Method for producing and purifying monomethyl auristatin E compound

Abstract

This invention relates to a method for producing and purifying MMAE. By employing a purification method using back titration crystallization of toluene and n-heptane instead of the column chromatography purification procedure used in conventional techniques, production efficiency is significantly improved and solvent usage is effectively reduced. Furthermore, the MMAE produced by this production and purification method has high yield and purity, effectively reducing production costs, and the method is stable, making it suitable for scale-up production.

Description

Technical Field

[0001] The present invention belongs to the field of methods for synthesizing compounds, and specifically relates to a method for producing and purifying monomethyl auristatin E (i.e., MMAE) compounds.

Background Art

[0002] MMAE (Monomethyl Auristantin E, also called monomethyl auristatin E) is a totally synthetic derivative of auristatin, and can effectively suppress cell division by inhibiting the polymerization of tubulin. Therefore, it is widely used as a cytotoxic small molecule payload in the development of antibody drug conjugates (ADCs) for cancer treatment.

[0003] On page 29 of the specification of the Chinese patent application with the publication number CN105143199A, a method for producing MMAE is disclosed.

[0004]

Chemical

[0005] In that method, compound a was dissolved in acetonitrile and piperidine in an inert atmosphere, stirred overnight at ambient temperature, and then evaporated to dryness under reduced pressure. The residue was purified using a mixture of dichloromethane and methanol on a silica column to obtain MMAE in the form of a white solid.

Summary of the Invention

Problems to be Solved by the Invention

[0006] The yield obtained by this method was low, at only 68%. Furthermore, the purification method used was silica gel column chromatography, which was costly and required significant equipment and operator skill. Additionally, the limited purification capacity of silica gel columns led to reduced efficiency in the production process. Therefore, there is an urgent need to develop synthesis and purification methods that offer high yield, high purity, high efficiency, and are suitable for scale-up production. [Means for solving the problem]

[0007] In view of the above problems, the present invention provides a method for producing and purifying MMAE that is simple, highly efficient, has high production efficiency, is environmentally friendly, and has advantages such as high quality of the final product (high purity, high yield). Specifically, the present invention provides a method for producing and purifying a compound represented by the following formula (I) (i.e., MMAE):

[0008] [ka]

[0009] The manufacturing route in the aforementioned manufacturing and purification method is as follows:

[0010] [ka]

[0011] [However, in the formula, R is an amino protecting group.] The above manufacturing and purification method is Step A, remove the amino protecting group R from compound 1 under alkaline conditions. Step B involves adding an appropriate amount of water to the reaction system from Step A, stirring, filtering, and collecting the filtrate. Step C involves adding an appropriate amount of the first organic solvent to the filtrate recovered in Step B, extracting, collecting and concentrating the organic phase, and obtaining concentrate a. Step D involves adding a second organic solvent to the concentrate a obtained in step C and dissolving it to obtain solution b, and Step E includes adding the solution b obtained in step D dropwise to a third organic solvent to precipitate a large amount of solid material, then filtering by suction, and recovering the filtered cake to obtain MMAE. however, The amino protecting group R is selected from the group consisting of an Fmoc protecting group and a trifluoroacetyl group. The first organic solvent is selected from the group consisting of ethyl acetate, dichloromethane, isopropyl acetate, chloroform, and toluene. The second organic solvent is selected from the group consisting of toluene, ethyl acetate, and acetone. The third organic solvent is selected from the group consisting of n-heptane, petroleum ether, n-hexane, cyclohexane, n-pentane, methylcyclohexane, and methyl tert-butyl ether.

[0012] In some specific embodiments, the amino protecting group R is selected from the group consisting of the following structures.

[0013] [Table 1]

[0014] In some specific embodiments, compound 1 is compound a or compound b represented by the following structure.

[0015] [Table 2]

[0016] In some specific embodiments, the first organic solvent is ethyl acetate. Alternatively, in some other specific embodiments, the first organic solvent is dichloromethane. In some other specific embodiments, the first organic solvent is isopropyl acetate. In some other specific embodiments, the first organic solvent is chloroform. In some other specific embodiments, the first organic solvent is toluene.

[0017] In some specific embodiments, the second organic solvent is toluene. Alternatively, in some other specific embodiments, the second organic solvent is ethyl acetate. In some other specific embodiments, the second organic solvent is acetone.

[0018] In some specific embodiments, the third organic solvent is n-heptane. Alternatively, in some other specific embodiments, the third organic solvent is petroleum ether. In some other specific embodiments, the third organic solvent is n-hexane. In some other specific embodiments, the third organic solvent is cyclohexane. In some other specific embodiments, the third organic solvent is n-pentane. In some other specific embodiments, the third organic solvent is methylcyclohexane. In some other specific embodiments, the third organic solvent is methyl tert-butyl ether.

[0019] It should be understood that the selection of the first organic solvent, the second organic solvent and the third organic solvent is independent of each other. That is, the organic solvents in each step (for example, the first organic solvent in step C, the second organic solvent in step D and the third organic solvent 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 may be the following combinations.

[0020] [Table 3] JPEG2026513745000008.jpg255169

[0021] In some specific embodiments, the water in step B is purified water.

[0022] In some specific embodiments, the dissolution method in step D is dissolution by stirring.

[0023] 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 to 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 some other specific embodiments, the weight-to-volume ratio of compound 1 in step A to the purified water in step B is not limited but may be other ratios within the range of 1:14.1, 1:14.2, 1:14.3, 1:14.4, 1:14.5, etc.

[0024] 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 to 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, or 10°C. Alternatively, in some specific embodiments, the temperature of the low-temperature stirring in step B may be any other temperature within the range. Alternatively, in some other specific embodiments, the temperature of the low-temperature stirring in step B may vary by 1 to 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, or 10°C, for example, -5°C, -4°C, -3°C, -2°C, -1°C, or 11°C, 12°C, 13°C, 14°C, or 15°C.

[0025] 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 some other specific embodiments, the weight-to-volume ratio of compound 1 in step A to the first organic solvent in step C may be any other ratio within the range.

[0026] 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. Alternatively, in some other specific embodiments, the weight-to-volume ratio of compound 1 in step A to the first organic solvent in step C may be any other ratio within those ranges.

