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WO2025232116A1 - Conjugué à effet ciblé, son procédé de préparation et son utilisation - Google Patents

Conjugué à effet ciblé, son procédé de préparation et son utilisation

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Publication number
WO2025232116A1
WO2025232116A1 PCT/CN2024/129461 CN2024129461W WO2025232116A1 WO 2025232116 A1 WO2025232116 A1 WO 2025232116A1 CN 2024129461 W CN2024129461 W CN 2024129461W WO 2025232116 A1 WO2025232116 A1 WO 2025232116A1
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WO
WIPO (PCT)
Prior art keywords
seq
sequence
variant
cdr
chain variable
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/129461
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English (en)
Chinese (zh)
Inventor
田强
张毅涛
叶健
刘万里
李怡乐
宋攀
周琴
龙虎
杨禹
袁晓曦
宋宏梅
葛均友
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Original Assignee
Sichuan Kelun Biotech Biopharmaceutical Co Ltd
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Application filed by Sichuan Kelun Biotech Biopharmaceutical Co Ltd filed Critical Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Publication of WO2025232116A1 publication Critical patent/WO2025232116A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • This invention relates to the field of targeted therapy, and more specifically to a conjugate with targeting effects.
  • ADCs Antibody-drug conjugates
  • cytotoxins are a novel type of anticancer therapy that combines the precise targeting capabilities of monoclonal antibodies with the cytotoxic effects of cytotoxins to precisely target cancer cells.
  • These drugs consist of three main components: a monoclonal antibody, a cytotoxin (usually a bioactive molecule), and a linker, where the cytotoxin forms a covalent bond with the antibody via the linker.
  • the antibody recognizes and binds to specific antigens on the surface of tumor cells, guiding the ADC into the tumor microenvironment and into the cancer cells, where it enters the cells via endocytosis. Inside the cell, the cytotoxin is released, effectively killing cancer cells through mechanisms such as inhibiting tubulin synthesis or directly damaging DNA, while minimizing impact on normal cells.
  • Pyrrolobenzodiazepine PBD is a class of compounds with high DNA binding selectivity, belonging to the anthraxmycin antibiotic family. Studies have shown that PBD dimers, formed by linking two PBD units through an alkylene chain, can form stable interchain crosslinks with DNA, exhibiting extremely high activity against various tumor cell lines, with activity levels reaching the pmol/L level.
  • the present invention aims to improve upon the aforementioned problems in existing pharmaceutical technologies, specifically by providing a class of PBD-type antibody-drug conjugates.
  • These antibody-drug conjugates exhibit good stability, homogeneity, hydrophilicity, efficacy, and safety.
  • this application provides an antibody-drug conjugate having the structure shown in the formula Ab-[MLED] x , wherein:
  • Ab is an antibody or its antigen-binding fragment that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family of receptor tyrosine kinases.
  • M is the linker site for binding to antibodies or their antigen fragments
  • L is a structural segment connecting connectors M and E;
  • E is a structural segment connecting L and D;
  • D is a cytotoxic drug fragment
  • X is between 1 and 10.
  • M is selected from the following substituted or unsubstituted structural segments:
  • M is selected from the following substituted or unsubstituted structural segments:
  • M is selected from the following substituted or unsubstituted structural segments:
  • L is selected from one or more of the following substituted or unsubstituted structural fragments or stereoisomers thereof: C1-6 alkylene, 6-10 aryl, 5-6 heteroaryl, 9-12 nitrogen-containing heterocyclic, -N(R')-, -NH(R'), -N(R') 2 , carbonyl, -O-, natural or non-natural amino acids and their analogs (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys( COCH2CH2 ( OCH2CH2 ) rOCH3 ) (), Lys(R'), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro
  • DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues
  • DOTAGA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, ⁇ -propionyl
  • NOA 1,4,7-triazacyclononane-N,N',N”-triacetic acid residues
  • L is selected from one or more substituted or unsubstituted structural fragments composed of: natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH 2 CH 2 (OCH 2 CH 2 ) r OCH 3 )), Lys(R'), and short peptides composed of amino acids (e.g., Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala).
  • natural or non-natural amino acids and their analogues e.g., Ala
  • R' is composed of one or more of the following groups, including but not limited to hydrogen, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues), DOTAGA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, ⁇ -propionyl), or NOA (1,4,7-triazacyclononane-N,N',N”-triacetic acid residues), wherein s is selected from an integer from 1 to 20, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably, s is 3-10, for example, s is 5, 8, or 10.
  • DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid residues
  • DOTAGA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra
  • L is selected from one or more substituted or unsubstituted structural fragments composed of: natural or non-natural amino acids and their analogues (e.g., Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala, Lys(COCH 2 CH 2 (OCH 2 CH 2 ) r OCH 3 )), and short peptides composed of amino acids (e.g., Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala).
  • natural or non-natural amino acids and their analogues e.g., Ala, Arg, Asn
  • r is selected from an integer from 1 to 20, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20;
  • s is selected from an integer from 1 to 20, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably, s is 3-10, for example, s is 5, 8, 9, or 10.
  • L is selected from one or more of the following substituted or unsubstituted structural fragments: Val, Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala, Where s is selected from an integer from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably, s is 3 to 10, for example, s is 5, 8, 9, or 10.
  • L is selected from one or more of the following substituted or unsubstituted structural fragments: Val, Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala, Where s is selected from an integer from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; preferably, s is 3 to 10, for example, s is 5, 8, 9, or 10.
  • L is selected from the following substituted or unsubstituted structures: Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala,
  • n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.
  • L is selected from the following substituted or unsubstituted structures: Gly-Lys, Val-Ala, Val-Cit, Val-Lys, Ala-Ala-Ala, Ala-D-Ala-Ala, D-Leu-Ala-Glu, Val-Lys-Gly, Gly-Gly-Val-Ala,
  • n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.
  • L is selected from the following substituted or unsubstituted structures:
  • n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.
  • E is a single bond, substituted or unsubstituted -NH- CH2- or
  • E is a single bond, substituted or unsubstituted -NH- CH2- .
  • E is a single bond or -NH- CH2- .
  • n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.
  • n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.
  • the cytotoxic agent is selected from anti-microtubule agents, DNA intercalating agents, DNA topoisomerase inhibitors, RNA polymerase inhibitors, and gene transcription inhibitors.
  • the anti-microtubule agent is an oliguriatin class, a maytansine class, or an eribulin class.
  • the DNA intercalating agent is a pyrrolobenzodiazepine (PBD) class, trabectedin, or rubotecan.
  • the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecone, or rubotecan) or a topoisomerase II inhibitor (e.g., doxorubicin, doxorubicin, PNU-159682 and its analogues, docarmicin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide).
  • the RNA polymerase inhibitor is ⁇ -amanitin.
  • the gene transcription inhibitor is triptolide and its pharmaceutically acceptable salts, esters, and analogs.
  • the cytotoxic drug is selected from pyrrolobenzodiazepine (PBD) compounds.
  • the cytotoxic drugs disclosed in this application typically contain multiple functional groups, such as hydroxyl (-OH), carboxyl (-COOH), primary amino ( -NH2 ), secondary amino ( -NRaH ), and tertiary amino ( -NRbRc ), where Ra , Rb , and Rc represent only non-hydrogen substituents on the nitrogen atom, or thiol groups (-SH).
  • functional groups such as hydroxyl (-OH), carboxyl (-COOH), primary amino ( -NH2 ), secondary amino ( -NRaH ), and tertiary amino ( -NRbRc ), where Ra , Rb , and Rc represent only non-hydrogen substituents on the nitrogen atom, or thiol groups (-SH).
  • the cytotoxic drug is linked to the E in the antibody-drug conjugate via a -OH, primary amino, secondary amino, or tertiary amino group, or a -SH group.
  • D is a monovalent structure obtained by losing an H from the -OH, -NH2 , or secondary amino group on the cytotoxic drug.
  • D is a monovalent structure obtained by losing an H from the -OH or -NH2 group on the cytotoxic drug.
