WO2023016488A1 - 一种基于微管抑制剂的抗体偶联药物 - Google Patents

一种基于微管抑制剂的抗体偶联药物 Download PDF

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WO2023016488A1
WO2023016488A1 PCT/CN2022/111443 CN2022111443W WO2023016488A1 WO 2023016488 A1 WO2023016488 A1 WO 2023016488A1 CN 2022111443 W CN2022111443 W CN 2022111443W WO 2023016488 A1 WO2023016488 A1 WO 2023016488A1
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compound
pharmaceutically acceptable
stereoisomer
acceptable salt
antibody
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PCT/CN2022/111443
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English (en)
French (fr)
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丁会
于海勇
许云雷
劳芳
刘岩
张西东
荣鹏飞
柯天一
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昆山新蕴达生物科技有限公司
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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
    • 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
    • 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 disclosure relates to antibody drug conjugates (ADCs) that bind human oncology antigen targets and/or provide anti-tubulin drug activity.
  • ADCs antibody drug conjugates
  • the present disclosure further relates to methods and compositions useful for treating cancers that express tumor antigen targets and/or are treatable by disrupting tubulin.
  • antibody-drug conjugates obtained by coupling monoclonal antibodies to toxin small molecules have become a hot spot in tumor-targeted therapy.
  • Antibody-drug conjugates (antibody-drug conjugates, ADCs) connect biologically active small-molecule drugs to monoclonal antibodies through a chemical link, and monoclonal antibodies serve as carriers to target and transport small-molecule drugs into target cells.
  • Research on ADC can be traced back to the 1980s, but it was not until 2000 that the first antibody-conjugated drug (trade name Mylotarg, developed by Pfizer) was approved by the FDA for the treatment of acute myeloid leukemia, and the fatal toxicity caused by immature technology was Withdrawal from the market in 2010.
  • brentuximabvedotin SGN-35, trade name Adcetris
  • Ado-trastuzumabemtansine T-DM1, trade name Kadcyla
  • Genentech/ImmunoGen was approved by the FDA for HER2-positive breast cancer, becoming the first antibody-conjugated drug for solid tumors.
  • ADCs there are 12 kinds of ADCs that have been approved for marketing, and the products whose effects have attracted attention include IMMU-132, Daiichi Sankyo's ds-8210a, etc.
  • ADC consists of antibodies, small molecule toxins and linkers. Small-molecule toxins are covalently coupled to antibodies through linkers; antibodies (such as monoclonal antibodies) can specifically recognize specific targets on the surface of tumor cells, and then guide ADCs to reach the surface of cancer cells, and allow ADCs to be internalized and absorbed. Kill tumor cells.
  • the small-molecule toxins used in ADC are divided into two categories: DNA damage agents and microtubule inhibitors.
  • the former includes camptothecins, and the latter includes dolastatin and its auristatins derivatives (MMAE, MMAF, MMAD).
  • MMAE, MMAF, MMAD dolastatin and its auristatins derivatives
  • DM1, DM2, DM3, DM4 maytansine and maytansinoid derivatives maytansinoids.
  • the small molecule toxin used by Kadcyla is DM1.
  • Eribulin belongs to the class of tubulin polymerization inhibitors, which is a synthetic analogue of halichondrin B, a substance discovered from black sponges growing off the coast of Japan in 2010 Approved by the US FDA for the first time for the treatment of metastatic breast cancer, it can effectively cure the tumor, and it is the only single-drug chemotherapy drug. In addition to its remarkable therapeutic effect on metastatic breast cancer, Eribulin can be used for multiple indications, so its late-stage development space is huge, and it is a drug with great ADC development value.
  • One of the most common attachment sites on antibodies for ADC conjugation is lysine, whose ⁇ -amino group can react with the activated carboxyl group of the linker to form an amide bond.
  • amide bonds are prone to hydrolysis under the action of enzymes in the body, resulting in the shedding of bioactive molecules and antibodies before reaching the target cells, which increases the toxicity while losing the targeting of ADC.
  • Another common attachment site is a cysteine residue.
  • the sulfur groups of antibody cysteine exist in the form of disulfide bonds. Opening disulfide bonds in antibodies can provide multiple free sulfhydryl groups as conjugation sites.
  • one method is Michael addition reaction between the free sulfhydryl group on the antibody and maleimide, or a specific substrate and the free sulfhydryl group on the antibody through two Michael addition reactions to form A structurally unique sulfur bridge.
  • Michael addition reaction between the free sulfhydryl group on the antibody and maleimide, or a specific substrate and the free sulfhydryl group on the antibody through two Michael addition reactions to form A structurally unique sulfur bridge.
  • the small molecule toxin eribulin is at least 10 times more toxic than DNA-damaging toxoids such as camptothecin derivatives, so when developing ADC drugs targeting eribulin, it is It is more necessary to consider the issue of its safety.
  • ADC drugs targeting eribulin it is It is more necessary to consider the issue of its safety.
  • the present disclosure provides an antibody-drug conjugate (hereinafter referred to as ADC) capable of inhibiting tumor cells.
  • ADC antibody-drug conjugate
  • the ADC has an improved linker part, which has an antibody coupling reaction group that is not easy to break, and a more specific enzyme cleavage site than the acid-base sensitive linker, thereby increasing the blood flow stability of the ADC and reducing off-target effects
  • DAR high drug loading capacity
  • the first aspect of the present disclosure provides the compound represented by formula (I), its pharmaceutically acceptable salt, its stereoisomer, or the solvent of said compound, its pharmaceutically acceptable salt or its stereoisomer compound,
  • L1 is L 2 is a single bond or -NH-R 1 -(CO)-,
  • L 2 is -R 1 -(CO)-;
  • R 1 is selected from -(CH 2 ) a -, -(CH 2 CH 2 O) b -(CH 2 ) c -, -CH 2 CH 2 -(OCH 2 CH 2 ) b -NH-(CO)-CH 2 -O-CH 2 -;
  • L3 is a peptide residue selected from glycine-glycine-phenylalanine-glycine (GGFG), glutamic acid-valine-citrulline (EVC), valine-citrulline (VC), natural Aspartic acid-valine-citrulline (DVC), glutamic acid-glycine-glycine-phenylalanine-glycine (EGGFG), aspartic acid-glycine-glycine-phenylalanine-glycine (DGGFG ), whose structural formulas are:
  • L 4 is selected from single bond, -NH-CH 2 -,
  • D is a drug linked to L4 through a chemical bond, and the drug is selected from eribulin or its derivatives, camptothecin drugs, auristatins (such as monomethyl auristatin E (MMAE), monomethyl auristatin E (MMAE), Auristatin F (MMAF));
  • auristatins such as monomethyl auristatin E (MMAE), monomethyl auristatin E (MMAE), Auristatin F (MMAF)
  • a is selected from an integer between 1-6, such as 1, 2, 3, 4, 5, 6;
  • b is independently selected from an integer between 1-20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, for example, an integer selected from 1-8;
  • c 1 or 2.
  • L3 is selected from GGFG, VEC, VC.
  • R 1 is -(CH 2 ) a -. In certain embodiments, R 1 is -(CH 2 CH 2 O) b -(CH 2 ) c -. In certain embodiments, R 1 is -CH 2 CH 2 -(OCH 2 CH 2 ) b -NH-(CO)-CH 2 -O-CH 2 -.
  • L 2 is a single bond. In certain embodiments, L2 is -NH- R1- (CO)-.
  • L3 is GGFG. In certain embodiments, L3 is VEC. In certain embodiments, L3 is VC.
  • L4 is -NH- CH2- . In certain embodiments, L4 is a single bond. In certain embodiments, L4 is
  • L is L 2 is -NH-R 1 -(CO)-, R 1 is -(CH 2 ) a -, and a is 2, 3, 4, 5 or 6.
  • L is L 2 is -NH-R 1 -(CO)-, R 1 is -(CH 2 CH 2 O) b -(CH 2 ) c -, b is 2, 3, 4, 5, 6, 7 or 8, c is 1 or 2.
  • L is L 2 is a single bond.
  • L is L 2 is -NH-R 1 -(CO)-, R 1 is -CH 2 CH 2 -(OCH 2 CH 2 ) b -NH-(CO)-CH 2 -O-CH 2 -, b is 2, 3, 4, 5, 6, 7 or 8.
  • L 1 -L 2 are Wherein, L 2 is -R 1 -(CO)-, R 1 is -CH 2 CH 2 -(OCH 2 CH 2 ) b -NH-(CO)-CH 2 -O-CH 2 -; in some implementations In the scheme, b is 2, 3, 4, 5, 6, 7 or 8.
  • L is L 2 is -NH-R 1 -(CO)-, R 1 is -(CH 2 ) a -, a is 2, 3, 4, 5 or 6, and L 3 is GGFG.
  • L is L 2 is -NH-R 1 -(CO)-, R 1 is -(CH 2 CH 2 O) b -(CH 2 ) c -, b is 2, 3, 4, 5, 6, 7 or 8, c is 1 or 2, L 3 is GGFG or EVC.
  • L is L 2 is a single bond and L 3 is GGFG.
  • L is L 2 is -NH-R 1 -(CO)-, R 1 is -CH 2 CH 2 -(OCH 2 CH 2 ) b -NH-(CO)-CH 2 -O-CH 2 -, b is 2, 3, 4, 5, 6, 7 or 8, L 3 is EVC.
  • L 1 -L 2 are Wherein, L 2 is -R 1 -(CO)-, R 1 is -CH 2 CH 2 -(OCH 2 CH 2 ) b -NH-(CO)-CH 2 -O-CH 2 -, b is 2, 3, 4, 5, 6, 7 or 8, L 3 is EVC.
  • L is L 2 is a single bond
  • L 3 is VC or GGFG.
  • L is selected from single bonds
  • L 1 -L 2 are
  • L3 is GGFG and L4 is a single bond.
  • L3 is GGFG and L4 is -NH- CH2- .
  • L 3 is EVC and L 4 is
  • L3 is GGFG and L4 is
  • D is eribulin or a derivative thereof. In certain embodiments, D is selected from
  • L4 is -NH- CH2- and D is d1.
  • L4 is -NH- CH2- and D is d2.
  • L4 is -NH- CH2- and D is d3.
  • L4 is a single bond and D is d2.
  • L4 is D is d2.
  • the compound shown in formula (I) is selected from:
  • the compound of formula (I) can be used as a linker-drug intermediate compound for the synthesis of antibody-drug conjugates. Therefore, the second aspect of the present disclosure provides an antibody-drug conjugate, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or the conjugate, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof.
  • the solvate of the isomer can be formed by linking the targeting moiety with the compound represented by formula (I) through a thioether bond.
  • the antibody-drug conjugate has a structure as shown in formula (II)
  • Ab is a targeting moiety selected from antibodies, antibody fragments, or antibody-based molecules or compounds;
  • L 1 , L 2 , L 3 , L 4 , D are as defined above,
  • p is an integer selected from 1-20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, for example, an integer selected from 1-8.
  • the Ab targeting moiety is an antibody, antibody fragment, bispecific or other multivalent antibody, or other antibody-based molecule or compound.
  • Antibodies may be of various isotypes, preferably human IgGl, IgG2, IgG3 or IgG4, more preferably comprising human IgGl hinge and constant region sequences.
  • Antibodies or fragments thereof may be chimeric human-mouse, chimeric human-primate, humanized (human framework and murine hypervariable (CDR) regions) or fully human antibodies and variants thereof, such as half IgG4 antibodies (referred to as "single antibody”), as described by van der Neut Kolfschoten et al. (Science 2007; 317:1554-1557).
  • antibodies or fragments thereof can be designed or selected to comprise human constant region sequences belonging to a particular allotype, which can result in reduced immunogenicity when the antibody or immunoconjugate is administered to a human subject.
  • Preferred allotypes for administration include non-G1m1 allotypes (nG1m1), such as G1m3, G1m3,1, G1m3,2 or G1m3,1,2. More preferably, the allotype is selected from the group consisting of nG1m1, G1m3, nG1m1,2 and Km3 allotypes.
  • Suitable antibodies can bind any disease-associated antigen known in the art.
  • the disease state is cancer
  • a number of antigens expressed by or otherwise associated with tumor cells are known in the art, including but not limited to carbonic anhydrase IX, alpha-fetoprotein , AFP), ⁇ -actinin-4 ( ⁇ -actinin-4), A3, antigen specific to A33 antibody, ART-4, B7, Ba 733, BAGE, BrE3 antigen, CA125, CAMEL, CAP -1, CASP-8/m,, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67
  • IGF-1 insulin-like growth factor 1
  • IGF-1R insulin-like growth factor 1
  • KS1-4 insulin-like growth factor 1
  • MIF macrophage migration inhibitory factor
  • MAGE MAGE-3
  • MART -1 MART-2
  • NY-ESO-1 NY-ESO-1
  • TRAG-3 mCRP
  • MCP-1 MIP-1 A.
  • MIP-1B MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, pancreatic cancer mucin, PD-1 receptor, PD -L1 receptor, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, S100, Survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptor, TNF- ⁇ , Tn antigen, Thomson-Friedenreich antigen , tumor necrosis antigen, VEGFR, ED-B fibronectin, WT-1, 17-1A antigen, complement factors C3, C3a, C3b, C5a, C5, angiogenesis markers, bcl-2, b
  • Exemplary antibodies that may be utilized include, but are not limited to, hR1 (anti-IGF-1R, U.S. Patent No. 13/688,812), hPAM4 (anti-Mucin, U.S. Patent No. 7,282,567), hA20 (anti-CD20, U.S. Patent No. 7,151,164), hA19 ( Anti-CD19, U.S. Patent No. 7,109,304), hIMMU31 (anti-AFP, U.S. Patent No. 7,300,655), hLL1 (anti-CD74, U.S. Patent No. 7,312,318), hLL2 (anti-CD22, U.S. Patent No.
