Camptothecin derivatives and conjugates thereof

Abstract

Camptothecin derivatives that are therapeutically effective in antibody drug conjugates (ADCs) are provided. The present invention provides camptothecins, camptothecin payloads, and camptothecin conjugates, methods for their preparation and use, and intermediates useful in their preparation. Also provided herein are methods for treating cancer and autoimmune diseases using the camptothecin conjugates described herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 981,197, filed February 25, 2020, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Antibody-drug conjugates (ADCs) have attracted significant interest as a new class of therapeutics. For example, ADCs utilize monoclonal antibodies (mAbs) for targeted delivery of cytotoxic agents to tumor cells, thereby enabling the use of highly cytotoxic drugs that could not be used using traditional non-targeted delivery methods. The design of ADCs, which typically feature the attachment of a cytotoxic agent to an antibody via a linker, involves consideration of various factors, including the presence of a conjugation handle on the agent for attachment to the linker and linker technology for attaching the agent to the antibody in a conditionally stable manner. Suboptimal design can result in reduced ADC efficacy, poor immunological specificity of the conjugate, and increased toxicity due to nonspecific release of the drug from the conjugate.

[0003] Camptothecin (CPT) is a pentacyclic quinoline alkaloid originally isolated from the wood and bark of a native Chinese tree, Camptotheca acuminata (Camptotheca) in Latin and xi shu in Chinese. Camptothecin is overexpressed in various tumor cell lines and exhibits significant antitumor activity by inhibiting topoisomerase I, an enzyme essential for DNA synthesis. Due to its broad antitumor activity and unique mechanism of action, considerable efforts have been directed toward the development of clinical analogs of camptothecin. However, most camptothecin and its derivatives have poor solubility and are inactive under physiological conditions, limiting the clinical development of suitable camptothecin analogs.

[0004] Therefore, camptothecin as a toxin for use in ADCs has the potential to overcome these limitations, and there remains a need for therapeutically effective camptothecin derivatives, as well as a need for new ADCs for therapeutic use. Summary of the Invention [Means for solving the problem]

[0005] New cytotoxic agents according to formula (I) are described herein. These cytotoxic agents can then be combined with a peptide linker to form a payload according to formula (II), which can be used to prepare a conjugate with a cell-binding agent (formula (III)). Compounds of formula (III) include ADCs that are useful for treating cell proliferative diseases such as cancer.

[0006] Thus, in one aspect, the present invention provides a compound of formula (I): D-L1-L2-Q (I) or a pharmaceutically acceptable salt thereof, wherein: D has the following structural formula: [ka] wherein: R 1 are independently -H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, silyl, C3-C6 cycloalkyl, C1-C6 halogenated alkyl, C2-C6 halogenated alkenyl, or C2-C6 halogenated alkynyl; R 2 are independently -H, -F, -N(R 4 )2, -N(R 4 )(R 5 ), -OR 4 , -SR 4 , -S(=O)R 5 , -SO2R 5 , C1-C6 alkyl, or C1-C6 fluoroalkyl; R 3are independently -H, -F, -CN, -OCH3, -CH3, or -CF3, or R 2 and R 3 together, the formula -O(CH2) n O- or -O(CF2) n O- group, where n is 1 or 2, R 4 are independently —H or C1-C4 alkyl; R 5 are independently C1-C4 alkyl; L1 can independently be absent or -(C1-C 10 alkylene)-, L2 is independently absent, -OCH2-L3-*, -SCH2-L3-*, -S(=O)-L3-*, -SO2-L3-*, -C(=O)-L3-*, -N(R 6 )CH2-L3-*, -N(R 6 )C(=O)-L3-*, -N(R 6 )C(=O)N(R 7 )-L3-*, -C(=O)N(R 6 )CH2-L3-*, -OC(=O)N(R 6 )CH2-L3-* or -N(R 6 )C(=O)OCH2-L3-*, where * indicates the site of covalent attachment to Q; L3 is independently -(C1-C 10 alkylene)-, -CH2OCH2CH2-, or -CH2CH2OCH2CH2-; Each R 6 and R 7 are independently —H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; Q is -OH or -SH; During the ceremony, R 2 and R 3 When they combine to form -OCHO-, R 1 is not -CH2CH2CH2CH3, R 1is -H or -CH2CH3, and R 2 is —OH or alkoxy, and R 3 is —H, then —L1-L2-Q is not —CH(R′)CH2OH or —CH(R′)(CH2)2OH, where R′ is —H, or C1-C6 alkyl, alkoxy, substituted alkyl, phenyl, or PhCH2—.

[0007] In embodiments, R 1 , R 2 , and R 3 At least one of is not -H.

[0008] In an embodiment, at least one of L1 and L2 is present.

[0009] In embodiments, R 1 is independently C1-C6 alkyl, silyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C1-C6 alkyl halide, alkene, or alkyne.

[0010] In embodiments, R 1 are independently --H or C1-C6 alkyl.

[0011] In embodiments, R 2 are independently -H, -F, -N(R 4 )2, -N(R 4 )(R 5 ), -OR 4 , -SR 4 , -S(=O)R 5 , -SO2R 5 , C1-C6 alkyl, or C1-C6 fluoroalkyl; R 3 are independently --H, -F, -CN, -OCH 3, -CH3, or -CF3.

[0012] In embodiments, R 2 is independently C1-C6 alkyl, C1-C6 fluoroalkyl, or --F.

[0013] In embodiments, R 3 are independently -H, -F, -CN, or -CF3.

[0014] In embodiments, R 3 are independently -F, -CN, -OCH3, -CH3, or -CF3.

[0015] In embodiments, R 2 and R 3 is bonded to -O(CH2) n O- or --O(CF2) n O-, where n is 1 or 2.

[0016] In embodiments, D is represented by one of the following structures: [Table 1]

[0017] In embodiments, R 1 is —H or C1-C6 alkyl.

[0018] In embodiments, D is represented by one of the following structures: [Table 2]

[0019] In embodiments, L is -(C 10 alkylene)-, and L2 is absent.

[0020] In embodiments, L is -(C 10 alkylene)- and L2 is -N(R 6 )CH2-L3-* or -N(R 6 )C(=O)-L3-*, where * indicates the site of covalent attachment to Q.

[0021] In embodiments, L1 is absent and L2 is -N(R6 )CH2-L3-* or -N(R 6 )C(=O)-L3-*, where * indicates the site of covalent attachment to Q.

[0022] In embodiments, L3 is -(C1-C 10 alkylene).

[0023] In embodiments, R 6 is -H or -CH3.

[0024] In embodiments, L1-L2 is -CH2-, -CH2CH2-, -CH2CH2CH2-, or -CH2CH2CH2CH2-.

[0025] In an embodiment, L1-L2 are selected from the group consisting of -OCH2CH2-*, -OCH2CH2OCH2CH2-*, -SCH2CH2-*, -SCH2CH2OCH2CH2-*, -S(=O)CH2-*, -SO2CH2-*, -C(=O)CH2-*, -NHCH2CH2-*, -N(CH3)CH2CH2-*, -N(CF3)CH2CH2-*, -NHC(=O)CH2-*, -CH2NHC(=O)CH2-*, -CH2CH2NHC(=O)CH2-*, CH2N(CH3)C(=O)CH2-*, -N(CH3)C(=O)CH2-*, -N(CH3)C(=O)CH2CH2-*, -C(=O)NHCH2CH2-*, -NHC(=O)NHCH2CH2-*, -NHC(=O)OCH2CH2-*, -CH2OC(=O)NHCH2CH2-*, or -C(=O)N(CH3)CH2CH2-*, where * indicates the site of covalent attachment to Q.

[0026] In embodiments, L1-L2-Q is -CH2CH2CH2CH2OH, -CH2CH2CH2OH, -CH2CH2OH, -CH2CH2OCH2CH2OH, -CH2SCH2CH2OH, -CH2NHC(=O)CH2OH, -CH2CH2NHC(=O)CH2OH, -CH2N( CH3)C(=O)CH2OH, -OCH2CH2OH, -OCH2CH2CH2OH, -SCH2CH2CH2OH, -SCH2CH2OH, -NHCH2CH2OH, -NHCH2CH2CH2OH, -N(CH3)CH2CH2OH, -C(=O)NHCH2CH2OH, -NHC( =O)CH2OH, -CH2S(=O)CH2OH, -CH2SO2CH2OH, -CH2CH2CH2CH2SH, -CH2CH2CH2SH, -CH2CH2SH, -CH2CH2OCH2CH2SH, -CH2SCH2CH2SH, -CH2NHC(=O)CH2SH, -OCH2 CH2CH2SH, -SCH2CH2CH2SH, -SCH2CH2SH, -NHCH2CH2CH2SH, -N(CH3)CH2CH2SH, -C(=O)NHCH2CH2SH, -NHC(=O)CH2SH, -CH2S(=O)CH2SH, or -CH2SO2CH2SH.

[0027] In an embodiment, D-L1-L2 is represented by the structure: [Table 3]

[0028] In embodiments, R 1 is —H or C1-C6 alkyl.

[0029] In embodiments, R 1 is H or -CH2CH3.

[0030] In embodiments, Q is —OH.

[0031] In embodiments, Q is —SH.

[0032] In embodiments, the compound has one of the following structures: [Table 4] or a pharmaceutically acceptable salt thereof.

[0033] In another aspect, the present invention provides a compound of formula (II): D-L1-L2-Q'-CH2-NH-EZ (II) or a pharmaceutically acceptable salt thereof, wherein: D has the following structural formula: [ka] wherein: R 1 are independently -H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, silyl, C3-C6 cycloalkyl, C1-C6 halogenated alkyl, C2-C6 halogenated alkenyl, or C2-C6 halogenated alkynyl; R 2 are independently -H, -F, -N(R 4 )2, -N(R 4 )(R 5 ), -OR 4 , -SR 4 , -S(=O)R 5 , -SO2R 5 , C1-C6 alkyl, or C1-C6 fluoroalkyl; R 3 is -H, -F, -CN, -OCH3, -CH3, -CF3, or R 2 and R 3 together, the formula -O(CH2) n O- or -O(CF2) n O- group, where n is 1 or 2, R 4 are independently —H or C1-C4 alkyl; R 5 are independently C1-C4 alkyl; L1 can independently be absent or -(C1-C 10 alkylene)-, L2 is independently absent, -OCH2-L3-*, -SCH2-L3-*, -S(=O)-L3-*, -SO2-L3-*, -C(=O)-L3-*, -N(R 6 )CH2-L3-*, -N(R 6 )C(=O)-L3-*, -N(R 6 )C(=O)N(R 7 )-L3-*, -C(=O)N(R 6 )CH2-L3-*, -OC(=O)N(R 6 )CH2-L3-* or -N(R 6 )C(=O)OCH2-L3-*, where * indicates the site of covalent attachment to Q'; L3 is independently -(C1-C 10 alkylene)-, -CH2OCH2CH2-, or -CH2CH2OCH2CH2-; Each R 6 and R 7 are independently —H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; Q' is -O- or -S-; E is a peptide comprising 2 to 10 amino acids, wherein E is optionally substituted with one or more polyols, and the N-terminus of the peptide is covalently linked to Z; Z is -C(=O)-L4-Y, [ka] wherein m represents an integer of 1 to 10; L4 is -(C1-C 10 Alkylene)-*, -CH2CH2(OCH2CH2) n N(R 8 )C(=O)-L5-*, or -CH2(OCH2CH2) n N(R 8 )C(═O)—L5-*, wherein n represents an integer of 1 to 10, and * represents the site of covalent bonding to Y; L5 is -(C1-C 10 alkylene)-, R 8 is -H or -CH3, Y is an electrophilic group; In the formula, R 2 and R 3 When they combine to form -OCHO-, R 1 is not -CH2CH2CH2CH3.

[0034] In embodiments, E is a peptide of 2, 3, or 4 amino acids, wherein each amino acid in the peptide is an L-amino acid, or at least one amino acid in the peptide is a D-amino acid.

[0035] In embodiments, E comprises one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, wherein the glutamine or glutamic acid is optionally substituted by a polyol.

[0036] In embodiments, E comprises an amino acid selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, wherein the glutamine or glutamic acid is optionally substituted by a polyol.

[0037] In embodiments, E comprises an amino acid having the following structure: [ka] In the formula, R 9 is —H or C1-C6 alkyl.

[0038] In embodiments, E comprises an amino acid having the following structure: [ka]

[0039] in embodiments、Eは、-Ala-Val-*、-Val-Ala-*、-Gly-Gly-*、-Val-Cit-*、-Cit-Val-*、-Leu-Ala-*、-Ala-Leu-*、 -Leu-Cit-*、-Cit-Leu-*、-Leu-Ala-*、-Ala-Leu-*、-Lys-Lys-*、-Ala-Lys-*、-Lys-Ala-*、-Val-Lys-*、-Lys-Val-*、-Tyr-Arg-*、-Arg-Tyr-*、-Arg-Arg-*、-Ala-Ala-*、-Phe-Lys-*、 -Lys-Phe-*、-Thr-Thr-*、-Thr-Met-*、-Met-Thr-*、-Met-Tyr-*、-Tyr-Met-*、-Phe-Gln-*、 -Gln-Phe-*、-Gly-Ser-*、-Leu-Gln-*、-Gln-Leu-*、-Ser-Ala-*、-Ser-Gly-*、-Val-Thr-*、-Thr-Val-*、-Val-Gln-*、-Ser-Val-*、-Val-Ser-*、-Ala-Met-*、-Met-Ala-*、-Val-Arg-*、 -Arg-Val-*、-Phe-Ala-*、-Ala-Phe-*、-Cit-Val-*、-Gln-Val-*、-Phe-Arg-*、-Arg-Phe-*、-Ala-Ala-Ala- * 、-Gly-Gly-Gly- * 、-Ala-Val-Ala- * 、-Gly-Val-Gly- * 、-Ala-Val-Gly- * 、 -Gly-Phe-Lys-*、-Lys-Phe-Gly-*、-Leu-Ala-Leu-*、-Val-Ala-Leu-*、-Leu-Ala-Val-*、-Val-Ala-Val-*、-Ala-Val-Ala-Gly- * 、-Gly-Phe-Gly-Gly- * 、-Gly-Gly-Phe-Gly-*、-Ala-Val-Gly-Gly-*、-Ala-Ala-Ala-Ala- * 、-Ala-Val-Ala-Ala- *, -Ala-Leu-Ala-Leu-*, -Leu-Ala-Leu-Ala-*, -Gly-Phe-Leu-Gly-*, and -Gly-Leu-Phe-Gly-*, where * indicates the N-terminus of the peptide covalently attached to Z.

[0040] In embodiments, E is -L-Ala-D-Val-*, -L-Val-D-Ala-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Val-D-Cit-*, -L-Val-D-Arg-*, -L-Val-D-Cit-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Arg-D-Arg-*, -L-Ala-D-Ala-*, -L-Ala-D-Lys-*, -L-Ala-D-Arg-*, -L-Ala-D-Ala-L-Ala- * , -L-Ala-D-Val-L-Ala- * , -L-Ala-D-Ala-Gly- * , and -L-Ala-D-Val-Gly- * wherein * indicates the N-terminus of the peptide covalently attached to Z.

[0041] In embodiments, -E-NH--CH 2-- has one of the following structures, where * indicates the N-terminus of the peptide covalently attached to Z: [ka]

[0042] In embodiments, L4 is -(C1-C 10 alkylene).

[0043] In embodiments, L4 is -CH2CH2(OCH2CH2) n N(R 8 )C(=O)-L5-* or -CH2(OCH2CH2) n N(R 8)C(=O)-L5-*, where n represents an integer of 1 to 10, and * represents the site of covalent bonding to Y.

[0044] In embodiments, L4 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, where * indicates the site of covalent attachment to Y.

[0045] In embodiments, Y is a Michael acceptor group, a succinimide, an epoxide, or a halogen.

[0046] In an embodiment, Y is [ka] and In the formula, R 10 and R 11 are each independently H or C1-C3 alkyl.

[0047] In embodiments, Z is -C(=O)-L4-Y.

[0048] In an embodiment, Z is [ka] In embodiments, m is 1. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4. In embodiments, m is 5. In embodiments, m is 6. In embodiments, m is 7. In embodiments, m is 8. In embodiments, m is 9. In embodiments, m is 10.

[0049] In an embodiment, Z is [ka] In embodiments, m is 1. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4. In embodiments, m is 5. In embodiments, m is 6. In embodiments, m is 7. In embodiments, m is 8. In embodiments, m is 9. In embodiments, m is 10.

[0050] In an embodiment, Z is: [ka]

[0051] In embodiments, ZE-NH--CH2- has one of the following structures: [ka]

[0052] In embodiments, R 1 is -H or -CH2CH3, and R 2 is —OH or alkoxy, and R 3 is —H, then —L1-L2-Q′- is not —CH(R′)CHO— or —CH(R′)(CH)O—, where R′ is —H, or C1-C6 alkyl, alkoxy, substituted alkyl, phenyl, or PhCH2—.

[0053] In an embodiment, at least one of L1 and L2 is present.

[0054] In embodiments, R 1 , R 2 , and R 3 At least one of is not -H.

[0055] In embodiments, R 1 is independently C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, silyl, C3-C6 cycloalkyl, C1-C6 alkyl halide, alkene, or alkyne.

[0056] In embodiments, R 1 are independently --H or C1-C6 alkyl.

[0057] In embodiments, R 2 are independently -H, -F, -N(R 4 )2, -N(R 4 )(R 5 ), -OR 4 , -SR 4 , -S(=O)R 5 , -SO2R 5 , C1-C6 alkyl, or C1-C6 fluoroalkyl; R 3 are independently --H, --F, --CN, --OCH3, --CH3, or --CF3.

[0058] In embodiments, R 2 is independently C1-C6 alkyl, C1-C6 fluoroalkyl, or --F.

[0059] In embodiments, R 3 are independently -H, -F, -CN, or -CF3.

[0060] In embodiments, R 3 are independently -F, -CN, -OCH3, -CH3, or -CF3.

[0061] In embodiments, R 2 and R 3 is bonded to -O(CH2) n O- or --O(CF2) n O-, where n is 1 or 2.

[0062] In embodiments, D is represented by one of the following structures: [Table 5]

[0063] In embodiments, R 1is —H or C1-C6 alkyl.

[0064] In embodiments, D is represented by one of the following structures: [Table 6]

[0065] In embodiments, L is -(C 10 alkylene)-, and L2 is absent.

[0066] In embodiments, L is -(C 10 alkylene)- and L2 is -N(R 6 )CH2-L3-* or -N(R 6 )C(=O)-L3-*, where * indicates the site of covalent attachment to Q'.

[0067] In embodiments, L1 is absent and L2 is -N(R 6 )CH2-L3-* or -N(R 6 )C(=O)-L3-*, where * indicates the site of covalent attachment to Q'.

[0068] In embodiments, L3 is -(C1-C 10 alkylene).

[0069] In embodiments, R 6 is -H or -CH3.

[0070] In embodiments, L1-L2 is -CH2-, -CH2CH2-, -CH2CH2CH2-, or -CH2CH2CH2CH2-.

[0071] In an embodiment, L1-L2 is -OCH2CH2-*, -OCH2CH2OCH2CH2-*, -SCH2CH2-*, -SCH2CH2OCH2CH2-*, -S(=O)CH2-*, -SO2CH2-*, -C(=O)CH2-*, -NHCH2CH2-*, -N(CH3)CH2CH2-*, -N(CF3)CH2CH2-*, -NHC(=O)CH2-*, -CH2NHC(=O)CH2-*, -CH2CH2NHC(=O)CH2-*, -CH2N(CH3)C(=O)CH2-*, -N(CH3)C(=O)CH2-*, -N(CH3)C(=O)CH2CH2-*, -C(=O)NHCH2CH2-*, -NHC(=O)NHCH2CH2-*, -NHC(=O)OCH2CH2-*, -CH2OC(=O)NHCH2CH2-*, or -C(=O)N(CH3)CH2CH2-*, where * indicates the site of covalent attachment to Q'.

[0072] In embodiments, L1-L2-Q' is -CH2CH2CH2CH2O-, -CH2CH2CH2O-, -CH2CH2O-, -CH2CH2OCH2CH2O-, -CH2SCH2CH2O-, -CH2NHC(=O)CH2O-, -CH2CH2NHC(=O)CH2O-, -CH2N (CH3)C(=O)CH2O-, -OCH2CH2O-, -OCH2CH2CH2O-, -SCH2CH2CH2O-, -SCH2CH2O-, -NHCH2CH2O-, -NHCH2CH2CH2O-, -N(CH3)CH2CH2O-, -C(=O)NHCH2CH2O-, -NHC (=O)CH2O-, -CH2S(=O)CH2O-, -CH2SO2CH2O-, -CH2CH2CH2CH2S-, -CH2CH2CH2S-, -CH2CH2S-, -CH2CH2OCH2CH2S-, -CH2SCH2CH2S-, -CH2NHC(=O)CH2S-, -OCH2 CH2CH2S-, -SCH2CH2CH2S-, -SCH2CH2S-, -NHCH2CH2CH2S-, -N(CH3)CH2CH2S-, -C(=O)NHCH2CH2S-, -NHC(=O)CH2S-, -CH2S(=O)CH2S-, or -CH2SO2CH2S-.

[0073] In an embodiment, D-L1-L2 is represented by the structure: [Table 7]

[0074] In embodiments, R 1 is —H or C1-C6 alkyl.

[0075] In embodiments, R 1 is -H or -CH2CH3.

[0076] In embodiments, Q' is -O-.

[0077] In embodiments, Q' is -S-.

[0078] In embodiments, D-L1-L2-Q'- has one of the following structures: [Table 8]

[0079] In embodiments, the compound has one of the following structures: [ka] [ka] [ka] or a pharmaceutically acceptable salt thereof.

[0080] In yet a further embodiment, the present invention provides a compound of formula (III): {D-L1-L2-Q'-CH2-NH-E-Z'} p -C (III) or a pharmaceutically acceptable salt thereof, wherein: D has the following structural formula: [ka] wherein: R 1are independently -H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, silyl, C3-C6 cycloalkyl, C1-C6 halogenated alkyl, C2-C6 halogenated alkenyl, or C2-C6 halogenated alkynyl; R 2 are independently -H, -F, -N(R 4 )2, -N(R 4 )(R 5 ), -OR 4 , -SR 4 , -S(=O)R 5 , -SO2R 5 , C1-C6 alkyl, or C1-C6 fluoroalkyl; R 3 is -H, -F, -CN, -OCH3, -CH3, or -CF3, or R 2 and R 3 together, the formula -O(CH2) n O- or -O (CF2) n O- group, where n is 1 or 2, R 4 are independently —H or C1-C4 alkyl; R 5 are independently C1-C4 alkyl; L1 can independently be absent or -(C1-C 10 alkylene)-, L2 is independently absent, -OCH2-L3-*, -SCH2-L3-*, -S(=O)-L3-*, -SO2-L3-*, -C(=O)-L3-*, -N(R 6 )CH2-L3-*, -N(R 6 )C(=O)-L3-*, -N(R 6 )C(=O)N(R 7 )-L3-*, -C(=O)N(R 6 )CH2-L3-*, -OC(=O)N(R 6 )CH2-L3-* or -N(R 6 )C(=O)OCH2-L3-*, where * indicates the site of covalent attachment to Q'; L3 is independently -(C1-C 10alkylene)-, -CH2OCH2CH2-, or -CH2CH2OCH2CH2-; Each R 6 and R 7 are independently —H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; Q' is -O- or -S-; E is a peptide comprising 2 to 10 amino acids, wherein E is optionally substituted with one or more polyols, and the N-terminus of the peptide is covalently linked to Z'; Z' is -C(=O)-L4-Y', [ka] In the formula, m represents an integer of 1 to 10, and * represents a site covalently bonded to the C; L4 is -(C1-C 10 Alkylene)-, -CH2CH2(OCH2CH2) n N(R 8 )C(=O)-L5-*, or -CH2(OCH2CH2) n N(R 8 )C(═O)—L5-*, wherein n represents an integer of 1 to 10, and * represents the site of covalent bonding to Y′; L5 is -(C1-C 10 alkylene)-, R 8 is -H or -CH3, C represents a cell-binding agent; Y' is a group formed by reaction of an electrophilic group with a reactive nucleophilic group present on the cell-binding agent; In the formula, R 2 and R 3 When they combine to form -OCHO-, R 1 is not -CH2CH2CH2CH3, p has a value of 1 to 18.

[0081] In embodiments, L4 is -(C1-C 10 alkylene).

[0082] In embodiments, L4 is -CH2CH2(OCH2CH2) n N(R 8 )C(=O)-L5-* or -CH2(OCH2CH2) n N(R 8 )C(=O)-L5-*, where n represents an integer of 1 to 10, and * represents the site of covalent bonding to Y'.

[0083] In embodiments, L4 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, where * indicates the site of covalent attachment to Y'.

[0084] In embodiments, Y' is formed from a Michael acceptor group, a succinimide, an epoxide, or a halogen.

[0085] In embodiments, Y' is [ka] wherein R 10 and R 11 are each independently —H or C1-C3 alkyl.

[0086] In embodiments, Y' is [ka] and In the formula, R 10 and R 11 are each independently -H or C1-C3 alkyl, and * indicates the site of covalent bonding to the C.

[0087] In an embodiment, Z' is formed from: [ka]

[0088] In embodiments, Z' is -C(=O)-L4-Y'.

[0089] In embodiments, Z' is [ka] wherein * indicates the site covalently bonded to said C. In embodiments, m is 1. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4. In embodiments, m is 5. In embodiments, m is 6. In embodiments, m is 7. In embodiments, m is 8. In embodiments, m is 9. In embodiments, m is 10.

[0090] In embodiments, Z' is [ka] wherein * indicates the site covalently bonded to said C. In embodiments, m is 1. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4. In embodiments, m is 5. In embodiments, m is 6. In embodiments, m is 7. In embodiments, m is 8. In embodiments, m is 9. In embodiments, m is 10.

[0091] In embodiments, Z' is [ka] and In the formula, * indicates the site of covalent attachment to C.

[0092] In embodiments, E is a peptide of 2, 3, or 4 amino acids, wherein each amino acid in said peptide is an L-amino acid, or at least one amino acid in said peptide is a D-amino acid.

[0093] In embodiments, E comprises one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, wherein the glutamine or glutamic acid is optionally substituted by a polyol.

[0094] In embodiments, E comprises an amino acid selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, wherein the glutamine or glutamic acid is optionally substituted by a polyol.

[0095] In embodiments, E comprises an amino acid having the following structure: [ka] In the formula, R 9 is —H or C1-C6 alkyl.

[0096] In embodiments, E comprises an amino acid having the following structure: [ka]

[0097] In an embodiment, E is selected from the group consisting of -Ala-Val-*, -Val-Ala-*, -Gly-Gly-*, -Val-Cit-*, -Cit-Val-*, -Leu-Ala-*, -Ala-Leu-*, -Leu-Cit-*, -Cit-Leu-*, -Leu-Ala-*, -Ala-Leu-*, -Lys-Lys-*, -Ala-Lys-*, -Lys-Ala-*, -Val-Lys-*, -Lys-Val-*, -Tyr-Arg-*, -Arg-Tyr-*, -Arg-Arg-*, -Ala-Ala-*, -Phe-Lys-*, -Lys-Phe-*, -Thr-Thr-*, -Thr-Met-*, -Met-Thr-*, -Met-Tyr-*, -Tyr-Met-*, -Phe-Gln-*, -Gln-Phe-*, -Gly-Ser-*, -Leu-Gln-*, -Gln-Leu-*, -Ser-Ala-*, -Ser-Gly-*, -Val-Thr-*, -Thr-Val-*, -Val-Gln-*, -Ser-Val-*, -Val-Ser-*, -Ala-Met-*, -Met-Ala-*, -Val-Arg-*, -Arg-Val-*, -Phe-Ala-*, -Ala-Phe-*, -Cit-Val-*, -Gln-Val-*, -Phe-Arg-*, -Arg-Phe-*, -Ala-Ala-Ala- * -Gly-Gly-Gly- * -Ala-Val-Ala- * -Gly-Val-Gly- * -Ala-Val-Gly- * 、 -Gly-Phe-Lys-*, -Lys-Phe-Gly-*, -Leu-Ala-Leu-*, -Val-Ala-Leu-*, -Leu-Ala-Val-*, -Val-Ala-Val-*, -Ala-Val-Ala-Gly- * -Gly-Phe-Gly-Gly- * -Gly-Gly-Phe-Gly-*, -Ala-Val-Gly-Gly-*, -Ala-Ala-Ala-Ala- * -Ala-Val-Ala-Ala- * selected from the group consisting of -Ala-Leu-Ala-Leu-*, -Leu-Ala-Leu-Ala-*, -Gly-Phe-Leu-Gly-*, and -Gly-Leu-Phe-Gly-*, wherein * represents the N-terminus of a peptide covalently linked to Z'.

[0098] In an embodiment, E is -L-Ala-D-Val-*, -L-Val-D-Ala-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Val-D-Cit-*, -L-Val-D-Arg-*, -L-Val-D-Cit-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Arg-D-Arg-*, -L-Ala-D-Ala-*, -L-Ala-D-Lys-*, -L-Ala-D-Arg-*, -L-Ala-D-Ala-L-Ala- * , -L-Ala-D-Val-L-Ala- * , -L-Ala-D-Ala-Gly- * , and -L-Ala-D-Val-Gly- * wherein * indicates the N-terminus of the peptide covalently attached to Z'.

[0099] In embodiments, -E-NH--CH 2-- has one of the following structures, where * indicates the N-terminus of the peptide covalently attached to Z': [ka]

[0100] In embodiments, Z'-E-NH--CH2 is formed from one of the following structures: [ka]

[0101] In embodiments, Z'-E-NH--CH2 is one of the following structures, where * indicates the point of attachment to C: [ka]

[0102] In embodiments, R 1 is -H or -CH2CH3, and R 2 is —OH or alkoxy, and R 3is -H, then -L1-L2-Q'- is not -CH(R')CHO- or -CH(R')(CH)O-, where R' is -H, or C1-C6 alkyl, alkoxy, substituted alkyl, phenyl, or PhCH2-. In embodiments, R 1 is -H or -CH2CH3, and R 2 is —OH or alkoxy, and R 3 is —H, then —L1-L2-Q′- is not —CH(R′)CHO— or —CH(R′)(CH)O—, where R′ is —H, or C1-C6 alkyl, alkoxy, substituted alkyl, phenyl, or PhCH2—.

[0103] In an embodiment, at least one of L1 and L2 is present.

[0104] In embodiments, R 1 , R 2 , and R 3 At least one of is not -H.

[0105] In embodiments, R 1 is independently C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, silyl, C3-C6 cycloalkyl, C1-C6 alkyl halide, alkene, or alkyne.

[0106] In embodiments, R 1 are independently --H or C1-C6 alkyl.

[0107] In embodiments, R 2 are independently -H, -F, -N(R 4 )2, -N(R 4 )(R 5 ), -OR 4 , -SR 4 , -S(=O)R 5 , -SO2R 5 , C1-C6 alkyl, or C1-C6 fluoroalkyl, R 3are independently --H, --F, --CN, --OCH3, --CH3, or --CF3.

[0108] In embodiments, R 2 is independently C1-C6 alkyl, C1-C6 fluoroalkyl, or --F.

[0109] In embodiments, R 3 are independently -H, -F, -CN, or -CF3.

[0110] In embodiments, R 3 are independently -F, -CN, -OCH3, -CH3, or -CF3.

[0111] In embodiments, R 2 and R 3 is bonded to -O(CH2) n O- or --O(CF2) n O-, where n is 1 or 2.

[0112] In embodiments, D is represented by one of the following structures: [Table 9]

[0113] In embodiments, R 1 is —H or C1-C6 alkyl.

[0114] In embodiments, D is represented by one of the following structures: [Table 10]

[0115] In embodiments, L is -(C 10 alkylene)-, and L2 is absent.

[0116] In embodiments, L is -(C 10alkylene)- and L2 is -N(R 6 )CH2-L3-* or -N(R 6 )C(=O)-L3-*, where * indicates the site of covalent attachment to Q'.

[0117] In embodiments, L1 is absent and L2 is -N(R 6 )CH2-L3-* or -N(R 6 )C(=O)-L3-*, where * indicates the site of covalent attachment to Q'.

[0118] In embodiments, L3 is -(C1-C 10 alkylene).

[0119] In embodiments, R 6 is -H or -CH3.

[0120] In embodiments, L1-L2 is -CH2-, -CH2CH2-, -CH2CH2CH2-, or -CH2CH2CH2CH2-.

[0121] In an embodiment, L1-L2 is -OCH2CH2-*, -OCH2CH2OCH2CH2-*, -SCH2CH2-*, -SCH2CH2OCH2CH2-*, -S(=O)CH2-*, -SO2CH2-*, -C(=O)CH2-*, -NHCH2CH2-*, -N(CH3)CH2CH2-*, -N(CF3)CH2CH2-*, -NHC(=O)CH2-*, -CH2NHC(=O)CH2-*, -CH2CH2NHC(=O)CH2-*, -CH2N(CH3)C(=O)CH2-*, -N(CH3)C(=O)CH2-*, -N(CH3)C(=O)CH2CH2-*, -C(=O)NHCH2CH2-*, -NHC(=O)NHCH2CH2-*, -NHC(=O)OCH2CH2-*, -CH2OC(=O)NHCH2CH2-*, or -C(=O)N(CH3)CH2CH2-*, where * indicates the site of covalent attachment to Q'.

[0122] In embodiments, L1-L2-Q' is -CH2CH2CH2CH2O-, -CH2CH2CH2O-, -CH2CH2O-, -CH2CH2OCH2CH2O-, -CH2SCH2CH2O-, -CH2NHC(=O)CH2O-, -CH2CH2NHC(=O)CH2O-*, -CH2N (CH3)C(=O)CH2O-, -OCH2CH2O-, -OCH2CH2CH2O-, -SCH2CH2CH2O-, -SCH2CH2O-, -NHCH2CH2O-, -NHCH2CH2CH2O-, -N(CH3)CH2CH2O-, -C(=O)NHCH2CH2O-, -NHC (=O)CH2O-, -CH2S(=O)CH2O-, -CH2SO2CH2O-, -CH2CH2CH2CH2S-, -CH2CH2CH2S-, -CH2CH2S-, -CH2CH2OCH2CH2S-, -CH2SCH2CH2S-, -CH2NHC(=O)CH2S-, -OCH2 CH2CH2S-, -SCH2CH2CH2S-, -SCH2CH2S-, -NHCH2CH2CH2S-, -N(CH3)CH2CH2S-, -C(=O)NHCH2CH2S-, -NHC(=O)CH2S-, -CH2S(=O)CH2S-, or -CH2SO2CH2S-.

[0123] In an embodiment, D-L1-L2 is represented by the structure: [Table 11]

[0124] In embodiments, R 1 is —H or C1-C6 alkyl.

[0125] In embodiments, R 1 is -H or -CH2CH3.

[0126] In embodiments, Q' is -O-.

[0127] In embodiments, Q' is -S-.

[0128] In embodiments, D-L1-L2-Q'- has one of the following structures: [Table 12]

[0129] In embodiments, D-L1-L2-Q'-CH2-NH-E-Z'- is formed from one of the following structures: [ka] [ka] [ka]

[0130] In an embodiment, {D-L1-L2-Q'-CH2-NH-E-Z'} p -C is one of the following structures, where C is a monoclonal antibody, p is the drug-to-antibody ratio (DAR), and p is an average number ranging from about 2 to 10, 4 to 8, 7 to 8, or 3.2 to 8.0: [ka] [ka] [ka]

[0131] In another aspect, the invention features a method for preparing a conjugate of Formula (III) including a cell-binding agent and a drug, the method including contacting the cell-binding agent with a compound of Formula (II) such that a covalent bond is formed between the cell-binding agent and the compound of Formula (II).

[0132] In yet another aspect, the invention features a conjugate including a cell-binding agent and a drug. In embodiments, the conjugate is prepared according to any method described herein.

[0133] In embodiments, the conjugate comprises a cell binding agent that is an antibody or an antigen-binding fragment thereof.

[0134] In embodiments, the conjugate comprises a cell binding agent that is a monoclonal antibody or an antigen-binding fragment thereof.

[0135] In embodiments, the cell binding agent is an antibody or antigen-binding fragment thereof, and p is the drug-to-antibody ratio (DAR) and has a value of 1 to 18. In embodiments, p is an average number ranging from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).

[0136] In embodiments, the cell binding agent is a monoclonal antibody or antigen-binding fragment thereof, and p is the drug-to-antibody ratio (DAR) and has a value of 1 to 18. In embodiments, p is an average number ranging from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).

[0137] In another aspect, the invention features a pharmaceutical composition including any of the conjugates described herein.

[0138] In yet another aspect, the invention features a method of treating a cell proliferative disease or disorder or inhibiting abnormal cell growth, the method comprising administering any of the conjugates described herein or any pharmaceutical composition comprising any of the conjugates described herein.

[0139] In another aspect, the invention features a pharmaceutical composition including any compound of Formula (III) described herein, or a pharmaceutically acceptable salt thereof.

[0140] In another aspect, the invention features a method of treating a cell proliferative disease or disorder or inhibiting abnormal cell growth, the method comprising administering any compound of Formula (III) described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising any compound of Formula (III) described herein or a pharmaceutically acceptable salt thereof.

[0141] In embodiments, the method is for treating cancer.

[0142] In embodiments, the cancer is adenocarcinoma, brain cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, CNS tumors, colon or colorectal cancer, diffuse pontine glioma (DIPG), endometrial cancer, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, blood cancer, Hodgkin's lymphoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, myelodysplastic syndrome (MDS), neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, pancreatic cancer, peritoneal cancer, prostate cancer, ovarian cancer, renal cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, or Wilms' tumor.

[0143] In embodiments, the cancer is breast cancer. [Brief explanation of the drawings]

[0144] [Figure 1] This figure shows the efficacy of antibody-drug conjugates (ADCs) in the NCI-N87 CDX model. MB-2a and MB-3a are ADCs encompassed by this formula and were studied along with vehicle control, trastuzumab, and the ADC DS-8201a. As shown in this figure, MB-2a (1 mg / kg and 4 mg / kg) and MB-3a (1 mg / kg and 4 mg / kg) demonstrated potent antitumor efficacy. [Figure 2]

[0039] Figure 1 shows the efficacy of antibody drug conjugates (ADCs) in the JIMT-1 CDX model. In this study, all three doses of MB-2a and MB-3a demonstrated significant anti-tumor efficacy. [Figure 3] A selection of data from Figure 2 is shown showing the antitumor efficacy using a single 2.5 mg / kg IV dose of MB-2a and MB-3a. [Figure 4] A selection of data from Figure 2 is shown showing the antitumor efficacy using a single 5 mg / kg IV dose of MB-2a and MB-3a. [Figure 5] A selection of data from Figure 2 is shown showing the antitumor efficacy using a single 10 mg / kg IV dose of MB-2a and MB-3a. DETAILED DESCRIPTION OF THE INVENTION

[0145] definition Unless otherwise specified, the following terms and phrases as used herein are intended to have the following meanings: When trade names are used herein, unless the context dictates otherwise, the trade name includes formulations of the product, generic drugs, and the active pharmaceutical ingredient of the trade name product.

