WO2024067754A1 - 一种带有高稳定性亲水连接单元的奥瑞他汀类药物及其偶联物 - Google Patents

一种带有高稳定性亲水连接单元的奥瑞他汀类药物及其偶联物 Download PDF

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WO2024067754A1
WO2024067754A1 PCT/CN2023/122381 CN2023122381W WO2024067754A1 WO 2024067754 A1 WO2024067754 A1 WO 2024067754A1 CN 2023122381 W CN2023122381 W CN 2023122381W WO 2024067754 A1 WO2024067754 A1 WO 2024067754A1
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antibody
compound
adc
drug conjugate
seq
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PCT/CN2023/122381
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French (fr)
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朱义
万维李
卓识
赖伟荣
杨秀娟
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成都百利多特生物药业有限责任公司
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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
    • 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
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • the present invention relates to the field of medical technology, and in particular to ligand-drug conjugates, linker-drug compounds, and preparation methods and uses thereof.
  • ADCs Antibody-Drug Conjugates
  • ADCs are a new type of targeted therapeutic drugs that combine the advantages of high selectivity of antibodies and high activity of cytotoxic drugs. They have the advantages of "high efficiency and low toxicity" and have become a research hotspot for targeted tumor therapy.
  • ADCs have developed rapidly and have developed to the third generation.
  • 13 ADCs have been approved for marketing, including Mylotarg, Adcetris, Kadcyla, Besponsa, Lumoxiti, Polivy, Enhertu, Padcev, Trodelvy, Blenrep, Zynlonta, Edixi and Tivdak, and more than 200 ADCs have entered clinical trials.
  • ADCs are playing an increasingly important role in the field of targeted tumor therapy.
  • ADCs Antibody-drug conjugates
  • mAbs antibodies
  • highly stable linkers highly efficient small molecule cytotoxic drugs
  • payload/warhead small molecule cytotoxic drugs
  • the antibody part of ADCs is most commonly IgG1
  • the warhead part is generally a cytotoxic drug that acts on microtubules, DNA or RNA, such as maytansines, auristatins, calicheamicin, camptothecins, pyrrolobenzodiazepines and amanita phalloidins.
  • linkers There are two main types of linkers: one is a cleavable linker and the other is a non-cleavable linker.
  • Auristatins are a class of tubulin inhibitors that can block tubulin from binding to GTP and block the binding of microtubules to vinblastine binding sites, thereby inducing cell apoptosis and inhibiting tumor growth.
  • MMAE US6884869
  • MMAF US7498298
  • Both MMAE and MMAF show good anti-tumor activity, but due to factors such as lack of selectivity and low therapeutic index, they cannot be used clinically as a single drug.
  • the high cytotoxicity of MMAE and MMAF makes them ideal warheads for ADC.
  • MMAE/MMAF has been widely used in the field of ADC.
  • ADC drugs with MMAE as a warhead and 1 with MMAF as a warhead on the market, namely Adcetris, Polivy, Padcev, Aidixi, Tivdak and Blenrep.
  • Adcetris Polivy
  • Padcev Padcev
  • Aidixi Tivdak
  • Blenrep auristatins
  • these ADCs with auristatins as toxins have some shortcomings.
  • ADCs with MMAE as toxins have adverse reactions such as neurotoxicity (peripheral neuropathy) and hematotoxicity (thrombocytopenia and neutropenia)
  • ADCs with MMAF as toxins have eye toxicity, etc., and their safety needs to be improved.
  • One of the technical problems to be solved by the present invention is to explore and discover better anti-tumor auristatin ADC drugs so that they have higher safety and effectiveness and better meet clinical needs.
  • the present invention connects auristatin or its derivatives to the enzymatic peptide unit from the hydroxyl site through the aminomethylene structure to form a corresponding linker-payload, which can increase the DAR value of ADC to 8, with better hydrophilicity at high drug loading, better plasma stability at high drug loading value, and more advantages in terms of drug efficacy.
  • positive drugs such as ADCs with Vc-MMAE as linker-payload
  • the present application provides the following inventions:
  • the present application provides a ligand-drug conjugate as shown in general formula I or a pharmaceutically acceptable salt or solvate thereof,
  • Ab is a ligand unit selected from an antibody, an antibody fragment, a targeting protein or an Fc-fusion protein;
  • M is a linker unit connected to Ab
  • A is selected from a peptide residue consisting of 2-7 amino acids, wherein, optionally, each of the amino acids is independently substituted by one or more substituents selected from the group consisting of: a deuterium atom, a halogen, a hydroxyl, a cyano group, an amino group, a nitro group, an alkyl group, a substituted alkyl group, an alkoxy group, a cycloalkyl group, and a substituted cycloalkyl group;
  • W represents an aminomethyleneoxy structural unit as shown in formula (i):
  • the wavy line on the left represents the connection site between the nitrogen atom and A in formula (i), and the wavy line on the right represents the connection site between the oxygen atom and drug D in formula (i), and the oxygen atom is a common group between drug D and W;
  • R 1 , R 2 and R 3 are each independently selected from a hydrogen atom, a deuterium atom, an alkyl group and a substituted alkyl group;
  • p is an integer or decimal selected from 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20);
  • Drug D is auristatin having a structure shown in formula D, or an isomer, mesoform, racemate, enantiomer or a mixture thereof, or a pharmaceutically acceptable salt thereof,
  • R 4 and R 5 are each independently selected from a hydrogen atom, a deuterium atom, an alkyl group and a deuterated alkyl group, or R 4 and R 5 are linked to form the following structure: -(CR 11 R 12 ) n -B-(CR 13 R 14 ) m -, wherein R 11 , R 12 , R 13 and R 14 are selected from a hydrogen atom, a deuterium atom, an alkyl group and a deuterated alkyl group; B is selected from O, NR 15 , CR 16 R 17 , wherein R 15 , R 16 , R 17 are selected from a hydrogen atom, a deuterium atom and an alkyl group; n and m are each independently selected from an integer of 0-8 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8); the nitrogen atom bonded to R 4 and R 5 forms a ring together with -(CR 11 R 12 ) n -B-(CR 13 R 14
  • R 6 , R 7 , R 8 , and R 9 are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an azido group, an alkyl group, and NR 18 R 19 , or any two of R 6 , R 7 , R 8 , and R 9 together with the atom to which they are bound form a cycloalkyl group, and the remaining two groups are each independently selected from a hydrogen atom, a halogen, an azido group, an alkyl group, and NR 18 R 19 , wherein R 18 and R 19 are selected from a hydrogen atom and an alkyl group;
  • R 10 is selected from aryl and heteroaryl, and the aryl or heteroaryl is optionally substituted by one or more substituents selected from hydrogen, halogen, alkyl, alkoxy, amino, and nitro;
  • the wavy line in formula D represents the connection site between the oxygen atom at position 1 and W in the structure of drug D, and the oxygen atom is a common group between drug D and W.
  • R 4 and R 5 are each independently selected from a hydrogen atom and a C 1 -C 4 alkyl group.
  • R 4 and R 5 are linked to form the following structure: -(CH 2 ) 2 -B-(CH 2 ) 2 -, B is selected from O and NH, and the nitrogen atom bonded to R 4 and R 5 forms a ring together with -(CH 2 ) 2 -B-(CH 2 ) 2 -.
  • R 6 , R 7 , R 8 , and R 9 in Formula D are each a hydrogen atom.
  • one of R 6 , R 7 , R 8 , and R 9 in Formula D is selected from halogen, azido, and amino, and the remaining three are each hydrogen.
  • R 10 in Formula D is phenyl, optionally, the phenyl is substituted by one or more of the substituents. In certain embodiments, the substituents are selected from amino and nitro.
  • drug D is selected from, but not limited to, the following compounds:
  • drug D is linked to W via the hydroxyl group.
  • the linker unit M comprises maleimide.
  • the ligand-drug conjugate can be hydrolyzed under hydrolysis conditions, and the hydrolysis site is the maleimide portion of the linker unit.
  • the ligand contains multiple linkers-drugs, the following situations may occur depending on the degree of hydrolysis:
  • maleimide is not hydrolyzed at all, that is, maleimide is in a closed ring form
  • Maleimide is not completely hydrolyzed, that is, part of the maleimide is in a closed ring form The other part of maleimide is in the open ring form
  • maleimide is completely hydrolyzed, that is, maleimide is in the open ring form
  • these maleimide groups can all be in a closed-ring form, some in an open-ring form, or all in an open-ring form.
  • the wavy line on the left represents the connection site to Ab
  • the wavy line on the right represents other structures in M.
  • the ligand-drug conjugate has the structure shown in Formula Ia:
  • Z is selected from -C 1 -C 10 alkylene-, -C 3 -C 8 carbocycle-, -arylene-, -C 1 -C 10 alkylene-arylene-, -arylene-C 1 -C 10 -alkylene-, -C 1 -C 10 alkylene-(C 3 -C 8 carbocycle)-, -(C 3 -C 8 carbocycle)-C 1 -C 10 alkylene-, 3-8 membered heterocycle-, -C 1 -C 10 alkylene-(3-8 membered heterocycle)-, -(3-8 membered heterocycle)-C 1 -C 10 alkylene-, -(CH 2 CH 2 O) r -, -(CH 2 CH 2 O) r -CH 2 - or wherein X is selected from -C 1 -C 10 alkylene-, -C 3 -C 8 carbocycle-, -arylene-, -C 1 -
  • n1 , n2 , n3 are independently selected from integers or decimals between 0 and 20, n1 , n2 , n3 are not 0 at the same time, and n1 + n2 + n3 ⁇ 20 , for example, 1 ⁇ n1 +n2+ n3 ⁇ 2 , or 7 ⁇ n1 + n2 + n3 ⁇ 8 .
  • n1 , n2, and n3 are independently selected from 0, 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 (e.g., 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 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, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4,
  • a in Formula I or Formula Ia is a polypeptide residue formed by 2-7 amino acids selected from phenylalanine (F), glycine (G), valine (V), lysine (K), alanine (A), citrulline, serine (S), glutamic acid (E) or aspartic acid (D).
  • A is a peptide residue formed by 2-4 amino acids selected from phenylalanine and glycine. In certain embodiments, A is a tetrapeptide residue consisting of glycine-glycine-phenylalanine-glycine.
  • Z is selected from -C 1 -C 10 alkylene-, such as -C 4 -C 6 alkylene-, such as -C 5 alkylene-.
  • Z is Wherein, q is selected from an integer between 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8), for example, 1.
  • R 1 , R 2 and R 3 are each independently selected from a hydrogen atom, a deuterium atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group.
  • R 1 , R 2 and R 3 are simultaneously hydrogen atoms or deuterium atoms.
  • R 1 , R 2 and R 3 are hydrogen atoms at the same time.
  • the ligand-drug conjugate has a structure as shown in Formula Ib,
  • the ligand-drug conjugate has a structure as shown in Formula Ic,
  • Ac is a hydrophilic structural unit having a structure shown in formula c:
  • Ac is selected from glycine, (D/L) alanine, (D/L) leucine, (D/L) isoleucine, (D/L) valine, (D/L) phenylalanine, (D/L) proline, (D/L) tryptophan, (D/L) serine, (D/L) tyrosine, (D/L) cysteine, (D/L) cystine, (D/L) arginine, (D/L) histidine, (D/L) methionine, (D/L) Asparagine, (D/L) glutamine, (D/L) threonine, (D/L) aspartic acid, (D/L) glutamic acid, natural or unnatural amino acid derivatives or the following structures,
  • the ligand-drug conjugates of the present invention can be selected from the following structures without limitation:
  • the configuration of the chiral carbon at position 2 is R-type or S-type.
  • the present application provides a linker-drug compound as shown in Formula II, or a pharmaceutically acceptable salt or solvate thereof,
  • Z, A, R1 , R2 , R3, R4 , R5 , R11 , R12 , R13 , R14 , B, R15 , R16 , R17 , n, m, R6 , R7 , R8 , R9 , R18 , R19 , and R10 are as defined in any of the above items.
  • the linker-drug compound has the structure shown in Formula IIa,
  • the linker-drug compound has the structure shown in Formula IIb,
  • Ac is a hydrophilic structural unit having a structure shown in formula c:
  • linker-drug compounds of the present invention can be selected from the following structures without limitation:
  • the configuration of the chiral carbon at position 2 is R-type or S-type.
  • the ligand unit Ab can be selected from antibodies, antibody fragments or proteins, wherein the antibody is selected from mouse antibodies, rabbit antibodies, phage display antibodies, yeast display antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies and multispecific antibodies.
  • the antibody is a monoclonal antibody, selected from, but not limited to, anti-EGFRvIII antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD70 antibody, anti-MUC16 antibody, anti-ENPP3 antibody, anti-TDGF1 antibody, anti-ETBR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC15 antibody, anti-LIV-1 antibody, anti-CanAg/AFP antibody, anti-claudin 18.2 antibody , Anti-Mesothelin Antibody, Anti-HER2 (ErbB2) Antibody, Anti-EGFR Antibody, Anti-c-MET Antibody, Anti-SLITRK6 Antibody, Anti-KIT/CD117 Antibody, Anti-STEAP1 Antibody, Anti-SLAMF7/CS1 Antibody, Anti-NaPi2B/SLC34A2 Antibody, Anti-GPNMB Antibody, Anti-HER3 (ErbB3) Antibody, Anti-EGFRv
  • the antibody is an anti-Trop2 antibody.
  • the antibody is composed of a light chain and a heavy chain, the light chain comprises CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequences thereof are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.
  • the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3, and the amino acid sequences thereof are shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.
  • the light chain comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 7. In some embodiments, the light chain further comprises a light chain constant region having an amino acid sequence of SEQ ID NO: 8. In certain embodiments, the amino acid sequence of the light chain is SEQ ID NO:9.
  • the light chain comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 10. In certain embodiments, the light chain further comprises a light chain constant region having an amino acid sequence of SEQ ID NO: 11. In certain embodiments, the amino acid sequence of the light chain is SEQ ID NO: 12.
  • the heavy chain comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 13. In certain embodiments, the heavy chain further comprises a heavy chain constant region having an amino acid sequence of SEQ ID NO: 14. In certain embodiments, the amino acid sequence of the heavy chain is SEQ ID NO: 15.
  • the antibody is an anti-Trop2 antibody.
  • the antibody is composed of a light chain and a heavy chain, the light chain comprises CDR-L1, CDR-L2 and CDR-L3, and the nucleic acid coding sequences thereof are shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.
  • the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3, and the nucleic acid coding sequences thereof are shown in SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively.
  • the light chain comprises a light chain variable region whose nucleic acid encoding sequence is SEQ ID NO: 22. In certain embodiments, the light chain further comprises a light chain constant region whose nucleic acid encoding sequence is SEQ ID NO: 23. In certain embodiments, the nucleic acid encoding sequence of the light chain is SEQ ID NO: 24.
  • the light chain comprises a light chain variable region whose nucleic acid encoding sequence is SEQ ID NO: 25. In certain embodiments, the light chain further comprises a light chain constant region whose nucleic acid encoding sequence is SEQ ID NO: 26. In certain embodiments, the light chain nucleic acid encoding sequence is SEQ ID NO: 27.
  • the heavy chain comprises a heavy chain variable region whose nucleic acid encoding sequence is SEQ ID NO: 28. In certain embodiments, the heavy chain further comprises a heavy chain constant region whose nucleic acid encoding sequence is SEQ ID NO: 29. In certain embodiments, the nucleic acid encoding sequence of the heavy chain is SEQ ID NO: 30.
  • the antibody is an anti-Trop2 antibody.
  • the antibody is composed of a light chain and a heavy chain, the light chain comprises CDR-L1, CDR-L2 and CDR-L3, and the amino acid sequences thereof are shown in SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively.
  • the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3, and the amino acid sequences thereof are shown in SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively.
  • the light chain comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 37. In certain embodiments, the light chain further comprises a light chain constant region having an amino acid sequence of SEQ ID NO: 38. In certain embodiments, the amino acid sequence of the light chain is SEQ ID NO: 39.
  • the heavy chain comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 40. In certain embodiments, the heavy chain further comprises a heavy chain constant region having an amino acid sequence of SEQ ID NO: 41. In certain embodiments, the amino acid sequence of the heavy chain is SEQ ID NO: 42.
  • the antibody is an anti-Trop2 antibody.
  • the antibody is composed of a light chain and a heavy chain, the light chain comprises CDR-L1, CDR-L2 and CDR-L3, and the nucleic acid coding sequences thereof are shown in SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 45, respectively.
  • the heavy chain comprises CDR-H1, CDR-H2, and CDR-H3, and the nucleic acid coding sequences thereof are shown in SEQ ID NO: 46, SEQ ID NO: 47, and SEQ ID NO: 48, respectively.
  • the light chain comprises a light chain variable region whose nucleic acid encoding sequence is SEQ ID NO: 49. In certain embodiments, the light chain further comprises a light chain constant region whose nucleic acid encoding sequence is SEQ ID NO: 50. In certain embodiments, the nucleic acid encoding sequence of the light chain is SEQ ID NO: 51.
  • the heavy chain comprises a heavy chain variable region whose nucleic acid encoding sequence is SEQ ID NO: 52. In certain embodiments, the heavy chain further comprises a heavy chain constant region whose nucleic acid encoding sequence is SEQ ID NO: 53. In certain embodiments, the nucleic acid encoding sequence of the heavy chain is SEQ ID NO: 54.
  • the present application also provides the use of the ligand-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof, or the linker-drug compound of the present invention or its isomer, mesomorph, racemate, enantiomer or a mixture thereof, or a pharmaceutically acceptable salt or solvate thereof for preparing a drug for treating or preventing tumors.
  • the tumor is selected from a solid tumor or a non-solid tumor, such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma and leukemia.
  • a solid tumor or a non-solid tumor such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanom
  • the present application also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of the ligand-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof, or the linker-drug compound of the present invention or its isomer, mesomorph, racemate, enantiomer or a mixture thereof, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the present application also provides a method for treating or preventing tumors, comprising administering to a subject in need thereof an effective amount of the ligand-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof, or a linker-drug compound of the present invention or an isomer, meso-racemate, racemate, enantiomer or mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.
  • the subject is a mammal, such as a human.
  • the present application also provides the use of the linker-drug compound of the present invention or its isomer, meso-racemate, racemate, enantiomer or mixture thereof, or its pharmaceutically acceptable salt or solvate for preparing a ligand-drug conjugate or its pharmaceutically acceptable salt or solvate.
  • the ligand-drug conjugate is selected from the ligand-drug conjugate of the present invention.
  • the present application also provides a method for preparing the ligand-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof, the method comprising: coupling a reduced antibody or an antigen-binding fragment thereof with a linker-drug compound of the present invention or an isomer, mesomorph, racemate, enantiomer or a mixture thereof, or a pharmaceutically acceptable salt or solvate thereof to obtain the ligand-drug conjugate.
  • the antibody or the antigen-binding fragment thereof can be reduced by a thiol reducing agent (e.g., tris(2-carboxyethyl)phosphine (TCEP)).
  • TCEP tris(2-carboxyethyl)phosphine
  • ligand is a targeting agent that specifically binds to a target moiety.
  • the ligand can specifically bind to a cell component or to other target molecules of interest.
  • the target moiety or target is usually on the cell surface.
  • the role of the ligand is to deliver the drug unit to a specific target cell population with which the ligand unit interacts.
  • the ligand includes, but is not limited to, proteins, polypeptides and peptides, and non-proteins such as sugars.
  • Suitable ligand units include, for example, antibodies, such as full-length (complete) antibodies and antigen-binding fragments thereof.
  • the ligand unit is a non-antibody targeting agent
  • it can be a peptide or polypeptide, or a non-protein molecule.
  • targeting agents include interferons, lymphokines, hormones, growth factors and colony stimulating factors, vitamins, nutrient transport molecules, or any other cell binding molecules or substances.
  • the linker is covalently attached to the sulfur atom of the ligand.
  • the sulfur atom is a sulfur atom of a cysteine residue that forms an interchain disulfide bond of an antibody.
  • the sulfur atom is a sulfur atom of a cysteine residue that has been introduced into the ligand unit that forms an interchain disulfide bond of an antibody.
  • the sulfur atom is a sulfur atom of a cysteine residue introduced into the Ligand unit by, for example, site-directed mutagenesis or chemical reaction.
  • drug refers to cytotoxic drugs, that is, molecules that have a strong ability to disrupt the normal growth of tumor or cancer cells. Cytotoxic drugs can kill tumor cells in principle at sufficiently high concentrations, but due to the lack of specificity, while killing tumor or cancer cells, they can also cause apoptosis of normal cells, which can easily lead to serious side effects.
  • ligand-drug conjugate refers to a molecule formed by connecting a ligand to a drug via a stable linker.
  • the "ligand-drug conjugate” is preferably an antibody-drug conjugate (ADC), which refers to a monoclonal antibody or a functional antibody fragment or a targeted protein connected to a cytotoxic drug via a stable linker.
  • ADC antibody-drug conjugate
  • antibody or “functional antibody fragment” includes within its scope any portion of the antibody structure. This unit can bind, reactively associate or complex with a receptor, antigen or other receptor unit possessed by the target cell population.
  • the antibody can be any protein or protein-like molecule that can bind, complex or react with a portion of the cell population to be treated or bioengineered.
  • the antibodies of the present invention include, but are not limited to, murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, preferably humanized antibodies and fully human antibodies.
  • natural amino acid refers to an amino acid that can be synthesized biologically. Natural amino acids are generally L-form, but There are a few exceptions, such as glycine, both natural and biosynthesized.
  • unnatural amino acid refers to an amino acid that can only be synthesized by artificial methods.
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 or 1 to 4 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl,
  • lower alkyl groups having 1 to 6 e.g., 1 to 4) carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like.
  • 1 to 6
  • the alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment.
  • the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, and oxo.
  • substituted alkyl refers to an alkyl radical in which a hydrogen atom is replaced by a substituent.
  • R', R" and R'"' each represent hydrogen, C 1-8 alkyl, aryl, aryl substituted by 1-3 halogens, C 1-8 alkyl substituted by 1-3 halogens, C 1-8 alkoxy or C 1-8 thioalkoxy, or unsubstituted aryl-C 1-4 alkyl.
  • R' and R" When R' and R" are attached to the same nitrogen atom, they can form a 3-, 4-, 5-, 6- or 7-membered ring with the nitrogen atom.
  • -NR'R" includes 1-pyrrolidinyl and 4-morpholinyl.
  • heteroalkyl refers to a group in which one or more carbon atoms on an alkyl group are replaced by N, O or S.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon group, the ring of which contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc.; polycyclic cycloalkyls include spirocyclic, condensed and bridged cycloalkyls.
  • alkoxy refers to-O-(alkyl) and-O-(cycloalkyl), wherein the definition of alkyl or cycloalkyl is as described above.
  • alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy.
  • Alkoxy can be optionally substituted or unsubstituted, and when substituted, substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.
  • heterocycle refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon containing 3 to 20 ring atoms, wherein one or more (e.g., 1, 2, 3 or 4) ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) m (wherein m is 0, 1 or 2), and the remaining ring atoms are carbon.
  • ring atoms e.g., 1, 2, 3 or 4
  • it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, it contains 3 to 10 or 3 to 8 ring atoms.
  • Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc.
  • Polycyclic heterocyclic groups include spirocyclic, fused ring and bridged heterocyclic groups.
  • aryl refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated ⁇ electron system, preferably 6-10 members, such as phenyl.
  • the aryl group may be substituted or unsubstituted, and when substituted, the substituent may be one or more of the following groups, selected from, but not limited to, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, deuterium atom, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio or heterocycloalkylthio.
  • heteroaryl includes 5-8 membered monocyclic heteroaryl and 8-12 membered fused heteroaryl.
  • 5-8 membered monocyclic heteroaryl refers to a monocyclic cyclic group having aromaticity and containing 5-8 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom).
  • the ring atoms such as carbon atoms, nitrogen atoms or sulfur atoms
  • "5-8 membered monocyclic heteroaryl” includes, for example, "5-7 membered monocyclic heteroaryl", “5-6 membered monocyclic heteroaryl”, “5-6 membered monocyclic nitrogen-containing heteroaryl", “6 membered monocyclic nitrogen-containing heteroaryl", etc.
  • the heteroatom in the "nitrogen-containing heteroaryl group” contains at least one nitrogen atom, for example, contains only 1 or 2 nitrogen atoms, or contains one nitrogen atom and other 1 or 2 heteroatoms (for example, oxygen atoms and/or sulfur atoms), or contains 2 nitrogen atoms and other 1 or 2 heteroatoms (for example, oxygen atoms and/or sulfur atoms).
  • 5-8 membered monocyclic heteroaryl include, but are not limited to, furanyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, pyridyl, 2-pyridonyl, 4-pyridonyl, pyrimidinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, azacycloheptatrienyl, 1,3-diazacycloheptatrienyl
  • 8-12 membered fused heteroaryl refers to an unsaturated aromatic cyclic structure formed by two or more cyclic structures sharing two adjacent atoms, containing 8-12 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom).
  • the ring atoms such as carbon atoms, nitrogen atoms or sulfur atoms
  • the cyclic structure may be oxo-substituted.
  • “8-12 membered fused heteroaryl” includes “8-10 membered fused heteroaryl”, “8-9 membered fused heteroaryl” and the like; specific examples include, but are not limited to, pyrrolopyrrole, pyrrolofuran, pyrazolopyrrole, pyrazolothiophene, furathiophene, pyrazolooxazole, benzofuranyl, benzisofuranyl, benzothiophenyl, indolyl, isoindolyl, benzoxazolyl, benzimidazolyl, indazolyl, benzotriazolyl, quinolyl, 2-quinolinone, 4-quinolinone, 1-isoquinolinone, isoquinolyl, acridinyl, phenanthridinyl, benzopyridazinyl, phthalazinyl, quinazolinyl, quinoxalinyl, purinyl, naphthy
  • haloalkyl refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.
  • deuterated alkyl refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.
  • hydroxy refers to an -OH group.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • amino refers to -NH2 .
  • nitro refers to -NO2 .
  • derivative refers to a substance that has a chemical structure similar to that of a compound but also contains at least one chemical group not present in the compound and/or lacks at least one chemical group present in the compound.
  • the compound to which the derivative is compared is called the "parent” compound.
  • a “derivative” can be produced from a parent compound in one or more chemical reaction steps.
  • pharmaceutically acceptable salt refers to a pharmaceutically acceptable organic or inorganic salt of a compound (e.g., a drug, a linker-drug compound or a ligand-drug conjugate).
  • the compound or conjugate may contain at least one amino or carboxyl group and thus may form addition salts with the corresponding acids or bases.
  • Exemplary salts include, but are not limited to, sulfates, trifluoroacetates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, hydrogen sulfates, Salt, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, salicylate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, potassium salt, sodium salt, etc.
  • solvate refers to a linker-drug compound or ligand-drug conjugate of the present invention formed with one or more solvent molecules, including but not limited to water, ethanol, acetonitrile, isopropanol, DMSO, ethyl acetate, etc.
  • pharmaceutical composition refers to a mixture containing one or more compounds of the present invention or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and/or excipients.
  • the purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredients, and thus exert biological activity.
  • carrier refers to a system that can change the way a drug enters the human body and its distribution in the body, control the release rate of the drug, and deliver the drug to the target site.
  • Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and improve bioavailability.
  • excipient refers to the additives or auxiliary materials in pharmaceutical preparations other than the main drug.
  • binders, fillers, disintegrants, and lubricants in tablets for example, binders, fillers, disintegrants, and lubricants in tablets; matrix backup in semi-solid preparations such as ointments and creams; preservatives, antioxidants, flavoring agents, aromatics, cosolvents, emulsifiers, permeability enhancers, osmotic pressure regulators, colorants, etc. in liquid preparations can all be called excipients.
  • diluent or "filler” is mainly used to increase the weight and/or volume of the preparation.
  • the addition of diluent not only ensures a certain volume, but also reduces the dosage deviation of the main ingredients, improves the compression moldability of the drug, etc.
  • the present invention provides an auristatin drug conjugate with a high-stability hydrophilic linking unit.
  • the conjugate introduces a linker by forming an ether bond between an aminomethyl group with a high-stability hydrophilic linking unit and a hydroxyl group.
  • the conjugate can carry multiple toxins, has good plasma stability, good water solubility, uniformity and safety, can specifically bind to receptors highly expressed in tumor cells, and release toxins in tumor cells. It has good anti-tumor activity and can be used to prevent or treat diseases such as tumors.
  • FIG1 is the LC-MS spectrum of compound LP-1a.
  • Figure 2 is the LC-MS spectrum of VcMMAE.
  • FIG3 is the SEC-HPLC detection result of ADC-1-1a.
  • FIG4 is the SEC-HPLC detection result of ADC-2-1a.
  • FIG5 is the SEC-HPLC detection result of ADC-1-2a.
  • FIG6 is the SEC-HPLC detection result of ADC-2-2a.
  • FIG. 7 shows the mass spectrometry detection results of the light chain of the reduced antibody TR005.
  • FIG8 and FIG9 are the mass spectrometry detection results of the light chain of ADC-2-1a after reduction.
