Camptothecin derivatives

Abstract

To provide therapeutically effective camptothecin derivatives that have increased solubility, potency, lactone stability, and bioavailability.SOLUTION: Disclosed herein are novel cytotoxic compounds, and cytotoxic conjugates comprising these cytotoxic compounds and cell-binding agents. More specifically, this disclosure relates to novel camptothecin derivatives thereof, intermediates thereof, conjugates thereof, and pharmaceutically acceptable salts thereof, which are useful as medicaments, in particular as anti-proliferative agents (anticancer agents). In one aspect, the invention provides a compound of Formula I, or a pharmaceutically acceptable salt thereof: Z-L1-D (Formula I), wherein: D is represented by the following structural formula (R1 is -F, -CH3, or -CF3; R2 is -H, -F or the like; -L1-Z is -H, -(C1 to C6 alkylene)-H or the like).SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application Nos. 62 / 839,440, filed April 26, 2019, 62 / 875,169, filed July 17, 2019, and 62 / 978,159, filed February 18, 2020. Each of the foregoing applications is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The above ASCII copy, created on March 27, 2020, is named 000219-0002-WO1_-_Sequence_Listing.txt and is 61,926 bytes in size.

[0003] Field of the Application Disclosed herein are novel compounds and conjugates thereof. More specifically, the present disclosure relates to novel camptothecin derivatives, intermediates, metabolites, and conjugates thereof, as well as pharmaceutically acceptable salts thereof, which are useful as pharmaceutical agents, particularly antiproliferative agents (anticancer agents). [Background technology]

[0004] Cell-binding agent-drug conjugates, including antibody-drug conjugates (ADCs), have emerged as a powerful class of agents with efficacy across a range of abnormal cell proliferation or proliferative disorders (e.g., cancer). Cell-binding agent-drug conjugates (such as ADCs) typically consist of three distinct components: a cell-binding agent (e.g., an antibody); a linker; and a cytotoxic moiety.

[0005] Camptothecin (CPT) is a pentacyclic alkaloid isolated from the bark and stem of Camptotheca acuminata (Camptotheca, Happy tree), a tree native to China. Camptothecin inhibits topoisomerase I, which causes cell death. Due to its cytotoxic mechanism and broad-spectrum antitumor activity, considerable 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. Furthermore, camptothecin exists in equilibrium between its active lactone form and its inactive hydrolyzed carboxylate salt form, thereby limiting its therapeutic efficacy.

[0006] There is a need for therapeutically effective camptothecin derivatives with increased solubility, potency, lactone stability, and bioavailability. Summary of the Invention

[0007] In one aspect, the present invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof: ZL 1 -D (Formula I), During the ceremony: D is represented by the following structural formula: [ka] R 1 is -F, -CH3, or -CF3; R 2 -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 is that together with the carbon atom to which they are attached form a methylenedioxy or difluoromethylenedioxy ring; R3 is H or C1-C6 alkyl; R 4 is C1-C6 alkyl; L 1 is absent or -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, -X 1’ -(C1-C6 alkylene)-*, or -(C1-C6 alkylene)-X 1 -L 2 -*; where * is the moiety covalently bonded to Z. and; X 1 -O-, -S-, -S(O)-, -S(O)2-, -C(=O)-, -NR 5 - , -NR 5 C(=O)- or -C(=O)NR 5 - and; X 1’ is -O-, -S-, -S(O)-, or -S(O)2-; L 2 is phenylene; Each R 5 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; Z is -H or -X 2 and; X 2 -OR 6 , -SR 6 , -S(O)R 6 , -S(O)2R 6 , -SSR 6 ,or -N(R 6 )2; Each R 6 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; L 1 and L 2 are each independently a halogen, -CN, or -OR7 , -SR 7 , -N(R 7 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 Heterocycloalkyl, aryl, or heteroaryl optionally substituted with 1 to 4 substituents selected from Each R 7 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3- C6 cycloalkyl, aryl, heteroaryl, or benzyl; However, this is the case if R 1 If 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 -*; where * is the site of covalent attachment to Z; and Z is -X 2 provided that do However, this is the case if R 1 is F and R 2 If -OMe, then -L 1 With the proviso that -Z cannot be -NH2.

[0008] In some embodiments, the compound of Formula I has the proviso that if R 1 is F and R 2 If is -Me, then -L 1 With the further proviso that -Z cannot be -CH2OH.

[0009] In some embodiments, R 1 is -H or -F. In some embodiments, R 1 is -F. In some embodiments, R 2-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 is -F. In some embodiments, R 2 is -OMe, -SMe, -S(O)Me, or methyl. In some embodiments, R 2 is methyl. In some embodiments, R 1 is -F and R 2 is -F. In some embodiments, R 1 is methyl and R 2 is -F. In some embodiments, R 1 is -F and R 2 is -methyl.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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)-SR6 In some embodiments, -L 1 -Z is -(C1-C5 alkylene)-S-(C1-C5 alkylene)-SSR 6 is.

[0014] In some embodiments, -L 1 -Z is -CH2NHC(=O)CH2OH, -CH2NHC(=O)(CH2)2OH, -CH2NHC(=O)(CH2)3OH, -CH2NHC(=O)(CH2)4OH, -CH2N HC(=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)3S H, -CH2NHC(=O)(CH2)4SH, -CH2NHC(=O)(CH2)5SH, -CH2NHC(=O)CH2SMe, -CH2NHC(=O)(CH2)2SMe, -CH2N HC(=O)(CH2)3SMe, -CH2NHC(=O)(CH2)4SMe, -CH2NHC(=O)(CH2)5SMe, -CH2SCH2OH, -CH2S(CH2)2OH, -C H2S(CH2)3OH, -CH2S(CH2)4OH, -CH2S(CH2)5OH, -CH2SCH2OMe, -CH2S(CH2)2OMe, -CH2S(CH2)3OMe, -CH2 S(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.

[0015] In some embodiments, each R 5 is independently —H, methyl, or benzyl. In some embodiments, each R 5is independently —H. In some embodiments, each R 5 is methyl. In some embodiments, each R 5 is benzyl.

[0016] 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 6 is methyl. In some embodiments, each R 6 is benzyl.

[0017] 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, -OCH2SMe, -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.

[0018] In some embodiments, -L 1 -Z is -(C1-C6 alkylene)-X l -L 2 -X 2 In some embodiments, -L 1 -Z is [ka] is. In some embodiments, -L 1 -Z is [ka] In some embodiments, -L 1 -Z is [ka] is.

[0019] In some embodiments, the compound is any one of the compounds selected from the following: [ka] is.

[0020] In some embodiments, the compound is any one of the compounds selected from the following: [ka] .

[0021] In some embodiments, the compound is any one of the compounds selected from Table 1B.

[0022] In another aspect, the present invention provides a compound of formula II, or a pharmaceutically acceptable salt thereof: EA-Z'-L 1 -D (Formula II) During the ceremony: D is represented by the following structural formula: [ka] R 1 is -H, -F, -CH3, or -CF3; R 2-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 together with the carbon atom to which they are attached form a methylenedioxy or difluoromethylenedioxy ring; provided that R 1 and R 2 provided that both cannot be -H; R 3 is H or C1-C6 alkyl; R 4 is C1-C6 alkyl; L 1 is absent or -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, X 1’ -(C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; where * is the moiety covalently bonded to Z'; X 1 -O-, -S-, -S(O)-, -S(O)2-, -C(=O)-, -NR 5 -, -NR 5 C(=O)- or -C(=O)NR 5 - and; X 1’ is -O-, -S-, -S(O)-, or -S(O)2-; L 2 is phenylene; Each R 5 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; Z' is -O-CH2-NR 8 -*, -S-CH2-NR 8 -*, -NR 8 -*, where * is the site of covalent attachment to A; Each R 8 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; L 1 and L 2 are each independently a halogen, -CN, or -OR 7 , -SR 7 , -N(R 7 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 optionally substituted with 1 to 4 substituents selected from heterocycloalkyl, aryl, or heteroaryl; and Each R 7 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; A is a peptide containing 2 to 10 amino acids; wherein A is optionally substituted with one or more polyols; and E is -C(=O)-L 3 -X 3 and; L 3 is -(C1-C 10 alkylene)- or -Y 1 -(C1-C 10 alkylene)-X 4 -Y 2 -(C1-C 10 alkylene)-*; where * is X 3 is a moiety covalently bound to; Y 1 does not exist or is -(CR a R b O) n - or -(CR a R b CR a’ R b’ O) m - and; X 4 is -NR 9 C(=O)- or -C(=O)NR 9 - and; Y 2 does not exist or is -(CR c R d O) o - or -(CR c R d CR c’ R d’ O) p - and; n, m, o, and p are each independently 1 to 10; Each R a , R b , R a’ , R b’ , R c , R d , R c’ , and R d’ are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; L in the ceremony 3 is 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 optionally substituted with 0 to 4 substituents selected from heterocycloalkyl, aryl, heteroaryl, and polyol; Each R 11 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; X 3 teeth, [ka] -C(=O)-CR bb R cc -W', -NR ee -C(=O)-CR bb R cc -W', or -SR 10 and; 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 and; q is 1 to 24; Each R aa , R bb , R cc , R ee , and R ff is independently —H or optionally substituted C1-C6 alkyl; Each R yy and R xx are independently —H or C1-C6 alkyl; R gg are each independently —H or C1-C6 alkyl; and R 9 and R 10 are each independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl.

[0023] In some embodiments, R 1 is -H or -F. In some embodiments, R 1 is -F. 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 is -F. In some embodiments, R 2 is -OMe, -SMe, -S(O)Me, or methyl. In some embodiments, R 2 is methyl. In some embodiments, R 1 is -F and R 2 is -F. In some embodiments, R 1 is methyl and R 2is -F. In some embodiments, R 1 is -F and R 2 is -methyl.

[0024] 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 -*.

[0025] In some embodiments, -L 1 -Z'-* stands for -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-*.

[0026] 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 -*.

[0027] In some embodiments, -L 1-Z'-* stands for -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-*.

[0028] 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 is methyl. In some embodiments, each R 5 is benzyl. 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 is methyl. In some embodiments, each R 8 is benzyl.

[0029] 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 -*.

[0030] 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-NH-, -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-, or -S(O)2(CH2)4-NH-.

[0030] In some embodiments, -L 1 -Z'-* is -(C1-C6 alkylene)-X 1 -L 2-Z'-*. In some embodiments, -L 1 Z'-* is [ka] is. In some embodiments, -L 1 -Z'- * teeth, [ka] is. In some embodiments, -L 1 -Z'- * teeth, [ka] is.

[0031] As used herein, -L 1 In various embodiments disclosing -Z'-*, * is the site of covalent attachment to A.

[0032] 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.

[0033] In some embodiments, A is -(AA 1 )-(AA 2 ) a1 -*, where * is the site of covalent attachment to E; AA 1 and A.A. 2 are each independently an amino acid residue; and a1 is an integer of 1 to 9.

[0034] In some embodiments, -AA 1 -(AA 2 ) al-* 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-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-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-*.

[0035] In some embodiments, -AA 1 -(AA 2 ) al-* stands for -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-A la-*, -L-Ala-L-Val-*, -L-Gln-L-Val-*, -L-Gln-L-Leu-*, or -L-Ser-L-Val-*.

[0036] In some embodiments, -AA 1 -(AA 2 ) al -* is: -Ala-Ala-*, -Ala-Val-*, -Val-Ala-*, -Gln-Leu-*, -Leu-Gln-*, -Ala-Ala-Ala-*, -Ala-Ala-Ala-Ala-*, -G ly-Ala-Gly-Gly-*, -Gly-Gly-Ala-Gly-*, -Gly-Val-Gly-Gly-*, -Gly-Gly-Val-Gly-*, -Gly-Phe-Gly-Gly-*, or -Gly-Gly-Phe-Gly-*.

[0037] In some embodiments, -AA 1 -(AA 2 ) al-* 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-G ln-*, -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-*.

[0038] In some embodiments, -AA 1 -(AA 2 ) al -* 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-*.

[0039] -AA herein 1 -(AA 2 ) al In various embodiments disclosing -*, * is a site covalently attached to E.

[0040] 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)-X5 -Y 3 wherein: X 5 is -NR 12 C(=O)- or -C(=O)NR 12 -Y 3 is -C1-C 10 alkyl, wherein Y 3 is substituted with 0 to 10 OH groups; and R 12 is substituted with -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl.

[0041] In some embodiments, wherein the polyol is [ka] where R 12 is H or methyl.

[0042] In some embodiments, E is —C(═O)—(C-C 10 alkylene)-X 3 In some embodiments, E is [ka] is.

[0043] In some embodiments, E is —C(═O)—Y 1 -(C1-C 10 alkylene)-X 4 -(C1-C 10 alkylene)-X 3 and; Y 1 is -(CR a R b O) n - or -(CR a R b CR a’ R b’ O) m - and; X 4 is -NR 9 C(=O)—; and X3 is [ka] -C(=O)-CR bb R cc -W', NR ee -C(=O)-CR bb R cc -W', or -SR 10 is.

[0044] In some embodiments, E is —C(═O)—Y 1 -(CH2)2-X 4 -(CH2)2-X 3 is; Y 1 is -(CHO) n -or-(CH2CH2O) m - and; X 4 is -NHC(=O)-; n is 2; m is 2 to 6; X 3 teeth, [ka] -C(=O)-CR bb R cc -W', NR ee -C(=O)-CR bb R cc -W', or -SR 10 is.

[0045] In some embodiments, the compound is any one of the compounds selected from Table 2.

[0046] In another aspect, the present invention provides a compound of formula III, or a pharmaceutically acceptable salt thereof: CBA-E'-A-Z'-L 1 -D (Formula III) During the ceremony: D is represented by the following structural formula: [ka] R 1 is -H, -F, -CH3, or -CF3; R 2 -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 together with the carbon atom to which they are attached form a methylenedioxy or difluoromethylenedioxy ring; provided that R 1 and R 2 is under conditions where -H is not possible; R 3 is H or C1-C6 alkyl; R 4 is C1-C6 alkyl; L 1 is absent or -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, X 1’ -(C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; where * is the moiety covalently bonded to Z'; X 1 -O-, -S-, -S(O)-, -S(O)2-, -C(=O)-, -NR 5 -, -NR 5 C(=O)- or -C(=O)NR 5 - and; X 1’ is -O-, -S-, -S(O)-, or -S(O)2-; L 2 is phenylene; Each R 5 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; Z' is -O-CH2-NR 8 -*, -S-CH2-NR 8-*, -NR 8 -*, where * is the site of covalent attachment to A; Each R 8 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; L 1 and L 2 are each independently a halogen, -CN, or -OR 7 , -SR 7 , -N(R 7 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 optionally substituted with 1 to 4 substituents selected from heterocycloalkyl, aryl, or heteroaryl; and Each R 7 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 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 -*; where * is the site of covalent attachment to the CBA; L 3 is -(C1-C 10 alkylene)- or -Y 1 -(C1-C 10 alkylene)-X 4 -Y 2 -(C1-C 10 alkylene)-*; where * is X 6 is a moiety covalently bound to; Y 1 does not exist or is -(CR a R b O) n -or- (CR a R b CR a‘ R b‘ O) m - and; X 4 is -NR 9 C(=O)- or -C(=O)NR 9 - and; Y 2 does not exist or is -(CR c R d O) o - or -(CR c R d CR c‘ R d‘ O) p - and; n, m, o, and p are each independently 1 to 10; Each R a , R b , R a‘ , R b‘ , R c , R d , R c‘ , and R d‘ are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; L in the ceremony 3 is 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 optionally substituted with 0 to 4 substituents selected from heterocycloalkyl, aryl, heteroaryl, and polyol; Each R 11 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; X 6 teeth, [ka] -C(=O)-CR bb R cc -* or -NR ee -C(=O)-CR bb R cc-*; where * is the site of covalent attachment to the CBA; Each R aa , R bb , R cc , and R ee is independently —H or optionally substituted C1-C6 alkyl; Each R yy and R xx are independently —H or C1-C6 alkyl; R 9 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; and CBA is a cell binding agent.

[0047] In some embodiments, R 1 is -H or -F. In some embodiments, R 1 is -F. 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 is -F. In some embodiments, R 2 is -OMe, -SMe, -S(O)Me, or methyl. In some embodiments, R 2 is methyl. In some embodiments, R 1 is -F and R 2 is -F. In some embodiments, R 1 is methyl and R 2 is -F. In some embodiments, R 1 is -F and R 2 is -methyl.

[0048] In some embodiments, -L 1 -Z'-* is -(C1-C4 alkylene)-O-CH2-NR 8 -*, -(C1-C4 alkylene)-S-CH2-NR8 -*, 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 -*.

[0049] In some embodiments, -L 1 -Z'-* stands for -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- be.

[0050] 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 -*.

[0051] In some embodiments, -L 1 -Z'-* stands for -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-*.

[0052] 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 5is methyl. In some embodiments, each R 5 is benzyl. 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 is methyl. In some embodiments, each R 8 is benzyl.

[0053] 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 -*.

[0054] In some embodiments, -L 1 -Z'-* stands for -OCH2O-CH2-NH-*, -O(CH2)2O-CH2-NH-*, -O(CH2)3O-CH2-NH-*, -O(CH2)4O-CH2-NH-*, -SCH2O-CH2-N H-*, -S(CH2)2O-CH2-NH-*, -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-N H-*, -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(C H2)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-*.

[0055] In some embodiments, -L 1 -Z'-* is -(C1-C6 alkylene)-X 1 -L 2 -Z'-*. In some embodiments, -L 1 -Z'-* is [ka] is. In some embodiments, -L 1 -Z'-* is [ka] is. In some embodiments, -L 1 -Z'-* is [ka] is.

[0056] As used herein, -L 1 In various embodiments disclosing -Z'-, * is the site of covalent attachment to A.

[0057] In some embodiments, A is a peptide containing 2 to 8 amino acids. In some embodiments, A is a peptide comprising 2-4 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.

[0058] In some embodiments, A is -(AA 1 )-(AA 2 ) al -*, where * is the point of attachment to E', and AA 1 and A.A. 2 are each independently an amino acid residue; and al is an integer of 1 to 9.

[0059] In some embodiments, -AA 1 -(AA 2 ) al-* 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-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-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-*.

[0060] In some embodiments, -AA 1 -(AA 2 ) al-* stands for -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-A la-*, -L-Ala-L-Val-*, -L-Gln-L-Val-*, -L-Gln-L-Leu-*, or -L-Ser-L-Val-*.

[0061] In some embodiments, -AA 1 -(AA 2 ) al -* stands for -Ala-Ala-*, -Ala-Val-*, -Val-Ala-*-Gln-Leu-*, -Leu-Gln-*, -Ala-Ala-Ala-*, -Ala-Ala-Ala-Ala-*, -Gly-Ala-Gly-G ly-*, -Gly-Gly-Ala-Gly-*, -Gly-Val-Gly-Gly-*, -Gly-Gly-Val-Gly-*, -Gly-Phe-Gly-Gly-*, or -Gly-Gly-Phe-Gly-*.

[0062] In some embodiments, -AA 1 -(AA 2 ) al -* stands for -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-V al-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-*.

[0063] In some embodiments, -AA 1 -(AA 2 ) al -* 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-*.

[0064] As used herein, -AA 1 -(AA 2 ) al In various embodiments disclosing -*, * is the site of covalent attachment to E'.

[0065] 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 wherein: X 5 is -NR12 C(=O)- or -C(=O)NR 12 -Y 3 is -C1-C 10 alkyl, wherein Y 3 is substituted with 0 to 10 OH groups; and R 12 is —H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl.

[0066] In some embodiments, the polyol is [ka] where R 12 is H or methyl.

[0067] In some embodiments, E' is -C(=O)-(C-C 10 alkylene)-X 6 In some embodiments, E' is [ka] , -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 the CBA.

[0068] In some embodiments, E' is -C(=O)-Y 1 -(C1-C 10 alkylene)-X 4 -(C1-C 10 alkylene)-X 6 -* and; Y 1 is -(CR a R b O) n - or -(CR a R b CRa’ R b’ O) m - and; X 4 is -NR 9 C(=O)—; and X 6 teeth, [ka] , -C(=O)-CR bb R cc -* or -NR ee -C(=O)-CR bb R cc -*; where * is the site of covalent attachment to the CBA.

[0069] In some embodiments, E' is -C(=O)-Y 1 -(CH2)2-X 4 -(CH2)2-X 6 -* and; Y 1 is -(CHO) n - or -(CH2CH2O) m - and; X 4 is -NHC(=O)-; n is 2; m is 2 to 6; X 6 teeth, [ka] , -C(=O)-CR bb R cc -* or -NR ee -C(=O)-CR bb R cc -*; where * is the site of covalent attachment to the CBA.

[0070] In some embodiments, CBA comprises an —SH group that is covalently attached to E′: [ka] , -C(=O)-CR bb Rcc -S-CBA, or -NR ee -C(=O)-CR bb R cc -S-CBA is produced.

[0071] In some embodiments, the CBA is an antibody and is -E'-A-Z'-L 1 -D is a drug linker structure, and the average number of drug linker structures conjugated per antibody ranges from 2 to 10.

[0072] In some embodiments, the average number of drug linker structures conjugated per antibody ranges from 2 to 10. In some embodiments, the average number of drug linker structures conjugated per antibody ranges from 6 to 8. In some embodiments, the average number of drug linker structures conjugated per antibody is 8.

[0073] 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.

[0074] In some embodiments, the CBA is a target cell selected from a tumor cell, a virus-infected cell, a microorganism-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; 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, CD H6, 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-alpha, FSH, GCC, GD2, GD3, GloboH, 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 class I chain-related proteins A and B (MICA and MICB), MT1-MMP, MTX3, MTX5, MUC1, MUC16, NaPi2b, Nectin-4, NOTCH3, OAcGD2, OXOOIL, p-cadherin, PD-L1, phosphatidylserine (PS), polymorphic epithelial mucin (PEM) mucin (PEM), prolactin receptor (PRLR), PSMA, PTK7, RNF43, ROR1, ROR2, SAIL, SLAMF7, SLC44A4, SLITRK6, SSTR2, STEAP-1, STING, STn, TIM-1, TM4SF1, TNF-alpha, TRA, TROP-2, tumor-associated glycoprotein 72 (TAG-72), tumor-specific epitope of mucin-1 (TA-MUC1), CD5, TIM-3, UPK2, or UPK1b antigens.

[0075] In some embodiments, the cell binding agent is an anti-folate receptor antibody or antibody fragment thereof, an anti-EGFR antibody or antibody fragment thereof, an anti-CD33 antibody or antibody fragment thereof, an anti-EpCAM antibody or antibody fragment thereof, an anti-CD19 antibody or antibody fragment thereof, an anti-Muc1 antibody or antibody fragment thereof, or an anti-CD37 antibody or antibody fragment thereof.

[0076] The present invention also includes compositions (e.g., pharmaceutical compositions) comprising the cytotoxic compounds or conjugates of the invention described herein and a carrier (a pharmaceutically acceptable carrier). The compounds, conjugates, or compositions of the invention are useful 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 kidney disease in a mammal (e.g., a human).

[0077] The compounds, conjugates, and compositions of the invention are useful for treating cancer in a subject in need thereof, hi 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's lymphoma (NHL), Mantel 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 lymphatic system cancer. In some embodiments, the lung cancer is non-small cell lung cancer or small cell lung cancer.

[0078] Also included in the invention is the use of a cytotoxic compound, conjugate, or composition of the invention for the manufacture of a medicament for inhibiting abnormal cell proliferation or for 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 kidney disease in a mammal (e.g., a human).

[0079] The compounds, conjugates, or compositions of the invention are useful for the manufacture of a medicament for treating cancer in a subject in need thereof, hi 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's lymphoma (NHL), Mantel 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 lymphatic system cancer. In some embodiments, the lung cancer is non-small cell lung cancer or small cell lung cancer. [Brief explanation of the drawings]

[0080] [Figure 1] 1 shows the first part of the synthesis of camptothecin building blocks. [Figure 2] 1 shows the second part of the synthesis of camptothecin building blocks. [Figure 3] The first part of the synthesis of the side chain is shown. [Figure 4] The second part of the synthesis of the side chain is shown. [Figure 5] The first moiety that connects the camptothecin building block to the side chain is shown. [Figure 6] A second moiety that connects the camptothecin building block to the side chain is shown. [Figure 7] A third moiety that connects the camptothecin building block to the side chain is shown. [Figure 8] The synthesis of additional camptothecin metabolites is shown. [Figure 9] The attachment to the side chain of the camptothecin building block is shown. [Figure 10] 1 shows the synthesis of additional camptothecin compounds. [Figure 11] The compounds used for comparison are shown, including the general Ab-999 structure of an ADC bearing 999 moieties linked via reduced interchain disulfides of an antibody. [Figure 12] The cytotoxicity of sulfide-containing compound 8c and its sulfoxide 34a and sulfone 34b is shown. [Figure 13] Figure 1 shows the pharmacokinetics of ML66-999 in mice. The top panel shows plots of the concentration (μg / mL) of the mAb component (mean) and payload component versus time at 2 minutes, 1 day, and 3 days after administration to mice. The bottom panel shows plots of the concentration (μg / mL) of the ADC versus time (mean) and retained bioactivity (pooled samples) of the mAb component at 2 minutes, 1 day, and 3 days after administration to mice. [Figure 14] Pharmacokinetics of ML66-22a in mice. The top panel shows plots of the concentration (μg / mL) of the mAb component (mean) and payload component versus time at 2 minutes, 1 day, and 3 days after administration to mice. The bottom panel shows plots of the concentration (μg / mL) of the ADC versus time (mean) and retained bioactivity (pooled samples) of the mAb component at 2 minutes, 1 day, and 3 days after administration to mice. [Figure 15]Figure 1 shows the in vitro cytotoxicity of ADCs against Ag+ and Ag- cells. ADC standard of ADC-containing formulation (standard) or serum (pooled) collected 2 minutes, 1 day, or 3 days after administration of ML66-999 to mice. [Figure 16] Figure 1 shows the in vitro cytotoxicity of ADCs against Ag+ and Ag- cells. ADC standard of ADC-containing formulation (standard) or serum (pooled) collected 2 minutes, 1 day, or 3 days after administration of ML66-22a to mice. [Figure 17] Efficacy of the ADC in an HSC-2 xenograft model is shown. Dosage is based on payload (75 μg / kg and 250 μg / kg are approximately 3 mg / kg and 10 mg / kg based on antibody). [Figure 18] Efficacy of the ADC in the FaDu xenograft model is shown. Dosage is based on payload (75 μg / kg and 250 μg / kg are approximately 3 mg / kg and 10 mg / kg based on antibody). [Figure 19] 1 shows the tolerance of the ML66-999, ML66-22a and ML66-28a ADCs in mice. [Figure 20] The antitumor activity of the ADC in an H1703 mouse xenograft model is shown. Dosages are based on payload (75 μg / kg and 250 μg / kg are approximately 3 mg / kg and 10 mg / kg based on antibody). [Figure 21] Shows mouse tolerance of a non-cross-reactive ADC at a payload dose of 5000 μg / kg (approximately 200 mg / kg based on Ab component). AbF = humanized anti-folate receptor antibody. DETAILED DESCRIPTION OF THE INVENTION

[0081] In order to provide a thorough understanding of the invention described herein, the following detailed description is set forth. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention.

[0082] The term "herein" means the entire application.

