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WO2024255873A1 - Method for preparing antibody-drug conjugate - Google Patents

Method for preparing antibody-drug conjugate Download PDF

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Publication number
WO2024255873A1
WO2024255873A1 PCT/CN2024/099341 CN2024099341W WO2024255873A1 WO 2024255873 A1 WO2024255873 A1 WO 2024255873A1 CN 2024099341 W CN2024099341 W CN 2024099341W WO 2024255873 A1 WO2024255873 A1 WO 2024255873A1
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Prior art keywords
formula
alkyl
group
substituted
antibody
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Pending
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PCT/CN2024/099341
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French (fr)
Chinese (zh)
Inventor
周伟
朱会凯
陈宇航
王珍珍
谭小钉
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangsu Mabwell Health Pharmaceutical R & D Co Ltd
Mabwell Shanghai Bioscience Co Ltd
Original Assignee
Jiangsu Mabwell Health Pharmaceutical R & D Co Ltd
Mabwell Shanghai Bioscience Co Ltd
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Publication of WO2024255873A1 publication Critical patent/WO2024255873A1/en
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68033—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a maytansine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention belongs to the field of biomedicine, and more specifically, the present invention relates to a new method for preparing antibody-drug conjugates.
  • Antibody-drug conjugates are a new form of drug that connects small molecule drugs to monoclonal antibodies. They use the targeting function of monoclonal antibodies to transport small molecule drugs to target sites to exert their efficacy, reduce toxic side effects, and increase the therapeutic window. In recent years, antibody-drug conjugates have been one of the hot directions in precision tumor treatment, bringing hope to tumor treatment.
  • Antibody-drug conjugates are composed of antibody drugs targeting specific antigens and small molecule cytotoxic drugs coupled through linkers. They have both the powerful killing effect of traditional small molecule chemotherapy and the tumor targeting of antibody drugs. So far, more than a dozen ADC drugs have been approved for marketing worldwide.
  • Antibody-drug conjugates consist of several parts, such as antibodies, linkers, and payloads. Among them, antibodies and drug-linkers can be connected in different coupling modes.
  • the coupling methods are mainly divided into non-fixed site coupling and fixed site coupling. In the early days, non-fixed site coupling methods were used, mainly lysine coupling and cysteine coupling, which directly coupled drugs to amino acid residues on antibodies using chemical methods, without involving antibody transformation or modification. The number of toxin molecules and coupling sites coupled to them cannot be determined, and the uniformity is poor.
  • the currently commonly used fixed-site coupling method is to perform specific coupling through genetic engineering sites or special connectors to achieve more uniform coupling, and can achieve the connection of cytotoxins at specific sites.
  • Antibody-drug conjugates produced by fixed-site coupling can reduce the fluctuations in efficacy, pharmacokinetics, and quality control caused by different coupling sites and coupling numbers.
  • common fixed-site coupling methods include THIOMAB technology, non-natural amino acid coupling technology, glutamine enzymatic coupling technology, Sortase transpeptidase coupling technology, and Thiobridge technology.
  • the modification of antibodies by antibody engineering or enzymatic coupling may have a certain impact on the structural stability of the antibody, and at the same time have certain requirements for CMC.
  • the Thiobridge technology using chemical coupling also has certain defects, such as the replacement of DBM (dibromomaleimides)-type linkers and other thiol-containing biological groups, which are unstable in plasma, resulting in reduced efficacy and increased toxic side effects (Chem.-Eur.J, 2019, 25, 43-59.).
  • DBM dibromomaleimides
  • the drug-to-antibody ratio is a unique and important quality attribute of ADC drugs, which represents the average number of small molecule drugs coupled to antibodies.
  • the degree of drug coupling will affect the stability and aggregation tendency of ADC drugs. Therefore, the DAR value is a key quality attribute for ADC drug analysis and an important quality control link in the ADC drug development process.
  • Fixed-site coupling technology can achieve fixed-site and quantitative coupling of antibodies and small molecule toxins.
  • the ADC obtained by this technology has a suitable drug-to-antibody ratio (DAR), high uniformity, good stability, high batch-to-batch reproducibility, better activity and pharmacokinetic properties, and is also more suitable for large-scale production of ADC.
  • ADC drugs use site-specific coupling methods to couple highly active small molecule drugs to antibodies to form site-specific DAR2 ADCs, such as the approved targeted CD19-ADC (loncastuximab tesirine, Zynlonta) that couples PBD small molecules to antibodies through glycosylation site-specific coupling technology to achieve site-specific DAR2, as well as targeted HER2 ADC (ARX788) and targeted FR ⁇ ADC (MORAb-202). All of them use the method of non-natural amino acid mutation to achieve site-specific DAR2.
  • these methods either require the use of enzymes or the modification of antibody sequences, and the manufacturing process is complicated.
  • WO2022253033A1 discloses the following method for preparing an ADC conjugate: dilute the antibody with a solution of disodium edetate, then adjust the pH with a Na 2 HPO 4 solution, add a TCEP (tri(2-carboxyethyl)phosphine) solution and mix well, and leave at room temperature for 2 hours. Add 5-10 times the amount of the antibody to the above solution system, mix well, and let stand at room temperature for 20 hours. After completion, use a NAP-5 gel column (Cytiva) to replace the buffer with a 10mM histidine buffer solution at pH 6.0 to obtain an ADC product. According to sample measurement, the DAR of the ADC conjugate prepared by the above non-site-specific coupling method in this patent is 4, and there is no record of a DAR of 2.
  • WO2022078524A2 discloses a site-specific coupling method for antibody conjugates. After reducing and incubating the antibody with a variety of zinc complexes and TCEP, a payload/linker complex is introduced. Cysteine is then added to consume excess TCEP and excess payload. Free thiol groups in the protein are oxidized. Finally, the reaction mixture is purified using a desalting column or UF/DF or ion exchange chromatography. The results of the DAR analysis of the conjugates using HIC-HPLC showed that the proportion of conjugates with a DAR of 4 was about 60-70%, while the proportion of conjugates with a DAR of 2 was only 5-10%. It can be seen that the DAR of the antibody conjugates prepared by this patented method is mainly concentrated in 4, while the conjugate products with a DAR of 2 account for only a minority.
  • WO2020164561A1 prepares an antibody-drug conjugate (ADC) with improved homogeneity, and the coupling method used comprises the following steps:
  • a reducing agent e.g., tris(2-carboxyethyl)phosphine (TCEP)
  • TCEP tris(2-carboxyethyl)phosphine
  • a buffer system e.g., Hepes, histidine buffer, PBS, MES, etc.
  • transition metal ions e.g., Zn2+, etc.
  • a payload carrying a reactive group e.g., a maleimide-linked drug
  • an oxidant e.g., dehydroascorbic acid (DHAA)
  • the DAR value is the key quality attribute of ADC.
  • high DAR can bring more small molecule drugs to the target site and may have a better therapeutic effect
  • the design of high DAR will sacrifice the stability and uniformity of ADC drugs, which may cause the aggregation of antibodies, thereby affecting the efficacy of ADC drugs and causing greater toxic side effects.
  • the preparation of ADCs with high uniformity and/or purity of DAR2 can avoid the adverse effects of high DAR and can be beneficial to the stability and efficacy of ADC drugs.
  • the prior art can achieve fixed-point DAR2 through glycosylation fixed-point coupling technology. However, these methods either require the use of enzymes or the modification of antibody sequences, and the manufacturing process is complicated.
  • the chemical fixed-point coupling method currently reported for the preparation of ADC although the process is relatively simple, can only achieve ADC products with a main DAR of 4.
  • the present invention provides a novel method for preparing an antibody-drug conjugate, which obtains highly homogeneous antibody conjugate molecules through a specific chemical site-specific coupling method, and the DAR2 main peak accounts for more than 99%.
  • the method is simple to operate and is conducive to industrial amplification.
  • the prepared product has few impurities and high purity, which significantly improves the safety of medication and reduces production costs.
  • the anti-tumor drug prepared by the coupling method of the present invention has excellent anti-tumor effect and safety (including higher stability and less side effects).
  • the present invention provides a method for preparing an antibody-drug conjugate (ADC), the method comprising the following steps:
  • the metal in the metal salt or metal complex described in step (a) can be selected from Zn, Cd, Hg, and the like.
  • the metal salt or metal complex in step (a) is selected from one or more of the following: hydrochloride or sulfate of Zn, Cd and Hg,
  • the Zn salts or Zn complexes disclosed in WO2022078524A2 include but are not limited to
  • the metal salt or metal complex in step (a) is selected from ZnCl 2 , CdCl 2 , HgCl 2 , One or more of .
  • the reducing agent in step (a) is selected from tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), mercaptoethylamine and mercaptoethanol, preferably TCEP.
  • TCEP tris(2-carboxyethyl)phosphine
  • DTT dithiothreitol
  • mercaptoethylamine mercaptoethanol
  • the concentration of the antibody in step (a) is 0.5-150 mg/mL, such as 1-75 mg/mL, 1-50 mg/mL, 2-40 mg/mL, 3-30 mg/mL, 4-30 mg/mL or 5-15 mg/mL, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120 or 150 mg/mL.
  • the amount of the reducing agent used in step (a) is 1-6 times the antibody molar equivalent, such as 2-6, 3-5, 3.5-5.0 or 4.0-4.5 times the antibody molar equivalent, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 times the antibody molar equivalent.
  • the amount of the metal salt or metal complex used in step (a) is 1-5 times the antibody molar equivalent, such as 1-4, 1-3 or 1.5-2.5 times the antibody molar equivalent, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 times the antibody molar equivalent.
  • the buffer solution in step (a) has a pH of 5-8, such as a pH of 5.5-7.5, 6.5-7.5 or 6.5-7.4, such as a pH of 5.0, 5.5, 6.0, 6.5, 6.9, 7.0, 7.5 or 8.0, preferably a pH of 6.9.
  • the buffer solution in step (a) is selected from phosphate buffer, acetate buffer, citrate buffer, succinate buffer, preferably disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.
  • the buffer concentration in the buffer solution is 5-100mM, such as 10-90mM, 20-80mM, 30-70mM, 40-60mM, 10-30 or mM, such as 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100mM.
  • the incubation in step (a) is performed at 0-30°C, such as 0-15°C or 5-15°C.
  • the incubation in step (a) is performed for 1-48 hours, such as 2-24 hours, 2-12 hours, 3-10 hours, 4-8 hours or 5-24 hours, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 35 hours.
  • the amount of the drug-containing linker in step (b) is 1-8 times the antibody molar equivalent, such as 2-6, 2.5-5.5, 3-5 or 3.5-4.5 times the antibody molar equivalent, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7 or 7.5 times the antibody molar equivalent.
  • the coupling reaction temperature in step (b) is 0-30°C, such as 0-15°C or 3-15°C.
  • the reaction solution in step (b) has a pH of 5-8, such as a pH of 5.5-7.5, 6.5-7.5, or 6.5-7.4, such as a pH of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0.
  • the drug-containing linker is provided as a DMA solution.
  • the coupling reaction of step (b) is carried out for 0.2-24 hours, such as 0.5-12 hours, 1-10 hours, 1.5-5 hours, 1-3 hours or 1-4 hours, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 hours. Hour.
  • the hydrophobic gel filler in step (c) is selected from Butyl-650M, Butyl-4FF, Butyl-650S, Butyl-ImpRes and Phenyl Sepharose HP.
  • the type of salt in step (c) is selected from sulfates, chlorides, etc., such as alkali metal (such as lithium, sodium, potassium) salts of sulfuric acid or hydrochloric acid, alkaline earth metal (such as calcium, magnesium) salts, salts formed by amines or ammonia and sulfuric acid or hydrochloric acid, preferably ammonium sulfate and sodium chloride; preferably, the salt concentration is 0.1-5 mol/L, such as 0.2-4 mol/L, 0.3-3 mol/L, 0.5-2 mol/L, 0.5-1.5 mol/L, 0.6-1.2 mol/L or 0.6-1.0 mol/L, such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5 or 2.0 mol/L.
  • alkali metal such as lithium, sodium, potassium
  • alkaline earth metal such as calcium, magnesium
  • salt concentration is 0.1-5 mol/L, such as 0.2-4 mol
  • the salt solution described in step (c) is used as mobile phase A for hydrophobic interaction chromatography.
  • the hydrophobic interaction chromatography in step (c) uses phosphate buffer as mobile phase B, preferably, the phosphate buffer has a phosphate concentration of 5-200 mM and a pH of 7.0-7.6, such as 7.1, 7.2, 7.3, 7.4 or 7.5.
  • mobile phase A and/or mobile phase B may further contain other solvents such as isopropanol.
  • step (b) is followed by step (b'): adding a metal ion chelator such as ethylenediaminetetraacetic acid (EDTA) to react.
  • a metal ion chelator such as ethylenediaminetetraacetic acid (EDTA)
  • step (b') is performed at room temperature, e.g., for 5 min-5 hours, 10 min-45 min, or 30 min.
  • the amount of metal ion chelator used is 1-8 times the antibody molar equivalent, such as 2-6, 2.5-5.5, 3-5 or 3.5-4.5 times the antibody molar equivalent, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7 or 7.5 times the antibody molar equivalent.
  • step (b') is followed by step (b"): adding dehydroascorbic acid (DHAA) to react.
  • DHAA dehydroascorbic acid
  • step (b") is performed at 25-35°C, for example 0.2-24 hours, for example 0.5-12 hours, 1-10 hours, 1.5-5 hours, 1-3 hours or 1-4 hours, for example 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 hours.
  • the amount of DHAA used is 1-16 times the antibody molar equivalent, such as 2-10, 2-6, 6-16 or 6-12 times the antibody molar equivalent, such as 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14 or 16 times the antibody molar equivalent.
  • the DAR2-ADC obtained by the method of the present invention has a purity greater than 90%, such as greater than 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, as measured, for example, by HIC-HPLC.
  • the antibody is selected from the group consisting of a humanized antibody, a murine antibody, a human antibody, a chimeric antibody, a single chain antibody, and a bispecific antibody.
  • the antibody is a monoclonal antibody or a polyclonal antibody.
  • the antibody is an antibody that binds to the following antigens: HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, tissue factor, c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C, Mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, Claudin 18.2.
  • the antibody is an anti-HER2 antibody.
  • the antibody comprises one or more CDRs (preferably 3 CDRs, i.e., HCDR1, HCDR2H and HCDR3; or LCDR1, LCDR2 and LCDR3, more preferably 6 CDRs, i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) of adalimumab, bevacizumab, cetuximab, trastuzumab, pertuzumab, nimotuzumab, rituximab, h23-12 or hH2L1, or comprises the VH and/or VL of the antibody, or comprises the heavy chain and/or light chain of the antibody.
  • CDRs preferably 3 CDRs, i.e., HCDR1, HCDR2H and HCDR3; or LCDR1, LCDR2 and LCDR3, more preferably 6 CDRs, i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and
  • the antibody is selected from adalimumab, bevacizumab, cetuximab, trastuzumab, pertuzumab, nimotuzumab, rituximab, h23-12, and hH2L1.
  • the antibody comprises one or more CDRs (preferably 3 CDRs, i.e., HCDR1, HCDR2H and HCDR3; or LCDR1, LCDR2 and LCDR3, more preferably 6 CDRs, i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) of trastuzumab, Pertuzumab, h23-12 or hH2L1, or comprises the VH and/or VL of trastuzumab, Pertuzumab, h23-12 or hH2L1, or comprises the heavy chain and/or light chain of the antibody.
  • CDRs preferably 3 CDRs, i.e., HCDR1, HCDR2H and HCDR3; or LCDR1, LCDR2 and LCDR3, more preferably 6 CDRs, i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3
  • trastuzumab preferably 3 CDRs
  • the antibody is selected from trastuzumab, pertuzumab, h23-12, and hH2L1.
  • the drug-containing linker in step (b) is a compound represented by "L 1 -AL 2 -D" or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.
  • L1 represents a linker unit for connecting to an antibody
  • A represents one or more (e.g. 1, 2, 3 or 4) amino acids
  • L2 represents a linker unit for connecting to a small molecule drug
  • D is a small molecule drug.
  • the drug-containing linker in step (b) is a compound represented by Formula AI or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof,
  • E is selected from the following groups, wherein The bond of represents the attachment site to M:
  • n is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • R 6 and R 7 are independently halogen or Ar'S-
  • Ar' is phenyl or phenyl substituted by one or more substituents, in the substituted phenyl, the substituents are selected from alkyl (e.g. C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), 5-6 membered heterocyclic group -CO-, halogen, ester group, amide group and cyano group.
  • Ar' is phenyl, 4-methylcarbamoylphenyl or 4-formylmorpholine substituted phenyl
  • M is a phenylene group or a phenylene group substituted by one or more substituents, or a chemical bond; in the substituted phenylene group, the substituent group is selected from an alkyl group (e.g., a C1-C6 alkyl group, preferably a C1-C4 alkyl group), a haloalkyl group (e.g., a halo C1-C6 alkyl group, preferably a halo C1-C4 alkyl group, such as a trifluoromethyl group), an alkoxy group (e.g., a C1-C6 alkoxy group, preferably a C1-C4 alkoxy group, preferably a methoxy group), a halogen group, an ester group, an amide group and a cyano group; preferably, M is a halogen-substituted phenylene group.
  • an alkyl group e.g., a C1
  • SP 1 is selected from C1-8 alkylene, C1-8 alkylene phenylene-, C1-8 cycloalkylene or C1-21 (preferably C1-16, more preferably C1-11) straight chain heteroalkylene, the C1-21 straight chain heteroalkylene containing 1-11 (preferably 1-6) heteroatoms selected from N, O or S, wherein the C1-8 alkylene, C1-8 cycloalkylene and C1-21 straight chain heteroalkylene are each independently optionally substituted by one or more substituents selected from hydroxy, oxo, amino, sulfonic acid and cyano;
  • SP 2 is selected from -NH(CH2CH2O)aCH2CH2CO-, -NH(CH2CH2O)aCH2CO-, -S(CH2)aCO- or a chemical bond, wherein a is an integer of 1-20, preferably an integer of 1-10, more preferably an integer of 1-6;
  • A represents 2 to 4 amino acids, wherein when A represents 2 amino acids, it can be NH-Phe-Lys-CO, NH-Val-Ala-CO, NH-Val-Lys-CO, NH-Ala-Lys-CO, NH-Val-Cit-CO, NH-Phe-Cit-CO, NH-Leu-Cit-CO, NH-Phe-Arg-CO or NH-Gly-Val-CO, preferably NH-Phe-Lys-CO, NH-Val-Ala-CO or NH-Val-Cit-CO.
  • A when A represents 3 amino acids, it can be NH-Glu-Val-Ala-CO, NH-Glu-Val-Cit-CO or NH-Ala-Ala-Ala-CO, preferably NH-Glu-Val-Ala-CO or NH-Ala-Ala-Ala-CO; when A represents 4 amino acids, it can be NH-Gly-Gly-Phe-Gly-CO or NH-Gly-Phe-Gly-Gly-CO, preferably NH-Gly-Gly-Phe-Gly-CO.
  • A is NH-Val-Ala-CO, NH-Gly-Gly-Phe-Gly-CO or NH-Ala-Ala-Ala-CO;
  • A is selected from the group consisting of NH-Val-Cit-CO, NH-Val-Ala-CO and NH-Gly-Gly-Phe-Gly-CO;
  • NH on the left side of A is the amino group in the left amino acid in A
  • CO on the right side of A is the carbonyl group in the right amino acid in A
  • L 2 is selected from In some embodiments, M is selected from
  • q is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • p is an integer selected from 1-20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;
  • D is selected from the group consisting of camptothecins, calicheamicins, maytansinoids, dolastatins, auristatins, and trichothecenes.
  • the maytansinoid derivative is selected from DM1, DM3 and DM4; the auristatin compound is selected from MMAE and MMAF; the camptothecin compound is selected from camptothecin, 10-hydroxycamptothecin, exatecan, SN-38, topotecan and camptothecin derivatives.
  • the drug-containing linker is a compound of Formula III or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.
  • E, M, SP 1 , SP 2 and A are as defined above, and CPT is a camptothecin compound.
  • the drug-containing linker is a compound represented by Formula III' or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof,
  • E, M, SP 1 , SP 2 and A are as defined above, and D' is an auristatin compound.
  • the compound of formula III is further a compound of formula IIIA:
  • R 6 and R 7 are independently halogen or Ar'S-
  • Ar' is phenyl or phenyl substituted by one or more substituents, in the substituted phenyl, the substituent is selected from alkyl (e.g. C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), 5-6 membered heterocyclic group -CO-, halogen, ester group, amide group and cyano group.
  • Ar' is phenyl, 4-methylcarbamoylphenyl or 4-morpholinoformylphenyl wherein M, SP 1 , SP 2 , A and CPT are as defined above.
  • the compound of formula III' is further a compound of formula IIIA':
  • R 6 , R 7 , M, SP 1 , SP 2 , A and D′ are as defined above.
  • CPT is a compound as shown in Formula I or Formula IA below, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, and the corresponding specific compound:
  • R 1 , R 2 , R 3 , R 4 are independently hydrogen, halogen, Hydroxyl, C1-6 alkoxy, amino or substituted amino, C1-7 alkyl or substituted C1-7 alkyl, or any two of R 1 , R 2 , R 3 , R 4 together with the carbon atoms to which they are connected constitute a C3-6 cyclic alkyl.
  • R 1 , R 2 , R 3 , R 4 are independently C1-6 alkoxy
  • the C1-6 alkoxy includes a straight or branched C1-6 alkoxy, preferably a straight or branched C1-3 alkoxy, more preferably a methoxy.
  • R 1 , R 2 , R 3 , R 4 are independently substituted amino
  • the substituted amino is an amino substituted by one or more substituents selected from methyl and ethyl.
  • R 1 , R 2 , R 3 , and R 4 are independently C1-7 alkyl or substituted C1-7 alkyl
  • the C1-7 alkyl or substituted C1-7 alkyl includes linear or branched C1-7 alkyl or substituted C1-7 alkyl
  • the substituted C1-7 alkyl is C1-7 alkyl substituted by one or more substituents selected from cyclopropyl and cyclobutyl; or, the linear or branched C1-7 alkyl or substituted C1-7 alkyl is preferably C1-3 alkyl or substituted C1-3 alkyl, such as methyl, halogenated methyl (preferably trifluoromethyl).
  • G is hydrogen, halogen, methyl or methoxy.
  • G is hydrogen, fluorine or chlorine.
  • Y is oxygen, sulfur, sulfone, sulfoxide, methylene or substituted methylene.
  • the substituted methylene may be a methylene in which one hydrogen is substituted or two hydrogens are substituted simultaneously, and the substituent may be a benzyl or an alkyl group; when the substituent is an alkyl group, the alkyl group and R 3 and/or R 4 and the carbon atoms to which they are attached may form a C3-6-membered cyclic or spirocyclic structure.
  • Y is a substituted methylene group
  • the substituent of the substituted methylene group is preferably an alkyl group, more preferably a linear or branched C1-4 alkyl group.
  • Y is oxygen, sulfur, sulfone or sulfoxide; or, preferably, Y is oxygen, sulfur or methylene.
  • X is oxygen or sulfur
  • n 0 or 1.
  • R 1 , R 2 , R 3 , and R 4 are independently hydrogen, halogen (e.g., fluorine), C1-7 alkyl, or substituted C1-7 alkyl, or any two of R 1 , R 2 , R 3 , and R 4 together with the carbon atoms to which they are attached constitute a C3-6 cyclic alkyl (e.g., a C3-5 cyclic alkyl).
  • R 1 and R 2 may be the same; and/or, R 3 and R 4 may be the same.
  • Y is a methylene group substituted by an alkyl group, and the alkyl group, R3 and/or R4, and the carbon atoms to which they are connected can form a C3-6-membered cyclic or spirocyclic structure.
  • X may be oxygen
  • X is oxygen
  • G is hydrogen
  • halogen eg fluorine or chlorine
  • Y and R 1 , R 2 , R 3 , R 4 are as defined above.
  • X is oxygen
  • G is fluorine
  • Y is methylene or substituted methylene, oxygen or sulfur
  • R 1 , R 2 , R 3 and R 4 are as defined above.
  • X is oxygen
  • G is chlorine
  • Y is methylene or substituted methylene, oxygen or sulfur
  • R 1 , R 2 , R 3 and R 4 are as defined above.
  • X is oxygen
  • G is methyl
  • Y is methylene or substituted methylene, oxygen or sulfur
  • R 1 , R 2 , R 3 and R 4 are as defined above.
  • X is oxygen
  • G is methoxy
  • Y is methylene or substituted methylene, oxygen or sulfur
  • R 1 , R 2 , R 3 and R 4 are as defined above.
  • X is oxygen
  • G is hydrogen
  • Y is oxygen, sulfone or sulfoxide
  • R 1 , R 2 , R 3 , R 4 are as defined above.
  • X is oxygen
  • G is hydrogen
  • Y is sulfone or sulfoxide
  • R 1 , R 2 , R 3 , R 4 are as defined above.
  • the compound represented by formula I is further represented by formula IA:
  • R 1 , R 2 , R 3 , and R 4 are the same as defined in formula I above.
  • R 1 , R 2 , R 3 , and R 4 may be hydrogen at the same time or not at the same time.
  • the camptothecin compound is a compound represented by Formula II or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
  • R 5 is C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted by one or more substituents, phenyl or substituted phenyl.
  • R 5 is C1-5 alkyl or substituted C1-5 alkyl
  • the C1-5 alkyl includes straight or branched C1-5 alkyl.
  • R 5 is C1-4 straight chain alkyl.
  • R 5 When R 5 is substituted C1-5 alkyl or substituted C3-6 cyclic alkyl, the substituent is selected from halogen, hydroxyl, methoxy, trifluoromethyl, amino or substituted amino, mesyl and C3-6 cyclic alkyl; and wherein the substituted amino is an amino substituted by one or more substituents selected from methyl and ethyl.
  • R 5 When R 5 is substituted phenyl, the substituent is selected from alkyl (e.g. C1-6 alkyl, preferably C1-3) or halogen.
  • G is hydrogen, halogen (eg fluorine), methyl or methoxy.
  • G is hydrogen, fluorine or chlorine.
  • X is oxygen or sulfur.
  • n 0 or 1.
  • the compound represented by formula II is further represented by formula IIA:
  • R 5 is the same as the definition of the group R 5 in Formula II above.
  • R 5 may not be n-butyl.
  • R 5 may be n-butyl.
  • camptothecin compound (CPT) has the following structure:
  • the compound represented by Formula I or Formula IA is connected to the carboxyl group of A through an amide bond via its amino group (see Formula I or Formula IA, respectively), that is, the amino group of the compound represented by Formula I or Formula IA forms an amide bond with the carboxyl group of A in Formula III or IIIA.
  • the compound represented by formula II or formula IIA is connected to the carboxyl group of A through an amide bond via its amino group (see formula II or formula IIA, respectively), that is, the amino group of the compound represented by formula II or formula IIA forms an amide bond with the carboxyl group of A in formula III or IIIA.
  • CPT pharmaceutically acceptable salts, stereoisomers, solvates, or prodrugs thereof set forth above
  • CPT pharmaceutically acceptable salts, stereoisomers, solvates, or prodrugs thereof set forth above
  • the amino group is linked to the carboxyl group of A in formula III or IIIA via an amide bond.
  • CPT in Formula III and Formula IIIA, can be an exteacan derivative shown in Formula IV or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
  • R 8 is hydrogen, trifluoromethyl, C 1-5 alkyl or C 1-5 alkyl substituted by one or more substituents, C 3-6 cyclic alkyl or C 3-6 cyclic alkyl substituted by one or more substituents, or halogen.
  • R8 is substituted C1-5 alkyl or substituted C3-6 cyclic alkyl
  • the substituent is selected from halogen, hydroxy, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl and C3-6 cyclic alkyl; and wherein the substituted amino is an amino substituted by one or more substituents selected from methyl and ethyl.
  • the compound represented by formula IV is connected to the carboxyl group of A through a self-releasing structure via its hydroxyl group connected to the same carbon as R 8 (see formula IV), and the self-releasing structure is, for example
  • the solid line indicates the site of attachment to the carboxyl group of A in Formula III or Formula IIIA, and the wavy line indicates the site of attachment to the hydroxyl group in Formula IV.
  • M is preferably a phenylene or substituted phenylene
  • SP1 is a C1-21 (preferably C1-16, more preferably C1-11) straight chain heteroalkylene, wherein the straight chain heteroalkylene contains 1-11 (preferably 1-6) heteroatoms selected from N, O or S.
  • D' is a compound as shown in the following Formula I' or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, and the corresponding specific compound:
  • Ra1 , Ra2 , Ra3 , Ra4 , Ra5 and Ra8 are each independently selected from C1-8 alkyl; preferably C1-4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl;
  • R a6 and R a7 are each independently selected from C 1-8 alkoxy, such as methoxy, ethoxy or propoxy;
  • R a9 is selected from C 1-8 alkyl and COOH; preferably C 1-4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, Isobutyl or sec-butyl;
  • R a10 is selected from OH and H.
  • the compound of formula I' is further a compound of formula IA':
  • R a1 -R a10 are as defined above;
  • the compound of Formula I' is selected from:
  • the compound represented by formula I’ is connected to the carboxyl group of A via an amide bond via its amino group, that is, the left secondary amino group of the compound represented by formula I’ forms an amide bond with the carboxyl group of A in formula III’ or IIIA’.
  • the compound represented by Formula III and Formula IIIA is further represented by Formula V.
  • R 6 and R 7 are independently Ar'S
  • Ar' is phenyl or phenyl substituted by one or more substituents, in the substituted phenyl, the substituent is selected from alkyl (e.g. C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), 5-6 membered heterocyclic group -CO-, halogen, ester group, amide group and cyano group.
  • Ar' is phenyl, 4-methylcarbamoylphenyl or 4-morpholinoformylphenyl
  • Xh and Yh are independently hydrogen, halogen, haloalkyl (e.g. haloC1-C6 alkyl, preferably haloC1-C4 alkyl, such as trifluoromethyl) or alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, such as methoxy).
  • haloalkyl e.g. haloC1-C6 alkyl, preferably haloC1-C4 alkyl, such as trifluoromethyl
  • alkoxy e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, such as methoxy
  • m is any integer from 1 to 10, preferably from 1 to 5, more preferably from 3 to 5.
  • A represents 2 to 4 amino acids, as defined above.
  • the compound of formula IIIA' is further a compound of formula V',
  • R 6 , R 7 , Xh, Yh, m and A are as defined above (especially in formula V), and D′ is as defined above.
  • the compound of formula V' is further a compound of formula V-A',
  • the compound of formula V is further a compound of formula VA:
  • G is hydrogen, fluorine or chlorine.
  • Y is methylene, sulfur or oxygen.
  • the compound represented by Formula VA is further represented by Formula VA-1:
  • the compound represented by formula VB is further represented by formula VB-1:
  • A represents 2 to 4 amino acids, as defined above.
  • CPT is a camptothecin compound.
  • the definition of CPT and its connection relationship in Formula VI are the same as those of CPT in Formula III and Formula IIIA, as defined above.
  • the compound represented by Formula VI provided by the present invention is further represented by Formula VI-A:
  • G is hydrogen, fluorine or chlorine.
  • Y is methylene, sulfur or oxygen.
  • the compound represented by formula VI-A is further represented by formula VI-A-1:
  • formula VC can be further structured as shown in formula VI-C.
  • the drug-containing linker is selected from the following structures:
  • the present invention also relates to the specific methods disclosed in the embodiments of the present invention.
  • the technical solutions obtained by replacing or combining certain technical features in the specific embodiments of the present invention with the technical features in the above-mentioned embodiments are also considered in the present invention. middle.
  • Some small molecule drugs, drug-containing linkers and/or intermediates thereof, and/or antibody-drug conjugates in the present invention are recorded in Chinese patent application 202211627164.8, and the entire content of the patent application is introduced into this application as part of the content of this application.
  • the steps of the above preparation method are interrelated and interact with each other to produce a technical effect of improving the purity of the DAR2-ADC conjugate, and this technical effect is produced by the steps as a whole.
  • the present invention provides an antibody drug conjugate having the general formula The structure shown, wherein mAb represents an antibody or antibody fragment, such as the antibody defined above, and groups M, SP1 , SP2 , A, L2 and D are as defined above in the first aspect.
  • y is 1-10, preferably 1-8 (such as 1-5), more preferably 1-3, and most preferably 2.
  • EL is selected from the following groups, wherein, Indicates that it is linked to the cysteine in the mAb, and the other The key indicates connection with M:
  • n is as defined above in variable E.
  • mAb may be an IgG type antibody or a fragment thereof, preferably an IgG1 subtype antibody or a fragment thereof.
  • the antibody drug conjugate is prepared by the method of the present invention as described in the first aspect.
  • the antibody drug conjugate has an average DAR of about 2.
  • the antibody drug conjugate has a purity greater than 90%, such as greater than 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, such as measured by HIC-HPLC.
  • the antibody drug conjugate has the structure shown in the following general formula VII, VII', VIII, VIII':
  • y is 1-10, preferably 1-8 (eg, 1-5), and more preferably 1, 2 or 3.
  • the present invention provides the small molecule drugs, drug-containing linkers and/or intermediates thereof (including but not limited to formula A-I, I, I', I-A, IA', II, II-A, III, III', III-A, III-A', V, V-A, V-A-1, V-B, V-B-1, V-C, VI, VI-A, VI-A-1, VI-B, VI-B-1, VI-C) described in the preparation method of the first aspect of the present invention, preferably including but not limited to the compounds disclosed in the embodiments.
  • formula A-I, I, I', I-A, IA', II, II-A, III, III', III-A, III-A', V, V-A, V-A-1, V-B, V-B-1, V-C, VI, VI-A, VI-A-1, VI-B, VI-B-1, VI-C described in the preparation method of the first aspect of the present invention, preferably including but not limited to the compounds disclosed in
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the small molecule drug, drug-containing linker, intermediate and/or antibody-drug conjugate described in the first to third aspects of the present invention or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.
  • the pharmaceutical composition is a liquid preparation or a lyophilized preparation.
  • the pharmaceutical composition comprises a buffer, a stabilizer, and a surfactant.
  • the present invention provides use of a compound according to the present invention or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, an antibody-drug conjugate or a pharmaceutical composition thereof in the preparation of a medicament for treating a tumor.
  • the tumor is cancer.
  • the tumor is associated with tumor-associated antigens such as HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor, c-MET, FGFR, Nectin 4, AG It is associated with positive or high expression of S-16, guanylate cyclase C, mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, and C
  • the tumor is colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, liver cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, melanoma, leukemia, lymphoma, glioma, glioblastoma.
  • the present invention provides a method for treating tumors using a compound according to the present invention or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, an antibody-drug conjugate or a pharmaceutical composition thereof, the method comprising administering the compound or antibody-drug conjugate to a subject in need thereof.
  • the tumor is cancer.
  • the tumor is associated with tumor-associated antigens such as HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor, c-MET, FGFR, Nectin 4, AG It is associated with positive or high expression of S-16, guanylate cyclase C, mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, and C
  • the tumor is colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, liver cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, melanoma, leukemia, lymphoma, glioma, glioblastoma.
  • the subject is a mammal, preferably a primate, more preferably a human.
  • the method and/or product of the present invention has the following advantages and/or benefits:
  • the method of the present invention can provide highly homogeneous antibody conjugate molecules, the main peak of which accounts for more than 99% of DAR2;
  • the preparation method of the present invention has few steps and is simple to operate, which is conducive to industrial scale-up production. At the same time, the prepared product has few impurities and high purity, which significantly improves the safety of drug use and reduces production costs; and/or
  • the anti-tumor drug prepared by the coupling method of the present invention has excellent anti-tumor effect and safety, including high stability. Qualitative and smaller side effects, expand the treatment window, improve treatment effects and reduce toxic reactions.
  • ADC antibody drug conjugate
  • the term "antibody drug conjugate” or "ADC” refers to a substance obtained by connecting a bioactive compound fragment (small molecule drug) to an antibody or its antigen-binding fragment.
  • the bioactive compound fragment is connected to the targeting portion through a linker.
  • the linker can be broken in a specific environment (e.g., a low pH environment in the cell) or under a specific action (e.g., the action of a lysosomal protease), thereby separating the bioactive compound fragment from the antibody or its antigen-binding fragment.
  • the linker comprises a cleavable or non-cleavable unit, such as a peptide or a disulfide bond.
  • the bioactive compound fragment is directly connected to the antibody or its antigen-binding fragment through a covalent bond, and the covalent bond can be broken under a specific environment or action, thereby separating the bioactive compound fragment from the antibody or its antigen-binding fragment.
  • linker refers to a fragment that connects a biologically active compound fragment (small molecule drug) to an antibody portion.
  • the linker has a functional group that can form a bond with a functional group of the antibody or its antigen-binding fragment before being attached to the antibody or its antigen-binding fragment (i.e., a linker precursor).
  • small molecule drug refers to a low molecular weight compound that can regulate biological processes.
  • Small molecule is defined as a molecule with a molecular weight less than 10kD, usually less than 2kD and preferably less than 10kD.
  • Small molecules include but are not limited to inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics and antibody mimics. As therapeutic agents, small molecules can be more permeable to cells, less susceptible to degradation and less prone to eliciting immune responses than macromolecules.
  • Small molecule drugs include but are not limited to cytotoxic agents, such as camptothecin compounds, calicheamicin (calicheamicin), maytansinoids (maytansinoids), dolastatin (dolastatin), auristatin compounds and trichothecene (trichothecene).
  • cytotoxic agents such as camptothecin compounds, calicheamicin (calicheamicin), maytansinoids (maytansinoids), dolastatin (dolastatin), auristatin compounds and trichothecene (trichothecene).
  • the maytansinoid derivative is selected from DM1, DM3 and DM4;
  • the auristatin compound is selected from the auristatin compounds mentioned in the embodiments of the present invention, including but not limited to MMAE and MMAF;
  • the camptothecin compound is selected from camptothecin, 10-hydroxycamptothecin, exitecan, SN-38 and topotecan and the camptothecin compounds mentioned in the embodiments of the present invention.
  • a small molecule drug when a small molecule drug is a part of an ADC or a drug-containing linker, it refers to a structural fragment from a small molecule drug, such as a structural fragment formed by bonding a hydroxyl group or an amino group of a small molecule drug to a linker, which can form a part (fragment or radical) of a biologically active drug (such as a small molecule cytotoxic drug, wherein the drug includes a group after losing an atom or a group of atoms) or a derivative (such as a precursor thereof) after the linker is cleaved/degraded/enzymatically cleaved between tumor tissues or in tumor cells.
  • a biologically active drug such as a small molecule cytotoxic drug, wherein the drug includes a group after losing an atom or a group of atoms
  • a derivative such as a precursor thereof
  • drug-containing linker refers to a compound formed by linking a bioactive compound fragment (small molecule drug) to a linker, which is sometimes referred to herein as a "drug-linker conjugate”.
  • Metal ion chelators are compounds that, through their strong binding with metal ions, encapsulate metal ions into the interior of the chelator, turning them into stable compounds with larger molecular weight, thereby preventing the metal ions from taking effect.
  • Metal ion chelators include but are not limited to ethylenediaminetetraacetic acid,
  • composition refers to a composition that is in a form that permits the active ingredient contained therein to be effective for biological activity, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.
  • prevention includes inhibition of the occurrence or development of a disease or disorder or symptoms of a particular disease or disorder.
  • subjects with a family history of cancer are candidates for preventive regimens.
  • prevention refers to the administration of a drug before the signs or symptoms of cancer occur, particularly in a subject at risk for cancer.
  • alkyl refers to a straight chain, branched, fully saturated hydrocarbon group, preferably C1 - C10 , more preferably C1 - C8 , C1 - C6 , or C1 - C4 alkyl.
  • alkyl groups are methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl or n-decyl, and the like.
  • alkylene refers to a divalent group formed by removing a hydrogen atom from an alkyl group as defined above.
  • cycloalkyl refers to a cyclic alkyl group, preferably a C3 - C15 cycloalkyl, such as a C3 - C8 cycloalkyl, a C3 - C6 cycloalkyl.
  • Examples of cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexyl or cycloheptyl and the like.
  • cycloalkylene refers to a divalent group formed by removing a hydrogen atom from a cycloalkyl group as defined above.
  • heteroalkylene refers to an alkyl group as defined above, which contains one or more heteroatoms selected from N, O or S in the chain. It should be understood that heteroalkylene does not include a group formed by two or more N, O or S heteroatoms directly connected to each other.
  • heteroalkylene is a C1-21 (e.g., C1-10, C1-6) straight chain heteroalkylene, which contains 1-11 (preferably 1-6, 1-3) heteroatoms selected from N, O or S.
  • heterocyclyl refers to a partially unsaturated or fully unsaturated 3-10-membered cyclic group, preferably a 3-10-membered cyclic group, more preferably a 3-6-membered cyclic group, and more preferably a 5-6-membered cyclic group, which may be optionally substituted and contains one or more (e.g., 1, 2, 3 or 4 ) heteroatoms selected from N, O, S, SO, SO2 or P; the heterocyclyl group contains 1-9 carbon atoms, preferably 2-8 carbon atoms, and more preferably 3-5 carbon atoms.
  • heterocyclyl groups include: 1,3-dioxolanyl, 1,3-dioxanyl, 1,4-dioxanyl, imidazolinyl, indolinyl, morpholinyl, pyridone, 2-pyrrolidone, piperazinyl, homopiperazinyl, piperidinyl, pyrazinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydroquinolinyl, tetrahydrothienyl, oxanyl, oxathiolanyl, thianyl, and the like.
  • “Chemical bond” refers to a bond connecting two groups on both sides. For example, for the group -AL 2 -D, when L 2 is a chemical bond, the group can be written as -AD.
  • halogen refers to F, Cl, Br, I.
  • reducing agent refers to an agent capable of selectively reducing disulfide bonds.
  • the reducing agent is selected from but not limited to tris(2-carboxyethyl)phosphine (TCEP) (including tris(2-carboxyethyl)phosphine hydrochloride), dithiothreitol (DTT), mercaptoethylamine, mercaptoethanol, and combinations thereof.
  • TCEP tris(2-carboxyethyl)phosphine
  • DTT dithiothreitol
  • mercaptoethylamine mercaptoethanol
  • antibody molar equivalent refers to the molar equivalent relative to the antibody.
  • DAR drug to antibody ratio
  • D drug to antibody ratio
  • mAb antibody moiety
  • the DAR of an ADC can be in the range of 1 to 10, but higher loadings are possible depending on the number of attachment sites on the antibody.
  • DAR can be used when referring to the number of drugs loaded onto a single antibody.
  • the DAR can also be calculated as the average DAR of a population of molecules in a product, i.e., the overall ratio (molar ratio) of the small molecule drug moiety (D) coupled to the mAb moiety described herein to the Ab moiety in a product measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and/or HPLC), which DAR is referred to herein as the average DAR.
  • a detection method e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and/or HPLC
  • the average DAR value of the antibody drug conjugate of the invention is 1.0-10.0, such as 4.0-10.0, 5.0-9.0, 6.0-8.0, 1.0-8.0, 2.0-6.0, such as 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.
  • DAR2-ADC refers to an ADC with a DAR of 2, i.e., an ADC in which an antibody is linked to two small molecule drugs.
  • the term “comprising” or “including” means including the elements, integers or steps described, but does not exclude any other elements, integers or steps.
  • the term “comprising” or “including” when used, unless otherwise specified, it also covers the combination of the elements, integers or steps described.
  • an antibody variable region “comprising” a specific sequence when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.
  • the disclosure is further described below by describing specific embodiments, but this is not a limitation of the disclosure. Those skilled in the art can make various modifications or improvements based on the teachings of the disclosure without departing from the basic idea and scope of the disclosure.
  • the instruments or equipment used without indicating the source are all products that can be obtained commercially.
  • the reagents used without indicating the source are products that can be obtained commercially or synthesized by conventional methods.
  • MWC-1 was prepared using B-1 as an intermediate. It was a yellow solid.
  • LC-MS (ESI): [M+1]+ 404
  • the BL20E compound is prepared by the method disclosed in WO2018/095422A1 and the BL linker compound VC-PABC-MMAE is obtained by condensation, and the synthetic route is as follows:
  • the MWD-L1 compound is synthesized from the BL linker compound and GGFG-Dxd.
  • the synthesis route is as follows:
  • BL linker compound (857 mg, 1 mmol), GGFG-Dxd [synthesized according to the method described in the patent disclosure document (US20190151328A1)] (840 mg, 1 mmol, 1 eq), DIPEA (323 mg, 2.5 mmol, 2.5 eq), HATU (570 mg, 1.5 mmol, 1.5 eq), dissolved in 30 ml DCM, stirred for 2 h.
  • the reaction solution was cooled to 5-10 ° C, 1N hydrochloric acid (20 ml) was added, and stirred for 0.5 h.
  • the liquid was separated, the aqueous phase was extracted with DCM (30 ml * 2), and the organic phases were combined.
  • BL linker compound (857mg, 1mmol), HoSu (138mg, 1.2mmol, 1.2eq), DCC (310mg, 1.5mmol, 1.5eq) were dissolved in 30ml DCM, stirred at room temperature for 3h, and the reaction solution was filtered. The filtrate was the DCM solution of A-Osu. The filtrate was added to the mixed solution of the crude intermediate 2-2, DIPEA (323mg, 2.5mmol, 2.5eq) and DCM (30ml), and the reaction was stirred for 3h. The reaction solution was cooled to 10°C, 1N hydrochloric acid (20ml) was added, and stirred for 0.5h.
  • Boc-Gly-OH (175 mg, 1 mmol), A1 compound (176 mg, 1 mmol, 1 eq), DIPEA (322 mg, 2.5 mmol, 2.5 eq), HATU (456 mg, 1.2 mmol, 1.2 eq) were dissolved in 30 ml DCM and stirred for 2 h.
  • BL linker compound (318 mg, 0.37 mmol), HoSu (51 mg, 0.44 mmol, 1.2 eq), DCC (114 mg, 0.56 mmol, 1.5 eq) were dissolved in 30 ml DCM, stirred at room temperature for 3 h, and the reaction solution was filtered. The filtrate was added to a mixed solution of the crude intermediate 3-4, DIPEA (120 mg, 0.93 mmol, 2.5 eq), and DCM (30 ml), and the reaction was stirred for 3 h. The reaction solution was cooled to 10 ° C, 1N hydrochloric acid (20 ml) was added, and stirred for 0.5 h.
  • MWF-L6 was prepared by replacing compound A1 with compound A7.
  • the product was an orange-red solid.
  • LC-MS (ESI): M+1 1450.
  • compound A7-2 (2.45 g, 7.7 mmol, 1 eq) was dissolved in tetrahydrofuran (200 ml), and the reaction solution was cooled to -70 to -60 ° C with a dry ice-acetone bath, and KHMDS (1 M, 31 ml, 31.0 mmol, 4 eq) was slowly added dropwise. After the addition, the mixture was stirred at -70 to -60 ° C for 10 minutes; a tetrahydrofuran solution of NFSI (7.35 g, 23 mmol, 3 eq) was added dropwise.
  • DT-2-A compound was used to replace the BL linker compound to synthesize DT-2, which was a light yellow solid.
  • LC-MS (ESI): [M+1]+ 1339.7, wherein the DT-2-A compound was synthesized according to the literature Chem. Sci., 2022, 13, 8781–8790.
  • MC-VC-PAB-MMAE used in the examples was purchased from MCE.
  • the trastuzumab antibody solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution at pH 6.5-7.4 and diluted to 5-15mg/mL.
  • Different metal ion aqueous solutions and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solutions were added in sequence and reacted at 0-15°C overnight.
  • the reaction equivalents are shown in Table 1-1.
  • the concentration of the above reaction solution was adjusted to about 5-15 mg/mL, 8 times the antibody equivalent of dehydroascorbic acid (DHAA) was added, and the solution was changed to 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4 after reacting at 25-35°C for 2 hours.
  • the antibody drug conjugate product was obtained. Samples were taken for analysis, and the antibody coupling ratio (DAR) was determined. The test results are shown in Table 1-1.
  • the trastuzumab antibody was exchanged into a 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 6.5-7.4 and diluted to 5-15 mg/mL.
  • a ZnCl 2 aqueous solution of 2 times the antibody molar equivalent and a tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solution of 4.0-4.5 times the antibody molar equivalent were added in sequence, and after overnight reaction at 0-15°C, the solution was exchanged into a 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution of different pH values.
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • a DMA solution of BL20E (C-3) of 3 times the antibody molar equivalent was added to the above reaction solution.
  • the coupling reaction temperature and the pH of the coupling solution are shown in Table 1-2.
  • the solution was exchanged into a 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution of pH 6.5-7.4.
  • the concentration of the above reaction solution was adjusted to about 5-15 mg/mL, 4 times the antibody equivalent of ethylenediaminetetraacetic acid (EDTA) was added, and the solution was changed to 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with a pH of 7.4-8.0 after reacting at room temperature for 30 minutes.
  • EDTA ethylenediaminetetraacetic acid
  • the concentration of the above reaction solution was adjusted to about 5-15 mg/mL, 8 times the antibody molar equivalent of dehydroascorbic acid (DHAA) was added, and the solution was changed to 50 mM sodium dihydrogen phosphate-disodium dihydrogen phosphate buffer with a pH of 7.4 after reacting at 25-35°C for 2 hours.
  • the antibody drug conjugate product was obtained. Samples were taken for analysis, and the antibody coupling ratio (DAR) and purity were determined. The test results are shown in Table 1-2.
  • the HIC peak value of DAR2-ADC can be achieved to be 61-78% by using the above coupling reaction temperature and coupling solution pH in the preparation method of the present application.
  • the trastuzumab antibody solution was changed to 20 mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 6.5-7.4 and diluted to 5-15 mg/mL.
  • a 2-fold antibody molar equivalent of ZnCl 2 aqueous solution and a 4.0-4.5-fold antibody molar equivalent of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solution were added in sequence and reacted at 0-15°C overnight.
  • the concentration of the above reaction solution was adjusted to about 5-15 mg/mL, and 8 times the antibody equivalent of dehydroascorbic acid (DHAA) was added, and the reaction was carried out at 25-35°C for 2 hours. After the reaction was completed, the solution was changed to a 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with a pH of 7.5, diluted to about 5-20 mg/mL, and ammonium sulfate solution was added to adjust the conductivity.
  • the antibody drug conjugate product was purified by hydrophobic interaction chromatography (HIC). The purification conditions are shown in Table 1-3. The purified product was sampled for HIC analysis. The HIC test results are shown in Table 1-3.
  • the antibody solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 6.5-7.4 and diluted to 5-15mg/mL.
  • a ZnCl 2 aqueous solution with a molar equivalent of 2 times the antibody and a tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solution with a molar equivalent of 4.0-4.5 times the antibody were added in sequence, and reacted at 0-15°C overnight.
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • a DMA solution containing a drug linker with a molar equivalent of 2.5-5.5 times the antibody was added to the above reaction solution, and the solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 6.5-7.4 after reacting at 0-15°C for 2h.
  • the concentration of the above reaction solution was adjusted to about 5-15mg/mL, and ethylenediaminetetraacetic acid (EDTA) with a molar equivalent of 4 times the antibody was added.
  • EDTA ethylenediaminetetraacetic acid
  • antibody h23-12 was prepared according to the method described in patent WO2021068949A1
  • antibody hH2L1 was prepared according to the method described in patent CN113527486A.
  • reaction mixture was purified using a desalting column (Model: 40K, 0.5 mL, REF: 87766, Lot: SJ251704, Thermo).
  • Sample injection analysis For samples with a concentration exceeding 5 mg/mL, dilute the sample with 50% phase A solution, filter through a 0.2 ⁇ m filter, and then perform sample injection analysis; for samples with a concentration of 5 mg/mL or less, filter directly before sample injection analysis.
  • the method was determined according to the monoclonal antibody molecular size variant determination method in General Chapter 3127 of the Chinese Pharmacopoeia Volume IV.
  • This experiment used human gastric cancer cells NCI-N87 (purchased from ATCC). After adjusting the density of NCI-N87 cells to 15 ⁇ 10 4 with complete medium (RPMI 1640 medium 45ml, 5ml FBS, mixed before use), 100 ⁇ l/well was added to the cell culture plate and cultured overnight. On the second day, the ADC samples were diluted with the above complete medium to 50ug/ml, and then diluted 4 times in a gradient, with a total of 9 gradients plus zero point, and all samples were set up with 3 replicates. Set up negative control (cells + culture medium) and blank control (no cells, pure culture medium); add the above diluted ADC samples to the cell culture plate cultured overnight in sequence, 100 ⁇ l/well.
  • complete medium RPMI 1640 medium 45ml, 5ml FBS, mixed before use

