Preparation method of connexon-drug

Abstract

The present invention relates to a linker-drug preparation method, and particularly provides a target compound preparation method, which comprises: reacting a compound represented by a formula a with Lewis acid to obtain a crude product; and purifying the crude product to obtain the compound as shown in the formula A. The preparation method can reach the quality control standard of the product.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a method for preparing a linker-drug. Background Art

[0002] Antibody-Drug Conjugates (ADCs) are molecules with specific targeted killing effects obtained by connecting small molecule toxins with cell-killing effects to antibodies through linkers. They are mainly used in the treatment of tumors and other diseases. The antibodies used in antibody-drug conjugates can specifically bind to proteins on the surface of tumor cells, so they have tumor specificity and potential that traditional drugs cannot achieve. At present, 12 ADC drugs have been approved for marketing worldwide, and hundreds of projects are undergoing clinical trials.

[0003] Linker-drug is a compound formed by linking a linker to a small molecule toxin. It is an intermediate in the preparation of ADC and plays a key role in determining the overall success of antibody-drug conjugates. Summary of the invention

[0004] The object of the present invention is to provide a new method for preparing the linker-drug represented by formula A.

[0005]

[0006] Specifically, in the first aspect of the present invention, the present invention provides a method for preparing a compound represented by formula A, comprising:

[0007]

[0008] The compound represented by formula a is reacted with Lewis acid to obtain a crude product; the crude product is purified to obtain a compound represented by formula A;

[0009]

[0010] In some embodiments, the Lewis acid is a bromide, TMSI (trimethylsilyl iodide), or TMSOTf (trimethylsilyl trifluoromethanesulfonate).

[0011] In some embodiments, the Lewis acid is zinc bromide.

[0012] The inventors have found that the impurities that may be produced when compound a is subjected to an acidic deprotection agent include:

[0013] 1) Single desorption product impurities

[0014] 2) Impurities in Compound A

[0015] 3) Drug unit impurities

[0016] The generation of the above impurities will affect the drug loading rate (DAR) of the final ADC prepared. Therefore, in the production process of the target compound finished product, the content of the above impurities must be strictly controlled (the mono-dehydrogenated product must be controlled within 10%, the compound a must be controlled within 2%, and the drug unit must be controlled within 5%) to ensure the coupling effect of the target compound and the antibody.

[0017] The inventors of the present application have found through experimental research that in the above reaction, compared with non-Lewis acids (such as trifluoroacetic acid, etc.) as acidic deprotection reagents, when Lewis acids (such as zinc bromide) are used as acidic deprotection reagents, the impurity content is significantly reduced, especially the content of drug unit impurities, which can be as low as no more than 5%, or even as low as no more than 2%, meeting the quality control standards, or even far exceeding the quality control standards. In particular, when zinc bromide is used as an acidic deprotection reagent, the impurity content is significantly reduced, far exceeding the quality control standards.

[0018] Furthermore, after the above reaction is completed, when the crude product is purified, the inventors found that the impurities that may be produced include:

[0019]

[0020] 1) Drug unit impurities

[0021] 2) Impurities in the product of the ring opening of the connector

[0022] The generation of the above impurities will significantly affect the drug loading (DAR) of the ADC finally prepared (specifically, on the one hand, the succinimide structure contained in the linker-drug connector shown in formula A undergoes Michael addition with the antibody thiol group through a double bond to form ADC. It can be seen that the generated open-loop product impurities can no longer be coupled with the antibody, thereby affecting the drug loading. On the other hand, the generation of drug unit impurities causes it to be separated from the linker. The separated linker will still be coupled with the antibody but will no longer produce drug efficacy, thereby affecting the drug loading). Therefore, in the production process of the target compound finished product, the content of the above impurities must be strictly controlled (the open-loop product must be controlled within 10%, and the drug unit must be controlled within 5%) to ensure the coupling effect of the target compound and the antibody.

[0023] The inventors of the present application found through experimental research that in the above purification, by combining preparative liquid chromatography purification under certain conditions with a nanofiltration step, the impurity content is significantly reduced, and the quality control standard of the product can be achieved (the open-loop product is controlled within 10%, and the drug unit is controlled within 5%), or even far better than the quality control standard (the open-loop product is as low as within 3%, and the drug unit is as low as within 2%).

[0024] In some embodiments, the purification comprises the following steps:

[0025] (2-1) purifying the crude product by preparative liquid chromatography to obtain a preparative solution;

[0026] (2-2) concentrating and drying the prepared solution to obtain a compound represented by formula A;

[0027] Optionally, after obtaining the preparation solution in step (2-1) and before step (2-2), an extraction step is further included;

[0028] Optionally, after obtaining the preparation solution in step (2-1) and before step (2-2), the following steps are further included:

[0029] (2-i) extracting the prepared solution with an organic solvent, discarding the organic phase, and obtaining an aqueous phase;

[0030] Wherein, in step (2-1), when purification is performed by preparative liquid chromatography, mobile phase A is TFA / water and mobile phase B is TFA / acetonitrile; in step (2-2), the concentration is nanofiltration concentration.

[0031] Those skilled in the art will appreciate that after obtaining the aqueous phase in step (2-i), the subsequent step (2-2) will be adaptively adjusted to: concentrating and drying the aqueous phase to obtain the compound represented by formula A.

[0032] In some embodiments, the purification comprises the following steps:

[0033] (2-1) purifying the crude product by preparative liquid chromatography to obtain a preparative solution;

[0034] (2-2) concentrating and drying the prepared solution to obtain a compound represented by formula A;

[0035] Wherein, in step (2-1), when purification is performed by preparative liquid chromatography, mobile phase A is TFA / water and mobile phase B is TFA / acetonitrile; in step (2-2), the concentration is nanofiltration concentration.

[0036] In some embodiments, the purification comprises the following steps:

[0037] (2-1) purifying the crude product by preparative liquid chromatography to obtain a preparative solution;

[0038] (2-i) extracting the prepared solution with an organic solvent, discarding the organic phase, and obtaining an aqueous phase;

[0039] (2-2) concentrating and drying the aqueous phase to obtain a compound represented by formula A;

[0040] Wherein, in step (2-1), when purification is performed by preparative liquid chromatography, mobile phase A is TFA / water and mobile phase B is TFA / acetonitrile; in step (2-2), the concentration is nanofiltration concentration.

[0041] In some embodiments, in step (2-1), when purification is performed by preparative liquid chromatography, mobile phase A is 0.2% TFA / water and mobile phase B is 0.1% TFA / acetonitrile; in step (2-2), the concentration is nanofiltration concentration.

[0042] In some embodiments, in step (2-1), when purification is performed by preparative liquid chromatography, gradient elution is used.

[0043] In some embodiments, the gradient conditions are:

[0044] Time / min Mobile phase A / % Mobile phase B / % 0～5 90～75 10～25 5～32 75～60 25～40 32～39 10～0 90～100 39～50 90～75 10～25 .

[0045] In some embodiments, the gradient conditions are:

[0046] Time / min Mobile phase A / % Mobile phase B / % 0 80 20 3 80 20 6 75 25 10 75 25 15 70 30 15.5 63 37 25 63 37 29 62 38 31 62 38 31.5 2 98 39 2 98 39.5 80 20 43 80 20 .

