Self-immolative linkers and conjugates thereof
Alternative self-immolative linkers with ester or thioester bonds in ADCs address the issues of lipophilicity and aggregation in conventional ADCs, enhancing drug delivery efficiency and solubility.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- ENLAZA THERAPEUTICS INC
- Filing Date
- 2025-11-12
- Publication Date
- 2026-05-21
AI Technical Summary
Conventional antibody-drug conjugates (ADCs) face issues with lipophilicity and poor drug-antibody ratio due to the use of para-amino benzylcarbamate (PABC) linkers, leading to ADC aggregation and inefficient payload release, particularly for lipophilic drugs like auristatins and camptothecins.
Introduction of alternative self-immolative groups, such as ester or thioester linkages, with lower lipophilicity, allowing for more efficient payload release through cleavable peptides that are recognized by enzymes like cathepsin B, enabling higher drug-antibody ratios and reduced aggregation.
The new linkers improve payload release kinetics, enhance aqueous solubility, and increase drug-antibody ratios, addressing the limitations of PABC linkers by providing tunable and efficient drug delivery.
Smart Images
Figure US2025055190_21052026_PF_FP_ABST
Abstract
Description
WSGR Docket No. 60801-718.601SELF-IMMOLATIVE LINKERS AND CONJUGATES THEREOFCROSS REFERENCE TO OTHER APPLICATIONS
[0001] This international patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 719,829, filed on November 13, 2024, which is incorporated herein by reference in its entirety.BACKGROUND
[0002] Conjugates formed by linking a payload (e.g., a drug) to a targeting group (e.g., a binder) via a linker have seen an explosion in popularity in the 21stcentury, with several antibody-drug conjugates (ADCs) having been approved for medical use since the year 2000. Identifying new ways to conjugate payloads to targeting groups with cleavable linker groups has been an area of active research.SUMMARY
[0003] A general format for conjugates such as ADCs is Ab-[L-D]zwherein Ab is an antibody, L is a linker, D is a drug, and z reflects the drug-antibody ratio (DAR). When L comprises a peptide that is a substrate to an enzyme, particularly an enzyme that is overexpressed in cancer cells or in a tumor microenvironment, the drug can be released, thereby producing its (typically cytotoxic) therapeutic effects at the site of release.
[0004] The present disclosure relates to cleavable peptides and cleavable peptide-based linkers. Chemical spacer groups capable of releasing a payload have been explored and utilized in the field of antibody-drug conjugates, with a para-amino benzylcarbamate (“PABC”) group having been utilized for releasing a N-linked monomethyl auristatin E (“MMAE”) payload in the drug brentuximab vedotin (sold under brand name Adcetris®). The name “vedotin” refers to the following MC-Val-Cit-PABC-MMAE linker-payload structure:MC Val-Cit PABC MMAE
[0005] Vedotin and analogues thereof leverage the ability of cathepsin B to cleave between citrulline and a PABC group, which quickly decomposes following cleavage of the adjacentWSGR Docket No. 60801-718.601amide bond, thereby releasing the drug to which it is attached, as illustrated in the following scheme:
[0006] However, one drawback to the PABC self-immolative group is the lipophilicity it imparts on the linker-payload, which may result in ADC aggregation and / or poor coupling to an antibody (low drug-antibody ratio, or DAR). This feature is particularly problematic for lipophilic payloads including, but not limited to, auristatins and camptothecins. Moreover, the PABC group is typically bonded to a nitrogen atom of the drug in a carbamate bond, meaning the payload should have a functionalizable primary or secondary amine. Carbonate linkages to PABA are not commonly utilized in conventional ADCs, potentially due to the lability of the carbonate bond. Provided herein are alternative and improved self-immolative groups with lower lipophilicity than PABC, and advantageously, the adaptability to handle payloads conjugated through an ester or a thioester linkage.
[0007] One aspect of the disclosure described herein is a compound of Formula (I):Formula (I)or a salt or stereoisomer thereof, or a mixture of stereoisomers thereof, wherein: P1is a payload (e.g., bonded via an ester bond or a thioester bond),R1is H, Ci-Ce alkyl, wherein Ci-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, -OH, -NH2, -SH, and - SCH3,R2is Ci-C6alkyl, -[CH2]i-3[OCH2CH2]i-i2OH, -[CH2]I-3[OCH2CH2]I-I2OCH3, - [CH2CON(CH3)]I-I2CH3, or -[CH2CON(CH3)]I-I2CH2COOH;R3is H, Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)-NHC(=O)NH2, -(Ci-C6alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)-NHC(=NH)NH2, -(Ci-C6alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O-NHC(=NH)NH2, or -(Ci-C6alkylene)-O-NHC(=NH)NHCH3, wherein Ci-Ce alkyl is optionally substituted with one, two, or three groupsWSGR Docket No. 60801-718.601selected from the group consisting of -F, phenyl, -C(=O)NH2, -C(=O)OH, - OH, -NH2, -SH, and -SCH3,or R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, wherein when R1and R2form a pyrrolidine, then R3is Ci- Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)- NHC(=O)NH2, -(C1-C6 alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)- NHC(=NH)NH2, -(CI-C6alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O- NHC(=NH)NH2, or -(Ci-C6alkylene)-O-NHC(=NH)NHCH3, wherein Ci-C6alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NH2, -SH, and -SCH3,L1is -L3-[AA]a- wherein:L3is a spacer,each AAis independently an amino acid,a is 2, 3, 4, or 5,R4is H, C1-C12 alkyl, PEG1-20 (e g., -[CH2]i-3[OCH2CH2]i-i2OH or-[CH2]i-L2is a bond or a spacer, andR5is a moiety capable of reacting with a thiol or an amine side-chain of a peptide or polypeptide (e.g., a cysteine or a lysine side-chain of an antibody or antigenbinding fragment).
[0008] Another embodiment of the disclosure described herein is a compound of Formula (II):Formula (II)or a salt or stereoisomer thereof, or a mixture of stereoisomers thereof, wherein: P1, R1, R2, R3, R4, L1, L2, are as defined in Formula (I),n is an integer from 1 to 20,WSGR Docket No. 60801-718.601P2is a targeting moiety, andL5is a bond, Ci-6 alkylene, -CO-, -NHCO-CI-6 alkylene, -NHCO-CI-6 alkenylene, or 5-6 membered heterocycloalkylene, wherein the Ci-6 alkylene, Ci-6 alkenylene, and heterocycloalkylene are substituted with one, two, three, or four groups selected from oxo (=0), -C00H, and CH2COOH.
[0009] In some embodiments, the compound is of Formula (la) or (Ila):Formula (Ila)or a salt or stereoisomer thereof, or a mixture of stereoisomers thereof, wherein:P1is a payload bonded via an ester bond or a thioester bond,R1is H, Ci-Ce alkyl, wherein C1-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, -OH, -NH2, -COOH, - CONH2, -NHC(=0)NH2, -NHC(=NH)NH2, -SH, and -SCH3, or two R1groups together form a C3-6 cycloalkyl;R2is Ci-C6alkyl, -[CH2]i-3[OCH2CH2]i-i2OH, -[CH2]i-3[OCH2CH2]i-i2OCH3, - [CH2CON(CH3)]i-i2CH3, or -[CH2CON(CH3)]i-i2CH2COOH, R3is H, Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)-NHC(=O)NH2, -(Ci-C6alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)-NHC(=NH)NH2, -(Ci-C6alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O-NHC(=NH)NH2, or -(Ci-C6alkylene)-O-NHC(=NH)NHCH3, wherein Ci-Ce alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, -C(=O)NH2, -C(=O)OH, - OH, -NH2, -SH, and -SCH3,or R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, wherein when R1and R2form a pyrrolidine, then R3is Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)-WSGR Docket No. 60801-718.601NHC(=O)NH2, -(C1-C6 alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)- NHC(=NH)NH2, -(CI-C6alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O- NHC(=NH)NH2, or -(Ci-C6alkylene)-O-NHC(=NH)NHCH3, wherein Ci-C6alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NH2, -SH, and -SCH3,L1is -L3-[AA]a-,wherein:L3is a first spacer,each AAis independently an amino acid,a is 2, 3, 4, or 5,R4is H, Ci-Ci2alkyl, PEGI-2O (i.e., -[CH2]I-3[OCH2CH2]I-I2OH or -[CH2]i-L2is a bond or a second spacer,R5is a moiety (e.g., an electrophile) capable of reacting with a thiol (-SH) or an amine (-NH2) side-chain of a peptide or polypeptide (e.g., a cysteine or a lysine side-chain of an antibody or antigen-binding fragment) P2is a targeting moiety, andn is an integer from 1 to 20.
[0010] In some embodiments of Formula (II) or (Ila), P2is a peptide or polypeptide that binds to a target. In some embodiments of Formula (II) or (Ila), P2is an antibody or antigen-binding peptide that binds to a target. In some embodiments of Formula (II) or (Ila), P2is an antigenbinding moiety that binds to a target.
[0011] In some embodiments of Formula (I) (la), (II), or (Ila), R1is H or Ci-Ce alkyl; R2is Ci-Ce alkyl; and R3is H, Ci-Ce alkyl, (C1-C4 alkylene)-OH, -(C2-Ce alkylene)-NH2, -(C2-Ce alkylene)-NHC(=O)NH2, -(C2-C6alkylene)-NHC(=O)NHCH3, -(C2-C6alkylene)-NHC(=NH)NH2, -(C2-C6 alkylene)-NHC(=NH)NHCH3, -(C2-C6alkylene)-O-NHC(=NH)NH2, or -(C2-C6 alkylene)-O-NHC(=NH)NHCH3. In some embodiments of Formula (I) or Formula (II), R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine; and R3is Ci-Ce alkyl, (C1-C4 alkylene)-OH, -(C2-Ce alkylene)-NH2, -(C2-C6alkylene)-NHC(=O)NH2, -(C2-C6alkylene)-NHC(=O)NHCH3, -(C2-C6alkylene)-NHC(=NH)NH2, -(C2-C6alkylene)-NHC(=NH)NHCH3, -(C2-C6alkylene)-O-WSGR Docket No. 60801-718.601NHC(=NH)NH2, or -(C2-C6 alkylene)-0-NHC(=NH)NHCH3. In some embodiments, of Formula (I) or Formula (II), R1is H, -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH(CH3)2, -C(CH3)2-, -CH2CH(CH3)CH2CH3, or -CH2CH(CH3)2, R2is -CH3, and R3is H, -CH3, -CH2OH, -CH2CH2OH, or -CH(CH3)0H. In some embodiments, of Formula (I) or Formula (II), R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine; and R3is -CH2OH, -CH2CH2OH, or -CH(CH3)0H.
[0012] In some embodiments, the compound of Formula (I) or Formula (II) is further characterized according to Formula (lb) or Formula (lib), respectively:Formula (lb) Formula (lib).
[0013] In some embodiments, the compound of Formula (I) or Formula (II) is further characterized according to Formula (Ic) or Formula (lie), respectively:F ormul a (Ic) F ormul a (lie) .
[0014] Each [AA] in Formula (I) or (II) may be independently selected from the group consisting of glycine (Gly), alanine (Ala), valine (Vai), leucine (Leu), isoleucine (He), proline (Pro), citrulline (Cit), lysine (Lys), arginine (Arg), glutamate (Glu), aspartate (Asp), asparagine (Asn), glutamine (Gin), phenylalanine (Phe), histidine (His), tryptophan (Trp), tyrosine (Tyr), methionine (Met), serine (Ser), and threonine (Thr). Preferably [AA]arepresents an enzymatically cleavable peptide comprising 2, 3, 4, or 5 amino acids, bonded via an amide bond to the self-immolative dipeptide group of Formula (I) or (II). For example, [AA]amay be a Cathepsin B substrate, a legumain substrate, or a TACE substrate. In some embodiments, a is 2 or 3, and the C-terminal [AA] is Cit, Asn, or Arg. In some embodiments, [AA]ais Val-WSGR Docket No. 60801-718.601Arg*, Gly-Arg*, Ile-Arg*, Ala-Arg*, Glu-Arg*, Arg-Arg*, Val-Cit*, Gly-Cit, Ile-Cit*, Ala-Cit*, Glu-Cit*, Glu-Val-Arg*, Gly-Val-Arg*, Gly-Gly-Arg*, Gly-Glu-Val-Arg*, Glu- Val-Cit*, Glu-Gly-Cit, Gly-Val-Cit*, Gly-Gly-Cit*, Gly-Glu-Val-Cit*, or Glu-Arg-Arg*; wherein in each instance, * indicates the C-terminal amino acid residue. In some embodiments of Formula (I) or (II), the term [AA]ais the peptide: Gly-Asn*, Ala-Asn*, Gly-Gly-Asn*, Ala-Ala-Asn*, or Ala-(D) Ala-Asn*; wherein in each instance, * indicates the C-terminal amino acid residue. In some embodiments, [AA]ais Leu-Asp-Leu* or Ala-Gin-Ala*; wherein in each instance, * indicates the C-terminal amino acid residue. In some embodiments, [AA]ais Val-Ala*, Gly-Ala*, Glu-Ala*, Ala-Ala*, Phe-Ala*, Leu-Ala*, His-Ala*, Trp-Ala*, Pro- Ala*, Val-Arg*, Gly-Arg*, Ile-Arg*, Ala-Arg*, Glu-Arg*, Tyr-Arg*, Arg-Arg*, Gly-Asn*, Ala-Asn*, Asn-Asn*, Arg-Asn*, Gly-Asp*, Glu-Asp*, His-Asp*, Asp-Asp*, Pro-Asp*, Val-Cit*, Gly-Cit*, Ile-Cit*, Ala-Cit*, Glu-Cit*, Glu-Glu*, Ala-Glu*, Tyr-Glu*, Asp-Glu*, Val-Glu*, Val-Gly*, Gly-Gly*, Glu-Gly*, Ile-Gly*, Ala-Gly*, Phe-Gly*, Leu-Gly*, Asp-Gly*, Cys-Gly*, Val-His*, Gly-His*, Ala-His*, Val-Ile*, Gly-Ile*, Ile-Ile*, Leu-Ile*, Gly-Leu*, Phe-Leu*, Lys-Leu*, Leu-Leu*, Asp-Leu*, Phe-Lys*, Leu-Lys*, His-Lys*, Vai-Met*, Val-Phe*, Gly-Phe*, Ala-Phe*, Phe-Phe*, Leu-Phe*, His-Phe*, Asp-Phe*, Val-Pro*, Gly-Pro*, Ala-Pro*, Phe-Pro*, Leu-Pro*, Gly-Ser*, Glu-Ser*, Ala-Ser*, Phe-Ser*, Tyr-Ser*, Trp-Ser*, Ser-Ser*, Asp-Ser*, Asn-Ser*, Val-Thr*, Gly-Thr*, Lys-Thr*, Tyr-Thr*, Trp-Thr*, Thr-Thr*, Ser-Thr*, Asp-Thr*, Gln-Thr*, Val-Trp*, Ile-Trp*, Phe-Trp*, Trp-Trp*, Val-Tyr*, Leu-Tyr*, His-Tyr*, Glu-Val*, Val-Val*, Gly-Val*, Ala-Vai*, Glu-Val-Arg*, Gly-Val-Arg*, Gly-Gly-Arg*, Gly-Glu-Val-Arg*, Glu- Val-Cit*, Gly-Val-Cit*, Gly-Gly-Cit*, Gly-Glu- Val-Cit*, Glu-Arg-Arg*, Gly-Gly-Asn*, Ala-Ala-Asn*, or Ala-(D)Ala-Asn*, Leu-Asp-Leu*, or Ala-Gin- Ala*; wherein in each instance, * indicates the C-terminal amino acid residue.
[0015] In some embodiments, provided herein is a compound of Formula (I), (la), (lb), (Ic), (II), (Ila), (lib), or (lie), wherein L3is -(L3a)i-2o-, wherein each L3ais independently -C1-2 alkylene-O-, -O-C1-2 alkylene-, -C(O)NH-, -NHC(O)-, - C1-2 alkylene-C(O)NH-, - C1-2 alkylene-NHC(O)-, -O-, -S-, phenylene, -NH-, -NHC(O)O-, -OC(O)NH-, or -(CR'R , wherein each R1and R2is independently hydrogen, C1-6 alkyl, -C1-5 alkylene-OH, -C1-5 alkylene-NH2, -C1-5 alkylene-CONH2, -C1-5 alkylene-COOH, or -OH, -NH2, -CONH2, -COOH, and v is 1-20.
[0016] In some embodiments, L3is -(L3a)i-io-, each L3ais independently -C1-2 alkylene-O-, -O-C1-2 alkylene-, -C(O)NH-, -NHC(O)-, - C1-2 alkylene-C(O)NH-, - C1-2 alkylene-NHC(O)-, -O-, -S-, phenylene, -NH-, -NHC(O)O-, -OC(O)NH-, or -(CRJR2)v-, wherein each R1and R2isWSGR Docket No. 60801-718.601independently hydrogen or -CH2NH2 and v is 1-6. In some embodiments, L3is -(L3a)i-4-, -(L3a)i-8-, -(L3a)i-io-, -(L3a)i-12-, -(L3a)2-20-, or -(L3a)2-8-.
[0017] In some embodiments of Formula (I) or Formula (II), L3is:wherein * indicates a bond to -[AA]a-;each u is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; andeach Z is independently selected from O, and NRZ; and each Rzis independently hydrogen or Ci-C4alkyl.
[0018] In some embodiments, provided herein is a compound of Formula (I), (la), (lb), (Ic), (II), (Ila), (lib), or (lie), wherein L2is independently -C1-2 alkylene-O-, -O-C1-2 alkylene-, -C(O)NH-, -NHC(O)-, - C1-2 alkyl ene-C(O)NH-, - C1-2 alkylene-NHC(O)-, -O-, -S-, phenylene, -NH-, -NHC(O)O-, -OC(O)NH-, or -(CRJR2)v-, wherein each R1and R2is independently hydrogen, C1-6 alkyl, -C1-5 alkylene-OH, -C1-5 alkylene-NFb, -C1-5 alkylene-CONFb, -C1-5 alkylene-COOH, or -OH, -NH2, -CONH2, -COOH, and v is 1-20.
[0019] In some embodiments, L2is -(L2a)i-io-, each L2ais independently -C1-2 alkylene-O-, -O-C1-2 alkylene-, -C(O)NH-, -NHC(O)-, - C1-2 alkylene-C(O)NH-, - C1-2 alkylene-NHC(O)-, -O-, -S-, phenylene, -NH-, -NHC(O)O-, -OC(O)NH-, or -(CRJR2)v-, wherein each R1and R2is independently hydrogen or -CH2NH2 and v is 1-6. In some embodiments, L2is -(L2a)i-4-, -(L2a)i-8-, -(L2a)i-io-, - (L2a)i-12-, - (L2a)2-20-, or -(L2a)2-8-.
[0020] In some embodiments of Formula (I) or Formula (II), L2is:WSGR Docket No. 60801-718.601wherein* indicates a bond to NR4;each m is independently 0, 1, 2, 3, 4, or 5;each u is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; andeach Z is independently O, NH, or N(CH3).
[0021] In some embodiments, the compound (of Formula (I)) has the structure of FormulaFormula (F)wherein P1, R1, R2, R3, R4, R5, L2, and L3are as defined in Formula (I), or any one of the preceding embodiments,a is 0, 1, or 2; andeach of R6, R7, and R8is independently hydrogen or Ci-6 alkyl, wherein each Ci-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NH2, -NHC(=O)NH, -SH, -SCH3, -NHC(=O)NHCH3, -NHC(=NH)NH2, -NHC(=NH)NHCH3, -O- NHC(=NH)NH2, and -O-NHC(=NH)NHCH3.
[0022] In some embodiments, the compound (of Formula (II)) has the structure of Formula (IF):Formula (IF)wherein P1, P2, R2, R3, R4, L2, L3, and L5are as defined in Formula (II),a is 0, 1, or 2; andeach of R1, R6, R7, and R8is independently hydrogen or C1-6 alkyl, wherein each Ci- 6 alkyl is optionally substituted with one, two, or three groups selected from theWSGR Docket No. 60801-718.601group consisting of phenyl, -C(=0)NH2, -C(=O)OH, -OH, -NH2, -NHC(=O)NH, -SH, -SCH3, -NHC(=O)NHCH3, -NHC(=NH)NH2, -NHC(=NH)NHCH3, -O- NHC(=NH)NH2, and -O-NHC(=NH)NHCH3; or two R1together form C3-6 cycloalkyl.
[0023] In some embodiments, each R3is H or -CH2OH. In some embodiments, each R3is -CH2OH. In some embodiments, each R3is H. In some embodiments, the peptide or polypeptide (P2) comprises an antibody fragment, or an antigen binding domain. In some embodiments, the peptide or polypeptide (P2) comprises a nanobody, an antigen binding domain. In some embodiments, the peptide or polypeptide (P2) comprises a single domain antibody, a Fab, or an ScFv. In some embodiments, the peptide or polypeptide (P2) is capable of binding covalently to the target. In some embodiments, the peptide or polypeptide (P2) binds to one or more tumor antigen receptors selected from the group consisting of 5T4, B7-H3, B7-H4, BCMA, CAIX, CD 123, CD 19, CD20, CD22, CD30, CD33, CD79b, CEA, DLL3, EGFR, EGFRviii, FAP, FcRH5, GPR20, GUCY2C(GCC), HER2, HER3, KAAG1, LIV-1, MICA, MSLN, Mucl, PSMA, ROR1, SEZ6, and SLAMF7. In some embodiments, the peptide or polypeptide (P2) comprises an unnatural amino acid residue configured to form a covalent bond with a histidine, lysine, or tyrosine residue of the target. In some embodiments, the peptide or polypeptide (P2) comprises an unnatural amino acid of the structure:
[0024] In some embodiments, provided herein is a method of delivering a payload to a target cell, the method comprising contacting the target cell with the compound of Formula (II) or Formula (IF). In some embodiments, the target cell is a mammalian cell. In some embodiments, provided herein is a method of delivering a therapeutic payload to an intracellular target within a tumor cell of a mammal, the method comprising administering to the tumor cell an effective amount of a conjugate of a therapeutic payload, wherein the therapeutic payload is conjugated to a cleavable peptide via a self-immolative group comprising the sequence Ser-Sar. In some embodiments, provided herein is a method of delivering a therapeutic payload to an intracellular target within a tumor cell of a mammal, the method comprising administering to the tumor cell an effective amount of the compound of Formula (II) or Formula (IF). In some embodiments, provided herein is a method of treating cancer in a mammal, the method comprising administering to the mammal an effective amount of a composition comprising theWSGR Docket No. 60801-718.601compound of Formula (II) or Formula (IF). In some embodiments, provided herein is a method of eradicating a tumor or killing a tumor cell in a subj ect in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising the compound of Formula (II) or Formula (IF).
[0025] In some embodiments, provided herein is a compound of Formula (III):R3R2OH2N'X^^N'Y^'P1O R1Formula (III)wherein:P1is a chemotoxin,R1is H, Ci-Ce alkyl, wherein Ci-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, -OH, -NH2, -SH, and - SCH3,R2is C1-C6 alkyl,R3is H, Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)-NHC(=O)NH2, -(Ci-C6alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)-NHC(=NH)NH2, -(Ci-C6alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O-NHC(=NH)NH2, or -(Ci-C6alkylene)-O-NHC(=NH)NHCH3, wherein Ci-Ce alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NH2, -SH, and -SCH3,or R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, and R3is Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)-NHC(=O)NH2, -(Ci-Ce alkylene)- NHC(=O)NHCH3, -(C1-C6 alkylene)-NHC(=NH)NH2, -(Ci-C6alkylene)- NHC(=NH)NHCH3, -(CI-C6alkylene)-O-NHC(=NH)NH2, or -(Ci-C6alkylene)- O-NHC(=NH)NHCH3, wherein Ci-Ce alkyl is optionally substituted with one, two, or three groups selected from -F, phenyl, -C(=O)NH2, -C(=O)OH, -OH, - NH2, -SH, and -SCH3.
[0026] In some embodiments, provided herein is a compound of Formula (Illa):HO> CH3OH2N P’O R1WSGR Docket No. 60801-718.601Formula (Illa)
[0027] In some embodiments, the compound of Formula (III) is further characterized according to Formula (Illb)." "Formula (Illb)wherein:P1is a chemotoxin,R1is H, Ci-Ce alkyl, wherein Ci-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, -COOH, -CONH2, -OH, -NH2, -NHC(=O)NH2, -NHC(=NH)NH2, -SH, and -SCH3,; or two R1groups together form a C3-6 cycloalkyl;R2is Ci-Ce alkyl;R3is H, Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)- NHC(=O)NH2, -(CI-C6alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)- NHC(=NH)NH2, -(CI-C6alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O- NHC(=NH)NH2, or -(Ci-C6alkylene)-O-NHC(=NH)NHCH3, wherein Ci-C6alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NH2, -SH, and -SCH3;or R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, and R3is Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)-NHC(=O)NH2, -(Ci-C6alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)-NHC(=NH)NH2, -(C1-C6 alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O-NHC(=NH)NH2, or -(C1-C6 alkylene)-O-NHC(=NH)NHCH3, wherein Ci-C6alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NH2, -SH, and -SCH3. In some embodiments, R1is hydrogen, R2is C1-6 alkyl, and R3is CH2OH.
[0028] In some embodiments, the compound of Formula (III) has the structure:WSGR Docket No. 60801-718.601
[0029] In some embodiments, R3is -CH2OH. In some embodiments, R3is hydrogen. In some embodiments, R3is -CH2OH, and R3is hydrogen.
[0030] In some embodiments of Formula (I), (II), or (III), R3is an amino acid side-chain of a natural or unnatural amino acid. In some embodiments, R1is an unsubstituted C1-6 alkyl. In some embodiments, R1is substituted C1-6 alkyl, wherein the C1-6 alkyl is substituted with COOH, -CONH2, -OH, -NH2, -NHC(=O)NH2, -NHC(=NH)NH2, -SH, or -SCH3. In some embodiments, R1is an aromatic amino acid side-chain (e.g., benzyl, phenolyl, indolyl, or imidazolyl, corresponding to Phe, Tyr, Trp, and His respectively. In some embodiments, R1is a side-chain ofN(Me)Glu, N(Me)Lys, N(Me)Asp, N(Me)Arg, N(Me)Gln, orN(Me)Asn. In some embodiments, R3is -CH2OH. In some embodiments, R3is substituted C1-6 alkyl.
[0031] In some embodiments, P1is or comprises a maytansinoid, taxane, auristatin, camptothecin, vinca alkaloid, tubulysin, or epothilone. In some embodiments, P1is or comprises maytansine, mertansine (DM1), DM4, paclitaxel, docetaxel, ellipticine, a vinca alkaloid, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, utidelone, camptothecin, 7-ethyl camptothecin, exatecan, topotecan, irinotecan, belotecan, SN-38, Dxd, auristatin E, monomethyl auristatin E, auristatin F, or monomethyl auristatin F. In some embodiments, P1is:wherein:Ralis H, CH3, or CH2OH;Rblis H or F;Rb2is H, CH3, OH, or OC(=O)-G-G; wherein each G is piperidine;or Rbland Rb2taken together form a methylenedioxy or ethylenedioxy group; Rb3is H or Ci-Ce alkylene-N(CH3)2; andWSGR Docket No. 60801-718.601Rb4is H, Ci-Ce alkyl, or Ci-6 alkylene-NH-CH(CH3)2;or Rb3and Rb4taken together form a 6-membered carbocycle substituted with - NH2or -NHCOCH2OH; andeach Rcis independently H or CH3.INCORPORATION BY REFERENCE
[0032] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS
[0033] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:
[0034] FIG. 1 illustrates the hydrolytic stability of Compounds 1-4 in PBS (pH 7.8, 37 °C).
[0035] FIG. 2 illustrates the enzymatic payload release kinetics of Compound 5 and Compound 6 in comparison to a PABC reference Compound Rl, in the presence of papain.
[0036] FIG. 3 illustrates the enzymatic payload release kinetics of Compound 5 and Compound 6 in comparison to PABC reference Compound Rl, in the presence of cathepsin B.
[0037] FIG. 4 illustrates the cytotoxicity of example ADC 1, ADC 2, and ADC Rl, each conjugated to a cell surface tumor antigen #1 (Ag#l) in a cell line expressing the cell surface tumor antigen# 1 (Ag#l -positive).
[0038] FIG. 5 illustrates the cytotoxicity of example ADC 1, ADC 2, and ADC Rl, each conjugated to a cell surface tumor antigen #1 (Ag#l) in a cell line that does not substantially express the cell surface tumor antigen#l (Ag#l -negative).
[0039] FIG. 6 shows hydrolytic stability of SerSar-AE (Compound 3) relative to SerPro-AE (Compound 20) and GlyPro-AE (Compound 18).
[0040] FIG. 7 illustrates the impact of P2’ substitutions on hydrolytic stability for Compounds 3, 22, 24, and 26.
[0041] FIG. 8 illustrates the impact of N-acetylated variants of P2’ substitutions on hydrolytic stability for Compounds 4, 23, 25, and 27.WSGR Docket No. 60801-718.601
[0042] FIG. 9 illustrates the release of AE from of P2’ cyclopropyl-substituted sarcosine for Compounds 3, 24, and 28.
[0043] FIG. 10 illustrates the hydrolytic stability of N-linked SerSar-auristatins for Compounds 23 and 35 at pH 5 and pH 7.4.
[0044] FIG. 11 illustrates the impact of N-methylation at P2’ on payload release for Compounds 1, 3, 41, and 42.
[0045] FIG. 12 shows comparable payload release for MMAE and exatecan for Compounds 8, 37, and R3.
[0046] FIG. 13 shows enzymatic payload release when P2’ Sar is replaced with (NMe)Glu for Compounds 9, 33, and R4.
[0047] FIG. 14 illustrates the tumor xenograft activity for vehicle, reference ADC 34 containing PABC linker (compound Rl), and ADC 35 containing SerSar linker (Compound 9).DETAILED DESCRIPTION
[0048] Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, medicinal chemistry, and pharmacology described herein are those well-known and commonly employed in the art. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
[0049] One aspect of the present disclosure is the provision of alternative self-immolative groups to the PABC group, comprising a dipeptide group disposed between an oxygen atom of a payload and a cleavable peptide. A key feature of the dipeptide moiety is that cleavage of the cleavable peptide triggers cyclization of the dipeptide, thereby releasing the payload as the hydroxyl-bearing metabolite following ester cleavage.
[0050] Advantageously, disclosed herein certain dipeptides with particularly fast (or slow) cleavage kinetics, enabling the skilled artisan to accelerate or decelerate the release of a payload from the dipeptide following cleavage of the cleavable peptide. For example, one aspect of the disclosure is the discovery that certain serine dipeptides autocatalyze cleavage of a proximal ester bond, thereby achieving release kinetics suitable for drug delivery. The dipeptides may be selected to tailor the release kinetics of the payload, enabling more efficient and / or tunable payload release.WSGR Docket No. 60801-718.601
[0051] As an additional advantage over the PABC group, the dipeptides provided herein reduce lipophilicity compared to PABC, thereby increasing aqueous solubility, reducing aggregation, and enabling higher drug-to-antibody ratio.
[0052] Another aspect of the disclosure is the discovery that cathepsin B, and potentially other lysosomal proteases, recognize and cleave the peptide bond between citrulline and a variety of adjacent amino acids disclosed herein (including, for example, between Cit and Ser, whose cleavage is unexpectedly more facile than between Cit and Gly), whereas the vedotin structure is cleaved by between citrulline and PABC.Compounds
[0053] An aspect of the present disclosure is a compound of Formula (I)Formula (I)wherein:P1is a payload, preferably bonded through an ester bond or a thioester bond, R1is H, Ci-Ce alkyl, wherein Ci-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, -OH, -NH2, -SH, and - SCH3,R2is Ci-C6alkyl, -[CH2]i-3[OCH2CH2]i-i2OH, -[CH2]I-3[OCH2CH2]I-I2OCH3, - [CH2CON(CH3)]I-I2CH3; or -[CH2CON(CH3)]i-i2CH2COOH;R3is H, Ci-Ce alkyl, (Ci-Ce alkylene)-OH , -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)-NHC(=O)NH2, -(Ci-C6alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)-NHC(=NH)NH2, -(Ci-C6alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O-NHC(=NH)NH2, or -(Ci-C6alkylene)-O-NHC(=NH)NHCH3, wherein Ci-Ce alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, -C(=O)NH2, -C(=O)OH, - OH, -NH2, -SH, and -SCH3,or R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, and R3is Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)-NHC(=O)NH2, -(Ci-Ce alkylene)- NHC(=O)NHCH3, -(C1-C6 alkylene)-NHC(=NH)NH2, -(Ci-C6alkylene)- NHC(=NH)NHCH3, -(CI-C6alkylene)-O-NHC(=NH)NH2, or -(Ci-C6WSGR Docket No. 60801-718.601alkylene)-O-NHC(=NH)NHCH3, wherein Ci-Ce alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NH2, -SH, and -SCH3, L1is -L3-[AA]a- wherein:L3is a spacer,each AAis independently an amino acid,a is 2, 3, 4, or 5,R4is H, Ci-Ci2alkyl, PEGI-2O (i.e., -[CH2]I-3[OCH2CH2]I-I2OH or -[CH2]i-L2is a bond or a spacer, andR5is a moiety (e.g., an electrophile) capable of reacting with a thiol (-SH) or an amine (-NH2) side-chain of a peptide or polypeptide (e.g., a cysteine or a lysine side-chain of an antibody or antigen-binding fragment).
[0054] An aspect of the present disclosure is a compound of Formula (la)Formula (la)wherein:P1is a payload, preferably bonded through an ester bond or a thioester bond, R1is H, Ci-Ce alkyl, wherein Ci-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, -OH, -NH2, -COOH, - CONH2, -NHC(=O)NH2, -NHC(=NH)NH2, -SH, and -SCH3, or two R1groups together form a 3- to 6-membered cycloalkyl;R2is Ci-C6alkyl, -[CH2]I-3[OCH2CH2]I-I2OH, -[CH2]I-3[OCH2CH2]I-I2OCH3, - [CH2CON(CH3)]I-I2CH3; or -[CH2CON(CH3)]I-I2CH2COOH;R3is H, Ci-Ce alkyl, (Ci-Ce alkylene)-OH , -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)-NHC(=O)NH2, -(Ci-C6alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)-NHC(=NH)NH2, -(Ci-C6alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O-NHC(=NH)NH2, or -(Ci-C6alkylene)-O-NHC(=NH)NHCH3, wherein Ci-Ce alkyl is optionally substituted with one, two, or three groupsWSGR Docket No. 60801-718.601selected from the group consisting of -F, phenyl, -C(=0)NH2, -C(=O)OH, - OH, -NH2, -SH, and -SCH3,or R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, and R3is Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)-NHC(=0)NH2, -(Ci-Ce alkylene)- NHC(=O)NHCH3, -(C1-C6 alkylene)-NHC(=NH)NH2, -(Ci-C6alkylene)- NHC(=NH)NHCH3, -(CI-C6alkylene)-O-NHC(=NH)NH2, or -(Ci-C6alkylene)-O-NHC(=NH)NHCH3, wherein Ci-Ce alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NH2, -SH, and -SCH3, L1is -L3-[AA]a- wherein:L3is a first spacer,each AAis independently an amino acid,a is 2, 3, 4, or 5,R4is H, C1-C12 alkyl, PEG1-20 (i.e., -[CH2]i-3[OCH2CH2]i-i2OH or -[CH2]I-L2is a bond or a second spacer, andR5is a reactive group.
[0055] In some embodiments, R5is a moiety (e.g., an electrophile) capable of reacting with a thiol (-SH) or an amine (-NH2) side-chain of a peptide or polypeptide (e.g., a cysteine or a lysine side-chain of an antibody or antigen-binding fragment). In some embodiments, R5is a reactive group capable of reacting with a thiol (-SH), an amine (-NH2), an azide, (-N3), or an alkyne. In some embodiments, the thiol, amine, azide, or alkyne is on a side-chain of an amino acid or polypeptide. In some embodiments, the reactive group is capable of reacting with a thiol or amine of a cysteine or a lysine side-chain of an antibody or antigen-binding fragment. In some embodiments, the reactive group is capable of reacting with an alkyne or an azide in a strain-promoted alkyne-azide conjugation reaction. In some embodiments, the reactive group is capable of reacting with an alkyne or an azide in a strain-promoted alkyneazide conjugation reaction.
[0056] Another aspect of the present disclosure is a compound according to Formula (II) or (Ila):WSGR Docket No. 60801-718.601F ormul a (II) F ormul a (Ila) wherein each of L2, R4, L1, R3, R2, R1, P1, and n are as defined in Formula (I) or (la) respectively, or as further defined in the subsequent embodiments; and L5is a bond, Ci-6 alkylene, -CO-, -NHCO-CI-6 alkylene, -NHCO-CI-6 alkenylene, or 5-6 membered heterocycloalkylene, wherein the Ci-6 alkylene, Ci-6 alkenylene, and heterocycloalkylene are substituted with one, two, three, or four groups selected from oxo (=0), -C00H, and CH2COOH; andP2is a targeting agent.
[0057] In some embodiments of Formula (II) or (Ila), P2is a peptide or polypeptide that binds to a target. In some embodiments of Formula (II) or (Ila), P2is an antibody or antigen-binding peptide that binds to a target. In some embodiments of Formula (II) or (Ila), P2is an antigenbinding moiety that binds to a target. In some embodiments, the antigen-binding moiety is a small molecule antigen binder. In some embodiments, the antigen-binding moiety is a peptide antigen binder. In some embodiments, the antigen-binding moiety is an antibody. In some embodiments, the antigen-binding moiety is a nanobody. In some embodiments, the antigenbinding moiety is a small molecule tumor antigen binder.
[0058] In some embodiments, the compound of Formula (I) or Formula (II) is further characterized according to Formula (lb) or Formula (lib), respectively:Formula (lb) Formula (lib).
[0059] In some embodiments, the compound of Formula (I) or Formula (II) is further characterized according to Formula (Ic) or Formula (lie), respectively:WSGR Docket No. 60801-718.601F ormul a (Ic) F ormul a (lie) .
[0060] In some embodiments, P1is a payload bonded via an ester bond or a thioester bond. In some embodiments, P1is a payload, bonded via an oxygen atom or a sulfur atom of P1to the carbonyl of Formula (I) or (II), forming an ester or thioester bond.
[0061] In some embodiments, P1is a payload bonded via an ester bond. In some embodiments, P1is a payload, bonded via an oxygen atom of P1to the carbonyl of Formula (I) or (II), forming an ester bond.
[0062] In some embodiments, P1is a payload bonded via a thioester bond. In some embodiments, P1is a payload, bonded via a sulfur atom of P1to the carbonyl of Formula (I) or (II), forming a thioester bond.
[0063] In some embodiments, P1is a payload bonded via an amide bond.
[0064] In some embodiments, P1is a therapeutic payload and / or a detectable agent.
[0065] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:R1is H, Ci-Ce alkyl, wherein Ci-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, -OH, -NH2, -COOH, - CONH2, -NHC(=O)NH2, -NHC(=NH)NH2, -SH, and -SCH3,R2is Ci-C6alkyl, -[CH2]i-3[OCH2CH2]i-i2OH, -[CH2]i-3[OCH2CH2]i-i2OCH3, - [CH2CON(CH3)]i-i2CH3; or -[CH2CON(CH3)]i-i2CH2COOH;R3is H, Ci-Ce alkyl, (Ci-Ce alkylene)-OH , -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)-NHC(=O)NH2, -(Ci-C6alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)-NHC(=NH)NH2, -(Ci-C6alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O-NHC(=NH)NH2, or -(Ci-C6alkylene)-O-NHC(=NH)NHCH3, wherein Ci-Ce alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, -C(=O)NH2, -C(=O)OH, - OH, -NH2, -SH, and -SCH3,WSGR Docket No. 60801-718.601or R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine,wherein when R1and R2form a pyrrolidine, then R3is Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)-NHC(=0)NH2, -(Ci- Ce alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)-NHC(=NH)NH2, -(Ci-C6alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O-NHC(=NH)NH2, or -(Ci- Ce alkylene)-O-NHC(=NH)NHCH3, wherein Ci-Ce alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NH2, -SH, and -SCH3
[0066] In some embodiments, R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, and R3is a side-chain of Vai, Ser, Lys, or Cit. In some embodiments, R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, and R3is a side-chain of Ser. In some embodiments, R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, and R3is a side-chain of Vai.
[0067] In some embodiments, R1is a side-chain of Gly, He, Glu, Arg. In some embodiments, R1is a side-chain of Gly, Ala, Vai, Leu, or He. In some embodiments, R1is a side-chain of Gly. In some embodiments, R1is a side-chain of Ala or Vai. In some embodiments, R1is H. In some embodiments, R1is Ci-6 alkyl. In some embodiments, R1is Ci-6 alkyl substituted with -F, -OH, -NH2, -COOH, -CONH2, -NHC(=O)NH2, -NHC(=NH)NH2, -SH, and -SCH3. In some embodiments, R1is C1-6 alkyl substituted with -COOH, -CONH2, or -NHC(=NH)NH2.
[0068] In some embodiments, R2is alkyl, [PEG]i-i2OH, [PEG]i-i2OCH3, [Sar]i-i2-N(CH3)2. or [Sar]i-i2-COOH; wherein PEG is polyethylene glycol (-OCH2CH2- or -CH2OCH2- or -CH2CH2O-) and Sar is sarcosine (-CH2CON(CH3)- or -COCH2N(CH3)- or -CON(CH3)CH2-). In some embodiments, R2is alkyl, [PEG]i-i2OH, [PEG]i-i2OCH3, [Sar]i-i2-N(CH3)2, or [Sar]i-12-COOH; wherein PEG is polyethylene glycol and Sar is sarcosine. In some embodiments, R2is alkyl, [PEG]i-i2OH, [PEG]i-i2OCH3, [Sar]i-i2-N(CH3)2. or [Sar]i-i2-COOH; wherein PEG is -OCH2CH2- and Sar is -CH2CON(CH3)-.
[0069] In some embodiments, R3is a side-chain of Gly, Ala, Vai, Leu, He, Ser, Lys, or Cit. In some embodiments, R3is a side-chain of Gly, Ala, Vai, or Ser. In some embodiments, R3is a side-chain of Gly or Ser. In some embodiments, R3is a side-chain of Ser. In some embodiments, R3is a side-chain of Gly. In some embodiments, R3is a side-chain of Lys.WSGR Docket No. 60801-718.601
[0070] In some embodiments, R1is a side-chain of Gly, Ala, Vai, Leu, or He; R2is alkyl, [PEG]i-i2OH, [PEG]i-i2OCH3, [Sar]i-i2-N(CH3)2, or [Sar]i-i2-COOH; and R3is a side-chain of Gly, Ala, Vai, Leu, He, Ser, Lys, or Cit.
[0071] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:R1is H or Ci-Ce alkyl;R2is Ci-Ce alkyl; andR3is H, Ci-Ce alkyl, (C1-C4 alkylene)-OH, -(C2-C6 alkylene)-NH2, -(C2-C6 alkylene)- NHC(=O)NH2, -(C2-C6 alkylene)-NHC(=O)NHCH3, -(C2-C6 alkylene)- NHC(=NH)NH2, -(C2-C6 alkylene)-NHC(=NH)NHCH3, -(C2-C6 alkylene)-O- NHC(=NH)NH2, or -(C2-C6 alkylene)-O-NHC(=NH)NHCH3.
[0072] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine; andR3is Ci-Ce alkyl, (C1-C4 alkylene)-OH, -(C2-C6 alkylene)-NH2, -(C2-C6 alkylene)- NHC(=O)NH2, -(C2-C6 alkylene)-NHC(=O)NHCH3, -(C2-C6 alkylene)- NHC(=NH)NH2, -(C2-C6 alkylene)-NHC(=NH)NHCH3, -(C2-C6 alkylene)-O- NHC(=NH)NH2, or -(C2-C6 alkylene)-O-NHC(=NH)NHCH3.
