Preparation and purification method of methyl aurestatin E compound intermediate

Abstract

The invention relates to a preparation and purification method of a methyl aurestatin E compound intermediate, and the preparation route of the compound is as follows: the preparation and purification method comprises the following steps: A, dissolving a compound 1, a compound 2, a polypeptide condensing agent and organic alkali in an organic solvent 1, and obtaining a solution 1 after the reaction is finished; b, adding an organic solvent 2 and water into the solution 1, and extracting; and C, collecting an organic phase, and concentrating to obtain the compound shown in the formula (I), wherein R is an amino protecting group. The product purification method is simple, the target product with high purity and yield can be obtained only through one-step extraction and concentration, production steps can be effectively saved, production consumption and investment are reduced, the yield of the product can be effectively improved, the requirements for equipment and the environment are not high, good stability is achieved among multiple batches, and the method is suitable for industrial production. And the final product has better color and is more suitable for large-scale production.

Description

A method for preparing and purifying a methyl auristatin E compound intermediate Technical Field The present invention relates to the field of compound synthesis methods, and in particular to a method for preparing and purifying a methyl auristatin E compound (i.e. Monomethyl Auristantin E, MMAE). Background Art Antibody drug conjugate (ADC) is a type of anti-tumor drug, which includes three components: antibody, linker and toxin. The antibody and toxin are connected by the linker. Its mechanism of action is to use the targeting of the antibody to transport the drug to the target cells (such as tumor cells) and then release the toxin to kill the tumor cells. Although a large number of natural and chemically synthesized cytotoxins are known to exist, only a very small number of drug structures can be applied to ADC. This is mainly because the toxins that can be used as ADC loads must have complex characteristics such as extremely high cytotoxicity, intracellular targets and small molecular structures. Among them, auristatins (such as MMAE, MMAF, etc.) are cytotoxic molecules that have been successfully developed and applied in multiple ADC drugs. MMAE (also known as methyl auristatin E) is a chemically synthesized derivative of auristatin, which can effectively inhibit mitosis by inhibiting tubulin polymerization. The current market price of MMAE is very expensive. One reason is that the currently known MMAE synthesis route contains multiple steps, the synthesis route is relatively complicated, and the yield is not high. In addition, the purification process of MMAE involved in the prior art is complicated, the steps are cumbersome, and more reagents are used. Most reagents cannot be recycled, or a separate recycling process needs to be added, resulting in an increase in production costs. At present, the more common preparation route of MMAE is as follows: Among them, compound d is the main precursor compound for synthesizing MMAE. The Chinese patent with publication number CN105143199A discloses a method for preparing compound d: in an inert atmosphere, compound b and compound c are dissolved in DCM, the solution is cooled to 0°C, DEPC and DIEA are then added dropwise, the reaction mixture is stirred at 0°C for 2 hours, and then stirred at ambient temperature overnight. The reaction mixture is diluted with 100 mL of water and then extracted three times with 50 mL of DCM. The organic phases are combined, washed once with KHSO4, NaHCO3, and NaCl, then dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound d. The yield of the preparation method of compound d disclosed in the above method is relatively low, only 39% (see CN105143199A specification

