heated in a 114 ° C oil bath for 3 h. The heating bath was removed and a thermometer was placed in the flask after 4 min. After cooling to 50 ° C, the reaction mixture was poured into deionized water (50 mL) and then extracted with ethyl acetate (2×60 mL). The organic layer was separated, dried over anhydrous Na2SO4, and then the solvent was removed by rotary evaporation under vacuum. The residue was dissolved in a minimum volume of ethyl acetate and then purified on an 80 g silica column eluted with 92:8 hexane:ethyl acetate. Fractions containing the pure desired product were combined and the solvent removed by rotary evaporation under vacuum to give 0.69 g of the desired product 2b as a yellow solid (46% yield). MS (M+H) 233.8; 1 H NMR (400 MHz, chloroform-d) δ 7.79 (d, J = 7.9 Hz, 1H), 6.40 (d, J = 11.0 Hz, 1H), 4.62 (s, 3H), 2.39 (s, 3H), 1.54 (s, 4H).

[0798]

[0799] 2e: To a solution of compound 2a (155 mg, 0.77 mmol) in anhydrous dichloromethane (1.5 mL) was added benzylamine (0.68 mL, 6.2 mmol) and the reaction mixture was stirred at room temperature for 6 hours. The reaction solution was stripped under reduced pressure and the residue was purified by silica gel chromatography (12 g silica column, CH2Cl2 / MeOH, 0-10% MeOH in 16 minutes) to give 156 mg of 2e (74% yield). MS (ESI): m / z 273.4 (M+H) + .

[0800]

[0801] 2c: To a flask containing anhydrous 1,2-dichloroethane (50 mL) was added dichloromethane (9.95 mL, 9.95 mmol) containing 1 M boron trichloride, which was then cooled to 0 ° C with an ice-water bath. 1a (1.56 g, 12.4 mmol) was added in portions, then stirred at 0 ° C for 10 minutes, 5-bromovaleronitrile (1.72 mL, 14.9 mmol) was added, followed by aluminum chloride (2.16 g, 16.2 mmol). The ice bath was removed and the reaction solution was gradually warmed to room temperature. After stirring at room temperature for 10 minutes, the reaction mixture was heated under reflux for 39.5 hours. The reaction solution was cooled to room temperature and cold water (25 mL) was slowly added, followed by a 5% aqueous HCl solution. After 30 minutes, it was diluted with dichloromethane (50 mL). The organic layer was washed with water and brine, dried over anhydrous sodium sulfate and filtered. The filtrate was stripped under reduced pressure and the residue was purified by reverse phase HPLC (100 g C18 column, CH3CN / H2O, 25% CH3CN for 5 minutes, then 95% CH3CN over 15 minutes, then 95% CH3CN for 5 minutes) to give compound 2c (1.42 g, 40% yield) as an off-white solid. MS (ESI): m / z 289.2 (M+H) + .

[0802]

[0803] 2d: Boron trichloride (4.73 mL, 4.73 mmol, 1 M in CH2Cl2) was added to a dry flask containing anhydrous 1,2-dichloroethane (40 mL) and cooled to 0 ° C with an ice-water bath. Compound 1b (0.93 g, 5.92 mmol) was added in portions and stirred at 0 ° C for 10 minutes, followed by 5-bromovaleronitrile (0.819 mL, 7.1 mmol) and aluminum chloride (1.025 g, 7.69 mmol). The ice bath was removed and the reaction solution was gradually warmed to room temperature. After stirring at room temperature for 10 minutes, the reaction mixture was heated under reflux for 45 hours. The reaction solution was cooled to room temperature and cold water (25 mL) was slowly added, followed by 5 mL of 5% HCl aqueous solution. After 30 minutes, it was diluted with dichloromethane (50 mL). The organic layer was washed with water and brine, dried over anhydrous sodium sulfate and filtered. The filtrate was stripped under reduced pressure and the residue was purified by reverse phase HPLC (100 g C18 column, CH3CN / H2O, 25% CH3CN for 3 minutes, then 95% CH3CN over 15 minutes, then 95% CH3CN for 5 minutes) to give compound 2d (0.776 g, 41% yield) as an off-white solid. MS (ESI): m / z 321.0 (M+H) + .

[0804] Example 2. General procedure for compounds 4a-4d

[0805]

[0806] 4b:Compound 3 (777mg, 2.95mmol), 2b (690mg, 2.95mmol) and PPTS (37mg, 0.15mmol) are suspended in a 50mL flask equipped with a reflux condenser containing anhydrous toluene (10mL). The reaction is heated to reflux overnight under magnetic stirring under an argon atmosphere and then allowed to cool to room temperature. The mixture is vacuum filtered and the solid is washed with toluene (5mL) to obtain 1.08g of the desired product 4b (79% yield) as a brown solid. MS (M+H) + 461, MS (MH) 459.

[0807]

[0808] 4a: Prepared by the general procedure, a solution of compound 2a (3.15 g, 15.64 mmol) and compound 3 (3.92 g, 14.89 mmol) in toluene (200 mL) containing PPTS (187 mg, 0.75 mmol) was refluxed for 40 hours to afford 4a (4.74 g) in 74% yield. MS (ESI): m / z 429.2.431.0 (M+H) + . 1 H NMR(400MHz, DMSO-d6)δ8.37(d,J=8.3,1.2Hz,1H),8.10-7.90(m,1H),7.33(d,J=2.1Hz,1H),6.55 (d,J=4.8Hz,1H),5.85-5.20(m,6H),2.61-2.45(m,3H),1.98-1.76(m,2H),0.89(t,J=7.3Hz,3H). HRMS(M+H) + Calculated value 429.1017, experimental value 429.1023.

[0809]

[0810] 4c: Following the general procedure, a solution of compound 2c (1.695 g, 5.88 mmol) and compound 3 (1.548 g, 5.88 mmol) in toluene (50 mL) containing PPTS (0.074 g, 0.294 mmol) was refluxed for 21.5 hours. The solvent was evaporated and the residue was dissolved in a minimum volume of 10% methanol in CHCl and then purified by silica gel chromatography (40 g silica column, CHCl / MeOH, 0-10% MeOH over 20 min) to give 4c (2.56 g) in 84% yield after silica gel chromatography (40 g silica column, CHCl / MeOH, 0-10% MeOH over 20 min). MS (ESI): m / z 515.2 and 517.1 (M+H) + ,513.1 and 515.1 (MH) - . 1 H NMR (400MHz, DMSO-d6) δ8.39-8.17(m,1H),7.89(d,J=10.9Hz,1H),7.31(s,1H),6.52(d,J=6.2Hz,1H),5.44(s,2H),5.31(s,2H ),3.65(t,J=6.7Hz,2H),3.28-3.09(m,2H),2.51(t,J=1.8Hz,3H),2.15-1.95(m,2H),1.95-1.71(m,4H),0.88(t,J=7.4Hz,3H). HRMS(M+H) + Calculated value 515.0981, experimental value 515.0992.

[0811]

[0812] 4d: Following the general procedure, a solution of compound 2d (776 mg, 2.42 mmol) and compound 3 (670 mg, 2.54 mmol) in toluene (20 mL) containing PPTS (30 mg, 0.12 mmol) was refluxed for 24 h to afford 4d (1.02 g) in 77% yield after silica gel chromatography (40 g silica column, CH2Cl2 / MeOH, 0-10% MeOH in 23 min). MS (ESI): m / z 547.0 and 548.9 (M+H) + ,544.8 and 546.8 (MH) - .

[0813]

[0814] 4e: To a solution of 6a (10 mg, 0.024 mmol) in DMF (0.5 mL) was added NMM (3 μL, 0.027 mmol) and 6-bromohexanoic acid (7.2 mg, 0.037 mmol) and the solution was cooled to 0°C with an ice bath and deionized water containing DMTMM (13.5 mg, 0.049 mmol) was added. The ice bath was removed and the reaction mixture was stirred at room temperature for 3 hours. The reaction solution was stripped under reduced pressure and the residue was purified by silica gel chromatography (4 g column, CH2Cl2 / MeOH, 0-10% MeOH in 15 minutes) to give 15 mg of 4e (yield 98%). MS (ESI): m / z 586.1 (M+H) + ,588.0(M+H) + .

[0815] Example 3. Synthesis of Compounds 8a-8e and 8p

[0816]

[0817] 8a: A solution of compound 4c (860 mg, 1,67 mmol) in HMPA (5 mL) and deionized water (0.9 mL) was heated at 101 ° C for 18 hours. The reaction mixture was cooled to room temperature and the solution was loaded on a C18 cartridge and purified by reverse phase HPLC (30 g C18 column, CH3CN / H2O, 25% CH3CN for 3 minutes, then reaching 95% CH3CN within 15 minutes, then 95% CH3CN for 5 minutes) to obtain 8a as a solid, which was contaminated by impurities. It was further purified by silica gel chromatography (CH2Cl2 / MeOH, 0-20% MeOH) to obtain 392 mg of 8a as an off-white solid (51% yield). MS (ESI): m / z 453.2 (M+H) + ,451.1(MH) - . 1 H NMR (400 MHz, methanol-d4) δ 8.00 (dd, J = 8.2, 1.1 Hz, 1H), 7.52-7.33 (m, 2H), 5.53 (d, J = 16.1 Hz, 1H), 5.33 (d, J = 16.1 Hz, 1H), 5.18-4.96 (m, 2H), 3.66 (q, J = 7.2, 6.7 Hz, 2H), 3.24-3.06 (m, 2H), 2.45 (dd, J = 20.1, 2.7 Hz, 3H), 1.94 (tdd, J = 7.1, 6.0, 2.0 Hz, 2H), 1.88-1.66 (m, 4H), 1.00 (td, J = 7.4, 2.5 Hz, 3H). 13C NMR (101 MHz, methanol-d4) δ 173.32, 163.68, 161.19, 157.56, 151.44, 151.09, 148.68, 148.56, 146.06, 144.46, 128.10, 127.89, 127.22, 125.66, 125.60, 124.26, 118.92, 111.91, 111.69, 97.96, 72.74, 65.28, 60.97, 49.40, 32.15, 30.80, 29.00, 25.96, 14.09, 14.06, 6.75; HRMS (M+H) + Calculated value 453.1826, experimental value 453.1832.

[0818]

[0819] 8b: Prepared from 4d using the procedure of 8a. Yield (48% yield). MS (ESI): m / z (M+H) + 485.7.

[0820]

[0821] 8c: Compound 4a (100 mg, 0.23 mmol) was dissolved in anhydrous DMF (2 mL), to which was added 2-mercaptoethanol (0.2 mL, 2.8 mmol), followed by TEA (0.13 mL, 0.94 mmol) and magnetic stirring for 20 min. The reaction mixture was injected onto a 250 g medium pressure C18 column, which was pre-equilibrated with 95:5 deionized water containing 0.2% formic acid:acetonitrile. The column was eluted with 95:5 deionized water containing 0.2% formic acid:acetonitrile at 50 mL / min for 5 min, followed by a linear gradient elution from 5% acetonitrile at 5 min to 95% acetonitrile at 35 min. The fractions containing the desired product were combined, frozen and lyophilized to give 85 mg of 8c (78% yield). MS (ESI): m / z 471.4 (M+H) + . 1H NMR (400 MHz, DMSO-d6) δ 8.25 (d, J = 8.2 Hz, 1H), 7.78 (d, J = 10.8 Hz, 1H), 7.27 (s, 1H), 6.52 (s, 1H), 5.42 (s, 2H), 5.22 (s, 2H), 4.87 (t, J = 5.4 Hz, 1H), 4.47-4.28 (m, 2H), 3.56 (q, J = 6.3 Hz, 2H), 3.33 (s, 2H), 2.62 (t, J = 6.6 Hz, 2H), 2.48-2.44 (m, 3H), 1.87 (septet, J = 7.1, Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H). 13 CNMR(101MHz,DMSO-d6)δ172.44,163.07,160.58,156.69,152.16,149.97,148.66,148.53,145.48,140.06,128.18,128.16,127.28,1 27.07,126.61,126.54,123.59,119.09,112.56,112.35,96.77,72.36,65.26,60.83,49.69,34.38,30.31,28.78,15.24,15.20,7.75. HRMS(M+H) + Calculated value 471.1390, experimental value 471.1393.

[0822] 8d: Prepared similarly to 8c by reaction of 1,3-propanedithiol with 4a (60% yield). MS (ESI): m / z 501.7 (M+H) +

[0823]

[0824] 8e: Prepared similarly to 8c by reaction of 1,2-ethanedithiol with 4a (47% yield). MS (ESI): (M+H) + Calculated value 487.1, experimental value 487.3.

[0825]

[0826] 8p:Compound 8e (28mg, 0.057mmol) is dissolved in anhydrous THF (1mL), and mineral oil (5mg, 2.1mmol) containing 60% NaH emulsion is added thereto under magnetic stirring.After 2min, iodoethane (20 μ L, 0.32mmol) is added.After 20min, solvent is evaporated under vacuum and residue is dissolved in anhydrous DMF (1mL), then by medium pressure C18 chromatography purification.Post 100gC18, 50mL / min, deionized water containing 0.2% formic acid, has 5% acetonitrile from 0-5min, then has 5%-95% linear gradient from 5min-28min.The elution fraction containing the desired product is merged, frozen and lyophilized to obtain 15mg 8p (54% yield) as a yellow solid. 1 H NMR(400MHz,DMSO-d6)δ8.40 -8.27(m,1H),7.89(d,J=10.8Hz,1H),7.31(s,1H),6.52(s,1H),5.43(s,2H),5.39(s,1H),5.32(s,1H),4.57-4.39 (m,2H),2.84-2.69(m,3H),2.44(m,1H),2.07(m,1H),2.02(s,3H),1.87(m,2H),1.23(s,1H),0.87(t,J=7.3Hz,3H). HRMS(M+H) + Calculated value 501.1318; experimental value 501.1322.

[0827] Example 4. Synthesis of Compound 5a

[0828]

[0829] 5a: A solution of 4a (2 g, 4.66 mmol) and sodium azide (0.455 g, 7.0 mmol) in 25 mL of anhydrous DMSO was stirred at room temperature for 6 h and then diluted with deionized water (200 mL). The product precipitated and was collected by vacuum filtration and then dried under vacuum to give 2.03 g of 5a (100% yield). MS (ESI): (M+H) + Calculated value 435.1, found value 436.2, MS (ESI): (MH) - Calculated value 433.1, experimental value 434.2.

[0830] Example 5. Synthesis of Compound 6a

[0831]

[0832] 6a:To a solution of 5a (2.03g, 4.66mmol) in anhydrous benzene (60mL), triethyl phosphite (1.94g, 11.66mmol) is added and the solution is flushed with argon, then heated under reflux for 4 hours. The reaction is cooled to room temperature and 3M methanol HCl (30mL) is added and heated under reflux (80 DEG C of baths) for 38h. The reaction is cooled to room temperature and then vacuum filtered to obtain 6a (1.016g) in the form of a pale solid. The filtrate is concentrated and the residue is purified by silica gel chromatography (12g silica columns, CH2Cl2 / MeOH, 0-20% MeOH in 16 minutes) to obtain additional 6a (0.079g). The combination 6a (57% yield) of 1.09g is obtained. 1 H NMR (400 MHz, DMSO-d6) δ 8.62 (s, 3H), 8.43 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 10.7 Hz, 1H), 7.35 (s, 1H), 5.59 (s, 2H), 5.45 (s, 2H), 4.70 (d, J = 6.0 Hz, 2H), 2.55 (s, 3H), 1.88 (septet, J = 7.1 Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H). MS (ESI): m / z calcd 410.2, found 410.4 (M+H). + ; 408.1(MH) - .

[0833] Example 6. Synthesis of Compound 7a

[0834]

[0835] 7a: To a solution of 6a (7.5 mg, 0.018 mmol) in DMF (0.5 mL) was added triethylamine (2.6 μL, 0.018 mml) and glycolic acid (1.5 mg, 0.020 mmol). The solution was cooled to 0°C with an ice bath and deionized water (0.1 mL) containing DMTMM (10.1 mg, 0.037 mmol) was added. The ice bath was removed and the reaction mixture was stirred at room temperature for 2 hours. The reaction solution was stripped under reduced pressure and the residue was purified by silica gel chromatography (4 g silica column, CH2Cl2 / MeOH, 0-20% MeOH in 15 minutes) to give the desired product 7a (8 mg, 93% yield). MS (ESI): m / z 468.2 (M+H) + ,466.1(MH) - . 1H NMR (400MHz, DMSO-d6) δ0.88(t,J=7.3Hz,3H),1.79-1.98(m,3H),2.55(s,0H),3.84(d,J=5.6Hz,3H),4.85(d,J=6.0Hz,2H),5.44(s,3H) ,5.51(s,2H),5.58(t,J=5.7Hz,1H),6.53(s,1H),7.32(s,1H),7.90(d,J=10.8Hz,1H),8.48(d,J=1.2,8.3Hz,1H),8.76(t,J=6.0Hz,1H). 13 C NMR (101MHz, DMSO-d6) δ172.95,172.89,157.23,152.85,150.44,149.07,145.85,140.12,129.27,127. 97,124.28,119.63,97.23,72.83,65.74,61.91,54.06,50.54,49.58,44.06,37.34,30.72,15.74,8.22. HRMS(M+H) + Calculated value 468.1571, experimental value 468.1593.

[0836] Example 7. Synthesis of Compound 6c

[0837]

[0838] 6c:Compound 2e (129mg, 0.47mmol), compound 3 (125mg, 0.47mmol) and PPTS (131mg, 0.52mmol) in anhydrous toluene (10mL) are briefly placed under vacuum and then heated under reflux for 20 hours. The reaction solution is cooled to room temperature and stripped and the residue is purified by silica gel chromatography (12g column, CH2Cl2 / MeOH, 0-20% MeOH in 16 minutes) to obtain 196mg of impure product in the form of a black solid. It is further purified by reverse phase HPLC (30gC18 column, CH3CN / H2O, 20%-60% CH3CN in 15 minutes, then 95% CH3CN for 5 minutes) to obtain 6c (31mg, 13% yield). MS (ESI): m / z 500.4 (M+H) + ,498.3(MH) - .