  • antibody molecules with a monomer rate greater than 95% were replaced with an ultrafiltration centrifuge tube to a phosphate buffer containing EDTA at a concentration of 10 mg/mL.
  • TCEP 10 times the number of antibody moles was added and reacted at room temperature for 6 hours.
  • the interchain disulfide bonds of the antibody were opened, and the number of free thiols was determined by the Ellman method to determine whether all disulfide bonds were opened.
  • a payload 10 times the number of antibody moles was added and reacted at room temperature for 6 hours.
  • antibody molecules with a monomer rate greater than 95% were replaced with an ultrafiltration centrifuge tube to a phosphate buffer containing EDTA at a concentration of 10 mg/mL.
  • TCEP was added at 8 times the number of moles of the antibody and reacted at room temperature for 3 hours.
  • the solution was replaced with a pH 6.5 phosphate buffer using an ultrafiltration centrifuge tube, and DHAA (dehydroascorbic acid) was added at 8 times the number of moles of the antibody and reacted at room temperature for 3 hours. Then 5 times the number of moles of the antibody payload was added and reacted at room temperature for 3 hours.
  • RP mobile phase A 0.1% TFA aqueous solution
  • RP mobile phase B 0.1% TFA acetonitrile solution.
  • the chromatographic column model is Proteomix RP-1000 (4.6*100mm, 5 ⁇ m), Sepax.
  • the detection wavelengths are 214nm and 280nm.
  • the running method is edited as follows:
  • the ADC was analyzed using hydrophobic interaction chromatography (HIC).
  • HIC hydrophobic interaction chromatography
  • the elution was performed with 0-100% mobile phase B (MPB), where mobile phase A (MPA) consisted of 1.5 M ammonium sulfate and 0.025 M sodium phosphate, and MPB consisted of 0.025 M sodium phosphate and 25% isopropanol.
  • MPB mobile phase B
  • the sample loading was about 20 ⁇ g, and the gradient elution was completed in 15 minutes.
  • the detection wavelength was 280 nm, and the more hydrophobic the sample, the later the peak.
  • ADC sample Take a certain amount of ADC sample and add it to human plasma from which IgG has been removed. Repeat three tubes for each ADC and place them in a 37°C water bath for incubation. After 0 days, 3 days, 7 days and 21 days, take out the ADC sample, add ProteinA (MabSelectSuReTMLX Lot:#10257475GE) to each tube, take 100 ⁇ l washed with PBS and shake it for adsorption on a vertical mixer for 2h, and after washing and elution steps, obtain the incubated ADC. Perform RP-HPLC detection on the ADC samples incubated for a specific time. Determine the plasma stability of the sample.
  • ProteinA MebSelectSuReTMLX Lot:#10257475GE
  • Auristatin E (826 mg, 1.128 mmol, 1.0 eq), ki-1 (831.4 mg, 2.256 mmol, 2.0 eq, the synthesis method refers to the synthesis of compound 1 in CN111686259A), zinc acetate (414.2 mg, 5.64 mmol, 2.0 eq) and toluene (15 mL) were added to a 50 mL single-mouth round-bottom flask in sequence, and nitrogen was replaced 3 times. The reaction was refluxed at 115 ° C for 4 h, and the reaction was stopped. The mixture was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. The mixture was purified by reverse phase preparative column and freeze-dried to obtain a white solid (605 mg, 51.55%). LC-MS m/z (ES + ): [M+H] + : 1041.3.
  • Z-Gly-Gly-Phe-OH (240 mg, 0.581 mmol, 1.0 eq) was added in sequence.
  • HATU 264 mg, 0.697 mmol, 1.2 eq
  • HOBt 94 mg, 0.697 mmol, 1.2 eq
  • DMF 2 mL
  • reaction solution 1 In a 25 mL single-necked round-bottom flask, compound 3 (487 mg, 0.402 mmol, 1.0 eq), 5% Pd/C (48.7 mg) and DMF (5 mL) were added in sequence. After hydrogen replacement three times, the reaction was carried out at room temperature for 1 h. The reaction was monitored by HPLC. The starting material disappeared and a new peak was generated, which was compound 4, recorded as reaction solution 1.
  • reaction solution 1 was filtered into a new 25mL single-mouth round-bottom flask, DIEA (73 ⁇ L, 0.442mmol, 1.1eq) was added under ice-water bath, the reaction solution 2 was filtrated, and the temperature was raised to room temperature for 1h after addition, and the reaction was monitored by HPLC.
  • the reaction solution was filtered, and the filtrate was added to the reaction solution containing compound 4 under stirring in an ice bath, and then added, the ice bath was removed, and the reaction was reacted at room temperature for 1h, and HPLC was monitored.
  • the reaction solution was directly purified by reverse phase preparation, and the preparation solution was lyophilized to obtain a white solid product (320mg, 54.53%).
  • MMAE 2.0 g, 2.79 mmol, 1.0 eq
  • (Boc) 2 O (1.21 g, 5.57 mmol, 2.0 eq) were added to a 100 mL single-mouth bottle, and DCM (20 mL) was used to dissolve the mixture.
  • TEA 563 mg, 5.57 mmol, 2.0 eq
  • the mixture was heated to room temperature and reacted for 72 h.
  • reaction solution 1 DIEA (103.4mg, 0.8mmol, 1.0eq) in an ice-water bath, and after addition, warm to room temperature to react for 1h, and monitor the reaction by HPLC.
  • the reaction solution is purified by reverse phase preparation, and the preparation solution is lyophilized to obtain a white solid (960mg, 92.4%).
  • reaction solution 1 was filtered into a 50 mL single-mouth bottle, and DIEA (105 mg, 0.81 mmol, 1.1 eq) was added under an ice-water bath.
  • the reaction solution 2 was filtrated and heated to room temperature for 1 h after addition.
  • the reaction was monitored by HPLC.
  • the reaction solution was purified by reverse phase preparation, and the preparation solution was lyophilized to obtain a white solid (895 mg, 78.5%).
  • compound 29 was synthesized using compound 28 (synthesized according to patent US2017014524A1) as a raw material.
  • compound 36 was synthesized using compound 35 (synthesized by referring to patent US20210346523A1) as a raw material.
  • ADC-1-1a was prepared by combining the corresponding linker-payload (LP-1a, whose structure is shown in the structural formula of LP-1, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A, and the sequence information of TR000 is shown in Table 1.
  • the SEC-HPLC detection results are shown in Figure 3.
  • ADC-2-1a was prepared by combining the corresponding linker-payload (LP-2a, whose structure is shown in the structural formula of LP-2, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • the SEC-HPLC detection results are shown in Figure 4.
  • ADC-C-1a was prepared by reacting compound VcMMAE with anti-Trop2 antibody TR000 according to the method of general procedure A.
  • ADC-3-1a was prepared by combining the corresponding linker-payload (LP-10) with the anti-Trop2 antibody TR000 according to the method of general procedure A.
  • ADC-4-1a was prepared by combining the corresponding linker-payload (LP-37) with the anti-Trop2 antibody TR000 according to the method of general procedure A.
  • ADC-5-1a was prepared by combining the corresponding linker-payload (LP-16) with the anti-Trop2 antibody TR000 according to the method of general procedure A.
  • ADC-6-1a was prepared by combining the corresponding linker-payload (LP-11) with the anti-Trop2 antibody TR000 according to the method of general procedure A.
  • ADC-7-1a was prepared by combining the corresponding linker-payload (LP-12) with the anti-Trop2 antibody TR000 according to the method of general procedure A.
  • ADC-8-1a was prepared by combining the corresponding linker-payload (LP-13) with the anti-Trop2 antibody TR000 according to the method of general procedure A.
  • ADC-9-1a was prepared by combining the corresponding linker-payload (LP-14) with the anti-Trop2 antibody TR000 according to the method of general procedure A.
  • ADC-10-1a was prepared by combining the corresponding linker-payload (LP-15) with the anti-Trop2 antibody TR000 according to the method of general procedure A.
  • ADC-11-1a was prepared by combining the corresponding linker-payload (LP-17) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-12-1a was prepared by combining the corresponding linker-payload (LP-18) with the anti-Trop2 antibody TR000 according to the method of general procedure A.
  • ADC-13-1a was prepared by combining the corresponding linker-payload (LP-3a, whose structure is shown in the structural formula of LP-3, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-14-1a was prepared by combining the corresponding linker-payload (LP-7a, whose structure is shown in the structural formula of LP-7, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-15-1a was prepared by combining the corresponding linker-payload (LP-3sa, whose structure is shown in the structural formula of LP-3s, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-16-1a was prepared by combining the corresponding linker-payload (LP-4a, whose structure is shown in the structural formula of LP-4, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-17-1a was prepared by combining the corresponding linker-payload (LP-5a, whose structure is shown in the structural formula of LP-5, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-18-1a was prepared by combining the corresponding linker-payload (LP-6a, whose structure is shown in the structural formula of LP-6, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-19-1a was prepared by combining the corresponding linker-payload (LP-8a, whose structure is shown in the structural formula of LP-8, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-20-1a was prepared by combining the corresponding linker-payload (LP-9a, whose structure is shown in the structural formula of LP-9, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-21-1a was prepared by combining the corresponding linker-payload (LP-28a, whose structure is shown in the structural formula of LP-28, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-22-1a was prepared by combining the corresponding linker-payload (LP-29a, whose structure is shown in the structural formula of LP-29, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-23-1a was prepared by combining the corresponding linker-payload (LP-30a, whose structure is shown in the structural formula of LP-30, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-24-1a was prepared by combining the corresponding linker-payload (LP-31a, whose structure is shown in the structural formula of LP-31, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-25-1a was prepared by combining the corresponding linker-payload (LP-32a, whose structure is shown in the structural formula of LP-32, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-26-1a was prepared by combining the corresponding linker-payload (LP-33a, whose structure is shown in the structural formula of LP-33, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-27-1a was prepared by combining the corresponding linker-payload (LP-35a, whose structure is shown in the structural formula of LP-35, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-28-1a was prepared by combining the corresponding linker-payload (LP-36a, whose structure is shown in the structural formula of LP-36, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-29-1a was prepared by combining the corresponding linker-payload (LP-39a, whose structure is shown in the structural formula of LP-39, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-30-1a was prepared by combining the corresponding linker-payload (LP-34a, whose structure is shown in the structural formula of LP-34, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-31-1a was prepared by combining the corresponding linker-payload (LP-19a, whose structure is shown in the structural formula of LP-19, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-35-1a was prepared by combining the corresponding linker-payload (LP-22a, whose structure is shown in the structural formula of LP-22, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-39-1a was prepared by combining the corresponding linker-payload (LP-26a, whose structure is shown in the structural formula of LP-26, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-45-1a was prepared by combining the corresponding linker-payload (LP-20a, whose structure is shown in the structural formula of LP-20, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-48-1a was prepared by combining the corresponding linker-payload (LP-27a, whose structure is shown in the structural formula of LP-27, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR000 according to the method of general step A.
  • ADC-1-2a was prepared by combining the corresponding linker-payload (LP-1a, whose structure is shown in the structural formula of LP-1, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • the sequence information of TR005 is shown in Table 1.
  • the SEC-HPLC detection results are shown in Figure 5.
  • ADC-2-2a was prepared by combining the corresponding linker-payload (LP-2a, whose structure is shown in the structural formula of LP-2, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • the SEC-HPLC detection results are shown in Figure 6.
  • ADC-C-2a was prepared by reacting compound VcMMAE with anti-Trop2 antibody TR005 according to the method of general procedure B.
  • ADC-3-2a was prepared by combining the corresponding linker-payload (LP-10) with the anti-Trop2 antibody TR005 according to the method of general procedure B.
  • ADC-4-2a was prepared by combining the corresponding linker-payload (LP-37) with the anti-Trop2 antibody TR005 according to the method of general procedure B.
  • ADC-5-2a was prepared by combining the corresponding linker-payload (LP-16) with the anti-Trop2 antibody TR005 according to the method of general procedure B.
  • ADC-6-2a was prepared by combining the corresponding linker-payload (LP-11) with the anti-Trop2 antibody TR005 according to the method of general procedure B.
  • ADC-7-2a was prepared by combining the corresponding linker-payload (LP-12) with the anti-Trop2 antibody TR005 according to the method of general procedure B.
  • ADC-8-2a was prepared by combining the corresponding linker-payload (LP-13) with the anti-Trop2 antibody TR005 according to the method of general procedure B.
  • ADC-9-2a was prepared by combining the corresponding linker-payload (LP-14) with the anti-Trop2 antibody TR005 according to the method of general procedure B.
  • ADC-10-2a was prepared by combining the corresponding linker-payload (LP-15) with the anti-Trop2 antibody TR005 according to the method of general procedure B.
  • ADC-11-2a was prepared by combining the corresponding linker-payload (LP-17) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-12-2a was prepared by combining the corresponding linker-payload (LP-18) with the anti-Trop2 antibody TR005 according to the method of general procedure B.
  • ADC-13-2a was prepared by combining the corresponding linker-payload (LP-3a, whose structure is shown in the structural formula of LP-3, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-14-2a was prepared by combining the corresponding linker-payload (LP-7a, whose structure is shown in the structural formula of LP-7, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-15-2a was prepared by combining the corresponding linker-payload (LP-3sa, whose structure is shown in the structural formula of LP-3s, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-16-2a was prepared by combining the corresponding linker-payload (LP-4a, whose structure is shown in the structural formula of LP-4, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-17-2a was prepared by combining the corresponding linker-payload (LP-5a, whose structure is shown in the structural formula of LP-5, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-18-2a was prepared by combining the corresponding linker-payload (LP-6a, whose structure is shown in the structural formula of LP-6, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-19-2a was prepared by combining the corresponding linker-payload (LP-8a, whose structure is shown in the structural formula of LP-8, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-20-2a was prepared by combining the corresponding linker-payload (LP-9a, whose structure is shown in the structural formula of LP-9, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-21-2a was prepared by combining the corresponding linker-payload (LP-28a, whose structure is shown in the structural formula of LP-28, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-22-2a was prepared by combining the corresponding linker-payload (LP-29a, whose structure is shown in the structural formula of LP-29, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-23-2a was prepared by combining the corresponding linker-payload (LP-30a, whose structure is shown in the structural formula of LP-30, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-24-2a was prepared by combining the corresponding linker-payload (LP-31a, whose structure is shown in the structural formula of LP-31, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-25-2a was prepared by combining the corresponding linker-payload (LP-32a, whose structure is shown in the structural formula of LP-32, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-26-2a was prepared by combining the corresponding linker-payload (LP-33a, whose structure is shown in the structural formula of LP-33, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-27-2a was prepared by combining the corresponding linker-payload (LP-35a, whose structure is shown in the structural formula of LP-35, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-28-2a was prepared by combining the corresponding linker-payload (LP-36a, whose structure is shown in the structural formula of LP-36, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-29-2a was prepared by combining the corresponding linker-payload (LP-39a, whose structure is shown in the structural formula of LP-39, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-30-2a was prepared by combining the corresponding linker-payload (LP-34a, whose structure is shown in the structural formula of LP-34, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-31-2a was prepared by combining the corresponding linker-payload (LP-19a, whose structure is shown in the structural formula of LP-19, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-35-2a was prepared by combining the corresponding linker-payload (LP-22a, whose structure is shown in the structural formula of LP-22, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-39-2a was prepared by combining the corresponding linker-payload (LP-26a, whose structure is shown in the structural formula of LP-26, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-45-2a was prepared by combining the corresponding linker-payload (LP-20a, whose structure is shown in the structural formula of LP-20, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-48-2a was prepared by combining the corresponding linker-payload (LP-27a, whose structure is shown in the structural formula of LP-27, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody TR005 according to the method of general step B.
  • ADC-1-3a was prepared by combining the corresponding linker-payload (LP-1a, whose structure is shown in the structural formula of LP-1, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • the sequence information of hu7F11 is shown in Table 2.
  • ADC-2-3a was prepared by combining the corresponding linker-payload (LP-2a, whose structure is shown in the structural formula of LP-2, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-C-3a was prepared by reacting compound VcMMAE with anti-Trop2 antibody hu7F11 according to the method of general procedure A.
  • ADC-3-3a was prepared by combining the corresponding linker-payload (LP-10) with the anti-Trop2 antibody hu7F11 according to the method of general procedure A.
  • ADC-4-3a was prepared by combining the corresponding linker-payload (LP-37) with the antibody according to the method of general procedure A.
  • the Trop2 antibody hu7F11 was prepared.
  • ADC-5-3a was prepared by combining the corresponding linker-payload (LP-16) with the anti-Trop2 antibody hu7F11 according to the method of general procedure A.
  • ADC-6-3a was prepared by combining the corresponding linker-payload (LP-11) with the anti-Trop2 antibody hu7F11 according to the method of general procedure A.
  • ADC-7-3a was prepared by combining the corresponding linker-payload (LP-12) with the anti-Trop2 antibody hu7F11 according to the method of general procedure A.
  • ADC-8-3a was prepared by combining the corresponding linker-payload (LP-13) with the anti-Trop2 antibody hu7F11 according to the method of general procedure A.
  • ADC-9-3a was prepared by combining the corresponding linker-payload (LP-14) with the anti-Trop2 antibody hu7F11 according to the method of general procedure A.
  • ADC-10-3a was prepared by combining the corresponding linker-payload (LP-15) with the anti-Trop2 antibody hu7F11 according to the method of general procedure A.
  • ADC-11-3a was prepared by combining the corresponding linker-payload (LP-17) with the anti-Trop2 antibody hu7F11 according to the method of general procedure A.
  • ADC-12-3a was prepared by combining the corresponding linker-payload (LP-18) with the anti-Trop2 antibody hu7F11 according to the method of general procedure A.
  • ADC-13-3a was prepared by combining the corresponding linker-payload (LP-3a, whose structure is shown in the structural formula of LP-3, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-14-3a was prepared by combining the corresponding linker-payload (LP-7a, whose structure is shown in the structural formula of LP-7, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-15-3a was prepared by combining the corresponding linker-payload (LP-3sa, whose structure is shown in the structural formula of LP-3s, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-16-3a was prepared by combining the corresponding linker-payload (LP-4a, whose structure is shown in the structural formula of LP-4, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-17-3a was prepared by combining the corresponding linker-payload (LP-5a, whose structure is shown in the structural formula of LP-5, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-18-3a was prepared by combining the corresponding linker-payload (LP-6a, whose structure is shown in the structural formula of LP-6, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-19-3a was prepared by combining the corresponding linker-payload (LP-8a, whose structure is shown in the structural formula of LP-8, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-20-3a was prepared by combining the corresponding linker-payload (LP-9a, whose structure is shown in the structural formula of LP-9, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-21-3a was prepared by combining the corresponding linker-payload (LP-28a, whose structure is shown in the structural formula of LP-28, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-22-3a was prepared by combining the corresponding linker-payload (LP-29a, whose structure is shown in the structural formula of LP-29, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-23-3a was prepared by combining the corresponding linker-payload (LP-30a, whose structure is shown in the structural formula of LP-30, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-24-3a was prepared by combining the corresponding linker-payload (LP-31a, whose structure is shown in the structural formula of LP-31, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-25-3a was prepared by combining the corresponding linker-payload (LP-32a, whose structure is shown in the structural formula of LP-32, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-26-3a was prepared by combining the corresponding linker-payload (LP-33a, whose structure is shown in the structural formula of LP-33, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-27-3a was prepared by combining the corresponding linker-payload (LP-35a, whose structure is shown in the structural formula of LP-35, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-28-3a was prepared by combining the corresponding linker-payload (LP-36a, whose structure is shown in the structural formula of LP-36, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-29-3a was prepared by combining the corresponding linker-payload (LP-39a, whose structure is shown in the structural formula of LP-39, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-30-3a was prepared by combining the corresponding linker-payload (LP-34a, whose structure is shown in the structural formula of LP-34, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-31-3a was prepared by combining the corresponding linker-payload (LP-19a, whose structure is shown in the structural formula of LP-19, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-35-3a was prepared by combining the corresponding linker-payload (LP-22a, whose structure is shown in the structural formula of LP-22, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-39-3a was prepared by combining the corresponding linker-payload (LP-26a, whose structure is shown in the structural formula of LP-26, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-45-3a was prepared by combining the corresponding linker-payload (LP-20a, whose structure is shown in the structural formula of LP-20, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • ADC-48-3a was prepared by combining the corresponding linker-payload (LP-27a, whose structure is shown in the structural formula of LP-27, wherein the configuration of the chiral carbon at position 2 is S-type) with the anti-Trop2 antibody hu7F11 according to the method of general step A.
  • the average drug/antibody ratio DAR of the corresponding ADCs measured by reverse phase high performance liquid chromatography in general step C is shown in Table 2 below.
  • DAR average drug/antibody ratio
  • the aggregation degree of each ADC can be obtained by general step D.
  • the SEC-HPLC peak diagrams are shown in Figures 3 to 6. As shown in the following table, the monomer rates of ADC-1-1a, ADC-2-1a, ADC-1-2a and ADC-2-2a were all greater than 95%, and the aggregation and degradation levels were low.
  • ADCs ADC-1-1a and ADC-2-1a
  • acidic stable linkers introduced from hydroxyl groups
  • traditional linker ADC-C-1a i.e., MC-VC-PAB-MMAE
  • ADCs were analyzed by hydrophobic interaction chromatography HIC according to general procedure E.
  • ADCs with greater hydrophobicity or a larger number of drugs/molecules elute at a later retention time. The experimental results are shown in the following table.
  • ADC-1-1a and ADC-2-1a with acidic stable linkers introduced from hydroxyl groups have relatively short retention times in HIC, while ADC-C-1a with MC-VC-PAB linkers introduced from amino groups has the longest retention time.
  • the experimental results show that ADC-1-1a and ADC-2-1a with acidic stable linkers introduced from hydroxyl groups have better hydrophilicity.
  • the plasma stability of ADC was studied according to general procedure F. The results are shown in the table below.
  • the experimental results show that the ADC with an acidic stable linker introduced on the hydroxyl group of the toxin has almost no drug loss during the plasma incubation process, while the DAR of the classic MC linker ADC (ADC-C-1a) is significantly reduced after 72 hours of incubation.
  • the experimental results prove that the introduction of an acidic stable linker on the hydroxyl group of the toxin can significantly improve the plasma stability of the ADC drug.
  • Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) was used to analyze the molecular weight of the light and heavy chains of ADC-2-1a.
  • the hydrolysis of ADC-2-1a was characterized based on the changes in the molecular weight of the light and heavy chains.
  • Sample preparation Take 100 ⁇ g of ADC-2-1a, add 2 ⁇ L of 1M DTT solution at the ratio of 4 ⁇ L of 1M DTT solution to every 50 ⁇ g of protein, mix well, centrifuge briefly (10-20 seconds), and place at room temperature for 20 minutes.
  • RP mobile phase A 0.05% TFA in water
  • RP mobile phase B 0.05% TFA in acetonitrile
  • the detection wavelength is 280nm
  • the sample running method is edited as follows:
  • Injection volume 1 mg/mL, 10 ⁇ L, i.e. 20 ⁇ g;
  • Detection range 1000-3500Da; output range: 20000-120000Da.
  • Figure 7 shows the mass spectrometry results of the light chain of the reduced antibody
  • Figures 8 and 9 show the mass spectrometry results of the light chain of ADC-2-1a after reduction.
  • the molecular weight of the light chain (LC) of the antibody TR000 is 23372.7721Da. Since the molecular weight of compound 13 is 1289.5400Da, the molecular weight of its ring-opening hydrolysis product is 1307.5550Da. Therefore, when an antibody is connected to 8 drugs, one of its light chains is coupled to 1 compound, and its theoretical molecular weight is 24662.3121Da when no hydrolysis occurs.
  • the molecular weights of the light chains of ADC actually detected include 24663.0000Da and 24680.0000Da, of which 24680.0000Da differs from the theoretical molecular weight by 17.6879Da.
  • the light chain of ADC is hydrolyzed and one maleimide is hydrolyzed, its theoretical molecular weight is 24680.3271Da.
  • the relative deviation is within the error range of the instrument. It is inferred that ADC-2-1a is partially hydrolyzed, resulting in an increase of nearly 18Da in molecular weight. It can be seen that the maleimide group in ADC has both open-ring and closed-ring forms.
  • DMEM medium and RMPI1640 medium were purchased from Yuanpei Biotechnology, and fetal bovine serum was purchased from ExCellBio.
  • Human skin squamous cell carcinoma cell line A431 (Trop2-positive cells), human in situ pancreatic adenocarcinoma cell line BxPC-3 (Trop2-positive cells), human gastric cancer cell line NCI-N87 (Trop2-positive cells), and human colon cancer cell line SW620 (negative control cells) were purchased from ATCC (American type culture collection) and used the literature. The recommended method was used for culture, and the ADCs tested were ADC-1-1a, ADC-2-1a, ADC-C-1a, ADC-1-2a, ADC-2-2a, and ADC-C-2a.
  • AQueous One Solution Cell Proliferation Assay was purchased from Promega.
  • Human skin squamous cell carcinoma cell line A431, human pancreatic adenocarcinoma cell line BxPC-3, human gastric cancer cell line NCI-N87 and human colon cancer cell line SW620 in logarithmic growth phase were taken.
  • a certain number of tumor cell lines were inoculated in 96-well plates, and gradient dilutions of the test antibodies and corresponding ADC drugs were added to the cells for 5 days.
  • Cell viability was detected by MTS, and the inhibitory effect of the test antibodies and ADCs on tumor cell lines was evaluated by calculating IC 50.
  • the initial concentration of ADC drug was 500nM, the dilution multiple was 7 times, a total of 8 concentration points, and the treatment lasted for 5 days.
  • the ADC drug described in the present invention showed good anti-tumor activity in multiple Trop2-positive tumor cell lines and has great clinical application value.
  • an A431 tumor-bearing mouse model was established to evaluate the in vivo efficacy of ADC.
  • 3 ⁇ 10 6 A431 cells were subcutaneously injected into the right side of BALB/c nude mice of 4 to 6 weeks of age.
  • the mice were randomly divided into groups, with 5 mice in each group.
  • blank control buffer solution blank
  • antibody drug conjugates ADC-1-1a, ADC-2-1a, ADC-C-1a, ADC-1-2a, ADC-2-2a, and ADC-C-2a were given intravenously at a dose of 2 mg/kg.
  • the tumor volume measurement data were displayed as the average tumor volume at the time of measurement, and the changes in the weight of the mice were recorded at the same time to observe the initial toxicity of the ADC drug in vivo.
  • the results are shown in the following table. It can be seen from the table that, under the same payload, high DAR ADC-1-1a, ADC-2-1a and ADC-C-1a have stronger tumor inhibition effects than low DAR ADC-1-2a, ADC-2-2a and ADC-C-2a, among which ADC-2-1a has the strongest tumor inhibition effect. Under the same DAR, ADC-1-1a and ADC-2-1a with acid-stable linkers introduced from hydroxyl groups have stronger tumor inhibition effects than ADC-C-1a with MC-VC-PAB linkers.
  • the ADC with an acid-stable linker introduced from the hydroxyl group has better in vivo antitumor activity than the ADC using the MC-VC-PAB linker, and the same tumor inhibition effect can be achieved with a lower dose. Since the ADC of the present invention can achieve a good antitumor effect at a lower dosage and has improved blood stability, the blood toxicity and neurotoxicity are lower.