[0328] In addition, the purification process of compound d disclosed in the above method is relatively complicated. The purification process needs to be washed with KHSO4, NaHCO3 and NaCl in sequence after extraction, which undoubtedly brings great inconvenience to large-scale production and post-treatment, and increases production costs, making it unsuitable for industrial promotion. Summary of the invention In view of the above problems, the present invention provides a method for preparing and purifying a methyl auristatin E compound intermediate with high yield, simplified purification steps, high purity and stable product quality. Specifically, the present invention provides a method for preparing and purifying a compound represented by formula (I) (i.e., the intermediate of the methyl auristatin E compound of the present invention): The preparation route of the compound is as follows: The preparation and purification method comprises the following steps: A. dissolving compound 1, compound 2, a polypeptide condensing agent and an organic base in an organic solvent 1 for reaction; B. After the reaction of step A is completed, adding organic solvent 2 and water to the solution after the reaction to extract; C. Collect the organic phase and concentrate it to obtain the compound of formula (I). Wherein, the R is an amino protecting group, and the amino protecting group of the present invention refers to a group introduced into the molecule for the purpose of protecting the amino group. In some preferred embodiments, the R is selected from the group consisting of Fmoc protecting group, Boc protecting group, and Cbz protecting group, such as: The organic solvent 1 is selected from any one or more of N,N-dimethylformamide (CAS No.: 127-19-5), N-methylpyrrolidone (CAS No.: 872-50-4), methanol, ethanol, isopropanol, acetonitrile, acetone, tetrahydrofuran, acetonitrile, and 1,4-dioxane solvents. The organic solvent 2 is selected from any one or more of n-heptane, petroleum ether, n-hexane, cyclohexane, n-pentane, methylcyclohexane, and methyl tert-butyl ether. The polypeptide condensing agent refers to a reaction auxiliary agent added during the condensation reaction of carboxylic acid and amino group. In some preferred embodiments, the polypeptide condensing agent is selected from any one or more of HATU (CAS No.: 148893-10-1, N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) uronium hexafluorophosphate), HBTU (CAS No.: 94790-37-1, benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate), and TBTU (CAS No.: 125700-67-6, O-benzotriazole-N,N,N′,N′-tetramethyluronium tetrafluoroborate); The organic base refers to an organic compound containing an amino group in the molecule. In some preferred embodiments, the organic base is selected from any one or more of DIPEA (CAS No.: 7087-68-5, N,N-diisopropylethylamine), triethylamine (CAS No.: 121-44-8), 4-methylmorpholine (CAS No.: 109-02-4), and pyridine (CAS No.: 110-86-1). The step A is carried out under the protection of an inert gas, preferably, the inert gas is nitrogen, argon or helium. In some preferred embodiments, the organic solvent 1 is N,N-dimethylformamide; in other preferred embodiments, the organic solvent 1 is N-methylpyrrolidone; in other preferred embodiments, the organic solvent 1 is methanol; in other preferred embodiments, the organic solvent 1 is ethanol; in other preferred embodiments, the organic solvent 1 is isopropanol; in other preferred embodiments, the organic solvent 1 is acetonitrile; in other preferred embodiments, the organic solvent 1 is acetone; in other preferred embodiments, the organic solvent 1 is tetrahydrofuran; in other preferred embodiments, the organic solvent 1 is acetonitrile; in other preferred embodiments, the organic solvent 1 is 1,4-dioxane solvent; in other preferred embodiments, In the embodiment, the organic solvent 1 is any one or more of N,N-dimethylformamide, N-methylpyrrolidone, methanol, ethanol, isopropanol, acetonitrile, acetone, tetrahydrofuran, acetonitrile, and 1,4-dioxane solvents, such as (including but not limited to) a mixed solution of N,N-dimethylformamide and N-methylpyrrolidone (volume ratio of 1:1, 1:2, 1:3... etc.), a mixed solution of methanol and ethanol (volume ratio of 1:1, 1:2, 1:3... etc.), a mixed solution of isopropanol and acetonitrile (volume ratio of 1:1, 1:2, 1:3... etc.), a mixed solution of acetone and tetrahydrofuran (volume ratio of 1:1, 1:2, 1:3... etc.), a mixed solution of acetonitrile and 1,4-dioxane (volume ratio of 1:1, 1:2, 1:3... etc.), and the like. In some preferred embodiments, the organic solvent 2 is n-heptane; in other preferred embodiments, the organic solvent 2 is petroleum ether; in other preferred embodiments, the organic solvent 2 is n-hexane; in other preferred embodiments, the organic solvent 2 is cyclohexane; in other preferred embodiments, the organic solvent 2 is n-pentane; in other preferred embodiments, the organic solvent 2 is methylcyclohexane; in other preferred embodiments, the organic solvent 2 is methyl tert-butyl ether. In some preferred embodiments, organic solvent 1 and organic solvent 2 can be selected independently. In some preferred embodiments, the polypeptide condensing agent is HATU; in other preferred embodiments, the polypeptide condensing agent is HBTU; in other preferred embodiments, the polypeptide condensing agent is TBTU; in other preferred embodiments, the polypeptide condensing agent is a mixture of HATU and HBTU, HATU and TBTU, HBTU and TBTU in any ratio; in other preferred