[0027] In some preferred embodiments, the weight-to-volume ratio (g / ml) of concentrate a in step C to the second organic solvent in step D is 1:3 to 15. In some specific embodiments, the weight-to-volume ratio (g / ml) of 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 some other specific embodiments, the weight-to-volume ratio of concentrate a in step C to the second organic solvent in step D may be any other ratio within the above range.

[0028] In some more preferred embodiments, the weight-to-volume ratio (g / ml) of concentrate a in step C to the second organic solvent in step D is 1:5 to 10. In some specific embodiments, the weight-to-volume ratio (g / ml) of 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 some other specific embodiments, the weight-to-volume ratio of concentrate a in step C to the second organic solvent in step D may be any other ratio within the above range.

[0029] In some preferred embodiments, the weight-to-volume ratio (g / ml) of concentrate a in step C to the third organic solvent in step E is 1:15 to 45. In some specific embodiments, the weight-to-volume ratio (g / ml) of 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-volume ratio of concentrate a in step C to the third organic solvent in step E is any other ratio within the above range.

[0030] In some more preferred embodiments, the weight-to-volume ratio (g / ml) of concentrate a in step C to the third organic solvent in step E is 1:20 to 40. In some specific embodiments, the weight-to-volume ratio (g / ml) of 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 some other specific embodiments, the weight-to-volume ratio of concentrate a in step C to the third organic solvent in step E is any other ratio within the above range.

[0031] Alternatively, in some more preferred embodiments, the weight-to-volume ratio (g / ml) of concentrate a in step C to the third organic solvent in step E is 1:25 to 35. In some other specific embodiments, the weight-to-volume ratio (g / ml) of 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 some other specific embodiments, the weight-to-volume ratio of concentrate a in step C to the third organic solvent in step E is any other ratio within the above range.

[0032] It should be understood that concentrate a in step C is actually unpurified MMAE, and the concentration in step C refers to vacuum concentration, that is, to concentrating the collected organic phase to a certain extent by vacuum concentration. In some preferred embodiments, in step C, the collected organic phase is evaporated to dryness by vacuum concentration, i.e., concentrate a in solid form is obtained.

[0033] In some preferred embodiments, the base used in the step of "removing the amino protecting group R under alkaline conditions" may include, but is not limited to, piperidine, diethylamine, and DBU. In some specific embodiments, piperidine is used to remove the amino protecting group from compound 1. In some other specific embodiments, diethylamine is used to remove the amino protecting group from compound 1. Or, in some other specific embodiments, DBU is used to remove the amino protecting group from compound 1.

[0034] In some specific embodiments, the amino protecting group is removed from compound 1 using piperidine. The specific process is not limited, but compound 1 may be dissolved in an appropriate amount of a fourth organic solvent (the fourth organic solvent may be selected from the group consisting of acetonitrile, ethanol, and methanol, but is not limited), and after stirring and dissolution, piperidine may be added to initiate the reaction. In some preferred embodiments, the weight-to-volume ratio (g / ml) of compound 1 to the fourth organic solvent is 1:2 to 12 (for example, non-integer ratios 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 1:2.4, 1:2.6, 1:2.8, ..., 1:3.7, ..., 1:5.5, ..., 1:10.4, ...). The equivalent ratio of compound 1 to piperidine is 1:2 to 8 (for example, non-integer ratios such as 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, or 1:2.4, 1:2.6, 1:2.8, ..., 1:3.7, ..., 1:5.5, ...). In some more specific embodiments, the specific process for removing the amino protecting group from compound 1 using piperidine is not limited, but compound 1 may be dissolved in an appropriate amount of acetonitrile, stirred and dissolved, and then piperidine may be added to initiate the reaction. In this case, the equivalent ratio of compound 1 to piperidine is 1:3, and after the reaction is complete, the next reaction is carried out. In some other more specific embodiments, the specific process for removing the amino protecting group from compound 1 using piperidine is not limited, but compound 1 may be dissolved in an appropriate amount of methanol, stirred and dissolved, and then piperidine may be added to initiate the reaction. In this case, the equivalent ratio of compound 1 to piperidine is 1:4, and after the reaction is complete, the next reaction is carried out. Alternatively, in another, more specific embodiment, the specific process for removing the amino protecting group from compound 1 using piperidine is not limited, but compound 1 may be dissolved in an appropriate amount of ethanol, stirred and dissolved, and then piperidine may be added to initiate the reaction. In this case, the equivalent ratio of compound 1 to piperidine is 1:5, and after the reaction is complete, the following reaction procedure is carried out.

[0035] In some other specific embodiments, the amino protecting group is removed from compound 1 using diethylamine. The specific process is not limited, but compound 1 may be dissolved in an appropriate amount of a fourth organic solvent (the fourth organic solvent may be selected from the group consisting of acetonitrile, ethanol, and methanol, but is not limited), and after stirring and dissolution, piperidine may be added to initiate the reaction. In some preferred embodiments, the weight-to-volume ratio (g / ml) of compound 1 to the fourth organic solvent is 1:2 to 12 (for example, non-integer ratios 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 1:2.4, 1:2.6, 1:2.8, ..., 1:3.7, ..., 1:5.5, ..., 1:10.4, ...). The equivalent ratio of compound 1 to diethylamine is 1:2 to 8 (for example, non-integer ratios such as 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, or 1:2.4, 1:2.6, 1:2.8, ..., 1:3.7, ..., 1:5.5, ...). In some more specific embodiments, the specific process for removing the amino protecting group from compound 1 using diethylamine is not limited, but compound 1 may be dissolved in an appropriate amount of methanol, stirred and dissolved, and then diethylamine may be added to initiate the reaction. In this case, the equivalent ratio of compound 1 to diethylamine is 1:3, and after the reaction is complete, the following reaction procedure is carried out. In some more specific embodiments, the specific process for removing the amino protecting group from compound 1 using diethylamine is not limited, but compound 1 may be dissolved in an appropriate amount of ethanol, stirred and dissolved, and then diethylamine may be added to initiate the reaction. In this case, the equivalent ratio of compound 1 to diethylamine is 1:3, and after the reaction is complete, the following reaction procedure is carried out. In some other, more specific embodiments, the specific process for removing the amino protecting group from compound 1 using diethylamine is not limited, but may involve dissolving compound 1 in an appropriate amount of acetonitrile, stirring to dissolve, and then adding diethylamine to initiate the reaction. In this case, the equivalent ratio of compound 1 to diethylamine is 1:4, and after the reaction is complete, the following reaction procedure is carried out.

