Preparation method of ADC connexon

Abstract

The invention provides a preparation method of a compound L. The preparation method comprises the following step S8: under the condition of acid, a compound K and glycolic acid react in a solvent to generate the compound L. # imgabs0 # R1 is an amino protecting group.

Description

Technical Field

[0001] The present invention relates to a preparation method of an ADC linker, belonging to the field of organic synthesis. Background Art

[0002] Antibody-drug conjugates (ADCs) are a class of innovative targeted therapeutic drugs, whose structure contains three core parts. Among them, the linker plays a bridging role, precisely binding the cytotoxic payload to the monoclonal antibody (mAb) and ensuring the in vivo circulation stability of the ADC. The chemical properties of the linker and its binding sites have an important impact on the overall performance of the ADC, including drug stability, pharmacokinetic behavior, pharmacodynamic properties, and optimization of the therapeutic window. Therefore, it is a key element in the design and development of ADCs.

[0003] The structure of the linker (linker) MC-Gly-Gly-Phe-Gly-NH-CH2-O-CH2COOH (CAS No.: 1599440-25-1) is as follows:

[0004]

[0005] The prior art WO2024109949A1, specification Page75 discloses a preparation method of the compound (CAS No.: 1599440-25-1), and the reaction route is as follows:

[0006]

[0007] In this route, steps 9e to 9g require two-step reactions. Among them, the yield of preparing 9f in the fifth step is 48%, and the crude product is obtained in the sixth step of preparing 9h. All steps of the whole route result in crude products.

[0008] Although significant progress has been made in the overall technical route of ADC drugs, the research on the preparation method of ADC linkers is relatively limited. Therefore, it is of great practical significance to further explore the preparation process of ADC linkers, improve their yield, reduce impurity generation, and lower production costs, so as to expand the production scale and enhance economic benefits. Therefore, there is a current need to develop a new preparation method for the synthetic linker (CAS No.: 1599440-25-1). Summary of the Invention

[0009] The present invention provides a new method for preparing a linker.

[0010] In the first aspect of the present invention, a preparation method of a compound L is provided, and the preparation method of the compound L includes the following step S8:

[0011] Step S8: Under acidic conditions, compound K reacts with glycolic acid in a solvent to form compound L.

[0012]

[0013] As described above, R1 is an amino protecting group, and R1 independently selected from one of benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, tert-butoxycarbonyl, pivaloyl, acetyl, trityl, allyloxycarbonyl, benzyl, trimethylsilylethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phthaloyl, p-methoxybenzyl, trifluoroacetyl, 2-nitrobenzenesulfonyl, 2,4-dinitrobenzenesulfonyl, 2-nitrophenoxycarbonyl, 2-nitrobenzyl, 2-nitro phenethyl, methanesulfonyl, p-toluenesulfonyl, methoxycarbonyl or ethoxycarbonyl;

[0014] In a certain preferred embodiment, in step S8, the acid is selected from one or any combination of boron trifluoride diethyl ether, methanesulfonic acid, camphorsulfonic acid, p-toluenesulfonic acid, scandium trifluoromethanesulfonate, zinc chloride, acetic acid, etc., for example, methanesulfonic acid;

[0015] In a certain preferred embodiment, in step S8, the molar ratio of compound K to glycolic acid is 1:1 to 20, for example, 1:10;

[0016] In a certain preferred embodiment, in step S8, the molar ratio of compound K to the acid is 1:0.1 to 1.0, for example, 1:0.2;

[0017] In a certain preferred embodiment, in step S8, the mass-volume ratio of compound K to the solvent is 1:10 to 15 g / mL, for example, 1:10 g / mL;

[0018] In a certain preferred embodiment, in step S8, the reaction temperature is 30 to 45 °C;

[0019] In a certain preferred embodiment, in step S8, the reaction time is 3 to 5 h, for example, 3 h;

[0020] In a certain preferred embodiment, in step S8, the solvent is selected from any one or more of tetrahydrofuran, dichloromethane, dimethyl sulfoxide, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone, etc., for example, N,N-dimethylacetamide;

[0021] In certain embodiments, in step S8, after the reaction, the following post-treatment steps are further included, and the post-treatment steps are conventional post-treatment steps for such reactions in the art, for example, one or more of quenching, crystallization, filtration, washing, rotary evaporation, drying or column chromatography.

[0022] In a preferred embodiment, the method for preparing compound L further includes the method for preparing compound K, and the method for preparing compound K includes the following step S7;

[0023] Step S7: In a solvent, compound J reacts in the presence of acetic acid, lead tetraacetate, and cupric acetate anhydrous to form compound K;

[0024]

[0025] wherein R1 is as described in step S8 of the first aspect above;

[0026] In a preferred embodiment, in step S7, the molar ratio of compound J to acetic acid is 1:1 to 4, such as 1:2.2;

[0027] In a preferred embodiment, in step S7, the molar ratio of compound J to lead tetraacetate is 1:2 to 4, such as 1:2;

[0028] In a preferred embodiment, in step S7, the molar ratio of compound J to cupric acetate anhydrous is 1:1 to 4, such as 1:1;

[0029] In a preferred embodiment, in step S7, the mass-volume ratio of compound J to the solvent is 1:10 to 15 g / mL, such as 1:10 g / mL;

[0030] In a preferred embodiment, in step S7, the reaction temperature is 30 to 55 °C;

[0031] In a preferred embodiment, in step S7, the reaction time is 0.5 to 4 h, such as 1 h;

[0032] In a preferred embodiment, in step S7, the solvent is selected from any one or more of N,N-dimethylacetamide, N,N-dimethylformamide, and N-methyl-2-pyrrolidone, such as N,N-dimethylacetamide.

[0033] In a preferred embodiment, the method for preparing compound L further includes the method for preparing compound J, and the method for preparing compound J includes the following steps S5 and S6:

[0034] Step S5: Compound G condenses with HOSU to form compound H,

[0035]

[0036] wherein R1 is as described in step S8 of the first aspect above;

[0037] In a preferred embodiment, in step S5, the molar ratio of compound G to HOSU is 1:1 to 3, such as 1:1.5;

[0038] In a preferred embodiment, in step S5, the condensing agent for the reaction is selected from carbodiimides, imidazolinium reagents, phosphonium salts, organophosphorus reagents, uronium salts, pyridinium reagents, and coupling reaction reagents of phosphonic acids, and more particularly, any single or combined condensing agent selected from EDCI, DIC, DCC, HBTU, PyBOP, BOP, HATU, HOBT, TBTU, DIC / HOBT, DCC / DMAP, EDCI / DMAP, EDCI / HOBT, HATU / HOBT, such as EDCI;

[0039] In a preferred embodiment, in step S5, the molar ratio of compound G to the condensing agent is 1:1 to 3, such as 1:2;

[0040] In a preferred embodiment, in step S5, the reaction temperature is 0 to 30 °C, such as 20 °C;

[0041] In a preferred embodiment, in step S5, the reaction time is 0.5 to 16 h, such as 5 h;

[0042] In a preferred embodiment, in step S5, the solvent is selected from any one or more of N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone, tetrahydrofuran, dichloromethane, acetonitrile, benzonitrile, phenylacetonitrile, ether, methyl tert-butyl ether, 1,4-dioxane, diisopropyl ether, ethyl acetate, and acetone, such as N,N-dimethylacetamide.

[0043] Step S6: Under basic conditions, compound H reacts with compound I to form compound J,

[0044]

[0045] wherein R1 is as described in step S8 of the first aspect above;

[0046] In a preferred embodiment, in step S6, the molar ratio of compound H to compound I is 1:1 to 2, such as 1:1.2;

[0047] In a preferred embodiment, in step S6, the base is selected from inorganic bases or organic bases; the inorganic bases are selected from one or more of sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, calcium carbonate, tripotassium phosphate, trisodium phosphate, dipotassium hydrogen phosphate, and disodium hydrogen phosphate, such as sodium bicarbonate; the organic bases are selected from any one of triethylamine, N-methylmorpholine, pyridine, and diisopropylethylamine, such as diisopropylethylamine;

[0048] In a preferred embodiment, in step S6, the molar ratio of compound H to the base is 1:1.5 to 5, such as 1:2;

[0049] In a preferred embodiment, in step S6, the reaction temperature is 0 to 30 °C, such as 20 °C;

[0050] In a preferred embodiment, in step S6, the reaction time is 0.5 to 5 h, such as 1 h;

[0051] In a preferred embodiment, in step S6, the solvent is selected from any one or more of ethylene glycol dimethyl ether, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone, tetrahydrofuran, dichloromethane, acetonitrile, 1,4-dioxane, and water, such as a mixed solvent of ethylene glycol dimethyl ether and water.

[0052] In a preferred embodiment, the preparation method of the compound L further includes the preparation method of the compound G, and the preparation method of the compound G includes the following steps S3 and S4:

[0053] Step S3: The compound D is condensed with HOSU to form the compound E,

[0054]

[0055] wherein R1 is the same as described in step S8 of the first aspect above;

[0056] In a preferred embodiment, in step S3, the molar ratio of the compound D to HOSU is 1:1 to 3, such as 1:1.5;

[0057] In a preferred embodiment, in step S3, the condensing agent for the reaction is selected from carbodiimide, imidazolinium reagent, phosphonium salt, organic phosphorus reagent, uronium salt, pyridinium reagent, and coupling reagent of phosphonic acid, and more particularly any single condensing agent or composite condensing agent among EDCI, DIC, DCC, HBTU, PyBOP, BOP, HATU, HOBT, TBTU, DIC / HOBT, DCC / DMAP, EDCI / DMAP, EDCI / HOBT, HATU / HOBT, such as EDCI, DIC or DCC;

[0058] In a preferred embodiment, in step S3, the molar ratio of the compound D to the condensing agent is 1:1 to 3, such as 1:1.5;

[0059] In a preferred embodiment, in step S3, the reaction temperature is 0 to 30 °C, such as 20 °C;

[0060] In a preferred embodiment, in step S3, the reaction time is 1 to 24 h, such as 20 h;

[0061] In a preferred embodiment, in step S3, the solvent is selected from any one or more of N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone, tetrahydrofuran, dichloromethane, acetonitrile, benzonitrile, phenylacetonitrile, diethyl ether, methyl tert-butyl ether, 1,4-dioxane, diisopropyl ether, ethyl acetate, and acetone, such as N,N-dimethylacetamide.

[0062] Step S4: Under basic conditions, compound E reacts with compound F to form compound G,

[0063]

[0064] wherein R1 is the same as described in step S8 of the first aspect above;

[0065] In a preferred embodiment, in step S4, the molar ratio of compound E to compound F is 1:1 to 2, such as 1:1.2;

[0066] In a preferred embodiment, in step S4, the base is selected from inorganic bases or organic bases; the inorganic bases are selected from one or more of sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, calcium carbonate, tripotassium phosphate, trisodium phosphate, dipotassium hydrogen phosphate, and disodium hydrogen phosphate, such as sodium bicarbonate; the organic bases are selected from any one of triethylamine, N-methylmorpholine, pyridine, and diisopropylethylamine, such as diisopropylethylamine;

[0067] In a preferred embodiment, in step S4, the molar ratio of compound E to the base is 1:1.5 to 5, such as 1:2;

[0068] In a preferred embodiment, in step S4, the reaction temperature is 0 to 30 °C, such as 20 °C;

[0069] In a preferred embodiment, in step S4, the reaction time is 0.5 to 3 h, such as 0.5 h;

[0070] In a preferred embodiment, in step S4, the solvent is selected from any one or more of ethylene glycol dimethyl ether, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone, tetrahydrofuran, dichloromethane, acetonitrile, and 1,4-dioxane, such as ethylene glycol dimethyl ether.