[0047] In some embodiments, the gradient conditions are:

[0048]

[0049] .

[0051] In some embodiments, in step (2-1), when purification is performed by preparative liquid chromatography, the chromatographic column is UniSil10-100C18 450mm*100mm.

[0052] In some embodiments, in step (2-1), when purification is performed by preparative liquid chromatography, the detection wavelength is 200-365 nm, for example, 200 nm, 220 nm, 250 nm, 270 nm, 300 nm, 350 nm or 365 nm, or such as 200-230 nm.

[0053] In some embodiments, in step (2-1), when purification is performed by preparative liquid chromatography, the detection wavelength is 214 nm.

[0054] In some embodiments, in step (2-2), the nanofiltration is performed at a temperature ≤ 30°C.

[0055] In some embodiments, the nanofiltration is achieved by a nanofiltration device.

[0056] In some embodiments, in step (2-2), the drying is freeze-drying.

[0057] In some embodiments, in step (2-i), the organic solvent is mixed solvent 1 and / or mixed solvent 2, the mixed solvent 1 is a mixed solvent of ethyl acetate and n-hexane, and the mixed solvent 2 is a mixed solvent of dichloromethane and n-hexane.

[0058] In some embodiments, step (2-i) is performed as follows: sequentially extracting the prepared solution with mixed solvent 1 and mixed solvent 2, respectively, discarding the organic phase, and obtaining an aqueous phase.

[0059] In some embodiments, the volume ratio of the mixed solvent 1 to the preparation solution is (1-5):1, such as 1:1, 2:1, 3:1, 4:1 or 5:1, preferably 1:1.

[0060] In some embodiments, the volume ratio of the preparation solution to the mixed solvent 2 is (1-5):1, such as 1:1, 2:1, 3:1, 4:1 or 5:1, preferably 5:2.

[0061] In some embodiments, in the mixed solvent 1, the volume ratio of the ethyl acetate to the n-hexane is (1-5):1, for example, 1:1, 2:1, 3:1, 4:1 or 5:1, preferably 3:1.

[0062] In some embodiments, in the mixed solvent 2, the volume ratio of the dichloromethane to the n-hexane is (1-5):1, such as 1:1, 2:1, 3:1, 4:1 or 5:1, preferably 3:1.

[0063] In some embodiments, the molar ratio of the Lewis acid to the compound of formula a is not less than 10:1, for example 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1 or 45:1, preferably 20:1.

[0064] In some embodiments, the molar ratio of the Lewis acid to the compound represented by formula a is (10-30):1, for example 10:1, 15:1, 20:1, 25:1 or 30:1, preferably 20:1.

[0065] In some embodiments, the reaction is carried out in the presence of a solvent, and the solvent is selected from nitroalkanes (such as nitromethane, nitroethane), halogenated alkanes (such as dichloromethane, dichloroethane, chloroform), water, ketones (such as acetone, butanone, etc.), toluenes (such as toluene, trifluorotoluene), esters (such as ethyl acetate, dimethyl carbonate), acetonitrile, and a mixed solvent obtained by mixing two or more of the above solvents in any proportion, preferably selected from nitromethane, dimethyl carbonate, dichloromethane, a mixed solution of dichloromethane / ketones, a mixed solution of dichloromethane / water, and a mixed solution of dichloromethane / esters.

[0066] In some embodiments, the solvent is selected from nitromethane, dichloromethane / water, and dimethyl carbonate. Among them, those skilled in the art can understand that, for example, "dichloromethane / water" refers to a mixture of dichloromethane and water.

[0067] In some embodiments, the mass ratio of the compound represented by formula a to the volume ratio of the solvent is 1 g / (5-50 mL), for example, 1 g / 5 mL, 1 g / 10 mL, 1 g / 15 mL, 1 g / 20 mL, 1 g / 25 mL, 1 g / 30 mL, 1 g / 35 mL, 1 g / 40 mL, 1 g / 45 mL or 1 g / 50 mL, preferably 1 g / (5-35 mL).

[0068] In some embodiments, the solvent is nitromethane.

[0069] In some embodiments, the mass ratio of the compound represented by formula a to the volume ratio of the nitromethane is 1 g / (20-50 mL), for example, 1 g / 20 mL, 1 g / 25 mL, 1 g / 30 mL, 1 g / 35 mL, 1 g / 40 mL, 1 g / 45 mL or 1 g / 50 mL, preferably 1 g / 35 mL.

[0070] In some embodiments, the solvent is dichloromethane / water.

[0071] In some embodiments, the mass ratio of the compound represented by formula a to the volume ratio of dichloromethane / water is 1 g / (10-30 mL), for example, 1 g / 10 mL, 1 g / 15 mL, 1 g / 16 mL, 1 g / 20 mL, 1 g / 25 mL or 1 g / 30 mL, preferably 1 g / (15-16 mL).

[0072] In some embodiments, in the dichloromethane / water, the volume ratio of the dichloromethane to the water is (500-1000):1, for example, 500:1, 550:1, 600:1, 650:1, 700:1, 750:1, 800:1, 850:1, 900:1, 950:1 or 1000:1, preferably 750:1.

[0073] In some embodiments, the solvent is dimethyl carbonate.

[0074] In some embodiments, the mass ratio of the compound represented by formula a to the volume ratio of dimethyl carbonate is 1 g / (5-30 mL), for example, 1 g / 5 mL, 1 g / 10 mL, 1 g / 15 mL, 1 g / 20 mL, 1 g / 25 mL or 1 g / 30 mL, preferably 1 g / (5-15 mL).

[0075] In some embodiments, the reaction is carried out at a temperature of 20-110°C (eg, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, 100°C, or 110°C).

[0076] In some embodiments, the reaction is carried out at a temperature of 20-50°C (eg, 20°C, 30°C, 35°C, 40°C, 45°C or 50°C, preferably 40°C).

[0077] In some embodiments, the reaction is carried out at a temperature of 30-50°C (eg, 30°C, 35°C, 40°C, 45°C or 50°C, preferably 40°C).

[0078] In some embodiments, the reaction is carried out for 20 min-24 h (e.g., 20 min, 30 min, 40 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, or 24 h).

[0079] In some embodiments, the reaction is carried out for 20-120 min (e.g., 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, or 120 min). In some embodiments, the reaction is carried out for 20-60 min. In some embodiments, the reaction is carried out for 30-60 min. In some embodiments, the reaction is carried out for 30 min.

[0080] In some embodiments, after the compound represented by formula a is reacted with Lewis acid, a post-treatment step is further included before purification.

[0081] In some embodiments, the post-treatment comprises: concentrating the obtained reactant under reduced pressure to obtain a crude product.

[0082] In some embodiments, the post-treatment includes: crystallizing, filtering, and drying the obtained reactants to obtain a crude product.

[0083] In some embodiments, the concentration under reduced pressure is carried out at a temperature of 30-55°C (eg, 30°C, 35°C, 40°C, 45°C, 50°C or 55°C, preferably 45°C).