[0073] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:R1is H, -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH(CH3)2, -C(CH3)2-, - CH2CH(CH3)CH2CH3, or -CH2CH(CH3)2,R2is -CEE, or R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine;R3is H, -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH(CH3)2, -C(CH3)2-, - CH2CH(CH3)2, -CH2OH, -CH2CH2OH, -CH(CH3)OH, -CH2CH2CH2NH2, - CH2CH2CH2CH2NH2, -CH2CH2CH2CH2CH2NH2, -CH2CH2CH2CH2CH2NH2, - CH2CH2NHC(=O)NH2, -CH2CH2CH2NHC(=O)NH2, - CH2CH2CH2CH2NHC(=O)NH2, -CH2CH2-NHC(=O)NHCH3, -CH2CH2CH2- NHC(=O)NHCH3, -CH2CH2CH2CH2-NHC(=O)NHCH3, -CH2CH2- NHC(=NH)NH2, -CH2CH2CH2-NHC(=NH)NH2, -CH2CH2CH2CH2- NHC(=NH)NH2, -CH2CH2-NHC(=NH)NHCH3, -CH2CH2CH2- NHC(=NH)NHCH3, -CH2CH2CH2CH2-NHC(=NH)NHCH3, -CH2CH2-O-WSGR Docket No. 60801-718.601NHC(=NH)NH2, -CH2CH2CH2-O-NHC(=NH)NH2, -CH2CH2CH2CH2-O- NHC(=NH)NH2, -CH2CH2-O-NHC(=NH)NHCH3, -CH2CH2CH2-O- NHC(=NH)NHCH3, or -CH2CH2CH2CH2-O-NHC(=NH)NHCH3,wherein when R1and R2form a pyrrolidine, then R3is -CH3, -CH2CH3, - CH2CH2CH3, -CH2CH2CH2CH3, -CH(CH3)2, -C(CH3)2-, -CH2CH(CH3)2, - CH2OH, -CH2CH2OH, -CH(CH3)OH, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, - CH2CH2CH2CH2CH2NH2, -CH2CH2CH2CH2CH2NH2, -CH2CH2NHC(=O)NH2, - CH2CH2CH2NHC(=O)NH2, -CH2CH2CH2CH2NHC(=O)NH2, -CH2CH2- NHC(=O)NHCH3, -CH2CH2CH2-NHC(=O)NHCH3, -CH2CH2CH2CH2- NHC(=O)NHCH3, -CH2CH2-NHC(=NH)NH2, -CH2CH2CH2-NHC(=NH)NH2, - CH2CH2CH2CH2-NHC(=NH)NH2, -CH2CH2-NHC(=NH)NHCH3, -CH2CH2CH2- NHC(=NH)NHCH3, -CH2CH2CH2CH2-NHC(=NH)NHCH3, -CH2CH2-O- NHC(=NH)NH2, -CH2CH2CH2-O-NHC(=NH)NH2, -CH2CH2CH2CH2-O- NHC(=NH)NH2, -CH2CH2-O-NHC(=NH)NHCH3, -CH2CH2CH2-O- NHC(=NH)NHCH3, or -CH2CH2CH2CH2-O-NHC(=NH)NHCH3.
[0074] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:R1is H, -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH(CH3)2, -C(CH3)2-, - CH2CH(CH3)CH2CH3, or -CH2CH(CH3)2,R2is -CH3,R3is H, -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH(CH3)2, -C(CH3)2-, - CH2CH(CH3)2, -CH2OH, -CH2CH2OH, -CH(CH3)OH, -CH2CH2CH2NH2, - CH2CH2CH2CH2NH2, -CH2CH2CH2CH2CH2NH2, -CH2CH2CH2CH2CH2NH2, - CH2CH2NHC(=O)NH2, -CH2CH2CH2NHC(=O)NH2, - CH2CH2CH2CH2NHC(=O)NH2, -CH2CH2-NHC(=O)NHCH3, -CH2CH2CH2- NHC(=O)NHCH3, -CH2CH2CH2CH2-NHC(=O)NHCH3, -CH2CH2- NHC(=NH)NH2, -CH2CH2CH2-NHC(=NH)NH2, -CH2CH2CH2CH2- NHC(=NH)NH2, -CH2CH2-NHC(=NH)NHCH3, -CH2CH2CH2- NHC(=NH)NHCH3, -CH2CH2CH2CH2-NHC(=NH)NHCH3, -CH2CH2-O- NHC(=NH)NH2, -CH2CH2CH2-O-NHC(=NH)NH2, -CH2CH2CH2CH2-O- NHC(=NH)NH2, -CH2CH2-O-NHC(=NH)NHCH3, -CH2CH2CH2-O- NHC(=NH)NHCH3, or -CH2CH2CH2CH2-O-NHC(=NH)NHCH3.
[0075] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:WSGR Docket No. 60801-718.601two R1taken together form a cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl group,R2is -CH3,R3is H, -CH3, -CH2CH3, -CH2CH2CJL, -CH2CH2CH2CJL, -CH(CH3)2, -C(CH3)2-, - CH2CH(CH3)2, -CH2OH, -CH2CH2OH, -CH(CH3)OH, -CH2CH2CH2NH2, - CH2CH2CH2CH2NH2, -CH2CH2CH2CH2CH2NH2, -CH2CH2CH2CH2CH2NH2, - CH2CH2NHC(=O)NH2, -CH2CH2CH2NHC(=O)NH2, - CH2CH2CH2CH2NHC(=O)NH2, -CH2CH2-NHC(=O)NHCH3, -CH2CH2CH2- NHC(=O)NHCH3, -CH2CH2CH2CH2-NHC(=O)NHCH3, -CH2CH2- NHC(=NH)NH2, -CH2CH2CH2-NHC(=NH)NH2, -CH2CH2CH2CH2- NHC(=NH)NH2, -CH2CH2-NHC(=NH)NHCH3, -CH2CH2CH2- NHC(=NH)NHCH3, -CH2CH2CH2CH2-NHC(=NH)NHCH3, -CH2CH2-O- NHC(=NH)NH2, -CH2CH2CH2-O-NHC(=NH)NH2, -CH2CH2CH2CH2-O- NHC(=NH)NH2, -CH2CH2-O-NHC(=NH)NHCH3, -CH2CH2CH2-O- NHC(=NH)NHCH3, or -CH2CH2CH2CH2-O-NHC(=NH)NHCH3.
[0076] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine; andR3is -CH3, -CH2CH3, -CH2CH2CIL, -CH2CH2CH2CIL, -CH(CH3)2, -C(CH3)2-, - CH2CH(CH3)2, -CH2OH, -CH2CH2OH, -CH(CH3)OH, -CH2CH2CH2NH2, - CH2CH2CH2CH2NH2, -CH2CH2CH2CH2CH2NH2, -CH2CH2CH2CH2CH2NH2, - CH2CH2NHC(=O)NH2, -CH2CH2CH2NHC(=O)NH2, - CH2CH2CH2CH2NHC(=O)NH2, -CH2CH2-NHC(=O)NHCH3, -CH2CH2CH2- NHC(=O)NHCH3, -CH2CH2CH2CH2-NHC(=O)NHCH3, -CH2CH2- NHC(=NH)NH2, -CH2CH2CH2-NHC(=NH)NH2, -CH2CH2CH2CH2- NHC(=NH)NH2, -CH2CH2-NHC(=NH)NHCH3, -CH2CH2CH2- NHC(=NH)NHCH3, -CH2CH2CH2CH2-NHC(=NH)NHCH3, -CH2CH2-O- NHC(=NH)NH2, -CH2CH2CH2-O-NHC(=NH)NH2, -CH2CH2CH2CH2-O- NHC(=NH)NH2, -CH2CH2-O-NHC(=NH)NHCH3, -CH2CH2CH2-O- NHC(=NH)NHCH3, or -CH2CH2CH2CH2-O-NHC(=NH)NHCH3.
[0077] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:WSGR Docket No. 60801-718.601R1is H, -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH(CH3)2, -C(CH3)2-, - CH2CH(CH3)CH2CH3, or -CH2CH(CH3)2,R2is -CH3, or R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine,R3is H, -CH3, -CH2OH, -CH2CH2OH, or -CH(CH3)OH,wherein when R1and R2form a pyrrolidine, then R3is -CH2OH, -CH2CH2OH, or - CH(CH3)OH.
[0078] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:R1is H, -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH(CH3)2, -C(CH3)2-, - CH2CH(CH3)CH2CH3, or -CH2CH(CH3)2,R2is -CH3, andR3is H, -CH3, -CH2OH, -CH2CH2OH, or -CH(CH3)OH.
[0079] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, andR3is -CH2OH, -CH2CH2OH, or -CH(CH3)OH.
[0080] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:R1is H, -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH(CH3)2, -C(CH3)2-, - CH2CH(CH3)CH2CH3, or -CH2CH(CH3)2,R2is -CH3, andR3is -CH2OH.
[0081] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:R1is H,R2is -CH3, andR3is -CH2OH.
[0082] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:R1is -CH3,R2is -CH3, andR3is -CH2OH.WSGR Docket No. 60801-718.601
[0083] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:R1is -CH(CH3)2, -CH2CH(CH3)CH2CH3, or -CH2CH(CH3)2,R2is -CH3, andR3is -CH2OH.
[0084] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:R1is H,R2is -CH2CH3, andR3is -CH2OH.
[0085] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:R1is H, -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH(CH3)2, -C(CH3)2-, - CH2CH(CH3)CH2CH3, or -CH2CH(CH3)2,R2is -CH3, andR3is H.
[0086] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:R1is H,R2is -CH3, andR3is H.
[0087] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:R1is -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH(CH3)2, -C(CH3)2-, - CH2CH(CH3)CH2CH3, or -CH2CH(CH3)2,R2is -CH3, andR3is H.
[0088] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein R3is -H, R2is -CH3, and R1is H, substituted -Ci-Ce alkyl, or unsubstituted -C2-Ce alkyl, wherein the C1-6 alkyl is substituted with NHC(=NH)NH2, -COOH, or two R1groups together form a 3- to 6-membered cycloalkyl. In some embodiments, provided herein is a compound of Formula (I) or (II), wherein R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, and R3is substituted or unsubstituted Ci-Ce alkyl.
[0089] In some embodiments, the compound of Formula (I) or (II), isWSGR Docket No. 60801-718.601wherein R1is Ci-Ce alkyl, optionally substituted with -OH, -NH2, -COOH, -CONH2, -NHC(=0)NH2, -NHC(=NH)NH2, -SH, or -SCH3, and other variables are as defined in (I) or (II). In some embodiments, R2is methyl. In some embodiments, R3is -CH2OH.
[0090] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein L1is -L3-[AA]a-, wherein L3is a first spacer, each AA is independently an amino acid, and a is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, a is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, a is 2, 3, 4, or 5. In some embodiments, a is 2, 3, or 4. In some embodiments, a is 2 or 3. In some embodiments, a is 2. In some embodiments, a is 3.
[0091] In some embodiments, each [AA] is independently selected from the group consisting of glycine (Gly), alanine (Ala), valine (Vai), leucine (Leu), isoleucine (He), proline (Pro), citrulline (Cit), lysine (Lys), arginine (Arg), glutamate (Glu), aspartate (Asp), asparagine (Asn), glutamine (Gin), phenylalanine (Phe), histidine (His), tryptophan (Trp), tyrosine (Tyr), methionine (Met), serine (Ser), and threonine (Thr).
[0092] In some embodiments, [AA]ais a Cathepsin B substrate, a legumain substrate, or a tumor necrosis factor (TNF) alpha converting enzyme (“TACE”) substrate. In some embodiments, [AA]ais a matriptase substrate, a matrix metalloproteinase substrate, a furin substrate, a prostate-specific membrane antigen, (“PSMA,” also known as glutamate carboxypeptidase II) substrate, a dipeptidyl peptidase substrate, kallikrein substrate, a fibroblast activation protein (“FAP”) substrate, a cathepsin family members substrate, aWSGR Docket No. 60801-718.601plasmin substrate, a carboxypeptidase A substrate, a proteinase III substrate, or a neutrophil elastase substrate. In some embodiments, [AA]ais a Cathepsin B substrate. In some embodiments, [AA]ais a legumain substrate. In some embodiments, [AA]ais a tumor necrosis factor alpha converting enzyme (TACE) substrate.
[0093] In some embodiments, [AA]ais a cathepsin B substrate, a legumain substrate, a TACE substrate, a matriptase substrate, a MMP substrate, a furin substrate, a PSMA substrate, a dipeptidyl peptidase substrate, a kallikrein substrate, a FAP substrate, a cathepsin substrate, a plasmin substrate, a carboxypeptidase A substrate, a proteinase III substrate, or a neutrophil elastase substrate.
[0094] In some embodiments, provided herein is a compound of Formula (I), wherein a is 1, and the C-terminal [AA] is any naturally-occurring amino acid, standard unnatural amino acid, or a stereoisomer thereof. In some embodiments, provided herein is a compound of Formula (I), wherein a is 1, and the C-terminal [AA] is Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Pro, Pyl, Ser, Sec, Thr, Trp, Tyr, or Vai. In some embodiments, provided herein is a compound of Formula (I), wherein a is 1, and the [AA] is Asn. In some embodiments, provided herein is a compound of Formula (I), wherein a is 1, and the [AA] is Asp.
[0095] In some embodiments, provided herein is a compound of Formula (I), wherein a is 2, and the C-terminal [AA] is any naturally-occurring amino acid, standard unnatural amino acid, or a stereoisomer thereof. In some embodiments, provided herein is a compound of Formula (I), wherein a is 2, and the C-terminal [AA] is Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Pro, Pyl, Ser, Sec, Thr, Trp, Tyr, or Vai. In some embodiments, the C-terminal [AA] is citrulline (Cit). In some embodiments, provided herein is a compound of Formula (I), wherein a is 2, and the C-terminal [AA] is Cit, Asn, or Arg.
[0096] In some embodiments, provided herein is a compound of Formula (I), wherein a is 3, and the C-terminal [AA] is any naturally-occurring amino acid, standard unnatural amino acid, or a stereoisomer thereof. In some embodiments, provided herein is a compound of Formula (I), wherein a is 3, and the C-terminal [AA] is Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Pro, Pyl, Ser, Sec, Thr, Trp, Tyr, or Vai. In some embodiments, the C-terminal [AA] is citrulline (Cit). In some embodiments, provided herein is a compound of Formula (I), wherein a is 3, and the C-terminal [AA] is Cit, Asn, or Arg.
[0097] In some embodiments, [AA]ais Val-Arg*, Gly-Arg*, Ile-Arg*, Ala-Arg*, Glu-Arg*, Arg-Arg*, Val-Cit*, Gly-Cit, Ile-Cit*, Ala-Cit*, Glu-Cit*, Glu- Val-Arg*, Gly-Val-Arg*, Gly-Gly-Arg*, Gly-Glu- Val-Arg*, Glu- Val-Cit*, Glu-Gly-Cit*, Gly- Val-Cit*, Gly-Gly-Cit*, Gly-WSGR Docket No. 60801-718.601Glu-Val-Cit*, or Glu-Arg-Arg*; wherein in each instance, * indicates the C-terminal amino acid residue. In some embodiments, [AA]ais Gly-Asn*, Ala-Asn*, Gly-Gly-Asn*, Ala-Ala-Asn*, or Ala-(D)Ala-Asn*; wherein in each instance, * indicates the C-terminal amino acid residue. In some embodiments, [AA]ais Leu-Asp-Leu* or Ala-Gin-Ala*; wherein in each instance, * indicates the C-terminal amino acid residue. In some embodiments, [AA]ais Val-Ala*, Gly-Ala*, Glu-Ala*, Ala-Ala*, Phe-Ala*, Leu-Ala*, His-Ala*, Trp-Ala*, Pro- Ala*, Val-Arg*, Gly-Arg*, Ile-Arg*, Ala-Arg*, Glu-Arg*, Tyr-Arg*, Arg-Arg*, Gly-Asn*, Ala-Asn*, Asn-Asn*, Arg-Asn*, Gly-Asp*, Glu-Asp*, His-Asp*, Asp-Asp*, Pro-Asp*, Val-Cit*, Gly-Cit*, Ile-Cit*, Ala-Cit*, Glu-Cit*, Glu-Glu*, Ala-Glu*, Tyr-Glu*, Asp-Glu*, Val-Glu*, Val-Gly*, Gly-Gly*, Glu-Gly*, Ile-Gly*, Ala-Gly*, Phe-Gly*, Leu-Gly*, Asp-Gly*, Cys-Gly*, Val-His*, Gly-His*, Ala-His*, Val-Ile*, Gly-Ile*, Ile-Ile*, Leu-Ile*, Gly-Leu*, Phe-Leu*, Lys-Leu*, Leu-Leu*, Asp-Leu*, Phe-Lys*, Leu-Lys*, His-Lys*, Vai-Met*, Val-Phe*, Gly-Phe*, Ala-Phe*, Phe-Phe*, Leu-Phe*, His-Phe*, Asp-Phe*, Val-Pro*, Gly-Pro*, Ala-Pro*, Phe-Pro*, Leu-Pro*, Gly-Ser*, Glu-Ser*, Ala-Ser*, Phe-Ser*, Tyr-Ser*, Trp-Ser*, Ser-Ser*, Asp-Ser*, Asn-Ser*, Val-Thr*, Gly-Thr*, Lys-Thr*, Tyr-Thr*, Trp-Thr*, Thr-Thr*, Ser-Thr*, Asp-Thr*, Gln-Thr*, Val-Trp*, Ile-Trp*, Phe-Trp*, Trp-Trp*, Val-Tyr*, Leu-Tyr*, His-Tyr*, Glu-Val*, Val-Val*, Gly-Val*, Ala-Vai*, Glu-Val-Arg*, Gly-Val-Arg*, Gly-Gly-Arg*, Gly-Glu-Val-Arg*, Glu-Val-Cit*, Gly- Val-Cit*, Gly-Gly-Cit*, Gly-Glu- Val-Cit*, Glu-Arg-Arg*, Gly-Gly-Asn*, Ala-Ala-Asn*, or Ala-(D)Ala-Asn*, Leu-Asp-Leu*, or Ala-Gin- Ala*; wherein in each instance, * indicates the C-terminal amino acid residue.
[0098] In some embodiments, the compound is of Formula (I’) or (II’):Formula (II’)wherein R1, R2, and R3are as described in any of the preceding embodiments;L2, L3, P1, P2, R4, R5, and n are as defined in Formula (I) and Formula (II);a’ is 0, 1, or 2; andWSGR Docket No. 60801-718.601each of R6, R7, and R8is independently hydrogen or Ci-6 alkyl, wherein each Ci-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NH2, -NHC(=O)NH, - SH, -SCH3, -NHC(=O)NHCH3, -NHC(=NH)NH2, -NHC(=NH)NHCH3, -O- NHC(=NH)NH2, and -O-NHC(=NH)NHCH3.
[0099] In some embodiments, the compound is of Formula (I’):Formula (F)wherein R1, R2, and R3are as described herein;L2, L3, P1, R4, and R5, are as defined in Formula (I);a’ is 0, 1, or 2; andeach of R6, R7, and R8is independently hydrogen or C1-6 alkyl, wherein each C1-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NH2, -NHC(=O)NH, - SH, -SCH3, -NHC(=O)NHCH3, -NHC(=NH)NH2, -NHC(=NH)NHCH3, -O- NHC(=NH)NH2, and -O-NHC(=NH)NHCH3.
[0100] In some embodiments, L3is a first spacer. In some embodiments, L3is a bond. In some embodiments, L3consists of one or more amino acids and / or PEG groups.
[0101] In some embodiments, L3is:L3e-, -L3b-L3c-L3d-, -L3c-L3d-L3e, -L3a-L3b-L3c-L3d-L3e-, or a combination thereof; each L3ais independently a bond, unsubstituted or substituted alkylene, unsubstituted or substituted heteroalkylene, unsubstituted or substituted alkenylene, unsubstituted or substituted alkynylene, unsubstituted or substituted cycloalkylene, unsubstituted or substituted heterocycloalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene, one or more amino acids, -(CH2)q-, - (CH2)P-C(=O)-, -C(=O)-(CH2)P-, -(CH2)P-C(=O)-(CH2)P-, -C(=O)NH-,-C(=O)NH- (CH2)P-, -(CH2)P-C(=O)NH-, -(CH2)p-C(=O)NH-(CH2)P-, each p is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12;each L3bis independently a bond, -O-, -S-, -S(=O)-, -S(=O)2-, -NH-, -CH(OH)-, - NHC(=O)-, -C(=O)O-, -OC(=O)-, -CH(=N)-, -CH(=N-NH)-, -CCH3(=N)-, -WSGR Docket No. 60801-718.601CCH3(=N-NH)-, -OC(=O)NH-, -NHC(=O)NH-, -NHC(=O)O-, -(CH2)P-, -C(=0)- (CH2CH2Z)P-, or -(CH2CH2Z)p-, each p is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12;each L3Cis independently a bond, unsubstituted or substituted alkylene, unsubstituted or substituted heteroalkylene, unsubstituted or substituted alkenylene, unsubstituted or substituted alkynylene, unsubstituted or substituted cycloalkylene, unsubstituted or substituted heterocycloalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene, one or more amino acids, -(CH2)q-, - (CH2CH2Z)q-, or -(ZCH2CH2)q-, each q is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12;each L3dis independently a bond, -O-, -S-, -S(O)-, -S(O)2-, -NH-, -CH(OH)-, -C(=O)-, -C(=O)NH-, -NHC(=O)-, -C(=O)O-, -OC(=O)-, -OC(=O)NH-, -NHC(=O)NH-, or -NHC(=O)O-;each L3eis independently absent, unsubstituted or substituted alkylene, or unsubstituted or substituted heteroalkylene;each Z is independently selected from O, S, and NRZ; and each Rzis independently selected from hydrogen, Ci-C4alkyl and -CH2C(=O)OH.
[0102] In some embodiments, the first spacer, L3is:wherein * indicates a bond to -[AA]a-;each u is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; andeach Z is independently selected from O, and NRZ; and each Rzis independently hydrogen or Ci-C4alkyl.
[0103] In some embodiments, L3is:WSGR Docket No. 60801-718.601whereineach Z is independently selected from O, and NRZ;each Rzis independently selected from hydrogen, -C1-C4 alkyl and -CH2C(=0)0H; and * indicates a bond to -[AA]a-.
[0104] In some embodiments, L3is:WSGR Docket No. 60801-718.601WSGR Docket No. 60801-718.601wherein indicates a bond to -[AA]a-. In some embodiments, L is:wherein * indicates a bond to the N-terminus of -[AA]a-. In some embodiments, L3is:, wherein * indicates a bond to the -NH-CHR8group of Formula (F) or (ir).
[0105] In some embodiments, L2is unsubstituted or substituted alkylene, unsubstituted or substituted heteroalkylene, unsubstituted or substituted alkenylene, unsubstituted or substituted alkynylene, unsubstituted or substituted cycloalkylene, unsubstituted or substituted heterocycloalkylene, unsubstituted or substituted arylene, unsubstituted or substituted heteroarylene, one or more amino acids, -C(=O)-, -(CH2)q-, -(CH2)q-C(=O)-, -C(=O)-(CH2)q-, -(CH2CH2O)q-, -(CH2CH2S)q-, -(CH2CH2NH)q-, -(CH2)q-C(=O)-(CH2)q-, -C(=O)NH-,-C(=O)NH-(CH2)q-, -(CH2)q-C(=O)NH-, or -(CH2)q-C(=O)NH-(CH2)q-, wherein each q is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.
[0106] In some embodiments, provided herein is a compound according to Formula (I), (I’), (II), or (IF), wherein L2is:WSGR Docket No. 60801-718.601* indicates a bond to NR4;each m is independently 0, 1, 2, 3, 4, or 5;each u is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; andeach Z is independently O, NH, or N(CHs).
[0107] In some embodiments, L2is:wherein* indicates a bond to NR4;each m is independently 0, 1, or 2;each u is independently 0, 1, 2, 3, or 4; andeach Z is independently O or N(CH3).* indicates a bond to NR4; m is 0, 1, or 2; and u is 0, 1, 2, 3, or 4. In some embodiments, m is 0 or 1. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, u is 0, 1, 2, or 3. In some embodiments, u is 1, 2, or 3. In some embodiments, u is 1. In some embodiments, u is 2. In some embodiments, u is 3. In some embodiments, L2is:WSGR Docket No. 60801-718.601wherein * indicates a bond to NR4.
[0108] In some embodiments, provided herein is a compound of Formula (I), (I’), (II), or (IF), wherein P1is monomethyl auristatin E (MMAE), auristatin E (AE), dasatinib, a maytansinoid mertansine (DM1), a camptothecin, or an anthracy cline. In some embodiments, provided herein is a compound of Formula (I), (F), (II), or (IF), wherein P1is:WSGR Docket No. 60801-718.601WSGR Docket No. 60801-718.601wherein:Ralis H, CH3, or CH2OH;Rblis H or F;Rb2is H, CH3, OH, or OC(=O)-G-G; wherein each G is piperidine;Rb3is H or Ci-Ce alkylene-N(CH3)2; andRb4is H, Ci-Ce alkyl, or C1-6 alkylene-NH-CH(CH3)2;or Rb3and Rb4taken together form a 6-membered carbocycle substituted with - NH2 or -NHCOCH2OH.
[0109] In some embodiments, provided herein is a compound of Formula (I), (F), (II), or (IF), wherein P1is:wherein:Ralis H, CH3, or CH2OH;Rblis H or F;Rb2is H, CH3, OH, or OC(=O)-G-G; wherein each G is piperidine;WSGR Docket No. 60801-718.601or Rbland Rb2taken together form a methylenedioxy or ethylenedioxy group; Rb3is H or Ci-Ce alkylene-N(CH3)2; andRb4is H, Ci-C6alkyl, or Ci-6 alkylene-NH-CH(CH3)2;or Rb3and Rb4taken together form a 6-membered carbocycle substituted with - NH2or -NHCOCH2OH.
[0110] In some embodiments, provided herein is a compound of Formula (I), (I’), (II), or (IF), wherein P1is:wherein: Ralis H, CH3, or CH2OH.
[0111] In some embodiments, provided herein is a compound of Formula (I), (F), (II), or (IF), wherein P1is:wherein: Ralis H or CH3.
[0112] In some embodiments, provided herein is a compound of Formula (I), (F), (II), or (IF), wherein P1is:wherein:Rblis H or F;Rb2is H, CH3, OH, or OC(=O)-G-G; wherein each G is piperidine;or Rbland Rb2taken together form a methylenedioxy or ethylenedioxy group; Rb3is H or Ci-Ce alkylene-N(CH3)2; andRb4is H, Ci-Ce alkyl, or C1-6 alkylene-NH-CH(CH3)2;or Rb3and Rb4taken together form a 6-membered carbocycle substituted with - NH2or -NHCOCH2OH.
[0113] In some embodiments,Rblis H or F;WSGR Docket No. 60801-718.601Rb2is H, CH3, OH, or OC(=O)-G-G; wherein each G is piperidine;and Rb3and Rb4taken together form a 6-membered carbocycle substituted with - NH2 or -NHCOCH2OH.
[0114] In some embodiments,Rblis H or F;Rb2is H, CH3, OH, or OC(=O)-G-G; wherein each G is piperidine;Rb3is H or Ci-Ce alkylene-N(CH3)2; andRb4is H, Ci-Ce alkyl, or C1-6 alkylene-NH-CH(CH3)2.
[0115] In some embodiments,Rbland Rb2taken together form a methylenedioxy or ethylenedioxy group;Rb3is H; andRb4is Ci-C6alkyl.
[0116] In some embodiments, each P1is independently selected from the group consisting of:WSGR Docket No. 60801-718.601wherein each Rcis independently hydrogen or Ci-6 alkyl (e.g., H or CH3).
[0118] In some embodiments, P1is:wherein each Rcis independently hydrogen or C1-6 alkyl (e.g., H or CH3).
[0119] In some embodiments, P1is:WSGR Docket No. 60801-718.601WSGR Docket No. 60801-718.601or a stereoisomer or mixture of stereoisomers thereof.or a stereoisomer or mixture of stereoisomers thereof.
[0123] In some embodiments, -R5is:each R5aand R5bis independently selected from H or halogen, R5Cis halogen, andR5dis H orNO2.
[0124] In some embodiments, -L2-R5is:WSGR Docket No. 60801-718.601whereinm is 0, 1, 2, 3, 4, or 5;u is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12;each R5aand R5bis independently selected from H or halogen, andR5Cis halogen, azide, or cyclooctyne (e.g. DBCO or analogous conjugating groups for use in SPAAC or Click chemistry conjugations); andR5dis H orNO2.
[0125] In some embodiments, provided herein is a compound of Formula (I) or (I’), whereinWSGR Docket No. 60801-718.601
[0126] In some embodiments, provided herein is a compound of Formula (I) or (I’), whereinWSGR Docket No. 60801-718.601
[0127] In some embodiments, provided herein is a compound of Formula (I) or (I’), wherein
[0128] In some embodiments, the compound of Formula (I) or Formula (F) is:wherein:WSGR Docket No. 60801-718.601each m is independently 0, 1, 2, 3, 4, or 5;each u is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8;each a is 0, 1, or 2;each R5aand R5bis H or halogen;each of R6, R7, and R8is independently hydrogen or Ci-6 alkyl, wherein each Ci-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NH2, -NHC(=O)NH, - SH, -SCH3, -NHC(=O)NHCH3, -NHC(=NH)NH2, -NHC(=NH)NHCH3, -O- NHC(=NH)NH2, and -O-NHC(=NH)NHCH3;each R3is H or CH2OH;each Ralis H, CH3, or CH2OH,each Rblis H or F;each Rb2is H, CH3, OH, or OC(=O)-G-G; wherein each G is piperidine;or Rbland Rb2taken together form a methylenedioxy or ethylenedioxy group; each Rb3is H or Ci-Ce alkylene-N(CH3)2; andeach Rb4is H, Ci-Ce alkyl, or C1-6 alkylene-NH-CH(CH3)2;or Rb3and Rb4taken together form a 6-membered carbocycle substituted with -NH2or -NHCOCH2OH.
[0129] In some embodiments, the compound of Formula (I) or Formula (F) is:WSGR Docket No. 60801-718.601wherein:each m is independently 0, 1, 2, 3, 4, or 5;each u is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8;each a is 0, 1, or 2;each R5aand R5bis H or halogen;each of R6, R7, and R8is independently hydrogen or C1-6 alkyl, wherein each C1-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NH2, -NHC(=O)NH, - SH, -SCH3, -NHC(=O)NHCH3, -NHC(=NH)NH2, -NHC(=NH)NHCH3, -O- NHC(=NH)NH2, and -O-NHC(=NH)NHCH3;each R3is H or CH2OH;each Ralis H, CH3, or CH2OH,each Rblis H or F;each Rb2is H, CH3, OH, or OC(=O)-G-G; wherein each G is piperidine;each Rb3is H or Ci-Ce alkylene-N(CH3)2; andeach Rb4is H, Ci-Ce alkyl, or C1-6 alkylene-NH-CH(CH3)2;or Rb3and Rb4taken together form a 6-membered carbocycle substituted with -NH2 or -NHC0CH20H.
[0130] In some embodiments, R4is hydrogen.
[0131] In some embodiments, the compound of Formula (I) or Formula (F) is:WSGR Docket No. 60801-718.601wherein:R3is H or OH,Ralis H, CH3, or CH2OH,Rblis H or F;Rb2is H, CH3, OH, or OC(=O)-G-G; wherein each G is piperidine;Rb3is H or Ci-Ce alkylene-N(CH3)2; andRb4is H, Ci-Ce alkyl, or C1-6 alkylene-NH-CH(CH3)2;or Rb3and Rb4taken together form a 6-membered carbocycle substituted with -NH2 or -NHCOCH2OH.
[0132] In some embodiments, the compound of Formula (I) or Formula (F) is:or a salt or stereisomer or mixture of stereoisomers thereof, wherein:each Rcis independently selected from -H and C1-6 alkyl (e.g., H or -CH3).
[0133] In some embodiments, the compound of Formula (I) or Formula (F) is a compound of Table 1. In some embodiments, the compound of Formula (la) is a compound of Table 1.WSGR Docket No. 60801-718.601
[0134] Table 1. DARI thiol-reactive linker-drug conjugatesWSGR Docket No. 60801-718.601WSGR Docket No. 60801-718.601WSGR Docket No. 60801-718.601or a salt or stereoisomer thereof, or a mixture of stereoisomers thereof."
[0135] In some embodiments,
[0136] In some embodiments, the compound of Formula (I) or Formula (F) is a compound of Table 2. In some embodiments, the compound of Formula (la) is a compound of Table 2.
[0137] Table 2. DAR2 thiol-reactive linker-drug conjugatesWSGR Docket No. 60801-718.601WSGR Docket No. 60801-718.601WSGR Docket No. 60801-718.601WSGR Docket No. 60801-718.601
[0138] Another embodiment of the present disclosure is a compound of Formula (II)Formula (II)wherein:n is an integer from 1 to 20,P2is a peptide or polypeptide that binds to a target,P1is a payload bonded via an ester bond or a thioester bond,WSGR Docket No. 60801-718.601R1is H, Ci-Ce alkyl, wherein Ci-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, -OH, -NH2, -SH, and - SCH3,R2is Ci-C6alkyl, -[CH2]i-3[OCH2CH2]i-i2OH, -[CH2]I-3[OCH2CH2]I-I2OCH3, - [CH2CON(CH3)]I-I2CH3, or -[CH2CON(CH3)]I-I2CH2COOH;R3is H, Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)-NHC(=O)NH2, -(Ci-C6alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)-NHC(=NH)NH2, -(Ci-C6alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O-NHC(=NH)NH2, or -(Ci-C6alkylene)-0-NHC(=NH)NHCH3, wherein Ci-Ce alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, -C(=O)NH2, -C(=O)OH, - OH, -NH2, -SH, and -SCH3,or R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, wherein when R1and R2form a pyrrolidine, then R3is Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)- NHC(=O)NH2, -(C1-C6 alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)- NHC(=NH)NH2, -(CI-C6alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O- NHC(=NH)NH2, or -(Ci-C6alkylene)-O-NHC(=NH)NHCH3, wherein Ci-C6alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NH2, -SH, and -SCH3,L1is -L3-[AA]a- wherein:L3is a spacer,each AAis independently an amino acid,a is 2, 3, 4, or 5,R4is H, Ci-Ci2alkyl, PEGI-2O (i.e., -[CH2]I-3[OCH2CH2]I-I2OH or -[CH2]I-<wherein L1, R1, R2, R3, and P1are as defined above;L2is a bond or a spacer, andL5is a bond, C1-6 alkylene, -CO-, -NHCO-C1-6 alkylene, -NHCO-C1-6 alkenylene, or 5-6 membered heterocycloalkylene, wherein the C1-6 alkylene, C1-6 alkenylene,WSGR Docket No. 60801-718.601and heterocycloalkylene are substituted with one, two, three, or four groups selected from oxo (=0), -C00H, and CH2COOH.
[0139] In some embodiments, provided herein is a compound of Formula (Ila)Formula (Ila)wherein:n is an integer from 1 to 20,P2is a targeting moiety (e.g., a peptide or polypeptide that binds to a target), P1is a payload bonded via an ester bond or a thioester bond,R1is an amino acid side-chain of a natural, unnatural, or D-amino acid (e.g., H, Ci-Ce alkyl, wherein C1-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, -OH, -NH2, -COOH, - CONH2, -NHC(=0)NH2, -NHC(=NH)NH2, -SH, -SCH3, phenyl, phenol, imidazole, or indole, or two R1groups together form a C3-6 cycloalkyl);R2is Ci-C6alkyl, -[CH2]i-3[OCH2CH2]i-i2OH, -[CH2]i-3[OCH2CH2]i-i2OCH3, - [CH2CON(CH3)]i-i2CH3, or -[CH2CON(CH3)]i-i2CH2COOH;R3is an amino acid side-chain of a natural, unnatural, or D-amino acid (e.g., H, Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)- NHC(=0)NH2, -(C1-C6 alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)- NHC(=NH)NH2, -(CI-C6alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O- NHC(=NH)NH2, or -(Ci-C6alkylene)-O-NHC(=NH)NHCH3, wherein Ci-C6alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, imidazole, -C(=O)NH2, -C(=O)OH, -OH, - NH2, -SH, and -SCH3),or R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, wherein when R1and R2form a pyrrolidine, then R3is Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)- NHC(=0)NH2, -(C1-C6 alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)- NHC(=NH)NH2, -(CI-C6alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O- NHC(=NH)NH2, or -(Ci-C6alkylene)-O-NHC(=NH)NHCH3, wherein Ci-C6alkyl is optionally substituted with one, two, or three groups selected from theWSGR Docket No. 60801-718.601group consisting of -F, phenyl, -C(=0)NH2, -C(=O)OH, -OH, -NH2, -SH, and -SCH3,L1is -L3-[AA]a- wherein:L3is a first spacer,each AAis independently an amino acid,a is 2, 3, 4, or 5,R4is H, C1-C12 alkyl, PEG1-20 (i.e., -[CH2]i-3[OCH2CH2]i-i2OH or -[CH2]I-wherein L1, R1, R2, R3, and P1are as defined above;L2is a bond or a second spacer, andL5is a bond, C1-6 alkylene, -CO-, -NHCO-C1-6 alkylene, -NHCO-C1-6 alkenylene, or 5-6 membered heterocycloalkylene, wherein the C1-6 alkylene, C1-6 alkenylene, and heterocycloalkylene are substituted with one, two, three, or four groups selected from oxo (=0), -C00H, and CH2COOH.
[0140] In some embodiments, provided herein is a compound of Formula (II), wherein each of P2, L2, R4, L1, R3, R2, R1, P1, and n are as defined in Formula (I), or as further defined in the preceding embodiments.
[0141] In some embodiments, the compound is of Formula (IF):(Formula (IF))wherein R1, R2, and R3are as described above;L2, L3, P1, P2, R4, R5, and n are as defined in Formula (I) and Formula (II);a’ is 0, 1, or 2; andeach of R6, R7, and R8is independently hydrogen or C1-6 alkyl, wherein each C1-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NH2, -NHC(=O)NH, - SH, -SCH3, -NHC(=O)NHCH3, -NHC(=NH)NH2, -NHC(=NH)NHCH3, -O- NHC(=NH)NH2, and -O-NHC(=NH)NHCH3.WSGR Docket No. 60801-718.601
[0142] In some embodiments, provided herein is a compound of Formula (II) or (II’), wherein P1is a drug and P2is an antibody or antigen-binding fragment thereof. In some embodiments, the compound of Formula (II) or (IF) is an antibody-drug conjugate (ADC).
[0143] In some embodiments, provided herein is a compound of Formula (II) or (IF), wherein: -L5- is a bond or:wherein * indic-1-1a bisected bond11 indicates a bond to one of the two adjacent carbon atoms.
[0144] In some embodiments, provided herein is a compound of Formula (II) or (IF), wherein:-L2-L5- is:WSGR Docket No. 60801-718.601whereinm is 0, 1, 2, 3, 4, or 5,each u is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12;* indicates a bond to NR4, anda bisected bondindicates a bond to one of the two adjacent carbon atoms.
[0145] In some embodiments, provided herein is a compound of Formula (II) or (II’), wherein:-L2-L5- is:WSGR Docket No. 60801-718.601WSGR Docket No. 60801-718.601of the two adjacent carbon atoms.WSGR Docket No. 60801-718.601
[0146] In some embodiments, provided herein is a compound of Formula (I) or (I’), wherein, wherein each * denotes the targeting moiety, and wherein the S represents a sulfur atom of the targeting moiety, and the nitrogen atoms of the fused triazole represent an azide of the targeting moiety.
[0147] In some embodiments, provided herein is a compound of Formula (II) or (IF), wherein:-L2-L5- is:wherein * indicates a bond to NR4.
[0148] In some embodiments, the compound of Formula (II) or (IF) is selected from Table 3. In some embodiments, the compound of Formula (Ila) is a compound of Table 3.
[0149] Table 3: DARI Binder-Linker-Drug Conjugate Compounds of Formula (II) orWSGR Docket No. 60801-718.601WSGR Docket No. 60801-718.601WSGR Docket No. 60801-718.601n is an integer from 1 to 20; andP2is a peptide or polypeptide that binds to a target.
[0150] In some embodiments, the compound of Formula (II) or (II’) is selected from Table 4. In some embodiments, the compound of Formula (Ila) is a compound of Table 4.
[0151] Table 4: DAR2 Binder-Linker-Drug Conjugate Compounds of Formula (II) or (II’)WSGR Docket No. 60801-718.601WSGR Docket No. 60801-718.601whereinWSGR Docket No. 60801-718.601n is an integer from 1 to 20; andP2is a peptide or polypeptide that binds to a target.
[0152] In some embodiments, P2is an antibody, or antigen-binding fragment thereof. In some embodiments, P2is a single domain antibody, a Fab, or an ScFv. In some embodiments, P2binds (preferably specifically) to a cell surface receptor. In some embodiments, P2binds (preferably specifically) to a tumor-specific target. In some embodiments, the tumor-specific target comprises an antigen on a cell surface of a tumor cell. In some embodiments, the target is present in the tumor microenvironment. In some embodiments, P2is capable of binding covalently to the target. In some embodiments, P2binds to one or more tumor antigen receptors selected from the group consisting of 5T4, B7-H3, B7-H4, BCMA, CAIX, CD123, CD19, CD20, CD22, CD30, CD33, CD79b, CEA, DLL3, EGFR, EGFRviii, FAP, FcRH5, GPR20, GUCY2C(GCC), HER2, HER3, KAAG1, LIV-1, MICA, MSLN, Mucl, PSMA, ROR1, SEZ6, and SLAMF7. In some embodiments, P2binds specifically to one of the tumor antigen receptors selected from the group consisting of 5T4, B7-H3, B7-H4, BCMA, CAIX, CD123, CD19, CD20, CD22, CD30, CD33, CD79b, CEA, DLL3, EGFR, EGFRviii, FAP, FcRH5, GPR20, GUCY2C(GCC), HER2, HER3, KAAG1, LIV-1, MICA, MSLN, Mucl, PSMA, ROR1, SEZ6, and SLAMF7.
[0153] Another aspect of the disclosure is a compound of Formula (III):R3R2OH2N''J^N'Y^P1O R1Formula (III)wherein:P1is a chemotoxin,R1is H, Ci-Ce alkyl, wherein Ci-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, -OH, -NH2, -SH, and - SCH3,R2is C1-C6 alkyl,R3is H, Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)-NHC(=O)NH2, -(Ci-C6alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)-NHC(=NH)NH2, -(Ci-C6alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O-NHC(=NH)NH2, or -(Ci-C6alkylene)-O-NHC(=NH)NHCH3, wherein Ci-Ce alkyl is optionally substituted with one, two, or three groupsWSGR Docket No. 60801-718.601selected from the group consisting of -F, phenyl, -C(=0)NH2, -C(=O)OH, - OH, -NH2, -SH, and -SCH3,or R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, and R3is Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)-NHC(=0)NH2, -(Ci-Ce alkylene)- NHC(=O)NHCH3, -(C1-C6 alkylene)-NHC(=NH)NH2, -(Ci-C6alkylene)- NHC(=NH)NHCH3, -(CI-C6alkylene)-O-NHC(=NH)NH2, or -(Ci-C6alkylene)-O-NHC(=NH)NHCH3, wherein Ci-Ce alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NHz, -SH, and -SCH3.
[0154] Another aspect of the disclosure is a compound of Formula (Illa):Formula (Illa)wherein:P1is a chemotoxin,R1is H, Ci-Ce alkyl, wherein Ci-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, -COOH, -CONH2, -OH, -NH2, -NHC(=O)NH2, -NHC(=NH)NH2, -SH, and -SCH3,; or two R1groups together form a C3-6 cycloalkyl;R2is Ci-Ce alkyl;R3is H, Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)- NHC(=O)NH2, -(CI-C6alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)- NHC(=NH)NH2, -(CI-C6alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O- NHC(=NH)NH2, or -(Ci-C6alkylene)-O-NHC(=NH)NHCH3, wherein Ci-C6alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NH2, -SH, and -SCH3;or R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, and R3is Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)-NHC(=O)NH2, -(Ci-Ce alkylene)- NHC(=O)NHCH3, -(C1-C6 alkylene)-NHC(=NH)NH2, -(Ci-C6alkylene)- NHC(=NH)NHCH3, -(CI-C6alkylene)-O-NHC(=NH)NH2, or -(Ci-C6WSGR Docket No. 60801-718.601alkylene)-0-NHC(=NH)NHCH3, wherein Ci-Ce alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NH2, -SH, and -SCH3.
[0155] In some embodiments, R1is hydrogen, R2is Ci-6 alkyl; and R3is CH2OH. In some embodiments, R1is hydrogen, R2is Ci-6 alkyl; and R3is hydrogen. In some embodiments, R1is Ci-6 alkyl optionally substituted with -COOH, -CONH2, -OH, -NH2, -NHC(=O)NH2, or -NHC(=NH)NH2; R2is Ci-6 alkyl; and R3is CH2OH. In some embodiments, R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, and R3is CH2OH. In some embodiments, two R1groups together form a C3-6 cycloalkyl; R2is C1-6 alkyl; and R3is CH2OH.
[0156] In some embodiments, the compound of Formula (III) has the structure:
[0157] In some embodiments, R3is -CH2OH. In some embodiments, R3is hydrogen.
[0158] In some embodiments, P1is a chemotoxin bonded via an amide, ester, thioester, carbamate, or carbonate bond. In some embodiments, P1is a chemotoxin bonded via an ester bond. In some embodiments, P1of Formula (III) is the same as defined for P1of Formula (I) or (II).
[0159] In some embodiments, R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, and R3is a side-chain of Vai, Ser, Lys, or Cit. In some embodiments, R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, and R3is a side-chain of Ser. In some embodiments, R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, and R3is a side-chain of Vai.
[0160] In some embodiments, R1is a side-chain of Gly, Ala, Vai, Leu, or He. In some embodiments, R1is a side-chain of Gly. In some embodiments, R1is a side-chain of Ala or Vai.