[0036] In some other specific embodiments, the amino protecting group is removed from compound 1 using DBU. The specific process is not limited, but compound 1 may be dissolved in an appropriate amount of a fourth organic solvent (the fourth organic solvent may be selected from the group consisting of acetonitrile, ethanol, and methanol, but is not limited), and after stirring and dissolution, piperidine may be added to initiate the reaction. In some preferred embodiments, the weight-to-volume ratio (g / ml) of compound 1 to the fourth organic solvent is 1:2 to 12 (for example, non-integer ratios 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 1:2.4, 1:2.6, 1:2.8, ..., 1:3.7, ..., 1:5.5, ..., 1:10.4, ...). The equivalent ratio of compound 1 to DBU is 1:2 to 8 (for example, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, or non-integer ratios such as 1:2.4, 1:2.6, 1:2.8, ..., 1:3.7, ..., 1:5.5, ...). In some more specific embodiments, the specific process for removing the amino protecting group from compound 1 using DBU is not limited, but compound 1 may be dissolved in an appropriate amount of methanol, stirred and dissolved, and then DBU may be added to initiate the reaction. In this case, the equivalent ratio of compound 1 to DBU is 1:3, and after the reaction is complete, the following reaction procedure is carried out. In some more specific embodiments, the specific process for removing the amino protecting group from compound 1 using DBU is not limited, but compound 1 may be dissolved in an appropriate amount of ethanol, stirred and dissolved, and then DBU may be added to initiate the reaction. In this case, the equivalent ratio of compound 1 to DBU is 1:3, and after the reaction is complete, the following reaction procedure is carried out. In some other, more specific embodiments, the specific process for removing the amino protecting group from compound 1 using DBU is not limited, but may involve dissolving compound 1 in an appropriate amount of acetonitrile, stirring to dissolve, and then adding DBU to initiate the reaction. In this case, the equivalent ratio of compound 1 to DBU is 1:4, and after the reaction is complete, the following reaction procedure is carried out.

[0037] The present invention further provides the use of back titration in the production and purification of MMAE. The production route is as follows:

[0038] [ka]

[0039] In the formula, R is an amino protecting group.

[0040] The aforementioned back titration method refers to a method in which the MMAE-containing reaction product to be purified, produced according to the aforementioned production route, is dissolved in a second organic solvent, the resulting solution is then added dropwise to a third organic solvent, and the solution is subsequently filtered and dried to obtain purified MMAE. Here, the second organic solvent is selected from the group consisting of toluene, ethyl acetate, and acetone, and the third organic solvent is selected from the group consisting of n-heptane, petroleum ether, n-hexane, cyclohexane, n-pentane, methylcyclohexane, and methyl tert-butyl ether.

[0041] The aforementioned back titration method includes the following steps: Step (1): Dissolve the MMAE-containing reaction product to be purified in a second organic solvent to obtain solution 1; Step (2): The obtained solution 1 is added dropwise to the third organic solvent, causing a large amount of solid matter to precipitate. This is then filtered by suction, and the filtered cake is collected to obtain purified MMAE.

[0042] The above-mentioned "MMAE-containing reaction product to be purified, manufactured according to the above-mentioned manufacturing route" or "MMAE-containing reaction product to be purified" refers to crude MMAE produced by the above-mentioned manufacturing route and not purified. For example, in some embodiments of the present invention, "MMAE-containing reaction product to be purified, manufactured according to the above-mentioned manufacturing route" or "MMAE-containing reaction product to be purified" refers to the crude product, i.e., concentrate a, obtained by removing the amino protecting group from compound 1 and performing filtration, extraction, and vacuum concentration.

[0043] The "back titration method" mentioned above refers to a technique in which a solvent is added to a solution containing the product, in a manner consistent with conventional organic chemistry experiments. Specifically, in this invention, it refers to "dissolving the MMAE-containing reaction product to be purified, produced according to the aforementioned manufacturing route, in a second organic solvent, and then dropping the resulting solution into a third organic solvent."

[0044] In some preferred embodiments, the weight-to-volume ratio (g / ml) of the MMAE-containing reaction product to be purified in step (1) to the second organic solvent is 1:3 to 15, and preferably, the weight-to-volume ratio (g / ml) of the MMAE-containing reaction product to be purified in step (1) to the second organic solvent is 1:5 to 10.

[0045] In some preferred embodiments, the weight-to-volume ratio (g / ml) of the MMAE-containing reaction product to be purified in step (1) to the third organic solvent in step (2) is 1:15 to 45, preferably 1:20 to 40, and more preferably 1:25 to 35.

[0046] In some preferred embodiments, the weight-to-volume ratio (g / ml) of the MMAE-containing reaction product to be purified in step (1) to the second organic solvent is 1:3 to 15. In some specific embodiments, the weight-to-volume ratio (g / ml) of the MMAE-containing reaction product to be purified in step (1) to the second organic solvent 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 some other specific embodiments, the weight-to-volume ratio of the MMAE-containing reaction product to be purified in step (1) to the second organic solvent may be any other ratio within the above range.

[0047] In some more preferred embodiments, the weight-to-volume ratio (g / ml) of the MMAE-containing reaction product to be purified in step (1) to the second organic solvent is 1:5 to 10. In some specific embodiments, the weight-to-volume ratio (g / ml) of the MMAE-containing reaction product to be purified in step (1) to the second organic solvent is 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some other specific embodiments, the weight-to-volume ratio of the MMAE-containing reaction product to be purified in step (1) to the second organic solvent may be any other ratio within the above range.

[0048] In some preferred embodiments, the weight-to-volume ratio (g / ml) of the MMAE-containing reaction product to be purified in step (1) to 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 MMAE-containing reaction product to be purified in step (1) to 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-volume ratio of the MMAE-containing reaction product to be purified in step (1) to the third organic solvent in step (2) may be any other ratio within the above range.