  • the antibody is IMMU-31 (anti-AFP), hRS7 (anti-Trop-2), hMN-14 (anti-CEACAM5), hMN-3 (anti-CEACAM6), hMN-15 (anti-CEACAM6), hLL1 (anti-CD74 ), hLL2 (anti-CD22), hL243 or IMMU-114 (anti-HLA-DR), hA19 (anti-CD19) or hA20 (anti-CD20).
  • the terms epratuzumab and hLL2 are interchangeable, as are the terms veltuzumab and hA20, hL243g4P, hL243 ⁇ 4P, and IMMU-114.
  • the antibody is an anti-Trop-2 antibody such as hRS7; an anti-ROR1 antibody such as 99961 (Chinese Patent No. 104662044).
  • Suitable alternative antibodies include, but are not limited to, abciximab (anti-glycoprotein IIb/IIIa), alemtuzumab (anti-CD52), bevacizumab (anti-VEGF), Western Cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab (anti-CD20), panitumumab (anti-EGFR), Rituximab (anti-CD20), tositumomab (anti-CD20), trastuzumab (anti-ErbB2), lambrolizumab (anti-PD1 receptor ), atezolizumab (anti-PD-L1), MEDI4736 (anti-PD-L1), nivolumab (anti-PD-1 receptor), ipilimumab (anti-PD-1 CTLA-4), abagovomab (anti-CA-125), adecatumumab (anti-EpCAM), atlizumab (anti
  • the targeting moiety is an hRS7 antibody.
  • the light chain and heavy chain amino acid sequences of the hRS7 antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and the nucleotide sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • the drug moiety conjugated to the subject antibody is selected from eribulin or a derivative thereof, eg, the mesylate salt of eribulin.
  • eribulin refers to a synthetic analog of halichondrin B, a macrocyclic compound originally isolated from the sponge Halichondria okadais. Eribulin, an inhibitor of microtubule dynamics, is thought to bind tubulin and cause cell cycle arrest in G2/M phase by inhibiting mitotic spindle assembly.
  • eribulin mesylate refers to the mesylate salt of eribulin, which is sold under the tradename Halaven TM .
  • the drug part can also choose a water-soluble camptothecin compound or its derivatives, such as exatecan, irinotecan, gemnotecan, dxd, gemotecan, SN-38 or CPT-11.
  • a water-soluble camptothecin compound or its derivatives such as exatecan, irinotecan, gemnotecan, dxd, gemotecan, SN-38 or CPT-11.
  • the drug part can also choose auristatin (for example, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF)).
  • auristatin for example, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF)
  • the antibody or fragment thereof is linked to at least one chemotherapeutic moiety; for example 1 to 5 (eg 1, 2, 3, 4 or 5) drug moieties, 5 to 8 (eg 5, 6 , 7 or 8) drug moieties or 8 to 12 (eg 8, 9, 10, 11 or 12) drug moieties.
  • chemotherapeutic moiety for example 1 to 5 (eg 1, 2, 3, 4 or 5) drug moieties, 5 to 8 (eg 5, 6 , 7 or 8) drug moieties or 8 to 12 (eg 8, 9, 10, 11 or 12) drug moieties.
  • the present disclosure provides the compound, pharmaceutically acceptable salt, stereoisomer or solvate of the first aspect, and the antibody-drug conjugate and pharmaceutically acceptable salt of the second aspect , A method for the preparation of a stereoisomer or a solvate.
  • the method comprises the following steps:
  • the method comprises the following steps:
  • L 1 -L 2 can be prepared first, and then synthesized by referring to the above three methods.
  • L 1 -L 2 can be formed through the condensation reaction of carboxyl and amino groups to form amide bonds, or can be formed through the click reaction of azide-alkynyl groups to form L 1 -L 2 .
  • an antibody-drug conjugate in which an antibody and a linker structure are linked via a thioether can be produced by the following method.
  • L 1 , L 2 , L 3 , L 4 , D, Ab, and S are as described in the description of the present invention.
  • the antibody-drug conjugate represented by formula (II) is described in the form of a structure in which one structural part from the drug to the end of the linker is linked to one antibody, but in fact, relatively A plurality of such structural parts are often linked to one antibody molecule.
  • 2 to 8, preferably 4 to 8, and more preferably 6 to 8 linker-drug intermediate compounds are linked to one antibody molecule.
  • the average number of linker-drugs linked to each antibody molecule is represented by the average number of drug links.
  • the antibody-drug conjugate represented by the formula (II) can be produced by reacting the above-mentioned linker-drug intermediate compound of the present invention with the antibody Ab-SH having a sulfhydryl group.
  • a reducing agent 1 to 7 molar equivalents of TCEP are used per one hinge disulfide bond in the antibody, and reacted with the antibody in a buffer containing the chelating agent EDTA, thereby obtaining a partial Or an antibody (AB-SH) carrying a sulfhydryl group obtained by completely reducing the disulfide in the hinge part of the antibody.
  • a chelating agent ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), etc. are mentioned, for example. They can be used at a concentration of 1 mM to 20 mM.
  • the buffer solution sodium phosphate, sodium borate, sodium acetate solution or the like can be used.
  • antibody AB-SH having a sulfhydryl group can be obtained by reacting the antibody with TCEP at 4°C to 37°C for 1 to 4 hours.
  • each antibody Ab-SH having a sulfhydryl group 2 to 20 molar equivalents of the compound represented by formula (I) can be used to produce an antibody-drug conjugate in which one antibody is linked with two to eight drugs ( II).
  • a solution in which the compound represented by the formula (I) is dissolved is added to a buffer containing the antibody Ab-SH having a sulfhydryl group, and reacted.
  • the buffer sodium acetate solution, sodium phosphate, sodium borate, or the like can be used as the buffer.
  • the pH at the time of reaction is 5-9, and it is more preferable to react around pH7.
  • organic solvents such as dimethylsulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), and N-methyl-2-pyridone (NMP) can be used .
  • DMSO dimethylsulfoxide
  • DMF dimethylformamide
  • DMA dimethylacetamide
  • NMP N-methyl-2-pyridone
  • the organic solvent solution in which the compound represented by the formula (I) is dissolved can be added to the buffer containing the antibody Ab-SH having a sulfhydryl group at 1 to 20% v/v and reacted.
  • the reaction temperature is 0-37°C, more preferably 10-25°C, and the reaction time is 0.5-2 hours.
  • the reaction can be terminated by inactivating the reactivity of the unreacted compound represented by formula (I) with a thiol-containing reagent.
  • Thiol-containing reagents are, for example, cysteine or N-acetyl-L-cysteine (NAC). More specifically, the reaction is completed by adding 1 to 2 molar equivalents of cysteine to the compound represented by the formula (I) used and incubating at room temperature for 10 to 30 minutes.
  • cysteine or N-acetyl-L-cysteine (NAC). More specifically, the reaction is completed by adding 1 to 2 molar equivalents of cysteine to the compound represented by the formula (I) used and incubating at room temperature for 10 to 30 minutes.
  • centrifuge to perform centrifugation (for example, centrifuge at 2000G-3800G for 5-20 minutes) to concentrate the antibody or antibody-drug conjugate solution.
  • Antibody concentration was measured using a UV analyzer according to the method specified by the manufacturer.
  • phosphate buffer e.g. PBS
  • sodium chloride e.g., 137 mM
  • EDTA ethylenediaminetetraacetic acid
  • a PD-10 column using Sephadex G-25 carrier was equilibrated.
  • 1 mL of an antibody aqueous solution was loaded, and then a fraction (3.5 mL) eluted with PBS/EDTA 2 mL was separated. This fraction was concentrated by common procedure A, and the antibody concentration was measured by common procedure B, and then adjusted to 10 mg/mL using PBS/EDTA.
  • Phosphate buffer eg, 50 mM, pH 6.5, also referred to as PBS6.5/EDTA in this specification
  • sodium chloride eg, 50 mM
  • EDTA eg, 2 mM
  • a PD-10 column using Sephadex G-25 carrier was equilibrated.
  • 1 mL of antibody aqueous solution was loaded, and then the fraction (3.5 mL) eluted with PBS6.5/EDTA 2 mL was separated and obtained. This fraction was concentrated by common procedure A, and the antibody concentration was measured by common procedure B, and then the antibody concentration was adjusted to 5 mg/mL using PBS6.5/EDTA.
  • phosphate buffer eg, PBS7.4
  • sodium phosphate buffer eg, 10 mM, pH6.5; also referred to as PBS6.5 in this specification
  • Acetic acid buffer e.g., 10 mM, pH 5.5; also referred to as ABS in this specification
  • sorbitol e.g., 5%
  • An antibody-drug conjugate reaction aqueous solution (for example, about 1 mL) was packed in this column, and the antibody fraction was separated and obtained by eluting with an amount of buffer solution specified by the manufacturer.
  • Drug linkers, antibody-drug conjugates of low molecular weight compounds tris(2-carboxyethyl)phosphine hydrochloride (TCEP), cysteine, dimethyl sulfoxide, etc.
  • Phosphate buffer eg, PBS7.4
  • sodium phosphate buffer eg, 10 mM, pH6.5; also referred to as PBS6.5 in this specification
  • sodium chloride eg, 137 mM
  • sorbitol-containing for example, 5%
  • acetate buffer for example, 10mM, pH5.5; also referred to as ABS in this specification
  • MES 25mM pH6.5 or any buffer solution in His 10mM pH 5.5 AKTA column ( Filler: Sephadex G 25) Balanced.
  • the injector is loaded with an antibody-drug conjugate reaction aqueous solution (eg, about 2 mL), and eluted with an amount of buffer solution specified by the manufacturer, thereby separating and obtaining antibody fractions.
  • an antibody-drug conjugate in which unlinked drug linkers and low-molecular compounds (tris(2-carboxyethyl)phosphine hydrochloride (TCEP), cysteine, dimethyl sulfoxide, etc.) were removed was obtained. United things.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the compound, pharmaceutically acceptable salt, stereoisomer, or solvate described in the first aspect, or the conjugated compound described in the second aspect substances, pharmaceutically acceptable salts, stereoisomers, or solvates, and one or more pharmaceutical adjuvants, such as carriers and/or excipients.
  • the pharmaceutical composition can be made into any pharmaceutically acceptable dosage form.
  • the pharmaceutical composition may also be administered to an individual in need of such treatment by any suitable means of administration, such as oral, parenteral, rectal or pulmonary administration.
  • the pharmaceutical composition can be made into conventional solid preparations, such as tablets, capsules, pills, granules, etc.; it can also be made into oral liquid preparations, such as oral solutions, oral suspensions , syrup, etc.
  • suitable fillers, binders, disintegrants, lubricants and the like can be added.
  • parenteral administration the pharmaceutical composition can be made into injections, including injections, sterile powders for injections and concentrated solutions for injections.
  • the pharmaceutical composition When making injections, it can be produced by conventional methods in the existing pharmaceutical field. When preparing injections, no additives can be added, and suitable additives can also be added according to the properties of the medicine.
  • the pharmaceutical composition For rectal administration, the pharmaceutical composition can be made into suppositories and the like.
  • the pharmaceutical composition When used for pulmonary administration, can be made into inhalants or sprays and the like.
  • the present disclosure provides the compound, pharmaceutically acceptable salt, stereoisomer, or solvate described in the first aspect, or the conjugate, pharmaceutically acceptable salt, Use of stereoisomers or solvates in the preparation of medicines for treating diseases related to abnormal cell activity (such as cancer diseases).
  • the present disclosure provides the compound, pharmaceutically acceptable salt, stereoisomer, or solvate described in the first aspect, or the conjugate, pharmaceutically acceptable salt, Stereoisomers, or solvates, or the use of the pharmaceutical composition of the fourth aspect for treating diseases related to abnormal cell activity (such as cancer diseases).
  • the present disclosure provides a method for treating a disease related to abnormal cell activity (such as cancer disease), including providing an effective dose of the present disclosure to an individual in need of the compound described in the first aspect, a pharmaceutically acceptable salt , stereoisomer, or solvate, or the conjugate, pharmaceutically acceptable salt, stereoisomer, or solvate of the second aspect, or the pharmaceutical composition of the fourth aspect.
  • Dosage regimens may be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Dosage values may vary with the type and severity of the condition to be alleviated and may comprise single or multiple doses. It is further understood that for any given individual, the particular dosing regimen may be adjusted over time according to the needs of the individual and the professional judgment of the person administering the composition or supervising the administration of the composition.
  • Various embodiments may relate to using the subject methods and compositions to treat cancer, including but not limited to metastatic breast cancer, non-small cell lung cancer, Burkitt lymphoma, Hodgkin's lymphoma, acute myeloid Leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytes, skin cancer, oral cancer, esophagus cancer, gastrointestinal tract cancer, lung cancer, lung cancer, stomach cancer, colon cancer, rectal cancer, triple-negative breast cancer, Ovarian cancer, prostate cancer, uterine cancer, endometrial cancer, cervical cancer, bladder cancer, pancreatic cancer, bone cancer, brain cancer, connective tissue cancer, thyroid cancer, liver cancer, gallbladder cancer, bladder (urothelial) cancer, kidney cancer, skin cancer, central nervous system cancer and testicular cancer.
  • metastatic breast cancer non-small cell lung cancer, Burkitt lymphoma, Hodgkin's lymphoma, acute myeloid Leukemia, chronic myelogenous leuk
  • the antibody or immunoconjugate can be combined with surgery, radiation therapy, chemotherapy, immunotherapy with naked antibodies including checkpoint inhibitory antibodies, radioimmunotherapy, immunomodulators, vaccines and other combinations.
  • the antibody or immunoconjugate is used in combination with a PARP inhibitor, microtubule inhibitor, Bruton kinase inhibitor and/or PI3K inhibitor.
  • combination therapies may allow lower doses of each therapeutic agent to be administered in the context of the combination, thus reducing certain serious side effects and potentially reducing the required course of treatment.
  • the full doses of each can also be administered in the absence or presence of minimal overlapping toxicity.