  • the cytotoxic drug is selected from compounds with the structure shown in formula (I) or pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs thereof:
  • R1 and R2 are each independently selected from hydrogen, -CN, halogen, -OR ⁇ , -NH2 , -NH ( C1-6 alkyl), -N( C1-6 alkyl) 2 , C1-6 alkyl, C2-6 alkenyl, C2-6 ynyl, C3-6 cycloalkyl, 3-6 heterocyclic, C6-10 aryl, and 5-10 heteroaryl; wherein the alkyl, alkenyl, ynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally substituted by one or more substituents selected from -CN, halogen, -OH, -NH2 , C1-6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 ynyl, C3-6 cycloalkyl, 3-6 heterocyclic, C6-10 aryl, and 5-10 heteroaryl groups;
  • R4 and R4 ' are each independently selected from hydrogen, halogen, -OH, -NH2 , C1-6 alkyl, halo- C1-6 alkyl and C1-6 alkoxy, and R5 and R5 ' are both hydrogen;
  • R4 and R4 ' together form a carbon-carbon double bond or a 3-6 membered carbon ring with the carbon atom connecting R4 and R4 ' , and R5 and R5 ' are both hydrogen atoms;
  • R4 and R5 may be bonded together or together form a 3-6 membered carbon ring with the carbon atom connecting R4 and R5 , and R5 ' and R4 ' may be independently selected from hydrogen, halogen, -NH2, C1-6 alkyl, halo -C1-6 alkyl and C1-6 alkoxy;
  • R6 is hydrogen, C1-6 alkyl, or...
  • R6 ' , R7 , and R7 ' are all hydrogen; or R6 ' is bonded to R7 , and R7 ' is hydrogen.
  • Ra is selected from H, C1-6 alkyl, or C3-6 cycloalkyl
  • Ring A is selected from 3-6-membered cycloalkyl, C6-10 aryl, 5-10-membered heteroaryl, and 3-6-membered heterocyclic groups; the 3-6-membered cycloalkyl, C6-10 aryl, 5-10-membered heteroaryl, and 3-6-membered heterocyclic groups are optionally substituted by one or more of the following substituents: hydrogen, -CN, halogen, -OR ⁇ , -NH2 , -NH( C1-6 alkyl), -N( C1-6 alkyl) 2 , C1-6 alkyl, C3-6 cycloalkyl, 3-6-membered heterocyclic group, C6-10 aryl, and 5-10-membered heteroaryl; the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally selected by one or more substituents selected from -CN, halogen, -OH
  • R3 is selected from hydrogen, -CN, halogen, -OR ⁇ , -NH2 , -NH ( C1-6 alkyl), -N( C1-6 alkyl) 2 , C1-6 alkyl, C3-6 cycloalkyl, 3-6 -membered heterocyclic, C6-10 aryl, and 5-10-membered heteroaryl; the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally substituted by one or more substituents selected from -CN, halogen, -OH, -NH2 , C1-6 alkyl, C1-6 alkoxy, C3-6 cycloalkyl, 3-6-membered heterocyclic, C6-10 aryl, and 5-10-membered heteroaryl groups;
  • X and Y are independently -CH 2- and -CD 2- , respectively. and -C(O)-;
  • t is selected from 1 to 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • R3 is not methoxy
  • R1 is selected from hydrogen, -CN, halogen, -ORa , and C1-6 alkyl, wherein Ra is selected from C1-6 alkyl, deuterated C1-6 alkyl, and C3-6 cycloalkyl.
  • R1 is selected from -ORa , wherein Ra is selected from C1-6 alkyl, deuterated C1-6 alkyl and C3-6 cycloalkyl.
  • R1 is selected from methoxy, trideuteroxy, and -O-cyclopropyl.
  • R1 is selected from hydrogen, -CN, halogen, -ORa , and C1-6 alkyl, wherein Ra is a C1-6 alkyl or C3-6 cycloalkyl.
  • R1 is -ORa , where Ra is a C1-6 alkyl or C3-6 cycloalkyl.
  • R1 is methoxy or -O-cyclopropyl.
  • R2 is selected from hydrogen, -CN, halogen, -ORa , and C1-6 alkyl, wherein Ra is a C1-6 alkyl or C3-6 cycloalkyl.
  • R2 is -ORa , where Ra is a C1-6 alkyl group.
  • R2 is a methoxy group.
  • R4 is hydrogen or a halogen
  • R4 ' is selected from hydrogen, halogen, -OH, -NH2 , C1-6 alkyl, halo- C1-6 alkyl, and C1-6 alkoxy
  • R5 and R5 ' are both hydrogen
  • R4 and R4 ' together form a carbon-carbon double bond or a 3-6 membered carbon ring with the carbon atom connecting R4 and R4 '
  • R5 and R5 ' are both hydrogen
  • R4 and R5 are bonded together or together form a 3-6 membered carbon ring with the carbon atom connecting R4 and R5
  • R5 ' and R4 ' are both hydrogen.
  • R4 is hydrogen or fluorine
  • R4 ' is selected from hydrogen, fluorine, -OH, -NH2 , methyl, trifluoromethyl, and methoxy
  • R5 and R5 ' are both hydrogen
  • R4 and R4 ' together form a carbon-carbon double bond or a 3-6 membered carbon ring with the carbon atom connecting R4 and R4 '
  • R5 and R5' are both hydrogen
  • R4 and R5 are bonded together or together form a 3 membered carbon ring with the carbon atom connecting R4 and R5 , and R5' and R4 ' are both hydrogen.
  • R4 is hydrogen or fluorine
  • R4 ' is selected from hydrogen, fluorine, -OH, -NH2 , methyl, trifluoromethyl, and methoxy
  • R5 and R5 ' are both hydrogen
  • R4 and R4 ' together form a carbon-carbon double bond with the carbon atom connecting R4 and R4 '
  • R5 and R5 ' are both hydrogen
  • R4 and R5 are bonded together or together form a 3-membered carbon ring with the carbon atom connecting R4 and R5 , in which R5 ' and R4 ' are both hydrogen.
  • R4 and R4 ' form a ternary carbon ring with the carbon atom connecting R4 and R4 ' , and R5 and R5 ' are both hydrogen atoms.
  • R6 is
  • Ring A is selected from 3-6 membered cycloalkyl, C6-10 aryl, or 5-10 membered heteroaryl;
  • R3 is selected from hydrogen, -CN, halogen, -OR ⁇ , -NH2 , -NH ( C1-6 alkyl), -N( C1-6 alkyl) 2 , C1-6 alkyl, C3-6 cycloalkyl, 3-6 -membered heterocyclic, C6-10 aryl, and 5-10-membered heteroaryl; the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, and heteroaryl groups are optionally substituted by one or more substituents selected from -CN, halogen, -OH, -NH2 , C1-6 alkyl, C1-6 alkoxy, C3-6 cycloalkyl, 3-6-membered heterocyclic, C6-10 aryl, and 5-10-membered heteroaryl groups;
  • Ra is selected from H and C1-6 alkyl groups.
  • R6 is
  • Ring A is selected from cyclopropyl, phenyl, furanyl, thiophene, pyrimidinyl, and pyridinyl;
  • R3 is selected from hydrogen, fluorine, -OH, methoxy, -NH2 and hydroxymethyl.
  • R6 is
  • Ring A is phenyl
  • R3 is selected from hydrogen, fluorine, -OH, methoxy, -NH2 and hydroxymethyl.
  • ring A is C6-10 aryl or 5-10 heteroaryl.
  • ring A is selected from phenyl, pyrimidinyl, pyridinyl, furanyl, and thiophenyl.
  • R3 is selected from -OH, methoxy, -NH2 and hydroxymethyl.
  • R6 is selected from phenyl, pyridyl,
  • R6 is selected from phenyl, pyridyl,
  • R6 ' and R7 are bonded together, and R7 ' is hydrogen.
  • R6 is
  • R6 ' , R7 and R7 ' are all hydrogen.
  • t is 3-8, such as 3, 4, 5, 6, 7 or 8.
  • X is -CH2- or -CD2- ; optionally, Y is selected from -CH2- , -CD2- , And -C(O)-.
  • X is -CH2- and Y is -CH2- ;
  • X is -CH2- and Y is -CD2- or -C(O)-; or
  • X is -CD2- and Y is -CH2- .
  • X is -CH 2 - and Y is -CH 2 -.
  • R1 is selected from methoxy, trideuteroxy, and -O-cyclopropyl
  • R2 is a methoxy group
  • R4 is hydrogen or fluorine
  • R4 ' is selected from hydrogen, fluorine, -OH, -NH2 , trifluoromethyl and methoxy
  • R5 and R5 ' are both hydrogen
  • R4 and R4 ' form a carbon-carbon double bond or a 3-membered carbon ring with the carbon atom connecting R4 and R4 '
  • R5 and R5 ' are both hydrogen
  • R4 and R5 are bonded together or together form a 3-membered carbon ring with the carbon atom connecting R4 and R5
  • R5 ' and R4 ' are both hydrogen.
