CN116897149A

CN116897149A - Preparation method of linker drug conjugate and intermediate thereof

Info

Publication number: CN116897149A
Application number: CN202180093749.3A
Authority: CN
Inventors: 鲍彬; 邱雪飞; 杨彤; 张文伯
Original assignee: SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL CO LTD
Current assignee: SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL CO LTD
Priority date: 2021-03-30
Filing date: 2021-03-30
Publication date: 2023-10-17
Also published as: WO2022204947A1

Abstract

A preparation method of a linker drug conjugate and an intermediate thereof, in particular discloses a preparation method of a compound shown in a formula (I), wherein R is ¹ Is C ₁ ～C ₆ Alkyl, one or more R ^1‑3 S(O) ₂ -substituted C ₁ ～C ₆ Alkyl, or one or more N (R) ^1‑1 )(R ^1‑2 ) -substituted C ₁ ～C ₆ An alkyl group; r is R ² And R is ³ Each independently is C ₁ ～C ₆ Alkyl, one or more halogen-substituted C ₁ ～C ₆ Alkyl, or halogen; r is R ^1‑1 、R ^1‑2 And R is ^1‑3 Each independently is C ₁ ～C ₄ An alkyl group. The preparation method has one or more of the following advantages: simple operation, good yield, easy control of product quality and suitability for industrial production.

Description

Preparation method of linker drug conjugate and intermediate thereof

Technical Field

The invention belongs to the field of drug synthesis, and particularly relates to a preparation method of a linker drug conjugate and an intermediate thereof.

Background

Antibody-conjugated drugs (ADCs) are one of the hot spots of recent interest in the pharmaceutical industry. Because of the inadequate clinical efficacy of many antibody drugs, many industries are increasingly turning their eyes to ADC drugs. The basic module of the ADC medicine comprises an antibody, a connector and an effector molecule, and the effector molecule is transmitted to a tumor site for enrichment by using the antibody, so that tumor cells are killed. The traditional effector molecules are mostly high-activity tubulin inhibitors, and have large toxic and side effects, so that the application of the ADC is limited. Recently, the company Immunometics invented a novel ADC drug IMMU-132 (ZL 200980156218) which takes a camptothecin compound as an effector molecule, and the novel ADC drug IMMU-132 shows better anti-tumor effect, and the company firstly invented another camptothecin compound as an effector molecule, namely an ADC drug DS-8201a (ZL 201380053256), and the novel ADC drug IMMU-132 also shows better anti-tumor effect.

WO2020259258A1 discloses an ADC compound with a camptothecin derivative Dxd as an effector molecule, which also shows a good antitumor effect. Wherein the target ADC compound can be obtained by coupling a camptothecin derivative shown in a formula I with an antibody, wherein the linker drug conjugate shown in the formula I can be prepared by the following synthesis route 1 or synthesis route 2.

Route 1:

the synthetic method of scheme 1 includes: the compound 1-1 reacts with 4-amino benzyl alcohol, the obtained compound reacts with di (p-nitrobenzene) carbonic ester and then reacts with substituted alkylamine to obtain a compound 1-2, the compound 1-2 reacts with paraformaldehyde and trimethylchlorosilane to obtain a compound 1-3, the compound 1-3 reacts with tert-butyl glycolate, the tert-butyl is removed under the action of trifluoroacetic acid to obtain a compound 1-4, the compound 1-4 reacts with Exatecan mesylate to obtain a compound 1-5, fmoc protection on deamination under the action of DBU is removed, and then the compound 1-4 undergoes coupling reaction with 6- (maleimide) caproic acid succinimidyl ester to obtain the target compound I.

Route 2:

the synthetic method of scheme 2 includes: reacting compound 2-1 with paraformaldehyde and trimethylchlorosilane, reacting the obtained compound with tert-butyl glycolate to obtain compound 2-2, removing tert-butyl from compound 2-2 under the action of trifluoroacetic acid, reacting with Exatecan mesylate to obtain compound 2-3, and reducing azide from compound 2-3 under the action of triethylphosphine

Amino is formed to obtain a compound 2-4, and the compound 2-4 and MC-V undergo a coupling reaction to obtain a target compound I.

However, the synthetic methods of the above routes 1 and 2 still have the problem that the purity of the final product is not acceptable by using the method of column chromatography purification in the actual production scale-up process, so that a new route needs to be explored to make the purity of the final product meet the requirement.

Disclosure of Invention

The invention aims to solve the technical problem that the preparation method of the linker drug conjugate shown in the formula I in the prior art is difficult to obtain the end product with qualified purity, and provides a novel preparation method and an intermediate of the linker drug conjugate shown in the formula I. The preparation method of the invention has one or more of the following advantages: simple operation, good yield, easy control of product quality and suitability for industrial production.

The present invention provides a compound of formula III:

wherein R is ¹ Is C ₁ ～C ₆ Alkyl, one or more R ^1-3 S(O) ₂ -substituted C ₁ ～C ₆ Alkyl, or one or more N (R) ^1-1 )(R ^1-2 ) -substituted C ₁ ～C ₆ An alkyl group;

R ² and R is ³ Each independently is C ₁ ～C ₆ Alkyl, one or more halogen-substituted C ₁ ～C ₆ Alkyl, or halogen;

R ^1-1 、R ^1-2 and R is ^1-3 Each independently is C ₁ ～C ₄ An alkyl group.

In some embodiments, R ¹ Is R ^1-3 S(O) ₂ -substituted C ₁ ～C ₆ An alkyl group;

R ² is C ₁ ～C ₆ An alkyl group;

R ³ is halogen;

R ^1-3 is C ₁ ～C ₄ An alkyl group.

In some embodiments, at R ¹ 、R ² And R is ³ In the definition of (C), said C ₁ ～C ₆ The alkyl group may be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl or ethyl.

In some embodiments, at R ¹ 、R ² And R is ³ In the definition of (2), the halogen may be fluorine, chlorine, bromine or iodine, preferably fluorine.

In some embodiments, at R ^1-1 、R ^1-2 And R is ^1-3 In the definition of (C), said C ₁ ～C ₄ The alkyl group may be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl.

In some embodiments, R ¹ Is R ^1-3 S(O) ₂ -substituted C ₁ ～C ₆ Alkyl or one-NR ^1-1 R ^1-2 Substituted C ₁ ～C ₆ Alkyl is preferably methylsulfonylethyl or N, N-dimethylethyl, more preferably methylsulfonylethyl.

In some embodiments, R ² Is C ₁ ～C ₆ Alkyl is preferably methyl.

In some embodiments, R ³ Halogen is preferably fluorine or chlorine, more preferably fluorine.

In some embodiments, R ¹ Is R ^1-3 S(O) ₂ -substituted C ₁ ～C ₆ Alkyl groups such as methylsulfonylethyl;

R ² is C ₁ ～C ₆ Alkyl groups such as methyl;

R ³ halogen, such as fluorine;

R ^1-3 is C ₁ ～C ₄ Alkyl groups such as ethyl.

In some embodiments, R ¹ R is as defined in any one of the preceding schemes ² Is methyl, and R ³ Is fluorine.

In some embodiments, R ¹ Is methylsulfonylethyl, R ² Is methyl, and R ³ Is fluorine.

In some embodiments, the compound of formula III is

The invention also provides a preparation method of the compound shown in the formula III, which comprises the following steps: carrying out substitution reaction on a compound of formula IV and a compound of formula IV' in a solvent and in the presence of alkali to obtain a compound of formula III;

wherein R is ¹ 、R ² And R is ³ As previously described.

In some embodiments, in the methods of preparing the compounds of formula III, preferably R ² Is methyl, R ³ Is fluorine.

In some embodiments, the compound of formula III may be prepared in a molar ratio of 1:1 to 5:1, preferably 2:1 to 4:1, and more preferably 3.0:1 to 3.5:1.

In some embodiments, the base may be a base conventional in such reactions in the art, such as an organic base, an inorganic base, or mixtures thereof, preferably an organic base; wherein the organic base is preferably tert-butyl potassium, triethylamine, DMAP, pyridine, pan Bi pyridine or a mixture of any two or more of the above, and is further preferably panulidine; the inorganic base is preferably an alkali metal hydroxide, an alkali metal carbonate, an alkali metal phosphate, or a mixture of any two or more thereof, and more preferably potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate, or a mixture of any two or more thereof. In some embodiments, the base is pantidine in the process for preparing the compound of formula III.

In some embodiments, the molar ratio of the base to the compound of formula III' may be 3:1 to 6:1, preferably 4.5:1 to 5.5:1, and more preferably 4.8:1.

In some embodiments, the solvent may be a solvent conventional in such reactions in the art, preferably an aprotic organic solvent, such as an ether solvent, a chlorinated alkane solvent, a nitrile solvent, or a mixture of any two or more thereof, preferably an ether solvent; the ether solvent can be tetrahydrofuran, diethyl ether, 1, 4-dioxane, anisole, methyl tertiary butyl ether or a mixture of any two or more of the above, preferably 1, 4-dioxane; the chlorinated alkane solvent is preferably dichloromethane, dichloroethane, chloroform or a mixture of any two or more of the above; the nitrile solvent is preferably acetonitrile. In some embodiments, the solvent is 1, 4-dioxane in the process for preparing the compound of formula III.

In some embodiments, the temperature of the substitution reaction in the preparation of the compound of formula III may be conventional in the art for such reactions, for example, 20-80 ℃, preferably 40-60 ℃, and more preferably 60 ℃.