[0083] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used in connection with the compounds, compositions and methods described herein is that which is well known and commonly used in the art.

[0084] It is understood that any of the embodiments described herein, including those described under different aspects of the invention and in different parts of this specification (including embodiments described by way of example only), may be combined with one or more other embodiments of the invention, unless expressly disclaimed or inappropriate, and that combinations of embodiments are not limited to those specific combinations claimed via multiple dependent claims.

[0085] Chemical terms used herein are used in accordance with conventional usage in the art, as set forth in "The McGraw-Hill Dictionary of Chemical Terms," ​​Parker S., Ed., McGraw-Hill, San Francisco, Calif. (1985).

[0086] All of the above, as well as any other publications, patents, and published patent applications mentioned in this application, are specifically incorporated herein by reference. Any information from any material incorporated by reference herein is incorporated by reference only to the extent that no conflict exists between such information and other descriptions and drawings set forth herein. In the event of such a conflict, any such conflicting information incorporated by such reference material not specifically incorporated herein by reference, will be governed by the present specification, including its specific definitions.

[0087] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated integer (or component) or group of integers (or components), but not the exclusion of any other integer (or component) or group of integers (or components).

[0088] Throughout this specification, when a composition is described as having, including, or comprising particular components (or variations thereof), it is contemplated that the composition may also consist essentially of, or consist of, the recited components. Similarly, when a method or process is described as having, including, or comprising particular process steps, the process may also consist essentially of, or consist of, the recited processing steps. Furthermore, it is understood that the order of steps or order for performing certain actions is immaterial so long as the compositions and methods described herein are operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0089] The term "including" is used herein to mean that "including but not limited to," "including," and "including but not limited to" are used interchangeably.

[0090] As used herein, "about" or "approximately" means within an acceptable error range for a 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.

[0091] The use of the terms "a," "an," and "the" and similar referents in the context of describing elements (particularly in the context of the claims which follow) should be construed to cover both the singular and the plural unless otherwise stated herein or clearly contradicted by context.

[0092] As used herein, the term "or" should be understood to mean "and / or" unless the context clearly indicates otherwise.

[0093] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each individual value within the range, unless otherwise stated herein, and each individual value is incorporated herein as if set forth individually. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided herein, or the use of exemplary language (e.g., "etc.") are intended merely to better clarify embodiments and do not impose limitations on the scope of the claims unless otherwise expressly stated. No language in this specification should be construed as indicating any non-claimed element as essential.

[0094] definition As used herein, the term "alkyl" or "linear or branched alkyl" refers to a saturated, linear or branched monovalent hydrocarbon radical. In preferred embodiments, a straight-chain or branched alkyl has 30 or fewer carbon atoms (e.g., C1-C6 for a straight-chain alkyl group). 30 and C3-C for branched alkyl 30), more preferably 20 or fewer carbon atoms. Even more preferably, a straight or branched chain alkyl has 10 or fewer carbon atoms (i.e., C1-C for straight chain alkyl groups). 10 and for branched alkyls, C3 to C 10 In other embodiments, a straight or branched chain alkyl has six or fewer carbon atoms (i.e., C1-C6 for straight chain alkyl groups, or C3-C6 for branched chain alkyl groups). Examples of alkyl include, but are not limited to, methyl, Examples include ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, -CHCH(CH), 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, etc. Furthermore, the term "alkyl," as used throughout the specification, examples, and claims, is intended to include both "unsubstituted alkyl" and "substituted alkyl," the latter referring to alkyl moieties 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-xx Alkyl means a straight or branched alkyl having x-xx carbon atoms.

[0095] As used herein, the term "alkylene" refers to a saturated, linear or branched, divalent hydrocarbon radical. In preferred embodiments, a straight-chain or branched-chain alkylene has 30 or fewer carbon atoms (e.g., C1-C5 for a straight-chain alkylene group). 30 and C3-C for branched alkylene groups. 30), more preferably 20 or fewer carbon atoms. Even more preferably, the straight or branched chain alkylene has 10 or fewer carbon atoms (i.e., C1-C5 for straight chain alkylene groups). 10 , C3-C for branched alkylene 10 In other embodiments, a straight chain or branched chain alkylene has six or fewer carbon atoms (i.e., C1-C6 for a straight chain alkylene group, or C3-C6 for a branched chain alkylene). As used herein, (C x -C xx ) alkylene or C x-xx Alkylene means a linear or branched alkylene having x-xx carbon atoms.

[0096] The terms "alkenyl" or "linear or branched alkenyl" refer to a linear or branched monovalent hydrocarbon radical of 2 to 20 carbon atoms having at least one site of unsaturation, i.e., a carbon-carbon double bond; this alkenyl radical includes radicals having the "cis" and "trans" orientations, or alternatively, the "E" and "Z" orientations. Examples include, but are not limited to, ethylenyl or vinyl (-CH=CH), allyl (-CHCH=CH), and the like. Preferably, the alkenyl has 2 to 10 carbon atoms. More preferably, the alkyl has 2 to 4 carbon atoms.

[0097] The term "alkynyl" or "linear or branched alkynyl" refers to a linear or branched monovalent hydrocarbon radical of 2 to 20 carbon atoms 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 has 2 to 10 carbon atoms. More preferably, the alkynyl has 2 to 4 carbon atoms.

[0098] The terms "cyclic alkyl" and "cycloalkyl" can be used interchangeably. As used herein, this term refers to a radical of a saturated carbocyclic ring. In preferred embodiments, cycloalkyls have from 3 to 10 carbon atoms in their ring structure, and more preferably 5-7 carbon atoms in the ring structure. In some embodiments, two cyclic rings can have more than one atom in common; for example, the rings are "fused rings." Suitable cycloalkyls include, but are not limited to, cycloheptyl, cyclohexyl, cyclopentyl, cyclobutyl, and cyclopropyl. In some embodiments, a cycloalkyl is a monocyclic group. In some embodiments, a cycloalkyl is a bicyclic group. In some embodiments, a cycloalkyl is a tricyclic group.

[0099] The term "cycloalkylalkyl" refers to any of the above groups substituted with a cycloalkyl group. Refers to an alkyl group.

[0100] The term "cyclic alkenyl" refers to a carbocyclic ring radical that has at least one double bond in the ring structure.

[0101] The term "cyclic alkynyl" refers to a carbocyclic ring radical that has at least one triple bond in the ring structure.

[0102] 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 "polycyclyl," "polycyclic," and "polycyclic" ring systems having two or more rings in which two or more atoms are common to two adjacent rings, e.g., the rings are "fused rings," and at least one of the rings is aromatic; for example, the other cyclic ring may be a cycloalkyl, cycloalkenyl, cycloalkynyl, or aromatic ring. In some preferred embodiments, a polycyclic ring has two or three rings. In certain preferred embodiments, a polycyclic ring system has two cyclic rings in which both rings are aromatic. Each ring of a polycyclic ring may be substituted or unsubstituted. In certain embodiments, each ring of a polycyclic ring contains 3 to 10 carbon atoms, preferably 5 to 7 carbon atoms. For example, aryl groups include, but are 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, an aryl is a monocyclic aromatic group. In some embodiments, an aryl is a bicyclic aromatic group. In some embodiments, an aryl is a tricyclic aromatic group.

[0103] The term "heteroalkyl" refers to an alkyl group in which one or more skeletal atoms of the alkyl are selected from atoms other than carbon, e.g., O, S, N (e.g., -NH, -N(alkyl)-), or combinations thereof. The heteroalkyl is attached to the remainder of the molecule at a carbon atom of the heteroalkyl. In one aspect, a heteroalkyl is a C-C 30 Heteroalkyl. C1-C 30 Heteroalkyl refers to an alkyl group having 1 to 30 carbon atoms and 1 to 15 heteroatoms. 30Examples 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)2]O). 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 provides C1-C 30 Heteroalkyl groups are also contemplated, where one of the 1 to 15 heteroatoms occupies a terminal position of the alkyl group, resulting in, for example, an alcohol (i.e., OH), a thiol (i.e., SH), or an amine (e.g., -NH) at the terminal position of the moiety.

[0104] The term "heteroalkenyl" refers to an alkenyl group, as defined herein, Heteroalkenyl refers to an alkenyl group in which one or more carbon atoms are replaced by a heteroatom, such as O, S, or N (e.g., -NH, -N(alkyl)-). A heteroalkenyl is attached to the rest of the molecule at a carbon atom of the heteroalkenyl. In one aspect, heteroalkenyl refers to a C-C 30 Heteroalkenyl. C1-C 30Heteroalkenyl refers to an alkenyl group having 1 to 30 carbon atoms and 1 to 15 heteroatoms, for example, 1 to 10 heteroatoms or 1 to 5 heteroatoms. 30 Heteroalkenyl groups are contemplated, in which one of the 1 to 15 heteroatoms occupies a terminal portion of the alkenyl group, resulting in, for example, an alcohol (i.e., OH), a thiol (i.e., SH), an amine (e.g., -NH), or an imine (-C=N) at the terminal position of the moiety.

[0105] The term "heteroalkynyl" refers to an alkenyl group, as defined herein, in which one or more carbon atoms are replaced by a heteroatom, e.g., O, S, N (e.g., -NH, -N(alkyl)-). A heteroalkynyl is attached to the rest of the molecule at a carbon atom of the heteroalkynyl. In one aspect, a heteroalkynyl is a C-C 30 Heteroalkynyl. C1-C 30 Heteroalkenyl refers to an alkynyl group having 1 to 30 carbon atoms and 1 to 15 heteroatoms, for example, 1 to 10 heteroatoms or 1 to 5 heteroatoms. 30 Also contemplated are heteroalkynyl groups in which one of the 1 to 15 heteroatoms occupies a terminal portion of the alkynyl group, resulting in, for example, an alcohol (i.e., OH), a thiol (i.e., SH), an amine (e.g., -NH), or a nitrile (-C≡N) at the terminal position of the moiety.

[0106] As used herein, the terms "heterocycle," "heterocyclyl," and "heterocyclic ring" refer to a substituted or unsubstituted non-aromatic ring structure having 3 to 18 members, preferably 3 to 10 members, and more preferably 3 to 7 members, and containing at least one heteroatom, preferably 1 to 4 heteroatoms, and more preferably 1 or 2 heteroatoms. In certain embodiments, the ring structure can have two cyclic rings. In some embodiments, the two cyclic rings can have two or more atoms in common, e.g., the rings are "fused rings." Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like. Heterocycles are described in Paquette, Leo A. "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968), specifically, 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), specifically 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, tetrahydrothiene, tetrahydropyran, dihydropyran, tetrahydrothiopyran, thiomorpholine, thioxane, homopiperazine, azetidine, oxetane, thietane, homopiperidine, piperidine, piperazine, pyrrolidine, morpholine, oxepane, thiepane, oxazepine, diazepine, thiazol-1-one ... Examples of heterocyclic groups include azepine, 2-pyrroline, 3-pyrroline, indoline, 2H-pyran, 4H-pyran, dioxane, 1,3-dioxolane, pyrazoline, dithiane, dithiolane, dihydropyran, dihydrothien, dihydrofuran, pyrazolidinylimidazoline, 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 in which a ring atom is replaced with an oxo (=O) moiety are pyrimidinone and 1,1-dioxo-thiomorpholine.

[0107] 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 contains at least one heteroatom (e.g., O, N, or S), preferably 1 to 4 or 1 to 3 heteroatoms, more preferably 1 or 2 heteroatoms. When two or more heteroatoms are present in a heteroaryl ring, they may be the same or different. The term "heteroaryl" also includes "polycyclyl," "polycyclic," and "polycyclic" ring systems having two or more cyclic rings in which two or more ring atoms are common to two adjacent rings, e.g., rings are "fused rings," where at least one of the rings is heteroaromatic; e.g., the other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaromatic, and / or heterocyclyl. In some preferred embodiments, polycyclic heteroaryls have two to three rings. In certain embodiments, preferred polycyclic heteroaryls have two cyclic rings, both of which are aromatic. In certain embodiments, each ring of the polycyclic ring contains 3 to 10 atoms in the ring, preferably 5 to 7 atoms in the ring. For example, heteroaryl groups include, but are 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, and the like. In some embodiments, the heteroaryl is a monocyclic aromatic group. In some embodiments, the heteroaryl is a bicyclic aromatic group. In some embodiments, the heteroaryl is a tricyclic aromatic group.

[0108] The heterocycle or heteroaryl group may be bonded to carbon (carbon bond) or nitrogen (nitrogen bond) where possible. By way of example and not limitation, the carbon-bonded heterocycle or heteroaryl is bonded to the 2, 3, 4, 5, or 6 position of pyridine, the 3, 4, 5, or 6 position of pyridazine, the 2, 4, 5, or 6 position of pyrimidine, the 2, 3, 5, or 6 position of pyrazine, the 2, 3, 4, or 5 position of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole, the 2, 4, or 5 position of oxazole, imidazole, or thiazole, the 3, 4, or 5 position of isoxazole, pyrazole, or isothiazole, the 2 or 3 position of aziridine, the 2, 3, or 4 position of azetidine, the 2, 3, 4, 5, 6, 7, or 8 position of quinoline, or the 1, 3, 4, 5, 6, 7, or 8 position of isoquinoline.

[0109] By way of example and not limitation, a nitrogen-linked heterocycle or heteroaryl can be bonded to 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 at the 1-position, isoindole, or isoindoline at the 2-position, morpholine at the 4-position, and carbazole at the 9-position, or O-carboline.

[0110] Heteroatoms present in heteroaryl or heterocyclyl include oxidized forms such as NO, SO, and SO2.

[0111] In some embodiments, the heteroaromatic ring is a 5-18 membered ring.

[0112] 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, as defined herein, substituted by one or more halo groups, as defined herein. A haloalkyl can be a monohaloalkyl, dihaloalkyl, or polyhaloalkyl. A monohaloalkyl can have one fluoro, chloro, bromo, or iodo substituent. A dihaloalkyl or polyhaloalkyl may be substituted with two or more of the same halo atoms or a combination of different halo groups. Examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoroisopropyl, dichloroethyl, and dichloropropyl.

[0113] The term "alkoxy" as used herein refers to alkyl-O-, where alkyl is defined herein above. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.

[0114] The above alkyl, haloalkyl, alkoxy, alkenyl, alkynyl, cyclic alkyl, cyclic alkenyl, cyclic alkynyl, carbocyclyl, aryl, heterocyclyl and heteroaryl may be optionally substituted with one or more (e.g., 2, 3, 4, 5, 6 or more) substituents.

[0115] Unless specifically stated as "unsubstituted," references to chemical moieties herein are understood to include substituted variants. For example, reference to an "alkyl" group or moiety implicitly includes both substituted and unsubstituted variants. Examples of substituents on chemical moieties include, but are not limited to, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl, 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.

[0116] "Optional" or "optionally" means that the subsequently described circumstance may or may not occur, and consequently, the present application includes cases where the circumstance occurs and cases where it 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 present application includes structures where a non-hydrogen substituent is present and structures where a non-hydrogen substituent is not present.

[0117] The term "substituted" refers to moieties having substituents replacing a hydrogen on one or more carbon, nitrogen, oxygen, or sulfur atoms. "Substituted" or "substituted" includes the implicit proviso that such substitution is in accordance with the permissible valences of the replacing atoms and substituents, and it is understood that the substitution results in a stable compound that does not spontaneously undergo transformation, for example, by rearrangement, cyclization, elimination, and the like. As used herein, the term "substituted" is considered to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valence of the heteroatom. Substituents include those described herein. Optional substituents include, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkoxyl, alkylthio, acyloxy, phosphoryl, phosphate, phosphonate, amino, amido, amidine, imine, cyano, nitro, azide, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moiety. For the purpose of explanation, monofluoroalkyl is alkyl substituted with a fluoro substituent, and difluoroalkyl is alkyl substituted with two fluoro substituents. When a substituent has multiple substitutions, it should be recognized that each non-hydrogen substituent may be the same or different (unless otherwise specified).

[0118] When a carbon of a substituent is described as being optionally substituted with one or more of a list of substituents, one or more of the hydrogens on the carbon (to the extent present) may be replaced separately and / or together with any independently selected substituents. When a nitrogen of a substituent is described as being optionally substituted with one or more of a list of substituents, one or more of the hydrogens on the nitrogen (to the extent present) may each be replaced with any independently selected substituent. One exemplary substituent may be depicted as -NR'R", where R' and R" together with the nitrogen atom to which they are attached may form a heterocycle. The heterocycle 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 heterocycle consists of 3 to 7 atoms. In other embodiments, the heterocycle is selected from pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, pyridyl, and thiazolyl.

[0119] This specification uses the terms "substituent," "radical," and "group" interchangeably.

[0120] When a group of substituents is collectively described as being optionally substituted by one or more of the listed substituents, the group can include (1) unsubstitutable substituents, (2) substitutable substituents that are not substituted by any substituents, and / or (3) substitutable substituents that are substituted by one or more optional substituents.

[0121] When a substituent is described as being optionally substituted with up to a specified number of non-hydrogen substituents, the substituent is either (1) unsubstituted; or (2) substituted with up to the specified number of non-hydrogen substituents or up to the maximum number of substitutable positions on the substituent, whichever is less. Thus, for example, if a substituent is described as heteroaryl optionally substituted with up to three non-hydrogen substituents, any heteroaryl having fewer than three substitutable positions is optionally substituted only with up to the same number of non-hydrogen substituents as the heteroaryl has substitutable positions. Such substituents include, by way of non-limiting example, linear, branched, or cyclic alkyl, alkenyl, or alkynyl having 1 to 10 carbon atoms, aryl, heteroaryl, heterocyclyl, halogen, guanidinium [—NH(C═NH)NH], —OR 100 , N.R. 101 R 102 , -NO2, -NR 101 COR 102 , -SR 100 , -SOR 101 Sulfoxides represented by -SO2R 101 Sulfones, sulfonates -SO3M, sulfates -OSO3M, -SO2NR 101 R 102 Sulfonamide, cyano, azido, -COR 101 , -OCOR 101 , -OCONR 101 R 102 and polyethylene glycol units (-OCH2CH2) n R 101 (M is H or cation (Na + or K + may be selected from; R 101 , R 102 and R 103 are each H, a linear, branched or cyclic alkyl, alkenyl or alkynyl (having 1 to 10 carbon atoms), a polyethylene glycol unit (-OCH2CH2) n -R 104 (where n is an integer from 1 to 24), aryls having 6 to 10 carbon atoms, heterocyclic rings having 3 to 10 carbon atoms, and heterocyclic rings having 5 to 10 carbon atoms. and R 104 is H or a straight or branched alkyl having 1 to 4 carbon atoms, where R 100 , R 101 , R 102 , R 103 and R 104 The alkyl, alkenyl, alkynyl, aryl, heteroaryl, and heterocyclyl in the group represented by the formula (I) are optionally substituted with one or more (e.g., 2, 3, 4, 5, 6, or more) substituents independently selected from halogen, —OH, —CN, —NO2, and unsubstituted straight-chain or branched alkyl having 1 to 4 carbon atoms. Preferably, the substituents for the above-mentioned optionally substituted alkyl, alkenyl, alkynyl, cyclic alkyl, cyclic alkenyl, cyclic alkynyl, carbocyclyl, aryl, heterocyclyl, and heteroaryl include halogen, —CN, —NR 102 R 103 , -CF3, -OR 101 , aryl, heteroaryl, heterocyclyl, -SR 101 , -SOR 101 , -SO2R 101 and -SO3M.

[0122] As indicated in the preceding paragraph -SOR 101 For sulfoxides represented by: both optical isomers (R and S configurations at the sulfoxide group sulfur atom) are included.

[0123] The number of carbon atoms in a group is indicated herein by the prefix "C x-xx " or "C x -C xx " where x and xx are integers. For example, "C 1-4 "Alkyl" or "C1-C4 alkyl" is an alkyl group having from 1 to 4 carbon atoms.

[0124] The terms "compound" or "cytotoxic compound," "cytotoxic dimer," and "cytotoxic dimeric compound" are used interchangeably. They are intended to include compounds whose structures or formulas, or any derivatives thereof, are disclosed in the present invention or incorporated by reference, or any derivatives thereof. The term also includes stereoisomers, geometric isomers, tautomers, solvates, metabolites, salts (e.g., pharmaceutically acceptable salts), and prodrugs, as well as prodrug salts, of all formulae of compounds disclosed in the present invention. The term also includes any solvates, hydrates, and polymorphs of any of the foregoing. The specific reference to a "stereoisomer," "geometric isomer," "tautomer," "solvate," "metabolite," "salt," "prodrug," "prodrug salt," "conjugate," "conjugate salt," "solvate," "hydrate," or "polymorph" should not be construed as an intentional omission of these forms in other aspects of the invention where, in certain aspects of the invention described in this application, the term "compound" is used without reference to these other forms.

[0125] As used herein, the term "conjugate" refers to a compound described herein or a derivative thereof linked to a cell binding agent.

[0126] The term "chiral" refers to molecules that have the property of being non-superimposable on their mirror image partners, while the term "achiral" refers to molecules that are superimposable on their mirror image partners.

[0127] The term "stereoisomers" refers to compounds that have identical chemical constitution and connectivity but different orientations of their atoms in space that cannot be interconverted by rotation about a single bond.

[0128] The term "diastereomer" refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers can be separated by high-resolution analytical procedures such as crystallization, electrophoresis, and chromatography.

[0129] The term "enantiomers" refers to two stereoisomers of a compound which are non-superimposable mirror images of one another.

[0130] Stereochemical definitions and conventions used herein generally follow those of S.P. 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. All stereoisomeric forms of the compounds of the present invention, including, but not limited to, diastereomers, enantiomers, and atropisomers, as well as mixtures thereof, such as racemic mixtures, are intended to form part of the present invention. Many organic compounds exist in optically active forms, i.e., have the ability to rotate the plane of plane-polarized light. In describing optically active compounds, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are used to indicate the sign of rotation of plane-polarized light by a compound; (-) or 1 means the compound is levorotatory. Compounds beginning with the prefix (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Specific stereoisomers are sometimes called enantiomers, and mixtures of such isomers are often called enantiomeric mixtures. A 50:50 mixture of enantiomers is called a racemic mixture or racemate and can occur when there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species that lacks optical activity.

[0131] 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 via reorganization of some of the bonding electrons.

[0132] As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound of the present invention. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, acid tartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate "mesylate", ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may include 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 of the parent compound. Furthermore, a pharmaceutically acceptable salt may have two or more charged atoms in its structure. If multiple charged atoms are part of the pharmaceutically acceptable salt, it may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0133] When the compound of the present invention is a base, the desired pharmaceutically acceptable salt can be prepared by any method known in the art. It can be prepared by any suitable method available, 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, and 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 pyranosidyl acid such as glucuronic acid or galacturonic acid, an alpha hydroxy acid such as citric acid or tartaric acid, an amino acid such as aspartic acid or glutamic acid, an aromatic acid such as benzoic acid or cinnamic acid, a sulfonic acid such as p-toluenesulfonic acid or ethanesulfonic acid, and the like.

[0134] 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.

[0135] 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 a compound are readily prepared by adding at least one molar equivalent of a hydroxylic solvent, such as methanol, ethanol, 1-propanol, 2-propanol, or water, to the compound to effect solvation or hydration of the imine moiety.

[0136] The phrase "pharmaceutically acceptable" indicates that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients comprising the formulation and / or the mammal being treated therewith.

[0137] The term "leaving group" refers to a charged or uncharged moiety that is displaced during substitution or replacement. Such leaving groups are well known in the art and include, but are not limited to, halogens, esters, alkoxy, hydroxyl, tosylates, triflates, mesylates, nitriles, azides, carbamates, disulfides, thioesters, thioethers, and diazonium compounds.

[0138] The term "reactive ester" refers to an ester having a readily displaceable leaving group that can easily 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.

[0139] The term "reactive group" refers to a group that can react with a moiety located on another molecule, such as a cell-binding agent or a cytotoxic compound, to form a covalent bond. Reactive groups include, but are not limited to, amine-reactive groups and thiol-reactive groups.

[0140] 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, halo groups, Examples of the amine-reactive group include an acyl halide, a sulfonyl halide, an imide ester, or a reactive thioester group. 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-hydroxysulfosuccinimide ester.

[0141] 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, alloacetamide, vinyl sulfone, vinyl sulfonamide, or vinyl pyridine. In one embodiment, the thiol-reactive group is maleimide.

[0142] The term "bifunctional crosslinker," "bifunctional linker," or "crosslinker" refers to a modifying agent having two reactive groups, one of which can react with a cell-binding agent, while the other reacts with a cytotoxic compound to link the two moieties together. Such bifunctional crosslinkers are well known in the art (see, e.g., Isalm and Dent, Bioconjugation chapter 5, pp. 218-363, Groves Dictionaries Inc. New York, 1999). For example, bifunctional crosslinkers that allow conjugation via a thioether bond include N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) to introduce a maleimide group or N-succinimidyl-4-(iodoacetyl)-aminobenzoic acid (SIAB) to introduce an iodoacetyl group. Other bifunctional crosslinkers that introduce maleimide or haloacetyl groups into cell-binding agents are well known in the art (Pierce Biotechnology Inc. PO Box 117, Rockland, IL 61105,See U.S. Patent Application Nos. 2008 / 0050310 and 20050169933, available from the U.S. Patent Office, San Diego, CA 942149, USA), and include, but are not limited to, bis-maleimide polyethylene glycol (BMPEO), BM(PEO)2, BM(PEO)3, N-(β-maleimidopropyloxy)succinimide ester (BMPS), γ-maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), 5-maleimidovaleric acid NHS, HBVS, the “long chain” analog of SMCC (LC-SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxy-(6-amidocaproate), 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), κ-maleimidoundecanoic acid N-succinimidyl ester (KMUA), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), succinimidyl-6-(β-maleimidopropionamido)hexanoate (SMPH), succinimidyl-(4-vinylsulfonyl)benzoate (SVSB), dithiobis-maleimidoethane (DTME), 1,4-bis-maleimidobutane (BMB), 1,4-bismaleimidoyl-2,Examples include 3-dihydroxybutane (BMDB), bis-maleimidohexane (BMH), bis-maleimidoethane (BMOE), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), sulfosuccinimidyl (4-iodo-acetyl)aminobenzoate (sulfo-SIAB), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS), N-(γ-maleimidobutryloxy)sulfosuccinimide ester (sulfo-GMBS), N-(ε-maleimidocaproyloxy)sulfosuccinimide ester (sulfo-EMCS), N-(κ-maleimidoundecanoyloxy)sulfosuccinimide ester (sulfo-KMUS), and sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB).

[0143] Heterobifunctional crosslinkers are bifunctional crosslinkers that have two different reactive groups. Heterobifunctional crosslinkers containing both an anion-reactive N-hydroxysuccinimide group (NHS group) and a carbonyl-reactive hydrazine group may be used to link the cytotoxic compounds described herein to cell-binding agents (e.g., antibodies). Examples of such commercially available heterobifunctional crosslinkers include succinimidyl 6-hydrazinonicotinamide acetone hydrazone (SANH), succinimidyl 4-hydrazide terephthalate hydrochloride (SHTH), and succinimidyl hydrazinium nicotinate hydrochloride (SHNH). Conjugates with acid-labile bonds can also be prepared using the hydrazine-containing benzodiazepine derivatives of the present invention. Examples of bifunctional crosslinkers that can be used include succinimidyl-p-formylbenzoate (SFB) and succinimidyl-p-formylphenoxyacetate (SFPA).