[0161] In some embodiments, R2is alkyl, [PEG]I-I2OH, [PEG]I-I2OCH3, [Sar]i-i2-N(CH3)2, or [Sar]i-i2-COOH; wherein PEG is polyethylene glycol (-OCH2CH2- or -CH2OCH2- or -CH2CH2O-) and Sar is sarcosine (-CH2CON(CH3)- or -COCH2N(CH3)- or -CON(CH3)CH2-). In some embodiments, R2is alkyl, [PEG]I-I2OH, [PEG]I-I2OCH3, [Sar]i-i2-N(CH3)2, or [Sar]i-I2-COOH; wherein PEG is polyethylene glycol and Sar is sarcosine. In some embodiments,WSGR Docket No. 60801-718.601R2is alkyl, [PEG]i-i20H, [PEGJi-nOCHs, [Sar]i-i2-N(CH3)2, or [Sar]i-i2-COOH; wherein PEG is -OCH2CH2- and Sar is -CH2CON(CH3)-.
[0162] In some embodiments, R3is a side-chain of Gly, Ala, Vai, Leu, He, Ser, Lys, or Cit. In some embodiments, R3is a side-chain of Gly, Ala, Vai, or Ser. In some embodiments, R3is a side-chain of Gly, Vai, or Ser. In some embodiments, R3is a side-chain of Gly or Ser. In some embodiments, R3is a side-chain of Ser. In some embodiments, R3is a side-chain of Gly. In some embodiments, R3is a side-chain of Lys.
[0163] In some embodiments, R1is a side-chain of Gly, Ala, Vai, Leu, or He; R2is alkyl, [PEG]i-i2OH, [PEG]i-i2OCH3, [Sar]i-i2-N(CH3)2, or [Sar]i-i2-COOH; and R3is a side-chain of Gly, Ala, Vai, Leu, He, Ser, Lys, or Cit.
[0164] In some embodiments, provided herein is a compound of Formula (III), wherein:R1is H, Ci-Ce alkyl, wherein C1-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, -OH, -NH2, -SH, and - SCH3,R2is Ci-C6alkyl, -[CH2]i-3[OCH2CH2]i-i2OH, -[CH2]i-3[OCH2CH2]i-i2OCH3, - [CH2CON(CH3)]i-i2CH3; or -[CH2CON(CH3)]i-i2CH2COOH;R3is H, Ci-Ce alkyl, (Ci-Ce alkylene)-OH , -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)-NHC(=O)NH2, -(Ci-C6alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)-NHC(=NH)NH2, -(Ci-C6alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O-NHC(=NH)NH2, or -(Ci-C6alkylene)-O-NHC(=NH)NHCH3, wherein Ci-Ce alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, -C(=O)NH2, -C(=O)OH, - OH, -NH2, -SH, and -SCH3,or R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine,wherein when R1and R2form a pyrrolidine, then R3is Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)-NHC(=O)NH2, -(Ci- Ce alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)-NHC(=NH)NH2, -(Ci-C6alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O-NHC(=NH)NH2, or -(Ci- Ce alkylene)-O-NHC(=NH)NHCH3, wherein Ci-Ce alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NH2, -SH, and -SCH3
[0165] In some embodiments, provided herein is a compound of Formula (III), wherein:R1is H or Ci-Ce alkyl;WSGR Docket No. 60801-718.601R2is Ci-Ce alkyl; andR3is H, Ci-Ce alkyl, (C1-C4 alkylene)-OH, -(C2-C6 alkylene)-NH2, -(C2-C6 alkylene)- NHC(=O)NH2, -(C2-C6 alkylene)-NHC(=O)NHCH3, -(C2-C6 alkylene)- NHC(=NH)NH2, -(C2-C6 alkylene)-NHC(=NH)NHCH3, -(C2-C6 alkylene)-O- NHC(=NH)NH2, or -(C2-C6 alkylene)-O-NHC(=NH)NHCH3.
[0166] In some embodiments, provided herein is a compound of Formula (III), wherein:R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine; andR3is Ci-Ce alkyl, (C1-C4 alkylene)-OH, -(C2-C6 alkylene)-NH2, -(C2-C6 alkylene)- NHC(=O)NH2, -(C2-C6 alkylene)-NHC(=O)NHCH3, -(C2-C6 alkylene)- NHC(=NH)NH2, -(C2-C6 alkylene)-NHC(=NH)NHCH3, -(C2-C6 alkylene)-O- NHC(=NH)NH2, or -(C2-C6 alkylene)-O-NHC(=NH)NHCH3.
[0167] In some embodiments, provided herein is a compound of Formula (III), wherein:R1is H, -CH3, -CH2CH3, -CH2CH2CIL, -CH2CH2CH2CIL, -CH(CH3)2, -C(CH3)2-, - CH2CH(CH3)CH2CH3, or -CH2CH(CH3)2,R2is -CH3, or R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine;R3is H, -CH3, -CH2CH3, -CH2CH2CIL, -CH2CH2CH2CIL, -CH(CH3)2, -C(CH3)2-, - CH2CH(CH3)2, -CH2OH, -CH2CH2OH, -CH(CH3)OH, -CH2CH2CH2NH2, - CH2CH2CH2CH2NH2, -CH2CH2CH2CH2CH2NH2, -CH2CH2CH2CH2CH2NH2, - CH2CH2NHC(=O)NH2, -CH2CH2CH2NHC(=O)NH2, - CH2CH2CH2CH2NHC(=O)NH2, -CH2CH2-NHC(=O)NHCH3, -CH2CH2CH2- NHC(=O)NHCH3, -CH2CH2CH2CH2-NHC(=O)NHCH3, -CH2CH2- NHC(=NH)NH2, -CH2CH2CH2-NHC(=NH)NH2, -CH2CH2CH2CH2- NHC(=NH)NH2, -CH2CH2-NHC(=NH)NHCH3, -CH2CH2CH2- NHC(=NH)NHCH3, -CH2CH2CH2CH2-NHC(=NH)NHCH3, -CH2CH2-O- NHC(=NH)NH2, -CH2CH2CH2-O-NHC(=NH)NH2, -CH2CH2CH2CH2-O- NHC(=NH)NH2, -CH2CH2-O-NHC(=NH)NHCH3, -CH2CH2CH2-O- NHC(=NH)NHCH3, or -CH2CH2CH2CH2-O-NHC(=NH)NHCH3,wherein when R1and R2form a pyrrolidine, then R3is -CH3, -CFbCHs, - CH2CH2CIL, -CH2CH2CH2CIL, -CH(CH3)2, -C(CH3)2-, -CH2CH(CH3)2, - CH2OH, -CH2CH2OH, -CH(CH3)OH, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, - CH2CH2CH2CH2CH2NH2, -CH2CH2CH2CH2CH2NH2, -CH2CH2NHC(=O)NH2, - CH2CH2CH2NHC(=O)NH2, -CH2CH2CH2CH2NHC(=O)NH2, -CH2CH2-WSGR Docket No. 60801-718.601NHC(=O)NHCH3, -CH2CH2CH2-NHC(=O)NHCH3, -CH2CH2CH2CH2- NHC(=O)NHCH3, -CH2CH2-NHC(=NH)NH2, -CH2CH2CH2-NHC(=NH)NH2, - CH2CH2CH2CH2-NHC(=NH)NH2, -CH2CH2-NHC(=NH)NHCH3, -CH2CH2CH2- NHC(=NH)NHCH3, -CH2CH2CH2CH2-NHC(=NH)NHCH3, -CH2CH2-O- NHC(=NH)NH2, -CH2CH2CH2-O-NHC(=NH)NH2, -CH2CH2CH2CH2-O- NHC(=NH)NH2, -CH2CH2-O-NHC(=NH)NHCH3, -CH2CH2CH2-O- NHC(=NH)NHCH3, or -CH2CH2CH2CH2-O-NHC(=NH)NHCH3.
[0168] In some embodiments, provided herein is a compound of Formula (III), wherein:R1is H, -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH(CH3)2, -C(CH3)2-, - CH2CH(CH3)CH2CH3, or -CH2CH(CH3)2,R2is -CH3,R3is H, -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH(CH3)2, -C(CH3)2-, - CH2CH(CH3)2, -CH2OH, -CH2CH2OH, -CH(CH3)OH, -CH2CH2CH2NH2, - CH2CH2CH2CH2NH2, -CH2CH2CH2CH2CH2NH2, -CH2CH2CH2CH2CH2NH2, - CH2CH2NHC(=O)NH2, -CH2CH2CH2NHC(=O)NH2, - CH2CH2CH2CH2NHC(=O)NH2, -CH2CH2-NHC(=O)NHCH3, -CH2CH2CH2- NHC(=O)NHCH3, -CH2CH2CH2CH2-NHC(=O)NHCH3, -CH2CH2- NHC(=NH)NH2, -CH2CH2CH2-NHC(=NH)NH2, -CH2CH2CH2CH2- NHC(=NH)NH2, -CH2CH2-NHC(=NH)NHCH3, -CH2CH2CH2- NHC(=NH)NHCH3, -CH2CH2CH2CH2-NHC(=NH)NHCH3, -CH2CH2-O- NHC(=NH)NH2, -CH2CH2CH2-O-NHC(=NH)NH2, -CH2CH2CH2CH2-O- NHC(=NH)NH2, -CH2CH2-O-NHC(=NH)NHCH3, -CH2CH2CH2-O- NHC(=NH)NHCH3, or -CH2CH2CH2CH2-O-NHC(=NH)NHCH3.
[0169] In some embodiments, provided herein is a compound of Formula (III), wherein:R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine; andR3is -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH(CH3)2, -C(CH3)2-, - CH2CH(CH3)2, -CH2OH, -CH2CH2OH, -CH(CH3)OH, -CH2CH2CH2NH2, - CH2CH2CH2CH2NH2, -CH2CH2CH2CH2CH2NH2, -CH2CH2CH2CH2CH2NH2, - CH2CH2NHC(=O)NH2, -CH2CH2CH2NHC(=O)NH2, - CH2CH2CH2CH2NHC(=O)NH2, -CH2CH2-NHC(=O)NHCH3, -CH2CH2CH2- NHC(=O)NHCH3, -CH2CH2CH2CH2-NHC(=O)NHCH3, -CH2CH2- NHC(=NH)NH2, -CH2CH2CH2-NHC(=NH)NH2, -CH2CH2CH2CH2- NHC(=NH)NH2, -CH2CH2-NHC(=NH)NHCH3, -CH2CH2CH2-WSGR Docket No. 60801-718.601NHC(=NH)NHCH3, -CH2CH2CH2CH2-NHC(=NH)NHCH3, -CH2CH2-O- NHC(=NH)NH2, -CH2CH2CH2-O-NHC(=NH)NH2, -CH2CH2CH2CH2-O- NHC(=NH)NH2, -CH2CH2-O-NHC(=NH)NHCH3, -CH2CH2CH2-O- NHC(=NH)NHCH3, or -CH2CH2CH2CH2-O-NHC(=NH)NHCH3.
[0170] In some embodiments, provided herein is a compound of Formula (III), wherein:R1is H, -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH(CH3)2, -C(CH3)2-, - CH2CH(CH3)CH2CH3, or -CH2CH(CH3)2,R2is -CH3, or R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine,R3is H, -CH3, -CH2OH, -CH2CH2OH, or -CH(CH3)OH,wherein when R1and R2form a pyrrolidine, then R3is -CH2OH, -CH2CH2OH, or - CH(CH3)OH.
[0171] In some embodiments, provided herein is a compound of Formula (III), wherein:R1is H, -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH(CH3)2, -C(CH3)2-, - CH2CH(CH3)CH2CH3, or -CH2CH(CH3)2,R2is -CH3, andR3is H, -CH3, -CH2OH, -CH2CH2OH, or -CH(CH3)OH.
[0172] In some embodiments, provided herein is a compound of Formula (III), wherein:R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, andR3is -CH2OH, -CH2CH2OH, or -CH(CH3)OH.
[0173] In some embodiments,R1is C1-6 alkyl,R2is Ci -6 alkyl, andR3is -CH2OH.
[0174] In some embodiments, provided herein is a compound of Formula (III), wherein:R1is H, -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH(CH3)2, -C(CH3)2-, - CH2CH(CH3)CH2CH3, or -CH2CH(CH3)2,R2is -CH3, andR3is -CH2OH.
[0175] In some embodiments,R1is hydrogen,R2is C1-6 alkyl, andR3is -CH2OH.WSGR Docket No. 60801-718.601
[0176] In some embodiments, provided herein is a compound of Formula (III), wherein:R1is H,R2is -CH3, andR3is -CH2OH.
[0177] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:R1is -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -CH2CH(CH3)CH2CH3, or - CH2CH(CH3)2,R2is -CH3, andR3is -CH2OH.
[0178] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:R1is H, -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH(CH3)2, -C(CH3)2-, - CH2CH(CH3)CH2CH3, or -CH2CH(CH3)2,R2is -CH3, andR3is H.
[0179] In some embodiments,R1is hydrogen,R2is C1-6 alkyl, andR3is hydrogen.
[0180] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:R1is H,R2is -CH3, andR3is H.
[0181] In some embodiments, provided herein is a compound of Formula (I) or (II), wherein:R1is -CH3, -CH2CH3, -CH2CH2CH3, -CH2CH2CH2CH3, -CH(CH3)2, -C(CH3)2-, - CH2CH(CH3)CH2CH3, or -CH2CH(CH3)2,R2is -CH3, andR3is H.
[0182] In some embodiments, P1is a chemotoxin.
[0183] In some embodiments, the compound of Formula (III) is:WSGR Docket No. 60801-718.601or a pharmaceutically acceptable salt thereof.
[0184] In some embodiments, the compound of Formula (III) is:or a pharmaceutically acceptable salt thereof.
[0185] In some embodiments, provided herein is a compound of Formula (I), (I)’, (II), (IF), or (III), wherein P1is a chemotoxin.
[0186] In some embodiments, the chemotoxin comprises an antimitotic, DNA-damaging agent, transcriptional inhibitor, kinase inhibitor or combination thereof. In some embodiments, provided herein is a compound of Formula (I), (I)’, (II), (IF), or (III), wherein P1is an antimitotic, DNA-damaging agent, transcriptional inhibitor, kinase inhibitor or combination thereof.
[0187] In some embodiments, P1is an antimitotic. In some embodiments, P1is an DNA-damaging agent. In some embodiments, P1is a transcriptional inhibitor. In some embodiments,WSGR Docket No. 60801-718.601P1is a kinase inhibitor. In some embodiments, the antimitotic is a maytansinoid, taxane, auristatin, alkaloid, tub lysin, or epothilone. In some embodiments, P1is a maytansinoid, taxane, auristatin, alkaloid, tubulysin, or epothilone. In some embodiments, P1is a maytansinoid. In some embodiments, P1is maytansine, DM1 (mertansine), or DM4, bonded to the linker at a hydroxy group (ROH) via an ester bond (-C(=O)-OR), or at a thiol group (RSH)
[0188] In some embodiments, P1is a taxane. In some embodiments, P1is an auristatin. In some embodiments, P1is an alkaloid. In some embodiments, P1is a tubulysin. In some embodiments, P1is an epothilone.
[0189] In some embodiments, the DNA-damaging agent, transcriptional inhibitor, or combination thereof is a DNA polymerase inhibitor, DNA replication inhibitor, topoisomerase inhibitor, or cytotoxic antibiotic. In some embodiments, P1is a DNA polymerase inhibitor, DNA replication inhibitor, topoisomerase inhibitor, or cytotoxic antibiotic. In some embodiments, P1is a DNA polymerase inhibitor. In some embodiments, P1is a DNA replication inhibitor. In some embodiments, P1is a topoisomerase inhibitor. In some embodiments, P1is a cytotoxic antibiotic.
[0190] In some embodiments, the kinase inhibitor is an inhibitor of a cytoplasmic tyrosine kinase (CTK), a serine / threonine kinase (S / T Kinase), a lipid kinase (LK), or a receptor tyrosine kinase (RTK). In some embodiments, P1is an inhibitor of a cytoplasmic tyrosine kinase (CTK), a serine / threonine kinase (S / T Kinase), a lipid kinase (LK), or a receptor tyrosine kinase (RTK). In some embodiments, P1is an inhibitor of a cytoplasmic tyrosine kinase (CTK). In some embodiments, P1is an inhibitor of a serine / threonine kinase (S / T Kinase). In some embodiments, P1is an inhibitor of a lipid kinase (LK). In some embodiments, P1is an inhibitor of a receptor tyrosine kinase (RTK).
[0191] In some embodiments, the chemotoxin (P1) comprises mertansine, emtansine, paclitaxel, docetaxel, ellipticine, a vinca alkaloid, epothilone A, epothilone B, epothilone C,WSGR Docket No. 60801-718.601epothilone D, epothilone E, epothilone F, utidelone, monomethyl auristatin E or monomethyl auri statin F.
[0192] In some embodiments, the chemotoxin (P1) is or comprises a maytansinoid, taxane, auristatin, camptothecin, vinca alkaloid, tubulysin, or epothilone. In some embodiments, the chemotoxin (P1) is or comprises maytansine, mertansine (DM1), DM4, paclitaxel, docetaxel, ellipticine, a vinca alkaloid, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, utidelone, camptothecin, 7-ethyl camptothecin, exatecan, topotecan, irinotecan, belotecan, SN-38, Dxd, auristatin E, monomethyl auristatin E, auristatin F, or monomethyl auristatin F.
[0193] In some embodiments, P1is monomethyl auristatin E (MMAE), auristatin E (AE), dasatinib, maytansine, mertansine, DM4, a camptothecin, or an anthracycline. In some embodiments, P1is:WSGR Docket No. 60801-718.601wherein:Ralis H, CH3, or CH2OH;Rblis H or F;Rb2is H, CH3, OH, or OC(=O)-G-G; wherein each G is piperidine;Rb3is H or Ci-Ce alkylene-N(CH3)2; andRb4is H, Ci-C6alkyl, or C1-6 alkylene-NH-CH(CH3)2;or Rb3and Rb4taken together form a 6-membered carbocycle substituted with - NH2 or -NHCOCH2OH.
[0194] In some embodiments, each P1is independently selected from the group consisting of:WSGR Docket No. 60801-718.601WSGR Docket No. 60801-718.601WSGR Docket No. 60801-718.601
[0195] In some embodiments, each P1is independently selected from the group consistingDefinitionsWSGR Docket No. 60801-718.601
[0197] To facilitate understanding of the disclosure set forth herein, a number of terms are defined below.
[0198] As used herein, Ci-Cxincludes C1-C2, C1-C3 . . . Ci-Cx. By way of example only, a group designated as "Ci-Ce" indicates that there are one to six carbon atoms in the moiety, e.g., groups containing 1 carbon atom, 2 carbon atoms, 3 carbon atoms or 4 carbon atoms. Thus, by way of example only, "C1-C4 alkyl" indicates that there are one to four carbon atoms in the alkyl group, e.g., the alkyl group is selected from among methyl, ethyl, propyl, Ao-propyl, n-butyl, Ao-butyl, .scc-butyl, and / -butyl.
[0199] An “alkyl” group refers to an aliphatic hydrocarbon group. The alkyl group is branched or straight chain. In some embodiments, the “alkyl” group has 1 to 10 carbon atoms, e.g., a Ci-Cioalkyl. Whenever it appears herein, a numerical range such as “1 to 10” refers to each integer in the given range; e.g., “1 to 10 carbon atoms” means that the alkyl group consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, although the present definition also covers the occurrence of the term “alkyl” where no numerical range is designated. In some embodiments, an alkyl is a Ci-Cealkyl. In one aspect the alkyl is methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, or t-butyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tertiary butyl, pentyl, neopentyl, or hexyl.
[0200] An “alkylene” group refers to a divalent alkyl radical. Any of the above mentioned monovalent alkyl groups may be an alkylene by abstraction of a second hydrogen atom from the alkyl. In some embodiments, an alkylene is a Ci-Cealkylene. In other embodiments, an alkylene is a Ci-C4alkylene. Typical alkylene groups include, but not limited to, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, and the like. In some embodiments, an alkylene is -CH2-.
[0201] An “alkoxy” group refers to a -O(alkyl) group, where alkyl is as defined herein. Examples of alkoxy groups include -OCH3, -OCH2CH3, -OCH2CH2CH3, -OC(CH3)3, and the like.
[0202] An “hydroxyalkyl” refers to an alkyl in which one hydrogen atom is replaced by a hydroxyl. In some embodiments, a hydroxyalkyl is a Ci-C4hydroxy alkyl. Typical hydroxyalkyl groups include, but not limited to, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, -CH2CH2CH2CH2OH, -C(CH3)2OH, and the like.
[0203] The term “alkylamine” refers to the -N(alkyl)xHygroup, where x is 0 and y is 2, or where x is 1 and y is 1, or where x is 2 and y is 0.WSGR Docket No. 60801-718.601
[0204] An “aminoalkyl” refers to an alkyl in which one hydrogen atom is replaced by an amino. In some embodiments, aminoalkyl is a Ci-C4aminoalkyl. Typical aminoalkyl groups include, but not limited to, -CH2NH2, -CH2CH2NH2, -CH2CH2CH2NH2, -CH2CH2CH2CH2NH2, -C(CH3)2NH2, and the like.
[0205] The term “subject” refers to an animal, including, but not limited to, a primate (e.g., human), cow, pig, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. The terms “subject” and “patient” are used interchangeably herein in reference, for example, to a mammalian subject, such as a human subject, in one embodiment, a human.
[0206] The terms “treat,” “treating,” and “treatment” are meant to include alleviating or abrogating a disorder, disease, or condition, or one or more of the symptoms associated with the disorder, disease, or condition; or alleviating or eradicating the cause(s) of the disorder, disease, or condition itself.
[0207] The terms “prevent,” “preventing,” and “prevention” are meant to include a method of delaying and / or precluding the onset of a disorder, disease, or condition, and / or its attendant symptoms; barring a subject from acquiring a disorder, disease, or condition; or reducing a subject’s risk of acquiring a disorder, disease, or condition.
[0208] The terms “therapeutically effective amount” and “effective amount” are meant to include the amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disorder, disease, or condition being treated. The terms “therapeutically effective amount” or “effective amount” also refer to the amount of a compound that is sufficient to elicit the biological or medical response of a biological molecule (e.g., a protein, enzyme, RNA, orDNA), cell, tissue, system, animal, or human, which is being sought by a researcher, veterinarian, medical doctor, or clinician.
[0209] The terms “pharmaceutically acceptable carrier,” “pharmaceutically acceptable excipient,” “physiologically acceptable carrier,” and “physiologically acceptable excipient” refer to a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. In one embodiment, each component is “pharmaceutically acceptable” in the sense of being compatible with other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. See, Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 5th Edition, Rowe el al.,WSGR Docket No. 60801-718.601Eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd Edition, Ash and Ash Eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd Edition, Gibson Ed., CRC Press LLC: Boca Raton, FL, 2009.
[0210] The terms “about” and “approximately” mean an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the terms “about” and “approximately” mean within 1, 2, 3, or 4 standard deviations. In certain embodiments, the terms “about” and “approximately” mean within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of a given value or range.
[0211] As used herein, the term “payload” generally refers a chemical group that is covalently bonded to a targeting moiety, where the payload can be delivered to a target by virtue of its conjugation to said targeting moiety. In the context of the present disclosure, a payload may be a drug (i.e., a therapeutic payload) or a detectable agent as defined herein.
[0212] As used herein, the terms “ADC” and “antibody -drug conjugate” generally refer to conjugates of a small molecule or peptide compound, conjugated via a linker described herein, to a peptide or polypeptide that binds a target protein (e.g., a cell-surface or transmembrane receptor / protein). The term “ADC” may be used generically to encompass conjugates that do not possess the secondary and / or quaternary structure of a prototypical antibody-drug conjugate. For example, ADC is intended to encompass single-domain antibodies (sdAbs) and single-domain antibody-drug conjugates (sdADCs). Analogously, ADC also encompasses nanobody-drug conjugates, wherein the “antibody” is a nanobody. Moreover, the term is intended to encompass both naturally occurring peptides and polypeptides (e.g., nanobodies, sdAbs, antibodies), and those comprising unnatural amino acids (UAAs). In certain uses, the term ADC refers to a conjugate of a compound disclosed or described herein with a nanobody or sdAb comprising an unnatural amino acid within or proximal to CDR or binding site -particularly an UAA that covalently reacts with a target residue within or proximal to the binding site interface (e.g., as described in WO2023 / 122753).
[0213] The terms “drug,” and “therapeutic payload,” generally refer to a compound, or a pharmaceutical composition thereof, which is administered to a subject for treating, preventing, or ameliorating one or more symptoms of a disorder, disease, or condition, such as a hyperproliferative disease or disorder. The therapeutic payload may be, for example, a cytotoxic or cytostatic compound. Therapeutic payloads may be effective in killing or slowing the growth of cancer cells. In some embodiments, the therapeutic payload is aWSGR Docket No. 60801-718.601chemotherapeutic agent. In some embodiments, the therapeutic payload is an auristatin or camptothecin compound. In some embodiments, the therapeutic payload is a chemotoxin. Specific examples of therapeutic payloads used in accordance with the disclosure are provided throughout the present disclosure.
[0214] As used herein, the term “spacer” generally refers to a chemical group that conjoins or links two or more covalently bonded groups. Unless otherwise specified, a spacer could be a bivalent group comprising alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups, or any combination thereof. A spacer may, for example, be a heteroalkyl ring comprising a polyether (e.g., PEG), polyamine, polyamide, or alkylene group. Unless otherwise specified, a spacer may be a bond. A spacer linking two elements may have between 1 and 100 non-hydrogen atoms selected from C, N, O, S, and P. A spacer may comprise a substituted or unsubstituted Ci-ioo alkyl or 1 to 100-membered heteroalkyl chain, each of which may be substituted by carbonyls, hydroxyls, amines, acids, amides, or other side-chains as functional modifications to the spacer. A spacer may comprise, for example, a Ci-ioo alkylene main chain, optionally substituted by oxo (=0), amino (-NH2), alkylamino (C1-6 alkyl-NEb), hydroxyalkyl (C1-6 alkyl-OH), or -COOH. A spacer may comprise a heteroalkyl chain (e.g., a combination of alkylene units and heteroatoms selected from O, N, S, and P, and optionally substituted with one or more oxo groups, which may be standalone as carbonyls, or adjacent to heteroatoms thereby forming amides, esters, thioesters, carbamates, carboxamides, and the like. In some embodiments, a spacer comprises a PEG1-12 group, and optionally additional spacer elements such as alkylene, carbonyl, or amide units, to conjoin two (or more) chemical groups. As used herein, the term “first spacer” and “L3” may be substituted unless otherwise specified, just as “second spacer” and “L2” may similarly be substituted unless otherwise specified. In some embodiments, the “spacer” or “first spacer” or “second spacer” is -(LS)I-2O-, each Lsis independently -C1-2 alkylene-O-, -O-C1-2 alkylene-, -C(0)NH-, -NHC(O)-, - C1-2 alkylene-C(O)NH-, - C1-2 alkylene-NHC(O)-, -O-, -S-, phenylene, -NH-, -NHC(O)O-, -OC(O)NH-, or -(CRS2)V-, wherein each Rsis independently hydrogen, C1-6 alkyl, -C1-5 alkylene-OH, -C1-5 alkylene-NEb, -C1-5 alkylene-CONEE, -C1-5 alkylene-COOH, or -OH, -NH2, -CONH2, -COOH, and v is 1-20.
[0215] As used herein, a “reactive group” or “reactive moiety” may refer to a “moiety capable of reacting with a thiol or an amine side-chain of a peptide or polypeptide.” In some embodiments, the reactive group is an electrophilic group. In some embodiments, the reactive group corresponds to R5, which forms a linker (L5) with a targeting moiety (e.g., a peptide or polypeptide) after reacting with (conjugating to), for example, a thiol of a cysteine side-chainWSGR Docket No. 60801-718.601or amine of a lysine side-chain of the targeting peptide or polypeptide. Alternatively, a reactive group may comprise a cyclooctyne or azide, each of which may be suitable reactive groups for strain-promoted azide-alkyne conjugation (SPAAC) chemistry. In some embodiments, the reactive group comprises an alkyne, and the targeting group is an antibody or polypeptide comprising one or more amino acids comprising an azide side-chain. In some embodiments, the reactive group comprises a halogen (e.g., an acyl halogen). For example, in some embodiments, the reactive group is a acyl halide selected from C(O)CH2Br, C(0)CH2l, and C(O)CH2C1. In some embodiments, the reactive group comprises or is an oxime (-O-NH2) group. In some embodiments, the reactive group is an N-hydroxysuccinimide ester (e.g.,, wherein * is an amine of the targeting moiety, e.g., an amine of a lysine of an antibody).
[0216] In some embodiments, the reactive groups comprises a maleimide group. In some embodiments, the maleimide group is also referred to as a maleimide spacer. In some embodiments, the maleimide group further comprises a caproic acid, forming maleimidocaproyl (me). In some embodiments, the linker comprises maleimidocaproyl (me). In some embodiments, linker is maleimidocaproyl (me). In some embodiments, the maleimide group comprises a maleimidomethyl group, such as succinimidyl-4-(N-maleimidomethyl)cyclohexane-l -carboxylate (sMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-l -carboxylate (sulfo-sMCC) described above. In some embodiments, maleimido is abbreviated MA. For example, in MA-EVCit-SerSar-MMAE (Compound 8) and analogs thereof, MA corresponds to a maleimido group.
[0217] In some embodiments, the maleimido group is or comprises. In someembodiments, the maleimido group or MA group is or comprises:In some embodiments, the maleimido group is or comprisesIn someWSGR Docket No. 60801-718.601embodiments, the maleimido group or MDPR group is or comprises:
[0218] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some embodiments, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide to provide intramolecular catalysis of thiosuccinimide ring hydrolysis, thereby eliminating maleimide from undergoing an elimination reaction through a retro-Michael reaction. In some embodiments, the selfstabilizing maleimide is a maleimide group described in Lyon, et al., “Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates,” Nat. Biotechnol. 32(10): 1059-1062 (2014). In some embodiments, the linker comprises a self-stabilizing maleimide. In some embodiments, the linker is a self-stabilizing maleimide.
[0219] In some embodiments, a reactive group comprises an alkyne. In some embodiments, a reactive group comprises a strained alkyne, e.g., a cyclooctyne or a fused cyclooctyne. In some embodiments, the reactive group comprises a dibenzoannulated cyclooctyne (DIBO), dibenzocyclooctyne (DBCO) amine, aza-dibenzocyclooctyne (DIBAC), or bicyclononyne (BCN). In some embodiments, a reactive group comprises an azide. In some embodiments, the reactive group comprises a strained cycloalkyne group capable of reacting with an azide sidechain of a targeting moiety. In some embodiments, the reactive group comprises a SPAAC-reactive group. In some embodiments, the reactive group comprises a thiol -reactive group.
[0220] In some embodiments, the reactive group is as shown in the following scheme:" "WSGR Docket No. 60801-718.601wherein the black circle is a targeting moiety,the reactive group is the maleimide or cyclooctyne (where X is nitrogen or carbon); and L2 is a second spacer of Formula (I), (la), (II), or (Ila).
[0221] As used herein, the term “detectable agent” generally refers to a compound or a monovalent radical thereof (e.g., bonded to a linker group disclosed herein), capable of being detected by standard medical or laboratory means (e.g., spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means such as color contrast, fluorescence, luminescence, phosphorescence, radioactive decay, etc.).
[0222] For example, useful detectable agents include 18F, 32P, 33P, 45Ti, 47Sc, 52Fe, 59Fe, 62Cu, 64Cu, 67Cu, 67Ga, 68Ga, 77As, 86Y, 90Y, 89Sr, 89Zr, 94Tc, 99mTc, 99Mo, 105Pd, 105Rh, lllAg, Ulin, 1231, 1241, 1251, 1311, 142Pr, 149Pm, 153Sm, 161Tb, 166Dy, I66H0, 169Er, 175Lu, 177Lu, 186Re, 188Re, 189Re, 194Ir, 198Au, 199Au, 211 At, 211Pb, 212Bi, 212Pb, 213Bi, 223Ra, 225Ac, 153Sm, 177Lu, 90Y, 1311, 149Tb, 212Pb / 212Bi, 213Bi, 227Th, Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, 32P, fluorophore (e.g. fluorescent dyes), electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, paramagnetic molecules, paramagnetic nanoparticles, ultrasmall superparamagnetic iron oxide ("USPIO") nanoparticles, USPIO nanoparticle aggregates, superparamagnetic iron oxide ("SPIO") nanoparticles, SPIO nanoparticle aggregates, monocrystalline iron oxide nanoparticles, monocrystalline iron oxide, nanoparticle contrast agents, liposomes or other delivery vehicles containing Gadolinium chelate ("Gd-chelate") molecules, Gadolinium, radioisotopes, radionuclides (e.g. carbon-1 1, nitrogen-13, oxygen-15, fluorine-18, rubidium-82), fluorodeoxyglucose (e.g. fluorine-18 labeled), any gamma ray emitting radionuclides, positron emitting radionuclide, radiolabeled glucose, radiolabeled water, radiolabeled ammonia, biocolloids, microbubbles (e.g. including microbubble shells including albumin, galactose, lipid, and / or polymers; microbubble gas core including air, heavy gas(es), perfluorocarbon, nitrogen, octafluoropropane, perflexane lipid microsphere, perflutren, etc.), iodinated contrast agents (e.g. iohexol, iodixanol, ioversol, iopamidol, ioxilan, iopromide, diatrizoate, metrizoate, ioxaglate), barium sulfate, thorium dioxide, gold, gold nanoparticles, gold nanoparticle aggregates, fluorophores, two-photon fluorophores, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide. A detectable moiety is a monovalent detectable agent or a detectable agent capable of forming a bond with another composition.WSGR Docket No. 60801-718.601
[0223] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may in embodiments be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. A polypeptide or peptide comprises at least one amino acid, which is generally bonded at both the N- and C-termini to an adjacent amino acid, thereby forming the peptide or polypeptide. In sufficiently large, functional assemblies, polypeptides are referred to as proteins. In some embodiments, a peptide or polypeptide refers to an antibody, or to an antigen-binding fragment of an antibody. In some embodiments, polypeptide or peptide refers to a cysteine or a lysine amino acid, or to an unnatural amino acid (UAA) such as those disclosed herein, bonded on either end to an adjacent amino acid, thereby forming said peptide or polypeptide (or antibody or antigen-binding fragment, as the case may be). In some embodiments, the peptide or polypeptide that binds to a target is a single domain antibody (sdAb). In some embodiments, the peptide or polypeptide that binds to a target is a single domain antibody (sdAb) described in WO2023122753 Al, which is herein incorporated by reference in its entirety. In some instances, a conjugate comprises a single domain antibody (sdAb), and at least one unnatural amino acid (UAA) within or in the proximity of a CDR region within the sdAb. In some instances, a conjugate comprises a single domain antibody (sdAb), and at least one unnatural amino acid (UAA) within or in the proximity of a CDR region within the sdAb configured to covalently react with the target (i.e., at a residue proximal to the binding site). In some embodiments, the antibody (Ab) or single-domain antibody (sdAb) is a nanobody.
[0224] Targeting domains (e.g., the peptide or polypeptide that binds to a target) may guide payloads attached to the conjugate to a target. In some instances, the targeting domain comprises an antibody or fragment thereof. In some instances, a targeting domain comprises a monospecific Fab2, bispecific Fab2, trispecific Fab3, monovalent IgG, scFv, bispecific diabody, trispecific triabody, scFv-Fc, nanobody (i.e., single domain antibody, sdAb), minibody, IgNAR, V-NAR, hcIgG, VhH, or peptibody, DARPin, monobody / FN3, VNAR, Repebody, Darpin. In some instances, the targeting domain comprises a nanobody. In some embodiments, the targeting domain comprises a single domain antibody (sdAb). In some instances, a targeting domain comprises one or more CDR regions. In some embodiments, the targeting domain binds to a cell surface molecule. In some instances, a conjugate is biparatopic and comprises a first targeting domain and a second targeting domain. In some embodiments,WSGR Docket No. 60801-718.601the first targeting domain and the second targeting domain each comprise an antibody or antigen binding fragment, and the structure of such antibody or antigen binding fragment may be the same or different. In some instances, the first targeting domain can be a single chain (e.g., Fv) antibody fragment and the second targeting domain can be a sdAb, or in other instances, the first targeting domain can be a sdAb and the second targeting domain can be a sdAb. In some instances, the targeting domain (e.g., the first targeting domain and / or the second targeting domain) is an antibody mimic such as an affibody, DARPin or a mini binder.
[0225] As used herein, the term “antibody” or “antibodies” generally refers to large, complex proteins with a specific structure that imbues it with certain functional properties, particularly with respect to its ability to bind to a target antigen. A natural antibody molecule contains two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. Each light chain and heavy chain in turn consists of two regions: a variable ("V") region involved in binding the target antigen, and a constant ("C") region that interacts with other components of the immune system. The light and heavy chain variable regions come together in 3 -dimensional space to form a variable region that binds the antigen (for example, a receptor on the surface of a cell). Within each light or heavy chain variable region, there are three short segments (averaging 10 amino acids in length) called the complementarity determining regions ("CDRs"). The six CDRs in an antibody variable domain (three from the light chain and three from the heavy chain) fold up together in 3 -dimensional space to form the actual antibody binding site which docks onto the target antigen. The position and length of the CDRs have been precisely defined by Kabat, E. et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1983, 1987. The part of a variable region not contained in the CDRs is called the framework ("FR"), which forms the environment for the CDRs. The term "antibody" is used according to its commonly known meaning in the art. Antibodies exist, e.g., as intact immunoglobulins or as a number of well -characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragmentsWSGR Docket No. 60801-718.601either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (e.g., McCafferty et al., Nature, 348:552-554 (1990)).
[0226] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively. The Fc (i.e. fragment crystallizable region) is the “base” or "tail" of an immunoglobulin and is typically composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. By binding to specific proteins the Fc region ensures that each antibody generates an appropriate immune response for a given antigen. The Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins.
[0227] An “antigen binding fragment” as provided herein refers to a polypeptide capable of binding to an antigen and including one or more structural domains of an antibody or fragment thereof. Nonlimiting examples of antibody variants include single-domain antibodies (nanobodies), affibodies (polypeptides smaller than monoclonal antibodies (e.g., about 6kDA) and capable of binding antigens with high affinity and imitating monoclonal antibodies, monospecific Fab2, bispecific Fab2, trispecific Fab3, monovalent IgGs, scFv, bispecific diabodies, trispecific triabodies, scFv-Fc, minibodies, IgNAR, V-NAR, hcIgG, VhH, or peptibodies. A “peptibody” as provided herein refers to a peptide moiety attached (through a covalent or non-covalent linker) to the Fc domain of an antibody. Further non-limiting examples of antibody variants known in the art include antibodies produced by cartilaginous fish or camelids. A general description of antibodies from camelids and the variable regions thereof and methods for their production, isolation, and use may be found in references WO 97 / 49805 and WO 97 / 49805, which are incorporated, by reference herein in their entirety and for all purposes. Likewise, antibodies from cartilaginous fish and the variable regions thereof and methods for their production, isolation, and use may be found in W02005 / 118629, which is incorporated by reference herein in its entirety and for all purposes.
[0228] A “single-domain antibody” or “nanobody” interchangeably refers to an antibody fragment having a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. In some embodiments, the single domain antibodyWSGR Docket No. 60801-718.601is a human or humanized single domain antibody. In some embodiments, the single domain antibody is a camelid single domain antibody.
[0229] The term "antigen" as provided herein refers to molecules capable of binding to the antibody binding domain provided herein. An "antigen binding domain" as provided herein is a region of an antibody that binds to an antigen (epitope). As described above, the antigen binding domain may include one constant and one variable domain of each of the heavy and the light chain (VL, VH, CL and CHI, respectively). In embodiments, the antigen binding domain includes a light chain variable domain and a heavy chain variable domain. In embodiments, the antigen binding domain includes light chain variable domain and does not include a heavy chain variable domain and / or a heavy chain constant domain. The paratope or antigen-binding site is formed on the N-terminus of the antigen binding domain. The two variable domains of an antigen binding domain may bind the epitope of an antigen. Antibodies exist, for example, as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)’2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)’2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)’2 dimer into an Fab’ monomer. The Fab’ monomer is essentially the antigen binding portion with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries.
[0230] As used herein, a “chemotoxin” may be used interchangeably with terms such as “chemotherapeutic” or “cytotoxin,” all of which generally refer to a compound that is toxic or inhibitory to the growth of a cell, preferably a cancer cell of a mammalian subject. As used herein, a chemotoxin may or may not be specific or selective for targeting diseased cells. Generally, a chemotoxin is conjugated to a peptide that binds to a target, such as an antibody, thereby limiting the toxicity of the chemotoxin to cells expressing a target to which the peptide binds.
[0231] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to theWSGR Docket No. 60801-718.601naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, g-carboxy glutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine, citrulline) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
[0232] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
[0233] The term “amino acid side chain” refers to the functional substituent contained on amino acids. For example, an amino acid side chain may be the side chain of a naturally occurring amino acid. Naturally occurring amino acids are those encoded by the genetic code (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine), as well as those amino acids that are later modified, e.g., hydroxyproline, g-carboxyglutamate, and O-phosphoserine. In aspects, the amino acid side chain may be a non-natural amino acid side chain. In aspects, the amino acid side chain is H,
[0234] Examples of unnatural amino acid side chains, as used herein, include but are not limited to the following:WSGR Docket No. 60801-718.601
[0235] The term “non-natural amino acid ” or “unnatural amino acid ” or “Uaa”, or “non naturally occurring amino acid” refers to the functional substituent of compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium, allylalanine, 2-aminoisobutryric acid. The term may refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature, e.g. amino acid residues containing arylamide, vinyl sulfonamide, sulfonyl fluoride, aryl fluoro sulfate, aryl sulfonyl fluoride, aryl fluorosulfate, and 4-sulfotetrafluorophenyl (STP) esters. Non-natural amino acids are non-proteinogenic amino acids that either occur naturally or are chemically synthesized. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Non-limiting examples include exo-cis-3-aminobicyclo[2.2.1]hept-5-ene-2-carboxylic acid hydrochloride, cis-2-aminocycloheptanecarboxylic acid hydrochloride, cis-6-Amino-3- cyclohexene-1 -carboxylic acid hydrochloride, cis-2-amino-2-methylcyclohexanecarboxylic acid hydrochloride, cis-2-amino-2-methylcyclopentanecarboxylic acid hydrochloride, 2-(Bocaminomethyl), benzoic acid, 2-(Boc-amino)octanedioic acid, Boc-4,5-dehydro-Leu-OH (dicyclohexylammonium), Boc-4-(Fmoc-amino)-L-phenylalanine, Boc-P-Homopyr-OH, Boc- (2-indanyl)-Gly-OH, 4-Boc-3 -morpholineacetic acid, 4-Boc-3 -morpholineacetic acid, Boc-pentafluoro- D-phenyl alanine, Boc-pentafluoro-L-phenylalanine, Boc-Phe(2-Br)-OH, Boc-Phe(4-Br)-OH, Boc-D-Phe(4-Br)-OH, Boc-D-Phe(3-Cl)-OH, Boc-Phe(4-NH2)-OH, Boc-Phe(3-NH2)-OH, Boc-Phe(3,5-F2)-OH, 2-(4-Boc-piperazino)-2-(3,4-dimethoxyphenyl)acetic acid purum, 2-(4-Boc-piperazino)-2-(2-fluorophenyl)acetic acid purum, 2-(4-Boc-piperazino)-2-(3-fluorophenyl)acetic acid purum, 2-(4-Boc-piperazino)-2-(4-fluorophenyl)acetic acid purum, 2-(4-Boc-piperazino)-2-(4-methoxyphenyl)acetic acid purum, 2-(4-Boc-piperazino)-2-phenylacetic acid purum, 2-(4-Boc-piperazino)-2-(3-pyridyl)acetic acid purum, 2-(4-Bocpiperazino)-2-[4-(trifluoromethyl)phenyl]acetic acid purum, Boc-P-(2-quinolyl)-Ala-OH, NBoc-l,2,3,6-tetrahydro-2-pyridinecarboxylic acid, Boc-P-(4-thiazolyl)-Ala-OH, Bo -b-(2-thienyl)-D-Ala-OH, Fmoc-N-(4-Boc-aminobutyl)-Gly-OH, Fmoc-N-(2-Boc-aminoethyl)-Gly-OH, Fmoc-N-(2,4-dimethoxybenzyl)-Gly-OH, Fmoc-(2-indanyl)-Gly-OH, Fmoc-WSGR Docket No. 60801-718.601pentafluoro-L-phenylalanine, Fmoc-Pen(Trt)-OH, Fmoc-Phe(2-Br)-OH, Fmoc-Phe(4-Br)-OH, Fmoc-Phe(3,5-F2)-OH, Fmoc-P-(4-thiazolyl)-Ala-OH, Fmoc-P-(2-thienyl)-Ala-OH, 4-(Hydroxymethyl)-D-phenylalanine.
[0236] The terms "polypeptide," "peptide", and "protein" may be used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may in embodiments be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
[0237] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
[0238] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site ncbi.nlm.nih.gov / BLAST / or the like). Such sequences are then said to be "substantially identical." This definition also refers to, or may be applied to, the complement of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the algorithms can account for gaps and the like. Identity exists over a region that is at least about 25 amino acids or nucleotides in length, or over a region that is 50-100 amino acids or nucleotides in length.