In some embodiments, the process for preparing the compound of formula III, the substitution reaction may be carried out as is conventional in the art for such reactions, for example, comprising the steps of: stirring the mixture of the compound of formula IV, the compound of formula IV', the solvent and the base to perform substitution reaction.

In some embodiments, the progress of the substitution reaction may be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC or NMR, etc.), typically by no longer detecting the compound of formula IV or the compound of formula IV' as the end point of the reaction. The reaction time of the substitution reaction may be 2 to 12 hours, more preferably 2 to 3 hours, still more preferably 2 hours.

In some embodiments, the preparation method of the compound of formula III may further include the following post-treatment steps after the substitution reaction is completed: the solvent in the reaction solution was removed, the organic phase was washed, and the residue after the solvent was removed from the organic phase obtained by washing was purified. Wherein the organic phase in the washing may be ethyl acetate or dichloromethane, preferably dichloromethane. The aqueous phase in the washing can be aqueous acid solution, water and/or saturated saline; the aqueous acid solution may be 0.1N hydrochloric acid, 0.05N sulfuric acid or a mixture thereof, preferably 0.1N hydrochloric acid. Preferably, the washing may comprise washing the organic phase with an aqueous acid solution, water and saturated brine in sequence, preferably with 0.1N dilute acid, water and saturated brine in sequence. In some embodiments, the purification may be performed by methods conventional in the art, such as beating, crystallization, preparative chromatography or silica gel column chromatography, preferably by column silica gel column chromatography, using an eluent preferably a mixture of dichloromethane and methanol, preferably at an elution gradient of 100:1 to 10:1, more preferably 60:1 to 10:1.

In some embodiments, the substitution reaction is preferably performed under anhydrous conditions in the process for preparing the compound of formula III.

The preparation method of the compound of the formula III can further comprise a preparation method of the compound of the formula IV, and the preparation method can comprise the following steps: reacting a compound of formula V with paraformaldehyde and trimethylchlorosilane in a solvent to obtain a compound of formula IV;

wherein R1 is as previously described.

In some embodiments, the molar ratio of paraformaldehyde to compound of formula V, in terms of formaldehyde, is from 3:1 to 12:1, preferably from 3:1 to 4:1, and more preferably 3.1:1.

In some embodiments, the molar ratio of the trimethylchlorosilane to the compound of formula V in the process for preparing the compound of formula IV is 3:1 to 12:1, preferably 3:1 to 4:1, and more preferably 3.9:1.

In some embodiments, the solvent may be a solvent conventional in such reactions in the art, preferably an aprotic organic solvent, such as an ether solvent, a chlorinated alkane solvent, or a mixture thereof, preferably an ether solvent; the ether solvent can be tetrahydrofuran, diethyl ether, 1, 4-dioxane, anisole, methyl tertiary butyl ether or a mixture of any two or more of the above, preferably tetrahydrofuran; the chlorinated alkane solvent is preferably dichloromethane, dichloroethane, chloroform or a mixture of any two or more of them. In some embodiments, the method for preparing the compound of formula IV, the solvent is tetrahydrofuran.

In some embodiments, the reaction temperature in the preparation of the compounds of formula IV may be conventional in the art for such reactions, e.g., 10-40 ℃, preferably 25-40 ℃, and more preferably 25-30 ℃.

In some embodiments, the reaction is preferably carried out under anhydrous conditions in the process for the preparation of the compound of formula IV.

In some embodiments, the process for preparing the compound of formula IV, the reaction may be carried out as is conventional in the art for such reactions, for example comprising the steps of: the trimethylchlorosilane is added in portions (e.g., dropwise) to a mixture of the compound of formula IV, paraformaldehyde and solvent, and the reaction is carried out with stirring.

In some embodiments, the progress of the reaction in the preparation of the compound of formula IV may be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC or NMR, etc.), typically with no more detection of the compound of formula V as the end point of the reaction. The reaction time of the reaction may be 1 to 24 hours, preferably 12 to 20 hours, more preferably 16 to 20 hours, still more preferably 16 hours.

In some embodiments, the preparation method of the compound of formula IV may further include the following post-treatment steps after the substitution reaction is completed: the reaction liquid was subjected to solid-liquid separation, the solvent was removed from the resulting liquid phase, and the resulting residue was directly used for the next reaction.

The preparation method of the compound of the formula IV can further comprise a preparation method of the compound of the formula V, and the preparation method can comprise the following steps: reacting a compound of formula VI with a sulfonyl azide in the presence of a base and a catalyst in a solvent to obtain the compound of formula V;

wherein R is ¹ As previously described.

In some embodiments, the sulfonyl azide may be 1H-imidazole-1-sulfonyl azide hydrochloride, 2-azido-1, 3-dimethylimidazole hexafluorophosphate, trifluoro sulfonyl azide, p-toluenesulfonyl azide, or methanesulfonyl azide, preferably 1H-imidazole-1-sulfonyl azide hydrochloride.

In some embodiments, the sulfonyl azide compound of formula V is prepared in a molar ratio of 1.0:1 to 1.5:1, preferably 1.0:1 to 1.2:1, more preferably 1.02:1.

In some embodiments, the base may be a base conventional in such reactions in the art, such as an organic base, an inorganic base, or mixtures thereof, preferably an inorganic base; wherein the inorganic base is preferably an alkali metal hydroxide, an alkali metal carbonate, an alkali metal phosphate or a mixture of any two or more thereof, more preferably potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate or a mixture of any two or more thereof, still more preferably potassium carbonate; the organic base is preferably tert-butyl potassium, triethylamine, DMAP, pyridine, panprine, 2, 6-lutidine or a mixture of any two or more of the above. In some embodiments, the base is an alkali metal carbonate, such as potassium carbonate, in the process for preparing the compound of formula V.

In some embodiments, the molar ratio of the base to the compound of formula V is 1.5:1 to 3.0:1, preferably 2.0:1 to 2.5:1, more preferably 2.0:1.

In some embodiments, the catalyst may be a catalyst conventional in such reactions in the art, such as a ketone salt, preferably copper sulfate, more preferably copper sulfate pentahydrate.

In some embodiments, the molar ratio of the ketone salt to the compound of formula V may be from 0.1:1 to 0.5:1, preferably from 0.1:1 to 0.3:1, more preferably from 0.1:1 to 0.2:1, and most preferably 0.1:1.

In some embodiments, the solvent may be a solvent conventional in the art for such reactions, preferably a mixed solvent of an organic solvent and water, and the organic solvent may be an alcohol solvent, a chlorinated alkane solvent, an ether solvent or a mixture of any two or more thereof, preferably a mixture of an alcohol solvent and a chlorinated alkane solvent; the alcohol solvent can be methanol, ethanol, isopropanol or a mixture thereof, preferably methanol; the chlorinated alkane solvent can be dichloromethane, chloroform, dichloroethane or a mixture of any two or more of the dichloromethane, preferably dichloromethane; the ether solvent is preferably tetrahydrofuran, diethyl ether, 1, 4-dioxane, anisole, methyl tertiary butyl ether or a mixture of any two or more of the above. In some embodiments, the method for preparing the compound of formula IV, the solvent is a mixed solvent of methanol, dichloromethane and water.

In some embodiments, the reaction temperature in the preparation of the compounds of formula V may be conventional in the art for such reactions, e.g., from 10 to 40 ℃, preferably from 25 to 40 ℃, and more preferably from 25 to 30 ℃.

In some embodiments, the process for preparing the compound of formula V, the reaction may be carried out as is conventional in the art for such reactions, for example comprising the steps of: the sulfonyl azide is added to the mixture of the compound of formula VI, base, catalyst and solvent (preferably, the sulfonyl azide is added after the mixture is clarified) and the reaction is carried out with stirring.

In some embodiments, the progress of the reaction in the preparation of the compound of formula V may be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC or NMR, etc.), typically with no more detection of the compound of formula VI as the end point of the reaction. The reaction time of the reaction may be 1 to 24 hours, preferably 12 to 20 hours, more preferably 16 to 20 hours, still more preferably 16 hours.

In some embodiments, the preparation method of the compound of formula V may further include the following post-treatment steps after the reaction is completed: the organic solvent in the reaction solution is removed, extraction (for example, extraction with methylene chloride) is performed, and the organic phase obtained by extraction is recrystallized by ethanol and activated carbon to obtain the compound of the formula V.

The preparation method of the compound of the formula V can further comprise a preparation method of the compound of the formula VI, which can comprise the following steps: carrying out Fmoc removal reaction on a compound of a formula VII in the presence of alkali and an organic solvent to obtain the compound of the formula VI;

wherein R is ¹ As previously described.

In some embodiments, the base may be a base conventional in such reactions in the art, such as an organic base, an inorganic base, or mixtures thereof, preferably an organic base; wherein the organic base is preferably diethylamine, tert-butylpotassium, triethylamine, DMAP, pyridine, panpridine, 2, 6-lutidine or a mixture of any two or more of them, and further preferably ethylenediamine; the inorganic base is preferably an alkali metal hydroxide, an alkali metal carbonate, an alkali metal phosphate, or a mixture of any two or more thereof, and more preferably potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate, or a mixture of any two or more thereof. In some embodiments, the base is diethylamine in the preparation of the compound of formula VI.

In some embodiments, the volume ratio of the base to the organic solvent in the process for preparing the compound of formula VI may be from 0.1:1 to 0.5:1, preferably from 0.2:1 to 0.3:1, and more preferably 0.2:1.

