Preparation method of exemestane EP pharmacopoeia C impurity

Abstract

The invention discloses a preparation method of exemestane EP pharmacopoeia C impurity. According to the preparation method, a low-cost intermediate is used as a starting material, a common formaldehyde aqueous solution, p-toluenesulfonic acid and enzyme catalytic reaction are used, high-toxicity and high-harm reagents are reduced, and side reactions are reduced; 6-bit and 16-bit methylene structure construction is completed in one step by heating and pressurizing, the post-treatment workload is reduced, the subsequent purification difficulty is reduced, the yield is improved, and the method has remarkable significance in quality control of exemestane.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical impurity preparation technology, specifically a method for preparing exemestane EP pharmacopoeia C impurity. Background Technology

[0002] Breast cancer is a common malignant tumor among women, with approximately 1.2 million women diagnosed with it globally each year, and about 500,000 dying from the disease. In the United States, the lifetime risk of a woman developing breast cancer is as high as one in eight, making it the second leading cause of death from malignant tumors. The incidence of breast cancer in my country is rising annually, reaching 56 per 100,000 in major cities like Beijing and Shanghai, making it the leading cause of death among women from malignant tumors.

[0003] Breast cancer treatment drugs can be classified into four categories based on their mechanisms of action: anti-estrogens, aromatase inhibitors (A), luteinizing hormone-releasing hormone (LHRH) analogs, and progestins, with anti-estrogens and aromatase inhibitors being the most dominant. Androstenedione derivatives belong to the steroid class of aromatase inhibitors. These drugs have structures similar to the endogenous substrates of aromatase, androstenedione and testosterone, and irreversibly bind to them via covalent bonds, causing permanent inactivation of the enzyme and thus inhibiting estrogen synthesis. With the widespread clinical application of aromatase inhibitors, new varieties are constantly being introduced, with exemestane and anastrozole becoming the main clinical drugs currently used.

[0004] Exemestane, chemically known as 6-methylene-androst-1,4-diene-3,17-dione, is an irreversible third-generation aromatase inhibitor developed by the Italian company Pharmacia & Upjohn. It was first launched in the UK in 1999.

[0005] 6,16-Dimethylene-androst-1,4-diene-3,17-dione is a C-impurity in the European Pharmacopoeia of exemestane (hereinafter referred to as C-impurity). The presence of this impurity can have a significant impact on the quality of the exemestane product. Therefore, it is necessary to analyze the properties of this impurity in a more targeted manner to provide ideas for controlling the generation and removal of this impurity, thereby further improving the product stability.

[0006] CN106928302A provides a heterosynthetic route for exemestane EP (epistemitetan) in Pharmacopoeia C:

[0007] This route uses exemestane as a raw material, which undergoes a Mannich reaction with paraformaldehyde and dimethylamine hydrochloride to obtain C-hexane. Experiments showed that the purity and yield of C-hexane obtained by this method were both low.

[0008] Therefore, designing a high-yield and high-purity preparation method for exemestane (European Pharmacopoeia C) is of great significance for the quality control of exemestane. Summary of the Invention

[0009] This invention provides a method for preparing exemestane (European Pharmacopoeia C grade) to improve yield and purity, and to better apply it to the quality control of exemestane.

[0010] In view of this, the solution of the present invention is as follows: A method for preparing exemestane EP (epistemtans) pharmacopoeia C (excipient), comprising the following steps: S1. After compound 1 is dissolved, N-methylaniline, formaldehyde aqueous solution and p-toluenesulfonic acid are added. The mixture is heated and pressurized until compound 1 has reacted completely. Then hydrochloric acid is added and the mixture is heated to obtain compound 2. S2. Water, menaquinone, polyethylene glycol-300, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate were added to compound 2. After controlling the pH and raising the temperature, KstD enzyme solution was added, and the mixture was sealed with pure oxygen to pressurize it. The reaction yielded C-impurities. The preparation method involves the following reaction process:

[0011] Preferably, in step S1, the temperature of the heating and pressurizing reaction is 40~60℃, more preferably 50~55℃; the pressure is 0.15~0.2MPa, and the reaction time is 5~8h.

