[go: up one dir, main page]

WO2024114531A1 - 杂环化合物及其制备方法和用途 - Google Patents

杂环化合物及其制备方法和用途 Download PDF

Info

Publication number
WO2024114531A1
WO2024114531A1 PCT/CN2023/134038 CN2023134038W WO2024114531A1 WO 2024114531 A1 WO2024114531 A1 WO 2024114531A1 CN 2023134038 W CN2023134038 W CN 2023134038W WO 2024114531 A1 WO2024114531 A1 WO 2024114531A1
Authority
WO
WIPO (PCT)
Prior art keywords
gly
ala
alkyl
drug
antibody
Prior art date
Application number
PCT/CN2023/134038
Other languages
English (en)
French (fr)
Other versions
WO2024114531A9 (zh
Inventor
田强
袁晓曦
张毅涛
宋宏梅
葛均友
Original Assignee
四川科伦博泰生物医药股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 四川科伦博泰生物医药股份有限公司 filed Critical 四川科伦博泰生物医药股份有限公司
Priority to CN202380079300.0A priority Critical patent/CN120202203A/zh
Priority to KR1020257016035A priority patent/KR20250106279A/ko
Priority to AU2023404000A priority patent/AU2023404000A1/en
Publication of WO2024114531A1 publication Critical patent/WO2024114531A1/zh
Publication of WO2024114531A9 publication Critical patent/WO2024114531A9/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—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 organic compound
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K47/54—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 organic compound
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • 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
    • 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 present application relates to the field of compounds, and in particular to a heterocyclic compound, which can be used to prepare a conjugate, such as an antibody-drug conjugate.
  • ADC drugs have attracted global attention for their targeting properties.
  • ADC is a new type of anti-tumor drug that connects toxin drugs to antibodies. It is usually composed of antibodies, toxin drugs and linkers. It exerts anti-tumor effects by leveraging the targeting recognition of antibodies and the high activity of toxins. It is more specific and effective than traditional small molecule drugs.
  • ADC After ADC enters the body, the toxin is transported to the tumor tissue through antibodies with antigen targeting function. ADC binds to the antigen on the surface of tumor cells, and the cells engulf ADC, which is then broken down in lysosomes to release cytotoxins, destroy DNA or prevent cell division, thereby achieving the effect of killing cells.
  • the linker ensures the stability of ADC in the blood, while the toxin plays a killing role after reaching the target.
  • the toxins of ADC mainly include microtubule inhibitors, DNA damaging agents and RNA polymerase inhibitors.
  • the linker also includes various types of structures such as cleavable and non-cleavable types. The structure of the toxin drug and the linker is crucial to the efficacy and safety of ADC. Therefore, the development of drug linker compounds with excellent activity and safety is still a problem that needs to be solved in this field.
  • the present application relates to a drug-linker compound and a preparation method and use thereof.
  • the drug-linker compound can be used to form a conjugate with other molecules such as antibodies.
  • the present invention provides a drug-linker compound having a structure shown in the formula GM-[LED] x , wherein:
  • G is a functional group or leaving group capable of reacting with a specific amino acid or sugar group
  • M is a linker connected to G, wherein M is
  • M 1 is selected from a single bond and C 1-20 alkylene, C 2-20 alkenylene, C 2-20 alkynylene or amine, wherein the C 1-20 alkylene, C 2-20 alkenylene, C 2-20 alkynylene or amine is optionally substituted by one or more suitable substituents;
  • L is a linker connecting the linkers M and E, and L is selected from one or more of the following structures: C 1-6 alkylene, -N(R')-, carbonyl, -O-, natural amino acids or non-natural amino acids and their analogs, and amino acids
  • R' represents hydrogen, C 1-6 alkyl or a polyethylene glycol fragment containing 1-10 EO units; s is selected from an integer of 1-20;
  • E is a structural fragment connecting L and D, wherein E is a single bond, -NHCH 2 - or is selected from the following structures:
  • D is a cytotoxic drug fragment
  • x is selected from 1 to 10.
  • the G is a functional group or a leaving group that can react with a specific amino acid or sugar group in an antibody.
  • G is selected from halogen, halogenated C 1-6 alkyl, C 1-6 sulfonyl, halogenated C 1-6 sulfonyl, halogenated sulfonyl, C 1-6 sulfonate, halogenated C 1-6 sulfonate, C 1-6 sulfinate, C 1-6 sulfoxide, nitro, azido, cyano, alkenyl, alkynyl and alkynyl-containing structural fragments, and the halogenated C 1-6 alkyl, C 1-6 sulfonyl, halogenated C 1-6 sulfonyl, halogenated sulfonyl, C 1-6 sulfonate, halogenated C 1-6 sulfonate, C 1-6 sulfinate, C 1-6 sulfoxide , alkenyl, alkynyl and alkynyl-containing structural fragments are optionally
  • G is selected from halogen, halogenated C 1-6 alkyl, C 1-6 sulfonyl, halogenated C 1-6 sulfonyl, halogenated sulfonyl, C 1-6 sulfonate, halogenated C 1-6 sulfonate, C 1-6 sulfinate, C 1-6 sulfoxide , nitro, azide, cyano, alkenyl, alkynyl and alkynyl-containing structural fragments.
  • the M is Wherein ring A is a 5-membered alicyclic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more (e.g., 2) 6-membered heteroaromatic rings to a benzene ring or a 6-membered heteroaromatic ring via a single bond, wherein the alicyclic heterocycle is optionally substituted by one or more groups selected from oxy ( ⁇ O), halogen, and C 1-4 alkyl; M 1 is selected from a single bond, a C 1-20 alkylene group, a C 2-20 alkenylene group, a C 2-20 alkynylene group, or an amine group, wherein the C 1-20 alkylene group, the C 2-20 alkenylene group, the C 2-20 alkynylene group, or an amine group is optionally substituted by one or more suitable substituents.
  • M 1 is selected from a single bond, a C 1-20 alkylene group,
  • ring A is a 5-membered alicyclic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more (e.g., 2) 6-membered heteroaromatic rings to a benzene ring or a
  • the M is wherein ring A is selected from M 1 is selected from a single bond and a C 1-6 alkylene group, a C 2-6 alkenylene group, a C 2-6 alkynylene group or an amine group, wherein the C 1-6 alkylene group, the C 2-6 alkenylene group, the C 2-6 alkynylene group or the amine group is optionally substituted with one or more suitable substituents.
  • the M is selected from
  • the M is selected from
  • the M is selected from
  • the M is selected from
  • L is selected from one or more of the following structures: C 1-6 alkylene, -N(R')-, carbonyl, -O-, Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys(COCH 2 CH 2 (OCH 2 CH 2 ) r OCH 3 )) ⁇ Ala-Ala ⁇ Ala-Lys ⁇ Ala-Lys(Ac) ⁇ Ala-Pro ⁇ Gly-Glu ⁇ Gly-Gly ⁇ Phe-Lys ⁇ Phe-Lys(Ac) ⁇ Val-Ala ⁇ Val-Lys ⁇ Val-Lys(Ac) ⁇ Val-Cit ⁇ Ala-Ala-Ala-Asn ⁇ Leu-Ala-Glu ⁇ Gly-Gly-Arg ⁇ Gly-Glu-Gly ⁇ Gly- G
  • R′ represents hydrogen, C 1-6 alkyl or a polyethylene glycol fragment containing 1-10 EO units; s is selected from an integer of 1-20, for example 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
  • L is selected from one or more of the following structures: 1-6 Alkylene, carbonyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-Gly-
  • L is selected from one or more of the following structures:
  • the L is selected from the following structures:
  • the L is selected from the following structures:
  • the L is selected from the following structures:
  • the L is selected from the following structures:
  • E is a single bond, -NHCH 2 -, In some embodiments, the E is a single bond.
  • the E is -NHCH 2 -.
  • E 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-phenyl
  • E 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-phenyl
  • the cytotoxic drug is selected from the group consisting of a tubulin inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor, and an RNA polymerase inhibitor.
  • the microtubulin inhibitor is an auristatin compound or a maytansine compound;
  • the DNA intercalator is a pyrrolobenzodiazepine PBD;
  • the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide);
  • the RNA polymerase inhibitor is ⁇ -amanitin or a pharmaceutically acceptable salt, ester or analog thereof.
  • the cytotoxic drugs disclosed in the present application generally contain a variety of functional groups, such as hydroxyl (-OH), carboxyl (-COOH), sulfhydryl (-SH), primary amino (-NH 2 ), secondary amine (-NR A H) or tertiary amine (-NR B RC ), wherein RA , RB , RC here merely represent non-hydrogen substituents on N, and the cytotoxic drugs can be connected to the linker in the conjugate through these functional groups.
  • functional groups such as hydroxyl (-OH), carboxyl (-COOH), sulfhydryl (-SH), primary amino (-NH 2 ), secondary amine (-NR A H) or tertiary amine (-NR B RC ), wherein RA , RB , RC here merely represent non-hydrogen substituents on N, and the cytotoxic drugs can be connected to the linker in the conjugate through these functional groups.
  • the cytotoxic drug is linked to E in the antibody drug conjugate via -OH, -SH, primary amino, secondary amine or tertiary amine groups on the cytotoxic drug.
  • the cytotoxic drug is selected from a compound of Formula I or Formula II, or a pharmaceutically acceptable salt, ester, stereoisomer, tautomer or prodrug of a compound of Formula I or Formula II:
  • R 1 and R 2 are each independently selected from C 1-6 alkyl and halogen;
  • R 3 is selected from H and -CO-CH 2 OH
  • R4 and R5 are each independently selected from H, halogen and hydroxyl; or R4 and R5 are connected to the connected carbon atom to form a 5-6 membered oxygen-containing heterocyclic ring;
  • R 6 is selected from hydrogen or -C 1-4 alkylene-NR a R b ;
  • R7 is selected from C1-6 alkyl and -C1-4 alkylene - NRaRb ;
  • Ra , Rb at each occurrence are independently selected from H, C1-6 alkyl, -SO2 - C1-6 alkyl and -CO- C1-6 alkyl.
  • the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of the compounds:
  • the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of the compounds:
  • the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable derivatives of the compounds: Acceptable salts, esters, stereoisomers, tautomers or prodrugs of:
  • the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of the compounds:
  • the corresponding fragment of the cytotoxic drug obtained after the cytotoxic drug is connected to the linker is D in the general formula; preferably, D is a monovalent structure obtained by losing one H from the -OH, -NH2 or secondary amine group on the cytotoxic drug.
  • D is selected from the following structures:
  • the drug linker compound provided by the present invention has a structure shown in the formula GM-[LED] x , wherein:
  • G is a functional group or leaving group capable of reacting with a specific amino acid or sugar group in the antibody or antigen-binding fragment;
  • G is preferably selected from halogen, halogenated C 1-6 alkyl, C 1-6 sulfonyl, halogenated C 1-6 sulfonyl, halogenated sulfonyl, C 1-6 sulfonate, halogenated C 1-6 sulfonate, C 1-6 sulfinate, C 1-6 sulfoxide, nitro, azido, cyano, alkenyl, alkynyl and alkynyl-containing structural fragments;
  • M is a connector connected to G.
  • M 1 is selected from a single bond and C 1-20 alkylene, C 2-20 alkenylene, C 2-20 alkynylene or amine;
  • M is wherein Ring A is a 5-membered alicyclic heterocyclic ring, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more (e.g., 2) 6-membered heteroaromatic rings to a benzene ring or a 6-membered heteroaromatic ring via a single bond, wherein the alicyclic heterocyclic ring is optionally substituted by one or more groups selected from oxy ( ⁇ O), halogen, and C 1-4 alkyl; M 1 is selected from a single bond, C 1-20 alkylene, C 2-20 alkenylene, C 2-20 alkynylene, or amine;
  • M is wherein ring A is selected from M1 is selected from a single bond and a C1-6 alkylene group, a C2-6 alkenylene group, a C2-6 alkynylene group or an amine group;
  • M is selected from
  • M is selected from
  • M is selected from
  • M is selected from
  • L is a linker connecting the linkers M and E, and L is selected from one or more of the following structures: C 1-6 alkylene, -N(R')-, carbonyl, -O-, natural amino acids or non-natural amino acids and their analogs (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Lys (COCH 2 CH 2 (OCH 2 CH 2 ) r OCH 3 )), and short peptides composed of amino acids (such as Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val-Ala, Val-Lys, Val-Lys(Ac), Val-Cit, Ala-Ala-Ala, Ala
  • R' represents hydrogen, C 1-6 alkyl or a polyethylene glycol fragment containing 1-10 EO units; s is selected from an integer of 1-20;
  • L is selected from the following structures consisting of one or more: C 1-6 Alkylene, carbonyl, -NH-, Ala-Ala, Ala-Lys, Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Val-Ala, Val-Lys, Val-Cit, Ala-Ala-Ala, Ala-Ala-Asn, Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-Val-Cit, Ser-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Val-Ala, Gly-Phe-Leu-Gly, Glu-Ala-Ala-Ala, Gly-Gly-Gly-Gly-G
  • L is selected from one or more of the following structures:
  • L is selected from the following structures:
  • L is selected from the following structures:
  • L is selected from the following structures:
  • L is selected from the following structures:
  • E is a structural fragment connecting L and D, wherein E is a single bond, -NHCH 2 - or is selected from the following structures:
  • E is a single bond, -NHCH 2 -,
  • E is -NHCH 2 - or
  • E is -NHCH 2 -;
  • E is a single bond
  • D is a cytotoxic drug fragment, wherein the cytotoxic drug is selected from a microtubule inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor and an RNA polymerase inhibitor; preferably, the microtubule inhibitor is an auristatin compound or a maytansine compound; preferably, the DNA intercalator is a pyrrolobenzodiazepine (PBD); preferably, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); preferably, the RNA polyme
  • the cytotoxic drug is selected from the compounds of Formula I, Formula II, or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of the compounds of Formula I, Formula II:
  • R 1 and R 2 are each independently selected from C 1-6 alkyl and halogen;
  • R 3 is selected from H and -CO-CH 2 OH
  • R4 and R5 are each independently selected from H, halogen and hydroxyl; or R4 and R5 are connected to the connected carbon atom to form a 5-6 membered oxygen-containing heterocyclic ring;
  • R 6 is selected from hydrogen or -C 1-4 alkylene-NR a R b ;
  • R7 is selected from C1-6 alkyl and -C1-4 alkylene - NRaRb ;
  • Ra , Rb are each independently selected from H, C1-6 alkyl, -SO2 - C1-6 alkyl and -CO- C1-6 alkyl at each occurrence;
  • the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of the compounds:
  • the corresponding fragment of the cytotoxic drug obtained after the cytotoxic drug is connected to the linker is D in the general formula; preferably, D is a monovalent structure obtained by losing one H from -OH, -NH2 or a secondary amine group on the cytotoxic drug;
  • the cytotoxic drug is selected from the following compounds or pharmaceutically acceptable salts, esters, stereoisomers, tautomers or prodrugs of the compounds:
  • x is selected from 1 to 10;
  • the present invention provides a drug-linker having a structure shown in the formula GM-[LED] x , wherein x is selected from 1 to 10, wherein:
  • GM is G is a leaving group for a nucleophilic substitution reaction (e.g.,
  • M 1 is selected from a single bond, C 1-20 alkylene, C 2-20 alkenylene, C 2-20 alkynylene or amine;
  • the L, E and D structures are as defined above.
  • GM is G is a methylsulfonyl group, or G forms a carbon-carbon double bond with the adjacent atom on ring A;
  • M 1 is selected from a single bond, a C 1-20 alkylene group, a C 2-20 alkenylene group, a C 2-10 alkynylene group or an amine group.
  • GM is Selected from M 1 is selected from a single bond, a C 1-20 alkylene group, a C 2-20 alkenylene group, a C 2-20 alkynylene group or an amine group.
  • GM is
  • GM is selected from
  • GM is selected from
  • x is selected from 1 to 10.
  • the drug linker compound is selected from A-01 to A-34, B-01 to B-07 shown below, C-01 ⁇ C-28:
  • the drug linker compound of the present invention is selected from:
  • the drug linker compounds described above may be optionally substituted with one or more suitable substituents.
  • the drug linker compound described above has the following structure:
  • R10 , R11 , R12 are independently selected from hydrogen, C1-6 alkyl, C3-6 cycloalkyl, 5-12 membered heterocyclyl, C6-10 aryl, 5-12 membered heteroaryl, -C1-6 alkyl- C6-10 aryl and -C1-6 alkyl-5-12 membered heteroaryl; the alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally substituted with one or more substituents selected from hydroxy, CN, halogen, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C6-10 aryl, 5-12 membered heteroaryl;
  • R 13 and R 14 are each independently selected from hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl and 4-6 membered heterocyclyl; the alkyl, cycloalkyl and heterocyclyl are optionally substituted by one or more substituents selected from hydroxy, CN, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 6-10 aryl, 5-12 membered heteroaryl;
  • R 15 is selected from hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl, halogenated C 1-6 alkyl, C 1-6 alkyl-OC 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl and 3-6 membered heterocycloalkyl;
  • R 16 is H; or, R 15 , R 16 and the atoms to which they are connected together form a 4-7 membered ring;
  • the 4-7 membered ring is optionally substituted by one or more substituents selected from hydroxy, CN, halogen, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 6-10 aryl, and 5-12 membered heteroaryl.
  • R 10 , R 11 , and R 12 are independently selected from hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl, C 6-10 aryl, benzyl, hydroxy-substituted benzyl, and indolyl-C 1-6 alkyl;
  • R 13 and R 14 are each independently selected from hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl and 4-6 membered heterocyclyl;
  • R 15 is selected from hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl, halogenated C 1-6 alkyl, C 1-6 alkyl-OC 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl and 3-6 membered heterocycloalkyl;
  • R 16 is H; or, R 15 , R 16 and the atoms to which they are attached together form a 4-7 membered ring.
  • R 10 , R 11 , and R 12 are independently selected from hydrogen, C 1-4 alkyl, C 3-6 cycloalkyl, phenyl, benzyl, p-hydroxybenzyl, and indolylmethyl.
  • R 13 and R 14 are each independently selected from hydrogen, C 1-4 alkyl, C 3-6 cycloalkyl, and 4-6 membered heterocyclyl.
  • R 13 and R 14 are each independently selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and 4-6 membered heterocyclyl.
  • R 15 is selected from hydrogen, C 1-4 alkyl, C 3-6 cycloalkyl, halogenated C 1-4 alkyl, C 1-4 alkyl-OC 1-4 alkyl, C 2-6 alkenyl, C 2-6 alkynyl and 3-6 membered heterocycloalkyl; or, R 15 , R 16 and the atoms to which they are attached together form a 4-7 membered heterocycloalkyl or a 4-7 membered heteroaryl.
  • R 15 is selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, halogenated C 1-4 alkyl, C 1-4 alkyl-OC 1-4 alkyl , C 2-6 alkenyl, C 2-6 alkynyl and 3-6 membered heterocycloalkyl; or, R 15 , R 16 and the atoms to which they are attached together form a 4-7 membered heterocycloalkyl or a 4-7 membered heteroaryl.
  • R 10 , R 11 , and R 12 are independently selected from hydrogen, C 1-4 alkyl, C 3-6 cycloalkyl, phenyl, benzyl, p-hydroxybenzyl, and indolylmethyl;
  • R 13 and R 14 are each independently selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and 4-6 membered heterocyclyl;
  • R 15 is selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, halogenated C 1-4 alkyl, C 1-4 alkyl-OC 1-4 alkyl, C 2-6 alkenyl, C 2-6 alkynyl and 3-6 membered heterocycloalkyl; or, R 15 , R 16 and the atoms to which they are connected together form a 4-7 membered heterocycloalkyl or a 4-7 membered heteroaryl.
  • the present invention provides an intermediate compound having the following structure:
  • X is selected from benzyloxycarbonyl, tert-butyloxycarbonyl, fluorenylmethoxycarbonyl, allyloxycarbonyl, trimethylsilylethoxycarbonyl, methoxycarbonyl, ethoxycarbonyl, phthaloyl, p-toluenesulfonyl, trifluoroacetyl, nitrobenzenesulfonyl, benzoyl, pivaloyl, trityl, 4-methoxyphenyldiphenylmethyl, dimethoxytrityl, 2,4-dimethoxybenzyl, p-methoxybenzyl and benzyl; a is an integer from 1 to 10, preferably an integer from 3 to 8; and R 1 , R 2 and D are as defined above.
  • X is selected from fluorenylmethoxycarbonyl (Fmoc).
  • D is selected from:
  • R 1 and R 2 are each independently selected from C 1-6 alkyl and halogen;
  • R 3 is selected from H and -CO-CH 2 OH
  • R4 and R5 are each independently selected from H, halogen and hydroxyl; or R4 and R5 are connected to the connected carbon atom to form a 5-6 membered oxygen-containing heterocyclic ring;
  • R 6 is selected from hydrogen or -C 1-4 alkylene-NR a R b ;
  • R7 is selected from C1-6 alkyl and -C1-4 alkylene - NRaRb ;
  • Ra , Rb at each occurrence are independently selected from H, C1-6 alkyl, -SO2 - C1-6 alkyl and -CO- C1-6 alkyl.
  • the present invention provides an intermediate compound having the following structure:
  • the present invention provides an antibody-drug conjugate having a structure shown in the formula Ab-[MLED] x , wherein M, L, E, D, and x are as defined above, and Ab is an antibody or an antigen-binding fragment thereof that specifically binds to an antigen.
  • the antibody-drug conjugate is selected from ADC A-01 to ADC A-34, ADC B-01 to ADC B-07, ADC C-01 to ADC C-28 shown below:
  • the definition of Ab in the following diagram is as described above, wherein the thiol group on the antibody and the drug linker compound form a thioether bond through an addition reaction or a substitution reaction to obtain a complete antibody-drug conjugate, and x represents the number of drug loads:
  • compositions which may include a plurality of ADCs described herein.
  • Each antibody molecule in the composition may be coupled to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 compounds of the invention. Therefore, the composition is characterized in that the "drug-antibody ratio" (DAR) is in the range of about 1 to about 10.
  • DAR drug-antibody ratio
  • the DAR value (drug-antibody conjugate ratio) of the antibody drug conjugate is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4- 10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10, preferably 3-9, for example, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 3.0-5.5, 3.0-6.0, 3.5-4.0, 3.5-4.5, 3.5-5.0, 3.5-5.5, 3.5 ⁇ 6.0,3.5 ⁇ 6.5,3.5 ⁇ 7.0,3.5 ⁇ 7.5,3.5 ⁇ 8.0,4.0 ⁇ 4.5,4.0 ⁇ 5.0,4.0 ⁇ 5.5,4.0 ⁇ 6.0
  • the DAR value of the antibody-drug conjugate is 4 to 8.
  • the antibody-drug conjugate described in the present application can be prepared modularly. For example, a free form of a "drug-linker" (which can be understood as GM-[LED] x , wherein GM is the structural form before covalently linking to an antibody or an antigen-binding fragment thereof) is first obtained, and then covalently linked to an antibody or an antigen-binding fragment thereof to obtain the antibody-drug conjugate described in the present application.
  • GM-[LED] x wherein GM is the structural form before covalently linking to an antibody or an antigen-binding fragment thereof
  • GM is linked to one or more thiol (-SH), amino (-NH 2 ) or carboxyl (-COOH) groups on the antibody or its antigen-binding fragment by a substitution reaction (e.g., removal of structures such as -SO 2 Me or -Br thereon) or by an addition reaction, and x is selected from 1 to 10.
  • a substitution reaction e.g., removal of structures such as -SO 2 Me or -Br thereon
  • x is selected from 1 to 10.
  • compositions of antibody drug conjugates and/or drug-linkers are provided.
  • the present application provides a pharmaceutical composition comprising the antibody-drug conjugate described in any of the foregoing or optionally the drug-linker described in any of the foregoing, and one or more pharmaceutical excipients.
  • the antibody drug conjugates described herein are usually formulated in a unit injectable form together with a pharmaceutically acceptable parenteral vehicle for parenteral use, such as bolus injection, intravenous injection, intratumoral injection, etc.
  • a pharmaceutically acceptable parenteral vehicle for parenteral use, such as bolus injection, intravenous injection, intratumoral injection, etc.
  • the antibody drug conjugate having the desired purity is mixed with a pharmaceutically acceptable diluent, carrier, excipient or stabilizer in the form of a lyophilized agent or solution (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.).
  • the antibody drug conjugates described herein or pharmaceutical compositions containing the antibody drug conjugates can be administered by any route suitable for the individual to be treated.
  • the pharmaceutical composition may further comprise an additional pharmaceutically active agent.
  • the additional pharmaceutically active agent is a drug with anti-tumor activity.
  • the additional pharmaceutically active agent is selected from B7-H3 inhibitors, EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met or VEGF inhibitors, chemotherapeutic drugs or any combination thereof.
  • the antibody-drug conjugates, drug-linkers or pharmaceutical compositions described herein can be used to treat a variety of diseases or conditions, such as B7-H3-positive tumors and Her2-positive tumors.
  • the present application provides the use of any of the above-mentioned antibody-drug conjugates, drug-linkers, or pharmaceutical compositions containing the same in the preparation of drugs for preventing and/or treating and/or assisting in the treatment of positive tumors.
  • the present application provides the use of any of the above-mentioned antibody-drug conjugates, drug-linkers, or pharmaceutical compositions containing the same in the preparation of drugs for preventing and/or treating and/or assisting in the treatment of B7-H3-positive tumors.
  • the present application provides use of any of the above-mentioned antibody-drug conjugates, drug-linkers, or pharmaceutical compositions containing the same in the preparation of drugs for preventing and/or treating and/or assisting in the treatment of HER2-positive tumors.
  • the present application also provides a method for preventing and/or treating and/or adjuvant treating positive tumors, which comprises the step of administering an effective amount of the antibody-drug conjugate, drug linker, or pharmaceutical composition containing the same as described in any of the above items to a subject in need thereof.
  • the present application also provides a method for preventing and/or treating and/or adjuvanting treatment of B7-H3 positive tumors, which comprises the step of administering an effective amount of any of the above-described antibody-drug conjugates, drug linkers, or pharmaceutical compositions containing the same to a subject in need thereof.
  • the present application also provides a method for preventing and/or treating and/or adjuvanting HER2-positive tumors, which comprises the step of administering an effective amount of the antibody-drug conjugate, drug linker, or pharmaceutical composition containing the same as described in any of the foregoing items to a subject in need thereof.
  • the present application also provides use of any of the above-mentioned antibody-drug conjugates, drug linkers or drug compositions in inhibiting the proliferation of B7-H3-positive tumor cells.
  • the present application also provides use of any of the above-mentioned antibody-drug conjugates, drug linkers or drug compositions in inhibiting the proliferation of Her2-positive tumor cells.
  • the antibody-drug conjugate, drug linker or drug composition is administered to cells in vitro or in vivo in a subject; for example, applied to a subject to inhibit the proliferation of tumor cells in the subject; or, applied to in vitro tumor cells (e.g., cell lines or cells from a subject) to inhibit the proliferation of tumor cells in vitro.
  • B7-H3 positive tumors include solid tumors or hematological malignancies, such as colorectal cancer, gastric cancer, breast cancer, prostate cancer, head and neck squamous cell carcinoma, melanoma, neuroblastoma, sarcoma, lung cancer (e.g., small cell Lung cancer, non-small cell lung cancer, etc.), kidney cancer, bladder cancer, thyroid cancer, mesothelioma, pancreatic cancer, ovarian cancer, endometrial cancer, esophageal cancer, liver cancer, salivary gland cancer, bile duct cancer, meningioma.
  • solid tumors or hematological malignancies such as colorectal cancer, gastric cancer, breast cancer, prostate cancer, head and neck squamous cell carcinoma, melanoma, neuroblastoma, sarcoma, lung cancer (e.g., small cell Lung cancer, non-small cell lung cancer, etc.), kidney cancer, bladder cancer, thyroid cancer, mesotheli
  • the subject is preferably a mammal, such as bovine, equine, porcine, canine, feline, rodent, primate; for example, human.
  • the method further comprises administering a second therapy to the subject, the second therapy being selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, viral therapy, adjuvant therapy, and any combination thereof.
  • the second therapy can be applied simultaneously, separately, or sequentially with the above method.
  • the tumor involved in the pharmaceutical composition of the present invention is selected from breast cancer, colorectal cancer, head and neck cancer, renal clear cell carcinoma, renal papillary cell carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, prostate cancer, gastric adenocarcinoma, thyroid cancer or any combination thereof.
  • the drug linker described above in the present invention is used to prepare a conjugate.
  • the conjugate of the present invention includes an antibody drug conjugate and the like.
  • the intermediate compounds described above in the present invention are used to prepare drug linker compounds.
  • the present invention also provides a method for preparing the drug linker compound, comprising the step of deprotecting the intermediate compound.
  • the method 1 includes a method for preparing compound A-05a, which includes the step of coupling with a compound of formula I;
  • R 1 and R 2 are each independently selected from C 1-6 alkyl and halogen;
  • R 3 is selected from H and -CO-CH 2 OH
  • R4 and R5 are each independently selected from H, halogen and hydroxyl; or R4 and R5 are connected to the connected carbon atom to form a 5-6 membered oxygen-containing heterocyclic ring;
  • R 6 is selected from hydrogen or -C 1-4 alkylene-NR a R b ;
  • R7 is selected from C1-6 alkyl and -C1-4 alkylene - NRaRb ;
  • Ra , Rb at each occurrence are independently selected from H, C1-6 alkyl, -SO2 - C1-6 alkyl and -CO- C1-6 alkyl.
  • R 1 and R 2 are each independently selected from C 1-4 alkyl, F, Cl, Br and I.
  • R 1 is selected from halogen, methyl, ethyl, propyl, and butyl, such as methyl and Cl.
  • R 2 is selected from F, Cl, Br and I, such as F and Cl.
  • R1 is selected from methyl and Cl
  • R2 is selected from F and Cl.
  • the method 2 includes a method for preparing compound A-14-a, which includes the step of deprotecting IM-5-a to obtain a compound of formula IM-6-a;
  • X is as defined in the intermediate compound described above, and R1 and R2 are as defined in method one.
  • the deprotection reaction is as follows: the solvent is selected from N,N-dimethylformamide, and an alkylamine compound, such as diethylamine, is added.
  • the deprotection is carried out at room temperature, and the reaction time is 1-5 h, such as 1-3 h.
  • the method 2 further comprises the step of reacting IM-6-a with IM-2 to obtain compound A-14-a.
  • the solvent of the reaction is selected from N,N-dimethylformamide and N,N-dimethylacetamide.
  • the reaction further comprises the step of adding N,N-diisopropylethylamine.
  • the method three includes a method for preparing compound B-02-a, which includes the step of deprotecting B-02-4a;
  • the method four includes a method for preparing compound C-07-a, the method comprising the steps of reacting C-07-6 with C-07-8a;
  • R1 and R2 are as defined in method 1.
  • C-07-8a in the method four is prepared by deprotection of C-07-7a;
  • the method five includes a method for preparing compound C-10a, the method comprising the steps of reacting C-10-5a with C-07-8a;
  • R 1 , R 2 and a are as defined above.
  • C-10-5a in the method five is prepared by deprotection of C-10-4a;
  • C-10-4a in the method five is prepared by C-10-3 and compound A;
  • the method six includes a method for preparing compound C-17a, the method comprising the steps of reacting C-17-3a with C-07-8a;
  • R 1 , R 2 and a are as defined above; preferably, R 1 is methyl, R 2 is Cl and a is 3 or 8; and RX is selected from CH or N.
  • C-17-3a in the method six is prepared by oxidation reaction of C-17-2a;
  • C-17-2a in the method six is prepared by deprotection of C-17-1a;
  • C-17-1a in the method six is prepared by coupling reaction of C-10-2 or C-19-3 with compound A.
  • antibody refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC).
  • Antibody light chains can be classified as ⁇ (kappa) and ⁇ (lambda) light chains.
  • Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and define the isotype of the antibody as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of three domains (CH1, CH2, and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • VH and VL regions can also be subdivided into regions with high variability (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs).
  • CDRs complementary determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions (VH and VL) of each heavy chain/light chain pair form antigen binding sites, respectively.
  • the allocation of amino acids in each region or domain can follow various numbering systems known in the art.
  • CDR complementarity determining region
  • the variable region of the heavy chain and light chain each contains three CDRs, designated CDR1, CDR2, and CDR3.
  • CDR1, CDR2, and CDR3 The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al.
  • the CDR contained in the antibody or its antigen-binding fragment can be determined according to various numbering systems known in the art, such as by Kabat, Chothia, IMGT or AbM numbering systems. In certain embodiments, the CDR contained in the antibody or its antigen-binding fragment is defined by the Chothia numbering system.
  • framework region or "FR” residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.
  • antigen-binding fragment of an antibody refers to a polypeptide of a fragment of an antibody, such as a polypeptide of a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen bound by the full-length antibody and/or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion".
  • Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab)' 2 fragments, F(ab)' 3 fragments, Fd, Fv, scFv, di-scFv, (scFv) 2 , disulfide-stabilized Fv proteins ("dsFv"), single domain antibodies (sdAb, nanobodies), and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.
  • Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.
  • Fd means an antibody fragment consisting of a VH and CH1 domains
  • dAb fragment means an antibody fragment consisting of a VH domain (Ward et al., Nature 341:544-546 (1989))
  • Fab fragment means an antibody fragment consisting of a VL, VH, CL and CH1 domains
  • F(ab') 2 fragment means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • Fab'fragment means a fragment obtained after reducing the disulfide bonds linking two heavy chain fragments in a F(ab') 2 fragment, consisting of a complete light chain and the Fd fragment (consisting of VH and CH1 domains) of the heavy chain.
  • Fv means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody.
  • the Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen binding site. It is generally believed that the six CDRs confer antigen binding specificity to an antibody. However, even a single variable region (e.g., a Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of a complete binding site.
  • Fc means an antibody fragment formed by the second and third constant regions of the first heavy chain of an antibody and the second and third constant regions of the second heavy chain of an antibody bound via disulfide bonds.
  • the Fc fragment of an antibody has a variety of different functions but does not participate in antigen binding.
  • scFv refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315). (1994)).
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 may be used, but variants thereof may also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol.
  • a disulfide bond may also exist between the VH and VL of the scFv.
  • the VH and VL domains may be positioned relative to each other in any suitable arrangement.
  • a scFv comprising NH 2 -VH-VH-COOH, NH 2- VL-VL-COOH.
  • single-domain antibody has the meaning generally understood by those skilled in the art, and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region) that retains the ability to specifically bind to the same antigen as the full-length antibody (Holt, L. et al., Trends in Biotechnology, 21(11):484-490, 2003). Single-domain antibodies are also called nanobodies.
  • Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody, and/or competes with the full-length antibody for specific binding to the antigen.
  • antibody includes not only intact antibodies but also antigen-binding fragments of antibodies.
  • Antibody antigen-binding fragments can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antibody antigen-binding fragments can be screened for specificity in the same manner as for intact antibodies.
  • murine antibody refers to antibodies obtained by the following method: fusing B cells of immunized mice with myeloma cells, screening out mouse hybrid fusion cells that can both proliferate indefinitely and secrete antibodies, and then performing screening, antibody preparation and antibody purification; or refers to antibodies secreted by plasma cells formed by differentiation and proliferation of B cells after antigens invade the mouse body.
  • humanized antibody refers to a non-human antibody that has been genetically engineered, and its amino acid sequence has been modified to increase the homology with the sequence of a human antibody.
  • CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and/or constant region) comes from a human immunoglobulin (recipient antibody).
