A method for preparing a linker-drug conjugate and intermediates thereof

Abstract

The application discloses a preparation method of a linker drug conjugate and an intermediate thereof, and particularly discloses a preparation method of a compound of formula (I), wherein R 1 is C1-C6 alkyl, one or more R 1‑3 S(O)2-substituted C1-C6 alkyl, or one or more N(R 1‑1 )(R 1‑2 )-substituted C1-C6 alkyl; R 2 and R 3 are each independently C1-C6 alkyl, one or more halogen-substituted C1-C6 alkyl, or halogen; R 1‑1 , R 1‑2 and R 1‑3 are each independently C1-C4 alkyl. The preparation method has one or more advantages of simple operation, good yield, easy control of product quality and suitability for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of drug synthesis, specifically relating to a method for preparing a linker drug conjugate and its intermediates. Background Technology

[0002] Antibody-drug conjugates (ADCs) have become a hot topic in the pharmaceutical industry in recent years. Due to the unsatisfactory clinical efficacy of many antibody drugs, many industry giants are increasingly turning their attention to ADCs. The basic components of an ADC include an antibody, a linker, and an effector molecule. The antibody delivers the effector molecule to the tumor site for accumulation, thereby killing tumor cells. Traditional effector molecules are mostly highly active microtubule inhibitors, which often have significant toxic side effects, limiting the application of ADCs. Recently, Immunomedics invented a novel ADC drug, IMMU-132 (ZL200980156218), using camptothecin as the effector molecule, which has shown good anti-tumor effects. Daiichi Sankyo invented another ADC drug, DS-8201a (ZL201380053256), also using camptothecin as the effector molecule, which has similarly shown good anti-tumor effects.

[0003] WO2020259258A1 discloses an ADC compound with a camptothecin derivative Dxd as the effector molecule, which also exhibits good antitumor effects. The target ADC compound can be obtained by conjugating the camptothecin derivative shown in Formula I with an antibody, wherein the linker drug conjugate shown in Formula I can be prepared via either synthetic route 1 or synthetic route 2.

[0004] Route 1:

[0005]

[0006] The synthetic method of Route 1 includes: reacting compound 1-1 with 4-aminobenzyl alcohol, reacting the resulting compound with di(p-nitrobenzene) carbonate and then with a substituted alkylamine to obtain compound 1-2, reacting compound 1-2 with paraformaldehyde and trimethylchlorosilane to obtain compound 1-3, reacting compound 1-3 with tert-butyl glycolate and then removing the tert-butyl group under the action of trifluoroacetic acid to obtain compound 1-4, reacting compound 1-4 with Exatecan methanesulfonate to obtain compound 1-5, removing the Fmoc protection on the amino group under the action of DBU, and then undergoing a coupling reaction with succinimide 6-(maleimide)hexanoate to obtain target compound I.

[0007] Route 2:

[0008]

[0009] The synthetic method of Route 2 includes: reacting compound 2-1 with paraformaldehyde and trimethylchlorosilane, reacting the resulting compound with tert-butyl glycolate to obtain compound 2-2, removing the tert-butyl group from compound 2-2 in the presence of trifluoroacetic acid, and then reacting it with Exatecan methanesulfonate to obtain compound 2-3, which is then reduced by triethylphosphine.

[0010] The amino group was formed to give compound 2-4, and compound 2-4 was coupled with MC-V to give target compound I.

[0011] However, in actual production scale-up, the synthesis methods of Route 1 and Route 2 still suffer from unacceptable purity of the final product when purified by column chromatography. Therefore, it is necessary to explore a new route to ensure that the purity of the final product meets the requirements. Summary of the Invention

[0012] The technical problem to be solved by this invention is that it is difficult to obtain a final product with qualified purity using existing methods for preparing linker-based drug conjugates of Formula I. This invention provides a new method for preparing linker-based drug conjugates of Formula I and its intermediates. The preparation method of this invention has one or more of the following advantages: simple operation, good yield, easy control of product quality, and suitability for industrial production.

[0013] This invention provides a compound of formula III:

[0014]

[0015] Among them, R 1 It is a C1 to C6 alkyl group, or one or more R 1-3 S(O)2-substituted C1-C6 alkyl group, or one or more N(R) 1-1 (R) 1-2 )-substituted C1- to C6 alkyl groups;

[0016] R 2 and R 3 Each is independently a C1-C6 alkyl group, a C1-C6 alkyl group substituted with one or more halogens, or a halogen;

[0017] R 1-1 R 1-2 and R 1-3 Each is independently a C1 to C4 alkyl group.

[0018] In some implementation schemes, R 1 For an R 1-3 S(O)2-substituted C1-C6 alkyl groups;

[0019] R 2 It is a C1 to C6 alkyl group;

[0020] R 3 It is a halogen;

[0021] R 1-3 It is a C1 to C4 alkyl group.

[0022] In some implementations, in R 1 R 2 and R 3 In the definition, the C1 to C6 alkyl group can be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl or ethyl.

[0023] In some implementations, in R 1 R 2 and R 3 In the definition, the halogen can be fluorine, chlorine, bromine or iodine, preferably fluorine.

[0024] In some implementations, in R 1-1 R 1-2 and R 1-3 In the definition, the C1 to C4 alkyl group can be methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably methyl.

[0025] In some implementation schemes, R 1 For an R 1-3 S(O)2-substituted C1-C6 alkyl group or one -NR 1-1 R 1-2 The substituted C1 to C6 alkyl group is preferably methyl sulfone ethyl or N,N-dimethyl ethyl, more preferably methyl sulfone ethyl.

[0026] In some implementation schemes, R 2 It is a C1 to C6 alkyl group, preferably methyl.

[0027] In some implementation schemes, R 3 It is a halogen, preferably fluorine or chlorine, and more preferably fluorine.

[0028] In some implementation schemes, R 1 For an R 1-3 S(O)2-substituted C1-C6 alkyl groups, such as methyl sulfone ethyl;

[0029] R 2 It is a C1 to C6 alkyl group, such as methyl;

[0030] R 3 Halogens, such as fluorine;

[0031] R 1-3 It is a C1 to C4 alkyl group, such as ethyl.

[0032] In some implementation schemes, R 1 As defined in any of the aforementioned schemes, R 2 It is methyl, and R 3 It is fluorine.

[0033] In some implementation schemes, R 1 It is methyl sulfone ethyl, R 2 It is methyl, and R 3 It is fluorine.

[0034] In some embodiments, the compound of formula III is

[0035]

[0036] The present invention also provides a method for preparing a compound of formula III, comprising the following steps: subjecting a compound of formula IV and a compound of formula IV' to a substitution reaction in a solvent and in the presence of a base to obtain the compound of formula III;

[0037]

[0038] Among them, R 1 R 2 and R 3 As mentioned above.

[0039] In some embodiments, in the method for preparing the compound of formula III, preferably, R 2 It is methyl, R 3 It is fluorine.

[0040] In some embodiments, in the method for preparing the compound of formula III, the molar ratio of the compound of formula IV to the compound of formula IV' can be 1:1-5:1, preferably 2:1-4:1, and more preferably 3.0:1-3.5:1.

[0041] In some embodiments, in the method for preparing the compound of formula III, the base can be a base conventional to such reactions in the art, such as an organic base, an inorganic base, or a mixture thereof, preferably an organic base; wherein, the organic base is preferably potassium tert-butyl, triethylamine, DMAP, pyridine, panpididine, or a mixture of any two or more thereof, more preferably panpididine; 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. In some embodiments, in the method for preparing the compound of formula III, the base is panpididine.

[0042] In some embodiments, in the method for preparing the compound of formula III, the molar ratio of the base to the compound of formula IV' can be 3:1-6:1, preferably 4.5:1-5.5:1, and more preferably 4.8:1.

[0043] In some embodiments, in the method for preparing the compound of formula III, the solvent can be a solvent conventional to such reactions in the art, preferably an aprotic organic solvent, such as ether solvents, chloroalkane solvents, nitrile solvents, or any mixture of two or more thereof, preferably an ether solvent; the ether solvent can be tetrahydrofuran, diethyl ether, 1,4-dioxane, anisole, methyl tert-butyl ether, or any mixture of two or more thereof, preferably 1,4-dioxane; the chloroalkane solvent is preferably dichloromethane, dichloroethane, chloroform, or any mixture of two or more thereof; the nitrile solvent is preferably acetonitrile. In some embodiments, in the method for preparing the compound of formula III, the solvent is 1,4-dioxane.

[0044] In some embodiments, the temperature of the substitution reaction in the preparation method of the compound of formula III can be a conventional temperature for such reactions in the art, for example, 20-80°C, preferably 40-60°C, and more preferably 60°C.

[0045] In some embodiments, the substitution reaction in the method for preparing the compound of formula III can be a conventional operation of such reactions in the art, for example including the following steps: stirring the mixture of the compound of formula IV, the compound of formula IV', the solvent and the base to carry out the substitution reaction.

