Preparation and purification method of methyl aurestatin E compound

Abstract

The invention relates to a preparation and purification method of MMAE, which mainly uses a purification method of toluene and n-heptane back drop crystallization to replace the operation of column chromatography purification in a method involved in the prior art, greatly improves the production efficiency, and effectively reduces the solvent dosage. Besides, the MMAE prepared by utilizing the provided preparation and purification method is relatively high in yield and purity, the production cost can be effectively reduced, and the method is good in stability and is more suitable for large-scale production.

Description

A preparation and purification method of methyl auristatin E compound Technical Field

[0001] The present invention relates to the field of compound synthesis methods, and in particular to methods for preparing and purifying a methyl auristatin E compound (ie, MMAE). Background Art

[0002] MMAE (Monomethyl Auristantin E, also known as methyl auristatin E) is a fully synthetic derivative of auristatin.

[0003] It can effectively inhibit mitosis by inhibiting tubulin polymerization and is currently widely used as a cytotoxic small molecule payload for the development of antibody drug conjugates (ADCs) for the treatment of cancer.

[0004] Chinese patent publication number CN105143199A discloses a method for preparing MMAE on page 29 of the specification:

[0005] The method is as follows: in an inert atmosphere, compound a is dissolved in acetonitrile and piperidine, stirred at ambient temperature overnight, and evaporated to dryness under reduced pressure. The residue is purified on a silica gel column with a mixture of dichloromethane and methanol to obtain MMAE in the form of a white solid. The yield of this method is relatively low, only 68%. In addition, the purification method of this method is silica gel column chromatography, which is relatively expensive and has very high requirements for equipment and operating skills of operators. In addition, due to the limited purification capacity of the silica gel column, the working efficiency in the production process is relatively low. Therefore, there is an urgent need for a synthesis and purification method with high yield, high purity, high efficiency and suitable for scaled-up production.

[0006] Summary of the Invention

[0007] In response to the above problems, the present invention provides a method for preparing and purifying MMAE, which has a simple and efficient purification method, high production efficiency, environmental friendliness, and high quality (high purity and high yield) of the final product.

[0008] Specifically, the present invention provides a method for preparing and purifying a compound represented by formula (I) (i.e., MMAE):

[0009] The preparation route of the preparation and purification method is as follows:

[0010] The R is an amino protecting group, and the preparation and purification method comprises the following steps:

[0011] A. removing the amino protecting group R from compound 1 under alkaline conditions;

[0012] B. After the reaction in step A is completed, add an appropriate amount of water to the reaction system in step A, stir, filter, and collect the filtrate;

[0013] C. adding an appropriate amount of the first organic solvent to the filtrate collected in step B, extracting, collecting and concentrating the organic phase to obtain a concentrate a;

[0014] D. adding a second organic solvent to the concentrate a obtained in step C, and dissolving the concentrate a to obtain a solution b;

[0015] E. adding the solution b obtained in step D dropwise to the third organic solvent, and when a large amount of solid precipitates, filtering and collecting the filter cake to obtain MMAE;

[0016] in:

[0017] The amino protecting group R is selected from Fmoc protecting group and trifluoroacetyl group;

[0018] The first organic solvent is selected from ethyl acetate, dichloromethane, isopropyl acetate, chloroform, and toluene;

[0019] The second organic solvent is selected from toluene, ethyl acetate, and acetone;

[0020] The third organic solvent is selected from n-heptane, petroleum ether, n-hexane, cyclohexane, n-pentane, methylcyclohexane, and methyl tert-butyl ether.

[0021] In some specific embodiments, the amino protecting group R is selected from the following structures:

[0022] In some specific embodiments, the compound 1 is selected from the following structures:

[0023] In some specific embodiments, the first organic solvent is ethyl acetate; or in other specific embodiments, the first organic solvent is dichloromethane; in other specific embodiments, the first organic solvent is isopropyl acetate; in other specific embodiments, the first organic solvent is chloroform; in other specific embodiments, the first organic solvent is toluene.

[0024] In some specific embodiments, the second organic solvent is toluene; or in other specific embodiments, the second organic solvent is ethyl acetate; in other specific embodiments, the second organic solvent is acetone.

[0025] In some specific embodiments, the third organic solvent is n-heptane; or in other specific embodiments, the third organic solvent is petroleum ether; in other specific embodiments, the third organic solvent is n-hexane; in other specific embodiments, the third organic solvent is cyclohexane; in other specific embodiments, the third organic solvent is n-pentane; in other specific embodiments, the third organic solvent is methylcyclohexane; in other specific embodiments, the third organic solvent is methyl tert-butyl ether.

[0026] It is understood that the first organic solvent, the second organic solvent, and the third organic solvent are selected independently of each other, that is, the organic solvents involved in each step (such as the first organic solvent involved in step C, the second organic solvent involved in step D, and the third organic solvent involved in step E) are selected independently of each other. In a more specific embodiment, the first organic solvent, the second organic solvent, and the third organic solvent can be the following combination:

[0027] In some specific embodiments, the water in step B is purified water.

[0028] In some specific embodiments, the dissolving method in step D is dissolving by stirring.

[0029] In some preferred embodiments, the weight-to-volume ratio (g / ml) of compound 1 in step A to the purified water in step B is 1:4-20. In some specific embodiments, the weight-to-volume ratio (g / ml) of compound 1 in step A to the purified water in step B is 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19 or 1:20. In other specific embodiments, the weight-to-volume ratio of compound 1 in step A to the purified water in step B can also be other ratios within the range, such as, but not limited to, 1:14.1, 1:14.2, 1:14.3, 1:14.4, 1:14.5, etc.

[0030] In some preferred embodiments, the stirring in step B is low-temperature stirring. In some more preferred embodiments, the temperature of the low-temperature stirring in step B is 0-10°C. In some specific embodiments, the temperature of the low-temperature stirring in step B is 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C; or in some specific embodiments, the temperature of the low-temperature stirring in step B can be other temperatures within the range; or in some specific embodiments, the temperature of the low-temperature stirring in step B can fluctuate by 1-5°C within the range of 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, such as -5°C, -4°C, -3°C, -2°C, -1°C or 11°C, 12°C, 13°C, 14°C, 15°C, etc.

