Aureastatin derivatives and their conjugates
Patent Information
- Application Number
- JP2025504427
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2022-07-27
- Filing Date
- 2023-07-26
- Publication Date
- 2025-08-01
Smart Images
Figure 2025524986000001_ABST
Abstract
Description
Technical Field
[0001] Cross-reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 392,806, filed Jul. 27, 2022, which is hereby incorporated by reference in its entirety.
Background Art
[0002] Antibody-drug conjugates (ADCs) have received significant attention as a new class of therapeutic agents. For example, ADCs can utilize monoclonal antibodies (mAbs) for the targeted delivery of cytotoxic agents to tumor cells, thereby enabling the use of highly cytotoxic agents that could not be used with conventional non-targeted modes. The design of ADCs - typically characterized by the attachment of a cytotoxic agent to an antibody via a linker - requires consideration of various factors, including the presence of a chemical binding site on the agent for attachment to the linker and linker technology for attaching the agent to the antibody in a conditionally stable manner.
[0003] Described herein are new cytotoxic agents that are microtubule inhibitors and can form a payload in combination with a peptide linker. The payload is useful for the preparation of single-drug conjugates and / or dual-drug conjugates when combined with a cell-binding agent. The ADCs described herein are useful for the treatment of cell proliferative diseases such as cancer.
Summary of the Invention
[0004] New cytotoxic agents according to formula (I) are described herein. These cytotoxic agents can, in combination with a peptide linker, form a payload according to formula (II). The resulting payload can then be used to prepare a cell-binding agent and a single-agent conjugate of formula (III), and / or a cell-binding agent and a dual-agent conjugate of formula (IV) when combined with another payload having a different formula. The compounds of formula (III) and formula (IV) include ADCs useful for the treatment of cell proliferative diseases such as cancer. Furthermore, by incorporating two classes of payloads (e.g., two classes of payloads with different mechanisms of action (MOA)) into the ADC, the efficacy can be increased and the therapeutic window of the drug can be expanded.
[0005] Accordingly, in one aspect, the invention provides a compound of formula (I) D-Q(I) or a pharmaceutically acceptable salt thereof, wherein D is represented by the following structural formula
Chemical formula
[0006] In a plurality of embodiments, R 1 is -H.
[0007] In multiple embodiments, R 1 is -OH.
[0008] In multiple embodiments, R 2 is -CH3.
[0009] In multiple embodiments, R 2 is -C(=O)OH.
[0010] In multiple embodiments, R 2 is -C(=O)NHCH2CH2CH2OH.
[0011] In multiple embodiments, R 2 is
Chemical formula
[0012] In multiple embodiments, R 1 is -H, and R 2 is -C(=O)OH.
[0013] In multiple embodiments, R 1 is -H, and R 2 is -C(=O)NHCH2CH2CH2OH.
[0014] In multiple embodiments, R 1 is -H, and R 2 is
Chemical formula
[0015] In multiple embodiments, R 1 is -OH, and R 2 is -CH3.
[0016] In multiple embodiments, R 3 and R 4 are both -H.
[0017] In multiple embodiments, R3 and R 4 are both -CH3.
[0018] In multiple embodiments, n is 1.
[0019] In multiple embodiments, Q is -H.
[0020] In multiple embodiments, Q is -CH3.
[0021] In multiple embodiments, D is represented by one of the following structures:
Chemical formula
[0022] In multiple embodiments, the compound has one of the following structures
Chemical formula
[0023] In another aspect, the present invention provides a compound of formula (II) D-CH2-NH-E-Z (II) or a pharmaceutically acceptable salt thereof, wherein D is represented by the following structural formula
Chemical formula
Chemical formula
[0024] In a plurality of embodiments, R 1 is -H.
[0025] In a plurality of embodiments, R 1 is -OH.
[0026] In a plurality of embodiments, R 2 is -CH3.
[0027] In a plurality of embodiments, R 2 is -C(=O)OH.
[0028] In multiple embodiments, R 2 is -C(=O)NHCH2CH2CH2OH.
[0029] In multiple embodiments, R 2 is
Chemical formula
[0030] In multiple embodiments, R 1 is -H, and R 2 is -C(=O)OH.
[0031] In multiple embodiments, R 1 is -H, and R 2 is -C(=O)NHCH2CH2CH2OH.
[0032] In multiple embodiments, R 1 is -H, and R 2 is
Chemical formula
[0033] In multiple embodiments, R 1 is -OH, and R 2 is -CH3.
[0034] In multiple embodiments, R 3 and R 4 are both -H.
[0035] In multiple embodiments, R 3 and R 4 are both -CH3.
[0036] In multiple embodiments, n is 1.
[0037] In multiple embodiments, 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. [[ID=l]]
[0038] In multiple embodiments, E contains one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, and the glutamine or glutamic acid may optionally be substituted by a polyol.
[0039] In multiple embodiments, E contains an amino acid having the following structure:
Chemical formula
[0040] In multiple embodiments, E contains an amino acid having the following structure.
Chemical formula
[0041] In multiple embodiments, 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- * selected from the group consisting of, wherein * represents the N-terminus of the peptide covalently attached to Z.
[0042] In a plurality of embodiments, E is -L-Ala-L-Val- * , -L-Val-L-Ala- * , -L-Val-L-Lys- * , -L-Val-L-Arg- * , -L-Val-L-Cit- * , -L-Ala-L-Val-L-Glu- * , -L-Ala-L-Ala-L-Ala- * , -L-Ala-L-Val-L-Ala- * , -L-Ala-L-Ala-Gly- * , -L-Ala-L-Val-Gly- * , -Gly-Gly-L-Glu- * , -Gly-L-Phe-Gly-Gly- * , -Gly-L-Glu-Gly-Gly- * selected from the group consisting of * represents the N-terminus of the peptide covalently attached to Z.
[0043] In a plurality of embodiments, Z is -C(=O)-L-Y.
[0044] In multiple embodiments, Z is
Chemical formula
[0045] In multiple embodiments, Z is
Chemical formula
[0046] In multiple embodiments, L is -(C1-C 10 alkylene)-.
[0047] In multiple embodiments, L is, -CH2(OCH2CH2) j - * , -CH2CH2(OCH2CH2) j -, or -(OCH2CH2) j - wherein j represents an integer from 1 to 10, * indicates the site covalently bonded to Y.
[0048] In multiple embodiments, L is, -CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1- * , or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1- * wherein j represents an integer from 1 to 10, * indicates the site covalently bonded to Y.
[0049] In multiple embodiments, L1 is, -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2- * or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2- * wherein, * indicates the site covalently bonded to Y.
[0050] In multiple embodiments, Y is a Michael acceptor group, succinimide, epoxide, or halogen.
[0051] In multiple embodiments, Y is
Chemical formula
[0052] In multiple embodiments, Z is
Chemical formula
[0053] In multiple embodiments, -E-NH-CH2- has one of the following structures, wherein * represents the N-terminus of the peptide that covalently binds to Z.
Chemical formula
[0054] In multiple embodiments, Z-E-NH-CH2- has one of the following structures.
Chemical formula
[0055] In multiple embodiments, D is represented by one of the following structures.
Chemical formula
[0056] In multiple embodiments, the compound has one of the following structures
Chemical formula
[0057] In a further aspect, the present invention relates to a compound of formula (III) {D-CH2-NH-E-Z’} p -C(III) characterized in that wherein D is represented by the following structural formula:
Chemical formula
Chemical formula
[0058] In a plurality of embodiments, R 1 is -H.
[0059] In a plurality of embodiments, R 1 is -OH.
[0060] In a plurality of embodiments, R 2 is -CH3.
[0061] In a plurality of embodiments, R 2 is -C(=O)OH.
[0062] In a plurality of embodiments, R 2 is -C(=O)NHCH2CH2CH2OH.
[0063] In a plurality of embodiments, R 2 is
Chemical formula
[0064] In a plurality of embodiments, R 1 is -H and R 2 is -C(=O)OH.
[0065] In a plurality of embodiments, R 1 is -H and R 2is -C(=O)NHCH2CH2CH2OH.
[0066] In multiple embodiments, R 1 is -H, and R 2 is
Chemical formula
[0067] In multiple embodiments, R 1 is -OH, and R 2 is -CH3.
[0068] In multiple embodiments, R 3 and R 4 are both -H.
[0069] In multiple embodiments, R 3 and R 4 are both -CH3.
[0070] In multiple embodiments, n is 1.
[0071] In multiple embodiments, 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.
[0072] In multiple embodiments, E contains one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, and the glutamine or glutamic acid may optionally be substituted by a polyol.
[0073] In multiple embodiments, E contains an amino acid having the following structure
Chemical formula
[0074] In multiple embodiments, E includes an amino acid having the following structure.
Chemical formula
[0075] In multiple embodiments, 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- * selected from the group consisting of, wherein * represents the N-terminus of the peptide that covalently binds to Z'.
[0076] In multiple embodiments, E is -L-Ala-L-Val- * , -L-Val-L-Ala- * , -L-Val-L-Lys- * , -L-Val-L-Arg- * , -L-Val-L-Cit- * , -L-Ala-L-Val-L-Glu- * , -L-Ala-L-Ala-L-Ala- * , -L-Ala-L-Val-L-Ala- * , -L-Ala-L-Ala-Gly- * , -L-Ala-L-Val-Gly- * , -Gly-Gly-L-Glu- * , -Gly-L-Phe-Gly-Gly- * , -Gly-L-Glu-Gly-Gly- * selected from the group consisting of, wherein * represents the N-terminus of the peptide covalently bonded to Z'.
[0077] In multiple embodiments, Z' is -C(=O)-L-Y'-.
[0078] In multiple embodiments, Z' is
Chemical formula
[0079] In multiple embodiments, Z' is
Chemical formula
[0080] In multiple embodiments, L is -(C1-C 10 alkylene)-.
[0081] In a plurality of embodiments, L is -CH2(OCH2CH2) j - * , -CH2CH2(OCH2CH2) j -, or -(OCH2CH2) j -, where j represents an integer from 1 to 10, and where * represents the site for covalent attachment to Y'.
[0082] In a plurality of embodiments, L is -CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1- * or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1- * , where j represents an integer from 1 to 10, and where * represents the site for covalent attachment to Y'.
[0083] In a plurality of embodiments, L1 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2- * or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2- * , where * represents the site for covalent attachment to Y'.
[0084] In a plurality of embodiments, Y' is a group formed by the reaction of an electrophilic group and a reactive nucleophilic group present on the cell binding agent.
[0085] In a plurality of embodiments, Y' is
Chemical formula
[0086] In a plurality of embodiments, Y' is [Chemical formula] and is wherein, R 7 and R 8 are each independently -H or a C1-C3 alkyl group, * indicates the site covalently bonded to said C.
[0087] In multiple embodiments, Z’ is [Chemical formula] formed from
[0088] In multiple embodiments, Z’ is [Chemical formula] and is, wherein * indicates the site covalently bonded to C.
[0089] In multiple embodiments, -E-NH-CH2- has one of the following structures, wherein * indicates the N-terminus of the peptide covalently bonded to Z’. [Chemical formula]
[0090] In multiple embodiments, -Z’-E-NH-CH2- is formed from one of the following structures. [Chemical formula]
[0091] In multiple embodiments, -Z’-E-NH-CH2- is one of the following structures, wherein * indicates the bonding point to C. [Chemical formula]
[0092] In multiple embodiments, D is represented by one of the following structures: [Chemical Formula]
[0093] In multiple embodiments, D-CH2-NH-E-Z’- is formed from one of the following structures. [Chemical Formula]
[0094] In multiple embodiments, {D-CH2-NH-E-Z’} p -C is one of the following structures, where C is a monoclonal antibody and p is the drug-to-antibody ratio (DAR), and the average value of p ranges from about 2 to 8, 4 to 8, or 7 to 8. [Chemical Formula] [Chemical Formula]
[0095] In multiple embodiments, the average value of p ranges from about 3 to 8 (e.g., 3.2 to 8.0), or 4 to 8.
[0096] In multiple embodiments, the average value of p is about 4.
[0097] In multiple embodiments, p is 4.
[0098] In multiple embodiments, the average value of p is about 7.5.
[0099] In multiple embodiments, the average value of p is about 8.
[0100] In multiple embodiments, p is 8.
[0101] In yet a further aspect, the present invention relates to formula (IV) {D-CH2-NH-E-Z’} p’ -C-{W} t (IV) characterized by the compound of wherein D is represented by the following structural formula [Chemical formula] wherein R 1 is -H or -OH R 2 is C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH, or heteroaryl R 3 and R 4 are independently -H or C1-C3 alkyl n is an integer from 1 to 6 E is a peptide containing 2 to 10 amino acids, wherein E may be optionally substituted with one or more polyols, and the N-terminus of the peptide is covalently bonded to Z’ Z’ is -C(=O)-L-Y’- [Chemical formula] wherein m represents an integer from 1 to 10 * indicates the site covalently bonded to said C L is -(C1-C 10 alkylene)- * , -CH2(OCH2CH2) j - * , -CH2CH2(OCH2CH2) j -, -(OCH2CH2) j -, -CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1- * , or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1- *wherein j represents an integer from 1 to 10, * represents a site covalently bonded to Y’, L1 is -(C1-C 10 alkylene)-, R 5 is -H or -CH3, C represents a cell-binding agent, Y’ is a group formed by the reaction of an electrophilic group with a reactive nucleophilic group present on the cell-binding agent, W is a group formed by the reaction of compound W’ with a reactive nucleophilic group present on C, and W’ is a cell-killing agent linked to a linker such that W’ can be conjugated to C, p’ and t have values from 1 to 10, p’ and t may be the same or different numbers, p’:t is about 1:1, about 1:2, or about 2:1, and p’:t is 1:1, or 1:2, or 2:1.
[0102] In a plurality of embodiments, R 1 is -H.
[0103] In a plurality of embodiments, R 1 is -OH.
[0104] In a plurality of embodiments, R 2 is -CH3.
[0105] In a plurality of embodiments, R 2 is -C(=O)OH.
[0106] In a plurality of embodiments, R 2 is -C(=O)NHCH2CH2CH2OH.
[0107] In a plurality of embodiments, R 2 is
Chemical formula
[0108] In a plurality of embodiments, R 1is -H, and R 2 is -C(=O)OH.
[0109] In multiple embodiments, R 1 is -H, and R 2 is -C(=O)NHCH2CH2CH2OH.
[0110] In multiple embodiments, R 1 is -H, and R 2 is
Chemical formula
[0111] In multiple embodiments, R 1 is -OH, and R 2 is -CH3.
[0112] In multiple embodiments, R 3 and R 4 are both -H.
[0113] In multiple embodiments, R 3 and R 4 are both -CH3.
[0114] In multiple embodiments, n is 1.
[0115] In multiple embodiments, 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.
[0116] In multiple embodiments, E contains one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, and the glutamine or glutamic acid may optionally be substituted by a polyol.
[0117] In multiple embodiments, E contains an amino acid having the following structure, [Chemical formula] In the formula, R 6 is -H or a C1-C6 alkyl group.
[0118] In multiple embodiments, E includes an amino acid having the following structure. [Chemical formula]
[0119] In multiple embodiments, 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- * selected from the group consisting of, wherein * represents the N-terminus of the peptide covalently bound to Z'.
[0120] In a plurality of embodiments, E is -L-Ala-L-Val- * , -L-Val-L-Ala- * , -L-Val-L-Lys- * , -L-Val-L-Arg- * , -L-Val-L-Cit- * , -L-Ala-L-Val-L-Glu- * , -L-Ala-L-Ala-L-Ala- * , -L-Ala-L-Val-L-Ala- * , -L-Ala-L-Ala-Gly- * , -L-Ala-L-Val-Gly- * , -Gly-Gly-L-Glu- * , -Gly-L-Phe-Gly-Gly- * , -Gly-L-Glu-Gly-Gly- * selected from the group consisting of, wherein * represents the N-terminus of the peptide covalently bound to Z'.
[0121] In a plurality of embodiments, Z' is -C(=O)-L-Y'.
[0122] In a plurality of embodiments, Z' is
Chemical formula
[0123] In a plurality of embodiments, Z' is
Chemical formula
[0124] In multiple embodiments, L is -(C1-C 10 alkylene)-
[0125] In multiple embodiments, L is -CH2(OCH2CH2) j - * , -CH2CH2(OCH2CH2) j -, or -(OCH2CH2) j -, where j represents an integer from 1 to 10, and * represents the site covalently bonded to Y'.
[0126] In multiple embodiments, L is -CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1- * or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1- * , where j represents an integer from 1 to 10, and where * represents the site covalently bonded to Y'.
[0127] In multiple embodiments, L1 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2- * or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2- * , where * represents the site covalently bonded to Y'.
[0128] In multiple embodiments, Y' is a group formed by the reaction of an electrophilic group and a reactive nucleophilic group present on the cell binding agent.
[0129] In multiple embodiments, Y' is
Chemical formula
[0130] In multiple embodiments, Y’ is
Chemical formula
[0131] In multiple embodiments, Z’ is
Chemical formula
[0132] In multiple embodiments, Z’ is
Chemical formula
[0133] In multiple embodiments, -E-NH-CH2- has one of the following structures, wherein * indicates the N-terminus of the peptide covalently bonded to Z’.
Chemical formula
[0134] In multiple embodiments, -Z’-E-NH-CH2- is formed from one of the following structures.
Chemical formula
[0135] In multiple embodiments, -Z’-E-NH-CH2- is one of the following structures, wherein * indicates the bonding point to C. [Chemical]
[0136] In multiple embodiments, D is represented by one of the following structures: [Chemical]
[0137] In multiple embodiments, D-CH2-NH-E-Z’- is formed from one of the following structures. [Chemical] [Chemical]
[0138] In multiple embodiments, W is formed by covalently bonding compound W’ to C.
[0139] In multiple embodiments, W’ is any molecule that can be covalently bonded to C.
[0140] In multiple embodiments, D-CH2-NH-E-Z’- is formed from PL1, and W is formed from PL2.
[0141] In multiple embodiments, D-CH2-NH-E-Z’- is formed from PL3, and W is formed from PL4.
[0142] In multiple embodiments, D-CH2-NH-E-Z’- is formed from PL5, and W is formed from PL6.
[0143] In multiple embodiments, D-CH2-NH-E-Z’- is formed from PL7, and W is formed from PL8.
[0144] In multiple embodiments, D-CH2-NH-E-Z’- is formed from PL9, and W is formed from PL10.
[0145] In multiple embodiments, W’ is any compound described in International Publication No. WO2021 / 173773 A1 that can be covalently attached to C.
[0146] In multiple embodiments, W’ is any compound described in U.S. Patent Application No. 2021 / 0283125 A1 that can be covalently attached to C.
[0147] In multiple embodiments, W’ is meditecan as described in International Publication No. 2021 / 173773 A1.
[0148] In multiple embodiments, {D-CH2-NH-E-Z’} p’ -C-{W} t is one of the following structures, where C is a monoclonal antibody, p’ and t are the drug-to-antibody ratio (DAR), p’:t is 1:1 or about 1:1, and the average of p’ and t is in the range of about 1 to 7, or about 2, about 3, about 4, about 5, or about 6, respectively.
Chemical formula
[0149] In multiple embodiments, both p’ and t are 4, or the average of 4, or about 4.
[0150] In multiple embodiments, p’:t is about 1:1, about 1:2, or about 2:1.
[0151] In multiple embodiments, p’:t is 1:1, 1:2, or 2:1.
[0152] In another aspect, the present invention is a method for preparing a conjugate of formula (III) comprising a cell-binding agent and a drug, the method comprising contacting the cell-binding agent with a compound of formula (II), whereby a covalent bond is formed between the cell-binding agent and the compound of formula (II).
[0153] In yet another aspect, the present invention is a method for preparing a dual-drug conjugate of formula (IV) comprising a cell-binding agent and two different drugs, the method comprising contacting the cell-binding agent with a compound of formula (II) and another compound of a different formula, whereby a covalent bond is formed between the cell-binding agent and the compound of formula (II) and the other compound of a different formula.
[0154] In yet another aspect, the present invention features a conjugate comprising a cell-binding agent and a drug. In multiple embodiments, the conjugate is prepared according to any of the methods described herein.
[0155] In multiple embodiments, the conjugate comprises a cell-binding agent that is an antibody or an antigen-binding fragment thereof.
[0156] In multiple embodiments, the conjugate comprises a cell-binding agent that is a monoclonal antibody or an antigen-binding fragment thereof.
[0157] In multiple embodiments, the cell-binding agent is an antibody or an antigen-binding fragment thereof, and p is the drug-to-antibody ratio (DAR) and has a value from 1 to 18. In multiple embodiments, the average value of p ranges from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).
[0158] In multiple embodiments, the cell-binding agent is a monoclonal antibody or an antigen-binding fragment thereof, and p is the drug-to-antibody ratio (DAR) and has a value from 1 to 18. In multiple embodiments, the average value of p ranges from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).
[0159] In multiple embodiments, the cell-binding agent is an antibody or an antigen-binding fragment thereof, and p' and t are the drug-to-antibody ratio (DAR), having values from 1 to 10. In multiple embodiments, the average value of p' ranges from about 2 to 6. In multiple embodiments, the average value of t ranges from about 2 to 6. In multiple embodiments, p':t is about 1:1. In multiple embodiments, p':t is about 1:2. In multiple embodiments, p':t is about 2:1. In multiple embodiments, p':t is 1:1. In multiple embodiments, p':t is 1:2. In multiple embodiments, p':t is 2:1.
[0160] In multiple embodiments, the cell-binding agent is a monoclonal antibody or an antigen-binding fragment thereof, and p' and t are the drug-to-antibody ratio (DAR), having values from 1 to 10. In multiple embodiments, the average value of p' ranges from about 2 to 6. In multiple embodiments, the average value of t ranges from about 2 to 6. In multiple embodiments, p':t is about 1:1. In multiple embodiments, p':t is about 1:2. In multiple embodiments, p':t is about 2:1. In multiple embodiments, p':t is 1:1. In multiple embodiments, p':t is 1:2. In multiple embodiments, p':t is 2:1.
[0161] In multiple embodiments, the compound of formula (IV) is trastuzumab-MB0324. [Chemical formula]
[0162] In multiple embodiments, the compound of formula (IV) is trastuzumab-MB0326. [Chemical formula]
[0163] In another aspect, the present invention features a pharmaceutical composition comprising any conjugate described herein.
[0164] In yet another aspect, the present invention is a method of treating a cell proliferative disease or disorder or inhibiting abnormal cell proliferation, the method comprising administering any conjugate described herein or any pharmaceutical composition comprising any conjugate described herein.
[0165] In another aspect, the present invention features a pharmaceutical composition comprising any compound of formula (III) described herein.
[0166] In another aspect, the present invention is a method of treating a cell proliferative disease or disorder or inhibiting abnormal cell proliferation, the method comprising administering any compound of formula (III) described herein or any pharmaceutical composition comprising any compound of formula (III) described herein.
[0167] In another aspect, the present invention features a pharmaceutical composition comprising any compound of formula (IV) described herein.
[0168] In another aspect, the present invention is a method of treating a cell proliferative disease or disorder or inhibiting abnormal cell proliferation, the method comprising administering any compound of formula (IV) described herein or any pharmaceutical composition comprising any compound of formula (IV) described herein.
[0169] In multiple embodiments, this method is for treating cancer.
[0170] In multiple embodiments, the cancer is adenocarcinoma, brain cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, CNS tumor, colon cancer or colorectal cancer, diffuse intrinsic pontine glioma (DIPG), endometrial cancer, esophageal cancer, Ewing sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, blood cancer, Hodgkin lymphoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, myelodysplastic syndrome (MDS), neuroblastoma, non-Hodgkin lymphoma, osteosarcoma, pancreatic cancer, peritoneal cancer, prostate cancer, ovarian cancer, renal cell carcinoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, or Wilms tumor.
[0171] In multiple embodiments, the cancer is breast cancer.
Brief Description of the Drawings
[0172]
Figure 1
Figure 2
Figure 3
Figure 4
Best Mode for Carrying Out the Invention
[0173] Definitions Unless otherwise specified, the following terms and phrases used in this specification are intended to have the following meanings. When a trademark is used in this specification, the trademark includes, unless otherwise suggested by the context, the product formulation of the product with the trademark name, generic pharmaceuticals, and pharmaceutical active ingredients.
[0174] 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 immunoglobulin structural elements sufficient 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 mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody may contain one or more sequence elements that are humanized, primatized, chimeric, etc., as known in the art. In many embodiments, the term "antibody" is used to refer to one or more of the constructs or forms known or developed in the art for exploiting the structural and functional characteristics of antibodies in alternative presentations.For example, in multiple embodiments, the antibodies utilized in accordance with the present invention include, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., Zybodies®); 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; camelid-like antibodies, masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (SMIPsTM); single-chain or tandem diabodies (TandAb®); VHH; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPIN®; Avimers®; DART, TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; microproteins; Fynomers®; Centyrins®; and KALBITOR®. In some embodiments, the antibody may lack covalent modifications (e.g., glycosylation) that it would have if produced naturally. In some embodiments, the antibody may contain covalent modifications (e.g., the attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.], or another pendant group [e.g., polyethylene glycol, etc.]).In many embodiments, an antibody is a polypeptide that includes or comprises one or more structural elements whose amino acid sequences are recognized by those of skill in the art as complementarity determining regions (CDRs), and in some embodiments, an antibody is a polypeptide that includes or comprises at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) whose amino acid sequence is substantially identical to that found in a reference antibody. In some embodiments, an antibody agent is a polypeptide that includes or comprises a structural element whose amino acid sequence is recognized by those of skill in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent is a polypeptide protein having a binding domain that is homologous or mostly homologous to an immunoglobulin binding domain.
[0175] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and are 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 and is not to be construed as requiring production of the antibody by any particular method.
[0176] As used herein, the term “human antibody” is intended to include antibodies having variable and constant regions generated (or assembled) from human immunoglobulin sequences. In some embodiments, an antibody (or antibody component) may be considered “human” even if it contains residues or elements not encoded by human germline immunoglobulin sequences (e.g., sequence variations that may be introduced (initially) in one or more CDRs, particularly CDR3, by random or site-directed mutagenesis in vitro or by somatic mutation in vivo).
[0177] As is known in the art, the term "humanized" is used to refer to an antibody (or antibody component) whose amino acid sequence generally includes the sequences of the VH and VL regions of a reference antibody cultured in a non-human species (e.g., mouse), but which has those sequences modified relative to the reference antibody to make them more "human-like," i.e., more similar to human germline sequences. In some embodiments, a "humanized" antibody (or antibody component) immunospecifically binds to a target antigen and has a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementarity determining region (CDR) having substantially the amino acid sequence of a non-human antibody. A humanized antibody includes at least one, and typically two, substantially all of the variable domains (Fab, Fab’, F(ab’)2, FabC, Fv), with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin (i.e., the donor immunoglobulin) and all or substantially all of the framework regions being those of a human immunoglobulin consensus sequence. In some embodiments, a humanized antibody also includes at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin constant region. In some embodiments, a humanized antibody contains both a light chain and at least the variable domains of the heavy chain. The antibody also includes CH1, hinge, CH2, CH3 of the heavy chain constant region and may optionally include the CH4 region. In some embodiments, a humanized antibody includes only the humanized V L region only. In some embodiments, a humanized antibody includes only the humanized V H region only. In some embodiments, a humanized antibody includes the humanized V H region and the V L region.
[0178] An "intact antibody" includes an antigen-binding variable region, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2, CH3, and CH4 appropriate for the antibody class. The constant domains may be the native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof.
[0179] An "antibody fragment" includes a part of an intact antibody that includes its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments, diabodies, triabodies, tetra-bodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multi-specific antibody fragments formed from antibody fragment(s), fragments produced by a Fab expression library, or any of the above epitope-binding fragments that immunospecifically bind to a target antigen (e.g., a cancer cell antigen, a viral antigen, or a microbial antigen).
[0180] An "antigen" is an entity to which an antibody specifically binds.
[0181] As used herein, the term "binding" will be understood to typically refer to a non-covalent associative interaction between two or more entities. "Direct" binding involves physical contact between entities or moieties, and indirect binding involves a physical interaction through physical contact with one or more intermediate entities. The binding between two or more entities can be evaluated in any of a variety of contexts, typically where the interacting entities or moieties are studied alone 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 to which the immunoglobulin is specific. The strength of an immunological binding interaction, i.e., the affinity, can be expressed in terms of the dissociation constant (K d ) of the interaction, with a smaller K d indicating a greater 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 rates depend on the concentrations of the complex partners, the affinity of the interaction, and the geometric parameters that equally affect the rates in both directions. Thus, the "on-rate constant" (K on) and "off-rate constant" (K off ) can both be determined by concentration and calculation of the actual association and dissociation rates. (See Nature 361:186-87 (1993)). K off / K on The ratio allows cancellation of all parameters not related to affinity and is equal to the dissociation constant K d . (Generally, see Davies et al. (1990) Annual Rev Biochem 59:439-473).)
[0182] The terms "specific binding" and "specifically binds" mean that an antibody or antibody derivative binds in a highly selective manner to its corresponding epitope of the target antigen rather than to a number of other antigens. Typically, an antibody or antibody derivative binds with an affinity of at least about 1×10 -7 M, preferably 10 -8 M to 10 -9 M, 10 -10 M, 10 -11 M, or 10 -12 M and binds to the given antigen with an affinity at least 2-fold higher than the affinity for binding to non-specific antigens (e.g., BSA, casein) other than the given antigen or closely related antigens. The term "specificity" refers to the ability of a cell-binding agent (e.g., an antibody or a fragment thereof as described herein) to specifically bind (e.g., immunoreact) to a given target antigen, e.g., a human target antigen.
[0183] 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., it may be a glycoprotein), and / or may be processed or modified in other ways. One of ordinary skill in the art will understand that a protein can be a complete polypeptide chain produced by a cell (with or without a signal sequence), or a functional portion thereof. One of ordinary skill in the art will further understand that a protein can comprise two or more polypeptide chains that are linked, for example, by one or more disulfide bonds, or associated by other means.
[0184] The terms "inhibit" or "inhibition of" mean to reduce by a measurable amount or to prevent completely.
[0185] The terms "substantial" or "substantially" refer to a majority, i.e., more than 50%, preferably 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% of a mixture or population of samples.