[0012] Further, in step S1, the mass ratio of N-methylaniline, formaldehyde aqueous solution, p-toluenesulfonic acid and compound 1 is (1~3):(1~2.5):(0.01~0.02):1; the mass concentration of the formaldehyde aqueous solution is 37%.

[0013] Preferably, the mass ratio of N-methylaniline, formaldehyde aqueous solution, p-toluenesulfonic acid, and compound 1 is (2.1~2.3):(1.9~2.2):(0.01~0.02):1.

[0014] Further, in step S1, the hydrochloric acid has a mass concentration of 36% and a mass ratio of (2.5~3.5):1 with compound 1; And / or, the temperature at which hydrochloric acid is added and the reaction is raised is 50~60℃, and the reaction time is 15~22h.

[0015] Furthermore, in step S1, the solvent used in the dissolution process of compound 1 is tetrahydrofuran, and the volume amount is 4-5 times the mass of compound 1.

[0016] Further, in step S2, the mass ratio of the amount of water, menadione, polyethylene glycol-300, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate added to compound 2 is (4~6):(0.01~0.02):(0.5~0.8):(0.01~0.05):(0.1~0.5):1.

[0017] Further, in step S2: the reaction pH is 7~8; and / or the reaction temperature is 30~40℃, preferably 33~37℃, and the reaction time is 18~22h.

[0018] Furthermore, in step S2, the reaction process pressure is 0.2~0.5MPa.

[0019] Further, in step S2, the KstD enzyme is a common 3-sterone-1,2-dehydrogenase, preferably the 3-sterone-1,2-dehydrogenase mutant KstD-M disclosed in patent document CN117070482A. 55 The ratio of its dosage to the mass of the substrate is 0.5~2 U / g.

[0020] Furthermore, the preparation method also includes a step of separating compound 2 obtained from the reaction: after cooling the reaction solution to 20~30°C, water is added to continue cooling to 5~15°C, the mixture is stirred evenly, filtered, and dried to obtain compound 2.

[0021] Furthermore, the preparation method also includes the step of separating the C impurities obtained from the reaction: after the reaction is completed, the pressure is released, the reaction solution is filtered, the filter cake is heated to dissolve and the solvent is evaporated, and the obtained product is recrystallized to obtain the C impurities.

[0022] In the above preparation method, the reaction process is as follows:

[0023] Compound 1 is 3-ethoxy-androst-3,5-dien-17-one, CAS: 972-46-3.

[0024] Compared with the prior art, the present invention has the following beneficial effects: The preparation method provided by this invention adopts a novel synthetic route, using low-cost intermediates as starting materials, and employing common formaldehyde aqueous solution, p-toluenesulfonic acid, and enzyme catalysis to reduce highly toxic and hazardous reagents and side reactions. The method achieves the construction of the 6- and 16-methylene structures in a single step under temperature and pressure, reducing post-processing workload, simplifying subsequent purification, and increasing yield. Exemestane EP (Pharmacopoeia C) with a purity of over 99% can be obtained through simple purification steps.

[0025] The preparation method provided by this invention uses excipients that are all used in the synthetic route of exemestane, eliminating the need to use other uncommon materials and avoiding new pressure on downstream environmental treatment; in addition, the reaction system has few difficult-to-handle solvents and is easy to process, further reducing environmental pressure. Detailed Implementation

[0026] The technical solution of the present invention will now be clearly and completely described in conjunction with preferred embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Unless otherwise specified, the reagents used in the embodiments are commercially available reagents, and the experimental methods used are all methods that are well known to those skilled in the art.

[0028] In the examples, compound 1 was a commercially available reagent with a purity ≥98%; the KstD enzyme was the 3-sterone-1,2-dehydrogenase mutant KstD-M55 disclosed in patent document CN117070482A.

[0029] Example 1

[0030] 1. Take 10g of compound 1 and add it to 50ml of tetrahydrofuran. Dissolve it at 25℃. Add 7.5g of N-methylaniline, 21g of 37% formaldehyde aqueous solution, and 0.13g of p-toluenesulfonic acid. Control the temperature at 50℃ and the pressure at 0.15MPa and react for about 7h. After the reaction is complete, add 30g of 36% hydrochloric acid dropwise. Raise the temperature to 58℃ and react for about 18h. After the reaction is complete, cool down to 25℃, add 200ml of water, and continue to cool down to 10℃. Stir well, filter, and dry to obtain 9.81g of off-white solid, which was identified as compound 2, with a yield of 99.37% and an HPLC purity of 97.971%.