  • the antibody or immunoconjugate can be administered as a periodic bolus injection, in alternative embodiments, the antibody or immunoconjugate can be administered by continuous infusion.
  • continuous infusion can be administered, eg, through an indwelling catheter.
  • indwelling catheter Such devices are known in the art, such as or catheter (see, eg, Skolnik et al., The Drug Monit 32:741-48, 2010), and any such known indwelling catheter can be used.
  • a variety of continuous infusion pumps are also known in the art, and any such known infusion pump can be used.
  • the antibody or immunoconjugate and schedule of administration may be effective in patients resistant to standard therapy.
  • hRS7-eribulin immunoconjugates can be administered to patients who have not responded to previous therapy with eribulin.
  • the ability of immunoconjugates to specifically target tumor tissue can overcome tumor resistance due to improved targeting and enhanced delivery of therapeutic agents.
  • a particularly preferred subject may have Trop-2 positive breast cancer, ovarian cancer, cervical cancer, endometrial cancer, lung cancer, prostate cancer, colon cancer, rectal cancer, gastric cancer, esophageal cancer, bladder (urothelial) cancer , kidney cancer, pancreatic cancer, brain cancer, thyroid cancer, epithelial cancer, or head and neck cancer.
  • the cancer is metastatic cancer. More preferably, the patient has previously failed treatment with at least one standard anticancer therapy.
  • the present disclosure provides a pharmaceutical preparation comprising the compound, pharmaceutically acceptable salt, stereoisomer, or solvate described in the first aspect, or the conjugate described in the second aspect , a pharmaceutically acceptable salt, stereoisomer, or solvate, or the pharmaceutical composition of the fourth aspect.
  • the present disclosure provides the compound, pharmaceutically acceptable salt, stereoisomer, or solvate described in the first aspect, or the conjugate or pharmaceutically acceptable salt described in the second aspect , stereoisomers, or solvates, or the pharmaceutical composition of the fourth aspect for the preparation of pharmaceutical preparations.
  • the present disclosure provides a kit comprising the compound, pharmaceutically acceptable salt, stereoisomer, or solvate described in the first aspect, or the conjugate described in the second aspect , a pharmaceutically acceptable salt, stereoisomer, or solvate, or the pharmaceutical composition of the fourth aspect, or the pharmaceutical preparation of the eighth aspect.
  • the compounds, conjugates, pharmaceutically acceptable salts, stereoisomers, or solvates, pharmaceutical preparations or kits provided in this disclosure can be used to inhibit the growth, proliferation or migration of cancer cells.
  • the compounds, conjugates, pharmaceutically acceptable salts, stereoisomers, or solvates, pharmaceutical formulations or kits are for in vivo or in vitro administration; e.g., administered into a subject, or administered in vitro Cells (eg cell lines or cells from an individual such as cancer cells).
  • the present disclosure provides a method for inhibiting the growth, proliferation or migration of cancer cells, which comprises administering to cancer cells an effective amount of the compound, pharmaceutically acceptable salt, stereoisomeric Conformer, or solvate, or the conjugate, pharmaceutically acceptable salt, stereoisomer, or solvate described in the second aspect, or the pharmaceutical composition of the fourth aspect, or the pharmaceutical composition of the eighth aspect Pharmaceutical preparations.
  • linker refers to a chemical structural fragment or bond that is connected to an antibody at one end and a drug (drug compound) at the other end, and other linkers can also be connected It is then linked to a drug compound.
  • linker structure of the present invention can be synthesized by methods known in the art, and can also be synthesized using the methods described in the present invention.
  • the "antibody-drug conjugate" in the present invention refers to that the targeting moiety is linked to a drug with biological activity through a stable linking unit.
  • “pharmaceutically acceptable salt” refers to a relatively non-toxic acid addition salt or base addition salt of the conjugate of the present invention.
  • the acid addition salt is a salt formed by the conjugate of the present invention and a suitable inorganic acid or organic acid, and these salts can be carried out by making the conjugate of the present invention and a suitable organic acid or inorganic acid in a suitable solvent reaction to prepare.
  • Representative acid addition salts include hydrobromide, hydrochloride, sulfate, bisulfate, sulfite, acetate, oxalate, valerate, oleate, palmitate, stearate Salt, Luurosilicate, Borate, Benzoate, Lactate, Nitrate, Phosphate, Phosphate, Carbonate, Bicarbonate, Toluate, Citrate, Maleic Acid Salt, fumarate, succinate, malate, ascorbate, tannate, pamoate, alginate, naphthalenesulfonate, tartrate, benzoate, methanesulfonate, p-toluene Sulfonate, Gluconate, Lactobionate and Lauryl Sulfonate etc.
  • the base addition salt is a salt formed by the conjugate of the present invention and a suitable inorganic base or organic base, and these salts can be carried out by making the conjugate of the present invention and a suitable inorganic base or organic base in a suitable solvent reaction to prepare.
  • Representative base addition salts include, for example, salts with alkali metal, alkaline earth metal, quaternary ammonium cations, such as sodium, lithium, potassium, calcium, magnesium, tetramethylquaternary ammonium, tetraethylquaternary ammonium Salts, etc.; amine salts, including salts formed with ammonia (NH3), primary amines, secondary amines or tertiary amines, such as methylamine salts, dimethylamine salts, trimethylamine salts, triethylamine salts, ethylamine salts, etc.
  • quaternary ammonium cations such as sodium, lithium, potassium, calcium, magnesium, tetramethylquaternary ammonium, tetraethylquaternary ammonium Salts, etc.
  • amine salts including salts formed with ammonia (NH3), primary amines, secondary amines or tertiary amines, such as methylamine salts
  • the conjugate of the present invention may exist in a specific geometric or stereoisomer form.
  • the chiral center may exist in the drug, in the linker structure, or in the antibody and its derivatives. in things.
  • all such compounds including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, are included in the present invention within the range.
  • Optically active (R)- and (S)-isomers as well as D and L-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a conjugate of the invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide Pure desired enantiomer.
  • a diastereoisomeric salt is formed with an appropriate optically active acid or base, and then a diastereomeric salt is formed by a conventional method known in the art. Diastereomeric resolution is performed and the pure enantiomers are recovered. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using chiral stationary phases, optionally in combination with chemical derivatization methods (e.g. amines to amino groups formate).
  • solvates such as hydrates of the compounds, conjugates, pharmaceutically acceptable salts, and stereoisomers of the present invention are also within the scope of the present invention.
  • suitable solvates include solvates of the conjugate of the present invention with acetone, 2-butanol, 2-propanol, ethanol, ethyl acetate, tetrahydrofuran, diethyl ether, and the like. Hydrates or ethanolates are also mentioned.
  • treating an individual suffering from a disease or condition means that the individual's symptoms are partially or completely relieved, or remain unchanged after treatment.
  • treatment includes prophylaxis, treatment and/or cure.
  • Prevention refers to preventing an underlying disease and/or preventing worsening of symptoms or development of a disease.
  • Treatment also includes any pharmaceutical use of the provided ADCs and the pharmaceutical compositions, pharmaceutical formulations provided herein.
  • therapeutic effect means the effect resulting from the treatment of an individual, which alters, usually ameliorates or improves the symptoms of a disease or condition, or cures the disease or condition.
  • “individual” includes human or non-human animal.
  • exemplary human subjects include human subjects suffering from a disease (eg, a disease described herein) (referred to as a patient) or normal subjects.
  • non-human animals include all vertebrates, such as non-mammals (such as birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and/or domesticated animals (such as sheep, dogs, cats, cows, pigs, etc.).
  • compositions and methods are not to be limited to the particular compositions and methods described and/or illustrated herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting. Compositions and methods claimed.
  • compositions and methods of using the compositions refers to compositions and methods of using the compositions.
  • present disclosure describes or claims features or embodiments in relation to a composition, such features or embodiments apply equally to methods of using said compositions.
  • present disclosure describes or claims a feature or embodiment in relation to a method of using the composition, such feature or embodiment applies equally to said composition.
  • compositions and methods which are described herein in the context of separate embodiments, are, for clarity, also provided in combination in a single embodiment. Conversely, various features of the disclosed compositions and methods that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.
  • Fig. 1 is the SEC-HPLC result of the conjugate ADC-4 synthesized in Example 2.
  • Fig. 2 is the statistical curve of antitumor activity in Example 5.
  • ADC-137 is only used as a control, in which the linker-toxin small molecule part is purchased from Changzhou Chenhong Biotechnology Co., Ltd., CAS#: 1279680-68-0, and the antibody coupling method is the same as ADC-4 below .
  • the structure of ADC-137 is as follows:
  • hRS7 antibody was produced in CHO cells.
  • the expression vectors containing the hRS7 antibody gene were constructed by conventional molecular biology methods.
  • the amino acid sequences of the hRS7 antibody light chain and heavy chain are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively, and the corresponding nucleotide sequences Shown in SEQ ID NO:3 and SEQ ID NO:4 respectively.
  • Insert the above two sequences into the same expression vector extract a large number of transfection plasmids, and transfect them into CHO-K1 cells (ATCC CCL-61).
  • the specific transfection and antibody preparation processes are as follows:
  • Cell culture CHO-K1 cells were grown in suspension in ActiPro (GE HyClone) medium, and cultured at 37°C, 7% CO 2 , 140 rpm, and 90% relative humidity;
  • the highly expressed cell fluid cultured in shake flasks was collected and purified by protein A affinity purification (GE, Mab Select SuRe) and ion exchange purification (GE, Capto S).
  • SDS-PAGE and SEC-HPLC were used to analyze the molecular weight and purity of the purified antibody.
  • the results of SDS-PAGE showed that the molecular weight of the prepared hRS7 was as expected, and the purity of the antibody measured by SEC-HPLC was 98.8%.
  • Embodiment 2 Preparation of conjugate ADC-4
  • DTT dithiothreitol
  • MDA-MB-468 (ATCC HTB-132) cells were cultured in a single layer in vitro.
  • the culture conditions were L-15 medium plus 10% heat-inactivated fetal bovine serum, and cultured in an incubator at 37°C without CO2 air. Change the fluid 2-3 times a week.
  • the cells were in the exponential growth phase, they were digested with 0.25% trypsin, passaged at a ratio of 1:2 to 1:4, and the cells that had grown to the logarithmic growth phase were collected, counted, and inoculated.
  • the P6 tumor tissue was used to evaluate the antitumor activity of the test product.
  • the P5 tumor grows to 600-800 mm 3
  • the tumor-bearing mice are anesthetized with CO 2 and killed, the tumor mass is removed, and the surrounding necrotic tissue is removed, and the tumor mass in good condition is cut into small tumors of 20-30 mm 3
  • Blocks were inoculated to the right shoulder blade of formal experimental mice, and a total of 40 mice were inoculated. Thirteen days after tumor block inoculation, when the average tumor volume reached about 135 mm 3 , the mice with too small or too large tumor volume were excluded, and the remaining 20 mice were randomly grouped according to the tumor volume and started to be administered. See the table below for the dosing regimen.
  • Only one time refers to no administration after the first administration on the day of grouping; the dosage is calculated according to the dosage of eribulin in ADC.
  • tumor volume (mm 3 ) 1/2 ⁇ (a ⁇ b 2 ) (where a represents the long diameter and b represents the short diameter).
  • the relative tumor proliferation rate, T/C% is the percentage value of the relative tumor volume or tumor weight between the treatment group and the control group at a certain time point.
  • tumors were taken from all mice, weighed and photographed.
  • Figure 2 is the statistical curve of anti-tumor activity. As shown in the figure, in this experiment, the tumor inhibitory activity of ADC-4 and ADC-5 is better than that of ADC-137.