[0071] Furthermore, the second aspect of the present invention also provides a method for preparing compound C, and the method for preparing compound C includes the following steps S1 and S2:

[0072] Step S1: Under basic conditions, compound A reacts with glycine to form compound B,

[0073]

[0074] In a preferred embodiment, in step S1, the molar ratio of compound A to glycine is 1:1 to 2, such as 1:1.5;

[0075] In a preferred embodiment, in step S1, the base is selected from any one of 2,4-dimethylpyridine, 2,4,6-trimethylpyridine, 4-dimethylaminopyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene, triethylamine, N-methylmorpholine, pyridine, diisopropylethylamine, such as 2,4,6-trimethylpyridine;

[0076] In a preferred embodiment, in step S1, the molar ratio of compound A to the base is 1:1.2 to 3, such as 1:1.5;

[0077] In a preferred embodiment, in step S1, the reaction temperature is 0 to 30 °C, such as 10 to 20 °C;

[0078] In a preferred embodiment, in step S1, the reaction time is 3 to 12 h, such as 4 h;

[0079] In a preferred embodiment, in step S1, the solvent is selected from any one or more of acetonitrile, benzonitrile, phenylacetonitrile, water, such as a mixed solvent of acetonitrile and water;

[0080] In a preferred embodiment, in step S1, the volume ratio of the solvent to water in terms of mass is 1:1 to 1.5 g / mL, such as 1:1.2 g / mL;

[0081] Step S2: Compound B is condensed with HOSU to form compound C,

[0082]

[0083] In a preferred embodiment, in step S2, the molar ratio of compound B to HOSU is 1:1 to 3, such as 1:1.2;

[0084] In a preferred embodiment, in step S2, the condensing agent for the reaction is selected from carbodiimide, imidazolinium reagent, phosphonium salt, organophosphorus reagent, uronium salt, pyridinium reagent and coupling reagent of phosphonic acid, more particularly any single condensing agent or composite condensing agent selected from EDCI, DIC, DCC, HBTU, PyBOP, BOP, HATU, HOBT, TBTU, DIC / HOBT, DCC / DMAP, EDCI / DMAP, EDCI / HOBT, HATU / HOBT, such as EDCI;

[0085] In a preferred embodiment, in step S2, the molar ratio of compound B to the condensing agent is 1:1 to 3, such as 1:1.5;

[0086] In a preferred embodiment, in step S2, the reaction temperature is 0 to 30 °C, such as 15 to 25 °C;

[0087] In a preferred embodiment, in step S2, the reaction time is 1 to 24 h, such as 3 h;

[0088] In a preferred embodiment, in step S2, the solvent is selected from any one or more of N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone, tetrahydrofuran, dichloromethane, acetonitrile, benzonitrile, phenylacetonitrile, tetrahydrofuran, diethyl ether, methyl tert-butyl ether, 1,4-dioxane, diisopropyl ether, ethyl acetate, acetone, such as acetone.

[0089] Furthermore, the third aspect of the present invention also provides a method for preparing compound N, and the method for preparing compound N includes the following steps S9 and S10:

[0090] Step S9: Deprotecting compound L to obtain compound M,

[0091]

[0092] wherein R1 is the same as described in step S8 of the first aspect above;

[0093] In a preferred embodiment, in step S9, the deprotecting reagent is a conventional amino deprotecting reagent, and different reagents can be selected for deprotection according to specific different protecting groups;

[0094] In a preferred embodiment, when the protecting group in step S9 is 9-fluorenylmethyloxycarbonyl, the deprotecting reagent in step S9 is selected from piperidine, triethylamine, tetrabutylammonium fluoride, 1,8-diazabicyclo[5.4.0]undec-7-ene, such as triethylamine;

[0095] In a preferred embodiment, in step S9, the molar ratio of compound L to the deprotecting reagent is 1:3 to 10, such as 1:10;

[0096] In a preferred embodiment, in step S9, the solvent is selected from any one or more of cyclopentyl methyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, methyl tert-butyl ether, 1,4-dioxane, propyl ether, isopropyl ether, diethyl ether, ethylene glycol dimethyl ether or anisole, such as a mixed solvent of tetrahydrofuran and water;

[0097] In a preferred embodiment, in step S9, the mass-volume ratio of the solvent to water is 1 to 20:1 g / mL, such as 20:1 g / mL;

[0098] In a preferred embodiment, in step S9, the reaction temperature is 5 to 40 °C, such as 20 to 25 °C;

[0099] In a preferred embodiment, in step S9, the reaction time is 3 to 24 h, such as 4 h;

[0100] Step S10: Under alkaline conditions, compound M reacts with compound C to form compound N.

[0101]

[0102] In a preferred embodiment, in step S10, the molar ratio of compound M to compound C is 1:1 to 1.2, such as 1:1;

[0103] In a preferred embodiment, in step S10, the base is selected from inorganic bases or organic bases; the inorganic bases are selected from one or more of sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, calcium carbonate, tripotassium phosphate, trisodium phosphate, dipotassium hydrogen phosphate, and disodium hydrogen phosphate, such as sodium bicarbonate; the organic bases are selected from any one of triethylamine, N-methylmorpholine, pyridine, and diisopropylethylamine, such as triethylamine;

[0104] In a preferred embodiment, in step S10, the molar ratio of compound M to the base is 1:0.5 to 3, such as 1:1;

[0105] In a preferred embodiment, in step S10, the solvent is a nitrile, ether, sulfone solvent, or a mixed solvent composed of an amide and water; preferably a mixed solvent composed of a nitrile and water;

[0106] In a preferred embodiment, in step S10, the volume ratio of the nitrile solvent to water is 2 to 3:1, such as 2:1;

[0107] In a preferred embodiment, in step S10, the nitrile solvent is selected from any one or more of acetonitrile, propionitrile, butyronitrile, isobutyronitrile, benzonitrile, or phenylacetonitrile, such as acetonitrile; the ether solvent is selected from any one or more of tetrahydrofuran, ethylene glycol dimethyl ether, anisole, 1,4-dioxane, and diethyl ether; the sulfone solvent is selected from dimethyl sulfoxide; the amide solvent is selected from any one or more of N,N-dimethylformamide, N-methyl-2-pyrrolidone, and N,N-dimethylacetamide, such as N,N-dimethylformamide;

[0108] In a preferred embodiment, in step S10, the reaction temperature is 5 to 30 °C, such as 15 to 25 °C;

[0109] In a preferred embodiment, in step S10, the reaction time is 1 to 3 h, such as 2 h;

[0110] In the fourth aspect of the present invention, a new compound H is provided, and its structural formula is as follows:

[0111]

[0112] Wherein, R1 is the same as described in step S8 of the above first aspect, and R1 is not benzyloxycarbonyl or tert-butoxycarbonyl;

[0113] Further, preferably, R1 is 9-fluorenylmethoxycarbonyl, and the structural formula of the new compound H-1 is as follows:

[0114]

[0115] In the fifth aspect of the present invention, a preparation method of a drug-linker complex is provided, which includes the following steps:

[0116] (1) Preparation method of compound N: It includes the following step S10: Under alkaline conditions, compound M reacts with compound C to form compound N,

[0117]

[0118] The conditions and operations of the preparation method of the compound N can be as described in any aspect of the present invention;

[0119] (2) Using compound N as a raw material to prepare the drug-linker complex; The drug can be selected from calicheamicin, MMAF, MMAE, doxorubicin, SN-38, Dxd, and amanitin.

[0120] In the sixth aspect of the present invention, a preparation method of an ADC conjugate is provided, which includes the following steps:

[0121] (1) Preparation method of compound N: It includes the following step S10: Under alkaline conditions, compound M reacts with compound C to form compound N,

[0122]

[0123] The conditions and operations of the preparation method of the compound N can be as described in any aspect of the present invention;

[0124] (2) Using compound N as a raw material to prepare the ADC conjugate; The ADC conjugate includes but is not limited to the following drugs: trastuzumab deruxtecan, datopotamab deruxtecan, patritumab deruxtecan, ifinatamab deruxtecan.

[0125] In the seventh aspect of the present invention, a preparation method of a compound of formula 7 is provided, including the following steps:

[0126] Step S1: In a solvent, the compound of Formula 1 reacts under acidic conditions to obtain the compound of Formula 2;

[0127] Step S2: In a solvent, the compound of Formula 2 reacts with the compound of Formula 3 to obtain the compound of Formula 4;

[0128] Step S3: Deprotect the compound of Formula 4 to obtain the compound of Formula 5;

[0129] Step S4: The compound of Formula 5 undergoes a condensation reaction with the compound of Formula 6 to obtain the compound of Formula 7;

[0130]

[0131] The R1 is an amino protecting group, especially an amino protecting group that can be removed under reducing conditions, preferably Cbz, Bn, Alloc, more preferably Cbz;

[0132] The R2 is selected from optionally substituted or unsubstituted C1-C 12 alkyl, optionally substituted or unsubstituted C1-C8 cycloalkyl; Preferably, the R2 is optionally substituted or unsubstituted methylene; More preferably, the R2 is methylene.

[0133] The R3 is a carboxyl protecting group; especially a carboxyl protecting group that can be removed under palladium-carbon catalysis conditions; Preferably, the carboxyl protecting group is benzyl or optionally substituted benzyl; Preferably, the R3 is benzyl.

[0134] The R4 is a linker, and the linker is selected from Preferably, the R4 is

[0135] The L is an optionally substituted or unsubstituted PEG 1-12 chain, optionally substituted or unsubstituted C1-C 12 alkyl chain; The PEG 1-12 chain is represented as For example, PEG 1-11 、PEG 1-10 、PEG 1-9 、PEG 1-8 、PEG 1-7 、PEG 1-6 、PEG 1-5 、PEG 1-4 、PEG 1-3 、PEG 1-2 、PEG 12 、PEG 11 、PEG 10, PEG9, PEG8, PEG7, PEG6, PEG5, PEG4, PEG3, PEG2, (PEG); Preferably, the L is a C2 alkyl chain, a C3 alkyl chain, a C4 alkyl chain, a C5 alkyl chain, a PEG2 chain, a PEG4 chain, a PEG6 chain, a PEG8 chain.

[0136] The R5 is an active ester, selected from Preferably, the R5 is

[0137]

[0138] In certain embodiments, in step S1, the solvent is selected from one or more of toluene, tetrahydrofuran, N,N-dimethylformamide DMF, dioxane, ethyl acetate; preferably DMF.

[0139] In certain embodiments, in step S1, the catalyst for the reaction is an acetate. Preferably, the catalyst is lead tetraacetate and / or copper acetate; preferably, the catalyst is lead tetraacetate.

[0140] In certain embodiments, in step S1, the reaction time is 1 - 3 h, preferably 2 h.

[0141] In certain embodiments, in step S1, the reaction progress can be monitored by conventional monitoring methods in the art, such as HPLC, TLC or LCMS. Generally, the disappearance or no longer reaction of the compound of formula 1 is used as the reaction end point.

[0142] In certain preferred embodiments, in step S1, after the reaction, the following post-treatment steps are further included, such as one or more of pulping, washing, filtering, drying.

[0143] In certain preferred embodiments, in step S2, the reaction is carried out under basic conditions; the base is selected from one or more of potassium tert-butoxide, sodium tert-butoxide, sodium methoxide, sodium ethoxide, potassium carbonate, lithium hydroxide, sodium hydroxide, potassium hydroxide; preferably, the base is lithium hydroxide; preferably, the molar ratio of the compound of formula 2 to the base is 1 - 1.5;

[0144] In certain preferred embodiments, the preparation method of step S2 includes the following steps: dissolving the compound of formula 2 and the compound of formula 3 in an organic solvent, cooling, dissolving the base in water, slowly dropping it into the reaction flask, and after monitoring the reaction is completed, performing post-treatment to obtain the product.

[0145] In certain preferred embodiments, the post-treatment in step S2 is selected from one of extraction, washing, drying, pulping.

[0146] In certain preferred embodiments, the preparation method described in step S3 includes the following steps: adding the compound of formula 4, a catalyst, and a reaction solvent into a reaction flask, purging with hydrogen, reacting overnight, monitoring until the raw materials are completely reacted, stopping the reaction, and performing post-treatment to obtain the product; preferably, the catalyst is selected from one of palladium on carbon, palladium hydroxide on carbon, and platinum on carbon; preferably, when R1 is Alloc, the protecting group can be selectively removed step by step according to conventional methods in the art; preferably, the Alloc can be removed using tetrakis(triphenylphosphine)palladium.

[0147] In certain preferred embodiments, in step S3, the reaction solvent is selected from one or more of methanol, tetrahydrofuran, dimethylformamide, acetone, toluene, dichloromethane, and water; preferably, the reaction solvent is a combination of methanol and water, preferably, the volume ratio of methanol to water is (1 - 3):1; more preferably, the volume ratio of methanol to water is 2:1.

[0148] In certain preferred embodiments, in step S3, the reaction progress can be monitored by conventional monitoring methods in the art, such as HPLC, TLC, or LCMS, and generally, the disappearance of the compound of formula 4 or the cessation of the reaction is used as the reaction endpoint.

