Preparation method for biologically active compound

Abstract

The present application provides a preparation method for a biologically active compound. The method has the advantages of low costs, mild reaction conditions, high product yield and simple operation, thereby facilitating process scale-up.

Description

Preparation methods of biologically active compounds

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to CN application No. 202510123075.7 filed on January 24, 2025 and CN application No. 202510954249.4 filed on July 10, 2025, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This application relates to the field of pharmaceutical chemicals, specifically to a method for preparing a biologically active compound. Background Technology

[0004] Antibody-drug conjugates (ADCs) are novel biotherapeutic agents that link monoclonal antibodies with cytotoxic drugs via chemical linkers. The concept was first proposed by Paul Ehrlich in the early 20th century, who described them as "magic bullets." Linkers are a crucial component of ADCs, and their design influences their stability and drug release efficiency. Early linkers were mostly non-cleavable, but later, various cleavable linkers were developed, such as the cathepsin B-sensitive Val-Cit linker, which can cleave in specific intracellular environments to release cytotoxic drugs. The selection and optimization of the payload are critical to the efficacy of ADCs. From the initial vinblastine and DNA damaging agents to calcipomycin, orestane, and maytansine, and now to PBD and camptothecin derivatives, the variety of payloads has continuously increased, resulting in stronger tumor-killing effects and better stability. With continuous technological advancements and innovation, ADC therapy has a very broad application prospect in the field of cancer treatment, and is expected to provide patients with more treatment options.

[0005] The load linker in an ADC (Antibody-Drug Concentrate) is a crucial component, determining not only the drug's stability in vivo but also its release efficiency within tumor cells. Significant progress has been made in the synthesis of load linkers in recent years. The synthesis process of load linkers is a key factor affecting the commercial production of ADC drugs, including impurity control and process scale-up, all of which directly impact the production cost of antitumor drugs.

[0006] Currently, the synthetic methods for loaded linkers have many shortcomings that require improvement, such as harsh reaction conditions, high equipment requirements, long reaction times, low yields of target products, difficulty in isomer control, and high production costs. Therefore, there is still a need to develop a synthetic route for loaded linker compounds that offers high yields, low costs, high product purity, and fewer side reactions. Summary of the Invention

[0007] To address the aforementioned technical problems, this application provides a method for preparing the compound of formula (I) or an acceptable pharmaceutical salt thereof:

[0008] Wherein: Z1 is selected from C 1-6 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 3-8 Cycloalkylene, 6-10 aryl and 5-14 heteroaryl;

[0009] Z2 is selected from oxygen or sulfur;

[0010] Ring A is selected from C 6-10 Aryl, 5-10 heteroaryl, C 3-6 Cycloalkyl and 5-10 membered heterocyclic groups; said ring A is optionally substituted by one or more of the following substituents: halogen (e.g., F, Cl or Br), nitro, cyano, oxo (=O), C 1-6 Alkyl and C 1-6 Alkoxy;

[0011] m can be independently 0, 1, 2, 3, 4, 5, or 6.

[0012] E is selected from the following groups optionally substituted with one or more R1s: 6-10 aryl, 5-14 heteroaryl; wherein R1 is independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-6 Alkyl and Halogenated C 1-6 alkyl;

[0013] G is the leaving group for nucleophilic substitution reactions;

[0014] The method includes preparing the compound shown in formula (I) by means of a compound of formula (I-1):

[0015] In some embodiments, the compound of formula (I-1) is prepared by a condensation reaction to form the compound of formula (I).

[0016] In some embodiments, the condensation reaction is carried out in the presence of one of the reagents in NHS or PFP.

[0017] In some embodiments, the condensation reaction is carried out in the presence of one or both of the reagents DIC and DCC (N,N'-dicyclohexylcarboimide).

[0018] In some embodiments, the condensation reaction is carried out in the presence of one or more reagents of NHS, DIC, and DCC.

[0019] In some embodiments, the condensation reaction is carried out in the presence of NHS and DIC.

[0020] In some embodiments, the condensation reaction is carried out in the presence of one or more reagents of PFP, DIC, and DCC.

[0021] In some embodiments, the condensation reaction is carried out in the presence of PFP and DIC.

[0022] In some embodiments, the molar ratio of the NHS reagent to the compound of formula (I-1) is (0.1–5):1. In some embodiments, the molar ratio of the NHS reagent to the compound of formula (I-1) is (0.8–3):1. In some embodiments, the molar ratio of the NHS reagent to the compound of formula (I-1) is (1–2):1. In some embodiments, the molar ratio of the NHS reagent to the compound of formula (I-1) is 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, or 1.8:1.

[0023] In some embodiments, the molar ratio of the PFP reagent to the compound of formula (I-1) is (0.1–5):1. In some embodiments, the molar ratio of the PFP reagent to the compound of formula (I-1) is (0.8–3):1. In some embodiments, the molar ratio of the PFP reagent to the compound of formula (I-1) is (0.8–1.2):1. In some embodiments, the molar ratio of the PFP reagent to the compound of formula (I-1) is 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, or 1.3:1.

[0024] In some embodiments, the condensation reaction is carried out using an NHS reagent and at -10 to 20°C; in other embodiments, the condensation reaction is carried out using an NHS reagent and at 0 to 10°C.

[0025] In some embodiments, the condensation reaction uses a PFP reagent and is carried out at -20 to 25°C. In some embodiments, the condensation reaction uses a PFP reagent and is carried out at -10 to 15°C. In some embodiments, the condensation reaction uses a PFP reagent and is carried out at -5 to 10°C.

[0026] In some embodiments, the condensation reaction uses a PFP reagent and is carried out at -10 to 25°C. In other embodiments, the condensation reaction uses a PFP reagent and is carried out at 0 to 15°C.

[0027] In some embodiments, the condensation reaction is carried out in the presence of an organic solvent selected from one or more of dichloromethane, chloroform, ethyl acetate, tetrahydrofuran, and dioxane. In some embodiments, the condensation reaction is carried out in the presence of dichloromethane.

[0028] In some embodiments, the condensation reaction is carried out under stirring.

[0029] In some embodiments, the stirring time is 5 to 48 hours. In some embodiments, the stirring time is 10 to 36 hours. In some embodiments, the stirring time is 15 to 24 hours.

[0030] In some embodiments, the method for preparing the compound shown in formula (I) further includes a post-processing step.

[0031] In some embodiments, the post-processing steps of the compound represented by formula (I) include filtration, rinsing with an organic solvent, and concentration.

[0032] In some embodiments, the organic solvent used for rinsing is selected from one or more of dichloromethane, chloroform, ethyl acetate, tetrahydrofuran, and dioxane. In some embodiments, the organic solvent used for rinsing is dichloromethane.

[0033] In some implementation schemes, Z1 is selected from C. 1-6 Alkylene, C 2-10 imidene group, C 2-10 Ethyne group, C 3-8 Cycloalkylene.

[0034] In some implementation schemes, Z1 is selected from C. 1-4 Alkylene, C 2-6 imidene group, C 2-6 Ethyne group, C 3-6 Cycloalkylene.

[0035] In some implementation schemes, Z1 is C. 1-6 Alkylene. In some embodiments, Z1 is C. 2-4 Alkylene. In some embodiments, Z1 is propylene.

[0036] In some implementations, Z2 is oxygen.

[0037] In some implementation schemes, when ring A is C 6-10 When aryl or 5-10 heteroaryl, the aryl group is selected from one or more halogens, nitro groups, cyano groups, C6 groups, etc. 1-6 Alkyl and C 1-6 The alkoxy group is substituted. In some embodiments, when ring A is C... 3-6When the cycloalkyl or 5-10 membered heterocyclic group is used, the cycloalkyl or heterocyclic group is replaced by one or more groups selected from halogen, nitro, cyano, oxo (=O), C 1-6 Alkyl and C 1-6 The alkoxy group is replaced by a substituent.

[0038] In some implementations, ring A is C. 6-10 Aryl or 5-10 membered heterocyclic group.

[0039] In some implementation schemes, when ring A is C 6-10 In the case of an aryl group, the aryl group is substituted with one or more halogens. In some embodiments, when ring A is a 5-10 membered heterocyclic group, the heterocyclic group is substituted with one or more oxo groups (=O).

[0040] In some embodiments, ring A is a phenyl or a 5-6 membered heterocyclic group.

[0041] In some embodiments, ring A is a phenyl group or a 5-6 membered nitrogen-containing heterocyclic group.

[0042] In some embodiments, ring A is a phenyl or a 5-6 member nitrogen-containing heterocyclic group, wherein the phenyl group is substituted with one or more halogens; and the heterocyclic group is substituted with one or more oxo groups (=O).

[0043] In some implementation schemes, for

[0044] In some implementations, m is 0, 1, 2, or 3. In some implementations, m is 1.

[0045] In some embodiments, E is selected from 5-10-membered heteroaryl groups optionally substituted with one or more R1 groups; wherein R1 is independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-4 Alkyl and Halogenated C 1-4 alkyl.

[0046] In some embodiments, E is selected from the following groups optionally substituted with one or more R1s: pyrimidine, quinazoline, and pyrrolo[2,3-d]pyrimidine; wherein R1 is independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-2 Alkyl and Halogenated C 1-2 alkyl.

[0047] In some embodiments, E is selected from pyrimidines optionally substituted with one or more R1s; wherein R1 is independently selected from H (hydrogen) and D (deuterium).

[0048] In some implementations, E is pyrimidine.

[0049] In some embodiments, G is selected from halogens, OMs, OTs, OTf, nitro, and optionally the following groups substituted with one or more R2: alkyl thioether, aryl thioether, heteroaryl thioether, alkyl sulfoxide, aryl sulfoxide, heteroaryl sulfoxide, alkyl sulfonyl, aryl sulfonyl, heteroaryl sulfonyl; wherein R2 is independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, C 1-6 Alkoxy, 6-10 aryl and 5-12 heteroaryl.

[0050] In some embodiments, G is selected from F, Cl, Br, I, OMs, OTs, OTf, methanesulfonyl, ethanesulfonyl, p-toluenesulfonyl, and naphthalenesulfonyl.

[0051] In some implementations, G is selected from F, Cl, Br, OMs, OTs, methanesulfonyl, and p-toluenesulfonyl.

[0052] In some implementations, G is selected from Cl and methanesulfonyl.

[0053] In some implementations, G is methanesulfonyl.

[0054] In some embodiments, the compound of formula (I) has the following structure:

[0055] In some embodiments, the method for preparing compound 2 as shown below further includes a purification step, which includes heating, cooling, stirring while maintaining the temperature, separating the solid, and rinsing with an organic solvent:

[0056] In some embodiments, the refining step includes adding the product obtained from the post-treatment to an organic solvent and then heating it. In some embodiments, the organic solvent is methanol.

[0057] In some implementation schemes, the cooling process of the cooling step is as follows: stirring to cool down to 50℃~55℃, water bath to cool down to 25℃~30℃, and then continuing to cool down to 15℃~20℃.

[0058] In some embodiments, the heat preservation and stirring time is 5–120 minutes. In some embodiments, the heat preservation and stirring time is 10–60 minutes. In some embodiments, the heat preservation and stirring time is 10 minutes. In some embodiments, the heat preservation and stirring time is 20 minutes. In some embodiments, the heat preservation and stirring time is 30 minutes. In some embodiments, the heat preservation and stirring time is 40 minutes.

[0059] In some implementations, the solid separation process is carried out by filtration or centrifugation.