[0031] In some preferred embodiments, the weight-to-volume ratio (g / ml) of compound 1 in step A to the first organic solvent in step C is 1:5 to 25. In some specific embodiments, the weight-to-volume ratio (g / ml) of compound 1 in step A to the first organic solvent in step C is 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24 or 1:25; in other specific embodiments, the weight-to-volume ratio of compound 1 in step A to the first organic solvent in step C can be other ratios within the range.

[0032] In some more preferred embodiments, the weight-to-volume ratio (g / ml) of compound 1 in step A to the first organic solvent in step C is 1:5 to 15. In some specific embodiments, the weight-to-volume ratio (g / ml) of compound 1 in step A to the first organic solvent in step C is 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14 or 1:15; or in other specific embodiments, the weight-to-volume ratio of compound 1 in step A to the first organic solvent in step C can be other ratios within the range.

[0033] In some preferred embodiments, the weight-to-volume ratio (g / ml) of the concentrate a in step C to the second organic solvent in step D is 1:3-15. In some specific embodiments, the weight-to-volume ratio (g / ml) of the concentrate a in step C to the second organic solvent in step D is 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, or 1:15; in other specific embodiments, the weight-to-volume ratio of the concentrate a in step C to the second organic solvent in step D may also be other ratios within the above range.

[0034] In some more preferred embodiments, the weight-to-volume ratio (g / ml) of the concentrate a in step C to the second organic solvent in step D is 1:5-10. In some specific embodiments, the weight-to-volume ratio (g / ml) of the concentrate a in step C to the second organic solvent in step D is 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10; in other specific embodiments, the weight-to-volume ratio of the concentrate a in step C to the second organic solvent in step D may also be other ratios within the above range.

[0035] In some preferred embodiments, the weight-to-volume ratio (g / ml) of the concentrate a in step C to the third organic solvent in step E is 1:15-45. In some specific embodiments, the weight-to-volume ratio (g / ml) of the concentrate a in step C to the third organic solvent in step E is 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44 or 1:45. Alternatively, in some other specific embodiments, the weight-to-volume ratio of the concentrate a in step C to the third organic solvent in step E is another ratio within the above range.

[0036] In some more preferred embodiments, the weight-to-volume ratio (g / ml) of the concentrate a in step C to the third organic solvent in step E is 1:20-40. In some specific embodiments, the weight-to-volume ratio (g / ml) of the concentrate a in step C to the third organic solvent in step E is 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, or 1:40. Alternatively, in other specific embodiments, the weight-to-volume ratio of the concentrate a in step C to the third organic solvent in step E is another ratio within the above range.

[0037] Alternatively, in some more preferred embodiments, the weight-to-volume ratio (g / ml) of the concentrate a in step C to the third organic solvent in step E is 1:25-35. In other specific embodiments, the weight-to-volume ratio (g / ml) of the concentrate a in step C to the third organic solvent in step E is 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, or 1:35. Alternatively, in other specific embodiments, the weight-to-volume ratio of the concentrate a in step C to the third organic solvent in step E is another ratio within the above range.

[0038] It can be understood that the concentrate a in step C is actually unpurified MMAE. The concentration described in step C refers to reduced pressure concentration, that is, the collected organic phase is concentrated to a certain degree by reduced pressure concentration. In some preferred embodiments, in step C, the collected organic phase is evaporated to dryness by reduced pressure concentration to obtain a solid concentrate a.

[0039] In some preferred embodiments, the base used in the "removal of the amino protecting group R under alkaline conditions" includes but is not limited to piperidine, diethylamine, and DBU. In some specific embodiments, piperidine is used to remove the amino protecting group of compound 1. In other specific embodiments, diethylamine is used to remove the amino protecting group of compound 1. In other specific embodiments, DBU is used to remove the amino protecting group of compound 1.

[0040] In some specific embodiments, piperidine is used to remove the amino protecting group of compound 1. The specific process can be non-limiting as follows: dissolving compound 1 in an appropriate amount of a fourth organic solvent (the fourth organic solvent can be selected from acetonitrile, ethanol, and methanol without limitation), stirring and dissolving, and then adding piperidine to start the reaction. In some preferred embodiments, the weight-to-volume ratio (g / ml) of compound 1 and the fourth organic solvent is 1:2 to 12 (such as 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, or other non-integer ratios, such as 1:2.4, 1:2.6, 1:2.8, ..., 1:3.7, ..., 1:5.5, ..., 1:10.4, ..., etc.). The equivalent ratio of compound 1 to piperidine is 1:2 to 8 (such as 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8 or other non-integer ratios, such as 1:2.4, 1:2.6, 1:2.8, ..., 1:3.7, ..., 1:5.5, ..., etc.). In some more specific embodiments, the specific process of removing the amino protecting group of compound 1 using piperidine is non-limiting as follows: dissolving compound 1 in an appropriate amount of acetonitrile, stirring and dissolving, and then adding piperidine to start the reaction, wherein the equivalent ratio of compound 1 to piperidine is 1:3, and after the reaction is completed, the next reaction is carried out. In other more specific embodiments, the specific process of removing the amino protecting group of compound 1 using piperidine is non-limiting as follows: dissolving compound 1 in an appropriate amount of methanol, stirring and dissolving, and then adding piperidine to start the reaction, wherein the equivalent ratio of compound 1 to piperidine is 1:4, and after the reaction is completed, the next reaction is carried out. Alternatively, in other more specific embodiments, the specific process of using piperidine to remove the amino protecting group of compound 1 is non-limiting as follows: dissolving compound 1 in an appropriate amount of ethanol, stirring to dissolve, and then adding piperidine to start the reaction, wherein the equivalent ratio of compound 1 to piperidine is 1:5. After the reaction is completed, the next step of the reaction is carried out.

