CN118027061A

CN118027061A - Maytansine DM1 crystal form A and crystal form B and preparation methods thereof

Info

Publication number: CN118027061A
Application number: CN202211361249.6A
Authority: CN
Inventors: 胡范; 贾侃彦; 曹杰; 马鹏程
Original assignee: Hangzhou Zhongmei Huadong Pharmaceutical Co Ltd
Current assignee: Hangzhou Zhongmei Huadong Pharmaceutical Co Ltd
Priority date: 2022-11-02
Filing date: 2022-11-02
Publication date: 2024-05-14

Abstract

The invention provides a new crystal form A and a new crystal form B of maytansine DM1, fills the technical blank, improves the stability of the compound by the two crystal forms, can be stored at room temperature, greatly reduces the transportation pressure, has high purity, is easy to post-treat and process, and is better used for preparing antibody drug conjugates, polypeptide drug conjugates or micromolecular drug conjugates.

Description

Maytansine DM1 crystal form A and crystal form B and preparation methods thereof

Technical Field

The invention belongs to the technical field of synthesis of pharmaceutical intermediates, in particular to the technical field of HPAPI manufacturing, and particularly relates to research on a new crystal form of maytansine DM 1.

Background

Maytansine DM1, N2 '-deacetylated-N-2' (3-mercapto-1-oxypropyl) -maytansine, 1, cas no: 139504-50-0 has the chemical structural formula as follows:

Maytansine DM1 (139504-50-0, mertansine) has antitumor activity and tubulin modulator effect. It is an alpha-amino acid ester, carbamate, epoxide, organic heterocyclic tetracyclic compound, organic chlorine compound, thiol and maytansinoid. Maytansine DM1 is derived from maytansine. Maytansine DM1 is a cytotoxic moiety in an antibody conjugate, which is produced by a sulfhydryl group attached to SPP (N-succinimidyl 4- (2-pyridyldithio)) or SMCC (4- (3-mercapto-dioxo-1 pyrrolidinylmethyl) -cyclohexanecarboxylic acid).

The synthesis processes of maytansinoid DM1 reported so far are all used for obtaining a final product through concentration, and the inventors repeatedly obtain a crystal form of DM1 in a method of synthesizing novel anticancer drug Kadcyla effector molecule DM-1, such as Du Deping, by the disclosed concentration process, wherein the crystal form is shown in figure 8, and the DM1 under the crystal form has the advantages of yellow appearance, caking, low purity, poor stability and harsh storage condition (-20 ℃).

Drug polymorphism is a common phenomenon in drug development and is an important factor affecting drug quality. Different crystal forms of the same medicine may have obvious differences in appearance, solubility, melting point, dissolution rate, bioavailability and the like, and also have different effects on the stability, bioavailability and curative effect of the medicine. The polymorphic forms of a drug may exhibit different physical and mechanical properties including hygroscopicity, particle shape, density, flowability, compressibility, etc., which may further affect the preparation of bulk drugs and formulations. Therefore, in drug development, the problem of polymorphism of drugs should be comprehensively considered. Therefore, there is a need to re-develop synthetic routes suitable for industrial production and obtain crystalline forms with better stability.

Disclosure of Invention

Aiming at the defects and the blank existing in the prior art, the invention provides a novel crystal form of maytansine DM1 and a preparation method thereof.

The present invention provides a crystalline form of maytansinoid DM1, which crystalline form is referred to herein as DM1 form a, the single crystal structure of which is: orthorhombic, P212121 (19 #) space group, unit cell parameters are: z=4, formula: 4 (C ₃₅H₄₈ClN₃O10S)·2(C₄H₈O₂)·(H₂ O).

In the single crystal structure of the crystal, the minimum asymmetric unit contains four DM1 molecules, two ethyl acetate molecules and one water molecule, and the minimum asymmetric unit is a solvate of ethyl acetate and water.

The crystal form A uses Cu-K alpha radiation, and an X-ray powder diffraction pattern expressed by a2 theta angle has characteristic peaks at 8.6+/-0.2 degrees, 11.3+/-0.2 degrees, 19.6+/-0.2 degrees and 22.0+/-0.2 degrees.

As a specific embodiment, the crystal form a uses Cu-ka radiation, and has an X-ray powder diffraction pattern expressed as 2θ angle with at least one characteristic peak at 3.7±0.2°,9.9±0.2°, and 10.8±0.2°.

