Specific topoisomerase inhibitors and their use in antibody-drug conjugates and methods for their preparation

Abstract

The present invention discloses specific topoisomerase inhibitors and can be used for antibody-drug conjugates and their preparation methods, belonging to the technical field of medicinal chemistry. The inhibitor is compound A or its tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof. The structure of compound A is Such a compound can further be prepared into an antibody-drug conjugate; the antibody-drug conjugate of the present invention has good solubility and drugability, and no precipitation occurs during the conjugation process; the ADC exhibits obvious in vivo antitumor activity and can show significantly stronger antitumor activity compared with the control sample.

Description

Technical Field

[0001] The present invention relates to a conjugate of a specific topoisomerase inhibitor and a drug linker for use in antibody-drug conjugates, and to a method for preparing the compound and related antibody-conjugated drugs and their use in the preparation of drugs for treating cancer, belonging to the technical field of medicinal chemistry. Background Art

[0002] The basic modules of antibody-drug conjugates include antibodies, linkers, and toxin molecules. Antibodies are used to transport toxin molecules to tumor sites for enrichment, thereby killing tumor cells. Traditional toxin molecules are mostly highly active tubulin inhibitors or direct DNA-targeting cytotoxic drugs, usually with large toxic side effects, which limits the application of ADCs.

[0003] Recently, Immunomedics has developed a novel ADC drug IMMU-132 with camptothecin compounds as warhead molecules, showing good anti-tumor effects. Daiichi Sankyo has developed another ADC drug DS-8201a with a camptothecin compound as a warhead molecule, also showing good anti-tumor effects.

[0004] In existing ADC technologies, camptothecin compounds are mainly linked to antibodies by modifying existing linker technologies. Generally speaking, an ideal linker in ADCs needs to meet the following requirements: First, it ensures that small molecule drugs do not detach from antibodies in plasma, and after entering cells, the linker breaks under appropriate conditions to rapidly release active small molecule drugs; second, the linker also needs to have good physicochemical properties to be able to form conjugates with antibodies; third, the linker should be easy to prepare to lay a foundation for the large-scale production of ADCs. IMMU-132 uses a pH-sensitive linker with poor stability. DS-8201a uses a structure containing a glycine-glycine-phenylalanine-glycine (GGFG) tetrapeptide, which has good stability. The toxins released by the above ADC drugs are SN38 and Dxd, both of which are Pgp substrates, and there are still problems such as tumor multi-drug resistance.

[0005] Therefore, there is still a need to further develop camptothecin derivatives and ADC drugs with better efficacy and / or safety. Summary of the Invention

[0006] To overcome the above technical deficiencies, the present invention provides an inhibitor compound or its tautomer, meso form, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, the compound comprising the structure shown in formula (A):

[0007]

[0008] Wherein: X is hydrogen or fluorine; Q is a linker capable of conjugating with an antibody, and L1 is a linker-drug amino group linking group.

[0009] Further, in the above technical solution, Q is a group capable of conjugating with the mercapto group on the antibody, and is selected from maleimide.

[0010] Further, in the above technical solution, L1 is a linker-drug amino group linking group, and is selected from L2 is an optionally substituted C3-C7 alkylene group, a C3-C8 cycloalkyl group, an optionally substituted diethylene glycol to octaethylene glycol acyl group, AA is a peptide segment composed of 2 to 4 amino acids, and M is a methylene group, a C1-C6 alkyl or cycloalkyl-substituted methylene group, a trifluoromethyl-substituted methylene group, or a C3-C6 cycloalkyl group.

[0011] Further, in the above technical solution, L1 is a linker-drug amino group linking group, and is selected from The AA polypeptide residue is selected from: NH -Phe-Lys- C＝O 、 NH -Val-Cit- C＝O 、 NH -Val-Ala- C＝O 、 NH -Phe-Cit- C＝O 、 NH -Gly-Val- C＝O 、 NH -Ala-Lys- C＝O 、 NH -Ala-Ala-Ala- C＝O 、 NH -Glu-Val-Ala- C＝O 、 NH -Glu-Val-Cit- C＝O 、 NH -Gly-Gly-Phe-Gly- C＝O 。

[0012] Further, in the above technical solution, L1 is a linker-drug amino group linking group, and is selected from L2 is an optionally substituted C3-C7 alkylene group, a C3-C8 cycloalkyl group, an optionally substituted diethylene glycol to octaethylene glycol acyl group.

[0013] Further, in the above technical solution, the specific molecular structure of formula A is as follows:

[0014]

[0015]

[0016]

[0017]

[0018]

[0019] The present invention also provides an antibody-drug conjugate or a tautomer, mesomer, racemate, enantiomer, diastereoisomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, and the compound comprises the structure shown in formula (B):

[0020]

[0021] Wherein: X is hydrogen, fluorine; Q is a linker capable of coupling with a mercapto group, L1 is a linker-drug amino group linking group, Ab is a ligand, and n = 1-8.

[0022] Further, in the above technical solution, the Q comprises a linker after coupling with a mercapto group, and is selected from

[0023] Further, in the above technical solution, the L1 is a linker-drug amino group linking group, and is selected from L2 is an optionally substituted C3-C7 alkylene group, a C3-C8 cycloalkyl group, an optionally substituted diethylene glycol to octaethylene glycol acyl group, AA is a peptide segment composed of 2 to 4 amino acids, and M is a methylene group, a C1-C6 alkyl group or a cycloalkyl-substituted methylene group, a trifluoromethyl-substituted methylene group, a C3-C6 cycloalkyl group.

[0024] Further, in the above technical solution, the L1 is a linker-drug amino group linking group, and is selected from The AA polypeptide residue is selected from: NH -Phe-Lys- C＝O 、 NH -Val-Cit- C＝O 、 NH -Val-Ala- C＝O 、 NH -Phe-Cit- C＝O 、 NH -Gly-Val- C＝O 、 NH -Ala-Lys- C＝O 、 NH -Ala-Ala-Ala- C＝O 、 NH -Glu-Val-Ala- C＝O 、 NH -Glu-Val-Cit- C＝O 、 NH-Gly-Gly-Phe-Gly- C＝O 。

[0025] Further, in the above technical solution, L1 is a linker and a drug amino linking group, selected from L2 is an optionally substituted C3-C7 alkylene group, a C3-C8 cycloalkyl group, or an optionally substituted diethylene glycol to octaethylene glycol acyl group.

[0026] Further, in the above technical solution, the specific structure of Formula B is as follows:

[0027]

[0028]

[0029]

[0030]

[0031] Wherein: Ab is a ligand, and n = 1-8.

[0032] Further, in the above technical solution, the Ab is selected from a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0033] Further, in the above technical solution, the antibody includes a monoclonal antibody.

[0034] Further, in the above technical solution, the antibody includes a bispecific antibody.

[0035] Further, in the above technical solution, the antibody can bind to tumor-associated antigens such as HER2, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD79b, CD138, CD147, CD223, EpCAM, Mucin1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor, c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C, Mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, ROR1, PD-L1, CD27L, 5T4, Mucin 16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3 or Claudin 18.2.

[0036] The present invention also provides a pharmaceutical composition, comprising: (a) the above antibody-drug conjugate; and (b) a pharmaceutically acceptable diluent, carrier or excipient.

[0037] The present invention also provides the use of the above antibody-drug conjugate in the preparation of a drug for treating tumors.

[0038] The present invention also provides a method for preparing the antibody-drug conjugate, comprising the following steps:

[0039] a. React the antibody with a reducing agent in a buffer to obtain a reduced antibody;

[0040] b. Crosslink the linker-drug conjugate (A) with the reduced antibody obtained in step a in a mixture of a buffer and an organic solvent to obtain the antibody-drug conjugate. Detailed implementation manners

[0041] The following specific embodiments illustrate the implementation manners of the invention of the present application. Those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

[0042] Term definitions

[0043] In the present invention, the term "pharmaceutically acceptable" component refers to a substance that is suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, a substance with a reasonable benefit / risk ratio.

[0044] In the present invention, the term "effective amount" refers to the amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or preventive effect. The precise effective amount for a particular subject depends on the subject's size and health, the nature and severity of the disorder, and the therapeutic agent and / or combination of therapeutic agents selected for administration. Accordingly, it is not useful to specify an exact effective amount in advance. However, for a given condition, the effective amount can be determined by routine experimentation and can be judged by a clinician.

[0045] Unless otherwise specified, in the present invention, all occurrences of a compound are intended to include all possible optical isomers, such as compounds of a single chirality, or mixtures of various different chiral compounds (i.e., racemates). Among all compounds of the present invention, each chiral carbon atom can optionally be of the R configuration or the S configuration, or a mixture of the R and S configurations.

[0046] As used herein, the term "compounds of the present invention" refers to compounds of formula I. The term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates, or solvates of the compounds of formula I.

[0047] As used herein, the term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention formed with acids or bases that are suitable for use as pharmaceuticals. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts are salts of the compounds of the present invention formed with acids. Acids suitable for forming salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid; organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid; and acidic amino acids such as aspartic acid, glutamic acid.

[0048] Unless otherwise specified, the "amino acids" used herein are intended to include any conventional amino acids, such as aspartic acid, glutamic acid, cysteine, asparagine, phenylalanine, glutamine, tyrosine, serine, methionine, tryptophan, glycine, valine, leucine, alanine, isoleucine, proline, threonine, histidine, lysine, arginine.

[0049] When a trade name is used herein, the trade name is intended to include the trade name product formulation, its corresponding generic drug, and the active pharmaceutical ingredient of the trade name product.

[0050] The term "antibody" as used herein is used in its broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided that they exhibit the desired biological activity (Miller et al. (2003) Journal of Immunology 170:4854 - 4861). Antibodies can be murine, human, humanized, chimeric, or from other species. An antibody is a protein produced by the immune system that is capable of recognizing and binding a specific antigen (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) ImmunoBiology, 5th Ed., Garland Publishing, New York). A target antigen generally has a large number of binding sites, also called epitopes, recognized by the CDRs of multiple antibodies. Each antibody that specifically binds a different epitope has a different structure. Thus, an antigen can have more than one corresponding antibody. Antibodies include full - length immunoglobulin molecules or the immunologically active portions of full - length immunoglobulin molecules, i.e., molecules that contain an antigen or a portion thereof that specifically binds a target of interest, such targets including, but not limited to, cancer cells or cells that produce autoantibodies associated with autoimmune diseases. The immunoglobulins disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. Immunoglobulins can be from any species. However, in one aspect, the immunoglobulins are from human, murine, or rabbit.