[0041] In other specific embodiments, diethylamine is used to remove the amino protecting group of compound 1. The specific process can be non-limiting as follows: dissolving compound 1 in an appropriate amount of a fourth organic solvent (the fourth organic solvent can be selected from acetonitrile, ethanol, and methanol without limitation), stirring and dissolving, and then adding piperidine to start the reaction. In some preferred embodiments, the weight-to-volume ratio (g / ml) of compound 1 and the fourth organic solvent is 1:2 to 12 (such as 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, or other non-integer ratios, such as 1:2.4, 1:2.6, 1:2.8, ..., 1:3.7, ..., 1:5.5, ..., 1:10.4, ..., etc.). The equivalent ratio of compound 1 to diethylamine is 1:2 to 8 (e.g., 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8 or other non-integer ratios, such as 1:2.4, 1:2.6, 1:2.8, ..., 1:3.7, ..., 1:5.5, ..., etc.). In some more specific embodiments, the specific process of removing the amino protecting group of compound 1 using diethylamine is non-limiting as follows: dissolving compound 1 in an appropriate amount of methanol, stirring to dissolve, and then adding diethylamine to start the reaction, wherein the equivalent ratio of compound 1 to diethylamine is 1:3. After the reaction is completed, the next reaction is carried out. In some more specific embodiments, the specific process of removing the amino protecting group of compound 1 using diethylamine is non-limiting as follows: dissolving compound 1 in an appropriate amount of ethanol, stirring to dissolve, and then adding diethylamine to start the reaction, wherein the equivalent ratio of compound 1 to diethylamine is 1:3. After the reaction is completed, the next reaction is carried out. In other more specific embodiments, the specific process of removing the amino protecting group of compound 1 using diethylamine is non-limiting as follows: dissolving compound 1 in an appropriate amount of acetonitrile, stirring to dissolve, and then adding diethylamine to start the reaction, wherein the equivalent ratio of compound 1 to diethylamine is 1:4. After the reaction is completed, the next reaction is carried out.

[0042] In other specific embodiments, DBU is used to remove the amino protecting group of compound 1. The specific process can be non-limiting as follows: dissolving compound 1 in an appropriate amount of a fourth organic solvent (the fourth organic solvent can be selected from acetonitrile, ethanol, and methanol without limitation), stirring to dissolve, and then adding piperidine to start the reaction. In some preferred embodiments, the weight-to-volume ratio (g / ml) of compound 1 and the fourth organic solvent is 1:2 to 12 (such as 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, or other non-integer ratios, such as 1:2.4, 1:2.6, 1:2.8, ..., 1:3.7, ..., 1:5.5, ..., 1:10.4, ..., etc.). The equivalent ratio of compound 1 to DBU is 1:2 to 8 (e.g., 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8 or other non-integer ratios, such as 1:2.4, 1:2.6, 1:2.8, ..., 1:3.7, ..., 1:5.5, ..., etc.). In some more specific embodiments, the specific process of using DBU to remove the amino protecting group of compound 1 is non-limiting as follows: dissolving compound 1 in an appropriate amount of methanol, stirring to dissolve, and then adding DBU to start the reaction, wherein the equivalent ratio of compound 1 to DBU is 1:3. After the reaction is completed, the next reaction is carried out. In some more specific embodiments, the specific process of using DBU to remove the amino protecting group of compound 1 is non-limiting as follows: dissolving compound 1 in an appropriate amount of ethanol, stirring to dissolve, and then adding DBU to start the reaction, wherein the equivalent ratio of compound 1 to DBU is 1:3. After the reaction is completed, the next reaction is carried out. In other more specific embodiments, the specific process of using DBU to remove the amino protecting group of compound 1 is non-limiting as follows: dissolving compound 1 in an appropriate amount of acetonitrile, stirring to dissolve, and then adding DBU to start the reaction, wherein the equivalent ratio of compound 1 to DBU is 1:4. After the reaction is completed, the next reaction is carried out.

[0043] The present invention also provides the application of the reverse dropwise addition method in the preparation and purification of MMAE, wherein the preparation route is:

[0044] The R is an amino protecting group;

[0045] The reverse addition method is to dissolve the reaction product containing MMAE to be purified prepared according to the preparation route in a second organic solvent, and then reversely add the resulting solution dropwise to a third organic solvent, followed by filtration and drying to obtain purified MMAE; wherein:

[0046] The second organic solvent is selected from toluene, ethyl acetate, and acetone;

[0047] The third organic solvent is selected from n-heptane, petroleum ether, n-hexane, cyclohexane, n-pentane, methylcyclohexane, and methyl tert-butyl ether.

[0048] The reverse addition method comprises the following steps:

[0049] (1) dissolving the reaction product containing MMAE to be purified in a second organic solvent to obtain solution 1;

[0050] (2) The obtained solution 1 is added dropwise to a third organic solvent, and a large amount of solid is precipitated. The solid is filtered and the filter cake is collected to obtain the purified MMAE.

[0051] As the name implies, the "reaction product comprising MMAE to be purified, prepared according to the preparation route" or "reaction product comprising MMAE to be purified" refers to crude, unpurified MMAE obtained by the preparation route. For example, in certain embodiments of the present invention, the "reaction product comprising MMAE to be purified, prepared according to the preparation route" or "reaction product comprising MMAE to be purified" refers to the crude product obtained after removal of the amino protecting group of Compound 1, followed by filtration, extraction, and concentration under reduced pressure, i.e., concentrate a.

[0052] The "reverse addition method" described above is relative to the conventional organic experimental operation of adding a solvent to a solution containing a product. Specifically, in the present invention, it means "first dissolving the reaction product containing MMAE to be purified prepared according to the preparation route in a second organic solvent, and then reversely adding the resulting solution dropwise to a third organic solvent."

[0053] In some preferred embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified and the second organic solvent in step (1) is 1:3-15; preferably, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified and the second organic solvent in step (1) is 1:5-10.

[0054] In some preferred embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified in step (1) and the third organic solvent described in step (2) is 1:15-45; preferably, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified in step (1) and the third organic solvent described in step (2) is 1:20-40; preferably, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified in step (1) and the third organic solvent described in step (2) is 1:25-35.

[0055] In some preferred embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified and the second organic solvent in step (1) is 1:3 to 15. In some specific embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified and the second organic solvent in step (1) is 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14 or 1:15; in other specific embodiments, the weight-to-volume ratio of the reaction product containing MMAE to be purified and the second organic solvent in step (1) may also be other ratios within the above range.

[0056] In some more preferred embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified and the second organic solvent in step (1) is 1:5 to 10. In some specific embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified and the second organic solvent in step (1) is 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10; in other specific embodiments, the weight-to-volume ratio of the reaction product containing MMAE to be purified and the second organic solvent in step (1) may also be other ratios within the above range.

[0057] In some preferred embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified in step (1) and the third organic solvent in step (2) is 1:15 to 45. In some specific embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified in step (1) and the third organic solvent in step (2) is 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44 or 1:45. Alternatively, in some other specific embodiments, the weight-to-volume ratio of the reaction product containing MMAE to be purified in step (1) and the third organic solvent in step (2) is another ratio within the above range.