  • donor antibody non-human antibody
  • non-CDR region e.g., variable region FR and/or constant region
  • human immunoglobulin residual antibody
  • Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc.
  • the donor antibody can be a mouse, rat, rabbit or non-human primate (e.g., cynomolgus monkey) antibody with the expected properties (e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and/or ability to enhance immune response).
  • non-human primate e.g., cynomolgus monkey
  • expected properties e.g., antigen specificity, affinity, reactivity, ability to increase immune cell activity and/or ability to enhance immune response.
  • identity is used to refer to the matching of sequences between two polypeptides or between two nucleic acids.
  • a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine)
  • the molecules are identical at that position.
  • the "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions being compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
  • two sequences are compared when they are aligned to produce maximum identity.
  • Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.).
  • the percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • variant in the context of a polypeptide (including a polypeptide), also refers to a polypeptide or peptide comprising an amino acid sequence that has been changed by introducing amino acid residue substitutions, deletions or additions. In some cases, the term “variant” also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to a polypeptide or peptide).
  • a polypeptide can be modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection/blocking groups, proteolytic cleavage, connection to a cell ligand or other protein, etc.
  • Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.
  • a variant has a function similar, identical or improved to the polypeptide or peptide from which it is derived.
  • the term "specific binding” refers to a non-random binding reaction between two molecules, such as an antibody and its antigen.
  • the strength or affinity of a specific binding interaction can be expressed as the equilibrium dissociation constant (KD) or half-maximal effect concentration (EC50 ) of the interaction.
  • the specific binding properties between two molecules can be determined using methods known in the art.
  • One method involves measuring the rate of formation and dissociation of the antigen binding site/antigen complex.
  • Both the "association rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rate of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187).
  • the ratio of kdis/kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473).
  • KD, kon and kdis values can be measured by any effective method.
  • the dissociation constant can be measured using bioluminescence interferometry (e.g., ForteBio Octet method).
  • the dissociation constant can be measured using surface plasmon resonance technology (e.g., Biacore) or Kinexa.
  • conservative substitution means an amino acid substitution that does not adversely affect or change the expected properties of the protein/polypeptide comprising the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions in which amino acid residues are substituted with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, and histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • amino acids are generally represented by single-letter and three-letter abbreviations known in the art.
  • alanine can be represented by A or Ala.
  • alkyl refers to a group obtained by removing one hydrogen atom from a straight or branched hydrocarbon group, for example, “ C1-20 alkyl”, “ C1-10 alkyl”, “ C1-6 alkyl”, “ C1-4 alkyl”, “ C1-3 alkyl”, etc.
  • Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.
  • alkylene refers to a group obtained by removing two hydrogen atoms from a straight or branched hydrocarbon group, for example, “C 1-20 alkylene”, “C 1-10 alkylene”, “C 3-10 alkylene”, “C 5-8 alkylene”, “C 1-6 alkylene”, “C 1-4 alkylene", “C 1-3 Specific examples include, but are not limited to, methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene or 1,6-hexylene.
  • alkenylene refers to a divalent group obtained by losing two hydrogen atoms from a straight or branched hydrocarbon group containing at least one carbon-carbon double bond, including, for example, "C 2-20 alkenylene”, “C 3-10 alkenylene”, “C 5-8 alkenylene”, etc.
  • Examples include, but are not limited to, vinylene, 1-propenylene, 2-propenylene, 1-butenylene, 2-butenylene, 1,3-butadienylene, 1-pentenylene, 2-pentenylene, 3-pentenylene, 1,3-pentadienylene, 1,4-pentadienylene, 1-hexenylene, 2-hexenylene, 3-hexenylene, 1,4-hexadienylene, etc.
  • alkynylene refers to a divalent group obtained by losing two hydrogen atoms from a straight or branched hydrocarbon group containing at least one carbon-carbon triple bond. Examples include, for example, “C 2-20 alkynylene”, “C 3-10 alkynylene”, “C 5-8 alkynylene”, etc.
  • Examples include, but are not limited to, ethynylene, 1-propynylene, 2-propynylene, 1-butynylene, 2-butynylene, 1,3-butadiynylene, 1-pentynylene, 2-pentynylene, 3-pentynylene, 1,3-pentadiynylene, 1,4-pentadiynylene, 1-hexynylene, 2-hexynylene, 3-hexynylene, 1,4-hexadiynylene, etc.
  • aliphatic heterocycle refers to a saturated or partially saturated cyclic structure containing at least one (e.g., 1, 2, or 3) ring member selected from N, O, and S. Specific examples include, but are not limited to, 5-6 membered aliphatic heterocycles, 5-6 membered nitrogen-containing aliphatic heterocycles, 5-6 membered oxygen-containing aliphatic heterocycles, and the like, such as tetrahydrofuran, pyrrolidine, piperidine, tetrahydropyran, and the like.
  • heteromatic ring refers to an aromatic ring structure containing at least one ring member selected from N, O and S.
  • specific examples include, but are not limited to, 5-6 membered aromatic heterocycles, 5-6 membered nitrogen-containing aromatic heterocycles, 5-6 membered oxygen-containing aromatic heterocycles, and the like, such as furan, thiophene, pyrrole, thiazole, isothiazole, thiadiazole, oxazole, isoxazole, oxadiazole, imidazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, pyridine, pyrimidine, pyridazine, pyrazine, 1,2,3-triazine, 1,3,5-triazine, 1,2,4,5-tetrazine,
  • aromatic ring system refers to a monocyclic or polycyclic system comprising at least one aromatic ring (e.g., a benzene ring, etc.) or a heteroaromatic ring (e.g., a 5-6 membered aromatic heterocycle, e.g., a 5-6 membered nitrogen-containing aromatic heterocycle, e.g., a pyrimidine ring, etc.), two or more aromatic rings and/or heteroaromatic rings may form a fused ring or be connected by a single bond (e.g., dipyrimidinylphenyl, etc.), and the aromatic ring system may be divalent or higher valent (e.g., trivalent or tetravalent), e.g., a 5-20 membered aromatic ring system.
  • aromatic ring system may be divalent or higher valent (e.g., trivalent or tetravalent), e.g., a 5-20 membered aromatic ring system.
  • suitable substituents refers to modifications that one skilled in the art can make to a compound according to the needs of the compound's substituents.
  • Suitable substituents include oxy ( ⁇ O), halogen, cyano, NR 8 R 9 , carboxyl, thiol, hydroxyl, ester (e.g.
  • R 8 and R 9 are each independently selected from H, C 1-6 alkyl, C 3-6 cycloalkyl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl, C 6-10 aryl, benzyl, hydroxy-substituted benzyl, indolylmethylene and C 1-6 haloalkoxy, R 8 and R 9 are each independently selected from H, C 1-6 alkyl, C 3-6 cycloalkyl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl, C 6-10 aryl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 1-6 haloalkoxy, halogen, hydroxy, carboxyl and ester groups (e.g. -
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives.
  • pH adjusters include, but are not limited to, phosphate buffers.
  • Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc.
  • Stabilizers have the meanings generally understood by those skilled in the art, which can stabilize the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc.
  • sugars such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose
  • amino acids such as glutamic acid, glycine
  • proteins such as dried whey, albumin or casein
  • degradation products such as lactalbumin hydrolysate
  • prevention refers to a method implemented in order to prevent or delay the occurrence of a disease or illness or symptom (e.g., a tumor) in a subject.
  • treatment refers to a method implemented in order to obtain a beneficial or desired clinical result.
  • beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable.
  • treatment can also refer to, compared to the expected survival period (if not receiving treatment), extending the survival period.
  • the term "subject” refers to a mammal, such as a primate mammal, such as a human.
  • the subject eg, human
  • an effective amount refers to an amount sufficient to obtain or at least partially obtain the desired effect.
  • an effective amount for preventing a disease e.g., a tumor
  • an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art.
  • an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.
  • cancer and “tumor” are used interchangeably to refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Disease. Unregulated cell division may lead to the formation of malignant tumors or cells that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers as well as dormant tumors or micrometastases. Cancer also includes hematological malignancies.
  • lymphomas includes lymphomas, leukemias, myelomas or lymphoid malignancies, as well as spleen cancer and lymph node tumors.
  • exemplary lymphomas include B-cell lymphomas and T-cell lymphomas.
  • B-cell lymphomas include, for example, Hodgkin's lymphoma.
  • T-cell lymphomas include, for example, cutaneous T-cell lymphomas.
  • Hematological malignancies also include leukemias, such as secondary leukemias or acute lymphocytic leukemias.
  • myeloma e.g., multiple myeloma
  • other hematological and/or B-cell or T-cell related cancers include myeloma (e.g., multiple myeloma) and other hematological and/or B-cell or T-cell related cancers.
  • FR Antibody framework region amino acid residues in the variable region of an antibody other than CDR residues
  • IMGT Immunoglobulin G
  • Kabat The immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
  • Chothia The immunoglobulin numbering system proposed by Chothia et al. is a classical rule for identifying CDR region boundaries based on the location of structural loop regions (see, e.g., Chothia & Lesk (1987) J.
  • EC50 is the concentration that produces 50% efficacy or binding.
  • IC50 is the concentration that produces 50% inhibition
  • NMR nuclear magnetic resonance
  • MS Mass spectrometry
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% trifluoroacetic acid)
  • reaction solution was poured into a mixture of DCM (600 mL), IPA (60 mL), and water (100 mL) and stirred for 10 minutes, the DCM phase was separated, washed with brine (100 ml), concentrated to obtain a crude product, purified by preparative HPLC, and freeze-dried to obtain 0.98 g of compound A-05.
  • A-05 separation and purification method is as follows:
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • A-05 structural characterization data are as follows:
  • A-07 separation and purification method is as follows:
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • A-07 structural characterization data are as follows:
  • the preparation method is as follows:
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • the preparation method is as follows:
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • IM-6 (170 mg, 0.226 mmol) and compound IM-2 (90.83 mg, 0.249 mmol) were dissolved in N,N-dimethylformamide (10 mL), and N,N-diisopropylethylamine (29.21 mg, 0.226 mmol) was added. The reaction solution was stirred at room temperature for 16 hours. The reaction solution was directly purified by preparative HPLC and freeze-dried to obtain 50.56 mg of compound A-14.
  • the preparation method is as follows:
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • compound B-01-1 (413.40 mg, 0.251 mmol, its synthesis reference patent CN111295389B) was dissolved in dimethyl sulfoxide and water (2.0 mL: 0.5 mL), and cuprous bromide (72.95 mg, 0.503 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-hex-5-ynamide (95.10 mg, 0.302 mmol) were added. The reaction was stirred for 1 hour and then filtered. The filtrate was purified by preparative HPLC (conditions as follows) to obtain 30.00 mg of compound B-01-2.
  • Mobile phase A acetonitrile
  • Mobile phase B water
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% trifluoroacetic acid)
  • Example 6 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxy-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamido)benzyl((1S,9R)-9-ethyl-5-fluoro-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-02)
  • the mesylate of 1-1 (30.00 mg, 56.44 ⁇ mol) was dissolved in N,N-dimethylformamide (1 mL), and 1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate (58.74 mg, 112.88 ⁇ mol), N,N-diisopropylethylamine (43.76 mg, 338.63 ⁇ mol) and 2-((tert-butyldiphenylsilyl)oxy)acetic acid (26.62 mg, 84.66 ⁇ mol) were added in sequence. The reaction was maintained at 25°C for 1 hour, and the reaction was monitored by LC-MS.
  • B-02-1 (20 mg, 27.33 ⁇ mol) was dissolved in dichloromethane (2 mL), and a dichloromethane solution (0.5 mL) of 4-dimethylaminopyridine (26.71 mg, 218.61 ⁇ mol) and triphosgene (8.11 mg, 27.33 ⁇ mol) were added in sequence.
  • B-02-2 (250.00 mg, 137.51 ⁇ mol) was dissolved in a mixed solvent of DMSO (2 mL) and water (0.4 mL), and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide (62.98 mg, 206.26 ⁇ mol) and cuprous bromide (39.45 mg, 275.01 ⁇ mol) were added.
  • reaction was maintained at 25°C for 1 hour; the reaction was monitored by LC-MS chromatography; after the reaction was completed, the reaction solution was purified by preparative HPLC (conditions as follows), and the preparative solution was lyophilized to obtain 150.00 mg of B-02-3 compound.
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • the reaction was maintained at 25°C for 0.5 hours, and the reaction was monitored by LC-MS chromatography. After the reaction was completed, the reaction solution was purified by preparative HPLC (conditions as follows), and the preparative solution was lyophilized to obtain 50.00 mg of B-02-4 compound.
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • B-02-4 50 mg, 26.52 ⁇ mol was dissolved in dichloromethane (1 mL), trifluoroacetic acid (60.49 mg, 530.49 ⁇ mol) was added, and the reaction was maintained at 25°C for 0.5 hours; the reaction was monitored by LC-MS chromatography; after the reaction was completed, the reaction solution was concentrated, and the crude product was purified by preparative HPLC (conditions as follows), and the preparative solution was lyophilized to obtain 23.69 mg of B-02 compound.
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • the preparation method is as follows:
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • the preparation method is as follows:
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • the preparation method is as follows:
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • the preparation method is as follows:
  • Chromatographic column Phenomenex Luna C18 200*40mm*10um.
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% hydrochloric acid)
  • the preparation method is as follows:
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • the preparation method is as follows:
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • the preparation method is as follows:
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • C-17-2 (56 mg, 97.61 ⁇ mol) was added to acetonitrile (6 mL) and water (3 mL), and then sodium periodate (208.79 mg, 976.15 ⁇ mol) and ruthenium trichloride hydrate (8.10 mg, 39.05 ⁇ mol) were added to the reaction system. The reaction was stirred at 25 °C for 30 minutes. The reaction was monitored by LC-MS. Water and ethyl acetate were added for extraction and concentrated to obtain C-17-3 (60 mg).
  • IM-6 (20 mg, 25.06 ⁇ mol), C-17-3 (16 mg, 25.06 ⁇ mol), HATU (19.05 mg, 50.11 ⁇ mol), and DIPEA (16.19 mg, 125.28 ⁇ mol) were added to DMF (3 mL) in sequence, and the reaction system was reacted at 25 ° C for 1 hour.
  • the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain C-17 (16 mg).
  • the preparation method is as follows:
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • C-19-2 (5.26 g, 13.7 mmol) was dissolved in THF (30.0 mL), MeOH (30.0 mL) and water (30.0 mL), and LiOH ⁇ H 2 O (1.72 g, 40.9 mmol) was added, and stirred at 25°C for 2 hours.
  • the reaction solution was adjusted to pH 3 with 1N hydrochloric acid aqueous solution, and solid precipitated, which was filtered and the filter cake was dried in vacuo to obtain C-19-3 (4.20 g).
  • the preparation method is as follows:
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • IM-6 (20.0 mg, 0.025 mmol) and C-19-6 (16.0 mg, 0.025 mmol) were added to DMF (1 mL) and stirred to dissolve.
  • HATU (19.0 mg, 0.050 mmol) and DIPEA (12.9 mg, 0.100 mmol) were added and reacted at room temperature for 2 hours.
  • the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain C-19 (20.4 mg).
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • the preparation method is as follows:
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • IM-6 27.91 mg, 34.97 ⁇ mol
  • C-21-3 30 mg, 34.97 ⁇ mol
  • HATU 26.59 mg, 69.93 ⁇ mol
  • DIPEA 22.60 mg, 174.84 ⁇ mol
  • the preparation method is as follows:
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • the aqueous phase was extracted twice with ethyl acetate (25.0 mL x 2), and the organic phases were combined and dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product C-23-1 (3.00 g), which was directly used in the next step.
  • the preparation method is as follows:
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • IM-6 (20.0 mg, 0.025 mmol) and C-23-3 (21.5 mg, 0.025 mmol) were dissolved in DMF (1 mL), and HATU (19.0 mg, 0.050 mmol) and DIPEA (12.9 mg, 0.100 mmol) were added and reacted at room temperature for 2 hours.
  • the reaction solution was directly purified by preparative HPLC and freeze-dried to obtain C-23 (17.0 mg).
  • the preparation method is as follows:
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Example 13 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl((1S,9R)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-03)
  • Step 1 Preparation of ethyl 2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoacetate (B-03-1)
  • Step 2 2-(((1S,9S)-9-(((4-((S)-35-azido-2-(4-(4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamido)benzyl)oxy)carbonyl)
  • Step 3 Preparation of 4-((S)-35-azido-2-(4-((4-methoxyphenyl)benzhydryl)amino)butyl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-03-3)
  • Step 4 Preparation of 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamido)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-03-4)
  • Step 5 Preparation of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl((1S,9R)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-03)
  • the preparation high performance liquid chromatography method is as follows:
  • Mobile phase A acetonitrile
  • Mobile phase B water (0.05% formic acid)
  • Human B7-H3-4Ig-His protein was used to immunize fully human mice, and the serum titer was monitored by ELISA and cell flow cytometry. The best mice were selected according to the titer results, and spleen cells were fused, screened and subcloned. The activities of different monoclonal clones binding to human ⁇ monkey proteins and cells were detected, and the preferred clone 20G11G6/2# was obtained.
  • the antibody sequence was modified by PTM site removal, PI reduction, and heavy chain constant region ADCC removal, and finally the fully human antibody 2#8890 (heavy chain variable region, SEQ ID NO: 3; light chain variable region, SEQ ID NO: 4) and human IgG1 heavy chain constant region removal were obtained.
  • the ADCC activity modified sequence (SEQ ID NO:31) and the human ⁇ light chain constant region (SEQ ID NO:32) were combined to form a complete humanized antibody (see Table 1).
  • the antibody was entrusted to Nanjing GenScript Biotechnology Co., Ltd. for codon optimization and gene synthesis, constructed into the pTT5 plasmid, and the heavy chain and light chain plasmids were simultaneously transfected into CHO-S cells.
  • the expressed antibody in the supernatant was purified using Protein A to obtain the corresponding antibody protein 2#8890.
  • the heavy chain amino acid sequence and light chain amino acid sequence of 2#8890 are shown in SEQ ID NO:42 and SEQ ID NO:43, respectively.
  • hIgG1 is an anti-chicken lysozyme antibody.
  • the heavy chain variable region is fused to the mutated human IgG1 heavy chain constant region (SEQ ID NO: 31), and the light chain variable region is fused to the wild-type human kappa light chain constant region (SEQ ID NO: 32).
  • the antibody hIgG1 was expressed and purified according to the above method.
  • the B7-H3 control antibody DS7300 is from patent CN 103687945A. After codon optimization, the antibody heavy chain variable region nucleotide sequence was synthesized and cloned into the human IgG1 heavy chain constant region containing a mutation (SEQ ID NO: 31), and the light chain variable region nucleotide sequence was synthesized into the pTT5 vector containing the wild-type kappa light chain constant region (SEQ ID NO: 32). The antibody DS7300 was expressed and purified according to the above method.
  • the antibodies 2#8890, DS7300, and hIgG1 involved in the antibody-drug conjugates prepared in the following examples are the corresponding antibodies described in the second part above.
  • 3.052ml 2#8890 antibody (9.83mg/mL) was taken and diluted with 153uL 20mM PB+0.1M EDTA (pH 7.60), and then the pH was adjusted to 7.60 with 1M Na 2 HPO 4 solution, and 10mM TCEP (tri(2-carboxyethyl)phosphine, 114.5uL, pH 7.60) solution was added and mixed, and the mixture was placed at room temperature for 1.5h. Then, 10 times the amount of the substance was added, and the A-05 (219.1uL, 10mM) solution dissolved in dimethyl sulfoxide was mixed, and the mixture was placed at room temperature for 2h.
  • TCEP tri(2-carboxyethyl)phosphine, 114.5uL, pH 7.60
  • the buffer was replaced with a 20mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain an antibody drug conjugate, namely ADC 5 (2#8890-A-05).
  • the DAR value was determined by mass spectrometry to be 7.90.
  • hIgG1 antibody (18.3 mg/mL) was diluted with 95.6 uL 20 mM PB + 0.1 M EDTA (pH 7.60), and then the pH was adjusted to 7.60 with 1 M Na 2 HPO 4 solution, and 10 mM TCEP (tris (2-carboxyethyl) phosphine, 66.86 uL, pH 7.60) solution was added and mixed, and the mixture was placed at room temperature for 1.5 hours. Then, 10 times the amount of the substance was added, and the A-14 (260.53 uL, 10 mM) solution dissolved in dimethyl sulfoxide was mixed, and the mixture was placed at room temperature for 2 hours.
  • A-14 260.53 uL, 10 mM
  • the buffer was replaced with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain an antibody drug conjugate, namely ADC 8 (hIgG1-A-14).
  • ADC 8 hIgG1-A-14.
  • the DAR value was determined by mass spectrometry to be 8.0.
  • 3.6788ml 2#8890 antibody (10.873mg/mL) was taken and diluted with 183.94uL 20mM PB+0.1M EDTA (pH 7.60), and then the pH was adjusted to 7.60 with 1M Na 2 HPO 4 solution, and 10mM TCEP (tris(2-carboxyethyl)phosphine, 152uL, pH 7.60) solution was added and mixed, and allowed to stand at room temperature for 1.5h. Then, 10 times the amount of substance was added to the A-14 (292.26uL, 10mM) solution dissolved in dimethyl sulfoxide and mixed, and allowed to stand at room temperature for 2h.
  • the buffer was replaced with a 20mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain an antibody drug conjugate, namely ADC 9 (2#8890-A-14).
  • the DAR value was determined by mass spectrometry to be 7.22.
  • 1.533 ml of hIgG1 antibody (19.57 mg/mL) was diluted with 76.65 uL 20 mM PB + 0.1 M EDTA (pH 7.60), and then the pH was adjusted to 7.60 with 1 M Na 2 HPO 4 solution, and 10 mM TCEP (tris (2-carboxyethyl) phosphine, 114.5 uL, pH 7.60) solution was added and mixed, and the mixture was placed at room temperature for 1.5 hours. Then, 10 times the amount of B-01 (212.4 uL, 10 mM) dissolved in dimethyl sulfoxide was added and mixed, and the mixture was placed at room temperature for 2 hours.
  • B-01 212.4 uL, 10 mM
  • the buffer was replaced with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain an antibody drug conjugate, namely ADC 10 (hIgG1-B-01).
  • ADC 10 hIgG1-B-01.
  • the DAR value determined by mass spectrometry was 8.03.
  • the ADC samples after coupling were subjected to LC-MS molecular weight analysis.
  • Liquid chromatography column Thermo MAbPac RP 3.0*100mm;
  • Mobile phase A 0.1% FA/H 2 O
  • Mobile phase B 0.1% FA/ACN
  • Mass spectrometer model AB Sciex Triple TOF 5600+;
  • the ADC samples after coupling were subjected to LC-MS molecular weight analysis.
  • Liquid chromatography column ACQUITY UPLC MAbPac BEH SEC;
  • Mass spectrometer model AB Sciex Triple TOF 5600+;
  • the buffer was replaced with a 20mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain an antibody drug conjugate, namely ADC 16 (2#8890-C-21).
  • the DAR value determined by mass spectrometry was 3.92.
  • the antibody 19F6_Hu35v1 involved in the following examples is the 19F6_Hu35v1 antibody described in the international patent application WO2022253035A1, and is prepared by the method described in Example 2 of the patent.
  • the sample coupling preparation is as follows:
  • ADC 20 (Trastuzumab-A-05, DAR 8)
  • the buffer was replaced with a 20mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain an antibody-drug conjugate (i.e., Trastuzumab-A-05).
  • the DAR value was determined by mass spectrometry to be 8.04.
  • ADC 21 (Trastuzumab-A-14, DAR 8)
  • ADC 22 (Trastuzumab-A-24, DAR 8)
  • ADC23 (Trastuzumab-A-32, DAR 8)
  • ADC24 (Trastuzumab-C-21, DAR 4)
  • the conjugate to be tested was diluted to 5 ⁇ g/ml with PBST (0.02% Tween-20), and human B7-H3-4Ig-his, human B7-H3-2Ig-his, rat B7-H3-his and monkey B7-H3-his proteins were gradiently diluted to 200nM, 100nM, 50nM, 25nM, 12.50nM, 6.25nM, 3.125nM, 0nM, and then the conjugate to be tested was captured by Protein A Sensor (Pall life sciences) in PBST (0.02% Tween-20) solution for 60s, and then bound to the four proteins for 60s, and then dissociated for 180s.
  • PBST 0.02% Tween-20
  • a flow cytometer (Beckman, model Cytoflex) was used to detect the affinity of the anti-human B7-H3 fully human antibody conjugate to human colon cancer cells HT29 (Cell Bank of the Chinese Academy of Sciences), human gastric cancer cells NCI-N87 (ATCC), human breast squamous cancer cells HCC1806 (ATCC) and human non-small cell lung cancer cells HCC827 (ATCC); the affinity of the anti-human B7-H3 fully human antibody conjugate to CHOS-human B7-H3-4Ig and CHOS-human B7-H3-2Ig was detected; the species cross-reaction of the anti-human B7-H3 fully human antibody conjugate was detected: affinity with CHOS-rat B7-H3 and CHOS-monkey B7-H3.
  • Anti-human B7-H3 antibody conjugates are affinity-bound to HT29, NCI-N87, HCC1806, and HCC827 tumor cells
  • the EC50 of ADC17 for HT29 tumor cells is 3.213ng/ml
  • the EC50 for HCC827 tumor cells is 8.054ng/ml.
  • the affinity results of the other antibody conjugates are shown in Table 3, the affinity results of the anti-human B7-H3 fully human antibody conjugate for CHOS-human B7-H3-4Ig and CHOS-human B7-H3-2IG cells are shown in Table 4, and the affinity results of the anti-human B7-H3 antibody conjugate for CHOS-rat B7-H3 and CHOS-monkey B7-H3 cells are shown in Table 5.
  • the results show that the conjugate prepared by the drug linker compound of the present invention binds to monkey B7-H3 overexpressing cells, but not to rat B7-H3 overexpressing cells.
  • the endocytic activity of anti-human B7-H3 antibody conjugates on human gastric cancer cells NCI-N87 (ATCC), human breast squamous carcinoma cells HCC1806 (ATCC) and human non-small cell lung cancer cells HCC827 (ATCC) was detected by flow cytometry (Thermo, model Attune NxT).
  • the cells growing on the wall were digested with Trypsin-EDTA (0.25%) (Thermo) solution and counted, and the cell density was adjusted to 1 ⁇ 10 5 cells/ml with complete medium.
  • the Median YL-1H value was exported and then imported into GraphPad Prism 6 software to calculate EC 50 .
  • the results are shown in Table 6.
  • the antibody-drug conjugates (eg, ADC 5 and ADC 9) prepared by the drug linker compounds of the present invention all have good cellular endocytosis activity.
  • the adherent A375, Calu6-B7-H3 and U87MG-B7-H3 cells were digested and counted with Trypsin-EDTA (0.25%) (Thermo) solution, and the cell density was adjusted to 1 ⁇ 10 4 , 5 ⁇ 10 4 , and 1 ⁇ 10 4 /ml respectively with complete medium, and 100 ⁇ l of cell suspension was added to each well of a 96-well plate (the number of cells was 1000, 5000, and 1000 per well, respectively).
  • the 96-well plate was placed in a 37°C, CO 2 constant temperature incubator for 24 hours.
  • the ADC to be tested was diluted with complete medium, starting with a final concentration of 3333.3 nM, and a 4-fold gradient dilution was performed, with a total of 12 concentration points; 100 ⁇ l of the diluted ADC was added to a 96-well plate, and cultured at 37°C, 5% CO 2 for 4 to 7 days. The 96-well plate was taken out, 20 ⁇ l of CCK8 reagent was added to each well, and the plate was incubated at 37° C. for 2 to 3 h. The OD 450 nm signal value was detected using an enzyme reader, and then imported into GraphPad Prism 6 software to calculate IC 50 . The results are shown in Table 7.
  • Human breast squamous cell carcinoma cell HCC1806 (ATCC) was cultured in RPMI1640 medium with 10% fetal bovine serum at 37°C in an incubator containing 5% CO2 air. When the cells were in exponential growth phase, the culture medium was taken for mycoplasma detection, and the cells were collected and counted. 2 ⁇ 10 6 HCC1806 cells were subcutaneously inoculated at the right shoulder of each mouse and suspended in 0.1 ml PBS.
  • mice with too small or too large tumor volume were eliminated, and the remaining mice were randomly divided into 6 groups according to tumor volume and animal weight, with 6 mice in each group, and a single dose was injected into the tail vein (DAR4 administration group: 10 mg/kg, DAR8 administration group: 5 mg/kg). Tumor volume and body weight were measured twice a week after administration. The specific results are shown in Table 8.
  • mice in the drug-treated group was stable and well tolerated. 95 days after a single dose, the tumors of mice in the ADC 5 group completely disappeared. This indicates that the antibody-drug conjugate prepared by the drug linker compound of the present invention has a significant tumor inhibitory effect.
  • Human breast squamous cell carcinoma cell HCC1806 (ATCC) was cultured in RPMI1640 medium with 10% fetal bovine serum at 37°C in an incubator containing 5% CO2 air. When the cells were in the exponential growth phase, the culture medium was taken for mycoplasma detection, and the cells were collected and counted. 2 ⁇ 106 HCC1806 cells were subcutaneously inoculated at the right scapula of each mouse and suspended in 0.1ml PBS. When the average tumor volume grew to about 100-200mm3 , mice with too small or too large tumor volume were eliminated, and the remaining mice were randomly divided into 12 groups according to tumor volume and animal weight, with 6 mice in each group, and a single dose was given by tail vein injection. Tumor volume and body weight were measured twice a week after administration. The specific results are shown in Table 9.
  • NCI-N87 Human gastric cancer cells NCI-N87 (ATCC), cultured in RPMI1640 medium with 10% fetal bovine blood The cells were cleared and cultured in an incubator at 37°C with 5% CO2 air. When the cells were in the exponential growth phase, the culture medium was taken for mycoplasma detection, and the cells were collected and counted. 5 ⁇ 106 NCI-N87 cells were subcutaneously inoculated at the right scapula of each mouse and suspended in 0.1ml PBS matrix gel.
  • mice with tumors that were too small or too large were eliminated, and the remaining mice were randomly divided into 8 groups according to tumor volume and animal weight, with 6 mice in each group, and the drugs were injected into the tail vein, QW twice. Tumor volume and body weight were measured twice a week after administration. The specific results are shown in Table 10.
  • mice in each drug administration group was stable and well tolerated, indicating that the antibody-drug conjugate prepared by the drug linker compound of the present invention has a significant tumor inhibitory effect and good safety.
  • ELISA and LC-MS/MS methods were used to quantitatively detect ADC (ADC 5, ADC 9), total antibody (TAb), and Payload in cynomolgus monkey serum.
  • the standard curve quantitative range of ADC (ADC 5, ADC 9) and total antibody (TAb) methods was 11.72-3000.00 ng/mL, and the linear range of Payload method was 0.1-40 ng/mL.
  • Both ADC (ADC 5, ADC 9) and total antibody (TAb) methods used B7-H3 protein as the capture protein, which was coated in a 96-well ELISA plate, and then total antibody (Tab) used Goat Anti-Human IgG-HRP as the detection antibody; ADC (ADC 5, ADC 9) used Anti-Toxin mouse antibody and Goat Anti-Mouse IgG as the secondary antibody and detection antibody, respectively. The color was developed by the action of enzyme and substrate, and read on the SpectraMax i3x (Molecular Devices) microplate reader. The standard curve was fitted using the 4-P parameter method and the concentration of each sample was calculated.
  • ADC ADC 5, ADC 9
  • total antibody Tb
  • LC-MS/MS was performed by Shimadzu LC 30-AD liquid phase system coupled with SCIEX QTRAP 5500+ (SCIEX) mass spectrometer, using (+) ESI ionization mode and selecting multiple reaction monitoring mode (MRM) analysis.
  • the chromatographic column was Xbridge C18 50*4.6mm, 5 ⁇ m, the ion pair of the test compound 1-10 was 510.2/435.2, and acetonitrile was used for protein precipitation in sample pretreatment.
  • the repeated-dose toxicity study included four repeated intravenous injections of ADC5 and ADC 9 in crab-eating monkeys.
  • This study set up 3 groups, 1 animal/group/sex, and intravenously injected 30mg/kg of ADC5, ADC 9 and normal saline (volume: 10mL/kg), once a week, for a total of 2 times; then the dose was increased to 50mg/kg of ADC 5, ADC 9 and normal saline, once a week, for a total of 2 times. No death or dying related to the test product was observed during the experiment. During the administration period, the animals in the ADC 5 administration group showed loss of appetite, hair loss, skin pigmentation and weight loss. In female monkeys, WBC, NEUT, LYM and MONO were reduced.
  • HNSTD serious toxicity dose
  • Antibody-drug conjugates inhibit tumor growth in a mouse subcutaneous transplant tumor model
  • the preparations containing the ADC of the present invention were respectively administered to CDX models of mice subcutaneously transplanted with human breast squamous cell carcinoma cells HCC1806 by tail vein injection, and the tumor volume and animal body weight changes were measured twice a week to calculate the tumor inhibition efficacy of the ADC of the present invention on tumor-bearing mice.
  • mice Balb/c Nude mice (Chengdu Yaokang Biotechnology Co., Ltd.)
  • HCC1806 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO 2. HCC1806 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and inoculated subcutaneously in female Balb/c-nude mice to establish a breast squamous cell carcinoma model. When the average tumor volume was about 200 mm 3 , the mice were randomly divided into groups according to the tumor size and administered separately. The groups and their dosages were as follows: vehicle control group (i.e.
  • V Tend the mean tumor volume of the treatment group at the end of the experiment
  • V T0 Mean tumor volume at the start of drug administration in the treatment group
  • V C end mean tumor volume of negative control group at the end of the experiment
  • V C0 Mean tumor volume of the negative control group at the beginning of drug administration
  • T/C (V T end /V T0 )/(V C end /V C0 ).
  • the ADC of the present invention has a significant inhibitory effect on the tumor growth of the HCC1806 breast squamous cell carcinoma transplanted tumor model.
  • TGI tumor growth inhibition rates
  • TGI tumor growth inhibition rate
  • T/C relative tumor proliferation rate
  • Cell inhibition rate 1-(sample RLU-background RLU)/(cell control RLU-background RLU) ⁇ 100%, and the half-maximal inhibitory concentration (IC 50 ) of ADC 19 is calculated according to the four-parameter model fitting curve.
  • the half maximal inhibitory concentration (IC 50 ) of ADC 19 on the NCI-H1975 cell line was 5.00 ⁇ g/mL; the half maximal inhibitory concentration (IC 50 ) of ADC 19 on the HT-29 cell line was 4.56 ⁇ g/mL.
  • NCI-N87 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum at 37°C and 5% CO 2. NCI-N87 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and inoculated subcutaneously in female Balb/c-nu mice to establish a gastric cancer model. When the average tumor volume was about 160 mm3, the mice were randomly divided into groups according to the tumor size, in order: vehicle control group (i.e., negative control, Vehicle group), Trastuzumab-A-05 1 mg/kg group of the present invention, Trastuzumab-A-14 1 mg/kg group, and Trastuzumab-A-24 1 mg/kg group.
  • vehicle control group i.e., negative control, Vehicle group
  • Trastuzumab-A-05 1 mg/kg group of the present invention Trastuzumab-A-14 1 mg/kg group
  • the ADC of the present invention has a significant inhibitory effect on the tumor growth of the NCI-N87 gastric cancer transplant model.
  • the tumor growth inhibition rates (TGI) of the Trastuzumab-A-05 1mg/kg group, the Trastuzumab-A-14 1mg/kg group and the Trastuzumab-A-24 1mg/kg group of the present invention were 81.62%, 107.38% and 61.43%, respectively. No animal died and the animal weight decreased significantly in each treatment group on Day 30, and no obvious drug toxicity was observed.
  • the mice had good tolerance to the ADC prepared by the drug linker compound of the present invention. The specific results are shown in Table 17.
  • TGI tumor growth inhibition rate
  • T/C relative tumor proliferation rate