[0031] Spectral characteristics of compound 2 identified by 1H NMR spectrum: 1H NMR (CHLOROFORM-d, 600 MHz) δ 6.06 (s, 1H), 5.98 (s, 1H), 5.224 (s, 1H), 5.14 (s, 1H), 4.99 (s, 1H), 2.72 (d, 1H, J=14.1Hz), 2.57 (dd. 1H,J=3.5, 13.5 Hz),2.4-2.5 (m. 2H), 2.0-2.2 (m 1H), 1.9-2.0(m,3H), 1.7-1.8 (m, 2H), 1.5-1.6(m. 1H),1.47 (br dd, 1H, J=4.1, 12.9 Hz), 1.3-14(m, 1.23 (br dd, 1H, J=3.8, 11.4 Hz), 1.01 (s, 3H), 0.88 (s, 3H).

[0032] 2. Take 9g of the solid product from step 1, add 45g of water, maintain the temperature at 15℃, stir well, add 0.18g of menaquinone and polyethylene glycol-300. 6.5g of potassium dihydrogen phosphate, 0.4g of potassium dihydrogen phosphate, and 4.5g of dipotassium hydrogen phosphate were added. After the addition was complete, the air was replaced with oxygen, and the temperature was raised to 35℃. KstD enzyme solution was added at a rate of 0.6 U / g of substrate. The oxygen pressure was increased to 0.3 MPa, and the reaction was maintained at 35℃ for 21 h. During the reaction, the pH of the reaction system was controlled at 7.5. After the reaction was completed, the pressure was released to atmospheric pressure, and the mixture was filtered. 90 ml of acetonitrile was added to the filter cake, and the mixture was heated to 85℃ and stirred for 1.5 h. The mixture was filtered while hot, and 9 ml of acetonitrile was used to wash the filter cake. 63 ml of the filtrate was distilled off under negative pressure at 50-60℃. The mixture was allowed to cool naturally to 30℃, and then cooled to 5℃. The mixture was filtered, and the filter cake was recrystallized by repeating the process of heating and cooling to completely dissolve the solid with 45 ml of acetonitrile. After filtration and drying, 8.05 g of a white solid was obtained, which was exemestane EP pharmacopoeia C impurity. The yield was 88.86%, and the HPLC purity was 98.971%.

[0033] Example 2

[0034] 1. Take 10g of compound 1 and add 40ml of tetrahydrofuran. Dissolve the compound at 25℃. Add 7.5g of N-methylaniline, 22g of 37% formaldehyde aqueous solution, and 0.15g of p-toluenesulfonic acid. Control the temperature at 55℃ and the pressure at 0.15MPa and react for about 6 hours. After the reaction is complete, add 35g of 36% hydrochloric acid dropwise. Increase the temperature to 60℃ and react for about 16 hours. After the reaction is complete, cool down to 30℃, add 200ml of water, and continue to cool down to 10℃. Stir well, filter, and dry to obtain 9.85g of off-white solid, which was identified as compound 2 with a yield of 99.77% and an HPLC purity of 97.822%.

[0035] 2. Take 9g of the solid product from step 1, add 54g of water, maintain the temperature at 10℃, stir well, add 0.15g of menaquinone and polyethylene glycol-300. 4.5g of potassium dihydrogen phosphate, 0.1g of potassium dihydrogen phosphate, and 0.9g of dipotassium hydrogen phosphate were added. After the addition was complete, the air was replaced with oxygen, and the temperature was raised to 35℃. KstD enzyme solution was added at a rate of 0.6 U / g of substrate. The oxygen pressure was increased to 0.4 MPa, and the reaction was maintained at 33℃ for 22 hours. During the reaction, the pH of the reaction system was controlled at 8. After the reaction was completed, the pressure was released to atmospheric pressure, and the mixture was filtered. 90ml of acetonitrile was added to the filter cake, and the mixture was heated to 90℃ and stirred for 1 hour. The mixture was filtered while hot, and 95ml of acetonitrile was used to wash the filter cake. 63ml of the filtrate was distilled off under negative pressure at 50-60℃. The mixture was allowed to cool naturally to 20℃, and then cooled to 5℃. The mixture was filtered, and the filter cake was recrystallized by repeating the process of heating and cooling to completely dissolve the solid with 45ml of acetonitrile. After filtration and drying, 8.12g of a white solid was obtained, which was exemestane EP pharmacopoeia C impurity. The yield was 89.63%, and the HPLC purity was 99.213%.