[0058] In some more preferred embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified in step (1) and the third organic solvent in step (2) is 1:20-40. In some specific embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified in step (1) and the third organic solvent in step (2) is 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39 or 1:40. Alternatively, in some other specific embodiments, the weight-to-volume ratio of the reaction product containing MMAE to be purified in step (1) and the third organic solvent in step (2) is other ratios within the above range.

[0059] Alternatively, in some more preferred embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified in step (1) and the third organic solvent in step (2) is 1:25 to 35. In other specific embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified in step (1) and the third organic solvent in step (2) is 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34 or 1:35. Alternatively, in other specific embodiments, the weight-to-volume ratio of the reaction product containing MMAE to be purified in step (1) and the third organic solvent in step (2) is another ratio within the above range.

[0060] The present invention also provides an application of a reverse addition method in purifying MMAE, wherein the reverse addition method comprises first dissolving the reaction product containing MMAE to be purified in a second organic solvent, then reversely adding the resulting solution dropwise to a third organic solvent, followed by filtering and drying to obtain purified MMAE; wherein:

[0061] The second organic solvent is selected from toluene, ethyl acetate, and acetone;

[0062] The third organic solvent is selected from n-heptane, petroleum ether, n-hexane, cyclohexane, n-pentane, methylcyclohexane, and methyl tert-butyl ether.

[0063] In some preferred embodiments, the reverse dripping method comprises the following steps:

[0064] (a) dissolving the reaction product containing MMAE to be purified in a second organic solvent to obtain solution 2;

[0065] (b) The obtained solution 2 is added dropwise to a third organic solvent, and a large amount of solid is precipitated. The solid is filtered and the filter cake is collected to obtain purified MMAE.

[0066] In some preferred embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified and the second organic solvent in step (a) is 1:3-15; preferably, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified and the second organic solvent in step (a) is 1:5-10.

[0067] In some preferred embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified in step (a) and the third organic solvent in step (b) is 1:15-45; preferably, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified in step (a) and the third organic solvent in step (b) is 1:20-40; preferably, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified in step (a) and the third organic solvent in step (b) is 1:25-35.

[0068] In some preferred embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified and the second organic solvent in step (a) is 1:3 to 15. In some specific embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified and the second organic solvent in step (a) is 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14 or 1:15; in other specific embodiments, the weight-to-volume ratio of the reaction product containing MMAE to be purified and the second organic solvent in step (a) may also be other ratios within the above range.

[0069] In some more preferred embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified and the second organic solvent in step (a) is 1:5 to 10. In some specific embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified and the second organic solvent in step (a) is 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10; in other specific embodiments, the weight-to-volume ratio of the reaction product containing MMAE to be purified and the second organic solvent in step (a) may also be other ratios within the above range.

[0070] In some preferred embodiments, the weight-to-volume ratio (g / ml) of the reaction product comprising MMAE to be purified in step (a) and the third organic solvent in step (b) is 1:15 to 45. In some specific embodiments, the weight-to-volume ratio (g / ml) of the reaction product comprising MMAE to be purified in step (a) and the third organic solvent in step (b) is 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44 or 1:45. Alternatively, in some other specific embodiments, the weight-to-volume ratio of the reaction product containing MMAE to be purified in step (a) and the third organic solvent in step (b) is another ratio within the above range.

[0071] In some more preferred embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified in step (a) and the third organic solvent in step (b) is 1:20-40. In some specific embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified in step (a) and the third organic solvent in step (b) is 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39 or 1:40. Alternatively, in other specific embodiments, the weight-to-volume ratio of the reaction product containing MMAE to be purified in step (a) and the third organic solvent in step (b) is other ratios within the above ranges.

[0072] Alternatively, in some more preferred embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified in step (a) and the third organic solvent in step (b) is 1:25-35. In other specific embodiments, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified in step (a) and the third organic solvent in step (b) is 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, or 1:35. Alternatively, in other specific embodiments, the weight-to-volume ratio of the reaction product containing MMAE to be purified in step (a) and the third organic solvent in step (b) is another ratio within the above range.

[0073] Application of the above-mentioned MMAE preparation and / or purification method in the preparation of antibody-drug conjugates with MMAE as a toxin.

[0074] The application of the above-mentioned MMAE preparation and / or purification method in the preparation of antibody-drug conjugate intermediates using MMAE as a toxin.

[0075] The preparation and purification method for MMAE provided by the present invention utilizes a purification method using back-titration crystallization with toluene and n-heptane, replacing the column chromatography purification step used in prior art methods. This significantly improves production efficiency, effectively reduces solvent usage, and lowers production costs. Furthermore, the MMAE produced using the preparation and purification method provided by the present invention exhibits significantly improved yield and purity, and the method exhibits excellent stability, making it more suitable for scale-up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] FIG1 is a chromatogram of purified MMAE prepared by the method provided in Example 1;

[0077] FIG2 is a chromatogram of purified MMAE prepared by the method provided in Example 2;

[0078] FIG3 is a chromatogram of purified MMAE prepared by the method provided in Example 3;

[0079] FIG4 is a chromatogram of purified MMAE prepared by the method provided in Example 4;

[0080] FIG5 is a chromatogram of purified MMAE prepared by the method provided in Example 5;

[0081] FIG6 is a chromatogram of purified MMAE prepared by the method provided in Example 6;

[0082] FIG7 is a chromatogram of purified MMAE prepared by the method provided in Example 7;

[0083] FIG8 is a chromatogram of purified MMAE prepared by the method provided in Example 8;

[0084] FIG9 is a chromatogram of purified MMAE prepared by the method provided in Example 9;

[0085] Figure 10 is a chromatogram of purified MMAE prepared by the method provided in Example 10;

[0086] Figure 11 is a chromatogram of purified MMAE prepared by the method provided in Example 11;

[0087] Figure 12 is a chromatogram of purified MMAE prepared by the method provided in Example 12;

[0088] Figure 13 is a chromatogram of purified MMAE prepared by the method provided in Example 13;

[0089] Figure 14 is a chromatogram of purified MMAE prepared by the method provided in Example 14;

[0090] Figure 15 is a chromatogram of purified MMAE prepared by the method provided in Example 15;

[0091] Figure 16 is a chromatogram of purified MMAE prepared by the method provided in Example 16;

[0092] Figure 17 is a chromatogram of purified MMAE prepared by the method provided in Comparative Example 1;

[0093] FIG18 is a chromatogram of MMAE prepared and purified by the method provided in Comparative Example 2. DETAILED DESCRIPTION

[0094] The technical solution of the present invention is further described in detail below 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. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and they are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included within the scope of protection of the present invention.