[0049] In some more preferred embodiments, the weight-to-volume ratio (g / ml) of the MMAE-containing reaction product to be purified in step (1) to the third organic solvent in step (2) is 1:20 to 40. In some specific embodiments, the weight-to-volume ratio (g / ml) of the MMAE-containing reaction product to be purified in step (1) to 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 MMAE-containing reaction product to be purified in step (1) to the third organic solvent in step (2) may be any other ratio within the above range.

[0050] Alternatively, in some more preferred embodiments, the weight-to-volume ratio (g / ml) of the MMAE-containing reaction product to be purified in step (1) to the third organic solvent in step (2) is 1:25 to 35. In some other specific embodiments, the weight-to-volume ratio (g / ml) of the MMAE-containing reaction product to be purified in step (1) to 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 some other specific embodiments, the weight-to-volume ratio of the MMAE-containing reaction product to be purified in step (1) to the third organic solvent in step (2) may be any other ratio within the above range.

[0051] The present invention further provides the use of a back titration method in the purification of MMAE. The back titration method refers to first dissolving the MMAE-containing reaction product to be purified in a second organic solvent, then adding the resulting solution dropwise to a third organic solvent, and then filtering and drying to obtain purified MMAE. Here, the second organic solvent is selected from the group consisting of toluene, ethyl acetate, and acetone, and the third organic solvent is selected from the group consisting of n-heptane, petroleum ether, n-hexane, cyclohexane, n-pentane, methylcyclohexane, and methyl tert-butyl ether.

[0052] In some preferred embodiments, the back titration method includes the following steps: Step (a): Dissolve the MMAE-containing reaction product to be purified in a second organic solvent to obtain solution 2; Step (b): The obtained solution 2 is added dropwise to a third organic solvent to precipitate a large amount of solid matter, then filtered by suction, and the filtered cake is collected to obtain purified MMAE.

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

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

[0055] In some preferred embodiments, the weight-to-volume ratio (g / ml) of the MMAE-containing reaction product to be purified in step (a) to the second organic solvent is 1:3 to 15. In some specific embodiments, the weight-to-volume ratio (g / ml) of the MMAE-containing reaction product to be purified in step (a) to the second organic solvent 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 some other specific embodiments, the weight-to-volume ratio of the MMAE-containing reaction product to be purified in step (a) to the second organic solvent may be any other ratio within the above range.

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

[0057] In some preferred embodiments, the weight-to-volume ratio (g / ml) of the MMAE-containing reaction product to be purified in step (a) to 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 MMAE-containing reaction product to be purified in step (a) to 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-volume ratio of the MMAE-containing reaction product to be purified in step (a) to the third organic solvent in step (b) may be any other ratio within the above range.

[0058] In some more preferred embodiments, the weight-to-volume ratio (g / ml) of the MMAE-containing reaction product to be purified in step (a) to the third organic solvent in step (b) is 1:20 to 40. In some specific embodiments, the weight-to-volume ratio (g / ml) of the MMAE-containing reaction product to be purified in step (a) to 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 some other specific embodiments, the weight-to-volume ratio of the MMAE-containing reaction product to be purified in step (a) to the third organic solvent in step (b) may be any other ratio within the above range.

[0059] Alternatively, in some more preferred embodiments, the weight-to-volume ratio (g / ml) of the MMAE-containing reaction product to be purified in step (a) to the third organic solvent in step (b) is 1:25 to 35. In some other specific embodiments, the weight-to-volume ratio (g / ml) of the MMAE-containing reaction product to be purified in step (a) to 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 some other specific embodiments, the weight-to-volume ratio of the MMAE-containing reaction product to be purified in step (a) to the third organic solvent in step (b) may be any other ratio within the above range.

[0060] Further information provides the use of the above-mentioned method for producing and / or purifying MMAE in the production of antibody-drug conjugates using MMAE as a toxin.

[0061] Further information provides the use of the above-mentioned method for producing and / or purifying MMAE in the production of an intermediate for an antibody-drug conjugate using MMAE as a toxin.

[0062] The present invention provides a method for producing and purifying MMAE, which, by employing a purification method using back titration crystallization of toluene and n-heptane instead of the conventional column chromatography purification procedure, significantly improves production efficiency, effectively reduces solvent usage, and lowers production costs. Furthermore, the MMAE produced by the present invention has significantly higher yield and purity, and exhibits good method stability, making it more suitable for scale-up production. [Brief explanation of the drawing]

[0063] [Figure 1] This is a chromatogram of MMAE prepared and purified by the method of Example 1. [Figure 2] This is a chromatogram of MMAE prepared and purified by the method of Example 2. [Figure 3]This is a chromatogram of MMAE prepared and purified by the method of Example 3. [Figure 4] This is a chromatogram of MMAE prepared and purified by the method of Example 4. [Figure 5] This is a chromatogram of MMAE prepared and purified by the method of Example 5. [Figure 6] This is a chromatogram of MMAE prepared and purified by the method of Example 6. [Figure 7] This is a chromatogram of MMAE prepared and purified by the method of Example 7. [Figure 8] This is a chromatogram of MMAE prepared and purified by the method of Example 8. [Figure 9] This is a chromatogram of MMAE prepared and purified by the method of Example 9. [Figure 10] This is a chromatogram of MMAE prepared and purified by the method of Example 10. [Figure 11] This is a chromatogram of MMAE prepared and purified by the method of Example 11. [Figure 12] This is a chromatogram of MMAE prepared and purified by the method of Example 12. [Figure 13] This is a chromatogram of MMAE prepared and purified by the method of Example 13. [Figure 14] This is a chromatogram of MMAE prepared and purified by the method of Example 14. [Figure 15] This is a chromatogram of MMAE prepared and purified by the method of Example 15. [Figure 16] This is a chromatogram of MMAE prepared and purified by the method of Example 16. [Figure 17] This is a chromatogram of MMAE produced and purified using the method of Comparative Example 1. [Figure 18] This is a chromatogram of MMAE produced and purified by the method of Comparative Example 2. [Modes for carrying out the invention]

[0064] The technical concept of the present invention will be described in more detail below, in a non-limiting manner, along with specific embodiments. It should be noted that the following embodiments are merely for illustrating the technical idea and features of the present invention and are intended to enable those skilled in the art to understand and implement the invention accordingly, and are not intended to limit the scope of protection. Equivalent changes or modifications made in accordance with the spirit of the present invention should be included within the scope of protection. (Example 1)

[0065] [ka]

[0066] The Fmoc protecting group in compound a was removed by the alkaline method. The specific procedure was as follows: 100.0 g of compound a (i.e., Fmoc-MMAE, hereafter the same) and 500 ml of acetonitrile were added to a three-necked flask, and after stirring to dissolve, 36.25 g of piperidine was added to start the reaction.