[0084] In some embodiments, the compound represented by formula a is prepared by the following method:

[0085] (3-1-1) The compound represented by formula b is reacted with a base (such as triethylamine) to obtain a compound

[0086]

[0087] (3-1-2) Compound With compound Reaction to obtain compound

[0088] (3-1-3) Compound Reaction with exitecan mesylate to obtain a compound represented by formula a;

[0089] Alternatively, the compound represented by formula a is prepared by the following method:

[0090] (3-2-1) The compound represented by formula b is reacted with a base (such as triethylamine) to obtain a compound

[0091]

[0092] (3-2-2) Compound Reaction with pentafluorophenol and DCC to obtain compound

[0093]

[0094] (3-2-3) Compound With compound Reaction to obtain compound

[0095] (3-2-4) Compound The compound is reacted with exitecan mesylate to obtain a compound represented by formula a.

[0096] In some embodiments, the compound represented by formula b is prepared by the following method:

[0097] (4-1) Compound Reaction with thionyl chloride and benzyl alcohol to obtain compound

[0098]

[0099] (4-2) Compound With compound Reaction to obtain compound

[0100]

[0101] (4-3) Compound React with (Boc)2O to obtain compound

[0102]

[0103] (4-4) Compound React with hydrogen in the presence of Pd / C to obtain compound

[0104] (4-5) Compound The reaction is carried out with maleic anhydride to obtain a compound shown in formula b.

[0105] In some embodiments, step (4-1) is performed as follows:

[0106] (4-1-1) Add thionyl chloride dropwise to benzyl alcohol, and then add compound to react;

[0107] (4-1-2) Add isopropyl ether to the reaction product obtained in step (4-1-1) to crystallize and filter, and dry the filter cake to obtain compound

[0108] In some embodiments, step (4-4) is performed as follows:

[0109] (4-4-1) Compound Reacting with hydrogen in the presence of Pd / C;

[0110] (4-4-2) Filtering the reaction product obtained in step (4-4-1), and concentrating the filtrate under reduced pressure to obtain a crude product;

[0111] (4-4-3) The crude product is recrystallized with isopropanol / water to obtain the compound

[0112] Preferably, during the recrystallization, the volume ratio of the isopropanol to water is (1-5):1, preferably 3:1.

[0113] In some embodiments, steps (4-5) are performed as follows:

[0114] (4-5-1) Compound reacting with maleic anhydride;

[0115] (4-5-2) Add n-hexane to the reactant obtained in step (4-5-1), perform crystallization, filter, and dry the filter cake to obtain a compound represented by formula b.

[0116] In another aspect of the present invention, the present invention also provides a method for preparing the compound represented by formula A, comprising:

[0117]

[0118] (1-1) mixing the compound represented by formula a with nitromethane and zinc bromide and reacting them to obtain a crude product;

[0119]

[0120] (1-2) purifying the crude product by preparative liquid chromatography to obtain a preparative solution;

[0121] (1-3) extracting the prepared solution with an organic solvent, discarding the organic phase, and obtaining an aqueous phase;

[0122] (1-4) concentrating and drying the aqueous phase to obtain a compound represented by formula A;

[0123] Wherein, in step (1-2), when purification is performed by preparative liquid chromatography, mobile phase A is 0.2% TFA / water and mobile phase B is 0.1% TFA / acetonitrile; in step (1-4), the concentration is nanofiltration concentration.

[0124] In some embodiments, in step (1-2), when purification is performed by preparative liquid chromatography, gradient elution is used.

[0125] In some embodiments, the gradient conditions are:

[0126] Time / min Mobile phase A / % Mobile phase B / % <![CDATA[Flow rate / mL·min -1 > 0 80 20 200 3 80 20 200 6 75 25 200 10 75 25 200 15 70 30 190 15.5 63 37 160 25 63 37 160 29 62 38 160 31 62 38 160 31.5 2 98 200 39 2 98 200 39.5 80 20 200 43 80 20 200

[0127] In some embodiments, in step (1-2), when purification is performed by preparative liquid chromatography, the chromatography column is UniSil10-100C18 450mm*100mm.

[0128] In some embodiments, in step (1-2), when purification is performed by preparative liquid chromatography, the detection wavelength is 214 nm.

[0129] In some embodiments, in step (1-4), the nanofiltration is performed at a temperature ≤ 30°C.

[0130] In some embodiments, the nanofiltration is achieved by a nanofiltration device.

[0131] In some embodiments, in step (1-4), the drying is freeze-drying.

[0132] In some embodiments, in step (1-3), the organic solvent is mixed solvent 1 and / or mixed solvent 2, the mixed solvent 1 is a mixed solvent of ethyl acetate and n-hexane, and the mixed solvent 2 is a mixed solvent of dichloromethane and n-hexane.

[0133] In some embodiments, step (1-3) is performed as follows: sequentially extracting the prepared solution with mixed solvent 1 and mixed solvent 2, respectively, discarding the organic phase, and obtaining an aqueous phase.

[0134] In some embodiments, the volume ratio of the mixed solvent 1 to the preparation solution is (1-5):1, such as 1:1, 2:1, 3:1, 4:1 or 5:1, preferably 1:1.

[0135] In some embodiments, the volume ratio of the preparation solution to the mixed solvent 2 is (1-5):1, such as 1:1, 2:1, 3:1, 4:1 or 5:1, preferably 5:2.

[0136] In some embodiments, in the mixed solvent 1, the volume ratio of the ethyl acetate to the n-hexane is (1-5):1, for example, 1:1, 2:1, 3:1, 4:1 or 5:1, preferably 3:1.

[0137] In some embodiments, in the mixed solvent 2, the volume ratio of the dichloromethane to the n-hexane is (1-5):1, such as 1:1, 2:1, 3:1, 4:1 or 5:1, preferably 3:1.

[0138] In some embodiments, in step (1-1), the molar ratio of zinc bromide to the compound represented by formula a is (10-30):1, for example 10:1, 15:1, 20:1, 25:1 or 30:1, preferably 20:1.

[0139] In some embodiments, in step (1-1), the volume ratio of the compound represented by formula a to the nitromethane is 1 g / (20-50 mL), for example, 1 g / 20 mL, 1 g / 25 mL, 1 g / 30 mL, 1 g / 35 mL, 1 g / 40 mL, 1 g / 45 mL or 1 g / 50 mL, preferably 1 g / 35 mL.

[0140] In some embodiments, in step (1-1), the reaction is carried out at a temperature of 30-50°C (eg, 30°C, 35°C, 40°C, 45°C or 50°C, preferably 40°C).

[0141] In some embodiments, in step (1-1), the reaction is carried out for 20-60 min (e.g., 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min or 60 min, preferably 30 min).

[0142] In some embodiments, in step (1-1), the reaction after mixing the compound represented by formula a with nitromethane and zinc bromide is carried out in the following manner:

[0143] (1-1-1) Mix the compound represented by formula a with nitromethane, and then add zinc bromide to react after dissolving;

[0144] (1-1-2) The reaction product obtained in step (1-1-1) is concentrated under reduced pressure to obtain a crude product.

[0145] In some embodiments, the concentration under reduced pressure is carried out at a temperature of 30-55°C (eg, 30°C, 35°C, 40°C, 45°C, 50°C or 55°C, preferably 45°C).

[0146] In some embodiments, the compound represented by formula a is prepared by the following method:

[0147] (2-1) The compound represented by formula b is reacted with triethylamine to obtain a compound

[0148]

[0149] (2-2) Compound Reaction with pentafluorophenol and DCC to obtain compound

[0150]

[0151] (2-3) Compound With compound Reaction to obtain compound

[0152] (2-4) Compound The compound is reacted with exitecan mesylate to obtain a compound represented by formula a.