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Abstract

The present invention relates to a method for preparing an antibody-drug conjugate. According to the method, a highly homogeneous antibody conjugate molecule is obtained by means of a specific chemical site-directed conjugation method, and the proportion of a DAR2 main peak thereof reaches 99% or above. The preparation method of the present invention has few steps, and is easy to operate, and is thus beneficial for industrial scaled-up production. In addition, the prepared product has few impurities and a high purity, the medication safety is obviously improved, and the production cost is also reduced.

Description

一种制备抗体药物偶联物的方法A method for preparing antibody drug conjugate 技术领域Technical Field

本发明属于生物医药领域，更具体地，本发明涉及一种制备抗体药物偶联物的新方法。The present invention belongs to the field of biomedicine, and more specifically, the present invention relates to a new method for preparing antibody-drug conjugates.

背景概述Background Overview

抗体-药物偶联物(ADC)是一种将小分子药物连接到单克隆抗体上的一种新药物形式，利用单克隆抗体的靶向功能，将小分子药物运输到靶部位发挥药效，减少毒副作用，提高了治疗窗口。近年来，抗体偶联药物是肿瘤精准治疗的热门方向之一，为肿瘤治疗带来希望。抗体-偶联药物(Antibody-Drug Conjugates,ADC)由靶向特异性抗原的抗体药物和小分子细胞毒药物通过连接子偶联而成,兼具传统小分子化疗的强大杀伤效应及抗体药物的肿瘤靶向性。截止到目前，全球已有十多款ADC药物获批上市。Antibody-drug conjugates (ADCs) are a new form of drug that connects small molecule drugs to monoclonal antibodies. They use the targeting function of monoclonal antibodies to transport small molecule drugs to target sites to exert their efficacy, reduce toxic side effects, and increase the therapeutic window. In recent years, antibody-drug conjugates have been one of the hot directions in precision tumor treatment, bringing hope to tumor treatment. Antibody-drug conjugates (ADCs) are composed of antibody drugs targeting specific antigens and small molecule cytotoxic drugs coupled through linkers. They have both the powerful killing effect of traditional small molecule chemotherapy and the tumor targeting of antibody drugs. So far, more than a dozen ADC drugs have been approved for marketing worldwide.

抗体偶联药物由抗体、连接子、有效载荷(payload)等几部分组成。其中抗体和药物-连接体可以以不同的偶联方式连接。偶联方法主要分为非定点偶联和定点偶联。早期使用的是非定点偶联法，主要由赖氨酸偶联和半胱氨酸偶联，利用化学方法直接将药物与抗体上氨基酸残基进行偶联，不涉及抗体的改造或修饰，其偶联的毒素分子个数和偶联位点都不能确定，均一性欠佳。目前常用的定点偶联方式即通过基因工程位点或者特殊的连接头进行特异性偶联，实现更均一地偶联，能在特定位点实现细胞毒素的连接。定点偶联生产的抗体偶联药物可以减少因偶联位点和偶联个数不同而造成药效、药代以及质控的波动。目前常见的定点偶联方式有THIOMAB技术、非天然氨基酸偶联技术、谷氨酰胺酶促偶联技术、Sortase转肽酶偶联技术以及Thiobridge技术等。其中，利用抗体工程化或者酶促偶联对抗体的改造修饰可能会对抗体的结构稳定性有一定影响，同时对CMC有一定要求。另外，利用化学偶联的Thiobridge技术也有一定的缺陷，例如DBM(dibromomaleimides)类连接子和其他含巯基的生物基团发生置换作用，而在血浆中不稳定，造成药效降低，毒副作用增加(Chem.-Eur.J,2019,25,43-59.)。Antibody-drug conjugates consist of several parts, such as antibodies, linkers, and payloads. Among them, antibodies and drug-linkers can be connected in different coupling modes. The coupling methods are mainly divided into non-fixed site coupling and fixed site coupling. In the early days, non-fixed site coupling methods were used, mainly lysine coupling and cysteine coupling, which directly coupled drugs to amino acid residues on antibodies using chemical methods, without involving antibody transformation or modification. The number of toxin molecules and coupling sites coupled to them cannot be determined, and the uniformity is poor. The currently commonly used fixed-site coupling method is to perform specific coupling through genetic engineering sites or special connectors to achieve more uniform coupling, and can achieve the connection of cytotoxins at specific sites. Antibody-drug conjugates produced by fixed-site coupling can reduce the fluctuations in efficacy, pharmacokinetics, and quality control caused by different coupling sites and coupling numbers. At present, common fixed-site coupling methods include THIOMAB technology, non-natural amino acid coupling technology, glutamine enzymatic coupling technology, Sortase transpeptidase coupling technology, and Thiobridge technology. Among them, the modification of antibodies by antibody engineering or enzymatic coupling may have a certain impact on the structural stability of the antibody, and at the same time have certain requirements for CMC. In addition, the Thiobridge technology using chemical coupling also has certain defects, such as the replacement of DBM (dibromomaleimides)-type linkers and other thiol-containing biological groups, which are unstable in plasma, resulting in reduced efficacy and increased toxic side effects (Chem.-Eur.J, 2019, 25, 43-59.).

药物抗体比DAR(drug-to-antibody ratio)是ADC药物独特的重要质量属性，它代表抗体偶联小分子药物的平均数量。药物偶联的程度会影响ADC药物的稳定性和聚集倾向。因此，DAR值是进行ADC药物分析的关键质量属性，是ADC药物研发过程重要的质控环节。定点偶联技术可以实现抗体与小分子毒素定点、定量偶联，通过该技术获得的ADC具有合适的药抗比DAR，均一性高，稳定性好，批次间重现性高，具有更好的活性和药动学特性，同时也更适用于ADC的大规模生产。目前的ADC药物的研发趋势是采用定点偶联方法将高活性小分子药物偶联到抗体上，形成定点DAR2的ADC，如批准上市的靶向CD19-ADC(loncastuximab tesirine，Zynlonta)通过糖基化定点偶联技术将PBD小分子偶联到抗体上，实现定点DAR2，以及靶向HER2ADC(ARX788)以及靶向FRαADC(MORAb-202)， 均采用非天然氨基酸突变的方法实现定点DAR2。但这些方法要么需要使用酶，要么需对抗体序列进行改造，制造工艺复杂。The drug-to-antibody ratio (DAR) is a unique and important quality attribute of ADC drugs, which represents the average number of small molecule drugs coupled to antibodies. The degree of drug coupling will affect the stability and aggregation tendency of ADC drugs. Therefore, the DAR value is a key quality attribute for ADC drug analysis and an important quality control link in the ADC drug development process. Fixed-site coupling technology can achieve fixed-site and quantitative coupling of antibodies and small molecule toxins. The ADC obtained by this technology has a suitable drug-to-antibody ratio (DAR), high uniformity, good stability, high batch-to-batch reproducibility, better activity and pharmacokinetic properties, and is also more suitable for large-scale production of ADC. The current development trend of ADC drugs is to use site-specific coupling methods to couple highly active small molecule drugs to antibodies to form site-specific DAR2 ADCs, such as the approved targeted CD19-ADC (loncastuximab tesirine, Zynlonta) that couples PBD small molecules to antibodies through glycosylation site-specific coupling technology to achieve site-specific DAR2, as well as targeted HER2 ADC (ARX788) and targeted FRα ADC (MORAb-202). All of them use the method of non-natural amino acid mutation to achieve site-specific DAR2. However, these methods either require the use of enzymes or the modification of antibody sequences, and the manufacturing process is complicated.

WO2022253033A1公开了如下ADC偶联物制备方法：用依地酸二钠的溶液稀释抗体，然后用Na2HPO4溶液调pH，加入TCEP(三(2-羧乙基)膦)溶液混匀，室温放置2h。向上述溶液体系加入抗体的5-10倍物质的量的溶解在DMSO的药物-连接体，混匀，室温静置20h，完毕后采用NAP-5凝胶柱(Cytiva)将缓冲液置换为pH 6.0的10mM组氨酸缓冲溶液，得到ADC产物。经样品测定，该专利采用以上非定点偶联的方式制备的ADC偶联物的DAR为4，未见DAR为2的结果记载。WO2022253033A1 discloses the following method for preparing an ADC conjugate: dilute the antibody with a solution of disodium edetate, then adjust the pH with a Na 2 HPO 4 solution, add a TCEP (tri(2-carboxyethyl)phosphine) solution and mix well, and leave at room temperature for 2 hours. Add 5-10 times the amount of the antibody to the above solution system, mix well, and let stand at room temperature for 20 hours. After completion, use a NAP-5 gel column (Cytiva) to replace the buffer with a 10mM histidine buffer solution at pH 6.0 to obtain an ADC product. According to sample measurement, the DAR of the ADC conjugate prepared by the above non-site-specific coupling method in this patent is 4, and there is no record of a DAR of 2.

WO2022078524A2公开了抗体偶联物的定点偶联方法。其采用多种锌配合物和TCEP对抗体进行还原孵育后，引入有效载荷/接头复合物。然后加入半胱氨酸以消耗多余的TCEP和多余的有效载荷。氧化蛋白质中的游离硫醇基团。最后使用脱盐柱或UF/DF或离子交换色谱纯化反应混合物。采用HIC-HPLC对于偶联物DAR分析的结果显示，DAR为4偶联物占比为60-70％左右，而DAR为2偶联物的占比仅为5-10％。可见，采用该专利方法所制备的抗体偶联物DAR主要集中在4，而DAR为2的偶联物产物仅占少数。WO2022078524A2 discloses a site-specific coupling method for antibody conjugates. After reducing and incubating the antibody with a variety of zinc complexes and TCEP, a payload/linker complex is introduced. Cysteine is then added to consume excess TCEP and excess payload. Free thiol groups in the protein are oxidized. Finally, the reaction mixture is purified using a desalting column or UF/DF or ion exchange chromatography. The results of the DAR analysis of the conjugates using HIC-HPLC showed that the proportion of conjugates with a DAR of 4 was about 60-70%, while the proportion of conjugates with a DAR of 2 was only 5-10%. It can be seen that the DAR of the antibody conjugates prepared by this patented method is mainly concentrated in 4, while the conjugate products with a DAR of 2 account for only a minority.