[0239] “Antibodies” generally refer to large, complex proteins with an intricate internal structure. A natural antibody molecule contains two identical pairs of polypeptide chains, eachWSGR Docket No. 60801-718.601pair having one light chain and one heavy chain. Each light chain and heavy chain in turn consists of two regions: a variable ("V") region involved in binding the target antigen, and a constant ("C") region that interacts with other components of the immune system. The light and heavy chain variable regions come together in 3 -dimensional space to form a variable region that binds the antigen (for example, a receptor on the surface of a cell). Within each light or heavy chain variable region, there are three short segments (averaging 10 amino acids in length) called the complementarity determining regions ("CDRs"). The six CDRs in an antibody variable domain (three from the light chain and three from the heavy chain) fold up together in 3 -dimensional space to form the actual antibody binding site which docks onto the target antigen. The position and length of the CDRs have been precisely defined by Kabat, E. et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1983, 1987. The part of a variable region not contained in the CDRs is called the framework ("FR"), which forms the environment for the CDRs. Antibodies may be full-length (e.g., 150 kDa IgG antibodies), or may be antigen-binding fragments thereof, including antigen-binding peptides and / or nanobodies.
[0240] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively. The Fc (i.e. fragment crystallizable region) is the “base” or "tail" of an immunoglobulin and is typically composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. By binding to specific proteins the Fc region ensures that each antibody generates an appropriate immune response for a given antigen. The Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins.
[0241] An “antigen binding fragment” as provided herein refers to a polypeptide capable of binding to an antigen and including one or more structural domains of an antibody or fragment thereof. Nonlimiting examples of antibody variants include single-domain antibodies (nanobodies), affibodies (polypeptides smaller than monoclonal antibodies (e.g., about 6kDa) and capable of binding antigens with high affinity and imitating monoclonal antibodies, monospecific Fab2, bispecific Fab2, trispecific Fab3, monovalent IgGs, scFv, bispecific diabodies, trispecific triabodies, scFv-Fc, minibodies, IgNAR, V-NAR, hcIgG, VhH, or peptibodies. A “peptibody” as provided herein refers to a peptide moiety attached (through aWSGR Docket No. 60801-718.601covalent or non-covalent linker) to the Fc domain of an antibody. Further non-limiting examples of antibody variants known in the art include antibodies produced by cartilaginous fish or camelids. A general description of antibodies from camelids and the variable regions thereof and methods for their production, isolation, and use may be found in references WO 97 / 49805 and WO 97 / 49805, which are incorporated, by reference herein in their entirety and for all purposes. Likewise, antibodies from cartilaginous fish and the variable regions thereof and methods for their production, isolation, and use may be found in W02005 / 118629, which is incorporated by reference herein in its entirety and for all purposes.
[0242] A “single-domain antibody” or “nanobody” interchangeably refers to an antibody fragment having a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. In some embodiments, the single domain antibody is a human or humanized single domain antibody. In some embodiments, the single domain antibody is a camelid single domain antibody.
[0243] The term "antigen" as provided herein refers to molecules capable of binding to the antibody binding domain provided herein. An "antigen binding domain" as provided herein is a region of an antibody that binds to an antigen (epitope). As described above, the antigen binding domain may include one constant and one variable domain of each of the heavy and the light chain (VL, VH, CL and CHI, respectively). In embodiments, the antigen binding domain includes a light chain variable domain and a heavy chain variable domain. In embodiments, the antigen binding domain includes light chain variable domain and does not include a heavy chain variable domain and / or a heavy chain constant domain. The paratope or antigen-binding site is formed on the N-terminus of the antigen binding domain. The two variable domains of an antigen binding domain may bind the epitope of an antigen. Antibodies exist, for example, as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)’2, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)’2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)’2 dimer into an Fab’ monomer. The Fab’ monomer is essentially the antigen binding portion with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or thoseWSGR Docket No. 60801-718.601synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature, 348:552-554 (1990)).Targets
[0244] As used herein, the term “target” generally refers to a polypeptide or protein that is expressed on or in a mammalian cell. In some embodiments, the target is a protein. In some embodiments, a target is an antigen. In some embodiments, the target is a cell surface molecule present on an exterior surface of a tumor cell. In some embodiments, the target is a cell surface molecule present on or in a tumor cell. In some embodiments, the target is a molecule, peptide, or protein expressed by a mammalian tumor or cancer cell. In some embodiments, the target is overexpressed by a cancer or tumor cell. In some embodiments, a target is selectively overexpressed by a particular cancer or tumor.
[0245] An aspect of the present disclosure is a compound (alternatively, a “conjugate” or, in some instances, an “antibody-drug conjugate or ADC”) of Formula (II):Formula (II)wherein P2is a targeting moiety.
[0246] In some embodiments of Formula (II), the targeting moiety is a small molecule or a peptide or polypeptide that binds a target. Generally, compounds of Formula (II), being conjugated to a targeting moiety, do not inhibit or otherwise substantially affect the binding of the moiety to its respective target. For example, compounds of Formula (II), being conjugated to a peptide or polypeptide that binds to a target, do not inhibit or otherwise substantially affect the binding of the peptide or polypeptide to its respective target. In such embodiments, it can also be said that the compound of Formula (II) binds to a target. In some embodiments, the peptide or polypeptide, or the compound of Formula (II), binds selectively to a target. In some embodiments, the target is a cell surface molecule present on a tumor cell. In some embodiments, the target comprises a cell surface molecule. In some embodiments, the target comprises 5T4, ADAM-9, AG-7, ALK, ALPP, ALPPL2, ALPV, AMHR2, ASCT2, AXL, B1R, B7-H3, B7-H4, BCMA, C4.4a, CA6, CAIX, CanAg, CAXII, CCR2, CCR4, CCR7, CD103, CD123, CD13, CD138, CD142, CD147, CD155, CD16, CD166, CD171, CD19, CD2, CD20, CD205, CD206, CD22, CD228, CD24, CD248, CD25, CD30, CD300f, CD33, CD34,WSGR Docket No. 60801-718.601CD352, CD36, CD37, CD38, CD40, CD44v6, CD45, CD46, CD47, CD48, CD51, CD56, CD66, CD66, CD70, CD71, CD73, CD74, CD79b, CD8, CD99, CDCP1, CDH17, CDH6, CEA, CEACAM6, cKIT, CLDN18.2, CLDN6, CLDN9, CLL-1, cMET, CSP-1, CSPG4, CXCR3, CXCR4, CXCR5, DCLK1, DLK1, DLL1, DLL3, Doppel, DPEP3, DPP4, DR5, DUX4, Dysadherin, EDB fibronectin, EGFR, EGFRviii, EMP2, endol80, Endoglin, ENPP3, EpCAM, EphA2, EphA3, EphA4, ETBR, FAP, FcRH5, FGFR2, FGFR3, FLT3, FolRa, GD2, GD3, GloboH, gpA33, GPC-1, GPC3, GPNMB, GPR20, GPRC5d, GRPR, GSPT1, GUCY2C(GCC ), HER2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-13Ra2, IL-1RAP, IL-7R, IL-7RILT-3, ILT-3, IntegrinalOpi, ITGaVb3, ITGaVb6, ITGB4, KAAG1, KIF20A, LAMP-1, Lewis Y antigen, LGR5, LIV-1, LRP5, LRP6, LRRC15, LSR, Ly6E, Ly6G6D, Macrophage mannose receptor 1, MAGE, MC1R, MerTK, MICA, MICB, MRC2, MSLN, MT1-MMP, MTX7, Mucl, Mucl6, NaPi2b, NECTIN4, NKG2DL, NOTCH3rec, NTS1, OXOOIL, pCadherin, PD-L1, PD-L2, Podocalyxin, PRLR, Prostaglandin F2 Receptor Negative Regulator, PSCA, PSMA, PT1, PTK7, RET, RON, R0R1, R0R2, SAIL, SEA, SEZ6, SIRPa, SLAMF7, SLC44A4, SLITRK6, S SEA-4, SSTR2, STEAP1, STT3, STT4, Survivin, TEM8, TFR, TIM-1, tissue factor, TM4SF1, TMEFF2, TNFSF12, TNFSF12A, TRA-1-60, TREM2, Trk, TROP-2, TRP1, TSLPR, UCHT1, VCAM-1, VEGF, VEGFR2, VpreB, VPREB1, orxCT.
[0247] In some instances, the target comprises 5T4, ADAM-9, AG-7, ALK, ALPP, ALPPL2, ALPV, AMHR2, ASCT2, AXL, B1R, B7-H3, B7-H4, BCMA, C4.4a, CA6, CAIX, CanAg, CAXII, CD123, CD13, CD138, CD142, CD147, CD155, CD166, CD171, CD19, CD2, CD20, CD205, CD22, CD228, CD24, CD248, CD30, CD33, CD34, CD36, CD38, CD44v6, CD45, CD46, CD47, CD48, CD56, CD66, CD70, CD71, CD73, CD74, CD79b, CD99, CDCP1, CDH17, CDH6, CEA, CEACAM6, cKIT, CLDN18.2, CLDN6, CLDN9, cMET, CSP-1, CSPG4, CXCR3, CXCR4, CXCR5, DCLK1, DLK1, DLL1, DLL3, Doppel, DPEP3, DPP4, DR5, DUX4, Dysadherin, EDB fibronectin, EGFR, EGFRviii, EMP2, endol80, Endoglin, ENPP3, EpCAM, EphA2, EphA3, EphA4, ETBR, FAP, FcRH5, FGFR2, FGFR3, FolRa, GD2, GD3, GloboH, gpA33, GPC-1, GPC3, GPNMB, GPR20, GPRC5d, GRPR, GSPT1, GUCY2C(GCC ), HER2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-13Ra2, IL-1RAP, IL-7R, ILT-3, Integrin aioPi, ITGaVb3, ITGaVb6, ITGB4, KAAG1, KIF20A, LAMP-1, Lewis Y antigen, LGR5, LIV-1, LRP5, LRP6, LRRC15, LSR, Ly6E, Ly6G6D, MAGE, MC1R, MerTK, MICA, MICB, MRC2, MSLN, MT1-MMP, MTX7, Mucl, Mucl 6, NaPi2b, NECTIN4, NKG2DL, NOTCH3rec, NTS1, OXOOIL, pCadherin, PD-L1, PD-L2, Podocalyxin, PRLR, Prostaglandin F2 Receptor Negative Regulator, PSCA, PSMA, PT1, PTK7, RET, RON, ROR1, ROR2, SAIL, SEA, SEZ6, SIRPa, SLAMF7, SLC44A4, SLITRK6, SSEA-4, SSTR2,WSGR Docket No. 60801-718.601STEAP1, STT3, STT4, Survivin, TEM8, TfR, TIM-1, tissue factor, TM4SF1, TMEFF2, TNFSF12, TRA-1-60, Trk, TROP-2, TRP1, TSLPR, UCHT1, VCAM-1, VEGF, VEGFR2, VpreBl, or xCT.
[0248] In some instances, the target is selected from the group consisting of PSMA, EGFR, EGFRviii, MSLN, CEA, DLL3, FAP, CD33, HER3, PD-L1, EphA2, EphA4, HER2, SIRPa, DLK1, Mucl6, LRP5, LRP6, endol80, LIV-1, SLAMF7, PTK7, GPR20, CDH6, CSP-1, CD71, PRLR, SEZ6, DLL1, NOTCH3 rec, NaPi2b, CD 16, GCC, SSTR2, CAIX, CAXII, MC1R, CXCR4, B1R, GRPR, STEAP1, CD70, CD46, CD166, CLL-1, ADAM9, cKIT, CD36, CD73, ITGaVb3, ITGaVb6, GPC-1, CD38, CD51, FGFR3, Ly6E, CD44v6, ENPP3, CXCR3, CXCR5, FcRH5, VEGF, VEGFR2, CD45, CCR4, CD25, 5T4, ROR1, TROP-2, NECTIN4, cMET, CD19, CD22, CD30, CD33, CD123, BCMA, CD79b, AXL, RON, B7-H3, B7-H4, KAAG1, Mucl, ADAM-9, GPNMB, EDB fibronectin, tissue factor, GPNMB, FolRa, ALPP, ALPPL2, MT1-MMP, CLDN18.2, CLDN6, CLDN9, pCadherin, CEACAM6, CD47, and EpCAM.
[0249] In some instances, the target comprises 5T4, B7-FI3, B7-4, BCMA, CAIX, CD123, CD19, CD20, CD22, CD30, CD33, CD79b, CEA, DLL3, EGFR, EGFRviii, FAP, FcRH5, GPR20, GUCY2C(GCC), HER2, HER3, KAAG1, LIV-1, MICA, MSLN, Mucl, BCMA, C4.4a, CA6, CAIX, CanAg, CAXII, CCR2, CCR4, CCR7, CD103, CD123, CD13, CD138, CD142, ENPP3, EpCAM, EphA2, EphA3, EphA4, ETBR, FAP, FcRH5, FGFR2, FGFR3, FLT3, FolRa, GD2, GD3, MRC2, MSLN, MT1-MMP, MTX7, Mucl, Mucl 6, NaPi2b, NECTIN4, UCHT1, VCAM-1, VEGF, VEGFR2, VpreB, VPREB1, orxCT.
[0250] In some embodiments, the target comprises 5T4, B7-H3, B7-H4, BCMA, CAIX, CD 123, CD 19, CD20, CD22, CD30, CD33, CD79b, CEA, DLL3, EGFR, EGFRviii, FAP, FcRH5, GPR20, GUCY2C(GCC), HER2, HER3, KAAG1, LIV-1, MICA, MSLN, Mucl, PSMA, ROR1, SEZ6, or SLAMF7.
[0251] In some embodiments, the target comprises a cytokine. In some embodiments, the cytokine comprises CD2, CD13, CD19, CD20, CD22, CD24, CD30, CD33, CD34, CD36, CD38, CD44v6, CD45, CD46, CD47, CD48, CD56, CD66, CD70, CD71, CD73, CD74, CD79b, CD99, CD123, CD138, CD142, CD147, CD155, CD166, CD171, CD205, CD228, or CD248.
[0252] In some embodiments, the binding of the peptide or polypeptide, or compound of Formula (II), to a target may be characterized by an affinity constant (KA), which may be expressed as a dissociation constant (KD). In some instances, the conjugate comprises a targeting domain configured to bind to a target on a cell. In some instances, the binding of theWSGR Docket No. 60801-718.601targeting domain to the target may be characterized by KD. In some instances, KD ranges from about 1 pM to about 100 mM, from 10 nM to about 1 mM, from about 100 nM to about 50 pM, from about 50 nM to about 100 pM, from about 1 pM to about 100 pM. In some instances, KD ranges from about 1 nM to about 1 pM, from 10 nM to about 500 nM, from about 50 nM to about 250 nM, from about 100 nM to about 500 nM, or from about 1 nM to about 100 nM. In some instances, Kois at least about 1 nM, about 10 nM, about 25 nM, 50 nM, about 100 nM, about 250 nM, about 500 nM, about 1 pM, about 50 pM, about 100 pM, about 250 pM, about 500 pM, or about 1 mM. In some instances, when the first targeting domain comprises at least one UAA, the at least one UAA may covalently bind to a first target. In some cases, when the at least one UAA covalently binds to the first target, KD is about 0 pM, about 10 pM, about 20 pM, about 50 pM, or about 100 pM.
[0253] In some embodiments, the peptide or polypeptide comprises an antibody fragment, or an antigen binding domain. In some embodiments, the peptide or polypeptide comprises a single domain antibody, a Fab, or an ScFv. In some embodiments, the target comprises a cell surface receptor. In some embodiments, the target comprises a molecule or protein expressed on the extracellular surface of a tumor or cancer cell. In some embodiments, the target comprises one or more tumor antigen receptors. In some embodiments, the target comprises a tumor-specific target. In some embodiments, the tumor-specific target comprises an antigen on a cell surface of a tumor cell. In some embodiments, the target is present in the tumor microenvironment. In some embodiments, the target is expressed on a noncancerous cell present in a tumor microenvironment. In some embodiments, the peptide or polypeptide is capable of binding covalently to the target.
[0254] In some embodiments, the peptide or polypeptide binds to one or more tumor antigen receptors selected from the group consisting of 5T4, B7-H3, B7-H4, BCMA, CAIX, CD123, CD19, CD20, CD22, CD30, CD33, CD79b, CEA, DLL3, EGFR, EGFRviii, FAP, FcRH5, GPR20, GUCY2C(GCC), HER2, HER3, KAAG1, LIV-1, MICA, MSLN, Mucl, PSMA, ROR1, SEZ6, and SLAMF7.
[0255] In some embodiments, the peptide or polypeptide comprises at least one unnatural amino acid (UAA).WSGR Docket No. 60801-718.601
[0256] In some embodiments, at least one UAA comprises:.TIn someembodiments, the at least one LTAA has the structure:
[0257] In some embodiments, at least one UAA comprises:In some embodiments, at least one UAA has the structure:
[0258] In some embodiments, at least one UAA comprises:. In someembodiments, at least one UAA has the structure:
[0259] In some embodiments, at least one UAA has a structure:wherein, each X is independently O or NR’; Y is a bond, -O-, -NR-, or -N=; A is a bond or -(CH2)n-; m is 1 or 2; n is an integer from 1 to 4; each R and R’, when present, is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycloalkyl; R1is hydrogen, fluoro, or iodo; R2is hydrogen or methyl; L is -(CH2)P- or -C(O)NH-(CH2)P-; p is an integer from 1 to 6; and wherein when Y is -O- or -NR-, m is 1; when Y is -N=, m is 2.
[0260] In some embodiments, P2comprises an unnatural amino acid of the structure:WSGR Docket No. 60801-718.601Methods of Treatment
[0261] In another aspect of the present disclosure, provided herein is a method of forming a compound of the structure:in a mammal diagnosed with cancer, the method comprising administering to the mammal a composition comprising a compound of Formula (II) or (III):Formula (II) Formula (III) wherein R1is H, R2is -CH3, R3is -CH2OH; and P2, L5, L2, R4, and L1are as defined herein for Formula (II) and its embodiments. In some embodiments, the compound is any one of Compounds 1-50. In some embodiments, the compound of Formula (II) or (Ila) is a compound of Table 1 or Table 2. In some embodiments, the compound is a DARI comprising a thiol-reactive linker-drug compound of Table 1. In some embodiments, the compound is a DAR2 comprising a thiol -reactive linker-drug compound of Table 2.
[0262] Also provided herein is a method of delivering a payload to a target cell, the method comprising contacting the target cell with a compound of Formula (II) or Formula (III). In some embodiments, the target cell is a mammalian cell. In some embodiments, provided herein is a method of delivering a therapeutic payload to an intracellular target within a tumor cell of a mammal, the method comprising administering to the tumor cell an effective amount of a conjugate of a therapeutic payload, wherein the therapeutic payload is conjugated to a cleavable peptide via a self-immolative group comprising the sequence Ser-Sar. In some embodiments, the conjugate of a therapeutic payload is a compound of Formula (II) or Formula (III). In some embodiments, provided herein is a method of delivering a therapeutic payload to an intracellular target within a tumor cell of a mammal, the method comprising contacting the tumor cell with an effective amount of a compound of Formula (II) or (III).WSGR Docket No. 60801-718.601
[0263] In another aspect, provided herein is a method of treating a disease or disorder in a mammal, the method comprising administering to the mammal an effective amount of a composition comprising a compound of Formula (II).
[0264] Compounds provided herein may be used to treat conditions and / or diseases. In some instances, the disease or disorder is a (hyper)proliferative disease or disorder. In some instances, the disease or disorder is a cancer. In some embodiments, the cancer comprises tumor cells. In some embodiments, the cancer comprises one or more solid tumors. In some embodiments, a compound of Formula (II) is administered to kill or inhibit growth of a rapidly dividing cell, such as a tumor cell.
[0265] In some embodiments, a method of treating a proliferative disease or condition in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a compound described herein. In some embodiments, the solid tumor cancer is bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer. In some instances, the disease comprises PCa (prostate cancer), CRPCa (castration resistant prostate cancer), solid tumors (neovasculature), NSCLC (non-small cell lung cancer), HNSCC (head and neck squamous cell carcinoma), ESCC (esophageal cancer) GC (gastric cancer), CRC (colorectal cancer), SCLC (small cell lung cancer), MPM (mesothelioma), PDAC (Pancreatic ductal adenocarcinoma), ALL (Acute Lymphoblastic Leukemia), AML (Acute Myeloid Leukemia), MDS (Myelodysplastic syndromes), MS high tumors, melanoma, DLBCL (diffuse large B cell lymphoma), endometrial cancer, cervical cancer, bladder cancer, BrCa (breast cancer), TNBC (triple negative breast cancer), NE-PCa (Neuroendocrine prostate cancer), GBM (glioblastoma), and RCC (Renal cell carcinoma).
[0266] In some instances, tumor cells targeted herein overexpress one or more targets. In some instances, the target comprises surface markers or receptors. In some instances, the target is selected from one or more of 5T4, ADAM-9, AG-7, ALK, ALPP, ALPPL2, ALPV, AMHR2, ASCT2, AXL, B1R, B7-H3, B7-H4, BCMA, C4.4a, CA6, CAIX, CanAg, CAXII, CCR2, CCR4, CCR7, CD103, CD123, CD13, CD138, CD142, CD147, CD155, CD16, CD166, CD171, CD19, CD2, CD20, CD205, CD206, CD22, CD228, CD24, CD248, CD25, CD30, CD300f, CD33, CD34, CD352, CD36, CD37, CD38, CD40, CD44v6, CD45, CD46, CD47, CD48, CD51, CD56, CD66, CD66, CD70, CD71, CD73, CD74, CD79b, CD8, CD99, CDCP1, CDH17, CDH6, CEA, CEACAM6, cKIT, CLDN18.2, CLDN6, CLDN9, CLL-1, cMET, CSP-1, CSPG4, CXCR3, CXCR4, CXCR5, DCLK1, DLK1, DLL1, DLL3, Doppel, DPEP3, DPP4,WSGR Docket No. 60801-718.601DR5, DUX4, Dysadherin, EDB fibronectin, EGFR, EGFRviii, EMP2, endol80, Endoglin, ENPP3, EpCAM, EphA2, EphA3, EphA4, ETBR, FAP, FcRH5, FGFR2, FGFR3, FLT3, FolRa, GD2, GD3, GloboH, gpA33, GPC-1, GPC3, GPNMB, GPR20, GPRC5d, GRPR, GSPT1, GUCY2C(GCC ), HER2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-13Ra2, IL-1RAP, IL-7R, IL-7RILT-3, ILT-3, Integrin aioPi, ITGaVb3, ITGaVb6, ITGB4, KAAG1, KIF20A, LAMP-1, Lewis Y antigen, LGR5, LIV-1, LRP5, LRP6, LRRC15, LSR, Ly6E, Ly6G6D, Macrophage mannose receptor 1, MAGE, MC1R, MerTK, MICA, MICB, MRC2, MSLN, MT1-MMP, MTX7, Mucl, Mucl6, NaPi2b, NECTIN4, NKG2DL, NOTCH3rec, NTS1, OXOOIL, pCadherin, PD-L1, PD-L2, Podocalyxin, PRLR, Prostaglandin F2 Receptor Negative Regulator, PSCA, PSMA, PT1, PTK7, RET, RON, R0R1, R0R2, SAIL, SEA, SEZ6, SIRPa, SLAMF7, SLC44A4, SLITRK6, S SEA-4, SSTR2, STEAP1, STT3, STT4, Survivin, TEM8, TFR, TIM-1, tissue factor, TM4SF1, TMEFF2, TNFSF12, TNFSF12A, TRA-1-60, TREM2, Trk, TROP-2, TRP1, TSLPR, UCHT1, VCAM-1, VEGF, VEGFR2, VpreB, VPREB1, or xCT. In some instances, the first target comprises 5T4, ADAM-9, AG-7, ALK, ALPP, ALPPL2, ALPV, AMHR2, ASCT2, AXL, B1R, B7-H3, B7-H4, BCMA, C4.4a, CA6, CAIX, CanAg, CAXII, CD123, CD13, CD138, CD142, CD147, CD155, CD166, CD171, CD19, CD2, CD20, CD205, CD22, CD228, CD24, CD248, CD30, CD33, CD34, CD36, CD38, CD44v6, CD45, CD46, CD47, CD48, CD56, CD66, CD70, CD71, CD73, CD74, CD79b, CD99, CDCP1, CDH17, CDH6, CEA, CEACAM6, cKIT, CLDN18.2, CLDN6, CLDN9, cMET, CSP-1, CSPG4, CXCR3, CXCR4, CXCR5, DCLK1, DLK1, DLL1, DLL3, Doppel, DPEP3, DPP4, DR5, DUX4, Dysadherin, EDB fibronectin, EGFR, EGFRviii, EMP2, endol80, Endoglin, ENPP3, EpCAM, EphA2, EphA3, EphA4, ETBR, FAP, FcRH5, FGFR2, FGFR3, FolRa, GD2, GD3, GloboH, gpA33, GPC-1, GPC3, GPNMB, GPR20, GPRC5d, GRPR, GSPT1, GUCY2C (GCC), HER2, HER3, HLA-DR, ICAM-1, IGF-1R, IL-13Ra2, IL-1RAP, IL-7R, ILT-3, Integrin aioPi, ITGaVb3, ITGaVb6, ITGB4, KAAG1, KIF20A, LAMP-1, Lewis Y antigen, LGR5, LIV-1, LRP5, LRP6, LRRC15, LSR, Ly6E, Ly6G6D, MAGE, MC1R, MerTK, MICA, MICB, MRC2, MSLN, MT1-MMP, MTX7, Mucl, Mucl6, NaPi2b, NECTIN4, NKG2DL, NOTCH3rec, NTS1, 0X00 IL, pCadherin, PD-L1, PD-L2, Podocalyxin, PRLR, Prostaglandin F2 Receptor Negative Regulator, PSCA, PSMA, PT1, PTK7, RET, RON, ROR1, ROR2, SAIL, SEA, SEZ6, SIRPa, SLAMF7, SLC44A4, SLITRK6, SSEA-4, SSTR2, STEAP1, STT3, STT4, Survivin, TEM8, TfR, TIM-1, tissue factor, TM4SF1, TMEFF2, TNFSF12, TRA-1-60, Trk, TROP-2, TRP1, TSLPR, UCHT1, VCAM-1, VEGF, VEGFR2, VpreBl, or xCT. In some instances, the first target comprises 5T4, B7-H3, B7-4, BCMA, CAIX, CD 123, CD 19, CD20, CD22, CD30, CD33, CD79b, CEA, DLL3,WSGR Docket No. 60801-718.601EGFR, EGFRviii, FAP, FcRH5, GPR20, GUCY2C(GCC), HER2, HER3, KAAG1, LIV-1, MICA, MSLN, Mucl, BCMA, C4.4a, CA6, CAIX, CanAg, CAXII, CCR2, CCR4, CCR7, CD 103, CD 123, CD 13, CD 138, CD 142, ENPP3, EpCAM, EphA2, EphA3, EphA4, ETBR, FAP, FcRH5, FGFR2, FGFR3, FLT3, FolRa, GD2, GD3, MRC2, MSLN, MT1-MMP, MTX7, Mucl, Mucl6, NaPi2b, NECTIN4, UCHT1, VCAM-1, VEGF, VEGFR2, VpreB, VPREB1, or xCT. In some instances, the first target comprises 5T4, B7-H3, B7-H4, BCMA, CAIX, CD 123, CD 19, CD20, CD22, CD30, CD33, CD79b, CEA, DLL3, EGFR, EGFRviii, FAP, FcRH5, GPR20, GUCY2C(GCC), HER2, HER3, KAAG1, LIV-1, MICA, MSLN, Mucl, PSMA, ROR1, SEZ6, or SLAMF7. In some instances, the first target comprises a lymphocyte antigen. In some instances, the lymphocyte antigen comprises BAFFR, CCR2, CCR4, CCR7, CD103, CD155, CD16, CD2, CD205, CD206, CD25, CD300f, CD34, CD352, CD36, CD37, CD38, CD40, CD46, CD47, CD48, CD51, CD56, CD66, CD70, CD8, CLL-1, CXCR4, FcRH5, FLT3, GPRC5d, HLA-DR, HLA-DR, IL-13Ra2, IL-1RAP, IL-7RILT-3, Ly6E, Ly6G6D, Macrophage mannose receptor 1, MerTK, NKG2DL, PD-L1, PD-L2, SAIL, SIRPa, TFR, TIM-1, TNFSF12A, TREM2, TSLPR, VpreB, or VPREB1.
[0267] Pharmaceutical compositions are administered in a manner appropriate to the disease to be treated (or prevented). An appropriate dose and a suitable duration and frequency of administration will be determined by such factors as the condition of the patient, the type and severity of the patient's disease, the particular form of the active ingredient, and the method of administration. In general, an appropriate dose and treatment regimen provides the composition(s) in an amount sufficient to provide therapeutic and / or prophylactic benefit (e.g., an improved clinical outcome), or a lessening of symptom severity. Optimal doses are generally determined using experimental models and / or clinical trials. The optimal dose depends upon the body mass, weight, or blood volume of the patient.
[0268] In one embodiment, an injectable pharmaceutical composition described herein, is used in the preparation of medicaments for the treatment of diseases or conditions in a mammal that would benefit from administration of any one of the injectable pharmaceutical compositions of the compounds disclosed herein (i.e., of Formula (II) or (III)).
[0269] Methods for treating any of the diseases or conditions described herein in a mammal in need of such treatment, may involve, for example, administration of a pharmaceutical composition comprising at least one compound of Formula (II) described herein or a pharmaceutically acceptable salt thereof, in therapeutically effective amounts to said mammal.
[0270] In certain embodiments, the compositions containing the compound(s) described herein are administered for prophylactic and / or therapeutic treatments. In certain therapeuticWSGR Docket No. 60801-718.601applications, the compositions are administered to a patient already suffering from a disease or condition, in an amount sufficient to cure or at least partially arrest at least one of the symptoms of the disease or condition. Amounts effective for this use depend on the severity and course of the disease or condition, previous therapy, the patient's health status, weight, and response to the drugs, and the judgment of the treating physician. Therapeutically effective amounts are optionally determined by methods including, but not limited to, a dose escalation and / or dose ranging clinical trial.
[0271] When used in patients, effective amounts for this use will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician. In one aspect, prophylactic treatments include administering to a mammal, in which the mammal previously experienced at least one symptom of the disease being treated and is currently in remission, a pharmaceutical composition comprising a compound described herein, or a pharmaceutically acceptable salt thereof, in order to prevent a return of the symptoms of the disease or condition.
[0272] In certain embodiments, wherein the patient's condition does not improve, upon the doctor's discretion the administration of the compounds is administered chronically, that is, for an extended period of time, including throughout the duration of the patient's life in order to ameliorate or otherwise control or limit the symptoms of the patient's disease or condition.
[0273] Additional Embodiments
[0274] In some embodiments,Compound 1 corresponds to GlySar-AE,Compound 2 corresponds to NAc-GlySar-AE,Compound 3 corresponds to SerSar-AE,Compound 4 corresponds to NAc-SerSar-AE,Compound 5 corresponds to MA-EVCit-GlySar-AE,Compound 6 corresponds to MA-EVCit-SerSar-AE,Compound 7 corresponds to MA-(EVCit-SerSar-AE)2,Compound 8 corresponds to MA-EVCit-SerSar-MMAECompound 9 corresponds to MA-(EVCit-SerSar-MMAE)2,Compound 10 corresponds to MDPR-LDL-SerSar-AE,Compound 11 corresponds to MA-EVCit-GlySar-MMAE,Compound 12 corresponds to MDPR-LDL-SerSar-MMAE,Compound 13 corresponds to MA-EVCit-GlySar- CPT,WSGR Docket No. 60801-718.601Compound 14 corresponds to MA-EVCit-SerSar- 7EtCPT, Compound 15 corresponds to MA-(EVCit-SerSar-7EtCamptothecin)2, Compound 16 corresponds to MA-(EVCit-GlySar-7EtCamptothecin)2, Compound 17 corresponds to MA-EVCit-SerSar-S-DMl, Compound 18 corresponds to H-GlyPro-AECompound 19 corresponds to NAc-GlyPro-AECompound 20 corresponds to H-SerPro-AECompound 21 corresponds to NAc-SerPro-AECompound 22 corresponds to H-Ser(NMe)Ile-AECompound 23 corresponds to NAc-Ser(NMe)Ile-AECompound 24 corresponds to H-Ser(NMe)Glu-AECompound 25 corresponds to NAc-Ser(NMe)Glu-AECompound 26 corresponds to H-Ser(NMe)Arg-AECompound 27 corresponds to NAc-Ser(NMe)Arg-AECompound 28 corresponds to H-SerSar(cyclopropyl)-AE Compound 29 corresponds to NAc-SerSar(cyclopropyl)-AE Compound 30 corresponds to MA-GluValLysSerSar-MMAE Compound 31 corresponds to MA-GluValCitSer(NMe)Glu-MMAE Compound 32 corresponds to MA-GluValCitSer(NMe)Ile-MMAE Compound 33 corresponds to MA-(GluValCit-Ser(NMe)Glu-MMAE)2 Compound 34 corresponds to N-linked H-SerSar-MMAE Compound 35 corresponds to N-linked H-SerSar-desmethyl-AE Compound 36 corresponds to N-linked MA-GluValLysSerSar-MMAE Compound 37 corresponds to MA-GluValCit-SerSar-exatecan Compound 38 corresponds to MA-GluValCit-SerSar-MDOCPT Compound 39 corresponds to MA-(GluValCit-SerSar-exatecan)2 Compound 40 corresponds to MA-(GluValCit-SerSar-MDOCPT)2 Compound 41 corresponds to GlyGly-AECompound 42 corresponds to SerGly-AECompound 43 corresponds to MA-GluValLys-Ser(NMe)Glu-MMAE Compound 44 corresponds to MA-(GluValCit-Ser(NMe)Glu-MDO-CPT)2 Compound 45 corresponds to MA-(GluValCit-Ser(NMe)Ile-MDO-CPT)2 Compound 46 corresponds to MA-(GluValCit-Ser(NMe)Ile-MMAE)2 Compound 47 corresponds to MA-(GluValCit-Ser(NMe)Ile-exatecan)2WSGR Docket No. 60801-718.601Compound 48 corresponds to MA-GluValCit-Ser(NMe)Ile-MDO-CPT Compound 49 corresponds to MA-GluValCit-Ser(NMe)Ile-exatecan Compound 50 corresponds to MA-(GluValCit-Ser(NMe)Glu-exatecan)2 Compound R1 corresponds to MA-EVCit-PABC-MMAE.Numbered ADC EmbodimentsADC 1 corresponds to aAg#l-MA-EVCit-GlySar-AE (Compound 5)ADC 2 corresponds to aAg#l-MA-EVCit-SerSar-AE (Compound 6)ADC 3 corresponds to aAg#l-MA-(EVCit-SerSar-AE)2 (Compound 7)ADC 4 corresponds to aAg#l-MA-(EVCit-SerSar-MMAE)2 (Compound 9) ADC 5 corresponds to sdAbl- MA-EVCit-PABC-MMAE (Compound Rl) ADC 6 corresponds to sdAbl - MA-EVCit-SerSar-MMAE (Compound 8)ADC 7 corresponds to sdAbl - MA-(EVCit-SerSar-MMAE)2 (Compound 9) ADC 8 corresponds to sdAb2- MA-EVCit-PABC-MMAE (Compound Rl) ADC 9 corresponds to sdAb2- MA-EVCit-SerSar-MMAE (Compound 8)ADC 10 corresponds to sdAb2- MA-(EVCit-SerSar-MMAE)2 (Compound 9) ADC 11 corresponds to sdAb3- MA-EVCit-PABC-MMAE (Compound Rl) ADC 12 corresponds to sdAb3- MA-EVCit-SerSar-MMAE (Compound 8) ADC 13 corresponds to sdAb3- MA-(EVCit-SerSar-MMAE)2 (Compound 9) ADC 14 corresponds to sdAb4- MA-EVCit-PABC-MMAE (Compound Rl) ADC 15 corresponds to sdAb4- MA-EVCit-SerSar-MMAE (Compound 8) ADC 16 corresponds to sdAb4- MA-(EVCit-SerSar-MMAE)2 (Compound 9) ADC 17 corresponds to sdAb5- MA-EVCit-PABC-MMAE (Compound Rl) ADC 18 corresponds to sdAb5- MA-EVCit-SerSar-MMAE (Compound 8) ADC 19 corresponds to sdAb5- MA-(EVCit-SerSar-MMAE)2 (Compound 9) ADC 20 corresponds to sdAbl - MDPR-EVCit-PABC-exatecan (Compound R2) ADC 21 corresponds to sdAbl - MA- MA-EVCit-SerSar-exatecan (Compound 37) ADC 22 corresponds to sdAbl - MA-MA-EVCit-SerSar-MDOCPT (Compound 38) ADC 23 corresponds to sdAbl - MA-(EVCit-SerSar-MDOCPT)2 (Compound 40) ADC 24 corresponds to sdAbl - MA-(GluValCit-Ser(NMe)Glu-MMAE)2 (Compound 33) ADC 25 corresponds to aAg#l-MDPR-LDL-SerSar-AE (Compound 10)ADC 26 corresponds to aAg#l-MA-EVCit-SerSar-MMAE (Compound 8) ADC 27 corresponds to aAg#l-MA-EVCit-GlySar-MMAE (Compound 11) ADC 28 corresponds to aAg#l-MDPR-LDL-SerSar-MMAE (Compound 12)WSGR Docket No. 60801-718.601ADC 29 corresponds to aAg#l-MA-EVCit-GlySar- CPT (Compound 13)ADC 30 corresponds to aAg#l-MA-EVCit-SerSar- 7EtCPT (Compound 14)ADC 31 corresponds to aAg#l-MA-(EVCit-SerSar-7EtCamptothecin)2 (Compound 15) ADC 32 corresponds to aAg#l-MA-(EVCit-GlySar-7EtCamptothecin)2 (Compound 16) ADC 33 corresponds to aAg#l-MA-EVCit-SerSar-S-DMl (Compound 17)ADC 34 corresponds to Ab6-MA-EVCit-PABC-MMAE (Compound Rl)ADC 35 corresponds to Ab6-MA-(EVCit-SerSar-MMAE)2 (Compound 9)ADC Rl corresponds to aAg#l-MA-EVCit-PABC-MMAE (Compound Rl).EXAMPLESA. Chemistry Examples
[0275] General procedures
[0276] LC / MS Method #!: 5 -95AB_3.5min: The column used for chromatography was a Luna C182.0x30mm 3um. Detection methods are diode array (DAD). MS mode was positive electrospray ionization. MS range was 50-2000. Mobile phase A was 0.04% trifluoroacetic acid in water, and mobile phase B was 0.02% trifluoroacetic acid in HPLC grade acetonitrile. The gradient was 5-95% B in 3.50 min. 5% B in 0.01 min, 5-95% B (0.01-2.50 min) with a hold at 95% B for 0.50 min, 95-5% B (3.00 -3.01 min) with a hold at 5% B for 0.49 min. The flow rate was 1 mL / min (0.01-3.00 min)-1.2 mL / min (3.01-3.50 min).
[0277] LC / MS Method #2: 5_95AB_6min-220-254: The gradient was 5%B in 0.40min and 5-95% B in 2.60 min, hold on 95% B in 1. OOmin, and then 95-5%B in O.Olmin, the flow rate was 1.0 ml / min. Mobile phase A was 0.04% trifluoroacetic Acid in water, mobile phase B was 0.02% trifluoroacetic Acid in acetonitrile. The column used for chromatography was a Luna C18 50x2.0mm column (5 pm particles). Detection methods are diode array (DAD) detection. MS mode was positive electrospray ionization. MS range was 100-1000.
[0278] LC / MS Method #3: 5_95AB_6min-220-254-MS2000: The gradient was 5%B in 0.40min and 5-95% B in 2.60 min, hold on 95% B in 1.00 min, and then 95-5%B in 0.01 min, the flow rate was 1.0 ml / min. Mobile phase A was 0.04% trifluoroacetic Acid in water, mobile phase B was 0.02% trifluoroacetic Acid in acetonitrile. The column used for chromatography was a Luna C 1850x2.0mm column (5 pm particles). Detection methods are diode array (DAD) detection. MS mode was positive electrospray ionization. MS range was 100-2000.
[0279] LC / MS Method #4: 5_95AB_6min-220-254-MS1000: The gradient was 5%B in 0.40min and 5-95% B in 2.60 min, hold on 95% B in 1.00 min, and then 95-5%B in 0.01 min, the flow rate was 1.0 ml / min. Mobile phase A was 0.04% trifluoroacetic Acid in water, mobileWSGR Docket No. 60801-718.601phase B was 0.02% trifluoroacetic Acid in acetonitrile. The column used for chromatography was a Luna C18 50x2.0mm column (5 pm particles). Detection methods are diode array (DAD) detection. MS mode was positive electrospray ionization. MS range was 100-1000.
[0280] LC / MS Method #5: 5_95AB_6min-220-254-ELSD-MS2000: The gradient was 5%B in 0.40min and 5-95% B in 2.60 min , hold on 95% B in 1.00 min, and then 95-5%B in 0.01 min, the flow rate was 1.0 ml / min. Mobile phase A was 0.04% Trifluoroacetic Acid in water, mobile phase B was 0.02% Trifluoroacetic Acid in acetonitrile. The column used for chromatography was a Luna C18 50*2.0mm column (5 pm particles). Detection methods are diode array (DAD) and evaporative light scattering (ELSD) detection. MS mode was positive electrospray ionization. MS range was 100-2000.
[0281] LC / MS Method #6: 10-100AB 1MIN: The column used for chromatography was a Halo C183.0*30mm,5um. Detection methods are diode array (DAD). MS mode was positive electrospray ionization. MS range was 50-2000. Mobile phase A was 0.04% TFA in water, and mobile phase B was 0.02% TFA in HPLC grade acetonitrile. The gradient was 10-100% B in 0.90 min. 10% B in 0.01 min, 10-100% B (0.01-0.50 min) with a hold at 100% B for 0.40 min. The flow rate was 2.0 mL / min.
[0282] LC / MS Method #7: 5-95AB_3.5min: The column used for chromatography was a Halo 5pm C18 90A, 30*3.0mm. Detection methods are diode array (DAD). MS mode was positive electrospray ionization. MS range was 50-2000. Mobile phase A was 0.04% TFA in water, and mobile phase B was 0.02% TFA in HPLC grade acetonitrile. The gradient was 5-95% B in 3.50 min. 5% B in 0.01 min, 5-95% B (0.01-2.50 min) with a hold at 95% B for 0.50 min, 95-5% B (3.00 -3.01 min) with a hold at 5% B for 0.49 min. The flow rate was 1 mL / min(0.01-3.00 min)-1.2 mL / min(3.01-3.50 min).
[0283] LC / MS Method #8: 10-80_AB_20 min: LCMS (The gradient was 10-80% B in 16.00 min, 80-100%B in 3.00 min, 100-10% B in 0.01 min, and then held at 10% for 1.0 min (1.2ml / min flow rate). Mobile phase A was 0.04% Trifluoroacetic Acid in water, mobile phase B was 0.02% Trifluoroacetic Acid in acetonitrile. The column used for chromatography was a 4.6 x 150 mm XBridge C18 column (3.5 pm particles). Detection methods are diode array (DAD). MS mode was positive electrospray ionization. MS range was 50-2000.
[0284] LC / MS Method #9: Description: Mobile Phase: 0.04% TFA in water(solvent A) and mobile phase B was 0.02% TFA in HPLC grade acetonitrile (solvent B), using the elution gradient 5%-95% (solvent B) over 2.5 minutes and holding at 95% for 0.5 minutes at a flow rate of 1 mL / min (0.01-3. OOmin). Column: Agilent Poroshell SB-C183.0*30mm, 4.0 pmWSGR Docket No. 60801-718.601
[0285] LC / MS Method #10: 5_95AB_lmin_220&254: LC / MS (The column used for chromatography was a Halo C18 3.0*30mm (5 pm particles). Detection methods are diode array (DAD). MS mode was positive electrospray ionization. MS range was 50-2000.Mobile phase A was 0.04% TFA in water, and mobile phase B was 0.02% TFA in HPLC grade acetonitrile. The gradient was 5-95% B in 0.90 min. 5% B in 0.01 min, 5-95% B (0.01-0.50 min) with a hold at 95% B for 0.40 min. The flow rate was 2.0 mL / min (0.00-0.90 min).
[0286] LC / MS Method #11: Description: Mobile Phase: 0.04% TFA in water (solvent A) and mobile phase B was 0.02% TFA in HPLC grade acetonitrile (solvent B), using the elution gradient 5%-95% (solvent B) over 2.5 minutes and holding at 95% for 0.5 minutes at a flow rate of 1 mL / min (0.01-3. OOmin). ColummAgilent Poroshell SB-C183.0*30mm, 4.0pm. Wavelength: UV 220nm&254nm.
[0287] LC / MS Method #12: 10-100AB_3.5MIN: Description: Mobile Phase: 0.04% TFA in water(solvent A) and mobile phase B was 0.02% TFA in HPLC grade acetonitrile (solvent B), using the elution gradient 5%-95% (solvent B) over 2.5 minutes and holding at 95% for 0.5 minutes at a flow rate of 1 mL / min(0.01-3.00min)Column:Halo C18 5pm,3.0*30mm, Wavelength: UV 220nm&254nm, column temperature: 40 C; MS ionization.