In some embodiments, in the preparation method of the compound of formula VI, the organic solvent may be DMF, DMSO, tetrahydrofuran, 1, 4-dioxane, or a mixture of any two or more thereof, preferably DMF.

In some embodiments, the temperature of the Fmoc removal reaction in the process for the preparation of the compound of formula VI may be conventional in the art for such reactions, e.g., from 10 to 40 ℃, preferably from 25 to 40 ℃, and more preferably from 25 to 30 ℃.

In some embodiments, the Fmoc removal reaction may be performed as is conventional in the art for such reactions, for example, comprising the steps of: the Fmoc removal reaction is carried out by stirring the mixed system of the compound of formula VII, the base and the organic solvent.

In some embodiments, the progress of the Fmoc removal reaction in the preparation of the compound of formula VI may be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC or NMR, etc.), typically to no longer detect the compound of formula VII as the end point of the reaction. The reaction time of the reaction may be 1 to 24 hours, preferably 4 to 12 hours, more preferably 4 to 6 hours, still more preferably 4 hours.

In some embodiments, the preparation method of the compound of formula VI may further include the following post-treatment steps after the reaction is completed: and (3) carrying out solid-liquid separation on the reaction liquid, and removing the solvent in the obtained liquid phase to obtain a crude product. The post-processing step may further comprise the steps of: the crude product is slurried to obtain a solid product of the compound of formula VI. The solvent used for beating may be an ether solvent such as tetrahydrofuran, diethyl ether, 1, 4-dioxane, anisole, methyl tert-butyl ether or a mixture of any two or more thereof, preferably methyl tert-butyl ether. The post-processing step may further comprise the steps of: and (3) recrystallizing and purifying the solid product of the compound of the formula VI obtained by beating. The solvent used for recrystallization may be an alcohol solvent such as methanol, ethanol, isopropanol or a mixture of any two or more thereof, and ethanol is preferable.

The preparation method of the compound of the formula VI can further comprise a preparation method of the compound of the formula VII, which can comprise the following steps: carrying out coupling reaction on a compound of formula VIII and N-Fmoc-L-valine N-butadiene amine imine ester in a solvent to obtain the compound of formula VII;

Wherein R is ¹ As previously described.

In some embodiments, the molar ratio of the N-Fmoc-L-valine N-butadienaminimide ester to the compound of formula VIII may be in the range of from 0.8:1 to 5:1, preferably from 0.8:1 to 1.2:1, more preferably 1:1.

In some embodiments, the solvent may be DMF, DMSO, acetonitrile, dichloromethane, dichloroethane, or a mixture of any two or more thereof, preferably dichloromethane.

In some embodiments, the temperature of the coupling reaction in the preparation of the compound of formula VII may be conventional in the art for such reactions, e.g., from 10 to 40 ℃, preferably from 35 to 40 ℃, and more preferably 40 ℃.

In some embodiments, the coupling reaction is preferably carried out under gas protection in the process for the preparation of the compound of formula VII. The gas in the gas shield does not participate in the reaction, such as argon, helium or nitrogen, and also such as nitrogen.

In some embodiments, the process for preparing the compound of formula VII, the operation of the coupling reaction may be conventional in the art for such reactions, e.g., comprising the steps of: the mixed system of the compound of the formula VIII, the N-Fmoc-L-valine N-butadiene amine imine ester and the solvent is stirred for coupling reaction.

In some embodiments, the progress of the coupling reaction may be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC or NMR, etc.), typically with no more detection of the compound of formula VIII as the reaction endpoint. The reaction time of the coupling reaction may be 1 to 24 hours, preferably 12 to 20 hours, more preferably 16 to 20 hours, still more preferably 16 hours.

In some embodiments, the method for preparing a compound of formula VII may further include a post-treatment step after the coupling reaction is completed, the post-treatment step may include: adding alcohol solvent (such as methanol, ethanol, isopropanol or mixture of any two or more thereof, preferably methanol) into the reaction system, stirring (stirring temperature may be 20-40deg.C, preferably 35-40deg.C, more preferably 40deg.C, stirring time may be 1-24 hr, preferably 4-12 hr, more preferably 4-6 hr, more preferably 4 hr), and separating the solid in the system to obtain compound of formula VII.

The preparation method of the compound of the formula VII can further comprise the preparation method of the compound of the formula VIII, which can comprise the following steps: subjecting a compound of formula IX to Fmoc removal in the presence of a base and in a solvent to obtain a compound of formula VIII;

Wherein R is ¹ As previously described.

In some embodiments, the base may be a base conventional in such reactions in the art, such as an organic base, an inorganic base, or mixtures thereof, preferably an organic base; wherein the organic base is preferably diethylamine, tert-butylpotassium, triethylamine, DMAP, pyridine, panpridine, 2, 6-lutidine or a mixture of any two or more of them, and further preferably ethylenediamine; the inorganic base is preferably an alkali metal hydroxide, an alkali metal carbonate, an alkali metal phosphate, or a mixture of any two or more thereof, and more preferably potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate, or a mixture of any two or more thereof. In some embodiments, the base is diethylamine in the preparation of the compound of formula VIII.

In some embodiments, in the method for preparing the compound of formula VIII, the solvent may be DMF, DMSO, tetrahydrofuran, 1, 4-dioxane, or a mixture of any two or more thereof, preferably DMF.

In some embodiments, the volume ratio of the base to the solvent in the process for preparing the compound of formula VIII may be from 0.2:1 to 0.5:1, preferably from 0.3:1 to 0.4:1, and more preferably 0.3:1.

In some embodiments, the reaction temperature of the Fmoc removal reaction in the described process for the preparation of a compound of formula VIII may be conventional in the art for such reactions, e.g., from 10 to 40 ℃, preferably from 25 to 40 ℃, and more preferably from 25 to 30 ℃.

In some embodiments, in the method for preparing the compound of formula VIII, the Fmoc removal reaction may be performed as is conventional in the art for such reactions, for example, comprising the steps of: the Fmoc removal reaction is carried out by stirring the mixture of the compound of formula IX, base and solvent.

In some embodiments, the progress of the Fmoc removal reaction in the preparation of the compound of formula VIII may be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC or NMR, etc.), typically to no longer detect the compound of formula VII as the end point of the reaction. The reaction time of the reaction may be 1 to 24 hours, preferably 2 to 12 hours, more preferably 2 to 6 hours, still more preferably 2 hours.

In some embodiments, the method for preparing a compound of formula VIII may further include a post-treatment step after the Fmoc removal reaction is completed, wherein the post-treatment step may include: the solvent in the reaction liquid is removed, and the obtained residue is pulped to obtain a solid which is the compound of the formula VIII. The solvent used for beating may be an ether solvent such as tetrahydrofuran, diethyl ether, 1, 4-dioxane, anisole, methyl tert-butyl ether or a mixture of any two or more thereof, preferably methyl tert-butyl ether.

The process for preparing the compound of formula VIII may further comprise a process for preparing a compound of formula IX, which may comprise the steps of: coupling the compound of formula X with an amino compound R1NH2 in the presence of a base and in a solvent to obtain a compound of formula IX;

wherein R is ¹ As previously described.

In some embodiments, in the process for preparing the compound of formula IX, the amino compound R ¹ NH ₂ The molar ratio to the compound of the formula X may be from 1.0:1 to 3.0:1, preferably from 1.1:1 to 1.5:1, more preferably 1.1:1.

In some embodiments, the base may be a base conventional in such reactions in the art, such as an organic base, an inorganic base, or mixtures thereof, preferably an organic base; wherein the organic base is preferably diethylamine, tert-butylpotassium, triethylamine, DMAP, pyridine, panpridine, 2, 6-lutidine or a mixture of any two or more thereof, and further preferably DMAP; the inorganic base is preferably an alkali metal hydroxide, an alkali metal carbonate, an alkali metal phosphate, or a mixture of any two or more thereof, and more preferably potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate, or a mixture of any two or more thereof. In some embodiments, the base may be DMAP in the preparation of the compound of formula IX.

In some embodiments, the molar ratio of the base to the compound of formula X may be from 2.0:1 to 4.0:1, preferably from 2.5:1 to 3.0:1, and more preferably 2.5:1.

In some embodiments, in the preparation method of the compound of formula IX, the solvent may be DMF, DMSO, dichloromethane, dichloroethane, tetrahydrofuran, 1, 4-dioxane or a mixture of any two or more thereof, preferably dichloromethane.

In some embodiments, the process for preparing the compound of formula IX may be carried out at a temperature conventional in the art for such reactions, for example, from 10 to 40 ℃, preferably from 25 to 40 ℃, and more preferably from 25 to 30 ℃.

In some embodiments, the process for preparing the compound of formula IX, the operation of the coupling reaction may be conventional in the art for such reactions, e.g., comprising the steps of: stirring the compound X and the amino compound R ¹ NH ₂ And (3) carrying out coupling reaction on the mixed system of the alkali and the solvent.

In some embodiments, the progress of the coupling reaction in the preparation of the compound of formula IX may be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC or NMR, etc.), typically with no more detection of the compound of formula VII as the reaction endpoint. The reaction time of the reaction may be 1 to 24 hours, preferably 12 to 20 hours, more preferably 12 to 16 hours, still more preferably 12 hours.