WO2020164561A1制备了具有改善的同质性的抗体-药物缀合物(ADC)，所用的偶联方法包括以下步骤：WO2020164561A1 prepares an antibody-drug conjugate (ADC) with improved homogeneity, and the coupling method used comprises the following steps:

(a)将还原剂(例如，三(2-羧乙基)膦(TCEP))和待缀合的抗体在有效量的过渡金属离子(例如，Zn2+等)的存在下在缓冲体系(例如，Hepes、组氨酸缓冲液、PBS、MES等)中温育，以选择性地还原抗体内链间二硫键；(b)将过量的携带有反应性基团的有效载荷(例如，马来酰亚胺连接的药物)与步骤(a)得到的还原的巯基基团反应；以及(c)加入有效量的氧化剂(例如，脱氢抗坏血酸(DHAA))以将未反应的巯基再氧化，然后回收获得的抗体-药物缀合物。虽然该偶联方法产生的抗体-药物缀合物(ADC)的同质性可以得到显著改善，但是其ADC产品的DAR2重量占比仅为10-28％，该比例远小于DAR4的重量比数值。(a) Incubate a reducing agent (e.g., tris(2-carboxyethyl)phosphine (TCEP)) and an antibody to be conjugated in a buffer system (e.g., Hepes, histidine buffer, PBS, MES, etc.) in the presence of an effective amount of transition metal ions (e.g., Zn2+, etc.) to selectively reduce intrachain disulfide bonds in the antibody; (b) react an excess of a payload carrying a reactive group (e.g., a maleimide-linked drug) with the reduced thiol group obtained in step (a); and (c) add an effective amount of an oxidant (e.g., dehydroascorbic acid (DHAA)) to reoxidize the unreacted thiol group, and then recover the obtained antibody-drug conjugate. Although the homogeneity of the antibody-drug conjugate (ADC) produced by this coupling method can be significantly improved, the weight ratio of DAR2 of its ADC product is only 10-28%, which is much smaller than the weight ratio of DAR4.

对于ADC来讲，DAR值是ADC的关键质量属性，高DAR虽然能将更多的小分子药物带到靶部位，可能起到更好的治疗效果，但是高DAR的设计会牺牲ADC药物的稳定性和均一性，可能引起抗体的集聚，从而影响ADC药物的药效，带来较大的毒副作用。制备高均一性和/或纯度的DAR2的ADC可避免高DAR带来的不利影响，并可有利于ADC药物的稳定和疗效。然而，如上所述，现有技术可通过糖基化定点偶联技术实现定点DAR2。但这些方法要么需要使用酶，要么需对抗体序列进行改造，制造工艺复杂。目前报道的用于制备ADC的化学定点偶联法，虽然工艺相对简单，但只能实现主要DAR为4的ADC产物。For ADC, the DAR value is the key quality attribute of ADC. Although high DAR can bring more small molecule drugs to the target site and may have a better therapeutic effect, the design of high DAR will sacrifice the stability and uniformity of ADC drugs, which may cause the aggregation of antibodies, thereby affecting the efficacy of ADC drugs and causing greater toxic side effects. The preparation of ADCs with high uniformity and/or purity of DAR2 can avoid the adverse effects of high DAR and can be beneficial to the stability and efficacy of ADC drugs. However, as mentioned above, the prior art can achieve fixed-point DAR2 through glycosylation fixed-point coupling technology. However, these methods either require the use of enzymes or the modification of antibody sequences, and the manufacturing process is complicated. The chemical fixed-point coupling method currently reported for the preparation of ADC, although the process is relatively simple, can only achieve ADC products with a main DAR of 4.

因此，本领域迫切需要一种更简便有效的定点DAR2-ADC制备方法。Therefore, there is an urgent need in the art for a simpler and more effective method for preparing site-directed DAR2-ADC.

发明内容Summary of the invention

本发明提供一种抗体－药物偶联物的新制备方法，其通过特定的化学定点偶联方法，获得了高度均质的抗体偶联物分子，其DAR2主峰占比达到99％以上。本发明的制备方法步骤 少，操作简单，利于工业放大生产，同时制备的产品杂质少、纯度高，明显提高用药安全性，也降低了生产成本。通过本发明的偶联方法制备的抗肿瘤药具有优异的抗肿瘤效果和安全性(包括较高的稳定性和较小的副作用)。The present invention provides a novel method for preparing an antibody-drug conjugate, which obtains highly homogeneous antibody conjugate molecules through a specific chemical site-specific coupling method, and the DAR2 main peak accounts for more than 99%. The method is simple to operate and is conducive to industrial amplification. At the same time, the prepared product has few impurities and high purity, which significantly improves the safety of medication and reduces production costs. The anti-tumor drug prepared by the coupling method of the present invention has excellent anti-tumor effect and safety (including higher stability and less side effects).

第一方面First aspect

本发明提供用于制备抗体-药物偶联物(ADC)的方法，该方法包括以下步骤：The present invention provides a method for preparing an antibody-drug conjugate (ADC), the method comprising the following steps:

(a)在含抗体的缓冲溶液中，加入金属盐或金属配合物、还原剂，然后进行孵育；(a) adding a metal salt or a metal complex and a reducing agent to a buffer solution containing an antibody, followed by incubation;

(b)向(a)中的反应溶液中加入含药连接子进行偶联；和(b) adding a drug-containing linker to the reaction solution in (a) for coupling; and

(c)使用疏水作用色谱方法，采用疏水凝胶型填料和盐溶液对于偶联物产物进行纯化，以获得高纯度的DAR2-ADC偶联物。(c) Using hydrophobic interaction chromatography, a hydrophobic gel-type filler and a salt solution to purify the conjugate product to obtain a high-purity DAR2-ADC conjugate.

在一些实施方案中，步骤(a)中所述的金属盐或金属配合物中的金属可以选自Zn、Cd和Hg等。In some embodiments, the metal in the metal salt or metal complex described in step (a) can be selected from Zn, Cd, Hg, and the like.

在一些实施方案中，步骤(a)中所述的金属盐或金属配合物选自下述中的一种或多种：Zn、Cd和Hg的盐酸盐或硫酸盐、以及WO2022078524A2公开的Zn盐或Zn配合物包括但不限于
In some embodiments, the metal salt or metal complex in step (a) is selected from one or more of the following: hydrochloride or sulfate of Zn, Cd and Hg, The Zn salts or Zn complexes disclosed in WO2022078524A2 include but are not limited to

Zn(NH2CH3)2 2+，Zn(NH2CH2CH3)2 2+，Zn(NH2CH2CH2CH3)2 2+，Zn(NH2CH(CH3)2)2 2+，Zn(NH 2 CH 3 ) 2 2+ , Zn(NH 2 CH 2 CH 3 ) 2 2+ , Zn(NH 2 CH 2 CH 2 CH 3 ) 2 2+ , Zn(NH 2 CH(CH 3 ) 2 ) 2 2+ ,

Zn(NH2C(CH3)3)2 2+，Zn(NH2CH2C(CH3)3)2 2+，Zn(NH(CH3)2)2 2+，Zn(NH(CH2CH3)2)2 2+，Zn(NH 2 C(CH 3 ) 3 ) 2 2+ , Zn(NH 2 CH 2 C(CH 3 ) 3 ) 2 2+ , Zn(NH(CH 3 ) 2 ) 2 2+ , Zn(NH(CH 2 CH 3 ) 2 ) 2 2+ ,

Zn(NH(CH(CH3)2)2)2 2+，Zn(NH(C(CH3)3)2)2 2+，Zn(NH(CH(CH2CH3)2)2)2 2+，Zn(NH(CH(CH 3 ) 2 ) 2 ) 2 2+ , Zn(NH(C(CH 3 ) 3 ) 2 ) 2 2+ , Zn(NH(CH(CH 2 CH 3 ) 2 ) 2 ) 2 2+ ,

Zn(NH(CH2C(CH3)3)2)2 2+，Zn(NH(CH2C(CH2CH3)3)2)2 2+，Zn(NH(CH2CH2C(CH3)3)2)2 2+，Zn(NH(CH 2 C(CH 3 ) 3 ) 2 ) 2 2+ , Zn(NH(CH 2 C(CH 2 CH 3 ) 3 ) 2 ) 2 2+ , Zn(NH(CH 2 CH 2 C( CH 3 ) 3 ) 2 ) 2 2+ ,

Zn(NH2CH2CH2OH)2 2+，Zn(NH(CH2CH2OH)2)2 2+，Zn(N(CH2CH2OH)3)2 2+，Zn(NH 2 CH 2 CH 2 OH) 2 2+ , Zn(NH(CH 2 CH 2 OH) 2 ) 2 2+ , Zn(N(CH 2 CH 2 OH) 3 ) 2 2+ ,

Zn(NH2CH2COOH)2 2+，Zn(NH2CH2CONH2)2 2+，Zn(NH2CH2COOCH3)2 2+，Zn(NH 2 CH 2 COOH) 2 2+ , Zn(NH 2 CH 2 CONH 2 ) 2 2+ , Zn(NH 2 CH 2 COOCH 3 ) 2 2+ ,

Zn(NH2CH2COOCH2CH3)2 2+，Zn(NH2CH2COOC(CH3)3)2 2+，Zn(NH2CH2COOCH(CH3)2)2 2+，Zn(NH 2 CH 2 COOCH 2 CH 3 ) 2 2+ , Zn(NH 2 CH 2 COOC(CH 3 ) 3 ) 2 2+ , Zn(NH 2 CH 2 COOCH(CH 3 ) 2 ) 2 2+ ,

Zn(NH2CH2CH2COOH)2 2+，Zn(NH(CH2COOH)2)2 2+，Zn(N(CH2CH2COOH)3)2 2+，Zn(NH2CH3)4 2+，Zn(NH 2 CH 2 CH 2 COOH) 2 2+ , Zn(NH(CH 2 COOH) 2 ) 2 2+ , Zn(N(CH 2 CH 2 COOH) 3 ) 2 2+ , Zn(NH 2 CH 3 ) 4 2+ ,

Zn(NH2CH2CH3)4 2+，Zn(NH2CH2CH2CH3)4 2+，Zn(NH2CH(CH3)2)4 2+，Zn(NH2C(CH3)3)4 2+，Zn(NH 2 CH 2 CH 3 ) 4 2+ , Zn(NH 2 CH 2 CH 2 CH 3 ) 4 2+ , Zn(NH 2 CH(CH 3 ) 2 ) 4 2+ , Zn(NH 2 C(CH 3 ) 3 ) 4 2+ ,

Zn(NH2CH2C(CH3)3)4 2+，Zn(NH(CH3)2)4 2+，Zn(NH(CH2CH3)2)4 2+，Zn(NH(CH(CH3)2)2)4 2+，Zn(NH 2 CH 2 C(CH 3 ) 3 ) 4 2+ , Zn(NH(CH 3 ) 2 ) 4 2+ , Zn(NH(CH 2 CH 3 ) 2 ) 4 2+ , Zn(NH(CH (CH 3 ) 2 ) 2 ) 4 2+ ,

Zn(NH(C(CH3)3)2)4 2+，Zn(NH(CH(CH2CH3)2)2)4 2+，Zn(NH(CH2C(CH3)3)2)4 2+，Zn(NH(C(CH 3 ) 3 ) 2 ) 4 2+ , Zn(NH(CH(CH 2 CH 3 ) 2 ) 2 ) 4 2+ , Zn(NH(CH 2 C(CH 3 ) 3 ) 2 ) 4 2+ ,

Zn(NH(CH2C(CH2CH3)3)2)4 2+，Zn(NH(CH2CH2C(CH3)3)2)4 2+，Zn(NH2CH2CH2OH)4 2+，。 Zn(NH(CH 2 C(CH 2 CH 3 ) 3 ) 2 ) 4 2+ , Zn(NH(CH 2 CH 2 C(CH 3 ) 3 ) 2 ) 4 2+ , Zn(NH 2 CH 2 CH 2 OH) 4 2+ ,.

在一些实施方案中，步骤(a)中所述的金属盐或金属配合物选自ZnCl2、CdCl2、HgCl2、中的一种或多种。In some embodiments, the metal salt or metal complex in step (a) is selected from ZnCl 2 , CdCl 2 , HgCl 2 , One or more of .

在一些实施方案中，步骤(a)中所述的还原剂选自三(2-羧乙基)膦(TCEP)、二硫苏糖醇(DTT)、巯基乙胺和巯基乙醇，优选TCEP。In some embodiments, the reducing agent in step (a) is selected from tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), mercaptoethylamine and mercaptoethanol, preferably TCEP.

在一些实施方案中，步骤(a)中的抗体的浓度为0.5-150mg/mL，例如1-75mg/mL、1-50mg/mL、2-40mg/mL、3-30mg/mL、4-30mg/mL或5-15mg/mL，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、20、30、40、50、60、70、80、90、100、120或150mg/mL。In some embodiments, the concentration of the antibody in step (a) is 0.5-150 mg/mL, such as 1-75 mg/mL, 1-50 mg/mL, 2-40 mg/mL, 3-30 mg/mL, 4-30 mg/mL or 5-15 mg/mL, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120 or 150 mg/mL.

在一些实施方案中，步骤(a)中所述的还原剂的用量是1-6倍抗体摩尔当量，例如2-6、3-5、3.5-5.0或4.0-4.5倍抗体摩尔当量，例如1、1.5、2、2.5、3、3.5、4、4.5、5、5.5倍抗体摩尔当量。In some embodiments, the amount of the reducing agent used in step (a) is 1-6 times the antibody molar equivalent, such as 2-6, 3-5, 3.5-5.0 or 4.0-4.5 times the antibody molar equivalent, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 times the antibody molar equivalent.

在一些实施方案中，步骤(a)中所述的金属盐或金属配合物的用量为1-5倍抗体摩尔当量，例如1-4、1-3或1.5-2.5倍抗体摩尔当量，例如1、1.5、2、2.5、3、3.5、4、4.5或5倍抗体摩尔当量。In some embodiments, the amount of the metal salt or metal complex used in step (a) is 1-5 times the antibody molar equivalent, such as 1-4, 1-3 or 1.5-2.5 times the antibody molar equivalent, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 times the antibody molar equivalent.

在一些实施方案中，步骤(a)中的所述缓冲溶液具有5-8的pH，例如5.5-7.5、6.5-7.5或6.5-7.4的pH，例如5.0、5.5、6.0、6.5、6.9、7.0、7.5或8.0的pH，优选pH为6.9。In some embodiments, the buffer solution in step (a) has a pH of 5-8, such as a pH of 5.5-7.5, 6.5-7.5 or 6.5-7.4, such as a pH of 5.0, 5.5, 6.0, 6.5, 6.9, 7.0, 7.5 or 8.0, preferably a pH of 6.9.

在一些实施方案中，步骤(a)中的所述缓冲溶液选自磷酸盐缓冲液、醋酸盐缓冲液、柠檬酸盐缓冲液、琥珀酸盐缓冲液，优选磷酸氢二钠-磷酸二氢钠缓冲液。优选地，所述缓冲溶液中的缓冲剂浓度为5-100mM，例如10-90mM、20-80mM、30-70mM、40-60mM、10-30或mM，例如10、20、30、40、50、60、70、80、90或100mM。In some embodiments, the buffer solution in step (a) is selected from phosphate buffer, acetate buffer, citrate buffer, succinate buffer, preferably disodium hydrogen phosphate-sodium dihydrogen phosphate buffer. Preferably, the buffer concentration in the buffer solution is 5-100mM, such as 10-90mM, 20-80mM, 30-70mM, 40-60mM, 10-30 or mM, such as 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100mM.

在一些实施方案中，步骤(a)中所述的孵育在0～30℃，例如0～15℃或5-15℃进行。In some embodiments, the incubation in step (a) is performed at 0-30°C, such as 0-15°C or 5-15°C.

应当理解，本领域技术人员可以容易地确定本发明各反应的终点，从而确定反应进行的时间。It should be understood that those skilled in the art can easily determine the endpoint of each reaction of the present invention, thereby determining the time for the reaction to proceed.

在一些实施方案中，步骤(a)中所述的孵育进行1-48小时，例如2-24小时、2-12小时、3-10小时、4-8小时或5-24小时，例如1、2、3、4、5、6、7、8、9、10、15、20、25、30、或35小时。In some embodiments, the incubation in step (a) is performed for 1-48 hours, such as 2-24 hours, 2-12 hours, 3-10 hours, 4-8 hours or 5-24 hours, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 35 hours.

在一些实施方案中，步骤(b)中的含药连接子的用量是1-8倍抗体摩尔当量，例如2-6、2.5-5.5、3-5或3.5-4.5倍抗体摩尔当量，例如1、1.5、2、2.5、3、3.5、4、4.5、5、5.5、6、6.5、7或7.5倍抗体摩尔当量。In some embodiments, the amount of the drug-containing linker in step (b) is 1-8 times the antibody molar equivalent, such as 2-6, 2.5-5.5, 3-5 or 3.5-4.5 times the antibody molar equivalent, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7 or 7.5 times the antibody molar equivalent.

在一些实施方案中，步骤(b)中的偶联反应温度为0～30℃，例如0～15℃或3-15℃。In some embodiments, the coupling reaction temperature in step (b) is 0-30°C, such as 0-15°C or 3-15°C.

在一些实施方案中，步骤(b)中反应溶液具有5-8的pH，例如5.5-7.5、6.5-7.5或6.5-7.4的pH，例如5.0、5.5、6.0、6.5、7.0、7.5或8.0的pH。In some embodiments, the reaction solution in step (b) has a pH of 5-8, such as a pH of 5.5-7.5, 6.5-7.5, or 6.5-7.4, such as a pH of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0.

在一些实施方案中，含药连接子以DMA溶液形式提供。In some embodiments, the drug-containing linker is provided as a DMA solution.

在一些实施方案中，步骤(b)的偶联反应进行0.2-24小时，例如0.5-12小时、1-10小时、1.5-5小时、1-3小时或1-4小时，例如1、1.5、2、2.5、3、3.5、4、5、6、7、8、9或10 小时。In some embodiments, the coupling reaction of step (b) is carried out for 0.2-24 hours, such as 0.5-12 hours, 1-10 hours, 1.5-5 hours, 1-3 hours or 1-4 hours, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 hours. Hour.

在一些实施方案中，步骤(c)中所述疏水凝胶填料选自Butyl-650M、Butyl-4FF、Butyl-650S、Butyl-ImpRes和Phenyl Sepharose HP。In some embodiments, the hydrophobic gel filler in step (c) is selected from Butyl-650M, Butyl-4FF, Butyl-650S, Butyl-ImpRes and Phenyl Sepharose HP.

在一些实施方案中，步骤(c)中的盐种类选自硫酸盐、氯化盐等，例如硫酸或盐酸的碱金属(例如锂、钠、钾)盐、碱土金属(例如钙、镁)盐，胺或氨与硫酸或盐酸形成的盐，优选硫酸铵、氯化钠；优选地，盐浓度为0.1-5mol/L，例如0.2-4mol/L、0.3-3mol/L、0.5-2mol/L、0.5-1.5mol/L、0.6-1.2mol/L或0.6-1.0mol/L，例如0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0、1.2、1.5或2.0mol/L。In some embodiments, the type of salt in step (c) is selected from sulfates, chlorides, etc., such as alkali metal (such as lithium, sodium, potassium) salts of sulfuric acid or hydrochloric acid, alkaline earth metal (such as calcium, magnesium) salts, salts formed by amines or ammonia and sulfuric acid or hydrochloric acid, preferably ammonium sulfate and sodium chloride; preferably, the salt concentration is 0.1-5 mol/L, such as 0.2-4 mol/L, 0.3-3 mol/L, 0.5-2 mol/L, 0.5-1.5 mol/L, 0.6-1.2 mol/L or 0.6-1.0 mol/L, such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5 or 2.0 mol/L.

在一些实施方案中，步骤(c)所述的盐溶液用作疏水作用色谱的流动相A。In some embodiments, the salt solution described in step (c) is used as mobile phase A for hydrophobic interaction chromatography.

在一些实施方案中，步骤(c)中的疏水作用色谱使用磷酸盐缓冲液作为流动相B，优选地，所述磷酸盐缓冲液具有5-200mM的磷酸盐浓度，具有7.0-7.6例如7.1、7.2、7.3、7.4或7.5的pH。In some embodiments, the hydrophobic interaction chromatography in step (c) uses phosphate buffer as mobile phase B, preferably, the phosphate buffer has a phosphate concentration of 5-200 mM and a pH of 7.0-7.6, such as 7.1, 7.2, 7.3, 7.4 or 7.5.

在一些实施方案中，流动相A和/或流动相B还可含有其它溶剂例如异丙醇等。In some embodiments, mobile phase A and/or mobile phase B may further contain other solvents such as isopropanol.

在一些实施方案中，在步骤(b)后包含步骤(b’):加入金属离子螯合剂例如乙二胺四乙酸(EDTA)进行反应。In some embodiments, step (b) is followed by step (b'): adding a metal ion chelator such as ethylenediaminetetraacetic acid (EDTA) to react.

在一些实施方案中，步骤(b’)在室温下进行，例如5min-5小时、10min–45min或30min。In some embodiments, step (b') is performed at room temperature, e.g., for 5 min-5 hours, 10 min-45 min, or 30 min.

在一些实施方案中，金属离子螯合剂的用量是1-8倍抗体摩尔当量，例如2-6、2.5-5.5、3-5或3.5-4.5倍抗体摩尔当量，例如1、1.5、2、2.5、3、3.5、4、4.5、5、5.5、6、6.5、7或7.5倍抗体摩尔当量。In some embodiments, the amount of metal ion chelator used is 1-8 times the antibody molar equivalent, such as 2-6, 2.5-5.5, 3-5 or 3.5-4.5 times the antibody molar equivalent, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7 or 7.5 times the antibody molar equivalent.

在一些实施方案中，在步骤(b’)后包含步骤(b”):加入脱氢抗坏血酸(DHAA)进行反应。In some embodiments, step (b') is followed by step (b"): adding dehydroascorbic acid (DHAA) to react.

在一些实施方案中，步骤(b”)在25～35℃下进行，例如0.2-24小时，例如0.5-12小时、1-10小时、1.5-5小时、1-3小时或1-4小时，例如1、1.5、2、2.5、3、3.5、4、5、6、7、8、9或10小时。In some embodiments, step (b") is performed at 25-35°C, for example 0.2-24 hours, for example 0.5-12 hours, 1-10 hours, 1.5-5 hours, 1-3 hours or 1-4 hours, for example 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 hours.

在一些实施方案中，DHAA的用量是1-16倍抗体摩尔当量，例如2-10、2-6、6-16或6-12倍抗体摩尔当量，例如1、2、3、4、5、6、7、8、10、12、14或16倍抗体摩尔当量。In some embodiments, the amount of DHAA used is 1-16 times the antibody molar equivalent, such as 2-10, 2-6, 6-16 or 6-12 times the antibody molar equivalent, such as 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14 or 16 times the antibody molar equivalent.

在一些实施方案中，本发明方法获得的DAR2-ADC具有大于90％的纯度，例如大于92％、93％、94％、95％、96％、97％、98％、99％或99.5％的纯度，所述纯度例如通过HIC-HPLC测得。In some embodiments, the DAR2-ADC obtained by the method of the present invention has a purity greater than 90%, such as greater than 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, as measured, for example, by HIC-HPLC.

在一些实施方案中，其中所述抗体选自人源化抗体、鼠源抗体、人抗体、嵌合抗体、单链抗体和双特异性抗体。In some embodiments, the antibody is selected from the group consisting of a humanized antibody, a murine antibody, a human antibody, a chimeric antibody, a single chain antibody, and a bispecific antibody.

在一些实施方案中，其中所述抗体是单克隆抗体或多克隆抗体。In some embodiments, the antibody is a monoclonal antibody or a polyclonal antibody.

在一些实施方案中，所述抗体是与下述抗原结合的抗体：HER2、B7H3、HER3、CD19、CD20、CD22、CD30、CD33、CD37、CD45、CD56、CD66e、CD70、CD74、CD73、CD79b、CD138、CD147、CD223、EpCAM、粘蛋白1(Mucin 1)、STEAP1、GPNMB、FGF2、FOLR1、 EGFR、EGFRvIII、组织因子(Tissuefactor)、c-MET、FGFR、Nectin 4、AGS-16、鸟苷酸环化酶C(Guanylyl cyclase C)、间皮素(Mesothelin)、SLC44A4、PSMA、EphA2、AGS-5、GPC-3、c-KIT、RoR1、PD-L1、CD27L、5T4、Mucin16、NaPi2b、STEAP、SLITRK6、ETBR、BCMA、Trop-2、CEACAM5、SC-16、SLC39A6、Delta-like protein3、Claudin 18.2。In some embodiments, the antibody is an antibody that binds to the following antigens: HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, tissue factor, c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C, Mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, Claudin 18.2.

在一些实施方案中，所述抗体是抗HER2抗体。In some embodiments, the antibody is an anti-HER2 antibody.

在一些实施方案中，所述抗体包含阿达木单抗、贝伐单抗、西妥昔单抗、曲妥珠单抗(trastuzumab)、帕妥珠单抗(Pertuzumab)、尼妥珠单抗、利妥昔单抗、h23-12或hH2L1的一个或多个CDR(优选3个CDR，即HCDR1、HCDR2H和HCDR3；或LCDR1、LCDR2和LCDR3，更优选6个CDR，即HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3)，或包含所述抗体的VH和/或VL，或包含所述抗体的重链和/或轻链。In some embodiments, the antibody comprises one or more CDRs (preferably 3 CDRs, i.e., HCDR1, HCDR2H and HCDR3; or LCDR1, LCDR2 and LCDR3, more preferably 6 CDRs, i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) of adalimumab, bevacizumab, cetuximab, trastuzumab, pertuzumab, nimotuzumab, rituximab, h23-12 or hH2L1, or comprises the VH and/or VL of the antibody, or comprises the heavy chain and/or light chain of the antibody.

在一些实施方案中，所述抗体选自阿达木单抗、贝伐单抗、西妥昔单抗、曲妥珠单抗(trastuzumab)、帕妥珠单抗(Pertuzumab)、尼妥珠单抗、利妥昔单抗、h23-12和hH2L1。In some embodiments, the antibody is selected from adalimumab, bevacizumab, cetuximab, trastuzumab, pertuzumab, nimotuzumab, rituximab, h23-12, and hH2L1.

在一些实施方案中，所述抗体包含trastuzumab、Pertuzumab、h23-12或hH2L1的一个或多个CDR(优选3个CDR，即HCDR1、HCDR2H和HCDR3；或LCDR1、LCDR2和LCDR3，更优选6个CDR，即HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3)，或包含trastuzumab、Pertuzumab、h23-12或hH2L1的VH和/或VL，或包含所述抗体的重链和/或轻链。In some embodiments, the antibody comprises one or more CDRs (preferably 3 CDRs, i.e., HCDR1, HCDR2H and HCDR3; or LCDR1, LCDR2 and LCDR3, more preferably 6 CDRs, i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) of trastuzumab, Pertuzumab, h23-12 or hH2L1, or comprises the VH and/or VL of trastuzumab, Pertuzumab, h23-12 or hH2L1, or comprises the heavy chain and/or light chain of the antibody.

在一些实施方案中，所述抗体选自trastuzumab、Pertuzumab、h23-12和hH2L1。In some embodiments, the antibody is selected from trastuzumab, pertuzumab, h23-12, and hH2L1.

在一些实施方案中，步骤(b)中的含药连接子为“L1-A-L2-D”所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药，In some embodiments, the drug-containing linker in step (b) is a compound represented by "L 1 -AL 2 -D" or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.

其中L1表示用于与抗体连接的接头单元，Wherein L1 represents a linker unit for connecting to an antibody,

A表示一个或多个(例如1、2、3或4个)氨基酸，A represents one or more (e.g. 1, 2, 3 or 4) amino acids,

L2表示用于与小分子药物连接的接头单元，且 L2 represents a linker unit for connecting to a small molecule drug, and

D为小分子药物。D is a small molecule drug.

在一些实施方案中，步骤(b)中的含药连接子为式A-I所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药，
In some embodiments, the drug-containing linker in step (b) is a compound represented by Formula AI or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof,

在结构式A-I中，并且在本发明的上下文中如无其他说明，E选自如下基团，其中，带有的键表示与M的连接位点：
In the structural formula AI, and in the context of the present invention, if not otherwise specified, E is selected from the following groups, wherein The bond of represents the attachment site to M:

n为选自1-10的整数，例如为1、2、3、4、5、6、7、8、9或10；n is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

R6、R7独立地为卤素或Ar’S-，Ar’为苯基或由一个或多个取代基取代的苯基，在取代的苯基中，所述取代基选自烷基(例如C1-C6烷基、优选C1-C4烷基)、烷氧基(例如C1-C6烷氧基、优选C1-C4烷氧基，优选甲氧基)、5-6元杂环基-CO-、卤素、酯基、酰胺基和氰基。优选地，Ar’为苯基、4-甲基氨基甲酰基苯基或4-甲酰基吗啉取代苯基 R 6 and R 7 are independently halogen or Ar'S-, Ar' is phenyl or phenyl substituted by one or more substituents, in the substituted phenyl, the substituents are selected from alkyl (e.g. C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), 5-6 membered heterocyclic group -CO-, halogen, ester group, amide group and cyano group. Preferably, Ar' is phenyl, 4-methylcarbamoylphenyl or 4-formylmorpholine substituted phenyl

M为亚苯基或由一个或多个取代基取代的亚苯基，或化学键；在取代的亚苯基中，所述取代基选自烷基(例如C1-C6烷基、优选C1-C4烷基)、卤代烷基(例如卤代C1-C6烷基、优选卤代C1-C4烷基，例如三氟甲基)、烷氧基(例如C1-C6烷氧基、优选C1-C4烷氧基，优选甲氧基)、卤素、酯基、酰胺基和氰基；优选地，M为卤素取代的亚苯基。 M is a phenylene group or a phenylene group substituted by one or more substituents, or a chemical bond; in the substituted phenylene group, the substituent group is selected from an alkyl group (e.g., a C1-C6 alkyl group, preferably a C1-C4 alkyl group), a haloalkyl group (e.g., a halo C1-C6 alkyl group, preferably a halo C1-C4 alkyl group, such as a trifluoromethyl group), an alkoxy group (e.g., a C1-C6 alkoxy group, preferably a C1-C4 alkoxy group, preferably a methoxy group), a halogen group, an ester group, an amide group and a cyano group; preferably, M is a halogen-substituted phenylene group.

SP1选自C1-8亚烷基、C1-8亚烷基亚苯基-、C1-8亚环烷基或C1-21(优选C1-16、更优选C1-11)直链亚杂烷基，所述C1-21直链亚杂烷基包含1-11个(优选1-6个)选自N、O或S的杂原子，其中所述C1-8亚烷基、C1-8亚环烷基和C1-21直链亚杂烷基各自独立地任选被选自羟基、氧代、氨基、磺酸基和氰基的一个或多个取代基取代；SP 1 is selected from C1-8 alkylene, C1-8 alkylene phenylene-, C1-8 cycloalkylene or C1-21 (preferably C1-16, more preferably C1-11) straight chain heteroalkylene, the C1-21 straight chain heteroalkylene containing 1-11 (preferably 1-6) heteroatoms selected from N, O or S, wherein the C1-8 alkylene, C1-8 cycloalkylene and C1-21 straight chain heteroalkylene are each independently optionally substituted by one or more substituents selected from hydroxy, oxo, amino, sulfonic acid and cyano;

SP2选自-NH(CH2CH2O)aCH2CH2CO-、-NH(CH2CH2O)aCH2CO-、-S(CH2)aCO-或化学键，其中a为1-20的整数，优选1-10的整数，更优选1-6的整数；SP 2 is selected from -NH(CH2CH2O)aCH2CH2CO-, -NH(CH2CH2O)aCH2CO-, -S(CH2)aCO- or a chemical bond, wherein a is an integer of 1-20, preferably an integer of 1-10, more preferably an integer of 1-6;

A表示2-4个氨基酸，其中，A表示2个氨基酸时，可以为NH-Phe-Lys-CO、NH-Val-Ala-CO、NH-Val-Lys-CO、NH-Ala-Lys-CO、NH-Val-Cit-CO、NH-Phe-Cit-CO、NH-Leu-Cit-CO、NH-Phe-Arg-CO或NH-Gly-Val-CO，优选为NH-Phe-Lys-CO、NH-Val-Ala-CO或NH-Val-Cit-CO；A表示3个氨基酸时，可以为NH-Glu-Val-Ala-CO、NH-Glu-Val-Cit-CO或NH-Ala-Ala-Ala-CO，优选为NH-Glu-Val-Ala-CO或NH-Ala-Ala-Ala-CO；A表示4个氨基酸时，可以为NH-Gly-Gly-Phe-Gly-CO或NH-Gly-Phe-Gly-Gly-CO，优选为NH-Gly-Gly-Phe-Gly-CO。优选地，A为NH-Val-Ala-CO、NH-Gly-Gly-Phe-Gly-CO或NH-Ala-Ala-Ala-CO；A represents 2 to 4 amino acids, wherein when A represents 2 amino acids, it can be NH-Phe-Lys-CO, NH-Val-Ala-CO, NH-Val-Lys-CO, NH-Ala-Lys-CO, NH-Val-Cit-CO, NH-Phe-Cit-CO, NH-Leu-Cit-CO, NH-Phe-Arg-CO or NH-Gly-Val-CO, preferably NH-Phe-Lys-CO, NH-Val-Ala-CO or NH-Val-Cit-CO. t-CO; when A represents 3 amino acids, it can be NH-Glu-Val-Ala-CO, NH-Glu-Val-Cit-CO or NH-Ala-Ala-Ala-CO, preferably NH-Glu-Val-Ala-CO or NH-Ala-Ala-Ala-CO; when A represents 4 amino acids, it can be NH-Gly-Gly-Phe-Gly-CO or NH-Gly-Phe-Gly-Gly-CO, preferably NH-Gly-Gly-Phe-Gly-CO. Preferably, A is NH-Val-Ala-CO, NH-Gly-Gly-Phe-Gly-CO or NH-Ala-Ala-Ala-CO;

优选地，A选自：NH-Val-Cit-CO、NH-Val-Ala-CO和NH-Gly-Gly-Phe-Gly-CO；Preferably, A is selected from the group consisting of NH-Val-Cit-CO, NH-Val-Ala-CO and NH-Gly-Gly-Phe-Gly-CO;

应当理解，A左侧的NH是A中左侧氨基酸中氨基，A右侧的CO是A中右侧氨基酸中的羰基。It should be understood that the NH on the left side of A is the amino group in the left amino acid in A, and the CO on the right side of A is the carbonyl group in the right amino acid in A.