[0839] Example 8. Synthesis of Compound 7c

[0840]

[0841] 7c: To a solution of 6c (8.5 mg, 0.017 mmol) in DMF (0.5 mL) was added triethylamine (2.4 μL, 0.017 mml) and glycolic acid (1.9 mg, 0.026 mmol). The solution was cooled to 0°C with an ice bath and deionized water (0.1 mL) containing DMTMM (9.4 mg, 0.034 mmol) was added. The ice bath was removed and the reaction mixture was stirred at room temperature for 3 hours. The reaction solution was stripped under reduced pressure (35°C bath) and the residue was purified by semi-preparative HPLC (C18 column, CH3CN / H2O, 25%-65% CH3CN in 23 minutes) to give 2.5 mg 7c (26% yield). MS (ESI): m / z 558.5 (M+H) + ,556.3(MH) - .

[0842] Example 9. Synthesis of side chains

[0843]

[0844] 11: N-methyl-D-glucamine 9 (3.94 g, 20.19 mmol) and ZL-Glu-OBn 10 (5 g, 13.46 mmol) were weighed into a 200 mL flask, to which DMF (50 mL) was added and magnetically stirred. DIPEA (2.351 mL, 13.46 mmol) was then added, followed by a suspension of DMTMM (5.22 g, 18.85 mmol) in DMF (30 mL) and deionized water (10 mL). After 2 h, one-third of the reactants were loaded onto a 450 g C18 cartridge pre-equilibrated with 95:5 deionized water:acetonitrile. The column was eluted with 95:5 deionized water:acetonitrile at 100 mL / min for 5 min, followed by a linear gradient of 5% acetonitrile to 95% acetonitrile over 38 min. The chromatography was repeated twice more and the fractions containing the pure desired product were combined. The solvent was removed by rotary evaporation under vacuum. Methanol (100 mL) was added and evaporated twice to give 5 g of the desired product 11 (68% yield) as a viscous colorless oil. MS (ESI): MS (M+H) + Calculated value 549.2, found value 549.3, MS (ESI): (MH) - Calculated value 547.2, experimental value 547.2.

[0845]

[0846] 12: Compound 11 (5 g, 9.1 mmol) was suspended in 95:5 methanol: deionized water (100 mL) in a 250 mL PARR shaker flask, to which was added 10% Pd-C (0.13 g, 1.222 mmol) and hydrogenated at 30 PSI H2 for 45 min, with H2 added periodically to reestablish pressure. The solution was vacuum filtered through celite filter aid. The clear, colorless filtrate was concentrated by rotary evaporation under vacuum and then left under vacuum overnight to give 2.8 g of the desired product 12 as a white solid (94% yield). 1 H NMR (400MHz, DMSO-d6) δ4.09-3.98(m,1H),3.84(dt,J=6.1,3.1Hz,1H),3.79(d,J=6.0Hz,1H),3.75(ddd,J=7.5,4.2,2.3Hz,1H),3 .72-3.59(m,3H),3.56-3.51(m,1H),3.48(dd,J=15.0,3.6Hz,1H),3.13(s,2H),2.97(s,1H),2.78-2.49(m,2H),2.22-2.06(m,2H). MS(ESI):MS(MH) - Calculated value 323.1, experimental value 323.4.

[0847]

[0848] 14b: Compound 13b (1.4, 4.5 mmol) was added to a magnetically stirred solution of 12 (1.4 g, 4.3 mmol) in DMF (10 mL) and DIPEA (1.1 mL, 6.3 mmol). After stirring for 15 min, the reaction was injected onto a medium pressure 350 g C18 column pre-equilibrated with 98:2 deionized water containing 0.1% formic acid:acetonitrile. The column was eluted with 2% acetonitrile at 100 mL / min for 5 min, followed by a linear gradient elution from 2% acetonitrile at 5 min to 60% acetonitrile at 35 min, with detection at 214 and 306 nm. The fractions containing pure product were combined, frozen and lyophilized to give 1.3 g of the desired product 14b as a white solid (58% yield). MS (M+H)+ 518.5, 1H NMR (400MHz, DMSO-d6) δ8.03(dd,J=15.3,7.7Hz,1H),6.99(s,3H),4.79(d,J=65.9Hz,1H),4.33(d,J =30.0Hz,1H),4.22-4.07(m,1H),3.75(p,J=4.2Hz,1H),3.57(ddt,J=11.4,6.7,3.6Hz,1H),3.52-3.3 9(m,2H),3.39-3.30(m,3H),3.30-3.19(m,1H),2.97(s,2H),2.80(s,2H),2.45-2.21(m,1H),2.08(h ,J=3.4Hz,2H),2.01-1.85(m,1H),1.76(qd,J=8.5,3.9Hz,1H),1.59-1.39(m,3H),1.30-1.08(m,2H).

[0849]

[0850] 14a: Compound 13a (1.4, 5.3 mmol) was added to a magnetically stirred solution of 12 (1.8 g, 5.5 mmol) in DMF (10 mL) and DIPEA (1.1 mL, mmol). After stirring for 15 min, the reaction was injected onto a medium-pressure 350 g C18 column pre-equilibrated with 98:2 deionized water containing 0.1% formic acid:acetonitrile. The column was eluted with 2% acetonitrile at 100 mL / min for 5 min, followed by a linear gradient elution from 2% acetonitrile at 5 min to 60% acetonitrile at 35 min, with detection at 214 and 306 nm. The fractions containing pure product were combined, frozen, and lyophilized to afford 1.4 g of 14a as a white solid (55% yield). 1 H NMR(400MHz, DMSO-d6)δ8.33(dd,J=9.2,7.7Hz,1H),7.08(d,J=1.1Hz,2H),4.24(q d,J=8.8,4.8Hz,1H),3.86(dt,J=8.2,4.0Hz,1H),3.76-3.63(m,4H),3.62-3.43(m ,4H),3.41-3.29(m,1H),3.08(s,2H),2.91(s,2H),2.61(q,J=1.8Hz,1H),2.40(dd ,J=10.0,6.4Hz,1H),2.02(tdd,J=12.6,10.3,9.1,5.8Hz,1H),1.93-1.76(m,1H). MS(ESI):MS(M+H) + Calculated value 476.2, experimental value 476.4.

[0851] Fmoc-protected peptides 15a, 15b, 15c, and 15d were prepared by solid phase synthesis using standard procedures.

[0852] Example 10. Method for converting Fmoc-peptide-Gly-OH to Fmoc-peptide-NHCH2OAc (Compounds 16a-16d)

[0853]

[0854] 16a: Compound 15a (2.5 g, 4.9 mmol) was dissolved in anhydrous DMF (40 mL) and magnetically stirred in a 100 mL flask. Copper (II) acetate (0.334 g, 1.84 mmol), acetic acid (0.64 mL, 11.1 mmol) and lead tetraacetate (2.5 g, 5.6 mmol) were added. The flask was heated in a 60°C oil bath for 15 min. The oil bath was removed and the reaction was allowed to cool to room temperature. Approximately 1 / 2 of the mixture was purified on a 350 g medium pressure C18 column equilibrated with 90:10 deionized water containing 0.3% formic acid:acetonitrile. The column was eluted with 10% acetonitrile at 100 mL / min for 5 min, followed by a linear gradient elution from 10% acetonitrile over 5 min to 95% acetonitrile over 38 min. This procedure was repeated for another 1 / 2 of the reaction mixture and the fractions containing the desired product 16a were combined, frozen and lyophilized to give 1.2 g of a white semisolid (62% yield). 1 H NMR(400MHz,DMSO-d6)δ8.86(t,J=6.9Hz,1H),7.97(dd,J=16.1,7.4Hz,2H),7 .89(dt,J=7.6,0.9Hz,2H),7.72(t,J=7.1Hz,2H),7.53(d,J=7.5Hz,1H),7.42( td,J=7.5,1.2Hz,2H),7.33(td,J=7.5,1.2Hz,2H),5.13-5.01(m,2H),4.30-4. 17(m,4H),4.06(t,J=7.3Hz,1H),3.32(s,1H),1.99(s,3H),1.27-1.13(m,9H). MS (ESI): MS (M+Na) + Calculated value 547.2, experimental value 547.5.

[0855] 16b was prepared from Fmoc-Ala-Ala-Gly-OH 15b in 55% yield. MS (M+Na)+ 476.7.

[0856] 16c was prepared from Fmoc-Leu-Gln-Gly-OH 15c in 46% yield. MS (M+Na)+ 575.6.

[0857] 16d was prepared from Fmoc-Ala-D-Ala-Ala-Gly-OH 15d in 52% yield. MS (M+Na)+ 547.3.

[0858] Example 11. Method for reacting benzyl-2-hydroxyacetate with Fmoc-peptide-NHCH2OAc (Compounds 17a-17d)

[0859]

[0860] 17a: Compound 16a (142 mg, 0.27 mmol) and benzyl-2-hydroxyacetate (226 mg, 1.36 mmol) were suspended in a solution of 20% TFA in dichloromethane (7 mL) and magnetically stirred at room temperature for 30 min. The solvent was rotary evaporated under vacuum and the residue was dissolved in a minimum volume of DMF and then purified on a 200 g C18 medium pressure column pre-equilibrated with 90:10 deionized water containing 0.1% formic acid:acetonitrile. The column was then eluted with 10% acetonitrile at 60 mL / min for 5 min, followed by a linear gradient elution from 10% acetonitrile at 5 min to 95% acetonitrile at 38 min. The fractions containing the desired product were combined, frozen and lyophilized to give 102 mg of 17a as a white solid (59% yield). MS (ESI): MS (M+Na) + Calculated value 653.3, experimental value 653.5.

[0861] 17b was prepared from 16b in 61% yield. MS (M+Na)+ 582.7.

[0862] 17c was prepared from 16c in 52% yield. MS (M+H) + 659.5.

[0863] 17d was prepared from 16d in 56% yield. MS (M+Na)+ 653.4.

[0864] Example 12. Conversion of Fmoc-peptide-NHCH2OCH2COOBn to H-peptide-NHCH2OCH2COOBn (Compounds 18a-18d)

[0865]

[0866] 18a: Compound 17a (100 mg, 0.16 mmol) was dissolved in DMF (4 mL), morpholine (0.6 mL, 6.9 mmol) was added thereto and stirred magnetically. After 1 h, the reaction mixture was purified on a 200 g C18 medium pressure column pre-equilibrated with 95:5 deionized water containing 0.1% formic acid:acetonitrile. The column was then eluted with 5% acetonitrile at 60 mL / min for 5 min, followed by a linear gradient elution from 5% acetonitrile at 5 min to 70% acetonitrile at 38 min. The fractions containing the desired product were combined, frozen and lyophilized to give 50 mg of 18a as a white solid (76% yield). MS (ESI): MS (M+H) + Calculated value 409.2, experimental value 409.6.

[0867] 18b was prepared from 17b in 80% yield. MS (M+H) + 338.3.

[0868] 18c was prepared from 17c in 61% yield. MS (M+H) + 437.6.

[0869] 18d was prepared from 17d in 61% yield. MS (M+H) + 409.6.

[0870] Example 13. Conversion of H-peptide-NHCH2OCH2COOBn to H-peptide-NHCH2OCH2COOH (Compounds 19a-19d)

[0871]

[0872] 19a: In a 100 mL PARR shaker flask, H-Ala-Ala-Ala-NHCHOCHCOOBn 18a (50 mg, 0.12 mmol) was dissolved in 5:95 deionized water: methanol (50 mL), 10% palladium on carbon (0.1 g) was added and the reaction was hydrogenated in a PARR shaker under 30 PSI H for 1 h. The solution was vacuum filtered through celite filter aid and the solvent was removed from the filtrate by rotary evaporation under vacuum to give 35 mg of the desired product 19a as a viscous oil (91% yield). MS (ESI): MS (M+H) + Calculated value 319.2, experimental value 319.3.

[0873] 19b was prepared from 18b in 92% yield. MS (M+H) + 248.3.

[0874] 19c was prepared from 18c in 83% yield. MS (M+H) + 347.4.

[0875] 19d was prepared from 18d in 81% yield. MS (M+H) + 319.5.

[0876] Example 14. Method for preparing Mal-(CH2)5-CO-peptide NHCH2OCH2COOH compounds (Compounds 20a-20d)

[0877]

[0878] 20a: Mal-(CH2)5-COONHS13b (46 mg, 0.15 mmol) was dissolved in anhydrous DMF (2 mL) to which was added DIPEA (0.1 mL, 0.31 mmol) and H-Ala-Ala-Ala-NHC2OCH2COOH 19a (30 mg, 0.093 mmol). The reaction was stirred magnetically for 15 min and then purified on a 50 g C18 medium pressure column pre-equilibrated with 95:5 deionized water containing 0.1% formic acid:acetonitrile. The column was then eluted with 5% acetonitrile at 40 mL / min for 5 min, followed by a linear gradient of 5% acetonitrile over 5 min to 90% acetonitrile over 38 min. The fractions containing the desired product were combined, frozen, and lyophilized to afford 30 mg of 20a as a white solid (63% yield). MS (ESI): MS (MH) - Calculated value 510.2, experimental value 510.1. 1 H NMR(400MHz,DMSO-d6)δ12.16(s,1H),8.64-8.48(m,1H),7.97(dd,J=7.3,3 .2Hz,2H),7.89(d,J=7.5Hz,1H),7.00(s,2H),4.3-4.7(m,4H),4.21(dt,J= 10.3,7.2Hz,4H),3.55(t,J=6.4Hz,2H),3.37(t,J=7.1Hz,2H),2.41(t,J=6 .4Hz, 2H), 2.08 (t, J = 7.4Hz, 2H), 1.47 (p, J = 7.2Hz, 4H), 1.27-1.10 (m, 6H).

[0879] 20b was prepared from 19b in 83% yield. MS (M+H) + 441.4.

[0880] 20c was prepared from 19c in 83% yield. MS (MH) - 655.4.

[0881]

[0882] 20d:To a solution of compound 18d (99.7 mg, 0.24 mmol) in methanol (3 mL), Pd (10% / carbon, 26 mg, 0.024 mmol) was added and the reaction flask was purged with hydrogen. It was hydrogenated with a hydrogen balloon at room temperature for 3 hours and then filtered. The filtrate was stripped to give compound 19d (78 mg, 100% yield) as a white solid. 41.9 mg (0.13 mmol) was taken out and dissolved in anhydrous DMF (0.5 mL) and 6-maleimidohexanoic acid N-hydroxysuccinimide ester 13b (40.6 mg, 0.13 mmol) was added. The obtained colorless clear solution was stirred at room temperature for 24 hours. It was stripped under reduced pressure and the residue was purified by reverse phase HPLC (C18 column and eluted with CH3CN / H2O, 10-50% CH3CN in 15 minutes, then 95% CH3CN for 5 minutes) to give compound 20d (30.8 mg, 45% yield). MS (ESI): m / z 512.4 (M+H) + ,510.4(MH) - .

[0883] Example 15. Synthesis of Compounds 21a and 21b

[0884]

[0885] 21a: Compound 16a (300 mg, 0.57 mmol) and 6-mercaptohexanoic acid (254 mg, 1.7 mmol) were suspended in a solution of 20% TFA in dichloromethane (10 mL) and magnetically stirred at room temperature for 30 min. The solvent was rotary evaporated under vacuum and the residue was dissolved in a minimum volume of DMF and then purified on a 200 g C18 medium pressure column pre-equilibrated with 95:5 deionized water containing 0.1% formic acid:acetonitrile. The column was then eluted with 10% acetonitrile at 60 mL / min for 5 min, followed by a linear gradient elution from 5% acetonitrile at 5 min to 95% acetonitrile at 38 min. The fractions containing the desired product were combined, frozen, and lyophilized to give 202 mg of 21a as a white solid (58% yield). MS (M+Na)+ 613.9.

[0886] 21b was prepared similarly to 21a from 16d and 6-mercaptohexanoic acid in 60% yield. MS (M+H) + 613.7.

[0887] Example 16. Synthesis of Compound 22a

[0888]

[0889] 22a: Compound 6a (20 mg, 0.049 mmol) and DMTMM (18 mg, 0.065 mmol) in 85:15 DMF:deionized water (0.8 mL) were magnetically stirred while adding 20a (35 mg, 0.068 mmol) and TEA (0.04 mL, 0.28 mmol) sequentially. After 1 h, the reaction mixture was loaded onto a 50 g medium-pressure silica column equilibrated with dichloromethane and run at 30 mL / min with dichloromethane using a linear gradient of 0% to 100% dichloromethane containing 20% ​​methanol over 40 min. Fractions containing the pure product were combined and the solvent removed by rotary evaporation under vacuum to yield 16 mg of 22a as a brown solid (36% yield). MS (M+Na)+ 925.6. 1 H NMR(400MHz, DMSO-d6)δ0.88(t,J=7.3Hz,3H),1.11-1.23(m,15H),1.46(p,J=7.3Hz,5H),1.79-1.95(m, 2H),2.07(t,J=7.4Hz,2H),3.04-3.16(m,2H),3.88(s,2H),4.18(dd,J=7.1,11.0Hz,2H),4.50-4.65(m, 2H),4.86(d,J=5.9Hz,2H),5.44(s,2H),5.49(s,2H),6.53(s,1H),7.00(s,2H),7.32(s,1H),7.91(d,J= 10.0Hz, 1H), 7.97 (d, J = 7.0Hz, 2H), 8.43 (d, J = 8.3Hz, 1H), 8.65 (t, J = 6.6Hz, 1H), 8.73 (t, J = 5.9Hz, 1H). 13 C NMR(101MHz,DMSO-d6)δ180.20,173.96,172.96,172.89,172.56,172.36,171.54,170.03 ,163.99,157.27,156.21,152.90,150.48,145.85,139.87,134.92,129.28,128.06,124.2 4,119.65,97.28,72.84,70.18,67.29,65.74,50.53,48.81,48.68,48.60,46.26,41.01, 37.44,35.26,30.71,28.24,26.23,25.09,18.33,18.24,18.18,15.77,15.74,9.12,8.21. HRMS(M+H) + Calculated value 903.3688, experimental value 903.3676.