[0153] In some embodiments, the compound represented by formula b is prepared by the following method:

[0154] (3-1) Compound Reaction with thionyl chloride and benzyl alcohol to obtain compound

[0155]

[0156] (3-2) Compound With compound Reaction to obtain compound

[0157]

[0158] (3-3) Compound React with (Boc)2O to obtain compound

[0159]

[0160] (3-4) Compound React with hydrogen in the presence of Pd / C to obtain compound

[0161] (3-5) Compound The reaction is carried out with maleic anhydride to obtain a compound shown in formula b.

[0162] In some embodiments, step (3-1) is performed as follows:

[0163] (3-1-1) Add thionyl chloride dropwise to benzyl alcohol, and then add compound to react;

[0164] (3-1-2) Add isopropyl ether to the reaction product obtained in step (3-1-1) to crystallize and filter, and dry the filter cake to obtain compound

[0165] In some embodiments, steps (3-4) are performed as follows:

[0166] (3-4-1) Compound Reacting with hydrogen in the presence of Pd / C;

[0167] (3-4-2) Filtering the reaction product obtained in step (3-4-1), and concentrating the filtrate under reduced pressure to obtain a crude product;

[0168] (3-4-3) The crude product is recrystallized with isopropanol / water to obtain the compound

[0169] In some embodiments, during the recrystallization, the volume ratio of isopropanol to water is (1-5):1, such as 1:1, 2:1, 3:1, 4:1 or 5:1, preferably 3:1.

[0170] In some embodiments, steps (3-5) are performed as follows:

[0171] (3-5-1) Compound reacting with maleic anhydride;

[0172] (3-5-2) Add n-hexane to the reactant obtained in step (3-5-1), perform crystallization, filter, and dry the filter cake to obtain a compound represented by formula b. BRIEF DESCRIPTION OF THE DRAWINGS

[0173] Figure 1 The impurity detection result of the first step of Example 1 of the present invention is shown;

[0174] Figure 2 The impurity detection result of the first step of Example 2 of the present invention is shown;

[0175] Figure 3 The impurity detection result of the first step of Example 3 of the present invention is shown;

[0176] Figure 4 The impurity detection results of the first step of Comparative Example 1 of the present invention are shown;

[0177] Figure 5 The impurity detection result of the second step of Example 1 of the present invention is shown;

[0178] Figure 6 The impurity detection results of the second step of Comparative Example 2 of the present invention are shown;

[0179] Figure 7 The impurity detection results of the second step of Comparative Example 3 of the present invention are shown;

[0180] Figure 8 The impurity detection results of the second step of Comparative Example 4 of the present invention are shown;

[0181] Fig. 9 The impurity detection results of the second step of Comparative Example 5 of the present invention are shown;

[0182] Fig.10The impurity detection results of the second step of Comparative Example 6 of the present invention are shown;

[0183] Fig.11 The impurity detection results of the second step of Comparative Example 7 of the present invention are shown;

[0184] Fig.12 The impurity detection results of the second step of Comparative Example 8 of the present invention are shown;

[0185] Fig.13 The impurity detection results of the second step of Comparative Example 9 of the present invention are shown. DETAILED DESCRIPTION

[0186] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments.

[0187] In the following experimental steps, raw materials not otherwise specified can be obtained commercially.

[0188] Preparatory Example 1:

[0189] Compound Preparation

[0190] Synthesis route:

[0191]

[0192] Step 1: Ac002

[0193] Add 2.8L benzyl alcohol to a 5L three-necked flask, cool to below 10°C, add thionyl chloride (36mL, 0.50mol, 2.0eq) dropwise, and heat to room temperature for 2h. Add 3-amino-N-Cbz-L-alanine (60g, 0.25mol, 1.0eq), react at room temperature for 16h, and the raw material (Ac001) basically disappears when monitored by TLC, and the reaction is terminated. Add 12L isopropyl ether to the system, crystallize for 2h, filter, and dry the filter cake at 45°C with air blowing to obtain 55.2gAc001, with a yield of 60.2%.

[0194] Step 2: Ac003

[0195] Add Ac002 (55g, 0.15mol, 1.0eq), 300mL acetonitrile, 33mL purified water to a 1L reaction bottle, stir and dissolve, add potassium carbonate (31.2g, 0.225mol, 1.5eq), add tert-butyl bromoacetate (26.42g, 0.135mol, 0.9eq), and react at room temperature for 20h. TLC monitoring shows that the raw material (Ac002) disappears, and the reaction is terminated. Add purified water (1.5L) to the system, extract with ethyl acetate (1L), separate the liquids, wash the organic layer with 10% sodium chloride solution, dry over anhydrous sodium sulfate, filter and concentrate under reduced pressure to obtain a crude product. The crude product is purified by column chromatography (petroleum ether / ethyl acetate) to obtain 44.4gAc003 with a yield of 66%.

[0196] Step 3: Ac004

[0197] Add Ac003 (44.2 g, 0.1 mol, 1.0 eq) and 100 mL of dichloromethane to a 500 mL reaction bottle, stir to dissolve, add triethylamine (15 mL, 0.11 mol, 1.1 eq), dropwise add Boc anhydride (22.9 g, 0.105 mol, 1.05 eq), react at room temperature for 20 h, and the raw material (Ac003) is basically disappeared by TLC monitoring, and the reaction is terminated. Add purified water (100 mL) to the system, separate the liquids, extract the water layer with dichloromethane (40 mL), combine the organic layers, wash with purified water, dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain 55.3 g of yellow oil, which is directly used in the next step.

[0198] Step 4: Ac005

[0199] Add the yellow oil (55.3 g) from the third step and 200 mL of methanol to a 1L single-mouth bottle, stir to dissolve, then add 5% Pd / C (8.3 g, 15% m / m), hydrogenate for 4 hours, and the raw material (Ac004) is basically gone after TLC monitoring, and the reaction is terminated. The reaction solution is filtered, and the filtrate is concentrated under reduced pressure to obtain a crude product. The crude product is recrystallized from isopropanol / water (volume 3 / 1) to obtain 18.4 g of Ac005, with a yield of 58% (two steps).

[0200] Step 5: SM-2

[0201] Add Ac005 (18 g, 0.056 mol, 1.0 eq), 100 mL of dichloromethane, and 17 mL of glacial acetic acid to a 500 mL single-mouth bottle, stir to dissolve, add maleic anhydride (6 g, 0.062 mol, 1.1 eq), react at room temperature for 4 h, and the raw material (Ac005) is basically gone by TLC monitoring, and the reaction is terminated. Add n-hexane (80 mL) to the system, cool to 10 ° C, crystallize for 2 h, filter, and dry the filter cake at 50 ° C for 4 h to obtain 20.6 g of SM-2, with a yield of 87%.