[0149] In certain preferred embodiments, in step S3, the post-treatment of the reaction is selected from one or more of filtration, washing, and concentration.

[0150] In certain preferred embodiments, in step S3, the washing reagent is a combination of methanol and water.

[0151] In certain preferred embodiments, in step S4, the molar ratio of the compound of formula 5 to the compound of formula 6 is 1:(1 - 3); preferably, the molar ratio of the compound of formula 5 to the compound of formula 6 is 1:1.

[0152] In certain preferred embodiments, in step S4, the solvent for the condensation reaction is selected from one or more of N,N-dimethylacetamide, N,N-dimethylformamide, N-ethyl-2-pyrrolidone, N-methyl-2-pyrrolidone, and dimethyl sulfoxide; more preferably, the solvent is N,N-dimethylacetamide.

[0153] In some preferred embodiments, in step S4, the condensation reaction is carried out under basic conditions; the base is selected from one or more of pyridine, 2,6-dimethylpyridine, trimethylpyridine, triethylamine, quinoline, N,N-dimethylaminopyridine, 2,3-dimethylpyridine, 3,4-dimethylpyridine, 1,3,5-trimethylpyridine, 2,3,5-trimethylpyridine, 2,4,6-trimethylpyridine, 2,6-di-tert-butylpyridine; preferably, the base is 2,6-dimethylpyridine; more preferably, the molar ratio of the compound of formula 5 to the base is 1:(1-3); preferably, the molar ratio of the compound of formula 5 to the base is 1:(1-2).

[0154] In some preferred embodiments, the method for preparing the compound of formula 6 comprises the following step S5: The compound of formula 8 and compound A undergo a condensation reaction to obtain the compound of formula 6;

[0155]

[0156] wherein the definitions of R4, L, and R5 are as described in any embodiment of the present invention.

[0157] In some preferred embodiments, in step S5, the condensing agent for the condensation reaction is selected from any single condensing agent or composite condensing agent of EDCI, DIC, DCC, HBTU, PyBOP, BOP, HATU, HOBT, HOSu, TBTU, DIC / HOBT, DCC / DMAP, EDCI / DMAP, EDCI / HOBT, HATU / HOBT; preferably, the condensing agent is preferably DCC; preferably, the molar ratio of the compound of formula 8 to the condensing agent is 1:(1-3); preferably, the molar ratio of the compound of formula 8 to the condensing agent is 1:(1-2); more preferably, the molar ratio of the compound of formula 8 to the condensing agent is 1:1.5.

[0158] In some preferred embodiments, in step S5, compound A for the condensation reaction is selected from one of tetrafluorophenol, pentafluorophenol, N-hydroxysuccinimide (HOSu), p-nitrophenol; preferably, compound A is tetrafluorophenol; preferably, the molar ratio of the compound of formula 8 to compound A is 1:(1-1.4); more preferably, the molar ratio of the compound of formula 8 to compound A is 1:1.

[0159] In some preferred embodiments, in step S5, the reaction temperature of the condensation reaction is room temperature.

[0160] In some preferred embodiments, in step S5, the reaction time of the condensation reaction is 1-3 h, more preferably, the reaction time is 2 h.

[0161] In certain embodiments, in step S5, the reaction progress of the reaction can be monitored by conventional monitoring methods in the art, such as HPLC, TLC or LCMS. Generally, the disappearance or non-reaction of the compound of formula 8 is taken as the reaction end point.

[0162] In certain preferred embodiments, in step S5, after the reaction, the following post-treatment steps are further included, such as one or more of extraction, washing, drying, and column purification; preferably, the solvent for extraction is a combination of ethyl acetate and water; preferably, the solvent for column purification is a combination of petroleum ether and ethyl acetate, and more preferably, the volume ratio of petroleum ether to ethyl acetate is 2:1 to 1:2.

[0163] The present invention provides a method for preparing a compound of formula 6, and the method for preparing the compound of formula 6 includes the following step S5: a compound of formula 8 and compound A are subjected to a condensation reaction to obtain a compound of formula 6;

[0164]

[0165] wherein the definitions of R4, L, and R5 are as described in any embodiment of the present invention;

[0166] The conditions and operations of the method for preparing the compound of formula 6 can be as described in any item of the present invention.

[0167] The present invention provides a method for preparing a compound of formula 4, and the method for preparing the compound of formula 4 includes the following step S2: in a solvent, a compound of formula 2 and a compound of formula 3 are reacted to obtain a compound of formula 4;

[0168]

[0169] The definitions of R1, R2, and R3 are as described in any embodiment of the present invention;

[0170] The conditions and operations of the method for preparing the compound of formula 4 can be as described in any item of the present invention.

[0171] The present invention provides a method for preparing a compound of formula 5, and the method for preparing the compound of formula 5 includes step S3: deprotecting the compound of formula 4 to obtain a compound of formula 5;

[0172]

[0173] The definitions of R1, R2, and R3 are as described in any embodiment of the present invention;

[0174] The conditions and operations of the method for preparing the compound of formula 5 can be as described in any item of the present invention.

[0175] The present invention provides a method for preparing a compound of formula 7, and the method for preparing the compound of formula 7 includes the following step S4: a compound of formula 5 and a compound of formula 6 are subjected to a condensation reaction to obtain a compound of formula 7;

[0176]

[0177] The definitions of R2, R4, R5, and L are as described in any aspect of the present invention;

[0178] The conditions and operations of the method for preparing the compound of formula 7 can be as described in any item of the present invention.

[0179] The present invention provides a compound of formula 4, a compound of formula 5, or a compound of formula 6, and their structures are as shown below:

[0180]

[0181] The definitions of R1, R2, R3, R4, R5, and L are as described in any aspect of the present invention.

[0182] In some preferred embodiments, the structures of the compound of formula 4, the compound of formula 5, or the compound of formula 6 are as shown below:

[0183]

[0184] The present invention provides an application of a compound of formula 7. The compound of formula 7 can be used to prepare an ADC antibody-drug conjugate; preferably, the compound of formula 7 can be conjugated with a toxin and an antibody having an amino group at the end to be used to prepare an ADC antibody-drug conjugate; preferably, the toxin is selected from one of MMAE, MMAF, Exatecan, and Eribulin.

[0185] In the eighth aspect of the present invention, a method for preparing compound 10 is provided, including the following steps:

[0186] Step S1: In a solution, compound 1 reacts with a resin to obtain compound 2;

[0187] Step S2: Compound 2 undergoes a deprotection reaction under basic conditions to obtain compound 3;

[0188] Step S3: In a solution, compound 3 and compound 4 undergo a condensation reaction to obtain compound 5;

[0189] Step S4: Compound 5 removes the resin under the action of an acid to obtain compound 6;

[0190] Step S5: In a solution, compound 6 is obtained under the action of a catalyst to obtain compound 7;

[0191] Step S6: Compound 7 reacts with compound 8 to obtain compound 9;

[0192] Step S7: Deprotect compound 9 to obtain compound 10;

[0193]

[0194] The R is an amino protecting group, especially an amino protecting group that can be removed under reducing conditions or basic conditions, preferably Fmoc, Cbz, Bn, Alloc; preferably Fmoc;

[0195] The R1 is an amino protecting group, especially an amino protecting group that can be removed under basic conditions, preferably Fmoc;

[0196] The n is 0 to 2; preferably, the n is 0, 1, 2;

[0197] The R2 is selected from any substituted or unsubstituted C1-C 12 alkyl, any substituted or unsubstituted C1-C8 cycloalkyl; preferably, the R2 is any substituted or unsubstituted methylene; more preferably, R2 is methylene.

[0198] The R3 is a carboxyl protecting group; especially a carboxyl protecting group that can be removed under the condition of palladium-carbon catalysis; preferably, the carboxyl protecting group is benzyl, any substituted benzyl or allyl, and preferably the carboxyl protecting group is benzyl.

[0199] In some preferred embodiments, in step S1, the resin is a chlorine resin; preferably, the CAS number of the chlorine resin is 934816-82-7.

[0200] In some preferred embodiments, in step S1, the molar ratio of the resin to compound 1 is 1:(1-5); preferably 1:(2-4).

[0201] In some preferred embodiments, in step S1, the reaction is carried out under basic conditions; preferably, the base is selected from one or more of N,N-diisopropylethylamine, triethylamine, N-methylmorpholine, pyridine; preferably, the base is N,N-diisopropylethylamine; preferably, the molar ratio of the resin to the base is 1:(1-7), preferably (3-6).

[0202] In some preferred embodiments, in step S2, the base is selected from one or more of sodium methoxide, sodium ethoxide, potassium ethoxide, sodium tert-butoxide, piperidine, sodium hydroxide, potassium hydroxide; preferably, the base is piperidine.

[0203] In some preferred embodiments, in step S3, the condensing agent for the condensation reaction is selected from any single condensing agent or composite condensing agent among DIC, DCC, HBTU, PyBOP, BOP, HATU, HOBT, HOSu, TBTU, DIC / HOBT, DCC / DMAP, EDCI / DMAP, EDCI / HOBT, HATU / HOBT, preferably HOBT / DIC.

[0204] In some preferred embodiments, in step S4, the acid is trifluoroacetic acid or hexafluoroisopropanol; preferably, the acid is hexafluoroisopropanol.

[0205] In some preferred embodiments, in step S5, the catalyst is acetate, preferably, the acetate is Pb(OAc)4 and / or Cu(OAc)2; preferably, the acetate is Pb(OAc)4, a combination of Pb(OAc)4 and Cu(OAc)2; preferably, the molar ratio of Pb(OAc)4 to Cu(OAc)2 is (3 - 7):1; preferably, the molar ratio of compound 6 to the acetate is 1:(1 - 5); preferably 1:(2 - 4).

[0206] In some preferred embodiments, in step S6, the molar ratio of compound 7 to compound 8 is 1:(1 - 3); preferably 1:1.

[0207] In some preferred embodiments, in step S6, the reaction is carried out under basic conditions, and the base is selected from one or more of lithium hydroxide, lithium carbonate, lithium bicarbonate, lithium acetate, sodium hydroxide, potassium hydroxide; preferably, the base is lithium hydroxide; preferably, the molar ratio of compound 7 to the base is 1:(1 - 2), preferably 1:(1 - 1.1).

[0208] In some preferred embodiments, step S7 can remove the protecting group according to conventional methods in the art.

[0209] In some preferred embodiments, in step S6, the reaction time of the reaction is 0.5 - 5 h.

[0210] The present invention provides a method for preparing an ADC linker, comprising the following steps:

[0211] Step S8: React compound 10 with a resin to obtain compound 11;

[0212] Step S9: Carry out a deprotection reaction on compound 11 under basic conditions to obtain compound 12;

[0213] Step S10: React compound 12 with compound 13 to obtain compound 14;

[0214] Step S11: Compound 14 is deprotected from the resin under acidic conditions to obtain Compound 15;

[0215]

[0216] The definitions of R1, n, and R2 are as described in any aspect of the present invention;

[0217] R4 is a linker, and the linker is selected from one of them; preferably, R4 is

[0218] L is an optionally substituted or unsubstituted PEG 1-12 chain, an optionally substituted or unsubstituted C1-C 12 alkyl chain; the PEG 1-12 chain is represented as For example, PEG 1-11 PEG 1-10 PEG 1-9 PEG 1-8 PEG 1-7 PEG 1-6 PEG 1-5 PEG 1-4 PEG 1-3 PEG 1-2 PEG 12 PEG 11 PEG 10 PEG9, PEG8, PEG7, PEG6, PEG5, PEG4, PEG3, PEG2, (PEG);

[0219] R5 is selected from hydroxy, one of them; preferably, R5 is hydroxy or

[0220] m is 0 to 2; preferably, m is 0, 1, 2.

[0221] In some preferred embodiments, n is 1 and m is 0; preferably, the structures of Compound 11, Compound 12, Compound 13, Compound 14, or Compound 15 are selected from the following compounds:

[0222]

[0223]

[0224] In some preferred embodiments, in step S8, the resin is a chlorine resin. Preferably, the CAS number of the chlorine resin is 934816-82-7.

[0225] In some preferred embodiments, in step S8, the molar ratio of the resin to compound 10 is 1:(1 - 5); preferably 1:(2 - 4).