  • Ring A is selected from cyclopropyl, phenyl, furanyl, thiophene, pyrimidinyl, and pyridinyl;
  • R3 is selected from hydrogen, fluorine, -OH, methoxy, -NH2 and hydroxymethyl;
  • R1 and R2 are both methoxy groups
  • R4 is hydrogen or fluorine
  • R4 ' is selected from hydrogen, fluorine, -OH, -NH2 , trifluoromethyl and methoxy
  • R5 and R5 ' are both hydrogen
  • R4 and R4 ' form a carbon-carbon double bond or a 3-membered carbon ring with the carbon atom connecting R4 and R4 '
  • R5 and R5 ' are both hydrogen
  • R4 and R5 are bonded together or together form a 3-membered carbon ring with the carbon atom connecting R4 and R5
  • R5 ' and R4 ' are both hydrogen.
  • the cytotoxic agent is selected from the following compounds or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs:
  • the cytotoxic agent is selected from the following compounds or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs:
  • the cytotoxic agent is selected from the following compounds or their pharmaceutically acceptable salts, esters, stereoisomers, polymorphs, solvates, nitrogen oxides, isotope-labeled substances, metabolites, or prodrugs:
  • the cytotoxic drug fragment (D) has the structure shown in formula (I'):
  • rings A, R1 , R2 , R4 , R4' , R5 , R5' , R6' , R7 , R7 ' , X, Y, and t are as described in any of the preceding items;
  • R 3A is selected from chemical bonds, -NH-, -N(C 1-6 alkyl)-, -O-, -C 1-6 alkylene-O-, and -C 1-6 alkylene-O-.
  • the cytotoxic drug fragment (D) has the structure shown below:
  • the antibody-drug conjugates described in this application can be prepared in a modular manner.
  • a compound in its free form as a "drug-linker” (which can be understood as M'-LED, where M' is the structural form of M before covalently linking it to an antibody or its antigen-binding fragment) can be obtained, and then covalently linked to an antibody or its antigen-binding fragment to obtain the antibody-drug conjugates described in this application.
  • M' is linked to one or more thiol groups (-SH) on the antibody or its antigen-binding fragment through a substitution reaction (e.g., removal of the -SO2Me structure).
  • M' is -M-Lg
  • Lg is selected from halogens (e.g., F, Cl, Br, I), halogenated C1-6 alkyl, C1-6 alkylsulfonyl, halogenated C1-6 alkylsulfonyl, halogenated C1-6 alkylsulfonate, halogenated C1-6 alkylsulfinate, C1-6 alkyl sulfoxide, halogenated phenoxy, hydroxy (-OH), mercapto (-SH), amino ( -NH2 ), nitro, azide, cyano, alkenyl, alkynyl, and alkynyl-containing structural fragments.
  • halogens e.g., F, Cl, Br, I
  • the 1-6 alkyl sulfoxide, halophenoxy, alkenyl, ynyl and ynyl-containing structural segments
  • Lg is selected from substituted or unsubstituted C1-6 alkylsulfonyl groups ( C1-6 alkyl- SO2- ), preferably, Lg is...
  • the free form of the "drug-connector" has the following structure:
  • n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.
  • the antibody or its antigen-binding fragment comprises:
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;
  • the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • CDR-L1 with sequence SEQ ID NO:8 or a variant thereof
  • CDR-L2 with sequence SEQ ID NO:9 or a variant thereof
  • CDR-L3 with sequence SEQ ID NO:10 or a variant thereof.
  • VH heavy chain variable region
  • CDR-H2 with the sequence of SEQ ID NO:34 or a variant thereof
  • CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof
  • VL light chain variable region
  • the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,
  • VH heavy chain variable region
  • VL light chain variable region
  • the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH Heavy chain variable region
  • VL Light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.
  • the antibody or its antigen-binding fragment comprises:
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;
  • the variant described in either (1a) or (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, or 3 amino acids) compared to the sequence from which it originates. (replacement, deletion, or addition); preferably, the replacement is a conservative replacement;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 with the sequence of SEQ ID NO:34 or a variant thereof
  • CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof
  • VL light chain variable region
  • the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,
  • VH heavy chain variable region
  • VL light chain variable region
  • the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:13 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:14 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:15 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:16 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:17 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,
  • VH heavy chain variable region
  • VL light chain variable region
  • the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.
  • the antibody or its antigen-binding fragment comprises:
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 of SEQ ID NO:5, CDR-H2 of SEQ ID NO:6, and CDR-H3 of SEQ ID NO:7; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 of SEQ ID NO:20, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:22; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 of SEQ ID NO:13, CDR-H2 of SEQ ID NO:14, and CDR-H3 of SEQ ID NO:15; and/or, comprising the following The light chain variable regions (VL) of the three CDRs: CDR-L1 with sequence SEQ ID NO:16, CDR-L2 with sequence SEQ ID NO:17, and CDR-L3 with sequence SEQ ID NO:10; or,
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or its antigen-binding fragment comprises:
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 of SEQ ID NO:20, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or its antigen-binding fragment comprises:
  • the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.
  • the antibody or its antigen-binding fragment comprises:
  • the antibody or its antigen-binding fragment comprises:
  • the antibody or its antigen-binding fragment comprises:
  • the antibody or its antigen-binding fragment further comprises:
  • the light chain constant region (CL) of human immunoglobulin or a variant thereof which has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids).
  • the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as the human IgG1 heavy chain constant region or the human IgG4 heavy chain constant region.
  • the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 35.
  • CH heavy chain constant region
  • the antibody or its antigen-binding fragment includes a heavy chain constant region (CH) as shown in SEQ ID NO: 35.
  • the antibody or its antigen-binding fragment comprises a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, or 5 amino acid substitutions) compared to SEQ ID NO: 36.
  • CL light chain constant region
  • the antibody or its antigen-binding fragment includes a light chain constant region (CL) as shown in SEQ ID NO: 36.
  • the antibody or its antigen-binding fragment comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 and a light chain constant region (CL) as shown in SEQ ID NO: 36.
  • CH heavy chain constant region
  • CL light chain constant region
  • the antibody or its antigen-binding fragment comprises:
  • a heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or
  • a heavy chain comprising the VH of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.
  • the antibody or its antigen-binding fragment is selected from Trastuzumab or Pertuzumab, the amino acid sequence of which has an IMGT accession number (IMGT/mAb-DB ID) of 97 and the amino acid sequence of which has an IMGT accession number (IMGT/mAb-DB ID) of 80.
  • the heavy chain constant domain may contain a C-terminal lysine residue or lack a C-terminal lysine residue or a C-terminal glycine-lysine dipeptide.
  • the N-terminal amino acid of the antibody or antigen-binding fragment thereof may be cyclized to pyroglutamic acid.
  • pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.
  • compositions comprising antibody or antigen-binding fragments disclosed herein are provided, wherein the various antibody or antigen-binding fragments may independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine and/or comprise an N-terminal glutamine or glutamic acid, an N-terminal amino acid cyclized to pyroglutamic acid or an N-terminal amino acid cyclized to pyroglutamate salt.
  • the antibody or antigen-binding fragments disclosed herein include antibodies or antigen-binding fragments that specifically bind to antigens and may include post-translational modifications thereof (e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate conversion to pyroglutamic acid or pyroglutamate salt in the heavy or light chain), which may occur during recombinant expression in host cells (e.g., CHO cells) or during purification/storage.
  • post-translational modifications thereof e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate conversion to pyroglutamic acid or pyroglutamate salt in the heavy or light chain
  • the N-terminal glutamine of the VH or variant thereof as shown in SEQ ID NO:1 or 3, or the heavy chain or variant thereof as shown in SEQ ID NO:37 or 39 undergoes cyclization to form pyroglutamic acid or pyroglutamic acid salt.
  • the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 35 or a variant thereof, or the heavy chain of the sequence shown in SEQ ID NO: 37 or 39 or a variant thereof lacks a C-terminal lysine residue.
  • the antibody-drug conjugate is selected from:
  • HA represents an antibody or its antigen-binding fragment that specifically binds to epidermal growth factor receptor 2 (Her2), a member of the ErbB family receptor tyrosine kinase.
  • n is an integer from 1 to 20; preferably an integer from 3 to 15, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; preferably, n is 5, 8, 9 or 10.