embodiments, the polypeptide condensing agent is a mixture of HATU, HBTU and TBTU in any ratio. In some preferred embodiments, the organic base is DIPEA; in other preferred embodiments, the organic base is triethylamine; in other preferred embodiments, the organic base is 4-methylmorpholine; in other preferred embodiments, the organic base is pyridine; in other preferred embodiments, the organic base is a mixture of DIPEA and triethylamine, DIPEA and 4-methylmorpholine, DIPEA and pyridine, triethylamine and 4-methylmorpholine, triethylamine and pyridine, or 4-methylmorpholine and pyridine in any proportion. Further, the compound 2 is selected from the following structures: Furthermore, the compound of formula (I) is selected from the following structures: Preferably, the preparation route of the compound (I-1) is as follows: Preferably, the preparation route of the compound (I-2) is as follows: Preferably, the preparation route of the compound (I-3) is as follows: Further, the molar ratio of compound 1 to compound 2 in step A is 1:1-2; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:2; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1.95; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1.9; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1.85; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1.8; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1.75; Preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1.7; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1.65; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1.6; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1.55; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1.5; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1.45; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1.4; Preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1.35; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1.3; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1.25; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1.2; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1.15; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1.1; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1.0 5; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:0.95; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:0.9; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:0.85; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:0.8; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:0.75; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:0.7. Further, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1-2; preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:2; preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1.95; preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1.9; preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1.85; preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1.8; preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1.75; Preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1.7; preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1.65; preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1.6; preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1.55; preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1.5; preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1.45; preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1.4; Preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1.35; preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1.3; preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1.25; preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1.2; preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1.15; preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1.1; preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1.0 5; preferably, the molar ratio of compound 1 and polypeptide condensing agent in step A is 1:1; preferably, the molar ratio of compound 1 and polypeptide condensing agent in step A is 1:0.95; preferably, the molar ratio of compound 1 and polypeptide condensing agent in step A is 1:0.9; preferably, the molar ratio of compound 1 and polypeptide condensing agent in step A is 1:0.85; preferably, the molar ratio of compound 1 and polypeptide condensing agent in step A is 1:0.8; preferably, the molar ratio of compound 1 and polypeptide condensing agent in step A is 1:1.75; preferably, the molar ratio of compound 1 and polypeptide condensing agent in step A is 1:0.7. Further, the molar ratio of compound 1 to the organic base in step A is 1:2-5; preferably, the molar ratio of compound 1 to the organic base in step A is 1:5; preferably, the molar ratio of compound 1 to the organic base in step A is 1:4.9; preferably, the molar ratio of compound 1 to the organic base in step A is 1:4.8; preferably, the molar ratio of compound 1 to the organic base in step A is 1:4.7; preferably, the molar ratio of compound 1 to the organic base in step A is 1:4.6; preferably, the molar ratio of compound 1 to the organic base in step A is 1:4.5; preferably, the molar ratio of compound 1 to the organic base in step A is 1:4.4; preferably, the step The molar ratio of compound 1 to the organic base in step A is 1:4.3; preferably, the molar ratio of compound 1 to the organic base in step A is 1:4.2; preferably, the molar ratio of compound 1 to the organic base in step A is 1:4.1; preferably, the molar ratio of compound 1 to the organic base in step A is 1:4; preferably, the molar ratio of compound 1 to the organic