[0046] In some embodiments, the process of the substitution reaction in the preparation method of the compound of formula III can be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC, or NMR), and the reaction endpoint is generally defined as the absence of detectable compound of formula IV or formula IV'. The reaction time of the substitution reaction can be 2 to 12 hours, more preferably 2 to 3 hours, and even more preferably 2 hours.

[0047] In some embodiments, the method for preparing the compound of formula III may further include the following post-processing steps after the substitution reaction: removing the solvent from the reaction solution, washing the organic phase, and purifying the residue of the washed organic phase after removing the solvent. The organic phase used in the washing process may be ethyl acetate or dichloromethane, preferably dichloromethane. The aqueous phase used in the washing process may be an aqueous solution of an acid, water, and / or saturated saline solution; the aqueous solution of the acid may be 0.1N hydrochloric acid, 0.05N sulfuric acid, or a mixture thereof, preferably 0.1N hydrochloric acid. Preferably, the washing process may include washing the organic phase sequentially with an aqueous solution of an acid, water, and saturated saline solution, preferably sequentially with 0.1N dilute acid, water, and saturated saline solution. In some embodiments, the purification may employ conventional purification methods in the art, such as pulping, crystallization, preparative chromatography, or silica gel column chromatography, preferably silica gel column chromatography. The eluent used is preferably a mixture of dichloromethane and methanol, and the elution gradient is preferably 100:1-10:1, more preferably 60:1-10:1.

[0048] In some embodiments, the substitution reaction is preferably carried out under anhydrous conditions in the preparation method of the compound of formula III.

[0049] The method for preparing the compound of formula III may further include a method for preparing the compound of formula IV, which may include the following steps: reacting the compound of formula V with paraformaldehyde and trimethylchlorosilane in a solvent to obtain the compound of formula IV;

[0050]

[0051] R1 is as described above.

[0052] In some embodiments, in the method for preparing the compound of formula IV, the molar ratio of paraformaldehyde to the compound of formula V, converted from formaldehyde, is 3:1-12:1, preferably 3:1-4:1, and more preferably 3.1:1.

[0053] In some embodiments, in the method for preparing the compound of formula IV, the molar ratio of trimethylchlorosilane to the compound of formula V is 3:1-12:1, preferably 3:1-4:1, and more preferably 3.9:1.

[0054] In some embodiments, in the method for preparing the compound of formula IV, the solvent can be a solvent conventional to such reactions in the art, preferably an aprotic organic solvent, such as ether solvents, chloroalkane solvents, or mixtures thereof, preferably ether solvents; the ether solvent can be tetrahydrofuran, diethyl ether, 1,4-dioxane, anisole, methyl tert-butyl ether, or a mixture of any two or more thereof, preferably tetrahydrofuran; the chloroalkane solvent is preferably dichloromethane, dichloroethane, chloroform, or a mixture of any two or more thereof. In some embodiments, in the method for preparing the compound of formula IV, the solvent is tetrahydrofuran.

[0055] In some embodiments, the reaction temperature in the preparation method of the compound of formula IV can be a conventional temperature for such reactions in the art, for example, 10-40°C, preferably 25-40°C, and more preferably 25-30°C.

[0056] In some embodiments, the reaction is preferably carried out under anhydrous conditions in the method for preparing the compound of formula IV.

[0057] In some embodiments, the method for preparing the compound of formula IV may be carried out in accordance with conventional procedures for such reactions in the art, for example, including the following steps: adding trimethylchlorosilane in batches (e.g., dropwise) to a mixture of the compound of formula IV, paraformaldehyde and solvent, and stirring to carry out the reaction.

[0058] In some embodiments, the reaction process in the preparation method of the compound of formula IV can be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC, or NMR), and the reaction endpoint is generally defined as the point at which the compound of formula V is no longer detected. The reaction time can be 1 to 24 hours, preferably 12 to 20 hours, more preferably 16 to 20 hours, and even more preferably 16 hours.

[0059] In some embodiments, the preparation method of the compound of formula IV may further include the following post-processing steps after the substitution reaction is completed: solid-liquid separation of the reaction liquid, removal of solvent from the obtained liquid phase, and direct use of the obtained residue for the next reaction.

[0060] The method for preparing compound IV may further include a method for preparing compound V, which may include the following steps: reacting compound VI with sulfonyl azide compound in a solvent in the presence of a base and a catalyst to obtain compound V;

[0061]

[0062] Among them, R 1 As mentioned above.

[0063] In some embodiments, in the method for preparing the compound of formula V, the sulfonyl azide compound may be 1H-imidazolium-1-sulfonyl azide hydrochloride, 2-azido-1,3-dimethylimidazolium hexafluorophosphate, trifluorosulfonyl azide, p-methylbenzenesulfonyl azide, or methanesulfonyl azide, preferably 1H-imidazolium-1-sulfonyl azide hydrochloride.

[0064] In some embodiments, in the method for preparing the compound of formula V, the molar ratio of the sulfonyl azide compound to the compound of formula VI is 1.0∶1-1.5∶1, preferably 1.0∶1-1.2∶1, and more preferably 1.02∶1.

[0065] In some embodiments, in the method for preparing the compound of formula V, the base can be a base conventional for such reactions in the art, such as an organic base, an inorganic base, or a mixture 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, and even more preferably potassium carbonate; the organic base is preferably tert-butylpotassium, triethylamine, DMAP, pyridine, panpididine, 2,6-dimethylpyridine, or a mixture of any two or more thereof. In some embodiments, in the method for preparing the compound of formula V, the base is an alkali metal carbonate, such as potassium carbonate.

[0066] In some embodiments, in the method for preparing compound V, the molar ratio of the base to compound VI is 1.5:1-3.0:1, preferably 2.0:1-2.5:1, and more preferably 2.0:1.

[0067] In some embodiments, in the method for preparing the compound of formula V, the catalyst can be a conventional catalyst for such reactions in the art, such as a ketone salt, preferably copper sulfate, and more preferably copper sulfate pentahydrate.

[0068] In some embodiments, in the method for preparing the compound of formula V, the molar ratio of the ketone salt and the compound of formula VI can be 0.1∶1-0.5∶1, preferably 0.1∶1-0.3∶1, more preferably 0.1∶1-0.2∶1, and most preferably 0.1∶1.

[0069] In some embodiments, in the method for preparing the compound of formula V, the solvent can be a solvent conventional to such reactions in the art, preferably a mixture of an organic solvent and water. The organic solvent can be an alcohol solvent, a chloroalkane solvent, an ether solvent, or a mixture of any two or more thereof, preferably a mixture of an alcohol solvent and a chloroalkane solvent. The alcohol solvent can be methanol, ethanol, isopropanol, or a mixture thereof, preferably methanol. The chloroalkane solvent can be dichloromethane, chloroform, dichloroethane, or a mixture of any two or more thereof, preferably dichloromethane. The ether solvent is preferably tetrahydrofuran, diethyl ether, 1,4-dioxane, anisole, methyl tert-butyl ether, or a mixture of any two or more thereof. In some embodiments, in the method for preparing the compound of formula IV, the solvent is a mixture of methanol, dichloromethane, and water.

[0070] In some embodiments, the reaction temperature in the preparation method of the compound of formula V can be a conventional temperature for such reactions in the art, for example, 10 to 40°C, preferably 25 to 40°C, and more preferably 25 to 30°C.

[0071] In some embodiments, the method for preparing compound V may be carried out using conventional methods for such reactions in the art, for example, including the following steps: adding the sulfonyl azide compound to a mixture of compound VI, base, catalyst and solvent (preferably, the sulfonyl azide compound is added after the mixture has been clarified), and stirring to carry out the reaction.

[0072] In some embodiments, the reaction process in the preparation method of compound V can be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC, or NMR), and the reaction endpoint is generally defined as the point at which compound VI is no longer detected. The reaction time can be 1–24 hours, preferably 12–20 hours, more preferably 16–20 hours, and even more preferably 16 hours.

[0073] In some embodiments, the preparation method of the compound of formula V may further include the following post-processing steps after the reaction is completed: removing the organic solvent from the reaction solution, extraction (e.g., extraction with dichloromethane), and recrystallizing the organic phase obtained from the extraction with ethanol and activated carbon to obtain the compound of formula V.

[0074] The method for preparing compound V may further include a method for preparing compound VI, which may include the following steps: performing a de-Fmoc reaction on compound VII in the presence of a base and an organic solvent to obtain compound VI;

[0075]

[0076] Among them, R 1 As mentioned above.

[0077] In some embodiments, in the method for preparing the compound of formula VI, the base can be a base conventional for such reactions in the art, such as an organic base, an inorganic base, or a mixture thereof, preferably an organic base; wherein, the organic base is preferably diethylamine, potassium tert-butyl, triethylamine, DMAP, pyridine, panpididine, 2,6-dimethylpyridine, or a mixture of any two or more thereof, more 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, more preferably potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate, or a mixture of any two or more thereof. In some embodiments, in the method for preparing the compound of formula VI, the base is diethylamine.

[0078] In some embodiments, in the method for preparing the compound of formula VI, the volume ratio of the base to the organic solvent can be 0.1∶1-0.5∶1, preferably 0.2∶1-0.3∶1, and more preferably 0.2∶1.