[0036] Example 3

[0037] 1. Take 100g of compound 1 and add 400ml of tetrahydrofuran. Heat to 25℃ to dissolve. Add 75g of N-methylaniline, 230g of 37% formaldehyde aqueous solution, and 2g of p-toluenesulfonic acid. Control the temperature at 52℃ and the pressure at 0.2MPa and react for about 6 hours. After the reaction is complete, add 250g of 36% hydrochloric acid dropwise. Heat to 52℃ and react for about 21 hours. After the reaction is complete, cool to 30℃, add 2000ml of water, and continue to cool to 10℃. Stir well, filter, and dry to obtain 98.32g of off-white solid, which was identified as compound 2, with a yield of 99.59% and an HPLC purity of 98.036%.

[0038] 2. Take 95g of the product from step 1, add 570g of water, maintain the temperature at 15℃, stir well, add 1.9g of menaquinone and polyethylene glycol-300. 76g of potassium dihydrogen phosphate, 4.5g of potassium dihydrogen phosphate, and 45g of dipotassium hydrogen phosphate were added. After the addition was complete, the air was replaced with oxygen, and the temperature was raised to 35℃. KstD enzyme solution was added at a rate of 0.6 U / g of substrate. The oxygen pressure was increased to 0.5 MPa, and the reaction was maintained at 37℃ for 20 h. During the reaction, the pH of the reaction system was controlled at 7.5. After the reaction was completed, the pressure was released to atmospheric pressure, and the mixture was filtered. 950 ml of acetonitrile was added to the filter cake, and the temperature was raised to 80℃ and stirred for 2 h. The mixture was filtered while hot, and 9 ml of acetonitrile was used to wash the filter cake. 665 ml of the filtrate was distilled off under negative pressure at 50-60℃. The mixture was allowed to cool naturally to 25℃, and then cooled to 5℃. The mixture was filtered, and the filter cake was recrystallized by repeating the process of heating and cooling to completely dissolve the solid with 475 ml of acetonitrile. After filtration and drying, 83.01 g of a white solid was obtained, which was exemestane EP pharmacopoeia C impurity. The yield was 91.63%, and the HPLC purity was 99.110%.

[0039] Comparative Example 1

[0040] 10g of compound 1 was added to 50ml of tetrahydrofuran and heated to 25℃ to dissolve. 7.5g of N-methylaniline, 21g of 37% formaldehyde aqueous solution, and 0.13g of p-toluenesulfonic acid were added. The temperature was controlled at 50℃ and the reaction was allowed to proceed for about 10 hours. 30g of 36% hydrochloric acid was added dropwise, and the temperature was raised to 58℃. The reaction was allowed to proceed for about 18 hours. After the reaction was completed, the temperature was lowered to 25℃, 200ml of water was added, and the temperature was further lowered to 10℃. The mixture was stirred until homogeneous, filtered, and dried to obtain 9.56g of a pale yellow solid with an HPLC purity of 5.628%. The main impurity was compound 3 of the following formula.

[0041]

[0042] The difference between this comparative example and Example 1 is that the pressure restriction was removed, the reaction time was extended until the reaction reached equilibrium, and acid was added subsequently for elimination reaction and post-treatment. The resulting product yielded only a small amount of methylated product at position 16, with most of the reaction yielding only methylated product at position 6.