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Peptides Or Proteins (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

本申请涉及医药领域，具体涉及一种药物连接子化合物，所述药物可以包括DNA拓扑异构酶抑制剂，通过药物连接子化合物制得的偶联物，例如抗体药物偶联物具有较优的药物抗体偶联比，对肺癌、黑色素瘤、乳腺癌、胃癌、结肠癌等肿瘤具有很好的靶向杀伤效果。此外，本申请进一步提供所述抗体药物-连接体分子的制备方法及其用途。

Description

杂环化合物及其制备方法和用途 技术领域
本申请涉及化合物领域，具体涉及一种杂环化合物，所述化合物可以用于制备偶联物，例如抗体药物偶联物。
背景技术
在抗肿瘤药物研发领域，ADC药物以其靶向性引起全球关注。ADC是将毒素药物连接在抗体上的新型抗肿瘤药物，通常由抗体、毒素药物和连接子构成，借助抗体靶向识别性和毒素的高活性发挥抗肿瘤作用，比传统小分子药物更具特异性和有效性。
ADC进入体内后，通过具有抗原靶向作用的抗体将毒素运输到肿瘤组织，ADC与肿瘤细胞表面抗原结合，细胞吞噬ADC，随后在溶酶体中分解释放细胞毒素，破坏DNA或阻止细胞分裂，达到杀伤细胞的效果。
其中连接子保证ADC在血液中的稳定性，而毒素在达到靶点后发挥杀伤作用。ADC的毒素主要包括微管类抑制剂、DNA损伤剂和RNA聚合酶抑制剂等类型。连接子方面也包括裂解型、不可裂解型等多种类型的结构。毒素药物及连接体的结构对ADC的药效和安全性至关重要，因此，研发出兼具优良活性及安全性的药物连接子化合物的仍是本领域需要解决的问题。
发明内容
本申请涉及一种药物-连接子化合物及其制备方法和用途，药物-连接子化合物可用于同其他分子例如抗体形成偶联物。
在一个方面，本发明提供一种药物-连接子化合物，其具有式G-M-[L-E-D]x所示结构，其中：
G是能够与特异性氨基酸或糖基发生反应的官能团或离去基团；
M是与G相连的接头，所述的M为
其中，环A为5-6元脂杂环、或5-20元芳香族环系，所述脂杂环和芳香族环系任选地被一个或多个选自氧基(＝O)、卤素、氰基、氨基、羧基、巯基和C1-6烷基的基团取代；M1选自单键和C1-20亚烷基、C2-20亚烯基、C2-20亚炔基或胺基，所述的C1-20亚烷基、C2- 20亚烯基、C2-20亚炔基或胺基任选地被一个或多个适合的取代基所取代；
L是连接接头M和E之间的连接子，L选自由下述的一个或多个组成的结构：C1-6亚烷基、-N(R’)-、羰基、-O-、天然氨基酸或非天然氨基酸及其类似物、以及氨基酸 组成的短肽、
其中R’代表氢、C1-6烷基或含1-10个EO单元的聚乙二醇片段；s选自1-20的整数；
E是连接L和D的结构片段，其中E为单键、-NHCH2-或选自以下结构：
D是细胞毒性药物片段；和/或
x选自1至10。
在一些实施方式中，所述的G是能够与抗体中的特异性氨基酸或糖基发生反应的官能团或离去基团。
在一些实施方式中，所述的G选自卤素、卤代C1-6烷基、C1-6磺酰基、卤代C1-6磺酰基、卤代磺酰基、C1-6磺酸酯基、卤代C1-6磺酸酯基、C1-6亚磺酸酯基、C1-6亚砜基、硝基、叠氮基、氰基、烯基、炔基及含炔基的结构片段，所述的卤代C1-6烷基、C1-6磺酰基、卤代C1-6磺酰基、卤代磺酰基、C1-6磺酸酯基、卤代C1-6磺酸酯基、C1-6亚磺酸酯基、C1-6亚砜基、烯基、炔基及含炔基的结构片段任选地被一个或多个适合的取代基所取代。
在一些实施方式中，所述的G选自卤素、卤代C1-6烷基、C1-6磺酰基、卤代C1-6磺酰基、卤代磺酰基、C1-6磺酸酯基、卤代C1-6磺酸酯基、C1-6亚磺酸酯基、C1-6亚砜基、硝基、叠氮基、氰基、烯基、炔基及含炔基的结构片段。
在一些实施方式中，所述的M为其中环A为5元脂杂环、6元杂芳环、或由一个以上(例如2个)的6元芳杂环与苯环或6元杂芳环通过单键连接形成的多环，所述脂杂环任选地被一个或多个选自氧基(＝O)、卤素和C1-4烷基的基团取代；M1选自单键、C1-20亚烷基、C2-20亚烯基、C2-20亚炔基或胺基，所述的C1-20亚烷基、C2-20亚烯基、C2-20亚炔基或胺基任选地被一个或多个适合的取代基所取代。
在一些实施方式中，所述的M为其中环A为5元脂杂环、6元杂芳环、或由一个以上(例如2个)的6元芳杂环与苯环或6元杂芳环通过单键连接形成的多环，所述脂杂环任选地被一个或多个选自氧基(＝O)、卤素和C1-4烷基的基团取代；M1选自单键、C1-20亚烷基、C2-20亚烯基、C2-20亚炔基或胺基。
在一些实施方式中，所述的M为其中环A选自 M1选自单键和C1-6亚烷基、C2-6亚烯基、C2-6亚炔基或胺基，所述的C1-6亚烷基、C2-6亚烯基、C2-6亚炔基或胺基任选地被一个或多个适合的取代基所取代。
在一些实施方式中，所述的M选自
在一些实施方式中，所述的M选自
在一些实施方式中，所述的M选自
在一些实施方式中，所述的M选自
在一些实施方式中，所述的L选自由下述的一个或多个组成的结构：C1-6亚烷基、-N(R’)-、羰基、-O-、Ala、Arg、Asn、Asp、Cit、Cys、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、Tyr、Val、Lys(COCH2CH2(OCH2CH2)rOCH3))、Ala-Ala、Ala-Lys、Ala-Lys(Ac)、Ala-Pro、Gly-Glu、Gly-Gly、Phe-Lys、Phe-Lys(Ac)、Val-Ala、Val-Lys、Val-Lys(Ac)、Val-Cit、Ala-Ala-Ala、Ala-Ala-Asn、Leu-Ala-Glu、Gly-Gly-Arg、Gly-Glu-Gly、Gly-Gly-Gly、Gly-Ser-Lys、Glu-Val-Ala、Glu-Val-Cit、Ser-Ala-Pro、Val-Leu-Lys、Val-Lys-Ala、Val-Lys-Gly、Gly-Gly-Phe-Gly、Gly-Gly-Val-Ala、Gly-Phe-Leu-Gly、Glu-Ala-Ala-Ala、Gly-Gly-Gly-Gly-Gly、
其中R’代表氢、C1-6烷基或含1-10个EO单元的聚乙二醇片段；s选自1-20的整数，例如1-15、1-10、1-9、1-8、1-7、1-6、1-5、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20。
在一些实施方式中，所述的L选自由下述的一个或多个组成的结构：C1-6亚烷基、羰基、-NH-、Ala-Ala、Ala-Lys、Ala-Pro、Gly-Glu、Gly-Gly、Phe-Lys、Val-Ala、Val-Lys、Val-Cit、Ala-Ala-Ala、Ala-Ala-Asn、Leu-Ala-Glu、Gly-Gly-Arg、Gly-Glu-Gly、Gly-Gly-Gly、Gly-Ser-Lys、Glu-Val-Ala、Glu-Val-Cit、Ser-Ala-Pro、Val-Leu-Lys、Val-Lys-Ala、Val-Lys-Gly、Gly-Gly-Phe-Gly、Gly-Gly-Val-Ala、Gly-Phe-Leu-Gly、Glu-Ala-Ala-Ala、Gly-Gly-Gly-Gly-Gly、 其中s选自1-20的整数。
在一些实施方式中，所述的L选自由下述的一个或多个组成的结构：

在一些实施方式中，所述的L选自以下结构：
在一些实施方式中，所述的L选自以下结构：

在一些实施方式中，所述的L选自以下结构：
在一些实施方式中，所述的L选自以下结构：
在一些实施方式中，所述的E为单键、-NHCH2-、在一些实施方式中，所述的E为单键。
在一些实施方式中，所述的E为-NHCH2-。
在一些实施方式中，所述的E为
在一些实施方式中，所述的E为
在一些实施方式中，选自以下结构：







在一些实施方式中，选自以下结构：


在一些实施方式中，所述细胞毒性药物选自微管蛋白抑制剂、DNA嵌入剂、DNA拓扑异构酶抑制剂和RNA聚合酶抑制剂。
在一些实施方式中，所述微管蛋白抑制剂为奥瑞他汀类化合物或美登素类化合物；所述DNA嵌入剂为吡咯并苯二氮卓PBD；所述DNA拓扑异构酶抑制剂为拓扑异构酶I抑制剂(例如，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、或卢比替康)或拓扑异构酶II抑制剂(例如，阿霉素、PNU-159682、多卡米星、柔红霉素、米托蒽醌、鬼臼毒素、或依托泊苷)；所述RNA聚合酶抑制剂为α-鹅膏草碱(α-amanitin)或其药学上可接受的盐、酯或类似物。
本申请中所公开的细胞毒性药物通常含有多种官能团，例如羟基(-OH)、羧基(-COOH)、巯基(-SH)、一级氨基(-NH2)、二级胺基(-NRAH)或三级胺基(-NRBRC)，其中RA、RB、RC在此仅代表N上的非氢取代基，所述细胞毒性药物可通过这些官能团与偶联物中的连接体连接。
在一些实施方案中，所述细胞毒性药物通过其上的-OH、-SH、一级氨基、二级胺基或三级胺基与所述抗体药物偶联物中的E连接。
在一些实施方式中，所述细胞毒性药物选自式I、式II所示化合物、或式I、式II所示化合物的药学上可接受的盐、酯、立体异构体、互变异构体或前药：
其中，R1,R2各自独立地选自C1-6烷基和卤素；
R3选自H和-CO-CH2OH；
R4和R5各自独立地选自H、卤素和羟基；或者R4和R5与相连碳原子连接成5-6元含氧杂环；
R6选自氢或-C1-4亚烷基-NRaRb；
R7选自C1-6烷基和-C1-4亚烷基-NRaRb；
其中Ra、Rb在每次出现时各自独立地选自H、C1-6烷基、-SO2-C1-6烷基和-CO-C1-6烷基。
在一些实施方式中，所述细胞毒性药物选自以下化合物或所述化合物的药学上可接受的盐、酯、立体异构体、互变异构体或前药：

在一些实施方式中，所述细胞毒性药物选自以下化合物或所述化合物的药学上可接受的盐、酯、立体异构体、互变异构体或前药：
在一些实施方式中，所述细胞毒性药物选自以下化合物或所述化合物的药学上可接 受的盐、酯、立体异构体、互变异构体或前药：
在一些实施方案中，所述细胞毒性药物选自以下化合物或所述化合物的药学上可接受的盐、酯、立体异构体、互变异构体或前药：
在一些实施方式中，所述细胞毒性药物与连接体连接后得到的该细胞毒性药物相应的片段即为通式中的D；优选地，D为所述细胞毒性药物上的-OH、-NH2或二级胺基失掉一个H得到的一价结构。
在一些实施方案中，D选自以下结构：