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Abstract

本发明涉及医药技术领域，涉及一种带有高稳定性亲水连接单元的奥瑞他汀类药物及其偶联物。特别地，本发明涉及一种配体‐药物偶联物或其药学上可接受的盐或溶剂化物、其制备方法和用途。本发明还涉及一种连接子-药物化合物或其异构体、内消旋体、外消旋体、对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化物，其制备方法和用途。

Description

一种带有高稳定性亲水连接单元的奥瑞他汀类药物及其偶联物 技术领域
本发明涉及医药技术领域，特别涉及配体‐药物偶联物、连接子-药物化合物、其制备方法和用途。
背景技术
抗体药物偶联物(Antibody-Drug Conjugates，ADCs)作为一类新型靶向治疗药物，结合了抗体的高选择性和细胞毒药物高活性的优点，具有“高效低毒”的优势，已成为肿瘤靶向治疗的研究热点。近年来，ADCs发展迅猛，已发展至第三代，目前已有13个ADCs药物被批准上市，分别是Mylotarg、Adcetris、Kadcyla、Besponsa、Lumoxiti、Polivy、Enhertu、Padcev、Trodelvy、Blenrep、Zynlonta、爱地希和Tivdak，200多个ADCs进入临床试验。ADCs在肿瘤靶向治疗领域正发挥着越来越重要的作用。
抗体药物偶联物(antibody-drug conjugates，ADCs)由三个部分组成：高特异性和亲和力的抗体(mAb)、高稳定性的接头(linker)和高效的小分子细胞毒药物(payload/warhead，弹头)。ADCs的抗体部分最常用IgG1，其弹头部分一般都是作用于微管、DNA或RNA的细胞毒药物，如美登素类、奥瑞他汀类、卡奇霉素、喜树碱类、吡咯并苯并二氮杂卓和毒伞肽等，其接头主要有两大类，一类是可裂解的接头，另一类是不可裂解的接头。
奥瑞他汀类(Auristatins)是一类微管蛋白抑制剂，其可阻断微管蛋白与GTP结合并阻断微管与长春碱结合位点的结合，从而诱导细胞凋亡，抑制肿瘤生长。目前，MMAE(US6884869)和MMAF(US7498298)是最常用的ADC弹头，两者均是基于海兔毒素10(Dolastatin 10)改构得到的五肽。MMAE和MMAF均展现出很好的抗肿瘤活性，但由于缺乏选择性以及治疗指数不高等因素，导致其不能以单药应用于临床。然而，MMAE和MMAF的高细胞毒性使其成为ADC的理想弹头。MMAE/MMAF在ADC领域已有广泛应用，目前已有5个以MMAE为弹头和1个以MMAF为弹头的ADC药物上市，分别为Adcetris、Polivy、Padcev、爱地希、Tivdak和Blenrep。然而，这些以奥瑞他汀类为毒素的ADC存在一些不足，如以MMAE为毒素的ADC存在神经毒性(周围神经病变)和血液毒性(血小板减少和中性粒细胞减少)等不良反应，以MMAF为毒素的ADC则存在眼毒性等，安全性有待提高。另外，这些ADCs使用的linker大都为VClinker，DAR最高为4，这是因为VClinker疏水性较强，然而，高DAR容易导致ADC 沉淀和聚集，故不能提高其DAR，增强药效。
进一步地，目前以MMAE为弹头的ADC，大多是从MMAE的N端的仲胺引入linker，从MMAE的羟基上引入linker的实例较少，仅有Seagen、丹麦技术大学、Mersana和上海新理念公司报道过。
Seagen在其专利申请US2005009751A1中，将MMAE类似物Auristatin E的羟基进行酯化反应后再引入带有腙键的linker，该类带有酯键和腙键的linker在人血浆中不稳定，会发生一定程度的水解，导致部分脱靶毒性(参见Nat Biotechnol.2003Jul；21(7):778-84)。Seagen在其专利CN105813653中，在MMAE的羟基中引入自消除基团——亚甲基烷氧基氨基甲酸酯(methylene alkoxy carbamate,MAC)后，再引入β-葡糖醛酸糖苷类linker，虽然带有该类linker的ADC在人血浆中的稳定性较好，但其弹头释放涉及到β-葡糖醛酸糖苷酶(β-Glucuronidase)的催化及后续的自消除过程，相对较复杂。
丹麦技术大学在其专利申请WO2020260597A1中，在MMAE类似物的羟基上引入linker的方式同Seagen类似，也是进行酯化反应后再引入linker，但引入linker后其酯键较难被酶水解，不易释放出弹头。Mersana在其专利申请CN110234357A中，从MMAE类似物的羟基引入linker的方式同Seagen类似，引入酯键或碳酸酯结构，但酯键较难被酶水解，而碳酸酯血浆稳定性较差，会降低药效。上海新理念公司在其专利申请CN106279352A中，在MMAE类似物的羟基上引入氨基甲酸酯后，再引入Mc-Vc-PAB linker，这种氨基甲酸酯在血浆中稳定性较差，有潜在的脱靶风险。
因此，开发出新的引入linker的方式，得到具有更高安全性和有效性的奥瑞他汀类ADC药物具有非常重要的临床意义。
发明内容
本发明所要解决的一个技术问题，在于探索发现更优的抗肿瘤奥瑞他汀类ADC药物，使其具有更高的安全性、有效性，更好地满足临床需求。
本发明将奥瑞他汀或其衍生物从羟基位点与酶切肽单元通过氨基亚甲基结构连接，形成相应的linker-payload，可以使ADC的DAR值提高到8，在高载药下亲水性更好、高载药值下血浆稳定性更好，药效方面更有优势。同阳性药(例如以Vc-MMAE为linker-payload的ADC)相比，使用更低的剂量即可达到同等肿瘤抑制效果，MTD有所提高，且神经毒性和血液毒性降低。由此，本申请提供了以下发明：
在第一方面，本申请提供了如通式I所示的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，
其中：
Ab为配体单元，选自抗体、抗体片段、靶向蛋白或Fc-融合蛋白；
M为与Ab连接的连接单元；
A选自由2-7个氨基酸构成的肽残基，其中，任选地，所述氨基酸各自独立地被一个或多个取代基所取代，所述取代基选自：氘原子、卤素、羟基、氰基、氨基、硝基、烷基、取代烷基、烷氧基、环烷基、取代环烷基；
W表示如式(i)所示的氨基亚甲基氧结构单元：
其中：
左侧波浪线表示式(i)中氮原子与A的连接位点，右侧波浪线表示式(i)中氧原子与药物D的连接位点，氧原子为药物D与W的共用基团；
R1、R2和R3各自独立地选自氢原子、氘原子、烷基和取代烷基；
p选自1-20的整数或小数(例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20)；
并且
药物D为具有式D所示结构的奥瑞他汀，或其异构体、内消旋体、外消旋体、对映异构体或其混合物形式，或其药学上可接受的盐，
其中：
R4、R5各自独立地选自氢原子、氘原子、烷基和氘代烷基，或者R4、R5相连形成以下结构：-(CR11R12)n-B-(CR13R14)m-，其中R11、R12、R13和R14选自氢原子、氘原子、烷基和氘代烷基；B选自O、NR15、CR16R17，其中R15、R16、R17选自氢原子、氘原子和烷基；n和m分别选自0-8的整数(例如0、1、2、3、4、5、6、7、8)；与R4和R5键合的氮原子与-(CR11R12)n-B-(CR13R14)m-一起形成环；
R6、R7、R8、R9各自独立地选自氢原子、氘原子、卤素、叠氮基、烷基和NR18R19，或者R6、R7、R8、R9之中的任意两个基团与所键合的原子一起形成环烷基，余下的两个基团各自独立地选自氢原子、卤素、叠氮基、烷基和NR18R19，其中R18、R19选自氢原子、烷基；
R10选自芳基、杂芳基，所述的芳基或杂芳基任选地被一个或多个取代基所取代，所述取代基选自：氢原子、卤素、烷基、烷氧基、氨基、硝基；
式D中的波浪线表示药物D结构中1位氧原子与W的连接位点，所述氧原子为药物D与W的共用基团。
在某些实施方案中，R4、R5各自独立地选自氢原子、C1-C4烷基。
在某些实施方案中，R4、R5相连形成以下结构：-(CH2)2-B-(CH2)2-，B选自O、NH，与R4和R5键合的氮原子与-(CH2)2-B-(CH2)2-一起形成环。
在某些实施方案中，式D中的R6、R7、R8、R9各自为氢原子。
在某些实施方案中，式D中R6、R7、R8、R9中的一个选自卤素、叠氮基、氨基，其余的三个各自为氢。
在某些实施方案中，式D中R6、R7、R8、R9之中的任意两个基团与其所键合的原子一起形成环丙基，余下的两个基团各自独立地为氢原子。
在某些实施方案中，式D中的R10为苯基，任选地，所述苯基被一个或多个所述取代基所取代。在某些实施方案中，所述取代基选自氨基、硝基。
在某些实施方案中，药物D非限制性地选自以下化合物：
在上述实施方案中，药物D通过羟基与W相连。
在某些实施方案中，连接单元M包含马来酰亚胺。在这些实施方案中，所述配体‐药物偶联物在易水解条件下可以发生水解，水解部位即为连接单元的马来酰亚胺部分。当配体中包含多个连接子-药物时，随水解程度不同，可出现以下情形：
马来酰亚胺完全不水解，即马来酰亚胺均为闭环形式
马来酰亚胺不完全水解，即部分马来酰亚胺为闭环形式而另一部分马来酰亚胺为开环形式
马来酰亚胺完全水解，即马来酰亚胺均为开环形式
因此，当ADC中同时存在多个含马来酰亚胺基团的连接单元M(即，Ab连接有多个含马来酰亚胺基团的药物-连接子)时，这些马来酰亚胺基团可以均为闭环形式、部分为开环形式或者全部为开环形式。
上述马来酰亚胺的各结构式中，左侧波浪线表示到Ab的连接位点，右侧波浪线表示到M中的其他结构。
在某些实施方案中，所述配体‐药物偶联物具有式Ia所示的结构：
其中：Z选自-C1-C10亚烷基-、-C3-C8碳环-、-亚芳基-、-C1-C10亚烷基-亚芳基-、-亚芳基-C1-C10-亚烷基-、-C1-C10亚烷基-(C3-C8碳环)-、-(C3-C8碳环)-C1-C10亚烷基-、3-8元杂环-、-C1-C10亚烷基-(3-8元杂环)-、-(3-8元杂环)-C1-C10亚烷基-、-(CH2CH2O)r-、-(CH2CH2O)r-CH2-或其中X选自-C1-C10亚烷基-、-C3-C8碳环-、-亚芳基-、-C1-C10亚烷基-亚芳基-、-亚芳基-C1-C10-亚烷基-、-C1-C10亚烷基-(C3-C8碳环)-、-(C3-C8碳环)-C1-C10亚烷基-、-3-8元杂环-、-C1-C10亚烷基-(3-8元杂环)-、-(3-8元杂环)-C1-C10亚烷基 -、-(CH2CH2O)r-、-(CH2CH2O)r-CH2；Y为亲水结构，选自羧基、磷酸、聚磷酸、亚磷酸、磺酸、亚磺酸或聚乙二醇(PEG)；所述杂环各自独立地包含选自N、O、S的1-3个原子；所述-C1-C10亚烷基-、-C3-C8碳环-、杂环各自独立地被一个或者多个取代基所取代，所述取代基选自氘原子、卤素、羟基、氰基、硝基、氨基、烷基、杂烷基、取代烷基、烷氧基、羧基或环烷基；中的左侧波浪线表示到马来酰亚胺上N的连接位点，右侧波浪线表示到羰基的连接位点；r选自1-10之间的整数(例如1、2、3、4、5、6、7、8、9、10)；q选自1-8之间的整数(例如1、2、3、4、5、6、7、8)；
n1、n2、n3独立地选自0～20之间的整数或小数，n1、n2、n3不同时为0，且n1+n2+n3≤20，例如1≤n1+n2+n3≤2，或7≤n1+n2+n3≤8。
在某些实施方案中，n1、n2、n3独立地选自0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20(例如0、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、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、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8、8.1、8.2、8.3、8.4、8.5、8.6、8.7、8.8、8.9、9、9.1、9.2、9.3、9.4、9.5、9.6、9.7、9.8、9.9或10)。
在某些实施方案中，式I或式Ia中的A选自苯丙氨酸(F)、甘氨酸(G)、缬氨酸(V)、赖氨酸(K)、丙氨酸(A)、瓜氨酸、丝氨酸(S)、谷氨酸(E)或者天冬氨酸(D)中的2-7个氨基酸形成的多肽残基。
在某些实施方案中，A为由2-4个选自苯丙氨酸和甘氨酸的氨基酸形成的肽残基。在某些实施方案中，A为甘氨酸-甘氨酸-苯丙氨酸-甘氨酸组成的四肽残基。
在某些实施方案中，Z选自-C1-C10亚烷基-，例如-C4-C6亚烷基-，例如-C5亚烷基-。
在某些实施方案中，Z为其中，q选自1-8之间的整数(例如1、2、3、4、5、6、7、8)，例如1。
在某些实施方案中，R1、R2和R3各自独立地选自氢原子、氘原子、烷基、卤代烷基、氘代烷基和羟烷基。
在某些实施方案中，R1、R2和R3同时为氢原子或氘原子。
在某些实施方案中，R1、R2和R3同时为氢原子。在某些实施方案中，所述配体‐药物偶联物具有如式Ib所示的结构，
在某些实施方案中，所述配体‐药物偶联物具有如式Ic所示的结构，
Ac为具有式c所示结构的亲水结构单元：
Ac通过氨基官能团与式Ie中已标示的2位亚甲基碳相连，X、Y如上文所定义。
在某些实施方案中，所述Ac选自甘氨酸、(D/L)丙氨酸、(D/L)亮氨酸、(D/L)异亮氨酸、(D/L)缬氨酸、(D/L)苯丙氨酸、(D/L)脯氨酸、(D/L)色氨酸、(D/L)丝氨酸、(D/L)酪氨酸、(D/L)半胱氨酸、(D/L)胱氨酸、(D/L)精氨酸、(D/L)组氨酸、(D/L)蛋氨酸、(D/L) 天冬酰胺、(D/L)谷氨酰胺、(D/L)苏氨酸、(D/L)天冬氨酸、(D/L)谷氨酸、天然或非天然氨基酸衍生物或以下结构，
本发明的配体‐药物偶联物非限制性地可以选自以下结构：

















其中，2位手性碳的构型为R型或S型。
在第二方面，本申请提供了如式II所示的连接子-药物化合物，或其药学上可接受的盐或溶剂化物，
其中：
Z、A、R1、R2、R3、R4、R5、R11、R12、R13、R14、B、R15、R16、R17、n、m、R6、R7、R8、R9、R18、R19、R10如上文任一项所定义。
在某些实施方案中，所述连接子-药物化合物具有式IIa所示的结构，
在某些实施方案中，所述连接子-药物化合物具有式IIb所示的结构，
其中，Ac为具有式c所示结构的亲水结构单元：
其中，X、Y如上文所定义，Ac通过-NH-与式IIb中已标示的2位亚甲基碳相连。
本发明的连接子‐药物化合物非限制性地可以选自以下结构：









其中，2位手性碳的构型为R型或S型。
本发明中，配体单元Ab可以选自抗体、抗体片段或蛋白，其中所述抗体选自鼠源抗体、兔源抗体、噬菌体展示来源抗体、酵母展示来源抗体、嵌合抗体、人源化抗体、全人源抗体、抗体片段、双特异性抗体及多特异性抗体。
在某些实施方案中，所述抗体为单克隆抗体，非限制性地选自：抗EGFRvIII抗体、抗PD-1抗体、抗PD-L1抗体、抗DLL-3抗体、抗PSMA抗体、抗CD70抗体、抗MUC16抗体、抗ENPP3抗体、抗TDGF1抗体、抗ETBR抗体、抗MSLN抗体、抗TIM-1抗体、抗LRRC15抗体、抗LIV-1抗体、抗CanAg/AFP抗体、抗claudin 18.2抗体、抗Mesothelin抗体、抗HER2(ErbB2)抗体、抗EGFR抗体、抗c-MET抗体、抗SLITRK6抗体、抗KIT/CD117抗体、抗STEAP1抗体、抗SLAMF7/CS1抗体、抗NaPi2B/SLC34A2抗体、抗GPNMB抗体、抗HER3(ErbB3)抗体、抗MUC1/CD227抗体、抗AXL抗体、抗CD166抗体、抗B7-H3(CD276)抗体、抗PTK7/CCK4抗体、抗PRLR抗体、抗EFNA4抗体、抗5T4抗体、抗NOTCH3抗体、抗Nectin 4抗体、抗Trop2抗体、抗CD142抗体、抗CA6抗体、抗GPR20抗体、抗CD174抗体、抗CD71抗体、抗EphA2抗体、抗LYPD3抗体、抗FGFR2抗体、抗FGFR3抗体、抗FRα抗体、抗CEACAMs抗体、抗GCC抗体、抗Integrin Av抗体、抗CAIX抗体、抗P-cadherin抗体、抗GD3抗体、抗Cadherin 6抗体、抗LAMP1抗体、抗FLT3抗体、抗BCMA抗体、抗CD79b抗体、抗CD19抗体、抗CD33抗体、抗CD56抗体、抗CD74抗体、抗CD22抗体、抗CD30抗体、抗CD37抗体、抗CD47抗体、抗CD138抗体、抗CD352抗体、抗CD25抗体或抗CD123抗体。
在某些实施方案中，所述抗体为抗Trop2抗体。在某些实施方案中，所述抗体由轻链和重链组成，所述轻链包含CDR-L1、CDR-L2和CDR-L3，其氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3所示。在某些实施方案中，所述重链包含CDR-H1、CDR-H2、CDR-H3，其氨基酸序列分别如SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6所示。
在某些实施方案中，所述轻链包含氨基酸序列为SEQ ID NO：7的轻链可变区。在某 些实施方案中，所述轻链还包含氨基酸序列为SEQ ID NO：8的轻链恒定区。在某些实施方案中，所述轻链的氨基酸序列为SEQ ID NO：9。
在某些实施方案中，所述轻链包含氨基酸序列为SEQ ID NO：10的轻链可变区。在某些实施方案中，所述轻链还包含氨基酸序列为SEQ ID NO：11的轻链恒定区。在某些实施方案中，所述轻链的氨基酸序列为SEQ ID NO：12。
在某些实施方案中，所述重链包含氨基酸序列为SEQ ID NO：13的重链可变区。在某些实施方案中，所述重链还包含氨基酸序列为SEQ ID NO：14的重链恒定区。在某些实施方案中，所述重链的氨基酸序列为SEQ ID NO：15。
在某些实施方案中，所述抗体为抗Trop2抗体。在某些实施方案中，所述抗体由轻链和重链组成，所述轻链包含CDR-L1、CDR-L2和CDR-L3，其核酸编码序列分别如SEQ ID NO：16、SEQ ID NO：17、SEQ ID NO：18所示。在某些实施方案中，所述重链包含CDR-H1、CDR-H2、CDR-H3，其核酸编码序列分别如SEQ ID NO：19、SEQ ID NO：20、SEQ ID NO：21所示。
在某些实施方案中，所述轻链包含核酸编码序列为SEQ ID NO：22的轻链可变区。在某些实施方案中，所述轻链还包含核酸编码序列为SEQ ID NO：23的轻链恒定区。在某些实施方案中，所述轻链的核酸编码序列为SEQ ID NO：24。
在某些实施方案中，所述轻链包含核酸编码序列为SEQ ID NO：25的轻链可变区。在某些实施方案中，所述轻链还包含核酸编码序列为SEQ ID NO：26的轻链恒定区。在某些实施方案中，所述轻链的核酸编码序列为SEQ ID NO：27。
在某些实施方案中，所述重链包含核酸编码序列为SEQ ID NO：28的重链可变区。在某些实施方案中，所述重链还包含核酸编码序列为SEQ ID NO：29的重链恒定区。在某些实施方案中，所述重链的核酸编码序列为SEQ ID NO：30。
在某些实施方案中，所述抗体为抗Trop2抗体。在某些实施方案中，所述抗体由轻链和重链组成，所述轻链包含CDR-L1、CDR-L2和CDR-L3，其氨基酸序列分别如SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：33所示。在某些实施方案中，所述重链包含CDR-H1、CDR-H2、CDR-H3，其氨基酸序列分别如SEQ ID NO：34、SEQ ID NO：35、SEQ ID NO：36所示。
在某些实施方案中，所述轻链包含氨基酸序列为SEQ ID NO：37的轻链可变区。在某些实施方案中，所述轻链还包含氨基酸序列为SEQ ID NO：38的轻链恒定区。在某些实施方案中，所述轻链的氨基酸序列为SEQ ID NO：39。
在某些实施方案中，所述重链包含氨基酸序列为SEQ ID NO：40的重链可变区。在 某些实施方案中，所述重链还包含氨基酸序列为SEQ ID NO：41的重链恒定区。在某些实施方案中，所述重链的氨基酸序列为SEQ ID NO：42。
在某些实施方案中，所述抗体为抗Trop2抗体。在某些实施方案中，所述抗体由轻链和重链组成，所述轻链包含CDR-L1、CDR-L2和CDR-L3，其核酸编码序列分别如SEQ ID NO：43、SEQ ID NO：44、SEQ ID NO：45所示。在某些实施方案中，所述重链包含CDR-H1、CDR-H2、CDR-H3，其核酸编码序列分别如SEQ ID NO：46、SEQ ID NO：47、SEQ ID NO：48所示。
在某些实施方案中，所述轻链包含核酸编码序列为SEQ ID NO：49的轻链可变区。在某些实施方案中，所述轻链还包含核酸编码序列为SEQ ID NO：50的轻链恒定区。在某些实施方案中，所述轻链的核酸编码序列为SEQ ID NO：51。
在某些实施方案中，所述重链包含核酸编码序列为SEQ ID NO：52的重链可变区。在某些实施方案中，所述重链还包含核酸编码序列为SEQ ID NO：53的重链恒定区。在某些实施方案中，所述重链的核酸编码序列为SEQ ID NO：54。
本发明涉及的部分序列的信息提供于表1中。
表1：序列的描述