base in step A is 1:3.9; preferably, the molar ratio of compound 1 to the organic base in step A is 1:3.8; preferably, the molar ratio of compound 1 to the organic base in step A is 1:3.7; preferably, the molar ratio of compound 1 to the organic base in step A is 1:3.6; preferably, compound 1 in step A and the organic base is 1:3.5; preferably, the molar ratio of compound 1 in step A to the organic base is 1:3.4; preferably, the molar ratio of compound 1 in step A to the organic base is 1:3.3; preferably, the molar ratio of compound 1 in step A to the organic base is 1:3.2; preferably, the molar ratio of compound 1 in step A to the organic base is 1:3.1; preferably, the molar ratio of compound 1 in step A to the organic base is 1:3; preferably, the molar ratio of compound 1 in step A to the organic base is 1:2.9; preferably, the molar ratio of compound 1 in step A to the organic base is 1:2.8; preferably, the step The molar ratio of compound 1 to the organic base in step A is 1:2.7; preferably, the molar ratio of compound 1 to the organic base in step A is 1:2.6; preferably, the molar ratio of compound 1 to the organic base in step A is 1:2.5; preferably, the molar ratio of compound 1 to the organic base in step A is 1:2.4; preferably, the molar ratio of compound 1 to the organic base in step A is 1:2.3; preferably, the molar ratio of compound 1 to the organic base in step A is 1:2.2; preferably, the molar ratio of compound 1 to the organic base in step A is 1:2.1; preferably, the molar ratio of compound 1 to the organic base in step A is 1:2. Further, the weight-to-volume ratio (g / ml) of compound 1 to organic solvent 1 in step A is 1:5-15. Preferably, the weight-to-volume ratio (g / ml) of compound 1 to organic solvent 1 in step A is 1:15; preferably, the weight-to-volume ratio (g / ml) of compound 1 to organic solvent 1 in step A is 1:14; preferably, the weight-to-volume ratio (g / ml) of compound 1 to organic solvent 1 in step A is 1:13; preferably, the weight-to-volume ratio (g / ml) of compound 1 to organic solvent 1 in step A is 1:12; preferably, the weight-to-volume ratio (g / ml) of compound 1 to organic solvent 1 in step A is 1:11; preferably ...1 Preferably, the weight-to-volume ratio (g / ml) of compound 1 in step A to the organic solvent 1 is 1:10; preferably, the weight-to-volume ratio (g / ml) of compound 1 in step A to the organic solvent 1 is 1:9; preferably, the weight-to-volume ratio (g / ml) of compound 1 in step A to the organic solvent 1 is 1:8; preferably, the weight-to-volume ratio (g / ml) of compound 1 in step A to the organic solvent 1 is 1:7; preferably, the weight-to-volume ratio (g / ml) of compound 1 in step A to the organic solvent 1 is 1:6; preferably, the weight-to-volume ratio (g / ml) of compound 1 in step A to the organic solvent 1 is 1:5. Furthermore, the reaction temperature of step A is 0-30°C. It can be understood that, in some preferred embodiments, the reaction temperature of step A may fluctuate within a certain range (eg, ±5°C, ±10°C, ±15°C). Preferably, the reaction temperature of step A is 0°C±5°C; preferably, the reaction temperature of step A is 1°C±5°C; preferably, the reaction temperature of step A is 2°C±5°C; preferably, the reaction temperature of step A is 3°C±5°C; preferably, the reaction temperature of step A is 4°C±5°C; preferably, the reaction temperature of step A is 5°C±5°C; preferably, the reaction temperature of step A is 6°C±5°C; preferably, the reaction temperature of step A is 7°C±5°C; preferably, the reaction temperature of step A is 8°C±5°C; preferably, the reaction temperature of step A is 9°C±5°C; preferably, the reaction temperature of step A is 10°C±5°C; preferably, the reaction temperature of step A is 11°C±5°C; preferably, the reaction temperature of step A is 12°C±5°C; preferably, the reaction temperature of step A is 13°C±5°C; preferably, the reaction temperature of step A is 14°C±5°C; preferably, the reaction temperature of step A is 1 Preferably, the reaction temperature of step A is 16°C±5°C; preferably, the reaction temperature of step A is 17°C±5°C; preferably, the reaction temperature of step A is 18°C±5°C; preferably, the reaction temperature of step A is 19°C±5°C; preferably, the reaction temperature of step A is 20°C±5°C; preferably, the reaction temperature of step A is 21°C±5°C; preferably, the reaction temperature of step A is 22°C±5°C; preferably, the reaction temperature of step A is 23°C±5°C; preferably, the reaction temperature of step A is 24°C±5°C; preferably, the reaction temperature of step A is 25°C±5°C; preferably, the reaction temperature of step A is 26°C±5°C; preferably, the reaction temperature of step A is 27°C±5°C; preferably, the reaction temperature of step A is 28°C±5°C; preferably, the reaction temperature of step A is 29°C±5°C; preferably, the reaction temperature of step A is 30°C±5°C. Preferably, the reaction temperature of step A is 0-5°C, 10-15°C, 20-30°C, 0-15°C, 0-10°C. Further, the weight-to-volume ratio (g / ml) of the compound 1 described in step A and the organic solvent 2 described in step B is 1:5-15. Preferably, the weight-to-volume ratio (g / ml) of the compound 1 described in step A and the organic solvent 2 described in step B is 1:5; preferably, the weight-to-volume ratio (g / ml) of the compound 1 described in step A and the organic solvent 2 described in step B is 1:6; preferably, the weight-to-volume ratio (g / ml) of the compound 1 described in step A and the organic solvent 2 described in step B is 1:7; preferably, the