[0144] Bifunctional crosslinkers that allow the linkage of cell-binding agents and cytotoxic compounds via disulfide bonds are known in the art, including N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), and N-succinimidyl-4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB), which introduce dithiopyridyl groups. Other bifunctional crosslinkers that can be used to introduce disulfide groups are known in the art and are disclosed in U.S. Patent Nos. 6,913,748, 6,716,821, and U.S. Patent Publication Nos. 20090274713 and 20100129314, all of which are incorporated herein by reference. Alternatively, cross-linking agents that introduce thiol groups, such as 2-iminothiolane, homocysteine ​​thiolactone, or S-acetylsuccinic anhydride, can also be used.

[0145] 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, together. Typically, a linker is substantially inert under the conditions under which the two groups it connects are linked. A bifunctional crosslinker may contain two reactive groups, one at each end of the linker moiety, such that one reactive group is first reacted with a cytotoxic compound to provide a compound with a linker moiety and a second reactive group, which can then react with a cell-binding agent. Alternatively, one end of the bifunctional crosslinker may be first reacted with a cell-binding agent to provide a cell-binding agent with a linker moiety and a second reactive group, which can then react with a cytotoxic compound. The linking moiety may contain a chemical bond that allows for release of the cytotoxic moiety at a specific site. Suitable chemical bonds are well known in the art, including disulfide bonds, thioether bonds, acid-labile bonds, photolabile bonds, peptidase-labile bonds, and esterase-labile bonds (see, for example, U.S. Patent Nos. 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).Preferred are disulfide bonds, thioether bonds, and peptidase-labile bonds. Other linkers that may be used in the present invention include non-cleavable linkers, such as those described in detail in U.S. Publication No. 20050169333, or charged or hydrophilic linkers, and are described in U.S. Patent Application Publication Nos. 2009 / 0274713, 2010 / 01293140, and WO2009 / 134976, each of which is expressly incorporated herein by reference.

[0146] The term "self-immolative linker" refers to a linker that allows for release of a cytotoxic compound when a remote site is activated. In certain embodiments, the linker comprises a p-aminobenzyl unit. In some such embodiments, the p-aminobenzyl alcohol is The linker is attached to the amino acid unit via an amide bond, and a carbamate, methylcarbamate, or carbonate is formed between benzyl alcohol and the drug (Hamann et al. (2005) Exprest Opin. Ther. Patents (2005) 15:1087-1103). In some embodiments, the linker comprises p-aminobenzyloxycarbonyl (PAB). Other examples of self-immolative linkers include, but are not limited to, 2-aminoimidazole-5-methanol derivatives (U.S. Pat. No. 7,375,078; Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and aromatic compounds electronically similar to the PAB group, such as ortho- or para-aminobenzyl acetals. 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 a drug to the α-carbon of a glycine residue is another example of a self-immolative linker that may be useful in ADCs (Kingsbury et al. (1984) J. Med. Chem. 27:1447).

[0147] The term "amino acid" refers to naturally occurring or non-naturally occurring amino acids. In some embodiments, an amino acid is an NH2-C(R aa’ R aa )-C(=O)OH, where R aa and R aa’are each independently H, optionally substituted linear, branched, or cyclic alkyl, alkenyl, or alkynyl of 1 to 10 carbon atoms, aryl, heteroaryl, or heterocyclyl, or R aa and the N-terminal nitrogen atom may together form a heterocycle (e.g., as in proline). The term "amino acid residue" refers to -NH-C(R aa’ R aa )-C(=O)- refers to the corresponding residue when one hydrogen atom is removed from the amine and / or carboxy terminus of an amino acid.

[0148] The term "peptide" refers to a short chain of amino acid monomers linked by peptide (amide) bonds. In some embodiments, a peptide contains 2-20 amino acid residues. In other embodiments, a peptide contains 2-10 or 2-8 amino acid residues. In still other embodiments, a peptide contains 2-5 amino acid residues. As used herein, when a peptide is part of a cytotoxic agent or a linker (represented by a specific sequence of amino acids, as described herein), the peptide can be connected in both directions to the remainder of the cytotoxic agent or linker.

[0149] The term "cation" refers to an ion that has a positive charge. A positive ion is a monovalent ion (e.g., Na + , K. + etc.), divalent (e.g., Ca 2+ , Mg 2+ ), or multivalent (e.g., Al 3+ etc.) Preferably, the cation is monovalent.

[0150] 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, through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" includes intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) variants, 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 containing an antigenic determinant of an antibody, and any other modified immunoglobulin molecule containing an antigen-recognition site, so long as the antibody exhibits the desired biological activity. An antibody may be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) (based on the identity of their heavy chain constant domains, called alpha, delta, epsilon, gamma, and mu, respectively). Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies may be naked or conjugated to other molecules, such as toxins, radioisotopes, etc. As used herein, "antibody" also includes activatable antibodies (e.g., probodies). Activatable means that the activatable antibody exhibits a first level of binding to the target when the activatable antibody is in an inhibited, masked, intact or uncleaved state (i.e., a first conformation), and a second level of binding to the target when the activatable antibody is 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.

[0151] 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.

[0152] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv 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 by the term "antigen-binding fragment" of an antibody include (but are not limited to): (i) Fab fragments, V L , V H , C L , and C H1 (ii) a monovalent fragment consisting of a V domain (e.g., papain digestion of an antibody produces three fragments: two antigen-binding Fab fragments and one Fc fragment that does not bind to antigen); L , V H , C L , and C H1 single-chain Fab (scFab), which is a fragment consisting of the C L and V H the domains are linked via a linker peptide; (iii) F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region (e.g., digestion of an antibody with pepsin produces two fragments: a bivalent antigen-binding F(ab')2 fragment, and a pFc' fragment that does not bind antigen) and its associated monovalent F(ab') unit; (iv) V H and C H1F domain (i.e., that portion of the heavy chain contained in Fab) d (v) single-chain V of an antibody L and V H F consisting of domains v Fragments and associated disulfide bonds F v ;(vi)V H (viii) single-chain variable fragments (scFv), consisting of V domains connected via a linker peptide; H and V L fragments consisting of domains; and (ix) tetravalent antibodies (which can include a variety of formats (structures) in which the antibody contains four antigen-binding sites).

[0153] The term "monoclonal antibody" refers to a homogeneous antibody population that is responsible for highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies directed against different antigenic determinants. The term "monoclonal antibody" refers to both intact and full-length monoclonal antibodies, as well as antibody fragments (Fab, Fab', F(ab'), F v etc.), single chain ( "monoclonal antibody" also includes antibody fragments (scFv), variants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, "monoclonal antibody" refers to such antibodies produced by a number of methods, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.

[0154] The term "humanized antibody" refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequence. Typically, humanized antibodies are human immunoglobulins in which residues from the complementarity-determining regions (CDRs) are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, hamster) having 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).

[0155] In some cases, human immunoglobulin F v Framework region (FR) residues are replaced by corresponding residues of an antibody from a non-human species having the desired specificity, affinity, and capacity. v Further modification can be made by substituting additional residues either within the framework regions and / or within the substituted non-human residues to refine and optimize the specificity, affinity, and / or potency of the antibody. Generally, a humanized antibody will contain substantially all of at least one, and typically two or three, variable domains, including all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FR regions are of human immunoglobulin consensus sequences. Humanized antibodies also contain immunoglobulin constant regions or domains (F c), typically those of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Patent Nos. 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 (all incorporated herein by reference). In some embodiments, a "humanized antibody" is a resurfaced antibody. In some embodiments, a "humanized antibody" is a CDR-grafted antibody.

[0156] The term "variable region" of an antibody, alone or in combination, refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain. The heavy and light chain variable regions each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), also known as hypervariable regions. The CDRs of each chain are held in close proximity by the FRs and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) an approach based on interspecies sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., 1991, National Institutes of Health, Bethesda, Md.); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., J. Molec. Biol. 273:927-948, 1997). Furthermore, a combination of these two approaches is sometimes used in the art to determine CDRs.

[0157] The Kabat numbering system is commonly used when referring to residues in the variable domain (residues 1-107 of the light chain and 1-113 of the heavy chain) (see, e.g., Kabat et al., Sequences of Immunological Interest, 5th Ed., Public Health Service, National Institute of Infectious Diseases, Vol. 1, No. 1, pp. 111-113, 2003). l Institutes of Health, Bethesda, Md. (1991)).

[0158] The numbering of amino acid positions as in Kabat refers to the numbering system used for the heavy or light chain variable domains of the antibody compilation in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., 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 shortening or insertion of the FRs or CDRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues can be determined for a given antibody by alignment of the region of homology of the antibody's sequence with the "standard" Kabat numbered sequence. Chothia instead refers to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987). When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies 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 neither 35A nor 35B is present, the loop ends at 32. If only 35A is present, the loop ends at 33. 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. [ka]

[0159] The EU index or EU index in the Kabat or EU numbering scheme refers to the human IgG1 Refers to a numbering system based on the Eu antibody.

[0160] The term "human antibody" means 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, a human antibody is free of non-human sequences. This definition of a human antibody includes intact or full-length antibodies, or antigen-binding fragments thereof.

[0161] The term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecules are derived from more than one species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions are homologous to antibody sequences derived from another species (usually human) to avoid or reduce eliciting an immune response in that species (e.g., human). In certain embodiments, a chimeric antibody can comprise an antibody or antigen-binding fragment thereof comprising at least one human heavy and / or light chain polypeptide, such as, for example, an antibody comprising a murine light chain and a human heavy chain polypeptide.

[0162] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to that portion of an antigen that can be recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed from both contiguous and non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids are usually retained upon denaturation of the protein, whereas epitopes formed by tertiary folding are usually lost upon denaturation of the protein. An epitope typically contains at least three, more commonly at least five or 8-10 amino acids in a unique spatial conformation.

[0163] "Binding affinity" generally refers to the strength of the sum 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 specified, as used herein, "binding affinity" refers to the 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 a partner Y is generally determined by the dissociation constant (K d ) or half the maximum effective concentration (EC 50 ) Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigens slowly and tend to dissociate easily, whereas high-affinity antibodies generally bind antigens quickly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Certain exemplary embodiments are described herein.

[0164] "Specifically binds" generally means that an antibody binds to an epitope via its antigen-binding domain and that the binding involves 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 via its antigen-binding domain more readily than it binds to a random, unrelated epitope. The term "specificity" is used herein to identify the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" may be considered to have higher specificity for a given epitope than antibody "B," or antibody "A" may be said to bind epitope "C" with higher specificity than to the related epitope "D."

[0165] As used herein, the term "immunoconjugate," "conjugate," or "ADC" refers to a compound or derivative thereof that is linked to a cell-binding agent (e.g., an antibody or antigen-binding fragment thereof).

[0166] The term "cysteine ​​engineered antibody" includes antibodies with at least one Cys not normally present at a given residue in the antibody's light or heavy chain. Such Cys, which may also be referred to as "engineered Cys," can be engineered using any conventional molecular biology or recombinant DNA technique (e.g., by replacing the coding sequence for a non-Cys residue at the target residue with the coding sequence for Cys). For example, if the original residue has the coding sequence 5'-UCU-3', If it is Ser, the coding sequence may be mutated (e.g., by site-directed mutagenesis) to 5'-UGU-3', which encodes Cys. In certain embodiments, Cys-engineered antibodies of the invention have an engineered Cys in the heavy chain. In certain embodiments, the engineered Cys is within or near the CH3 domain of the heavy chain. In certain embodiments, the engineered Cys is at residue 442 of the heavy chain (EU / OU numbering). The C442 residue can be conjugated to a cytotoxic drug / drug via the free thiol group of the C442 residue, such as through reaction with a thiol-reactive agent (e.g., a maleimide group) of the cytotoxic drug.

[0167] 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 one or more cells resulting from excessive cell growth or proliferation, which can be either benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions.

[0168] 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 lymphatic cancer. 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 carcinoma, non-squamous cell, adenocarcinoma, or large cell undifferentiated carcinoma), colorectal cancer (adenocarcinoma, gastrointestinal carcinoma-like 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, the cancer is lymphoma or leukemia. In certain embodiments, exemplary cancers include AML, CML, ALL (e.g., B-ALL), CLL, myelodysplastic syndromes, blastic plasmacytoid DC neoplasm (BPDCN) leukemia, B-cell lymphomas including non-Hodgkin's lymphoma (NHL), precursor B-cell lymphoblastic leukemia / lymphoma, and mature B-cell neoplasms such as B-cell chronic lymphocytic leukemia (B-CLL) / small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma, and leukemia. Examples of cancers include cutaneous lymphoma (MCL), follicular lymphoma (FL), including low-grade, intermediate-grade, and high-grade FL, cutaneous follicle center lymphoma, marginal zone B-cell lymphoma (MALT type, lymph node and splenic type), hairy cell leukemia (HCL), diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, anaplastic large cell lymphoma (ALCL), and Hodgkin's leukemia (HL). In certain embodiments, the cancer is BPDCN leukemia. In certain embodiments, the cancer is ALL. In other embodiments, the cancer is AML.

[0169] The term "subject" refers to any animal (e.g., mammal), which will be the recipient of a particular treatment, including, but not limited to, humans, non-human primates, rodents, etc. Typically, the terms "subject," "patient," and "individual" are used interchangeably herein in reference to a human subject.

[0170] The term "pharmaceutical composition" refers to a preparation that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered. Such compositions may be sterile.

[0171] As used herein, a "therapeutically effective amount" is an amount of a compound or composition sufficient to carry out a specifically stated purpose. The full therapeutic effect does not necessarily occur with a single administration, but may occur only after a series of administrations. The specific "therapeutically effective amount" will depend, for example, on the age, weight, and 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 routinely in relation to the stated purpose.

[0172] As used herein, the terms "treating," "treat," or "treatment" include reversing, alleviating, or arresting the symptoms, clinical signs, or underlying pathology of a condition in a manner that improves or stabilizes the subject's condition. As used herein and as well understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired results include, but are not limited to, prevention, alleviation, amelioration, or delay in progression of one or more symptoms or conditions associated with a condition, reduction in the extent of disease, stable disease, delay or slowing of disease progression, progression, improvement, or palliation, and remission (partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0173] compound In one aspect, the present invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof: ZL 1 -D (Formula I) During the ceremony: D is represented by the following structural formula: [ka] R 1 is -F, -CH3, or -CF3; R 2 -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 together with the carbon atom to which they are attached form a methylenedioxy or difluoromethylenedioxy ring; R 3 is H or C1-C6 alkyl; R 4 is C1-C6 alkyl; L 1 is absent or -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, -X 1 '-(C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; where * is the moiety covalently bonded to Z; X 1 -O-, -S-, -S(O)-, -S(O)2-, -C(=O)-, -NR 5 -, -NR 5 C(=O)- or -C(=O)NR 5 - and; X 1 ' is -O-, -S-, -S(O)-, or -S(O)2-; L 2 is phenylene; Each R 5 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; Z is -H or -X 2 and; X 2 -OR 6 , -SR 6 , -S(O)R 6 , -S(O)2R 6 , -SSR 6 , or -N(R 6 )2; Each R 6 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; L 1 and L 2 are each independently a halogen, -CN, or -OR 7 , -SR 7 , -N(R 7 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 optionally substituted with 1 to 4 substituents selected from heterocycloalkyl, aryl, or heteroaryl; and Each R 7 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; However, this is the case if R 1 If is F, then L 1 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 -*; where * is the site of covalent attachment to Z; and Z is -X 2 and However, this is the case if R 1 is F and R 2 If -OMe, then -L 1 With the proviso that -Z cannot be -NH2.

[0174] In some embodiments, the present invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof: ZL 1 -D (Formula I) During the ceremony: D is represented by the following structural formula: [ka] R 1 is -F, -CH3, or -CF3; R 2 -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 together with the carbon atom to which they are attached form a methylenedioxy or difluoromethylenedioxy ring; R 3 is H or C1-C6 alkyl; R 4 is C1-C6 alkyl; L 1 is absent or -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, -X 1 '-(C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; where * is the moiety covalently bonded to Z; X 1 -O-, -S-, -S(O)-, -S(O)2-, -C(=O)-, -NR 5 -, -NR 5 C(=O)- or -C(=O)NR 5 - and; X 1 ' is -O-, -S-, -S(O)-, or -S(O)2-; L 2 is phenylene; Each R 5are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; Z is -H or -X 2 and; X 2 -OR 6 , -SR 6 , -S(O)R 6 , -S(O)2R 6 , -SSR 6 , or -N(R 6 )2; Each R 6 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; L 1 and L 2 are each independently a halogen, -CN, or -OR 7 , -SR 7 , -N(R 7 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 optionally substituted with 1 to 4 substituents selected from heterocycloalkyl, aryl, or heteroaryl; and Each R 7 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; However, this is the case if R 1 If is F, then L 1 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 -*; where * is the site of covalent attachment to Z; and Z is -X 2 provided that: However, this is the case if R 1 is F and R2 If -OMe, then -L 1 -Z cannot be -NH; and However, this is the case if R 1 is F and R 2 If -Me, then -L 1 With the proviso that -Z cannot be -CH2OH.

[0175] In some embodiments, R 1 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 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)-SSR 6 , or -(C1-C6 alkylene)-N(R 6 )2. In some embodiments, R 1 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 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 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.

[0176] In some embodiments, R 1 is -H or -F. In some embodiments, R1 is -F. 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 is -F. In some embodiments, R 2 is -OMe, -SMe, -S(O)Me, or methyl. In some embodiments, R 2 is methyl. In some embodiments, R 1 is -F and R 2 is -F. In some embodiments, R 1 is methyl and R 2 is -F. In some embodiments, R 1 is -F and R 2 is -methyl.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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 is.

[0181] 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)3 SH, -CH2NHC(=O)(CH2)4SH, -CH2NHC(=O)(CH2)5SH, -CH2NHC(=O)CH2SMe, -CH2NHC(=O)(CH2)2SMe, -CH2 NHC(=O)(CH2)3SMe, -CH2NHC(=O)(CH2)4SMe, -CH2NHC(=O)(CH2)5SMe, -CH2SCH2OH, -CH2S(CH2)2OH, - CH2S(CH2)3OH, -CH2S(CH2)4OH, -CH2S(CH2)5OH, -CH2SCH2OMe, -CH2S(CH2)2OMe, -CH2S(CH2)3OMe, -CH 2S(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.

[0182] In some embodiments, each R 5 is independently —H, methyl, or benzyl. In some embodiments, each R 5is independently —H. In some embodiments, each R 5 is methyl. In some embodiments, each R 5 is benzyl.

[0183] 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 6 is methyl. In some embodiments, each R 6 is benzyl.

[0184] 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, -OCH2SMe, -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.

[0185] In some embodiments, -L 1 -Z is -(C1-C6 alkylene)-X l -L 2 -X 2 In some embodiments, -L 1 -Z is [ka] is. In some embodiments, -L 1 -Z is [ka] In some embodiments, -L 1 -Z is [ka] is.

[0186] In another aspect, the present invention provides a compound of formula II, or a pharmaceutically acceptable salt thereof: EA-Z'-L 1 -D (Formula II) During the ceremony: D is represented by the following structural formula: [ka] R 1 is -H, -F, -CH3, or -CF3; R 2 -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 together with the carbon atom to which they are attached form a methylenedioxy or difluoromethylenedioxy ring; provided that R 1 and R 2 provided that both cannot be -H; R 3 is H or C1-C6 alkyl; R 4 is C1-C6 alkyl; L 1is absent or -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, X 1 '-(C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; where * is the moiety covalently bonded to Z'; X 1 -O-, -S-, -S(O)-, -S(O)2-, -C(=O)-, -NR 5 -, -NR 5 C(=O)- or -C(=O)NR 5 - and; X 1 ' is -O-, -S-, -S(O)-, or -S(O)2-; L 2 is phenylene; Each R 5 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; Z' is -O-CH2-NR 8 -*, -S-CH2-NR 8 -*, -NR 8 -*, where * is the site of covalent attachment to A; Each R 8 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; L 1 and L 2 are each independently a halogen, -CN, or -OR 7 , -SR 7 , -N(R 7 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 optionally substituted with 1 to 4 substituents selected from heterocycloalkyl, aryl, or heteroaryl; and Each R 7are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; A is a peptide containing 2 to 10 amino acids; wherein A is optionally substituted with one or more polyols; and E is -C(=O)-L 3 -X 3 and; L 3 is -(C1-C 10 alkylene)- or -Y 1 -(C 1 -C 10 alkylene)-X 4 -Y 2 -(C 1 -C 10 alkylene)-*; where * is X 3 is a moiety covalently bound to; Y 1 does not exist or is -(CR a R b O) n - or -(CR a R b CR a’ R b’ O) m - and; X 4 is -NR 9 C(=O)- or -C(=O)NR 9 - and; Y 2 does not exist or is -(CR c R d O)0-, or -(CR c R d CR c’ R d’ O) p - and; n, m, o, and p are each independently 1 to 10; Each R a , R b , R a’ , R b’ , R c , R d , R c’ , and R d’are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; L in the ceremony 3 is 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 optionally substituted with 0 to 4 substituents selected from heterocycloalkyl, aryl, heteroaryl, and polyol; Each R 11 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; X 3 teeth, [ka] -C(=O)-CR bb R cc -W', or -NR ee -C(=O)-CR bb R cc -W', or -SR 10 and; 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 and; q is 1 to 24; Each R aa , R bb , R cc , R ee , and R ff is independently —H or optionally substituted C1-C6 alkyl; Each R yy and R xxare independently —H or C1-C6 alkyl; R gg are each independently —H or C1-C6 alkyl; and R 9 and R 10 are each independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl.

[0187] In some embodiments, R 1 is -H or -F. In some embodiments, R 1 is -F. 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 is -F. In some embodiments, R 2 is -OMe, -SMe, -S(O)Me, or methyl. In some embodiments, R 2 is methyl. In some embodiments, R 1 is -F and R 2 is -F. In some embodiments, R 1 is methyl and R 2 is -F. In some embodiments, R 1 is -F and R 2 is -methyl.

[0188] 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'- * denotes -(C1-C4 alkylene)-O-CH2-NR 8In 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 -*.

[0189] In some embodiments, -L 1 -Z'-* stands for -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-*.

[0190] 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 -*.

[0191] In some embodiments, -L 1 -Z'-* stands for -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-*.

[0192] 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 is methyl. In some embodiments, each R 5 is benzyl. In some embodiments, each R 8 is independently —H, methyl, or benzyl. In some embodiments, each R8 is independently —H. In some embodiments, each R 8 is methyl. In some embodiments, each R 8 is benzyl.

[0193] 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 -*. Some implementations In terms of form, 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 -*.

[0194] 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-NH-, -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-, or -S(O)2(CH2)4-NH-.

[0195] In some embodiments, -L 1 -Z'-* is -(C1-C6 alkylene)-X 1 -L2 -Z'-*. In some embodiments, -L 1 -Z' is [ka] is. In some embodiments, -L 1 -Z' is [ka] is. In some embodiments, -L 1 -Z' is [ka] is.

[0196] As used herein, -L 1 In various embodiments disclosing -Z'-, * is the site of covalent attachment to A.

[0197] 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.

[0198] In some embodiments, A is -(AA 1 )-(AA 2 ) a1 -*, where * is the site of covalent attachment to E. AA 1 and A.A. 2 are each independently an amino acid residue; and a1 is an integer from 1 to 9.

[0199] In some embodiments, -AA 1 -(AA 2 ) al-* 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-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-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-*.

[0200] In some embodiments, -AA 1 -(AA 2 ) al-* stands for -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-*.

[0201] In some embodiments, -AA 1 -(AA 2 ) al -* stands for -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-*.

[0202] In some embodiments, -AA 1 -(AA 2 ) al-* stands for -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-G ln-*, -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-*.

[0203] In some embodiments, -AA 1 -(AA 2 ) al -* 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-*.

[0204] As used herein, -AA 1 -(AA 2 ) al In various embodiments disclosing -*, * is a site covalently attached to E.

[0205] 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)-X5 -Y 3 wherein: X 5 is -NR 12 C(=O)- or -C(=O)NR 12 -Y 3 is -C1-C 10 alkyl, wherein Y 3 is substituted with 0 to 10 OH groups; and R 12 is —H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl.

[0206] In some embodiments, the polyol is [ka] where R 12 is H or methyl.

[0207] In some embodiments, the polyol is [ka] where R 12 is H or methyl.

[0208] In some embodiments, E is —C(═O)—(C-C 10 alkylene)-X 3 In some embodiments, E is [ka] is.

[0209] In some embodiments, E is —C(═O)—Y 1 -(C1-C 10 alkylene)-X 4 -(C1-C 10 alkylene)-X 3 and; Y 1 is -(CR a R b O)n - or -(CR a R b CR a’ R b’ O) m - and; X 4 is -NR 9 C(=O)—; and X 3 teeth, [ka] , -C(=O)-CR bb R cc -W', NR ee -C(=O)-CR bb R cc -W', or -SR 10 is.

[0210] In some embodiments, E is —C(═O)—Y 1 -(CH2)2-X 4 -(CH2)2-X 3 and; Y 1 is -(CHO) n -or-(CH2CH2O) m - and; X 4 is -NHC(=O)-; n is 2; m is 2 to 6; X 3 teeth, [ka] , -C(=O)-CR bb R cc -W', NR ee -C(=O)-CR bb R cc -W', or -SR 10 is.

[0211] In another aspect, the present invention provides a compound of formula III, or a pharmaceutically acceptable salt thereof: CBA-E'-A-Z'-L 1 -D (Formula III) During the ceremony: D is represented by the following structural formula: [ka] R 1 is -H, -F, -CH3, or -CF3; R 2 -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 together with the carbon atom to which they are attached form a methylenedioxy or difluoromethylenedioxy ring; provided that R 1 and R 2 provided that both cannot be -H; R 3 is H or C1-C6 alkyl; R 4 is C1-C6 alkyl; L 1 is absent or -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, X 1 '-(C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; where * is the moiety covalently bonded to Z'; X 1 -O-, -S-, -S(O)-, -S(O)2-, -C(=O)-, -NR 5 -, -NR 5 C(=O)- or -C(=O)NR 5 - and; X 1 ' is -O-, -S-, -S(O)-, or -S(O)2-; L 2 is phenylene; Each R 5are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; Z' is -O-CH2-NR 8 -*, -S-CH2-NR 8 -*, -NR 8 -*, where * is the site of covalent attachment to A; Each R 8 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; L 1 and L 2 are each independently a halogen, -CN, or -OR 7 , -SR 7 , -N(R 7 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 optionally substituted with 1 to 4 substituents selected from heterocycloalkyl, aryl, or heteroaryl; and Each R 7 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 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 -*; where * is a moiety operably linked to a CBA; L 3 is -(C1-C 10 alkylene)- or -Y 1 -(C1-C 10 alkylene)-X 4 -Y 2 -(C1-C 10 alkylene)-*; where * is X 6 is a moiety covalently bound to; Y 1does not exist or is -(CR a R b O) n -or- (CR a R b CR a’ R b’ O) m - and; X 4 is -NR 9 C(=O)- or -C(=O)NR 9 - and; Y 2 does not exist or is -(CR c R d O) o - or -(CR c R d CR c’ R d’ O) p - and; n, m, o, and p are each independently 1 to 10; Each R a , R b , R a’ , R b’ , R c , R d , R c’ , and R d’ are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl aryl, heteroaryl, or benzyl; L in the ceremony 3 is 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 optionally substituted with 0 to 4 substituents selected from heterocycloalkyl, aryl, heteroaryl, and polyol; Each R 11 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; X 6 teeth, [ka] , -C-(=O)-CR bb R cc -* or NR ee -C(=O)-CR bb R cc -*; where * is the site of covalent attachment to the CBA; Each R aa , R bb , R cc , and R ee is independently —H or optionally substituted C1-C6 alkyl; Each R YY and R XX are independently —H or C1-C6 alkyl; R 9 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; and CBA is a cell binding agent.

