Exatecan mesylate-based antibody-drug conjugate, preparation method therefor and use thereof

Abstract

An exatecan mesylate-based antibody–drug conjugate, a preparation method therefor and a use thereof, belonging to the field of pharmaceuticals. The linker of an antibody-drug conjugate using exatecan mesylate as the toxin comprises a structure represented by Formula I. The drug-linker assembly unit using exatecan mesylate as the toxin exhibits excellent conjugation efficiency. The antibody-drug conjugate obtained based on this drug-linker assembly unit shows significantly enhanced cytotoxicity against HER2-positive tumor cells, while exhibiting weak cytotoxicity against HER2-negative tumor cells, demonstrating that the antibody-drug conjugate has strong target selectivity and good clinical application prospects.

Description

An antibody-drug conjugate based on exatecan and a preparation method and use thereof TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to an antibody-drug conjugate based on exatecan and a preparation method and use thereof. BACKGROUND

[0002] Exatecan mesylate (DX-8951f) is a water-soluble camptothecin (CPT) derivative. Compared with camptothecin, exatecan has higher membrane permeability and can overcome P-glycoprotein-mediated multidrug resistance, and has stronger efficacy on various tumor implantation models. However, free exatecan has the problems of poor targeting and high toxicity in vivo, which limits its clinical application.

[0003] Antibody-drug conjugates (ADCs) are connectors coupled by a linker between a monoclonal antibody targeting a specific antigen and a toxin. Through this coupling mode, ADCs combine the targeting of monoclonal antibodies and the strong killing effect of toxins, utilize the specific binding of antigen-antibody to accurately deliver toxins to target cells, and realize specific killing of tumor cells and adjacent tumor stromal cells, tumor blood vessels and other cells supporting tumor growth through endocytosis and bystander effect, greatly reducing the toxic side effects of drugs, expanding the therapeutic window, improving the pharmacokinetic characteristics of drugs, and both making up for the poor efficacy of monoclonal antibodies alone and avoiding the regret of too strong toxins that cannot be made into drugs. ADCs have been proved to be a very promising treatment in the clinical treatment of hematological tumors and solid tumors, and have become a popular direction of antibody drug research and development at home and abroad.

[0004] However, the complex molecular design of ADCs not only has advantages but also brings many challenges to the development of the drug. It is of great significance to develop an antibody-drug conjugate with exatecan as a toxin with excellent coupling effect, safety, effectiveness and stability. SUMMARY

[0005] The purpose of the present application is to provide an antibody-drug conjugate based on exatecan and a preparation method and use thereof.

[0006] The present application provides a linker for a target linker-drug conjugate, a stereoisomer thereof, an optical isomer thereof, a salt thereof or a deuterated product thereof, wherein the linker comprises a structure shown in formula I:

[0007] T is a tethering group which is capable of being linked to a targeting linker; the targeting linker is a substance capable of targeting binding to a diseased site;

[0008] L is no, a cleavable or non-cleavable linker fragment;

[0009] a is selected from 0, 1, 2, 3, 4 or 5;

[0010] b is selected from 0, 1, 2, 3, 4 or 5;

[0011] c is selected from 0, 1, 2, 3 or 4;

[0012] R1is each independently selected from hydrogen, a carboxyl-containing group;

[0013] R2is each independently selected from hydrogen, a carboxyl-containing group.

[0014] Further, the linker comprises a structure shown in Formula II or Formula III:

[0015] Further, the R1is selected from hydrogen,

[0016] R2is selected from hydrogen,

[0017] wherein R3is an amino protecting group, R4is selected from hydrogen, C 1-3 alkyl, and R5is selected from hydrogen, C 1-3 alkyl.

[0018] Further, the R1is selected from hydrogen,

[0019] R2is selected from hydrogen,

[0020] Further, the L is A-B, wherein A is selected from no,

[0021] B is selected from no,

[0022] wherein R6is an amino protecting group.

[0023] Further, the L is A-B, wherein A is selected from no, B is selected from

[0024] Further, the linker comprises one of the following structures:

[0025] The application also provides a drug-linking assembly unit, a stereoisomer thereof, an optical isomer thereof, a salt thereof, or a deuterated product thereof, characterized in that the drug-linking assembly unit is connected by the linker and the drug.

[0026] Further, the drug is irinotecan.

[0027] Further, the drug-linking assembly unit is selected from one of the following structures:

[0028] The application also provides a targeting linker-drug conjugate, a stereoisomer thereof, an optical isomer thereof, a salt thereof, or a deuterated product thereof, characterized in that the targeting linker-drug conjugate is connected by the targeting linker and the drug-linking assembly unit, and the structure of the targeting linker-drug conjugate is shown in formula IV:

[0029] Ab is a targeting linker; q is an integer from 1 to 20;

[0030] The targeting linker is a substance capable of targeting and binding to a lesion site.

[0031] Further, the DAR value of the targeting linker-drug conjugate is 1.00-20.00, preferably 4.00-8.20.

[0032] Further, the targeting linker-drug conjugate is:

[0033] with a DAR value of 5.10-5.50, preferably 5.28;

[0034] Or, the targeting linker-drug conjugate is:

[0035] with a DAR value of 6.90-7.30, preferably 7.12;

[0036] Or, the targeting linker-drug conjugate is:

[0037] with a DAR value of 7.65-8.05, preferably 7.85;

[0038] Or, the targeting linker-drug conjugate is:

[0039] DAR value of 7.80-8.20, preferably 8.00;

[0040] or, the targeted linker-drug conjugate is:

[0041] DAR value of 7.70-8.10, preferably 7.89;

[0042] or, the targeted linker-drug conjugate is:

[0043] DAR value of 6.10-6.50, preferably 6.31;

[0044] or, the targeted linker-drug conjugate is:

[0045] DAR value of 6.90-7.30, preferably 7.11;

[0046] or, the targeted linker-drug conjugate is:

[0047] DAR value of 7.80-8.20, preferably 8.00;

[0048] or, the targeted linker-drug conjugate is:

[0049] DAR value of 7.80-8.20, preferably 8.00;

[0050] or, the targeted linker-drug conjugate is:

[0051] DAR value of 7.25-7.65, preferably 7.46.

[0052] Further, the targeted linker is an antibody, an antibody fragment, a protein, a small molecule polypeptide, a glycopeptide, a peptidomimetic, a small molecule compound, or a nucleic acid oligonucleotide aptamer;

[0053] Preferably, the antibody is an antibody against a cell surface receptor and a tumor-associated antigen;

[0054] More preferably, the antibody is an anti-HER3 antibody, an anti-B7H3 antibody, an anti-TROP2 antibody, an anti-HER2 antibody, or an anti-CEACAM5 antibody.

[0055] The present application also provides a pharmaceutical preparation for preventing and / or treating a tumor, characterized in that it is a preparation prepared by using the above-mentioned targeted linker-drug conjugate, stereoisomer thereof, optical isomer thereof, salt thereof, or deuterated product thereof as an active ingredient, and a pharmaceutically acceptable adjuvant.

[0056] The application also provides use of the above-mentioned targeted linker-drug conjugate, stereoisomer thereof, optical isomer thereof, salt thereof or deuterated product thereof in the preparation of a pharmaceutical preparation for preventing and / or treating tumors.

[0057] Further, the tumor is a HER3-positive, B7H3-positive, HER2-positive or CEACAM5-positive tumor.

[0058] Further, the tumor is selected from lung cancer, lung adenocarcinoma, urethral cancer, large intestine cancer, prostate adenocarcinoma, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma or sarcoma.

[0059] The application also provides a method for preparing the above-mentioned targeted linker-drug conjugate, stereoisomer thereof, optical isomer thereof, salt thereof or deuterated product thereof, characterized in that the method comprises the following steps: coupling a drug linker assembly unit with a targeting linker to obtain a targeted linker-drug conjugate.

[0060] Alternatively, the method comprises the following steps:

[0061] (1) coupling a drug with a linker to obtain a drug linker assembly unit;

[0062] (2) coupling the drug linker assembly unit with a targeting linker to obtain a targeted linker-drug conjugate;

[0063] Alternatively, the method comprises the following steps:

[0064] (1') coupling a targeting linker with a linker to obtain a targeting linker-linker unit;

[0065] (2') coupling the targeting linker-linker unit with a drug to obtain a targeted linker-drug conjugate.

[0066] Definitions of terms used in the application: unless otherwise specified, the initial definition of a group or term provided herein applies throughout the specification for that group or term; for terms not specifically defined herein, the meaning given to them by one of ordinary skill in the art, in light of the disclosure and context, should be given to them.

[0067] The amino protecting groups in the application include but are not limited to acetyl (Ac), benzyloxycarbonyl (Cbz), 9-fluorenylmethoxycarbonyl (Fmoc), trityl (Trt), benzoyl (Bz), tert-butyloxycarbonyl (Boc).

[0068] The drug-linking assembly unit of the present application can be connected with a targeting linker, which is a substance capable of targeting binding to a lesion site; the drug-linking assembly unit is composed of a linker and a drug.

[0069] The covalent connection of the targeting linker and 1-20 drug-linking assembly units can form a targeting linker-drug conjugate.

[0070] When the targeting linker is an antibody, the targeting linker-drug conjugate is an antibody-drug conjugate (ADC for short).

[0071] In the present application, the "DAR value" represents the average number of drug-linking assembly units conjugated to one targeting linker in the targeting linker-drug conjugate, which is equivalent to the average value of q. The DAR value can not be an integer.

[0072] The minimum and maximum values of the carbon content in the hydrocarbon group are represented by a prefix, for example, the prefix C a-b Alkyl refers to a straight-chain or branched alkyl group containing 1-3 carbon atoms. 1-3 Alkyl refers to a straight-chain or branched alkyl group containing 1-3 carbon atoms.

[0073] "Targeting linker-drug conjugate" refers to the conjugate obtained by coupling a drug with a targeting linker having the effect of targeting binding to a lesion site through a linker. For example, "antibody-drug conjugate" (ADC) is one of the targeting linker-drug conjugates of the present application, and its targeting linker is an antibody or an antibody fragment.