[0226] In some preferred embodiments, in step S8, the reaction is carried out under basic conditions; preferably, the base is selected from one or more of N,N-diisopropylethylamine, triethylamine, N-methylmorpholine, and pyridine; preferably, the base is N,N-diisopropylethylamine; preferably, the molar ratio of the resin to the base is 1:(1 - 7), preferably (3 - 6).

[0227] In some preferred embodiments, in step S9, the base is selected from one or more of sodium methoxide, sodium ethoxide, potassium ethoxide, sodium tert-butoxide, piperidine, sodium hydroxide, and potassium hydroxide; preferably, the base is piperidine.

[0228] In some preferred embodiments, in step S10, when R5 is a hydroxyl group and compound 13 is an acid compound, the reaction requires a condensing agent. Preferably, the condensing agent is selected from any single condensing agent or a combination of condensing agents such as DIC, DCC, HBTU, PyBOP, BOP, HATU, HOBT, HOSu, TBTU, DIC / HOBT, DCC / DMAP, EDCI / DMAP, EDCI / HOBT, HATU / HOBT; preferably HATU.

[0229] In some preferred embodiments, in step S10, when compound 13 is an ester compound, the molar ratio of compound 17 to compound 13 is 1:(1 - 3); preferably 1:1; preferably, the reaction is carried out under basic conditions, and the base is selected from one or more of N,N-diisopropylethylamine, triethylamine, N-methylmorpholine, and pyridine; preferably, the base is N,N-diisopropylethylamine; preferably, the molar ratio of the resin to the base is 1:(1 - 7), preferably (1 - 3).

[0230] In some preferred embodiments, in step S10, the reaction time of the reaction is 1 - 5 h; preferably 1 - 3 h.

[0231] In some preferred embodiments, in step S11, the acid is trifluoroacetic acid or hexafluoroisopropanol; preferably, the acid is hexafluoroisopropanol.

[0232] In some preferred embodiments, in step S11, the reaction time for removing the resin is 0.5 to 3 h; preferably, the reaction time is 1 to 2 h.

[0233] The present invention provides a preparation method of an ADC linker conjugated with a toxin, comprising the following steps:

[0234] Step S12: Compound 10 undergoes a condensation reaction with the toxin to obtain Compound 16;

[0235] Step S13: The deprotection reaction of Compound 16 is carried out to obtain Compound 17;

[0236] Step S14: Compound 17 reacts with Compound 13 to obtain Compound 18;

[0237]

[0238] The definitions of R1, n, R2, R4, R5, L, and m are as described in any aspect of the present invention;

[0239] R6 is a toxin containing an amino group at the end; the toxin is selected from one of MMAE, MMAF, Exatecan, and Eribulin; preferably, the toxin is Exatecan.

[0240] In some preferred embodiments, n is 1 and m is 0; preferably, the structure of Compound 16 or Compound 17 is selected from the compounds shown below:

[0241]

[0242] In some preferred embodiments, n is 0 and m is 1; preferably, the structure of Compound 16 or Compound 17 is selected from the compounds shown below:

[0243]

[0244] In some preferred embodiments, in step S12, the molar ratio of Compound 10 to the toxin is 1:(1 to 1.5); preferably 1:1.

[0245] In some preferred embodiments, in step S12, the condensing agent for the condensation reaction is selected from any single condensing agent or composite condensing agent among DIC, DCC, HBTU, PyBOP, BOP, HATU, HOBT, HOSu, TBTU, DIC / HOBT, DCC / DMAP, EDCI / DMAP, EDCI / HOBT, HATU / HOBT, preferably HATU, EDCI / HOBT; preferably, the molar ratio of compound 10 to the condensing agent is 1:(1-3); preferably, the molar ratio of compound 10 to the condensing agent is 1:(1-1.5).

[0246] In some preferred embodiments, in step S12, the condensation reaction also uses a base. Preferably, the base is selected from one or more of N,N-diisopropylethylamine, triethylamine, N-methylmorpholine, pyridine; preferably, the base is N,N-diisopropylethylamine; preferably, the molar ratio of compound 10 to the base is 1:(1-7), preferably (2-5).

[0247] In some preferred embodiments, in step S12, the reaction time of the condensation reaction is 1-3 h; preferably 1 h.

[0248] In some preferred embodiments, in step S13, the deprotection reaction is carried out under basic conditions; preferably, the base is selected from one or more of piperidine, triethylenediamine, triethylenetetramine, triethylamine, diisopropylethylamine, 4-dimethylaminopyridine; preferably, the base is triethylenediamine; preferably, the molar ratio of compound 16 to the base is 1:(2-12); preferably, the molar ratio of compound 16 to the base is 1:(5-10).

[0249] In some preferred embodiments, in step S13, the progress of the reaction can be detected by conventional monitoring methods for such reactions in the art (such as HPLC, TLC or LC-MS), and the disappearance or non-reaction of compound 16 is taken as the end point of the reaction; preferably, the reaction time of the reaction is 1-16 h.

[0250] In some preferred embodiments, in step S13, the post-treatment steps of the reaction are conventional post-treatment steps for such reactions in the art, selected from one or more of crystallization, filtration, reverse-phase purification; preferably, the solvent for crystallization is methyl tert-butyl ether.

[0251] In some preferred embodiments, in step S14, when R5 is a hydroxyl group and compound 13 is an acid compound, the molar ratio of compound 17 to compound 13 is 1:(1 - 3); a condensing agent is required for the reaction. Preferably, the condensing agent is selected from any single condensing agent or composite condensing agent such as DIC, DCC, HBTU, PyBOP, BOP, HATU, HOBT, HOSu, TBTU, DIC / HOBT, DCC / DMAP, EDCI / DMAP, EDCI / HOBT, HATU / HOBT, and preferably HATU; preferably, the molar ratio of compound 17 to the condensing agent is 1:(1 - 2); preferably 1:1.1; preferably, the reaction is carried out under basic conditions, and the base is selected from one or more of N,N - diisopropylethylamine, triethylamine, N - methylmorpholine, and pyridine; preferably, the base is N,N - diisopropylethylamine; preferably, the molar ratio of compound 17 to the base is 1:(1 - 7), preferably (1 - 3).

[0252] In some preferred embodiments, in step S14, when compound 13 is an ester compound, the molar ratio of compound 17 to compound 13 is 1:(1 - 3); preferably 1:1; preferably, the reaction is carried out under basic conditions, and preferably, the base is selected from one or more of N,N - diisopropylethylamine, triethylamine, N - methylmorpholine, and pyridine; preferably, the base is N,N - diisopropylethylamine; preferably, the molar ratio of compound 17 to the base is 1:(1 - 5), preferably (1 - 3).

[0253] The present invention provides compound 10, and its structure is as follows:

[0254]

[0255] The definitions of R1 and R2 are as described in any aspect of the present invention.

[0256] In some preferred embodiments, the structure of compound 10 is selected from the following compounds:

[0257]

[0258] The present invention provides the use of compound 18, and compound 18 can be used to prepare ADC antibody - conjugated drugs.

[0259] Advantages of the present invention:

[0260] 1. The first to sixth aspects of the present invention provide a brand - new method for preparing a linker (CAS No.: 1599440 - 25 - 1).

[0261] This method can improve the yield, reduce the generation of impurities, and lower the cost, thus being more conducive to expanding production and increasing product returns.

[0262] 2. In the first to sixth aspects of the present invention, in the step of preparing compound L, the prior art requires two-step reactions, while the present invention obtains product L in one step. This step directly uses glycolic acid for the reaction, without using benzyl-protected glycolic acid in the original route for the reaction, and also avoids the subsequent need to use palladium on carbon for deprotection. This preparation method is simple and feasible with strong operability.

[0263] 3. The seventh aspect of the present invention provides a completely new intermediate compound of formula 4, compound of formula 5, and compound of formula 6 for preparing ADC linker. This completely new series of intermediate compounds has made great contributions to the successful commercial preparation of ADC linker. The present invention overcomes the defects of the prior art and provides a new preparation method for ADC linker. Each step of this method has high selectivity, can obtain high-purity products, and the post-treatment operation is simple, which is superior to the methods reported in the prior art of ADC linker currently.

[0264] 4. The yield of the key step S5 ester formation in the synthesis method of the seventh aspect of the present invention is 72%, which is increased by about 30% compared with the prior art. The post-treatment is simple, and pure products can be obtained, which is conducive to carrying out the next reaction, thereby improving the overall yield and purity of the reaction and meeting the quality requirements of subsequent drugs.

[0265] 5. The seventh aspect of the present invention provides a preparation method for ADC linker and its intermediate. The preparation method of the present invention is convenient to operate, the reaction is easy to control, has high selectivity and high yield, reduces the production cost, ensures the quality of subsequent drugs, and avoids the defect of the need for multiple column purifications in the prior art, and is suitable for industrial scale-up production.

[0266] 6. The eighth aspect of the present invention overcomes the above-mentioned deficiencies in the prior art and provides a new solid-phase synthesis method for ADC linker according to the structural characteristics of ADC linker itself; at the same time, it also provides a new solid-phase synthesis method for coupling toxins of ADC linker.

[0267] 7. The eighth aspect of the present invention overcomes the defects of the prior art and provides a new solid-phase synthesis method. Each step of this method has high selectivity, the process is simple, can carry out continuous multi-step reactions, simplifies the post-treatment of the reaction, and is superior to the methods reported in the prior art of ADC linker currently.

[0268] 8. When performing the key step S13 of deprotecting the group in the synthesis method of the eighth aspect of the present invention, using a conventional base results in a relatively low reaction yield. The inventor unexpectedly found in the experiment that, based on the special structure of the reaction raw material compound 16, by preferably using a specific base and controlling the proportion of the raw material compound 16, the reaction yield can be effectively increased. Compared with the conventional reaction, the yield is increased by about 8% in two steps, and a pure product can be obtained, which is beneficial for the next reaction, thereby improving the overall yield and purity of the reaction and meeting the subsequent drug quality requirements.

[0269] 9. The eighth aspect of the present invention provides a preparation method of an ADC linker and its derivatives. The preparation method of the present invention is convenient to operate, has mild reaction conditions, is easy to control the reaction, has high selectivity and high yield, reduces production costs, ensures the quality of subsequent drugs, and avoids the defect of the need for multiple column purifications in the prior art, and is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0270] Figure 1 It is the HPLC chromatogram of compound H-1 in Example 5;

[0271] Figure 2 It is the HPLC chromatogram of compound J-1 in Example 6. SPECIFIC EXAMPLES

[0272] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following examples are usually carried out under conventional conditions.

[0273] Unless otherwise specified, the raw materials or reagents used in the examples are commercially available.

[0274] The room temperature mentioned in the examples all refers to 20 - 35 °C. Unless otherwise specified, the reagents are used directly without purification. All solvents are purchased from commercial suppliers, such as Aldrich, and can be used without treatment.

[0275] Example 1

[0276] A preparation method of a compound B, and its reaction formula is

[0277]

[0278] Add 200 mL of acetonitrile, 240 mL of water, and compound A (20 g, 64.874 mmol) to a reaction flask and stir until completely dissolved. Control the temperature of the reaction solution at 10 - 20 °C, add glycine (7.305 g, 97.311 mmol) and 2,4,6 - trimethylpyridine (11.79 g, 97.311 mmol), and react for 4 hours. After the reaction is completed, add 200 mL of 0.5 M hydrochloric acid solution to the reaction solution, stir, add 200 mL of a dichloromethane / isopropanol mixed solvent (DCM / IPA = 4 / 1) for extraction, separate the layers and wash, dry, and concentrate. Add 100 mL of n - hexane to the residue for pulping, filter, and dry the filter cake to obtain 15.2 g of a white - like powdery solid compound B, with a yield of 87.3%.

[0279] Example 2

[0280] A preparation method of a compound C, and its reaction formula is

[0281]

[0282] Add compound B (11.2 g, 42 mmol, 1.0 eq), HOSU (5.76 g, 50 mmol, 1.2 eq), and acetone (112 mL) to a reaction flask. Cool the reaction solution to 15 - 20 °C and add EDCI (12 g, 63 mmol, 1.5 eq). Stir and react at 15 - 25 °C for 2 - 3 hours, then add dichloromethane (112 mL, equivalent to 10 times the volume) to the reaction mixture. Next, wash the reaction solution with water (56 mL each time, repeated 4 times, a total of 20 times the volume) 230 mL, separate the layers, and dry the organic phase with anhydrous sodium sulfate. Mix the organic layer with anhydrous sodium sulfate for drying treatment. After drying is completed, concentrate the organic phase to remove the solvent, and finally obtain 13 g of a brown solid compound C, with a yield of 85%.