[0095] Example 1

[0096] The Fmoc protecting group in compound a was removed by alkaline method. The specific process was as follows: 100.0 g of compound a (i.e., Fmoc-MMAE, the same below) and 500 ml of acetonitrile were added to a three-necked flask, stirred and dissolved, and then 36.25 g of piperidine was added to start the reaction.

[0097] After the reaction was completed, 500 ml of purified water was added to the reaction solution, and the reaction bottle was placed in a low-temperature tank, stirred at 0-5°C for 0.5 h, and filtered.

[0098] 1000 ml of dichloromethane was added for extraction, and the organic phase was separated and concentrated to dryness under reduced pressure at 20-25°C to obtain 71.03 g of the concentrate (i.e., crude MMAE).

[0099] To the above concentrate (ie, crude MMAE), 710 ml of toluene was added to dissolve the product, and the product was transferred to a constant pressure dropping funnel.

[0100] Under stirring, the toluene solution of crude MMAE was added dropwise to 2131 ml of n-heptane. A large amount of solid precipitated and 67.6 g of pure MMAE was obtained by filtration with a yield of 88.5%, a purity of 99.97%, and a single impurity of 0.03%. The chromatogram is shown in Figure 1.

[0101] Example 2

[0102] The Fmoc protecting group in compound a was removed by alkaline method. The specific process was as follows: 2.0 g of compound a and 4 ml of acetonitrile were added to a three-necked flask, stirred and dissolved, and then 362 mg of piperidine was added to start the reaction.

[0103] After the reaction was completed, 8 ml of purified water was added to the reaction solution, and the reaction bottle was placed in a low-temperature tank, stirred at 0-5°C for 0.5 h, and filtered.

[0104] 10 ml of dichloromethane was added for extraction, and the organic phase was separated and concentrated to dryness under reduced pressure at 20-25°C to obtain 1.40 g of the concentrate (i.e., crude MMAE).

[0105] To the above concentrate (ie, crude MMAE), 4.2 ml of toluene was added to dissolve the product, and the product was transferred to a constant pressure dropping funnel.

[0106] Under stirring, the toluene solution of crude MMAE was added dropwise to 21 ml of n-heptane. A large amount of solid precipitated and 1.30 g of pure MMAE was obtained by filtration with a yield of 83.7%, a purity of 99.60%, and a single impurity of 0.21%. The chromatogram is shown in Figure 2.

[0107] Example 3

[0108] The Fmoc protecting group in compound a was removed by alkaline method. The specific process was as follows: 2.0 g of compound a and 4 ml of acetonitrile were added to a three-necked flask, stirred and dissolved, and then 362 mg of piperidine was added to start the reaction.

[0109] After the reaction was completed, 8 ml of purified water was added to the reaction solution, and the reaction bottle was placed in a low-temperature tank, stirred at 0-5°C for 0.5 h, and filtered.

[0110] 10 ml of dichloromethane was added for extraction, and the organic phase was separated and concentrated to dryness under reduced pressure at 20-25°C to obtain 1.42 g of the concentrate (i.e., crude MMAE).

[0111] To the above concentrate (ie, crude MMAE), 4.3 ml of toluene was added to dissolve the product, and the product was transferred to a constant pressure dropping funnel.

[0112] Under stirring, the toluene solution of crude MMAE was added dropwise to 28.4 ml of n-heptane. A large amount of solid precipitated and 1.30 g of pure MMAE was obtained by filtration. The yield was 85%, the purity was 99.58%, and the impurity content was 0.24%. The chromatogram is shown in Figure 3.

[0113] Example 4

[0114] The Fmoc protecting group in compound a was removed by alkaline method. The specific process was as follows: 2.0 g of compound a and 4 ml of acetonitrile were added to a three-necked flask, stirred and dissolved, and then 362 mg of piperidine was added to start the reaction.

[0115] After the reaction was completed, 8 ml of purified water was added to the reaction solution, and the reaction bottle was placed in a low-temperature tank, stirred at 0-5°C for 0.5 h, and filtered.

[0116] 10 ml of dichloromethane was added for extraction, and the organic phase was separated and concentrated to dryness under reduced pressure at 20-25°C to obtain 1.44 g of the concentrate (i.e., crude MMAE).

[0117] To the above concentrate (ie, crude MMAE), 4.3 ml of toluene was added to dissolve the product, and the product was transferred to a constant pressure dropping funnel.

[0118] Under stirring, the toluene solution of crude MMAE was added dropwise to 36 ml of n-heptane. A large amount of solid precipitated and 1.33 g of pure MMAE was obtained by filtration. The yield was 86.9%, the purity was 99.21%, and the impurity content was 0.35%. The chromatogram is shown in Figure 4.

[0119] Example 5

[0120] The Fmoc protecting group in compound a was removed by alkaline method. The specific process was as follows: 2.0 g of compound a and 24 ml of acetonitrile were added to a three-necked flask, stirred and dissolved, and then 1.45 g of piperidine was added to start the reaction.

[0121] After the reaction was completed, 40 ml of purified water was added to the reaction solution, and the reaction bottle was placed in a low-temperature tank, stirred at 0-5°C for 0.5 h, and filtered.

[0122] 50 ml of dichloromethane was added for extraction, and the organic phase was separated and concentrated to dryness under reduced pressure at 20-25°C to obtain 1.36 g of the concentrate (i.e., crude MMAE).

[0123] To the above concentrate (ie, crude MMAE), 20.4 ml of toluene was added to dissolve the product, and the product was transferred to a constant pressure dropping funnel.

[0124] Under stirring, the toluene solution of crude MMAE was added dropwise to 61.2 ml of n-heptane. A large amount of solid precipitated and 1.27 g of pure MMAE was obtained by filtration. The yield was 83%, the purity was 99.90%, and the impurity content was 0.06%. The chromatogram is shown in Figure 5.

[0125] Example 6

[0126] The Fmoc protecting group in compound a was removed by alkaline method. The specific process was as follows: 2.0 g of compound a and 24 ml of acetonitrile were added to a three-necked flask, stirred and dissolved, and then 1.45 g of piperidine was added to start the reaction.