[0890] Example 17. Synthesis of Compound 22c

[0891]

[0892] 22c: To a solution of 6a (8 mg, 0.02 mmol) in DMF (0.5 mL) was added NMM (2.2 μL, 0.02 mmol) and compound 20d (10 mg, 0.02 mmol). The solution was cooled to 0°C with an ice bath and deionized water (0.1 mL) containing DMTMM (10.8 mg, 0.04 mmol) was added. The ice bath was removed and the reaction mixture was stirred at room temperature for 3 hours. The reaction solution was stripped under reduced pressure (35°C bath) and the residue was purified by reverse phase HPLC (30 g C18 column, CH3CN / HO, 25% CH3CN for 3 minutes, then 95% CH3CN in 12 minutes, then 95% CH3CN for 5 minutes). The product fractions were combined and lyophilized to give a white solid. This was further purified by silica gel chromatography (4 g silica column, CH2Cl2 / MeOH, 0-20% MeOH in 15 min) to give the desired product 22c (9.8 mg, 55% yield). MS (ESI): m / z 903.9 (M+H) + ,901.9(MH) - ,947.9(M+HCOOH-H) - .

[0893] Example 18. Synthesis of Compound 22b

[0894]

[0895] 22b: To a solution of 6c (10.7 mg, 0.022 mmol) in DMF (0.5 mL) was added NMM (2.4 μL, 0.022 mmol) and compound 20a (11 mg, 0.022 mmol). The solution was cooled to 0°C with an ice bath and deionized water (0.1 mL) containing DMTMM (11.9 mg, 0.043 mmol) was added. The ice bath was removed and the reaction mixture was stirred at room temperature for 3 hours. The reaction solution was stripped under reduced pressure (35°C bath) and the residue was purified by reverse phase HPLC (30 g C18 column, CH3CN / H2O, 25% CH3CN for 3 minutes, then reaching 95% CH3CN within 12 minutes, then 95% CH3CN for 5 minutes) to give the desired product 22b (4 mg, 18% yield). MS (ESI): m / z 1015.9 (M+Na) + ,991.9(MH) -,1037.9(M+HCOOH-H) - .

[0896] Example 19. Synthesis of Compound 22d

[0897] 22d: Prepared analogously to 22c by reaction of 6c with 20d (22% yield). MS (ESI): m / z 1015.8 (M+Na) +

[0898] Example 20. Synthesis of Compound 22e

[0899]

[0900] 22e:To a solution of compound 18c (15.2 mg, 0.035 mmol) in methanol (2 mL), Pd (10% / carbon, 3.7 mg, 0.0035 mmol) was added and the reaction flask was purged with hydrogen. It was hydrogenated with a hydrogen balloon at room temperature for 3 hours and then filtered. The filtrate was stripped to obtain compound 19c in a colorless foamy form. It was dissolved in anhydrous DMF (0.3 mL) and 13d (15.9 mg, 0.03 mmol) was added. The obtained colorless clear solution was stirred at room temperature for 15 hours. It was diluted with DMF (0.2 mL), followed by the addition of 6a (15.3 mg, 0.037 mmol) and NMM (4.1 μL, 0.037 mmol). Deionized water (0.1 mL) containing DMTMM (20.7 mg, 0.075 mmol) was then added and the reaction solution was stirred at room temperature for 1.5 hours. The reaction mixture was stripped under reduced pressure and the residue was purified by silica gel chromatography (CH2Cl2 / MeOH, 0-20% MeOH) to give the desired 22e (10 mg, 26% yield). MS (ESI): m / z 1049.1 (M+H) + ,1093.2(M+HCOOH-H) - .

[0901] Example 21. Synthesis of Compound 23a

[0902]

[0903] 23a: To a solution of 6a (32 mg, 0.078 mmol) in DMF (0.8 mL) was added NMM (8.6 μL, 0.078 mmol) and Fmoc-protected L-Ala-L-Ala-L-Ala tripeptide linker 21a (53 mg, 0.078 mmol), followed by deionized water (0.16 mL) containing DMTMM (43 mg, 0.156 mmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction solution was stripped under reduced pressure and the residue was purified by silica gel chromatography (4 g column, CH2Cl2 / MeOH, 0-20% MeOH in 15 minutes) to give the desired compound 23a (78 mg, 99% yield). MS (ESI): m / z 1004.5 (M+H) + ,1026.6(M+Na) + ,1048.4(M+HCOOH-H) - .

[0904] Example 22. Synthesis of Compound 24a

[0905]

[0906] 24a: To a solution of compound 23a (78 mg, 0.078 mmol) in anhydrous DMF (1 mL) was added morpholine (0.24 mL, 2.7 mmol) and the reaction mixture was stirred at room temperature for 2 hours. The reaction solution was stripped under reduced pressure and the residue was purified by silica gel chromatography (4 g silica column, eluted with CH2Cl2 / MeOH, 0-20% MeOH in 9 minutes, then 20% MeOH for 11 minutes) to give the desired compound 24a (39.6 mg, 65% yield). MS (ESI): m / z 782.4 (M+H) + ,780.0(MH) - .

[0907] Example 23. Synthesis of Compound 25a

[0908]

[0909] 25a:To a solution of compound 24a (20 mg, 0.026 mmol) in anhydrous DMF (0.3 mL) was added 13b (9.6 mg, 0.031 mmol) and the reaction mixture was stirred at room temperature for 4 hours. The reaction solution was diluted with DMSO and purified by reverse phase semi-preparative HPLC (C18 column, eluted with CH3CN / H2O, 25%-55% CH3CN in 23 minutes, then 95% CH3CN for 7 minutes). The fractions containing 25a were combined and lyophilized to give 25a as a white solid. It was further purified by silica gel chromatography (4 g silica column, CH2Cl2 / MeOH, 0-20% MeOH in 15 minutes) to give the desired 25a (7.7 mg, 30% yield). MS (ESI): m / z 975.8 (M+H) + ,997.8(M+Na) + ,773.7(MH) - ,1019.7(M+HCOOH-H) - .

[0910] Example 24. Synthesis of Compound 23b

[0911]

[0912] 23b: To a solution of 6a (31 mg, 0.076 mmol) in DMF (0.8 mL) was added NMM (8.3 μL, 0.076 mmol) and 21b (46.4 mg, 0.076 mmol). The solution was cooled to 0°C with an ice bath and deionized water (0.16 mL) containing DMTMM (48 mg, 0.16 mmol) was added. The ice bath was removed and the reaction mixture was stirred at room temperature for 2.5 hours. The reaction solution was stripped under reduced pressure and the residue was purified by silica gel chromatography (4 g silica column, CH2Cl2 / MeOH, 0-20% MeOH in 15 minutes) to give the desired product, compound 23b (31 mg, 40% yield). MS (ESI): m / z 1004.6 (M+H) + ,1048.7(M+HCOOH-H) - .

[0913] Example 25. Synthesis of Compound 24b

[0914]

[0915] 24b: To a solution of 23b (31 mg, 0.031 mmol) in anhydrous DMF (0.4 mL) was added morpholine (95 μL, 1.08 mmol) and the reaction mixture was stirred at room temperature for 2.5 hours. The reaction solution was stripped under reduced pressure and the residue was purified by silica gel chromatography (4 g silica column, eluted with CH2Cl2 / MeOH, 0-20% MeOH in 15 minutes, then 20% MeOH for 5 minutes) to give the product compound 24b (18 mg, 75% yield). MS (ESI): m / z 782.5 (M+H) + ,780.2(MH) - ,826.4(M+HCOOH-H) - .

[0916] Example 26. Synthesis of Compound 25b

[0917]

[0918] 25b: To a solution of 24b (12.3 mg, 0.016 mmol) in anhydrous DMF (0.3 mL) was added 5-maleimidocaproic acid N-hydroxysuccinimide ester 13b (7.3 mg, 0.024 mmol) and the reaction mixture was stirred at room temperature for 4 hours. The reaction solution was stripped under reduced pressure and the residue was purified by silica gel chromatography (4 g column, CH2Cl2 / MeOH, 0-20% MeOH in 15 minutes) to give 3.2 mg 25b and an additional 6.5 mg of impure product. The impure product was further purified by semi-preparative reverse phase HPLC (C18 column, eluted with CH3CN / H2O, 25%-55% CH3CN in 23 minutes, then 95% CH3CN for 7 minutes) to give 2.8 mg 25b. A total of 6 mg 25b (39% yield) was isolated. MS (ESI): m / z 975.6 (M+H) + ,997.6(M+Na) + ,973.7(MH) - ,1019.6(M+HCOOH-H) - .

[0919] Example 27. Synthesis of Compound 26a

[0920]

[0921] 26a: To a stirred solution of 8a (58 mg, 0.106 mmol) and compound 16a (55.8 mg, 0.106 mmol) in anhydrous DMF (1.5 mL) was added HCl etherate (2 M HCl in ether, 64 μL, 0.128 mmol). After stirring at room temperature for 22 hours, the reaction solution was stripped under reduced pressure (35° C. heating bath). The residue was purified by reverse phase HPLC (30 g C18 column, CH 3 CN / H 2 O, 25% CH 3 CN for 3 minutes, then reaching 90% CH 3 CN within 12 minutes, then 90% CH 3 CN for 3 minutes) to give compound 26a (47 mg, 48% yield) as a white solid. MS (ESI): m / z calculated value 917.4, found value 917.6 (M + H) + ,961.5(M+HCOOH-H) - Unreacted 8a was also recovered (12 mg).

[0922] Example 28. Synthesis of Compound 27a

[0923]

[0924] 27a: To a stirred solution of compound 26a (57 mg, 0.062 mmol) in anhydrous DMF (0.8 mL) was added morpholine (27 μL, 0.31 mmol). After stirring at room temperature for 6 hours, the reaction solution was stripped under reduced pressure. The residue was purified by reverse phase HPLC (30 g C18 column and eluted with CH3CN / H2O, 20% CH3CN for 3 minutes, then reaching 90% CH3CN within 15 minutes, then 90% CH3CN for 3 minutes) to give compound 27a (35.9 mg, 83% yield) as an off-white solid. MS (ESI): m / z 695.5 (M+H) + ,739.3(M+HCOOH-H) - .

[0925] Example 29. Synthesis of Compound 28a

[0926]

[0927] 28a: To a solution of compound 27a (18 mg, 0.026 mmol) in anhydrous DMF (0.3 mL) was added 5-maleimidocaproic acid N-hydroxysuccinimide ester 13b (12 mg, 0.039 mmol) and NMM (3.1 μL, 0.028 mmol). After stirring at room temperature for 4 hours, the reaction solution was stripped under reduced pressure. The residue was purified by reverse phase HPLC (30 g C18 column, eluted with CH3CN / H2O, 18 minutes running, 20% CH3CN for 3 minutes, then 20%-90% CH3CN in 12 minutes, then 90% CH3CN for 3 minutes) to give the product 28a (11.9 mg, 51% yield) as a white solid. MS (ESI): m / z 888.5 (M+H) + ,932.5(M+HCOOH-H) - . 1 H NMR (400MHz, DMSO-d6) δ8.45(t,J=6.7Hz,1H),8.11(d,J=8.2Hz,1H),7.90(t,J=7.5Hz,2H),7.78(dd,J=15. 2,9.1Hz,2H),7.22(s,1H),6.92(s,2H),6.45(s,1H),5.36(s,2H),5.17(s,2H),4.54-4.38(m,2H),4.15-4.0 9(m,2H),3.38-3.33(m,1H),3.32-3.21(m,1H),3.15-3.06(m,1H),2.50-2.42(m,3H),1.99(t,J=7.4Hz,2H) ,1.79(h,J=7.0Hz,2H),1.67-1.55(m,6H),1.38(p,J=7.4Hz,5H),1.15-1.04(m,12H),0.81(t,J=7.4Hz,3H). 13C NMR(101MHz,DMSO-d6)δ174.54,173.86,171.34,171.26,170.84,170.79,170.47,170.15,161.42,158.95, 155.71,155.16,150.43,148.38,146.97,146.80,144.22,142.23,129.03,126.25,125.69,125.48,124.98, 124.50,122.77,122.48,117.29,111.01,110.78,95.09,70.75,70.67,67.55,65.17,63.64,48.03,46.55, 33.20,28.68,27.46,26.96,25.04,24.55,24.05,23.06,22.84,16.39,16.14,13.54,6.13,5.98; HRMS(M+H) + Calculated value 888.3943, experimental value 888.3966.

[0928] Example 30. Synthesis of Compound 26b

[0929]

[0930] 26b: To a stirred solution of 8a (73 mg, 0.14 mmol) and compound 16b (64 mg, 0.14 mmol) in anhydrous DMF (1.2 mL) was added HCl etherate (2 M HCl in ether, 0.14 mL, 0.28 mmol). After stirring at room temperature for 6 h, the reaction solution was stripped under reduced pressure (35 ° C heating bath). The residue was purified by reverse phase HPLC (30 g C18 column, CH3CN / H2O, 25% CH3CN for 3 minutes, then reaching 90% CH3CN within 12 minutes, then 90% CH3CN for 3 minutes) to give compound 26b (60 mg, 50% yield) as an off-white solid. MS (ESI): m / z 846.4 (M+H) + ,890.3(M+HCOOH-H) - .

[0931] Example 31. Synthesis of Compound 26c

[0932]

[0933] 26c: To a stirred solution of 8a (36 mg, 0.08 mmol) and 16c (40.2 mg, 0.084 mmol) in anhydrous DMF (0.6 mL) was added HCl etherate (2 M HCl in ether, 72 μL, 0.43 mmol). After stirring at room temperature for 15 h, the reaction solution was stripped under reduced pressure (35 ° C heating bath). The residue was purified by reverse phase HPLC (30 g C18 column, CH3CN / H2O, 25% CH3CN for 3 minutes, then reaching 95% CH3CN within 12 minutes, then 95% CH3CN for 3 minutes) to give compound 26c (41.9 mg, 60% yield) as a white solid. MS (ESI): m / z 874.4 (M+H) + ,918.5(M+HCOOH-H) - Unreacted 8a was also recovered (12 mg).

[0934] Example 32. Synthesis of Compound 27b

[0935]

[0936] 27b: To a stirred solution of compound 26b (60 mg, 0.071 mmol) in anhydrous DMF (0.4 mL) was added morpholine (31 μL, 0.36 mmol). After stirring at room temperature for 3.5 hours, the reaction solution was stripped under reduced pressure. The residue was purified by reverse phase HPLC (15.5 g C18 column and eluted with CH3CN / H2O, 10% CH3CN for 3 minutes, then reaching 90% CH3CN within 9 minutes, then 90% CH3CN for 3 minutes) to give compound 27b (36.7 mg, 83% yield) as an off-white solid. MS (ESI): m / z 624.5 (M+H) + ,668.3(M+HCOOH-H) - .

[0937] Example 33. Synthesis of Compound 27c

[0938]

[0939] 27c: To a stirred solution of compound 26c (41.9 mg, 0.048 mmol) in anhydrous DMF (0.4 mL) was added morpholine (21 μL, 0.24 mmol). After stirring at room temperature for 5 h, the reaction solution was stripped under reduced pressure. The residue was purified by reverse phase HPLC (30 g C18 column and eluted with CH3CN / H2O, 20% CH3CN for 3 min, then reaching 90% CH3CN within 12 min, then 90% CH3CN for 5 min) to give compound 27c (25.5 mg, 82% yield) as a white solid. MS (ESI): m / z 652.5 (M+H) + ,650.2(MH) - .696.2(M+HCOOH-H) - .

[0940] Example 34. Synthesis of Compound 28b

[0941]

[0942] 28b: To a stirred solution of compound 27a (18 mg, 0.026 mmol) and 14a (18.5 mg, 0.039 mmol) in anhydrous DMF (0.2 mL) was added DMTMM (17 mg, 0.052 mmol) and NMM (2.9 μL, 0.026 mmol) at room temperature. After stirring at room temperature for 19 h, the reaction solution was stripped under reduced pressure (bath temperature 35 ° C.) and the residue was purified by reverse phase HPLC (30 g C18 column, CH3CN / H2O, 20% CH3CN for 3 minutes, then reaching 90% CH3CN within 12 minutes, then 90% CH3CN for 3 minutes). The fractions containing the product were combined and lyophilized to give 28b (2.3 mg, 7% yield) as a white solid. MS (ESI): m / z 1152.5 (M+H) + ,1150.0(MH) - ,1196.5(M+HCOOH-H) - .

[0943] Example 35. Synthesis of Compound 28c

[0944]

[0945] 28c: To a stirred solution of 27b (36.7 mg, 0.059 mmol) and 14a (42 mg, 0.088 mmol) in anhydrous DMF (0.6 mL) was added DMTMM (39 mg, 0.12 mmol) and NMM (3.2 μL, 0.029 mmol) at room temperature. After stirring at room temperature for 5 h, the reaction solution was directly loaded onto a C18 cartridge and purified by reverse phase HPLC (30 g C18 column, CH3CN / H2O, 20% CH3CN for 3 minutes, then reaching 90% CH3CN within 12 minutes, then 90% CH3CN for 3 minutes). The fractions containing the product were combined and lyophilized to give 28c (9.9 mg, 15% yield) as a white solid. MS (ESI): m / z 1081.5 (M+H) + ,1079.2(MH) - ,1025.5(M+HCOOH-H) - .