[0146] As used herein, the term "antibody" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that contains sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibodies include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody may contain one or more constant region sequences characteristic of murine, rabbit, primate, or human antibodies. In some embodiments, an antibody may contain one or more sequence elements of humanized, primatized, chimeric, etc., sequences known in the art. In many embodiments, the term "antibody" is used to refer to one or more constructs or formats known or developed in the art that utilize the structural and functional characteristics of antibodies in alternative presentations. For example, in embodiments, antibodies utilized in accordance with the present invention include intact IgA, IgG, IgE, or IgM antibodies; bi- or multispecific antibodies (such as, for example, Zybody®); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fv; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies, such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probody®; Small Modular ImmunoPharmaceuticals ("SMIP™"); single chain or tandem diabodies (TandAb®); VHH; Anticalin®, Nanobody® minibodies; BiTE®; ankyrin repeat proteins or DARPIN®; Avimer®; DART; TCR-like antibodies; Adnectin®; Affilin®; Trans-body®; Affibody®; TrimerX®; MicroProtein; Fynomer®, Centyrin®, and KALBITOR®.In some embodiments, an antibody may lack covalent modifications (e.g., glycan attachments) that it would have if produced naturally. In some embodiments, an antibody may contain covalent modifications (e.g., attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant groups (e.g., poly-ethylene glycol, etc.). In many embodiments, an antibody is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as complementarity-determining regions (CDRs), and in some embodiments, an antibody is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to that found in a reference antibody. In some embodiments, an antibody agent is a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to that found in a reference antibody. In some embodiments, an antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain.

[0147] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, but is not to be construed as requiring production of the antibody by any particular method.

[0148] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions generated from (or assembled from) human immunoglobulin sequences. In some embodiments, an antibody (or antibody component) may be considered "human" even though its amino acid sequence, for example in one or more CDRs, and particularly CDR3, includes residues or elements that are not encoded by human germline immunoglobulin sequences (e.g., including sequence variations that may have been (initially) introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0149] As known in the art, the term "humanized" refers to a V antibody whose amino acid sequence is derived from a reference antibody made in a non-human species (e.g., mouse). H and V LThe term "humanized" is generally used to refer to an antibody (or antibody component) containing a region sequence, but also includes modifications of those sequences relative to a reference antibody intended to make the antibody more "human-like," i.e., more similar to human germline sequences. In some embodiments, a "humanized" antibody (or antibody component) is an antibody that immunospecifically binds to an antigen of interest and has framework (FR) regions with amino acid sequences substantially like those of a human antibody, and complementarity-determining regions (CDRs) with amino acid sequences substantially like those of a non-human antibody. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv), in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., a donor immunoglobulin) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. In some embodiments, a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin constant region. In some embodiments, a humanized antibody contains both a light chain and at least the variable domain of a heavy chain. The antibody may also include the CH1, hinge, CH2, CH3, and optionally CH4 regions of the heavy chain constant region. In some embodiments, a humanized antibody contains a humanized V L In some embodiments, the humanized antibody contains only a humanized V region. H In some particular embodiments, the humanized antibody contains only a humanized V region. H and V L Contains the region.

[0150] An "intact antibody" is an antibody that contains an antigen-binding variable region appropriate to the antibody class, as well as a light chain constant domain (C L ) and heavy chain constant domain, C H 1. C H 2. C H 3, and C H 4. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof.

[0151] "Antibody fragments" include portions of an intact antibody, including its antigen-binding and / or variable regions. Examples of antibody fragments include Fab, Fab', F(ab'), and Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragments, Fab-expressing antibodies, and the like. or an epitope-binding fragment of any of the above that immunospecifically binds to a target antigen (e.g., a cancer cell antigen, a viral antigen, or a microbial antigen).

[0152] An "antigen" is an entity to which an antibody specifically binds.

[0153] As used herein, the term "binding" will be understood to typically refer to a non-covalent association between two or more entities. "Direct" binding involves physical contact between the entities or moieties, while indirect binding involves a physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts, including when the interacting entities or moieties are studied separately or in the context of a more complex system (e.g., while covalently or otherwise associated with a carrier entity and / or in a biological system or cell). In some embodiments, "binding" refers to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity, of an immunological binding interaction can be determined by the dissociation constant (K d ), and K d A smaller K represents a higher affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, the "on rate constant" (K on) and the "off-rate constant" (K off Both the K and K can be determined by calculation of the concentration and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). off / K on The rate of dissociation allows for the removal of all parameters not related to affinity, and the dissociation constant K d (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473.)

[0154] The terms "specific binding" and "specifically bind" mean that an antibody or antibody derivative binds in a highly selective manner to its corresponding epitope of a target antigen, but not to a multitude of other antigens. Typically, an antibody or antibody derivative binds to at least about 1 x 10 -7 M, and preferably 10 -8 M~10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 and binds to a predetermined antigen with an affinity of M that is at least 2-fold higher than its affinity for binding to a nonspecific antigen other than the predetermined antigen or a closely related antigen (e.g., BSA, casein). The term "specificity" refers to the ability of a cell binding agent (e.g., as described herein, such as an antibody or fragment thereof) to specifically bind to (e.g., immunoreact with) a given target antigen, e.g., a human target antigen.

[0155] Generally, a "protein" is a polypeptide (i.e., a chain of at least two amino acids linked together by peptide bonds). A protein may contain moieties other than amino acids (e.g., may be a glycoprotein) and / or may be otherwise processed or modified. Those of skill in the art will understand that a "protein" may be the entire polypeptide chain produced by a cell (with or without a signal sequence), or a functional portion thereof. Those of skill in the art will further understand that a protein may comprise two or more polypeptide chains, for example, linked by one or more disulfide bonds or associated by other means.

[0156] The term "inhibit" or "inhibition of" means to reduce by a measurable amount or to prevent completely.

[0157] The term "substantial" or "substantially" refers to a majority of the population of a mixture or sample, i.e., greater than 50%, preferably greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the population.

[0158] The term "cytotoxic activity" refers to the cell-killing effect of a drug or a camptothecin conjugate or an intracellular metabolite of a camptothecin conjugate. Cytotoxic activity is measured by the IC 50 It may be expressed as a value, which is the concentration (molar or mass) per unit volume at which half of the cells survive.

[0159] The term "cytostatic activity" refers to the antiproliferative effect of a drug or a camptothecin conjugate or an intracellular metabolite of a camptothecin conjugate.

[0160] As used herein, the term "cytotoxic agent" refers to a substance that has cytotoxic activity and causes destruction of cells. The term is intended to include chemotherapeutic agents and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including synthetic analogs and derivatives thereof.

[0161] As used herein, the term "cytostatic agent" refers to a substance that inhibits cellular function, including cell growth or proliferation. Cytostatic agents include inhibitors such as protein inhibitors, e.g., enzyme inhibitors. Cytostatic agents have cytostatic activity.

[0162] The terms "cancer" and "cancerous" refer to or describe the physiological condition or disorder in mammals that is typically characterized by unregulated cell growth. A "tumor" contains one or more cancerous cells.

[0163] As used herein, "autoimmune disease" refers to a disease or disorder arising from and directed against an individual's own tissues or proteins.

[0164] As used herein, the term "patient" or "subject" refers to any organism to which a compound provided herein described is administered in accordance with the present invention, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals. The term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cow, primate, and / or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or parasites. In some embodiments, the animal may be a transgenic animal, a genetically engineered animal, and / or a clone. In embodiments, the animal is a mammal, insect, parasite, such as a mouse, rat, rabbit, non-human primate, and human. In embodiments, the subject is a human. In some embodiments, the subject may be suffering from and / or susceptible to a disease, disorder, and / or condition (e.g., cancer). As used herein, a "patient population" or a "population of subjects" refers to a plurality of patients or subjects.

[0165] As used herein, the term "normal," when used to modify the term "individual" or "subject," refers to an individual or group of individuals who do not have a particular disease or condition, and who are not carriers of that disease or condition. The term "normal" also refers to a biological specimen or sample isolated from a normal or wild-type individual or subject. As used herein (eg, "normal biological sample").

[0166] An individual "suffering from" a disease, disorder, and / or condition (e.g., any of the cancers described herein) has been diagnosed with or exhibits one or more symptoms of that disease, disorder, and / or condition.

[0167] An individual who is "susceptible to" a disease, disorder, and / or condition may not have been diagnosed with and / or exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., cancer) may be characterized by one of the following: (1) a genetic mutation associated with the development of the disease, disorder, and / or condition, (2) a genetic polymorphism associated with the development of the disease, disorder, and / or condition, (3) an increase and / or decrease in protein expression and / or activity associated with the disease, disorder, and / or condition, (4) habits and / or lifestyle associated with the development of the disease, disorder, and / or condition, (5) a family history of the disease, disorder, and / or condition, (6) a response to a particular bacteria or virus, or (7) exposure to a particular chemical. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0168] The terms "treat" or "treatment," unless otherwise indicated by context, refer to any administration of a therapeutic molecule (e.g., any compound described herein) that partially or completely alleviates, ameliorates, relieves, inhibits, delays the onset of, delays the progression of, reduces the severity of, and / or reduces the occurrence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition (e.g., cancer). Such treatment may be in subjects who do not exhibit signs of the relevant disease, disorder, and / or condition and / or in subjects who exhibit only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be in subjects who exhibit one or more established signs of the relevant disease, disorder, and / or condition. Alternatively, the pharmacological and / or physiological effect may be prophylactic, i.e., an effect that completely or partially prevents a disease or its symptoms (e.g., delays the onset of or slows the progression of a disease or its symptoms). In this regard, the methods of the present invention include administering a "prophylactically effective amount" of a binding agent. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Thus, treatments (including prophylactic treatments) whose purpose is to inhibit or slow (reduce) undesirable physiological changes or disorders, such as the onset or spread of cancer. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stable (i.e., not worsening) state of disease, delay or slowing of disease progression, improvement or palliation of the disease state, and remission (partial or complete), whether detectable or undetectable. Treatment can also include prolonging survival compared to expected survival if not treated. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder.

[0169] In the context of cancer, the term "treating" includes any or all of killing tumor cells, inhibiting the growth of tumor cells, cancer cells, or tumors, inhibiting the replication of tumor cells or cancer cells, reducing the total tumor burden or the number of cancerous cells, and ameliorating one or more symptoms associated with the disease.

[0170] In the context of autoimmune disease, the term "treating" refers to inhibiting the replication of cells associated with the autoimmune disease state, including, but not limited to, cells that produce autoimmune antibodies. The objectives of the present invention include any or all of the following: reducing the autoimmune antibody load; and ameliorating one or more symptoms of an autoimmune disease.

[0171] The term "therapeutically effective amount" or "effective amount" refers to an amount of a conjugate effective to treat or prevent a disease or disorder (e.g., as described herein) in a mammal. In the case of cancer, a therapeutically effective amount of a conjugate can reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow to some extent, preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent, preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more symptoms associated with cancer. To the extent a drug can inhibit growth and / or kill existing cancer cells, the drug may be cytostatic and / or cytotoxic. For cancer therapy, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0172] As used herein, the term "pharmaceutically acceptable form" refers to a form of a disclosed compound, including, but not limited to, pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, isomers, prodrugs, and isotopically labeled derivatives thereof. In one embodiment, "pharmaceutically acceptable form" includes, but is not limited to, pharmaceutically acceptable salts, esters, prodrugs, and isotopically labeled derivatives thereof. In embodiments, "pharmaceutically acceptable form" includes, but is not limited to, pharmaceutically acceptable isomers and stereoisomers, prodrugs, and isotopically labeled derivatives thereof.

[0173] In embodiments, the pharmaceutically acceptable form is a pharmaceutically acceptable salt. As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound (e.g., camptothecin, camptothecin payload, or camptothecin conjugate). In some aspects, the compound may contain at least one amino group, and accordingly, an acid addition salt may be formed with the amino group. Exemplary salts include, but are not limited to, sulfate, trifluoroacetate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)). Pharmaceutically acceptable salts may involve the inclusion of another molecule, such as acetate, succinate, or other counterion. Counter ions can be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, pharmaceutically acceptable salts can have two or more charged atoms in their structure. If multiple charged atoms are part of a pharmaceutically acceptable salt, it can have multiple counter ions. Therefore, pharmaceutically acceptable salts can have one or more charged atoms and / or one or more counter ions.

[0174] As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent (e.g., a compound according to any of Formulas (I)-(III) described herein) is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, a pharmaceutical composition may be specially formulated for administration in solid or liquid form, and is described below. Suitable for: oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, targeted for systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension or sustained release formulation; topical application, e.g., as a cream, ointment, or controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or rectally, e.g., as a pessary, cream, or foam; sublingually; intraocularly; transdermally; or intranasal, pulmonary, and other mucosal surfaces.

[0175] As used herein, "carrier" or "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered. In some exemplary embodiments, the carrier may include sterile liquids such as water and oils (oils of petroleum, animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.)). In some embodiments, the carrier is or includes one or more solid ingredients. In some embodiments, the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerin, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some cases, it may be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0176] As used herein, the term "kit" refers to any delivery system for delivering materials. Such delivery systems may include systems that allow for the storage, transport, or delivery of various diagnostic or therapeutic reagents (e.g., oligonucleotides, enzymes, etc. in appropriate containers) and / or supporting materials (e.g., buffers, written instructions for conducting an assay, etc.) from one location to another. For example, a kit may include one or more enclosures (e.g., boxes, cartridges, bottles, ampoules, etc.) containing relevant reaction reagents and / or supporting materials. As used herein, the term "fragmented kit" refers to a delivery system that includes two or more separate containers, each containing a subportion of the overall kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain an enzyme for use in an assay, while a second container contains oligonucleotides. The term "fragmented kit" is intended to encompass, but is not limited to, kits containing analyte-specific reagents (ASRs) regulated under Section 520(e) of the Federal Food, Drug, and Cosmetic Act. Indeed, any delivery system containing two or more separate containers, each containing a subportion of the overall kit components, is encompassed by the term "fragmented kit." In contrast, a "combined kit" refers to a delivery system containing all of the components in a single container (e.g., in a single box housing each of the desired components). The term "kit" includes both fragmented and combined kits.

[0177] As used herein, the term "administration" typically refers to the administration of a composition to a subject or system to achieve delivery of the composition or an agent contained therein. Those skilled in the art will recognize the various routes that may be utilized for administration to a subject, e.g., a human, in the appropriate circumstances. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. For example, in some embodiments, administration is ocular, oral, parenteral, topical, etc. In embodiments, administration is parenteral (e.g., intravenous administration). In embodiments, intravenous administration is intravenous infusion. In some particular embodiments, administration may be or include one or more of: bronchial (e.g., by bronchial infusion), oral, cutaneous (e.g., topical to the dermis, intradermal, interdermal, transdermal, etc.), intestinal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal infusion), vaginal, vitreous, etc.

[0178] As used herein, the term "nucleophile" refers to a reactive group that is electron-rich and has an unshared electron pair that acts as a reactive site and reacts with positively charged or electron-deficient sites. Examples of nucleophilic groups suitable for use in the present invention include, but are not limited to, amino groups (e.g., primary amines, secondary amines, hydroxylamine, and / or hydrazine), thiols, phenols, and alcohols. In embodiments, the nucleophilic functional group comprises amino, hydrazino, hydroxyamino, hydroxy, or thio. In embodiments, the nucleophilic functional group is carboxamide, N-hydroxycarboxamide, carboxylhydrazide, or guanidino. In embodiments, the nucleophilic group is or includes a thiol group. Certain nucleophilic groups must be activated with base to be able to react with electrophilic groups. For example, if there are nucleophilic thiol and hydroxyl groups in a multifunctional compound, the compound must be mixed with aqueous base to remove a proton and provide a thiolate or hydroxylate anion, allowing reaction with the electrophilic group. Non-nucleophilic bases are preferred unless it is desired that the base participate in the reaction. In some embodiments, the base may be present as a component of a buffer solution.

[0179] As used herein, the term "electrophilic" refers to a reactive group that is susceptible to nucleophilic attack, i.e., susceptible to reaction with an incoming nucleophilic group. The electrophilic group can be selected to be capable of reacting with the nucleophilic group of a counter reactant. For example, if the nucleophilic reactive group is an amino group, the electrophilic group can be selected to react with an amino group. Similarly, if the nucleophilic reactive group is a thiol moiety, the corresponding electrophilic group can be a thiol reactive group, etc. Examples of electrophilic groups suitable for use in the present invention include, but are not limited to, carboxylic acid esters, acid chloride groups, anhydrides, isocyanates, thioisocyanates, epoxides, activated hydroxyl groups, succinimidyl esters, sulfosuccinimidyl esters, maleimides, and ethenesulfonyl. In embodiments, the electrophilic group is an aldehyde, α-haloketone, maleimide, succinimide, hydroxysuccinimide, isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, tosylate ester, glyoxal, epoxide, oxirane, carbonate, imidoester, anhydride, fluorophenyl ester, hydroxymethylphosphine derivative, carbonate, haloacetyl, chlorotriazine, haloacetyl, alkyl halide, aziridine, acryloyl derivative, ketone, carboxylic acid, ester, acetyl chloride, or acetic anhydride. In embodiments, the electrophilic group is or includes a maleimide or succinimide group. The carboxylic acid group may be activated to react with a nucleophile, including reaction with a suitable hydroxyl-containing compound in the presence of a dehydrating agent such as dicyclohexylcarbodiimide (DCC) or dicyclohexylurea (DHU). For example, carboxylic acids can be reacted with alkoxy-substituted N-hydroxysuccinimides or N-hydroxysulfosuccinimides in the presence of DCC to form reactive electrophilic groups, N-hydroxysuccinimide esters and N-hydroxysulfosuccinimide esters, respectively. Carboxylic acids may also be activated by reaction with acyl halides, such as acyl chlorides (e.g., acetyl chloride), to provide reactive anhydride groups.In a further example, a carboxylic acid may be converted to an acid chloride group using, for example, thionyl chloride or an acyl chloride capable of undergoing an exchange reaction.

[0180] Unless otherwise indicated, the term "alkyl," by itself or as part of another term, refers to a substituted or unsubstituted straight or branched chain, saturated or unsaturated hydrocarbon having the designated number of carbon atoms (e.g., "-C1-C8 alkyl" or "-C1-C 10 ("Alkyl" refers to alkyl groups having 1 to 8 or 1 to 10 carbon atoms, respectively). If the number of carbon atoms is not specified, the alkyl group has 1 to 8 carbon atoms. Representative straight chain "-C1-C8 alkyl" groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, and -n-octyl, while branched chain -C3-C8 alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl, and unsaturated -C2-C8 alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl. Alkyl includes, but is not limited to, vinyl, allyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl, acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, and 3-methyl-1-butynyl. Alkyl groups can be unsubstituted. Alkyl groups can be substituted with one or more groups. In other embodiments, alkyl groups are saturated.

[0181] Unless otherwise indicated, "alkylene," by itself or as part of another term, refers to a substituted or unsubstituted saturated, branched or straight-chain, or cyclic hydrocarbon radical having a specified number of carbon atoms, typically 1 to 10 carbon atoms, and having two monovalent radical centers obtained by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent alkane. Typical alkylene radicals include, but are not limited to, methylene (-CH-), 1,2-ethylene (-CHCH-), 1,3-propylene (-CHCHCH-), 1,4-butylene (-CHCHCHCH-), and the like. In preferred embodiments, the alkylene is a branched or straight-chain hydrocarbon (i.e., not a cyclic hydrocarbon).

[0182] Unless otherwise indicated, "aryl," by itself or as part of another term, means a substituted or unsubstituted monovalent carbocyclic aromatic hydrocarbon radical of the specified number of carbon atoms, typically 6 to 20 carbon atoms, derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Some aryl groups are represented in the exemplary structures as "Ar." Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like. An exemplary aryl group is the phenyl group.

[0183] Unless otherwise indicated, an "arylene" by itself or as part of another term is an aryl group, as defined above, having two covalent bonds (i.e., is divalent) and which may be ortho, meta, or para oriented.

[0184] Unless otherwise indicated, "C3-C8 heterocycle," by itself or as part of another term, refers to a monovalent, substituted or unsubstituted, aromatic or non-aromatic, monocyclic or bicyclic ring system having 3 to 8 carbon atoms (also referred to as ring members) and 1 to 4 heteroatom ring members independently selected from N, O, P, or S, obtained by the removal of one hydrogen atom from a ring atom of the parent ring system. One or more N, C, or S atoms of a heterocycle may be oxidized. A ring containing a heteroatom may be aromatic or non-aromatic. A heterocycle in which all of the ring atoms participate in aromaticity is referred to as a heteroaryl; otherwise, it is referred to as a heterocarbocycle. Unless otherwise specified, a heterocycle is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. Thus, a heteroaryl may be attached through an aromatic carbon of its aromatic ring system, referred to as a C-linked heteroaryl, or through an N-linked heteroaryl. Nitrogen-containing heterocycles may be bonded through a non-double-bonded N atom (i.e., not =N-) of their aromatic ring system, referred to as a "C-linked" heterocycle. Thus, nitrogen-containing heterocycles may be C-linked or N-linked, and include pyrrole moieties such as pyrrol-1-yl (N-linked) and pyrrol-3-yl (C-linked), and imidazole moieties such as imidazol-1-yl and imidazol-3-yl (both N-linked), and imidazol-2-yl, imidazol-4-yl, and imidazol-5-yl moieties (all C-linked).

[0185] Unless otherwise specified, "C3-C8 heteroaryl" refers to an aromatic C3-C8 heterocycle, and the subscript indicates the total number of carbon atoms in the cyclic ring system of the heterocycle or the total number of aromatic carbon atoms in the aromatic ring system of the heteroaryl, without implying the size of the ring system or whether or not it is fused. Representative examples of C3-C8 heterocycles include, but are not limited to, pyrrolidinyl, azetidinyl, piperidinyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, pyrrolyl, thiophenyl (thiophene), furanyl, thiazolyl, imidazolyl, pyrazolyl, pyrimidinyl, pyridinyl, pyrazinyl, pyridazinyl, isothiazolyl, and isoxazolyl. When explicitly indicated, the size of the heterocycle or heteroaryl ring system is indicated by the total number of atoms in the ring. For example, a designation as a 5- or 6-membered heteroaryl indicates the total number of aromatic atoms in the heteroaromatic ring system of the heteroaryl (i.e., 5 or 6), but does not imply the number of aromatic heteroatoms or aromatic carbons in the ring system. Fused heteroaryls are explicitly stated or implied by the context and are typically indicated by the number of aromatic atoms in each aromatic ring that are fused together to form the fused heteroaromatic ring system. For example, a 5,6-membered heteroaryl is an aromatic 5-membered ring fused to an aromatic 6-membered ring, where one or both of the rings have an aromatic heteroatom or the heteroatom is shared between the two rings.

[0186] A heterocycle fused to an aryl or heteroaryl is an example of an optionally substituted heterocycle in which the heterocycle is substituted by ring fusion with an aryl or heteroaryl, such that the heterocycle remains non-aromatic and is part of a larger structure through attachment to the non-aromatic portion of the fused ring system. Similarly, an aryl or heteroaryl fused to a heterocycle or carbocycle that is part of a larger structure through attachment to the aromatic portion of the fused ring system is an example of an optionally substituted aryl or heterocycle in which the aryl or heterocycle is substituted by ring fusion with a heterocycle or carbocycle.

[0187] Unless otherwise indicated, "C3-C8 heterocyclo," by itself or as part of another term, refers to a C3-C8 heterocycle, as defined above, in which one of the heterocycle's hydrogen atoms is replaced with a bond (i.e., it is divalent). Unless otherwise indicated, "C3-C8 heteroarylene," by itself or as part of another term, refers to a C3-C8 heteroaryl group, as defined above, in which one of the heteroaryl group's hydrogen atoms is replaced with a bond (i.e., it is divalent).

[0188] Unless otherwise indicated, a "C3-C8 carbocycle," by itself or as part of another term, is a 3-, 4-, 5-, 6-, 7-, or 8-membered monovalent, substituted or unsubstituted, saturated or unsaturated, non-aromatic monocyclic or bicyclic carbocycle obtained by the removal of one hydrogen atom from a ring atom of a parent ring system. Representative -C3-C8 carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentadienyl, cyclohexyl, cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, cycloheptyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, cyclooctenyl, and cyclooctadienyl.

[0189] Unless otherwise indicated, "C3-C8 carbocyclo" may be used by itself or as another term. As part of the formula, refers to a C3-C8 carbocyclic group as defined above, in which another of the carbocyclic group's hydrogen atoms is replaced with a bond (i.e., is divalent).

[0190] Unless otherwise indicated, the term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable straight- or branched-chain hydrocarbon, or combination thereof, that is fully saturated or contains one to three degrees of unsaturation and consists of the specified number of carbon atoms and one to ten, preferably one to three, heteroatoms selected from the group consisting of O, N, Si, and S, where the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The O, N, and S heteroatoms may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. The Si heteroatom may be placed at any position of the heteroalkyl group, including the position at which the alkyl group is attached to the remainder of the molecule. Examples include -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -NH-CH2-CH2-NH-C(O)-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=NO-CH3, and -CH=CH-N(CH3)-CH3. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. Typically, a C1-C4 heteroalkyl or heteroalkylene has 1 to 4 carbon atoms and 1 or 2 heteroatoms, and a C1-C3 heteroalkyl or heteroalkylene has 1 to 3 carbon atoms and 1 or 2 heteroatoms. In some embodiments, the heteroalkyl or heteroalkylene is saturated.

[0191] Unless otherwise stated, the term "heteroalkylene," by itself or in combination with another term, means a divalent group derived from a heteroalkyl (as defined above), as exemplified by -CH-CH-S-CH-CH- and -CH-S-CH-CH-NH-CH-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini. Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied.

[0192] Unless otherwise indicated, "aminoalkyl," by itself or in combination with another term, means a heteroalkyl in which the alkyl portion, as defined herein, is substituted with an amino, alkylamino, dialkylamino, or cycloalkylamino group. Exemplary non-limiting aminoalkyls are -CHNH, -CHCHNH, -CHCHNHCH, and -CHCHN(CH), further including branched species such as -CH(CH)NH and -C(CH)CHNH in either the (R) or (S) configuration. Alternatively, an aminoalkyl may be a heteroalkyl in which no sp carbon other than the radical carbon is present. 3 carbon is replaced by an amino or alkylamino moiety, and the sp 3 Nitrogen is alkyl sp 3 Replaces carbon, but with at least one sp 3 is an alkyl moiety, group, or substituent as defined herein, provided that the carbon remains. When an aminoalkyl moiety is referred to as a substituent on a larger structure or another moiety, the aminoalkyl is covalently attached to that structure or moiety through a carbon radical on the alkyl portion of the aminoalkyl.

[0193] Unless otherwise indicated, "alkylamino" and "cycloalkylamino", by themselves or in combination with another term, refer to an alkyl or cycloalkyl radical in which the radical carbon is replaced with a nitrogen radical, provided that at least one sp 3" refers to an alkyl or cycloalkyl radical as described herein, provided that the carbon remains. When an alkylamino is substituted at the nitrogen with another alkyl moiety, the resulting substituted radical is referred to as a dialkylamino moiety, group, or substituent, and the nitrogen The alkyl moieties substituting are independently selected. Exemplary and non-limiting amino, alkylamino, and dialkylamino substituents include those having the structure —N(R′)2, where R′ in these examples is independently selected from hydrogen or C1-6 alkyl, typically hydrogen or methyl, while in cycloalkylamines contained within heterocycloalkyls, both R′, together with the nitrogen to which they are attached, define a heterocycle. When both R′ are hydrogen or alkyl, the moieties may be described as primary amino and tertiary amine groups, respectively. When one R′ is hydrogen and the other is alkyl, the moiety is described as a secondary amino group. Primary and secondary alkylamino moieties are more reactive as nucleophiles toward carbonyl-containing electrophilic centers, while tertiary amines are more basic.

[0194] "Substituted alkyl" and "substituted aryl" refer to alkyl and aryl, respectively, in which one or more hydrogen atoms, typically one, are each independently replaced with a substituent. Exemplary substituents include -X, -R', -OH, -OR', -SR', -N(R'), -N(R'), =NR', -CX, -CN, -NO, -NR'C(=O)R', -C(=O)R', -C(=O)N(R'), -S(=O)R', -S(=O)NR, -S(=O)R', -OP(=O)(OR'), -P(=O)(OR'), -PO=, POH, and -C(=O)R. and -C(=NR)N(R')2, where each X is independently selected from the group consisting of a halogen: -F, -CI, -Br, and -I; and where each R' is independently selected from the group consisting of -H, -CI-C 20Alkyl, -C6-C 20 Aryl, -C3-C 14 It is selected from the group consisting of a heterocycle, a protecting group, and a prodrug moiety.

[0195] More typically, the substituents are selected from the group consisting of -X, -R', -OH, -OR', -SR', -N(R'), -N(R'), =NR', -NR'C(=O)R, -C(=O)R', -C(=O)N(R'), -S(=O)R', -S(=O)NR', ​​-S(=O)R', -C(=O)R', -C(=S)R, -C(=O)N(R'), -C(=S)N(R'), and -C(=NR)N(R'), where each X is independently -F and and -CI, or selected from the group consisting of -X, -R, -OH, -OR', -N(R')2, -N(R')3, -NR'C(=O)R', -C(=O)N(R')2, -S(=O)2R', -S(=O)2NR', -S(=O)R', -C(=O)R', -C(=O)N(R')2, -C(=NR)N(R')2, protecting groups, and prodrug moieties, wherein each X is -F and wherein each R' is independently hydrogen, -C-C 20 Alkyl, -C6-C 20 Aryl, -C3-C 14 In some embodiments, the alkyl substituent is selected from the group consisting of -N(R')2, -N(R')3, and -C(=NR)N(R')2, where R is selected from the group consisting of hydrogen and -C1-C 20 In other embodiments, the alkyl is substituted with a series of ethyleneoxy moieties to define the PEG unit. The aforementioned alkylene, carbocycle, carbocyclo, arylene, heteroalkyl, heteroalkylene, heterocycle, heterocyclo, heteroaryl, and heteroarylene groups may also be similarly substituted.

[0196] As used herein, a "protecting group" means a moiety that prevents or reduces the ability of the atom or functional group to which it is attached to participate in undesired reactions. Exemplary protecting groups for atoms or functional groups are described in Greene (1999), "Protective Groups In Organic Synthesis, 3 RD Ed.”, Wiley Interscience. Protecting groups for heteroatoms such as oxygen, sulfur, and nitrogen are sometimes used to minimize or avoid their undesired reactions with electrophilic compounds. In other cases, protecting groups are used to reduce or eliminate the nucleophilicity and / or basicity of the unprotected heteroatom. Non-limiting examples of protected oxygens include -OR PR where R PR is a protecting group for hydroxyl, which is typically protected as an ester (e.g., acetate, propionate, or benzoate). Other protecting groups for hydroxyl avoid interfering with the nucleophilicity of organometallic or other highly basic reagents; hydroxyl is typically protected as an ether, including alkyl or heterocycloalkyl ethers (e.g., methyl or tetrahydropyranyl ethers), alkoxymethyl ethers (e.g., methoxymethyl or ethoxymethyl ethers), optionally substituted aryl ethers, and silyl ethers (e.g., trimethylsilyl (TMS), triethylsilyl (TES), tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl (TBS / TBDMS), triisopropylsilyl (TIPS), and [2-(trimethylsilyl)ethoxy]-methylsilyl (SEM)). Nitrogen protecting groups include -NHR PR or -N(R PR ) 2-, which is for primary or secondary amines, PR at least one of R is a nitrogen atom protecting group, or PR together contain a protecting group.

[0197] A protecting group is suitable if it can prevent or avoid undesired side reactions or premature loss of the protecting group under the reaction conditions required to effect a desired chemical transformation elsewhere in the molecule, and, if necessary, during purification of the newly formed molecule, and can be removed under conditions that do not adversely affect the structural or stereochemical integrity of the newly formed molecule. By way of example and not limitation, suitable protecting groups can include those described above for protecting functional groups. Suitable protecting groups can also be those used in peptide coupling reactions.

[0198] "Aromatic alcohol" refers to an aromatic ring system substituted with a hydroxyl functional group -OH, either by itself or as part of a larger structure. Thus, aromatic alcohol refers to any aryl, heteroaryl, arylene, and heteroarylene moiety described herein that has a hydroxyl functional group bonded to an aromatic carbon of the aromatic ring system. The aromatic alcohol may be part of a larger moiety, such as when the aromatic ring system is a substituent of the moiety, or may be embedded in the larger moiety by ring fusion, and may optionally be substituted with one or more other hydroxyl substituents as described herein. Phenolic alcohol is an aromatic alcohol that has a phenol group as the aromatic ring.

[0199] "Aliphatic alcohol" refers, by itself or as part of a larger structure, to a moiety having a non-aromatic carbon bonded to a hydroxyl functional group -OH. The hydroxy-containing carbon may be unsubstituted (i.e., methyl alcohol) or may have one, two, or three optionally substituted branched or unbranched alkyl substituents to define a primary alcohol, or a secondary or tertiary aliphatic alcohol within a linear or cyclic structure. When part of a larger structure, the alcohol may be a substituent of the structure by being bonded to the hydroxy-containing carbon through the hydroxy-containing carbon, through a carbon of an alkyl or other moiety described herein, or through a substituent of the alkyl or other moiety. Aliphatic alcohol contemplates non-aromatic cyclic structures (i.e., optionally substituted carbocyclic and hetero-carbocyclic rings) in which the hydroxy functional group is bonded to a non-aromatic carbon of the cyclic ring system.

[0200] As used herein, "arylalkyl" or "heteroarylalkyl" means a substituent, moiety, or group in which an aryl moiety is linked to an alkyl moiety, i.e., aryl-alkyl-, where the alkyl and aryl groups are as described above, e.g., CH-CH- or CH-CH(CH)CH-. alkyl or heteroarylalkyl is a group having sp in the alkyl portion. 3 Related to a larger structure or moiety through carbon.

[0201] As used herein, a "succinimide moiety" refers to an organic moiety comprised of a succinimide ring system, which is typically present in one type of Y' in a compound of Formula (III), further comprised of an alkylene-containing moiety attached to the imide nitrogen of the ring system. The succinimide moiety typically results from the Michael addition of a thiol group of a cell-binding agent to the maleimide ring system of a camptothecin payload compound (Formula II). Thus, the succinimide moiety is comprised of a thio-substituted succinimide ring system, and when present in a camptothecin conjugate, the imide nitrogen is replaced with the remainder of the cell-binding agent of the camptothecin conjugate and optionally with a substituent that was present on the maleimide ring system of the compound of Formula II.

[0202] As used herein, "acid-amide moiety" refers to a succinic acid having an amide substituent resulting from the thio-substituted succinimide ring system of the succinimide moiety, which has undergone hydrolysis to cleave one of its carbonyl-nitrogen bonds. Hydrolysis resulting in the succinic acid-amide moiety provides a linker that is less likely to suffer premature loss of the antibody-linked linker via elimination of the thio substituent. Hydrolysis of the succinimide ring system of the thio-substituted succinimide moiety is expected to result in regiochemical isomers of the acid-amide moiety due to differences in the reactivity of the two carbonyl carbons of the succinimide ring system, which are at least partially due to any substituents present in the maleimide ring system of the compound of Formula II and the thio substituent introduced by the targeting ligand.

[0203] As used herein, the term "prodrug" refers to a less biologically active or inactive compound that is transformed into a more biologically active compound in the body via a chemical or biological process (i.e., a chemical reaction or an enzymatic biotransformation). Typically, a biologically active compound is rendered less biologically active (i.e., converted to a prodrug) by chemically modifying the compound with a prodrug moiety. In some embodiments, the prodrug is a Type II prodrug, which is bioactivated extracellularly, e.g., in digestive fluids, or in the body's circulatory system, e.g., in blood. Exemplary prodrugs are esters and (β-D-glucopyranosides).

[0204] In many cases, the conjugates, linkers, and component assemblies described herein refer to reactive groups. A "reactive group" or RG is a group containing a reactive site (RS) that can form a bond with either a camptothecin payload or a linker component of a camptothecin conjugate, or camptothecin. RS is the reactive site within the reactive group (RG). Reactive groups include thiol groups that form disulfide or thioether bonds, aldehydes, ketones, or hydrazine groups that form hydrazone bonds, carboxylic acids or amino groups that form peptide bonds, carboxylic acids or hydroxy groups that form ester bonds, sulfonic acids that form sulfonamide bonds, alcohols that form carbamate bonds, and amines that form sulfonamide or carbamate bonds. The following table illustrates exemplary functional groups that can be formed after the reaction of reactive groups, reactive sites, and reactive sites. This table is not intended to be limiting. Those skilled in the art will understand that the R' and R" moieties described in the table are effectively any organic moiety (e.g., alkyl, aryl, heteroaryl, or substituted alkyl, aryl, or heteroaryl) compatible with the bond formation provided upon converting RG to one of the exemplary functional groups. Also, as applied to embodiments of the present invention, R' may optionally represent one or more components of a self-stabilizing linker or optional secondary linker, and R' may optionally represent one or more components of an optional secondary linker. It will be understood that the term may represent one or more components of the camptothecin, the stabilizing unit, or the detection unit. [Table 13]

[0205] Combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds. As used herein, the term "stable" refers to compounds that have sufficient stability to permit their manufacture and that maintain their integrity for a sufficient period of time to be useful for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject).

[0206] After their preparation, the compounds of the present invention are preferably isolated and purified to obtain compositions containing 95% or more by weight ("substantially pure"), which can then be used or formulated as described herein.

[0207] As used herein, the term "conjugate" refers to a compound described herein or a derivative thereof linked to a cell binding agent.

[0208] As used herein, the term "linkable to a cell binding agent" refers to a compound or derivative thereof that includes at least one linking group or precursor thereof suitable for linking the compound or derivative thereof described herein to a cell binding agent.