[0186] The term "cytotoxic activity" refers to the cell-killing effect of a drug or auristatin conjugate, or an intracellular metabolite of an auristatin conjugate. Cytotoxic activity may be expressed as an IC 50 value, which is the concentration (molar or mass) per unit volume at which half of the cells survive.
[0187] The term "cytostatic activity" refers to the anti-proliferative effect of a drug or auristatin conjugate or an intracellular metabolite of an auristatin conjugate.
[0188] As used herein, the term "cytotoxic agent" refers to a substance that has cytotoxic activity and causes cell destruction. This term is intended to include chemotherapeutic agents, as well as toxins such as low molecular weight toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including synthetic analogs and their derivatives.
[0189] As used herein, the term "cytostatic agent" refers to a substance that inhibits cell growth or a cell function that includes growth. Cytostatic agents include inhibitors such as protein inhibitors, for example enzyme inhibitors. Cytostatic agents have cytostatic activity.
[0190] As used herein, the term "payload" refers to a substance that can be conjugated to a cell binding agent. This consists of a cytotoxic agent and a linker.
[0191] As used herein, "linker", "linker moiety", or "linking group" refers to a moiety that joins two groups together, such as a cell binding agent and a cytotoxic compound. Typically, the linker is substantially inert under the conditions under which the two groups to which it is linked are joined. A bifunctional crosslinking agent may contain two reactive groups, one at each end of the linker moiety, such that one reactive group first reacts with the cytotoxic compound to provide a compound having the linker moiety and the second reactive group, which can then react with the cell binding agent. Alternatively, one end of the bifunctional crosslinking agent can first be reacted with the cell binding agent to provide a cell binding agent having the linker moiety and the second reactive group, which can then be reacted with the cytotoxic compound. The linking moiety may include a chemical bond that allows for the 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, photo-labile bonds, peptidase-labile bonds, and esterase-labile bonds (see, e.g., U.S. Pat. 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 can be used in the present invention include non-cleavable linkers, such as those described in U.S. Patent Application Publication No. 20050169933, or charged or hydrophilic linkers, which are described in detail in U.S. Patent Application Publication Nos. 2009 / 0274713, 2010 / 01293140, and International Publication No. 2009 / 134976, each of which is hereby expressly incorporated by reference herein.
[0192] The terms "abnormal cell proliferation" and "proliferative disorder" are used interchangeably in this application. As used herein, "abnormal cell proliferation" refers to cell proliferation independent of normal regulatory mechanisms (e.g., loss of contact inhibition), unless otherwise indicated. This includes, for example, the following abnormal proliferations: (1) tumor cells (tumors) that express mutant tyrosine kinases or proliferate by overexpression of receptor tyrosine kinases; (2) benign and malignant cells of other proliferative diseases in which abnormal tyrosine kinase activation occurs; (3) any tumor that proliferates by receptor tyrosine kinases; (4) any tumor that proliferates by abnormal serine / threonine kinase activation; (5) benign and malignant cells of other proliferative diseases in which abnormal serine / threonine kinase activation occurs.
[0193] The terms "cancer" and "cancerous" typically refer to or describe a physiological state or disorder in a mammal characterized by unregulated cell proliferation. A "tumor" includes one or more cancer cells and / or benign or precancerous cells.
[0194] As used herein, an "autoimmune disease" refers to a disease or disorder that arises from and targets an individual's own tissues or proteins.
[0195] As used herein, the terms "patient" or "subject" refer to any organism to which a compound provided herein is administered in accordance with the present invention for, e.g., 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 a human at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., 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 multiple embodiments, the animal is a mammal such as a mouse, rat, rabbit, non-human primate, and human, an insect, a parasite, etc. In multiple embodiments, the subject is a human. In some embodiments, the subject has and / or is susceptible to a disease, disorder, and / or condition (e.g., cancer). As used herein, "patient population" or "subject population" refers to a plurality of patients or subjects.
[0196] As used herein, the term "normal" when used to modify the term "individual" or "subject" refers to an individual or group of individuals that do not have a particular disease or condition and are not carriers of that disease or condition. Also, as used herein, the term "normal" is used herein to qualify a biological sample or sample isolated from a normal or wild-type individual or subject, e.g., a "normal" biological sample.
[0197] An individual "suffering from" a disease, disorder, and / or condition (e.g., any cancer described herein) has been diagnosed with or exhibits one or more symptoms of that disease, disorder, and / or condition.
[0198] An individual who is "susceptible to" a disease, disorder, and / or condition may not be diagnosed with and / or exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, a solid that is prone to a disease, individual, disorder, and / or condition (e.g., cancer) may be characterized by one or more of the following: (1) a genetic mutation associated with the occurrence of the disease, disorder, and / or condition, (2) a gene polymorphism associated with the occurrence of the disease, disorder, and / or condition, (3) an increase and / or decrease in the expression and / or activity of a protein associated with the occurrence of the disease, disorder, and / or condition, (4) a habit and / or lifestyle associated with the onset of the disease, disorder, and / or condition, (5) a family history of the disease, disorder, and / or condition, (6) a reaction to a specific bacterium or virus, (7) exposure to a specific chemical substance. 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.
[0199] The term "treating" or "treatment" refers to the administration of a therapeutic molecule (e.g., any compound described herein) that, unless otherwise indicated by context, partially or completely alleviates, ameliorates, mitigates, inhibits, delays onset, delays progression, reduces severity, and / or reduces incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition (e.g., cancer). Such treatment may be of a subject who exhibits no signs of the associated disease, disorder, and / or condition, and / or of a subject who exhibits only early signs of the disease, disorder, and / or condition. Alternatively, or additionally, such treatment may be of a subject who exhibits one or more established signs of the associated 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 or progression of a disease or its symptoms). In this regard, the methods of the invention include administering a "prophylactically effective amount" of a binder. "Prophylactically effective amount" refers to an amount effective at the dosage and for the period required to achieve the desired prophylactic result. Thus, the goal is treatment (including prophylactic treatment) that inhibits or slows (reduces) an undesirable physiological change or disorder such as the development or spread of cancer. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, stabilization of the disease state (i.e., does not worsen), delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (partial or complete). Treatment may also include prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those who already have a condition or disorder, as well as those who are predisposed to having a condition or disorder.
[0200] In the context of cancer, the term "treatment" 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 overall tumor mass, or reducing the number of cancer cells, and improving one or more of the symptoms associated with the disease.
[0201] In the context of autoimmune diseases, the term "treatment" includes any or all of inhibiting the replication of cells associated with the autoimmune disease state, including but not limited to cells that produce autoantibodies, reducing the burden of autoantibodies, and improving one or more of the symptoms of the autoimmune disease.
[0202] The term "therapeutically effective amount" or "effective amount" refers to the amount of conjugate effective to treat or prevent a disease or disorder in a mammal (e.g., as described herein). In the case of cancer, a therapeutically effective amount of the conjugate reduces the number of cancer cells, shrinks the tumor size, inhibits (i.e., slows to some extent, preferably stops) the invasion of cancer cells into peripheral organs, inhibits (i.e., slows to some extent, preferably stops) tumor metastasis, inhibits tumor growth to some extent, and / or reduces one or more of the symptoms associated with cancer to some extent. The agent can be cytostatic and / or cytotoxic to the extent that it can inhibit and / or kill the growth of existing cancer cells. For cancer therapies, efficacy can be determined, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (RR).
[0203] As used herein, the term "pharmaceutically acceptable form" refers to forms of the disclosed compounds, including but not limited to pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, isomers, prodrugs, and isotopically labeled derivatives. In one embodiment, "pharmaceutically acceptable form" includes but is not limited to pharmaceutically acceptable salts, esters, prodrugs, and isotopically labeled derivatives. In multiple embodiments, "pharmaceutically acceptable form" includes but is not limited to pharmaceutically acceptable isomers and stereoisomers, prodrugs, and their isotopically labeled derivatives.
[0204] In several 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., an auristatin, an auristatin payload, or an auristatin conjugate). In some embodiments, the compound can include at least one amino group, and thus, the acid addition salt can be formed with the amino group. Exemplary salts include, but are not limited to, sulfate, trifluoroacetate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphoric acid, phosphorous acid, isonicotinate, lactate, salicylate, citric acid, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate trisalt, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Pharmaceutically acceptable salts involve the inclusion of another molecule such as an acetate ion, a succinate ion, or other counterions. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. Further, a pharmaceutically acceptable salt can have two or more charged atoms in its structure. An instance where multiple charged atoms are part of a pharmaceutically acceptable salt can have multiple counterions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterions.
[0205] 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 dosage appropriate for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to the relevant population. In some embodiments, the pharmaceutical composition may be specially formulated for administration in solid or liquid form and may be adapted for: oral administration, such as a medicated drink (aqueous solution or non-aqueous solution or suspension), tablets, such as those targeted for buccal, sublingual, and systemic absorption, pills, powders, granules, pastes to be applied to the tongue; parenteral administration, such as by subcutaneous, intramuscular, intravenous, epidural injection (e.g., parenteral administration by subcutaneous, intramuscular, intravenous, epidural injection, e.g., a sterile solution or suspension, or a sustained release formulation; e.g., topical application by a cream, ointment, or controlled release patch or spray applied to the skin, lung, oral cavity; e.g., vaginal or rectal administration by a pessary, cream, or foam; sublingual; ocular; transdermal; or nasal, pulmonary, and other mucosal surfaces).
[0206] As used herein, "carrier" or "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or vehicle into which the composition is administered. In some exemplary embodiments, carriers can include, for example, sterile liquids such as water and oils, such as oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. In some embodiments, the carrier is or comprises one or more solid components. In some embodiments, the carrier can be a solvent or dispersion medium including, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by 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 in the composition isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride. Sustained absorption of injectable compositions can be brought about, for example, by including in the composition agents that delay absorption, such as aluminum monostearate and gelatin.
[0207] As used herein, the term "kit" refers to any delivery system for delivering materials. Such delivery systems can include systems that enable the storage, transport, or delivery of various diagnostic or therapeutic reagents (e.g., oligonucleotides, enzymes, etc. in appropriate containers) and / or auxiliary materials (e.g., buffers, written instructions for performing an assay, etc.) from one location to another. For example, a kit can include one or more containers (e.g., boxes, cartridges, bottles, ampules, etc.) containing the relevant reaction reagents and / or auxiliary materials. As used herein, the term "fragmented kit" refers to a delivery system comprising two or more separate containers, each of which contains a portion of the total kit components. The containers may be delivered together or separately to the intended recipient. For example, the first container may contain an enzyme for use in an assay, while the second container contains an oligonucleotide. The term "fragmented kit" is intended to, but not limited to, include kits containing analyte specific reagents (ASR) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act. In fact, any delivery system comprising two or more separate containers, each of which contains a portion of the total kit components, is included in the term "fragmented kit". In contrast, a "combined kit" refers to a delivery system that houses all of the components within a single container (e.g., a single box containing each of the desired components). The term "kit" includes both fragmented kits and combined kits.
[0208] As used herein, the term "administer" typically refers to administering a composition to a subject or system to achieve delivery of the composition or an agent contained in the composition. One of ordinary skill in the art will recognize that there are various routes available for administration to a subject, such as a human, under appropriate circumstances. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In multiple embodiments, administration is parenteral (e.g., intravenous administration). In multiple embodiments, intravenous administration is by infusion. In some specific embodiments, administration may be bronchial (e.g., by bronchial instillation), oral, dermal (e.g., one or more of topical to the dermis, intradermal, subcutaneous, transdermal), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a particular organ (e.g., within the liver), mucosal, nasal, oral, rectal, subcutaneous, sublingual, tracheal (e.g., by tracheal instillation), vaginal, intravitreal, etc.
[0209] As used herein, the term "nucleophilic" refers to a reactive group that is electron-rich, has a non-bonding electron pair that acts as a reactive site, and reacts with a positively charged or electron-deficient site. 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, hydroxyamines, and / or hydrazines), thiols, phenols, and alcohols. In a plurality of embodiments, the nucleophilic functional group comprises amino, hydrazino, hydroxyamino, hydroxy, or thio. In a plurality of embodiments, the nucleophilic functional group is carboxamide, N-hydroxycarboxamide, carboxylhydrazide, or guanidino. In a plurality of embodiments, the nucleophilic group is a thiol group or comprises a thiol group. Certain nucleophilic groups must be activated with a base so that they can react with an electrophilic group. For example, if a multifunctional compound has a nucleophilic thiol group and a hydroxyl group, the compound must be mixed with an aqueous base to remove a proton and provide a thiolate or hydroxyl anion to enable reaction with an electrophilic group. A non-nucleophilic base is preferred, except when it is desirable for the base to participate in the reaction. In some embodiments, the base may be present as a component of a buffer.
[0210] As used herein, the term "electrophilicity" refers to a reactive group that is susceptible to the influence of a nucleophilic attack, i.e., a reactive group that is susceptible to the influence of a reaction with an incoming nucleophilic group. The selection of the electrophilic group can be made such that the reaction with the nucleophilic group of the pair of reactants is possible. For example, when the nucleophilic reactive group is an amino group, the electrophilic group(s) can be selected to react with the amino group. Similarly, when the nucleophilic reactive group is a thiol moiety, the corresponding electrophilic group can be a thiol-reactive group or the like. Examples of electrophilic groups suitable for use in the present invention include, but are not limited to, carboxylic acid esters, acid chloride groups, anhydrides, isocyanato, thioisocyanato, epoxides, activated hydroxyl groups, succinimidyl esters, sulfosuccinimidyl esters, maleimides, and ethenesulfonyl. In multiple embodiments, the electrophilic group is an aldehyde, α-haloketone, maleimide, succinimide, hydroxysuccinimide, isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, tosylate, glyoxal, epoxide, oxirane, carbonate, imido ester, anhydride, fluorophenyl ester, hydroxymethylphosphine derivative, carbonate, haloacetyl, chlorotriazine, haloacetyl, alkyl halide, aziridine, acryloyl derivative, ketone, carboxylic acid, ester, acetyl chloride, or acetic anhydride. In multiple embodiments, the electrophilic group is a maleimide group or a succinimide group, or includes them. The carboxylic acid group can be activated to be reactive with a nucleophile, including reactions with suitable hydroxyl-containing compounds in the presence of a dehydrating agent such as dicyclohexylcarbodiimide (DCC) or dicyclohexylurea (DHU). For example, a carboxylic acid can be reacted with an alkoxy-substituted N-hydroxysuccinimide or N-hydroxysulfosuccinimide in the presence of DCC to form a reactive electrophilic group, an N-hydroxysuccinimide ester, and an N-hydroxysulfosuccinimide ester, respectively. The carboxylic acid can also be activated by reaction with an acyl halide, such as acetyl chloride (e.g., acetyl chloride), to provide a reactive anhydride group.In a further example, a carboxylic acid can be converted to an acid chloride group using, for example, thionyl chloride or an acyl chloride capable of an exchange reaction.
[0211] Unless otherwise indicated, the term "alkyl" by itself or as part of another term refers to a substituted or unsubstituted, straight-chain or branched, saturated or unsaturated hydrocarbon having the number of carbon atoms indicated (e.g., "-C1-C8 alkyl" or "-C1-C 10 " alkyl refers to an alkyl group having 1 to 8 or 1 to 10 carbon atoms, respectively). When the number of carbon atoms is not indicated, 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. On the other hand, branched -C3-C8 alkyls include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and -2-methylbutyl, and unsaturated -C2-C8 alkyls include -vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutyrinyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, -1-hexyl, 2-hexyl, -3-hexyl, -ethynyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl and -3-methyl-1-butynyl. The alkyl group may be unsubstituted. The alkyl group can be substituted with one or more groups. In other embodiments, the alkyl group is saturated.
[0212] Unless otherwise indicated, "alkylene" by itself or as part of another term refers to a substituted or unsubstituted saturated, branched, straight-chain or cyclic hydrocarbon radical having the indicated number of carbon atoms, typically from 1 to 10 carbon atoms, and having two monovalent radical centers derived by removal of two hydrogen atoms from the same or two different carbon atoms of the parent alkane. Typical alkylene radicals include, but are not limited to, methylene (-CH2-), 1,2-ethylene (-CH2CH2-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), etc. In a preferred embodiment, the alkylene is a branched or straight-chain hydrocarbon (i.e., not a cyclic hydrocarbon).
[0213] Unless otherwise indicated, "aryl" by itself or as part of another term means a substituted or unsubstituted monovalent carbocyclic aromatic hydrocarbon radical having the specified number of carbon atoms, typically from 6 to 20 carbon atoms, resulting from the removal of one hydrogen atom from a single carbon atom of the parent aromatic ring system. Some aryl groups are represented as "Ar" in exemplary structures. Typical aryl groups include, but are not limited to, groups derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, etc. An exemplary aryl group is the phenyl group.
[0214] Unless otherwise indicated, "arylene" by itself or as part of another term is an aryl group as defined above having two covalent bonds (i.e., divalent) and may have an ortho, meta, or para orientation.
[0215] Unless otherwise indicated, "C3-C8 heterocyclic ring", 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 called ring members) and 1 to 4 heteroatom ring members independently selected from N, O, P, or S, which are provided by removing one hydrogen atom from the ring atoms of the parent ring system. One or more N atoms, C atoms, or S atoms in the heterocyclic ring can be oxidized. The ring containing heteroatoms can be aromatic or non-aromatic. A heterocyclic ring in which all of the ring atoms are involved in aromaticity is termed a heteroaryl and otherwise is termed a heterocarbocycle. Unless otherwise stated, the heterocyclic ring is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. Thus, a heteroaryl can be attached via an aromatic carbon of its aromatic ring system, called a C-bonded heteroaryl, or via a non-double-bonded N atom (i.e., not =N-) of its aromatic ring system, called an N-bonded heteroaryl. Accordingly, a nitrogen-containing heterocyclic ring can be C-bonded or N-bonded and includes pyrrole moieties such as pyrrol-1-yl (N-bonded) and pyrrol-3-yl (C-bonded), as well as imidazole moieties such as imidazol-1-yl and imidazol-3-yl (both N-bonded), and imidazol-2-yl, imidazol-4-yl, and imidazol-5-yl moieties (all of which are C-bonded).
[0216] As used herein, the term "heteroaryl" refers to a monocyclic, bicyclic, or tricyclic carbocyclic ring system having a total of 6 to 14 ring members, the ring system having a single point of attachment to the remainder of the molecule, at least one ring in the system being aromatic, each ring in the system containing 4 to 7 ring members, and at least one ring atom being a heteroatom such as, but not limited to, nitrogen and oxygen.
[0217] Unless otherwise indicated, "C3-C8 heteroaryl" is an aromatic C3-C8 heterocycle, and the subscript indicates the total number of carbon atoms in the ring system of the heterocycle or the total number of aromatic carbon atoms in the aromatic ring system of the heteroaryl, and does not suggest the size of the ring system or the presence or absence of ring fusion. 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 given, the size of the ring system of the heterocycle or heteroaryl 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 (i.e., 5 or 6) in the heteroaromatic ring system of the heteroaryl, but does not imply the number of aromatic heteroatoms or aromatic carbons in that ring system. A fused heteroaryl is explicitly described or implied by such context and is typically indicated by the number of aromatic atoms in each aromatic ring 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 may have aromatic heteroatom(s) or the heteroatom is shared between the two rings.
[0218] A heterocycle fused to an aryl or heteroaryl such that the heterocycle remains non-aromatic and becomes part of a larger structure through attachment to the non-aromatic portion of a fused ring system is an example of an optionally substituted heterocycle, where the heterocycle is optionally substituted by ring fusion with the aryl or heteroaryl. Similarly, an aryl or heteroaryl fused to a heterocycle or carbocycle such that it becomes part of a larger structure by bonding to the aromatic portion of a fused ring system is an example of an aryl or heteroaryl that may be optionally substituted. In this case, the aryl or heterocycle is substituted by ring fusion with the heterocycle or carbocycle.
[0219] Unless otherwise indicated, "C3-C8 heterocycle", by itself or as part of another term, refers to a C3-C8 heterocyclic ring as defined above, where one of the hydrogen atoms of the heterocyclic ring is replaced by 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, where one of the hydrogen atoms of the heteroaryl group is replaced by a bond (i.e., it is divalent).
[0220] Unless otherwise indicated, "C3-C8 carbocycle" is a monovalent, substituted or unsubstituted, saturated or unsaturated, non-aromatic monocyclic or bicyclic carbocyclic ring having 3, 4, 5, 6, 7, or 8 members, derived by removal of one hydrogen atom from a ring atom of the parent ring system, by itself or as part of another term. 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-cycloheptatrieneyl, cyclooctyl, and cyclooctadienyl.
[0221] Unless otherwise indicated, "C3-C8 carbocyclo" refers to a C3-C8 carbocyclic group as defined above, by itself or as part of another term, where another hydrogen atom of the hydrogen atoms of the carbocyclic group is replaced by a bond (i.e., it is divalent).
[0222] Unless otherwise indicated, the term "heteroalkyl" by itself or in combination with another term means a stable straight or branched chain hydrocarbon, or combinations thereof, that is fully saturated or contains from 1 to 3 degrees of unsaturation, composed of the stated number of carbon atoms and heteroatoms selected from the group consisting of 1 to 10, preferably 1 to 3, O, N, Si, and S, where the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, and S may be placed at any internal position of the heteroalkyl group or at the position where the alkyl group is attached to the remainder of the molecule. The heteroatom Si may be placed at any position of the heteroalkyl group including the position where 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)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-O-CH3, and -CH=CH-N(CH3)-CH3. Up to two heteroatoms may be consecutive, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. Typically, C1-Cs heteroalkyl or heteroalkylene has from 1 to 4 carbon atoms and 1 or 2 heteroatoms, and C1-C3 heteroalkyl or heteroalkylene has from 1 to 3 carbon atoms and 1 or 2 heteroatoms. In some embodiments, the heteroalkyl or heteroalkylene is saturated.
[0223] Unless otherwise indicated, the term "heteroalkylene", by itself or in combination with other terms, means a divalent group derived from a heteroalkyl (as defined above) exemplified by -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, the heteroatom can also occupy either or both of the chain termini. Still further, for alkylene and heteroalkylene linking groups, the orientation of the linking group is not implied.
[0224] Unless otherwise suggested, the term "aminoalkyl", by itself or in combination with other terms, means a heteroalkyl, and the alkyl moiety as defined herein is substituted with an amino, alkylamino, dialkylamino, or cycloalkylamino group. Exemplary non-limiting aminoalkyls are -CH2NH2, -CH2CH2NH2, -CH2CH2NHCH3, and -CH2CH2N(CH3)2, and further includes branched species such as -CH(CH3)NH2 and -C(CH3)CH2NH2 in the (R) or (S) configuration. Alternatively, aminoalkyl is an alkyl moiety, group, or substituent as defined herein where a carbon other than the radical carbon is substituted with an amino moiety or an alkylamino moiety, and the sp 3 carbon is substituted with an amino moiety or an alkylamino moiety, and the sp 3 nitrogen is attached to at least one sp 3 carbon remaining, substituting the sp 3 carbon of the alkyl. When referring to an aminoalkyl moiety as a substituent of a larger structure or another moiety, the aminoalkyl is covalently bonded to the structure or moiety through the carbon radical of the alkyl portion of the aminoalkyl.
[0225] Unless otherwise indicated, the terms "alkylamino" and "cycloalkylamino", when used alone or in combination with other terms, mean an alkyl or cycloalkyl radical as described herein, and the radical carbon of the alkyl or cycloalkyl radical is attached to at least one sp 3It is substituted with nitrogen radicals, provided that carbon remains. When the alkylamino is substituted with another alkyl moiety at nitrogen, the resulting substituted radical is referred to as a dialkylamino moiety, group, or substituent, and the alkyl moieties substituting the nitrogen are independently selected. Exemplary and non-limiting amino, alkylamino, and dialkylamino substituents include those having the structure of -N(R’)2, where R’ in these examples is independently selected from hydrogen or C1-6 alkyl, typically hydrogen or methyl, and in cycloalkylamines contained in heterocycloalkyl, both R’ together with the nitrogen to which they are attached define a heterocyclic ring. When both R’ are hydrogen or alkyl, the moieties may be described as a primary amino group and a tertiary amine group, respectively. When one R’ is hydrogen and the other is alkyl, the moiety may be described as a secondary amino group. Primary alkylamino moieties and secondary alkylamino moieties are more reactive as nucleophiles towards carbonyl-containing electrophilic centers, and tertiary amines are more basic.
[0226] "Substituted alkyl" and "substituted aryl" mean alkyl and aryl, respectively, in which one or more hydrogen atoms, usually one hydrogen atom, are each independently substituted with a substituent. Exemplary substituents include, but are not limited to, -X, -R’, -OH, -OR’, -SR’, -N(R’)2, -N(R’)3, =NR’, -CX3, -CN, -NO2, -NR’C(=O)R’, -C(=O)R’, -C(=O)N(R’)2, -S(=O)2R’, -S(=O)2NR, -S(=O)R’, -OP(=O)(OR’)2, -P(=O)(OR’)2, -PO3=, PO3H2, -C(=O)R’, -C(=S)R’, -CO2R’, -CO2-, -C(=S)OR’, -C(=O)SR’, -C(=S)SR’, -C(=O)N(R’)2, -C(=S)N(R)2, and -C(=NR)N(R’)2, where X is selected independently from the group consisting of halogen: -F, -CI, -Br, and -I, and each R’ is -H, -C1-C 20 alkyl, -C6-C 20Aryl, -C3-C 14 is independently selected from the group consisting of a heterocyclic ring, a protecting group, and a prodrug moiety.
[0227] More typically, the substituent is selected from the group consisting of -X, -R', -OH, -OR', -SR', -N(R')2, -N(R')3, =NR', -NR'C(=O)R, -C(=O)R', -C(=O)N(R')2, -S(=O)2R', -S(=O)2NR', -S(=O)R', -C(=O)R', -C(=S)R, -C(=O)N(R')2, -C(=S)N(R')2, and -C(=NR)N(R')2, wherein each X is independently selected from the group consisting of -F and -CI, or -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, includes a protecting group, and a prodrug moiety, wherein each X is -F, wherein each R' is independently hydrogen, -C1-C 20 alkyl, -C6-C 20 aryl, -C3-C 14 is selected from the group consisting of a heterocyclic ring, a protecting group, and a prodrug moiety. 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, wherein R is hydrogen and -C1-C 20 is selected from the group consisting of alkyl. In other embodiments, the alkyl is substituted with a series of ethyleneoxy moieties, defining PEG units. The alkylene, carbocyclic, carbocyclo, arylene, heteroalkyl, heteroalkylene, heterocyclic ring, heterocyclo, heteroaryl, and heteroarylene groups described above can also be similarly substituted.
[0228] As used herein, "protecting group" means a moiety that prevents or reduces the ability of a bonded atom or functional group to participate in unwanted reactions. Typical 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 in some instances used to minimize or avoid their unwanted reactions with electrophilic compounds. In other instances, protecting groups are used to reduce or remove the nucleophilicity and / or basicity of unprotected heteroatoms. Non-limiting examples of protected oxygen are given by -OR PR , where R PR is a protecting group for hydroxyl, and hydroxyl is typically protected as an ester (e.g., acetate, propionate, or benzoate). Other protecting groups for hydroxyl avoid interfering with the nucleophilicity of organometallic reagents or other highly basic reagents, and hydroxyl is typically protected as an ether including alkyl or heterocycloalkyl ethers (e.g., methyl or tetrahydropyranyl ether), alkoxymethyl ethers (e.g., methoxymethyl or ethoxymethyl ether), 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)). Examples of nitrogen protecting groups include those for primary or secondary amines such as -NHR PR or -N(R PR )2-, where at least one of R PR is a nitrogen atom protecting group or both R PR together contain a protecting group.
[0229] A protecting group can prevent or avoid unwanted side reactions or premature loss of the protecting group during the reaction conditions necessary to effect the desired chemical transformation on other parts of the molecule and during purification of the newly formed molecule, if desired, 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 the protection of functional groups. Suitable protecting groups may also be those used in peptide coupling reactions.
[0230] "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, an aromatic alcohol refers to any aryl moiety, heteroaryl moiety, arylene moiety, and heteroarylene moiety described herein that has a hydroxyl functional group attached to an aromatic carbon of its aromatic ring system. The aromatic alcohol may be part of a larger moiety, as when its aromatic ring system is a substituent of this moiety, or may be embedded in a larger moiety by ring fusion, and may optionally be substituted with moieties described herein that contain one or more other hydroxyl substituents. A phenolic alcohol is an aromatic alcohol that has a phenol group as the aromatic ring.
[0231] "Aliphatic alcohol" refers to a moiety having a non-aromatic carbon bonded to a hydroxyl functional group -OH, either by itself or as part of a larger structure. The hydroxyl-bearing carbon, when unsubstituted (i.e., methyl alcohol), or having one, two, or three optionally substituted branched or unbranched alkyl substituents, may define a primary alcohol, or a secondary or tertiary aliphatic alcohol within a straight-chain or cyclic structure. When part of a larger structure, the alcohol may become a substituent of this structure by bonding to this hydroxyl-containing carbon through the carbon of the alkyl or other moiety described herein through the hydroxyl-containing carbon. Aliphatic alcohols contemplate non-aromatic cyclic structures (i.e., optionally substituted carbocyclic and heterocyclic carbocycles) in which the hydroxyl functional group is bonded to a non-aromatic carbon of its cyclic ring system.
[0232] As used herein, "arylalkyl" or "heteroarylalkyl" means a substituent, moiety, or group in which an aryl moiety is bonded to an alkyl moiety, i.e., aryl-alkyl-, and the alkyl and aryl groups are as described above, for example C6H5-CH2- or C6H5-CH(CH3)CH2-. An arylalkyl or heteroarylalkyl is associated with a larger structure or moiety through the sp 3 carbon of its alkyl moiety.
[0233] As used herein, the term "succinimide moiety" refers to an organic moiety consisting of a succinimide ring system, which is typically present as one of the Y' in the compound of formula (III) consisting of an alkylene-containing moiety bonded to the imide nitrogen of the ring system. The succinimide moiety is typically generated by Michael addition to the maleimide ring system of the thiol group of the cell-binding agent of the auristatin payload compound (formula II). Thus, the succinimide moiety consists of a thio-substituted succinimide ring system and, when present in an auristatin conjugate, has its imide nitrogen substituted with the remaining portion of the cell-binding agent of the auristatin conjugate and may be optionally substituted with the substituent(s) present on the maleimide ring system of the compound of formula II.