[0051] "Antibody fragment" includes a portion of a full - length antibody, generally its antigen - binding region or variable region. Examples of antibody fragments include: Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; linear antibodies; minibodies (Olafsen et al. (2004) Protein Eng. Design & Sel. 17(4):315 - 323); fragments prepared from Fab expression libraries; anti - idiotypic (anti - Id) antibodies; CDRs (complementary determining regions); and any of the above epitope - binding fragments that bind a cancer cell antigen, a viral antigen, or a microbial antigen in an immunospecific manner; single - chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0052] In the present invention, the antibody constituting the antibody-drug conjugate preferably retains its antigen-binding ability in its original wild state. Thus, the antibody in the present invention can, preferably specifically, bind to an antigen. The antigens involved include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, molecules related to tissue growth and differentiation (such as those known or predicted to have functionality), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules related to angiogenesis (such as the antigens known to be bound by antibodies can be one or a subset of the above classifications, while other subsets contain other molecules / antigens with special properties (compared to the target antigen).

[0053] Antibodies applied in antibody-drug conjugates include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well-known in the art and can be prepared by methods and information well-known in the art for antibody preparation. To develop effective cellular-level targets for cancer diagnosis and treatment, researchers strive to find transmembrane or other tumor-associated polypeptides. These targets can be specifically expressed on the surface of one or more cancer cells and are rarely or not expressed on the surface of one or more non-cancer cells. Generally, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells relative to the surface of non-cancer cells. Identifying such tumor-associated factors can greatly improve the specific targeting characteristics of antibody-based cancer treatment.

[0054] In the present application, the term "ligand" generally refers to a macromolecular compound that can recognize and bind to an antigen or receptor associated with a target cell. The function of the ligand can be to deliver a drug to a population of target cells that bind to the ligand, and these ligands include, but are not limited to, protein hormones, lectins, growth factors, antibodies, or other molecules that can bind to cells, receptors, and / or antigens. In the present application, the ligand can be represented as Ab, and the ligand antigen forms a linkage bond with the linking unit through a heteroatom on the ligand. It can be an antibody or an antigen-binding fragment thereof. The antibody can be selected from chimeric antibodies, humanized antibodies, fully human antibodies, or murine antibodies; the antibody can be a monoclonal antibody. For example, the antibody can be an antibody targeting the following targets: HER2, HER3, B7H, TROP2, Claudin 18.2, CD30, CD33, CD70, EGFR, 5T4, AGS-16, ANGPTL4, ApoE, CD19, CTGF, CXCR5, FGF2, MCPT8, MF12, MS4A7, NCA, Sema5b, SLITRK6, STC2, TGF, 0772P, ST4, ACTA2, ADGRE1, AG-7, AIF1, AKRIC1, AKR1C2, ASLG659, Axl, B7H3, BAFF-R, BCMA, BMPRIB, BNIP3, C1QA, C1QB, CA6, CADM1, CCD79b, CCL5, CCR5, CCR7, CD11c, CD123, CD138, CD142, CD147, CD166, CD19, CD22, CD21, CD20, CD205, CD22, CD223, CD228, CD25, CD30, CD33, CD37, CD38, CD40, CD45, CD45(PTPRC), CD46, CD47, CD49D(ITGA4), CD56, CD66e, CD70, CD71, CD72, CD74, CD79a, CD79b, CDS0, CDCP1, CDH11, CD11b, CEA, CEACAMS, c-Met, COL6A3, COL7A1, CRIPTO, CSF1R, CTSD, CTSS, CXCL11, CXCL10, DDIT4, DLL3, DLL4, DR5, E16, EFNA4, EGFR, EGFRVIII, EGLN, EGLN3, EMR2, ENPP3, EpCAM, EphA2, EphB2R, ETBR, FcRH2, FcRHI, FGFR2, FGFR3, FLT3, FOLR-ALPHA, GD2, GEDA, GPC-1, GPNMB, GPR20, GZMB, HER2, HER3, HLA-DOB, HMOX1, IFI6, IFNG, IGF-1R, IGFBP3,IL-13R, IL-2, IL20Ra, IL-3, IL-4, IL-6, IRTA2, KISS1R, KRT33A, LIV-1, LOX, LRP-1, LRRC15, LUM, LY64, LY6E, Ly86, LYPD3, MDP, MMP10, MMP14, MMP16, MPF, MSG783, MSLN, MUC-1, NaPi2b, Napi3b, Nectin-4, NOG, P2X5, pAD, P-Cadherin, PDGFRA, PDK1, PD-LI, PFKFB3, PGF, PGK1, PIK3AP1, PIK3CD, PLOD2, PSCA, PSCAh1g, PSMA, PTK7, P-cadherin, RNF43, NaPi2b, ROR1, ROR2, SERPINE1, SLC39A6, SLTRK6, STAT1, STEAP1, STEAP2, TCF4, TENB2, TGFB1, TGFB2, TGFBR1, TNFRSF21, TNFSF9, Trop-2, TrpM4, Tyro7, UPK1B, VEGFA, WNTSA, epidermal growth factor, short protein glycans, mesothelin, sodium phosphate cotransporter 2B, claudin 18.2, endodermal receptor, mucins (such as mucin 1 and mucin 16), guanylate cyclase C, integrin a4p7, integrin a5p6, trophoblast glycoprotein or tissue factor.,

[0055] Enzyme-labile linkers, such as peptide linkers, can better control drug release. Peptide linkers can be effectively cleaved by lysosomal proteases, such as cathepsin B or plasmin (the content of such enzymes increases in some tumor tissues). This peptide linkage is considered to be very stable in plasma circulation because the inappropriate extracellular pH value and serum protease inhibitors render proteases usually inactive. Given the high plasma stability and good intracellular cleavage selectivity and effectiveness, enzyme-labile linkers are widely used as cleavable linkers for antibody-drug conjugates. Typical enzyme-labile linkers include Val-Cit (VC), Phe-Lys, etc.

[0056] Self-immolative linkers are generally embedded between the cleavable linker and the active drug, or are themselves part of the cleavable linker. The mechanism of action of self-immolative linkers is that when the cleavable linker breaks under appropriate conditions, the self-immolative linker can spontaneously rearrange its structure, thereby releasing the active drug connected to it. Common self-immolative linkers include p-aminobenzyl alcohols (PAB) and β-glucuronides (β-Glucuronide), etc.

[0057] Preparation Method of Antibody-Drug Conjugate

[0058] The inter-chain disulfide bonds of the antibody are reduced to produce a total of 8 mercapto groups. The substituted maleimide linker drug conjugate crosslinks with the reduced antibody mercapto groups to generate the corresponding antibody-drug conjugate.

[0059] Dilute the antibody stock solution with reaction buffer to 2 - 10 mg / mL, add 6.0 - 20-fold excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), or add 140 - 200-fold excess molar ratio of dithiothreitol (DTT). Stir the reaction solution at 25 °C for 2 - 4 hours; herein, the reaction buffer is 50 mM potassium dihydrogen phosphate-sodium hydroxide (KH2PO4-NaOH) / 150 mM sodium chloride (NaCl) / 1 mM diethylenetriaminepentaacetic acid (DTPA), pH = 6 - 9; 50 mM disodium hydrogen phosphate-citric acid / 150 mM sodium chloride (NaCl) / 1 mM diethylenetriaminepentaacetic acid (DTPA), pH = 6 - 9; 50 mM boric acid-borax / 150 mM sodium chloride (NaCl) / 1 mM diethylenetriaminepentaacetic acid (DTPA), pH = 6 - 9; 50 mM histidine-sodium hydroxide / 150 mM sodium chloride (NaCl) / 1 mM diethylenetriaminepentaacetic acid (DTPA), pH 6 - 9 and PBS / / 1 mM diethylenetriaminepentaacetic acid (DTPA), pH = 6 - 9.

[0060] Cool the above reaction solution to 0 - 10 °C. If TCEP is used for reduction, the maleimide compound (previously dissolved at 10 mg / mL in dimethyl sulfoxide (DMSO), dimethylformamide (DMF) or diethylacetamide (DMA)) can be directly added without purification, and ensure that the volume ratio of the organic solvent in the reaction solution does not exceed 15%. The coupling reaction is stirred at 10 - 25 °C for 2 hours. If DTT is used for reduction, after the reduction reaction is completed, excess DTT needs to be removed by passing through a desalting column or ultrafiltration, and then the substituted maleimide compound is added for coupling.

[0061] Use a desalting column to purify the coupling reaction mixture by gel filtration with sodium succinate / 150 mM NaCl buffer or histidine-acetate / sucrose, and collect the eluted samples according to the UV280 absorbance value. Or ultrafilter several times. Then sterilize through a filtration device with a pore size of 0.22 μm and store at -80 °C.

[0062] The drug-antibody conjugate ratio (DAR8) of the obtained antibody-drug conjugate is relatively uniform. Using the drug linker compounds described in this patent for antibody-drug conjugates with certain differences in product uniformity, if a sample with better uniformity is needed, it can be further separated and purified by, but not limited to, the following methods: hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC), and ion exchange chromatography (IEC).

[0063] Drug Compositions and Methods of Administration

[0064] Because the antibody-drug conjugates provided by the present invention can target specific cell populations, bind to specific proteins (antigens) on the cell surface, and thus release the drug in an active form into the cell through conjugate endocytosis or drug infiltration, the antibody-drug conjugates of the present invention can be used to treat target diseases. The above-mentioned antibody-drug conjugates can be administered to a subject (such as a human) in a therapeutically effective amount by a suitable route. A subject in need of treatment can be a patient at risk or suspected of having a disorder related to the activity or expression level of a specific antigen. Such patients can be identified by routine medical examinations.

[0065] Conventional methods, known to those of ordinary skill in the medical art, can be used to administer the pharmaceutical composition to the subject, depending on the type of disease to be treated or the location of the disease. This composition can also be administered by other conventional routes, for example, orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or by implantation. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In addition, it can be administered by the depot injectable route, for example, using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.

[0066] Injectable compositions can contain various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies can be administered by drip method, whereby a pharmaceutical preparation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients can include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular preparations, for example, sterile preparations of a suitable soluble salt form of the antibody, can be dissolved and administered in a pharmaceutical excipient such as water for injection, 0.9% saline, or 5% dextrose solution.

[0067] When treating with the antibody-drug conjugates of the present invention, delivery can be carried out by conventional methods in the art. For example, it can be introduced into cells by using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the nucleic acid or vector can be delivered locally by direct injection or by using an infusion pump. Other methods include the use of various transport and carrier systems by using conjugates and biodegradable polymers.

[0068] The pharmaceutical composition of the present invention contains a safe and effective amount of the antibody-drug conjugate of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should be matched with the mode of administration. The pharmaceutical composition of the present invention can be made into a solution form, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. The said pharmaceutical composition should preferably be manufactured under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount.