Abstract

涉及医药技术领域，特别涉及一种基于微管抑制剂的抗体偶联药物。具体地，涉及式(I)所示化合物、其药学上可接受的盐、其立体异构体，或所述化合物、其药学上可接受的盐或其立体异构体的溶剂合物，还涉及由靶向部分与式(I)所示化合物、其药学上可接受的盐、其立体异构体，或所述偶联物、其药学上可接受的盐或其立体异构体的溶剂合物经硫醚键连接而成的抗体-药物偶联物。 L 1-L 2-L 3-L 4-D (I)

Description

一种基于微管抑制剂的抗体偶联药物 技术领域
本公开涉及结合人类肿瘤学抗原标靶和/或提供抗微管蛋白药物活性的抗体药物偶联物(ADC)。本公开进一步涉及可用于治疗表达肿瘤抗原靶标和/或通过破坏微管蛋白可治疗的癌症的方法和组合物。
背景技术
肿瘤治疗领域中，采用单克隆抗体偶联毒素小分子获得的抗体药物偶联物(ADC)已经成为而今肿瘤靶向治疗的热点。抗体药物偶联物(antibody-drug conjugate，ADC)是通过一个化学链接将具有生物活性的小分子药物连接到单抗上，单抗作为载体将小分子药物靶向运输到目标细胞中。对ADC的研究可以追溯到1980s，但是直到2000年，首个抗体偶联药物(商品名Mylotarg，Pfizer研发)才被FDA批准用于治疗急性粒细胞白血病，并因为技术不成熟产生的致命毒性于2010年撤市。随着Takeda/SeattleGenetics对原有技术进行改进开发了brentuximabvedotin(SGN-35，商品名Adcetris，)，于2011年被FDA批准用于治疗霍奇金淋巴瘤和系统性间变性大细胞淋巴瘤。2013年，Genentech/ImmunoGen联合开发的Ado-trastuzumabemtansine(T-DM1，商品名Kadcyla)被FDA批准用于HER2阳性乳腺癌，成为首个针对实体瘤的抗体偶联药物。目前，获批上市的ADC有12种，其中效果引发关注的产品有IMMU-132、第一三共的ds-8210a等。
ADC由抗体、小分子毒素及连接体(Linker)组成。小分子毒素通过连接体共价偶联到抗体上；抗体(例如单克隆抗体)能够特异性识别肿瘤细胞表面的特异性靶点，进而能够引导ADC到达癌细胞表面，并使ADC内吞进入并杀灭肿瘤细胞。
用于ADC的小分子毒素分为DNA损伤剂和微管抑制剂两类，前者包括喜树碱类，后者包括海兔毒素及其奥瑞他汀类衍生物auristatins(MMAE、MMAF、MMAD)，以及美登素及美登素类衍生物maytansinoids(DM1、DM2、DM3、DM4)。目前临床在研ADCs项目采用微管蛋白抑制剂的占绝大多数，如Kadcyla采用的小分子毒素即为DM1。2021年，卫材(Eisai)和百时美施贵宝(BMS)达成协议，共同开发和推广靶向叶酸受体α(FRα)的在研抗体偶联药物(ADC)MORAb-202，这款ADC使用的小分子毒素为艾日布林。艾日布林属于微管蛋白聚合抑制剂，它是一种合成的大田软海绵素(halichondrin B)类似物， Halichondrin B是一种从生长在日本沿海的黑色海绵上发现的物质，于2010年首次获美国FDA批准用于转移性乳腺癌的治疗，能够有效治愈肿瘤，该药是唯一的一种单药化疗药物。除了对转移性乳腺癌的治疗作用非常显著外，艾日布林可用于多个适应症，因此其后期开发的空间非常巨大，是一个极具ADC开发价值的药物。
抗体上用于ADC偶联的最常见连接位点，其中之一是赖氨酸，其ε-氨基可以与连接体的活化羧基反应，形成酰胺键。但是此类酰胺键，在体内酶的作用下，易发生水解，导致生物活性分子与抗体在未达到靶细胞时即发生脱落，在丧失ADC的靶向性的同时，增加了毒性。另外一种常见的连接位点是半胱氨酸残基。抗体半胱氨酸的硫基都是以二硫键的形式存在。打开抗体中的二硫键，可以提供多个自由的巯基作为偶联位点。与抗体巯基进行偶联，一种方法是抗体上自由的巯基与马来酰亚胺发生Michael加成反应，也可以是特定的底物与抗体上自由的巯基通过两次Michael加成反应，形成一个结构独特的硫桥键。但有文献报道Michael反应在体循环中发生逆Michael加成，从而造成毒素过早脱落，产生毒性反应。
如上所介绍的，小分子毒素艾日布林(eribulin)与DNA损伤类毒素如喜树碱衍生物相比，其毒性要强至少10倍，因此在针对艾日布林进行ADC药物开发时，则更需要考量其安全性的问题。而目前基于ADC抗体连接位点反应基团的改造和创新较少，尤其针对艾日布林的ADC连接子的开发仍有很大空间，即本领域仍有改进该连接位点反应基团以获得脱靶效应更小，有效性更高的ADC药物的需求。
发明内容
本公开提供一种具有抑制肿瘤细胞作用的抗体-药物偶联物(以下简称ADC)。所述ADC具有改进的连接子部分，其具有不易断裂的抗体偶联反应基团，以及比酸碱敏感性连接子更特异的酶切位点，从而在增加ADC血流稳定性、减少脱靶效应的同时，在到达肿瘤部位后具备快速特异的释放小分子毒素的能力，并具有很高的偶联效率和高载药量(DAR为5-8之间)，与目前上市或者临床热门的ADC相比，在某些肿瘤模型上，具有更好的治疗效果和治疗窗口。
本公开的第一方面，提供式(I)所示化合物、其药学上可接受的盐、其立体异构体，或所述化合物、其药学上可接受的盐或其立体异构体的溶剂合物，
L 1-L 2-L 3-L 4-D
(I)
其中，L 1为
Figure PCTCN2022111443-appb-000001
L 2为单键或-NH-R 1-(CO)-，
或者L 1-L 2为
Figure PCTCN2022111443-appb-000002
其中，L 2为-R 1-(CO)-；
R 1选自-(CH 2) a-、-(CH 2CH 2O) b-(CH 2) c-、-CH 2CH 2-(OCH 2CH 2) b-NH-(CO)-CH 2-O-CH 2-；
L 3为肽残基，选自甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(GGFG)、谷氨酸-缬氨酸-瓜氨酸(EVC)、缬氨酸-瓜氨酸(VC)、天冬氨酸-缬氨酸-瓜氨酸(DVC)、谷氨酸-甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(EGGFG)、天冬氨酸-甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(DGGFG)，其结构式分别为：
Figure PCTCN2022111443-appb-000003
Figure PCTCN2022111443-appb-000004
L 4选自单键、-NH-CH 2-、
Figure PCTCN2022111443-appb-000005
D为与L 4通过化学键相连的药物，所述药物选自艾日布林或其衍生物、喜树碱类药物、奥瑞他汀(例如单甲基奥瑞他汀E(MMAE)、单甲基奥瑞他汀F(MMAF))；
a选自1-6之间的整数，例如1、2、3、4、5、6；
b独立地选自1-20之间的整数，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20，例如选自1-8之间的整数；
c为1或2。
在某些实施方案中，L 3选自GGFG、VEC、VC。
在某些实施方案中，R 1为-(CH 2) a-。在某些实施方案中，R 1为-(CH 2CH 2O) b-(CH 2) c-。在某些实施方案中，R 1为-CH 2CH 2-(OCH 2CH 2) b-NH-(CO)-CH 2-O-CH 2-。
在某些实施方案中，L 2为单键。在某些实施方案中，L 2为-NH-R 1-(CO)-。
在某些实施方案中，L 3为GGFG。在某些实施方案中，L 3为VEC。在某些实施方案中，L 3为VC。
在某些实施方案中，L 4为-NH-CH 2-。在某些实施方案中，L 4为单键。在某些实施方案中，L 4为
Figure PCTCN2022111443-appb-000006
在某些实施方案中，L 1为
Figure PCTCN2022111443-appb-000007
L 2为-NH-R 1-(CO)-，R 1为-(CH 2) a-，a为2、3、4、5或6。
在某些实施方案中，L 1为
Figure PCTCN2022111443-appb-000008
L 2为-NH-R 1-(CO)-，R 1为-(CH 2CH 2O) b-(CH 2) c-，b为2、3、4、5、6、7或8，c为1或2。
在某些实施方案中，L 1为
Figure PCTCN2022111443-appb-000009
L 2为单键。
在某些实施方案中，L 1为
Figure PCTCN2022111443-appb-000010
L 2为-NH-R 1-(CO)-，R 1为-CH 2CH 2-(OCH 2CH 2) b-NH-(CO)-CH 2-O-CH 2-，b为2、3、4、5、6、7或8。
在某些实施方案中，L 1-L 2为
Figure PCTCN2022111443-appb-000011
其中，L 2为-R 1-(CO)-，R 1为-CH 2CH 2-(OCH 2CH 2) b-NH-(CO)-CH 2-O-CH 2-；在某些实施方案中，b为2、3、4、5、6、7或8。
在某些实施方案中，L 1为
Figure PCTCN2022111443-appb-000012
L 2为-NH-R 1-(CO)-，R 1为-(CH 2) a-，a为2、3、4、5或6，L 3为GGFG。
在某些实施方案中，L 1为
Figure PCTCN2022111443-appb-000013
L 2为-NH-R 1-(CO)-，R 1为-(CH 2CH 2O) b-(CH 2) c-，b为2、3、4、5、6、7或8，c为1或2，L 3为GGFG或EVC。
在某些实施方案中，L 1为
Figure PCTCN2022111443-appb-000014
L 2为单键，L 3为GGFG。
在某些实施方案中，L 1为
Figure PCTCN2022111443-appb-000015
L 2为-NH-R 1-(CO)-，R 1为-CH 2CH 2-(OCH 2CH 2) b-NH-(CO)-CH 2-O-CH 2-，b为2、3、4、5、6、7或8，L 3为EVC。
在某些实施方案中，L 1-L 2为
Figure PCTCN2022111443-appb-000016
其中，L 2为-R 1-(CO)-，R 1为-CH 2CH 2-(OCH 2CH 2) b-NH-(CO)-CH 2-O-CH 2-，b为2、3、4、5、6、7或8，L 3为EVC。
在某些实施方案中，L 1为
Figure PCTCN2022111443-appb-000017
L 2为单键，L 3为VC或GGFG。
在某些实施方案中，L 2选自单键、
Figure PCTCN2022111443-appb-000018
Figure PCTCN2022111443-appb-000019
在某些实施方案中，L 1-L 2为
Figure PCTCN2022111443-appb-000020
在某些实施方案中，L 3为GGFG，L 4为单键。
在某些实施方案中，L 3为GGFG，L 4为-NH-CH 2-。
在某些实施方案中，L 3为EVC，L 4为
Figure PCTCN2022111443-appb-000021
在某些实施方案中，L 3为GGFG，L 4为
Figure PCTCN2022111443-appb-000022
在某些实施方案中，D为艾日布林或其衍生物。在某些实施方案中，D选自
Figure PCTCN2022111443-appb-000023
在某些实施方案中，L 4为-NH-CH 2-，D为d1。
在某些实施方案中，L 4为-NH-CH 2-，D为d2。
在某些实施方案中，L 4为-NH-CH 2-，D为d3。
在某些实施方案中，L 4为单键，D为d2。
在某些实施方案中，L 4为
Figure PCTCN2022111443-appb-000024
D为d2。在某些实施方案中，式(I)所示化合物选自：
化合物1
Figure PCTCN2022111443-appb-000025
化合物2
Figure PCTCN2022111443-appb-000026
化合物3
Figure PCTCN2022111443-appb-000027
化合物4
Figure PCTCN2022111443-appb-000028
化合物5
Figure PCTCN2022111443-appb-000029
化合物6
Figure PCTCN2022111443-appb-000030
化合物7
Figure PCTCN2022111443-appb-000031
化合物8
Figure PCTCN2022111443-appb-000032
化合物9
Figure PCTCN2022111443-appb-000033
化合物10
Figure PCTCN2022111443-appb-000034
化合物11
Figure PCTCN2022111443-appb-000035
化合物12
Figure PCTCN2022111443-appb-000036
化合物13
Figure PCTCN2022111443-appb-000037
化合物14
Figure PCTCN2022111443-appb-000038
式(I)的化合物可作为接头-药物中间体化合物，用于抗体-药物偶联物的合成。因此，本公开的第二方面，提供一种抗体-药物偶联物、其药学上可接受的盐、其立体异构体，或所述偶联物、其药学上可接受的盐或其立体异构体的溶剂合物，其可由靶向部分与式(I)所示化合物经硫醚键连接而成。
在某些实施方案中，所述抗体-药物偶联物具有如式(II)所示的结构
Ab-(S-L 1-L 2-L 3-L 4-D) p
(II)
其中Ab为靶向部分，选自抗体、抗体片段、或基于抗体的分子或化合物；
-S-为硫醚键，
L 1、L 2、L 3、L 4、D如上文所定义，
p为选自1-20之间的整数，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20，例如选自1-8之间的整数。