[0209] The term "precursor" of a given group refers to any group that can be connected to that group by any deprotection, chemical modification, or coupling reaction.

[0210] The term "linked to a cell binding agent" refers to a conjugated molecule comprising at least one of the compounds described herein, or a derivative thereof, attached to a cell binding agent via a suitable linking group or precursor thereof.

[0211] The terms "abnormal cell growth" and "proliferative disorder" are used interchangeably in this application. As used herein, unless otherwise indicated, "abnormal cell growth" refers to cell growth that is independent of normal regulatory mechanisms (e.g., loss of contact inhibition). This includes, for example, the abnormal growth of (1) tumor cells (tumors) that grow due to the expression of mutant tyrosine kinases or the overexpression of receptor tyrosine kinases, (2) benign and malignant cells of other proliferative diseases in which abnormal tyrosine kinase activation occurs, (3) any tumors that grow due to receptor tyrosine kinases, (4) any tumors that grow due to abnormal serine / threonine kinase activation, and (5) benign and malignant cells of other proliferative diseases in which abnormal serine / threonine kinase activation occurs.

[0212] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" contains one or more cancerous cells and / or benign or pre-cancerous cells.

[0213] "Therapeutic agents" include both biological agents such as antibodies, peptides, proteins, enzymes, etc., or chemotherapeutic agents.

[0214] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer.

[0215] A "metabolite" is a product produced through metabolism in the body of a particular compound, its derivative, or conjugate, or its salt. Metabolites of a compound, its derivative, or conjugate may be identified using routine techniques known in the art, and their activity may be determined using tests such as those described herein. Such products may result, for example, from oxidation, hydroxylation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, etc., of the administered compound. Accordingly, the present invention includes metabolic products of a compound, its derivative, or conjugate of the present invention, including compounds, its derivative, or conjugate produced by a process comprising contacting the compound, its derivative, or conjugate with a mammal for a period of time sufficient to yield a metabolic product thereof.

[0216] As defined herein, a "linker," "linker moiety," or "linking group" refers 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 can first react with a cytotoxic compound to provide a compound having a linker moiety and a second reactive group, which can then react with a cell-binding agent. Alternatively, one end of the bifunctional crosslinker can first react with a cell-binding agent to provide a cell-binding agent having 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 and include 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). Disulfide bonds, thioethers, and peptidase labile bonds are preferred. Other linkers that may be used in the present invention include non-cleavable linkers, such as those described and detailed in U.S. Publication No. 2005 / 0169933, or charged or hydrophilic linkers, and are described in U.S. Publication No. US 2009 / 0274713, US 2010 / 01293140, and WO 2009 / 134976, each of which is expressly incorporated herein by reference.

[0217] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, selinocystiene, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., carbons bonded to hydrogen, carboxyl groups, amino groups, and R groups, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. One amino acid that can be particularly used is citrulline, a derivative of arginine that is involved in the formation of urea in the liver. Amino acid mimetics A non-naturally occurring amino acid refers to a compound that has a structure different from the general chemical structure of an amino acid but functions in a manner similar to a naturally occurring amino acid. The term "unnatural amino acid" is intended to refer to the "D" stereochemical form of the 20 naturally occurring amino acids listed above. It is further understood that the term non-naturally occurring amino acid includes homologs of natural amino acids or their D isomers, as well as synthetically modified forms of natural amino acids. Synthetically modified forms include, but are not limited to, amino acids with side chains shortened or lengthened by up to two carbon atoms, amino acids containing optionally substituted aryl groups, amino acids containing halogenated groups, preferably halogenated alkyl and aryl groups, and N-substituted amino acids, such as N-methyl-alanine. An amino acid or peptide can be attached to a linker / spacer or cell-binding agent through the terminal amine or terminal carboxylic acid of the amino acid or peptide. An amino acid can also be attached to a linker / spacer or cell-binding agent through a side chain reactive group, such as, but not limited to, the thiol group of cysteine, the epsilon amine of lysine, or the side chain hydroxyl of serine or threonine.

[0218] In embodiments, the amino acid is NH2-C(R aa 'R aaa )—C(═O)OH, where R aa and R aa Each R' is independently H, an optionally substituted linear, branched, or cyclic alkyl, alkenyl, or alkynyl having 1 to 10 carbon atoms, aryl, heteroaryl, or heterocyclyl, or R 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)O- refers to the corresponding residue when one hydrogen atom is removed from the amine and / or a hydroxyl group is removed from the carboxy terminus of an amino acid, such as -C(=O)O-.

[0219] As used herein, amino acids may be L or D isomers. Unless otherwise specified, when an amino acid is referred to, it may be L or D isomer or a mixture thereof. In embodiments, when a peptide is referred to by its amino acid sequence, each amino acid may be L or D isomer unless otherwise specified. If one of the amino acids in a peptide is designated as a D isomer, the other amino acids are L isomers unless otherwise specified. For example, peptide D-Ala-Ala means D-Ala-L-Ala.

[0220] Amino acids and peptides can be protected by blocking groups. Blocking groups are atoms or chemical moieties that protect the N-terminus of amino acids or peptides from undesired reactions and can be used during the synthesis of drug-ligand conjugates. They should remain attached to the N-terminus throughout the entire synthesis, and can be removed after the completion of the synthesis of drug conjugates by chemical or other conditions that selectively achieve their removal. Blocking groups suitable for N-terminal protection are well known in the art of peptide chemistry. Exemplary blocking groups include, but are not limited to, methyl ester, tert-butyl ester, 9-fluorenylmethylcarbamate (Fmoc), and carbobenzoxy (Cbz).

[0221] The term "protease-cleavable peptide" refers to a peptide containing a protease cleavage recognition sequence. As used herein, a protease is an enzyme capable of cleaving a peptide bond. A protease cleavage recognition sequence is a specific amino acid sequence recognized by a protease during proteolytic cleavage. Many protease cleavage sites are known in the art, and these and other cleavage sites can be included in the linker moiety. For example, see Matayoshi et al. Science 247:954 (1990), Dunn et al. Meth. Enzymol. 241:254 (1994), Seidah et al. Meth. Enzymol. 244:175 (1994), Thornberry, Meth. Enzymol. 244:615 ( 1994), Weber et al.Meth.Enzymol.244:595(1994), Smith et al.Meth.Enzymol.244:412(1994), Bouvier et al.Meth.Enzymol.248:614(1995)Hardy et al,AMYLOID PROTEIN PRECURSOR IN DEVELOPMENT,AGING,AND See ALZHEIMER'S DISEASE, ed. Masters et al. pp. 190-198 (1994).

[0222] The peptide sequence is selected based on its ability to be cleaved by a protease, non-limiting examples of which include cathepsin B, C, D, H, L, and S, and furin. Preferably, the peptide sequence is capable of being cleaved in vitro by a suitable isolated protease, which can be tested using in vitro protease cleavage assays known in the art.

[0223] In another embodiment, peptide sequence is selected based on its ability to be cut by lysosomal protease.Lysosomal protease is the protease that is mainly located in lysosome, but can also be located in endosome.Examples of lysosomal proteases include but are not limited to cathepsin B, C, D, H, L and S and furin.

[0224] In another embodiment, the peptide sequence is selected based on its ability to be cleaved by tumor-associated proteases, such as proteases found on the surface of cancerous cells or extracellularly in the vicinity of tumor cells, including, but not limited to, thimet oligopeptidase (TOP), CD10 (neprilysin), matrix metalloproteases (such as MMP2 or MMP9), type II transmembrane serine proteases (hepsin, testisin, TMPRSS4, or matriptase / MT-SP1), legumain, and enzymes described in the following references (Current Topics in Developmental Biology: Cell Surface Proteases, vol. 54 Zucker S. 2003, Boston, MA). The ability of a peptide to be cleaved by a tumor-associated protease can be tested using in vitro protease cleavage assays known in the art.

[0225] The term "cation" refers to an ion with a positive charge. A cation is a monovalent ion (e.g., Na + , K. + etc.), divalent (e.g., Ca 2+ , Mg2+ ), or multivalent (e.g., Al 3+ etc. In embodiments, the cation is monovalent.

[0226] Compounds of formula (I) In some aspects, the invention features metabolites, including camptothecin derivatives, that can exhibit desirable cytotoxic properties and can be used to prepare conjugates that include the cell-binding agents described herein.

[0227] In one aspect, the present invention provides a compound of formula (I): D-L1-L2-Q (I) or a pharmaceutically acceptable salt thereof, wherein: D has the following structural formula: [ka] wherein: R 1 are independently -H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, silyl, C3-C6 cycloalkyl, C1-C6 halogenated alkyl, C2-C6 halogenated alkenyl, or C2-C6 halogenated alkynyl; R 2 are independently -H, -F, -N(R 4 )2, -N(R 4 )(R 5 ), -OR 4 , -SR 4 , -S(=O)R 5 , -SO2R 5 , C1-C6 alkyl, or C1-C6 fluoroalkyl; R 3 are independently -H, -F, -CN, -OCH3, -CH3, or -CF3, or R 2 and R 3 together, the formula -O(CH2) n O- or -O(CF2) n O- group, where n is 1 or 2, R 4are independently —H or C1-C4 alkyl; R 5 are independently C1-C4 alkyl; L1 can independently be absent or -(C1-C 10 alkylene)-, L2 is independently absent, -OCH2-L3-*, -SCH2-L3-*, -S(=O)-L3-*, -SO2-L3-*, -C(=O)-L3-*, -N(R 6 )CH2-L3-*, -N(R 6 )C(=O)-L3-*, -N(R 6 )C(=O)N(R 7 )-L3-*, -C(=O)N(R 6 )CH2-L3-*, -OC(=O)N(R 6 )CH2-L3-* or -N(R 6 )C(=O)OCH2-L3-*, where * indicates the site of covalent attachment to Q; L3 is independently -(C1-C 10 alkylene)-, -CH2OCH2CH2-, or -CH2CH2OCH2CH2-; Each R 6 and R 7 are independently —H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; Q is —OH or —SH.

[0228] In embodiments, R 2 and R 3 When they combine to form -OCHO-, R 1 is not -CH2CH2CH2CH3.

[0229] In embodiments, R 1 is -H or -CH2CH3, and R 2 is —OH or alkoxy, and R 3is -H, then -L1-L2-Q is not -CH(R')CH2OH or -CH(R')(CH2)2OH, where R' is -H, or C1-C6 alkyl, alkoxy, substituted alkyl, phenyl, or PhCH2-. In embodiments, R 1 is -H or -CH2CH3, and R 2 is —OH or alkoxy, and R 3 is —H, then —L1-L2-Q is not —CH(R′)CH2OH or —CH(R′)(CH2)2OH, where R′ is —H, or C1-C6 alkyl, alkoxy, substituted alkyl, phenyl, or PhCH2—.

[0230] In embodiments, R 1 , R 2 , and R 3 At least one of is not -H.

[0231] In an embodiment, at least one of L1 and L2 is present.

[0232] In embodiments, R 1 is independently C1-C6 alkyl, silyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C1-C6 alkyl halide, alkene, or alkyne.

[0233] In embodiments, R 1 are independently --H or C1-C6 alkyl.

[0234] In embodiments, R 2 are independently -H, -F, -N(R 4 )2, -N(R 4 )(R 5 ), -OR 4 , -SR 4 , -S(=O)R 5 , -SO2R 5 , C1-C6 alkyl, or C1-C6 fluoroalkyl; R 3 are independently --H, --F, --CN, --OCH3, --CH3, or --CF3.

[0235] In embodiments, R 2 is independently C1-C6 alkyl, C1-C6 fluoroalkyl, or --F.

[0236] In embodiments, R 3 are independently -H, -F, -CN, or -CF3.

[0237] In embodiments, R 3 are independently -F, -CN, -OCH3, -CH3, or -CF3.

[0238] In embodiments, R 2 and R 3 is bonded to -O(CH2) n O- or --O(CF2) n O-, where n is 1 or 2.

[0239] In embodiments, D is represented by one of the following structures: [Table 14]

[0240] In an embodiment, D is: [Table 15]

[0241] In embodiments, D is (DI). In embodiments, D is (D-III). In embodiments, D is (D-IV).

[0242] In embodiments, D is (DV). In embodiments, D is (D-VI). In embodiments, D is (D-VII). In embodiments, D is (D-VIII).

[0243] In embodiments, R 1 is —H or C1-C6 alkyl.

[0244] In embodiments, D is represented by one of the following structures: [Table 16]

[0245] In an embodiment, D is: [Table 17]

[0246] In embodiments, D is (D1). In embodiments, D is (D2). In embodiments, D is (D4).

[0247] In embodiments, D is (D5). In embodiments, D is (D6). In embodiments, D is (D7). In embodiments, D is (D8).

[0248] In embodiments, D is (D9). In embodiments, D is (D10). In embodiments, D is (D11). In embodiments, D is (D12).

[0249] In embodiments, D is (D13). In embodiments, D is (D14). In embodiments, D is (D15). In embodiments, D is (D16).

[0250] In embodiments, L is -(C 10 alkylene)-, and L2 is absent.

[0251] In embodiments, L is -(C 10 alkylene)- and L2 is -N(R 6 )CH2-L3-* or -N(R 6 )C(=O)-L3-*, where * indicates the site of covalent attachment to Q.

[0252] In embodiments, L1 is absent and L2 is -N(R 6 )CH2-L3-* or -N(R 6 )C(=O)-L3-*, where * indicates the site of covalent attachment to Q.

[0253] In embodiments, L3 is -(C1-C 10 alkylene).

[0254] In embodiments, R 6 is -H or -CH3.

[0255] In embodiments, L1-L2 is -CH2-, -CH2CH2-, -CH2CH2CH2-, or -CH2CH2CH2CH2-.

[0256] In an embodiment, L1-L2 are selected from the group consisting of -OCH2CH2-*, -OCH2CH2OCH2CH2-*, -SCH2CH2-*, -SCH2CH2OCH2CH2-*, -S(=O)CH2-*, -SO2CH2-*, -C(=O)CH2-*, -NHCH2CH2-*, -N(CH3)CH2CH2-*, -N(CF3)CH2CH2-*, -NHC(=O)CH2-*, -CH2NHC(=O)CH2-*, -CH2CH2NHC(=O)CH2-*, CH2N(CH3)C(=O)CH2-*, -N(CH3)C(=O)CH2-*, -N(CH3)C(=O)CH2CH2-*, -C(=O)NHCH2CH2-*, -NHC(=O)NHCH2CH2-*, -NHC(=O)OCH2CH2-*, -CH2OC(=O)NHCH2CH2-*, or -C(=O)N(CH3)CH2CH2-*, where * indicates the site of covalent attachment to Q.

[0257] In embodiments, L1-L2-Q is -CH2CH2CH2CH2OH, -CH2CH2CH2OH, -CH2CH2OH, -CH2CH2OCH2CH2OH, -CH2SCH 2CH2OH, -CH2NHC(=O)CH2OH, -CH2CH2NHC(=O)CH2OH, -CH2N(CH3)C(=O)CH2OH, -OCH2CH2OH, -OCH2CH2CH2OH, -SCH2CH2CH2OH, -SCH2 CH2OH, -NHCH2CH2OH, -NHCH2CH2CH2OH, -N(CH3)CH2CH2OH, -C(=O)NHCH2CH2OH, -NHC(=O)CH2OH, -CH2S(=O)CH2OH, -CH2SO2CH2OH, -C H2CH2CH2CH2SH, -CH2CH2CH2SH, -CH2CH2SH, -CH2CH2OCH2CH2SH, -CH2SCH2CH2SH, -CH2NHC(=O)CH2SH, -OCH2CH2CH2SH, -SCH2CH2CH2 SH, -SCH2CH2SH, -NHCH2CH2CH2SH, -N(CH3)CH2CH2SH, -C(=O)NHCH2CH2SH, -NHC(=O)CH2SH, -CH2S(=O)CH2SH, or -CH2SO2CH2SH.

[0258] In an embodiment, D-L1-L2 is represented by the structure: [Table 18]

[0259] In an embodiment, D-L1-L2 is represented by the structure: [Table 19]

[0260] In embodiments, D-L1-L2 is represented by the structure (PI).

[0261] In an embodiment, D-L1-L2 is represented by the structure (P-III).

[0262] In an embodiment, D-L1-L2 is represented by the structure (P-IV).

[0263] In an embodiment, D-L1-L2 is represented by the structure (PV).

[0264] In embodiments, R 1 is —H or C1-C6 alkyl.

[0265] In embodiments, R 1 is -H or -CH2CH3.

[0266] In embodiments, Q is —OH.

[0267] In embodiments, Q is —SH.

[0268] In embodiments, the compound has one of the following structures: [Table 20] or a pharmaceutically acceptable salt thereof.

[0269] In embodiments, the compound is [Table 21] or a pharmaceutically acceptable salt thereof.

[0270] In an embodiment, the compound is compound P2, or a pharmaceutically acceptable salt thereof.

[0271] In embodiments, the compound is compound P3, or a pharmaceutically acceptable salt thereof.

[0272] In embodiments, the compound is compound P4, or a pharmaceutically acceptable salt thereof.

[0273] In an embodiment, the compound is compound P5, or a pharmaceutically acceptable salt thereof.

[0274] In an embodiment, the compound is compound P6, or a pharmaceutically acceptable salt thereof.

[0275] Compound of formula (II) In some embodiments, metabolites, including camptothecin derivatives, can include a peptide linker and a reactive group. Such compounds can be useful in preparing conjugates that include the cell-binding agents described herein.

[0276] In embodiments, such compounds are formed from or include a structure according to any embodiment of formula (I) described herein.

[0277] In another aspect, the present invention provides a compound of formula (II): D-L1-L2-Q'-CH2-NH-EZ (II) or a pharmaceutically acceptable salt thereof, wherein: D has the following structural formula: [ka] wherein: R 1 are independently -H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, silyl, C3-C6 cycloalkyl, C1-C6 halogenated alkyl, C2-C6 halogenated alkenyl, or C2-C6 halogenated alkynyl; R 2 are independently -H, -F, -N(R 4 )2, -N(R 4 )(R 5 ), -OR 4 , -SR 4 , -S(=O)R 5 , -SO2R 5 , C1-C6 alkyl, or C1-C6 fluoroalkyl; R 3 is -H, -F, -CN, -OCH3, -CH3, -CF3, or R 2 and R 3 together, the formula -O(CH2) n O- or -O(CF2) n O- group, where n is 1 or 2, R4 are independently —H or C1-C4 alkyl; R 5 are independently C1-C4 alkyl; L1 can independently be absent or -(C1-C 10 alkylene)-, L2 is independently absent, -OCH2-L3-*, -SCH2-L3-*, -S(=O)-L3-*, -SO2-L3-*, -C(=O)-L3-*, -N(R 6 )CH2-L3-*, -N(R 6 )C(=O)-L3-*, -N(R 6 )C(=O)N(R 7 )-L3-*, -C(=O)N(R 6 )CH2-L3-*, -OC(=O)N(R 6 )CH2-L3-* or -N(R 6 )C(=O)OCH2-L3-*, where * indicates the site of covalent attachment to Q'; L3 is independently -(C1-C 10 alkylene)-, -CH2OCH2CH2-, or -CH2CH2OCH2CH2-; Each R 6 and R 7 are independently —H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; Q' is -O- or -S-; E is a peptide comprising 2 to 10 amino acids, wherein E is optionally substituted with one or more polyols, and the N-terminus of the peptide is covalently linked to Z; Z is -C(=O)-L4-Y, [ka] wherein m represents an integer of 1 to 10; L4 is -(C1-C 10 Alkylene)-*, -CH2CH2(OCH2CH2) n N(R 8 )C(=O)-L5-*, or -CH2(OCH2CH2) n N(R 8 )C(═O)—L5-*, wherein n represents an integer of 1 to 10, and * represents the site of covalent bonding to Y; L5 is -(C1-C 10 alkylene)-, R 8 is -H or -CH3, Y is an electrophilic group; In the formula, R 2 and R 3 When they combine to form -OCHO-, R 1 is not -CH2CH2CH2CH3, or a pharmaceutically acceptable salt thereof.

[0278] In embodiments, E is a peptide of 2, 3, or 4 amino acids, wherein each amino acid in said peptide is an L-amino acid, or at least one amino acid in said peptide is a D-amino acid.

[0279] In embodiments, E comprises one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, wherein the glutamine or glutamic acid is optionally substituted by a polyol.

[0280] In embodiments, E comprises an amino acid selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, wherein the glutamine or glutamic acid is optionally substituted by a polyol.

[0281] In embodiments, E comprises an amino acid having the following structure: [ka] In the formula, R 9 is —H or C1-C6 alkyl.

[0282] In an embodiment, E includes an amino acid having the following structure. [Chemical formula]

[0283] In an embodiment, E is -Ala-Val-*, -Val-Ala-*, -Gly-Gly-*, -Val-Cit-*, -Cit-Val-*, -Leu-Ala-*, -Ala-Leu-*, -Leu-Cit-*, -Cit-Leu-*, -Leu-Ala-*, -Ala-Leu-*, -Lys-Lys-*, -Ala-Lys-*, -Lys-Ala-*, -Val-Lys-*, -Lys-Val-*, -Tyr-Arg-*, -Arg-Tyr-*, -Arg-Arg-*, -Ala-Ala-*, -Phe-Lys-*, -Lys-Phe-*, -Thr-Thr-*, -Thr-Met-*, -Met-Thr-*, -Met-Tyr-*, -Tyr-Met-*, -Phe-Gln-*, -Gln-Phe-*, -Gly-Ser-*, -Leu-Gln-*, -Gln-Leu-*, -Ser-Ala-*, -Ser-Gly-*, -Val-Thr-*, -Thr-Val-*, -Val-Gln-*, -Ser-Val-*, -Val-Ser-*, -Ala-Met-*, -Met-Ala-*, -Val-Arg-*, -Arg-Val-*, -Phe-Ala-*, -Ala-Phe-*, -Cit-Val-*, -Gln-Val-*, -Phe-Arg-*, -Arg-Phe-*, -Ala-Ala-Ala- * , -Gly-Gly-Gly- * , -Ala-Val-Ala- * , -Gly-Val-Gly- * , -Ala-Val-Gly- * , -Gly-Phe-Lys-*, -Lys-Phe-Gly-*, -Leu-Ala-Leu-*, -Val-Ala-Leu-*, -Leu-Ala-Val-*, -Val-Ala-Val-*, -Ala-Val-Ala-Gly- *, -Gly-Phe-Gly-Gly- * , -Gly-Gly-Phe-Gly-*, -Ala-Val-Gly-Gly-*, -Ala-Ala-Ala-Ala- * , -Ala-Val-Ala-Ala- * , -Ala-Leu-Ala-Leu-*, -Leu-Ala-Leu-Ala-*, -Gly-Phe-Leu-Gly-*, and -Gly-Leu-Phe-Gly-*, where * indicates the N-terminus of the peptide covalently attached to Z.

[0284] In embodiments, E is -L-Ala-D-Val-*, -L-Val-D-Ala-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Val-D-Cit-*, -L-Val-D-Arg-*, -L-Val-D-Cit-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Arg-D-Arg-*, -L-Ala-D-Ala-*, -L-Ala-D-Lys-*, -L-Ala-D-Arg-*, -L-Ala-D-Ala-L-Ala- * , -L-Ala-D-Val-L-Ala- * , -L-Ala-D-Ala-Gly- * , and -L-Ala-D-Val-Gly- * wherein * indicates the N-terminus of the peptide covalently attached to Z.

[0285] In embodiments, -E-NH--CH 2-- has one of the following structures, where * indicates the N-terminus of the peptide covalently attached to Z: [ka]

[0286] In embodiments, L4 is -(C1-C 10 alkylene).

[0287] In embodiments, L4 is -CH2CH2(OCH2CH2)n N(R 8 )C(=O)-L5-* or -CH2(OCH2CH2) n N(R 8 )C(=O)-L5-*, where n represents an integer of 1 to 10, and * represents the site of covalent bonding to Y.

[0288] In embodiments, L4 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, where * indicates the site of covalent attachment to Y.

[0289] In embodiments, Y is a Michael acceptor group, a succinimide, an epoxide, or a halogen.

[0290] In an embodiment, Y is [ka] and In the formula, R 10 and R 11 are each independently H or C1-C3 alkyl.

[0291] In embodiments, Z is -C(=O)-L4-Y.

[0292] In an embodiment, Z is [ka] In embodiments, m is 1. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4. In embodiments, m is 5. In embodiments, m is 6. In embodiments, m is 7. In embodiments, m is 8. In embodiments, m is 9. In embodiments, m is 10.

[0293] In an embodiment, Z is [ka] In embodiments, m is 1. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4. In embodiments, m is 5. In embodiments, m is 6. In embodiments, m is 7. In embodiments, m is 8. In embodiments, m is 9. In embodiments, m is 10.

[0294] In an embodiment, Z is: [ka]

[0295] In embodiments, ZE-NH--CH2- has one of the following structures: [ka]

[0296] In embodiments, R 1 is -H or -CH2CH3, and R 2 is —OH or alkoxy, and R 3 is -H, then -L1-L2-Q'- is not -CH(R')CHO- or -CH(R')(CH)O-, where R' is -H, or C1-C6 alkyl, alkoxy, substituted alkyl, phenyl, or PhCH2-. In embodiments, R 1 is -H or -CH2CH3, and R 2 is —OH or alkoxy, and R 3 is —H, then —L1-L2-Q′- is not —CH(R′)CHO— or —CH(R′)(CH)O—, where R′ is —H, or C1-C6 alkyl, alkoxy, substituted alkyl, phenyl, or PhCH2—.

[0297] In an embodiment, at least one of L1 and L2 is present.

[0298] In embodiments, R 1 , R 2 , and R 3 At least one of is not -H.

[0299] In embodiments, R 1 is independently C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, silyl, C3-C6 cycloalkyl, C1-C6 alkyl halide, alkene, or alkyne.

[0300] In embodiments, R 1 are independently --H or C1-C6 alkyl.

[0301] In embodiments, R 2 are independently -H, -F, -N(R 4 )2, -N(R 4 )(R 5 ), -OR 4 , -SR 4 , -S(=O)R 5 , -SO2R 5 , C1-C6 alkyl, or C1-C6 fluoroalkyl; R 3 are independently --H, --F, --CN, --OCH3, --CH3, or --CF3.

[0302] In embodiments, R 2 is independently C1-C6 alkyl, C1-C6 fluoroalkyl, or --F.

[0303] In embodiments, R 3 are independently -H, -F, -CN, or -CF3.

[0304] In embodiments, R 3 are independently -F, -CN, -OCH3, -CH3, or -CF3.

[0305] In embodiments, R 2 and R 3 is bonded to -O(CH2) nO- or --O(CF2) n O-, where n is 1 or 2.

[0306] In embodiments, D is represented by one of the following structures: [Table 22]

[0307] In an embodiment, D is: [Table 23]

[0308] In embodiments, D is (DI). In embodiments, D is (D-III). In embodiments, D is (D-IV).

[0309] In an embodiment, D is (DV). In an embodiment, D is (D-VI). In an embodiment, D is (D-VII). In an embodiment, D is (D-VIII).

[0310] In embodiments, R 1 is —H or C1-C6 alkyl.

[0311] In embodiments, D is represented by one of the following structures: [Table 24]

[0312] In an embodiment, D is: [Table 25]

[0313] In embodiments, D is (D1). In embodiments, D is (D2). In embodiments, D is (D4).

[0314] In embodiments, D is (D5). In embodiments, D is (D6). In embodiments, D is (D7). In embodiments, D is (D8).

[0315] In embodiments, D is (D9). In embodiments, D is (D10). In embodiments, D is (D11). In embodiments, D is (D12).

[0316] In embodiments, D is (D13). In embodiments, D is (D14). In embodiments, D is (D15). In embodiments, D is (D16).

[0317] In embodiments, L is -(C 10 alkylene)-, and L2 is absent.

[0318] In embodiments, L is -(C 10 alkylene)- and L2 is -N(R 6 )CH2-L3-* or -N(R 6 )C(=O)-L3-*, where * indicates the site of covalent attachment to Q'.

[0319] In embodiments, L1 is absent and L2 is -N(R 6 )CH2-L3-* or -N(R 6 )C(=O)-L3-*, where * indicates the site of covalent attachment to Q'.

[0320] In embodiments, L3 is -(C1-C 10 alkylene).

[0321] In embodiments, R 6 is -H or -CH3.

[0322] In embodiments, L1-L2 is -CH2-, -CH2CH2-, -CH2CH2CH2-, or -CH2CH2CH2CH2-.

[0323] In an embodiment, L1-L2 is -OCH2CH2-*, -OCH2CH2OCH2CH2-*, -SCH2CH2-*, -SCH2CH2OCH2CH2-*, -S(=O)CH2-*, -SO2CH2-*, -C(=O)CH2-*, -NHCH2CH2-*, -N(CH3)CH2CH2-*, -N(CF3)CH2CH2-*, -NHC(=O)CH2-*, -CH2NHC(=O)CH2-*, -CH2CH2NHC(=O)CH2-*, -CH2N(CH3)C(=O)CH2-*, -N(CH3)C(=O)CH2-*, -N(CH3)C(=O)CH2CH2-*, -C(=O)NHCH2CH2-*, -NHC(=O)NHCH2CH2-*, -NHC(=O)OCH2CH2-*, -CH2OC(=O)NHCH2CH2-*, or -C(=O)N(CH3)CH2CH2-*, where * indicates the site of covalent attachment to Q'.

[0324] In embodiments, L1-L2-Q' is -CH2CH2CH2CH2O-, -CH2CH2CH2O-, -CH2CH2O-, -CH2CH2OCH2CH2O-, -CH2SC H2CH2O-, -CH2NHC(=O)CH2O-, -CH2CH2NHC(=O)CH2O-, -CH2N(CH3)C(=O)CH2O-, -OCH2CH2O-, -OC H2CH2CH2O-, -SCH2CH2CH2O-, -SCH2CH2O-, -NHCH2CH2O-, -NHCH2CH2CH2O-, -N(CH3)CH2CH2O-, - C(=O)NHCH2CH2O-, -NHC(=O)CH2O-, -CH2S(=O)CH2O-, -CH2SO2CH2O-, -CH2CH2CH2CH2S-, -CH2CH 2CH2S-, -CH2CH2S-, -CH2CH2OCH2CH2S-, -CH2SCH2CH2S-, -CH2NHC(=O)CH2S-, -OCH2CH2CH2S-, -SCH2CH2CH2S-, -SCH2CH 2S-, -NHCH2CH2CH2S-, -N(CH3)CH2CH2S-, -C(=O)NHCH2CH2S-, -NHC(=O)CH2S-, -CH2S(=O)CH2S-, or -CH2SO2CH2S-.

[0325] In an embodiment, D-L1-L2 is represented by the structure: [Table 26]

[0326] In an embodiment, D-L1-L2 is represented by the structure: [Table 27]

[0327] In embodiments, D-L1-L2 is represented by the structure (PI).

[0328] In an embodiment, D-L1-L2 is represented by the structure (P-III).

[0329] In an embodiment, D-L1-L2 is represented by the structure (P-IV).

[0330] In an embodiment, D-L1-L2 is represented by the structure (PV).

[0331] In embodiments, R 1 is —H or C1-C6 alkyl.

[0332] In embodiments, R 1 is -H or -CH2CH3.

[0333] In embodiments, Q' is -O-.

[0334] In embodiments, Q' is -S-.

[0335] In embodiments, D-L1-L2-Q'- has one of the following structures: [Table 28]

[0336] In an embodiment, D-L1-L2-Q'- is: [Table 29]

[0337] In an embodiment, D-L1-L2-Q'- is (P2').

[0338] In an embodiment, D-L1-L2-Q'- is (P3').

[0339] In an embodiment, D-L1-L2-Q'- is (P4').

[0340] In an embodiment, D-L1-L2-Q'- is (P5').

[0341] In an embodiment, D-L1-L2-Q'- is (P6').

[0342] In embodiments, the compound has one of the following structures: [ka] [ka] [ka] [ka] or a pharmaceutically acceptable salt thereof.

[0343] In embodiments, the compound is [ka] or a pharmaceutically acceptable salt thereof.

[0344] In embodiments, the compound is [ka] or a pharmaceutically acceptable salt thereof.

[0345] In embodiments, the compound is (PL2), or a pharmaceutically acceptable salt thereof.

[0346] In an embodiment, the compound is (PL4), or a pharmaceutically acceptable salt thereof.

[0347] In an embodiment, the compound is (PL5), or a pharmaceutically acceptable salt thereof.

[0348] In an embodiment, the compound is (PL6), or a pharmaceutically acceptable salt thereof.

[0349] In an embodiment, the compound is (PL7), or a pharmaceutically acceptable salt thereof.

[0350] In an embodiment, the compound is (PL8), or a pharmaceutically acceptable salt thereof.

[0351] In an embodiment, the compound is (PL9), or a pharmaceutically acceptable salt thereof.

[0352] In an embodiment, the compound is (PL10), or a pharmaceutically acceptable salt thereof.

[0353] In an embodiment, the compound is (PL11), or a pharmaceutically acceptable salt thereof.

[0354] In an embodiment, the compound is (PL12), or a pharmaceutically acceptable salt thereof.

[0355] In an embodiment, the compound is (PL13), or a pharmaceutically acceptable salt thereof.

[0356] In an embodiment, the compound is (PL14), or a pharmaceutically acceptable salt thereof.

[0357] In another aspect, the invention features a method for preparing a conjugate including a cell-binding agent and a drug, the method including contacting the cell-binding agent with a compound of Formula (II) such that a covalent bond is formed between the cell-binding agent and the compound of Formula (II). In embodiments, the conjugate has a structure according to Formula (III), described herein.

[0358] In embodiments, the cell binding agent is an antibody or an antigen-binding fragment thereof.

[0359] In embodiments, the cell binding agent is a monoclonal antibody or an antigen-binding fragment thereof.

[0360] In another aspect, the invention features a conjugate including a cell-binding agent and a drug. In embodiments, the conjugate is prepared according to any method described herein.

[0361] In embodiments, the conjugate comprises a cell binding agent that is an antibody or an antigen-binding fragment thereof.

[0362] In embodiments, the conjugate comprises a cell binding agent that is a monoclonal antibody or an antigen-binding fragment thereof.

[0363] In another aspect, the invention features a pharmaceutical composition including any of the conjugates described herein.

[0364] In yet another aspect, the invention features a method of treating a cell proliferative disease or disorder or inhibiting abnormal cell growth, the method comprising administering any of the conjugates described herein or any pharmaceutical composition comprising any of the conjugates described herein.

[0365] In embodiments, the method is for treating cancer.

[0366] In embodiments, the cancer is adenocarcinoma, brain cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, CNS tumors, colon or colorectal cancer, diffuse pontine glioma (DIPG), endometrial cancer, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, blood cancer, Hodgkin's lymphoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, myelodysplastic syndrome (MDS), neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, pancreatic cancer, peritoneal cancer, prostate cancer, ovarian cancer, renal cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, or Wilms' tumor.

[0367] In embodiments, the cancer is breast cancer.

[0368] Compound of formula (III) In some aspects, the invention features a conjugate comprising a cell-binding agent and a camptothecin derivative. In embodiments, the conjugate moiety comprises a camptothecin derivative. is formed from or includes a structure according to any embodiment of formula (I) or formula (II) described herein.

[0369] In yet a further embodiment, the present invention provides a compound of formula (III): {D-L1-L2-Q'-CH2-NH-E-Z'} p -C (III) or a pharmaceutically acceptable salt thereof, wherein: D has the following structural formula: [ka] wherein: R 1 are independently -H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, silyl, C3-C6 cycloalkyl, C1-C6 halogenated alkyl, C2-C6 halogenated alkenyl, or C2-C6 halogenated alkynyl; R 2 are independently -H, -F, -N(R 4 )2, -N(R 4 )(R 5 ), -OR 4 , -SR 4 , -S(=O)R 5 , -SO2R 5 , C1-C6 alkyl, or C1-C6 fluoroalkyl; R 3 is -H, -F, -CN, -OCH3, -CH3, or -CF3, or R 2 and R 3 together, the formula -O(CH2) n O- or -O(CF2) n O- group, where n is 1 or 2, R 4 are independently —H or C1-C4 alkyl; R 5 are independently C1-C4 alkyl; L1 can independently be absent or -(C1-C 10 alkylene)-, L2 is independently absent, -OCH2-L3-*, -SCH2-L3-*, -S(=O)-L3-*, -SO2-L3-*, -C(=O)-L3-*, -N(R 6 )CH2-L3-*, -N(R 6 )C(=O)-L3-*, -N(R 6 )C(=O)N(R 7 )-L3-*, -C(=O)N(R 6 )CH2-L3-*, -OC(=O)N(R 6 )CH2-L3-* or -N(R 6)C(=O)OCH2-L3-*, where * indicates the site of covalent attachment to Q'; L3 is independently -(C1-C 10 alkylene)-, -CH2OCH2CH2-, or -CH2CH2OCH2CH2-; Each R 6 and R 7 are independently —H, C1-C6 alkyl, C1-C6 fluoroalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or benzyl; Q' is -O- or -S-; E is a peptide comprising 2 to 10 amino acids, wherein E is optionally substituted with one or more polyols, and the N-terminus of the peptide is covalently linked to Z'; Z' is -C(=O)-L4-Y', [ka] In the formula, m represents an integer of 1 to 10, and * represents a site covalently bonded to the C; L4 is -(C1-C 10 Alkylene)-, -CH2CH2(OCH2CH2) n N(R 8 )C(=O)-L5-*, or -CH2(OCH2CH2) n N(R 8 )C(=O) -L5-*, wherein n represents an integer of 1 to 10, and * represents a site covalently bonded to Y'; L5 is -(C1-C 10 alkylene)-, R 8 is -H or -CH3, C represents a cell-binding agent; Y' is a group formed by reaction of an electrophilic group with a reactive nucleophilic group present on the cell-binding agent; In the formula, R 2 and R 3 When they combine to form -OCHO-, R 1 is not -CH2CH2CH2CH3, p has a value of 1 to 18.

[0370] In embodiments, L4 is -(C1-C 10 alkylene).

[0371] In embodiments, L4 is -CH2CH2(OCH2CH2) n N(R 8 )C(=O)-L5-* or -CH2(OCH2CH2) n N(R 8 )C(=O)-L5-*, where n represents an integer of 1 to 10, and * represents the site of covalent bonding to Y'.

[0372] In embodiments, L4 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2-*, where * indicates the site of covalent attachment to Y'.

[0373] In embodiments, Y' is formed from a Michael acceptor group, a succinimide, an epoxide, or a halogen.

[0374] In embodiments, Y' is [ka] In the formula, R 10 and R 11 are each independently -H or C1-C3 alkyl.