另一方面，本发明提供的药物连接子化合物，其具有式G-M-[L-E-D]x所示结构，其中：
G是能够与抗体或抗原结合片段中特异性氨基酸或糖基发生反应的官能团或离去基团；G优选自卤素、卤代C1-6烷基、C1-6磺酰基、卤代C1-6磺酰基、卤代磺酰基、C1-6磺酸酯基、卤代C1-6磺酸酯基、C1-6亚磺酸酯基、C1-6亚砜基、硝基、叠氮基、氰基、烯基、炔基及含炔基的结构片段；
M是与G相连的接头，M为
其中，环A为5-6元脂杂环、或5-20元芳香族环系，所述脂杂环和芳香族环系任选地被一个或多个选自氧基(＝O)、卤素、氰基、氨基、羧基、巯基和C1-6烷基的基团取代；M1选自单键和C1-20亚烷基、C2-20亚烯基、C2-20亚炔基或胺基；
优选地，M为其中环A为5元脂杂环、6元杂芳环、或由一个以上(例如2个)的6元芳杂环与苯环或6元杂芳环通过单键连接形成的多环，所述脂杂环任选地被一个或多个选自氧基(＝O)、卤素和C1-4烷基的基团取代；M1选自单键、C1-20亚烷基、C2-20亚烯基、C2-20亚炔基或胺基；
优选地，M为其中环A选自 M1选自单键和C1-6亚烷基、C2-6亚烯基、C2-6亚炔基或胺基；
优选地，M选自
优选地，M选自
优选地，M选自
优选地，M选自
L是连接接头M和E之间的连接子，L选自由下述的一个或多个组成的结构：C1-6亚烷基、-N(R’)-、羰基、-O-、天然氨基酸或非天然氨基酸及其类似物(比如Ala、Arg、Asn、Asp、Cit、Cys、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、Tyr、Val、Lys(COCH2CH2(OCH2CH2)rOCH3))、以及氨基酸组成的短肽(比如Ala-Ala、Ala-Lys、Ala-Lys(Ac)、Ala-Pro、Gly-Glu、Gly-Gly、Phe-Lys、Phe-Lys(Ac)、Val-Ala、Val-Lys、Val-Lys(Ac)、Val-Cit、Ala-Ala-Ala、Ala-Ala-Asn、Leu-Ala-Glu、Gly-Gly-Arg、Gly-Glu-Gly、Gly-Gly-Gly、Gly-Ser-Lys、Glu-Val-Ala、Glu-Val-Cit、Ser-Ala-Pro、Val-Leu-Lys、Val-Lys-Ala、Val-Lys-Gly、Gly-Gly-Phe-Gly、Gly-Gly-Val-Ala、Gly-Phe-Leu-Gly、Glu-Ala-Ala-Ala、Gly-Gly-Gly-Gly-Gly)、

其中R’代表氢、C1-6烷基或含1-10个EO单元的聚乙二醇片段；s选自1-20的整数；
优选地，L选自由下述的一个或多个组成的结构：C1-6亚烷基、羰基、-NH-、Ala-Ala、Ala-Lys、Ala-Pro、Gly-Glu、Gly-Gly、Phe-Lys、Val-Ala、Val-Lys、Val-Cit、Ala-Ala-Ala、Ala-Ala-Asn、Leu-Ala-Glu、Gly-Gly-Arg、Gly-Glu-Gly、Gly-Gly-Gly、Gly-Ser-Lys、Glu-Val-Ala、Glu-Val-Cit、Ser-Ala-Pro、Val-Leu-Lys、Val-Lys-Ala、Val-Lys-Gly、Gly-Gly-Phe-Gly、Gly-Gly-Val-Ala、Gly-Phe-Leu-Gly、Glu-Ala-Ala-Ala、Gly-Gly-Gly-Gly-Gly、
其中s选自1-20的整数；
优选地，L选自由下述的一个或多个组成的结构：

优选地，L选自以下结构：

优选地，L选自以下结构：
优选地，L选自以下结构：
优选地，L选自以下结构：
E是连接L和D的结构片段，其中E为单键、-NHCH2-或选自以下结构：
优选地，E为单键、-NHCH2-、
优选地，E为-NHCH2-或
优选地，E为-NHCH2-；
优选地，E为单键；
优选地，E为
D是细胞毒性药物片段，所述细胞毒性药物选自微管蛋白抑制剂、DNA嵌入剂、DNA拓扑异构酶抑制剂和RNA聚合酶抑制剂；优选地，所述微管蛋白抑制剂为奥瑞他汀类化合物或美登素类化合物；优选地，所述DNA嵌入剂为吡咯并苯二氮卓(PBD)；优选地，所述DNA拓扑异构酶抑制剂为拓扑异构酶I抑制剂(例如，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、或卢比替康)或拓扑异构酶II抑制剂(例如，阿霉素、PNU-159682、多卡米星、柔红霉素、米托蒽醌、鬼臼毒素、或依托泊苷)；优选地，所述RNA聚合酶抑制剂为α-鹅膏草碱(α-amanitin)或其药学上可接受的盐、酯或类似物；
优选地，所述细胞毒性药物选自式I、式II所示化合物、或式I、式II所示化合物的药学上可接受的盐、酯、立体异构体、互变异构体或前药：
其中，R1,R2各自独立地选自C1-6烷基和卤素；
R3选自H和-CO-CH2OH；
R4和R5各自独立地选自H、卤素和羟基；或者R4和R5与相连碳原子连接成5-6元含氧杂环；
R6选自氢或-C1-4亚烷基-NRaRb；
R7选自C1-6烷基和-C1-4亚烷基-NRaRb；
其中Ra、Rb在每次出现时各自独立地选自H、C1-6烷基、-SO2-C1-6烷基和-CO-C1-6烷基；
优选地，所述细胞毒性药物选自以下化合物或所述化合物的药学上可接受的盐、酯、立体异构体、互变异构体或前药：

所述细胞毒性药物与连接体连接后得到的该细胞毒性药物相应的片段即为通式中的D；优选地，D为所述细胞毒性药物上的-OH、-NH2或二级胺基失掉一个H得到的一价结构；
优选地，所述细胞毒性药物选自以下化合物或所述化合物的药学上可接受的盐、酯、立体异构体、互变异构体或前药：
和/或
x选自1至10；
优选地，选自以下结构：







优选地，选自以下结构：



在另一个方面，本发明提供一种药物-连接体，其具有式G-M-[L-E-D]x所示结构，x选自1至10，其中：
G-M为G为亲核取代反应的离去基团(例如，
卤素、甲磺酰基、氟代苯酚基或)，或为羟基(-OH)、巯基(-SH)或氨基(-NH2)；或者，G与环A上相邻原子形成不饱和双键；环A为5-6元脂杂环、或5-20元芳香族环系，所述脂杂环和芳香族环系任选地被一个或多个选自氧基(＝O)、卤素、氰基、氨基、羧基、巯基和C1-6烷基的基团取代；M1选自单键、C1-20亚烷基、C2-20亚烯基、C2- 20亚炔基或胺基；
L、E和D结构如前述所定义。
在一些实施方式中，G-M为G为甲磺酰基，或者，G与环A上相邻原子形成碳碳双键；环A为5元脂杂环、6元杂芳环、或由一个以上的6元芳杂环与苯环或6元杂芳环通过单键连接形成的多环，所述脂杂环任选地被一个或多个选自氧基(＝O)、卤素和C1-4烷基的基团取代；M1选自单键、C1-20亚烷基、C2-20亚烯基、C2-10亚炔基或胺基。
在一些实施方式中，G-M为选自 M1选自单键、C1-20亚烷基、C2-20亚烯基、C2-20亚炔基或胺基。
在一些实施方式中，G-M为
在一些实施方式中，G-M选自
在一些实施方式中，G-M选自
在一些实施方式中，x选自1至10。
在一些实施方式中，所述的药物连接子化合物选自下示的A-01～A-34，B-01～B-07， C-01～C-28： 












在一些实施方式中，本发明的药物连接子化合物选自：

在一些实施方式中，前文所述的药物连接子化合物可任选地被一个或多个适合的取代基所取代。
在一些实施方式中，前文所述的药物连接子化合物具有如下结构：
R10、R11、R12独立地选自氢、C1-6烷基、C3-6环烷基、5-12元杂环基、C6-10芳基、5-12元杂芳基、-C1-6烷基-C6-10芳基和-C1-6烷基-5-12元杂芳基；所述烷基、环烷基、杂环基、芳基和杂芳基任选被一个或多个选自羟基、CN、卤素、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C6-10芳基、5-12元杂芳基的取代基所取代；
R13和R14各自独立选自氢、C1-6烷基、C3-6环烷基和4-6元杂环基；所述烷基、环烷基和杂环基任选被一个或多个选自羟基、CN、卤素、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C6- 10芳基、5-12元杂芳基的取代基所取代；
R15选自氢、C1-6烷基、C3-6环烷基、卤代C1-6烷基、C1-6烷基-O-C1-6烷基、C2-6烯基、C2- 6炔基和3-6元杂环烷基；R16为H；或者，R15、R16及其所连接的原子共同形成4-7元环；所 述4-7元环任选被一个或多个选自羟基、CN、卤素、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C6-10芳基、5-12元杂芳基的取代基所取代。
在一些实施方式中，R10、R11、R12独立地选自氢、C1-6烷基、C3-6环烷基、C6-10芳基、苄基、羟基取代的苄基和吲哚基-C1-6烷基；
R13和R14各自独立选自氢、C1-6烷基、C3-6环烷基和4-6元杂环基；且
R15选自氢、C1-6烷基、C3-6环烷基、卤代C1-6烷基、C1-6烷基-O-C1-6烷基、C2-6烯基、C2- 6炔基和3-6元杂环烷基；R16为H；或者，R15、R16及其所连接的原子共同形成4-7元环。
在一些实施方式中，R10、R11、R12独立地选自氢、C1-4烷基、C3-6环烷基、苯基、苄基、对羟基苄基和吲哚基甲基。
在一些实施方式中，R13和R14各自独立选自氢、C1-4烷基、C3-6环烷基和4-6元杂环基。
在一些实施方式中，R13和R14各自独立选自氢、甲基、乙基、丙基、异丙基、丁基、异丁基、叔丁基、环丙基、环丁基、环戊基、环己基和4-6元杂环基。
在一些实施方式中，R15选自氢、C1-4烷基、C3-6环烷基、卤代C1-4烷基、C1-4烷基-O-C1-4烷基、C2-6烯基、C2-6炔基和3-6元杂环烷基；或者，R15、R16及其所连接的原子共同形成4-7元杂环烷基或4-7元杂芳基。
在一些实施方式中，R15选自氢、甲基、乙基、丙基、异丙基、丁基、异丁基、叔丁基、环丙基、环丁基、环戊基、环己基、卤代C1-4烷基、C1-4烷基-O-C1-4烷基、C2-6烯基、C2-6炔基和3-6元杂环烷基；或者，R15、R16及其所连接的原子共同形成4-7元杂环烷基或4-7元杂芳基。
在一些实施方式中，R10、R11、R12独立地选自氢、C1-4烷基、C3-6环烷基、苯基、苄基、对羟基苄基和吲哚基甲基；
R13和R14各自独立选自氢、甲基、乙基、丙基、异丙基、丁基、异丁基、叔丁基、环丙基、环丁基、环戊基、环己基和4-6元杂环基；
R15选自氢、甲基、乙基、丙基、异丙基、丁基、异丁基、叔丁基、环丙基、环丁基、环戊基、环己基、卤代C1-4烷基、C1-4烷基-O-C1-4烷基、C2-6烯基、C2-6炔基和3-6元杂环烷基；或者，R15、R16及其所连接的原子共同形成4-7元杂环烷基或4-7元杂芳基。
另一个方面，本发明提供了具有如下结构的中间体化合物：

其中，X选自苄氧羰基、叔丁氧羰基、芴甲氧羰基、烯丙氧羰基、三甲基硅乙氧羰基、甲氧羰基、乙氧羰基、邻苯二甲酰基、对甲苯磺酰基、三氟乙酰基、硝基苯磺酰基、苯甲酰基、特戊酰基、三苯甲基、4-甲氧基苯基二苯基甲基、二甲氧基三苯甲基、2,4-二甲氧基苄基、对甲氧基苄基和苄基；a为1至10的整数，优选为3至8的整数；R1、R2和D如前文所定义。
在一些实施方式中，X选自芴甲氧羰基(Fmoc)。
在一些实施方式中，D选自：
其中，R1,R2各自独立地选自C1-6烷基和卤素；
R3选自H和-CO-CH2OH；
R4和R5各自独立地选自H、卤素和羟基；或者R4和R5与相连碳原子连接成5-6元含氧杂环；
R6选自氢或-C1-4亚烷基-NRaRb；
R7选自C1-6烷基和-C1-4亚烷基-NRaRb；
其中Ra、Rb在每次出现时各自独立地选自H、C1-6烷基、-SO2-C1-6烷基和-CO-C1-6烷基。
另一个方面，本发明提供了具有如下结构的中间体化合物：



另一个方面，本发明提供抗体药物偶联物，其具有式Ab-[M-L-E-D]x所示结构，M、L、E、D、x如前文所定义，Ab是与抗原特异性结合的抗体或其抗原结合片段。
在一些实施方案中，所述抗体药物偶联物选自下示的ADC A-01～ADC A-34，ADC B-01～ADC B-07，ADC C-01～ADC C-28：下述图示中的Ab定义如前所述，其中抗体上的巯基与药物连接子化合物通过加成反应或者取代反应形成硫醚键进而得到完整的抗体药物偶联物，x代表药物载荷数量：













其中，表示抗体或其抗原结合片段中的巯基与连接体的具体连接方式。
组合物
在另一方面，本申请提供了组合物，所述可包含多个本文所述的ADC。所述组合物中的每个抗体分子可以与1、2、3、4、5、6、7、8、9或10个本发明所述的化合物偶联。因此，所述组合物的特征在于“药物-抗体比”(DAR)在约1至约10的范围内。 测定DAR的方法是技术人员熟知的，包括使用反相色谱或HPLC-MS的方法。
在一些实施方案中，所述抗体药物偶联物的DAR值(药物抗体偶联比)为1-10，例如：1～2，1～3，1～4，1～5，1～6，1～7，1～8，1～9，1～10，2～3，2～4，2～5，2～6，2～7，2～8，2～9，2～10，3～4，3～5，3～6，3～7，3～8，3～9，3～10，4～5，4～6，4～7，4～8，4～9，4～10，5～6，5～7，5～8，5～9，5～10，6～7，6～8，6～9，6～10，7～8，7～9，7～10，8～9，8～10，或9～10，优选为3～9，例如，3.0～3.5，3.0～4.0，3.0～4.5，3.0～5.0，3.0～5.5，3.0～6.0，3.5～4.0，3.5～4.5，3.5～5.0，3.5～5.5，3.5～6.0，3.5～6.5，3.5～7.0，3.5～7.5，3.5～8.0，4.0～4.5，4.0～5.0，4.0～5.5，4.0～6.0，4.0～6.5，4.0～7.0，4.0～7.5，4.0～8.0，4.5～5.0，4.5～5.5，4.5～6.0，4.5～6.5，4.5～7.0，4.5～7.5，4.5～8.0，5.0～5.5，5.0～6.0，5.0～6.5，5.0～7.0，5.0～7.5，5.0～8.0，5.5～6.0，5.5～6.5，5.5～7.0，5.5～7.5，5.5～8.0，6.0～6.5，6.0～7.0，6.0～7.5，6.0～8.5，6.5～7.0，6.5～7.5，6.5～8.5，7.0～7.5，7.0～9.0或7.5～9.0。
在一些实施方案中，所述抗体药物偶联物的DAR值为4～8。药物-连接体(药物连接子化合物)本领域技术人员应当理解，可模块化制备本申请所述抗体-药物偶联物。例如，先获得游离形式的“药物-连接体”(可理解为G-M-[L-E-D]x，其中G-M为与抗体或其抗原结合片段共价连接前的结构形式)，而后将其与抗体或其抗原结合片段共价连接得到本申请所述抗体药物偶联物。相应地，所述游离形式的“药物-连接体”中G-M通过取代反应(例如脱除其上的-SO2Me或-Br等结构)或者通过加成反应等方式与抗体或其抗原结合片段上的一个或多个巯基(-SH)、氨基(-NH2)或羧基(-COOH)连接，x选自1至10。
药物组合物
抗体药物偶联物和/或药物-连接体的药物组合物
在另一个方面，本申请提供一种药物组合物，其含有前文任一项所述的抗体药物偶联物或任选的前文任一项所述的药物-连接体，以及一种或多种药用辅料。
本文所述抗体药物偶联物通常与药学上可接受的胃肠外媒介一起以单位可注射形式配制，供胃肠外使用，例如推注、静脉注射、肿瘤内注射等。任选地，以冻干剂或溶液剂的形式将具有期望纯度的抗体药物偶联物与药学上可接受的稀释剂、载体、赋型剂或稳定剂混合(Remington’s Pharmaceutical Sciences(1980)16th edition,Osol,A.Ed.)。可以通过对于要治疗的个体适宜的任何路径施用本文所述抗体药物偶联物或含有所述抗体药物偶联物的药物组合物。
在某些实施方案中，所述药物组合物还可以包含另外的药学活性剂。
在某些实施方案中，所述另外的药学活性剂是具有抗肿瘤活性的药物。在某些实施方 案中，所述另外的药学活性剂选自B7-H3抑制剂、EGFR抑制剂、HER2抑制剂、HER3抑制剂、HER4抑制剂、IGFR-1抑制剂、mTOR抑制剂、PI3激酶抑制剂、c-met或VEGF抑制剂、化疗药物或其任意组合。
应用
1、抗体药物偶联物和/或药物-连接体的治疗用途
本文所述抗体药物偶联物、药物-连接体或药物组合物可以用于治疗多种疾病或病症，例如B7-H3阳性肿瘤、Her2阳性肿瘤。
因此，本申请提供前文任一项所述的抗体药物偶联物、药物-连接体、或含有其的药物组合物在制备预防和/或治疗和/或辅助治疗阳性肿瘤的药物中的用途。
本申请提供前文任一项所述的抗体药物偶联物、药物-连接体、或含有其的药物组合物在制备预防和/或治疗和/或辅助治疗B7-H3阳性肿瘤的药物中的用途。
本申请提供前文任一项所述的抗体药物偶联物、药物-连接体、或含有其的药物组合物在制备预防和/或治疗和/或辅助治疗HER2阳性肿瘤的药物中的用途。
同时，本申请还提供一种预防和/或治疗和/或辅助治疗阳性肿瘤的方法，其包括向由此需要的受试者施用有效量的前文任一项所述的抗体药物偶联物、药物连接体、或含有其的药物组合物的步骤。
本申请还提供一种预防和/或治疗和/或辅助治疗B7-H3阳性肿瘤的方法，其包括向由此需要的受试者施用有效量的前文任一项所述的抗体药物偶联物、药物连接体、或含有其的药物组合物的步骤。
本申请还提供一种预防和/或治疗和/或辅助治疗HER2阳性肿瘤的方法，其包括向由此需要的受试者施用有效量的前文任一项所述的抗体药物偶联物、药物连接体、或含有其的药物组合物的步骤。
本申请还提供了前文任一项所述的抗体药物偶联物、药物连接体或药物组合物在抑制B7-H3阳性肿瘤细胞增殖中的用途。
本申请还提供了前文任一项所述的抗体药物偶联物、药物连接体或药物组合物在抑制Her2阳性肿瘤细胞增殖中的用途。
在某些实施方案中，所述抗体药物偶联物、药物连接体或药物组合物被施用于体外细胞或受试者体内细胞；例如应用于受试者体内，以抑制受试者体内肿瘤细胞的增殖；或者，应用于体外肿瘤细胞(例如细胞系或者来自受试者的细胞)，以抑制体外肿瘤细胞的增殖。
本申请中，B7-H3阳性肿瘤包括实体瘤或血液系统恶性肿瘤，例如结直肠癌，胃癌，乳腺癌，前列腺癌，头颈部鳞癌，黑色素瘤，神经母细胞瘤，肉瘤，肺癌(例如，小细胞 肺癌，非小细胞肺癌等)、肾癌、膀胱癌、甲状腺癌、间皮瘤、胰腺癌、卵巢癌，子宫内膜癌、食管癌，肝癌、唾液腺癌、胆管癌、脑膜瘤。
本申请中，受试者优选为哺乳动物，例如牛科动物、马科动物、猪科动物、犬科动物、猫科动物、啮齿类动物、灵长类动物；例如，人。
在某些实施方案中，所述方法还包括向所述受试者施用第二疗法，所述第二疗法选自手术、化疗、放疗、免疫疗法、基因疗法、DNA疗法、RNA疗法、纳米疗法、病毒疗法、辅助疗法及其任意组合。在某些实施方案中，所述第二疗法可以与上述方法同时、分开或相继应用。
在某些实施方案中，本发明药物组合物所涉及的肿瘤选自乳腺癌，结直肠癌，头颈癌，肾透明细胞癌，肾乳头状细胞癌，肝癌，肺腺癌，肺鳞癌，前列腺癌，胃腺癌，甲状腺癌或其任意组合。
2、药物-连接体在制备偶联物中的用途
本发明前文所述的药物连接子用于制备偶联物。本发明的偶联物包含抗体药物偶联物等。
3、中间体化合物在制备药物连接子化合物中的用途
本发明前文所述的中间体化合物用于制备药物连接子化合物。
制备方法
本发明还提供了药物连接子化合物的制备方法，包含对中间体化合物进行脱保护的步骤。
方法一
在部分实施方案中，所述方法一包括制备化合物A-05a的方法，所述方法包括与式I化合物进行偶联的步骤；
A-05a：
其中，R1,R2各自独立地选自C1-6烷基和卤素；
R3选自H和-CO-CH2OH；
R4和R5各自独立地选自H、卤素和羟基；或者R4和R5与相连碳原子连接成5-6元含氧杂环；
R6选自氢或-C1-4亚烷基-NRaRb；
R7选自C1-6烷基和-C1-4亚烷基-NRaRb；
其中Ra、Rb在每次出现时各自独立地选自H、C1-6烷基、-SO2-C1-6烷基和-CO-C1-6烷基。
在一些实施方式中，R1,R2各自独立地选自C1-4烷基、F、Cl、Br和I。
在一些实施方式中，R1选自卤素、甲基、乙基、丙基和丁基，例如甲基和Cl。
在一些实施方式中，R2选自F、Cl、Br和I，例如F和Cl。
在一些实施方式中，R1选自甲基和Cl，R2选自F和Cl。
方法二
在部分实施方案中，所述方法二包括制备化合物A-14-a的方法，所述方法包括IM-5-a进行脱保护得到式IM-6-a化合物的步骤；

其中，X如前文所述中间体化合物所定义，R1和R2如方法一中所定义。
在一些实施方式中，所述脱保护的反应为：溶剂选自N,N-二甲基甲酰胺，并加入烷胺类化合物，例如二乙胺。
在一些实施方式中，所述脱保护在室温条件下进行，反应时间为1-5h，例如1-3h。
在部分实施方案中，所述方法二还进一步包括IM-6-a与IM-2进行反应得到化合物A-14-a的步骤。
在一些实施方式中，所述反应的溶剂选自N,N-二甲基甲酰胺和N,N-二甲基乙酰胺。
在一些实施方式中，所述反应还包括加入N,N-二异丙基乙胺的步骤。
方法三
在部分实施方案中，所述方法三包括制备化合物B-02-a的方法，所述方法包括将B-02-4a进行脱保护的步骤；