[0127] After the reaction was completed, 20 ml of purified water was added to the reaction solution, and the reaction bottle was placed in a low-temperature tank, stirred at 0-5°C for 0.5 h, and filtered.

[0128] 30 ml of dichloromethane was added for extraction, and the organic phase was separated and concentrated to dryness under reduced pressure at 20-25°C to obtain 1.36 g of the concentrate (i.e., crude MMAE).

[0129] To the above concentrate (ie, crude MMAE), 13.6 ml of toluene was added to dissolve the product, and the product was transferred to a constant pressure dropping funnel.

[0130] Under stirring, the toluene solution of crude MMAE was added dropwise to 54.4 ml of n-heptane. A large amount of solid precipitated and 1.29 g of pure MMAE was obtained by filtration. The yield was 85%, the purity was 99.82%, and the impurity content was 0.11%. The chromatogram is shown in Figure 6.

[0131] Example 7

[0132] The Fmoc protecting group in compound a was removed by alkaline method. The specific process was as follows: 2.0 g of compound a and 24 ml of acetonitrile were added to a three-necked flask, stirred and dissolved, and then 1.45 g of piperidine was added to start the reaction.

[0133] After the reaction was completed, 20 ml of purified water was added to the reaction solution, and the reaction bottle was placed in a low-temperature tank, stirred at 0-5°C for 0.5 h, and filtered.

[0134] 30 ml of dichloromethane was added for extraction, and the organic phase was separated and concentrated to dryness under reduced pressure at 20-25°C to obtain 1.36 g of the concentrate (i.e., crude MMAE).

[0135] To the above concentrate (ie, crude MMAE), 13.6 ml of toluene was added to dissolve the product, and the product was transferred to a constant pressure dropping funnel.

[0136] Under stirring, the toluene solution of crude MMAE was added dropwise to 47.6 ml of n-heptane. A large amount of solid precipitated and 1.28 g of pure MMAE was obtained by filtration. The yield was 84%, the purity was 99.87%, and the impurity content was 0.13%. The chromatogram is shown in Figure 7.

[0137] Example 8

[0138] The Fmoc protecting group in compound a was removed by alkaline method. The specific process was as follows: 1.0 g of compound a and 5 ml of ethanol were added to a three-necked flask, stirred and dissolved, and then 453 mg of piperidine was added to start the reaction.

[0139] After the reaction was completed, 5 ml of purified water was added to the reaction solution, and the reaction bottle was placed in a low-temperature tank, stirred at 0-5°C for 0.5 h, and filtered.

[0140] 10 ml of ethyl acetate was added for extraction, and the organic phase was separated and concentrated to dryness under reduced pressure at 20-25°C to obtain 704 mg of a concentrate (i.e., crude MMAE).

[0141] To the above concentrate (ie, crude MMAE) was added 7 ml of ethyl acetate to dissolve the product, and the solution was transferred to a constant pressure dropping funnel.

[0142] Under stirring, the ethyl acetate solution of crude MMAE was added dropwise to 21.1 ml of petroleum ether. A large amount of solid precipitated and 634 mg of pure MMAE was obtained by filtration with a yield of 83%, a purity of 99.11%, and a single impurity of 0.25%. The chromatogram is shown in Figure 8.

[0143] Example 9

[0144] The Fmoc protecting group in compound a was removed by alkaline method. The specific process was as follows: 1.0 g of compound a and 5 ml of acetonitrile were added to a three-necked flask, stirred and dissolved, and then 453 mg of piperidine was added to start the reaction.

[0145] After the reaction was completed, 5 ml of purified water was added to the reaction solution, and the reaction bottle was placed in a low-temperature tank, stirred at 0-5°C for 0.5 h, and filtered.

[0146] 10 ml of isopropyl acetate was added for extraction, and the organic phase was separated and concentrated to dryness under reduced pressure at 20-25°C to obtain 639 mg of the concentrate (i.e., crude MMAE).

[0147] To the above concentrate (ie, crude MMAE), 6.4 ml of toluene was added to dissolve the product, and the product was transferred to a constant pressure dropping funnel.

[0148] Under stirring, the toluene solution of crude MMAE was added dropwise to 19.2 ml of cyclohexane. A large amount of solid precipitated and 588 mg of pure MMAE was obtained by filtration. The yield was 77%, the purity was 99.85%, and the impurity was 0.07%. The chromatogram is shown in Figure 9.

[0149] Example 10

[0150] The Fmoc protecting group in compound a was removed by alkaline method. The specific process was as follows: 1 g of compound a and 5 ml of acetonitrile were added to a three-necked flask, stirred and dissolved, and then 453 mg of piperidine was added to start the reaction.

[0151] After the reaction was completed, 5 ml of purified water was added to the reaction solution, and the reaction bottle was placed in a low-temperature tank, stirred at 0-5°C for 0.5 h, and filtered.

[0152] 10 ml of chloroform was added for extraction, and the organic phase was separated and concentrated to dryness under reduced pressure at 20-25°C to obtain 653 mg of the concentrate (i.e., crude MMAE).

[0153] To the above concentrate (ie, crude MMAE) was added 6.5 ml of ethyl acetate to dissolve the product, and the solution was transferred to a constant pressure dropping funnel.

[0154] Under stirring, the ethyl acetate solution of crude MMAE was added dropwise to 19.6 ml of n-pentane. A large amount of solid precipitated and 596 mg of pure MMAE was obtained by filtration. The yield was 78%, the purity was 99.63%, and the impurity content was 0.07%. The chromatogram is shown in Figure 10.

[0155] Example 11

[0156] The Fmoc protecting group in compound a was removed by alkaline method. The specific process was as follows: 1.0 g of compound a and 5 ml of acetonitrile were added to a three-necked flask, stirred and dissolved, and then 453 mg of piperidine was added to start the reaction.

[0157] After the reaction was completed, 5 ml of purified water was added to the reaction solution, and the reaction bottle was placed in a low-temperature tank, stirred at 0-5°C for 0.5 h, and filtered.

[0158] 10 ml of toluene was added for extraction, and the organic phase was separated and concentrated to dryness under reduced pressure at 20-25°C to obtain 649 mg of a concentrate (i.e., crude MMAE).

[0159] To the above concentrate (ie, crude MMAE) was added 6.5 ml of acetone to dissolve the product, and the product was transferred to a constant pressure dropping funnel.