[0067] After the reaction was complete, 500 ml of purified water was added to the reaction mixture, the reaction flask was placed in a low-temperature bath, stirred at 0-5°C for 0.5 hours, and then filtered.

[0068] Extraction was performed by adding 1000 ml of dichloromethane, and the organic phase was separated. The mixture was concentrated to dryness under reduced pressure at 20-25°C to obtain 71.03 g of concentrate (i.e., crude MMAE).

[0069] The above concentrate (i.e., crude MMAE) was dissolved in 710 ml of toluene and transferred to a constant-pressure dropping funnel.

[0070] Under stirring, a toluene solution of crude MMAE was added dropwise to 2131 ml of n-heptane, causing a large amount of solid material to precipitate. The solution was then filtered to obtain 67.6 g of pure MMAE. The yield was 88.5%, the purity 99.97%, and the single impurity content was 0.03%. The chromatogram is shown in Figure 1. (Example 2)

[0071] [ka]

[0072] The Fmoc protecting group in compound a was removed by the alkaline method. The specific procedure was as follows: 2.0 g of compound a and 4 ml of acetonitrile were added to a three-necked flask, stirred to dissolve, and then 362 mg of piperidine was added to start the reaction.

[0073] After the reaction was complete, 8 ml of purified water was added to the reaction mixture, the reaction flask was placed in a low-temperature bath and stirred at 0-5°C for 0.5 hours, and then filtered.

[0074] Extraction was performed by adding 10 ml of dichloromethane, and the organic phase was separated. The mixture was concentrated to dryness under reduced pressure at 20-25°C to obtain 1.40 g of concentrate (i.e., crude MMAE).

[0075] The above concentrate (i.e., crude MMAE) was dissolved in 4.2 ml of toluene and transferred to a constant-pressure dropping funnel.

[0076] Under stirring, a toluene solution of crude MMAE was added dropwise to 21 ml of n-heptane, causing a large amount of solid material to precipitate. The solution was then filtered by suction to obtain 1.30 g of pure MMAE. The yield was 83.7%, the purity was 99.60%, and the single impurity content was 0.21%. The chromatogram is shown in Figure 2. (Example 3)

[0077] [ka]

[0078] The Fmoc protecting group in compound a was removed by the alkaline method. The specific procedure was as follows: 2.0 g of compound a and 4 ml of acetonitrile were added to a three-necked flask, stirred to dissolve, and then 362 mg of piperidine was added to start the reaction.

[0079] After the reaction was complete, 8 ml of purified water was added to the reaction mixture, the reaction flask was placed in a low-temperature bath and stirred at 0-5°C for 0.5 hours, and then filtered.

[0080] Extraction was performed by adding 10 ml of dichloromethane, and the organic phase was separated. The mixture was concentrated to dryness under reduced pressure at 20-25°C to obtain 1.42 g of concentrate (i.e., crude MMAE).

[0081] The above concentrate (i.e., crude MMAE) was dissolved in 4.3 ml of toluene and transferred to a constant-pressure dropping funnel.

[0082] Under stirring, a toluene solution of crude MMAE was added dropwise to 28.4 ml of n-heptane, causing a large amount of solid material to precipitate. The solution was then filtered by suction to obtain 1.30 g of pure MMAE. The yield was 85%, the purity was 99.58%, and the single impurity content was 0.24%. The chromatogram is shown in Figure 3. (Example 4)

[0083] [ka]

[0084] The Fmoc protecting group in compound a was removed by the alkaline method. The specific procedure was as follows: 2.0 g of compound a and 4 ml of acetonitrile were added to a three-necked flask, stirred to dissolve, and then 362 mg of piperidine was added to start the reaction.

[0085] After the reaction was complete, 8 ml of purified water was added to the reaction mixture, the reaction flask was placed in a low-temperature bath and stirred at 0-5°C for 0.5 hours, and then filtered.

[0086] Extraction was performed by adding 10 ml of dichloromethane, and the organic phase was separated. The mixture was concentrated to dryness under reduced pressure at 20-25°C to obtain 1.44 g of concentrate (i.e., crude MMAE).

[0087] The above concentrate (i.e., crude MMAE) was dissolved in 4.3 ml of toluene and transferred to a constant-pressure dropping funnel.

[0088] Under stirring, a toluene solution of crude MMAE was added dropwise to 36 ml of n-heptane, causing a large amount of solid material to precipitate. The solution was then filtered by suction to obtain 1.33 g of pure MMAE. The yield was 86.9%, the purity was 99.21%, and the single impurity content was 0.35%. The chromatogram is shown in Figure 4. (Example 5)

[0089] [ka]

[0090] The Fmoc protecting group in compound a was removed by the alkaline method. The specific procedure was as follows: 2.0 g of compound a and 24 ml of acetonitrile were added to a three-necked flask, stirred to dissolve, and then 1.45 g of piperidine was added to start the reaction.

[0091] After the reaction was complete, 40 ml of purified water was added to the reaction mixture, the reaction flask was placed in a low-temperature bath, stirred at 0-5°C for 0.5 hours, and then filtered.

[0092] Extraction was performed by adding 50 ml of dichloromethane, and the organic phase was separated. The mixture was concentrated to dryness under reduced pressure at 20-25°C to obtain 1.36 g of concentrate (i.e., crude MMAE).

[0093] The above concentrate (i.e., crude MMAE) was dissolved in 20.4 ml of toluene and transferred to a constant-pressure dropping funnel.