[0069] The effective amount of the antibody-drug conjugate described in the present invention may vary with the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by those of ordinary skill in the art according to various factors (such as through clinical trials). The said factors include but are not limited to: the pharmacokinetic parameters of the bifunctional antibody conjugate such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the patient's body weight, the patient's immune status, the route of administration, etc. Generally, when the antibody-drug conjugate of the present invention is administered at a dose of about 0.0001 mg - 50 mg / kg of animal body weight per day (preferably 0.001 mg - 10 mg / kg of animal body weight), satisfactory effects can be obtained. For example, due to the urgency of the treatment situation, several separate doses may be administered per day, or the dose may be proportionally reduced.

[0070] The dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed with a physiologically acceptable carrier and any preservatives, buffer solutions, or propellants that may be required under aseptic conditions.

[0071] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable therapeutic agents.

[0072] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically recognized effective dosage. For a person with a body weight of 60 kg, the daily dosage is usually 1 - 2000 mg, preferably 5 - 500 mg. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health status, which are all within the scope of the skills of a skilled physician.

[0073] The present invention will be further illustrated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0074] Example 1: Synthesis and Preparation of Compounds

[0075] 1. Synthesis of Compound A1

[0076]

[0077]

[0078] First Step

[0079] Add glycylglycine A1-1 (2.0 g, 15.2 mmol), fluorenylmethoxycarbonyl chloride (4.7 g, 18.2 mmol), dioxane (20 mL), cool down to 0 - 5 °C, and dropwise add 1N sodium carbonate aqueous solution (18 mL). After dropping, restore to room temperature and stir the reaction for about 2 h. Cool down to 0 - 5 °C, adjust the pH = 2 with about 30 mL of 1M HCl, extract the aqueous phase with ethyl acetate (100 mL × 3), combine the organic phases, concentrate to dryness under reduced pressure to obtain 6.0 g of off-white solid. Add 19 mL of methyl tert-butyl ether, stir at room temperature for 20 min, filter, and concentrate the filter cake to dryness under reduced pressure to obtain A1-2 (4.3 g, yield: 81.3%). MS(ESI)(m / z): 355([M + H] + )

[0080] Second Step

[0081] Add tetrahydrofuran (450 mL), A1-2 (29.0 g, 81.9 mmol), acetic acid (90 mL), protect by nitrogen replacement, heat up to 40 °C, add lead tetraacetate (60.0 g, 135.4 mmol), heat up to reflux and keep the reaction for 16 h. Cool down to room temperature, filter through a diatomaceous earth cake, wash the filter cake with ethyl acetate, concentrate the filtrate to dryness under reduced pressure to obtain a crude oil product, and then purify by column chromatography to obtain A1-3 (25.0 g, yield: 82.8%). MS(ESI)(m / z): 391([M + Na] + )

[0082] Third Step

[0083] Add dichloromethane (112 mL), A1-3 (9.8 g, 26.6 mmol), A1-3X (17.6 g, 106.0 mmol), PPTS (1.3 g, 5.2 mmol), protect by nitrogen replacement, heat up to 45 °C and reflux for 2 h. Concentrate the reaction solution under reduced pressure and purify by column chromatography to obtain A1-4 (5.4 g, yield: 43.2%), MS(ESI)(m / z): 497([M + Na] + )

[0084] Fourth Step

[0085] Add A1-4 (2.8 g, 5.9 mmol), add N,N-dimethylformamide (14 mL), add DBU (0.9 g, 5.9 mmol). After purging with nitrogen for protection, stir at room temperature for 3 - 4 h. After the raw materials react completely, obtain the crude product A1-5, without further treatment, and directly proceed to the next reaction.

[0086] The fifth step

[0087] Add glacial acetic acid (39 mL), maleic anhydride (3.5 g, 36 mmol), 6-aminohexanoic acid (3.9 g, 30 mmol). Heat up to 120 °C and keep stirring for 4 h. After the reaction is complete, cool down. Concentrate the reaction solution under reduced pressure to dryness. Add water to the concentrate and extract with ethyl acetate. Wash the organic phase successively with water and saturated brine, and concentrate the organic phase under reduced pressure to dryness. Add 50 mL of water, stir at room temperature, filter, and dry under reduced pressure at 50 °C to obtain A1-8A (4.5 g, yield: 71.0%).

[0088] The sixth step

[0089] Add A1-8A (4.5 g, 21.3 mmol), acetonitrile (45 mL), N-hydroxysuccinimide (2.7 g, 23.4 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.9 g, 25.6 mmol), and stir at room temperature for reaction. Concentrate the system under reduced pressure to dryness, add 50 mL of saturated sodium bicarbonate aqueous solution, extract with dichloromethane, dry the organic phase, concentrate under reduced pressure, purify the crude product by column chromatography, and collect A1-8X (5.0 g, yield: 79.8%). MS(ESI)(m / z): 315.3([M+Na]+).

[0090] The seventh step

[0091] Add L-phenylalanine (5.2 g, 31.5 mmol), acetonitrile (56 mL), purified water (56 mL), triethylamine (3.7 g, 36.5 mmol), A1-2 (15.8 g, 44.6 mmol), and stir at room temperature for 16 h until the raw materials react completely. Concentrate under reduced pressure and purify by column chromatography to obtain the target product A1-6 (12.0 g, yield: 76.9%). MS(ESI)(m / z): 502([M+H] + )

[0092] The eighth step:

[0093] Add A1-6 (3.0 g, 6.0 mmol), N,N-dimethylformamide (15 mL), N,N-diisopropylethylamine (1.1 g), cool down to -5 to 0 °C under nitrogen protection, add 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.62 g, 6.9 mmol), and keep the temperature for reaction for 0.5 to 1 h. Add the crude product of A1-5 reserved in the fourth step. Restore to room temperature and stir the reaction until the raw materials react completely. Add 100 mL of water to quench the reaction, extract with ethyl acetate, combine the organic phases, wash with dilute acid water with pH = 3 - 5 and saturated brine, dry the organic phase, concentrate under reduced pressure to obtain the crude product, and purify it by column chromatography to obtain the target product A1-7 (1.6 g, two-step yield: 37.5%). MS(ESI)(m / z): 758([M+Na] + )。

[0094] Step 9:

[0095] Add A1-7 (1.6 g, 2.2 mmol), N,N-dimethylformamide (11 mL), DBU (0.3 g, 2 mmol), displace with nitrogen and stir the reaction at room temperature for 1.5 h. Take a sample to check if the raw materials react completely. Directly add 10% Pd / C (0.2 g), displace with hydrogen at atmospheric pressure, stir the reaction at room temperature for 2 h, filter through diatomaceous earth, add 20 mL of process purified water, add 10 mL of dichloromethane, stir, separate the layers, and concentrate the obtained aqueous phase to dryness under reduced pressure to obtain the target product A1-8 (0.9 g, yield: 98.0%).

[0096] Step 10

[0097] Add A1-8 (0.9 g, 2 mmol), acetonitrile (6.3 mL), water (6.3 mL), triethylamine (0.2 g, 2 mmol), A1-8X (0.5 g, 1.7 mmol), displace with nitrogen and stir at room temperature for 2 h. Add 20 mL of water, wash with 20 mL × 2 of ethyl acetate, adjust the pH of the aqueous phase to 4 - 5 with acetic acid, and concentrate the aqueous phase to dryness under reduced pressure to obtain A1-9 (1.0 g, yield 95.2%). MS(ESI)(m / z): 639([M+Na] + )。

[0098] Step 11

[0099] Add A1-9 (1.0 g, 1.6 mmol), dichloromethane (16 mL), 4-dimethylaminopyridine (0.26 g), and add A1-9X (0.68 g, 1.57 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.4 g) under stirring at room temperature, and stir at room temperature for 16 h. After the raw materials have reacted completely, the reaction solution is washed successively with 10% aqueous citric acid solution, washed with water, washed with saturated aqueous sodium carbonate solution, and washed with saturated brine. The organic phase is dried and concentrated under reduced pressure to dryness. The crude product is purified by column chromatography to obtain the target product A1 (1.2 g, yield: 73.0%). MS(ESI)(m / z): 1043([M+Na] + )。

[0100] 2. Synthesis of Compound A1 Fragment A1-9X

[0101]

[0102] The first step

[0103] Add A1-9X-1X (642 g, 4200 mmol), triphenylphosphine (1155 g, 4410 mmol), and acetonitrile (6 L), heat up to reflux and keep the reaction for 24 h. Cool down to room temperature, concentrate the reaction solution to dryness under reduced pressure, add 6 L of methyl tert-butyl ether, filter, wash the filter cake with methyl tert-butyl ether, and dry the filter cake under reduced pressure to obtain A1-9X-2X (1500 g, yield: 86.0%).

[0104] The second step

[0105] Add tetrahydrofuran (2.4 L), A1-9X-1 (426 g, 3000 mmol), and A1-9X-2X (1500 g, 3600 mmol), cool down to -5 °C under nitrogen protection, and then dropwise add sodium tert-butoxide (840 g, 7500 mmol) / tetrahydrofuran (2.4 L) solution, and keep the reaction for 5 - 8 h. Pour the system into 7.5 L of ice water, concentrate most of the THF under reduced pressure at 40 - 45 °C, and then extract the by-products with 5.0 L of MTBE in three portions. Adjust the pH of the aqueous phase to 4 - 5 with 6.0 N hydrochloric acid, and then extract the product with 4.0 L of EA in three portions. Combine the organic layers, wash with water, brine, dry over anhydrous sodium sulfate, filter, and concentrate to dryness to obtain A1-9X-2 (594.0 g, yield 100%).

[0106] The third step

[0107] A1-9X-2 (594.0, 3000 mmol) was added and dissolved in ethyl acetate (2500 mL). Then 10% Pd / C (50% water content, 75 g) was added. The system was purged with nitrogen and hydrogen respectively, and hydrogenation reaction was carried out at normal pressure at 25 - 35 °C for 16 h. The mixture was filtered through diatomite, and the filtrate was concentrated under reduced pressure to dryness to obtain A1-9X-3 (600.0 g, yield 100%).

[0108] The fourth step

[0109] A1-9X-3 (600.0 g, 3000 mmol) was added to concentrated sulfuric acid (3000 g). During the addition, the temperature was controlled below 35 °C. After the addition, the mixture was stirred at room temperature for 2 - 3 h. The reaction solution was slowly poured into 6.5 kg of ice water. Methyl tert-butyl ether (3.5 L) and ethyl acetate (0.5 L) were added for extraction and liquid separation. The combined organic layers were washed successively with saturated sodium bicarbonate and brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography to obtain A1-9X-4 (230.0 g, yield: 42.1%). 1 1H-NMR (400 MHz, CDCl3): δ 6.79 - 6.69 (m, 2H), 2.96 (t, J = 6.0 Hz, 2H), 2.64 (t, J = 6.0 Hz, 2H), 2.11 (m, 2H).