L2选自和化学键。在一些实施方案中，M选自 L 2 is selected from In some embodiments, M is selected from

在一些实施方案中，选自
In some embodiments, Selected from

其中q为选自1-10的整数，例如为1、2、3、4、5、6、7、8、9或10；wherein q is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

p为选自1-20的整数，例如为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、 16、17、18、19或20；p is an integer selected from 1-20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;

在一些实施方案中，选自
In some embodiments, Selected from

在一些是实施方案中，D选自喜树碱类化合物、加利车霉素(calicheamicin)、美登木素生物碱(maytansinoids)、多拉司他汀(dolastatin)、澳瑞他汀类化合物和单端孢霉素(trichothecene)。In some embodiments, D is selected from the group consisting of camptothecins, calicheamicins, maytansinoids, dolastatins, auristatins, and trichothecenes.

在一些是实施方案中，所述美登木素衍生物选自DM1、DM3和DM4；所述澳瑞他汀类化合物选自MMAE和MMAF；所述喜树碱类化合物选自喜树碱、10-羟基喜树碱、依喜替康(exatecan)、SN-38、托泊替康和喜树碱类衍生物。In some embodiments, the maytansinoid derivative is selected from DM1, DM3 and DM4; the auristatin compound is selected from MMAE and MMAF; the camptothecin compound is selected from camptothecin, 10-hydroxycamptothecin, exatecan, SN-38, topotecan and camptothecin derivatives.

在一些实施方案中，含药连接子为式III所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药，
In some embodiments, the drug-containing linker is a compound of Formula III or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.

其中E、M、SP1、SP2和A如上文所定义，CPT为喜树碱类化合物。wherein E, M, SP 1 , SP 2 and A are as defined above, and CPT is a camptothecin compound.

在一些实施方案中，含药连接子为式III'所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药，
In some embodiments, the drug-containing linker is a compound represented by Formula III' or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof,

其中E、M、SP1、SP2和A如上文所定义，D'为澳瑞他汀类化合物。wherein E, M, SP 1 , SP 2 and A are as defined above, and D' is an auristatin compound.

在一些实施方案中，式III化合物进一步为式IIIA化合物：
In some embodiments, the compound of formula III is further a compound of formula IIIA:

其中，R6、R7独立地为卤素或Ar’S-，Ar’为苯基或由一个或多个取代基取代的苯基，在取代的苯基中，所述取代基选自烷基(例如C1-C6烷基、优选C1-C4烷基)、烷氧基(例如C1-C6烷氧基、优选C1-C4烷氧基，优选甲氧基)、5-6元杂环基-CO-、卤素、酯基、酰胺基和氰基。优选地，Ar’为苯基、4-甲基氨基甲酰基苯基或4-吗啉代甲酰基苯基其中M、SP1、SP2、A和CPT如上文所定义。Wherein, R 6 and R 7 are independently halogen or Ar'S-, Ar' is phenyl or phenyl substituted by one or more substituents, in the substituted phenyl, the substituent is selected from alkyl (e.g. C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), 5-6 membered heterocyclic group -CO-, halogen, ester group, amide group and cyano group. Preferably, Ar' is phenyl, 4-methylcarbamoylphenyl or 4-morpholinoformylphenyl wherein M, SP 1 , SP 2 , A and CPT are as defined above.

在一些实施方案中，式III'化合物进一步为式IIIA'化合物：
In some embodiments, the compound of formula III' is further a compound of formula IIIA':

其中R6、R7、M、SP1、SP2、A和D’如上文所定义。wherein R 6 , R 7 , M, SP 1 , SP 2 , A and D′ are as defined above.

在一些实施方案中，在式III和式IIIA中，CPT为如下式I或式IA所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药，以及对应的具体化合物：
In some embodiments, in Formula III and Formula IIIA, CPT is a compound as shown in Formula I or Formula IA below, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, and the corresponding specific compound:

在式I中，并且在本发明的上下文中如无其他说明，R1、R2、R3、R4独立地为氢、卤素、 羟基、C1-6烷氧基、氨基或取代氨基、C1-7烷基或取代的C1-7烷基，或者R1、R2、R3、R4中的任意两个连同它们所连接的碳原子构成C3-6环状烷基。当R1、R2、R3、R4独立地为C1-6烷氧基时，所述C1-6烷氧基包括直链或支链的C1-6烷氧基，优选地为直链或支链的C1-3烷氧基，更优选甲氧基。当R1、R2、R3、R4独立地为取代氨基时，所述取代氨基为由选自甲基和乙基的一个或多个取代基取代的氨基。当R1、R2、R3、R4独立地为C1-7烷基或取代的C1-7烷基时，所述C1-7烷基或取代的C1-7烷基包括直链或支链的C1-7烷基或取代的C1-7烷基，并且，取代的C1-7烷基为由选自环丙基和环丁基的一个或多个取代基取代的C1-7烷基；或者，所述直链或支链的C1-7烷基或取代的C1-7烷基优选为C1-3烷基或取代的C1-3烷基，例如甲基、卤代甲基(优选三氟甲基)。In formula I, and unless otherwise stated in the context of the present invention, R 1 , R 2 , R 3 , R 4 are independently hydrogen, halogen, Hydroxyl, C1-6 alkoxy, amino or substituted amino, C1-7 alkyl or substituted C1-7 alkyl, or any two of R 1 , R 2 , R 3 , R 4 together with the carbon atoms to which they are connected constitute a C3-6 cyclic alkyl. When R 1 , R 2 , R 3 , R 4 are independently C1-6 alkoxy, the C1-6 alkoxy includes a straight or branched C1-6 alkoxy, preferably a straight or branched C1-3 alkoxy, more preferably a methoxy. When R 1 , R 2 , R 3 , R 4 are independently substituted amino, the substituted amino is an amino substituted by one or more substituents selected from methyl and ethyl. When R 1 , R 2 , R 3 , and R 4 are independently C1-7 alkyl or substituted C1-7 alkyl, the C1-7 alkyl or substituted C1-7 alkyl includes linear or branched C1-7 alkyl or substituted C1-7 alkyl, and the substituted C1-7 alkyl is C1-7 alkyl substituted by one or more substituents selected from cyclopropyl and cyclobutyl; or, the linear or branched C1-7 alkyl or substituted C1-7 alkyl is preferably C1-3 alkyl or substituted C1-3 alkyl, such as methyl, halogenated methyl (preferably trifluoromethyl).

在式I中，并且在本发明的上下文中如无其他说明，G为氢、卤素、甲基或甲氧基。优选地，G为氢、氟或氯。In formula I, and unless otherwise stated in the context of the present invention, G is hydrogen, halogen, methyl or methoxy. Preferably, G is hydrogen, fluorine or chlorine.

在式I中，并且在本发明的上下文中如无其他说明，Y为氧、硫、砜、亚砜、亚甲基或取代亚甲基。取代亚甲基可以是亚甲基的一个氢被取代，也可以是两个氢同时被取代，取代基可以为苄基或烷基；当取代基为烷基时，所述烷基与R3和/或R4以及连同它们所连接的碳原子可以构成C3-6元并环或螺环的结构。当Y为取代亚甲基时，取代亚甲基的取代基优选为烷基，更优选为直链或支链的C1-4烷基。优选地，Y为氧、硫、砜或亚砜；或者，优选地，Y为氧、硫或亚甲基。In formula I, and in the context of the present invention, unless otherwise specified, Y is oxygen, sulfur, sulfone, sulfoxide, methylene or substituted methylene. The substituted methylene may be a methylene in which one hydrogen is substituted or two hydrogens are substituted simultaneously, and the substituent may be a benzyl or an alkyl group; when the substituent is an alkyl group, the alkyl group and R 3 and/or R 4 and the carbon atoms to which they are attached may form a C3-6-membered cyclic or spirocyclic structure. When Y is a substituted methylene group, the substituent of the substituted methylene group is preferably an alkyl group, more preferably a linear or branched C1-4 alkyl group. Preferably, Y is oxygen, sulfur, sulfone or sulfoxide; or, preferably, Y is oxygen, sulfur or methylene.

在式I中，并且在本发明的上下文中如无其他说明，X为氧或硫。In formula I, and unless otherwise stated in the context of the present invention, X is oxygen or sulfur.

在式I中，并且在本发明的上下文中如无其他说明，n＝0或1。In formula I, and unless otherwise stated in the context of the present invention, n=0 or 1.

在式I的一些实施方案中，在R1、R2、R3、R4同时为氢、X为氧、n＝0的情况下，当Y为亚甲基时，G不可为氢或氟；而当Y为氧或硫时，G不可为氢。In some embodiments of formula I, when R 1 , R 2 , R 3 , and R 4 are simultaneously hydrogen, X is oxygen, and n=0, when Y is methylene, G cannot be hydrogen or fluorine; and when Y is oxygen or sulfur, G cannot be hydrogen.

优选地，R1、R2、R3、R4独立地为氢、卤素(例如氟)、C1-7烷基或取代的C1-7烷基，或者R1、R2、R3、R4中的任意两个连同它们所连接的碳原子构成C3-6环状烷基(例如C3-5环状烷基)。进一步地，R1、R2可以是相同的；和/或，R3、R4可以是相同的。Preferably, R 1 , R 2 , R 3 , and R 4 are independently hydrogen, halogen (e.g., fluorine), C1-7 alkyl, or substituted C1-7 alkyl, or any two of R 1 , R 2 , R 3 , and R 4 together with the carbon atoms to which they are attached constitute a C3-6 cyclic alkyl (e.g., a C3-5 cyclic alkyl). Further, R 1 and R 2 may be the same; and/or, R 3 and R 4 may be the same.

优选地，Y为由烷基取代的亚甲基，所述烷基与R3和/或R4以及连同它们所连接的碳原子可以构成C3-6元并环或螺环的结构。Preferably, Y is a methylene group substituted by an alkyl group, and the alkyl group, R3 and/or R4, and the carbon atoms to which they are connected can form a C3-6-membered cyclic or spirocyclic structure.

优选地，X可以为氧。Preferably, X may be oxygen.

优选地，X为氧，G为氢、卤素(例如氟或氯)、甲基或甲氧基，Y和R1、R2、R3、R4如上文所定义。Preferably, X is oxygen, G is hydrogen, halogen (eg fluorine or chlorine), methyl or methoxy, and Y and R 1 , R 2 , R 3 , R 4 are as defined above.

优选地，X为氧，G为氢，Y为亚甲基或取代亚甲基、氧或硫，R1、R2、R3、R4如上文所定义。Preferably, X is oxygen, G is hydrogen, Y is methylene or substituted methylene, oxygen or sulfur, and R 1 , R 2 , R 3 and R 4 are as defined above.

优选地，X为氧，G为氟，Y为亚甲基或取代亚甲基、氧或硫，R1、R2、R3、R4如上文所定义。Preferably, X is oxygen, G is fluorine, Y is methylene or substituted methylene, oxygen or sulfur, and R 1 , R 2 , R 3 and R 4 are as defined above.

优选地，X为氧，G为氯，Y为亚甲基或取代亚甲基、氧或硫，R1、R2、R3、R4如上文所定义。Preferably, X is oxygen, G is chlorine, Y is methylene or substituted methylene, oxygen or sulfur, and R 1 , R 2 , R 3 and R 4 are as defined above.

优选地，X为氧，G为甲基，Y为亚甲基或取代亚甲基、氧或硫，R1、R2、R3、R4如上文所定义。 Preferably, X is oxygen, G is methyl, Y is methylene or substituted methylene, oxygen or sulfur, and R 1 , R 2 , R 3 and R 4 are as defined above.

优选地，X为氧，G为甲氧基，Y为亚甲基或取代亚甲基、氧或硫，R1、R2、R3、R4如上文所定义。Preferably, X is oxygen, G is methoxy, Y is methylene or substituted methylene, oxygen or sulfur, and R 1 , R 2 , R 3 and R 4 are as defined above.

优选地，X为氧，G为氢，Y为氧、砜或亚砜，R1、R2、R3、R4如上文所定义。Preferably, X is oxygen, G is hydrogen, Y is oxygen, sulfone or sulfoxide, and R 1 , R 2 , R 3 , R 4 are as defined above.

优选地，X为氧，G为氢，Y为砜或亚砜，R1、R2、R3、R4如上文所定义。Preferably, X is oxygen, G is hydrogen, Y is sulfone or sulfoxide, and R 1 , R 2 , R 3 , R 4 are as defined above.

优选地，式I所示的化合物进一步为式IA所示的化合物：
Preferably, the compound represented by formula I is further represented by formula IA:

式IA中，基团R1、R2、R3、R4与上文式I中基团R1、R2、R3、R4的定义相同，R1、R2、R3、R4可以同时为氢或不同时为氢。 In formula IA, the groups R 1 , R 2 , R 3 , and R 4 are the same as defined in formula I above. R 1 , R 2 , R 3 , and R 4 may be hydrogen at the same time or not at the same time.

在一些实施方案中，所述喜树碱类化合物为式II所示的化合物或其药学上可接受的盐、立体异构体、溶剂化物或前药：
In some embodiments, the camptothecin compound is a compound represented by Formula II or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:

在式II中，并且在本发明的上下文中如无其他说明，R5为C1-5烷基或由一个或多个取代基取代的C1-5烷基、C3-6环状烷基或由一个或多个取代基取代的C3-6环状烷基、苯基或取代苯基。当R5为C1-5烷基或取代的C1-5烷基时，所述C1-5烷基包括直链或支链的C1-5烷基。进一步地，R5为C1-4直链烷基。当R5为取代的C1-5烷基或取代的C3-6环状烷基时，取代基选自卤素、羟基、甲氧基、三氟甲基、氨基或取代氨基、甲磺酰基和C3-6环状烷基；并且其中，取代氨基为由选自甲基和乙基的一个或多个取代基取代的氨基。当R5为取代苯基时，取代基选自烷基(例如C1-6烷基、优选C1-3)或卤素。In formula II, and in the context of the present invention, unless otherwise specified, R 5 is C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted by one or more substituents, phenyl or substituted phenyl. When R 5 is C1-5 alkyl or substituted C1-5 alkyl, the C1-5 alkyl includes straight or branched C1-5 alkyl. Further, R 5 is C1-4 straight chain alkyl. When R 5 is substituted C1-5 alkyl or substituted C3-6 cyclic alkyl, the substituent is selected from halogen, hydroxyl, methoxy, trifluoromethyl, amino or substituted amino, mesyl and C3-6 cyclic alkyl; and wherein the substituted amino is an amino substituted by one or more substituents selected from methyl and ethyl. When R 5 is substituted phenyl, the substituent is selected from alkyl (e.g. C1-6 alkyl, preferably C1-3) or halogen.

在式II中，并且在本发明的上下文中如无其他说明，G为氢、卤素(例如氟)、甲基或甲氧基。优选地，G为氢、氟或氯。In formula II, and unless otherwise stated in the context of the present invention, G is hydrogen, halogen (eg fluorine), methyl or methoxy. Preferably, G is hydrogen, fluorine or chlorine.

式II中，X为氧或硫。In formula II, X is oxygen or sulfur.

式II中，n＝0或1。In formula II, n=0 or 1.

优选地，式II中，当X为氧、G为氢、n＝0时，R5不可为正丁基。Preferably, in formula II, when X is oxygen, G is hydrogen, and n=0, R 5 cannot be n-butyl.

优选地，式II所示的化合物进一步为式IIA所示的化合物：
Preferably, the compound represented by formula II is further represented by formula IIA:

式IIA中，基团R5与上文式II中基团R5的定义相同，在一些实施方案中，R5不可为正丁基，在一些实施方案中，R5可为正丁基。In Formula IIA, the group R 5 is the same as the definition of the group R 5 in Formula II above. In some embodiments, R 5 may not be n-butyl. In some embodiments, R 5 may be n-butyl.

在一些实施方案中，所述喜树碱类化合物(CPT)具有如下结构：


In some embodiments, the camptothecin compound (CPT) has the following structure:


在式III和式IIIA中，式I或式IA所示的化合物经由其氨基(分别参见式I或式IA)与A的羧基通过酰胺键相连，即在式I或式IA所示化合物的氨基与式III或IIIA中A的羧基形成了酰胺键。In Formula III and Formula IIIA, the compound represented by Formula I or Formula IA is connected to the carboxyl group of A through an amide bond via its amino group (see Formula I or Formula IA, respectively), that is, the amino group of the compound represented by Formula I or Formula IA forms an amide bond with the carboxyl group of A in Formula III or IIIA.

又如，在式III和式IIIA中，CPT式II或式IIA所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药，以及对应的具体化合物。For example, in Formula III and Formula IIIA, the compound shown in CPT Formula II or Formula IIA or its pharmaceutically acceptable salt, stereoisomer, solvate or prodrug, and the corresponding specific compound.

在式III和式IIIA中，式II或式IIA所示的化合物经由其氨基(分别参见式II或式IIA)与A的羧基通过酰胺键连接，即式II或式IIA所示化合物的氨基与式III或IIIA中A的羧基形成酰胺键。In formula III and formula IIIA, the compound represented by formula II or formula IIA is connected to the carboxyl group of A through an amide bond via its amino group (see formula II or formula IIA, respectively), that is, the amino group of the compound represented by formula II or formula IIA forms an amide bond with the carboxyl group of A in formula III or IIIA.

上文提出的化合物1、2、3、4、5、6、7、8、9、10、11、12、13、14、14-P、15、16、17、18、19、20、21、22、23、24、25、26、27、31、32、33、34、35、36、37、38、39、40或其药学可接受的盐、立体异构体、溶剂化物或前药均可用作式III中的“CPT”，并通过其 氨基与式III或IIIA中A的羧基通过酰胺键连接。Compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14-P, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or pharmaceutically acceptable salts, stereoisomers, solvates, or prodrugs thereof set forth above may be used as "CPT" in Formula III and may be expressed by the formula The amino group is linked to the carboxyl group of A in formula III or IIIA via an amide bond.

又如，在式III和式IIIA中，CPT可以为式IV所示的依喜替康(exteacan)衍生物或其药学可接受的盐、立体异构体、溶剂化物或前药：
For example, in Formula III and Formula IIIA, CPT can be an exteacan derivative shown in Formula IV or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:

在式IV中，并且在本发明的上下文中如无其他说明，R8为氢、三氟甲基、C1-5烷基或由一个或多个取代基取代的C1-5烷基、C3-6环状烷基或由一个或多个取代基取代的C3-6环状烷基、或卤素。In formula IV, and unless otherwise specified in the context of the present invention, R 8 is hydrogen, trifluoromethyl, C 1-5 alkyl or C 1-5 alkyl substituted by one or more substituents, C 3-6 cyclic alkyl or C 3-6 cyclic alkyl substituted by one or more substituents, or halogen.

当R8为取代的C1-5烷基或取代的C3-6环状烷基时，所述取代基选自卤素、羟基、甲氧基、三氟甲基、氨基或取代氨基、甲磺酰基和C3-6环状烷基；并且其中，取代氨基为由选自甲基和乙基的一个或多个取代基取代的氨基。When R8 is substituted C1-5 alkyl or substituted C3-6 cyclic alkyl, the substituent is selected from halogen, hydroxy, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl and C3-6 cyclic alkyl; and wherein the substituted amino is an amino substituted by one or more substituents selected from methyl and ethyl.

在式III和式IIIA中，式IV所示的化合物经由其与R8连接于同一个碳的羟基(参见式IV)与A的羧基通过自释放结构相连，该自释放结构例如实线表示与式III或式IIIA中A的羧基连接的位点，波浪线表示与式IV中羟基连接的位点。In formula III and formula IIIA, the compound represented by formula IV is connected to the carboxyl group of A through a self-releasing structure via its hydroxyl group connected to the same carbon as R 8 (see formula IV), and the self-releasing structure is, for example The solid line indicates the site of attachment to the carboxyl group of A in Formula III or Formula IIIA, and the wavy line indicates the site of attachment to the hydroxyl group in Formula IV.

特别地，对于式IIIA所示的化合物，当R6、R7为Ar’S时，M优选为亚苯基或取代的亚苯基，同时SP1为C1-21(优选C1-16、更优选C1-11)直链亚杂烷基，所述直链亚杂烷基包含1-11个(优选1-6个)选自N、O或S的杂原子。In particular, for the compound represented by formula IIIA, when R 6 and R 7 are Ar'S, M is preferably a phenylene or substituted phenylene, and SP1 is a C1-21 (preferably C1-16, more preferably C1-11) straight chain heteroalkylene, wherein the straight chain heteroalkylene contains 1-11 (preferably 1-6) heteroatoms selected from N, O or S.

在一些实施方案中，在式III’和式IIIA’中，D’为如下式I’所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药，以及对应的具体化合物：
In some embodiments, in Formula III' and Formula IIIA', D' is a compound as shown in the following Formula I' or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, and the corresponding specific compound:

其中Ra1、Ra2、Ra3、Ra4、Ra5和Ra8各自独立的选自C1-8烷基；优选C1-4烷基，例如甲基、乙基、正丙基、异丙基、正丁基、异丁基或仲丁基；wherein Ra1 , Ra2 , Ra3 , Ra4 , Ra5 and Ra8 are each independently selected from C1-8 alkyl; preferably C1-4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl;

Ra6和Ra7各自独立地选自C1-8烷氧基，例如甲氧基、乙氧基或丙氧基；R a6 and R a7 are each independently selected from C 1-8 alkoxy, such as methoxy, ethoxy or propoxy;

Ra9选自C1-8烷基和COOH；优选C1-4烷基，例如甲基、乙基、正丙基、异丙基、正丁基、 异丁基或仲丁基；R a9 is selected from C 1-8 alkyl and COOH; preferably C 1-4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, Isobutyl or sec-butyl;

Ra10选自OH和H。R a10 is selected from OH and H.

在一些实施方案中，式I'化合物进一步为式IA'化合物：
In some embodiments, the compound of formula I' is further a compound of formula IA':

其中Ra1-Ra10如上文所定义；wherein R a1 -R a10 are as defined above;

在一些实施方案中，式I'化合物选自：
In some embodiments, the compound of Formula I' is selected from:

在III’和式IIIA’中，式I’所示的化合物经由其氨基与A的羧基通过酰胺键连接，即式I’所示化合物的左侧仲氨基与式III’或IIIA’中A的羧基形成酰胺键。In formula III’ and formula IIIA’, the compound represented by formula I’ is connected to the carboxyl group of A via an amide bond via its amino group, that is, the left secondary amino group of the compound represented by formula I’ forms an amide bond with the carboxyl group of A in formula III’ or IIIA’.

在一些实施方案中，式III和式IIIA所示的化合物进一步为式V所示的化合物。
In some embodiments, the compound represented by Formula III and Formula IIIA is further represented by Formula V.

式V中，R6、R7独立地为Ar’S，Ar’为苯基或由一个或多个取代基取代的苯基，在取代的苯基中，取代基选自烷基(例如C1-C6烷基、优选C1-C4烷基)、烷氧基(例如C1-C6烷氧基、优选C1-C4烷氧基，优选甲氧基)、5-6元杂环基-CO-、卤素、酯基、酰胺基和氰基。优选地，Ar’为苯基、4-甲基氨基甲酰基苯基或4-吗啉代甲酰基苯基 In formula V, R 6 and R 7 are independently Ar'S, Ar' is phenyl or phenyl substituted by one or more substituents, in the substituted phenyl, the substituent is selected from alkyl (e.g. C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), 5-6 membered heterocyclic group -CO-, halogen, ester group, amide group and cyano group. Preferably, Ar' is phenyl, 4-methylcarbamoylphenyl or 4-morpholinoformylphenyl

式V中，并且在本发明的上下文中如无其他说明，Xh和Yh独立地为氢、卤素、卤代烷基(例如卤代C1-C6烷基、优选卤代C1-C4烷基，例如三氟甲基)或烷氧基(例如C1-C6烷氧基、优选C1-C4烷氧基，例如甲氧基)。In formula V, and unless otherwise specified in the context of the present invention, Xh and Yh are independently hydrogen, halogen, haloalkyl (e.g. haloC1-C6 alkyl, preferably haloC1-C4 alkyl, such as trifluoromethyl) or alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, such as methoxy).

式V中，并且在本发明的上下文中如无其他说明，m为1～10、优选1～5、更优选3-5中任一整数。In formula V, and unless otherwise specified in the context of the present invention, m is any integer from 1 to 10, preferably from 1 to 5, more preferably from 3 to 5.

式V中，A表示2-4个氨基酸，如上文所定义。In formula V, A represents 2 to 4 amino acids, as defined above.

式V中，CPT的定义及其在式V中的连接关系与式III和式IIIA中CPT相同，如上文所定义。In Formula V, the definition of CPT and its linkage in Formula V are the same as CPT in Formula III and Formula IIIA, as defined above.

在一些实施方案中，式IIIA’化合物进一步为式V’化合物，
In some embodiments, the compound of formula IIIA' is further a compound of formula V',

其中R6、R7、Xh、Yh、m和A如上文(尤其式V)所定义，D'如上文所定义。wherein R 6 , R 7 , Xh, Yh, m and A are as defined above (especially in formula V), and D′ is as defined above.

在一些实施方案中，式V’化合物进一步为式V-A’化合物，
In some embodiments, the compound of formula V' is further a compound of formula V-A',

其中A和D'如上文所定义。wherein A and D' are as defined above.

在一些实施方案中，式V所示的化合物进一步为式V-A所示的化合物：
In some embodiments, the compound of formula V is further a compound of formula VA:

式V-A中，基团A、G、Y、R1、R2、R3、R4、X、n与上文式III或式IIIA中基团A、G、Y、R1、R2、R3、R4、X、n的定义相同。In formula VA, the groups A, G, Y, R1 , R2 , R3 , R4 , X, and n are the same as defined above in formula III or formula IIIA.

优选地，式V-A中，G为氢、氟或氯。Preferably, in formula V-A, G is hydrogen, fluorine or chlorine.

优选地，式V-A中，Y为亚甲基、硫或氧。Preferably, in formula V-A, Y is methylene, sulfur or oxygen.

式V-A中，并且在本发明的上下文中如无其他说明，“-A-NH-”表示所示氨基与A中的羧基形成酰胺键。In formula V-A, and unless otherwise specified in the context of the present invention, "-A-NH-" means that the amino group shown forms an amide bond with the carboxyl group in A.

在一些实施方案中，式V-A所示的化合物进一步为式V-A-1所示的化合物：
In some embodiments, the compound represented by Formula VA is further represented by Formula VA-1:

或者，式V所示的化合物进一步为式V-B所示的化合物：
Alternatively, the compound represented by formula V is further represented by formula VB:

式V-B中，基团A、G、R5、X、n与上文式III或式IIIA中基团A、G、R5、X、n的定义相同。In Formula VB, the groups A, G, R 5 , X, and n are the same as defined above in Formula III or Formula IIIA.

式V-B中，并且在本发明的上下文中如无其他说明，“-A-NH-”表示所示氨基与A中的羧基形成酰胺键。 In Formula VB, and in the context of the present invention, unless otherwise specified, "-A-NH-" indicates that the amino group indicated forms an amide bond with the carboxyl group in A.

优选地，式V-B所示的化合物进一步为式V-B-1所示的化合物：
Preferably, the compound represented by formula VB is further represented by formula VB-1:

或者，式V所示的化合物进一步为式V-C所示的化合物：
Alternatively, the compound represented by formula V is further represented by formula VC:

式V-C中，基团A、R8与上文式III或式IIIV中基团A、R8的定义相同。In formula VC, the groups A and R8 are the same as defined above in formula III or formula IIIV .

式V-C中，并且在本发明的上下文中如无其他说明，“-A-NH-”表示所示氨基与A中的羧基形成酰胺键。In formula V-C, and unless otherwise specified in the context of the present invention, "-A-NH-" means that the amino group indicated forms an amide bond with the carboxyl group in A.

式V-C中，并且在本发明的上下文中如无其他说明，与上文式III或式IIIV中的CPT为式IV时的自释放结构相同。In formula VC, and in the context of the present invention unless otherwise specified, The self-releasing structure is the same as when the CPT in the above formula III or formula IIIV is formula IV.

根据本发明的具体实施方式，式V所示的化合物进一步为式VI所示的化合物：
According to a specific embodiment of the present invention, the compound represented by formula V is further represented by formula VI:

式VI中，A表示2-4个氨基酸，如上文所定义。In formula VI, A represents 2 to 4 amino acids, as defined above.

式VI中，CPT为喜树碱类化合物。CPT的定义及其在式VI中的连接关系与式III和式IIIA中CPT相同，如上文所定义。In Formula VI, CPT is a camptothecin compound. The definition of CPT and its connection relationship in Formula VI are the same as those of CPT in Formula III and Formula IIIA, as defined above.

优选地，本发明提供的式VI所示的化合物进一步为式VI-A所示的化合物：
Preferably, the compound represented by Formula VI provided by the present invention is further represented by Formula VI-A:

式VI-A中，基团A、G、Y、R1、R2、R3、R4、X、n与上文式III或式IIIA中基团A、G、Y、R1、R2、R3、R4、X、n的定义相同。In Formula VI-A, the groups A, G, Y, R 1 , R 2 , R 3 , R 4 , X, and n are the same as defined above in Formula III or Formula IIIA.

优选地，式VI-A中，G为氢、氟或氯。Preferably, in Formula VI-A, G is hydrogen, fluorine or chlorine.

优选地，式VI-A中，Y为亚甲基、硫或氧。Preferably, in Formula VI-A, Y is methylene, sulfur or oxygen.

式VI-A中，并且在本发明的上下文中如无其他说明，“-A-NH-”表示所示氨基与A中的羧基形成酰胺键。In Formula VI-A, and in the context of the present invention, unless otherwise specified, "-A-NH-" means that the amino group indicated forms an amide bond with the carboxyl group in A.

优选地，式VI-A所示的化合物进一步为式VI-A-1所示的化合物：
Preferably, the compound represented by formula VI-A is further represented by formula VI-A-1:

或者，式VI所示的化合物进一步为式VI-B所示的化合物：
Alternatively, the compound represented by formula VI is further represented by formula VI-B:

式VI-B中，基团A、G、R5、X、n与上文式III或式IIIA中基团A、G、R5、X、n的定义相同。In Formula VI-B, the groups A, G, R 5 , X, and n are the same as defined above in Formula III or Formula IIIA.