在某些实施方案中，Ab靶向部分是抗体、抗体片段、双特异性或其它多价抗体、或其它基于抗体的分子或化合物。抗体可具有各种同种型，优选是人IgG1、IgG2、IgG3或IgG4，更优选包含人IgG1铰链和恒定区序列。抗体或其片段可为嵌合人-小鼠、嵌合人-灵长类动物、人源化(人框架和鼠高变(CDR)区)或完全人抗体以及其变型形式，诸如半IgG4抗体(被称为“单抗体”)，如由van der Neut Kolfschoten等人(Science 2007；317:1554-1557)所述。更优选地，可设计或选择抗体或其片段以包含属于特定同种异型的人恒定区序列，此可导致在向人受试者施用抗体或免疫缀合物时免疫原性降低。用于施用的优选同种异型包括非G1m1同种异型(nG1m1)，诸如G1m3、G1m3,1、G1m3,2或G1m3,1,2。更优选地，同种异型选自由nG1m1、G1m3、nG1m1,2和Km3同种异型组成的组。
适用的抗体可结合本领域中已知的任何疾病相关的抗原。当疾病状态是癌症时，举例来说，由肿瘤细胞表达或另外与肿瘤细胞相关的许多抗原在本领域中是已知的，包括但不限于碳酸酐酶IX、α-胎蛋白(alpha-fetoprotein，AFP)、α-辅肌动蛋白-4(α-actinin-4)、A3、对A33抗体具有特异性的抗原、ART-4、B7、Ba 733、BAGE、BrE3抗原、CA125、CAMEL、CAP-1、CASP-8/m、、CCL19、CCL21、CD1、CD1a、CD2、CD3、CD4、CD5、CD8、CD11A、CD14、CD15、CD16、CD18、CD19、CD20、CD21、CD22、CD23、CD25、CD29、CD30、CD32b、CD33、CD37、CD38、CD40、CD40L、CD44、CD45、CD46、CD52、CD54、CD55、CD59、CD64、CD66a-e、CD67、CD70、CD70L、CD74、CD79a、CD80、CD83、CD95、CD126、CD132、CD133、CD138、CD147、CD154、CDC27、CDK-4/m、CDKN2A、CTLA-4、CXCR4、CXCR7、CXCL12、HIF-1α、结肠特异性抗原p(CSAp)、CEA(CEACAM5)、CEACAM6、c-Met、DAM、EGFR、EGFRvIII、EGP-1(Trop-2)、EGP-2、ELF2-M、Ep-CAM、claudin 18.2、ROR1、ROR2、纤维母细胞生长因子(FGF)、Flt-1、Flt-3、叶酸受体、G250抗原、GAGE、gp100、GRO-β、HLA-DR、HM1.24、人绒毛膜促性腺激素(HCG)和它的亚基、HER2/neu、HMGB-1、缺氧诱导性因子(HIF-1)、HSP70-2M、HST-2、Ia、IGF-1R、IFN-γ、IFN-α、IFN-β、IFN-λ、IL-4R、IL-6R、IL-13R、IL-15R、IL-17R、IL-18R、IL-2、IL-6、IL-8、IL-12、IL-15、IL-17、IL-18、IL-23、IL-25、胰岛素样生长因子1(IGF-1)、IGF-1R、KS1-4、Le-Y、LDR/FUT、巨噬细胞迁移抑制因子(MIF)、MAGE、MAGE-3、MART-1、MART-2、NY-ESO-1、TRAG-3、mCRP、MCP-1、MIP-1A、MIP-1B、MIF、MUC1、MUC2、MUC3、MUC4、MUC5ac、MUC13、MUC16、MUM-1/2、MUM-3、NCA66、NCA95、NCA90、胰腺癌粘蛋白、PD-1受体、PD-L1受体、胎盘生长因子、p53、PLAGL2、前列腺酸性磷酸酶、PSA、PRAME、PSMA、PlGF、ILGF、ILGF-1R、IL-6、IL-25、RS5、RANTES、T101、SAGE、S100、存活素(survivin)、存活素-2B、TAC、TAG-72、腱生蛋白(tenascin)、TRAIL受体、TNF-α、Tn抗原、汤姆逊-弗雷登里希抗原(Thomson-Friedenreich antigen)、肿瘤坏死抗原、VEGFR、ED-B纤维连接蛋白(fibronectin)、WT-1、17-1A抗原、补体因子C3、C3a、C3b、C5a、C5、血管生成标志物、bcl-2、bcl-6、Kras、致癌基因标志物和致癌基因产物(参见例如Sensi等,Clin Cancer Res2006,12:5023-32；Parmiani等,J Immunol 2007,178:1975-79；Novellino等Cancer Immunol Immunother 2005,54:187-207)。优选地，抗体结合her2、her3、claudin 18.2、ROR-1、dll-3、muc1、muc-17、EGP-1(Trop-2)。
可利用的示例性抗体包括但不限于hR1(抗IGF-1R，美国专利号13/688,812)、hPAM4 (抗粘蛋白，美国专利号7,282,567)、hA20(抗CD20，美国专利号7,151,164)、hA19(抗CD19，美国专利号7,109,304)、hIMMU31(抗AFP，美国专利号7,300,655)、hLL1(抗CD74，美国专利号7,312,318)、hLL2(抗CD22，美国专利号5,789,554)、hMu-9(抗CSAp，美国专利号7,387,772)、hL243(抗HLA-DR，美国专利号7,612,180)、hMN-14(抗CEACAM5，美国专利号6,676,924)、hMN-15(抗CEACAM6，美国专利号8,287,865)、hRS7(抗EGP-1，美国专利号7,238,785)、hMN-3(抗CEACAM6，美国专利号7,541,440)、Ab124和Ab125(抗CXCR4，美国专利号7,138,496)，各引用的专利或申请的实施例部分以引用的方式并入本文。更优选地，抗体是IMMU-31(抗AFP)、hRS7(抗Trop-2)、hMN-14(抗CEACAM5)、hMN-3(抗CEACAM6)、hMN-15(抗CEACAM6)、hLL1(抗CD74)、hLL2(抗CD22)、hL243或IMMU-114(抗HLA-DR)、hA19(抗CD19)或hA20(抗CD20)。如本文所用，术语依帕珠单抗(epratuzumab)和hLL2可互换，术语维妥珠单抗(veltuzumab)和hA20、hL243g4P、hL243γ4P和IMMU-114也是如此。在一最优选实施方案中，抗体是抗Trop-2抗体诸如hRS7；抗ROR1抗体诸如99961(中国专利号104662044)。
适用的替代性抗体包括但不限于阿昔单抗(abciximab)(抗糖蛋白IIb/IIIa)、阿仑单抗(alemtuzumab)(抗CD52)、贝伐单抗(bevacizumab)(抗VEGF)、西妥昔单抗(cetuximab)(抗EGFR)、吉妥单抗(gemtuzumab)(抗CD33)、替伊莫单抗(ibritumomab)(抗CD20)、帕尼单抗(panitumumab)(抗EGFR)、利妥昔单抗(rituximab)(抗CD20)、托西莫单抗(tositumomab)(抗CD20)、曲妥珠单抗(trastuzumab)(抗ErbB2)、兰罗利珠单抗(lambrolizumab)(抗PD1受体)、阿特珠单抗(atezolizumab)(抗PD-L1)、MEDI4736(抗PD-L1)、纳武单抗(nivolumab)(抗PD-1受体)、伊匹单抗(ipilimumab)(抗CTLA-4)、阿巴伏单抗(abagovomab)(抗CA-125)、阿德木单抗(adecatumumab)(抗EpCAM)、阿利珠单抗(atlizumab)(抗IL-6受体)、贝那珠单抗(benralizumab)(抗CD125)、奥滨尤妥珠单抗(obinutuzumab)(GA101，抗CD20)、CC49(抗TAG-72)、AB-PG1-XG1-026(抗PSMA，美国专利申请11/983,372，以ATCC PTA-4405和PTA-4406保藏)、D2/B(抗PSMA，WO 2009/130575)、托珠单抗(tocilizumab)(抗IL-6受体)、巴利昔单抗(basiliximab)(抗CD25)、达利珠单抗(daclizumab)(抗CD25)、依法珠单抗(efalizumab)(抗CD11a)、GA101(抗CD20；GlycartRoche)、莫罗莫那(muromonab)-CD3(抗CD3受体)、那他珠单抗(natalizumab)(抗α4整联蛋白)、奥马珠单抗(omalizumab)(抗IgE)；抗TNF-α抗体诸如CDP571(Ofei等,2011,Diabetes 45:881-85)、MTNFAI、M2TNFAI、M3TNFAI、M3TNFABI、M302B、M303(ThermoScientific,Rockford,IL)、英夫利昔单抗 (infliximab)(Centocor,Malvern,PA)、聚乙二醇赛妥珠单抗(certolizumab pegol)(UCB,Brussels,Belgium)、抗CD40L(UCB,Brussels,Belgium)、阿达木单抗(adalimumab)(Abbott,Abbott Park,IL)、Benlysta(Human Genome Sciences)；用于阿尔茨海默氏病(Alzheimer's disease)的治疗的抗体诸如Alz 50(Ksiezak-Reding等,1987,J Biol Chem 263:7943-47)、甘特如单抗(gantenerumab)、苏兰珠单抗(solanezumab)和英夫利昔单抗；抗纤维蛋白抗体如59D8、T2G1s、MH1；抗CD38抗体诸如MOR03087(MorphoSys AG)、MOR202(Celgene)、HuMax-CD38(Genmab)或达雷木单抗(daratumumab)(Johnson&Johnson)；(抗HIV抗体诸如P4/D10(美国专利8,333,971)、Ab75、Ab76、Ab77(Paulik等,1999,Biochem Pharmacol 58:1781-90)以及由Polymun(Vienna,Austria)描述和销售的抗HIV抗体，也描述于美国专利5,831,034、美国专利5,911,989以及Vcelar等,AIDS 2007；21(16):2161-2170和Joos等,Antimicrob.Agents Chemother.2006；50(5):1773-9中，全都以引用的方式并入本文。
在某些实施方案中，靶向部分为hRS7抗体。hRS7抗体的轻链和重链氨基酸序列分别如SEQ ID NO:1和SEQ ID NO:2所示，核苷酸序列分别如SEQ ID NO:3和SEQ ID NO:4所示。
在某些实施方案中，缀合于主题抗体的药物部分选自艾日布林或其衍生物，例如艾日布林的甲磺酸盐。如本文所用，术语“艾日布林”是指软海绵素B(最初从海绵冈田软海绵(Halichondria okadais)分离的大环化合物)的合成类似物。艾日布林为微管动力学抑制剂，认为其结合微管蛋白并通过抑制有丝分裂纺锤体组件，在G2/M期引起细胞周期停滞。术语“艾日布林甲磺酸盐”是指艾日布林的甲磺酸盐，其以商品名Halaven TM出售。
药物部分还可以选择水溶性喜树碱化合物或其衍生物，如依沙替康，伊立替康，吉咪替康、dxd、吉马替康、SN-38或CPT-11。涉及CPT-11的药理学以及它在体内向活性SN-38转化的广泛临床数据是可用的(Iyer和Ratain,Cancer Chemother Pharmacol.42:S31-43(1998)；Mathijssen等,Clin Cancer Res.7:2182-2194(2002)；Rivory,Ann NY Acad Sci.922:205-215,2000))。活性形式SN-38的强力性比CPT-11大约2至3个数量级。
药物部分还可以选择奥瑞他汀(例如单甲基奥瑞他汀E(MMAE)、单甲基奥瑞他汀F(MMAF))。
在某些实施方案中，抗体或其片段连接于至少一个化学治疗部分；例如是1至5个(例如1、2、3、4或5个)药物部分、5至8个(例如5、6、7或8个)药物部分或8至12个(例如8、9、10、11或12个)药物部分。
在第三方面，本公开提供第一方面的化合物、药学上可接受的盐、立体异构体或溶剂合物、以及第二方面所述的抗体-药物偶联物、药学上可接受的盐、立体异构体或溶剂合物的制备方法。
(一)式(I)所示化合物的制备方法:
1、当L 2为单键且L 4为
Figure PCTCN2022111443-appb-000039
时，所述方法包括以下步骤：
Figure PCTCN2022111443-appb-000040
(1)使6-(2-(甲基磺酰基)嘧啶)-5-己炔酸与化合物i在DIEA和HATU存在的条件下发生缩合反应，生成化合物ii；
(2)使化合物ii与对硝基苯基氯甲酸酯在DIEA存在的条件下发生缩合反应，生成化合物iii；
(3)使化合物iii与艾日布林在DIEA存在的条件下发生缩合反应，生成式(I)所示的化合物(D为d2)。
2、当L 2为单键且L 4为-NH-CH 2-时，所述方法包括以下步骤：
Figure PCTCN2022111443-appb-000041
(1)使6-(2-(甲基磺酰基)嘧啶)-5-己炔酸与化合物IV在DIEA和HATU存在的条件下发生缩合反应，生成化合物V；
(2)使化合物V与艾日布林在DIEA和HATU存在的条件下发生缩合反应，生成式(I)所示的化合物(D为d2)。
3、当L 2为单键且L 4为单键时，所述方法包括以下步骤：
Figure PCTCN2022111443-appb-000042
(1)使6-(2-(甲基磺酰基)嘧啶)-5-己炔酸与化合物VI在DIEA和HATU存在的条件下发生缩合反应，生成化合物VII；
(2)使化合物VII与艾日布林在DIEA和HATU存在的条件下发生缩合反应，生成式(I)所示的化合物(D为d2)。
上述三种合成方法可适用于L 2为单键的化合物的合成。如果L 2不是单键，可先制备L 1-L 2，再参照上述三种方法进行合成。可通过羧基与氨基的缩合反应形成酰胺键生成L 1-L 2，或通过叠氮-炔基的点击反应生成L 1-L 2。
以下对上述三种合成方法进行示例性地说明。
Figure PCTCN2022111443-appb-000043