[0074] "Drug-linking assembly unit" is the product obtained by connecting the aforementioned linker and drug in the targeting linker-drug conjugate, and is an intermediate for preparing the targeting linker-drug conjugate. Coupling it with a targeting linker can obtain a targeting linker-drug conjugate.

[0075] "Drug" refers to a compound having a desired biological activity (such as a therapeutic effect). For example, it can be a cytotoxic compound for cancer treatment, or a biologically active protein or polypeptide.

[0076] "Targeting linker" refers to a substance having the effect of targeting binding to a lesion site, such as an antibody, an antibody fragment, a protein, a small molecular polypeptide, a glycopeptide, a mimetic peptide, a small molecular compound, or a nucleic acid oligonucleotide aptamer. The targeting linker can bind to a target (such as an antigen) on the lesion site, so that the targeting linker-drug conjugate can target bind to the lesion site.

[0077] An "antibody" refers to a protein produced by the body in response to an antigen that has a protective effect. When the targeting linker is an antibody or antibody fragment, the resulting targeting linker-drug conjugate is an antibody-drug conjugate. The antibody or antibody fragment of the present application can bind to an antigen at a diseased site, allowing the antibody-drug conjugate to target the diseased site. An antibody can be any protein or protein-like molecule that can bind to or complex with or react with a portion of the cell population to be treated or biologically modified.

[0078] In the present application, the antibody that makes up the antibody-drug conjugate preferably retains its antigen binding ability as in its original wild-type state. Thus, the antibody in the present application is capable of specifically binding to an antigen. The antigen involved includes, but is not limited to, tumor associated antigens (TAA), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, molecules associated with tissue growth or differentiation, lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and factors associated with angiogenesis. The tumor associated factor can also be a cluster differentiation factor (e.g., CD proteins). The antigen to which the antibody binds in the present application can be one or a subset of the above categories, while other subsets include other molecules / antigens with special properties.

[0079] Antibodies for use in the 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 known in the art. These targets are specifically expressed on the surface of one or more cancer cells, but are rarely or not expressed on the surface of one or more non-cancer cells. Typically, such tumor associated polypeptides are overexpressed on the surface of cancer cells relative to non-cancer cells. Identification of such tumor associated factors greatly enhances the specific targeting properties of antibody-based cancer therapies.

[0080] Tumor associated antigens include, but are not limited to, the tumor associated antigens listed below. Antibodies targeting the corresponding tumor associated antigens include all amino acid sequence variants and species that share at least 70%, 80%, 85%, 90%, or 95% homology with the sequences identified in the references, or that possess biological properties and characteristics identical to those of the tumor associated antigens of the references.

[0081] Tumor associated antigens are: BMPR1B (Genbank Accession No: NM-001203), E16 (Genbank Accession No: NM-003486), STEAP1 (Genbank Accession No: NM-012449), 0772P (Genbank Accession No: AF361486), MPF (Genbank Accession No: NM-005823), Napi3b (Genbank Accession No: NM-006424), Sema 5b (Genbank Accession No: AB040878), PSCA hlg (Genbank Accession No: AY358628), ETBR (Genbank Accession No: AY275463), MSG783 (Genbank Accession No: NM-017763), STEAP2 (Genbank Accession No: AF455138), TrpM4 (Genbank Accession No: NM-017636), CRIPTO (Genbank Accession No: NP-003203 or NM-003212), CD21 (Genbank Accession No: M26004), CD79B (Genbank Accession No: NM-000626), FcRH2 (Genbank Accession No: NM-030764), HER2 (Genbank Accession No: M11730), NCA (Genbank Accession No: M18728), MDP (Genbank Accession No: BC017023), IL20Ra (Genbank Accession No: AF184971), Brevican (Genbank Accession No: AF229053), EphB2R (Genbank Accession No: NM-004442), GEDA (Genbank Accession No: AY260763), BAFF-R (Genbank Accession No: AF1164546), CD22 (Genbank Accession No: AK026467), CD79a (Genbank Accession No: NP-001774.1), CXCR5 (Genbank Accession No: NP-001701.1), HLA-DOB (Genbank Accession No: NP-002111.1), P2X5 (Genbank Accession No: NP-002552.2), CD72 (Genbank Accession No: NP-001773.1), LY64 (Genbank Accession No: NP-005573.1), FcRH1 (Genbank Accession No: NP-443170.1), IRTA2 (Genbank Accession No: NP-112571.1), TENB2 (Genbank Accession No: AF179274), CEA, B7H3, Her3, 5T4, Claudin 18.2, FGFR2b, FRa, TROP2, B7H4, Nectin4, CD30, c-MET, EGFR, Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), ROR1, CD20, Hepatocyte growth factor receptor (HGFR), ROR2, IL3RA, Mesothelin receptor, CD74, PDL1.

[0082] The antibody used in the present application can be an antibody corresponding to the aforementioned antigen.

[0083] A "linker" is a substance used to connect a compound having a therapeutic effect with a targeting linker that binds to a lesion site. A "linker fragment" refers to a partial structure having a linking function in the linker.

[0084] According to the intracellular drug release mechanism, the linker can be divided into two categories: non-cleavable linkers and cleavable linkers.

[0085] For a targeting linker-drug conjugate (such as an antibody-drug conjugate) containing a non-cleavable linker, the drug release mechanism can be that after the conjugate binds to the antigen and is endocytosed by the cell, the antibody is enzymatically degraded in the lysosome, and an active small molecule composed of the linker and the amino acid residues of the antibody is released.

[0086] A targeting linker-drug conjugate (such as an antibody-drug conjugate) containing a cleavable linker can be cleaved in the target cell and release the drug (such as a small molecule drug itself). Cleavable linkers can be divided into two main categories: chemically unstable linkers and enzymatically unstable linkers. Chemically unstable linkers can be selectively cleaved due to the differences in plasma and cytoplasmic properties, such as pH, glutathione concentration, etc. Enzymatically unstable linkers, such as peptide linkers, can be effectively cleaved by lysosomal proteases, such as cathepsin. Peptide linkers are considered to be very stable in plasma.

[0087] The linker is the core part of the targeting linker-drug conjugate (such as an antibody-drug conjugate), which can greatly affect the pharmacokinetics, therapeutic index, and efficacy of the targeting linker-drug conjugate (such as an antibody-drug conjugate).

[0088] The targeting linker-drug conjugate provided by the embodiments of the present application comprises a targeting linker and 1-20 covalently linked drug linking assembly units, wherein the drug linking assembly units can be connected by a thiol produced by reduction of the interchain disulfide bond in the antibody and / or each linking assembly unit is connected to a thiol from a cysteine residue.

[0089] For ease of connection, the drug-linking assembly unit is usually constructed before being connected to the targeting linker. However, the order of construction can also be changed. For example, the assembly unit with a protecting group is first connected to the targeting linker, and the protecting group is removed after being connected to the targeting linker and other drug units are added.

[0090] The drug-linking assembly unit with exatecan as the toxin provided by the application has excellent coupling effect.

[0091] The antibody-drug conjugate obtained by the drug-linking assembly unit with exatecan as the toxin provided by the application has significantly improved killing effect on HER2 positive tumor cells, and weak killing effect on HER2 negative tumor cells, proving that the ADC has strong target selectivity and meets the design principle of ADC.

[0092] The antibody-drug conjugate with exatecan as the toxin provided by the application has excellent coupling effect, safety, effectiveness, stability, low polymer content and naked antibody percentage, suitable DAR value, and exhibits excellent plasma stability, storage stability and anti-tumor effect, and has good clinical application prospect.

[0093] Obviously, according to the above content of the application, according to the ordinary technical knowledge and common means in the art, other various forms of modifications, replacements or changes can be made without departing from the above technical idea of the application.

[0094] The above content of the application will be further described in detail through the specific embodiments in the form of examples. However, this should not be understood as the scope of the above subject matter of the application being limited to the following examples. Any technology realized based on the above content of the application belongs to the scope of the application. BRIEF DESCRIPTION OF DRAWINGS

[0095] Figure 1. Results of in vivo activity detection of antibody-drug conjugate. DETAILED DESCRIPTION

[0096] The raw materials and equipment used in the application are known products, which are obtained by purchasing commercially available products.

[0097] Examples 1-10 are methods for preparing the drug-linking assembly unit of the application.

[0098] Example 1, preparation of compound SMP-95614-L

[0099] Step 1: Preparation of compound 95614-1

[0100] Into a 250 mL round-bottom flask, was placed the reactants 6-nitrophthalide (2.0 g, 11.2 mmol), MeOH (60 mL), methylamine (20 mL, 33% in ethanol), and the reaction was stirred at room temperature for 2 h. LCMS monitoring indicated the reaction was complete. The target compound 95614-1 (2.3 g, 98.0% yield) was obtained after concentration.

[0101] MS (ESI) m / z: 211.0 [M+H] +

[0102] Step 2: Preparation of compound 95614-2

[0103] Into a 50 mL round-bottom flask, was placed the reactants 95614-1 (1.0 g, 4.8 mmol) and solvent THF (10 mL), borane-dimethyl sulfide (7.1 mL, 14.2 mmol), and the reaction was stirred at reflux for 5 h. LCMS monitoring indicated the reaction was complete. The reaction was quenched with methanol at zero degrees and the target compound 95614-2 (370 mg, 39.6% yield) was obtained after concentration and reverse phase separation.

[0104] MS (ESI) m / z: 196.8 [M+H] +

[0105] Step 3: Preparation of compound 95614-3

[0106] Into a 50 mL round-bottom flask, was placed the reactants compound 95614-2 (370 mg, 1.92 mmol), N-acetyl-L-glutamic acid 5-tert-butyl ester (462 mg, 1.92 mmol) and solvent DMF (20 mL), 2-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1075.5 mg, 2.84 mmol), N,N-diisopropylethylamine (0.9 mL, 5.62 mmol), and the reaction was stirred at room temperature for 2 h. LCMS monitoring indicated the reaction was complete. The target compound 95614-3 (700 mg, 87.7% yield) was obtained after reverse phase separation.