[0202] Preparatory Example 2:

[0203] Compound Preparation

[0204] 1. Synthesis of compound M3:

[0205] Synthesis route:

[0206]

[0207] Step 1: Compound M2

[0208] Compound SM-2 (40 g, 96 mmol, 1.0 eq), triethylamine (26.7 mL, 2.0 eq), and toluene (400 mL) were added to a 1000 mL single-mouth bottle, and the temperature was raised to 120 ° C and refluxed for 2 h. The reaction was basically complete under TLC monitoring, and the temperature was reduced to 50 ° C and the solvent was removed under reduced pressure. Dissolved with ethyl acetate (150 mL) and water (40 mL), adjusted to pH 2-3 with 1M HCl under ice bath stirring, and separated. The aqueous layer was extracted once more with ethyl acetate, and the organic layers were combined and dried over anhydrous sodium sulfate. After filtration, the crude product was concentrated to obtain a pale yellow oily crude product, which was purified by column chromatography (DCM: MeOH = 40: 1) to obtain compound M2 (26.6 g); LC-MS: [M+H] + =399.3.

[0209] Step 2: Compound M3

[0210] Compound M2 (26.5 g, 60.5 mmol, 1.0 eq), pentafluorophenol (12.2 g, 66.5 mmol, 1.1 eq), DCC (13.7 g, 66.5 mmol, 1.1 eq) and THF (300 mL) were added to a 1000 mL single-mouth bottle, and the mixture was reacted at room temperature for 30 min (monitored by TLC), and the insoluble matter was filtered off. The reaction solution was directly purified by preparative purification, and the preparative solution was concentrated by a water pump under reduced pressure in a water bath at 35°C to remove acetonitrile, and lyophilized to obtain compound M3 (31.5 g) with a yield of 64%; LC-MS: [M+H] + =565.1.

[0211] 2. Synthesis of compound 5e:

[0212] Synthesis route:

[0213] Solution 1:

[0214]

[0215] Option 2:

[0216]

[0217] Step 1: Compound 5a

[0218] M1 (500 mg, 1.4 mmol, 1.0 eq), p-toluenesulfonic acid monohydrate (26 mg, 0.1 mmol, 0.1 eq) and 10 mL THF were added to a 25 mL single-mouth bottle, stirred evenly, cooled to 0 °C, and then L-benzyl lactate (1.2 g, 7.0 mmol, 5 eq) was slowly added. After the addition, the temperature was raised to room temperature for reaction. After TLC monitoring, saturated NaHCO3 solution was added after the reaction was completed, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by reverse phase column to obtain 5a (400 mg);

[0219] LC-MS: [M+NH4] + =506.2.

[0220] 1 H NMR (400Mz, CDCl3 / CD3OD): 1.39 (3H, d, J = 6.8Hz), 3.78 (2H, t, J = 4.0Hz), 4.17-4.27 (2H, m), 4.42 (2H, d, J = 4.0Hz), 4.72-4.85 (2H, m), 5.11 -5.58(2H,m),5.43(1H,s),7.06(1H,t,J=8.0Hz),7.25-7.33(6H,m),7.38(2H,t,J=8.0Hz),7.57(2H,d,J=8.0Hz),7.75(2H,d,J=8.0Hz).

[0221] Step 2: Compound 5b

[0222] Add compound 5a (400 mg, 0.8 mmol, 1.0 eq) and 4 mL DMF to a 25 mL single-mouth bottle, stir evenly, cool to 0 °C, slowly add DBU (137 mg, 0.9 mmol, 1.1 eq), and warm to room temperature to react. Monitor by TLC, the reaction is complete, and is recorded as reaction solution ①;

[0223] In another 25mL single-mouth bottle, add M4 (372mg, 0.9mmol, 1.1eq), PyBOP (852mg, 1.6mmol, 2.0eq) and 3mL DMF, stir at room temperature for 5 minutes, add reaction solution ①, react at room temperature, and monitor by HPLC. After the reaction is completed, the reaction solution is purified by high-performance liquid chromatography to obtain compound 5b (326mg); LC-MS: [M+NH4] + =679.2.

[0224] Step 3: Compound 5c

[0225] Add 5b (4.0 g, 6.05 mmol, 1.0 eq) to a 100 mL single-mouth bottle, dissolve in DMF (60 mL), then add 5% Pd / C (4 g), and hydrogenate at room temperature for 4 h (HPLC was used to monitor the reaction progress). Filter the Pd / C, and place the filtrate in an ice-water bath (about 0°C) without concentration for later use.

[0226] Step 4: Compound 5d

[0227] Scheme 1: Place the crude product 5c in an ice-water bath, add DIPEA (1.1 mL, 1.1 eq), then add compound M3 (3.4 g, 6.05 mmol), and warm to room temperature for 2 h. After the addition, the reaction is completed by HPLC monitoring, and the reaction solution is purified by high-performance liquid chromatography to obtain a preparative solution, which is lyophilized to obtain 5d (3.15 g); LC-MS: [MH] - =816.3.

[0228] Scheme 2: Compound M2 (2.4 g, 6.05 mmol, 1.0 eq) was added to a single-mouth bottle and dissolved in acetonitrile (24 mL). The temperature was lowered to -10±5°C, EEDQ (1.49 g, 6.05 mmol, 1.0 eq) was added, and the mixture was kept warm for 2 h. The crude product 5c was then added to the system, and the mixture was kept warm for 1 h. After the reaction was completed, the reaction solution was purified by high-performance liquid chromatography to obtain a preparation solution. The preparation solution was extracted with dichloromethane and concentrated to obtain 5d (3.0 g).

[0229] Step 5: Compound 5e

[0230] 5d (2.07 g, 2.53 mmol, 1.0 eq), exitecan mesylate M5 (1.35 g, 2.53 mmol, 1.0 eq), PyBOP (1.98 g, 3.79 mmol, 1.5 eq), HOBt (0.51 g, 3.79 mmol, 1.5 eq) and DMF (40 mL) were added to a 100 mL single-mouth bottle, and DIPEA (1.05 mL, 1.5 eq) was added under an ice-water bath, and the mixture was heated to room temperature for 2 h (monitored by HPLC). The reaction solution was directly purified by preparative purification, and the preparative solution was concentrated at 35 ° C in a water bath under reduced pressure by a water pump to remove acetonitrile, and lyophilized to obtain compound 5e (1.92 g), with a yield of 61%; LC-MS: [M+H] + =1235.4.

[0231] Embodiment 1:

[0232] Target compound Preparation

[0233] Synthesis route:

[0234]

[0235] Step 1: Add compound 5e (1.0 g, 0.8 mmol, 1.0 eq) and 35 mL of nitromethane to a 100 mL single-mouth bottle, add zinc bromide (3.64 g, 16 mmol, 20.0 eq) after dissolution, react in an oil bath at 40 ° C (preheated in advance for stabilization) for 30 min (monitored by HPLC), and concentrate in a water bath at 45 ° C with a water pump to remove nitromethane to obtain a yellow residue solid.

[0236] Impurity detection method:

[0237] The target compound was tested for impurities by HPLC. The results are shown in Table 1 and Figure 1 shown.