在第三方面，本申请还提供了本发明的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，或本发明的连接子-药物化合物或其异构体、内消旋体、外消旋体、对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化物用于制备药物的用途，所述药物用于治疗或预防肿瘤。
在某些实施方案中，所述肿瘤选自实体瘤或非实体瘤，例如乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、肺癌、结肠癌、直肠癌、结直肠癌、骨癌、皮肤癌、甲状腺癌、胰腺癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、多形性胶质细胞瘤、肉瘤、淋巴瘤和白血病。
在第四方面，本申请还提供了一种药物组合物，其包含有效量的本发明的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，或本发明的连接子-药物化合物或其异构体、内消旋体、外消旋体、对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化物，以及药学上可接受的载体、稀释剂或赋形剂。
在第五方面，本申请还提供了一种治疗或预防肿瘤的方法，包括给有此需要的受试者施用有效量的本发明的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，或本发明的连接子-药物化合物或其异构体、内消旋体、外消旋体、对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化物。在某些实施方案中，所述受试者为哺乳动物，例如人。
在第六方面，本申请还提供了本发明的连接子-药物化合物或其异构体、内消旋体、外消旋体、对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化物用于制备配体‐药物偶联物或其药学上可接受的盐或溶剂化物的用途。在某些实施方案中，所述配体-药物偶联物选自本发明的配体‐药物偶联物。
在第七方面，本申请还提供了制备本发明的配体‐药物偶联物或其药学上可接受的盐或溶剂化物的方法，所述方法包括：通过还原的抗体或者其抗原结合片段与本发明的连接子-药物化合物或其异构体、内消旋体、外消旋体、对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化物进行偶联反应，得到所述配体‐药物偶联物。可以通过硫醇类还原剂(例如三(2-羧乙基)膦(TCEP))对抗体或者其抗原结合片段进行还原。
术语定义
除非另有限定，本文所用的所有技术和科学术语均与本发明所属领域普通技术人员的通常理解一致。虽然也可采用与本发明所述相似或等同的任何方法和材料实施或测试本发明，但本发明描述了优选的方法的材料。描述和要求保护本发明时，依据以下定义 使用下列术语。
当本发明中使用商品名时，申请人旨在包括该商品名产品的制剂、该商品名的非专利药和活性药物部分。
除非有相反陈述，在说明书和权利要求书中使用的术语具有下述含义。
术语“配体”是与靶标部分特异性结合的靶向剂。所述配体能够特异性结合至细胞组分或结合至其他感兴趣的靶标分子。靶标部分或靶标通常在细胞表面上。在一些方面中，配体的作用是将药物单元递送至配体单元与之相互作用的特定靶细胞群。配体包括但不限于蛋白质、多肽和肽，以及非蛋白质如糖。合适的配体单元包括，例如，抗体，例如全长(完整)抗体及其抗原结合片段。在配体单元是非抗体靶向试剂的实施方式中，其可以是肽或多肽，或非蛋白质分子。这类靶向试剂的示例包括干扰素、淋巴因子、激素、生长因子和集落刺激因子、维生素、营养转运分子、或任何其他细胞结合分子或物质。在一些实施方式中，连接子共价连接至配体的硫原子。在一些方面中，硫原子是半胱氨酸残基的硫原子，其形成抗体的链间二硫键。在另一方面中，硫原子是已经导入配体单元的半胱氨酸残基的硫原子，其形成抗体的链间二硫键。在另一方面中，硫原子是通过例如定点诱变或化学反应导入配体单元的半胱氨酸残基的硫原子。
术语“药物”是指细胞毒性药物，即，能在肿瘤或癌症细胞内具有较强破坏其正常生长的能力的分子。细胞毒性药物原则上在足够高的浓度下都可以杀死肿瘤细胞，但是由于缺乏特异性，在杀伤肿瘤或癌症细胞的同时，也会导致正常细胞的凋亡，容易导致严重的副作用。
术语“配体-药物偶联物”，指配体通过稳定的连接单元与药物相连形成的分子。在本发明中“配体-药物偶联物”优选为抗体-药物偶联物(Antibody drug conjugate,ADC)，指把单克隆抗体或者功能性的抗体片段或者具有靶向性的蛋白等通过稳定的连接单元与细胞毒性药物相连。
术语“抗体”或“功能性的抗体片段”在其所属的范围内，包括抗体结构的任何部分。这一单元可以结合、反应性关联或者络合一个受体、抗原或者靶向细胞群体具有的其它受体单元。抗体可以是任何蛋白或蛋白类分子，它可以结合、络合或者与待治疗或生物改造的细胞群体的一部分发生反应。
本发明的抗体包括但不限于鼠源抗体、嵌合抗体、人源化抗体和全人源抗体，优选人源化抗体和全人源抗体。
本发明所用氨基酸三字母代码和单字母代码如J.boil.Chem.1968,243,3558.中所述。
术语“天然氨基酸”指可由生物合成的氨基酸。天然氨基酸一般情况下是L-型的，但 也有少数例外，比如甘氨酸，包括天然的和生物体合成的。
术语“非天然氨基酸”指只能通过人工方法合成的氨基酸。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子的烷基，更优选含有1至10个碳原子的烷基，最优选含有1至6个或1至4个碳原子的烷基。非限制性实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各种支链异构体等。更优选的是含有1至6个(例如1至4个)碳原子的低级烷基，非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基。
术语“取代烷基”指烷基中的氢被取代基团取代，除非文中另有说明，烷基的取代基可以是选自下组的一种或多种基团：-卤素、-OR’、-NR’R”、-SR’、-SiR’R”R”’、-OC(O)R’、-C(O)R’、-CO2R’、-CONR’R”、-OC(O)NR’R”、-NR”C(O)R’、-NR’-C(O)NR”R”’、-NR”C(O)2R’、-NH-C(NH2)＝NH、-NR’C(NH2)＝NH、-NH-C(NH2)＝NR’、-S(O)R’、-S(O)2R’、-S(O)2NR’R”、-NR’S(O)2R”、-CN和-NO2，取代基数量为1至(2m’+1)，其中m’为该基团中碳原子的总数，例如1、2、3、4、5或6。R’、R”和R”’各自地指代氢、C1-8烷基、芳基、由1-3个卤素取代的芳基、由1-3个卤素取代的C1-8烷基、 C1-8烷氧基或C1-8硫代烷氧基、或未取代的芳基-C1-4烷基。R’和R”连接于同一个氮原子时，它们可与该氮原子一起形成3-，4-，5-，6-或7-元环。例如，-NR’R”包括1-吡咯烷基和4-吗啉基。
术语“杂烷基”指烷基上的一个或多个碳被N、O或S取代形成的基团。
术语“环烷基”指饱和或部分不饱和的单环或多环环状烃基，环烷基的环包含3至20个碳原子，优选包含3至12个碳原子，更优选包含3至10个碳原子，最优选包含3至8个碳原子。单环环烷基的非限制性实例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等；多环环烷基包括螺环、稠环和桥环的环烷基。
术语“烷氧基”指-O-(烷基)和-O-(环烷基)，其中烷基或环烷基的定义如上所述。烷氧基的非限制性实例包括：甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基。烷氧基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“杂环”指饱和或部分不饱和的单环或多环环状烃，其包含3至20个环原子，其中一个或多个(例如1、2、3或4个)环原子为选自氮、氧或S(O)m(其中m是0、1或2)的杂原子，其余环原子为碳。优选包含3至12个环原子，其中1～4个是杂原子；更优选包含3至10个或3至8个环原子。单环杂环基的非限制性实例包括吡咯烷基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等。多环杂环基包括螺环、稠环和桥环的杂环基。
术语“芳基”指具有共轭的π电子体系的6至14元全碳单环或稠合多环(即共享毗邻碳原子对的环)基团，优选为6-10元，例如苯基。芳基可以是取代的或非取代的，当被取代时，取代基可以为以下一个或多个基团，非限制性地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、氘原子、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基或杂环烷硫基。
术语“杂芳基”包括5-8元单环杂芳基和8-12元稠杂芳基。
术语“5-8元单环杂芳基”是指含有5-8个环原子(其中至少一个环原子为杂原子，例如氮原子、氧原子或硫原子)的具有芳香性的单环环状基团。任选地，环状结构中的环原子(例如碳原子、氮原子或硫原子)可以被氧代。“5-8元单环杂芳基”包括例如“5-7元单环杂芳基”、“5-6元单环杂芳基”、“5-6元单环含氮杂芳基”、“6元单环含氮杂芳基”等，所述 的“含氮杂芳基”中的杂原子至少含有一个氮原子，例如，仅包含1个或2个氮原子，或者，包含一个氮原子和其他的1个或2个杂原子(例如氧原子和/或硫原子)，或者，包含2个氮原子和其他的1个或2个杂原子(例如氧原子和/或硫原子)。“5-8元单环杂芳基”的具体实例包括但不仅限于呋喃基、噻吩基、吡咯基、噻唑基、异噻唑基、噻二唑基、噁唑基、异噁唑基、噁二唑基、咪唑基、吡唑基、1,2,3-三唑基、1,2,4-三唑基、1,2,3-噁二唑基、1,2,4-噁二唑基、1,2,5-噁二唑基、1,3,4-噁二唑基、吡啶基、2-吡啶酮基、4-吡啶酮基、嘧啶基、哒嗪基、吡嗪基、1,2,3-三嗪基、1,3,5-三嗪基、1,2,4,5-四嗪基、氮杂环庚三烯基、1,3-二氮杂环庚三烯基、氮杂环辛四烯基等。
术语“8-12元稠杂芳基”是指由两个或两个以上环状结构彼此共用两个相邻的原子所形成的、含有8-12个环原子(其中至少一个环原子为杂原子，例如氮原子、氧原子或硫原子)的、不饱和的具有芳香性的环状结构。任选地，环状结构中的环原子(例如碳原子、氮原子或硫原子)可以被氧代。“8-12元稠杂芳基”包括“8-10元稠杂芳基”，“8-9元稠杂芳基”等；具体实例包括但不限于：吡咯并吡咯、吡咯并呋喃、吡唑并吡咯、吡唑并噻吩、呋喃并噻吩、吡唑并噁唑、苯并呋喃基、苯并异呋喃基、苯并噻吩基、吲哚基、异吲哚基、苯并噁唑基、苯并咪唑基、吲唑基、苯并三唑基、喹啉基、2-喹啉酮基、4-喹啉酮基、1-异喹啉酮基、异喹啉基、吖啶基、菲啶基、苯并哒嗪基、酞嗪基、喹唑啉基、喹喔啉基、嘌呤基、萘啶基等。
术语“卤代烷基”指被一个或多个卤素取代的烷基，其中烷基如上所定义。
术语“氘代烷基”指被一个或多个氘原子取代的烷基，其中烷基如上所定义。
术语“羟基”指-OH基团。
术语“卤素”指氟、氯、溴或碘。
术语“氨基”指-NH2。
术语“硝基”指-NO2。
术语“衍生物”是指具有与化合物相似的化学结构但还含有至少一个化合物中不存在的化学基团和/或缺少至少一个化合物中存在的化学基团的物质。衍生物所比较的化合物被称为“母体”化合物。通常，“衍生物”可在一个或多个化学反应步骤中由母体化合物产生。
术语“药学上可接受的盐”指的是，化合物(例如，药物，连接子-药物化合物或配体-药物偶联物)的药学上可接受的有机或无机盐。该化合物或偶联物可含有至少一个氨基或羧基，并且因此可与相应的酸或碱形成加成盐。示例性的盐包括但不限于：硫酸盐、三氟乙酸盐、柠檬酸盐、乙酸盐、草酸盐、氯化物、溴化物、碘化物、硝酸盐、硫酸氢 盐、磷酸盐、酸性磷酸盐、异烟酸盐、乳酸盐、水杨酸盐、酸性柠檬酸盐、酒石酸盐、油酸盐、单宁酸盐、泛酸盐、酒石酸氢盐、抗坏血酸盐、水杨酸盐、甲酸盐、苯甲酸盐、谷氨酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐，钾盐、钠盐等。
术语“溶剂化物”指本发明中的连接子-药物化合物或配体-药物偶联物与一种或多种溶剂分子形成的，溶剂分子包括但不限于水、乙醇、乙腈、异丙醇、DMSO、乙酸乙酯等。
术语“药物组合物”是指含有一种或多种本发明所述化合物或其生理学上/可药用的盐或前体药物与其它化学组分的混合物，以及其它组分例如生理学上/可药用的载体和/或赋形剂。药物组合物的目的时促进对生物体的给药，有利于活性成分的吸收进而发挥生物活性。
术语“载体”是指能改变药物进入人体的方式和在体内的分布、控制药物的释放速度并将药物输送至靶点的体系。药物载体释放和靶向系统能够减少药物降解及损失，降低副作用，提高生物利用度。
术语“赋形剂”是指在药物制剂中除主药以外的附加物或辅料。如片剂中的粘合剂、填充剂、崩解剂、润滑剂；半固体制剂软膏剂、霜剂中的基质备份；液体制剂中的防腐剂、抗氧化剂、矫味剂、芳香剂、助溶剂、乳化剂、增透剂、渗透压调节剂、着色剂等均可称为赋形剂。
术语“稀释剂”或“填充剂”，主要用于增加制剂的重量和/或体积。稀释剂的加入不仅保证一定的体积大小，而且减少主要成分的剂量偏差，改善药物的压缩成型性等。
有益效果
本发明提供了一种带有高稳定性亲水连接单元的奥瑞他汀类药物偶联物，所述偶联物通过带有高稳定性亲水连接单元的氨基甲基与羟基形成醚键引入linker，其能携带多个毒素，具有良好的血浆稳定性，良好的水溶性、均一性和安全性，能够特异性地结合肿瘤细胞中高表达的受体，并在肿瘤细胞内释放出毒素，具有良好的抗肿瘤活性，可用于预防或治疗肿瘤等疾病。
附图说明
图1为化合物LP-1a的LC-MS谱图。
图2为VcMMAE的LC-MS谱图。
图3为ADC-1-1a的SEC-HPLC检测结果。
图4为ADC-2-1a的SEC-HPLC检测结果。
图5为ADC-1-2a的SEC-HPLC检测结果。
图6为ADC-2-2a的SEC-HPLC检测结果。
图7为被还原过后的抗体TR005的轻链质谱检测结果。
图8和图9为被还原过后的ADC-2-1a的轻链质谱检测结果。
具体实施方式
下面结合具体实施例，进一步阐述本发明，应理解，这些实施例只用于说明本发明，而不用于限制本发明的范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件或按照制造厂商所建议的条件。除非另外说明，否则所有的百分数、比例、比率、或份数按重量计。除非另行定义，文中所使用的所有专业和科学用于与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
本发明下列实施例中采用的通用步骤是：
通用步骤A
偶联制备ADC
将通过初步的纯化后单体率大于95％的抗体分子，使用超滤离心管换液至含有EDTA的磷酸盐缓冲液中，浓度10mg/mL。加入10倍于抗体摩尔分子数的TCEP，室温下反应6h。打开抗体链间二硫键，并用Ellman方法测定游离巯基数，判断二硫键是否全部打开。然后加入10倍于抗体摩尔分子数的payload，室温下反应6h。反应结束后，使用截留分子量为30KDa的超滤离心管换液至PBS中，并去除未偶联的payload得偶联ADC(DAR＝8)。
通用步骤B
定点偶联制备ADC
将通过初步的纯化后单体率大于95％的抗体分子，使用超滤离心管换液至含有EDTA的磷酸盐缓冲液中，浓度10mg/mL。加入8倍于抗体摩尔分子数的TCEP，室温下反应3h。使用超滤离心管换液至pH6.5的磷酸缓冲液中，再加入8倍于抗体摩尔分子数的DHAA(脱氢抗坏血酸)，室温下反应3h。然后加入5倍于抗体摩尔分子数的payload，室温下反应3h。反应结束后，使用截留分子量为30KDa的超滤离心管换液至PBS中，并去除未偶联的payload得定点偶联ADC(DAR＝2)。
通用步骤C
反相高效液相色谱(RP-HPLC)检测DAR
配制RP-HPLC流动相:
RP流动相A：0.1％TFA水溶液，RP流动相B：0.1％TFA乙腈溶液。
用样品稀释液将待测样品及对应的抗体对照稀释到1mg/mL，每98μl稀释好的样品中加入2μl DTT储液，同时配制98u1样品稀释液加2μl DTT储液的空白对照，各样品混匀后，于65℃金属浴中加热30min。将处理后的样品14000rpm离心5分钟或者用0.22μm滤器过滤以去除样品中大颗粒，并将内插管放于样品瓶中盖好盖子。
将装好样品的样品瓶放与样品板上，按照《UPLC使用标准操作规程》设置各样品对应的位置、进样体积、进样针数和进样方法。色谱柱型号为Proteomix RP-1000(4.6*100mm,5μm)，Sepax。
检测波长为214nm和280nm。跑样方法编辑如下：
通用步骤D
SEC检测抗体单体率
配制SEC流动相50mM PB+300mM NaCl+200mM Arg，IPA＝95:5，调节pH＝6.5(各缩写含义如下：PB：磷酸盐缓冲溶液，Arg：精氨酸，IPA：异丙醇)。将样品稀释到浓度在1～2mg/ml范围间，再将样品用14000rpm离心5分钟或者用0.22μm滤器过滤以去除样品中大颗粒。将样品瓶放于样品板上，按照《UPLC使用标准操作规程》设置各样品对应的位置、进样体积、进样针数和进样方法。色谱柱型号为：XBridge BEH SEC，1.7μm，4.6*150mm，Waters。
设定检测波长为214nm和280nm，编辑跑样方法为：
通用步骤E
疏水性相互作用色谱(HIC)测定
使用疏水相互作用色谱(HIC)来进行对ADC的分析。通过0-100％流动相B(MPB)洗脱，其中流动相A(MPA)由1.5M硫酸铵和0.025M磷酸钠组成，并且MPB由0.025M的磷酸钠、25％异丙醇组成。样品上样量约为20μg，梯度洗脱在15分钟完成。检测波长为280nm，疏水性越强的样品越晚出峰。
通用步骤F
血浆稳定性研究
取一定量的ADC样品，加入到已去除IgG的人血浆中,每种ADC重复三管，放置37℃水浴中孵育,分别孵育0天、3天、7天和21天后，取出ADC样品，每管加入ProteinA(MabSelectSuReTMLX Lot:#10257475GE)，取PBS洗涤过的100μl垂直混合仪晃动吸附2h,经过洗涤洗脱步骤,获得孵育后的ADC。对孵育特定时间的ADC样品进行RP-HPLC检测。判定样品的血浆稳定性。
实施例1化合物1的合成
于50mL单口圆底烧瓶中依次加入化合物Auristatin E(826mg,1.128mmol,1.0eq)，ki-1(831.4mg,2.256mmol,2.0eq，合成方法参照CN111686259A中化合物1的合成)，醋酸锌(414.2mg,5.64mmol,2.0eq)和甲苯(15mL)，置换氮气3次，115℃回流反应4h，停止反应，冷却至室温，过滤，滤液减压浓缩，反相制备柱纯化，冻干得白色固体(605mg，51.55％)。LC‐MS m/z(ES+)：[M+H]+:1041.3。
实施例2化合物3的合成
于10mL EP管中依次加入Z-Gly-Gly-Phe-OH(240mg,0.581mmol,1.0eq)， HATU(264mg,0.697mmol,1.2eq)，HOBt(94mg,0.697mmol,1.2eq)，和DMF(2mL)，室温搅拌备用。
另取25mL单口圆底烧瓶中依次加入化合物1(605mg,0.581mmol,1.0eq)和DMF(8mL)，室温搅拌溶解后，加入DBU(95.6μL,0.64mmol,1.1eq)，室温反应0.5h，TLC监控，原料消失，产生化合物2，然后将上述混合液中加至此瓶中，再加入DIEA(96μL,0.581mmol,1.0eq)。室温反应1h，HPLC监控。反应结束后，经反相制备柱纯化得白色固体产物(487mg，69.08％)。LC‐MS m/z(ES+)：[M+H]+:1213.6。
实施例3化合物5的合成
于25mL单口圆底烧瓶中依次加入化合物3(487mg,0.402mmol,1.0eq),5％Pd/C(48.7mg)和DMF(5mL)，氢气置换3次后，室温反应1h，HPLC监控反应，原料消失，产生一新峰，为化合物4，记为反应液①。
另取25mL单口瓶中加入ki-2(176mg,0.442mmol,1.1eq合成方法参照CN108452321A),五氟苯酚(81mg,0.442mmol,1.1eq),DCC(91mg,0.442mmol,1.1eq)和DMF(3mL)，室温反应30min，TLC监控，反应结束得到ki-3，记为反应液②。
然后把反应液①过滤到新的25mL单口圆底烧瓶中，冰水浴下加入DIEA(73μL,0.442mmol,1.1eq)，②的反应液滤液，加完后升至室温反应1h，HPLC监控反应，将反应液过滤，滤液在冰浴搅拌下加入到含有化合物4的反应液中，然后加入，撤去冰浴，室温反应1h，HPLC监控。反应液直接经反相制备纯化，制备液冻干，得到白色固体产物(320mg，54.53％)。LC‐MS m/z(ES+)：[M/2+H]+：730.4。
实施例4化合物LP-1a的合成
将化合物5(100mg,0.0685mmol,1.0eq)溶于10mL干燥二氯甲烷，4mL TFA，室温反应3h，HPLC监控。反应结束后，减压浓缩除去溶剂，经反相制备柱纯化，冻干制备液，得白色固体产物(21mg，29.78％)。LC‐MS m/z(ES+)：[M/2+H]+：652.4。LC-MS谱图见图1。
实施例5化合物6的合成
100mL单口瓶中加入MMAE(2.0g,2.79mmol，1.0eq)，(Boc)2O(1.21g,5.57mmol，2.0eq)，DCM(20mL)溶清，冰水浴下加入TEA(563mg,5.57mmol，2.0eq),加完后升至室温反应72h，TLC监控，原料MMAE反应完。后处理：反应液减压浓缩后，经柱层析纯化(洗脱剂：DCM/MeOH＝20/1)得到白色固体(2.28g，100％)。LC-MSm/z(ES+)：[M+H]+:818.4。
实施例6化合物7的合成
100mL单口瓶中依次加入化合物6(2.28g,2.79mmol，1.0eq)，ki-1(2.05g,5.57mmol，2.0eq),醋酸锌(1.02g,5.57mmol，2.0eq),甲苯(30mL)溶解，N2置换3次后，N2保护下升至115℃反应4h，停止反应，冷却至室温，过滤，滤液减压浓缩，反相制备柱纯化，制备液冻干得到白色固体产物(1.071g，34％)。LC-MSm/z(ES+)：[M+H]+:1126.4。
实施例7化合物9的合成
于50mL单口瓶中加入化合物7(900mg,0.8mmol,1.0eq)和DMF(9mL)，搅拌溶清后，冰水浴下滴加DBU(134mg,0.88mmol,1.1eq)，加完后升至室温反应30min。TLC监控，反应结束，记为反应液①；
另取50mL单口瓶中加入Z-Gly-Gly-Phe-OH(364mg,0.88mmol,1.1eq)，HATU(365mg,0.96mmol,1.2eq),HOBt(129.7mg,0.96mmol,1.2eq)和DMF(7mL)溶清后，冰水浴下依次滴加反应液①，DIEA(103.4mg,0.8mmol,1.0eq)，加完后升至室温反应1h，HPLC监控反应。反应液经反相制备纯化，制备液冻干得到白色固体(960mg，92.4％)。LC-MSm/z(ES+)：[M+H]+:1299.6。
实施例8化合物11的合成
于50mL单口瓶中加入化合物10(960mg,0.74mmol,1.0eq),5％Pd/C(960mg)和DMF(10mL)，H2置换3次后，室温反应1h，HPLC监控反应，记为反应液①；
另取25mL单口瓶中加入ki-2(322.7mg,0.81mmol,1.1eq)，DCC(167mg,0.81mmol,1.1eq)和DMF(5mL)溶清后，冰水浴下加入五氟苯酚(149mg,0.81mmol,1.1eq)，加完后升至室温反应30min，TLC监控反应，反应完，得到ki-3，记为反应液②。
将反应液①过滤至50mL单口瓶中，冰水浴下加入DIEA(105mg,0.81mmol,1.1eq)，②的反应液滤液，加完后升至室温反应1h，HPLC监控反应。反应液经反相制备纯化，制备液冻干，得到白色固体(895mg，78.5％)。LC-MSm/z(ES+)：[M/2+H]+：773.4。
实施例9化合物LP-2a的合成
将化合物12(400mg,0.259mmol，1.0eq)溶于20mL干燥二氯甲烷，8mL TFA，室温反应3h，HPLC监控。反应结束后，减压浓缩除去溶剂，粗品经反相制备柱纯化，制备液冻干，得到白色固体(248mg，74％)，LC-MSm/z(ES+)：[M/2+H]+：645.3。
实施例10 VcMMAE的合成
于25mL圆底烧瓶中，加入MMAE(120mg，0.167mmol，1.0eq)和MC-VC-PAB-PNP(186mg，0.25mmol，1.5eq)，用DMF(5mL)溶解后，依次加入HOBt(27.1mg,0.20mmol,1.2eq)和吡啶(1mL)，室温下搅拌过夜，HPLC监控。反应结束后，粗品经反相制备柱纯化，制备液冻干，得到白色固体(158.3mg，72％)，LC-MSm/z(ES+)：[M/2+H]+：659.0。LC-MS谱图见图2。
实施例11化合物13的合成
参照实施例5的合成方法，以化合物12(参照专利CN106279352中化合物17的合成)为原料合成化合物13。LC-MSm/z(ES+)：[M+H]+:873.6
实施例12化合物14的合成
参照实施例6的合成方法，以化合物13和ki-1为原料合成化合物14。LC-MSm/z (ES+)：[M+H]+:1180.7。
实施例13化合物16的合成
参照实施例7的合成方法，以化合物14为原料合成化合物16。LC-MSm/z(ES+)：[M+H]+:1353.9。
实施例14化合物18的合成
参照实施例8的合成方法，以化合物16为原料合成化合物18。LC-MSm/z(ES+)：[M+H]+:1600.9。
实施例15化合物LP-3a的合成
参照实施例9的合成方法，以化合物18为原料合成化合物LP-3a。LC-MSm/z(ES+)：[M+H]+：1344.7。
实施例16化合物23的合成
参照实施例10的合成方法，以化合物12和MC-VC-PAB-PNP为原料合成化合物19。LC-MS m/z(ES+)：[M+H]+：1371.8。
实施例17化合物21的合成
参照实施例5的合成方法，以化合物20(参照专利US2017014524A1合成)为原料合成化合物21。LC-MSm/z(ES+)：[M+H]+:933.6。
实施例18化合物22的合成
参照实施例6的合成方法，以化合物21和ki-1为原料合成化合物22。LC-MSm/z(ES+)：[M+H]+:1241.7。
实施例19化合物24的合成
参照实施例7的合成方法，以化合物22为原料合成化合物24。LC-MSm/z(ES+)：[M+H]+:1414.8。
实施例20化合物26的合成
参照实施例8的合成方法，以化合物24为原料合成化合物26。LC-MSm/z(ES+)：[M+H]+:1660.9。
实施例21化合物27(LP-3sa)的合成
参照实施例9的合成方法，以化合物26为原料合成化合物27(LP-3sa)。LC-MSm/z(ES+)：[M+H]+：1304.7。
实施例22化合物29的合成
参照实施例5的合成方法，以化合物28(参照专利US2017014524A1合成)为原料合成化合物29。LC-MSm/z(ES+)：[M+H]+:847.6。
实施例23化合物30的合成
参照实施例6的合成方法，以化合物29和ki-1为原料合成化合物30。LC-MSm/z(ES+)：[M+H]+:1155.7。
实施例24化合物32的合成
参照实施例7的合成方法，以化合物30为原料合成化合物32。LC-MSm/z(ES+)：[M+H]+:1328.8。
实施例25化合物34的合成
参照实施例8的合成方法，以化合物32为原料合成化合物34。LC-MSm/z(ES+)：[M+H]+:1574.9。
实施例26化合物LP-4a的合成
参照实施例9的合成方法，以化合物34为原料合成化合物LP-4a。LC-MSm/z(ES+)：[M+H]+：1318.7。
实施例27化合物36的合成
参照实施例5的合成方法，以化合物35(参照专利US20210346523A1合成)为原料合成化合物36。LC-MSm/z(ES+)：[M+H]+:863.5。
实施例28化合物37的合成
参照实施例6的合成方法，以化合物36和ki-1为原料合成化合物37。LC-MSm/z(ES+)：[M+H]+:1171.7。
实施例29化合物39的合成
参照实施例7的合成方法，以化合物37为原料合成化合物39。LC-MSm/z(ES+)：[M+H]+:1344.7。
实施例30化合物41的合成
参照实施例8的合成方法，以化合物39为原料合成化合物41。LC-MSm/z(ES+)：[M+H]+:1590.9。
实施例31化合物LP-5a的合成
参照实施例9的合成方法，以化合物41为原料合成化合物LP-5a。LC-MSm/z(ES+)：[M+H]+：1334.7。
实施例32化合物43的合成
参照实施例6的合成方法，以化合物42(参照专利US20210346523A1中化合物17的合成)和ki-1为原料合成化合物43。LC-MSm/z(ES+)：[M+H]+:1085.6。
实施例33化合物45的合成
参照实施例7的合成方法，以化合物43为原料合成化合物45。LC-MSm/z(ES+)：[M+H]+:1258.7。
实施例34化合物47的合成
参照实施例8的合成方法，以化合物45为原料合成化合物47。LC-MSm/z(ES+)：[M+H]+:1504.8。
实施例35化合物LP-6a的合成
参照实施例9的合成方法，以化合物47为原料合成化合物LP-6a。LC-MSm/z(ES+)：[M+H]+：1348.7。
实施例36化合物49的合成
参照实施例6的合成方法，以化合物48(参照专利CN106279352中化合物6的合成)和ki-1为原料合成化合物49。LC-MSm/z(ES+)：[M+H]+:1082.6。
实施例37化合物51的合成
参照实施例7的合成方法，以化合物49为原料合成化合物51。LC-MSm/z(ES+)：[M+H]+:1255.7。
实施例38化合物53的合成
参照实施例8的合成方法，以化合物51为原料合成化合物53。LC-MSm/z(ES+)：[M+H]+:1501.8。
实施例39化合物LP-7a的合成
参照实施例9的合成方法，以化合物53为原料合成化合物LP-7a。LC-MSm/z(ES+)： [M+H]+：1345.7。
实施例40化合物63的合成
参照实施例5的合成方法，以化合物54(参照专利CN113121639A中化合物1的合成)为原料合成化合物55。LC-MSm/z(ES+)：[M+H]+:830.6。
实施例41化合物56的合成
参照实施例6的合成方法，以化合物55和ki-1为原料合成化合物56。LC-MSm/z(ES+)：[M+H]+:1138.7。
实施例42化合物58的合成
参照实施例7的合成方法，以化合物56为原料合成化合物58。LC-MSm/z(ES+)：[M+H]+:1311.8。
实施例43化合物60的合成
参照实施例8的合成方法，以化合物58为原料合成化合物60。LC-MSm/z(ES+)： [M+H]+:1557.9。
实施例44化合物LP-8a的合成
参照实施例9的合成方法，以化合物60为原料合成化合物LP-8a。LC-MSm/z(ES+)：[M+H]+：1301.7。
实施例45化合物62的合成
参照实施例6的合成方法，以化合物61(参照专利CN113121639A中化合物1的合成)和ki-1为原料合成化合物62。LC-MSm/z(ES+)：[M+H]+:1052.6。
实施例46化合物63的合成
参照实施例7的合成方法，以化合物61为原料合成化合物63。LC-MSm/z(ES+)：[M+H]+:1225.7。
实施例47化合物65的合成
参照实施例8的合成方法，以化合物63为原料合成化合物65。LC-MSm/z(ES+)： [M+H]+:1471.8。
实施例48化合物LP-9a的合成
参照实施例9的合成方法，以化合物65为原料合成化合物LP-9a。LC-MSm/z(ES+)：[M+H]+：1315.7。
实施例49化合物66的合成
于50mL单口瓶中加入化合物9(960mg,0.74mmol,1.0eq),5％Pd/C(960mg)和DMF(10mL)，H2置换3次后，室温反应1h，HPLC监控反应，反应完成，记为反应液①；将反应液①过滤至50mL单口瓶中，冰水浴下依次加入MCOSU(274mg,0.89mmol,1.2eq)和DIEA(105mg,0.81mmol,1.1eq)，加完后升至室温反应1h，HPLC监控反应。反应液经反相制备纯化，制备液冻干，得到白色固体(847.5mg，84.3％)。LC-MSm/z(ES+)：[M+H]+：1358.2。
实施例50化合物LP-10的合成
参照实施例9的合成方法，以化合物66为原料合成化合物LP-10。LC-MSm/z(ES+)：[M/2+H]+：630.0。
实施例51化合物LP-11的合成
参照实施例49的合成方法，以化合物3为原料合成化合物LP-11。LC-MSm/z(ES+)：[M+H]+：1272.8。
实施例52化合物67的合成
参照实施例49的合成方法，以化合物28为原料合成化合物67。LC-MSm/z(ES+)：[M+H]+：1473.9。
实施例53化合物LP-12的合成
参照实施例9的合成方法，以化合物67为原料合成化合物LP-12。LC-MSm/z(ES+)：[M+H]+：1273.8。
实施例54化合物68的合成
参照实施例49的合成方法，以化合物36为原料合成化合物68。LC-MSm/z(ES+)：[M+H]+：1387.8。
实施例55化合物LP-13的合成
参照实施例9的合成方法，以化合物68为原料合成化合物LP-13。LC-MSm/z(ES+)：[M+H]+：1287.8。
实施例56化合物69的合成
参照实施例49的合成方法，以化合物39为原料合成化合物69。LC-MSm/z(ES+)：[M+H]+：1403.8。
实施例57化合物LP-14的合成
参照实施例9的合成方法，以化合物69为原料合成化合物LP-14。LC-MSm/z(ES+)：[M+H]+：1303.7。
实施例58化合物LP-15的合成
参照实施例49的合成方法，以化合物45为原料合成化合物LP-15。LC-MSm/z(ES+)：[M+H]+：1403.8。
实施例59化合物LP-16的合成
参照实施例49的合成方法，以化合物51为原料合成化合物LP-16。LC-MSm/z(ES+)：[M+H]+:1314.8。
实施例60化合物70的合成
参照实施例49的合成方法，以化合物58为原料合成化合物70。LC-MSm/z(ES+)：[M+H]+：1370.8。
实施例61化合物LP-17的合成
参照实施例9的合成方法，以化合物70为原料合成化合物LP-17。LC-MSm/z(ES+)：[M+H]+：1270.8。
实施例62化合物LP-18的合成
参照实施例49的合成方法，以化合物63为原料合成化合物LP-18。LC-MSm/z(ES+)：[M+H]+：1284.8。
实施例63化合物71的合成
于50mL单口瓶中加入化合物9(960mg,0.74mmol,1.0eq),5％Pd/C(960mg)和DMF(10mL)，H2置换3次后，室温反应1h，HPLC监控反应，反应完成，记为反应液①；将反应液①过滤至50mL单口瓶中，冰水浴下依次加入化合物M6(512.5mg,0.74mmol,1.0eq，化合物M6参照CN113827736中化合物M6的合成)和DIEA(105mg,0.81mmol,1.1eq)，加完后升至室温反应1h，HPLC监控反应。反应液经反相制备纯化，制备液冻干，得到白色固体(860mg，70％)。LC-MSm/z(ES+)：[M+H]+：1660.1。
实施例64化合物LP-19a的合成
参照实施例9的合成方法，以化合物71为原料合成化合物LP-19a。LC-MSm/z(ES+)：[M/2+H]+：674.4。
实施例65化合物72的合成
参照实施例63的合成方法，以化合物3为原料合成化合物72。LC-MSm/z(ES+)：[M/2+H]+：783.5。
实施例66化合物LP-20a的合成
参照实施例9的合成方法，以化合物72为原料合成化合物LP-20a。LC-MSm/z(ES+)：[M/2+H]+：681.4。
实施例67化合物73的合成
参照实施例63的合成方法，以化合物24为原料合成化合物73。LC-MSm/z(ES+)：[M/2+H]+：888.5。
实施例68化合物LP-21a的合成
参照实施例9的合成方法，以化合物73为原料合成化合物LP-21a。LC-MSm/z(ES+)：[M+H]+：1362.7。
实施例69化合物74的合成
参照实施例63的合成方法，以化合物32为原料合成化合物74。LC-MSm/z(ES+)：[M/2+H]+：845.1。
实施例70化合物LP-22a的合成
参照实施例9的合成方法，以化合物74为原料合成化合物LP-22a。LC-MSm/z(ES+)：[M+H]+：1376.8。
实施例71化合物75的合成