[0212] In some embodiments, R 1 is -H or -F. In some embodiments, R 1 is -F. 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 is -F. In some embodiments, R 2 is -OMe, -SMe, -S(O)Me, or methyl. In some embodiments, R 2 is methyl. In some embodiments, R 1 is -F and R 2 is -F. In some embodiments, R 1 is methyl and R 2 is -F. In some embodiments, R 1 is -F and R2 is -methyl.

[0213] 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 In some embodiments, -L 1 -Z'-* is -(C1-C4 alkylene)-NR 8 -*.

[0214] In some embodiments, -L 1 -Z'-* stands for -CH2O-CH2NH-*, -(CH2)2O-CH2NH-*, -(CH2)3O-CH2NH-*, -(CH2)4O-CH2NH-*, -CH2S-CH2NH-*, -(CH2)2 S-CH2NH-*, -(CH2)3S-CH2NH-*, -(CH2)4S-CH2NH-*, -CH2NH-*, -(CH2)2NH-*, -(CH2)3NH-*, or -(CH2)4NH-.

[0215] 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 -*.

[0216] In some embodiments, -L 1-Z'-* stands for -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-*.

[0217] 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 is methyl. In some embodiments, each R 5 is benzyl. 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 is methyl. In some embodiments, each R 8 is benzyl.

[0218] 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 alkyl Ren)-S-CH2-NR 8 -*. In some embodiments, -L 1 -Z'-* is -X 1 -(C1-C4 alkylene)-NR 8 -*.

[0219] 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-NH-*, -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-*.

[0220] In some embodiments, -L 1 -Z'-* is -(C1-C6 alkylene)-X 1 -L2 -Z'-*. In some embodiments, -L 1 -Z'-* is [ka] is. In some embodiments, -L 1 -Z'-* is [ka] is. In some embodiments, -L 1 -Z'-* is [ka] is.

[0221] Herein -L 1 In various embodiments disclosing -Z'-*, * is the site of covalent attachment to A.

[0222] 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.

[0223] In some embodiments, A is -(AA 1 )-(AA 2 ) al -*, where * is the point of attachment to E', and AA 1 and A.A. 2 are each independently an amino acid residue; a1 is an integer of 1 to 9.

[0224] In some embodiments, -AA 1 -(AA 2 ) al-* 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-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-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-*.

[0225] In some embodiments, -AA 1 -(AA 2 ) al-* stands for -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-A la-*, -L-Ala-L-Val-*, -L-Gln-L-Val-*, -L-Gln-L-Leu-*, or -L-Ser-L-Val-*.

[0226] In some embodiments, -AA 1 -(AA 2 ) al -* stands for -Ala-Ala-*, -Ala-Val-*, -Val-Ala-*-Gln-Leu-*, -Leu-Gln-*, -Ala-Ala-Ala-*, -Ala-Ala-Ala-Ala-*, -Gl -Ala-Gly-Gly-*, -Gly-Gly-Ala-Gly-*, -Gly-Val-Gly-Gly-*, -Gly-Gly-Val-Gly-*, -Gly-Phe-Gly-Gly-*, or -Gly-Gly-Phe-Gly-*.

[0227] In some embodiments, -AA 1 -(AA 2 ) al-* stands for -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-G ln-*, -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-*.

[0228] In some embodiments, -AA 1 -(AA 2 ) al -* 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-*.

[0229] In this specification, -AA 1 -(AA 2 ) al In various embodiments disclosing -*, * is the site of covalent attachment to E'.

[0230] 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)-X5 -Y 3 wherein: X 5 is -NR 12 C(=O)- or -C(=O)NR 12 -Y 3 is -C1-C 10 alkyl, wherein Y 3 is substituted with 0 to 10 OH groups; and R 12 is —H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl.

[0231] In some embodiments, the polyol is [ka] where R 12 is H or methyl.

[0232] In some embodiments, the polyol is [ka] where R 12 is H or methyl.

[0233] In some embodiments, E' is -C(=O)-(C-C 10 alkylene)-X 6 In some embodiments, E' is [ka] -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 the CBA. In some embodiments, R YY , R xx , R aa , R bbare each independently —H or C1-C6 alkyl.

[0234] In some embodiments, E' is -C(=O)-Y 1 -(C1-C 10 alkylene)-X 4 -(C1-C 10 alkylene)-X 6 -* and; Y 1 is -(CR a R b O) n - or -(CR a R b CR a’ R b’ O) m - and; X 4 is -NR 9 C(=O)—; and X 6 teeth, [ka] -C(=O)-CR bb R cc -* or -NR ee -C(=O)-CR bb R cc -*; where * is the site of covalent attachment to the CBA.

[0235] In some embodiments, E' is -C(=O)-Y 1 -(CH2)2-X 4 -(CH2)2-X 6 -* and; Y 1 is -(CHO) n - or -(CH2CH2O) m - and; X 4 is -NHC(=O)-; n is 2; m is 2 to 6; X 6 teeth, [ka] , -C(=O)-CR bb R cc -* or -NR ee -C(=O)-CR bb R cc -*; where * is the site of covalent attachment to the CBA.

[0236] In some embodiments, CBA comprises an —SH group, which is covalently attached to E′: [ka] , -C(=O)-CR bb R cc -S-CBA, or -NR ee -C(=O)-CR bb R cc -Brings S-CBA.

[0237] In another aspect, the present invention provides a compound of formula IV, or a pharmaceutically acceptable salt thereof: EA-Z'-L 1 -D (Formula IV) During the ceremony: D is represented by the following structural formula: [ka] R 1 is -H, -F, -CH3, or -CF3; R 2 -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 together with the carbon atom to which they are attached form a methylenedioxy or difluoromethylenedioxy ring; provided that R 1 and R 2 Both of these cannot be -H; R 3 is H or C1-C6 alkyl; R4 is C1-C6 alkyl; L 1 is absent or -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, X 1 '-(C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; where * is the moiety covalently bonded to Z'; X 1 -O-, -S-, -S(O)-, -S(O)2-, -C(=O)-, -NR 5 -, -NR 5 C(=O)- or -C(=O)NR 5 - and; X 1 ' is -O-, -S-, -S(O)-, or -S(O)2-; L 2 is phenylene; Each R 5 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; Z' is -O-CH2-NR 8 -*, -S-CH2-NR 8 -*, -NR 8 -*, where * is the site of covalent attachment to A; Each R 8 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; L 1 and L 2 are each independently a halogen, -CN, or -OR 7 , -SR 7 , -N(R 7 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 optionally substituted with 1 to 4 substituents selected from heterocycloalkyl, aryl, or heteroaryl; Each R 7 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 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)OL 3 -X 3 and; L 3 is -(C1-C 10 alkylene)-; Here, L 3 is 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 optionally substituted with 0 to 4 substituents selected from heterocycloalkyl, aryl, heteroaryl, and polyol; Each R 9 are independently H, C-C alkyl, C-C fluoroalkyl, C-C cycloalkyl, aryl, heteroaryl, or benzyl; and X 3 teeth, [ka] is.

[0238] In some embodiments, E is -C(=O)OL. 3 -X 3 and L 3 is -(C alkylene)-. In some embodiments, L 3 is -CH2-.

[0239] In another aspect, the present invention provides a compound of formula V, or a pharmaceutically acceptable salt thereof: A-Z'-L 1 -D (Formula V) During the ceremony: D is represented by the following structural formula: [ka] R 1 is -H, -F, -CH3, or -CF3; R 2 -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 together with the carbon atom to which they are attached form a methylenedioxy or difluoromethylenedioxy ring; provided that R 1 and R 2 provided that both cannot be -H; R 3 is H or C1-C6 alkyl; R 4 is C1-C6 alkyl; L 1 is absent or -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, X 1 '-(C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; where * is the moiety covalently bonded to Z'; X 1 -O-, -S-, -S(O)-, -S(O)2-, -C(=O)-, -NR 5 -, -NR 5 C(=O)- or -C(=O)NR 5 - and; X 1 ' is -O-, -S-, -S(O)-, or -S(O)2-; L 2 is phenylene; Each R 5are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; Z' is -O-CH2-NR 8 -*, -S-CH2-NR 8 -*, -NR 8 -*, where * is the site of covalent attachment to A; Each R 8 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; L 1 and L 2 are each independently a halogen, -CN, or -OR 7 , -SR 7 , -N(R 7 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 optionally substituted with 1 to 4 substituents selected from heterocycloalkyl, aryl, or heteroaryl; Each R 7 are independently H, C-C alkyl, C-C fluoroalkyl, C-C cycloalkyl, aryl, heteroaryl, or benzyl; and A is a peptide comprising 2 to 10 amino acids; wherein A is optionally substituted with one or more polyols.

[0240] In some embodiments, the compound is a compound of Formula I and is any one of the following compounds in the table below: [Table 1-1] [Table 1-2]

[0241] In some embodiments, the compound is a compound of Formula I and is any one of the following compounds in the table below: [Table 1-3] [Table 1-4] [Table 1-5]

[0242] In some embodiments, the compound is a compound of formula II and is any one of the following compounds in the table below: [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7]

[0243] In some embodiments, the compound comprises a sulfoxide, which is either in the R or S configuration. , which is one of the following: [ka]

[0244] In some embodiments, the compound is any one of the following: [ka] [ka]

[0245] Compound synthesis The compounds described herein are prepared by the general synthetic routes depicted in the schemes below.

[0246] In some embodiments, the compounds described herein are prepared as outlined in Scheme 1. Scheme 1. General synthetic scheme for preparing RNH2 compounds (A-6) [ka]

[0247] In some embodiments, the compounds described herein are prepared as outlined in Scheme 2. Scheme 2. General synthetic scheme for preparing ROH compounds (B-2) [ka]

[0248] In some embodiments, the compounds described herein are prepared as outlined in Scheme 3. Scheme 3.RNHR c1 General synthetic scheme for preparing compound (C-4) [ka]

[0249] In some embodiments, the compounds described herein are prepared as outlined in Scheme 4. Scheme 4. RS(CH2) n General synthetic scheme for preparing OH compounds (D-2) [ka]

[0250] In some embodiments, the compounds described herein are prepared as outlined in Scheme 5. Scheme 5. General method for preparing thiol-bearing camptothecin derivatives (E-4) [ka]

[0251] 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.

[0252] In some embodiments, the compounds described herein are prepared as outlined in Scheme 6. Scheme 6. General method for preparing thioaminal-peptide linkages that can be further derivatized to incorporate thiols or maleimides. [ka]

[0253] 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.

[0254] In some embodiments, the compounds described herein are prepared as outlined in Scheme 7. [ka]

[0255] 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.

[0256] In some embodiments, the compounds described herein are prepared as outlined in Scheme 8.

[0257] [ka]

[0258] In some embodiments, v is 1 to 10. In some embodiments, v is 1 to 5.

[0259] Cell-binding agents The cell-binding agents in the immunoconjugates of the invention can be of any type currently known or that become known, including peptides and non-peptides that bind to cells or cell components (e.g., receptors, proteins, DNA, RNA, etc.). Generally, they can be antibodies (such as polyclonal and monoclonal antibodies, particularly monoclonal antibodies) or fragments thereof, lymphokines, hormones, growth factors, vitamins (such as folic acid, which can bind to its cell surface receptor, e.g., the folate receptor), nutrient transport molecules (such as transferrin), probodies, nanobodies, or any other cell-binding molecule or substance.

[0260] In certain embodiments, the cell binding agent is an antibody, a single chain antibody, or a compound that specifically binds to a target cell. antibody fragment, monoclonal antibody, single-chain monoclonal antibody, monoclonal antibody fragment (or "antigen-binding portion" or "antigen-binding fragment") that specifically binds to a target cell, chimeric antibody, chimeric antibody fragment (or "antigen-binding portion" or "antigen-binding fragment") that specifically binds to a target cell, domain antibody (e.g., sdAb), or domain antibody fragment that specifically binds to a target cell.

[0261] 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").

[0262] 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").

[0263] In certain embodiments, the cell binding agent is an antibody or antigen-binding portion thereof (including antibody derivatives), and the CBA is capable of binding to a ligand on a target cell, such as a cell surface ligand including a cell surface receptor.

[0264] In certain embodiments, the cell binding agent (CBA) binds to a target cell selected from a tumor cell, a virus-infected cell, a microbe-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 is selected from the group consisting of 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, C D71, CD74, CD79b, CDH6, CEACAM5, CEACAM6, cKIT, CLDN18.2, CLDN6, CLL-1, c-MET, Cripto, CSP-1, CXCR5, DLK-1, DLL3, DPEP3, Disadherin (Dysadherin), EFNA4, EGFR, EGFRviii, ENPP3, EpCAM, EphA2, EphA3, ETBR, FGFR2, FGFR3, FLT3, FOLR-alpha, 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 class I chain-related proteins A and B (MICA and MICB), MT1-MMP, MTX3, MTX5, MUC1, MUC16, NaPi2b, nectin-4, NOTCH3, OAcGD2 , 0X00IL, 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.

[0265] In certain embodiments, the cell binding agent is selected from the group consisting of 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, CD3 52, 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, Dysadherin, EFNA4, EGFR, EGFRviii, ENPP3, EpCAM, EphA2, EphA3, ETBR, FGFR2, FGFR3, FLT3, FOLR-alpha, 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 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, SS A cysteine ​​engineered antibody or antigen-binding fragment thereof that specifically binds to cells expressing any one or more of the following antigens: TR2, STEAP-1, STING, STn, TIM-1, TM4SF1, TNF-alpha, TRA, TROP-2, tumor-associated glycoprotein 72 (TAG-72), tumor-specific epitope mucin-1 (TA-MUC1), CD5, TIM-3, UPK2, or UPK1b.

[0266] 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 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 disclosed in USPN 9,238,690, 8,790,649, and 9,125,896, the contents of which are incorporated herein by reference in their entirety).

[0267] In certain embodiments, the CBA is an anti-CD123 antibody or antigen-binding fragment thereof and may comprise: a) three consecutive complementarity-determining regions (CDRs) L 1. CDR L 2, and CDR L 3, or a fragment thereof, wherein each of the CDRs L 1 has the amino acid sequence of RASQDINSYLS (SEQ ID NO: 1), and CDR L 2 has the amino acid sequence RVNRLVD (SEQ ID NO: 2), and CDR L 3 has the amino acid sequence of LQYDAFPYT (SEQ ID NO: 3); and b) each of three consecutive complementarity-determining regions (CDRs) H 1. CDR H 2, and CDR H 3, wherein the CDRs are: H 1 has the amino acid sequence of SSIMH (SEQ ID NO: 4), and H 2 has the amino acid sequence of YIKPYNDGTKYNEKFKG (SEQ ID NO: 5), and CDR H 3 has the amino acid sequence EGGNDYYDTMDY (SEQ ID NO: 6).

[0268] In certain embodiments, the anti-CD123 antibody or antigen-binding fragment thereof is [ka] A heavy chain variable region (V) having the amino acid sequence of (SEQ ID NO: 7) H )(where CDR H 1. CDR H 2, and CDR H 3 is double underline); and [ka] A light chain variable region (V) having the amino acid sequence of (SEQ ID NO: 9) L )(where CDR L 1. CDR L 2, and CDR L 3 is a double underline).

[0269] In certain embodiments, the anti-CD123 antibody is [ka] The heavy chain full length sequence of (SEQ ID NO: 8) (wherein CDR H 1. CDR H 2, and CDR H 3 is double underline); and [ka] The full-length light chain sequence of (SEQ ID NO: 10) (wherein CDR L 1. CDR L 2, and CDR L 3 is underlined).

[0270] In certain embodiments, the anti-CD123 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-CD123 activatable antibody or activatable CD123 antibody-binding antibody fragment may be conjugated to a compound of Formula I.

[0271] In certain embodiments, the CBA is an anti-CD33 antibody or antigen-binding fragment thereof, as described in US Pat. Nos. 7,342,110 and 7,557,189, which are incorporated herein by reference.

[0272] In certain embodiments, an anti-CD33 antibody or antigen-binding fragment thereof may comprise: a) three consecutive complementarity-determining regions (CDRs) L 1. CDR L 2, and CD R L 3, or a fragment thereof, wherein each of the CDRs L 1 has the amino acid sequence of KSSQSVFFSSSQKNYLA (SEQ ID NO: 11), L 2 has the amino acid sequence of WASTRES (SEQ ID NO: 12), and CDR L 3 has the amino acid sequence HQYLSSRT (SEQ ID NO: 13); and; b) each of three consecutive complementarity-determining regions (CDRs) H 1. CDR H 2, and CDR H 3, wherein the CDRs are: H 1 has the amino acid sequence of SYYIH (SEQ ID NO: 14), and 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).

[0273] In certain embodiments, the anti-CD33 antibody or antigen-binding fragment thereof is [ka] (SEQ ID NO: 17) (wherein CDR H 1. CDR H 2, and CDR H 3 is double underlined) The heavy chain variable region (V H ) and [ka] (SEQ ID NO: 19) (wherein CDR L 1. CDR L 2, and CDR L The light chain variable region (V) has the amino acid sequence L ) is included.

[0274] In certain embodiments, the anti-CD33 antibody is [ka] (SEQ ID NO: 18) (wherein CDR H 1. CDR H 2, and CDR H The full-length heavy chain sequence of and [ka] (SEQ ID NO: 20) (wherein CDR L 1. CDR L 2, and CDR L 3 is double underlined).

[0275] In a specific embodiment, the anti-CD33 antibody is the huMy9-6 antibody.

[0276] 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.

[0277] In certain embodiments, the CBA is an anti-ADAM9 antibody or antigen-binding fragment thereof, such as those described in WO2018 / 119196 and U.S. Provisional Application Nos. 62 / 690052 and 62 / 691342 (each of which is incorporated herein by reference).

[0278] 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 cynoADAM9.

[0279] In certain embodiments, the humanized anti-ADAM9 antibody or ADAM9-binding fragment thereof is optimized to have at least a 100-fold enhancement in binding affinity to cynoADAM9 and retains high affinity binding to human ADAM9 compared to the chimeric or murine parent antibody.

[0280] In certain embodiments, the anti-ADAM9 antibody or antigen-binding fragment thereof (e.g., a humanized anti-ADAMP antibody or antigen-binding fragment thereof) comprises: a) three contiguous complementarity-determining regions (CDRs) L 1. CDR L 2, and CDR L 3, each of which comprises at least one light chain variable region or fragment thereof, wherein L 1 has the amino acid sequence of KASQSVDYSGDSYMN (SEQ ID NO: 21), and L 2 has the amino acid sequence of AASDLES (SEQ ID NO: 22), and L 3 has the amino acid sequence QQSHEDPFT (SEQ ID NO: 23); and b) three consecutive complementarity-determining regions (CDRs) CDR H 1. CDR H 2, and CDR H 3, each of which comprises at least one heavy chain variable region or fragment thereof, H 1 has the amino acid sequence of SYWMH (SEQ ID NO: 24), and H 2 has the amino acid sequence of EIIPIFGHTNYNEKFKS (SEQ ID NO: 25), and H 3 has the amino acid sequence GGYYYYPRQGFLDY (SEQ ID NO: 26).

[0281] In certain embodiments, the anti-ADAM9 antibody or antigen-binding fragment thereof (e.g., a humanized anti-ADAM9 antibody or antigen-binding fragment thereof) [ka] (SEQ ID NO: 27) (wherein CDR H 1. CDR H 2, and CDR H 3 is double underlined) The heavy chain variable region (V H ) and [ka] (SEQ ID NO: 28) (wherein CDR L 1. CDR L 2, and CDR L The light chain variable region (V) has the amino acid sequence L ) is included.

[0282] In certain embodiments, the anti-ADAM9 antibody is [ka] (SEQ ID NO: 29) (wherein CDR H 1. CDR H 2, and CDR H The full-length heavy chain sequence of and [ka] (SEQ ID NO: 30) (wherein CDR L 1. CDR L 2, and CDR L 3 is double underlined).

[0283] 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 may be conjugated to a compound of Formula I.

[0284] In certain embodiments, the CBA is an anti-folate receptor antibody (i.e., FOLR1 or FRα) (see, e.g., U.S. Pat. No. 8,709,432, U.S. Pat. No. 8,557,966, No. 6,299,499, and WO2011106528, all of which are incorporated herein by reference.

[0285] In certain embodiments, an anti-FRα antibody or antigen-binding fragment thereof may comprise: a) three consecutive complementarity-determining regions (CDRs) L 1. CDR L 2, and CDR L 3, or a fragment thereof, wherein each of the CDRs L 1 has the amino acid sequence of KASQSVSFAGTSLMH (SEQ ID NO: 31), L 2 has the amino acid sequence of RASNLEA (SEQ ID NO: 32), and CDR L 3 has the amino acid sequence of QQSREYPYT (SEQ ID NO: 33); and b) three consecutive complementarity-determining regions (CDRs) H 1. CDR H 2, and CDR H 3, or a fragment thereof, wherein each of the CDRs H 1 has the amino acid sequence of GYFMN (SEQ ID NO: 34) or GYTFTGYFMN (SEQ ID NO: 37), and H 2 has the amino acid sequence of RIHPYDGDTFYNQKFQG (SEQ ID NO: 35) or RIHPYDGDTF (SEQ ID NO: 38), and HIn certain embodiments, the anti-FRα antibody or antigen-binding fragment thereof comprises: a) a CDR having the amino acid sequence set forth in SEQ ID NO: 31; L a light chain variable region comprising: a 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 light chain variable region comprising a CDR having the amino acid sequence set forth 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 In certain embodiments, the anti-FRα antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising: a) a CDR having the amino acid sequence set forth 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 CDR having the amino acid sequence set forth 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, comprising a heavy chain variable region.

[0286] In certain embodiments, the anti-FRα antibody or antigen-binding fragment thereof [ka] (SEQ ID NO: 39) (wherein CDR H 1. CDR H 2, and CDR H 3 is double underlined) The heavy chain variable region (V H ) and [ka] (SEQ ID NO: 40), or [ka] (SEQ ID NO: 41) (wherein CDR L 1. CDR L 2, and CDR L The amino acid sequence of The light chain variable region (V L ) is included.

[0287] In certain embodiments, the anti-FRα antibody is [ka] (SEQ ID NO: 42) (wherein CDR H 1. CDR H 2, and CDR H The full-length heavy chain sequence of and [ka] (SEQ ID NO: 43), or [ka] (SEQ ID NO: 44) (wherein CDR L 1. CDR L 2, and CDR L 3 is double underlined).

[0288] In a specific embodiment, the anti-FRα antibody is a huMovl9 or M9346A antibody.

[0289] 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 embodiments, the anti-FRα activatable antibody or activatable FRα antibody-binding antibody fragment can be conjugated to a compound of Formula I.

[0290] In certain embodiments, the CBA is an anti-EpCAM antibody or antigen-binding fragment thereof. In certain embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof comprises: a) three consecutive complementarity-determining regions (CDRs) L 1. CDR L 2, and CDR L 3 it at least one light chain variable region or fragment thereof comprising each of the CDRs L 1 has the amino acid sequence RSSRSLLHSDGFTYLY (SEQ ID NO: 45), L 2 has the amino acid sequence of QTSNLAS (SEQ ID NO: 46), and CDR L 3 has the amino acid sequence of AQNLELPNT (SEQ ID NO: 47); and b) three consecutive complementarity-determining regions (CDRs) CDR H 1. CDR H 2, and CDR H 3, or a fragment thereof, wherein each of the CDRs H 1 has the amino acid sequence of NYYIH (SEQ ID NO: 48), and H 2 has the amino acid sequence of WIYPGNVYIQYNEKFKG (SEQ ID NO: 49), and H 3 may include one having the amino acid sequence of DGPWFAY (SEQ ID NO: 50).

[0291] In certain embodiments, the anti-EpCAM antibody or antigen-binding fragment thereof [ka] (SEQ ID NO: 52) (wherein CDR H 1. CDR H 2, and CDR H 3 is double underlined) The heavy chain variable region (V H ) and [ka] (SEQ ID NO: 51) (Here, CDR L 1. CDR L 2, and CDR L The light chain variable region (V) has the amino acid sequence L ) is included.

[0292] In certain embodiments, the anti-EpCAM antibody is [ka] (SEQ ID NO: 54) (wherein CDR H 1. CDR H 2, and CDR H The full-length heavy chain sequence of and [ka] (SEQ ID NO: 53) (wherein CDR L 1. CDR L 2, and CDR L 3 is double underlined).

[0293] In certain embodiments, the CBA is an anti-EpCAM antibody, or an antigen-binding fragment thereof, may comprise: a) three consecutive complementarity-determining regions (CDRs) L 1. CDR L 2 and CDR L at least one light chain variable (VL) region or fragment thereof comprising each of CDRs 1 to 3; L 1 has an amino acid sequence selected from SEQ ID NOs: 78, 45, 79, 80 and 82; L 2 has the amino acid sequence of SEQ ID NO: 46; and L 3 has an amino acid sequence selected from SEQ ID NOs: 47, 81, 83, 84, 85, 86, and 87, and b) three consecutive complementarity-determining regions (CDRs) H 1. CDR H 2 and CDR H at least one heavy chain variable (VH) region or fragment thereof comprising each of the CDRsH 1 has an amino acid sequence selected from SEQ ID NOs: 48, 57, 58, 60, 61, 62, 68, 69, 70, 77 and 88; H 2 has the amino acid sequence of SEQ ID NOs: 49, 56, 59, 63, and 64; and H 3 has an amino acid sequence selected from SEQ ID NOs: 55, 65, 66, 67, 71, 72, 73, 74, 75 and 76.

[0294] 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.

[0295] Variants of the CDRs of anti-EpCAM antibodies or antigen-binding fragments are summarized in Tables 3-6 below.

[0296] [Table 3]

[0297] [Table 4]

[0298] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0299] [Table 6-1] [Table 6-2]

[0300] In certain embodiments, the antibodies described herein are murine, non-human mammalian, chimeric, humanized, or human antibodies. For example, a humanized antibody may be a CDR-grafted or resurfaced antibody. In certain embodiments, the antibody is a full-length antibody. In certain embodiments, the antigen-binding fragment thereof may be a Fab, Fab', F(ab'), F d , single-chain Fab (scFab), single-chain Fv or scFv, disulfide-linked F v , V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, duobody, single domain antibody, DVD-Ig, Fcab, mAb2, (SCFV)2, or scFv-Fc.