[0946] Example 36. Synthesis of Compound 28d

[0947]

[0948] 28d: To a solution of 27c (10.5 mg, 0.016 mmol) in anhydrous DMF (0.2 mL) was added 5-maleimidocaproic acid N-hydroxysuccinimide ester 13b (6 mg, 0.019 mmol) and NMM (2.1 μL, 0.019 mmol). After stirring at room temperature for 4 h, the reaction solution was stripped under reduced pressure. The residue was purified by reverse phase HPLC (30 g C18 column, eluted with CH3CN / H2O, 18 min run, 20% CH3CN for 3 minutes, then 20%-90% CH3CN in 12 minutes, then 90% CH3CN for 3 minutes) to give the product 28d (8.7 mg, 63% yield) as a white solid. MS (ESI): m / z 845.4 (M+H) + ,989.4(M+HCOOH-H) - .

[0949] Example 37. Synthesis of Compound 29a

[0950]

[0951] 29a: Compound 16a (30 mg, 0.057 mmol) and 8d (50 mg, 0.1 mmol) were suspended in 20% TFA in dichloromethane (10 mL) and magnetically stirred at room temperature for 30 min. The solvent was rotary evaporated under vacuum and the residue was dissolved in a minimum volume of DMF and then purified on a 100 g C18 medium-pressure column pre-equilibrated with 90:10 deionized water:acetonitrile containing 0.1% formic acid. The column was then eluted with 10% acetonitrile at 30 mL / min for 5 min, followed by a linear gradient from 10% acetonitrile at 5 min to 95% acetonitrile at 38 min. The fractions containing the desired product were combined, frozen, and lyophilized to afford 31 mg of 29a as a white solid (56% yield). MS (M+Na)+ 987.5.

[0952] Example 38. Synthesis of Compound 30a

[0953]

[0954] 30a: Compound 29a (28 mg, 0.029 mmol) was dissolved in anhydrous DMF (0.8 mL) and magnetically stirred when morpholine (0.2 mL) was added. After 1 h, the reaction mixture was directly loaded onto a 100 g C18 cartridge, 25:75 CH3CN / H2O, running at 50 mL / min with 25% CH3CN for 3 minutes, followed by a linear gradient from 3-23 min to 90% CH3CN. The eluate fractions containing the desired product were combined, frozen, and lyophilized to give 18 mg (83% yield) of 30a as a yellow solid. MS (M+H) 743.5.

[0955] Example 39. Synthesis of Compound 32a

[0956]

[0957] 32a: Compound 30a (16 mg, 0.02 mmol) was dissolved in 84:16 DMF:deionized water (0.5 mL), to which DMTMM (15 mg, 0.054 mmol), TEA (0.02 mL, 0.14 mmol), and 14a (20 mg, 0.042 mmol) were quickly added with magnetic stirring. After 35 min, the reaction mixture was loaded onto a 100 g silica cartridge pre-equilibrated with dichloromethane and then run at 35 mL / min using a linear gradient of 0% to 100% 40:60 methanol:dichloromethane over 30 min. The fractions containing the desired product were combined and the solvent evaporated under vacuum to afford 7 mg (29% yield) of 32a as a viscous oil. MS (M+Na)+ 1223.0.

[0958] Example 40. Synthesis of Compound 33a

[0959]

[0960] 33a: To a solution of 4c (11 mg, 0.021 mmol) in anhydrous DMF (0.2 mL) was added sodium thiomethoxide (3.6 mg, 0.052 mmol) and the reaction mixture was stirred at room temperature for 18 hours. The reaction solution was directly loaded onto a 30 g C18 cartridge, 25:75 CH3CN / H2O, running at 20 mL / min, 25% CH3CN for 3 minutes, then a linear gradient from 3-12 min to 90% CH3CN. The fractions containing the desired product were combined and evaporated under vacuum to give compound 33a (3.1 mg, 30% yield) as a white solid. MS (ESI): m / z 483.4 (M+H) + ,481.3(MH) - .

[0961] Example 41. Synthesis of Compound 34a and Compound 34b

[0962]

[0963] 34a and 34b: Compound 8c (30 mg, 0.064 mmol) was magnetically stirred in DMF (1 mL) and vanadyl acetylacetonate (3 mg, 0.008 mmol) and a 5M solution of tert-butyl hydroperoxide in decane (0.05 mL, 0.25 mmol) were added. After 5 min, the solution was directly loaded onto a 30 g C18 cartridge, 25:75 CH3CN / H2O, running at 20 mL / min with 25% CH3CN for 3 minutes, followed by a linear gradient to 90% CH3CN from 3-12 min. The fractions containing pure 34a and 34b were combined separately and both were frozen and lyophilized to give 6 mg 34a (19% yield) and 15 mg 34b (47% yield), both as yellow solids. 34a MS(M+H)+487.4, 34b MS(M+H)+503.6.

[0964] Example 42. Synthesis of Compound 35a

[0965]

[0966] 35a: To a solution of compound 4e (17 mg, 0.025 mmol) in anhydrous DMF (0.3 mL) was added sodium thiomethoxide (7 mg, 0.1 mmol) and the reaction mixture was stirred at room temperature for 24 hours. The reaction solution was diluted with DMSO and injected into a semi-preparative HPLC (C18 column, CH3CN / H2O, 25%-65% CH3CN in 23 minutes) for purification to give the desired product 35a (2.7 mg, 19% yield). MS (ESI): m / z 554.4 (M+H) + ,552.5(MH) - .

[0967] Example 43. Synthesis of Compound 29b

[0968]

[0969] 29b: Compound 16a (30 mg, 0.057 mmol) and 8e (50 mg, 0.1 mmol) were suspended in a solution of 20% TFA in dichloromethane (10 mL) and magnetically stirred at room temperature for 30 min. The solvent was rotary evaporated under vacuum and the residue was dissolved in a minimum volume of DMF and then purified on a 100 g C18 medium pressure column pre-equilibrated with 90:10 deionized water:acetonitrile containing 0.1% formic acid. The column was then eluted with 10% acetonitrile at 30 mL / min for 5 min, followed by a linear gradient elution from 10% acetonitrile at 5 min to 95% acetonitrile at 38 min. The fractions containing the desired product were combined, frozen and lyophilized to give 31 mg of 29b as a white solid (56% yield). MS (ESI): MS (M+Na) + Calculated value 973.3, experimental value 973.7.

[0970] Example 44. Synthesis of Compound 30b

[0971]

[0972] 30b:Compound 29b (28mg, 0.029mmol) is dissolved in anhydrous DMF (0.8mL) and magnetically stirred when adding morpholine (0.2mL, 2.32mmol).After 1h, reaction mixture is directly loaded on 100g C18 cartridge, 25:75CH3CN / H2O, runs with 50mL / min, 25%CH3CN continues 3min, then uses the linear gradient that reaches 90%CH3CN from 3-23min.The elution fraction containing required product is merged, frozen and lyophilized, obtain 18mg (83% productive rate) 30b in the shape of yellow solid. 1H NMR (400MHz, DMSO-d6) δ8.52(t,J=6.3Hz,1H),8.31(d,J=8.3Hz,1H),8.11(d,J=7.4Hz,2H),7.87(t,J=9.1Hz, 2H),7.69(d,J=7.5Hz,1H),7.37(t,J=7.4Hz,1H),7.31(t,J=3.7Hz,2H),5.43(s,2H),5.31(s,2H),4.47(d,J= 2.6Hz,2H),4.32-4.09(m,4H),3.69(t,J=4.6Hz,2H),3.36(q,J=6.9Hz,1H),2.84(s,4H),2.61-2.54(m,3H),2 .46-2.42(m,4H),1.94-1.75(m,2H),1.17(dd,J=7.2,3.4Hz,6H),1.12(d,J=6.8Hz,3H),0.87(t,J=7.3Hz,3H). MS(ESI):MS(M+H) + Calculated value 729.3, experimental value 729.4.

[0973] Example 45. Synthesis of Compound 32b

[0974]

[0975] 32b: Compound 30b (16 mg, 0.02 mmol) was dissolved in 84:16 DMF: deionized water (0.5 mL), to which DMTMM (15 mg, 0.054 mmol), TEA (0.02 mL, 0.14 mmol) and 14a (20 mg, 0.042 mmol) were quickly added and magnetically stirred. After 35 min, the reaction mixture was loaded onto a 100 g silica cartridge pre-equilibrated with dichloromethane and then run at 35 mL / min using a linear gradient of 0%-100% 40:60 methanol: dichloromethane over 30 min. The fractions containing the desired product were combined and the solvent was evaporated under vacuum to give 7 mg of 32b (29% yield) as a viscous oil. 11H NMR (400 MHz, DMSO-d6) δ 7.86 - 7.83 (m, 1H), 7.71 (s, 1H), 7.69 (d, J = 1.1 Hz, 1H), 7.66 (d, J = 8.2 Hz, 1H), 7.51 - 7.47 (m, 2H), 7.28 (t, J = 1.0 Hz, 1H), 6.61 (s, 2H), 5.59 - 5.53 (m, 1H), 5.40 (dd, J = 3.3, 1.1 Hz, 2H), 5.29 - 5.26 (m, 2H), 4.72 (s, 2H), 4.52 - 4.46 (m, 1H), 4.38 (s, 1H), 4.35 (dd, J = 3.5, 1.8 Hz, 2H), 4.28 - 4.22 (m, 2H), 4.21 - 4.13 (m, 2H), 3.74 (t, J = 6.5 Hz, 2H), 3.70 (ddd, J = 11.9, 5.7, 4.8 Hz, 1H), 3.60 - 3.51 (m, 2H), 3.44 (dd, J = 11.9, 4.9 Hz, 1H), 3.3 (broad s, 8H), 3.20 (dd, J = 4.7, 3.7 Hz, 2H), 2.91 (s, 3H), 2.89 (s, 3H), 2.56 (td, J = 6.4, 1.7 Hz, 2H), 2.31 (s, 3), 2.37 - 2.21 (m, 2H), 2.07 - 1.82 (m, 3H), 1.72 (dq, J = 13.7, 8.1 Hz, 1H), 1.53 (d, J = 5.5 Hz, 6H), 1.45 (d, J = 5.9 Hz, 3H), 0.86 (t, J = 8.0 Hz, 3H). 13 13C NMR (101 MHz, DMSO-d6) δ 174.75, 174.58, 172.85, 172.75, 172.45, 171.62, 171.37, 169.30, 163.44, 160.52, 157.18, 149.96, 148.73, 147.39, 147.32, 142.26, 132.98, 131.74, 127.54, 127.38, 127.24, 126.88, 124.90, 124.84, 124.34, 122.88, 118.32, 116.83, 116.67, 97.97, 74.87, 73.80, 72.41, 72.34, 69.44, 65.10, 62.94, 53.02, 50.02, 49.92, 49.62, 48.46, 45.53, 42.38, 40.85, 35.32, 35.13, 34.00, 32.31, 32.24, 30.20, 26.63, 20.49, 17.81, 17.71, 17.61, 7.68. HRMS (M + H)+ Calculated value 1186.4237, experimental value 1186.4220.

[0976] Example 46. General method for preparing conjugates

[0977]

[0978] Humanized IgG1 antibodies were generated, such as anti-epidermal growth factor (ML66), anti-folate receptor alpha (FRα), and chimeric antibodies conjugated to Kunitz soybean trypsin inhibitor (KTI). Conjugation of the antibodies to maleimide-bearing payloads was performed as described for the preparation of ML66-22a. The resulting conjugates will be designated herein as target binder-payload, for example, ML66 conjugated to 22a is ML66-22a.

[0979] Example 47. Synthesis of ML66-22a

[0980] ML66-22a: 5 mg / ml of ML66 was treated with 7.0 equivalents of TCEP in 50 mM EPPS pH 7.4, 5 mM EDTA at 37 ° C for 1-1.5 hours. The mixture was then cooled to room temperature. The conjugation reaction between the antibody and the payload was performed at 2 mg / ml by adding 15-20 equivalents of 22a dissolved in DMSO in a buffer containing 20% ​​DMSO (50 mM EPPS, pH 7.4) and rotating on a tube rotator for 1.5-2.5 hours at room temperature. The reaction mixture was immediately purified using a NAP desalting column (Illustra Sephadex G-25, GE Healthcare) into formulation buffer (10 mM acetate, 9% sucrose, 0.01% Tween-20, pH 5.0). The drug: antibody ratio (DAR) of the resulting conjugate was 7.5, and 99% monomer was measured by size exclusion chromatography.

[0981] Biophysical evaluation of the interchain high DAR conjugates was performed, including determination of conjugate concentration, yield, DAR (drug:antibody ratio), free drug, percent monomer, and DAR distribution. Conjugate concentration was determined to have a final protein concentration of 4.5 mg / ml (via UV-Vis using extinction coefficient ε 280 =205520M -1 cm -1 ), DAR 7.5 (via UV-Vis using the extinction coefficient ε for 22a 280 =10764M -1 cm -1 , ε 370 =20982M -1 cm-1 ), 99% monomer (via size exclusion UPLC protein BEH SEC column), <1% free drug (via HiSEP HPLC column), and primarily homogeneous 8 drugs attached to each antibody (via Q-ToF mass spectrometry and Butyl-NPR HIC chromatography). The yield of this particular interchain conjugate ML66-22a was 75%.

[0982] UPLC Protein BEH SEC Method: On an Acquity UPLC Protein BEH SEC column ( Monomer percentage analysis was performed on a Waters Acquity UPLC H-Class system (1.7 μm, 4.6 mm×150 mm, part #186005225). The mobile phase was 400 mM sodium perchlorate, 50 mM sodium phosphate, 5% IPA, pH 7.0, with a flow rate of 0.30 mL / min and a run time of 20 min.

[0983] HiSep HPLC Method: Percent free drug analysis was performed on an Agilent HPLC system equipped with a Supelco analytical HiSep column (25 cm x 4.6 mm, 5 μm, catalog #58919). The mobile phase consisted of 0.1 M ammonium, pH 7.0 (solvent A) and acetonitrile (solvent B). The method was run at 0.70 mL / min with solvent A using a linear gradient from 25% solvent B to 40% solvent B over 0-25 min.

[0984] Example 48. In vitro cytotoxicity assay

[0985] As previously described (Kovtun YV et al., Antibody-maytansinoid conjugates designed to bypass multidrug resistance. Cancer Res 2010; 70(6): 2528-37), cytotoxicity was assessed using a water-soluble bisazo salt (WST-8)-based cell viability assay (Dojindo, Molecular Technologies, Inc.) in flat-bottom 96-well plates (Costar). Briefly, human tumor cells (1,000-5,000 cells / well, depending on the cell line) in appropriate culture medium were incubated with the conjugates in the presence or absence of excess corresponding unconjugated antibody or with metabolites at 37° C., 6% CO 2 for 5 days. Cell viability was determined from background-corrected WST-8 absorbance.

[0986] The results of this study are summarized in the following tables. Figure 12The cytotoxicity of sulfide-bearing compound 8c and its sulfoxide 34a and sulfone 34b are depicted.

[0987] Table 7A. In vitro cytotoxicity (IC 50 value)

[0988]

[0989] Table 7B. In vitro cytotoxicity (IC 50 value)

[0990]

[0991] N-Luc indicates Namalwa cells stably transfected with the luciferase gene

[0992] Example 49. Bystander Cell Killing Assay

[0993] Cell Titer Glo and One Glo reagents were purchased from Promega. The ability of ADCs to induce bystander killing was determined by one of two assays. Both assays were performed in U-bottom 96-well plates (Costar) to keep mixed antigen-negative (Ag-) and antigen-positive (Ag+) cells in close proximity to each other.

[0994] Namalwa-luciferase (N-Luc) Ag- cells (1000 cells / well) in appropriate culture medium were incubated with a specified number of MDA-MB-468Ag+ cells and ADC (1.1 nM) in a well of a U-bottom 96-well plate at 37°C, 6% CO2 for 5 days. The concentration of the conjugate used in the assay was high enough to kill all Ag+ cells, but not Ag- cells unless Ag+ cells were present. On day 5, cell viability was determined by Cell Titer Glo assay according to the manufacturer's protocol; luminescent signals were read using a Victor3 plate reader. The table below shows bystander killing of the in vitro cytotoxicity and compounds described herein obtained as described in the previous example.

[0995] Table 8. In vitro cytotoxicity and bystander killing of ADCs

[0996]

[0997] N-Luc indicates Namalwa cells stably transfected with the luciferase gene

[0998] Example 50. Method for determining in vivo efficacy in a xenograft model

[0999] 6-week-old female CB.17 SCID mice were received from Charles River Laboratories. All in vivo procedures were performed in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals. The in vivo efficacy of ADCs was evaluated in a given tumor xenograft model. Female SCID mice were subcutaneously inoculated in the right flank with the desired cell type in serum-free medium at a ratio of 1:1. The animals were then randomly distributed in groups of 6 or 8 mice per group. Control mice were treated with phosphate-buffered saline (vehicle). The desired concentration of ADC was prepared by diluting the stock sample with the vehicle. Xenografts grew to approximately 100 mm 3 Mice were then administered ADC or vehicle via intravenous (iv) injection (200 μL / mouse) via the tail vein. All dosing was based on the weight of the antibody component of the conjugate. Tumor size was measured twice weekly using a caliper, with tumor volume calculated in mm using the formula V = 1 / 2 (length × width × height). 3 In addition, body weight was measured twice weekly. Data from these studies were interpreted using standardized methods as previously described (Bissery MC et al., Experimental antitumor activity of taxotere (RP 56976, NSC 628503), a taxol analogue. Cancer Res 1991; 51(18): 4845-52).