[0079] In some embodiments, in the method for preparing 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.

[0080] In some embodiments, the temperature of the deFmoc reaction in the preparation method of the compound of formula VI can be a conventional temperature for such reactions in the art, for example, 10 to 40°C, preferably 25 to 40°C, and more preferably 25 to 30°C.

[0081] In some embodiments, the de-Fmoc reaction in the preparation method of the compound of formula VI can be a conventional operation of such reactions in the art, for example including the following steps: stirring a mixture of the compound of formula VII, a base and an organic solvent to carry out the de-Fmoc reaction.

[0082] In some embodiments, the progress of the deFmoc reaction in the preparation method of the compound of formula VI can be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC, or NMR), and the reaction endpoint is generally defined as the absence of detectable compound of formula VII. The reaction time can be 1 to 24 hours, preferably 4 to 12 hours, more preferably 4 to 6 hours, and even more preferably 4 hours.

[0083] In some embodiments, the method for preparing compound VI may further include the following post-processing step after the reaction: solid-liquid separation of the reaction solution to remove the solvent from the resulting liquid phase to obtain a crude product. The post-processing step may further include the following step: pulping the crude product to obtain a solid product of compound VI. The solvent used for pulping 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 include the following step: recrystallizing and purifying the solid product of compound VI obtained from pulping. The solvent used for recrystallization may be an alcohol solvent, such as methanol, ethanol, isopropanol, or a mixture of any two or more thereof, preferably ethanol.

[0084] The preparation method of the compound of formula VI may further include a preparation method of the compound of formula VII, which may include the following steps: coupling the compound of formula VIII with N-Fmoc-L-valine N-butadieneamine imine ester in a solvent to obtain the compound of formula VII;

[0085]

[0086] Among them, R 1 As mentioned above.

[0087] In some embodiments, in the method for preparing the compound of formula VII, the molar ratio of the N-Fmoc-L-valine N-butadieneamine imine ester to the compound of formula VIII can be 0.8:1-5:1, preferably 0.8:1-1.2:1, and more preferably 1:1.

[0088] In some embodiments, in the method for preparing the compound of formula VII, the solvent may be DMF, DMSO, acetonitrile, dichloromethane, dichloroethane, or a mixture of any two or more thereof, preferably dichloromethane.

[0089] In some embodiments, the coupling reaction temperature in the preparation method of the compound of formula VII can be a conventional temperature for such reactions in the art, for example, 10 to 40°C, preferably 35 to 40°C, and more preferably 40°C.

[0090] In some embodiments, the coupling reaction is preferably carried out under gas protection in the method for preparing the compound of formula VII. The gas in the gas protection does not participate in the reaction, such as argon, helium, or nitrogen, for example, nitrogen.

[0091] In some embodiments, the coupling reaction in the preparation method of the compound of formula VII can be a conventional operation of such reactions in the art, for example including the following steps: carrying out a coupling reaction of a mixture of compound of formula VIII, N-Fmoc-L-valine N-butadieneamine imine ester and solvent by stirring.

[0092] In some embodiments, the process of the coupling reaction in the preparation method of the compound of formula VII can be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC, or NMR), and the reaction endpoint is generally defined as the point at which the compound of formula VIII is no longer detected. The reaction time of the coupling reaction can be 1-24 hours, preferably 12-20 hours, more preferably 16-20 hours, and even more preferably 16 hours.

[0093] In some embodiments, the preparation method of the compound of formula VII may further include a post-processing step after the coupling reaction is completed. The post-processing step may include: adding an alcohol solvent (e.g., methanol, ethanol, isopropanol or a mixture of any two or more thereof, preferably methanol) to the reaction system, stirring (the stirring temperature may be 20-40°C, preferably 35-40°C, more preferably 40°C; the stirring time may be 1-24 hours, preferably 4-12 hours, more preferably 4-6 hours, and even more preferably 4 hours), and separating the solid in the system to obtain the compound of formula VII.

[0094] The method for preparing compound VII may further include a method for preparing compound VIII, which may include the following steps: performing a deFmoc reaction on compound IX in the presence of a base and a solvent to obtain compound VIII;

[0095]

[0096] Among them, R 1 As mentioned above.

[0097] In some embodiments, in the method for preparing the compound of formula VIII, the base can be a base conventional for such reactions in the art, such as an organic base, an inorganic base, or a mixture thereof, preferably an organic base; wherein, the organic base is preferably diethylamine, potassium tert-butyl, triethylamine, DMAP, pyridine, panpididine, 2,6-dimethylpyridine, or a mixture of any two or more thereof, more 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, more preferably potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate, or a mixture of any two or more thereof. In some embodiments, in the method for preparing the compound of formula VIII, the base is diethylamine.

[0098] 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.

[0099] In some embodiments, in the method for preparing the compound of formula VIII, the volume ratio of the base to the solvent can be 0.2∶1-0.5∶1, preferably 0.3∶1-0.4∶1, and more preferably 0.3∶1.

[0100] In some embodiments, the reaction temperature of the deFmoc reaction in the preparation method of the compound of formula VIII can be a conventional temperature for such reactions in the art, for example, 10-40°C, preferably 25-40°C, and more preferably 25-30°C.

[0101] In some embodiments, the de-Fmoc reaction in the preparation method of the compound of formula VIII can be a conventional operation of such reactions in the art, for example including the following steps: stirring a mixture of compound of formula IX, base and solvent to carry out the de-Fmoc reaction.

[0102] In some embodiments, the progress of the de-Fmoc reaction in the preparation method of the compound of formula VIII can be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC, or NMR), and the reaction endpoint is generally defined as the absence of detectable compound of formula VII. The reaction time can be 1-24 hours, preferably 2-12 hours, more preferably 2-6 hours, and even more preferably 2 hours.

[0103] In some embodiments, the method for preparing the compound of formula VIII may further include a post-processing step after the Fmoc removal reaction. This post-processing step may include removing the solvent from the reaction solution, and the resulting residue being pulped to obtain a solid, which is the compound of formula VIII. The solvent used for pulping 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.

[0104] The method for preparing compound VIII may further include a method for preparing compound IX, which may include the following steps: coupling compound X with amino compound R1NH2 in the presence of a base and in a solvent to obtain compound IX;

[0105]

[0106] Among them, R 1As mentioned above.

[0107] In some embodiments, in the method for preparing the compound of formula IX, the amino compound R 1 The molar ratio of NH2 to compound X can be 1.0∶1-3.0∶1, preferably 1.1∶1-1.5∶1, and more preferably 1.1∶1.

[0108] In some embodiments, in the method for preparing the compound of formula IX, the base can be a base conventional for such reactions in the art, such as an organic base, an inorganic base, or a mixture thereof, preferably an organic base; wherein, the organic base is preferably diethylamine, potassium tert-butyl, triethylamine, DMAP, pyridine, panpididine, 2,6-dimethylpyridine, or a mixture of any two or more thereof, more 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, more preferably potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate, or a mixture of any two or more thereof. In some embodiments, in the method for preparing the compound of formula IX, the base can be DMAP.

[0109] In some embodiments, in the method for preparing the compound of formula IX, the molar ratio of the base to the compound of formula X can be 2.0∶1-4.0∶1, preferably 2.5∶1-3.0∶1, and more preferably 2.5∶1.

[0110] In some embodiments, in the preparation method of the compound of formula IX, the solvent can be DMF, DMSO, dichloromethane, dichloroethane, tetrahydrofuran, 1,4-dioxane, or a mixture of any two or more thereof, preferably dichloromethane.

[0111] In some embodiments, the coupling reaction temperature in the preparation method of the compound of formula IX can be a conventional temperature for such reactions in the art, for example, 10-40°C, preferably 25-40°C, and more preferably 25-30°C.

[0112] In some embodiments, the coupling reaction in the method for preparing the compound of formula IX can be a conventional operation of such reactions in the art, for example including the following steps: stirring the compound X, amino compound R... 1 A coupling reaction is carried out in a mixed system of NH2, base and solvent.

[0113] In some embodiments, the process of the coupling reaction in the preparation method of the compound of formula IX can be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC, or NMR), and the reaction endpoint is generally defined as the point at which the compound of formula VII is no longer detected. The reaction time can be 1-24 hours, preferably 12-20 hours, more preferably 12-16 hours, and even more preferably 12 hours.