[0234] As used herein, the term "acid-amide moiety" refers to succinic acid having an amide substituent resulting from a thio-substituted succinimide ring system of a succinimide moiety in which one of its carbonyl-nitrogen bonds has been cleaved by hydrolysis. The hydrolysis that results in the acid-amide moiety provides a linker that is less likely to undergo early loss of the linker to which it is attached via removal of the antibody-thio substituent. Hydrolysis of the succinimide ring system of the thio-substituted succinimide moiety results in differences in the reactivity of the two carbonyl carbons of the succinimide ring system due at least in part to any substituents present on the maleimide ring system of the compound of formula II, and positional isomers of the acid-amide moiety are expected to be provided due to the thio substituents introduced by the targeting ligand.
[0235] As used herein, the term "prodrug" refers to a more biologically inactive or inert compound that is converted in vivo to a more biologically active compound via a chemical or biological process (i.e., a chemical reaction or enzymatic biotransformation). Typically, the biologically active compound is rendered biologically inactive (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 the blood. Exemplary prodrugs are esters and (β-D-glucopyranosides).
[0236] Often, the conjugates, linkers, and sets of components 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 linker of an auristatin payload or an auristatin conjugate, or any of the components of an auristatin. The 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 acid or amino groups that form peptide bonds, carboxylic acid 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 reactive groups, reactive sites, and exemplary functional groups that can be formed after reaction of the reactive sites. The table is not limiting. One of ordinary skill in the art will understand that the R' and R" portions of the descriptions in the table are substantially any organic moiety (e.g., an alkyl group, an aryl group, a heteroaryl group, or a substituted alkyl, aryl, or heteroaryl group) that is compatible with the bond formation provided when converting the RG to one of the exemplary functional groups. Of course, when applied to embodiments of the present invention, R' may, in some cases, represent one or more components of a self-stabilizing linker or an optional secondary linker, and R" may, in some cases, represent one or more components of any secondary linker, auristatin, stabilizing unit, or detection unit.
[0237]
Table 1
[0238] The combinations of substituents and variables contemplated by the present invention are only those that result in the formation of stable compounds. As used herein, the term "stable" refers to a compound having sufficient stability to allow for manufacture and maintaining its integrity for a period of time sufficient to be useful for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject).
[0239] Following their preparation, the compounds of the invention are preferably isolated and purified to give a composition containing them in an amount of 95% by weight or more ("substantially pure"), which is then used or formulated as described herein.
[0240] As used herein, the term "conjugate" refers to a compound or derivative thereof described herein linked to a cell binding agent.
[0241] As used herein, the term "capable of binding to a cell binding agent" refers to a compound or derivative thereof described herein, and at least one linking group or precursor thereof suitable for linking these compounds or derivatives to a cell binding agent.
[0242] The term "precursor" of a given group refers to any group that can give rise to that group by any deprotection, chemical modification, or coupling reaction.
[0243] The term "linked to a cell binding agent" refers to a conjugate molecule comprising at least one of the compounds or derivatives thereof described herein that is bound to a cell binding agent via a suitable linking group or precursor thereof.
[0244] "Therapeutic agent" encompasses both biological agents such as antibodies, peptides, proteins, enzymes, or chemotherapeutic agents.
[0245] "Chemotherapeutic agent" is a compound useful for the treatment of cancer.
[0246] A "metabolite" is a product formed by the metabolism in the body of a specific compound, its derivative, or its conjugate, or a salt thereof. The metabolites of a compound, its derivative, or its conjugate can be identified using routine techniques known in the art, and their activities are determined using tests as described herein. Such products can result, for example, from the oxidation, hydroxylation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, etc. of the administered compound. Accordingly, the present invention includes metabolites of the compounds, derivatives, or conjugates of the present invention, which are produced by a process comprising contacting the compounds, derivatives, or conjugates of the present invention with a mammal for a period sufficient to obtain the metabolites thereof, and which include the compounds, derivatives, or conjugates of the present invention.
[0247] The term "amino acid" refers to natural amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to natural amino acids. Natural amino acids include those encoded by the genetic code, as well as amino acids that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate, selenocysteine, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as natural amino acids, i.e., compounds having a carbon bonded to hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same basic chemical structure as natural amino acids. One amino acid that may be particularly useful is citrulline, a derivative of arginine that is involved in the formation of urea in the liver. Amino acid mimetics refer to chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to natural amino acids. The term "unnatural amino acid" is intended to represent the "D" stereochemical form of the above-described 20 natural amino acids. It is further understood that the term unnatural 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 amino acids having side chains that are shortened or lengthened by up to two carbon atoms, amino acids containing an optionally substituted aryl group, and amino acids containing a halogenated group, preferably a halogenated alkyl group and an aryl group, as well as N-substituted amino acids, such as N-methyl-alanine, but are not limited thereto. An amino acid or peptide can be attached to a linker / spacer or a cell binding agent via the terminal amine or terminal carboxylic acid of the amino acid or peptide. Amino acids can also be attached to a linker / spacer or a cell binding agent via side chain reactive groups 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.
[0248] In multiple embodiments, the amino acid is represented by NH2-C(R aa ’R aaa )-C(=O)OH, where R aa and R aa ’ are each independently H, a linear, branched, or cyclic alkyl optionally substituted, alkenyl or alkynyl having 1 to 10 carbon atoms, aryl, heteroaryl or heterocyclyl, or R and the N-terminal nitrogen atom can together form a heterocyclic ring (such as in the case of proline). The term “amino acid residue” 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 the amino acid, e.g., -NH-C(R aa ’R aa )-C(=O)O-.
[0249] As used herein, an amino acid can be an L-isomer or a D-isomer. Unless otherwise specified, when an amino acid is referred to, the amino acid can be an L-isomer or a D-isomer or a mixture thereof. In multiple embodiments, when a peptide is referred to by its amino acid sequence, each of the amino acids can be an L-isomer or a D-isomer unless otherwise specified. If one of the amino acids in the peptide is designated as a D-isomer, the other amino acid(s) are L-isomers unless otherwise specified. For example, the peptide D-Ala-Ala means D-Ala-L-Ala.
[0250] Amino acids and peptides can be protected by a blocking group. A blocking group is an atom or chemical moiety that protects the N-terminus of an amino acid or peptide from unwanted reactions and can be used during the synthesis of a drug-ligand conjugate. This remains attached to the N-terminus throughout the synthesis and can be removed after the synthesis of the drug conjugate is complete by chemical or other conditions that selectively achieve its 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-fluorenylmethyl carbamate (Fmoc), and carbobenzoxy (Cbz).
[0251] The term "peptide cleavable by a protease" refers to a peptide that contains 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 that is 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 portion. See, for example, Matayshi 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, in AMYLOID PROTEIN PRECURSOR IN DEVELOPMENT, AGING, AND ALZHEIMER’s Diseasee, ed. Masters et al.pp.190-198(1994).
[0252] The peptide sequences are selected based on their ability to be cleaved by proteases, non-limiting examples of which include cathepsin B, C, D, H, L, and S, and furin. Preferably, the peptide sequences can be cleaved by a suitable isolated protease in vitro, which can be tested using in vitro protease cleavage assays known in the art.
[0253] In another embodiment, the peptide sequences are selected based on their ability to be cleaved by lysosomal proteases. Lysosomal proteases are mainly located within lysosomes, but may also be located within endosomes. Examples of lysosomal proteases include, but are not limited to, cathepsin B, C, D, H, L, and S, and furin.
[0254] In another embodiment, the peptide sequences are selected based on their ability to be cleaved by tumor-associated proteases, such as proteases present extracellularly on the surface of cancer cells or in the vicinity of tumor cells. Non-limiting examples of proteases include thimet oligopeptidase (TOP), CD10 (neprilysin), matrix metalloproteases (such as MMP2 or MMP9), type II transmembrane serine proteases (e.g., hepsin, testisin, TMPRSS4, or matriptase / MT-SP1), legumain, and the 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.
[0255] The term "cation" refers to an ion having a positive charge. Cations can be monovalent (e.g., Na + , K + etc.), divalent (e.g., Ca 2+, Mg 2+ etc.), or polyvalent (e.g., Al 3+ etc.). In multiple embodiments, the cation is monovalent.
[0256] Compound of formula (I) In some aspects, the present invention features compounds (e.g., cytotoxic agents) that include auristatin derivatives. Such compounds can exhibit desirable cytotoxic properties and can be generated from the conjugates described herein after linker cleavage.
[0257] In one aspect, herein, formula (I) D-Q(I) provides a compound having the structure according to, or a pharmaceutically acceptable salt thereof, wherein D is represented by the following structural formula
Chemical formula
[0258] In multiple embodiments, R 1 is -H.
[0259] In multiple embodiments, R 1 is -OH.
[0260] In multiple embodiments, R 2is C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH, or heteroaryl.
[0261] In multiple embodiments, R 2 is C1-C3 alkyl. In multiple embodiments, R 2 is -CH3.
[0262] In multiple embodiments, R 2 is -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, or -C(=O)NHCH2CH2CH2OH. In multiple embodiments, R 2 is -C(=O)OH.
[0263] In multiple embodiments, R 2 is heteroaryl (e.g., C3-C8 heteroaryl). In multiple embodiments, R 2 is N-containing and / or S-containing heteroaryl. In multiple embodiments, R 2 is
Chemical formula
[0264] In multiple embodiments, R 1 is -H and R 2 is -C(=O)OH.
[0265] In multiple embodiments, R 1 is -H and R 2 is -C(=O)NHCH2CH2CH2OH.
[0266] In multiple embodiments, R 1 is -H and R 2 is
Chemical formula
[0267] In multiple embodiments, R 1 is -OH, and R 2 is -CH3.
[0268] In multiple embodiments, each R 3 and R 4 is independently -H or C1-C3 alkyl.
[0269] In multiple embodiments, R 3 and R 4 are both -H. In multiple embodiments, R 3 and R 4 are both C1-C3 alkyl. In multiple embodiments, R 3 and R 4 are both -CH3.
[0270] In multiple embodiments, n is an integer from 1 to 6. In multiple embodiments, n is 1. In multiple embodiments, n is 2. In multiple embodiments, n is 3. In multiple embodiments, n is 4. In multiple embodiments, n is 5. In multiple embodiments, n is 6. In multiple embodiments, n is 1.
[0271] In multiple embodiments, Q is -H.
[0272] In multiple embodiments, Q is -CH3.
[0273] In multiple embodiments, D is represented by one of the following structures:
Chemical formula
[0274] In multiple embodiments, D is (D-I).
[0275] In multiple embodiments, D is (D-II).
[0276] In a plurality of embodiments, D is (D-III).
[0277] In a plurality of embodiments, D is (D-IV).
[0278] In a plurality of embodiments, the compound is one of the following structures, [Chemical Formula] or a pharmaceutically acceptable salt thereof.
[0279] In a plurality of embodiments, the compound is compound D1, or a pharmaceutically acceptable salt thereof.
[0280] In a plurality of embodiments, the compound is compound D2, or a pharmaceutically acceptable salt thereof.
[0281] In a plurality of embodiments, the compound is compound D3, or a pharmaceutically acceptable salt thereof.
[0282] In a plurality of embodiments, the compound is compound D4, or a pharmaceutically acceptable salt thereof.
[0283] In a plurality of embodiments, the compound is compound D5, or a pharmaceutically acceptable salt thereof.
[0284] In a plurality of embodiments, the compound is compound D6, or a pharmaceutically acceptable salt thereof.
[0285] In a plurality of embodiments, the compound is compound D7 or a pharmaceutically acceptable salt thereof.
[0286] In a plurality of embodiments, the compound is compound D8 or a pharmaceutically acceptable salt thereof.
[0287] Compound of formula (II) In some embodiments, a compound comprising an auristatin derivative may comprise a peptide linker for forming a payload. Such compounds may be useful in the preparation of conjugates comprising a cell binding agent described herein.
[0288] In multiple embodiments, such compounds are formed from or comprise a structure according to any embodiment of formula (I) described herein.
[0289] In another aspect, the present invention relates to a compound of formula (II) D-CH2-NH-E-Z (II) or a pharmaceutically acceptable salt thereof, wherein D is represented by the following structural formula
Chemical formula
Chemical formula
[0290] In a plurality of embodiments, R 1 is -H.
[0291] In a plurality of embodiments, R 1 is -OH.
[0292] In a plurality of embodiments, R 2 is C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH, or heteroaryl.
[0293] In a plurality of embodiments, R 2 is C1-C3 alkyl. In a plurality of embodiments, R 2 is -CH3.
[0294] In a plurality of embodiments, R 2 is -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, or -C(=O)NHCH2CH2CH2OH. In a plurality of embodiments, R 2 is -C(=O)OH.
[0295] In a plurality of embodiments, R 2is heteroaryl (e.g., C3-C8 heteroaryl). In multiple embodiments, R 2 is N-containing and / or S-containing heteroaryl. In multiple embodiments, R 2 is
Chemical formula
[0296] In multiple embodiments, R 1 is -H and R 2 is -C(=O)OH.
[0297] In multiple embodiments, R 1 is -H and R 2 is -C(=O)NHCH2CH2CH2OH.
[0298] In multiple embodiments, R 1 is -H and R 2 is
Chemical formula
[0299] In multiple embodiments, R 1 is -OH and R 2 is -CH3.
[0300] In multiple embodiments, each R 3 and R 4 is independently -H or C1-C3 alkyl.
[0301] In multiple embodiments, R 3 and R 4 are both -H. In multiple embodiments, R 3 and R 4 are both C1-C3 alkyl. In multiple embodiments, R 3 and R 4 are both -CH3.
[0302] In multiple embodiments, n is an integer from 1 to 6. In multiple embodiments, n is 1. In multiple embodiments, n is 2. In multiple embodiments, n is 3. In multiple embodiments, n is 4. In multiple embodiments, n is 5. In multiple embodiments, n is 6. In multiple embodiments, n is 1.
[0303] In multiple embodiments, 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.
[0304] In multiple embodiments, E contains one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, and the glutamine or glutamic acid may optionally be substituted by a polyol.
[0305] In multiple embodiments, E contains an amino acid having the following structure
Chemical formula
[0306] In multiple embodiments, E contains an amino acid having the following structure.
Chemical formula
[0307] In multiple embodiments, 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- * wherein: * indicates the N-terminus of the peptide covalently attached to Z.
[0308] In some embodiments, E is -L-Ala-L-Val- * , -L-Val-L-Ala- * , -L-Val-L-Lys- * , -L-Val-L-Arg- * , -L-Val-L-Cit- * , -L-Ala-L-Val-L-Glu- * , -L-Ala-L-Ala-L-Ala- * , -L-Ala-L-Val-L-Ala- * , -L-Ala-L-Ala-Gly- * , -L-Ala-L-Val-Gly- * ,-Gly-Gly-L-Glu- * ,-Gly-L-Phe-Gly-Gly- * ,-Gly-L-Glu-Gly-Gly-* selected from the group consisting of, wherein * represents the N-terminus of the peptide covalently attached to Z.
[0309] In a plurality of embodiments, Z is -C(=O)-L-Y.
[0310] In a plurality of embodiments, Z is
Chemical formula
[0311] In a plurality of embodiments, Z is
Chemical formula
[0312] In a plurality of embodiments, L is -(C1-C 10 alkylene)-.
[0313] In a plurality of embodiments, L is, -CH2(OCH2CH2) j - * , -CH2CH2(OCH2CH2) j -, or -(OCH2CH2) j- and in the formula, j represents an integer from 1 to 10, * represents a site covalently bonded to Y.
[0314] In a plurality of embodiments, L is -CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1- * or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1- * and in the formula, j represents an integer from 1 to 10, and in the formula, * represents a site covalently bonded to Y.
[0315] In a plurality of embodiments, L1 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2- * or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2- * and in the formula, * represents a site covalently bonded to Y.
[0316] In a plurality of embodiments, Y is a Michael acceptor group, succinimide, epoxide, or halogen.
[0317] In a plurality of embodiments, Y is
Chemical formula
[0318] In a plurality of embodiments, Z is
Chemical formula
[0319] In a plurality of embodiments, -E-NH-CH2- has one of the following structures, and in the formula, *indicates the N-terminus of the peptide covalently bound to Z. [Chemical formula]
[0320] In multiple embodiments, Z-E-NH-CH2- has one of the following structures. [Chemical formula]
[0321] In multiple embodiments, D is represented by one of the following structures: [Chemical formula]
[0322] In multiple embodiments, D is (D-I).
[0323] In multiple embodiments, D is (D-II).
[0324] In multiple embodiments, D is (D-III).
[0325] In multiple embodiments, D is (D-IV).
[0326] In multiple embodiments, the compound has one of the following structures, [Chemical formula] or a pharmaceutically acceptable salt thereof.
[0327] In multiple embodiments, the compound is (PL1), or a pharmaceutically acceptable salt thereof.
[0328] In multiple embodiments, the compound is (PL2), or a pharmaceutically acceptable salt thereof.
[0329] In several embodiments, the compound is (PL3), or a pharmaceutically acceptable salt thereof.
[0330] In several embodiments, the compound is (PL4), or a pharmaceutically acceptable salt thereof.
[0331] In several embodiments, the compound is (PL5), or a pharmaceutically acceptable salt thereof.
[0332] In several embodiments, the compound is (PL6), or a pharmaceutically acceptable salt thereof.
[0333] In several embodiments, the compound is (PL7), or a pharmaceutically acceptable salt thereof.
[0334] In several embodiments, the compound is (PL8), or a pharmaceutically acceptable salt thereof.
[0335] In several embodiments, the compound is (PL9), or a pharmaceutically acceptable salt thereof.
[0336] In several embodiments, the compound is (PL10), or a pharmaceutically acceptable salt thereof.
[0337] Compounds of formula (III) and formula (IV) Compound of formula (III) In some aspects, the invention features conjugates (e.g., single-agent conjugates) that include a cell-binding agent and a payload (e.g., a payload that includes an auristatin derivative and a linker). In several embodiments, the portion of the conjugate that includes the auristatin derivative is formed from a structure according to any embodiment of formula (II) described herein.
[0338] In a further aspect, the invention features a compound of formula (III) {D-CH2-NH-E-Z’} p -C(III) wherein, In the formula, D is represented by the following structural formula, [Chemical formula] wherein, R 1 is -H or -OH, R 2 is C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH, or heteroaryl, R 3 and R 4 are independently -H or C1-C3 alkyl, n is an integer from 1 to 6, E is a peptide containing 2 to 10 amino acids. In the formula, E may be optionally substituted with one or more polyols. The N-terminus of the peptide is covalently bonded to Z’, Z’ is -C(=O)-L-Y’, [Chemical formula] wherein m represents an integer from 1 to 10, * indicates the site covalently bonded to the C, L is -(C1-C 10 alkylene)- * , -CH2(OCH2CH2)j- * , -CH2CH2(OCH2CH2)j-, -(OCH2CH2)j-, -CH2CH2(OCH2CH2)jN(R 5 )C(=O)-L1- * , or -CH2(OCH2CH2)jN(R 5 )C(=O)-L1- * wherein j represents an integer from 1 to 10, * indicates the site covalently bonded to Y’, L 1 is -(C1-C 10 alkylene)-, R 5 is -H or -CH3, C represents a cell-binding agent, Y’ is a group formed by the reaction of an electrophilic group with a nucleophilic group present on the cell-binding agent, p has a value from 1 to 18.
[0339] In a plurality of embodiments, R 1 is -H.
[0340] In a plurality of embodiments, R 1 is -OH.
[0341] In a plurality of embodiments, R 2 is C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH, or heteroaryl.
[0342] In a plurality of embodiments, R 2 is C1-C3 alkyl. In a plurality of embodiments, R 2 is -CH3.
[0343] In a plurality of embodiments, R 2 is -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, or -C(=O)NHCH2CH2CH2OH. In a plurality of embodiments, R 2 is -C(=O)OH.
[0344] In a plurality of embodiments, R 2 is heteroaryl (e.g., C3-C8 heteroaryl). In a plurality of embodiments, R 2 is N-containing and / or S-containing heteroaryl. In a plurality of embodiments, R 2 is
Chemical formula
[0345] In multiple embodiments, R 1 is -H, and R 2 is -C(=O)OH.
[0346] In multiple embodiments, R 1 is -H, and R 2 is -C(=O)NHCH2CH2CH2OH.
[0347] In multiple embodiments, R 1 is -H, and R 2 is
Chemical formula
[0348] In multiple embodiments, R 1 is -OH, and R 2 is -CH3.
[0349] In multiple embodiments, each R 3 and R 4 is independently -H, or C1-C3 alkyl.
[0350] In multiple embodiments, R 3 and R 4 are both -H. In multiple embodiments, R 3 and R 4 are both C1-C3 alkyl. In multiple embodiments, R 3 and R 4 are both -CH3.
[0351] In multiple embodiments, n is an integer from 1 to 6. In multiple embodiments, n is 1. In multiple embodiments, n is 2. In multiple embodiments, n is 3. In multiple embodiments, n is 4. In multiple embodiments, n is 5. In multiple embodiments, n is 6. In multiple embodiments, n is 1.
[0352] In multiple embodiments, 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.
[0353] In multiple embodiments, E contains one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, and the glutamine or glutamic acid may optionally be substituted by a polyol.
[0354] In multiple embodiments, E contains an amino acid having the following structure.
Chemical formula
[0355] In multiple embodiments, E contains an amino acid having the following structure.
Chemical formula
[0356] In multiple embodiments, 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- * selected from the group consisting of, wherein * represents the N-terminus of the peptide covalently attached to Z'.
[0357] In a plurality of embodiments, E is --L-Ala-L-Val- * , -L-Val-L-Ala- * , -L-Val-L-Lys- * , -L-Val-L-Arg- * , -L-Val-L-Cit- * , -L-Ala-L-Val-L-Glu- * , -L-Ala-L-Ala-L-Ala- * , -L-Ala-L-Val-L-Ala- * , -L-Ala-L-Ala-Gly- * , -L-Ala-L-Val-Gly- * , -Gly-Gly-L-Glu- * , -Gly-L-Phe-Gly-Gly- * , -Gly-L-Glu-Gly-Gly- selected from the group consisting of, wherein * represents the N-terminus of the peptide covalently attached to Z'.
[0358] In a plurality of embodiments, Z' is -C(=O)-L-Y'.
[0359] In multiple embodiments, Z’ is
Chemical formula
[0360] In multiple embodiments, Z’ is
Chemical formula
[0361] In multiple embodiments, L is -(C1-C 10 alkylene)-.
[0362] In multiple embodiments, L is -CH2(OCH2CH2) j - * , -CH2CH2(OCH2CH2) j -, or -(OCH2CH2) j -, where j represents an integer from 1 to 10, and where * represents a site covalently bonded to Y’.
[0363] In multiple embodiments, L is -CH2CH2(OCH2CH2) j N(R 5 )C(=O)-L1- * or -CH2(OCH2CH2) j N(R 5 )C(=O)-L1- * where j represents an integer from 1 to 10, and where * represents the site for covalent bonding to Y'.
[0364] In multiple embodiments, L1 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2- * or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2- * where * represents the site for covalent bonding to Y'.
[0365] In multiple embodiments, Y' is a group formed by the reaction of an electrophilic group and a reactive nucleophilic group present on the cell binding agent.
[0366] In multiple embodiments, Y' is formed from
Chemical formula
[0367] In multiple embodiments, Y' is
Chemical formula
[0368] In multiple embodiments, Z' is [Chemical formula] It is formed from
[0369] In multiple embodiments, Z’ is [Chemical formula] wherein * represents a site covalently bonded to C.
[0370] In multiple embodiments, -E-NH-CH2- has one of the following structures, wherein * represents the N-terminus of the peptide covalently bonded to Z’. [Chemical formula]
[0371] In multiple embodiments, -Z’-E-NH-CH2- is formed from one of the following structures. [Chemical formula]
[0372] In multiple embodiments, Z’-E-NH-CH2- is one of the following structures, wherein * represents the bonding point to C. [Chemical formula]
[0373] In multiple embodiments, D is represented by one of the following structures: [Chemical formula]
[0374] In multiple embodiments, D is (D-I).
[0375] In multiple embodiments, D is (D-II).
[0376] In multiple embodiments, D is (D-III).
[0377] In multiple embodiments, D is (D-IV).
[0378] In multiple embodiments, D-CH2-NH-E-Z’- is formed from one of the following structures. [Chemical formula]
[0379] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL1) or a pharmaceutically acceptable salt thereof.
[0380] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL2) or a pharmaceutically acceptable salt thereof.
[0381] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL3) or a pharmaceutically acceptable salt thereof.
[0382] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL4) or a pharmaceutically acceptable salt thereof.
[0383] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL5) or a pharmaceutically acceptable salt thereof.
[0384] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL6) or a pharmaceutically acceptable salt thereof.
[0385] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL7) or a pharmaceutically acceptable salt thereof.
[0386] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL8), or a pharmaceutically acceptable salt thereof.
[0387] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL9), or a pharmaceutically acceptable salt thereof.
[0388] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL10), or a pharmaceutically acceptable salt thereof.
[0389] In multiple embodiments, {D-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 the average value of p ranges from about 2 to 8, 4 to 8, or 7 to 8.
Chemical formula
Chemical formula
[0390] In multiple embodiments, p has a value from 1 to 18.
[0391] In multiple embodiments, the average value of p ranges from about 3 to 8 (e.g., 3.2 to 8.0), or 4 to 8.
[0392] In multiple embodiments, the average value of p is about 4.
[0393] In multiple embodiments, p is 4.
[0394] In multiple embodiments, the average value of p is about 7.5.
[0395] In multiple embodiments, the average value of p is about 8.
[0396] In multiple embodiments, p is 8.
[0397] In multiple embodiments, {D-CH2-NH-E-Z’} p -C is (PL1’), where C is a monoclonal antibody, p is the drug-to-antibody ratio (DAR), and the average value of p ranges from about 2 to 8, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).
[0398] In multiple embodiments, {D-CH2-NH-E-Z’} p -C is (PL2’), where C is a monoclonal antibody, p is the drug-to-antibody ratio (DAR), and the average value of p ranges from about 2 to 8, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).
[0399] In multiple embodiments, {D-CH2-NH-E-Z’} p -C is (PL3’), where C is a monoclonal antibody, p is the drug-to-antibody ratio (DAR), and the average value of p ranges from about 2 to 8, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).
[0400] In multiple embodiments, {D-CH2-NH-E-Z’} p -C is (PL4’), where C is a monoclonal antibody, p is the drug-to-antibody ratio (DAR), and the average value of p ranges from about 2 to 8, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).
[0401] In multiple embodiments, {D-CH2-NH-E-Z’} p -C is (PL5’), where C is a monoclonal antibody, p is the drug-to-antibody ratio (DAR), and the average value of p ranges from about 2 to 8, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).
[0402] In multiple embodiments, {D-CH2-NH-E-Z’} p -C is (PL6’), where C is a monoclonal antibody, p is the drug-to-antibody ratio (DAR), and the average value of p ranges from about 2 to 8, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).
[0403] In multiple embodiments, {D-CH2-NH-E-Z’} p -C is (PL7’), where C is a monoclonal antibody, p is the drug-to-antibody ratio (DAR), and the average value of p ranges from about 2 to 8, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).
[0404] In multiple embodiments, {D-CH2-NH-E-Z’} p -C is (PL8’), where C is a monoclonal antibody, p is the drug-to-antibody ratio (DAR), and the average value of p ranges from about 2 to 8, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).
[0405] In multiple embodiments, {D-CH2-NH-E-Z’} p -C is (PL9’), where C is a monoclonal antibody, p is the drug-to-antibody ratio (DAR), and the average value of p ranges from about 2 to 8, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).
[0406] In multiple embodiments, {D-CH2-NH-E-Z’} p -C is (PL10’), where C is a monoclonal antibody, p is the drug-to-antibody ratio (DAR), and the average value of p ranges from about 2 to 8, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).
[0407] Compound of formula (IV) In some aspects, the present invention features a conjugate (e.g., a dual-drug conjugate) comprising a cell-binding agent, a payload (e.g., a payload comprising an auristatin derivative and a linker), and another payload (e.g., another payload comprising a compound of a different formula). In multiple embodiments, the portion of the conjugate comprising the auristatin derivative is formed from formula (II) described herein.
[0408] Furthermore, considering the large variants of cancer cells, incorporating two classes of payloads with different mechanisms of action (MOA) into the ADC can increase efficacy and expand the therapeutic window of the drug. For example, anti-Her2 dual-drug ADCs are more effective than the corresponding single-drug ADCs and the co-administration of two single-drug variants in a xenograft mouse model [see, e.g., Yamazaki, C.M. & Tsuchikama, K. et al., Antibody-drug conjugates with dual payloads for combating breast tumor heterogeneity and drug resistance. Nat Commun 12, 3528 (2021). https: / / doi.org / 10.1038 / s41467-021-23793-7].
[0409] In yet a further aspect, the present invention features a compound of formula (IV) {D-CH2-NH-E-Z’} p’ -C-{W} t (IV) wherein in the formula, D is represented by the following structural formula
Chemical formula
Chemical formula
[0410] In multiple embodiments, R 1 is -H.
[0411] In multiple embodiments, R1 is -OH.
[0412] In multiple embodiments, R 2 is C1-C3 alkyl, -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, -C(=O)NHCH2CH2CH2OH, or heteroaryl.
[0413] In multiple embodiments, R 2 is C1-C3 alkyl. In multiple embodiments, R 2 is -CH3.
[0414] In multiple embodiments, R 2 is -C(=O)OH, -C(=O)OCH3, -C(=O)OCH2CH2OH, -C(=O)OCH2CH2CH2OH, -C(=O)NHCH2CH2OH, or -C(=O)NHCH2CH2CH2OH. In multiple embodiments, R 2 is -C(=O)OH.
[0415] In multiple embodiments, R 2 is heteroaryl (e.g., C3-C8 heteroaryl). In multiple embodiments, R 2 is N-containing and / or S-containing heteroaryl. In multiple embodiments, R 2 is
Chemical formula
[0416] In multiple embodiments, R 1 is -H, and R 2 is -C(=O)OH.