其中，X如前文中间体化合物中所述，R1、R2和R3如前文式一化合物中所述。
方法四
在部分实施方案中，所述方法四包括制备化合物C-07-a的方法，所述方法包括C-07-6与C-07-8a进行反应的步骤；
C-07-a：
其中，R1和R2如方法一中所定义。
在一些实施方式中，所述方法四中的C-07-8a通过C-07-7a脱保护制备得到；
C-07-7a：其中X如中间体化合物中所定义，R1和R2如方法一中所定义。
方法五
在部分实施方案中，所述方法五包括制备化合物C-10a的方法，所述方法包括C-10-5a与C-07-8a进行反应的步骤；
C-10-a：
其中，R1、R2和a如上文任意一项所定义。
在一些实施方式中，所述方法五中的C-10-5a通过C-10-4a脱保护制备得到；
在一些实施方式中，所述方法五中的C-10-4a通过C-10-3与化合物A制备得到；
方法六
在部分实施方案中，所述方法六包括制备化合物C-17a的方法，所述方法包括C-17-3a与C-07-8a进行反应的步骤；
其中，R1、R2和a如上文任意一项所定义；优选地，R1为甲基、R2为Cl且a为3或8；RX选自CH或N。
在一些实施方式中，所述方法六中的C-17-3a通过C-17-2a氧化反应制备得到；
在一些实施方式中，所述方法六中的C-17-2a通过C-17-1a脱保护制备得到；
在一些实施方式中，所述方法六中的C-17-1a通过C-10-2或C-19-3与化合物A进行偶联反应制备得到。
定义
除非在下文中另有定义，本文中所使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。并且，本文中所用的基因组学、核酸化学、分子生物学等实验室操作步骤均为相应领域内广泛使用的常规步骤。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。
术语“抗体”是指，通常由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成的免疫球蛋白分子。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循本领域已知的各种编号系统。
术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在重链和轻链的可变区中各含有三个CDRs，命名为CDR1、CDR2和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)、IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)或AbM编号系统(Martin ACR,Cheetham JC,Rees AR(1989)Modelling antibody hypervariable loops:A combined algorithm.Proc Natl Acad Sci USA 86:9268-9272)中的定义。对于给定的抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。
在本发明中，抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号系统确定，例如通过Kabat、Chothia、IMGT或AbM编号系统确定。在某些实施方案中，抗体或其抗原结合片段含有的CDR通过Chothia编号系统定义。
术语“构架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
术语抗体的“抗原结合片段”是指抗体的片段的多肽，例如全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括Fab片段、Fab'片段、F(ab)'2片段、F(ab)'3片段、Fd、Fv、scFv、di-scFv、(scFv)2、二硫键稳定的Fv蛋白(“dsFv”)、单结构域抗体(sdAb，纳米抗体)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。工程改造的抗体变体综述于Holliger等,2005；Nat Biotechnol,23:1126-1136中。
术语“Fd”意指由VH和CH1结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人,Nature 341:544 546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab’)2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段；术语“Fab’片段”意指还原连接F(ab’)2片段中两个重链片段的二硫键后所获片段，由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成。
术语“Fv”意指由抗体的单臂的VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为，六个CDRs赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDRs)也能够识别并结合抗原，尽管其亲和力可能低于完整的结合位点。
术语“Fc”意指，由抗体的第一重链的第二、第三恒定区与第二重链的第二、第三恒定区经二硫键结合而形成的抗体片段。抗体的Fc片段具有多种不同的功能，但不参与抗原的结合。
术语“scFv”是指，包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页 (1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键。在某些实施方案中，VH和VL结构域可以以任何合适的排列彼此相对定位。例如，包含NH2-VH-VH-COOH、NH2-VL-VL-COOH的scFv。
术语“单域抗体(single-domain antibody,sdAb)”具有本领域技术人员通常理解的含义，其是指由单个单体可变抗体结构域(例如单个重链可变区)所组成的抗体片段，其保持特异性结合全长抗体所结合的相同抗原的能力(Holt,L.等人,生物技术趋势(Trends in Biotechnology),21(11):484-490，2003)。单域抗体也称为纳米抗体(nanobody)。
上述各个抗体片段均保持了特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的抗体)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
术语“鼠源抗体”是指通过下述方法获得的抗体：融合免疫接种过的小鼠的B细胞与骨髓瘤细胞，筛选出既能无限增殖又能分泌抗体的鼠杂交融合细胞，继而进行筛选、抗体制备和抗体纯化；或者是指，由抗原侵入小鼠体内后B细胞分化增殖而形成浆细胞所分泌产生的抗体。
术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力、增强免疫应答的能力等。供体抗体可以是有预期性质(例如，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力和/或增强免疫应答的能力)的小鼠、大鼠、兔或非人灵长类动物(例如，食蟹猴)抗体。
术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中所使用的，术语“变体”，在多肽的情境中(包括多肽)也指包含已通过引入氨基酸残基置换、缺失或添加改变的氨基酸序列的多肽或肽。在某些情况下，术语“变体”还指已被修饰(即，通过将任何类型的分子共价连接至多肽或肽)的多肽或肽。例如，但非限制性地，多肽可以被修饰，例如通过糖基化、乙酰化、聚乙二醇化、磷酸化、酰胺化、通过已知保护/封闭基团进行的衍生化、蛋白水解切割、连接至细胞配体或其它蛋白质等。衍生多肽或肽可使用本领域技术人员已知的技术通过化学修饰来产生，所述技术包括但不限于特异性化学切割、乙酰化、甲酰化、衣霉素的代谢合成等。此外，变体具有与其所源自的多肽或肽相似、相同或改善的功能。
如本文中所使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以该相互作用的平衡解离常数(KD)或半最大效应浓度(EC50)表示。
两分子间的特异性结合性质可使用本领域公知的方法进行测定。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。“结合速率常数”(ka或kon)和“解离速率常数”(kdis或koff)两者都可通过浓度及缔合和解离的实际速率而计算得出(参见Malmqvist M,Nature,1993,361:186-187)。kdis/kon的比率等于解离常数KD(参见Davies等人,Annual  Rev Biochem,1990；59:439-473)。可用任何有效的方法测量KD、kon和kdis值。在某些实施方案中，可以使用生物发光干涉测量法(例如ForteBio Octet法)来测量解离常数。除此以外还可用表面等离子共振技术(例如Biacore)或Kinexa来测量解离常数。
术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
术语“包括”、“包含”、“具有”、“含有”或“涉及”及其在本文中的其它类似形式为包含性的(inclusive)或开放式的，且不排除其它未列举的元素或方法步骤。
术语“烷基”表示直链或支链烃基去掉1个氢原子得到的基团，例如“C1-20烷基”、“C1- 10烷基”、“C1-6烷基”、“C1-4烷基”、“C1-3烷基”等，具体实例包括但不限于：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、2-甲基丁基、新戊基、1-乙基丙基、正己基、异己基、3-甲基戊基、2-甲基戊基、1-甲基戊基、3,3-二甲基丁基、2,2-二甲基丁基、1,1-二甲基丁基、1,2-二甲基丁基、1,3-二甲基丁基、2,3-二甲基丁基、2-乙基丁基、1,2-二甲基丙基等。
术语“亚烷基”表示直链或支链烃基去掉2个氢原子得到的基团，例如“C1-20亚烷基”、“C1-10亚烷基”、“C3-10亚烷基”、“C5-8亚烷基”、“C1-6亚烷基”、“C1-4亚烷基”、“C1-3 亚烷基”等，具体实例包括但不限于：亚甲基、亚乙基、1,3-亚丙基、1,4-亚丁基、1,5-亚戊基或1,6-亚己基等。
术语“亚烯基”是指含有至少一个碳碳双键的直链或支链的烃基失去两个氢原子得到的二价基团，包括例如“C2-20亚烯基”、“C3-10亚烯基”、“C5-8亚烯基”等。其实例包括但不限于：亚乙烯基、1-亚丙烯基、2-亚丙烯基、1-亚丁烯基、2-亚丁烯基、1,3-亚丁二烯基、1-亚戊烯基、2-亚戊烯基、3-亚戊烯基、1,3-亚戊二烯基、1,4-亚戊二烯基、1-亚己烯基、2-亚己烯基、3-亚己烯基、1,4-亚己二烯基等。
术语“亚炔基”是指含有至少一个碳碳三键的直链或支链烃基失去两个氢原子得到的二价基团。包括例如“C2-20亚炔基”、“C3-10亚炔基”、“C5-8亚炔基”等。其实例包括但不限于：亚乙炔基、1-亚丙炔基、2-亚丙炔基、1-亚丁炔基、2-亚丁炔基、1,3-亚丁二炔基、1-亚戊炔基、2-亚戊炔基、3-亚戊炔基、1,3-亚戊二炔基、1,4-亚戊二炔基、1-亚己炔基、2-亚己炔基、3-亚己炔基、1,4-亚己二炔基等。
术语“脂杂环”是指含至少一个(例如1、2或3个)选自N、O和S的环成员的饱和或部分饱和的环状结构。具体实例包括但不限于5-6元脂杂环、5-6元含氮脂杂环、5-6元含氧脂杂环等，例如四氢呋喃、吡咯烷、哌啶、四氢吡喃等。
术语“杂芳环”是指含至少一个选自N、O和S的环成员的芳香环状结构。具体实例包括但不限于5-6元芳杂环、5-6元含氮芳杂环、5-6元含氧芳杂环等，例如呋喃、噻吩、吡咯、噻唑、异噻唑、噻二唑、噁唑、异噁唑、噁二唑、咪唑、吡唑、1,2,3-三唑、1,2,4-三唑、1,2,3-噁二唑、1,2,4-噁二唑、1,2,5-噁二唑、1,3,4-噁二唑、吡啶、嘧啶、哒嗪、吡嗪、1,2,3-三嗪、1,3,5-三嗪、1,2,4,5-四嗪等。
术语“芳香族环系”是指包含至少一个芳环(例如苯环等)或杂芳环(例如5-6元芳杂环，例如5-6元含氮芳杂环，例如嘧啶环等)的单环或多环体系，两个或更多个芳环和/或杂芳环可以形成稠合环或通过单键连接(例如二嘧啶基苯基等)，所述芳香族环系可以是二价或更高价态(例如三价或四价)，例如5-20元芳香族环系。
如本文中所使用的，术语“适合的取代基”是指本领域技术人员可根据化合物取代基的需要对化合物进行的变型。“适合的取代基”包括氧基(＝O)、卤素、氰基、NR8R9、羧基、巯基、羟基、酯基(例如-C1-6烷基-C(＝O)-OC1-6烷基)、C1-6烷基、C2-6烯基、C2-6炔基、C1-6烷基-O-C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C3-6环烷基、3-10元杂环基、5-10元杂芳基、C6-10芳基、苄基、羟基取代的苄基、吲哚基亚甲基和C1-6卤代烷氧基，R8、R9各自独立地选自H、C1-6烷基、C3-6环烷基、3-10元杂环基、5-10元杂芳基、C6-10芳基、C1- 6烷氧基、C1-6卤代烷基、C1-6卤代烷氧基、卤素、羟基、羧基和酯基(例如-C1-6烷基-C(＝O)- OC1-6烷基)。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。
如本文中所使用的，术语“预防”是指，为了阻止或延迟疾病或病症或症状(例如，肿瘤)在受试者体内的发生而实施的方法。如本文中所使用的，术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。为了本发明的目的，有益或所需的临床结果包括但不限于，减轻症状、缩小疾病的范围、稳定(即，不再恶化)疾病的状态，延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部)，无论是可检测或是不可检测的。此外，“治疗”还可以指，与期望的存活期相比(如果未接受治疗)，延长存活期。
如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如人。在某些实施方式中，所述受试者(例如人)患有肿瘤，或者，具有患有上述疾病的风险。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如，肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如，肿瘤)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
术语“癌症”“肿瘤”可互换使用，其是指以体内异常细胞的不受控生长为特征的一大类 疾病。不受管制的细胞分裂可能导致恶性肿瘤或侵入邻近组织的细胞的形成，并可能通过淋巴系统或血流转移到身体的远端部位。癌症包括良性和恶性癌症以及休眠肿瘤或微转移。癌症也包括血液学恶性肿瘤。
术语“血液学恶性肿瘤”包括淋巴瘤，白血病，骨髓瘤或淋巴恶性肿瘤，以及脾癌和淋巴结肿瘤。示例性淋巴瘤包括B细胞淋巴瘤和T细胞淋巴瘤。B细胞淋巴瘤，包括例如霍奇金淋巴瘤。T细胞淋巴瘤，包括例如皮肤T细胞淋巴瘤。血液学恶性肿瘤还包括白血病，例如继发性白血病或急性淋巴细胞性白血病。血液恶性肿瘤还包括骨髓瘤(例如多发性骨髓瘤)及其他血液学和/或B细胞或T细胞相关的癌症。
具体实施方式
以下通过具体实施方式的描述对本发明作进一步说明，但这并非是对本发明的限制。本领域技术人员根据本发明的教导，可以做出各种修改或改进，而不脱离本发明的基本思想和范围。
本发明涉及的序列的信息描述于下面的表中：





本文中所使用的缩写具有以下含义：
缩写       含义
CDR        免疫球蛋白可变区中的互补决定区
FR         抗体构架区：抗体可变区中除CDR残基以外的氨基酸残基
VH         抗体重链可变区
VL         抗体轻链可变区
IgG        免疫球蛋白G
IMGT       基于由Lefranc等人发起的国际免疫遗传学信息系统(The international ImMunoGeneTics information(IMGT))的编号系统，可参阅Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003。
Kabat      由Elvin A.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md., 1991)。
Chothia    由Chothia等人提出的免疫球蛋白编号系统，其是基于结构环区的位置鉴定CDR区边界的经典规则(参见，例如Chothia&Lesk(1987)J.
Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)。
AbM       CDR定义方式，来源于Martin的相关研究(Martin ACR,Cheetham JC,Rees AR(1989)Modelling antibody hypervariable loops:A combined algorithm.Proc Natl Acad Sci USA 86:9268-9272)。
mAb       单克隆抗体
EC50       产生50％功效或结合的浓度
IC50       产生50％抑制的浓度
ELISA     酶联免疫吸附测定
PCR       聚合酶链式反应
HRP       辣根过氧化物酶
KD        平衡解离常数
Ka        结合速率常数
Kd        解离速率常数
ADCC      抗体依赖性细胞介导的细胞毒性
CDC       补体依赖的细胞毒性
FACS      流式细胞仪技术
CDR-H1    免疫球蛋白重链可变区中的互补决定区1
CDR-H2    免疫球蛋白重链可变区中的互补决定区2
CDR-H3    免疫球蛋白重链可变区中的互补决定区3
CDR-L1    免疫球蛋白轻链可变区中的互补决定区1
CDR-L2    免疫球蛋白轻链可变区中的互补决定区2
CDR-L3    免疫球蛋白轻链可变区中的互补决定区3

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)的测定使用Bruker 400MHz核磁共振仪；氘代试剂为六氘代二甲基亚砜(DMSO-d6)；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、DMSO-d6：氘化二甲基亚砜。δ值用ppm值表示。
质谱(MS)的测定使用Agilent(ESI)质谱仪，型号为Agilent 6120B。
实施例一 N-((S)-10-苄基-1-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-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-6-(2,5-二氧代-2,5-二氢-1-H-吡咯- 1-基)己酰胺(M-01)
将化合物IM-1(0.40g,640.59μmol，其合成参考专利CN 111936169A)、依喜替康甲磺酸盐(0.37g,704.65μmol)溶于DMF(8mL)中，加入HATU(0.32g,832.77μmol)和DIPEA(0.25g,1.92mmol)，25℃反应4小时。减压除去DIPEA并加水冷冻干燥以除去大部分DMF得到粗品，粗品用制备高效液相色谱纯化(条件如下)得M-01化合物273mg。
色谱柱：Waters XBridge Prep C18 OBD 45mm×450mm×8.0μm
流动相A：乙腈；流动相B：水(0.05％三氟乙酸)
M-01结构表征数据如下：
ESI-MS(m/z):1034.4[M+H]+.
实施例二 N-((S)-10-苄基-1-(((1S,9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H，12H-苯并[de]吡喃[3',4':6,7]吲哚利嗪[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧基-5,8,11,14-四氮杂十六烷-16-基)-6-(2-(甲基磺基)嘧啶-5-基)己-5-酰胺(A-05)
氮气保护下，将2,5-二氧吡咯烷-1-基-6-(2-(甲磺酰基)嘧啶-5-基)己基-5-炔酸酯(IM-2，0.66g，1.80mmol)和(R)-16-氨基-10-苄基-6,9,12,15-四氧-3-氧-5,8,11,14-四氮杂十六酸(IM-3，0.75g,1.77mmol)加入DMF(19mL)中，升温至35℃反应16小时后向该体系中加入(1S，9S)-1-氨基-5-氯-9-乙基-9-羟基-4-甲基-1,2,3,9,12,15-六氢-10H,13H-苯并[de]吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-10,13-二酮(1-4，1.00g，1.77mmol)，冰水降温至5～15℃，加入DMTMM(0.98g,3.53mmol)，再滴入DIPEA(1.14g,8.84mmol)，25℃反应16小时。将反应液倒入DCM(600mL)、IPA(60mL)、水(100mL)混合液中搅拌10分钟，分出DCM相，盐水(100ml)洗，浓缩得到粗品，用制备高效液相色谱纯化后冷冻干燥得A-05化合物0.98g。
A-05分离纯化方法如下：
色谱柱：Waters SunFire Prep C18 OBD(5μm*19mm*150mm)
流动相A：乙腈；流动相B：水(0.05％甲酸)
A-05结构表征数据如下：
MS m/z(ESI):1107.3[M+H]+
1H NMR(400MHz,DMSO)δ9.10(s,2H),8.66-8.63(m,1H),8.51(d,J＝8.8Hz,1H),8.34-8.31(m,1H),8.21-8.19(m,1H),8.17-8.09(m,2H),8.08-8.04(m,1H),7.30(s,1H),7.26-7.15(m,5H),6.55(s,1H),5.56-5.55(m,1H),5.48-5.35(m,2H),5.25-5.10(m,2H),4.64(d,J＝6.4Hz,2H),4.45-4.44(m,1H),4.06-3.98(m,2H),3.77-3.52(m,6H),3.41( s,3H),3.25-3.12(m,2H),3.03-3.00(m,1H),2.83-2.72(m,1H),2.58-2.56(m,2H),2.48(s,3H),2.33-2.30(m,2H),2.21-2.13(m,2H),1.91-1.76(m,4H),0.87(t,J＝7.2Hz,3H).
实施例三 N-((S)-10-苄基-1-(((1S,9S)-5-氟-9-乙基-9-羟基-4-氯-10,13-二氧代-2,3,9,10,13,15-六氢-1H，12H-苯并[de]吡喃[3',4':6,7]吲哚利嗪[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧基-5,8,11,14-四氮杂十六烷-16-基)-6-(2-(甲基磺基)嘧啶-5-基)己-5-酰胺(A-07)
氮气保护下，将2,5-二氧吡咯烷-1-基-6-(2-(甲磺酰基)嘧啶-5-基)己基-5-炔酸酯(IM-2，21.6mg，0.059mmol)和(R)-16-氨基-10-苄基-6,9,12,15-四氧-3-氧-5,8,11,14-四氮杂十六酸(IM-3，24.5mg,0.058mmol)加入DMF(1mL)中，升温至35℃反应16小时后，向该体系中加入(1S,9S)-1-氨基-5-氟-9-乙基-9-羟基-4-氯-1,2,3,9,12,15-六氢-10H,13H-苯并[de]吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-10,13-二酮三氟乙酸盐(30.0mg，0.053mmol)，HATU(30mg,0.079mmol)和DIPEA(27.2mg,0.21mmol,)，反应体系于25℃反应16小时。反应液直接用制备高效液相色谱纯化后冷冻干燥得A-07化合物26.4mg。
A-07分离纯化方法如下：
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)
A-07结构表征数据如下：
ESI-MS(m/z):1111.3[M+H]+.
实施例四 N-((7S,10S,13S)-1-(((1S，9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧-2,3,9,10,13,15-六氢-1H，12H-苯并[de]吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-1-基)氨基)-7,10-二甲基-1,6,9,12-四氧-3-氧-5,8,11-三氮十四烷-13-基)-6-(2-(甲基磺基)嘧啶-5-基)己-5-酰胺(A-14)
步骤一：
将化合物IM-4(657mg,1.22mmol)和化合物1-4(500mg,1.11mmol)溶于N,N-二甲基甲酰胺(10mL)中，随后加入HATU(630.67mg,1.66mmol)和N,N-二异丙基乙胺(428mg,3.32mmol)，室温搅拌1小时。反应完毕后，反应液直接用制备高效液相色谱纯化后冷冻干燥得IM-5化合物700mg。
其制备方法如下：
色谱柱：Waters SunFire Prep C18 OBD(5μm*19mm*150mm)
流动相A：乙腈；流动相B：水(0.05％甲酸)
步骤二：
将化合物IM-5(500mg,0.513mmol)溶于N,N-二甲基甲酰胺(2mL)中，加入二乙胺(75.05mg,1.03mmol)，室温反应1小时。反应结束后，反应液直接用制备高效液相色谱纯化后冷冻干燥得IM-6化合物307mg。
其制备方法如下：
色谱柱：Waters SunFire Prep C18 OBD(5μm*19mm*150mm)
流动相A：乙腈；流动相B：水(0.05％甲酸)
步骤三：
将IM-6(170mg,0.226mmol)和化合物IM-2(90.83mg,0.249mmol)、溶解在N,N-二甲基甲酰胺(10mL)，加入N,N-二异丙基乙胺(29.21mg,0.226mmol)。反应液在室温条件下搅拌16小时。反应液直接用制备高效液相色谱纯化后冷冻干燥得A-14化合物50.56mg。
其结构表征数据如下：
MS m/z(ESI):1002.4[M+H]+
其制备方法如下：
色谱柱：Waters SunFire Prep C18 OBD(5μm*19mm*150mm)
流动相A：乙腈；流动相B：水(0.05％甲酸)
1H NMR(400MHz,DMSO)δ9.11(s,2H),8.68(t,J＝6.4Hz,1H),8.49(d,J＝8.8Hz,1H),8.16(s,1H),8.10(d,J＝7.2Hz,1H),8.01(d,J＝7.2Hz,1H),7.91(d,J＝6.8Hz,1H),7.31(s,1H),6.55(s,1H),5.65-5.55(m,1H),5.43(s,2H),5.21(s,2H),4.67-4.55(m,2H),4.29-4.15(m,3H),3.98(s,2H),3.41(s,3H),3.25-3.15(m,2H),2.57-2.56(m,2H),2.35-2.27(m,2H),2.22-2.12(m,2H),1.91-1.75(m,4H),1.23-1.09(m,9H),0.87(t,J＝7.2Hz,3H).
实施例五 4-((S)-2-(4-氨基丁基)-35-(4-((6-(2-(甲基磺酰基)嘧啶-5-基)己-5-炔酰胺基)甲基)-1H-1,2,3-三唑-1-基)-4,8-二氧代-6,12,15,18,21,24,27,30,33-九氧杂-3,9-二氮杂三十五烷酰胺基)苄基((S)-4-乙基-11-(2-(N-异丙基甲基磺酰胺基)乙基)-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-4-基)碳酸酯的合成(B-01)
步骤一：
室温下，将化合物B-01-1(413.40mg，0.251mmol,其合成参考专利CN111295389B)溶于二甲基亚砜和水(2.0mL：0.5mL)中，加入溴化亚铜(72.95mg，0.503mmol)和6-(2-(甲基磺酰基)嘧啶-5-基)-N-(丙-2-炔-1-基)-己-5-炔酰胺(95.10mg，0.302mmol)，搅拌反应1h后过滤，滤液经制备高效液相色谱纯化(条件如下)，得B-01-2化合物30.00mg。
色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μm
流动相A：乙腈；流动相B：水
步骤二：
将化合物B-01-2(30.00mg，0.02mmol)溶于二氯甲烷(1.0mL)反应液中加入三氟 乙酸(0.2mL)，室温反应30min。反应液减压浓缩后经制备高效液相色谱纯化(条件如下)，得B-01化合物的三氟乙酸盐20.00mg。
色谱柱：SunFire Prep C18 OBD 19mm×150mm×5.0μm
流动相A：乙腈；流动相B：水(0.05％三氟乙酸)
结构表征数据如下：ESI-MS(m/z):1631.7[M+H]+,816.0[M/2+H]+.
实施例六 4-((S)-2-(4-氨基丁基)-35-(4-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺基)甲基)-1H-1,2,3-三唑-1-基)-4,8-二氧基-6,12,15,18,21,24,27,30,33-九氧杂-3,9-二氮杂三十五烷酰胺基)苄基((1S,9R)-9-乙基-5-氟-1-(2-羟基乙酰胺基)-4-甲基-10,13-二氧基-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-9-基)碳酸酯(B-02)
步骤一:
25℃下,将1-1的甲磺酸盐(30.00mg,56.44μmol)溶于N,N-二甲基甲酰胺(1mL)中，依次加入1H-苯并三唑-1-基氧三吡咯烷基六氟磷酸盐(58.74mg,112.88μmol)、N,N-二异丙基乙胺(43.76mg,338.63μmol)和2-((叔丁基二苯基硅基)氧基)乙酸(26.62mg，84.66μmol)，保持25℃反应1小时，用液质联用色谱监测反应，反应完成后向反应液中加入水，乙酸乙酯萃取，有机相合并，经硫酸钠干燥后减压浓缩，粗品经薄层层析分离(二氯甲烷:甲醇＝15:1)得到B-02-1化合物27.00mg。
步骤二:
0℃下，将B-02-1(20mg,27.33μmol)溶于二氯甲烷(2mL)中，依次加入4-二甲氨基吡啶(26.71mg,218.61μmol)和三光气(8.11mg,27.33μmol)的二氯甲烷溶液(0.5 mL)，保持0℃反应0.5小时；用氮气置换掉残余三光气后滴加入(S)-2-(32-叠氮基-5-氧代-3,9,12,15,18,21,24,27,30-九氧杂-3,9-二氮杂三十五烷酰胺基)-N-(4-(羟甲基)苯基)-6-(((4-甲氧基苯基)二苯甲基)氨基)己酰胺(43.46mg,40.99μmol)的二氯甲烷溶液(1mL)，保持0℃反应0.5小时；用液质联用色谱监测反应，反应完成后将反应液浓缩，粗品经薄层层析分离(二氯甲烷:甲醇＝15:1)纯化得到B-02-2化合物30.00mg。
步骤三:
25℃下，将B-02-2(250.00mg,137.51μmol)溶于DMSO(2mL)和水(0.4mL)混合溶剂中，加入6-(2-(甲磺酰基)嘧啶-5-基)-N-(丙-2-炔-1-基)己-5-炔酰胺(62.98mg,206.26μmol)和溴化亚铜(39.45mg,275.01μmol)，保持25℃反应1小时；用液质联用色谱监测反应；反应完成后将反应液用制备高效液相色谱纯化(条件如下)，制备液冻干得到B-02-3化合物150.00mg。
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)
步骤四:
25℃下，将B-02-3(150mg,49.45μmol)溶于四氢呋喃(1mL)中,滴加入四丁基氟化铵(1M四氢呋喃溶液)/冰醋酸混合液(v/v＝13/1)(50uL),保持25℃反应0.5小时，用液质联用色谱监测反应；反应完成后将反应液用制备高效液相色谱纯化(条件如下)，制备液冻干得到B-02-4化合物50.00mg。
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)
步骤五:
25℃下，将B-02-4(50mg,26.52μmol)溶于二氯甲烷(1mL)中，加入三氟乙酸(60.49mg,530.49μmol)，保持25℃反应0.5小时；用液质联用色谱监测反应；反应完成后将反应液浓缩，粗品用制备高效液相色谱纯化(条件如下)，制备液冻干得到B-02化合物23.69mg。
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)
B-02结构表征数据如下:
ESI-MS(m/z):1613.6[M+H]+.
实施例七 N-((7S,10S,13S)-1-(((1S，9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧-2,3,9,10,13,15-六氢-1H，12H-苯并[de]吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-1-基)胺基)-7,10,13-三甲基-1,6,9,12,15-五氧-3,17,20,23-四氧-5,8,11,14-四氮五碳烷-25-基)-3,5-双(2-(甲基磺基)嘧啶-4-基)苯酰胺(C-07)