[0094] Under stirring, a toluene solution of crude MMAE was added dropwise to 61.2 ml of n-heptane, causing a large amount of solid material to precipitate. This was then filtered by suction to obtain 1.27 g of pure MMAE. The yield was 83%, the purity was 99.90%, and the single impurity content was 0.06%. The chromatogram is shown in Figure 5. (Example 6)

[0095] [ka]

[0096] The Fmoc protecting group in compound a was removed by the alkaline method. The specific procedure was as follows: 2.0 g of compound a and 24 ml of acetonitrile were added to a three-necked flask, stirred to dissolve, and then 1.45 g of piperidine was added to start the reaction.

[0097] After the reaction was complete, 20 ml of purified water was added to the reaction mixture, the reaction flask was placed in a low-temperature bath and stirred at 0-5°C for 0.5 hours, and then filtered.

[0098] Extraction was performed by adding 30 ml of dichloromethane, and the organic phase was separated. The mixture was concentrated to dryness under reduced pressure at 20-25°C to obtain 1.36 g of concentrate (i.e., crude MMAE).

[0099] The above concentrate (i.e., crude MMAE) was dissolved in 13.6 ml of toluene and transferred to a constant-pressure dropping funnel.

[0100] Under stirring, a toluene solution of crude MMAE was added dropwise to 54.4 ml of n-heptane, causing a large amount of solid material to precipitate. The solution was then filtered by suction to obtain 1.29 g of pure MMAE. The yield was 85%, the purity was 99.82%, and the single impurity content was 0.11%. The chromatogram is shown in Figure 6. (Example 7)

[0101] [ka]

[0102] The Fmoc protecting group in compound a was removed by the alkaline method. The specific procedure was as follows: 2.0 g of compound a and 24 ml of acetonitrile were added to a three-necked flask, stirred to dissolve, and then 1.45 g of piperidine was added to start the reaction.

[0103] After the reaction was complete, 20 ml of purified water was added to the reaction mixture, the reaction flask was placed in a low-temperature bath and stirred at 0-5°C for 0.5 hours, and then filtered.

[0104] Extraction was performed by adding 30 ml of dichloromethane, and the organic phase was separated. The mixture was concentrated to dryness under reduced pressure at 20-25°C to obtain 1.36 g of concentrate (i.e., crude MMAE).

[0105] The above concentrate (i.e., crude MMAE) was dissolved in 13.6 ml of toluene and transferred to a constant-pressure dropping funnel.

[0106] Under stirring, a toluene solution of crude MMAE was added dropwise to 47.6 ml of n-heptane, causing a large amount of solid material to precipitate. This was then filtered by suction to obtain 1.28 g of pure MMAE. The yield was 84%, the purity was 99.87%, and the single impurity content was 0.13%. The chromatogram is shown in Figure 7. (Example 8)

[0107] [ka]

[0108] The Fmoc protecting group was removed from compound a by the alkaline method. The specific procedure was as follows: 1.0 g of compound a and 5 ml of ethanol were added to a three-necked flask, stirred to dissolve, and then 453 mg of piperidine was added to start the reaction.

[0109] After the reaction was complete, 5 ml of purified water was added to the reaction mixture, the reaction flask was placed in a low-temperature bath, stirred at 0-5°C for 0.5 hours, and then filtered.

[0110] Extraction was performed by adding 10 ml of ethyl acetate, and the organic phase was separated. The mixture was concentrated to dryness under reduced pressure at 20-25°C to obtain 704 mg of concentrate (i.e., crude MMAE).

[0111] The above concentrate (i.e., crude MMAE) was dissolved in 7 ml of ethyl acetate and transferred to a constant-pressure dropping funnel.

[0112] Under stirring, an ethyl acetate solution of crude MMAE was added dropwise to 21.1 ml of petroleum ether, causing a large amount of solid material to precipitate. The solution was then filtered by suction to obtain 634 mg of pure MMAE. The yield was 83%, the purity was 99.11%, and the single impurity content was 0.25%. The chromatogram is shown in Figure 8. (Example 9)

[0113] [ka]

[0114] The Fmoc protecting group was removed from compound a by the alkaline method. The specific procedure was as follows: 1.0 g of compound a and 5 ml of acetonitrile were added to a three-necked flask, stirred to dissolve, and then 453 mg of piperidine was added to start the reaction.

[0115] After the reaction was complete, 5 ml of purified water was added to the reaction mixture, the reaction flask was placed in a low-temperature bath, stirred at 0-5°C for 0.5 hours, and then filtered.

[0116] Extraction was performed by adding 10 ml of isopropyl acetate, and the organic phase was separated. The mixture was concentrated to dryness under reduced pressure at 20-25°C to obtain 639 mg of concentrate (i.e., crude MMAE).

[0117] The above concentrate (i.e., crude MMAE) was dissolved in 6.4 ml of toluene and transferred to a constant-pressure dropping funnel.

[0118] Under stirring, a toluene solution of crude MMAE was added dropwise to 19.2 ml of cyclohexane, causing a large amount of solid material to precipitate. The solution was then filtered by suction to obtain 588 mg of pure MMAE. The yield was 77%, the purity was 99.85%, and the single impurity content was 0.07%. The chromatogram is shown in Figure 9. (Example 10)

[0119] [ka]

[0120] The Fmoc protecting group in compound a was removed by the alkaline method. The specific procedure was as follows: 1 g of compound a and 5 ml of acetonitrile were added to a three-necked flask, stirred to dissolve, and then 453 mg of piperidine was added to start the reaction.

[0121] After the reaction was complete, 5 ml of purified water was added to the reaction mixture, the reaction flask was placed in a low-temperature bath, stirred at 0-5°C for 0.5 hours, and then filtered.

[0122] Extraction was performed by adding 10 ml of chloroform, and the organic phase was separated. The mixture was concentrated to dryness under reduced pressure at 20-25°C to obtain a concentrate (i.e., crude MMAE) of 653 mg.

[0123] The above concentrate (i.e., crude MMAE) was dissolved in 6.5 ml of ethyl acetate and transferred to a constant-pressure dropping funnel.