[0107] MS (ESI) m / z: 423.8 [M+H] +

[0108] Step 4: Preparation of compound 95614-4

[0109] Into a 50-mL round-bottom flask, was added reactant 95614-3 (430 mg, 1.02 mmol), tert-butyldimethylsilyl chloride (230 mg, 1.51 mmol) and solvent DMF (10 mL), imidazole (207 mg, 3.03 mmol) sequentially, and stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction solution was poured into 100 mL of water, extracted with ethyl acetate three times, the organic phase was combined, washed with saturated brine three times, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated on a silica gel column to give the target compound 95614-4 (500.0 mg, yield 91.6%).

[0110] MS (ESI) m / z: 537.8 [M+H] +

[0111] Step 5: Preparation of compound 95614-5

[0112] Into a 50-mL round-bottom flask, was added reactant 95614-4 (500 mg, 0.93 mmol), MeOH (20 mL), Pd / C (50 mg, 10 wt%), the system was replaced with nitrogen three times, and then replaced with hydrogen three times, and stirred at room temperature for 2 h. The reaction was monitored by LCMS. The filtrate was concentrated to give the target compound 95614-5 (450 mg, purity 70%) (containing 30% by-product

[0378] + ).

[0113] MS (ESI) m / z: 507.8 [M+H] +

[0114] Step 6: Preparation of compound 95614-6

[0115] Into a 50-mL round-bottom flask, was added reactant 95614-5 (450 mg, 0.89 mmol), (S)-2-((S)-2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido (450 mg, 0.89 mmol) and solvent DMF (15 mL) sequentially, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (505 mg, 1.31 mmol), N,N-diisopropylethylamine (0.4 mL, 2.62 mmol), and stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction solution was separated by reverse phase to give the target compound 95614-6 (120 mg, yield 13.7%).

[0116] MS (ESI) m / z: 985.7 [M+H] +

[0117] Step 7: Preparation of compound 95614-7

[0118] Into a 50 mL round-bottom flask were added reactant 95614-6 (120 mg, 0.12 mmol), solvent THF (5 mL), tetrabutylammonium fluoride (0.24 mL, 1 mmol / L) sequentially, and stirred at 40 °C for 2 h. LCMS monitored the completion of the reaction. After concentration, the target compound 95614-7 (79.0 mg, 99.0%) was obtained by reverse phase preparation.

[0119] MS (ESI) m / z: 649.7 [M+H] +

[0120] Step 8: Preparation of compound 95614-8

[0121] Into a 4 mL reaction bottle were added reactant INT1 (27 mg, 0.07 mmol), 95614-7 (46 mg, 0.07 mmol) and solvent DMF (2 mL) sequentially, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (40 mg, 0.11 mmol), N,N-diisopropylethylamine (0.04 mL, 0.2 mmol), and stirred at room temperature for 2 h. LCMS monitored the completion of the reaction. The target compound 95614-8 (18 mg, yield 25.1%) was obtained by reverse phase separation of the reaction solution.

[0122] MS (ESI) m / z: 1014.7 [M+H] +

[0123] Step 9: Preparation of compound 95614-9

[0124] Into a 4 mL reaction bottle were added reactant 95614-8 (18 mg, 0.02 mmol), di(p-nitrophenyl) carbonate (8.1 mg, 0.03 mmol) and solvent DMF (2 mL) sequentially, N,N-diisopropylethylamine (0.009 mL, 0.05 mmol), and reacted at 60 °C for 16 h. LCMS monitored the completion of the reaction. The reaction solution was directly used in the next step. MS (ESI) m / z: 1179.6 [M+H] +

[0125] Step 10: Preparation of compound 95614-10

[0126] To the reaction solution of previous step, exatecan mesylate (7.4 mg, 0.02 mmol) was added and reacted at 60 °C for 2 h. LCMS was used to monitor the reaction. The reaction solution was separated by reverse phase to give the target compound 95614-10 (10 mg, yield 40.1%).

[0127] MS (ESI) m / z: 1475.9 [M+H] +

[0128] Step 11: Preparation of compound SMP-95614-L

[0129] To the reaction solution of previous step, exatecan mesylate (7.4 mg, 0.02 mmol) was added and reacted at 60 °C for 2 h. LCMS was used to monitor the reaction. The reaction solution was separated by reverse phase to give the target compound 95614-10 (10 mg, yield 40.1%).

[0130] MS (ESI) m / z: 1419.7 [M+H] +

[0131] Example 2, Preparation of compound SMP-40009-L

[0132] Step 1: Preparation of compound 40009-1

[0133] To the reaction solution of previous step, exatecan mesylate (7.4 mg, 0.02 mmol) was added and reacted at 60 °C for 2 h. LCMS was used to monitor the reaction. The reaction solution was separated by reverse phase to give the target compound 95614-10 (10 mg, yield 40.1%).

[0134] MS (ESI) m / z: 1124.7 [M+H] +

[0135] Step 2: Preparation of compound 40009-2

[0136] To the reaction solution of previous step, exatecan mesylate (7.4 mg, 0.02 mmol) was added and reacted at 60 °C for 2 h. LCMS was used to monitor the reaction. The reaction solution was separated by reverse phase to give the target compound 95614-10 (10 mg, yield 40.1%).

[0137] MS (ESI) m / z: 902.8 [M+H] +

[0138] Step 3: Preparation of compound SMP-40009-3

[0139] Compound 40009-2 (30 mg, 0.03 mmol) and INT3 (16.7 mg, 0.042 mmol) were dissolved in DMSO (3 mL), N,N-diisopropylethylamine (0.01 mL, 0.08 mmol) was added, followed by 2-(7-azobenzotriazol-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (16 mg, 0.04 mmol), after completion, the reaction was stirred at room temperature for 1 hour. LCMS monitoring reaction was completed, the reaction was prepared by reversed phase, and 40009-3 (18 mg, yield 42.3%) was obtained as a yellow solid after freeze-drying.

[0140] MS (ESI) m / z: 1280.9 [M+H] +

[0141] Step 4: Preparation of compound SMP-40009-L

[0142] The reactants 40009-3 (18 mg, 0.01 mmol) were added to a 4 mL reaction bottle, trifluoroacetic acid (2 mL) was added at zero degrees, and the reaction was stirred at zero degrees for 1 h. LCMS monitoring reaction was completed. The reaction was separated by reversed phase to obtain the target compound 40009-L (6.3 mg, yield 34.9%).

[0143] MS (ESI) m / z: 1224.9 [M+H] +

[0144] Example 3, Preparation of SMP-78968-L

[0145] First step: Preparation of compound 78968-1

[0146] To a solution of Fmoc-N-trityl-L-aspartamide (500 mg, 0.84 mmol) in tetrahydrofuran (10 mL) was added 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (249 mg, 1.01 mmol) and p-aminobenzyl alcohol (114 mg, 0.92 mmol), and stirred at room temperature overnight, LCMS monitoring reaction was completed. Concentration, the concentrated liquid was purified by high pressure liquid chromatography to obtain the target compound 78968-1 (500 mg, Y = 85.1%).

[0147] MS m / z (ESI): 684.2 [(M-17) + H] +

[0148] Second Step: Preparation of compound 78968-2

[0149] To the solution of 78968-1 (100 mg, 0.14 mmol) in N,N-dimethylformamide (3 mL) was added di(p-nitrophenyl) carbonate (64 mg, 0.21 mmol) and N,N-diisopropylethylamine (36 mg, 0.28 mmol), stirred at room temperature overnight, LCMS monitored the reaction was completed. The reaction liquid was purified by high pressure liquid chromatography to obtain the target compound 78968-2 (50 mg, Y = 41.3%).

[0150] MS m / z (ESI): 867.3 [M + H] +

[0151] Third Step: Preparation of compound 78968-3

[0152] To the solution of 78968-2 (20 mg, 0.023 mmol) and exatecan mesylate (13.44 mg, 0.025 mmol) in N,N-dimethylformamide (3 mL) was added N,N-diisopropylethylamine (7.42 mg, 0.058 mmol), stirred at room temperature for 4 hours, LCMS monitored the reaction was completed. The reaction liquid was purified by high pressure liquid chromatography to obtain the target compound 78968-3 (20 mg, Y = 74.4%).

[0153] MS m / z (ESI): 1163.4 [M + H] +

[0154] Fourth Step: Preparation of compound 78968-4

[0155] To the solution of 78968-3 (20 mg, 0.017 mmol) in N,N-dimethylformamide (4 mL) was added diethylamine (1 mL), stirred at room temperature for 30 minutes, LCMS monitored the reaction was completed. The reaction liquid was purified by high pressure liquid chromatography to obtain the target compound 78968-4 (14 mg, Y = 87.5%).

[0156] MS m / z (ESI): 941.3 [M + H] +

[0157] Fifth Step: Preparation of compound 78968-5

[0158] To a solution of 78968-4 (14 mg, 0.015 mmol) and Fmoc-O-tert-butyl-L- glutamic acid (7.66 mg, 0.018 mmol) in N,N-dimethylformamide (3 mL) was added N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-l-yl) uronium hexafluorophosphate (6.84 mg, 0.018 mmol) and N,N-diisopropylethylamine (3.87 mg, 0.03 mmol), stirred at room temperature for 1 hour, LCMS monitored the reaction was completed. The reaction liquid was purified by high pressure liquid chromatography to obtain the target compound 78968-5 (15 mg, Y = 75.1%).

[0159] MS m / z (ESI): 1348.5 [M+H] +

[0160] Sixth step: preparation of compound 78968-6

[0161] To a solution of 78968-5 (15 mg, 0.011 mmol) in N,N-dimethylformamide (4 mL) was added diethylamine (1 mL), stirred at room temperature for 30 minutes, LCMS monitored the reaction was completed. The reaction liquid was purified by high pressure liquid chromatography to obtain the target compound 78968-6 (11 mg, Y = 88.1%).