[0375] In embodiments, Y' is [ka] where R 10 and R 11 are each independently -H or C1-C3 alkyl, and * indicates the site of covalent bonding to the C.

[0376] In an embodiment, Z' is formed from: [ka]

[0377] In embodiments, Z' is -C(=O)-L4-Y'.

[0378] In embodiments, Z' is [ka] wherein * indicates the site covalently bonded to said C. In embodiments, m is 1. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4. In embodiments, m is 5. In embodiments, m is 6. In embodiments, m is 7. In embodiments, m is 8. In embodiments, m is 9. In embodiments, m is 10.

[0379] In embodiments, Z' is [ka] wherein * indicates the site covalently bonded to said C. In embodiments, m is 1. In embodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4. In embodiments, m is 5. In embodiments, m is 6. In embodiments, m is 7. In embodiments, m is 8. In embodiments, m is 9. In embodiments, m is 10.

[0380] In embodiments, Z' is [ka] and In the formula, * indicates the site of covalent attachment to C.

[0381] In embodiments, E is a peptide of 2, 3, or 4 amino acids, wherein each amino acid in said peptide is an L-amino acid, or at least one amino acid in said peptide is a D-amino acid.

[0382] In embodiments, E comprises one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, wherein the glutamine or glutamic acid is optionally substituted by a polyol.

[0383] In embodiments, E comprises an amino acid selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, wherein the glutamine or glutamic acid is optionally substituted by a polyol.

[0384] In embodiments, E comprises an amino acid having the following structure: [ka] In the formula, R 9 is —H or C1-C6 alkyl.

[0385] In embodiments, E comprises an amino acid having the following structure: [ka]

[0386] In an embodiment, E is selected from the group consisting of -Ala-Val-*, -Val-Ala-*, -Gly-Gly-*, -Val-Cit-*, -Cit-Val-*, -Leu-Ala-*, -Ala-Leu-*, -Leu-Cit-*, -Cit-Leu-*, -Leu-Ala-*, -Ala-Leu-*, -Lys-Lys-*, -Ala-Lys-*, -Lys-Ala-*, -Val-Lys-*, -Lys-Val-*, -Tyr-Arg-*, -Arg-Tyr-*, -Arg-Arg-*, -Ala-Ala-*, -Phe-Lys-*, -Lys-Phe-*, -Thr-Thr-*, -Thr-Met-*, -Met-Thr-*, -Met-Tyr-*, -Tyr-Met-*, -Phe-Gln-*, -Gln-Phe-*, -Gly-Ser-*, -Leu-Gln-*, -Gln-Leu-*, -Ser-Ala-*, -Ser-Gly-*, -Val-Thr-*, -Thr-Val-*, -Val-Gln-*, -Ser-Val-*, -Val-Ser-*, -Ala-Met-*, -Met-Ala-*, -Val-Arg-*, -Arg-Val-*, -Phe-Ala-*, -Ala-Phe-*, -Cit-Val-*, -Gln-Val-*, -Phe-Arg-*, -Arg-Phe-*, -Ala-Ala-Ala * 、-Gly-Gly-Gly * 、-Ala-Val-Ala * 、-Gly-Val-Gly * 、-Ala-Val-Gly * 、 -Gly-Phe-Lys-*, -Lys-Phe-Gly-*, -Leu-Ala-Leu-*, -Val-Ala-Leu-*, -Leu-Ala-Val-*, -Val- Ala-Val-*, -Ala-Val-Ala-Gly * 、-Gly-Phe-Gly-Gly * 、-Gly-Gly-Phe-Gly-*, -Ala-Val-Gly-Gly-*, -Ala-Ala-Ala-Ala * 、-Ala-Val-Ala-Ala * 、-Ala-Leu-Ala-Leu-*, -Leu-Ala-Leu-Ala-*, -Gly-Phe-Leu-Gly-*, and -Gly-Leu-Phe-Gly-* selected from the group consisting of, wherein * represents the N-terminus of the peptide covalently bound to Z'.

[0387] In an embodiment, E is -L-Ala-D-Val-*, -L-Val-D-Ala-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Val-D-Cit-*, -L-Val-D-Arg-*, -L-Val-D-Cit-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Arg-D-Arg-*, -L-Ala-D-Ala-*, -L-Ala-D-Lys-*, -L-Ala-D-Arg-*, -L-Ala-D-Ala-L-Ala- * , -L-Ala-D-Val-L-Ala- * , -L-Ala-D-Ala-Gly- * , and -L-Ala-D-Val-Gly- * wherein * indicates the N-terminus of the peptide covalently attached to Z'.

[0388] In embodiments, -E-NH--CH 2-- has one of the following structures, where * indicates the N-terminus of the peptide covalently attached to Z': [ka]

[0389] In embodiments, Z'-E-NH--CH2 is formed from one of the following structures: [ka]

[0390] In embodiments, Z'-E-NH--CH2 is one of the following structures, where * indicates the point of attachment to C: [ka]

[0391] In embodiments, R 1 is -H or -CH2CH3, and R 2 is —OH or alkoxy, and R 3is -H, then -L1-L2-Q'- is not -CH(R')CHO- or -CH(R')(CH)O-, where R' is -H, or C1-C6 alkyl, alkoxy, substituted alkyl, phenyl, or PhCH2-. In embodiments, R 1 is -H or -CH2CH3, and R 2 is —OH or alkoxy, and R 3 is —H, then —L1-L2-Q′- is not —CH(R′)CHO— or —CH(R′)(CH)O—, where R′ is —H, or C1-C6 alkyl, alkoxy, substituted alkyl, phenyl, or PhCH2—.

[0392] In an embodiment, at least one of L1 and L2 is present.

[0393] In embodiments, R 1 , R 2 , and R 3 At least one of is not -H.

[0394] In embodiments, R 1 is independently C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, silyl, C3-C6 cycloalkyl, C1-C6 alkyl halide, alkene, or alkyne.

[0395] In embodiments, R 1 are independently --H or C1-C6 alkyl.

[0396] In embodiments, R 2 are independently -H, -F, -N(R 4 )2, -N(R 4 )(R 5 ), -OR 4 , -SR 4 , -S(=O)R 5 , -SO2R 5 , C1-C6 alkyl, or C1-C6 fluoroalkyl; R 3are independently --H, --F, --CN, --OCH3, --CH3, or --CF3.

[0397] In embodiments, R 2 is independently C1-C6 alkyl, C1-C6 fluoroalkyl, or --F.

[0398] In embodiments, R 3 are independently -H, -F, -CN, or -CF3.

[0399] In embodiments, R 3 are independently -F, -CN, -OCH3, -CH3, or -CF3.

[0400] In embodiments, R 2 and R 3 is bonded to -O(CH2) n O- or --O(CF2) n O-, where n is 1 or 2.

[0401] In embodiments, D is represented by one of the following structures: [Table 30]

[0402] In an embodiment, D is: [Table 31]

[0403] In embodiments, D is (DI). In embodiments, D is (D-III). In embodiments, D is (D-IV).

[0404] In embodiments, D is (DV). In embodiments, D is (D-VI). In embodiments, D is (D-VII). In embodiments, D is (D-VIII).

[0405] In embodiments, R 1 is —H or C1-C6 alkyl.

[0406] In embodiments, D is represented by one of the following structures: [Table 32]

[0407] In an embodiment, D is: [Table 33]

[0408] In embodiments, D is (D1). In embodiments, D is (D2). In embodiments, D is (D4).

[0409] In embodiments, D is (D5). In embodiments, D is (D6). In embodiments, D is (D7). In embodiments, D is (D8).

[0410] In embodiments, D is (D9). In embodiments, D is (D10). In embodiments, D is (D11). In embodiments, D is (D12).

[0411] In embodiments, D is (D13). In embodiments, D is (D14). In embodiments, D is (D15). In embodiments, D is (D16).

[0412] In embodiments, L is -(C 10 alkylene)-, and L2 is absent.

[0413] In embodiments, L is -(C 10 alkylene)- and L2 is -N(R 6 )CH2-L3-* or -N(R 6 )C(=O)-L3-*, where * indicates the site of covalent attachment to Q'.

[0414] In embodiments, L1 is absent and L2 is -N(R 6 )CH2-L3-* or -N(R 6 )C(=O)-L3-*, where * indicates the site of covalent attachment to Q'.

[0415] In embodiments, L3 is -(C1-C 10 alkylene).

[0416] In embodiments, R 6 is -H or -CH3.

[0417] In embodiments, L1-L2 is -CH2-, -CH2CH2-, -CH2CH2CH2-, or -CH2CH2CH2CH2-.

[0418] In an embodiment, L1-L2 is -OCH2CH2-*, -OCH2CH2OCH2CH2-*, -SCH2CH2-*, -SCH2CH2OCH2CH2-*, -S(=O)CH2-*, -SO2CH2-*, -C(=O)CH2-*, -NHCH2CH2-*, -N(CH3)CH2CH2-*, -N(CF3)CH2CH2-*, -NHC(=O)CH2-*, -CH2NHC(=O)CH2-*, -CH2CH2NHC(=O)CH2-*, -CH2N(CH3)C(=O)CH2-*, -N(CH3)C(=O)CH2-*, -N(CH3)C(=O)CH2CH2-*, -C(=O)NHCH2CH2-*, -NHC(=O)NHCH2CH2-*, -NHC(=O)OCH2CH2-*, -CH2OC(=O)NHCH2CH2-*, or -C(=O)N(CH3)CH2CH2-*, where * indicates the site of covalent attachment to Q'.

[0419] In embodiments, L1-L2-Q' is -CH2CH2CH2CH2O-, -CH2CH2CH2O-, -CH2CH2O-, -CH2CH2OCH2CH2O-, -CH2SCH2CH2O-, -CH2NHC(=O)CH2O-, -CH2CH2NHC(=O)CH2O-, -CH2N (CH3)C(=O)CH2O-, -OCH2CH2O-, -OCH2CH2CH2O-, -SCH2CH2CH2O-, -SCH2CH2O-, -NHCH2CH2O-, -NHCH2CH2CH2O-, -N(CH3)CH2CH2O-, -C(=O)NHCH2CH2O-, -NHC (=O)CH2O-, -CH2S(=O)CH2O-, -CH2SO2CH2O-, -CH2CH2CH2CH2S-, -CH2CH2CH2S-, -CH2CH2S-, -CH2CH2OCH2CH2S-, -CH2SCH2CH2S-, -CH2NHC(=O)CH2S-, -OCH2 CH2CH2S-, -SCH2CH2CH2S-, -SCH2CH2S-, -NHCH2CH2CH2S-, -N(CH3)CH2CH2S-, -C(=O)NHCH2CH2S-, -NHC(=O)CH2S-, -CH2S(=O)CH2S-, or -CH2SO2CH2S-.

[0420] In an embodiment, D-L1-L2 is represented by the structure: [Table 34]

[0421] In an embodiment, D-L1-L2 is represented by the structure: [Table 35]

[0422] In embodiments, D-L1-L2 is represented by the structure (PI).

[0423] In an embodiment, D-L1-L2 is represented by the structure (P-III).

[0424] In an embodiment, D-L1-L2 is represented by the structure (P-IV).

[0425] In an embodiment, D-L1-L2 is represented by the structure (PV).

[0426] In embodiments, R 1 is —H or C1-C6 alkyl.

[0427] In embodiments, R 1 is -H or -CH2CH3.

[0428] In embodiments, Q' is -O-.

[0429] In embodiments, Q' is -S-.

[0430] In embodiments, D-L1-L2-Q'- has one of the following structures: [Table 36]

[0431] In an embodiment, D-L1-L2-Q'- is: [Table 37]

[0432] In an embodiment, D-L1-L2-Q'- is (P2').

[0433] In an embodiment, D-L1-L2-Q'- is (P3').

[0434] In an embodiment, D-L1-L2-Q'- is (P4').

[0435] In an embodiment, D-L1-L2-Q'- is (P5').

[0436] In an embodiment, D-L1-L2-Q'- is (P6').

[0437] In embodiments, D-L1-L2-Q'-CH2-NH-E-Z'- is formed from one of the following structures: [ka] [ka] [ka]

[0438] In embodiments, the compound is [ka] or a pharmaceutically acceptable salt thereof.

[0439] In embodiments, the compound is [ka] or a pharmaceutically acceptable salt thereof.

[0440] In embodiments, the compound is (PL2), or a pharmaceutically acceptable salt thereof.

[0441] In an embodiment, the compound is (PL4), or a pharmaceutically acceptable salt thereof.

[0442] In an embodiment, the compound is (PL5), or a pharmaceutically acceptable salt thereof.

[0443] In an embodiment, the compound is (PL6), or a pharmaceutically acceptable salt thereof.

[0444] In an embodiment, the compound is (PL7), or a pharmaceutically acceptable salt thereof.

[0445] In an embodiment, the compound is (PL8), or a pharmaceutically acceptable salt thereof.

[0446] In an embodiment, the compound is (PL9), or a pharmaceutically acceptable salt thereof.

[0447] In an embodiment, the compound is (PL10), or a pharmaceutically acceptable salt thereof.

[0448] In an embodiment, the compound is (PL11), or a pharmaceutically acceptable salt thereof.

[0449] In an embodiment, the compound is (PL12), or a pharmaceutically acceptable salt thereof.

[0450] In an embodiment, the compound is (PL13), or a pharmaceutically acceptable salt thereof.

[0451] In an embodiment, the compound is (PL14), or a pharmaceutically acceptable salt thereof.

[0452] In an embodiment, {D-L1-L2-Q'-CH2-NH-E-Z'} p -C is one of the following structures, where C is a monoclonal antibody, p is the drug-to-antibody ratio (DAR), and p is an average number ranging from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0). [ka] [ka] [ka]

[0453] In an embodiment, {D-L1-L2-Q'-CH2-NH-E-Z'} p -C is [ka] where C is a monoclonal antibody and p is the drug-to-antibody ratio (DAR). In embodiments, p is an average number ranging from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).

[0454] In an embodiment, {D-L1-L2-Q'-CH2-NH-E-Z'} p -C is [ka] where C is a monoclonal antibody and p is the drug-to-antibody ratio (DAR). In embodiments, p is an average number ranging from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).

[0455] In an embodiment, {D-L1-L2-Q'-CH2-NH-E-Z'} p -C is (PL2'), where C is a monoclonal antibody and p is the drug-to-antibody ratio (DAR). In embodiments, p is an average number ranging from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).

[0456] In an embodiment, {D-L1-L2-Q'-CH2-NH-E-Z'} p -C is (PL4'), where C is a monoclonal antibody and p is the drug-to-antibody ratio (DAR). In embodiments, p is an average number ranging from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).

[0457] In an embodiment, {D-L1-L2-Q'-CH2-NH-E-Z'} p -C is (PL5'), where C is a monoclonal antibody and p is the drug-to-antibody ratio (DAR). In embodiments, p is an average number ranging from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).

[0458] In an embodiment, {D-L1-L2-Q'-CH2-NH-E-Z'} p-C is (PL6'), where C is a monoclonal antibody and p is the drug-to-antibody ratio (DAR). In embodiments, p is an average number ranging from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).

[0459] In an embodiment, {D-L1-L2-Q'-CH2-NH-E-Z'} p -C is (PL7'), where C is a monoclonal antibody and p is the drug-to-antibody ratio (DAR). In embodiments, p is an average number ranging from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).

[0460] In an embodiment, {D-L1-L2-Q'-CH2-NH-E-Z'} p -C is (PL8'), where C is a monoclonal antibody and p is the drug-to-antibody ratio (DAR). In embodiments, p is an average number ranging from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).

[0461] In an embodiment, {D-L1-L2-Q'-CH2-NH-E-Z'} p -C is (PL9'), where C is a monoclonal antibody and p is the drug-to-antibody ratio (DAR). In embodiments, p is an average number ranging from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).

[0462] In an embodiment, {D-L1-L2-Q'-CH2-NH-E-Z'} p -C is (PL10'), where C is a monoclonal antibody and p is the drug-to-antibody ratio (DAR). In embodiments, p is an average number ranging from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).

[0463] In an embodiment, {D-L1-L2-Q'-CH2-NH-E-Z'} p-C is (PL11'), where C is a monoclonal antibody and p is the drug-to-antibody ratio (DAR). In embodiments, p is an average number ranging from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).

[0464] In an embodiment, {D-L1-L2-Q'-CH2-NH-E-Z'} p -C is (PL12'), where C is a monoclonal antibody and p is the drug-to-antibody ratio (DAR). In embodiments, p is an average number ranging from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).

[0465] In an embodiment, {D-L1-L2-Q'-CH2-NH-E-Z'} p -C is (PL13'), where C is a monoclonal antibody and p is the drug-to-antibody ratio (DAR). In embodiments, p is an average number ranging from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).

[0466] In an embodiment, {D-L1-L2-Q'-CH2-NH-E-Z'} p -C is (PL14'), where C is a monoclonal antibody and p is the drug-to-antibody ratio (DAR). In embodiments, p is an average number ranging from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).

[0467] In another aspect, the invention features a method for preparing a conjugate of Formula (III) including a cell-binding agent and a drug, the method including contacting the cell-binding agent with a compound of Formula (II) such that a covalent bond is formed between the cell-binding agent and the compound of Formula (II).

[0468] In yet another aspect, the invention features a conjugate including a cell-binding agent and a drug. In embodiments, the conjugate is prepared according to any method described herein.

[0469] In embodiments, the conjugate comprises a cell binding agent that is an antibody or an antigen-binding fragment thereof.

[0470] In embodiments, the conjugate comprises a cell binding agent that is a monoclonal antibody or an antigen-binding fragment thereof.

[0471] In embodiments, the cell binding agent is an antibody or antigen-binding fragment thereof, and p is the drug-to-antibody ratio (DAR) and has a value of 1 to 18. In embodiments, p is an average number ranging from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).

[0472] In embodiments, the cell binding agent is a monoclonal antibody or antigen-binding fragment thereof, and p is the drug-to-antibody ratio (DAR) and has a value of 1 to 18. In embodiments, p is an average number ranging from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).

[0473] In another aspect, the invention features a pharmaceutical composition including any of the conjugates described herein.

[0474] In yet another aspect, the invention features a method of treating a cell proliferative disease or disorder or inhibiting abnormal cell growth, the method comprising administering any of the conjugates described herein or any pharmaceutical composition comprising any of the conjugates described herein.

[0475] In another aspect, the invention features a pharmaceutical composition including any compound of Formula (III) described herein, or a pharmaceutically acceptable salt thereof.

[0476] In another aspect, the invention features a method of treating a cell proliferative disease or disorder or inhibiting abnormal cell growth, the method comprising administering any compound of Formula (III) described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising any compound of Formula (III) described herein or a pharmaceutically acceptable salt thereof.

[0477] In embodiments, the method is for treating cancer.

[0478] In embodiments, the cancer is adenocarcinoma, brain cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, CNS tumors, colon or colorectal cancer, diffuse pontine glioma (DIPG), endometrial cancer, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, blood cancer, Hodgkin's lymphoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, myelodysplastic syndrome (MDS), neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, pancreatic cancer, peritoneal cancer, prostate cancer, ovarian cancer, renal cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, or Wilms' tumor.

[0479] In embodiments, the cancer is breast cancer.

[0480] Subscript "p" The conjugates described herein (e.g., any compound according to formula (III)) can include the covalent attachment of at least a camptothecin derivative (e.g., any compound according to formula (II) described herein, such as that formed from any compound according to formula (I) described herein).

[0481] In embodiments, the subscript p represents the number of camptothecin payload moieties (e.g., as formed from a compound according to Formula (II)) on the cell-binding agent and has a value of 1 to 18, 1 to 12, or 1 to 8. An individual camptothecin conjugate may also be referred to as a camptothecin-conjugate compound. In embodiments herein, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 camptothecin payload moieties conjugated to the cell-binding agent of an individual camptothecin conjugate.

[0482] In embodiments, the populations of individual camptothecin conjugates are substantially identical except for the number of camptothecin payload moieties attached to each cell-binding agent (i.e., camptothecin conjugate composition), such that p represents the average number of camptothecin payload moieties attached to the cell-binding agents of the camptothecin conjugate composition. In that group of embodiments, p is 1 to about 18, 1 to about 10, or 1 to about 8, 2 to about 6, 3 to about 5, or or an average number ranging from 6 to about 8. In embodiments, p is an average number ranging from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0). In embodiments, p is about 2. In embodiments, p is about 4. In embodiments, p is about 6. In embodiments, p is about 8. In embodiments, p is about 10. In embodiments, p is about 12. In embodiments, p is 2. In embodiments, p is 4. In embodiments, p is 8. In embodiments, p has a value of 3 to 4. In embodiments, p has a value of 4 to 5. In embodiments, p has a value of 5 to 6. In embodiments, p has a value of 6 to 7. In embodiments, p has a value of 7 to 8. In embodiments, p has a value of 7.4 to 8. In embodiments, the p value refers to the average drug loading as well as the drug loading of the primary ADC in the composition.

[0483] In embodiments, the conjugation (e.g., as found in any compound according to Formula (III) described herein) can be from about 1 to about 8, or 3 to 5, or 6 to 8 camptothecin payload compounds (e.g., any compound according to Formula (II) described herein or formed from a compound of Formula (I) described herein) conjugated to a cell-binding agent via reduced interchain disulfides.

[0484] In embodiments, conjugation (e.g., as found in any compound according to formula (III) described herein) can occur via introduced cysteine ​​residues and reduced interchain disulfides, with 1 to 8, or 1 to 10, or 1 to 12, or 1 to 18 camptothecin payload compounds (e.g., any compound according to formula (II) described herein or formed from a compound of formula (I) described herein) conjugated to a cell-binding agent.

[0485] In embodiments, the conjugation (e.g., as found in any compound according to formula (III) described herein) is via an introduced cysteine ​​residue, and there are two, four, six, or eight camptothecin payload compounds (e.g., any compound according to formula (II) described herein or formed from a compound of formula (I) described herein) conjugated to a cell-binding agent.

[0486] In embodiments, conjugation (e.g., as found in any compound according to Formula (III) described herein) is via a lysine residue, and there can be 1 to 10, or 1 to 12, or 1 to 14, or 1 to 18 camptothecin payload compounds (e.g., any compound according to Formula (II) described herein or formed from a compound of Formula (I) described herein) conjugated to a cell-binding agent.

[0487] Reactive groups on cell-binding agents for covalent attachment In embodiments, a cell-binding agent is attached to a peptide releasable linker of a compound of formula (II) to form a conjugate such as one according to formula (III). As mentioned above, additional linking moieties in formula (II) can be present in the conjugates described herein to serve the purpose of providing additional space between the camptothecin compound and the cell-binding agent. In embodiments, the cell-binding agent is attached to the linker unit in formula (II) via a heteroatom of the cell-binding agent.

[0488] Heteroatoms that may be present on the cell-binding agent for attachment include sulfur (in one embodiment, from a thiol group of the targeting ligand), oxygen (in one embodiment, from a carboxyl or hydroxyl group of the targeting ligand), and optionally substituted nitrogen (in one embodiment, from a primary or secondary amine functional group of the targeting ligand, or in another embodiment, from an optionally substituted amide nitrogen). These heteroatoms may be present on the targeting ligand in its native state of the cell-binding agent, for example, in a naturally occurring antibody, or Alternatively, it may be introduced into the targeting ligand via chemical modification or biological engineering.

[0489] In one embodiment, the cell-binding agent has a thiol functional group such that the cell-binding agent is attached to a camptothecin payload compound (e.g., any compound according to formula (II) described herein or formed from a compound of formula (I) described herein) via the sulfur atom of the thiol functional group.

[0490] In another embodiment, the cell-binding agent has one or more lysine residues that can react with an activated ester (such esters include, but are not limited to, N-hydroxysuccinimide, pentafluorophenyl, and p-nitrophenyl esters) of a camptothecin payload compound (e.g., any compound according to formula (II) described herein or formed from a compound of formula (I) described herein), thus providing an amide bond consisting of a nitrogen atom of the cell-binding agent and a C=O group of the compound of formula (II).

[0491] In yet another aspect, the cell-binding agent has one or more lysine residues that can be chemically modified to introduce one or more thiol groups. In these embodiments, the cell-binding agent is covalently attached to a camptothecin payload compound (e.g., any compound according to Formula (II) described herein or formed from a compound of Formula (I) described herein) via the sulfur atom of the thiol functional group. Reagents that can be used to modify lysines in this manner include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-iminothiolane hydrochloride (Traut's reagent).

[0492] In another embodiment, the cell-binding agent has one or more carbohydrate groups that can be modified to provide one or more thiol functional groups. The chemically modified cell-binding agent in the camptothecin conjugate is attached to a camptothecin payload compound (e.g., any compound according to Formula (II) described herein or formed from a compound of Formula (I) described herein) via the sulfur atom of the thiol functional group.

[0493] In yet another embodiment, the cell-binding agent has one or more carbohydrate groups that can be oxidized to provide an aldehyde (—CHO) functional group (see, e.g., Laguzza, et al., 1989, J. Med. Chem. 32(3):548-55). In these embodiments, the corresponding aldehyde interacts with a reactive site on the camptothecin payload compound (e.g., according to Formula (II) described herein or in any compound formed from the compound of Formula (I) described herein) to form a bond between the camptothecin payload compound (e.g., according to Formula (II) described herein or in any compound formed from the compound of Formula (I) described herein) and the cell-binding agent. Reactive sites on the camptothecin payload compound (e.g., according to Formula (II) described herein or in any compound formed from the compound of Formula (I) described herein) that can interact with a reactive carbonyl-containing functional group on the targeting ligand include, but are not limited to, hydrazine and hydroxylamine.

[0494] In some aspects, the cell-binding agent can form a bond by interacting with a reactive functional group Y (e.g., in any compound according to Formula (II)) to form a covalent bond between Y' in Formula (III) and the cell-binding agent corresponding to the targeting ligand. The functional group Y capable of interacting with the targeting ligand depends on the nature of the cell-binding agent. In embodiments, the reactive group is a maleimide present on the camptothecin payload compound prior to its attachment to form the cell-binding agent. Covalent attachment of the cell-binding agent to the camptothecin payload compound can be achieved by reacting the payload compound (e.g., a compound according to Formula (II) described herein) with a maleimide group. or in any compound formed from a compound of Formula (I) described herein) to form a thio-substituted succinimide. The thiol functionality may be present on the cell-binding agent in its native state, e.g., in a naturally occurring residue, or may be introduced into the cell-binding agent via chemical modification or by biological engineering.

[0495] In yet another embodiment, the cell-binding agent is an antibody and the thiol group is generated by reduction of the antibody's interchain disulfide. Thus, in embodiments, a camptothecin payload compound is conjugated to a cysteine ​​residue from the reduced interchain disulfide.

[0496] In yet another embodiment, the cell-binding agent is an antibody and the thiol functional group is chemically introduced into the antibody, for example, by the introduction of a cysteine ​​residue. Thus, in embodiments, the camptothecin payload compound is conjugated to the cell-binding agent via the introduced cysteine ​​residue of the cell-binding agent.

[0497] It has been observed that the site of drug conjugation for bioconjugation can affect many parameters, including ease of conjugation, drug-linker stability, the impact on the biophysical properties of the resulting bioconjugate, and in vitro cytotoxicity. Regarding drug-linker stability, the site of conjugation of the drug-linker moiety to the cell-binding agent can affect the ability of the conjugated drug-linker moiety to undergo elimination reactions, potentially causing premature release of the free drug. Sites of conjugation on targeting ligands include, for example, reduced interchain disulfides and selected cysteine ​​residues at engineered sites. In embodiments, the conjugation method for forming camptothecin conjugates described herein uses a thiol residue at an engineered site (e.g., position 239 according to the EU index as described in Kabat) that is less susceptible to elimination reactions than conjugation methods that use thiol residues from reduced disulfide bonds. In other embodiments, the conjugation methods for forming camptothecin conjugates described herein use thiol residues at sites more susceptible to elimination reactions (e.g., resulting from interchain disulfide reduction).

[0498] Cell-binding agent (C) In embodiments of the invention, a cell-binding agent is present that acts to target and present the camptothecin or drug component containing the camptothecin to a specific target cell population with which the cell-binding agent interacts, due to the presence of its targeting component or molecule, allowing for subsequent release of free drug within (i.e., intracellularly) or in the vicinity of (i.e., extracellularly) the target cell.

[0499] In embodiments, the cell binding agent may be a ligand that binds to a moiety on a target cell, such as a cell surface receptor. In embodiments, the ligand may be a growth factor or a fragment thereof that binds to a growth factor receptor. In embodiments, the ligand may be a cytokine or a fragment thereof that binds to a cytokine receptor. In embodiments, the growth factor receptor or cytokine receptor is a cell surface receptor.

[0500] Thus, the therapeutic use of camptothecin conjugates (eg, compounds according to formula (III) described herein) may be followed by the appropriate selection of a cell binding agent.

[0501] Cell binding agents include, but are not limited to, proteins, polypeptides, and peptides. Suitable cell binding agents include, for example, antibodies (e.g., polyclonal antibodies and Examples of cell-binding agents include antibodies, including full-length antibodies and antigen-binding fragments thereof, including monoclonal antibodies, interferons, lymphokines, hormones, growth factors, colony-stimulating factors, vitamins (e.g., folate), nutrient transport molecules (e.g., but not limited to, transferrin), or any other cell-binding molecule or substance. In embodiments, the cell-binding agent is an antibody or a non-antibody protein targeting agent.

[0502] Antigens targeted by cell-binding agents In embodiments, exemplary antigens or ligands include renin, growth hormone (eg, human growth hormone and bovine growth hormone), growth hormone releasing factor, parathyroid hormone, or thyroid stimulating hormone, or fragments thereof.

[0503] In embodiments, exemplary antigens or ligands include lipoproteins, alpha-1-antitrypsin, insulin A chain, insulin B chain, proinsulin, follicle-stimulating hormone, calcitonin, luteinizing hormone, or glucagon, or fragments thereof.

[0504] In embodiments, exemplary antigens or ligands include coagulation factors (e.g., factor VMC, factor IX, tissue factor, and von Willebrand factor), anticoagulants (e.g., protein C), atrial natriuretic factor, pulmonary surfactant, plasminogen activators (e.g., urokinase, human urinary or tissue-type plasminogen activator), bombesin, thrombin, or hematopoietic growth factors, or fragments thereof.

[0505] In embodiments, exemplary antigens or ligands include tumor necrosis factor-alpha and -beta, or fragments thereof.

[0506] In embodiments, exemplary antigens or ligands include enkephalinase, RANTES (i.e., regulated on activation, normal T cell expressed and secreted), human macrophage inflammatory protein-1-alpha, serum albumin (human serum albumin), Müllerian inhibiting substance, relaxin A chain, relaxin B chain, prorelaxin, mouse gonadotropin-related peptide, microbial protein (beta-lactamase), DNase, IgE, inhibin, or activin, or fragments thereof.

[0507] In embodiments, exemplary antigens or ligands include cytotoxic T lymphocyte-associated antigens (eg, CTLA-4) or fragments thereof.

[0508] In embodiments, exemplary antigens or ligands include vascular endothelial growth factor or fragments thereof.

[0509] In embodiments, exemplary antigens or ligands include receptors for hormones or growth factors, protein A or D, rheumatoid factor, neurotrophic factors (e.g., bone-derived neurotrophic factor, neurotrophin-3, -4, -5, or -6), nerve growth factor (e.g., NGF-b), platelet-derived growth factor, fibroblast growth factors (e.g., aFGF and bFGF), fibroblast growth factor receptor 2, epidermal growth factor, transforming growth factors (e.g., TGF-alpha, TGF-bI, TGF-p2, TGF-p3, TGF-p4, and TGF-p5), insulin-like growth factor-I and -II, des(1-3)-IGF-I (brain IGF-I), or insulin-like growth factor binding protein, or fragments thereof.

[0510] In embodiments, exemplary antigens or ligands include melanotransferrin, CA6, CAK1, CALLA, CAECAM5, GD3, FLT3, PSMA, PSCA, MU C1, STEAP, CEA, TENB2, EphA receptor, EphB receptor, folate receptor, FOLR1, mesothelin, Crypto, alpha v beta6, or integrin, or fragments thereof.

[0511] In embodiments, exemplary antigens or ligands include VEGF or VEGFR, or fragments thereof.

[0512] In embodiments, exemplary antigens or ligands include EGFR or fragments thereof.

[0513] In embodiments, exemplary antigens or ligands include FGFR3, LAMP1, p-cadherin, or transferrin receptor, or fragments thereof.

[0514] In embodiments, exemplary antigens or ligands include IRTA1, IRTA2, IRTA3, IRTA4, IRTA5, or fragments thereof.

[0515] In embodiments, exemplary antigens or ligands include tyrosine-protein kinase transmembrane receptors (eg, ROR1 and ROR2), or fragments thereof.

[0516] In embodiments, exemplary antigens or ligands include CD proteins (e.g., CD2, CD3, CD4, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80, CD81, CD103, CD105, CD123, CD134, CD137, CD138, CD152, and CD276), or fragments thereof.

[0517] In embodiments, exemplary antigens or ligands include one or more tumor-associated antigens or cell surface receptors (see U.S. Publication No. 2008 / 0171040 or U.S. Publication No. 2008 / 0305044, which are incorporated by reference in their entireties), or fragments thereof.

[0518] In embodiments, exemplary antigens or ligands include erythropoietin or fragments thereof.

[0519] In embodiments, exemplary antigens or ligands include osteoinductive factors or fragments thereof.

[0520] In embodiments, exemplary antigens or ligands include immunotoxins or fragments thereof.

[0521] In embodiments, exemplary antigens or ligands include bone morphogenetic proteins or fragments thereof.

[0522] In embodiments, exemplary antigens or ligands include interferons (eg, interferon-alpha, -beta, and -gamma).

[0523] In embodiments, exemplary antigens or ligands include colony stimulating factors (eg, M-CSF, GM-CSF, and G-CSF), or fragments thereof.

[0524] In embodiments, exemplary antigens or ligands include interleukins (e.g., IL- IL-1 to IL-10), or fragments thereof.

[0525] In embodiments, exemplary antigens or ligands include superoxide dismutase or fragments thereof.

[0526] In embodiments, exemplary antigens or ligands include T cell receptors or fragments thereof.

[0527] In embodiments, exemplary antigens or ligands include surface membrane proteins or fragments thereof.

[0528] In embodiments, exemplary antigens or ligands include decay-accelerating factors or fragments thereof.

[0529] In embodiments, exemplary antigens or ligands include viral antigens (eg, portions of the HIV envelope), or fragments thereof.

[0530] In embodiments, exemplary antigens or ligands include transport proteins or fragments thereof.

[0531] In embodiments, exemplary antigens or ligands include homing receptors or fragments thereof.

[0532] In embodiments, exemplary antigens or ligands include addressins or fragments thereof.

[0533] In embodiments, exemplary antigens or ligands include regulatory proteins or fragments thereof.

[0534] In embodiments, exemplary antigens or ligands include integrins (e.g., CD11a, CD11b, CD11c, CD18, ICAM, VLA-4, and VCAM), or fragments thereof.

[0535] In embodiments, exemplary antigens or ligands include tumor-associated antigens (eg, HER2, HER3, and HER4 receptors), or fragments thereof.

[0536] In embodiments, exemplary antigens or ligands include endoglin, c-Met, c-kit, 1GF1R, PSGR, NGEP, PSMA, PSCA, TMEFF2, LGR5, B7H4, TROP-2, DLL-3, CDH6, AXL, SLITRK6, ENPP3, BCMA, tissue factor, or CD352, or fragments thereof.

[0537] In embodiments, the cell binding agent is selected from the group consisting of Apo2, BAFF-R, bone morphogenetic protein receptor, IGF-IR, CA125, CanAg, E16, ErbB2, MUC1, MUC16, Napi3b, TF, EpCAM, FcRH2, C242, CD2, CD3, CD4, CD5, CD6, CD11, CD18, CD19, CD20, CD21, CD22, CD26, CD30, CD33, CD37, CD38 , CD40, CD44, CD56, CD70, CD72, CD79, CD90, CD138, CRIPTO, CXCR5, LY64, TDGF1, endothelin B receptor, EphA receptor, EphB receptor, endothelin, FCRH1, HER2, HER2 / neu, HER3, MHC class II molecule Ia antigen, integrin, IRTA2, LIV-1, MPF, NaPi2b, PDL1, FLJ10 372, KIAA1445, Mm42015, SEMA5B, SEMAG, prostate 6 transmembrane epithelial antigen 1, IPCA-1, PCANP1, STMP, prostate antigen, insulin growth factor receptor, or folate receptor.

[0538] In embodiments, the cell binding agent targets GPNMB, NCAM (CD56), TACSTD2 (TROP-2), folate receptor alpha, tissue factor, ENPP3, CD70, P-cadherin, mesothelin, STEA1, CEACAM5, mucin 1, nectin 4, guanylyl cyclase C, SLC44A4, PSMA, LIV1 (ZIP6), SLITRK6, 5T4, or SC-16.

[0539] In embodiments, the cell binding agent targets HER2 or EGFR.

[0540] In embodiments, the cell binding agent targets fibronectin extra-domain B (EDB), endothelial receptor ETB, PSMA, VEGFR2 (CD309), tissue factor, or ROBO4.

[0541] In embodiments, the cell binding agent targets collagen IV, periostin, or tenascin c.

[0542] In embodiments, the cell binding agent targets CD30, CD22, CD79b, CD19, CD138, CD74, CD37, CD33, CD19, or CD98.

[0543] In embodiments, the cell binding agent targets HER2.

[0544] In embodiments, the cell binding agent targets EGFR.

[0545] In embodiments, the cell binding agent targets CD70.

[0546] In embodiments, the cell binding agent targets CD33.

[0547] In embodiments, the cell binding agent targets CD30.

[0548] In embodiments, the cell binding agent targets CD22.

[0549] In embodiments, the cell binding agent targets CD19.

[0550] In embodiments, the cell binding agent targets Mucl.

[0551] In embodiments, the cell binding agent targets CD37.

[0552] In embodiments, the cell binding agent targets CD123.

[0553] Non-protein cell-binding agents In embodiments, the cell-binding agent is not a protein. For example, in embodiments, the cell-binding agent may be a vitamin that binds to a vitamin receptor, such as a cell surface receptor. In this regard, vitamin A binds to retinol-binding protein (RBP) to form a complex, which then binds to the STRA6 receptor with high affinity, increasing vitamin A uptake. In another example, folic acid / folate / vitamin B9 binds to cell surface folate receptors (FRs), such as FRa, with high affinity. Folic acid or antibodies that bind to FRa can be used to target folate receptors expressed on ovarian and other tumors. Furthermore, vitamin Vitamin D and its analogs bind to the vitamin D receptor.