步骤一：
将原料C-07-1(4.80g,16.33mmol)、三丁基(2-甲基磺胺基嘧啶-4-基)锡(16.27g,39.19mmol)和双三苯基磷二氯化钯(2.29g,3.27mmol)溶于1，4-二氧六环(100mL)中，反应体系在氮气氛围下于110℃搅拌反应5小时，LC-MS监控反应，浓缩反应体系，经柱层析纯化(EA/PE＝0-50％)得C-07-2化合物1.36g。
步骤二：
将化合物C-07-2(510mg，1.33mol)、NaOH(212.24mg,5.31mmol)溶于THF(12.5mL)、MeOH(12.5mL)和H2O(2.5mL)中。25℃搅拌反应2小时，LC-MS监控反应，用3N HCl调节体系pH约为2，析出大量固体，过滤，收集滤饼，干燥，得到C-07-3化合物380mg。
步骤三：
将化合物C-07-3(315mg,850.32μmol)、2-[2-[2-(2-氨基乙氧基)乙氧基]乙氧基乙酸叔丁酯(246.31mg,935.35μmol)、HATU(484.99mg,1.28mmol)，DIPEA(329.69mg,2.55mmol)加到DMF(3mL)中，25℃反应2h。LC-MS监控反应。反应液用制备高效液相色谱纯化后冷冻干燥得C-07-3化合物40mg。
其制备方法如下：
色谱柱：Waters XBridge Prep C18OBD(5μm*19mm*150mm)
流动相A：乙腈；流动相B：水(0.05％甲酸)
步骤四：
将化合物C-07-4(40mg,64.96μmol)溶于DCM(3mL)和TFA(1.5mL)中，25℃反应1.5小时，LC-MS监控反应，浓缩反应体系至干得C-07-5化合物36mg。
步骤五：
将化合物C-07-5(26mg,46.46μmol)、高碘酸钠(99.37mg,464.57μmol)、RuCl3·H2O(9.64mg,46.46μmol)，溶于ACN(15mL)和水(7.5mL)中，25℃反应40分钟，LC-MS监控反应，加入水和乙酸乙酯萃取，乙酸乙酯层浓缩得C-07-6化合物28mg。
步骤六：
将化合物依喜替康甲磺酸盐(600mg,1.13mmol)、(5S，8S，11S)-1-(9H-芴-9-基)-5,8,11-三甲基-3,6,9,12-四氧基-2,15-二氧基-4,7,10,13-四氮杂庚烷-17-酸(IM-4，610.17mg,1.13mmol)、HATU(643.81mg,1.69mmol)，DIPEA(437.65mg,3.39mmol)加到DMF(6mL)中，25℃反应16h。LC-MS监控反应。向反应液中加入水，析出大量固体，过滤，收集固体，DCM溶解固体，浓缩得粗品，经柱层析纯化(DCM/MeOH＝0-10％)，得C-07-7化合物660mg。
步骤七：
将化合物C-07-7(660mg,688.94μmmol)溶于N,N-二甲基甲酰胺(6mL)中，加入二乙胺(251.94mg,3.44mmol)，室温反应1小时。反应结束后，反应液直接用制备高效液相色谱纯化后冷冻干燥得C-07-8化合物325mg。
其制备方法如下：
色谱柱：Waters SunFire Prep C18 OBD(5μm*19mm*150mm)
流动相A：乙腈；流动相B：水(0.05％甲酸)
步骤八：
将化合物C-07-6(15.95mg,25.58μmol)、C-07-8(20mg,25.58μmol)、HATU(14.59mg,38.37μmol)，DIPEA(9.92mg,76.75μmol)加到DMF(3mL)中，25℃反应2h。LC-MS监控反应。反应液用制备高效液相色谱纯化后冷冻干燥得C-07化合物7mg。
其结构表征数据如下：
ESI-MS(m/z):1342.4[M+H]+.
其制备方法如下：
色谱柱：Waters XBridge Prep C18OBD(5μm*19mm*150mm)
流动相A：乙腈；流动相B：水(0.05％甲酸)
实施例八 N-((7S,10S,13S)-1-(((1S，9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧-2,3,9,10,13,15-六氢-1H，12H-苯并[de]吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-1-基)胺基)-7,10,13-三甲基-1,6,9,12,15-五氧-3,17,20,23-四氧-5,8,11,14-四氮五碳烷-25-基)-3,5-双(2-(甲基磺基)嘧啶-5-基)苯酰胺(C-10)
步骤一：
将原料C-10-1(720mg，2.45mmol)、2-甲硫基嘧啶-5-硼酸(874mg，5.14mmol)XPhosPd G3(207mg,245μmol)、K3PO4(1.56g,7.35mmol)加到dioxane(12mL)and H2O(4mL)中，反应体系在氮气氛围下于90℃搅拌反应3小时，LC-MS监控反应，垫硅藻土过滤，往滤液中加水和乙酸乙酯，萃取，浓缩，得到粗品，经柱层析纯化(EA/PE＝0-25％)得到C-10-1化合物710mg。
步骤二：
将化合物C-10-1(650mg，1.69mol)、氢氧化锂(121mg,5.07mmol)溶于THF(2mL)、MeOH(2mL)和H2O(2mL)中。25℃搅拌反应2小时，LC-MS监控反应，用1N HCl调节体系pH约为2，析出大量固体，过滤收集滤饼，干燥得C-10-2化合物560mg。
步骤三：
将化合物C-10-2(450.80mg,1.22mmol)溶于DCM(10mL)中，将m-CPBA(2.46g,12.1mmol,纯度为85％)加到反应体系中，25℃反应12小时，LC-MS监控反应。氮气流吹 干溶剂得到粗品，粗品经制备高效液相色谱纯化后冷冻干燥得C-10-3化合物153mg。
其制备方法如下：
色谱柱：Phenomenex Luna C18 200*40mm*10um。
流动相A：乙腈；流动相B：水(0.05％盐酸)
流动相：[water(HCl)-ACN]；B％:13％-43％,10min)。
步骤四：
将化合物C-10-3(140mg,322.25μmol)、2-[2-[2-(2-氨基乙氧基)乙氧基]乙氧基乙酸叔丁酯(84.86mg,322.25μmol)、HATU(183.80mg,483.37μmol)，DIPEA(124.94mg,966.75μmol)加到DMF(4mL)中，25℃反应2h。LC-MS监控反应。反应液用制备高效液相色谱纯化后冷冻干燥得C-10-4化合物51mg。
其制备方法如下：
色谱柱：Waters XBridge Prep C18OBD(5μm*19mm*150mm)
流动相A：乙腈；流动相B：水(0.05％甲酸)
步骤五：
将化合物C-10-4(50mg,73.56μmol)加到DCM(2mL)和TFA(1mL)中，25℃反应1h，LC-MS监控反应，浓缩反应体系至干得到C-10-5化合物45mg。
步骤六：
将化合物C-10-5(31.91mg,51.17μmol)、C-07-8(40mg,51.17μmol)、HATU(29.18mg,76.75μmol)，DIPEA(19.84mg,153.50μmol)加到DMF(3mL)中，25℃反应2h。LC-MS监控反应。反应液用制备高效液相色谱纯化后冷冻干燥得C-10化合物13mg。
其结构表征数据如下：
ESI-MS(m/z):1342.5[M+H]+.
其制备方法如下：
色谱柱：Waters XBridge Prep C18OBD(5μm*19mm*150mm)
流动相A：乙腈；流动相B：水(0.05％甲酸)
实施例九 N-((7S,10S,13S)-1-(((1S，9S)-9-乙基-5-氯-9-羟基-4-甲基-10,13-二氧-2,3,9,10,13,15-六氢-1H，12H-苯并[de]吡喃[3',4':6,7]吲哚嗪[1,2-b]喹啉-1-基)胺基)-7,10,13-三甲基-1,6,9,12,15-五氧-3,17,20,23-四氧-5,8,11,14-四氮五碳烷-25-基)-3,5-双(2-(甲基磺基)嘧啶-5-基)苯酰胺(C-17)
步骤一：
将C-10-2(3.00g,8.10mmol)和3-[2-[2-(2-氨基乙氧基)乙氧基]乙氧基]-丙酸叔丁酯(2.25g,8.10mmol)加入到DMF(3mL)，依次加入HOBt(3.28g,24.3mmol),EDCI(4.66g,24.3mmol)和DIPEA(4.19g,32.4mmol,5.64mL)，升至60℃反应2小时。向反应液中加入水(50mL)，用乙酸乙酯萃取(30mL x 3)，合并有机相用无水硫酸钠干燥,过滤浓缩得C-17-1粗品(3.8g,4.75mmol)，未经纯化直接用于下一步.
步骤二：
将C-17-1(3.40g,5.40mmol)溶于二氯甲烷(30mL)中，加入三氟乙酸(10.8g,94.2mmol,7mL)。反应液25℃搅拌2小时.将反应液直接浓缩后，经制备高效液相色谱纯化后冷冻干燥得C-17-2(2.09g,3.64mmol)。
其制备方法如下：
色谱柱：Phenomenex luna C18(250mm*70mm*10μm)
流动相A：乙腈；流动相B：水(0.05％甲酸)
步骤三：
将C-17-2(56mg,97.61μmol)加到乙腈(6mL)和水(3mL)中，再将高碘酸钠(208.79mg,976.15μmol)和三氯化钌水合物(8.10mg,39.05μmol)加到反应体系中，25℃搅拌反应30分钟，LC-MS监控反应，加入水和乙酸乙酯萃取，浓缩得C-17-3(60mg)。
步骤四：
将IM-6(20mg,25.06μmol)、C-17-3(16mg,25.06μmol)、HATU(19.05mg,50.11μmol)、DIPEA(16.19mg,125.28μmol)依次加入到DMF(3mL)中，反应体系于25℃反应1小时。反应液直接用制备高效液相色谱纯化后冷冻干燥得C-17(16mg)。
其结构表征数据如下：
ESI-MS(m/z):1371.4[M+H]+.
其制备方法如下：
色谱柱：Waters XBridge Prep C18OBD(5μm*19mm*150mm)
流动相A：乙腈；流动相B：水(0.05％甲酸)
实施例十 N-((7S,10S,13S)-1-(((1S,9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-1-基)氨基)-7,10,13-三甲基-1,6,9,12,15-五氧代-3,18,21,24-四氧代-5,8,11,14-四氮杂六烷-26-基)-2,6-双(2-(甲基磺酰基)嘧啶-5-基)异烟酰胺(C-19)
步骤一：
将C-19-1(5.00g,16.9mmol),(2-(甲硫基)嘧啶-5-基)硼酸(6.34g,37.3mmol),XPhos Pd G3(1.44g,1.70mmol),磷酸钾(10.80g,50.9mmol)加入到1,4-二氧六环(51.0mL)和水(17.0mL)中，反应体系经氮气置换3次后于100℃反应5小时。待反应体系冷却至室温后，向反应液中加入水(50.0mL)，过滤，滤液浓缩得粗品，用石油醚打浆后再次过滤，滤饼真空干燥后得C-19-2(5.65g)。
步骤二：
将C-19-2(5.26g,13.7mmol)溶于THF(30.0mL)、MeOH(30.0mL)和水(30.0mL)中，加入LiOH·H2O(1.72g,40.9mmol)，于25℃搅拌2小时。反应液用1N盐酸水溶液调节pH至3，有固体析出，过滤，滤饼真空干燥得C-19-3(4.20g)。
步骤三：
将C-19-3(1.50g,4.04mmol)和3-(2-(2-氨基乙氧基)乙氧基乙氧基乙基)丙酸 叔丁酯(1.12g,4.04mmol)溶于DMF(20.0mL)中，依次加入HOBt(1.64g,12.1mmol)、EDCI(2.32g,12.1mmol)和DIPEA(2.09g,16.2mmol)，升温至60℃搅拌2小时。待反应体系冷却至室温，向反应液中加入水(10.0mL)和乙酸乙酯(20.0mL)，水相用乙酸乙酯(25.0mL*2)萃取两次，合并有机相用无水硫酸钠干燥,过滤，滤液浓缩得C-19-4粗品(2.50g)，未经纯化直接用于下一步。
步骤四：
将C-19-4(2.50g,3.96mmol)溶于二氯甲烷(3.00mL)中，加入TFA(4.61g,40.4mmol)后，反应体系于25℃搅拌12小时。将反应液直接浓缩后，经制备高效液相色谱纯化后冷冻干燥得C-19-5(1.20g)。
其制备方法如下：
色谱柱：Phenomenex luna C18(150mm*25mm*10μm)
流动相A：乙腈；流动相B：水(0.05％甲酸)
步骤五：
将C-19-5(1.10g,1.91mmol)溶于乙腈(30mL)和水(15mL)的混合溶剂中,加入三氯化钌水合物(39.70mg,0.19mmol)和高碘酸钠(4.09g,19.14mmol)，反应体系于25℃反应1小时后，经水(50mL)和乙酸乙酯(80mL)萃取，有机相浓缩得到粗品，粗品经柱层析纯化(MeOH/DCM＝10～20％)，浓缩得C-19-6(130mg)。
步骤六：
将IM-6(20.0mg,0.025mmol)和C-19-6(16.0mg,0.025mmol)加入DMF(1mL)中搅拌溶解,加入HATU(19.0mg,0.050mmol)和DIPEA(12.9mg,0.100mmol)，室温反应2小时，反应液直接用制备高效液相色谱纯化后冷冻干燥得C-19(20.4mg)。
其结构表征数据如下：
ESI-MS(m/z):1372.4[M+H]+.
色谱柱：Waters XBridge Prep C18OBD(5μm*19mm*150mm)
流动相A：乙腈；流动相B：水(0.05％甲酸)
实施例十一 N-((7S,10S,13S)-1-(((1S,9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-1-基)氨基)-7,1,13-三甲基-1,6,9,12,15-五氧代-3,18,21,24,27,30,33,36,39-壬氧基-5,8,11,14-四氮杂基四烷-41-基)-3,5-双(2-(甲基磺酰基)嘧啶-5-基)苯甲酰胺(C-21)
步骤一：
将C-10-2(3.00g,8.10mmol)和1-氨基-3,6,9,12,15,18,21,24-八氧杂庚烷-27-酸叔丁酯(4.03g,8.10mmol)加入到DMF(40mL)中，依次加入HOBt(3.28g,24.3mmol),EDCI(4.66g,24.3mmol)和DIPEA(4.19g,32.4mmol,5.64mL)，反应体系于60℃搅拌2小时。向反应液中加入水(100mL)和乙酸乙酯萃取(60mL x 3)，有机相合并后用无水硫酸钠干燥,过滤，浓缩得C-21-1(4.20g,4.14mmol)，未经纯化直接用于下一步。
步骤二：
将C-21-1(3.60g,4.24mmol)溶于二氯甲烷(30mL)中，加入TFA(15.3g,134mmol,10mL)，反应体系于25℃搅拌6小时。向反应液加入水(60mL)和乙酸乙酯萃取(40mL x 3)。有机相合并后用无水硫酸钠干燥,过滤，浓缩得粗品，粗品经制备高 效液相色谱纯化后冷冻干燥得C-21-2(2.93g,3.63mmol)。
其制备方法如下：
色谱柱：Phenomenex luna C18(250mm*70mm*10μm)
流动相A：乙腈；流动相B：水(0.05％甲酸)
步骤三：
将C-21-2(148mg，0.186mmol)加到乙腈(15mL)和水(7.5mL)中，再将高碘酸钠(398.71mg，1.86mmol)和三氯化钌水合物(15.47mg，74.56μmol)加到反应体系中，25℃搅拌反应30分钟，反应体系经水和乙酸乙酯萃取，浓缩得C-21-3(155mg)。
步骤四：
将IM-6(27.91mg,34.97μmol)、C-21-3(30mg,34.97μmol)、HATU(26.59mg,69.93μmol)，DIPEA(22.60mg,174.84μmol)加到DMF(3mL)中，反应体系于25℃反应1小时。反应液经高效液相色谱纯化后冷冻干燥得C-21(15mg)。
其结构表征数据如下：
ESI-MS(m/z):1591.7[M+H]+.
其制备方法如下：
色谱柱：Waters XBridge Prep C18OBD(5μm*19mm*150mm)
流动相A：乙腈；流动相B：水(0.05％甲酸)
实施例十二 N-((7S,10S,13S)-1-(((1S,9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-1-基)氨基)-7,1,13-三甲基-1,6,9,12,15-五氧代-3,18,21,24,27,30,33,36,39-壬氧基-5,8,11,14-四氮杂四烷-4-基)-2,6-双(2-(甲基磺酰基)嘧啶-5-基)异烟酰胺(C-23)
步骤一：
将C-19-3(1.50g,4.04mmol)和1-氨基-3,6,9,12,15,18,21,24-八氧杂庚烷-27-酸叔丁酯(2.01g,4.04mmol)加入到DMF(20.0mL)中，依次加入HOBt(1.64g,12.1mmol)、EDCI(2.32g,12.1mmol和DIEA(2.09g,16.2mmol)，升温至60℃搅拌2小时。冷却到室温，向反应液中加入水(10.0mL)和乙酸乙酯(20.0mL)分液，水相用乙酸乙酯(25.0mL x 2)萃取两次，合并有机相用无水硫酸钠干燥,过滤浓缩得粗品C-23-1(3.00g)，直接用于下一步。
步骤二：
将C-23-1(3.00g,3.53mmol)加入到二氯甲烷(10.0mL)中，加入TFA(15.4g,134mmol)后于25℃搅拌12小时。反应液直接浓缩得粗品，粗品经制备高效液相色谱纯化后冷冻干燥得C-23-2(1.20g)。
其制备方法如下：
色谱柱：Welch Ultimate C18(150mm*25mm*5μm)
流动相A：乙腈；流动相B：水(0.05％甲酸)
步骤三：
将C-23-2(500mg,0.63mmol)加到乙腈(10mL)和水(5mL)中，加入三氯化钌水合物(13.0mg,0.063mmol)和高碘酸钠(1.35g,6.29mmol)，体系于25℃反应1小时后用水(10ml)和乙酸乙酯(40ml)萃取，有机相浓缩得到粗品，粗品经柱层析纯化(MeOH/DCM＝10～20％)并浓缩得C-23-3(350mg)。
步骤四：
将IM-6(20.0mg,0.025mmol)和C-23-3(21.5mg,0.025mmol)溶于DMF(1mL)，加入HATU(19.0mg,0.050mmol)和DIPEA(12.9mg,0.100mmol)后室温反应2小时，反应液直接用制备高效液相色谱纯化后冷冻干燥得C-23(17.0mg)。
其结构表征数据如下：
ESI-MS(m/z):1592.6[M+H]+.
其制备方法如下：
色谱柱：Waters XBridge Prep C18OBD(5μm*19mm*150mm)
流动相A：乙腈；流动相B：水(0.05％甲酸)
实施例十三 4-((S)-2-(4-氨基丁基)-35-(4-((6-(2-(甲基磺酰基)嘧啶-5-基)己基-5-炔酰胺基)甲基)-1H-1,2,3-三唑-1-基)-4,8-二氧代-6,12,15,18,24,27,30,33-壬氧基-3,9-二氮杂五氮杂三酰氨基)苄基((1S,9R)-5-氯-9-乙基-1-(2-羟基乙酰胺基)-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[d]吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-9-基)碳酸酯(B-03)
步骤一:2-(((1S,9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[d]吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-1-基)氨基)-2-氧代乙酸乙酯(B-03-1)的制备
将(1S,9S)-1-氨基-5-氯-9-乙基-9-羟基-4-甲基-1,2,3,9,12,15-六氢-10H,13H-苯并[d]吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-10,13-二酮(2g,3.65mmol)溶于DMF(50mL),滴入DIPEA(1.18g,9.12mmol,1.59mL),冰浴冷却搅拌下滴入乙酰氧基乙酰氯(548.12mg,4.01mmol,431.59μL)，继续搅拌反应1小时。将反应液加入0.1M稀盐酸水溶液中析出固体，过滤。将滤饼溶于二氯甲烷和甲醇中，无水硫酸钠干燥后过滤浓缩得粗品，经硅胶柱纯化(甲醇/二氯甲烷＝0％～5％)再次浓缩得标题化合物(1.7g，3.077mmol)
其结构表征数据如下：
ESI-MS(m/z):552.2[M+1]+.
步骤二:2-(((1S,9S)-9-(((4-((S)-35-叠氮基-2-(4-(4-甲氧基苯基)二苯基甲基)氨基)丁基)-4,8-二氧代-6,12,15,18,21,24,27,30,33-壬氧基-3,9-二氮杂五苯并三酰氨基)苄基)氧基)羰基) 氧基-5-氯-9-乙基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲唑嗪[1,2-b]喹啉-1-基)氨基)-2-氧代乙酸乙酯(B-03-2)的制备
将2-(((1S,9S)-5-氯-9-乙基-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[d]吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-1-基)氨基)-2-氧代乙酸乙酯(500mg,0.905mmol)和DMAP(885.33mg,7.25mmol)溶于干燥二氯甲烷(5mL)中，氮气保护下冷却至0℃，滴入三光气(268.81mg,0.905mmol)的二氯甲烷溶液(5mL)，保温搅拌反应0.5小时。缓慢滴入(S)-2-(32-叠氮基-5-氧代-3,9,12,15,18,21,24,27,30-壬氧基-6-氮杂三硝基氨基)-N-(4-(羟甲基)苯基)-6-(((4-甲氧基苯基)二苯基甲基)氨基)己酰胺(1.44g,1.36mmol)的二氯甲烷溶液，自然恢复室温反应4小时。加水淬灭反应，用二氯甲烷萃取3次(100ml x 3)后合并有机相，饱和食盐水洗涤后干燥浓缩。硅胶柱纯化(MeOH/DCM＝0％～5％)得标题化合物(498mg，0.304mmol)
其结构表征数据如下：
ESI-MS(m/z):1352.8[M+1]+.
步骤三:4-((S)-35-叠氮基-2-(4-((4-甲氧基苯基)二苯甲基)氨基)丁基)-4,8-二氧代-6,12,15,18,24,27,30,33-壬氧基-3,9-二氮杂五氮杂三酰氨基)苄基((1S,9S)-5-氯-9-乙基-1-(2-羟基乙酰胺基)-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]氮茚并[1,2-b]喹啉-9-基)碳酸酯(B-03-3)的制备
将2-(((1S,9S)-9-(((4-((S)-35-叠氮基-2-(4-(4-甲氧基苯基)二苯基甲基)氨基)丁基)-4,8-二氧代-6,12,15,18,21,24,27,30,33-壬氧基-3,9-二氮杂五苯并三酰氨基)苄基)氧基)羰基)氧基-5-氯-9-乙基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲唑嗪[1,2-b]喹啉-1-基)氨基)-2-氧代乙酸乙酯(200mg,0.122mmol)溶于THF(3mL)和MeOH(3mL)中，搅拌下滴入碳酸钠(25.88mg,0.224mmol)水溶液(1mL)，滴加完毕后继续搅拌1小时。向反应液中滴入稀盐酸中和反应，减压浓缩后直接进行下一步。
其结构表征数据如下：
ESI-MS(m/z):1596.7[M+1]+.
步骤四:4-((S)-2-(4-氨基丁基)-35-叠氮基-4,8-二氧代-6,12,15,18,21,24,27,30,33-壬氧基-3,9-二氮杂五苯并三酰氨基)苄基((1S,9S)-5-氯-9-乙基-1-(2-羟基乙酰胺基)-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-9-基)碳酸酯(B-03-4)的制备
将4-((S)-35-叠氮基-2-(4-((4-甲氧基苯基)二苯甲基)氨基)丁基)-4,8-二氧代-6,12,15,18,24,27,30,33-壬氧基-3,9-二氮杂五氮杂三酰氨基)苄基((1S,9S)-5-氯-9-乙基-1-(2-羟基乙酰胺基)-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]氮茚并[1,2-b]喹啉-9-基)碳酸酯(190mg,119.04μmol)溶于二氯甲烷(5mL)，加入三氟乙酸(0.5mL)后继续反应1小时。向反应液中滴入饱和碳酸氢钠水溶液中和后分液，将有机相浓缩得粗品。经反相柱色谱(乙腈/1％甲酸水溶液＝0％～50％)纯化后冷冻干燥得标题化合物(95mg,69.35μmol)。
其结构表征数据如下：
ESI-MS(m/z):1323.6[M+1]+.
步骤五:4-((S)-2-(4-氨基丁基)-35-(4-((6-(2-(甲基磺酰基)嘧啶-5-基)己基-5-炔酰胺基)甲基)-1H-1,2,3-三唑-1-基)-4,8-二氧代-6,12,15,18,24,27,30,33-壬氧基-3,9-二氮杂五氮杂三酰氨基)苄基((1S,9R)-5-氯-9-乙基-1-(2-羟基乙酰胺基)-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[d]吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-9-基)碳酸酯(B-03)的制备
将4-((S)-2-(4-氨基丁基)-35-叠氮基-4,8-二氧代-6,12,15,18,21,24,27,30,33-壬氧基-3,9-二氮杂五苯并三酰氨基)苄基((1S,9S)-5-氯-9-乙基-1-(2-羟基乙酰胺基)-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]中氮茚并[1,2-b]喹啉-9-基)碳酸酯(90mg,0.066mmol)和6-(2-(甲基磺酰基)嘧啶-5-基)-N-(丙-2-炔-1-基)己-5-炔酰胺(24.07mg,0.079mmol)溶于DMSO(2mL)和水(0.2mL)中，加入溴化亚铜(9.42mg,0.066mmol)后继续搅拌2小时。将反应液直接过滤，将反应液直接过滤浓缩的粗品，经制备高效液相色谱纯化后冷冻干燥得标题化合物(42.2mg，24.69μmol)。
其结构表征数据如下：
ESI-MS(m/z):1628.7[M+1]+.
其制备高效液相色谱方法如下：
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)
二、抗体的制备
采用人B7-H3-4Ig-His蛋白免疫全人源小鼠，通过ELISA、细胞流式监测血清效价，根据效价结果选择最优小鼠取脾脏细胞融合、筛选并进行亚克隆，检测不同单克隆结合人\猴蛋白、细胞等活性，获得优选克隆20G11G6/2#，并对抗体的序列进行PTM位点去除改造，PI降低改造，以及重链恒定区ADCC去除改造后，最终得到全人源抗体2#8890(重链可变区,SEQ ID NO:3；轻链可变区,SEQ ID NO:4)、人IgG1重链恒定区去除ADCC活性改造的序列(SEQ ID NO:31)、人κ轻链恒定区(SEQ ID NO:32)，形成完整的人源化抗体(见表1)，抗体委托南京金斯瑞生物科技有限公司进行密码子优化与基因合成，构建至pTT5质粒中，重链和轻链质粒同时转染到CHO-S细胞中，采用Protein A对上清中的表达抗体进行纯化，从而获得相应抗体蛋白2#8890。2#8890的重链氨基酸序列和轻链氨基酸序列分别如SEQ ID NO：42和SEQ ID NO：43所示。
hIgG1为抗鸡溶菌酶抗体，重链可变区融合至突变的人IgG1重链恒定区(SEQ ID NO:31)，轻链可变区融合至野生型的人kappa轻链恒定区(SEQ ID NO:32)，按上述方法表达纯化,获得抗体hIgG1。
B7-H3对照抗体DS7300来自专利CN 103687945A中，进行密码子优化后，抗体重链可变区核苷酸序列分别合成克隆到含有突变的人IgG1重链恒定区(SEQ ID NO:31)，轻链可变区核苷酸序列合成至含有野生型kappa轻链恒定区(SEQ ID NO:32)的pTT5载体，按上述方法表达纯化,获得抗体DS7300。
表1：2#8890可变区及CDR氨基酸序列