As a specific embodiment, the form a has an X-ray powder diffraction pattern as shown in fig. 1.

The DSC spectrum of the DM1 crystal form A is shown by the detection of differential scanning calorimetric analysis (DSC), and endothermic peaks appear in the range of 110-202 ℃.

As a specific embodiment, the DSC spectrum of the DM1 crystal form A shows endothermic peaks in the range of 110-202 ℃; further, endothermic peaks appear successively at 110.8.+ -. 2 ℃, 152.1.+ -. 2 ℃, 170.6.+ -. 2 ℃, 201.9.+ -. 2 ℃;

As a specific embodiment, the DSC profile of DM1 form a is shown in fig. 2.

As a specific embodiment, the TGA profile of DM1 form a loses 9.67±0.2% when heated to 180±2 ℃.

As a specific embodiment, the TGA profile of DM1 form a is shown in fig. 2.

The invention also provides a preparation method of the DM1 crystal form A, which comprises the following steps:

according to the invention, the specific preparation method comprises the following steps:

a) Hydrolysis reaction: adding a good solvent and DM1-B00 into a reaction tank, stirring and dissolving, then adding a buffer solution with pH=7.6+/-0.2 containing dithiothreitol, and stirring and reacting. And (3) after the reaction is finished, regulating the pH value to be neutral by using a dilute hydrochloric acid solution, extracting the reaction liquid by using an extraction solvent to obtain an organic phase, washing by using purified water and saline solution, drying, concentrating under reduced pressure until solid is separated out, slowly dropwise adding an antisolvent for crystallization, filtering, and drying to obtain the DM1 crystal form A.

In a specific embodiment, the reaction solvent is one or more selected from ethyl acetate, methanol, DCM, THF, acetone, ethanol and isopropanol, preferably ethyl acetate.

As a specific embodiment, the extraction solvent is selected from a mixture of water and an organic solvent selected from ethyl acetate, DCM and 2-MeTHF.

In a specific embodiment, the antisolvent is one or more selected from n-hexane, cyclohexane, heptane, cyclopentane, and methyl tert-butyl ether, preferably n-hexane.

Furthermore, the invention also provides a method for carrying out single crystal diffraction on the maytansinoid DM1 crystal form A to confirm the absolute configuration of the maytansinoid DM1 single crystal.

Furthermore, the invention also provides application of the maytansinoid DM1 crystal form A to preparation of crystal form B.

The present invention also provides a solvent-free crystalline form of maytansinoid DM1, which crystalline form is referred to herein as DM1 form B;

the crystal form B uses Cu-K alpha radiation, and an X-ray powder diffraction pattern expressed in terms of 2 theta angles is 9.8+/-0.2 degrees, 11.3+/-0.2 degrees and 16.3+/-0.2 degrees; a characteristic peak at 19.7+/-0.2 degrees;

As a specific embodiment, the crystal form B, which uses Cu-ka radiation, has an X-ray powder diffraction pattern expressed in terms of 2θ angles with characteristic peaks at 8.6±0.2°,11.2±0.2°,22.0±0.2°.

As a specific embodiment, the crystal form B, which uses Cu-ka radiation, has an X-ray powder diffraction pattern expressed in terms of 2θ angles with characteristic peaks at 13.4±0.2°,16.3±0.2°, and 19.7±0.2°.

As a specific embodiment, the crystal form B, which uses Cu-ka radiation, has an X-ray powder diffraction pattern expressed in terms of 2θ angles with characteristic peaks at 3.7±0.2°,6.4±0.2°,6.5±0.2°,8.1±0.2°,8.3±0.2°, and 19.7±0.2°.

As a specific embodiment, the X-ray powder diffraction pattern of form B is shown in fig. 4.

The DSC spectrum of the DM1 crystal form B is shown by the detection of differential scanning calorimetric analysis (DSC), and endothermic peaks appear in the range of 197-210 ℃.

As a specific embodiment, the maytansinoid DM1 form B has a DSC profile, and an endothermic peak at 197.97 ±0.2 ℃.

As a specific embodiment, the maytansinoid DM1 form B has a DSC profile as shown in fig. 5.