[0110] The fifth step

[0111] A1-9X-4 (125 g, 686.8 mmol) was added to concentrated sulfuric acid (1200 g). During the addition, the temperature was controlled below 5 °C. Then sodium nitrate (70 g, 823.5 mmol) was added portionwise. After the addition, the mixture was stirred at room temperature for 2 - 3 h. The reaction solution was slowly poured into 6.5 kg of ice water. The mixture was extracted with a mixed solution of 1.5 L of methyl tert-butyl ether and 150 mL of ethyl acetate. The combined organic layers were washed successively with saturated sodium bicarbonate and brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by column chromatography to obtain A1-9X-5 (71.0 g, yield: 45.5%).

[0112] The sixth step

[0113] Ethanol (2.1 L), water (0.3 L), ammonium chloride (70 g, 1308 mmol), and A1-9X-5 (100 g, 440 mmol) were added and stirred until dissolved. Then iron powder (200 g, 3571 mmol) was added slowly. The temperature was raised to 80 °C and the reaction was carried out for 1 - 2 h. After cooling, the mixture was filtered. Most of the ethanol in the filtrate was removed under reduced pressure. Then water and 2.6 L of dichloromethane were added for extraction. The combined organic layers were washed successively with saturated sodium bicarbonate and brine, dried over anhydrous sodium sulfate to obtain the extraction solution of A1-9X-6, which was directly used for the next step.

[0114] The seventh step

[0115] Pyridine (150 g, 1900 mmol) and 4-dimethylaminopyridine (1.0 g) were added to the A1-9X-6 extract, and acetic anhydride (99 g, 968 mmol) was slowly added dropwise while controlling the temperature below 15 °C. The reaction was carried out at room temperature for 2 - 3 h. The reaction solution was slowly poured into 1.5 kg of ice water. The organic layer was washed with 1.0 N dilute hydrochloric acid until acidic, and then washed with saturated sodium bicarbonate and brine respectively. The organic phase was concentrated to dryness under reduced pressure. 1.0 L of methanol was added to the concentrate to dissolve it, and the pH was adjusted to 11 - 12 with 25.0% NaOH. Hydrolysis was carried out at 35 - 45 °C for 1 - 3 hours, and the organic solvent was concentrated off under reduced pressure. Dichloromethane and water were added for extraction and layering. The organic layer was concentrated to dryness under reduced pressure. The concentrate was slurried with methyl tert-butyl ether, filtered, and the filter cake was dried to obtain A1-9X-7 (55.0 g, yield: 52.3%). 1 1H-NMR (400 MHz, CDCl3): 6.83 (m, 1H), 2.86 (t, J = 6.0 Hz, 2H), 2.64 (t, J = 6.0 Hz, 2H), 2.25 (s, 3H), 2.06 (m, 2H).

[0116] The eighth step

[0117] A1-9X-7 (9.0 g, 37.6 mmol) and dimethyl sulfoxide (150 mL) were added. 25% ammonia water (250 g) was slowly added with stirring. The reaction solution was transferred to an autoclave and heated to 65 - 75 °C for reaction for 16 h. After cooling, an appropriate amount of water was added, and it was extracted with ethyl acetate multiple times. The organic layers were combined, washed successively with water and saturated brine, dried, and concentrated to dryness under reduced pressure. The crude product was purified by column chromatography to obtain A1-9X-8 (4.5 g, yield: 50.6%). 1 1H-NMR (400 MHz, DMSO-d6): 9.09 (s, 1H), 6.43 (d, J = 12.4 Hz, 1H), 2.66 (t, J = 6.0 Hz, 2H), 2.50 (m, 2H), 2.00 (s, 3H), 1.87 (t, J = 6.0 Hz, 2H). LC-MS: (m / z): 237.1 [M + H] + 。

[0118] The ninth step

[0119] A1-9X-8 (4 g, 17 mmol), A1-10X-8B (4.9 g, 18.6 mmol), PPTS (4.8 g, 17 mmol), and toluene (800 mL) were added. Under nitrogen protection, the temperature was raised to 120 °C and the reaction was kept for 8 h. After cooling, the reaction solution was concentrated under reduced pressure. The concentrate was slurried with 50 mL of acetone, cooled to 0 - 5 °C, filtered by suction, and the filter cake was dried under reduced pressure to obtain A1-9X-9 (6.5 g, yield: 82.5%). LC-MS: (m / z): 464.2 [M + H]+ .

[0120] The tenth step

[0121] Add A1-9X-9 (5.0 g, 10.8 mmol), concentrated hydrochloric acid (25 mL), and water (25 mL). Heat up to 80 °C and hold the reaction for 1 h. Concentrate the reaction solution to dryness under reduced pressure, add 30 mL of acetone for pulping, and filter to obtain A1-9X (3.1 g, yield: 68.1%). LC-MS: (m / z): 422.1 [M+H] + .

[0122] 3. Synthesis of Compounds A2 - A6

[0123] Referring to the synthesis of Compound A1, replace A1-3X with the following respectively:

[0124]

[0125] 4. Synthesis of Compounds A7 and A8

[0126] Referring to the synthesis of Compounds A1 and A5, replace A1-9X with A1-10X:

[0127]

[0128] 5. Synthesis of Fragment A1-10X of Compounds A7 and A8

[0129]

[0130] The first step

[0131] Add dichloromethane (6 L), A1-10X-1 (914 g, 6220 mmol), and triethylamine (754 g, 7464 mmol). Cool down to 0 °C. Dropwise add acetic anhydride (698 g, 6842 mmol), and finish dropping in about 1 h. Continue the reaction for 30 min. Add 1 L of water to the system, let it stand for liquid separation. Extract the aqueous layer with 800 mL of dichloromethane once, and combine the organic phases. Adjust the pH of the organic phase to about 2 with 1 N hydrochloric acid, separate out the organic phase, extract the aqueous layer with dichloromethane once, and combine the organic phases. Wash the organic phase with 1 L of saturated sodium bicarbonate solution, and concentrate the filtrate to dryness under reduced pressure to obtain A1-10X-2 (1150 g, yield: 97.8%).

[0132] The second step

[0133] Add sulfuric acid (2 L) and A1-10X-2 (300 g, 1580 mmol). Cool down to 0 °C and add sodium nitrate (134.3 g, 1580 mmol) in portions within 2 h. React for 30 min. Slowly pour the reaction solution into 8 L of ice water under stirring, and perform suction filtration. Wash the filter cake twice with ice water. Dissolve the filter cake in 1 L of dichloromethane, wash with saturated sodium bicarbonate solution until alkaline, dry the organic phase, and concentrate it to dryness under reduced pressure. Add 400 mL of methyl tert-butyl ether for pulping, cool down to 0 - 5 °C, perform suction filtration, and dry the filter cake under reduced pressure to obtain A1-10X-3 (264 g, yield: 71.3%).

[0134] The third step

[0135] Add acetone (5 L) and A1-10X-3 (160 g, 680 mmol). Then dissolve magnesium sulfate (111.6 g, 930 mmol) in water (620 mL) and add it to the system. Cool down to 0 °C and add potassium permanganate (323 g, 2050 mmol) in portions within 3 h. After addition, let it warm up naturally. React for 1.5 h. Dropwise add the prepared 40% sodium thiosulfate solution (2 L) to the reaction system until no oxidizing property is detected. Perform suction filtration, concentrate the filtrate, add 6 L of dichloromethane, wash the organic phase with water, saturated sodium bicarbonate solution, and saturated brine, dry it, and concentrate it to dryness under reduced pressure to obtain the crude product. Purify it by column chromatography to obtain A1-10X-4 (100 g, yield: 59.2%).

[0136] The fourth step

[0137] Add A1-10X-4 (80 g, 320 mmol) and concentrated hydrochloric acid (500 mL), heat to 90 - 100 °C and keep the reaction for 2 h. Cool down to 0 - 10 °C, pour the reaction solution into 2 L of ice water, filter, and wash the filter cake with ice water to obtain A1-10X-5 (66 g, yield: 100%).

[0138] The fifth step

[0139] Add A1-10X-5 (66 g, 320 mmol) and dichloromethane (1.5 L), stir to dissolve, add pyridine (52 mL, 650 mmol), cool down to 0 °C, and dropwise add trifluoroacetic anhydride (94 mL, 676 mmol) within 40 min. After dropping, continue the reaction for 20 min. Concentrate the reaction solution to dryness under reduced pressure, then add 1 L of dichloromethane to dissolve, wash with 1 N hydrochloric acid until there is no pyridine, then wash with saturated sodium bicarbonate solution until alkaline, and wash with saturated brine. Dry the organic phase and concentrate it to dryness under reduced pressure to obtain A1-10X-6 (62 g, yield: 64.1%).

[0140] The sixth step

[0141] Add methanol (1 L), formic acid (50 mL), and water (50 mL). Dissolve A1-10X-6 (54 g, 180 mmol) in dichloromethane (100 mL) and add it to the system. Cool the temperature to 0 °C, and add zinc powder (150 g, 2290 mmol) in portions within 50 min. Continue the reaction for 1.5 h. Perform suction filtration, separate the liquid in the filtrate, wash the organic phase with saturated sodium bicarbonate solution and saturated brine, dry the organic phase, and concentrate it under reduced pressure to obtain A1-10X-7 (42 g, yield: 85.7%).

[0142] The seventh step

[0143] Add dichloromethane (1.6 L), A1-10X-7 (40 g, 147.0 mmol), and triethylamine (49 mL, 352.5 mmol). Cool the temperature to 0 - 5 °C, dropwise add acetyl chloride (27 mL, 380.0 mmol), continue the reaction for 50 min, and add 600 mL of water. Perform suction filtration, and wash the filter cake with water to obtain A1-10X-8 (46.2 g, yield: 100%).

[0144] The eighth step

[0145] Add methanol (2 L), A1-10X-8 (30 g, 96 mmol), and water (150 mL). Heat the temperature to 50 °C, add potassium carbonate (50 g, 360 mmol), and continue the reaction for 20 min. Cool to room temperature, concentrate the reaction solution to dryness under reduced pressure, add 400 mL of water to make a slurry, perform suction filtration, and wash the filter cake with 300 mL of water and then perform suction filtration to obtain A1-10X-9 (7.8 g, yield: 37.3%).

[0146] The ninth step

[0147] Add A1-10X-9 (6 g, 27.5 mmol), A1-10X-8B (8 g, 30.3 mmol), PPTS (6.9 g, 27.5 mmol), and toluene (1 L). Protect with nitrogen, heat the temperature to 120 °C and keep the reaction for 4 h. Cool to 0 - 5 °C, perform suction filtration, dissolve the filter cake with dichloromethane and methanol, and concentrate to dryness under reduced pressure. Concentrate the concentrate with 50 mL of acetone at 0 - 5 °C to make a slurry, perform suction filtration, and obtain A1-10X-10 (12.2 g, yield: 100%).

