WO2022253033A1 - 一类化学偶联连接子及其用途 - Google Patents

一类化学偶联连接子及其用途 Download PDF

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WO2022253033A1
WO2022253033A1 PCT/CN2022/094540 CN2022094540W WO2022253033A1 WO 2022253033 A1 WO2022253033 A1 WO 2022253033A1 CN 2022094540 W CN2022094540 W CN 2022094540W WO 2022253033 A1 WO2022253033 A1 WO 2022253033A1
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compound
formula
methyl
antibody
group
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PCT/CN2022/094540
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English (en)
French (fr)
Inventor
田强
汪小蓓
李德亮
张毅涛
叶健
胡瑞斌
宋宏梅
葛均友
王晶翼
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四川科伦博泰生物医药股份有限公司
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Priority to EP22815080.1A priority Critical patent/EP4349832A1/en
Priority to KR1020237037967A priority patent/KR20240017343A/ko
Priority to CN202280032554.2A priority patent/CN117255790A/zh
Priority to JP2023567997A priority patent/JP2024520283A/ja
Publication of WO2022253033A1 publication Critical patent/WO2022253033A1/zh

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • 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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/44—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members
    • C07D207/444—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5
    • C07D207/448—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide
    • C07D207/452—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide with hydrocarbon radicals, substituted by hetero atoms, directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/79—Acids; Esters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/32—One oxygen, sulfur or nitrogen atom
    • C07D239/38—One sulfur atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/58—Two sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D251/00—Heterocyclic compounds containing 1,3,5-triazine rings
    • C07D251/02—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings
    • C07D251/12—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D251/14—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hydrogen or carbon atoms directly attached to at least one ring carbon atom
    • C07D251/16—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hydrogen or carbon atoms directly attached to at least one ring carbon atom to only one ring carbon atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D271/00—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms
    • C07D271/02—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms not condensed with other rings
    • C07D271/10—1,3,4-Oxadiazoles; Hydrogenated 1,3,4-oxadiazoles
    • C07D271/107—1,3,4-Oxadiazoles; Hydrogenated 1,3,4-oxadiazoles with two aryl or substituted aryl radicals attached in positions 2 and 5
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • 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
    • C07D403/02—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 two hetero rings
    • C07D403/12—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 two hetero rings linked by a chain containing hetero atoms as chain links
    • 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

Definitions

  • the invention relates to a new type of linker used for chemical conjugation, antibody-conjugated drugs and related compositions prepared from the linker, as well as its preparation method and application in the treatment of tumor-related diseases.
  • ADC Antibody-Drug Conjugates
  • ADC are composed of monoclonal antibody drugs targeting specific antigens and small-molecule cytotoxic drugs coupled through linkers, which have the powerful killing effect of traditional small-molecule chemotherapy and antibody drugs. tumor targeting.
  • 12 ADC drugs have been approved for marketing worldwide, of which 7 are used to treat hematological tumors and 5 are used to treat solid tumors.
  • Antibody-drug conjugates are composed of antibody, linker, payload, and conjugation methods.
  • the coupling mode is the connection mode of the antibody and the drug-linker.
  • Coupling methods are mainly divided into non-point-specific coupling and point-specific coupling.
  • the non-site-specific coupling method was used, which mainly consisted of lysine coupling and cysteine coupling.
  • Chemical methods were used to directly couple the drug to the amino acid residues on the antibody, which did not involve the transformation or modification of the antibody. The number of toxin molecules linked and the coupling site cannot be determined, and the uniformity is not good.
  • the commonly used fixed-point coupling method is to perform specific coupling through genetic engineering sites or special linkers to achieve more uniform coupling and to realize the connection of cytotoxins at specific sites.
  • Antibody-conjugated drugs produced by site-directed coupling can reduce fluctuations in drug efficacy, pharmacokinetics, and CMC quality control caused by differences in the number of coupling sites and couplings.
  • One purpose of this application is to provide a novel linker for chemical conjugation.
  • This type of linker has high reactivity, mild coupling conditions, easy operation, and can realize site-specific coupling. Good stability, clear efficacy in vivo and in vitro.
  • the application provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotopically labeled compound thereof , wherein the compound has the structure of formula I:
  • X is selected from leaving groups such as Cl, Br, I, OMs, OTs, OTf or
  • Y is absent or selected from carbonyl
  • Ring A is selected from substituted or unsubstituted C 6-10 aromatic rings, 5-12 membered aromatic heterocycles or 5-12 membered heterocycles;
  • Z 1 is absent or selected from -CH 2 - or C 2-6 alkynylene
  • W 1 does not exist or is selected from one or more of C 1-10 alkylene, -(CH 2 CH 2 O)p-, and -(OCH 2 CH 2 )p-;
  • J 1 is selected from -COOH, -NH 2 , 3-10 membered nitrogen-containing heterocyclic group, sulfonylurea group or hydroxyl group;
  • p is an integer of 1-10.
  • X is selected from one or more of Cl, Br, I, OMs, OTs and OTf.
  • Y is absent.
  • Z is absent or is C2-6 alkynylene.
  • W 1 does not exist or is selected from one or more of C 1-10 alkylene and -(CH 2 CH 2 O)p-.
  • W 1 is C 1-10 alkylene; preferably C 1-6 alkylene, more preferably C 1-3 alkylene.
  • J 1 is -COOH.
  • ring A is selected from substituted or unsubstituted 5-12 membered nitrogen-containing aromatic heterocycles or 5-12 membered nitrogen-containing heterocycles; preferably, ring A is selected from unsubstituted or oxo groups or- COOH-substituted 5-12 membered nitrogen-containing aromatic heterocycle or 5-12 membered nitrogen-containing heterocycle.
  • p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • X is selected from Cl, Br, I, OMs, OTs, OTf and
  • Ring A is selected from 5-12 membered nitrogen-containing aromatic heterocycles or 5-12 membered nitrogen-containing heterocycles that are unsubstituted or substituted by oxo or -COOH;
  • Z 1 is absent or is -CH 2 -;
  • W 1 does not exist or is selected from one or more of C 1-10 alkylene and -(CH 2 CH 2 O)p-;
  • J 1 is -COOH
  • p is an integer of 1-10.
  • formula I is selected from the following structures:
  • the compound of formula I has the following structure:
  • J 1 is -COOH or -NH 2 .
  • the compound of formula I has the following structure:
  • the application provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof , wherein the compound has the structure of formula II:
  • B 1 is each independently selected from a single bond or a 5-12 membered nitrogen-containing aromatic heterocycle at each occurrence;
  • Each occurrence of Y 1 , Y 2 and Y 3 is independently selected from CH and N;
  • W 2 does not exist or is selected from one or more of C 1-10 alkylene, -(CH 2 CH 2 O) p - or -(OCH 2 CH 2 ) p -;
  • J 2 is selected from -COOH, -NH 2 , 3-10 membered nitrogen-containing heterocyclic group, sulfonylurea group or hydroxyl group;
  • p is an integer of 1-10.
  • each occurrence of B 1 is independently selected from a single bond or a 5-6 membered nitrogen-containing aromatic heterocycle; preferably, each occurrence of B 1 is independently selected from a single bond Or a 5-6 membered nitrogen-containing aromatic heterocyclic ring; further preferably, each occurrence of B 1 is independently selected from a single bond or a pyrimidine ring.
  • Y 1 , Y 2 and Y 3 are all N; or Y 1 is CH, Y 2 and Y 3 are all N; or Y 1 is N, Y 2 and Y 3 are all CH; or Y 1 , Y 2 and Y 3 are all CH.
  • Z is absent or is C alkynylene .
  • W 2 is absent or is C 1-10 alkylene.
  • J 2 is selected from -COOH or -NH 2 .
  • each occurrence of B is independently selected from a single bond or a pyrimidine ring
  • Y 1 , Y 2 and Y 3 are all CH;
  • W 2 does not exist or is selected from one or more of C 1-10 alkylene, -(CH 2 CH 2 O) p ;
  • J 2 is -COOH
  • p is an integer of 1-10.
  • the compound of formula II has the following structure:
  • p is an integer of 1-10.
  • p is an integer of 1-8; more preferably, p is an integer of 1-5.
  • the compound of formula II has the following structure:
  • the compound of formula II has the following structure:
  • the present application provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof , wherein the compound has the structure of formula III:
  • V 1 is the group formed when J 1 is connected to L in the compound of formula I in the first aspect of the application; preferably, V 1 is selected from -CO-, -N(R 1 )-, -O-, 3-10 members Nitrogen-containing heterocyclic group or sulfonylurea group, wherein R 1 is H, C 1-6 alkyl or C 2-6 alkoxyalkyl; further preferably, V 1 is selected from -C(O)- and -N (R 1 )-, wherein R 1 is H, C 1-6 alkyl or C 2-6 alkoxyalkyl;
  • L is a linker between V1 and E';
  • E' is selected from H, -NHCH 2 -Lg, -COOH, Wherein, Lg represents a leaving group, such as Cl, Br, I, OMs, OTs, OTf or
  • X, Y, A, Q, Z 1 , W 1 are as defined in any one of the first aspect above.
  • L is selected from one or more of the following groups: C 1-6 alkylene, -N(R 6 )-, carbonyl, -O-, Val, Cit, Phe, Lys, D -Val, Leu, Gly, Phe, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys , Gly-Gly-Arg, Ala-Ala-Asn, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly, Wherein R 6 is H, C 1-6 alkyl or C 2-6 alkoxyalkyl, and s is an integer of 1-10.
  • L is selected from one or more of the following groups: Val, Cit, Gly, Phe, Ala, Val-Cit, Val-Ala, Gly-Gly-Phe-Gly,
  • L is selected from
  • L is selected from
  • the present application provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof , wherein the compound has the structure of formula IV:
  • V 2 is the group obtained after J 2 is connected to L in the compound of formula II in the second aspect of the application; preferably, V 2 is selected from -CO-, -N(R 2 )-, -O-, 3-10 members Nitrogen-containing heterocyclic group or sulfonylurea group, wherein R 2 is H, C 1-6 alkyl or C 2-6 alkoxyalkyl; further preferably, V 2 is selected from -C(O)- and -N (R 2 )-, wherein R 2 is H, C 1-6 alkyl or C 2-6 alkoxyalkyl;
  • B 1 , Y 1 , Y 2 , Y 3 , Z 2 and W 2 are as defined in any one of the preceding aspects of the second aspect;
  • L and E' are as defined in any one of the third aspects above.
  • the present application provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound , wherein the compound has the structure of formula V:
  • E is selected from a single bond, -NH-CH 2 -, or the following structures:
  • D is a fragment of a biologically active molecule (such as a cytotoxic drug);
  • X, Y, A, Q, Z 1 , W 1 are as defined in any one of the first aspect above;
  • V and L are as defined in any one of the third aspect.
  • the bioactive molecule is selected from the group consisting of anti-tubulin agents, DNA intercalators, DNA topoisomerase inhibitors and RNA polymerase inhibitors.
  • the biologically active molecule is selected from the group consisting of tubulin inhibitors auristatins and maytansinoids; DNA intercalator pyrrolobenzodiazepines (PBD); DNA topoisomerization Enzyme inhibitors, such as topoisomerase I inhibitors (such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubotecan ) or topoisomerase II inhibitors (such as doxorubicin, doxorubicin, PNU-159682, doxorubicin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); RNA polymerization Enzyme inhibitor ⁇ -amanitin, etc.; and pharmaceutically acceptable salts, esters and analogs thereof.
  • PPD DNA intercalator pyrrolobenzodiazepines
  • the biologically active molecule is selected from: topoisomerase I inhibitors (such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan , belotecan, rubitecan), MMAE and MMAE derivatives.
  • topoisomerase I inhibitors such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan , belotecan, rubitecan
  • MMAE MMAE derivatives.
  • the bioactive molecule is selected from: MMAE and MMAE derivatives.
  • the D is selected from:
  • the compound of formula V is selected from:
  • the present application provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer, polymorph, solvate, N-oxide or isotope-labeled compound thereof , wherein the compound has the structure of formula VI:
  • B 1 , Y 1 , Y 2 , Y 3 , Z 2 and W 2 are as defined in any one of the preceding aspects of the second aspect;
  • V 2 is as defined in any one of the fourth aspect above;
  • the compound of formula VI is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • the present application provides a biologically active conjugate, the structure of which is shown in formula VII:
  • Ab is a targeting moiety (such as small molecule ligands, proteins (such as antibodies), polypeptides, non-protein reagents (such as sugars, RNA or DNA)); n is selected from integers or decimals between 1-10;
  • V 1 is selected from -C(O)- or -N(R 1 )-, wherein R 1 is H, C 1-6 alkyl or C 2-6 alkoxyalkyl;
  • L is a linker between V1 and E;
  • E is a structural fragment connecting L and D;
  • D is a fragment of a biologically active molecule (such as a cytotoxic drug);
  • Another aspect of the present invention provides a biologically active conjugate, the structure of which is shown in formula VIII:
  • Ab is a targeting moiety (such as small molecule ligands, proteins (such as antibodies), polypeptides, non-protein reagents (such as sugars, RNA or DNA)); n is selected from integers or decimals between 1-10;
  • V 2 is selected from -C(O)- or -N(R 2 )-, wherein R 2 is H, C 1-6 alkyl or C 2-6 alkoxyalkyl;
  • L is a linker between V2 and E;
  • E is a structural fragment connecting L and D;
  • D is a fragment of a biologically active molecule (such as a cytotoxic drug);
  • the Ab is selected from Epidermal Growth Factor, Trop-2, CD37, HER2, CD70, EGFRvIII, Mesothelin, Folate receptor1, Mucin 1, CD138, CD20, CD19, CD30, SLTRK6, Nectin 4, Tissue factor, Mucin16 , Endothelin receptor, STEAP1, SLC39A6, Guanylylcyclase C, PSMA, CCD79b, CD22, Sodium phosphate cotransporter 2B, GPNMB, Trophoblast glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, TACSTD2, DR5, E16, STEAP1, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP,
  • the Ab is selected from anti-Her2 antibody trastuzumab or anti-Trop2 antibody sacituzumab or anti-ROR1 antibody 19F6_Hu35V1.
  • L is selected from one or more of the following groups: C 1-6 alkylene, -N(R 6 )-, carbonyl, -O-, Val, Cit, Phe, Lys, D -Val, Leu, Gly, Phe, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys , Gly-Gly-Arg, Ala-Ala-Asn, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly, Wherein R 6 is H, C 1-6 alkyl or C 2-6 alkoxyalkyl, s is an integer of 1-10;
  • L is selected from the following structures:
  • the E is a single bond, -NH-CH 2 -, or the following structure:
  • the E is -NH-CH 2 -.
  • the bioactive molecule is selected from the group consisting of anti-tubulin agents, DNA intercalators, DNA topoisomerase inhibitors and RNA polymerase inhibitors.
  • the biologically active molecule is selected from the group consisting of tubulin inhibitors auristatins and maytansinoids; DNA intercalator pyrrolobenzodiazepines (PBD); DNA topoisomerization Enzyme inhibitors, such as topoisomerase I inhibitors (such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubotecan ) or topoisomerase II inhibitors (such as doxorubicin, doxorubicin, PNU-159682, doxorubicin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); RNA polymerization Enzyme inhibitor ⁇ -amanitin, etc.; and pharmaceutically acceptable salts, esters and analogs thereof.
  • PPD DNA intercalator pyrrolobenzodiazepines
  • the biologically active molecule is selected from: topoisomerase I inhibitors (such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan , belotecan, rubitecan), MMAE and MMAE derivatives.
  • topoisomerase I inhibitors such as camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan , belotecan, rubitecan
  • MMAE MMAE derivatives.
  • the bioactive molecule is selected from: MMAE and MMAE derivatives.
  • the D is selected from:
  • n is 1-8; more preferably n is 3-5.
  • the structure of the biologically active conjugate is as follows, wherein Ab is selected from anti-Her2 antibody trastuzumab or anti-Trop2 antibody sacituzumab or anti-ROR1 antibody 19F6_Hu35V1, n 1 is 1-8; more preferably 3- 5:
  • the structure of the biologically active conjugate is as follows, wherein Ab is selected from anti-Her2 antibody trastuzumab or anti-Trop2 antibody sacituzumab or anti-ROR1 antibody 19F6_Hu35V1, n 1 is 1-8; more preferably 3- 5:
  • alkylene means a saturated divalent hydrocarbon group, preferably a saturated divalent hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms, such as methylene, ethylene, Propylene or Butylene.
  • alkyl is defined as a linear or branched saturated aliphatic hydrocarbon.
  • the alkyl group has 1 to 12, eg, 1 to 6 carbon atoms.
  • C 1-6 alkyl refers to a linear or branched aliphatic hydrocarbon group of 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n- butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl or n-hexyl), which are optionally substituted by 1 or more (such as 1 to 3) suitable (e.g.
  • halogen in which case the group is referred to as "haloalkyl”
  • haloalkyl e.g. CH 2 F, CHF 2 , CF 3 , CCl 3 , C 2 F 5 , C 2 Cl 5 , CH 2 CF 3 , CH 2 Cl or -CH 2 CH 2 CF 3 etc.
  • C 1-4 alkyl refers to a linear or branched aliphatic hydrocarbon group of 1 to 4 carbon atoms (i.e. methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec. butyl or tert-butyl).
  • alkoxy is defined as -O-alkyl, said alkyl being as defined above.
  • C 1-6 alkoxy refers to -OC 1-6 alkyl.
  • alkoxyalkyl is defined as an alkyl group substituted with an alkoxy group, the alkyl group being as defined above.
  • C 2-6 alkoxyalkyl refers to an alkyl group having 2 to 6 carbon atoms substituted with an alkoxy group.
  • alkynylene denotes a divalent hydrocarbon radical comprising at least one carbon-carbon triple bond, preferably having 1, 2, 3, 4, 5 or 6 carbon atoms, for example ethynylene, propynylene group or butynylene.
  • heterocyclyl refers to a saturated or partially unsaturated ring atom in which at least one ring atom is a heteroatom selected from N, O, and S and the remaining ring atoms are C (i.e. A cyclic group having one or more double bonds and/or triple bonds within the ring).
  • a “5-12 membered heterocycle (group)” has 4-11 (such as 2, 3, 4, 5, 6, 7, 8 or 9) ring carbon atoms and one or more (such as 1 , 2, 3 or 4) a saturated or partially unsaturated heterocyclic group of heteroatoms independently selected from N, O and S.
  • a “5-12 membered nitrogen-containing heterocycle (group)” is a heterocycle (group) in which at least one ring-forming atom is N.
  • heterocyclic groups include, but are not limited to: oxiranyl, aziridinyl, azetidinyl (azetidinyl), oxetanyl (oxetanyl), tetrahydrofuranyl, dioxolyl ( dioxolinyl), pyrrolidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, Piperazinyl or trithianyl.
  • the heterocyclic group can be optionally substituted by one or more (for example, 1, 2, 3 or 4) suitable substituents, and can be optionally combined with one or more aromatic rings, heteroaryl rings
  • aromatic ring or “aryl” refers to a monocyclic or polycyclic aromatic ring system having, for example, 5, 6, 8, 9, 10, 11, 12, 13 or 14 ring-forming Carbon atoms, especially 1 or 2 or 3 or 4 or 5 or 6 or 9 or 10 carbon atoms.
  • heteroaryl refers to a monocyclic or polycyclic aromatic ring system having, for example, 5, 6, 8, 9, 10, 11, 12, 13 or 14 ring atoms, in particular 1 or 2 or 3 or 4 or 5 or 6 or 9 or 10 carbon atoms and which contain at least one heteroatom which may be the same or different (the heteroatom is for example oxygen, nitrogen or sulfur), And, additionally in each case may be benzo-fused.
  • halogen includes F, Cl, Br or I.
  • substituted means that one or more (e.g., one, two, three or four) hydrogens on the designated atom are replaced by a selection from the indicated group, provided that no more than the designated atom is present.
  • the normal valences of the cases and such substitutions result in stable compounds. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
  • substituent can be (1) unsubstituted or (2) substituted. If a carbon of a substituent is described as being optionally substituted with one or more of the list of substituents, one or more hydrogens on the carbon (to the extent of any hydrogen present) may be independently and/or together Selected optional substituents are substituted. If the nitrogen of a substituent is described as being optionally substituted with one or more of the list of substituents, one or more hydrogens on the nitrogen (to the extent of any hydrogen present) may each be independently selected Substituent substitution.
  • each substituent is selected independently of the other. Accordingly, each substituent may be the same as or different from another (other) substituent.