将6-(2-(甲基磺酰基)嘧啶)-5-己炔酸与HATU(1.5当量)加入到反应瓶中用适量DMF溶解后，再加入DIEA(2.0当量)反应半小时左右，再将化合物VC-PAB-OH(1.0当量)加入继续反应3小时，纯化得到目标产物A2。将连接子A2与对硝基苯基氯甲酸酯(1.5当量)加入到适量的DMF溶剂中，完全溶解后，再加入2当量的DIEA进行反应3小时左右，纯化后得到目标产物A3。将A3与等物质的量的eribulin加入到反应瓶中用适量DMF溶解后，再加入DIEA(2.0当量)反应3小时，纯化得到目标产物A。实施例3中的中间体E即参照此方法合成。
Figure PCTCN2022111443-appb-000044
将6-(2-(甲基磺酰基)嘧啶)-5-己炔酸与等物质的量的HATU加入到反应瓶中用适量DMF溶解后，再加入DIEA(2.0当量)反应2小时左右，再将化合物B1或者C1(1.0当量)加入继续反应3小时，纯化得到目标产物B2或C2。将B2或C2与HATU(1.5当量)加入到反应瓶中用适量DMF溶解后，再加入DIEA(2.0当量)反应半小时左右，再将化合物eribulin(1.0当量)加入继续反应3小时，纯化得到目标产物B或C。实施例2中的中间体D和实施例4中的中间体F即参照此方法合成。
(二)式(II)所示抗体-药物偶联物的制备方法
本发明中，经由硫醚而将抗体和接头结构连接的抗体-药物偶联物可通过下述的方法来制造。
Figure PCTCN2022111443-appb-000045
即，使作为接头-药物中间体的第一方面所述的化合物、药学上可接受的盐、立体异构体或溶剂合物与靶向部分(例如抗体)反应，以通过由靶向部分的铰链部的二硫键部分形成的硫醚键，从而将接头-药物中间体与靶向部分连接，制备得到式(II)表示的抗体-药物偶联物。
其中，L 1、L 2、L 3、L 4、D、Ab、S的定义如本发明说明书所述。
为了便于说明，式(II)所示的抗体-药物偶联物中，以1个从药物至接头末端的结构部分与1个抗体连接而成的结构的形式进行了记载，但实际上，相对于1个抗体分子而言连接有多个该结构部分的情况较多。例如，如上所述，在一个抗体分子上连接2～8个、优选为4～8个、更优选为6～8个接头-药物中间体化合物。该情况在以下的制造方法的说明中也同样。实际上，如上所述，本发明中，以平均药物连接数表示连接于每一个分子抗体的接头-药物的平均数目。
即，如上所示，通过使本发明上述的接头-药物中间体化合物与具有巯基的抗体Ab-SH反应，可制造式(II)所示的抗体-药物偶联物。
具体而言，作为还原剂，相对于每一个抗体内铰链部二硫键，使用1～7摩尔当量TCEP，在含有螯合剂EDTA的缓冲液中，使其与抗体反应，由此，可得到部分或完全地将抗体内铰链部二硫醚还原而得到的携带巯基的抗体(AB-SH)。作为螯合剂，可举出例如乙二胺四乙酸(EDTA)、二亚乙基三胺五乙酸(DTPA)等。可以以1mM～20mM的浓度使用它们。作为缓冲液，可使用磷酸钠、硼酸钠、乙酸钠溶液等。在具体的例子中，于4℃～37℃使抗体与TCEP反应1～4小时，由此，可得到部分或完全还原而具有巯基的抗体AB-SH。
相对于每一个具有巯基的抗体Ab-SH，可使用2～20摩尔当量的式(I)所示的化合物，制造1个抗体连接2个-8个药物而成的抗体-药物偶联物(II)。具体而言，在含有具有巯基的抗体Ab-SH的缓冲液中，添加溶解有式(I)所示的化合物的溶液并使其进行反应。此处，作为缓冲液，可使用乙酸钠溶液、磷酸钠、硼酸钠等。反应时的pH为5～9，更优选在pH7附近反应。作为溶解化合物的溶剂，可使用二甲基亚砜(DMSO)、二甲基甲酰胺(DMF)、二甲基乙酰胺(DMA)、N-甲基-2-吡啶酮(NMP)等有机溶剂。可以以1～20％v/v将溶解有式(I)所 示的化合物的有机溶剂溶液添加到含有具有巯基的抗体Ab-SH的缓冲液中并进行反应。反应温度为0～37℃、更优选为10～25℃，反应时间为0.5～2小时。可通过利用含硫醇试剂使未反应的式(I)所示的化合物的反应性失活而结束反应。含硫醇试剂例如为半胱氨酸或N-乙酰-L-半胱氨酸(NAC)。更具体而言，添加相对于使用的式(I)所示的化合物而言1～2摩尔当量的半胱氨酸，于室温孵育10～30分钟，由此使反应结束。
对于制造的抗体-药物偶联物(II)，可利用以下的共通操作，进行浓缩、缓冲液交换、纯化等的操作。
共通操作A：抗体或抗体-药物偶联物水溶液的浓缩
在容器内，放入抗体或抗体-药物偶联物溶液，使用离心机进行离心操作(例如，以2000G～3800G离心5～20分钟)，将抗体或抗体-药物偶联物溶液浓缩。
共通操作B：抗体的浓度测定
使用UV测定器，按照制造商规定的方法，进行抗体浓度的测定。
此时，使用随着抗体不同而显示不同的280nm吸光系数(1.3mLmg -1cm -1～1.8mLmg -1cm - 1)。
共通操作C-1：抗体的缓冲液交换
按照制造商说明书的方法，用含有氯化钠(例如，137mM)及乙二胺四乙酸(EDTA)，例如，5mM)的磷酸缓冲液(例如PBS)(本说明书中也称为PBS/EDTA)。将使用了Sephadex G-25载体的PD-10柱平衡化。针对一根该PD-10柱，装填1mL抗体水溶液，然后分离用PBS/EDTA 2mL洗脱的级分(3.5mL)。利用共通操作A将该级分浓缩，使用共通操作B，进行抗体浓度的测定，然后使用PBS/EDTA，将抗体浓度调整为10mg/mL。
共通操作C-2：抗体的缓冲液交换
按照制造商规定的方法，用含有氯化钠(例如，50mM)及EDTA(例如，2mM)的磷酸缓冲液(例如，50mM，pH6.5，本说明书中也称为PBS6.5/EDTA)。将使用了Sephadex G-25载体的PD-10柱平衡化。针对一根该PD-10柱，装填1mL抗体水溶液，然后分离获取用PBS6.5/EDTA 2mL洗脱的级分(3.5mL)。利用共通操作A将该级分浓缩，使用共通操作B进行抗体浓度的测定，然后使用PBS6.5/EDTA，将抗体浓度调整为5mg/mL。
共通操作D-1：抗体-药物偶联物的纯化
使用市售的磷酸缓冲液(例如，PBS7.4)、含有氯化钠(例如，137mM)的磷酸钠缓冲液(例如，10mM，pH6.5；本说明书中也称为PBS6.5)，或含有山梨糖醇(例如，5％)的乙酸 缓冲液(例如，10mM，pH5.5；本说明书中也称为ABS)或MES 25mM pH6.5，或His 10mM pH 5.5中的任一种缓冲液将PD-10柱平衡化。在该柱中装填抗体-药物偶联物反应水溶液(例如，约1mL)，用制造商规定的量的缓冲液洗脱，由此分离获取抗体级分，由此，得到了除去了未连接的药物接头、低分子化合物(三(2-羧基乙基)膦盐酸盐(TCEP)，半胱氨酸，二甲基亚砜等)的抗体-药物偶联物。
共通操作D-2：抗体-药物偶联物的纯化
磷酸缓冲液(例如，PBS7.4)、含有氯化钠(例如，137mM)的磷酸钠缓冲液(例如，10mM，pH6.5；本说明书中也称为PBS6.5)，或含有山梨糖醇(例如，5％)的乙酸缓冲液(例如，10mM，pH5.5；本说明书中也称为ABS)或MES 25mM pH6.5，或His 10mM pH 5.5中的任一种缓冲液将AKTA柱(填料：sephadex G 25)平衡化。进样器装载抗体-药物偶联物反应水溶液(例如，约2mL)，用制造商规定的量的缓冲液洗脱，由此分离获取抗体级分。由此，得到了除去了未连接的药物接头、低分子化合物(三(2-羧基乙基)膦盐酸盐(TCEP)，半胱氨酸，二甲基亚砜等)的抗体-药物偶联物。
在第四方面，本公开提供一种药物组合物，其包含第一方面所述的化合物、药学上可接受的盐、立体异构体、或溶剂合物，或第二方面所述的偶联物、药学上可接受的盐、立体异构体、或溶剂合物，以及一种或多种药用辅料，例如载体和/或赋形剂。
所述药物组合物可以制成药学上可接受的任一剂型。所述药物组合物还可以以任何合适的给药方式，例如口服、肠胃外、直肠或经肺给药等方式施用于需要这种治疗的个体。用于口服给药时，所述药物组合物可制成常规的固体制剂，如片剂、胶囊剂、丸剂、颗粒剂等；也可制成口服液体制剂，如口服溶液剂、口服混悬剂、糖浆剂等。制成口服制剂时，可以加入适宜的填充剂、粘合剂、崩解剂、润滑剂等。用于肠胃外给药时，所述药物组合物可制成注射剂，包括注射液、注射用无菌粉末与注射用浓溶液。制成注射剂时，可采用现有制药领域中的常规方法生产，配制注射剂时，可以不加入附加剂，也可根据药物的性质加入适宜的附加剂。用于直肠给药时，所述药物组合物可制成栓剂等。用于经肺给药时，所述药物组合物可制成吸入剂或喷雾剂等。
第五方面，本公开提供第一方面所述的化合物、药学上可接受的盐、立体异构体、或溶剂合物，或第二方面所述的偶联物、药学上可接受的盐、立体异构体、或溶剂合物在制备治疗与细胞活动异常相关的疾病(例如癌症疾病)的药物中的用途。
第六方面，本公开提供第一方面所述的化合物、药学上可接受的盐、立体异构体、或溶剂合物，或第二方面所述的偶联物、药学上可接受的盐、立体异构体、或溶剂合物，或第四方面的药物组合物用于治疗与细胞活动异常相关的疾病(例如癌症疾病)中的用途。
第七方面，本公开提供治疗与细胞活动异常相关的疾病(例如癌症疾病)的方法，包括给有此需要的个体有效剂量的本公开提供第一方面所述的化合物、药学上可接受的盐、立体异构体、或溶剂合物，或第二方面所述的偶联物、药学上可接受的盐、立体异构体、或溶剂合物，或第四方面的药物组合物。可调整给药方案以提供最佳所需响应。例如，可给药单次推注，可随时间给药数个分剂量，或可如治疗情况的急需所表明而按比例减少或增加剂量。剂量值可随要减轻的病况的类型及严重性而变化，且可包括单次或多次剂量。要进一步理解，对于任何特定个体，具体的给药方案可根据个体需要及给药组合物或监督组合物的给药的人员的专业判断来随时间调整。
各种实施方案可涉及使用主题方法和组合物来治疗癌症，包括但不限于转移性乳腺癌、非小细胞肺癌、伯基特淋巴瘤(Burkitt lymphoma)、霍奇金氏淋巴瘤、急性骨髓性白血病、慢性骨髓性白血病、急性淋巴细胞性白血病、慢性淋巴细、皮肤癌、口腔癌、食道癌、胃肠道癌、肺道癌、肺癌、胃癌、结肠癌、直肠癌、三阴性乳腺癌、卵巢癌、前列腺癌、子宫癌、子宫内膜癌、子宫颈癌、膀胱癌、胰腺癌、骨癌、脑癌、结缔组织癌、甲状腺癌、肝癌、胆囊癌、膀胱(尿道上皮)癌、肾癌、皮肤癌、中枢神经系统癌和睾丸癌。
在涉及治疗癌症的某些实施方案中，抗体或免疫缀合物可与手术、放射疗法、化学疗法、用包括检查点抑制抗体的裸抗体进行的免疫疗法、放射免疫疗法、免疫调节剂、疫苗等组合使用。最优选地，抗体或免疫缀合物与PARP抑制剂、微管抑制剂、布鲁顿激酶抑制剂和/或PI3K抑制剂组合使用。这些组合疗法可允许在所述组合的情况下给予较低剂量的各治疗剂，因此降低某些重度副作用，以及潜在缩减所需疗程。当不存在或存在最小重叠毒性时，也可给予各自的完全剂量。
尽管抗体或免疫缀合物可以定期弹丸注射液形式施用，但在替代性实施方案中，抗体或免疫缀合物可通过连续输注来施用。为使抗体或免疫缀合物在血液中的Cmax增加以及延长抗体或免疫缀合物在血液中的PK，可例如通过留置导管来施用连续输注。此类装置在本领域中是已知的，诸如
Figure PCTCN2022111443-appb-000046
或
Figure PCTCN2022111443-appb-000047
导管(参见例如Skolnik等,Ther Drug Monit 32:741-48,2010)，并且可使用任何所述已知留置导管。多种连续输注泵在本领域中也是已知的，并且可使用任何所述已知输注泵。
在特别优选实施方案中，在对标准疗法具有抗性的患者中，抗体或免疫缀合物和给药时程可为有效的。举例来说，可向尚未对用艾日布林进行的先前疗法响应的患者施用hRS7-艾日布林免疫缀合物。免疫缀合物特异性靶向肿瘤组织的能力可由于治疗剂的靶向性改进和递送增强而克服肿瘤抗性。一特定优选受试者可为患有Trop-2阳性乳腺癌、卵巢癌、子宫颈癌、子宫内膜癌、肺癌、前列腺癌、结肠癌、直肠癌、胃癌、食道癌、膀胱(尿道上皮)癌、肾癌、胰腺癌、脑癌、甲状腺癌、上皮癌、或头颈部癌的患者。优选地，癌症是转移性癌症。更优选地，患者用至少一种标准抗癌疗法进行的治疗先前已失败。
第八方面，本公开提供了一种药物制剂，其包含第一方面所述的化合物、药学上可接受的盐、立体异构体、或溶剂合物，或第二方面所述的偶联物、药学上可接受的盐、立体异构体、或溶剂合物，或第四方面的药物组合物。
第九方面，本公开提供了第一方面所述的化合物、药学上可接受的盐、立体异构体、或溶剂合物，或第二方面所述的偶联物、药学上可接受的盐、立体异构体、或溶剂合物，或第四方面的药物组合物用于制备药物制剂的用途。
第十方面，本公开提供了一种试剂盒，其包括第一方面所述的化合物、药学上可接受的盐、立体异构体、或溶剂合物，或第二方面所述的偶联物、药学上可接受的盐、立体异构体、或溶剂合物，或第四方面的药物组合物，或第八方面的药物制剂。
本公开提供的化合物、偶联物、药学上可接受的盐、立体异构体、或溶剂合物、药物制剂或试剂盒可用于抑制癌细胞生长、增殖或迁移。所述化合物、偶联物、药学上可接受的盐、立体异构体、或溶剂合物、药物制剂或试剂盒用于体内或者体外施用；例如，被施用至个体体内，或者被施用至体外细胞(例如细胞系或者来自个体的细胞例如癌症细胞)。
因此，第十一方面，本公开提供了一种抑制癌细胞生长、增殖或迁移的方法，其包括给癌细胞施用有效量的第一方面所述的化合物、药学上可接受的盐、立体异构体、或溶剂合物，或第二方面所述的偶联物、药学上可接受的盐、立体异构体、或溶剂合物，或第四方面的药物组合物，或第八方面的药物制剂。
除非另有定义，本文中所用的所有技术术语和科学术语的含义意图与本领域技术人员通常所理解的相同。提及本文中使用的技术意图指在本领域中通常所理解的技术，包括那些对本领域技术人员显而易见的技术的变化或等效技术的替换。虽然相信以下术语对于本领域技术人员很好理解，但仍然阐述以下定义以更好地解释本发明。
本发明所述“接头”、“接头结构”或“连接子”或“连接单元”是指一端与抗体连接而另一端与药物(药物化合物)相连的化学结构片段或键，也可以连接其它接头后再与药物化合物相连。本发明的接头结构可以通过本领域已知方法合成，也可使用本发明所述的方法进行合成。
本发明所述“抗体-药物偶联物”，即ADC，指靶向部分通过稳定的连接单元与具有生物活性的药物相连。