[0301] In certain embodiments, the cell-binding agent is an alternative protein scaffold, for example, a peptide targeting the somatostatin receptor (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;2013:926-295), inhibitor cystine knots (also known as ICKs or Knottins: 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), duobodies (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), hexabodies (de Jong RN, Beurskens FJ, Verploegen S, Strumane K, van Kampen MD, Voorhorst M, et al. Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface PLoS Biol 2016;14(1):e1002344); single-chain Fab (scFab) fragments (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 PaiPai MY, Lomenick B, Hwang H, Schiessel R,McBride W,Loo JA,et al.Drug affinity 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 type III fibronectin (FN3) repeats; see U.S. Patent Publications 2010 / 0255056, 2010 / 0216708, and 2011 / 0274623, which are incorporated herein by reference), ankyrin repeat proteins (e.g., designed ankyrin repeat proteins known as DARPins; see U.S. Patent Publication Nos. 2004 / 0132028, 2009 / 0082274, 2011 / 0118146, and 2011 / 0224100, which are incorporated herein by reference; see also C. Zahnd et al., Cancer Res. (2010) 70:1595-1605; Zahnd et al. al., J. Biol. Chem. (2006) 281(46):35167-35175; and Binz, HK, Amstutz, P. & 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 Nos. 2007 / 0238667; U.S. Patent No. 7,101,675; WO2007 / 147213; and WO2007 / 062466, incorporated herein by reference), adnectins (fibronectin domain scaffold proteins; see U.S. Patent Publication Nos. 2007 / 0082365; 2008 / 0139791, incorporated herein by reference), knottins (small disulfide-rich proteins characterized by disulfides linked via disulfide knots), bicyclic peptides (also called bicycles; Heinis C, Rutherford T, Freund S, Winter G.Phage-encoded combinatorial chemical libraries based on bicyclic peptides.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 Bicyclic Peptide Plasma Kallikrein Inhibitors for the Treatment of Diabetic. Macular Edema. J Med Chem 2018;61(7):2823-36), avibodies (including diabodies, triabodies, and tetrabodies; 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)).

[0302] Activable CBA In additional embodiments, the provided CBAs are activatable antibodies or activatable antigen-binding antibody fragments (collectively referred to as AAs). In some embodiments, the activatable antibodies or activatable antigen-binding antibody fragments comprise antibodies or antigen-binding antibody fragments (e.g., antibodies or antigen-binding antibody fragments described herein) that specifically bind to a ligand on a target cell (or "target") conjugated to a masking moiety (MM), such that conjugation 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 conjugated via a sequence comprising a substrate for a protease, e.g., a protease that is active in diseased tissue and / or a protease that co-localizes with the target at a treatment site of interest. Activatable antibodies are preferably stable in circulation, are activated at the intended site of treatment and / or diagnosis, but not in normal, e.g., healthy tissue or other tissues not targeted for treatment and / or diagnosis, and, upon activation, exhibit binding to the target that is at least comparable to that of the corresponding unmodified antibody. In some embodiments, the AA is one described in WO2009 / 025846, WO2010 / 081173, WO2015 / 048329, WO2015 / 116933, and WO2016 / 118629, each of which is incorporated by reference in its entirety.

[0303] In some embodiments, the activatable antibody or antibody fragment is: (a) a cleavable moiety (CM) attached to an antibody or antibody fragment (collectively "AB"), wherein the CM is a polypeptide that functions as a substrate for a protease; (b) a masking moiety (MM) attached 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; Here, the uncleaved, activatable antibody has the following structural configuration from N-terminus to C-terminus: (MM)-(CM)-(AB) or (AB)-(CM)-(MM).

[0304] In some embodiments, a masking moiety (or "mask") is an amino acid sequence that is attached to or otherwise attached to an antibody and is positioned within an activatable antibody construct such that the masking moiety reduces the antibody's ability to specifically bind to a 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 WO2009 / 025846, the contents of which are incorporated herein by reference in their entirety.

[0305] K of MM-modified AB against target d is the K of the unmodified AB or the parent AB directed to the target. d is 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, or 50,000,000 or more, or is between 5 and 10, 10 and 100, 10 between 1,000, 10 between 10, 10 and 100, 10 between ...,000, 10 between 100, 10 between 1,000, 10 between 1,000, 10 between 1,000, 10 between 10, 10,000, 10 between 100, 10 between 1,000, 10 between 1,000, 10 between 1,000, 10 between 10, 10,000, 10 between 1,000, 10 between 10, 10,000, 10 between 1,000, 10 between 10, 10,000, 10 between 1,000, , 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000, 1,000-10,000,000, 1,000-10,000,000, 1,000-100,000, 1,000-10,000,000, 10,000-10,000,000, 10,000-10,000,000, or 100,000-10,000,000 times greater. Conversely, the binding affinity of an AB modified with MM for a target may be greater than that of an AB not modified with MM, or the parent molecule directed towards the target. the binding affinity of the AB is 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 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 10-10,000,000 or 100-1 ,000, 100 to 10,000, 100 to 100,000, 100 to 1,000,000, 100 to 10,000,000, 1,000 to 10,000,000, 1,000 to 10,000,000, 1000 to 10,000,000, 10,000 to 100,000, 10,000 to 1,000,000, 100,000 to 1,000,000, or 100,000 to 10,000,000 times less.

[0306] The dissociation constant of MM towards AB (K d ) is generally the K of AB towards the target d K of MM against AB is larger than d is the K of AB against the target d The binding affinity of the MM to the AB may 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 greater than the binding affinity of the MM to the AB. Conversely, the binding affinity of the MM to the AB is generally lower than the binding affinity of the AB to the target. The binding affinity of the MM to the AB may 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 lower than the binding affinity of the AB to the target.

[0307] When an AB is modified with a MM and in the presence of a target, the specific binding of the AB to the target may be reduced or inhibited compared to the specific binding of an AB that is not modified with a MM or the specific binding of the parent AB to the target. When compared to the binding of an AB that is not modified with an MM, or the binding of the parent AB to a target, the ability of the AB to bind to a target when modified with an MM may be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% over 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 more, as measured in an in vivo or in vitro immunoabsorption assay for target displacement as described in WO2010 / 081173.

[0308] The MM may inhibit binding of the AB to its target. The MM may bind to the antigen-binding domain of the AB to inhibit binding of the AB to its target. The MM may sterically inhibit binding of the AB to its target. The MM may allosterically inhibit binding of the AB to its target. In these embodiments, when the AB is modified or attached to the MM, and in the presence of the target, the MM may be activated in vivo or by target displacement, as described in WO2010 / 081173. No or substantially no binding of the AB to the target as measured in an in vitro immunosorbent assay, or 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% binding of the AB to the target 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 more compared to binding of the unmodified AB, the parent AB, or an AB not conjugated to the MM to the target.

[0309] When an AB is conjugated or modified to a MM, the MM controls the specific binding of the AB to its target. When an AB is conjugated or modified to a MM, such conjugation or modification may result in a conformational change that reduces or inhibits the ability of the AB to specifically bind to its target.

[0310] The AB conjugated with or modified with MM can be represented by the following formula (order from amino terminal region (N) to carboxyl terminal region (C)): (MM)-(AB) (AB)-(MM) (MM)-L-(AB) (AB)-L-(MM) where MM is a masking moiety, Ab is an antibody or target-binding antigen fragment, and L is a linker. In many embodiments, it may be desirable to insert one or more linkers, e.g., flexible linkers, into the composition to thereby provide flexibility.

[0311] 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 no more than 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 no more than 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 no more than 25% identical to any natural binding partner of the Ab. In some embodiments, the MM is no more than 50% identical to any natural binding partner of the Ab. In some embodiments, the MM is no more than 20% identical to any natural binding partner of the Ab. In some embodiments, the MM is no more than 10% identical to any natural binding partner of the Ab.

[0312] The activatable antibodies provided herein may also comprise a cleavable moiety (CM). Such an AA exhibits an activatable / switchable bond to the target of the AB. The AA generally comprises an antibody or antibody fragment (AB) modified or conjugated with a masking moiety (MM) and a modifiable or cleavable moiety (CM). In some embodiments, the CM comprises an amino acid sequence that functions as a substrate for a protease of interest. In other embodiments, the CM provides a cysteine-cysteine ​​disulfide bond that can be cleaved by reduction. In yet other embodiments, the CM provides a photolytic substrate that can be activated by photolysis.

[0313] 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 the methods described in U.S. Pat. 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. When the CM is cleaved by a cleavage agent (e.g., a protease substrate in the CM is cleaved by a protease and / or a cysteine-cysteine ​​disulfide bond is disrupted by reduction upon exposure to a reducing agent), in the presence of the target, a cleaved state results, and the Ab binds to the target, and in the uncleaved state, in the presence of the target, The CM is positioned such that binding of the Ab to the target is inhibited by the MM. Note that the amino acid sequence of the CM may overlap with or be contained within the MM, such that all or part of the CM facilitates "masking" of the Ab when the activatable antibody is in an uncleaved conformation.

[0314] In some embodiments, CMs can be selected based on the protease co-localized in the tissue with the desired target of the activatable antibody. A variety of different conditions are known in which the target of interest co-localizes with the protease, where 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. There are many reports in the literature of increased levels of proteases with known substrates in many cancers, such as solid tumors. See, for example, La Rocca et al., (2004) British J. of Cancer 90(7):1414-1421.

[0315] Exemplary substrates may include, but are not limited to, substrates cleavable 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.

[0316] In some embodiments, the CM is a polypeptide up to 15 amino acids in length.

[0317] In some embodiments, a CM is a polypeptide comprising a first cleavable portion (CM1) that is a substrate for at least one matrix metalloprotease (MMP) and a second cleavable portion (CM2) that is a substrate for at least one serine protease (SP). In some embodiments, each of the CM1 and CM2 substrate sequences of a CM1-CM2 substrate is independently a polypeptide up to 15 amino acids in length.

[0318] In some embodiments, the CM is a substrate for legumain, plasmin, TMPRSS-3 / 4, MMP-9, MT1-MMP, cathepsin, caspase, human neutrophil elastase, beta-secretase, uPA, or PSA.

[0319] K of MM and CM modified Abs against targets d is the K of Ab unmodified with MM and CM d, or at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or more than the parent Ab against the target, or 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 10 It can be up to 1,000, 100 to 10,000, 100 to 100,000, 100 to 1,000,000, 100 to 10,000,000, 1,000 to 10,000, 1,000 to 10,000,000, 1000 to 10,000,000, 10,000 to 100,000, 10,000 to 1,000,000, 100,000 to 1,000,000, or more than 100,000 to 10,000,000 times. Conversely, the binding affinity of the MM and CM modified Ab for the target is 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, or 5,000,000 times greater than the binding affinity of the non-MM and CM modified Ab or parent Ab for the target. , 10,000,000, 50,000,000 or more, or 5 to 10, 10 to 100, 10 to 1,000, 10 to 10,000, 10 to 100,000, 10 to 1,000,000, 10 to 10,000,000, 100 to 1,000, 100 to 10,000, 100 to 100,000, 100 to 1,000,000, 100 to 10,000,000 00, 1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000, 10,000-10,000,000, 10,000-10,000,000, 100,000-1,000,000, or even 100,000-10,000,000 times lower.

[0320] When an Ab is modified with MM and CM and in the presence of a target but not in the presence of a modifying agent (e.g., enzyme, protease, reducing agent, light), the specific binding of the Ab to its target may be reduced or inhibited compared to the specific binding of the parent Ab to the target or Ab not modified with MM and CM. The target binding ability of an Ab when modified with MM and CM, when compared to the binding of the parent Ab or the binding of an Ab not modified with MM and CM to its target, may be reduced or inhibited in vivo or after target displacement, as described in WO201008117, which is incorporated herein by reference in its entirety. Displacement) as measured by an in vitro immunosorbent assay may be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% over 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 more.

[0321] As used herein, the term "cleaved state" refers to the state of an AA after modification of the CM by a protease and / or reduction of the cysteine-cysteine ​​disulfide, binding of the CM, and / or photoactivation. As used herein, the term "uncleaved state" refers to the state of an AA in the absence of cleavage of the CM by a protease and / or reduction of the cysteine-cysteine ​​disulfide bond of the CM, and / or in the absence of light. As noted above, the term "AA" is used herein to refer to both the uncleaved (native) and cleaved states of an AA. It will be apparent to those skilled in the art that in some embodiments, a cleaved AA may lack MM due to cleavage of the CM by a protease, resulting in release of at least MM (e.g., when the MM is not covalently attached to the AA (e.g., a disulfide bond between cysteine ​​residues)).

[0322] Activatable or switchable 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 a second level of binding to the target when in an inhibited, unmasked, and / or cleaved state (i.e., a second conformation), where the second level of target binding is greater than the first level of binding. Generally, the access of the target to the Ab of the AA is greater in the presence of a cleaving agent capable of cleaving the CM than in the absence of such a cleaving agent. Thus, when the AA is in the uncleaved state, the Ab can be inhibited or masked from target binding (i.e., the first conformation is such that the Ab cannot bind to the target), and in the cleaved state, the AB is not inhibited or masked from target binding.

[0323] The CM and AB of the AA can be selected such that AB represents a binding moiety for a target of interest and the CM represents a substrate for a protease that co-localizes with the target at a treatment site of interest. Alternatively, or in addition, the CM is a cysteine-cysteine ​​disulfide bond that is cleavable as a result of reduction of this disulfide bond. The AA comprises at least one of a protease-cleavable CM or a cysteine-cysteine ​​disulfide bond, and in some embodiments, Both types of CM are included. Alternatively or additionally, the AAs may comprise a photolabile substrate activatable by a light source. The AAs disclosed herein find particular use, for example, when a protease capable of cleaving a moiety of CM is present at a relatively higher level in target-containing tissue at a treatment site (e.g., diseased tissue; e.g., for therapeutic or diagnostic treatment) than in tissue at a non-treatment site (e.g., healthy tissue). The AAs disclosed herein also find particular use, for example, when a reducing agent capable of reducing a moiety of CM is present at a relatively higher level in target-containing tissue at a treatment or diagnostic site than in tissue at a non-treatment, non-diagnostic site. The AAs disclosed herein also find particular use, for example, when a light source, e.g., a laser, capable of photolyzing a moiety in the CM is introduced into target-containing tissue at a treatment or diagnostic site.

[0324] In some embodiments, an AAs may provide for a reduction in toxicity and / or adverse side effects that could result from Ab binding at non-treatment sites if the Ab's binding to its target were not masked or inhibited. If an AAs contains a CM cleavable by a reducing agent that promotes disulfide bond reduction, the AB of such an AAs may be selected to take advantage of Ab activation where the target of interest is present at a desired treatment site characterized by elevated levels of reducing agent, such that this environment is of a higher reduction potential than, for example, the environment at the non-treatment site.

[0325] In general, an AA can be designed by selecting an Ab of interest and constructing the remainder of the AA so that, when conformationally constrained, the MM provides masking of the Ab or reduced binding of the Ab to its target. Structural design criteria should be considered to provide this functional feature.

[0326] AAs are provided that exhibit a desired dynamic range of switchable phenotypes for target binding in inhibited versus uninhibited conformations. Dynamic range generally refers to the ratio between (a) the maximum detectable level of a parameter under a first set of conditions and (b) the minimum detectable value of that parameter under a second set of conditions. For example, in the context of an AA, dynamic range refers to the ratio between (a) the maximum detectable level of target protein binding to the AA in the presence of a protease capable of cleaving the CM of the AA and (b) the minimum detectable level of target protein binding 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 of treatment with an AA cleaving agent (e.g., enzyme) to the equilibrium dissociation constant of treatment with the AA cleaving agent. The larger the dynamic range of the AA, the better the switchable phenotype of the AA. AAs with a relatively high dynamic range value (e.g., greater than 1) exhibit a more desirable switchable phenotype, such that target protein binding by the AA occurs more frequently (e.g., primarily) in the presence of a cleaving agent (e.g., enzyme) capable of cleaving the CM of the AA than in the absence of the cleaving agent.

[0327] AA can be provided in a variety of structural configurations. An exemplary formula for AA is shown below. It is specifically contemplated that within an AA, the order of AB, MM, and CM from N-terminus to C-terminus can be reversed. It is also specifically contemplated that CM and MM can overlap in amino acid sequence, e.g., such that CM is contained within MM.

[0328] For example, AA may be represented by the following formula (from amino terminal region (N) to carboxyl terminal region (C): (MM)-(CM)-(AB) (AB)-(CM)-(MM), where MM is a masking moiety, CM is a cleavable moiety, and AB is an antibody or fragment thereof. Note that although MM and CM are shown as separate components in the formula above, in all exemplary embodiments (including formulas) disclosed herein, it is contemplated that the amino acid sequences of MM and CM may overlap, e.g., such that CM is fully or partially contained within MM. Furthermore, the above The formulas above provide additional amino acid sequences that can be placed at the N-terminus or C-terminus of the AA element.

[0329] 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 MM-CM junctions, CM-AB junctions, or both. For example, the AB, MM, and / or CM may not contain a sufficient number of residues (e.g., Gly, Ser, Asp, Asn, particularly Gly and Ser, especially Gly) to provide the desired flexibility. Thus, the switchable phenotype of such an AA construct may benefit from the introduction of one or more amino acids to provide a flexible linker. Furthermore, as described below, when the AA is provided as a conformationally constrained construct, a flexible linker may be operably inserted to facilitate the formation and maintenance of a cyclic structure in the uncleaved AA.

[0330] For example, in certain embodiments, AA comprises one of the following formulas (the following formulas represent the amino acid sequence in either the N to C terminal or C to N terminal direction): (MM)-L1-(CM)-(AB) (MM)-(CM)-L1-(AB) (MM)-L1-(CM)-L2-(AB) CyClo[L1-(MM)-L2-(CM)-L3-(AB)] wherein MM, CM, and AB are as defined above; and wherein L1, L2, and L3 are each independently the same or different flexible linkers, optionally present or absent, containing at least one flexible amino acid (e.g., Gly). Wherein, when Cyclo is present, AA is in the form of a cyclic structure due to the presence of a disulfide bond between a pair of cysteines within AA. Furthermore, the above formula provides additional amino acid sequences that can be placed at the N- or C-terminus of the AA element. It should be understood that in the formula Cyclo[L1-(MM)-L2-(CM)-L3-(AB)], the cysteines involved in the disulfide bond can be placed at the AA to allow for one or two tails, which can generate a lasso or omega structure when the AA is in a disulfide-bonded structure (and thus conformationally constrained). The amino acid sequence of the tail(s) can provide additional AA functions, such as binding to a target receptor to facilitate AA localization or increasing the serum half-life of the AA. a targeting moiety (e.g., a ligand for a receptor on cells present in the target tissue) and a serum half-life extending moiety (e.g., a polypeptide that binds to a serum protein such as an immunoglobulin (e.g., IgG) or serum albumin (e.g., human serum albumin (HSA))).

[0331] Linkers suitable for use with the AAs described herein generally provide flexibility to the modified AB or AA to facilitate inhibition of AB binding to a target. Such linkers are commonly referred to as flexible linkers. Suitable linkers can be easily selected and can be of any suitable length, including 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, etc., for example, 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0332] Exemplary flexible linkers include glycine polymers (G) n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n and (GGGS) n where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and may therefore function as neutral tethers between components. Glycine has access to significantly more phi-psi space than alanine and is much less restricted than residues with longer side chains (Scheraga, Rev. (See 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-Ser-Gly-Gly-Gly, Gly-Ser-Ser-Gly, Gly-Ser-Ser-Ser-Gly, and the like. Those skilled in the art will recognize that the design of an AA can include a linker that is fully or partially flexible, such that the linker can include a flexible linker as well as one or more moieties that confer a less flexible structure to provide the desired AA structure.

[0333] In addition to the above elements, modified AB and AA may contain additional elements, such as amino acid sequences at the N- or C-terminus of the AA. For example, the AA may contain a targeting moiety to facilitate delivery to a cell or tissue of interest. Furthermore, the AA may be provided in the context of a scaffold protein to facilitate display of the AA on the cell surface.

[0334] In some embodiments, the activatable antibody also comprises a signal peptide. In some embodiments, the signal peptide is conjugated 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, the spacer is directly attached to the MM of the activatable antibody in the N- to C-terminal structural arrangement of spacer-MM-CM-AB.

[0335] 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, e.g., WO2016 / 179285 (e.g., pages 52-53), the contents of which are incorporated herein by reference in their entirety.

[0336] In some embodiments, the activatable antibody is exposed to a protease and cleaved by the protease, such that in the activated or cleaved state, the activated antibody comprises a light chain sequence that includes at least a portion of the LP2 and / or CM sequence after the protease cleaves the CM.

[0337] CM is approximately 0.001 to 1500 x 10 4 M -1 S -1or 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 by at least one protease 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 ~Approx. 1×10 6 M -1 S -1 (i.e., about 1×10 2 ~Approx. 1×10 6 M -1 S -1 ) for specific cleavage by the enzyme. Contact between the enzyme and the CM is required. When an activatable antibody, including an Ab (e.g., an antibody or EpCAM-binding antibody fragment) bound to the MM and CM, is in the presence of the target and sufficient enzymatic activity, the CM can be cleaved. Sufficient enzymatic activity may refer to the ability of the enzyme to contact the CM and effect cleavage. It is easy to imagine that the enzyme may be near the CM but unable to cleave it due to other cellular factors or protein modifications of the enzyme.

[0338] Generation of cell-binding agent-drug conjugates To attach any one of the cytotoxic compounds or derivatives thereof described herein to a cell-binding agent, the cytotoxic compound can include a linking moiety having a reactive group attached thereto. These compounds can be directly attached to a cell-binding agent. Cytotoxic compounds with reactive groups attached Representative processes for conjugating entities with cell-binding agents to produce cell-binding agent-cytotoxic agent conjugates are described in the Examples.

[0339] In some embodiments, a bifunctional crosslinking reagent can be first reacted with a cytotoxic compound to provide a compound having a linking moiety with one reactive group attached thereto (i.e., a drug-linker compound), which can then react with a cell-binding agent. Alternatively, one end of the bifunctional crosslinking reagent can be first reacted with a cell-binding agent to provide a cell-binding agent having a linking moiety with one reactive group attached thereto, which can then react with a cytotoxic compound. The linking moiety can include a chemical bond that allows for release of the cytotoxic moiety at a specific site. Suitable chemical bonds are well known in the art, including disulfide bonds, thioether bonds, acid labile bonds, photolabile bonds, peptidase labile bonds, and esterase labile bonds (see, for example, U.S. Patent Nos. 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).Preferred are disulfide bonds, thioether bonds, and peptidase labile bonds. Other linkers that may be used in the present invention include non-cleavable linkers, such as those described in detail in U.S. Publication No. 2005 / 0169933, or charged or hydrophilic linkers, and are described in U.S. Publication No. 2009 / 0274713, U.S. Publication No. 2010 / 01293140, and WO2009 / 134976 (each of which is expressly incorporated herein by reference).

[0340] In some embodiments, a solution of a cell-binding agent (e.g., an antibody) in an aqueous buffer may be incubated with a molar excess of a bifunctional crosslinker, such as N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), or N-succinimidyl-4-(2-pyridyldithio)2-sulfobutanoate (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 generate a disulfide-linked cell-binding agent-cytotoxic agent conjugate of the invention.

[0341] In another embodiment, the thiol-containing cytotoxic compounds described herein may be reacted with a bifunctional crosslinker, such as N-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), or N-succinimidyl-4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB), to form a cytotoxic agent-linker compound, which is then reacted with a cytotoxic agent to produce a disulfide-linked cell-binding agent-cytotoxic agent conjugate of the invention. The cytotoxic agent-linker compound may be prepared in situ without purification before reaction with the cell-binding agent. Alternatively, the cytotoxic agent-linker compound may be purified before reaction with the cell-binding agent.

[0342] 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 of which are incorporated herein by reference). For example, the cell-binding agent-cytotoxic agent conjugate can be purified using tangential flow filtration, adsorption chromatography, adsorptive filtration, selective precipitation, non-adsorptive filtration, or a combination thereof. Preferably, tangential flow filtration (TFF, also known as cross-flow filtration, ultrafiltration, and diafiltration) and / or adsorptive chromatography resins are used to purify the conjugate.

[0343] The number of cytotoxic molecules conjugated per antibody molecule can be determined spectrophotometrically by measuring the ratio of absorbance at 280 nm and 330 nm. In some embodiments, an average of 1-10 cytotoxic compounds / antibody molecule(s) can be conjugated by the methods described herein. In some embodiments, the average number of cytotoxic compounds conjugated 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 to 8. In some embodiments, the DAR value is 7.6 to 8. In some embodiments, the DAR value is 7.7 to 8. In some embodiments, the DAR value is 7.8 to 8. In some embodiments, the DAR value is 7.9 to 8. In some embodiments, a composition (e.g., a pharmaceutical composition) comprising a conjugate of the invention has a DAR value of between 2 and 12, between 2 and 10, or more particularly between 6 and 8. In some embodiments, the average number of linked cytotoxic compounds per antibody molecule (DAR) is 7 to 8, or more particularly 8.

[0344] In some embodiments, when an antibody is linked to a cytotoxic agent via a cysteine ​​thiol group, the conjugate has one cytotoxic compound per antibody molecule. In some embodiments, the conjugate has one or two cytotoxic compounds per antibody molecule. In some embodiments, the conjugate has two cytotoxic compounds per antibody molecule. In some embodiments, the average number of cytotoxic compounds linked per antibody molecule (DAR) is 1.5 to 2.5, more specifically 1.8 to 2.2. In some embodiments, compositions (e.g., pharmaceutical compositions) comprising the conjugates of the invention have 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.

[0345] Exemplary processes for preparing the cell-binding agent-drug conjugates of the invention are described in U.S. Patent Application Publication No. 2012 / 0238731 and U.S. Patent Application Publication No. 8,765,740, the entire teachings of which are incorporated herein by reference.

[0346] composition The present invention includes compositions (e.g., 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).

[0347] The pharmaceutical compositions described herein can be administered in a number of ways for either local or systemic treatment. Administration can be topical (such as 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 powders or aerosols, including by nebulizers; 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). In some specific embodiments, administration is intravenous. The pharmaceutical compositions described herein can also be used in vitro or ex vivo.

[0348] Suitable pharmaceutically acceptable carriers, diluents, and excipients are well known and can be determined by those skilled in the art, as the clinical situation warrants. Examples of suitable carriers, diluents, and / or excipients include: (1) Dulbecco's phosphate buffered saline, pH about 7.4, with or without about 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 include antioxidants such as tryptamine and stabilizers such as Tween 20.

[0349] How to use The compositions are useful for inhibiting abnormal cell growth or treating a proliferative disorder in a mammal (eg, a human).

[0350] The present invention includes a method of inhibiting abnormal cell proliferation or treating a proliferative disorder, an autoimmune disorder, a destructive bone disorder, an infectious disease, a viral disease, a fibrotic disease, a neurodegenerative disorder, pancreatitis, or a kidney disease in a mammal (e.g., a human), comprising administering to said mammal a therapeutically effective amount of a cytotoxic compound, derivative, or conjugate thereof (and / or solvates and salts thereof) or composition thereof, as described herein.

[0351] In certain embodiments, the proliferative disorder in the mammal is cancer, including hematological cancer, leukemia, or lymphoma. In certain embodiments, the proliferative disorder is cancer of the lymphoid organs or a hematological malignancy. In some embodiments, the cancer is lymphoma or leukemia.

[0352] For example, the cancer may be acute myeloid leukemia (including AML, CD33-low AML, P-glycoprotein positive AML, relapsed AML, or refractory AML), chronic myeloid leukemia (CML), including the blastic phase of CML and the Abelson oncogene associated with CML (Bcr-ABL translocation), 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, but not limited to, Richter's syndrome or The tumor may be selected from the group consisting of Richter's transformation of CLL, hairy cell leukemia (HCL), acute myeloid leukemia (APL), B-cell chronic lymphoproliferative disorder (B-CLPD), atypical chronic lymphocytic leukemia (preferably with prominent CD11c expression), diffuse large B-cell lymphoma (DLBCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin's lymphoma (NHL), such as Mantel cell leukemia (MCL) and small lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis, and Burkitt's lymphoma.