  • one or more means 1 or more than 1, such as 2, 3, 4, 5 or 10, under reasonable conditions.
  • the point of attachment of a substituent may be from any suitable position of the substituent.
  • the present invention also includes all pharmaceutically acceptable isotopically labeled compounds which are identical to the compounds of the present invention except that one or more atoms have been labeled with the same atomic number but an atomic mass or mass number different from the atomic mass prevailing in nature. or mass number of atomic substitutions.
  • isotopes suitable for inclusion in compounds of the invention include, but are not limited to, isotopes of hydrogen (e.g., deuterium ( 2H ), tritium ( 3H )); isotopes of carbon (e.g. , 11C , 13C , and 14C ).
  • isotopes of chlorine such as 36 Cl
  • isotopes of fluorine such as 18 F
  • isotopes of iodine such as 123 I and 125 I
  • isotopes of nitrogen such as 13 N and 15 N); , 17 O and 18 O
  • phosphorus isotopes eg 32 P
  • sulfur isotopes eg 35 S
  • stereoisomer means isomers formed as a result of at least one asymmetric center.
  • compounds with one or more (e.g., one, two, three or four) asymmetric centers which can give rise to racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereoisomers.
  • Certain individual molecules may also exist as geometric isomers (cis/trans).
  • compounds of the present invention may exist as mixtures of two or more structurally distinct forms (commonly referred to as tautomers) in rapid equilibrium.
  • tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers Wait. It is to be understood that the scope of this application encompasses all such ratios in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) %) isomers or mixtures thereof.
  • Solid lines can be used in this article solid wedge or imaginary wedge Depicting the carbon-carbon bonds of the compounds of the invention.
  • the use of a solid line to delineate a bond to an asymmetric carbon atom is intended to indicate that all possible stereoisomers at that carbon atom are included (eg, specific enantiomers, racemic mixtures, etc.).
  • the use of solid or dashed wedges to delineate bonds to asymmetric carbon atoms is intended to indicate that the stereoisomers shown exist. When present in a racemic mixture, solid and imaginary wedges are used to define relative rather than absolute stereochemistry.
  • the compounds of the present invention are intended to be stereoisomers (which include cis and trans isomers, optical isomers (such as R and S enantiomers), diastereomers, Geometric isomers, rotamers, conformational isomers, atropisomers and mixtures thereof).
  • the compounds of the invention may exhibit more than one type of isomerism and consist of mixtures thereof, such as racemic mixtures and pairs of diastereoisomers.
  • the present invention covers all possible crystalline forms or polymorphs of the compounds of the present invention, which may be a single polymorph or a mixture of more than one polymorph in any proportion.
  • compositions of the present invention may exist in free form for use in therapy, or, where appropriate, as pharmaceutically acceptable derivatives thereof.
  • pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, N-oxides, metabolites or prodrugs, which are administered to patients in need thereof Following administration, the compound of the invention or its metabolites or residues can be provided directly or indirectly. Therefore, when a "compound of the present invention" is referred to herein, it is also intended to cover the above-mentioned various derivative forms of the compound.
  • the pharmaceutically acceptable salts of the compounds of the present invention include acid addition salts and base addition salts thereof.
  • Suitable acid addition salts are formed from acids which form pharmaceutically acceptable salts. Examples include aspartate, benzoate, bicarbonate/carbonate, glucoheptonate, gluconate, nitrate, orotate, palmitate and other similar salts.
  • Suitable base addition salts are formed from bases which form pharmaceutically acceptable salts. Examples include aluminum salts, arginine salts, choline salts, magnesium salts and other similar salts.
  • esters means an ester derived from each of the compounds of the general formula in this application, including physiologically hydrolyzable esters (hydrolyzable under physiological conditions to release the free acid or alcohol form of the present invention) compound).
  • the compounds of the invention may also themselves be esters.
  • the compounds of the invention may exist in the form of solvates, preferably hydrates, wherein the compounds of the invention comprise a polar solvent, such as water, methanol or ethanol in particular, as a structural element of the crystal lattice of the compound.
  • a polar solvent such as water, methanol or ethanol in particular, as a structural element of the crystal lattice of the compound.
  • the amount of polar solvent, especially water, may be present in stoichiometric or non-stoichiometric ratios.
  • nitrogen-containing heterocycles are capable of forming N-oxides since nitrogen requires available lone pairs of electrons to oxidize to oxides; nitrogen-containing heterocycle.
  • tertiary amines are capable of forming N-oxides.
  • N-oxides of heterocycles and tertiary amines are well known to those skilled in the art and include the use of peroxyacids such as peracetic acid and m-chloroperbenzoic acid (MCPBA), hydrogen peroxide, alkyl Hydrogen peroxides such as t-butyl hydroperoxide, sodium perborate and dioxiranes such as dimethyldioxirane are used to oxidize heterocycles and tertiary amines.
  • MCPBA m-chloroperbenzoic acid
  • hydrogen peroxide alkyl Hydrogen peroxides such as t-butyl hydroperoxide
  • sodium perborate and dioxiranes such as dimethyldioxirane
  • metabolites of the compounds of the present invention ie substances formed in vivo upon administration of the compounds of the present invention. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc., of the administered compound. Accordingly, the invention includes metabolites of the compounds of the invention, including compounds produced by contacting a compound of the invention with a mammal for a time sufficient to produce a metabolite thereof.
  • the present invention further includes within its scope prodrugs of the compounds of the invention, which are certain derivatives of the compounds of the invention which themselves may have little or no pharmacological activity when administered into or on the body. can be converted to a compound of the invention having the desired activity by, for example, hydrolytic cleavage. Typically such prodrugs will be functional group derivatives of the compound which are readily converted in vivo into the desired therapeutically active compound. Additional information on the use of prodrugs can be found in "Pro-drugs as Novel Delivery Systems", Volume 14, ACS Symposium Series (T. Higuchi and V. Stella).
  • prodrugs of the present invention can be obtained, for example, by using certain moieties known to those skilled in the art as "pro-moiety (such as described in "Design of Prodrugs", H. Bundgaard (Elsevier, 1985))". Prepared by substituting appropriate functional groups present in the compounds of the invention.
  • the invention also encompasses compounds of the invention which contain protecting groups.
  • protecting groups such as those described in T.W. Greene & P.G.M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, which references are incorporated herein by reference.
  • Protecting groups may be removed at an appropriate subsequent stage using methods known in the art.
  • Another aspect of the present invention provides a kind of preparation method of formula I compound, described method comprises the steps:
  • the compounds of formula I-TM1 and formula I-TM2 in the present invention can be synthesized by the following synthetic route.
  • X, Z 1 , W 1 , J 1 are as defined in the general formula above;
  • M is a leaving group that undergoes a substitution reaction, including but not limited to halogen, trifluoromethanesulfonate, p-toluenesulfonate, preferably halogen.
  • a compound of formula I-IM1 is obtained by a substitution reaction between a compound of formula I-SM1 and a compound of MZ 1 -W 1 -J 1 ;
  • the reaction is carried out under basic conditions
  • the reaction is carried out at a suitable temperature, and the temperature is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 20°C;
  • the reaction is carried out in a suitable solvent, including but not limited to acetone, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, preferably acetone.
  • a suitable solvent including but not limited to acetone, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, preferably acetone.
  • a compound of formula I-IM2 is obtained by a substitution reaction between a compound of formula I-IM1 and sodium azide;
  • the reaction is carried out at a suitable temperature, and the temperature is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 20°C;
  • the reaction is carried out in a suitable solvent, including but not limited to acetone, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, preferably acetone.
  • a suitable solvent including but not limited to acetone, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl Sulfoxide, preferably acetone.
  • the compound of formula I-IM3 is obtained through a reduction reaction of the compound of formula I-IM2;
  • this step is carried out in the presence of a suitable reducing agent, which can be selected from palladium catalysts, platinum catalysts, rhodium catalysts, preferably palladium catalysts;
  • this step is carried out at a suitable temperature, the temperature being 20°C, 25°C, 50°C, 60°C, 100°C, preferably 20°C;
  • this step is carried out in a suitable organic solvent, which can be selected from tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide , n-heptane, n-hexane, ethyl acetate, preferably ethyl acetate.
  • a suitable organic solvent which can be selected from tetrahydrofuran, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide , n-heptane, n-hexane, ethyl acetate, preferably ethyl acetate.
  • this step is carried out at a suitable temperature, the temperature being 20°C, 25°C, 50°C, 60°C, 100°C, preferably 20°C;
  • this step is carried out in a suitable organic solvent, which can be selected from acetonitrile, ethanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, n- Heptane, n-hexane, ethyl acetate, preferably acetonitrile.
  • a suitable organic solvent which can be selected from acetonitrile, ethanol, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, n- Heptane, n-hexane, ethyl acetate, preferably acetonitrile.
  • the compound of formula I-TM2 is obtained by the hydrolysis reaction of the compound of formula I-TM1;
  • the reaction is carried out under alkaline conditions, including but not limited to PB buffer with a pH of 7.0, 7.4 or 8.0;
  • the reaction is carried out at a suitable temperature, the temperature is 20°C, 25°C, 50°C, 60°C, 100°C, preferably 20°C;
  • the reaction is carried out in a suitable solvent, including but not limited to acetonitrile, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, preferably acetonitrile .
  • a suitable solvent including but not limited to acetonitrile, tetrahydrofuran, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, preferably acetonitrile .
  • the compound of formula I-TM3 in the present invention can be synthesized by the following synthetic route.
  • L is a leaving group that undergoes a substitution reaction, including but not limited to halogen, trifluoromethanesulfonate, p-toluenesulfonate, preferably halogen, OTf.
  • a compound of formula I-IM4 is obtained by a compound of formula I-IM1 through a coupling reaction
  • the coupling reagents include but are not limited to methylboronic acid, trimethylboroxane, preferably trimethylboroxane;
  • the coupling reaction is carried out in alkaline conditions
  • the base includes but not limited to triethylamine, DIPEA, NMM, sodium tert-butoxide, potassium acetate, sodium acetate, cesium fluoride, fluoride Potassium, potassium carbonate, sodium carbonate, sodium bicarbonate, cesium carbonate, potassium phosphate, potassium dihydrogenphosphate, preferably cesium fluoride.
  • the coupling reaction is carried out in the presence of a catalyst, the catalyst including but not limited to tetrakistriphenylphosphine palladium, palladium acetate, Pd 2 (dba) 3 , Pd(PPh 3 ) 2 Cl 2 , Pd(PPh 3 ) 2 Cl 2 dichloromethane complex, Pd(dppf)Cl 2 , Pd(Amphos)Cl 2 , preferably tetrakistriphenylphosphine palladium.
  • a catalyst including but not limited to tetrakistriphenylphosphine palladium, palladium acetate, Pd 2 (dba) 3 , Pd(PPh 3 ) 2 Cl 2 , Pd(PPh 3 ) 2 Cl 2 dichloromethane complex, Pd(dppf)Cl 2 , Pd(Amphos)Cl 2 , preferably tetrakistriphenylphosphine palladium.
  • the coupling reaction is carried out at a temperature of 0-200°C, preferably at a temperature of 50-150°C.
  • the reaction is carried out in a suitable solvent, including but not limited to 1,4-dioxane, water, toluene, tetrahydrofuran, N,N-dimethylformamide, N- Methylpyrrolidone, dimethylsulfoxide and any combination thereof, preferably 1,4-dioxane.
  • a suitable solvent including but not limited to 1,4-dioxane, water, toluene, tetrahydrofuran, N,N-dimethylformamide, N- Methylpyrrolidone, dimethylsulfoxide and any combination thereof, preferably 1,4-dioxane.
  • the halogenating reagent includes but not limited to bromine, N-iodosuccinimide, N-bromosuccinimide, N-chlorosuccinimide, preferably N-bromosuccinimide;
  • the coupling reaction is performed in the presence of a catalyst, and the catalyst is benzoyl peroxide.
  • the coupling reaction is carried out at a temperature of 0-200°C, preferably at a temperature of 50-150°C.
  • the reaction is carried out in a suitable solvent, including but not limited to halogenated hydrocarbons (such as carbon tetrachloride, methylene chloride, chloroform, 1,2-dichloroethane, etc.) , methanol, ethanol, DMF, acetonitrile, ethers (such as ethylene glycol dimethyl ether, tetrahydrofuran, dioxane), aromatic hydrocarbons (such as toluene, benzene, xylene), water and any combination thereof, preferably tetrachloro carbonized.
  • halogenated hydrocarbons such as carbon tetrachloride, methylene chloride, chloroform, 1,2-dichloroethane, etc.
  • ethers such as ethylene glycol dimethyl ether, tetrahydrofuran, dioxane
  • aromatic hydrocarbons such as toluene, benzene, xylene
  • water and any combination thereof preferably t
  • the compound of formula II-TM1 in the present invention can be synthesized by the following synthetic route.
  • Y 1 , Y 2 , Y 3 , B 1 , J 2 are as defined above in the general formula
  • LG is a leaving group for coupling reaction, including but not limited to halogen, trifluoromethanesulfonate, preferably halogen.
  • a compound of formula II-IM2 is obtained by a compound of formula II-IM1 through a coupling reaction
  • the coupling reaction reagent is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • the coupling reaction is carried out in the presence of a catalyst, the catalyst including but not limited to tetrakistriphenylphosphine palladium, palladium acetate, Pd 2 (dba) 3 , Pd(PPh 3 ) 2 Cl 2 , Pd(PPh 3 ) 2 Cl 2 dichloromethane complex, Pd(dppf)Cl 2 , Pd(Amphos)Cl 2 , preferably tetrakistriphenylphosphine palladium.
  • a catalyst including but not limited to tetrakistriphenylphosphine palladium, palladium acetate, Pd 2 (dba) 3 , Pd(PPh 3 ) 2 Cl 2 , Pd(PPh 3 ) 2 Cl 2 dichloromethane complex, Pd(dppf)Cl 2 , Pd(Amphos)Cl 2 , preferably tetrakistriphenylphosphine palladium.
  • the coupling reaction is carried out at a temperature of 0-200°C, preferably at a temperature of 50-150°C.
  • the reaction is carried out in a suitable solvent, including but not limited to 1,4-dioxane, water, toluene, tetrahydrofuran, N,N-dimethylformamide, N- Methylpyrrolidone, dimethylsulfoxide and any combination thereof, preferably 1,4-dioxane.
  • a suitable solvent including but not limited to 1,4-dioxane, water, toluene, tetrahydrofuran, N,N-dimethylformamide, N- Methylpyrrolidone, dimethylsulfoxide and any combination thereof, preferably 1,4-dioxane.
  • the compound of formula II-TM1 is obtained by oxidation reaction of the compound of formula II-IM2;
  • the coupling reaction is carried out in the presence of an oxidizing agent, and the catalyst is m-chloroperoxybenzoic acid.
  • the coupling reaction is carried out at a temperature of 0-120°C, preferably at a temperature of 50-80°C.
  • the reaction is carried out in a suitable solvent, including but not limited to halogenated hydrocarbons (such as carbon tetrachloride, methylene chloride, chloroform, 1,2-dichloroethane, etc.) , methanol, ethanol, DMF, acetonitrile, ethers (such as ethylene glycol dimethyl ether, tetrahydrofuran, dioxane), aromatic hydrocarbons (such as toluene, benzene, xylene), water and any combination thereof, preferably methanol.
  • halogenated hydrocarbons such as carbon tetrachloride, methylene chloride, chloroform, 1,2-dichloroethane, etc.
  • methanol such as ethylene glycol dimethyl ether, tetrahydrofuran, dioxane
  • aromatic hydrocarbons such as toluene, benzene, xylene
  • the compounds of the present invention can also be prepared in a variety of ways known to those skilled in the art of organic synthesis.
  • the compounds of the present invention can be synthesized using the methods described below as well as synthetic methods known in the art of synthetic organic chemistry or variations thereof known to those skilled in the art. Preferred methods include, but are not limited to, those described above.
  • the reactions can be performed in a solvent or mixture of solvents appropriate to the reagents and materials employed and suitable to effect the transformations.
  • Those skilled in the art of organic synthesis will appreciate that the functional groups present on the molecule should be consistent with the proposed transformation. This will sometimes require judgment in modifying the order of synthetic steps or choosing a particular process route over another to obtain the desired compound of the invention.
  • the present invention also provides a pharmaceutical composition, which contains the bioactive conjugate described in the present invention and one or more pharmaceutically acceptable carriers.
  • the pharmaceutical excipients mentioned in this article refer to the excipients and additives used in the production of drugs and the formulation of prescriptions, and refer to the active ingredients that have been reasonably evaluated in terms of safety and are included in pharmaceutical preparations substance.
  • the pharmaceutical composition may be administered in any form as long as it achieves prevention, alleviation, prevention or cure of symptoms of human or animal patients.
  • various appropriate dosage forms can be prepared according to the route of administration.
  • the present application also provides a kit product, which contains the biologically active conjugate of the present invention, or the pharmaceutical composition, and optional drug instructions.
  • Another aspect of the present application provides the use of the biologically active conjugate in the preparation of a medicament for preventing or treating tumor diseases.
  • Another aspect of the present application provides the biologically active conjugate, which is used for preventing or treating tumor diseases.
  • Another aspect of the present application provides a method for preventing or treating tumor diseases, which includes administering an effective amount of the biologically active conjugate of the ZL, or a drug containing the biologically active conjugate to a subject in need thereof. pharmaceutical composition.
  • the tumor disease is a solid tumor or hematological malignancy; for example, selected from colon cancer, gastric cancer, breast cancer, lung cancer (for example, non-small cell lung cancer, specifically lung adenocarcinoma), lymphoma .
  • an effective amount refers to an amount of a conjugate which, when administered, alleviates to some extent one or more symptoms of the condition being treated.
  • treating means reversing, alleviating the applied disorder or condition, or the progression of one or more symptoms of such a disorder or condition.
  • an “individual” or “subject” as used herein includes a human or non-human animal.
  • exemplary human subjects include human subjects suffering from a disease (eg, a disease described herein) (referred to as a patient) or normal subjects.
  • Non-human animals in the present invention include all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and/or domesticated animals (e.g., sheep, dogs, , cats, cows, pigs, etc.).
  • the mouse antibody 19F6 was obtained by immunizing Balb/c, C57Bl/6, NZB and A/J mice through hybridoma screening, and the humanized antibody sequence 19F6_Hu35v1 (heavy chain variable region, SEQ ID NO: 1; light chain variable region, SEQ ID NO: 2), the heavy chain constant region of the above humanized antibody is human IgG1 heavy chain constant region (SEQ ID NO: 18), the light chain constant region is human kappa Light chain constant region (SEQ ID NO: 19).
  • the nuclear magnetic resonance ( 1 H NMR) measuring instrument used a Bruker 400MHz nuclear magnetic resonance instrument; deuterated chloroform (CDCl 3 ); the internal standard substance was tetramethylsilane (TMS).
  • NMR nuclear magnetic resonance
  • the measuring instrument of mass spectrometry uses Agilent (ESI) mass spectrometer, model is Agilent 6120B.
  • Step 1 Synthesis of tert-butyl 2-(3,4-diazido-2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)acetate (1-2)
  • Dissolve compound 1-1 (0.40g, 1.08mmol, synthetic method refer to patent WO2019057964) and sodium azide (141.00mg, 2.17mmol) in acetone (10mL), react at 25°C for 8h, and use high performance liquid chromatography mass spectrometry The reaction was monitored by chromatography. Water was added to the reaction liquid and extracted with ethyl acetate, the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 0.28 g of the crude product of the title compound, which was directly used in the next reaction without purification.
  • Step 2 Synthesis of tert-butyl 2-(3,4-diamino-2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)acetate (1-3)
  • Dissolve compound 1-2 (0.15g, 0.51mmol) and 10% palladium carbon (15.00mg) in ethanol (20mL), replace with hydrogen three times, react at 25°C for 3h under hydrogen atmosphere, and monitor with HPLC-MS reaction.
  • the reaction solution was filtered with celite, and the filtrate was concentrated under reduced pressure to obtain 101.00 mg of the crude product of the title compound, which was directly used in the next reaction without purification.