[0417] In multiple embodiments, R 1 is -H, and R 2 is -C(=O)NHCH2CH2CH2OH.
[0418] In multiple embodiments, R 1 is -H, and R 2 is
Chemical formula
[0419] In multiple embodiments, R 1 is -OH, and R 2 is -CH3.
[0420] In multiple embodiments, each R 3 and R 4 is independently -H, or C1-C3 alkyl.
[0421] In multiple embodiments, R 3 and R 4 are both -H. In multiple embodiments, R 3 and R 4 are both C1-C3 alkyl. In multiple embodiments, R 3 and R 4 are both -CH3.
[0422] In multiple embodiments, n is an integer from 1 to 6. In multiple embodiments, n is 1. In multiple embodiments, n is 2. In multiple embodiments, n is 3. In multiple embodiments, n is 4. In multiple embodiments, n is 5. In multiple embodiments, n is 6. In multiple embodiments, n is 1.
[0423] In multiple embodiments, 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.
[0424] In multiple embodiments, E contains one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, and the glutamine or glutamic acid may optionally be substituted by a polyol.
[0425] In multiple embodiments, E includes an amino acid having the following structure,
Chemical formula
[0426] In multiple embodiments, E includes an amino acid having the following structure.
Chemical formula
[0427] In multiple embodiments, 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- * selected from the group consisting of, wherein * represents the N-terminus of the peptide covalently attached to Z'.
[0428] In multiple embodiments, E is -L-Ala-L-Val- * 、 -L-Val-L-Ala- * 、 -L-Val-L-Lys- * 、 -L-Val-L-Arg- * 、 -L-Val-L-Cit- * 、 -L-Ala-L-Val-L-Glu- * 、 -L-Ala-L-Ala-L-Ala- * 、 -L-Ala-L-Val-L-Ala- * 、 -L-Ala-L-Ala-Gly- * 、 -L-Ala-L-Val-Gly- * 、 -Gly-Gly-L-Glu- * 、 -Gly-L-Phe-Gly-Gly- * 、 -Gly-L-Glu-Gly-Gly- * selected from the group consisting of, wherein * represents the N-terminus of the peptide covalently attached to Z'.
[0429] In multiple embodiments, Z' is -C(=O)-L-Y'.
[0430] In multiple embodiments, Z' is
Chemical formula
[0431] In a plurality of embodiments, Z’ is
Chemical formula
[0432] In a plurality of embodiments, L is -(C1-C 10 alkylene)-.
[0433] In a plurality of embodiments, L is, -CH2(OCH2CH2) j - * , -CH^2CH^2(OCH^2CH^2) j , or -(OCH^2CH^2) j -, where j represents an integer from 1 to 10, and where * indicates the site covalently bonded to Y’.
[0434] In a plurality of embodiments, L is, -CH^2CH^2(OCH^2CH^2) j N(R 5 )C(=O)-L1- * , or -CH^2(OCH^2CH^2) j N(R It should be noted that there may be some inaccuracies in the original text with the "CH^2" notations which might be a formatting issue. It should likely be "CH₂" for proper chemical notation. The translation has been done as accurately as possible based on the provided text.5 )C(=O)-L1- * where j represents an integer from 1 to 10, * indicates the site covalently bonded to Y'.
[0435] In multiple embodiments, L1 is -CH2CH2CH2CH2CH2-, -CH2CH2-, -CH2-, -CH2CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2- * or -CH2OCH2CH2OCH2CH2NHC(=O)CH2CH2- * wherein, * indicates the site covalently bonded to Y'.
[0436] In multiple embodiments, Y' is a group formed by the reaction of an electrophilic group and a reactive nucleophilic group present on the cell-binding agent.
[0437] In multiple embodiments, Y' is formed from
Chemical formula
[0438] In multiple embodiments, Y' is
Chemical formula
[0439] In multiple embodiments, Z' is
Chemical formula
[0440] In multiple embodiments, Z' is [Chemical formula] wherein, * represents a site covalently bonded to C.
[0441] In multiple embodiments, -E-NH-CH2- has one of the following structures, wherein, * represents the N-terminus of a peptide covalently bonded to Z'. [Chemical formula]
[0442] In multiple embodiments, -Z'-E-NH-CH2- is formed from one of the following structures. [Chemical formula]
[0443] In multiple embodiments, -Z'-E-NH-CH2- is one of the following structures, wherein, * represents a bonding point to C. [Chemical formula]
[0444] In multiple embodiments, D is represented by one of the following structures: [Chemical formula]
[0445] In multiple embodiments, D is (D-I).
[0446] In multiple embodiments, D is (D-II).
[0447] In multiple embodiments, D is (D-III).
[0448] In multiple embodiments, D is (D-IV).
[0449] In multiple embodiments, D-CH2-NH-E-Z’- is formed from one of the following structures.
Chemical formula
Chemical formula
[0450] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL1), or a pharmaceutically acceptable salt thereof.
[0451] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL2), or a pharmaceutically acceptable salt thereof.
[0452] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL3), or a pharmaceutically acceptable salt thereof.
[0453] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL4), or a pharmaceutically acceptable salt thereof.
[0454] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL5), or a pharmaceutically acceptable salt thereof.
[0455] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL6), or a pharmaceutically acceptable salt thereof.
[0456] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL7), or a pharmaceutically acceptable salt thereof.
[0457] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL8), or a pharmaceutically acceptable salt thereof.
[0458] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL9), or a pharmaceutically acceptable salt thereof.
[0459] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL10), or a pharmaceutically acceptable salt thereof.
[0460] In multiple embodiments, W is formed from compound W’. In multiple embodiments, W is formed by covalently bonding compound W’ to C.
[0461] In multiple embodiments, W’ is a compound capable of covalently bonding to C. In multiple embodiments, W’ is a compound described in International Patent Publication No. 2021 / 173773 A1 (e.g., a compound capable of covalently bonding to C). In multiple embodiments, W’ is a medicament of the compound described in International Patent Publication No. 2021 / 173773 A1. In multiple embodiments, W’ is a compound described in U.S. Patent Application Publication No. 2021 / 0283125 A1 (e.g., a compound capable of covalently bonding to C).
[0462] In multiple embodiments, W’ is of formula (A) D w -L w -Q w ’-CH2-NH-E w -Z w (A) or a pharmaceutically acceptable salt thereof, wherein, D w is represented by the following structural formula,
Chemical formula
Chemical formula
[0463] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL1), and W is formed from (PL2).
[0464] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL3), and W is formed from (PL4).
[0465] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL5), and W is formed from (PL6).
[0466] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL7), and W is formed from (PL8).
[0467] In multiple embodiments, D-CH2-NH-E-Z’- is formed from (PL9), and W is formed from (PL10).
[0468] In multiple embodiments, {D-CH2-NH-E-Z’}p’-C-{W} t is one of the following structures, where C is a monoclonal antibody, p’ and t are the drug-to-antibody ratio (DAR), p’:t is 1:1 or about 1:1, and the average values of p’ and t are each in the range of about 1 to 7, or about 2, about 3, about 4, about 5, or about 6.
Chemical formula
[0469] In multiple embodiments, both p’ and t are 4, or the average value is 4, or about 4.
[0470] In multiple embodiments, p’:t is about 1:1, about 1:2, or about 2:1.
[0471] In multiple embodiments, p’:t is 1:1, 1:2, or 2:1.
[0472] In multiple embodiments, {D-CH2-NH-E-Z’} p’ -C-{W} t is (PL1’’), wherein C is a monoclonal antibody, p’ and t are drug-to-antibody ratios (DARs), p’:t is 1:1 (or about 1:1), and the average of p’ and t ranges from about 1 to 7 (e.g., average about 2, about 3, about 4, about 5, or about 6).
[0473] In multiple embodiments, {D-CH2-NH-E-Z’} p’ -C-{W} t is (PL2’’), wherein C is a monoclonal antibody, p’ and t are drug-to-antibody ratios (DARs), p’:t is 1:1 (or about 1:1), and the average of p’ and t ranges from about 1 to 7 (e.g., average about 2, about 3, about 4, about 5, or about 6).
[0474] In another aspect, the present invention is a method for preparing a conjugate of formula (III) comprising a cell-binding agent and a drug, comprising contacting the cell-binding agent with a compound of formula (II), whereby a covalent bond is formed between the cell-binding agent and the compound of formula (II).
[0475] In yet another aspect, the present invention is a method for preparing a dual-drug conjugate of formula (IV) comprising a cell-binding agent and two different drugs, comprising contacting the cell-binding agent with a compound of formula (II) and another compound of a different formula, whereby a covalent bond is formed between the cell-binding agent and the compound of formula (II) and the other compound of a different formula of formula (II).
[0476] In yet another aspect, the present invention features a conjugate comprising a cell-binding agent and a drug. In multiple embodiments, the conjugate is prepared according to any method described herein.
[0477] In multiple embodiments, the conjugate comprises a cell-binding agent that is an antibody or an antigen-binding fragment thereof.
[0478] In multiple embodiments, the conjugate comprises a cell-binding agent that is a monoclonal antibody or an antigen-binding fragment thereof.
[0479] In multiple embodiments, the cell-binding agent is an antibody or an antigen-binding fragment thereof, p is the drug-to-antibody ratio (DAR), and has a value from 1 to 18. In multiple embodiments, the average value of p ranges from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).
[0480] In multiple embodiments, the cell-binding agent is a monoclonal antibody or an antigen-binding fragment thereof, p is the drug-to-antibody ratio (DAR), and has a value from 1 to 18. In multiple embodiments, the average value of p ranges from about 2 to 10, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0).
[0481] In multiple embodiments, the cell-binding agent is an antibody or an antigen-binding fragment thereof, p' and t are the drug-to-antibody ratio (DAR), and have values from 1 to 10. In multiple embodiments, the average value of p' ranges from about 2 to 6. In multiple embodiments, the average value of t ranges from about 2 to 6. In multiple embodiments, p':t is about 1:1. In multiple embodiments, p':t is about 1:2. In multiple embodiments, p':t is about 2:1. In multiple embodiments, p':t is 1:1. In multiple embodiments, p':t is 1:2. In multiple embodiments, p':t is 2:1.
[0482] In multiple embodiments, the cell-binding agent is a monoclonal antibody or an antigen-binding fragment thereof, p' and t are the drug-to-antibody ratio (DAR), and have values from 1 to 10. In multiple embodiments, the average value of p' ranges from about 2 to 6. In multiple embodiments, the average value of t ranges from about 2 to 6. In multiple embodiments, p':t is about 1:1. In multiple embodiments, p':t is about 1:2. In multiple embodiments, p':t is about 2:1. In multiple embodiments, p':t is 1:1. In multiple embodiments, p':t is 1:2. In multiple embodiments, p':t is 2:1.
[0483] In another aspect, the present invention features a pharmaceutical composition comprising any conjugate described herein.
[0484] In yet another aspect, the present invention is a method of treating a cell proliferative disease or disorder or inhibiting abnormal cell proliferation, the method comprising administering any conjugate described herein or any pharmaceutical composition comprising any conjugate described herein.
[0485] In another aspect, the present invention features a pharmaceutical composition comprising any compound of formula (III) described herein.
[0486] In another aspect, the present invention is a method of treating a cell proliferative disease or disorder or inhibiting abnormal cell proliferation, the method comprising administering any compound of formula (III) described herein or any pharmaceutical composition comprising any compound of formula (III) described herein.
[0487] In another aspect, the present invention features a pharmaceutical composition comprising any compound of formula (IV) described herein.
[0488] In another aspect, the present invention is a method of treating a cell proliferative disease or disorder or inhibiting abnormal cell proliferation, the method comprising administering any compound of formula (IV) described herein or any pharmaceutical composition comprising any compound of formula (IV) described herein.
[0489] In a plurality of embodiments, the method is for treating cancer.
[0490] In multiple embodiments, the cancer is adenocarcinoma, brain cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, CNS tumor, colon cancer or colorectal cancer, diffuse intrinsic pontine glioma (DIPG), endometrial cancer, esophageal cancer, Ewing sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, hematologic cancer, Hodgkin lymphoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, myelodysplastic syndrome (MDS), neuroblastoma, non-Hodgkin 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.
[0491] In multiple embodiments, the cancer is breast cancer.
[0492] Subscripts “p”, “p”, and “t” The conjugates described herein [e.g., any compound according to formula (III) or formula (IV)] can form a covalent bond with at least an auristatin derivative (e.g., any compound according to formula (II) described herein, e.g., formed from any compound according to formula (I) described herein).
[0493] In multiple embodiments, the subscript p (or p') represents the number of auristatin payload moieties on the cell binding agent [e.g., formed from a compound according to formula (II)] and has a value of 1 to 18, 1 to 12, 1 to 10, or 1 to 8. Individual auristatin conjugates may also be referred to as auristatin conjugate compounds. In multiple embodiments herein, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 auristatin payload moieties conjugated to the cell binding agent of the individual auristatin conjugate.
[0494] In multiple embodiments, the subscript t represents the number of other payload moieties (e.g., W payload moieties). In multiple embodiments herein, there may be 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) W payload moieties conjugated to the cell binding agent of an individual auristatin conjugate.
[0495] In multiple embodiments, a population of individual auristatin conjugates is substantially identical except for the number of auristatin payload moieties (and / or the number of W payload moieties) conjugated to each cell binding agent (i.e., the auristatin conjugate composition). In multiple embodiments, p and / or p' represent the average number of auristatin payload moieties conjugated to the cell binding agent of the auristatin conjugate composition. In that group of multiple embodiments, the average value of p (or p') ranges from 1 to about 18, 1 to about 10, 1 to about 8, or 1 to about 7, 2 to about 6, 3 to about 5, or 6 to about 8. In multiple embodiments, the average value of p (or p') ranges from about 2 to 10, 2 to 8, 4 to 8, or 7 to 8 (e.g., 3.2 to 8.0). In multiple embodiments, p (or p') is about 2. In multiple embodiments, p (or p') is about 4. In multiple embodiments, p (or p') is about 6. In multiple embodiments, p (or p') is about 8. In multiple embodiments, p (or p') is about 10. In multiple embodiments, p (or p') is about 12. In multiple embodiments, p (or p') is 2. In multiple embodiments, p (or p') is 4. In multiple embodiments, p (or p') is 8. In multiple embodiments, p (or p') has a value of 3 - 4. In multiple embodiments, p (or p') has a value of 4 - 5. In multiple embodiments, p (or p') has a value of 5 - 6. In multiple embodiments, p (or p') has a value of 6 - 7. In multiple embodiments, p (or p') has a value of 7 - 8. In multiple embodiments, p (or p') has a value of 7.4 - 8. In multiple embodiments, the p (or p') value refers to the average drug load and the drug load of the predominant ADC in the composition.
[0496] In multiple embodiments, the population of individual auristatin conjugates is substantially identical except for the number of W payload moieties (and / or the number of auristatin payload moieties) attached to each cell binding agent (i.e., the auristatin conjugate composition). In multiple embodiments, t represents the average number of W payload moieties attached to the cell binding agent of the auristatin conjugate composition. In that group of multiple embodiments, the average value of t ranges from 1 to about 10, 1 to about 8, or 1 to about 7, 2 to about 6, 3 to about 5, or 6 to about 8. In multiple embodiments, the average value of t ranges from about 1 to 10 or 1 to 7. In multiple embodiments, t is about 2. In multiple embodiments, t is about 3. In multiple embodiments, t is about 4. In multiple embodiments, t is about 5. In multiple embodiments, t is about 6. In multiple embodiments, t is 2. In multiple embodiments, t is 3. In multiple embodiments, t is 4. In multiple embodiments, t is 5. In multiple embodiments, t is 6. In multiple embodiments, t is 4. In multiple embodiments, the t value refers to the average drug loading and the drug loading of the major ADC in the composition.
[0497] In multiple embodiments, p' and t independently have values of 1 to 10, or 1 to 7 (e.g., about 2, 3, 4, 5, 6, or 7). In multiple embodiments, the values are average values. In multiple embodiments, p' and t are the same. In multiple embodiments, p' and t are different. In multiple embodiments, p':t is about 1:1, about 1:2, or about 2:1. In multiple embodiments, p':t is about 1:1. In multiple embodiments, both p' and t are about 4.
[0498] In multiple embodiments, the conjugation [(such as found in any compound according to formula (III) described herein] is via a reduced interchain disulfide and is about 1 to 18, 1 to 12, 1 to 10, 1 to 8, 2 to 8, 4 to 8, or 7 to 8 auristatin payload compounds (e.g., any compound formed from a compound according to formula (II) described herein or a compound of formula (I) described herein) complexed to a cell binding agent.
[0499] In multiple embodiments, the conjugation (such as found in any compound according to formula (IV) described herein) is via a reduced interchain disulfide and is about 1 to 10 or 1 to 7 auristatin payload compounds [e.g., any compound formed from a compound according to formula (II) described herein or a compound of formula (I) described herein] conjugated to a cell binding agent, and may be about 1 to 10 or 1 to 7 W' payload compounds.
[0500] In multiple embodiments, the conjugation [such as found in any compound according to formula (III) described herein] is via an introduced cysteine residue and a reduced interchain disulfide and is about 1 to 18, 1 to 12, 1 to 10, 1 to 8, 2 to 8, 4 to 8, or 7 to 8 auristatin payload compounds [e.g., any compound formed from a compound according to formula (II) described herein or a compound of formula (I) described herein] conjugated to a cell binding agent.
[0501] In multiple embodiments, the conjugation [such as found in any compound according to formula (IV) described herein] is via an introduced cysteine residue and a reduced interchain disulfide and is about 1 to 10 or 1 to 7 auristatin payload compounds [e.g., any compound formed from a compound according to formula (II) described herein or a compound of formula (I) described herein] conjugated to a cell binding agent, and there may be about 1 to 10 or 1 to 7 W' payload compounds.
[0502] In multiple embodiments, the conjugation [such as that found in any compound according to formula (III) described herein] is via an introduced cysteine residue and about 1 to 18, 1 to 12, 1 to 10, 1 to 8, 2 to 8, 4 to 8, or 7 to 8 auristatin payload compounds [such as any compound according to formula (II) described herein or formed from a compound of formula (I) described herein] conjugated to a cell binding agent may be present.
[0503] In multiple embodiments, the conjugation [such as that found in any compound according to formula (IV) described herein] is via an introduced cysteine residue and about 1 to 10, or 1 to 7 auristatin payload compounds [such as any compound according to formula (II) described herein or formed from a compound of formula (I) described herein] conjugated to a cell binding agent, as well as about 1 to 10, or 1 to 7 W' payload compounds may be present.
[0504] In multiple embodiments, the conjugation [such as that found in any compound according to formula (III) described herein] is via a lysine residue and about 1 to 18, 1 to 12, 1 to 10, 1 to 8, 2 to 8, 4 to 8, or 7 to 8 auristatin payload compounds (such as any compound according to formula (II) described herein or formed from a compound of formula (I) described herein) conjugated to a cell binding agent may be present.
[0505] In multiple embodiments, the conjugation [such as that found in any compound according to formula (IV) described herein] is via a lysine residue and about 1 to 10, or 1 to 7 auristatin payload compounds [such as any compound according to formula (II) described herein or formed from a compound of formula (I) described herein] conjugated to a cell binding agent, as well as about 1 to 10, or 1 to 7 W' payload compounds may be present.
[0506] Reactive groups on the cell-binding agent for covalent bond In multiple embodiments, the cell binding agent binds to the peptide-releasable linker in the compound of formula (II) to form a conjugate such as those according to formula (III) or formula (IV). As described above, yet other linking components in formula (II) may be present in the conjugates described herein for the purpose of providing additional space between the auristatin compound and the cell binding agent. In multiple embodiments, the cell binding agent binds to the linker unit of formula (II) via a heteroatom of the cell binding agent.
[0507] Heteroatoms that may be present on the cell binding agent for such binding include sulfur (derived from the thiol group of the targeting ligand in one embodiment), oxygen (derived from the carboxyl group or hydroxyl group of the targeting ligand in one embodiment), and nitrogen, which may be optionally substituted (derived from the primary amine functional group or secondary amine functional group of the targeting ligand in one embodiment, or in another embodiment, from an optionally substituted amide nitrogen). These heteroatoms may be present on the native targeting ligand of the cell binding agent, e.g., in a naturally occurring antibody, or may be introduced into the targeting ligand via chemical modification or biological engineering.
[0508] In one embodiment, the cell binding agent has a thiol functional group, and the cell binding agent binds to an auristatin payload compound (e.g., any compound according to formula (II) described herein, or a compound formed from the compound of formula (I) described herein) via the sulfur atom of the thiol functional group.
[0509] In another embodiment, the cell-binding agent has one or more lysine residues that can react with an activating ester of an auristatin payload compound (e.g., any compound according to formula (II) described herein, or a compound formed from a compound of formula (I) described herein), such esters including, but not limited to, N-hydroxysuccinimide, pentafluorophenyl, and p-nitrophenyl esters, thereby providing an amide bond consisting of the nitrogen atom of the cell-binding agent and the C=O group of the compound of formula (II).
[0510] In yet another aspect, the cell-binding agent has one or more lysine residues capable of chemical modification to introduce one or more thiol groups. In those embodiments, the cell-binding agent is covalently attached to an auristatin payload compound (e.g., any compound according to formula (II) described herein, or a compound 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 lysine in that manner include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-iminothiolane hydrochloride (Traut's reagent).
[0511] 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 auristatin conjugate is attached to an auristatin payload compound [e.g., any compound according to formula (II) described herein, or a compound formed from a compound of formula (I) described herein] via the sulfur atom of the thiol functional group.
[0512] 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, for example, Laguzza, et al., 1989, J. Med. Chem. 32(3):548-55]. In these embodiments, the corresponding aldehyde interacts with the reactive site of the auristatin payload compound [e.g., in any compound according to formula (II) described herein or formed from a compound of formula (I) described herein] to form a bond between the auristatin payload compound and the cell binding agent [e.g., in any compound according to formula (II) described herein or formed from a compound of formula (I) described herein]. Reactive sites on the auristatin payload compound [e.g., in any compound according to formula (II) described herein or formed from a compound of formula (I) described herein] that can interact with the reactive carbonyl-containing functional group on the targeting ligand include, but are not limited to, hydrazine and hydroxylamine.
[0513] In some embodiments, the cell binding agent forms a bond by interacting with the reactive functional group Y in an auristatin payload compound (e.g., any compound according to formula (II)), and can form a covalent bond between Y' in formula (III) or formula (IV) and the cell binding agent corresponding to the targeting ligand. The functional group Y having the ability to interact with the targeting ligand depends on the nature of the cell binding agent. In a plurality of embodiments, the reactive group is a maleimide that is present on the auristatin payload compound prior to its binding to form the cell binding agent. The covalent bond of the cell binding agent to the auristatin payload compound is achieved via the thiol functional group of the cell binding agent that interacts with the maleimide functional group Y of the payload compound (e.g., in any compound according to formula (II) described herein, or any compound formed from the compounds of formula (I) described herein), and a thio-substituted succinimide is formed. The thiol functional group can also be present on the cell binding agent in its native state, e.g., in a naturally occurring residue, or can be introduced into the cell binding agent by chemical modification or biological engineering.
[0514] In yet another embodiment, the cell binding agent is an antibody, and the thiol group is generated by reduction of the inter-chain disulfide of the antibody. Thus, in a plurality of embodiments, the auristatin payload compound is bound to the cysteine residue(s) from the reduced inter-chain disulfide(s).
[0515] 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 introduction of a cysteine residue. Thus, in a plurality of embodiments, the auristatin payload compound is bound to the cell binding agent via the introduced cysteine residue of the cell binding agent.
[0516] The binding site of the drug conjugate has been observed to affect many parameters, such as ease of binding, stability of the drug linker, influence on the biophysical properties of the resulting bioconjugate, and in vitro cytotoxicity. Regarding the stability of the drug linker, the binding site of the drug linker moiety to the cell binder may, in some cases, affect the ability of the bound drug linker moiety to undergo an elimination reaction and, in some cases, cause premature release of the free drug. Sites for conjugation on the targeting ligand include, for example, reduced interchain disulfides and cysteine residues selected at engineered sites. In a plurality of embodiments for forming the auristatin conjugates described herein, the conjugation method uses a genetically engineered site (e.g., position 239 according to the EU index described in Kabat) that is less susceptible to the effects of elimination reactions compared to conjugation methods that use thiol residues from reduced disulfide bonds. In other embodiments of the method for conjugation for forming the auristatin conjugates described herein, thiol residues are used at sites that are susceptible to the effects of elimination reactions (e.g., resulting from interchain disulfide reduction).
[0517] Cell-binding agent (C) In a plurality of embodiments of the present invention, a cell binder is present. The cell binder serves to target and present an auristatin or a drug component containing auristatin to a specific target cell population that interacts due to the presence of its targeted component or molecule, thereby enabling the release of the free drug within or in the vicinity of the target cell (i.e., intracellularly or extracellularly).
[0518] In multiple embodiments, the cell binding agent can be a ligand that binds to a moiety on a target cell such as a cell surface receptor. In multiple embodiments, the ligand can be a growth factor or a fragment thereof that binds to a growth factor receptor. In multiple embodiments, the ligand can be a cytokine or a fragment thereof that binds to a cytokine receptor. In multiple embodiments, the growth factor receptor or cytokine receptor is a cell surface receptor.
[0519] Accordingly, the therapeutic use of auristatin conjugates [e.g., compounds of formula (III) or formula (IV) described herein] can be achieved by appropriate selection of a cell binding agent.
[0520] Cell binding agents include, but are not limited to, proteins, polypeptides, and peptides. Suitable cell binding agents include, for example, antibodies (e.g., full-length antibodies and antigen-binding fragments thereof including polyclonal and monoclonal antibodies), interferons, lymphokines, hormones, growth factors, colony stimulating factors, vitamins (e.g., folic acid), nutrient transport molecules (e.g., but not limited to transferrin), or any other cell binding molecule or substance. In multiple embodiments, the cell binding agent is an antibody or a non-antibody protein targeting agent.
[0521] Antigen targeted by the cell-binding agent In multiple embodiments, exemplary antigens or ligands include renin, growth hormone (e.g., human growth hormone and bovine growth hormone), growth hormone releasing factor, parathyroid hormone, or thyroid stimulating hormone, or fragments thereof.
[0522] In multiple 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.
[0523] In multiple embodiments, exemplary antigens or ligands include coagulation factors (e.g., Factor VIIIc, Factor IX, tissue factor, and von Willebrand factor), anticoagulation factors (e.g., protein C), atrial natriuretic factor, pulmonary surfactant, plasminogen activators (e.g., urokinase, human urine, or tissue-type plasminogen activator), bombesin, thrombin, or hematopoietic growth factors, or fragments thereof.
[0524] In multiple embodiments, exemplary antigens or ligands include tumor necrosis factor alpha and -beta, or fragments thereof.
[0525] In multiple 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), mupellarin inhibitor, relaxin A chain, relaxin B chain, prorelaxin, mouse gonadotropin-related peptide, microbial proteins (beta-lactamase), DNase, IgE, inhibin, or activin, or fragments thereof.
[0526] In multiple embodiments, exemplary antigens or ligands include cytotoxic T lymphocyte-associated antigen (e.g., CTLA-4), or fragments thereof.
[0527] In multiple embodiments, exemplary antigens or ligands include vascular endothelial growth factor or fragments thereof.
[0528] In multiple embodiments, exemplary antigens or ligands include receptors for hormones or growth factors; Protein A or D; rheumatoid factors, neurotrophic factors (e.g., bone-derived neurotrophic factor, neurotrophin-3, -4, -5, or -6), nerve growth factors (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-betaI, TGF-beta2, TGF-beta3, TGF-beta4, and TGF-beta5), insulin-like growth factor-I and -II, des(1-3)-IGF-I (brain IGF-I), or insulin-like growth factor binding proteins, or fragments thereof.
[0529] In multiple embodiments, exemplary antigens or ligands include melanotransferrin, CA6, CAK1, CALLA, CAECAM5, GD3; FLT3, PSMA, PSCA, MUC1, STEAP, CEA, TENB2, EphA receptor, EphB receptor, folate receptor, FOLR1, mesothelin, crypt, alpha v beta6, or integrin, or fragments thereof.
[0530] In multiple embodiments, exemplary antigens or ligands include VEGF or VEGFR, or fragments thereof.
[0531] In multiple embodiments, exemplary antigens or ligands include EGFR or fragments thereof.
[0532] In multiple embodiments, exemplary antigens or ligands include FGFR3; LAMP1, p-cadherin, or transferrin receptor, or fragments thereof.
[0533] In multiple embodiments, exemplary antigens or ligands include IRTA1, IRTA2, IRTA3, IRTA4, IRTA5, or fragments thereof.
[0534] In multiple embodiments, exemplary antigens or ligands include tyrosine-protein kinase transmembrane receptors (e.g., ROR1 and ROR2), or fragments thereof.
[0535] In multiple 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.
[0536] In multiple embodiments, exemplary antigens or ligands include one or more tumor-associated antigens or cell surface receptors (see US Application Publication No. 2008 / 0171040 or US Application Publication No. 2008 / 0305044, which are incorporated by reference in their entirety), or fragments thereof.
[0537] In multiple embodiments, exemplary antigens or ligands include erythropoietin or fragments thereof.
[0538] In multiple embodiments, exemplary antigens or ligands include osteogenic factors or fragments thereof.
[0539] In multiple embodiments, exemplary antigens or ligands include immunotoxins or fragments thereof.
[0540] In multiple embodiments, exemplary antigens or ligands include bone morphogenetic proteins or fragments thereof.
[0541] In multiple embodiments, exemplary antigens or ligands include interferons (e.g., interferon-alpha, -beta, and -gamma).
[0542] In multiple embodiments, exemplary antigens or ligands include colony-stimulating factors (e.g., M-CSF, GM-CSF, and G-CSF), or fragments thereof.
[0543] In multiple embodiments, exemplary antigens or ligands include interleukins (e.g., IL-1 to IL-10), or fragments thereof.
[0544] In multiple embodiments, exemplary antigens or ligands include superoxide dismutase or fragments thereof.