[0114] In some embodiments, the preparation method of the compound of formula IX may further include a post-processing step after the coupling reaction. This post-processing step may include: performing solid-liquid separation on the reaction solution, and washing the solid obtained after 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) as a portion of the compound of formula IX. The post-processing step may further include: extracting and washing the liquid phase obtained from the solid-liquid separation, removing the solvent from the obtained organic phase, and then slurrying the resulting solid to obtain another portion of the compound of formula IX. The extraction and washing process can use an aqueous solution of acid, an aqueous solution of alkali, water, and a saturated saline solution, preferably using an aqueous solution of acid, an aqueous solution of alkali, water, and a saturated saline solution sequentially; more preferably, the process involves washing once with an aqueous solution of acid, twice with an aqueous solution of alkali, once with water, and once with saturated saline solution. The aqueous solution of acid can be an aqueous solution of hydrochloric acid, an aqueous solution of sulfuric acid, or an aqueous solution of phosphoric acid, preferably 1N hydrochloric acid, 0.5N sulfuric acid, or 0.33N phosphoric acid, more preferably 1N hydrochloric acid; the aqueous solution of alkali can be an aqueous solution of sodium hydroxide, an aqueous solution of potassium hydroxide, or a mixture thereof, preferably 1N sodium hydroxide, 1N potassium hydroxide, or a mixture thereof, more preferably 1N sodium hydroxide. The solvent for pulping can be dichloromethane, ethyl acetate, or a mixture thereof, preferably dichloromethane; the pulping temperature can be 10-40℃, preferably 25-40℃, more preferably 25-30℃.

[0115] The method for preparing compound IX may further include a method for preparing compound X, which may include the following steps: reacting compound XI and compound XII in a base and a solvent to obtain compound X;

[0116]

[0117] In some embodiments, in the method for preparing compound X, the molar ratio of compound XII to compound XI can be 3.0∶1-1.2∶1, preferably 2.0∶1-1.5∶1, and more preferably 1.5∶1.

[0118] In some embodiments, in the method for preparing the compound of formula X, the base can be a base conventional to such reactions in the art, such as an organic base, an inorganic base, or a mixture thereof, preferably an organic base; wherein, the organic base is preferably diethylamine, potassium tert-butyl, triethylamine, DMAP, pyridine, panpididine, 2,6-dimethylpyridine, or a mixture of any two or more thereof, more preferably pyridine; the inorganic base is preferably an alkali metal hydroxide, an alkali metal carbonate, an alkali metal phosphate, or a mixture thereof, more preferably potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate, or a mixture of any two or more thereof. In some embodiments, in the method for preparing the compound of formula X, the base is pyridine.

[0119] In some embodiments, in the method for preparing compound X, the molar ratio of the base to compound XI can be 1.0∶1-4.0∶1, preferably 2.0∶1-3.0∶1, and more preferably 2.0∶1.

[0120] In some embodiments, in the method for preparing compound X, the solvent may be DMF, DMSO, dichloromethane, dichloroethane, tetrahydrofuran, 1,4-dioxane, or a mixture of any two or more thereof, preferably dichloromethane.

[0121] In some embodiments, the reaction temperature in the preparation method of the compound of formula X can be a conventional temperature for such reactions in the art, for example, 10 to 40°C, preferably 25 to 40°C, and more preferably 25 to 30°C.

[0122] In some embodiments, the preparation method of the compound of formula X can be carried out by conventional operations of such reactions in the art, for example, including the following steps: adding the compound of formula XII in batches (which can be divided into 6-3 batches, preferably 5-4 batches) to a mixture of the compound of formula XI, base and solvent (the temperature of the mixture can be controlled at 0-20°C, preferably 10-0°C, and more preferably 0-5°C during the addition process), and stirring to carry out the reaction.

[0123] In some embodiments, the reaction process in the preparation method of the compound of formula X can be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC, or NMR), and the reaction endpoint is generally defined as the point at which the compound of formula XI is no longer detected. The reaction time can be 1-24 hours, preferably 2-12 hours, more preferably 4-8 hours, and even more preferably 4 hours.

[0124] In some embodiments, the method for preparing compound X may further include a post-processing step after the reaction is completed. This post-processing step may include: solid-liquid separation of the reaction solution, with the solid obtained after solid-liquid separation being a portion of the compound X product. The post-processing step may further include: rinsing the liquid phase obtained after solid-liquid separation, removing the solvent from the obtained organic phase, and then slurrying the resulting solid to obtain another portion of the compound X product. The rinsing may use an aqueous solution of acid, an aqueous solution of alkali, water, and a saturated saline solution, preferably sequentially using an aqueous solution of acid, an aqueous solution of alkali, water, and saturated saline solution; more preferably, the rinsing may be performed once with an aqueous solution of acid, twice with an aqueous solution of alkali, once with water, and once with saturated saline solution; the aqueous solution of acid may be an aqueous solution of hydrochloric acid, an aqueous solution of sulfuric acid, or an aqueous solution of phosphoric acid, preferably 1N hydrochloric acid, 0.5N sulfuric acid, or 0.33N phosphoric acid, more preferably 1N hydrochloric acid; the aqueous solution of alkali may be an aqueous solution of sodium hydroxide, an aqueous solution of potassium hydroxide, or a mixture thereof, preferably 1N sodium hydroxide, 1N potassium hydroxide, or a mixture thereof, more preferably 1N sodium hydroxide. The solvent used for pulping can be 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.

[0125] The present invention also provides a compound of formula II:

[0126]

[0127] Among them, R 1 R 2 and R 3 As defined in the aforementioned compound of formula III;

[0128] The premise is: when R 1 It is methyl, sulfone, ethyl, or dimethyl ethyl, R 2 When it is methyl, R 3 It is not fluorine.

[0129] The present invention also provides a method for preparing a compound of formula II, comprising the following steps: reducing a compound of formula III and a reducing agent in an organic solvent and in the presence of an acid buffer to obtain the compound of formula II;

[0130]

[0131] Among them, R 1 R 2 and R 3 As defined in the aforementioned compound of formula III.

[0132] In some embodiments, in the method for preparing the compound of formula II, the reducing agent may be a reducing agent conventional to such reactions in the art, preferably triphenylphosphine, tri-tert-butylphosphine or trimethylphosphine, and more preferably trimethylphosphine.

[0133] In some embodiments, in the method for preparing the compound of formula II, the molar ratio of the reducing agent to the compound of formula III can be 1:1-5:1, preferably 2:1-4:1, more preferably 2.9:1-3.5:1, and even more preferably 2.9:1-3.0:1.

[0134] In some embodiments, in the method for preparing the compound of formula II, the organic solvent may be a conventional organic solvent for 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 more preferably tetrahydrofuran.

[0135] In some embodiments, in the method for preparing the compound of formula II, the volume-to-mass ratio of the organic solvent to the compound of formula II can be 5-50 mL / g, preferably 10-20 mL / g, and more preferably 13-14 mL / g.

[0136] In some embodiments, in the method for preparing the compound of formula II, the acid buffer can be a conventional acid buffer for such reactions in the art, such as acetate buffer or formic acid buffer, preferably acetate buffer; the pH of the acetate buffer used can be 4.0-6.0, preferably 4.5-5.5, and more preferably 5.0.

[0137] In some embodiments, in the method for preparing the compound of formula II, the volume ratio of the organic solvent to the acid buffer can be 1:1-5:1, preferably 1:1-2:1, more preferably 1.25:1-1.35:1, and most preferably 1.28:1-1.30:1.

[0138] In some embodiments, in the method for preparing the compound of formula II, the temperature of the reduction reaction can be a temperature conventional for such reactions in the art, for example, 0-20°C, preferably 0-10°C, and more preferably 0-5°C.

[0139] In some embodiments, the reduction reaction in the method for preparing the compound of formula II can be performed using conventional methods for such reactions in the art, for example, including the steps of stirring a mixture of an organic solvent, an acid buffer, the compound of formula III, and a reducing agent to carry out the reduction reaction.

[0140] In some embodiments, the process of the reduction reaction in the preparation method of the compound of formula II can be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC, or NMR), and the reaction endpoint is generally defined as the absence of detectable compound of formula III. The reaction time of the reduction reaction is 1-24 hours, preferably 2-5 hours, and more preferably 2 hours.

[0141] In some embodiments, the method for preparing the compound of formula II may further include the following post-processing steps after the reduction reaction: extracting the reaction solution, and purifying the residue of the extracted organic phase after removing the solvent to obtain the compound of formula II. The purification can be performed using conventional purification methods in the art, such as pulping, crystallization, preparative chromatography, or silica gel column chromatography, preferably silica gel column chromatography. The eluent used is preferably a mixture of dichloromethane and methanol, and the elution gradient is preferably 50:1-10:1, more preferably 20:1-10:1.

[0142] The method for preparing the compound of formula II may further include the method for preparing the compound of formula III as described herein.

[0143] The present invention provides a method for preparing a compound of formula I, comprising the following steps: coupling a compound of formula II and 6-(maleimide)hexanoic acid succinimide ester in a solvent to obtain the compound of formula I;

[0144]

[0145] Among them, R 1 R 2 and R 3 As defined in the aforementioned compound of formula III.

[0146] In some embodiments, in the preparation method of the compound of formula I, the molar ratio of 6-(maleimide)hexanoic acid succinimide ester to the compound of formula II can be 1.0∶1-5.0∶1, preferably 1.0∶1-2.0∶1, more preferably 1.9∶1-2.0∶1, and even more preferably 1.96∶1.