[0288] LC / MS Method #13: 5_95AB_3.5min_220&254_ELSD: LC / MS (The column was a Luna C18 30*2.0mm, 3um. Detection methods are diode array (DAD) and evaporative light scattering (ELSD). MS mode was positive electrospray ionization. MS range was 50-2000. Mobile phase A was 0.04% TFA in water, and mobile phase B was 0.02% TFA in HPLC grade acetonitrile. The gradient was 5-95% B in 3.50 min. 5% B in 0.01 min, 5-95% B (0.01-2.50 min) with a hold at 95% B for 0.50 min, 95-5% B (3.00 -3.01 min) with a hold at 5% B for 0.49 min. The flow rate was 1 mL / min (0.01-3. OOmin) - 1.2 mL / min (3.01-3.50min).
[0289] LC / MS Method #14: 50-100_AB_20min: LCMS (The gradient was 50-100% B in 16.00 min with a hold at 100% B for 3. OOmin, 100-50% B in 0.01 min, and then held at 50% for l.Omin (1.2ml / min flow rate). Mobile phase A was 0.04% Trifluoroacetic Acid in water, mobile phase B was 0.02% Trifluoroacetic Acid in acetonitrile. The column used for chromatography was a 4.6*150mm Xbridge C18 column (5 pm particles). Detection methods are diode array (DAD). MS mode was positive electrospray ionization. MS range was 50-2000.
[0290] NMR: Proton NMR spectra were collected on a 400 MHz Bruker nuclear magnetic resonance spectrometer.WSGR Docket No. 60801-718.601
[0291] Example Al. Synthesis of Compound 1, GlySar-AE prodrugCompound 1, GlySar-AE dipeptideScheme 1, Synthesis of GlySar-AE dipeptide prodrug
[0292] Synthesis of Boc-Sar-AE (2). To a solution of 1, auristatin E (500 mg, 683.06 pmol, 1 eq) in dichloromethane (5 mL) was added 2-[tert-butoxycarbonyl (methyl) amino] acetic acid (142.16 mg, 751.36 pmol, 1.1 eq), DMAP (8.34 mg, 68.31 pmol, 0.1 eq), DIC (129.30 mg, 1.02 mmol, 158.65 pL, 1.5 eq) and molecular sieves (4A). The mixture was stirred at 25°C for 12 h. LC-MS showed that 1 was consumed completely and one main peak with desired mass was detected. The reaction mixture was filtered to remove molecular sieves, then mother liquor was partitioned between dichloromethane (10 mL) and H2O (10 mL). The organic phase was separated, washed with aqueous NaCl (10 mL), dried over Na2SC>4, filtered and concentrated under reduced pressure to give a residue. The crude product 2, Boc-Sar-AE (600 mg, 87% yield, 90% purity) was used in the next step without further purification. LCMS (ESI+, Method #1): m / z found 903.5 (M+l), calculated 903.6, RT: 1.681 min.
[0293] Synthesis of Sar-AE (3). To a solution of 2 (600 mg, 597.88 pmol, 1 eq) in dichloromethane (5 mL) was added trifluoroacetic acid (1 mL). The mixture was stirred at 20°C for 2 h. LC-MS showed 2 was consumed completely and one main peak with desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by Prep-HPLC (FA condition; column: Phenomenex luna C18 100 x 40mm 5 um; mobile phase: [H2O(0.2% FA)-ACN]; gradient: l%-45% B over 8.0 min). Target 3, Sar-AE (400 mg, 81% yield, 98% purity) was obtained. LCMS (ESI+, Method #1): m / z found 803.4 (M+l), calculated 803.6, RT: 1.197 min.
[0294] Synthesis of Boc-GlySar-AE (4). To a solution of 3 (100 mg, 122.03 pmol, 1 eq) in N, N-dimethylformamide (1 mL) was added 2-(tert-butoxycarbonylamino)-acetic acid (25.65 mg, 146.44 pmol, 1.2 eq), triethylamine (37.05 mg, 366.09 pmol, 3 eq) and HATU (69.60 mg,WSGR Docket No. 60801-718.601183.05 pmol, 1.5 eq). The mixture was stirred at 25°C for 2 h. LC-MS showed 4 was consumed completely and one main peak with desired mass was detected. The residue was purified by Prep-HPLC (FA condition; column: Phenomenex luna Cl 8 100 x 40mm x 5 urn; mobile phase: [H2O(0.2% FA)-ACN]; gradient:20%-50% B over 8.0 min). Intermediate 4, Boc-GlySar-AE (60 mg, 96% purity, 47% yield) was obtained. LCMS (ESI+, Method #2): m / z found 960.7 (M+l) calculated 960.6, RT: 2.286 min.
[0295] Synthesis of GlySar-AE dipeptide prodrug Compound 1. To a solution of intermediate 4 (55 mg, 52.69 pmol, 1 eq) in dichloromethane (0.5 mL) was added trifluoroacetic acid (0.1 mL). The mixture was stirred at 20°C for 2 h. LC-MS showed starting material 4 was consumed completely and one main peak with desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by Prep-HPLC (TFA condition; column: 3_Phenomenex Luna Cl 8 75 x 30mm x 3um; mobile phase: [H2O(0.1% TFA)-ACN]; gradient: 15%-45% B over 8.0 min). GlySar-AE dipeptide, Compound 1 (40 mg, 88% yield, 100% purity) was obtained. LCMS (ESI+, Method #3): m / z found 860.5 (M+l), calculated 860.6, RT: 1.916 min. 'HNMR (400 MHz, DMSO-d6) 8 ppm 0.70 - 1.14 (m, 24 H) 1.20 - 1.63 (m, 3 H) 1.64 - 1.94 (m, 3 H) 1.95 - 2.17 (m, 2 H) 2.20 - 2.39 (m, 2 H) 2.44 (br d, 7=9.30 Hz, 1 H) 2.78 (br d, 7=14.78 Hz, 6 H) 2.90 (s, 1 H) 2.95 - 3.10 (m, 4 H) 3.12 - 3.41 (m, 9 H) 3.69 - 3.86 (m, 2 H) 3.88 - 4.12 (m, 4 H) 4.18 - 4.35 (m, 2 H) 4.45 - 4.79 (m, 2 H) 5.66 - 5.91 (m, 1 H) 7.20 - 7.45 (m, 5 H) 7.71 -8.25 (m, 4 H) 8.93 (br d, 7=5.01 Hz, 1 H) 9.53 - 9.74 (m, 1 H).
[0296] Example A2. Synthesis of Compound 2, NAc-GlySar-AE prodrugScheme 2, Synthesis of NAc-GlySar-AE dipeptide prodrug
[0297] Synthesis of NAc-GlySar-AE dipeptide prodrug Compound 2. To a solution of intermediate 3 (50 mg, 61.02 pmol, 1 eq) in N, N-dimethylformamide (0.5 mL) was added 2-acetamidoacetic acid (8.57 mg, 73.22 pmol, 1.2 eq), triethylamine (18.52 mg, 183.05 pmol, 25.48 pL, 3 eq) and HATU (34.80 mg, 91.52 pmol, 1.5 eq). The mixture was stirred at 25°C for 2 h. LC-MS showed starting material 3 was consumed completely and one main peak with desired mass was detected. The residue was purified by Prep-HPLC (TFA condition; column: 3_Phenomenex Luna C18 75x30mmx3um; mobile phase: [H2O(0.1% TFA)-ACN];WSGR Docket No. 60801-718.601gradient: 15%-45% B over 8.0 min). NAc-GlySar-AE dipeptide, Compound 2 (12 mg, 20% yield, 95.2% purity) was obtained. LCMS (ESI+, Method #3): m / z found 902.6 (M+l), calculated 902.6, RT: 2.025 min. 'H NMR (400 MHz, DMSO-d6) 6 ppm 0.70 - 1.13 (m, 24 H) 1.21 - 1.52 (m, 2 H) 1.53 - 1.82 (m, 3 H) 1.82 - 1.93 (m, 4 H) 1.95 - 2.20 (m, 2 H) 2.22 -2.47 (m, 3 H) 2.66 - 2.90 (m, 7 H) 2.96 - 3.10 (m, 4 H) 3.14 - 3.22 (m, 4 H) 3.23 - 3.31 (m, 3 H) 3.38 (br d, J=10.37 Hz, 1 H) 3.43 - 3.54 (m, 2 H) 3.67 - 3.80 (m, 3 H) 3.82 - 3.92 (m, 1 H) 3.95 - 4.06 (m, 2 H) 4.16 (br s, 1 H) 4.20 - 4.29 (m, 1 H) 4.50 - 4.80 (m, 2 H) 5.65 - 5.94 (m, 1 H) 7.23 - 7.42 (m, 5 H) 7.76 - 8.27 (m, 2 H) 8.91 (br d, 7=7.63 Hz, 1 H) 9.38 - 9.63 (m, 1 H).
[0298] Example A3. Synthesis of Compound 3, SerSar-AE prodrugCompound 3, SerSar-AE dipeptideScheme 3, Synthesis of SerSar-AE dipeptide prodrug
[0299] Synthesis of Boc-SerSar-AE dipeptide prodrug (7). To a solution of starting material 3 (100 mg, 122.03 pmol, 1 eq) in N, N-dimethylformamide (1 mL) was added (2S)-2-(tert-butoxycarbonylamino)-3-hydroxy-propanoic acid (30.05 mg, 146.44 pmol, 1.2 eq), triethylamine (37.05 mg, 366.09 pmol, 3 eq) andHATU (69.60 mg, 183.05 pmol, 1.5 eq). The mixture was stirred at 25°C for 2 h. LC-MS showed starting material 3 was consumed completely and one main peak with desired mass was detected. The residue was purified by Prep-HPLC (FA condition; column: Phenomenex luna C18 100x40mm><5 um; mobile phase: [H2O(0.2% FA)-ACN]; gradient:20%-50% B over 8.0 min). AE peptide 7 (30 mg, 94% purity, 20% yield) was obtained. LCMS (ESI+, Method #4): m / z found 990.7 (M+l), calculated 990.6, RT: 2.212 min.
[0300] Synthesis of SerSar-AE dipeptide prodrug Compound 3. To a solution of 7 (25 mg, 21.46 pmol, 1 eq) in dichloromethane (0.5 mL) was added trifluoroacetic acid (0.1 mL). The mixture was stirred at 20°C for 2 h. LC-MS showed starting material 7 was consumed completely and one main peak with desired mass was detected. The reaction mixture was concentrated under reduced pressure to remove solvent. The residue was purified by Prep-WSGR Docket No. 60801-718.601HPLC (TFA condition; column: 3_Phenomenex Luna C18 75 x 30mm 3 pm; mobile phase: [H2O(0.1% TFA)-ACN]; gradient: 15%-45% B over 8.0 min). Target SerSar-AE dipeptide Compound 3 (12 mg, 62% yield, 99% purity) was obtained. LCMS (ESI+, Method #3): m / z found 890.5 (M+l), calculated 890.6, RT: 1.900 min. 'H NMR (400 MHz, DMSO-d6) 6 ppm 0.71 - 1.12 (m, 24 H) 1.20 - 1.59 (m, 3 H) 1.62 - 1.90 (m, 3 H) 1.93 - 2.13 (m, 2 H) 2.18 - 2.37 (m, 2 H) 2.38 - 2.46 (m, 1 H) 2.77 (br d, 7=14.19 Hz, 6 H) 2.90 (s, 1 H) 3.00 (br s, 2 H) 3.07 -3.30 (m, 11 H) 3.57 (br dd, 7=11.44, 7.27 Hz, 3 H) 3.67 - 3.87 (m, 3 H) 3.91 - 4.07 (m, 1 H) 4.13 - 4.33 (m, 2 H) 4.37 - 4.49 (m, 1 H) 4.53 - 4.79 (m, 2 H) 5.64 - 5.85 (m, 1 H) 7.20 - 7.42 (m, 5 H) 7.73 - 8.26 (m, 4 H) 8.92 (br d, 7=8.23 Hz, 1 H) 9.47 - 9.72 (m, 1 H).
[0301] Example A4. Synthesis of Compound 4, NAc-SerSar-AE prodrugScheme 4, Synthesis of NAc-SerSar-AE dipeptide prodrug
[0302] Synthesis of NAc-SerSar-AE dipeptide prodrug Compound 4. To a solution of starting material 3 (50 mg, 61.02 pmol, 1 eq) inN, N-dimethylformamide (0.5 mL) was added (2S)-2-acetamido-3-hydroxy-propanoic acid (10.77 mg, 73.22 pmol, 1.2 eq), triethylamine (18.52 mg, 183.05 pmol, 25.48 pL, 3 eq) and HATU (34.80 mg, 91.52 pmol, 1.5 eq). The mixture was stirred at 25°C for 2 h. LC-MS showed 3 was consumed completely and one main peak with desired mass was detected. The residue was purified by Prep-HPLC (TFA condition; column: 3_Phenomenex Luna C18 75x30mmx3pm; mobile phase: [H2O(0.1% TFA)-ACN]; gradient: 15%-45% B over 8.0 min). Target NAc-SerSar-AE dipeptide (14 mg, 24.% yield, 99.8% purity) was obtained. LCMS (ESI+, Method #3): m / z found 932.6 (M+l), calculated 932.6, RT: 2.049 min. 'H NMR (400 MHz, DMSO-d6) 8 ppm 0.70 - 1.12 (m, 24 H) 1.22 -1.66 (m, 3 H) 1.72 (br s, 1 H) 1.76 - 1.92 (m, 4 H) 1.94 - 2.19 (m, 2 H) 2.21 - 2.47 (m, 3 H) 2.65 - 2.91 (m, 7 H) 2.95 - 3.09 (m, 2 H) 3.12 - 3.22 (m, 6 H) 3.26 (br d, 7=9.30 Hz, 3 H) 3.35 - 3.46 (m, 2 H) 3.48 - 3.60 (m, 5 H) 3.83 - 3.91 (m, 1 H) 4.01 (br s, 1 H) 4.09 - 4.31 (m, 3 H) 4.39 - 4.78 (m, 3 H) 4.80 - 4.96 (m, 1 H) 5.67 - 5.88 (m, 1 H) 7.20 - 7.41 (m, 5 H) 7.75 - 8.22 (m, 2 H) 8.91 (br d, 7=7.99 Hz, 1 H) 9.50 (br d, J=3.10 Hz, 1 H).
[0303] Example A5. Synthesis of Compound 5, MA-EVCit-GlySar-AEWSGR Docket No. 60801-718.601Scheme 5, Synthesis of MA-EVCIt-GlySar-AE linker-payload
[0304] Synthesis of Fmoc-aminoethoxypropionate NHS ester (11). To a solution of 10 (10 g, 28.14 mmol, 1 eq) in tetrahydrofuran (150 mL) was added DCC (8.71 g, 42.21 mmol, 1.5 eq), HOSu (4.86 g, 42.21 mmol, 1.5 eq). The mixture was degassed and purged with N2 for 3 times and stirred at 25°C for 12 h. LCMS showed starting material 10 was consumed completely and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give desired product 11 (10 g, crude). LCMS (ESI+, Method #1): m / z found 453.2 (M+l), calculated 453.2, RT: 1.724 min.
[0305] Synthesis of Fmoc-aminoethoxypropionate-Glu(OtBu)-OH (12). To a solution 11 (9 g, 19.89 mmol, 1 eq) in N, N-Dimethylformamide (90 mL) was added (2S)-2-amino-5-tert-butoxy-5-oxo-pentanoic acid (4.04 g, 19.89 mmol, 1 eq) and DIPEA (7.71 g, 59.67 mmol, 3 eq). The mixture was degassed and purged with N2 for 3 times, and stirred at 25°C for 3 h.WSGR Docket No. 60801-718.601LCMS showed starting material 11 was consumed completely and desired mass was detected. The reaction mixture was purified by Prep-HPLC (column: Phenomenex Luna C18 80 x30mm x 3um; mobile phase: [A: H2O (0.1% FA); B: ACN]; B%: 30.00%-50.00%, 10.00 min) to give product 12 (8 g, yield 66%). LCMS (ESI+, Method #1): m / z found 541.3 (M+l), calculated 541.3, RT: 1.774 min.
[0306] Synthesis of Fmoc-aminoethoxypropionate-Glu(OtBu)-OSu (13). To a solution of 12 (7 g, 12.95 mmol, 1 eq) in tetrahydrofuran (105 mL) was added DCC (4.01 g, 19.42 mmol, 1.5 eq), HOSu (2.24 g, 19.42 mmol, 1.5 eq). The mixture was degassed and purged with N2 for 3 times and stirred at 25°C for 12 h. LCMS showed starting material 12 was consumed completely and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give desired product 13 (6 g, crude). LCMS (ESI+, Method #1): m / z found 638.3 (M+l) calculated 638.3, RT: 1.937 min.
[0307] Synthesis of Fmoc-aminoethoxypropionate-Glu(OtBu)-Val-Cit-OH (14). To a solution starting material 13 (5 g, 7.84 mmol, 1 eq) in N, N-Dimethylformamide (50 mL) was added (2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5-ureido-pentanoic acid (3.23 g, 11.76 mmol, 1.5 eq) and DIPEA (3.04 g, 23.52 mmol, 3 eq). The mixture was degassed and purged with N2 for 3 times and stirred at 25°C for 3 h. LCMS showed 13 was consumed completely and desired mass was detected. The reaction mixture was purified by Prep-HPLC (column: Phenomenex Luna C18 80 x 30mm x 3um; mobile phase: [A: H2O (0.1% FA); B: ACN]; B%: 30.00%-50.00%, 10.00 min) to give desired product 14 (4 g, yield 64%). LCMS (ESI+, Method #1): m / z found 797.5 (M+l), calculated 797.4, RT: 1.596 min.
[0308] Synthesis of Fmoc-aminoethoxypropionate-Glu(OtBu)ValCit-GlySar-AE (15).To a solution of starting material 14 (30 mg, 34.88 pmol, 1 eq) in dimethylformamide (0.3 mL) was added Compound 1 (27.79 mg, 34.88 pmol, 1 eq), HATU (15.91 mg, 41.85 pmol, 1.5 eq) and DIPEA (9.02 mg, 69.76 pmol, 2 eq) at 0°C. The mixture was degassed and purged with N2 3 times, and stirred at 0°C for 1 hr. LCMS showed starting material 14 was consumed completely and desired mass was detected. Another five additional vials were set up as described above. All six reaction mixtures were combined. The reaction mixture was purified by Prep-HPLC, (column: 3_Phenomenex Luna C18 75 x 30 mm x 3 urn; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 30%-60% B over 8.0 min) to give desired product 15 (190 mg, yield 53%). LCMS (ESI+, Method #1): m / z found 820.5 ((M+2) / 2), calculated 820.6, RT: 1.753 min.WSGR Docket No. 60801-718.601
[0309] Synthesis of H-aminoethoxypropionate-Glu(OtBu)ValCit-GlySar-AE (16). To a solution of starting material 15 (190 mg, 111.29 pmol, 1 eq) in DMF (1.9 mL) was added piperidine (28.43 mg, 333.86 pmol, 32.97 pL, 3 eq). The mixture was stirred at 25°C for 1 hour. LCMS showed the starting material was consumed and one main peak with desired m / z was detected. The reaction mixture was purified by prep-HPLC (TFA condition: column: 3_Phenomenex Luna C18 75 * 30 mm * 3 um; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 10%-40% B over 8.0 min) to give desired product 16 (130 mg, 74% yield). LCMS (ESI+, Method #1): m / z found 709.4 ((M+2 / 2)), calculated 709.4, RT: 1.280 min.
[0310] Synthesis of MA-aminoethoxypropionate-Glu(OtBu)ValCit-GlySar-AE (17). To a solution of 16 (130 mg, 82.58 pmol, 1 eq) in dimethylformamide (1.3 mL) was added DIEA (21.35 mg, 165.16 pmol, 2 eq) and MA-OSu (24.99 mg, 90.10 pmol, 1.2 eq). The mixture was degassed and purged with N2 3 times, and stirred at 25°C for 1 hr. LCMS showed starting material 16 was consumed completely and desired mass was detected. The reaction mixture was purified by Prep-HPLC (column: Phenomenex Luna C18 75 x 30 mm x 3 um; mobile phase: [H2O (0.1% TFA)- ACN]; gradient: 15%- 45% B over 8.0 min) to give desired product 17 (100 mg, yield 73%). LCMS (ESI+, Method #1): m / z found 778.0 ((M+2) / 2), calculated 778.0, RT: 1.405 min.
[0311] Synthesis of MA-EVCit-GIySar-AE linker-payload Compound 5. To a solution of starting material 17 (100 mg, 60.49 pmol, 1 eq) in dichloromethane (0.8 mL) and TFA (0.2 mL) was stirred at 25°C for 2 hours. LC-MS showed the starting material was consumed completely and one main peak with desired m / z was detected. The reaction mixture was purified by prep-HPLC (TFA condition: column: 3_Phenomenex Luna C18 75 * 30 mm * 3 um; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 10%-40% B over 8.0 min) to give final product Compound 5 MA-EVCit-GIySar-AE (76 mg, 80% yield). LCMS (ESI+, Method #5): m / z found 749.4 ((M+2) / 2), calculated 749.9, RT: 1.989 min. HRMS (ES-): m / z found 1495.83 (M-l), calculated 1495.85.XH NMR (400 MHz, DMSO-d6) 8 ppm 0.71 - 1.11 (m, 30 H) 1.12 - 1.58 (m, 6 H) 1.59 - 2.20 (m, 9 H) 2.21 - 2.46 (m, 7 H) 2.71 - 2.88 (m, 7 H) 2.89 -2.97 (m, 2 H) 2.97 - 3.08 (m, 4 H) 3.18 (dd, J=12.26, 5.13 Hz, 6 H) 3.23 - 3.41 (m, 8 H) 3.69 - 3.75 (m, 3 H) 3.80 - 3.96 (m, 3 H) 3.97 - 4.08 (m, 4 H) 4.08 - 4.34 (m, 6 H) 4.35 - 4.50 (m, 1 H) 4.53 - 4.62 (m, 1 H) 4.64 - 4.80 (m, 1 H) 5.00 - 5.60 (m, 2 H) 5.62 - 5.84 (m, 1 H) 5.86 -6.00 (m, 1 H) 7.08 (s, 2 H) 7.13 - 7.48 (m, 5 H) 7.66 - 7.77 (m, 1 H) 7.81 - 7.93 (m, 1 H) 8.02 - 8.13 (m, 2 H) 8.19 (br t, J=5.19 Hz, 1 H) 8.91 (br d, J=8.13 Hz, 1 H) 9.32 - 9.71 (m, 1 H).
[0312] Example A6. Synthesis of Compound 6, MA-EVCit-SerSar-AEWSGR Docket No. 60801-718.601Scheme 6, Synthesis of MA-EVCit-SerSar-AE linker-payload
[0313] Synthesis of Fmoc-aminoethoxypropionate-Glu(OtBu)ValCit-SerSar-AE (19). To a solution of linker intermediate 14 (89.52 mg, 112.34 pmol, 1 eq) and prodrug Compound 3 (100 mg, 112.34 pmol, 1 eq) in DMF (1 mL) was added HATU (51.26 mg, 134.81 pmol, 1.2 eq) and DIPEA (29.04 mg, 224.68 pmol, 39.13 pL, 2 eq). The mixture was stirred at 0°C for 1 hour. LCMS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was purified by prep-HPLC (TFA condition: column: Phenomenex Luna C1875*30mm*3um; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 30%-60% B over 8.0 min) to give desired product 19 (120 mg, 64% yield). LCMS (ESI+, Method #1): m / z found 835.6 ((M+2) / 2), calculated 835.6, RT: 1.694 min.
[0314] Synthesis of H-aminoethoxypropionate-Glu(OtBu)ValCit-SerSar-AE (20). To a solution of 19 (100 mg, 59.91 pmol, 1 eq) in DMF (1 mL) was added piperidine (15.30 mg, 179.74 pmol, 17.75 pL, 3 eq). The mixture was stirred at 20°C for 1 hour. LC-MS showedWSGR Docket No. 60801-718.601the starting material was consumed completely and desired mass was detected. The reaction mixture was purified by prep-HPLC (TFA condition: column: 3_Phenomenex Luna Cl 8 75*30mm*3um; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 5%-40% B over 8.0 min) to give free amine product 20 (88 mg, 84% yield). LCMS (ESI+, Method #1): m / z found 724.4 ((M+2 / 2)), calculated 724.4, RT: 1.267 min.
[0315] Synthesis of MA-aminoethoxypropionate-Glu(OtBu)ValCit-SerSar-AE (21). To a solution of starting material 20 (88 mg, 60.82 pmol, 1 eq) in DMF (0.8 mL) was added DIPEA (23.58 mg, 182.47 pmol, 31.78 pL, 3 eq) and MA-OSu (23.01 mg, 91.24 pmol, 1.5 eq). The mixture was stirred at 20°C for 1 hour. LCMS showed the starting material was consumed completely and one main peak with desired mass was detected. The reaction mixture was purified directly by prep-HPLC (TFA condition: column: 3_Phenomenex Luna C18 75*30mm*3um; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 15%-45% B over 8.0 min) to give linker-payload 21 (50 mg, 52% yield). LCMS (ESI+, Method #1): m / z found 793.0 ((M+2) / 2), calculated 793.0, RT: 1.384 min.
[0316] Synthesis of MA-EVCit-SerSar-AE linker-payload Compound 6. To a solution of linker-payload intermediate 21 (50 mg, 31.57 pmol, 1 eq) in di chloromethane (0.4 mL) and TFA (0.1 mL) was stirred at 20°C for 1 hour. LCMS showed the reaction was completed. The reaction mixture was purified directly by prep-HPLC (TFA condition: column: 3_Phenomenex Luna C1875*30mm*3um; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 10%-40% B over 8.0 min) to give final product Compound 6, MA-EVCit-SerSar-AE (28 mg, 58% yield). LCMS (ESI+, Method #5): m / z found 764.4 ((M+2) / 2), calculated 764.9, RT: 1.966 min. HRMS (ES-): m / z found 1526.85 (M-l), calculated 1526.82. 'H NMR (400 MHz, DMSO-d6) 8 ppm 6 = 9.59 - 9.45 (m, 1H), 8.96 - 8.88 (m, 1H), 8.20 (br t, J = 5.1 Hz, 1H), 8.12 - 8.06 (m, 1H), 8.05 - 7.95 (m, 1H), 7.84 - 7.71 (m, 1H), 7.37 - 7.25 (m, 5H), 7.09 (s, 2H), 5.95 - 5.85 (m, 1H), 5.82 - 5.67 (m, 1H), 5.52 - 5.48 (m, 1H), 5.51 - 5.29 (m, 1H), 4.87 - 4.58 (m, 3H), 4.39 - 4.15 (m, 5H), 4.07 - 3.98 (m, 3H), 3.78 - 3.69 (m, 1H), 3.65 - 3.54 (m, 5H), 3.27 (br s, 4H), 3.25 (br d, J = 4.4 Hz, 4H), 3.22 - 3.17 (m, 8H), 3.14 (br d, J = 7.0 Hz, 3H), 3.00 (s, 2H), 2.96 - 2.87 (m, 4H), 2.84 - 2.73 (m, 7H), 2.27 - 2.19 (m, 3H), 2.18 - 2.01 (m, 3H), 1.98 - 1.93 (m, 1H), 1.91 - 1.83 (m, 2H), 1.81 - 1.69 (m, 3H), 1.64 - 1.54 (m, 2H), 1.53 - 1.41 (m, 2H), 1.41 - 1.27 (m, 4H), 1.10 - 0.80 (m, 30H).
[0317] Example A7. Synthesis of branched activated ester linker intermediate 26WSGR Docket No. 60801-718.601Scheme 7, Synthesis of branched activated ester linker intermediate 26
[0318] Synthesis orthogonally protected branched spacer unit (24). To a solution off free amine 23 (2.13 g, 6.01 mmol, 1 eq) in dichloromethane (21.3 mL) was added DIPEA (2.33 g, 18.02 mmol, 3.14 mL, 3 eq), HOBt (1.62 g, 12.01 mmol, 2 eq) and EDCI (2.30 g, 12.01 mmol, 2 eq) at 0°C. The mixture was stirred at 25°C for 3 h. LCMS showed the reaction was completed. The reaction mixture was quenched by addition water (50 mL), and then extracted with dichloromethane (20 mL x 3). The combined organic layers were washed with brine (50 mL), dried over Na2SC>4, filtered and concentrated under reduced pressure to give desired product 24 (3 g, 63% yield). LCMS (ESI+, Method #6): m / z found 787.5 (M+l), calculated 787.4, RT: 0.633 min.
[0319] Synthesis Fmoc-protected branched spacer unit di-acid (25). To a solution of 24 (3 g, 3.81 mmol, 1 eq) in di chloromethane (25 mL) was added TFA (5 mL). The mixture was stirred at 25°C for 3 hour. LCMS showed the reaction was completed. The reaction mixture was concentrated under high vacuum to give a residue. The residue obtained was purified by Prep-HPLC (TFA condition: column: Phenomenex Titank Cl 8 Bulk 250*70mm 10u;mobile phase: [H2O(0.1%TFA)-ACN];gradient:38%-68% B over 20.0 min) to give bis free acid 25 (2.3 g, 89% yield). LCMS (ESI+, Method #6): m / z found 675.4 (M+l), calculated 675.3, RT: 0.437 min.
[0320] Synthesis Fmoc-protected branched spacer unit bis-NHS activated ester (26).To a solution of di-acid 25 (500 mg, 741.03 pmol, 1 eq) in di chloromethane (5 mL) was added HOSu (255.85 mg, 2.22 mmol, 3 eq) and EDCI (426.17 mg, 2.22 mmol, 3 eq). The mixture was stirred at 25°C for 1 h. LCMS showed the starting material was consumedWSGR Docket No. 60801-718.601completely and one main peak with desired m / z. The reaction mixture was washed with IN HC1 (5 mL x 2) and brine (5 mL x 2), the organic phase was dried over Na2SC>4, filtered and concentrated under reduced pressure to give bis-activated ester product 26 (500 mg, 78% yield). LCMS (ESI+, Method #7): m / z found 869.4 (M+l), calculated 869.3, RT: 1.844 min.
[0321] Example A8. Synthesis of Compound 7, MA-(EVCit-SerSar-AE)2WSGR Docket No. 60801-718.601
[0322] Synthesis of Boc-Glu(OtBu)-OSu activated ester (28). To a solution of Boc-Glu(OtBu)-OH free acid 27 (1 g, 3.30 mmol, 1 eq) in dichloromethane (10 mL) was added HOSu (417.32 mg, 3.63 mmol, 1.1 eq) and EDCI (758.33 mg, 3.96 mmol, 1.2 eq). The mixture was stirred at 20°C for 1 hour. LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was washed with IN HC1 (5 mL x 2) and H2O (5 mL x 2), dried over Na2SC>4, filtered and concentrated under reduced pressure to give desired product 28 (1.2 g, 91% yield) which was used to next step directly without purification. LCMS (ESI+, Method #7): m / z found 423.2 (M+23), calculated 423.2, RT: 1.740 min.
[0323] Synthesis of Boc-Glu(OtBu)-Val-Cit-OH tripeptide (29). To a solution of activated ester 28 (1.2 g, 3.00 mmol, 1 eq) and (2S)-2-[[(2S)-2-amino-3-methyl-butanoyl] amino]-5-ureido-pentanoic acid (931.35 mg, 3.00 mmol, 1 eq, HC1) in dioxane (19.2 mL) and H2O (4.8 mL) was added NaHCCh (503.51 mg, 5.99 mmol, 233.21 pL, 2 eq). The mixture was stirred at 25°C for 2 hours. LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was decreased to pH=2~3 by addition IN HC1 (30 mL) at 20°C, and then extracted with ethyl acetate (50 mL x 3). The combined organic layers were washed with aqueous NaCl (100 mL), dried over Na2SC>4, filtered and concentrated under reduced pressure to give a crude reaction residue. The residue obtained was purified by flash silica gel chromatography (ISCO®; 4 g SepaFlash® Silica Flash Column, Eluent of 5-10% methanol / ethyl acetate @ 80 mL / min) to give tripeptide product 29 (450 mg, 27% yield). LCMS (ESI+, Method #7): m / z found 560.4 (M+l), calculated 560.3, RT: 1.361 min.
[0324] Synthesis of Boc-Glu(OtBu)-ValCit-SerSar-AE linker intermediate (30). To a solution of tripeptide 29 (300 mg, 536.05 pmol, 1 eq) and SerSar-AE prodrug Compound 3 (477.17 mg, 536.05 pmol, 1 eq) in DMF (3 mL) was added DIPEA (103.92 mg, 804.07 pmol, 140.05 pL, 1.5 eq) and HATU (244.59 mg, 643.26 pmol, 1.2 eq). The mixture was stirred at 0°C for 1 hour. LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction was purified by prep-HPLC (TFA condition: column:3_Phenomenex Luna C18 75*30mm*3um; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 20%-50% B over 8.0 min) to give protected tripeptide-SerSar-AE intermediate 30 (590 mg, 77% yield). LCMS (ESI+, Method #7): m / z found 716.8 ((M+2) / 2), calculated 716.9, RT: 1.521 min.
[0325] Synthesis of deprotected H-GluValCit-SerSar-AE linker intermediate (31). To a solution of Boc- and t-butyl ester protected starting material 30 (600 mg, 419.05 pmol, 1WSGR Docket No. 60801-718.601eq) in TFA (1.2 mL) and dichloromethane (4.8 mL) was stirred at 25°C for 3 hours. LC-MS showed the starting material was consumed and desired mass was detected. The reaction mixture was purified by prep-HPLC (TFA condition: column: 3_Phenomenex Luna Cl 8 75*30mm*3um; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 5%-35% B over 8.0 min) to give EVCit-SerSar-AE linker intermediate 31 (300 mg, 56% yield) as a yellow solid. LCMS (ESI+, Method #7): m / z found 638.6 ((M+2) / 2), calculated 638.8, RT: 1.159 min.
[0326] Synthesis of Fmoc-protected branched spacer DAR2 EVCit-SerSar-AE linkerpayload intermediate (32). To a solution of bis activated ester spacer intermediate 26 (65 mg, 74.81 pmol, 1 eq) and EVCit-SerSar-AE linker intermediate 31 (286.27 mg, 224.43 pmol, 3 eq) in DMF (0.65 mL) was added DIPEA (48.34 mg, 374.05 pmol, 65.15 pL, 5 eq) and the mixture was stirred at 25°C for 1 hour. LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was purified by prep-HPLC (TFA condition: column: Phenomenex Luna C18 80*30mm*3um; mobile phase:[H2O (0.1%TFA)-ACN]; gradient: 20%-50% B over 8.0 min) to give desired product 32 (160 mg, 67% yield). LCMS (ESI+, Method #7): m / z found 1595.5 ((M+2) / 2), calculated 1596.0, RT: 1.502 min.
[0327] Synthesis of free amine-terminated branched DAR2 EVCit-SerSar-AE linkerpayload intermediate (33). To a solution of Fmoc-protected starting material 32 (160 mg, 50.16 pmol, 1 eq) in DMF (1.6 mL) was added piperidine (64.06 mg, 752.39 pmol, 74.30 pL, 15 eq). The mixture was stirred at 25°C for 1 hour, after which time LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was purified by prep-HPLC (TFA condition: column: 3_Phenomenex Luna Cl 8 75*30mm*3um; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 10%-40% B over 8.0 min) to give free amine product 33 (120 mg, 80% yield). LCMS (ESI+, Method #7): m / z found 1484.4 ((M+2) / 2), calculated 1484.9, RT: 1.320 min.
[0328] Synthesis of MA-(EVCit-SerSar-AE)2 linker-payload Compound 7. To a solution off free amine starting material 33 (120 mg, 40.44 pmol, 1 eq) in DMF (1.2 mL) was added DIPEA (52.26 mg, 404.37 pmol, 70.43 pL, 10 eq) and MA-OSu (12.24 mg, 48.52 pmol, 1.2 eq). The mixture was stirred at 25°C for 1 hour, at which time LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was purified by prep-HPLC twice (TFA condition: column: 3_Phenomenex Luna C18 75*30mm*3um; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 15%-45% B over 8.0 min, column: Phenomenex Kinetex EVO C18 150*30mm 5um; mobile phase: [H2O (0.1%TFA)-ACN]; gradient: 15%-40% B over 20.0 min) to give MA-(EVCit-SerSar-AE)2WSGR Docket No. 60801-718.601linker-payload Compound 7 (25 mg, 20% yield). LCMS (ESI+, Method #5): m / z found 1552.8 ((M+2) / 2), calculated 1553.4, RT: 2.097 min. HRMS (ES-): m / z found 3103.78 (M-1), calculated 3103.76. ‘HNMR (400 MHz, DMSO-d6) 6 ppm 6 = 9.63 - 9.45 (m, 1H), 8.98 - 8.85 (m, 1H), 8.23 - 7.93 (m, 6H), 7.91 - 7.65 (m, 3H), 7.39 - 7.16 (m, 10H), 7.08 (s, 2H), 5.95 - 5.66 (m, 3H), 5.53 - 5.21 (m, 3H), 4.91 - 4.79 (m, 2H), 4.78 - 4.53 (m, 5H), 4.41 - 4.07 (m, 11H), 4.06 - 3.94 (m, 4H), 3.85 (br d, J = 9.2 Hz, 1H), 3.77 - 3.66 (m, 4H), 3.63 - 3.54 (m, 14H), 3.40 - 3.35 (m, 8H), 3.29 - 3.10 (m, 25H), 3.00 (s, 4H), 2.90 (br dd, J = 4.1, 10.9 Hz, 5H), 2.85 - 2.71 (m, 13H), 2.60 (br t, J = 6.6 Hz, 3H), 2.46 - 2.37 (m, 3H), 2.37 - 2.17 (m, 9H), 2.16 - 2.00 (m, 3H), 1.99 - 1.81 (m, 6H), 1.80 - 1.66 (m, 5H), 1.65 - 1.52 (m, 4H), 1.52 -1.41 (m, 3H), 1.39 - 1.22 (m, 7H), 1.13 - 0.72 (m, 60H).
[0329] Example A9. Synthesis of Compound 8, MA-EVCit-SerSar-MMAEWSGR Docket No. 60801-718.601
[0330] Synthesis of Boc-MMAE (36). To a solution of MMAE, 35 (8 g, 11.14 mmol, 1 eq) in acetonitrile (160 mL) was added DIPEA (2.74 g, 21.17 mmol, 1.9 eq), (Boc)2O (4.62 g, 21.17 mmol, 1.9 eq). The mixture was degassed and purged with N2 three times and stirred at 25°C for 12 h. LCMS showed MMAE was consumed completely and desired mass was detected. The reaction mixture was concentrated under reduced pressure to give residue. The crude product was triturated with 10 volumes of 1:1 petroleum: ethyl acetate at 25°C for 2 h. The crude product was filtered and concentrated under reduced pressure to give Boc-MMAE (8 g, yield 87%). LCMS (ESI+, Method #8): m / z found 818.7 (M+l), calculated 818.6, RT: 15.529 min.
[0331] Synthesis of Boc-MMAE-Sar-Fmoc (37). To a solution of Boc-MMAE, 36 (6.2 g, 7.58 mmol 1 eq) in dichloromethane (62 mL) was added DIC (1.43 g, 11.37 mmol, 1.5 eq), DMAP (92.59 mg, 757.86 pmol, 0.1 eq), 2-[9H-fluoren-9-ylmethoxycarbonyl (methyl) amino] acetic acid (2.36 g, 7.58 mmol, 1 eq). The mixture was degassed and purged with N2 three times and stirred at 25°C for 1 h. LCMS showed that the starting material 36 was consumed completely and desired mass was detected. The reaction mixture was quenched by addition H2O (50 mL) at 25°C, and extracted with dichloromethane (50 mL x 2). The combined organic layers were washed with saturated NaCl (50 mL), dried over Na2SC>4, filtered and concentrated under reduced pressure to give desired product Boc-MMAE-Sar-Fmoc, 37 (7 g, yield 83%). The crude product was used into the next step without further purification. LCMS (ESI+, Method #8): m / z foundllll.7 (M+l), calculated 1111.7, RT: 12.872 min.
[0332] Synthesis of Boc-MMAE-Sar (38). To a solution of starting material 37 (7 g, 6.30 mmol, 1 eq) in N, N-Dimethylformamide (70 mL) was added piperidine (1.61 g, 18.89 mmol, 3 eq). The mixture was degassed and purged with N2 three times and stirred at 25°C for 1 h. LCMS showed 37 was consumed completely and desired mass was detected by LC / MS. The reaction mixture was purified by Prep-HPLC, (column: Phenomenex Luna C18 80 x 30 mm x 3 urn; mobile phase: [A: H2O (0.1% TFA); B: ACN]; B%: 30.00%-50.00%, 10.00 min) to provide desired product Boc-MMAE-Sar, 38 (4 g, yield 71%). LCMS (ESI+, Method #8): m / z found 889.7 (M+l), calculated 889.6, RT: 13.224 min.
[0333] Synthesis of Fmoc-aminoethoxypropionate-Glu(OtBu)ValCit-OSu activated ester (39). To a solution of linker intermediate 14 (3 g, 3.76 mmol, 1 eq) in tetrahydrofuran (20 mL), N, N-dimethylformamide (10 mL) was added DCC (1.17 g, 5.65 mmol, 1.5 eq), N-hydroxysuccinimide (649.88 mg, 19.42 mmol, 1.5 eq). The mixture was degassed and purged with N2 three times and stirred at 25°C for 1 h. LCMS showed starting material 14 was consumed completely and desired mass was detected. The residue was filtered andWSGR Docket No. 60801-718.601concentrated under reduced pressure to give desired product 39 (2.5 g, crude) and carried forward without further purification. LCMS (ESI+, Method #1): m / z found 894.5 (M+l), calculated 894.4, RT: 1.719 min.
[0334] Synthesis of Fmoc-aminoethoxypropionate-Glu(OtBu)ValCit-Ser-OH (40). To a solution of activated ester 39 (2.5 g, 2.80 mmol, 1 eq) in N, N-dimethylformamide (25 mL) was added DIPEA (722.85 mg, 5.59 mmol, 2 eq), and (S)-serine (323.28 mg, 3.08 mmol, 1.1 eq). The mixture was degassed, purged with N2 three times, and stirred at 25°C for 2 h. LCMS showed activated ester 39 was consumed completely and desired mass was detected. The reaction mixture was purified by Prep-HPLC, (column: Phenomenex Luna C18 80 * 30 mm x 3 um; mobile phase: [A: H2O (0.1% TFA); B: ACN]; B%: 30.00%-50.00%, 10.00 min) to give desired product 40 (1.4 g, yield 46%). LCMS (ESI+, Method #8): m / z found 884.5 (M+l) calculated 884.4, RT: 11.278 min.
[0335] Synthesis of Fmoc-aminoethoxypropionate-Glu(OtBu)ValCit-SerSar-MMAE-Boc (41). To a solution of linker intermediate 40 (1.3 g, 1.47 mmol, 1 eq) in N, N-Dimethylformamide (13 mL) was added DIPEA (380.13 mg, 2.94 mmol, 2 eq), HCTU (730.05 mg, 1.76 mmol, 1.2 eq), and Boc-MMAE-Sar 38 (1.3 g, 1.47 mmol, 1 eq). The mixture was degassed, purged with N2 three times, and stirred at 0°C for 1 h. LCMS showed 40 was consumed completely and desired mass was detected. The reaction mixture was purified by Prep-HPLC, (column: Phenomenex Luna C18 80 x 30 mm x 3 um; mobile phase: [A: H2O (0.1% TFA); B: ACN]; B%: 30.00%-50.00%, 10.00 min) to give linker-payload intermediate 41 (1 g, yield 38%). LCMS (ESI+, Method #8): m / z found 878.5 ((M+2) / 2), calculated 878.6, RT: 9.165 min.
[0336] Synthesis of aminoethoxypropionate-Glu(OtBu)ValCit-SerSar-MMAE-Boc (42).To a solution of linker-payload intermediate 41 (1 g, 569.76 pmol, 1 eq) in N, N-dimethylformamide (10 mL) was added piperidine (145.54 mg, 1.71 mmol, 3 eq). The mixture was degassed, and purged with N2 three times, and stirred at 25°C for 1 h. LCMS showed starting material 41 was consumed completely and desired mass was detected. The reaction mixture was purified by Prep-HPLC, (column: Phenomenex Luna C18 80 x 30 mm x 3 um; mobile phase: [A: H2O (0.1% TFA); B: ACN]; B%: 30.00%-50.00%, 10.00 min) to give free amine 42 (800 mg, yield 91%). LCMS (ESI+, Method #8): m / z found 767.4 ((M+2) / 2), calculated 767.5, RT: 12.785 min.