[0353] 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 lymphatic cancer. In some embodiments, the lung cancer is non-small cell lung cancer or small cell lung cancer. In further embodiments, the compounds of the present invention may be useful in treating non-small cell lung cancer (squamous cell, non-squamous cell, adenocarcinoma, or large cell undifferentiated carcinoma), colorectal cancer (adenocarcinoma, gastrointestinal carcinoma tumor, gastrointestinal stromal tumor, primary colorectal lymphoma, leiomyosarcoma, or squamous cell carcinoma), or breast cancer (e.g., triple-negative breast cancer (TNBC)).

[0354] 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. [Example]

[0355] Example The following abbreviations are used for the following terms: DAR drug-to-antibody ratio; DIPEA diisopropylethylamine; DMF dimethylformamide; DMSO dimethyl sulfoxide; DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride; EDTA ethylenediaminetetraacetic acid; EPPS 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid; Fmoc 9-fluoroenylmethoxycarbonyl; HMPA hexamethylphosphoramide; iv intravenous; NMM N-methylmorpholine; PPTS pyridinium p-toluenesulfonate; SEC size exclusion chromatography; SEM SEM standard error of the mean; TCEP 3,3',3"-phosphinetriyltripropanoic acid hydrochloride; TEA triethylamine; and TFA Trifluoroacetic acid.

[0356] Figures 1 and 2 show the synthesis of camptothecin building blocks. Figures 3, 4, and 5 show the synthesis of side chains. Figures 6 and 7 show the attachment of camptothecin building blocks to side chains. Figure 8 shows the synthesis of additional camptothecin metabolites. Figure 9 shows the attachment of camptothecin building blocks to side chains. Figure 10 shows the synthesis of additional camptothecin compounds.

[0357] Figure 11 shows the compounds used for comparison, including the general Ab-999 structure of an ADC with a 999 moiety linked via a reduced interchain disulfide of an antibody. Compounds 998 and 999 were prepared as described in U.S. Patent 2016 / 0297890A1 and references therein. [Example]

[0358] Example 1. Synthesis of Compounds 2a-2e [ka]

[0359] 2a: To a flask containing anhydrous 1,2-dichloroethane (80 mL), 1 M boron trichloride in dichloromethane (16 mL, 16 mmol) was added and cooled to 0 °C in an ice bath. 1a (2.5 g, 20 mmol) was added in portions and stirred at 0 °C for 10 minutes. Chloroacetonitrile (2.7 mL, 23.5 mmol) was added, followed by aluminum chloride (3.5 g, 26 mmol). The ice bath was removed, and the reaction solution was allowed to warm gradually to room temperature. After stirring at room temperature for 10 minutes, the reaction mixture was heated to reflux for 39 hours. The reaction solution was cooled to room temperature, and cold water (40 mL) was slowly added, followed by 5% aqueous HCl. After 30 minutes, 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 9 min). Purification by 5% CH3CN for 22 min, then 95% CH3CN for 5 min) gave compound 2a as an off-white solid (1.52 g, 38% yield). MS (ESI): (M+H). + Calculated value 202.0, measured value 202.2. [ka]

[0360] 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 nitrogen, followed by 1 M BCl3 in toluene (3.85 mL, 7.6 mmol). 2-Chloroacetonitrile (1.7 mL, 26.6 mmol) was added dropwise over approximately 2 min, and the flask was then fitted with a reflux condenser and heated in a 114 °C oil bath for 3 h. The heating bath was removed, and after 4 min, a thermometer was placed in the flask. 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 and dried over anhydrous Na2SO4, and the solvent was then removed by rotary evaporation under vacuum. The residue was taken up in a minimal amount 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). [ka]

[0361] 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 h. The reaction solution was stripped under reduced pressure, and the residue was purified by silica gel chromatography (12 g silica column, CHCl / MeOH, 0-10% MeOH in 16 min) to give 156 mg of 2e (74% yield). MS (ESI): m / z 273A (M+H). + . [ka]

[0362] 2c: To a flask containing anhydrous 1,2-dichloroethane (50 mL) was added 1 M boron trichloride in dichloromethane (9.95 mL, 9.95 mmol) and then cooled to 0 °C in an ice bath. 1a (1.56 g, 12.4 mmol) was added in portions, followed by stirring at 0 °C for 10 min. 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 allowed to warm gradually to room temperature. After stirring at room temperature for 10 min, the reaction mixture was heated to reflux for 39.5 h. The reaction solution was cooled to room temperature, and cold water (25 mL) was slowly added, followed by the addition of 5% aqueous HCl. After 30 min, 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 / HO, 25% CH3CN, 5 min, then up to 95% CH3CN for 15 min, then 95% CH3CN, 5 min) to give compound 2c as an off-white solid (1.42 g, 40% yield). MS (ESI): m / z 289.2 (M+H). + . [ka]

[0363] 2d: To a dry flask containing anhydrous 1,2-dichloroethane (40 mL), boron trichloride (4.73 mL, 4.73 mmol, 1 M in CHCl) was added and cooled to 0 °C in an ice-water bath. Compound 1b (0.93 g, 5.92 mmol) was added in portions and stirred for 10 minutes at 0 °C. After that, 5-bromovaleronitrile (0.819 mL, 7.1 mmol) was added, followed by aluminum chloride (1.025 g, 7.69 mmol). The ice bath was removed, and the reaction mixture was allowed to warm gradually to room temperature. After stirring for 10 minutes at room temperature, the reaction mixture was heated to reflux for 45 hours. The reaction mixture was cooled to room temperature, and cold water (25 mL) was slowly added, followed by 5 mL of 5% aqueous HCl. 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 min, then up to 95% CH3CN for 15 min, then 95% CH3CN for 5 min) to give compound 2d as an off-white solid (0.776 g, 41% yield). MS (ESI): m / z 321.0 (M+H). + . [Example]

[0364] Example 2. General procedure for compounds 4a-4d [ka]

[0365] 4b: Compound 3 (777 mg, 2.95 mmol), 2b (690 mg, 2.95 mmol), and PPTS (37 mg, 0.15 mmol) were suspended in a 50 mL flask equipped with a reflux condenser containing anhydrous toluene (10 mL). The reaction was heated to reflux with magnetic stirring under an argon atmosphere overnight and then allowed to cool to room temperature. The mixture was reduced Pressure filtration and washing of the solid with toluene (5 mL) gave 1.08 g of the desired product 4b as a brown solid (79% yield): MS (M+H)+ 461, MS (MH) 459. [ka]

[0366] 4a: A solution of compound 2a (3.15 g, 15.64 mmol) and compound 3 (3.92 g, 14.89 mmol) in toluene (200 mL) of PPTS (187 mg, 0.75 mmol), prepared by the general procedure. The mixture (containing 20 mmol) was reperfused for 40 h to give 4a (4.74 g) in 74% yield. MS (ESI): m / z 429.2, 431.0 (M+H). + . 1 HNMR(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),6.5 5(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, measured value: 429.1023. [ka]

[0367] 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 h. The solvent was evaporated, and the residue was taken up 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. MS (ESI): m / z 515.2 and 517.1 (M+H). + , 513.1 and 515.1(MH) - . 1 HN MR(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, measured value: 515.0992. [ka]

[0368] 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 give 4d (1.02 g) in 77% yield after silica gel chromatography (40 g silica column, CHCl / MeOH, 0-10% MeOH in 23 min). MS (ESI): m / z 547.0 and 548.9 (M+H). +, 544.8 and 546.8 (MH) - . [ka]

[0369] 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-bromohexalic acid (7.2 mg, 0.037 mmol). The solution was cooled to 0 °C in an ice bath, and DMTMM (13.5 mg, 0.049 mmol) in deionized water was added. The ice bath was removed, and the reaction mixture was stirred at room temperature for 3 h. The reaction solution was stripped under reduced pressure, and the residue was purified by silica gel chromatography (4 g column, CHCl / MeOH, 0–10% MeOH in 15 min) to give 15 mg of 4e (98% yield). MS (ESI): m / z 586.1 (M+H). + ,588.0(M+H) + . [Example]

[0370] Synthesis of compounds 8a-8e and 8p [ka]

[0371] 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 h. The reaction mixture was cooled to room temperature, and the solution was loaded onto a C18 cartridge and purified by reverse-phase HPLC (30 g C18 column, CHCN / HO, 25% CH3CN for 3 min, then 95% CH3CN for 15 min, then 95% CH3CN for 5 min) to remove contaminated impurities. 8a was obtained as a stained solid, which was further purified by silica gel chromatography (CH2Cl2 / MeOH, 0-20% MeOH) to give 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 (400MHz, methanol-d4)δ 8.00(dd,J=8.2,1.1Hz,1H),7.52-7.33(m,2H),5.53(d,J=16.1Hz,1H),5.33(d,J=16.1Hz,1H),5.18-4.96(m,2H),3.66(q,J=7.2,6.7Hz ,2H),3.24-3.06(m,2H),2.45(dd,J=20.1,2.7Hz,3H),1.94(tdd,J=7.1,6.0,2.0Hz,2H),1.88-1.66(m,4H),1.00(td,J=7.4,2.5Hz,3H). 13 C NMR(1 01MHz, methanol-d4) δ 173.32, 163.68, 161.19, 15 7.56,151.44,151.09,148.68,148.56,146.06,144.46,128.10,127.89,127.22,125.66,125.60,124.26,118.92 ,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, measured value: 453.1832. [ka]

[0372] 8b: Prepared from 4d using procedure 8a. Yield (48% yield). MS (ESI): m / z (M+H)+ 485.7. [ka]

[0373] 8c: Compound 4a (100 mg, 0.23 mmol) was dissolved in anhydrous DMF (2 mL) and 2-mercaptoethanol (0.2 mL, 2.8 mmol) was added, followed by TEA (0.13 mL, 0.94 mmol) and magnetically stirred for 20 min. The reaction mixture was injected onto a 250 g medium-pressure C18 column 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 from 5% acetonitrile (5 min) to 95% acetonitrile (35 min). 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). + . 1 H NMR(400MHz,DMSO-d6)δ8.25(d,J=8.2Hz,1H),7.78(d,J=10.8Hz,1H),7.27(s,1H),6.52(s,1H), 5.42(s,2H),5.22(s,2H),4.87(t,J=5.4Hz,1H),4.47-4.28(m,2H),3.56(q,J=6.3Hz,2H),3.33(s ,2H),2.62(t,J=6.6Hz,2H),2.48-2.44(m,3H),1.87(hept,J=7.1,Hz,2H),0.88(t,J=7.3Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ 172.44,163. 07,160.58,156.69,152.16,149.97,148.66,148.53,145.48,140.06,128.18,128.16,127.28,127.07,126.61,126.54, 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, measured value: 471.1393.

[0374] 8d: Prepared similarly to 8c by reaction of 4a with 1,3-propanedithiol (60% yield). MS (ESI): m / z 501.7 (M+H). + . [ka]

[0375] 8e: Prepared similarly to 8c by reaction of 4a with 1,2-ethanedithiol (47% yield). MS (ESI): (M+H). + Calculated value: 487.1, measured value: 487.3. [ka]

[0376] 8p: Compound 8e (28 mg, 0.057 mmol) was dissolved in anhydrous THF (1 mL) and to this was added 60% NaH emulsion in mineral oil (5 mg, 2.1 mmol) with magnetic stirring. After 2 min, iodomethane (20 μL, 0.32 mmol) was added. After 20 min, the solvent was evaporated under reduced pressure, and the residue was taken up in anhydrous DMF (1 mL) and then purified by medium-pressure C18 chromatography. A 100g C18 column was run at 50 mL / min with 0.2% formic acid in deionized water (containing 5% acetonitrile) from 0 to 5 min, followed by a linear gradient from 5% to 95% acetonitrile from 5 to 28 min. Fractions containing the desired product were combined, frozen, and lyophilized to give 15 mg of 8p as a yellow solid (54% yield). 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 501.1 318;Actual value 501.1322. [Example]

[0377] Synthesis of compound 5a [ka]

[0378] 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, collected by vacuum filtration, and then dried under reduced pressure 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, measured value: 434.2. [Example]

[0379] Synthesis of compound 6a [ka]

[0380] 6a: To a solution of 5a (2.03 g, 4.66 mmol) in anhydrous benzene (60 mL) was added phosphorous acid (1.94 g, 11.66 mmol), the solution was flushed with argon, and then heated to reflux for 4 h. The reaction was cooled to room temperature, and 3 M methanolic HCl (30 mL) was added and heated to reflux (80°C bath) for 38 h. The reaction was allowed to cool to room temperature and then vacuum filtered to give 6a (1.016 g) as an off-white solid. The filtrate was concentrated, and the residue was purified by silica gel chromatography (12 g silica column, CHCl / MeOH, 0 to 20% MeOH in 16 min) to give additional 6a (0.079 g). 1.09 g of combined 6a was obtained (57% yield). 1H NMR(400MHz,DMSO-d6)δ 8.62(s,3H),8.43(d,J=8.0Hz,1H),7.99(d,J=10.7Hz,1H),7.35(s,1H),5.59(s,2H),5.45( s,2H),4.70(d,J=6.0Hz,2H),2.55(s,3H),1.88(hept,J=7.1Hz,2H),0.88(t,J=7.3Hz,3H). MS(ESI): m / z calculated value 410.2, actual value 410.4 (M+H) + ;408.1(MH) - . [Example]

[0381] Synthesis of compound 7a [ka]

[0382] 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 mmol) and glycolic acid (1.5 mg, 0.020 mmol). The solution was cooled to 0 °C in an ice bath, and DMTMM (10.1 mg, 0.037 mmol) in deionized water (0.1 mL) was added. The ice bath was removed, and the reaction mixture was stirred at room temperature for 2 h. The reaction solution was stripped under reduced pressure, and the residue was purified by silica gel chromatography (4 g silica column, CHCl / MeOH, 0–20% MeOH in 15 min) to give the desired product 7a (8 mg, 93% yield). MS (ESI): m / z 468.2 (M+H). + ,466.1( MH) - . 1 H NMR(400MHz,DMSO-d6)δ0.88(t,J=7. 3Hz,3H),1.79-1.98(m,3H),2.55(s,OH),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. 23,152.85,150.44,149.07,145.85,140.12,129.27,127.97,124.28,119.63,97.23,7 2.83,65.74,61.91,54.06,50.54,49.58,44.06,37.34,30.72,15.74,8.22.HRMS(M+H) + Calculated value: 468.1571, measured value: 468.1593. [Example]

[0383] Synthesis of compound 6c [ka]

[0384] 6c: A stirred solution of compound 2e (129 mg, 0.47 mmol), compound 3 (125 mg, 0.47 mmol), and PPTS (131 mg, 0.52 mmol) in anhydrous toluene (10 mL) was briefly placed under reduced pressure and then heated to reflux for 20 h. The reaction solution was cooled to room temperature, stripped, and the residue purified by silica gel chromatography (12 g column, CHCl / MeOH, 0–20% MeOH in 16 min) to give 196 mg of impure product as a black solid. This was further purified by reverse-phase HPLC (30 g C column, CHCN / HO, 20%–60% CHCN in 15 min, then 95% CHCN in 5 min) to give 6c (31 mg, 13% yield). MS(ESI): m / z 500.4(M+H) +,498.3(MH) - . [Example]

[0385] Synthesis of compound 7c [ka]

[0386] 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 mmol) and glycolic acid (1.9 mg, 0.026 mmol). The solution was cooled to 0 °C using an ice bath, and DMTMM (9.4 mg, 0.034 mmol) in deionized water (0.1 mL) was added. The ice bath was removed, and the reaction mixture was stirred at room temperature for 3 h. The reaction solution was stripped under reduced pressure (bath at 35 °C), and the residue was purified by semi-preparative HPLC (C18 column, CHCN / HO, 25% to 65% CHCN in 23 min) to give 2.5 mg of 7c (26% yield). MS (ESI): m / z 558.5 (M+H). + ,556.3(MH) - . [Example]

[0387] Side chain synthesis [ka]

[0388] 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 the addition of 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 reaction mixture was loaded onto a 450 g C18 cartridge pre-equilibrated with 95:5 deionized water:acetonitrile. The column was eluted at 100 mL / min with 95:5 deionized water:acetonitrile for 5 min, followed by a linear gradient from 5% acetonitrile to 95% acetonitrile over 38 min. The chromatography was repeated three more times and the fractions containing the pure desired product were pooled. The solvent was removed by rotary evaporation under reduced pressure. Methanol (100 mL) was added and evaporated twice to give 5 g of the desired product 11 as a viscous, colorless oil (68% yield). MS (ESI): MS (M+H) + Calculated value 549.2, Found value 549.3, MS(ESI): (MH) - Calculated value 547.2, measured value 547.2. [ka]

[0389] 12: Compound 11 (5 g, 9.1 mmol) was dissolved in 95:5 methanol:deionized water. (100 mL) in a 250 mL Parr shaker flask, to which 10% Pd-C (0.13 g, 1.222 mmol) was added and hydrogenated at 30 PSI of H for 45 min, periodically adding H to re-establish pressure. The solution was vacuum filtered through Celite filter aid. The clear, colorless filtrate was concentrated by rotary evaporation under reduced pressure and then placed under vacuum overnight to give 2.8 g of the desired product 12 as a white solid (94% yield). 1 H NMR (400 MHz, 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, measured value: 323.4." [ka]

[0390] 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 15 min of stirring, 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 at 100 mL / min with 2% acetonitrile for 5 min, followed by a linear gradient from 2% acetonitrile (5 min) to 60% acetonitrile (35 min) with detection at 214 and 306 nm. 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, 1 H NMR (400 MHz, DMSO-d6) δ 8.03(dd,J=15.3,7.7Hz,1H),6.99(s,3H),4.79(d,J=65.9Hz,1H),4.33(d,J=30.0H z,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) ,3.52-3.39(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=8.5,3.9H z,1H),1.59-1.39(m,3H),1.30-1.08(m,2H). [ka]

[0391] 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 15 min of stirring, 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 at 100 mL / min with 2% acetonitrile for 5 min, followed by a linear gradient from 2% acetonitrile (5 min) to 60% acetonitrile (35 min) with detection at 214 and 306 nm. Fractions containing pure product were combined, frozen, and lyophilized to give 1.4 g of product 14a as a white solid (55% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.33(dd,J=9.2,7.7Hz,1H),7.08(d,J=1.1Hz,2H),4.24(qd,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, measured value: 476.4.

[0392] Fmoc-protected peptides 15a, 15b, 15c and 15d were prepared by solid phase synthesis using standard procedures. [Example]

[0393] Methods for the Conversion of Fmoc-Peptide-Gly-OH to Fmoc-Peptide-NHCHOAC (Compounds 16a-16d) [ka]

[0394] 16a: Compound 15a (2.5 g, 4.9 mmol) was dissolved in anhydrous DMF (40 mL) and magnetically stirred in a 100 mL flask while 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 half 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 at 100 mL / min with 10% acetonitrile for 5 min, followed by a linear gradient from 10% acetonitrile (5 min) to 95% acetonitrile (38 min). This procedure was repeated with the other half of the reaction mixture. The fractions containing the desired product 16a were combined, frozen, and lyophilized to give 1.2 g of a white semi-solid (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,2 H),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, measured value: 547.5.

[0395] 16b was prepared from Fmoc-Ala-Ala-Gly-OH 15b in 55% yield. MS (M+Na)+ 476.7.

[0396] 16c was prepared from Fmoc-Leu-Gln-Gly-OH 15c in 46% yield. MS (M+Na) 575.6.

[0397] 16d was prepared from Fmoc-Ala-D-Ala-Ala-Gly-OH 15d in 52% yield. MS (M+Na) 547.3. [Example]

[0398] Method for reacting benzyl-2-hydroxyacetic acid with Fmoc-peptide-NHCHOAc (compounds 17a-17d) [ka]

[0399] 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 reduced pressure, and the residue was taken up 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 at 60 mL / min with 10% acetonitrile for 5 min, followed by a linear gradient from 10% acetonitrile (5 min) to 95% acetonitrile (38 min). Fractions containing the desired product were combined, frozen, and lyophilized to give 102 mg of a white solid, 17a (59% yield). MS (ESI): MS (M+Na) + Calculated value: 653.3, measured value: 653.5.

[0400] 17b was prepared from 16b in 61% yield. MS (M+Na)+ 582.7.

[0401] 17c was prepared from 16c in 52% yield. MS (M+H) + 659.5.

[0402] 17d was prepared from 16d in 56% yield. MS (M+Na)+ 653.4. [Example]

[0403] Conversion of Fmoc-peptide-NHCHOCHCOOBn to H-peptide-NHCHOCHCOOBn (compounds 18a-18d) [ka]

[0404] 18a: Compound 17a (100 mg, 0.16 mmol) was dissolved in DMF (4 mL) and morpholine (0.6 mL, 6.9 mmol) was added with magnetic stirring. 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 from 5% acetonitrile (5 min) to 70% acetonitrile (38 min). Fractions containing the desired product were combined, frozen, and lyophilized to give 50 mg of a white solid, 18a (76% yield). MS (ESI): MS (M+H) + Calculated value: 409.2, measured value: 409.6.

[0405] 18b was prepared from 17b in 80% yield. MS (M+H)+ 338.3.

[0406] 18c was prepared from 17c in 61% yield. MS (M+H) + 437.6.

[0407] 18d was prepared from 17d in 61% yield, MS (M+H)+ 409.6. [Example]

[0408] Example 13. Conversion of H-peptide-NHCH2OCH2COOBn to H-peptide-NHCH2OCH2COOH (Compounds 19a-19d) [ka]

[0409] 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). To this was added 10% palladium on carbon (0.1 g). The reaction was hydrogenated at 30 PSI H2 in a PARR shaker for 1 hour. The solution was vacuum filtered through Celite filter aid, and the solvent was removed from the filtrate by rotary evaporation under reduced pressure to give 35 mg of the desired product 19a as a thick oil (91% yield). MS(ESI):MS(M+H) + Calculated value: 319.2, measured value: 319.3.

[0410] 19b was prepared from 18b in 92% yield. MS (M+H) + 248.3.

[0411] 19c was prepared from 18c in 83% yield. MS (M+H) + 347.4.

[0412] 19d was prepared from 18d in 81% yield, MS (M+H)+ 319.5. [Example]

[0413] Methods for preparing Mal-(CH2)5-CO-peptide NHCH2OCH2COOH compounds (compounds 20a-20d) [ka]

[0414] 20a: Mal-(CH2)5-COONHS 13b (46 mg, 0.15 mmol) was dissolved in anhydrous DMF (2 mL) to which DIPEA (0.1 mL, 0.31 mmol) and H-Ala-Ala-Ala-NHCHOCH2COOH 19a (30 mg, 0.093 mmol) were added. The reaction was magnetically stirred 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 at 40 mL / min with a linear gradient of 5% acetonitrile over 5 min, from 5% acetonitrile in 5 min to 90% acetonitrile in 38 min. Fractions containing the desired product were combined, frozen, and lyophilized to give 30 mg of a white solid 20a (63% yield). MS(ESI):MS(MH) - Calculated value 510.2, measured value 510.l. 1 H NMR (400 MHz, DMSO-d6) δ1 2.16(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).

[0415] 20b was prepared from 19b in 83% yield. MS (M+H)+ 441.4.

[0416] 20c was prepared from 19c in 83% yield. MS(MH)-655.4. [ka]

[0417] 20d: To a solution of compound 18d (99.7 mg, 0.24 mmol) in methanol (3 mL), Pd (10% on carbon, 26 mg, 0.024 mmol) was added, and the reaction flask was purged with hydrogen. This was hydrogenated with a hydrogen balloon at room temperature for 3 hours, then filtered. The filtrate was stripped to give compound 19d as a white solid (78 mg, 100% yield). 41.9 mg (0.13 mmol) was taken 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 resulting clear, colorless solution was stirred at room temperature for 24 hours. This was stripped under reduced pressure, and the residue was purified by reverse-phase HPLC (C18 column, eluting with CHCN / H2O, 10-50% CH3CN in 15 min, then 95% CH3CN in 5 min) to give compound 20d (30.8 mg, 45% yield). MS (ESI): m / z 512.4 (M+H). + , 510.4(MH) - .

[0418] Example 15. Synthesis of compounds 21a and 21b [ka]

[0419] 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 reduced pressure, and the residue was taken up 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 from 5% acetonitrile in 5 min to 95% acetonitrile in 38 min. Fractions containing the desired product were combined, frozen, and lyophilized to give 202 mg of white solid 21a (58% yield). MS (M+Na) 613.9.

[0420] 21b was prepared similarly to 21a from 16d and 6-mercaptohexanoic acid in 60% yield, MS (M+H) + 613.7.

[0421] Example 16. Synthesis of Compound 22a [ka]

[0422] 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 20a (35 mg, 0.068 mmol) and TEA (0.04 mL, 0.28 mmol) were added 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 over 40 min using a linear gradient of 20% methanol in dichloromethane from 0% to 100%. Fractions containing pure product were combined, and the solvent was removed by rotary evaporation under reduced pressure to give 16 mg of a brown solid, 22a (36% yield). MS (M+Na) 925.6. 1 H NMR (400 MHz, DMSO-d6) δ 0.88 (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=7.4H z,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). 13C NMR(101MHz,DMSO-d6)δ180.20,173.9 6, 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, 124.24, 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, measured value: 903.3676.

[0423] Example 17. Synthesis of Compound 22c [ka]

[0424] 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 using an ice bath, and DMTMM (10.8 mg, 0.04 mmol) in deionized water (0.1 mL) was added. The ice bath was removed, and the reaction mixture was stirred at room temperature for 3 h. The reaction solution was stripped under reduced pressure (bath at 35 °C), and the residue was purified by reverse-phase HPLC (30 g C18 column, CHCN / HO, 25% CHCN for 3 min, then 95% CHCN for 12 min, then 95% CHCN for 5 min). 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) - .

[0425] Example 18. Synthesis of Compound 22b [ka] 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 using an ice bath, and DMTMM (11.9 mg, 0.043 mmol) in deionized water (0.1 mL) was added. The ice bath was removed, and the reaction mixture was stirred at room temperature for 3 h. The reaction solution was stripped under reduced pressure (bath at 35 °C), and the residue was purified by reverse-phase HPLC (30 g C18 column, CHCN / HO, 25% CHCN in 3 min, then 95% CHCN in 12 min, then 95% CHCN in 5 min) 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) - .

[0426] Example 19. Synthesis of compound 22d 22d: Prepared similarly to 22c by reaction of 6c with 20d (22% yield). MS(ESI):m / z 1015.8(M+Na) +

[0427] Example 20. Synthesis of Compound 22e [ka] 22e: To a solution of compound 18c (15.2 mg, 0.035 mmol) in methanol (2 mL) was added Pd (10% on carbon, 3.7 mg, 0.0035 mmol), and the reaction flask was purged with hydrogen. This was hydrogenated with a hydrogen balloon at room temperature for 3 hours and then filtered. The filtrate was stripped to give compound 19c as a colorless foam. This was dissolved in anhydrous DMF (0.3 mL), and 13d (15.9 mg, 0.03 mmol) was added. The resulting colorless, clear solution was stirred at room temperature for 15 hours. This 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). Then, DMTMM (20.7 mg, 0.075 mmol) in deionized water (0.1 mL) was added, and the reaction solution was stirred at room temperature for 1.5 h. The reaction mixture was stripped under reduced pressure, and the residue was purified by silica gel chromatography (CHCl / 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) - .