[0060] In some embodiments, the organic solvent used in the rinsing step is methanol, ethanol, acetonitrile, acetone, or isopropanol. In some embodiments, the organic solvent used in the rinsing step is methanol.

[0061] In some embodiments, the method for preparing compound 7 shown below further includes one or more purification steps, said purification steps including heating, cooling, separation of solids, and rinsing with an organic solvent:

[0062] In some embodiments, the first purification of compound 7 includes heating, cooling, separating solids, and rinsing with an organic solvent.

[0063] In some embodiments, the heating step of the first refining process includes adding the product obtained from the post-treatment to an organic solvent and then heating it. In some embodiments, the organic solvent is isopropanol.

[0064] In some embodiments, the heating temperature is 50–100°C. In some embodiments, the heating temperature is 60–80°C. In some embodiments, the heating temperature is 65–70°C.

[0065] In some implementation schemes, the cooling process is as follows: cool down to 40°C to 50°C, precipitate solid, keep warm and stir for 0.5 to 1 hour, continue cooling down to 15°C to 25°C, keep warm and stir for 0.5 to 1 hour.

[0066] In some implementations, the solid separation process is carried out by filtration or centrifugation.

[0067] In some embodiments, the organic solvent used in the rinsing step is methanol, ethanol, acetonitrile, acetone, or isopropanol. In some embodiments, the organic solvent used in the rinsing step is isopropanol.

[0068] In some implementation schemes, a second purification is included after the first purification, which includes: (1) heating, (2) adsorption, (3) cooling, and (4) separation of solids.

[0069] In some embodiments, the second purification step (1) before heating includes adding the compound to be purified to an organic solvent.

[0070] In some embodiments, the weight-to-volume ratio (g / mL) of the compound to be further purified to the organic solvent is 1:0.1 to 1:10. In some embodiments, the weight-to-volume ratio of the compound to be further purified to the organic solvent is 1:1 to 1:5. In some embodiments, the weight-to-volume ratio of the compound to be further purified to the organic solvent is 1:2 to 1:4. In some embodiments, the weight-to-volume ratio of the compound to be further purified to the organic solvent is 1:3 to 1:4. In some embodiments, the weight-to-volume ratio of the compound to be further purified to the organic solvent is 1:3.

[0071] In some embodiments, the organic solvent is selected from ester solvents, such as isopropyl acetate; alcohol solvents, such as methanol; aromatic solvents, such as toluene; and ether solvents, such as methyl tert-butyl ether. In some embodiments, the organic solvent is an ester solvent, and in some embodiments it may be isopropyl acetate.

[0072] In some embodiments, the temperature is raised to 45–85°C in the first step of the second refining process. In some embodiments, the temperature is raised to 50–75°C in the first step of the second refining process. In some embodiments, the temperature is raised to 55–65°C, for example, 60°C in the first step of the second refining process.

[0073] In some embodiments, the adsorption step (2) of the second purification includes the addition of an adsorbent. In some embodiments, the adsorbent is activated carbon.

[0074] In some embodiments, the amount of adsorbent added is 1% to 100% of the mass of the compound to be further purified. In some embodiments, the amount of adsorbent added is 3% to 20% of the mass of the compound to be further purified. In some embodiments, the amount of adsorbent added is 5% to 15% of the mass of the compound to be further purified. In some embodiments, the amount of adsorbent added is about 8%, 9%, 10%, 11%, or 12% of the mass of the compound to be further purified.

[0075] In some embodiments, the second step of adsorption in the second purification process further includes a step of separating solids after the addition of the adsorbent; for example, separating solids by filtration.

[0076] In some embodiments, the adsorption step (2) of the second purification process further includes a stirring step before the solids are separated. In some embodiments, the stirring lasts for 1 to 60 minutes. In some embodiments, the stirring lasts for 5 to 20 minutes, for example, 10 minutes.

[0077] In some implementations, before the cooling step (3) of the second refining, the product obtained in step (2) is further heated to dissolve it.

[0078] In some embodiments, the third step of the second refining process involves lowering the temperature to 0–15°C, or in some embodiments, to 0–10°C, for example, to 5°C.

[0079] In some implementations, the fourth step of the second refining process uses filtration to separate the solids.

[0080] In some embodiments, step (4) of the second purification further includes washing the filter cake with an organic solvent. The organic solvent is selected from hydrocarbon solvents, such as n-heptane or petroleum ether; or from alcohol solvents, such as ethanol or isopropanol; in some embodiments, the organic solvent is an ether solvent, such as methyl tert-butyl ether (MTBE).

[0081] In some embodiments, step (4) of the second refining process further includes a drying step. In some embodiments, the drying is vacuum drying.

[0082] In some embodiments, the compound of formula (I-1) has the following structure:

[0083] In some embodiments, the present invention provides a method for preparing a compound of formula (A) as shown below from the compound of formula (I):

[0084] Wherein, s is selected from integers from 1 to 20; L is selected from natural amino acids or non-natural amino acids and their analogues (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala), and short peptides composed of amino acids (such as Gly-Lys, Asp-Gly-Gly-Phe-Gly (DGGFG, SEQ ID NO:1), Glu-Gly-Gly-Phe-Gly (EGGFG, SEQ ID NO:1), and Glu-Gly-Gly-Phe-Gly (EGGFG, SEQ ID NO:1). NO:2), Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val -Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gl y, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-V al-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG, SEQ ID NO: 3), Gly-Gly-Val-Ala (GGVA, SEQ ID NO: 4), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO: 5), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO: 6), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO: 7));

[0085] Z1, E, G, and m are as described in any of the above.

[0086] In some implementations, L is selected from the following structures:

[0087] In some implementations, L is selected from the following structures:

[0088] In some implementations, s is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0089] In some embodiments, the compound of formula (I) is further reacted with the compound of formula (II) as shown below to form the compound of formula (A):

[0090] Wherein, L and s are each independently as described in any of the above items.

[0091] In some embodiments, the molar ratio of compound (I) to compound (II) is (0.1–10):1. In some embodiments, the molar ratio of compound (I) to compound (II) is (0.3–3):1. In some embodiments, the molar ratio of compound (I) to compound (II) is (0.5–2):1. In some embodiments, the molar ratio of compound (I) to compound (II) is (0.7–1.1):1.

[0092] In some embodiments, the compound of formula (I) and the compound of formula (II) react at 0–100°C. In some embodiments, the compound of formula (I) and the compound of formula (II) react at 20–90°C. In some embodiments, the compound of formula (I) and the compound of formula (II) react at 25–80°C. In some embodiments, the compound of formula (I) and the compound of formula (II) react at 36–40°C or 55–65°C.

[0093] In some embodiments, the reaction that produces the compound of formula (A) is carried out in a DMF, DMSO, or acetonitrile-water mixture.

[0094] In some embodiments, compound (A) is formed by reacting compound 2 and compound 3 in a molar ratio of (0.6–1.3):1. In some embodiments, the molar ratio is (0.7–1.1):1.

[0095] In some embodiments, compound 2 is purified and then reacted with compound 3. In some embodiments, the purification solvent is methanol. In some embodiments, the solvent volume is 10 times the volume of the compound. In some embodiments, the temperature is 55–65°C. In some embodiments, compound 2 and compound 3 react at 20–60°C. In some embodiments, the temperature is 30–50°C. Further, in some embodiments, the temperature is 35–45°C.

[0096] In some embodiments, the reaction of compound 2 and compound 3 further includes a post-processing step.

[0097] In some implementations, the post-processing step includes the following operations:

[0098] (1) Cooling down;

[0099] (2) Add reagents;

[0100] (3) Separating solids; and

[0101] (4) Rinse.

[0102] In some embodiments, step (1) involves cooling to 0–40°C. In some embodiments, step (1) involves cooling to 0–35°C. Further, in some embodiments, step (1) involves cooling to 20–30°C.

[0103] In some embodiments, an ether reagent is added in step (2). Further, in some embodiments, a methyl tert-butyl ether (MTBE) reagent is added in step (2).

[0104] In some implementations, the separation of solids in step (3) is carried out by filtration, for example by vacuum filtration.

[0105] In some embodiments, the organic solvent used for rinsing in step (4) is an ether-based reagent. Further, in some embodiments, the organic solvent used for rinsing in step (4) is an MTBE reagent.

[0106] In some implementations, a drying step is included after step (4).

[0107] In some embodiments, the reaction of compound 2 and compound 3 further includes a purification step.

[0108] In some implementations, the refining step is performed after the post-processing step.

[0109] In some implementations, the refining step includes:

[0110] (1) Mix with organic solvents;

[0111] (2) Add organic reagents; and

[0112] (3) Separate solids.

[0113] In some embodiments, the organic solvent in step (1) of the purification step is acetonitrile, DMSO, NMP, or DMF. In other embodiments, the organic solvent in step (1) of the purification step is DMF.

[0114] In some embodiments, the organic solvent added in step (2) of the purification step is an ether reagent, such as MTBE.

[0115] In some implementations, the separation of solids in step (3) of the purification step is carried out by filtration, for example by vacuum filtration.

[0116] In some implementations, a washing step is included after step (3) of the refining step.

[0117] In some embodiments, the washing step is performed using an ether solvent, and in other embodiments, MTBE.

[0118] In some embodiments, the washing process is followed by a drying step, for example, in some embodiments, the drying step is vacuum drying.

[0119] In some embodiments, the compound of formula (A) is formed by reacting the following compound 7 and the following compound 8.

[0120] The molar ratio of compound 7 to compound 8 is (0.8–1.6):1. In some embodiments, compound (A) is formed by reacting compound 7 and compound 8, wherein the molar ratio of compound 7 to compound 8 is (0.9–1.2):1. In some embodiments, compound (A) is formed by reacting compound 7 and compound 8, wherein the molar ratio of compound 7 to compound 8 is (1–1.1):1.

[0121] In some embodiments, compound 7 is recrystallized and then reacted with compound 8, using isopropanol or methanol as the recrystallization solvent. In some embodiments, isopropanol is used as the recrystallization solvent. In some embodiments, the volume of the recrystallization solvent is 25 times its volume. In some embodiments, the recrystallization temperature is 65–75°C.

[0122] In some embodiments, the reaction of compound 8 with compound 7 further includes a step of purifying compound 8.

[0123] In some embodiments, compound 8 is purified using preparative liquid chromatography.

[0124] In some embodiments, compound 8 is purified by dynamic axial compression (DAC) liquid chromatography; in some embodiments, a DAC80 column is used.

[0125] In some embodiments, the chromatographic column uses reversed-phase chromatographic column packing, such as a C18 packed column, a T3 packed column, or a HILIC packed column. In some embodiments, a C18 packed column or a T3 packed column is used.

[0126] In some embodiments, the mobile phase of the liquid chromatography is water.

[0127] In some embodiments, the liquid chromatography system begins receiving eluent when the absorbance reaches 1000–2300 mAu and stops receiving eluent when the absorbance reaches 200–1500 mAu. In some embodiments, the liquid chromatography system begins receiving eluent when the absorbance reaches 1500–2300 mAu and stops receiving eluent when the absorbance reaches 500–1500 mAu. In some embodiments, the liquid chromatography system begins receiving eluent when the absorbance reaches 1800–2200 mAu and stops receiving eluent when the absorbance reaches 800–1500 mAu. In some embodiments, the liquid chromatography system begins receiving eluent when the absorbance reaches 2000 mAu and stops receiving eluent when the absorbance reaches 1000 mAu.