[0148] The tenth step

[0149] Add A1-10X-10 (12.2 g, 27.5 mmol), concentrated hydrochloric acid (80 mL), and water (80 mL). Heat the temperature to 80 °C and keep the reaction for 2.5 h. Cool to room temperature, add a small amount of methanol, concentrate the reaction solution to dryness under reduced pressure. Concentrate the concentrate with 30 mL of acetone at room temperature to make a slurry, perform suction filtration, and dry the filter cake under reduced pressure to obtain A1-10X (11.1 g, yield: 100%). LC-MS: (m / z): 404.1 [M + H]+

[0150] 6. Synthesis of Compound A10

[0151]

[0152] The First Step

[0153] Add A10-1 (5.0 g, 66.6 mmol), fluorenylmethyloxycarbonyl chloride (20.6 g, 79.6 mmol), and dioxane (50 mL). Cool the temperature to 0 - 5 °C, and slowly add 1N aqueous sodium carbonate solution (60 mL). After the addition, restore the temperature to room temperature and stir the reaction for about 2 h. Then cool the temperature to 0 - 5 °C, adjust the pH to 2 with about 60 mL of 1M HCl, extract the aqueous phase with ethyl acetate (100 mL × 3), combine the organic phases, and concentrate under reduced pressure to obtain a solid of 15 g. Add methyl tert-butyl ether (50 mL), stir at room temperature for 30 min, filter, and concentrate the filter cake under reduced pressure to obtain A10-2 (17.9 g, yield: 90.3%). MS(ESI)(m / z): 298([M + H]+).

[0154] The Second Step

[0155] Add A10-2 (17.9 g, 60.2 mmol), acetonitrile (180 mL), N-hydroxysuccinimide (7.7 g, 66.9 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (13.8 g, 72.0 mmol), and stir the reaction at room temperature. Concentrate the system to dryness under reduced pressure, add 270 mL of saturated aqueous sodium bicarbonate solution, extract with dichloromethane, dry the organic phase, and concentrate to dryness under reduced pressure. Purify the crude product by column chromatography to collect A10-3 (19.2 g, yield: 84.3%). MS(ESI)(m / z): 401([M + Na]+).

[0156] The Third Step

[0157] Add A10-4 (5.0 g, 43.4 mmol) and methanol (75 mL). Cool the temperature to -5 - 0 °C, slowly add thionyl chloride (10.3 g, 86.6 mmol), and after the addition, restore the temperature to room temperature and stir for 1 h. Then heat to reflux and keep the reaction for 1 h. Cool down, concentrate the reaction solution to dryness under reduced pressure, and purify by column chromatography to obtain A10-5 (5.16 g, yield: 92.0%).

[0158] The Fourth Step

[0159] Add A10-5 (5.0 g, 38.7 mmol), acetonitrile (50 mL), water (50 mL), triethylamine (8.2 g, 81.0 mmol), and A10-3 (17.6 g, 46.5 mmol). Protect with nitrogen replacement and stir at room temperature for 2 h. Add 80 mL of water and wash twice with 50 mL of ethyl acetate each time. Adjust the pH of the aqueous phase to 4 - 5 with acetic acid, and concentrate the aqueous phase under reduced pressure to dryness to obtain A10-6 (11.9 g, yield 75.3%). MS(ESI)(m / z): 431([M+Na]+).

[0160] The fifth step

[0161] Add A10-6 (11 g, 26.9 mmol), N,N-dimethylformamide (55 mL), and DBU (4.1 g, 26.9 mmol). Protect with nitrogen replacement and stir at room temperature for 3 - 4 h. Without further treatment of the reacted raw materials, directly use the crude product of A10-7 for the next step.

[0162] The sixth step

[0163] Add A1-6 (13.5 g, 26.9 mmol), N,N-dimethylformamide (55 mL), N,N-diisopropylethylamine (4.9 g, 37.9 mmol). Cool to -5 - 0 °C under nitrogen protection, add 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (11.8 g, 31.0 mmol), and keep the temperature for reaction for 0.5 - 1 h. Add the entire batch of the reaction solution of A10-7 prepared in the fifth step. Restore to room temperature and stir the reaction until the raw materials are completely reacted. Quench the reaction by adding 200 mL of water, extract with ethyl acetate, combine the organic phases, wash with dilute acid water with pH = 3 - 5 and saturated brine successively, dry the organic phase, concentrate under reduced pressure, and purify the obtained crude product by column chromatography to obtain A10-8 (8.1 g, two-step yield: 45.0%). MS(ESI)(m / z): 693([M+Na]+).

[0164] The seventh step

[0165] Add A10-8 (8.0 g, 11.9 mmol), N,N-dimethylformamide (40 mL), and DBU (1.8 g, 11.8 mmol). Protect with nitrogen replacement and stir at room temperature for 3 - 4 h. After the raw materials are completely reacted, directly use the crude product of A10-9 for the next step without further treatment.

[0166] The eighth step

[0167] Add A1-8A (2.5 g, 11.8 mmol), N,N-dimethylformamide (12.5 mL), N,N-diisopropylethylamine (2.2 g, 17.0 mmol), cool down to -5 to 0 °C under nitrogen protection, add 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.2 g, 13.7 mmol), and keep the temperature for reaction for 0.5 to 1 h. Add the whole batch of the reaction solution of A10-9 prepared in the ninth step. Restore to room temperature and stir the reaction until the raw materials are completely reacted. Add 100 mL of water to quench the reaction, extract with ethyl acetate, combine the organic phases, wash with dilute acid water with pH = 3 - 5 and saturated brine, dry the organic phase, concentrate under reduced pressure to obtain the crude product, and purify it by column chromatography to obtain A10-10 (4.78 g, two-step yield: 62.8%). MS(ESI)(m / z): 663([M+Na]+).

[0168] The ninth step

[0169] Under nitrogen protection, add A10-10 (4.7 g, 7.3 mmol), dry methanol (50 mL), and stir at room temperature until dissolved and clear. Add anhydrous lithium hydroxide (0.63 g, 26.3 mmol), continue to stir the reaction at room temperature for 2 h, adjust the pH to 4.5 with acetic acid, and concentrate to dryness under reduced pressure to obtain A10-11 (4.1 g, yield: 89.1%).

[0170] The tenth step

[0171] Add A10-11 (4.0 g, 6.4 mmol), dichloromethane (50 mL), 4-dimethylaminopyridine (1.1 g, 9.0 mmol), add A1-9X (2.7 g, 6.4 mmol) under stirring at room temperature, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.5 g), and stir at room temperature for 16 h. After the raw materials are completely reacted, wash the reaction solution successively with 10% aqueous citric acid solution, water wash, saturated sodium carbonate aqueous solution wash, and saturated brine wash, dry the organic phase and concentrate under reduced pressure, and purify the crude product by column chromatography to obtain A10 (4.0 g, yield: 60.6%). MS(ESI)(m / z): 1053([M+Na]+).

[0172] 7. Synthesis of compounds A9, A11, and A12

[0173] Referring to the synthesis of compound A10, replace A10-4 with:

[0174]

[0175]

[0176] 8. Synthesis of compound A13

[0177] With reference to the synthesis of reference compound A11, replace A1-9X with A1-10X:

[0178]

[0179] 9. Synthesis of Compound A15

[0180]

[0181]

[0182] First Step

[0183] Add A15-3X-2 (50 g, 342 mmol), dichloromethane (500 mL), stir to dissolve, add pyridine (13.5 g, 171 mmol), cool down to 0 °C, and dropwise add trifluoroacetic anhydride (35.9 g, 171 mmol) within 40 min. After dropping, continue the reaction for 20 min. Concentrate the reaction solution to dryness under reduced pressure, then add 1 L of dichloromethane to dissolve, wash with 1 N hydrochloric acid until there is no pyridine, then wash with saturated sodium bicarbonate solution until alkaline, and wash with saturated brine. Dry the organic phase, concentrate to dryness under reduced pressure, and purify by column chromatography to obtain A15-3X (13.2 g, yield: 32.0%).

[0184] Second Step

[0185] Add A15-3X (10 g, 41.3 mmol), acetonitrile (100 mL), water (100 mL), triethylamine (41.8 g, 41.3 mmol), A14-3 (17.4 g, 41.3 mmol), displace and protect with nitrogen, and stir at room temperature for 2 h. Add 200 mL of water, wash with 200 mL * 2 of ethyl acetate. Adjust the pH of the aqueous phase to 4 - 5 with acetic acid, and concentrate the aqueous phase to dryness under reduced pressure to obtain A15-4 (17.7 g, yield 76.3%).

[0186] Third Step

[0187] Add A15-4 (17.7 g, 31.4 mmol), acetonitrile (180 mL), N-hydroxysuccinimide (4.0 g, 34.5 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (7.2 g, 37.7 mmol), and stir at room temperature for reaction. Concentrate the system to dryness under reduced pressure, add 100 mL of saturated sodium bicarbonate aqueous solution, extract with dichloromethane, dry the organic phase, concentrate to dryness under reduced pressure, purify the crude product by column chromatography, and collect A15-5 (16.4 g, yield: 81.3%).

[0188] Fourth Step

[0189] Add glycine (1.9 g, 24.8 mmol), acetonitrile (50 mL), water (50 mL), triethylamine (2.5 g, 24.8 mmol), A15-5 (16.0 g, 24.8 mmol), protect with nitrogen replacement, and stir at room temperature for 1.5 h. Add 80 mL of water, wash with 50 mL×2 of ethyl acetate, adjust the pH of the aqueous phase to 4 - 5 with acetic acid, and concentrate the aqueous phase to dryness under reduced pressure to obtain A15-6 (13.2 g, yield 85.6%).

[0190] The fifth step

[0191] Add tetrahydrofuran (90 mL), A14-6 (13.0 g, 20.9 mmol), acetic acid (55 mL), protect with nitrogen replacement, heat up to 40 °C, add lead tetraacetate (37.1 g, 83.6 mmol), heat up to reflux and keep the reaction for 16 h. Cool down to room temperature, filter through a diatomaceous earth cake, wash the filter cake with ethyl acetate, concentrate the filtrate to dryness under reduced pressure, and purify by column chromatography to obtain A15-7 (13.2 g, yield: 100%).

[0192] The sixth step

[0193] Add dichloromethane (100 mL), A15-7 (13.0 g, 20.5 mmol), glycolic acid (6.2 g, 82 mmol), PPTS (1.0 g, 4.1 mmol), protect with nitrogen replacement, heat up to 45 °C and reflux for 2 h. Concentrate the reaction solution under reduced pressure and purify by column chromatography to obtain A15-8 (6.8 g, yield: 51.1%).