[0554] Protein and Polypeptide Cell-Binding Agents In other embodiments, the cell binding agent is a protein or polypeptide, including an antibody, a non-antibody protein, or a polypeptide, or a compound that includes a protein or polypeptide.

[0555] In embodiments, the cell binding agent may be a lymphokine, hormone, growth factor, colony-stimulating factor, or nutrient transport molecule.

[0556] In embodiments, GM-CSF, a ligand / growth factor that binds to myeloid cells, may be used as a cell binding agent to diseased cells from acute myeloid leukemia.

[0557] In embodiments, IL-2, which binds to activated T cells, may be used to prevent transplant rejection, treat and prevent graft-versus-host disease, and treat acute T-cell leukemia.

[0558] In embodiments, MSH, which binds to melanocytes, can be used to treat melanoma, as can antibodies directed against melanoma.

[0559] In embodiments, epidermal growth factor can be used to target squamous cell carcinomas, such as lung, and head and neck, and gingival squamous cell carcinomas.

[0560] In embodiments, somatostatin can be used to target neuroblastoma and other tumor types.

[0561] In embodiments, estrogen (or estrogen analogs) can be used to target breast cancer.

[0562] In embodiments, androgens (or androgen analogs) can be used to target the testes.

[0563] In embodiments, the cell binding agent is an ankyrin repeat protein, centirin, or an antibody mimetic such as an adnectin / monobody.

[0564] In embodiments, the camptothecin conjugate includes a non-immunoreactive protein, polypeptide, or peptide as its cell-binding agent. Thus, in embodiments, the cell-binding agent is a non-immunoreactive protein, polypeptide, or peptide. Examples include, but are not limited to, transferrin, epidermal growth factor ("EGF"), bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, transforming growth factors ("TGF") such as TGF-α and TGF-β, vaccinia growth factor ("VGF"), insulin and insulin-like growth factors I and II, somatostatin, lectins, and apoproteins derived from low-density lipoproteins.

[0565] Antibodies and related cell-binding agents In embodiments, the cell binding agent is an antibody or an antigen-binding fragment thereof. In any of the embodiments described herein, the cell binding agent may be an antibody.

[0566] In embodiments, when the cell binding agent is an antibody or antigen-binding portion thereof (including antibody derivatives), or a specific antibody mimetic, the cell binding agent can bind to a ligand on a target cell, such as a cell surface ligand including a cell surface receptor.

[0567] Suitable antibodies also include, but are not limited to, human antibodies, primatized antibodies, chimeric antibodies, bispecific antibodies, humanized antibodies, conjugated antibodies (i.e., antibodies conjugated or fused to other proteins, radiolabels, cytotoxins), Small Modular ImmunoPharmaceuticals ("SMIPs™"), and antibody fragments.

[0568] For example, antibodies include immunoglobulins (Ig) and fragments thereof, which are specifically reactive with designated proteins or peptides, or fragments thereof. In embodiments, antibodies include intact monoclonal antibodies, polyclonal antibodies, single domain antibodies (e.g., shark single domain antibodies (e.g., IgNAR or fragments thereof)), and antibody fragments, so long as they exhibit the desired biological activity. In embodiments, the antibody is IgG, IgA, IgE, IgD, or IgM. In embodiments, the antibody is IgG1, IgG2, IgG3, or IgG4. In embodiments, the antibody is IgA1 or IgA2.

[0569] In embodiments, the cell binding agent is a resurfaced antibody, a resurfaced single chain antibody, a resurfaced antibody fragment (or "antigen-binding portion"), or a bispecific antibody.

[0570] In embodiments, the cell binding agent is a minibody, avibody, diabody, tribody, tetrabody, nanobody, probody, domain antibody, or unibody.

[0571] Antibody fragments can include portions of intact antibodies, such as the antigen-binding or variable regions of the antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, triabodies, tetrabodies, linear antibodies, and single-chain antibody molecules. Antibody fragments can also be any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. For example, antibody fragments can include isolated fragments, "Fv" fragments consisting of the variable regions of the heavy and light chains, recombinant single-chain polypeptide molecules in which the variable regions of the light and heavy chains are connected by a peptide linker ("ScFv proteins"), and minimal recognition units consisting of amino acid residues mimicking the hypervariable regions.

[0572] Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of immunized animals. Useful monoclonal antibodies are homogeneous populations of antibodies against a particular antigenic determinant (e.g., a cancer cell antigen, a viral antigen, a microbial antigen, a protein, a peptide, a carbohydrate, a chemical, a nucleic acid, or a fragment thereof). Monoclonal antibodies (mAbs) against an antigen of interest can be prepared using any technique known in the art that provides for the production of antibody molecules by continuous cell lines in culture.

[0573] In embodiments, the cell binding agent is a monoclonal antibody or an antigen-binding fragment thereof.

[0574] Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, or chimeric human-mouse (or other species) monoclonal antibodies. Antibodies include full-length antibodies and antigen-binding fragments thereof. Human monoclonal antibodies can be produced by any of a number of techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; and Olsson et al., 1982, Meth. Enzymol. 92:3-16).

[0575] In embodiments, antibodies suitable for the present invention may include humanized or human antibodies. Humanized forms of non-human antibodies are chimeric Igs, Ig chains, or fragments (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of Abs) that contain minimal sequence derived from non-human Igs. Generally, humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, typically taken from an "import" variable domain. Humanization is achieved by substituting rodent complementarity-determining regions (CDRs) or CDR sequences for the corresponding sequences of a human antibody (Riechmann et al., Nature 332(6162):323-7, 1988; Verhoeyen et al., Science. 239(4847):1534-6, 1988). Such "humanized" antibodies are chimeric antibodies in which substantially less intact human variable domains have been replaced by corresponding sequences from a non-human species (U.S. Pat. No. 4,816,567, 1989). In embodiments, the CDRs of a non-human antibody (e.g., murine) targeting a human antigen are grafted onto the framework regions of the variable domain of a human Ig. Various techniques known in the art are suitable for CDR grafting, including, for example, site-directed mutagenesis. In embodiments, humanized antibodies are typically human antibodies in which some CDR residues, and possibly some FR residues, have been replaced by residues from analogous sites in a rodent Ab. Humanized antibodies include human Igs (recipient antibodies) in which residues from the recipient CDRs have been replaced by residues from CDRs of a non-human species (donor antibody) such as mouse, rat, or rabbit with the desired specificity, affinity, and capacity. In embodiments, the monospecific and bispecific antibodies described herein are cross-reactive with non-human primate common antigens. In some instances, corresponding non-human residues replace Fv framework residues of the human Ig. Humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, with most, if not all, of the CDR regions corresponding to those of a non-human Ig, and most, if not all, of the FR regions being those of a human Ig consensus sequence. Optimally, the humanized antibody will also comprise at least a portion of an Ig constant region (Fc), typically that of a human Ig (Riechmann et al., Nature 332(6162):323-7, 1988; Verhoeyen et al., Science. 239(4847):1534-6, 1988).

[0576] Human antibodies can also be produced using a variety of techniques, including phage display libraries (Hoogenboom et al., Mol Immunol. (1991) 28(9):1027-37; Marks et al., J Mol Biol. (1991) 222(3):581-97), and preparation of human monoclonal antibodies (Reisfeld and Sell, 1985, Cancer Surv. 4(1):271-90). Similarly, human antibodies can be synthesized using introduction of human Ig genes into transgenic animals in which the endogenous Ig genes have been partially or completely inactivated. After challenge, human antibody production was observed, which closely resembled that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire (Fishwild et al., High-avidity human IgG kappa monoclonal antibodies from a novel strain of minilocus transgenic mice, Nat Biotechnol. 1996 July;14(7):845-51; Lonberg et al., Antigen-specific human antibodies from mice comprising four distinct genetic modifications, Nature 1994 April 28;368(6474):856-9; Lonberg and Huszar, Human antibodies from transgenic mice, Int. Rev. Immuno l.1995;13(1):65-93, Marks et al., By-passing immunization: building high affinity human antibodies by chain shuffling.Biotechnology(NY).1992 July;10(7):779-83).

[0577] The antibody may be a functionally active fragment, derivative, or analog of an antibody that immunospecifically binds to a target cell (e.g., a cancer cell antigen, a viral antigen, or a microbial antigen), or to another antibody bound to a tumor cell or substrate. In this context, "functionally active" means that the fragment, derivative, or analog can immunospecifically bind to a target cell. To determine which CDR sequence binds to an antigen, synthetic peptides containing the CDR sequence can be used in binding assays with the antigen by any binding assay method known in the art (e.g., BIAcore assay) (see, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md.; Kabat E et al., 1980, J. Immunology 125(3):961-969).

[0578] Other useful antibodies include fragments of antibodies, such as, but not limited to, F(ab')2 fragments, Fab fragments, Fv, single chain antibodies, diabodies, triabodies, tetrabodies, scFv, scFv-Fv, or any other molecule with the same specificity as an antibody.

[0579] Another form of antibody fragment is the peptide that encodes a single CDR.CDR peptide ("minimal recognition unit") can be obtained by constructing the gene that encodes the CDR of the antibody of interest.For example, such gene is prepared by synthesizing variable region from the RNA of antibody-producing cells using polymerase chain reaction. See, for example, Larrick et al., Methods: A Companion to Methods in Enzymology 2:106 (1991); Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering And Clinical Application, Ritter et al. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles And Applications, Birch et al., (eds.), pages 137-185 (Wiley-Liss, Inc. 1995).

[0580] Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, containing both human and non-human portions, can be produced using standard recombinant DNA techniques and are useful antibodies. Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having a variable region derived from a mouse monoclonal and a human immunoglobulin constant region. (See, e.g., U.S. Pat. Nos. 4,816,567 and 4,816,397, which are incorporated herein by reference in their entireties.) Humanized antibodies are antibody molecules from non-human species that have one or more complementarity-determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., U.S. Pat. No. 5,585,089, which is incorporated herein by reference in its entirety.) Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example, in WO 87 / 02671, EP 0 184 544, and the like. 187, European Patent Publication No. 0 171 496, European Patent Publication No. 0 173 494, International Publication No. 86 / 01533, US Patent No. 4,816,567, European Patent Publication No. 012 023, Berter et al.,1988, Science 240:1041-1043, Liu et al. al.,1987,Proc.Natl.Acad.Sci.USA 84:3439-3443, Liu et al.,1987,J.Immunol.139:3521-3526, Sun et al,1987,Proc.Natl.Acad.Sci.USA 84:214-218, Nishimura et al. al,1987,Cancer.Res.47:999-1005, Wood et al. al, 1985, Nature 314:446-449, and Shaw et al, 1988, J. Natl. Cancer Inst. 80:1553-1559, Morrison, 1985, Science 229:1202-1207, Oi et al, 1986, BioTechniques 4:214, U.S. Patent No. 5,225,539, Jones et al, 1986, Nature 321:552-525, Verhoeyan et al, 1988, Science 239:1534, and Beidler et al, 1988, J. Immunol. 141:4053-4060 (each of which is incorporated herein by reference in its entirety).

[0581] In some cases (e.g., when immunogenicity to non-human or chimeric antibodies is possible), fully human antibodies may be more desirable and can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but which can express human heavy and light chain genes.

[0582] Antibodies include analogs and derivatives that have been modified, i.e., by the covalent attachment of any type of molecule, so long as such covalent attachment allows the antibody to retain its antigen-binding immunospecificity. For example, without limitation, antibody derivatives and analogs include those that have been further modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular antibody units or other proteins, etc. Any of a number of chemical modifications can be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Additionally, analogs or derivatives can contain one or more unnatural amino acids.

[0583] The antibody can have modifications (e.g., substitutions, deletions, or additions) in amino acid residues that interact with Fc receptors. In particular, the antibody can have modifications in amino acid residues identified as being involved in the interaction between the anti-Fc domain and the FcRn receptor (see, e.g., WO 97 / 34631, which is incorporated herein by reference in its entirety).

[0584] Antibodies can be produced using methods well known in the art. For example, protocols for antibody production are described by Harlow and Lane, Antibodies: A Laboratory Manual, (1988). Typically, antibodies can be produced in mice, rats, guinea pigs, hamsters, camels, llamas, sharks, or other suitable hosts. Alternatively, antibodies can be made in chickens to produce IgY molecules (Schade et al., (1996) ALTEX 13(5):80-85). In embodiments, antibodies suitable for the present invention are near-human primate antibodies. For example, general techniques for making therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al., International Patent Publication No. 91 / 11465 (1991) and Losman et al., Int. J. Cancer 46:310 (1990). In embodiments, monoclonal antibodies can be produced using hybridoma methods (Milstein et al., 1996). and Cuello, (1983) Nature 305(5934):537-40). In embodiments, monoclonal antibodies may also be made by recombinant methods (U.S. Pat. No. 4,166,452, 1979).

[0585] Many of the difficulties associated with generating monoclonal antibodies through B cell immortalization can be overcome by using phage display to engineer and express antibody fragments in E. coli. To ensure the recovery of high-affinity monoclonal antibodies, combinatorial immunoglobulin libraries typically must contain large repertoire sizes. A typical strategy utilizes mRNA obtained from lymphocytes or spleen cells of immunized mice to synthesize cDNA using reverse transcriptase. Heavy and light chain genes are amplified separately by PCR and ligated into phage cloning vectors. Two distinct libraries are produced: one containing heavy chain genes and one containing light chain genes. Phage DNA is isolated from each library, and the heavy and light chain sequences are ligated together and packaged to form a combinatorial library. Each phage contains random pairs of heavy and light chain cDNAs and, upon infection of E. coli, induces the expression of antibody chains in infected cells. To identify antibodies that recognize the antigen of interest, the phage library is plated, and the antibody molecules present in the plaques are transferred to a filter. The filter is incubated with radioactively labeled antigen and then washed to remove excess unbound ligand. Radioactive spots on the autoradiogram identify plaques containing antibodies that bind to the antigen. Cloning and expression vectors useful for producing human immunoglobulin phage libraries can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, Calif.).

[0586] Similar strategies can be used to obtain high affinity scFvs. See, e.g., Vaughn et al., Nat. Biotechnol., 14:309-314 (1996). Large repertoire scFv libraries can be constructed that contain all known VFvs. H , V k , and V λ It can be constructed by isolating V genes from unimmunized human donors using PCR primers corresponding to the gene family. k and Vλ The pools are combined to form one pool. These fragments are ligated into a phagemid vector. The scFv linker, (Gly4, Ser)3, is then inserted into the V L The fragment is ligated into the phagemid upstream of V. H Fragment and Linker-V L The fragments are amplified and assembled onto the JH region. H -Linker-V L The fragments are ligated into a phagemid vector. Phagemid libraries can be panned using filters as described above or using immunotubes (Nunc; Maxisorp™). Similar results can be achieved by constructing combinatorial immunoglobulin libraries from lymphocytes or spleen cells of immunized rabbits and expressing the scFv constructs in P. pastoris. See, e.g., Ridder et al., Biotechnology, 13:255-260 (1995). Furthermore, after isolation of suitable scFvs, antibody fragments with higher binding affinities and slower dissociation rates can be obtained through affinity maturation processes such as CDR3 mutagenesis and chain shuffling. See, e.g., Jackson et al., Br. J. Cancer, 78:181-188 (1998); Osbourn et al., Immunotechnology, 2:181-196 (1996).

[0587] In embodiments, the conjugates described herein (e.g., any compound according to Formula (III)) include a cell binding agent, which is an antibody that targets an antigen that is overexpressed in cancer cells.

[0588] In embodiments, the conjugates described herein (e.g., any compound according to Formula (III)) comprise a cell-binding agent, which is an antibody that recognizes a specific tumor-associated antigen (TAA). be.

[0589] Antibodies immunospecific for cancer cell antigens can be obtained commercially or produced by any method known to those of skill in the art, such as recombinant expression techniques. Nucleotide sequences encoding antibodies immunospecific for cancer cell antigens can be obtained, for example, from the GenBank database or a database like it, literature publications, or routine cloning and sequencing.

[0590] In certain embodiments, known antibodies for the treatment of cancer may be used.

[0591] In another specific embodiment, antibodies for the treatment of autoimmune diseases are used in accordance with the compositions and methods of the invention.

[0592] In embodiments, useful antibodies are capable of binding to a receptor or receptor complex expressed on activated lymphocytes, which may include an immunoglobulin gene superfamily member, a TNF receptor superfamily member, an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin, or a complement control protein.

[0593] In embodiments, the conjugates described herein (e.g., any compound according to Formula (III)) are selected from the group consisting of Apo2, BAFF-R, bone morphogenetic protein receptor, IGF-IR, CA125, CanAg, E16, ErbB2, MUC1, MUC16, Napi3b, TF, EpCAM, FcRH2, C242, CD2, CD3, CD4, CD5, CD6, CD11, CD18, CD19, CD20, CD21, CD22, CD26, CD30, CD33, CD37, CD38, CD40, CD44, CD56, CD70, CD72, CD79, CD90, CD138, CRI and cell binding agents (e.g., antibodies or fragments thereof) that target PTO, CXCR5, LY64, TDGF1, endothelin B receptor, EphA receptor, EphB receptor, endothelin, FCRH1, HER2, HER2 / neu, HER3, MHC class II molecule Ia antigen, integrin, IRTA2, LIV-1, MPF, NaPi2b, PDL1, FLJ10372, KIAA1445, Mm42015, SEMA5B, SEMAG, prostate 6 transmembrane epithelial antigen 1, IPCA-1, PCANP1, STMP, prostate antigen, insulin growth factor receptor, or folate receptor.

[0594] In embodiments, a conjugate described herein (e.g., any compound according to Formula (III)) comprises a cell-binding agent (e.g., an antibody or fragment thereof) that targets GPNMB, NCAM (CD56), TACSTD2 (TROP-2), folate receptor alpha, tissue factor, ENPP3, CD70, P-cadherin, mesothelin, STEA1, CEACAM5, mucin 1, nectin 4, guanylyl cyclase C, SLC44A4, PSMA, LIV1 (ZIP6), SLITRK6, 5T4, or SC-16.

[0595] In embodiments, a conjugate described herein (e.g., any compound according to Formula (III)) includes a cell binding agent (e.g., an antibody or fragment thereof) that targets HER2 or EGFR.

[0596] In embodiments, a conjugate described herein (e.g., any compound according to Formula (III)) comprises a cell binding agent (e.g., an antibody or fragment thereof) that targets fibronectin extra-domain B (EDB), endothelial receptor ETB, PSMA, VEGFR2 (CD309), tissue factor, or ROBO4.

[0597] In embodiments, a conjugate described herein (e.g., according to formula (III) Any compound) includes a cell binding agent (eg, an antibody or fragment thereof) that targets collagen IV, periostin, or tenascin c.

[0598] In embodiments, a conjugate described herein (e.g., any compound according to Formula (III)) includes a cell binding agent (e.g., an antibody or fragment thereof) that targets CD30, CD22, CD79b, CD19, CD138, CD74, CD37, CD33, CD19, or CD98.

[0599] In embodiments, a conjugate described herein (e.g., any compound according to Formula (III)) includes a cell binding agent (e.g., an antibody or fragment thereof) that targets HER2.

[0600] In embodiments, a conjugate described herein (e.g., any compound according to Formula (III)) includes a cell binding agent (e.g., an antibody or fragment thereof) that targets EGFR.

[0601] In embodiments, a conjugate described herein (e.g., any compound according to Formula (III)) includes a cell-binding agent (e.g., an antibody or fragment thereof) that targets CD70.

[0602] In embodiments, a conjugate described herein (e.g., any compound according to Formula (III)) includes a cell binding agent (e.g., an antibody or fragment thereof) that targets CD33.

[0603] In embodiments, a conjugate described herein (e.g., any compound according to Formula (III)) includes a cell binding agent (e.g., an antibody or fragment thereof) that targets CD30.

[0604] In embodiments, a conjugate described herein (e.g., any compound according to Formula (III)) includes a cell binding agent (e.g., an antibody or fragment thereof) that targets CD22.

[0605] In embodiments, a conjugate described herein (e.g., any compound according to Formula (III)) includes a cell-binding agent (e.g., an antibody or fragment thereof) that targets CD19.

[0606] In embodiments, a conjugate described herein (eg, any compound according to Formula (III)) includes a cell binding agent (eg, an antibody or fragment thereof) that targets Mucl.

[0607] In embodiments, a conjugate described herein (e.g., any compound according to Formula (III)) includes a cell binding agent (e.g., an antibody or fragment thereof) that targets CD37.

[0608] In embodiments, a conjugate described herein (e.g., any compound according to Formula (III)) includes a cell-binding agent (e.g., an antibody or fragment thereof) that targets CD123.

[0609] Synthesis method A compound described herein (e.g., of Formula (I), Formula (II), or Formula (III) Compounds according to any one of the above methods can be prepared according to methods known in the art. Exemplary methods are described herein.

[0610] In an embodiment, Scheme 1 provides an exemplary method for the synthesis of the described compound MB-1 (P1). [ka]

[0611] In an embodiment, Scheme 2 provides an alternative method for the synthesis of the described compound MB-1 (P1). [ka]

[0612] In an embodiment, Scheme 3 provides an exemplary method for the synthesis of the described compound (P2). [ka]

[0613] In an embodiment, Scheme 4 provides an exemplary method for the synthesis of the described compound (P3). [ka]

[0614] In an embodiment, Scheme 5 provides an exemplary method for the synthesis of the described compound (P4). [ka]

[0615] In an embodiment, Scheme 6 provides an exemplary method for the synthesis of the described compound (P5). [ka]

[0616] In an embodiment, Scheme 7 provides an exemplary method for the synthesis of the described compound (P6). [ka]

[0617] In embodiments, Scheme 8 provides exemplary synthetic methods for preparing the described linkable payloads (PL1), (PL2), (PL4), and (PL7). [ka]

[0618] In an embodiment, Scheme 9 provides an exemplary method for the synthesis of the described compound MB-2 (PL1). [ka]

[0619] In embodiments, Scheme 10 provides an exemplary method for the synthesis of the described compound MB-3 (meditecan) (PL3). [ka]

[0620] In embodiments, Scheme 11 provides an alternative method for the synthesis of the described linkable payload MB-3 (meditecan) (PL3). [ka]

[0621] In embodiments, Scheme 12 provides an exemplary method for the synthesis of the described linkable payload (PL9). [ka]

[0622] In embodiments, Scheme 13 provides an exemplary method for the synthesis of the described compound PL12. do. [ka]

[0623] In an embodiment, Scheme 14 provides an exemplary method for the synthesis of the described compound PL13. [ka]

[0624] In embodiments, Scheme 15 provides an exemplary general method for preparing the conjugate (PL'). [ka]

[0625] In embodiments, an exemplary experimental procedure for preparing a conjugate (PL') with a drug-to-antibody ratio (DAR) of 7-8 or 8: Antibody C is treated with 8 equivalents (2 equivalents per disulfide bond) of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in 50 mM pH 7.4 phosphate buffer and 10 mM DTPA (diethylenetriaminepentaacetic acid) for 2 hours at 25 °C, followed by the addition of 12 equivalents of payload (PL) in DMSO (the volume of DMSO is approximately 12-15% of the volume of phosphate buffer). The resulting reaction solution is rotated on a tube rotator for 1 hour at 25 °C. The reaction mixture is immediately purified using ultrafiltration tubes (30 KD) through several cycles with formulation buffer. The resulting conjugate (PL') typically has a drug-to-antibody ratio (DAR) of 7-8 or 8 and is greater than 95% monomeric, as determined by size exclusion chromatography.

[0626] In embodiments, the antibody-drug conjugate is MB-2a. In embodiments, Scheme 16 provides an exemplary method for synthesizing the described antibody-drug conjugate MB-2a. [ka]

[0627] In an embodiment, the antibody-drug conjugate is MB-3a (trastuzumab meditecan). In an embodiment, Scheme 17 provides an exemplary method for synthesizing the described antibody-drug conjugate MB-3a (trastuzumab meditecan). [ka]

[0628] Camptothecin conjugate mixtures and compositions The present invention provides camptothecin conjugate mixtures and pharmaceutical compositions comprising any of the camptothecin conjugates (Formula III) described herein. The mixtures and pharmaceutical compositions comprise multiple conjugates. In some embodiments, the mixtures or compositions Each conjugate in a mixture or composition is identical or substantially identical. However, the distribution of drug linkers on the cell-binding agents in the mixture or composition may vary, as may the drug loading. For example, the conjugation technique used to conjugate a drug linker to an antibody as a targeting ligand may result in a composition or mixture that is heterogeneous with respect to the distribution of camptothecin payload compounds on the antibody (cell-binding agent) within the mixture and / or composition. In some embodiments, the loading of the camptothecin payload compound on each antibody molecule in such a mixture or composition of molecules is an integer ranging from 1 to 18.

[0629] In these embodiments, when referring to the composition as a whole, the drug linker loading is a number ranging from 1 to about 18. A small percentage of unconjugated antibody may also be present within the composition or mixture. The average number of drug linkers per cell-binding agent in the mixture or composition (i.e., average drug loading) is an important attribute in determining the maximum amount of drug that can be delivered to a target cell. The average drug loading can be about 1, 2 or about 2, 3 or about 3, 4 or about 4, 5 or about 5, 6 or about 6, 7 or about 7, 8 or about 8, 9 or about 9, 10 or about 10, 11 or about 11, 12 or about 12, 13 or about 13, 14 or about 14, 15 or about 15, 16 or about 16, 17 or about 17, 18 or about 18.

[0630] In some aspects, the mixtures and pharmaceutical compositions contain multiple conjugates (i.e., populations). However, the conjugates are identical or substantially identical and are substantially homogeneous with respect to the distribution of drug linkers on the ligand molecules within the mixture and / or composition and the drug linker load on the cell-binding agent molecules within the mixture and / or composition. In some such aspects, the drug linker load on the antibody is 2 or 4. A small percentage of unconjugated antibodies may also be present within the composition or mixture. The average drug load in such embodiments is about 2 or about 4. Typically, such compositions and mixtures result from the use of site-specific conjugation techniques, and conjugation is via an introduced cysteine ​​residue.

[0631] The average number of camptothecin (Formula I) or camptothecin payload compound (Formula II) per cell-binding agent in preparations from conjugation reactions can be characterized by conventional means, such as HIC, UV, LC-MS, ELISA assays, etc. The quantitative distribution of camptothecin conjugates with respect to p can also be determined. In some cases, isolation, purification, and characterization of homogeneous camptothecin conjugates can be achieved by means such as reverse-phase HPLC or electrophoresis.

[0632] In some embodiments, the composition is a pharmaceutical composition comprising a camptothecin conjugate described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is in liquid form. In some embodiments, the pharmaceutical composition is solid. In some embodiments, the pharmaceutical composition is a lyophilized powder.

[0633] Compositions, including pharmaceutical compositions, may be provided in purified form. As used herein, "purified" means that when isolated, the isolate contains at least 95%, and in other embodiments at least 98%, of the conjugate by weight of the isolate.

[0634] How to use Compositions and Methods of Administration In another aspect, the invention features a pharmaceutical composition including any compound described herein (e.g., any compound of Formula (I), Formula (II), or (Formula III)) or a pharmaceutically acceptable salt thereof described herein. In embodiments, the pharmaceutical composition includes a pharmaceutically acceptable carrier.

[0635] In an embodiment, the pharmaceutical composition comprises a conjugate according to formula (III).

[0636] In embodiments, the present invention provides a pharmaceutical composition comprising a camptothecin conjugate described herein and a pharmaceutically acceptable carrier. The camptothecin conjugate may be in any form that allows the compound to be administered to a patient for the treatment of a disorder associated with the expression of an antigen to which the cell-binding agent binds. For example, the conjugate may be in liquid or solid form. A preferred route of administration is parenteral. Parenteral administration includes subcutaneous injection, intravenous, intramuscular, intrasternal injection, or infusion techniques. In one aspect, the composition is administered parenterally. In one aspect, the conjugate is administered intravenously. Administration may be by any convenient route, for example, by infusion or bolus injection.

[0637] The pharmaceutical composition can be formulated to allow the compound to be bioavailable upon administration of the composition to a patient. The composition can be in the form of one or more dosage units.

[0638] The materials used in preparing pharmaceutical compositions can be non-toxic in the amounts used.The optimal dosage of the active ingredient in pharmaceutical compositions will be clear to those skilled in the art, depending on various factors.Relevant factors include, but are not limited to, the type of animal (e.g., human), the specific form of the compound, the method of administration, and the composition used.

[0639] Composition can be, for example, in liquid form.Liquid can be useful for injection delivery.In the composition for injection administration, it can also contain one or more of surfactant, preservative, wetting agent, dispersant, suspending agent, buffer, stabilizer and isotonic agent.

[0640] Liquid compositions, whether in solution, suspension, or other similar form, may also contain one or more of the following: sterile diluents such as water for injection; saline solution, preferably saline; Ringer's solution; isotonic sodium chloride; fixed oils such as synthetic mono- or di-silsesquioxanes that can serve as solvents or suspending media; polyethylene glycol, glycerin, cyclodextrin, propylene glycol, or other solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as amino acids, acetates, citrates, or phosphates; detergents such as nonionic surfactants and polyols; and tonicity adjusters such as sodium chloride or dextrose. Parenteral compositions can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass, plastic, or other materials. Physiological saline is an exemplary adjuvant. Injectable compositions are preferably sterile.

[0641] The amount of the conjugate that is effective in treating a particular disorder or condition depends on the nature of the disorder or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can be optionally used to help identify optimal dosage ranges. The exact dose to be used in the composition also depends on the route of administration and the severity of the disease or disorder, and should be determined according to the judgment of the practitioner and each patient's circumstances.

[0642] The compositions contain an effective amount of the compound such that a suitable dosage will be obtained, typically this amount being at least about 0.01% of the compound by weight of the composition.

[0643] For intravenous administration, the composition can contain about 0.01 to about 100 mg of camptothecin conjugate per kg of animal body weight. In one embodiment, the composition can contain about 1 to about 100 mg of camptothecin conjugate per kg of animal body weight. In another embodiment, the amount administered is in the range of about 0.1 to about 25 mg of the compound per kg of body weight. Depending on the drug used, the dosage may be even lower, for example, 1.0 μg / kg to 5.0 mg / kg, 4.0 mg / kg, 3.0 mg / kg, 2.0 mg / kg, or 1.0 μg / kg, or 1.0 μg / kg to 500.0 μg / kg of the subject's body weight.

[0644] In general, the dosage of the conjugate administered to a patient is typically about 0.01 mg / kg to about 100 mg / kg of the subject's body weight, or 1.0 μg / kg to 5.0 mg / kg of the subject's body weight. In embodiments, the dosage administered to a patient is about 0.01 mg / kg to about 15 mg / kg of the subject's body weight. In embodiments, the dosage administered to a patient is about 0.1 mg / kg to about 15 mg / kg of the subject's body weight. In embodiments, the dosage administered to a patient is about 0.1 mg / kg to about 20 mg / kg of the subject's body weight. In embodiments, the dosage administered is about 0.1 mg / kg to about 5 mg / kg or about 0.1 mg / kg to about 10 mg / kg of the subject's body weight. In embodiments, the dosage administered is about 1 mg / kg to about 15 mg / kg of the subject's body weight. In embodiments, the dosage administered is about 1 mg / kg to about 10 mg / kg of the subject's body weight. In embodiments, the dosage administered is about 0.1-4 mg / kg, even more preferably 0.1-3.2 mg / kg, or even more preferably 0.1-2.7 mg / kg of the subject's body weight over a treatment cycle.

[0645] The term "carrier" refers to a diluent, adjuvant, or excipient that is administered with a compound. Such pharmaceutical carriers can be liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Carriers can be saline, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, and urea. In addition, auxiliary agents, stabilizers, thickeners, lubricants, and coloring agents can be used. In one embodiment, when administered to a patient, the compound or composition and pharmaceutically acceptable carrier are sterilized.

[0646] Water is the exemplary carrier when compound is administered intravenously.Saline solution and aqueous dextrose and glycerol solution can also be used as liquid carrier, especially for injection solution.Suitable pharmaceutical carrier also includes starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol and other excipients.If desired, the composition can also contain a small amount of wetting agent or emulsifier, or pH buffering agent.

[0647] In one embodiment, the conjugate is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to animals, particularly humans. Typically, the carrier or vehicle for intravenous administration is a sterile isotonic aqueous buffer solution. If necessary, the composition can also include a solubilizing agent. Compositions for intravenous administration can optionally include a local anesthetic, such as lignocaine, to alleviate pain at the injection site. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example, as a lyophilized powder or water-free concentrate in a sealed container, such as an ampoule or sachet, indicating the quantity of active ingredient. When the conjugate is administered by infusion, it can be dispensed, for example, using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0648] Pharmaceutical compositions are generally formulated as sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the US Food and Drug Administration.

[0649] Cancer treatment The compounds described herein (e.g., any compound according to any one of Formula (I), Formula (II), or Formula (III)) can be effective in selectively inducing cell death in particular populations (e.g., cells that overexpress particular antigens, including those described herein, such as tumor-associated antigens).

[0650] In vitro cytotoxicity assay: The cytotoxic potential of compounds was assessed in flat-bottom 96-well cell culture plates (Corning Costar) using a Cell Counting Kit-8 (CCK-8) assay (Shanghai Life Lab Biotech Co., Ltd.). Briefly, human tumor cells (2,000–10,000 cells / well, depending on the cell line) in appropriate culture medium were incubated with compounds or conjugates in the presence or absence of excess corresponding unconjugated antibody for 120 h at 37°C and 5% CO2.

[0651] For example, for any compound according to formula (III), appropriate selection of the cell binding agent can result in effective and highly selective targeting of cancer cells, which is useful.

[0652] The camptothecin conjugates described herein (e.g., any compound according to Formula (III)) are useful for inhibiting abnormal cell proliferation (e.g., of tumor or cancer cells, causing apoptosis in tumor or cancer cells) or for treating cancer in a patient. Accordingly, provided herein are methods of treating cancer in a subject in need thereof, the methods comprising administering to the subject one or more camptothecin conjugates described herein.

[0653] In embodiments, the invention features a method of treating a cell proliferative disease or disorder or inhibiting abnormal cell growth, the method comprising administering any compound of Formula (III) described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising any compound of Formula (III) described herein or a pharmaceutically acceptable salt thereof.

[0654] Thus, the compounds described herein (e.g., any compound according to Formula (III)) can be used accordingly to treat various cancers. In embodiments, camptothecin conjugates can be used to deliver drugs to tumor or cancer cells. Without being bound by theory, in one embodiment, the cell-binding agent of the camptothecin conjugate binds to or associates with a cancer cell or tumor cell-associated antigen, and the camptothecin conjugate can be taken up (internalized) into the tumor or cancer cell via receptor-mediated endocytosis or other internalization mechanisms. The antigen can be attached to the tumor or cancer cell or an extracellular matrix protein associated with the tumor or cancer cell. Once inside the cell, the drug is released via intracellular peptide cleavage. In an alternative embodiment, the free drug is released from the camptothecin conjugate outside the tumor or cancer cell, after which the free drug penetrates the cell.

[0655] In one embodiment, the cell binding agent binds to tumor or cancer cells.

[0656] In another embodiment, the cell binding agent binds to a tumor cell or cancer cell antigen on the surface of the tumor or cancer cell.

[0657] In another embodiment, the cell binding agent binds to a tumor cell or cancer cell antigen that is an extracellular matrix protein associated with tumor or cancer cells.

[0658] The specificity of a cell-binding agent for a particular tumor or cancer cell can be important for determining which tumor or cancer is most effectively treated.

[0659] The cancers that can be treated with camptothecin conjugate include, but are not limited to, hematopoietic cancers such as lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma) and leukemia, and solid tumors.Examples of hematopoietic cancers include follicular lymphoma, anaplastic large cell lymphoma, mantle cell lymphoma, acute myeloblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, diffuse large B-cell lymphoma and multiple myeloma. Examples of solid tumors include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer, pharyngeal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, and breast cancer. These include head adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung cancer, bladder cancer, lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, and retinoblastoma.

[0660] In embodiments, the cancer is adenocarcinoma, brain cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, CNS tumors, colon or colorectal cancer, diffuse pontine glioma (DIPG), endometrial cancer, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, blood cancer, Hodgkin's lymphoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, myelodysplastic syndrome (MDS), neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, pancreatic cancer, peritoneal cancer, prostate cancer, ovarian cancer, renal cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, or Wilms' tumor.

[0661] In embodiments, the cancer is breast cancer.

[0662] Multidisciplinary cancer therapy Cancer, including but not limited to tumors, metastases, or other diseases or disorders characterized by uncontrolled cell growth, can be treated or inhibited by administration of camptothecin conjugates.

[0663] In another embodiment, a method for treating cancer is provided, comprising administering to a patient in need thereof an effective amount of a camptothecin conjugate and a chemotherapeutic agent. In one embodiment, the chemotherapeutic agent is one that has not yet proven refractory to treatment of the cancer therewith. In another embodiment, the chemotherapeutic agent is one that has proven refractory to treatment of the cancer therewith. The camptothecin conjugate may be administered to a patient who has also undergone surgery as a treatment for the cancer.

[0664] In embodiments, the patient also receives an additional treatment, such as radiation therapy. In certain embodiments, the camptothecin conjugate is administered simultaneously with a chemotherapeutic agent or radiation therapy. In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered before or after administration of the camptothecin conjugate.

[0665] The chemotherapy agents may be administered over a series of sessions. Any one or combination of chemotherapy agents, such as standard of care chemotherapy agents, may be administered.

[0666] Furthermore, methods of treating cancer with camptothecin conjugates are provided as an alternative to chemotherapy or radiation therapy, which, for example, have proven or may prove excessively toxic to the treated subject, e.g., resulting in unacceptable or intolerable side effects. The treated patient can optionally be treated with another cancer therapy, such as surgery, radiation therapy, or chemotherapy, depending on which therapy is acceptable or tolerable.

[0667] Treatment of autoimmune diseases Camptothecin conjugates are useful for killing or inhibiting the unwanted replication of cells that lead to autoimmune disease or for treating autoimmune disease.

[0668] Camptothecin conjugates can be used accordingly in various settings for the treatment of autoimmune diseases in patients. Camptothecin conjugates can be used to deliver drugs to target cells. Without being bound by theory, in one embodiment, the camptothecin conjugate associates with an antigen on the surface of proinflammatory or inappropriately stimulated immune cells, and then the camptothecin conjugate is taken up into the target cells via receptor-mediated endocytosis. Once inside the cells, the cell-binding agent is cleaved, resulting in the release of camptothecin. The released camptothecin then moves freely through the cytosol and induces cytotoxic or cytostatic activity. In an alternative embodiment, the drug is cleaved from the camptothecin conjugate outside the target cells, and then the camptothecin penetrates the cells.