  • Step 1 Synthesis of tert-butyl 2-(3,4-dimethyl-2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)acetate (2-1)
  • Step 2 Synthesis of tert-butyl 2-(3,4-bis(bromomethyl)-2,5-dioxy-2,5-dihydro-1H-pyrrol-1-yl)acetate (2-2 )
  • compound 2-1 72.00mg, 0.30mmol
  • NBS 118.00mg, 0.66mmol
  • dibenzoyl peroxide 7.00mg, 0.03mmol
  • Example 3 4-((S)-2-((S)-2-(2-(3,4-bis(bromomethyl)-2,5-dioxo-2,5-dihydro-1H -pyrrol-1-yl)acetamido)-3-methylbutylamino)-5-ureidopentamido)benzyl ((S)-1-((3R,4S,5S)-1-( (S)-2-((1R,2R)-3-((1S),2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl -3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1 -oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (drug-linker 3)
  • Step 1 4-((S)-2-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutylamino)-5- Ureadopentamido)benzyl ((S)-1-((S)-1-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-( (1S,2R)-1-Hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3 -Methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl Synthesis of -1-oxobutan-2-yl)(methyl)carbamate (3-2)
  • Step 2 4-((S)-2-((S)-2-amino-3-methylbutylamino)-5-ureidopentanamido)benzyl ((S)-1-((S) )-1-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((1S,2R)-1-hydroxy-1-phenylpropane-2- Base) amino)-1-methoxy-2-methyl-3-oxypropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl) (Methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl(methyl)carbamate Synthesis (3-3)
  • Step 3 4-((S)-2-((S)-2-(2-(3,4-bis(bromomethyl)-2,5-dioxo-2,5-dihydro-1H -pyrrol-1-yl)acetamido)-3-methylbutylamino)-5-ureidopentamido)benzyl ((S)-1-((3R,4S,5S)-1-( (S)-2-((1R,2R)-3-((1S),2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl -3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxohept-4-yl)(methyl)amino)-3-methyl-1 Synthesis of -oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)carbamate (3)
  • Step 1 (9H-fluoren-9-yl)methyl((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-sec-butyl)-3-(2-( (S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl -3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12,17,20,23 , Synthesis of 26-heptaoxo-2,14-dioxa-5,8,11,16,19,22,25-heptylpimelicamide-27-yl) carbamate (4-2)
  • Step 2 (S)-2-((2S,13S)-19-amino-13-benzyl-2-isopropyl-3-methyl-4,9,12,15,18-pentaoxo- 6-oxa-3,8,11,14,17-heptanezone)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3- (((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)- Synthesis of 3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide (4-3)
  • Step 3 (S)-2-((2S,13S)-13-benzyl-22-(3,4-bis(bromomethyl)-2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)-2-isopropyl-3-methyl-4,9,12,15,18,21-hexaoxo-6-oxa-3,8,11,14,17 ,20-Hexaazadocosaneamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R) -1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropane)pyrrolidin-1-yl)-3-methoxy-5 Synthesis of -methyl-1-oxoheptan-4-yl)-N,3-dimethylbutanamide (4)
  • Step 1 (9H-fluoren-9-yl)methyl((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-sec-butyl)-3-(2-( (S)-2–(((1R,2R)-3-(((1S,2R)-1-hydroxyl-1-phenylpropan-2-yl)amino)-1-methoxy-2-methanol Base-3-oxopropyl)pyrrolidin-1-yl)-2-oxoethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12,17,20 ,23,26,29-Octaoxo-2,14,32,35,38,41-Hexaoxa-5,8,11,16,19,22,25,28-Octaazapentacosane Synthesis of -43-yl) carbamate (5-2)
  • Step 2 1-amino-N-(((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-sec-butyl)-3-(2-((S)- 2-((1R,2R)-3-(((((S,2R)-1-hydroxy-1-phenylpropanyl-2-yl)amino)-1-methoxy-2-methyl-3 -Oxypropyl)pyrrolidin-1-yl)-2-oxyethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12,17,20,23,26 -Heptaoxo-2,14-dioxa-5,8,11,16,19,22,25-heptaazaheptacosan-27-yl)-3,6,9,12-tetraoxo Synthesis of Heteropentadecane-15-amide (5-3)
  • Step 3 N-(((3R,4S,7S,10S,21S)-21-benzyl-4-((S)-sec-butyl)-3-(2-((S)-2-(( 1R,2R)-3-((((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrole Alk-1-yl)-2-oxyethyl)-7,10-diisopropyl-5,11-dimethyl-6,9,12,17,20,23,26-heptaoxo-2 ,14-Dioxa-5,8,11,16,19,22,25-heptaazaheptacos-27-yl)-1-(2-(3,4-bis(bromomethyl) Synthesis of -2,5-dioxa-2-,5-dihydro-1H-pyrrol-1-yl)acetamido)-3
  • Example 8 4-((2S,5S)-41-(3,4-bis(bromomethyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)- 5-isopropyl-2-methyl-4,7,11,40-tetraoxo-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12 ,39-tetraazapentadecanoyl)phenyl
  • Step 1 (S)-2-(32-Azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatritetradecamide Synthesis of )-N-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)-3-methylbutylamine (8-3)
  • Step 2 4-((2S,5S)-38-azido-5-isopropyl-2-methyl-4,7,11-trioxo-9,15,18,21,24,27, Synthesis of 30,33,36-nonaoxa-3,6,12-triazatricoctadecanoylamido)benzyl(4-nitrophenyl)carbonate (8-4)
  • Step 3 4-((2S,5S)-38-azido-5-isopropyl-2-methyl-4,7,11-trioxo-9,15,18,21,24,27 ,30,33,36-Naoxa-3,6,12-Triazatricoctadecanoylamido)benzyl ((S)-1-(((S)-1-(((3R,4S ,5S)-1-((S)-2-((1R,2R)-3)-((1S,2R)-1-hydroxyl-1-phenylpropan-2-yl)amino)-1- Methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl) Amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl) carbonate )
  • Step 4 4-((2S,5S)-38-amino-5-isopropyl-2-methyl-4,7,11-trioxo-9,15,18,21,24,27,30 ,33,36-Naoxa-3,6,12-Triazatricoctadecanoylamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S )-1-((S)-2-((1R,2R)-3)-((1S,2R)-1-hydroxyl-1-phenylpropan-2-yl)amino)-1-methoxy Base-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino) Synthesis of -3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)
  • Step 5 4-((2S,5S)-41-(3,4-bis(bromomethyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)- 5-isopropyl-2-methyl-4,7,11,40-tetraoxo-9,15,18,21,24,27,30,33,36-nonaoxa-3,6,12 ,39-tetraazapentadecanoyl)phenyl((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R ,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropane)pyrrolidine-1 -yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1
  • Step 2 2-(2-(2-(2-(3,4-diazido-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy ) Synthetic (9-3) of ethoxy) tert-butyl acetate
  • Step 4 2-(2-(2-(2-(2-(2,3-bis(bromomethyl)-5,7-dioxo-5,7-dihydro-6H-pyrrole[3,4-b ] pyrazin-6-yl) ethoxy) ethoxy) acetic acid synthesis (I-3)
  • Example 10 4-((2S,5S)-17-(2,3-bis(bromomethyl)-5,7-dioxo-5,7-dihydro-6H-pyrrole[3,4-b ]pyrazin-6-yl)-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-9,12,15-trioxa-3,6-diaze Heptadecanamido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)- 3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl )-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-o
  • Embodiment Eleven 3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)benzoic acid (II-19)
  • Example 12 4-((31S,34S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)phenyl)-31-isopropyl-1,29,32 -Trioxo-34-(3-ureidopropyl))-5,8,11,14,17,20,23,26-octaoxa-2,30,33-triazapentatetradecane -35-amido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3 -(((1S,2R)-1-Hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl) -3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-
  • Step 1 4-((34S,37S)-34-isopropyl-2,2-dimethyl-4,32,35-trioxo-37-(3-ureidopropyl)-3,8 ,11,14,17,20,23,26,29-Naoxa-5,33,36-Triazaoctadecane-38-amido)benzyl ((S)-1-(((S )-1-((((3R,4S,5S)-1-((S)-)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-benzene Propan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxo Heptane-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-ox
  • Step 2 4-((29S,32S)-1-amino-29-isopropyl-27,30-dioxo-32-(3-ureidopropyl)-3,6,9,12,15 ,18,21,24-Octoxa-28,31-diazatritetradecane-33-amido)benzyl ((S)-1-(((S)-1-(((3R, 4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1- Methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxobutan-4-yl)(methyl)amino )-3-methyl-1-oxobutan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)(methyl)
  • Step 3 4-((31S,34S)-1-(3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)phenyl)-31-isopropyl-1,29,32- Trioxo-34-(3-ureidopropyl))-5,8,11,14,17,20,23,26-octaoxa-2,30,33-triazapentatetradecane- 35-amido)benzyl ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3- (((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)- 3-methoxy-5-methyl-1-oxoheptan-4-yl)(methyl)amino)-3-methyl-1-oxobutan
  • Example 13 N-((S)-10-benzyl-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxyl-4-methyl-10,13-dioxy -1,2,3,9,10,12,13,15-Octahydrobenzo[de]pyrano[3',4':6,7]indoleazino[1,2-b]quino Lin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-1- (2-(2,3-bis(bromomethyl)-5,7-dioxo-5H-pyrrolo[3,4-b]pyrazin-6(7H)-yl)acetamide)-3, 6,9,12-tetraoxapentadecanamide (drug-linker 13)
  • Step 1 (9H-fluoro-9-yl)methyl((S)-10-benzyl-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl -10,13-Dioxo-1,2,3,9,10,12,13,15-Octahydrobenzo[de]pyrano[3',4':6,7]indoleazino [1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecane Synthesis of -16-yl)) carbamate (13-3)
  • Step 2 (S)-2-(2-aminoacetamido)acetamido)-N-(2-((2-(((1S,9S)-9-ethyl-5-fluoro-9- Hydroxy-4-methyl-10,13-dioxocode-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6, 7] Synthesis of indolazino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)-2-oxo-3-phenylpropanamide (13- 4)
  • Step 3 (9H-fluoren-9-yl)methyl((S)-10-benzyl-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl -10,13-Dioxy-1,2,3,9,10,12,15-octahydrobenzo[de]pyrano[3',4':6,7]indoleazino[1 ,2-b]quinolin-1-yl)amino)-1,6,9,9,15,18-hexaoxo-3,21,24,27,30-pentaoxa-5,8,11 , Synthesis of 14,17-pentaazatricarboxylate (13-6)
  • Step 4 1-amino-N-((S)-10-benzyl-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13- Dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indoleazino[1,2- b] quinolin-1-yl)amino-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)- Synthesis of 3,6,9,12-tetraoxa-15-decaneamide(13-7)
  • Step 5 N-((S)-10-benzyl-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy- 1,2,3,9,10,12,13,15-Octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline -1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-1-( 2-(2,3-bis(bromomethyl)-5,7-dioxo-5H-pyrrolo[3,4-b]pyrazin-6(7H)-yl)acetamide)-3,6 , Synthesis of 9,12-tetraoxapentadecanamide (13)
  • Step 1 1-(3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)phenyl)-1-oxo-5,8,11-trioxa-2-azatridecane Synthesis of tert-butyl alkane-13-oleate (15-2)
  • Step 1 (9H-fluoro-9-yl)methyl((S)-10-benzyl-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl -10,13-Dioxo-1,2,3,9,10,12,13,15-Octahydrobenzo[de]pyrano[3',4':6,7]indoleazino [1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,21,24,27,30,33,36,39,42 Synthesis of -nonoxa-5,8,11,14,17-pentaazatetradec-44-yl)carbamate (16-2)
  • the mesylate salt of compound 13-4 (132.00 mg, 148.84 ⁇ mol) was dissolved in DMF (5 mL), and HATU (90.55 mg, 238.14 ⁇ mol), compound 16-1 (148.19 mg, 223.26 ⁇ mol) and DIPEA (96.18 mg , 744.19 ⁇ mol, 132.48 ⁇ L), reacted at 25°C for 2 hours.
  • the reaction solvent was dried under reduced pressure, and the concentrate was directly prepared by high performance liquid phase, and freeze-dried to obtain the title compound 16-2 (110.00 mg).
  • Step 2 1-amino-N-((S)-10-benzyl-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13- Dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indoleazino[1,2- b] quinolin-1-yl)amino-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-3 , Synthesis of 6,9,12,15,18,21,2-octaoxapane-27-amide (16-3)
  • Step 3 N-((S)-10-benzyl-1-((1R,9R)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo- 1,2,3,9,10,12,13,15-Octahydrobenzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline -1-yl)amino)-1,6,9,12,15,18,46-heptaoxo-3,21,24,27,30,33,36,39,42,48,51,54- Dodecyloxa-5,8,11,14,17,45-hexaazahexapentan-56-yl)-3,5-bis(2-(methylsulfonyl)pyrimidin-4-yl)benzene Synthesis of Formamide(16)
  • anti-ROR1 antibody 19F6_Hu35V1, 3-20mg/mL, referred to as 19F6 in Table 1) dilute with 0.1M edetate disodium solution (pH 7.60), and then adjust with 1M Na 2 HPO 4 solution When the pH reached 7.60, 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.60) was added to mix well, and left at room temperature for 2 h.
  • TCEP tris(2-carboxyethyl)phosphine
  • the molecular weight of ADC samples was determined by SEC-MS, and the drug/antibody ratio (DAR) was calculated.
  • Sample chamber temperature 8°C; column temperature: no column temperature control; UV: 280nm;
  • Mobile phase 20mM ammonium acetate, flow rate: 0.1ml/min, 20min, sample injection volume: 50ug;
  • Mass spectrometer model AB Sciex Triple TOF 5600+;
  • the SEC-MS molecular weight analysis of the coupled 19F6-ADC III-5-A is shown in Table 2, and the DAR is 3.87.
  • the SEC-MS molecular weight analysis of the coupled 19F6-ADC III-6-A is shown in Table 3, and the DAR is 4.42.
  • the SEC-MS molecular weight analysis of the coupled 19F6-ADC IV-1-A is shown in Table 4, and the DAR is 3.74.
  • the SEC-MS molecular weight analysis of the coupled 19F6-ADC IV-1-B is shown in Table 5, and the DAR is 3.75.
  • the SEC-MS molecular weight analysis of the coupled 19F6-ADC IV-1-C is shown in Table 6, and the DAR is 3.82.
  • the SEC-MS molecular weight analysis of the coupled 19F6-ADC IV-1-D is shown in Table 7, and the DAR is 3.87.
  • the SEC-MS molecular weight analysis of the coupled 19F6-ADC IV-7-A is shown in Table 8, and the DAR is 3.98.
  • Co-incubation of the compound of the present invention and tumor cells after the cells are attached to the wall, the culture medium in the cells is removed, and the diluted bioactive molecule (the compound of the present invention) is added to the wells of the above plate, and incubated for 96 hours.
  • ADC formed by the new conjugation method can kill tumor cells, and the application of the new conjugation method to ADC molecules is effective.