[0128] In some embodiments, the liquid chromatography uses C18 packing material; and eluent is received when the absorbance reaches 1000–2300 mAu, and eluent reception is stopped when the absorbance reaches 200–2000 mAu; in some embodiments, the liquid chromatography starts receiving eluent when the absorbance reaches 1500–2300 mAu, and stops receiving eluent when the absorbance reaches 500–1500 mAu; in some embodiments, the liquid chromatography starts receiving eluent when the absorbance reaches 1800–2200 mAu, and stops receiving eluent when the absorbance reaches 800–1500 mAu; for example, the liquid chromatography starts receiving eluent when the absorbance reaches 2000 mAu, and stops receiving eluent when the absorbance reaches 1000 mAu.

[0129] In some embodiments, the liquid chromatography system begins receiving eluent when the absorbance reaches 100–1200 mAu and stops receiving eluent when the absorbance reaches 100–1200 mAu. In some embodiments, the liquid chromatography system begins receiving eluent when the absorbance reaches 300–900 mAu and stops receiving eluent when the absorbance reaches 300–900 mAu. In some embodiments, the liquid chromatography system begins receiving eluent when the absorbance reaches 400–800 mAu and stops receiving eluent when the absorbance reaches 400–800 mAu. In some embodiments, the liquid chromatography system begins receiving eluent when the absorbance reaches 600 mAu and stops receiving eluent when the absorbance reaches 600 mAu.

[0130] In some embodiments, the liquid chromatography uses T3 packing material; and eluent is received when the absorbance reaches 100–1200 mAu, and eluent reception is stopped when the absorbance reaches 100–1200 mAu. In some embodiments, the liquid chromatography starts receiving eluent when the absorbance reaches 300–900 mAu, and stops receiving eluent when the absorbance reaches 300–900 mAu. In some embodiments, the liquid chromatography starts receiving eluent when the absorbance reaches 400–800 mAu, and stops receiving eluent when the absorbance reaches 400–800 mAu. In some embodiments, the liquid chromatography starts receiving eluent when the absorbance reaches 600 mAu, and stops receiving eluent when the absorbance reaches 600 mAu.

[0131] In some embodiments, the eluent from the liquid chromatography is further subjected to a lyophilization step.

[0132] In some embodiments, the freeze-drying step is followed by a drying step, such as vacuum drying.

[0133] In some embodiments, the vacuum drying is performed for 1 to 24 hours. In some embodiments, the vacuum drying is performed for 2 to 12 hours. In some embodiments, the vacuum drying is performed for 6 to 7 hours.

[0134] In some embodiments, the vacuum drying is carried out at 5–80°C. In some embodiments, the vacuum drying is carried out at 10–75°C. In some embodiments, the vacuum drying is carried out at 40–60°C.

[0135] In some embodiments, compound 7 and compound 8 react at 40–80°C. In some embodiments, compound 7 and compound 8 react at 50–70°C. In some embodiments, compound 7 and compound 8 react at 55–65°C.

[0136] In some embodiments, compounds 7 and 8 react at 40–80°C, for example, 50–70°C or 55–65°C.

[0137] In some embodiments, the reaction of compound 7 and compound 8 further includes a post-processing step.

[0138] In some implementations, the post-processing step includes the following operations:

[0139] (1) Filter and collect the filtrate;

[0140] (2) Add reagents;

[0141] (3) Filter and collect solids;

[0142] (4) Add organic solvent; and

[0143] (5) Filtering.

[0144] In some implementations, the first step of filtration is performed while the filter is still hot.

[0145] In some embodiments, step (2) involves adding an ether reagent, such as MTBE.

[0146] In some embodiments, step (2) is followed by a stirring operation. In some embodiments, the stirring is performed for 0.1 to 5 hours. In some embodiments, the stirring is performed for 0.1 to 3 hours. In some embodiments, the stirring is performed for 0.5 to 1 hour.

[0147] In some embodiments, step (3) is followed by a solid washing operation. In some embodiments, the washing is performed using an ether solvent. In some embodiments, the ether solvent is MTBE.

[0148] In some embodiments, the organic solvent added in step (4) is dichloromethane, chloroform, ethyl acetate, or dioxane. In some embodiments, the organic solvent added in step (4) is ethyl acetate.

[0149] In some embodiments, step (4) includes adding the solid to the organic solvent.

[0150] In some implementations, step (4) is followed by steps of heating and cooling.

[0151] In some implementations, step (4) is further followed by a heating and stirring process followed by a cooling and stirring process followed by a heating and stirring process.

[0152] In some embodiments, the holding temperature for heating followed by stirring is 20°C to 50°C. In some embodiments, the holding temperature for heating followed by stirring is 30°C to 40°C. In some embodiments, the stirring time is 0.5 to 5 hours. In some embodiments, the stirring time is 1 to 3 hours.

[0153] In some embodiments, the temperature for cooling and then stirring is 5°C to 35°C or 15°C to 25°C; and the stirring time is 0.1 to 3 hours or 0.5 to 2 hours.

[0154] In some embodiments, step (5) filtration is followed by an organic solvent washing step. In some embodiments, the organic solvent is dichloromethane, chloroform, ethyl acetate, or dioxane. For example, in some embodiments, the organic solvent is ethyl acetate.

[0155] In some embodiments, the reaction of compounds 7 and 8 further includes a purification step.

[0156] In some implementations, the refining step is performed after the post-processing step.

[0157] In some implementations, the refining step includes:

[0158] (1) Mix with organic solvents;

[0159] (2) Filter and collect the filtrate;

[0160] (3) Concentrate the filtrate;

[0161] (4) Add organic solvent; and

[0162] (5) Separate solids.

[0163] In some embodiments, the organic solvent in step (1) of the purification step is selected from one or more of dichloromethane, chloroform, ethyl acetate, dioxane, tetrahydrofuran, and acetonitrile. In some embodiments, the organic solvent in step (1) of the purification step is a mixed solvent of isopropanol and dichloromethane. In some embodiments, the volume ratio of isopropanol to dichloromethane in the mixed solvent is 10:1.

[0164] In some embodiments, step (2) of the refining process further includes a heat preservation and stirring step. In some embodiments, the heat preservation temperature is 0–50°C, and the stirring time is 0.1–5 h or 0.5–3 h. In some embodiments, the heat preservation temperature is 20–35°C, and the stirring time is 0.1–5 h or 0.5–3 h.

[0165] In some implementations, step (2) of the refining process further includes a step of washing the filter cake.

[0166] In some embodiments, the solvent used in the filter cake washing step is selected from one or more of dichloromethane, chloroform, ethyl acetate, dioxane, tetrahydrofuran, and acetonitrile. In some embodiments, the filter cake washing step uses a mixed solvent of isopropanol and dichloromethane. In some embodiments, the volume ratio of isopropanol to dichloromethane in the mixed solvent is 10:1.

[0167] In some implementations, step (3) employs a reduced pressure concentration method.

[0168] In some embodiments, the vacuum concentration is performed below 50°C. In some embodiments, the vacuum concentration is performed below 40°C. In some embodiments, the vacuum concentration is performed below 35°C.

[0169] In some embodiments, the addition of an ether-based organic solvent in step (4) may be MTBE.

[0170] In some embodiments, step (4) is followed by a stirring operation. In some embodiments, step (4) is followed by a stirring operation at 0–50°C. In some embodiments, step (4) is followed by a stirring operation at 15–30°C. In these embodiments, the stirring time is 0.1–5 h, and in some embodiments, it can be 0.5–3 h.

[0171] In some implementations, the separation of solids in step (5) is carried out by filtration.

[0172] In some embodiments, step (5) is followed by a step of washing the filter cake. In some embodiments, step (5) is followed by a step of washing the filter cake with an ether reagent. In some embodiments, step (5) is followed by a step of washing the filter cake with MTBE.

[0173] In some embodiments, the washing of the filter cake is followed by a drying step.

[0174] In some embodiments, the compound of formula (A) is formed by reacting the following compound 2 with the following compound 8.

[0175] The molar ratio of compound 2 to compound 8 is (1-2):1. In some embodiments, compound (A) is formed by reacting compound 2 and compound 8, wherein the molar ratio of compound 2 to compound 8 is (1.2-1.8):1. In some embodiments, compound (A) is formed by reacting compound 2 and compound 8, wherein the molar ratio of compound 2 to compound 8 is 1.5:1.

[0176] .

[0177] In some embodiments, the reaction of compound 2 and compound 8 is carried out in an acetonitrile-water mixture.

[0178] In some embodiments, the reaction of compound 2 and compound 8 includes dispersing compound 8 in water, dispersing compound 2 in acetonitrile, and then mixing the two.

[0179] In some embodiments, the reaction of compounds 2 and 8 further includes a post-treatment step, which includes pH adjustment, filtration, pH readjustment, and filtration again. In some embodiments, the post-treatment includes:

[0180] (1) Adjust the pH value to 5-8; for example, adjust the pH value to 6-7.

[0181] (2) Filter and collect the filtrate;

[0182] (3) Adjust the pH value to: 2-5; for example, adjust the pH value to: 3-4; and

[0183] (4) Filter to obtain solids.

[0184] In some embodiments, the post-treatment step (1) after adjusting the pH value further includes a stirring step. In some embodiments, the stirring is performed at 0–40°C. In some embodiments, the stirring is performed at 0–30°C. In some embodiments, the stirring is performed at 0–20°C.

[0185] In some embodiments, the stirring lasts for 3 to 12 hours. In some embodiments, the stirring lasts for 5 to 8 hours.

[0186] In some embodiments, the post-treatment step (3) after adjusting the pH value further includes a stirring step. In some embodiments, the stirring is performed at -10 to 30°C. In some embodiments, the stirring is performed at 0 to 20°C. In some embodiments, the stirring is performed at 0 to 10°C.

[0187] In some implementations, the post-processing step (4) after filtration also includes a drying step.

[0188] In some embodiments, the compound of formula (A) has the following structure:

[0189] In some embodiments, the compound of formula (II) has the following structure:

[0190] In some embodiments, the present invention provides a method for reacting a compound of formula (A) with a cytotoxic drug to generate a compound of formula (B) as shown below:

[0191] Wherein: D is the cytotoxic drug portion; L, Z1, E, G, s, and m are as described in any of the above.

[0192] In some embodiments, the method of reacting the compound of formula (A) with a cytotoxic drug to generate the compound of formula (B) is carried out in the presence of one of the following condensing agents: DMTMM, CDTM (e.g., used in combination with an organic base, such as CDTM / N-methylmorpholine), HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate), HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate), EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) / HOBT (1-hydroxybenzotriazole), DCC, or PyBOP, further for example, DMTMM, or a combination of CDTM / N-methylmorpholine.

[0193] In some embodiments, the method for generating compound (B) is carried out in the presence of one or more bases selected from N,N-diisopropylethylamine, N-methylmorpholine, triethylamine, and morpholine. In some embodiments, the method for generating compound (B) is carried out in the presence of N,N-diisopropylethylamine or N-methylmorpholine.

[0194] In some embodiments, the method for generating the compound of formula (B) is carried out in an aprotic organic solvent. In some embodiments, the solvent is selected from one or more of DMF, DMSO, DMAc, and NMP. In some embodiments, the solvent is DMF. In some embodiments, the solvent is DMSO.

[0195] In some embodiments, the molar ratio of the compound of formula (A) to the cytotoxic drug is (0.1-10):1; for example, (0.2-5):1. In some embodiments, the molar ratio of the compound of formula (A) to the cytotoxic drug is (1-2):1. In some embodiments, the molar ratio of the compound of formula (A) to the cytotoxic drug is 1:1.

[0196] In some embodiments, the method for generating the compound of formula (B) is carried out under nitrogen protection.