[0147] In some embodiments, in the method for preparing the compound of formula I, the solvent may be a solvent conventional to such reactions in the art, such as an amide solvent, a chloroalkane solvent, an ether solvent, a nitrile solvent, or a mixture of any two or more thereof, preferably an amide solvent, a chloroalkane solvent, or a mixture thereof, more preferably an amide solvent or a chloroalkane solvent, and even more preferably a chloroalkane solvent; wherein the amide solvent is preferably DMF, DMAC, or a mixture thereof, more preferably DMF; the chloroalkane solvent is preferably dichloromethane, chloroform, dichloroethane, or a mixture of any two or more thereof, more preferably dichloromethane; the ether solvent is preferably tetrahydrofuran, diethyl ether, 1,4-dioxane, anisole, methyl tert-butyl ether, or a mixture of any two or more thereof, more preferably tetrahydrofuran; the nitrile solvent is preferably acetonitrile. In some embodiments, in the method for preparing the compound of formula I, the solvent is a chloroalkane solvent, such as dichloromethane.

[0148] In some embodiments, in the method for preparing the compound of formula I, the volume-to-mass ratio of the solvent to the compound of formula II can be 20-200 mL / g, preferably 30-80 mL / g, and more preferably 35-40 mL / g.

[0149] In some embodiments, the reaction temperature in the preparation method of the compound of formula I can be a temperature conventional for such reactions in the art, for example, 0-50°C, preferably 25-40°C, and more preferably 40°C.

[0150] In some embodiments, the coupling reaction in the preparation method of the compound of formula I can be a conventional operation of such reactions in the art, for example including the following steps: stirring a mixture of the compound of formula II, 6-(maleimide)hexanoyl compound and solvent to carry out the coupling reaction.

[0151] In some embodiments, the reaction progress in the preparation method of the compound of formula I can be monitored using conventional testing methods in the art (e.g., TLC, GC, HPLC, or NMR), and the reaction endpoint is generally defined as the point at which the compound of formula II is no longer detected. The reaction time of the coupling reaction can be 1-24 hours, preferably 12-20 hours, and more preferably 16 hours.

[0152] In some embodiments, the preparation method of the compound of formula I may further include the following post-processing step after the coupling reaction is completed: removing the solvent from the reaction solution, and purifying the resulting residue to obtain the compound of formula I. The purification may employ conventional purification methods in the art, such as pulping, crystallization, preparative chromatography, or silica gel column chromatography, preferably silica gel column chromatography. The eluent used is preferably a mixture of dichloromethane and methanol, and the elution gradient is preferably 50:1-10:1, more preferably 50:1-15:1.

[0153] The method for preparing the compound of formula I may further include the method for preparing the compound of formula II as described herein.

[0154] This invention also provides a compound of formula IV:

[0155]

[0156] Among them, R 1 As defined in the aforementioned compound of formula III.

[0157] The present invention also provides a method for preparing a compound of formula IV, comprising the following steps: reacting a compound of formula V with paraformaldehyde and trimethylchlorosilane in a solvent to obtain the compound of formula IV;

[0158]

[0159] Among them, R 1 As defined in the aforementioned compound of formula III.

[0160] In the preparation method of the compound of formula IV, the reaction conditions can all be as described above. The preparation method of the compound of formula IV may further include the preparation method of the compound of formula V described herein.

[0161] In some embodiments, the compound of formula IV is

[0162]

[0163] This invention also provides a compound of formula V:

[0164]

[0165] Among them, R 1 As defined in the aforementioned compound of formula III.

[0166] In some embodiments, the compound of formula V is

[0167]

[0168] The present invention also provides a method for preparing a compound of formula V, comprising the following steps: reacting a compound of formula VI with a sulfonyl azide compound in a solvent in the presence of a base and a catalyst to obtain the compound of formula V;

[0169]

[0170] Among them, R 1 As defined in the aforementioned compound of formula III.

[0171] In the preparation method of compound V, the reaction conditions can all be as described above. The preparation method of compound V may further include the preparation method of compound VI described herein.

[0172] This invention also provides a compound of formula VI:

[0173]

[0174] Among them, R 1 As defined in the aforementioned compound of formula III.

[0175] In some embodiments, the compound of formula VI is

[0176] The present invention also provides a method for preparing a compound of formula VI, comprising the following steps: performing a deFmoc reaction on a compound of formula VII in the presence of a base and an organic solvent to obtain the compound of formula VI;

[0177]

[0178] Among them, R 1 As defined in the aforementioned compound of formula III.

[0179] In the preparation method of the compound of formula VI, the reaction conditions can all be as described above. The preparation method of the compound of formula VI may further include the preparation method of the compound of formula VII described herein.

[0180] The present invention also provides a method for preparing a compound of formula VII, comprising the following steps: coupling a compound of formula VIII with N-Fmoc-L-valine N-butadieneamine imine ester in a solvent to obtain the compound of formula VII;

[0181]

[0182] Among them, R 1 As defined in the aforementioned compound of formula III.

[0183] In the preparation method of the compound of formula VII, the reaction conditions can all be as described above. The preparation method of the compound of formula VII may further include the preparation method of the compound of formula VIII described herein.

[0184] The present invention also provides a compound of formula VIII:

[0185]

[0186] Among them, R 1 As defined in the aforementioned compound of formula III.

[0187] In some embodiments, the compound of formula VIII is

[0188]

[0189] The present invention also provides a method for preparing a compound of formula VIII, which includes the following steps: performing a deFmoc reaction on a compound of formula IX in the presence of a base and a solvent to obtain a compound of formula VIII;

[0190]

[0191] Among them, R 1 As defined in the aforementioned compound of formula III.

[0192] In the preparation method of the compound of formula VIII, the reaction conditions can all be as described above. The preparation method of the compound of formula VIII may further include the preparation method of the compound of formula IX described herein.

[0193] This invention also provides a compound of formula IX:

[0194]

[0195] Among them, R 1 As defined in the aforementioned compound of formula III.

[0196] In some embodiments, the compound of formula IX is

[0197]

[0198] The present invention also provides a method for preparing a compound of formula IX, comprising the following steps: reacting a compound of formula X with an amino compound R. 1 NH2 undergoes a coupling reaction with a solvent in the presence of a base to give the compound of formula IX;

[0199]

[0200] Among them, R 1As defined in the aforementioned compound of formula III.

[0201] In the preparation method of the compound of formula IX, the reaction conditions can all be as described above. The preparation method of the compound of formula IX may further include the preparation method of the compound of formula X described herein.

[0202] This invention also provides a compound of formula X:

[0203]

[0204] The present invention also provides a method for preparing a compound of formula X, comprising the following steps: reacting a compound of formula XI and a compound of formula XII in a base and a solvent as follows to obtain the compound of formula X;

[0205]

[0206] In the preparation method of compound X, the reaction conditions can all be as described above.

[0207] definition

[0208] In this invention, the term "C1-C6 alkyl" refers to a saturated straight-chain or branched alkyl group comprising 1-6, particularly 1-4, carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, etc., particularly methyl or ethyl.

[0209] In this invention, the term "halogen" means fluorine, chlorine, bromine or iodine, especially fluorine or chlorine.

[0210] Table 1. Abbreviations

[0211] EMCS 6-(maleimide)hexanoic acid succinimide ester DMF N,N-Dimethylformamide DMAC N,N-Dimethylacetamide DMSO Dimethyl sulfoxide Fmoc 9-fluorenylmethoxycarbonyl protecting group

[0212] 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

[0213] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0214] The reagents and raw materials used in this invention are all commercially available.

[0215] The positive and progressive effects of this invention are: it provides a novel method for preparing the linker drug conjugate shown in Formula I and its intermediates. This preparation method has one or more of the following advantages: simple operation, high yield, easy control of product quality, and suitability for industrial production. Detailed Implementation

[0216] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0217] In the following examples, mass spectrometry was performed using a Waters Acquity Xevo G2-XS QTof UPLC / MS ultra-high performance liquid chromatography-high resolution mass spectrometry system. 1 H-NMR was performed using a Bruker AVANCE III 400MHz NMR spectrometer or a Bruker AVANCE III HD 300MHz NMR spectrometer, and HPLC was performed using an Agilent 1260 high-performance liquid chromatograph.

[0218] Example 1: Synthesis method of compound 1

[0219] Step 1: Synthesis of Compound 11

[0220]

[0221] Compound 13 (10 g, 32.2 mmol) and compound 14 (4 g, 32.5 mmol, 1.0 equivalent) were dispersed in 200 mL of dichloromethane at room temperature. EEDQ (9.5 g, 38.4 mmol, 1.2 equivalent) was added in three portions. After the addition was complete, the reaction mixture was mechanically stirred at room temperature for 12 hours, during which a large amount of white solid precipitated. After the reaction was confirmed to be complete by TLC, the reaction mixture was directly filtered, and the filter cake was dried to obtain compound 11 (11 g, yield 82%) as a white solid.

[0222] 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).