参照实施例63的合成方法，以化合物39为原料合成化合物75。LC-MSm/z(ES+)：[M/2+H]+：853.1。
实施例72化合物LP-23a的合成
参照实施例9的合成方法，以化合物75为原料合成化合物LP-23a。LC-MSm/z(ES+)：[M+H]+：1392.7。
实施例73化合物76的合成
参照实施例63的合成方法，以化合物45为原料合成化合物76。LC-MSm/z(ES+)：[M/2+H]+：810.1。
实施例74化合物LP-24a的合成
参照实施例9的合成方法，以化合物76为原料合成化合物LP-24a。LC-MSm/z(ES+)：[M+H]+：1406.8。
实施例75化合物77的合成
参照实施例63的合成方法，以化合物51为原料合成化合物77。LC-MSm/z(ES+)：[M/2+H]+：808.5。
实施例76化合物LP-25a的合成
参照实施例9的合成方法，以化合物77为原料合成化合物LP-25a。LC-MSm/z(ES+)：[M+H]+：1403.8。
实施例77化合物78的合成
参照实施例63的合成方法，以化合物58为原料合成化合物78。LC-MSm/z(ES+)：[M/2+H]+：836.5。
实施例78化合物LP-26a的合成
参照实施例9的合成方法，以化合物78为原料合成化合物LP-26a。LC-MSm/z(ES+)：[M+H]+：1359.7。
实施例79化合物79的合成
参照实施例63的合成方法，以化合物63为原料合成化合物79。LC-MSm/z(ES+)：[M/2+H]+：793.5。
实施例80化合物LP-27a的合成
参照实施例9的合成方法，以化合物79为原料合成化合物LP-27a。LC-MSm/z(ES+)：[M+H]+：1373.8。
实施例81化合物80的合成
于50mL单口瓶中加入化合物9(960mg,0.74mmol,1.0eq),5％Pd/C(960mg)和DMF(10mL)，H2置换3次后，室温反应1h，HPLC监控反应，反应完成，记为反应液①；将反应液①过滤至50mL单口瓶中，冰水浴下依次加入化合物M8(497.7mg,0.74mmol,1.0eq，化合物M8参照CN113827736中化合物M8的合成)和DIEA(105mg,0.81mmol, 1.1eq)，加完后升至室温反应1h，HPLC监控反应。反应液经反相制备纯化，制备液冻干，得到白色固体(918mg，75％)。LC-MSm/z(ES+)：[M/2+H]+：827.5。
实施例82化合物LP-28a的合成
参照实施例9的合成方法，以化合物80为原料合成化合物LP-28a。LC-MSm/z(ES+)：[M/2+H]+：671.4。
实施例83化合物81的合成
参照实施例81的合成方法，以化合物3为原料合成化合物81。LC-MSm/z(ES+)：[M/2+H]+：784.5。
实施例84化合物LP-29a的合成
参照实施例9的合成方法，以化合物81为原料合成化合物LP-29a。LC-MSm/z(ES+)：[M/2+H]+：678.4。
实施例85化合物82的合成
参照实施例81的合成方法，以化合物24为原料合成化合物82。LC-MSm/z(ES+)：[M/2+H]+：885.0。
实施例86化合物LP-30a的合成
参照实施例9的合成方法，以化合物82为原料合成化合物LP-30a。LC-MSm/z(ES+)：[M/2+H]+：678.9。
实施例87化合物83的合成
参照实施例81的合成方法，以化合物32为原料合成化合物83。LC-MSm/z(ES+)：[M/2+H]+：842.0。
实施例88化合物LP-31a的合成
参照实施例9的合成方法，以化合物83为原料合成化合物LP-31a。LC-MSm/z(ES+)：[M/2+H]+：685.9。
实施例89化合物84的合成
参照实施例81的合成方法，以化合物39为原料合成化合物84。LC-MSm/z(ES+)：[M/2+H]+：850.1。
实施例90化合物LP-32a的合成
参照实施例9的合成方法，以化合物84为原料合成化合物LP-32a。LC-MSm/z(ES+)：[M/2+H]+：693.9。
实施例91化合物85的合成
参照实施例81的合成方法，以化合物45为原料合成化合物85。LC-MSm/z(ES+)：[M/2+H]+：806.9。
实施例92化合物LP-33a的合成
参照实施例9的合成方法，以化合物85为原料合成化合物LP-33a。LC-MSm/z(ES+)：[M/2+H]+：700.9。
实施例93化合物86的合成
参照实施例81的合成方法，以化合物51为原料合成化合物86。LC-MSm/z(ES+)：[M/2+H]+：805.5。
实施例94化合物LP-34a的合成
参照实施例9的合成方法，以化合物86为原料合成化合物LP-34a。LC-MSm/z(ES+)：[M/2+H]+：699.4。
实施例95化合物87的合成
参照实施例81的合成方法，以化合物58为原料合成化合物87。LC-MSm/z(ES+)： [M/2+H]+：833.5。
实施例96化合物LP-35a的合成
参照实施例9的合成方法，以化合物87为原料合成化合物LP-35a。LC-MSm/z(ES+)：[M/2+H]+：677.4。
实施例97化合物88的合成
参照实施例81的合成方法，以化合物63为原料合成化合物88。LC-MSm/z(ES+)：[M/2+H]+：790.5。
实施例98化合物LP-36a的合成
参照实施例9的合成方法，以化合物88为原料合成化合物LP-36a。LC-MSm/z(ES+)：[M/2+H]+：670.4。
实施例99化合物89的合成
参照实施例49的合成方法，以化合物16为原料合成化合物89。LC-MSm/z(ES+)：[M/2+H]+：707.4。
实施例100化合物LP-37的合成
参照实施例9的合成方法，以化合物89为原料合成化合物LP-37。LC-MSm/z(ES+)：[M+H]+：1313.8。
实施例101化合物90的合成
参照实施例63的合成方法，以化合物16为原料合成化合物90。LC-MSm/z(ES+)：[M/2+H]+：858.0。
实施例102化合物LP-38a的合成
参照实施例9的合成方法，以化合物90为原料合成化合物LP-38a。LC-MSm/z(ES+)：[M/2+H]+：701.9。
实施例103化合物91的合成
参照实施例81的合成方法，以化合物16为原料合成化合物91。LC-MSm/z(ES+)：[M/2+H]+：855.0。
实施例104化合物LP-39a的合成
参照实施例9的合成方法，以化合物91为原料合成化合物LP-39a。LC-MSm/z(ES+)：[M/2+H]+：698.9。
实施例105抗体药物偶联物ADC-1-1a的制备
ADC-1-1a的制备是相应的linker-payload(LP-1a，其结构如LP-1的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到，TR000的序列信息见表1。SEC-HPLC检测结果见图3。
实施例106抗体药物偶联物ADC-2-1a的制备
ADC-2-1a的制备是相应的linker-payload(LP-2a，其结构如LP-2的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。SEC-HPLC检测结果见图4。
实施例107抗体药物偶联物ADC-C-1a的制备
ADC-C-1a的制备是化合物VcMMAE按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例108抗体药物偶联物ADC-3-1a的制备
ADC-3-1a的制备是相应的linker-payload(LP-10)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例109抗体药物偶联物ADC-4-1a的制备
ADC-4-1a的制备是相应的linker-payload(LP-37)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例110抗体药物偶联物ADC-5-1a的制备
ADC-5-1a的制备是相应的linker-payload(LP-16)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例111抗体药物偶联物ADC-6-1a的制备
ADC-6-1a的制备是相应的linker-payload(LP-11)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例112抗体药物偶联物ADC-7-1a的制备
ADC-7-1a的制备是相应的linker-payload(LP-12)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例113抗体药物偶联物ADC-8-1a的制备
ADC-8-1a的制备是相应的linker-payload(LP-13)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例114抗体药物偶联物ADC-9-1a的制备
ADC-9-1a的制备是相应的linker-payload(LP-14)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例115抗体药物偶联物ADC-10-1a的制备
ADC-10-1a的制备是相应的linker-payload(LP-15)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例116抗体药物偶联物ADC-11-1a的制备
ADC-11-1a的制备是相应的linker-payload(LP-17)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例117抗体药物偶联物ADC-12-1a的制备
ADC-12-1a的制备是相应的linker-payload(LP-18)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例118抗体药物偶联物ADC-13-1a的制备
ADC-13-1a的制备是相应的linker-payload(LP-3a，其结构如LP-3的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例119抗体药物偶联物ADC-14-1a的制备
ADC-14-1a的制备是相应的linker-payload(LP-7a，其结构如LP-7的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例120抗体药物偶联物ADC-15-1a的制备
ADC-15-1a的制备是相应的linker-payload(LP-3sa，其结构如LP-3s的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例121抗体药物偶联物ADC-16-1a的制备
ADC-16-1a的制备是相应的linker-payload(LP-4a，其结构如LP-4的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例122抗体药物偶联物ADC-17-1a的制备
ADC-17-1a的制备是相应的linker-payload(LP-5a，其结构如LP-5的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例123抗体药物偶联物ADC-18-1a的制备
ADC-18-1a的制备是相应的linker-payload(LP-6a，其结构如LP-6的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例124抗体药物偶联物ADC-19-1a的制备
ADC-19-1a的制备是相应的linker-payload(LP-8a，其结构如LP-8的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例125抗体药物偶联物ADC-20-1a的制备
ADC-20-1a的制备是相应的linker-payload(LP-9a，其结构如LP-9的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例126抗体药物偶联物ADC-21-1a的制备
ADC-21-1a的制备是相应的linker-payload(LP-28a，其结构如LP-28的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例127抗体药物偶联物ADC-22-1a的制备
ADC-22-1a的制备是相应的linker-payload(LP-29a，其结构如LP-29的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例128抗体药物偶联物ADC-23-1a的制备
ADC-23-1a的制备是相应的linker-payload(LP-30a，其结构如LP-30的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例129抗体药物偶联物ADC-24-1a的制备
ADC-24-1a的制备是相应的linker-payload(LP-31a，其结构如LP-31的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例130抗体药物偶联物ADC-25-1a的制备
ADC-25-1a的制备是相应的linker-payload(LP-32a，其结构如LP-32的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例131抗体药物偶联物ADC-26-1a的制备
ADC-26-1a的制备是相应的linker-payload(LP-33a，其结构如LP-33的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例132抗体药物偶联物ADC-27-1a的制备
ADC-27-1a的制备是相应的linker-payload(LP-35a，其结构如LP-35的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例133抗体药物偶联物ADC-28-1a的制备
ADC-28-1a的制备是相应的linker-payload(LP-36a，其结构如LP-36的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例134抗体药物偶联物ADC-29-1a的制备
ADC-29-1a的制备是相应的linker-payload(LP-39a，其结构如LP-39的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例135抗体药物偶联物ADC-30-1a的制备
ADC-30-1a的制备是相应的linker-payload(LP-34a，其结构如LP-34的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例136抗体药物偶联物ADC-31-1a的制备
ADC-31-1a的制备是相应的linker-payload(LP-19a，其结构如LP-19的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例137抗体药物偶联物ADC-35-1a的制备
ADC-35-1a的制备是相应的linker-payload(LP-22a，其结构如LP-22的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例138抗体药物偶联物ADC-39-1a的制备
ADC-39-1a的制备是相应的linker-payload(LP-26a，其结构如LP-26的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例139抗体药物偶联物ADC-45-1a的制备
ADC-45-1a的制备是相应的linker-payload(LP-20a，其结构如LP-20的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例140抗体药物偶联物ADC-48-1a的制备
ADC-48-1a的制备是相应的linker-payload(LP-27a，其结构如LP-27的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体TR000制备得到。
实施例141抗体药物偶联物ADC-1-2a的制备
ADC-1-2a的制备是相应的linker-payload(LP-1a，其结构如LP-1的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到，TR005的序列信息见表1。SEC-HPLC检测结果见图5。
实施例142抗体药物偶联物ADC-2-2a的制备
ADC-2-2a的制备是相应的linker-payload(LP-2a，其结构如LP-2的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。SEC-HPLC检测结果见图6。
实施例143抗体药物偶联物ADC-C-2a的制备
ADC-C-2a的制备是化合物VcMMAE按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例144抗体药物偶联物ADC-3-2a的制备
ADC-3-2a的制备是相应的linker-payload(LP-10)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例145抗体药物偶联物ADC-4-2a的制备
ADC-4-2a的制备是相应的linker-payload(LP-37)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例146抗体药物偶联物ADC-5-2a的制备
ADC-5-2a的制备是相应的linker-payload(LP-16)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例147抗体药物偶联物ADC-6-2a的制备
ADC-6-2a的制备是相应的linker-payload(LP-11)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例148抗体药物偶联物ADC-7-2a的制备
ADC-7-2a的制备是相应的linker-payload(LP-12)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例149抗体药物偶联物ADC-8-2a的制备
ADC-8-2a的制备是相应的linker-payload(LP-13)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例150抗体药物偶联物ADC-9-2a的制备
ADC-9-2a的制备是相应的linker-payload(LP-14)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例151抗体药物偶联物ADC-10-2a的制备
ADC-10-2a的制备是相应的linker-payload(LP-15)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例152抗体药物偶联物ADC-11-2a的制备
ADC-11-2a的制备是相应的linker-payload(LP-17)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例153抗体药物偶联物ADC-12-2a的制备
ADC-12-2a的制备是相应的linker-payload(LP-18)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例154抗体药物偶联物ADC-13-2a的制备
ADC-13-2a的制备是相应的linker-payload(LP-3a，其结构如LP-3的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例155抗体药物偶联物ADC-14-2a的制备
ADC-14-2a的制备是相应的linker-payload(LP-7a，其结构如LP-7的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例156抗体药物偶联物ADC-15-2a的制备
ADC-15-2a的制备是相应的linker-payload(LP-3sa，其结构如LP-3s的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例157抗体药物偶联物ADC-16-2a的制备
ADC-16-2a的制备是相应的linker-payload(LP-4a，其结构如LP-4的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例158抗体药物偶联物ADC-17-2a的制备
ADC-17-2a的制备是相应的linker-payload(LP-5a，其结构如LP-5的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例159抗体药物偶联物ADC-18-2a的制备
ADC-18-2a的制备是相应的linker-payload(LP-6a，其结构如LP-6的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例160抗体药物偶联物ADC-19-2a的制备
ADC-19-2a的制备是相应的linker-payload(LP-8a，其结构如LP-8的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例161抗体药物偶联物ADC-20-2a的制备
ADC-20-2a的制备是相应的linker-payload(LP-9a，其结构如LP-9的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例162抗体药物偶联物ADC-21-2a的制备
ADC-21-2a的制备是相应的linker-payload(LP-28a，其结构如LP-28的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例163抗体药物偶联物ADC-22-2a的制备
ADC-22-2a的制备是相应的linker-payload(LP-29a，其结构如LP-29的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例164抗体药物偶联物ADC-23-2a的制备
ADC-23-2a的制备是相应的linker-payload(LP-30a，其结构如LP-30的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例165抗体药物偶联物ADC-24-2a的制备
ADC-24-2a的制备是相应的linker-payload(LP-31a，其结构如LP-31的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例166抗体药物偶联物ADC-25-2a的制备
ADC-25-2a的制备是相应的linker-payload(LP-32a，其结构如LP-32的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例167抗体药物偶联物ADC-26-2a的制备
ADC-26-2a的制备是相应的linker-payload(LP-33a，其结构如LP-33的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例168抗体药物偶联物ADC-27-2a的制备
ADC-27-2a的制备是相应的linker-payload(LP-35a，其结构如LP-35的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例169抗体药物偶联物ADC-28-2a的制备
ADC-28-2a的制备是相应的linker-payload(LP-36a，其结构如LP-36的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例170抗体药物偶联物ADC-29-2a的制备
ADC-29-2a的制备是相应的linker-payload(LP-39a，其结构如LP-39的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例171抗体药物偶联物ADC-30-2a的制备
ADC-30-2a的制备是相应的linker-payload(LP-34a，其结构如LP-34的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例172抗体药物偶联物ADC-31-2a的制备
ADC-31-2a的制备是相应的linker-payload(LP-19a，其结构如LP-19的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例173抗体药物偶联物ADC-35-2a的制备
ADC-35-2a的制备是相应的linker-payload(LP-22a，其结构如LP-22的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例174抗体药物偶联物ADC-39-2a的制备
ADC-39-2a的制备是相应的linker-payload(LP-26a，其结构如LP-26的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例175抗体药物偶联物ADC-45-2a的制备
ADC-45-2a的制备是相应的linker-payload(LP-20a，其结构如LP-20的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例176抗体药物偶联物ADC-48-2a的制备
ADC-48-2a的制备是相应的linker-payload(LP-27a，其结构如LP-27的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤B的方法与抗Trop2抗体TR005制备得到。
实施例177抗体药物偶联物ADC-1-3a的制备
ADC-1-3a的制备是相应的linker-payload(LP-1a，其结构如LP-1的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到，hu7F11的序列信息见表2。
实施例178抗体药物偶联物ADC-2-3a的制备
ADC-2-3a的制备是是相应的linker-payload(LP-2a，其结构如LP-2的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例179抗体药物偶联物ADC-C-3a的制备
ADC-C-3a的制备是化合物VcMMAE按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例180抗体药物偶联物ADC-3-3a的制备
ADC-3-3a的制备是相应的linker-payload(LP-10)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例181抗体药物偶联物ADC-4-3a的制备
ADC-4-3a的制备是相应的linker-payload(LP-37)按照通用步骤A的方法与抗 Trop2抗体hu7F11制备得到。
实施例182抗体药物偶联物ADC-5-3a的制备
ADC-5-3a的制备是相应的linker-payload(LP-16)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例183抗体药物偶联物ADC-6-3a的制备
ADC-6-3a的制备是相应的linker-payload(LP-11)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例184抗体药物偶联物ADC-7-3a的制备
ADC-7-3a的制备是相应的linker-payload(LP-12)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例185抗体药物偶联物ADC-8-3a的制备
ADC-8-3a的制备是相应的linker-payload(LP-13)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例186抗体药物偶联物ADC-9-3a的制备
ADC-9-3a的制备是相应的linker-payload(LP-14)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例187抗体药物偶联物ADC-10-3a的制备
ADC-10-3a的制备是相应的linker-payload(LP-15)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例188抗体药物偶联物ADC-11-3a的制备
ADC-11-3a的制备是相应的linker-payload(LP-17)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例189抗体药物偶联物ADC-12-3a的制备
ADC-12-3a的制备是相应的linker-payload(LP-18)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例190抗体药物偶联物ADC-13-3a的制备
ADC-13-3a的制备是相应的linker-payload(LP-3a，其结构如LP-3的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例191抗体药物偶联物ADC-14-3a的制备
ADC-14-3a的制备是相应的linker-payload(LP-7a，其结构如LP-7的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例192抗体药物偶联物ADC-15-3a的制备
ADC-15-3a的制备是相应的linker-payload(LP-3sa，其结构如LP-3s的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例193抗体药物偶联物ADC-16-3a的制备
ADC-16-3a的制备是相应的linker-payload(LP-4a，其结构如LP-4的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例194抗体药物偶联物ADC-17-3a的制备
ADC-17-3a的制备是相应的linker-payload(LP-5a，其结构如LP-5的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例195抗体药物偶联物ADC-18-3a的制备
ADC-18-3a的制备是相应的linker-payload(LP-6a，其结构如LP-6的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例196抗体药物偶联物ADC-19-3a的制备
ADC-19-3a的制备是相应的linker-payload(LP-8a，其结构如LP-8的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例197抗体药物偶联物ADC-20-3a的制备
ADC-20-3a的制备是相应的linker-payload(LP-9a，其结构如LP-9的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例198抗体药物偶联物ADC-21-3a的制备
ADC-21-3a的制备是相应的linker-payload(LP-28a，其结构如LP-28的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例199抗体药物偶联物ADC-22-3a的制备
ADC-22-3a的制备是相应的linker-payload(LP-29a，其结构如LP-29的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例200抗体药物偶联物ADC-23-3a的制备
ADC-23-3a的制备是相应的linker-payload(LP-30a，其结构如LP-30的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例201抗体药物偶联物ADC-24-3a的制备
ADC-24-3a的制备是相应的linker-payload(LP-31a，其结构如LP-31的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例202抗体药物偶联物ADC-25-3a的制备
ADC-25-3a的制备是相应的linker-payload(LP-32a，其结构如LP-32的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例203抗体药物偶联物ADC-26-3a的制备
ADC-26-3a的制备是相应的linker-payload(LP-33a，其结构如LP-33的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例204抗体药物偶联物ADC-27-3a的制备
ADC-27-3a的制备是相应的linker-payload(LP-35a，其结构如LP-35的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例205抗体药物偶联物ADC-28-3a的制备
ADC-28-3a的制备是相应的linker-payload(LP-36a，其结构如LP-36的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例206抗体药物偶联物ADC-29-3a的制备
ADC-29-3a的制备是相应的linker-payload(LP-39a，其结构如LP-39的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例207抗体药物偶联物ADC-30-3a的制备
ADC-30-3a的制备是相应的linker-payload(LP-34a，其结构如LP-34的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例208抗体药物偶联物ADC-31-3a的制备
ADC-31-3a的制备是相应的linker-payload(LP-19a，其结构如LP-19的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例209抗体药物偶联物ADC-35-3a的制备
ADC-35-3a的制备是相应的linker-payload(LP-22a，其结构如LP-22的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例210抗体药物偶联物ADC-39-3a的制备
ADC-39-3a的制备是相应的linker-payload(LP-26a，其结构如LP-26的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例211抗体药物偶联物ADC-45-3a的制备
ADC-45-3a的制备是相应的linker-payload(LP-20a，其结构如LP-20的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例212抗体药物偶联物ADC-48-3a的制备
ADC-48-3a的制备是相应的linker-payload(LP-27a，其结构如LP-27的结构式所示，其中，2位手性碳的构型为S型)按照通用步骤A的方法与抗Trop2抗体hu7F11制备得到。
实施例213反相高效液相色谱(RP-HPLC)检测ADC的DAR
经通用步骤C的反相高效液相色谱测定，相应ADC的平均药物/抗体比DAR如下表2所示，对于ADC-C-1a、ADC-C-2a、ADC-C-3a，其DAR＝n，除此之外的其余ADC，DAR＝n1+n2+n3。
表2