[0197] In some embodiments, the compound of formula (B) has the following structure:

[0198] In some embodiments, the reaction that generates compound 6 further includes a post-processing step, said step comprising:

[0199] (1) Quenching the reaction with water;

[0200] (2) Add solvent;

[0201] (3) Separation of the organic phase; and

[0202] (4) Concentration.

[0203] In some implementations, the post-processing is performed under nitrogen protection.

[0204] In some embodiments, the solvent added in step (2) is selected from one or more of dichloromethane, chloroform, ethyl acetate, isopropanol, and acetone. In some embodiments, the solvent added in step (2) is a mixed solution of dichloromethane and isopropanol.

[0205] In some embodiments, step (2) is followed by a stirring step. In some embodiments, the stirring is performed at 10-50°C. In some embodiments, the stirring is performed at 20-30°C. In some embodiments, the stirring time is 0.1-5 hours. In some embodiments, the stirring time is 0.5-1.5 hours.

[0206] In some implementations, steps (2) and (3) are repeated 1 to 5 times. In some implementations, steps (2) and (3) are repeated 1 to 3 times.

[0207] In some implementations, the concentration step (4) is a vacuum concentration step.

[0208] In some embodiments, the vacuum concentration operation of step (4) is repeated 1 to 8 times. In some embodiments, the vacuum concentration operation of step (4) is repeated 1 to 6 times.

[0209] In some embodiments, the reaction that generates compound 6 further includes a purification step following post-treatment, the purification step comprising:

[0210] (1) Mix the post-processed product with an organic solvent to obtain solution I;

[0211] (2) Add solution I to the organic reagent; and

[0212] (3) Filter to obtain solids.

[0213] In some embodiments, the organic solvent in step (1) is selected from one or more of dichloromethane, chloroform, ethyl acetate, isopropanol, and acetone. In some embodiments, the organic solvent in step (1) is a mixed solution of dichloromethane and isopropanol. In some embodiments, the volume ratio of isopropanol to dichloromethane in the mixed solution is 10:1.

[0214] In some implementations, step (2) involves adding solution I to the organic reagent by dropping it.

[0215] In some embodiments, the organic reagent in step (2) is an ether reagent. In some embodiments, the organic reagent in step (2) is methyl tert-butyl ether.

[0216] In some embodiments, step (3) after filtration further includes a step of rinsing with an organic solvent. In some embodiments, step (3) includes rinsing with methyl tert-butyl ether.

[0217] In some embodiments, the rinsing operation after filtration in step (3) is repeated 1 to 3 times. In some embodiments, the rinsing operation after filtration in step (3) is repeated 2 times.

[0218] In some implementations, steps (1) to (3) are repeated 1 to 3 times. In some implementations, steps (1) to (3) are repeated 2 times.

[0219] In some embodiments, the reaction to generate compound 10 further includes a post-processing step, said step comprising:

[0220] (1) Add an organic solvent;

[0221] (2) Washing;

[0222] (3) Separation of the organic phase;

[0223] (4) Separate the solids after drying;

[0224] (5) Washing; and

[0225] (6) Separate solids.

[0226] In some embodiments, the solvent added in step (1) is selected from one or more of dichloromethane, chloroform, ethyl acetate, isopropanol, and acetone. In some embodiments, the solvent added in step (1) is a mixed solution of dichloromethane and isopropanol. In some embodiments, the volume ratio of isopropanol to dichloromethane in the mixed solution is 10:1.

[0227] In some implementations, the washing step (2) may be performed using water or an aqueous solution of inorganic salts (e.g., an aqueous solution of sodium chloride).

[0228] In some implementations, steps (1) to (3) are repeated 1 to 5 times. In some implementations, steps (1) to (3) are repeated 1 to 3 times.

[0229] In some implementations, step (4) involves drying with an inorganic sodium salt (e.g., anhydrous sodium sulfate, anhydrous magnesium sulfate, etc.).

[0230] In some embodiments, the washing solvent used in step (5) is selected from one or more of dichloromethane, chloroform, ethyl acetate, isopropanol, acetone, and ether solvents. In some embodiments, the washing solvent used in step (5) is a dichloromethane-isopropanol mixture or MTBE. In some embodiments, the volume ratio of isopropanol to dichloromethane in the mixture is 10:1.

[0231] In some implementations, step (5) is repeated 1 to 2 times.

[0232] In some embodiments, the reaction that generates compound 10 further includes a purification step following post-treatment, namely silica gel column chromatography.

[0233] In some embodiments, the mobile phase of the column chromatography is one or more selected from dichloromethane, chloroform, ethyl acetate, petroleum ether, methanol, and acetonitrile. In some embodiments, the mobile phase of the column chromatography is a mixture of dichloromethane and methanol.

[0234] In some embodiments, the volume ratio of dichloromethane to methanol is 1:1 to 100:1. In some embodiments, the volume ratio of dichloromethane to methanol is 5:1 to 30:1. In some embodiments, the volume ratio of dichloromethane to methanol is 10:1. In some embodiments, the volume ratio of dichloromethane to methanol is 15:1. In some embodiments, the volume ratio of dichloromethane to methanol is 20:1.

[0235] In some embodiments, the column chromatography further includes concentration, filtration, elution, and drying steps.

[0236] In some embodiments, the cytotoxic drug is selected from compounds represented by Formula III or their acceptable pharmaceutical salts:

[0237] Among them, R5 and R6 are each independently selected from OH, -NH2, and -NH(C) 1-6 Alkyl), C 1-6 Alkyl and halogen; the C 1- 6-alkyl groups are optionally further oxidized by one or more elements selected from halogens, hydroxyl groups, and C64 groups. 1-6 Haloalkyl, C 3-6 Substituents of cycloalkyl groups;

[0238] R7 is selected from H, -OH, -NH2, and -NH(C). 1-6 alkyl), and -NH-CO-(C 1-6 alkylene)-OH; the C 1-6 Alkyl groups may optionally be further oxidized by one or more elements selected from halogens, hydroxyl groups, and C. 1-6Haloalkyl, C 3-6 Substituents of cycloalkyl groups;

[0239] q is 1 or 2;

[0240] In some implementations, R5 and R6 are each independently selected from OH, -NH2, and C. 1-4 Alkyl and halogen; the C 1-4 Alkyl groups may optionally be further oxidized by one or more elements selected from halogens, hydroxyl groups, and C. 1-4 Haloalkyl, C 3-6 Substituents of cycloalkyl groups.

[0241] In some implementations, R7 is selected from -H, -NH2, -NH(C) 1-4 alkyl), and -NH-CO-(C 1-4 alkylene)-OH; the C 1-4 Alkyl groups may optionally be further oxidized by one or more elements selected from halogens, hydroxyl groups, and C. 1-4 Haloalkyl, C 3-6 Substituents of cycloalkyl groups.

[0242] In some implementations, R5 and R6 are each independently selected from OH, -NH2, and C. 1-6 Alkyl and halogen;

[0243] R7 is selected from -H, -NH2, and -NH(C). 1-6 alkyl), and -NH-CO-(C 1-6 alkylene)-OH; and

[0244] q is 2.

[0245] In some implementations, R5 and R6 are each independently selected from OH, -NH2, and C. 1-4 Alkyl groups and halogens. In some embodiments, R5 and R6 are each independently selected from OH, -NH2, methyl, ethyl, n-propyl, isopropyl, and halogens. In some embodiments, R5 and R6 are each independently selected from OH, -NH2, methyl, fluorine, and chlorine.

[0246] In some implementations, R7 is selected from -H, -NH2, -NH(C) 1-4 alkyl), and -NH-CO-(C 1-4 (alkylene)-OH.

[0247] In some embodiments, R7 is selected from -H, -NH2, -NH(CH3), -NH(CH2CH3), -NH(CH(CH3)2), -NH-CO-CH2-OH, -NH-CO-CH2CH2-OH, -NH-CO-CH(CH3)-OH and -NH-CO-C(CH3)2-OH.

[0248] In some implementations, R7 is selected from -H, -NH2, and -NH-CO-CH2-OH.

[0249] In some embodiments, the cytotoxic drug is selected from the following compounds or their acceptable pharmaceutical salts:

[0250] In some implementations, the structure of D is shown in equation III-A:

[0251] Among them, R 7A Selected from chemical bonds, -O-, -NH-, -N(C) 1-6 alkyl)- and -NH-CO-(C 1-6 (alkylene)-O-; the alkyl and alkylene groups are optionally further converted by one or more elements selected from halogens, hydroxyl groups, C 1-6 Haloalkyl, C 3-6 Substituents of cycloalkyl groups;

[0252] R5~R6, q are as described in any of the above items.

[0253] In some implementation schemes, R 7A Selected from chemical bonds, -O-, -NH-, -N(C) 1-4 alkyl)- and -NH-CO-(C 1-4 (alkylene)-O-; the C 1-4 Alkyl groups may optionally be further oxidized by one or more elements selected from halogens, hydroxyl groups, and C. 1-4 Haloalkyl, C 3- It is substituted by a 6-cycloalkyl substituent.

[0254] In some implementation schemes, R 7A Selected from chemical bonds, -O-, -NH-, -N(CH3)-, -N(CH2CH3)-, -N(CH(CH3)2)-, -NH-CO-CH2-O-, -NH-CO-CH2CH2-O-, -NH-CO-CH(CH3)-O- and -NH-CO-C(CH3)2-O-.

[0255] In some implementation schemes, R 7A Selected from -NH-.

[0256] In some implementations, D is selected from the following structures:

[0257] Another aspect of the present invention provides the following synthetic intermediate compounds, or pharmaceutically acceptable salts, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites, or prodrugs thereof, said compounds having the following structures:

[0258] Among them, rings A, Z1, Z2, E, G, L, R3, m and s are each independently as described in any one of the above.

[0259] In some embodiments, the present invention provides the following compounds, or pharmaceutically acceptable salts, stereoisomers, tautomers, polymorphs, solvates, N-oxides, isotopically labeled compounds, metabolites, or prodrugs thereof, said compounds having the following structures:

[0260] Among them, rings A, Z1, Z2, E, G, L, m, and s are each independently described as described in any one of the above items.

[0261] In some embodiments, the structure of the synthetic intermediate compound is shown below:

[0262] In some embodiments, the structure of the synthetic intermediate compound is shown below:

[0263] As used herein, the term "cytotoxic drug" refers to substances that inhibit or prevent cellular function and / or cause cell death or destruction, such as: radioactive isotopes, such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and Lu; chemotherapeutic agents, such as methotrexate, adriamycin, vincaalkaloids, vincristine, vinblastine, and etoposide. Poside, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents; enzymes and fragments thereof, such as ribolysins; antibiotics; and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof; growth inhibitors; pharmaceutical modules; and compounds of formula (III) of the present invention, or pharmaceutically acceptable salts, solvates, hydrates, isomers, or any crystal forms or racemates thereof. The term “cytotoxic drug moiety” refers to the structural fragment formed after the dehydrogenation of the said cytotoxic drug.

[0264] The terms “comprising,” “including,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other unlisted elements or method steps, although such other unlisted elements or method steps may not necessarily exist (i.e., these terms also cover the terms “consistently made up of” and “composed of”).