As a specific embodiment, the TGA profile of crystalline form B of maytansinoid DM1 loses 0.34±0.2% upon heating to 250±2 ℃.

As a specific embodiment, the TGA profile of maytansinoid DM1 form B is shown in fig. 6.

The invention also provides a preparation method of the maytansine DM1 crystal form B, which comprises the following steps: adding a poor solvent into the crystal form A, stirring, and carrying out crystal transformation at a certain temperature;

In a specific embodiment, the poor solvent is one or more selected from n-hexane, cyclohexane, dichloromethane, ethyl acetate, methyl tert-butyl ether, cyclopentane, diethyl ether, acetone, and acetonitrile.

As a specific embodiment, the seeding temperature is-20 to 40 ℃, preferably 0 to 20 ℃.

The invention also provides application of the crystal form A or the crystal form B of the maytansinoid DM1 in preparing a drug conjugate, wherein the drug conjugate comprises an antibody drug conjugate, a polypeptide drug conjugate or a small molecule drug conjugate of cytotoxin of the crystal form A or the crystal form B of the maytansinoid DM 1.

The invention has the beneficial effects that:

Drawings

Fig. 1: XRPD pattern of maytansine DM1 form a;

Fig. 2: DSC and TGA profiles of maytansinoid DM1 form A;

Fig. 3: powder appearance diagram of maytansine DM1 form a;

Fig. 4: XRPD pattern of maytansine DM1 form B;

fig. 5: DSC profile of maytansine DM1 form B;

fig. 6: TGA profile of maytansinoid DM1 form B;

fig. 7: powder appearance diagram of maytansine DM1 form B;

fig. 8: powder appearance diagram of maytansinoid DM1 of example 3;

Fig. 9: XRPD pattern of maytansinoid DM1 of example 3.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The following examples are presented to illustrate the method and core concepts of the present invention and, as such, to those skilled in the art, any possible variations or alterations may be made without departing from the spirit of the present invention. The experimental method without specific conditions noted in the embodiments of the present invention is generally conventional conditions or conditions suggested by the manufacturer of the raw materials or goods; reagents of unspecified origin are typically conventional reagents commercially available.

Example1 preparation of DM1-B00

79.8Kg of dichloromethane, 2.5kg of DM1-SM1 and 01 kg of AP are added into a reaction tank and stirred for dissolution, 20.91kg of dicyclohexylcarbodiimide dichloromethane solution is added, the temperature is controlled at 20 ℃, and the stirring reaction is carried out for 30min. Adding 2.2L of zinc chloride (1.0 mol/L diethyl ether) into a reaction tank, controlling the temperature to 20 ℃, continuously stirring for reaction for 3 hours, adding 10.48kg of sodium bicarbonate solution after the reaction is finished, stirring, adding purified water, stirring, filtering, layering filtrate, adding anhydrous sodium sulfate into an organic phase for drying, filtering to obtain a DM1-B00 crude product, filtering the DM1-B00 crude product, performing high-pressure preparation, detecting eluent, and combining DM1-B00 preparation solutions with the purity (area normalization) of more than or equal to 95.0 percent. Concentrating: concentrating under reduced pressure, controlling water bath temperature (38+ -3deg.C, vacuum degree not less than 0.06MPa, concentrating to dry to obtain DM1-B00.

Example 2 preparation of crystalline form A of maytansinoid DM1

Adding 193.04kg of ethyl acetate, 84.028kg of absolute methanol and 1kg of DM1-B00 kg of absolute methanol into a reaction tank, stirring and dissolving, then adding a buffer solution containing dithiothreitol and having pH=7.6+/-0.2, stirring and reacting for 3 hours at 25 ℃, adding dilute hydrochloric acid into the reaction tank after the reaction is finished, and regulating the pH to 6.7+/-0.2. And (3) extracting, washing and drying: the reaction solution was extracted with 80kg of ethyl acetate and 230kg of purified water, the aqueous phase was discarded, and the mixture was washed with purified water, the aqueous phase was discarded, and then with sodium chloride solution, the aqueous phase was discarded, and finally with purified water, and the aqueous phase was discarded. And (3) adding anhydrous magnesium sulfate for drying, filtering, concentrating at least a certain amount of solid to precipitate under reduced pressure, slowly dropwise adding n-hexane for crystallization, filtering, and vacuum drying the obtained solid to obtain DM1 crystal form A, wherein the XRPD pattern of the solid is shown in figure 1.