[0194] The seventh step

[0195] Add A14-8 (6.5 g, 10.0 mmol), add N,N-dimethylformamide (32.5 mL), DBU (1.5 g, 10.0 mmol), protect with nitrogen replacement and stir at room temperature for 3 - 4 h. After the raw materials react completely, no post-treatment is carried out, and the crude product of A15-9 is directly fed into the next step.

[0196] The eighth step

[0197] Add A1-8X (2.9 g, 10.0 mmol), N,N-dimethylformamide (45 mL), N,N-diisopropylethylamine (1.8 g, 14.0 mmol). Cool to -5 to 0 °C under nitrogen protection, add 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (4.4 g, 11.5 mmol), and maintain the temperature for reaction for 0.5 to 1 h. Add the entire batch of reaction solution of A15-9. Restore to room temperature and stir the reaction until the raw materials are completely reacted. Add 100 mL of water to quench the reaction, extract with ethyl acetate, combine the organic phases, wash with dilute acid water with pH = 3 - 5 and saturated brine, dry the organic phase, concentrate under reduced pressure, and purify the obtained crude product by column chromatography to obtain A15-10 (3.1 g, two-step yield: 49.2%).

[0198] The ninth step

[0199] Under nitrogen protection, add A15-10 (3.0 g, 4.7 mmol), dry methanol (20 mL). After stirring and dissolving clearly at room temperature, add anhydrous lithium hydroxide (0.45 g, 18.8 mmol), continue to stir the reaction at room temperature for 3 h, add acetic acid to adjust pH = 4.5, and concentrate to dryness under reduced pressure to obtain A15-11 (2.7 g, yield: 93.4%).

[0200] The tenth step

[0201] Add A15-11 (2.5 g, 4.0 mmol), dichloromethane (20 mL), 4-dimethylaminopyridine (0.67 g, 5.5 mmol). Under stirring at room temperature, add A1-9X (1.7 g, 4.0 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.95 g, 5.0 mmol), and stir at room temperature for 16 h. After the raw materials are completely reacted, wash the reaction solution successively with 10% aqueous citric acid solution, water wash, saturated sodium carbonate aqueous solution wash, and saturated brine wash. Dry the organic phase and concentrate to dryness under reduced pressure. Purify the crude product by column chromatography to obtain A15-12 (2.8 g, yield: 68.7%).

[0202] The eleventh step

[0203] Add methanol (25 mL), A15-12 (2.5 g, 2.4 mmol), and water (12.5 mL). Heat to 50 °C, add potassium carbonate (1.3 g, 9.6 mmol), and continue the reaction for 20 min. Cool to room temperature, concentrate the reaction solution to dryness under reduced pressure, add 40 mL of water for pulping, filter, and pulp the filter cake with 30 mL of water again and filter to obtain A15 (0.83 g, yield: 37.3%).

[0204] 10. Synthesis of compound A14

[0205] Referring to the synthesis of reference compound A15, replace A15-3X with D-alanine:

[0206]

[0207] 11. Synthesis of compound A16

[0208] Referring to the synthesis of reference compound A15, replace A15-3X with D-alanine and A1-9X with A1-10X:

[0209]

[0210] 12. Synthesis of compound A17

[0211] Referring to the synthesis of reference compound A1, replace 6-aminohexanoic acid with 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propionic acid:

[0212]

[0213] 13. Synthesis of compound A18

[0214] Referring to the synthesis of reference compound A15, replace A15-3X with D-alanine and A1-9X with A1-10X,

[0215] and replace 6-aminohexanoic acid with 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propionic acid:

[0216]

[0217] 14. Synthesis of compound A20

[0218]

[0219] The first step

[0220] Add A20-1 (10.0 g, 67.5 mmol) and dichloromethane (500 mL), cool down to 0 - 5 °C, dropwise add Boc2O (7.4 g, 33.9 mmol), stir at this temperature for 5 h, then warm up to room temperature and stir overnight. After the raw material Boc2O has completely reacted, add 200 mL of water to wash the organic phase, extract the aqueous phase with 50 mL of DCM, combine the organic phases, dry and filter, and concentrate under reduced pressure to dryness to obtain A20-2 (8.4 g, yield: 100%).

[0221] The second step

[0222] Add A20-3 (9.7 g, 100 mmol), ethyl acetate (50 mL), N-methylmorpholine (11 mL, 100 mmol), cool down to 0-5 °C, and slowly add ethyl chloroformate (7.7 mL, 80.5 mmol). The heat release is not obvious during the addition. After the addition, restore to room temperature and stir for 1 h. Add 50 mL of water to the reaction solution, separate the layers, extract the aqueous phase with 30 mL of ethyl acetate twice, combine the organic phases, concentrate under reduced pressure to dryness, mix with silica gel and purify by column chromatography to collect A20-4 (7.3 g, yield: 54.0%).

[0223] The third step

[0224] Add A20-2 (3.0 g, 12.1 mmol), saturated aqueous sodium bicarbonate solution (90 mL), and stir at room temperature for 15 min. Filter, cool the filtrate to 0-5 °C, add A20-4 (2.0 g, 11.8 mmol), and stir at room temperature for 16 h. Concentrate the aqueous solution to dryness under reduced pressure and remove water with toluene. Add sodium acetate (10 g, 121.9 mmol) and acetic anhydride (20 mL, 220.0 mmol) to the concentrate, heat up to 120 °C and keep the reaction for 0.5 h. Cool down, concentrate the reaction solution to dryness under reduced pressure, add 50 mL of water, extract with 50 mL of dichloromethane twice, dry the organic phase, and concentrate under reduced pressure to obtain A20-5 (5.7 g, yield: 100%).

[0225] The fourth step

[0226] Add A20-5 (5.7 g, 17.4 mmol), dichloromethane (50 mL), and trifluoroacetic acid (10 g, 87.7 mmol), stir at room temperature for 2-3 h, precipitate solid, filter, and dry the filter cake under reduced pressure to obtain A20-6 (5.3 g, yield: 89.1%).

[0227] The fifth step

[0228] Under nitrogen protection, add A20-7 (15.0 g, 90.3 mmol), DMF (240 mL), and potassium bicarbonate (9.1 g, 90.9 mmol), cool down to 0-5 °C, dropwise add benzyl bromide (10.8 mL, 90.9 mmol), after dropping, restore to room temperature and stir for 16 h. After the raw materials react completely, pour the reaction solution into ice water, stir for 1 h, filter, dissolve the filter cake with 50 mL of ethyl acetate, dry the organic phase, filter, and concentrate under reduced pressure to dryness to obtain A20-8 (20.4 g, yield: 88.3%).

[0229] The sixth step

[0230] Add 40% hydrogen bromide / acetic acid solution (25 mL), cool down to 0 - 5 °C, and slowly add A20-9 (5.0 g, 13.3 mmol) portionwise. After addition, restore to room temperature, stir the reaction for 2 h, add 50 mL of toluene, concentrate the mixture under reduced pressure to dryness. Add 100 mL of ethyl acetate to the concentrate, wash the organic phase with 100 mL of saturated aqueous sodium bicarbonate solution. After washing, the pH of the aqueous phase is 8. Dry the organic phase over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to dryness to obtain A20-10 (5.1 g, yield: 96.6%).

[0231] The seventh step

[0232] Add A20-8 (2.69 g, 10.5 mmol), A20-10 (5.0 g, 12.6 mmol), acetonitrile (50 mL), activated molecular sieve (5.0 g), silver oxide (9.3 g, 40.1 mmol), displace and protect with nitrogen, stir at room temperature for 2 h, sample for TLC (PE / EA = 10 / 1, UV), and the raw materials have completely reacted. Add 100 mL of water, stir and filter through diatomaceous earth. Extract the filtrate with 150 mL × 3 of ethyl acetate. Combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to dryness to obtain A20-11 (6.0 g, yield: 100%). MS (ESI) (m / z): 595 ([M + Na]+).

[0233] The eighth step

[0234] Add a mixed solution of A20-11 (6.0 g, 10.5 mmol), isopropanol (18 mL) / dichloromethane (90 mL), silica gel (6.0 g, 200 - 300 mesh), cool down to 0 - 5 °C, and slowly add sodium borohydride (0.8 g, 21.1 mmol) portionwise. After addition, restore to room temperature and stir the reaction for 2 h. Add 100 mL of water, extract with ethyl acetate. Dry the organic phase, filter and concentrate under reduced pressure. Add anhydrous ethanol (275 mL) and 10% Pd / C (0.55 g) to the concentrate, displace with hydrogen and react at normal pressure and room temperature for 10 - 20 min. Filter through a diatomaceous earth cake, wash the filter cake with ethanol, and concentrate the filtrate under reduced pressure to dryness to obtain A20-12 (4.2 g, two-step yield: 81.9%).

[0235] The ninth step

[0236] Add A20-12 (2.8 g, 5.8 mmol), N,N-dimethylformamide (20 mL), stir and cool down to 0 - 5 °C, add triethylamine (1.7 g, 16.8 mmol), 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (3.5 g, 11.9 mmol), and A20-6 (2.0 g, 5.8 mmol). Stir the reaction at room temperature for 1 h, add 50 mL of water, extract with 50 mL * 3 of dichloromethane, dry and concentrate the organic phase, and purify the obtained crude product by column chromatography to obtain A20-13 (1.4 g, yield: 32.2%).

[0237] The tenth step

[0238] Add A1-9X (0.7 g, 1.7 mmol), acetonitrile (20 mL), 4-dimethylaminopyridine (0.26 g, 2.1 mmol), add triphosgene (0.5 g, 1.7 mmol) under stirring at room temperature, and stir at room temperature for 2 h. Add A20-13 (1.2 g, 1.7 mmol), stir at room temperature for 16 h, concentrate the reaction solution under reduced pressure to dryness, purify the crude product by column chromatography, and obtain A20 (1.15 g, yield: 68.8%).

[0239] 15. Synthesis of compound A19 fragment A19-4

[0240]

[0241] The first step

[0242] Add A19-1X (10.0 g, 166.4 mmol), dichloromethane (200 mL), cool down to 0 - 5 °C, dropwise add Boc2O (18.1 g, 83.2 mmol), keep stirring at this temperature for 3 h, warm up to room temperature and stir. After the raw material Boc2O reacts completely, add 100 mL of water to wash the organic phase, extract the aqueous phase with 50 mL of DCM again, combine the organic phases, dry, filter, and concentrate under reduced pressure to dryness to obtain A19-2X (12.1 g, yield: 90.8%).