[0283] Example 3

[0284] A preparation method of a compound E - 1, and its reaction formula is

[0285]

[0286] Example 3.1

[0287] Add DMAc (1000 mL) and compound D-1 (100 g, 336.349 mmol, 1.0 eq.) into a reaction flask, stir until dissolved and clear, adjust the temperature to 10 - 20 °C, add HOSU (58.07 g, 504.524 mmol, 1.5 eq.) and EDCI (96.71 g, 504.524 mmol, 1.5 eq.), and stir for 20 h. Add EtOAc and H₂O to the reaction solution, stir and separate the layers, dry, and concentrate. Subsequently, dissolve it in 2 L of THF, add 6 L of n-hexane dropwise, stir for 2 h, filter, and rinse the filter cake with n-hexane. Concentrate the filter cake to remove the solvent, and finally obtain the white powdery solid compound E-1, which is directly used for the next reaction.

[0288] Example 3.2

[0289] Add DMAc (10 mL, 10 V) and compound D-1 (1 g, 3.36 mmol, 1.0 eq.) into a reaction flask, stir until dissolved and clear, adjust the temperature to 10 - 20 °C, add HOSU (0.58 g, 5.04 mmol, 1.5 eq.) and DCC (1.04 g, 5.04 mmol, 1.5 eq.), and stir for 1 h. Add EA and H₂O to the reaction solution, stir and separate the layers, extract the aqueous phase with EA again, combine the organic phases, wash with water once, dry the organic phase and rotary evaporate. Dissolve it in THF, add n-hexane dropwise, stir for 2 h, filter, and rinse the filter cake with n-hexane. Next, thoroughly remove the solvent using an oil pump, and finally obtain 1.22 g of the white powdery solid compound E-1 with a yield of 92.1%.

[0290] Example 3.3

[0291] Add DMAc (10 mL, 10 V) and compound D-1 (1 g, 3.36 mmol, 1.0 eq.) into a reaction flask, stir until dissolved and clear, adjust the temperature to 10 - 20 °C, add HOSU (0.58 g, 5.04 mmol, 1.5 eq.) and DIC (0.637 g, 5.04 mmol, 1.5 eq.), and stir for 1 h. Add EA and H₂O to the reaction solution, stir and separate the layers, extract the aqueous phase with EA again, combine the organic phases, wash with water once, dry the organic phase and rotary evaporate. Dissolve it in THF, add n-hexane dropwise, stir for 2 h, filter, and rinse the filter cake with n-hexane. Next, thoroughly remove the solvent using an oil pump, and finally obtain 1.29 g of the white powdery solid compound E-1 with a yield of 97.1%.

[0292] Example 4

[0293] A preparation method of a compound G-1, and its reaction formula is

[0294]

[0295] Add DME (1000 mL) and Compound E-1 (100 g, 253.563 mmol, 1.0 eq.) into a reaction flask, stir evenly, adjust the temperature to 10 - 20 °C, add L-phenylalanine (50.26 g, 304.275 mmol, 1.2 eq.), and add NaHCO₃ (42.60 g, 507.125 mmol, 2.0 eq.) dissolved in H₂O (600 mL) into the reaction solution, and stir for 0.5 h. Add water and dimethyltetrahydrofuran to the reaction solution, then add 0.5 M hydrochloric acid solution to adjust the pH to 3 - 4, let it stand for liquid separation, dry the organic phase, concentrate to obtain the crude product. Add THF / n-hexane to the crude product for the first pulping, filter, and wash the filter cake with n-hexane; add acetonitrile / MTBE to the filter cake for the second pulping, filter, and wash the filter cake with MTBE; concentrate the filter cake to remove the solvent to obtain 76 g of white powder solid Compound G-1, with a two-step yield of 67.4%.

[0296] Example 5

[0297] A preparation method of a compound H-1, and its reaction formula is

[0298]

[0299] Add DCM (200 mL, 20 V) and G-1 (10 g, 22.5 mmol, 1.0 eq) into a reaction flask. Start stirring, add HOSU (3.88 g, 33.7 mmol, 1.5 eq) and EDCI (8.62 g, 45.0 mmol, 2.0 eq). Adjust the temperature to 10 - 20 °C and stir for 5 h. Add H₂O to the reaction solution, stir for liquid separation, and separate the organic phase and wash it with H₂O again. Add anhydrous sodium sulfate to dry the organic phase. Filter by suction, and wash the desiccant with DCM. Concentrate the filtrate until it hardly drips, and directly use it for the next step. LCMS detection [MS+H] = 542.2, and the LCMS spectrum is as Figure 1 shown.

[0300] Example 6

[0301] A preparation method of a compound J-1, and its reaction formula is

[0302]

[0303] Compound I (3.57 g, 27.0 mmol, 1.2 eq), NaHCO3 (3.78 g, 45.0 mmol, 2.0 eq) and H2O (60 mL, 6V) were added to a reaction flask and stirred until dissolved and clear. The temperature was adjusted to 10 - 20 °C, and compound H-1 (16.95 g, 22.5 mmol, 1.0 eq) and DME (60 mL) were added to the reaction flask. The mixture was stirred for 1 h until the reaction was complete. 0.5 M hydrochloric acid solution was added dropwise to the reaction solution to adjust the pH to 3 - 4, and the mixture was stirred for 0.5 h. The mixture was filtered to obtain 36.58 g of a white wet product, which was slurried with acetone (200 mL) for 1 h. After filtration, the filter cake was dried to obtain 8.34 g of a white solid product J-1, with a two-step yield of 66% and a purity of 95.7%. The HPLC chromatogram is as Figure 2 shown.

[0304] The NMR of compound J-1 is as follows:

[0305] 1H NMR (400 MHz, DMSO) δ 12.60 (s, 1H), 8.39 (s, 1H), 8.09 (d, J = 9.2 Hz, 2H), 7.91 (d, J = 7.5 Hz, 2H), 7.72 (d, J = 7.4 Hz, 2H), 7.57–7.12 (m, 10H), 4.57 (s, 1H), 4.26 (d, J = 11.0 Hz, 3H), 3.92–3.48 (m, 6H), 3.15–2.73 (m, 2H).

[0306] Example 7

[0307] A preparation method of a compound K-1, the reaction formula of which is

[0308]

[0309] DMAc 470 mL and compound J-1 (47 g, 84.140 mmol, 1.0 eq) were added to a reaction flask and stirred evenly. The temperature was adjusted to 10 - 20 °C, and lead tetraacetate (74.61 g, 168.281 mmol, 2.0 eq.), cupric acetate anhydrous (15.28 g, 84.140 mmol, 1.0 eq.) and AcOH (11.116 g, 185.109 mmol, 2.2 eq.) were added. The temperature was raised to 45 - 55 °C and stirred for 1 h. 2-Methyltetrahydrofuran and water were successively added to the reaction solution and stirred, and then liquid separation was carried out. The separated aqueous phase was used to wash the organic phase, and then the organic phase was washed with saturated brine. After the organic phase was dried, n-hexane was added and stirred for 2 h. After filtration, the filter cake was dried to obtain 43.3 g of a white powdery solid compound K-1, with a yield of 89.9%.

[0310] Example 8

[0311] A preparation method of a compound L-1, the trans form is,

[0312]

[0313] Add N,N-dimethylacetamide (DMAc, 15 mL), compound K-1 (1.5 g, 2.62 mmol) and glycolic acid (2.0 g, 26.20 mmol) into a reaction flask, and stir to dissolve. Add methanesulfonic acid (52 mg, 0.2 eq) to the reaction solution, adjust the temperature to 40 - 45 °C, and stir the reaction for 3 hours. After the reaction is completed, add the reaction solution to water (40 mL), and filter to precipitate a solid. Dry the filter cake to obtain 1.32 g of a white powder solid compound L-1, with a yield of 86%.

[0314] Example 9

[0315] A preparation method of a compound M, the trans form is,

[0316]

[0317] Add compound L-1 (13.8 g, 23.4 mmol, 1.0 eq) into a reaction flask, then add the mixture of tetrahydrofuran (138 mL) and water (7.2 mL) into the reaction system. Add triethylamine (TEA, 23.7 g, 234.4 mmol, 10 eq), and stir the reaction at 20 - 25 °C for 4 hours. After the reaction is completed, filter the reaction solution, and wash the filter cake with ethyl acetate. After drying, 8.0 g of a white solid compound M is obtained, with a yield of 93%

[0318] Example 10

[0319] A preparation method of a compound N, the trans form is,

[0320]

[0321] Add compound M (5.5 g, 15 mmol, 1.0 eq) and compound C (5.5 g, 15 mmol, 1.0 eq) into a reaction flask, then add acetonitrile (55 mL) and water (27.5 mL). Add triethylamine (TEA, 1.5 g, 15 mmol, 1.0 eq) to the reaction system, and stir the reaction at 15 - 25 °C for 2 hours. After the reaction is completed, add dichloromethane (DCM, 27.5 mL, 5V) to the reaction solution, separate the layers, and collect the lower aqueous phase. Add citric acid to the aqueous phase, dissolve it, extract it with a dichloromethane / isopropanol mixed solvent, collect the organic phase, dry it with anhydrous sodium sulfate, filter, and concentrate. Add acetonitrile to the concentrate for slurrying, filter, and drain to obtain 6 g of a white solid product compound N, with a yield of 65% and a purity of 97.5%.

[0322] Example 11

[0323] The preparation method of compound Deruxtecan, its trans form is,

[0324]

[0325] Under nitrogen protection, compound P (133 g, 250.4 mmol, 1.0 eq) was stirred and dispersed in tetrahydrofuran (THF, 1596 mL), then 5% aqueous sodium sulfate solution (1330 mL) and N-methylmorpholine (25.33 g, 250.4 mmol, 1.0 eq) were added. After the addition was completed, the reaction was stirred at 15 °C for 1 hour. Compound O (185.17 g, 300.48 mmol, 1.2 eq), EDCI (72 g, 375.6 mmol, 1.5 eq) and ethyl 2-oximinoacetate (17.79 g, 125.2 mmol, 0.5 eq) were successively added to the reaction system. After the addition was completed, the reaction was continued to stir at 15 °C for 2 hours. After the reaction was completed, water was added to the reaction solution, then a DCM / IPA mixed solvent was added for extraction, and the layers were separated. The organic phase was washed with 0.05 M hydrochloric acid solution to adjust the pH to 4-5, and after separation, the organic phase was washed with water again, the layers were separated, the organic phase was dried over anhydrous sodium sulfate, concentrated to obtain a crude product. The crude product was dissolved in a dichloromethane / methanol mixed solvent, mixed with silica gel and then purified by column chromatography. The chromatographic eluent was collected, concentrated and evaporated to dryness to obtain 192.5 g of a light yellow solid, with a yield of 76% and a purity of 98%.

[0326] Example 12

[0327]

[0328] Compound 1.1 (40 g, 150.23 mmol) was dissolved in DMF (400 mL), nitrogen was displaced, AcOH (1.80 g, 30.05 mmol) was added. After the raw materials were dissolved, Pb(OAc)4 (99.89 g, 225.3 mmol) was added. The reaction was stirred at room temperature for 2 h. After the reaction was monitored by LCMS and ended, MTBE was added for pulping. The filter cake was collected after filtration, washed with MTBE, filtered, and dried to obtain pure compound 2.1 (37 g, yield 88%).

[0329] 1H NMR (400 MHz, DMSO) δ8.96 (d, J = 6.7 Hz, 1H), 7.50 (t, J = 6.1 Hz, 1H), 7.41–7.34 (m, 5H), 5.12 (d, J = 6.9 Hz, 2H), 5.06 (s, 2H), 3.68 (d, J = 6.2 Hz, 2H), 2.02 (s, 3H).

[0330] Example 13

[0331]

[0332] Dissolve Compound 2.1 (10 g, 35.68 mmol) and Compound 3.1 (11.86 g, 71.36 mmol) in THF (100 mL). Cool the solution to 0 °C. Dissolve LiOH (0.90 g, 37.46 mmol) in water (1 mL) and slowly add it dropwise into the reaction flask. After monitoring the reaction by LCMS until it is completed, extract with ethyl acetate and water. Wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, concentrate, add MTBE for slurrying, filter and dry to obtain pure Compound 4.1 (12 g, yield 87%).