[0337] Synthesis of MA-aminoethoxypropionate-Glu(OtBu)ValCit-SerSar-MMAE-Boc (43). To a solution of linker-payload intermediate 42 (800 mg, 521.89 pmol, 1 eq) in N, N-dimethylformamide (8 mL) was added DIPEA (134.90 mg, 1.04 mmol, 2 eq) and MA-OSuWSGR Docket No. 60801-718.601(131.61 mg, 521.89 pmol, 1 eq). The mixture was degassed, purged with N2 three times, and stirred at 25°C for 1 h. LCMS showed starting material 42 was consumed completely and desired mass was detected. The reaction mixture was purified by Prep-HPLC (column: Phenomenex Luna C18 80 x 30 mm x 3 um; mobile phase: [A: H2O (0.1% TFA); B: ACN]; B%: 30.00%-50.00%, 10.00 min) to give penultimate linker-payload intermediate 43 (500 mg, yield 57%). LCMS (ESI+, Method #8): m / z found 835.9 ((M+2) / 2), calculated 836.0, RT: 5.479 min.
[0338] Synthesis of MA-EVCit-SerSar-MMAE Compound 8. To a solution of starting material 43 (500 mg, 297.01 pmol, 1 eq) in dichloromethane (5 mL) was added trifluoroacetic acid (1 mL, 13.35 mmol, 1 eq). The mixture was degassed, purged with N2 three times, and stirred at 25°C for 3 h. LCMS showed starting material 43 was consumed and the desired mass was detected. The reaction mixture was purified by Prep-HPLC (column: 3_Phenomenex Luna C1875 x 30 mm x 3 um; mobile phase: [H2O (0.1% TFA)- ACN]; gradient: 10%- 45% B over 8.0 min]; B%: 30.00%- 50.00%, 10.00 min) to give Compound 8, MA-EVCit-SerSar-MMAE (200 mg, yield 44%). LCMS (ESI+, Method #8): m / z found 757.8 ((M+2) / 2), calculated 757.9, RT: 8.062 min. HRMS (ES-): m / z found 1511.85 (M-l), calculated 1511.84. 'H NMR (400 MHz, DMSO-d6) 6 ppm 0.75 - 1.01 (m, 30 H) 1.06 - 1.11 (m, 2 H) 1.19 - 1.34 (m, 4 H) 1.43 - 1.65 (m, 6 H) 1.80 - 2.11 (m, 6 H) 2.16 - 2.30 (m, 4 H) 2.35 - 2.46 (m, 4 H) 2.69 - 2.83 (m, 10 H) 2.99 (s, 1 H) 3.12 - 3.27 (m, 14 H) 3.36 - 3.39 (m, 4 H) 3.59 (br t, J=6.25 Hz, 3 H) 3.82 (br d, J=8.50 Hz, 1 H) 4.02 (s, 2 H) 4.08 - 4.20 (m, 3 H) 4.26 - 4.35 (m, 2 H) 4.53 - 4.62 (m, 1 H) 4.79 - 4.88 (m, 1 H) 4.91 - 5.06 (m, 1 H) 5.57 - 5.95 (m, 1 H) 7.08 (s, 2 H) 7.22 - 7.37 (m, 5 H) 7.56 - 7.67 (m, 3 H) 7.68 - 7.72 (m, 1 H) 7.76 - 7.98 (m, 2 H) 8.00 - 8.08 (m, 2 H) 8.18 (br t, J=5.50 Hz, 1 H) 8.67 - 9.06 (m, 1 H) 9.39 - 9.67 (m, 1 H) 11.96 - 12.26 (m, 1 H).
[0339] Example A10. Synthesis of Compound 9, MA-(EVCit-SerSar-MMAE)2WSGR Docket No. 60801-718.601>Scheme 10, Synthesis of MA-(EVCit-SerSar-MMAE)2 linker-payload
[0340] Synthesis of Fmoc-Glu(OtBu)-OSu activated ester (46). To a solution of orthogonally protected glutamate starting material 45 (2 g, 4.70 mmol, 1 eq) inWSGR Docket No. 60801-718.601dichloromethane (20 mL) was added N-hydroxysuccinimide (540.98 mg, 4.70 mmol, 1 eq) and DCC (969.87 mg, 4.70 mmol, 950.85 pL, 1 eq). The mixture was stirred at 25°C for 4 hours. LCMS showed that starting material 45 was consumed and desired product mass was detected. The reaction mixture was filtered and filtrate was concentrated under reduced pressure to give activated ester 46 (2.2 g, 89% yield) which was used to next step reaction without purification. LCMS (ESI+, Method #9): m / z found 545.1 (M+23), calculated 545.2, RT: 2.099 min.
[0341] Synthesis of Fmoc-Glu(OtBu)-Val-Cit-OH tripeptide (47). To a solution of activated ester 46 (2.1 g, 4.02 mmol, 1 eq) and (2S)-2-[[(2S)-2- amino-3-methyl-butanoyl] amino]-5-ureido-pentanoic acid (1.43 g, 5.22 mmol, 1.3 eq) in dioxane (16 mL) and water (4 mL) was added sodium bicarbonate (2.03 g, 24.11 mmol, 938.23 pL, 6 eq). The mixture was stirred at 25°C for 3 hours. LCMS showed the starting material was consumed and desired mass for the product was detected. The reaction mixture was adjusted to pH=l-2 with HC1 (1 M, 10 mL) and then diluted with H2O (10 mL) and extracted with ethyl acetate (10 mL x 4). The combined organic layers were washed with brine (15 mL x 2), dried over Na2SC>4 filtered and concentrated under reduced pressure to give a residue. The residue was triturated with ethyl acetate (10 ml) at 25°C for 1 hour. The filtration solid was concentrated under reduced pressure to give tripeptide linker intermediate 47 (1.1 g, 40% yield). LCMS (ESI+, Method #6): m / z found 682.3 (M+l), calculated 682.3, RT: 0.494 min.
[0342] Synthesis of Fmoc-Glu(OtBu)-Val-Cit-OSu activated ester (48). To a solution of tripeptide 47 (1.1 g, 1.61 mmol, 1 eq) in N,N-dimethylformamide (11 mL) was added N-hydroxysuccinimide (278.54 mg, 2.42 mmol, 1.5 eq) and EDCI (463.95 mg, 2.42 mmol, 1.5 eq). The mixture was stirred at 25°C for 2 hours. LCMS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was filtered and filtrate was concentrated under reduced pressure to give activated ester 48 (1.1 g, 68% yield) which was used to next step reaction without purification. LCMS (ESI+, Method #9): m / z found 779.4 (M+l), calculated 779.4, RT: 1.802 min.
[0343] Synthesis of Fmoc-Glu(OtBu)-Val-Cit-Ser-OH tripeptide (49). To a solution of activated ester starting material 48 (1.1 g, 1.41 mmol, 1 eq) and (S)-serine (219.91 mg, 1.55 mmol, 1.1 eq) in dioxane (10 mL) and water (2 mL) was added sodium bicarbonate (237.29 mg, 2.82 mmol, 109.91 pL, 2 eq). The mixture was stirred at 25°C for 2 hours. The reaction mixture was adjusted to pH=l-2 with HC1 (1 M, 8 mL) and then diluted with water (10 mL) and extracted with ethyl acetate (8 mL x 4). The combined organic layers were washed with brine (10 mL x 2), dried over Na2SC>4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (TFA condition: column: PhenomenexWSGR Docket No. 60801-718.601Luna C18 (250*70mm,15 um);mobile phase: [H20(0.1%TFA)-ACN];gradient:30%-65% B over 20.0 min) to give desired product 49 (600 mg, 92% yield). LCMS (ESI+, Method #9): m / z found 769.4 (M+l), calculated 769.4, RT: 1.624 min.
[0344] Synthesis of Fmoc-Glu(OtBu)ValCit-SerSar-MMAE-Boc (50). To a solution of tetrapeptide linker intermediate 49 (600 mg, 780.39 pmol, 1 eq) and Boc-MMAE-Sar 38 (693.89 mg, 780.39 pmol, 1 eq) in N,N-dimethylformamide (6 mL) was added HATU (534.11 mg, 1.40 mmol, 1.8 eq) and DIPEA (201.72 mg, 1.56 mmol, 271.86 pL, 2 eq). The mixture was stirred at 0°C for 1 hour. LCMS showed the starting material was consumed and desired product mass was detected. The reaction mixture was filtered and filtrate was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (TFA condition: column: Waters Xbridge BEH C18 250*70mm*10pm ; mobile phase: [H2O (0.1%TFA)-ACN]; gradient: 60%-95% B over 20.0 min) to give desired product 50 (700 mg, 55% yield). LCMS (ESI+, Method #9): m / z found 1639.8 (M+l), calculated 1640.0, RT: 2.386 min.
[0345] Synthesis of H-Glu(OtBu)ValCit-SerSar-MMAE-Boc (51). To a solution of linkerpayload intermediate 50 (700 mg, 426.83 pmol, 1 eq) in N, N-dimethylformamide (7 mL) was added piperidine (363.43 mg, 4.27 mmol, 421.52 pL, 10 eq). The mixture was stirred at 25°C for 1 hour. LCMS showed the starting material was consumed and desired product mass was detected. The reaction mixture was filtered and filtrate was concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (TFA condition: Waters Xbridge BEHC18250*70mm*10pm ; mobilephase: [H2O (0.1%TFA)-ACN]; gradient: 35%-62% B over 18.0 min) to give free amine product 51 (400 mg, 66% yield). LCMS (ESI+, Method #9): m / z found 1417.9 (M+l), calculated 1417.9, RT: 1.753 min.
[0346] Synthesis of Fmoc-protected branched spacer DAR2 EVCit-SerSar-MMAE linker-payload intermediate (52). To a solution of linker-payload intermediate 51 (400 mg, 282 pmol, 2.5 eq) and linker intermediate 26 (98 mg, 112.8 pmol, 1 eq) in N, N-dimethylformamide (4 mL) was added DIPEA (43.76 mg, 338.56 pmol, 90.21 pL, 3 eq). The mixture was stirred at 25°C for 12 hours. LCMS showed the starting material was consumed and desired product mass was detected. The reaction mixture was filtered and filtrate concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (3_Phenomenex Luna C18 75*30mm*3um; mobile phase: [H2O (0.1% TFA)-ACN];gradient:60%-90% B over 8.0 min) to give branched linker-payload intermediate 52WSGR Docket No. 60801-718.601(300 mg, 76.52% yield). LCMS (ESI+, Method #9): m / z found 1738.0 ((M+2) / 2), calculated 1738.2, RT: 2.613 min.
[0347] Synthesis of free amine-terminated branched DAR2 EVCit-SerSar-MMAE linkerpayload intermediate (53). To a solution of Fmoc-protected linker-payload intermediate 52 (300 mg, 86.35 pmol, 1 eq) in DMF (3 mL) was added piperidine (110.29 mg, 1.30 mmol, 127.92 pL, 15 eq) at 25°C. After one hour, LCMS showed starting material was consumed and desired product was formed. The reaction was purified by prep-HPLC (TFA condition: column: 3_Phenomenex Luna C18 75*30mm*3um; mobile phase: [H2O (0.1% TFA)-ACN];gradient:45%-75% B over 8.0 min) to provide desired product 53 (200 mg, 71% yield). LCMS (ESI+, Method #9): m / z found 1084.9 ((M+3) / 3), calculated 1085.0, RT: 16.08 min.
[0348] Synthesis of t-butyl-protected MA-(EVCit-SerSar-MMAE)2 linker-payload intermediate (54). To a solution of starting material 53 (190 mg, 58.43 pmol, 1 eq) in DMF (1.9 mL) was added MA-OSu (17.68 mg, 70.11 pmol, 1.2 eq) and DIPEA (75.51 mg, 584.26 pmol, 101.77 pL, 10 eq). The reaction was stirred at 25°C for 0.5 h. LCMS showed starting material was consumed and a product with the desired mass was formed. The reaction was purified by pre-HPLC (TFA condition column: 3_Phenomenex Luna C18 75*30mm*3um;mobile phase: [H2O (0.1% TFA)-ACN];gradient:50%-80% B over 8.0 min) to give linker-payload intermediate 54 (100 mg, 47% yield, 95% purity). LCMS (ESI+, Method #8): m / z found 1130.6 ((M+3) / 3), calculated 1130.7, RT: 16.90 min.
[0349] Synthesis of MA-(EVCit-SerSar-MMAE)2 linker-payload Compound 9. To a solution of butyl-protected linker-payload starting material 54 (100 mg, 29.51 pmol, 1 eq) in DCM (1.6 mL) was added TFA (0.4 mL) at ambient temperature. The reaction was stirred at 25°C for 1 h. LCMS showed starting material was consumed and desired product was formed. The reaction was purified by pre-HPLC (TFA condition: column: 3_Phenomenex Luna Cl 8 75*30mm*3um;mobile phase: [H2O(0.1% TFA)-ACN];gradient:15%-45% B over 8.0 min) to give linker-payload Compound 9 (35 mg, 37% yield, 98% purity)f. LCMS (ESI+, Method #5): m / z found 1538.8 ((M+2) / 2), calculated 1538.4, RT: 2.077 min. HRMS (ES-): m / z found 3073.75 (M-l), calculated 3173.77. 'HNMR (400 MHz, DMSO-d6) 8 ppm 0.72 - 0.96 (m, 56 H) 1.01 (br d, J=6.88 Hz, 7 H) 1.07 (br d, J=6.50 Hz, 3 H) 1.29 - 1.37 (m, 6 H) 1.42 (br s, 3 H) 1.54 - 1.62 (m, 3 H) 1.66 - 1.76 (m, 5 H) 1.85 - 1.93 (m, 4 H) 1.98 - 2.16 (m, 7 H) 2.19 - 2.25 (m, 4 H) 2.29 - 2.36 (m, 4 H) 2.38 - 2.42 (m, 1 H) 2.45 (br s, 2 H) 2.60 (br t, J=6.69 Hz, 3 H) 2.83 - 3.01 (m, 10 H) 3.11 - 3.26 (m, 28 H) 3.45 - 3.49 (m, 15 H) 3.55 - 3.62 (m, 12 H) 3.84 (br d, J=9.76 Hz, 2 H) 4.02 (s, 3 H) 4.13 - 4.35 (m, 12 H) 4.44 - 4.77 (m, 7 H) 4.78 - 4.92 (m, 3 H) 5.29 - 5.49 (m, 3 H) 5.67 - 5.72 (m, 1 H) 5.79 (br d, J=6.75 Hz, 1 H) 5.85 - 5.95 (m, 2 H)WSGR Docket No. 60801-718.6017.08 (s, 2 H) 7.25 - 7.36 (m, 11 H) 7.67 - 7.82 (m, 3 H) 7.94 - 8.11 (m, 7 H) 8.19 (br t, J=5.32 Hz, 1 H) 8.58 - 8.71 (m, 2 H) 8.76 - 8.88 (m, 4 H).
[0350] Example All. Synthesis of Compound 18, H-GlyPro-AEScheme 11, Synthesis of GlyPro-AE dipeptide prodrug
[0351] Synthesis of Boc-Pro-AE (55). To a solution of AE, 1 (500 mg, 683.06 pmol, 1 eq) and (2S)-l-tert-butoxycarbonylpyrrolidine-2-carboxylic acid (176.43 mg, 819.67 pmol, 1.2 eq) in dichloromethane (5 mL) was added DMAP (8.34 mg, 68.31 pmol, 0.1 eq and DIC (129.30 mg, 1.02 mmol, 158.65 pL, 1.5 eq). The mixture was stirred at 25°C for 1 hour, at which time LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was diluted with H2O (10 mL) and extracted with dichloromethane (8 mL x 4). The combined organic layers were washed with aqueous NaCl (5 mL x 2), dried over Na2SC>4, filtered, and concentrated under reduced pressure to give Boc-Pro-AE, 55 (650 mg, 93% yield) as a white solid. LCMS (ESI+, Method #6): m / z found 929.7, calculated 929.6 (M+l), RT: 0.497 min.
[0352] Synthesis of Pro-AE (56). A solution of Boc-Pro-AE 55 (550 mg, 591.89 pmol, 1 eq) dissolved in dichloromethane (5 mL) and trifluoroacetic acid (1 mL) was stirred at 25°C for 1 hour. LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a reaction residue. The residue obtained was purified by prep-HPLC (TFA condition: column: Phenomenex Luna C18 100*30mm*5um; mobile phase: [H2O (0.04% HC1) - ACN]; gradient: 10%-40% B over 8.0 min) to give Pro-AE, 56 (530 mg, 92% yield) as a white solid. LCMS (ESI+, Method #1): m / z found 829.5, calculated 829.6 (M+l), RT: 1.182 min.WSGR Docket No. 60801-718.601
[0353] Synthesis of Boc-GlyPro-AE (57). To a solution of H-Pro-AE 56 (100 mg, 120.61 pmol, 1 eq and 2-(tert-butoxycarbonylamino)acetic acid (25.35 mg, 144.73 pmol, 1.2 eq) in DMF (1 mL) was added HATU (68.79 mg, 180.92 pmol, 1.5 eq) and DIPEA (15.59 mg, 120.61 pmol, 21.01 pL, 1 eq) at 0°C. The resulting mixture was stirred at 0°C for 1 hour, at which time LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was diluted with H2O (3 mL) and extracted with dichloromethane (5 mL x 3). The combined organic layers were washed with aqueous NaCl (2 mL x 3), dried over Na2SC>4, filtered and concentrated under reduced pressure to give Boc-GlyPro-AE 57 (110 mg, 92% yield) as a white solid. LCMS (ESI+, Method #9): m / z found 986.5, calculated 986.7 (M+l), RT: 1.523 min.
[0354] Synthesis of H-GlyPro-AE (compound 18). To a solution of Boc-GlyPro-AE 57 (80 mg, 81.11 pmol, 1 eq in dichloromethane (0.8 mL) was added trifluoroacetic acid (0.16 mL). The mixture was stirred at 25°C for 1 hour. LC-MS showed the starting material was consumed and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a reaction residue. The residue obtained was purified by prep-HPLC (TFA condition: column: 3_Phenomenex Luna C18 75*30mm*3um; mobile phase: [H2O (0.1% TFA) - ACN]; gradient: 10%-40% B over 8.0 min) to give Compound 18, H-GlyPro-AE (87 mg, 88% yield) as a white solid. LCMS (ESI+, Method #5): m / z found 886.5, calculated 886.6 (M+l), RT: 1.891 min. HRMS (ES+): m / z found 886.60 (M+l), calculated 886.59.1H NMR (400 MHz, CD3SOCD3, 298 K) 8 (ppm) = 9.69 -9.49 (m, 1H), 8.91 (br d, J= 4.5 Hz, 1H), 8.29 - 7.74 (m, 4H), 7.46 - 7.17 (m, 5H), 5.92 -5.66 (m, 1H), 4.80 - 4.63 (m, 1H), 4.61 - 4.53 (m, 1H), 4.52 - 4.43 (m, 1H), 4.35 - 4.16 (m, 1H), 4.09 - 3.94 (m, 1H), 3.92 - 3.79 (m, 2H), 3.71 (br d, J= 1.1 Hz, 1H), 3.64 - 3.47 (m, 4H), 3.25 (br d, J= 13.0 Hz, 4H), 3.21 - 3.13 (m, 4H), 3.11 - 2.92 (m, 2H), 2.86 - 2.70 (m, 6H), 2.35 - 2.23 (m, 3H), 2.17 - 1.65 (m, 8H), 1.62 - 1.43 (m, 2H), 1.42 - 1.21 (m, 2H), 1.14 -0.73 (m, 24H)
[0355] Example All. Synthesis of Compound 19, NAc-GlyPro-AEScheme 12, Synthesis of NAc-GlyPro-AE dipeptide prodrugWSGR Docket No. 60801-718.601
[0356] Synthesis of NAc-GlyPro-AE (compound 19). To a solution of H-Pro-AE 56 (60 mg, 72.37 pmol, 1 eq) and 2-acetamidoacetic acid (10.17 mg, 86.84 pmol, 1.2 eq) in DMF (0.6 mL) was added HATU (41.27 mg, 108.55 pmol, 1.5 eq) and DIPEA (9.35 mg, 72.37 pmol, 12.60 pL, 1 eq) at 0°C. The resulting mixture was stirred at 0°C for 1 hour, at which time LC-MS showed the starting material was consumed and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (neutral condition: column: Waters Xbridge BEH Cl 8 100*30mm*10pm ; mobile phase: [H2O (lOmM NH4HCO3) - ACN]; gradient: 20%-80% B over 8.0 min) to give Compound 19, NAc-GlyPro-AE (50 mg, 37% yield) as a white solid. LCMS (ESI+, Method #5): m / z found 928.5, calculated 928.6 (M+l), RT: 2.047 min. HRMS (ES+): m / z found 928.62 (M+l), calculated 926.60. 'H NMR (400 MHz, CD3SOCD3, 300 K) 8 (ppm) = 8.12 - 7.97 (m, 2H), 7.92 - 7.60 (m, 1H), 7.46 - 7.13 (m, 5H), 5.93 - 5.55 (m, 1H), 4.89 - 4.45 (m, 2H), 4.44 - 4.32 (m, 1H), 4.31 - 4.10 (m, 1H), 4.07 - 3.80 (m, 3H), 3.70 -3.45 (m, 3H), 3.44 - 3.35 (m, 2H), 3.29 - 3.22 (m, 4H), 3.22 - 3.15 (m, 5H), 2.99 (s, 2H), 2.65 - 2.60 (m, 1H), 2.46 - 2.35 (m, 2H), 2.31 - 2.06 (m, 9H), 2.04 - 1.68 (m, 10H), 1.64 - 1.17 (m, 3H), 1.16 - 0.69 (m, 24H).
[0357] Example A13. Synthesis of Compound 20, H-SerPro-AEScheme 13, Synthesis of SerPro-AE dipeptide prodrug
[0358] Synthesis of Boc-SerPro-AE (58). To a solution of (2S)-2-(tert-butoxycarbonylamino)-3-hydroxy-propanoic acid (59.40 mg, 289.46 pmol, 1.2 eq and H-Pro-AE, 56 (200 mg, 241.22 pmol, 1 eq) in DMF (2 mL) was added HATU (137.58 mg, 361.83 pmol, 1.5 eq) and DIPEA (31.18 mg, 241.22 pmol, 42.02 pL, 1 eq) at 0°C. The resulting mixture was stirred at 0°C for 1 hour. LC-MS showed the starting material was completely consumed and desired mass was detected. The reaction mixture was diluted with H2O (10 mL) and extracted with dichloromethane (8 mL x 4). The combined organic layersWSGR Docket No. 60801-718.601were washed with aqueous NaCl (5 mL x 2), dried over Na2SC>4, filtered and concentrated under reduced pressure to give Boc-SerPro-AE 58 (230 mg, 94% yield) as a white solid.LCMS (ESI+, Method #6): m / z found 1016.6, calculated 1016.7 (M+l), RT: 0.433 min.
[0359] Synthesis of H-SerPro-AE (Compound 20). A solution of Boc-SerPro-AE 58 (200 mg, 196.79 pmol, 1 eq) in di chloromethane (2 mL) and trifluoroacetic acid (0.4 mL) was stirred at 25°C for 1 hour. LC-MS showed the starting material was consumed completely and desired mass was detected for the product. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (TFA condition: column: Phenomenex Luna C18 80*30mm*3um; mobile phase:[H2O (0.1%TFA) - ACN]; gradient: 10%-40% B over 8.0 min) to give Compound 20, H-SerPro-AE (150 mg, 75.63% yield) as a white solid. LCMS (ESI+, Method #5): m / z found 916.4, calculated 916.6 (M+l), RT: 1.873 min. HRMS (ES+): m / z found 916.61 (M+l), calculated 916.60. 'HNMR (400 MHz, CD3SOCD3, 299 K) 8 (ppm) = 9.65 - 9.43 (m, 1H), 9.00 - 8.87 (m, 1H), 8.14 (br s, 2H), 8.05 - 7.66 (m, 1H), 7.39 - 7.22 (m, 4H), 5.81 - 5.49 (m, 2H), 4.81 - 4.54 (m, 2H), 4.50 - 4.41 (m, 1H), 4.34 - 4.14 (m, 2H), 4.11 - 3.93 (m, 1H), 3.91 -3.63 (m, 4H), 3.63 - 3.51 (m, 4H), 3.30 - 3.25 (m, 2H), 3.22 (d, J= 3.3 Hz, 3H), 3.20 - 3.13 (m, 3H), 3.12 - 2.95 (m, 2H), 2.88 - 2.72 (m, 5H), 2.44 (br s, 3H), 2.32 - 2.20 (m, 3H), 2.16 -1.99 (m, 2H), 1.97 - 1.60 (m, 6H), 1.59 - 1.23 (m, 3H), 1.14 - 0.71 (m, 24H).
[0360] Example A14. Synthesis of Compound 21, H-SerPro-AEScheme 14, Synthesis of NAc-SerPro-AE dipeptide prodrug
[0361] Synthesis of NAc-SerPro-AE (Compound 21). To a solution of (2S)-2-acetamido-3 -hydroxy -propanoic acid (10.65 mg, 72.37 pmol, 1 eq) and H-Pro-AE, 56 (60 mg, 72.37 pmol, 1 eq) in DMF (0.6 mL) was added HATU (41.27 mg, 108.55 pmol, 1.5 eq) and DIPEA (9.35 mg, 72.37 pmol, 12.60 pL, 1 eq) at 0°C. The resulting mixture was stirred for one hour at 0°C. LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (TFA condition: column: Phenomenex Luna C18 80*30mm*3um; mobile phase: [H2O (0.1%TFA) - ACN]; gradient: 10%-40% BWSGR Docket No. 60801-718.601over 8.0 min) to give Compound 21, NAc-SerPro-AE (27.5 mg, 38% yield) as a white solid. LCMS (ESI+, Method #5): m / z found 958.5, calculated 958.6 (M+l), RT: 2.024 min. HRMS (ES+): m / z found 958.63 (M+l), calculated 958.62. 'HNMR (400 MHz, CD3SOCD3, 299 K) 6 (ppm) = 9.70 - 9.51 (m, 1H), 8.97 - 8.87 (m, 1H), 8.28 - 7.62 (m, 1H), 7.41 - 7.21 (m, 4H), 5.82 - 5.60 (m, 1H), 5.10 - 4.52 (m, 3H), 4.42 - 3.95 (m, 2H), 3.87 - 3.79 (m, 1H), 3.77 - 3.66 (m, 2H), 3.64 - 3.53 (m, 2H), 3.51 - 3.44 (m, 4H), 3.28 - 3.26 (m, 3H), 3.24 (br d, J= 5.1 Hz, 3H), 3.22 - 3.19 (m, 3H), 3.19 - 3.15 (m, 3H), 3.00 (s, 1H), 2.83 - 2.71 (m, 6H), 2.15 - 1.66 (m, 13H), 1.64 - 1.19 (m, 5H), 1.13 - 0.70 (m, 24H).
[0362] Example A15. Synthesis of Compound 22, H-Ser(NMe)Ile-AE
[0363] Synthesis of Boc-(NMe)Ile-AE (59). To a solution of AE, 1 (1.6 g, 2.19 mmol 1 eq) in dichloromethane (16 mL) was added DIC (413.77 mg, 3.28 mmol, 1.5 eq), DMAP (26.70 mg, 218.58 pmol, 0.1 eq), Boc-N-methyl-isoleucine (643.45 mg, 2.62 mmol, 1.2 eq). The mixture was degassed and purged with nitrogen 3 times and stirred at 25°C for 12 h. LCMS showed starting material was consumed completely and desired mass was detected. The reaction mixture was quenched by addition H2O (25 mL) at 25°C, and extracted with dichloromethane (25 mL x 2). The combined organic layers were washed with saturated NaCl 50 mL x 1, dried over Na2SC>4, filtered and concentrated under reduced pressure to give product 59 (1.4 g, yield 67%) as a white solid. The crude product was used into the next step without further purification. LCMS (ESI+, Method #1): m / z found 959.8, calculated 959.7 (MS+1), RT: 1.902 min.
[0364] Synthesis of H-(NMe)Ile-AE (60). To a solution of Boc-(NMe)Ile-AE 59 (1.4 g, 1.46 mmol 1 eq) in dichloromethane (11 mL) was added Trifluoroacetic acid (3 mL). TheWSGR Docket No. 60801-718.601mixture was degassed and purged with N2 for 3 times and stirred at 25°C for 2 h. LCMS showed starting material was consumed completely and desired mass was detected. The reaction mixture was purified by Prep-HPLC (column: Phenomenex Luna C18 80 * 30 mm x 3 um; mobile phase: [A: H2O (0.1% FA); B: ACN]; B%: 30.00%-50.00%, 10.00 min) to give H-(NMe)Ile-AE 60 (1 g, yield 80%) as a white solid. LCMS (ESI+, Method #1): m / z found 859.4, calculated 859.6 (Ms+1), RT: 0.379 min.
[0365] Synthesis of Boc-Ser(NMe)Ile-AE (61). To a solution of 60, H-(NMe)Ile-AE (400 mg, 465.56 pmol, 1 eq) in DMF (4 mL) was added HATU (265.52 mg, 698.34 pmol, 1.5 eq), DIPEA (180.5 mg, 1.401 mmol, 3 eq) and Boc-Ser (124.20 mg, 605.22 pmol, 1.3 equiv). The mixture was degassed and purged with nitrogen three times and stirred at 0°C for 1 h. LCMS showed intermediate 60 was consumed completely and desired mass was detected. The reaction mixture was purified by Prep-HPLC, (column: Phenomenex Luna C18 80 x 30 mm x 3 um; mobile phase: [A: H2O (0.1% FA); B: ACN]; B%: 30.00%-50.00%, 10.00 min) to give Boc-Ser(NMe)Ile-AE 61 (100 mg, yield 40%) as a white solid. LCMS (ESI+, Method #1): m / z found 1046.8, calculated 1046.7 (MS+1), RT: 1.686 min.
[0366] Synthesis of H-Ser(NMe)Ile-AE (Compound 22). To a solution of intermediate 61 (100 mg, 47.78 pmol, 1 eq) in di chloromethane (0.8 mL) was added trifluoroacetic acid (0.2 mL). The mixture was degassed and purged with nitrogen three times, with stirring at 25°C for 2 h. LCMS showed 61 was consumed completely and desired mass was detected. The reaction mixture was purified by Prep-HPLC, (column: Phenomenex Luna C18 80 x 30 mm x 3 um; mobile phase: [A: H2O (0.1% FA); B: ACN]; B%: 30.00%-50.00%, 10.00 min) to give Compound 22, H-Serlle-AE (32 mg, yield 40%) as a white solid. LCMS (ESI+, Method #8): m / z found 474.1, calculated 473.8 (M+2 / 2), RT: 8.289 min. HRMS (ES+): m / z found 946.66 (M+l), calculated 946.65. 'H NMR (400 MHz, DMSO-d6) 8 ppm 0.75 - 1.10 (m, 30 H) 1.12 - 1.53 (m, 6 H) 1.58 - 1.89 (m, 4 H) 1.93 - 2.14 (m, 3 H) 2.27 (br dd, J= 13.82, 5.07 Hz, 1 H) 2.67 - 2.81 (m, 8 H) 2.88 (s, 1 H) 3.00 (s, 2 H) 3.13 - 3.23 (m, 6 H) 3.27 (br s, 2 H) 3.59 - 3.64 (m, 1 H) 3.70 (br s, 2 H) 3.76 - 3.84 (m, 1 H) 3.88 - 4.12 (m, 2 H) 4.13 - 4.23 (m, 1 H) 4.31 - 4.44 (m, 1 H) 4.48 - 4.61 (m, 1 H) 4.63 - 4.76 (m, 1 H) 4.83 - 4.96 (m, 1 H) 5.43 - 5.60 (m, 1 H) 5.61 - 5.87 (m, 1 H) 7.23 - 7.37 (m, 4 H) 7.82 - 8.37 (m, 3 H) 8.77 -9.06 (m, 1 H) 9.37 - 9.71 (m, 1 H).
[0367] Example A16. Synthesis of Compound 23, NAc-Ser(NMe)Be-AEWSGR Docket No. 60801-718.601Scheme 16, Synthesis of NAc-Ser(NMe)lle-AE dipeptide prodrug
[0368] Synthesis of NAc-Ser(NMe)Ile-AE (Compound 23). To a solution of Compound 22, H-Ser(NMe)Ile-AE (120 mg, 126.81 pmol, 1 eq) in dichloromethane (0.60 mL) was added AC2O (12.95 mg, 126.81 pmol, 11.91 pL, 1 eq) and TEA (25.66 mg, 253.63 pmol, 35.30 pL, 2 eq . The mixture was stirred at 25°C for 1 hour. LCMS showed the starting material was consumed and desired mass was detected. The residue so obtained was purified by prep-HPLC (TFA condition: column: WePure Biotech XP tC18 100*30*10pm ; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 20%-50% B over 8.0 min) to give Compound 23, NAc-Ser(NMe)Ile-AE (42.0 mg, 33% yield) as a white solid. LCMS (ESI+, Method #5): m / z found 495, calculated 494.8 (M+2 / 2), RT: 2.131 min. HRMS (ES+): m / z found 988.67 (M+l), calculated 988.66. 'H NMR (400 MHz, DMSO-d6) 8 ppm 0.66 - 1.11 (m, 27 H), 1.23 - 1.59 (m, 3 H), 1.62 - 1.75 (m, 1 H), 1.82 (s, 1 H), 1.79 - 1.83 (m, 1 H), 1.86 (br d, J=11.51 Hz, 2 H), 1.96 - 2.20 (m, 2 H), 2.22 - 2.42 (m, 2 H), 2.70 - 2.84 (m, 5 H), 2.86 - 3.04 (m, 3 H), 3.14 - 3.32 (m, 7 H), 3.36 - 3.63 (m, 5 H), 3.64 - 3.79 (m, 7 H), 3.84 - 4.05 (m, 9 H), 4.08 - 4.26 (m, 2 H), 4.45 - 4.80 (m, 3 H), 4.86 (br dd, J=10.76, 5.63 Hz, 1 H), 5.61 - 5.89 (m, 1 H), 7.16 - 7.39 (m, 4 H), 7.86 - 8.19 (m, 1 H), 8.93 (br d, J=8.25 Hz, 1 H), 9.47 - 9.66 (m, 1 H).
[0369] Example A17. Synthesis of Compound 24, H-Ser(NMe)Glu-AEWSGR Docket No. 60801-718.601
[0370] Synthesis of Fmoc-(NMe)Glu(OtBu)-AE (62). To a solution of AE, 1 (500 mg, 683.06 pmol, 1 eq in di chloromethane (5 mL) was added (2S)-5-tert-butoxy-2- [9H-fluoren-9-ylmethoxycarbonyl (methyl) amino]-5-oxo-pentanoic acid (450.31 mg, 1.02 mmol, 1.5 eq , DIC (129.30 mg, 1.02 mmol, 158.65 pL, 1.5 t^) andDMAP (41.72 mg, 341.53 pmol, 0.5 eq . The mixture was stirred at 20°C for 2 hours, at which time LC-MS showed the starting material was consumed completely and desired mass was detected. The mixture was washed with water (5 mL) three times, the organic layers were dried over Na2SC>4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (TFA condition: column: Phenomenex Luna C18 80*30mm*3um; mobile phase: [FLO (0.1% TFA)-ACN]; gradient: 50%-80% B over 8.0 min) to give intermediate 62 (610 mg, 77% yield) as a white solid. LCMS (ESI+, Method #7): m / z found 577.7, calculated 577.4 (M+2 / 2), RT: 2.248 min.
[0371] Synthesis of H-(NMe)-Glu(OtBu)-AE (63). To a solution of intermediate 62 (0.61 g, 528.83 pmol, 1 CY / ) in DMF (6.1 mL) was added piperidine (135.09 mg, 1.59 mmol, 156.68 pL, 3 eq . The mixture was stirred at 20°C for 2 hours. LC-MS showed the starting material was completely consumed and desired mass was detected. The reaction was purified by prep-HPLC (TFA condition: column: WePure Biotech XP tC18 100*30*10pm ; mobile phase: [FLO (0.1% TFA)-ACN]; gradient: 25%-55% B over 8.0 min) to give H-(NMe)-Glu(OtBu)-AE 63 (115 mg, 23% yield) as a white solid. LCMS (ESI+, Method #7): m / z found 466.6, calculated 466.3 (M+2 / 2), RT: 1.529 min.
[0372] Synthesis of Boc-Ser(NMe)-Glu(OtBu)-AE (64). To a solution of intermediate 63 (95 mg, 102.01 pmol, 1 eq in DMF (1 mL) was added (2S)-2-(tert-butoxycarbonylamino)-3-hydroxy-propanoic acid (20.93 mg, 102.01 pmol, 1 eq), HATU (46.55 mg, 122.42 pmol, 1.2 eq and DIPEA (65.92 mg, 510.07 pmol, 88.84 pL, 5 eq at 0°C. The mixture was stirred at 0°C for 2 hours. LC-MS showed the starting material was consumed completely and one main product peak with desired mass was detected. The reaction was purified by prep-HPLC (TFA condition: column: WePure Biotech XP tC18 100*30*10pm ; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 35%-65% B over 8.0 min) to give Boc-Ser(NMe)-Glu(OtBu)-AE 64 (50 mg, 43% yield) as a white solid. LCMS (ESI+, Method #7): m / z found 1119.1, calculated 1118.7 (M+l), RT: 1.811 min.
[0373] Synthesis of H-Ser(NMe)Glu-AE (Compound 24). To a solution of intermediate 64 (50 mg, 44.71 pmol, 1 eq in TFA (0.2 mL) and dichloromethane (0.8 mL) was stirred at 20°C for 1 hour. LC-MS showed the starting material was consumed completely and desired mass for the product was detected. The reaction was purified by prep-HPLC (TFA condition:WSGR Docket No. 60801-718.601column: WePure Biotech XPtC18 100*30*10pm ; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 10%-40% B over 8.0 min) to give Compound 24, H-Ser(NMe)Glu-AE (25 mg, 58% yield) as a white solid. LCMS (ESI+, Method #5): m / z found 481.9, calculated 481.8 (M+2 / 2), RT: 1.878 min. HRMS (ES+): m / z found 962.62 (M+l), calculated 962.61. 'H NMR(400 MHz, DMSO-d6) 6 ppm 0.67 - 1.12 (m, 24 H), 1.22 - 1.54 (m, 3 H), 1.59 - 1.90 (m, 3 H), 1.92 - 2.11 (m, 3 H), 2.14 - 2.34 (m, 5 H), 2.43 (br s, 1 H), 2.59 - 2.69 (m, 1 H), 2.72 - 2.82 (m, 5 H), 2.83 (s, 1 H), 2.89 (s, 1 H), 3.00 (s, 1 H), 3.19 - 3.23 (m, 3 H), 3.27 (s, 1 H), 3.36 - 3.63 (m, 9 H), 3.66 - 3.85 (m, 4 H), 3.95 - 4.06 (m, 1 H), 4.15 - 4.46 (m, 1 H), 4.16 - 4.28 (m, 1 H), 4.51 - 4.77 (m, 1 H), 4.57 (td, J=8.54, 3.81 Hz, 1 H), 5.06 - 5.19 (m, 1 H), 5.52 - 5.64 (m, 1 H), 5.65 - 5.90 (m, 1 H), 7.20 - 7.41 (m, 5 H), 7.73 - 8.28 (m, 2 H), 8.82 -9.02 (m, 1 H), 9.45 - 9.64 (m, 1 H), 12.10 - 12.37 (m, 1 H).
[0374] Example A18. Synthesis of Compound 25, NAc-SerGlu-AEScheme 18, Synthesis of NAc-Ser(NMe)Glu-AE dipeptide prodrug
[0375] Synthesis of NAc-Ser(NMe)Glu-AE (Compound 25). To a solution of Compound 24, H-Ser(NMe)Glu-AE (132 mg, 137.18 pmol, 1 eq) in dichloromethane (1 mL) was added AC2O (14.00 mg, 137.18 pmol, 12.88 pL, 1 eq) and triethylamine (27.76 mg, 274.37 pmol, 38.19 pL, 2 eq). The mixture was stirred at 20°C for 1 hour. LC-MS showed the starting material was consumed completely and desired product mass was detected. The reaction was purified by prep-HPLC (TFA condition: column: WePure Biotech XP tC18 100*30*10pm ; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 15%-45% B over 8.0 min) to give Compound 25, NAc-SerGlu-AE (26 mg, 18% yield) as a white solid. LCMS (ESI+, Method #5): m / z found 502.9, calculated 502.8 (M+2 / 2), RT: 1.971 min. HRMS (ES+): m / z found 1004.63 (M+l), calculated 1004.62. 'HNMR (400 MHz, DMSO-d6) 8 ppm 0.70 - 1.14 (m, 24 H), 1.22 - 1.38 (m, 1 H), 1.39 - 1.63 (m, 2 H), 1.64 - 1.91 (m, 6 H), 2.00 (br dd, J= 9.13, 5.50 Hz, 2 H), 2.05 - 2.23 (m, 4 H), 2.24 - 2.37 (m, 2 H), 2.44 (br s, 1 H), 2.62 (s, 1 H), 2.70 -2.86 (m, 6 H), 3.01 (br d, J= 5.38 Hz, 4 H), 3.12 - 3.22 (m, 4 H), 3.27 (s, 1 H), 3.41 (br d, J = 9.88 Hz, 1 H), 3.44 - 3.52 (m, 2 H), 3.54 - 3.64 (m, 2 H), 3.72 (br d, J= 5.25 Hz, 1 H), 3.85 (br d, J= 9.76 Hz, 1 H), 4.01 (br s, 2 H), 4.08 - 4.22 (m, 2 H), 4.56 (dt, J= 12.91, 8.49 Hz, 1 H), 4.61 - 4.69 (m, 1 H), 4.69 - 4.78 (m, 1 H), 4.78 - 4.91 (m, 2 H), 4.92 - 5.08 (m, 1 H), 5.64WSGR Docket No. 60801-718.601- 5.92 (m, 1 H), 7.12 - 7.44 (m, 5 H), 7.87 (br d, J= 7.63 Hz, 1 H), 8.02 - 8.26 (m, 1 H), 8.92 (br d, J= 7.88 Hz, 1 H), 9.59 (br d, J= 1.75 Hz, 1 H).
[0376] Example A19. Synthesis of Compound 26, H-Ser(NMe)Arg-AECompound 26, H-Ser(NMe)Arg-AEScheme 19, Synthesis of Ser(NMe)Arg-AE dipeptide prodrug
[0377] Synthesis of Fmoc-Arg(N-alloc)2 activated intermediate (66). To a solution of 65, Fmoc-Arg(N-alloc)2-OH (4 g, 7.06 mmol, 1 eq) in THF (40 mL) was added TsOH (121.57 mg, 705.96 pmol, 0.1 eq) and (HCHO)n (254.46 mg, 2.82 mmol, 0.4 eq). The mixture was stirred at 60°C for 3 hours. LC-MS showed the starting material was consumed and desired product mass was detected. The reaction mixture was diluted with brine (50 mL) and extracted with dichloromethane (50 mL x 3). The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Commercial hexanes: ethyl acetate= 100 / 90 to 4 / 1) to give 66 (1 g, 17% yield) as yellow oil. LCMS (ESI+, Method #7): m / z found 577.1, calculated 577.2 (M+l), RT: 2.176 min.
[0378] Synthesis of Fmoc-(NMe)Arg(N-alloc)2-OH (67). To a solution of intermediate 66 (1 g, 1.73 mmol, 1 eq) in CHCh (5 mL) and TFA (5 mL) was added triethylsilane (806.64 mg, 6.94 mmol, 1.11 mL, 4 eq). The mixture was stirred at 20°C for 4 hours, at which timeWSGR Docket No. 60801-718.601LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was quenched by addition of NaHCCh (10 mL), and then extracted with ethyl acetate (60 mL). The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiCh, ethyl acetate :methanol= 100 / 1 to 10 / 1) to give 67 (0.65 g, 55% yield) as white solid. LCMS (ESI+, Method #7): m / z found 579.5, calculated 579.2 (M+l), RT: 1.921 min.
[0379] Synthesis of Fmoc-(NMe)Arg(N-alloc)2-AE (68). To a solution of 67 (300 mg, 518.48 pmol, 1 eq) and 1, AE (379.53 mg, 518.48 pmol, 1 eq) in DCM (3 mL) was added DIC (98.15 mg, 777.72 pmol, 120.43 pL, 1.5 eq) and DMAP (31.67 mg, 259.24 pmol, 0.5 eq). The mixture was stirred at 20°C for 1 hour. LC-MS showed the starting material was consumed and desired mass was detected. The reaction mixture was quenched by addition IN HC1 (10 mL) and then extracted with di chloromethane (45 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by pre-HPLC (TFA condition: column:3_Phenomenex Luna C18 75*30mm*3um; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 40%-80% B over 8.0 min) to give 68 (0.65 g, 77% yield) as white solid. LCMS (ESI+, Method #7): m / z found 647.3, calculated 646.9 (M+2 / 2), RT: 2.213 min.
[0380] Synthesis of H-(NMe)Arg(N-alloc)2-AE (69). To a solution of 68 (650 mg, 502.86 pmol, 1 eq) in DMF (6.5 mL) was added piperidine (128.46 mg, 1.51 mmol, 148.99 pL, 3 eq). The mixture was stirred at 20°C for 1 hour. LC-MS showed the starting material was completely consumed and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by pre-HPLC (TFA condition: column: WePure Biotech XPtC18 100*30*10pm ; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 15%-55% B over 8.0 min) to give 69 (350 mg, 62% yield) as white solid. LCMS (ESI+, Method #8): m / z found 536.3, calculated 535.8 (M+2 / 2), RT: 9.754 min.