[0669] In one embodiment, the cell binding agent binds to an autoimmune antigen. In one aspect, the antigen is on the surface of a cell involved in an autoimmune condition.

[0670] In one embodiment, the cell binding agent binds to activated lymphocytes associated with an autoimmune disease state.

[0671] In a further embodiment, the camptothecin conjugate kills or inhibits the proliferation of cells that produce autoimmune antibodies associated with certain autoimmune diseases.

[0672] Specific types of autoimmune diseases that can be treated with camptothecin conjugates include, but are not limited to, Th2 lymphocyte-associated disorders (e.g., atopic dermatitis, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, and graft-versus-host disease); Th1 lymphocyte-associated disorders (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjogren's syndrome, Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis, Wegener's granulomatosis, and tuberculosis); and activated B lymphocyte-associated disorders (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes).

[0673] Multidrug therapy for autoimmune diseases Also disclosed is a method for treating an autoimmune disease comprising administering to a patient in need thereof an effective amount of a camptothecin conjugate and another therapeutic agent known to treat the autoimmune disease.

[0674] Method for preparing camptothecin conjugates The camptothecin conjugates described herein can be prepared either by sequential construction of the antibody, linker, and drug units, or in a convergent manner by assembly of moieties followed by completion of the assembly process.

[0675] In one group of embodiments, the camptothecin payload compounds provided herein are preferably The antibody is combined with a suitable cell-binding agent to facilitate covalent attachment of a camptothecin payload compound to the cell-binding agent. In embodiments, the cell-binding agent is an antibody having at least two, at least four, at least six, or eight thiols available for attachment of a camptothecin payload compound as a result of reduction of interchain disulfide linkages. In embodiments, the camptothecin payload compound is attached to the cell-binding agent via an engineered cysteine ​​moiety on the antibody.

[0676] Kits for therapeutic use In some embodiments, kits for use in cancer therapy and autoimmune disease therapy are provided. Such kits can include pharmaceutical compositions comprising the camptothecin conjugates described herein.

[0677] In embodiments, the kit can include instructions for use in any of the therapeutic methods described herein. The included instructions can provide instructions for administering the pharmaceutical composition to a subject to achieve the intended activity in the subject, for example, treatment of a disease or condition such as cancer. In embodiments, the instructions for using the pharmaceutical composition described herein can include information regarding the dosage, administration schedule, and route of administration for the intended treatment. The container can be a unit dose, bulk package (e.g., a multi-dose package), or partial unit dose. The instructions provided with the kit of the present disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical composition is used to treat, delay the onset of, and / or alleviate a disease or disorder in a subject.

[0678] In embodiments, the kits provided herein are suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, etc. Packaging for use in combination with specific devices, such as inhalers, nasal administration devices, or injection devices, is also contemplated. In embodiments, the kits can have a sterile access port (for example, the container can be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle).

[0679] In embodiments, the kits provided herein include additional therapeutic agents useful in treating the autoimmune diseases cancers described herein. [Example]

[0680] The following abbreviations are used in the following terms: ADC antibody-drug conjugate ACN Acetonitrile DAR Drug-to-Antibody Ratio DCC N,N'-dicyclohexylcarbodiimide DCM dichloromethane DIPA Diisopropylamine DIPEA Diisopropylethylamine DMF Dimethylformamide DMSO dimethyl sulfoxide DMTMM 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride DMTMMT 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate DTPA Diethylenetriaminepentaacetic acid HIC Hydrophobic Interaction Chromatography iv LC-MS Liquid Chromatography-Mass Spectrometry M mole nM nanomolar NMM N-methylmorpholine PPTS Pyridinium p-toluenesulfonate PTSA 4-methylbenzenesulfonic acid SEC Size Exclusion Chromatography TBS tert-butyldimethylsilyl TCEP 3,3',3''-phosphinetriyltripropane hydrochloride TEA Triethylamine TFA trifluoroacetic acid THF tetrahydrofuran TLC thin layer chromatography p-TsOH p-toluenesulfonic acid

[0681] Example 1. Exemplary synthesis of compound MB-1 (P1) General procedure for the preparation of 2,6-dibromo-4-fluorobenzaldehyde (2) [ka]

[0682] A solution of compound 1 (30 g, 79.0 mmol, 1 equiv.) in anhydrous toluene (180 mL) was cooled to −35° C., and i-PrMgCl (2 M in anhydrous THF, 51.3 mL, 1.3 equiv.) was added over 5 min, maintaining the internal temperature below −25° C. A clear brown solution was obtained. The reaction mixture was stirred at −30° C. to −25° C. for 1.5 h. Anhydrous DMF (17.32 g, 236.97 mmol, 18.2 mL, 3.3 equiv.) was then added dropwise over 5 min. The reaction mixture was warmed to 10° C. and stirred at this temperature for 1.5 h. TLC (petroleum ether / ethyl acetate = 10 / 1, Rf = 0.7) showed no starting material remained. The reaction was quenched with saturated aqueous NH4Cl (60 mL) and then filtered. The filtrate was dried over Na2SO4 and evaporated under reduced pressure to give a residue, which was purified by silica-gel column chromatography (petroleum ether / ethyl acetate = 50 / 1 to 10 / 1) to give 2,6-dibromo-4-fluoro-benzaldehyde (14.8 g, 47.3 mmol, yield 59.8%) as a yellow solid.1 H NMR (400 MHz, chloroform-d) δ 10.23 (s, 1H), 7.44 (d, J = 7.7 Hz, 2H).

[0683] General procedure for the preparation of 2-(2,6-dibromo-4-fluorophenyl)-1,3-dioxolane (3) [ka]

[0684] To a solution of compound 2 (14.8 g, 47.3 mmol, 1 equiv.) in 1,2-dichloroethane (240 mL), ethylene glycol (14.66 g, 236.25 mmol, 13.2 mL, 5 equiv.), diethoxymethoxyethane (6.02 g, 40.64 mmol, 6.76 mL, 0.86 equiv.), and 4-methylbenzenesulfonic acid (81.37 mg, 472.50 μmol, 0.01 equiv.) were added at 25 °C. The reaction mixture was stirred at 80 °C for 10 h. TLC (petroleum ether / ethyl acetate = 10 / 1, Rf = 0.45, UV and I2) showed that the starting material was consumed. The reaction mixture was cooled to 20 °C, washed successively with saturated NaHCO (100 mL), HO (2 × 100 mL), and brine (2 × 100 mL), dried over NaSO, and concentrated under reduced pressure to give 2-(2,6-dibromo-4-fluoro-phenyl)-1,3-dioxolane (15.4 g, 44.9 mmol, 100% yield) as a yellow solid. 1 H NMR (400 MHz, chloroform-d) δ 7.36 (d, J = 7.7 Hz, 2H), 6.36 (s, 1H), 4.36-4.31 (m, 2H), 4.11-4.06 (m, 2H).

[0685] General procedure for the preparation of 2-(2,6-dibromo-4-fluoro-3-methylphenyl)-1,3-dioxolane (4) [ka]

[0686] To a solution of diisopropylamine (6.50 g, 64.25 mmol, 9.08 mL, 1.36 equiv) in anhydrous THF (26 mL) was added n-butyllithium (2.5 M, 24.6 mL, 1.3 equiv) dropwise at −65° C. The reaction mixture was warmed to 0° C. and stirred for 20 minutes. The reaction was then cooled again to −65° C. A solution of compound 3 (15.4 g, 47.3 mmol, 1 equiv) in anhydrous THF (42 mL) was added dropwise, and the mixture was stirred at −65° C. for an additional hour. Iodomethane (8.05 g, 56.7 mmol, 3.5 mL, 1.2 equiv) was added dropwise at −65° C. The mixture was stirred at −65° C. for 2 hours, then warmed to 15° C. and stirred for 12 hours. TLC (petroleum ether / ethyl acetate, 10 / 1, Rf=0.48) showed that the starting material had been consumed. The reaction was quenched by the addition of water (50 mL) and extracted with ethyl acetate (2×80 mL). The combined organic layers were washed with saturated brine (2×50 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to give a residue that was purified by silica gel chromatography (petroleum ether / ethyl acetate, 50 / 1 to 10 / 1) to give 2-(2,6-dibromo-4-fluoro-3-methyl-phenyl)-1,3-dioxolane (6.5 g, 19.1 mmol, 40.5% yield) as a yellow solid. 1 H NMR (400 MHz, chloroform-d) δ 7.34 (d, J = 8.8 Hz, 1H), 6.44 (s, 1H), 4.37-4.32 (m, 2H), 4.11-4.07 (m, 2H), 2.33 (d, J = 2.4 Hz, 3H).

[0687] General procedure for the preparation of N-[3-bromo-2-(1,3-dioxolan-2-yl)-5-fluoro-4-methyl-phenyl]-1,1-diphenyl-methanimine (5) [ka]

[0688] To a solution of compound 4 (0.1 g, 294.13 μmol, 1 equiv.) in toluene (6 mL), compound 4a (63.97 mg, 352.96 μmol, 59.23 μL, 1.2 equiv.), sodium tert-butoxide (56.53 mg, 588.26 μmol, 2 equiv.), and Xantphos-Pd-G4 (14.14 mg, 14.71 μmol, 0.05 equiv.) were added under N2 protection. The reaction mixture was stirred at 100 °C for 12 h under N2 protection. TLC (petroleum ether / ethyl acetate = 10 / 1, Rf = 0.32) indicated that the starting material had been consumed. Nine additional vials were set up as described above, and all ten reaction mixtures were combined. The combined reaction mixture was filtered through a Celite pad, and the filter cake was washed with ethyl acetate (100 mL). The combined filtrate was concentrated under reduced pressure to give a residue, which was purified by silica gel chromatography (petroleum ether / ethyl acetate=100 / 1 to 10 / 1) to give N-[3-bromo-2-(1,3-dioxolan-2-yl)-5-fluoro-4-methyl-phenyl]-1,1-diphenyl-methanimine (0.7 g, 1.27 mmol, 43.24% yield) as a yellow solid. 1 H NMR(400MHz,chloroform-d)δ 7.77(br d,J=7.5Hz,2H),7.48(br d,J=7.1Hz,1H),7.45-7.37(m,3H),7.31(br d,J=4.3Hz,4H),6.48(s,1H),5.93(d,J=10.1Hz,1H),4.11-4.06(m,2H),3.96-3.91(m,2H),2.23(d,J=2.3Hz,3H).

[0689] General procedure for the preparation of 6-amino-2-bromo-4-fluoro-3-methylbenzaldehyde (6) [ka] To a solution of compound 5 (0.5 g, 1.14 mmol, 1 equiv.) in tetrahydrofuran (2 mL) was added HCl (12 M, 6.66 mL, 70.39 equiv.) at 0° C. The reaction mixture was stirred at 0° C. for 10 minutes. TLC (petroleum ether / ethyl acetate = 10 / 1, Rf = 0.35) showed that all starting material was consumed. The reaction mixture was neutralized by the addition of solid NaHCO3. The resulting solution was extracted with ethyl acetate (3 × 2 mL). Four additional reaction vials were set up as described above. The reaction mixtures were combined and neutralized by the addition of solid NaHCO3. The resulting solution was extracted with ethyl acetate. All of the organic layers were combined, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. This was purified by preparative TLC (petroleum ether / ethyl acetate=10 / 1) to give 6-amino-2-bromo-4-fluoro-3-methyl-benzaldehyde (0.12 g, 439.6 umol, 38.7% yield) as a yellow solid. 1 H NMR (400 MHz, chloroform-d) δ 10.40 (s, 1H), 6.52 (br s, 2H), 6.32 (d, J = 11.2 Hz, 1H), 2.26 (d, J = 2.2 Hz, 3H).

[0690] General procedure for the preparation of (S)-10-bromo-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione (8) [ka] A mixture of compound 6 (120 mg, 517.13 μmol, 1 equiv.) and compound 7 (122.52 mg, 465.42 μmol, 0.9 equiv.) in acetic acid (2 mL) was heated to 120° C. Then, HCl (12N, 100 μL, 2.32 equiv.) was added to the mixture. The reaction mixture was stirred at 120° C. for 12 hours. TLC (petroleum ether / ethyl acetate = 2 / 1, Rf = 0.2) indicated that all starting material had been consumed. Six additional reaction vials were set up as described above, and all seven reaction mixtures were combined. The combined reaction mixture was concentrated under reduced pressure to give a residue. The residue was triturated with methanol (6 mL) and filtered to give (S)-10-bromo-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione (600 mg, 1.18 mmol, 45.47% yield) as a brown solid. 1 H NMR(400MHz,DMSO-d6)δ 8.90(s,1H),8.01(d,J=10.6Hz,1H),7.33(s,1H),6.55(br s,1H),5.43(s,2H),5.29(s,2H),2.57(d,J=1.8Hz,3H),1.87(tt,J=7.1,14.6Hz,2H),0.88(t,J=7.3 Hz,3H).

[0691] General procedure for the preparation of (S)-10-(4-((tert-butyldimethylsilyl)oxy)but-1-yn-1-yl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione (9) [ka] To a solution of compound 8 (50 mg, 108.87 umol, 1 equiv.) in toluene (1.5 mL) was added compound 8a (100.35 mg, 544.35 umol, 5 equiv.), K2CO3 (75.23 mg, 544.35 umol, 5 equiv.), (±)-2,2'-bis(diphenylphosphino)-1,1'-binaphthalene (BINAP, 13.56 mg, 21.77 umol, 0.2 equiv.), and diacetoxypalladium (4.89 mg, 21.77 umol). ol, 0.2 equiv.) was added under N2 protection. The reaction mixture was stirred at 100°C for 12 hours. TLC (petroleum ether / ethyl acetate = 1 / 2, Rf = 0.35) showed that all starting material had been consumed. Three additional reaction vials were set up as above, and all four reaction mixtures were combined. The combined reaction mixtures were diluted with water (6 mL) and ethyl acetate (6 mL). The organic layer was separated, and the aqueous phase was extracted with ethyl acetate (2 x 6 mL). The combined organic layers were concentrated under reduced pressure to give a residue, which was purified by preparative TLC (petroleum ether / ethyl acetate=1 / 2) to give (S)-10-(4-((tert-butyldimethylsilyl)oxy)but-1-yn-1-yl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1H-pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione (60 mg, 90.6 μmol, 27.8% yield) as a light brown solid. 1 H NMR(400MHz,chloroform-d)δ 8.87-8.82(m,1H),7.77(d,J=10.3Hz,1H),7.68-7.64(m,1H),5.76(d,J=16.4Hz,1H),5.30(s,3H),3.99-3.91(m, 2H), 2.91-2.81 (m, 2H), 2.69-2.55 (m, 3H), 1.98-1.81 (m, 2H), 1.06 (s, 3H), 0.96-0.93 (m, 9H), 0.16-0.11 (m, 6H).

[0692] General procedure for the preparation of (S)-4-ethyl-8-fluoro-4-hydroxy-10-(4-hydroxybutyl)-9-methyl-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione MB-1 (P1) [ka] To a solution of compound 9 (30 mg, 53.31 μmol, 1 equiv.) in tetrahydrofuran (5 mL) was added PdCl (9.45 mg, 53.31 μmol, 1 equiv.). The reaction mixture was stirred at 15 °C for 15 min under H (10 psi). TLC (petroleum ether / ethyl acetate = 1 / 2, Rf = 0.45) indicated that the starting material had been consumed. The desired product MB-1 (P1) and the TBS-protected product were detected. The reaction mixture was then stirred at 25 °C for an additional 1 h. TLC (ethyl acetate / methanol = 8 / 1, Rf = 0.45) indicated that the TBS-protected product had been consumed, and the major product was compound MB-1 (P1). An additional reaction vial was set up as described above, and the two reaction mixtures were combined. The combined reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give a residue, which was purified by preparative HPLC under neutral conditions to give (S)-4-ethyl-8-fluoro-4-hydroxy-10-(4-hydroxybutyl)-9-methyl-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione (5 mg, 10.4% yield) as a yellow solid. 1 H NMR(400MHz,DMSO-d6)δ 8.87(s,1H),7.75(d,J=10.8Hz,1H),7.31(s,1H),6.52(br s,1H),5.42(s,2H),5.25(s,2H),3.49(br t,J=5.8Hz,2H),3.16(br s,1H),3.14(br s,2H),2.43(d,J=1.5Hz,3H),1.87(tt,J=7.0,14.4Hz,2H),1.62(br s,4H),0.88(t,J=7.3Hz,3H); 13C NMR(101MHz,DMSO-d6)δ 172.93,161.00,157.28,152.78,150.42,148.38(d,J=14.5Hz,1C),145.80,140.76(d,J=5.1Hz ,1C),129.70,128.78,125.53(d,J=18.9Hz,1C),124.48,119.49,110.64(br d,J=23.3Hz,1C),97.15,72.84,65.72,60.88,50.88,32.84,30.75,28.31,27.07,11.91,8.23;HRMS(ESI-TOF)m / z:[MH] - Calculated value 451.1650; measured value 451.1650.

[0693] Preparative HPLC Method (Gilson 281 Semi-Prep HPLC System): Mobile phase: A: H2O; B: Acetonitrile; Column: Welch Xtimate C18 150 x 25 mm x 5 μm Flow rate: 25 mL / min; monitor wavelengths: 220 and 254 nm; gradient: 20% to 45% B in 8 min, then 45% to 100% B in 0.2 min, then 100% B for 2 min, 100% to 20% B in 0.2 min, then 20% B for 1.5 min.

[0694] Example 2. Exemplary Alternative Synthesis of Compound MB-1 (P1) General procedure for the preparation of 2,6-dibromo-4-fluoro-3-methyl-aniline (1-2) [ka] To a stirred solution of compound 1-1 (70 g, 559.36 mmol, 1 equiv.) in CHCl / methanol (1:1, 1.2 L), a solution of Br (223.48 g, 1.40 mol, 72.09 mL, 2.5 equiv.) in CHCl / methanol (1:1, 200 mL) was added dropwise over 1.5 h at 15 °C using an addition funnel. The reaction mixture was stirred at 25 °C for 4 h, and TLC (petroleum ether / ethyl acetate = 6 / 1, Rf = 0.6) indicated that the starting material had been consumed. Three additional vials were set up as described above, and the mixtures from the four reactions were combined and concentrated. To the resulting residue, 1 N NaSO (1.5 L) and ethyl acetate (1.5 L) were added. The solution was stirred for 10 min and then carefully basified with 1 N NaCO (150 mL). It was transferred to a separatory funnel, and the organic layer was isolated. The aqueous layer was extracted with ethyl acetate (2 x 1 L). The combined organic layers were washed with 1N NaSO (1 L), followed by brine (1 L), and then dried over NaSO. It was filtered and concentrated under reduced pressure to give a residue. The residue was triturated with petroleum ether (1 L) and filtered to give product 1-2 (574 g, 1.93 mol, 86% yield, 95% purity) as a pale purple solid. 1 H NMR (400 MHz, chloroform-d) δ 7.18 (d, J = 8.6 Hz, 1H), 4.52-4.30 (m, 2H), 2.29 (d, J = 2.4 Hz, 3H).

[0695] General procedure for the preparation of 1,3-dibromo-5-fluoro-2-iodo-4-methyl-benzene (1-3) [ka] p-TsOH (90 g, 522.2 mmol, 3 equiv.) in acetonitrile (700 mL) To a solution of 1-2 (49.25 g, 174.07 mmol, 1 equiv.) was added compound 1-2 (49.25 g, 174.07 mmol, 1 equiv.). The resulting white suspension was cooled to 10-15 °C, and then a solution of NaNO (24.02 g, 348.14 mmol, 2 equiv.) and KI (73.22 g, 435.13 mmol, 2.5 equiv.) in water (105 mL) was slowly added. The suspension turned dark brown, and gas was released. The thick mixture was stirred at 10 °C for 10 min and then at 20 °C for an additional 1 h. TLC (petroleum ether / ethyl acetate = 6 / 1, Rf = 0.6) indicated that the starting material had been consumed. The reaction mixture was poured into water (400 mL). 1 N sodium bicarbonate solution (200 mL) was added to adjust the pH to 9-10, followed by the addition of a 2 N solution of sodium thiosulfate (200 mL). The resulting mixture was extracted with ethyl acetate (3 x 500 mL). Eleven additional vials were installed as described above. The combined organic layers from the 12 reactions were combined, dried over NaSO, and concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography, eluting with petroleum ether, to give the product 1-3 (504 g, 1.09 mol, 56% yield, 85% purity) as a yellow solid. 1 H NMR (400 MHz, chloroform-d) δ 7.41 (d, J = 8.8 Hz, 1H), 2.43 (d, J = 2.4 Hz, 3H).

[0696] General procedure for the preparation of 2,6-dibromo-4-fluoro-3-methyl-benzaldehyde (1-4) [ka] To a solution of compound 1-3 (50.4 g, 127.98 mmol, 1 equiv.) in anhydrous toluene (300 mL), a solution of chloro(isopropyl)magnesium (2 M in tetrahydrofuran, 76.80 mL, 1.2 equiv.) was added over 10 min, maintaining the internal temperature below −25° C. A clear, brown solution was obtained, and the mixture was stirred for 1.5 h. Subsequently, N,N-dimethylformamide (30.86 g, 422.33 mmol, 3.3 equiv.) was added over 10 min. The temperature of the reaction mixture rose to −19° C. after the addition. The reaction mixture was warmed to 20° C. over 0.5 h and stirred for 1.5 h. TLC (petroleum ether / ethyl acetate = 10 / 1, Rf = 0.45) indicated the reaction was complete. The reaction mixture was quenched with saturated aqueous NH4Cl (50 mL). Ten additional vials were set up as described above, and all 11 reaction mixtures were combined. The combined mixture was filtered, and the filtrate was evaporated under reduced pressure to give a residue, which was purified by silica gel column chromatography eluting with petroleum ether to give product 1-4 (253 g, 812.18 mmol, 60% yield, 95% purity) as a yellow solid. 1 H NMR (400 MHz, chloroform-d) δ 10.22 (s, 1H), 7.40 (d, J = 8.6 Hz, 1H), 2.37 (d, J = 2.4 Hz, 3H).

[0697] General procedure for the preparation of 2-(2,6-dibromo-4-fluoro-3-methyl-phenyl)-1,3-dioxolane (4) [ka] To a solution of compound 1-4 (50.6 g, 170.99 mmol, 1 equiv.) in 1,2-dichloroethane (430 mL), ethylene glycol (53.06 g, 878.58 mmol, 47.80 mL, 5 equiv.), triethyl orthoformate (25.34 g, 170.99 mmol, 28.44 mL, 1 equiv.), and p-toluenesulfonic acid (1.47 g, 8.55 mmol, 0.05 equiv.) were added. The reaction mixture was stirred at 80 °C for 3 h, and TLC (petroleum ether / ethyl acetate = 10 / 1, Rf = 0.59) indicated the reaction was complete. Four additional vials were set up as described above, and the reaction mixtures from the five reactions were combined. The combined reaction mixture was then washed with saturated aqueous Na2CO3 (1 L), saturated aqueous NHCl (1 L), and water (1 L). The organic layer was dried over NaSO, filtered, and evaporated under reduced pressure to give the crude product, which was triturated with petroleum ether at 20° C. for 15 minutes and filtered to give product 4 (280 g, 741.21 mmol, 84% yield, 90% purity) as a pale yellow solid. 1 H NMR (400 MHz, chloroform-d) δ 7.34 (d, J = 8.6 Hz, 1H), 6.44 (s, 1H), 4.37-4.31 (m, 2H), 4.11-4.06 (m, 2H), 2.34 (d, J = 2.4 Hz, 3H).

[0698] General procedure for the preparation of N-[3-bromo-2-(1,3-dioxolan-2-yl)-5-fluoro-4-methyl-phenyl]-1,1-diphenyl-methanimine (5) [ka] To a solution of compound 4 (53 g, 155.89 mmol, 1 equiv.) in toluene (100 mL), compound 4a (29.67 g, 163.69 mmol, 27.46 mL, 1.05 equiv.), sodium tert-butoxide (29.97 g, 311.78 mmol, 2 equiv.), palladium(II) acetate (3.5 g, 15.59 mmol, 0.1 equiv.), and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (9.02 g, 15.59 mmol, 0.1 equiv.) were added under N2 protection. The reaction mixture was stirred at 100 °C for 12 h under N2 protection, and TLC (petroleum ether / ethyl acetate = 10 / 1, Rf = 0.32) indicated the reaction was complete. Two additional vials were set up as described above, and all three reaction mixtures were combined and filtered through a Celite pad. The filter cake was washed with ethyl acetate (500 mL). The combined filtrate was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography, eluting with petroleum ether / ethyl acetate=10 / 1 to obtain product 5 (105 g, 214.62 mmol, yield 45.61%, purity 80%) as a yellow solid. The product was used in the next step without further purification. 1 H NMR(400MHz,chloroform-d)δ 7.77(br d,J=7.3Hz,2H),7.54-7.37(m,4H),7.31(br d,J=4.5Hz,3H),7.26-7.22(m,1H),6.48(s,1H),5.93(d,J=10.3Hz,1H),4.11-4.05(m,2H),3.96-3.91(m,2H),2.23(d,J=2.3Hz,3H).

[0699] General procedure for the preparation of N-[3-[4-[tert-butyl(dimethyl)silyl]oxybutyl]-2-(1,3-dioxolan-2-yl)-5-fluoro-4-methyl-phenyl]-1,1-diphenyl-methanimine (1-6) [ka] To a stirred mixture of compound 5a (5.3 g, 28.44 mmol, 1 equiv.) in toluene (80 mL) was added 9-BBN (0.5 M in tetrahydrofuran, 68.13 mL, 1.2 equiv.) under a nitrogen atmosphere at 10° C. The resulting mixture was stirred at 80° C. for 20 minutes under nitrogen protection, and TLC (petroleum ether / ethyl acetate=1 / 1, product Rf=0.2, I2) indicated the reaction was complete. A solution of NaOH (2.27 g, 56.78 mmol, 2 equiv.) in water (20 mL) was added to the above mixture under a nitrogen atmosphere at 10° C. The resulting mixture was stirred at 10 °C for 10 minutes, followed by the addition of compound 5 (10.00 g, 22.71 mmol, 0.8 equiv.), tetrabutylammonium iodide (524.31 mg, 1.42 mmol, 0.05 equiv.), and [1,1-bis(diphenylphosphino)ferrocene]palladium(II) dichloride dichloromethane adduct (463.7 mg, 568.8 μmol, 0.02 equiv.) under a nitrogen atmosphere at 10 °C. The resulting mixture was stirred at 80 °C under a nitrogen atmosphere for 15 hours, and LCMS (retention time = 3.620) indicated the reaction was complete. Seven additional vials were set up as described above, and all eight reaction mixtures were combined. The combined organic layer was washed with water (500 mL × 3), dried over Na SO , filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography (SiO, petroleum ether / ethyl acetate = 10 / 1 to 5 / 1) to give the crude product, which was further purified by reverse-phase HPLC to give product 1-6 (45 g, 82.15 mmol, 50% yield, 80% purity) as a yellow gum. 1 H NMR(400MHz,DMSO-d6)δ 7.72-7.61(m,2H),7.59-7.51(m,1H),7.50-7.42(m,2H),7.37-7.22(m,4H),7.19(br d,J=3.5Hz,1H),6.03(s,1H),5.89(s,1H),4.07-3.99(m,2H),3.93-3.78(m,2H),3.65-3.56(m, 2H), 2.75-2.64 (m, 2H), 2.01 (s, 2H), 1.64 (s, 1H), 1.59-1.44 (m, 4H), 0.87 (s, 9H), 0.03 (s, 6H).

[0700] (19S)-19-Ethyl-6-fluoro-19-hydroxy-8-(4-hydroxybutyl)-7-methyl-17-oxa-3,13-diazapentacyclo[11.8.0.0 2,11 .0 4,9 .0 15,20 ] General Procedure for the Preparation of Henicosa-1(21),2,4,6,8,10,15(20)-Heptaene-14,18-dione (MB-1) (P1) [ka] Compound 1-6 (500 mg, 912.79 μmol, 1 equiv.) in ethanol (5 mL) To a solution of 7 (144.17 mg, 547.67 μmol, 0.6 equiv.) and concentrated hydrochloric acid (12 M, 0.5 mL, 6.57 equiv.) were added at 20 °C. The reaction mixture was stirred at 80 °C for 2 h. TLC (petroleum ether / ethyl acetate = 10 / 1, product Rf = 0; ethyl acetate / methanol = 10 / 1, product Rf = 0.2) indicated that most of compound 7 had been consumed and a new spot had formed. 29 additional vials were set up as described above, and all 30 reaction mixtures were combined. The combined reaction mixtures were concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography (ethyl acetate / methanol = 1 / 0 to 7 / 3) to give the crude product. Compound 7 (1.7 g, 4.52 mmol, 22.5% yield, 70% purity) was recovered as a brown solid after purification by column chromatography (SiO2, ethyl acetate / methanol = 1 / 0 to 7 / 3) and reverse-phase HPLC. The recovered compound 7 was subjected to the same reaction conditions in another 20 reactions. The reactions were worked up and purified as detailed above. The combined crude products were further triturated with ethyl acetate / methanol (1 / 1, 1 mL) to give product MB-1 (P1) (3.7 g, 8.18 mmol, 10% yield, 90% purity) as a dark brown solid. 1H NMR(400MHz,DMSO-d6)δ 8.89(s,1H),7.77(d,J=11.0Hz,1H),7.31(s,1H),6.54(br s,1H),5.53-5.33(m,2H),5.26(s,2H),4.80-4.06(m,1H),3.49(br t,J=5.9Hz,2H),3.19-3.11(m,2H),2.43(d,J=1.8Hz,3H),1.86(tt,J=7.2,14.5Hz,2H),1.62(br s,4H),0.88(t,J=7.3Hz,3H).

[0701] Example 3. Exemplary synthesis of compound MB-2 (PL1) C 17 H 14 General procedure for the preparation of NO4(S2). [ka]

[0702] A column packed with 2-(9H-fluoren-9-ylmethoxycarbonylamino)acetic acid (74.92 g, 252.10 mmol, 2 equiv.), Trt-Resin S1 (120.00 g, 126.05 mmol, 1 equiv.), and N,N-diisopropylethylamine (162.85 g, 1.26 mol, 219.47 mL, 10 equiv.) in dichloromethane (1500 mL) was bubbled with nitrogen at 20 °C for 12 h. After filtration, the residue was washed successively with dichloromethane (3 × 300 mL), dimethylformamide / methanol = 1 / 1 (3 × 300 mL), and dimethylformamide (3 × 300 mL). The residue was further dried under high vacuum to give crude resin-C. 17 H 14 NO4 (150 g, 123.66 mmol, 98.10% yield, crude purity) was obtained as a yellow solid. The product was used directly in the next step without purification. Resin-C in DMF (1200 mL) 17 H 14To a column packed with NO (150 g, 123.66 mmol, 1 equiv.) was added piperidine (105.30 g, 1.24 mol, 122.13 mL, 10 equiv.). The mixture was bubbled with N at 20 °C for 1 h. The resulting resin was filtered and subsequently washed with dimethylformamide (2 × 500 mL) and dichloromethane (2 × 500 mL). The resin was dried to give Resin-C H NO (S) (120 g, 121.21 mmol, 98.02% yield) as a yellow solid, which was used directly in the next step.

[0703] General Procedure for Preparation of Resin-C5H9N2O3 (S3) [ka] To a column packed with (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)propanoic acid (75.47 g, 242.42 mmol, 2 equiv.) and resin-CHNO(S) (120 g, 121.21 mmol, 1 equiv.) in dimethylformamide (1200 mL), HCTU (O-(6-chloro-1-hydroxybenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) (100.29 g, 242.42 mmol, 2 equiv.) and N,N-diisopropylethylamine (78.33 g, 606.06 mmol, 105.56 mL, 5 equiv.) were added. The mixture was bubbled with N at 20 °C for 1 h. The resulting resin was filtered and washed successively with dimethylformamide (2 x 500 mL) and dichloromethane (2 x 500 mL). It was dried and obtained as Resin-C. 20 H 19 N2O5 (150 g, crude) was obtained as a yellow solid, which was used directly in the next step. Resin-C in dimethylformamide (1200 mL) 20 H 19To a column packed with NO (150 g, 116.91 mmol, 1 equiv.) was added piperidine (99.55 g, 1.17 mol, 115.46 mL, 10 equiv.). The mixture was bubbled with N at 20 °C for 1 h. The resulting resin was filtered and washed successively with dimethylformamide (2 × 500 mL) and dichloromethane (2 × 500 mL). It was dried to give resin CHNO (S) (120 g, crude) as a yellow solid, which was used directly in the next step.

[0704] General procedure for the preparation of 2-[[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxy-carbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]acetic acid (1-7) [ka] To a column packed with resin CHNO (S3) (120 g, 112.99 mmol, 1 equiv.) and (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoic acid (76.70 g, 225.99 mmol, 2 equiv.) in dimethylformamide (200 mL), O-(6-chloro-1-hydroxybenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (93.49 g, 225.99 mmol, 2 equiv.) and N,N-diisopropyl-ethylamine (73.02 g, 564.97 mmol, 98.41 mL, 5 equiv.) were added. The mixture was bubbled with N at 20 °C for 12 h. The resulting resin was filtered and washed successively with dimethylformamide (2 × 500 mL) and dichloromethane (2 × 500 mL). The resin was quenched with trifluoroacetic acid / dichloromethane (10%, 3 × 500 mL). The organic layers were combined and concentrated under reduced pressure to give a residue. The residue was triturated with n-hexane at 20 °C for 12 hours. Filtration gave product 1-7 (60 g, 39.05 mmol, 34.56% yield, 90% purity) as a white solid. 1H NMR(400MHz,DMSO-d6)δ 8.17(br t,J=5.7Hz,1H),7.99(d,J=7.5Hz,1H),7.89(d,J=7.3Hz,2H),7.74(t,J=6. 6Hz,2H),7.45-7.38(m,3H),7.37-7.29(m,2H),4.39-4.19(m,4H),3.93-3.66 (m,3H),2.03-1.92(m,1H),1.22(d,J=7.1Hz,3H),0.85(dd,J=6.9,9.8Hz,6H).

[0705] General procedure for the preparation of methyl [[(2S)-2-[[(2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]acetate (1-8) [ka] A solution of compound 1-7 (15 g, 32.08 mmol, 1 equiv.), pyridine (3.04 g, 38.51 mmol, 1.2 equiv.), and lead(IV) acetate (17.07 g, 38.51 mmol, 1.2 equiv.) in tetrahydrofuran (192 mL) and toluene (38.4 mL) was stirred at 80 °C for 12 h, and LCMS (product retention time = 1.157) indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure to give a residue, which was triturated with ethanol (15 mL) and filtered to give product 1-8 (5 g, 8.31 mmol, 38.83% yield, 80% purity) as a brown solid. 1 H NMR(400MHz,DMSO-d6)δ 8.03-7.93(m,1H),7.89(d,J=7.5Hz,2H),7.74(br t,J=6.3Hz,2H),7.47-7.36(m,3H),7.36-7.28(m,2H),5.16-5.02(m,1H),4.39-4.26(m,2H),4.22(br d,J=3.9Hz,2H),3.93-3.82(m,1H),1.98(s,2H),1.78(s,1H),1.27-1.13(m,3H),0.92-0.75(m,6H).

[0706] 9H-Fluoren-9-ylmethyl N-[(1S)-1-[[(1S)-2-[4-[(19S)-19-ethyl-6-fluoro-19-hydroxy-7-methyl-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.0 2,11 .0 4,9 .0 15,20 General procedure for the preparation of ]henicosa-1(21),2,4,6,8,10,15(20)-heptaen-8-yl]butoxymethylamino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]carbamate (1-9) [ka] To a stirred solution of MB-1 (411 mg, 0.908 mmol, 1 equiv.) and compound 1-8 (555 mg, 1.15 mmol, 1.27 equiv.) in anhydrous N,N-dimethylformamide (6 mL), HCl / etherate (1.5 M HCl in diethyl ether, 1.18 mL, 2 equiv.) was added. The reaction mixture was stirred at 20°C for 15 hours. LCMS (product retention time = 2.415) indicated that most of the starting material had been consumed, and a new peak with the desired MS was detected. Eight additional vials were set up as described above, and all nine reaction mixtures were combined. The combined reaction mixture was heated under reduced pressure (35°C). The mixture was concentrated in a 500 ml bath to give a residue, which was redissolved in N,N-dimethylformamide (20 mL) and purified by preparative HPLC to give product 1-9 (2 g, 2.29 mmol, 25.19% yield, 90% purity) as a pale yellow solid. 1H NMR(400MHz,DMSO-d6)δ 8.85(br s,1H),8.62(br s,1H),8.02(br d,J=6.8Hz,1H),7.85(br d,J=6.8Hz,2H),7.77(br d,J=11.0Hz,1H),7.70(br s,2H),7.37(br d,J=7.3Hz,3H),7.33-7.22(m,3H),6.52(s,1H),5.49-5.35(m,2H),5.27(br s,2H),4.55(br d,J=4.9Hz,2H),4.31-4.12(m,4H),3.90-3.79(m,1H),3.58(br s,2H),3.13(br s,2H),2.42(br s,3H),1.88(td,J=7.0,13.8Hz,3H),1.68(br s,2H),1.57(br s,2H),1.19(br d,J=6.6Hz,3H),0.94-0.72(m,9H).

[0707] Preparative HPLC method: Column: Kromasil C18 (250 x 50 mm x 10 um); Mobile phase: A is H2O, B is acetonitrile; Gradient: 40% to 70% B in 20 min; Flow rate: 80 mL / min; Wavelength: 220 and 254 nm.