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Abstract

本发明涉及化学偶联连接子及其用途，以及通过所述化学偶联连接子制备得到的生物活性偶联物。本发明还涉及所述生物活性偶联物用于制备预防或治疗肿瘤疾病的药物的用途。

Description

一类化学偶联连接子及其用途
本申请是以CN申请号为202110615166.4，申请日为2021年6月2日的申请，以及CN申请号为202111225483.1，申请日为2021年10月21日的申请为基础，并主张其优先权，该CN申请的公开内容在此作为整体引入本申请中。
技术领域
发明涉及一类用于化学偶联的新型连接子、由该连接子制备的抗体偶联药物和相关组合物，以及其制备方法和在治疗肿瘤相关疾病中的应用。
背景技术
近年来，抗体偶联药物是肿瘤精准治疗的热门方向之一，为肿瘤治疗带来希望。抗体偶联药物(Antibody–Drug Conjugates，ADC)由靶向特异性抗原的单克隆抗体药物和小分子细胞毒药物通过连接子偶联而成，兼具传统小分子化疗的强大杀伤效应及抗体药物的肿瘤靶向性。截止到2021年04月Zynlonta的批准，全球已有12款ADC药物获批上市，其中7款用于治疗血液系统肿瘤，5款用于治疗实体瘤。
抗体偶联药物由抗体、linker、payload及偶联方式等几部分组成。其中偶联方式是抗体和药物-连接体的连接方式。偶联方法主要分为非定点偶联和定点偶联。早期使用的是非定点偶联法，主要由赖氨酸偶联和半胱氨酸偶联,利用化学方法直接将药物与抗体上氨基酸残基进行偶联，不涉及抗体的改造或修饰，其偶联的毒素分子个数和偶联位点都不能确定，均一性欠佳。目前常用的定点偶联方式即通过基因工程位点或者特殊的连接头进行特异性偶联，实现更均一地偶联，能在特定位点实现细胞毒素的连接。定点偶联生产的抗体偶联药物，可以减少因偶联位点和偶联个数不同而造成药效、药代以及CMC质控的波动。
目前常见的定点偶联方式THIOMAB技术、非天然氨基酸偶联技术、谷氨酰胺酶促偶联技术、Sortase转肽酶偶联技术以及ThioBridge技术等。其中，利用抗体工程化或者酶促偶联，对抗体的改造修饰，可能会对抗体的结构稳定性有一定影响，同时对CMC有一定要求。另外利用化学偶联的部分ThioBridge技术也有一定的缺陷，例如DBM(dibromomaleimides)类连接子和其他含巯基的生物基团发生置换作用，而在血浆中不稳定，造成药效降低，毒副作用增加(Chem.–Eur.J.,2019,25,43–59.)。因此，开发新型的连接子结构对于研发出具有良好药效及安全性的抗体偶联药物仍具有重要意义。
发明内容
本申请的一个目的在于提供用于化学偶联的新型连接子，该类连接子反应活性高，偶联条件温和、操作简便，可以实现定点偶联，得到的生物活性偶联物均一性和血浆稳定性较好，体内外药效明确。
化合物
在第一个方面，本申请提供化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、N-氧化物或同位素标记的化合物，其中所述化合物具有式I的结构：
Figure PCTCN2022094540-appb-000001
其中：
X选自离去基团，例如Cl、Br、I、OMs、OTs、OTf或
Figure PCTCN2022094540-appb-000002
Y不存在或选自羰基；
A环选自取代或非取代的C 6-10芳环、5-12元芳杂环或5-12元杂环；
Q不存在或为-C(＝O)-NH-；
Z 1不存在或者选自-CH 2-或C 2-6亚炔基；
W 1不存在或选自C 1-10亚烷基、-(CH 2CH 2O)p-、以及-(OCH 2CH 2)p-中的一种或多种；
J 1选自-COOH、-NH 2、3-10元含氮杂环基、磺酰脲基或羟基；
p为1-10的整数。
在部分实施方案中，X选自Cl、Br、I、OMs、OTs以及OTf中的一种或多种。
在部分实施方案中，Y不存在。
在部分实施方案中，Z 1不存在或者为C 2-6亚炔基。
在部分实施方案中，W 1不存在或选自C 1-10亚烷基和-(CH 2CH 2O)p-中的一种或多种。
在部分实施方案中，W 1为C 1-10亚烷基；优选为C 1-6亚烷基，进一步优选为C 1-3亚烷基。
在部分实施方案中，J 1为-COOH。
在部分实施方案中，A环选自取代或非取代的5-12元含氮芳杂环或5-12元含氮杂环；优选地，A环选自非取代或被氧代基或-COOH取代的5-12元含氮芳杂环或5-12元含氮杂环。
在部分实施方案中，p为1，2，3，4，5，6，7，8，9，或10。
在部分实施方案中，X选自Cl、Br、I、OMs、OTs、OTf和
Figure PCTCN2022094540-appb-000003
Y不存在；
A环选自非取代或被氧代基或-COOH取代的5-12元含氮芳杂环或5-12元含氮杂环；
Q不存在或为-C(＝O)-NH-；
Z 1不存在或者为-CH 2-；
W 1不存在或选自C 1-10亚烷基和-(CH 2CH 2O)p-中的一种或多种；
J 1为-COOH；
p为1-10的整数。
在部分实施方案中，式I选自如下结构：
Figure PCTCN2022094540-appb-000004
在部分实施方案中，式I化合物具有如下结构：
Figure PCTCN2022094540-appb-000005
其中p为1-10的整数，J 1为-COOH或-NH 2。
在部分实施方案中，式I化合物具有如下结构：
Figure PCTCN2022094540-appb-000006
在第二个方面，本申请提供化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、N-氧化物或同位素标记的化合物，其中所述化合物具有式II的结构：
Figure PCTCN2022094540-appb-000007
其中，B 1在每次出现时，各自独立地选自单键或5-12元含氮芳杂环；
Y 1，Y 2及Y 3在每次出现时，各自独立的选自CH和N；
Z 2不存在或者选自-NH-、-CH 2-、羰基、-C(＝O)NH-、-NHC(＝O)-或C 2-6亚炔基；优选地，Z 2不存在或者选自-NH-、-CH 2-、羰基或C 2-6亚炔基；
W 2不存在或选自C 1-10亚烷基、-(CH 2CH 2O) p-或-(OCH 2CH 2) p-中的一个或多个；
J 2选自-COOH、-NH 2、3-10元含氮杂环基、磺酰脲基或羟基；
p为1-10的整数。
在部分实施方案中，B 1在每次出现时，各自独立地选自单键或5-6元含氮芳杂环；优选地，B 1在每次出现时，各自独立地选自单键或5-6元含氮芳杂环；进一步优选地，B 1在每次出现时，各自独立地选自单键或嘧啶环。
在部分实施方案中，Y 1，Y 2及Y 3均为N；或者Y 1为CH、Y 2及Y 3均为N；或者Y 1为N、Y 2及Y 3均为CH；或者Y 1，Y 2及Y 3均为CH。
在部分实施方案中，Z 2不存在或者为C 2-6亚炔基。
在部分实施方案中，W 2不存在或为C 1-10亚烷基。
在部分实施方案中，J 2选自-COOH或-NH 2。
在部分实施方案中，B 1在每次出现时，各自独立地选自单键或嘧啶环；
Y 1，Y 2及Y 3均为CH；
Z 2不存在或者为-C(＝O)NH-；
W 2不存在或选自C 1-10亚烷基、-(CH 2CH 2O) p中的一个或多个；
J 2为-COOH；
p为1-10的整数。
在部分实施方案中，式II化合物具有如下结构：
Figure PCTCN2022094540-appb-000008
Figure PCTCN2022094540-appb-000009
Figure PCTCN2022094540-appb-000010
Figure PCTCN2022094540-appb-000011
Figure PCTCN2022094540-appb-000012
Figure PCTCN2022094540-appb-000013
p为1-10的整数。
在部分实施方案中，p为1-8的整数；进一步优选p为1-5的整数。
在部分实施方案中，式II化合物具有如下结构：
Figure PCTCN2022094540-appb-000014
在部分实施方案中，式II化合物具有如下结构：
Figure PCTCN2022094540-appb-000015
在第三个方面，本申请提供化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、N-氧化物或同位素标记的化合物，其中所述化合物具有式III的结构：
Figure PCTCN2022094540-appb-000016
其中：
V 1为本申请第一方面式I化合物中J 1与L连接时形成的基团；优选地，V 1选自-CO-、-N(R 1)-、-O-、3-10元含氮杂环基或磺酰脲基，其中R 1为H、C 1-6烷基或C 2-6烷氧烷基；进一步优选地，V 1选自-C(O)-和-N(R 1)-，其中R 1为H、C 1-6烷基或C 2-6烷氧烷基；
L是连接V 1和E’之间的连接子；
E’选自H、-NHCH 2-Lg、-COOH、
Figure PCTCN2022094540-appb-000017
其中，Lg表示离去基团，例如Cl、Br、I、OMs、OTs、OTf或
Figure PCTCN2022094540-appb-000018
X、Y、A、Q、Z 1、W 1如前文第一方面任一项中所定义。
在部分实施方案中，L选自以下基团中的一个或多个：C 1-6亚烷基、-N(R 6)-、羰基、-O-、Val、Cit、Phe、Lys、D-Val、Leu、Gly、Phe、Ala、Asn、Val-Cit、Val-Ala、Val-Lys、Val-Lys(Ac)、Phe-Lys、Phe-Lys(Ac)、D-Val-Leu-Lys、Gly-Gly-Arg、Ala-Ala-Asn、Gly-Gly-Gly、Gly-Gly-Phe-Gly、Gly-Gly-Gly-Gly-Gly、
Figure PCTCN2022094540-appb-000019
Figure PCTCN2022094540-appb-000020
其中R 6为H、C 1-6烷基或C 2-6烷氧烷基，s为1-10的整数。
在部分实施方案中，L选自以下基团中的一个或多个：Val、Cit、Gly、Phe、Ala、 Val-Cit、Val-Ala、Gly-Gly-Phe-Gly、
Figure PCTCN2022094540-appb-000021
Figure PCTCN2022094540-appb-000022
在部分实施方案中，L选自
Figure PCTCN2022094540-appb-000023
Figure PCTCN2022094540-appb-000024
在部分实施方案中，L选自
Figure PCTCN2022094540-appb-000025
Figure PCTCN2022094540-appb-000026
在第四个方面，本申请提供化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、N-氧化物或同位素标记的化合物，其中所述化合物具有式IV的结构：
Figure PCTCN2022094540-appb-000027
其中，
V 2为本申请第二方面式II化合物中J 2与L连接后得到的基团；优选地，V 2选自-CO-、 -N(R 2)-、-O-、3-10元含氮杂环基或磺酰脲基，其中R 2为H、C 1-6烷基或C 2-6烷氧烷基；进一步优选地，V 2选自-C(O)-和-N(R 2)-，其中R 2为H、C 1-6烷基或C 2-6烷氧烷基；
B 1、Y 1、Y 2、Y 3、Z 2和W 2如前文第二方面任一项中所定义；
L和E’如前文第三方面任一项中所定义。
在第五个方面，本申请提供化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、N-氧化物或同位素标记的化合物，其中所述化合物具有式V的结构：
Figure PCTCN2022094540-appb-000028
其中：
E选自单键、-NH-CH 2-，或以下结构：
Figure PCTCN2022094540-appb-000029
D是生物活性分子(例如细胞毒性药物)片段；
X、Y、A、Q、Z 1、W 1如前文第一方面任一项中所定义；
V 1和L如第三方面任一项中所定义。
在部分实施方案中，所述生物活性分子选自下组：抗微管蛋白剂、DNA嵌入剂、DNA拓扑异构酶抑制剂和RNA聚合酶抑制剂。
在部分实施方案中，所述生物活性分子选自下组：微管蛋白抑制剂奥瑞他汀类化合物、美登素类化合物；DNA嵌入剂吡咯并苯二氮卓(PBD)；DNA拓扑异构酶抑制剂，例如拓扑异构酶I抑制剂(诸如喜树碱、羟基喜树碱、9-氨基喜树碱、SN-38、伊立替康、拓扑替康、贝洛替康、卢比替康)或拓扑异构酶II抑制剂(诸如阿霉素、多柔比星、PNU-159682、多卡米星、柔红霉素、米托蒽醌、鬼臼毒素或依托泊苷)；RNA聚合酶抑制剂α-鹅膏草碱(α-amanitin)等；及其药学上可接受的盐、酯和类似物。
在部分实施方案中，所述生物活性分子选自：拓扑异构酶I抑制剂(诸如喜树碱、羟基喜树碱、9-氨基喜树碱、SN-38、伊立替康、拓扑替康、贝洛替康、卢比替康)、MMAE以及MMAE衍生物。
在部分实施方案中，所述生物活性分子选自：MMAE以及MMAE衍生物。
在部分实施方案中，所述D选自：
Figure PCTCN2022094540-appb-000030
在部分实施方案中，式V化合物选自：
Figure PCTCN2022094540-appb-000031
Figure PCTCN2022094540-appb-000032
Figure PCTCN2022094540-appb-000033
在第六个方面，本申请提供化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、N-氧化物或同位素标记的化合物，其中所述化合物具有式VI的结构：
Figure PCTCN2022094540-appb-000034
B 1、Y 1、Y 2、Y 3、Z 2和W 2如前文第二方面任一项中所定义；
L如前文第三方面任一项中所定义；
V 2如前文第四方面任一项中所定义；
E和D如前文第五方面任一项中所定义。
在部分实施方案中，所述式VI化合物为
Figure PCTCN2022094540-appb-000035
Figure PCTCN2022094540-appb-000036
Figure PCTCN2022094540-appb-000037
在第七个方面，本申请提供一种生物活性偶联物，其结构如式VII所示：
Figure PCTCN2022094540-appb-000038
其中，Ab为靶向部分(例如小分子配体、蛋白质(例如抗体)、多肽、非蛋白质试剂(例如糖、RNA或DNA))；n选自1-10之间的整数或小数；
V 1选自-C(O)-或-N(R 1)-，其中R 1为H、C 1-6烷基或C 2-6烷氧烷基；
L是连接V 1和E之间的连接子；
E是连接L和D的结构片段；
D是生物活性分子(例如细胞毒性药物)片段；
所述偶联物中的
Figure PCTCN2022094540-appb-000039
表示靶向部分为抗体时，抗体中的巯基与偶联物其余部分的具体连接方式；
其余基团如前文任一方面任一项中所定义。
本发明的另一个方面提供一种生物活性偶联物，其结构如式VIII所示：
Figure PCTCN2022094540-appb-000040
其中，Ab为靶向部分(例如小分子配体、蛋白质(例如抗体)、多肽、非蛋白质试剂(例如糖、RNA或DNA))；n选自1-10之间的整数或小数；
V 2选自-C(O)-或-N(R 2)-，其中R 2为H、C 1-6烷基或C 2-6烷氧烷基；
L是连接V 2和E之间的连接子；
E是连接L和D的结构片段；
D是生物活性分子(例如细胞毒性药物)片段；
所述偶联物中的
Figure PCTCN2022094540-appb-000041
表示靶向部分为抗体时，抗体中的巯基与偶联物其余部分的具体连接方式；
其余基团如前文任一方面任一项中所定义。在部分实施方案中，Ab选自表皮生长因子、Trop-2、CD37、HER2、CD70、EGFRvIII、Mesothelin、Folate receoptor1、Mucin 1、CD138、CD20、CD19、CD30、SLTRK6、Nectin 4、Tissue factor、Mucin16、Endothelinreceoptor、STEAP1、SLC39A6、Guanylylcyclase C、PSMA、CCD79b、CD22、Sodium phosphate cotransporter 2B、GPNMB、Trophoblast glycoprotein、AGS-16、EGFR、CD33、CD66e、CD74、CD56、PD-L1、TACSTD2、DR5、E16、STEAP1、0772P、MPF、Napi3b、Sema 5b、PSCA hlg、ETBR、MSG783、STEAP2、TrpM4、CRIPTO、CD21、CD79b、FcRH2、NCA、MDP、IL20Rα、Brevican、EphB2R、ASLG659、PSCA、GEDA、BAFF-R、CD22、CD79a、CXCR5、HLA-DOB、P2X5、CD72、LY64、FcRH1、IRTA2、TENB2、整合素α5β6，α4β7、FGF2、FGFR2、Her3、CD70、CA6、DLL3、DLL4、P-cadherin、EpCAM、pCAD、CD223、LYPD3、LY6E、EFNA4、ROR1、SLITRK6、5T4、ENPP3、SLC39A6、Claudin18.2、BMPR1B、E16、STEAP1、Tyro7、0772P、MPF、Napi3b、Sema 5b、PSCA hlg、ETBR、MSG783、STEAP2、TrpM4、CRIPTO、CD21、CD79b、FcRH2、NCA、MDP、IL20Rα、Brevican、 EphB2R、ASLG659、PSCA、GEDA、CD22、CD79a、CXCR5、HLA-DOB、P2X5、CD72、LY64、FcRH1、IRTA2，c-Met，ApoE、CD1lc、CD40、CD45(PTPRC)、CD49D(ITGA4)、CD80、CSF1R、CTSD、GZMB、Ly86、MS4A7、PIK3AP1、PIK3CD、CCR5、IFNG、IL10RA1、IL-6、ACTA2、COL7A1、LOX、LRRC15、MCPT8、MMP10、NOG、SERPINEl、STAT1、TGFBR1、CTSS、PGF、VEGFA、C1QA、C1QB、ANGPTL4、EGLN、ANGPTL4、EGLN3、BNIP3、AIF1、CCL5、CXCL10、CXCL11、IFI6、PLOD2、KISS1R、STC2、DDIT4、PFKFB3、PGK1、PDK1、AKR1C1、AKR1C2、CADM1、CDH11、COL6A3、CTGF、HMOX1、KRT33A、LUM、WNT5A、IGFBP3、MMP14、CDCP1、PDGFRA、TCF4、TGF、TGFB1、TGFB2、CDl lb、ADGRE1、EMR2、TNFRSF21、UPK1B、TNFSF9、MMP16、MFI2、IGF-1R、RNF43、NaPi2b、BCMA、B7H3和TENB2；
优选地，Ab选自anti-Her2抗体trastuzumab或anti-Trop2抗体sacituzumab或anti-ROR1抗体19F6_Hu35V1。
在部分实施方案中，L选自以下基团中的一个或多个：C 1-6亚烷基、-N(R 6)-、羰基、-O-、Val、Cit、Phe、Lys、D-Val、Leu、Gly、Phe、Ala、Asn、Val-Cit、Val-Ala、Val-Lys、Val-Lys(Ac)、Phe-Lys、Phe-Lys(Ac)、D-Val-Leu-Lys、Gly-Gly-Arg、Ala-Ala-Asn、Gly-Gly-Gly、Gly-Gly-Phe-Gly、Gly-Gly-Gly-Gly-Gly、
Figure PCTCN2022094540-appb-000042
Figure PCTCN2022094540-appb-000043
其中R 6为H、C 1-6烷基或C 2-6烷氧烷基，s为1-10的整数；
优选地，L选自以下结构：
Figure PCTCN2022094540-appb-000044
在部分实施方案中，所述的E为单键、-NH-CH 2-，或以下结构：
Figure PCTCN2022094540-appb-000045
在部分实施方案中，所述的E为-NH-CH 2-。
在部分实施方案中，所述生物活性分子选自下组：抗微管蛋白剂、DNA嵌入剂、DNA拓扑异构酶抑制剂和RNA聚合酶抑制剂。
在部分实施方案中，所述生物活性分子选自下组：微管蛋白抑制剂奥瑞他汀类化合物、美登素类化合物；DNA嵌入剂吡咯并苯二氮卓(PBD)；DNA拓扑异构酶抑制剂，例如拓扑异构酶I抑制剂(诸如喜树碱、羟基喜树碱、9-氨基喜树碱、SN-38、伊立替康、拓扑替康、贝洛替康、卢比替康)或拓扑异构酶II抑制剂(诸如阿霉素、多柔比星、PNU-159682、多卡米星、柔红霉素、米托蒽醌、鬼臼毒素或依托泊苷)；RNA聚合酶抑制剂α-鹅膏草碱(α-amanitin)等；及其药学上可接受的盐、酯和类似物。
在部分实施方案中，所述生物活性分子选自：拓扑异构酶I抑制剂(诸如喜树碱、羟基喜树碱、9-氨基喜树碱、SN-38、伊立替康、拓扑替康、贝洛替康、卢比替康)、MMAE以及MMAE衍生物。
在部分实施方案中，所述生物活性分子选自：MMAE以及MMAE衍生物。
在部分实施方案中，所述的D选自：
Figure PCTCN2022094540-appb-000046
在部分实施方案中，n为1-8；进一步优选n为3-5。
在部分实施方案中，所述生物活性偶联物结构如下，其中Ab选自anti-Her2抗体trastuzumab或anti-Trop2抗体sacituzumab或anti-ROR1抗体19F6_Hu35V1，n 1为1-8；进一步优选为3-5：
Figure PCTCN2022094540-appb-000047
Figure PCTCN2022094540-appb-000048
在部分实施方案中，所述生物活性偶联物结构如下，其中Ab选自anti-Her2抗体trastuzumab或anti-Trop2抗体sacituzumab或anti-ROR1抗体19F6_Hu35V1，n 1为 1-8；进一步优选为3-5：
Figure PCTCN2022094540-appb-000049
Figure PCTCN2022094540-appb-000050
Figure PCTCN2022094540-appb-000051
Figure PCTCN2022094540-appb-000052
定义
除非在下文中另有定义，本文中所用的所有技术术语和科学术语的含义意图与本领域技术人员通常所理解的相同。提及本文中使用的技术意图指在本领域中通常所理解的技术，包括那些对本领域技术人员显而易见的技术的变化或等效技术的替换。虽然相信以下术语对于本领域技术人员很好理解，但仍然阐述以下定义以更好地解释本发明。
如本文中所使用，术语“亚烷基”表示饱和二价烃基，优选表示具有1、2、3、4、5或6个碳原子的饱和二价烃基，例如亚甲基、亚乙基、亚丙基或亚丁基。