[0381] Synthesis of Boc-Ser(NMe)Arg(N-alloc)2-AE (70). To a solution of 69 (230 mg, 214.88 pmol, 1 eq) and (2S)-2-(tert-butoxycarbonylamino)-3-hydroxy-propanoic acid (44.10 mg, 214.88 pmol, 1 eq) in DMF (2.3 mL) was added HATU (122.56 mg, 322.32 pmol, 1.5 eq) and DIPEA (111.08 mg, 859.52 pmol, 149.71 pL, 4 eq). The mixture was stirred at 0°C for 1 hour, at which time LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by pre-HPLC (TFA condition: column:WSGR Docket No. 60801-718.601Phenomenex Luna C18 80 * 30mm * 3um; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 25%-55% B over 8.0 min) to give 70 (80 mg, 29% yield) as white solid. LCMS (ESI+, Method #7): m / z found 629.9, calculated 629.4 (M+2 / 2), RT: 1.736 min.
[0382] Synthesis of H-Ser(NMe)Arg(N-alloc)2-AE (71). To a solution of 70 (80 mg, 63.62 pmol, 1 eq) in DCM (1.6 mL) was added TMSOTf (28.28 mg, 127.23 pmol, 22.99 pL, 2 eq). The mixture was stirred at 25°C for 1 hour. LC-MS showed the starting material was consumed and desired product mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give 71 (80 mg, 89% yield) as yellow solid. LCMS (ESI+, Method #7): m / z found 579.7, calculated 579.4 (M+2 / 2), RT: 1.581 min.
[0383] Synthesis of H-Ser(NMe)Arg-AE (Compound 26). To a solution of 71 (80 mg, 56.68 pmol, 1 eq) in DMF (0.8 mL) was added Pd(PPh3)4 (19.65 mg, 17.00 pmol, 0.3 eq) and 1, 3 -dimethylhexahydropyrimidine-2, 4, 6-trione (44.25 mg, 283.38 pmol, 5 eq). The mixture was stirred at 25°C for 1 hour. LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a crude residue. The residue was purified by prep-HPLC (TFA condition: column: Phenomenex Luna C18 80*30mm*3um; mobile phase:[H2O (0.1% TFA)-ACN]; gradient: 5%-35% B over 8.0 min) to give Compound 26, H-Ser(NMe)Arg-AE (23 mg, 38% yield) as white solid. LCMS (ESI+, Method #5): m / z found 495.5, calculated 495.3 (M+2 / 2), RT: 1.807 min. HRMS (ES+): m / z found 989.68 (M+l), calculated 989.67. 'H NMR (400 MHz, DMSO-i / s) 6 ppm 9.61 (br s, 1 H) 8.92 (br d, J= 5.63 Hz, 1 H) 8.08 - 8.32 (m, 3 H) 7.72 - 7.87 (m, 2 H) 7.24 - 7.38 (m, 5 H) 6.91 - 7.21 (m, 2 H) 5.74 - 5.87 (m, 1 H) 5.61 - 5.69 (m, 1 H) 5.14 (ddd, J= 16.38, 11.13, 4.75 Hz, 1 H) 4.61 -4.85 (m, 1 H) 4.56 (q,J= 8.80 Hz, 1 H) 4.41 (br s, 1 H) 4.15 - 4.32 (m, 1 H) 3.89 - 4.08 (m, 2 H) 3.68 - 3.81 (m, 4 H) 3.31 - 3.46 (m, 3 H) 3.25 - 3.27 (m, 1 H) 3.06 - 3.22 (m, 8 H) 2.98 -3.05 (m, 2 H) 2.87 - 2.90 (m, 1 H) 2.80 - 2.84 (m, 2 H) 2.63 - 2.79 (m, 5 H) 2.38 - 2.47 (m, 1 H) 2.19 - 2.34 (m, 2 H) 1.96 - 2.11 (m, 2 H) 1.91 (br d, J= 6.50 Hz, 1 H) 1.67 - 1.83 (m, 3 H) 1.55 - 1.65 (m, 1 H) 1.36 - 1.52 (m, 3 H) 1.15 - 1.35 (m, 2 H) 0.74 - 1.11 (m, 24 H).
[0384] Example A20. Synthesis of Compound 27, NAc-Ser(NMe)Arg-AEWSGR Docket No. 60801-718.601Compound 27, NAc-Ser(NMe)Arg-AEScheme 20, Synthesis of NAc-Ser(NMe)Arg-AE dipeptide prodrug
[0385] Synthesis of NAc-Ser(NMe)Arg(N-alloc)2-AE (72). To a solution of 71 (110 mg, 95.04 pmol, 1 eq) in DCM (1.1 mL) was added acetic anhydride (9.70 mg, 95.04 pmol, 8.93 pL, 1 eq) and triethylamine (19.23 mg, 190.08 pmol, 26.46 pL, 2 eq). The mixture was stirred at 25°C for 1 hour. LC-MS showed the starting material was consumed and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue, which was purified by pre-HPLC (TFA condition: column:Phenomenex Luna C18 80*30mm*3um; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 15%-50% B over 8.0 min) to give 72 (50 mg, 43% yield) as white solid. LCMS (ESI+, Method #7): m / z found 600.4, calculated 600.4 (M+2 / 2), RT: 1.507 min.
[0386] Synthesis of NAc-Ser(NMe)Arg-AE (Compound 27). To a solution of 72 (50 mg, 41.68 pmol, 1 eq) in DMF (0.5 mL) was added Pd(PPh3)4 (14.45 mg, 12.51 pmol, 0.3 eq) and 1, 3 -dimethylhexahydropyrimidine-2, 4, 6-trione (32.54 mg, 208.42 pmol, 5 eq). The mixture was stirred at 25°C for 1 hour, at which time LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a reaction residue. The residue was purified by pre-HPLC (TFA condition: column: WePure Biotech XP tC18 100*30*10pm ; mobile phase:[H2O (0.1% TFA)-ACN]; gradient: 10%-40% B over 8.0 min) to give Compound 27, NAc-Ser(NMe)Arg-AE (25 mg, 53% yield) as a white solid. LCMS (ESI+, Method #5): m / z found 516.5, calculated 516.3 (M+2 / 2), RT: 1.902 min. HRMS (ES+): m / z found 1031.69 (M+l), calculated 1031.68. ‘HNMR (400 MHz, DMSO-fifc) 6 ppm 9.63 (br s, 1 H) 8.86 -9.00 (m, 1 H) 8.00 - 8.31 (m, 2 H) 7.70 - 7.90 (m, 1 H) 7.58 - 7.69 (m, 1 H) 7.16 - 7.38 (m, 6 H) 6.82 - 7.16 (m, 2 H) 5.62 - 5.85 (m, 1 H) 4.90 - 5.05 (m, 1 H) 4.48 - 4.83 (m, 3 H) 4.18WSGR Docket No. 60801-718.601(dt, J= 12.41, 6.36 Hz, 1 H) 3.94 - 4.06 (m, 2 H) 3.56 - 3.61 (m, 2 H) 3.35 - 3.53 (m, 4 H) 3.23 - 3.29 (m, 2 H) 3.13 - 3.23 (m, 6 H) 3.04 - 3.12 (m, 2 H) 2.99 (br d, J= 10.13 Hz, 2 H) 2.94 (s, 1 H) 2.70 - 2.83 (m, 6 H) 2.61 (br d, J= 8.25 Hz, 1 H) 2.38 - 2.46 (m, 1 H) 2.20 -2.36 (m, 2 H) 2.06 - 2.16 (m, 1 H) 1.98 - 2.06 (m, 1 H) 1.84 - 1.90 (m, 1 H) 1.83 (br d, J= 4.25 Hz, 3 H) 1.71 - 1.79 (m, 2 H) 1.43 - 1.57 (m, 2 H) 1.40 (br d, J= 7.25 Hz, 2 H) 1.24 -1.34 (m, 1 H) 0.73 - 1.11 (m, 24 H)
[0387] Example All. Synthesis of Compound 28, H-SerSar(cyclopropyl)-AE
[0388] Synthesis of Boc-Gly(cyclopropyl)-AE (74). To a solution of 1, AE (2 g, 2.73 mmol, 1 eq) and 73 (1.65 g, 8.20 mmol, 3 eq) in dichloromethane (20 mL) was added DMAP (166.89 mg, 1.37 mmol, 0.5 eq) and DIC (1.03 g, 8.20 mmol, 1.27 mL, 3 eq). The mixture was stirred at 25°C for 2 hours. LCMS showed the starting material was completely consumed and desired mass corresponding to product was detected. The reaction mixture was diluted with H2O (50 mL) and extracted with di chloromethane (80 mL x 3). The combined organic layers were washed with aqueous NaCl (50 mL x 3), dried over Na2SC>4, filtered and concentrated under reduced pressure to give 74 (2.2 g, 88% yield) as yellow solid. LCMS (ESI+, Method #10): m / z found 915.4, calculated 915.6 (M+l), RT: 0.487 min.
[0389] Synthesis of H-Gly(cyclopropyl)-AE (75). To a solution of 74 (2.2 g, 2.40 mmol, 1 eq) in dichloromethane (18 mL) was added trifluoroacetic acid (4.5 mL). The mixture wasWSGR Docket No. 60801-718.601stirred at 25°C for three hours. LCMS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue, which was purified by prep-HPLC (TFA condition: column: Welch Xtimate C18250*70mm*10pm ; mobile phase: [H2O (0.1% TFA) - ACN]; gradient: 0%-40% B over 20.0 min) to give 75 (1.8 g, 92% yield) as a white solid. LCMS (ESI+, Method #10): m / z found 815.6, calculated 815.6 (M+l), RT: 0.419 min.
[0390] Synthesis of formyl-Gly(cyclopropyl)-AE (76). To a solution of 75 (1.8 g, 2.21 mmol, 1 eq) in di chloromethane (18 mL) and formic acid (18 mL) was added acetyl acetate (2.71 g, 26.50 mmol, 2.49 mL, 12 eq). The mixture was stirred at 25°C for 12 h, at which time LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (TFA condition: column: Welch Xtimate C18250*70mm*10pm ; mobile phase: [H2O (0.1% TFA) - ACN]; gradient: 5%-45% B over 18.0 min) to give 76 (1.6 g, 86% yield) as a white solid. LCMS (ESI+, Method #7): m / z found 843.5, calculated 843.6 (M+l), RT: 1.324 min.
[0391] Synthesis of H-Sar(cyclopropyl)-AE (77). To a solution of 76 (650 mg, 770.96 pmol, 1 eq) in tetrahydrofuran (6.5 mL) was added dropwise BH3 THF (1 M, 3.85 mL, 5 eq) at 0°C. The resulting mixture was stirred at 25°C for 6 hours. LCMS showed the starting material was consumed and desired mass was detected. Another two additional vials were set up as described above, and all three reaction mixtures were combined. The reaction mixture was quenched by addition water (15 mL) at 0°C, and then extracted with ethyl acetate (30 mL x 3). The combined organic layers were washed with aqueous NaCl (13 mL x 3), dried over Na2SC>4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (TFA condition: column: WePure Biotech XP tC18250*70* 10pm ; mobile phase: [H2O (0.1% TFA) -ACN]; gradient: 15%-45% B over 18.0 min) to give 77 (1 g, 64% yield) as a white solid. LCMS (ESI+, Method #8): m / z found 829.6, calculated 829.6 (M+l), RT: 7.820 min.
[0392] Synthesis of Boc-Ser(OTBS)Sar(cyclopropyl)-AE (78). To a solution of 77 (400 mg, 482.44 pmol, 1 eq) and (2S)-2-(tert-butoxycarbonylamino)-3-[tert-butyl(dimethyl)silyl]oxy-propanoic acid (385.31 mg, 1.21 mmol, 2.5 eq) in DMF (4 mL) was added HATU (275.16 mg, 723.66 pmol, 1.5 eq) and DIPEA (124.70 mg, 964.88 pmol, 168.06 pL, 2 eq) at 0°C. The resulting mixture was stirred at 0°C for 1 h, at which time LCMS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a crudeWSGR Docket No. 60801-718.601residue. The residue was purified by prep-HPLC (TFA condition: column: WePure Biotech XP tC18 100*30*10pm ; mobile phase: [H2O (0.1% TFA) - ACN]; gradient: 45%-75% B over 8.0 min) to give 78 (250 mg, 37% yield) as a white solid. LCMS (ESI+, Method #7): m / z found 566.2, calculated 565.9 (M+2 / 2), RT: 2.119 min.
[0393] Synthesis of H-SerSar(cyclopropyl)-AE (Compound 28). To a solution of 78 (250 mg, 221.13 pmol, 1 eq) in di chloromethane (2.3 mL) and TFA (0.23 mL) was stirred at 25°C for 1 hour. LC-MS showed the starting material was completely consumed and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (TFA condition: column: WePure Biotech XPtC18 100*30*10pm ; mobile phase: [H2O (0.1% TFA) - ACN]; gradient: 10%-40% B over 8.0 min) to give Compound 28, H-SerSar(cyclopropyl)-AE (141 mg, 70% yield) as a white solid. LCMS (ESI+, Method #8): m / z found 459.2, calculated 458.8 (M+2 / 2), RT: 8.069 min. HRMS (ES+): m / z found 916.61 (M+l), calculated 916.60. 'H NMR(400 MHz, DMSO-d6) 6 ppm 0.73 - 1.12 (m, 24 H) 1.19 - 1.44 (m, 4 H) 1.47 - 1.88 (m, 6 H) 1.95 - 2.17 (m, 2 H) 2.23 - 2.36 (m, 2 H) 2.78 (br s, 6 H) 2.87 - 2.95 (m, 1 H) 2.98 -3.10 (m, 5 H) 3.18 (br d, J=17.39 Hz, 6 H) 3.25 (br d, J=10.13 Hz, 5 H) 3.53 - 3.64 (m, 2 H) 3.67 - 3.83 (m, 2 H) 3.91 - 4.08 (m, 1 H) 4.11 - 4.24 (m, 1 H) 4.29 - 4.42 (m, 1 H) 4.53 - 4.77 (m, 2 H) 5.57 - 5.77 (m, 1 H) 7.28 (br s, 4 H) 7.69 - 7.89 (m, 1 H) 8.04 - 8.27 (m, 3 H) 8.72 -9.04 (m, 1 H) 9.43 - 9.81 (m, 1 H).
[0394] Example A22. Synthesis of Compound 29, NAc-SerSar(cyclopropyl)-AECompound 29, NAc-SerSar(cy)-AEScheme 22, Synthesis of NAc-SerSar(cy)-AE dipeptide prodrug
[0395] Synthesis of NAc-SerSar(cyclopropyl)-AE (Compound 29). To a solution of 77 (400 mg, 482.44 pmol, 1 eq) and (2S)-2-acetamido-3-[tert-butyl(dimethyl)silyl]oxy-propanoic acid (315.26 mg, 1.21 mmol, 2.5 eq) in DMF (4 mL) was added HATU (275.16 mg, 723.66 pmol, 1.5 eq) and DIPEA (124.70 mg, 964.88 pmol, 168.06 pL, 2 eq) at 0°C. The resulting mixture was stirred at 0°C for 1 hour, at which time LCMS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a crude residue. The residue wasWSGR Docket No. 60801-718.601purified by prep-HPLC (TFA condition: column: Xbridge OBD C18 100*30*5 & Kinetex EVO C18 100*30*5; mobile phase: [H2O (0.1% TFA) - ACN]; gradient: 15%-45% B over 15.0 min) to give Compound 29, NAc-SerSar(cyclopropyl)-AE (85 mg, 17% yield) as a white solid. LCMS (ESI+, Method #8): m / z found 958.6, calculated 958.6 (M+l), RT: 8.967 min. HRMS (ES+): m / z found 958.63 (M+l), calculated 958.62. 'H NMR (400 MHz, DMSO-t / e) 6 ppm 0.72 - 1.02 (m, 24 H) 1.02 - 1.12 (m, 4 H) 1.14 - 1.24 (m, 1 H) 1.26 - 1.37 (m, 2 H) 1.38 - 1.50 (m, 2 H) 1.54 - 1.63 (m, 1 H) 1.73 - 1.91 (m, 6 H) 1.95 - 2.06 (m, 1 H) 2.06 - 2.21 (m, 1 H) 2.25 - 2.36 (m, 2 H) 2.40 - 2.46 (m, 1 H) 2.75 (br d, J=3.25 Hz, 1 H) 2.78 (br s, 4 H) 2.82 - 2.93 (m, 1 H) 3.00 (s, 1 H) 3.12 - 3.21 (m, 7 H) 3.22 - 3.30 (m, 5 H) 3.54 - 3.66 (m, 2 H) 3.68 - 3.79 (m, 1 H) 3.80 - 3.92 (m, 1 H) 3.97 - 4.22 (m, 2 H) 4.52 - 4.61 (m, 1 H) 4.61 - 4.89 (m, 2 H) 5.54 - 5.90 (m, 1 H) 7.21 - 7.37 (m, 5 H) 7.72 - 8.35 (m, 2 H) 8.81 - 9.02 (m, 1 H) 9.52 (br d, J=3.75 Hz, 1 H).WSGR Docket No. 60801-718.601[0Scheme 23, Synthesis of MA-EVK-SerSar-MMAE linker-payload
[0397] Synthesis of Bn-SerLys(Boc)OBz (80). To a solution of 79 (5 g, 13.14 mmol, 1 eq) and benzyl (2 S)-2-amino-3 -hydroxy -propanoate (3.04 g, 13.14 mmol, 1 eq, HC1) in dichloromethane (50 mL) was added HATU (7.50 g, 19.71 mmol, 1.5 eq) and DIPEA (3.40 g, 26.29 mmol, 4.58 mL, 2 eq) at 0°C. The resulting mixture was stirred at 0°C for 1 hour, at which time LC-MS showed the starting material was consumed and desired mass was detected. The reaction mixture was diluted with water (75 mL) and extracted with ethyl acetate (100 mL x 3). The combined organic layers were washed with aqueous NaCl (60 mL x 2), dried over sodium sulfate, filtered and concentrated under reduced pressure to give a crude residue. The crude product was triturated with ethyl acetate (50 mL) at 25°C for 30WSGR Docket No. 60801-718.601minutes to give 80 (5.6 g, 76% yield) as a white solid. LCMS (ESI+, Method #11): m / z found 558.3, calculated 558.3 (M+l), RT: 1.779 min.
[0398] Synthesis of H-SerLys(Boc)OH (81). To a solution of 80 (5.6 g, 10.04 mmol, 1 eq in EtOH (112 mL) was added Pd / C (320.61 mg, 301.27 pmol, 10% purity, 0.03 eq) under an argon atmosphere. The suspension was degassed and purged with hydrogen three times. The mixture was stirred under hydrogen (15 Psi) at 25°C for 12 hours. LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give 81 (2.1 g, 63% yield) as a yellow oil. LCMS (ESI+, Method #7): m / z found 334.1, calculated 334.2 (M+l), RT: 0.724 min.
[0399] Synthesis of Fmoc-stretcher-Glu(OtBu)Val-OH (82). To a solution of intermediate 13 (5 g, 7.84 mmol, 1 eq) and (2S)-2-amino-3-methyl-butanoic acid (1.10 g, 9.41 mmol, 1.2 eq) in dioxane (80 mL) and water (20 mL) was added NaHCCh (1.32 g, 15.68 mmol, 610.18 pL, 2 eq). The mixture was stirred at 25°C for 4 hours. LC-MS showed the starting material was completely consumed and desired mass was detected. The reaction was adjusted to pH=l~2 by IN HC1 (IM, 30 mL), and then diluted with water (100 mL) and extracted with ethyl acetate (180 mL x 4). The combined organic layers were washed with aqueous NaCl (150 mL x 2), dried over sodium sulfate, filtered and concentrated under reduced pressure to give a crude reaction residue. The residue was purified by prep-HPLC (TFA condition: column: 3_Phenomenex Luna C1875*30mm*3um; mobile phase: [H2O (0.1% TFA) -ACN]; gradient: 35%-65% B over 8.0 min) to give 82 (5.3 g, 75% yield) as a white solid. LCMS (ESI+, Method #8): m / z found 640.5, calculated 640.3 (M+l), RT: 13.618 min.
[0400] Synthesis of Fmoc-stretcher-Glu(OtBu)Val-OSu (83). To a solution of 82 (4.8 g, 7.50 mmol, 1 eq) in tetrahydrofuran (72 mL) was added HOSu (1.30 g, 11.25 mmol, 1.5 eq) and DCC (2.32 g, 11.25 mmol, 2.28 mL, 1.5 eq) at 0°C. The resulting mixture was stirred at 25°C for 4 hours, at which time LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a crude residue 83 (4.96 g, 90% yield) as a white solid. LCMS (ESI+, Method #1): m / z found 759.2, calculated 759.3 (M+23), RT: 1.864 min.
[0401] Synthesis of Fmoc-stretcher-Glu(OtBu)ValLys(Boc)Ser (84). To a solution of intermediate 81 (1.80 g, 5.39 mmol, 0.8 eq) and intermediate 83 (4.96 g, 6.73 mmol, 1 eq) in dioxane (80 mL) and H2O (20 mL) was added NaHCCh (1.13 g, 13.46 mmol, 523.86 pL, 2 eq). The mixture was stirred at 25°C for 4 hours. LC-MS showed the starting material was consumed and desired mass was detected. The reaction was adjusted to pH=l~2 by HC1WSGR Docket No. 60801-718.601(IM, 28 mL) and then diluted with water (60 mL) and extracted with ethyl acetate (100 mL x 4). The combined organic layers were washed with aqueous NaCl (50 mL x 2) dried over sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 5-20% Ethyl acetate / MeOH / ether gradient @ 100 mL / min) to give 84 (1.4 g, 22% yield) as a white solid. LCMS (ESI+, Method #8): m / z found 955.5, calculated 955.5 (M+l), RT: 13.625 min.
[0402] Synthesis of Fmoc-stretcher-Glu(OtBu)ValLys(Boc)SerSar-MMAE-Boc (85). To a solution of intermediate 84 (1.4 g, 1.47 mmol, 1 eq) and 38, Boc-MMAE-Sar (1.30 g, 1.47 mmol, 1 eq) in DMF (14 mL) was added HATU (836.02 mg, 2.20 mmol, 1.5 eq) and DIPEA (284.17 mg, 2.20 mmol, 382.98 pL, 1.5 eq) at 0°C. The resulting mixture was stirred at 0°C for 1 hour. LC-MS showed the starting material was consumed and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a crude reaction residue. The residue was purified by prep-HPLC (TFA condition: column: 3_Phenomenex Luna C18 75*30mm*3um; mobile phase: [H2O (0.1% TFA) - ACN]; gradient: 60%-90% B over 8.0 min) to give 85 (800 mg, 30% yield) as a white solid. LCMS (ESI+, Method #8): m / z found 914.0, calculated 913.5 (M+2 / 2), RT: 18.994 min.
[0403] Synthesis of H-stretcher-Glu(OtBu)ValLys(Boc)SerSar-MMAE-Boc (86). To a solution of 85 (800 mg, 438.06 pmol, 1 eq) in DMF (8 mL) was added piperidine (111.90 mg, 1.31 mmol, 129.78 pL, 3 eq). The mixture was stirred at 25°C for 1 hour. LC-MS showed the starting material was consumed and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (TFA condition: column: Phenomenex Luna Cl 8 80*30mm*3um; mobile phase: [H2O (0.1%TFA) - ACN]; gradient: 35%-65% B over 8.0 min) to give 86 (670 mg, 95% yield) as a white solid. LCMS (ESI+, Method #8): m / z found 802.9, calculated 802.5 (M+2 / 2), RT: 14.342 min.
[0404] Synthesis of MA-Glu(OtBu)ValLys(Boc)SerSar-MMAE-Boc (87). To a solution of 86 (670 mg, 417.70 pmol, 1 eq) and MA-OSu (126.40 mg, 501.24 pmol, 1.2 eq) in DMF (6.7 mL) was added DIPEA (161.95 mg, 1.25 mmol, 218.27 pL, 3 eq). The mixture was stirred at 25°C for 1 hour, at which time LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a crude reaction residue. The residue was purified by prep-HPLC (TFA condition: column: 3_Phenomenex Luna C18 75*30mm*3um; mobile phase: [H2O (0.1% TFA) - ACN]; gradient: 45%-75% B over 8.0 min) to give 87 (400WSGR Docket No. 60801-718.601mg, 55% yield) as a white solid. LCMS (ESI+, Method #8): m / z found 871.4, calculated 871.0 (M+2 / 2), RT: 16.033 min.
[0405] Synthesis of MA-GluValLysSerSar-MMAE (Compound 30). To a solution of 87 (380 mg, 218.25 pmol, 1 eq) in di chloromethane (3 mL) was added TFA (1.2 mL). The mixture was stirred at 25°C for 1 hour. LC-MS showed the starting material was consumed and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a crude residue. The residue was purified by prep-HPLC (TFA condition: column: Xbridge OBD C18 100*30*5 &Kinetex EVO C18 100*30*5; mobile phase: [FEO (0.1%TFA) - ACN]; gradient: 10%-40% B over 15.0 min) to give Compound 30, MA-GluValLysSerSar-MMAE (170 mg, 50% yield) as a white solid. LCMS (ESI+, Method #8): m / z found 743.3, calculated 742.8 (M+2 / 2), RT: 7.098 min. HRMS (ES+): m / z found 1484.86 (M+l), calculated 1484.85. 'HNMR (400 MHz, CD3SOCD3, 299 K) 8 (ppm) = 12.10 (br dd, J= 1.6, 4.1 Hz, 1H), 8.88 - 8.59 (m, 3H), 8.19 (br t, J= 5.3 Hz, 1H), 8.11 - 7.78 (m, 4H), 7.75 - 7.53 (m, 4H), 7.42 - 7.25 (m, 5H), 7.09 (s, 2H), 5.90 - 5.64 (m, 1H), 5.04 - 4.78 (m, 2H), 4.77 - 4.47 (m, 3H), 4.37 - 4.08 (m, 6H), 4.07 - 3.95 (m, 3H), 3.91 -3.79 (m, 1H), 3.74 - 3.63 (m, 2H), 3.63 - 3.52 (m, 4H), 3.29 - 3.09 (m, 13H), 2.99 (s, 2H), 2.87 (br d, J= 11.0 Hz, 1H), 2.80 - 2.69 (m, 2H), 2.44 - 2.35 (m, 2H), 2.30 - 2.16 (m, 3H), 2.15 - 1.56 (m, 11H), 1.55 - 1.19 (m, 8H), 1.17 - 0.64 (m, 32H).
[0406] Example A24. Compound 31, MA-GluValCitSer(NMe)Glu-MMAECompound 31, MA-EVCit-Ser(NMe)Glu-MMAEWSGR Docket No. 60801-718.601
[0407] Example A25. Compound 32, MA-GluValCitSer(NMe)Ile-MMAECompound 32, MA-EVCit-Ser(NMe)lle-MMAEWSGR Docket No. 60801-718.601
[0408] Example A26. Synthesis of Compound 33, MA-(GluValCit-Ser(NMe)Glu- MMAE)2
[0409] Synthesis of Fmoc-Glu(OtBu)-MMAE-Boc (254). To a solution of Boc-MMAE 36 (4.2 g, 5.13 mmol, 1 eq) and (2S)-5-tert-butoxy-2-[9H-fluoren-9-ylmethoxycarbonyl(methyl)amino]-5-oxo-pentanoic acid (2.71 g, 6.16 mmol, 1.2 eq) in dichloromethane (42 mL) was added DIC (971.84 mg, 7.70 mmol, 1.19 mL, 1.5 eq) and DMAP (62.72 mg, 513.39 qmol, 0.1 eq). The mixture was stirred at 25°C for 12 hours.WSGR Docket No. 60801-718.601Another 2.2 g batch was set up as described above and the reaction mixtures were combined. LCMS showed the starting material was consumed and desired mass was detected. The reaction mixture was diluted with water (10 mL) and extracted with dichloromethane (15 mL x 3). The combined organic layers were washed with aqueous NaCl (10 mL x 2), dried over sodium sulfate, filtered and concentrated under reduced pressure to give 254 (8.5 g, 90% yield) as a white solid. LCMS (ESI+, Method #10): m / z found 1239.6, calculated 1239.8 (M+l), RT: 0.843 min.
[0410] Synthesis of H-Glu(OtBu)-MMAE-Boc (255). To a solution of 254 (6 g, 484 mmol, 1 eq) in DMF (60 mL) was added piperidine (1.24 g, 14.52 mmol, 1.43 mL, 3 eq). The mixture was stirred at 25°C for 1 hour. Another 2.5 g batch was set up as described above and both of two reaction mixtures were combined. LCMS showed the starting material was completely consumed and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a crude reaction residue. The residue obtained was purified by prep-HPLC (TFA condition: column: Waters Xbridge BEH C18 250*70mm*10pm ; mobile phase: [H2O (0.1% TFA) - ACN]; gradient: 35%-70% B over 18.0 min) to give 255 (6 g, 84% yield) as a white solid. LCMS (ESI+, Method #7): m / z found 1017.4, calculated 1017.7 (M+l), RT: 2.004 min.
[0411] Synthesis of Fmoc-Glu(OtBu)ValCit-Ser(NMe)Glu(OtBu)-MMAE-Boc (245). To a solution of 49 (6 g, 5.90 mmol, 1 eq) and Boc-MMAE-Glu(OtBu)-OH (5.44 g, 7.08 mmol, 1.2 eq) in DMF (60 mL) was added TPTU (4.56 g, 15.33 mmol, 2.6 eq), DIPEA (4.57 g, 35.39 mmol, 6.16 mL, 6 eq) and HOBt (2.39 g, 17.69 mmol, 3 eq). The mixture was stirred at 25°C for 2 hours, at which time LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue, which was purified by prep-HPLC (TFA condition: column: WePure Biotech XP tC18250*70* 10pm ; mobile phase: [H2O (0.1% TFA) - ACN]; gradient: 65%-95% B over 18.0 min) to give 245 (2.3 g, 22% yield) as a white solid. LCMS (ESI+, Method #13): m / z found 884.9, calculated 884.5 ([M+2] / 2), RT: 2.485 min.
[0412] Synthesis of H-Glu(OtBu)ValCit-Ser(NMe)Glu(OtBu)-MMAE-Boc (246). To a solution of 245 (2.3 g, 1.30 mmol, 1 eq) in DMF (23 mL) was added piperidine (332.27 mg, 3.90 mmol, 385.38 pL, 3 eq). The mixture was stirred at 25°C for 1 hour, at which time LCMS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue, which was purified by prep-HPLC (TFA condition: column: WePure Biotech XPWSGR Docket No. 60801-718.601tC 18250*70*10pm ; mobile phase: [H2O (0.1% TFA) - ACN]; gradient: 25%-60% B over 18.0 min) to give 246 (1.69 g, 77% yield) as a white solid. LCMS (ESI+, Method #8): m / z found 773.9, calculated 773.5 ([M+2] / 2), RT: 14.686 min.
[0413] Synthesis of Fmoc-stretcher-(Glu(OtBu)ValCit-Ser(NMe)Glu(OtBu)-MMAE-Boc)2 (247). To a solution of branched stretcher intermediate 26 (350 mg, 402.82 pmol, 1 eq) and 246 (1.56 g, 1.01 mmol, 2.5 eq) in DMF (3.5 mL) was added DIPEA (416.49 mg, 3.22 mmol, 561.31 pL, 8 eq). The mixture was stirred at 25°C for 2 hours, at which time LCMS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue, which was purified by prep-HPLC (TFA condition: column: WePure Biotech XP tC18250*70*10pm ; mobile phase: [H2O (0.1% TFA) - ACN: THF=1:1]; gradient: 50%-80% B over 15.0 min) to give 247 (1.1 g, 68% yield) as a white solid. LCMS (ESI+, Method #14): m / z found 1866.1, calculated 1865.1 ([M+2] / 2), RT: 13.833 min.
[0414] Synthesis of H-stretcher-(Glu(OtBu)ValCit-Ser(NMe)Glu(OtBu)-MMAE-Boc)2 (248). To a solution of 247 (1 g, 268.06 pmol, 1 eq) in DMF (10 mL) was added piperidine (68.47 mg, 804.17 pmol, 79.42 pL, 3 eq). The mixture was stirred at 25°C for 1 hour. LCMS showed the starting material was consumed and desired product mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue, which was purified by prep-HPLC (TFA condition: column: WePure Biotech XP tC18 250*70* 10pm ; mobile phase: [H2O (0.1% TFA) - ACN]; gradient: 45%-85% B over 20.0 min) to give 248 (650 mg, 69% yield) as a white solid. LCMS (ESI+, Method #8): m / z found 1754.9, calculated 1754.1 ([M+2] / 2), RT: 18.034 min.
[0415] Synthesis of MA-(Glu(OtBu)ValCit-Ser(NMe)Glu(OtBu)-MMAE-Boc)2 (249).To a solution of 248 (650 mg, 185.27 pmol, 1 eq) and MA-OSu (70.08 mg, 277.91 pmol, 1.5 eq) in DMF (6.5 mL) was added DIPEA (71.84 mg, 555.82 pmol, 96.81 pL, 3 eq). The mixture was stirred at 25°C for 1 hour, at which time LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue, which was purified by prep-HPLC (TFA condition: column: WePure Biotech XP tC18250*70*10pm ; mobile phase: [H2O (0.1% TFA) - ACN]; gradient: 40%-90% B over 25.0 min) to give 249 (480 mg, 66% yield) as a white solid. LCMS (ESI+, Method #8): m / z found 1823.5, calculated 1822.6 ([M+2] / 2), RT: 19.053 min.
[0416] Synthesis of MA-(GluValCit-Ser(NMe)Glu-MMAE)2 (Compound 33). A solution of 249 (480 mg, 131.67 pmol, 1 eq) in di chloromethane (4 mL) was added trifluoroaceticWSGR Docket No. 60801-718.601acid (0.8 mL). The mixture was stirred at 25°C for 2 hours. LC-MS showed the starting material was consumed and the desired product mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue, which was purified by prep-HPLC (TFA condition: column: Wuxi Phenyl-Hexyl 100*30* 10pm ; mobile phase:[H2O (0.1% TFA) - ACN: THF=1:1]; gradient: 20%-35% B over 20.0 min), to give compound 33 (80 mg, 19% yield) as a white solid. LCMS (ESI+, Method #5): m / z found 1074.4, calculated 1073.9 (M+2 / 2), RT: 2.101 min. HRMS (ES+): m / z found 3219.82 (M+l), calculated 3219.81. ‘HNMR (400 MHz, DMSO4) 60.67 - 1.12 (m, 60 H) 1.19 -1.63 (m, 16 H) 1.65 - 1.91 (m, 13 H) 2.04 - 2.16 (m, 7 H) 2.17 - 2.27 (m, 7 H) 2.36 - 2.44 (m, 5 H) 2.46 (br s, 5 H) 2.56 - 2.63 (m, 5 H) 2.87 - 3.01 (m, 14 H) 3.03 - 3.14 (m, 6 H) 3.14 -3.22 (m, 16 H) 3.22 - 3.28 (m, 16 H) 3.54 - 3.64 (m, 17 H) 3.65 - 3.90 (m, 8 H) 3.96 - 4.06 (m, 4 H) 4.08 - 4.42 (m, 9 H) 4.50 - 4.88 (m, 6 H) 4.89 - 5.20 (m, 4 H) 5.30 - 5.49 (m, 3 H) 5.62 - 5.76 (m, 1 H) 5.79 - 5.99 (m, 3 H) 7.02 - 7.15 (m, 2 H) 7.20 - 7.38 (m, 9 H) 7.55 - 7.79 (m, 2 H) 7.81 - 8.29 (m, 8 H) 8.58 - 8.89 (m, 5 H).
[0417] Example A27. Synthesis of Compound 34, N-linked H-SerSar-MMAE
[0418] Synthesis of Cbz-SerSar-OtBu (201). To a solution of 200 (25 g, 104.50 mmol, 1 eq and tert-butyl 2-(methylamino)acetate;hydrochloride (28.48 g, 156.76 mmol, 1.5 eq) in DMF (250 mL) was added DIPEA (33.77 g, 261.26 mmol, 45.51 mL, 2.5 eq) and HATU (59.60 g, 156.76 mmol, 1.5 eq at 0°C. The reaction was stirred at 0°C for 1 h, at which time LCMS showed starting material was consumed and desired product was formed. The reaction mixture was concentrated under reduced pressure to give a crude residue. The residue was purified by prep-HPLC (column: WePure Biotech XP tC18250*70* 10pm ; mobile phase: [H2O (lOmM NH4HCC>3)-ACN];gradient: 25%-55% B over 20.0 min) to give 201 (18 g, 47% yield) as white solid. LCMS (ESI+, Method #6): m / z found 367.2, calculated 367.2 (M+l), RT: 0.465 min.WSGR Docket No. 60801-718.601
[0419] Synthesis of Cbz-SerSar-OH (202). To a solution of 201 (18 g, 49.13 mmol, 1 eq in dichloromethane (90 mL) was added TFA (90 mL) at 20°C. The reaction was stirred at 20°C for 1 h. LCMS showed starting material was completely consumed and desired product was formed. The reaction mixture was concentrated under reduced pressure to give a crude residue. The residue was purified by prep-HPLC (column: Phenomenex luna Cl 8 (250*70mm, 15 um);mobile phase: [H2O(0.1%TFA)-ACN]; gradient: 5%-45% B over 20.0 min) to give 202 (10 g, 66% yield) as white solid. LCMS (ESI+, Method #12): m / z found 311.2, calculated 311.1 (M+l), RT: 1.032 min.
[0420] Synthesis of N-linked Cbz-SerSar-MMAE (203). To a solution of 202 (1 00 g, 3 22 mmol, 1 eq) and 35, MMAE (2.31 g, 3.22 mmol, 1 eq in DMF (10 mL) was added HATU (1.84 g, 4.83 mmol, 1.5 eq) and DIPEA (1.04 g, 8.06 mmol, 1.40 mL, 2.5 eq) at O°C. The reaction was stirred at 0°C for 1 h. LCMS showed starting material was consumed and desired product formed. The reaction mixture was partitioned between DCM (30 mL) and cold water (20 mL *3). The organic phase was separated, dried over Na2SC>4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by pre-HPLC (column: 3_Phenomenex Luna C18 75*30mm*3um; mobile phase: [FLO (0.1% TFA)-ACN];gradient:35%-65% B over 8.0 min) to give 203 (1 g, 31% yield) as white solid.LCMS (ESI+, Method #8): m / z found 1010.5, calculated 1010.6 (M+l), RT: 12.220 min.
[0421] Synthesis of N-linked H-SerSar-MMAE (Compound 34). To a solution of 203 (1 g, 989.84 pmol, 1 eq) in DCM (10 mL) was added PdCh (26.33 mg, 148.48 pmol, 0.15 eq) and TEA (15.02 mg, 148.48 pmol, 20.67 pL, 0.15 eq and EtsSiH (161.13 mg, 1.39 mmol, 221.34 pL, 1.4 eq at 40°C. The reaction was stirred at 40°C for 5 h, at which time LCMS showed starting material was consumed and desired product mass was formed. The reaction mixture was concentrated under reduced pressure to give a crude residue. The residue was purified by prep-HPLC (column: WePure Biotech XP tC18250*70* 10pm ;mobile phase: [H2O(10mM NH4HCC>3)-ACN];gradient:25%-55% B over 20.0 min)to give compound 34 (0.5 g, 57% yield) as white solid. LCMS (ESI+, Method #5): m / z found 876.6, calculated 876.6 (M+l), RT: 2.040 min. HRMS (ES+): m / z found 876.58 (M+l), calculated 876.57. 'H NMR (400 MHz, DMSO-d6) 8 ppm 0.70 - 0.94 (m, 18 H) 0.96 - 1.11 (m, 6 H) 1.29 (br d, J=2.13 Hz, 1 H) 1.35 - 1.58 (m, 2 H) 1.60 - 1.86 (m, 3 H) 1.88 - 2.03 (m, 1 H) 2.04 - 2.19 (m, 2 H) 2.21 - 2.32 (m, 1 H) 2.34 - 2.44 (m, 1 H) 2.79 - 2.85 (m, 1 H) 2.85 - 2.92 (m, 1 H) 2.94 -3.04 (m, 5 H) 3.04 - 3.26 (m, 9 H) 3.29 (br d, J=8.25 Hz, 1 H) 3.42 - 3.62 (m, 3 H) 3.64 - 3.88 (m, 3 H) 3.91 - 4.04 (m, 2 H) 4.05 - 4.26 (m, 1 H) 4.34 - 4.49 (m, 3 H) 4.54 (td, J=10.91, 8.19 Hz, 1 H) 4.60 - 4.78 (m, 1 H) 7.08 - 7.36 (m, 5 H).WSGR Docket No. 60801-718.601
[0422] Example A28. Synthesis of Compound 35, N-linked H-SerSar-desmethyl-AEScheme 28, Synthesis of N-linked SerSar-desmethyl-AE prodrug
[0423] Synthesis of Cbz-SerSarVal-OtBu (204). To a solution of 202 (8 g, 25.78 mmol, 1 eq and tert-butyl (2S)-2-amino-3-methyl-butanoate;hydrochloride (6.49 g, 30.94 mmol, 1.2 eq) in DMF (80 mL) was added HATU (14.70 g, 38.67 mmol, 1.5 eq and DIPEA (5.00 g, 38.67 mmol, 6.74 mL, 1.5 eq . The reaction was stirred at 0°C for 1 h. LCMS showed starting material was consumed and desired product was detected. The reaction was concentrated to give a crude residue. The residue was purified by prep-HPLC (column:Welch Xtimate C18250*100mm#10pm ; mobile phase: [H2O(10mM NH4HCO3)-ACN];gradient:30%-62% B over 20.0 min) to give 204 (11.8 g, 98% yield) as white solid. LCMS (ESI+, Method #7): m / z found 446.3, calculated 466.3 (M+l), RT: 1.675 min.
[0424] Synthesis of Cbz-SerSarVal-OH (205). To a solution of 204 (11.8 g, 25.35 mmol, 1 eq) in DCM (118 mL) was added TFA (118 mL). The reaction was stirred at 20°C for 1 h, at which time LCMS showed starting material was consumed and desired product detected. The reaction was concentrated to give a crude residue. The residue was purified by prep-HPLC (column: Phenomenex Luna C18250*150mm* 15um;mobile phase: [H2O(0.1% TFA)-ACN];gradient:10%-40% B over 20.0 min) to give 205 (9 g, 86% yield) as white solid.LCMS (ESI+, Method #8): m / z found 410.2, calculated 410.2 (M+l), RT: 7.482 min.
[0425] Synthesis of Cbz-ValDil-OtBu (208). To a solution of 206 (1 g, 3.98 mmol, 1 eq) in DCM (20 mL) was added DIPEA (771.50 mg, 5.97 mmol, 1.04 mL, 1.5 eq and 2-bromo-l-WSGR Docket No. 60801-718.601ethyl-pyridin-l-ium; tetrafluoroborate (1.09 g, 3.98 mmol, 1 eq) and tert-butyl (3R,4S,5S)-3-methoxy-5-methyl -4-(methylamino)heptanoate, dolaisoleuine-OtBu (Dil, 207) (1.03 g, 3.98 mmol, 1 eq). The reaction was stirred at 0°C for 1 h. LCMS showed starting material was consumed and desired product was detected. The reaction was then concentrated to give a crude residue. The residue was purified by prep-HPLC (column: WePure Biotech XP tC18 150*40*7um;mobile phase: [H20(0.05% NH3H2O+10mM NH4HCO3)-ACN] gradient: 65%-95% B over 8.0 min) to give 208 (1.5 g, 76% yield) as white solid. LCMS (ESI+, Method #7): m / z found 493.2, calculated 493.3 (M+l), RT: 1.567 min.
[0426] Synthesis of Cbz-ValDil-OH (209). To a solution of 208 (0.5 g, 1.01 mmol, 1 eq) in DCM (5 mL) was added TFA (5 mL). The reaction was stirred at 0°C for 1 h, at which time LCMS showed starting material was completely consumed and desired product was detected. The reaction was concentrated to give 209 (0.4 g, 90% yield) as white solid and was used to next step reaction without purification. LCMS (ESI+, Method #7): m / z found 437.2, calculated 437.3 (M+l), RT: 1.684 min.
[0427] Synthesis of Cbz-ValDilDapNor (211). To a solution of 209 (400 mg, 595.59 pmol, 1 eq) and (2R,3R)-N-[(lR,2S)-2-hydroxy-l-methyl-2-phenyl-ethyl]-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propenamide, 210, DapNor (190.84 mg, 595.59 pmol, 1 eq) in DCM (4 mL) was added DIPEA (153.95 mg, 1.19 mmol, 207.48 pL, 2 eq) and 2-bromo-l-ethyl-pyridin-l-ium;tetrafluoroborate (244.67 mg, 893.39 pmol, 1.5 eq). The reaction was stirred at 0°C for 1 h. LCMS showed starting material was consumed and product was detected. The reaction was filtered and concentrated to give a crude reaction residue. The residue was purified by prep-HPLC (column: WePure Biotech XP tC18 100*30*10pm ;mobile phase: [H2O(0.1% TFA)-ACN];gradient:45%-75% B over 8.0 min) to give 211 (350 mg, 79% yield) as white solid. LCMS (ESI+, Method #10): m / z found 739.5, calculated 739.5 (M+l), RT: 0.571 min.