In some embodiments, the method for preparing a compound of formula IX may further comprise a post-treatment step after the coupling reaction is completed, the post-treatment step may comprise: the reaction solution is subjected to solid-liquid separation, and the solid obtained by the solid-liquid separation (the solid may be washed with an organic solvent such as diethyl ether, ethyl acetate or a mixture thereof, preferably ethyl acetate after the solid-liquid separation) is a part of the product of the compound of formula IX. The post-processing step may further include: and (3) carrying out extraction washing on a liquid phase obtained by solid-liquid separation, removing a solvent from an obtained organic phase, and pulping the obtained solid to obtain another part of the product of the compound shown in the formula IX. The extraction and washing can use aqueous acid solution, aqueous alkali solution, water and saturated saline solution, and preferably the extraction and washing are sequentially carried out by using the aqueous acid solution, the aqueous alkali solution, the water and the saturated saline solution; further preferably, washing with an aqueous acid solution 1 time, washing with an aqueous alkali solution 2 times, washing with water 1 time, and washing with saturated brine 1 time in this order; the aqueous acid solution can be hydrochloric acid aqueous solution, sulfuric acid aqueous solution or phosphoric acid aqueous solution, preferably 1N hydrochloric acid, 0.5N sulfuric acid or 0.33N phosphoric acid, and more preferably 1N hydrochloric acid; the aqueous alkali solution may be an aqueous sodium hydroxide solution, an aqueous potassium hydroxide solution or a mixture thereof, preferably 1N sodium hydroxide, 1N potassium hydroxide or a mixture thereof, and more preferably 1N sodium hydroxide. The beating solvent can be dichloromethane, ethyl acetate or a mixture thereof, preferably dichloromethane; the beating temperature may be 10-40 ℃, preferably 25-40 ℃, further preferably 25-30 ℃.

The preparation method of the compound of the formula IX can further comprise a preparation method of the compound of the formula X, which can comprise the following steps: reacting a compound of formula XI with a compound of formula XII in the presence of a base and in a solvent to give a compound of formula X;

in some embodiments, the molar ratio of the compound of formula XII to the compound of formula XI may be 3.0:1 to 1.2:1, preferably 2.0:1 to 1.5:1, and more preferably 1.5:1.

In some embodiments, the base may be a base conventional in such reactions in the art, for example, an organic base, an inorganic base, or a mixture thereof, preferably an organic base; wherein the organic base is preferably diethylamine, tert-butylpotassium, triethylamine, DMAP, pyridine, panpridine, 2, 6-lutidine or a mixture of any two or more thereof, and more preferably pyridine; the inorganic base is preferably an alkali metal hydroxide, an alkali metal carbonate, an alkali metal phosphate or a mixture thereof, and more preferably potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate or a mixture of any two or more thereof. In some embodiments, the base is pyridine in the process for preparing the compound of formula X.

In some embodiments, the molar ratio of the base to the compound of formula XI may be from 1.0:1 to 4.0:1, preferably from 2.0:1 to 3.0:1, and more preferably 2.0:1.

In some embodiments, in the preparation method of the compound of formula X, the solvent may be DMF, DMSO, dichloromethane, dichloroethane, tetrahydrofuran, 1, 4-dioxane or a mixture of any two or more thereof, preferably dichloromethane.

In some embodiments, the reaction temperature in the preparation of the compounds of formula X may be conventional in the art for such reactions, e.g., from 10 to 40 ℃, preferably from 25 to 40 ℃, and more preferably from 25 to 30 ℃.

In some embodiments, the process for preparing the compound of formula X described herein, the reaction may be carried out as is conventional in the art for such reactions, for example, comprising the steps of: the compound of formula XII is added in portions (divided into 6 to 3 portions, preferably 5 to 4 portions) to a mixed system of the compound of formula XI, a base and a solvent (the addition process can control the temperature of the mixed system to be 0 to 20 ℃, preferably 10 to 0 ℃ C., further preferably 0 to 5 ℃ C.), and the reaction is carried out with stirring.

In some embodiments, the progress of the reaction in the preparation of the compound of formula X may be monitored using conventional test methods in the art (e.g., TLC, GC, HPLC or NMR, etc.), typically with no more detection of the compound of formula XI as the end point of the reaction. The reaction time of the reaction may be 1 to 24 hours, preferably 2 to 12 hours, more preferably 4 to 8 hours, still more preferably 4 hours.

In some embodiments, the method for preparing a compound of formula X may further include a post-treatment step after the reaction is completed, and the post-treatment step may include: and (3) carrying out solid-liquid separation on the reaction liquid, wherein the solid obtained through the solid-liquid separation is a part of products of the compound of the formula X. The post-processing step may further include: and (3) carrying out extraction and washing on a liquid phase obtained by solid-liquid separation, removing a solvent from an obtained organic phase, and pulping the obtained solid to obtain another part of the product of the compound shown in the formula X. The extraction and washing can use aqueous acid solution, aqueous alkali solution, water and saturated saline solution, and preferably the extraction and washing are sequentially carried out by using the aqueous acid solution, the aqueous alkali solution, the water and the saturated saline solution; further preferably, washing with an aqueous acid solution 1 time, washing with an aqueous alkali solution 2 times, washing with water 1 time, and washing with saturated brine 1 time in this order; the aqueous acid solution can be hydrochloric acid aqueous solution, sulfuric acid aqueous solution or phosphoric acid aqueous solution, preferably 1N hydrochloric acid, 0.5N sulfuric acid or 0.33N phosphoric acid, and more preferably 1N hydrochloric acid; the aqueous alkali solution may be an aqueous sodium hydroxide solution, an aqueous potassium hydroxide solution or a mixture thereof, preferably 1N sodium hydroxide, 1N potassium hydroxide or a mixture thereof, and more preferably 1N sodium hydroxide. The beating solvent can be tetrahydrofuran, diethyl ether, 1, 4-dioxane, anisole, methyl tertiary butyl ether or a mixture of any two or more of the above, preferably methyl tertiary butyl ether.

The present invention also provides a compound of formula II:

wherein R is ¹ 、R ² And R is ³ As defined in the foregoing compounds of formula III;

the precondition is that: when R is ¹ Is methyl, methylsulfonylethyl or dimethylethyl, R ² When methyl, R ³ Is not fluorine.

The invention also provides a preparation method of the compound shown in the formula II, which comprises the following steps: carrying out reduction reaction on a compound of the formula III and a reducing agent in an organic solvent and in the presence of an acid buffer solution to obtain the compound of the formula II;

wherein R is ¹ 、R ² And R is ³ As defined for the compounds of formula III hereinbefore.

In some embodiments, the reducing agent may be a reducing agent conventional in such reactions in the art, preferably triphenylphosphine, tri-t-butylphosphine or trimethylphosphine, more preferably trimethylphosphine.

In some embodiments, the molar ratio of the reducing agent to the compound of formula II may be from 1:1 to 5:1, preferably from 2:1 to 4:1, more preferably from 2.9:1 to 3.5:1, and even more preferably from 2.9:1 to 3.0:1.

In some embodiments, the organic solvent may be an organic solvent conventional in such reactions in the art, preferably an ether solvent, such as tetrahydrofuran, diethyl ether, 1, 4-dioxane, anisole, methyl tert-butyl ether or a mixture of any two or more thereof, and further preferably tetrahydrofuran.

In some embodiments, the volume to mass ratio of the organic solvent to the compound of formula II may be 5-50mL/g, preferably 10-20mL/g, and more preferably 13-14mL/g.

In some embodiments, the acid buffer may be an acid buffer conventional in such reactions in the art, such as an acetate buffer, a formate buffer, preferably an acetate buffer; the pH of the acetate buffer used may be 4.0 to 6.0, preferably 4.5 to 5.5, more preferably 5.0.

In some embodiments, the volume ratio of the organic solvent to the acid buffer may be 1:1 to 5:1, preferably 1:1 to 2:1, more preferably 1.25:1 to 1.35:1, and most preferably 1.28:1 to 1.30:1.

In some embodiments, the temperature of the reduction reaction in the preparation of the compound of formula II may be a temperature conventional in such reactions in the art, for example, 0-20deg.C, preferably 0-10deg.C, and more preferably 0-5deg.C.

In some embodiments, the process for preparing the compound of formula II, the operation of the reduction reaction may be conventional in the art for such reactions, e.g., comprising the steps of: the mixture of organic solvent, acid buffer, compound of formula III and reducing agent is stirred to effect the reduction reaction.

In some embodiments, the progress of the reduction reaction in the preparation of the compound of formula II may be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC or NMR, etc.), typically to no longer detect the compound of formula III as the end point of the reaction. The reaction time of the reduction reaction is 1 to 24 hours, preferably 2 to 5 hours, and more preferably 2 hours.

In some embodiments, the method for preparing the compound of formula II may further include the following post-treatment steps after the reduction reaction is completed: the reaction liquid is extracted, and residues obtained by the extraction after the solvent is removed are purified to obtain the compound of the formula II. The purification may be carried out by a method conventional in the art, such as beating, crystallization, preparative chromatography or silica gel column chromatography, preferably by column silica gel column chromatography, using an eluent preferably a mixture of methylene chloride and methanol, preferably at an elution gradient of 50:1 to 10:1, further preferably at 20:1 to 10:1.

The methods of preparing the compounds of formula II may further include methods of preparing compounds of formula III as described herein.

The invention provides a preparation method of a compound shown in a formula I, which comprises the following steps: coupling the compound of the formula II and 6- (maleimide) caproic acid succinimidyl ester in a solvent to obtain the compound of the formula I;

In some embodiments, the molar ratio of succinimidyl 6- (maleimido) hexanoate to the compound of formula II may be 1.0:1 to 5.0:1, preferably 1.0:1 to 2.0:1, more preferably 1.9:1 to 2.0:1, and even more preferably 1.96:1.