本发明中，“药学上可接受的盐”是指相对无毒的本发明的偶联物的酸加成盐或碱加成盐。所述酸加成盐为本发明的偶联物与合适的无机酸或者有机酸形成的盐，这些盐可通过使本发明的偶联物与适宜的有机酸或无机酸在适当的溶剂中进行反应来制备。代表性酸加成盐包括氢溴酸盐、盐酸盐、硫酸盐、硫酸氢盐、亚硫酸盐、乙酸盐、草酸盐、戊酸盐、油酸盐、棕榈酸盐、硬脂酸盐、月硅酸盐、硼酸盐、苯甲酸盐、乳酸盐、硝酸盐、磷酸盐、磷酸氢盐、碳酸盐、碳酸氢盐、甲苯甲酸盐、柠檬酸盐、马来酸盐、富马酸盐、琥珀酸盐、苹果酸盐、抗坏血酸盐、鞣酸盐、扑酸盐、藻酸盐、萘磺酸盐、酒石酸盐、苯甲酸盐、甲磺酸盐、对甲苯磺酸盐、葡萄糖酸盐、乳糖酸盐和月桂基磺酸盐等。所述碱加成盐为本发明的偶联物与合适的无机碱或者有机碱形成的盐，这些盐可通过使本发明的偶联物与适宜的无机碱或者有机碱在适当的溶剂中进行反应来制备。代表性碱加成盐包括例如与碱金属、碱土金属、季铵阳离子形成的盐，例如钠盐、锂盐、钾盐、钙盐、镁盐、四甲基季铵盐、四乙基季铵盐等；胺盐，包括与氨(NH3)、伯胺、仲胺或叔胺形成的盐，如甲胺盐、二甲胺盐、三甲胺盐、三乙胺盐、乙胺盐等。
本发明的偶联物可以存在特定的几何或立体异构体形式，本发明的偶联物中，手性中心可以存在于药物中，可以存在于接头结构中，还可以存在于抗体及其衍生物中。本发明中，所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，均包括在本发明的范围之内。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本发明的偶联物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是 通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。
本发明中，本发明的化合物、偶联物、药学上可接受的盐、立体异构体的溶剂合物(例如水合物)也在本发明的范围内。作为适当的溶剂合物，具体而言，可以列举本发明的偶联物与丙酮、2-丁醇、2-丙醇、乙醇、乙酸乙酯、四氢呋喃、二乙醚等形成的溶剂合物。还可以列举出水合物或乙醇化物。
本发明中，“治疗”患有疾病或疾病状况的个体表示所述个体的症状部分或全部缓解，或者在治疗后保持不变。因此，治疗包括预防、治疗和/或治愈。预防指防止潜在疾病和/或防止症状恶化或疾病发展。治疗还包括所提供的ADC以及本文所提供的药物组合物、药物制剂的任何药学用途。
本发明中，“疗效”表示由个体的治疗所导致的效果，其改变、通常改良或改善疾病或疾病状况的症状，或者治愈疾病或疾病状况。
本发明中，“个体”包括人或非人动物。示例性人个体包括患有疾病(例如本文所述的疾病)的人个体(称为患者)或正常个体。本发明中，“非人动物”包括所有脊椎动物，例如非哺乳动物(例如鸟类、两栖动物、爬行动物)和哺乳动物，例如非人灵长类、家畜和/或驯化动物(例如绵羊、犬、猫、奶牛、猪等)。
应了解所公开的组合物和方法不局限于本文所述和/或所示的特定组合物和方法，且本文所用的术语仅仅出于借助于实例描述特定实施方案的目的，且不意图限制所要求的组合物和方法。
本文通篇，描述提及组合物和使用所述组合物的方法。在本公开描述或要求与组合物有关的特征或实施方案的情况下，这类特征或实施方案同等适用于使用所述组合物的方法。同样，在本公开描述或要求与使用组合物的方法有关的特征或实施方案的情况下，这类特征或实施方案同等适用于所述组合物。
当表述值的范围时，其包括使用所述范围内的任何特定值的实施方案。此外，提及范围内所述的值包括所述范围内的每个值。所有范围都包括其终点且可以组合。当通过利用前面的“约”，将值表述为近似值时，应了解特定值形成另一实施方案。除非上下文另外清楚规定，否则提及特定数值至少包括所述特定值。除非所使用的特定上下文另外规定，否则使用“或”将意指“和/或”。本文中引用的所有参考文献都以引用的方式并入以达成任何目的。在参考文献与本说明书冲突的情况下，将以本说明书为主。
应了解，为清楚起见，在本文中单独实施方案的上下文中描述的所公开组合物和方法的某些特征也可以组合提供于单个实施方案。相反，为简便起见，在单个实施方案的上下文中描述的所公开的组合物和方法的各种特征也可以分开或呈任何子组合提供。
本文中提及的文献均以其整体援引加入本文中。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是，本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1为实施例2合成的偶联物ADC-4的SEC-HPLC结果。
图2为实施例5中的抑瘤活性统计曲线。
具体实施方式
下面结合具体实施例，对本发明的方案进行解释。应理解，这些实施例仅用于举例说明本发明而不用于限制本发明的范围。下列实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
以下实施例中，ADC-137仅作为对照，其中连接子-毒素小分子部分购自常州辰鸿生物科技有限公司，CAS#:1279680-68-0，抗体偶联方法同下述的ADC-4。ADC-137结构如下：
Figure PCTCN2022111443-appb-000048
实施例1：hRS7抗体的制备和检测
1.基因合成、转染和抗体制备
hRS7抗体在CHO细胞产生。含hRS7抗体基因的表达载体分别用常规的分子生物学方法构建，hRS7抗体轻链和重链的氨基酸序列分别如SEQ ID NO:1和SEQ ID NO:2所示，其对应的核苷酸序列分别如SEQ ID NO:3和SEQ ID NO:4所示。将上述两序列插入到同一表达载体中，大量提取制备转染质粒，并转染到CHO-K1细胞(ATCC CCL-61)中，具体转染和抗体制备的过程如下：
(1)细胞培养：CHO-K1细胞悬浮生长于ActiPro(GE HyClone)培养基，于37℃，7％CO 2，140rpm，90％相对湿度进行培养；
(2)转染：在进入对数生长期后，取细胞离心，重悬于新鲜的ActiPro培养基，计数并调节细胞密度到1.2×10 7个/毫升，转500μl细胞悬液到电击杯中，然后加入40μg构建好的质粒，将细胞与质粒混匀，然后用电转方式导入质粒(Bio-rad电转仪)；
(3)亚克隆：电转后的细胞用37℃的ActiPro培养基重悬，每孔100μl分装于96孔板。测定细胞上清以测定抗体的表达水平。将表达水平较高的克隆从96孔板转移到24孔板培养，而后再转入6孔板培养，测定细胞的抗体产量和产率，选择表达量最高的4个克隆进行亚克隆，而后转入摇瓶，放在培养箱中继续培养。
2抗体的纯化
收集摇瓶培养的高表达的细胞液，用蛋白A亲和纯化(GE，Mab Select SuRe)和离子交换纯化(GE，Capto S)。采用SDS-PAGE和SEC-HPLC对纯化后的抗体进行分子量和纯度分析。SDS-PAGE测定结果表明制备的hRS7分子量符合预期，用SEC-HPLC法测得抗体纯度为98.8％。
实施例2：制备偶联物ADC-4
Figure PCTCN2022111443-appb-000049
1中间体D的制备
Figure PCTCN2022111443-appb-000050
1.1在6-(2-(甲基磺酰基)嘧啶)-5-己炔酸(39.88mg)D2的DMF(4mL)溶液中加入化合物HATU(56.52mg)和DIEA(38.43mg)，反应液在室温条件下搅拌2小时后加入化合物D1(50mg)，将反应液于室温条件下搅拌3h。HPLC显示有新峰生成，将反应液送Pre-HPLC纯化后冻干得产物大约(21mg)的白色固体D3，产率大约24.08％.LCMS：[M+1]+＝587.4。
Figure PCTCN2022111443-appb-000051
1.2在化合物D3(21mg)的DMF(4mL)溶液中加入化合物HATU(20.42mg)和DIEA(9.25mg)，反应液在室温条件下搅拌半小时后加入化合物eribulin A2(26.13mg)，将反应液于室温条件下搅拌3h。HPLC显示有新峰生成，将反应液送Pre-HPLC纯化后冻干得产物大约(11mg)的白色固体D，产率大约23.66％.LCMS：[M+1]+＝1298.9；
2.偶联粗产物ADC-4的合成
Figure PCTCN2022111443-appb-000052
抗体在10mg/mL的pH 7.4 PBS/EDTA 5mM溶液中置于冰水浴，当蛋白溶液温度达 到4℃时，搅拌混合的同时加入3.5倍的物质的量的等体积TCEP溶液，溶液置25℃水浴锅静置反应1小时。反应液使用25℃水浴锅，当温度降低至25℃时，接着向抗体溶液中搅拌混合的同时加入7倍的物质的量的化合物D的40％DMSO溶液，25℃摇床摇动反应90分钟，最后反应液搅拌混合的同时加入8倍的物质的量的半胱氨酸，置于25℃水浴锅静置反应10min，得到偶联产物ADC-4。
3.偶联粗产物ADC-4的检测
偶联反应粗产品用SEC检测，
SEC色谱条件如下：
色谱柱型号：TSKgel G4000SWxl 7.8mmI.D.*30cm,8μm
检测器波长：280nm/220nm
柱温：30℃
流速：1mL/min
洗脱方式：等度洗脱
进样体积：10μL
运行时间：55min
4.偶联反应产品纯化：
通过PD-10脱盐柱(填料：sephadex G 25)脱盐纯化后，得偶联物，偶联物再换液到MES 25mM pH5.5海藻糖5％的溶液中，SEC检测，小分子已经完全除掉。纯化后的ADC-4的SEC-HPLC结果见图1。
5.DAR的测定
ADC-4和单抗溶液中加入等体积50mM二硫苏糖醇(DTT)溶液，涡旋混匀，37℃水浴30min。还原后的ADC和抗体用RP-HPLC分析偶联和未偶联的抗体轻链和重链。通过分析其组成确定DAR值为4.2。
实施例3 制备偶联物ADC-5
1中间体E的制备
Figure PCTCN2022111443-appb-000053
1.在E1(141.4mg)的DMF(8mL)溶液中加入化合物HATU(300.6mg)和DIEA(136.24mg)，反应液在室温条件下搅拌半小时后加入VC-PAB-OH E2(200mg)，将反应液于室温条件下搅拌3h。HPLC显示反应完全，并有新峰生成，Pre-HPLC纯化后冻干得产物大约(155mg)的白色固体E3，产率大约46.7％.LCMS：[M+1]+＝630.4；
Figure PCTCN2022111443-appb-000054
2.在E3(155mg)的DMF(6mL)溶液中加入化合物对硝基苯基氯甲酸酯E4
(74.42mg)和DIEA(63.62mg)，将反应液于室温条件下搅拌3h。HPLC显示反应完全，并有新峰生成，将反应液送Pre-HPLC纯化后冻干得产物大约(80mg)的白色固体E5，产率大约40.89％.LCMS：[M+1]+＝795.4；
Figure PCTCN2022111443-appb-000055
3.在E5(25mg)的DMF(3mL)溶液中加入化合物eribulin A2(23mg)和DIEA(8.13mg)，反应液在室温条件下搅拌3小时，HPLC显示反应完全，并有新峰生成，LCMS显示有目标产物生成，将反应液送Pre-HPLC纯化后冻干得产物大约(11mg)的白色固体E，产率大约25.24％.LCMS：[M+1]+＝1385.9；
4.E与实施例1制备的hRS7抗体的偶联、纯化、检测步骤同实施例3中的步骤2-5，获得的ADC-5的DAR值为4.2。即，n为4.2。
实施例4 制备偶联物ADC-6
Figure PCTCN2022111443-appb-000056
1.在6-(2-(甲基磺酰基)嘧啶)-5-己炔酸E1(31.68mg)的DMF(4mL)溶液中加入化合物HATU(44.9mg)和DIEA(30.52mg)，反应液在室温条件下搅拌2小时后加入化合物F1(50mg)，将反应液于室温条件下搅拌3h。HPLC显示有新峰生成，将反应液送Pre-HPLC纯化后冻干得产物大约(27mg)的白色固体F2，产率大约33.94％.LCMS：[M+1]+＝674.4。
Figure PCTCN2022111443-appb-000057
2.在化合物F2(27mg)的DMF(4mL)溶液中加入化合物HATU(22.86mg)和DIEA(10.36mg)，反应液在室温条件下搅拌半小时后加入化合物eribulin A2(29.25mg)，将反应液于室温条件下搅拌3h。HPLC显示有新峰生成，将反应液送Pre-HPLC纯化后冻干得产物大约(13mg)的白色固体F，产率大约23.41％.LCMS：[M+1]+＝1385.9；
3.F与实施例1制备的hRS7抗体的偶联、纯化、检测步骤同实施例3中的步骤2-5，获得的ADC-6
的DAR值为4.3。即，n为4.3。
实施例5 ADC对乳腺癌的抑瘤效果
实验方法
1.1细胞培养
MDA-MB-468(ATCC HTB-132)细胞体外单层培养，培养条件为L-15培养基中加10％热灭活胎牛血清，于37℃、无CO2空气的培养箱中培养。一周换2-3次液。当细胞呈指数生 长期时，用0.25％胰酶进行消化处理，按1:2～1:4传代，将长到对数生长期的细胞进行收取，计数，接种。
1.2肿瘤细胞接种及瘤块传代
将4.5.0×10 6MDA-MB-468肿瘤细胞悬浮于0.1ml PBS与Matrigel混合物(1:1)，接种于5只裸鼠右侧肩胛处(P1代)。待肿瘤长至600-800mm 3时，将荷瘤小鼠用CO 2麻醉处死，取出瘤块，去除周围坏死的组织，将瘤块切成20-30mm 3的小瘤块，接种到新的一批裸鼠(P2代)。
1.3瘤块接种种及分组给药
本试验使用P6代肿瘤组织进行受试品的抗肿瘤活性评价。待P5代肿瘤长至600-800mm 3时，将荷瘤小鼠用CO 2麻醉处死，取出瘤块，去除周围坏死的组织，将状态较好的将瘤块切成20-30mm 3的小瘤块，接种到正式实验用鼠的右侧肩胛处，一共接种40只鼠。瘤块接种13天后肿瘤平均体积达到约135mm 3时，剔除瘤体积过小或过大的小鼠，将剩余的20只小鼠根据瘤体积随机分组并开始给药。给药方案见下表。
Figure PCTCN2022111443-appb-000058
注：“仅一次”指分组当天一次给药后不再给药；用药剂量是按照ADC中艾日布林的剂量计算的。
1.4实验观察和数据收集
肿瘤细胞接种后，除了观察肿瘤生长情况，还对药物治疗对动物行为的影响进行监测，包括：实验动物的活动性，摄食和饮水，体重变化(体重每周测量2次)，眼睛、被毛及其它异常情况。实验过程中观察到的临床症状均记录在原始数据中。肿瘤体积计算方法为：肿瘤体积(mm 3)＝1/2×(a×b 2)(其中a表示长径，b表示短径)。
当单只动物的体重下降超过15％时(BWL>15％)，给予相应单只动物停药处理，体重下 降恢复到10％以内，恢复给药。当单只小鼠体重下降>20％，按照动物福利对其实施安乐死。