[0223] Step 2: Synthesis of Compound 10

[0224]

[0225] Compound 11 (11 g, 26.4 mmol) was dispersed in 200 mL of dichloromethane at room temperature, and pyridine (4.2 mL, 52.8 mmol, 2.0 equivalent) was added. The resulting mixture was cooled to 0 °C in an ice bath, and p-nitrophenol chloroformate (4 times, 8.0 g, 39.6 mmol, 1.5 equivalent) was added in portions under ice bath conditions. After the addition was complete, the reaction solution was stirred at room temperature for 4 hours. After the reaction was completed by TLC monitoring, the reaction solution was filtered, the filter cake was collected and dried to obtain the first batch of solids. The filtrate was washed successively with 1N hydrochloric acid, 1N sodium hydroxide (twice), water and saturated brine, dried and concentrated, and then slurried with methyl tert-butyl ether for 1 hour. The mixture was filtered, the filter cake was collected and dried to obtain the second batch of solids. The two batches of solids were combined to obtain a pale yellow solid compound 10 (12 g, yield 78%).

[0226] Step 3: Synthesis of Compound 9

[0227]

[0228] At room temperature, compound 10 (12 g, 20.6 mmol) and methyl sulfone ethylamine hydrochloride (3.6 g, 22.6 mmol, 1.1 equivalents) were dispersed in 200 mL of dichloromethane. DMAP (6.3 g, 51.6 mmol, 2.5 equivalents) was added in three portions. The resulting reaction solution was stirred at room temperature for 12 hours, resulting in the precipitation of a large amount of pale yellow solid. After the reaction was completed by TLC monitoring, the mixture was filtered. The filter cake was washed twice with ethyl acetate (100 mL each time) and dried to obtain the first batch of white solid, 7.6 g. The filtrate was evaporated to dryness, dissolved in 150 mL of ethyl acetate, and washed successively with 1N hydrochloric acid (50 mL), 1N sodium hydroxide (twice, 50 mL each time), water, and saturated brine (100 mL). After drying and concentration, the mixture was slurried with dichloromethane (20 mL), filtered, and dried to obtain the second batch of white solid, 2 g. The two batches of solid were combined to obtain white solid compound 9 (9.6 g, yield 82%). 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).

[0229] Step 4: Synthesis of Compound 8

[0230]

[0231] Compound 9 (9.6 g, 17.0 mmol) was dissolved in 50 mL of LDM at room temperature, and 15 mL of diethylamine was added. The mixture was stirred for 2 hours at room temperature, and the reaction was monitored by TLC until the starting material was completely reacted. The reaction solution was concentrated to remove the solvent, and the residue was slurried with methyl tert-butyl ether (50 mL) to give compound 8 (5.2 g, 89% yield) as a white solid. MS: m / z = 344.1 (M+H). 1 H-NMR (400MHz, DMSO-d6) 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).

[0232] Step 5: Synthesis of Compound 7

[0233]

[0234] At room temperature, N-Fmoc-L-valine N-butadieneamine imine ester (5 g, 11.5 mmol) and amino compound 8 (4 g, 11.6 mmol) were dispersed in 100 mL LCM. The resulting reaction solution was stirred overnight at 40 °C under nitrogen protection, and a white insoluble substance remained in the system. Then, 5 mL of methanol was added to the reaction system, and stirring was continued at 40 °C for 4 hours. The resulting reaction solution was filtered, and the filter cake was collected and dried to obtain compound 7, which was directly used in the next step. MS: m / z = 665.3 (M+H). 1 H-NMR (400MHz, DMSO-d6) 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, 2 H), 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).

[0235] Step 6: Synthesis of Compound 6

[0236]

[0237] At room temperature, compound 7 obtained in step 5 was dissolved in 50 mL of LDM, and 10 mL of diethylamine was added. The resulting mixture was stirred at room temperature for 4 hours, and the reaction was monitored by TLC until the starting material was completely reacted. At this point, a white insoluble impurity was present in the reaction solution. The impurity was removed by filtration, and the filtrate was evaporated to dryness using an oil pump. The resulting pale yellow oily substance was thoroughly stirred and slurried with methyl tert-butyl ether (50 mL) for about 2 hours, and then filtered to obtain a white solid. The white solid was purified by recrystallization from ethanol to obtain a white solid compound 6 (5 g, two-step yield 97%). MS: m / z = 443.2 (M+H).

[0238] Step 5: Synthesis of Compound 5

[0239]

[0240] Compound 6 (5.0 g, 11.3 mmol) was dispersed in 50 mL of methanol, 10 mL of dichloromethane, and 25 mL of water at room temperature. Potassium carbonate (3.1 g, 22.4 mmol, 2.0 equivalent) and copper sulfate pentahydrate (0.30 g, 1.2 mmol, 0.1 equivalent) were added. After the mixture became clear, 1H-imidazolium-1-sulfonyl azidohydrochloride (CAS: 952234-36-5, 2.4 g, 11.5 mmol, 1.02 equivalent) was added, and the reaction mixture was stirred overnight at room temperature. After the reaction was completed by TLC, 25 mL of water was added, and most of the methanol was removed under reduced pressure. The resulting mixture was extracted twice with dichloromethane (50 mL each time). The combined organic phases were washed with saturated brine, dried, and recrystallized from 40 mL 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(d q, J=13.4, 6.7Hz, 1H), 1.33 (d, J=7.1Hz, 3H), 0.93 (dd, J=10.2, 6.7Hz, 6H).

[0241] Step 6: Synthesis of Compound 4

[0242]

[0243] Compound 5 (150 g, 0.32 mol) synthesized according to step 5 and paraformaldehyde (29 g, 1.00 mol, 3.1 equivalents based on formaldehyde) were added to 3 L of ultra-dry tetrahydrofuran. Trimethylchlorosilane (122 mL, 1.24 mmol, 3.9 equivalents) was added dropwise to the resulting mixture. After the addition was complete, the mixture was stirred overnight at room temperature. The reaction solution was then directly filtered. The solvent in the filtrate was removed by vacuum evaporation and then evacuated under vacuum for 2 hours to obtain bubbly solid compound 4 (for direct use in the next step).

[0244] Step 7: Synthesis of Compound 3

[0245]

[0246] The crude compound 4 obtained in step 6 was dissolved in 2 L of ultra-dry 1,4-dioxane, and then Dxd (50 g, 0.10 mol, commercially available) and Panpidine (87 mL, 0.48 mol) were added. The resulting mixture was heated to 60 °C and stirred for 2 hours. The solvent in the resulting reaction solution was removed by vacuum distillation, and the crude product was dissolved in 1.5 L of dichloromethane. The crude product solution was then washed successively with 0.1 mol / L dilute hydrochloric acid, water, and saturated brine. The resulting organic phase was dried overnight with anhydrous sodium sulfate. After the solvent was removed from the dried solution, it was subjected to silica gel column chromatography, eluting with dichloromethane / methanol at a ratio of 60:1-10:1 to obtain compound 3 (95 g, directly used in the next step).

[0247] Step 8: Synthesis of Compound 2

[0248]

[0249] Compound 3 obtained in step 7 was dissolved in 600 mL of tetrahydrofuran, then 400 mL of tetrahydrofuran and 1000 mL of acetate buffer (pH 5.0, 100 mM) were added, followed by 290 mL of a 1 M trimethylphosphine tetrahydrofuran solution (290 mmol). The resulting mixture was stirred at 0–5 °C for 2 hours. After the reaction was complete, 1000 mL of saturated saline solution was added to the resulting reaction solution, followed by extraction with 2.5 L of dichloromethane. The resulting organic phase was dried over anhydrous sodium sulfate overnight, and the solvent was removed by vacuum distillation. The crude product was subjected to silica gel column chromatography, eluted with dichloromethane / methanol at a ratio of 20:1–10:1 to give compound 2 (50 g, purity 97%, yield 53% based on Dxd).

[0250] Step 9: Synthesis of Compound 1

[0251]

[0252] Compound 2 (50 g, 53.0 mmol) was mixed with compound EMCS (commercially available, 32 g, 0.104 mmol) and dissolved in 2 L of dichloromethane. The resulting mixture was stirred overnight at 40 °C. After the reaction was complete, the solvent was evaporated under reduced pressure. The residue was subjected to silica gel column chromatography, eluted with dichloromethane / methanol at a ratio of 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), 6.9 9 (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, 4 H), 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).

[0253] Example 2: Comparison of final product purity across several process routes

[0254] The purity of the final product of compound 1 synthesized according to Example 1 was compared with that of the final product of compound 1 obtained according to route 1 (i.e., the synthesis of LE14 in Example 7) and route 2 (i.e., the synthesis of LE14 in Example 10) disclosed in WO2020259258A1 by high performance liquid chromatography. The results are shown in Table 3 below.

[0255] The liquid chromatography conditions used were as follows: Phase A was 0.1% formic acid aqueous solution, Phase B was 0.1% formic acid acetonitrile solution, the detection wavelength was 370 nm, the instrument was an Agilent 1260, and the chromatographic column was a ZORBAX Eclipse Plus C18, 3.5 μm, 4.6 × 150 mm. The gradient settings are shown in Table 2 below.