[0162] MS m / z (ESI): 1126.4 [M+H] +

[0163] Seventh step: preparation of compound 78968-7

[0164] To a solution of 78968-6 (11 mg, 0.010 mmol) and INT3 (4.50 mg, 0.012 mmol) in N,N-dimethylformamide (2 mL) was added N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-l-yl) uronium hexafluorophosphate (5.47 mg, 0.014 mmol) and N,N-diisopropylethylamine (3.10 mg, 0.024 mmol), stirred at room temperature for 1 hour, LCMS monitored the reaction was completed. The reaction liquid was purified by high pressure liquid chromatography to obtain the target compound 78968-7 (12 mg, Y = 80.5%).

[0165] MS m / z (ESI): 1491.2 [M+H] +

[0166] Eighth step: preparation of compound SMP-78968-L

[0167] To the reaction flask of 78968-7 (12 mg, 0.0081 mmol) was added trifluoroacetic acid (2 mL), stirred at room temperature for 1 hour, the reaction was monitored by LCMS. The reaction solution was concentrated at low temperature, the concentrate was purified by high pressure liquid chromatography to obtain the target compound SMP-78968-L (8 mg, Y = 82.8%).

[0168] MS m / z (ESI): 1193.3 [M+H] +

[0169] Example 4, Preparation of compound SMP-98516-L

[0170] Step 1: Preparation of compound 98516-1

[0171] To a 25 mL reaction flask was added A (50 mg, 0.046 mmol), 1-hydroxybenzotriazole (3 mg, 0.023 mmol), exetecan (20 mg, 0.046 mmol) N, N-diisopropyl ethylamine (12 mg, 0.092 mmol), N, N-dimethylformamide (3 mL) in turn, stirred at room temperature until clear, then continue to stir for 30 min after the reaction was complete, the reaction solution was distilled under reduced pressure to remove most of the solvent, the residue was purified by HPLC to obtain compound 98516-1, weight 50 mg, yield 78%.

[0172] MS (ESI) m / z: 1376.5 [M+H] + .

[0173] Step 2: Preparation of compound 98516-2

[0174] To a 25 mL reaction flask was added 98516-1 (50 mg, 0.036 mmol), N, N-dimethylformamide (2 mL) in turn, stirred at room temperature until clear, then added diethylamine (0.2 mL) continue to stir for 30 min after the reaction was complete, the reaction solution was distilled under reduced pressure to remove the solvent, the residue was purified by HPLC to obtain compound 98516-2, weight 35 mg, yield 83.6%.

[0175] MS (ESI) m / z: 1154.3 [M+H] + .

[0176] Step 3: Preparation of compound 98516-3

[0177] Into a 10 mL reaction vial, was placed intermediate INT1 (11.5 mg, 0.03 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl) uronium hexafluorophosphate (17 mg, 0.045 mmol), N,N-dimethylformamide (3 mL), N,N-diisopropyl ethylamine (7.8 mg, 0.061 mmol). After stirring the reaction at room temperature for 5 min, compound 98516-2 (35 mg, 0.03 mmol) was added and the reaction was continued for 25 min. The reaction was monitored by LCMS and was complete. The reaction was stopped and the reaction mixture was purified by HPLC to give compound 98516-3, 28 mg, 60.8% yield.

[0178] MS (ESI) m / z: 1519.7 [M+H]+.

[0179] Step 4: Preparation of compound SMP-98516-L

[0180] Into a 25 mL reaction vial, was placed compound 98516-3 (28 mg, 0.018 mmol), trifluoroacetic acid (2 mL). The reaction was stirred at 0 °C until clear, then stirred for 30 min. The reaction was monitored by LCMS and was complete. The reaction was stopped and the reaction mixture was evaporated under reduced pressure to remove the solvent. The residue was purified by HPLC to give compound SMP-98516-L, 18 mg, 66.7% yield.

[0181] MS (ESI) m / z: 1463.5 [M+H] + .

[0182] Example 5, Preparation of compound SMP-14303-L

[0183] Step 1: Preparation of compound 14303-1

[0184] Into a 25 mL reaction vial, was placed C (50 mg, 0.048 mmol), 1-hydroxybenzotriazole (3.2 mg, 0.023 mmol), and compound exatecan (20.9 mg, 0.048 mmol) N,N-diisopropyl ethylamine (12.4 mg, 0.096 mmol), N,N-dimethylformamide (3 mL). The reaction was stirred at room temperature until clear, then stirred for 30 min. The reaction was monitored by LCMS and was complete. The reaction was stopped and the reaction mixture was evaporated under reduced pressure to remove most of the solvent. The residue was purified by HPLC to give compound 14303-1, 50 mg, 78% yield.

[0185] MS (ESI) m / z: 1348.5 [M+H] + .

[0186] Step 2: Preparation of compound 14303-2

[0187] Into a 25 mL reaction vial was added 14303-1 (50 mg, 0.037 mmol), N,N- dimethylformamide (2 mL), stirred at room temperature until clear, then diethylamine (0.2 mL) was added, the reaction was stirred for another 30 min until complete by LCMS. The reaction was concentrated under reduced pressure to remove the solvent, the residue was purified by HPLC to give compound 14303-2, 37 mg in 88.7% yield.

[0188] MS (ESI) m / z: 1126.3 [M+H] + .

[0189] Step 3: Preparation of compound 14303-3

[0190] Into a 10 mL reaction vial was added sequentially intermediate INT1 (12.6 mg, 0.033 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl) uronium hexafluorophosphate (17 mg, 0.045 mmol), N,N-dimethylformamide (3 mL), N,N- diisopropylethylamine (8.5 mg, 0.066 mmol). The reaction was stirred at room temperature for 5 min until clear, then compound 14303-2 (37 mg, 0.033 mmol) was added, the reaction was stirred for another 25 min until complete by LCMS. The reaction was stopped, the reaction was purified by HPLC to give compound 14303-3, 30 mg in 61% yield.

[0191] MS (ESI) m / z: 1491.6 [M+H]+.

[0192] Step 4: Preparation of compound SMP-14303-L

[0193] Into a 25 mL reaction vial was added sequentially 14303-3 (30 mg, 0.02 mmol), trifluoroacetic acid (2 mL), stirred at 0 °C until clear, then the reaction was stirred for another 30 min until complete by LCMS. The reaction was concentrated under reduced pressure to remove the solvent, the residue was purified by HPLC to give compound SMP-14303-L, 20 mg in 72.2% yield.

[0194] MS (ESI) m / z: 1379.4 [M+H] + .

[0195] Example 6, Preparation of SMP-03539-L

[0196] Step 1: Preparation of compound 03539-1

[0197] Compound VKP (72 mg, 0.10 mmol) was dissolved in N,N-dimethylformamide (3.5 mL), formic acid (1 mL) was added at room temperature, after completion of the addition, the reaction was allowed to proceed at room temperature for 3 hours. The system was filtered, and prepared by reverse phase chromatography, and white solid 03539-1 (52.0 mg, yield 83.7%) was obtained after freeze-drying.

[0198] MS m / z (ESI): 577.6 [M+H] + .

[0199] Step 2: Preparation of compound 03539-2

[0200] Compound 03539-1 (52.0 mg, 0.09 mmol) was dissolved in N,N-dimethylformamide (4 mL), Fmoc-tert-butyl glutamate (49.5 mg, 0.12 mmol), N,N-diisopropyl ethylamine (34.8 mg, 0.27 mmol), 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (44.4 mg, 0.12 mmol) were added at room temperature, and the system was stirred at room temperature for 60 minutes. The system was filtered, and prepared by reverse phase chromatography, and white solid 03539-2 (48.0 mg, yield 54.1%) was obtained after freeze-drying.

[0201] MS m / z (ESI): 985.5 [M+H] + .

[0202] Step 3: Preparation of compound 03539-3

[0203] Compound 03539-2 (48.0 mg, 0.048 mmol) was dissolved in N,N-dimethylformamide (2.5 mL), diethylamine (0.25 mL) was added, and the reaction was allowed to proceed at room temperature for 1 hour. Prepared by reverse phase chromatography, and white solid 03539-3 (36.0 mg, yield 99.1%) was obtained after freeze-drying.

[0204] MS m / z (ESI): 762.5 [M+H] + .

[0205] Step 4: Preparation of compound 03539-4

[0206] Compound 03539-3 (36.0 mg, 0.047 mmol) was dissolved in N,N- dimethylformamide (2 mL), INT1 (23.5 mg, 0.061 mmol), N,N- diisopropylethylamine (34.8 mg, 0.141 mmol), 2-(7-azabenzotriazol-1- yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (23.4 mg, 0.061 mmol) were added successively at room temperature. After stirring at room temperature for 60 minutes, the system was filtered, and white solid 03539-4 (42.0 mg, yield 78.9%) was obtained by reverse phase chromatography preparation and freeze-drying.

[0207] MS m / z (ESI): 1127.4 [M+H] + .

[0208] Fifth step: Preparation of compound 03539-5

[0209] Compound 03539-4 (42.0 mg, 0.037 mmol), N,N-dimethylformamide (2 mL), N,N-diisopropylethylamine (14.3 mg, 0.111 mmol), diphenyldiacylurea (4-nitrophenyl) (22.4 mg, 0.074 mmol) were added successively into a 25 mL vial, and stirred at room temperature for 4 hours. After the system was filtered, white solid 03539-5 (15.0 mg, yield 31.3%) was obtained by reverse phase chromatography preparation and freeze-drying.

[0210] MS m / z (ESI): 1293.4 [M+H] + .

[0211] Sixth step: Preparation of compound 03539-6

[0212] Compound 03539-5 (15.0 mg, 0.011 mmol), N,N-dimethylformamide (1 mL), N,N-diisopropylethylamine (4.2 mg, 0.033 mmol), exatecan (6.5 mg, 0.022 mmol) were added successively into a 5 mL vial, and stirred at room temperature for 4 hours. After the system was filtered, yellow solid 03539-6 (7.0 mg, yield 37.9%) was obtained by reverse phase chromatography preparation and freeze-drying.