[1000] Example 51. Tolerance of ML66-999, ML66-22a, and ML-28a ADCs in Mice

[1001] The tolerability of the ADC was evaluated in female CD-1 mice by performing daily body weight measurements and clinical observations for 2 weeks after injection of the ADC. A 10 mg / kg pre-dose of naked ML66 antibody was administered intravenously to 32 7-week-old female CD-1 mice because it was shown to alleviate the acute infusion reaction caused by doses of ML66 alone greater than 20 mg / kg. After this injection of naked antibody, one mouse died. Two hours later, three groups of seven to eight mice were administered with an intravenous bolus of 1500 μg / kg payload ML66-999 (58 mg / kg antibody), ML66-22a (59 mg / kg antibody), or ML66-28a (69 mg / kg antibody). The maximum tolerated dose (MTD) was defined as the highest dose at which the animal did not die or require euthanasia due to >20% weight loss or signs of distress or morbidity (hunched back, hypoactivity, inability to eat or drink, or signs of pain / distress). Based on these criteria, all three ADCs were well tolerated at a 1500 μg / kg payload dose. Figure 19Tolerability of mice to ML66-999, ML66-22a, and ML66-28a ADCs is depicted.

[1002] Example 52. Method for determining ADC pharmacokinetic (PK) parameters in mice

[1003] Eleven 7-week-old female CD-1 mice were each administered a single intravenous bolus of 10 mg / kg naked antibody or ADC. Terminal blood samples were collected from 3 mice at 2 minutes, 3 mice at 24 hours, and 5 mice at 72 hours after the injection of each ADC. The blood was processed into serum and the ADC was purified using affinity capture anti-human Fc beads. The samples were analyzed by anti-human Fc ELISA to determine the concentration of the antibody component (regardless of the payload load). The samples were also analyzed by size exclusion chromatography (SEC) and mass spectrometry (MS). The partial loss of the linker-payload from the captured ADC was measured at all time points by a complete MS method. The ADC concentration was calculated based on the antibody concentration and drug:antibody ratio (DAR).

[1004] Figure 13 Table 9 and Table 9 show the pharmacokinetics of ML66-999 in mice. Figure 14 The pharmacokinetics of ML66-999 in mice are shown in Table 10. Table 11 summarizes the binding of ADC or naked antibody to cell lines expressing the corresponding antigen. Figure 15 and Figure 16 The in vitro cytotoxicity of ML66-999 and ML66-22a against Ag+ and Ag- cells was shown. The fraction of ADCs in plasma samples that retained bioactivity over time was determined using Ag+ or Ag- cells in the cytotoxicity assay. The ADCs retained most of their activity against Ag+ cells at each time point, while still being more than 200-fold less active than Ag- cells, indicating that the cytotoxicity was due to the intact ADC, with little contribution from any released payload.

[1005] Table 9.

[1006]

[1007] Table 10.

[1008]

[1009] Table 11. Binding of ADC or naked antibodies to cell lines expressing the corresponding antigens

[1010]

[1011]

[1012] Example 53. Antitumor Activity of Anti-EGFR Antibody Drug Conjugates in Nude Mice Bearing HSC-2 Human Head and Neck Squamous Cell Carcinoma Xenografts

[1013] The antitumor activity of ML66-999 and ML66-22a at 1, 3, and 10 mg / kg was evaluated in female nude mice bearing HSC-2 cells, a xenograft model of human head and neck squamous cell carcinoma.

[1014] Mice were inoculated subcutaneously with 1×10 cells in 0.1 ml of 50% Matrigel / 50% serum-free medium. 7 HSC-2 cells were obtained from female athymic Foxn1 nu Mice (6 weeks old) were observed for 9 days after receipt before the start of the study. Animals showed no signs of illness or disease upon arrival or before treatment.

[1015] Forty-eight mice were randomly divided into 8 groups (6 mice per group) according to tumor volume. Tumor volume ranged from 68.48 to 118.26 (93.42 ± 11.25, mean ± SD) mm 3 The mice were measured, randomly divided into groups, and dosed based on the tumor volume on the 4th day after implantation (11 / 12 / 18). The body weight of the mice was in the range of 19.46 to 25.77 (22.98 ± 1.50, mean ± SD) grams. The mice in each group were identified by the perforation method. The test agent and vehicle were administered intravenously using a 1.0 ml syringe equipped with a 27-gauge, 1 / 2 inch needle. The antibody drug conjugate test agent was administered at 1, 3 or 10 mg / kg qdx1, wherein 75 μg / kg or 250 μg / kg based on payload was associated with approximately 3 or 10 mg / kg based on antibody concentration. The groups included: a control group dosed with vehicle (PBS, 200 μL), a control group dosed with non-targeted KTI-999 at 10 mg / kg, ML66-999 dosed at 1, 3, and 10 mg / kg based on antibody concentration, and ML66-22a dosed at 1, 3, and 10 mg / kg based on antibody concentration.

[1016] Tumor size was measured twice weekly using calipers. Tumor volume was calculated using the formula: volume = length x width x height x 1 / 2 in mm. 3 Mice were considered to have partial regression (PR) when tumor volume was reduced by 50% or more and complete tumor regression (CR) when no palpable tumor could be detected. Tumor volume was determined by StudyLog software.

[1017] Tumor growth inhibition (%T / C) is measured at a predetermined time (e.g., when the median TV of control tumors reaches a maximum tumor volume of approximately 1000 mm 3 The ratio of the median tumor volume (TV) of the treated group (T) to the median TV of the control group (C) is calculated on day 22 after inoculation, when the median TV of the control group reached 1038 mm 3 According to NCI criteria, T / C ≤ 42% is the lowest level of antitumor activity and T / C < 10% is considered a high antitumor activity level.

[1018] The body weight (BW) of all mice was measured twice a week as a rough index of drug toxicity and was determined by StudyLog software. The body weight of the mice was expressed as the percentage change in body weight compared to the body weight before treatment as follows: BW change % = [(BW after / BW before) - 1] × 100, where BW after is the weight after treatment and BW before is the starting weight before treatment. The body weight loss percentage (BWL) is expressed as the average change in body weight after treatment. If the tumor volume is greater than 1000 mm 3 Animals were euthanized if necrosis of the tumor occurred, mice lost >20% of their initial body weight, or mice became moribund at any time during the study.

[1019] Figure 17Table 12 and Table 13 depict the efficacy of ADC in the HSC-2 xenograft model. ML66-999 and ML66-22a conjugates have similar anti-tumor activity. ML66-KTI administered at 10 mg / kg had a T / C value of 70% (inactive) with no tumor regression. This demonstrates that the activity of the ML66 conjugate is EGFR-targeted, as the control ADC is inactive. ML66-999 administered at 1 mg / kg had a T / C value of 49% (inactive) with no tumor regression. ML66-999 administered at 3 mg / kg had a T / C value of 7% (highly active), with 4 of 6 mice experiencing partial tumor regression and 1 experiencing complete regression. ML66-999 administered at 10 mg / kg had a T / C value of 3% (highly active), with 6 of 6 mice experiencing partial tumor regression and 2 experiencing complete regression. ML66-22a dosed at 1 mg / kg had a T / C value of 39% (active), with 1 of 6 mice experiencing partial tumor regression and no complete regression. ML66-22a dosed at 3 mg / kg had a T / C value of 5% (highly active), with 3 of 6 mice experiencing partial tumor regression and 1 experiencing complete regression. ML66-22a dosed at 10 mg / kg had a T / C value of 2% (highly active), with 6 of 6 mice experiencing partial tumor regression and 5 experiencing complete regression. No significant weight loss was observed for any of the conjugates at any of the doses indicated, indicating that the conjugates were well tolerated. The results of this study demonstrate that both the ML66-999 and ML66-22a conjugates demonstrated dose-dependent anti-tumor activity and were effective in the HSC-2 head and neck squamous cell carcinoma tumor xenograft model.

[1020] Table 12.

[1021]

[1022]

[1023] Example 54. Antitumor activity of the conjugate in nude mice bearing non-small cell lung cancer (NSCLC) xenografts

[1024] The antitumor activity of the conjugate was determined in nude mice bearing non-small cell lung cancer (NSCLC) xenografts in a manner similar to that described in Example 53 for HSC-2 cells.

[1025] Six-week-old female athymic nude mice (Foxn1 nuAll in vivo procedures were performed in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals. The antitumor activity of the ADC was evaluated in a non-small cell lung cancer (NSCLC) squamous H1703 tumor xenograft model. Female nude mice were subcutaneously inoculated in the right flank with the desired cell type (5×10 cells / mL) in a 1:1 ratio of serum-free medium to Matrigel. 6 Three-dimensional tumor volume (TV) was measured twice weekly using calipers, where tumor volume was calculated in mm using the formula TV = 1 / 2 (length × width × height). 3 Xenografts grew to approximately 100 mm 3 The mice were screened based on their TV at day 16 after cell inoculation (with 116.0 + / - 18.5 mm 3 [Mean + / - SD] TV) were randomly distributed in groups of 6 mice per group. Stock ADC was diluted with conjugate dilution buffer and mice were dosed according to subject body weight. Mice received a single intravenous (IV) bolus of vehicle (phosphate buffered saline (PBS), 200 μL / mouse) or ADC at a payload of 75 μg / kg or 250 μg / kg (based on an antibody concentration of approximately 3 or 10 mg / kg) in a dose volume of 5 mL / kg. Tumor growth inhibition (T / C) was measured at a predetermined time (e.g., when the median TV of the control tumors reached a maximum tumor volume of approximately 1000 mm 3 The ratio of the median tumor volume (TV) of the treatment group (T) to the median TV of the control group (C) (the time at which the mice were euthanized) was calculated. According to NCI criteria, T / C ≤ 42% is the lowest level of anti-tumor activity and T / C < 10% is considered a high level of anti-tumor activity. When TV is reduced by 50% or more, the mouse is considered to have partial regression (PR) and when no palpable tumor can be detected, the mouse is considered to have complete regression (CR). T / C, PR, and CR for both efficacy studies are listed in the table below. In addition, body weight was measured twice a week as a rough index of drug toxicity.

[1026] Figure 20 The efficacy of the ADC in the H1703 xenograft model is depicted in Table 14. The results showed that both ML66-999 and ML66-22a conjugates demonstrated dose-dependent anti-tumor activity and were effective in the H1703 tumor xenograft model.

[1027] Table 14. H1703 efficacy study results

[1028]

[1029]

[1030] Example 55. Antitumor Activity of Anti-EGFR Antibody Drug Conjugates in Nude Mice Bearing FaDu Human Head and Neck Squamous Cell Carcinoma Xenografts

[1031] The antitumor activity of ML66-999 and ML66-22a at 1, 3, and 10 mg / kg was evaluated in female nude mice bearing FaDu cells, a xenograft model of human head and neck squamous cell carcinoma.

[1032] Mice were inoculated subcutaneously with 1×10 cells in 0.1 ml of 50% Matrigel / 50% serum-free medium. 7 Obtain female athymic Foxn1 nu Mice (6 weeks old) were observed for 7 days after receipt before the start of the study. Animals showed no signs of illness or disease upon arrival or before treatment.

[1033] Sixty-four mice were randomly divided into 8 groups (8 mice per group) according to tumor volume. Tumor volume ranged from 74.07 to 128.73 (104.66 ± 15.70, mean ± SD) mm 3 The mice were measured, randomly divided into groups, and administered based on the tumor volume on the 6th day after implantation (11 / 19 / 18). The body weight of mice was in the range of 20.48 to 25.77 (23.55 ± 1.25, mean ± SD) grams. The mice in each group were identified by perforation method. The test agent and vehicle were administered intravenously using a 1.0 ml syringe equipped with a 27-gauge, 1 / 2 inch needle. The antibody drug conjugate test agent was administered at 1, 3 or 10 mg / kg qdx1. The group included: a control group administered with vehicle (PBS, 200 μL), a control group administered with non-targeted KTI-999 at 10 mg / kg, ML66-999 administered at 1, 3 and 10 mg / kg, and ML66-22a administered at 1, 3 and 10 mg / kg.

[1034] Tumor size was measured twice weekly using calipers. Tumor volume was calculated using the formula: volume = length x width x height x 1 / 2 in mm. 3 Mice were considered to have partial regression (PR) when tumor volume was reduced by 50% or more and complete tumor regression (CR) when no palpable tumor could be detected. Tumor volume was determined by StudyLog software.

[1035] Tumor growth inhibition (%T / C) is measured at a predetermined time (e.g., when the median TV of control tumors reaches a maximum tumor volume of approximately 1000 mm 3The ratio of the median tumor volume (TV) of the treated group (T) to the median TV of the control group (C) is calculated on day 21 after inoculation, when the median TV of the control group reached 749 mm 3 According to NCI criteria, T / C ≤ 42% is the lowest level of antitumor activity and T / C < 10% is considered a high antitumor activity level.

[1036] The body weight (BW) of all mice was measured twice a week as a rough index of drug toxicity and was determined by StudyLog software. The body weight of the mice was expressed as the percentage change in body weight compared to the body weight before treatment as follows: BW change % = [(BW after / BW before) - 1] × 100, where BW after is the weight after treatment and BW before is the starting weight before treatment. The body weight loss percentage (BWL) is expressed as the average change in body weight after treatment. If the tumor volume is greater than 1000 mm 3 Animals were euthanized if necrosis of the tumor occurred, mice lost >20% of their initial body weight, or mice became moribund at any time during the study.

[1037] Figure 18Table 13 and Table 14 depict the efficacy of the ADC in the FaDu xenograft model. The ML66-999 and ML66-22a conjugates have similar anti-tumor activity. ML66-KTI administered at 10 mg / kg had a T / C value of 20% (active), with 2 of 8 mice experiencing partial tumor regression and 2 experiencing complete regression. This demonstrates that some of the anti-tumor activity in this model is non-targeted. ML66-999 administered at 1 mg / kg had a T / C value of 12% (active), with 4 of 8 mice experiencing partial tumor regression and 2 experiencing complete regression. ML66-999 administered at 3 mg / kg had a T / C value of 2% (highly active), with 8 of 8 mice experiencing partial tumor regression and 4 experiencing complete regression. ML66-999 administered at 10 mg / kg had a T / C value of 0% (highly active), with 8 of 8 mice experiencing partial tumor regression and 8 experiencing complete regression. ML66-22a dosed at 1 mg / kg had a T / C value of 13% (active), with 4 of 8 mice experiencing partial tumor regression and 3 experiencing complete regression. ML66-22a dosed at 3 mg / kg had a T / C value of 0% (highly active), with 8 of 8 mice experiencing partial tumor regression and 8 experiencing complete regression. ML66-22a dosed at 10 mg / kg had a T / C value of 0% (highly active), with 8 of 8 mice experiencing partial tumor regression and 8 experiencing complete regression. No significant body weight loss was observed for any of the conjugates at any of the doses indicated, indicating that the conjugates were well tolerated. The results of this study demonstrate that both the ML66-999 and ML66-22a conjugates demonstrated dose-dependent anti-tumor activity and were effective in the FaDu head and neck squamous cell carcinoma tumor xenograft model.

[1038] Table 13.

[1039]

[1040] Example 56.Ab F -999 and Ab F Tolerability of -22a ADC in mice

[1041] The tolerability of non-cross-reactive ADCs was evaluated in female CD-1 mice by performing daily body weight measurements and clinical observations for 2 weeks after ADC injection. F -22a (184 mg / kg based on antibody) or Ab F-999 (198 mg / kg antibody) was administered as a 5000 μg / kg IV bolus. The maximum tolerated dose (MTD) was defined as the highest dose at which the animal did not die or require euthanasia due to >20% body weight loss or signs of distress or morbidity (hunched back, hypoactivity, inability to eat or drink, or signs of pain / distress). GraphPad was used for statistical analysis of body weights in each group (two-way ANOVA with Tukey's multiple comparison test was used to show Ab F -999 group was significantly different from vehicle and Ab F -22a group, p<0.05). Figure 21 Describe these results.

[1042] While certain embodiments have been illustrated and described, it will be understood that changes and modifications may be made therein in accordance with ordinary skill in the art without departing from the technology in its broader aspects as defined in the following claims.

[1043] The embodiments illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein.

[1044] The present disclosure is not limited to the specific embodiments described in this application. Many modifications and variations can be made without departing from its spirit and scope, which will be apparent to those skilled in the art. Based on the foregoing description, in addition to those enumerated herein, functionally equivalent methods and compositions within the scope of the present disclosure will be apparent to those skilled in the art. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is only limited by the terms of the appended claims and the full scope of equivalents granted by these claims. It should be understood that the present disclosure is not limited to specific methods, reagents, compounds or compositions, which may of course vary. It should also be understood that the terms used herein are only used to describe the purpose of specific embodiments and are not intended to be limiting.

[1045] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[1046] As will be understood by those skilled in the art, for any and all purposes, particularly with respect to providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be readily considered to fully describe the range and enable the same range to be decomposed into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily decomposed into a lower third, a middle third, and an upper third, etc. As will also be understood by those skilled in the art, all language su...