[0256] Table 2. Mobile phase gradient settings

[0257] 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

[0258] Table 3. Comparison of final product purity data for different process routes

[0259]

Claims

1. A compound of formula III: ； in, R 1 For an R 1-3 S(O)2-substituted C1~C6 alkyl groups; R 2 It is a C1~C6 alkyl group; R 3 It is a halogen; R 1-3 It is a C1~C4 alkyl group.

2. The compound of formula III as claimed in claim 1, characterized in that, R 1 It is methyl sulfone ethyl; And / or, R 2 It is methyl; And / or, R 3 It is fluorine; And / or, R 1-3 It is an ethyl group.

3. The compound of formula III as claimed in claim 1, characterized in that, The compound of formula III is 。 4. A method for preparing a compound of formula III as described in any one of claims 1-3, characterized in that, The process includes the following steps: subjecting compounds of formula IV and IV' to a substitution reaction in a solvent and in the presence of a base to obtain compound of formula III; ； Among them, R 1 R 2 and R 3 As described in any one of claims 1-3.

5. The method for preparing the compound of formula III as described in claim 4, characterized in that, In the preparation method of the compound of formula III, the molar ratio of the compound of formula IV to the compound of formula IV' is 2:1-4:1; And / or, in the method for preparing the compound of formula III, the base is an organic base, an inorganic base, or a mixture thereof; And / or, in the method for preparing the compound of formula III, the molar ratio of the base to the compound of formula IV' is 3:1-6:1; And / or, in the method for preparing the compound of formula III, the solvent is an ether solvent, a chloroalkane solvent, a nitrile solvent, or a mixture of any two or more thereof; And / or, in the method for preparing the compound of formula III, the temperature of the substitution reaction is 20-80°C; And / or, in the method for preparing the compound of formula III, the substitution reaction is carried out under anhydrous conditions.

6. The method for preparing the compound of formula III as described in claim 5, characterized in that, In the preparation method of the compound of formula III, the molar ratio of the compound of formula IV to the compound of formula IV' is 3.0:1-3.5:1; And / or, in the method for preparing the compound of formula III, the organic base is tert-butylpotassium, triethylamine, DMAP, pyridine, panpididine, or a mixture of any two or more thereof; And / or, in the alkali, the inorganic alkali is an alkali metal hydroxide, an alkali metal carbonate, an alkali metal phosphate, or a mixture of any two or more thereof; And / or, in the method for preparing the compound of formula III, the molar ratio of the base to the compound of formula IV' is 4.5:1-5.5:1; And / or, in the method for preparing the compound of formula III, the ether solvent is tetrahydrofuran, diethyl ether, 1,4-dioxane, anisole, methyl tert-butyl ether, or a mixture of any two or more thereof; And / or, in the method for preparing the compound of formula III, the chloroalkane solvent is dichloromethane, dichloroethane, chloroform, or a mixture of any two or more thereof; And / or, in the method for preparing the compound of formula III, the nitrile solvent is acetonitrile; And / or, in the method for preparing the compound of formula III, the temperature of the substitution reaction is 40-60°C.

7. The method for preparing the compound of formula III as described in claim 6, characterized in that, In the preparation method of the compound of formula III, the inorganic base is potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate, or a mixture of any two or more thereof.

8. The method for preparing the compound of formula III as described in claim 4, characterized in that, It further includes a method for preparing a compound of formula IV, the method comprising the following steps: reacting a compound of formula V with paraformaldehyde and trimethylchlorosilane in a solvent to obtain the compound of formula IV; ； Among them, R 1 As described in claim 4.

9. The method for preparing the compound of formula III as described in claim 8, characterized in that, In the preparation method of the compound of formula IV, the molar ratio of the paraformaldehyde to the compound of formula V, converted from formaldehyde, is 3:1-12:

1. And / or, in the method for preparing the compound of formula IV, the molar ratio of trimethylchlorosilane to the compound of formula V is 3:1-12:1; And / or, in the method for preparing the compound of formula IV, the solvent is an ether solvent, a chloroalkane solvent, or a mixture thereof; And / or, in the method for preparing the compound of formula IV, the reaction temperature is 10-40℃; And / or, in the method for preparing the compound of formula IV, the reaction is carried out under anhydrous conditions.

10. The method for preparing the compound of formula III as described in claim 9, characterized in that, In the preparation method of the compound of formula IV, the molar ratio of paraformaldehyde to compound of formula V, converted from formaldehyde, is 3:1-4:

1. And / or, in the method for preparing the compound of formula IV, the molar ratio of trimethylchlorosilane to the compound of formula V is 3:1-4:1; And / or, in the method for preparing the compound of formula IV, the ether solvent is tetrahydrofuran, diethyl ether, 1,4-dioxane, anisole, methyl tert-butyl ether, or a mixture of any two or more thereof; And / or, in the method for preparing the compound of formula IV, the chloroalkane solvent is dichloromethane, dichloroethane, chloroform, or a mixture of any two or more thereof; And / or, in the preparation method of the compound of formula IV, the reaction temperature is 25-40℃.

11. The method for preparing the compound of formula III as described in claim 8, characterized in that, It further includes a method for preparing compound V, the method comprising the following steps: reacting compound VI with sulfonyl azide compound in a solvent in the presence of a base and a catalyst to obtain compound V; ; Optionally, the method for preparing the compound of formula III further includes a method for preparing the compound of formula VI, wherein the method for preparing the compound of formula VI includes the following steps: performing a de-Fmoc reaction on the compound of formula VII in the presence of a base and an organic solvent to obtain the compound of formula VI; ; Among them, R 1 As described in claim 8.

12. The method for preparing the compound of formula III as described in claim 11, characterized in that, In the preparation method of compound V, the sulfonyl azide compound is 1H-imidazolium-1-sulfonyl azide hydrochloride, 2-azido-1,3-dimethylimidazolium hexafluorophosphate, trifluorosulfonyl azide, p-methylbenzenesulfonyl azide, or methanesulfonyl azide; And / or, in the method for preparing compound V, the molar ratio of the sulfonyl azide compound to compound VI is 1.0:1-1.5:1; And / or, in the method for preparing the compound of formula V, the base is an organic base, an inorganic base, or a mixture thereof; And / or, in the method for preparing compound V, the molar ratio of the base to compound VI is 1.5:1-3.0:1; And / or, in the method for preparing compound V, the catalyst is a copper salt; And / or, in the method for preparing compound V, the solvent is a mixture of organic solvent and water; And / or, in the method for preparing the compound of formula V, the reaction temperature is 10~40℃; And / or, in the method for preparing the compound of formula VI, the base is an organic base, an inorganic base, or a mixture thereof; And / or, in the method for preparing the compound of formula VI, the volume ratio of the base to the organic solvent is 0.1:1-0.5:1; And / or, in the method for preparing the compound of formula VI, the organic solvent is DMF, DMSO, tetrahydrofuran, 1,4-dioxane, or a mixture of any two or more thereof; And / or, in the preparation method of the compound of formula VI, the temperature of the deFmoc reaction is 10~40℃.

13. The method for preparing the compound of formula III as described in claim 12, characterized in that, In the preparation method of compound V, the sulfonyl azide compound is 1H-imidazolium-1-sulfonyl azide hydrochloride; And / or, in the method for preparing compound V, the molar ratio of the sulfonyl azide compound to compound VI is 1.0:1-1.2:1; And / or, in the method for preparing compound V, the inorganic base is an alkali metal hydroxide, an alkali metal carbonate, an alkali metal phosphate, or a mixture of any two or more thereof; And / or, in the method for preparing compound V, the organic base is tert-butylpotassium, triethylamine, DMAP, pyridine, panpididine, 2,6-dimethylpyridine, or a mixture of any two or more thereof; And / or, in the method for preparing compound V, the molar ratio of the base to compound VI is 2.0:1-2.5:1; And / or, in the method for preparing compound V, the copper salt is copper sulfate; And / or, in the method for preparing compound V, the molar ratio of the copper salt to compound VI is 0.1:1-0.5:1; And / or, in the method for preparing compound V, the organic solvent is an alcohol solvent, a chloroalkane solvent, an ether solvent, or a mixture of any two or more thereof; And / or, in the method for preparing compound V, the solvent is methanol, ethanol, isopropanol or a mixture thereof; And / or, in the method for preparing compound V, the chloroalkane solvent is dichloromethane, chloroform, dichloroethane, or a mixture of any two or more thereof; And / or, in the method for preparing compound V, the solvent is tetrahydrofuran, diethyl ether, 1,4-dioxane, anisole, methyl tert-butyl ether, or a mixture of any two or more thereof; And / or, in the method for preparing the compound of formula V, the reaction temperature is 25~40℃; And / or, in the method for preparing the compound of formula VI, the base is an organic base; And / or, in the method for preparing the compound of formula VI, the organic base is diethylamine, tert-butylpotassium, triethylamine, DMAP, pyridine, panpididine, 2,6-dimethylpyridine, or a mixture of any two or more thereof; And / or, in the method for preparing the compound of formula VI, the inorganic base is an alkali metal hydroxide, an alkali metal carbonate, an alkali metal phosphate, or a mixture of any two or more thereof; And / or, in the method for preparing the compound of formula VI, the volume ratio of the base to the organic solvent is 0.2:1-0.3:1; And / or, in the method for preparing the compound of formula VI, the organic solvent is DMF; And / or, in the preparation method of the compound of formula VI, the temperature of the deFmoc reaction is 25~40℃.