  • the HA in each antibody-drug conjugate includes:
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:5 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:6 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;
  • the variant described in either (1a) or (1b) has at least 70% and at least 80% of the content of the sequence from which it originates. At least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, or the variant having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to its source sequence; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 with the sequence of SEQ ID NO:34 or a variant thereof
  • CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof
  • VL light chain variable region
  • the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,
  • VH heavy chain variable region
  • CDR-H2 with sequence SEQ ID NO:27 or a variant thereof
  • CDR-H3 with sequence SEQ ID NO:22 or a variant thereof
  • VL light chain variable region
  • Its variant CDR-L1 has the sequence SEQ ID NO:24 or its variant
  • CDR-L2 has the sequence SEQ ID NO:25 or its variant CDR-L3;
  • the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:13 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:14 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:15 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:16 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:17 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,
  • VH heavy chain variable region
  • VL light chain variable region
  • the variant described in any one of (4a) and (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.
  • the HA in each antibody-drug conjugate includes:
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO:5 or a variant thereof, CDR-H2 with sequence SEQ ID NO:6 or a variant thereof, and CDR-H3 with sequence SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO:8 or a variant thereof, CDR-L2 with sequence SEQ ID NO:9 or a variant thereof, and CDR-L3 with sequence SEQ ID NO:10 or a variant thereof. Its variant of CDR-L3; or,
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:20 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:21 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:23 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:24 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:25 or a variant thereof;
  • the variant described in any one of (1a) and (1b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • CDR-H2 with the sequence of SEQ ID NO:34 or a variant thereof
  • CDR-H3 with the sequence of SEQ ID NO:22 or a variant thereof
  • VL light chain variable region
  • the variant described in any of (2a) and (2b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:11 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:12 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:7 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:8 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:9 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,
  • VH heavy chain variable region
  • VL light chain variable region
  • the variant described in any one of (3a) and (3b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with respect to the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO:13 or a variant thereof, CDR-H2 with the sequence of SEQ ID NO:14 or a variant thereof, and CDR-H3 with the sequence of SEQ ID NO:15 or a variant thereof; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with the sequence of SEQ ID NO:16 or a variant thereof, CDR-L2 with the sequence of SEQ ID NO:17 or a variant thereof, and CDR-L3 with the sequence of SEQ ID NO:10 or a variant thereof; or,
  • VH heavy chain variable region
  • VL light chain variable region
  • the variant described in either (4a) or (4b) has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, or the variant having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to the sequence from which it originated; preferably, the substitutions are conservative substitutions.
  • the HA in each antibody-drug conjugate includes:
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 of SEQ ID NO:5, CDR-H2 of SEQ ID NO:6, and CDR-H3 of SEQ ID NO:7; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:9, and CDR-L3 of SEQ ID NO:10; or,
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 of SEQ ID NO:20, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:22; and/or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO:11, CDR-H2 of SEQ ID NO:12, and CDR-H3 of SEQ ID NO:7; and/or, a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:8, CDR-L2 of SEQ ID NO:7, and CDR-H3 of SEQ ID NO:8; CDR-L2 of SEQ ID NO: 9, has the sequence CDR-L3 of SEQ ID NO: 10; or,
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • the HA in each antibody-drug conjugate includes:
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • a heavy chain variable region comprising the following three CDRs: CDR-H1 of SEQ ID NO:20, CDR-H2 of SEQ ID NO:21, and CDR-H3 of SEQ ID NO:22; and a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO:23, CDR-L2 of SEQ ID NO:24, and CDR-L3 of SEQ ID NO:25;
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable regions
  • VL light chain variable regions
  • VH heavy chain variable region
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • the HA in each antibody-drug conjugate includes:
  • the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence from which it originates, or the variant has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids) compared to the sequence from which it originates; preferably, the substitutions are conservative substitutions.
  • the HA in each antibody-drug conjugate includes:
  • the HA in each antibody-drug conjugate includes:
  • the HA in each antibody-drug conjugate includes:
  • the HA in each antibody-drug conjugate further comprises:
  • the light chain constant region (CL) of human immunoglobulin or a variant thereof which has one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids).
  • the heavy chain constant region is an IgG heavy chain constant region, such as the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region, such as the human IgG1 heavy chain constant region or the human IgG4 heavy chain constant region.
  • the HA in each antibody-drug conjugate comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 35 or a variant thereof, said variant having a conserved position of up to 20 amino acids compared to SEQ ID NO: 35.
  • Replacement e.g., conservative replacement of up to 15, 10, or 5 amino acids; e.g., conservative replacement of 1, 2, 3, 4, or 5 amino acids).
  • the HA in each antibody-drug conjugate includes a heavy chain constant region (CH) as shown in SEQ ID NO: 35.
  • the HA in each antibody-drug conjugate contains a light chain constant region (CL) as shown in SEQ ID NO: 36 or a variant thereof, the variant having up to 20 conserved substitutions of amino acids compared to SEQ ID NO: 36 (e.g., up to 15, up to 10, or up to 5 conserved substitutions of amino acids; e.g., 1, 2, 3, 4, or 5 conserved substitutions of amino acids).
  • CL light chain constant region
  • the HA in each antibody-drug conjugate includes a light chain constant region (CL) as shown in SEQ ID NO: 36.
  • the HA in each antibody-drug conjugate includes a heavy chain constant region (CH) as shown in SEQ ID NO: 35 and a light chain constant region (CL) as shown in SEQ ID NO: 36.
  • CH heavy chain constant region
  • CL light chain constant region
  • the HA in each antibody-drug conjugate includes:
  • a heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or
  • a heavy chain comprising the VH of the sequence shown in SEQ ID NO: 3 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL of the sequence shown in SEQ ID NO: 4 and the light chain constant region (CL) shown in SEQ ID NO: 36.
  • the HA in each antibody-drug conjugate is selected from Trastuzumab or Pertuzumab, wherein the amino acid sequence of Trastuzumab has a lookup accession number (IMGT/mAb-DB ID) of 97 in the IMGT database, and the amino acid sequence of Pertuzumab has a lookup accession number (IMGT/mAb-DB ID) of 80 in the IMGT database.
  • the HA in each antibody-drug conjugate may independently contain C-terminal lysine, lack C-terminal lysine, lack C-terminal glycine-lysine and/or contain N-terminal glutamine or glutamate, cyclize the N-terminal amino acid to pyroglutamic acid or cyclize the N-terminal amino acid to pyroglutamate salt.
  • the HA in each antibody-drug conjugate may include its post-translational modifications (e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate in the heavy or light chain being converted to pyroglutamate or pyroglutamate salt), which may occur during recombinant expression in host cells (e.g., CHO cells) or during purification/storage.
  • post-translational modifications e.g., C-terminal lysine cleavage in the heavy chain, N-terminal glutamine or glutamate in the heavy or light chain being converted to pyroglutamate or pyroglutamate salt
  • the N-terminal glutamine of the VH or variant thereof, as shown in SEQ ID NO:1 or 3, or the heavy chain or variant thereof, as shown in SEQ ID NO:37 or 39 undergoes cyclization to form pyroglutamic acid or Pyroglutamic acid salt.
  • the heavy chain constant region (CH) of the sequence shown in SEQ ID NO: 35 or a variant thereof, or the heavy chain of the sequence shown in SEQ ID NO: 37 or 39 or a variant thereof lacks a C-terminal lysine residue.
  • HA in each antibody-drug conjugate represents trastuzumab, pertuzumab, or their antigen-binding fragments.
  • the HA in each antibody-drug conjugate represents the following antibody or antigen-binding fragment:
  • a heavy chain comprising the VH region of the sequence shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 35, and a light chain comprising the VL region of the sequence shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 36; or
  • x is 1-10, for example 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10.
  • x is 5-8, such as 5-6, 5-7, 5-8, 6-7, 6-8, or 7-8.
  • x is 1-6, such as 2-6, 3-6, 4-6.
  • x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the antibody-drug conjugates of the present invention may optionally be substituted with one or more suitable substituents.
  • the present invention relates to the following synthetic intermediates:
  • the present invention relates to the following synthetic intermediates:
  • compositions of antibody-drug conjugates as described herein.
  • Such compositions may comprise a plurality of ADCs as described herein, wherein each ADC comprises a drug-linker as described herein, wherein x independently is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • each antibody molecule in the composition may be conjugated to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drug-linkers. Therefore, the composition is characterized by being “drug- The antibody-to-antibody ratio (DAR) ranges from about 1 to about 10. Methods for determining DAR are well known to those skilled in the art, including methods using reversed-phase chromatography or HPLC-MS.