式VI-B中，并且在本发明的上下文中如无其他说明，“-A-NH-”表示所示CPT中的氨基与A中的羧基形成酰胺键。In Formula VI-B, and unless otherwise specified in the context of the present invention, "-A-NH-" indicates that the amino group in the CPT shown forms an amide bond with the carboxyl group in A.

更优选地，式VI-B所示化合物进一步为式VI-B-1所示的化合物：
More preferably, the compound represented by formula VI-B is further represented by formula VI-B-1:

或者，式VI所示的化合物进一步为式VI-C所示的化合物：
Alternatively, the compound represented by formula VI is further represented by formula VI-C:

式VI-C中，基团A、R8的定义与上文式III或式IIIA中基团A、R8的定义相同。In Formula VI-C, the definitions of Group A and R8 are the same as those of Group A and R8 in Formula III or IIIA above.

式VI-C中，并且在本发明的上下文中如无其他说明，“-A-NH-”表示所示氨基与A中的羧基形成酰胺键。In Formula VI-C, and in the context of the present invention, unless otherwise specified, "-A-NH-" indicates that the amino group indicated forms an amide bond with the carboxyl group in A.

当式V-C中自释放结构为时，式V-C可进一步如式VI-C所示结构。When the self-releasing structure in VC is When, formula VC can be further structured as shown in formula VI-C.

在一些实施方案中，所述含药连接子选自以下结构：







In some embodiments, the drug-containing linker is selected from the following structures:







应当理解，本发明还涉及本发明实施例中公开的具体方法。另外，本发明具体实施例中某些技术特征用上述实施方案中的技术特征替换或组合后得到的技术方案也在本发明的考虑 中。It should be understood that the present invention also relates to the specific methods disclosed in the embodiments of the present invention. In addition, the technical solutions obtained by replacing or combining certain technical features in the specific embodiments of the present invention with the technical features in the above-mentioned embodiments are also considered in the present invention. middle.

本发明中的一些小分子药物、含药连接子和/或其中间体、和/或抗体-药物偶联物等记载于中国专利申请202211627164.8中，将该专利申请以其全部内容引入本申请中作为本申请内容的一部分。Some small molecule drugs, drug-containing linkers and/or intermediates thereof, and/or antibody-drug conjugates in the present invention are recorded in Chinese patent application 202211627164.8, and the entire content of the patent application is introduced into this application as part of the content of this application.

以上制备方法的各步骤彼此相互关联、相互作用产生了提高DAR2-ADC偶联物纯度的技术效果，该技术效果是各步骤作为整体所产生的。The steps of the above preparation method are interrelated and interact with each other to produce a technical effect of improving the purity of the DAR2-ADC conjugate, and this technical effect is produced by the steps as a whole.

第二方面Second aspect

本发明提供了抗体药物偶联物，其具有通式所示结构，其中mAb表示抗体或抗体片段，例如上文定义的抗体，基团M、SP1、SP2、A、L2及D如上文第一方面中所定义。y为1～10、优选1～8(例如1～5)、更优选1～3，最优选2。The present invention provides an antibody drug conjugate having the general formula The structure shown, wherein mAb represents an antibody or antibody fragment, such as the antibody defined above, and groups M, SP1 , SP2 , A, L2 and D are as defined above in the first aspect. y is 1-10, preferably 1-8 (such as 1-5), more preferably 1-3, and most preferably 2.

其中，EL选自如下基团，其中，表示与mAb中半胱氨酸相连，另外的带有的键表示与M相连：Wherein, EL is selected from the following groups, wherein, Indicates that it is linked to the cysteine in the mAb, and the other The key indicates connection with M:

和/或 其中n如上面变量E中所定义。 and/or where n is as defined above in variable E.

在所述通式结构中，mAb可以是IgG型抗体或其片段，优选为的IgG1亚型抗体或其片段。In the general structure, mAb may be an IgG type antibody or a fragment thereof, preferably an IgG1 subtype antibody or a fragment thereof.

在一些实施方案中，所述抗体药物偶联物是通过第一方面所述的本发明的方法制备的。In some embodiments, the antibody drug conjugate is prepared by the method of the present invention as described in the first aspect.

在一些实施方案中，所述抗体药物偶联物具有约2的平均DAR。In some embodiments, the antibody drug conjugate has an average DAR of about 2.

在一些实施方案中，所述抗体药物偶联物(DAR2-ADC)具有大于90％的纯度，例如大于92％、93％、94％、95％、96％、97％、98％、99％或99.5％的纯度，所述纯度例如通过HIC-HPLC测得。In some embodiments, the antibody drug conjugate (DAR2-ADC) has a purity greater than 90%, such as greater than 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, such as measured by HIC-HPLC.

在一些实施方案中，所述抗体药物偶联物具有如下通式VII、VII'、VIII、VIII’所示的结构：

In some embodiments, the antibody drug conjugate has the structure shown in the following general formula VII, VII', VIII, VIII':

和/或and/or

其中y为1～10、优选1～8(例如1～5)、更优选1、2或3。Here, y is 1-10, preferably 1-8 (eg, 1-5), and more preferably 1, 2 or 3.

其中，基团A、CPT、D’与上文第一方面中定义相同。Wherein, the groups A, CPT, and D' are the same as defined in the first aspect above.

第三方面The third aspect

在第三方面，本发明提供了本发明第一方面的制备方法中所述的小分子药物、含药连接子和/或其中间体(包括但不限于式A-I、I、I'、I-A、IA'、II、II-A、III、III'、III-A、III-A'、V、V-A、V-A-1、V-B、V-B-1、V-C、VI、VI-A、VI-A-1、VI-B、VI-B-1、VI-C)，优选地包括但不限于实施例中公开的化合物。In the third aspect, the present invention provides the small molecule drugs, drug-containing linkers and/or intermediates thereof (including but not limited to formula A-I, I, I', I-A, IA', II, II-A, III, III', III-A, III-A', V, V-A, V-A-1, V-B, V-B-1, V-C, VI, VI-A, VI-A-1, VI-B, VI-B-1, VI-C) described in the preparation method of the first aspect of the present invention, preferably including but not limited to the compounds disclosed in the embodiments.

第四方面The fourth aspect

本发明提供了药物组合物，其包含本发明第一至第三方面述及的小分子药物、含药连接子、其中间体和/或抗体药物偶联物或其药学上可接受的盐、立体异构体、溶剂化物或前药。The present invention provides a pharmaceutical composition comprising the small molecule drug, drug-containing linker, intermediate and/or antibody-drug conjugate described in the first to third aspects of the present invention or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.

在一些实施方案中，所述药物组合物为液体制剂或冻干制剂。In some embodiments, the pharmaceutical composition is a liquid preparation or a lyophilized preparation.

在一些实施方案中，所述药物组合物包含缓冲剂、稳定剂和表面活性剂。 In some embodiments, the pharmaceutical composition comprises a buffer, a stabilizer, and a surfactant.

第五方面本发明提供根据本发明的化合物或其药学上可接受的盐、立体异构体、溶剂化物或前药、抗体药物偶联物或药物组合物在制备用于治疗肿瘤的药物中的用途。 In a fifth aspect, the present invention provides use of a compound according to the present invention or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, an antibody-drug conjugate or a pharmaceutical composition thereof in the preparation of a medicament for treating a tumor.

优选地，所述肿瘤为癌症。优选地，所述肿瘤与肿瘤相关抗原例如HER2、B7H3、HER3、CD19、CD20、CD22、CD30、CD33、CD37、CD45、CD56、CD66e、CD70、CD74、CD73、CD79b、CD138、CD147、CD223、EpCAM、粘蛋白1(Mucin 1)、STEAP1、GPNMB、FGF2、FOLR1、EGFR、EGFRvIII、组织因子(Tissuefactor)、c-MET、FGFR、Nectin 4、AGS-16、鸟苷酸环化酶C(Guanylyl cyclase C)、间皮素(Mesothelin)、SLC44A4、PSMA、EphA2、AGS-5、GPC-3、c-KIT、RoR1、PD-L1、CD27L、5T4、Mucin16、NaPi2b、STEAP、SLITRK6、ETBR、BCMA、Trop-2、CEACAM5、SC-16、SLC39A6、Delta-like protein3、Claudin 18.2阳性或高表达相关。Preferably, the tumor is cancer. Preferably, the tumor is associated with tumor-associated antigens such as HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor, c-MET, FGFR, Nectin 4, AG It is associated with positive or high expression of S-16, guanylate cyclase C, mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, and Claudin 18.2.

优选地，所述肿瘤为结肠直肠癌、膀胱癌、乳腺癌、胰腺癌、肝癌、卵巢癌、子宫内膜癌、输卵管癌、胃癌、前列腺癌、小细胞肺癌、非小细胞肺癌、食道鳞状细胞癌、头颈部鳞状细胞癌、黑色素瘤、白血病、淋巴瘤、神经胶质瘤、胶质母细胞瘤。Preferably, the tumor is colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, liver cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, melanoma, leukemia, lymphoma, glioma, glioblastoma.

第六方面本发明提供采用根据本发明的化合物或其药学上可接受的盐、立体异构体、溶剂化物或前药、抗体药物偶联物或药物组合物治疗肿瘤的方法，所述方法包括给有此需要的受试者施用所述化合物或抗体药物偶联物。 In a sixth aspect, the present invention provides a method for treating tumors using a compound according to the present invention or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, an antibody-drug conjugate or a pharmaceutical composition thereof, the method comprising administering the compound or antibody-drug conjugate to a subject in need thereof.

优选地，所述肿瘤为癌症。优选地，所述肿瘤与肿瘤相关抗原例如HER2、B7H3、HER3、CD19、CD20、CD22、CD30、CD33、CD37、CD45、CD56、CD66e、CD70、CD74、CD73、CD79b、CD138、CD147、CD223、EpCAM、粘蛋白1(Mucin 1)、STEAP1、GPNMB、FGF2、FOLR1、EGFR、EGFRvIII、组织因子(Tissuefactor)、c-MET、FGFR、Nectin 4、AGS-16、鸟苷酸环化酶C(Guanylyl cyclase C)、间皮素(Mesothelin)、SLC44A4、PSMA、EphA2、AGS-5、GPC-3、c-KIT、RoR1、PD-L1、CD27L、5T4、Mucin16、NaPi2b、STEAP、SLITRK6、ETBR、BCMA、Trop-2、CEACAM5、SC-16、SLC39A6、Delta-like protein3、Claudin 18.2阳性或高表达相关。Preferably, the tumor is cancer. Preferably, the tumor is associated with tumor-associated antigens such as HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor, c-MET, FGFR, Nectin 4, AG It is associated with positive or high expression of S-16, guanylate cyclase C, mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, and Claudin 18.2.

优选地，所述肿瘤为结肠直肠癌、膀胱癌、乳腺癌、胰腺癌、肝癌、卵巢癌、子宫内膜癌、输卵管癌、胃癌、前列腺癌、小细胞肺癌、非小细胞肺癌、食道鳞状细胞癌、头颈部鳞状细胞癌、黑色素瘤、白血病、淋巴瘤、神经胶质瘤、胶质母细胞瘤。Preferably, the tumor is colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, liver cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, melanoma, leukemia, lymphoma, glioma, glioblastoma.

优选地，所述受试者为哺乳动物，优选灵长类动物，更优选为人。Preferably, the subject is a mammal, preferably a primate, more preferably a human.

本发明的方法和/产品具有以下优势和/或益处：The method and/or product of the present invention has the following advantages and/or benefits:

(1)本发明的方法可提供高度均质的抗体偶联物分子，其DAR2主峰占比达到99％以上；(1) The method of the present invention can provide highly homogeneous antibody conjugate molecules, the main peak of which accounts for more than 99% of DAR2;

(2)本发明的制备方法步骤少，操作简单，利于工业放大生产，同时制备的产品杂质少、纯度高，明显提高用药安全性，也降低了生产成本；和/或(2) The preparation method of the present invention has few steps and is simple to operate, which is conducive to industrial scale-up production. At the same time, the prepared product has few impurities and high purity, which significantly improves the safety of drug use and reduces production costs; and/or

(3)通过本发明的偶联方法制备的抗肿瘤药具有优异的抗肿瘤效果和安全性,包括高的稳 定性和较小的副作用，扩大治疗窗口，提升治疗效果，减少毒性反应。(3) The anti-tumor drug prepared by the coupling method of the present invention has excellent anti-tumor effect and safety, including high stability. Qualitative and smaller side effects, expand the treatment window, improve treatment effects and reduce toxic reactions.

定义：definition:

在下文详细描述本发明前，应理解本发明不限于本文中描述的特定方法学、方案和试剂，因为这些可以变化。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本发明的范围，其仅会由所附权利要求书限制。除非另外定义，本文中使用的所有技术和科学术语与本发明所属领域中普通技术人员通常的理解具有相同的含义。Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, scheme and reagent described herein, because these can change. It should also be understood that the terms used in this article are only for describing specific embodiments, and are not intended to limit the scope of the present invention, which will only be limited by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs.

为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。要理解，本文所用的术语仅是为了描述具体的实施方案，并且不意欲是限制性的。To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

术语“抗体药物偶联物”或“ADC”是指生物活性化合物片段(小分子药物)与抗体或其抗原结合片段部分连接得到的物质。生物活性化合物片段与靶向部分通过连接子相连。在一些情况中，所述连接子在特定环境(例如胞内低pH值环境)中或特定作用(例如溶酶体蛋白酶的作用)下能够断裂，从而使生物活性化合物片段与抗体或其抗原结合片段分离。在本公开的部分实施方案中，所述连接子包含可切割或不可切割的单元，例如肽或二硫键。在本公开的部分实施方案中，生物活性化合物片段与抗体或其抗原结合片段直接通过共价键相连，所述共价键在特定环境或作用下能够断裂，从而使生物活性化合物片段与抗体或其抗原结合片段部分分离。The term "antibody drug conjugate" or "ADC" refers to a substance obtained by connecting a bioactive compound fragment (small molecule drug) to an antibody or its antigen-binding fragment. The bioactive compound fragment is connected to the targeting portion through a linker. In some cases, the linker can be broken in a specific environment (e.g., a low pH environment in the cell) or under a specific action (e.g., the action of a lysosomal protease), thereby separating the bioactive compound fragment from the antibody or its antigen-binding fragment. In some embodiments of the present disclosure, the linker comprises a cleavable or non-cleavable unit, such as a peptide or a disulfide bond. In some embodiments of the present disclosure, the bioactive compound fragment is directly connected to the antibody or its antigen-binding fragment through a covalent bond, and the covalent bond can be broken under a specific environment or action, thereby separating the bioactive compound fragment from the antibody or its antigen-binding fragment.

术语“连接子”是指将生物活性化合物片段(小分子药物)与抗体部分连接起来的片段。在这点上，连接子在连接至抗体或其抗原结合片段之前(即连接子前体)具有可与抗体或其抗原结合片段的官能团形成键的官能团。The term "linker" refers to a fragment that connects a biologically active compound fragment (small molecule drug) to an antibody portion. In this regard, the linker has a functional group that can form a bond with a functional group of the antibody or its antigen-binding fragment before being attached to the antibody or its antigen-binding fragment (i.e., a linker precursor).

术语“小分子药物”是指低分子量的能够调节生物过程的化合物。“小分子”被定义为分子量小于10kD、通常小于2kD和优选小于1kD的分子。小分子包括但不限于无机分子、有机分子、含无机组分的有机分子、含放射性原子的分子、合成分子、肽模拟物和抗体模拟物。作为治疗剂，小分子可以比大分子更能透过细胞、对降解更不易感和更不易于引发免疫应答。小分子药物包括但不限于细胞毒性剂，例如喜树碱类化合物、加利车霉素(calicheamicin)、美登木素生物碱(maytansinoids)、多拉司他汀(dolastatin)、澳瑞他汀类化合物和单端孢霉素(trichothecene)。优选地，所述美登木素衍生物选自DM1、DM3和DM4；所述澳瑞他汀类化合物选自本发明实施方案中提及的澳瑞他汀类化合物，包括但不限于MMAE和MMAF；所述喜树碱类化合物选自喜树碱、10-羟基喜树碱、依喜替康、SN-38和托泊替康以及本发明实施方案中提及的喜树碱类化合物。The term "small molecule drug" refers to a low molecular weight compound that can regulate biological processes. "Small molecule" is defined as a molecule with a molecular weight less than 10kD, usually less than 2kD and preferably less than 10kD. Small molecules include but are not limited to inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics and antibody mimics. As therapeutic agents, small molecules can be more permeable to cells, less susceptible to degradation and less prone to eliciting immune responses than macromolecules. Small molecule drugs include but are not limited to cytotoxic agents, such as camptothecin compounds, calicheamicin (calicheamicin), maytansinoids (maytansinoids), dolastatin (dolastatin), auristatin compounds and trichothecene (trichothecene). Preferably, the maytansinoid derivative is selected from DM1, DM3 and DM4; the auristatin compound is selected from the auristatin compounds mentioned in the embodiments of the present invention, including but not limited to MMAE and MMAF; the camptothecin compound is selected from camptothecin, 10-hydroxycamptothecin, exitecan, SN-38 and topotecan and the camptothecin compounds mentioned in the embodiments of the present invention.

本公开中，当小分子药物为ADC或含药连接子的一部分时，其指来自小分子药物的结构片段，例如小分子药物的羟基或氨基与连接子键合形成的结构片段，在肿瘤组织间或肿瘤细胞内连接子裂解/降解/酶切后，能够形成具有生物活性的药物(例如小分子细胞毒药物，所述药物包括其失去一个原子或原子团后的基团)或其衍生物(例如其前体)的部分(片段或基 团)。为了避免歧义，“小分子药物”并非仅指已获得医药监管部门审批的“药品”，还包括临床中，或者研发和学术研究中任何有潜在治疗生物活性的化合物。In the present disclosure, when a small molecule drug is a part of an ADC or a drug-containing linker, it refers to a structural fragment from a small molecule drug, such as a structural fragment formed by bonding a hydroxyl group or an amino group of a small molecule drug to a linker, which can form a part (fragment or radical) of a biologically active drug (such as a small molecule cytotoxic drug, wherein the drug includes a group after losing an atom or a group of atoms) or a derivative (such as a precursor thereof) after the linker is cleaved/degraded/enzymatically cleaved between tumor tissues or in tumor cells. To avoid ambiguity, “small molecule drugs” do not only refer to “drugs” that have been approved by the pharmaceutical regulatory authorities, but also include any compounds with potential therapeutic biological activity in clinical practice, or in R&D and academic research.

术语“含药连接子”是指生物活性化合物片段(小分子药物)与连接子连接形成的化合物。在本文中有时也称为“药物连接子偶联物”。The term "drug-containing linker" refers to a compound formed by linking a bioactive compound fragment (small molecule drug) to a linker, which is sometimes referred to herein as a "drug-linker conjugate".

金属离子螯合剂是指通过其与金属离子的强结合作用，将金属离子包合到螯合剂内部，变成稳定的、分子量更大的化合物，从而阻止金属离子起作用的化合物。金属离子螯合剂包括但不限于乙二胺四乙酸、Metal ion chelators are compounds that, through their strong binding with metal ions, encapsulate metal ions into the interior of the chelator, turning them into stable compounds with larger molecular weight, thereby preventing the metal ions from taking effect. Metal ion chelators include but are not limited to ethylenediaminetetraacetic acid,

术语“药物组合物”指这样的组合物，其以允许包含在其中的活性成分以生物学活性有效的形式存在，并且不包含对施用所述组合物的受试者具有不可接受的毒性的另外的成分。The term "pharmaceutical composition" refers to a composition that is in a form that permits the active ingredient contained therein to be effective for biological activity, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

术语“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性。The terms "treat," ...

术语“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。在一些实施方式中，具有癌症家族病史的受试者是预防性方案的候选。通常，在癌症的背景中，术语“预防”是指在癌症的病征或症状发生前，特别是在具有癌症风险的受试者中发生前的药物施用。The term "prevention" includes inhibition of the occurrence or development of a disease or disorder or symptoms of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for preventive regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the signs or symptoms of cancer occur, particularly in a subject at risk for cancer.

术语“烷基”是指直链、支链完全饱和的烃基，优选C1-C10，更优选C1-C8、C1-C6，或者C1-C4烷基。烷基实例是甲基、乙基、丙基、异丙基、正丁基、仲丁基、异丁基、叔丁基、正戊基、正己基、正庚基、正辛基、正壬基或正癸基等等。The term "alkyl" refers to a straight chain, branched, fully saturated hydrocarbon group, preferably C1 - C10 , more preferably C1 - C8 , C1 - C6 , or C1 - C4 alkyl. Examples of alkyl groups are methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl or n-decyl, and the like.

术语“亚烷基”是指如上所定义的烷基进一步去掉一个氢原子形成的二价基团。The term "alkylene" refers to a divalent group formed by removing a hydrogen atom from an alkyl group as defined above.

术语“环烷基”是指环状的烷基基团，优选C3-C15环烷基，例如C3-C8环烷基、C3-C6环烷基。环烷基实例是环丙基、环丁基、环戊基、环己基、环己基或环庚基等等。The term "cycloalkyl" refers to a cyclic alkyl group, preferably a C3 - C15 cycloalkyl, such as a C3 - C8 cycloalkyl, a C3 - C6 cycloalkyl. Examples of cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexyl or cycloheptyl and the like.

术语“亚环烷基”是指如上定义的环烷基进一步去掉一个氢原子形成的二价基团。The term "cycloalkylene" refers to a divalent group formed by removing a hydrogen atom from a cycloalkyl group as defined above.

术语“亚杂烷基”是指如上定义的烷基，其在链中包含1个或多个选自N、O或S的杂原子。应当理解，亚杂烷基不包括两个或更多个N、O或S杂原子直接相互连接形成的基团。优选地，亚杂烷基为C1-21(例如C1-10、C1-6)直链亚杂烷基，其包含1-11个(优选1-6个、1-3个)选自N、O或S的杂原子。The term "heteroalkylene" refers to an alkyl group as defined above, which contains one or more heteroatoms selected from N, O or S in the chain. It should be understood that heteroalkylene does not include a group formed by two or more N, O or S heteroatoms directly connected to each other. Preferably, heteroalkylene is a C1-21 (e.g., C1-10, C1-6) straight chain heteroalkylene, which contains 1-11 (preferably 1-6, 1-3) heteroatoms selected from N, O or S.

术语“杂环基”是指可以任选地被取代的含有一个或多个(例如1、2、3或4个)选自N、O、S、SO、SO2或P的杂原子的部分不饱和或完全不饱和的3-10元环状基团，优选3-10元环状基团，更优选3-6元环状基团；更加优选5-6元环状基团；所述杂环基含有1-9个碳原子，优选含有2-8个碳原子，更优选含有3-5个碳原子。杂环基的实例包括：1,3-二氧杂环戊烷基、1,3-二噁烷基、1,4-二噁烷基、咪唑啉基、吲哚啉基、吗啉基、吡啶酮、2-吡咯烷酮、哌嗪基、高哌嗪基、哌啶基、吡嗪基、四氢呋喃基、四氢吡喃基、四氢喹啉基、四氢噻吩基、噁烷基、氧硫杂环戊烷基、噻烷基等等。The term "heterocyclyl" refers to a partially unsaturated or fully unsaturated 3-10-membered cyclic group, preferably a 3-10-membered cyclic group, more preferably a 3-6-membered cyclic group, and more preferably a 5-6-membered cyclic group, which may be optionally substituted and contains one or more (e.g., 1, 2, 3 or 4 ) heteroatoms selected from N, O, S, SO, SO2 or P; the heterocyclyl group contains 1-9 carbon atoms, preferably 2-8 carbon atoms, and more preferably 3-5 carbon atoms. Examples of heterocyclyl groups include: 1,3-dioxolanyl, 1,3-dioxanyl, 1,4-dioxanyl, imidazolinyl, indolinyl, morpholinyl, pyridone, 2-pyrrolidone, piperazinyl, homopiperazinyl, piperidinyl, pyrazinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydroquinolinyl, tetrahydrothienyl, oxanyl, oxathiolanyl, thianyl, and the like.

“化学键”是指连接两侧基团的键，例如对于基团-A-L2-D，当L2为化学键时，所述基团可写作-A-D。"Chemical bond" refers to a bond connecting two groups on both sides. For example, for the group -AL 2 -D, when L 2 is a chemical bond, the group can be written as -AD.

术语“卤素”是指F、Cl、Br、I。The term "halogen" refers to F, Cl, Br, I.

术语“还原剂”是指能够选择性还原二硫键的试剂。还原剂选自但不限于三(2-羧乙基)膦 (TCEP)(包括三(2-羧乙基)膦盐酸盐)、二硫苏糖醇(DTT)、巯基乙胺、巯基乙醇及其组合。The term "reducing agent" refers to an agent capable of selectively reducing disulfide bonds. The reducing agent is selected from but not limited to tris(2-carboxyethyl)phosphine (TCEP) (including tris(2-carboxyethyl)phosphine hydrochloride), dithiothreitol (DTT), mercaptoethylamine, mercaptoethanol, and combinations thereof.

术语“抗体摩尔当量”是指相对于抗体而言的摩尔当量。The term "antibody molar equivalent" refers to the molar equivalent relative to the antibody.

应当理解，对于本申请的通式结构中的二价基团，所列举的基团以所显示的方向与两侧基团相连接，除非如此进行连接得到的结构不合理或偏离本发明、或与上下文矛盾。例如当L2为时，L2左侧的氨基与A连接，L2右侧的羰基与小分子药物(D)连接。It should be understood that for the divalent groups in the general structure of the present application, the listed groups are connected to the two side groups in the direction shown, unless the structure obtained by such connection is unreasonable or deviates from the present invention, or contradicts the context. For example, when L 2 is When , the amino group on the left side of L2 is connected to A, and the carbonyl group on the right side of L2 is connected to the small molecule drug (D).

术语“药物与抗体比率”或“DAR”是指偶联于本文所述的抗体部分(mAb)的小分子药物部分(D)与抗体部分的数量比例。ADC的DAR可以在1到10的范围内，但是取决于抗体上的连接位点的数量，更高的负载也是可能的。提及负载到单个抗体上的药物的数量时，可以使用术语DAR。DAR也可被计算为产品中分子群体的平均DAR，即通过检测方法(例如通过常规方法如质谱法、ELISA测定、电泳和/或HPLC)测得的产品中偶联于本文所述的mAb部分的小分子药物部分(D)与Ab部分的总体比例(摩尔比例)，此DAR在文中称为平均DAR。在一些实施方案中，本发明的抗体药物偶联物的平均DAR值是1.0-10.0，例如4.0-10.0、5.0-9.0、6.0-8.0、1.0-8.0，2.0-6.0，例如1.0、1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8.0、7.9、8、8.1、8.2、8.3、8.4、8.5、8.6、8.7、8.8、8.9、9.0、9.1、9.2、9.3、9.4、9.5、9.6、9.7、9.8、9.9和10.0，以这些数值中的两个作为端点的范围。The term "drug to antibody ratio" or "DAR" refers to the ratio of the number of small molecule drug moieties (D) coupled to the antibody moiety (mAb) described herein to the antibody moiety. The DAR of an ADC can be in the range of 1 to 10, but higher loadings are possible depending on the number of attachment sites on the antibody. The term DAR can be used when referring to the number of drugs loaded onto a single antibody. The DAR can also be calculated as the average DAR of a population of molecules in a product, i.e., the overall ratio (molar ratio) of the small molecule drug moiety (D) coupled to the mAb moiety described herein to the Ab moiety in a product measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and/or HPLC), which DAR is referred to herein as the average DAR. In some embodiments, the average DAR value of the antibody drug conjugate of the invention is 1.0-10.0, such as 4.0-10.0, 5.0-9.0, 6.0-8.0, 1.0-8.0, 2.0-6.0, such as 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6. 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9,

术语“DAR2-ADC”是指DAR为2的ADC，即抗体连接有2个小分子药物的ADC。The term “DAR2-ADC” refers to an ADC with a DAR of 2, i.e., an ADC in which an antibody is linked to two small molecule drugs.

术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小10％的下限和比指定数字数值大10％的上限的范围内的数字数值。The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 10% less than the specified numerical value and an upper limit that is 10% greater than the specified numerical value.

如本文所用，术语“和/或”意指可选项中的任一项或可选项的两项或多项。As used herein, the term "and/or" means any one of the alternatives or two or more of the alternatives.

如本文所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组合的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。As used herein, the term "comprising" or "including" means including the elements, integers or steps described, but does not exclude any other elements, integers or steps. In this article, when the term "comprising" or "including" is used, unless otherwise specified, it also covers the combination of the elements, integers or steps described. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

具体实施方式DETAILED DESCRIPTION

以下通过具体实施方式的描述对本披露作进一步说明，但这并非是对本披露的限制。本领域技术人员根据本披露的教导，可以做出各种修改或改进，而不脱离本披露的基本思想和范围。所用仪器或设备未注明来源的，均为可以通过市购获得的产品。所用试剂未注明来源的，为可以通过市购获得的或通过常规方法合成的产品。 The disclosure is further described below by describing specific embodiments, but this is not a limitation of the disclosure. Those skilled in the art can make various modifications or improvements based on the teachings of the disclosure without departing from the basic idea and scope of the disclosure. The instruments or equipment used without indicating the source are all products that can be obtained commercially. The reagents used without indicating the source are products that can be obtained commercially or synthesized by conventional methods.