[0160] Under stirring, the acetone solution of crude MMAE was added dropwise to 19.5 ml of methylcyclohexane. A large amount of solid precipitated and 580 mg of pure MMAE was obtained by filtration. The yield was 76%, the purity was 99.13%, and the impurity content was 0.21%. The chromatogram is shown in Figure 11.

[0161] Example 12

[0162] The Fmoc protecting group in compound a was removed by alkaline method. The specific process was as follows: 1.0 g of compound a and 5 ml of acetonitrile were added to a three-necked flask, stirred and dissolved, and then 453 mg of piperidine was added to start the reaction.

[0163] After the reaction was completed, 5 ml of purified water was added to the reaction solution, and the reaction bottle was placed in a low-temperature tank, stirred at 0-5°C for 0.5 h, and filtered.

[0164] 10 ml of dichloromethane was added for extraction, and the organic phase was separated and concentrated to dryness under reduced pressure at 20-25°C to obtain 693 mg of the concentrate (i.e., crude MMAE).

[0165] To the above concentrate (ie, crude MMAE), 6.9 ml of toluene was added to dissolve the product, and the product was transferred to a constant pressure dropping funnel.

[0166] Under stirring, the toluene solution of crude MMAE was added dropwise to 20.8 ml of methyl tert-butyl ether. A large amount of solid precipitated and 603 mg of pure MMAE was obtained by filtration. The yield was 79%, the purity was 99.77%, and the impurity content was 0.12%. The chromatogram is shown in Figure 12.

[0167] Example 13

[0168] 10.0 g of compound a and 50 ml of acetonitrile were added to a three-necked flask, stirred and dissolved, and then 2.71 g of piperidine was added to start the reaction.

[0169] After the reaction was completed, 80 ml of purified water was added to the reaction solution, and the reaction bottle was placed in a low-temperature tank at 0-5°C and stirred for 0.5 h.

[0170] 50 ml of chloroform was added for extraction, and the organic phase was separated and concentrated to dryness under reduced pressure at 20-25°C to obtain 6.70 g of crude MMAE.

[0171] 67 ml of toluene was added to the above crude MMAE to dissolve it, and the mixture was transferred to a constant pressure dropping funnel.

[0172] Under stirring, the toluene solution of crude MMAE was added dropwise to 201 ml of n-hexane. A large amount of solid precipitated and 6.49 g of pure MMAE was obtained by filtration. The yield was 85%, the purity was 99.96%, and the maximum single impurity was 0.04%. The chromatogram is shown in Figure 13.

[0173] Example 14

[0174] 1.0 g of compound a and 5 ml of acetonitrile were added to a three-necked flask, stirred and dissolved, and then 271 mg of piperidine was added to start the reaction.

[0175] After the reaction was completed, 10 ml of purified water was added to the reaction solution, and the reaction bottle was placed in a low-temperature tank at 0-5°C and stirred for 0.5 h.

[0176] 15 ml of dichloromethane was added for extraction, and the organic phase was separated and concentrated to dryness under reduced pressure at 20-25°C to obtain 683 mg of crude MMAE.

[0177] Add 6.8 ml of acetone to the above crude MMAE to dissolve it, and transfer it to a constant pressure dropping funnel.

[0178] Under stirring, the acetone solution of crude MMAE was added dropwise to 20.5 ml of n-heptane. A large amount of solid precipitated and 620 mg of pure MMAE was obtained by filtration. The yield was 81%, the purity was 99.44%, and the maximum single impurity was 0.25%. The chromatogram is shown in Figure 14.

[0179] Example 15

[0180] 1200 mg of compound a and 10 ml of methanol were added to a three-necked flask, stirred to dissolve, and then 544 mg of piperidine was added to start the reaction.

[0181] After the reaction was completed, 9.6 ml of purified water was added to the reaction solution, and the reaction bottle was placed in a low-temperature tank at 0-5°C and stirred for 0.5 h.

[0182] 12 ml of toluene was added for extraction, and the organic phase was separated and concentrated to dryness under reduced pressure at 20-25°C to obtain 770 mg of crude MMAE.

[0183] 11.6 ml of ethyl acetate was added to the above crude MMAE to dissolve it, and the mixture was transferred to a constant pressure dropping funnel.

[0184] Under stirring, the ethyl acetate solution of crude MMAE was added dropwise to 30.8 ml of n-hexane. A large amount of solid precipitated and 724 mg of pure MMAE was obtained by filtration. The yield was 79%, the purity was 99.20%, and the maximum single impurity was 0.38%. The chromatogram is shown in Figure 15.

[0185] Example 16

[0186] 10.0 g of compound a and 100 ml of acetonitrile were added to a three-necked flask, stirred to dissolve, and then 9.05 g of piperidine was added to start the reaction.

[0187] After the reaction was completed, 100 ml of purified water was added to the reaction solution, and the reaction bottle was placed in a low-temperature tank at 0-5°C and stirred for 0.5 h.

[0188] 150 ml of dichloromethane was added for extraction, and the organic phase was separated and concentrated to dryness under reduced pressure at 20-25°C to obtain 6.53 g of crude MMAE.

[0189] 98 ml of toluene was added to the above crude MMAE to dissolve it, and the mixture was transferred to a constant pressure dropping funnel.

[0190] Under stirring, the toluene solution of crude MMAE was added dropwise to 294 ml of n-heptane. A large amount of solid precipitated and 6.31 g of pure MMAE was obtained by filtration. The yield was 82.6%, the purity was 99.53%, and the impurity content was 0.07%. The chromatogram is shown in Figure 16.

[0191] Comparative Example 1

[0192] 10.0 g of compound a and 70 ml of dichloromethane were added to a three-necked flask, stirred to dissolve, and then 3.1 g of diethylamine was added to start the reaction.

[0193] After the reaction was completed, 100 ml of purified water and 70 ml of dichloromethane were added to the reaction solution for extraction, and the organic phase was separated and concentrated to dryness under reduced pressure at 20-25°C to obtain 6.68 g of crude MMAE.

[0194] The crude MMAE was purified by column chromatography, and the solvent was evaporated to obtain 5.11 g of pure MMAE with a yield of 66.9%, a purity of 98.43%, and a maximum single impurity of 0.22%. The chromatogram is shown in Figure 17.

[0195] Comparative Example 2

[0196] 1.0 g of compound a and 10 ml of dichloromethane were added to a three-necked flask, stirred to dissolve, and then 485 mg of DBU was added to start the reaction.

[0197] After the reaction was completed, the mixture was concentrated to dryness under reduced pressure at 20-25°C to obtain 1.52 g of crude MMAE.