  • the ADC composition described herein has a DAR of about 1 to about 10 or any subrange therebetween, such as: about 1 to 2, about 1 to 3, about 1 to 4, about 1 to 5, about 1 to 6, about 1 to 7, about 1 to 8, about 1 to 9, about 1 to 10, about 2 to 3, about 2 to 4, about 2 to 5, about 2 to 6, about 2 to 7, about 2 to 8, about 2 to 9, about 2 to 10, about 3 to 4, about 3 to 5, about 3 to 6, about 3 to 7, about 3 to 8, about 3 to 9, about 3 to 10, about 4 to 5, about 4 to 6, about 4 to 7, about 4 to 8, about 4 to 9, about 4 to 10, about 5 to 6, about 5 to 7, about 5 to 8, about 5 to 9, about 5 to 10, about 6 to 7, about 6 to 8, about 6 to 9, about 6 to 10, about 7 to 8, about 7 to 9, about 7 to 10, about 8 to 9, about 8 to 10 or about 9 to 10.
  • the DAR of the ADC compositions described herein is about 3 to 9, such as about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 3.0 to 6.0, about 3.5 to 4.0, about 3.5 to 4.5, about 3.5 to 5.0, about 3.5 to 5.5, about 3.5 to 6.0, about 3.5 to 6.5, about 4.0 to 4.5, about 4.0 to 5.0, about 4.0 to 5.5, about 4.0 to 6.0, about 4.0 to 6.5, about 4.0 to 7.0, about 4.0 to 8.
  • the DAR of the ADC composition described herein is about 3 to 8, for example, about 3.0 to 3.5, about 3.0 to 4.0, about 3.0 to 4.5, about 3.0 to 5.0, about 6.0 to 6.5, about 6.0 to 7.0, about 6.0 to 7.5, about 6.0 to 8.0, about 6.0 to 8.5, about 6.5 to 7.0, about 6.5 to 7.5, about 6.5 to 8.0, about 6.5 to 8.5, about 7.0 to 7.5, about 7.0 to 8.0, or about 7.5 to 8.0.
  • the DAR of the ADC composition described herein is about 3.5 to 5.0, for example about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0.
  • the DAR of the ADC compositions described herein is from about 3.0 to 6.0, for example, about 3.0, about 3.01, about 3.02, about 3.03, about 3.04, about 3.05, about 3.06, about 3.07, about 3.08, about 3.09, about 3.1, about 3.11, about 3.12, about 3.13, about 3.14, about 3.15, about 3.16, about 3.17, about 3.18, about 3.19, about 3.2, about 3.21, about 3.22, about 3.23, about 3.24, about 3.25, etc.
  • the DAR of the ADC composition described herein is 7.5 to 8.5, for example, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5.
  • the DAR of the ADC compositions described herein is about 6.0 to 9.0, preferably about 6.0-8.0, for example about 6.0, about 6.01, about 6.02, about 6.03, about 6.04, about 6.05, about 6.06, about 6.07, about 6.08, about 6.09, about 6.1, about 6.11, about 6.12, about 6.13, about 6.14, about 6.15, about 6.16, about 6.17, about 6.18, about 6.19, about 6.2, about 6.21, about 6.22, and about 6.23.
  • this application provides a pharmaceutical composition comprising any of the antibody-drug conjugates or compositions described in any of the preceding claims, and one or more pharmaceutical excipients.
  • the antibody-drug conjugates or compositions described herein are typically formulated in a single injectable form with a pharmaceutically acceptable parenteral medium for parenteral use, such as bolus injection, intravenous injection, intratumoral injection, etc.
  • a pharmaceutically acceptable parenteral medium for parenteral use, such as bolus injection, intravenous injection, intratumoral injection, etc.
  • antibody-drug conjugates of desired purity are mixed with pharmaceutically acceptable diluents, carriers, excipients, or stabilizers in the form of lyophilized or solution formulations (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A.Ed.).
  • the antibody-drug conjugates described herein or pharmaceutical compositions containing said antibody-drug conjugates can be administered via any route appropriate for the individual to be treated.
  • the antibody-drug conjugates, compositions, or pharmaceutical compositions thereof described herein can be used to treat a variety of diseases or conditions, such as Her2-expressing cancers, including solid tumors or hematologic malignancies, such as urothelial carcinoma, gastric cancer, and breast cancer.
  • Her2-expressing cancers including solid tumors or hematologic malignancies, such as urothelial carcinoma, gastric cancer, and breast cancer.
  • Lung cancer e.g., non-small cell lung cancer, specifically lung adenocarcinoma
  • lymphoma e.g., lymphoma.
  • this application provides the use of any of the antibody-drug conjugates, compositions, or pharmaceutical compositions containing the thereof described in the foregoing in the preparation of a medicament for treating Her2-expressing cancers.
  • this application provides the use of any of the antibody-drug conjugates, compositions, or pharmaceutical compositions containing the conjugates described above in a medicament for treating Her2-expressing cancer.
  • this application provides a method for treating Her2-expressing cancer, comprising the step of administering an effective amount of any of the preceding antibody-drug conjugates, compositions, or pharmaceutical compositions containing the conjugates to a subject in need of such treatment.
  • the antibody-drug conjugate, compound, drug-linker, or pharmaceutical composition containing it is sufficient (e.g., in a subject):
  • the cancer is selected from solid tumors or hematologic malignancies; for example, it is selected from gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma) and urothelial carcinoma.
  • gastric cancer breast cancer
  • lung cancer e.g., non-small cell lung cancer, specifically lung adenocarcinoma
  • urothelial carcinoma e.g., non-small cell lung cancer, specifically lung adenocarcinoma
  • antibody refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as ⁇ (kappa) and ⁇ (lambda) light chains. Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and the heavy chain also contains "D" regions of approximately 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of three domains (CH1, CH2, and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the constant region of the light chain consists of a single domain, CL. This constant domain does not directly participate in antibody-antigen binding but exhibits various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity-determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus.
  • the variable regions (VH and VL) of each heavy/light chain pair form antigen-binding sites.
  • the amino acid assignment in each region or domain can follow various numbering systems known in the art.
  • antibody also includes embodiments in which the heavy chain constant region contains a C-terminal lysine, or lacks a C-terminal lysine, or a C-terminal glycine-lysine dipeptide.
  • the term also includes embodiments in which the N-terminal amino acid of the antibody variable region has been cyclized into a pyroglutamate salt. Therefore, in compositions comprising the antibodies disclosed herein, various antibodies may independently contain a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and/or contain N-terminal glutamine or glutamate, or have an N-terminal amino acid cyclized into pyroglutamate.
  • CDR complementarity-determining region
  • CDR1 complementarity-determining region
  • CDR2 complementarity-determining region
  • CDR3 complementarity-determining region
  • the precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991) and the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; C).
  • the CDR contained in the antibody or its antigen-binding fragment can be determined according to various numbering systems known in the art, such as the Kabat, Chothia, IMGT, or AbM numbering systems. In some embodiments, the CDR contained in the antibody or its antigen-binding fragment is defined using the Chothia numbering system.
  • the entire amino acid sequence of VH is typically numbered according to Kabat, while the three CDRs within the variable region can be defined according to any of the aforementioned numbering systems.
  • the amino acid sites in VH can be sequentially numbered starting from amino acid site 1 until the end of the sequence, or they can be numbered according to Kabat. Unless otherwise stated, the amino acid sites in VH and VL described herein are defined according to sequential numbering.
  • the amino acid sites in the heavy chain constant region can be numbered sequentially from amino acid site 1 to the end of the sequence, or they can be numbered according to Eu.
  • the amino acid sequence of the IgG1 heavy chain constant region has 330 amino acids, numbered sequentially from 1 to 330.
  • the corresponding sequence numbered according to Eu starts from site 118 and ends at site 447. Unless otherwise stated, the amino acid sites of the heavy and light chains described herein are defined according to sequential numbering.
  • framework region or "FR” residues refers to those residues in the antibody variable region other than the CDR residues defined above. Amino acid residues.
  • antigen-binding fragment in antibody refers to a fragment of the antibody polypeptide, such as a fragment of the full-length antibody polypeptide, which retains the ability to specifically bind to the same antigen bound by the full-length antibody, and/or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the "antigen-binding moiety".
  • Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies.
  • Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab)' 2 fragments, F(ab)' 3 fragments, Fd, Fv, scFv, di-scFv, (scFv) 2 , disulfide-stabilized Fv proteins (“dsFv”), single-domain antibodies (sdAb, nanobodies), and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide.
  • Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.
  • Fd refers to an antibody fragment composed of VH and CH1 domains
  • dAb fragment refers to an antibody fragment composed of VH domain (Ward et al., Nature 341:544 546 (1989))
  • Fab fragment refers to an antibody fragment composed of VL, VH, CL and CH1 domains
  • F(ab') 2 fragment refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region
  • Fab'fragment refers to the fragment obtained by reducing the disulfide bonds of the two heavy chain fragments in the F(ab') 2 fragment, which consists of a complete light chain and heavy chain Fd fragment (composed of VH and CH1 domains).