本文中的缩写具有以下含义，未提供含义的缩写具有本领域通常理解的含义：The abbreviations herein have the following meanings, and abbreviations without meanings provided have the meanings commonly understood in the art:

缩写  名称Abbreviation Name

KHMDS  六甲基二硅基氨基钾KHMDS  Potassium hexamethyldisilazide

NFSI  N-氟代双苯磺酰亚胺NFSI  N-Fluorobis(benzenesulfonyl)imide

DIPEA  N,N-二异丙基乙胺DIPEA  N,N-Diisopropylethylamine

PE  石油醚PE  Petroleum ether

EA  乙酸乙酯EA  Ethyl acetate

eq  当量eq  equivalent

MeOH  甲醇MeOH  Methanol

DMF  二甲基甲酰胺DMF  Dimethylformamide

PB  磷酸氢二钠和磷酸二氢钠缓冲溶液PB  Disodium hydrogen phosphate and sodium dihydrogen phosphate buffer solution

IPA  异丙醇IPA  Isopropyl alcohol

MTS  Abcam,货号ab223881MTS  Abcam, Catalog Number ab223881

实施例1小分子的合成Example 1 Synthesis of small molecules

实施例1.1化合物MWC-1的合成
 Example 1.1 Synthesis of Compound MWC-1

按照WO2020200880A1专利方法，以B-1为中间体，制备得到MWC-1，为黄色固体，LC-MS(ESI)：[M+1]+＝404According to the patent method of WO2020200880A1, MWC-1 was prepared using B-1 as an intermediate. It was a yellow solid. LC-MS (ESI): [M+1]+=404

实施例1.2化合物3的合成
 Example 1.2 Synthesis of Compound 3

步骤1Step 1

于500ml三口烧瓶中，氮气保护下，将化合物B(2.45g，7.7mmol，1eq)溶于四氢呋喃(200ml)中，用干冰-丙酮浴将反应液冷却至-70～-60℃，慢慢滴加KHMDS(1M，31ml，31.0mmol，4eq)，滴毕，于-70～-60℃搅拌10分钟；滴加NFSI的四氢呋喃溶液(将NFSI(7.35g，23mmol，3eq)溶于四氢呋喃(70ml))，滴毕，于-70～-60℃搅拌10分钟，自然升温至20～30℃并搅3小时；加入饱和氯化铵水溶液(80ml)淬灭反应，反应液用乙酸乙酯(150ml*3)萃取，合并有机相，饱和食盐水洗涤，浓缩至干得粗品，经柱层析纯化，洗脱剂为石油醚：乙酸乙酯＝1：0-1：3，得化合物3a，黄色固体，0.85g，收率31.2％。LC-MS(ESI)：[M+1]+＝355.8In a 500 ml three-necked flask, under nitrogen protection, compound B (2.45 g, 7.7 mmol, 1 eq) was dissolved in tetrahydrofuran (200 ml), and the reaction solution was cooled to -70 to -60 ° C with a dry ice-acetone bath, and KHMDS (1 M, 31 ml, 31.0 mmol, 4 eq) was slowly added dropwise. After the addition, the mixture was stirred at -70 to -60 ° C for 10 minutes; a tetrahydrofuran solution of NFSI (NFSI (7.35 g, 23 mmol, 3 eq) was added dropwise. ) was dissolved in tetrahydrofuran (70 ml), and after the addition, the mixture was stirred at -70 to -60 °C for 10 minutes, and the temperature was naturally raised to 20 to 30 °C and stirred for 3 hours; a saturated aqueous solution of ammonium chloride (80 ml) was added to quench the reaction, and the reaction solution was extracted with ethyl acetate (150 ml*3), the organic phases were combined, washed with saturated brine, and concentrated to dryness to obtain a crude product, which was purified by column chromatography with an eluent of petroleum ether: ethyl acetate = 1:0-1:3 to obtain compound 3a as a yellow solid, 0.85 g, with a yield of 31.2%. LC-MS (ESI): [M+1]+ = 355.8

1H NMR(400MHz,CDCl3)δ11.49(s,1H),8.64(d,J＝9.2Hz,1H),7.74(d,J＝8.5Hz,1H),6.06(s,1H),3.01(t,J＝6.4Hz,2H),2.55–2.39(m,2H),2.19(s,3H),1.44(s,9H) 1H NMR (400MHz, CDCl3) δ11.49(s,1H),8.64(d,J＝9.2Hz,1H),7.74(d,J＝8.5Hz,1H) ,6.06(s,1H),3.01(t,J＝6.4Hz,2H),2.55–2.39(m,2H),2.19(s,3H),1.44(s,9H)

步骤2Step 2

于100ml单口烧瓶中，将化合物3a(0.85g，2.4mmol)，与6N盐酸(30ml)混合，加热回流反应4小时。停止加热，待反应液冷却至室温后，用碳酸氢钠调节pH至7～8，用二氯甲烷(30ml*3)萃取，合并有机相，饱和食盐水洗涤，浓缩至干得粗品，经柱层析纯化，洗脱剂为石油醚：乙酸乙酯＝1：0～1：3，得化合物3b，棕色固体0.25g，收率49.1％。LC-MS(ESI)：[M+1]+＝213.9In a 100ml single-necked flask, compound 3a (0.85g, 2.4mmol) was mixed with 6N hydrochloric acid (30ml) and heated under reflux for 4 hours. The heating was stopped, and after the reaction solution was cooled to room temperature, the pH was adjusted to 7-8 with sodium bicarbonate, extracted with dichloromethane (30ml*3), the organic phases were combined, washed with saturated brine, and concentrated to dryness to obtain a crude product, which was purified by column chromatography with an eluent of petroleum ether: ethyl acetate = 1:0-1:3 to obtain compound 3b, 0.25g of brown solid, with a yield of 49.1%. LC-MS (ESI): [M+1]+ = 213.9

步骤3Step 3

于50ml单口反应瓶中，氮气保护下，将化合物3b(120mg，0.59mmol，1eq)、CDE三环化合物(180mg，0.68mmol，1.2eq)和PPTS(35mg,0.14mmol，0.25eq)混合于15ml乙酸中，加热回流反应3～4小时；减压蒸馏，蒸除大部分乙酸，残留物经柱层析粗分离，洗脱剂为二氯甲烷：甲醇＝15:1，得到粗品80mg，再经制备液相分离纯化，得喜树碱类化合物3，为土黄色固体，LC-MS(ESI)：[M+1]+＝440.8In a 50 ml single-mouth reaction bottle, under nitrogen protection, compound 3b (120 mg, 0.59 mmol, 1 eq), CDE tricyclic compound (180 mg, 0.68 mmol, 1.2 eq) and PPTS (35 mg, 0.14 mmol, 0.25 eq) were mixed in 15 ml of acetic acid and heated under reflux for 3 to 4 hours; most of the acetic acid was distilled off under reduced pressure, and the residue was crudely separated by column chromatography with an eluent of dichloromethane:methanol=15:1 to obtain 80 mg of a crude product, which was then separated and purified by preparative liquid phase separation to obtain camptothecin compound 3 as a khaki solid. LC-MS (ESI): [M+1]+=440.8

1H NMR(400MHz,Acetone)δ7.89(d,J＝9.1Hz,1H),7.48(d,J＝9.1Hz,1H),7.37(s,1H),7.31(d,J＝8.9Hz,0H),5.59–5.26(m,5H),3.11–3.05(m,4H),2.73–2.59(m,3H),1.98(dt,J＝14.0,7.0Hz,1H),1.05–0.98(m,3H) 1H NMR (400MHz, Acetone) δ7.89(d,J＝9.1Hz,1H),7.48(d,J＝9.1Hz,1H),7.37(s,1H),7.31(d,J＝8.9Hz,0H) , 5.59–5.26(m,5H),3.11–3.05(m,4H),2.73–2.59(m,3H),1.98(dt,J＝14.0,7.0Hz,1H),1.05–0.98(m,3H)

1H NMR(400MHz,DMSO)δ7.87(d,J＝9.1Hz,0H),7.41(d,J＝9.1Hz,0H),7.21(s,0H),6.50(s,0H),6.15(s,1H),5.36(d,J＝46.6Hz,1H),2.93(t,J＝6.5Hz,1H),2.66–2.54(m,1H),1.97–1.68(m,3H),1.24(s,1H),0.88(t,J＝7.3Hz,3H) 1H NMR (400MHz, DMSO) δ7.87(d,J＝9.1Hz,0H),7.41(d,J＝9.1Hz,0H),7.21(s,0H),6.50(s,0H),6.15(s, 1H),5.36(d ,J＝46.6Hz,1H),2.93(t,J＝6.5Hz,1H),2.66–2.54(m,1H),1.97–1.68(m,3H),1.24(s,1H),0.88(t, J＝7.3Hz,3H)

化合物B的合成
Synthesis of compound B

化合物B-1按照文献Jornal of Medcinal Chemstry,1998,41(13),2308-2318方法合成，化合物B-1为棕色固体，LC-MS(ESI)：[M+1]+＝177Compound B-1 was synthesized according to the method of Jornal of Medcinal Chemistry, 1998, 41(13), 2308-2318. Compound B-1 was a brown solid. LC-MS (ESI): [M+1]+=177

1H NMR(400MHz,DMSO-d6)δ6.76(d,J＝8.7Hz,1H),6.67(bs,2H),6.42(d,J＝8.7Hz,1H),4.20(bs,2H),2.56(t,J＝6.2Hz,2H),2.48–2.41(m,2H),1.96–1.84(m,2H) 1H NMR(400MHz,DMSO-d6)δ6.76(d,J＝8.7Hz,1H),6.67(bs,2H),6.42(d,J＝8.7Hz, 1H),4.20(bs,2H),2.56(t,J＝6.2Hz,2H),2.48–2.41(m,2H),1.96–1.84(m,2H)

于250ml三口烧瓶中，将化合物B-1(2.18g,124mmol，1eq)溶于四氢呋喃(100ml)中，加入Boc酸酐(8.2g，376mmol，3eq)，于40～45℃搅拌反应5小时；将反应液直接浓缩干，经柱层析纯化，洗脱剂为石油醚：乙酸乙酯＝1：0～1：3，得化合物B-2，黄色固体3.0g，收率90％。In a 250ml three-necked flask, compound B-1 (2.18g, 124mmol, 1eq) was dissolved in tetrahydrofuran (100ml), and Boc anhydride (8.2g, 376mmol, 3eq) was added. The mixture was stirred at 40-45°C for 5 hours. The reaction solution was directly concentrated to dryness and purified by column chromatography with the eluent being petroleum ether: ethyl acetate = 1:0-1:3 to obtain compound B-2 as a yellow solid (3.0g). The yield was 90%.

1H NMR(400MHz,CDCl3)δ7.25(s,1H),6.41(t,J＝10.2Hz,3H),5.90(s,1H),2.73(t,J＝6.2Hz,2H),2.61–2.47(m,2H),2.02–1.86(m,3H),1.49–1.36(m,9H) 1H NMR (400MHz, CDCl3) δ7.25 (s, 1H), 6.41 (t, J = 10.2Hz, 3H), 5.90 (s, 1H), 2.7 3(t,J＝6.2Hz,2H),2.61–2.47(m,2H),2.02–1.86(m,3H),1.49–1.36(m,9H)

于250ml三口烧瓶中，将化合物B-2(3.0g，10.8mmol，1eq)和DIPEA(3.5g，27.1mmol，1.5eq)混于二氯甲烷(125ml)中，用冰盐浴将反应液冷却至-5～0℃，滴加乙酰氯(1.3g，16.5mmol，2eq)，滴毕，撤除冰盐浴，自然升温，于室温下搅拌反应4小时；将反应液浓缩干，残留物经柱层析纯化，洗脱剂为石油醚：乙酸乙酯＝1：0～1：3，得化合物B，淡黄色固体，3.48g，收率100％。In a 250ml three-necked flask, compound B-2 (3.0g, 10.8mmol, 1eq) and DIPEA (3.5g, 27.1mmol, 1.5eq) were mixed in dichloromethane (125ml), and the reaction solution was cooled to -5~0°C with an ice-salt bath, and acetyl chloride (1.3g, 16.5mmol, 2eq) was added dropwise. After the addition was completed, the ice-salt bath was removed, the temperature was naturally raised, and the reaction was stirred at room temperature for 4 hours; the reaction solution was concentrated to dryness, and the residue was purified by column chromatography with the eluent being petroleum ether: ethyl acetate = 1:0~1:3 to obtain compound B as a light yellow solid, 3.48g, with a yield of 100%.

1H NMR(400MHz,CDCl3)δ12.01(s,1H),8.55(d,J＝9.1Hz,1H),7.57(t,J＝36.2Hz,1H),6.09(s,1H),2.80(t,J＝6.2Hz,2H),2.65–2.58(m,2H),2.15(s,3H),2.07–1.98(m,2H),1.44(s,9H) 1H NMR (400MHz, CDCl3) δ12.01(s,1H),8.55(d,J＝9.1Hz,1H),7.57(t,J＝36.2Hz,1H),6.09(s, 1H),2.80(t,J＝6.2Hz,2H),2.65–2.58(m,2H),2.15(s,3H),2.07–1.98(m,2H),1.44(s,9H)

实施例2新型含药连接子的合成Example 2 Synthesis of novel drug-containing linkers

实施例2.1含药连接子BL20E(C-3)的合成
 Example 2.1 Synthesis of drug-containing linker BL20E (C-3)

BL20E化合物按照WO2018/095422A1公开的方法，由BL接头化合物与 VC-PABC-MMAE缩合得到，合成路线如下：
The BL20E compound is prepared by the method disclosed in WO2018/095422A1 and the BL linker compound VC-PABC-MMAE is obtained by condensation, and the synthetic route is as follows:

BL20E化合物为黄色固体，LC-MS(ESI):[M+1]+为1963(C-3)。The BL20E compound was a yellow solid. LC-MS (ESI): [M+1]+ was 1963 (C-3).

实施例2.2含药连接子MWD-L1的合成
 Example 2.2 Synthesis of drug-containing linker MWD-L1

MWD-L1化合物由BL接头化合物与GGFG-Dxd合成得到，合成路线如下：
The MWD-L1 compound is synthesized from the BL linker compound and GGFG-Dxd. The synthesis route is as follows:

BL接头化合物(857mg，1mmol)、GGFG-Dxd[按照专利公开文件(US20190151328A1)中所述方法合成得到](840mg，1mmol，1eq)、DIPEA(323mg，2.5mmol，2.5eq)，HATU(570mg，1.5mmol，1.5eq)，溶于30ml DCM中，搅拌反应2h。将反应液冷却至5～10℃，加入1N盐酸(20ml)，搅拌0.5h。分液，水相用DCM(30ml*2)萃取，合并有机相。有机相用饱和食盐水洗涤，无水硫酸钠干燥，抽滤，旋干。柱层析纯化，洗脱剂：DCM：MeOH＝50：1～10：1，得含药接头MWD-L1为黄色固体，500mg，收率29.8％，LC-MS(ESI):M+1＝1681，(M+1)/2＝840.9。BL linker compound (857 mg, 1 mmol), GGFG-Dxd [synthesized according to the method described in the patent disclosure document (US20190151328A1)] (840 mg, 1 mmol, 1 eq), DIPEA (323 mg, 2.5 mmol, 2.5 eq), HATU (570 mg, 1.5 mmol, 1.5 eq), dissolved in 30 ml DCM, stirred for 2 h. The reaction solution was cooled to 5-10 ° C, 1N hydrochloric acid (20 ml) was added, and stirred for 0.5 h. The liquid was separated, the aqueous phase was extracted with DCM (30 ml * 2), and the organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and dried. Purification by column chromatography, eluent: DCM:MeOH=50:1-10:1, to obtain drug-containing linker MWD-L1 as a yellow solid, 500 mg, yield 29.8%, LC-MS (ESI): M+1=1681, (M+1)/2=840.9.

实施例2.3含药连接子MWC-L2的合成
 Example 2.3 Synthesis of drug-containing linker MWC-L2

步骤1Step 1

Boc-Val-Ala-OH(288mg，1mmol)、A1化合物[根据文献Journal of Medicinal Chemistry,1998,vol.41,#13,p.2308-2318方法合成得到](176mg，1mmol，1eq)、DIPEA(322mg，2.5mmol，2.5eq)、HATU(456mg，1.2mmol，1.2eq)溶于30ml DCM中，搅拌反应2h。反应液旋干，柱层析纯化，洗脱剂：PE：EA＝10：1～5：1，定量地得到中间体2-1，为灰绿色固体(450mg)。Boc-Val-Ala-OH (288 mg, 1 mmol), A1 compound [synthesized according to the method of Journal of Medicinal Chemistry, 1998, vol. 41, #13, p. 2308-2318] (176 mg, 1 mmol, 1 eq), DIPEA (322 mg, 2.5 mmol, 2.5 eq), HATU (456 mg, 1.2 mmol, 1.2 eq) were dissolved in 30 ml DCM and stirred for 2 h. The reaction solution was dried and purified by column chromatography, eluent: PE: EA = 10: 1 to 5: 1, and intermediate 2-1 was quantitatively obtained as a gray-green solid (450 mg).

步骤2Step 2

中间体2-1(450mg，1mmol)、CDE三环化合物(263mg，1mmol，1eq)、对甲苯磺酸吡啶盐(PPTS)(251mg，1mmol，1eq)悬浮于30ml甲苯中，加热回流反应2小时。停止加热，待反应液冷却后，收集固体沉淀物即为中间体2-2粗品，棕色固体750mg。LC-MS(ESI):M+1＝574。不经纯化直接下一步反应。Intermediate 2-1 (450 mg, 1 mmol), CDE tricyclic compound (263 mg, 1 mmol, 1 eq), p-toluenesulfonic acid pyridinium salt (PPTS) (251 mg, 1 mmol, 1 eq) were suspended in 30 ml of toluene and heated under reflux for 2 hours. Heating was stopped, and after the reaction solution was cooled, the solid precipitate was collected as the crude intermediate 2-2, 750 mg of brown solid. LC-MS (ESI): M+1=574. The product was directly used for the next step without purification.

步骤3Step 3

BL接头化合物(857mg，1mmol)、HoSu(138mg，1.2mmol，1.2eq)、DCC(310mg，1.5mmol，1.5eq)溶于30ml DCM中，室温搅拌3h，反应液抽滤，滤液即为A-Osu的DCM溶液。将滤液加入到中间体2-2粗品、DIPEA(323mg，2.5mmol，2.5eq)、DCM(30ml)的混合溶液中，搅拌反应3h。将反应液冷却至10℃下，加入1N盐酸(20ml)，搅拌0.5h。分液，水相用DCM(30ml*2)萃取，合并有机相。有机相用饱和食盐水洗涤，无水硫酸钠干燥，抽滤，旋干。柱层析纯化，洗脱剂：DCM：MeOH＝50：1～10：1，得连接子MWC-L2为橘红色固体，325mg，收率23.0％，LC-MS(ESI):M+1＝1414，(M+1)/2＝707 BL linker compound (857mg, 1mmol), HoSu (138mg, 1.2mmol, 1.2eq), DCC (310mg, 1.5mmol, 1.5eq) were dissolved in 30ml DCM, stirred at room temperature for 3h, and the reaction solution was filtered. The filtrate was the DCM solution of A-Osu. The filtrate was added to the mixed solution of the crude intermediate 2-2, DIPEA (323mg, 2.5mmol, 2.5eq) and DCM (30ml), and the reaction was stirred for 3h. The reaction solution was cooled to 10°C, 1N hydrochloric acid (20ml) was added, and stirred for 0.5h. The liquid was separated, the aqueous phase was extracted with DCM (30ml*2), and the organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and spin-dried. Column chromatography purification, eluent: DCM: MeOH = 50: 1 to 10: 1, the linker MWC-L2 was obtained as an orange-red solid, 325 mg, yield 23.0%, LC-MS (ESI): M+1 = 1414, (M+1)/2 = 707

实施例2.4含药连接子MWC-L3的合成
 Example 2.4 Synthesis of drug-containing linker MWC-L3

步骤1Step 1

Boc-Gly-OH(175mg，1mmol)、A1化合物(176mg，1mmol，1eq)、DIPEA(322mg，2.5mmol，2.5eq)、HATU(456mg，1.2mmol，1.2eq)溶于30ml DCM中，搅拌反应2h。反应液旋干，柱层析纯化，洗脱剂：PE：EA＝10：1～5：1，定量地得到中间体3-1为淡绿色固体335mg。Boc-Gly-OH (175 mg, 1 mmol), A1 compound (176 mg, 1 mmol, 1 eq), DIPEA (322 mg, 2.5 mmol, 2.5 eq), HATU (456 mg, 1.2 mmol, 1.2 eq) were dissolved in 30 ml DCM and stirred for 2 h. The reaction solution was dried and purified by column chromatography, eluent: PE: EA = 10: 1 to 5: 1, and intermediate 3-1 was quantitatively obtained as a light green solid 335 mg.

步骤2Step 2

中间体3-1(335mg，1mmol)、CDE三环化合物(263mg，1mmol，1eq)、PPTS(251mg，1mmol，1eq)悬浮于30ml甲苯中，加热回流反应2小时。停止加热，待反应液冷却后，收集固体沉淀物即为中间体3-2粗品，棕色固体560mg。LC-MS(ESI):M+1＝461。不经纯化直接下一步反应。Intermediate 3-1 (335 mg, 1 mmol), CDE tricyclic compound (263 mg, 1 mmol, 1 eq), PPTS (251 mg, 1 mmol, 1 eq) were suspended in 30 ml of toluene and heated under reflux for 2 hours. Heating was stopped, and after the reaction solution was cooled, the solid precipitate was collected as the crude intermediate 3-2, 560 mg of brown solid. LC-MS (ESI): M+1=461. The product was directly used for the next step without purification.

步骤3Step 3

Fmoc-Gly-Gly-Phe-OH(501mg，1mmol)、中间体3-2粗品(560mg)、DIPEA(322mg，2.5mmol，2.5eq)、HATU(456mg，1.2mmol，1.2eq)溶于20ml DMF中，搅拌反应3h。加入1N盐酸(30ml)，搅拌0.5h。用DCM(30ml*2)萃取，合并有机相。有机相用饱和食盐水洗涤，无水硫酸钠干燥，抽滤，旋干。柱层析纯化，洗脱剂：DCM：MeOH＝50：1～10：1，得中间体3-3为棕色固体，350mg，LC-MS(ESI):M+1＝945Fmoc-Gly-Gly-Phe-OH (501 mg, 1 mmol), crude intermediate 3-2 (560 mg), DIPEA (322 mg, 2.5 mmol, 2.5 eq), HATU (456 mg, 1.2 mmol, 1.2 eq) were dissolved in 20 ml DMF and stirred for 3 h. 1N hydrochloric acid (30 ml) was added and stirred for 0.5 h. Extracted with DCM (30 ml*2) and the organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered and dried. Purified by column chromatography, eluent: DCM: MeOH = 50: 1 ~ 10: 1, intermediate 3-3 was obtained as a brown solid, 350 mg, LC-MS (ESI): M+1 = 945

步骤4Step 4

中间体3-3(350mg，0.37mmol)溶于甲醇(20ml)中，加入二乙胺(2ml)，搅拌反应3h。旋干反应液，得中间体3-4粗品，为棕色粘稠物，LC-M(ESI):M+1＝722，粗品不经纯化直接下一步。Intermediate 3-3 (350 mg, 0.37 mmol) was dissolved in methanol (20 ml), diethylamine (2 ml) was added, and the mixture was stirred for 3 h. The reaction solution was spin-dried to obtain a crude intermediate 3-4 as a brown viscous substance, LC-M (ESI): M+1=722, and the crude product was directly used for the next step without purification.

步骤5 Step 5

BL接头化合物(318mg，0.37mmol)、HoSu(51mg，0.44mmol，1.2eq)、DCC(114mg，0.56mmol，1.5eq)溶于30ml DCM中，室温搅拌3h，反应液抽滤。将滤液加入到中间体3-4粗品、DIPEA(120mg，0.93mmol，2.5eq)、DCM(30ml)的混合溶液中，搅拌反应3h。将反应液冷却至10℃下，加入1N盐酸(20ml)，搅拌0.5h。分液，水相用DCM(30ml*2)萃取，合并有机相。有机相用饱和食盐水洗涤，无水硫酸钠干燥，抽滤，旋干。柱层析纯化，洗脱剂：DCM：MeOH＝50：1～10：1，得连接子MWC-L3，为橘红色固体120mg，收率20.8％，LC-MS(ESI):M+1＝1562，(M+1)/2＝781BL linker compound (318 mg, 0.37 mmol), HoSu (51 mg, 0.44 mmol, 1.2 eq), DCC (114 mg, 0.56 mmol, 1.5 eq) were dissolved in 30 ml DCM, stirred at room temperature for 3 h, and the reaction solution was filtered. The filtrate was added to a mixed solution of the crude intermediate 3-4, DIPEA (120 mg, 0.93 mmol, 2.5 eq), and DCM (30 ml), and the reaction was stirred for 3 h. The reaction solution was cooled to 10 ° C, 1N hydrochloric acid (20 ml) was added, and stirred for 0.5 h. The liquid was separated, the aqueous phase was extracted with DCM (30 ml * 2), and the organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and spin-dried. Column chromatography purification, eluent: DCM: MeOH = 50: 1 to 10: 1, to obtain linker MWC-L3, an orange-red solid 120 mg, yield 20.8%, LC-MS (ESI): M+1 = 1562, (M+1)/2 = 781

实施例2.5含药连接子MWF-L6的合成
 Example 2.5 Synthesis of drug-containing linker MWF-L6

根据MWC-L2的合成方法，由化合物A7代替化合物A1制备得到MWF-L6，为橘红色固体，LC-MS(ESI):M+1＝1450。According to the synthesis method of MWC-L2, MWF-L6 was prepared by replacing compound A1 with compound A7. The product was an orange-red solid. LC-MS (ESI): M+1=1450.

其中化合物A7按如下方法合成得到
Compound A7 was synthesized as follows:

于250ml三口烧瓶中，将A1化合物(2.18g,124mmol，1eq)溶于四氢呋喃(100ml)中，加入Boc酸酐(8.2g，376mmol，3eq)，于40～45℃搅拌反应5小时；将反应液直接浓缩干，经柱层析纯化，洗脱剂为石油醚：乙酸乙酯＝1：0～1：3，得A7-1化合物，黄色固体3.0g，收率90％。In a 250 ml three-necked flask, compound A1 (2.18 g, 124 mmol, 1 eq) was dissolved in tetrahydrofuran (100 ml), and Boc anhydride (8.2 g, 376 mmol, 3 eq) was added. The mixture was stirred at 40-45 °C for 5 hours. The reaction solution was directly concentrated to dryness and purified by column chromatography with petroleum ether: ethyl acetate = 1:0-1:3 as the eluent to obtain compound A7-1 as a yellow solid (3.0 g). The yield was 90%.

1H NMR(400MHz,CDCl3)δ7.25(s,1H),6.41(t,J＝10.2Hz,3H),5.90(s,1H),2.73(t,J＝6.2Hz,2H),2.61–2.47(m,2H),2.02–1.86(m,3H),1.49–1.36(m,9H) 1H NMR (400MHz, CDCl3) δ7.25 (s, 1H), 6.41 (t, J = 10.2Hz, 3H), 5.90 (s, 1H), 2.7 3(t,J＝6.2Hz,2H),2.61–2.47(m,2H),2.02–1.86(m,3H),1.49–1.36(m,9H)

于250ml三口烧瓶中，将A7-1化合物(3.0g，10.8mmol，1eq)和DIPEA(3.5g，27.1mmol，1.5eq)混于二氯甲烷(125ml)中，用冰盐浴将反应液冷却至-5～0℃，滴加乙酰氯(1.3g，16.5mmol，2eq)，滴毕，撤除冰盐浴，自然升温，于室温下搅拌反应4小时；将反应液浓缩干，残留物经柱层析纯化，洗脱剂为石油醚：乙酸乙酯＝1：0～1：3，得A7-2化合物，淡黄 色固体，3.48g，收率100％。In a 250ml three-necked flask, compound A7-1 (3.0g, 10.8mmol, 1eq) and DIPEA (3.5g, 27.1mmol, 1.5eq) were mixed in dichloromethane (125ml), and the reaction solution was cooled to -5 to 0°C with an ice-salt bath, and acetyl chloride (1.3g, 16.5mmol, 2eq) was added dropwise. After the addition was completed, the ice-salt bath was removed, the temperature was naturally raised, and the reaction was stirred at room temperature for 4 hours; the reaction solution was concentrated to dryness, and the residue was purified by column chromatography with the eluent being petroleum ether: ethyl acetate = 1:0 to 1:3 to obtain compound A7-2, which was light yellow. Color solid, 3.48 g, yield 100%.

1H NMR(400MHz,CDCl3)δ12.01(s,1H),8.55(d,J＝9.1Hz,1H),7.57(t,J＝36.2Hz,1H),6.09(s,1H),2.80(t,J＝6.2Hz,2H),2.65–2.58(m,2H),2.15(s,3H),2.07–1.98(m,2H),1.44(s,9H) 1H NMR (400MHz, CDCl3) δ12.01(s,1H),8.55(d,J＝9.1Hz,1H),7.57(t,J＝36.2Hz,1H),6.09(s, 1H),2.80(t,J＝6.2Hz,2H),2.65–2.58(m,2H),2.15(s,3H),2.07–1.98(m,2H),1.44(s,9H)

于500ml三口烧瓶中，氮气保护下，将A7-2化合物(2.45g，7.7mmol，1eq)溶于四氢呋喃(200ml)中，用干冰-丙酮浴将反应液冷却至-70～-60℃，慢慢滴加KHMDS(1M，31ml，31.0mmol，4eq)，滴毕，于-70～-60℃搅拌10分钟；滴加NFSI的四氢呋喃溶液(将NFSI(7.35g，23mmol，3eq)溶于四氢呋喃(70ml))，滴毕，于-70～-60℃搅拌10分钟，自然升温至20～30℃并搅3小时；加入饱和氯化铵水溶液(80ml)淬灭反应，反应液用乙酸乙酯(150ml*3)萃取，合并有机相，饱和食盐水洗涤，浓缩至干得粗品，经柱层析纯化，洗脱剂为石油醚：乙酸乙酯＝1：0-1：3，得A7-3化合物，黄色固体，0.85g，收率31.2％。LC-MS(ESI)：[M+1]+＝355.8In a 500 ml three-necked flask, under nitrogen protection, compound A7-2 (2.45 g, 7.7 mmol, 1 eq) was dissolved in tetrahydrofuran (200 ml), and the reaction solution was cooled to -70 to -60 ° C with a dry ice-acetone bath, and KHMDS (1 M, 31 ml, 31.0 mmol, 4 eq) was slowly added dropwise. After the addition, the mixture was stirred at -70 to -60 ° C for 10 minutes; a tetrahydrofuran solution of NFSI (7.35 g, 23 mmol, 3 eq) was added dropwise. ) was dissolved in tetrahydrofuran (70 ml), and after the addition, the mixture was stirred at -70 to -60 °C for 10 minutes, and the temperature was naturally raised to 20 to 30 °C and stirred for 3 hours; a saturated aqueous solution of ammonium chloride (80 ml) was added to quench the reaction, and the reaction solution was extracted with ethyl acetate (150 ml*3), the organic phases were combined, washed with saturated brine, and concentrated to dryness to obtain a crude product, which was purified by column chromatography with an eluent of petroleum ether: ethyl acetate = 1:0-1:3 to obtain compound A7-3 as a yellow solid, 0.85 g, with a yield of 31.2%. LC-MS (ESI): [M+1]+ = 355.8

1H NMR(400MHz,CDCl3)δ11.49(s,1H),8.64(d,J＝9.2Hz,1H),7.74(d,J＝8.5Hz,1H),6.06(s,1H),3.01(t,J＝6.4Hz,2H),2.55–2.39(m,2H),2.19(s,3H),1.44(s,9H) 1H NMR (400MHz, CDCl3) δ11.49(s,1H),8.64(d,J＝9.2Hz,1H),7.74(d,J＝8.5Hz,1H) ,6.06(s,1H),3.01(t,J＝6.4Hz,2H),2.55–2.39(m,2H),2.19(s,3H),1.44(s,9H)

于100ml单口烧瓶中，将A7-3化合物(0.85g，2.4mmol)，与6N盐酸(30ml)混合，加热回流反应4小时。停止加热，待反应液冷却至室温后，用碳酸氢钠调节pH至7～8，用二氯甲烷(30ml*3)萃取，合并有机相，饱和食盐水洗涤，浓缩至干得粗品，经柱层析纯化，洗脱剂为石油醚：乙酸乙酯＝1：0～1：3，得A7化合物，棕色固体0.25g，收率49.1％。LC-MS(ESI)：[M+1]+＝213.9In a 100ml single-necked flask, compound A7-3 (0.85g, 2.4mmol) was mixed with 6N hydrochloric acid (30ml) and heated under reflux for 4 hours. The heating was stopped, and after the reaction solution was cooled to room temperature, the pH was adjusted to 7-8 with sodium bicarbonate, extracted with dichloromethane (30ml*3), the organic phases were combined, washed with saturated brine, and concentrated to dryness to obtain a crude product, which was purified by column chromatography with an eluent of petroleum ether: ethyl acetate = 1:0-1:3 to obtain compound A7, a brown solid of 0.25g, with a yield of 49.1%. LC-MS (ESI): [M+1]+ = 213.9

实施例2.6含药连接子DT-1的合成
 Example 2.6 Synthesis of drug-containing linker DT-1

根据CN114106088A的方法合成得到DT-1，为黄色固体，LC-MS(ESI)：[M+1]+＝1474.4DT-1 was synthesized according to the method of CN114106088A as a yellow solid, LC-MS (ESI): [M+1]+=1474.4

实施例2.7含药连接子DT-2的合成
 Example 2.7 Synthesis of drug-containing linker DT-2

根据BL20E化合物合成方法，由DT-2-A化合物代替BL接头化合物，合成得到DT-2，为淡黄色固体，LC-MS(ESI)：[M+1]+＝1339.7其中DT-2-A化合物按照文献Chem.Sci.,2022,13,8781–8790合成得到。According to the synthesis method of BL20E compound, DT-2-A compound was used to replace the BL linker compound to synthesize DT-2, which was a light yellow solid. LC-MS (ESI): [M+1]+=1339.7, wherein the DT-2-A compound was synthesized according to the literature Chem. Sci., 2022, 13, 8781–8790.