Claims

1. A compound of formula I or a pharmaceutically acceptable salt thereof, Z—L 1 —D (Formula I) in: D is represented by the following structural formula: R 1 Yes -F, -CH 3 or -CF 3 ; R 2 Yes -H, -F, -OR 3 、-SR 3 、-S(O)R 4 、-S(O) 2 R 4 , C 1 -C 6 Alkyl or C 1 -C 6 Fluoroalkyl; or R 1 and R 2 The carbon atoms to which they are attached together form a methylenedioxy or difluoromethylenedioxy ring; R 3 Is H or C 1 -C 6 alkyl; R 4 It is C 1 -C 6 alkyl; L 1 Does not exist, is -(C 1 -C 6 Alkylene)-, -(C 1 -C 6 Alkylene)-X 1 -(C 1 -C 6 Alkylene)-, -X 1 '-(C 1 -C 6 Alkylene)-* or -(C 1 -C 6 Alkylene)-X 1 -L 2 -*; in * is the site of covalent attachment to Z; X 1 is -O-, -S-, -S(O)-, -S(O) 2 -, -C(=O)-, -NR 5 -, -NR 5 C(=O)- or -C(=O)NR 5 -; X 1 ' is -O-, -S-, -S(O)-, or -S(O) 2 -; L 2 is phenylene; Each R 5 Independently -H, C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl or benzyl; Z is -H or -X 2 ; X 2 is - OR 6 , - SR 6 , - S(O)R 6 , - S(O) 2 R 6 , - SSR 6 or - N(R 6 ) 2 ; Each R 6 Independently -H, C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl or benzyl; L 1 and L 2 Each independently optionally substituted by 1-4 selected from halogen, -CN, -OR 7 、-SR 7 、-N(R 7 ) 2 , C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 1 -C 6 Heteroalkyl, C 3 -C 6 Cycloalkyl, C 2 -C 10 Substitution of a heterocycloalkyl, aryl or heteroaryl substituent; and Each R 7 Independently H, C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl or benzyl; The condition is that if R 1 is F, then L 1 Yes-(C 1 -C 6 Alkylene)-, -(C 1 -C 6 Alkylene)-X 1 -(C 1 -C 6 Alkylene)-, -X 1 '-(C 1 -C 6 Alkylene)-* or -(C 1 -C 6 Alkylene)-X 1 -L 2 -*; in * is the site of covalent attachment to Z; and Z is -X 2 ;and The condition is that if R 1 is F and R 2 is -OMe, then -L 1 -Z cannot be -NH 2 .

2. A compound of formula I or a pharmaceutically acceptable salt thereof, Z—L 1 —D (Formula I) in: D is represented by the following structural formula: R 1 Yes -F, -CH 3 or -CF 3 ; R 2 Yes -H, -F, -OR 3 、-SR 3 、-S(O)R 4 、-S(O) 2 R 4 , C 1 -C 6 Alkyl or C 1 -C 6 Fluoroalkyl; or R 1 and R 2 The carbon atoms to which they are attached together form a methylenedioxy or difluoromethylenedioxy ring; R 3 Is H or C 1 -C 6 alkyl; R 4 It is C 1 -C 6 alkyl; L 1 Does not exist, is -(C 1 -C 6 Alkylene)-, -(C 1 -C 6 Alkylene)-X 1 -(C 1 -C 6 Alkylene)-, -X 1 '-(C 1 -C 6 Alkylene)-* or -(C 1 -C 6 Alkylene)-X 1 -L 2 -*; in * is the site of covalent attachment to Z; X 1 is -O-, -S-, -S(O)-, -S(O) 2 -, -C(=O)-, -NR 5 -, -NR 5 C(=O)- or -C(=O)NR 5 -; X 1 ' is -O-, -S-, -S(O)-, or -S(O) 2 -; L 2 is phenylene; Each R 5 Independently -H, C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl or benzyl; Z is -H or -X 2 ; X 2 is - OR 6 、-SR 6 、-S(O)R 6 、-S(O) 2 R 6 、-SSR 6 or - N(R 6 ) 2 ; Each R 6 Independently -H, C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl or benzyl; L 1 and L 2 Each independently optionally substituted by 1-4 selected from halogen, -CN, -OR 7 、-SR 7 、-N(R 7 ) 2 , C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 1 -C 6 Heteroalkyl, C 3 -C 6 Cycloalkyl, C 2 -C 10 Substitution of a heterocycloalkyl, aryl or heteroaryl substituent; and Each R 7 Independently H, C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl or benzyl; The condition is that if R 1 is F, then L 1 Yes-(C 1 -C 6 Alkylene)-, -(C 1 -C 6 Alkylene)-X 1 -(C 1 -C 6 Alkylene)-, -X 1 '-(C 1 -C 6 Alkylene)-* or -(C 1 -C 6 Alkylene)-X 1 -L 2 -*; in * is the site of covalent attachment to Z; and Z is -X 2 ; The condition is that if R 1 is F and R 2 is -OMe, then -L 1 -Z cannot be -NH 2 ;and The condition is that if R 1 is F and R 2 If it is -Me, then -L 1 -Z cannot be -CH 2 OH.

3. The compound according to claim 1 or claim 2, in: (i)R 1 is -H or -F; or (ii) R 1 Yes -F.

4. The compound according to claim 1 or claim 2, in: (i)R 2 Yes -H, -F, -OCF 3 , -CF 3 , -OMe, -OEt, -SMe, -S(O)Me, -S(O) 2 Me, -SEt, -S(O)Et, -S(O 2 )Et, methyl or ethyl; (ii) R 2 Yes - F; (iii) R 2 is -OMe, -SMe, -S(O)Me or methyl; or (iv) R 2 It's methyl.

5. The compound according to claim 1 or claim 2, in: (i)R 1 Yes -F and R 2 Yes - F; (ii) R 1 is methyl and R 2 Yes - F; or (iii) R 1 Yes -F and R 2 It is -methyl.

6. The compound according to claim 1 or claim 2, in: (i)-L 1 -Z is -H; (ii)-L 1 -Z is -(C 1 -C 6 Alkylene)-H or -(C 1 -C 6 Alkylene)-X 2 ; (iii)-L 1 -Z is methyl, ethyl, propyl or butyl; (iv)-L 1 -Z is -(C 1 -C 4 Alkylene)-OR 6 、-(C 1 -C 4 Alkylene)-SR 6 or -(C 1 -C 4 Alkylene)-N(R 6 ) 2 ; (v)-L 1 -Z is -CH 2 OH, -(CH 2 ) 2 OH, -(CH 2 ) 3 OH, -(CH 2 ) 4 OH, -CH 2 OMe, -(CH 2 ) 2 OMe, -(CH 2 ) 3 OMe, -(CH 2 ) 4 OMe, -CH 2 SH, -(CH 2 ) 2 SH, -(CH 2 ) 3 SH, -(CH 2 ) 4 SH, -CH 2 SMe, -(CH 2 ) 2 SMe, -(CH 2 ) 3 SMe, -(CH 2 ) 4 SMe, -CH 2 NH 2 , -(CH 2 ) 2 NH 2 , -(CH 2 ) 3 NH 2 , -(CH 2 ) 4 NH 2 ; (vi)-L 1 -Z is -(C 1 -C 5 Alkylene)-NR 5 C(=O)-(C 1 -C 5 Alkylene)-OR 6 、-(C 1 -C 5 Alkylene)-NR 5 C(=O)-(C 1 -C 5 Alkylene)-SR 6 、-(C 1 -C 5 Alkylene)-S-(C 1 -C 5 Alkylene)-SR 6 or -(C 1 -C 5 Alkylene)-S-(C 1 -C 5 Alkylene)-SSR 6 ; (vii)-L 1 -Z is -CH 2 NHC(=O)CH 2 OH, -CH 2 NHC(=O)(CH 2 ) 2 OH, -CH 2 NHC(=O)(CH 2 ) 3 OH, -CH 2 NHC(=O)(CH 2 ) 4 OH, -CH 2 NHC(=O)(CH 2 ) 5 OH, -CH 2 NHC(=O)CH 2 OMe, -CH 2 NHC(=O)(CH 2 ) 2 OMe, -CH 2 NHC(=O)(CH 2 ) 3 OMe, -CH 2 NHC(=O)(CH 2 ) 4 OMe, -CH 2 NHC(=O)(CH 2 ) 5 OMe, -CH 2 NHC(=O)CH 2 SH, -CH 2 NHC(=O)(CH 2 ) 2 SH, -CH 2 NHC(=O)(CH 2 ) 3 SH, -CH 2 NHC(=O)(CH 2 ) 4 SH, -CH 2 NHC(=O)(CH 2 ) 5 SH, -CH 2 NHC(=O)CH 2 SMe, -CH 2 NHC(=O)(CH 2 ) 2 SMe, -CH 2 NHC(=O)(CH 2 ) 3 SMe, -CH 2 NHC(=O)(CH 2 ) 4 SMe, -CH 2 NHC(=O)(CH 2 ) 5 SMe, -CH 2 SCH 2 OH, -CH 2 S(CH 2 ) 2 OH, -CH 2 S(CH 2 ) 3 OH, -CH 2 S(CH 2 ) 4 OH, -CH 2 S(CH 2 ) 5 OH, -CH 2 SCH 2 OMe, -CH 2 S(CH 2 ) 2 OMe, -CH 2 S(CH 2 ) 3 OMe, -CH 2 S(CH 2 ) 4 OMe, -CH 2 S(CH 2 ) 5 OMe, -CH 2 SCH 2 SH, -CH 2 S(CH 2 ) 2 SH, -CH 2 S(CH 2 ) 3 SH, -CH 2 S(CH 2 ) 4 SH, -CH 2 S(CH 2 ) 5 SH, -CH 2 SCH 2 SMe, -CH 2 S(CH 2 ) 2 SMe, -CH 2 S(CH 2 ) 3 SMe, -CH 2 S(CH 2 ) 4 SMe or -CH 2 S(CH 2 ) 5 SMe; (viii)-L 1 -Z is -X 1 '-(C 1 -C 4 Alkylene)-X 2 ; (ix)-L 1 -Z is -OCH 2 OH, -O(CH 2 ) 2 OH, -O(CH 2 ) 3 OH, -O(CH 2 ) 4 OH, -SCH 2 OH, -S(CH 2 ) 2 OH, -S(CH 2 ) 3 OH, -S(CH 2 ) 4 OH, -S(O)CH 2 OH, -S(O)(CH 2 ) 2 OH, -S(O)(CH 2 ) 3 OH, -S(O)(CH 2 ) 4 OH, -S(O) 2 CH 2 OH, -S(O) 2 (CH 2 ) 2 OH, -S(O) 2 (CH 2 ) 3 OH, -S(O) 2 (CH 2 ) 4 OH, -OCH 2 SMe, -O(CH 2 ) 2 SMe, -O(CH 2 ) 3 SMe, -O(CH 2 ) 4 SMe, -SCH 2 SMe, -S(CH 2 ) 2 SMe, -S(CH 2 ) 3 SMe, -S(CH 2 ) 4 SMe, -S(O)CH 2 SMe, -S(O)(CH 2 ) 2 SMe, -S(O)(CH 2 ) 3 SMe, -S(O)(CH 2 ) 4 SMe, -S(O) 2 CH 2 SMe, -S(O) 2 (CH 2 ) 2 SMe, -S(O) 2 (CH 2 ) 3 SMe or -S(O) 2 (CH 2 ) 4 SMe; (x)-L 1 -Z is -(C 1 -C 6 Alkylene)-X 1 -L 2 -X 2 ;or (xi)-L 1 -Z is 7. The compound of claim 1 or claim 2, wherein each R 5 is independently -H, methyl or benzyl.

8. The compound of claim 1 or claim 2, wherein each R 6 is independently -H, methyl or benzyl.

9. The compound according to claim 1, in: (i) The compound is any one selected from the following compounds: (ii) The compound is any one selected from the compounds in Table 1B.

10. A compound of formula II or a pharmaceutically acceptable salt thereof, E—A—Z'—L 1 —D (Formula II) in: D is represented by the following structural formula: R 1 Yes -H, -F, -CH 3 or -CF 3 ; R 2 Yes -H, -F, -OR 3 、-SR 3 、-S(O)R 4 、-S(O) 2 R 4 , C 1 -C 6 Alkyl or C 1 -C 6 Fluoroalkyl; or R 1 and R 2 The carbon atoms to which they are attached together form a methylenedioxy or difluoromethylenedioxy ring; provided that R 1 and R 2 Cannot be -H at the same time; R 3 Is H or C 1 -C 6 alkyl; R 4 It is C 1 -C 6 alkyl; L 1 Does not exist, is -(C 1 -C 6 Alkylene)-, -(C 1 -C 6 Alkylene)-X 1 -(C 1 -C 6 Alkylene)-, X 1 '-(C 1 -C 6 Alkylene)-* or -(C 1 -C 6 Alkylene)-X 1 -L 2 -*; in * is the site of covalent attachment to Z'; X 1 is -O-, -S-, -S(O)-, -S(O) 2 -, -C(=O)-, -NR 5 -, -NR 5 C(=O)- or -C(=O)NR 5 -; X 1 ' is -O-, -S-, -S(O)-, or -S(O) 2 -; L 2 is phenylene; Each R 5 Independently -H, C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl or benzyl; Z' is -O-CH 2 -NR 8 -*, -S-CH 2 -NR 8 -*, -NR 8 -*; in * is the site of covalent attachment to A; Each R 8 Independently -H, C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl or benzyl; L 1 and L 2 Each independently optionally substituted by 1-4 selected from halogen, -CN, -OR 7 、-SR 7 、-N(R 7 ) 2 , C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 1 -C 6 Heteroalkyl, C 3 -C 6 Cycloalkyl, C 2 -C 10 Substitution of a heterocycloalkyl, aryl or heteroaryl substituent; and Each R 7 Independently H, C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl or benzyl; A is a peptide comprising 2 to 10 amino acids; wherein A is optionally substituted with one or more polyols; and E is -C(=O)-L 3 -X 3 ; L 3 Yes-(C 1 -C 10 Alkylene)- or -Y 1 -(C 1 -C 10 Alkylene)-X 4 -Y 2 -(C 1 -C 10 Alkylene)-*; wherein * is covalently linked to X 3 The location of Y 1 Does not exist, is - (CR a R b O) n -or-(CR a R b CR a 'R b 'O) m -; X 4 Yes - NR 9 C(=O)- or -C(=O)NR 9 -; Y 2 Does not exist, is - (CR c R d O) o -or-(CR c R d CR c 'R d 'O) p -; n, m, o and p are each independently 1-10; Each R a , R b , R a '、R b '、R c , R d , R c ' and R d 'Independently -H, C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl or benzyl; Where L 3 Optionally substituted by 0-4 selected from halogen, -CN, -OR 11 、-SR 11 、-N(R 11 ) 2 , C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 1 -C 6 Heteroalkyl, C 3 -C 6 Cycloalkyl, C 2 -C 10 Substitution of heterocycloalkyl, aryl, heteroaryl and polyol substituents; Each R 11 Independently H, C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl or benzyl; X 3 yes -C(=O)-CR bb R cc -W', -NR ee -C(=O)-CR bb R cc -W' or -SR 10 ; Each W' is independently -H, -N(R gg ) 2 , C 1 -C 10 Alkyl, C 1 -C 10 Alkenyl, C 1 -C 10 Alkynyl, C 3 -C 6 Cycloalkyl, aryl, heteroaryl or -(CH 2 CH 2 O) q -R ff ; q is 1 to 24; Each R aa , R bb , R cc , R ee and R ff is independently -H or optionally substituted C 1 -C 6 alkyl; Each R YY and R XX Independently -H or C 1 -C 6 alkyl; R gg Each independently is -H or C 1 -C 6 Alkyl; and R 9 and R 10 Each independently is -H, C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl or benzyl.

11. The compound according to claim 10, in: (i)R 1 is -H or -F; or (ii) R 1 Yes -F.

12. The compound according to claim 10, in: (i)R 2 Yes -H, -F, -OCF 3 , -CF 3 , -OMe, -OEt, -SMe, -S(O)Me, -S(O) 2 Me, -SEt, -S(O)Et, -S(O 2 )Et, methyl or ethyl; (ii) R 2 Yes - F; (iii) R 2 is -OMe, -SMe, -S(O)Me or methyl; or (iv) R 2 It's methyl.

13. The compound according to claim 10, in: (i)R 1 Yes -F and R 2 Yes - F; (ii) R 1 is methyl and R 2 Yes - F; or (iii) R 1 Yes -F and R 2 It is -methyl.