实施例214 SEC检测ADC单体率
通过通用步骤D可得出各ADC的聚集程度，SEC-HPLC峰图见图3-图6，数据总 结如下表所示，ADC-1-1a、ADC-2-1a、ADC-1-2a和ADC-2-2a的单体率均大于95％，聚集和降解程度低。
表3
实施例215疏水性相互作用色谱(HIC)测定
按照通用步骤A将IgG1抗体全部还原到每抗体8硫醇后，通过疏水作用色谱对每抗体8个药物的从羟基引入酸性稳定接头的ADC(ADC-1-1a和ADC-2-1a)和传统接头ADC-C-1a(即MC-VC-PAB-MMAE)进行进一步分离纯化，并按照通用步骤E，用疏水性相互作用色谱HIC对ADC分析。具有更大疏水性，或更大数量的药物/分子的ADC在更晚的保留时间洗脱。实验结果如下表所示，从羟基引入酸性稳定接头的ADC-1-1a和ADC-2-1a在HIC中具有相对较短的保留时间，而从氨基引入MC-VC-PAB接头的ADC-C-1a具有最长的保留时间。实验结果表明，从羟基引入酸性稳定接头的ADC-1-1a和ADC-2-1a具有更好的亲水性。
表4
实施例216体外血浆稳定性
按通用步骤F进行ADC的血浆稳定性研究，结果如下表所示，实验结果表明,在毒素的羟基上引入酸性稳定接头的ADC在血浆孵育过程中几乎没有损失药物，而经典的MC接头ADC(ADC-C-1a)在孵育72h后DAR降低非常显著。实验结果证明，在毒素的羟基上引入酸性稳定接头可显著提高ADC药物血浆稳定性。
表5
实施例217 ADC的水解分析
采用超高效液相串联高分辨质谱仪(UPLC-MS)对ADC-2-1a进行轻重链分子量检 测，根据轻重链分子量的变化情况来对ADC-2-1a的水解情况进行表征。
样品准备：取ADC-2-1a 100μg，按照每50μg蛋白加入1M DTT溶液4μL的比例分别加入1M DTT 2μL，混匀并短暂离心(10～20s)后于室温下放置20min，即得。
仪器:WatesAcquity UPLC H-Class，Wates Xevo G2-XS QTof with UNIFI；
色谱柱：Sepax RP-1000,4.6*100mm,5μm
柱温：60℃
RP流动相A：0.05％TFA水溶液，RP流动相B：0.05％TFA乙腈溶液
检测波长280nm，跑样方法编辑如下：
表6
进样量：1mg/mL，10μL，即20μg；
质谱扫描设置：低质量端(m/z)：500；高质量端(m/z)：4000；
检测范围：1000-3500Da；输出范围：20000-120000Da。
图7为被还原过后的抗体的轻链质谱检测结果，图8和图9为被还原过后的ADC-2-1a的轻链质谱检测结果。可知抗体TR000轻链(LC)的分子量为23372.7721Da。由于化合物13的分子量为1289.5400Da，其开环水解产物的分子量为1307.5550Da。故当一个抗体连接8个药物时，其一条轻链偶联1个化合物，在未发生水解的情况下，其理论分子量为24662.3121Da。如图8和图9所示，实际检测到ADC的轻链的分子量包括24663.0000Da、24680.0000Da，其中24680.0000Da与理论分子量相差17.6879Da。当ADC的轻链发生水解且水解1个马来酰亚胺时，其理论分子量为24680.3271Da，与实测值比，相对偏差在仪器的误差范围内，推测ADC-2-1a发生了部分水解，导致分子量增加了近18Da。由此可知，ADC中的马来酰亚胺基团既有开环形式，又有闭环形式。
实施例218 ADC抗肿瘤细胞活性测试
DMEM培养基、RMPI1640培养基购自源培生物、胎牛血清购自ExCellBio公司。人皮肤鳞癌细胞系A431(Trop2阳性表达细胞)、人原位胰腺腺癌细胞系BxPC-3(Trop2阳性表达细胞)、人胃癌细胞系NCI-N87(Trop2阳性表达细胞)和人结肠癌细胞系SW620(阴性对照细胞)均购于ATCC(American type culture collection)，并采用文献 推荐的方法进行培养，所用测试ADC为ADC-1-1a、ADC-2-1a、ADC-C-1a、ADC-1-2a、ADC-2-2a、ADC-C-2a。CellTiterAQueous One Solution Cell Proliferation Assay购买自Promega。
取对数生长期的人皮肤鳞癌细胞系A431、人原位胰腺腺癌细胞系BxPC-3、人胃癌细胞系NCI-N87和人结肠癌细胞系SW620。接种一定数目的肿瘤细胞系于96孔板中，向细胞中加入梯度稀释的受试抗体及对应的ADC药物，处理5天，利用MTS检测细胞活力，通过计算IC50评价受试抗体及ADC对肿瘤细胞系的抑制效果。ADC药物起始浓度为500nM，稀释倍数为7倍，共8个浓度点，处理5天。最终算法按照存活率＝(实验组-空白)/(对照组-空白组)×100％，随后利用Graph Pad Prism拟合曲线，计算半数抑制浓度(IC50)。
表7 ADC药物的体外细胞毒性(120h)