weight-to-volume ratio (g / ml) of the compound 1 described in step A and the organic solvent 2 described in step B is 1:8; preferably, the weight-to-volume ratio (g / ml) of the compound 1 described in step A and the organic solvent 2 described in step B is 1:9; preferably, the weight-to-volume ratio (g / ml) of the compound 1 described in step A and the organic solvent 2 described in step B is 1:10. Preferably, the weight-to-volume ratio (g / ml) of the compound 1 in step A to the organic solvent 2 in step B is 1:10; preferably, the weight-to-volume ratio (g / ml) of the compound 1 in step A to the organic solvent 2 in step B is 1:11; preferably, the weight-to-volume ratio (g / ml) of the compound 1 in step A to the organic solvent 2 in step B is 1:12; preferably, the weight-to-volume ratio (g / ml) of the compound 1 in step A to the organic solvent 2 in step B is 1:13; preferably, the weight-to-volume ratio (g / ml) of the compound 1 in step A to the organic solvent 2 in step B is 1:14; preferably, the weight-to-volume ratio (g / ml) of the compound 1 in step A to the organic solvent 2 in step B is 1:15. Further, the weight-to-volume ratio (g / ml) of the compound 1 in step A to the water in step B is 1:5-15. Preferably, the weight-to-volume ratio (g / ml) of the compound 1 in step A to the water in step B is 1:5; preferably, the weight-to-volume ratio (g / ml) of the compound 1 in step A to the water in step B is 1:6 ... The weight volume ratio (g / ml) of the compound 1 in step A and the water in step B is 1:7; preferably, the weight volume ratio (g / ml) of the compound 1 in step A and the water in step B is 1:8; preferably, the weight volume ratio (g / ml) of the compound 1 in step A and the water in step B is 1:9; preferably, the weight volume ratio (g / ml) of the compound 1 in step A and the water in step B is 1:10; preferably, the weight volume ratio (g / ml) of the compound 1 in step A and the water in step B is 1:10. The volume ratio (g / ml) is 1:11; preferably, the weight volume ratio (g / ml) of the compound 1 in step A and the water in step B is 1:12; preferably, the weight volume ratio (g / ml) of the compound 1 in step A and the water in step B is 1:13; preferably, the weight volume ratio (g / ml) of the compound 1 in step A and the water in step B is 1:14; preferably, the weight volume ratio (g / ml) of the compound 1 in step A and the water in step B is 1:15. Preferably, the water in step B is ice water. Preferably, the concentration in step C is concentration under reduced pressure. The present invention also provides the use of any of the above-mentioned preparation and purification methods in the preparation of MMAE. The present invention also provides the use of any of the above-mentioned preparation and purification methods in the preparation of MMAE intermediates. The use of the above-mentioned preparation and / or purification method of the methyl auristatin E compound intermediate (i.e., the compound represented by formula (I)) in the preparation of an antibody-drug conjugate with MMAE as a toxin. The use of the above-mentioned preparation and / or purification method of the methyl auristatin E compound intermediate (i.e., the compound represented by formula (I)) in the preparation of an antibody-drug conjugate intermediate using MMAE as a toxin. The preparation and purification method of the methyl auristatin E compound intermediate (i.e., the compound shown in formula (I)) provided by the present invention has a simple product purification method, and only needs to be extracted and concentrated in one step to obtain a target product with a high purity (>99%), and the preparation and purification method of the methyl auristatin E compound intermediate (i.e., the compound shown in formula (I)) provided by the present invention has a high yield (up to 95% to 105%), which can not only effectively save production steps, reduce production consumption and investment, but also effectively improve the yield of the product, and obtain a high yield and high purity target product while achieving energy saving and cost saving, greatly improving production efficiency, effectively reducing the use of reagents, and reducing production costs. In addition, since the preparation and purification method provided by the present invention has streamlined steps, low requirements on equipment and environment, high process stability, good stability between multiple batches, the final product color is better, the product quality is more stable, and it is more suitable for scaled-up production. BRIEF DESCRIPTION OF THE DRAWINGS FIG1 is a chromatogram of compound (I-1) prepared in Example 1; FIG2 is a chromatogram of compound (I-2) prepared in Example 2; FIG3 is a chromatogram of compound (I-3) prepared in Example 3; FIG4 is a chromatogram of compound (I-1) prepared in Example 4 FIG5 is a chromatogram of compound (I-1) prepared in Example 5; FIG6 is a chromatogram of compound (I-1) prepared in Comparative Example 1. DETAILED DESCRIPTION The technical scheme of the present invention is further described in detail in a non-limiting manner in conjunction with specific embodiments. It should be pointed out that the following embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention. Example 1 Preparation of Compound (I-1) Compound 1 (i.e. (2R, 3R)-N-((1S, 2R)-1-hydroxy-1-phenylpropan-2-yl)-3-methoxy-2-methyl-3-((S)-pyrrolidin-2-yl)propanamide hydrochloride, 1.0 g, 2.8 mmol), compound 2-1 (i.e. (5S, 8S, 11S, 12R)-11-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6, 9-trioxo-2-oxa-4,7,10-triazatetradecane-14-oic acid, 1.88 g, 2.95 mmol), HATU (i.e. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1.28 g, 3.4 mmol), DIPEA (i.e. N,N-diisopropylethylamine, 1.09 g, 8.4 mmol) and DMF (i.e. N,N-dimethylformamide, 9 ml, 9 vol) were reacted at 0-5°C under nitrogen protection. After the reaction was completed, the reaction solution was transferred to a separating funnel, and methyl tert-butyl ether (10 ml) and ice water (10 ml) were added in sequence for extraction, and the organic phase was separated. The mixture was concentrated under reduced pressure in a 100 mL single-necked round-bottom flask until foaming, and vacuum dried for no less than 1 h until the weight no longer changed, to give 2.59 g of compound I-1 (i.e., (9H-fluorene-9-yl)methyl ((S)-1-(((S)-1-(3R, 4S, 5S)-1-(S)-2-((1R, 2R)-3-((1S, 2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl (methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)3-methyl-1-oxobutan-2-yl)) (methyl) The product is white in color, and the purity is 99.67% after sampling and testing, and the maximum single impurity is 0.20%. The chromatogram is shown in Figure 1. Example 2 Preparation of Compound (I-2) Compound 1 (i.e. (2R, 3R)-N-((1S, 2R)-1-hydroxy-1-phenylpropan-2-yl)-3-methoxy-2-methyl-3-((S)-pyrrolidin-2-yl)propanamide hydrochloride, 20.0 g, 56 mmol), compound 2-2 (i.e. (6S, 9S, 12S, 13R)-12-((S)-sec-butyl)-6,9-diisopropyl-13-methoxy-2,2,5,11-tetramethyl-4,7,10-trioxo)-1,4-dihydro-2-nitro-1-phenylpropan-2-yl)-3-methoxy-2-methyl-3-((S)-pyrrolidin-2-yl)propanamide hydrochloride, 20.0 g, 56 mmol), -3-oxa-5,8,11-triazapentadecan-15-enoic acid, 34.68 g, 67.2 mmol), HATU (i.e. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 23.44 g, 61.6 mmol), DIPEA (i.e. N,N-diisopropylethylamine, 28.97 g, 224 mmol) and N-methylpyrrolidone (280 ml, 14 vol) were reacted at 10-15 °C under nitrogen protection. After the reaction was completed, the reaction solution was transferred to a separatory funnel, and petroleum ether (280 ml) and ice water (280 ml) were added in sequence for extraction, and the organic phase was separated. The mixture was concentrated under reduced pressure in a 1000 mL single-necked round-bottom flask until foaming, and vacuum dried for no less than 1 h until the weight no longer changed, to give 45.75 g of compound I-2 (i.e., tert-butyl (S)-1-(S)-1-(3R, 4S, 4S, 5S, 5S)-1-(S)-2-((1R, 2R, 2R)-3-(1S, 2Rs)-1-hydroxy-1-phenylpropane-2-phenylpropane-2-phenylpropane-2-phenylpropane-1-methoxymethylmethoxy-1-methoxymethylmethyl-1-methoxyethylethyl-1-methoxymethoxy-3-methoxy-5-methyl-1-oxoheptane-4-methylmethylmethyl-3-methoxymethyl-3-methyl-1-methyl-1-oxobutan-2-yl)(methyl)carbamate), with a yield of 99.8%. The product is off-white in color. Samples were taken to test its purity, which was 99.44% and the maximum single impurity was 0.26%. The chromatogram is shown in Figure 2. Example 3 Preparation of Compound (I-3) Compound 1 (i.e. (2R, 3R)-N-((1S, 2R)-1-hydroxy-1-phenylpropan-2-yl)-3-methoxy-2-methyl-3-((S)-pyrrolidin-2-yl)propanamide hydrochloride, 5.0 g, 14 mmol), compound 2-3 (i.e. (5S, 8S, 11S, 12R)-11-((S)-sec-butyl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6,9- trioxo-1-phenyl-2-oxa-4,7,10-triazatetradecane-14 acid, 11.55 g, 21 mmol), HATU (i.e. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 5.33 g, 14 mmol), DIPEA (i.e. N,N-diisopropylethylamine, 9.05 g, 70 mmol) and methanol (25 ml, 5 vol) were reacted at 20-30 °C under nitrogen protection. After the reaction was completed, the reaction solution was transferred to a separatory funnel, and n-heptane (40 ml) and ice water (40 ml) were added in sequence for extraction, and the organic phase was separated. The mixture was concentrated under reduced pressure in a 250 mL single-necked round-bottom flask until foaming, and vacuum dried for no less than 1 h until the weight no longer changed to give 11.59 g of compound I-3 (i.e., (S)-1-(((S)-1-[(3R, 4S, 5S)-1-(S)-2-(1R, 2R)-3-(((1S, 2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl)(methyl)amino)-3-methyl-1-oxobutan-2-yl)-3-methyl 1-oxobutan-2-yl-(methyl)carbamate)(methyl)carbamate), with a yield of 97.1%. The product is off-white in color. Samples were taken to test its purity, which was 99.46% and the maximum single impurity was 0.27%. The chromatogram is shown in Figure 3. Example 4 Preparation of Compound (I-1) Compound 1 (i.e. (2R, 3R)-N-((1S, 2R)-1-hydroxy-1-phenylpropan-2-yl)-3-methoxy-2-methyl-3-((S)-pyrrolidin-2-yl)propanamide hydrochloride, 2.0 g, 5.6 mmol), compound 2-1 (i.e. (5S, 8S, 11S, 12R)-11-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3, 6,9-trioxo-2-oxa-4,7,10-triazatetradecane-14-oic acid, 3.57 g, 5.6 mmol), HATU (i.e. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 2.13 g, 5.6 mmol), DIPEA (i.e. N,N-diisopropylethylamine, 1.45 g, 11.2 mmol) and DMF (i.e. N,N-dimethylformamide, 10 ml) were reacted at 0-15°C under nitrogen protection. After the reaction was completed, the reaction solution was transferred to a separating funnel, and methyl tert-butyl ether (10 ml) and ice water (10 ml) were added in sequence for extraction, and the organic phase was separated. The mixture was concentrated under reduced pressure in a 100 mL single-necked round-bottom flask until foaming and vacuum dried for no less than 1 h until the weight no longer changed to give 5.03 g of compound I-1 (i.e., (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-(3R, 4S, 5S)-1-(S)-2-((1R, 2R)-3-((1S, 2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl (methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)3-methyl-1-oxobutan-2-yl)(methyl)carbamate), with a yield of 95.6%. The product is white in color. Samples were taken to test its purity, which was 99.61% and the maximum single impurity was 0.23%. The chromatogram is shown in Figure 4. Example 5 Preparation of Compound (I-1) Compound 1 (i.e. (2R, 3R)-N-((1S, 2R)-1-hydroxy-1-phenylpropan-2-yl)-3-methoxy-2-methyl-3-((S)-pyrrolidin-2-yl)propanamide hydrochloride, 2.0 g, 5.6 mmol), compound 2-1 (i.e. (5S, 8S, 11S, 12R)-11-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-5,8-diisopropyl-12-methoxy-4,10-dimethyl-3,6 ,9-trioxo-2-oxa-4,7,10-triazatetradecane-14-oic acid, 7.14 g, 11.2 mmol), HATU (i.e. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 4.26 g, 11.2 mmol), DIPEA (i.e. N,N-diisopropylethylamine, 2.9 g, 22.4 mmol) and DMF (i.e. N,N-dimethylformamide, 30 ml), react at 0-10°C under nitrogen protection. After the reaction was completed, the reaction solution was transferred to a separating funnel, and methyl tert-butyl ether (30 ml) and ice water (30 ml) were added in sequence for extraction, and the organic phase was separated. The mixture was concentrated under reduced pressure in a 100 mL single-necked round-bottom flask until foaming and vacuum dried for no less than 1 h until the weight no longer changed to give 5.24 g of compound I-1 (i.e., (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-(3R, 4S, 5S)-1-(S)-2-((1R, 2R)-3-((1S, 2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl (methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)3-methyl-1-oxobutan-2-yl)(methyl)carbamate), with a yield of 99.6%. The product is off-white in color. Samples were taken to test its purity, which was 99.53% and the maximum single impurity was 0.32%. The chromatogram is shown in Figure 5. Comparative Example 1 Compound b (i.e. (2R, 3R)-N-((1S, 2R)-1-hydroxy-1-phenylpropan-2-yl)-3-methoxy-2-methyl-3-((S)-pyrrolidin-2-yl)propionamide trifluoroacetate, 1.0 g, 2.8 mmol), compound 2-1 (i.e. (5S, 8S, 11S, 12R)-11-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-5,8-diisopropyl-1 2-methoxy-4,10-dimethyl-3,6,9-trioxo-2-oxa-4,7,10-triazatetradecane-14-oic acid, 1.60 g, 2.94 mmol), DEPC (i.e. diethyl pyrocarbonate, 0.47 g, 3.4 mmol), DIPEA (i.e. N,N-diisopropylethylamine, 1.24 g, 11.2 mmol) and dichloromethane (10 ml, 10 vol) were reacted at 0-5 °C under argon protection. After the reaction is completed, the reaction solution is transferred to a separatory funnel, and ice water (15 ml) and dichloromethane (15 ml) are added in sequence for extraction, and the organic phase is separated and extracted twice with dichloromethane (15 ml). The organic phases are combined, washed once with KHSO4 aqueous solution (15 ml), NaHCO3 aqueous solution (15 ml), and NaCl aqueous solution (15 ml) in sequence, and then dried over anhydrous sodium sulfate (1 g), filtered, and concentrated under reduced pressure in a 100 mL single-mouth round-bottom flask to a foaming state, and vacuum dried for not less than 1 hour and the weight does not change, to obtain 1.02 g of compound I-1 (i.e., (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-(3R, 4S, 5S)-1-(S)-2-((1R, 2R)-3-((1S, 2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptane-4-yl(methyl)amino)-3-methyl-1-oxobutane-2-yl)amino)3-methyl-1-oxobutan-2-yl)(methyl)carbamate), yield 45.2%. The product color is off-white, and the purity is tested by sampling, the purity is 97.69%, the maximum single impurity is 0.34%, and the chromatogram is shown in Figure 6. Table 1 Comparison of product yield and purity between each embodiment and comparative example It can be seen from Table 1 that the yield of the preparation and purification process of the methyl auristatin E compound intermediate provided by the present invention is very high, up to more than 95% (even 99.8%), and in addition, the product purity obtained by the preparation and purification process provided by the present invention is high, up to more than 99%. The preparation and purification process provided by the prior art has a yield of only 45.2% and a low purity of only 97%, which cannot meet the needs of further production of drugs. Therefore, the present application has obvious technical advantages over the prior art. In addition, from the perspective of the reaction process and reaction / production / purification raw materials, the preparation and purification process steps involved in this application are simplified, and the raw materials involved are relatively few. Compared with the prior art, a large amount of input materials and equipment can be saved, thereby saving a large amount of production costs. And because the preparation and purification process steps involved in this application are simplified, there are no special equipment and environmental requirements, and the product approval is stable and suitable for industrial promotion. The present invention has been illustrated by various specific embodiments. However, it will be understood by those skilled in the art that the present invention is not limited to the specific embodiments, and that those skilled in the art can make various changes or modifications within the scope of the present invention, and that the various technical features mentioned in various places in this specification can be combined with each other without departing from the spirit and scope of the present invention. Such changes and modifications are all within the scope of the present invention.