[0265] As used herein, the term "alkyl" is defined as a linear or branched saturated aliphatic hydrocarbon. In some embodiments, the alkyl group has 1 to 12, for example, 1 to 6 carbon atoms or 1 to 4 carbon atoms. For example, as used herein, the term "C 1-6 "alkyl" and "C" 1-4 "Alkyl" refers to a linear or branched group (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, or n-hexyl) having 1-6 carbon atoms and 1-4 carbon atoms, respectively, optionally substituted by one or more (e.g., 1 to 3) suitable substituents such as halogens (in which case the group is called "haloalkyl") (e.g., CH2F, CHF2, CF3, CCl3, C2F5, C2Cl5, CH2CF3, CH2Cl, or -CH2CH2CF3, etc.). The term "C" refers to a linear or branched group having 1-6 carbon atoms and 1-4 carbon atoms respectively. 1-4"Alkyl" refers to a linear or branched aliphatic hydrocarbon chain having 1 to 4 carbon atoms (i.e., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl).

[0266] As used herein, the term "haloalkyl" refers to an alkyl group substituted with one or more (such as 1 to 3) identical or different halogen atoms, and the term "C" refers to an alkyl group substituted with one or more (such as 1 to 3) identical or different halogen atoms. 1-8 "halogenated alkyl", "C" 1-6 "Halogenated alkyl" and "C" 1-4 "Halogenated alkyl" refers to alkyl haloatoms having 1 to 8 carbon atoms, 1 to 6 carbon atoms, and 1 to 4 carbon atoms, such as -CF3, -C2F5, -CHF2, -CH2F, -CH2CF3, -CH2Cl, or -CH2CH2CF3.

[0267] As used herein, the term "alkoxy" means -O-alkyl, where the alkyl group is as defined above, for example, C 1-8 Alkoxy, C 1-6 Alkoxy, C 1-4 Alkoxy or C 1-3 Alkyl group. C 1-6 Representative examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy, hexoxy, etc., wherein the alkoxy group is optionally substituted by one or more (such as 1 to 3) identical or different substituents. The term "haloalkoxy" refers to an alkoxy group in which the hydrogen atom is substituted by one or more (such as 1 to 3) identical or different halogen atoms.

[0268] As used herein, the term "cycloalkyl" refers to a saturated or unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon cycloalkyl group, including but not limited to monocyclic alkyl groups (such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, etc.) and bicyclic alkyl groups, including spirocyclic, fused-ring (fused-ring) or bridged-ring systems (i.e., spirocyclic alkyl, fused-ring (fused-ring) alkyl, and bridged-ring alkyl groups, such as bicyclic [1.1.1]pentyl, bicyclic [2.2.1]heptyl, etc.). In this invention, the cycloalkyl group is optionally substituted with one or more (such as 1 to 3) identical or different substituents. The carbon atom on the cycloalkyl group is optionally substituted with an oxo group (i.e., forming C=O). The term "C 3-8 "Cycloalkyl" refers to a cycloalkyl group having 3 to 8 cyclic carbon atoms, such as C10. 3-6 Cycloalkyl groups can be monocycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl, or they can be bicycloalkyl, such as C10, C20, C30, C40, C50, C60, C7 ...70, C60, C70, C70, C70, C70, C70, C70, C70, C 5-8 Spirocycloalkyl, C 5-8 Bridged cycloalkyl, C 5-8 fused cycloalkyl, C 5-6Spirocycloalkyl, C 5-6 Bridged cycloalkyl or C 5-6 Fused cycloalkyl groups.

[0269] As used herein, the term "cycloalkoxy" refers to -O-cycloalkyl, where the cycloalkyl group is as defined above. Representative examples of cycloalkoxy groups include, but are not limited to, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy.

[0270] As used herein, the term "heterocyclic group" or "heterocycle" refers to a saturated or unsaturated aliphatic monocyclic or polycyclic (e.g., fused, spirocyclic, or bridged) group having two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) carbon atoms and one or more (e.g., 1, 2, 3, or 4) heteroatoms, said heteroatoms including, but not limited to, oxygen, nitrogen, and sulfur atoms, wherein the carbon atoms and heteroatoms on said heterocyclic group are optionally substituted with oxo groups (e.g., forming C=O, S(=O) or S(=O)2), or optionally with one or more (such as 1 to 3) independently selected from halogens and C. 1-3 Alkyl substituents. The term "saturated heterocycle" refers to a fully saturated heterocycle, such as tetrahydrofuran ring, piperidine ring, morpholine ring, tetrahydropyran ring, piperazine ring, etc. The term "partially saturated heterocycle" refers to a heterocycle that contains both saturated single bonds and unsaturated double bonds, such as 3,4-dihydro-2H-pyran, 1,2,3,4-tetrahydropyridine, 4,5-dihydroisoxazolyl, 4,5-dihydrooxazolyl, 2,5-dihydrooxazolyl, 2,3-dihydrooxazolyl, etc. As used herein, the term "4-11 membered heterocyclic group" refers to a heterocyclic group containing 4-11 ring atoms, including but not limited to 4-10 membered heterocyclic groups, 4-9 membered heterocyclic groups, 4-8 membered heterocyclic groups, 4-7 membered heterocyclic groups, 5-6 membered heterocyclic groups, 3-8 membered heterocyclic groups, 3-7 membered heterocyclic groups, 4-7 membered nitrogen-containing heterocyclic groups, 4-7 membered oxygen-containing heterocyclic groups, 4-7 membered sulfur-containing heterocyclic groups, 5-6 membered nitrogen-containing heterocyclic groups, 5-6 membered oxygen-containing heterocyclic groups, 5-6 membered sulfur-containing heterocyclic groups, etc., wherein each of the "nitrogen-containing heterocyclic group," "oxygen-containing heterocyclic group," and "sulfur-containing heterocyclic group" optionally also contains one or more other heteroatoms independently selected from oxygen, nitrogen, and sulfur. Examples of 4-11 membered heterocyclic groups include, but are not limited to, ethylene oxide, aziridinyl, aziridine, oxobutyl, tetrahydrofuranyl, pyrrolylyl, pyrrolidone (e.g., ... (Imidazolyl, pyrazolyl, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl). As used herein, the term "3-8 membered heterocyclic group" means a heterocyclic group containing 3-8 ring atoms, including but not limited to 3-8 membered heterocyclic groups, 3-7 membered heterocyclic groups, 3-6 membered heterocyclic groups, 4-8 membered heterocyclic groups, 4-7 membered heterocyclic groups, 4-6 membered heterocyclic groups, 5-6 membered heterocyclic groups, 4-7 membered nitrogen-containing heterocyclic groups, 4-7 membered oxygen-containing heterocyclic groups, 4-7 membered sulfur-containing heterocyclic groups, 5-6 membered nitrogen-containing heterocyclic groups, 5-6 membered oxygen-containing heterocyclic groups, 5-6 membered sulfur-containing heterocyclic groups, etc., wherein each of the "nitrogen-containing heterocyclic group", "oxygen-containing heterocyclic group" and "sulfur-containing heterocyclic group" optionally also contains one or more other heteroatoms independently selected from oxygen, nitrogen and sulfur. Examples of 3-8 membered heterocyclic groups include, but are not limited to, ethylene oxide, aziridinyl, aziridine, oxetidine, tetrahydrofuranyl, pyrrolylyl, and pyrrolidone (e.g.) ), imidazoalkyl, pyrazolyl, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl.

[0271] In this invention, the heterocyclic group can form a fused ring structure with a heterocyclic group or a cycloalkyl group. The connection point of the fused ring structure with other groups can be on any heterocyclic group or on a cycloalkyl group. Therefore, the heterocyclic group of this invention also includes (but is not limited to) heterocyclic fused heterocyclic groups, heterocyclic fused cycloalkyl groups, monoheterocyclic fused monoheterocyclic groups, and monoheterocyclic fused monocycloalkyl groups, such as 3-7 membered (mono)heterocyclic fused 3-7 membered (mono)heterocyclic groups, 3-7 membered (mono)heterocyclic fused (mono)cycloalkyl groups, and 3-7 membered (mono)heterocyclic fused C 4-6 (Mono)cycloalkyl groups, examples of which include, but are not limited to, pyrrolidinylcyclopropyl, cyclopentylazirylpropyl, pyrrolidinylcyclobutyl, pyrrolidinylpyrrolidinyl, pyrrolidinylpiperidinyl, pyrrolidinylpiperazinyl, and piperidinylmorpholinyl.

[0272] In this invention, the heterocyclic group also includes bridged heterocyclic groups and spiroheterocyclic groups.

[0273] As used herein, the terms "aryl," "phenyl," or "aromatic ring" refer to an all-carbon monocyclic or fused polycyclic aromatic group having a conjugated π-electron system. As used herein, the term "C" refers to a carbon-based monocyclic or fused polycyclic aromatic group. 6-10 "Aryl (aromatic ring)" refers to an aryl (aromatic ring) containing 6 to 10 carbon atoms, such as a phenyl (benzene ring) or a naphthyl (naphthalene ring). The aryl group is optionally substituted by one or more (such as 1 to 3) identical or different substituents (e.g., halogen, OH, CN, NO2, C1-C6 alkyl, etc.). The "aryl" group can be monovalent or polyvalent (e.g., divalent).

[0274] As used herein, the term "heteroaryl" or "heteroaromatic ring" refers to a monocyclic or polycyclic aromatic group containing one or more identical or different heteroatoms, including monocyclic heteroaryl and bicyclic or polycyclic ring systems containing at least one heteroaromatic ring (an aromatic ring system containing at least one heteroatom), which may have 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 ring atoms, for example 5, 6, 7, 8, 9, or 10 ring atoms, and in each case may also be benzofused. The heteroatom may be oxygen, nitrogen, or sulfur. The carbon atom and heteroatom on the heteroaryl group are optionally substituted with an oxo group (e.g., forming C=O, S(=O), or S(=O)2). The "heteroaryl" may be monovalent or polyvalent (e.g., divalent).

[0275] As used herein, the terms "5-10-membered heteroaryl" or "5-10-membered heteroaryl ring" refer to a heteroaryl (heteroaryl ring) containing 5 to 10 (e.g., 5 to 6) ring atoms, including 5-10-membered nitrogen-containing heteroaryl, 5-10-membered oxygen-containing heteroaryl, 5-10-membered sulfur-containing heteroaryl, 5-6-membered nitrogen-containing heteroaryl, 5-6-membered oxygen-containing heteroaryl, 5-6-membered sulfur-containing heteroaryl, etc. Each of the "nitrogen-containing heteroaryl," "oxygen-containing heteroaryl," and "sulfur-containing heteroaryl" may optionally contain one or more other heteroatoms independently selected from oxygen, nitrogen, and sulfur. Examples of these groups include, but are not limited to, thiophene, furanyl, pyrrole, oxazolyl, thiazolyl, imidazole, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, tetrazolyl, oxadiazolyl, thiadiazolyl, etc., or pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, etc., as well as 5-10 fused cyclic groups containing these groups.

[0276] In this invention, a heteroaryl group (e.g., a monoheteroaryl group) can share two adjacent atoms with an aryl group (e.g., a monocyclic aryl group, such as a phenyl group), a heterocyclic group (e.g., a monoheterocyclic group), a cycloalkyl group (e.g., a monocycloalkyl group), or another heteroaryl group (e.g., another monoheteroaryl group) to form a fused ring structure. The connection point can be on any heteroaryl ring or other rings, including but not limited to (mono)heteroaryl fused (mono)heteroaryl, (mono)heteroaryl fused (monocyclic) aryl, (mono)heteroaryl fused (mono)heterocyclic, and (mono)heteroaryl fused (mono)cycloalkyl, such as 5-6 membered (mono)heteroaryl fused 5-6 membered (mono)heteroaryl, 5-6 membered (mono)heteroaryl fused phenyl, 5-6 membered (mono)heteroaryl fused 5-6 membered (mono)heterocyclic, or 5-6 membered (mono)heteroaryl fused C 4-6 (Mono)cycloalkyl groups (e.g., 5-6-membered heteroarylcyclobutyl, 5-6-membered heteroarylcyclopentyl, or 5-6-membered heteroarylcyclohexyl), examples of which include, but are not limited to, benzothiazolyl, indolyl, isoyindolyl, indolyl, benzimidazole, quinolinyl, isoquinolinyl, wait.