三、包含细胞生物活性分子和连接体的化合物与抗体的偶联
以下实施例所制备得到的抗体药物偶联物中所涉及的抗体2#8890，DS7300，以及hIgG1为上述第二部分中所述的对应抗体。
1、ADC 1(DS7300-M-01，DAR 4)的制备
取38.041ml自制DS7300抗体(26.287mg/mL)，用1M Na2HPO4溶液调pH至7.4，用20mM PB将抗体稀释至3mg/mL，并于冰浴中向其中依次加入4mM ZnCl2(3.413mL)，10mM TCEP(三(2-羧乙基)膦，4.096mL，pH 7.4)溶液混匀，4℃放置过夜。再加入6倍物质的量的溶解在二甲基亚砜的M-01(4.18mL，10mM)溶液混匀，4℃反应4h，再向其中加入半胱氨酸溶液(10mM，6.826mL)，反应1.5h后将反应液移至室温，并向其中加入EDTA溶液(10mM，6.826mL)，30分钟后再加入DHAA溶液(10mM，6.826mL)，继续反应30分钟。完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物，即ADC 1(DS7300-M-01)。质谱法测定DAR值为3.8。
2、ADC 2(hIgG1-M-01,DAR 4)的制备
取1.2245ml hIgG1抗体(24.5mg/mL)，用1M Na2HPO4溶液调pH至7.3，用20mM PB将抗体稀释至3mg/mL，并于冰浴中向其中依次加入4mM ZnCl2(52.04uL)，10mM TCEP(三(2-羧乙基)膦，124.89uL，pH 7.3)溶液混匀，4℃放置过夜。再加入6倍物质的量的溶解在二甲基亚砜的171(127.44uL，10mM)溶液混匀，4℃反应4h，再向其中加入半胱氨酸溶液(10mM，208.15uL)，反应1.5h后将反应液移至室温，并向其中加入EDTA溶液(10mM，208.15uL)，30分钟后再加入DHAA溶液(10mM，208.15uL)，继续反应30分钟。完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到ADC 2(即hIgG1-M-01)。质 谱法测定DAR值为4.46。
3、ADC 3(2#8890-M-01,DAR 4)的制备
取9.36ml 2#8890抗体(3.205mg/mL)，用1M Na2HPO4溶液调pH至7.4，用20mM PB将抗体稀释至3mg/mL，并于冰浴中向其中依次加入4mM ZnCl2(57.31uL)，10mM TCEP(三(2-羧乙基)膦，124.9uL，pH 7.4)溶液混匀，4℃放置过夜。再加入6倍物质的量的溶解在二甲基亚砜的M-01(124.9uL，10mM)溶液混匀，4℃反应4h，再向其中加入半胱氨酸溶液(10mM，208.2uL)，反应1.5h后将反应液移至室温，并向其中加入EDTA溶液(10mM，208.2uL)，30分钟后再加入DHAA溶液(10mM，208.2uL)，继续反应30分钟。完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物，即ADC3(2#8890-M-01)。质谱法测定DAR值为3.89。
4、ADC 4(hIgG1-A-05,DAR 8)的制备
取0.943ml hIgG1抗体(11mg/mL)，用47uL 20mM PB+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.60，加入10mM TCEP(三(2-羧乙基)膦，57uL，pH 7.60)溶液混匀，室温放置1.5h。再加入10倍物质的量的溶解在二甲基亚砜的A-05(103uL，10mM)溶液混匀，室温静置2h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物，即ADC 4(hIgG1-A-05)。质谱法测定DAR值为8.03。
5、ADC 5(2#8890-A-05,DAR 8)的制备
取3.052ml 2#8890抗体(9.83mg/mL)，用153uL 20mM PB+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.60，加入10mM TCEP(三(2-羧乙基)膦，114.5uL，pH 7.60)溶液混匀，室温放置1.5h。再加入10倍物质的量的溶解在二甲基亚砜的A-05(219.1uL，10mM)溶液混匀，室温静置2h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物，即ADC 5(2#8890-A-05)。质谱法测定DAR值为7.90。
6、ADC 6(hIgG1-A-07,DAR 8)的制备
取0.518ml hIgG1抗体(19.3mg/mL)，用25.9uL 20mM PB+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.60，加入10mM TCEP(三(2-羧乙基)膦，38.1uL，pH 7.60)溶液混匀，室温放置1.5h。再加入10倍物质的量的溶解在二甲基亚砜的A-07(69.2uL，10mM)溶液混匀，室温静置2h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物， 即ADC 6(hIgG1-A-07)。质谱法测定DAR值为8.04。
7、ADC 7(2#8890-A-07,DAR 8)的制备
取1.017ml 2#8890抗体(9.83mg/mL)，用50.85uL 20mM PB+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.60，加入10mM TCEP(三(2-羧乙基)膦，38.2uL，pH 7.60)溶液混匀，室温放置1.5h。再加入12倍物质的量的溶解在二甲基亚砜的A-07(87.6uL，10mM)溶液混匀，室温静置2h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物，即ADC 7(2#8890-A-07)。质谱法测定DAR值为7.76。
8、ADC 8(hIgG1-A-14,DAR 8)的制备
取1.9126ml hIgG1抗体(18.3mg/mL)，用95.6uL 20mM PB+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.60，加入10mM TCEP(三(2-羧乙基)膦，66.86uL，pH 7.60)溶液混匀，室温放置1.5h。再加入10倍物质的量的溶解在二甲基亚砜的A-14(260.53uL，10mM)溶液混匀，室温静置2h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物，即ADC 8(hIgG1-A-14)。质谱法测定DAR值为8.0。
9、ADC 9(2#8890-A-14,DAR 8)的制备
取3.6788ml 2#8890抗体(10.873mg/mL)，用183.94uL 20mM PB+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.60，加入10mM TCEP(三(2-羧乙基)膦，152uL，pH 7.60)溶液混匀，室温放置1.5h。再加入10倍物质的量的溶解在二甲基亚砜的A-14(292.26uL，10mM)溶液混匀，室温静置2h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物，即ADC 9(2#8890-A-14)。质谱法测定DAR值为7.22。
10、ADC 10(hIgG1-B-01,DAR 8)的制备
取1.533ml hIgG1抗体(19.57mg/mL)，用76.65uL 20mM PB+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.60，加入10mM TCEP(三(2-羧乙基)膦，114.5uL，pH 7.60)溶液混匀，室温放置1.5h。再加入10倍物质的量的溶解在二甲基亚砜的B-01(212.4uL，10mM)溶液混匀，室温静置2h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物，即ADC 10(hIgG1-B-01)。质谱法测定DAR值为8.03。
11、ADC 11(2#8890-B-01,DAR 8)的制备
取3.052ml 2#8890抗体(9.83mg/mL)，用152.6uL 20mM PB+0.1M EDTA (pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.60，加入10mM TCEP(三(2-羧乙基)膦，114.5uL，pH 7.60)溶液混匀，室温放置1.5h。再加入10倍物质的量的溶解在二甲基亚砜的B-01(212.4uL，10mM)溶液混匀，室温静置2h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物，即ADC 11(2#8890-B-01)。质谱法测定DAR值为7.75。
对偶联后的ADC样品进行LC-MS分子量分析。
色谱测定条件：
液相色谱柱：Thermo MAbPac RP 3.0*100mm；
流动相A：0.1％FA/H2O；流动相B：0.1％FA/ACN；
流速：0.25ml/min；样品室温度：8℃；柱温：60℃；进样量：2μl；
质谱测定条件：
质谱型号：AB Sciex Triple TOF 5600+；
GS1 35；GS2 35；CUR 30；TEM 350；ISVF 5500；DP 200；CE 10；Accumulation time 0.5s；
m/z 600-4000；Time bins to sum 40。
12、ADC 12(2#8890-C-07,DAR 4)的制备
取0.2274ml 2#8890抗体(10.994mg/mL)，用11.4uL 20mM PB+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.60，加入10mM TCEP(三(2-羧乙基)膦，9.5uL，pH 7.60)溶液混匀，室温放置1.5h。再缓慢加入8倍物质的量的溶解在二甲基亚砜的C-07(14.6uL，10mM)溶液混匀，室温静置过夜，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物，即ADC 12(2#8890-C-07)。质谱法测定DAR值为4.12。
13、ADC 13(2#8890-C-10,DAR 4)的制备
取0.2274ml 2#8890抗体(10.994mg/mL)，用11.4uL 20mM PB+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.60，加入10mM TCEP(三(2-羧乙基)膦，9.5uL，pH 7.60)溶液混匀，室温放置1.5h。再缓慢加入8倍物质的量的溶解在二甲基亚砜的C-10(14uL，10mM)溶液混匀，室温静置过夜，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得 到抗体药物偶联物，即ADC 13(2#8890-C-10)。质谱法测定DAR值为4.17。
对偶联后的ADC样品进行LC-MS分子量分析。
色谱测定条件：
液相色谱柱：ACQUITY UPLC MAbPac BEH SEC；
流动相A：20mM NH4Ac；
流速：0.1ml/min；样品室温度：8℃；柱温：60℃；进样量：2μl；
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；Accumulation time 0.5s；m/z 900-7000；Time bins to sum 40。
14、ADC 14(2#8890-C-17,DAR 4)的制备
取0.292mL 2#8890抗体(8.565mg/mL)，用14.6uL 20mM PB+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.60，加入20mM TCEP(三(2-羧乙基)膦，4.77uL，pH 7.60)溶液混匀，室温放置1.5h。再缓慢加入5倍物质的量的溶解在二甲基亚砜的C-17(8.76uL，10mM)溶液混匀，室温静置过夜，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物，即ADC 14(2#8890-C-17)。质谱法测定DAR值为3.86。
15、ADC 15(2#8890-C-19,DAR 4)的制备
取0.584mL 2#8890抗体(8.565mg/mL)，用29.2uL 20mM PB+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.60，加入20mM TCEP(三(2-羧乙基)膦，9.54uL，pH 7.60)溶液混匀，室温放置1.5h。再缓慢加入5.5倍物质的量的溶解在二甲基亚砜的C-19(17.9uL，10mM)溶液混匀，室温静置过夜，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物，即ADC 15(2#8890-C-19)。质谱法测定DAR值为4.05。
16、ADC 16(2#8890-C-21,DAR 4)的制备
取0.584mL 2#8890抗体(8.565mg/mL)，用29.2uL 20mM PB+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.60，加入20mM TCEP(三(2-羧乙基)膦，9.54uL，pH 7.60)溶液混匀，室温放置1.5h。再缓慢加入5.5倍物质的 量的溶解在二甲基亚砜的C-21(17.9uL，10mM)溶液混匀，室温静置过夜，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物，即ADC 16(2#8890-C-21)。质谱法测定DAR值为3.92。
17、ADC 17(2#8890-B-03，DAR 8)的制备
取0.867ml 2#8890抗体(34.6mg/mL)，用93.4μL 20mM PB+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.60，加入10mM TCEP(三(2-羧乙基)膦，112.65μL，pH 7.60)溶液混匀，室温放置1.5h。再加入11倍物质的量的溶解在二甲基亚砜的B-03(227.58uL，10mM)溶液混匀，室温静置2h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物，即ADC 17(2#8890-B-03，DAR 8)。质谱法测定DAR值为7.82。
18、ADC 18(2#8890-B-03，DAR 8)的制备
取0.867ml 2#8890抗体(34.6mg/mL)，用58.35μL 20mM PB+0.1M EDTA(pH 7.60)稀释，然后用1M Na2HPO4溶液调pH至7.60，加入10mM TCEP(三(2-羧乙基)膦，112.65μL，pH 7.60)溶液混匀，室温放置1.5h。再加入10倍物质的量的溶解在二甲基亚砜的B-03(211.1uL，10mM)溶液混匀，室温静置2h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液，得到抗体药物偶联物，即ADC 18(2#8890-B-03，DAR 8)。质谱法测定DAR值为7.23。
19、ADC 19(19F6-B-02)的制备
以下实施例所涉及的抗体19F6_Hu35v1即为国际专利申请WO2022253035A1所记载的19F6_Hu35v1抗体，采用该专利实施例二所记载的方法制备。
样品的偶联制备如下：
取0.46ml 19F6_Hu35v1抗体(11.0mg/mL)，用0.1M依地酸二钠的溶液(pH7.7)稀释，然后用1M Na2HPO4溶液调pH至7.7，加入10mM TCEP(三(2-羧乙基)膦)溶液混匀，室温放置90min。向上述溶液体系加入10倍物质的量的溶解在二甲基亚砜的B-02，混匀，室温静置2h，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，然后添加蔗糖和吐温20，混匀，得到抗体药物偶联物，即ADC 19(19F6-B-02)。质谱法测定DAR值为7.92。
20、ADC 20(Trastuzumab-A-05，DAR 8)
取2.469mL Trastuzumab抗体(16.2mg/mL),用123uL 20mM PB+0.1M EDTA(pH 7.60)稀释,然后用1M Na2HPO4溶液调pH至7.60,加入10mM TCEP(三(2-羧乙基)膦,151.57uL,pH 7.60)溶液混匀,室温放置1.5h。再缓慢加入10倍物质的量的溶解 在二甲基亚砜中的A-05(290.09uL,10mM)溶液混匀,室温静置2h,完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液,得到抗体药物偶联物(即Trastuzumab-A-05)。质谱法测定DAR值为8.04。
21、ADC 21(Trastuzumab-A-14，DAR 8)
取2.564mL Trastuzumab抗体(15.6mg/mL),用128.2uL 20mM PB+0.1M EDTA(pH 7.60)稀释,然后用1M Na2HPO4溶液调pH至7.60,加入10mM TCEP(三(2-羧乙基)膦,151.57uL,pH 7.60)溶液混匀,室温放置1.5h。再缓慢加入10倍物质的量的溶解在二甲基亚砜中的A-14(290.1uL,10mM)溶液混匀,室温静置2h,完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液,得到抗体药物偶联物(即Trastuzumab-A-14)。质谱法测定DAR值为8.02。
22、ADC 22(Trastuzumab-A-24，DAR 8)
取0.617mL Trastuzumab抗体(16.2mg/mL),用30.86uL 20mM PB+0.1M EDTA(pH 7.60)稀释,然后用1M Na2HPO4溶液调pH至7.60,加入10mM TCEP(三(2-羧乙基)膦,37.89uL,pH 7.60)溶液混匀,室温放置1.5h。再缓慢加入14倍物质的量的溶解在二甲基亚砜的A-24(98.42uL,10mM)溶液混匀,室温静置2h,完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液,得到抗体药物偶联物(即Trastuzumab-A-24)。质谱法测定DAR值为7.37。
23、ADC23(Trastuzumab-A-32，DAR 8)
取0.423mL Trastuzumab抗体(4.73mg/mL),用21.17uL 20mM PB+0.1M EDTA(pH 7.60)稀释,然后用1M Na2HPO4溶液调pH至7.60,加入10mM TCEP(三(2-羧乙基)膦,7.58uL,pH 7.60)溶液混匀,室温放置1.5h。再加入10倍物质的量的溶解在二甲基亚砜的A-32(13.92uL,10mM)溶液混匀,室温静置2h,完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液,得到抗体药物偶联物(即Trastuzumab-A-32)。质谱法测定DAR值为6.77。
24、ADC24(Trastuzumab-C-21，DAR 4)
取0.617mL Trastuzumab抗体(16.2mg/mL),用30.86uL 20mM PB+0.1M EDTA(pH 7.60)稀释,然后用1M Na2HPO4溶液调pH至7.60,加入19.83mM TCEP(三(2-羧乙基)膦,19.1uL,pH 7.60)溶液混匀,室温放置1.5h。再依次加入5.5倍物质的量的溶解在二甲基亚砜的C-21(38.28uL,10mM)溶液混匀,室温静置18h,完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的20mM组氨酸缓冲溶液,得到抗体药物偶联物(即Trastuzumab-C-21)。质谱法测定DAR值为4.41,其中DAR4占比60.06％。
四、检测抗体药物偶联物活性
1.抗人B7-H3抗体偶联物动态亲和力检测
用ForteBio(Pall life sciences)检测抗人B7-H3抗体偶联物与人B7-H3-4Ig-his、人B7-H3-2Ig-his、大鼠B7-H3-his和猴B7-H3-his蛋白的动态亲和力。具体方法如下：用PBST(0.02％Tween-20)将待测偶联物稀释到5μg/ml，人B7-H3-4Ig-his、人B7-H3-2Ig-his、大鼠B7-H3-his和猴B7-H3-his蛋白梯度稀释到200nM、100nM、50nM、25nM、12.50nM、6.25nM、3.125nM、0nM，随后在PBST(0.02％Tween-20)溶液中用Protein A Sensor(Pall life sciences)分别捕获待测偶联物60s，随后分别与4个蛋白结合60s，然后解离180s，测得结果在Data Analysis 11.0软件中打开，选用1:1模式，global fitting，对结果进行分析，获得亲和力常数。结果如表2所示，结果显示本发明药物连接子化合物所制备的偶联物与猴B7-H3结合，不与大鼠B7-H3结合。
表2：抗人B7-H3抗体偶联物动态亲和力检测结果
2.抗人B7-H3抗体偶联物的细胞亲和力检测
用流式仪(Beckman，型号Cytoflex)检测抗人B7-H3全人源抗体偶联物与人结肠癌细胞HT29(中国科学院细胞库)、人胃癌细胞NCI-N87(ATCC)、人乳腺鳞状癌细胞HCC1806(ATCC)和人非小细胞肺癌细胞HCC827(ATCC)的亲和力；检测抗人B7-H3全人源抗体偶联物与CHOS-人B7-H3-4Ig、CHOS-人B7-H3-2Ig的亲和力；检测抗人B7-H3全人源抗体偶联物的种属交叉情况：与CHOS-大鼠B7-H3和CHOS-猴B7-H3的亲和力。用Trypsin-EDTA(0.25％)(Thermo)溶液消化贴壁生长的细胞，计数并调整细胞密度至4.0×106/ml，用1％BSA洗涤两次，重悬于1％BSA溶液中，每孔加入50μl细胞悬液到96孔尖底板中(细胞数目为2×105个/孔)；用1％BSA稀释抗体偶联物(终浓度10μg/ml起始，3倍梯度稀释，共稀释11个浓度点),hIgG1抗体偶联物作为对照(终浓度10ug/ml)，取50μl稀释好的抗体到含有细胞的尖底板中，4度孵育60min；1％BSA洗涤细胞两次，每孔加入50μl稀释好的二抗，混匀，4度孵育30min；1％BSA洗涤细胞两次，将细胞重悬于200μl 1％BSA中，流式上机检测。数据处理：导出Median PE数值，然后导入GraphPad Prism 6软件，计算EC50。
抗人B7-H3抗体偶联物的HT29、NCI-N87、HCC1806、HCC827肿瘤细胞亲和 力结果中，ADC17对HT29肿瘤细胞亲和力的EC50为3.213ng/ml；对HCC827肿瘤细胞亲和力的EC50为8.054ng/ml，其余抗体偶联物的亲和力结果分别如表3所示，抗人B7-H3全人源抗体偶联物对CHOS-人B7-H3-4Ig、CHOS-人B7-H3-2IG细胞的亲和力结果如表4所示，抗人B7-H3抗体偶联物对CHOS-大鼠B7-H3和CHOS-猴B7-H3细胞的亲和力结果如表5所示，结果显示本发明药物连接子化合物所制备的偶联物与猴B7-H3过表达细胞结合，不与大鼠B7-H3过表达细胞结合。
表3：ADC肿瘤细胞亲和力测定结果
表4：CHOS-人B7-H3-4Ig、CHOS-人B7-H3-2Ig细胞亲和力测定结果
表5：CHOS-大鼠B7-H3和CHOS-猴B7-H3细胞亲和力测定结果
3.抗人B7-H3抗体偶联物细胞内吞活性检测
用流式仪(Thermo，型号Attune NxT)检测抗人B7-H3抗体偶联物对人胃癌细胞NCI-N87(ATCC)、人乳腺鳞状癌细胞HCC1806(ATCC)和人非小细胞肺癌细胞HCC827(ATCC)的内吞活性。用Trypsin-EDTA(0.25％)(Thermo)溶液消化贴壁生长的细胞并计数，用完全培养基调整细胞密度至1×105个/ml，每孔加入100μl细胞悬液到96孔板中(细胞数目为1×104个/孔)，将96孔板放置于37℃，CO2恒温培养箱孵育培养24h。取出96孔板，吸弃培养基，每孔加入50μl新鲜完全培养基；采用完全培养基稀释待测抗体偶联物，共计6个浓度点；完全培养基稀释300μg/ml的pHrodo试剂(Thermo，Cat#Z25612)至12μg/ml(pHrodo的终浓度为3μg/ml)；将梯度稀释的待测抗体与稀释后的pHrodo试剂1:1混合均匀(30μl:30μl)，在室温避光孵育30min；取50μl待测抗体和pHrodo试剂混合物加入96孔板中，37℃、5％CO2培养24h；取出96孔板，吸弃培养基，无菌PBS洗涤1次，每孔加入100μl的Trypsin-EDTA(0.25％)消化细胞，再加入100μl完全培养基中和；将孔里细胞吹打分散后， FACS上机检测。数据处理：导出Median YL-1H数值，然后导入GraphPad Prism 6软件，计算EC50，结果如表6所示，本发明药物连接子化合物所制备的抗体药物偶联物(例如ADC 5和ADC 9)均具有较好的细胞内吞活性。
表6：ADC细胞内吞活性测定结果
4.抗人B7-H3抗体偶联药物体外细胞杀伤检测
用Trypsin-EDTA(0.25％)(Thermo)溶液消化贴壁生长的A375、Calu6-B7-H3和U87MG-B7-H3细胞并计数，用完全培养基分别调整细胞密度至1×104、5×104、1×104个/ml，每孔加入100μl细胞悬液到96孔板中(细胞数目分别为1000、5000、1000个/孔)，将96孔板放置于37℃，CO2恒温培养箱孵育培养24h。采用完全培养基稀释待测ADC，终浓度3333.3nM起始，4倍梯度稀释，共计12个浓度点；取100μl稀释好的ADC加入96孔板中，37℃、5％CO2培养4～7天。取出96孔板，每孔加20μl CCK8试剂，37℃孵育2～3h，使用酶标仪检测OD450nm信号值，然后导入GraphPad Prism 6软件，计算IC50，结果如表7所示。
表7 ADC体外细胞杀伤结果
结果显示，本发明药物连接子化合物所制备的抗体药物偶联物对于肿瘤细胞具有明确的杀伤作用。
5. 2#8890不同偶联药物HCC1806模型体内药效检测
人乳腺鳞状癌细胞HCC1806(ATCC)，培养条件为RPMI1640培养基中加10％胎牛血清，于37℃、含5％CO2空气的培养箱中培养。当细胞呈指数生长期时，取培养液进行支原体检测后，收集细胞并计数。每只小鼠右侧肩胛处皮下接种2×106个HCC1806细胞，悬浮于0.1ml PBS。待平均肿瘤体积生长至约100～200mm3时，剔除瘤体积过小或过大的小鼠，剩余小鼠根据肿瘤体积和动物体重随机分为6组，每组6只，尾静脉注射单次给药(DAR4给药组：10mg/kg，DAR8给药组：5mg/kg)。给药后每周2次测量肿瘤体积和体重，具体结果见表8。
表8 2#8890不同偶联药物对HCC1806细胞荷瘤鼠模型的疗效分析
注：*P<0.05，**P<0.01，***P<0.001表示与溶媒组相比有显著差异。
结果显示：给药组小鼠体重稳定，耐受性良好。单次给药后95天，ADC 5组小鼠肿瘤完全消退。说明本发明药物连接子化合物所制备的抗体药物偶联物具有明显的肿瘤抑制作用。
6.不同剂量抗体偶联药物HCC1806模型药效检测
人乳腺鳞状癌细胞HCC1806(ATCC)，培养条件为RPMI1640培养基中加10％胎牛血清，于37℃、含5％CO2空气的培养箱中培养。当细胞呈指数生长期时，取培养液进行支原体检测后，收集细胞并计数。每只小鼠右侧肩胛处皮下接种2×106个HCC1806细胞，悬浮于0.1ml PBS。待平均肿瘤体积生长至约100～200mm3时，剔除瘤体积过小或过大的小鼠，剩余小鼠根据肿瘤体积和动物体重随机分为12组，每组6只，尾静脉注射单次给药。给药后每周2次测量肿瘤体积和体重，具体结果见表9。
表9抗体偶联药物不同剂量HCC1806细胞荷瘤鼠模型的疗效分析
注：*P<0.05，**P<0.01，***P<0.001表示与溶媒组相比有显著差异。
结果显示：ADC 5与ADC 9组均具有较好的药效，且小鼠体重稳定，耐受性良好，说明本发明药物连接子化合物所制备的抗体药物偶联物具有明显的肿瘤抑制作用以及良好的安全性。
7.不同抗体偶联药物NCI-N87模型药效检测
人胃癌细胞NCI-N87(ATCC)，培养条件为RPMI1640培养基中加10％胎牛血 清，于37℃、含5％CO2空气的培养箱中培养。当细胞呈指数生长期时，取培养液进行支原体检测后，收集细胞并计数。每只小鼠右侧肩胛处皮下接种5×106个NCI-N87细胞，悬浮于0.1ml PBS基质胶中。待平均肿瘤体积生长至约100～200mm3时，剔除瘤体积过小或过大的小鼠，剩余小鼠根据肿瘤体积和动物体重随机分为8组，每组6只，尾静脉注射给药，QW给药两次。给药后每周2次测量肿瘤体积和体重，具体结果见表10。
表10不同抗体偶联药物NCI-N87细胞荷瘤鼠模型的疗效分析
注：*P<0.05，**P<0.01，***P<0.001表示与溶媒组相比有显著差异。
结果显示：各给药组小鼠体重稳定，耐受性良好，说明本发明药物连接子化合物所制备的抗体药物偶联物具有明显的肿瘤抑制作用以及良好的安全性。
8.抗人B7-H3抗体偶联药物与Fc受体结合活性检测
用ForteBio(Pall life sciences)检测2#8890抗体偶联药物与人Fc受体蛋白CD16a、CD32a、CD32b、C1q、FcRn动态亲和力。具体方法如下：在PBST溶液中用SA Sensor(Pall life sciences)分别捕获待测生物素化蛋白，用PBST将待测抗体及偶联物稀释到起始浓度5000nM并2倍稀释7个浓度点，结合、解离并将测得结果在Data Analysis11.0软件中打开，选用1:1模式，global fitting，对结果进行分析，获得结合速率、解离速率及亲和力常数，结果如表11所示。
表11抗体及偶联物结合Fc受体活性检测
结果表明，本发明药物连接子化合物所制备的抗体药物偶联物不结合Fc受体CD16a、CD32a、CD32b、C1q蛋白，可降低Fc受体介导的非特异性杀伤，提高药物 安全性，同时这些偶联物保留了FcRn蛋白结合活性，不影响药物的半衰期。
9.食蟹猴多次静脉注射给予抗人B7-H3抗体偶联药物后血清中总抗体(Tab)、ADC、Payload药代动力学研究
采用ELISA和LC-MS/MS方法来进行食蟹猴血清中ADC(ADC 5、ADC 9)、总抗体(TAb)、Payload的定量检测。ADC(ADC 5、ADC 9)和总抗体(TAb)方法的标准曲线定量范围均为11.72～3000.00ng/mL，Payload的方法线性范围为0.1～40ng/mL。ADC(ADC 5、ADC 9)和总抗体(TAb)方法均采用B7-H3蛋白作为捕获蛋白，包被于96孔酶标板中，随后总抗体(Tab)使用Goat Anti-Human IgG-HRP作为检测抗体；ADC(ADC 5、ADC 9)则先后分别使用Anti-Toxin mouse antibody和Goat Anti-Mouse IgG作为第二抗体和检测抗体。通过酶和底物的作用进行显色，于SpectraMax i3x(Molecular Devices)酶标仪上读取，采用4-P参数法拟合出标准曲线并计算出各样本浓度。ADC(ADC 5、ADC 9)和总抗体(TAb)浓度与颜色的深浅呈正相关。LC-MS/MS通过岛津LC 30-AD液相系统联用SCIEX QTRAP 5500+(SCIEX公司)质谱仪，采用(+)ESI离子化方式，选取多反应监测模式(MRM)分析。其中色谱柱为Xbridge C18 50*4.6mm,5μm，待测物化合物1-10离子对为510.2/435.2，样品前处理使用乙腈进行蛋白沉淀。结果：食蟹猴多次静脉注射给予ADC5和ADC 9后，测试结果显示，本申请的药物连接子化合物所制备的ADC分子(例如ADC 5及ADC 9)体现出良好的药代动力学特性，于体循环中较为稳定，游离毒素释放量少。
表12 ADC末次静脉注射50mg/kg后血清中TAb的药代参数
表13 ADC末次静脉注射50mg/kg后血清中ADC的药代参数
表14 ADC末次静脉注射50mg/kg后血清中Payload的药代参数
10.重复给药毒性试验
重复给药毒性试验包括食蟹猴静脉注射ADC5和ADC 9的给药4次的重复毒性试验。
本试验共设3个组，1只/组/性别，分别静脉注射给予30mg/kg的ADC5、ADC 9以及生理盐水(体积：10mL/kg)，每周给药1次，共给药2次；之后提高剂量至50mg/kg的ADC 5、ADC 9以及生理盐水，每周给药1次，共给药2次。试验中，未见供试品相关的死亡或濒死。ADC 5给药组给药期间可见动物食欲减退、脱毛、皮肤色素沉着和体重降低。雌猴可见WBC、NEUT、LYM、MONO降低。雄猴可见RBC、HGB、HCT降低，FGB升高，恢复期有恢复趋势。ADC 9给药组给药期间可见动物食欲减退。雌猴可见WBC、NEUT、LYM、MONO降低和FBG升高。雄猴可见FBG升高、RBC、HGB、HCT降低。恢复期间，另见给药局部色素沉着和轻微脱毛，其他改变均有恢复趋势。在本实验条件下，食蟹猴每周1次，连续2周，静脉注射30mg/kg的ADC 5和ADC 9；随后提高剂量，每周1次，连续2周，静脉注射50mg/kg的ADC 5和ADC 9。所有动物均可耐受。最高无严重毒性剂量(HNSTD)为50mg/kg。
11.抗体-药物偶联物对小鼠皮下移植瘤模型上的肿瘤生长抑制作用
分别通过尾静脉注射给予皮下移植人乳腺鳞状癌细胞HCC1806小鼠CDX模型含有本发明ADC的制剂，每周2次测定肿瘤体积和动物体重变化，计算本发明ADC对荷瘤小鼠的抑瘤疗效。
实验动物：Balb/c Nude小鼠(成都药康生物科技有限公司)
细胞系：人乳腺鳞状癌细胞HCC1806(ATCC)
实验方法：
用含10％胎牛血清的RPMI 1640培养液，在37℃，5％CO2的条件下培养HCC1806细胞。收集指数生长期的HCC1806细胞，PBS重悬至适合浓度，接种于雌性Balb/c-nude小鼠皮下建立乳腺鳞状癌模型。待肿瘤平均体积约200mm3左右时，根据肿瘤大小随机分组并分别给药，各组别及其给药剂量依次为：溶媒对照组(即阴性对照,Vehicle组)：给予0.9％Nacl注射液；ADC 5：给药剂量为3mg/kg；ADC 9：给药剂量为3mg/kg；ADC 11：给药剂量为3.16mg/kg组；ADC 17：给药剂量为3.32mg/kg。各组均采用尾静脉注射(i.v.)，在Day 0给药,共给药1次。给药后每周2次测量小鼠体重以及用游标卡尺测量肿瘤长径和短径，并按如下计算公式计算肿瘤体积：V＝0.5a×b2，其中a和b分别表示肿瘤的长径和短径，每天观察记录动物死亡情况。
采用以下公式计算肿瘤生长抑制率TGI(％)：
VT末>VT0,TGI(％)＝[1-(VT末-VT0)/(VC末-VC0)]*100％或VT末≤VT0,TGI(％)＝[1-(VT末-
VT0)/VT0]*100％。
其中VT末:治疗组实验结束时肿瘤体积均值
VT0:治疗组给药开始时肿瘤体积均值
VC末:阴性对照组实验结束时肿瘤体积均值
VC0:阴性对照组给药开始时肿瘤体积均值
采用以下公式计算肿瘤相对增殖率T/C(％)：
T/C＝(VT末/VT0)/(VC末/VC0)。
本发明ADC对HCC1806乳腺鳞状癌移植瘤模型的肿瘤生长抑制作用显著。在第14天，与Vehicle组相比，本发明ADC 5，ADC 9，ADC 11和ADC17的肿瘤生长抑制率(TGI)分别为96.68％、98.50％、82.61％、86.69％，与对照组相比具有显著性差异。治疗期间各治疗组无动物死亡及显著地动物体重降低，未见明显的药物毒性反应，小鼠对本发明ADC耐受性良好。具体结果见表15。
表15不同抗体药物偶联物对HCC1806细胞荷瘤鼠模型的疗效分析
注:TGI为肿瘤生长抑制率，T/C为相对肿瘤增殖率。
12.抗体药物偶联物对体外细胞活性的抑制作用
(1)细胞铺板：首先采用相应的培养基培养肿瘤细胞NCI-H1975、HT-29，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16。
表16：肿瘤细胞来源
待细胞贴壁后，移除细胞中培养基，将稀释好的抗体药物偶联物ADC 19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-LiteTM 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。通过加入Cell Counting-LiteTM，不含细胞的培养基孔获得背景RLU,有细胞但不含化合物的培养孔获得对照RLU。细胞抑制率＝1-(样品RLU-背景RLU)/(细胞对照RLU-背景RLU)×100％，按照四参数模型拟合曲线，计算ADC 19的半数抑制浓度(IC50)。
ADC 19对NCI-H1975细胞系的半数抑制浓度(IC50)5.00μg/mL；ADC 19对HT-29细胞系的半数抑制浓度(IC50)4.56μg/mL。
结果说明本发明的药物连接体所形成的抗体药物偶联物(例如ADC19)具有明显的肿瘤细胞杀伤活性。
13.抗人Her2抗体偶联药物在NCI-N87模型的药效检测
用含10％胎牛血清的RPMI1640培养液,在37℃,5％CO2的条件下培养NCI-N87细胞。收集指数生长期的NCI-N87细胞,PBS重悬至适合浓度,接种于雌性Balb/c-nu小鼠皮下建立胃癌模型。待肿瘤平均体积约160mm3左右时,根据肿瘤大小随机分组,依次为:溶媒对照组(即阴性对照,Vehicle组),本发明Trastuzumab-A-05 1mg/kg组,Trastuzumab-A-14 1mg/kg组,Trastuzumab-A-24 1mg/kg组。各组均采用尾静脉注射(i.v.),在Day0、Day7、Day14给药,共给药3次。给药后每周2次测量小鼠体重以及用游标卡尺测量肿瘤长径和短径,并按如下计算公式计算肿瘤体积:V＝0.5a×b2,其中a和b分别表示肿瘤的长径和短径,每天观察记录动物死亡情况。
本发明ADC对NCI-N87胃癌移植瘤模型的肿瘤生长抑制作用显著。与溶媒对照组相比,本发明Trastuzumab-A-05 1mg/kg组,Trastuzumab-A-14 1mg/kg组和Trastuzumab-A-24 1mg/kg组的肿瘤生长抑制率(TGI)分别为81.62％、107.38％和61.43％。Day30各治疗组无动物死亡及显著地动物体重降低,未见明显的药物毒性反应,治疗期间小鼠对本发明药物连接子化合物所制备的ADC耐受性良好。具体结果见表17。
表17人胃癌细胞NCI-N87 CDX模型
注:TGI为肿瘤生长抑制率,T/C为相对肿瘤增殖率。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解，根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (38)