  • Fv refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.
  • Fc refers to an antibody fragment formed by disulfide bonds connecting the second and third constant regions of the first heavy chain to the second and third constant regions of the second heavy chain.
  • the Fc fragment of an antibody has various functions but does not participate in antigen binding.
  • scFv refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have a general structure: NH2 - VL-linker-VH-COOH or NH2 - VH-linker-VL-COOH.
  • Suitable prior art linkers consist of a repeating GGGGS (SEQ ID NO:46) amino acid sequence or a variant thereof.
  • a linker having the amino acid sequence (GGGGS) 4 (SEQ ID NO:47) can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). This can be used in the present invention.
  • Other joints are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al.
  • disulfide bonds may also exist between the VH and VL domains of the scFv.
  • the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, scFvs containing NH2 -VH-VH-COOH and NH2 - VL-VL-COOH.
  • single-domain antibody has the meaning commonly understood by those skilled in the art, referring to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that maintains the ability to specifically bind to the same antigen bound by a full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also known as nanobodies.
  • Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and/or competes with the full-length antibody for specific binding to the antigen.
  • antibody includes not only the complete antibody but also the antigen-binding fragment of the antibody.
  • Antigen-binding fragments e.g., the antibody fragments described above
  • a given antibody e.g., the antibody provided in this invention
  • Antigen-binding fragments of a given antibody can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.
  • identity is used to refer to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are considered identical at that position when a position is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine).
  • the "percentage identity” between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared ⁇ 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match).
  • two sequences are compared to produce the maximum identity.
  • Such comparisons can be made using methods conveniently performed, for example, by computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453.
  • the percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4.
  • the Needleman and Wunsch J MoI Biol.
  • conservative substitution refers to an amino acid substitution that does not adversely affect or alter the intended properties of a protein/peptide containing an amino acid sequence.
  • conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, and histidine
  • acidic side chains e.g., aspartic acid and glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine
  • ⁇ -branched side chains e.g., threonine, valine, and isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, and histidine
  • amino acids are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.
  • amino acids are generally represented by single-letter and three-letter abbreviations known in the art.
  • alanine can be represented as A or Ala.
  • linker refers to a structural segment that links a cytotoxic drug to an antibody or antigen-binding fragment. For example, it refers to the -MLE- structural segment in the formula Ab-[MLED] x .
  • drug-linker refers to the structure of the cytotoxic drug and linker described in this invention before they are covalently linked to an antibody or its antigen-binding fragment.
  • drug-linker refers to M'-L-E-D, where M' is the structural form of M before it is covalently linked to an antibody or its antigen-binding fragment.
  • drug-linker is covalently linked to an antibody or its antigen-binding fragment to obtain the antibody-drug conjugate described in this application.
  • drug-linker also includes all pharmaceutically acceptable isotopically labeled compounds that are identical to the “drug-linker” compounds of the present invention, except that one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature.
  • isotopes suitable for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2H , 3H , deuterium D, tritium T); isotopes of carbon (e.g., 11C , 13C , and 14C ); isotopes of chlorine (e.g., 37Cl ); isotopes of fluorine (e.g., 18F ); isotopes of iodine (e.g., 123I and 125I ); isotopes of nitrogen (e.g., 13N and 15N ); isotopes of oxygen (e.g., 15O , 17O , and 18O ); and isotopes of sulfur (e.g., 35S ).
  • isotopes of hydrogen e.g., 2H , 3H , deuterium D, tritium T
  • isotopes of carbon e.g., 11C
  • isotopically labeled compound means that the compound has the same structure as the compound of the present invention, except that one or more atoms are replaced by atoms having the same atomic number but different atomic mass or mass number from the dominant atomic mass or mass number in nature.
  • isotopes suitable for inclusion in the present invention include, but are not limited to, isotopes of hydrogen (e.g., 2H , 3H , deuterium D, tritium T); isotopes of carbon (e.g., 11C , 13C , and 14C ); isotopes of chlorine (e.g., 37Cl ); isotopes of fluorine (e.g., 18F ); isotopes of iodine (e.g., 123I and 125I ); isotopes of nitrogen (e.g., 13N and 15N ); isotopes of oxygen (e.g., 15O , 17O , and 18O ); and isotopes of sulfur (e.g., 35S ).
  • isotopes of hydrogen e.g., 2H , 3H , deuterium D, tritium T
  • isotopes of carbon e.g., 11C
  • substitution refers to the replacement of one or more (e.g., 1, 2, 3, 4, or 5) hydrogen atoms on a specified compound or structural segment by a substituent, provided that the substitution does not exceed the normal valence of the specified atom in the present case and the substitution forms a stable compound. Combinations of substituents and/or variables are permitted only if such combinations form a stable compound.
  • a functional group or structural segment is described as “substituted or unsubstituted”, then the functional group or structural segment may be (1) unsubstituted or (2) substituted.
  • FIG. 1 Efficacy of antibody-drug conjugate (ADC) in a mouse model of NCI-N87 subcutaneous tumor-bearing cells.
  • NMR nuclear magnetic resonance
  • MS Mass spectrometry
  • Step 1 Preparation of (S)-8-(benzyloxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-5-2)
  • reaction solution was concentrated, water (50 mL) was added, and the mixture was extracted twice with ethyl acetate (50 mL x 2). The combined organic phases were washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product of the title compound (1.82 g, 4.99 mmol), which was used directly in the next step without purification.
  • Step 2 Preparation of (S)-8-hydroxy-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-5-3)
  • Step 3 Preparation of (S)-(4-(8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxy-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (7-5-5)
  • Tetra(triphenylphosphine)palladium (114 mg, 0.099 mmol) was added, and the mixture was heated at 80 °C for 3 hours under nitrogen purging twice and protection.
  • the reaction solution was then added to ethyl acetate (20 mL) and water (10 mL), stirred, filtered, and the filtrate was allowed to stand and separated.
  • Step 4 Preparation of tert-butyl carbamate (7-5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-5,11-dioxo-5,10,11,11a-tetrahydro-1-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate (7-5-6)
  • Step 5 Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-5-7)
  • Step Six Preparation of (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-5)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Preparation of (S)-2-(4-(benzyloxy)phenyl)-8-((5-bromopentyl)oxy)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-3-1)
  • the reaction was carried out at 80 °C for 3 hours. After the reaction was completed, the mixture was extracted with water and ethyl acetate. The organic phases were combined, dried, and concentrated to give a crude product. The crude product was purified by silica gel column chromatography to give the title compound (303 mg, 461.04 ⁇ mol).
  • Step 2 Preparation of (S)-2-(4-(benzyloxy)phenyl)-8-((5-bromopentyl)oxy)-7-methoxy-10-(2-(trimethylsilyl)ethoxy)methyl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-3-2)
  • Step 3 Preparation of (S)-2-(4-(benzyloxy)phenyl)-8-((5-bromopentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrole[1,2-a][1,4]diaza-5-one (7-3-3)
  • reaction was carried out at room temperature for 16 hours. After the reaction was complete, formic acid (0.5 mL) was added to quench the reaction. Stirring was continued for 10 minutes. The intermediate state conversion was confirmed. The reaction solution was prepared and purified to obtain the title compound (14 mg, 23.03 ⁇ mol).
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 4 Preparation of (S)-8-((5-(((S)-2-(4-(benzyloxy)phenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-3-4)
  • the reaction was carried out at room temperature for 4 hours. After the reaction was completed, the reaction was quenched with water, extracted with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried, and concentrated. The crude product was purified by silica gel column chromatography to obtain the title compound (10 mg, 12.32 ⁇ mol).
  • Step 5 Preparation of (S)-8-((5-(((S)-2-(4-hydroxyphenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrole[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrole[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-3)
  • Mobile phase A Acetonitrile
  • Mobile phase B Water (0.05% ammonium bicarbonate)
  • Step 1 Preparation of 8,8”-(pentane-1,5-diylbis(oxy))(11aS,11a”S)-bis(7-methoxy-5-oxo-11,11a-dihydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-10(5H)-carboxylic acid diallyl ester)(7-9-2)
  • Step 2 Preparation of (11aS,11a”S)-8,8”-(pentane-1,5-diylbis(oxy))bis(7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo][e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one)(7-9)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 3 Preparation of methyl 5-(methoxy-d3)-2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboron-2-yl)benzoate (7-10-4)
  • Methyl 5-(methoxy-d3)-2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboron-2-yl)benzoate (1.1 g, 3.23 mmol) was dissolved in tetrahydrofuran (30 mL), and glacial acetic acid (3.75 mL) was added at 0 °C. Hydrogen peroxide (7 mL) was slowly added dropwise, and the reaction was carried out at room temperature for 3 hours. Saturated sodium sulfite solution (30 mL) was added to the reaction solution, and the mixture was stirred for 1 hour. The mixture was concentrated under reduced pressure until no tetrahydrofuran residue was found.