[0277] As used herein, the term “halogenated” or “halogenated” is defined as including F, Cl, Br, or I.

[0278] The term "substitution" refers to the selective replacement of one or more (e.g., one, two, three, or four) hydrogen atoms on a specified atom by a designated group, provided that the substitution does not exceed the normal valence of the specified atom in the present case and that the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only if such combinations form a stable compound.

[0279] If a substituent is described as “optionally substituted by one or more…”, then the substituent may be (1) unsubstituted or (2) substituted. If the carbon of the substituent is described as being optionally substituted by one or more of the substituents in the list, then one or more hydrogens on the carbon (to the extent that any hydrogens are present) may be substituted individually and / or together by independently selected optional substituents. If the nitrogen of the substituent is described as being optionally substituted by one or more of the substituents in the list, then one or more hydrogens on the nitrogen (to the extent that any hydrogens are present) may each be substituted by independently selected optional substituents.

[0280] If a substituent is described as being “independently selected” from a group, then each substituent is selected independently of the others. Therefore, each substituent may be the same as or different from another (other) substituent.

[0281] As used herein, the term "one or more" means one or more under reasonable conditions, such as two, three, four, five, or ten.

[0282] Unless otherwise specified, as used herein, the connection point of a substituent may be derived from any suitable location of the substituent.

[0283] When the bond of a substituent is such that it passes through the ring and connects two atoms, then such a substituent can be bonded to any cyclic atom in the substituted ring.

[0284] This invention also includes all pharmaceutically acceptable isotopically labeled compounds that are identical to the compounds of this invention, except that one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the compounds of this invention include (but are not limited to) isotopes of hydrogen (e.g., deuterium). 2 H), tritium ( 3 H); carbon isotopes (e.g., ... 11 C 13 C and 14 C); isotopes of chlorine (e.g.) 36 Cl); isotopes of fluorine (e.g., Cl); 18F); isotopes of iodine (e.g., F); 123 I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 15 O、 17 O and 18 O); isotopes of phosphorus (e.g., O); phosphorus isotopes (e.g., O); 32 P); and isotopes of sulfur (e.g., ... 35 S). Certain isotope-labeled compounds of the present invention (e.g., those doped with radioactive isotopes) can be used in drug and / or substrate tissue distribution studies (e.g., analysis). Radioactive isotope tritium (i.e. 3 H) and carbon-14 (i.e. 14 C) It is particularly suitable for this purpose due to its ease of incorporation and detection. Using positron-emitting isotopes (e.g.) 11 C 18 F, 15 O and 13 Substitution of N) can be used in positron emission tomography (PET) studies to examine substrate acceptor occupancy. The isotopically labeled compounds of the present invention can be prepared by methods similar to those described in the accompanying routes and / or examples and preparations, by using a suitable isotopically labeled reagent instead of the previously used unlabeled reagent. Pharmaceutically acceptable solvates of the present invention include those in which the crystallization solvent can be isotopically substituted, for example, D2O, acetone-d6, or DMSO-d6.

[0285] The term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds having one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, diastereomer mixtures, and single diastereomers can be produced. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. For example, nitroso-oximes can exist in equilibrium in solution in the following tautomer forms:

[0286] It should be understood that the scope of this application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0287] Solid lines may be used in this article. solid wedge Or virtual wedge The chemical bonds of the compounds of the present invention are depicted. Solid lines are used to depict bonds to asymmetric carbon atoms to indicate all possible stereoisomers (e.g., specific enantiomers, racemic mixtures, etc.) at that carbon atom. Solid or dashed wedges are used to depict bonds to asymmetric carbon atoms to indicate the presence of the indicated stereoisomers. When present in racemic mixtures, solid and dashed wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise specified, the compounds of the present invention are intended to exist as stereoisomers (including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, trans-blocking isomers, and mixtures thereof). The compounds of the present invention may exhibit more than one type of isomerism and may consist of mixtures thereof (e.g., racemic mixtures and diastereomer pairs).

[0288] This invention covers all possible crystalline forms or polymorphs of the compounds of this invention, which may be a single polymorph or a mixture of more than one polymorph in any proportion.

[0289] Cocrystal refers to the combination of active pharmaceutical molecules and other physiologically acceptable acids, bases, salts, and nonionic compound molecules in the same crystal lattice via hydrogen bonds, π-π stacking interactions, van der Waals forces, and other non-covalent bonds.

[0290] It should also be understood that certain compounds of the present invention may exist in their free form for therapeutic purposes, or, where appropriate, in their pharmaceutically acceptable derivative forms. In the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, esters, solvates, N-oxides, metabolites, or prodrugs, which, upon administration to a patient in need, can directly or indirectly provide the compounds of the present invention or their metabolites or residues. Therefore, when referring to "compounds of the present invention" herein, it is also intended to encompass the various derivative forms of the compounds described above.

[0291] Pharmaceutically acceptable salts of the compounds of the present invention include their acid addition salts and base addition salts.

[0292] Pharmaceutically acceptable salts of the compounds of this invention include their acid addition salts and base addition salts. Examples include hexafluorophosphates and meglumine salts. For a review of suitable salts, see Stahl and Wermuth's "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002).

[0293] As used herein, the term "ester" means an ester derived from the various general formula compounds of this application, including physiologically hydrolyzable esters (the compounds of the present invention that can be hydrolyzed under physiological conditions to release free acids or alcohols). The compounds of the present invention may themselves also be esters.

[0294] In this article, amino acid analogs refer to compounds that are chemically similar to natural amino acids (usually referring to the 20 protein amino acids) or non-natural amino acids, but in which one or more atoms, chemical groups or stereoconfigurations have been modified or replaced.

[0295] The compounds of the present invention may exist in the form of solvates (e.g., hydrates), wherein the compounds of the present invention contain a polar solvent, particularly, for example, water, methanol, or ethanol, as a structural element of the lattice of the compound. The amount of the polar solvent, particularly water, may be stoichiometric or non-stoichiometric.

[0296] Those skilled in the art will understand that not all nitrogen-containing heterocycles can form N-oxides because nitrogen requires available lone pairs of electrons to be oxidized to oxides. Those skilled in the art will identify nitrogen-containing heterocycles capable of forming N-oxides. Those skilled in the art will also recognize that tertiary amines can form N-oxides. Synthetic methods for preparing N-oxides of heterocycles and tertiary amines are well known to those skilled in the art, including but not limited to the oxidation of heterocycles and tertiary amines using peroxy acids such as peracetic acid and m-chloroperoxybenzoic acid (MCPBA), hydrogen peroxide, alkyl peroxides such as tert-butyl peroxide, sodium perborate, and dioxiranes such as dimethyldioxirane. These methods for preparing N-oxides have been extensively described and reviewed in the literature, see, for example: T.L. Gilchrist, Comprehensive Organic Synthesis, vol. 7, pp. 748-750; A.R. Katritzky and A.J. Boulton, Eds., Academic Press; and G.W. H. Heeseman and E.S. G. Wierstiuk, Advances in Heterocyclic Chemistry, vol. 22, pp. 390-392, A.R. Katritzky and A.J. Boulton, Eds., Academic Press.

[0297] The scope of this invention also includes metabolites of the compounds of this invention, i.e., substances formed in the body when the compounds of this invention are administered. Such products can be generated, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc., of the administered compound. Therefore, this invention includes metabolites of the compounds of this invention, including compounds obtained by methods that expose the compounds of this invention to mammals for a time sufficient to produce their metabolites.

[0298] This invention further includes, within its scope, prodrugs of the compounds of this invention, which are certain derivatives of the compounds of this invention that may themselves have little or no pharmacological activity, and which, when administered to or onto the body, can be converted, for example, by hydrolysis and cleavage into the compounds of this invention having the desired activity. Typically, such prodrugs are functional group derivatives of the compounds that are readily converted in vivo into the compounds with the desired therapeutic activity. Further information regarding the use of prodrugs can be found in “Pro-drugs as Novel Delivery Systems,” Vol. 14, ACS Symposium Series (T. Higuchi and V. Stella). The prodrugs of this invention can be prepared, for example, by replacing suitable functional groups present in the compounds of this invention with certain portions known to those skilled in the art as “pro-moiety” (e.g., as described in “Design of Prodrugs,” H. Bundgaard (Elsevier, 1985)).

[0299] This invention also covers compounds of the invention containing protecting groups. In any process of preparing the compounds of the invention, protection of sensitive or reactive groups on any relevant molecule may be necessary and / or desired, thereby forming a form of chemical protection for the compounds of the invention. This can be achieved by conventional protecting groups, for example, those described in T.W. Greene & P. ​​G. W. M. Uts, *Protective Groups in Organic Synthesis*, John Wiley & Sons, 1991, which are incorporated herein by reference. Protecting groups can be removed at appropriate subsequent stages using methods known in the art.

[0300] The term “about” means within ±10% of the value, for example, within ±5% or ±2%.

[0301] The sequence information involved in this invention is described in Table 1 below: Detailed Implementation

[0302] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0303] The abbreviations and English terms used in this article have the meanings described in Table 2 below:

[0304] Example 1: Preparation of Compound 6

[0305] Step 1: Compound 1 (100 g, 372.73 mmol) and NHS (55.77 g, 484.55 mmol) were added to a 2000 L three-necked flask, followed by 1000 mL of DCM. The system was cooled to 0 °C using a cold trap. DIC (61.15 g, 484.55 mmol) was added dropwise to the stirred system, maintaining an internal temperature <10 °C. After the addition was complete, the mixture was stirred at this temperature for 15–24 hours. The concentration of compound 1 was determined by high-performance liquid chromatography (HPLC) to be no greater than 0.5%, at which point stirring was stopped. Post-processing: The reaction solution was filtered, and the filter cake was washed with DCM (140 mL × 3). The filtrates were combined and concentrated under reduced pressure at a temperature not exceeding 40 °C until no significant liquid distillation was observed, yielding the crude solid compound 2.

[0306] Purification: Add 1L of methanol, stir, heat to slight reflux, and slurry for 2 hours; stir and cool to 50℃~55℃, cool in a water bath to 25℃~30℃, then continue to cool to 15℃~20℃, keep warm and stir for 30 minutes, filter, wash the filter cake with anhydrous methanol (140mL×3), dry the solid to obtain about 120g of wet product, dry under reduced pressure at 35℃~40℃ for 15 hours~24 hours; collect the material to obtain 114.20g of compound 2 solid (yield: 84%, HPLC: 99.4%, QNMR: 102%).

[0307] Step 2: Add compound 2 (73.78 g, 201.94 mmol) and compound 3 (95 g, 224.36 mmol) to a 2000 mL three-necked flask, and add DMF (590 mL). Heat the system to 38 °C ± 2 °C with stirring, and stir for 16 to 24 hours; when HPLC analysis shows that compound 3 is not greater than 3%, stop stirring.

[0308] Post-processing: Cool to 20℃~30℃, and add the reaction solution dropwise to MTBE (5200mL) under stirring, causing a solid to precipitate. Continue stirring for 1 hour after the addition is complete. Filter, wash the filter cake with MTBE (300mL×2), and dry under vacuum to obtain a wet product. Transfer to a vacuum oven and dry under reduced pressure at no higher than 40℃ for 10~15 hours. Collect the material to obtain 158g of compound 4 solid.