[0428] Example 21. Synthesis of Compound 23a [ka] 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 the 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 h. The reaction solution was stripped under reduced pressure, and the residue was purified by silica gel chromatography (4 g column, CHCl / MeOH, 0–20% MeOH in 15 min) 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) - .

[0429] Example 22. Synthesis of Compound 24a [ka]

[0430] 24a: Compound 23a (78 mg, 0.078 mmol) in anhydrous DMF (1 mL) To the solution was added morpholine (0.24 mL, 2.7 mmol), and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was stripped under reduced pressure, and the residue was purified by silica gel chromatography (4 g silica column, CHCl / MeOH, eluting with 0-20% MeOH in 9 min, then 20% MeOH in 11 min) to give the desired compound 24a (39.6 mg, 65% yield). MS (ESI): m / z 782.4 (M+H). + ,780.0(MH) - .

[0431] Example 23. Synthesis of Compound 25a [ka]

[0432] 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 h. The reaction solution was diluted with DMSO and purified by reverse-phase semi-preparative HPLC (C18 column, CHCN / H2O, 25% to 55% CH3CN in 23 min, then 95% CH3CN in 7 min). Fractions containing 25a were combined and lyophilized to give 25a as a white solid. This was further purified by silica gel chromatography (4 g silica column, CHCl2 / MeOH, 0 to 20% MeOH in 15 min) 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) - .

[0433] Example 24. Synthesis of Compound 23b [ka] 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 using an ice bath, and DMTMM (48 mg, 0.16 mmol) in deionized water (0.16 mL) was added. The ice bath was removed, and the reaction mixture was stirred at room temperature for 2.5 h. The reaction solution was stripped under reduced pressure, and the residue was purified by silica gel chromatography (4 g silica column, CHCl / MeOH, 0-20% MeOH in 15 min) to give the desired product compound 23b (31 mg, 40% yield). MS (ESI): m / z 1004.6 (M+H). ) + , 1048.7(M+HCOOH-H) - .

[0434] Example 25. Synthesis of Compound 24b [ka]

[0435] 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 h. The reaction solution was stripped under reduced pressure, and the residue was purified by silica gel chromatography (4 g silica column, CHCl / MeOH, eluting with 0-20% MeOH in 15 min, then 20% MeOH in 5 min) 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) - .

[0436] Synthesis of compound 25b [ka] 25b: To a solution of 24b (12.3 mg, 0.016 mmol) in anhydrous DMF (0.3 mL) was added 5-maleimidohexanoic acid N-hydroxysuccinimide ester 13b (7.3 mg, 0.024 mmol), and the reaction mixture was stirred at room temperature for 4 h. The reaction solution was stripped under reduced pressure, and the residue was purified by silica gel chromatography (4 g column, CHCl / MeOH, 0–20% MeOH in 15 min) to give 3.2 mg of 25b and another 6.5 mg of impure product. The impure product was further purified by semi-preparative reverse-phase HPLC (C18 column, CHCN / HO, eluting with 25%–55% CHCN in 23 min, then 95% CHCN in 7 min) to give 2.8 mg of 25b. A total of 6 mg of 25b was isolated (39% yield). MS(ESI): m / z 975.6(M+H) + ,997.6(M+Na) + ,973.7(MH) - ,1019.6(M+HCOOH-H) - .

[0437] Example 27. Synthesis of Compound 26a [ka]

[0438] 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 diethyl ether, 64 μL, 0.128 mmol). After stirring at room temperature for 22 hours, the reaction solution was stripped under reduced pressure (heating bath at 35°C). The residue was purified by reverse-phase HPLC (30 g C18 column, CHCN / H2O, 25% CH3CN in 3 min, then 90% CH3CN in 12 min, then 90% CH3CN in 3 min) to give compound 26a as a white solid (47 mg, 48% yield). MS (ESI): m / z calculated 917.4, found 917.6 (M+H). + , 961.5(M+HCOOH-H) - Unreacted 8a was also recovered (12 mg).

[0439] Example 28. Synthesis of Compound 27a [ka]

[0440] 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 h, the reaction solution was stripped under reduced pressure. The residue was purified by reverse-phase HPLC (30 g C18 column and CHCN / HO, eluting with 20% CHCN in 3 min, then 90% CHCN in 15 min, then 90% CHCN in 3 min) to give compound 27a as an off-white solid (35.9 mg, 83% yield). MS (ESI): m / z 695.5 (M+H). + ,739.3(M+HCOOH-H) - .

[0441] Example 29. Synthesis of Compound 28a [ka]

[0442] 28a: To a solution of compound 27a (18 mg, 0.026 mmol) in anhydrous DMF (0.3 mL) was added 5-maleimidohexanoic 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 h, the reaction solution was stripped under reduced pressure. The residue was purified by reverse-phase HPLC (30 g C18 column, CHCN / HO, 18 min run, elution with 20% CHCN for 3 min, then 20% to 90% CHCN in 12 min, then 90% CHCN for 3 min) to give product 28a as a white solid (11.9 mg, 51% yield). MS (ESI): m / z 888.5 (M+H). + ,932.5(M+ HCOOH-H) - . 1 H NMR(400MHz,DMSO-d6)δ 8.45(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),4.15-4.09(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)δ 174.54,173.86,171.34,171.26,170.84,170.79,170.47,170.15,161.42,158.95,155.71,155.16,1 50.43,148.38,146.97,146.80,144.22,142.23,129.03,126.25,125.69,125.48,124.98,124.50,122 .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, measured value: 888.3966.

[0443] Example 30. Synthesis of compound 26b [ka]

[0444] 26b: To a stirred solution of 8a (73 mg, 0.14 mmol) and 16b (64 mg, 0.14 mmol) in anhydrous DMF (1.2 mL) was added HCl etherate (2 M HCl in diethyl 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, CHCN / HO, 25% CHCN in 3 min, then 90% CHCN in 12 min, then 90% CHCN in 3 min) to give 26b as an off-white solid (60 mg, 50% yield). MS (ESI): m / z 846.4 (M+H). + , 890.3 (M+HCOOH-H) - .

[0445] Example 31. Synthesis of Compound 26c [ka]

[0446] 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 diethyl 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, CHCN / HO, 25% CHCN for 3 min, then 95% CHCN for 12 min, then 95% CHCN for 3 min) to give compound 26c as a white solid (41.9 mg, 60% yield). MS (ESI): m / z 874.4 (M+H). + , 918.5(M+HCOOH-H) - . Unreacted 8a also returns The yield was 12 mg.

[0447] Example 32. Synthesis of compound 27b [ka] 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 h, the reaction solution was stripped under reduced pressure. The residue was purified by reverse-phase HPLC (15.5 g C18 column and CHCN / HO, eluting with 10% CHCN in 3 min, then 90% CHCN in 9 min, then 90% CHCN in 3 min) to give compound 27b as an off-white solid (36.7 mg, 83% yield). MS (ESI): m / z 624.5 (M+H). + ,668.3(M+HCOOH-H) - .

[0448] Example 33. Synthesis of compound 27c [ka]

[0449] 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 CHCN / HO eluting with 20% CHCN in 3 min, then 90% CHCN in 12 min, then 90% CHCN in 5 min) to give compound 27c as a white solid (25.5 mg, 82% yield). MS (ESI): m / z 652.5 (M+H). + ,650.2(MH) - 696.2 (M+HCOOH-H) - .

[0450] Example 34. Synthesis of compound 28b [ka]

[0451] 28b: Compound 27a (18 mg, 0.026 mmol) in anhydrous DMF (0.2 mL) To a stirred solution of 14a (18.5 mg, 0.039 mmol) and 14b (18.5 mg, 0.039 mmol), DMTMM (17 mg, 0.052 mmol) and NMM (2.9 μL, 0.026 mmol) were added 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, CHCN / HO, 20% CHCN in 3 min, then 90% CHCN in 12 min, then 90% CHCN in 3 min). Product-containing fractions were combined and lyophilized to give 28b as a white solid (2.3 mg, 7% yield). MS (ESI): m / z 1152.5 (M+H) + ,1150.0(MH) - , 1196.5(M+HCOOH-H) - .

[0452] Example 35. Synthesis of Compound 28c [ka]

[0453] 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 for 5 h at room temperature, the reaction solution was directly loaded onto a C18 cartridge and purified by reverse-phase HPLC (30 g C18 column, CH3CN / HO, 20% CH3CN in 3 min, then 90% CH3CN in 12 min, then 90% CH3CN in 3 min). The product-containing fractions were combined and lyophilized to give 28c as a white solid (9.9 mg, 15% yield). MS (ESI): m / z 1081.5 (M+H). + ,1079 .2(MH) - ;1025.5(M+HCOOH-H) - .

[0454] Example 36. Synthesis of compound 28d

[0455] [ka] 28d: To a solution of 27c (10.5 mg, 0.016 mmol) in anhydrous DMF (0.2 mL) was added 5-maleimidohexanoic 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, CHCN / HO, 18 min run, 20% CHCN for 3 min, then 20% to 90% CHCN in 12 min, then 90% CHCN for 3 min) to give product 28d as a white solid (8.7 mg, 63% yield). MS (ESI): m / z 845.4 (M+H). + ,989.4(M+HCOOH -H) - .

[0456] Example 37. Synthesis of compound 29a [ka]

[0457] 29a: Compounds 16a (30 mg, 0.057 mmol) and 8d (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 minutes. The solvent was rotary evaporated under reduced pressure, and the residue was taken up in a minimum volume of DMF and then purified on a 100 g C18 medium-pressure column pre-equilibrated with 90:10 deionized water containing 0.1% formic acid:acetonitrile. The column was then eluted at 30 mL / min with 10% acetonitrile for 5 minutes, followed by a linear gradient from 10% acetonitrile in 5 minutes to 95% acetonitrile in 38 minutes. Fractions containing the desired product were combined, frozen, and lyophilized to give 31 mg of white solid 29a (56% yield). MS (M+Na) 987.5.

[0458] Example 38. Synthesis of Compound 30a [ka]

[0459] 30a: Compound 29a (28 mg, 0.029 mmol) was dissolved in anhydrous DMF (0.8 mL) and magnetically stirred while morpholine (0.2 mL) was added. After 1 h, the reaction mixture was loaded directly onto a 100 g C18 cartridge (25:75 CH3CN / HO) and run at 50 mL / min for 3 min at 25% CH3CN, followed by a linear gradient of CH3CN to 90% CH3CN from 3 to 23 min. Fractions containing the desired product were combined, frozen, and lyophilized to afford 18 mg (83% yield) of 30a as a yellow solid. MS (M+H) 743.5.

[0460] Example 39. Synthesis of Compound 32a [ka]

[0461] 32a: Compound 30a (16 mg, 0.02 mmol) was dissolved in 84:16 DMF:deionized water (0.5 mL) and to this was added DMTMM (15 mg, 0.054 mmol), TEA (0.02 mL, 0.14 mmol), and 14a (20 mg, 0.042 mmol) rapidly with magnetic stirring. After 35 min, the reaction mixture was cooled to room temperature with dichloromethane. The mixture was loaded onto a 100 g silica cartridge pre-equilibrated with ethanol and then run at 35 mL / min with a linear gradient of 0% to 100% methanol:dichloromethane over 30 min. Fractions containing the desired product were combined and the solvent evaporated under reduced pressure to give 7 mg (29% yield) of 32a as a thick oil. MS (M+Na) 1223.0.

[0462] Example 40. Synthesis of Compound 33a [ka]

[0463] 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 h. The reaction solution was directly loaded onto a 30 g C18 cartridge 25:75 CHCN / HO and run at 20 mL / min with 25% CHCN for 3 min, followed by a linear gradient of CHCN to 90% for 3–12 min. Fractions containing the desired product were combined and evaporated under reduced pressure to give compound 33a as a white solid (3.1 mg, 30% yield). MS (ESI): m / z 483.4 (M+H) ) + ,481.3(MH) - .

[0464] Example 41. Synthesis of Compound 34a and Compound 34b [ka]

[0465] 34a and 34b: Compound 8c (30 mg, 0.064 mmol) was magnetically stirred in DMF (1 mL) to which vanadium acetylacetonate (3 mg, 0.008 mmol) and a 5 M 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 / HO) and run at 20 mL / min with 25% CH3CN for 3 min, followed by a linear gradient of CH3CN to 90% CH3CN over 3–12 min. Fractions containing pure 34a were separately combined from fractions containing pure 34b, and both were separately frozen and lyophilized to give 6 mg of 34a (19% yield) and 15 mg of 34b (47% yield), both as yellow solids. 34a MS(M+H)+487.4, 34b MS(M+H)+503.6.

[0466] Example 42. Synthesis of Compound 35a [ka]

[0467] 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 h. The reaction solution was diluted with DMSO and injected into semi-preparative HPLC for purification (C18 column, CHCN / HO, 25% to 65% CHCN in 23 min) to give the desired product 35a (2.7 mg, 19% yield). MS (ESI): m / z 554.4 (M+H). + ,552.5(MH) - .

[0468] Example 43. Synthesis of compound 29b [ka]

[0469] 29b: Compounds 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 reduced pressure, and the residue was taken up in a minimum volume of DMF and then purified on a 100 g C18 medium-pressure column pre-equilibrated with 90:10 deionized water containing 0.1% formic acid:acetonitrile. The column was then eluted at 30 mL / min with 10% acetonitrile for 5 min, followed by a linear gradient from 10% acetonitrile in 5 min to 95% acetonitrile in 38 min. Fractions containing the desired product were combined, frozen, and lyophilized to give 31 mg of white solid 29b (56% yield). MS (ESI): MS (M+Na) + Calculated value: 973.3, measured value: 973.7.

[0470] Example 44. Synthesis of Compound 30b [ka]

[0471] 30b: Compound 29b (28 mg, 0.029 mmol) was dissolved in anhydrous DMF (0.8 mL) and magnetically stirred while morpholine (0.2 mL, 2.32 mmol) was added. After 1 h, the reaction mixture was loaded directly onto a 100 g C18 cartridge containing 25:75 CH3CN / HO and washed with 25% CH3CN at 50 mL / min for 3 min, then 3 h. A linear gradient of CHCN to 90% was run from 3 to 23 min. Fractions containing the desired product were combined, frozen, and lyophilized to give 18 mg (83% yield) of 30b as a yellow solid. 1 H NMR (400 MHz, DMSO-d) δ 8.52 (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),1.9 4-1.75(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, measured value: 729.4.

[0472] Example 45. Synthesis of Compound 32b [ka]

[0473] 32b: Compound 30b (16 mg, 0.02 mmol) was dissolved in 84:16 DMF:deionized water (0.5 mL) and to this was rapidly added DMTMM (15 mg, 0.054 mmol), TEA (0.02 mL, 0.14 mmol), and 14a (20 mg, 0.042 mmol) 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 with a 30 min linear gradient of 40:60 methanol:dichloromethane from 0% to 100%. Fractions containing the desired product were combined, and the solvent was evaporated under reduced pressure to give 7 mg of 32b (29% yield) as a thick oil. 1 H NMR (400 MHz,DMSO-d6)δ7.86-7.83(m,1H),7.71(s,1H),7.69(d,J=1.1Hz,1H),7.66(d,J=8.2Hz,1H),7.51-7.47(m,2H),7.28(t,J=1.0Hz,1H),6.61(s,2H),5.59-5.53(m,1H),5.40(dd,J=3.3,1.1Hz,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=3.5,1.8Hz,2H),4.28-4.22(m,2H),4.21-4.13(m,2H),3.74(t,J=6.5Hz,2H),3.70(ddd,J=11.9,5.7,4.8Hz,1H),3.60-3.51(m,2H),3.44(dd,J=11.9,4.9Hz,1H),3.3(ブロード s,8H),3.20(dd,J=4.7,3.7Hz,2 H),2.91(s,3H),2.89(s,3H),2.56(td,J=6.4,1.7Hz,2H),2.31(s,3),2.37-2.21(m,2H),2.07-1.82(m,3H),1.72(dq,J=13.7,8.1Hz,1H),1.53(d,J=5.5Hz,6H),1.45(d,J=5.9Hz,3H),0.86(t,J=8.0Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ174 .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, 53.02,50.02,49.92,49.62,48.46,45.53,42.38,40.85,35.32,35.13,34.0 0,32.31,32.24,30.20,26.63,20.49,17.81,17.71,17.61,7.68.HRMS(M+H) + Calculated value: 1186.4237, measured value: 1186.4220.

[0474] Example 46. General method for preparation of conjugates [ka]

[0475] Chimeric antibodies that bind to humanized IgG1 antibodies, such as anti-epidermal growth factor (ML66), anti-folate receptor alpha (FRα), and Kunitz soybean trypsin inhibitor (KTI), were generated. Conjugation of the antibodies to maleimide-bearing payloads was carried out as described for the preparation of ML66-22a. The resulting conjugates are referred to herein as target binder-payloads; for example, ML66 conjugated to 22a is ML66-22a.

[0476] Example 47. Synthesis of ML66-22a 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 payload was carried out 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 spinning on a tube rotator for 1.5-2 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 resulting conjugate had a drug-to-antibody ratio (DAR) of 7.5 and was 99% monomeric as determined by size exclusion chromatography.

[0477] Biophysical evaluation of the interchain high DAR conjugates was performed, including determination of conjugate concentration, yield, DAR (drug-to-antibody ratio), free drug, percent monomer, and DAR distribution. The conjugate concentration was determined using a final protein concentration of 4.5 mg / ml (extinction coefficient ε 280 =205520M -1 cm -1 via UV-Vis using DAR7.5 (22a In this case, the extinction coefficient ε 280 =10764M -1 cm -1 , ε 370 =20982M -1 cm -1 The resulting antibody was confirmed to have 99% monomer (via UV-Vis using a size-exclusion UPLC protein BEH SEC column), <1% free drug (via a HiSEP HPLC column), and nearly uniform eight drugs linked per antibody (via Q-ToF mass spectrometry and butyl-NPR HIC chromatography). The yield of this particular interchain conjugate ML66-22a was 75%.

[0478] UPLC Protein BEH SEC Method: Percent monomer analysis was performed on a Waters Acquity UPLC H-Class system equipped with an Acquity UPLC Protein BEH SEC column (200 Å, 1.7 μm, 4.6 mm × 150 mm, part number 18605225). The mobile phase was 400 mM sodium perchloride, 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.

[0479] HiSep HPLC method: Percentage free drug analysis was performed on an Agilent HPLC system equipped with a Supelco analytical HiSep column (25 cm × 4.6 mm, 5 μm, catalog number 58919). The mobile phase consisted of 0.1 M ammonium hydroxide, pH 7.0 (solvent A) and acetonitrile (solvent B). The method was run using a 0-25 min linear gradient starting at 25% solvent B and going to 40% solvent B at 0.70 mL / min using solvent A.

[0480] Example 48. In vitro cytotoxicity assay Cytotoxicity was assessed in flat-bottom 96-well plates (Costar) using a water-soluble tetrazonium salt (WST-8)-based cell viability assay (Dojindo, Molecular Technologies, Inc.) as previously described (Kovtun YV, et al. Antibody-maytansinoid conjugates designed to bypass multidrug resistance. Cancer Res 2010;70(6):2528-37). Briefly, human tumor cells (1,000–5,000 cells / well, depending on the cell line) in appropriate medium were incubated with the conjugate or metabolite in the presence or absence of excess corresponding unconjugated antibody for 5 days at 37°C and 6% CO2. Cell viability was determined from background-corrected WST-8 absorbance.

[0481] The results of this study are summarized in the table below: Figure 12 shows the cytotoxicity of sulfide-containing compound 8c and its sulfoxide 34a and sulfone 34b. [Table 7] N-Luc denotes Namalwa cells stably transfected with the luciferase gene.

[0482] Example 49. Bystander cell killing assay 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.

[0483] Namalwa-luciferase (N-Luc) Ag- cells (1000 cells per well) in appropriate culture medium were incubated in wells of a U-bottom 96-well plate with the indicated number of MDA-MB-468 Ag+ cells and ADC (1.1 nM) at 37°C and 6% CO2 for 5 days. The concentration of conjugate used in the assay was high enough to kill all Ag+ cells but was unable to kill Ag- cells unless Ag+ cells were also present. On day 5, cell viability was determined by Cell Titer Glo assay according to the manufacturer's protocol. Luminescence signals were read using a Victor3 plate reader. The following table shows the in vitro cytotoxicity and bystander killing of the compounds described herein obtained in the previous examples. [Table 8] N-Luc denotes Namalwa cells stably transfected with the luciferase gene.

[0484] Example 50. Methods for determining in vivo efficacy in xenograft models Six-week-old female CB.17 SCID mice were obtained 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 specific tumor xenograft models. Female SCID mice were subcutaneously inoculated in the right flank with the desired cell type in a 1:1 ratio in serum-free medium. Animals were then randomly distributed into groups of 6 or 8 mice per group. Control mice were treated with phosphate-buffered saline (vehicle). The required concentration of ADC was created by diluting the stock sample with vehicle. Xenografts were placed in a volume of approximately 100 mm. 3 After growth to 100 μg / mouse, mice were administered ADC or vehicle via tail vein intravenous (iv) injection (200 μL / mouse). All dosing was based on the weight of the antibody component of the conjugate. Tumor size was calculated in mm using the formula V = 1 / 2 (length x width x height). 3 Tumor volumes, expressed as r, were measured twice weekly in three dimensions using calipers. Body weight was also measured twice weekly. Data from these studies were interpreted using a standardized method previously described (Bissery MC et al. Experimental antitumor activity of tumors (RP)). 56976, NSC 628503), a taxol analogue. Cancer Res1991;51(18):4845-52).

[0485] Example 51. Tolerance of ML66-999, ML66-22a, and ML-28a ADCs in mice The tolerability of the ADC was evaluated in female CD-1 mice by daily weight measurement and clinical observation for 2 weeks after injection of the ADC. Pre-administration of 10 mg / kg of naked ML66 antibody, which has been shown to attenuate acute infusion reactions caused by ML66 alone at doses above 20 mg / kg, was administered intravenously to 32 7-week-old female CD-1 mice. The ADCs were administered intravenously. One mouse died after this naked antibody injection. Two hours later, three groups of 7–8 mice received an intravenous bolus injection of a 1500 μg / kg payload of 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 animals did not die or require euthanasia due to >20% weight loss or signs of distress or morbidity (hunching, lack of movement, inability to eat or drink, or signs of pain / distress). Based on these criteria, all three ADCs were well tolerated at the 1500 μg / kg payload dose. Figure 19 shows the mouse tolerance of the ML66-999, ML66-22a, and ML66-28a ADCs.

[0486] Example 52. Method for determining ADC pharmacokinetic (PK) parameters in mice Three groups of 11 7-week-old female CD-1 mice were administered a single intravenous bolus injection of 10 mg / kg of naked antibody or ADC, respectively. For each ADC, terminal blood samples were collected from three mice at 2 min, three at 24 h, and five at 72 h postinjection. Blood was processed to serum, and ADCs were purified using affinity capture with anti-human Fc beads. Samples were analyzed by anti-human Fc ELISA to determine the concentration of antibody components (regardless of payload loading). Samples were also analyzed by size exclusion chromatography (SEC) and mass spectrometry (MS). Partial loss of linker payload from captured ADCs at all time points was measured by intact MS methods. ADC concentrations were calculated based on the total antibody concentration and drug-to-antibody ratio (DAR).

[0487] Figure 13 and Table 9 show the pharmacokinetics of ML66-999 in mice. Figure 14 and Table 10 show the pharmacokinetics of ML66-999 in mice. Table 11 summarizes the binding of the ADC or naked antibody to the corresponding antigen-expressing cell lines. Figures 15 and 16 show the in vitro cytotoxicity of ML66-999 and ML66-22a against Ag+ and Ag- cells. The percentage of retained bioactivity of the ADC in plasma samples over time was determined in a cytotoxicity assay using Ag+ or Ag- cells. The ADC retained most of its activity against Ag+ cells at each time point, but was more than 200-fold less active against Ag- cells, indicating that the cytotoxicity was due to the intact ADC, with little or no contribution from the released payload. [Table 9] [Table 10] [Table 11]

[0488] Example 53. Antitumor activity of anti-EGFR antibody drug conjugates in nude mice bearing HSC-2 human head and neck squamous cell carcinoma xenografts. 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 human head and neck squamous cell carcinoma xenograft model.

[0489] 1 × 10 mice in 0.1 ml of 50% Matrigel / 50% serum-free medium by subcutaneous injection in the right posterior abdominal region. 7 HSC-2 cells were inoculated into female athymic Foxn1 mice. nu Mice (6 weeks old) were obtained. After receipt, the animals were observed for 9 days before the start of the study. The animals showed no signs of illness or disease upon arrival or prior to treatment.

[0490] Forty-eight mice were randomized into eight groups (six mice per group) according to tumor volume, ranging from 68.48 to 118.26 (93.42 ± 11.25, mean ± SD) mm. 3 The mice were measured, randomized, and dosed based on tumor volume on day 4 after implantation (11 / 12 / 18). The weight of the mice ranged from 19.46 to 25.77 grams (22.98 ± 1.50, mean ± SD). Mice in each group were identified by punching. Test agent and vehicle administration was performed intravenously using a 1.0 ml syringe with a 27-gauge, 1 / 2-inch needle. Antibody-drug conjugates The test agent was qdx1 administered at 1, 3, or 10 mg / kg, where 75 μg / kg or 250 μg / kg based on payload correlates to approximately 3 or 10 mg / kg based on antibody concentration. Groups included: a control group administered vehicle (PBS, 200 μL), a control group administered 10 mg / kg of non-targeted KTI-999, ML66-999 administered at 1, 3, and 10 mg / kg based on antibody concentration, and ML66-22a administered at 1, 3, and 10 mg / kg based on antibody concentration.

[0491] Tumor size was measured twice weekly in three dimensions using calipers. Tumor volume was calculated in mm using the formula: volume = length × width × height × ½. 3 The tumor volume was expressed as . Mice were considered to have partial regression (PR) if tumor volume decreased by 50% or more, and complete regression (CR) if no palpable tumor was detected. Tumor volume was determined by StudyLog software.

[0492] Tumor growth inhibition (T / C (%)) is measured at a given time (e.g., when the median TV of control tumors reaches a maximum tumor volume of approximately 1000 mm). 3 T / C (%) is the ratio of the mean tumor volume (TV) of the treatment group (T) to the mean TV of the control group (C) at 22 days post-inoculation, when the median TV of the control group reached 1038 mm, which is when the mice were euthanized. 3According to the NCI criteria, T / C≦42% is the minimum level of antitumor activity, and T / C<10% is considered a high level of antitumor activity.

[0493] As a rough indicator of drug toxicity, the body weight (BW) of all mice was measured twice a week and determined by StuyLog software. Mouse weight was expressed as the percentage change in body weight from pre-treatment weight as follows: % BW change = [(post-BW / pre-BW) - 1] × 100, where post-BW is the post-treatment weight and pre-BW is the starting weight before treatment. Body weight loss (BWL) was expressed as the mean change in body weight after treatment. For tumors with a volume of 1000 mm, the BWL was calculated as the mean change in body weight after treatment. 3 Animals were euthanized at any time during the study if they became larger, if the tumor became necrotic, if the mice lost more than 20% of their initial body weight, or if the mice became moribund.