[0243] The second step

[0244] A19-2X (12.0 g, 74.9 mmol), N,N-dimethylformamide (100 mL), N,N-diisopropylethylamine (48.5 g, 374.9 mmol), A1-8A (15.8 g, 74.9 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (34.3 g, 90.1 mmol) were added. The reaction was stirred at room temperature for 15 min. The reaction solution was concentrated to dryness under reduced pressure. 100 mL of dichloromethane was added. The organic phase was washed with water and saturated brine, dried, and concentrated to dryness under reduced pressure to obtain A19-3X (16.3 g, yield: 61.6%). MS(ESI) (m / z): 354 ([M+H] + ).

[0245] The third step

[0246] A19-3X (16.0 g, 45.3 mmol) and dichloromethane (160 mL) were added. Trifluoroacetic acid (32 g, 280.7 mmol) was added slowly. The mixture was stirred at room temperature for 1 h. The reaction solution was concentrated to dryness under reduced pressure to obtain A19-4X (10.9 g, yield: 95.6%).

[0247] 16. Synthesis of compound A19

[0248] Referring to the synthesis of compound A20, A1-9X was replaced with A1-10X, and A20-6 was replaced with A19-4X.

[0249]

[0250] 17. Synthesis of compound A21

[0251] Referring to the synthesis of compound A20, A20-6 was replaced with A19-4X.

[0252]

[0253] 18. Synthesis of compound A22

[0254] Referring to the synthesis of compound A20, A20-1 was replaced with amino-monoethylene glycol-carboxylic acid.

[0255]

[0256] 19. Synthesis of compound A23

[0257] Referring to the synthesis of compound A20, A1-9X was replaced with A1-10X, and A20-1 was replaced with amino-monoethylene glycol-carboxylic acid.

[0258]

[0259] 20. Synthesis of Compound A24

[0260] Referring to the synthesis of Compound A20, replace A20-1 with 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propionic acid.

[0261]

[0262] 21. Synthesis of Compound A25

[0263] Referring to the synthesis of Compound A20, replace A20-1 with 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]propionic acid and replace A1-9X with A1-10X.

[0264]

[0265] 22. Synthesis of Intermediate A1-10X

[0266]

[0267] First Step

[0268] Add dichloromethane (6 L), A1-10X-1 (914 g, 6220 mmol), and triethylamine (754 g, 7464 mmol), and cool the temperature to 0 °C. Dropwise add acetic anhydride (698 g, 6842 mmol), and finish dropping in about 1 h. Continue the reaction for 30 min. Add 1 L of water to the system, let it stand for liquid separation, extract the aqueous layer with 800 mL of dichloromethane, and combine the organic phases. Adjust the pH of the organic phase to 2 with 1 N hydrochloric acid, separate out the organic phase, extract the aqueous layer with dichloromethane, and combine the organic phases. Wash the organic phase with 1 L of saturated sodium bicarbonate solution, and concentrate the filtrate under reduced pressure to dryness to obtain A1-10X-2 (1150 g, yield: 97.8%).

[0269] Second Step

[0270] Add sulfuric acid (2 L) and A1-10X-2 (300 g, 1580 mmol), and cool the temperature to 0 °C. Add sodium nitrate (134.3 g, 1580 mmol) in portions within 2 h, and react for 30 min. Slowly pour the reaction solution into 8 L of ice water under stirring, filter by suction, and wash the filter cake twice with ice water. Dissolve the filter cake in 1 L of dichloromethane, wash it with saturated sodium bicarbonate solution until it is alkaline, dry the organic phase, and concentrate it to dryness under reduced pressure. Pulp with 400 mL of methyl tert-butyl ether, cool the temperature to 0 - 5 °C, filter by suction, and dry the filter cake under reduced pressure to obtain A1-10X-3 (264 g, yield: 71.3%).

[0271] Third Step

[0272] Add acetone (5 L) and A1-10X-3 (160 g, 680 mmol). Dissolve magnesium sulfate (111.6 g, 930 mmol) in water (620 mL) and add it to the system. Cool the temperature to 0 °C and add potassium permanganate (323 g, 2050 mmol) in portions over 3 h. After addition, let the temperature rise naturally. React for 1.5 h. Dropwise add the prepared 40% sodium thiosulfate solution (2 L) to the reaction system until no oxidizing property is detected. Filter by suction, concentrate the filtrate, add 6 L of dichloromethane. Wash the organic phase with water, saturated sodium bicarbonate solution, and saturated brine, dry it, and concentrate it under reduced pressure to dryness to obtain the crude product. Purify by column chromatography to obtain A1-10X-4 (100 g, yield: 59.2%). LC-MS: (m / z): 274.3 [M+Na] + 。

[0273] Step 4

[0274] Add A1-10X-4 (80 g, 320 mmol) and concentrated hydrochloric acid (500 mL). Heat to 90 - 100 °C and keep the reaction for 2 h. Cool to 0 - 10 °C, pour the reaction solution into 2 L of ice water, filter, and wash the filter cake with ice water to obtain A1-10X-5 (66 g, yield: 100%).

[0275] Step 5

[0276] Add A1-10X-5 (66 g, 320 mmol) and dichloromethane (1.5 L), stir to dissolve, add pyridine (52 mL, 650 mmol), cool to 0 °C, and dropwise add trifluoroacetic anhydride (94 mL, 676 mmol) within 40 min. After dropping, continue the reaction for 20 min. Concentrate the reaction solution to dryness under reduced pressure, then add 1 L of dichloromethane to dissolve it. Wash with 1N hydrochloric acid until no pyridine remains, wash with saturated sodium bicarbonate solution until alkaline, and wash with saturated brine. Dry the organic phase and concentrate it to dryness under reduced pressure to obtain A1-10X-6 (62 g, yield: 64.1%).

[0277] Step 6

[0278] Add methanol (1 L), formic acid (50 mL), and water (50 mL). Dissolve A1-10X-6 (54 g, 180 mmol) in dichloromethane (100 mL) and add it to the system. Cool to 0 °C and add zinc powder (150 g, 2290 mmol) in portions within 50 min. Continue the reaction for 1.5 h. Filter by suction, separate the layers of the filtrate. Wash the organic phase with saturated sodium bicarbonate solution and saturated brine. Dry the organic phase and concentrate it under reduced pressure to obtain A1-10X-7 (42 g, yield: 85.7%).

[0279] Step 7

[0280] Add dichloromethane (1.6 L), A1-10X-7 (40 g, 147.0 mmol), triethylamine (49 mL, 352.5 mmol), cool down to 0 - 5 °C, dropwise add acetyl chloride (27 mL, 380.0 mmol), continue the reaction for 50 min, add 600 mL of water. Filter by suction, slurry the filter cake with water to obtain A1-10X-8 (46.2 g, yield: 100%). 1 1H-NMR (400 MHz, DMSO-d6): 13.30 (s, 1H), 9.60 (s, 1H), 8.31 (d, J = 9.2 Hz, 1H), 7.72 (d, J = 9.2 Hz, 1H), 2.86 (t, J = 7.0 Hz, 1H), 2.74 (t, J = 1.6 Hz, 1H), 2.00 (m, 1H). LC-MS: (m / z): 315.1 [M + H] + 。

[0281] The eighth step

[0282] Add methanol (2 L), A1-10X-8 (30 g, 96 mmol), water (150 mL), heat up to 50 °C, add potassium carbonate (50 g, 360 mmol), continue the reaction for 20 min. Cool down to room temperature, concentrate the reaction solution to dryness under reduced pressure, add 400 mL of water to slurry, filter by suction, slurry the filter cake with another 300 mL of water, and filter by suction to obtain A1-10X-9 (7.8 g, yield: 37.3%). LC-MS: (m / z): 219.1 [M + H] + 。

[0283] The ninth step

[0284] Add A1-10X-9 (6 g, 27.5 mmol), A1-10X-8B (8 g, 30.3 mmol), PPTS (6.9 g, 27.5 mmol), toluene (1 L), under nitrogen protection, heat up to 120 °C and keep the reaction for 4 h. Cool down to 0 - 5 °C, filter by suction, dissolve the filter cake with dichloromethane and methanol, and concentrate to dryness under reduced pressure. Slurry the concentrate with 50 mL of acetone at 0 - 5 °C, filter by suction to obtain A1-10X-10 (12.2 g, yield: 100%). LC-MS: (m / z): 446.2 [M + H] + 。

[0285] The tenth step

[0286] Add A1-10X-10 (12.2 g, 27.5 mmol), concentrated hydrochloric acid (80 mL), and water (80 mL). Heat up to 80 °C and hold the reaction for 2.5 h. Cool down to room temperature, add a small amount of methanol, and concentrate the reaction solution to dryness under reduced pressure. The concentrate is slurried with 30 mL of acetone at room temperature, and the filter cake is dried under reduced pressure to obtain A1-10X (11.1 g, yield: 100%). LC-MS: (m / z): 404.1 [M+H] + 。

[0287] 23. Synthesis of Intermediate A1-9X

[0288]

[0289] The First Step

[0290] Add tetrahydrofuran (2.4 L), A1-9X-1 (426 g, 3000 mmol), and A1-9X-1X (1500 g, 3600 mmol). Under nitrogen protection, cool down to -5 °C, and then dropwise add a solution of sodium tert-butoxide (840 g, 7500 mmol) / tetrahydrofuran (2.4 L). Hold the reaction for 5 - 8 h. Pour the system into 7.5 L of ice water, concentrate most of the THF under reduced pressure at 40 - 45 °C, extract the by-products with 5.0 L of MTBE in three portions, adjust the pH of the aqueous phase to 4 - 5 with 6.0 N hydrochloric acid, extract the product with 4.0 L of EEA in three portions, combine the organic layers, wash with water, brine, dry over anhydrous sodium sulfate, filter, and concentrate to dryness to obtain A1-9X-2 (594.0 g, yield 100%).

[0291] The Second Step

[0292] Dissolve A1-9X-2 (594.0, 3000 mmol) in ethyl acetate (2500 mL), add 10% Pd / C (containing 50% water, 75 g), displace with nitrogen and hydrogen respectively, and carry out hydrogenation reaction at atmospheric pressure at 25 - 35 °C for 16 h. Filter through diatomaceous earth, and concentrate the filtrate to dryness under reduced pressure to obtain A1-9X-3 (600.0 g, yield 100%).

[0293] The Third Step

[0294] Add A1-9X-3 (600.0 g, 3000 mmol) to concentrated sulfuric acid (3000 g), control the temperature below 35 °C during the addition, and react at room temperature for 2 - 3 h after addition. Slowly pour the reaction solution into 6.5 kg of ice water, add 3.5 L of methyl tert-butyl ether and 0.5 L of ethyl acetate for extraction and separation. Combine the organic layers, wash successively with saturated sodium bicarbonate and brine, dry the organic phase, concentrate to dryness under reduced pressure, and purify the crude product by column chromatography to obtain A1-9X-4 (230.0 g, yield: 42.1%). 11H-NMR (400 MHz, CDCl3): δ 6.79 - 6.69 (m, 2H), 2.96 (t, J = 6.0 Hz, 2H), 2.64 (t, J = 6.0 Hz, 2H), 2.11 (m, 2H).