[0309] Purification: Add the solid to DMF (474 ​​mL), stir until dissolved, then add MTBE (3800 mL) dropwise. The solid precipitates out. After the addition is complete, continue stirring for 1 hour. Filter. Wash the filter cake with MTBE (250 mL * 2), dry under vacuum to obtain a wet product, transfer to a vacuum oven, and dry under reduced pressure at no higher than 40°C for 15 to 20 hours. Collect the material and weigh to obtain 153.03 g of solid (yield: 112%, HPLC: 89%, QNMR: 85%).

[0310] Step 3: After purging the reactor with nitrogen and applying micro-nitrogen protection, add DMF (1.1 kg), then add compound 5 (230 g, 419.70 mmol); then add compound 4 (300 g, 445.31 mmol), and cool. Add DMTMM (140 g, 475.01 mmol) while stirring. After the reaction system is thoroughly mixed, add N,N-diisopropylethylamine (70 g, 541.63 mmol) dropwise. Maintain the temperature and stir for approximately 2 hours, then raise the temperature to approximately 20°C and continue stirring for approximately 5 hours. HPLC analysis shows that compound 5 concentration is no greater than 0.5%, then stop stirring.

[0311] Post-treatment: Slowly add water (600g, 33.33mol) to quench the reaction, controlling the internal temperature below 20℃. Add dichloromethane isopropanol solution (10 volumes (V): 1V, 12kg) and water (5kg), and stir at 20-30℃ for 0.5-1.5 hours under nitrogen protection. Allow to stand and separate the organic phase, then add water (2.2kg) to the organic phase. Stir at 20-30℃ for 0.5-1.5 hours, allow to stand, separate the organic phase, add dichloromethane isopropanol solution (10V: 1V, 6kg) and water (2.4kg), and stir for 0.5-1.5 hours. Allow to stand, separate the organic phase, add dichloromethane isopropanol solution (10V: 1V, 9.2kg) and water (2.4kg), and stir for 0.5-1.5 hours. Allow to stand, collect the organic phase to obtain the crude solution. Concentrate under reduced pressure to approximately 1L. Concentration: After purging the reactor with nitrogen and applying micro-nitrogen protection, add the solution containing the crude compound 6 obtained above, heat to a temperature not exceeding 45°C, and concentrate under reduced pressure to 0.5–1.4 L. Then add 3.2 kg of dichloromethane and continue concentrating to 0.5–1.4 L, then add another 3.5 kg of dichloromethane and continue concentrating under reduced pressure to 0.5–1.4 L.

[0312] Purification: Add dichloromethane (3.3 kg) and isopropanol (0.9 kg). Under nitrogen protection, cool to 20–30 °C and stir for 0.5–1.5 hours to obtain a solution. Add methyl tert-butyl ether (10.4 kg) to a crystallization vessel and add the above solution dropwise over a period of at least 2 hours while stirring. After the addition is complete, stir under nitrogen protection at 15–30 °C for 0.5–3.0 hours. Filter the solution until no obvious liquid flows out. Wash once with methyl tert-butyl ether (1.4 kg), filter until no obvious liquid flows out, then wash once again with methyl tert-butyl ether (1.6 kg), filter until no obvious liquid flows out, transfer the solid, weigh, and obtain compound 6 wet product. After purging the reactor with nitrogen and applying a micro-nitrogen protection layer, add dichloromethane (2.2 kg) and isopropanol (1.7 kg), stir until homogeneous, then add the wet product of compound 6. Using 5.2 kg of dichloromethane and under nitrogen protection, stir at 20–30°C for 0.5–1.5 hours to obtain a solution containing compound 6. Slowly add this solution dropwise to methyl tert-butyl ether (10.2 kg) under stirring, controlling the internal temperature at 15–30°C, for at least 2 hours, and stir at 20–30°C for 0.5–3.0 hours under nitrogen protection. Filter the solution until no obvious liquid flows out, then rinse once with methyl tert-butyl ether (1.6 kg), filter again until no obvious liquid flows out, then rinse once more with methyl tert-butyl ether (1.5 kg), filter again until no obvious liquid flows out, and transfer the solid to obtain the purified wet product of compound 6. The wet sample was transferred to a vacuum oven and dried under reduced pressure at 35–45 °C for 24–26 hours to obtain 6363.24 g of the compound (yield 76%, HPLC: 98.9%, QNMR: 98.4%).

[0313] Example 2: Preparation of Compound 10

[0314] Step 1: Preparation of Compound 7

[0315] Compound 1 (180.25 g, 671.85 mmol), pentafluorophenol (PFP) (135.84 g, 738.01 mmol), and dichloromethane (1.8 L) were added to a 3000 mL three-necked flask. The mixture was stirred to dissolve and cooled to -5 °C to 10 °C. DIC (93.15 g, 738.11 mmol) was then added dropwise while maintaining the internal temperature at 0 °C to 15 °C. After the addition was complete, the mixture was kept warm and stirred. When the concentration of compound 1 was found to be below 1.0% by HPLC, stirring was stopped.

[0316] Post-processing: Filter, wash the filter cake with dichloromethane (270mL×3), and collect the filtrate; concentrate the above filtrate under reduced pressure at no higher than 40℃ to remove the solvent, and obtain 329g of solid crude product.

[0317] First purification: 329 g of the crude product was transferred to a 5 L three-necked flask, and 4.50 L of isopropanol was added. Stirring was started, and the mixture was heated in an oil bath at an internal temperature of 65℃~70℃ until completely dissolved. Heating was then stopped, and the temperature was lowered to 40℃~50℃, causing a solid to precipitate. The mixture was kept at this temperature and stirred for 0.5 to 1 hour, then further cooled to 15℃~25℃ and stirred for another 0.5 to 1 hour before filtration. The filter cake was washed with isopropanol (360 mL × 3), and dried under vacuum until no obvious droplets flowed down, yielding a wet solid product. This product was then vacuum dried at 40℃~50℃ for 18~24 hours. 7267.30 g of solid compound was collected (total yield 91%, HPLC: 99.32%, QNMR: 95%).

[0318] Second purification: 24.63 g of the obtained solid compound 7 was added to a 100 mL three-necked flask, followed by 75 mL of isopropyl acetate. The mixture was stirred and heated to 60 °C. After dissolving, 2.5 g of activated carbon was added, and stirring was continued for 10 minutes. The mixture was filtered, and a solid precipitated from the filtrate. The solid was then heated to dissolve and cooled to 15 °C–25 °C. The mixture was then cooled to approximately 5 °C in an ice-water bath and stirred for 0.5 hours. The mixture was filtered, and the filter cake was washed with MTBE (12 mL × 2). The mixture was then filtered under vacuum until no obvious liquid dripped out, yielding a wet solid product. The product was dried under reduced pressure at 40 °C–50 °C for at least 6 hours, and the solid compound 7 was collected as 20.12 g (yield 81%, HPLC: 99.76%, QNMR: 99.2%). HNMR (400M, CDCl3) δ2.12-2.16 (2H, m), δ2.70 (2H, t, J=6.8), δ2.89 (2H, t, J=7.2), δ3.36 (3H, s), δ8.89 (2H, s).

[0319] ESI-MS (m / z): 435.0 [M+H] + ,891.1[2M+Na] +

[0320] Step 2: Preparation of Compound 9

[0321] Method 1:

[0322] Purification method 1 for compound 8: Take 34 g of compound 8, add 680 mL of purified water, stir, dissolve, filter, and send to HPLC for preparation.

[0323] Instrument: DAC80, 250*80mm

[0324] Filler: PrePulite C18 10μm

[0325] Wavelength: 210 nm; Flow rate: 200 mL / min; Single injection volume: 40 mL; Mobile phase A: pure water;

[0326] Measure 40 mL (2.0 g) of sample and inject it through the sample loading port using a syringe. Run the equipment and elute the sample with 100% purified water. Start collecting the eluent when the absorbance rises to 2000 mAu and stop collecting the eluent when the absorbance drops to 1000 mAu. Repeat the operation. After preparation, combine about 3 L of eluent and freeze-dry for 41 hours to obtain about 20 g of solid. Place it in a vacuum drying oven and continue drying under reduced pressure at 40℃~60℃ for 6~7 hours. The solid weight obtained is 19.54 g (overall yield 57.5%, HPLC: 99%, QNMR: 98.3%).

[0327] Purification method 2 for compound 8: Take 30g of compound 8, add 600mL of purified water, stir, dissolve, filter, and send to HPLC for preparation.

[0328] Instrument: Dynamic Axial Compression 80 (DAC80), 250*80mm;

[0329] Filler: PrePulite T3 10μm;

[0330] Wavelength: 210 nm; Flow rate: 200 mL / min; Single injection volume: 40 mL; Mobile phase A: pure water;

[0331] Measure 40 mL (2.0 g) of sample and inject it through the sample loading port using a syringe. Run the equipment and elute the sample with 100% purified water. Start collecting the eluent when the absorbance rises to 600 mAu and stop collecting the eluent when the absorbance drops to 600 mAu. Repeat the operation. After preparation, combine approximately 6.7 L of eluent. Take 2.3 L of the prepared solution and concentrate it to near dryness under reduced pressure at 60 °C. Add 5 V of ethanol, stir, filter, and dry under vacuum to obtain approximately 8.3 g of solid. Place it in a vacuum drying oven and dry under reduced pressure at 40 °C–60 °C for 6–7 hours. The solid weight is 8.1 g (yield 78.7%, HPLC: 99.7%, QNMR: 103%).

[0332] Compound 8 (176.05 g, 553.04 mmol) was added to a 3000 mL three-necked flask, followed by DMSO (1230 mL). Stirring was started, and the temperature was raised to 60 °C. Compound 7 (252.18 g, 580.61 mmol) was dissolved in DMSO (530 mL) and transferred to a constant pressure dropping funnel for later use. The internal temperature was maintained at 55–65 °C, and the mixture was stirred for 20–30 minutes. The DMSO solution of compound 7 was then added dropwise over a period of at least 2.5 hours. HPLC analysis showed that compound 8 was no more than 3%. Stirring was then stopped.

[0333] Post-processing: The reaction solution was filtered while hot, dried under vacuum, and the filtrate was collected. The filtrate was slowly added dropwise to 14 L of stirred MTBE at 20–30 °C, causing solid precipitation. After the addition was complete, a large amount of solid precipitated, and stirring was continued for 0.5–1 hour to allow crystallization. The mixture was then filtered, dried under vacuum, and the filter cake was washed with 1.75 L × 2 of MTBE until no obvious droplets flowed out. Filtration was continued for 10–20 minutes to obtain the solid. The solid was added to 1.4 L of ethyl acetate, stirred, and heated to 30–40 °C, maintaining the temperature and stirring for 2 hours. The temperature was then lowered to 15–25 °C, maintaining the temperature and stirring for 1 hour. The mixture was filtered, and the filter cake was washed with 352 mL of ethyl acetate, and dried under vacuum until no droplets flowed out. The wet product was vacuum dried at 35–45 °C for 17–21 hours, and the product was collected to obtain 310.89 g of crude compound 9.