如本文中所使用，术语“烷基”定义为线性或支化饱和脂肪族烃。在一些实施方案中，烷基具有1至12个，例如1至6个碳原子。例如，如本文中所使用，术语“C 1-6烷基”指1至6个碳原子的线性或支化的脂肪族烃基(例如甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、异戊基、新戊基或正己基)，其任选地被1或多个(诸如1至3个)适合的取代基(如卤素)取代(此时该基团被称作“卤代烷基”)(例如CH 2F、CHF 2、CF 3、CCl 3、C 2F 5、C 2Cl 5、CH 2CF 3、CH 2Cl或-CH 2CH 2CF 3等)。术语“C 1-4烷基”指1至4 个碳原子的线性或支化的脂肪族烃基(即甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基或叔丁基)。
如本文中所使用，术语“烷氧基”定义为-O-烷基，所述烷基根据上文所定义。例如，如本文中所使用，术语“C 1-6烷氧基”指-O-C 1-6烷基。
如本文中所使用，术语“烷氧烷基”定义为被烷氧基取代的烷基，所述烷基根据上文所定义。例如，如本文中所使用，术语“C 2-6烷氧烷基”指具有2至6个碳原子的被烷氧基取代的烷基基团。
如本文中所使用，术语“亚炔基”表示包括至少一个碳碳三键的二价烃基，优选具有1、2、3、4、5或6个碳原子，例如亚乙炔基、亚丙炔基或亚丁炔基。
如本文中所使用，术语“杂环基”、“杂环”是指其中至少一个环原子是选自N、O和S的杂原子且其余环原子是C的饱和或部分不饱和的(即在环内具有一个或多个双键和/或三键)环状基团。例如，“5-12元杂环(基)”是具有4-11个(如2、3、4、5、6、7、8或9个)环碳原子和一个或多个(例如1个、2个、3个或4个)独立地选自N、O和S的杂原子的饱和或部分不饱和杂环基。“5-12元含氮杂环(基)”是成环原子中至少一个为N的杂环(基)。杂环基的实例包括但不限于：环氧乙烷基、氮丙啶基、氮杂环丁基(azetidinyl)、氧杂环丁基(oxetanyl)、四氢呋喃基、二氧杂环戊烯基(dioxolinyl)、吡咯烷基、吡咯烷酮基、咪唑烷基、吡唑烷基、吡咯啉基、四氢吡喃基、哌啶基、吗啉基、二噻烷基(dithianyl)、硫吗啉基、哌嗪基或三噻烷基(trithianyl)。所述杂环基可任选地被一个或多个(例如1个、2个、3个或4个)适合的取代基取代，并且可以任选地和一个或多个芳环、杂芳环形成并环结构。
如本文中所使用，术语“芳环”或“芳基”指单环或多环芳族环系，其具有例如5、6、8、9、10、11、12、13或14个成环碳原子，特别是1或2或3或4或5或6或9或10个碳原子。
如本文中所使用，术语“芳杂环”或“杂芳基”指单环或多环芳族环系，其具有例如5、6、8、9、10、11、12、13或14个环原子，特别是1或2或3或4或5或6或9或10个碳原子，且其包含至少一个可以相同或不同的杂原子(所述杂原子是例如氧、氮或硫)，并且，另外在每一种情况下可为苯并稠合的。
如本文中所使用，术语“卤素”包括F、Cl、Br或I。
如本文中所使用，术语“磺酰脲基”指-SO 2-NH-(C＝O)-NH 2或-NH-(C＝O)-NH-SO 2H。
术语“取代”指所指定的原子上的一个或多个(例如一个、两个、三个或四个)氢被从所指出的基团的选择代替，条件是未超过所指定的原子在当前情况下的正常原子价并且所述取代形成稳定的化合物。取代基和/或变量的组合仅仅当这种组合形成稳定的化合物时 才是允许的。
如果取代基被描述为“任选地被……取代”，则取代基可(1)未被取代或(2)被取代。如果取代基的碳被描述为任选地被取代基列表中的一个或多个取代，则碳上的一个或多个氢(至存在的任何氢的程度)可单独和/或一起被独立地选择的任选的取代基替代。如果取代基的氮被描述为任选地被取代基列表中的一个或多个取代，则氮上的一个或多个氢(至存在的任何氢的程度)可各自被独立地选择的任选的取代基替代。
如果取代基被描述为“独立地选自”一组基团，则各取代基独立于另一者被选择。因此，各取代基可与另一(其他)取代基相同或不同。
如本文中所使用，术语“一个或多个”意指在合理条件下的1个或超过1个，例如2个、3个、4个、5个或10个。
除非指明，否则如本文中所使用，取代基的连接点可来自取代基的任意适宜位置。
当取代基的键显示为穿过环中连接两个原子的键时，则这样的取代基可键连至该可取代的环中的任一成环原子。
本发明还包括所有药学上可接受的同位素标记的化合物，其与本发明的化合物相同，除了一个或多个原子被具有相同原子序数但原子质量或质量数不同于在自然界中占优势的原子质量或质量数的原子替代。适合包含入本发明的化合物中的同位素的实例包括(但不限于)氢的同位素(例如氘( 2H)、氚( 3H))；碳的同位素(例如 11C、 13C及 14C)；氯的同位素(例如 36Cl)；氟的同位素(例如 18F)；碘的同位素(例如 123I及 125I)；氮的同位素(例如 13N及 15N)；氧的同位素(例如 15O、 17O及 18O)；磷的同位素(例如 32P)；及硫的同位素(例如 35S)。
术语“立体异构体”表示由于至少一个不对称中心形成的异构体。在具有一个或多个(例如一个、两个、三个或四个)不对称中心的化合物中，其可产生外消旋混合物、单一对映异构体、非对映异构体混合物和单独的非对映异构体。特定个别分子也可以几何异构体(顺式/反式)存在。类似地，本发明的化合物可以两种或更多种处于快速平衡的结构不同的形式的混合物(通常称作互变异构体)存在。互变异构体的代表性实例包括酮-烯醇互变异构体、苯酚-酮互变异构体、亚硝基-肟互变异构体、亚胺-烯胺互变异构体等。要理解，本申请的范围涵盖所有这样的以任意比例(例如60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％)的异构体或其混合物。
本文中可使用实线
Figure PCTCN2022094540-appb-000053
实楔形
Figure PCTCN2022094540-appb-000054
或虚楔形
Figure PCTCN2022094540-appb-000055
描绘本发明的化合物的碳-碳键。使用实线以描绘键连至不对称碳原子的键欲表明，包括该碳原子处的所有可能的立体异构体(例如，特定的对映异构体、外消旋混合物等)。使用实或虚楔形以描绘键连至不对称碳原子的键欲表明，存在所示的立体异构体。当存在于外消旋混合物中时，使用实及虚楔形以定义相对立体化学，而非绝对立体化学。除非另外指明，否则本发明的 化合物意欲可以立体异构体(其包括顺式及反式异构体、光学异构体(例如R及S对映异构体)、非对映异构体、几何异构体、旋转异构体、构象异构体、阻转异构体及其混合物)的形式存在。本发明的化合物可表现一种以上类型的异构现象，且由其混合物(例如外消旋混合物及非对映异构体对)组成。
本发明涵盖本发明的化合物的所有可能的结晶形式或多晶型物，其可为单一多晶型物或多于一种多晶型物的任意比例的混合物。
还应当理解，本发明的某些化合物可以游离形式存在用于治疗，或适当时，以其药学上可接受的衍生物形式存在。在本发明中，药学上可接受的衍生物包括但不限于，药学上可接受的盐、酯、溶剂合物、N-氧化物、代谢物或前药，在将它们向需要其的患者给药后，能够直接或间接提供本发明的化合物或其代谢物或残余物。因此，当在本文中提及“本发明的化合物”时，也意在涵盖化合物的上述各种衍生物形式。
本发明的化合物的药学上可接受的盐包括其酸加成盐及碱加成盐。
适合的酸加成盐由形成药学可接受盐的酸来形成。实例包括天冬氨酸盐、苯甲酸盐、碳酸氢盐/碳酸盐、葡庚糖酸盐、葡糖酸盐、硝酸盐、乳清酸盐、棕榈酸盐及其它类似的盐。
适合的碱加成盐由形成药学可接受盐的碱来形成。实例包括铝盐、精氨酸盐、胆碱盐、镁盐及其它类似的盐。
适合的盐的综述参见Stahl及Wermuth的“Handbook of Pharmaceutical Salts:Properties,Selection,and Use”(Wiley-VCH,2002)。用于制备本发明的化合物的药学上可接受的盐的方法为本领域技术人员已知的。
如本文中所使用，术语“酯”意指衍生自本申请中各个通式化合物的酯，其包括生理上可水解的酯(可在生理条件下水解以释放游离酸或醇形式的本发明的化合物)。本发明的化合物本身也可以是酯。
本发明的化合物可以溶剂合物(优选水合物)的形式存在，其中本发明的化合物包含作为所述化合物晶格的结构要素的极性溶剂，特别是例如水、甲醇或乙醇。极性溶剂特别是水的量可以化学计量比或非化学计量比存在。
本领域技术人员会理解，由于氮需要可用的孤对电子来氧化成氧化物，因此并非所有的含氮杂环都能够形成N-氧化物；本领域技术人员会识别能够形成N-氧化物的含氮杂环。本领域技术人员还会认识到叔胺能够形成N-氧化物。用于制备杂环和叔胺的N-氧化物的合成方法是本领域技术人员熟知的，包括用过氧酸如过氧乙酸和间氯过氧苯甲酸(MCPBA)、过氧化氢、烷基过氧化氢如叔丁基过氧化氢、过硼酸钠和双环氧乙烷(dioxirane)如二甲基双环氧乙烷来氧化杂环和叔胺。这些用于制备N-氧化物的方法已在文献中得到广泛描述 和综述，参见例如：T.L.Gilchrist,Comprehensive Organic Synthesis,vol.7,pp 748-750；A.R.Katritzky和A.J.Boulton,Eds.,Academic Press；以及G.W.H.Cheeseman和E.S.G.Werstiuk，Advances in Heterocyclic Chemistry,vol.22,pp 390-392,A.R.Katritzky和A.J.Boulton,Eds.,Academic Press。
在本发明的范围内还包括本发明的化合物的代谢物，即在给药本发明的化合物时体内形成的物质。这样的产物可由例如被给药的化合物的氧化、还原、水解、酰胺化、脱酰胺化、酯化、酶解等产生。因此，本发明包括本发明的化合物的代谢物，包括通过使本发明的化合物与哺乳动物接触足以产生其代谢产物的时间的方法制得的化合物。
本发明在其范围内进一步包括本发明的化合物的前药，其为自身可具有较小药理学活性或无药理学活性的本发明的化合物的某些衍生物当被给药至身体中或其上时可通过例如水解裂解转化成具有期望活性的本发明的化合物。通常这样的前药会是所述化合物的官能团衍生物，其易于在体内转化成期望的治疗活性化合物。关于前药的使用的其他信息可参见“Pro-drugs as Novel Delivery Systems”，第14卷，ACS Symposium Series(T.Higuchi及V.Stella)。本发明的前药可例如通过用本领域技术人员已知作为“前-部分(pro-moiety)(例如“Design of Prodrugs”，H.Bundgaard(Elsevier,1985)中所述)”的某些部分替代本发明的化合物中存在的适当官能团来制备。
本发明还涵盖含有保护基的本发明的化合物。在制备本发明的化合物的任何过程中，保护在任何有关分子上的敏感基团或反应基团可能是必需的和/或期望的，由此形成本发明的化合物的化学保护的形式。这可以通过常规的保护基实现，例如，在T.W.Greene&P.G.M.Wuts,Protective Groups in Organic Synthesis,John Wiley&Sons,1991中所述的那些保护基，这些参考文献通过援引加入本文。使用本领域已知的方法，在适当的后续阶段可以移除保护基。
制备方法
本发明的另一方面提供一种式I化合物的制备方法，所述方法包括如下步骤：
当Y不存在时，本发明中的式I-TM1和式I-TM2化合物可由下述合成路线进行合成制备。
Figure PCTCN2022094540-appb-000056
其中：
X、Z 1、W 1、J 1如前文通式所定义；
M为发生取代反应的离去基团，包括但不限于卤素、三氟甲烷磺酸酯、对甲苯磺酸酯，优选为卤素。
步骤一
由式I-SM1化合物与M-Z 1-W 1-J 1化合物发生取代反应得到式I-IM1化合物；
在部分实施方案中，所述反应在碱性条件下进行；
在部分实施方案中，所述反应在合适的温度下进行，所述温度为20℃、25℃、50℃、60℃、100℃，优选20℃；
在部分实施方案中，所述反应在合适的溶剂下进行，所述溶剂包括但不限于丙酮、四氢呋喃、二氯甲烷、N,N-二甲基甲酰胺、N-甲基吡咯烷酮、二甲基亚砜，优选为丙酮。
步骤二
由式I-IM1化合物与叠氮化钠发生取代反应得到式I-IM2化合物；
在部分实施方案中，所述反应在合适的温度下进行，所述温度为20℃、25℃、50℃、60℃、100℃，优选20℃；
在部分实施方案中，所述反应在合适的溶剂下进行，所述溶剂包括但不限于丙酮、四氢呋喃、二氯甲烷、N,N-二甲基甲酰胺、N-甲基吡咯烷酮、二甲基亚砜，优选为丙酮。
步骤三
通过式I-IM2化合物发生还原反应得到式I-IM3化合物；
在部分实施方案中，该步骤在合适的还原剂存在下进行，所述还原剂可选自钯催化剂、铂催化剂、铑催化剂，优选为钯催化剂；
在部分实施方案中，该步骤在适合的温度下进行，所述温度为20℃、25℃、50℃、60℃、100℃，优选为20℃；
在部分实施方案中，该步骤在适合的有机溶剂中进行，所述有机溶剂可选自四氢呋喃、二氯甲烷、N,N-二甲基甲酰胺、N-甲基吡咯烷酮、二甲基亚砜、正庚烷、正己烷、乙酸乙酯，优选为乙酸乙酯。
步骤四
通过式I-IM3化合物与
Figure PCTCN2022094540-appb-000057
发生脱水关环反应得到式I-TM1化合物；
在部分实施方案中，该步骤在适合的温度下进行，所述温度为20℃、25℃、50℃、60℃、100℃，优选为20℃；
在部分实施方案中，该步骤在适合的有机溶剂中进行，所述有机溶剂可选自乙腈、乙醇、四氢呋喃、二氯甲烷、N,N-二甲基甲酰胺、二甲基亚砜、正庚烷、正己烷、乙酸乙酯，优选为乙腈。
步骤五
由式I-TM1化合物发生水解反应得到式I-TM2化合物；
在部分实施方案中，所述反应在碱性条件下进行，所述碱性条件包括但不限于pH为7.0、7.4或8.0的PB缓冲液；
在部分实施方案中，所述反应在合适的温度下进行，所述温度为20℃、25℃、50℃、60℃、100℃，优选为20℃；
在部分实施方案中，所述反应在合适的溶剂下进行，所述溶剂包括但不限于乙腈、四氢呋喃、二氯甲烷、N,N-二甲基甲酰胺、二甲基亚砜，优选为乙腈。
当Y不存在时，本发明中的式I-TM3化合物可由下述合成路线进行合成制备。
Figure PCTCN2022094540-appb-000058
其中：
X、Z 1、W 1、J 1如前文通式中所定义；
L为发生取代反应的离去基团，包括但不限于卤素、三氟甲烷磺酸酯、对甲苯磺酸酯，优选为卤素、OTf。
步骤一
由式I-IM1化合物通过偶联反应得到式I-IM4化合物；
在部分实施方案中，所述偶联反应试剂包括但不限于甲基硼酸、三甲基环三硼氧烷，优选为三甲基环三硼氧烷；
在部分实施方案中，所述偶联反应在碱性条件中进行，所述碱包括但不限于三乙胺、DIPEA、NMM、叔丁醇钠、醋酸钾、醋酸钠、氟化铯、氟化钾、碳酸钾、碳酸钠、碳酸氢钠、碳酸铯、磷酸钾、磷酸二氢钾，优选为氟化铯。
在部分实施方案中，所述偶联反应在催化剂存在下进行，所述催化剂包括但不限于四三苯基膦钯、醋酸钯、Pd 2(dba) 3、Pd(PPh 3) 2Cl 2、Pd(PPh 3) 2Cl 2二氯甲烷络合物、Pd(dppf)Cl 2、Pd(Amphos)Cl 2，优选为四三苯基膦钯。
在部分实施方案中，所述偶联反应在温度为0-200℃的条件下进行，优选在温度为50-150℃的条件下进行。
在部分实施方案中，所述反应在合适的溶剂下进行，所述溶剂包括但不限于1,4-二氧六环、水、甲苯、四氢呋喃、N,N-二甲基甲酰胺、N-甲基吡咯烷酮、二甲基亚砜及其任意组合，优选为1,4-二氧六环。
步骤二
由式I-IM4化合物经卤代反应得到式I-TM3化合物；
在部分实施方案中，所述卤代试剂包括但不限于溴素、N-碘代丁二酰亚胺、N-溴代丁二酰亚胺、N-氯代丁二酰亚胺，优选为N-溴代丁二酰亚胺；
在部分实施方案中，所述偶联反应在催化剂存在下进行，所述催化剂为过氧苯甲酰。
在部分实施方案中，所述偶联反应在温度为0-200℃的条件下进行，优选在温度为50-150℃的条件下进行。
在部分实施方案中，所述反应在合适的溶剂下进行，所述溶剂包括但不限于卤代烃类(例如四氯化碳、二氯甲烷、氯仿、1,2-二氯乙烷等)、甲醇、乙醇、DMF、乙腈、醚类(例如乙二醇二甲醚、四氢呋喃、二氧六环)、芳烃类(例如甲苯、苯、二甲苯)、水及其任意组合，优选为四氯化碳。
当Z 2、W 2不存在时，本发明中的式II-TM1化合物可由下述合成路线进行合成制备。
Figure PCTCN2022094540-appb-000059
其中：
Y 1、Y 2、Y 3、B 1、J 2如前文通式中所定义；
LG为发生偶联反应的离去基团，包括但不限于卤素、三氟甲烷磺酸酯，优选为卤素。
步骤一
由式II-IM1化合物通过偶联反应得到式II-IM2化合物；
在部分实施方案中，所述偶联反应试剂为
Figure PCTCN2022094540-appb-000060
在部分实施方案中，所述偶联反应在催化剂存在下进行，所述催化剂包括但不限于四三苯基膦钯、醋酸钯、Pd 2(dba) 3、Pd(PPh 3) 2Cl 2、Pd(PPh 3) 2Cl 2二氯甲烷络合物、Pd(dppf)Cl 2、Pd(Amphos)Cl 2，优选为四三苯基膦钯。
在部分实施方案中，所述偶联反应在温度为0-200℃的条件下进行，优选在温度为50-150℃的条件下进行。
在部分实施方案中，所述反应在合适的溶剂下进行，所述溶剂包括但不限于1,4-二氧六环、水、甲苯、四氢呋喃、N,N-二甲基甲酰胺、N-甲基吡咯烷酮、二甲基亚砜及其任意组合，优选为1,4-二氧六环。
步骤二
由式II-IM2化合物经氧化反应得到式II-TM1化合物；
在部分实施方案中，所述偶联反应在氧化剂存在下进行，所述催化剂为间氯过氧苯甲酸。
在部分实施方案中，所述偶联反应在温度为0-120℃的条件下进行，优选在温度为50-80℃的条件下进行。
在部分实施方案中，所述反应在合适的溶剂下进行，所述溶剂包括但不限于卤代烃类(例如四氯化碳、二氯甲烷、氯仿、1,2-二氯乙烷等)、甲醇、乙醇、DMF、乙腈、醚类(例如乙二醇二甲醚、四氢呋喃、二氧六环)、芳烃类(例如甲苯、苯、二甲苯)、水及其任意组合，优选为甲醇。
另外，本发明的化合物还可以由有机合成领域的技术人员已知的多种方式制备。本发明的化合物可使用下文描述的方法以及合成有机化学领域中已知的合成方法或本领域技术人员所了解的其变化形式来合成。优选方法包括(但不限于)上文所述那些。反应可在适于所使用的试剂和材料且适合于实现转化的溶剂或溶剂混合物中进行。有机合成领域的技术人员应了解，分子上存在的官能团应与所提出的转化一致。这有时将需要以下判断：修改合成步骤的顺序或相对于一种方法路线选择另一特定方法路线以获得本发明的所需化合物。
还应认识到，本领域中设计任何合成途径的另一主要考虑因素是正确选择用于保护本发明中所述化合物中存在的反应性官能团的保护基团。向受过训练的相关人士描述许多替代方案的权威说明为Greene等人(Protective Groups in Organic Synthesis，第4版，Wiley-Interscience(2006))。
除非另外说明，上述路线中化合物的取代基如本发明所定义。本领域技术人员会明白， 根据期望获得的产物结构，可省略以上路线中的一个或多个步骤。本领域技术人员也可根据需要适当地调整反应步骤的顺序。
药物组合物
本发明还提供一种药物组合物，其含有本发明所述的生物活性偶联物，以及一种或多种药用上可接受的载体。
本文中所述药用辅料是指生产药品和调配处方时，使用的的赋形剂和附加剂，是指除活性成分外，在安全性方面已进行了合理的评估，并且包含在药物制剂中的物质。
所述药物组合物可以以任意形式施用，只要其实现预防、减轻、防止或者治愈人类或动物患者的症状。例如，可根据给药途径制成各种适宜的剂型。
本申请还提供一种药盒产品，其含有本发明所述的生物活性偶联物，或者所述药物组合物，以及任选的药品说明书。
治疗方法和用途
本申请的另一方面提供所述生物活性偶联物在制备用于预防或治疗肿瘤疾病的药物中的用途。
本申请的另一方面提供所述生物活性偶联物，其用于预防或治疗肿瘤疾病。
本申请的另一方面提供一种预防或治疗肿瘤疾病的方法，其包括向有此需要的受试者施用有效量的所述化生物活性偶联物、或包含所述生物活性偶联物的药物组合物。
在本发明的一个实施方案，所述肿瘤疾病为实体瘤或血液系统恶性肿瘤；例如选自结肠癌，胃癌，乳腺癌，肺癌(例如，非小细胞肺癌，具体如肺腺癌)，淋巴癌。
如本文中所使用的术语“有效量”指被给药后会在一定程度上缓解所治疗病症的一或多种症状的偶联物的量。
除非另外说明，否则如本文中所使用，术语“治疗”意指逆转、减轻所应用的病症或病况或者这样的病症或病况的一或多种症状的进展。
如本文所使用的“个体”或“受试者”包括人或非人动物。示例性人个体包括患有疾病(例如本文所述的疾病)的人个体(称为患者)或正常个体。本发明中“非人动物”包括所有脊椎动物，例如非哺乳动物(例如鸟类、两栖动物、爬行动物)和哺乳动物，例如非人灵长类、家畜和/或驯化动物(例如绵羊、犬、猫、奶牛、猪等)。
具体实施方式
以下列举实施例和试验例，进而详细地说明本发明，但它们不限制本发明的范围，另 外在不脱离本发明的范围下可进行变化。
1.本发明涉及的anti-ROR1抗体19F6_Hu35V1的制备。
前期通过免疫Balb/c，C57Bl/6，NZB和A/J小鼠，通过杂交瘤筛选获得鼠源抗体19F6，分别经人源化后获得人源化抗体序列19F6_Hu35v1(重链可变区,SEQ ID NO:1；轻链可变区,SEQ ID NO:2)，上述人源化抗体的重链恒定区均为人IgG1重链恒定区(SEQ ID NO:18)，轻链恒定区均为人kappa轻链恒定区(SEQ ID NO:19)。合成上述人源化抗体的编码DNA序列并进行密码子优化后，克隆到pcDNA3.4质粒中，将每一人源化抗体重链和轻链对应的pcDNA3.4质粒同时转染到Expi293F细胞中，采用蛋白A对上清中的表达抗体进行纯化，从而获得相应抗体。
19F6_Hu35v1的序列信息表：
Figure PCTCN2022094540-appb-000061
2.本发明涉及的药物-连接体合成实施例。
如本文中所使用的缩写具有以下含义：
Figure PCTCN2022094540-appb-000062
以下的实施例中记载的化合物的结构通过核磁共振( 1H NMR)或质谱(MS)来确定。
核磁共振( 1H NMR)的测定仪器使用Bruker 400MHz核磁共振仪；氘代氯仿(CDCl 3)；内标物质为四甲基硅烷(TMS)。
实施例中使用的核磁共振(NMR)图谱中的缩写示于以下。
s：单峰(singlet)、d：二重峰(doublet)、t：三重峰(triplet)、q：四重峰(quartet)、m：多重峰(multiplet)、br：宽峰(broad)、J：偶合常数、Hz：赫兹、CDCl 3：氘代氯仿。δ值用ppm值表示。
质谱(MS)的测定仪器使用Agilent(ESI)质谱仪，型号为Agilent 6120B。
化学合成实施例
实施例一 2-(2,3-双(溴甲基)-5,7-二氧基-5,7-二氢-6H-吡咯并[3,4-b]吡嗪-6-基)乙酸(I-1)
Figure PCTCN2022094540-appb-000063
步骤一：2-(3,4-二叠氮基-2,5-二氧基-2,5-二氢-1H-吡咯-1-基)乙酸叔丁酯的合成(1-2)
将化合物1-1(0.40g,1.08mmol,合成方法参考专利WO2019057964)和叠氮化钠(141.00mg,2.17mmol)溶于丙酮(10mL)中，25℃反应8h，用高效液相质谱联用色谱监测反应。向反应液中加水并用乙酸乙酯萃取，合并有机相用无水硫酸钠干燥，过滤，滤液经减压浓缩，得标题化合物粗品0.28g，未经纯化直接用于下一步反应。
结构表征数据如下：
ESI-MS(m/z):311.0[M+18] +.