Example 3 preparation of maytansine DM1 with reference to the literature-in-the-background method step 10

Adding 193.04kg of ethyl acetate, 84.028kg of absolute methanol and 1kg of DM1-B00 kg of absolute methanol into a reaction tank, stirring and dissolving, then adding a buffer solution containing dithiothreitol and having pH=7.6+/-0.2, stirring and reacting for 3 hours at 25 ℃, adding a dilute hydrochloric acid solution into the reaction tank after the reaction is finished, and regulating the pH to 6.7+/-0.2. And (3) extracting, washing and drying: the reaction solution was extracted with 80kg of ethyl acetate and 230kg of purified water, the aqueous phase was discarded, and the mixture was washed with purified water, the aqueous phase was discarded, and then with sodium chloride solution, the aqueous phase was discarded, and finally with purified water, and the aqueous phase was discarded. The DM1 organic phase was obtained, then dried over anhydrous magnesium sulfate, filtered, and concentrated to a foamy solid with an XRPD as shown in fig. 8 under repeated reduced pressure.

Example 4 preparation of maytansinoid DM1 Crystal form B

N-hexane (10 mL), form A (1 g), were added to the reaction flask, stirred at 25℃for 3 hours, then filtered, and dried to give form B, whose XRPD pattern is shown in FIG. 4.

Example 5 stability test of crystalline form

Placing the crystal form A in a vacuum drying oven, drying at 45 ℃ under reduced pressure for 48 hours, wherein solvate cannot be removed, and XRPD shows that the crystal form is unchanged;

The long-term test results of form B at 5.+ -. 3 ℃ and 25.+ -. 3 ℃ are shown in tables 1 and 2.

Table 1 long term stability data (lot number: 210501 AB) lot: 0.048kg investigation conditions: 5+ -3 DEG C

Table 2 long term stability data (lot number: 210501 AB) lot: 0.048kg investigation conditions: 25+ -3 DEG C

The long term stability data for the crystalline form of example 3 at 5±3 ℃ are shown in table 3.

Table 3 long term stability data investigation conditions: 5+ -3 DEG C

Stability data show that the crystal form B has better stability at 2-8 ℃ even at room temperature.

Claims

1. A crystalline form a of maytansine DM1, characterized by: the crystal is orthorhombic, the space group of P212121 (19#) and the unit cell parameters are as follows: z=4, formula: 4 (C ₃₅H₄₈ClN₃O10S)·2(C₄H₈O₂)·(H₂ O).

2. Form a of claim 1, wherein: in the single crystal structure of the crystal, the minimum asymmetric unit contains four maytansine DM1 molecules, two ethyl acetate molecules and one water molecule, and the minimum asymmetric unit is a solvate of ethyl acetate and water.

3. A crystalline form a of maytansine DM1, characterized by: the crystal form A uses Cu-K alpha radiation, an X-ray powder diffraction pattern expressed by a2 theta angle has a characteristic peak at least one position in 8.6+/-0.2 degrees, 11.3+/-0.2 degrees, 19.6+/-0.2 degrees and 22.0+/-0.2 degrees, and/or an X-ray powder diffraction pattern expressed by a2 theta angle has a characteristic peak at least one position in 3.7+/-0.2 degrees, 9.9+/-0.2 degrees and 10.8+/-0.2 degrees; further, the form a has an X-ray powder diffraction pattern as shown in fig. 1.

4. Form a of claim 1, wherein: the DSC spectrum of the maytansine DM1 crystal form A is detected by differential scanning calorimetric analysis (DSC), and endothermic peaks appear in the range of 110-202 ℃; further, endothermic peaks appear successively at 110.8.+ -. 2 ℃, 152.1.+ -. 2 ℃, 170.6.+ -. 2 ℃, 201.9.+ -. 2 ℃; still further, the DSC profile is shown in FIG. 2.

5. Form a of claim 1, wherein: the TGA spectrum of the DM1 crystal form A loses weight by 9.67+/-0.2% when heated to 180+/-2 ℃; further, the TGA profile is shown in fig. 2.