[0295] Step 4

[0296] A1-9X-4 (125 g, 686.8 mmol) was added to concentrated sulfuric acid (1200 g) while controlling the temperature below 5 °C. Then, sodium nitrate (70 g, 823.5 mmol) was added in batches. After addition, the reaction was carried out at room temperature for 2 - 3 h. The reaction solution was slowly poured into 6.5 kg of ice water, and extracted with a mixed solution of 1.5 L of methyl tert-butyl ether and 150 mL of ethyl acetate. The organic layers were combined, washed successively with saturated sodium bicarbonate and brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product obtained was purified by column chromatography to give A1-9X-5 (71.0 g, yield: 45.5%).

[0297] Step 5

[0298] Ethanol (2.1 L), water (0.3 L), ammonium chloride (70 g, 1308 mmol), and A1-9X-5 (100 g, 440 mmol) were added. After stirring and dissolving, iron powder (200 g, 3571 mmol) was slowly added, and the temperature was raised to 80 °C for reaction for 1 - 2 h. After cooling, the mixture was filtered. Most of the ethanol in the filtrate was removed under reduced pressure. Then, water and 2.6 L of dichloromethane were added for extraction. The organic layers were combined, washed successively with saturated sodium bicarbonate and brine, dried over anhydrous sodium sulfate, and the resulting A1-9X-6 extract was obtained and directly used for the next step without further treatment. LC-MS: (m / z): 198.1 [M+H] + 。

[0299] Step 6

[0300] Pyridine (150 g, 1900 mmol) and 4-dimethylaminopyridine (1.0 g) were added to the A1-9X-6 extract. While controlling the temperature below 15 °C, acetic anhydride (99 g, 968 mmol) was slowly added dropwise. The reaction was carried out at room temperature for 2 - 3 h. The reaction solution was slowly poured into 1.5 kg of ice water. The organic layer was washed with 1.0 N dilute hydrochloric acid until acidic, and then successively washed with saturated sodium bicarbonate and brine. The organic phase was concentrated to dryness under reduced pressure. The concentrate was dissolved in 1.0 L of methanol, and the pH was adjusted to 11 - 12 with 25.0% NaOH. Hydrolysis was carried out at 35 - 45 °C for 1 - 3 h, and the organic solvent was removed under reduced pressure. Dichloromethane and water were added for extraction, and the layers were separated. The organic layer was concentrated to dryness under reduced pressure. The concentrate was triturated with methyl tert-butyl ether, filtered, and the filter cake was dried to give A1-9X-7 (55.0 g, yield: 52.3%). 1H-NMR(400MHz, CDCl3): δ 6.83 (m, 1H), 2.86 (t, J = 6.0 Hz, 2H), 2.64 (t, J = 6.0 Hz, 2H), 2.25 (s, 3H), 2.06 (m, 2H).

[0301] The seventh step

[0302] Add A1-9X-7 (9.0 g, 37.6 mmol), dimethyl sulfoxide (150 mL), and slowly add 25% ammonia water (250 g) under stirring. Transfer the reaction solution to an autoclave, heat up to 65 - 75 °C and react for 16 h. Cool down, add an appropriate amount of water, extract with ethyl acetate multiple times, combine the organic layers, wash successively with water and saturated brine, dry the organic layer, and concentrate it to dryness under reduced pressure. Purify the crude product by column chromatography to obtain A1-9X-8 (4.5 g, yield: 50.6%). 1 H-NMR(400MHz, DMSO-d6): δ 9.09 (s, 1H), 6.43 (d, J = 12.4 Hz, 1H), 2.66 (t, J = 6.0 Hz, 2H), 2.50 (m, 2H), 2.00 (s, 3H), 1.87 (t, J = 6.0 Hz, 2H). LC-MS: (m / z): 237.1 [M + H] + 。

[0303] The eighth step

[0304] Add A1-9X-8 (4 g, 17 mmol), A1-10X-8B (4.9 g, 18.6 mmol), PPTS (4.8 g, 17 mmol), and toluene (800 mL). Under nitrogen protection, heat up to 120 °C and keep the temperature for 8 h. Cool down, concentrate the reaction solution under reduced pressure, slurry the concentrate with 50 mL of acetone, cool to 0 - 5 °C, filter by suction, and dry the filter cake under reduced pressure to obtain A1-9X-9 (6.5 g, yield: 82.5%). LC-MS: (m / z): 464.2 [M + H] + 。

[0305] The ninth step

[0306] Add A1-9X-9 (5.0 g, 10.8 mmol), concentrated hydrochloric acid (25 mL), and water (25 mL), heat up to 80 °C, and keep the temperature for 1 h. Concentrate the reaction solution to dryness under reduced pressure, slurry with 30 mL of acetone, filter by suction to obtain A1-9X (3.1 g, yield: 68.1%). LC-MS: (m / z): 422.1 [M + H] + 。

[0307] Example 2. Preparation of antibody conjugate

[0308] The targeted HER2 antibody Trastuzumab was replaced into 50 mM PB / 1.0 mM EDTA buffer (pH 7.0) using a G25 desalting column. 8 equivalents of TECP were added, and the mixture was stirred at 37 °C for 2 hours to completely open the interchain disulfide bonds of the antibody. Subsequently, the pH of the reduced antibody solution was adjusted to 6.0 using phosphoric acid, and the water bath temperature was lowered to 25 °C for the conjugation reaction. The linker-drug conjugate and GGFG-Dxd (control compound) prepared according to the method of Example 1 above were each dissolved in DMA. 12 equivalents of the linker-drug conjugate were pipetted dropwise into the reduced antibody solution, and DMA was added to make its final concentration 10% (V / V). The reaction was stirred at 25 °C for 0.5 hours. After the reaction was completed, the sample was filtered through a 0.22 μm membrane. The excess conjugated small molecules were removed by purification using a tangential flow ultrafiltration system, and the buffer was 50 mM PB / 1.0 mM EDTA solution (pH = 6.0). After purification, 6% sucrose was added at the final concentration and stored in a -20 °C refrigerator. The absorbance values were measured at 280 nm and 370 nm using the UV method, and the DAR value was calculated. In this technical solution, most of the linker-drug conjugates did not produce precipitation during the conjugation process. The DAR values of the conjugates were all 6 - 8. The DAR value was measured by HIC-HPLC, RP-HPLC or LCMS. The aggregate ratio of the conjugate was detected by SEC-HPLC to be in the normal range, indicating that the antibody-drug conjugate of the present invention has good solubility and drug-forming properties, and no precipitation occurs during the conjugation process.

[0309] Example 3 In Vitro Cytotoxic Activity Test

[0310] Stably transfected SK-BR-3 and BT-474 human breast cancer cells with high expression of Her2 and NCI-N87 human gastric cancer cells were selected as the cell lines for in vitro activity detection in this experiment to observe the dose-effect of different antibody-drug conjugates on cell killing. The initial seeding density of each type of cell was preliminarily selected: 2×10 3 cells / well. After 16 - 24 hours, the cytotoxic activity was measured. Secondly, the final concentration of the antibody-drug conjugate prepared in Example 2 was set at 5000 nM as the starting concentration, and 10 concentrations in a series (4 - 10-fold serial dilution) from 5000 - 0.006 nM were designed. The killing (or inhibition) changes were observed for 120 hours. After chemiluminescent staining (Luminescent Cell Viability Assay), the fluorescence data was read and the IC 50 .

[0311] All linkers in the present invention are cleavable linkers. Therefore, the in vitro cytotoxic activity of the released camptothecin analogues was first tested, and the results are shown in the following table. The test results show that the cell killing activity of most compounds is better than that of the control compound Dxd.

[0312]

[0313]

[0314] Furthermore, the cytotoxic activity of the conjugated ADC drug was tested in vitro. From the results of the activity test, the ADCs prepared from the compounds of the present application all showed certain anti-tumor activity, and the IC 50 reached 10 -6 -10 -10 M, showing significantly stronger anti-tumor activity compared with the control sample.

[0315] Example 4 Determination of Anti-Tumor Efficacy in Vivo

[0316] The efficacy of the combination of the present invention was measured in vivo, that is, allografts or xenografts of cancer cells were implanted in rodents, and the tumors were treated with the said combination. The test mice were treated with the drug or the control, and monitored for several weeks or longer to measure the time to tumor doubling, log cell kill, and tumor inhibition.

[0317] 1) Experimental Animals

[0318] BALB / cA-nude nude mice, 6-7 weeks old, female, purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0319] 2) Experimental Procedures

[0320] The nude mice were subcutaneously inoculated with human gastric cancer NCI-N87 cells. After the tumors grew to 100-250 mm 3 , the animals were randomly grouped (D0). The tumor volume was measured 2-3 times a week, the body weight of the mice was weighed, and the data were recorded. The formula for calculating the tumor volume (V) is: V = 1 / 2 × a × b 2 where a and b represent the length and width respectively. T / C(%) = (T - T0) / (C - C0) × 100%, where T and C are the tumor volumes at the end of the experiment for the test group and the control group respectively; T0 and C0 are the tumor volumes at the start of the experiment for the test group and the control group respectively.

[0321] From the results of the activity test, the ADCs prepared from the compounds of the present application all showed certain anti-tumor activity in vivo, and could show significantly stronger anti-tumor activity compared with the control sample. The tumor-bearing mice could well tolerate the above drugs, and no symptoms such as weight loss occurred.

[0322] The foregoing detailed description is provided by way of explanation and example, and is not intended to limit the scope of the appended claims. At present, various changes to the embodiments listed in the present application are obvious to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.

Claims

1. An inhibitor compound or a pharmaceutically acceptable salt thereof, characterized in that: The specific molecular structure of the compound is as follows:

2. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, characterized in that: An antibody-drug conjugate, the structure of which comprises the following: Wherein: Ab is an antibody, and n = 1-8.

3. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 2, wherein: The Ab is selected from murine antibodies, chimeric antibodies, humanized antibodies or fully human antibodies.

4. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 3, wherein: The antibody comprises monoclonal antibodies.

5. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 4, wherein: The antibody comprises bispecific antibodies.

6. The antibody-drug conjugate according to claim 5, wherein: The antibody is capable of binding to a tumor-associated antigen.

7. A pharmaceutical composition, characterized in that, Comprising: (a) The antibody-drug conjugate according to any one of claims 2-6; and (b) a pharmaceutically acceptable diluent, carrier or excipient.

8. Use of the antibody-drug conjugate according to any one of claims 2-7 in the preparation of a medicament for treating gastric cancer.

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