In some embodiments, the solvent may be a solvent conventional in the art for such reactions, for example, an amide solvent, a chlorinated alkane solvent, an ether solvent, a nitrile solvent, or a mixture of any two or more thereof, preferably an amide solvent, a chlorinated alkane solvent, or a mixture thereof, more preferably an amide solvent or a chlorinated alkane solvent, and even more preferably a chlorinated alkane solvent; wherein the amide solvent is preferably DMF, DMAC or a mixture thereof, and further preferably DMF; the chlorinated alkane solvent is preferably dichloromethane, chloroform, dichloroethane or a mixture of any two or more of them, and more preferably dichloromethane; the ether solvent is preferably tetrahydrofuran, diethyl ether, 1, 4-dioxane, anisole, methyl tertiary butyl ether or a mixture of any two or more of them, and further preferably tetrahydrofuran; the nitrile solvent is preferably acetonitrile. In some embodiments, the method of preparing the compound of formula I, the solvent is a chlorinated alkane solvent, such as methylene chloride.

In some embodiments, the volume to mass ratio of the solvent to the compound of formula I may be 20-200mL/g, preferably 30-80mL/g, more preferably 35-40mL/g.

In some embodiments, the reaction temperature in the preparation of the compounds of formula I may be a temperature conventional in such reactions in the art, for example, from 0 to 50 ℃, preferably from 25 to 40 ℃, and more preferably 40 ℃.

In some embodiments, the process for preparing the compound of formula I, the operation of the coupling reaction may be conventional in the art for such reactions, e.g., comprising the steps of: the mixture of the compound of formula II, 6- (maleimide) hexanoyl compound and solvent is stirred to carry out the coupling reaction.

In some embodiments, the progress of the reaction in the preparation of the compound of formula I may be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC or NMR, etc.), typically with no more detection of the compound of formula II as the end point of the reaction. The reaction time of the coupling reaction may be 1 to 24 hours, preferably 12 to 20 hours, and more preferably 16 hours.

In some embodiments, the preparation method of the compound of formula I may further comprise the following post-treatment steps after the coupling reaction is completed: the solvent in the reaction solution is removed, and the obtained residue is purified to obtain the compound of the formula I. The purification may be carried out by a method conventional in the art, such as beating, crystallization, preparative chromatography or silica gel column chromatography, preferably by column silica gel column chromatography, using an eluent preferably a mixture of methylene chloride and methanol, preferably at an elution gradient of 50:1 to 10:1, further preferably at 50:1 to 15:1.

The methods of preparing the compounds of formula I may further include methods of preparing the compounds of formula II as described herein.

The present invention also provides a compound of formula IV:

wherein R is ¹ As defined for the compounds of formula III hereinbefore.

The invention also provides a preparation method of the compound shown in the formula IV, which comprises the following steps: reacting a compound of formula V with paraformaldehyde and trimethylchlorosilane in a solvent to obtain a compound of formula IV;

wherein R is ¹ As defined for the compounds of formula III hereinbefore.

In the preparation method of the compound of the formula IV, the reaction conditions can be as described above. The process for preparing the compound of formula IV may further comprise a process for preparing a compound of formula V as described herein.

In some embodiments, the compound of formula IV is

The invention also provides a compound of formula V:

wherein R is ¹ As defined for the compounds of formula III hereinbefore.

In some embodiments, the compound of formula V is

The invention also provides a preparation method of the compound shown in the formula V, which comprises the following steps: reacting a compound of formula VI with a sulfonyl azide in the presence of a base and a catalyst in a solvent to obtain the compound of formula V;

Wherein R is ¹ As defined for the compounds of formula III hereinbefore.

In the preparation method of the compound of the formula V, the reaction conditions can be as described above. The methods of preparing the compounds of formula V may further include methods of preparing the compounds of formula VI described herein.

The invention also provides a compound of formula VI:

wherein R is ¹ As defined for the compounds of formula III hereinbefore.

In some embodiments, the compound of formula VI is

The invention also provides a preparation method of the compound shown in the formula VI, which comprises the following steps: carrying out Fmoc removal reaction on a compound of a formula VII in the presence of alkali and an organic solvent to obtain the compound of the formula VI;

wherein R is ¹ As defined for the compounds of formula III hereinbefore.

In the preparation method of the compound of the formula VI, the reaction conditions can be as described above. The methods of preparing the compounds of formula VI may further include methods of preparing the compounds of formula VII described herein.

The invention also provides a preparation method of the compound of formula VII, which comprises the following steps: carrying out coupling reaction on a compound of formula VIII and N-Fmoc-L-valine N-butadiene amine imine ester in a solvent to obtain the compound of formula VII;

Wherein R is ¹ As defined for the compounds of formula III hereinbefore.

In the preparation method of the compound of the formula VII, the reaction conditions can be as described above. The process for preparing the compound of formula VII may further comprise a process for preparing a compound of formula VIII as described herein.

The invention also provides a compound of formula VIII:

wherein R is ¹ As defined for the compounds of formula III hereinbefore.

In some embodiments, the compound of formula VIII is

The invention also provides a preparation method of the compound of the formula VIII, which comprises the following steps: subjecting a compound of formula IX to Fmoc removal in the presence of a base and in a solvent to obtain a compound of formula VIII;

wherein R is ¹ As defined for the compounds of formula III hereinbefore.

In the preparation method of the compound of the formula VIII, the reaction conditions can be as described above. The process for preparing the compound of formula VIII may further comprise a process for preparing a compound of formula IX as described herein.

The invention also provides a compound of formula IX:

wherein R is ¹ As defined for the compounds of formula III hereinbefore.

In some embodiments, the compound of formula IX is

The invention also provides a preparation method of the compound shown in the formula IX, which comprises the following steps: by reacting a compound of formula X with an amino compound R ¹ NH ₂ Carrying out coupling reaction in the presence of alkali and in a solvent to obtain a compound of a formula IX;

wherein R is ¹ As defined for the compounds of formula III hereinbefore.

In the preparation method of the compound of the formula IX, the reaction conditions can be as described above. The process for preparing the compound of formula IX may further comprise a process for preparing a compound of formula X as described herein.

The invention also provides a compound of formula X:

the invention also provides a preparation method of the compound of the formula X, which comprises the following steps: reacting a compound of formula XI with a compound of formula XII in the presence of a base and in a solvent to give a compound of formula X;

in the preparation method of the compound of the formula X, the reaction conditions can be as described above.

Definition of the definition

In the present invention, the term "C1-C6 alkyl" means a saturated straight-chain or branched alkyl group comprising 1 to 6, in particular 1 to 4, carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl and the like, in particular methyl or ethyl.

In the present invention, the term "halogen" means fluorine, chlorine, bromine or iodine, in particular fluorine or chlorine.

TABLE 1 abbreviations

EMCS	6- (Maleimidyl) caproic acid succinimidyl ester
DMF	N, N-dimethylformamide
DMAC	N, N-dimethylacetamide
DMSO	Dimethyl sulfoxide
Fmoc	9-fluorenylmethoxycarbonyl protecting group

TLC	Thin layer chromatography
GC	Gas chromatography
HPLC	High performance liquid chromatography
NMR	Nuclear magnetic resonance
DMAP	4-dimethylaminopyridine
EEDQ	2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline

The above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present invention can be obtained.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that: a novel method for preparing a linker drug conjugate of formula I and intermediates thereof are provided. The preparation method has one or more of the following advantages: simple operation, good yield, easy control of product quality and suitability for industrial production.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

In the following examples, wa was used for mass spectrometryters Acquity Xevo G2-XS QTof UPLC/MS ultra-high performance liquid chromatography and high resolution mass spectrometry combined system, ¹ H-NMR was performed using a Bruker AVANCE III MHz NMR spectrometer or a Bruker AVANCE III HD MHz NMR spectrometer, and HPLC was performed using an Agilent 1260 high performance liquid chromatograph.

Example 1: synthesis method 1 of Compound 1

Step 1: synthesis of Compound 11

Compound 13 (10 g,32.2 mmol) and compound 14 (4 g,32.5mmol,1.0 eq.) are dispersed in 200mL of dichloromethane at room temperature and EEDQ (9.5 g,38.4mmol,1.2 eq.) is added in 3 portions. After the addition, the reaction solution was stirred mechanically at room temperature for 12 hours, and a large amount of white solid was precipitated in the system. After the TLC monitoring of the completion of the reaction of the starting materials, the reaction solution was directly suction-filtered, and the cake was dried to give compound 11 (11 g, yield 82%) as a white solid.

¹ H NMR(400MHz，DMSO-d6)δ9.95(s，1H)，7.88(t，J＝9.9Hz，2H)，7.80-7.65(m，3H)，7.55 (t，J＝7.7Hz，2H)，7.46-7.19(m，6H)，5.20-5.02(m，1H)，4.43(t，J＝7.7Hz，2H)，4.32-4.06(m，4H)，1.41-1.21(m，3H)，MS：m/z＝417.2(M+H)。

Step 2: synthesis of Compound 10

Compound 11 (11 g,26.4 mmol) was dispersed in 200mL of dichloromethane at room temperature and pyridine (4.2 mL,52.8mmol,2.0 eq.) was added. The resulting mixture was cooled to 0℃with an ice bath, and p-nitrophenyl chloroformate (4 times, 8.0g,39.6mmol,1.5 eq.) was added in portions under ice bath incubation, and after the addition was complete, the resulting reaction mixture was stirred at room temperature for 4 hours. After TLC monitoring that the raw materials are completely reacted, filtering the reaction liquid, collecting a filter cake and drying to obtain a 1 st batch of solid; the filtrate is washed with 1N hydrochloric acid, 1N sodium hydroxide (2 times), water and saturated common salt in sequence, dried, concentrated, pulped for 1 hour with methyl tertiary butyl ether, filtered, and the filter cake is collected and dried to obtain the 2 nd batch of solids. The two solid batches were combined to give compound 10 (12 g, 78% yield) as a pale yellow solid.