  • Methyl 4-(benzyloxy)-5-(methoxy-d3)-2-nitrobenzoate (688 mg, 2.15 mmol) was dissolved in tetrahydrofuran (5 mL) and water (1 mL). Lithium hydroxide (103.1 mg, 4.3 mmol) was added, and the mixture was reacted at room temperature for 3 hours. The pH of the system was adjusted to 5 with dilute hydrochloric acid, and a solid precipitated out. The solid was extracted with 30 mL of ethyl acetate, and after separation, the layers were washed with saturated brine. The organic phase was dried over anhydrous sodium sulfate and filtered to obtain the title compound (400 mg, 1.31 mmol).
  • Step 7 Preparation of (S)-5-(4-(benzyloxy)-5-(methoxy-d3)-2-nitrobenzoyl)-5-azaspiro[2.4]heptane-6-carboxylic acid methyl ester (7-10-8)
  • Step 8 Preparation of (S)-8-(benzyloxy)-7-(methoxy-d3)-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5,11(10H)-dione (7-10-9)
  • Methyl (S)-5-(4-(benzyloxy)-5-(methoxy-d3)-2-nitrobenzoyl)-5-azaspiro[2.4]heptane-6-carboxylate (570 mg, 1.29 mmol) was dissolved in methanol (30 mL), and 10% ammonium chloride aqueous solution (4 mL) and zinc powder (1.28 g, 19.51 mmol) were added. The mixture was heated to 40 °C and reacted for 1 hour, then heated to 80 °C and reacted overnight. The crude product was concentrated and dissolved in 35 mL of ethyl acetate. The mixture was washed three times with saturated brine.
  • the organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product.
  • Step 11 Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((S)-7-(methoxy-d3)-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-10-12)
  • Step Twelve Preparation of (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-(methoxy-d3)-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-10)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 2 Preparation of methyl 5-cyclopropoxy-2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboron-2-yl)benzoate (7-11-2)
  • Methyl 5-cyclopropoxy-2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxoboron-2-yl)benzoate (1.6 g, 4.41 mmol) was dissolved in tetrahydrofuran (40 mL), and glacial acetic acid (5 mL) was added at 0 °C. Hydrogen peroxide (10 mL) was slowly added dropwise, and the reaction was carried out at room temperature for 3 hours. Saturated sodium sulfite solution (50 mL) was added to the reaction solution and stirred for 1 hour. The mixture was concentrated under reduced pressure until no tetrahydrofuran residue was found. 30 mL of ethyl acetate was added for extraction.
  • Step 7 Preparation of (S)-8-(benzyloxy)-7-cyclopropoxy-1,11a-dihydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5,11(10H)-dione (7-11-7)
  • Methyl (S)-5-(4-(benzyloxy)-5-cyclopropoxy-2-nitrobenzoyl)-5-azaspiro[2.4]heptane-6-carboxylate 910 mg, 1.95 mmol was dissolved in methanol (30 mL), and 10% ammonium chloride aqueous solution (6 mL) and zinc powder (1.28 g, 19.51 mmol) were added. The mixture was heated to 40 °C and reacted for 1 hour, then heated to 80 °C and reacted overnight. The crude product was concentrated and dissolved in 35 mL of ethyl acetate. The mixture was washed three times with saturated brine.
  • the organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product.
  • Step 8 Preparation of (S)-8-(benzyloxy)-7-cyclopropoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-11-8)
  • Step 11 Preparation of (S)-2-(4-aminophenyl)-8-((5-(((S)-7-cyclopropoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-11)
  • tert-butyl carbamate (4-((S)-8-((5-((((S)-7-cyclopropoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (0.6 mL) was added dropwise with stirring.
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Preparation of (S)-8-(benzyloxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one-11,11-d2(7-21-1)
  • Step 2 Preparation of (S)-8-hydroxy-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one-11,11-d2(7-21-2)
  • Step 4 Preparation of (S)-8-((5-(((S)-2-(4-aminophenyl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrole[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrole[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one-11,11-d2(7-21)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 3 Preparation of (S)-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-22-4)
  • Step 5 Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-22)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Preparation of (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-hydroxypyrrolidine-2-carboxylic acid methyl ester (7-23-2)
  • Step 2 Preparation of (2R,11aS)-8-(benzyloxy)-2-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-23-3)
  • Step 3 Preparation of (2R,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-23-4)
  • Step 4 Preparation of (2R,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrole[1,2-a][1,4]diaza-5-one (7-23-5)
  • Step 5 Preparation of (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-23-6)
  • Step Six Preparation of (4-((S)-8-((5-(((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5),10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (7-23-7)
  • Step 7 Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2R,11aS)-2-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-23)
  • tert-butyl carbamate (4-((S)-8-((5-((((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5),10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (3 mL) was added dropwise with stirring.
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Preparation of (2S,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-hydroxypyrrolidine-2-carboxylic acid methyl ester (7-24-2)
  • Step 2 Preparation of (2S,11aS)-8-(benzyloxy)-2-hydroxy-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-24-3)
  • the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product.
  • Step 3 Preparation of (2S,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-24-4)
  • Step 4 Preparation of (2S,11aS)-8-(benzyloxy)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrole[1,2-a][1,4]diaza-5--(7-24-5)
  • Step 5 Preparation of (2S,11aS)-2-((tert-butyldimethylsilyl)oxy)-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-24-6)
  • Step 7 Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-24)
  • tert-butyl carbamate (4-((S)-8-((5-((((2S,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate tert-butyl carbamate was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (2 mL) was added dropwise with stirring.
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 3 Preparation of (2R,11aS)-8-(benzyloxy)-2,7-dimethoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazazo-5,11(10H)-dione (7-25-4)
  • Step 4 Preparation of (2R,11aS)-8-(benzyloxy)-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-25-5)
  • Step 5 Preparation of (2R,11aS)-8-hydroxy-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-25-6)
  • Step Six Preparation of (4-((S)-8-((5-(((2R,11aS)-2,7-dimethoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H)-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (7-26-7)
  • Step 7 Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2S,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-25)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 3 Preparation of (2S,11aS)-8-(benzyloxy)-7-methoxy-2-(methoxy)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-26-4)
  • Step 4 Preparation of (2S,11aS)-8-(benzyloxy)-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-26-5)
  • Step 5 Preparation of (2S,11aS)-8-hydroxy-2,7-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-26-6)
  • Step Six Preparation of (4-((S)-8-((5-(((2S,11aS)-2,7-dimethoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (7-26-7)
  • Step 7 Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2,7-dimethoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-26)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Preparation of (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-((tert-butoxycarbonyl)amino)pyrrolidine-2-carboxylic acid methyl ester (7-27-2)
  • Step 2 Preparation of ((2R,11aS)-8-(benzyloxy)-7-methoxy-5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrole[1,2-a][1,4]diaza-2-yl)tert-butyl carbamate (7-27-3)
  • Step 3 Preparation of ((2R,11aS)-8-(benzyloxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)tert-butyl carbamate (7-27-4)
  • tert-butyl carbamate ((2R,11aS)-8-(benzyloxy)-7-methoxy-5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrole[1,2-a][1,4]diaza-2-yl) in tetrahydrofuran (100 mL), add sodium borohydride (843.15 mg, 11.55 mmol), and then add trifluoroacetic acid (877.8 mg, 7.7 mmol) dropwise at room temperature until no large amount of gas is released. Then, reflux the mixture at 75 °C for 5 hours.
  • reaction solution was concentrated, water (50 mL) was added, and the mixture was extracted twice with ethyl acetate (50 mL x 2). The combined organic phases were washed with saturated sodium chloride aqueous solution, dried with anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product of the title compound (1.4 g, 3.09 mmol), which was used directly in the next step without purification.
  • Step 4 Preparation of ((2R,11aS)-8-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)tert-butyl carbamate (7-27-5)
  • Step Six Preparation of (S)-8-((5-(((2R,11aS)-2-amino-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-2-(4-aminophenyl)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-27)
  • tert-butyl carbamate (4-((S)-8-((5-((((2R,11aS)-2-((tert-butoxycarbonyl)amino)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)carbamate was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (0.5 mL) was added dropwise with stirring.