[0124] Under stirring, an ethyl acetate solution of crude MMAE was added dropwise to 19.6 ml of n-pentane, and a large amount of solid material precipitated. The solution was then filtered by suction to obtain 596 mg of pure MMAE. The yield was 78%, the purity was 99.63%, and the single impurity content was 0.07%. The chromatogram is shown in Figure 10. (Example 11)

[0125] [ka]

[0126] The Fmoc protecting group in compound a was removed by the alkaline method. The specific procedure was as follows: 1.0 g of compound a and 5 ml of acetonitrile were added to a three-necked flask, stirred to dissolve, and then 453 mg of piperidine was added to start the reaction.

[0127] After the reaction was complete, 5 ml of purified water was added to the reaction mixture, the reaction flask was placed in a low-temperature bath, stirred at 0-5°C for 0.5 hours, and then filtered.

[0128] Extraction was performed by adding 10 ml of toluene, and the organic phase was separated. The mixture was concentrated to dryness under reduced pressure at 20-25°C to obtain a concentrate (i.e., crude MMAE) of 649 mg.

[0129] The above concentrate (i.e., crude MMAE) was dissolved in 6.5 ml of acetone and transferred to a constant-pressure dropping funnel.

[0130] Under stirring, an acetone solution of crude MMAE was added dropwise to 19.5 ml of methylcyclohexane, causing a large amount of solid material to precipitate. The solution was then filtered by suction to obtain 580 mg of pure MMAE. The yield was 76%, the purity was 99.13%, and the single impurity content was 0.21%. The chromatogram is shown in Figure 11. (Example 12)

[0131] [ka]

[0132] The Fmoc protecting group in compound a was removed by the alkaline method. The specific procedure was as follows: 1.0 g of compound a and 5 ml of acetonitrile were added to a three-necked flask, stirred to dissolve, and then 453 mg of piperidine was added to start the reaction.

[0133] After the reaction was complete, 5 ml of purified water was added to the reaction mixture, the reaction flask was placed in a low-temperature bath, stirred at 0-5°C for 0.5 hours, and then filtered.

[0134] Extraction was performed by adding 10 ml of dichloromethane, and the organic phase was separated. The mixture was concentrated to dryness under reduced pressure at 20-25°C to obtain 693 mg of concentrate (i.e., crude MMAE).

[0135] The above concentrate (i.e., crude MMAE) was dissolved in 6.9 ml of toluene and transferred to a constant-pressure dropping funnel.

[0136] Under stirring, a toluene solution of crude MMAE was added dropwise to 20.8 ml of methyl tert-butyl ether, causing a large amount of solid material to precipitate. The solution was then filtered by suction to obtain 603 mg of pure MMAE. The yield was 79%, the purity was 99.77%, and the single impurity content was 0.12%. The chromatogram is shown in Figure 12. Example 13)

[0137] [ka]

[0138] 10.0 g of compound a and 50 ml of acetonitrile were added to a three-necked flask, and after stirring to dissolve, 2.71 g of piperidine was added to start the reaction.

[0139] After the reaction was complete, 80 ml of purified water was added to the reaction mixture, and the reaction flask was placed in a low-temperature bath and stirred at 0-5°C for 0.5 hours.

[0140] Extraction was performed by adding 50 ml of chloroform, and the organic phase was separated. The mixture was concentrated to dryness under reduced pressure at 20-25°C to obtain 6.70 g of crude MMAE.

[0141] The crude MMAE mentioned above was dissolved in 67 ml of toluene and transferred to a constant-pressure dropping funnel.

[0142] Under stirring, a toluene solution of crude MMAE was added dropwise to 20 ml of n-hexane, causing a large amount of solid material to precipitate. The solution was then filtered by suction to obtain 6.49 g of pure MMAE. The yield was 85%, the purity was 99.96%, and the maximum single impurity was 0.04%. The chromatogram is shown in Figure 13. (Example 14)

[0143] [ka]

[0144] 1.0 g of compound a and 5 ml of acetonitrile were added to a three-necked flask, and after stirring to dissolve, 271 mg of piperidine was added to start the reaction.

[0145] After the reaction was complete, 10 ml of purified water was added to the reaction mixture, and the reaction flask was placed in a low-temperature bath and stirred at 0-5°C for 0.5 hours.

[0146] Extraction was performed by adding 15 ml of dichloromethane, and the organic phase was separated. The mixture was concentrated to dryness under reduced pressure at 20-25°C to obtain a crude product of 683 mg of MMAE.

[0147] The crude MMAE described above was dissolved in 6.8 ml of acetone and transferred to a constant-pressure dropping funnel.

[0148] Under stirring, an acetone solution of crude MMAE was added dropwise to 20.5 ml of n-heptane, and after a large amount of solid material precipitated, 620 mg of pure MMAE was obtained by suction 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. (Example 15)

[0149] [ka]

[0150] 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.

[0151] After the reaction was complete, 9.6 ml of purified water was added to the reaction mixture, and the reaction flask was placed in a low-temperature bath and stirred at 0-5°C for 0.5 hours.

[0152] Extraction was performed by adding 12 ml of toluene, and the organic phase was separated. The mixture was concentrated to dryness under reduced pressure at 20-25°C to obtain 770 mg of crude MMAE.

[0153] The crude MMAE described above was dissolved in 11.6 ml of ethyl acetate and transferred to a constant-pressure dropping funnel.

[0154] Under stirring, an ethyl acetate solution of crude MMAE was added dropwise to 30.8 ml of n-hexane, causing a large amount of solid material to precipitate. The solution was then filtered by suction to obtain 724 mg of pure MMAE. The yield was 79%, the purity was 99.20%, and the maximum single impurity was 0.38%. The chromatogram is shown in Figure 15. (Example 16)

[0155] [ka]

[0156] 10.0 g of compound a and 100 ml of acetonitrile were added to a three-necked flask, and after stirring to dissolve, 9.05 g of piperidine was added to start the reaction.

[0157] After the reaction was complete, 100 ml of purified water was added to the reaction mixture, and the reaction flask was placed in a low-temperature bath and stirred at 0-5°C for 0.5 hours.

[0158] Extraction was performed by adding 150 ml of dichloromethane, and the organic phase was separated. The mixture was concentrated to dryness under reduced pressure at 20-25°C to obtain 6.53 g of crude MMAE.

[0159] The crude MMAE mentioned above was dissolved in 98 ml of toluene and transferred to a constant-pressure dropping funnel.