[0334] Purification: The crude product was added to a reaction flask, and a dichloromethane solution of isopropanol (10V:1V, 12.6L) was added. Stirring was started, and the temperature was raised to 25℃~30℃ and stirred for 1 hour. Diatomaceous earth was placed on the flask, the mixture was filtered, and the product was dried under vacuum. The filter cake was washed with a dichloromethane solution of isopropanol (10V:1V, 1.2L×2), and the filtrates were combined. The mixture was concentrated under reduced pressure at a temperature not exceeding 35℃ until no obvious distillate was distilled off. MTBE (1.5L) was added, and stirring was started. The mixture was stirred at 15℃~30℃ for 1 hour. The mixture was filtered, and the filter cake was washed with MTBE (250mL×2). The product was dried under vacuum until no liquid droplets flowed out, and then the vacuum was continued for 10 minutes to obtain a solid wet product. The product was transferred to a vacuum drying oven at 40℃ and dried for 17~20 hours. The product was collected to obtain a solid compound 9230.08g (total yield 69%, HPLC: 96.42%, QNMR: 94%).

[0335] Method 2: Compound 8 (10.01 g, 31.41 mmol) was dispersed in purified water (200 mL) and stirred for 5–10 minutes. Compound 2 (17.23 g, 47.12 mmol) was dispersed in acetonitrile (100 mL) and added dropwise to the stirring system. A solid precipitated. The pH of the system was adjusted to 6–7 with saturated sodium bicarbonate aqueous solution. The temperature was controlled not to exceed 20 °C, and the mixture was stirred for 5–8 hours. The mixture was filtered, and the filtrate was collected. The pH was adjusted to 3–4 with 3N HCl aqueous solution at a temperature below 10 °C. The mixture was stirred for 1 hour, and a solid precipitated. The solid was filtered and dried under reduced pressure at 40–50 °C for 6–20 hours to obtain 13.17 g of compound 9 solid (HPLC 91.10%, QNMR 86%, yield 60%).

[0336] Step 3: Preparation of Compound 10

[0337] Compound 9 (101.44 g, QNMR purity 94%, 167.68 mmol) was added to a 2 L three-necked flask, along with 600 mL of DMSO. The mixture was stirred and heated to 35 °C–45 °C until the solid was completely dissolved. The temperature was then lowered to 20 °C–30 °C. Compound 5 (91.04 g, QNMR 99%, 164.47 mmol) was added and stirred for 10–20 minutes. DMTMM (58.25 g, QNMR purity 85%, 168.00 mmol) was added and stirred for 5 minutes. DIEA (32.00 g, 247.60 mmol) was added dropwise to the system, and the mixture was stirred at 25–30 °C for 1–2 hours. When the HPLC analysis showed that compound 9 was not greater than 1%, stirring was stopped.

[0338] Post-treatment: Add isopropanol in dichloromethane solution (10V:1V, 4550mL) to the reaction solution, then add purified water (910mL×2) and stir for 5 minutes. Let stand, separate the liquids, collect the organic phase, wash with 10% sodium chloride aqueous solution (910mL×2) and stir for 5 minutes. Let stand and separate the organic phase. Extract the aqueous phase with isopropanol in dichloromethane solution (10V:1V, 910mL×2), combine the organic phases, add anhydrous sodium sulfate (170g), stir and dry for 15-20 minutes; filter. The filter cake was washed with a dichloromethane solution of isopropanol (10V:1V, 1820mL), and the filtrate was concentrated under reduced pressure at 35℃ until no fractions were distilled off. MTBE (1820mL) was added, and stirring was started. The mixture was slurried at room temperature for 1 hour. The mixture was filtered, and the filter cake was washed with MTBE (910mL). The mixture was dried under vacuum until no liquid droplets flowed out, and then the mixture was filtered for another 20-30 minutes. The solid was obtained and dried under vacuum at 40℃ to obtain 170.23g of crude solid (yield 95.3%, HPLC: 94.5%, QNMR: 92.3%).

[0339] Purification: 150 g of crude compound 10 was added to a mixed solution of DCM:MeOH = 5:1 to 10:1 (700 mL), and the mixture was stirred and heated to 35 °C to dissolve. A 7.3 L DCM silica gel column (1.5 kg) was used for wet loading. After loading, elution was performed according to the procedure shown in Table 3 below:

[0340] HPLC analysis was performed, and fractions with a purity greater than 97% were combined (UV254 2000nm~1000nm). The fractions were concentrated under reduced pressure at a temperature not exceeding 30℃. After no obvious liquid distillation, MTBE (200mL×2) was added and distilled until no obvious distillation occurred. Then, MTBE (1000mL) was added and stirred for 0.5~1 hour. The mixture was filtered, and the filter cake was washed with MTBE (100mL). The wet product was dried under reduced pressure at 35℃~45℃ for 18~22 hours. The product was collected to obtain 124.89g of compound 10 solid (yield 85%, HPLC 98.99%, QNMR 95%).

[0341] Example 3: Equivalent fraction screening test for preparing compound 7

[0342] Using compound 1 as a substrate, 300 mg was added to each reaction and reacted with pentafluorophenol in the presence of DIC condensing agent to prepare target compound 7. The reaction progress was monitored by HPLC, and the results after 3.5 hours of reaction are shown in Table 4 below:

[0343] The data in Table 4 above show that the raw materials at 1.1 to 1.3 eq can be basically completely converted, which can be used as a relatively optimal condition for further optimization.

[0344] Example 4: Partial Screening Test of Reaction Temperature for the Preparation of Compound 7

[0345] Using compound 1 as a substrate, 300 mg of each reaction was fed with pentafluorophenol in the presence of the condensing agent DIC to prepare target compound 7. The reaction progress was monitored by HPLC, and the results after 4 hours of reaction are shown in Table 5 below.

[0346] The data in Table 5 above show that the raw material is completely converted at 15℃ with the fewest impurities, which can be used as a better condition for further optimization.

[0347] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A method for preparing a compound of formula (I) or an acceptable pharmaceutical salt thereof: wherein: Z1is selected from C 1-6 alkylene, C 2-10 alkenylene, C 2-10 alkynylene, C 3-8 cycloalkylene, 6-10 membered aryl and 5-14 membered heteroaryl; Z2 is selected from oxygen or sulfur; Ring A is selected from C 6-10 Aryl, 5-10 heteroaryl, C 3-6 Cycloalkyl and 5-10 membered heterocyclic groups; said ring A is optionally substituted by one or more of the following substituents: halogen, nitro, cyano, oxo (=O), C 1-6 Alkyl and C 1- 6-alkoxy; m can be independently 0, 1, 2, 3, 4, 5, or 6. E is selected from the following groups optionally substituted with one or more R1s: 6-10 aryl, 5-14 heteroaryl; wherein R1 is independently selected from H (hydrogen), D (deuterium), halogen, CN, nitro, C 1-6 Alkyl and Halogenated C 1-6 alkyl; G is the leaving group for nucleophilic substitution reactions; The method includes preparing the compound of formula (I) by means of the following compound (I-1):

2. According to the method of claim 1, the compound represented by formula (I) has the following structure:

3. The method according to claim 1 or 2, wherein it meets one or more of the following conditions: (1) The method is carried out in the presence of one or more reagents in NHS and DIC, DCC; (2) The method is carried out in the presence of one or more reagents among PFP and DIC, DCC; (3) In the method described, the molar ratio of NHS reagent to compound of formula (I-1) is (0.1-5):1; (4) In the method described, the molar ratio of PFP reagent to compound of formula (I-1) is (0.1-5):1; (5) The method is carried out in the presence of an organic solvent selected from one or more of dichloromethane, chloroform, ethyl acetate, tetrahydrofuran and dioxane.

4. The method according to any one of claims 1 to 3, wherein the method further comprises a post-processing step and / or a refining step.

5. The method according to claim 4, wherein the refining step comprises one or more refining processes.

6. The method according to claim 5, wherein the first purification includes heating, cooling, solid separation, and rinsing with an organic solvent, and / or the second purification includes: (1) Heating, (2) Adsorption, (3) Cooling, and (4) Separation of solids.

7. A method for preparing a compound of formula (A) as shown below by means of a compound of formula (I) as described in claim 1 or 2: wherein s is selected from integers from 1 to 20; L is selected from natural amino acids or non-natural amino acids and their analogues (such as Ala, Arg, Asn, Asp, Cit, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, D-Val, D-Leu, D-Ala), and short peptides composed of amino acids (such as Gly-Lys, Asp-Gly-Gly-Phe-Gly (DGGFG, SEQ ID NO:1), Glu-Gly-Gly-Phe-Gly (EGGFG, SEQ ID NO:1), Glu-Gly-Gly-Phe-Gly (EGGFG, SEQ ID NO:1)). NO:2), Ala-Ala, Ala-Lys, Ala-Lys(Ac), Ala-Pro, Gly-Glu, Gly-Gly, Phe-Lys, Phe-Lys(Ac), Val -Ala, Val-Cit, Val-Lys, Val-Lys(Ac), Ala-Ala-Ala, Ala-D-Ala-Ala, Ala-Ala-Asn, Ala-Ala-Gl y, D-Leu-Ala-Glu, Gly-Gly-Arg, Gly-Glu-Gly, Gly-Gly-Gly, Gly-Ser-Lys, Glu-Val-Ala, Glu-V al-Cit, Ser-D-Ala-Pro, Val-Leu-Lys, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Phe-Gly (GGFG, SEQ ID NO:3), Gly-Gly-Val-Ala (GGVA, SEQ ID NO:4), Gly-Phe-Leu-Gly (GFLG, SEQ ID NO:5), Glu-Ala-Ala-Ala (EAAA, SEQ ID NO:6), Gly-Gly-Gly-Gly-Gly (GGGGG, SEQ ID NO:7)); and Z1, E, G and m as described in claim 1.

8. The preparation method according to claim 7, wherein the structure of compound (A) is as follows:

9. A method for preparing a compound of formula (I) as described in claim 1 or 2 by reacting it with a compound of formula (II) as shown below: wherein L and s are each independently as described in claim 7.

10. The preparation method according to claim 9, wherein the compound of formula (II) undergoes a purification step before reacting with the compound of formula (I).

11. The preparation method according to claim 10, wherein the purification of the compound of formula (II) is performed by preparative liquid chromatography.

12. The preparation method according to claim 11, wherein the liquid chromatography is a dynamic axial compression (DAC) liquid chromatography method.

13. The preparation method according to any one of claims 9 to 12, wherein the compound of formula (II) has the following structure:

14. The method according to any one of claims 9 to 13, wherein it meets one or more of the following conditions: (1) The molar ratio of the compound of formula (I) and the compound of formula (II) is (0.1 to 10):1; (2) The compound of formula (I) and the compound of formula (II) react under conditions of 0 to 100 °C; (3) The method is carried out in a DMF, DMSO or acetonitrile-water mixture.

15. The method according to any one of claims 7 to 14, wherein the method further comprises a post-processing step and / or a refining step.

16. A method for reacting a compound of formula (A) as described in claim 7 or 8 with a cytotoxic drug to generate a compound of formula (B) as shown below: in: The D portion is the cytotoxic drug component; the L, Z1, E, G, s, and m are each independently as described in claim 1 or 7.

17. The method according to claim 16, wherein it meets one or more of the following conditions: (1) The method is carried out in the presence of one of the condensing agent reagents DMTMM, CDTM, HATU, HBTU, EDCI, DCC or HOBT; (2) The method is carried out in the presence of one or more bases selected from N,N-diisopropylethylamine, N-methylmorpholine, triethylamine, and morpholine; (3) The method is carried out in an aprotic organic solvent.

18. A compound, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, N-oxide, isotopically labeled compound, metabolite, or prodrug thereof, said compound having the following structure: Rings A, Z1, Z2, E, G, L, m, and s are each independently as described in claim 1 or 7.

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