  1. 一种药物连接子化合物，其具有式G-M-[L-E-D]x所示结构，其中：
    G是能够与特异性氨基酸或糖基发生反应的官能团或离去基团；
    M是与G相连的接头，所述的M为
    其中，环A为5-6元脂杂环、或5-20元芳香族环系，所述脂杂环和芳香族环系任选地被一个或多个选自氧基(＝O)、卤素、氰基、氨基、羧基、巯基和C1-6烷基的基团取代；M1选自单键和C1-20亚烷基、C2-20亚烯基、C2-20亚炔基或胺基，所述的C1-20亚烷基、C2- 20亚烯基、C2-20亚炔基或胺基任选地被一个或多个适合的取代基所取代；
    L是连接接头M和E之间的连接子，L选自由下述的一个或多个组成的结构：C1-6亚烷基、-N(R’)-、羰基、-O-、天然氨基酸或非天然氨基酸及其类似物、以及氨基酸组成的短肽、
    其中R’代表氢、C1-6烷基或含1-10个EO单元的聚乙二醇片段；s选自1-20的整数；
    E是连接L和D的结构片段，其中E为单键、-NHCH2-或选自以下结构：
    D是细胞毒性药物片段；和/或
    x选自1至10。
  2. 权利要求1所述的药物连接子化合物，所述的G选自卤素、卤代C1-6烷基、C1- 6磺酰基、卤代C1-6磺酰基、卤代磺酰基、C1-6磺酸酯基、卤代C1-6磺酸酯基、C1-6亚磺酸酯基、C1-6亚砜基、硝基、叠氮基、氰基、烯基、炔基及含炔基的结构片段，所述的卤代C1-6烷基、C1-6磺酰基、卤代C1-6磺酰基、卤代磺酰基、C1-6磺酸酯基、卤代C1-6磺酸酯基、C1-6亚磺酸酯基、C1-6亚砜基、烯基、炔基及含炔基的结构片段 任选地被一个或多个适合的取代基所取代。
  3. 权利要求1或2所述的药物连接子化合物，所述的M为其中环A为5元脂杂环、6元杂芳环、或由一个以上(例如2个)的6元芳杂环与苯环或6元杂芳环通过单键连接形成的多环，所述脂杂环任选地被一个或多个选自氧基(＝O)、卤素和C1-4烷基的基团取代；M1选自单键、C1-20亚烷基、C2-20亚烯基、C2-20亚炔基或胺基，所述的C1-20亚烷基、C2-20亚烯基、C2-20亚炔基或胺基任选地被一个或多个适合的取代基所取代。
  4. 权利要求1-3任一项所述的药物连接子化合物，所述的M为其中环A选自 M1选自单键和C1-6亚烷基、C2-6亚烯基、C2-6亚炔基或胺基，所述的C1-6亚烷基、C2-6亚烯基、C2-6亚炔基或胺基任选地被一个或多个适合的取代基所取代。
  5. 权利要求1-4任一项所述的药物连接子化合物，所述的M选自
  6. 权利要求5所述的药物连接子化合物，所述的M选自
  7. 权利要求1-4任一项所述的药物连接子化合物，所述的M选自
  8. 权利要求1-4任一项所述的药物连接子化合物，所述的M选自
  9. 权利要求1-8任一项所述的药物连接子化合物，所述的L选自由下述的一个或多个组成的结构：C1-6亚烷基、-N(R’)-、羰基、-O-、Ala、Arg、Asn、Asp、Cit、Cys、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、Tyr、Val、Lys(COCH2CH2(OCH2CH2)rOCH3))、Ala-Ala、Ala-Lys、Ala-Lys(Ac)、Ala-Pro、Gly-Glu、Gly-Gly、Phe-Lys、Phe-Lys(Ac)、Val-Ala、Val-Lys、Val-Lys(Ac)、Val-Cit、Ala-Ala-Ala、Ala-Ala-Asn、Leu-Ala-Glu、Gly-Gly-Arg、Gly-Glu-Gly、Gly-Gly-Gly、Gly-Ser-Lys、Glu-Val-Ala、Glu-Val-Cit、Ser-Ala-Pro、Val-Leu-Lys、Val-Lys-Ala、Val-Lys-Gly、Gly-Gly-Phe-Gly、Gly-Gly-Val-Ala、Gly-Phe-Leu-Gly、Glu-Ala-Ala-Ala、Gly-Gly-Gly-Gly-Gly、
    其中R’代表氢、C1-6烷基或含1-10个EO单元的聚乙二醇片段；s选自1-20的整数。
  10. 权利要求1-9任一项所述的药物连接子化合物，所述的L选自由下述的一个或多个组成的结构：C1-6亚烷基、羰基、-NH-、Ala-Ala、Ala-Lys、Ala-Pro、Gly-Glu、Gly-Gly、Phe-Lys、Val-Ala、Val-Lys、Val-Cit、Ala-Ala-Ala、Ala-Ala-Asn、Leu-Ala-Glu、Gly-Gly-Arg、Gly-Glu-Gly、Gly-Gly-Gly、Gly-Ser-Lys、Glu-Val-Ala、Glu-Val-Cit、Ser-Ala-Pro、Val-Leu-Lys、Val-Lys-Ala、Val-Lys-Gly、Gly-Gly-Phe-Gly、Gly-Gly-Val-Ala、Gly-Phe-Leu-Gly、Glu-Ala-Ala-Ala、Gly-Gly-Gly-Gly-Gly、 其中s选自1-20的整数。
  11. 权利要求1-10任一项所述的药物连接子化合物，所述的L选自由下述的一个或多个组成的结构：

  12. 权利要求1-11任一项所述的药物连接子化合物，所述的L选自以下结构：
  13. 权利要求1-12任一项所述的药物连接子化合物，所述的L选自以下结构：
  14. 权利要求1-13任一项所述的药物连接子化合物，所述的L选自以下结构：
  15. 权利要求1-12任一项所述的药物连接子化合物，所述的L选自以下结构：
  16. 权利要求1-15任一项所述的药物连接子化合物，所述的E为单键、-NHCH2-、
  17. 权利要求16所述的药物连接子化合物，所述的E为-NHCH2-。
  18. 权利要求1-17任一项所述的药物连接子化合物，选自以下结构：







  19. 权利要求1-18任一项所述的药物连接子化合物，选自以下结构：


  20. 权利要求1-19任一项所述的药物连接子化合物，所述细胞毒性药物选自微管蛋白抑制剂、DNA嵌入剂、DNA拓扑异构酶抑制剂和RNA聚合酶抑制剂。
  21. 权利要求1-20任一项所述的药物连接子化合物，所述微管蛋白抑制剂为奥瑞他汀类化合物或美登素类化合物；所述DNA嵌入剂为吡咯并苯二氮卓PBD；所述DNA拓扑异构酶抑制剂为拓扑异构酶I抑制剂(例如，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、或卢比替康)或拓扑异构酶II抑制剂(例如，阿霉素、PNU-159682、多卡米星、柔红霉素、米托蒽醌、鬼臼毒素、或依托泊苷)；所述RNA聚合酶抑制剂为α-鹅膏草碱(α-amanitin)或其药学上可接受的盐、酯或类似物。
  22. 权利要求1-21任一项所述的药物连接子化合物，所述细胞毒性药物选自式I、式II所示化合物、或式I、式II所示化合物的药学上可接受的盐、酯、立体异构体、互变异构体或前药：
    其中，R1,R2各自独立地选自C1-6烷基和卤素；
    R3选自H和-CO-CH2OH；
    R4和R5各自独立地选自H、卤素和羟基；或者R4和R5与相连碳原子连接成5-6元含氧杂环；
    R6选自氢或-C1-4亚烷基-NRaRb；
    R7选自C1-6烷基和-C1-4亚烷基-NRaRb；
    其中Ra、Rb在每次出现时各自独立地选自H、C1-6烷基、-SO2-C1-6烷基和-CO-C1-6烷基。
  23. 权利要求1-22任一项所述的药物连接子化合物，所述细胞毒性药物选自以下化合物或所述化合物的药学上可接受的盐、酯、立体异构体、互变异构体或前药：

  24. 权利要求1-23任一项所述的药物连接子化合物，所述细胞毒性药物选自以下化合物或所述化合物的药学上可接受的盐、酯、立体异构体、互变异构体或前药：
  25. 权利要求1-24任一项所述的药物连接子化合物，D选自以下结构：

  26. 权利要求1-25任一项所述的药物连接子化合物，选自下示的A-01～A-34，B-01～B-07，C-01～C-28：










  27. 权利要求1-26任一项所述的药物连接子化合物，所述的药物连接子化合物可任选地被一个或多个适合的取代基所取代。
  28. 权利要求27所述的药物连接子化合物，具有如下结构：
    其中，R10、R11、R12独立地选自氢、C1-6烷基、C3-6环烷基、5-12元杂环基、C6-10芳基、5-12元杂芳基、-C1-6烷基-C6-10芳基和-C1-6烷基-5-12元杂芳基；所述烷基、环烷基、杂环基、芳基和杂芳基任选被一个或多个选自羟基、CN、卤素、C1-6烷基、C1-6卤代烷基、C1- 6烷氧基、C6-10芳基、5-12元杂芳基的取代基所取代；
    R13和R14各自独立选自氢、C1-6烷基、C3-6环烷基和4-6元杂环基；所述烷基、环烷基和杂环基任选被一个或多个选自羟基、CN、卤素、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C6- 10芳基、5-12元杂芳基的取代基所取代；
    R15选自氢、C1-6烷基、C3-6环烷基、卤代C1-6烷基、C1-6烷基-O-C1-6烷基、C2-6烯基、C2- 6炔基和3-6元杂环烷基；R16为H；或者，R15、R16及其所连接的原子共同形成4-7元环；所述4-7元环任选被一个或多个选自羟基、CN、卤素、C1-6烷基、C1-6卤代烷基、C1-6烷氧基、C6-10芳基、5-12元杂芳基的取代基所取代。
  29. 权利要求28所述的药物连接子化合物，具有如下结构：
    R10、R11、R12独立地选自氢、C1-6烷基、C3-6环烷基、C6-10芳基、苄基、羟基取代的苄基和吲哚基-C1-6烷基-；
    R13和R14各自独立选自氢、C1-6烷基、C3-6环烷基和4-6元杂环基；
    R15选自氢、C1-6烷基、C3-6环烷基、卤代C1-6烷基、C1-6烷基-O-C1-6烷基、C2-6烯基、C2- 6炔基和3-6元杂环烷基；R16为H；或者，R15、R16及其所连接的原子共同形成4-7元环。
    。
  30. 权利要求1-29任一项所述的药物连接子化合物在制备偶联物(例如抗体药物偶联物)中的用途。
  31. 化合物或其药学上可接受的盐，具有如下结构：


    其中，X选自苄氧羰基、叔丁氧羰基、芴甲氧羰基、烯丙氧羰基、三甲基硅乙氧羰基、甲氧羰基、乙氧羰基、邻苯二甲酰基、对甲苯磺酰基、三氟乙酰基、硝基苯磺酰基、苯甲酰基、特戊酰基、三苯甲基、4-甲氧基苯基二苯基甲基、二甲氧基三苯甲基、2,4-二甲氧基苄基、对甲氧基苄基和苄基；a为1至10的整数，优选为3至8的整数；R1、R2和D如权利要求1或20-25任一项中所定义。
  32. 化合物，具有如下结构：



  33. 权利要求32所述的化合物在制备权利要求1-29任一项所述的药物连接子化合物中的用途。
  34. 一种化合物的制备方法，所述方法包括IM-5-a进行脱保护得到式IM-6-a化合物的步骤：
    其中，X、R1和R2如权利要求31或32所定义。
  35. 权利要求34所述的制备方法，所述脱保护的反应在以下一项或多项条件下进行：
    (1)溶剂选自N,N-二甲基甲酰胺；
    (2)反应体系中加入烷胺类化合物；
    (3)室温条件下进行；
    (4)反应时间为1-5h。
  36. 权利要求34所述的制备方法，所述方法进一步包括IM-6-a与IM-2进行反应得到化合物A-14-a的步骤：
    其中，R1和R2如权利要求31或32所定义。
  37. 权利要求36所述的制备方法，所述反应在以下一项或两项条件下进行：
    (1)反应的溶剂选自N,N-二甲基甲酰胺和N,N-二甲基乙酰胺；
    (2)所述反应还包括加入N,N-二异丙基乙胺的步骤。
  38. 一种化合物的制备方法，所述化合物为以下结构：

    其中，R1和R2如权利要求31或32所定义；a为1～10的整数；优选地，a为3或8。
PCT/CN2023/134038 2022-11-29 2023-11-24 杂环化合物及其制备方法和用途 WO2024114531A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CN202380079300.0A CN120202203A (zh) 2022-11-29 2023-11-24 杂环化合物及其制备方法和用途
KR1020257016035A KR20250106279A (ko) 2022-11-29 2023-11-24 헤테로고리 화합물 및 이의 제조방법과 용도
AU2023404000A AU2023404000A1 (en) 2022-11-29 2023-11-24 Heterocyclic compound, and preparation method therefor and use thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202211510297 2022-11-29
CN202211510297.7 2022-11-29
CN202211573191 2022-12-08
CN202211573191.1 2022-12-08

Publications (2)

Publication Number Publication Date
WO2024114531A1 true WO2024114531A1 (zh) 2024-06-06
WO2024114531A9 WO2024114531A9 (zh) 2024-10-03

Family

ID=91218681

Family Applications (3)

Application Number Title Priority Date Filing Date
PCT/CN2023/130235 WO2024114318A1 (zh) 2022-11-29 2023-11-07 药物连接子化合物及其制备方法和用途
PCT/CN2023/133966 WO2024114523A1 (zh) 2022-11-29 2023-11-24 抗体药物偶联物及其制备方法和用途
PCT/CN2023/134038 WO2024114531A1 (zh) 2022-11-29 2023-11-24 杂环化合物及其制备方法和用途

Family Applications Before (2)

Application Number Title Priority Date Filing Date
PCT/CN2023/130235 WO2024114318A1 (zh) 2022-11-29 2023-11-07 药物连接子化合物及其制备方法和用途
PCT/CN2023/133966 WO2024114523A1 (zh) 2022-11-29 2023-11-24 抗体药物偶联物及其制备方法和用途

Country Status (5)

Country Link
KR (1) KR20250106279A (zh)
CN (3) CN118105508A (zh)
AU (2) AU2023404000A1 (zh)
TW (1) TW202421632A (zh)
WO (3) WO2024114318A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025087401A1 (zh) * 2023-10-27 2025-05-01 四川科伦博泰生物医药股份有限公司 稠环化合物及其制备方法和用途

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103687945A (zh) 2011-04-25 2014-03-26 第一三共株式会社 抗-b7-h3抗体
CN111936169A (zh) 2018-04-06 2020-11-13 西雅图遗传学公司 喜树碱肽缀合物
CN111295389B (zh) 2017-12-15 2021-10-22 四川科伦博泰生物医药股份有限公司 生物活性物偶联物及其制备方法和用途
WO2022102695A1 (ja) * 2020-11-12 2022-05-19 第一三共株式会社 抗ｂ７－ｈ３抗体－薬物コンジュゲート投与による中皮腫の治療
WO2022166762A1 (zh) * 2021-02-05 2022-08-11 四川科伦博泰生物医药股份有限公司 喜树碱类化合物及其制备方法和应用
WO2022170971A1 (zh) * 2021-02-09 2022-08-18 苏州宜联生物医药有限公司 生物活性物偶联物及其制备方法和用途
CN114929284A (zh) * 2019-10-04 2022-08-19 西根公司 喜树碱肽缀合物
CN115160403A (zh) * 2022-07-05 2022-10-11 上海彩迩文生化科技有限公司 特异性拓扑异构酶抑制剂和可用于抗体药物偶联物及其制备方法
WO2022253033A1 (zh) * 2021-06-02 2022-12-08 四川科伦博泰生物医药股份有限公司 一类化学偶联连接子及其用途
WO2022253035A1 (zh) 2021-06-02 2022-12-08 四川科伦博泰生物医药股份有限公司 抗体药物偶联物及其制备方法和用途
WO2023207710A1 (zh) * 2022-04-29 2023-11-02 四川科伦博泰生物医药股份有限公司 一类抗体药物偶联物、其药物组合物及应用
WO2023216956A1 (zh) * 2022-05-13 2023-11-16 四川科伦博泰生物医药股份有限公司 喜树碱类化合物及其制备方法和应用

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR112015006521B1 (pt) * 2012-10-11 2022-03-03 Daiichi Sankyo Company, Limited Conjugado anticorpo-fármaco, seu método de preparação e seu uso, fármacos e composição farmacêutica
EP4364754A3 (en) * 2016-06-08 2025-02-12 AbbVie Inc. Anti-b7-h3 antibodies and antibody drug conjugates
US12312406B2 (en) * 2018-11-09 2025-05-27 Shanghai Fudan-Zhangjiang Bio-Pharmaceutical Co., Ltd. Anti-B7-H3 antibody, preparation method therefor, conjugate and application thereof
CN110642948B (zh) * 2019-10-09 2021-06-29 达石药业(广东)有限公司 B7-h3纳米抗体、其制备方法及用途
MX2022008474A (es) * 2020-01-22 2022-08-02 Jiangsu Hengrui Medicine Co Conjugado de anticuerpo anti-trop-2-analogo de exatecan y uso medico del mismo.
US20230226207A1 (en) * 2020-06-08 2023-07-20 Sichuan Baili Pharm Co., Ltd Camptothecin Drug Having High-Stability Hydrophilic Connecting Unit And Conjugate Thereof
CN111944050B (zh) * 2020-08-19 2022-05-13 苏州普乐康医药科技有限公司 一种抗b7-h3抗体及其应用
EP4227309A4 (en) * 2020-10-12 2024-11-27 Sichuan Baili Pharmaceutical Co. Ltd. DEUTERATED CAMPTOTHECIN DERIVATIVE AND ANTIBODY-DRUG CONJUGATE THEREOF
KR20250004686A (ko) * 2022-04-29 2025-01-08 쓰촨 케룬-바이오테크 바이오파마수티컬 컴퍼니 리미티드 항체-약물 접합체, 및 이의 제조 방법 및 용도

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103687945A (zh) 2011-04-25 2014-03-26 第一三共株式会社 抗-b7-h3抗体
CN111295389B (zh) 2017-12-15 2021-10-22 四川科伦博泰生物医药股份有限公司 生物活性物偶联物及其制备方法和用途
CN111936169A (zh) 2018-04-06 2020-11-13 西雅图遗传学公司 喜树碱肽缀合物
CN114929284A (zh) * 2019-10-04 2022-08-19 西根公司 喜树碱肽缀合物
WO2022102695A1 (ja) * 2020-11-12 2022-05-19 第一三共株式会社 抗ｂ７－ｈ３抗体－薬物コンジュゲート投与による中皮腫の治療
WO2022166762A1 (zh) * 2021-02-05 2022-08-11 四川科伦博泰生物医药股份有限公司 喜树碱类化合物及其制备方法和应用
WO2022170971A1 (zh) * 2021-02-09 2022-08-18 苏州宜联生物医药有限公司 生物活性物偶联物及其制备方法和用途
WO2022253033A1 (zh) * 2021-06-02 2022-12-08 四川科伦博泰生物医药股份有限公司 一类化学偶联连接子及其用途
WO2022253035A1 (zh) 2021-06-02 2022-12-08 四川科伦博泰生物医药股份有限公司 抗体药物偶联物及其制备方法和用途
WO2023207710A1 (zh) * 2022-04-29 2023-11-02 四川科伦博泰生物医药股份有限公司 一类抗体药物偶联物、其药物组合物及应用
WO2023216956A1 (zh) * 2022-05-13 2023-11-16 四川科伦博泰生物医药股份有限公司 喜树碱类化合物及其制备方法和应用
CN115160403A (zh) * 2022-07-05 2022-10-11 上海彩迩文生化科技有限公司 特异性拓扑异构酶抑制剂和可用于抗体药物偶联物及其制备方法

Non-Patent Citations (26)

* Cited by examiner, † Cited by third party
Title
"Fundamental Immunology", 1989, RAVEN PRESS
"Remington's Pharmaceutical Sciences", 1995, MACK PUBLISHING COMPANY
ALFTHAN ET AL., PROTEIN ENG., vol. 8, 1995, pages 725 - 731
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426
BRUMMELL ET AL., BIOCHEM., vol. 32, 1993, pages 1180 - 1187
BURKS ET AL., PROC. NATL ACAD. SET USA, vol. 94, 1997, pages 412 - 417
CHOI ET AL., EUR. J. IMMUNOL., vol. 31, 2001, pages 94 - 106
CHOTHIA ET AL., NATURE, vol. 341, 1989, pages 544 - 546
CHOTHIALESK, J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
DAVIES ET AL., ANNUAL REV BIOCHEM, vol. 59, 1990, pages 439 - 473
E. MEYERSW. MILLER, COMPUT. APPL BIOSCI., vol. 4, 1988, pages 11 - 17
HOLLIGER ET AL., NAT BIOTECHNOL, vol. 23, 2005, pages 1126 - 1136
HOLLIGER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 6444 - 6448
HOLT, L. ET AL., TRENDS IN BIOTECHNOLOGY, vol. 21, no. 11, 2003, pages 484 - 490
HU ET AL., CANCER RES., vol. 56, 1996, pages 3055 - 3061
HUSTON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 5879 - 5883
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, PUBLIC HEALTH SERVICE
KIPRIYANOV ET AL., J. MOL. BIOL., vol. 293, 1999, pages 41 - 56
KOBAYASHI ET AL., PROTEIN ENG., vol. 12, no. 10, 1999, pages 879 - 884
LEFRANC ET AL., DEV. COMPARAT. IMMUNOL., vol. 27, 2003, pages 55 - 77
MALMQVIST M, NATURE, vol. 361, 1993, pages 186 - 187
MARTIN ACR, CHEETHAM JC, REES AR: " Modelling antibody A combined algorithm", PROC NATL ACAD SCI USA, vol. 86, 1989, pages 9268 - 9272
NEEDLEMAN ET AL., J. MOL. BIOL., vol. 48, 1970, pages 443 - 453
NEEDLEMANWUNSCH, J MOL BIOL., vol. 48, 1970, pages 444 - 453
PLUCKTHUN: "The Pharmacology of Monoclonal Antibodies", vol. 113, 1994, SPRINGER-VERLAG, pages: 269 - 315
ROOVERS ET AL., CANCER IMMUNOL, 2001

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025087401A1 (zh) * 2023-10-27 2025-05-01 四川科伦博泰生物医药股份有限公司 稠环化合物及其制备方法和用途

Also Published As

Publication number Publication date
KR20250106279A (ko) 2025-07-09
WO2024114523A9 (zh) 2025-01-02
AU2023403987A1 (en) 2025-05-29
AU2023404000A1 (en) 2025-05-29
WO2024114531A9 (zh) 2024-10-03
WO2024114318A1 (zh) 2024-06-06
WO2024114523A1 (zh) 2024-06-06
CN120202203A (zh) 2025-06-24
CN120202027A (zh) 2025-06-24
CN118105508A (zh) 2024-05-31
TW202421632A (zh) 2024-06-01
WO2024114318A9 (zh) 2024-10-03

Similar Documents

Publication Publication Date Title
KR20240016249A (ko) 항체 약물 접합체, 이의 제조 방법, 및 이의 용도
WO2023208216A1 (en) Antibody-drug conjugates and preparation methods and use thereof
CN109600993A (zh) 抗egfr抗体药物偶联物
JP2019521975A (ja) 抗ｅｇｆｒ抗体薬物コンジュゲート
WO2023143263A1 (zh) 一种针对Her3的抗体，偶联物及其用途
WO2024078586A1 (en) Antibody-drug conjugate binding to human ptk7 and method for preparation and use thereof
WO2024114318A1 (zh) 药物连接子化合物及其制备方法和用途
KR20250113401A (ko) 항체 약물 접합체 및 이의 제조 방법과 용도
WO2025045015A1 (zh) 抗体-药物缀合物及其制备方法和用途
WO2024235133A1 (zh) 多环类药物偶联物及其制备方法和用途
TW202408590A (zh) 抗體藥物偶聯物及其製備方法和用途
WO2024235134A9 (zh) 具有靶向作用的偶联物及其制备方法和用途
WO2024235131A1 (zh) 大环类药物偶联物及其制备方法和用途
WO2025139993A1 (zh) 一类抗体药物偶联物、其药物组合物及应用
CN119053607A (zh) 喜树碱类化合物及其偶联物、其制备方法和用途
KR20250048561A (ko) Msln 항체-약물 접합체
CN120000805A (zh) 靶向EGFR/c-MET的抗体-药物缀合物及其制备方法和用途
TW202515913A (zh) Nectin-4抗體及抗體藥物結合物

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23896682

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 821548

Country of ref document: NZ

Ref document number: 2023896682

Country of ref document: EP

Ref document number: AU2023404000

Country of ref document: AU

ENP Entry into the national phase

Ref document number: 2023896682

Country of ref document: EP

Effective date: 20250515

ENP Entry into the national phase

Ref document number: 2023404000

Country of ref document: AU

Date of ref document: 20231124

Kind code of ref document: A

WWP Wipo information: published in national office

Ref document number: 821548

Country of ref document: NZ

WWE Wipo information: entry into national phase

Ref document number: 11202503306X

Country of ref document: SG

WWP Wipo information: published in national office

Ref document number: 11202503306X

Country of ref document: SG