[0213] MS m / z (ESI): 1588.9 [M+H] + .

[0214] Seventh step: Preparation of compound SMP-03539-L

[0215] Compound 03539-6 (7.00 mg, 0.004 mmol) was dissolved in trifluoroacetic acid (0.5 mL) under ice-bath. After stirring for 30 minutes under ice-bath, the system was concentrated. After preparation by reverse phase chromatography, SMP-03539-L (1.85 mg, yield 30.5%) was obtained as a yellow solid after lyophilization.

[0216] MS m / z (ESI): 1476.6 [M+H] + .

[0217] Example 7: Preparation of compound SMP-65279-L

[0218] Step 1: Preparation of 65279-2

[0219] In a 100 mL single-necked flask, intermediate 65279-1 (200 mg, 0.28 mmol), bis(p-nitrophenyl) carbonate (130 mg, 0.42 mmol), N,N-dimethylformamide (5 mL), N,N-diisopropyl ethylamine (110 mg, 0.85 mmol) were added in turn. After stirring for 30 minutes at room temperature, the reaction was monitored by LCMS and found to be substantially complete. The reaction solution was prepared by HPLC to obtain compound 65279-2 (200 mg), a white solid, with a yield of 80.9%.

[0220] MS (ESI) m / z: 867.6 [M+H] + .

[0221] Step 2: Preparation of compound 65279-3

[0222] In a 100 mL single-necked flask, intermediate 65279-2 (200 mg, 0.230 mmol), exetecan (146 mg, 0.276 mmol), N,N-dimethylformamide (5 mL), N,N-diisopropyl ethylamine (6.7 mg, 0.05 mmol) were added in turn. After stirring for 30 minutes at room temperature, the reaction was monitored by LCMS and found to be substantially complete. The reaction solution was used directly in the next step without treatment.

[0223] MS (ESI) m / z: 1163.8 [M+H] + .

[0224] Step 3: Preparation of compound 65279-4

[0225] In a 100 mL single neck flask, diethylamine (1 mL) was added and stirred at room temperature for 30 minutes. LCMS monitoring showed that the starting material was substantially reacted. The reaction solution was prepared by HPLC to obtain compound 65279-4 (150 mg), yellowish solid, and the total yield of two steps was 69.1%.

[0226] MS (ESI) m / z: 931.2 [M+H] + .

[0227] Step 4: Preparation of compound 65279-5

[0228] In a 10 mL single neck flask, intermediate 65279-4 (40 mg, 0.043 mmol), INT1 (19.5 mg, 0.051 mmol), N,N-dimethylformamide (1 mL), N,N-diisopropylethylamine (16.48 mg, 0.128 mmol) were added in turn, and then N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl) uronium hexafluorophosphate (24.2 mg, 0.064 mmol) was added. After stirring at room temperature for 30 minutes, LCMS monitoring showed that the starting material was substantially reacted. The reaction solution was prepared by HPLC to obtain compound 65279-5 (30 mg), yellowish solid, and the yield was 54.0%.

[0229] MS (ESI) m / z: 1305.2 [M+H] + .

[0230] Step 5: Preparation of compound SMP-65279-L

[0231] In a 25 mL single neck flask, trifluoroacetic acid (2 mL) was added, and 65279-5 (30 mg, 0.023 mmol) was added. After stirring at room temperature for one hour, the trifluoroacetic acid was removed under reduced pressure, and the residual solution was purified by preparative HPLC to obtain compound SMP-65279-L (10 mg), and the yield was 40.9%.

[0232] MS (ESI) m / z: 1063.4 [M+H] + .

[0233] Example 8: Preparation of compound SMP-70067-L

[0234] Step 1: Preparation of compound SMP-70067-1

[0235] Into a 25 mL vial, was added intermediate D (40 mg, 0.036 mmol), INT1 (13.8 mg, 0.036 mmol), N,N-dimethylformamide (1 mL), N,N-diisopropylethylamine (11.3 mg, 0.087 mmol), followed by N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (20.5 mg, 0.054 mmol). After stirring at room temperature for 30 min, LCMS monitoring showed that the starting material was almost completely consumed. The reaction mixture was prepared by HPLC to give compound 70067-1 (30 mg) as a light yellow solid in a yield of 56.1%.

[0236] MS (ESI) m / z: 1448.2 [M+H] + , 724.6 [M / 2+H] + .

[0237] Step 2: Preparation of compound SMP-70067-L

[0238] Into a 25 mL vial, was added trifluoroacetic acid (2 mL), followed by 70067-1 (30 mg, 0.020 mmol). After stirring at room temperature for 1 h, the trifluoroacetic acid was removed under reduced pressure. The residue was purified by preparative HPLC to give compound SMP-70067-L (14.38 mg) in a yield of 49.8%.

[0239] MS (ESI) m / z: 1392.2 [M+H] + .

[0240] Example 9: Preparation of compound SMP-32491-L

[0241] Step 1: Preparation of compound 32491-1

[0242] Into a 100 mL vial, was added intermediate 32491-0 (3 g, 20.1 mmol), benzyl oxycarbonyl succinimide (10 g, 40.1 mmol), ethanol (50 mL), followed by stirring at 80 °C for 2 h. LCMS monitoring showed that the starting material was almost completely consumed. The reaction mixture was dried by an oil pump and directly used in the next step.

[0243] MS (ESI) m / z: 284.2 [M+H] + .

[0244] Step 2: Preparation of compound 32491-2

[0245] In a 100 mL single-necked flask, intermediate 32491-1 (5.7 g, 20.1 mmol), sodium hydroxide (2.4 g, 60.3 mmol), methanol (40 mL) and water (40 mL) were added successively. After being stirred at 60 °C for 2 h, the reaction was monitored by LCMS and the starting material was almost consumed. The reaction solution was prepared by HPLC to obtain compound 32491-2 (2.1 g), white solid, in a yield of 65.8%.

[0246] MS (ESI) m / z: 302.2 [M+H] + .

[0247] Step 3: Preparation of compound 32491-3

[0248] In a 100 mL single-necked flask, intermediate 32491-2 (1.5 g, 4.98 mmol), imidazole (0.67 g, 9.96 mmol), tert-butyldimethylsilyl chloride (1.12 g, 7.47 mmol), N,N-dimethylformamide (20 mL) were added successively. After being stirred at room temperature for 1 h, the starting material was still remained. The reaction solution was prepared by HPLC to obtain compound 32491-3 (0.8 g), white solid, in a yield of 64.4%.

[0249] MS (ESI) m / z: 416.2 [M+H] + .

[0250] Step 4: Preparation of compound 32491-4

[0251] In a 100 mL single-necked flask, intermediate 32491-3 (800 mg, 1.92 mmol), L-glutamic acid di-tert-butyl ester hydrochloride (681 mg, 2.30 mmol), N,N-dimethylformamide (10 mL), N,N-diisopropylethylamine (743 mg, 5.76 mmol) were added successively, and then N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (1.09 g, 2.88 mmol) was added. After being stirred at room temperature for 60 min, the starting material was almost consumed. The reaction solution was prepared by HPLC to obtain compound 32491-4 (900 mg), white solid, in a yield of 71.1%.

[0252] MS (ESI) m / z: 657.2 [M+H] + .

[0253] Step 5: Preparation of compound 32491-5

[0254] In a 100 mL single-necked flask, intermediate 32491-4 (900 mg, 1.37 mmol), 10% palladium carbon (50 mg), methanol (15 mL) were added successively, stirred at room temperature for 30 minutes under hydrogen atmosphere, LCMS monitoring showed that the starting material was substantially completely reacted, the reaction liquid was prepared by HPLC to obtain compound 32491-5 (550 mg), white solid, yield 76.7%.

[0255] MS (ESI) m / z: 523.4 [M+H] + .

[0256] Step 6: Preparation of compound 32491-6

[0257] In a 100 mL single-necked flask, intermediate 32491-5 (200 mg, 0.38 mmol), tetrabutylammonium fluoride (1 mL, 1M solution in tetrahydrofuran), tetrahydrofuran (5 mL) were added successively, stirred at room temperature for 30 minutes, LCMS monitoring showed that the starting material was substantially completely reacted, the reaction liquid was prepared by HPLC to obtain compound 32491-6 (120 mg), white solid, yield 76.7%.

[0258] MS (ESI) m / z: 409.2 [M+H] + .

[0259] Step 7: Preparation of compound 32491-7

[0260] In a 100 mL single-necked flask, intermediate 32491-6 (90 mg, 0.22 mmol), (S)-2-((S)-2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido (131 mg, 0.26 mmol), tetrahydrofuran (5 mL), N,N-diisopropylethylamine (85 mg, 0.66 mmol) were added successively, then N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)urea hexafluorophosphate (125.4 mg, 0.33 mmol) was added, stirred at room temperature for 2 h, LCMS monitoring showed that the starting material was substantially completely reacted, the reaction liquid was prepared by HPLC to obtain compound 32491-7 (100 mg), white solid, yield 51.1%.

[0261] MS (ESI) m / z: 887.4 [M+H] + .

[0262] Step 8: Preparation of compound 32491-8

[0263] In a 100 mL single necked flask, was added intermediate 32491-7 (100 mg, 0.11 mmol), bis(p-nitrophenyl) carbonate (50.2 mg, 0.17 mmol), N,N-dimethylformamide (2 mL), N,N-diisopropyl ethylamine (28.4 mg, 0.22 mmol), stirred at room temperature for 30 minutes, LCMS monitoring showed the starting material was almost reacted completely, the reaction solution was prepared by HPLC to obtain compound 32491-8 (75 mg), white solid, yield 63.2%.

[0264] MS (ESI) m / z: 1052.4 [M+H] + .