步骤二：2-(3,4-二氨基-2,5-二氧基-2,5-二氢-1H-吡咯-1-基)乙酸叔丁酯的合成(1-3)
将化合物1-2(0.15g,0.51mmol)和10％钯碳(15.00mg)溶于乙醇(20mL)中，氢气置换三次，氢气环境下25℃反应3h，用高效液相质谱联用色谱监测反应。将反应液用硅藻土过滤，滤液经减压浓缩，得标题化合物粗品101.00mg，未经纯化直接用于下一步反应。
结构表征数据如下：
ESI-MS(m/z):185.9[M-56] +.
步骤三：2-(2,3-双(溴甲基)-5,7-二氧基-5,7-二氢-6H-吡咯并[3,4-b]吡嗪-6-基)乙酸叔丁酯的合成(1-5)
将化合物1-3(50.00mg,0.21mmol)和化合物1-4(54.00mg,0.21mmol)溶于乙腈(5mL)中，25℃反应3h，用高效液相质谱联用色谱监测反应。反应液直接用快速硅胶柱纯化(石油醚:乙酸乙酯＝5:1)，得标题化合物40.00mg。
结构表征数据如下：
ESI-MS(m/z):466.8[M+18] +.
1H NMR(400MHz,CDCl 3):δ4.88(s,4H),4.47(s,2H),1.48(s,9H).
步骤四：2-(2,3-双(溴甲基)-5,7-二氧基-5,7-二氢-6H-吡咯并[3,4-b]吡嗪-6-基)乙酸的合成(I-1)
将化合物1-5(20.00mg,0.05mmol)溶于二氯甲烷(5mL)和三氟乙酸(1mL)中，25℃反应5h，用高效液相质谱联用色谱监测反应。反应液直接经减压浓缩，得标题化合物粗品15.00mg，未经纯化直接用于下一步反应。
结构表征数据如下：
ESI-MS(m/z):410.8[M+18] +.
实施例二 2-(3,4-双(溴甲基)-2,5-二氧基-2,5-二氢-1H-吡咯-1-基)乙酸(I-2)
Figure PCTCN2022094540-appb-000064
步骤一：2-(3,4-二甲基-2,5-二氧基-2,5-二氢-1H-吡咯-1-基)乙酸叔丁酯的合成(2-1)
氮气保护下，将化合物1-1(0.20g,0.54mmol)、三甲基环三硼氧烷(0.34g,2.71mmol)、氟化铯(0.41g,2.71mmol)和四三苯基膦钯(63.00mg,0.05mmol)溶于1,4-二氧六环(20mL)中，110℃反应3h，用高效液相质谱联用色谱监测反应。反应液直接用快速硅胶柱纯化(石油醚:乙酸乙酯＝5:1)，得标题化合物80.00mg。
结构表征数据如下：
ESI-MS(m/z):184.0[M+H-56] +.
步骤二：2-(3,4-双(溴甲基)-2,5-二氧基-2,5-二氢-1H-吡咯-1-基)乙酸叔丁酯的合成(2-2)
氮气保护下，将化合物2-1(72.00mg,0.30mmol)、NBS(118.00mg,0.66mmol)和过氧化二苯甲酰(7.00mg,0.03mmol)溶于四氯化碳(8mL)中，85℃反应12h，用高效液相质谱联用色谱监测反应。反应液直接用快速硅胶柱纯化(石油醚:乙酸乙酯＝10:1)，得标题化合物65.00mg。
结构表征数据如下：
ESI-MS(m/z):414.8[M+18] +.
1H-NMR(400MHz,CDCl 3):δ4.28(s,4H),4.20(s,2H),1.45(s,9H).
步骤三：2-(3,4-双(溴甲基)-2,5-二氧基-2,5-二氢-1H-吡咯-1-基)乙酸的合成(I-2)
将化合物2-2(60.00mg,0.15mmol)溶于二氯甲烷(10mL)和三氟乙酸(2mL)中，25℃反应5h，用高效液相质谱联用色谱监测反应。反应液直接经减压浓缩，得标题化合物粗品50.00mg，未经纯化直接用于下一步反应。
结构表征数据如下：
ESI-MS(m/z):358.9[M+18] +.
实施例三 4-((S)-2-((S)-2-(2-(3,4-双(溴甲基)-2,5-二氧代-2,5-二氢-1H-吡咯-1-基)乙酰胺基)-3-甲基丁胺基)-5-脲基五酰胺基)苄基((S)-1-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((1S),2R)-1-羟基-1-苯基丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-氧代丙基)吡咯烷-1-基)-3-甲氧基-5-甲基-1-氧代庚-4-基)(甲基)氨基)-3-甲基-1-氧代丁烷-2-基)氨基)-3-甲基-1-氧代丁烷-2-基)(甲基)氨基甲酸酯(药物-连接体3)
Figure PCTCN2022094540-appb-000065
步骤一：4-((S)-2-((S)-2-(((9H-芴-9-基)甲氧基)羰基)氨基)-3-甲基丁胺基)-5-脲基戊酰胺基)苄基((S)-1-((S)-1-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((1S,2R)-1-羟基-1-苯基丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-氧代丙基)吡咯烷-1-基)-3-甲氧基-5-甲基-1-氧代庚-4-基)(甲基)氨基)-3-甲基-1-氧代丁烷-2-基)氨基)-3-甲基-1-氧代丁烷-2-基)(甲基)氨基甲酸酯的合成(3-2)
25℃下，将(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((1S,2R)-1-羟基-1-苯基丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-氧丙基)吡咯烷-1-基)-3-甲氧基-5-甲基-1-氧庚-4-基)-N,3-二甲基-2-((S)-3-甲基-2-(甲胺基)丁酰胺(30.00mg,0.04mmol)和化合物3-1(38.50mg,0.05mmol)溶于DMF(3mL)中，加入HOBt(8.46mg,0.06mmol)和DIPEA(10.80mg,0.08mmol)，保持25℃反应2h，用高效液相质谱联用色谱检测反应。反应液不经处理直接用于下一步反应。
结构表征数据如下：
ESI-MS(m/z):1345.2[M+H] +.
步骤二：4-((S)-2-((S)-2-氨基-3-甲基丁胺基)-5-脲基戊酰胺基)苄基((S)-1-((S)-1-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((1S,2R)-1-羟基-1-苯基丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-氧丙基)吡咯烷-1-基)-3-甲氧基-5-甲基-1-氧庚-4-基)(甲基)氨基)-3-甲基-1-氧代丁烷-2-基)氨基)-3-甲基-1-氧代丁烷-2-基(甲基)氨基甲酸酯的合成(3-3)
25℃下，将二乙胺(0.30mL)加入化合物3-2的反应液中，保持25℃反应2h，用高效液相质谱联用色谱监测反应。反应液经减压浓缩，浓缩物直接用制备高效液相色谱纯化，制备液冷冻干燥得标题化合物的甲酸盐36.30mg。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 30 70 28
18.00 90 10 28
结构表征数据如下：
ESI-MS(m/z):1123.2[M+H] +.
步骤三：4-((S)-2-((S)-2-(2-(3,4-双(溴甲基)-2,5-二氧代-2,5-二氢-1H-吡咯-1-基)乙酰胺基)-3-甲基丁胺基)-5-脲基五酰胺基)苄基((S)-1-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((1S),2R)-1-羟基-1-苯基丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-氧代丙基)吡咯烷-1-基)-3-甲氧基-5-甲基-1-氧代庚-4-基)(甲基)氨基)-3-甲基-1-氧代丁烷-2-基)氨基)-3-甲基-1-氧代丁烷-2-基)(甲基)氨基甲酸酯的合成(3)
将化合物I-2(12.75mg,0.04mmol)溶于二氯甲烷(3mL)中，加入DIC(2.36mg,0.02mmol)和化合物3-3(21.00mg,0.02mmol)，25℃反应1h，用高效液相质谱联用色谱监测反应。反应液经减压浓缩，浓缩物直接用制备高效液相色谱纯化，制备液冷冻干燥得标题化合物14.30mg。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 30 70 28
17.00 90 10 28
结构表征数据如下：
ESI-MS(m/z):1445.9[M+H] +.
实施例四 (S)-2-((2S,13S)-13-苄基-22-(3,4-双(溴甲基)-2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-2-异丙基-3-甲基-4,9,12,15,18,21-六氧代-6-氧杂-3,8,11,14,17,20-六氮杂二十二烷酰胺基)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-羟基-1-苯基丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-氧代丙烷)吡咯烷-1-基)-3-甲氧基-5-甲基-1-氧代庚烷-4-基)-N,3-二甲基丁酰胺(药物-连接体4)
Figure PCTCN2022094540-appb-000066
步骤一：(9H-芴-9-基)甲基((3R,4S,7S,10S,21S)-21-苄基-4-((S)-仲丁基)-3-(2-((S)-2-((1R,2R)-3-(((1S,2R)-1-羟基-1-苯基丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-氧丙基)吡咯烷-1-基)-2-氧乙基)-7,10-二异丙基-5,11-二甲基-6,9,12,17,20,23,26-七氧代-2,14-二氧杂-5,8,11,16,19,22,25-庚基庚二酰胺-27-基)氨基甲酸酯的合成(4-2)
称取化合物4-1(50.00mg,0.08mmol)和(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-羟基-1-苯基丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-氧丙烷)吡咯烷-1-基)-3-甲氧基-5-甲基-1-氧庚烷-4- 基)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰氨基)丁酰胺(55.60mg,0.08mmol)溶于DMF(1mL)中，然后加入HATU(32.37mg,85.18μmol)和DIPEA(20.02mg,154.88μmol)，加毕，室温反应1h，用高效液相质谱联用色谱监测反应。反应液直接用制备高效液相色谱纯化得标题化合物35.00mg。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 40 60 28
2.00 40 60 28
18.00 90 10 28
结构表征数据如下：
ESI-MS(m/z):1345.1[M+H] +.
步骤二：(S)-2-((2S,13S)-19-氨基-13-苄基-2-异丙基-3-甲基-4,9,12,15,18-五氧代-6-氧杂-3,8,11,14,17-庚烷氮酮)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-羟基-1-苯基丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-氧丙基)吡咯烷-1-基)-3-甲氧基-5-甲基-1-氧代庚烷-4-基)-N,3-二甲基丁酰胺的合成(4-3)
将化合物4-2(20.00mg,0.02mmol)溶于二氯甲烷(2mL)中，然后加入二乙胺(1mL)，加毕，室温反应1h，用高效液相质谱联用色谱监测反应。反应液经减压浓缩，浓缩物直接用制备高效液相色谱纯化，制备液冷冻干燥得标题化合物的甲酸盐15.00mg。
色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 10 90 28
2.00 10 90 28
18.00 90 10 28
结构表征数据如下：
ESI-MS(m/z):1123.1[M+H] +.
步骤三：(S)-2-((2S,13S)-13-苄基-22-(3,4-双(溴甲基)-2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-2-异丙基-3-甲基-4,9,12,15,18,21-六氧代-6-氧杂-3,8,11,14,17,20-六氮杂二十二烷酰胺 基)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-羟基-1-苯基丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-氧代丙烷)吡咯烷-1-基)-3-甲氧基-5-甲基-1-氧代庚烷-4-基)-N,3-二甲基丁酰胺的合成(4)
将化合物I-2(4.00mg,12.0μmol)溶于二氯甲烷(3mL)中，加入DIC(1.12mg,8.00μmol)和化合物4-3(6.58mg,6.00μmol)，25℃反应1h，用高效液相质谱联用色谱监测反应。反应液经减压浓缩，浓缩物直接用制备高效液相色谱纯化，制备液冷冻干燥得标题化合物4.77mg。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 35 65 28
18.00 90 10 28
结构表征数据如下：
ESI-MS(m/z):1445.9[M+H] +.
实施例五 N-(((3R,4S,7S,10S,21S)-21-苄基-4-((S)-仲丁基)-3-(2-((S)-2-((1R,2R)-3-((((1S,2R)-1-羟基-1-苯丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-氧丙基)吡咯烷-1-基)-2-氧乙基)-7,10-二异丙基-5,11-二甲基-6,9,12,17,20,23,26-七氧代-2,14-二氧杂-5,8,11,16,19,22,25-七氮杂二十七烷-27-基)-1-(2-(3,4-双(溴甲基)-2,5-二氧杂-2-，5-二氢-1H-吡咯-1-基)乙酰氨基)-3,6,9,12-四氧十五烷十五酰胺(药物-连接体5)
Figure PCTCN2022094540-appb-000067
步骤一：(9H-芴-9-基)甲基((3R,4S,7S,10S,21S)-21-苄基-4-((S)-仲丁基)-3-(2-((S)-2–(((1R,2R)-3-(((1S,2R)-1-羟基-1-苯基丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-氧丙基)吡咯烷-1-基)-2-氧代乙基)-7,10-二异丙基-5,11-二甲基-6,9,12,17,20,23,26,29-八氧代-2,14,32,35,38,41-六氧杂-5,8,11,16,19,22,25,28-八氮杂二十五烷-43-基)氨基甲酸酯的合成(5-2)
称取化合物5-1(5.21mg,11.00μmol)和4-3(10.00mg,9.00μmol)溶于DMF(1mL)中，然后加入HATU(4.06mg,11.00μmol)和DIPEA(2.30mg,18.00μmol)，加毕，室温反应1h，用高效液相质谱联用色谱监测反应。反应液直接用制备高效液相色谱纯化得标题化合物9.00mg。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 20 80 28
16.00 90 10 28
结构表征数据如下：
ESI-MS(m/z):1592.8[M+H] +.
步骤二：1-氨基-N-(((3R,4S,7S,10S,21S)-21-苄基-4-((S)-仲丁 基)-3-(2-((S)-2-((1R,2R)-3-(((((S,2R)-1-羟基-1-苯丙烷基-2-基)氨基)-1-甲氧基-2-甲基-3-氧丙基)吡咯烷-1-基)-2-氧乙基)-7,10-二异丙基-5,11-二甲基-6,9,12,17,20,23,26-七氧代-2,14-二氧杂-5,8,11,16,19,22,25-七氮杂二十七烷-27-基)-3,6,9,12-四氧杂十五烷-15-酰胺的合成(5-3)
将化合物5-2(10.00mg,6.00μmol)溶于二氯甲烷(2mL)中，然后加入二乙胺(1mL)，加毕，室温反应1h，用高效液相质谱联用色谱监测反应。反应液经减压浓缩，浓缩物直接用制备高效液相色谱纯化，制备液冷冻干燥得标题化合物的甲酸盐7.00mg。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 10 90 28
20.00 90 10 28
结构表征数据如下：
ESI-MS(m/z):1370.9[M+H] +.
步骤三：N-(((3R,4S,7S,10S,21S)-21-苄基-4-((S)-仲丁基)-3-(2-((S)-2-((1R,2R)-3-((((1S,2R)-1-羟基-1-苯丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-氧丙基)吡咯烷-1-基)-2-氧乙基)-7,10-二异丙基-5,11-二甲基-6,9,12,17,20,23,26-七氧代-2,14-二氧杂-5,8,11,16,19,22,25-七氮杂二十七烷-27-基)-1-(2-(3,4-双(溴甲基)-2,5-二氧杂-2-，5-二氢-1H-吡咯-1-基)乙酰氨基)-3,6,9,12-四氧十五烷十五酰胺的合成(5)
将化合物I-2(4.00mg,12.00μmol)溶于二氯甲烷(2mL)中，加入DIC(1.12mg,8.00μmol)和化合物5-3(8.04mg,6.00μmol)，25℃反应1h，用高效液相质谱联用色谱监测反应。反应液经减压浓缩，浓缩物直接用制备高效液相色谱纯化，制备液冷冻干燥得标题化合物4.68mg。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 35 65 28
18.00 90 10 28
结构表征数据如下：
ESI-MS(m/z):1693.7[M+H] +.
实施例六 (S)-2-((2S,13S)-13-苄基-22-(2,3-双(溴甲基)-5,7-二氧代-5,7-二氢-6H-吡咯并[3,4-b]吡嗪-6-基)-2-异丙基-3-甲基-4,9,12,15,18,21-六氧代-6-氧杂-3,8,11,14,17,20-六氮杂二十二烷酰胺)-N–(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-羟基-1-苯基丙烷-2-基)氨基))-1-甲氧基-2-甲基-3-氧丙基)吡咯烷-1-基)-3-甲氧基-5-甲基-1-氧庚基-4-基)-N,3-二甲基丁酰胺(药物-连接体6)
Figure PCTCN2022094540-appb-000068
将化合物I-1(6.03mg,15.00μmol)溶于二氯甲烷(2mL)中，加入DIC(1.45mg,12.00μmol)和化合物4-3(8.62mg,8.00μmol)，25℃反应1h，用高效液相质谱联用色谱监测反应。反应液经减压浓缩，浓缩物直接用制备高效液相色谱纯化，制备液冷冻干燥得标题化合物4.21mg。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 30 70 28
18.00 90 10 28
结构表征数据如下：
ESI-MS(m/z):1498.5[M+H] +.
实施例七 N-(((3R,4S,7S,10S,21S)-21-苄基-4-((S)-仲丁基)-3-(2-((S)-2-((1R,2R)-3-((((1S,2R)-1-羟基-1-苯丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-氧代丙基)吡咯烷-1-基)-2-氧代乙基)-7,10-二异丙基-5,11-二甲基-6,9,12,17,20,23,26-七氧代-2,14-二氧杂-5,8,11,16,19,22,25-七氮杂二十七烷-27–基)-1-(2-(2,3-双(溴甲基)-5,7-二氧代-5,7-二氢-6H-吡咯并[3,4-b]吡嗪-6-基)乙酰氨基)-3,6,9,12-四氧杂十五烷-15-酰胺(药物-连接体7)
Figure PCTCN2022094540-appb-000069
将化合物I-1(12.06mg,0.03mmol)溶于二氯甲烷(4mL)中，加入DIC(3.00mg,0.02mmol)和化合物5-3(20.00mg,15.00μmol)，25℃反应1h，用高效液相质谱联用色谱监测反应。反应液经减压浓缩，浓缩物直接用制备高效液相色谱纯化，制备液冷冻干燥得标题化合物12.00mg。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 30 70 28
2.00 30 70 28
18.00 90 10 28
结构表征数据如下：
ESI-MS(m/z):1745.8[M+H] +.
实施例八 4-((2S,5S)-41-(3,4-二(溴甲基)-2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-5-异丙基-2-甲基-4,7,11,40-四氧代-9,15,18,21,24,27,30,33,36-九氧杂-3,6,12,39-四氮杂十五酰胺基)苯基
((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-羟基-1-苯丙烷-2-基)氨基)-1-甲氧基-2-甲-3-氧代丙烷)吡咯烷-1-基)-3-甲氧基-5-甲基-1-氧代庚烷-4-基)(甲基)氨基)-3甲基-1-氧代丁烷-2-基)氨基)-3-甲基-1-氧代丁烷-2-基)(甲基)碳酸酯(药物-连接体8)
Figure PCTCN2022094540-appb-000070
步骤一：(S)-2-(32-叠氮-5-氧代-3,9,12,15,18,21,24,27,30-九氧杂-6-氮杂三十四酰胺)-N-((S)-1-((4-(羟甲基)苯基)氨基)-1-氧代丙烷-2-基)-3-甲基丁胺的合成(8-3)
将化合物8-2(3.60g,12.27mmol)溶于二氯甲烷(30mL)中，加入化合物8-1(7.49g,13.50mmol)和EEDQ(6.07g,24.54mmol)，25℃反应4小时。将反应液减压浓缩，浓缩物经反相C18柱分离(70％乙腈/0.1％甲酸水溶液)，得标题化合物8-3(7.80g)。
结构表征数据如下：
ESI-MS(m/z):830.4[M+H] +.