[0708] (2S)-2-amino-N-[(1S)-2-[4-[(19S)-19-ethyl-6-fluoro-19-hydroxy-7-methyl-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.0 2,11 .0 4,9 .0 15,20 General Procedure for the Preparation of]henicosa-1(21),2,4,6,8,10,15(20)-heptaen-8-yl]butoxymethylamino]-1-methyl-2-oxoethyl]-3-methyl-butanamide (1-10) [ka] To a stirred solution of compound 1-9 (400 mg, 0.458 mmol, 1 equiv.) in anhydrous N,N-dimethylformamide (4 mL) was added morpholine (199.37 mg, 2.29 mmol, 200 μL, 5 equiv.). The reaction mixture was stirred at 15 °C for 4 h. LCMS (product retention time = 1.812) indicated that all starting material had been consumed and a new peak with the desired MS was detected. Four additional vials were set up as described above. The five reaction mixtures were combined after the reaction was complete. The combined reaction mixtures were concentrated under reduced pressure to give a residue. The residue was redissolved in N,N-dimethylformamide and purified by preparative HPLC to give product 1-10 (990 mg, 1.47 mmol, 77.10% yield, 90% purity) as a white solid. 1 H NMR(400MHz,DMSO-d6)δ 8.87(s,1H),8.68(br s,1H),8.09(br s,1H),7.77(br d,J=10.6Hz,1H),7.32(s,1H),6.51(s,1H),5.43(s,2H),5.29(br s,2H),4.55(br s,2H),4.28(br s,1H),3.46(br s,1H),3.45-3.42(m,1H),3.14(br s,2H),3.02(br s,1H),2.43(br s,3H),1.88(br dd,J=7.9,14.8Hz,3H),1.70(br s,2H),1.58(br s,2H),1.19(br d,J=6.8Hz,3H),0.88(br t,J=7.2Hz,3H),0.82(br d,J=6.6Hz,3H),0.73(br d,J=6.6Hz,3H) .

[0709] Preparative HPLC method: Column: Kromasil C18 (250 x 50 mm x 10 um); Mobile phase: A is H2O, B is acetonitrile; Gradient: 10% to 45% B in 20 min; Flow rate: 80 mL / min; Wavelength: 220 and 254 nm.

[0710] 6-(2,5-dioxopyrrol-1-yl)-N-[(1S)-1-[[(1S)-2-[4-[(19S)-19-ethyl-6-fluoro-19-hydroxy-7-methyl-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.0 2,11 .0 4,9 .0 15,20 General Procedure for the Preparation of]henicosa-1(21),2,4,6,8,10,15(20)-heptan-8-yl]butoxymethylamino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]hexanamide (MB-2) (PL1) [ka] To a solution of compound 1-10 (400 mg, 613.8 μmol, 1 equiv.) in N,N-dimethylformamide (10 mL) was added compound 10A (283.8 mg, 920.6 μmol, 1.5 equiv.). The reaction mixture was stirred at 15 °C for 12 h. LCMS (product retention time = 2.080) showed that all of compound 1-10 was consumed, and a new peak with the desired MS was detected. The reaction mixture was filtered, and the filtrate was purified by preparative HPLC using acetonitrile and deionized water as the mobile phase to give product MB-2 (PL1) (173 mg, 203.9 μmol, 33.36% yield, 95.74% purity) as a white solid. 1H NMR(400MHz,DMSO-d6)δ 8.87(s,1H),8.57(t,J=6.4Hz,1H),7.97(d,J=7.2Hz,1H),7.81-7.71(m,2H),7.32(s,1H),6. 99(s,2H),6.52(s,1H),5.43(s,1H),5.49-5.37(m,1H),5.30(s,2H),4.54(dq,J=6.6,10.1Hz ,2H),4.21(quin,J=7.1Hz,1H),4.10(dd,J=6.8,8.4Hz,1H),3.48-3.41(m,2H),3.37-3.34(m ,2H),3.20-3.08(m,2H),2.43(d,J=2.0Hz,3H),2.18-2.01(m,2H),1.95-1.79(m,3H),1.68(br d,J=7.0Hz,2H),1.58(br s,2H),1.51-1.38(m,4H),1.20-1.10(m,5H),0.88(t,J=7.3Hz,3H),0.76(dd,J=6.8,9.3Hz,6H). 13 C NMR(101MHz,DMSO-d6)δ 173.09,172.54,172.26,171.09,170.85,160.54,156.86,152.36,150.00,147.85,145.38,140.1 3,134.46,129.33,128.33,125.02,124.04,119.05,110.14,96.73,72.40,69.18,66.72,65.27(br s,1C),57.44,50.49(br s,1C),48.29,37.02,34.88,30.31(br s,1C),29.00,27.78,27.62(br s,1C),26.73(br s,1C),25.78,24.89,19.18,18.03(br d,J=5.8Hz,1C),11.46,7.80. HRMS(ESI-TOF)m / z:[M+H] + The calculated value is 845.39; the measured value is 845.3859.

[0711] Separation HPLC method: Gilson 281 semi-preparative HPLC system and Phenomenex Gemini C18 column (75 × 40 mm × 3 μm); mobile phase: acetonitrile and water; flow rate: 25 mL / min; monitor wavelengths: 220 and 254 nm. Gradient: 30% to 50% acetonitrile in 8 min, 50% to 100% acetonitrile in 0.2 min, 100% acetonitrile in 2 min, 100% to 30% acetonitrile in 0.1 min, then 30% acetonitrile in 1.2 min.

[0712] Example 4. Synthesis of MB-3 (Meditecan) (PL3) General procedure for the preparation of benzyl (2S)-2-(benzyloxycarbonylamino)-5-oxo-5-[[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino]pentanoate (1-12) [ka] To a solution of compound 11A (6.03 g, 16.27 mmol, 1.1 equiv.) in N,N-dimethylformamide (27 mL), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate (DMTMMT) (9.7 g, 29.59 mmol, 2 equiv.) and triethylamine (2.24 g, 22.19 mmol, 1.5 equiv.) were added sequentially. After stirring at 25 °C for 0.5 h, compound 1-11 (2.68 g, 14.79 mmol, 1 equiv.) was added, and the reaction mixture was stirred at 25 °C for 12 h. LCMS (product retention time = 0.253) indicated that the starting material had been consumed and a new peak with the desired MS was detected. The reaction mixture was diluted with water (50 mL) and extracted with dichloromethane (6 × 50 mL). The combined organic layers were washed with brine (3 × 130 mL), dried over NaSO, and filtered. The filtrate was concentrated under reduced pressure, and the residue was subjected to high vacuum to give an oil. The oil was purified by reverse-phase HPLC (3 kg Agela C18 column, CHCN / HO, 300 mL / min, gradient: 30% CHCN in 10 min, 30% to 45% CHCN in 30 min, 45% CHCN in 35 min; approximately 15 grams of crude product was dissolved in 70 mL of DMF and loaded onto the column) to give product 1-12 (4 g, 6.74 mmol, 46.1% yield, 99% purity) as a white solid. 1 H NMR(400MHz,DMSO-d6)δ 1.72-1.85(m,1H)1.91-2.03(m,1H)2.18(br t,J=7.44Hz,2H)2.96-3.03(m,1H)3.24(dt,J=13.16,5.17Hz,2H)3.37-3.40(m,2H)3.44(br s,2H)4.04-4.11(m,1H)4.29(d,J=6.38Hz,1H)4.39-4.45(m,2H)4.51(d,J=5.63Hz ,1H)4.75(d,J=4.63Hz,1H)4.96-5.14(m,4H)7.19-7.46(m,10H)7.68-7.85(m,2H).

[0713] General procedure for the preparation of (2S)-2-amino-5-oxo-5-[[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino]pentanoic acid (1-13) [ka] To a solution of compound 1-12 (4 g, 7.48 mmol, 1 equiv.) in water (192 mL) and tetrahydrofuran (48 mL), Pd / C (15.86 g, 14.96 mmol, 10 wt.%, 2 equiv.) was added. The mixture was stirred under H (15 psi) at 25 °C for 12 h. LCMS (product retention time = 0.137) indicated that the starting material was consumed and the desired product was detected. The mixture was filtered through a Celite pad, and the filtrate was concentrated to give product 1-13 (2 g, 6.26 mmol, 83.6% yield, 97.1% purity) as a white solid. 1 H NMR(400MHz, heavy water)δ 3.84(dt,J=7.76,4.74Hz,1H)3.70-3.80(m,4H)3.58-3.64(m,2H)3.41(dd,J=14.06,4.03Hz,1H)3.25(dd,J=14.06,7.83Hz,1H)2.41(br s,2H)2.10(br s,2H).

[0714] General procedure for the preparation of (2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-5-oxo-5-[[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino]pentanoic acid (1-14) [ka] To a solution of compound 1-13 (1 g, 3.22 mmol, 1 equiv.) and compound 10A (993.51 mg, 3.22 mmol, 1 equiv.) in N,N-dimethylformamide (10 mL) was added triethylamine (3.26 g, 32.23 mmol, 4.49 mL, 10 equiv.). The mixture was stirred at 20 °C for 12 h. LCMS (product retention time = 1.054) indicated that the starting material had been consumed and the desired product was detected. As described above, one additional vial was installed. After the reactions were complete, the reaction mixtures from the two reactions were combined and diluted with water (15 mL). This was directly purified by preparative HPLC to give product 1-14 (700 mg, 1.39 mmol, 21.57% yield, 100% purity) as a white solid. 1 H NMR(400MHz, heavy water)δ 6.78(s,1H)4.30(dd,J=9.11,5.07Hz,1H)3.83(dt,J=7.89,4.74Hz,1H)3.79-3.74(m,1H) 3.74-3.68(m,2H)3.65-3.57(m,2H)3.46(t,J=6.91Hz,2H)3.40(dd,J=14.06,4.16Hz,1H) 3.23(dd,J=14.00,7.89Hz,1H)2.39-2.30(m,2H)2.24(t,J=7.27Hz,2H)2.20-2.08(m,1H) 2.04-1.89(m,1H)1.55(dquin,J=14.04,7.19,7.19,7.19,7.19Hz,4H)1.28-1.17(m,2H).

[0715] Preparative HPLC method: Column: Phenomenex Luna C18 250mm x 100mm x 15um Mobile phase: A is H2O (0.075% trifluoroacetic acid), B is acetonitrile Gradient: 1% to 30% B in 20 min; flow rate: 250 mL / min; wavelength: 220 and 254 nm.

[0716] General procedure for the preparation of (2,5-dioxopyrrolidin-1-yl)(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-5-oxo-5-[[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]amino]pentanoate (14A) [ka] To a solution of compound 1-14 (100 mg, 198.61 μmol, 1 equiv.) and N-hydroxysuccinimide (45.71 mg, 397.22 μmol, 2 equiv.) in N,N-dimethylformamide (2 mL) was added N,N-dicyclohexylcarbodiimide (DCC) (81.96 mg, 397.22 μmol, 80.35 μL, 2 equiv.). The reaction mixture was stirred at 20 °C for 6 h. LCMS (retention time = 1.418) indicated that most of the starting material had been consumed, with approximately 65% ​​product with the desired MS. Four additional vials were set up as described above, and another vial was set up on an 80 mg scale. The reaction mixtures from the six reactions were combined. This was filtered to remove solids, and the filtrate containing product 14A was used directly in the next step. LCMS (ESI+): m / z 601.3 (M+H). + , RT: 1.418 minutes.

[0717] (2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-N-[(1S)-1-[[(1S)-2-[4-[(19S)-19-ethyl-6-fluoro-19-hydroxy-7-methyl-14,18-dioxo-17-oxa-3,13-diazapentacyclo[11.8.0.0 2,11 .0 4,9 .0 15,20 General Procedure for the Preparation of]henicosa-1(21),2,4,6,8,10,15(20)-heptan-8-yl]butoxymethylamino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]-N'-[(2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl]pentanediamide (MB-3) (Meditecan) (PL3) [ka] A mixture of compound 1-10 (83.9 mg, 128.79 μmol, 0.65 equiv.) and compound 14A in 2 mL of N,N-dimethylformamide (crude product from the above reaction, 119 mg, 198.14 μmol) was stirred at 25° C. for 15 h. LCMS (retention time = 1.729) indicated that most of the starting material had been consumed and product with the desired MS was detected. Four additional vials were set up as described above, and another vial was set up on a 67.12 mg scale. The reaction solutions from the six reactions were combined and filtered. The filtrate was purified by preparative HPLC to give product MB-3 (meditecan) (PL3) (260 mg, 221.18 μmol, 18.6% yield, 96.74% purity) as an off-white solid. 1H NMR(400MHz,DMSO-d6)δ 8.87(s,1H),8.60(br s,1H),8.08(br d,J=6.2Hz,1H),8.00(br d,J=7.7Hz,1H),7.77(br d,J=11.5Hz,1H),7.72(br s,1H),7.63(br d,J=9.3Hz,1H),7.32(s,1H),6.99(s,2H),6.53(s,1H),5.43(br s,2H),5.29(br s,2H),4.74(br s,1H),4.57(br s,1H),4.52(br d,J=6.6Hz,1H),4.47(br d,J=5.5Hz,1H),4.38(br d,J=5.1Hz,1H),4.33(br s,1H),4.26(br d,J=6.4Hz,1H),4.23(br d,J=6.6Hz,2H),4.13(br s,1H),3.55(br d,J=4.4Hz,3H),3.45(br s,7H),3.14(br s,3H),3.00(br s,1H),2.42(br s,3H),2.08(br d,J=5.7Hz,4H),1.96-1.78(m,1H),1.96-1.78(m,4H),1.69(br s,3H),1.58(br s,2H),1.45(br s,4H),1.18(br d,J=6.4Hz,5H),0.88(br t,J=7.1Hz,3H),0.76-0.76(m,1H),0.76(br dd,J=6.9,11.4Hz,5H)。 13C NMR(101MHz,DMSO-d6)δ 173.14,172.49(d,J=10.3Hz,1C),172.08,171.53,171.14,170.58,163.06,160.59,156.93,152.35,150. 07,147.96(d,J=13.9Hz,1C),145.41,140.24,134.48,129.34,128.39,125.08,124.09,119.08,110.24(br d,J=22.7Hz,1C),96.83,72.44,72.10,71.78,71.55,69.67,69.25,66.80,65.33,63.38,57 .25,52.34,50.52,48.41,42.09,37.02,34.99,31.98,30.69,30.36,29.01,27.81,27.65(br s,1C),26.75,25.82,24.80,19.12,17.88(d,J=11.7Hz,1C),11.44(d,J=5.9Hz,1C),7.81. HRMS(ESI-TOF)m / z:[M+H] + Calculated value 1137.52; measured value 1137.5140.

[0718] Preparative HPLC method: Equipment: Gilson 281 semi-preparative HPLC system; Column: Phenomenex Gemini-NX 150 x 30 mm x 5 um; Mobile phase: A: HO; B: acetonitrile; Flow rate: 25 mL / min; Monitor wavelengths: 220 and 254 nm; Gradient: 20% to 50% B in 10 min, 50% B in 0.1 min, 50% to 100% B in 0.1 min, 100% B in 2 min, 100% to 20% B in 0.1 min, and 20% B in 1.2 min.

[0719] Example 5. Exemplary synthesis of antibody-drug conjugates MB-2a and MB-3a (trastuzumab-meditecan) General procedure for the preparation of trastuzumab-drug conjugate MB-2a [ka] 50 mM conjugation buffer (pH 7.4): 1 liter contains 6.86 g Na 2HPO4·2H2O and 1.58 g of NaH2PO4·H2O.

[0720] 10 mM DTPA (pentetic acid) solution: 1 liter contains 3.90 g DTPA and 1.20 g NaOH.

[0721] 25 mM His / His-HCl formulation buffer (pH 5.5): 1 liter contains 0.90 g L-histidine and 4.04 g L-histidine hydrochloride monohydrate.

[0722] Antibody preparation: 452 mg of lyophilized trastuzumab powder was dissolved in 22 mL of purified water. The resulting antibody solution was dialyzed using an ultrafiltration tube (30 KD) with 50 mM conjugation buffer for four cycles to obtain an antibody concentration of 8.63 mg / mL (the extinction coefficient of trastuzumab, ε 280 =213380M -1 cm -1 was used).

[0723] Antibody reduction: To a tube containing 12.2 mL (105 mg, 0.000724 mmol of trastuzumab) of the trastuzumab solution prepared above, 6.2 mL of 50 mM conjugation buffer was added, followed by 579.2 μl of 10 mM TCEP and 2.1 mL of 10 mM DTPA. The tube was placed in a thermomixer, and the reduction reaction was carried out at 25 °C for 2 hours.

[0724] Conjugation between antibody and payload: To the above trastuzumab reduction solution, a solution of MB-2 (PL1) (7.45 mg, 0.00882 mmol) in DMSO (1.76 mL) was added. The tube was placed in a thermomixer, and the conjugation reaction was carried out at 25°C for 1 hour.

[0725] Purification: The above conjugation reaction solution was subjected to purification using an ultrafiltration tube (30 KD) for six cycles with 25 mM His / His-HCl formulation buffer to obtain 5.5 mL (15.1 mg / mL, antibody yield = 83 mg, yield = 79%) of MB-2a in formulation buffer.

[0726] Physicochemical characterization of MB-2a (payload extinction coefficient ε 280 =4546M -1 cm -1 and ε 360 =17513M -1 cm -1 used) (Table 1): [Table 38]

[0727] General Procedure for the Preparation of Trastuzumab-Drug Conjugate MB-3a (Trastuzumab Meditecan) [ka] 50 mM conjugation buffer (pH 7.4): 1 liter contains 6.86 g NaHPO·2H0 and 1.58 g NaHPO·H0.

[0728] 10 mM DTPA (pentetic acid) solution: 1 liter contains 3.90 g DTPA and 1.20 g NaOH.

[0729] 25 mM His / His-HCl formulation buffer (pH 5.5): 1 liter contains 0.90 g L-histidine and 4.04 g L-histidine hydrochloride monohydrate.

[0730] Antibody preparation: 452 mg of lyophilized trastuzumab powder was dissolved in 22 mL of purified water. The resulting antibody solution was dialyzed using an ultrafiltration tube (30 KD) with 50 mM conjugation buffer for four cycles to obtain an antibody concentration of 8.63 mg / mL (the extinction coefficient of trastuzumab, ε 280=213380M -1 cm -1 was used).

[0731] Antibody reduction: To a tube containing 12.2 mL (105 mg, 0.000724 mmol of trastuzumab) of the trastuzumab solution prepared above, 6.2 mL of 50 mM conjugation buffer was added, followed by 579.2 μl of 10 mM TCEP and 2.1 mL of 10 mM DTPA. The tube was placed in a thermomixer, and the reduction reaction was carried out at 25 °C for 2 hours.

[0732] Conjugation between antibody and payload: To the above trastuzumab reduction solution, a solution of MB-3 (meditecan) (PL3) (10.02 mg, 0.00886 mmol) in DMSO (1.77 mL) was added. The tube was placed in a thermomixer, and the conjugation reaction was carried out at 25 °C for 1 hour.

[0733] Purification: The above conjugation reaction solution was subjected to purification using an ultrafiltration tube (30 KD) for six cycles with 25 mM His / His-HCl formulation buffer to obtain 6.2 mL (14.6 mg / mL, antibody yield = 90.5 mg, yield = 86%) of MB-3a (trastuzumab meditecan) in formulation buffer.

[0734] Physicochemical characterization of MB-3a (trastuzumab-meditecan) (extinction coefficient ε of the payload) 280 =4546M -1 cm -1 and ε 360 =17513M -1 cm -1 used) (Table 2): [Table 39]

[0735] Example 6. Exemplary synthesis of compound P2 General procedure for the preparation of 1-(6-amino-2-bromo-4-fluoro-3-methyl-phenyl)propan-1-ol (6a) [ka] To a stirred solution of ethylmagnesium bromide in THF (3 M, 2.01 mL, 2 equiv.) was added compound 6 (700 mg, 3.02 mmol, 1 equiv.) in tetrahydrofuran (5 mL) at 0 °C. The resulting suspension was warmed to 20 °C and stirred for 4 h. The reaction was carefully quenched with saturated aqueous NH Cl solution (4 mL) at 0 °C and extracted with ethyl acetate (4 mL × 3). The combined organic layers were washed with brine, dried over Na SO , filtered, and concentrated in vacuo to give a residue. The residue was purified by preparative TLC (petroleum ether / ethyl acetate = 5 / 1) to give product 6a (240 mg, 18.21% yield, 60% purity) as a yellow oil. 1 H NMR (400 MHz, chloroform-d) δ = 6.39 (d, J = 10.8 Hz, 1H), 4.02 (br s, 2H), 2.93-2.86 (m, 2H), 2.22 (d, J = 2.2 Hz, 3H), 1.23-1.18 (m, 3H).

[0736] General procedure for the preparation of 1-(6-amino-2-bromo-4-fluoro-3-methyl-phenyl)propan-1-ol (10) [ka] A mixture of compound 6a (80 mg, 0.305 mmol, 1 equiv.) and 2-iodoxybenzoic acid (213.66 mg, 0.763 mmol, 2.5 equiv.) in ethyl acetate (3 mL) was stirred at 80 °C for 4 hours. The reaction was complete based on TLC (petroleum ether / ethyl acetate = 8 / 1, Rf = 0.31). Two additional vials were set up as described above, and all three reaction mixtures were combined. The combined mixture was filtered, and the filter cake was washed with ethyl acetate. The filtrate was concentrated under reduced pressure to give a residue. The residue was purified by preparative TLC (petroleum ether / ethyl acetate = 8 / 1) to give the crude product, which was further purified by preparative HPLC under neutral conditions to give product 10 (80 mg, 276.81 μmol, 30.23% yield, 90% purity) as a yellow oil. 1 H NMR (400 MHz, chloroform-d) δ = 6.39 (d, J = 10.6 Hz, 1H), 4.02 (br s, 2H), 2.90 (q, J = 7.3 Hz, 2H), 2.22 (d, J = 2.0 Hz, 3H), 1.22 (t, J = 7.2 Hz, 3H).

[0737] Preparative HPLC method: Column: Kromasil C18 (250 x 50 mm x 10 μm) Mobile phase: A is H2O (10 mM NH4HCO3), and B is acetonitrile Gradient: 45% to 65% B in 20 min; Flow rate: 80 mL / min

[0738] (19S)-8-Bromo-10,19-diethyl-6-fluoro-19-hydroxy-7-methyl- -Methyl-17-oxa-3,13-diazapentacyclo[11.8.0.0 2,11 .0 4,9 .0 15,20 ] General procedure for the preparation of henicosa-1(21),2,4,6,8,10,15(20)-heptaene-14,18-dione (11) [ka] A mixture of compound 10 (80 mg, 0.307 mmol, 1 equiv.) and compound 7 (80.97 mg, 0.307 mmol, 1 equiv.) in AcOH (2.5 mL) was heated to 120° C., and then 12 N HCl (0.125 mL, 4.8 equiv.) was added. The reaction mixture was stirred at 120° C. for 12 hours. TLC (petroleum ether / ethyl acetate=2 / 1, product Rf=0.2) showed that the starting material was consumed. The reaction mixture was concentrated under reduced pressure to give a residue, which was purified by preparative TLC (petroleum ether / ethyl acetate=1 / 1, product Rf=0.5) to give product 11 (80 mg, 0.131 mmol, 42.7% yield, 80% purity) as a brown solid. 1 H NMR(400MHz,DMSO-d6)δ=8.09(d,J=9.5Hz,1H),7.35(s,1H),5.45(s,2H),5.35(s,2H),3.61(br d,J=8.2Hz,2H),2.67(s,3H),1.90-1.84(m,2H),1.37(q,J=7.6Hz,3H),0.92-0.87(m,3H).

[0739] (19S)-8-[4-[tert-butyl(dimethyl)silyl]oxybut-1-ynyl]-10,19-diethyl-6-fluoro-19-hydroxy-7-methyl-17-oxa-3,13-diazapentacyclo[11.8.0.0 2,11 .0 4,9 .0 15,20 ] General procedure for the preparation of henicosa-1(21),2,4,6,8,10,15(20)-heptaene-14,18-dione (12) [ka] To a solution of compound 11 (40 mg, 0.0821 mmol, 1 equiv.) in toluene (2 mL), compound 11A (60.53 mg, 0.328 mmol, 4 equiv.), Pd(OAc) (7.37 mg, 0.0328 mmol, 0.4 equiv.), (+ / -)-2,2-bis(diphenylphosphino)-1,1-dinaphthalene (25.56 mg, 0.0410 mmol, 0.5 equiv.), and KCO (56.72 mg, 0.410 mmol, 5 equiv.) were added under N protection. The reaction mixture was stirred at 100 °C for 12 h under N protection. TLC (petroleum ether / ethyl acetate = 1 / 2, Rf = 0.5) indicated that most of the starting material had been consumed. One additional vial was set up as described above. The reaction mixtures from the two reactions were combined and concentrated under reduced pressure to give a residue which was purified by preparative TLC (petroleum ether / ethyl acetate=1 / 2, product Rf=0.5) to give product 12 (30 mg, 0.0508 mmol, 30.9% yield) as a brown solid. LCMS (ESI+): m / z (M+H) + , calculated value 591.3, measured value 591.4.

[0740] (19S)-10,19-diethyl-6-fluoro-19-hydroxy-8-(4-hydroxybenzoate) (hydroxybutyl)-7-methyl-17-oxa-3,13-diazapentacyclo[11.8.0.0 2,11 .0 4,9 .0 15,20 ] General procedure for the preparation of henicosa-1(21),2,4,6,8,10,15(20)-heptaene-14,18-dione (P2) [ka] To a solution of compound 12 (10 mg, 0.0169 mmol, 1 equiv.) in tetrahydrofuran (2 mL), PdCl2 (4.50 mg, 0.0254 mmol, 1.5 equiv.) was added under H2 (15 psi) protection. The reaction mixture was stirred at 25 °C for 2 h. TLC (petroleum ether / ethyl acetate = 1 / 1, product Rf = 0, ethyl acetate / methanol = 10 / 1, product Rf = 0.45) and LCMS (retention time = 1.204) indicated that the starting material had been consumed and a new peak with the desired MS was detected. As described above, one additional vial was installed. The reaction mixtures from the two reactions were combined and filtered through a Celite pad. The filtrate was concentrated under reduced pressure to give a residue, which was purified by preparative HPLC to give product P2 (2.5 mg, 19.7% yield, 97.45% purity) as a white solid. 1 H NMR (400MHz, methanol-d4) δ=7.60(s,1H),7.37(d,J=10.6Hz,1H),5.61(d,J=16. 3Hz,1H),5.41(d,J=16.3Hz,1H),5.38(s,1H),3.60(t,J=6.2Hz,2H),3.35(br s,2H),3.25(br s,2H),2.73(d,J=2.2Hz,3H),2.02-1.94(m,2H),1.76(br d,J=7.5Hz,2H),1.72-1.63(m,2H),1.40(t,J=7.5Hz,3H),1.02(t,J=7.4Hz,3H). 13 C NMR (101 MHz, methanol-d4) δ = 188.26-187.89 (m, 1C), 188.02, 174.99, 157.34, 153.76, 152.96, 148.59, 146.32, 135.47, 121.49, 121.23, 120.19, 119.52, 111.49, 99.28, 86.57, 74.50, 66.91, 62.65, 51.49, 37.51, 33.44, 32.24, 30.61, 27.20, 15.71, 10.14, 8.33. HRMS (ESI-TOF) m / z: [M+H] + Calculated value 481.21; Measured value 481.2103

[0741] Preparative HPLC method: Equipment: Gilson 281 semi-preparative HPLC system Mobile phase: A: HO; B: acetonitrile; Column: Waters Xbridge BEH C18 100 x 25 mm x 5 um; Flow rate: 25 mL / min; Gradient: 30% to 60% B in 10 min, 60% to 100% B in 0.2 min, 100% B for 2 min, 100% to 30% B in 0.1 min, then 30% B for 1.2 min.

[0742] Example 7. In vitro cytotoxicity assays of toxins and ADCs 175 μL of cell suspension was dispensed into a 96-well plate at 1500 cells per well and incubated in a humidified incubator (37°C, 5% CO2) for 24 hours. For antibody blocking, 2 × 10 cells were added. -6 Cells (15,000 cells / mL) were incubated with 1 μM trastuzumab (final concentration 1 μM). 25 μL of various concentrations of compounds were added as 5× solutions to cell culture medium (fetal bovine serum, Invitrogen) in the plates. The plates were incubated in an incubator for 120 hours. CCK-8 was thawed on the benchtop or in a 37°C water bath, and 10 μL of CCK-8 was added to each well of the incubated plate (taking care not to introduce air bubbles into the wells, as this would interfere with the OD reading). The plates were then further incubated for 1–4 hours in an incubator. Absorbance was measured at 450 nm using a SpectraMax i3x Microplate Reader, and the percent cell inhibition was calculated. IC 50 The curves were analyzed using GraphPad Prism software. 50 Generated with the value.

[0743] The results of in vitro cytotoxicity assays of the toxins (predicted metabolites of the ADC) are summarized below in Table 3. The cytotoxicity of metabolite MB-1 is comparable to that of DXd, a metabolite of DS-8201a (Enhertu), in multiple cell lines, except for the moderately Her-2 expressing and trastuzumab-resistant cell line JIMT-1, where MB-1 is 10-fold more potent than DXd. [Table 40]

[0744] The results of the in vitro cytotoxicity assays of the ADCs are summarized in Table 4 below. In addition to exemplary compounds of Formula III, such as MB-2a and MB-3a (trastuzumab meditecan), the activity of trastuzumab and the ADC trastuzumab deruxtecan (DS-8201a, Enhertu) was also evaluated for comparison. As shown in Table 4, the trastuzumab ADCs MB-2a and MB-3a exhibited the same efficacy as DS-8201a in the Her2-highly expressing cell line, NCI-N87. However, blocking the Her2 antigen with trastuzumab reduced the ADC's ability to inhibit cell growth. Furthermore, the ADCs were not as potent in the Her2-negative cell line, MDA-MB-468, demonstrating the specificity of the ADCs for Her2-expressing cells. Although the ADCs are not sensitive in in vitro assays in JIMT-1 cells, which have a moderate level of Her2 expression, MB-2a and MB-3a are still relatively more potent than DS-8201a in this cell line. [Table 41] [ka]

[0745] Example 8. In vivo efficacy of ADCs in the NCI-N87 CDX model The right flank of each mouse (female Balb / c-Nude from Vital Rivers) was inoculated with NCI-N87 tumor cells (5 × 10 cells) mixed with Matrigel in 0.2 mL of PBS for tumor development. 6 ) (50:50) were subcutaneously inoculated. Animals were randomly divided into groups 6 days after tumor inoculation, and the mean tumor volume was approximately 160 mm 3 Treatment for efficacy studies began when tumor-bearing mice reached 100 mg / kg. Each group contained 8 mice. Test and control articles were administered to tumor-bearing mice via the tail vein at a volume of 5 mL / kg.

[0746] Tumor size was measured twice weekly in two dimensions using calipers, and volume was calculated in mm using the following formula: 3 Expressed as: V = 0.5a × b 2 (where a and b were the major and minor dimensions of the tumor, respectively.) Results were expressed as the mean and standard error (mean±SEM).

[0747] Statistical analysis: Two-way ANOVA was performed to compare tumor volumes between the two groups. All data were analyzed using Graphpad Prism 6.0, and P less than 0.05 was considered statistically significant. Both statistical analysis and biological observations were taken into account.

[0748] Tumor growth inhibition: Tumor size was used to calculate the T / C value. The relative tumor growth rate T / C (%) was calculated using the formula: T / C (%) = (Ti / T0) / (Vi / V0) × 100%. Relative tumor growth inhibition was calculated by the formula: TGI (%) = [1-(Ti / T0) / (Vi / V0)] × 100%. Ti refers to the mean tumor volume of the treatment group measured at each indicated time point after treatment, T0 refers to the tumor volume of the treatment group at the time of grouping, Vi refers to the mean tumor volume of the vehicle control group measured at each indicated time point after treatment, and V0 refers to the tumor volume of the vehicle control group at the time of grouping. If T / C is greater than 40%, there is no efficacy, and if T / C is less than 40% and the p-value is less than 0.05, there is tumor inhibition.

[0749] The antitumor efficacy of the ADCs in the NCI-N87 CDX model is shown in Figure 1 and Table 5. As shown in Figure 1, both MB-2a (1 mg / kg and 4 mg / kg) and MB-3a (trastuzumab meditecan) (doses of 1 mg / kg and 4 mg / kg) demonstrated potent antitumor efficacy and were more effective than DS-8201a (Enhertu). [Table 42]

[0750] Example 9. In vivo efficacy of ADCs in the JIMT-1 CDX model The right flank of each mouse (Scid-Beige from Shanghai Lingchang Biotech) was inoculated with JIMT-1 tumor cells (1 × 10 ) mixed with Matrigel in 0.2 mL of PBS for tumor development. 7 ) (50:50) were subcutaneously inoculated. Animals were randomly divided into groups 6 days after tumor inoculation, and the mean tumor volume was approximately 175 mm 3 Treatment for efficacy studies began when tumor-bearing mice reached 100 mg / kg. Each group contained 8 mice. Test and control articles were administered to tumor-bearing mice via the tail vein at a volume of 5 mL / kg.

[0751] Tumor size was measured twice weekly in two dimensions using calipers, and volume was calculated in mm using the following formula: 3 Expressed as: V = 0.5a × b 2 (where a and b were the major and minor dimensions of the tumor, respectively.) Results were expressed as the mean and standard error (mean±SEM).

[0752] Statistical analysis: Two-way ANOVA was performed to compare tumor volumes between the two groups. All data were analyzed using Graphpad Prism 6.0, and P less than 0.05 was considered statistically significant. Both statistical analysis and biological observations were taken into account.

[0753] Tumor growth inhibition: Tumor size was used to calculate the T / C value. The relative tumor growth rate T / C (%) was calculated using the formula: T / C (%) = (Ti / T0) / (Vi / V0) × 100%. Relative tumor growth inhibition was calculated by the formula: TGI (%) = [1-(Ti / T0) / (Vi / V0)] × 100%. Ti refers to the mean tumor volume of the treatment group measured at each indicated time point after treatment, T0 refers to the tumor volume of the treatment group at the time of grouping, Vi refers to the mean tumor volume of the vehicle control group measured at each indicated time point after treatment, and V0 refers to the tumor volume of the vehicle control group at the time of grouping. If T / C is greater than 40%, there is no efficacy, and if T / C is less than 40% and the p-value is less than 0.05, there is tumor inhibition.

[0754] The antitumor effects of ADCs in the JIMT-1 CDX model are shown in Figures 2-5 and Table 6. Figure 2 shows the effects of antibody-drug conjugates (ADCs) in the JIMT-1 CDX model at three different doses. In this study, all three doses of MB-2a and MB-3a (trastuzumab-meditecan) studied demonstrated significant antitumor effects. MB-2a and MB-3a were more effective than DS-8201a (Enhertu) at low and medium doses. The different doses studied in these experiments are also shown separately in Figure 3 (2.5 mg / kg, single intravenous dose), Figure 4 (5 mg / kg, single intravenous dose), and Figure 5 (10 mg / kg, single intravenous dose). [Table 43]

[0755] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the following claims.

Claims

1. A compound of formula (I), D-L １ -L ２ -Q (I) or a pharmaceutically acceptable salt thereof, wherein: D has the following structural formula: 【Chemistry 1】 wherein: R １ are independently —H, C １ -C ６ Alkyl, C ２ -C ６ Alkenyl, C ２ -C ６ Alkynyl, silyl, C ３ -C ６ Cycloalkyl, C １ -C ６ Alkyl halides, C ２ -C ６ halogenated alkenyl, or C ２ -C ６ is an alkynyl halide, R ２ are independently —H, —F, —N(R ４ ) ２ , -N(R ４ ) (R ５ ), -OR ４ , -SR ４ , -S(=O)R ５ , -SO ２ R ５ , C １ -C ６ Alkyl or C １ -C ６ fluoroalkyl, and R ３ are independently —H, —F, —CN, or —OCH ３ , -CH ３ , or -CF ３ or R ２ and R ３ together form a compound of the formula -O(CH ２ ) ｎ O- or -O(CF ２ ) ｎ O— group, where n is 1 or 2, R ４ are independently —H or C １ -C ４ is alkyl, R ５ is independent, C １ -C ４ is alkyl, L １ are independently absent or -(C １ -C １０ alkylene)-, L ２ are independently absent or —OCH ２ -L ３ -*, -SCH ２ -L ３ -*, -S(=O)-L ３ -*, -SO ２ -L ３ -*, -C(=O)-L ３ -*, -N(R ６ ) CH ２ -L ３ -*, -N(R ６ ) C(=O)-L ３ -*, -N(R ６ )C(=O)N(R ７ )-L ３ -*, -C(=O)N(R ６ ) CH ２ -L ３ -*, -OC(=O)N(R ６ ) CH ２ -L ３ -* or -N(R ６ )C(=O)OCH ２ -L ３ -*, where * indicates the site covalently bonded to Q; L ３ are independently -(C １ -C １０ alkylene)-, -CH ２ OCH ２ CH ２ - or -CH ２ CH ２ OCH ２ CH ２ - and Each R ６ and R ７ are independently —H, C １ -C ６ Alkyl, C １ -C ６ Fluoroalkyl, C ３ -C ６ cycloalkyl, aryl, heteroaryl, or benzyl; Q is —OH or —SH; During the ceremony, R ２ and R ３ is bonded to -OCH ２ When forming O-, R １ is -CH ２ CH ２ CH ２ CH ３ Instead, R １ is -H or -CH ２ CH ３ and R ２ is —OH or alkoxy, and R ３ When is -H, -L １ -L ２ -Q is -CH(R')CH ２ OH or —CH(R′)(CH ２ ) ２ Instead of OH, R' is -H, or C １ -C ６ Alkyl, alkoxy, substituted alkyl, phenyl, or PhCH ２ -, or a pharmaceutically acceptable salt thereof.

2. R １ , R ２ , and R ３ The compound of claim 1 , wherein at least one of

3. L １ and L ２ 3. The compound of claim 1, wherein at least one of:

4. R １ became independent and C １ -C ６ Alkyl, silyl, C ２ -C ６ Alkenyl, C ２ -C ６ Alkynyl, C ３ -C ６ Cycloalkyl, C １ -C ６ The compound of any one of claims 1 to 3, which is an alkyl halide, an alkene, or an alkyne.

5. R １ are independently —H or C １ -C ６ The compound of any one of claims 1 to 3, which is alkyl.

6. R ２ are independently —H, —F, —N(R ４ ) ２ , -N(R ４ ) (R ５ ), -OR ４ , -SR ４ , -S(=O)R ５ , -SO ２ R ５ , C １ -C ６ Alkyl, or C １ -C ６ fluoroalkyl, and R ３ are independently —H, —F, —CN, or —OCH ３ , -CH ３ , or -CF ３ The compound according to any one of claims 1 to 5,

7. R ２ became independent and C １ -C ６ Alkyl, C １ -C ６ The compound of any one of claims 1 to 6, wherein the fluoroalkyl group is -F.