14. The method for preparing the compound of formula III as described in claim 13, characterized in that, In the preparation method of compound V, the inorganic base is potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate, or a mixture of any two or more thereof. And / or, in the method for preparing compound V, the copper salt is copper sulfate pentahydrate; And / or, in the method for preparing compound V, the molar ratio of the copper salt to compound VI is 0.1:1-0.3:1; And / or, in the method for preparing the compound of formula VI, the inorganic base is potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate, or a mixture of any two or more thereof.

15. The method for preparing the compound of formula III as described in claim 11, characterized in that, It further includes a method for preparing compound VII, the method comprising the following steps: coupling compound VIII with N-Fmoc-L-valine N-butadieneamine imine ester in a solvent to obtain compound VII; ； Optionally, the method for preparing the compound of formula III further includes a method for preparing the compound of formula VIII, the method for preparing the compound of formula VIII comprising the following steps: performing a deFmoc reaction on the compound of formula IX in the presence of a base and in a solvent to obtain the compound of formula VIII; ； Optionally, the method for preparing the compound of formula III further includes a method for preparing the compound of formula IX, the method for preparing the compound of formula IX comprising the following steps: reacting the compound of formula X with an amino compound R 1 NH2 undergoes a coupling reaction with a solvent in the presence of a base to give the compound of formula IX; ； Optionally, the method for preparing the compound of formula III further includes a method for preparing the compound of formula X, wherein the method for preparing the compound of formula X includes the following steps: reacting the compound of formula XI and the compound of formula XII in the presence of a base and a solvent to obtain the compound of formula X; ； Among them, R 1 As described in claim 11.

16. The method for preparing the compound of formula III as described in claim 15, characterized in that, In the preparation method of the compound of formula VII, the molar ratio of the N-Fmoc-L-valine N-butadieneamine imine ester to the compound of formula VIII is 0.8:1-5:1; And / or, in the method for preparing the compound of formula VII, the solvent is DMF, DMSO, acetonitrile, dichloromethane, dichloroethane, or a mixture of any two or more thereof; And / or, in the method for preparing the compound of formula VII, the coupling reaction temperature is 10~40℃; And / or, in the method for preparing the compound of formula VII, the coupling reaction is carried out under gas protection; And / or, in the method for preparing the compound of formula VIII, the base is an organic base, an inorganic base, or a mixture thereof; And / or, in the method for preparing the compound of formula VIII, the solvent is DMF, DMSO, tetrahydrofuran, 1,4-dioxane, or a mixture of any two or more thereof; And / or, in the method for preparing the compound of formula VIII, the volume ratio of the base to the solvent is 0.2:1-0.5:1; And / or, in the method for preparing the compound of formula VIII, the reaction temperature for the deFmoc reaction is 10-40℃; And / or, in the method for preparing the compound of formula IX, the amino compound R 1 The molar ratio of NH2 to compound X is 1.0:1-3.0:1; And / or, in the method for preparing the compound of formula IX, the base is an organic base, an inorganic base, or a mixture thereof; And / or, in the method for preparing the compound of formula IX, the molar ratio of the base to the compound of formula X is 2.0:1-4.0:1; And / or, in the method for preparing the compound of formula IX, the solvent is DMF, DMSO, dichloromethane, dichloroethane, tetrahydrofuran, 1,4-dioxane, or a mixture of any two or more thereof; And / or, in the preparation method of the compound of formula IX, the coupling reaction temperature is 10-40℃; And / or, in the method for preparing compound X, the molar ratio of compound XII to compound XI is 3.0:1-1.2:1; And / or, in the method for preparing the compound of formula X, the base is an organic base, an inorganic base, or a mixture thereof; And / or, in the method for preparing compound X, the molar ratio of the base to compound XI is 1.0:1-4.0:1; And / or, in the method for preparing compound X, the solvent is DMF, DMSO, dichloromethane, dichloroethane, tetrahydrofuran, 1,4-dioxane, or a mixture of any two or more thereof; And / or, in the preparation method of the compound of formula X, the reaction temperature is 10~40℃.

17. The method for preparing the compound of formula III as described in claim 16, characterized in that, In the preparation method of the compound of formula VII, the molar ratio of the N-Fmoc-L-valine N-butadieneamine imine ester to the compound of formula VIII is 0.8:1-1.2:1; And / or, in the method for preparing the compound of formula VII, the solvent is dichloromethane; And / or, in the method for preparing the compound of formula VII, the coupling reaction temperature is 35~40℃; And / or, in the method for preparing the compound of formula VIII, the organic base is diethylamine, tert-butylpotassium, triethylamine, DMAP, pyridine, panpididine, 2,6-dimethylpyridine, or a mixture of any two or more thereof; And / or, in the method for preparing the compound of formula VIII, the inorganic base is an alkali metal hydroxide, an alkali metal carbonate, an alkali metal phosphate, or a mixture of any two or more thereof; And / or, in the method for preparing the compound of formula VIII, the solvent is DMF; And / or, in the method for preparing the compound of formula VIII, the volume ratio of the base to the solvent is 0.3:1-0.4:1; And / or, in the method for preparing the compound of formula VIII, the reaction temperature for the deFmoc reaction is 25-40°C; And / or, in the method for preparing the compound of formula IX, the amino compound R 1 The molar ratio of NH2 to compound X is 1.1:1 to 1.5:1; And / or, in the method for preparing the compound of formula IX, the organic base is diethylamine, potassium tert-butyl, triethylamine, DMAP, pyridine, panpididine, 2,6-dimethylpyridine, or a mixture of any two or more thereof; And / or, in the method for preparing the compound of formula IX, the inorganic base is an alkali metal hydroxide, an alkali metal carbonate, an alkali metal phosphate, or a mixture of any two or more thereof; And / or, in the method for preparing the compound of formula IX, the molar ratio of the base to the compound of formula X is 2.5:1-3.0:1; And / or, in the method for preparing the compound of formula IX, the solvent is dichloromethane; And / or, in the preparation method of the compound of formula IX, the coupling reaction temperature is 25-40℃; And / or, in the method for preparing compound X, the molar ratio of compound XII to compound XI is 2.0:1-1.5:1; And / or, in the method for preparing compound X, the organic base is diethylamine, tert-butylpotassium, triethylamine, DMAP, pyridine, panpididine, 2,6-dimethylpyridine, or a mixture of any two or more thereof; And / or, in the method for preparing compound X, the inorganic base is an alkali metal hydroxide, an alkali metal carbonate, an alkali metal phosphate, or a mixture thereof; And / or, in the method for preparing compound X, the molar ratio of the base to compound XI is 2.0:1-3.0:1; And / or, in the method for preparing compound X, the solvent is dichloromethane; And / or, in the preparation method of the compound of formula X, the reaction temperature is 25~40℃.

18. The method for preparing the compound of formula III as described in claim 17, characterized in that, In the method for preparing the compound of formula VIII, the organic base is ethylenediamine. And / or, in the method for preparing the compound of formula VIII, the inorganic base is potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate, or a mixture of any two or more thereof; And / or, in the method for preparing the compound of formula IX, the organic base is DMAP; And / or, in the method for preparing the compound of formula IX, the inorganic base is potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate, or a mixture of any two or more thereof; And / or, in the method for preparing compound X, the organic base is pyridine; And / or, in the method for preparing compound X, the inorganic base is potassium phosphate, potassium carbonate, potassium hydroxide, cesium carbonate, or a mixture of any two or more thereof.

19. A method for preparing a compound of formula II, characterized in that, The process includes the following steps: reducing a compound of formula III with a reducing agent in an organic solvent and in the presence of an acid buffer to obtain a compound of formula II; ； Among them, R 1 R 2 and R 3 As described in any one of claims 1-3.

20. A method for preparing a compound of formula I, comprising the following steps: 1) Compound II is prepared according to the method for preparing compound II as described in claim 19; 2) The compound of formula II obtained in step 1) and succinimide 6-(maleimide)hexanoate succinimide ester are coupled in a solvent to obtain the compound of formula I. ； in, R 1 R 2 and R 3 As described in claim 19.

21. A compound that is a compound of formula II or IV; or ； in, R 1 R 2 and R 3 As described in any one of claims 1-3; The premise is that, in compounds of formula II, when R 1 It is methyl sulfone ethyl, R 2 When it is methyl, R 3 It is not fluorine.

22. The compound of claim 21, wherein, The compound of formula IV is 。 23. A method for preparing a compound of formula IV, characterized in that, The preparation method of compound IV includes the following steps: reacting compound V with paraformaldehyde and trimethylchlorosilane in a solvent to obtain compound IV; ； Among them, R 1 For an R 1-3 S(O)2-substituted C1~C6 alkyl groups; R 1-3 It is a C1~C4 alkyl group.

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