实施例2.8含药连接子DT-3的合成
 Example 2.8 Synthesis of drug-containing linker DT-3

根据WO2022253033A的方法合成得到DT-3，为淡黄色固体，LC-MS(ESI)：[M+1]+＝1631.5。DT-3 was synthesized according to the method of WO2022253033A as a light yellow solid, LC-MS (ESI): [M+1]+=1631.5.

实施例2.9含药连接子DT-4的合成
 Example 2.9 Synthesis of drug-containing linker DT-4

根据CN103933575B的方法合成得到DT-4，为淡黄色固体，LC-MS(ESI)：[M+1]+＝1414.7DT-4 was synthesized according to the method of CN103933575B as a light yellow solid. LC-MS (ESI): [M+1]+=1414.7

实施例2.10含药连接子DT-5的合成
 Example 2.10 Synthesis of drug-containing linker DT-5

根据CN109232464A的方法合成得到DT-5，为黄色固体，LC-MS(ESI)：[M+1]+＝1591.8DT-5 was synthesized according to the method of CN109232464A as a yellow solid, LC-MS (ESI): [M+1]+=1591.8

实施例2.11含药连接子DT-6的合成
 Example 2.11 Synthesis of drug-containing linker DT-6

根据KR2019014324A的方法合成得到DT-6，为黄色固体，LC-MS(ESI)：[M+1]+＝1619.6DT-6 was synthesized according to the method of KR2019014324A as a yellow solid, LC-MS (ESI): [M+1]+=1619.6

实施例2.12含药连接子DT-7的合成
 Example 2.12 Synthesis of drug-containing linker DT-7

根据JP2018138538A的方法合成得到DT-7，为黄色固体，LC-MS(ESI)：[M+1]+＝1733.7DT-7 was synthesized according to the method of JP2018138538A as a yellow solid, LC-MS (ESI): [M+1]+=1733.7

实施例中使用的MC-VC-PAB-MMAE购自MCE。MC-VC-PAB-MMAE used in the examples was purchased from MCE.

实施例3偶联工艺研究Example 3 Coupling process study

实施例3.1抗体还原条件及金属离子的筛选Example 3.1 Antibody reduction conditions and metal ion screening

将trastuzumab抗体换液至pH6.5～7.4 20mM磷酸氢二钠-磷酸二氢钠缓冲液中，并稀释至5-15mg/mL。依次加入不同金属离子水溶液与三(2-羧乙基)膦盐酸盐(TCEP)水溶液，并在0～15℃过夜反应，反应当量如表1-1所示。然后，向上述反应液中加入3倍抗体摩尔当量的BL20E(C-3)的DMA溶液，0～15℃反应2h后换液至pH6.5～7.4的20mM磷酸二氢钠-磷酸氢二钠缓冲溶液中。调节上述反应液浓度至约5-15mg/mL，加入4倍抗体当量乙二胺四乙酸(EDTA)，室温下反应30min后换液至pH 7.4～8.0的20mM磷酸二氢钠-磷酸氢二钠缓冲溶液中。调节上述反应液浓度至约5-15mg/mL，加入8倍抗体当量脱氢抗坏血酸(DHAA)，并在25～35℃反应2h后换液至pH 7.4的50mM磷酸氢二钠-磷酸二氢钠缓冲液中。获得抗体药物偶联物产物。取样分析，测定抗体偶联比率(DAR)，检测结果见表1-1。The trastuzumab antibody solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution at pH 6.5-7.4 and diluted to 5-15mg/mL. Different metal ion aqueous solutions and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solutions were added in sequence and reacted at 0-15°C overnight. The reaction equivalents are shown in Table 1-1. Then, 3 times the antibody molar equivalent of BL20E (C-3) DMA solution was added to the above reaction solution, reacted at 0-15°C for 2h, and then the solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution at pH 6.5-7.4. The concentration of the above reaction solution was adjusted to about 5-15mg/mL, 4 times the antibody equivalent of ethylenediaminetetraacetic acid (EDTA) was added, and the solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution at pH 7.4-8.0 after reacting at room temperature for 30min. The concentration of the above reaction solution was adjusted to about 5-15 mg/mL, 8 times the antibody equivalent of dehydroascorbic acid (DHAA) was added, and the solution was changed to 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4 after reacting at 25-35°C for 2 hours. The antibody drug conjugate product was obtained. Samples were taken for analysis, and the antibody coupling ratio (DAR) was determined. The test results are shown in Table 1-1.

表1-1.不同的还原剂当量及不同金属离子的筛选结果

Table 1-1. Screening results of different reducing agent equivalents and different metal ions

可见，在本申请的制备方法中采用以上金属离子或金属配合物以及反应物当量可以实现DAR2-ADC的HIC峰值为61-74％。It can be seen that the use of the above metal ions or metal complexes and reactant equivalents in the preparation method of the present application can achieve a HIC peak value of 61-74% for DAR2-ADC.

实施例3.2抗体偶联条件的筛选Example 3.2 Screening of antibody coupling conditions

将trastuzumab抗体换液至pH6.5～7.4 20mM磷酸氢二钠-磷酸二氢钠缓冲液中，并稀释至5-15mg/mL。依次加入2倍抗体摩尔当量的ZnCl2水溶液与4.0～4.5倍抗体摩尔当量的三(2-羧乙基)膦盐酸盐(TCEP)水溶液，并在0～15℃下过夜反应后换液至不同pH的20mM磷酸二氢钠-磷酸氢二钠缓冲溶液中。然后，向上述反应液中加入3倍抗体摩尔当量的BL20E(C-3)的DMA溶液，偶联反应温度及偶联溶液的pH如表1-2所示。上述偶联反应结束后换液至pH6.5～7.4的20mM磷酸二氢钠-磷酸氢二钠缓冲溶液中。调节上述反应液浓度至约5-15mg/mL，加入4倍抗体当量乙二胺四乙酸(EDTA)，室温下反应30min后换液至pH 7.4～8.0的20mM磷酸二氢钠-磷酸氢二钠缓冲溶液中。调节上述反应液浓度至约5-15mg/mL，加入8倍抗体摩尔当量脱氢抗坏血酸(DHAA)，并在25～35℃℃下反应2h后换液至pH 7.4的50mM磷酸氢二钠-磷酸二氢钠缓冲液中。获得抗体药物偶联物产物。取样分析，测定抗体偶联比率(DAR)及纯度，检测结果见表1-2。The trastuzumab antibody was exchanged into a 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 6.5-7.4 and diluted to 5-15 mg/mL. A ZnCl 2 aqueous solution of 2 times the antibody molar equivalent and a tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solution of 4.0-4.5 times the antibody molar equivalent were added in sequence, and after overnight reaction at 0-15°C, the solution was exchanged into a 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution of different pH values. Then, a DMA solution of BL20E (C-3) of 3 times the antibody molar equivalent was added to the above reaction solution. The coupling reaction temperature and the pH of the coupling solution are shown in Table 1-2. After the above coupling reaction was completed, the solution was exchanged into a 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution of pH 6.5-7.4. The concentration of the above reaction solution was adjusted to about 5-15 mg/mL, 4 times the antibody equivalent of ethylenediaminetetraacetic acid (EDTA) was added, and the solution was changed to 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with a pH of 7.4-8.0 after reacting at room temperature for 30 minutes. The concentration of the above reaction solution was adjusted to about 5-15 mg/mL, 8 times the antibody molar equivalent of dehydroascorbic acid (DHAA) was added, and the solution was changed to 50 mM sodium dihydrogen phosphate-disodium dihydrogen phosphate buffer with a pH of 7.4 after reacting at 25-35°C for 2 hours. The antibody drug conjugate product was obtained. Samples were taken for analysis, and the antibody coupling ratio (DAR) and purity were determined. The test results are shown in Table 1-2.

表1-2.不同偶联反应温度及不同偶联溶液pH的筛选结果
Table 1-2. Screening results of different coupling reaction temperatures and different coupling solution pH

可见，在本申请的制备方法中采用以上偶联反应温度和偶联溶液pH可以实现DAR2-ADC的HIC峰值为61-78％。It can be seen that the HIC peak value of DAR2-ADC can be achieved to be 61-78% by using the above coupling reaction temperature and coupling solution pH in the preparation method of the present application.

实施例3.3抗体纯化条件的筛选Example 3.3 Screening of antibody purification conditions

将trastuzumab抗体换液至pH6.5～7.4 20mM磷酸氢二钠-磷酸二氢钠缓冲液中，并稀释至5-15mg/mL。依次加入2倍抗体摩尔当量的ZnCl2水溶液与4.0～4.5倍抗体摩尔当量的三(2-羧乙基)膦盐酸盐(TCEP)水溶液，并在0～15℃下过夜反应。然后，向上述反应液中加入3 倍抗体摩尔当量的BL20E(C-3)的DMA溶液，0～15℃下反应2h后换液至pH6.5～7.4的20mM磷酸二氢钠-磷酸氢二钠缓冲溶液中。调节上述反应液浓度至约10mg/mL，加入4倍抗体当量乙二胺四乙酸(EDTA)，室温下反应30min后换液至pH 7.4～8.0的20mM磷酸二氢钠-磷酸氢二钠缓冲溶液中。调节上述反应液浓度至约5-15mg/mL，加入8倍抗体当量脱氢抗坏血酸(DHAA)，并在25～35℃下反应2h。反应结束后换液至pH 7.5的50mM磷酸二氢钠-磷酸氢二钠缓冲溶液中，稀释至约5～20mg/mL，加入硫酸铵溶液调节电导，对于抗体药物偶联物产物采用疏水作用色谱(HIC)进行上样纯化，纯化条件见表1-3。对于纯化后的产物取样进行HIC分析，HIC检测结果见表1-3。The trastuzumab antibody solution was changed to 20 mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 6.5-7.4 and diluted to 5-15 mg/mL. A 2-fold antibody molar equivalent of ZnCl 2 aqueous solution and a 4.0-4.5-fold antibody molar equivalent of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solution were added in sequence and reacted at 0-15°C overnight. Then, 3 The DMA solution of BL20E (C-3) with a molar equivalent of 100 times the antibody was added, and the reaction was carried out at 0-15°C for 2 hours, and then the solution was changed to a 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with a pH of 6.5-7.4. The concentration of the above reaction solution was adjusted to about 10 mg/mL, and 4 times the antibody equivalent of ethylenediaminetetraacetic acid (EDTA) was added. After reacting at room temperature for 30 minutes, the solution was changed to a 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with a pH of 7.4-8.0. The concentration of the above reaction solution was adjusted to about 5-15 mg/mL, and 8 times the antibody equivalent of dehydroascorbic acid (DHAA) was added, and the reaction was carried out at 25-35°C for 2 hours. After the reaction was completed, the solution was changed to a 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with a pH of 7.5, diluted to about 5-20 mg/mL, and ammonium sulfate solution was added to adjust the conductivity. The antibody drug conjugate product was purified by hydrophobic interaction chromatography (HIC). The purification conditions are shown in Table 1-3. The purified product was sampled for HIC analysis. The HIC test results are shown in Table 1-3.

表1-3.不同纯化条件的筛选结果
Table 1-3. Screening results of different purification conditions

可见，在本申请的制备方法中采用以上填料、盐种类和盐浓度(作为A相)可以实现DAR2-ADC的HIC峰值为98-100％。It can be seen that the use of the above fillers, salt types and salt concentrations (as phase A) in the preparation method of the present application can achieve a HIC peak value of 98-100% for DAR2-ADC.

实施例3.4不同含药连接子与不同抗体偶联实验Example 3.4 Conjugation experiments of different drug-containing linkers and different antibodies

将抗体换液至pH6.5～7.4 20mM磷酸氢二钠-磷酸二氢钠缓冲液中，并稀释至5-15mg/mL。依次加入2倍抗体摩尔当量的ZnCl2水溶液与4.0～4.5倍抗体摩尔当量的三(2-羧乙基)膦盐酸盐(TCEP)水溶液，并在0～15℃下过夜反应。然后，向上述反应液中加入2.5～5.5倍抗体摩尔当量的含药连接子的DMA溶液，0～15℃下反应2h后换液至pH 6.5～7.4的20mM磷酸二氢钠-磷酸氢二钠缓冲溶液中。调节上述反应液浓度至约5-15mg/mL，加入4倍抗体摩尔当量乙二胺四乙酸(EDTA)，室温下反应30min后换液至pH 7.4～8.0的20mM磷酸二氢钠-磷酸氢二钠缓冲溶液中。调节上述反应液浓度至约5-15mg/mL，加入8倍抗体摩尔当量脱氢抗坏血酸(DHAA)，并在25～35℃下反应2h。反应结束后换液至pH 7.5的50mM磷酸 二氢钠-磷酸氢二钠缓冲溶液中，稀释至约5～20mg/mL，加入硫酸铵溶液调节电导，使用pH7.5的50mM磷酸二氢钠-磷酸氢二钠+0.45～0.75M硫酸铵溶液作为A相，pH 7.5的50mM磷酸二氢钠-磷酸氢二钠溶液作为B相，使用Butyl 650M填料进行纯化，得到的纯化溶液浓缩换液至pH 7.4的20mM磷酸二氢钠-磷酸氢二钠缓冲溶液中。纯化后取样分析，测定所得抗体药物偶联物产物中的抗体偶联比率(DAR)及纯度，检测结果见表1-4。The antibody solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 6.5-7.4 and diluted to 5-15mg/mL. A ZnCl 2 aqueous solution with a molar equivalent of 2 times the antibody and a tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solution with a molar equivalent of 4.0-4.5 times the antibody were added in sequence, and reacted at 0-15°C overnight. Then, a DMA solution containing a drug linker with a molar equivalent of 2.5-5.5 times the antibody was added to the above reaction solution, and the solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 6.5-7.4 after reacting at 0-15°C for 2h. The concentration of the above reaction solution was adjusted to about 5-15mg/mL, and ethylenediaminetetraacetic acid (EDTA) with a molar equivalent of 4 times the antibody was added. After reacting at room temperature for 30min, the solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 7.4-8.0. Adjust the concentration of the above reaction solution to about 5-15 mg/mL, add 8 times the molar equivalent of antibody dehydroascorbic acid (DHAA), and react at 25-35°C for 2 hours. After the reaction is completed, change the solution to 50 mM phosphoric acid at pH 7.5. Dilute to about 5-20 mg/mL in sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution, add ammonium sulfate solution to adjust conductivity, use 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate + 0.45-0.75M ammonium sulfate solution at pH 7.5 as phase A, 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate solution at pH 7.5 as phase B, use Butyl 650M filler for purification, and the obtained purified solution is concentrated and replaced with 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution at pH 7.4. After purification, samples were taken for analysis, and the antibody coupling ratio (DAR) and purity in the obtained antibody drug conjugate product were determined. The test results are shown in Table 1-4.

表1-4.不同抗体偶联不同小分子实验结果
Table 1-4. Experimental results of different antibodies coupled to different small molecules

其中，抗体h23-12是根据专利WO2021068949A1描述方法制备的，抗体hH2L1是根据专利CN113527486A描述方法制备的。Among them, antibody h23-12 was prepared according to the method described in patent WO2021068949A1, and antibody hH2L1 was prepared according to the method described in patent CN113527486A.

实施例3.5对比实施例Example 3.5 Comparative Example

按照专利WO2020/164561A1公开的制备方法，制备抗体药物偶联物Trastuzumab-MC-VC-PAB-MMAE。简述如下：According to the preparation method disclosed in patent WO2020/164561A1, the antibody drug conjugate Trastuzumab-MC-VC-PAB-MMAE was prepared. The brief description is as follows:

(1)将Zncl2(0.04mM)和TCEP(0.08mM)加入到Trastuzumab抗体溶液中，4℃下过夜反应；(1) Add ZnCl2 (0.04 mM) and TCEP (0.08 mM) to the Trastuzumab antibody solution and react overnight at 4°C;

(2)向上述(1)反应液中加入MC-VC-PAB-MMAE的DMA溶液(0.12mM)中,4℃下反应2h；抗体与含药连接子摩尔当量比为1:3。(2) Add DMA solution (0.12 mM) of MC-VC-PAB-MMAE to the reaction solution of (1) above and react at 4°C for 2 h; the molar equivalent ratio of antibody to drug-containing linker is 1:3.

(3)向上述(2)反应液中加入半胱氨酸(0.08mM)以除去过量的MC-VC-PAB-MMAE；(3) adding cysteine (0.08 mM) to the reaction solution of (2) above to remove excess MC-VC-PAB-MMAE;

(4)向上述(3)反应液中加入EDTA(0.08mM)，随后加入DHAA(0.16mM)；(4) Add EDTA (0.08 mM) to the reaction solution in (3) above, followed by DHAA (0.16 mM);

(5)使用脱盐柱(型号：40K，0.5mL，REF：87766，Lot：SJ251704，Thermo)纯化上述反应混合液。 (5) The reaction mixture was purified using a desalting column (Model: 40K, 0.5 mL, REF: 87766, Lot: SJ251704, Thermo).

表1-5.HIC分析对比结果
Table 1-5. HIC analysis comparison results

实施例4抗体药物偶联物的理化表征Example 4 Physicochemical Characterization of Antibody Drug Conjugates

实施例4.1抗体药物偶联物的理化表征Example 4.1 Physicochemical Characterization of Antibody Drug Conjugates

a、疏水作用色谱(HIC-HPLC)测定DARa. Determination of DAR by hydrophobic interaction chromatography (HIC-HPLC)

流动相A：2.0M(NH4)2SO4+50mM PB，pH 7.0Mobile phase A: 2.0M (NH 4 ) 2 SO 4 + 50mM PB, pH 7.0

流动相B：50mM PB+15％IPA，pH 7.0Mobile phase B: 50 mM PB + 15% IPA, pH 7.0

流速：0.6mL/minFlow rate: 0.6mL/min

进样浓度：3～5mg/mLInjection concentration: 3-5 mg/mL

进样体积：10μLInjection volume: 10 μL

分析柱：TOSOH Butyl NPR，4.6*100mm,2.5μm，PN0042168，CN081D00130DAnalytical column: TOSOH Butyl NPR, 4.6*100mm, 2.5μm, PN0042168, CN081D00130D

柱温：25℃Column temperature: 25°C

检测波长：280nmDetection wavelength: 280nm

梯度：
gradient:

进样分析：对于浓度超过5mg/mL的样品，用50％A相溶液稀释样品，0.2μm滤头过滤后进样分析；对于5mg/mL或以下样品，可直接过滤后进样分析。Sample injection analysis: For samples with a concentration exceeding 5 mg/mL, dilute the sample with 50% phase A solution, filter through a 0.2 μm filter, and then perform sample injection analysis; for samples with a concentration of 5 mg/mL or less, filter directly before sample injection analysis.

DAR＝∑(加权峰面积)/100，即DAR＝(D0峰面积比×0+D1峰面积比×1+D2峰面积比×2+D3峰面积比×3+D4峰面积比×4+D5峰面积比×5+D6峰面积比×6+D7峰面积比×7+D8峰面积比×8)/100。DAR = ∑ (weighted peak area) / 100, that is, DAR = (D0 peak area ratio × 0 + D1 peak area ratio × 1 + D2 peak area ratio × 2 + D3 peak area ratio × 3 + D4 peak area ratio × 4 + D5 peak area ratio × 5 + D6 peak area ratio × 6 + D7 peak area ratio × 7 + D8 peak area ratio × 8) / 100.

b.非还原毛细管电泳NR-CE-SDS测定纯度 b. Purity determination by non-reducing capillary electrophoresis NR-CE-SDS

根据《中国药典四部》通则3127单抗分子大小变异体测定法测定。The method was determined according to the monoclonal antibody molecular size variant determination method in General Chapter 3127 of the Chinese Pharmacopoeia Volume IV.

实施例5抗体药物偶联物的生物活性分析Example 5 Bioactivity Analysis of Antibody Drug Conjugates

5.1抗体药物偶联物体外杀伤作用研究5.1 Study on the killing effect of antibody-drug conjugates in vitro

示例性地，使用以下方法进行实验。Exemplarily, the following method was used to conduct the experiment.

本实验利用人胃癌细胞NCI-N87(购自：ATCC)。用完全培养基(RPMI 1640培养基45ml，5ml FBS，混匀后使用)将NCI-N87细胞密度调到15×104之后，100μl/孔加入细胞培养板中，过夜培养。第二天，用上述完全培养基稀释ADC样品，分别稀释到50ug/ml，之后再4倍梯度稀释，共9个梯度加零点，所有样品均设3个复孔。设阴性对照(细胞+培养基)和空白对照(无细胞，纯培养基)；将上述稀释后的ADC样品依次加入到过夜培养的细胞培养板中，100μl/孔。然后置于细胞培养箱中孵育120h。取出细胞培养板，40μl/孔加入MTS(abcam，货号ab223881)后，37℃培养箱反应2-4h；取出细胞板，在490nm处读取OD值。This experiment used human gastric cancer cells NCI-N87 (purchased from ATCC). After adjusting the density of NCI-N87 cells to 15×10 4 with complete medium (RPMI 1640 medium 45ml, 5ml FBS, mixed before use), 100μl/well was added to the cell culture plate and cultured overnight. On the second day, the ADC samples were diluted with the above complete medium to 50ug/ml, and then diluted 4 times in a gradient, with a total of 9 gradients plus zero point, and all samples were set up with 3 replicates. Set up negative control (cells + culture medium) and blank control (no cells, pure culture medium); add the above diluted ADC samples to the cell culture plate cultured overnight in sequence, 100μl/well. Then place it in a cell culture incubator and incubate for 120h. Take out the cell culture plate, add MTS (abcam, catalog number ab223881) at 40μl/well, and react in a 37℃ incubator for 2-4h; take out the cell plate and read the OD value at 490nm.

表1-6不同抗体药物偶联物活性对比研究结果
Table 1-6 Comparative study results of the activity of different antibody-drug conjugates

Claims (26)