P<0.05，差异具有统计学意义。
经过以上ADC细胞活性测试，本发明所述的ADC药物，在多个Trop2阳性肿瘤细胞系中均表现出良好的抗肿瘤活性，具有极大的临床应用价值。
实施例56 ADC体内药效测试
本发明中建立了A431荷瘤小鼠模型，以评价ADC的体内药效。以3×106A431细胞通过皮下注射到4～6周鼠龄的BALB/c裸鼠右侧，待小鼠肿瘤平均大小生长至140～150mm3，随机分组，每组5只，在第0,7,14,21天分别给予空白对照(缓冲溶液空白)、抗体药物偶联物ADC-1-1a、ADC-2-1a、ADC-C-1a、ADC-1-2a、ADC-2-2a和ADC-C-2a，均以2mg/kg剂量进行静脉给药。肿瘤体积测量数据显示为测量时肿瘤平均体积，同时记录小鼠体重变化情况，用以观察ADC药物的体内初步毒性。结果如下表所示，由表可知，在payload相同的情况下，高DAR的ADC-1-1a、ADC-2-1a和ADC-C-1a相比其低DAR的ADC-1-2a、ADC-2-2a和ADC-C-2a具有更强的肿瘤抑制效果,其中ADC-2-1a的肿瘤抑制效果最强。而在DAR基本相同的情况下，从羟基引入酸性稳定接头的ADC-1-1a和ADC-2-1a相比使用MC-VC-PAB接头的ADC-C-1a具有更强的肿瘤抑制效果。另一方面，可以看到，所有给药组小鼠的体重均无明显变化，这说明ADC-1-1a、ADC-2-1a、ADC-C-1a、ADC-1-2a、ADC-2-2a和ADC-C-2a均无明显毒性。
上述结果表明，从羟基引入酸性稳定接头的ADC相比使用MC-VC-PAB接头的ADC具有更好的体内抗肿瘤活性，使用更低的剂量即可达到同等肿瘤抑制效果。由于本发明的ADC以较低的使用剂量即可达到良好的抗肿瘤效果，并且具有改善的血液稳定性，因此血液毒性和神经毒性更低。
表8
表9
可以理解的是，以上关于本发明的具体描述，仅用于说明本发明而并非受限于本发明实施例所描述的技术方案，本领域的普通技术人员应当可以理解，仍然可以对本发明进行修改或等同替换，以达到相同的技术效果；上述修改或等同替换都在本发明的保护范围之内。