1.5疗效评价标准
相对肿瘤增殖率，T/C％，即在某一时间点，治疗组和对照组的相对肿瘤体积或瘤重的百分比值。计算公式如下：T/C％＝T RTV/C RTV×100％(T RTV：治疗组平均RTV；C RTV：溶媒对照组平均RTV；RTV＝V t/V 0，V 0为分组时该动物的瘤体积，V t为治疗后该动物的瘤体积)；或T/C％＝T TW/C TW×100％(T TW：治疗组实验终结时平均瘤重；C TW：溶媒对照组实验终结时平均瘤重)。
1.6实验终点
最后一次给药1周后，所有小鼠取肿瘤，并称重、拍照。
1.7统计分析
本实验用one-way ANOVA进行各组间肿瘤均值的比较。方差齐性分析得出F值有显著性差异，在ANOVA分析之后用Dunnet’s T3(方差不齐)法再进行多重比较。用SPSS 17.0进行所有数据分析。p<0.05认为有显著性差异。
实验结果
图2为抑瘤活性统计曲线。如图所示，本实验中，ADC-4和ADC-5的抑瘤活性要好于ADC-137。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (17)

  1. 式(I)所示化合物、其药学上可接受的盐、其立体异构体，或所述化合物、其药学上可接受的盐或其立体异构体的溶剂合物，
    L 1-L 2-L 3-L 4-D
    (I)
    其中，L 1为
    Figure PCTCN2022111443-appb-100001
    L 2为单键或-NH-R 1-(CO)-，
    或者L 1-L 2为
    Figure PCTCN2022111443-appb-100002
    其中，L 2为-R 1-(CO)-；
    R 1选自-(CH 2) a-、-(CH 2CH 2O) b-(CH 2) c-、-CH 2CH 2-(OCH 2CH 2) b-NH-(CO)-CH 2-O-CH 2-；
    L 3为肽残基，选自甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(GGFG)、谷氨酸-缬氨酸-瓜氨酸(EVC)、缬氨酸-瓜氨酸(VC)、天冬氨酸-缬氨酸-瓜氨酸(DVC)、谷氨酸-甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(EGGFG)、天冬氨酸-甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(DGGFG)，其结构式分别为：
    Figure PCTCN2022111443-appb-100003
    Figure PCTCN2022111443-appb-100004
    L 4选自单键、-NH-CH 2-、
    Figure PCTCN2022111443-appb-100005
    D为与L 4通过化学键相连的药物，所述药物选自艾日布林或其衍生物、喜树碱类药物、奥瑞他汀(例如单甲基奥瑞他汀E(MMAE)、单甲基奥瑞他汀F(MMAF))；
    a选自1-6之间的整数，例如1、2、3、4、5、6；
    b独立地选自1-20之间的整数，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20，例如选自1-8之间的整数；
    c为1或2。
  2. 权利要求1的化合物、药学上可接受的盐、立体异构体，或所述化合物、其药学上可接受的盐或其立体异构体的溶剂合物，其中，L 3选自GGFG、EVC、VC。
  3. 权利要求1或2的化合物、药学上可接受的盐、立体异构体，或所述化合物、其药学上可接受的盐或其立体异构体的溶剂合物，其中，
    L 1为
    Figure PCTCN2022111443-appb-100006
    L 2为-NH-R 1-(CO)-，R 1为-(CH 2) a-，a为2、3、4、5或6；或者
    L 1为
    Figure PCTCN2022111443-appb-100007
    L 2为-NH-R 1-(CO)-，R 1为-(CH 2CH 2O) b-(CH 2) c-，b为2、3、4、5、6、7或8，c为1或2；或者
    L 1为
    Figure PCTCN2022111443-appb-100008
    L 2为单键；或者
    L 1为
    Figure PCTCN2022111443-appb-100009
    L 2为-NH-R 1-(CO)-，R 1为-CH 2CH 2-(OCH 2CH 2) b-NH-(CO)-CH 2-O-CH 2-，b为2、3、4、5、6、7或8；或者
    L 1-L 2为
    Figure PCTCN2022111443-appb-100010
    其中，L 2为-R 1-(CO)-，R 1为-CH 2CH 2-(OCH 2CH 2) b-NH-(CO)-CH 2-O-CH 2-；在某些实施方案中，b为2、3、4、5、6、7或8；或者
    L 1为
    Figure PCTCN2022111443-appb-100011
    L 2为-NH-R 1-(CO)-，R 1为-(CH 2) a-，a为2、3、4、5或6，L 3为GGFG；或者
    L 1为
    Figure PCTCN2022111443-appb-100012
    L 2为-NH-R 1-(CO)-，R 1为-(CH 2CH 2O) b-(CH 2) c-，b为2、3、4、5、6、7或8，c为1或2，L 3为GGFG或EVC；或者
    L 1为
    Figure PCTCN2022111443-appb-100013
    L 2为单键，L 3为GGFG；或者
    L 1为
    Figure PCTCN2022111443-appb-100014
    L 2为-NH-R 1-(CO)-，R 1为-CH 2CH 2-(OCH 2CH 2) b-NH-(CO)-CH 2-O-CH 2-，b为2、3、4、5、6、7或8，L 3为EVC；或者
    L 1-L 2为
    Figure PCTCN2022111443-appb-100015
    其中，L 2为-R 1-(CO)-，R 1为-CH 2CH 2-(OCH 2CH 2) b-NH-(CO)-CH 2-O-CH 2-，b为2、3、4、5、6、7或8，L 3为EVC；或者
    L 1为
    Figure PCTCN2022111443-appb-100016
    L 2为单键，L 3为VC或GGFG。
  4. 权利要求1-3任一项的化合物、药学上可接受的盐、立体异构体，或所述化合物、其药学上可接受的盐或其立体异构体的溶剂合物，其中，
    L 2选自单键、
    Figure PCTCN2022111443-appb-100017
    Figure PCTCN2022111443-appb-100018
  5. 权利要求1或2的化合物、药学上可接受的盐、立体异构体，或所述化合物、其药学上可接受的盐或其立体异构体的溶剂合物，其中，
    L 1-L 2为
    Figure PCTCN2022111443-appb-100019
  6. 权利要求1-5任一项的化合物、药学上可接受的盐、立体异构体，或所述化合物、其药学上可接受的盐或其立体异构体的溶剂合物，其中，
    L 3为GGFG，L 4为单键；或
    L 3为GGFG，L 4为-NH-CH 2-；或
    L 3为EVC，L 4为
    Figure PCTCN2022111443-appb-100020
    或
    L 3为GGFG，L 4为
    Figure PCTCN2022111443-appb-100021
  7. 权利要求1-6任一项的化合物、药学上可接受的盐、立体异构体，或所述化合物、其药学上可接受的盐或其立体异构体的溶剂合物，其中，D为艾日布林或其衍生物；
    优选地，D选自
    Figure PCTCN2022111443-appb-100022
  8. 权利要求1-5、7任一项的化合物、药学上可接受的盐、立体异构体，或所述化合物、其药学上可接受的盐或其立体异构体的溶剂合物，其中，
    L 4为-NH-CH 2-，D为d1；或
    L 4为-NH-CH 2-，D为d3；或
    L 4为单键，D为d2；或
    L 4为
    Figure PCTCN2022111443-appb-100023
    D为d2。
  9. 权利要求1-8任一项的化合物、其药学上可接受的盐、其立体异构体，或所述化合物、其药学上可接受的盐或其立体异构体的溶剂合物，所述化合物选自：
    Figure PCTCN2022111443-appb-100024
    Figure PCTCN2022111443-appb-100025
    Figure PCTCN2022111443-appb-100026
    Figure PCTCN2022111443-appb-100027
  10. 一种抗体-药物偶联物、其药学上可接受的盐、其立体异构体，或所述偶联物、其药学上可接受的盐或其立体异构体的溶剂合物，所述抗体-药物偶联物由靶向部分与式(I)所示化合物、其药学上可接受的盐、其立体异构体，或所述偶联物、其药学上可接受的盐或其立体异构体的溶剂合物经硫醚键连接而成；
    优选地，所述抗体-药物偶联物具有如式(II)所示的结构
    Ab-(S-L 1-L 2-L 3-L 4-D) p
    (II)
    其中Ab为靶向部分，选自抗体、抗体片段、或基于抗体的分子或化合物；
    -S-为硫醚键，
    L 1、L 2、L 3、L 4、D如权利要求1-8任一项所定义，
    p为选自1-20之间的整数，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20，例如选自1-8之间的整数；
    优选地，Ab是抗体、抗体片段、双特异性或其它多价抗体、或其它基于抗体的分子或化合物。
  11. 制备权利要求1-9任一项的化合物、其药学上可接受的盐、其立体异构体，或所述化合物、其药学上可接受的盐或其立体异构体的溶剂合物的方法:
    当L2为单键且L4为
    Figure PCTCN2022111443-appb-100028
    时，所述方法按照以下步骤进行：
    Figure PCTCN2022111443-appb-100029
    (1)使6-(2-(甲基磺酰基)嘧啶)-5-己炔酸与化合物i在DIEA和HATU存在的条件下发生缩合反应，生成化合物ii；
    (2)使化合物ii与对硝基苯基氯甲酸酯在DIEA存在的条件下发生缩合反应，生成化合物iii；
    (3)使化合物iii与艾日布林在DIEA存在的条件下发生缩合反应，生成式(I)所示的化合物(D为d2)；
    当L2为单键且L4为-NH-CH 2-时，所述方法按照以下步骤进行：
    Figure PCTCN2022111443-appb-100030
    (1)使6-(2-(甲基磺酰基)嘧啶)-5-己炔酸与化合物IV在DIEA和HATU存在的条件下发生缩合反应，生成化合物V；
    (2)使化合物V与艾日布林在DIEA和HATU存在的条件下发生缩合反应，生成式(I)所示的化合物(D为d2)；
    当L2为单键且L4为单键时，所述方法按照以下步骤进行：
    Figure PCTCN2022111443-appb-100031
    (1)使6-(2-(甲基磺酰基)嘧啶)-5-己炔酸与化合物VI在DIEA和HATU存在的条件下发生缩合反应，生成化合物VII；
    (2)使化合物VII与艾日布林在DIEA和HATU存在的条件下发生缩合反应，生成式(I)所示的化合物(D为d2)。
  12. 权利要求10所述的抗体-药物偶联物、其药学上可接受的盐、其立体异构体，或所述偶联物、其药学上可接受的盐或其立体异构体的溶剂合物的制备方法，包括使式(I)所示的化合物、其药学上可接受的盐、其立体异构体，或所述化合物、其药学上可接受的盐或其立体异构体的溶剂合物与靶向部分(例如抗体)反应，以通过由靶向部分的铰链部的二硫键部分形成硫醚键。
  13. 一种药物组合物，其包含权利要求1-9任一项所述的化合物、其药学上可接受的盐、其立体异构体，或所述化合物、其药学上可接受的盐或其立体异构体的溶剂合物，或权利要求10所述的抗体-药物偶联物、其药学上可接受的盐、其立体异构体，或所述偶联物、其药学上可接受的盐或其立体异构体的溶剂合物，以及一种或多种药用辅料，例如载体和/或赋形剂。
  14. 权利要求1-9任一项所述的化合物、其药学上可接受的盐、其立体异构体，或所述化合物、其药学上可接受的盐或其立体异构体的溶剂合物，或权利要求10所述的抗体-药物偶联物、其药学上可接受的盐、其立体异构体，或所述偶联物、其药学上可接受的盐或其立体异构体的溶剂合物，在制备治疗与细胞活动异常相关的疾病(例如癌症疾病)的药物中的用途。
  15. 一种药物制剂，其包含权利要求1-9任一项所述的化合物、其药学上可接受的盐、其立体异构体，或所述化合物、其药学上可接受的盐或其立体异构体的溶剂合物，或权利要求10所述的抗体-药物偶联物、其药学上可接受的盐、其立体异构体，或所述偶联物、其药学上可接受的盐或其立体异构体的溶剂合物。
  16. 权利要求1-9任一项所述的化合物、其药学上可接受的盐、其立体异构体，或所述化合物、其药学上可接受的盐或其立体异构体的溶剂合物，或权利要求10所述的抗体-药物偶联物、其药学上可接受的盐、其立体异构体，或所述偶联物、其药学上可接受的盐或其立体异构体的溶剂合物，权利要求13所述的药物组合物，用于制备药物制剂的用途。
  17. 一种试剂盒，其包括权利要求1-9任一项所述的化合物、其药学上可接受的盐、其立体异构体，或所述化合物、其药学上可接受的盐或其立体异构体的溶剂合物，或权利要求10所述的抗体-药物偶联物、其药学上可接受的盐、其立体异构体，或所述偶联物、其药学上可接受的盐或其立体异构体的溶剂合物，权利要求13所述的药物组合物，或权利要求15所述的药物制剂。
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