[0545] In multiple embodiments, exemplary antigens or ligands include T cell receptors or fragments thereof.
[0546] In multiple embodiments, exemplary antigens or ligands include surface membrane proteins or fragments thereof.
[0547] In multiple embodiments, exemplary antigens or ligands include decay-accelerating factor or fragments thereof.
[0548] In multiple embodiments, exemplary antigens or ligands include viral antigens (e.g., a portion of the HIV envelope), or fragments thereof.
[0549] In multiple embodiments, exemplary antigens or ligands include transport proteins or fragments thereof.
[0550] In multiple embodiments, exemplary antigens or ligands include homing receptors or fragments thereof.
[0551] In multiple embodiments, exemplary antigens or ligands include adenosine or fragments thereof.
[0552] In multiple embodiments, exemplary antigens or ligands include regulatory proteins or fragments thereof.
[0553] In multiple embodiments, exemplary antigens or ligands include integrins (e.g., CDlla, CDllb, CDllc, CD18, ICAM, VLA-4, and VCAM), or fragments thereof.
[0554] In multiple embodiments, exemplary antigens or ligands include tumor-associated antigens (e.g., HER2, HER3, and HER4 receptors), or fragments thereof.
[0555] In multiple embodiments, exemplary antigens or ligands include endoglin, c-Met, c-kit, 1GF1R, PSGR, NGEP, PSMA, PSCA, TMEFF2, LGR5, B7H4, TROP-2, DLL-3, CDH6, AML, SLITRK6, ENPP3, BCMA, tissue factor, or CD352, or fragments thereof.
[0556] In multiple embodiments, the cell binding agent targets 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, FLJ10372, KIAA1445, Mm42015, SEMA5B, SEMAG, prostate six transmembrane epithelial antigen 1, IPCA-1, PCANP1, STMP, prostate antigen, insulin growth factor receptor, or folate receptor.
[0557] In multiple 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 (VDC6), SLITRK6, 5T4, or SC-16.
[0558] In multiple embodiments, the cell binding agent targets HER2 or EGFR.
[0559] In multiple embodiments, the cell binding agent targets fibronectin extra domain B (EDB), endothelial receptor ETB, PSMA, VEGFR2 (CD309), tissue factor, or ROBO4.
[0560] In multiple embodiments, the cell binding agent targets collagen IV, periostin, or tenascin c.
[0561] In multiple embodiments, the cell binding agent targets CD30, CD22, CD79b, CD19, CD138, CD74, CD37, CD33, CD19, or rCD98.
[0562] In multiple embodiments, the cell binding agent targets HER2.
[0563] In multiple embodiments, the cell binding agent targets EGFR.
[0564] In multiple embodiments, the cell binding agent targets CD70.
[0565] In multiple embodiments, the cell binding agent targets CD33.
[0566] In multiple embodiments, the cell binding agent targets CD30.
[0567] In multiple embodiments, the cell binding agent targets CD22.
[0568] In multiple embodiments, the cell binding agent targets CD19.
[0569] In multiple embodiments, the cell binding agent targets Mucl.
[0570] In multiple embodiments, the cell binding agent targets CD37.
[0571] In multiple embodiments, the cell binding agent targets CD123.
[0572] Non-protein cell-binding agent In multiple embodiments, the cell binding agent is not a protein. For example, in multiple 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, and the complex binds to the STRA6 receptor with high affinity, increasing the uptake of vitamin A. In other examples, folic acid / folate / vitamin B9 binds with high affinity to the cell surface folate receptor (FR), such as FRα. Folic acid or an antibody that binds to FRα can be used to target folate receptors expressed on ovaries and other tumors. Additionally, vitamin D and its analogs bind to the vitamin D receptor.
[0573] Protein and polypeptide cell-binding agents In other embodiments, the cell binding agent is a protein or polypeptide, or a compound comprising an antibody, non-antibody protein, or polypeptide.
[0574] In multiple embodiments, the cell binding agent can be a lymphokine, hormone, growth factor, colony-stimulating factor, or nutrient transport molecule.
[0575] In multiple embodiments, GM-CSF, a ligand / growth factor that binds to bone marrow cells, can be used as a cell binding agent for diseased cells derived from acute myeloid leukemia.
[0576] In multiple embodiments, IL-2, which binds to activated T cells, can be used for the prevention of graft rejection, the treatment and prevention of graft-versus-host disease, and the treatment of acute T cell leukemia.
[0577] In multiple embodiments, MSH, which binds to melanocytes, can be used for the treatment of melanoma, similar to an antibody against melanoma.
[0578] In multiple embodiments, epidermal growth factor can be used to target squamous cell carcinomas such as lung cancer, head and neck cancer, and gingival squamous cell carcinoma.
[0579] In multiple embodiments, somatostatin can be used to target neuroblastoma and other tumor types.
[0580] In multiple embodiments, estrogen (or an estrogen analog) can be used to target breast cancer.
[0581] In multiple embodiments, androgen (or an androgen analog) can be used to target the testis.
[0582] In multiple embodiments, the cell binding agent is an antibody mimetic such as an ankyrin repeat protein, centyrin, or adnectin / monobody.
[0583] In multiple embodiments, the auristatin conjugate includes, as its cell binding agent, a non-immunoreactive protein, polypeptide, or peptide. Thus, in multiple 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, lectin, and apolipoproteins derived from low density lipoprotein.
[0584] Antibodies and related cell-binding agents In multiple 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 can be an antibody.
[0585] In embodiments where the cell binding agent is an antibody or an antigen-binding portion thereof (including antibody derivatives), or a particular antibody mimetic, the cell binding agent can bind to a ligand on the target cell such as a cell surface ligand that includes a cell surface receptor.
[0586] Suitable antibodies 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 immuno pharmaceuticals ( S mall M odular[[ID=E]] I mmuno P harmaceuticals) (SMIPs (trademark)), and antibody fragments.
[0587] For example, antibodies include immunoglobulins (Ig) and fragments thereof, which are specifically reactive with a designated protein or peptide, or a fragment thereof. In multiple 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, as long as they exhibit the desired biological activity. In multiple embodiments, the antibody is IgG, IgA, IgE, IgD, or IgM. In multiple embodiments, the antibody is IgG1, IgG2, IgG3, or IgG4. In multiple embodiments, the antibody is IgAl or IgA2.
[0588] In multiple embodiments, the cell binding agent is a resurfaced antibody, resurfaced single-chain antibody, resurfaced antibody fragment (or antigen-binding portion), or bispecific antibody.
[0589] In multiple embodiments, the cell binding agent is a minibody, avibody, diabody, tribody, tetrabody, nanobody, probody, domain antibody, or unibody.
[0590] Examples of antibody fragments include portions of intact antibodies, such as the antigen-binding or variable regions of an antibody. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments, tribodies, tetrabodies, linear antibodies, 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 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 joined by a peptide linker (“ScFv protein”), and minimal recognition units consisting of amino acid residues that mimic hypervariable regions.
[0591] 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 specific antigenic determinants (e.g., cancer cell antigens, viral antigens, microbial antigens, proteins, peptides, carbohydrates, chemicals, nucleic acids, or fragments thereof). Monoclonal antibodies (mAbs) against a target antigen can be prepared by using any technique known in the art that provides for the production of antibody molecules by a continuous cell line in culture.
[0592] In a plurality of embodiments, the cell binding agent is a monoclonal antibody or an antigen-binding fragment thereof.
[0593] 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).
[0594] In some embodiments, antibodies suitable for the present invention can include humanized antibodies or human antibodies. Humanized forms of non-human antibodies are chimeric Igs, Ig chains, or fragments [e.g., Fv, Fab, Fab’, F(ab’)2, or other antigen-binding subsequences of Abs] that contain minimal sequences derived from non-human Igs. Generally, humanized antibodies have one or more amino acid residues introduced from non-human sources. These non-human amino acid residues are typically often referred to as “import” residues and typically are derived from the “import” variable domain. Humanization is achieved by substituting rodent complementarity-determining regions (CDRs) or CDR sequences with 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 Abs (U.S. Patent No. 4,816,567, 1989), with substantially fewer than intact human variable domains being replaced by the corresponding sequences from non-human species. In some embodiments, the CDRs of non-human antibodies (e.g., mouse) targeting human antigens are grafted onto the framework regions of the variable domains of human Igs. Various techniques known in the art are suitable for CDR grafting, including, for example, site-directed mutagenesis. In some embodiments, humanized antibodies are typically human antibodies in which some CDR residues and in some cases some FR residues are replaced by residues from analogous sites of rodent Abs. Examples of humanized antibodies include human Igs (recipient antibodies) in which residues from the CDRs of non-human species such as mouse, rat, or rabbit (donor antibodies) with the desired specificity, affinity, and potency replace residues from the recipient's CDRs. In some embodiments, the monospecific and bispecific antibodies described herein have cross-reactivity with non-human primate common antigens. In some examples, the corresponding non-human residues replace Fv framework residues of human Igs. Humanized antibodies may contain residues not found in either the CDR sequences or the framework sequences imported into the recipient antibody.Generally, humanized antibodies contain substantially all, or at least one, and typically two variable domains, with most of the CDR regions corresponding to the variable domains of non-human Igs and most of the FR regions being the variable domains of human Ig consensus sequences. Humanized antibodies optimally consist of at least a part of the Ig constant region (Fc), usually the Fc region of human Ig [Riechmann et al., Nature 332(6162):323-7, 1988; Verhoeyen et al., Science. 239(4847):1534-6, 1988].
[0595] 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 the preparation of human monoclonal antibodies [Reisfeld and Sell, 1985, Cancer Surv. 4(1):271-90]. Similarly, human antibodies can be synthesized by introducing human Ig genes into transgenic animals in which the endogenous Ig genes have been partially or completely inactivated. When challenged, human antibody production is observed, which is very similar to that seen in humans in all respects, including gene rearrangement, assembly, and the antibody repertoire [Fishwild et al., High-avidity human IgG kappa monoclonal 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, nt.Rev.Immunol. 1995;13(1):65-93; Marks et al., By-passing immunization:building high affinity human antibodies by chain shuffling.Biotechnology (N Y). 1992 July;10(7):779-83].
[0596] An antibody can 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 another antibody bound to a tumor cell or matrix. In this regard, "functionally active" means that the fragment, derivative, or analog can immunospecifically bind to the target cell. To determine which CDR sequences bind to the antigen, a synthetic peptide containing the CDR sequences can be used in a binding assay with the antigen by any binding assay method known in the art (e.g., BIAcore assay) [see, e.g., 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].
[0597] Other useful antibodies include, but are not limited to, fragments of antibodies such as F(ab’)2 fragments, Fab fragments, Fv, single-chain antibodies, diabodies, triabodies, tetra-bodies, scFv, scFv-FV, or any other molecule having the same specificity as the antibody.
[0598] Another form of antibody fragment is a peptide encoding a single CDR. CDR peptides (the "minimal recognition units") can be obtained by constructing a gene encoding the CDR of the antibody of interest. Such genes are prepared, for example, by synthesizing the 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).
[0599] Furthermore, recombinant antibodies, such as chimeric monoclonal antibodies and humanized monoclonal antibodies, which can be produced using standard recombinant DNA techniques and contain both human and non-human portions, are useful antibodies. Chimeric antibodies are molecules composed of different portions from different animal species, for example, those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. (See, e.g., U.S. Patent Nos. 4,816,567 and 4,816,397, which are incorporated herein by reference in their entirety.) Humanized antibodies are antibody molecules derived from non-human species that have one or more complementarity-determining regions (CDRs) from non-human species and framework regions from human immunoglobulin molecules. (See, e.g., U.S. Patent No. 5,585,089, which is incorporated herein by reference in its entirety.)) Such chimeric monoclonal antibodies and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example, each of which is incorporated herein by reference in its entirety: WO 87 / 02671, EP 0 184 187, EP 0 171 496, EP 0 173 494, WO 86 / 01533, U.S. Pat. No. 4,816,567, EP 012 023, Berter et al., 1988, Science 240:1041-1043, Liu et 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, 1987, Cancer. Res. 47:999-1005, Wood et al, 1985, Nature 314:446-449, and Shaw et al, 1988, J. Natl. Cancer Inst. 80:1553-1559, Morison, 1985, Science 229:1202-1207, Oi et al, 1986, BioTechniques 4:214, U.S. Pat. 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, by the methods described therein.
[0600] In some instances (e.g., where immunogenicity against non-human antibodies or chimeric antibodies may occur), fully human antibodies are more desirable and can be produced using transgenic mice that are unable to express endogenous immunoglobulin heavy and light chain genes but can express human heavy and light chain genes.
[0601] Antibodies include modified analogs and derivatives, i.e., any type of covalent modification of a molecule as long as the antibody can maintain antigen-binding immunospecificity. For example, but not limited to, derivatives and analogs of antibodies include those that have been further modified, for example, by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting groups / blocking groups, proteolytic cleavage, conjugation to cell antibody units or other proteins, etc. Any of a number of chemical modifications can be carried out by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Further, an analog or derivative can contain one or more non-natural amino acids.
[0602] An antibody can have a modification (e.g., substitution, deletion, or addition) to an amino acid residue that interacts with an Fc receptor. In particular, an antibody can have a modification to an amino acid residue that has been 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).
[0603] Antibodies can be generated using methods well known in the art. For example, for protocols of antibody production, see Harlow and Lane, Antibodies: A Laboratory Manual (1988). Typically, antibodies can be generated in mice, rats, guinea pigs, hamsters, camels, llamas, sharks, or other suitable hosts. Alternatively, antibodies can be made in chickens that produce IgY molecules [Schade et al., (1996) ALTEX 13(5):80-85]. In a plurality of embodiments, antibodies suitable for the present invention are non-human primate antibodies. For example, for general techniques for raising therapeutically useful antibodies in baboons, see, for example, Goldenberg et al., International Patent Publication No. 91 / 11465 (1991), and Losman et al., Int. J. Cancer 46:310 (1990). In a plurality of embodiments, monoclonal antibodies can be prepared using the hybridoma method [Milstein and Cuello, (1983) Nature 305(5934):537-40]. In a plurality of embodiments, monoclonal antibodies can also be produced by recombinant methods (U.S. Patent No. 4,166,452, 1979).
[0604] Many of the difficulties associated with the production of monoclonal antibodies by B cell immortalization can be overcome by manipulating and expressing antibody fragments in E. coli using the phage display method. To ensure the recovery of high affinity monoclonal antibodies, combinatorial immunoglobulin libraries typically must contain a large repertoire size. In a typical strategy, mRNA obtained from lymphocytes or spleen cells of immunized mice is utilized to synthesize cDNA using reverse transcriptase. The heavy chain gene and the light chain gene are amplified separately by PCR and ligated into a phage cloning vector. Two different libraries are created, one containing the heavy chain gene and one containing the light chain gene. Phage DNA is isolated from each library, the heavy chain and light chain sequences are ligated together, packaged, and a combinatorial library is formed. Each phage contains a random pair of heavy and light chain cDNAs and, when infecting E. coli, induces the expression of the antibody chains in the infected cells. To identify antibodies that recognize the target antigen, the phage library is plated and the antibody molecules present in the plaques are transferred to a filter. The filter is incubated with a radio-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 vectors and expression vectors useful for creating human immunoglobulin phage libraries can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, CA).
[0605] Using a similar strategy, high affinity scFv can be obtained. See, for example, Vaughn et al., Nat. Biotechnol., 14: 309 314 (1996). An scFv library with a large repertoire can be constructed by isolating V genes from non-immunized human donors using PCR primers corresponding to all known V H 、V k 、and Vλ gene families. After amplification, V kCombine the pool and the Vλ pool to form one pool. These fragments are ligated into a phagemid vector. Then, ligate the scFv linker (Gly4, Ser)3 upstream of the V L fragment into the phagemid. Amplify the V H fragment and the linker-V L fragment and assemble them over the JH region. Ligate the resulting V H -linker-V L fragment into the phagemid vector. The phagemid library can be panned using the filters described above or using an immunotube (Nunc; Maxisorp™). Similar results can be achieved by constructing a combinatorial immunoglobulin library from immunized rabbit lymphocytes or spleen cells and by expressing the scFv construct in P. pastoris. See, for example, Ridder et al., Biotechnology, 13: 255-260 (1995). Further, after isolating a suitable scFv, antibody fragments with higher binding affinity and slower dissociation rates can be obtained via affinity maturation processes such as CDR3 mutagenesis and chain shuffling. See, for example, Jackson et al., Br. J. Cancer, 78: 181-188 (1998); Osbourn et al., Immunotechnology, 2:181-196 (1996).
[0606] In multiple embodiments, the conjugates described herein [e.g., any compound according to formula (III) or formula (IV)] include a cell-binding agent that is an antibody targeting an antigen overexpressed in cancer cells.
[0607] In multiple embodiments, the conjugates described herein (e.g., any compound according to formula (III) or formula (IV)) include a cell-binding agent that is an antibody that recognizes a specific tumor-associated antigen (TAA).
[0608] Antibodies that are immunospecific for cancer cell antigens are commercially available or can be produced by any method known to those skilled 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 similar database, published literature, or by routine cloning and sequencing.
[0609] In certain embodiments, known antibodies for the treatment of cancer can be used.
[0610] In another particular embodiment, antibodies for the treatment of autoimmune diseases are used in accordance with the compositions and methods of the present invention.
[0611] In multiple embodiments, useful antibodies can bind to receptors or receptor complexes expressed on activated lymphocytes. The receptor or receptor complex can include immunoglobulin gene superfamily members, TNF receptor superfamily members, integrins, cytokine receptors, chemokine receptors, major histocompatibility proteins, lectins, or complement control proteins.
[0612] In multiple embodiments, the conjugates described herein [e.g., any compound according to formula (III) or formula (IV)] are cell-binding agents (e.g., an antibody or a fragment thereof) targeting 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, FLJ10372, KIAA1445, Mm42015, SEMA5B, SEMAG, prostate six transmembrane epithelial antigen 1, IPCA-1, PCANP1, STMP, prostate antigen, insulin growth factor receptor, or folate receptor.
[0613] In multiple embodiments, the conjugates described herein [e.g., any compound according to formula (III) or formula (IV)] are cell-binding agents (e.g., an antibody or a fragment thereof) targeting GPNMB, NCAM (CD56), TACSTD2 (TROP-2), folate receptor alpha, tissue factor, ENPP3, CD70P-cadherin, mesothelin, STEA1, CEACAM5, mucin 1, nectin 4, guanylyl cyclase C, SLC44A4, PSMA, LIV1 (SIP6), SLITRK6, 5T4, or SC-16.
[0614] In multiple embodiments, the conjugates described herein (e.g., any compound according to formula (III) or formula (IV)) are cell-binding agents (e.g., an antibody or a fragment thereof) targeting HER2 or EGFR.
[0615] In multiple embodiments, the conjugates described herein (e.g., any compound according to formula (III) or formula (IV)) comprise a cell-binding agent (e.g., an antibody or a fragment thereof) that targets fibronectin extra domain B (EDB), endothelial receptor ETB, PSMA, VEGFR2 (CD309), tissue factor, or ROBO4.
[0616] In multiple embodiments, the conjugates described herein (e.g., any compound according to formula (III) or formula (IV)) comprise a cell-binding agent (e.g., an antibody or a fragment thereof) that targets collagen IV, periostin, or tenascin c.
[0617] In multiple embodiments, the conjugates described herein (e.g., any compound according to formula (III) or formula (IV)) comprise a cell-binding agent (e.g., an antibody or a fragment thereof) that targets CD30, CD22, CD79b, CD19, CD138, CD74, CD37, CD33, CD19, or CD98.
[0618] In multiple embodiments, the conjugates described herein (e.g., any compound according to formula (III) or formula (IV)) comprise a cell-binding agent (e.g., an antibody or a fragment thereof) that targets HER2.
[0619] In multiple embodiments, the conjugates described herein (e.g., any compound according to formula (III) or formula (IV)) comprise a cell-binding agent (e.g., an antibody or a fragment thereof) that targets ROR1 or ROR2.
[0620] In multiple embodiments, the conjugates described herein (e.g., any compound according to formula (III) or formula (IV)) comprise a cell-binding agent (e.g., an antibody or a fragment thereof) that targets FOLR1.
[0621] In multiple embodiments, the conjugates described herein (e.g., any compound according to formula (III) or formula (IV)) comprise a cell-binding agent (e.g., an antibody or a fragment thereof) that targets nectin-4.
[0622] In multiple embodiments, the conjugates described herein (e.g., any compound according to formula (III) or formula (IV)) comprise a cell-binding agent (e.g., an antibody or a fragment thereof) that targets Trop2.
[0623] In multiple embodiments, the conjugates described herein (e.g., any compound according to formula (III) or formula (IV)) comprise a cell-binding agent (e.g., an antibody or a fragment thereof) that targets EGFR.
[0624] In multiple embodiments, the conjugates described herein (e.g., any compound according to formula (III) or formula (IV)) comprise a cell-binding agent (e.g., an antibody or a fragment thereof) that targets CD70.
[0625] In multiple embodiments, the conjugates described herein (e.g., any compound according to formula (III) or formula (IV)) comprise a cell-binding agent (e.g., an antibody or a fragment thereof) that targets CD33.
[0626] In multiple embodiments, the conjugates described herein (e.g., any compound according to formula (III) or formula (IV)) comprise a cell-binding agent (e.g., an antibody or a fragment thereof) that targets CD30.
[0627] In multiple embodiments, the conjugates described herein (e.g., any compound according to formula (III) or formula (IV)) comprise a cell-binding agent (e.g., an antibody or a fragment thereof) that targets CD22.
[0628] In multiple embodiments, the conjugates described herein (e.g., any compound according to formula (III) or formula (IV)) include a cell-binding agent that targets CD19 (e.g., an antibody or a fragment thereof).
[0629] In multiple embodiments, the conjugates described herein (e.g., any compound according to formula (III) or formula (IV)) include a cell-binding agent that targets Mucl (e.g., an antibody or a fragment thereof).
[0630] In multiple embodiments, the conjugates described herein (e.g., any compound according to formula (III) or formula (IV)) include a cell-binding agent that targets CD37 (e.g., an antibody or a fragment thereof).
[0631] In multiple embodiments, the conjugates described herein (e.g., any compound according to formula (III) or formula (IV)) include a cell-binding agent that targets CD123 (e.g., an antibody or a fragment thereof).
[0632] Synthetic methods The compounds described herein [e.g., a compound according to any one of formula (I), formula (II), formula (III), or formula (IV)] can be prepared according to methods known in the art.
[0633] In multiple embodiments, Scheme 1 provides an exemplary synthetic method of the described compound D1 (MB-25).
Chemical formula
[0634] In multiple embodiments, Scheme 2 provides an exemplary synthetic method of the described compound D2 (MB-23).
Chemical formula
[0635] In multiple embodiments, Scheme 3 provides an exemplary synthetic method of the described compound PL1 (MB-26).
Chemical formula
[0636] In multiple embodiments, Scheme 4 provides an exemplary synthetic method of the described compound D5.
Chemical formula
[0637] In multiple embodiments, Scheme 5 provides an exemplary synthetic method of the described compound D6.
Chemical formula
[0638] In multiple embodiments, Scheme 6 provides an exemplary synthetic method of the described compound PL2 (MB-24).
Chemical formula
[0639] In multiple embodiments, Scheme 7 provides an exemplary general method for preparing a conjugate (PL’).
Chemical formula
[0640] In multiple embodiments, Scheme 8 provides an exemplary general method for preparing a dual-drug conjugate (PL’’).
Chemical formula
[0641] In multiple embodiments, exemplary experimental procedures for preparing a conjugate (PL’) with a drug-to-antibody ratio (DAR) of 7-8 or 8:
[0642] Antibody C was reacted in 50 mM pH 7.4 phosphate buffer (conjugation buffer) and 10 mM DTPA (diethylenetriaminepentaacetic acid) solution with 8 equivalents (2 equivalents per disulfide bond) of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) at 25 °C for 2 hours, and then 12 equivalents of payload (PL) in DMSO was added (the volume of DMSO was about 12 - 15% of the volume of the phosphate buffer). The resulting reaction solution was rotated on a tube rotator at 25 °C for 1 hour. The reaction mixture was immediately purified using an ultrafiltration tube (30KD) for several cycles with formulation buffer. The resulting conjugate (PL’) usually has a drug-to-antibody ratio (DAR) of 7 - 8 or 8, and the proportion of monomers measured by size exclusion chromatography is over 95%.
[0643] Exemplary experimental procedure for preparing a conjugate (PL’) with a drug-to-antibody ratio (DAR) of about 4 in multiple embodiments:
[0644] Antibody C was treated with 4 equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in 50 mM pH 7.4 phosphate buffer (conjugation buffer) containing 2 equivalents of ZnCl2 at 25 °C for 3 hours (reference: JI, Ao, Sun, Chuchu; He, Wenxu, “Process for preparing - drug conjugates with improved homogeneity”, International Publication No. WO 2020164561), and then 6 equivalents of payload PL in DMSO was added (the volume of DMSO was about 12 - 15% of the volume of the phosphate buffer). The resulting reaction solution was rotated on a tube rotator at 25 °C for 1 hour. The reaction was stopped with 4 equivalents of cysteine, followed by the addition of 10 mM DTPA (4 equivalents) and 10 mM DHAA (8 equivalents). This was purified using an ultrafiltration tube (30KD) over several cycles with formulation buffer. The resulting conjugate usually has a drug-to-antibody ratio (DAR) of about 4, and the proportion of monomers measured by size exclusion chromatography is over 95%.
[0645] In multiple embodiments, the antibody-drug conjugate is trastuzumab-MB24. In multiple embodiments, Scheme 9 provides an exemplary synthetic method of trastuzumab-MB24, which is the antibody-drug conjugate described.
Chemical formula
[0646] In multiple embodiments, the antibody-drug conjugate is trastuzumab-MB26. In multiple embodiments, Scheme 10 provides an exemplary synthetic method of trastuzumab-MB26, which is the antibody-drug conjugate described.
Chemical formula
[0647] Exemplary experimental procedures for preparing the dual drug conjugate (PL''):
[0648] Antibody C was treated with 4 equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) in 50 mM phosphate buffer (conjugation buffer) at pH 7.4 containing 2 equivalents of ZnCl2 at 25 °C for 3 hours (reference: JI, Ao; Sun, Chuchu; He, Wenxu, “Process for preparing antibody-drug conjugates with improved homogeneity”, International Application Publication No. WO 2020 / 164561), and then 6 equivalents of the first payload PL in DMSO was added (the volume of DMSO is about 12-15% of the volume of the phosphate buffer). The resulting reaction solution was rotated on a tube rotator at 25 °C for 1 hour. The reaction was stopped with 4 equivalents of cysteine, followed by the addition of 10 mM DTPA (4 equivalents) and 10 mM DHAA (8 equivalents). This was purified using an ultrafiltration tube (30KD) with conjugation buffer for several cycles. The resulting conjugate usually has a drug-to-antibody ratio (DAR) of about 4, which was treated with 5 equivalents of TCEP and 10 mM DTPA in 50 mM phosphate buffer at pH 7.4 at 25 °C for 2 hours, followed by the addition of 6 equivalents of the second payload W’ in DMSO (the volume of DMSO is about 12-15% of the volume of the phosphate buffer). The resulting reaction solution was rotated on a tube rotator at 25 °C for 1 hour. The reaction mixture was immediately purified using an ultrafiltration tube (30KD) with formulation buffer for several cycles to provide a dual-drug conjugate (PL”). The DAR ratio of PL” corresponding to the two payloads is usually about 1:1, and the dual-drug conjugate has a monomer ratio of more than 95% as measured by size exclusion chromatography.
[0649] In multiple embodiments, the antibody dual-drug conjugate is trastuzumab-MB0324. In multiple embodiments, Scheme 11 provides an exemplary synthesis method of trastuzumab-MB0324, which is the antibody dual-drug conjugate described.
Chemical Structure
[0650] In multiple embodiments, the antibody-drug conjugate is trastuzumab-MB0326. In multiple embodiments, Scheme 12 provides an exemplary synthetic method of trastuzumab-MB0326, which is the antibody-drug conjugate described.
Chemical formula
[0651] Auristatin conjugate mixtures and compositions The present invention provides conjugate mixtures and pharmaceutical compositions comprising any of the conjugates (Formula III or Formula IV) described herein. The mixtures and pharmaceutical compositions comprise a plurality of conjugates. In some embodiments, each of the conjugates in the mixture or composition is identical or substantially identical, but the distribution of drug linkers on the cell-binding agent in the mixture or composition can vary, similar to the drug load. For example, conjugation techniques used to attach a drug linker to an antibody as a targeting ligand can result in a composition or mixture that is heterogeneous with respect to the distribution of payload compounds on the antibody (cell-binding agent) within the mixture and / or composition. In some embodiments, the loading of the payload compound onto each of the antibody molecules in such a mixture or composition of molecules is an integer in the range of 1 to 18.
[0652] In these embodiments, when referring to the entire composition, the loading of the drug linker is a number in the range of 1 to about 18. There may also be a small percentage of unbound antibodies in the composition or mixture. The average number of drug linkers per cell-binding agent in the mixture or composition (i.e., the average drug load) is an important attribute in determining the maximum amount of agent that can be delivered to target cells. The average drug load 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.
[0653] In some embodiments, the mixtures and pharmaceutical compositions include a plurality (i.e., a population) of conjugates, where the conjugates are identical or substantially identical and are substantially homogeneous with respect to the distribution of drug linkers on ligand molecules within the mixture and / or composition, and with respect to the loading of drug linkers on cell binding agent molecules within the mixture and / or composition. In some such embodiments, the loading of drug linkers on the antibody is 2, or 3, or 4, or 5, or 6, or 7, or 8. There may also be a small percentage of unbound antibody present in the composition or mixture. The average drug loading of such embodiments is about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 7.5, or about 8. Typically, such compositions and mixtures result from the use of site-specific conjugation techniques, and conjugation is due to introduced cysteine residues.
[0654] The average number of payload compounds per cell binding agent in a preparation from a conjugation reaction can be characterized by conventional means such as HIC, UV, LC-MS, ELISA assays. The quantitative distribution of conjugates from the perspective of p or p’ or t can also be determined. In some examples, the separation, purification, and characterization of homogeneous conjugates can be achieved by means such as reverse-phase HPLC or electrophoresis.