[0198] Add 15 ml of methanol to dissolve the crude MMAE and transfer it to a constant pressure dropping funnel.

[0199] Under stirring, the methanol solution of crude MMAE was added dropwise to 45.6 ml of methyl tert-butyl ether. Solid precipitated and 557 mg of pure MMAE was obtained by filtration. The yield was 73%, the purity was 89.37%, and the impurity content was 7.10%. The chromatogram is shown in Figure 18.

[0200] Table 1 Comparison of product yield and purity between the examples and the comparative examples

[0201] As can be seen from Table 1, the preparation and purification method of MMAE provided by the present invention uses a reverse titration crystallization purification method instead of the column chromatography purification operation in the prior art method, greatly improving production efficiency and reducing production costs. Most importantly, while improving product purity, the product yield is significantly improved. Not only does the final product purity increase from 98.43% to 99.97%, but the yield is also significantly increased from 66.9% to 88.5%, greatly reducing production costs. In addition, the MMAE prepared using the preparation and purification method provided by the present invention has good method stability, is more suitable for scale-up production, and has unexpected technical effects.

[0202] The present invention has been illustrated through various specific embodiments. However, those skilled in the art will appreciate that the present invention is not limited to these specific embodiments. Those skilled in the art may make various modifications and variations within the scope of the present invention, and the various technical features described throughout this specification may be combined without departing from the spirit and scope of the present invention. Such modifications and variations are 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: The R is an amino protecting group, characterized in that: The preparation and purification method comprises the following steps: A. removing the amino protecting group R from compound 1 under alkaline conditions; B. After the reaction of step A is completed, add an appropriate amount of water to the reaction system of step A, stir, filter, and collect the filtrate; C. adding an appropriate amount of the first organic solvent to the filtrate collected in step B, extracting, collecting and concentrating the organic phase to obtain a concentrate a; D. adding a second organic solvent to the concentrate a obtained in step C, dissolving the concentrate a to obtain a solution b; E. adding the solution b obtained in step D dropwise into the third organic solvent, and filtering after a large amount of solid precipitates, collecting the filter cake, and obtaining MMAE; in: The amino protecting group R is selected from Fmoc protecting group and trifluoroacetyl group; The first organic solvent is selected from ethyl acetate, dichloromethane, isopropyl acetate, chloroform, and toluene; The second organic solvent is selected from toluene, ethyl acetate and acetone; The third organic solvent is selected from n-heptane, petroleum ether, n-hexane, cyclohexane, n-pentane, methylcyclohexane, and methyl tert-butyl ether.

2. The preparation and purification method according to claim 1, characterized in that: The compound 1 is selected from the following structures:

3. The preparation and purification method according to claim 1, characterized in that: The weight-to-volume ratio (g / ml) of compound 1 in step A to the water in step B is 1:4-20; preferably, the weight-to-volume ratio (g / ml) of compound 1 in step A to the water in step B is 1:4-10; preferably, the stirring in step B is low-temperature stirring; further preferably, the temperature of the low-temperature stirring is 0-10°C.

4. The preparation and purification method according to claim 1, characterized in that: The weight-to-volume ratio (g / ml) of compound 1 in step A to the first organic solvent described in step C is 1:5-25; preferably, the weight-to-volume ratio (g / ml) of compound 1 in step A to the first organic solvent described in step C is 1:5-15.

5. The preparation and purification method according to claim 1, characterized in that: The weight-to-volume ratio (g / ml) of the concentrate a in step C and the second organic solvent in step D is 1:3-15; preferably, the weight-to-volume ratio (g / ml) of the concentrate a in step C and the second organic solvent in step D is 1:5-10.

6. The preparation and purification method according to claim 1, characterized in that: The weight-to-volume ratio (g / ml) of the concentrate a in step C and the third organic solvent in step E is 1:15-45; preferably, the weight-to-volume ratio (g / ml) of the concentrate a in step C and the third organic solvent in step E is 1:20-40; preferably, the weight-to-volume ratio (g / ml) of the concentrate a in step C and the third organic solvent in step E is 1:25-35.

7. The preparation and purification method according to claim 1, characterized in that: The removal of the amino protecting group R from compound 1 under alkaline conditions described in step A comprises the following steps: dissolving compound 1 in an appropriate amount of a fourth organic solvent, stirring to dissolve, and then adding a base to react and remove the amino protecting group R; preferably, the fourth organic solvent is selected from acetonitrile, ethanol, and methanol; further preferably, the base is selected from piperidine, diethylamine, and DBU.

8. The preparation and purification method according to claim 7, characterized in that: The weight-to-volume ratio (g / ml) of the compound 1 and the fourth organic solvent is 1:2-12, and / or the equivalent ratio of the compound 1 and the base is 1:2-8.

9. The preparation and purification method according to claim 1, characterized in that: The water in step B is pure water, and / or the dissolving method in step D is dissolving by stirring.

10. Application of reverse dropping method in the preparation and purification of MMAE, wherein the preparation route of the MMAE is: The R is an amino protecting group; The reverse dropping method refers to dissolving the reaction product containing MMAE to be purified prepared according to the preparation route in a second organic solvent, and then reversely dropping the obtained solution into a third organic solvent, followed by filtering and drying to obtain purified MMAE; wherein: The second organic solvent is selected from toluene, ethyl acetate and acetone; The third organic solvent is selected from n-heptane, petroleum ether, n-hexane, cyclohexane, n-pentane, methylcyclohexane, and methyl tert-butyl ether.

11. The use according to claim 10, characterized in that: The reverse dropwise addition method comprises the following steps: (1) dissolving the reaction product containing MMAE to be purified in a second organic solvent to obtain a solution 1; (2) Add the obtained solution 1 dropwise into the third organic solvent, wait for a large amount of solid to precipitate, filter by suction, collect the filter cake, and obtain the purified MMAE.

12. The use according to claim 11, characterized in that: The weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified and the second organic solvent described in step (1) is 1:3 to 15; preferably, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified and the second organic solvent described in step (1) is 1:5 to 10.

13. The use according to claim 12, characterized in that: The weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified in step (1) and the third organic solvent in step (2) is 1:15-45; preferably, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified in step (1) and the third organic solvent in step (2) is 1:20-40; preferably, the weight-to-volume ratio (g / ml) of the reaction product containing MMAE to be purified in step (1) and the third organic solvent in step (2) is 1:25-35.

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