8. R ３ are independently —H, —F, —CN, or —CF ３ The compound according to any one of claims 1 to 7,

9. R ３ are independently —F, —CN, —OCH ３ , -CH ３ , or -CF ３ The compound according to any one of claims 1 to 7,

10. R ２ and R ３ is bonded to form -O(CH ２ ) ｎ O- or --O(CF ２ ) ｎ 6. The compound of claim 1, wherein n is 1 or 2.

11. 2. The compound of claim 1, wherein D is represented by one of the following structures: Table 44

12. R １ is —H or C １ -C ６ The compound of claim 11 , wherein the compound is alkyl.

13. 12. The compound of claim 11, wherein D is represented by one of the following structures: Table 45

14. L １ But -(C １ -C １０ alkylene)-, and L ２ The compound according to any one of claims 1 to 13, wherein is absent.

15. L １ But -(C １ -C １０ alkylene)-, and L ２ But -N(R ６ ) CH ２ -L ３ -* or -N(R ６ ) C(=O)-L ３ The compound of any one of claims 1 to 13, wherein * represents the site of covalent attachment to Q.

16. L １ But it doesn't exist, L ２ But -N(R ６ ) CH ２ -L ３ -* or -N(R ６ ) C(=O)-L ３ The compound of any one of claims 1 to 13, wherein * represents the site of covalent attachment to Q.

17. L ３ But -(C １ -C １０ 17. The compound of claim 15 or 16, wherein the aryl group is aryl, ...

18. R ６ is —H or —CH ３ The compound according to any one of claims 15 to 17,

19. L １ -L ２ But -CH ２ -, -CH ２ CH ２ -, -CH ２ CH ２ CH ２ - or --CH ２ CH ２ CH ２ CH ２ The compound according to any one of claims 1 to 13, wherein

20. L １ -L ２ Yes, -OCH ２ CH ２ -*、 -OCH ２ CH ２ OCH ２ CH ２ -*、-SCH ２ CH ２ -*、-SCH ２ CH ２ OCH ２ CH ２ -*、-S(=O)CH ２ -*、-S ２ CH ２ -*、 -C(=0)CH ２ -*、-NHCH ２ CH ２ -*、-N(CH ３ )CH ２ CH ２ -*、-N(CF ３ )CH ２ CH ２ -*、-NHC(=O)CH ２ -*、-CH ２ NHC(=0)CH ２ -*、-CH ２ CH ２ NHC(=0)CH ２ -*、CH ２ N(CH) ３ )C(=O)CH ２ -*、-N(CH ３ )C(=O)CH ２ -*、-N(CH ３ )C(=O)CH ２ CH ２ -*、-C(=O)NHCH ２ CH ２ -*、-NHC(=O)NHCH ２ CH ２ -*、-NHC(=O)OCH ２ CH ２ -*、-CH ２ OC(=O)NHCH ２ CH ２ -*、または -C(=O)N(CH ３ ) CH ２ CH ２ The compound of any one of claims 1 to 13, wherein * represents the site of covalent attachment to Q.

21. L １ -L ２ -Qが、-CH ２ CH ２ CH ２ CH ２ OH、-H ２ CH ２ CH ２ OH、-H ２ CH ２ OH、-H ２ CH ２ OCH ２ CH ２ OH、-H ２ SCH ２ CH ２ OH、-H ２ NHC(=O)H ２ OH、-H ２ CH ２ NHC(=O)H ２ OH、-H ２ N(CH) ３ )C(=O)CH ２ OH、-OCH ２ CH ２ OH、-OCH ２ CH ２ CH ２ OH、-SCH ２ CH ２ CH ２ OH、-SCH ２ CH ２ OH、-NHCH ２ CH ２ OH、-NHCH ２ CH ２ CH ２ OH、-N(H) ３ )CH ２ CH ２ OH、-C(=O)NHCH ２ CH ２ OH、-NHC(=O)CH ２ OH、-H ２ S (=O) H ２ OH、-H ２ SO ２ CH ２ OH、-H ２ CH ２ CH ２ CH ２ SH、-SH ２ CH ２ CH ２ SH, -CH ２ CH ２ SH, -CH ２ CH ２ OCH ２ CH ２ SH, -CH ２ SCH ２ CH ２ SH, -CH ２ NHC(=O)CH ２ SH, -OCH ２ CH ２ CH ２ SH, -SCH ２ CH ２ CH ２ SH, -SCH ２ CH ２ SH, -NHCH ２ CH ２ CH ２ SH, -N(CH ３ ) CH ２ CH ２ SH, -C(=O)NHCH ２ CH ２ SH, -NHC(=O)CH ２ SH, -CH ２ S(=O)CH ２ SH, or -CH ２ SO ２ CH ２ The compound according to any one of claims 1 to 13, which is SH.

22. D-L １ -L ２ 2. The compound of claim 1, wherein: Table 46

23. R １ is —H or C １ -C ６ 23. The compound of claim 22, wherein the compound is alkyl.

24. R １ is —H or —CH ２ CH ３ 24. The compound of claim 23, wherein:

25. The compound of any one of claims 1 to 20 and 22 to 24, wherein Q is -OH.

26. 25. The compound of any one of claims 1 to 20 and 22 to 24, wherein Q is -SH.

27. The compound has one of the following structures: Table 47 10. The compound of claim 1, wherein:

28. A compound of formula (II), D-L １ -L ２ -Q’-CH ２ -NH-E-Z (II) or a pharmaceutically acceptable salt thereof, wherein: D has the following structural formula: 【Chemistry 2】 wherein: R １ are independently —H, C １ -C ６ Alkyl, C ２ -C ６ Alkenyl, C ２ -C ６ Alkynyl, silyl, C ３ -C ６ Cycloalkyl, C １ -C ６ Alkyl halides, C ２ -C ６ halogenated alkenyl, or C ２ -C ６ is an alkynyl halide, R ２ are independently —H, —F, —N(R ４ ) ２ , -N(R ４ ) (R ５ ), -OR ４ , -SR ４ , -S(=O)R ５ , -SO ２ R ５ , C １ -C ６ Alkyl or C １ -C ６ fluoroalkyl, and R ３ is -H, -F, -CN, -OCH ３ , -CH ３ , -CF ３ or R ２ and R ３ together form a compound of the formula -O(CH ２ ) ｎ O- or -O(CF ２ ) ｎ O— group, where n is 1 or 2, R ４ are independently —H or C １ -C ４ is alkyl, R ５ is independent, C １ -C ４ is alkyl, L １ are independently absent or -(C １ -C １０ alkylene)-, L ２ are independently absent or —OCH ２ -L ３ -*, -SCH ２ -L ３ -*, -S(=O)-L ３ -*, -SO ２ -L ３ -*, -C(=O)-L ３ -*, -N(R ６ ) CH ２ -L ３ -*, -N(R ６ ) C(=O)-L ３ -*, -N(R ６ )C(=O)N(R ７ )-L ３ -*, -C(=O)N(R ６ ) CH ２ -L ３ -*, -OC(=O)N(R ６ ) CH ２ -L ３ -* or -N(R ６ )C(=O)OCH ２ -L ３ -*, where * indicates the site covalently bonded to Q'; L ３ are independently -(C １ -C １０ alkylene)-, -CH ２ OCH ２ CH ２ - or -CH ２ CH ２ OCH ２ CH ２ - and Each R ６ and R ７ are independently —H, C １ -C ６ Alkyl, C １ -C ６ Fluoroalkyl, C ３ -C ６ cycloalkyl, aryl, heteroaryl, or benzyl; Q' is -O- or -S-; E is a peptide comprising 2 to 10 amino acids, wherein E is optionally substituted with one or more polyols, and the N-terminus of said peptide is covalently linked to Z; Zは、-C(=O)-L ４ -Y、 【Transformation 3】 wherein m represents an integer from 1 to 10; L ４ is -(C １ -C １０ alkylene)-*, -CH ２ CH ２ (OCH ２ CH ２ ) ｎ N (R ８ ) C(=O)-L ５ -*,or -CH ２ (OCH ２ CH ２ ) ｎ N (R ８ ) C(=O)-L ５ -*, where n represents an integer from 1 to 10, and * represents the site covalently bonded to Y; L ５ is -(C １ -C １０ alkylene)-, R ８ is -H or -CH ３ and Y is an electrophilic group; In the formula, R ２ and R ３ is bonded to -OCH ２ When forming O-, R １ is -CH ２ CH ２ CH ２ CH ３ A compound of formula (II) which is not: or a pharmaceutically acceptable salt thereof.

29. 29. The compound of claim 28, wherein E is a peptide of two, three, or four amino acids. Each amino acid in the peptide is an L-amino acid, or at least one amino acid in the peptide is a D-amino acid.

30. 30. The compound of claim 28 or 29, wherein E comprises one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, wherein the glutamine or glutamic acid is optionally substituted by a polyol.

31. 31. The compound of claim 30, wherein E comprises an amino acid selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, wherein the glutamine or glutamic acid is optionally substituted by a polyol.

32. E comprises an amino acid having the structure: 【Chemistry 4】 In the formula, R ９ is -H or C １ -C ６ The compound of any one of claims 28 to 31, which is alkyl.

33. 33. The compound of claim 32, wherein E comprises an amino acid having the following structure: 【Transformation 5】

34. E is -Ala-Val-*, -Val-Ala-*, -Gly-Gly-*, -Val-Cit-*, -Cit-Val-*, -Leu-Ala-*, -Ala-Leu-*, -Leu-Cit-*, -Cit-Leu-*, -Leu-Ala-*, -Ala-Leu-*, -Lys-Lys-*, -Ala-Lys-*, -Lys-Ala-*, -Val-Lys-*, -Lys-Val-*, -Tyr-Arg-*, -Arg-Tyr-*, -Arg-Arg-*, -Ala-Ala-*, -Phe-Lys-*, -Lys-Phe-*, -Thr-Thr-*, -Thr-Met-*, -Met-Thr-*, -Met-Tyr-*, -Tyr-Met-*, -Phe-Gln-*, -Gln-Phe-*, -Gly-Ser-*, -Leu-Gln-*, -Gln-Leu-*, -Ser-Ala-*, -Ser-Gly-*, -Val-Thr-*, -Thr-Val-*, -Val-Gln-*, -Ser-Val-*, -Val-Ser-*, -Ala-Met-*, -Met-Ala-*, -Val-Arg-*, -Arg-#--*、-Ae-Al-*、-Al-0h-*、-&----*、--.------------ ＊ 、-Gly-Gly-Gly- ＊ ,-Ala-Val-Ala- ＊ 、-Gly-Val-ｧly- ＊ 、-Ala-Val-Gly- ＊ 、 -Gly-Phe-Lys-*, -Lys-Phe-Gly-*, -Leu-Ala-Leu-*, -Val-Ala-Leu-*, -Leu-Ala-Val-*, -Val-Ala-Val-*, -Ala-Val-Ala-Gly- ＊ , -Gly-Phe-Gly-Gly- ＊ , -Gly-Gly-Phe-Gly-*, -Ala-Val-Gly-Gly-*, -Ala-Ala-Ala-Ala- ＊ , -Ala-Val-Ala-Ala- ＊ 30. The compound of claim 28 or 29, wherein * indicates the N-terminus of the peptide covalently attached to Z.

35. E is -L-Ala-D-Val-*, -L-Val-D-Ala-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Val-D-Cit-*, -L-Val-D-Arg-*, -L-Val-D-Cit-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Arg-D- Arg-*, -L-Ala-D-Ala-*, -L-Ala-D-Lys-*, -L-Ala-D-Arg-*, -L-Ala-D-Ala-L-Ala- ＊ , -L-Ala-D-Val-L-Ala- ＊ , -L-Ala-D-Ala-Gly- ＊ , and -L-Ala-D-Val-Gly- ＊ 35. The compound of claim 34, wherein the compound is selected from the group consisting of: wherein * indicates the N-terminus of the peptide covalently attached to Z.

36. -E-NH--CH ２－－ 29. The compound of claim 28, wherein: has one of the following structures, where * indicates the N-terminus of the peptide covalently attached to Z: 【Transformation 6】

37. L ４ But -(C １ -C １０ The compound of any one of claims 28 to 36, wherein the aryl group is aryl, ...

38. L ４ But -CH ２ CH ２ (OCH ２ CH ２ ) ｎ N (R ８ ) C(=O)-L ５ -* or -CH ２ (OCH ２ CH ２ ) ｎ N (R ８ ) C(=O)-L ５ 37. The compound of any one of claims 28 to 36, wherein n represents an integer from 1 to 10, and * represents the site of covalent attachment to Y.

39. L ４ が、-CH ２ CH ２ CH ２ CH ２ CH ２ -、-CH ２ CH ２ -、-CH ２ -、-CH ２ CH ２ OCH ２ CH ２ OCH ２ CH ２ NHC(=O)H ２ CH ２ -*、or -CH ２ OCH ２ CH ２ OCH ２ CH ２ NHC(=O)CH ２ CH ２ 37. The compound of any one of claims 28 to 36, wherein * represents the site of covalent attachment to Y.

40. 40. The compound of any one of claims 28 to 39, wherein Y is a Michael acceptor group, a succinimide, an epoxide, or a halogen.

41. Y is, 【Transformation 7】 and In the formula, R １０ and R １１ are each independently —H or C １ -C ３ The compound of any one of claims 28 to 40, which is alkyl.

42. 37. The compound of any one of claims 28 to 36, wherein Z is: 【Transformation 8】

43. Z-E-NH--CH ２ 29. The compound of claim 28, wherein - has one of the following structures: 【Chemistry 9】

44. R １ is -H or -CH ２ CH ３ and R ２ is —OH or alkoxy, and R ３ When is -H, -L １ -L ２ -Q'- is -CH(R')CH ２ O- or -CH(R')(CH ２ ) ２ Instead of O—, R′ is —H, or C １ -C ６ Alkyl, alkoxy, substituted alkyl, phenyl, or PhCH ２ The compound according to any one of claims 28 to 43, wherein

45. L １ and L ２ 45. The compound of any one of claims 28 to 44, wherein at least one of:

46. R １ , R ２ , and R ３ The compound of any one of claims 28 to 45, wherein at least one of is not -H.

47. R １ became independent and C １ -C ６ Alkyl, C ２ -C ６ Alkenyl, C ２ -C ６ Alkynyl, silyl, C ３ -C ６ Cycloalkyl, C １ -C ６ 47. The compound of any one of claims 28 to 46, which is an alkyl halide, an alkene, or an alkyne.

48. R １ are independently —H or C １ -C ６ The compound of any one of claims 28 to 46, which is alkyl.

49. R ２ are independently —H, —F, —N(R ４ ) ２ , -N(R ４ ) (R ５ ), -OR ４ , -SR ４ , -S(=O)R ５ , -SO ２ R ５ , C １ -C ６ Alkyl, or C １ -C ６ fluoroalkyl, and R ３ are independently —H, —F, —CN, or —OCH ３ , -CH ３ , or -CF ３ The compound according to any one of claims 28 to 48, wherein

50. R ２ became independent and C １ -C ６ Alkyl, C １ -C ６ 50. The compound of any one of claims 28 to 49, wherein the fluoroalkyl is -F.

51. R ３ are independently —H, —F, —CN, or —CF ３ The compound according to any one of claims 28 to 50, wherein

52. R ３ are independently —F, —CN, —OCH ３ , -CH ３ , or -CF ３ The compound according to any one of claims 28 to 50, wherein

53. R ２ and R ３ is bonded to form -O(CH ２ ) ｎ O- or --O(CF ２ ) ｎ 49. The compound of any one of claims 28 to 48, which forms O-, wherein n is 1 or 2.

54. 47. The compound of any one of claims 28-46, wherein D is represented by one of the following structures: Table 48

55. R １ is —H or C １ -C ６ 55. The compound of claim 54, which is alkyl.

56. 55. The compound of claim 54, wherein D is represented by one of the following structures: Table 49

57. L １ But -(C １ -C １０ alkylene)-, and L ２ The compound of any one of claims 28 to 56, wherein is absent.

58. L １ But -(C １ -C １０ alkylene)-, and L ２ But -N(R ６ ) CH ２ -L ３ -* or -N(R ６ ) C(=O)-L ３ 57. The compound of any one of claims 28 to 56, wherein * represents the site covalently bonded to Q'.

59. L １ But it doesn't exist, L ２ But -N(R ６ ) CH ２ -L ３ -* or -N(R ６ ) C(=O)-L ３ 57. The compound of any one of claims 28 to 56, wherein * represents the site covalently bonded to Q'.

60. L ３ But -(C １ -C １０ 60. The compound of claim 58 or 59, wherein:

61. R ６ is —H or —CH ３ The compound according to any one of claims 58 to 60, wherein

62. L １ -L ２ But -CH ２ -, -CH ２ CH ２ -, -CH ２ CH ２ CH ２ - or --CH ２ CH ２ CH ２ CH ２ The compound according to any one of claims 28 to 56, wherein

63. L １ -L ２ Yes, -OCH ２ CH ２ -*、 -OCH ２ CH ２ OCH ２ CH ２ -*、-SCH ２ CH ２ -*、-SCH ２ CH ２ OCH ２ CH ２ -*、-S(=O)CH ２ -*、-S ２ CH ２ -*、 -C(=0)CH ２ -*、-NHCH ２ CH ２ -*、-N(CH ３ )CH ２ CH ２ -*、-N(CF ３ )CH ２ CH ２ -*、-NHC(=O)CH ２ -*、-CH ２ NHC(=0)CH ２ -*、-CH ２ CH ２ NHC(=0)CH ２ -*、-CH ２ N(CH) ３ )C(=O)CH ２ -*、 -N(CH) ３ )C(=O)CH ２ -*、-N(CH ３ )C(=O)CH ２ CH ２ -*、-C(=O)NHCH ２ CH ２ -*、-NHC(=O)NHCH ２ CH ２ -*、-NHC(=O)OCH ２ CH ２ -*、-CH ２ OC(=O)NHCH ２ CH ２ -*、または -C(=O)N(CH ３ ) CH ２ CH ２ 57. The compound of any one of claims 28 to 56, wherein * represents the site covalently bonded to Q'.

64. L １ -L ２ -Q'が、-CH ２ CH ２ CH ２ CH ２ O-CH ２ CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ OCH ２ CH ２ O-CH ２ SCH ２ CH ２ O-CH ２ NHC(=0)CH ２ O-CH ２ CH ２ NHC(=0)CH ２ O-CH ２ N(CH) ３ )C(=O)CH ２ O-OCH ２ CH ２ O-OCH ２ CH ２ CH ２ O-SCH ２ CH ２ CH ２ O-SCH ２ CH ２ O-NHCH ２ CH ２ O-NHCH ２ CH ２ CH ２ O-N (CH) ３ )CH ２ CH ２ O-、-C(=O)NHCH ２ CH ２ O-,-NHC(=O)CH ２ O-CH ２ S(=0)CH ２ O-CH ２ SO ２ CH ２ O-CH ２ CH ２ CH ２ CH ２ S-CH ２ CH ２ CH ２ S-, -CH ２ CH ２ S-, -CH ２ CH ２ OCH ２ CH ２ S-, -CH ２ SCH ２ CH ２ S-, -CH ２ NHC(=O)CH ２ S-, -OCH ２ CH ２ CH ２ S-, -SCH ２ CH ２ CH ２ S-, -SCH ２ CH ２ S-, -NHCH ２ CH ２ CH ２ S-, -N(CH ３ ) CH ２ CH ２ S-, -C(=O)NHCH ２ CH ２ S-, -NHC(=O)CH ２ S-, -CH ２ S(=O)CH ２ S- or -CH ２ SO ２ CH ２ The compound of any one of claims 28 to 56, which is S-.

65. D-L １ -L ２ 47. The compound of any one of claims 28-46, wherein: Table 50

66. R １ is —H or C １ -C ６ 66. The compound of claim 65, which is alkyl.

67. R １ is —H or —CH ２ CH ３ 67. The compound of claim 66, wherein:

68. The compound of any one of claims 28-64 and 66-68, wherein Q' is -O-.

69. The compound of any one of claims 28-64 and 66-68, wherein Q' is -S-.

70. D-L １ -L ２ The compound of any one of claims 28 to 46, wherein -Q'- has one of the following structures: Table 51

71. The compound has one of the following structures: 【Chemistry 10-1】 【Chemistry 10-2】 【Chemistry 10-3】 29. The compound of claim 28, wherein:

72. A compound of formula (III), {D-L １ -L ２ -Q’-CH ２ -NH-E-Z’} ｐ -C (III) or a pharmaceutically acceptable salt thereof, wherein: D has the following structural formula: 【Chemistry 11】 wherein: R １ are independently —H, C １ -C ６ Alkyl, C ２ -C ６ Alkenyl, C ２ -C ６ Alkynyl, silyl, C ３ -C ６ Cycloalkyl, C １ -C ６ Alkyl halides, C ２ -C ６ halogenated alkenyl, or C ２ -C ６ is an alkynyl halide, R ２ are independently —H, —F, —N(R ４ ) ２ , -N(R ４ ) (R ５ ), -OR ４ , -SR ４ , -S(=O)R ５ , -SO ２ R ５ , C １ -C ６ Alkyl or C １ -C ６ fluoroalkyl, and R ３ is -H, -F, -CN, -OCH ３ , -CH ３ , or -CF ３ or R ２ and R ３ together form a compound of the formula -O(CH ２ ) ｎ O- or -O(CF ２ ) ｎ O— group, where n is 1 or 2, R ４ are independently —H or C １ -C ４ is alkyl, R ５ is independent, C １ -C ４ is alkyl, L １ are independently absent or -(C １ -C １０ alkylene)-, L ２ are independently absent or —OCH ２ -L ３ -*, -SCH ２ -L ３ -*, -S(=O)-L ３ -*, -SO ２ -L ３ -*, -C(=O)-L ３ -*, -N(R ６ ) CH ２ -L ３ -*, -N(R ６ ) C(=O)-L ３ -*, -N(R ６ )C(=O)N(R ７ )-L ３ -*, -C(=O)N(R ６ ) CH ２ -L ３ -*, -OC(=O)N(R ６ ) CH ２ -L ３ -* or -N(R ６ )C(=O)OCH ２ -L ３ -*, where * indicates the site covalently bonded to Q'; L ３ are independently -(C １ -C １０ alkylene)-, -CH ２ OCH ２ CH ２ - or -CH ２ CH ２ OCH ２ CH ２ - and Each R ６ and R ７ are independently —H, C １ -C ６ Alkyl, C １ -C ６ Fluoroalkyl, C ３ -C ６ cycloalkyl, aryl, heteroaryl, or benzyl; Q' is -O- or -S-; E is a peptide comprising 2 to 10 amino acids, wherein E is optionally substituted with one or more polyols, and the N-terminus of said peptide is covalently linked to Z'; Z'は、-C(=O)-L ４ -Y'、 【Chemistry 12】 In the formula, m represents an integer of 1 to 10, and * represents a site covalently bonded to C, L ４ is -(C １ -C １０ alkylene)-, -CH ２ CH ２ (OCH ２ CH ２ ) ｎ N (R ８ ) C(=O)-L ５ -* or -CH ２ (OCH ２ CH ２ ) ｎ N (R ８ ) C(=O)-L ５ -*, where n represents an integer from 1 to 10, and * represents the site covalently bonded to Y'; L ５ is -(C １ -C １０ alkylene)-, R ８ is -H or -CH ３ and C represents a cell binding agent; Y' is a group formed by reaction of an electrophilic group with a reactive nucleophilic group present on the cell-binding agent; In the formula, R ２ and R ３ is bonded to -OCH ２ When forming O-, R １ is -CH ２ CH ２ CH ２ CH ３ Instead, A compound of formula (III), or a pharmaceutically acceptable salt thereof, wherein p has a value of 1 to 18.

73. L ４ But -(C １ -C １０ 73. The compound of claim 72, wherein:

74. L ４ But -CH ２ CH ２ (OCH ２ CH ２ ) ｎ N (R ８ ) C(=O)-L ５ -* or -CH ２ (OCH ２ CH ２ ) ｎ N (R ８ ) C(=O)-L ５ 73. The compound of claim 72, wherein n represents an integer from 1 to 10, and * represents the site of covalent attachment to Y'.

75. L ４ が、-CH ２ CH ２ CH ２ CH ２ CH ２ -、-CH ２ CH ２ -、-CH ２ -、-CH ２ CH ２ OCH ２ CH ２ OCH ２ CH ２ NHC(=O)H ２ CH ２ -*、or -CH ２ OCH ２ CH ２ OCH ２ CH ２ NHC(=O)CH ２ CH ２ 73. The compound of claim 72, wherein * represents the site covalently bonded to Y'.

76. 76. The compound of any one of claims 72 to 75, wherein Y' is formed from a Michael acceptor group, a succinimide, an epoxide, or a halogen.

77. Y' is 【Chemistry 13】 is formed from In the formula, R １０ and R １１ are each independently —H or C １ -C ３ Alkyl, 76. The compound according to any one of claims 72 to 75.

78. Y' is 【Chemistry 14】 and In the formula, R １０ and R １１ are each independently —H or C １ -C ３ 76. The compound of any one of claims 72 to 75, wherein: C is alkyl and * indicates the site of covalent bonding to said C.

79. 79. The compound of any one of claims 72 to 78, wherein Z' is formed from: 【Chemistry 15】

80. Z' is 【Chemistry 16】 and 79. The compound of any one of claims 72 to 78, wherein * indicates the site of covalent attachment to C.

81. 81. The compound of any one of claims 72-80, wherein E is a peptide of 2, 3, or 4 amino acids. Each amino acid in the peptide is an L-amino acid, or at least one amino acid in the peptide is a D-amino acid.

82. 82. The compound of any one of claims 72-81, wherein E comprises one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, wherein the glutamine or glutamic acid is optionally substituted by a polyol.

83. 83. The compound of claim 82, wherein E comprises an amino acid selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, wherein the glutamine or glutamic acid is optionally substituted by a polyol.

84. E comprises an amino acid having the structure: 【Chemistry 17】 In the formula, R ９ is -H or C １ -C ６ 84. The compound of any one of claims 72 to 83, which is alkyl.

85. 85. The compound of claim 84, wherein E comprises an amino acid having the following structure: [Chemistry 18]

86. E is -Ala-Val-*, -Val-Ala-*, -Gly-Gly-*, -Val-Cit-*, -Cit-Val-*, -Leu-Ala-*, -Ala-Leu-*, -Leu-Cit-*, -Cit-Leu-*, -Leu-Ala-*, -Ala-Leu-*, -Lys-Lys-*, -Ala-Lys-*, -Lys-Ala-*, -Val-Lys-*, -Lys-Val-*, -Tyr-Arg-*, -Arg-Tyr-*, -Arg-Arg-*, -Ala-Ala-*, -Phe-Lys-*, -Lys-Phe-*, -Thr-Thr-*, -Thr-Met-*, -Met-Thr-*, -Met-Tyr-*, -Tyr-Met-*, -Phe-Gln-*, -Gln-Phe-*, -Gly-Ser-*, -Leu-Gln-*, -Gln-Leu-*, -Ser-Ala-*, -Ser-Gly-*, -Val-Thr-*, -Thr-Val-*, -Val-Gln-*, -Ser-Val-*, -Val-Ser-*, -Ala-Met-*, -Met-Ala-*, -Val-Arg-*, -Arg-#--*、-Ph-Al-*,-Al-Pe-*、-Cit----*、--e---*- ＊ 、-Gly-Gly-Gly- ＊ ,-Ala-Val-Ala- ＊ 、-Gly-Val-ｧly- ＊ 、-Ala-Val-Gly- ＊ 、 -Gly-Phe-Lys-*, -Lys-Phe-Gly-*, -Leu-Ala-Leu-*, -Val-Ala-Leu-*, -Leu-Ala-Val-*, -Val-Ala-Val-*, -Ala-Val-Ala-Gly- ＊ , -Gly-Phe-Gly-Gly- ＊ , -Gly-Gly-Phe-Gly-*, -Ala-Val-Gly-Gly-*, -Ala-Ala-Ala-Ala- ＊ , -Ala-Val-Ala-Ala- ＊ , -Ala-Leu-Ala-Leu-*, -Leu-Ala-Leu-Ala-*, -Gly-Phe-Leu-Gly-*, and -Gly-Leu-Phe-Gly-*, wherein * is the N-terminus of said peptide covalently attached to Z'. The compound according to any one of claims 72 to 80,

87. E is -L-Ala-D-Val-*, -L-Val-D-Ala-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Val-D-Cit-*, -L-Val-D-Arg-*, -L-Val-D-Cit-*, -L-Val-D-Lys-*, -L-Val-D-Arg-*, -L-Arg-D- Arg-*, -L-Ala-D-Ala-*, -L-Ala-D-Lys-*, -L-Ala-D-Arg-*, -L-Ala-D-Ala-L-Ala- ＊ , -L-Ala-D-Val-L-Ala- ＊ , -L-Ala-D-Ala-Gly- ＊ , and -L-Ala-D-Val-Gly- ＊ 87. The compound of claim 86, wherein the compound is selected from the group consisting of: wherein * indicates the N-terminus of the peptide covalently attached to Z'.

88. -E-NH--CH ２－－ 73. The compound of claim 72, wherein: has one of the following structures, where * indicates the N-terminus of the peptide covalently attached to Z': 【Chemistry 19】

89. Z'-E-NH--CH ２ 73. The compound of claim 72, wherein: 【Chemistry 20】

90. Z'-E-NH--CH ２ 73. The compound of claim 72, wherein: is one of the following structures, where * indicates the point of attachment to said C: 【Chemistry 21】

91. R １ is -H or -CH ２ CH ３ and R ２ is —OH or alkoxy, and R ３ When is -H, -L １ -L ２ -Q'- is -CH(R')CH ２ O- or -CH(R')(CH ２ ) ２ Instead of O—, R′ is —H, or C １ -C ６ Alkyl, alkoxy, substituted alkyl, phenyl, or PhCH ２ The compound according to any one of claims 72 to 90, wherein

92. L １ and L ２ 92. The compound of any one of claims 72 to 91, wherein at least one of:

93. R １ , R ２ , and R ３ The compound of any one of claims 72 to 92, wherein at least one of: is not -H.

94. R １ became independent and C １ -C ６ Alkyl, C ２ -C ６ Alkenyl, C ２ -C ６ Alkynyl, silyl, C ３ -C ６ Cycloalkyl, C １ -C ６ 94. The compound of any one of claims 72 to 93, which is an alkyl halide, an alkene, or an alkyne.

95. R １ are independently —H or C １ -C ６ 94. The compound of any one of claims 72 to 93, which is alkyl.

96. R ２ are independently —H, —F, —N(R ４ ) ２ , -N(R ４ ) (R ５ ), -OR ４ , -SR ４ , -S(=O)R ５ , -SO ２ R ５ , C １ -C ６ Alkyl, or C １ -C ６ fluoroalkyl, and R ３ are independently —H, —F, —CN, or —OCH ３ , -CH ３ , or -CF ３ The compound according to any one of claims 72 to 95,

97. R ２ became independent and C １ -C ６ Alkyl, C １ -C ６ 96. The compound of any one of claims 72 to 95, wherein the fluoroalkyl is --F.

98. R ３ are independently —H, —F, —CN, or —CF ３ The compound according to any one of claims 72 to 97,

99. R ３ are independently —F, —CN, —OCH ３ , -CH ３ , or -CF ３ That is, The compound according to any one of claims 72 to 97.

100. R ２ and R ３ is bonded to form -O(CH ２ ) ｎ O- or --O(CF ２ ) ｎ 96. The compound of any one of claims 72 to 95, which forms O-, wherein n is 1 or 2.

101. 94. The compound of any one of claims 72-93, wherein D is represented by one of the following structures: Table 52

102. R １ is —H or C １ -C ６ 102. The compound of claim 101, which is alkyl.

103. 102. The compound of claim 101, wherein D is represented by one of the following structures: Table 53

104. L １ But -(C １ -C １０ alkylene)-, and L ２ The compound according to any one of claims 72 to 103, wherein is absent.

105. L １ But -(C １ -C １０ alkylene)-, and L ２ But -N(R ６ ) CH ２ -L ３ -* or -N(R ６ ) C(=O)-L ３ The compound of any one of claims 72 to 103, wherein * represents the site covalently bonded to Q'.

106. L １ But it doesn't exist, L ２ But -N(R ６ ) CH ２ -L ３ -* or -N(R ６ ) C(=O)-L ３ The compound of any one of claims 72 to 103, wherein * represents the site covalently bonded to Q'.

107. L ３ But -(C １ -C １０ The compound of claim 105 or 106, wherein the aryl group is aryl, ...

108. R ６ is —H or —CH ３ The compound according to any one of claims 105 to 107,

109. L １ -L ２ But -CH ２ -, -CH ２ CH ２ -, -CH ２ CH ２ CH ２ - or --CH ２ CH ２ CH ２ CH ２ The compound according to any one of claims 72 to 103, wherein

110. L １ -L ２ Yes, -OCH ２ CH ２ -*、 -OCH ２ CH ２ OCH ２ CH ２ -*、-SCH ２ CH ２ -*、-SCH ２ CH ２ OCH ２ CH ２ -*、-S(=O)CH ２ -*、-S ２ CH ２ -*、 -C(=0)CH ２ -*、-NHCH ２ CH ２ -*、-N(CH ３ )CH ２ CH ２ -*、-N(CF ３ )CH ２ CH ２ -*、-NHC(=O)CH ２ -*、-CH ２ NHC(=0)CH ２ -*、-CH ２ CH ２ NHC(=0)CH ２ -*、-CH ２ N(CH) ３ )C(=O)CH ２ -* -N(CH) ３ )C(=O)CH ２ -*、-N(CH ３ )C(=O)CH ２ CH ２ -*、-C(=O)NHCH ２ CH ２ -*、-NHC(=O)NHCH ２ CH ２ -*、-NHC(=O)OCH ２ CH ２ -*、-CH ２ OC(=O)NHCH ２ CH ２ -*、または -C(=O)N(CH ３ ) CH ２ CH ２ The compound of any one of claims 72 to 103, wherein * represents the site covalently bonded to Q'.

111. L １ -L ２ -Q'が、-CH ２ CH ２ CH ２ CH ２ O-CH ２ CH ２ CH ２ O-CH ２ CH ２ O-CH ２ CH ２ OCH ２ CH ２ O-CH ２ SCH ２ CH ２ O-CH ２ NHC(=0)CH ２ O-CH ２ CH ２ NHC(=0)CH ２ O-CH ２ N(CH) ３ )C(=O)CH ２ O-OCH ２ CH ２ O-OCH ２ CH ２ CH ２ O-SCH ２ CH ２ CH ２ O-SCH ２ CH ２ O-NHCH ２ CH ２ O-NHCH ２ CH ２ CH ２ O-N (CH) ３ )CH ２ CH ２ O-、-C(=O)NHCH ２ CH ２ O-,-NHC(=O)CH ２ O-CH ２ S(=0)CH ２ O-CH ２ SO ２ CH ２ O-CH ２ CH ２ CH ２ CH ２ S-CH ２ CH ２ CH ２ S-, -CH ２ CH ２ S-, -CH ２ CH ２ OCH ２ CH ２ S-, -CH ２ SCH ２ CH ２ S-, -CH ２ NHC(=O)CH ２ S-, -OCH ２ CH ２ CH ２ S-, -SCH ２ CH ２ CH ２ S-, -SCH ２ CH ２ S-, -NHCH ２ CH ２ CH ２ S-, -N(CH ３ ) CH ２ CH ２ S-, -C(=O)NHCH ２ CH ２ S-, -NHC(=O)CH ２ S-, -CH ２ S(=O)CH ２ S- or -CH ２ SO ２ CH ２ The compound of any one of claims 72 to 103, which is S-.

112. D-L １ -L ２ 104. The compound of any one of claims 72-103, wherein: Table 54

113. R １ is —H or C １ -C ６ 113. The compound of claim 112, which is alkyl.

114. R １ is —H or —CH ２ CH ３ 114. The compound of claim 113, wherein:

115. The compound of any one of claims 72 to 110 and 112 to 114, wherein Q' is -O-.

116. The compound of any one of claims 72-110 and 112-114, wherein Q' is -S-.

117. D-L １ -L ２ The compound of any one of claims 72 to 103, wherein -Q'- has one of the following structures: Table 55

118. D-L １ -L ２ -Q'-CH ２ 73. The compound of claim 72, wherein -NH-E-Z'- is formed from one of the following structures: 【Chemistry 22-1】 【Chemistry 22-2】 【Chemistry 22-3】

119. {D-L １ -L ２ -Q'-CH ２ -NH-E-Z'} ｐ 73. The compound of claim 72, wherein C is one of the following structures: C is a monoclonal antibody; p is a drug-to-antibody ratio (DAR); and p is an average number ranging from about 2 to 10, 4 to 8, 7 to 8, or 3.2 to 8.

0. 【Chemistry 23-1】 【Chemistry 23-2】 【Chemistry 23-3】 [Chemistry 23-4]

120. 120. Any of claims 72 to 119, wherein the cell binding agent is an antibody or an antigen-binding fragment thereof.

3. The compound according to claim 1.

121. 121. The compound of claim 120, wherein the cell binding agent is a monoclonal antibody or an antigen-binding fragment thereof.

122. A pharmaceutical composition comprising a compound according to any one of claims 72 to 121.

123. 72. A method of preparing a conjugate comprising a cell-binding agent and a drug, the method comprising contacting the cell-binding agent with a compound of any one of claims 28 to 71, such that a covalent bond is formed between the cell-binding agent and the compound.

124. 124. The method of claim 123, wherein the cell binding agent is an antibody or an antigen-binding fragment thereof.

125. 125. The method of claim 124, wherein the cell binding agent is a monoclonal antibody or an antigen-binding fragment thereof.

126. A conjugate comprising a cell-binding agent and a drug, the conjugate being prepared according to the method of any one of claims 123 to 125.

127. 127. The conjugate of claim 126, comprising a cell binding agent that is an antibody or an antigen-binding fragment thereof.

128. 128. The conjugate of claim 127, comprising a cell-binding agent that is a monoclonal antibody or an antigen-binding fragment thereof.

129. 122. A method of treating a cell proliferative disease or disorder or inhibiting abnormal cell growth, comprising administering to a subject in need thereof a compound of any one of claims 72 to 121 or a pharmaceutical composition of claim 122.

130. 130. The method of claim 129, wherein the method is for treating cancer.

131. 131. The method of claim 130, wherein the cancer is adenocarcinoma, brain cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, CNS tumor, colon or colorectal cancer, diffuse pontine glioma (DIPG), endometrial cancer, esophageal cancer, Ewing's sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, blood cancer, Hodgkin's lymphoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, myelodysplastic syndrome (MDS), neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, pancreatic cancer, peritoneal cancer, prostate cancer, ovarian cancer, renal cancer, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, or Wilms' tumor.

132. 132. The method of claim 131, wherein the cancer is breast cancer.

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