[0238] Instrument: High performance liquid chromatography

[0239] Chromatographic column: YMC-Triart C18-5μm-12nm 4.6mm*250mm

[0240] Mobile phase: A—0.05% TFA / water (add 0.5 mL TFA to 1 L purified water)

[0241] Mobile phase: B—0.05% TFA / acetonitrile (add 0.5 mL TFA to 1 L acetonitrile)

[0242] Detection wavelength: 214nm

[0243] Flow rate: 1mL / min

[0244] Column temperature: 30°C

[0245] Gradient conditions:

[0246] Time / min Mobile phase A / % Mobile phase B / % 0 70 30 20 55 45 20.01 2 98 30 2 98 30.01 70 30 40 70 30

[0247] Step 2: Prepare and purify by acid method (mobile phase: 0.2% TFA / water-0.1% TFA / acetonitrile) to obtain a preparation solution (volume V) of the target compound, and extract the organic solvent in the preparation solution with ethyl acetate (0.75V) / n-hexane (0.25V) and dichloromethane (0.3V) / n-hexane (0.1V) in sequence, discard the organic phase, obtain the aqueous phase, and then use nanofiltration equipment to concentrate at low temperature (≤30°C), and freeze-dry to obtain the target compound.

[0248] LC-MS: [M+H] + =1079.4;

[0249] 1H NMR(400MHz, DMSO-d6)δ9.39-9.02(m,1H),8.70(t,J=6.5Hz,1H),8.64(t,J=5.7Hz,1H),8.56(d,J=8.8Hz,1H),8 .34(t,J=5.7Hz,1H),8.16(d,J=8.2Hz,1H),8.01(t,J=5.5Hz,1H),7.71(d,J=10.9Hz,1H),7.30(s,1H),7.28-7. 15(m,4H),7.14(s,2H),5.53(dd,J=14.5,6.4Hz,1H),5.49-5.34(m,2H),5.22(d,J=18.8Hz,1H),5.09(d,J=18.7 Hz,1H),5.03(dd,J=9.6,3.9Hz,1H),4.73(dd,J=9.9,6.9Hz,1H),4.59(dd,J=10.1,6.5Hz,1H),4.49(ddd,J=13.2 ,8.6,4.4Hz,1H),4.14(dd,J=13.3,6.6Hz,2H),3.93(s,2H),3.84(dd,J=16.5,6.3Hz,1H),3.76(dd,J=16.9,5.7 Hz,2H),3.70(d,J=5.2Hz,2H),3.60(dd,J=16.7,5.4Hz,1H),3.52(dd,J=16.4,5.1Hz,1H),3.45(dd,J=12.8,10.1 Hz,1H),3.25-3.15(m,1H),3.14-3.05(m,1H),3.01(dd,J=13.7,4.1Hz,1H),2.73(dd,J=13.5,9.8Hz,1H),2.54- 2.47(m,1H),2.33(s,2H),2.17(d,J=5.5Hz,2H),1.91-1.79(m,2H),1.33(d,J=6.6Hz,2H),0.87(t,J=7.3Hz,2H).

[0250] The specific conditions for preparation and purification are as follows:

[0251] Instrument: Preparative liquid chromatography

[0252] Chromatographic column: UniSil 10-100C18 450mm*100mm

[0253] Mobile phase: A—0.2% TFA / water (add 2 mL TFA to 1 L purified water)

[0254] Mobile phase: B—0.1% TFA / acetonitrile (1 mL TFA in 1 L acetonitrile)

[0255] Detection wavelength: 214nm

[0256] Gradient conditions:

[0257] Time / min Mobile phase A / % Mobile phase B / % <![CDATA[Flow rate / mL·min -1 > 0 80 20 200 3 80 20 200 6 75 25 200 10 75 25 200 15 70 30 190 15.5 63 37 160 25 63 37 160 29 62 38 160 31 62 38 160 31.5 2 98 200 39 2 98 200 39.5 80 20 200 43 80 20 200

[0258] Impurity detection method:

[0259] The target compound was tested for impurities by HPLC, and the results are shown in Table 2 and Figure 5 shown.

[0260] The HPLC detection conditions for impurity detection are as follows:

[0261] Instrument: High performance liquid chromatography

[0262] Chromatographic column: YMC-Triart C18-5μm-12nm 4.6mm*250mm

[0263] Mobile phase: A—0.05% TFA / water (add 0.5 mL TFA to 1 L purified water)

[0264] Mobile phase: B—0.05% TFA / acetonitrile (add 0.5 mL TFA to 1 L acetonitrile)

[0265] Detection wavelength: 214nm

[0266] Flow rate: 1mL / min

[0267] Column temperature: 30°C

[0268] Gradient conditions:

[0269] Time / min Mobile phase A / % Mobile phase B / % 0 70 30 20 55 45 20.01 2 98 30 2 98 30.01 70 30 40 70 30

[0270] Embodiment 2:

[0271] Synthesis route:

[0272] Compared with Example 1, the only difference is the first step, and the second step is the same as Example 1. Specifically, the first step: compound 5e (1.0 g, 0.8 mmol, 1.0 eq) and 15 mL of dichloromethane were added to a 100 mL single-mouth bottle, and 20 uL of water was added. After dissolution, zinc bromide (3.64 g, 16 mmol, 20.0 eq) was added, and the mixture was reacted in an oil bath at 40° C. (preheated in advance for stabilization) for 1 h (monitored by HPLC), and the dichloromethane was removed by concentrating with a water pump at 45° C. in a water bath under reduced pressure to obtain a yellow solid residue.

[0273] The first step of impurity detection method:

[0274] The impurity detection method in the first step is the same as in Example 1, and the results are shown in Table 1 and Figure 2 shown.

[0275] Embodiment 3:

[0276] Synthesis route:

[0277] Compared with Example 1, the only difference is the first step, and the second step is the same as Example 1. Specifically, the first step: compound 5e (1.0 g, 0.8 mmol, 1.0 eq) and 15 mL of dimethyl carbonate were added to a 100 mL single-mouth bottle, and zinc bromide (3.64 g, 16 mmol, 20.0 eq) was added after dissolution, and the mixture was reacted in an oil bath at 40° C. (preheated in advance for stabilization) for 30 min (monitored by HPLC), and after the reaction was completed, 20 mL of dichloromethane was added for crystallization, and after crystallization, the mixture was filtered and dried to obtain a crude solid product.

[0278] The first step of impurity detection method:

[0279] The impurity detection method in the first step is the same as in Example 1, and the results are shown in Table 1 and Figure 3 shown.

[0280] Comparative Example 1:

[0281] Synthesis route:

[0282] Compared with Example 1, the only difference is the first step, and the second step is the same as Example 1. Specifically, the first step: compound 5e (1.0 g, 0.8 mmol, 1.0 eq) and 10 mL of dichloromethane were added to a 100 mL single-mouth bottle, and 1 ml of trifluoroacetic acid was added after dissolution, and the mixture was reacted at room temperature for 30 min (monitored by HPLC), and after the reaction was completed, the mixture was concentrated under reduced pressure to obtain a crude product.

[0283] The first step of impurity detection method:

[0284] The impurity detection method in the first step is the same as in Example 1, and the results are shown in Table 1 and Figure 4 shown.

[0285] Table 1: Impurity test results

[0286]

[0287] From the comparison of the results of the above Examples 1-3 and Comparative Example 1, it can be intuitively seen that in the production process of the target compound, only the preparation method for which protection is sought in the present application can meet the quality standards of the products (the mono-de-product must be controlled within 10%, the compound a must be controlled within 2%, and the drug unit must be controlled within 5%), and is even far superior to the quality standards (the drug unit is as low as within 2%).