一种用于制备抗体-药物偶联物(ADC)的方法，该方法包括以下步骤：A method for preparing an antibody-drug conjugate (ADC), the method comprising the following steps: (a)在含抗体的缓冲溶液中，加入金属盐或金属配合物、还原剂，然后进行孵育；(a) adding a metal salt or a metal complex and a reducing agent to a buffer solution containing an antibody, followed by incubation; (b)向(a)中的反应溶液中加入含药连接子进行偶联；和(b) adding a drug-containing linker to the reaction solution in (a) for coupling; and (c)使用疏水作用色谱方法，采用疏水凝胶型填料和盐溶液对于偶联物产物进行纯化，以获得高纯度的DAR2-ADC偶联物。(c) Using hydrophobic interaction chromatography, a hydrophobic gel-type filler and a salt solution to purify the conjugate product to obtain a high-purity DAR2-ADC conjugate. 权利要求1所述的方法，其中步骤(a)中所述的金属盐或金属配合物中的金属可以选自Zn、Cd和Hg等，The method according to claim 1, wherein the metal in the metal salt or metal complex in step (a) can be selected from Zn, Cd and Hg, etc. 优选地，步骤(a)中所述的金属盐或金属配合物选自下述中的一种或多种：Zn、Cd和Hg的盐酸盐或硫酸盐、 Preferably, the metal salt or metal complex in step (a) is selected from one or more of the following: hydrochlorides or sulfates of Zn, Cd and Hg, Zn(NH2CH3)2 2+，Zn(NH2CH2CH3)2 2+，Zn(NH2CH2CH2CH3)2 2+，Zn(NH2CH(CH3)2)2 2+，Zn(NH2C(CH3)3)2 2+，Zn(NH2CH2C(CH3)3)2 2+，Zn(NH(CH3)2)2 2+，Zn(NH(CH2CH3)2)2 2+，Zn(NH(CH(CH3)2)2)2 2+，Zn(NH(C(CH3)3)2)2 2+，Zn(NH(CH(CH2CH3)2)2)2 2+，Zn(NH(CH2C(CH3)3)2)2 2+，Zn(NH(CH2C(CH2CH3)3)2)2 2+，Zn(NH(CH2CH2C(CH3)3)2)2 2+，Zn(NH2CH2CH2OH)2 2+，Zn(NH(CH2CH2OH)2)2 2+，Zn(N(CH2CH2OH)3)2 2+，Zn(NH2CH2COOH)2 2+，Zn(NH2CH2CONH2)2 2+，Zn(NH2CH2COOCH3)2 2+，Zn(NH2CH2COOCH2CH3)2 2+，Zn(NH2CH2COOC(CH3)3)2 2+，Zn(NH2CH2COOCH(CH3)2)2 2+，Zn(NH2CH2CH2COOH)2 2+，Zn(NH(CH2COOH)2)2 2+，Zn(N(CH2CH2COOH)3)2 2+，Zn(NH2CH3)4 2+，Zn(NH2CH2CH3)4 2+，Zn(NH2CH2CH2CH3)4 2+，Zn(NH2CH(CH3)2)4 2+，Zn(NH2C(CH3)3)4 2+，Zn(NH2CH2C(CH3)3)4 2+，Zn(NH(CH3)2)4 2+，Zn(NH(CH2CH3)2)4 2+，Zn(NH(CH(CH3)2)2)4 2+，Zn(NH(C(CH3)3)2)4 2+，Zn(NH(CH(CH2CH3)2)2)4 2+，Zn(NH(CH2C(CH3)3)2)4 2+，Zn(NH(CH2C(CH2CH3)3)2)4 2+，Zn(NH(CH2CH2C(CH3)3)2)4 2+，Zn(NH2CH2CH2OH)4 2+， Zn(NH 2 CH 3 ) 2 2+ , Zn(NH 2 CH 2 CH 3 ) 2 2+ , Zn(NH 2 CH 2 CH 2 CH 3 ) 2 2+ , Zn(NH 2 CH(CH 3 ) 2 ) 2 2+ , Zn(NH 2 C(CH 3 ) 3 ) 2 2+ , Zn(NH 2 CH 2 C(CH 3 ) 3 ) 2 2+ , Zn(NH(CH 3 ) 2 ) 2 2+ , Zn(NH(CH 2 CH 3 ) 2 ) 2 2+ , Zn(NH(CH(CH 3 ) 2 ) 2 ) 2 2+ , Zn(NH(C(CH 3 ) 3 ) 2 ) 2 2+ , Zn(NH(CH(CH 2 CH 3 ) 2 ) 2 ) 2 2+ , Zn(NH(CH 2 C(CH 3 ) 3 ) 2 ) 2 2+ , Zn(NH(CH 2 C(CH 2 CH 3 ) 3 ) 2 ) 2 2 + ,Zn(NH(CH 2 CH 2 C(CH 3 ) 3 ) 2 ) 2 2+ ,Zn(NH 2 CH 2 CH 2 OH) 2 2+ , Zn(NH(CH 2 CH 2 OH) 2 ) 2 2+ , Zn(N(CH 2 CH 2 OH) 3 ) 2 2+ , Zn(NH 2 CH 2 COOH) 2 2+ ,Zn(NH 2 CH 2 CONH 2 ) 2 2+ ,Zn(NH 2 CH 2 COOCH 3 ) 2 2+ , Zn(NH 2 CH 2 COOCH 2 CH 3 ) 2 2+ , Zn(NH 2 CH 2 COOC(CH 3 ) 3 ) 2 2+ , Zn(NH 2 CH 2 COOCH(CH 3 ) 2 ) 2 2+ , Zn(NH 2 CH 2 CH 2 COOH) 2 2+ , Zn(NH(CH 2 COOH) 2 ) 2 2+ , Zn(N(CH 2 CH 2 COOH) 3 ) 2 2+ , Zn(NH 2 CH 3 ) 4 2+ , Zn(NH 2 CH 2 CH 3 ) 4 2+ , Zn( NH 2 CH 2 CH 2 CH 3 ) 4 2+ , Zn(NH 2 CH(CH 3 ) 2 ) 4 2+ , Zn(NH 2 C(CH 3 ) 3 ) 4 2+ , Zn(NH 2 CH 2 C(CH 3 ) 3 ) 4 2+ , Zn(NH(CH 3 ) 2 ) 4 2+ , Zn(NH( CH 2 CH 3 ) 2 ) 4 2+ , Zn(NH(CH(CH 3 ) 2 ) 2 ) 4 2+ , Zn(NH(C(CH 3 ) 3 ) 2 ) 4 2+ , Zn(NH(CH(CH 2 CH 3 ) 2 ) 2 ) 4 2+ , Zn(NH(CH 2 C(CH 3 ) 3 ) 2 ) 4 2+ , Zn(NH(CH 2 C(CH 2 CH 3 ) 3 ) 2 ) 4 2+ ,Zn(NH(CH 2 CH 2 C(CH 3 ) 3 ) 2 ) 4 2+ ,Zn(NH 2 CH 2 CH 2 OH) 4 2+ , 更优选地，步骤(a)中所述的金属盐或金属配合物选自ZnCl2、CdCl2、HgCl2、中的一种或多种。More preferably, the metal salt or metal complex in step (a) is selected from ZnCl 2 , CdCl 2 , HgCl 2 , One or more of . 权利要求1或2所述的方法，其中，步骤(a)中所述的还原剂选自三(2-羧乙基)膦 (TCEP)、二硫苏糖醇(DTT)、巯基乙胺和巯基乙醇，优选TCEP。The method according to claim 1 or 2, wherein the reducing agent in step (a) is selected from tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), mercaptoethylamine and mercaptoethanol, preferably TCEP. 权利要求1-3中任何一项所述的方法，其中步骤(a)中的抗体的浓度为0.5-150mg/mL，例如4-30mg/mL，The method according to any one of claims 1 to 3, wherein the concentration of the antibody in step (a) is 0.5-150 mg/mL, such as 4-30 mg/mL, 步骤(a)中所述的还原剂的用量是1-6倍抗体摩尔当量，例如3.5-5.0倍抗体摩尔当量，The amount of the reducing agent used in step (a) is 1-6 times the antibody molar equivalent, for example 3.5-5.0 times the antibody molar equivalent, 步骤(a)中所述的金属盐或金属配合物的用量为1-5倍抗体摩尔当量，例如1-3倍抗体摩尔当量，The amount of the metal salt or metal complex in step (a) is 1-5 times the molar equivalent of the antibody, for example 1-3 times the molar equivalent of the antibody. 步骤(a)中的所述缓冲溶液具有5-8的pH，例如6.5-7.5的pH，优选pH为6.9，和/或The buffer solution in step (a) has a pH of 5-8, such as a pH of 6.5-7.5, preferably a pH of 6.9, and/or 步骤(a)中的所述缓冲溶液选自磷酸盐缓冲液、醋酸盐缓冲液、柠檬酸盐缓冲液、琥珀酸盐缓冲液，优选磷酸氢二钠-磷酸二氢钠缓冲液，优选地，所述缓冲溶液中的缓冲剂浓度为5-100mM，例如10-30或mM。The buffer solution in step (a) is selected from phosphate buffer, acetate buffer, citrate buffer, succinate buffer, preferably disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, preferably, the buffer concentration in the buffer solution is 5-100mM, such as 10-30 or mM. 权利要求1-4中任何一项所述的方法，其中步骤(b)中的含药连接子的用量是1-8倍抗体摩尔当量，例如3-5倍抗体摩尔当量，The method according to any one of claims 1 to 4, wherein the amount of the drug-containing linker in step (b) is 1 to 8 times the molar equivalent of the antibody, such as 3 to 5 times the molar equivalent of the antibody, 优选地，步骤(b)中反应溶液具有5-8的pH，例如6.5-7.5的pH。Preferably, the reaction solution in step (b) has a pH of 5-8, such as a pH of 6.5-7.5. 权利要求1-5中任何一项所述的方法，其中步骤(c)中所述疏水凝胶填料选自Butyl-650M、Butyl-4FF、Butyl-650S、Butyl-ImpRes和Phenyl Sepharose HP，和/或The method according to any one of claims 1 to 5, wherein the hydrophobic gel filler in step (c) is selected from Butyl-650M, Butyl-4FF, Butyl-650S, Butyl-ImpRes and Phenyl Sepharose HP, and/or 步骤(c)中的盐种类选自硫酸盐、氯化盐，优选硫酸铵、氯化钠；优选地，盐浓度为0.1-5mol/L，例如0.6-1.2mol/L。The salt type in step (c) is selected from sulfate and chloride, preferably ammonium sulfate and sodium chloride; preferably, the salt concentration is 0.1-5 mol/L, such as 0.6-1.2 mol/L. 权利要求1-6中任何一项所述的方法，其中在步骤(b)后包含步骤(b’):加入金属离子螯合剂例如乙二胺四乙酸(EDTA)进行反应，The method according to any one of claims 1 to 6, wherein after step (b), step (b') is included: adding a metal ion chelating agent such as ethylenediaminetetraacetic acid (EDTA) to react, 优选地，金属离子螯合剂的用量是1-8倍抗体摩尔当量，例如3-5倍抗体摩尔当量。Preferably, the amount of the metal ion chelator used is 1-8 times the molar equivalent of the antibody, such as 3-5 times the molar equivalent of the antibody. 权利要求1-7中任何一项所述的方法，其中，在步骤(b’)后包含步骤(b”):加入脱氢抗坏血酸(DHAA)进行反应，The method according to any one of claims 1 to 7, wherein after step (b'), step (b") is included: adding dehydroascorbic acid (DHAA) to react, 优选地，DHAA的用量是1-16倍抗体摩尔当量，例如6-12倍抗体摩尔当量。Preferably, the amount of DHAA used is 1-16 times the antibody molar equivalent, such as 6-12 times the antibody molar equivalent. 权利要求1-8中任何一项所述的方法，其中，所述方法获得的DAR2-ADC具有大于90％的纯度，所述纯度例如通过HIC-HPLC测得。The method according to any one of claims 1 to 8, wherein the DAR2-ADC obtained by the method has a purity greater than 90%, such as measured by HIC-HPLC. 权利要求1-9中任何一项所述的方法，其中所述抗体选自人源化抗体、鼠源抗体、人抗体、嵌合抗体、单链抗体和双特异性抗体，The method of any one of claims 1 to 9, wherein the antibody is selected from the group consisting of a humanized antibody, a murine antibody, a human antibody, a chimeric antibody, a single-chain antibody, and a bispecific antibody, 优选地，所述抗体是与下述抗原结合的抗体：HER2、B7H3、HER3、CD19、CD20、CD22、CD30、CD33、CD37、CD45、CD56、CD66e、CD70、CD74、CD73、CD79b、CD138、CD147、CD223、EpCAM、粘蛋白1(Mucin 1)、STEAP1、GPNMB、FGF2、FOLR1、EGFR、EGFRvIII、组织因子(Tissuefactor)、c-MET、FGFR、Nectin 4、AGS-16、鸟苷酸环化酶C(Guanylyl cyclase C)、间皮素(Mesothelin)、SLC44A4、PSMA、EphA2、AGS-5、GPC-3、c-KIT、RoR1、PD-L1、CD27L、5T4、Mucin16、NaPi2b、STEAP、SLITRK6、ETBR、BCMA、Trop-2、CEACAM5、SC-16、SLC39A6、Delta-like protein3、Claudin 18.2；Preferably, the antibody is an antibody that binds to the following antigens: HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor, c-MET, FGFR, Necti n 4, AGS-16, Guanylyl cyclase C, Mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, Claudin 18.2; 优选地，所述抗体包含阿达木单抗、贝伐单抗、西妥昔单抗、曲妥珠单抗(trastuzumab)、帕妥珠单抗(Pertuzumab)、尼妥珠单抗、利妥昔单抗、h23-12或hH2L1的一个或多个CDR(优选3个CDR，即HCDR1、HCDR2H和HCDR3；或LCDR1、LCDR2和LCDR3，更优选6 个CDR，即HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3)，或包含所述抗体的VH和/或VL，或包含所述抗体的重链和/或轻链；Preferably, the antibody comprises one or more CDRs (preferably 3 CDRs, i.e., HCDR1, HCDR2H and HCDR3; or LCDR1, LCDR2 and LCDR3, more preferably 6 CDRs) of adalimumab, bevacizumab, cetuximab, trastuzumab, pertuzumab, nimotuzumab, rituximab, h23-12 or hH2L1. CDRs, i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3), or VH and/or VL of the antibody, or heavy chain and/or light chain of the antibody; 优选地，所述抗体选自trastuzumab、Pertuzumab、h23-12和hH2L1。Preferably, the antibody is selected from trastuzumab, pertuzumab, h23-12 and hH2L1. 权利要求1-10中任何一项所述的方法，其中步骤(b)中的含药连接子为式A-I所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药，
The method according to any one of claims 1 to 10, wherein the drug-containing linker in step (b) is a compound represented by formula AI or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof,
其中，E选自如下基团：
Wherein, E is selected from the following groups:
其中，带有的键表示与M的连接位点：Among them, with The bond of represents the attachment site to M: n为选自1-10的整数，例如为1、2、3、4、5、6、7、8、9或10；n is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; R6、R7独立地为卤素或Ar’S-，Ar’为苯基或由一个或多个取代基取代的苯基，在取代的苯基中，所述取代基选自烷基(例如C1-C6烷基、优选C1-C4烷基)、烷氧基(例如C1-C6烷氧基、优选C1-C4烷氧基，优选甲氧基)、5-6元杂环基-CO-、卤素、酯基、酰胺基和氰 基，优选地，Ar’为苯基、4-甲基氨基甲酰基苯基或4-甲酰基吗啉取代苯基 R 6 and R 7 are independently halogen or Ar'S-, Ar' is phenyl or phenyl substituted by one or more substituents, in the substituted phenyl, the substituents are selected from alkyl (e.g. C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), 5-6 membered heterocyclic group -CO-, halogen, ester group, amide group and cyano group. Preferably, Ar' is phenyl, 4-methylcarbamoylphenyl or 4-formylmorpholine substituted phenyl M为亚苯基或由一个或多个取代基取代的亚苯基，或化学键；在取代的亚苯基中，所述取代基选自烷基(例如C1-C6烷基、优选C1-C4烷基)、卤代烷基(例如卤代C1-C6烷基、优选卤代C1-C4烷基，例如三氟甲基)、烷氧基(例如C1-C6烷氧基、优选C1-C4烷氧基，优选甲氧基)、卤素、酯基、酰胺基和氰基；优选地，M为卤素取代的亚苯基；M is a phenylene group or a phenylene group substituted by one or more substituents, or a chemical bond; in the substituted phenylene group, the substituent group is selected from an alkyl group (e.g., a C1-C6 alkyl group, preferably a C1-C4 alkyl group), a haloalkyl group (e.g., a halo C1-C6 alkyl group, preferably a halo C1-C4 alkyl group, such as a trifluoromethyl group), an alkoxy group (e.g., a C1-C6 alkoxy group, preferably a C1-C4 alkoxy group, preferably a methoxy group), a halogen, an ester group, an amide group, and a cyano group; preferably, M is a phenylene group substituted by a halogen; SP1选自C1-8亚烷基、C1-8亚烷基亚苯基-、C1-8亚环烷基或C1-21(优选C1-16、更优选C1-11)直链亚杂烷基，所述C1-21直链亚杂烷基包含1-11个(优选1-6个)选自N、O或S的杂原子，其中所述C1-8亚烷基、C1-8亚环烷基和C1-21直链亚杂烷基各自独立地任选被选自羟基、氧代、氨基、磺酸基和氰基的一个或多个取代基取代；SP 1 is selected from C1-8 alkylene, C1-8 alkylene phenylene-, C1-8 cycloalkylene or C1-21 (preferably C1-16, more preferably C1-11) straight chain heteroalkylene, the C1-21 straight chain heteroalkylene containing 1-11 (preferably 1-6) heteroatoms selected from N, O or S, wherein the C1-8 alkylene, C1-8 cycloalkylene and C1-21 straight chain heteroalkylene are each independently optionally substituted by one or more substituents selected from hydroxy, oxo, amino, sulfonic acid and cyano; SP2选自-NH(CH2CH2O)aCH2CH2CO-、-NH(CH2CH2O)aCH2CO-、-S(CH2)aCO-或化学键，其中a为1-20的整数，优选1-10的整数，更优选1-6的整数；SP 2 is selected from -NH(CH2CH2O)aCH2CH2CO-, -NH(CH2CH2O)aCH2CO-, -S(CH2)aCO- or a chemical bond, wherein a is an integer of 1-20, preferably an integer of 1-10, more preferably an integer of 1-6; A表示2-4个氨基酸，其中，A表示2个氨基酸时，可以为NH-Phe-Lys-CO、NH-Val-Ala-CO、NH-Val-Lys-CO、NH-Ala-Lys-CO、NH-Val-Cit-CO、NH-Phe-Cit-CO、NH-Leu-Cit-CO、NH-Phe-Arg-CO或NH-Gly-Val-CO，优选为NH-Phe-Lys-CO、NH-Val-Ala-CO或NH-Val-Cit-CO；A表示3个氨基酸时，可以为NH-Glu-Val-Ala-CO、NH-Glu-Val-Cit-CO或NH-Ala-Ala-Ala-CO，优选为NH-Glu-Val-Ala-CO或NH-Ala-Ala-Ala-CO；A表示4个氨基酸时，可以为NH-Gly-Gly-Phe-Gly-CO或NH-Gly-Phe-Gly-Gly-CO，优选为NH-Gly-Gly-Phe-Gly-CO，优选地，A为NH-Val-Ala-CO、NH-Gly-Gly-Phe-Gly-CO或NH-Ala-Ala-Ala-CO；A represents 2 to 4 amino acids, wherein when A represents 2 amino acids, it can be NH-Phe-Lys-CO, NH-Val-Ala-CO, NH-Val-Lys-CO, NH-Ala-Lys-CO, NH-Val-Cit-CO, NH-Phe-Cit-CO, NH-Leu-Cit-CO, NH-Phe-Arg-CO or NH-Gly-Val-CO, preferably NH-Phe-Lys-CO, NH-Val-Ala-CO or NH-Val-Cit-CO; when A represents 3 amino acids, it can be NH-Glu-Val-Ala-CO. la-CO, NH-Glu-Val-Cit-CO or NH-Ala-Ala-Ala-CO, preferably NH-Glu-Val-Ala-CO or NH-Ala-Ala-Ala-CO; when A represents 4 amino acids, it can be NH-Gly-Gly-Phe-Gly-CO or NH-Gly-Phe-Gly-Gly-CO, preferably NH-Gly-Gly-Phe-Gly-CO, preferably, A is NH-Val-Ala-CO, NH-Gly-Gly-Phe-Gly-CO or NH-Ala-Ala-Ala-CO; 优选地，A选自：NH-Val-Cit-CO、NH-Val-Ala-CO和NH-Gly-Gly-Phe-Gly-CO；Preferably, A is selected from the group consisting of NH-Val-Cit-CO, NH-Val-Ala-CO and NH-Gly-Gly-Phe-Gly-CO; L2选自和化学键；L 2 is selected from and chemical bonds; D为小分子药物。D is a small molecule drug. 权利要求11所述的方法，其中The method of claim 11, wherein M选自 M is selected from 权利要求11或12所述的方法，其中The method of claim 11 or 12, wherein 选自
 Selected from
其中q为选自1-10的整数；wherein q is an integer selected from 1-10; p为选自1-20的整数；p is an integer selected from 1-20; 优选地，选自
Preferably, Selected from
权利要求11-13中任何一项所述的方法，其中D选自喜树碱类化合物、加利车霉素(calicheamicin)、美登木素生物碱(maytansinoids)、多拉司他汀(dolastatin)、澳瑞他汀类化合物和单端孢霉素(trichothecene)；The method of any one of claims 11 to 13, wherein D is selected from the group consisting of camptothecins, calicheamicin, maytansinoids, dolastatins, auristatins and trichothecenes; 优选地，所述美登木素衍生物选自DM1、DM3和DM4；所述澳瑞他汀类化合物选自MMAE和MMAF；所述喜树碱类化合物选自喜树碱、10-羟基喜树碱、依喜替康、SN-38、托泊替康和喜树碱类衍生物。Preferably, the maytansinoid derivative is selected from DM1, DM3 and DM4; the auristatin compound is selected from MMAE and MMAF; the camptothecin compound is selected from camptothecin, 10-hydroxycamptothecin, exitecan, SN-38, topotecan and camptothecin derivatives. 权利要求11-14中任何一项所述的方法，其中，含药连接子为式III所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药，
The method according to any one of claims 11 to 14, wherein the drug-containing linker is a compound represented by formula III or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof,
其中E、M、SP1、SP2和A如权利要求11-14之一所定义，CPT为喜树碱类化合物；或者wherein E, M, SP 1 , SP 2 and A are as defined in any one of claims 11 to 14, and CPT is a camptothecin compound; or 为式III'所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药，
is a compound represented by formula III' or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof,
其中E、M、SP1、SP2和A如权利要求11-14之一所定义，D'为澳瑞他汀类化合物。wherein E, M, SP 1 , SP 2 and A are as defined in any one of claims 11 to 14, and D' is an auristatin compound. 权利要求15所述的方法，其中式III化合物进一步为式IIIA化合物：
The method of claim 15, wherein the compound of formula III is further a compound of formula IIIA:
其中，R6、R7独立地为卤素或Ar’S-，Ar’为苯基或由一个或多个取代基取代的苯基，在取代的苯基中，所述取代基选自烷基(例如C1-C6烷基、优选C1-C4烷基)、烷氧基(例如C1-C6烷氧基、优选C1-C4烷氧基，优选甲氧基)、5-6元杂环基-CO-、卤素、酯基、酰胺基和氰基，优选地，Ar’为苯基、4-甲基氨基甲酰基苯基或4-吗啉代甲酰基苯基 Wherein, R 6 and R 7 are independently halogen or Ar'S-, Ar' is phenyl or phenyl substituted by one or more substituents, in the substituted phenyl, the substituent is selected from alkyl (e.g. C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), 5-6 membered heterocyclic group -CO-, halogen, ester group, amide group and cyano group, preferably, Ar' is phenyl, 4-methylcarbamoylphenyl or 4-morpholinoformylphenyl 其中M、SP1、SP2、A和CPT如权利要求15中所定义；且wherein M, SP 1 , SP 2 , A and CPT are as defined in claim 15; and 式III'化合物进一步为式IIIA'化合物：
The compound of formula III' is further a compound of formula IIIA':
其中R6、R7、M、SP1、SP2、A和D’如权利要求15中所定义。 wherein R 6 , R 7 , M, SP 1 , SP 2 , A and D′ are as defined in claim 15 . 权利要求15或16所述的方法，其中CPT为如下式I或式IA所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药，以及对应的具体化合物：
The method of claim 15 or 16, wherein CPT is a compound represented by the following formula I or formula IA or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, and the corresponding specific compound:
其中R1、R2、R3、R4独立地为氢、卤素、羟基、C1-6烷氧基、氨基或取代氨基、C1-7烷基或取代的C1-7烷基，或者R1、R2、R3、R4中的任意两个连同它们所连接的碳原子构成C3-6环状烷基，当R1、R2、R3、R4独立地为C1-6烷氧基时，所述C1-6烷氧基包括直链或支链的C1-6烷氧基，优选地为直链或支链的C1-3烷氧基，更优选甲氧基，当R1、R2、R3、R4独立地为取代氨基时，所述取代氨基为由选自甲基和乙基的一个或多个取代基取代的氨基，当R1、R2、R3、R4独立地为C1-7烷基或取代的C1-7烷基时，所述C1-7烷基或取代的C1-7烷基包括直链或支链的C1-7烷基或取代的C1-7烷基，并且，取代的C1-7烷基为由选自环丙基和环丁基的一个或多个取代基取代的C1-7烷基；或者，所述直链或支链的C1-7烷基或取代的C1-7烷基优选为C1-3烷基或取代的C1-3烷基，例如甲基、卤代甲基(优选三氟甲基)；wherein R 1 , R 2 , R 3 , and R 4 are independently hydrogen, halogen, hydroxy, C1-6 alkoxy, amino or substituted amino, C1-7 alkyl or substituted C1-7 alkyl, or any two of R 1 , R 2 , R 3 , and R 4 together with the carbon atom to which they are attached constitute a C3-6 cyclic alkyl; when R 1 , R 2 , R 3 , and R 4 are independently C1-6 alkoxy, the C1-6 alkoxy includes a straight chain or branched C1-6 alkoxy, preferably a straight chain or branched C1-3 alkoxy, more preferably a methoxy; when R 1 , R 2 , R 3 , and R 4 are independently substituted amino, the substituted amino is an amino substituted by one or more substituents selected from methyl and ethyl; when R 1 , R 2 , R 3 , and R 4 are independently substituted amino, the substituted amino is an amino substituted by one or more substituents selected from methyl and ethyl; When 4 is independently C1-7 alkyl or substituted C1-7 alkyl, the C1-7 alkyl or substituted C1-7 alkyl includes linear or branched C1-7 alkyl or substituted C1-7 alkyl, and the substituted C1-7 alkyl is C1-7 alkyl substituted by one or more substituents selected from cyclopropyl and cyclobutyl; or, the linear or branched C1-7 alkyl or substituted C1-7 alkyl is preferably C1-3 alkyl or substituted C1-3 alkyl, such as methyl, halogenated methyl (preferably trifluoromethyl); G为氢、卤素、甲基或甲氧基，优选地，G为氢、氟或氯；G is hydrogen, halogen, methyl or methoxy, preferably, G is hydrogen, fluorine or chlorine; Y为氧、硫、砜、亚砜、亚甲基或取代亚甲基，取代亚甲基可以是亚甲基的一个氢被取代，也可以是两个氢同时被取代，取代基可以为苄基或烷基；当取代基为烷基时，所述烷基与R3和/或R4以及连同它们所连接的碳原子可以构成C3-6元并环或螺环的结构，当Y为取代亚甲基时，取代亚甲基的取代基优选为烷基，更优选为直链或支链的C1-4烷基，优选地，Y为氧、硫、砜或亚砜；或者，优选地，Y为氧、硫或亚甲基；Y is oxygen, sulfur, sulfone, sulfoxide, methylene or substituted methylene. The substituted methylene may be a methylene in which one hydrogen is substituted or two hydrogens are substituted simultaneously. The substituent may be a benzyl or an alkyl group. When the substituent is an alkyl group, the alkyl group and R3 and/or R4 and the carbon atoms to which they are connected may form a C3-6 membered cyclic or spirocyclic structure. When Y is a substituted methylene group, the substituent of the substituted methylene group is preferably an alkyl group, more preferably a linear or branched C1-4 alkyl group. Preferably, Y is oxygen, sulfur, sulfone or sulfoxide. Alternatively, preferably, Y is oxygen, sulfur or methylene. X为氧或硫；且X is oxygen or sulfur; and n＝0或1；n = 0 or 1; 优选地，式I所示的化合物进一步为式IA所示的化合物：
Preferably, the compound represented by formula I is further represented by formula IA:
式IA中，基团R1、R2、R3、R4与式I中基团R1、R2、R3、R4的定义相同。 In formula IA, the groups R 1 , R 2 , R 3 , and R 4 have the same definitions as the groups R 1 , R 2 , R 3 , and R 4 in formula I. 权利要求15或16所述的方法，其中所述喜树碱类化合物为式II所示的化合物或其药学上可接受的盐、立体异构体、溶剂化物或前药：
The method of claim 15 or 16, wherein the camptothecin compound is a compound represented by formula II or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
其中R5为C1-5烷基或由一个或多个取代基取代的C1-5烷基、C3-6环状烷基或由一个或多个取代基取代的C3-6环状烷基、苯基或取代苯基，当R5为C1-5烷基或取代的C1-5烷基时，所述C1-5烷基包括直链或支链的C1-5烷基，进一步地，R5为C1-4直链烷基，当R5为取代的C1-5烷基或取代的C3-6环状烷基时，取代基选自卤素、羟基、甲氧基、三氟甲基、氨基或取代氨基、甲磺酰基和C3-6环状烷基；并且其中，取代氨基为由选自甲基和乙基的一个或多个取代基取代的氨基，当R5为取代苯基时，取代基选自烷基(例如C1-6烷基、优选C1-3)或卤素；wherein R 5 is C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted by one or more substituents, phenyl or substituted phenyl; when R 5 is C1-5 alkyl or substituted C1-5 alkyl, the C1-5 alkyl includes a straight chain or branched C1-5 alkyl; further, R 5 is C1-4 straight chain alkyl; when R 5 is substituted C1-5 alkyl or substituted C3-6 cyclic alkyl, the substituent is selected from halogen, hydroxyl, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl and C3-6 cyclic alkyl; and wherein the substituted amino is an amino substituted by one or more substituents selected from methyl and ethyl; when R 5 is substituted phenyl, the substituent is selected from alkyl (e.g. C1-6 alkyl, preferably C1-3) or halogen; G为氢、卤素(例如氟)、甲基或甲氧基，优选地，G为氢、氟或氯；G is hydrogen, halogen (such as fluorine), methyl or methoxy, preferably, G is hydrogen, fluorine or chlorine; X为氧或硫；且X is oxygen or sulfur; and n＝0或1；n = 0 or 1; 优选地，式II所示的化合物进一步为式IIA所示的化合物：
Preferably, the compound represented by formula II is further represented by formula IIA:
式IIA中，基团R5与式II中基团R5的定义相同。In formula IIA, the group R 5 has the same definition as the group R 5 in formula II. 权利要求15或16所述的方法，其中，所述喜树碱类化合物(CPT)具有如下结构：



The method according to claim 15 or 16, wherein the camptothecin compound (CPT) has the following structure:



权利要求14-19中任一项所述的方法，其中喜树碱类化合物的氨基与A的羧基形成了酰胺键。The method according to any one of claims 14 to 19, wherein the amino group of the camptothecin compound forms an amide bond with the carboxyl group of A. 权利要求15-20中任一项所述的方法，其中CPT为式IV所示的依喜替康衍生物或其药学可接受的盐、立体异构体、溶剂化物或前药：
The method according to any one of claims 15 to 20, wherein CPT is an exitecan derivative represented by formula IV or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
其中R8为氢、三氟甲基、C1-5烷基或由一个或多个取代基取代的C1-5烷基、C3-6环状烷基或由一个或多个取代基取代的C3-6环状烷基、或卤素；wherein R 8 is hydrogen, trifluoromethyl, C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted by one or more substituents, or halogen; 当R8为取代的C1-5烷基或取代的C3-6环状烷基时，所述取代基选自卤素、羟基、甲氧基、三氟甲基、氨基或取代氨基、甲磺酰基和C3-6环状烷基；并且其中，取代氨基为由选自甲基和乙基的一个或多个取代基取代的氨基；When R 8 is a substituted C1-5 alkyl or a substituted C3-6 cyclic alkyl, the substituent is selected from halogen, hydroxy, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl and C3-6 cyclic alkyl; and wherein the substituted amino is an amino substituted by one or more substituents selected from methyl and ethyl; 优选地，式IV所示的化合物经由其与R8连接于同一个碳的羟基(参见式IV)与A的羧基通过自释放结构相连，该自释放结构例如实线表示与式III或式IIIA中A的羧基连接的位点，波浪线表示与式IV中羟基连接的位点。Preferably, the compound represented by formula IV is connected to the carboxyl group of A through a self-releasing structure via its hydroxyl group connected to the same carbon as R 8 (see formula IV), and the self-releasing structure is, for example The solid line indicates the site of attachment to the carboxyl group of A in Formula III or Formula IIIA, and the wavy line indicates the site of attachment to the hydroxyl group in Formula IV. 权利要求15-21中任一项所述的方法，其中，在式III’和式IIIA’中，D’为如下式I’所示的化合物或其药学可接受的盐、立体异构体、溶剂化物或前药，以及对应的具体化合物：
The method according to any one of claims 15 to 21, wherein in Formula III' and Formula IIIA', D' is a compound represented by the following Formula I' or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, and the corresponding specific compound:
其中Ra1、Ra2、Ra3、Ra4、Ra5和Ra8各自独立的选自C1-8烷基；优选C1-4烷基，例如甲基、乙基、正丙基、异丙基、正丁基、异丁基或仲丁基；wherein Ra1 , Ra2 , Ra3 , Ra4 , Ra5 and Ra8 are each independently selected from C1-8 alkyl; preferably C1-4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl; Ra6和Ra7各自独立地选自C1-8烷氧基，例如甲氧基、乙氧基或丙氧基；R a6 and R a7 are each independently selected from C 1-8 alkoxy, such as methoxy, ethoxy or propoxy; Ra9选自C1-8烷基和COOH；优选C1-4烷基，例如甲基、乙基、正丙基、异丙基、正丁基、异丁基或仲丁基；R a9 is selected from C 1-8 alkyl and COOH; preferably C 1-4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl; Ra10选自OH和H；R a10 is selected from OH and H; 优选地，式I'化合物进一步为式IA'化合物：
Preferably, the compound of formula I' is further a compound of formula IA':
其中Ra1-Ra10如对式I'中所定义；wherein R a1 -R a10 are as defined for Formula I'; 更优选地，式I'化合物选自：
More preferably, the compound of formula I' is selected from:
优选地，在III’和式IIIA’中，式I’所示化合物的左侧仲氨基与式III’或IIIA’中A的羧基形成酰胺键。Preferably, in III' and IIIA', the left secondary amino group of the compound represented by formula I' forms an amide bond with the carboxyl group of A in formula III' or IIIA'. 权利要求15-22中任一项所述的方法，其中The method of any one of claims 15 to 22, wherein 式III和式IIIA所示的化合物进一步为式V所示的化合物，
The compounds represented by formula III and formula IIIA are further represented by formula V,
式V中，R6、R7独立地为Ar’S，Ar’为苯基或由一个或多个取代基取代的苯基，在取代的苯基中，取代基选自烷基(例如C1-C6烷基、优选C1-C4烷基)、烷氧基(例如C1-C6烷氧基、优选C1-C4烷氧基，优选甲氧基)、5-6元杂环基-CO-、卤素、酯基、酰胺基和氰基，优选地，Ar’为苯基、4-甲基氨基甲酰基苯基或4-吗啉代甲酰基苯基 In formula V, R 6 and R 7 are independently Ar'S, Ar' is phenyl or phenyl substituted by one or more substituents, in the substituted phenyl, the substituent is selected from alkyl (e.g. C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), 5-6 membered heterocyclic group -CO-, halogen, ester group, amide group and cyano group, preferably, Ar' is phenyl, 4-methylcarbamoylphenyl or 4-morpholinoformylphenyl Xh和Yh独立地为氢、卤素、卤代烷基(例如卤代C1-C6烷基、优选卤代C1-C4烷基，例如三氟甲基)或烷氧基(例如C1-C6烷氧基、优选C1-C4烷氧基，例如甲氧基)；Xh and Yh are independently hydrogen, halogen, haloalkyl (e.g. haloC1-C6 alkyl, preferably haloC1-C4 alkyl, such as trifluoromethyl) or alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, such as methoxy); m为1～10的整数；m is an integer from 1 to 10; A和CPT如权利要求15-22之一所定义；A and CPT are as defined in any one of claims 15 to 22; 且式IIIA’化合物进一步为式V’化合物，
And the compound of formula IIIA' is further a compound of formula V',
其中R6、R7、Xh、Yh、m、A和D'如权利要求16-22之一所定义；wherein R 6 , R 7 , Xh, Yh, m, A and D′ are as defined in any one of claims 16 to 22; 优选地，式V’化合物进一步为式V-A’化合物，
Preferably, the compound of formula V' is further a compound of formula V-A',
其中A和D'如如权利要求16-22之一所定义。wherein A and D' are as defined in any one of claims 16-22. 权利要求23所述的方法，其中，式V所示的化合物进一步为式V-A所示的化合物：
The method of claim 23, wherein the compound represented by formula V is further represented by formula VA:
其中各变量如权利要求23中所定义；wherein the variables are as defined in claim 23; 优选地，式V-A所示的化合物进一步为式V-A-1所示的化合物：
Preferably, the compound represented by formula VA is further represented by formula VA-1:
其中各变量如权利要求23中所定义；wherein the variables are as defined in claim 23; 或者，式V所示的化合物进一步为式V-B所示的化合物：
Alternatively, the compound represented by formula V is further represented by formula VB:
其中各变量如权利要求23中所定义；wherein the variables are as defined in claim 23; 优选地，式V-B所示的化合物进一步为式V-B-1所示的化合物：
Preferably, the compound represented by formula VB is further represented by formula VB-1:
其中各变量如权利要求23中所定义；wherein the variables are as defined in claim 23; 或者，式V所示的化合物进一步为式V-C所示的化合物：
Alternatively, the compound represented by formula V is further represented by formula VC:
其中各变量如权利要求23中所定义。The variables are as defined in claim 23. 根据权利要求23所述的方法，其中式V所示的化合物进一步为式VI所示的化合物：
The method according to claim 23, wherein the compound represented by formula V is further represented by formula VI:
其中各变量如权利要求23中所定义；wherein the variables are as defined in claim 23; 优选地，式VI所示的化合物进一步为式VI-A所示的化合物：
Preferably, the compound represented by formula VI is further represented by formula VI-A:
其中各变量如权利要求23中所定义；wherein the variables are as defined in claim 23; 优选地，式VI-A所示的化合物进一步为式VI-A-1所示的化合物：
Preferably, the compound represented by formula VI-A is further represented by formula VI-A-1:
其中各变量如权利要求23中所定义；wherein the variables are as defined in claim 23; 或者，式VI所示的化合物进一步为式VI-B所示的化合物：
Alternatively, the compound represented by formula VI is further represented by formula VI-B:
其中各变量如权利要求23中所定义； wherein the variables are as defined in claim 23; 更优选地，式VI-B所示化合物进一步为式VI-B-1所示的化合物：
More preferably, the compound represented by formula VI-B is further represented by formula VI-B-1:
其中各变量如权利要求23中所定义；wherein the variables are as defined in claim 23; 或者，式VI所示的化合物进一步为式VI-C所示的化合物：
Alternatively, the compound represented by formula VI is further represented by formula VI-C:
其中各变量如权利要求23中所定义Wherein the variables are as defined in claim 23 式VI-C中，基团A、R8的定义与上文式III或式IIIA中基团A、R8的定义相同。In Formula VI-C, the definitions of Group A and R8 are the same as those of Group A and R8 in Formula III or IIIA above. 根据权利要求1-25中任何一项所述的方法，其中所述含药连接子选自以下化合物或其药学可接受的盐、立体异构体、溶剂化物或前药：








The method according to any one of claims 1 to 25, wherein the drug-containing linker is selected from the following compounds or pharmaceutically acceptable salts, stereoisomers, solvates or prodrugs thereof:








PCT/CN2024/099341 2023-06-14 2024-06-14 Method for preparing antibody-drug conjugate Pending WO2024255873A1 (en)

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CN109600993A (en) * 2016-06-08 2019-04-09 艾伯维公司 Anti-EGFR Antibody Drug Conjugates
CN113423730A (en) * 2019-02-15 2021-09-21 上海药明生物技术有限公司 Method for preparing antibody-drug conjugates with improved homogeneity
WO2022078524A2 (en) * 2021-11-03 2022-04-21 Hangzhou Dac Biotech Co., Ltd. Specific conjugation of an antibody

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105899235A (en) * 2014-01-10 2016-08-24 斯索恩生物制药有限公司 Method for purifying CYS-linked antibody-drug conjugates
CN106729743A (en) * 2015-11-23 2017-05-31 四川科伦博泰生物医药股份有限公司 Anti-ErbB 2 antibodies-drug conjugates and combinations thereof, preparation method and application
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