[0333] 1 H NMR (400 MHz, DMSO) δ 8.78 (t, J = 6.6 Hz, 1H), 7.52 (t, J = 6.0 Hz, 1H), 7.45–7.27 (m, 10H), 5.17 (d, J = 7.2 Hz, 2H), 5.05 (s, 2H), 4.66 (d, J = 6.7 Hz, 2H), 4.15 (d, J = 20.2 Hz, 2H), 3.66 (d, J = 6.1 Hz, 2H).

[0334] Example 14

[0335]

[0336] Add Compound 4.1 (12 g, 31.06 mmol), Pd / C (1.2 g, 10%), methanol (80 mL), and water (40 mL) into the reaction flask. Replace the air with hydrogen and react at room temperature overnight. After monitoring the reaction by LCMS until the raw materials are completely reacted, stop the reaction. Filter the reaction solution through diatomaceous earth. Wash the filter cake alternately with methanol and water, filter and concentrate. A large amount of white solid precipitates when methanol is added. Filter and dry to obtain pure Compound 5.1 (3.8 g, 75%). 1 HNMR (400 MHz, D2O) δ 4.74 (s, 2H), 3.94–3.90 (m, 2H), 3.84 (s, 2H).

[0337] Example 15

[0338]

[0339] Dissolve compound 8.1 (8 g, 16.93 mmol) in DCM (80 mL). Slowly add a solution of DCC (5.24 g, 25.40 mmol) in DCM (50 mL) dropwise into the reaction flask. Then add a solution of tetrafluorophenol (2.81 g, 16.93 mmol) in DCM (20 mL) dropwise into the reaction flask. Stir the reaction at room temperature for 2 h. Monitor the reaction by LCMS until the raw materials are completely reacted. Stop the reaction, add ethyl acetate and water for extraction. Wash the organic phase with saturated brine, dry it over anhydrous sodium sulfate, concentrate it, and then perform column chromatography (petroleum ether:ethyl acetate = 2:1 to 1:2) to obtain the pure compound 6.1 (7.56 g, 72%).

[0340] Example 16

[0341]

[0342] Dissolve compound 5.1 (0.47 g, 2.90 mmol) and compound 6.1 (1.80 g, 2.90 mmol) in DMA (20 mL). Replace the nitrogen, add 2,6-dimethylpyridine (0.62 g, 5.8 mmol) dropwise, and stir the reaction at room temperature overnight. LCMS shows that the reaction is complete. Perform reverse-phase purification (0.01% TFA system) to obtain the pure compound 7.1 (1.36 g, 76%).

[0343] 1H NMR (400 MHz, DMSO) δ 12.62 (s, 1H), 8.57 (s, 1H), 8.32 (s, 1H), 8.14 (d, J = 8.1 Hz, 1H), 8.09 (s, 1H), 8.02 (s, 1H), 7.27–7.19 (m, 5H), 7.01 (s, 2H), 4.63 (d, J = 6.7 Hz, 2H), 4.52 (td, J = 9.3, 4.6 Hz, 1H), 4.00 (s, 2H), 3.80–3.71 (m, 3H), 3.69 (d, J = 5.6 Hz, 2H), 3.65–3.58 (m, 1H), 3.39 (s, 2H), 3.06 (d, J = 4.4 Hz, 1H), 2.84 (d, J = 9.7 Hz, 1H), 2.13 (t, J = 7.4 Hz, 2H), 1.49 (dd, J = 14.8, 7.4 Hz, 4H), 1.21 (t, J = 7.6 Hz, 2H).

[0344] Example 17

[0345]

[0346] Step S1.1: Weigh chlororesin (10 g, 10.50 mmol) in a 500 mL polypeptide tube, add a DMF (200 mL) solution of compound 1.1 (11.16 g, 31.50 mmol, 3 eq), add DIEA (6.79 g, 52.5 mmol, 5 eq), shake on a shaker for 17 h. After the reaction is completed, add MeOH (30 mL) to the polypeptide tube, shake for 1 h and then dry by suction. Wash successively with DMF, MeOH, and DMF to obtain compound 2.1;

[0347] Step S2.1: Add the compound 2.1 obtained in step S1.1 to a 500 mL polypeptide tube, then pour 20% piperidine DMF solution (150 mL) into the polypeptide tube. After purging with nitrogen for 15 min, dry by suction. Pour 20% piperidine DMF solution (150 mL) again, purge with nitrogen for 15 min and then dry by suction. Wash successively with DMF, MeOH, and DMF to obtain compound 3.1;

[0348] Step S3.1: Add compound 4.1 (15.80 g, 31.5 mmol, 3 eq), HOBT (7.09 g, 52.50 mmol, 5 eq), and DMF (150 mL) to the compound 3.1 obtained in step S2.1. Under nitrogen purging, add DIC (6.63 g, 52.50 mmol, 5 eq), purge with nitrogen for 3 h. Take a small sample and add it to ninhydrin for color development. If it does not show blue color, wash successively with DMF, MeOH, and DMF, and dry to obtain compound 5.1;

[0349] Step S4.1: Slowly pour the compound 5.1 obtained in step S3.1 into a hexafluoroisopropanol / DCM solution (1 / 4, 100 mL), stir for 1 h, filter. After concentrating the mother liquor, add ether to precipitate a white solid, filter to obtain compound 6.1 (5.62 g, 4-step yield 87%). LCMS (m / z) + = 616.2

[0350] Example 18

[0351]

[0352] To a solution of compound 6.1 (2 g, 3.25 mmol, 1 eq) in DMF (20 mL) was added Pb(OAc)4 (4.32 g, 9.75 mmol, 3 eq) and Cu(OAc)2 (0.30 g, 1.62 mmol, 0.5 eq). After protecting with nitrogen, the mixture was heated to 45 °C and reacted for 3 h. The reaction was monitored by LCMS. After the reaction was completed, the reaction solution was poured into MTBE (200 mL). The obtained solid was dissolved in MeOH and DCM for sample mixing and then purified by column chromatography (MeOH:DCM = 1:9). After concentration, compound 7.1 (1.70 g, 83%) was obtained.

[0353] 1 H NMR (400 MHz, DMSO) δ 8.83 (t, J = 6.9 Hz, 1H), 8.32 (t, J = 5.7 Hz, 1H), 8.14 (d, J = 8.2 Hz, 1H), 8.00 (t, J = 5.6 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.71 (d, J = 7.5 Hz, 2H), 7.58 (t, J = 5.9 Hz, 1H), 7.41 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 7.28–7.21 (m, 4H), 7.18 (d, J = 6.3 Hz, 1H), 5.10 (d, J = 7.9 Hz, 2H), 4.52 (d, J = 3.4 Hz, 1H), 4.29 (d, J = 6.1 Hz, 2H), 4.23 (d, J = 6.7 Hz, 1H), 3.82–3.69 (m, 3H), 3.61 (t, J = 8.9 Hz, 3H), 3.05 (dd, J = 13.9, 4.2 Hz, 1H), 2.84–2.75 (m, 1H), 1.91 (s, 2H).

[0354] Example 19

[0355]

[0356] To a solution of compound 7.1 (1.63 g, 2.59 mmol, 1 eq) and compound 8.1 (430 mg, 2.59 mmol, 1 eq) in THF (16 mL) was added a solution of LiOH (62.03 mg, 2.59 mmol, 1 eq) in H2O (1 mL). The reaction was carried out at room temperature for 3 h. The reaction was monitored by LCMS. When 10% of the raw materials remained unreacted, LiOH (12.4 mg, 0.2 eq) was added and the reaction was continued for 1 h. After the reaction was monitored by LCMS to be completed, the reaction solution was directly concentrated for sample mixing and then purified by column chromatography (MeOH:EA = 1:11.5 - 1:19). After concentration, compound 9.1 (1.35 g, 69%) was obtained.

[0357] 11H NMR (400 MHz, DMSO) δ 8.59 (t, J = 6.6 Hz, 1H), 8.32 (s, 1H), 8.14 (d, J = 8.1 Hz, 1H), 8.01 (s, 1H), 7.89 (d, J = 7.4 Hz, 2H), 7.70 (d, J = 7.6 Hz, 2H), 7.58 (t, J = 5.8 Hz, 1H), 7.44–7.30 (m, 9H), 7.28–7.21 (m, 4H), 7.17 (d, J = 6.6 Hz, 1H), 5.14 (s, 2H), 4.63 (d, J = 6.7 Hz, 2H), 4.51 (s, 1H), 4.29 (d, J = 6.1 Hz, 2H), 4.25–4.19 (m, 1H), 4.15 (s, 2H), 3.82–3.68 (m, 3H), 3.62 (d, J = 5.7 Hz, 3H), 3.05 (dd, J = 13.8, 4.4 Hz, 1H), 2.79 (dd, J = 14.1, 10.0 Hz, 1H).

[0358] Example 20

[0359]

[0360] To a solution of Compound 9.1 (0.7 g, 0.93 mmol, 1 eq) in DCM / MeOH (5 / 1, 7.2 mL) was added Pd / C (140 mg). After replacing the hydrogen three times, the reaction was carried out at room temperature for 3 h. The reaction was monitored by LCMS. After completion, the reaction solution was filtered through diatomaceous earth, and the filter cake was rinsed with DCM / MeOH (5 / 1). The mother liquor was concentrated to obtain Compound 10.1 (570 mg, 95%). LCMS (m / z) [M+Na] + = 668.2

[0361] Example 21

[0362]

[0363] Step S8.1: Add chlororesin (50 mg, 0.05 mmol) to the peptide tube, then add a solution of Compound 10.1 (97 mg, 0.15 mmol, 3 eq) in DMF (10 mL) and DIEA (32 mg, 0.25 mmol, 5 eq). Shake on a shaker at room temperature for 16 h. Add methanol to the peptide tube and shake for 1 h, then wash the resin with DMF, MeOH, and DMF in sequence to obtain Compound 11.1;

[0364] Step S9.1: Pour 20% piperidine DMF solution into the compound 11.1 obtained in step S8.1, blow with nitrogen for 15 min and then dry by suction. Pour 20% piperidine DMF solution (10 mL) again, blow with nitrogen for 15 min and then dry by suction. Wash the resin with DMF, MeOH, and DMF in sequence to obtain compound 12.1;

[0365] Step S10.1: Add a DMF (10 mL) solution of compound 13.1 (23 mg, 0.08 mmol, 1.5 eq) and DIEA (13 mg, 0.10 mmol, 2 eq) to the compound 12.1 obtained in step S9.1, blow with nitrogen for 3 h. Take a small sample and add it to ninhydrin for color development. If it does not show blue color, dry by suction, wash the resin with DMF, MeOH, and DMF in sequence, and dry to obtain compound 14.1;

[0366] Step S11.1: Slowly pour the compound 14.1 obtained in step S10.1 into hexafluoroisopropanol / DCM (1 / 1, 1 mL), stir for 1 h, filter. After concentrating the mother liquor, add ether and white solid precipitates. Filter to obtain compound 15.1 (14.8 mg, 4-step yield 48%, characterization data consistent with the literature).

[0367] Example 22

[0368]

[0369] Step S12.1: Add DIEA (0.18 g, 1.38 mmol, 3 eq) to a DMF (6 mL) solution of compound 10.1 (0.3 g, 0.46 mmol), Exatecan (0.2 g, 0.46 mmol, 1 eq, CAS: 171335 - 80 - 1), and HATU (0.17 g, 0.46 mmol, 1 eq), and react at room temperature for 1 h; Monitor the reaction by LCMS until it ends. Drop the reaction solution into HCl (1 mol / L, 60 mL) solution, and solid precipitates. Filter, wash the filter cake with clear water and then lyophilize to obtain the crude compound 16.1;

[0370] Step S13.1: Mix the compound 16.1 obtained in step S12.1 with a DMF (4 mL) solution, add piperidine (0.39 g, 4.6 mmol, 10 eq), and react at room temperature for 1 h. Monitor the reaction by LCMS until it ends. Drop the reaction solution into MTBE (40 mL) and solid precipitates. After filtering, dissolve it with DMF (6 mL), purify by reverse phase (0.01% TFA water: acetonitrile = 1:3), and lyophilize to obtain compound 17.1 (170 mg, 2-step yield 44%). LCMS (m / z) [M + H] + = 841.3

[0371] Example 23

[0372]

[0373] To a solution of Compound 17.1 (70 mg, 0.08 mmol, 1 eq) and Compound 13.1 (24.66 mg, 0.08 mmol, 1 eq) in DMF (1.5 mL), DIEA (20.68 mg, 0.16 mmol, 2 eq) was added. The reaction was carried out at room temperature for 1 h. The reaction was monitored by LCMS until completion. After adjusting the pH of the reaction solution to 5 - 6 with acetic acid, it was purified by reverse phase (0.01% TFA in water:acetonitrile = 3:2), and then lyophilized to obtain Compound 18.1 (41.88 mg, 50%).