Claims

1. A method for preparing and purifying a compound represented by formula (I): The preparation route of the compound is as follows: It is characterized in that The preparation and purification method comprises the following steps: A. dissolving compound 1, compound 2, a polypeptide condensing agent, and an organic base in an organic solvent 1, and obtaining a solution 1 after the reaction is completed; B. adding organic solvent 2 and water to the solution 1 for extraction; C. collecting the organic phase and concentrating it to obtain the compound of formula (I); Wherein, the R is an amino protecting group; The organic solvent 1 is selected from any one or more of N,N-dimethylformamide, N-methylpyrrolidone, methanol, ethanol, isopropanol, acetonitrile, acetone, tetrahydrofuran, acetonitrile, and 1,4-dioxane solvents; The organic solvent 2 is selected from any one or more of n-heptane, petroleum ether, n-hexane, cyclohexane, n-pentane, methylcyclohexane, and methyl tert-butyl ether; The polypeptide condensing agent is selected from any one or more of HATU, HBTU and TBTU; The organic base is selected from any one or more of DIPEA, triethylamine, 4-methylmorpholine and pyridine.

2. The preparation and purification method according to claim 1, characterized in that: The R group is selected from Fmoc protecting group, Boc protecting group, Cbz protecting group, and the compound 2 is selected from the following structures:

3. The preparation and purification method according to claim 2, characterized in that: The compound of formula (I) is selected from the following structures: 4.-The preparation and purification method according to any one of claims 1 to 3, characterized in that The step A is carried out under the protection of an inert gas; preferably, the inert gas is nitrogen, argon or helium.

5. The preparation and purification method according to any one of claims 1 to 4, characterized in that: The molar ratio of compound 1 to compound 2 in step A is 1:1-2; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1-1.5; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1-1.2; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1-1.1; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1-1.05; preferably, the molar ratio of compound 1 to compound 2 in step A is 1:1.

05.

6. The preparation and purification method according to any one of claims 1 to 5, characterized in that: The molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1-2; preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1-1.5; the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1-1.4; the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1-1.3; the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1-1.2; preferably, the molar ratio of compound 1 to the polypeptide condensing agent in step A is 1:1.

2.

7. The preparation and purification method according to any one of claims 1 to 6, characterized in that: The molar ratio of compound 1 in step A to the organic base is 1:2-5; preferably, the molar ratio of compound 1 in step A to the organic base is 1:2-3.5; preferably, the molar ratio of compound 1 in step A to the organic base is 1:2-3.4; preferably, the molar ratio of compound 1 in step A to the organic base is 1:2-3.3; preferably, the molar ratio of compound 1 in step A to the organic base is 1:2-3.2; preferably, the molar ratio of compound 1 in step A to the organic base is 1:2-3.1; preferably, the molar ratio of compound 1 in step A to the organic base is 1:2-3; preferably, the molar ratio of compound 1 in step A to the organic base is 1:

3.

8. The preparation and purification method according to any one of claims 1 to 7, characterized in that: The weight-to-volume ratio (g / ml) of the compound 1 in step A to the organic solvent 1 is 1:5-15; preferably, the weight-to-volume ratio (g / ml) of the compound 1 in step A to the organic solvent 1 is 1:5-14; preferably, the weight-to-volume ratio (g / ml) of the compound 1 in step A to the organic solvent 1 is 1:5-13; preferably, the weight-to-volume ratio (g / ml) of the compound 1 in step A to the organic solvent 1 is 1:5-12; preferably, the weight-to-volume ratio (g / ml) of the compound 1 in step A to the organic solvent 1 is 1:5-11; preferably, the weight-to-volume ratio (g / ml) of the compound 1 in step A to the organic solvent 1 is 1:5-10; preferably, the weight-to-volume ratio (g / ml) of the compound 1 in step A to the organic solvent 1 is 1:5-9; preferably, the weight-to-volume ratio (g / ml) of the compound 1 in step A to the organic solvent 1 is 1:

9.

9. The preparation and purification method according to any one of claims 1 to 8, characterized in that: The reaction temperature of step A is 0-30°C.

10. The preparation and purification method according to any one of claims 1 to 9, characterized in that: The weight-to-volume ratio (g / ml) of the compound 1 described in step A and the organic solvent 2 described in step B is 1:5-15; preferably, the weight-to-volume ratio (g / ml) of the compound 1 described in step A and the organic solvent 2 described in step B is 1:5-14; preferably, the weight-to-volume ratio (g / ml) of the compound 1 described in step A and the organic solvent 2 described in step B is 1:5-13; preferably, the weight-to-volume ratio (g / ml) of the compound 1 described in step A and the organic solvent 2 described in step B is 1:5-12; preferably, the weight-to-volume ratio (g / ml) of the compound 1 described in step A and the organic solvent 2 described in step B is 1:5-11; preferably, the weight-to-volume ratio (g / ml) of the compound 1 described in step A and the organic solvent 2 described in step B is 1:5-10; preferably, the weight-to-volume ratio (g / ml) of the compound 1 described in step A and the organic solvent 2 described in step B is 1:

10.

11. The preparation and purification method according to any one of claims 1 to 10, characterized in that: The weight-to-volume ratio (g / ml) of the compound 1 described in step A and the water described in step B is 1:5-15; preferably, the weight-to-volume ratio (g / ml) of the compound 1 described in step A and the water described in step B is 1:5-14; preferably, the weight-to-volume ratio (g / ml) of the compound 1 described in step A and the water described in step B is 1:5-13; preferably, the weight-to-volume ratio (g / ml) of the compound 1 described in step A and the water described in step B is 1:5-12; preferably, the weight-to-volume ratio (g / ml) of the compound 1 described in step A and the water described in step B is 1:5-11; preferably, the weight-to-volume ratio (g / ml) of the compound 1 described in step A and the water described in step B is 1:5-10; preferably, the weight-to-volume ratio (g / ml) of the compound 1 described in step A and the water described in step B is 1:10; further preferably, the water in step B is ice water.

12. The preparation and purification method according to any one of claims 1 to 11, characterized in that: The concentration described in step C is concentration under reduced pressure.

13. Use of the preparation and purification method according to any one of claims 1 to 12 in the preparation of MMAE intermediates.

14. Use of the preparation and purification method according to any one of claims 1 to 12 in the preparation of MMAE.

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