[0265] Step 9: Preparation of compound 32491-9

[0266] In a 100 mL single necked flask, was added intermediate 32491-8 (75 mg, 0.071 mmol), exatecan (30.9 mg, 0.071 mmol), N,N-dimethylformamide (1 mL), N,N-diisopropyl ethylamine (27.5 mg, 0.213 mmol), stirred at room temperature for 30 minutes, LCMS monitoring showed the starting material was almost reacted completely, the reaction solution was directly used for the next step without treatment.

[0267] MS (ESI) m / z: 1348.2 [M+H] + .

[0268] Step 10: Preparation of compound 32491-10

[0269] In a 100 mL single necked flask of step 9, was added diethylamine (1 mL), stirred for 30 minutes. LCMS monitoring showed the starting material was almost reacted completely, the reaction solution was prepared by HPLC to obtain compound 32491-10 (35 mg), yellowish solid, yield 32.7%.

[0270] MS (ESI) m / z: 1126.2 [M+H] + .

[0271] Step 11: Preparation of compound 32491-11

[0272] Into a 10 mL vial, was added intermediate 32491-10 (35 mg, 0.031 mmol), INT1 (14.3 mg, 0.037 mmol), N,N-dimethylformamide (1 mL), N,N-diisopropylethylamine (12.0 mg, 0.093 mmol), followed by N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl) uronium hexafluorophosphate (17.7 mg, 0.046 mmol). After stirring at room temperature for 30 min, LCMS monitoring showed that the starting material was almost completely consumed. The reaction mixture was directly subjected to HPLC to give compound 32491-11 (25 mg) as a yellow solid in 53.9% yield.

[0273] MS (ESI) m / z: 1491.2 [M+H] + .

[0274] Step 12: Preparation of compound SMP-32491-L

[0275] Into a 25 mL vial, was added trifluoroacetic acid (2 mL), followed by 32491-11 (25 mg, 0.016 mmol). After stirring at room temperature for 1 h, the trifluoroacetic acid was removed under reduced pressure. The residue was purified by HPLC to give compound SMP-32491-L (6.8 mg) in 29.4% yield.

[0276] MS (ESI) m / z: 1379.2 [M+H] + .

[0277] Example 10: Preparation of compound SMP-74694-L

[0278] Step 1: Preparation of compound 74694-1

[0279] Into a 100 mL vial, was added intermediate x (130 mg, 0.155 mmol), exetecan (75 mg, 0.170 mmol), N,N-dimethylformamide (2 mL), N,N-diisopropylethylamine (60 mg, 0.46 mmol). After stirring at room temperature for 30 min, LCMS monitoring showed that the starting material was almost completely consumed. The reaction mixture was directly subjected to the next step.

[0280] MS (ESI) m / z: 1134.4 [M+H]+.

[0281] Step 2: Preparation of compound 74694-2

[0282] In a 100 mL single neck flask, add diethylamine (1 mL) and stir for 30 minutes. Monitor the reaction by LCMS until the starting material is substantially consumed. Prepare compound 74694-2 (70 mg) as a yellowish solid by HPLC purification. Yield: 49.4%.

[0283] MS (ESI) m / z: 912.2 [M+H] + .

[0284] Step 3: Preparation of compound 74694-3

[0285] In a 50 mL single neck flask, add sequentially intermediate 74694-2 (70 mg, 0.077 mmol), N-acetyl-L-glutamic acid 5-tert-butyl ester (22.6 mg, 0.092 mmol), N,N-dimethylformamide (1 mL), N,N-diisopropylethylamine (29.8 mg, 0.23 mmol), and then N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-l-yl)uronium hexafluorophosphate (43.9 mg, 0.116 mmol). Stir the reaction at room temperature for 30 minutes. Monitor the reaction by LCMS until the starting material is substantially consumed. Prepare compound 74694-3 (45 mg) as a yellowish solid by HPLC purification. Yield: 51.4%.

[0286] MS (ESI) m / z: 1139.2 [M+H] + .

[0287] Step 4: Preparation of compound 74694-4

[0288] In a 25 mL single neck flask, add trifluoroacetic acid (2 mL) and then 74694-3 (45 mg, 0.039 mmol). Stir the reaction at room temperature for one hour. Remove the trifluoroacetic acid under reduced pressure. Purify the residue by preparative HPLC to obtain compound 74694-4 (10 mg). Yield: 64.4%.

[0289] MS (ESI) m / z: 983.4 [M+H] + .

[0290] Step 5: Preparation of compound SMP-74694-L

[0291] In a 25 mL single neck flask, intermediate 74694-4 (25 mg, 0.025 mmol), INT1 (10.5 mg, 0.027 mmol), N,N-dimethylformamide (1 mL), N,N-diisopropylethylamine (9.6 mg, 0.075 mmol) were added successively, then N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl) uronium hexafluorophosphate (14.3 mg, 0.037 mmol) was added. After stirring at room temperature for 30 minutes, the reaction was monitored by LCMS and found to be substantially complete. The reaction solution was subjected to HPLC preparation to obtain compound SMP-74694-L (4.4 mg), a yellowish solid, in a yield of 12.8%.

[0292] MS (ESI) m / z: 1348.2 [M+H] + .

[0293] Example 11 is a method for preparing the antibody-drug conjugate of the present application.

[0294] Example 11: Preparation of antibody-drug conjugate

[0295] The antibody-drug conjugate was prepared by using the disulfide bond on the antibody to couple the drug linker assembly unit obtained from Examples 1-10. The coupling method is as follows:

[0296] First, the antibody was prepared into a solution with a concentration of 20 mg / mL. 0.1 mL of the antibody was taken into a 1.5 mL centrifuge tube, and PBS (pH 7.4) / DTPA solution (293 uL) was added. Then 5 mM TCEP aqueous solution (6.4 uL, 2.4 equivalents relative to one molecule of antibody) was added, and the solution was incubated at 25°C for 2 hours to reduce the disulfide bond in the hinge part of the antibody to a thiol group. Then 10% medical DMSO solution containing 10 mM drug linker assembly unit (13.3 uL, 10 equivalents relative to one molecule of antibody) was added to the above solution, and the solution was shaken (at a speed of 400 revolutions per minute) at 25°C for 2 hours to link the drug linker assembly unit and the antibody, thereby obtaining the antibody-drug conjugate. The characterization results are shown in Table 1.

[0297] The antibody used in this example is an anti-HER2 antibody, specifically trastuzumab or pertuzumab.

[0298] Pertuzumab

[0299] Light chain

[0300] Trastuzumab

[0301] Light chain

[0302] Examples 1-4 are methods of preparing control drug-linking assembly units. Example 1, preparation of compound SMP-74392-1

[0303] Step 1: Preparation of compound 74392-1

[0304] Into a 10 mL vial, was added sequentially intermediate 74392-0 (200 mg, 0.353 mmol), Exatecan (153 mg, 0.353 mmol), N,N-dimethylformamide (3 mL), N,N- diisopropylethylamine (137 mg, 1.06 mmol), the mixture was cooled at 0 °C for 10 min, then N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (134 mg, 0.353 mmol) was added, the stirring was continued for 30 min, LCMS monitoring showed the starting material was almost consumed. The reaction mixture was prepared by HPLC to give compound 74392-1 (290 mg), white solid, yield 83.6%.

[0305] MS (ESI) m / z: 985.4 [M+H] + .

[0306] Step 2: Preparation of compound 74392-2

[0307] Into a 10 mL vial, was added sequentially intermediate 74392-1 (290 mg, 0.294 mmol), N,N-dimethylformamide (4 mL), diethylamine (0.4 mL), the stirring was continued for 30 min, LCMS monitoring showed the starting material was almost consumed. The reaction mixture was prepared by HPLC to give compound 74392-2 (200 mg), white solid, yield 89.2%.

[0308] MS (ESI) m / z: 763.4 [M+H] + .

[0309] Step 3: Preparation of compound 74392-3

[0310] Into a 10-mL vial, were added Intermediate 74392-2 (200 mg, 0.262 mmol), N-acetyl-L-glutamic acid-5-tert-butyl ester (64 mg, 0.262 mmol), N,N-dimethylformamide (3 mL), N,N-diisopropyl ethylamine (101 mg, 0.786 mmol), the mixture was cooled at 0 °C for 10 min, then N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-l-yl)uronium hexafluorophosphate (100 mg, 0.262 mmol) was added, the stirring was continued for 30 min, LCMS monitoring showed the starting material was almost consumed. The reaction mixture was prepared by HPLC to give compound 74392-3 (180 mg) as a white solid in 69.2% yield.

[0311] MS (ESI) m / z: 990.5 [M+H] + .

[0312] Step 4: Preparation of compound 74392-4

[0313] Into a 25-mL vial, were added Intermediate 74392-3 (180 mg, 0.182 mmol) and trifluoroacetic acid (2.0 mL), the mixture was stirred at room temperature for 30 min. The trifluoroacetic acid was removed by concentration, the residue was prepared by HPLC to give compound 74392-4 (100 mg) as a colorless liquid in 65.8% yield.

[0314] MS (ESI) m / z: 834.4 [M+H] + .

[0315] Step 5: Preparation of compound SMP-74392-L

[0316] Into a 10-mL vial, were added Intermediate 74392-4 (40 mg, 0.048 mmol), INT3 (18.4 mg, 0.048 mmol), N,N-dimethylformamide (2 mL), N,N-diisopropyl ethylamine (18.6 mg, 0.144 mmol), the mixture was cooled at 0 °C for 10 min, then N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-l-yl)uronium hexafluorophosphate (18.2 mg, 0.048 mmol) was added, the stirring was continued for 30 min, LCMS monitoring showed the starting material was almost consumed. The reaction mixture was prepared by HPLC to give compound SMP-74392-L (4.3 mg) as a white solid in 7.6% yield.

[0317] MS (ESI) m / z: 1199.5 [M+H] + .