[0374] 1 H NMR (400 MHz, DMSO) δ 8.51 (s, 1H), 8.39 (d, J = 8.8 Hz, 1H), 8.15 (s, 1H), 7.99 (d, J = 8.2 Hz, 1H), 7.94 (s, 1H), 7.89 (s, 1H), 7.78 (d, J = 11.0 Hz, 1H), 7.35 (s, 1H), 7.28–7.17 (m, 5H), 6.97 (s, 2H), 6.44 (s, 1H), 5.61 (s, 1H), 5.43 (q, J = 16.3 Hz, 2H), 5.24 (d, J = 3.6 Hz, 2H), 4.67 (d, J = 6.6 Hz, 2H), 4.50 (dd, J = 13.2, 8.8 Hz, 1H), 4.05 (s, 2H), 3.79–3.61 (m, 6H), 3.39 (t, J = 7.1 Hz, 2H), 3.18–3.12 (m, 1H), 3.09–3.03 (m, 1H), 2.83 (dd, J = 13.9, 9.6 Hz, 1H), 2.50–2.00 (m, 8H), 1.90 (dt, J = 13.5, 6.9 Hz, 2H), 1.50 (dt, J = 14.7, 7.5 Hz, 4H), 1.23 (dd, J = 15.0, 7.7 Hz, 2H), 0.91 (t, J = 7.3 Hz, 3H).

[0375] Example 24

[0376]

[0377] Step S1.2: Weigh chlororesin (10 g, 10.50 mmol) in a peptide tube, add a DMF (200 mL) solution of Compound 1.2 (11.16 g, 31.50 mmol, 3 eq), add DIEA (6.79 g, 52.50 mmol, 5 eq), shake on a shaker for 17 h. After the reaction is completed, add MeOH (30 mL) to the peptide tube, shake for 1 h and then dry by suction. Wash successively with DMF, MeOH, and DMF to obtain Compound 2.2;

[0378] Step S2.2: Add Compound 2.2 obtained in Step S1.2 to the peptide tube, then pour 20% piperidine DMF solution (100 mL) into the peptide tube. After purging with nitrogen for 15 min, dry by suction. Pour 20% piperidine DMF solution (100 mL) again, purge with nitrogen for 15 min and then dry by suction. Wash successively with DMF, MeOH, and DMF to obtain Compound 3.2;

[0379] Step S3.2: Add Compound 4.2 (9.33 g, 21.00 mmol, 2 eq), HOBT (2.84 g, 21.00 mmol, 2 eq), and DMF (100 mL) to Compound 3.2 obtained in Step S2.2. Add DIC (2.65 g, 21.00 mmol, 2 eq) under nitrogen purging, and purge with nitrogen for 3 h. Take a small sample and add it to ninhydrin for color development. If it does not show blue color, wash successively with DMF, MeOH, and DMF, and dry to obtain Compound 5.2;

[0380] Step S4.2: Slowly pour Compound 5.1 obtained in Step S3.2 into a hexafluoroisopropanol / DCM solution (1 / 4, 100 mL), stir for 1 h, filter. After concentrating the mother liquor, add ether to precipitate white solid, filter to obtain Compound 6.2 (4.99 g, 85% yield in 4 steps). LCMS (m / z) [[M+H]] + = 559.2

[0381] Example 25

[0382]

[0383] To a solution of compound 6.2 (5.24 g, 9.38 mmol, 1 eq) in DMF (20 mL) was added Pb(OAc)4 (12.48 g, 28.14 mmol, 3 eq) and Cu(OAc)2 (0.85 g, 4.69 mmol, 0.5 eq). The reaction was carried out at room temperature for 3 h. When the reaction was monitored by LCMS to be complete, a mixed solution of DCM and water was added to the reaction solution. After stirring for 30 min, the mixture was filtered through diatomaceous earth. The filtrate was collected and extracted with DCM and water. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated to obtain compound 7.2 (4.3 g, 80%). LCMS (m / z) + = 595.2

[0384] Example 26

[0385]

[0386] Compound 7.2 (1.82 g, 3.18 mmol, 1 eq) and compound 8.1 (0.63 g, 3.82 mmol, 1.2 eq) were dissolved in THF (19 mL). The temperature was lowered to 0 °C. LiOH (0.08 g, 3.34 mmol, 1.05 eq) was dissolved in H2O (1.5 mL) and added dropwise to the reaction flask. The reaction was carried out at room temperature for 0.5 h. When the reaction was monitored by LCMS to be complete, the reaction solution was extracted with EA and water. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated and then triturated with MTBE. The solid was filtered to obtain compound 9.2 (1.5 g, 70%). LCMS (m / z) + = 701.2

[0387] Example 27

[0388]

[0389] To a solution of compound 9.2 (1.2 g, 1.77 mmol, 1 eq) in DCM / MeOH (5 / 1, 14.4 mL) was added Pd / C (240 mg). After replacing the gas with hydrogen three times, the reaction was carried out at room temperature for 3 h. When the reaction was monitored by LCMS to be complete, the reaction solution was filtered through diatomaceous earth. The filter cake was rinsed with DCM / MeOH (5 / 1). The mother liquor was concentrated to obtain compound 10.2 (0.98 g, 94%).

[0390] Example 28

[0391]

[0392] Step S12.2: To a solution of Compound 10.2 (725 mg, 1.23 mmol, 1 eq), Exatecan (536 mg, 1.23 mmol, 1 eq) and HATU (515 mg, 1.35 mmol, 1.1 eq) in DMF (7.5 ml), DIEA (477 mg, 3.69 mmol, 3 eq) was added. The reaction was carried out at room temperature for 1 h. After the reaction was monitored by LCMS to completion, the reaction solution was added to MTBE (15 mL), and a solid precipitated. After washing and filtration, the crude product Compound 16.2 was obtained; LCMS (m / z) [M+H] + = 1007.2

[0393] Step S13.2: Compound 16.2 obtained in Step S12.2 and triethylenediamine (690 mg, 6.15 mmol, 5 eq) were added to a solution of DMF (11 ml). The reaction was carried out at room temperature overnight. After the reaction was monitored by LCMS to completion, the reaction solution was dropped into MTBE (60 mL) to precipitate a solid. After filtration, it was dissolved in DMF (10 mL) and purified by reverse-phase column (0.01% TFA water:acetonitrile = 1:3), and then lyophilized to obtain Compound 17.2 (500 mg, 52%). LCMS (m / z) [M+H] + = 784.2

[0394] Example 29

[0395]

[0396] Compound 17.2 (218 mg, 0.26 mmol), Compound 13.2 (80 mg, 0.26 mmol, 1 eq) and HATU (109 mg, 0.29 mmol, 1.1 eq) were dissolved in a solution of DMF (2 ml). DIEA (50 mg, 0.39 mmol, 1.5 eq) was added. The reaction was carried out at room temperature for 1 h. After the reaction was monitored by LCMS to completion, the reaction was adjusted to pH 5 - 6 with acetic acid and then purified by reverse-phase column (0.01% TFA water:acetonitrile = 3:2), and then lyophilized to obtain Compound 18.1 (117.37 mg, 45%).

[0397] 11H NMR (400 MHz, DMSO) δ 8.51 (s, 1H), 8.39 (d, J = 8.8 Hz, 1H), 8.15 (s, 1H), 7.99 (d, J = 8.2 Hz, 1H), 7.94 (s, 1H), 7.89 (s, 1H), 7.78 (d, J = 11.0 Hz, 1H), 7.35 (s, 1H), 7.28–7.17 (m, 5H), 6.97 (s, 2H), 6.44 (s, 1H), 5.61 (s, 1H), 5.43 (q, J = 16.3 Hz, 2H), 5.24 (d, J = 3.6 Hz, 2H), 4.67 (d, J = 6.6 Hz, 2H), 4.50 (dd, J = 13.2, 8.8 Hz, 1H), 4.05 (s, 2H), 3.79–3.61 (m, 6H), 3.39 (t, J = 7.1 Hz, 2H), 3.18–3.12 (m, 1H), 3.09–3.03 (m, 1H), 2.83 (dd, J = 13.9, 9.6 Hz, 1H), 2.50–2.00 (m, 8H), 1.90 (dt, J = 13.5, 6.9 Hz, 2H), 1.50 (dt, J = 14.7, 7.5 Hz, 4H), 1.23 (dd, J = 15.0, 7.7 Hz, 2H), 0.91 (t, J = 7.3 Hz, 3H).

[0398] Example 30

[0399]

[0400] Compound A (1 g, 3.24 mmol) and glycine (0.24 g, 3.24 mmol, 1 eq) were dissolved in a mixed solution of acetonitrile and water (2 mL, acetonitrile / water = 1 / 1). 2,6-Dimethylpyridine (0.69 g, 6.48 mmol, 2 eq) was added, and the reaction was stirred at room temperature overnight. The reaction was monitored by LCMS until completion and then purified by reverse-phase column (0.01% TFA in water:acetonitrile = 3:2) to obtain compound 13.2 (600 mg, 69%). LCMS (m / z) + = 269.2.

Claims

1. A method for preparing compound L, characterized in that: The preparation method of the compound L comprises the following steps S8: Step S8: Under acidic conditions, compound K reacts with glycolic acid in a solvent to generate compound L. R1 is an amino protecting group.

2. The preparation method according to claim 1, characterized in that The amino protecting group is selected from one of benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, tert-butyloxycarbonyl, pivaloyl, acetyl, trityl, allyloxycarbonyl, benzyl, trimethylsilylethoxycarbonyl, trichloroethoxycarbonyl, phthaloyl, p-methoxybenzyl, trifluoroacetyl, 2-nitrobenzenesulfonyl, 2,4-dinitrobenzenesulfonyl, 2-nitrobenzyloxycarbonyl, 2-nitrobenzyl, 2-nitrobenzyl, 2-nitrophenethyl, methanesulfonyl, p-toluenesulfonyl, methoxycarbonyl or ethoxycarbonyl.

3. The preparation method according to claim 1, characterized in that: The preparation method of the compound L satisfies at least one of the following conditions: (1) In step S8, the acid is selected from one of boron trifluoride etherate, methanesulfonic acid, camphorsulfonic acid, p-toluenesulfonic acid, scandium trifluoromethanesulfonate, zinc chloride or acetic acid, or any combination thereof; (2) In step S8, the molar ratio of the compound K to the compound glycolic acid is 1:1 to 20, for example 1:10; (3) In step S8, the molar ratio of the compound K to the acid is 1:(0.1-1.0), for example, 1:0.2; (4) In step S8, the mass volume ratio of the compound K to the solvent is 1:10 to 15 g / mL, for example 1:10 g / mL; (5) In step S8, the reaction temperature is 30-45°C; (6) In step S8, the reaction time is 3 to 5 hours, for example 3 hours; (7) In step S8, the solvent is selected from any one or more of tetrahydrofuran, dichloromethane, dimethyl sulfoxide, N,N-dimethylacetamide, N,N-dimethylformamide, and N-methyl-2-pyrrolidone.

4. The preparation method according to claim 1, characterized in that: The preparation method of the compound K comprises the following steps S7: in a solvent, compound J reacts in the presence of acetic acid, lead tetraacetate, and anhydrous copper acetate to generate compound K; Wherein R1 is defined as a substituent as in claim 1; Preferably, the preparation method of compound K satisfies at least one of the following conditions: (1) In step S7, the molar ratio of compound J to acetic acid is 1:1 to 4, for example, 1:2.2; (2) In step S7, the molar ratio of the compound J to lead tetraacetate is 1:2 to 4, for example 1:2; (3) In step S7, the molar ratio of compound J to anhydrous copper acetate is 1:1 to 4, for example 1:1; (4) In step S7, the mass volume ratio of the compound J to the solvent is 1:10 to 15 g / mL, for example 1:10 g / mL; (5) In step S7, the reaction temperature is 30 to 55° C.; (6) In step S7, the reaction time is 0.5 to 4 hours, for example, 1 hour; (7) In step S7, the solvent is selected from any one or more of N,N-dimethylacetamide, N,N-dimethylformamide, and N-methyl-2-pyrrolidone, for example, N,N-dimethylacetamide.