[0428] Synthesis of H-ValDilDapNor (212). To a solution of 211 (200 mg, 270.65 pmol, 1 eq) in methanol (10 mL) was added Pd / C (28.80 mg, 27.07 pmol, 10% purity, 0.1 eq). The reaction was stirred at 20°C for 2 h. LCMS showed starting material was consumed and desired product was detected. The reaction was filtered and concentrated to give 212 (0.15 g, 91% yield) as white solid, which was used to next step reaction without purification. LCMS (ESI+, Method #10): m / z found 605.4, calculated 605.4 (M+l), RT: 0.390 min.
[0429] Synthesis of N-linked Cbz-SerSar-desmethyl-AE (213). To a solution of 212 (150 mg, 248.01 pmol, 1 eq) and 205 (101.54 mg, 248.01 pmol, 1 eq) in DMF (1.5 mL) was addedWSGR Docket No. 60801-718.601DIPEA (64.11 mg, 496.02 pmol, 86.40 pL, 2 eq) and HATU (141.45 mg, 372.01 pmol, 1.5 eq). The reaction was stirred at 0°C for 1 h and then filtered and concentrated to give a residue. The residue was purified by prep-HPLC (column: WePure Biotech XP tC18 100*30* 10pm ;mobile phase: [H2O(0.1% TFA)-ACN];gradient:35%-75% B over 8.0 min) to give 213 (0.2 g, 80% yield) as white solid. LCMS (ESI+, Method #10): m / z found 996.9, calculated 996.6 (M+l), RT: 0.505 min.
[0430] Synthesis of N-linked H-SerSar-desmethyl-AE (Compound 35). To a solution of 213 (0.2 g, 200.76 pmol, 1 eq) in methanol (10 mL) was added Pd / C (21.36 mg, 20.08 pmol, 10% purity, 0.1 eq). The reaction was stirred at 20°C for 2 h, at which time LCMS showed starting material was consumed and desired product was detected. The reaction was filtered and concentrated to give a crude reaction residue. The residue was purified by prep-HPLC (column: WePure Biotech XP tC18 100*30*10pm ;mobile phase: [H2O(0.1% TFA)-ACN];gradient:35%-75% B over 8.0 min) to give compound 35 (42 mg, 24% yield) as white solid. LCMS (ESI+, Method #8): m / z found 862.2, calculated 862.6 (M+l), RT: 8.287 min. HRMS (ES+): m / z found 862.57 (M+l), calculated 862.56. 'HNMR (400 MHz, DMSO-d6) 8 ppm 0.71 - 0.93 (m, 18 H) 0.95 - 1.06 (m, 6 H) 1.23 - 1.37 (m, 1 H) 1.39 - 1.61 (m, 2 H) 1.65 - 1.86 (m, 3 H) 1.86 - 2.06 (m, 2 H) 2.07 - 2.18 (m, 1 H) 2.22 - 2.32 (m, 1 H) 2.41 (br d, J=16.38 Hz, 1 H) 2.52 - 2.60 (m, 1 H) 2.80 (d, J=8.76 Hz, 1 H) 2.98 (s, 1 H) 3.04 (d, J=6.50 Hz, 2 H) 3.07 - 3.18 (m, 3 H) 3.19 - 3.26 (m, 5 H) 3.31 (br d, J=10.26 Hz, 1 H) 3.35 - 3.52 (m, 1 H) 3.52 - 3.64 (m, 2 H) 3.68 - 3.86 (m, 2 H) 3.89 - 4.03 (m, 2 H) 4.05 - 4.13 (m, 1 H) 4.14 - 4.22 (m, 1 H) 4.22 - 4.34 (m, 1 H) 4.35 - 4.47 (m, 2 H) 4.47 - 4.58 (m, 1 H) 4.59 - 4.77 (m, 1 H) 5.35 - 5.48 (m, 1 H) 7.12 - 7.35 (m, 4 H) 7.14 - 7.21 (m, 1 H) 7.63 (d, J=8.38 Hz, 1 H) 7.87 - 7.94 (m, 1 H) 7.97 - 8.10 (m, 1 H) 8.10 - 8.19 (m, 3 H) 8.21 - 8.32 (m, 1 H).
[0431] Example A29. Synthesis of Compound 36, N-linked MA-GluValLysSerSar-MMAEWSGR Docket No. 60801-718.601Scheme 29, Synthesis of N-linked MA-EVCit-SerSar-MMAE
[0432] Synthesis of Fmoc-stretcher-Glu(OMe)-OSu (215). To a solution of 214 (3 g, 6.02 mmol, 1 eq in tetrahydrofuran (30 mL) was added HOSu (1.04 g, 9.03 mmol, 1.5 eq) and DCC (1.86 g, 9.03 mmol, 1.83 mL, 1.5 eq). The mixture was stirred at 25°C for 12 hours, at which time LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give 215 (3.58 g, crude, 100% yield) as a yellow oil which was used into the next step without further purification. LCMS (ESI+, Method #7): m / z found 596.3, calculated 596.2 (M+l), RT: 1.774 min.
[0433] Synthesis of Fmoc-stretcher-Glu(OMe)ValCit-OH (216). To a solution of 215 (3.48 g, 5.84 mmol, 1 eq) in dioxane (61.12 mL) and FLO (15.28 mL) was added (2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5-ureido-pentanoic acid (1.60 g, 5.84 mmol, 1 eq), NaHCCb (2.45 g, 29.21 mmol, 1.14 mL, 5 eq) at 0°C. The mixture was stirred at 20°C for 12 hours. LC-MS showed the starting material was consumed and desired mass was detected. The reaction was adjusted to pH = 5-6 with IM HC1 and the mixture was lyophilized to give the residue. The residue was purified by column chromatography (SiO2, Tetrahydrofuran: Methanol= 100 / 0 to 1 / 1) to give 216 (2.1 g, 47% yield) as a white solid. LCMS (ESI+, Method #7): m / z found 755.5, calculated 755.4 (M+l), RT: 1.555 min.
[0434] Synthesis of Fmoc-stretcher-Glu(OMe)ValCitSerSar-MMAE (217). To a solution of 216 (650 mg, 861.13 pmol, 1 eq) in DMF (6.5 mL) was added intermediate 34 (754.46WSGR Docket No. 60801-718.601mg, 861.13 pmol, 1 eq), HATU (392.91 mg, 1.03 mmol, 1.2 eq) and DIEA (111.29 mg, 861.13 pmol, 149.99 pL, 1 eq) at 0°C. The mixture was stirred at 0°C for 2 hours, at which time LC-MS showed the starting material was consumed completely and one main peak with desired mass was detected. The reaction was purified by prep-HPLC (TFA condition: column: Waters Xbridge BEH C18250*70mm*10pm ; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 30%-70% B over 20.0 min) to give 217 (738 mg, 53% yield) as a white solid. LCMS (ESI+, Method #7): m / z found 807.5, calculated 807.0 (M+2 / 2), RT: 1.888 min.
[0435] Synthesis of HO-stretcher-GluValCitSerSar-MMAE (218). To a solution of 217 (738 mg, 457.55 pmol, 1 eq) in methanol (7 mL) and H2O (1.5 mL) was added LiOH (65.75 mg, 2.75 mmol, 6 eq). The mixture was stirred at 20°C for 3 hours. LC-MS showed the starting material was consumed and desired mass was detected. The reaction was purified by prep-HPLC (TFA condition: column: WePure Biotech XP tC18 100*30*10pm ; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 15%-45% B over 8.0 min) to give 218 (190 mg, 30% yield) as a white solid. LCMS (ESI+, Method #7): m / z found 689.4, calculated 688.9 (M+2 / 2), RT: 1.428 min.
[0436] Synthesis of N-linked MA-GluValLysSerSar-MMAE (Compound 36). To a solution of 218 (170 mg, 123.49 pmol, 1 eq) in DMF (0.2 mL) was added MA-OSu (37.37 mg, 148.18 pmol, 1.2 eq) and DIPEA (47.88 mg, 370.46 pmol, 64.53 pL, 3 eq). The mixture was stirred at 20°C for 1 hour. LC-MS showed the starting material was consumed and desired mass was detected. The reaction was purified by prep-HPLC (TFA condition: column: WePure Biotech XPtC18 100*30*10pm ; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 25%-55% B over 8.0 min) to give compound 36 (65 mg, 34% yield) as a white solid. LCMS (ESI+, Method #5): m / z found 757.5, calculated 757.4 (M+2 / 2), RT: 2.209 min. HRMS (ES+): m / z found 1513.85 (M+l), calculated 1513.84. 'HNMR (400 MHz, DMSO-d6) 8 ppm 0.67 - 1.13 (m, 30 H), 1.22 - 1.39 (m, 3 H), 1.41 - 1.66 (m, 4 H), 1.69 - 2.02 (m, 2 H), 1.69 - 1.86 (m, 4 H), 2.07 - 2.16 (m, 2 H), 2.19 - 2.29 (m, 3 H), 2.35 - 2.42 (m, 2 H), 2.63 - 2.74 (m, 1 H), 2.80 - 3.06 (m, 8 H), 3.10 - 3.18 (m, 4 H), 3.22 - 3.27 (m, 4 H), 3.32 (br d, J = 9.88 Hz, 1 H), 3.35 - 3.40 (m, 2 H), 3.41 - 3.51 (m, 2 H), 3.52 - 3.66 (m, 4 H), 3.73 - 3.91 (m, 2 H), 3.92 - 4.08 (m, 5 H), 4.19 (br d, J= 8.75 Hz, 3 H), 4.24 - 4.62 (m, 12 H), 4.69 -4.79 (m, 1 H), 4.85 (br dd, J= 12.32, 7.44 Hz, 1 H), 5.83 - 6.16 (m, 1 H), 7.09 (s, 2 H), 7.17 (br t, J= 5.63 Hz, 1 H), 7.22 - 7.36 (m, 4 H), 7.62 (br d, J= 8.25 Hz, 1 H), 7.74 (br d, J = 4.88 Hz, 1 H), 7.89 (br d, J= 5.00 Hz, 1 H), 7.93 - 8.11 (m, 2 H), 8.14 - 8.29 (m, 1 H), 8.52 -9.32 (m, 1 H).WSGR Docket No. 60801-718.601
[0437] Example A30. Synthesis of Compound 37, MA-GluValCit-SerSar-exatecan
[0438] Synthesis of Fmoc-exatecan (220). To a solution of 219, exatecan (5 g, 11.48 mmol, 1 eq) and sodium bicarbonate (3.86 g, 45.93 mmol, 1.79 mL, 4 eq) in water (10 mL) and acetonitrile (40 mL) was added Fmoc-OSu (5.81 g, 17.22 mmol, 1.5 eq) at 25°C. Thereaction was stirred at 25°C for 12 h, at which time LCMS showed starting material was consumed and desired product was formed. The reaction mixture was concentrated to give 220 (7.4 g, 70% yield, 72% purity) as white solid and was used to next step reaction without purification. LCMS (ESI+, Method #6): m / z found 658.2, calculated 658.2 (M+l), RT: 0.566 min.
[0439] Synthesis of Boc-Sar-exatecan-Fmoc (221). To a solution of 220 (7.4 g, 11.25 mmol, 1 eq and 2-[tert-butoxycarbonyl(methyl)amino]acetic acid (3.19 g, 16.88 mmol, 1.5 eq in dichloromethane (74 mL) was added DMAP (274.92 mg, 2.25 mmol, 0.2 eq and DIC (2.13 g, 16.88 mmol, 2.61 mL, 1.5 eq and 4 A molecular sieve (7.4 g, 11.25 mmol, 1 eq at 25°C. The reaction was stirred at 25°C for 2 h, at which time LCMS showed starting material was consumed and desired product was formed. The reaction mixture was filtered and concentrated to give a crude residue. The residue was purified by column chromatography (SiCh, petroleum ether / ethyl acetate=99 / l to 20 / 80) to give 221 (9 g, 89%WSGR Docket No. 60801-718.601yield, 93% purity) as white solid. LCMS (ESI+, Method #7): m / z found 829.4, calculated 829.3 (M+l), RT: 2.357 min.
[0440] Synthesis of H-Sar-exatecan-Fmoc (222). To a solution of 221 (9 g, 10.86 mmol, 1 eq in di chloromethane (80 mL) was added TFA (10 mL) at 25°C. The reaction was stirred at 25°C for 0.5 h. LCMS showed starting material was completely consumed and desired product was formed. The reaction mixture was concentrated to give a crude residue. The reaction was purified by prep-HPLC (TFA condition: column: 3_Phenomenex Luna C18 75*30mm*3um;mobile phase: [H2O(0.1% TFA)-ACN];gradient:30%-60% B over 8.0 min) to give 222 (5 g, 61% yield, 97% purity) as white solid. LCMS (ESI+, Method #6): m / z found 729.3, calculated 729.3 (M+l), RT: 0.457 min.
[0441] Synthesis of Fmoc-stretcher-Glu(OtBu)ValCit-SerSar-exatecan-Fmoc (223). To a solution of linker intermediate 40 (1.68 g, 1.90 mmol, 1 eq) and 222 (1.66 g, 2.28 mmol, 1.2 eq in DMF (16.8 mL) was added DIPEA (368.43 mg, 2.85 mmol, 496.54 pL, 1.5 eq and HATU (1.08 g, 2.85 mmol, 1.5 eq) at 0°C. The reaction was stirred at 0°C for 1 h, at which time LCMS showed starting material was consumed and desired product was formed. The reaction mixture was concentrated to give a crude reaction residue. The residue was purified by prep-HPLC (TFA condition: column: Waters Xbridge BEH C18250*70mm*10pm ;mobile phase: [H20(0.1%TFA)-ACN];gradient:50%-85% B over 18.0 min) to give 223 (1.7 g, 56% yield) as white solid. LCMS (ESI+, Method #6): m / z found 1595.6, calculated 1594.7 (M+l), RT: 0.595 min.
[0442] Synthesis of H-stretcher-Glu(OtBu)ValCit-SerSar-exatecan (224). To a solution of 223 (1.7 g, 1.07 mmol, 1 eq) in DMF (17 mL) was added piperidine (272.30 mg, 3.20 mmol, 315.82 pL, 3 eq at 25°C. The reaction was stirred at 25°C for 1 h, at which time LCMS showed starting material was consumed and desired product was formed. The reaction mixture was concentrated to give a crude residue. The residue was purified by prep-HPLC (column: Waters Xbridge BEH C18250*70mm*10pm ;mobile phase:[H20(0.1%TFA)-ACN];gradient:l%-40% B over 18.0 min) to give 224 (0.57 g, 46% yield) as white solid. LCMS (ESI+, Method #8): m / z found 576.1, calculated 575.8 ([M+2] / 2), RT: 7.046 min.
[0443] Synthesis of MA-Glu(OtBu)ValCit-SerSar-exatecan (225). To a solution of 224 (570 mg, 495.54 pmol, 1 eq in DMF (5.7 mL) was added DIPEA (115.28 mg, 891.98 pmol, 155.37 pL, 1.8 eq and (2,5-dioxopyrrolidin-l-yl) 2-(2,5-di oxopyrrol- l-yl)acetate (112.47 mg, 445.99 pmol, 0.9 eq at 25°C. The reaction was stirred at 25°C for 1 h, at which timeWSGR Docket No. 60801-718.601LCMS showed starting material was consumed and desired product was formed. The reaction mixture was concentrated to give a crude reaction residue. The residue obtained was purified by prep-HPLC (column: Waters Xbridge BEH Cl 8250*70mm*10pm ; mobile phase: [H2O (10 mMNH4HCO3)-ACN];gradient:15%-45% B over 15.0 min) to give 225 (200 mg, 31% yield) as white solid. LCMS (ESI+, Method #5): m / z found 1288.6, calculated 1287.6 (M+l), RT: 0.408 min.
[0444] Synthesis of MA-GluValCit-SerSar-exatecan (Compound 37). To a solution of 225 (200 mg, 155.36 pmol, 1 eq) in dichloromethane (1.6 mL) was added TFA (0.4 mL) at 25°C. The reaction was stirred at 25°C for 1 h, at which time LCMS showed starting material was completely consumed and desired product was formed. The reaction mixture was concentrated to give a crude residue. The residue was purified by prep-HPLC (TFA condition: column: Xbridge OBD C18 100*30*5 and Kinetex EVO C18 100*30*5;mobile phase: [H20(0.1%TFA)-ACN];gradient:5%-30% B over 20.0 min) to give compound 37 (85 mg, 44% yield) as yellow solid. LCMS (ESI+, Method #5): m / z found 616.4, calculated 616.3 ([M+2] / 2), RT: 1.880 min. HRMS (ES+): m / z found 1231.50 (M+l), calculated 1231.50.1H NMR (400 MHz, DMSO-76) 6 ppm 0.69 - 0.98 (m, 9 H) 1.11 - 1.58 (m, 4 H) 1.64 - 1.77 (m, 1 H) 1.80 - 2.01 (m, 2 H) 2.08 - 2.28 (m, 5 H) 2.32 - 2.43 (m, 6 H) 2.67 (br d, J=1.50 Hz, 4 H) 2.79 - 3.00 (m, 2 H) 3.12 (s, 3 H) 3.16 - 3.20 (m, 2 H) 3.49 - 3.53 (m, 1 H) 3.59 (br t, 7=5.50 Hz, 2 H) 3.70 (br dd, 7=10.13, 7.13 Hz, 1 H) 4.02 (s, 2 H) 4.10 - 4.25 (m, 2 H) 4.26 - 4.41 (m, 3 H) 4.79 - 4.86 (m, 1 H) 4.87 - 5.04 (m, 1 H) 5.11 (br s, 1 H) 5.37 - 5.46 (m, 2 H) 5.48 - 5.55 (m, 2 H) 5.67 - 5.93 (m, 2 H) 6.99 - 7.34 (m, 3 H) 7.70 (br d, 7=7.63 Hz, 1 H) 7.88 (br d, 7=10.51 Hz, 1 H) 7.93 - 8.14 (m, 3 H) 8.20 (br t, 7=5.32 Hz, 1 H) 8.57 (br s, 3 H) 11.64 - 12.41 (m, 1 H).
[0445] Example A31. Synthesis of Compound 38, MA-GluValCit-SerSar-MDOCPTWSGR Docket No. 60801-718.601
[0446] Synthesis of Boc-Sar-MDOCPT (227). To a solution of known camptothecin analogue 226, MDOCPT (5 g, 11.15 mmol, 1 eq and 2-[tert-butoxycarbonyl(methyl)amino] acetic acid (2.53 g, 13.38 mmol, 1.2 eq) in DCM (50 mL) was added DMAP (136.21 mg, 1.11 mmol, 0.1 eq and DIC (2.11 g, 16.72 mmol, 2.59 mL, 1.5 eq and 4Amol sieves (1 g, 1 eq at 20°C. The reaction was stirred at 20°C for 1 h, at which time LCMS showed starting material was consumed and desired product was formed. The reaction mixture was diluted with dichloromethane (100 mL) and extracted with water 300 mL (100 mL x 3). The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure to give 227 (6.9 g, 99% yield) as white solid and used to next step reaction without purification. LCMS (ESI+, Method #7): m / z found 620.3, calculated 620.3 (M+l), RT: 1.965 min.
[0447] Synthesis of H-Sar-MDOCPT (228). To a solution of 227 (6.8 g, 10.97 mmol, 1 eq) in dichloromethane (61.2 mL) was added TFA (6.8 mL) at 25°C.The reaction was stirred atWSGR Docket No. 60801-718.60125°C for 1 h, at which time LCMS showed starting material was consumed and desired product was formed. The reaction mixture was concentrated to give a crude residue. The reaction was purified by prep-HPLC (TFA condition: column: 3_Phenomenex Luna Cl 8 75*30mm*3um; mobile phase: [H2O(0.1% TFA)-ACN];gradient:45%-75% B over 8.0 min) to give 228 (5.5 g, 85% yield, 89% purity) as white solid. LCMS (ESI+, Method #7): m / z found 520.3, calculated 520.2 (M+l), RT: 1.362 min.
[0448] Synthesis of Boc-Glu(OtBu)-OSu (230). To a solution of 229, Boc-Glu(OtBu) (25 g, 82.41 mmol, 1 eq in dichloromethane (250 mL) was added HOSu (10.43 g, 90.65 mmol, 1.1 eq) and DCC (18.70 g, 90.65 mmol, 18.34 mL, 1.1 eq at 20°C. The reaction was stirred at 20°C for 1 h. LCMS showed starting material was completely consumed and desired product was formed. The reaction mixture was filtered to give filtrate, which was then concentrated to give 230 (31 g, 93% yield) as white solid and used to next step reaction without purification. LCMS (ESI+, Method #7): m / z found 423.0, calculated 423.2 (M+23), RT: 1.771 min.
[0449] Synthesis of Boc-Glu(OtBu)ValCit-OH (231). To a solution of 230 (10 g, 2497 mmol, 1 eq and (2S)-2-[[(2S)-2-amino-3-methyl-butanoyl]amino]-5-ureido-pentanoic acid (6.85 g, 24.97 mmol, 1 eq in dioxane (80 mL) and water (20 mL) was added sodium bicarbonate (14.69 g, 174.82 mmol, 6.80 mL, 7 eq at 20°C. The reaction was stirred at 20°C for 2 h, at which time LCMS showed starting material was consumed and desired product was formed. The reaction mixture was filtered to give filtrate, which was concentrated and purified by prep-HPLC (TFA condition : column: Welch Xtimate Cl 8 250*100mm#10pm ;mobile phase: [H2O(0.1%TFA)-ACN];gradient:25%-55% B over 20.0 min) to give 231 (9.3 g, 64% yield) as white solid. LCMS (ESI+, Method #7): m / z found 560.0, calculated 560.3 (M+l), RT: 1.358 min.
[0450] Synthesis of Boc-Glu(OtBu)ValCit-OSu (232). To a solution of 1-hydroxypyrrolidine-2, 5-dione (2.87 g, 24.93 mmol, 1.5 eq and 231 (9.3 g, 16.62 mmol, 1 eq in DMF (93 mL) was added EDCI (4.78 g, 24.93 mmol, 1.5 eq at 20°C. The reaction was stirred at 20°C for 2 h. LCMS showed starting material was consumed and desired product was formed. The reaction mixture was concentrated to give 232 (10 g, 64% yield, 70% purity) as white solid and was used to next step reaction without purification. LCMS (ESI+, Method #7): m / z found 657.4, calculated 657.3 (M+l), RT: 1.494 min.
[0451] Synthesis of Boc-Glu(OtBu)ValCit-Ser (233). To a solution of 232 (10 g, 15.23 mmol, 1 eq and (2 S)-2-amino-3 -hydroxy- propanoic acid (1.60 g, 15.23 mmol, 1 eq inWSGR Docket No. 60801-718.601dioxane (80 mL) and water (20 mL) was added sodium bicarbonate (8.95 g, 106.59 mmol, 4.15 mL, 7 eq) at 25°C. The reaction was stirred at 25°C for 2 h, at which time LCMS showed starting material was consumed and desired product was formed. The reaction mixture was concentrated to give a crude reaction residue. The residue was purified by column chromatography (SiCh, di chloromethane: methanol = 99:1 to 50:50) to give 233 (5.4 g, 54% yield) as white solid. LCMS (ESI+, Method #6): m / z found 647.4, calculated 647.4 (M+l), RT: 0.397 min.
[0452] Synthesis of Boc-Glu(OtBu)ValCit-SerSar-MDOCPT (234). To a solution of 233 (2 g, 3.09 mmol, 1 eq) and 228, Sar-MDOCPT (1.61 g, 3.09 mmol, 1 eq) in DMF (20 mL) was added DIPEA (399.68 mg, 3.09 mmol, 538.65 pL, 1 eq and HATU (1.76 g, 4.64 mmol, 1.5 eq) at 0°C. The reaction was stirred at 0°C for 1 h, at which time LCMS showed starting material was consumed and desired product was formed. The reaction mixture was concentrated to give a residue, which was purified by column chromatography (SiCh, tetrahydrofuran / ethyl acetate = 0 / 100 to 1 / 0) to give 234 (4 g, 76% yield) as white solid. LCMS (ESI+, Method #7): m / z found 1148.7, calculated 1148.5 (M+l), RT: 1.821 min.
[0453] Synthesis of H-GluValCit-SerSar-MDOCPT (235). To a solution of 234 (4 g, 237 mmol, 1 eq in dichloromethane (32 mL) was added TFA (8 mL) at 25°C. The reaction was stirred at 25°C for 1 h, at which time LCMS showed starting material was completely consumed and desired product was formed. The reaction mixture was concentrated to give a residue, which was purified by prep-HPLC (TFA condition: 3_Phenomenex Luna Cl 8 75*30mm*3um;mobile phase: [H2O(0.1% TFA)-ACN];gradient:10%-40% B over 8.0 min) to give 235 (1.06 g, 45% yield) as yellow solid. LCMS (ESI+, Method #7): m / z found 497.0, calculated 496.7 ([M+2] / 2), RT: 1.286 min.
[0454] Synthesis of Fmoc-stretcher-GluValCit-SerSar-MDOCPT (236). To a solution of 235 (400 mg, 403.21 pmol, 1 eq) in H2O (0.8 mL) and dioxane (3.2 mL) was added stretcher intermediate 11 (273.65 mg, 604.82 pmol, 1.5 eq) and sodium bicarbonate (237.11 mg, 2.82 mmol, 109.82 pL, 7 eq) at 25°C. The reaction was stirred at 25°C for 1 h, at which time LCMS showed starting material was consumed and desired product was formed. The reaction mixture was concentrated to give a residue, which was then purified by prep-HPLC (TFA condition: 3_Phenomenex Luna C18 75*30mm*3um;mobile phase: [H2O(0.1% TFA)-ACN];gradient:30%-60% B over 8.0 min) to give 236 (0.3 g, 55.97% yield) as yellow solid. LCMS (ESI+, Method #6): m / z found 665.5, calculated 665.3 ([M+2] / 2), RT: 0.501 min.
[0455] Synthesis of H-stretcher-GluValCit-SerSar-MDOCPT (237). To a solution of 236 (100 mg, 75.22 pmol, 1 eq) in DMF (1 mL) was added piperidine (19.21 mg, 225.66 pmol,WSGR Docket No. 60801-718.60129 pL, 3 eq) at 25°C. The reaction was stirred at 25°C for 1 h, at which time LCMS showed starting material was consumed and desired product was formed. The reaction mixture obtained was purified by prep-HPLC (TFA condition : Xbridge OBD C18 100*30*5 Kinetex EVO C18 100*30*5;mobile phase: [H2O (0.1%TFA)-ACN];gradient:25%-35% B over 22.0 min) to give 237 (0.08 g, 96% yield) as yellow solid. LCMS (ESI+, Method #7): m / z found 554.6, calculated 554.2 ([M+2] / 2), RT: 1.275 min.
[0456] Synthesis of MA-GluValCit-SerSar-MDOCPT (Compound 38). To a solution of 237 (50 mg, 45.16 pmol, 1 eq) in DMF (0.5 mL) was added (2,5-dioxopyrrolidin-l-yl) 2-(2,5-dioxopyrrol-l-yl)acetate (13.67 mg, 54.19 pmol, 1.2 eq) and DIPEA (17.51 mg, 135.48 pmol, 23.60 pL, 3 eq) at 25°C. The reaction was stirred at 25°C for 1 h, at which time LCMS showed starting material was consumed and desired product was formed. The reaction mixture was concentrated to give a crude residue, which was purified by prep-HPLC (column: 3_Phenomenex Luna C18 75*30mm*3um;mobile phase: [H2O(0.1% TFA)-ACN];gradient:20%-50% B over 8.0 min) to give compound 38 (30 mg, 47% yield) as off white solid. LCMS (ESI+, Method #5): m / z found 622.8, calculated 622.8 ([M+2] / 2), RT: 2.237 min. HRMS (ES+): m / z found 1244.51 (M+l), calculated 1244.50. 'HNMR (400 MHz, DMSO-tL) 6 ppm 0.69 - 0.98 (m, 12 H) 1.21 - 1.37 (m, 2 H) 1.45 (dt, 7=14.51, 7.25 Hz, 3 H) 1.54 - 1.76 (m, 4 H) 1.83 - 2.01 (m, 2 H) 2.08 - 2.28 (m, 4 H) 2.31 - 2.43 (m, 2 H) 2.79 - 2.99 (m, 3 H) 3.05 - 3.14 (m, 4 H) 3.18 (q, 7=5.71 Hz, 2 H) 3.32 - 3.40 (m, 2 H) 3.42 -3.53 (m, 1 H) 3.59 (br t, 7=6.13 Hz, 2 H) 3.72 (br dd, 7=10.51, 6.75 Hz, 1 H) 4.02 (s, 2 H) 4.11 - 4.16 (m, 1 H) 4.20 - 4.27 (m, 2 H) 4.32 (br dd, 7=13.26, 8.13 Hz, 2 H) 4.41 (br d, 7=17.26 Hz, 2 H) 4.78 - 4.88 (m, 3 H) 5.16 - 5.30 (m, 3 H) 5.45 - 5.52 (m, 2 H) 5.82 - 6.03 (m, 1 H) 6.29 (s, 2 H) 7.00 (s, 1 H) 7.08 (s, 2 H) 7.39 - 7.50 (m, 1 H) 7.61 (s, 1 H) 7.72 (br d, 7=8.50 Hz, 1 H) 8.01 (br d, 7=7.25 Hz, 1 H) 8.06 (br d, 7=7.63 Hz, 1 H) 8.19 (br t, 7=5.25 Hz, 1 H).
[0457] Example A32. Synthesis of Compound 39, MA-(GluValCit-SerSar-exatecan)2WSGR Docket No. 60801-718.601
[0458] Synthesis of Boc-Glu(OtBu)ValCit-SerSar-exatecan-Fmoc (238). To a solution of 222 (3.10 g, 4.25 mmol, 1.1 eq) and linker intermediate 233 (2.5 g, 3.87 mmol, 1 eq) in DMF (25 mL) was added HATU (2.20 g, 5.80 mmol, 1.5 eq) and DIPEA (749.40 mg, 5.80 mmol, 1.01 mL, 1.5 eq). The mixture was stirred at 0°C for 1 hour, at which time LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction was filtered and concentrated under reduced pressure to give a crude reaction residue. The residue obtained was purified by prep-HPLC (TFA condition: column: Waters Xbridge BEH Cl 8250*70mm*10pm ; mobile phase: [H2O (0.2% FA)-ACN]; gradient: 45%-65% B over 22.0 min) to give 238 (1.35 g, 24% yield) as yellow solid. LCMS (ESI+, Method #8): m / z found 679.8, calculated 679.3 ([M+2] / 2), RT: 14.428 min.
[0459] Synthesis of H-GluValCit-SerSar-exatecan-Fmoc (239). A solution of 238 (1.35 g, 994.49 pmol, 1 eq) in TFA (1.35 mL) and dichloromethane (12.15 mL) was stirred at 25°C for 1 hour. LC-MS showed the starting material was consumed completely and desired product mass was detected. The reaction was filtered and concentrated under reduced pressure to give a crude residue, which was purified by prep-HPLC (TFA condition: column: Waters Xbridge BEH C18250 * 70 mm * 10 um; mobile phase: [H2O (0.2% FA)-ACN];WSGR Docket No. 60801-718.601gradient: 20%-50% B over 20.0 min) to give 239 (400 mg, 31% yield) as yellow solid.LCMS (ESI+, Method #8): m / z found 601.6, calculated 601.2 ([M+2] / 2), RT: 10.002 min.
[0460] Synthesis of Fmoc-stretcher-(GluValCit-SerSar-exatecan-Fmoc)2 (240). To a solution of 239 (350 mg, 273.88 pmol, 2 eq) and branched PEG stretcher 26 (129.33 mg, 136.94 pmol, 1 eq) in DMF (3.5 mL) was added DIPEA (35.40 mg, 273.88 pmol, 47.70 pL, 2 eq). The mixture was stirred at 20°C for 1 hour, at which time LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction was filtered and concentrated under reduced pressure to give a residue, which was purified by prep-HPLC (TFA condition: column: Phenomenex Luna C18 80 * 30 mm * 3 um; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 40%-70% B over 8.0 min) to give 240 (100 mg, 21% yield) as yellow solid. LCMS (ESI+, Method #7): m / z found 1014.8, calculated 1014.1 ([M+3] / 3), RT: 2.15 min.
[0461] Synthesis of H-stretcher-(GluValCit-SerSar-exatecan)2 (241). To a solution of 240 (100 mg, 29.59 pmol, 1 eq) in DMF (1 mL) was added piperidine (7.56 mg, 88.78 pmol, 8.77pL, 3 eq). The mixture was stirred at 20°C for 1 hour, at which time LC-MS showed the starting material was consumed completely and desired mass was detected. The reaction was filtered and concentrated under reduced pressure to give a residue, which was purified by prep-HPLC (TFA condition: column: Phenomenex Luna C18 80 * 30 mm * 3 um; mobile phase: [H2O (0.1% TFA)-ACN]; gradient: 10%-40% B over 8.0 min) to give 241 (25 mg, 35% yield) as yellow solid. LCMS (ESI+, Method #7): m / z found 792.5, calculated 792.0 ([M+3] / 3), RT: 1.286 min.
[0462] Synthesis of MA-(GluValCit-SerSar-exatecan)2 (Compound 39). To a solution of 241 (25 mg, 10.53 pmol, 1 eq) and MA_OSu (2.66 mg, 10.53 pmol, 1 eq) in DMF (0.25 mL) was added DIPEA (2.72 mg, 21.06 pmol, 3.67 pL, 2 eq). The mixture was stirred at 25°C for 1 hour, at which time LC-MS showed the starting material was consumed completely and ...
Claims
WSGR Docket No. 60801-718.601CLAIMSWhat is claimed is:
1. A compound of F ormula (la) or (Ila) :>Formula (Ila)or a salt or stereoisomer thereof, or a mixture of stereoisomers thereof, wherein:P1is a payload bonded via an ester bond or a thioester bond,R1is H, Ci-Ce alkyl, wherein Ci-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, -OH, -NH2, -COOH, - CONH2, -NHC(=O)NH2, -NHC(=NH)NH2, -SH, and -SCH3, or two R1groups together form a C3-6 cycloalkyl;R2is Ci-Ce alkyl,R3is H, Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)-NHC(=O)NH2, -(Ci-C6alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)-NHC(=NH)NH2, -(Ci-C6alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O-NHC(=NH)NH2, or -(Ci-C6alkylene)-0-NHC(=NH)NHCH3, wherein Ci-Ce alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, -C(=0)NH2, -C(=O)OH, - OH, -NH2, -SH, and -SCH3,or R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, wherein when R1and R2form a pyrrolidine, then R3is Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)- NHC(=O)NH2, -(C1-C6 alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)- NHC(=NH)NH2, -(CI-C6alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O- NHC(=NH)NH2, or -(Ci-C6alkylene)-O-NHC(=NH)NHCH3, wherein Ci-C6alkyl is optionally substituted with one, two, or three groups selected from theWSGR Docket No. 60801-718.601group consisting of -F, phenyl, -C(=0)NH2, -C(=O)OH, -OH, -NH2, -SH, and -SCH3,L1is -L3-[AA]a-,wherein:L3is a first spacer,each AAis independently an amino acid,a is an integer from 1 to 10,R4is H, C1-C12 alkyl, PEG1-20,L2is a bond or a second spacer,L5is a bond, C1-6 alkylene, -CO-, -NHCO-C1-6 alkylene, -NHCO-C1-6 alkenylene, 5-6 membered heterocycloalkylene, or fused triazole group, wherein the C1-6 alkylene, C1-6 alkenylene, and heterocycloalkylene are substituted with one, two, three, or four groups selected from oxo (=0), -COOH, and CH2COOH, R5is a reactive group capable of reacting with a thiol, an amine, an azide, or an alkyne,P2is a targeting moiety, andn is an integer from 1 to 20.
2. The compound of claim 1, wherein each R3is -H, -Ci-Ce alkyl, -(C1-C4 alkylene)-OH, -(C2-C6 alkylene)-NH2, -(C2-C6 alkylene)-NHC(=O)NH2, or -(C2-C6 alkylene)- NHC(=NH)NH2.
3. The compound of claim 1, wherein each R3is H or -(C1-C4 alkylene)-OH.
4. The compound of claim 1, according to Formula (lb) or (lib):Formula (lb) Formula (lib) whereinP1is a payload bonded via an ester bond or a thioester bond,WSGR Docket No. 60801-718.601R1is H or substituted or unsubstituted Ci-Ce alkyl;L1is -L3-[AA]a-, wherein L3is a first spacer, each AA is independently an amino acid, and a is an integer from 1 to 10,L2is a bond or a second spacer,L5is a bond, a succinimide group, a succinic acid group, or a fused triazole group. R5is a reactive group capable of reacting with a thiol, an amine, or an azide,P2is small molecule or polypeptide targeting moiety, andn is an integer from 1 to 12.
5. The compound of claim 1, according to Formula (Ic) or (lie):Formula (Ic) Formula (lie) whereinP2is an antibody or antigen-binding peptide, andn is an integer from 1 to 8.
6. The compound of any one of claims 1-5, wherein L2is -(L2a)i-2o-, each L2ais independently -C1-2 alkylene-O-, -O-C1-2 alkylene-, -C(O)NH-, -NHC(O)-, - C1-2 alkylene-C(O)NH-, - C1-2 alkylene-NHC(O)-, -O-, -S-, phenylene, -NH-, -NHC(O)O-, -OC(O)NH-, or -(CR'R , wherein each R1and R2is independently hydrogen, C1-6 alkyl, -C1-5 alkylene-OH, -C1-5 alkylene-NBfc, -C1-5 alkylene-CONBfc, -C1-5 alkylene- COOH, or -OH, -NH2, -CONH2, -COOH, and v is 1-20.WSGR Docket No. 60801-718.6017. The compound of any one of claims 1-6, wherein R1is H or -Ci-Ce alkyl, wherein the Ci-6 alkyl is unsubstituted or is substituted with -NHC(=NH)NH2 or -COOH.
8. The compound of any one of claims 1-7, wherein each R1is H.
9. The compound of any one of claims 1-7, wherein R1is -Ci-Ce alkyl.
10. The compound of claim any one of claims 1-9, wherein [AA]ais Asn-, Asp-, Arg-, Lys-, Ala-, Gly-, Val-Arg*, Gly-Arg*, Ile-Arg*, Ala-Arg*, Glu-Arg*, Arg-Arg*, Val- Ala*, Val-Cit*, Val-Gly*, Gly-Cit*, Ile-Cit*, Ala-Cit*, Glu-Cit*, Glu- Val-Arg*, Glu- Val-Cit*, Glu-Val-Lys*, Glu-Gly-Cit, Glu-Val-Ala, Gly-Val-Arg*, Gly-Gly-Arg*, Gly-Glu- Val-Arg*, Gly-Val-Cit*, Gly-Gly-Cit*, Gly-Glu- Val-Cit*, or Glu-Arg-Arg*; wherein in each instance, * indicates the C-terminal amino acid residue.
11. The compound of claim 1, according to Formula (Id) or (lid) :Formula (lid)whereina’ is 0, 1, or 2; andeach of R6and R7is independently hydrogen or Ci-6 alkyl, wherein each Ci-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NH2, -NHC(=O)NH, -SH, - SCH3, -NHC(=O)NHCH3, -NHC(=NH)NH2, -NHC(=NH)NHCH3, -O- NHC(=NH)NH2, and -O-NHC(=NH)NHCH3.WSGR Docket No. 60801-718.60112. The compound of any one of claims 1-5, or 11, wherein R4iswherein R1is H or substituted or unsubstituted -Ci-Ce alkyl.
13. The compound of claim 1, wherein P1is:wherein:Ralis H, CH3, or CH2OH;Rblis H or F;Rb2is H, CH3, OH, or OC(=O)-G-G; wherein each G is piperidine;or Rbland Rb2taken together form a methylenedioxy or ethylenedioxy group; Rb3is H or Ci-Ce alkylene-N(CH3)2; andRb4is H, Ci-Ce alkyl, or C1-6 alkylene-NH-CH(CH3)2;or Rb3and Rb4taken together form a 6-membered carbocycle substituted with - NH2or -NHCOCH2OH; andeach Rcis independently H or CH3.
14. The compound of claim 1, according to any one of the following formulae:WSGR Docket No. 60801-718.601wherein:each m is independently 0, 1, 2, 3, 4, or 5;each u is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8;each a’ is 0, 1, or 2;each R5aand R5bis H or halogen;each of R1, R6, R7, and R8is independently hydrogen or Ci-6 alkyl, wherein each Ci-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of phenyl, -CONH2, -COOH, -OH, -NH2, -NHC(=O)NH, - NHC(=NH)NH2, -SH, -SCH3; or two R1together form C3-6 cycloalkyl;each R3is H or CH2OH;each Ralis H, CH3, or CH2OH,each Rblis H or F;each Rb2is H, CH3, OH, or OC(=O)-G-G; wherein each G is piperidine;WSGR Docket No. 60801-718.601or Rbland Rb2taken together form a methylenedioxy or ethylenedioxy group; each Rb3is H or Ci-Ce alkylene-N(CH3)2; andeach Rb4is H, Ci-Ce alkyl, or Ci-6 alkylene-NH-CH(CH3)2;or Rb3and Rb4taken together form a 6-membered carbocycle substituted with -NH2 or -NHCOCH2OH.
15. The compound of any one of claims 1-14, wherein the compound is selected from any one of Tables 1-4, or any one of Compounds 1-50.
16. The compound of any one of claims 1-15, wherein the peptide or polypeptide comprises an unnatural amino acid of the structure:
17. A method of delivering a payload to a target cell, the method comprising contacting the target cell with the compound of any one of claims 1-16.
18. A method of treating cancer in a mammal, the method comprising administering to the mammal an effective amount of a composition comprising the compound of any one of claims 1-16.
19. A compound of Formula (Illa):Formula (Illa)wherein:P1is a chemotoxin,R1is H, Ci-Ce alkyl, wherein C1-6 alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, -COOH, -CONH2, -OH, -NH2, -NHC(=O)NH2, -NHC(=NH)NH2, -SH, and -SCH3,; or two R1groups together form a C3-6 cycloalkyl;WSGR Docket No. 60801-718.601R2is Ci-Ce alkyl;R3is H, Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)- NHC(=O)NH2, -(C1-C6 alkylene)-NHC(=O)NHCH3, -(Ci-C6alkylene)- NHC(=NH)NH2, -(CI-C6alkylene)-NHC(=NH)NHCH3, -(Ci-C6alkylene)-O- NHC(=NH)NH2, or -(Ci-C6alkylene)-O-NHC(=NH)NHCH3, wherein Ci-C6alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NH2, -SH, and -SCH3;or R1and R2are taken together with the intervening atoms connecting R1and R2to form a pyrrolidine, and R3is Ci-Ce alkyl, (Ci-Ce alkylene)-OH, -(Ci-Ce alkylene)-NH2, -(Ci-Ce alkylene)-NHC(=O)NH2, -(Ci-Ce alkylene)- NHC(=O)NHCH3, -(C1-C6 alkylene)-NHC(=NH)NH2, -(Ci-C6alkylene)- NHC(=NH)NHCH3, -(CI-C6alkylene)-O-NHC(=NH)NH2, or -(Ci-C6alkylene)-O-NHC(=NH)NHCH3, wherein Ci-Ce alkyl is optionally substituted with one, two, or three groups selected from the group consisting of -F, phenyl, -C(=O)NH2, -C(=O)OH, -OH, -NH2, -SH, and -SCH3.R3CH3O H2N / ^^N'^X^P120. The compound of claim 19, having the structure: °21. The compound of claim 19, wherein R1is hydrogen, R2is Ci-6 alkyl; and R3is CH2OH.
22. The compound of claim 19, wherein R1is hydrogen, R2is Ci-6 alkyl; and R3is hydrogen.
23. The compound of claim 19, having the structure:
24. The compound of claim 19, wherein R1is Ci-6 alkyl optionally substituted with - COOH, -CONH2, -OH, -NH2, -NHC(=O)NH2, or -NHC(=NH)NH2; R2is Ci-6 alkyl; and R3is CH2OH.WSGR Docket No. 60801-718.60125. The compound of claim 19, having the structure:
26. The compound of any one of claims 19-25, wherein the chemotoxin comprises a maytansinoid, taxane, auristatin, camptothecin, vinca alkaloid, tubulysin, or epothilone.
27. The compound of any one of claims 1-26, wherein P1is:wherein:Ralis H, CH3, or CH2OH;Rblis H or F;Rb2is H, CH3, OH, or OC(=O)-G-G; wherein each G is piperidine;or Rbland Rb2taken together form a methylenedioxy or ethylenedioxy group; Rb3is H or Ci-Ce alkylene-N(CH3)2; andRb4is H, Ci-Ce alkyl, or C1-6 alkylene-NH-CH(CH3)2;or Rb3and Rb4taken together form a 6-membered carbocycle substituted with - NH2or -NHCOCH2OH; andeach Rcis independently H or CH3.