Step 3: synthesis of Compound 9

Compound 10 (12 g,20.6 mmol) and methylsulfonylethylamine hydrochloride (3.6 g,22.6mmol,1.1 eq.) are dispersed in 200mL of methylene chloride at room temperature and DMAP (6.3 g,51.6mmol,2.5 eq.) is added in 3 portions. The resulting reaction mixture was stirred at room temperature for 12 hours, and a large amount of pale yellow solid was precipitated. After TLC monitoring the completion of the reaction of the starting materials, suction filtration was carried out, and the filter cake was washed 2 times with ethyl acetate (100 mL each) and dried to give a first white solid, 7.6g. The filtrate was dried by spinning, dissolved in 150mL of ethyl acetate, washed successively with 1N hydrochloric acid (50 mL), 1N sodium hydroxide (2 times, 50mL each), water and saturated brine (100 mL), dried and concentrated, slurried with methylene chloride (20 mL), and filtered and dried to give 2g of a 2 nd white solid. The two solid portions were combined to give compound 9 (9.6 g, 82% yield) as a white solid. ¹ H-NMR(400MHz，DMSO-d6)δ10.04(s，1H)，7.88(t，J＝10.8Hz，2H)，7.79-7.66(m，3H)，7.59(d，J＝8.3Hz，2H)，7.51-7.22(m，6H)，4.97(s，2H)，4.37-4.05(m，4H)，3.41(dd，J＝12.8，6.6Hz，2H)，3.30-3.18(m，2H)，2.99(s，3H)，1.31(d，J＝7.1Hz，3H).MS：m/z＝566.2(M+H)。

Step 4: synthesis of Compound 8

Compound 9 (9.6 g,17.0 mmol) was dissolved in 50mL of LDMF at room temperature, and 15mL of diethylamine was added to the solutionThe mixture was stirred at room temperature for 2 hours and TLC monitored complete reaction of starting materials. The reaction solution was concentrated to remove the solvent, and the obtained residue was slurried with methyl tert-butyl ether (50 mL) to give compound 8 (5.2 g, yield 89%) as a white solid. MS: m/z= 344.1 (m+h), ¹ H-NMR(400MHz，DMSO-d ₆ )δ7.63(d，J＝8.5Hz，2H)，7.45(t，J＝5.6Hz，1H)，7.28(d，J＝8.4Hz，2H)，4.97(s，2H)，3.41(qd，J＝7.0，3.2Hz，3H)，3.29-3.18(m，2H)，2.99(s，3H)，1.20(d，J＝6.9Hz，3H)。

Step 5: synthesis of Compound 7

N-Fmoc-L-valine N-butamine imido ester (5 g,11.5 mmol) and amino compound 8 (4 g,11.6 mmol) were dispersed in 100mL DCM at room temperature, and the resulting reaction solution was stirred overnight at 40℃under nitrogen protection, and the system remained white insoluble. Next, 5mL of methanol was added to the reaction system, and stirring was continued at 40℃for 4 hours. The reaction solution was filtered off with suction, and the filter cake was collected and dried to give compound 7 which was used directly in the next step. MS: m/z= 665.3 (m+h), ¹ H-NMR(400MHz，DMSO-d ₆ )δ10.07(d，J＝34.2Hz，1H)，8.16(t，J＝23.9Hz，1H)，7.89(d，J＝7.5Hz，2H)，7.80-7.69(m，2H)，7.58(d，J＝8.4Hz，2H)，7.52-7.37(m，3H)，7.31(dd，J＝17.6，8.0Hz，4H)，4.97(s，2H)，4.42(p，J＝6.8Hz，1H)，4.34-4.17(m，3H)，3.98-3.83(m，1H)，3.41(dd，J＝13.3，6.2Hz，2H)，3.29-3.19(m，2H)，2.99(s，3H)，1.99(dq，J＝13.5，6.7Hz，1H)，1.31(d，J＝7.1Hz，3H)，1.01-0.74(m，6H)。

step 6: synthesis of Compound 6

Compound 7 obtained in step 5 was dissolved in 50mL of ldmf at room temperature, 10mL of diethylamine was added, and the resulting mixture was stirred for 4 hours at room temperature, and TLC monitored complete reaction of the starting materials. At this time, the reaction solution had white insoluble impurities. The impurities were removed by filtration, the filtrate was dried by oil pump, and the pale yellow oily substance was slurried with methyl tert-butyl ether (50 mL) under stirring for about 2 hours, and then filtered to obtain a white solid. The obtained white solid was purified by recrystallization from ethanol to give Compound 6 (5 g, yield in two steps: 97%) as a white solid. MS: m/z= 443.2 (m+h).

Step 5: synthesis of Compound 5

Compound 6 (5.0 g,11.3 mmol) was dispersed in 50mL of methanol, 10mL of dichloromethane and 25mL of water at room temperature, and potassium carbonate (3.1 g,22.4mmol,2.0 eq.) and copper sulfate pentahydrate (0.30 g,1.2mmol,0.1 eq.) were added. After the resulting mixture was clarified by stirring, 1H-imidazole-1-sulfonylazide hydrochloride (CAS: 952234-36-5,2.4g,11.5mmol,1.02 eq.) was added and the resulting reaction solution was stirred at room temperature overnight. After TLC monitored complete reaction of the starting materials, 25mL of water was added and most of the methanol was removed under reduced pressure, and the resulting mixture was extracted 2 times with dichloromethane (50 mL each). The organic phases were combined, washed with saturated brine, dried and recrystallized from 40mL of ethanol and 0.3 g of activated carbon to give Compound 5 (3.3 g, yield 62%) as a white solid. MS: m/z= 469.2 (m+h), ¹ H NMR(400MHz，DMSO)δ10.13(s，1H)，8.52(d，J＝6.9Hz，1H)，7.58(d，J＝8.5Hz，2H)，7.46(t，J＝5.6Hz，1H)，7.29(d，J＝8.4Hz，2H)，4.97(s，2H)，4.44(p，J＝7.0Hz，1H)，3.53-3.37(m，3H)，3.25(t，J＝6.9Hz，2H)，2.99(s，3H)，2.05(dq，J＝13.4，6.7Hz，1H)，1.33(d，J＝7.1Hz，3H)，0.93(dd，J＝10.2，6.7Hz，6H)。

Step 6: synthesis of Compound 4

Compound 5 (150 g,0.32 mol) and paraformaldehyde (29 g, 1.00mol in terms of formaldehyde, 3.1 eq) synthesized according to the method of step 5 were added to 3L of ultra-dry tetrahydrofuran, and trimethylchlorosilane (122 mL,1.24mmol,3.9 eq) was added dropwise to the resulting mixture, and after the addition was completed, the resulting mixture was stirred at room temperature overnight, then the reaction solution was directly filtered, and the resulting filtrate was subjected to evaporation of the solvent under reduced pressure and vacuum-pumped on a high vacuum pump for 2 hours to give a foamy solid compound 4 (directly used in the next step).

Step 7: synthesis of Compound 3

The crude product of compound 4 obtained in step 6 was dissolved with 2L of ultra-dry 1, 4-dioxane, and Dxd (50 g,0.10mol, commercially available) and panobidine (87 mL,0.48 mol) were added thereto, and the resulting mixture was heated to 60℃and stirred for 2 hours. The solvent was distilled off from the resultant reaction solution under reduced pressure, and the crude product was dissolved in 1.5L of methylene chloride, and then the crude solution was washed with 0.1 mol/L of dilute hydrochloric acid, water and saturated brine in this order, and the resultant organic phase was dried over anhydrous sodium sulfate overnight. The dried solution was distilled off to remove the solvent and then subjected to silica gel column chromatography eluting with methylene chloride/methanol=60:1 to 10:1 to give compound 3 (95 g, which was used directly in the next step).

Step 8: synthesis of Compound 2

Compound 3 from step 7 was dissolved in 600mL of tetrahydrofuran, then 400mL of tetrahydrofuran and acetic acid buffer (1000 mL,100 mM) at pH5.0 were added, followed by 1M solution of trimethylphosphine in tetrahydrofuran (290 mL,290 mmol). The resulting mixture was stirred at 0-5℃for 2 hours, and after completion of the reaction, 1000mL of saturated brine was added to the reaction mixture, followed by extraction with 2.5L of methylene chloride. The resulting organic phase was dried over anhydrous sodium sulfate overnight, the solvent was distilled off under reduced pressure, and the crude product was chromatographed on a silica gel column, eluting with dichloromethane/methanol 20:1-10:1 to give compound 2 (50 g, purity 97%, 53% yield based on Dxd).