[0655] In some embodiments, the composition is a pharmaceutical composition comprising a 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.
[0656] Compositions comprising a pharmaceutical composition can be provided in purified form. As used herein, “purified” means that when isolated, the isolate contains at least 95%, in another embodiment at least 98% of the conjugate on a weight basis of the isolate.
[0657] Methods of use Compositions and methods of administration In another aspect, the present invention features a pharmaceutical composition comprising any of the compounds described herein [e.g., any compound of formula (I), formula (II), formula (III), or formula (IV) described herein] or a pharmaceutically acceptable salt thereof. In a plurality of embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier.
[0658] In a plurality of embodiments, the pharmaceutical composition comprises a conjugate according to formula (III).
[0659] In a plurality of embodiments, the pharmaceutical composition comprises a conjugate according to formula (IV).
[0660] In a plurality of embodiments, the present invention provides a pharmaceutical composition comprising an auristatin conjugate described herein and a pharmaceutically acceptable carrier. The auristatin conjugate can be in any form that enables administration of the compound 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 can be in liquid or solid form. The preferred route of administration is parenteral. Parenteral administrations include 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 can be by any convenient route, such as by infusion or bolus injection.
[0661] The pharmaceutical composition can be formulated to make the compound bioavailable upon administration of the composition to a patient. The composition can take the form of one or more dosage units.
[0662] The materials used in the preparation of the pharmaceutical composition can be non-toxic in the amounts used. It will be apparent to those skilled in the art that the optimal dosage of the active ingredient(s) in the pharmaceutical composition will depend on various factors. Relevant factors include, but are not limited to, the type of animal (e.g., human), the particular form of the compound, the method of administration, and the composition being used.
[0663] The composition may be, for example, in liquid form. The liquid may be useful for delivery by injection. Compositions for administration by injection may also contain one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, and an isotonic agent.
[0664] When the liquid composition is in the form of a solution, suspension or other similar form, it can function as a sterile diluent such as water for injection, physiological saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride, a fixed oil such as synthetic mono- or diglycerides that can function as a solvent or suspending medium, polyethylene glycol, glycerin, cyclodextrin, propylene glycol or other solvents, an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium sulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as an amino acid, acetate, citrate or phosphate; a surfactant such as a nonionic surfactant, a polyol; and one or more of an agent for adjusting tonicity such as sodium chloride or dextrose. The parenteral composition may be enclosed in an ampoule, a disposable syringe, or a multi-dose vial made of glass, plastic, or other materials. Physiological saline is an exemplary adjuvant. The composition for injection is preferably sterilized.
[0665] The amount of the conjugate effective for 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 optionally be used to help identify the optimal dosage range. The exact dosage 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 physician's judgment and the circumstances of each patient.
[0666] The composition contains an effective amount of the compound so as to obtain an appropriate dosage. Typically, this amount is at least about 0.01% of the compound based on the weight of the composition.
[0667] For intravenous administration, the composition can contain from about 0.01 to about 100 mg of auristatin conjugate per kg of animal body weight. In one embodiment, the composition can contain from about 1 to about 100 mg of auristatin conjugate per kg of animal body weight. In another embodiment, the amount administered will be in the range of about 0.1 to about 25 mg per kg of body weight of the compound. Depending on the agent used, the dosage can 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, based on the weight of the subject. Generally, the dosage of the conjugate administered to a patient is typically from about 0.01 mg / kg to about 100 mg / kg based on the weight of the subject, or from about 1.0 μg / kg to 5.0 mg / kg based on the weight of the subject. In a plurality of embodiments, the dosage administered to the patient is from about 0.01 mg / kg to about 15 mg / kg based on the weight of the subject. In a plurality of embodiments, the dosage administered to the patient is from about 0.1 mg / kg to about 15 mg / kg based on the weight of the subject. In a plurality of embodiments, the dosage administered to the patient is from about 0.1 mg / kg to about 20 mg / kg based on the weight of the subject. In a plurality of embodiments, the dosage is from about 0.1 mg / kg to about 5 mg / kg or from about 0.1 mg / kg to about 10 mg / kg based on the weight of the subject. In a plurality of embodiments, the dosage is from about 1 mg / kg to about 15 mg / kg based on the weight of the subject. In a plurality of embodiments, the dosage is from about 1 mg / kg to about 10 mg / kg based on the weight of the subject. In a plurality of embodiments, the dosage is from about 0.1 to 4 mg / kg, more preferably 0.1 to 3.2 mg / kg, or even more preferably 0.1 to 2.7 mg / kg, based on the weight of the subject, over the course of a treatment cycle.
[0668] The term "carrier" refers to a diluent, adjuvant, or excipient to which a compound is administered. Such pharmaceutical carriers are liquids such as water and oil, including those of petroleum, animal, plant, and synthetic origin, and can be peanut oil, soybean oil, mineral oil, sesame oil, etc. The carrier can be physiological saline, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea. Further, adjuvants, stabilizers, thickeners, lubricants, and coloring agents can be used. In one embodiment, when administered to a patient, the compound or composition and the pharmaceutically acceptable carrier are sterile.
[0669] Water is an exemplary carrier when the compound is administered intravenously. Physiological saline and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol. The composition may contain, if desired, small amounts of wetting or emulsifying agents, or pH buffering agents.
[0670] In one embodiment, the conjugate is formulated according to routine procedures as a pharmaceutical composition suitable for intravenous administration to animals, particularly humans. Typically, the carrier or vehicle for intravenous administration is a sterile isotonic aqueous buffer. Optionally, the composition may also contain solubilizing agents. The composition for intravenous administration may optionally contain a local anesthetic such as lignocaine to reduce the pain at the injection site. Generally, the components are supplied separately or mixed together in unit dosage form as a dry lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or sachet indicating the amount of the active agent. When the conjugate is administered by infusion, it can be dispensed in an infusion bottle containing sterile pharmaceutical grade water or physiological saline. When the conjugate is administered by injection, an ampoule of sterile water for injection or physiological saline may be provided so that the components can be mixed before administration.
[0671] Pharmaceutical compositions are generally formulated to be sterile, substantially isotonic, and in full compliance with the manufacturing and quality control regulations (GMP) of all drugs of the US Food and Drug Administration and drugs and cosmetics of the Pharmaceutical Affairs Bureau.
[0672] Treatment of cancer The compounds described herein [e.g., any compound according to any one of formula (I), formula (II), formula (III), or formula (IV)] may be effective in selectively inducing cell death in a specific population (e.g., cells that overexpress a specific antigen, including those described herein such as tumor-associated antigens).
[0673] In vitro cytotoxicity assay: The cytotoxic performance of the compound is evaluated in a flat-bottom 96-well cell culture plate (Corning Costar) using the Cell Counting Kit-8 (CCK-8) assay (Shanghai Life Lab Biotech Co., Ltd.). Briefly, human tumor cells (1,000 - 10,000 cells / well depending on the cell line) in appropriate culture medium are incubated with the compound, or in the presence or absence of an excess of the corresponding unbound antibody, together with the conjugate at 37 °C and 5% CO2 for 120 hours.
[0674] For example, for any compound according to formula (III) or formula (IV), an appropriate selection of the cell binder can result in effective and highly selective targeting of cancer cells, which would be useful for the treatment of cancer.
[0675] The auristatin conjugates described herein [e.g., any compound of formula (III) or formula (IV)] are useful for inhibiting abnormal cell proliferation (e.g., causing apoptosis of tumor cells or cancer cells) or treating cancer in a patient. Accordingly, provided herein is a method of treating cancer in a subject in need of treatment for cancer, the method comprising administering to the subject one or more auristatin conjugates described herein.
[0676] In several embodiments, the invention features a method of treating a cell proliferative disease or disorder or inhibiting abnormal cell proliferation, the method comprising administering a pharmaceutical composition comprising any compound of formula (III) or formula (IV) described herein, or a pharmaceutically acceptable salt thereof, or any compound of formula (III) or formula (IV) described herein, or a pharmaceutically acceptable salt thereof.
[0677] Thus, the compounds described herein [e.g., any compound of formula (III) or formula (IV)] can be suitably used for treating various cancers. In several embodiments, an auristatin conjugate can be used to deliver a drug to tumor cells or cancer cells. Without being bound by theory, in one embodiment, the cell binding agent of the auristatin conjugate binds to or associates with a cancer cell or tumor cell-associated antigen, and the auristatin conjugate can be taken up (internalized) into the tumor cell or cancer cell via receptor-mediated endocytosis or other internalization mechanisms. The antigen can be a protein that binds to the tumor cell or cancer cell or an extracellular matrix protein associated with the tumor cell 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 auristatin conjugate outside the tumor cell or cancer cell, and then the free drug penetrates the cell.
[0678] In one embodiment, the cell binding agent binds to the tumor cell or cancer cell.
[0679] In another embodiment, the cell binding agent binds to a tumor cell or cancer cell antigen on the surface of a tumor cell or cancer cell.
[0680] In another embodiment, the cell binding agent binds to a tumor cell or cancer cell antigen that is an extracellular matrix protein associated with a tumor cell or cancer cell.
[0681] The specificity of the cell binding agent for a particular tumor cell or cancer cell can be important for determining the tumor or cancer that is most effectively treated.
[0682] Cancers that can be treated with auristatin conjugates include, for example, hematopoietic cancers such as lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma), as well as leukemia and solid tumors, but are not limited thereto. 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, osteogenic sarcoma, chondroma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, gastric cancer, oral cancer, nasal cancer, pharyngeal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, 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.
[0683] In multiple embodiments, the cancer is adenocarcinoma and is brain cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, CNS tumor, colon cancer or colorectal cancer, diffuse intrinsic pontine glioma (DIPG), endometrial cancer, esophageal cancer, Ewing sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, hematologic cancer, Hodgkin lymphoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma myelodysplastic syndrome (MDS), neuroblastoma, non-Hodgkin 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.
[0684] In multiple embodiments, the cancer is breast cancer.
[0685] Multimodal therapy for cancer
[0686] 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 an auristatin conjugate.
[0687] In other embodiments, provided is a method of treating cancer, comprising administering to a patient in need of treatment for cancer an effective amount of an auristatin conjugate and a chemotherapeutic agent. In one embodiment, the chemotherapeutic agent is one for which it is not known that the treatment of the cancer is refractory. In another embodiment, the chemotherapeutic agent is one for which it is known that the treatment of the cancer is refractory. The auristatin conjugate can be administered to a patient who has also undergone surgery as a treatment for cancer.
[0688] In multiple embodiments, the patient also receives additional treatments such as radiation therapy. In certain embodiments, the auristatin conjugate is administered concurrently with the chemotherapeutic agent or radiation therapy. In another certain embodiment, the chemotherapeutic agent or radiation therapy is administered before or after administration of the auristatin conjugate.
[0689] Chemotherapeutic agents can be administered over a series of sessions. The chemotherapeutic agents can be administered in any combination of one or more, such as chemotherapeutic agents that are standard treatments.
[0690] Furthermore, methods of treating cancer using auristatin conjugates are provided as an alternative to chemotherapy or radiation therapy when chemotherapy or radiation therapy has been demonstrated or can be demonstrated to be too toxic, such as causing unacceptable or intolerable side effects to the subject being treated. The patient being treated can optionally be treated with another cancer treatment, such as surgery, radiation therapy, or chemotherapy, depending on what treatments are acceptable or tolerable.
[0691] Treatment of autoimmune diseases Auristatin conjugates are useful for suppressing or inhibiting unwanted replication of cells that cause autoimmune diseases or for treating autoimmune diseases.
[0692] Auristatin conjugates can be used as appropriate in a variety of situations for treating a patient's autoimmune disease. Auristatin conjugates can be used to deliver a drug to target cells. Without being bound by theory, in one embodiment, the auristatin conjugate associates with an antigen on the surface of an inflammation-promoting or inappropriately stimulated immune cell, and then the auristatin conjugate is taken up into the target cell via receptor-mediated endocytosis. Once inside the cell, the cell-binding agent is cleaved, and as a result, auristatin is released. The released auristatin then freely moves within the cytosol and induces cytotoxic or cytostatic activity. In an alternative embodiment, the drug is cleaved from the auristatin conjugate outside the target cell, and the auristatin then penetrates into the cell.
[0693] In one embodiment, the cell binding agent binds to an autoimmune antigen. In one aspect, the antigen is on the surface of cells involved in an autoimmune condition.
[0694] In one embodiment, the cell binding agent binds to activated lymphocytes associated with an autoimmune disease state.
[0695] In a further embodiment, the auristatin conjugate suppresses or inhibits the proliferation of cells that produce autoantibodies associated with a particular autoimmune disease.
[0696] Specific types of autoimmune diseases that can be treated with an auristatin conjugate include, but are not limited to, Th2 lymphocyte-related disorders (e.g., atopic dermatitis, atopic asthma, rhinitis, allergic rhinitis, Wiskott-Aldrich syndrome, systemic sclerosis, and graft-versus-host disease); Th1 lymphocyte-related 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-related disorders (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes).
[0697] Method for preparing auristatin conjugate The auristatin conjugates described herein can be prepared convergently by any of the continuous construction of an antibody, linker, and drug unit, or by assembling parts followed by a completed assembly step.
[0698] In some embodiments, the auristatin payload compounds provided herein are combined with a suitable cell binding agent to facilitate covalent attachment of the auristatin payload compound to the cell binding agent. In multiple embodiments, the cell binding agent is an antibody that has at least 2, at least 4, at least 6, or 8 thiols available for attachment of the auristatin payload compound as a result of reducing the interchain disulfide bonds. In multiple embodiments, the auristatin payload compound binds to the cell binding agent via a cysteine moiety introduced onto the antibody.
[0699] Therapeutic kit In some aspects, kits are provided for use in treating cancer and treating autoimmune diseases. Such kits can include a pharmaceutical composition comprising an auristatin conjugate described herein.
[0700] In multiple embodiments, the kit can include instructions for use in any of the treatment methods described herein. The instructions included can indicate administration of the pharmaceutical composition to a subject to achieve the intended activity in the subject, e.g., treatment of a disease or condition such as cancer. In multiple embodiments, the instructions regarding the use of the pharmaceutical composition described herein can include information regarding dosage, dosing schedule, and route of administration for the intended treatment. The container can be a unit dose, a bulk package (e.g., a multi-dose package), or a sub-unit dose. The instructions provided with the kits 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 for treatment, delay in the onset, and / or alleviation of a disease or disorder in a subject.
[0701] In multiple embodiments, the kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, flasks, flexible packaging, etc. Also contemplated are packages for use in combination with certain devices such as inhalers, nasal delivery devices, or infusion devices. In multiple embodiments, the kit may comprise a sterile access port (e.g., the container can be a stoppered vial that can be pierced with an intravenous solution bag or a hypodermic needle).
[0702] In multiple embodiments, the kits provided herein include additional therapeutic agents useful for treating the cancers or autoimmune diseases described herein.
[0703] Examples While the specific compounds, compositions, and methods of the invention have been described as specific according to certain embodiments, the following examples function only to illustrate the compounds of the invention and are not intended to limit the same.
[0704] From the description of the specification, those skilled in the art can easily identify the essential features of the present invention and, without departing from its spirit and scope, make various changes and modifications to the present invention to adapt it to various uses and conditions.
[0705] All references, patents, or applications, whether domestic or foreign, cited in this application are hereby incorporated by reference as if fully set forth herein. In the event of any inconsistencies, the materials disclosed herein literally shall prevail.
[0706] The following abbreviations are used for the following terms. ADC Antibody-drug conjugate ACN Acetonitrile DAR Drug-to-antibody ratio DCC N,N'-Dicyclohexylcarbodiimide DCM Dichloromethane DHAA Dehydroascorbic acid DIPA Diisopropylamine DIPEA Diisopropylethylamine DMF N,N-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 EDCI 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate HIC Hydrophobic interaction chromatography HOBt Hydroxybenzotriazole HOPO 2-Hydroxypyridine-N-oxide i.v. Intravenous LC-MS Liquid chromatography mass spectrometry M Mole nM Nanomole NMM N-Methylmorpholine PPTS Pyridinium p-toluenesulfonate PTSA 4-Methylbenzenesulfonic acid PyBOP Benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate SEC Size exclusion chromatography TBAF Tetrabutylammonium fluoride TBS tert-Butyldimethylsilyl TBSCl tert-Butyldimethylsilyl chloride TCEP 3,3’,3’’-Phosphinetriyltripropanoic acid hydrochloride TEA Triethylamine TFA Trifluoroacetic acid THF Tetrahydrofuran TLC Thin layer chromatography TrOH Triphenylmethanol Tr Triphenylmethyl p-TsOH p-Toluenesulfonic acid
[0707] Example 1. Exemplary synthesis of compound MB-25 (D1) General procedure for the preparation of 2-(tritylthio)acetaldehyde (2) [Chemical formula]
[0708] To a solution of triphenylmethanol (29.0 g, 111 mmol) in dichloromethane (170 mL) were successively added 1,4-dithiane-2,5-diol (Compound 1, 17.0 g, 111 mmol) and trifluoroacetic acid (12.3 g, 108 mmol, 8.02 mL). The reaction mixture was stirred at 25 °C for 2 h. TLC (petroleum ether / ethyl acetate = 10 / 1, Rf = 0.3) indicated the completion of the reaction. The reaction mixture was quenched by the addition of aqueous NaHCO3 solution (100 mL) and extracted with dichloromethane (3 × 300 mL). The combined organic layers were washed with H2O (2 × 300 mL), dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo to give a residue, which was purified by column chromatography (SiO2, petroleum ether / ethyl acetate, 1 / 0 - 15 / 1) to afford Compound 2 (8.5 g, 8.0 mmol, 7.2% yield) as a white solid. 1 H NMR (400 MHz, DMSO-d6), δ ppm 3.15 (d, J = 2.25 Hz, 2H), 7.14 - 7.39 (m, 15 H), 8.90 (t, J = 2.38 Hz, 1H).
[0709] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-Hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(2-(tritylthio)ethyl)amino)butanamide)butanamide (3) Preparation of the general procedure.
Chemical formula
[0710] To a mixture of MMAE (5.0 g, 6.95 mmol) and Compound 2 (9.25 g, 13.9 mmol) in methanol (100 mL) was added acetic acid (2.09 g, 34.8 mmol). After stirring at 25 °C for 30 minutes under nitrogen protection, sodium cyanoborohydride (2.18 g, 34.8 mmol) was added to the reaction mixture all at once. The reaction was stirred at 25 °C for 12 hours. LCMS indicated that the reaction was complete. The reaction mixture was purified by preparative HPLC [column: Welch Xtimate C18 (250×100 mm×10 μm), mobile phase A: water (NH4HCO3), B: acetonitrile, B%: 75% - 99%, 20 minutes] to obtain Compound 3 (4.0 g, yield 56.2%) as a white solid. 11H NMR (400 MHz, methanol-d4), δ ppm 0.79 - 0.91 (m, 6 H), 0.92 - 1.07 (m, 13 H), 1.16 (br t, J=7.52 Hz, 3 H), 1.21 (br d, J=5.87 Hz, 3 H), 1.30 - 1.52 (m, 2 H), 1.54 - 1.67 (m, 1 H), 1.68 - 2.10 (m, 6 H), 2.14 (br d, J=10.76 Hz, 3 H), 2.21 - 2.47 (m, 4 H), 2.48 - 2.56 (m, 2 H), 2.57 - 2.69 (m, 2 H), 3.14 (s, 1 H), 3.23 (dt, J=11.62, 7.21 Hz, 1 H), 3.33 (br d, J=4.28 Hz, 5 H), 3.38 (s, 4 H), 3.40 - 3.49 (m, 1 H), 3.66 - 3.81 (m, 1 H), 4.18 - 4.32 (m, 2 H), 4.50 - 4.67 (m, 3 H), 4.71 (br d, J=8.19 Hz, 1 H), 4.80 (br s, 1 H), 7.20 - 7.46 (m, 20 H).
[0711] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-Hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-((2-mercaptoethyl)(methyl)amino)-3-methylbutanamide)-N,3-dimethylbutanamide (MB-25) (D1) General procedure for the preparation of [Chemical formula]
[0712] To a solution of compound 3 (150 mg, 147 μmol) in trifluoroacetic acid (2.23 g, 19.5 mmol) was added chlorotriisopropylsilane (384 mg, 2.43 mmol) and H2O (402 mg, 22.3 mmol). The reaction mixture was stirred at 25 °C for 1 h. LCMS indicated that the reaction was complete. This was diluted with acetonitrile (0.20 mL) and filtered. The filtrate was purified by preparative HPLC [column: C18 (150 × 30 mm × 5 μm); mobile phase A: water (NH4HCO3), B: acetonitrile; B%: 60% - 90%, 10 min] to give compound MB-25 (D1) (23.0 mg, yield 20.1%) as a white solid. MS (ESI+): m / z calcd for 778.52 (M+H) + , found 778.56. 1 H NMR (400 MHz, methanol-d4), δ ppm 0.77 - 0.91 (m, 6 H), 0.93 - 1.09 (m, 13 H), 1.09 - 1.16 (m, 3 H), 1.18 (br d, J = 6.44 Hz, 3 H), 1.31 - 1.64 (m, 3 H), 1.65 - 2.27 (m, 7 H), 2.32 (br d, J = 11.92 Hz, 3 H), 2.45 - 2.85 (m, 7 H), 3.11 - 3.24 (m, 2 H), 3.34 - 3.46 (m, 6 H), 3.52 - 3.92 (m, 2 H), 4.05 - 4.31 (m, 2 H), 4.45 - 4.81 (m, 5 H), 7.18 - 7.44 (m, 5 H).
[0713] Example 2. Exemplary synthesis of compound MB-23 (D2) (S)-2-((2R,3R)-3-((S)-1-((6S,9S,12S,13R)-12-((S)-sec-Butyl)-6,9-diisopropyl-13-methoxy-5,11-dimethyl-7,10-dioxo-1,1,1-triphenyl-2-thia-5,8,11-triazapentadecan-15-yl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamide)-3-phenylpropanoic acid (4) General procedure for the preparation. [Chemical]
[0714] To a suspension of MMAF (5.0 g, 6.85 mmol) and Compound 2 (10.0 g, 20.5 mmol) in methanol (100 mL), 4 Å molecular sieves (5.0 g) and acetic acid (2.05 g, 34.1 mmol) were added. After stirring at 25 °C for 30 minutes, sodium cyanoborohydride (2.15 g, 34.1 mmol) was added to the reaction mixture all at once. The reaction was stirred at 25 °C for 12 hours. LCMS indicated that the reaction was complete. This was quenched with water (500 mL) and extracted with ethyl acetate (3 × 500 mL). The organic phase was dried over sodium sulfate, filtered, and the filtrate was concentrated to give a residue, which was purified by preparative HPLC [column: Welch Xtimate C18 (250 × 70 mm × 10 μm), mobile phase A: water (NH4HCO3), B: acetonitrile, B%: 45% - 75%, 16 minutes] to give Compound 4 (4.0 g, yield 56.6%) as a white solid. MS (ESI+) m / z, calculated value 1034.60 (M+H) + , found 1034.70. 11H NMR (400 MHz, methanol-d4), δ ppm 0.74 - 0.88 (m, 6H), 0.90 - 1.09 (m, 13H), 1.18 (br dd, J = 18.30, 5.54 Hz, 3H), 1.27 - 1.69 (m, 4H), 1.71 - 1.98 (m, 4H), 2.10 (br d, J = 3.34 Hz, 3H), 2.15 - 2.52 (m, 6H), 2.59 (br dd, J = 9.54, 4.77 Hz, 2H), 2.89 - 3.04 (m, 1H), 3.08 - 3.28 (m, 5H), 3.35 (br d, J = 7.15 Hz, 3H), 3.38 - 3.45 (m, 1H), 3.46 - 3.58 (m, 1H), 3.62 - 3.76 (m, 1H), 3.84 (br d, J = 3.58 Hz, 1H), 4.17 (br s, 1H), 4.59 (br d, J = 8.70 Hz, 1H), 4.73 (br d, J = 7.75 Hz, 1H), 7.16 - 7.32 (m, 14H), 7.35 - 7.45 (m, 6H).
[0715] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-((2-Mercaptoethyl)(methyl)amino)-3-methylbutanamide)-N,3-dimethylbutanamide)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamide)-3-phenylpropanoic acid (MB-23) (D2) General procedure for the preparation.
Chemical Structure
[0716] To a suspension of compound 4 (2.0 g, 1.93 mmol, 1.0 equiv) in trifluoroacetic acid (29.2 g, 256 mmol, 19.0 mL) was added chlorotriisopropylsilane (5.05 g, 31.9 mmol, 6.55 mL) and H2O (5.29 g, 293 mmol). The reaction mixture was stirred at 25 °C for 1 h under N2 protection 、 and monitored by LCMS to show completion of the reaction. The reaction mixture was concentrated in vacuo to give a residue, which was purified by preparative HPLC [column: Phenomenex Luna C18 (80×30 mm×3 μm); mobile phase A: water, B: acetonitrile, B%: 15% - 45%, 8 min] to afford the product MB-23 (D2) (1.50 g, yield 77.0%) as a white solid. HRMS (ESI+), calculated for m / z 792.49 (M+H) + , found 792.4944. 1 H NMR (400 MHz, methanol-d4), δ ppm 0.75 - 0.91 (m, 6 H), 0.93 - 1.07 (m, 11 H), 1.07 - 1.22 (m, 5 H), 1.26 - 1.47 (m, 2 H), 1.50 - 1.67 (m, 1 H), 1.70 - 1.94 (m, 3 H), 1.98 - 2.18 (m, 2 H), 2.19 - 2.53 (m, 6 H), 2.75 - 3.03 (m, 5 H), 3.14 (s, 1 H), 3.18 - 3.29 (m, 4 H), 3.33 - 3.45 (m, 6 H), 3.46 - 3.56 (m, 1 H), 3.62 - 3.76 (m, 1 H), 3.85 (dd, J=8.50, 1.65 Hz, 1 H), 4.02 - 4.18 (m, 1 H), 4.63 - 4.76 (m, 4 H), 7.13 - 7.31 (m, 5 H).
[0717] Example 3. Exemplary synthesis of compound MB-26 (PL1) (9H-Fluoren-9-yl)methyl ((3R,4S,7S,10S,18S,21S)-4-((S)-sec-butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11,18,22-tetramethyl-6,9,17,20-tetraoxo-2-oxa-14-thia-5,8,11,16,19-pentaazatricosane-21-yl)carbamate (6) Preparation of general procedure.
Chemical formula
[0718] To a solution of compound MB-25 (1.95 g, 2.15 mmol) and compound 5 (2.48 g, 3.87 mmol) in N,N-dimethylformamide (19.5 mL) was added HCl / diethyl ether (6.40 M, 1.32 mL). The reaction mixture was stirred at 25 °C for 12 h. LCMS indicated that the reaction was complete. This was concentrated under reduced pressure to remove HCl / diethyl ether. The residue was purified by preparative HPLC [column: Phenomenex luna C18 (250×50 mm×10 μm), mobile phase A: water (TFA), B: acetonitrile, B%: 30% - 70%, 10 min] to give compound 6 (1.65 g, yield 52.3%) as a white solid. 11H NMR (400 MHz, methanol-d4), δ ppm 0.82 - 1.21 (m, 17 H), 1.31 - 1.49 (m, 3 H), 1.52 - 2.25 (m, 4 H), 2.33 - 2.58 (m, 2 H), 2.78 - 3.06 (m, 3 H), 3.09 - 3.14 (m, 1 H), 3.20 (dt, J=11.63, 7.25 Hz, 1 H), 3.33 - 3.38 (m, 2 H), 3.38 - 3.50 (m, 1 H), 3.50 - 3.58 (m, 1 H), 3.64 - 3.94 (m, 2 H), 4.02 - 4.11 (m, 1 H), 4.14 - 4.33 (m, 3 H), 4.34 - 4.42 (m, 1 H), 4.46 - 4.58 (m, 1 H), 4.59 - 4.64 (m, 1 H), 4.66 - 4.72 (m, 1 H), 4.73 - 4.80 (m, 1 H), 7.17 - 7.45 (m, 5 H), 7.67 (br d, J=7.50 Hz, 1 H), 7.81 (d, J=7.50 Hz, 1 H), 7.95 - 8.03 (m, 1 H), 8.20 - 8.28 (m, 1 H), 8.50 - 8.62 (m, 1 H).
[0719] (S)-2-((2S,10S,13S)-13-Amino-2-isopropyl-3,10,14-trimethyl-9,12-dioxo-6-thia-3,8,11-triazapentadecanamide)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide (7) Preparation of the general procedure.
Chemical Structure
[0720] To a solution of compound 6 (1.65 g, 1.38 mmol) in N,N-dimethylformamide (15.0 mL) was added morpholine (599 mg, 6.88 mmol, 605 μL). The reaction mixture was stirred at 25 °C for 12 h under N2 protection. LCMS indicated that the reaction was complete. This was concentrated under reduced pressure to obtain a residue, which was purified by preparative HPLC [column: C18 (250×50 mm×10 μm), mobile phase A: water (NH4HCO3), B: acetonitrile, B%: 35% - 65%, 10 min] to give compound 7 (880 mg, yield 65.4%) as a white solid. 1 H NMR (400 MHz, methanol-d4), δ ppm 0.74 - 1.04 (m, 24 H), 1.05 - 1.17 (m, 6 H), 1.27 - 1.45 (m, 4 H), 1.48 - 1.60 (m, 1 H), 1.61 - 2.23 (m, 8 H), 2.29 (br d, J=12.35 Hz, 3 H), 2.38 - 2.55 (m, 2 H), 2.59 - 2.89 (m, 5 H), 3.06 - 3.18 (m, 3 H), 3.27 (br d, J=1.22 Hz, 7 H), 3.39 (br d, J=9.29 Hz, 1 H), 3.47 - 3.74 (m, 2 H), 3.75 - 3.91 (m, 1 H), 3.96 - 4.41 (m, 5 H), 4.45 - 4.64 (m, 3 H), 4.71 (br dd, J=18.28, 8.99 Hz, 2 H), 7.07 - 7.45 (m, 5 H).