步骤二：4-((2S,5S)-38-叠氮-5-异丙基-2-甲基-4,7,11-三氧代-9,15,18,21,24,27,30,33,36-九氧杂-3,6,12-三氮杂三十八烷酰胺基)苄基(4-硝基苯基)碳酸酯的合成(8-4)
将化合物8-3(2.50g,3.01mmol)溶于二氯甲烷(20mL)中，加入二(对硝基苯)碳酸酯(3.66g,12.05mmol)和DIPEA(1.56g,12.05mmol)，25℃反应4小时。将反应液直接经硅胶柱(乙酸乙酯-二氯甲烷:甲醇＝84:16)纯化，得标题化合物8-4(2.07g)。
结构表征数据如下：
ESI-MS(m/z):995.4[M+H] +.
步骤三：4-((2S,5S)-38-叠氮基-5-异丙基-2-甲基-4,7,11-三氧代-9,15,18,21,24,27,30,33,36-九氧杂-3,6,12-三氮杂三十八烷酰胺基)苄基((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3)-(((1S,2R)-1-羟基-1-苯基丙-2-基)氨基)-1-甲氧基-2-甲基-3-氧代丙基)吡咯烷-1-基)-3-甲氧基-5-甲基-1-氧代庚烷-4-基)(甲基)氨基)-3-甲基-1-氧代丁烷-2-基)氨基)-3-甲基-1-氧代丁烷-2-基)(甲基)碳酸酯的合成(8-5)
将(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-((1S,2R)-1-羟基-1-苯基丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-氧丙基)吡咯烷-1-基)-3-甲氧基-5-甲基-1-氧庚-4-基)-N,3-二甲基-2-((S)-3-甲基-2-(甲胺基)丁酰胺(100mg,139.28μmol)、化合物8-4(180.17mg,181.06μmol)和HOBt(56.46mg,417.84μmol)用DMF(3mL)溶解，加入DIPEA(54.00mg,417.84μmol)，25℃反应6小时。将反应液减压浓缩，浓缩物经反相C18柱分离(55％乙腈/0.1％甲酸水溶液)，得标题化合物8-5(60.00mg)。
结构表征数据如下：
ESI-MS(m/z):1573.9[M+H] +.
步骤四：4-((2S,5S)-38-氨基-5-异丙基-2-甲基-4,7,11-三氧代-9,15,18,21,24,27,30,33,36-九氧杂-3,6,12-三氮杂三十八烷酰胺基)苄基((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3)-(((1S,2R)-1-羟基-1-苯基丙-2-基)氨基)-1-甲氧基-2-甲基-3-氧代丙基)吡咯烷-1-基)-3-甲氧基-5-甲基-1-氧代庚烷-4-基)(甲基)氨基)-3-甲基-1-氧代丁烷-2-基)氨基)-3-甲基-1-氧代丁烷-2-基)(甲基)碳酸酯的合成(8-6)
将化合物8-5(0.60g,381.22μmol)溶于THF(10mL)中，加入三苯基磷(232.02mg,762.44μmol)，再加入水(10mL)，50℃反应6小时。加水并用二氯甲烷萃取，无水硫酸钠干燥，过滤，滤液经减压浓缩。经硅胶柱(二氯甲烷:甲醇＝84:16)纯化，得标题化合物8-6(50.00mg)。
结构表征数据如下：
ESI-MS(m/z):1548.9[M+H] +.
步骤五：4-((2S,5S)-41-(3,4-二(溴甲基)-2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-5-异丙基-2-甲基-4,7,11,40-四氧代-9,15,18,21,24,27,30,33,36-九氧杂-3,6,12,39-四氮杂十五酰胺基)苯基((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-羟基-1-苯丙烷-2-基)氨基)-1-甲氧基-2-甲-3-氧代丙烷)吡咯烷-1-基)-3-甲氧基-5-甲基-1-氧代庚烷-4-基)(甲基)氨基)-3甲基-1-氧代丁烷-2-基)氨基)-3-甲基-1-氧代丁烷-2-基)(甲基)碳酸酯的合成(8)
将化合物I-2(10.00mg,0.03mmol)溶于二氯甲烷(4mL)中，加入DIC(3.70mg,0.03mmol)和化合物8-6(23.37mg,15.00μmol)，25℃反应1h，用高效液相质谱联用色谱监测反应。反应液经减压浓缩，浓缩物直接用制备高效液相色谱纯化，制备液冷冻干燥得标题化合物8(12.88mg)。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 35 65 28
2.00 35 65 28
18.00 95 5 28
结构表征数据如下：
ESI-MS(m/z):1871.8[M+H] +.
实施例九 2-(2-(2-(2-(2,3-双(溴甲基)-5,7-二氧代-5,7-二氢-6H-吡咯[3,4-b]吡嗪-6-基)乙氧基)乙氧基)乙酸(I-3)
Figure PCTCN2022094540-appb-000071
步骤一：2-(2-(2-(2-(3,4-二溴-2,5-二氧代-2,5-二氢-1H-吡咯-1-基)乙氧基)乙氧 基)乙酸叔丁酯的合成(9-2)
0℃下，将化合物9-1(523.00mg,1.98mmol)和三苯基膦(518.97mg,1.98mmol)用四氢呋喃(8mL)溶解，加入DIAD(400.11mg,1.98mmol,389.59μL)后搅拌5分钟，加入3,4-二溴吡咯-2,5-二酮(504.30mg,1.98mmol)，0℃反应3小时。加入饱和氯化铵水溶液并用乙酸乙酯萃取，无水硫酸钠干燥，过滤，滤液经减压浓缩。浓缩物经硅胶柱层析分离(石油醚:乙酸乙酯＝1:1)，得标题化合物9-2(0.90g)。
结构表征数据如下：
ESI-MS(m/z):519.0[M+18] +.
步骤二：2-(2-(2-(2-(3,4-二叠氮基-2,5-二氧代-2,5-二氢-1H-吡咯-1-基)乙氧基)乙氧基)乙酸叔丁酯的合成(9-3)
将化合物9-2(0.20g,399.07μmol)用丙酮(8mL)溶解，加入叠氮化钠(51.89mg,798.14μmol)，25℃反应8小时。加水并用乙酸乙酯萃取，无水硫酸钠干燥，过滤，滤液经减压浓缩，得标题化合物9-3的粗品(160.00mg)。
结构表征数据如下：
ESI-MS(m/z):443.1[M+18] +.
步骤三：2-(2-(2-(2-(3,4-二氨基-2,5-二氧代-2,5-二氢-1H-吡咯-1-基)乙氧基)乙氧基)乙酸叔丁酯的合成(9-4)
将化合物9-3(170.00mg,399.63μmol)和10％钯碳(85.00mg)用乙醇(34mL)溶解，25℃下氢气氛围反应8小时。将反应液过滤，减压抽干滤液，得标题化合物9-4的粗品(140.00mg)。
结构表征数据如下：
ESI-MS(m/z):391.3[M+18] +.
步骤四：2-(2-(2-(2-(2,3-双(溴甲基)-5,7-二氧代-5,7-二氢-6H-吡咯[3,4-b]吡嗪-6-基)乙氧基)乙氧基)乙酸的合成(I-3)
将化合物9-4(70.00mg,187.47μmol)和化合物1-4(45.72mg,187.47μmol)用乙腈(5mL)溶解，25℃反应2小时。减压抽干反应溶剂，浓缩物经硅胶柱层析分离(石油醚:乙酸乙酯＝1:1)，得标题化合物I-3(26.00mg)。
结构表征数据如下：
ESI-MS(m/z):391.3[M+18] +.
实施例十 4-((2S,5S)-17-(2,3-二(溴甲基)-5,7-二氧代-5,7-二氢-6H-吡咯[3,4-b]吡嗪-6-基)-5-异丙基-4,7-二氧代-2-(3-脲基丙基)-9,12,15-三氧杂-3,6-二氮杂十七烷酰胺基)苄基 ((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-羟基-1-苯丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-氧代丙基)吡咯烷-1-基)-3-甲氧基-5-甲基-1-氧代庚烷-4-基)(甲基)氨基)-3-甲基-1-氧代丁烷-2-基)氨基)-3-甲基-1-氧代丁烷-2-基)(甲基)碳酸酯(药物-连接体10)
Figure PCTCN2022094540-appb-000072
将化合物I-3(13.47mg,25.65μmol)用二氯甲烷(4mL)溶解，加入DIC(2.43mg,19.24μmol,2.98μL)，搅拌20min后加入化合物10-1的甲酸盐(15.00mg,12.83μmol)，20℃反应2小时。减压抽干溶剂，浓缩物直接用制备高效液相色谱纯化，制备液冷冻干燥得标题化合物10(9.00mg)。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 35 65 28
2.00 35 65 28
16.00 90 10 28
结构表征数据如下：
ESI-MS(m/z):1630.4[M+H] +.
实施例十一 3,5-二(2-(甲磺酰)嘧啶-4-基)苯甲酸(II-19)
Figure PCTCN2022094540-appb-000073
步骤一：3,5-二(2-(甲硫基)嘧啶-4-基)苯甲酸甲酯的合成(11-3)
将3,5-二溴苯甲酸甲酯(1.00g,3.40mmol)，4-三丁基甲锡烷基-2-硫代甲基嘧啶(3.11g,7.48mmol)和四(三苯基膦)钯(393.00mg,0.034mmol)溶于1,4-二氧六环(10mL)，氮气置换后微波110℃反应6小时。将反应液减压浓缩，浓缩物经硅胶柱纯化(石油醚:乙酸乙酯＝5:1)，得化合物11-3(398.00mg)。
结构表征数据如下：
ESI-MS(m/z):385.0[M+H] +.
步骤二：3,5-二(2-(甲硫基)嘧啶-4-基)苯甲酸的合成(11-4)
将化合物11-3(398.00mg,1.04mmol)溶于甲醇(5mL)、四氢呋喃(5mL)和水(1mL)中，加入氢氧化钠(166.00mg,4.14mmol)，搅拌反应1小时。滴入3N盐酸水溶液中和反应，减压浓缩后加水搅拌，过滤，固体水洗后真空干燥得标题化合物11-4(0.38g)。
步骤三：3,5-二(2-(甲磺酰)嘧啶-4-基)苯甲酸的合成(II-19)
将化合物11-4(0.38g,1.03mmol)溶于甲醇(20mL)，搅拌下加入间氯过氧苯甲酸(1.25g，80％，6.15mmol)，升至60℃反应4小时。氮气吹干溶剂，固体溶于二氯甲烷后直接经硅胶柱纯化(二氯甲烷:甲醇＝10:1)，得标题化合物II-19(0.25g)。
结构表征数据如下：
ESI-MS(m/z):452.0[M+18] +.
实施例十二 4-((31S,34S)-1-(3,5-双(2-(甲磺酰基)嘧啶-4-基)苯基)-31-异丙基-1,29,32-三氧代-34-(3-脲基丙基))-5,8,11,14,17,20,23,26-八氧杂-2,30,33-三氮杂五十四烷-35-酰胺基)苄基((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-羟基-1-苯丙烷 -2-基)氨基)-1-甲氧基-2-甲基-3-氧代丙基)吡咯烷-1-基)-3-甲氧基-5-甲基-1-氧代庚烷-4-基)(甲基)氨基)-3-甲基-1-氧代丁烷-2-基)氨基)-3-甲基-1-氧代丁烷-2-基)(甲基)碳酸酯(药物-连接体12)
Figure PCTCN2022094540-appb-000074
步骤一：4-((34S,37S)-34-异丙基-2,2-二甲基-4,32,35-三氧代-37-(3-脲基丙基)-3,8,11,14,17,20,23,26,29-九氧杂-5,33,36-三氮杂十八烷-38-酰胺基)苄基((S)-1-(((S)-1-((((3R,4S,5S)-1-((S)-)-2-((1R,2R)-3-(((1S,2R)-1-羟基-1-苯基丙-2-基)氨基)-1-甲氧基-2-甲基-3-氧代丙基)吡咯烷-1-基)-3-甲氧基-5-甲基-1-氧代庚烷-4-基)(甲基)氨基)-3-甲基-1-氧代丁烷-2-基)氨基)-3-甲基-1-氧代丁烷-2-基)(甲基)氨基甲酸酯的合成(12-2)
将化合物3-3的甲酸盐(200.00mg,171.02μmol)、HATU(91.04mg,239.43μmol)和化合物12-1(120.42mg,222.33μmol)溶于DMF(6mL)中，然后加入DIPEA(66.31mg,513.06μmol)，加毕室温反应0.5小时。减压抽干溶剂，浓缩物直接用制备高效液相色谱纯化，制备液冷冻干燥得标题化合物12-2(210.00mg)。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 30 70 28
2.00 30 70 28
18.00 90 10 28
结构表征数据如下：
ESI-MS(m/z):824.0[(M+H)/2] +.
步骤二：4-((29S,32S)-1-氨基-29-异丙基-27,30-二氧代-32-(3-脲基丙基)-3,6,9,12,15,18,21,24-八氧杂-28,31-二氮杂三十四烷-33-酰胺基)苄基((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-羟基-1-苯基丙-2-基)氨基)-1-甲氧基-2-甲基-3-氧丙基)吡咯烷-1-基)-3-甲氧基-5–甲基-1-氧代丁烷-4-基)(甲基)氨基)-3-甲基-1-氧代丁烷-2-基)氨基)-3-甲基-1-氧代丁烷-2-基)(甲基)碳酸酯的合成(12-3)
将化合物12-2(140.00mg,85.00μmol)用三氟乙酸(0.5mL)和二氯甲烷(5mL)溶解，0℃反应2小时。减压抽干溶剂，得标题化合物12-3的甲酸盐粗品73.00mg。
结构表征数据如下：
ESI-MS(m/z):774.1[(M+H)/2] +.
步骤三：4-((31S,34S)-1-(3,5-双(2-(甲磺酰基)嘧啶-4-基)苯基)-31-异丙基-1,29,32-三氧代-34-(3-脲基丙基))-5,8,11,14,17,20,23,26-八氧杂-2,30,33-三氮杂五十四烷-35-酰胺基)苄基((S)-1-(((S)-1-(((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-羟基-1-苯丙烷-2-基)氨基)-1-甲氧基-2-甲基-3-氧代丙基)吡咯烷-1-基)-3-甲氧基-5-甲基-1-氧代庚烷-4-基)(甲基)氨基)-3-甲基-1-氧代丁烷-2-基)氨基)-3-甲基-1-氧代丁烷-2-基)(甲基)碳酸酯的合成(12)
将化合物II-19(5.45mg,12.56μmol)、HATU(14.32mg,37.67μmol)和化合物12-3的甲酸盐(20.00mg,12.56μmol)溶于DMF(2mL)中，然后加入DIPEA(8.11mg,62.78μmol,11.18μL)，加毕室温反应2小时。减压抽干溶剂。浓缩物直接用制备高效液相色谱纯化，制备液冷冻干燥得标题化合物12(6.50mg)。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 40 60 28
2.00 40 60 28
18.00 80 20 28
结构表征数据如下：
ESI-MS(m/z):982.5[(M+H)/2] +.
实施例十三 N-((S)-10-苄基-1-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧基-1,2,3,9,10,12,13,15-八氢苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六烷-16-基)-1-(2-(2,3-二(溴甲基)-5,7-二氧代-5H-吡咯并[3,4-b]吡嗪-6(7H)-基)乙酰胺)-3,6,9,12-四氧杂十五烷酰胺(药物-连接体13)
Figure PCTCN2022094540-appb-000075
步骤一：(9H-氟-9-基)甲基((S)-10-苄基-1-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-1,2,3,9,10,12,13,15-八氢苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六烷-16-基))氨基甲酸酯的合成(13-3)
将化合物13-1(1.00g,1.55mmol)溶于DMF(5mL)中，依次加入HATU(647.40mg,1.70mmol)、化合物依喜替康13-2的甲磺酸盐(1.00g,1.55mmol)和DIPEA(400.34mg,3.10mmol)，25℃反应2小时。减压抽干反应溶剂，浓缩物直接经高效液相制备，冷冻干燥得标题化合物13-3(1.05g)。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 30 70 28
18.00 90 10 28
结构表征数据如下：
ESI-MS(m/z):1063.4[M+H] +.
步骤二：(S)-2-(2-氨基乙酰胺基)乙酰胺基)-N-(2-((2-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代码-1,2,3,9,10,12,13,15-八氢苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-2-氧代乙氧基)甲基)-2-氧代-3-苯丙酰胺的合成(13-4)
将化合物13-3(1.05g,987.69μmol)溶于二氯甲烷(100mL)中，加入二乙胺(20mL)，25℃反应1小时。减压抽干反应溶剂，浓缩物直接经高效液相制备，冷冻干燥得标题化合物13-4的甲酸盐(285.00mg)。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 30 70 28
18.00 90 10 28
结构表征数据如下：
ESI-MS(m/z):841.2[M+H] +.
步骤三：(9H-芴-9-基)甲基((S)-10-苄基-1-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧基-1,2,3,9,10,12,15-八氢苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,9,15,18-六氧代-3,21,24,27,30-五氧杂-5,8,11,14,17-五氮杂三甲酸酯的合成(13-6)
将化合物13-4的甲酸盐(83.00mg,98.71μmol)和HATU(45.04mg,118.45μmol)、化合物13-5(57.75mg,118.45μmol)溶于DMF(2mL)中，然后加入DIPEA(25.51mg,197.42μmol,35.14μL)，加毕室温反应0.5小时。减压抽干反应溶剂，浓缩物直 接经高效液相制备，冷冻干燥得标题化合物13-6(30.00mg)。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 30 70 28
16.00 90 10 28
结构表征数据如下：
ESI-MS(m/z):1310.5[M+H] +.
步骤四：1-氨基-N-((S)-10-苄基-1-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-1,2,3,9,10,12,13,15-八氢苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六烷-16-基)-3,6,9,12-四氧杂-15-癸烷酰胺的合成(13-7)
将化合物13-6(30.00mg,22.89μmol)溶于二乙胺(1mL)和DMF(2mL)中，室温反应1小时。反应液溶剂经减压抽干，浓缩物直接经高效液相制备，冷冻干燥得标题化合物13-7的甲酸盐(16.00mg)。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 5 95 28
18.00 90 10 28
结构表征数据如下：
ESI-MS(m/z):1088.4[M+H] +.
步骤五：N-((S)-10-苄基-1-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧基-1,2,3,9,10,12,13,15-八氢苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六烷-16-基)-1-(2-(2,3-二(溴甲基)-5,7-二氧代-5H-吡咯并[3,4-b]吡嗪-6(7H)-基)乙酰胺)-3,6,9,12-四氧杂十五烷酰胺的合成(13)
将化合物13-7的甲酸盐(16.00mg,14.70μmol)、化合物I-3(11.56mg,29.41μmol)和DIC(2.60mg,20.59μmol,3.19μL)用二氯甲烷(4mL)溶解，20℃反应1小时。减压抽干反应溶剂，浓缩物直接经高效液相制备，冷冻干燥得标题化合物13(5.33mg)。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 30 70 28
3.00 30 70 28
18.00 95 5 28
结构表征数据如下：
ESI-MS(m/z):1463.3[M+H] +.
实施例十四 (S)-2-(17-(2,3-二(溴甲基)-5,7-二氧代-5H-吡咯并[3,4-b]吡嗪-6(7H)-基)-4,7-二氧代-9,12,15-三氧杂-3,6-二氮七烷酰胺)-N-(2-((2-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代)-1,2,3,9,10,12,13,15-八氢苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-2-氧乙氧基(甲基)氨基)-2-氧乙基)-3-苯丙酰胺(药物-连接体14)
Figure PCTCN2022094540-appb-000076
将化合物I-3(40.26mg,76.67μmol)用二氯甲烷(5mL)溶解，加入DIC(9.68mg,76.67μmol)，搅拌20分钟后加入化合物13-4的甲酸盐(34.00mg,38.34μmol)，20℃反应2小时。减压抽干反应溶剂，浓缩物直接经高效液相制备，冷冻干燥得标题化合物14(5.33mg)。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 30 70 28
2.00 30 70 28
18.00 90 10 28
结构表征数据如下：
ESI-MS(m/z):1348.3[M+H] +.