[0494] Figure 17 and Table 12 show the efficacy of the ADCs in the HSC-2 xenograft model. The ML66-999 and ML66-22a conjugates had similar antitumor activity. The T / C value for ML66-KTI administered at 10 mg / kg was 70% (inactive), resulting in no tumor regression. This demonstrated that the activity of the ML66 conjugate targets EGFR, as the control ADC was inactive. The T / C value for ML66-999 administered at 1 mg / kg was 49% (inactive), resulting in no tumor regression. The T / C value for ML66-999 administered at 3 mg / kg was 7% (highly active), resulting in four partial tumor regressions and one complete tumor regression from six mice. The T / C value for ML66-999 administered at 10 mg / kg was 3% (highly active), with six partial tumor regressions and two complete regressions in six mice. The T / C value for ML66-22a administered at 1 mg / kg was 39% (active), with one partial tumor regression and no complete regressions in six mice. The T / C value for ML66-22a administered at 3 mg / kg was 5% (highly active), with three partial tumor regressions and one complete regression in six mice. The T / C value for ML66-22a administered at 10 mg / kg was 2% (highly active), with six partial tumor regressions and five complete regressions in six mice. No significant weight loss was observed for any of the conjugates at any of the doses shown, indicating that the conjugates were well tolerated. The results of this study suggest that both the ML66-999 and ML66-22a conjugates exhibited dose-dependent antitumor activity and were effective in the HSC-2 head and neck squamous cell carcinoma tumor xenograft model. [Table 12]

[0495] Example 54. Antitumor activity of the conjugate in nude mice bearing non-small cell lung cancer (NSCLC) xenografts The antitumor activity of the conjugates in nude mice bearing non-small cell lung cancer (NSCLC) xenografts was determined in a similar manner as for HSC-2 cells, as described in Example 53.

[0496] Six-week-old female athymic nude mice (Foxn1 nu ) 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 antitumor activity of the ADC was evaluated in a non-small cell lung cancer (NSCLC) squamous H1703 tumor xenograft model. Female nude mice were cultured in serum-free medium: Matrigel (5 × 10 6 The desired cell type was inoculated subcutaneously into the right flank at a 1:1 ratio of 100 cells / mouse. Tumor volume (TV) was calculated using the formula TV = 1 / 2 (length x width x height) mm. 3 Tumor volumes were measured twice weekly in three dimensions using calipers. Xenografts were grown to approximately 100 mm 3 The cells were grown until 16 days after cell inoculation (116.0 + / - 18.5 mm 3 Mice were randomly distributed into groups of 6 mice per group based on TV (mean + / - SD TV). Stock ADC was diluted in conjugate dilution buffer and administered to mice by individual body weight. Mice received a single intravenous (IV) bolus injection of vehicle (phosphate-buffered saline (PBS) at 200 μL / mouse) or 75 μg / kg or 250 μg / kg of ADC based on payload (approximately 3 or 10 mg / kg based on antibody concentration) at a dose of 5 mL / kg. Tumor growth inhibition (T / C) was measured as the time to tumor growth (T / C) over a given time period (e.g., for control tumors, the median TV was measured at a maximum tumor volume of approximately 1000 mm). 3T / C is the ratio of the median tumor volume (TV) of the treatment group (T) to the median TV of the control group (C) in the 100-well group (when the mice were euthanized). According to NCI criteria, a T / C ≤ 42% is the minimum level of antitumor activity, and a T / C < 10% is considered a high level of antitumor activity. Mice were considered to have exhibited partial regression (PR) if the TV decreased by 50% or more, and complete regression (CR) if no palpable tumor was detected. The T / C, PR, and CR for both efficacy studies are shown in the table below. Body weight was also measured twice weekly as a rough indicator of drug toxicity.

[0497] Figure 20 and Table 14 show the efficacy of the ADCs in the H1703 xenograft model. The results suggest that both the ML66-999 and ML66-22a conjugates exhibited dose-dependent antitumor activity and were effective in the H1703 tumor xenograft model. [Table 13]

[0498] Example 55. Antitumor activity of anti-EGFR antibody drug conjugates in nude mice bearing FaDu human head and neck squamous cell carcinoma xenografts. 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.

[0499] Mice were injected with 1 x 10 mice in 0.1 ml of 50% Matrigel / 50% serum-free medium by subcutaneous injection in the right posterior abdominal region. 7 FaDu cells were inoculated into female athymic Foxn1 mice. nu Mice (6 weeks old) were obtained. After receipt, animals were observed for 7 days before the start of the study. Animals showed no signs of illness or disease upon arrival or prior to treatment.

[0500] Sixty-four mice were randomized into eight groups (eight mice per group) according to tumor volume, ranging from 74.07 to 128.73 (104.66 ± 15.70, mean ± SD) mm 3 Mice were measured, randomized, and dosed based on tumor volume on day 6 post-implantation (11 / 19 / 18). Mouse weights ranged from 20.48 to 25.77 (23.55 ± 1.25, mean ± SD) grams. Mice in each group were identified by punch method. Test agent and vehicle administration was performed intravenously using a 1.0 ml syringe with a 27-gauge, 1 / 2-inch needle. Antibody-drug conjugate test agents were administered qdx1 at 1, 3, or 10 mg / kg. Groups included: a control group administered vehicle (PBS, 200 μL); a control group administered 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.

[0501] Tumor size was measured twice weekly in three dimensions using calipers. Tumor volume was calculated in mm using the formula: volume = length × width × height × ½. 3 The tumor volume was expressed as . Mice were considered to have partial regression (PR) if tumor volume decreased by 50% or more, and complete regression (CR) if no palpable tumor was detected. Tumor volume was determined by StudyLog software.

[0502] Tumor growth inhibition (T / C (%)) is measured at a given time (e.g., the median TV of control tumors increases from 0.1 to 0.25 times the maximum tumor volume of approximately 1000 mm). 3 T / C (%) is the ratio of the median tumor volume (TV) of the treatment group (T) to the median TV of the control group (C) at 21 days post-inoculation, when the median TV of the control group reached 749 mm, which is when the mice were euthanized. 3 According to the NCI criteria, T / C≦42% is the minimum level of antitumor activity, and T / C<10% is considered a high level of antitumor activity.

[0503] The body weight (BW) of all mice was measured twice a week as a rough indicator of drug toxicity and determined by StuyLog software. Mouse weight was expressed as the percentage change in body weight from pre-treatment weight as follows: BW change % = [(post-BW / pre-BW) - 1] × 100, where post-BW is the post-treatment weight and pre-BW is the starting weight before treatment. Body weight loss (BWL) was expressed as the mean change in body weight after treatment. For tumors with a volume of 1000 mm 3 Animals were euthanized at any time during the study if they became larger, if the tumor became necrotic, if the mice lost more than 20% of their initial body weight, or if the mice became moribund.

[0504] Figure 18 and Table 13 show the efficacy of ADCs in the FaDu xenograft model. ML66-999 and ML66-22a conjugates had similar antitumor activity. The T / C value for ML66-KTI administered at 10 mg / kg was 20% (active), with two partial tumor regressions and two complete regressions in eight mice. This indicated that some of the antitumor activity in this model was off-target. The T / C value for ML66-999 administered at 1 mg / kg was 12% (active), with four partial tumor regressions and two complete regressions in eight mice. The T / C value for ML66-999 administered at 3 mg / kg was 2% (highly active), with eight partial tumor regressions and four complete regressions in eight mice. The T / C value for ML66-999 administered at 10 mg / kg was 0% (highly active), with 8 partial tumor regressions and 8 complete regressions in 8 mice. The T / C value for ML66-22a administered at 1 mg / kg was 13% (active), with 4 partial tumor regressions and 3 complete regressions in 8 mice. The T / C value for ML66-22a administered at 3 mg / kg was 0% (highly active), with 8 partial tumor regressions and 8 complete regressions in 8 mice. The T / C value for ML66-22a administered at 10 mg / kg was 0% (highly active), with 8 partial tumor regressions and 8 complete regressions in 8 mice. No significant weight loss was observed for any of the conjugates at any of the doses shown, indicating that the conjugates were well tolerated. The results of this study suggest that both the ML66-999 and ML66-22a conjugates exhibited dose-dependent antitumor activity and were effective in the FaDu head and neck squamous cell carcinoma tumor xenograft model. [Table 14]

[0505] Example 56. Ab F -999 and Ab F Mouse tolerability of -22a ADC: Tolerance of the non-cross-reactive ADC was assessed by daily weight measurement and clinical observation for 2 weeks after injection of the ADC. The efficacy of AbF-999 in female CD-1 mice was evaluated by performing a 5000 μg / kg IV bolus injection of AbF-22a (184 mg / kg antibody) or AbF-999 (198 mg / kg antibody) payload. The maximum tolerated dose (MTD) was defined as the highest dose at which animals did not die or require euthanasia due to greater than 20% weight loss or signs of distress or morbidity (hunching, lack of movement, inability to eat or drink, or signs of pain / distress). GraphPad was used for statistical analysis of the body weights of each group (two-way ANOVA with Tukey's multiple comparison test showed that the AbF-999 group was significantly different from both the vehicle and AbF-22a groups, p<0.05). Figure 21 shows these results.

[0506] While particular embodiments have been shown and described, it is to be understood that changes and modifications may be made by those skilled in the art without departing from the technology in its broader aspects as defined in the following claims.

[0507] The embodiments illustratively described herein may suitably be practiced in the absence of any element(s), limitation(ies), not specifically disclosed herein.

[0508] The present disclosure should not be limited with respect to the specific embodiments described in this application. As will be apparent to those skilled in the art, many modifications and variations may be made without departing from its spirit and scope. Functionally equivalent methods and compositions within the scope of the present disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims and the full scope of equivalents to which such claims are entitled. It is to be understood that the present disclosure is not limited to particular methods, reagents, compounds, or compositions, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0509] Furthermore, when features or aspects of the present disclosure are described in terms of a Markush group, those skilled in the art will recognize that the present disclosure is also described thereby in terms of any individual member or subgroup of members of the Markush group.

[0510] 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 thereof. Any recited range can be readily recognized as fully descriptive and allowing for reductions of the same range by at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range described herein can be readily broken down into a lower third, middle third, upper third, etc. Additionally, as will be understood by those skilled in the art, all terms such as "up to," "at least," "greater than," "less than," etc., refer to ranges that are inclusive of the recited numerical values ​​and that may subsequently be divided into subranges as described above. Finally, as will be understood by those skilled in the art, a range includes each individual member.

[0511] All publications, patent applications, issued patents, and other documents mentioned herein are incorporated by reference herein as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions contained in the text incorporated by reference are excluded to the extent they conflict with definitions in the present disclosure.

[0512] Embodiments of the present disclosure

[0513] 1. A compound of formula I, or a pharmaceutically acceptable salt thereof: ZL 1 -D (Formula I) wherein: D is represented by the following structural formula: [ka] R 1 is -F, -CH3, or -CF3; R 2 -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 together with the carbon atom to which they are attached form a methylenedioxy or difluoromethylenedioxy ring; R 3 is H or C1-C6 alkyl; R 4 is C1-C6 alkyl; L 1 is absent or -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, -X 1 '-(C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; where * is the moiety covalently bonded to Z; X1 -O-, -S-, -S(O)-, -S(O)2-, -C(=O)-, -NR 5 -, -NR 5 C(=O)- or -C(=O)NR 5 - and; X 1 ' is -O-, -S-, -S(O)-, or -S(O)2-; L 2 is phenylene; Each R 5 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; Z is -H or -X 2 and; X 2 -OR 6 , -SR 6 , -S(O)R 6 , -S(O)2R 6 , -SSR 6 , or -N(R 6 )2; Each R 6 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; L 1 and L 2 are each independently a halogen, -CN, or -OR 7 , -SR 7 , -N(R 7 )2, C1-C6 alkyl, C1-C6 fluoroalkyl, C1-C6 heteroalkyl, C3-C6 cycloalkyl, C2-C 10 optionally substituted with 1 to 4 substituents selected from heterocycloalkyl, aryl, or heteroaryl; and Each R 7 are independently H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; However, this is the case if R 1 If 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 -*; where * is the site of covalent attachment to Z; and Z is -X 2 and However, this is the case if R 1 is F and R 2 If -OMe, then -L 1 With the proviso that -Z cannot be -NH2.

[0514] 2. A compound of formula I, or a pharmaceutically acceptable salt thereof: ZL 1 -D (Formula I) wherein: D has the following structural formula: It is shown by [ka] R 1 is -F, -CH3, or -CF3; R 2 -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 together with the carbon atom to which they are attached form a methylenedioxy or difluoromethylenedioxy ring; R 3 is H or C1-C6 alkyl; R 4 is C1-C6 alkyl; L 1 is absent or -(C1-C6 alkylene)-, -(C1-C6 alkylene)-X 1 -(C1-C6 alkylene)-, -X 1'-(C1-C6 alkylene)-* or -(C1-C6 alkylene)-X 1 -L 2 -*; where * is the moiety covalently bonded to Z; X 1 -O-, -S-, -S(O)-, -S(O)2-, -C(=O)-, -NR 5 -, -NR 5 C(=O)- or -C(=O)NR 5 - and; X 1 ' is -O-, -S-, -S(O)-, or -S(O)2-; L 2 is phenylene; Each R 5 are independently -H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; Z is -H or -X 2 and; X 2 ...

Claims

1. A compound of formula I, or a pharma- ceutically acceptable salt thereof: Z-L １ -D (Formula I) wherein: D is represented by the following structural formula: 【Chemistry 1】 R １ is -F, -CH ３ , or -CF ３ and R ２ is -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 together with the carbon atom to which they are attached form a methylenedioxy or difluoromethylenedioxy ring; R ３ is H or C １ -C ６ is alkyl; R ４ is C １ -C ６ is alkyl; L １ does not exist or -(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 -*; where * is the moiety covalently attached to Z; X １ -O-, -S-, -S(O)-, -S(O) ２ -, -C(=O)-, -NR ５ --, --NR ５ C(=O)- or -C(=O)NR ５ - and X １ ' is -O-, -S-, -S(O)-, or -S(O) ２ - and L ２ is phenylene; Each R ５ are 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 and X ２ is -OR ６ , -SR ６ , -S(O)R ６ , -S(O) ２ R ６ , -SSR ６ , or -N(R ６ ) ２ and Each R ６ are 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 represents a halogen, -CN, or -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 optionally substituted with 1 to 4 substituents selected from heterocycloalkyl, aryl, or heteroaryl; and Each R ７ are independently H, C １ -C ６ Alkyl, C １ -C ６ Fluoroalkyl, C ３ -C ６ cycloalkyl, aryl, heteroaryl, or benzyl; However, this is the case if R １ If is F, then L １ But -(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 -*; where * is the moiety covalently attached to Z; and Z is -X ２ and However, this is the case if R １ is F and R ２ If -OMe, then -L １ -Z is -NH ２ This is under the condition that it is not possible.

2. In the formula, R １ is -F and R ２ The compound of claim 1 , wherein is -methyl.

3. In the formula, R １ and R ２ The compound of claim 1 , wherein together with the carbon atom to which they are attached form methylenedioxy.

4. In the formula, -L １ -Z is -(C １ -C ４ alkylene)-OR ６ The compound according to any one of claims 1 to 3,

5. In the formula, -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, or -(CH ２ ) ４ The compound of claim 4 which is OMe.

6. In the formula, R １ is F, and -L １ -Z is -(C １ -C ６ alkylene)-X １ - (C １ -C ６ alkylene)-OR ６ , -(C １ -C ６ alkylene)-X １ - (C １ -C ６ (alkylene)-SR ６ , -(C １ -C ６ alkylene)-X １ - (C １ -C ６ (alkylene)-S(O)R ６ , -(C １ -C ６ alkylene)-X １ - (C １ -C ６ alkylene)-S(O) ２ R ６ , -(C １ -C ６ alkylene)-X １ - (C １ -C ６ (alkylene)-SSR ６ , or -(C １ -C ６ alkylene)-X １ - (C １ -C ６ alkylene)-N(R ６ ) ２ 3. The compound according to claim 1 or 2,

7. In the formula, -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 The compound according to any one of claims 1 to 3,

8. wherein, -L １ -Z is -CH ２ NH C(=O)CH ２ OH, -CH ２ NH C(=O)(CH ２ ). ２ OH, -CH ２ NH C(=O)(CH ２ ). ３ OH, -CH ２ NH C(=O)(CH ２ ). ４ OH, -CH ２ NH C(=O)(CH ２ ). ５ OH, -CH ２ NH C(=O)CH ２ OMe, -CH ２ NH C(=O)(CH ２ ). ２ OMe, -CH ２ NH C(=O)(CH ２ ). ３ OMe, -CH ２ NH C(=O)(CH ２ ). ４ OMe, -CH ２ NH C(=O)(CH ２ ). ５ OMe, -CH ２ NH C(=O)CH ２ SH, -CH ２ NH C(=O)(CH ２ ). ２ SH, -CH ２ NH C(=O)(CH ２ ). ３ SH, -CH ２ NH C(=O)(CH ２ ). ４ SH, -CH ２ NH C(=O)(CH ２ ). ５ SH, -CH ２ NH C(=O)CH ２ SMe, -CH ２ NH C(=O)(CH ２ ). ２ SMe, -CH ２ NH C(=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 The compound according to any one of claims 1 to 3, which is SMe.

9. wherein the compound is: 【Chemistry 2】 2. The compound of claim 1 ,

10. A compound of formula II, or a pharma- ceutically acceptable salt thereof: E-A-Z'-L １ -D (Formula II) wherein: D is represented by the following structural formula: 【Chemistry 3】 R １ is -H, -F, -CH ３ , or -CF ３ and R ２ is -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 together with the carbon atom to which they are attached form a methylenedioxy or difluoromethylenedioxy ring; provided that R １ and R ２ cannot both be -H; R ３ is H or C １ -C ６ is alkyl; R ４ is C １ -C ６ is alkyl; L １ does not exist or -(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 -*; where * is the moiety covalently attached 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 - and X １ ' is -O-, -S-, -S(O)-, or -S(O) ２ - and L ２ is phenylene; Each R ５ are 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 -*; where * is the site of covalent attachment to A; Each R ８ are 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 represents a halogen, -CN, or -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 optionally substituted with 1 to 4 substituents selected from heterocycloalkyl, aryl, or heteroaryl; and Each R ７ are 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 containing 2 to 10 amino acids; where A is optionally substituted with one or more polyols; and E is -C(=O)-L ３ -X ３ and L ３ is -(C １ -C １０ alkylene)- or -Y １ - (C １ -C １０ alkylene)-X ４ -Y ２ - (C １ -C １０ alkylene)-*; wherein * is X ３ is a moiety covalently attached to Y １ is not present or -(CR ａ R ｂ O) ｎ - or - (CR ａ R ｂ CR ａ’ R ｂ’ O) ｍ - and X ４ is -NR ９ C(=O)- or -C(=O)NR ９ - and Y ２ is not present or -(CR ｃ R ｄ O) ｏ - or - (CR ｃ R ｄ CR ｃ’ R ｄ’ O) ｐ - and n, m, o, and p are each independently 1 to 10; Each R ａ , R ｂ , R ａ’ , R ｂ’ , R ｃ , R ｄ , R ｃ’ , and R ｄ’ are independently -H, C １ -C ６ Alkyl, C １ -C ６ Fluoroalkyl, C ３ -C ６ cycloalkyl, aryl, heteroaryl, or benzyl; L in the ceremony ３ is 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 optionally substituted with 0 to 4 substituents selected from heterocycloalkyl, aryl, heteroaryl, and polyol; Each R １１ are independently H, C １ -C ６ Alkyl, C １ -C ６ Fluoroalkyl, C ３ -C ６ cycloalkyl, aryl, heteroaryl, or benzyl; X ３ teeth, 【Chemistry 4】 -C(=O)-CR ｂｂ R ｃｃ -W’、-NR ｅｅ -C(=O)-CR ｂｂ R ｃｃ -W’、 or -SR １０ and 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 and q is 1 to 24; Each R ａａ , R ｂｂ , R ｃｃ , R ｅｅ , and R ｆｆ are independently -H or optionally substituted C １ -C ６ is alkyl; Each R ｙｙ and R ｘｘ are independently -H or C １ -C ６ is alkyl; R gg are each independently -H or C １ -C ６ is alkyl; and R ９ and R １０ are each independently -H, C １ -C ６ Alkyl, C １ -C ６ Fluoroalkyl, C ３ -C ６ It is cycloalkyl, aryl, heteroaryl, or benzyl.

11. In the formula, R １ is -F and R ２ The compound of claim 10, wherein is -methyl.

12. In the formula, R １ and R ２ The compound of claim 10, wherein together with the carbon atom to which they are attached form methylenedioxy.

13. In the formula, -L １ -Z'-* is -(C １ -C ４ (alkylene)-O-CH ２ -NR ８ The compound according to any one of claims 10 to 12, wherein * is the site of covalent attachment to A.

14. In the formula, -L １ -Z'-* is -CH ２ O-CH ２ NH-*, -(CH ２ ) ２ O-CH ２ NH-*, -(CH ２ ) ３ O-CH ２ NH-* or -(CH ２ ) ４ O-CH ２ The compound of claim 13, which is NH-*.

15. In the formula, -L １ -Z'-* is -(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 The compound according to any one of claims 10 to 12, wherein * is the site of covalent attachment to A.

16. wherein, -L １ -Z'-*, is -CH ２ NH C(=O)CH ２ O-CH ２ -NH-*, -CH ２ NH C(=O)(CH ２ ) ２ O-CH ２ -NH-*, -CH ２ NH C(=O)(CH ２ ) ３ O-CH ２ -NH-*, -CH ２ NH C(=O)(CH ２ ) ４ O-CH ２ -NH-*, -CH ２ NH C(=O)(CH ２ ) ５ O-CH ２ -NH-*, -CH ２ NH C(=O)CH ２ S-CH ２ -NH-*, -CH ２ NH C(=O)(CH ２ ) ２ S-CH ２ -NH-*, -CH ２ NH C(=O)(CH ２ ) ３ S-CH ２ -NH-*, -CH ２ NH C(=O)(CH ２ ) ４ S-CH ２ -NH-*, -CH ２ NH C(=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 The compound according to any one of claims 10 to 12, wherein the compound is -NH-*, and * is the site of covalent attachment to A.

17. 1. A compound of formula III, or a pharma- ceutically acceptable salt thereof: CBA - E’ - A - Z’ - L １ -D (Formula III) During the ceremony: D has the following structural formula: 【Chemistry 5】 is shown by R １ is -H, -F, -CH ３ , or -CF ３ and R ２ is -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 together with the carbon atom to which they are attached form a methylenedioxy or difluoromethylenedioxy ring; provided that R １ and R ２ is a condition where both cannot be -H; R ３ is H or C １ -C ６ is alkyl; R ４ is C １ -C ６ is alkyl; L １ does not exist or -(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 -*; where * is the moiety covalently attached 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 - and X １ ' is -O-, -S-, -S(O)-, or -S(O) ２ - and L ２ is phenylene; Each R ５ are 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 -*; where * is the site of covalent attachment to A; Each R ８ are 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 represents a halogen, -CN, or -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 optionally substituted with 1 to 4 substituents selected from heterocycloalkyl, aryl, or heteroaryl; and Each R ７ are 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 containing 2 to 10 amino acids; where A is optionally substituted with one or more polyols; E' is -C(=O)-L ３ -X ６ -*; where * is a moiety operably linked to a CBA; L ３ is -(C １ -C １０ alkylene)- or -Y １ - (C １ -C １０ alkylene)-X ４ -Y ２ - (C １ -C １０ alkylene)-*; wherein * is X ６ is a moiety covalently attached to Y １ is not present or -(CR ａ R ｂ O) ｎ -or- (CR ａ R ｂ CR ａ’ R ｂ’ O) ｍ - and X ４ is -NR ９ C(=O)- or -C(=O)NR ９ - and Y ２ is not present or -(CR ｃ R ｄ O) ｏ - or - (CR ｃ R ｄ CR ｃ’ R ｄ’ O) ｐ - and n, m, o, and p are each independently 1 to 10; Each R ａ , R ｂ , R ａ’ , R ｂ’ , R ｃ , R ｄ , R ｃ’ , and R ｄ’ are independently -H, C １ -C ６ Alkyl, C １ -C ６ Fluoroalkyl, C ３ -C ６ cycloalkyl, aryl, heteroaryl, or benzyl; L in the ceremony ３ is 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 optionally substituted with 0 to 4 substituents selected from heterocycloalkyl, aryl, heteroaryl, and polyol; Each R １１ are independently H, C １ -C ６ Alkyl, C １ -C ６ Fluoroalkyl, C ３ -C ６ cycloalkyl, aryl, heteroaryl, or benzyl; X ６ teeth, 【Chemistry 6】 , -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 are independently -H or optionally substituted C １ -C ６ is alkyl; Each R ｙｙ and R ｘｘ are independently -H or C １ -C ６ is alkyl; R ９ are 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.

18. In the formula, R １ is -F and R ２ The compound of claim 17, wherein is -methyl.

19. In the formula, R １ and R ２ 18. The compound of claim 17, wherein together with the carbon atom to which they are attached form methylenedioxy.

20. In the formula, -L １ -Z'-* is -(C １ -C ４ (alkylene)-O-CH ２ -NR ８ -*, where * is the site of covalent attachment to A; The compound according to any one of claims 17 to 19.

21. In the formula, -L １ -Z'-* is -CH ２ O-CH ２ NH-*, -(CH ２ ) ２ O-CH ２ NH-*, -(CH ２ ) ３ O-CH ２ NH-* or -(CH ２ ) ４ O-CH ２ The compound of claim 20, which is NH-*.

22. In the formula, -L １ -Z'-* is -(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 The compound according to any one of claims 17 to 19, wherein * is the site of covalent attachment to A.

23. wherein, -L １ -Z'-*, is -CH ２ NH C(=O)CH ２ O-CH ２ -NH-*, -CH ２ NH C(=O)(CH ２ ２ O-CH ２ -NH-*, -CH ２ NH C(=O)(CH ２ ３ O-CH ２ -NH-*, -CH ２ NH C(=O)(CH ２ ４ O-CH ２ -NH-*, -CH ２ NH C(=O)(CH ２ ５ O-CH ２ -NH-*, -CH ２ NH C(=O)CH ２ S-CH ２ -NH-*, -CH ２ NH C(=O)(CH ２ ２ S-CH ２ -NH-*, -CH ２ NH C(=O)(CH ２ ３ S-CH ２ -NH-*, -CH ２ NH C(=O)(CH ２ ４ S-CH ２ -NH-*, -CH ２ NH C(=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 The compound according to any one of claims 17 to 19, which is -NH-*.

24. 24. A pharmaceutical composition comprising a compound according to any one of claims 1 to 23, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

25. 24. A pharmaceutical composition for use in treating cancer in a subject in need thereof, comprising a compound according to any one of claims 1 to 23, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

26. 26. The pharmaceutical composition of claim 25, comprising: (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), Mantel cell leukemia (MCL), small lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis, or 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, or lymphatic system cancer; or (iv) The cancer is lung cancer, and the lung cancer is non-small cell lung cancer or small cell lung cancer.

26. The pharmaceutical composition of claim 25.

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