5. The preparation method according to claim 4, characterized in that: The preparation method of the compound J comprises the following steps S5 and S6: Step S5: Compound G is condensed with HOSU to generate compound H. Step S6: Under alkaline conditions, compound H reacts with compound I to generate compound J. Wherein R1 is defined as a substituent as in claim 1; Preferably, at least one of the following conditions is met in step S5: (1) In step S5, the molar ratio of compound G to HOSU is 1:1-3, for example, 1:1.5; (2) in step S5, the condensing agent of the reaction is selected from the coupling reaction reagents of carbodiimide, imidazolinium reagent, phosphonium salt, organophosphorus reagent, uronium salt, pyridinium reagent and phosphonic acid, more particularly any single or composite condensing agent selected from EDCI, DIC, DCC, HBTU, PyBOP, BOP, HATU, HOBT, TBTU, DIC / HOBT, DCC / DMAP, EDCI / DMAP, EDCI / HOBT, HATU / HOBT, such as EDCI; (3) In step S5, the molar ratio of compound G to the condensing agent is 1:1 to 3, for example, 1:2; (4) In step S5, the reaction temperature is 0 to 30° C., for example, 20° C.; (5) In step S5, the reaction time is 0.5 to 16 hours, for example, 5 hours; (6) In step S5, the solvent is selected from any one or more of N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone, tetrahydrofuran, dichloromethane, acetonitrile, benzonitrile, benzyl cyanide, diethyl ether, methyl tert-butyl ether, 1,4-dioxane, diisopropyl ether, ethyl acetate, and acetone; Preferably, step S6 satisfies at least one of the following conditions: (1) In step S6, the molar ratio of compound H to compound I is 1:1-2, for example, 1:1.2; (2) in step S6, the base is selected from an inorganic base or an organic base; the inorganic base is selected from one or more of sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, calcium carbonate, tripotassium phosphate, trisodium phosphate, dipotassium hydrogen phosphate and disodium hydrogen phosphate; the organic base is selected from any one or more of triethylamine, N-methylmorpholine, pyridine and diisopropylethylamine; (3) In step S6, the molar ratio of the compound H to the base is 1:1.5-5, for example 1:2; (4) In step S6, the reaction temperature is 0 to 30° C., for example, 20° C.; (5) In step S6, the reaction time is 0.5 to 5 hours, for example, 1 hour; (6) In step S6, the solvent is selected from any one or more of ethylene glycol dimethyl ether, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone, tetrahydrofuran, dichloromethane, acetonitrile, 1,4-dioxane, and water.

6. The preparation method according to claim 5, characterized in that: The preparation method of the compound G comprises the following steps S3 and S4: Step S3: Compound D is condensed with HOSU to generate compound E. Step S4: Under alkaline conditions, compound E reacts with compound F to generate compound G. Wherein R1 is defined as a substituent as in claim 1, Preferably, at least one of the following conditions is met in step S3: (1) In step S3, the molar ratio of compound D to HOSU is 1:1-3, for example, 1:1.5; (2) in step S3, the condensing agent of the reaction is selected from the coupling reagent reaction of carbodiimide, imidazolinium reagent, phosphonium salt, organophosphorus reagent, uronium salt, pyridinium reagent and phosphonic acid, more particularly any single condensing agent or composite condensing agent selected from EDCI, DIC, DCC, HBTU, PyBOP, BOP, HATU, HOBT, TBTU, DIC / HOBT, DCC / DMAP, EDCI / DMAP, EDCI / HOBT, HATU / HOBT, such as EDCI, DIC or DCC; (3) In step S3, the molar ratio of the compound D to the condensing agent is 1:1 to 3, for example, 1:1.5; (4) In step S3, the reaction temperature is 0 to 30° C., for example, 20° C.; (5) In step S3, the reaction time is 1 to 24 hours, for example 20 hours; (6) In step S3, the solvent is selected from any one or more of N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone, tetrahydrofuran, dichloromethane, acetonitrile, benzonitrile, benzyl cyanide, diethyl ether, methyl tert-butyl ether, 1,4-dioxane, diisopropyl ether, ethyl acetate, and acetone; Preferably, the compound G in step S4 satisfies at least one of the following conditions: (1) In step S4, the molar ratio of compound E to compound F is 1:1-2, for example, 1:1.2; (2) in step S4, the base is selected from an inorganic base or an organic base; the inorganic base is selected from one or more of sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate, calcium carbonate, tripotassium phosphate, trisodium phosphate, dipotassium hydrogen phosphate and disodium hydrogen phosphate; the organic base is selected from one or more of triethylamine, N-methylmorpholine, pyridine and diisopropylethylamine; (3) In step S4, the molar ratio of the compound E to the base is 1:1.5-5, for example 1:2; (4) In step S4, the reaction temperature is 0 to 30° C., for example, 20° C.; (5) In step S4, the reaction time is 0.5 to 3 h, for example 0.5 h; (6) In step S4, the solvent is selected from any one or more of ethylene glycol dimethyl ether, N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone, tetrahydrofuran, dichloromethane, acetonitrile, and 1,4-dioxane.

7. The preparation method according to claim 1, characterized in that: Compound L is used to prepare compound N, and the preparation method of compound N comprises the following steps S9 and S10: Step S9: Deprotecting compound L to obtain compound M, Step S10: Under alkaline conditions, compound M reacts with compound C to generate compound N. Wherein R1 is defined as a substituent as in claim 1, Preferably, the preparation method of compound M in step S9 satisfies at least one of the following conditions: (1) The deprotection reagent in step S9 is an amino deprotection reagent, and different reagents can be selected for deprotection according to specific different protecting groups; (2) When the protecting group in step S9 is 9-fluorenylmethoxycarbonyl, the deprotecting agent in step S9 is selected from piperidine, triethylamine, tetrabutylammonium fluoride, and 1,8-diazabicyclo[5.4.0]undec-7-ene; (3) In step S9, the molar ratio of the compound L to the deprotection agent is 1:3 to 10, for example 1:10; (4) In step S9, the solvent is selected from any one or more of cyclopentyl methyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, methyl tert-butyl ether, 1,4-dioxane, propyl ether, isopropyl ether, ethyl ether, ethylene glycol dimethyl ether or anisole; (5) In step S9, the mass volume ratio of the ether solvent to water is 1 to 20:1 g / mL, for example 20:1 g / mL; (6) In step S9, the reaction temperature is 5 to 40° C., for example, 20 to 25° C.; (7) In step S9, the reaction time is 3 to 24 hours, for example, 4 hours; Preferably, the preparation method of compound N in step S10 satisfies one or more of the following conditions: (1) In step S10, the molar ratio of compound M to compound C is 1:1 to 1.2, for example 1:1; (2) In step S10, the base is selected from an inorganic base or an organic base; the inorganic base is selected from one or more of sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate and calcium carbonate, tripotassium phosphate, trisodium phosphate, dipotassium hydrogen phosphate and disodium hydrogen phosphate; for example, sodium bicarbonate, and the organic base is selected from any one of triethylamine, N-methylmorpholine, pyridine and diisopropylethylamine; (3) In step S10, the molar ratio of the compound M to the base is 1:0.5-3, for example 1:1; (4) In step S10, the solvent is a mixed solvent of nitrile, ether, sulfone solvent or amide and water; preferably a mixed solvent of nitrile and water; (5) In step S10, the volume ratio of the nitrile solvent to water is 2 to 3:1, for example, 2:1; (6) In step S10, the nitrile solvent is selected from any one or more of acetonitrile, propionitrile, butyronitrile, isobutyronitrile, benzonitrile or benzyl cyanide, preferably acetonitrile; the ether solvent is selected from any one or more of tetrahydrofuran, ethylene glycol dimethyl ether, anisole, 1,4-dioxane, and diethyl ether; the sulfone solvent is selected from dimethyl sulfoxide; the amide solvent is selected from any one or more of N,N-dimethylformamide, N-methyl-2-pyrrolidone and N,N-dimethylacetamide; (7) In step S10, the reaction temperature is 5 to 30° C., for example, 15 to 25° C.; (8) In step S10, the reaction time is 1 to 3 hours, for example, 2 hours.

8. The preparation method according to claim 7, characterized in that: The preparation method of the compound C comprises the following steps S1 and S2: Step S1: Under alkaline conditions, compound A reacts with glycine to generate compound B. Step S2: Compound B is condensed with HOSU to generate compound C. Preferably, the preparation method of compound B in step S1 satisfies one or more of the following conditions: (1) In step S1, the molar ratio of compound A to glycine is 1:1-2, for example, 1:1.5; (2) In step S1, the base is selected from any one of 2,4-lutidine, 2,4,6-trimethylpyridine, 4-dimethylaminopyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene, triethylamine, N-methylmorpholine, pyridine, and diisopropylethylamine; (3) In step S1, the molar ratio of compound A to base is 1:1.2-3, for example, 1:1.5; (4) In step S1, the reaction temperature is 0 to 30°C, for example, 10 to 20°C; (5) In step S1, the reaction time is 3 to 12 hours, for example, 4 hours; (6) In step S1, the solvent is selected from any one or more of acetonitrile, benzonitrile, benzyl cyanide, and water; (7) In step S1, the mass volume ratio of the solvent to water is 1:1 to 1.5 g / mL, for example 1:1.2 g / mL; Preferably, the preparation method of compound C in step S2 satisfies one or more of the following conditions: (1) In step S2, the molar ratio of compound B to HOSU is 1:1-3, for example, 1:1.2; (2) in step S2, the condensing agent of the reaction is selected from the coupling reagent reaction of carbodiimide, imidazolinium reagent, phosphonium salt, organophosphorus reagent, uronium salt, pyridinium reagent and phosphonic acid, more particularly any single condensing agent or composite condensing agent selected from EDCI, DIC, DCC, HBTU, PyBOP, BOP, HATU, HOBT, TBTU, DIC / HOBT, DCC / DMAP, EDCI / DMAP, EDCI / HOBT, HATU / HOBT, such as EDCI; (3) In step S2, the molar ratio of compound B to the condensing agent is 1:1 to 3, for example, 1:1.5; (4) In step S2, the reaction temperature is 0 to 30°C, for example, 15 to 25°C; (5) In step S2, the reaction time is 1 to 24 hours, for example, 3 hours; (6) In step S2, the solvent is selected from any one or more of N,N-dimethylacetamide, N,N-dimethylformamide, N-methyl-2-pyrrolidone, tetrahydrofuran, dichloromethane, acetonitrile, benzonitrile, benzyl cyanide, tetrahydrofuran, diethyl ether, methyl tert-butyl ether, 1,4-dioxane, diisopropyl ether, ethyl acetate, and acetone.

9. A compound H having the following structural formula: Wherein R1 is one of 9-fluorenylmethoxycarbonyl, pivaloyl, acetyl, trityl, allyloxycarbonyl, benzyl, trimethylsilylethoxycarbonyl, trichloroethoxycarbonyl, phthaloyl, p-methoxybenzyl, trifluoroacetyl, 2-nitrobenzenesulfonyl, 2,4-dinitrobenzenesulfonyl, 2-nitrobenzyloxycarbonyl, 2-nitrobenzyl, 2-nitrobenzyl, methylsulfonyl, p-toluenesulfonyl, methoxycarbonyl or ethoxycarbonyl.

10. A method for preparing a drug-linker complex or ADC conjugate, comprising the following steps: (1) A method for preparing compound N, comprising the following steps S10: under alkaline conditions, compound M reacts with compound C to generate compound N, The conditions and operations of the preparation method of the compound N may be as described in any one of claims 1 to 7; (2) preparing the drug-linker complex using compound N as a raw material; The drug includes the following compounds: calicheamicin, MMAF, MMAE, doxorubicin, SN-38, Dxd or amanitin; Or using compound N as a raw material to prepare the ADC conjugate; The ADC conjugates include: trastuzumab deruxtecan, datopotamab deruxtecan, patritumab deruxtecan or ifinatamab deruxtecan.

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