[0721] (S)-N1-((3R,4S,7S,10S,18S,21S)-4-((S)-sec-Butyl)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-Hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11,18,22-tetramethyl-6,9,17,20-tetraoxo-2-oxa-14-thia-5,8,11,16,19-pentaazatricosane-21-yl)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide)-N5-((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)pentanediamide (MB-26) (PL1) Preparation of general procedure.
Chem.
[0722] To a solution of compound 8 (245 mg, 0.487 mmol) and dicyclohexylcarbodiimide (201 mg, 0.974 mmol) in N,N-dimethylformamide (4.0 mL) was added N-hydroxysuccinimide (112 mg, 0.974 mmol). After stirring at 25 °C for 6 h, the reaction mixture was filtered to remove the precipitate. The filtrate was added to a solution of compound 7 (500 mg, 0.511 mmol) in N,N-dimethylformamide (7.50 mL). The resulting reaction mixture was stirred at 25 °C for an additional 12 h. LCMS indicated that the reaction was complete. This was filtered and the filtrate was loaded directly onto preparative HPLC (column: Phenomenex C18 (75×30 mm×3 μm), mobile phase A: water, B: acetonitrile, B%: 30% - 65%, 8 min) for purification, and the product MB-26 (PL1) (85.0 mg, yield 11.6%) was obtained as a white solid. HRMS (ESI+): m / z calcd 1462.85 (M+H) + , found 1462.8763. 11H NMR (400 MHz, methanol-d4), δ ppm 0.80 - 0.91 (m, 6 H), 0.92 - 1.08 (m, 18 H), 1.09 - 1.20 (m, 6 H), 1.22 - 1.49 (m, 10 H), 1.61 (ddd, J=14.87, 11.77, 7.45 Hz, 5 H), 2.15 (br d, J=3.81 Hz, 10 H), 2.20 - 2.36 (m, 7 H), 2.45 - 2.55 (m, 2 H), 2.66 - 2.85 (m, 5 H), 3.11 - 3.17 (m, 2 H), 3.35 (s, 5 H), 3.43 (br dd, J=9.83, 4.11 Hz, 3 H), 3.47 - 3.51 (m, 3 H), 3.60 - 3.81 (m, 7 H), 3.88 (br dd, J=7.81, 4.35 Hz, 1 H), 4.15 - 4.29 (m, 3 H), 4.31 - 4.37 (m, 3 H), 4.51 - 4.57 (m, 1 H), 4.60 - 4.67 (m, 1 H), 4.74 (br d, J=8.46 Hz, 1 H), 6.74 - 6.84 (m, 2 H), 7.13 - 7.43 (m, 5 H).
[0723] Example 4. Exemplary synthesis of compound MB-24 (PL2) (S)-2-((2R,3R)-3-((S)-1-((5S,8S,16S,19S,22S,23R)-22-((S)-sec-Butyl)-1-(9H-fluoren-9-yl)-5,16,19-triisopropyl-23-methoxy-8,15,21-trimethyl-3,6,9,17,20-pentaoxo-2-oxa-12-thia-4,7,10,15,18,21-hexaazapentacosane-25-yl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamide)-3-phenylpropanoic acid (9) Preparation of the general procedure. [Chemical formula]
[0724] To a mixture of compound MB-23 (900 mg, 1.13 mmol) and compound 5 (1.09 g, 1.70 mmol) in N,N-dimethylformamide (3 mL) was added HCl / diethyl ether (6.40 M, 236 μL). The reaction mixture was stirred at 25 °C for 12 h. LCMS indicated that the reaction was complete. This was concentrated under reduced pressure to remove diethyl ether. The residue was purified by preparative HPLC [column: Phenomenex luna C18 (250×50 mm×10 μm), mobile phase A: water (TFA), B: acetonitrile, B%: 30% - 60%, 10 min] to give compound 9 (450 mg, yield 32.6%) as a white solid. LCMS (ESI+): m / z calculated 1213.7 (M+H) + , found 1213.8. 11H NMR: (400 MHz, methanol-d4), δ ppm, 0.81 - 0.89 (m, 3 H), 0.90 - 1.05 (m, 18 H), 1.06 - 1.17 (m, 6 H), 1.20 (d, J=6.72 Hz, 2 H), 1.24 - 1.47 (m, 6 H), 1.49 - 1.65 (m, 1 H), 1.69 - 1.94 (m, 3 H), 1.99 - 2.14 (m, 2 H), 2.15 - 2.28 (m, 1 H), 2.29 - 2.40 (m, 1 H), 2.45 (br s, 2 H), 2.89 (br s, 6 H), 3.09 - 3.14 (m, 1 H), 3.16 - 3.29 (m, 4 H), 3.34 (d, J=4.40 Hz, 4 H), 3.37 - 3.52 (m, 3 H), 3.62 - 3.74 (m, 1 H), 3.88 (br d, J=7.09 Hz, 3 H), 4.03 - 4.18 (m, 1 H), 4.24 (br d, J=6.24 Hz, 3 H), 4.32 - 4.46 (m, 2 H), 4.51 (s, 2 H), 4.67 - 4.78 (m, 2 H), 7.14 - 7.35 (m, 7 H), 7.36 - 7.43 (m, 2 H), 7.67 (br d, J=7.09 Hz, 2 H), 7.77 - 7.85 (m, 2 H).
[0725] (S)-2-((2R,3R)-3-((S)-1-((3S,6S,14S,17S,20S,21R)-3-Amino-20-((S)-sec-butyl)-14,17-diisopropyl-21-methoxy-2,6,13,19-tetramethyl-4,7,15,18-tetraoxo-10-thia-5,8,13,16,19-pentaazatricosane-23-yl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamide)-3-phenylpropanoic acid (10) Preparation of the general procedure.
Chemical Structure
[0726] To a solution of Compound 9 (800 mg, 0.659 mmol) in N,N-dimethylformamide (20.0 mL) was added morpholine (373 mg, 4.28 mmol). The reaction mixture was stirred at 25 °C for 12 hours. LCMS indicated that the reaction was complete. This was concentrated in vacuo to give a residue, which was purified by preparative HPLC [column: C18 (250×50 mm×10 μm); mobile phase, A: water (NH4HCO3), B: acetonitrile; B%: 20% - 50%, 10 min] to afford Product 10 (625 mg, yield 87.0%) as a yellow solid. LCMS (ESI+): calculated m / z 991.6 (M+H) + , found 991.7. 1H NMR (400 MHz, methanol-d4), δ ppm 0.77 - 0.91 (m, 5H), 0.93 - 1.10 (m, 18H), 1.16 (br s, 1H), 1.19 (br d, J = 6.60 Hz, 2H), 1.24 - 1.33 (m, 1H), 1.39 (br d, J = 7.70 Hz, 1H), 1.42 (br d, J = 7.09 Hz, 2H), 1.46 - 1.68 (m, 2H), 1.72 - 1.94 (m, 3H), 2.00 - 2.13 (m, 2H), 2.15 - 2.27 (m, 2H), 2.31 (br s, 1H), 2.33 (s, 2H), 2.38 - 2.53 (m, 2H), 2.65 - 2.77 (m, 3H), 2.79 (br d, J = 9.78 Hz, 1H), 2.83 - 3.02 (m, 2H), 3.25 (br s, 4H), 3.33 - 3.38 (m, 5H), 3.38 - 3.54 (m, 2H), 3.69 (br d, J = 5.87 Hz, 2H), 3.74 - 3.89 (m, 1H), 4.07 - 4.21 (m, 1H), 4.25 - 4.33 (m, 1H), 4.38 (br s, 2H), 4.52 - 4.73 (m, 3H), 4.79 (br d, J = 8.19 Hz, 3H), 7.09 - 7.30 (m, 5H).
[0727] (S)-2-((2R,3R)-3-((S)-1-((3R,4S,7S,10S,18S,21S, 24S,30S,31R,32R,33R)-4-((S)-sec-Butyl)-24-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-30,31,32,33,34-pentahydroxy-7,10,21-triisopropyl-3-methoxy-5,11,18-trimethyl-6,9,17,20,23,27-hexaoxo-14-thia-5,8,11,16,19,22,28-heptaazatetratriacontan-1-yl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid (MB-24) (PL2) General procedure for the preparation.
Chem.
[0728] To a solution of compound 8 (50.0 mg, 99.3 μmol) and dicyclohexylcarbodiimide (40.9 mg, 198 μmol) in DMF (0.40 mL) was added N-hydroxysuccinimide (22.8 mg, 198 μmol). After stirring at 25 °C for 6 h, a solution of compound 10 (103 mg, 104 μmol) in DMF (0.40 mL) was added to the above reaction mixture and stirring was continued at 25 °C for an additional 12 h. LCMS indicated that the reaction was complete. This was filtered and, for purification, the filtrate was directly loaded onto preparative HPLC [column: Phenomenex Luna C18 (80×30 mm×3 μm); mobile phase A: water, B: acetonitrile, B%: 15% - 45%, 8 min] and purified to give the product MB-24 (PL2) (200.6 mg, yield 13.9%) as a white solid. HRMS (ESI+), m / z, calculated value 1476.83 (M+H) + , found value 1476.8430. 11H NMR (400 MHz, methanol-d4), δ ppm 0.82 - 0.91 (m, 6H), 0.93 - 1.06 (m, 17H), 1.13 (dd, J = 16.03, 6.74 Hz, 3H), 1.20 (d, J = 6.79 Hz, 2H), 1.23 - 1.48 (m, 8H), 1.49 - 1.69 (m, 5H), 1.70 - 2.00 (m, 4H), 2.01 - 2.18 (m, 4H), 2.19 - 2.36 (m, 5H), 2.37 - 2.54 (m, 5H), 2.61 - 3.08 (m, 7H), 3.14 (s, 1H), 3.27 (br d, J = 14.90 Hz, 4H), 3.33 - 3.56 (m, 9H), 3.59 - 3.91 (m, 8H), 4.03 - 4.22 (m, 2H), 4.25 - 4.44 (m, 4H), 4.64 - 4.71 (m, 1H), 4.75 - 4.82 (m, 2H), 6.80 (d, J = 1.19 Hz, 2H), 7.13 - 7.20 (m, 1H), 7.21 - 7.30 (m, 4H).
[0729] Example 5. Exemplary synthesis of compound D5 General procedure for the preparation of 2-(methylthio)acetaldehyde. [Chemical formula]
[0730] A solution of 1,1-dimethoxy-2-methylsulfanyl-ethane (2.00 g, 14.7 mmol, 1.96 mL) in aqueous HCl solution (0.32 M, 5.70 mL) was stirred at 50 °C for 0.5 h. The mixture was concentrated under reduced pressure to give a yellowish oil, which was distilled at 60 °C to give 2-methylsulfanylacetaldehyde (550 mg, 6.10 mmol, yield 41.5%) as a yellowish oil. 11H NMR (400 MHz, CDCl3) δ 2.03 (s, 3 H), 3.15 (d, J=3.50 Hz, 2 H), 9.46 (t, J=3.56 Hz, 1 H).
[0731] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-Hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(2-(methylthio)ethyl)amino)butanamide)butanamide (D5) General procedure for the preparation. [Chemical formula]
[0732] (2S)-N-[(1S)-1-[[(1S,2R)-4-[(2S)-2-[(1R,2R)-3-[[(1R,2S)-2-hydroxy-1-methyl-2-phenylethyl]amino]-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl]-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl]-methyl-carbamoyl]-2-methylpropyl]-3-methyl-2-(methylamino)butanamide (MMAE, 100 mg, 139 μmol), 2-methylsulfanylacetaldehyde (25.1 mg, 279 μmol) and acetic acid (41.8 mg, 696 μmol) were stirred at 25 °C for 20 min, followed by quenching by the addition of sodium cyanoborohydride (43.8 mg, 696 μmol) all at once. The reaction mixture was stirred at 25 °C for 12 h. LCMS indicated that the reaction was complete. The reaction mixture was filtered and the filtrate was purified by preparative HPLC (column: Waters Xbridge BEH C18 100×30 mm×10um, mobile phase: [H2O(10 mM NH4HCO3)-acetonitrile], gradient: 40% - 75% B over 8.0 min) to afford Compound D5 (21.9 mg, yield 19.9%) as a white solid. LCMS (ESI + ): m / z 792.3 [M+H] + . 11H NMR (400 MHz, DMSO-d6) δ 0.67 - 0.80 (m, 6 H), 0.80 - 0.95 (m, 13 H), 0.96 - 1.09 (m, 6 H), 1.20 - 1.38 (m, 1 H), 1.42 - 1.61 (m, 2 H), 1.61 - 1.86 (m, 3 H), 1.86 - 2.01 (m, 2 H), 2.02 - 2.08 (m, 3 H), 2.08 - 2.18 (m, 1 H), 2.19 - 2.34 (m, 4 H), 2.36 - 2.46 (m, 1 H), 2.52 - 2.79 (m, 5 H), 2.95 - 3.11 (m, 2 H), 3.13 - 3.28 (m, 8 H), 3.32 - 3.39 (m, 1 H), 3.41 - 3.63 (m, 1 H), 3.78 (dd, J=9.38, 1.75 Hz, 1 H), 3.88 - 4.11 (m, 2 H), 4.38 - 4.61 (m, 2 H), 4.61 - 4.83 (m, 1 H), 5.27 - 5.48 (m, 1 H), 7.08 - 7.21 (m, 1 H), 7.23 - 7.39 (m, 4 H), 7.53 - 7.99 (m, 1 H), 8.07 - 8.26 (m, 1 H).
[0733] Example 6. Exemplary synthesis of compound D6 ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methyl(2-(methylthio)ethyl)amino)butanamide)butanamide)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (D6) Preparation of general procedure.
Chemical Structure
[0734] To a mixture of ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (MMAF, 110 mg, 150 μmol) in methanol (2.00 mL) was added acetic acid (45.2 mg, 751 μmol) and 2-methylsulfanylacetaldehyde (27.1 mg, 301 μmol, 2.00 equiv), followed by sodium cyanoborohydride (47.2 equiv, 751 μmol)). The reaction mixture was stirred at 25 °C for 12 h. LCMS indicated that the reaction was complete. The reaction mixture was filtered and the filtrate was purified by preparative HPLC (column: Waters Xbridge BEH C18 100×30 mm×10 um, mobile phase: [H2O(10 mM NH4HCO3)-acetonitrile], gradient: 40% - 75% B over 8.0 min) to give Compound D6 (57.4 mg, 46.9% yield) as a white solid. LCMS (ESI + ): m / z 806.4 [M+H] + . 11H NMR (400 MHz, methanol-d4) δ 0.81 - 0.92 (m, 6 H), 0.95 - 1.06 (m, 11 H), 1.08 - 1.23 (m, 4 H), 1.12 - 1.14 (m, 1 H), 1.26 - 1.47 (m, 2 H), 1.49 - 1.67 (m, 1 H), 1.70 - 1.94 (m, 3 H), 1.99 - 2.18 (m, 5 H), 2.21 - 2.34 (m, 1 H), 2.31 - 2.33 (m, 1 H), 2.35 - 2.53 (m, 5 H), 2.59 - 2.79 (m, 3 H), 2.80 - 2.98 (m, 3 H), 3.14 (s, 1 H), 3.19 (dt, J=11.91, 7.30 Hz, 1 H), 3.24 - 3.28 (m, 2 H), 3.29 (s, 1 H), 3.32 - 3.45 (m, 7 H), 3.47 - 3.56 (m, 1 H), 3.61 - 3.78 (m, 1 H), 3.85 (dd, J=8.50, 2.13 Hz, 1 H), 4.01 - 4.21 (m, 1 H), 4.60 - 4.71 (m, 1 H), 4.72 - 4.84 (m, 2 H), 7.13 - 7.31 (m, 5 H).
[0735] Example 7. Exemplary synthesis of antibody-drug conjugate trastuzumab-MB24
Chemical formula
[0736] 50 mM conjugation buffer (pH 7.4): 1 L of the solution contains 6.86 g of Na2HPO4·2H2O and 1.58 g of NaH2PO4·H2O.
[0737] 10 mM aqueous DTPA solution: 1 L of the solution contains 3.90 g of DTPA and 1.20 g of NaOH.
[0738] 10 mM aqueous TCEP solution: 1 L of the solution contains 2.866 g of TCEP.
[0739] Formulation buffer (pH 6.0) (10 mM His / His-HCl): 1 L of the solution contains 0.73 g of His and 1.12 g of His-HCl.
[0740] Antibody preparation: 627 mg of lyophilized trastuzumab powder was dissolved in 22 mL of purified water. The resulting antibody solution was dialyzed 4 cycles against 50 mM conjugation buffer using an ultrafiltration tube (30 KD) to obtain an antibody concentration of 11.97 mg / ml (trastuzumab ε280 absorption coefficient = 213380 M -1 cm -1 was used).
[0741] Reduction of trastuzumab: To a tube containing 2.5 mL (30 mg, 0.000207 mmol of trastuzumab) of the trastuzumab solution prepared above, 3.4 mL of 50 mM conjugation buffer was added, followed by 87 μl of 10 mM TCEP (0.00087 mmol, 4.2 equivalents) and 41.4 μL of 10 mM ZnCl2 solution (0.000414 mmol, 2 equivalents). The tube was placed in a thermomixer and the reduction reaction was carried out at 25 °C for 3 hours.
[0742] Conjugation between antibody and payload: 5.68 mg (0.00385 mmol) of payload MB-24 (PL2) was dissolved in 769 μL of DMSO to obtain a 5 mM solution. To the above trastuzumab reduction solution, 331 μL (0.00166 mmol, 8 equivalents) of 5 mM MB-24 in DMSO solution was added. The tube was placed in a thermomixer and the conjugation reaction was carried out at 25 °C for 1 hour, followed by the addition of 5 mM cysteine solution (166 μL). Then, 10 mM DTPA solution (83 μL) and 10 mM DHAA solution (166 μL) were added to remove zinc ions.
[0743] Purification: The above conjugation reaction solution was subjected to purification using an ultrafiltration tube (30KD) for 8 cycles with 10 mM His / His-HCl formulation buffer, and 2.6 mL (8.2 mg / mL) of trastuzumab-MB24 (21.3 mg, yield = 68%, DAR = 4.3) was obtained in the formulation buffer.
[0744] Physicochemical characterization of trastuzumab-MB24 (extinction coefficient of payload MB-24: ε2 80 = 764.4 M -1 cm -1 and ε260 = 1280.1 M -1 cm -1 ). Additional characterization data are provided in Table 1.
[0745]
Table 2
[0746] Example 8. Exemplary synthesis of antibody-drug conjugate trastuzumab-MB26
Chemical formula
[0747] Reduction of trastuzumab: To a tube containing 2.9 mL (35 mg, 0.000240 mmol of trastuzumab) of trastuzumab solution (11.97 mg / mL), 4 mL of 50 mM conjugation buffer was added, followed by 92 μl of 10 mM TCEP (0.00092 mmol, 3.8 equivalents) and 48 μL of 10 mM ZnCl2 solution (0.00048 mmol, 2 equivalents). The tube was placed in a thermomixer, and the reduction reaction was carried out at 25 °C for 3 hours.
[0748] Conjugation between antibody and payload: 8.43 mg (0.00576 mmol) of payload MB-26 (PL1) was dissolved in 1153 μL of DMSO to obtain a 5 mM solution. To the above trastuzumab reduction solution, 384 μL (0.00192 mmol, 8 equivalents) of 5 mM MB-24 in DMSO solution was added. The tube was placed in a thermomixer, and the conjugation reaction was carried out at 25 °C for 1 hour, followed by the addition of 5 mM cysteine solution (192 μL). Then, 10 mM DTPA solution (96 μL) and 10 mM DHAA solution (192 μL) were added to remove zinc ions.
[0749] Purification: The above conjugation reaction solution was subjected to purification using an ultrafiltration tube (30 KD) with 8 cycles using 10 mM His / His-HCl formulation buffer to obtain 3.7 mL (5.7 mg / mL) of trastuzumab-MB26 (21.1 mg, yield = 58%, DAR = 4.3) in the formulation buffer.
[0750] Physicochemical characterization of trastuzumab-MB26 (absorption coefficient of payload MB-26: ε2 80 = 639.5 M -1 cm -1 and ε260 = 1006.3 M -1 cm -1 ). Additional characterization data are provided in Table 2.
[0751]
Table 3
[0752] Example 9. Exemplary synthesis of dual-drug conjugate trastuzumab-MB0324
Chemical formula
[0753] Reduction of trastuzumab: To a tube containing 4.1 mL (9.83 mg / mL, 40.3 mg of trastuzumab) of trastuzumab in conjugation buffer, 3.7 mL of 50 mM conjugation buffer was added, followed by the addition of 116 μL of 10 mM TCEP (0.00116 mmol, 4.2 equivalents) and 55 μL of 10 mM ZnCl2 (2 equivalents). The tube was placed in a thermomixer and the reduction reaction was carried out at 25 °C for 3 hours.
[0754] Conjugation between the antibody and the first payload: 4.02 mg (0.00272 mmol) of payload MB-24 was dissolved in 544 μL of DMSO to obtain a 5 mM solution. To the above trastuzumab reduction solution, 442 μL of 5 mM MB-24 in DMSO solution (0.00221 mmol, 8 equivalents per antibody molecule) was added. The tube was placed in a thermomixer and the conjugation reaction was carried out at 25 °C for 1 hour, followed by the addition of 221 μL of cysteine solution (5 mM), 110 μL of DTPA solution (10 mM), and 221 μL of DHAA solution (10 mM).
[0755] Purification: The above conjugation reaction solution was subjected to purification using an ultrafiltration tube (30KD) over 4 cycles with conjugation buffer (containing 10% 10 mM DTPA) to obtain 3.8 mL (8.48 mg / mL) of trastuzumab-MB24 (32.2 mg) in conjugation buffer. The DAR value was 4.2 based on HIC-HPLC.
[0756] Reduction of trastuzumab-MB24: To a tube containing the above trastuzumab-MB24 in conjugation buffer (3.8 mL, 8.48 mg / mL), 4.1 mL of 50 mM conjugation buffer (containing 10% 10 mM DTPA solution) and 110 μL of 10 mM TCEP solution (5 equivalents) were added. The tube was placed in a thermomixer and the reduction reaction was carried out at 25 °C for 2 hours.
[0757] Conjugation with the second payload: 4.33 mg (0.0081 mmol) of the payload medicament (MB-3) was dissolved in 761 μL of DMSO to obtain a 5 mM solution. To the above trastuzumab-MB24 reduction solution, 264 μL (0.00132 mmol, 6 equivalents) of 5 mM medicament was added as a DMSO solution. The tube was placed in a thermomixer, and the conjugation reaction was carried out at 25 °C for 1 hour.
[0758] Purification: The above conjugation reaction solution was subjected to purification using an 8-cycle ultrafiltration tube (30KD) with 10 mM His / His-HCl formulation buffer to obtain 1.6 mL (15.5 mg / mL) of trastuzumab-MB0324 (24.8 mg, yield = 62%, DAR = 3.9 for the payload medicament, DAR = 4.1 for the payload MB-24) in the formulation buffer.
[0759] Physicochemical characterization of trastuzumab-MB0324 (absorption coefficient of medicament: ε280 = 4546 M -1 cm -1 and ε360 = 17513 M -1 cm -1 , absorption coefficient of MB-24: ε280 = 764.4 M -1 cm -1 and ε260 = 1280.1 M -1 cm -1 ), and further characterization data are provided in Table 3.
[0760]
Table 4
[0761] Example 10. Exemplary synthesis of dual-drug conjugate trastuzumab-MB0326
Chemical formula
[0762] Reduction of Trastuzumab: To a tube containing 3.2 mL of trastuzumab in conjugation buffer (the concentration of trastuzumab in conjugation buffer was 15.6 mg / mL), 3.7 mL of 50 mM conjugation buffer was added, followed by the addition of 131 μL (3.8 equivalents) of 10 mM TCEP and 69 μL (2 equivalents) of 10 mM ZnCl2. The tube was placed in a thermomixer, and the reduction reaction was carried out at 25 °C for 3 hours.
[0763] Conjugation between the antibody and the first payload: 40.8 mg (0.0279 mmol) of payload MB-26 was dissolved in 5.6 mL of DMSO to obtain a 5 mM solution. To the above trastuzumab reduction solution, 552 μL (0.00276 mmol, 8 equivalents per antibody molecule) of 5 mM MB-26 in DMSO solution was added. The tube was placed in a thermomixer, and the conjugation reaction was carried out at 25 °C for 1 hour, followed by the addition of 276 μL of cysteine solution (5 mM), 138 μL of DTPA solution (10 mM), and 276 μL of DHAA solution (10 mM).
[0764] Purification: The above conjugation reaction solution was subjected to purification using an ultrafiltration tube (30KD) with conjugation buffer (containing 10% 10 mM DTPA) for 4 cycles to obtain 7.8 mL (6 mg / mL) of trastuzumab-MB26 (46.8 mg) in conjugation buffer. The DAR value was 4.4 based on HIC-HPLC.
[0765] Reduction of trastuzumab-MB26: To a tube containing the above trastuzumab-MB26 in conjugation buffer (7.8 mL, 6 mg / mL), 1.4 mL of 50 mM conjugation buffer (containing 10% 10 mM DTPA solution) and 129 μL of 10 mM TCEP solution (4 equivalents) were added. The tube was placed in a thermomixer, and the reduction reaction was carried out at 25 °C for 2 hours.
[0766] Conjugation with the second payload: 15.6 mg (0.0137 mmol) of payload medicament (MB-3) was dissolved in 2740 μL of DMSO to obtain a 5 mM solution. To the above trastuzumab-MB26 (0.000309 mmol) reduction solution, 371 μL (0.00185 mmol, 6 equivalents) of 5 mM medicament in DMSO solution was added. The tube was placed in a thermomixer, and the conjugation reaction was carried out at 25 °C for 1 hour.
[0767] Purification: The above conjugation reaction solution was subjected to purification using an ultrafiltration tube (30KD) with 8 cycles using 10 mM His / His-HCl formulation buffer to obtain 4 mL (10.1 mg / mL) of trastuzumab-MB0326 (40.4 mg, yield = 75%, DAR = 3.6, DAR = 4.4 for payload MB-26) in the formulation buffer. 80 μL of 1% polysorbate 80 was added to the solution for final storage.
[0768] Physicochemical characterization of trastuzumab-MB0326 (absorption coefficient of medicament: ε280 = 4546 M -1 cm -1 and ε360 = 17513 M -1 cm -1 , absorption coefficient of MB-26: ε280 = 639.5 M -1 cm -1 and ε260 = 1006.3 M -1 cm -1 ), and further characterization data are provided in Table 4.
[0769]
Table 5
[0770] Example 11. In vitro assay of toxins and ADCs Dispense 175 μL of cell suspension into a 96-well plate at 1500 cells per well and incubate in a humidified incubator (37 °C, 5% CO2) for 24 hours. Add 25 μL of various concentrations of the compound as a 5-fold solution to the cell culture medium (fetal bovine serum, Invitrogen) in the plate and incubate in the incubator for 120 hours. Thaw CCK-8 on the bench top or in a 37 °C water bath and add 10 μL of CCK-8 to each well of the incubated plate (take care not to introduce air bubbles into the wells as they interfere with O.D. readings), then incubate in the incubator for an additional 1 - 4 hours. Measure the absorbance at 450 nm using a SpectraMax i3x microplate reader and calculate the cell inhibition rate. IC 50 curves were generated using GraphPad Prism software along with the IC 50 values.
[0771] Toxins (predicted metabolites of the ADC) and the in vitro cytotoxicity of the ADC are presented in Table 5.
[0772]
Table 6
[0773] Example 12. In vivo efficacy of ADCs in NCI-N87 CDX model To each mouse (female Balb / c-Nude from Vital Rivers), NCI-N87 tumor cells (5 × 10 6 ) mixed with Matrigel (50:50) in 0.2 mL of PBS for tumor generation were subcutaneously inoculated into the right flank. The animals were randomly grouped on the 6th day after tumor inoculation and treatment was initiated for the efficacy study when the average tumor volume reached approximately 160 mm 3 . Each group contained 8 mice. The test article and the control article were administered to the tumor-bearing mice via the tail vein at a volume of 5 mL / kg.
[0774] Tumor size was measured two-dimensional twice a week using calipers and the volume was calculated in mm using the following formula 3It was expressed as V = 0.5a × b. 2 (In the formula, a and b were the long dimension and the short dimension of the tumor, respectively.) The results were expressed by the mean and the standard error (mean ± SEM).
[0775] Statistical analysis: Two-way ANOVA was performed to compare the tumor volumes between the two groups. All data were analyzed using GraphPad Prism 6.0, and P < 0.05 was considered statistically significant. Both statistical analysis and biological observations were taken into account.
[0776] Tumor growth inhibition: The tumor size was used for the calculation of the T / C value. The T / C (%) of the relative tumor growth rate was calculated using the following formula: T / C (%) = (Ti / T0) / (Vi / V0) × 100%. The 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. When T / C > 40%, there is no efficacy. When T / C = < 40% and the p-value < 0.05, there is tumor inhibition.
[0777] The anti-tumor effects of ADCs in the NCI-N87 CDX model are shown in Figures 1 and 2 and Tables 6 and 7. As shown in Figure 1, dose-dependent anti-tumor activities (0.3, 1, and / or 3 mg / kg) were shown in all three ADCs (trastuzumab-MB24-DAR4, trastuzumab-MB3-DAR8, and trastuzumab-MB0324). Similarly, trastuzumab-MB26-DAR4, trastuzumab-MB3-DAR4, and trastuzumab-MB0326 showed dose-dependent anti-tumor activities (1 and 3 mg / kg) that induced more than 90% TGI at a dose of 3 mg / kg, as shown in Figure 2. The dual-drug ADC (trastuzumab-MB0326) showed equivalent activity to the single-drug ADCs (trastuzumab-MB03-DAR4 and trastuzumab-MB26-DAR4).
[0778]
Table 7
[0779]
Table 8
[0780] Example 13. In vivo efficacy of ADCs in JIMT-1 CDX model Each mouse (Scid-Beige from Shanghai Lingchang Biotech) was subcutaneously inoculated with JIMT-1 tumor cells (1×10 7 ) mixed with Matrigel (50:50) in 0.2 mL of PBS in the right flank for tumor development. Animals were randomly grouped on the 6th day after tumor inoculation, and treatment was initiated for efficacy studies when the average tumor volume reached approximately 175 mm 3 . Each group contained 8 mice. The test article and the control article were administered to tumor-bearing mice via the tail vein at a volume of 5 mL / kg.