Step 9: synthesis of Compound 1

Compound 2 (50 g,53.0 mmol) was mixed with compound EMCS (commercially available, 32g,0.104 mmol) and dissolved in 2L dichloromethane and the resulting mixture was stirred overnight at 40 ℃. After the completion of the reaction, the solvent was distilled off under reduced pressure, and the obtained residue was subjected to silica gel column chromatography and eluted with methylene chloride/methanol=50:1 to 15:1 to give compound 1 (39 g, purity 98%, yield 64%). ESI-MS m/z:1141.4 (M+H), ¹ H NMR(400MHz，DMSO)δ9.98(s，1H)，8.55(s，1H)，8.31-8.11(m，1H)，7.88-7.72(m，2H)，7.63-7.50(m，2H)，7.28(s，3H)，6.99(s，2H)，6.51(s，1H)，5.59(s，1H)，5.50-5.32(m，2H)，5.17(s，2H)，4.98(s，2H)，4.85(d，J＝17.3Hz，2H)，4.43-4.33(m，1H)，4.25-4.11(m，1H)，4.03(s，2H)，3.74-3.64(m，2H)，3.20-3.03(m，3H)，3.01-2.81(m，4H)，2.36(s，3H)，2.23-2.09(m，4H)，2.01-1.91(m，1H)，1.90-1.76(m，2H)，1.55-1.39(m，4H)，1.30(d，J＝6.7Hz，3H)，1.22-1.11(m，2H)，0.93-0.77(m，9H)。

EXAMPLE 2 comparison of the purity of the end products of several Process routes

The purity of the final product of compound 1 obtained by synthesis according to example 1 was compared with the purity of the final product of compound 1 obtained by synthesis according to scheme 1 (i.e. synthesis of LE14 in example 7) and scheme 2 (i.e. synthesis of LE14 in example 10) disclosed in WO2020259258A1 by high performance liquid phase, and the results are given in table 3 below.

The liquid phase conditions used were: phase A is 0.1% formic acid aqueous solution, phase B is 0.1% formic acid acetonitrile solution, detection wavelength is 370nm, instrument is Agilent 1260, chromatographic column is ZORBAX Eclipse Plus C, 3.5 μm, 4.6X150 mm. The gradient settings are as follows in table 2.

TABLE 2 gradient setup of mobile phases

Time (min)	Mobile phase A%	Mobile phase B%
0.00	80.0	20.0
10.00	60.0	40.0
25.00	60.0	40.0
35.00	30.0	70.0
41.00	30.0	70.0
41.10	80.0	20.0
43.00	80.0	20.0

TABLE 3 comparison data of the final product purities of the different process routes

Claims

A compound of formula III:

wherein R is ¹ Is C ₁ ～C ₆ Alkyl, one or more R ^1-3 S(O) ₂ -substituted C ₁ ～C ₆ Alkyl, or one or more N (R) ^1-1 )(R ^1-2 ) -substituted C ₁ ～C ₆ An alkyl group;

R ² and R is ³ Each independently is C ₁ ～C ₆ Alkyl, one or more halogen-substituted C ₁ ～C ₆ Alkyl, or halogen;

R ^1-1 、R ^1-2 and R is ^1-3 Each independently is C ₁ ～C ₄ An alkyl group.
A compound of formula III according to claim 1, wherein R ¹ Is R ^1-3 S(O) ₂ -substituted C ₁ ～C ₆ Alkyl groups such as methylsulfonylethyl;

and/or R ² Is C ₁ ～C ₆ Alkyl groups such as methyl;

and/or R ³ Halogen, such as fluorine;

and/or R ^1-3 Is C ₁ ～C ₄ Alkyl groups such as ethyl.
The compound of formula III according to claim 1, wherein the compound of formula III is
A process for the preparation of a compound of formula III according to any one of claims 1 to 3, comprising the steps of: carrying out substitution reaction on a compound of formula IV and a compound of formula IV' in a solvent and in the presence of alkali to obtain a compound of formula III;

wherein R is ¹ 、R ² And R is ³ A method according to any one of claims 1 to 3.
The process for the preparation of a compound of formula III according to claim 4, wherein the molar ratio of the compound of formula IV to the compound of formula IV' is from 2:1 to 4:1, preferably from 3.0:1 to 3.5:1;

and/or, in the preparation method of the compound of the formula III, the alkali is organic alkali, inorganic alkali or a mixture thereof; wherein the organic base is preferably tert-butyl potassium, triethylamine, DMAP, pyridine, pan Bi pyridine or a mixture of any two or more of the above; the inorganic base is preferably alkali metal hydroxide, alkali metal carbonate, alkali metal phosphate or a mixture of any two or more of them, preferably potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate or a mixture of any two or more of them;

And/or, in the process for the preparation of the compound of formula III, the molar ratio of base to compound of formula IV' is from 3:1 to 6:1, preferably from 4.5:1 to 5.5:1;

and/or, in the preparation method of the compound shown in the formula III, the solvent is an ether solvent, a chlorinated alkane solvent, a nitrile solvent or a mixture of any two or more of the solvents; the ether solvent can be tetrahydrofuran, diethyl ether, 1, 4-dioxane, anisole, methyl tertiary butyl ether or the mixture of any two or more of the above; the chlorinated alkane solvent is preferably dichloromethane, dichloroethane, chloroform or a mixture of any two or more of the above; the nitrile solvent is preferably acetonitrile;

and/or, in the preparation method of the compound of the formula III, the temperature of the substitution reaction is 20-80 ℃, preferably 40-60 ℃;

and/or, in the preparation method of the compound of the formula III, the substitution reaction is carried out under anhydrous condition.
The method of preparing a compound of formula III according to claim 4, wherein the method of preparing a compound of formula IV comprises the steps of: reacting a compound of formula V with paraformaldehyde and trimethylchlorosilane in a solvent to obtain a compound of formula IV;

Wherein R is ¹ As claimed in claim 4.
The process for the preparation of a compound of formula III according to claim 6, wherein the molar ratio of paraformaldehyde to a compound of formula V is from 3:1 to 12:1, preferably from 3:1 to 4:1, in terms of formaldehyde;

and/or, in the preparation method of the compound of the formula IV, the molar ratio of the trimethylchlorosilane to the compound of the formula V is 3:1-12:1, preferably 3:1-4:1;

and/or, in the preparation method of the compound of the formula IV, the solvent is an ether solvent, a chlorinated alkane solvent or a mixture thereof; the ether solvent is preferably tetrahydrofuran, diethyl ether, 1, 4-dioxane, anisole, methyl tertiary butyl ether or the mixture of any two or more of the above; the chlorinated alkane solvent is preferably dichloromethane, dichloroethane, chloroform or a mixture of any two or more of the above;

and/or, in the preparation method of the compound of the formula IV, the reaction temperature is 10-40 ℃, preferably 25-40 ℃;

and/or, in the preparation method of the compound of the formula IV, the reaction is carried out under anhydrous condition.
The method of preparing a compound of formula III according to claim 6, wherein the method of preparing a compound of formula V comprises the steps of: reacting a compound of formula VI with a sulfonyl azide in the presence of a base and a catalyst in a solvent to obtain the compound of formula V;

Optionally, the method for preparing the compound of formula VI comprises the steps of: carrying out Fmoc removal reaction on a compound of a formula VII in the presence of alkali and an organic solvent to obtain the compound of the formula VI;

wherein R is ¹ As claimed in claim 6.
A process for the preparation of a compound of formula III according to claim 8, wherein in the process for the preparation of a compound of formula V the sulfonyl azide is 1H-imidazole-1-sulfonyl azide hydrochloride, 2-azido-1, 3-dimethylimidazole hexafluorophosphate, trifluorosulfonyl azide, p-toluenesulfonyl azide or methanesulfonyl azide, preferably 1H-imidazole-1-sulfonyl azide hydrochloride;

and/or, in the preparation method of the compound of the formula V, the molar ratio of the sulfonyl azide compound to the compound of the formula VI is 1.0:1-1.5:1, preferably 1.0:1-1.2:1;

and/or, in the preparation method of the compound of the formula V, the alkali is organic alkali, inorganic alkali or a mixture thereof; wherein the inorganic base is preferably alkali metal hydroxide, alkali metal carbonate, alkali metal phosphate or a mixture of any two or more thereof, preferably potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate or a mixture of any two or more thereof; the organic base is preferably tert-butyl potassium, triethylamine, DMAP, pyridine, panprine, 2, 6-lutidine or a mixture of any two or more of the above;

And/or, in the process for the preparation of the compound of formula V, the molar ratio of base to compound of formula VI is from 1.5:1 to 3.0:1, preferably from 2.0:1 to 2.5:1;

and/or, in the preparation method of the compound of the formula V, the catalyst is a ketone salt, preferably copper sulfate, more preferably copper sulfate pentahydrate; the molar ratio of the ketone salt to the compound of formula VI may be from 0.1:1 to 0.5:1, preferably from 0.1:1 to 0.3:1;

and/or, in the preparation method of the compound shown in the formula V, the solvent is a mixed solvent of an organic solvent and water, and the organic solvent can be an alcohol solvent, a chlorinated alkane solvent, an ether solvent or a mixture of any two or more of the solvents; the alcohol solvent can be methanol, ethanol, isopropanol or a mixture thereof; the chlorinated alkane solvent can be dichloromethane, chloroform, dichloroethane or the mixture of any two or more of the dichloromethane, chloroform, dichloroethane; the ether solvent is preferably tetrahydrofuran, diethyl ether, 1, 4-dioxane, anisole, methyl tertiary butyl ether or the mixture of any two or more of the above;

and/or, in the preparation method of the compound of the formula V, the reaction temperature is 10-40 ℃, preferably 25-40 ℃;