[0318] Preparation of Comparative Example 2, SMP-98545-L

[0319] First Step: Preparation of Compound SMP-98545-1

[0320] Compound Exatecan (100 mg, 0.23 mmol) was dissolved in N,N- dimethylformamide (4 mL), Fmoc-L-aspartic acid (106 mg, 0.30 mmol), N,N- diisopropylethylamine (90 mg, 0.69 mmol), 2-(7-azabenzotriazolyl)-N,N,N',N'- tetramethyluronium hexafluorophosphate (114 mg, 0.30 mmol) were added at room temperature. After stirring at room temperature for 60 minutes, the system was filtered and prepared by reverse phase chromatography to obtain yellow solid 98545-1 (110 mg, yield 62.0%) after lyophilization.

[0321] MS m / z (ESI): 772.2 [M+H] + .

[0322] Second Step: Preparation of Compound 98545-2

[0323] Compound 98545-1 (110 mg, 0.142 mmol) was dissolved in N,N- dimethylformamide (2.5 mL), diethylamine (0.25 mL) was added, and after completion, the reaction was carried out at room temperature for 1 hour. Prepared by reverse phase chromatography, and yellow solid 98545-2 (75 mg, yield 96.2%) was obtained after lyophilization.

[0324] MS m / z (ESI): 550.1 [M+H] + .

[0325] Third Step: Preparation of Compound 98545-3

[0326] Compound 98545-2 (45 mg, 0.081 mmol) was dissolved in N,N- dimethylformamide (4 mL), Fmoc-L-glutamic acid tert-butyl ester (45 mg, 0.11 mmol), N,N-diisopropylethylamine (31 mg, 0.24 mmol), 2-(7-azabenzotriazolyl)-N,N,N',N'- tetramethyluronium hexafluorophosphate (40 mg, 0.11 mmol) were added at room temperature. After stirring at room temperature for 60 minutes, the system was filtered and prepared by reverse phase chromatography to obtain yellow solid 98545-3 (43 mg, yield 55.4%) after lyophilization.

[0327] MS m / z (ESI): 957.2 [M+H]+ .

[0328] Fourth Step: Preparation of compound 98545-4

[0329] Compound 98545-3 (43 mg, 0.044 mmol) was dissolved in N,N-dimethylformamide (2.5 mL), diethylamine (0.25 mL) was added, and after completion, the reaction was allowed to proceed at room temperature for 1 hour. After preparation by reverse phase chromatography, a yellow solid 98545-4 (25.0 mg, yield 77.2%) was obtained after lyophilization.

[0330] MS m / z (ESI): 735.3 [M+H] + .

[0331] Fifth Step: Preparation of compound 98545-5

[0332] Compound 98545-4 (25 mg, 0.034 mmol) was dissolved in N,N-dimethylformamide (4 mL), INT3 (17 mg, 0.044 mmol), N,N-diisopropylethylamine (13 mg, 0.10 mmol), 2-(7-azabenzotriazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (17 mg, 0.044 mmol) were added at room temperature, and after stirring at room temperature for 1.5 hours, the system was filtered, and after preparation by reverse phase chromatography, a yellow solid 98545-5 (15 mg, yield 40.1%) was obtained after lyophilization.

[0333] MS m / z (ESI): 1100.4 [M+H] + .

[0334] Sixth Step: Preparation of compound SMP-98545-L

[0335] Compound 98545-5 (15 mg, 0.013 mmol) was dissolved in trifluoroacetic acid (0.5 mL) under ice bath, and after stirring in an ice bath for 30 minutes, the system was filtered, and after preparation by reverse phase chromatography, a yellow solid SMP-98545-L (3.62 mg, yield 26.6%) was obtained after lyophilization.

[0336] MS m / z (ESI): 1044.4 [M+H] + .

[0337] Preparation of compound SMP-00840-L

[0338] First Step: Preparation of compound SMP-00840-L

[0339] Compound SMP-98545-2 (30 mg, 0.054 mmol) was dissolved in N,N- dimethylformamide (3 mL), B (48.9 mg, 0.070 mmol), N,N-diisopropylethylamine (21 mg, 0.162 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (36 mg, 0.070 mmol) were added at room temperature. After stirring for 60 minutes at room temperature, the system was filtered, and compound SMP-00840-L (1.7 mg, 2.5% yield) was obtained by reverse phase chromatography preparation and lyophilization as a yellow solid.

[0340] MS m / z (ESI): 1229.2 [M+H] + .

[0341] Preparation of Comparative Example 4, SMP-89369-L

[0342] Step 1: Preparation of compound SMP-89369-1

[0343] Into a 25 mL vial, intermediate D (40 mg, 0.036 mmol), 6-maleimidocaproic acid (7.6 mg, 0.036 mmol), N,N-dimethylformamide (1 mL), N,N- diisopropylethylamine (11.3 mg, 0.087 mmol) were added in sequence, and then N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (20.5 mg, 0.054 mmol) was added. After stirring for 30 minutes at room temperature, the reaction was monitored by LCMS and found to be substantially complete. The reaction solution was purified by HPLC to obtain compound 89369-1 (20 mg) as a light yellow solid in a yield of 58.8%.

[0344] MS (ESI) m / z: 1275.9 [M+H]+

[0345] Step 2: Preparation of compound SMP-89369-L

[0346] Into a 25 mL vial, trifluoroacetic acid (2 mL) was added, and then 89369-1 (20 mg, 0.020 mmol) was added. After stirring for one hour at room temperature, the trifluoroacetic acid was removed under reduced pressure, and the residue was purified by preparative HPLC to obtain compound SMP-89369-L (12.1 mg) in a yield of 63.3%.

[0347] MS (ESI) m / z: 1219.9 [M+H] + .

[0348] Comparative Example 5 is a method for preparing a control antibody-drug conjugate.

[0349] Comparative Example 5: Preparation of an antibody-drug conjugate

[0350] A control antibody-drug conjugate was prepared according to the method of Example 11 above, except that the drug-linker assembly unit was replaced by the product obtained in Comparative Example 1-4. The results of the characterization of the control antibody-drug conjugate obtained are shown in Table 1.

[0351] To demonstrate the beneficial effects of the present application, the following experimental examples are provided.

[0352] Experimental Example 1: Detection of conjugation efficiency of antibody-drug conjugates

[0353] 1. Experimental method

[0354] Test sample: antibody-drug conjugates prepared in Examples and Comparative Examples, using trastuzumab as the anti-HER2 antibody.

[0355] Molecular exclusion high performance liquid chromatography (SE-HPLC) was used to detect the content of aggregates (high molecular components) in the antibody-drug conjugates. A Biocore SEC column (7.8 x 300 mm, 5 μm) was used, with a mobile phase of 50 mM phosphate (pH 6.8) containing 300 mM sodium chloride: isopropanol = 95:5 (V / V), a flow rate of 0.5 mL / min, a sample of 50 μg, a sample volume of 10 μl, and UV absorbance at 280 nm observed for at least 30 min. The content of aggregates in the antibody-drug conjugates was calculated by area normalization.

[0356] Reversed phase high performance liquid chromatography-mass spectrometry (RP-HPLC-MS) was used to detect the drug-to-antibody conjugation ratio (also referred to as the antibody-drug ratio, abbreviated as DAR) of the antibody-drug conjugates. The ADC sample was diluted to 1 mg / ml with 50 mmol / L Tris buffer (pH 8.0), and freshly prepared dithiothreitol (DTT) stock solution was added to give a final DTT concentration of 50 mmol / L, and incubated at 37°C for 30 min. A PLRP-S column (5 μm, 21.4 x 250 mm) was used, with a mobile phase of 50 mmol / L Tris buffer (pH 8.0) containing 0.1% trifluoroacetic acid, a flow rate of 0.5 mL / min, a sample of 50 μg, a sample volume of 10 μl, and UV absorbance at 280 nm observed for at least 30 min. The DAR of the antibody-drug conjugates was calculated by area normalization. Poroshell 120 EC-C18, 2.1 x 50 mm, 5 μm), column temperature 70 °C. Flow rate 0.25 mL / min. Injection 10-20 ug, mobile phase A 0.1% formic acid (V / V) and 0.025% trifluoroacetic acid (V / V) in water, mobile phase B 0.1% formic acid (V / V) and 0.025% trifluoroacetic acid (V / V) in acetonitrile, 0-3 min, 27% mobile phase B for 3 min, 3-25 min mobile phase B from 27% to 49%, 25-26 min mobile phase B from 49% to 95%, 26-31 min mobile phase B at 95%, 30-31.5 min mobile phase B from 95% to 27%, 31.5-45 min, mobile phase B at 27%. UV absorbance was observed at 280 nm. Mass spectrometry dry gas temperature 350 °C, dry gas flow 7.0 L / min, nebulizer pressure 40 psi, capillary voltage positive 3000 V negative 3000 V, mass range 500-1600 m / z, the molecular weight of each mass peak was calculated by deconvolution (range 20000-70000) and compared to the light and heavy chain molecular weight of the unconjugated antibody, the drug loading of each peak was calculated and the corresponding UV peak loading was determined. The area percentage of each heavy and light chain UV peak area was calculated by integration and combined with the drug loading of each peak to calculate the weighted average DAR of the antibody-drug conjugate.

[0357] The content of naked antibody in the antibody-drug conjugate was determined using hydrophobic interaction chromatography (HIC). A Biocore HIC-Butyl column (4.6 x 150 mm, 5 μm) was used. Mobile phase A was 100 mM phosphate buffer (pH 7.0) containing 2 M ammonium sulfate, mobile phase B was 100 mM phosphate buffer (pH 7.0): isopropanol = 80:20 (V / V). Injection sample 50 ug, injection 10 ul. Flow rate 1 mL / min, mobile phase A at 100% for 3 min. 3-25 min, mobile phase B from 0% to 100%, 25.1 mobile phase B decreased to 0%, run to 30 min. UV absorbance was observed at 280 nm. The retention time of naked antibody peak was located by the antibody of the antibody-drug conjugate, and the naked antibody content of the antibody-drug conjugate was calculated according to the area normalization method.

[0358] 2. Experimental results

[0359] Table 1. Detection results of antibody-drug conjugates

[0360] It can be seen that all the drug linker assembly units of the embodiments of the present application have excellent conjugation characteristics.