14. The compound according to claim 10, in: (i)-L 1 -Z'-*Yes-(C 1 -C 4 Alkylene)-O-CH 2 -NR 8 -*、-(C 1 -C 4 Alkylene)-S-CH 2 -NR 8 -* or -(C 1 -C 4 Alkylene)-NR 8 -*, where * is the site of covalent attachment to A; (ii)-L 1 -Z'-* is -CH 2 O-CH 2 NH-*, -(CH 2 ) 2 O-CH 2 NH-*, -(CH 2 ) 3 O-CH 2 NH-*, -(CH 2 ) 4 O-CH 2 NH-*, -CH 2 S-CH 2 NH-*, -(CH 2 ) 2 S-CH 2 NH-*, -(CH 2 ) 3 S-CH 2 NH-*, -(CH 2 ) 4 S-CH 2 NH-*, -CH 2 NH-*, -(CH 2 ) 2 NH-*, -(CH 2 ) 3 NH-* or -(CH 2 ) 4 NH-; (iii)-L 1 -Z'-*Yes-(C 1 -C 5 Alkylene)-NR 5 C(=O)-(C 1 -C 5 Alkylene)-O-CH 2 -NR 8 -*、-(C 1 -C 5 Alkylene)-NR 5 C(=O)-(C 1 -C 5 Alkylene)-S-CH 2 -NR 8 -*、-(C 1 -C 5 Alkylene)-S-(C 1 -C 5 Alkylene)-S-CH 2 -NR 8 -* or -(C 1 -C 5 Alkylene)-S-(C 1 -C 5 Alkylene)-SS-CH 2 -NR 8 -*, where * is the site of covalent attachment to A; (iv)-L 1 -Z'-* is -CH 2 NHC(=O)CH 2 O-CH 2 -NH-*, -CH 2 NHC(=O)(CH 2 ) 2 O-CH 2 -NH-*, -CH 2 NHC(=O)(CH 2 ) 3 O-CH 2 -NH-*, -CH 2 NHC(=O)(CH 2 ) 4 O-CH 2 -NH-*, -CH 2 NHC(=O)(CH 2 ) 5 O-CH 2 -NH-*, -CH 2 NHC(=O)CH 2 S-CH 2 -NH-*, -CH 2 NHC(=O)(CH 2 ) 2 S-CH 2 -NH-*, -CH 2 NHC(=O)(CH 2 ) 3 S-CH 2 -NH-*, -CH 2 NHC(=O)(CH 2 ) 4 S-CH 2 -NH-*, -CH 2 NHC(=O)(CH 2 ) 5 S-CH 2 -NH-*, -CH 2 SCH 2 O-CH 2 -NH-*, -CH 2 S(CH 2 ) 2 O-CH 2 -NH-*, -CH 2 S(CH 2 ) 3 O-CH 2 -NH-*, -CH 2 S(CH 2 ) 4 O-CH 2 -NH-*, -CH 2 S(CH 2 ) 5 O-CH 2 -NH-*, -CH 2 SCH 2 S-CH 2 -NH-*, -CH 2 S(CH 2 ) 2 S-CH 2 -NH-*, -CH 2 S(CH 2 ) 3 S-CH 2 -NH-*, -CH 2 S(CH 2 ) 4 S-CH 2 -NH-* or -CH 2 S(CH 2 ) 5 S-CH 2 -NH-*, where * is the site of covalent attachment to A; (v)-L 1 -Z'-* is -X 1 '-(C 1 -C 4 Alkylene)-O-CH 2 -NR 8 -*, -X 1 '-(C 1 -C 4 Alkylene)-S-CH 2 -NR 8 -* or -X 1 '-(C 1 -C 4 Alkylene)-NR 8 -*, where * is the site of covalent attachment to A; (vi)-L 1 -Z'-* is -OCH 2 O-CH 2 -NH-*, -O(CH 2 ) 2 O-CH 2 -NH-*, -O(CH 2 ) 3 O-CH 2 -NH-*, -O(CH 2 ) 4 O-CH 2 -NH-*, -SCH 2 O-CH 2 -NH-*, -S(CH 2 ) 2 O-CH 2 -NH-*, -S(CH 2 ) 3 O-CH 2 -NH-*, -S(CH 2 ) 4 O-CH 2 -NH-*, -S(O)CH 2 O-CH 2 -NH-*, -S(O)(CH 2 ) 2 O-CH 2 -NH-*, -S(O)(CH 2 ) 3 O-CH 2 -NH-*, -S(O)(CH 2 ) 4 O-CH 2 -NH-*, -S(O) 2 CH 2 O-CH 2 -NH-*, -S(O) 2 (CH 2 ) 2 O-CH 2 -NH-*, -S(O) 2 (CH 2 ) 3 O-CH 2 -NH-*, -S(O) 2 (CH 2 ) 4 O-CH 2 -NH-*, -OCH 2 S-CH 2 -NH-*, -O(CH 2 ) 2 S-CH 2 -NH-*、-O(CH 2 ) 3 S-CH 2 -NH-*、-O(CH 2 ) 4 S-CH 2 -NH-*、-SCH 2 S-CH 2 -NH-*、-S(CH 2 ) 2 S-CH 2 -NH-*、-S(CH 2 ) 3 S-CH 2 -NH-*、-S(CH 2 ) 4 S-CH 2 -NH-*、-S(O)CH 2 S-CH 2 -NH-*、-S(O)(CH 2 ) 2 S-CH 2 -NH-*、-S(O)(CH 2 ) 3 S-CH 2 -NH-*、-S(O)(CH 2 ) 4 S-CH 2 -NH-*、-S(O) 2 CH 2 S-CH 2 -NH-*、-S(O) 2 (CH 2 ) 2 S-CH 2 -NH-*、-S(O) 2 (CH 2 ) 3 S-CH 2 -NH-*、-S(O) 2 (CH 2 ) 4 S-CH 2 -NH-*、-OCH 2 -NH-*、-O(CH 2 ) 2 -NH-*、-O(CH 2 ) 3 -NH-*、-O(CH 2 ) 4 S-NH-*、-SCH 2 -NH-*、-S(CH 2 ) 2 -NH-*、-S(CH 2 ) 3 -NH-*、-S(CH 2 ) 4 -NH-*、-S(O)CH 2 -NH-*、-S(O)(CH 2 ) 2 -NH-*、-S(O)(CH 2 ) 3 -NH-*、-S(O)(CH 2 ) 4 -NH-*, -S(O) 2 CH 2 -NH-*, -S(O) 2 (CH 2 ) 2 -NH-*, -S(O) 2 (CH 2 ) 3 -NH-* or -S(O) 2 (CH 2 ) 4 -NH-*, where * is the site of covalent attachment to A; (vii)-L 1 -Z'-*Yes-(C 1 -C 6 Alkylene)-X 1 -L 2 -Z'-*, wherein * is the site of covalent attachment to A; or (viii)-L 1 -Z'-*Yes where * is the site of covalent attachment to A.

15. The compound of claim 10, wherein each R 5 is independently -H, methyl or benzyl.

16. The compound of claim 10, wherein each R 8 is independently -H, methyl or benzyl.

17. The compound according to claim 10, in: (i) A is a peptide comprising 2 to 8 amino acids; or (ii) A is a peptide comprising 2 to 4 amino acids.

18. The compound according to claim 17, in: (i) at least one amino acid in the peptide is an L amino acid; (ii) every amino acid in the peptide is an L amino acid; or (iii) at least one amino acid in the peptide is a D amino acid.

19. The compound of claim 10, wherein A is -(AA 1 )-(AA 2 ) a1 -*, where * is the site of covalent attachment to E; AA 1 and AA 2 are each independently an amino acid residue; and a1 is an integer from 1 to 9.

20. The compound according to claim 19, in: (i) - AA1-(AA2)a1-* is - Gly - Gly - Gly - *, - Ala - Val - *, - Val - Ala - *, - Val - Cit - *, - Val - Lys - *, - Lys - Val - *, - Phe - Lys - *, - Lys - Phe - *, - Lys - Lys - *, - Ala - Lys - *, - Lys - Ala - *, - Phe - Cit - *, - Cit - Phe - *, - Leu - Cit - *, - Cit - Leu - * - Ile - Cit - *, - Phe - Ala - *, - Ala - Phe - *, - Phe - N9 - tosyl - Arg - *, - N9 - tosyl - Arg - Phe - *, - Phe - N9 - nitro - Arg - *, - N9 - nitro - Arg - Phe*, - Phe - Phe - Lys - *, - Lys - Phe - Phe - *, - Gly - Phe - Lys - *, Lys - Phe - Gly - *, - Leu - Ala - Leu - *, - Ile - Ala - Leu - *, - Leu - Ala - Ile - *, - Val - Ala - Val - *, - Ala - Leu - Ala - Leu - *, - Leu - Ala - Leu - Ala - *, - β - Ala - Leu - Ala - Leu - *, - Gly - Phe - Leu - Gly - *, - Gly - Leu - Phe - Gly - *, - Val - Arg - *, - Arg - Val - *, - Arg - Arg - *, - Ala - Ala - *, - Ala - Met - *, - Met - Ala - *, - Thr - Thr - *, - Thr - Met - *, - Met - Thr - *, - Leu - Ala - *, - Ala - Leu - *, - Cit - Val - *, - Gln - Val - *, - Val - Gln - *, - Ser - Val - *, - Val - Ser - *, - Ser - Ala - *, - Ser - Gly - *, - Ala - Ser - *, - Gly - Ser - *, - Leu - Gln - *, - Gln - Leu - *, - Phe - Arg - *, - Arg - Phe - *, - Tyr - Arg - *, - Arg - Tyr - *, - Phe - Gln - *, - Gln - Phe - *, - Val - Thr - *, - Thr - Val - *, - Met - Tyr - * and - Tyr - Met - *; (ii)-AA 1 -(AA 2 ) a1 -*Yu-Val-D-Lys-*、-Val-D-Arg-*、-L-Val-Cit-*、-L-Val-Lys-*、-L-Val-Arg-*、-L-Val-D-Cit-*、-L-Phe-Phe-Lys-*、-L-Val-D-Lys-*、-L-Val-D-Arg-*、-L-Arg- D-Arg-*、-L-Ala-Ala-*、-L-Ala-D-Ala-*、-Ala-D-Ala-*、-Val-D-Cit-*、-L-Ala-L-Ala-*、-L-Ala-L-Val-*、-L-Gln-L-Val-*、-L-Gln-L-Leu-*、-L-Ser-L-Val-*; (iii)-AA 1 -(AA 2 ) a1 -*Yes: - Ala - Ala - * - Ala - Val - * - Val - Ala - * - Gln - Leu - * - Leu - Gln - * - Ala - Ala - Ala - * - Ala - Ala - Ala - Ala - * - Gly - Ala - Gly - Gly - * - Gly - Gly - Ala - Gly - * -Gly-Val-Gly-Gly-*, -Gly-Gly-Val-Gly-*, -Gly-Phe-Gly-Gly-* or -Gly-Gly-Phe-Gly-*; (iv)-AA 1 -(AA 2 ) a1 -* is: -L-Ala-L-Ala-*, -L-Ala-D-Ala-*, -L-Ala-L-Val-*, -L-Ala-D-Val-*, -L-Val-L-Ala-*, -L-Val-D-Ala-* -L-Gln-L-Leu-*, -L-Gln-D-Leu-*, -L-Leu-L-Gln-*, -L-Leu-D-Gln-*, -L-Ala-L-Ala- L-Ala-*, -L-Ala-D-Ala-L-Ala-*, -L-Ala-L-Ala-D-Ala-*, -L-Ala-L-Ala-L-Ala-L-Ala -*, -L-Ala-D-Ala-L-Ala-L-Ala-*, -L-Ala-L-Ala-D-Ala-L-Ala-*, -L-Ala-L-Ala-L-A la-D-Ala-*, -Gly-L-Ala-Gly-Gly-*, -Gly-Gly-L-Ala-Gly-*, -Gly-D-Ala-Gly-Gly-*, -Gly-Gly-D-Ala-Gly-*, -Gly-L-Val-Gly-Gly-*, -Gly-Gly-L-Val-Gly-*, -Gly-D-Val-Gly-Gly-*, -Gly-Gly-D-Val-Gly-*, -Gly-L-Phe-Gly-Gly-* or -Gly-Gly-L-Phe-Gly-*; or (v)-AA 1 -(AA 2 ) a1 -*Yes: -L-Ala-L-Ala-*, -L-Ala-D-Ala-LAla-*, -L-Ala-L-Ala-L-Ala-* or -L-Ala-L-Ala-L-Ala-L-Ala-*.

21. The compound of claim 10, wherein A is substituted with one or more polyols.

22. The compound of claim 10, wherein E is substituted with one or more polyols.

23. The compound according to claim 10, in: (i) The polyol is -(C 1 -C 6 Alkylene)-X 5 -Y 3 , X 5 Yes - NR 12 C(=O)- or -C(=O)NR 12 -, Y 3 Yes-C 1 -C 10 Alkyl, where Y 3 is substituted with 0-10 OH groups, and R 12 Yes - H, C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl or benzyl; or (ii) Polyols are Where R 12 It is H or methyl.

24. The compound according to claim 10, in: (i) E is -C(=O)-(C 1 -C 10 Alkylene)-X 3 ; (ii) E is (iii) E is -C(=O)-Y 1 -(C 1 -C 10 Alkylene)-X 4 -(C 1 -C 10 Alkylene)-X 3 ; Y 1 is -(CR a R b O) n - or -(CR a R b CR a 'R b 'O) m -; X 4 Yes - NR 9 C(=O)-; and X 3 yes -C(=O)-CR bb R cc -W', NR ee -C(=O)-CR bb R cc -W' or -SR 10 ;or (iv) E is -C(=O)-Y 1 -(CH 2 ) 2 -X 4 -(CH 2 ) 2 -X 3 ; Y 1 is -(CH 2 O) n - or -(CH 2 CH 2 O) m -; X 4 is -NHC(=O)-; n is 2; m is 2 to 6; X 3 yes -C(=O)-CR bb R cc -W', NR ee -C(=O)-CR bb R cc -W' or -SR 10 .

25. The compound of claim 10, wherein the compound is any one selected from the compounds of Table 2.

26. A compound of formula III or a pharmaceutically acceptable salt thereof, CBA—E'—A—Z'—L 1 —D (Formula III) in: D is represented by the following structural formula: R 1 Yes -H, -F, -CH 3 or -CF 3 ; R 2 Yes -H, -F, -OR 3 、-SR 3 、-S(O)R 4 、-S(O) 2 R 4 , C 1 -C 6 Alkyl or C 1 -C 6 Fluoroalkyl; or R 1 and R 2 The carbon atoms to which they are attached together form a methylenedioxy or difluoromethylenedioxy ring; provided that R 1 and R 2 Cannot be -H at the same time; R 3 Is H or C 1 -C 6 alkyl; R 4 It is C 1 -C 6 alkyl; L 1 Does not exist, is -(C 1 -C 6 Alkylene)-, -(C 1 -C 6 Alkylene)-X 1 -(C 1 -C 6 Alkylene)-, X 1 '-(C 1 -C 6 Alkylene)-* or -(C 1 -C 6 Alkylene)-X 1 -L 2 -*; in * is the site of covalent attachment to Z'; X 1 is -O-, -S-, -S(O)-, -S(O) 2 -, -C(=O)-, -NR 5 -, -NR 5 C(=O)- or -C(=O)NR 5 -; X 1 ' is -O-, -S-, -S(O)-, or -S(O) 2 -; L 2 is phenylene; Each R 5 Independently -H, C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl or benzyl; Z' is -O-CH 2 -NR 8 -*, -S-CH 2 -NR 8 -*, -NR 8 -*; in * is the site of covalent attachment to A; Each R 8 Independently -H, C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl or benzyl; L 1 and L 2 Each independently optionally substituted by 1-4 selected from halogen, -CN, -OR 7 、-SR 7 、-N(R 7 ) 2 , C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 1 -C 6 Heteroalkyl, C 3 -C 6 Cycloalkyl, C 2 -C 10 Substitution of a heterocycloalkyl, aryl or heteroaryl substituent; and Each R 7 Independently H, C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl or benzyl; A is a peptide comprising 2 to 10 amino acids; wherein A is optionally substituted with one or more polyols; E' is -C(=O)-L 3 -X 6 -*; wherein * is the site of covalent attachment to CBA; L 3 Yes-(C 1 -C 10 Alkylene)-or-Y 1 -(C 1 -C 10 Alkylene)-X 4 -Y 2 -(C 1 -C 10 Alkylene)-*; wherein * is covalently linked to X 6 The location of Y 1 Does not exist, is - (CR a R b O) n -or-(CR a R b CR a 'R b 'O) m -; X 4 Yes - NR 9 C(=O)- or -C(=O)NR 9 -; Y 2 Does not exist, is - (CR c R d O) o -or-(CR c R d CR c 'R d 'O) p -; n, m, o and p are each independently 1-10; Each R a , R b , R a '、R b '、R c , R d , R c ' and R d 'Independently -H, C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl or benzyl; Where L 3 Optionally substituted by 0-4 selected from halogen, -CN, -OR 11 、-SR 11 、-N(R 11 ) 2 , C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 1 -C 6 Heteroalkyl, C 3 -C 6 Cycloalkyl, C 2 -C 10 Substitution of heterocycloalkyl, aryl, heteroaryl and polyol substituents; Each R 11 Independently H, C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl or benzyl; X 6 is -C(=O)-CR bb R cc -* or -NR ee -C(=O)-CR bb R cc -*; Where * is the site of covalent attachment to CBA; Each R aa , R bb , R cc and R ee is independently -H or optionally substituted C 1 -C 6 alkyl; Each R YY and R XX Independently -H or C 1 -C 6 alkyl; R 9 Independently -H, C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl or benzyl; and CBA is a cell binding agent.

27. The compound according to claim 26, in: (i)R 1 is -H or -F; or (ii) R 1 Yes -F.

28. The compound according to claim 26, in: (i)R 2 Yes -H, -F, -OCF 3 , -CF 3 , -OMe, -OEt, -SMe, -S(O)Me, -S(O) 2 Me, -SEt, -S(O)Et, -S(O 2 )Et, methyl or ethyl; (ii) R 2 Yes - F; (iii) R 2 is -OMe, -SMe, -S(O)Me or methyl; or (iv) R 2 It's methyl.

29. The compound according to claim 26, in: (i)R 1 Yes -F and R 2 Yes - F; (ii) R 1 is methyl and R 2 Yes - F; or (iii) R 1 Yes -F and R 2 It is -methyl.