[0288] Comparative Example 2:

[0289] Synthesis route:

[0290] Compared with Example 1, the first step is the same as Example 1, and the difference is only in the second step. Specifically, the second step: prepare and purify (mobile phase: pure water-acetonitrile) to obtain a preparation solution of the target compound. Regarding the specific conditions for preparation and purification, except that the mobile phase A is replaced with pure water and the mobile phase B is replaced with acetonitrile, the other specific conditions for preparation and purification are the same as those in Example 1.

[0291] Second step impurity detection method:

[0292] The impurity detection method in the second step is the same as that in Example 1, and the results are shown in Table 2 and Figure 6 shown.

[0293] Comparative Example 3:

[0294] Synthesis route:

[0295] Compared with Example 1, the first step is the same as Example 1, and the only difference is the second step. Specifically, the second step: prepare and purify by acid method (mobile phase: 0.1% TFA / water-acetonitrile) to obtain a preparation solution (volume V) of the target compound, and extract the organic solvent in the preparation solution with ethyl acetate (0.75V) / n-hexane (0.25V) and dichloromethane (0.3V) / n-hexane (0.1V) in sequence, discard the organic phase, obtain the aqueous phase, and then use nanofiltration equipment to concentrate at low temperature (≤30°C), and freeze-dry to obtain the target compound. For the specific conditions of preparation and purification, except that the mobile phase A is replaced with 0.1% TFA / water and the mobile phase B is replaced with acetonitrile, the other specific conditions of preparation and purification are the same as those in Example 1.

[0296] Second step impurity detection method:

[0297] The impurity detection method in the second step is the same as that in Example 1, and the results are shown in Table 2 and Figure 7 shown.

[0298] Comparative Example 4:

[0299] Synthesis route:

[0300] Compared with Example 1, the first step is the same as Example 1, and the difference is only in the second step. Specifically, the second step: prepare and purify by acid method (mobile phase: 0.1% TFA / water-acetonitrile) to obtain a preparation solution (volume V) of the target compound, and extract the organic solvent in the preparation solution with ethyl acetate (0.75V) / n-hexane (0.25V) and dichloromethane (0.3V) / n-hexane (0.1V) in sequence, discard the organic phase, obtain the aqueous phase, and then concentrate the aqueous phase at 35°C in a water bath with an oil pump under reduced pressure, and freeze-dry to obtain the target compound. For the specific conditions of preparation and purification, except that the mobile phase A is replaced with 0.1% TFA / water and the mobile phase B is replaced with acetonitrile, the other specific conditions of preparation and purification are the same as those in Example 1.

[0301] Second step impurity detection method:

[0302] The impurity detection method in the second step is the same as that in Example 1, and the results are shown in Table 2 and Figure 8 shown.

[0303] Comparative Example 5:

[0304] Synthesis route:

[0305] Compared with Example 1, the first step is the same as Example 1, and the only difference is the second step. Specifically, the second step: prepare and purify by acid method (mobile phase: 0.1% TFA / water-0.1% TFA / acetonitrile) to obtain a preparation solution (volume V) of the target compound, and extract the organic solvent in the preparation solution with ethyl acetate (0.75V) / n-hexane (0.25V) and dichloromethane (0.3V) / n-hexane (0.1V) in sequence, discard the organic phase, obtain the aqueous phase, and then use nanofiltration equipment to concentrate at low temperature (≤30°C), and freeze-dry to obtain the target compound. For the specific conditions of preparation and purification, except that the mobile phase A is replaced with 0.1% TFA / water, the other specific conditions of preparation and purification are the same as those in Example 1.

[0306] Second step impurity detection method:

[0307] The impurity detection method in the second step is the same as that in Example 1, and the results are shown in Table 2 and Fig. 9 shown.

[0308] Comparative Example 6:

[0309] Synthesis route:

[0310] Compared with Example 1, the first step is the same as Example 1, and the difference is only in the second step. Specifically, the second step: prepare and purify by acid method (mobile phase: 0.1% TFA / water-0.1% TFA / acetonitrile) to obtain a preparation solution (volume V) of the target compound, and extract the organic solvent in the preparation solution with ethyl acetate (0.75V) / n-hexane (0.25V) and dichloromethane (0.3V) / n-hexane (0.1V) in sequence, discard the organic phase, obtain the aqueous phase, and then concentrate the aqueous phase at 35°C in a water bath with an oil pump under reduced pressure, and freeze-dry to obtain the target compound. For the specific conditions of preparation and purification, except that the mobile phase A is replaced with 0.1% TFA / water, the other specific conditions of preparation and purification are the same as those in Example 1.

[0311] Second step impurity detection method:

[0312] The impurity detection method in the second step is the same as that in Example 1, and the results are shown in Table 2 and Fig.10 shown.

[0313] Comparative Example 7:

[0314] Synthesis route:

[0315] Compared with Example 1, the first step is the same as Example 1, and the difference is only in the second step. Specifically, the second step: prepare and purify by acid method (mobile phase: 0.2% TFA / water-0.1% TFA / acetonitrile) to obtain a preparation solution (volume V) of the target compound, and extract the organic solvent in the preparation solution with ethyl acetate (0.75V) / n-hexane (0.25V) and dichloromethane (0.3V) / n-hexane (0.1V) in sequence, discard the organic phase, obtain the aqueous phase, and then concentrate the aqueous phase at 35°C in a water bath under reduced pressure with an oil pump, and freeze-dry to obtain the target compound. The specific conditions of preparation and purification are the same as those in Example 1.

[0316] Second step impurity detection method:

[0317] The impurity detection method in the second step is the same as that in Example 1, and the results are shown in Table 2 and Fig.11 shown.

[0318] Comparative Example 8:

[0319] Synthesis route:

[0320] Compared with Example 1, the first step is the same as Example 1, and the only difference is the second step. Specifically, the second step: prepare and purify by acid method (mobile phase: 0.2% TFA / water-0.2% TFA / acetonitrile) to obtain a preparation solution (volume V) of the target compound, and extract the organic solvent in the preparation solution with ethyl acetate (0.75V) / n-hexane (0.25V) and dichloromethane (0.3V) / n-hexane (0.1V) in sequence, discard the organic phase, obtain the aqueous phase, and then use nanofiltration equipment to concentrate at low temperature (≤30°C), and freeze-dry to obtain the target compound. For the specific conditions of preparation and purification, except that the mobile phase B is replaced with 0.2% TFA / acetonitrile, the other specific conditions of preparation and purification are the same as those in Example 1.

[0321] Second step impurity detection method:

[0322] The impurity detection method in the second step is the same as that in Example 1, and the results are shown in Table 2 and Fig.12 shown.

[0323] Comparative Example 9:

[0324] Synthesis route:

[0325] Compared with Example 1, the first step is the same as Example 1, and the difference is only in the second step. Specifically, the second step: prepare and purify by acid method (mobile phase: 0.2% TFA / water-0.2% TFA / acetonitrile) to obtain a preparation solution (volume V) of the target compound, and extract the organic solvent in the preparation solution with ethyl acetate (0.75V) / n-hexane (0.25V) and dichloromethane (0.3V) / n-hexane (0.1V) in sequence, discard the organic phase, obtain the aqueous phase, and then concentrate the aqueous phase at 35°C in a water bath with an oil pump under reduced pressure, and freeze-dry to obtain the target compound. For the specific conditions of preparation and purification, except that the mobile phase B is replaced with 0.2% TFA / acetonitrile, the other specific conditions of preparation and purification are the same as those in Example 1.