[0361] Experimental example 2: activity detection of antibody-drug conjugates

[0362] 1. Experimental method

[0363] Tested sample: antibody-drug conjugate prepared by the examples, and the anti-HER2 antibody used is trastuzumab.

[0364] Test method: On the first day of the experiment, cells in the logarithmic growth phase were counted and plated at 2000 / well, and then incubated in a 5% CO2 incubator overnight. The next day, sample stock solutions were prepared, the samples were diluted in a 3-fold gradient, and the diluted samples were added to the corresponding wells, and the same volume of PBS or DMSO solution was added to the negative control group, and then incubated in a 5% CO2 incubator for 120 hours. After adding 50 μL of CTG detection solution and shaking to mix, stand for 10 minutes, and then use an enzyme-labeled instrument to read the plate. Calculate the half-inhibitory concentration (IC 50 ) value.

[0365] 2. Experimental results

[0366] Table 2. IC 50 value test results of antibody-drug conjugate inhibition of tumor cells

[0367] From the cell activity results, it can be seen that the ADC obtained by the drug-linking assembly unit of the comparative example has almost no killing effect on HER2-positive tumor cells, and does not have the basic ADC drug-making conditions; the ADC obtained by the drug-linking assembly unit of the example has a killing effect on HER2-positive tumor cells that is about 100 times higher than that of the comparative example ADC, which shows that the ADC obtained by the drug-linking assembly unit with the specific structure of the example of the present application exhibits a significantly improved inhibitory effect on HER2-positive tumor cells.

[0368] In addition, the killing effect of the example ADC on HER2-negative tumor cells is weak, which proves that these ADCs have strong target selectivity, which meets the design principle of ADCs.

[0369] Experimental example 3: in vivo activity detection of antibody-drug conjugate

[0370] 1. Experimental method

[0371] Tested sample: antibody-drug conjugate prepared by the examples, and the anti-HER2 antibody used is trastuzumab. The HER2 ADC DS-8201a that has been marketed is used as a control, and DS-8201a is purchased from Daiichi Sankyo Deutschland GmbH.

[0372] 1.1 Establishment of tumor model:

[0373] Gastric cancer JIMT1 tumor cells were cultured in culture medium and maintained in a 37℃ saturated humidity incubator containing 5% CO2. Logarithmic growth phase tumor cells were collected and resuspended in basal medium to adjust the cell concentration to 1×10 8 / mL. Under sterile conditions, 0.1 mL of cell suspension was inoculated subcutaneously into the right armpit of mice at a concentration of 1×10 7 Cells / 0.1 mL / mouse.

[0374] 1.2 Grouping and administration observation

[0375] When the tumor volume reached ~110mm 3 , the animals were randomly grouped according to the tumor volume, the grouping day was recorded as Day 0, and the administration was performed according to the animal body weight. During the experiment, the tumor volume was measured twice a week, and the animal clinical symptoms were observed and recorded daily. After the last measurement, the remaining animals were euthanized and sacrificed with CO2.

[0376] 2. Experimental results

[0377] As can be seen from the experimental results (Figure 1), the ADC of the present application has good tumor growth inhibition activity in vivo, and the tumor inhibition effect of all ADCs is better than the currently marketed HER2 ADC DS-8201a at the same dose, and also has a good dose-effect relationship.

Claims

1. A linker for a targeted connector-drug conjugate, its stereoisomer, its optical isomer, its salt, or its deuterated derivative, characterized in that: the linker comprises a structure of Formula I: T is a tethering group capable of being linked to a targeting linker; L is a linker fragment which is absent, cleavable or non-cleavable; a is selected from 0, 1, 2, 3, 4 or 5; b is selected from 0, 1, 2, 3, 4 or 5; c is selected from 0, 1, 2, 3 or 4; R1is each independently selected from hydrogen, a carboxyl-containing group; R2is each independently selected from hydrogen, a carboxyl-containing group.

2. The linker of claim 1, a stereoisomer thereof, an optical isomer thereof, a salt thereof, or a deuterated form thereof, wherein: comprises a structure represented by Formula II or Formula III:

3. The linker of claim 1 or 2, a stereoisomer thereof, an optical isomer thereof, a salt thereof, or a deuterated form thereof, characterized in that: said R1is selected from hydrogen, R2is selected from hydrogen, wherein R3 is an amino protecting group, R4 is selected from hydrogen, C 1-3 alkyl, and R5 is selected from hydrogen, C 1-3 alkyl.

4. The linker of claim 3, a stereoisomer thereof, an optical isomer thereof, a salt thereof, or a deuterated form thereof, wherein: said R1is selected from hydrogen, R2is selected from hydrogen, 5. The linker of any one of claims 1-4, a stereoisomer thereof, an optical isomer thereof, a salt thereof, or a deuterated form thereof, wherein: The L is A-B, wherein A is selected from the group consisting of B is selected from the group consisting of no, R6is an amino protecting group.

6. The linker of claim 5, a stereoisomer thereof, an optical isomer thereof, a salt thereof, or a deuterated form thereof, wherein: The L is A-B, wherein A is selected from the group consisting of B is selected from 7. The linker of claim 1, a stereoisomer thereof, an optical isomer thereof, a salt thereof, or a deuterated form thereof, wherein: one of the following structures is included in the linker:

8. A pharmaceutical link assembly, stereoisomer thereof, optical isomer thereof, salt thereof, or deuterated form thereof, characterized in that: The drug-linking assembly unit is formed by linking the linker of any one of claims 1-7 and the drug.

9. The pharmaceutical link assembly unit of claim 8, stereoisomer thereof, optical isomer thereof, salt thereof, or deuterated thereof, characterized in that: The drug is irinotecan.

10. The pharmaceutical link assembly unit of claim 9, stereoisomer, optical isomer, salt or deuterated thereof, characterized in that: The drug link assembly unit is selected from one of the following structures:

11. A targeted linker-drug conjugate, stereoisomer thereof, optical isomer thereof, salt thereof, or deuterated form thereof, characterized in that: The targeting linker-drug conjugate is assembled by linking the targeting linker and the drug-linking assembly unit of any one of claims 8-10, and the structure of the targeting linker-drug conjugate is shown as Formula IV: Ab is a targeting linker; q is an integer from 1 to 20; The targeting linker is a substance capable of targeting binding to a lesion site.

12. The targeted linker-drug conjugate of claim 11, stereoisomer thereof, optical isomer thereof, salt thereof, or deuterated form thereof, characterized in that: The DAR value of the targeting linker-drug conjugate is 1.00-20.00, preferably 4.00-8.

20.

13. The targeted linker-drug conjugate of claim 12, stereoisomer thereof, optical isomer thereof, salt thereof, or deuterated form thereof, characterized in that: The targeting linker-drug conjugate is: with a DAR value of 5.10-5.50, preferably 5.28; Or, the targeting linker-drug conjugate is: with a DAR value of 6.90-7.30, preferably 7.12; Or, the targeting linker-drug conjugate is: with a DAR value of 7.65-8.05, preferably 7.85; Or, the targeting linker-drug conjugate is: with a DAR value of 7.80-8.20, preferably 8.00; Or, the targeting linker-drug conjugate is: with a DAR value of 7.70-8.10, preferably 7.89; Or, the targeting linker-drug conjugate is: with a DAR value of 6.10-6.50, preferably 6.31; Or, the targeting linker-drug conjugate is: with a DAR value of 6.90-7.30, preferably 7.11; Or, the targeting linker-drug conjugate is: with a DAR value of 7.80-8.20, preferably 8.00; Or, the targeting linker-drug conjugate is: with a DAR value of 7.80-8.20, preferably 8.00; Or, the targeting linker-drug conjugate is: with a DAR value of 7.25-7.65, preferably 7.

46.

14. The targeted linker-drug conjugate, stereoisomer, optical isomer, salt, or deuterated form thereof of any one of claims 11-13, wherein: The targeting linker is an antibody, an antibody fragment, a protein, a small molecule polypeptide, a glycopeptide, a peptidomimetic, a small molecule compound or a nucleic acid oligonucleotide aptamer; Preferably, the antibody is an antibody against a cell surface receptor and a tumor-associated antigen; More preferably, the antibody is an anti-HER3 antibody, an anti-B7H3 antibody, an anti-TROP2 antibody, an anti-HER2 antibody or an anti-CEACAM5 antibody.

15. A pharmaceutical preparation for preventing and / or treating a tumor, characterized by: It is a preparation made of the targeting linker-drug conjugate of any one of claims 11-14, a stereoisomer thereof, an optical isomer thereof, a salt thereof or a deuterated thereof as an active ingredient, plus a pharmaceutically acceptable adjuvant.

16. Use of the targeting linker-drug conjugate of any one of claims 11-14, a stereoisomer thereof, an optical isomer thereof, a salt thereof or a deuterated thereof in the preparation of a pharmaceutical preparation for preventing and / or treating a tumor.

17. Use according to claim 16, characterized in that: The tumor is a HER3-positive, B7H3-positive, HER2-positive or CEACAM5-positive tumor.

18. Use according to claim 16, characterized in that: The tumor is selected from lung cancer, lung adenocarcinoma, urethral cancer, large intestine cancer, prostate adenocarcinoma, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, colon cancer, rectal cancer, colorectal cancer, uterine cancer, salivary gland cancer, kidney cancer, vulvar cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma or sarcoma.

19. A method of preparing the targeted linker-drug conjugate, stereoisomer thereof, optical isomer thereof, salt thereof, or deuterated form thereof of any one of claims 11-14, characterized in that: The method comprises the following steps: coupling the drug-linker assembly unit with the targeting linker to obtain a targeting linker-drug conjugate; Or, the method comprises the following steps: (1) coupling a drug with a linker to obtain a drug-linker assembly unit; (2) coupling the drug-linker assembly unit with the targeting linker to obtain a targeting linker-drug conjugate; Or, the method comprises the following steps: (1') coupling a targeting linker with a linker to obtain a targeting linker-linker unit; (2') coupling the targeting linker-linker unit with a drug to obtain a targeting linker-drug conjugate.

Images