30. The compound of claim 26, in: (i)-L 1 -Z'-*Yes-(C 1 -C 4 Alkylene)-O-CH 2 -NR 8 -*、-(C 1 -C 4 Alkylene)-S-CH 2 -NR 8 -* or -(C 1 -C 4 Alkylene)-NR 8 -*, where * is the site of covalent attachment to A; (ii)-L 1 -Z'-*Yes-CH 2 O-CH 2 NH-*, -(CH 2 ) 2 O-CH 2 NH-*, -(CH 2 ) 3 O-CH 2 NH-*, -(CH 2 ) 4 O-CH 2 NH-*, -CH 2 S-CH 2 NH-*, -(CH 2 ) 2 S-CH 2 NH-*, -(CH 2 ) 3 S-CH 2 NH-*, -(CH 2 ) 4 S-CH 2 NH-*, -CH 2 NH-*, -(CH 2 ) 2 NH-*, -(CH 2 ) 3 NH-* or -(CH 2 ) 4 NH-, where * is the site of covalent attachment to A; (iii)-L 1 -Z'-*Yes-(C 1 -C 5 Alkylene)-NR 5 C(=O)-(C 1 -C 5 Alkylene)-O-CH 2 -NR 8 -*、-(C 1 -C 5 Alkylene)-NR 5 C(=O)-(C 1 -C 5 Alkylene)-S-CH 2 -NR 8 -*、-(C 1 -C 5 Alkylene)-S-(C 1 -C 5 Alkylene)-S-CH 2 -NR 8 -* or -(C 1 -C 5 Alkylene)-S-(C 1 -C 5 Alkylene)-SS-CH 2 -NR 8 -*, where * is the site of covalent attachment to A; (iv)-L 1 -Z'-* is -CH 2 NHC(=O)CH 2 O-CH 2 -NH-*, -CH 2 NHC(=O)(CH 2 ) 2 O-CH 2 -NH-*, -CH 2 NHC(=O)(CH 2 ) 3 O-CH 2 -NH-*, -CH 2 NHC(=O)(CH 2 ) 4 O-CH 2 -NH-*, -CH 2 NHC(=O)(CH 2 ) 5 O-CH 2 -NH-*, -CH 2 NHC(=O)CH 2 S-CH 2 -NH-*, -CH 2 NHC(=O)(CH 2 ) 2 S-CH 2 -NH-*, -CH 2 NHC(=O)(CH 2 ) 3 S-CH 2 -NH-*, -CH 2 NHC(=O)(CH 2 ) 4 S-CH 2 -NH-*, -CH 2 NHC(=O)(CH 2 ) 5 S-CH 2 -NH-*, -CH 2 SCH 2 O-CH 2 -NH-*, -CH 2 S(CH 2 ) 2 O-CH 2 -NH-*, -CH 2 S(CH 2 ) 3 O-CH 2 -NH-*, -CH 2 S(CH 2 ) 4 O-CH 2 -NH-*, -CH 2 S(CH 2 ) 5 O-CH 2 -NH-*, -CH 2 SCH 2 S-CH 2 -NH-*, -CH 2 S(CH 2 ) 2 S-CH 2 -NH-*, -CH 2 S(CH 2 ) 3 S-CH 2 -NH-*, -CH 2 S(CH 2 ) 4 S-CH 2 -NH-* or -CH 2 S(CH 2 ) 5 S-CH 2 -NH-*; (v)-L 1 -Z'-* is -X 1 '-(C 1 -C 4 Alkylene)-O-CH 2 -NR 8 -*, -X 1 '-(C 1 -C 4 Alkylene)-S-CH 2 -NR 8 -* or -X 1 '-(C 1 -C 4 Alkylene)-NR 8 -*, where * is the site of covalent attachment to A; (vi)-L 1 -Z'-* is -OCH 2 O-CH 2 -NH-*, -O(CH 2 ) 2 O-CH 2 -NH-*, -O(CH 2 ) 3 O-CH 2 -NH-*, -O(CH 2 ) 4 O-CH 2 -NH-*, -SCH 2 O-CH 2 -NH-*, -S(CH 2 ) 2 O-CH 2 -NH-*, -S(CH 2 ) 3 O-CH 2 -NH-*, -S(CH 2 ) 4 O-CH 2 -NH-*, -S(O)CH 2 O-CH 2 -NH-*, -S(O)(CH 2 ) 2 O-CH 2 -NH-*, -S(O)(CH 2 ) 3 O-CH 2 -NH-*, -S(O)(CH 2 ) 4 O-CH 2 -NH-*, -S(O) 2 CH 2 O-CH 2 -NH-*, -S(O) 2 (CH 2 ) 2 O-CH 2 -NH-*, -S(O) 2 (CH 2 ) 3 O-CH 2 -NH-*, -S(O) 2 (CH 2 ) 4 O-CH 2 -NH-*, -OCH 2 S-CH 2 -NH-*, -O(CH 2 ) 2 S-CH 2 -NH-*、-O(CH 2 ) 3 S-CH 2 -NH-*、-O(CH 2 ) 4 S-CH 2 -NH-*、-SCH 2 S-CH 2 -NH-*、-S(CH 2 ) 2 S-CH 2 -NH-*、-S(CH 2 ) 3 S-CH 2 -NH-*、-S(CH 2 ) 4 S-CH 2 -NH-*、-S(O)CH 2 S-CH 2 -NH-*、-S(O)(CH 2 ) 2 S-CH 2 -NH-*、-S(O)(CH 2 ) 3 S-CH 2 -NH-*、-S(O)(CH 2 ) 4 S-CH 2 -NH-*、-S(O) 2 CH 2 S-CH 2 -NH-*、-S(O) 2 (CH 2 ) 2 S-CH 2 -NH-*、-S(O) 2 (CH 2 ) 3 S-CH 2 -NH-*、-S(O) 2 (CH 2 ) 4 S-CH 2 -NH-*、-OCH 2 -NH-*、-O(CH 2 ) 2 -NH-*、-O(CH 2 ) 3 -NH-*、-O(CH 2 ) 4 S-NH-*、-SCH 2 -NH-*、-S(CH 2 ) 2 -NH-*, -S(CH 2 ) 3 -NH-*, -S(CH 2 ) 4 -NH-*, -S(O)CH 2 -NH-*, -S(O)(CH 2 ) 2 -NH-*, -S(O)(CH 2 ) 3 -NH-*, -S(O)(CH 2 ) 4 -NH-*, -S(O) 2 CH 2 -NH-*, -S(O) 2 (CH 2 ) 2 -NH-*, -S(O) 2 (CH 2 ) 3 -NH-* or -S(O) 2 (CH 2 ) 4 -NH-*; (vii)-L 1 -Z'-*Yes-(C 1 -C 6 Alkylene)-X 1 -L 2 -Z'-*, wherein * is the site of covalent attachment to A; or (viii)-L 1 -Z'-*Yes where * is the site of covalent attachment to A.

31. The compound of claim 26, wherein each R 5 is independently -H, methyl or benzyl.

32. The compound of claim 26, wherein each R 8 is independently -H, methyl or benzyl.

33. The compound of claim 26, in: (i) A is a peptide comprising 2 to 8 amino acids; or (ii) A is a peptide comprising 2 to 4 amino acids.

34. The compound of claim 33, in: (i) at least one amino acid in the peptide is an L amino acid; (ii) every amino acid in the peptide is an L amino acid; or (iii) at least one amino acid in the peptide is a D amino acid.

35. The compound of claim 26, wherein A is -(AA 1 )-(AA 2 ) a1 -*, where * is the point connected to E', AA 1 and AA 2 are each independently an amino acid residue; and a1 is an integer from 1 to 9.

36. The compound of claim 35, in: (i) - AA1 - (AA2)a1 - * is - Gly - Gly - Gly - *, - Ala - Val - *, - Val - Ala - *, - Val - Cit - *, - Val - Lys - *, - Lys - Val - *, - Phe - Lys - *, - Lys - Phe - *, - Lys - Lys - *, - Ala - Lys - *, - Lys - Ala - *, - Phe - Cit - *, - Cit - Phe - *, - Leu - Cit - *, - Cit - Leu - * - Ile - Cit - *, - Phe - Ala - *, - Ala - Phe - *, - Phe - N9 - tosyl - Arg - *, - N9 - tosyl - Arg - Phe - *, - Phe - N9 - nitro - Arg - *, - N9 - nitro - Arg - Phe *, - Phe - Phe - Lys - *, - Lys - Phe - Phe - *, - Gly - Phe - Lys - *, Lys - Phe - Gly - *, - Leu - Ala - Leu - *, - Ile - Ala - Leu - *, - Leu - Ala - Ile - *, - Val - Ala - Val - *, - Ala - Leu - Ala - Leu - *, - Leu - Ala - Leu - Ala - *, - β - Ala - Leu - Ala - Leu - *, - Gly - Phe - Leu - Gly - *, - Gly - Leu - Phe - Gly - *, - Val - Arg - *, - Arg - Val - *, - Arg - Arg - *, - Ala - Ala - *, - Ala - Met - *, - Met - Ala - *, - Thr - Thr - *, - Thr - Met - *, - Met - Thr - *, - Leu - Ala - *, - Ala - Leu - *, - Cit - Val - *, - Gln - Val - *, - Val - Gln - *, - Ser - Val - *, - Val - Ser - *, - Ser - Ala - *, - Ser - Gly - *, - Ala - Ser - *, - Gly - Ser - *, - Leu - Gln - *, - Gln - Leu - *, - Phe - Arg - *, - Arg - Phe - *, - Tyr - Arg - *, - Arg - Tyr - *, - Phe - Gln - *, - Gln - Phe - *, - Val - Thr - *, - Thr - Val - *, - Met - Tyr - * and - Tyr - Met - *; (ii)-AA 1 -(AA 2 ) a1 -*Yu-Val-D-Lys-*、-Val-D-Arg-*、-L-Val-Cit-*、-L-Val-Lys-*、-L-Val-Arg-*、-L-Val-D-Cit-*、-L-Phe-Phe-Lys-*、-L-Val-D-Lys-*、-L-Val-D-Arg-*、-L-Arg- D-Arg-*、-L-Ala-Ala-*、-L-Ala-D-Ala-*、-Ala-D-Ala-*、-Val-D-Cit-*、-L-Ala-L-Ala-*、-L-Ala-L-Val-*、-L-Gln-L-Val-*、-L-Gln-L-Leu-*、-L-Ser-L-Val-*; (iii)-AA 1 -(AA 2 ) a1 -*Yes: - Ala - Ala - * - Ala - Val - * - Val - Ala - * - Gln - Leu - * - Leu - Gln - * -Ala-Ala-Ala-*, -Ala-Ala-Ala-Ala-*, -Gly-Ala-Gly-Gly-*, -Gly-Gly-Ala-Gly-*, -Gly-Val-Gly-Gly-*, -Gly-Gly-Val-Gly-*, -Gly-Phe-Gly-Gly-* or -Gly-Gly-Phe-Gly-*; (iv)-AA 1 -(AA 2 ) a1 -* is: -L-Ala-L-Ala-*, -L-Ala-D-Ala-*, -L-Ala-L-Val-*, -L-Ala-D-Val-*, -L-Val-L-Ala-*, -L-Val-D-Ala-* -L-Gln-L-Leu-*, -L-Gln-D-Leu-*, -L-Leu-L-Gln-*, -L-Leu-D-Gln-*, -L-Ala-L-Ala-L-Ala-*, -L-Ala-D-Ala-L-Ala-*, -L-Ala-L-Ala-D-Ala-*, -L-Ala-L-Ala-L-Ala-L-Ala-*, -L-Ala-D-Ala-L-Ala-L-Ala-*, -L-Ala-L-Ala-D-Ala-L-Ala-*, -L-Ala-L-Ala-L-Ala-D-Ala-*, -Gly-L-Ala-Gly-Gly-*, -Gly-Gly-L-Ala-Gly-*, -Gly-D-Ala-Gly-Gly-*, -Gly-Gly-D-Ala-Gly-*, -Gly-L-Val-Gly-Gly-*, -Gly-Gly-L-Val-Gly-*, -Gly-D-Val-Gly-Gly-*, -Gly-Gly-L-Val-Gly-*, -Gly-L-Phe-Gly-Gly-* or -Gly-Gly-L-Phe-Gly-*; or (v)-AA 1 -(AA 2 ) a1 -* is: -L-Ala-L-Ala-*, -L-Ala-D-Ala-L-Ala-*, -L-Ala-L-Ala-L-Ala-*, or -L-Ala-L-Ala-L-Ala-L-Ala-*.

37. The compound of claim 26, wherein A is substituted with one or more polyols.

38. The compound of claim 26, wherein E' is substituted with one or more polyols.

39. The compound of claim 26, in: (i) The polyol is -(C 1 -C 6 Alkylene)-X 5 -Y 3 , X 5 Yes - NR 12 C(=O)- or -C(=O)NR 12 -, Y 3 Yes-C 1 -C 10 Alkyl, where Y 3 is substituted with 0-10 OH groups, and R 12 Yes - H, C 1 -C 6 Alkyl, C 1 -C 6 Fluoroalkyl, C 3 -C 6 cycloalkyl, aryl, heteroaryl or benzyl; or (ii) Polyols are Where R 12 It is H or methyl.

40. The compound of claim 26, in: (i) E' is -C(=O)-(C 1 -C 10 Alkylene)-X 6 -*; (ii) E' is -C(=O)CH 2 CH 2 -C(=O)-CR bb R cc -* or -C(=O)CH 2 CH 2 -NR ee -C(=O)-CR bb R cc -*; wherein * is the site of covalent attachment to CBA; (iii) E' is -C(=O)-Y 1 -(C 1 -C 10 Alkylene)-X 4 -(C 1 -C 10 Alkylene)-X 6 -*; Y 1 is -(CR a R b O) n - or -(CR a R b CR a 'R b 'O) m -; X 4 Yes - NR 9 C(=O)-; and X 6 yes -C(=O)-CR bb R cc -* or -NR ee -C(=O)-CR bb R cc -*; wherein * is the site of covalent attachment to CBA; or (iv) E' is -C(=O)-Y 1 -(CH 2 ) 2 -X 4 -(CH 2 ) 2 -X 6 -*; Y 1 is -(CH 2 O) n - or -(CH 2 CH 2 O) m -; X 4 is -NHC(=O)-; n is 2; m is 2 to 6; X 6 yes -C(=O)-CR bb R cc -* or -NR ee -C(=O)-CR bb R cc -*; wherein * is the site of covalent attachment to CBA.

41. The compound of claim 26, wherein the CBA comprises a -SH group covalently linked to E' to provide -C(=O)-CR bb R cc -S-CBA or -NR ee -C(=O)-CR bb R cc -S-CBA.

42. The compound of claim 26, wherein CBA is an antibody and -E'-A-Z'-L 1 —D is the drug-linker structure, in: (i) the average number of drug-linker structures conjugated to each antibody is in the range of 2 to 10; (ii) the average number of drug-linker structures conjugated to each antibody is in the range of 2 to 10; (iii) the average number of drug-linker structures conjugated per antibody is in the range of 6 to 8; or (iv) The average number of drug-linker structures conjugated to each antibody was 8.

43. The compound of claim 26, in: (i) the CBA is an antibody, a single-chain antibody, an antibody fragment that specifically binds to a target cell, a monoclonal antibody, a single-chain monoclonal antibody or a monoclonal antibody fragment that specifically binds to a target cell, a chimeric antibody, a chimeric antibody fragment that specifically binds to a target cell, a domain antibody, a domain antibody fragment that specifically binds to a target cell, a probody, a nanobody, a hexabody, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor or a nutrient transport molecule; (ii) the CBA binds to a target cell selected from the group consisting of tumor cells, virus-infected cells, microorganism-infected cells, parasite-infected cells, autoimmune cells, activated cells, myeloid cells, activated T cells, B cells or melanocytes; expressing 5T4, ADAM-9, ALK, AMHRII, ASCT2, Axl, B7-H3, BCMA, C4.4a, CA6, CA9, CanAg, CD123, CD138, CD142, CD166, CD184, CD19, CD20, CD205, CD22, CD248, CD25, CD3, CD30, CD33, CD352, CD37, CD38, CD40L, CD44v6, CD45, CD46, CD48, CD51, CD56, CD7, CD70, CD71, CD74, CD79b, CDH6, CEACAM5, CEACAM6, cKIT, CLDN18.2, CLDN6, CLL-1, c-MET, Cripto, CSP-1, CXCR5, DLK-1, DLL3, DPEP3, antiadhesin, EFNA4, EGFR, EGFRviii, ENPP3, EpCAM, EphA2, EphA3, ETBR, FGFR2, FGFR3, FLT3, FOLR-α, FSH, GCC, GD2, GD3, Globo H, GPC-1, GPC3, gpNMB, HER-2, HER-3, HLA-DR, HSP90, IGF-1R, IL-13R, IL1RAP, IL7R, interleukin-4 receptor (IL4R), KAAG-1, LAMP-1, LewisY antigen, LGALS3BP, LGR5, LH / hCG, LHRH, LIV-1, LRP-1, LRRC15, Ly6E, MAGE, mesothelin (MSLN), MET, MHC class I chain-related proteins A and B (MICA and MICB), MT1-MMP, MTX3, MTX5, MUC1, MUC16, NaPi2b, Nectin-4, NOTCH3, OAcGD2, OX001L, p-cadherin, PD-L1, phosphatidylserine (PS), polymorphic epithelial mucin (PEM), prolactin receptor (PRLR), PSMA, PTK7, RNF43, ROR1, ROR2, SAIL, SLAMF7, SLC44A4, SLITRK6, SSTR2, STEAP-1, STING, STn, TIM-1, TM4SF1, TNF-α, TRA, TROP-2, tumor-associated glycoprotein 72 (TAG-72), tumor-specific epitope of mucin-1 (TA-MUC1), CD5, TIM-3, UPK2, or UPK1b antigens; or (iii) the cell-binding agent is an anti-folate receptor antibody or an antibody fragment thereof, an anti-EGFR antibody or an antibody fragment thereof, an anti-CD33 antibody or an antibody fragment thereof, an anti-CD19 antibody or an antibody fragment thereof, an anti-Muc1 antibody or an antibody fragment thereof, an anti-CD37 antibody or an antibody fragment thereof, or an anti-EpCAM antibody or an antibody fragment thereof.

44. A pharmaceutical composition comprising the compound according to any one of claims 1 to 43 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

45. Use of the pharmaceutical composition of claim 44 in the preparation of a medicament for treating cancer in a subject, optionally, in: (i) the cancer is lymphoma or leukemia; (ii) the cancer is acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), acute B-lymphoblastic leukemia or B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), acute promyelocytic leukemia (APL), B-cell chronic lymphoproliferative disease (B-CLPD), atypical chronic lymphocytic leukemia, diffuse large B-cell lymphoma (DLBCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin's lymphoma (NHL), mantle cell leukemia (MCL), small lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis and Burkitt's lymphoma; (iii) the cancer is endometrial cancer, lung cancer, colorectal cancer, bladder cancer, gastric cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, esophageal cancer, breast cancer, head and neck cancer, uterine cancer, ovarian cancer, liver cancer, cervical cancer, thyroid cancer, testicular cancer, bone marrow cancer, melanoma and lymphoma; or (iv) the cancer is non-small cell lung cancer or small cell lung cancer.

46. ​​A method for preparing a compound of formula 4a: The method comprises the following steps (1) Compound 1a: Reaction with chloroacetonitrile to form a compound of formula 2a: (2) Compound 2a and compound 3: Reaction to form a compound of formula 4a.

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