[0781] Tumor size was measured two-dimensional twice a week using calipers, and the volume was expressed in mm 3 using the following formula. V = 0.5a × b2 (where a and b were the long and short dimensions of the tumor, respectively). The results were expressed as mean and standard error (mean ± SEM).
[0782] 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 < 0.05 was considered statistically significant. Both statistical analysis and biological observations were taken into account.
[0783] Tumor growth inhibition: The tumor size was used for the calculation of the T / C value. The T / C (%) of the relative tumor growth rate was calculated using the following formula: T / C (%) = (Ti / T0) / (Vi / V0) × 100%. The relative tumor growth inhibition was calculated using the formula: TGI (%) = [1 - (Ti / T0) / (Vi / V0)] × 100%. Ti refers to the average 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 average 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. When T / C > 40%, there is no efficacy. When T / C =< 40% and the p-value < 0.05, there is tumor inhibition.
[0784] The anti-tumor effects of ADCs in the JIMT-1 CDX model are shown in Figures 3 and 4 and Tables 8 and 9. As shown in Figure 3, all three ADCs (trastuzumab-MB24-DAR4, trastuzumab-MB3-DAR8, and trastuzumab-MB0324) showed dose-dependent anti-tumor activity (0.3, 1, and / or 3 mg / kg), and among them, trastuzumab-MB0324 at 3 mg / kg showed activity equivalent to that of trastuzumab-MB24-DAR4. Similarly, trastuzumab-MB26-DAR4, trastuzumab-MB3-DAR4, and trastuzumab-MB0326 showed dose-dependent anti-tumor activity (1 and 3 mg / kg) as shown in Figure 4. In this study, the dual-drug ADC (trastuzumab-MB0326) showed a slightly superior tumor growth inhibitory effect compared to the single-drug ADCs (trastuzumab-MB3-DAR4 and trastuzumab-MB26-DAR4) at either 1 or 3 mg / kg.
[0785]
Table 9
[0786]
Table 10
[0787] Equivalents One of ordinary skill 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-Q(I) or a pharmaceutically acceptable salt thereof, wherein D is represented by the following structural formula: 【Chemical 1】 wherein R １ is -H or -OH, and R ２ is C １ -C ３ alkyl, -C(=O)OH, -C(=O)OCH ３ -, -C(=O)OCH ２ CH ２ OH, -C(=O)OCH ２ CH ２ CH ２ OH, -C(=O)NHCH ２ CH ２ OH, -C(=O)NHCH ２ CH ２ CH ２ OH, or heteroaryl, and Each R ３ and R ４ are each independently -H or C １ -C ３ alkyl, n is an integer from 1 to 6, Q is -H or -CH ３ A compound, or a pharmaceutically acceptable salt thereof.
2. R １ The compound according to claim 1, wherein R is -H.
3. R １ The compound according to claim 1, wherein R is -OH.
4. R ２ is -CH ３ The compound according to any one of claims 1 to 3, wherein
5. R ２ The compound according to any one of claims 1 to 3, wherein R is -C(=O)OH.
6. R ２ is -C(=O)NHCH ２ CH ２ CH ２ OH, and the compound according to any one of claims 1 to 3.
7. R ２ is 【Chemical 2】 The compound according to any one of claims 1 to 3, wherein
8. R １ is -H, and R ２ is -C(=O)OH, the compound according to claim 1.
9. R １ is -H, and R ２ is -C(=O)NHCH ２ CH ２ CH ２ OH 、 The compound according to claim 1.
10. R １ is H, and R ２ is 【Chemical Formula 3】 The compound according to claim 1, wherein
11. R １ is - OH, and R ２ is - CH ３ as described in claim 1.
12. R ３ and R ４ are both -H, the compound according to any one of claims 1 to 11.
13. R ３ and R ４ are both -CH ３ and the compound according to any one of claims 1 to 11
14. The compound according to any one of claims 1 to 13, wherein n is 1.
15. The compound according to any one of claims 1 to 14, wherein Q is -H.
16. Q is -CH ３ The compound according to any one of claims 1 to 14, wherein Q is -CH
17. The compound according to claim 1, wherein D is represented by one of the following structures: 【Chemical Formula 4】
18. The compound according to claim 1, having one of the following structures [Chemical Formula 5] or a pharmaceutically acceptable salt thereof.
19. A compound of formula (II) D-CH ２ -NH-E-Z(II) or a pharmaceutically acceptable salt thereof, wherein D is represented by the following structural formula: 【Chemical Formula 6】 wherein R １ is -H or -OH, and R ２ is C １ -C ３ alkyl, -C(=O)OH, -C(=O)OCH ３ , -C(=O)OCH ２ CH ２ OH, -C(=O)OCH ２ CH ２ CH ２ OH, -C(=O)NHCH ２ CH ２ OH, -C(=O)NHCH ２ CH ２ CH ２ OH, or heteroaryl, and Each R ３ and R ４ are independently -H, or C １ -C ３ alkyl, n is an integer from 1 to 6, E is a peptide containing 2 to 10 amino acids, E may be optionally substituted with one or more polyols, and the N-terminus of said peptide is covalently bonded to Z, Z is -C(=O)-L-Y, [Chemical Formula 7] where m represents an integer from 1 to 10, L is -(C １ -C １０ -alkylene)- ＊ -CH ２ (OCH ２ CH ２ ) ｊ - ＊ -CH ２ CH ２ (OCH ２ CH ２ ) ｊ -(-(OCH ２ CH ２ ) ｊ -CH ２ CH ２ (OCH ２ CH ２ ) ｊ N(R ５ )C(=O)-L １ - ＊ -or -CH ２ (OCH ２ CH ２ ) ｊ N(R ５ )C(=O)-L １ - ＊ where j represents an integer from 1 to 10, ＊ indicates the site where it is covalently bonded to Y, L １ is -(C １ -C １０ -alkylene)-, and R ５ is -H or -CH ３ and Y is an electrophilic group or a nucleophilic group, the compound or a pharmaceutically acceptable salt thereof.
20. R １ The compound according to claim 19, wherein R is -H.
21. R １ The compound according to claim 19, wherein R is -OH.
22. R ２ is -CH ３ and the compound according to any one of claims 19 to 21.
23. R ２ The compound according to any one of claims 19 to 21, wherein R is -C(=O)OH.
24. R ２ is -C(=O)NHCH ２ CH ２ CH ２ OH, and the compound according to any one of claims 19 to 21.
25. R ２ is [Chemical Formula 8] The compound according to any one of claims 19 to 21, wherein
26. R １ is -H, and R ２ is -C(=O)OH, the compound according to claim 19.
27. R １ is -H, and R ２ is -C(=O)NHCH ２ CH ２ CH ２ OH 、 The compound according to claim 19.
28. R １ is - H, and R ２ is 【Chemical Formula 9】 The compound according to claim 19, wherein
29. R １ is -OH, and R ２ is -CH ３ as described in claim 19 of the compound
30. R ３ and R ４ The compound according to any one of claims 19 to 29, wherein both are -H.
31. R ３ and R ４ are both -CH ３ The compound according to any one of claims 19 to 29, wherein
32. The compound according to any one of claims 19 to 31, wherein n is 1.
33. The compound according to any one of claims 19 to 32, wherein E is a peptide of 2, 3 or 4 amino acids, 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.
34. E contains one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, and said glutamine or glutamic acid may optionally be substituted by a polyol, the compound according to any one of claims 19 to 33.
35. E is the following structure 【Chemical Formula 10】 comprising an amino acid having, wherein R ６ is -H or C １ -C ６ is an alkyl group, the compound according to any one of claims 19 to 34.
36. The compound according to claim 35, wherein E contains an amino acid having the following structure. 【Chemical Formula 11】
37. 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- ＊ selected from the group consisting of, ＊ wherein represents the N-terminus of the peptide that covalently binds to Z, the compound according to any one of claims 19 to 33.
38. E is -L-Ala-L-Val- ＊ , -L-Val-L-Ala- ＊ , -L-Val-L-Lys- ＊ , -L-Val-L-Arg- ＊ , -L-Val-L-Cit- ＊ , -L-Ala-L-Val-L-Glu- ＊ , -L-Ala-L-Ala-L-Ala- ＊ , -L-Ala-L-Val-L-Ala- ＊ , -L-Ala-L-Ala-Gly- ＊ , -L-Ala-L-Val-Gly- ＊ , -Gly-Gly-L-Glu- ＊ , -Gly-L-Phe-Gly-Gly- ＊ , -Gly-L-Glu-Gly-Gly- ＊ selected from the group consisting of, ＊ wherein is the N-terminus of the peptide that covalently binds to Z, the compound according to claim 37.
39. The compound according to any one of claims 19 to 38, wherein Z is -C(=O)-L-Y.
40. Z is 【Chemical Formula 12】 wherein m represents an integer from 1 to 10, the compound according to any one of claims 19 to 38.
41. Z is 【Chemical 13】 wherein m represents an integer from 1 to 10, the compound according to any one of claims 19 to 38.
42. L is -(C １ -C １０ -alkylene)-, and the compound according to any one of claims 19 to 39.
43. L is -CH ２ (OCH ２ CH ２ ) ｊ -, -CH ＊ CH ２ CH ２ (OCH ２ CH ２ ) ｊ -, or -(OCH ２ CH ２ ) ｊ -, wherein j represents an integer from 1 to 10, ＊ represents the site covalently bonded to Y, the compound according to any one of claims 19 to 39.
44. L is -CH ２ CH ２ (OCH ２ CH ２ ) ｊ N(R ５ )C(=O)-L １ - ＊ Or -CH ２ (OCH ２ CH ２ ) ｊ N(R ５ )C(=O)-L １ - ＊ And in the formula, j represents an integer from 1 to 10, ＊ Represents a site covalently bonded to Y. The compound according to any one of claims 19 to 39.
45. L １ is -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 NH C(=O)CH ２ CH ２ - ＊ or -CH ２ OCH ２ CH ２ OCH ２ CH ２ NH C(=O)CH ２ CH ２ - ＊ wherein ＊ represents the site covalently bonded to Y, the compound according to any one of claims 19 to 39 and 44.
46. The compound according to any one of claims 19 to 39, wherein Y is a Michael acceptor group, succinimide, epoxide, or halogen.
47. Y is 【Chemical 14】 In the formula, R ７ and R ８ are each independently H or C １ -C ３ an alkyl group, the compound according to claim 46.
48. Z is 【Chemical Formula 15】 The compound according to any one of claims 19 to 38.
49. -E-NH-CH ２ - has one of the following structures, wherein ＊ represents the N-terminus of the peptide that covalently binds to Z, the compound according to any one of claims 19 to 32. 【Chemical Formula 16】
50. Z-E-NH-CH ２ - is a compound according to any one of claims 19 to 32, having one of the following structures: 【Chemical 17】
51. The compound according to claim 19, wherein D is represented by one of the following structures. 【Chemical Formula 18】
52. The compound has one of the following structures 【Chemical Formula 19】 or a pharmaceutically acceptable salt thereof, the compound according to claim 19.
53. The compound of formula (III), {D-CH ２ -NH-E-Z'} ｐ -C(III) wherein D is represented by the following structural formula, 【Chemical 20】 wherein R １ is -H or -OH, and R ２ is C １ -C ３ alkyl, -C(=O)OH, -C(=O)OCH ３ -, -C(=O)OCH ２ CH ２ OH, -C(=O)OCH ２ CH ２ CH ２ OH, -C(=O)NHCH ２ CH ２ OH, -C(=O)NHCH ２ CH ２ CH ２ OH, or heteroaryl, and R ３ and R ４ are independently -H, or C １ -C ３ alkyl, n is an integer from 1 to 6, E is a peptide containing 2 to 10 amino acids, wherein E may optionally be substituted by one or more polyols, and the N-terminus of said peptide is covalently bonded to Z', Z' is -C(=O)-L-Y'-, 【Chemical 21】 wherein m represents an integer from 1 to 10, ＊ represents a site covalently bonded to the C, L is -(C １ -C １０ alkylene)- ＊ -CH ２ (OCH ２ CH ２ ) ｊ - ＊ -CH ２ CH ２ (OCH ２ CH ２ ) ｊ -、-(OCH ２ CH ２ ) ｊ -、-CH ２ CH ２ (OCH ２ CH ２ ) ｊ N(R ５ )C(=O)-L １ - ＊ 、or -CH ２ (OCH ２ CH ２ ) ｊ N(R ５ )C(=O)-L １ - ＊ wherein j represents an integer from 1 to 10, ＊ represents the site covalently bonded to Y', L １ is -(C １ -C １０ -alkylene)-, and R ５ is -H or -CH ３ and C represents a cell binding agent, Y' is a group formed by the reaction of an electrophilic group with a reactive nucleophilic group present on said cell binding agent, p has a value from 1 to 18, the compound.
54. R １ The compound according to claim 53, wherein R is -H.
55. R １ The compound according to claim 53, wherein R is -OH.
56. R ２ is -CH ３ The compound according to any one of claims 53 to 55, wherein
57. R ２ The compound according to any one of claims 53 to 55, wherein R is -C(=O)OH.
58. R ２ is -C(=O)NHCH ２ CH ２ CH ２ OH, the compound according to any one of claims 53 to 55.
59. R ２ is 【Chemical 22】 which is, the compound according to any one of claims 53 to 55.
60. R １ is -H, and R ２ is -C(=O)OH, the compound according to claim 53.
61. R １ is -H, and R ２ is -C(=O)NHCH ２ CH ２ CH ２ OH 、 The compound according to claim 53.
62. R １ is -H, and R ２ is 【Chemical 23】 which is, the compound according to claim 53.
63. R １ is -OH, and R ２ is -CH ３ The compound according to claim 53, wherein is
64. R ３ and R ４ wherein both are -H, the compound according to any one of claims 53 to 63.
65. R ３ and R ４ are both -CH ３ The compound according to any one of claims 53 to 63, wherein is.
66. The compound according to any one of claims 53 to 65, wherein n is 1.
67. The compound according to any one of claims 53 to 66, wherein E is a peptide of 2, 3, or 4 amino acids, and 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.
68. The compound according to any one of claims 53 to 67, wherein E contains one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, and the glutamine or glutamic acid may be optionally substituted by a polyol.
69. E contains an amino acid having the following structure 【Chemical 24】 In the formula, R ６ is -H or C １ -C ６ an alkyl group, and the compound according to any one of claims 53 to 67.
70. The compound according to claim 69, wherein E contains an amino acid having the following structure. 【Chemical Formula 25】
71. 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- ＊ selected from the group consisting of, wherein ＊ represents the N-terminus of the peptide that covalently binds to Z', the compound according to any one of claims 53 to 67.
72. E is -L-Ala-L-Val- ＊ , -L-Val-L-Ala- ＊ , -L-Val-L-Lys- ＊ , -L-Val-L-Arg- ＊ , -L-Val-L-Cit- ＊ , -L-Ala-L-Val-L-Glu- ＊ , -L-Ala-L-Ala-L-Ala- ＊ , -L-Ala-L-Val-L-Ala- ＊ , -L-Ala-L-Ala-Gly- ＊ , -L-Ala-L-Val-Gly- ＊ , -Gly-Gly-L-Glu- ＊ , -Gly-L-Phe-Gly-Gly- ＊ , -Gly-L-Glu-Gly-Gly- ＊ is selected from the group consisting of, wherein ＊ represents the N-terminus of the peptide that covalently binds to Z', the compound according to claim 71.
73. The compound according to any one of claims 53 to 72, wherein Z' is -C(=O)-L-Y'.
74. Z' is 【Chemical 26】 wherein m represents an integer of 1 to 10, ＊ is a site covalently bonded to the C, and the compound according to any one of claims 53 to 72.
75. Z' is 【Chemical 27】 wherein m represents an integer of 1 to 10, ＊ which represents a site covalently bonded to said C, a compound according to any one of claims 53 to 72.
76. L is -(C １ -C １０ -alkylene)-, and the compound according to any one of claims 53 to 73.
77. L is -CH ２ (OCH ２ CH ２ ) ｊ -, -CH ＊ CH ２ CH ２ (OCH ２ CH ２ ) ｊ -, or -(OCH ２ CH ２ ) ｊ -, where j represents an integer from 1 to 10, and in the formula, ＊ represents the site covalently bonded to Y', the compound according to any one of claims 53 to 73.
78. L is -CH ２ CH ２ (OCH ２ CH ２ ) ｊ N(R ５ )C(=O)-L １ - ＊ or -CH ２ (OCH ２ CH ２ ) ｊ N(R ５ )C(=O)-L １ - ＊ wherein j represents an integer from 1 to 10, and wherein ＊ represents a site covalently bonded to Y', a compound according to any one of claims 53 to 73.
79. L １ is -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)CH ２ CH ２ - ＊ or -CH ２ OCH ２ CH ２ OCH ２ CH ２ NHC(=O)CH ２ CH ２ - ＊ wherein ＊ represents a site that covalently binds to Y', the compound according to any one of claims 53 to 73 and 78.
80. The compound according to any one of claims 53 to 79, wherein Y' is a group formed by the reaction of an electrophilic group with a reactive nucleophilic group present on the cell-binding agent.
81. Y' is 【Chemical 28】 formed from In the formula, R ７ and R ８ are each independently H or C １ -C ３ an alkyl group, the compound according to claim 80.
82. Y' is 【Chemical 29】 and wherein R ７ and R ８ are each independently -H or C １ -C ３ is an alkyl group, ＊ represents a site covalently bonded to the C, and the compound according to claim 80.
83. Z' is 【Chemical Formula 30】 formed from, the compound according to any one of claims 53 to 72.
84. Z' is 【Chemical 31】 wherein, ＊ represents a site covalently bonded to C, and the compound according to any one of claims 53 to 72.
85. -E-NH-CH ２ - has one of the following structures, and in the formula, ＊ represents the N-terminus of the peptide that covalently binds to Z', the compound according to any one of claims 53 to 66. 【Chemical 32】
86. -Z'-E-NH-CH ２ - is formed from one of the following structures, the compound according to any one of claims 53 to 66. 【Chemical 33】
87. -Z'-ENH-CH ２ - is one of the following structures, wherein ＊ represents a bonding point to C, and the compound according to any one of claims 53 to 66. 【Chemical Formula 34】
88. The compound according to claim 53, wherein D is represented by one of the following structures. 【Chemical 35】
89. D-CH ２ The compound according to claim 53, wherein -NH-E-Z'- is formed from one of the following structures. 【Chemical 36】
90. {D-CH ２ -NH-E-Z'} ｐ -C is one of the following structures, wherein C is a monoclonal antibody, p is the drug-to-antibody ratio (DAR), and the average value of p is about 2-8, 4-8, or 7-8. The compound according to claim 53. 【Chemical 37】
91. The compound according to any one of claims 53 to 90, wherein the average value of p is in the range of about 3 to 8, or 4 to 8.
92. The compound according to any one of claims 53 to 90, wherein p is 8, or the average value is about 4, about 7.5, or about 8.
93. A compound of formula (IV) {D-CH ２ -NH-E-Z'} ｐ’ -C-{W} ｔ (IV) wherein in the formula D is represented by the following structural formula 【Chemical Formula 38】 in the formula R １ is -H or -OH, and R ２ is C １ -C ３ alkyl, -C(=O)OH, -C(=O)OCH ３ -, -C(=O)OCH ２ CH ２ OH, -C(=O)OCH ２ CH ２ CH ２ OH, -C(=O)NHCH ２ CH ２ OH, -C(=O)NHCH ２ CH ２ CH ２ OH, or heteroaryl, and R ３ and R ４ are independently -H, or C １ -C ３ alkyl, n is an integer from 1 to 6, E is a peptide containing 2 to 10 amino acids, in the formula, E may be optionally substituted by one or more polyols, and the N-terminus of the peptide is covalently bonded to Z', Z' is -C(=O)-L-Y', 【Chemical Formula 39】 wherein m represents an integer of 1 to 10, ＊ represents a site covalently bonded to said C, L is -(C １ -C １０ alkylene)- ＊ -CH ２ (OCH ２ CH ２ ) ｊ - ＊ -CH ２ CH ２ (OCH ２ CH ２ ) ｊ -,-(OCH ２ CH ２ ) ｊ -,-CH ２ CH ２ (OCH ２ CH ２ ) ｊ N(R ５ )C(=O)-L １ - ＊ -or -CH ２ (OCH ２ CH ２ ) ｊ N(R ５ )C(=O)-L １ - ＊ wherein j represents an integer from 1 to 10, ＊ indicates the site covalently bonded to Y', L １ is -(C １ -C １０ -alkylene)-, R ５ is -H or -CH ３ and C represents a cell-binding agent, Y' is a group formed by the reaction of an electrophilic group with a reactive nucleophilic group present on the cell-binding agent, W is a group formed by the reaction of a compound W' with a reactive nucleophilic group present on C, and W' is a cell-killing agent linked to a linker such that W' can be linked to C. A compound wherein p' and t have values of 1 to 10, p' and t may be the same or different numbers, and p':t is about 1:1, about 1:2, or about 2:1, and p':t is 1:1, or 1:2, or 2:
1. Claim 94 R １ The compound according to claim 93, wherein R is -H. Claim 95 R １ The compound according to claim 93, wherein R is -OH. Claim 96 R ２ is -CH ３ The compound according to any one of claims 93 to 95, wherein Claim 97 R ２ The compound according to any one of claims 93 to 95, wherein R is -C(=O)OH. Claim 98 R ２ is -C(=O)NHCH ２ CH ２ CH ２ OH, and the compound according to any one of claims 93 to 95. Claim 99 R ２ is 【Chemical Formula 40】 The compound according to any one of claims 93 to 95. Claim 100 R １ is -H, and R ２ is -C(=O)OH, the compound according to claim 93. Claim 101 R １ is -H, and R ２ is -C(=O)NHCH ２ CH ２ CH ２ OH 、 The compound according to claim 93. Claim 102 R １ is - H, and R ２ is 【Chemical 41】 The compound according to claim 93. Claim 103 R １ is -OH, and R ２ is -CH ３ as defined in claim 93. Claim 104 R ３ and R ４ are both -H, the compound according to any one of claims 93 to 103. Claim 105 R ３ and R ４ are both -CH ３ A compound according to any one of claims 93 to 103, wherein is. Claim 106 The compound according to any one of claims, 93 to 105, wherein n is 1. Claim 107 The compound according to any one of claims 93 to 106, wherein E is a peptide of 2, 3, or 4 amino acids, and 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. Claim 108 The compound according to any one of claims 93 to 107, wherein E contains one or more amino acids selected from glycine, alanine, valine, glutamine, glutamic acid, phenylalanine, and leucine, and the glutamine or glutamic acid may optionally be substituted by a polyol. Claim 109 E contains an amino acid having the following structure 【Chemical Formula 42】 wherein R ６ is -H or C １ -C ６ an alkyl group, a compound according to any one of claims [93] to [107]. Claim 110 The compound according to claim 109, wherein E contains an amino acid having the following structure. 【Chemical 43】 Claim 111 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- ＊ selected from the group consisting of, wherein ＊ represents the N-terminus of the peptide that covalently binds to Z', the compound according to any one of claims 93 to 107. Claim 112 E is -L-Ala-L-Val- ＊ , -L-Val-L-Ala- ＊ , -L-Val-L-Lys- ＊ , -L-Val-L-Arg- ＊ , -L-Val-L-Cit- ＊ , -L-Ala-L-Val-L-Glu- ＊ , -L-Ala-L-Ala-L-Ala- ＊ , -L-Ala-L-Val-L-Ala- ＊ , -L-Ala-L-Ala-Gly- ＊ , -L-Ala-L-Val-Gly- ＊ , -Gly-Gly-L-Glu- ＊ , -Gly-L-Phe-Gly-Gly- ＊ , -Gly-L-Glu-Gly-Gly- ＊ selected from the group consisting of, wherein ＊ represents the N-terminus of the peptide that covalently binds to Z', the compound according to claim 111. Claim 113 The compound according to any one of claims 93 to 112, wherein Z' is -C(=O)-L-Y'-. Claim 114 Z' is 【Chemical 44】 wherein m represents an integer of 1 to 10, ＊ which represents a site covalently bonded to said C, the compound according to any one of claims 93 to 112. Claim 115 Z' is 【Chemical 45】 wherein m represents an integer of 1 to 10, ＊ is a site covalently bonded to the C, and the compound according to any one of claims 93 to 112. Claim 116 L is -(C １ -C １０ -alkylene)-, the compound according to any one of claims 93 to 113. Claim 117 L is -CH ２ (OCH ２ CH ２ ) ｊ -, -CH ＊ CH ２ CH ２ (OCH ２ CH ２ ) ｊ -, or -(OCH ２ CH ２ ) ｊ -, wherein j represents an integer from 1 to 10, ＊ represents the site that covalently binds to Y', the compound according to any one of claims 93 to 113. Claim 118 L is -CH ２ CH ２ (OCH ２ CH ２ ) ｊ N(R ５ )C(=O)-L １ - ＊ 、 or -CH ２ (OCH ２ CH ２ ) ｊ N(R ５ )C(=O)-L １ - ＊ wherein j represents an integer from 1 to 10, ＊ represents a site covalently bonded to Y', the compound according to any one of claims 93 to 113. Claim 119 L １ is -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 NH C(=O)CH ２ CH ２ - ＊ or -CH ２ OCH ２ CH ２ OCH ２ CH ２ NH C(=O)CH ２ CH ２ - ＊ wherein ＊ represents a site covalently bonded to Y', a compound according to any one of claims 93 to 113 and 118. Claim 12o The compound according to any one of claims 93 to 119, wherein Y' is a group formed by the reaction of an electrophilic group with a reactive nucleophilic group present on the cell-binding agent. Claim 121 Y' is 【Chemical 46】 formed from, wherein R ７ and R ８ are each independently H or C １ -C ３ alkyl group, the compound according to claim 120. Claim 122 Y' is 【Chemical 47】 and in the formula, R ７ and R ８ are each independently -H or C １ -C ３ is an alkyl group, ＊ wherein represents the site covalently bonded to the C, the compound according to claim 120. Claim 123 Z' is 【Chemical Formula 48】 The compound according to any one of claims 93 to 112, formed from Claim 124 Z' is 【Chemical 49】 wherein, ＊ represents a site covalently bonded to C, and the compound according to any one of claims 93 to 112. Claim 125 -E-NH-CH ２ - has one of the following structures, and in the formula, ＊ represents the N-terminus of the peptide that covalently binds to Z', the compound according to any one of claims 93 to 106. 【Chemical Formula 50】 Claim 126 -Z'-E-NH-CH ２ - wherein is formed from one of the following structures, the compound according to any one of claims 93 to 106. 【Chemical 51】 Claim 127 -Z'-E-NH-CH ２ - is one of the following structures, wherein ＊ represents a bonding point to C, and the compound according to any one of claims 93 to 106. 【Chemical 52】 Claim 128 The compound according to claim 93, wherein D is represented by one of the following structures. 【Chemical Formula 53】
129. D-CH ２ The compound according to claim 93, wherein -NH-E-Z'- is formed from one of the following structures. 【Chemical 54】
130. The compound according to any one of claims 93 to 129, wherein W is formed by covalently bonding compound W' to C.
131. The compound according to any one of claims 93 to 129, wherein W' is any molecule capable of covalently bonding to C.
132. D-CH ２ The compound according to claim 93, wherein -NH-E-Z'- is formed from PL1 and W is formed from PL2.
133. D-CH ２ The compound according to claim 93, wherein -NH-E-Z'- is formed from PL3 and W is formed from PL4.
134. D-CH ２ The compound according to claim 93, wherein -NH-E-Z'- is formed from PL5 and W is formed from PL6.
135. D-CH ２ The compound according to claim 93, wherein -NH-E-Z'- is formed from PL7 and W is formed from PL8.
136. D-CH ２ The compound according to claim 93, wherein -NH-E-Z'- is formed from PL9 and W is formed from PL10.
137. {D-CH ２ -NH-E-Z'} ｐ’ -C-{W} ｔ is one of the following structures, wherein C is a monoclonal antibody, p' and t are the drug-to-antibody ratio (DAR), p':t is 1:1 or about 1:1, and p' and t are each the average number in the range of about 1 to 7, or the average number of about 2, about 3, about 4, about 5, or about 6, the compound according to claim 93. 【Chemical Formula 55】
138. The compound according to any one of claims 93 to 137, wherein the average of both p' and t is 4.
139. The compound according to any one of claims 93 to 137, wherein p':t is about 1:1, about 1:2, or about 2:
1.
140. The compound according to any one of claims 93 to 137, wherein p':t is 1:1, or 1:2, or 2:
1.
141. The compound according to any one of claims 53 to 140, wherein the cell binding agent is an antibody or an antigen-binding fragment thereof.
142. The compound according to claim 141, wherein the cell binding agent is a monoclonal antibody or an antigen-binding fragment thereof.
143. The compound according to claim 93, wherein the compound is trastuzumab-MB0324. 【Chemical Formula 56】
144. The compound according to claim 93, wherein the compound is trastuzumab-MB0326. 【Chemical 57】
145. A pharmaceutical composition comprising the compound according to any one of claims 53 to 144.
146. A method for treating a cell proliferative disease or disorder or inhibiting abnormal cell proliferation, the method comprising administering to a subject in need of treatment the compound according to any one of claims 53 to 144 or the pharmaceutical composition according to claim 145.
147. The method according to claim 146, wherein the method is for treating cancer.
148. The method according to claim 147, wherein the cancer is adenocarcinoma, brain cancer, bladder cancer, breast cancer, cervical cancer, choriocarcinoma, CNS tumor, colon cancer or colorectal cancer, diffuse intrinsic pontine glioma (DIPG), endometrial cancer, esophageal cancer, Ewing sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, glioblastoma, head and neck cancer, blood cancer, Hodgkin lymphoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, myelodysplastic syndrome (MDS), neuroblastoma, non-Hodgkin lymphoma, osteosarcoma, pancreatic cancer, peritoneal cancer, prostate cancer, ovarian cancer, renal cell carcinoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, or Wilms tumor.
149. The method according to claim 148, wherein the cancer is breast cancer.