And/or, in the preparation method of the compound of the formula VI, the alkali is an organic alkali, an inorganic alkali or a mixture thereof, preferably an organic alkali; wherein the organic base is preferably diethylamine, tert-butylpotassium, triethylamine, DMAP, pyridine, panpridine, 2, 6-lutidine or a mixture of any two or more of them; the inorganic base is preferably an alkali metal hydroxide, an alkali metal carbonate, an alkali metal phosphate, or a mixture of any two or more thereof, and more preferably potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate, or a mixture of any two or more thereof;

and/or, in the preparation method of the compound of the formula VI, the volume ratio of the alkali to the organic solvent is 0.1:1-0.5:1, preferably 0.2:1-0.3:1;

and/or, in the preparation method of the compound of the formula VI, the organic solvent is DMF, DMSO, tetrahydrofuran, 1, 4-dioxane or a mixture of any two or more of them, preferably DMF;

and/or, in the preparation method of the compound of the formula VI, the Fmoc removal reaction temperature is 10-40 ℃, preferably 25-40 ℃.
The process for the preparation of a compound of formula III according to claim 8, wherein the process for the preparation of a compound of formula VII comprises the steps of: carrying out coupling reaction on a compound of formula VIII and N-Fmoc-L-valine N-butadiene amine imine ester in a solvent to obtain the compound of formula VII;

Optionally, the method for preparing the compound of formula VIII comprises the steps of: subjecting a compound of formula IX to Fmoc removal in the presence of a base and in a solvent to obtain a compound of formula VIII;

optionally, the process for preparing the compound of formula IX comprises the steps of: by reacting a compound of formula X with an amino compound R ¹ NH ₂ Carrying out coupling reaction in the presence of alkali and in a solvent to obtain a compound of a formula IX;

optionally, the preparation method of the compound of the formula X comprises the following steps: reacting a compound of formula XI with a compound of formula XII in the presence of a base and in a solvent to give a compound of formula X;

wherein R is ¹ As claimed in claim 8.
The process according to claim 10, wherein the molar ratio of N-Fmoc-L-valine N-butadienaminimide ester to compound of formula VIII is 0.8:1 to 5:1, preferably 0.8:1 to 1.2:1;

and/or, in the preparation method of the compound of the formula VII, the solvent is DMF, DMSO, acetonitrile, dichloromethane, dichloroethane or a mixture of any two or more of them, preferably dichloromethane;

and/or, in the preparation method of the compound of the formula VII, the temperature of the coupling reaction is 10-40 ℃, preferably 35-40 ℃;

And/or, in the preparation method of the compound of the formula VII, the coupling reaction is carried out under the protection of gas;

and/or, in the preparation method of the compound of the formula VIII, the alkali is an organic alkali, an inorganic alkali or a mixture thereof; wherein the organic base is preferably diethylamine, tert-butylpotassium, triethylamine, DMAP, pyridine, panpridine, 2, 6-lutidine or a mixture of any two or more of them, and further preferably ethylenediamine; the inorganic base is preferably an alkali metal hydroxide, an alkali metal carbonate, an alkali metal phosphate, or a mixture of any two or more thereof, and more preferably potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate, or a mixture of any two or more thereof;

and/or, in the preparation method of the compound of the formula VIII, the solvent is DMF, DMSO, tetrahydrofuran, 1, 4-dioxane or a mixture of any two or more of them, preferably DMF;

and/or, in the preparation method of the compound of the formula VIII, the volume ratio of the alkali to the solvent is 0.2:1-0.5:1, preferably 0.3:1-0.4:1;

and/or, in the preparation method of the compound of the formula VIII, the reaction temperature of Fmoc removal reaction is 10-40 ℃, preferably 25-40 ℃;

And/or, in the preparation method of the compound of the formula IX, the amino compound R ¹ NH ₂ The molar ratio to the compound of formula X is from 1.0:1 to 3.0:1, preferably from 1.1:1 to 1.5:1;

and/or, in the preparation method of the compound of the formula IX, the alkali is an organic alkali, an inorganic alkali or a mixture thereof; wherein the organic base is preferably diethylamine, tert-butylpotassium, triethylamine, DMAP, pyridine, panpridine, 2, 6-lutidine or a mixture of any two or more thereof, and further preferably DMAP; the inorganic base is preferably an alkali metal hydroxide, an alkali metal carbonate, an alkali metal phosphate, or a mixture of any two or more thereof, and more preferably potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate, or a mixture of any two or more thereof;

and/or, in the process for the preparation of the compound of formula IX, the molar ratio of base to compound of formula X is from 2.0:1 to 4.0:1, preferably from 2.5:1 to 3.0:1;

and/or, in the preparation method of the compound of the formula IX, the solvent is DMF, DMSO, dichloromethane, dichloroethane, tetrahydrofuran, 1, 4-dioxane or a mixture of any two or more of them, preferably dichloromethane;

and/or, in the preparation method of the compound of the formula IX, the coupling reaction temperature is 10-40 ℃, preferably 25-40 ℃;

And/or, in the preparation method of the compound of the formula X, the molar ratio of the compound of the formula XII to the compound of the formula XI is 3.0:1-1.2:1, preferably 2.0:1-1.5:1;

and/or, in the preparation method of the compound of the formula X, the alkali is organic alkali, inorganic alkali or a mixture thereof; wherein the organic base is preferably diethylamine, tert-butylpotassium, triethylamine, DMAP, pyridine, panpridine, 2, 6-lutidine or a mixture of any two or more thereof, and more preferably pyridine; the inorganic base is preferably an alkali metal hydroxide, an alkali metal carbonate, an alkali metal phosphate or a mixture thereof, and more preferably potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate or a mixture of any two or more thereof;

and/or, in the process for the preparation of the compound of formula X, the molar ratio of base to compound of formula XI is from 1.0:1 to 4.0:1, preferably from 2.0:1 to 3.0:1;

and/or, in the preparation method of the compound of the formula X, the solvent is DMF, DMSO, dichloromethane, dichloroethane, tetrahydrofuran, 1, 4-dioxane or a mixture of any two or more of them, preferably dichloromethane;

and/or, in the preparation method of the compound of the formula X, the reaction temperature is 10-40 ℃, preferably 25-40 ℃.
A process for the preparation of a compound of formula II comprising the steps of: carrying out reduction reaction on a compound of the formula III and a reducing agent in an organic solvent and in the presence of an acid buffer solution to obtain the compound of the formula II;

wherein R is ¹ 、R ² And R is ³ A method according to any one of claims 1 to 3.
A process for the preparation of a compound of formula I comprising the steps of:

1) A compound of formula II prepared according to the process for the preparation of a compound of formula II as claimed in claim 12;

2) Coupling the compound of the formula II prepared in the step 1) and 6- (maleimide) caproic acid succinimidyl ester in a solvent to obtain the compound of the formula I;

wherein R is ¹ 、R ² And R is ³ The method of claim 12.
A compound of formula II, IV, V, VI, VIII, IX or X;

wherein R is ¹ 、R ² And R is ³ A method according to any one of claims 1 to 3;

provided that, in the compounds of formula II, when R ¹ Is methyl, methylsulfonylethyl or dimethylethyl, R ² When methyl, R ³ Not be ofFluorine.
The compound of claim 14, wherein the compound of formula IV is

And/or the compound of formula V is

And/or the compound of formula VI is

And/or, the compound of formula VIII is

And/or, the compound of formula IX is
A process for the preparation of a compound of formula IV, V, VI, VII, VIII, IX or X, wherein the process for the preparation of a compound of formula IV comprises the steps of: reacting a compound of formula V with paraformaldehyde and trimethylchlorosilane in a solvent to obtain a compound of formula IV;

the preparation method of the compound of the formula V comprises the following steps: reacting a compound of formula VI with a sulfonyl azide in the presence of a base and a catalyst in a solvent to obtain the compound of formula V;

the preparation method of the compound of the formula VI comprises the following steps: carrying out Fmoc removal reaction on a compound of a formula VII in the presence of alkali and an organic solvent to obtain the compound of the formula VI;

the preparation method of the compound of the formula VII comprises the following steps: carrying out coupling reaction on a compound of formula VIII and N-Fmoc-L-valine N-butadiene amine imine ester in a solvent to obtain the compound of formula VII;

the preparation method of the compound of the formula VIII comprises the following steps: subjecting a compound of formula IX to Fmoc removal in the presence of a base and in a solvent to obtain a compound of formula VIII;

a process for the preparation of a compound of formula IX comprising the steps of: by reacting a compound of formula X with an amino compound R ¹ NH ₂ Carrying out coupling reaction in the presence of alkali and in a solvent to obtain a compound of a formula IX;

A process for the preparation of a compound of formula X comprising the steps of: reacting a compound of formula XI with a compound of formula XII in the presence of a base and in a solvent to give a compound of formula X;

wherein R is ¹ Is C ₁ ～C ₆ Alkyl, one or more R ^1-3 S(O) ₂ -substituted C ₁ ～C ₆ Alkyl, or one or more N (R) ^1-1 )(R ^1-2 ) -substituted C ₁ ～C ₆ An alkyl group;

R ² and R is ³ Each independently is C ₁ ～C ₆ Alkyl, one or more halogen-substituted C ₁ ～C ₆ Alkyl, or halogen;

R ^1-1 、R ^1-2 and R is ^1-3 Each independently is C ₁ ～C ₄ An alkyl group.