6. A process for the preparation of form a according to any one of claims 1 to 5, characterized in that: the method comprises the following steps:

Dissolving DM1-B00 in a good solvent, adding a pH=7.6+/-0.2 buffer solution containing dithiothreitol, stirring for reaction, regulating the pH to be neutral by using a dilute hydrochloric acid solution after the reaction is finished, extracting the reaction solution by using an extraction solvent to obtain an organic phase, washing, drying, concentrating under reduced pressure until solid is separated out, adding an anti-solvent for crystallization, filtering, and drying to obtain a maytansinoid DM1 crystal form A;

further, the good solvent is selected from one or more of ethyl acetate, methanol, DCM, THF, acetone, ethanol and isopropanol, preferably ethyl acetate;

and/or the extraction solvent is selected from a mixed solution of water and an organic solvent of any one of ethyl acetate, DCM or 2-MeTHF, preferably ethyl acetate;

And/or the antisolvent is selected from one or more than two of normal hexane, cyclohexane, heptane, cyclopentane and methyl tertiary butyl ether, preferably normal hexane.

7. A crystalline form B of maytansine DM1, characterized by: the crystal form B uses Cu-K alpha radiation, and an X-ray powder diffraction pattern expressed in terms of 2 theta angles is 9.8+/-0.2 degrees, 11.3+/-0.2 degrees and 16.3+/-0.2 degrees; a characteristic peak at 19.7+/-0.2 degrees; and/or the X-ray powder diffraction pattern expressed in terms of 2 theta angles has characteristic peaks at 8.6 + -0.2 DEG, 11.2 + -0.2 DEG, 22.0 + -0.2 DEG; and/or an X-ray powder diffraction pattern expressed in terms of 2 theta angles has characteristic peaks at 13.4 + -0.2 DEG, 16.3 + -0.2 DEG, and 19.7 + -0.2 DEG; and/or an X-ray powder diffraction pattern expressed in terms of 2θ has characteristic peaks at 3.7±0.2°,6.4±0.2°,6.5±0.2°,8.1±0.2°,8.3±0.2°,19.7±0.2°, and further, the crystal form B has an X-ray powder diffraction pattern as shown in fig. 4.

8. Form B of claim 7, wherein: the DSC spectrum of the maytansine DM1 crystal form B is detected by differential scanning calorimetric analysis (DSC), and endothermic peaks appear successively in the range of 197-210 ℃, further, the DSC spectrum of the maytansine DM1 crystal form B appears in the range of 197.97 ℃, and still further, the DSC spectrum is shown in figure 5.

9. Form B of claim 7, wherein: the TGA spectrum of the maytansine DM1 crystal form B loses weight by 0.34+/-2% when heated to 250+/-2 ℃; further, the TGA profile is shown in fig. 6.

10. A process for the preparation of crystalline form B of maytansinoid DM1 as claimed in any one of claims 7 to 9, characterized in that: the preparation method comprises the following steps:

Adding a poor solvent into the maytansine DM1 crystal form A, stirring, and carrying out crystal transformation at a certain temperature to obtain a crystal form B; further, the poor solvent is selected from one or more of n-hexane, cyclohexane, dichloromethane, ethyl acetate, methyl tertiary butyl ether, cyclopentane, diethyl ether, acetone and acetonitrile; and/or the seeding temperature is-20 to 40 ℃, preferably 0 to 20 ℃.

11. A method for confirming the absolute configuration of maytansinoid DM1 single crystal by single crystal diffraction using maytansinoid DM1 crystal form a according to any one of claims 1 to 5 or the crystal form a obtained by the production method according to claim 6.

12. Use of crystalline form a of maytansinoid DM1 according to any one of claims 1 to 5 or crystalline form a obtained by the preparation method of claim 6 for the preparation of crystalline form B.

13. A pharmaceutical composition comprising one or more pharmaceutically acceptable carriers and an effective amount of crystalline form a of maytansinoid DM1 according to any one of claims 1 to 5 or crystalline form B according to any one of claims 7 to 9.

14. Use of a crystalline form a of maytansinoid DM1 according to any one of claims 1 to 5 or a crystalline form B of maytansinoid DM1 according to any one of claims 7 to 9 for the preparation of a pharmaceutical conjugate; further, the drug conjugates include antibody drug conjugates, polypeptide drug conjugates, or small molecule drug conjugates of cytotoxins of either form a or form B of maytansinoid DM 1.