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 2 Preparation of (S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4,4-difluoropyrrolidine-2-carboxylic acid methyl ester (7-29-3)
  • Step 3 Preparation of (S)-8-(benzyloxy)-2,2-difluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-29-4)
  • Step 4 Preparation of (S)-8-(benzyloxy)-2,2-difluoro-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-29-5)
  • Step 5 Preparation of (S)-2,2-difluoro-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazaphen-5-one (7-29-6)
  • Step Six Preparation of (4-((S)-8-((5-((((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro)-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yl)phenyl)tert-butyl carbamate (7-29-7)
  • Step 7 Preparation of (S)-2-(4-aminophenyl)-8-((5-(((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-29)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Preparation of (2S,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-fluoropyrrolidine-2-carboxylic acid methyl ester (7-30-2)
  • Step 2 Preparation of (2S,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazapheno-5,11(10H)-dione (7-30-3)
  • Step 3 Preparation of (2S,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-30-4)
  • Step 4 Preparation of (2S,11aS)-2-fluoro-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-30-5)
  • Step 5 (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H- Preparation of benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-30-6)
  • Step Six Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2S,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-30)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 2 Preparation of (2R,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-31-3)
  • Step 3 Preparation of (2R,11aS)-8-(benzyloxy)-2-fluoro-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-31-4)
  • Step 4 Preparation of (2R,11aS)-2-fluoro-8-hydroxy-7-methoxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-31-5)
  • Step 5 Preparation of (S)-2-(4-aminophenyl)-8-((5-(((2R,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-31-6)
  • Step Six (S)-2-(4-aminophenyl)-8-((5-(((2R,11aS)-2-fluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexadecyl) Preparation of hydrogen-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-31)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 2 Preparation of (2R,4S)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-(trifluoromethyl)pyrrolidine-2-carboxylic acid methyl ester (7-32-3)
  • Methyl (2S,4S)-4-(trifluoromethyl)pyrrolidine-2-carboxylate (820.79 mg, 3.51 mmol, CL) and 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (1.18 g, 3.88 mmol, FR) were dissolved in DMF (10 mL), and HATU (1.48 g, 3.88 mmol) and DIPEA (1.36 g, 10.59 mmol) were added. The mixture was reacted at room temperature for 1 hour. The reaction solution was added to ethyl acetate (100 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation.
  • Step 3 Preparation of (2S,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-32-4)
  • Step 4 Preparation of (2S,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-32-5)
  • Step 5 Preparation of (2S,11aS)-8-hydroxy-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-32-6)
  • Step Six Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2S,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-32-7)
  • Step 7 Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2S,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-32)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 2 Preparation of (2S,4R)-1-(4-(benzyloxy)-5-methoxy-2-nitrobenzoyl)-4-(trifluoromethyl)pyrrolidine-2-carboxylic acid methyl ester (7-33-3)
  • Methyl (2S,4R)-4-(trifluoromethyl)pyrrolidine-2-carboxylate (824.79 mg, 3.53 mmol) and 4-(benzyloxy)-5-methoxy-2-nitrobenzoic acid (1.18 g, 3.88 mmol) were dissolved in DMF (10 mL), and HATU (1.48 g, 3.88 mmol) and DIPEA (1.36 g, 10.59 mmol) were added. The mixture was reacted at room temperature for 1 hour. The reaction solution was added to ethyl acetate (100 mL) and saturated brine (100 mL), stirred, and allowed to stand before separation.
  • Step 3 Preparation of (2R,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-33-4)
  • Step 4 Preparation of (2R,11aS)-8-(benzyloxy)-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-33-5)
  • reaction solution was concentrated, water (50 mL) was added, and the mixture was extracted twice with ethyl acetate (50 mL x 2). The combined organic phases were washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude title compound (1.1 g, 2.71 mmol), which was used directly in the next step without purification.
  • Step 5 Preparation of (2R,11aS)-8-hydroxy-7-methoxy-2-(trifluoromethyl)-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-33-6)
  • Step Six Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2R,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-33-7)
  • Step 7 Preparation of (S)-2-(4-aminophenyl)-7-methoxy-8-((5-(((2R,11aS)-7-methoxy-5-oxo-2-(trifluoromethyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-33)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% trifluoroacetic acid)
  • Step 1 Preparation of (S)-8-((5-bromopentyl)oxy)-2-(furan-3-yl)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-36-1)
  • Furan-3-ylboronic acid (40.15 mg, 358.85 ⁇ mol), (S)-8-((5-bromopentyl)oxy)-7-methoxy-5,11-dioxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-2-yltrifluoromethanesulfonate (100 mg, 179.43 ⁇ mol), tetra(triphenylphosphine)palladium (20.73 mg, 17.94 ⁇ mol), and sodium carbonate (76.07 mg, 717.70 ⁇ mol) were mixed together, and water (0.5 mL), toluene (2 mL), and ethanol (0.5 mL) were added.
  • Step 2 Preparation of (S)-2-(furan-3-yl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-36-2)
  • Step 3 Preparation of (S)-8-((5-(((S)-2-(furan-3-yl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-36)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Preparation of (S)-8-((5-bromopentyl)oxy)-2-(furan-3-yl)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-37-1)
  • Step 2 Preparation of (S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrole)[1,2-a][1,4]diaza-2,1’-cyclopropyl]-8-yl)oxy)pentyl)oxy)-2-(thiophen-3-yl)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-37-2)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 3 Preparation of (S)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-2-(thiophen-3-yl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-1,10,11,11a-tetrahydro-3H,5H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-37)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Preparation of (S)-8-((5-bromopentyl)oxy)-2-(5-(hydroxymethyl)thiophen-3-yl)-7-methoxy-1,11a-dihydro-5H-benzo[e]pyrrole[1,2-a][1,4]diaza-5,11(10H)-dione (7-38-1)
  • Step 2 Preparation of (S)-2-(5-(hydroxymethyl)thiophene-3-yl)-7-methoxy-8-((5-(((S)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H,3H-spiro[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1'-cyclopropane]-8-yl)oxy)pentyl)oxy)-1,11a-dihydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-38-2)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 3 Preparation of (S)-8-((5-(((S)-2-(5-(hydroxymethyl)thiophene-3-yl)-7-methoxy-5-oxo-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-1,10,11,11a-tetrahydro-3H,5H-spirocyclic[benzo[e]pyrrolo[1,2-a][1,4]diaza-2,1’-cyclopropane]-5-one (7-38)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Preparation of allyl (S)-8-((5-(((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid ester (7-39-1)
  • Step 2 (S)-8-((5-(((S)-2,2-difluoro-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrole) Preparation of [1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-39)
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 1 Preparation of (S)-8-((5-(((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-5-oxo-11,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-10(5H)-carboxylic acid allyl ester (7-40-1)
  • Step 2 Preparation of (S)-8-((5-(((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-40-2)
  • Step 3 Preparation of (S)-8-((5-(((2R,11aS)-2-hydroxy-7-methoxy-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diaza-8-yl)oxy)pentyl)oxy)-7-methoxy-2-(4-methoxyphenyl)-1,10,11,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5-one (7-40)
  • the mixture was stirred for 1 hour.
  • the solution was diluted with ethyl acetate, washed twice with saturated ammonium chloride aqueous solution, and twice with saturated brine.
  • the solution was then concentrated under reduced pressure.
  • the title compound (17.11 mg, 24.24 ⁇ mol) was obtained by preparative high performance liquid chromatography purification followed by freeze drying.
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Step 3 Preparation of (S)-8-(benzyloxy)-7-methoxy-2-methylene-1,2,3,11a-tetrahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diaza-5,11(10H)-dione (7-41-4)

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Abstract

L'invention concerne un conjugué ayant un effet ciblé, son procédé de préparation et son utilisation. Le conjugué a une structure représentée par la formule Ab-[M-L-E-D]x, le médicament étant choisi parmi un intercalant d'ADN. Le conjugué anticorps-médicament préparé présente un rapport de conjugaison médicament-anticorps relativement bon et présente d'excellents effets de destruction ciblés sur des tumeurs solides telles que le cancer gastrique, le cancer du sein, le cancer du poumon et le carcinome urothélial.
PCT/CN2024/129461 2024-05-10 2024-11-01 Conjugué à effet ciblé, son procédé de préparation et son utilisation Pending WO2025232116A1 (fr)

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PCT/CN2024/129461 Pending WO2025232116A1 (fr) 2024-05-10 2024-11-01 Conjugué à effet ciblé, son procédé de préparation et son utilisation

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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