[0160] Under stirring, a toluene solution of crude MMAE was added dropwise to 294 ml of n-heptane, causing a large amount of solid material to precipitate. The solution was then filtered by suction to obtain 6.31 g of pure MMAE. The yield was 82.6%, the purity was 99.53%, and the single impurity content was 0.07%. The chromatogram is shown in Figure 16. (Comparative Example 1)

[0161] [ka]

[0162] 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.

[0163] After the reaction was complete, 100 ml of purified water and 70 ml of dichloromethane were added to the reaction mixture and extracted to separate the organic phase. The mixture was concentrated to dryness under reduced pressure at 20-25°C to obtain 6.68 g of crude MMAE.

[0164] The crude MMAE was purified by column chromatography, and the solvent was evaporated to dryness to obtain 5.11 g of pure MMAE. The yield was 66.9%, the purity was 98.43%, and the maximum single impurity was 0.22%. The chromatogram is shown in Figure 17. (Comparative Example 2)

[0165] [ka]

[0166] 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.

[0167] After the reaction was complete, the mixture was concentrated to dryness under reduced pressure at 20-25°C to obtain 1.52 g of crude MMAE.

[0168] 15 ml of methanol was added to the crude MMAE product described above and transferred to a constant-pressure dropping funnel.

[0169] Under stirring, a methanol solution of crude MMAE was added dropwise to 45.6 ml of methyl tert-butyl ether to precipitate solids, and then filtered by suction to obtain 557 mg of pure MMAE. The yield was 73%, the purity was 89.37%, and the single impurity content was 7.10%. The chromatogram is shown in Figure 18.

[0170] [Table 4]

[0171] As is clear from Table 1, the MMAE production and purification method according to the present invention significantly improves production efficiency and reduces production costs by employing a purification method by back titration crystallization instead of the column chromatography purification procedure used in conventional techniques. Most importantly, the purity of the product is improved, and the yield is significantly increased. Not only is the purity of the final product improved from 98.43% to 99.97%, but the yield is also significantly increased from 66.9% to 88.5%, resulting in a substantial reduction in production costs. Furthermore, the MMAE produced by the production and purification method according to the present invention exhibits good method stability, is suitable for scale-up production, and has unexpected technical advantages.

[0172] The present invention has been described through various specific embodiments. However, as those skilled in the art will understand, the present invention is not limited to these various specific embodiments, and a person of ordinary skill can make various changes and modifications within the scope of the invention, and the various technical features described herein can be combined with one another without departing from the spirit and scope of the invention. All such changes and modifications are within the scope of the invention.

Claims

1. A method for producing and purifying a compound represented by the following formula (I), 【Chemistry 1】 The aforementioned compound is produced by the following route: 【Chemistry 2】 [However, R in the formula represents an amino protecting group.] The above manufacturing and purification method consists of the following steps: Step A involves removing the amino protecting group R from compound 1 under alkaline conditions, Step B involves adding an appropriate amount of water to the reaction system from Step A, stirring, filtering, and collecting the filtrate. Step C involves adding an appropriate amount of the first organic solvent to the filtrate recovered in step B, extracting, collecting and concentrating the organic phase, and obtaining concentrate a. Step D involves adding a second organic solvent to the concentrate a obtained in step C, dissolving it, and obtaining a solution b. Step E includes adding the solution b obtained in step D dropwise to a third organic solvent to precipitate a large amount of solid matter, then filtering by suction, and recovering the filtered cake to obtain MMAE. however, The amino protecting group R is selected from the group consisting of an Fmoc protecting group and a trifluoroacetyl group. The first organic solvent is selected from the group consisting of ethyl acetate, dichloromethane, isopropyl acetate, chloroform, and toluene. The second organic solvent is selected from the group consisting of toluene, ethyl acetate, and acetone. The manufacturing and purification method is characterized in that the third organic solvent is selected from the group consisting of n-heptane, petroleum ether, n-hexane, cyclohexane, n-pentane, methylcyclohexane, and methyl tert-butyl ether. 【Request Item 2】 【Chemistry 3】 The manufacturing and purification method according to claim 1, characterized in that the compound 1 is compound a or compound b having the above structure.

3. The weight-to-volume ratio (g / ml) of compound 1 in step A to the water in step B is 1:4 to 20. Preferably, the weight-to-volume ratio (g / ml) of compound 1 in step A to the water in step B is 1:4 to 10. Preferably, the stirring in step B is low-temperature stirring, and more preferably, the temperature of the low-temperature stirring is 0 to 10°C, as described in claim 1.

4. 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. Preferably, the manufacturing 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 in step C is 1:5 to 15.

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

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

7. Step A, which removes the amino protecting group R from compound 1 under alkaline conditions, includes dissolving compound 1 in an appropriate amount of a fourth organic solvent, stirring to dissolve it, then adding a base and reacting to remove the amino protecting group R. Preferably, the fourth organic solvent is selected from the group consisting of acetonitrile, ethanol, and methanol. More preferably, the manufacturing and purification method according to claim 1, characterized in that the base is selected from the group consisting of piperidine, diethylamine, and DBU.

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

9. The manufacturing and purification method according to claim 1, characterized in that the water in step B is purified water and / or the dissolution in step D is by stirring.

10. The use of back titration in the manufacture and purification of MMAE, The MMAE is manufactured through the following process: 【Chemistry 4】 [However, R in the formula represents an amino protecting group.] The aforementioned back titration method involves first dissolving the MMAE-containing reaction product to be purified, produced according to the aforementioned route, in a second organic solvent, then dropping the resulting solution into a third organic solvent, and subsequently filtering and drying to obtain purified MMAE. however, The second organic solvent is selected from the group consisting of toluene, ethyl acetate, and acetone. The use is characterized in that the third organic solvent is selected from the group consisting of n-heptane, petroleum ether, n-hexane, cyclohexane, n-pentane, methylcyclohexane, and methyl tert-butyl ether.

11. The back titration method comprises the steps (1) of dissolving the MMAE-containing reaction product to be purified in a second organic solvent to obtain solution 1, The use according to claim 10, characterized by comprising the step (2) of adding the obtained solution 1 dropwise to a third organic solvent to precipitate a large amount of solid matter, then filtering by suction, and collecting the filtered cake to obtain purified MMAE.

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

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

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