实施例十五 1-(3,5-二(2-(甲磺酰基)嘧啶-4-基)苯基)-1-氧代-5,8,11-三氧杂-2-氮杂十三烷-13-油酸(15)
Figure PCTCN2022094540-appb-000077
步骤一：1-(3,5-二(2-(甲磺酰基)嘧啶-4-基)苯基)-1-氧代-5,8,11-三氧杂-2-氮杂十三烷-13-油酸叔丁酯的合成(15-2)
将化合物II-9(50.00mg,115.09μmol)用DMF(2mL)溶解，加入HATU(65.60mg,172.53μmol)，搅拌后加入化合物15-1(36.37mg,138.11μmol)和DIPEA(44.62mg,345.27μmol,61.46μL)，25℃反应2小时。减压抽干反应溶剂，浓缩物直接经高效液相制备，冷冻干燥得标题化合物15-2(25.00mg)。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 30 70 28
2.00 30 70 28
18.00 90 10 28
结构表征数据如下：
ESI-MS(m/z):697.2[M+18] +.
步骤二：1-(3,5-二(2-(甲磺酰基)嘧啶-4-基)苯基)-1-氧代-5,8,11-三氧杂-2-氮杂十三烷-13-油酸的合成(15)
将化合物15-2(20.00mg,29.42μmol)用三氟乙酸(1mL)和二氯甲烷(5mL)溶解，25℃反应2小时。减压抽干反应溶剂，浓缩物得标题化合物15的粗品(15.00mg)，未经纯化直接进行下一步。
结构表征数据如下：
ESI-MS(m/z):641.1[M+18] +.
实施例十六 N-((S)-10-苄基-1-((1R,9R)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧 代-1,2,3,9,10,12,13,15-八氢苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15,18,46-七氧代-3,21,24,27,30,33,36,39,42,48,51,54-十二烷氧杂-5,8,11,14,17,45-六氮杂六戊烷-56-基)-3,5-双(2-(甲磺酰)嘧啶-4-基)苯甲酰胺(药物-连接体16)
Figure PCTCN2022094540-appb-000078
步骤一：(9H-氟-9-基)甲基((S)-10-苄基-1-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-1,2,3,9,10,12,13,15-八氢苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15,18-六氧代-3,21,24,27,30,33,36,39,42-壬氧杂-5,8,11,14,17-五氮杂十四烷-44-基)氨基甲酸酯的合成(16-2)
将化合物13-4的甲磺酸盐(132.00mg,148.84μmol)用DMF(5mL)溶解，加入HATU(90.55mg,238.14μmol)、化合物16-1(148.19mg,223.26μmol)和DIPEA(96.18mg,744.19μmol,132.48μL)，25℃反应2小时。减压抽干反应溶剂，浓缩物直接经高效液相制备，冷冻干燥得标题化合物16-2(110.00mg)。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 20 80 28
3.00 20 80 28
17.00 90 10 28
结构表征数据如下：
ESI-MS(m/z):1486.5[M+1] +.
步骤二：1-氨基-N-((S)-10-苄基-1-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-1,2,3,9,10,12,13,15-八氢苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮十六烷-16-基)-3,6,9,12,15,18,21,2-八氧杂庚烷-27-酰胺的合成(16-3)
将化合物16-2(110.00mg,74.00μmol,FR)用二乙胺(1mL)和二氯甲烷(5mL)溶解，25℃反应2小时。减压抽干反应溶剂，浓缩物直接经高效液相制备，冷冻干燥得标题化合物16-3的甲酸盐(40.00mg)。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 10 90 28
2.00 10 90 28
18.00 90 10 28
结构表征数据如下：
ESI-MS(m/z):1264.6[M+1] +.
步骤三：N-((S)-10-苄基-1-((1R,9R)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-1,2,3,9,10,12,13,15-八氢苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15,18,46-七氧代-3,21,24,27,30,33,36,39,42,48,51,54-十二烷氧杂-5,8,11,14,17,45-六氮杂六戊烷-56-基)-3,5-双(2-(甲磺酰)嘧啶-4-基)苯甲酰胺的合成(16)
将化合物15(7.00mg,11.22μmol)用DMF(3mL)溶解，加入HATU(7.76mg,20.41μmol)、化合物16-3的甲酸盐(13.37mg,10.20μmol和DIPEA(3.96mg,30.61μmol,5.45μL)，25℃反应2小时。减压抽干反应溶剂，浓缩物直接经高效液相制备，冷冻干燥得标题化合物16(7.00mg)。
色谱柱：SunFire Prep C18 OBD 5μm 19x150mm
流动相A：乙腈；流动相B：水(0.05％甲酸)
时间[min] 流动相A[％] 流动相B[％] 流速[mL/min]
0.00 15 85 28
2.00 15 85 28
18.00 80 20 28
结构表征数据如下：
ESI-MS(m/z):1869.7[M+1] +.
3.生物活性偶联物的制备
偶联方法A:
取0.5mL抗体(anti-ROR1抗体19F6_Hu35V1，3-20mg/mL，表1中简称为19F6)，用0.1M依地酸二钠的溶液(pH 7.60)稀释，然后用1M Na 2HPO 4溶液调pH至7.60，加入10mM TCEP(三(2-羧乙基)膦)溶液(pH 7.60)混匀，室温放置2h。向上述溶液体系加入抗体的5-10倍物质的量的溶解在DMSO的药物-连接体(10mM)，混匀，室温静置20h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的10mM组氨酸缓冲溶液，得到的ADC产物，见表1。
偶联方法B:
取0.5mL抗体(anti-ROR1抗体19F6_Hu35V1，3-20mg/mL，表1中简称为19F6)，用0.1M依地酸二钠的溶液(pH 7.60)稀释，然后用1M Na 2HPO 4溶液调pH至7.60，加入10mM TCEP(三(2-羧乙基)膦)溶液(pH 7.60)混匀，室温放置2h。用NAP-5凝胶柱(Cytiva)除去TCEP，并向所得溶液体系加入抗体的5-10倍物质的量的溶解在DMSO的药物-连接体(10mM)，混匀，室温静置20h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的10mM组氨酸缓冲溶液，得到的ADC产物，见表1。
表1 ADC制备方法、编号和DAR
Figure PCTCN2022094540-appb-000079
Figure PCTCN2022094540-appb-000080
生物活性偶联物的药物/抗体比值(DAR)的测定
SEC-MS测定ADC样品分子量，计算药物/抗体比(DAR)。
色谱测定条件：
色谱柱:ACQUITY UPLC Protein BEH SEC Column，BTQR-18-016；
样品室温度：8℃；柱温：不控柱温；UV：280nm；
流动相：20mM醋酸铵，流速：0.1ml/min,20min，样品进样量：50ug；
质谱测定条件：
质谱型号：AB Sciex Triple TOF 5600+；
GS1 55；GS2 55；CUR 30；TEM 450；ISVF 5500；DP 75；CE 5；
m/z 900-7000；Time bins to sum 100。
(1).SEC-MS测定19F6-ADC III-5-A分子量，计算药物/抗体比值
对偶联后的19F6-ADC III-5-A进行SEC-MS分子量分析见表2，DAR为3.87。
Figure PCTCN2022094540-appb-000081
表2：19F6-ADC III-5-A实测分子量和DAR计算
Figure PCTCN2022094540-appb-000082
(2).SEC-MS测定19F6-ADC III-6-A分子量，计算药物/抗体比值
对偶联后的19F6-ADC III-6-A进行SEC-MS分子量分析见表3，DAR为4.42。
Figure PCTCN2022094540-appb-000083
表3：19F6-ADC III-6-A实测分子量和DAR计算
Figure PCTCN2022094540-appb-000084
(3).SEC-MS测定19F6-ADC IV-1-A分子量，计算药物/抗体比值
对偶联后的19F6-ADC IV-1-A进行SEC-MS分子量分析见表4，DAR为3.74。
Figure PCTCN2022094540-appb-000085
表4：19F6-ADC IV-1-A实测分子量和DAR计算
Figure PCTCN2022094540-appb-000086
Figure PCTCN2022094540-appb-000087
(4).SEC-MS测定19F6-ADC IV-1-B分子量，计算药物/抗体比值
对偶联后的19F6-ADC IV-1-B进行SEC-MS分子量分析见表5，DAR为3.75。
Figure PCTCN2022094540-appb-000088
表5：19F6-ADC IV-1-B实测分子量和DAR计算
Figure PCTCN2022094540-appb-000089
(5).SEC-MS测定19F6-ADC IV-1-C分子量，计算药物/抗体比值
对偶联后的19F6-ADC IV-1-C进行SEC-MS分子量分析见表6，DAR为3.82。
Figure PCTCN2022094540-appb-000090
表6：19F6-ADC IV-1-C实测分子量和DAR计算
Figure PCTCN2022094540-appb-000091
(6).SEC-MS测定19F6-ADC IV-1-D分子量，计算药物/抗体比值
对偶联后的19F6-ADC IV-1-D进行SEC-MS分子量分析见表7，DAR为3.87。
Figure PCTCN2022094540-appb-000092
表7：19F6-ADC IV-1-D实测分子量和DAR计算
Figure PCTCN2022094540-appb-000093
Figure PCTCN2022094540-appb-000094
(7).SEC-MS测定19F6-ADC IV-7-A分子量，计算药物/抗体比值
对偶联后的19F6-ADC IV-7-A进行SEC-MS分子量分析见表8，DAR为3.98。
Figure PCTCN2022094540-appb-000095
表8：19F6-ADC IV-7-A实测分子量和DAR计算
Figure PCTCN2022094540-appb-000096
4.检测生物活性偶联物对体外细胞活性的抑制作用
ADC化合物的细胞增殖抑制作用
(1)细胞铺板：首先采用相应的培养基培养肿瘤细胞N87，用胰酶消化细胞，离心后重悬细胞计数，调整细胞至合适的浓度进行铺板。肿瘤细胞来源见表9。
表9：肿瘤细胞来源
细胞名称 肿瘤类型 来源
NCI-N87 人胃癌细胞 ATCC
本发明化合物和肿瘤细胞共孵育：细胞待细胞贴壁后，移除细胞中培养基，将稀释好的生物活性分子(本发明化合物)加入到上述板孔中，孵育96小时。
体外细胞活性检测：孵育结束后，每孔加入Cell Counting-Lite TM 2.0试剂 (Vazyme/诺唯赞)50μL，避光振荡混匀，反应10min后即可进行检测，酶标仪(厂家：BMG,型号：PHERAStar-FS)读数。利用不含细胞的培养基的Cell Counting-Lite TM获得背景RLU,含有细胞的培养基的Cell Counting-Lite TM获得溶媒RLU。细胞抑制率＝1-(样品RLU-背景RLU)/(溶媒RLU-背景RLU)×100％，按照四参数模型拟合曲线，计算化合物的半数抑制浓度(IC 50)。
(2)数据结果：检测结果如表10所示。
表10：ADC偶联物对NCI-N87细胞系杀伤结果
Figure PCTCN2022094540-appb-000097
说明利用新的偶联方法形成的ADC，可对肿瘤细胞产生杀伤作用，新的偶联方法应用于ADC分子中是行之有效的。
除本文中描述的那些外，根据前述描述，本发明的各种修改对本领域技术人员而言会是显而易见的。这样的修改也意图落入所附权利要求书的范围内。本申请中所引用的各参考文献(包括所有专利、专利申请、期刊文章、书籍及任何其它公开)均以其整体援引加入本文。

Claims (18)

  1. 化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、N-氧化物或同位素标记的化合物，其中所述化合物具有式I的结构：
    Figure PCTCN2022094540-appb-100001
    其中：
    X选自离去基团，例如Cl、Br、I、OMs、OTs、OTf或
    Figure PCTCN2022094540-appb-100002
    Y不存在或选自羰基；
    A环选自取代或非取代的C 6-10芳环、5-12元芳杂环或5-12元杂环；
    Q不存在或为-C(O)-NH-；
    Z 1不存在或者选自-CH 2-或C 2-6亚炔基；
    W 1不存在或选自C 1-10亚烷基、-(CH 2CH 2O)p-、以及-(OCH 2CH 2)p-中的一种或多种；
    J 1选自-COOH、-NH 2、3-10元含氮杂环基、磺酰脲基或羟基；
    p为1-10的整数。
  2. 根据权利要求1所述的式I化合物，其具有如下结构：
    Figure PCTCN2022094540-appb-100003
    其中p为1-10的整数，J 1为-COOH或-NH 2；
    优选地，式I化合物结构选自：
    Figure PCTCN2022094540-appb-100004
  3. 化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、N-氧化物或同位素标记的化合物，其中所述化合物具有式II的结构：
    Figure PCTCN2022094540-appb-100005
    其中，B 1在每次出现时，各自独立地选自单键或5-12元含氮芳杂环；
    Y 1，Y 2及Y 3在每次出现时，各自独立的选自CH和N；
    Z 2不存在或者选自-NH-、-CH 2-、羰基、-C(＝O)NH-、-NHC(＝O)-或C 2-6亚炔基；
    W 2不存在或选自C 1-10亚烷基、-(CH 2CH 2O) p-或-(OCH 2CH 2) p-中的一个或多个；
    J 2选自-COOH、-NH 2、3-10元含氮杂环基、磺酰脲基或羟基；
    p为1-10的整数。
  4. 根据权利要求3所述的式II化合物，其具有如下结构：
    Figure PCTCN2022094540-appb-100006
    Figure PCTCN2022094540-appb-100007
    Figure PCTCN2022094540-appb-100008
    Figure PCTCN2022094540-appb-100009
    Figure PCTCN2022094540-appb-100010
    Figure PCTCN2022094540-appb-100011
    p为1-10的整数；
    优选地，式II化合物具有如下结构：
    Figure PCTCN2022094540-appb-100012
  5. 化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、N-氧化物或同位素标记的化合物，其中所述化合物具有式V的结构：
    Figure PCTCN2022094540-appb-100013
    其中：
    E选自单键、-NH-CH 2-，或以下结构：
    Figure PCTCN2022094540-appb-100014
    D是生物活性分子(例如细胞毒性药物)片段；
    X、Y、A、Q、Z 1、W 1如权利要求1或2中所定义；
    V 1为权利要求1或2所述的式I化合物中J 1与L连接时形成的基团；优选地，V 1选自-CO-、-N(R 1)-、-O-、3-10元含氮杂环基或磺酰脲基，其中R 1为H、C 1-6烷基或C 2-6烷氧烷基；进一步优选地，V 1选自-C(O)-和-N(R 1)-，其中R 1为H、C 1-6烷基或C 2-6烷氧烷基；
    L是连接V 1和E之间的连接子。
  6. 根据权利要求5所述的式V化合物，其具有如下结构：
    Figure PCTCN2022094540-appb-100015
    Figure PCTCN2022094540-appb-100016
    Figure PCTCN2022094540-appb-100017
  7. 化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、N-氧化物或同位素标记的化合物，其中所述化合物具有式VI的结构：
    Figure PCTCN2022094540-appb-100018
    B 1、Y 1、Y 2、Y 3、Z 2和W 2如权利要求3或4中所定义；
    L是连接V 2和E之间的连接子；
    V 2为权利要求3或4中的J 2与L连接时形成的基团；优选地，V 2选自-CO-、-N(R 2)-、-O-、3-10元含氮杂环基或磺酰脲基，其中R 2为H、C 1-6烷基或C 2-6烷氧烷基；进一步优选地，V 2选自-C(O)-和-N(R 2)-，其中R 2为H、C 1-6烷基或C 2-6烷氧烷基；
    E选自单键、-NH-CH 2-，或以下结构：
    Figure PCTCN2022094540-appb-100019
    D是生物活性分子(例如细胞毒性药物)片段。
  8. 根据权利要求7所述的化合物，其结构选自：
    Figure PCTCN2022094540-appb-100020
    Figure PCTCN2022094540-appb-100021
    Figure PCTCN2022094540-appb-100022
  9. 一种生物活性偶联物，其结构如式VII所示：
    Figure PCTCN2022094540-appb-100023
    其中，Ab为靶向部分(例如小分子配体、蛋白质(例如抗体)、多肽、非蛋白质试剂(例如糖、RNA或DNA))；n选自1-10之间的整数或小数；
    V 1选自-C(O)-或-N(R 1)-，其中R 1为H、C 1-6烷基或C 2-6烷氧烷基；
    L是连接V 1和E之间的连接子；
    E是连接L和D的结构片段；
    D是生物活性分子(例如细胞毒性药物)片段；
    所述偶联物中的
    Figure PCTCN2022094540-appb-100024
    表示靶向部分为抗体时，抗体中的巯基与偶联物其余部分的具体连接方式；
    其余基团如权利要求1-2任意一项所定义。
  10. 一种生物活性偶联物，其结构如式VIII所示：
    Figure PCTCN2022094540-appb-100025
    其中，Ab为靶向部分(例如小分子配体、蛋白质(例如抗体)、多肽、非蛋白质试剂(例如糖、RNA或DNA))；n选自1-10之间的整数或小数；
    V 2选自-C(O)-或-N(R 2)-，其中R 2为H、C 1-6烷基或C 2-6烷氧烷基；
    L是连接V 2和E之间的连接子；
    E是连接L和D的结构片段；
    D是生物活性分子(例如细胞毒性药物)片段；
    所述偶联物中的
    Figure PCTCN2022094540-appb-100026
    表示靶向部分为抗体时，抗体中的巯基与偶联物其余部分的具体连接方式；
    其余基团如权利要求3-4任意一项所定义。
  11. 根据权利要求9所述生物活性偶联物，其结构如下，其中Ab选自anti-Her2抗体trastuzumab或anti-Trop2抗体sacituzumab或anti-ROR1抗体19F6_Hu35V1(简称19F6)，n 1为1-8,优选为3-5：
    Figure PCTCN2022094540-appb-100027
    Figure PCTCN2022094540-appb-100028
    Figure PCTCN2022094540-appb-100029
  12. 根据权利要求10所述生物活性偶联物，其结构如下，其中Ab选自anti-Her2抗体trastuzumab或anti-Trop2抗体sacituzumab或anti-ROR1抗体19F6_Hu35V1(简称19F6)，n 1为1-8,优选为3-5：
    Figure PCTCN2022094540-appb-100030
    Figure PCTCN2022094540-appb-100031
    Figure PCTCN2022094540-appb-100032
    Figure PCTCN2022094540-appb-100033
  13. 一种药物组合物，其含有权利要求5-8任意一项所述的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、N-氧化物或同位素标记的化合物、或权利要求9-12任意一项所述的生物活性偶联物，以及一种或多种药用上可接受的载体。
  14. 一种药盒产品，其含有权利要求5-8任意一项所述的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、N-氧化物或同位素标记的化合物、或权利要求9-12任意一项所述的生物活性偶联物，或者权利要求13所述的药物组合物，以及任选的药品说明书。
  15. 权利要求5-8任意一项所述的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、N-氧化物或同位素标记的化合物、或权利要求9-12任意一项所述的生物活性偶联物在制备用于预防或治疗肿瘤疾病的药物中的用途。
  16. 权利要求5-8任意一项所述的化合物或其药学上可接受的盐、酯、立体异构体、 互变异构体、多晶型物、溶剂合物、N-氧化物或同位素标记的化合物、或权利要求9-12任意一项所述的生物活性偶联物，其用于预防或治疗肿瘤疾病。
  17. 一种预防或治疗肿瘤疾病的方法，其包括向有此需要的受试者施用有效量的权利要求5-8任意一项所述的化合物或其药学上可接受的盐、酯、立体异构体、互变异构体、多晶型物、溶剂合物、N-氧化物或同位素标记的化合物、或权利要求9-12任意一项所述的生物活性偶联物。
  18. 化合物的合成方法，其特征在于，所述方法包括以下步骤：
    Figure PCTCN2022094540-appb-100034
    其中：
    X、Z 1、W 1、J 1如权利要求1所定义；Y不存在；
    M为发生取代反应的离去基团，包括但不限于卤素、三氟甲烷磺酸酯、对甲苯磺酸酯，优选为卤素；
    或者，包括以下步骤：
    Figure PCTCN2022094540-appb-100035
    其中：
    X、Z 1、W 1、J 1如通式所定义；
    L为发生取代反应的离去基团，包括但不限于卤素、三氟甲烷磺酸酯、对甲苯磺酸酯，优选为卤素、OTf；
    或者，包括以下步骤：
    Figure PCTCN2022094540-appb-100036
    其中：
    Y 1、Y 2、Y 3、B 1、J 2如通式所定义；
    LG为发生偶联反应的离去基团，包括但不限于卤素、三氟甲烷磺酸酯，优选为卤素。
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