Claims (25)

  1. 如通式I所示的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，
    其中：
    Ab为配体单元，选自抗体、抗体片段、靶向蛋白或Fc-融合蛋白；
    M为与Ab连接的连接单元；
    A选自由2-7个氨基酸构成的肽残基，其中，任选地，所述氨基酸各自独立地被一个或多个取代基所取代，所述取代基选自：氘原子、卤素、羟基、氰基、氨基、硝基、烷基、取代烷基、烷氧基、环烷基、取代环烷基；
    W表示如式(i)所示的氨基亚甲基氧结构单元：
    其中：
    左侧波浪线表示式(i)中氮原子与A的连接位点，右侧波浪线表示式(i)中氧原子与药物D的连接位点，氧原子为药物D与W的共用基团；
    R1、R2和R3各自独立地选自氢原子、氘原子、烷基和取代烷基；
    p选自1-20的整数或小数；
    并且
    药物D为具有式D所示结构的奥瑞他汀，或其异构体、内消旋体、外消旋体、对映异构体或其混合物形式，或其药学上可接受的盐，
    其中：
    R4、R5各自独立地选自氢原子、氘原子、烷基和氘代烷基，或者R4、R5相连形成以下结构：-(CR11R12)n-B-(CR13R14)m-，其中R11、R12、R13和R14选自氢原子、氘原子、烷 基和氘代烷基；B选自O、NR15、CR16R17，其中R15、R16、R17选自氢原子、氘原子和烷基；n和m分别选自0-8的整数；与R4和R5键合的氮原子与-(CR11R12)n-B-(CR13R14)m-一起形成环；
    R6、R7、R8、R9各自独立地选自氢原子、氘原子、卤素、叠氮基、烷基和NR18R19，或者R6、R7、R8、R9之中的任意两个基团与所键合的原子一起形成环烷基，余下的两个基团各自独立地选自氢原子、卤素、叠氮基、烷基和NR18R19，其中R18、R19选自氢原子、烷基；
    R10选自芳基、杂芳基，所述的芳基或杂芳基任选地被一个或多个取代基所取代，所述取代基选自：氢原子、卤素、烷基、烷氧基、氨基、硝基；
    式D中的波浪线表示药物D结构中1位氧原子与W的连接位点，所述氧原子为药物D与W的共用基团。
  2. 如权利要求1所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其特征在于：所述配体单元Ab选自抗体、抗体片段或蛋白，其中所述抗体选自鼠源抗体、兔源抗体、噬菌体展示来源抗体、酵母展示来源抗体、嵌合抗体、人源化抗体、全人源抗体、抗体片段、双特异性抗体及多特异性抗体。
  3. 如权利要求1或2所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其特征在于：所述抗体为单克隆抗体，非限制性地选自：抗EGFRvIII抗体、抗PD-1抗体、抗PD-L1抗体、抗DLL-3抗体、抗PSMA抗体、抗CD70抗体、抗MUC16抗体、抗ENPP3抗体、抗TDGF1抗体、抗ETBR抗体、抗MSLN抗体、抗TIM-1抗体、抗LRRC15抗体、抗LIV-1抗体、抗CanAg/AFP抗体、抗claudin 18.2抗体、抗Mesothelin抗体、抗HER2(ErbB2)抗体、抗EGFR抗体、抗c-MET抗体、抗SLITRK6抗体、抗KIT/CD117抗体、抗STEAP1抗体、抗SLAMF7/CS1抗体、抗NaPi2B/SLC34A2抗体、抗GPNMB抗体、抗HER3(ErbB3)抗体、抗MUC1/CD227抗体、抗AXL抗体、抗CD166抗体、抗B7-H3(CD276)抗体、抗PTK7/CCK4抗体、抗PRLR抗体、抗EFNA4抗体、抗5T4抗体、抗NOTCH3抗体、抗Nectin 4抗体、抗Trop2抗体、抗CD142抗体、抗CA6抗体、抗GPR20抗体、抗CD174抗体、抗CD71抗体、抗EphA2抗体、抗LYPD3抗体、抗FGFR2抗体、抗FGFR3抗体、抗FRα抗体、抗CEACAMs抗体、抗GCC抗体、抗Integrin Av抗体、抗CAIX抗体、抗P-cadherin抗体、抗GD3抗体、抗 Cadherin 6抗体、抗LAMP1抗体、抗FLT3抗体、抗BCMA抗体、抗CD79b抗体、抗CD19抗体、抗CD33抗体、抗CD56抗体、抗CD74抗体、抗CD22抗体、抗CD30抗体、抗CD37抗体、抗CD47抗体、抗CD138抗体、抗CD352抗体、抗CD25抗体或抗CD123抗体。
  4. 权利要求1-3任一项的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，
    R4、R5各自独立地选自氢原子、C1-C4烷基，或者，R4、R5相连形成以下结构：-(CH2)2-B-(CH2)2-，B选自O、NH，与R4和R5键合的氮原子与-(CH2)2-B-(CH2)2-一起形成环。
  5. 权利要求1-4任一项的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，
    式D中的R6、R7、R8、R9各自为氢原子；或者，式D中R6、R7、R8、R9中的一个选自卤素、叠氮基、氨基，其余的三个各自为氢；或者，式D中R6、R7、R8、R9之中的任意两个基团与其所键合的原子一起形成环丙基，余下的两个基团各自独立地为氢原子。
  6. 权利要求1-5任一项的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，
    式D中的R10为苯基，任选地，所述苯基被一个或多个所述取代基所取代；优选地，所述取代基选自氨基、硝基；
    优选地，药物D选自以下化合物或其异构体、内消旋体、外消旋体、对映异构体或其混合物形式，或其药学上可接受的盐：
  7. 权利要求1-6任一项的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，所述配体‐药物偶联物具有式Ia所示的结构：
    其中：
    Z选自-C1-C10亚烷基-、-C3-C8碳环-、-亚芳基-、-C1-C10亚烷基-亚芳基-、-亚芳基-C1-C10-亚烷基-、-C1-C10亚烷基-(C3-C8碳环)-、-(C3-C8碳环)-C1-C10亚烷基-、-3-8元杂环-、-C1-C10亚烷基-(3-8元杂环)-、-(3-8元杂环)-C1-C10亚烷基-、-(CH2CH2O)r-、-(CH2CH2O)r-CH2-或其中X选自-C1-C10亚烷基-、-C3-C8碳环-、-亚芳基-、-C1-C10亚烷基-亚芳基-、-亚芳基-C1-C10-亚烷基-、-C1-C10亚烷基-(C3-C8碳环)-、-(C3-C8碳环)-C1-C10亚烷基-、-3-8元杂环-、-C1-C10亚烷基-(3-8元杂环)-、-(3-8元杂环)-C1-C10亚烷基-、-(CH2CH2O)r-、-(CH2CH2O)r-CH2；Y为亲水结构，选自羧基、磷酸、聚磷酸、亚磷酸、磺酸、亚磺酸或聚乙二醇(PEG)；所述杂环各自独立地包含选自N、O、S的1-3个原子；所述-C1-C10亚烷基-、-C3-C8碳环-、杂环各自独立地被一个或者多个取代基所取代，所述取代基选自氘原子、卤素、羟基、氰基、硝基、氨基、烷基、杂烷基、取代烷基、烷氧基、羧基或环烷基；中的左侧波浪线表示到马来酰亚胺上N的连接位点，右侧波浪线表示到羰基的连接位点；r选自1-10之间的整数；q选自1-8之间的整数；
    n1、n2、n3独立地选自0～20之间的整数或小数，n1、n2、n3不同时为0，且 n1+n2+n3≤20。
  8. 权利要求1-7任一项的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，
    A选自苯丙氨酸(F)、甘氨酸(G)、缬氨酸(V)、赖氨酸(K)、丙氨酸(A)、瓜氨酸、丝氨酸(S)、谷氨酸(E)或者天冬氨酸(D)中的2-7个氨基酸形成的多肽残基；
    优选地，A为由2-4个选自苯丙氨酸和甘氨酸的氨基酸形成的肽残基；
    优选地，A为甘氨酸-甘氨酸-苯丙氨酸-甘氨酸组成的四肽残基。
  9. 权利要求7或8的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，
    Z选自-C1-C10亚烷基-，例如-C4-C6亚烷基-，例如-C5亚烷基-；
    优选地，Z为其中，q选自1-8之间的整数。
  10. 权利要求7-9任一项的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，
    R1、R2和R3各自独立地选自氢原子、氘原子、烷基、卤代烷基、氘代烷基和羟烷基；
    优选地，R1、R2和R3同时为氢原子或氘原子；
    优选地，R1、R2和R3同时为氢原子。
  11. 权利要求1-10任一项的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，所述配体‐药物偶联物具有如式Ib所示的结构，
  12. 权利要求1-10任一项的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，所述配体‐药物偶联物具有如式Ic所示的结构，
    其中，Ac为具有式c所示结构的亲水结构单元：
    Ac通过氨基官能团与式Ie中已标示的2位亚甲基碳相连，X、Y如权利要求5所定 义；
    优选地，所述Ac选自甘氨酸、(D/L)丙氨酸、(D/L)亮氨酸、(D/L)异亮氨酸、(D/L)缬氨酸、(D/L)苯丙氨酸、(D/L)脯氨酸、(D/L)色氨酸、(D/L)丝氨酸、(D/L)酪氨酸、(D/L)半胱氨酸、(D/L)胱氨酸、(D/L)精氨酸、(D/L)组氨酸、(D/L)蛋氨酸、(D/L)天冬酰胺、(D/L)谷氨酰胺、(D/L)苏氨酸、(D/L)天冬氨酸、(D/L)谷氨酸、天然或非天然氨基酸衍生物或以下结构，
  13. 权利要求1-12任一项的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，所述配体‐药物偶联物选自以下结构：

















    其中，2位手性碳的构型为R型或S型。
  14. 权利要求1-13任一项的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，所述抗体为抗Trop2抗体；
    优选地，所述抗体由轻链和重链组成，所述轻链包含CDR-L1、CDR-L2和CDR-L3，其氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3所示；
    优选地，所述重链包含CDR-H1、CDR-H2、CDR-H3，其氨基酸序列分别如SEQ ID NO：4、SEQ ID NO：5、SEQ ID NO：6所示。
  15. 权利要求14的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，所述轻链包 含氨基酸序列为SEQ ID NO：7的轻链可变区；
    优选地，所述轻链还包含氨基酸序列为SEQ ID NO：8的轻链恒定区；
    优选地，所述轻链的氨基酸序列为SEQ ID NO：9。
  16. 权利要求14的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，所述轻链包含氨基酸序列为SEQ ID NO：10的轻链可变区；
    优选地，所述轻链还包含氨基酸序列为SEQ ID NO：11的轻链恒定区；
    优选地，所述轻链的氨基酸序列为SEQ ID NO：12。
  17. 权利要求15或16的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，所述重链包含氨基酸序列为SEQ ID NO：13的重链可变区；
    优选地，所述重链还包含氨基酸序列为SEQ ID NO：14的重链恒定区；
    优选地，所述重链的氨基酸序列为SEQ ID NO：15。
  18. 权利要求1-13任一项的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，所述抗体为抗Trop2抗体；所述抗体由轻链和重链组成，所述轻链包含CDR-L1、CDR-L2和CDR-L3，其氨基酸序列分别如SEQ ID NO：31、SEQ ID NO：32、SEQ ID NO：33所示；
    优选地，所述重链包含CDR-H1、CDR-H2、CDR-H3，其氨基酸序列分别如SEQ ID NO：34、SEQ ID NO：35、SEQ ID NO：36所示；
    优选地，所述轻链包含氨基酸序列为SEQ ID NO：37的轻链可变区；
    优选地，所述轻链还包含氨基酸序列为SEQ ID NO：38的轻链恒定区；
    优选地，所述轻链的氨基酸序列为SEQ ID NO：39；
    优选地，所述重链包含氨基酸序列为SEQ ID NO：40的重链可变区；
    优选地，所述重链还包含氨基酸序列为SEQ ID NO：41的重链恒定区；
    优选地，所述重链的氨基酸序列为SEQ ID NO：42。
  19. 如式II所示的连接子-药物化合物，或其异构体、内消旋体、外消旋体、对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化物，
    其中：
    Z、A、R1、R2、R3、R4、R5、R11、R12、R13、R14、B、R15、R16、R17、n、m、R6、R7、R8、R9、R18、R19、R10如权利要求1-10任一项所定义。
  20. 权利要求19的连接子-药物化合物，或其异构体、内消旋体、外消旋体、对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化物，
    所述连接子-药物化合物具有式IIa所示的结构，
  21. 权利要求20的连接子-药物化合物，或其异构体、内消旋体、外消旋体、对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化物，
    所述连接子-药物化合物具有式IIb所示的结构，
    其中，Ac为具有式c所示结构的亲水结构单元：
    其中，X、Y如权利要求5所定义，Ac通过-NH-与式IIb中已标示的2位亚甲基碳相连。
  22. 权利要求19-21任一项的连接子-药物化合物，或其异构体、内消旋体、外消旋 体、对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化物，所述连接子‐药物化合物选自以下结构：








    其中，2位手性碳的构型为R型或S型。
  23. 权利要求1-18任一项的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，或权利要求19-22任一项的连接子-药物化合物或其异构体、内消旋体、外消旋体、对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化物用于制备药物的用途，所述药物用于治疗或预防肿瘤；
    优选地，所述肿瘤选自实体瘤或非实体瘤，例如乳腺癌、卵巢癌、宫颈癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、肺癌、结肠癌、直肠癌、结直肠癌、骨癌、皮肤癌、甲状腺癌、胰腺癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、多形性胶质细胞瘤、肉瘤、淋巴瘤和白血病。
  24. 一种药物组合物，其包含有效量的权利要求1-18任一项的配体‐药物偶联物或其药学上可接受的盐或溶剂化物，或权利要求19-22任一项的连接子-药物化合物或其异构 体、内消旋体、外消旋体、对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化物，以及药学上可接受的载体、稀释剂或赋形剂。
  25. 权利要求19-22任一项的连接子-药物化合物或其异构体、内消旋体、外消旋体、对映异构体或其混合物形式，或其药学上可接受的盐或溶剂化物用于制备配体‐药物偶联物或其药学上可接受的盐或溶剂化物的用途；
    优选地，所述配体-药物偶联物选自权利要求1-18任一项的配体‐药物偶联物。
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