[0043] Comparative Example 2

[0044] 10g of compound 1 was added to 50ml of tetrahydrofuran and heated to 25℃ to dissolve. 7.5g of N-methylaniline, 21g of 37% formaldehyde aqueous solution, and 0.13g of p-toluenesulfonic acid were added. The reaction was carried out at 25~30℃ and 0.2MPa for about 16h. 30g of 36% hydrochloric acid was added dropwise, and the temperature was raised to 58℃ for about 18h. After the reaction was completed, the temperature was lowered to 25℃, 200ml of water was added, and the temperature was further lowered to 10℃. The mixture was stirred evenly, filtered, and dried to obtain 9.76g of an off-white solid with an HPLC purity of 1.354%. The main impurity was compound 3.

[0045] The difference between this comparative example and Example 1 is that the reaction temperature was lowered, the reaction time was extended until the reaction reached equilibrium, and acid was added subsequently for elimination reaction and post-treatment. The resulting product yielded only a very small amount of the 16-position methylene product, with the vast majority of the product being the 6-position methylene product.

[0046] From Examples 1-3 above, it is clear that compound 2 was prepared in high yield through step 1, and then exemestane EP pharmacopoeia C-type compound was obtained through KstD enzyme catalysis. The overall yield was high, and only simple post-processing was required to obtain high-purity exemestane EP pharmacopoeia C-type compound. Comparative Examples 1 and 2 changed the reaction conditions based on Example 1, but only the 6-position methyleneized product was obtained, indicating that the reaction conditions set in step 1 have an impact on the methyleneization at positions 6 and 16.

[0047] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A process for the preparation of Exemestane EP C8 impurity, characterized by the steps of The preparation method comprises the following steps: S1. After compound 1 is dissolved, N-methylaniline, an aqueous formaldehyde solution and p-toluenesulfonic acid are added, and a temperature and pressure reaction is carried out until compound 1 is completely reacted, then hydrochloric acid is added and a temperature reaction is carried out to obtain compound 2; S2. After water, menadione, polyethylene glycol-300, potassium dihydrogen phosphate and dipotassium hydrogen phosphate are added to compound 2, the pH is controlled and the temperature is increased, then KstD enzyme solution is added, the reaction is carried out under sealed pure oxygen pressure to obtain C heteroatom. The reaction process of the preparation method is as follows: 。 2. The production method according to claim 1, characterized by, In step S1, the temperature and pressure reaction temperature is 40-60 DEG C, the pressure is 0.1-0.2 MPa, and the reaction time is 5-8 h.

3. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of N-methylaniline, the aqueous formaldehyde solution, p-toluenesulfonic acid and compound 1 is (1-3):(1-2.5):(0.01-0.02):1; and the concentration of the aqueous formaldehyde solution is 37%.

4. The method of claim 1, wherein, In step S1, the concentration of the hydrochloric acid is 36%, and the mass ratio of the hydrochloric acid and compound 1 is (2.5-3.5):

1. And / or, the temperature reaction after the hydrochloric acid is added is 50-60 DEG C, and the reaction time is 15-22 h.

5. The preparation method according to claim 1, characterized in that, In step S1, the solvent used in the dissolution process of compound 1 is tetrahydrofuran, and the volume amount is 4-5 times the mass of compound 1.

6. The method of claim 1, wherein, In step S2, the mass ratio of the added water, menadione, polyethylene glycol-300, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and compound 2 is (4-6):(0.01-0.02):(0.5-0.8):(0.01-0.05):(0.1-0.5):

1.

7. The preparation method according to claim 1, characterized in that, In step S2, the reaction pH value is 7-8; and / or, the reaction temperature is 30-40 DEG C, and the reaction time is 18-22 h.

8. The method of claim 1, wherein, In step S2, the reaction process pressure is 0.2-0.5 MPa.

9. The method of claim 1, wherein, The preparation method further comprises the following steps of separating compound 2 obtained by the reaction: after the reaction liquid is reduced to 20-30 DEG C, water is added to continue reducing the temperature to 5-15 DEG C, then the mixture is uniformly stirred, filtered and dried to obtain compound 2.

10. The method of claim 1, wherein, The preparation method further comprises the following steps of separating C heteroatom obtained by the reaction: after the reaction is completed, the pressure is released, the reaction liquid is filtered, the filter cake is heated and dissolved, the solvent is evaporated, and the obtained product is recrystallized to obtain C heteroatom.

Images