[0326] Second step impurity detection method:

[0327] The impurity detection method in the second step is the same as that in Example 1, and the results are shown in Table 2 and Fig.13 shown.

[0328] Table 2: Impurity test results

[0329]

[0330]

[0331] From the comparison of the results of Example 1 and Comparative Examples 2-9 above, it can be intuitively seen that in the production process of the target compound, only the preparation method for which protection is sought in the present application can meet the quality standard of the product (the ring-opening product is controlled within 10%, and the drug unit is controlled within 5%), or even far exceeds the quality standard (the ring-opening product is as low as within 3%, and the drug unit is as low as within 2%).

[0332] It should be understood that the invention described herein is not limited to specific methodology, experimental protocols or reagents, as these may vary. The discussion and examples provided herein are presented only to describe specific embodiments and are not intended to limit the scope of the invention, which is limited only by the claims.

Claims

1. A method for preparing a compound of formula A, comprising: The compound represented by formula a is reacted with Lewis acid to obtain a crude product; the crude product is purified to obtain a compound represented by formula A; Preferably, the Lewis acid is bromide, TMSI or TMSOTf; More preferably, the Lewis acid is zinc bromide.

2. The method of claim 1, wherein: The purification comprises the following steps: (2-1) purifying the crude product by preparative liquid chromatography to obtain a preparative solution; (2-2) concentrating and drying the prepared solution to obtain a compound represented by formula A; Optionally, after obtaining the preparation solution in step (2-1) and before step (2-2), an extraction step is further included; Optionally, after obtaining the preparation solution in step (2-1) and before step (2-2), the following steps are further included: (2-i) extracting the prepared solution with an organic solvent, discarding the organic phase, and obtaining an aqueous phase; Wherein, in step (2-1), when purification is performed by preparative liquid chromatography, mobile phase A is TFA / water and mobile phase B is TFA / acetonitrile; in step (2-2), the concentration is nanofiltration concentration.

3. The method of claim 2, wherein: The method also has one or more technical features selected from the following (i)-(iii): (i) In step (2-1), when purification is performed by preparative liquid chromatography, gradient elution is used; The gradient conditions are: (ii) in step (2-1), when purification is performed by preparative liquid chromatography, the detection wavelength is 200-365 nm; (iii) in step (2-2), the nanofiltration is carried out at a temperature ≤ 30°C; Preferably, the nanofiltration is achieved by a nanofiltration device.

4. The method according to any one of claims 2 to 3, wherein: The method also has one or more technical features selected from the following (i)-(ii): ( i) in step (2-2), the drying is freeze-drying; (ii) in step (2-i), the organic solvent is mixed solvent 1 and / or mixed solvent 2, the mixed solvent 1 is a mixed solvent of ethyl acetate and n-hexane, and the mixed solvent 2 is a mixed solvent of dichloromethane and n-hexane; Preferably, step (2-i) is performed in the following manner: sequentially extracting the prepared solution with mixed solvent 1 and mixed solvent 2, discarding the organic phase, and obtaining an aqueous phase; Preferably, the volume ratio of the mixed solvent 1 to the preparation solution is (1-5):1, preferably 1:1; Preferably, the volume ratio of the preparation solution to the mixed solvent 2 is (1-5):1, preferably 5:2; Preferably, in the mixed solvent 1, the volume ratio of the ethyl acetate to the n-hexane is (1-5):1, preferably 3:1; Preferably, in the mixed solvent 2, the volume ratio of the dichloromethane to the n-hexane is (1-5):1, preferably 3:

1.

5. The method according to any one of claims 1 to 4, wherein The method also has one or more technical features selected from the following (i)-(iv): (i) the molar ratio of the Lewis acid to the compound represented by formula a is not less than 10:1; (ii) The reaction is carried out in the presence of a solvent, wherein the solvent is selected from nitroalkanes (such as nitromethane, nitroethane), halogenated alkanes (such as dichloromethane, dichloroethane, chloroform), water, ketones (such as acetone, butanone, etc.), toluenes (such as toluene, trifluorotoluene), esters (such as ethyl acetate, dimethyl carbonate), acetonitrile, and a mixed solvent obtained by mixing two or more of the above solvents in any proportion, preferably selected from nitromethane, dimethyl carbonate, dichloromethane, a mixed solution of dichloromethane / ketones, a mixed solution of dichloromethane / water, and a mixed solution of dichloromethane / esters. (iii) the reaction is carried out at a temperature of 20-110°C; (iv) The reaction is carried out for 20 min to 24 h.

6. The method according to any one of claims 1 to 5, wherein After the compound represented by formula a reacts with Lewis acid, before purification, a post-treatment step is also included; Preferably, the post-processing comprises (i) or (ii): (i) concentrating the obtained reactant under reduced pressure to obtain a crude product; (ii) crystallizing, filtering and drying the obtained reactant to obtain a crude product; Preferably, the reduced pressure concentration is carried out at a temperature of 30-55°C.

7. The method according to any one of claims 1 to 6, wherein The compound shown in formula a is prepared by the following method: (3-1-1) The compound represented by formula b is reacted with a base to obtain a compound (3-1-2) Compound With compound Reaction to obtain compound (3-1-3) Compound Reaction with exitecan mesylate to obtain a compound represented by formula a; Alternatively, the compound represented by formula a is prepared by the following method: (3-2-1) The compound represented by formula b is reacted with a base to obtain a compound (3-2-2) Compound Reaction with pentafluorophenol and DCC to obtain compound (3-2-3) Compound With compound Reaction to obtain compound (3-2-4) Compound The compound is reacted with exitecan mesylate to obtain a compound represented by formula a.

8. The method of claim 7, wherein: The compound shown in formula b is prepared by the following method: (4-1) Compound Reaction with thionyl chloride and benzyl alcohol to obtain compound (4-2) Compound With compound Reaction to obtain compound (4-3) Compound React with (Boc)2O to obtain compound (4-4) Compound React with hydrogen in the presence of Pd / C to obtain compound (4-5) Compound The reaction is carried out with maleic anhydride to obtain a compound shown in formula b.

9. The method of claim 8, wherein: Step (4-1) is carried out as follows: (4-1-1) Add thionyl chloride dropwise to benzyl alcohol, and then add compound to react; (4-1-2) Add isopropyl ether to the reaction product obtained in step (4-1-1) to crystallize and filter, and dry the filter cake to obtain compound 10. The method of claim 8, wherein: Step (4-4) is carried out in the following manner: (4-4-1) Compound Reacting with hydrogen in the presence of Pd / C; (4-4-2) Filtering the reaction product obtained in step (4-4-1), and concentrating the filtrate under reduced pressure to obtain a crude product; (4-4-3) The crude product is recrystallized with isopropanol / water to obtain the compound Preferably, during the recrystallization, the volume ratio of the isopropanol to water is (1-5):1, preferably 3:

1.

11. The method of claim 8, wherein: Steps (4-5) are performed as follows: (4-5-1) Compound reacting with maleic anhydride; (4-5-2) Add n-hexane to the reactant obtained in step (4-5-1), perform crystallization, filter, and dry the filter cake to obtain a compound represented by formula b.

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