Purification method of maxacalcitol

Abstract

The invention relates to a purification method of maxacalcitol. A maxacalcitol crude product prepared by chemical synthesis is subjected to supercritical fluid chromatography (SFC), dynamic axial compression preparation (DAC), refining and purification to obtain the maxacalcitol, and the maxacalcitol is mature in production process, mild in condition and suitable for industrial production. The purity of the maxacalcitol pure product prepared by the method is greater than 98%, preferably greater than 99%, and most preferably greater than 99.8%.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical pharmacy, and particularly relates to a method for purifying maxacalcitol. Background Art

[0002] Maxacalcitol, chemical name: (+)-(5Z,7E)-(1S,3R,20S)-20-(3-hydroxy-3-methylbutyloxy)-9,10-opened pregnan-5,7,10(19)-triene-1,3-diol, structural formula:

[0003]

[0004] Maxacalcitol is a new type of vitamin D agonist, a new third-generation active vitamin D drug developed by Chugai Pharmaceutical Co., Ltd. of Japan. Its injection was approved for marketing in 2000 under the trade name Oxarol, and is used to treat secondary hyperparathyroidism (SHPT) in patients with renal dialysis. After its launch, it has shown good therapeutic effects in clinical applications, and adverse reactions such as hypercalcemia are not obvious, and patients have high tolerance, making it suitable for clinical promotion. Its ointment was approved for marketing in Japan in 2001 for the treatment of dry moss skin diseases such as psoriasis.

[0005] Maxacalcitol has high physiological activity and a very small single injection dose, with a general therapeutic dose of only 2.5μg / time to 10μg / time. Maxacalcitol is extremely unstable. The JP interview form for injections mentions that it needs to be stored at -80°C, argon, and protected from light. In addition, because it contains multiple chiral centers and has a long chemical synthesis route, it is inevitable that some isomer impurities and other possible degradation impurities will be easily produced during the synthesis process. Conventional separation and purification techniques, such as column chromatography and recrystallization, are difficult to obtain APIs that meet drug quality requirements, so the synthesis process has a high threshold and is difficult.

[0006] The purification process of maxacalcitol in the present application is different from the conventional purification process method. The crude maxacalcitol obtained by chemical synthesis, after conventional post-processing purification such as column chromatography and recrystallization, contains multiple unknown impurities and impurity C that are difficult to remove, with the content exceeding 0.5%. TLC thin layer spot plate found that the polarity of the impurities is similar to that of maxacalcitol. The removal effect of conventional purification methods such as column chromatography and recrystallization is not obvious, and the yield is low, which brings high difficulty to the industrial production of the final product. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a method for purifying maxacalcitol, which is to subject the crude maxacalcitol obtained by photochemical synthesis to supercritical fluid chromatography (SFC), dynamic axial compression preparation (DAC), and refining and purification. The production process is mature and the conditions are mild, and it is suitable for industrial production.

[0008] The technical solution for realizing the present invention is:

[0009] The present invention provides a method for purifying maxacalcitol, comprising the following steps:

[0010] (1) Chemical synthesis process: (1α,3β,5E,7E,22E)-1,3-di(tert-butyldimethylsilyloxy)-9,10-secoerugosta-5,7,10(19),22-tetraene is used as the starting material, and maxacalcitol is obtained by eight-step chemical synthesis, and then purified by column chromatography to obtain a crude product of maxacalcitol;

[0011] (2) Supercritical fluid chromatography purification of crude product 1: The crude product 1 of maxacalcitol was dissolved in methanol, separated by SFC preparation, the preparation solution was collected, and concentrated under reduced pressure to obtain the crude product 2 of maxacalcitol;

[0012] (3) Dynamic axial compression preparation and recrystallization purification of crude product 2: The crude product 2 of maxacalcitol was dissolved in methanol, filtered, separated by DAC preparation, the preparation liquid was collected, concentrated to dryness under reduced pressure, and then recrystallized from 1.0% water acetonitrile solution, filtered and dried to obtain the crude product 3 of maxacalcitol.

[0013] In some embodiments, the eluent used for column chromatography separation in step (1) is ethyl acetate-n-hexane solution, and the specific method is: n-hexane / ethyl acetate = 5 / 1 to elute the impurities, and then change to n-hexane / ethyl acetate = 1 / 1 to elute the main product, TLC monitoring, after the elution is completed, the main point eluate is concentrated under reduced pressure at 40-50°C to dryness to obtain the crude product 1 of maxacalcitol.

[0014] In some embodiments, in step (2), the supercritical fluid chromatography adopts SFC200 supercritical carbon dioxide fluid chromatography system for preparative separation, and the chromatographic column is OJ column with a specification of 50*250mm. The specific method is: the crude product 1 is dissolved in methanol, filtered through a 0.45μm organic filter membrane and then injected; the mobile phase A is CO2, and the mobile phase B is methanol; the total flow rate is set to 150ml / min, the column temperature is 35°C, the mobile phase B injection ratio is 20%, and the detection wavelength is 265nm; the target solution is concentrated under the conditions of vacuum degree ≤-0.05MPa and water bath temperature of 30±5°C until no liquid drips out, and then concentrated for 20 to 30min to obtain the crude product 2.

[0015] In some embodiments, the dynamic axial compression in step (3) is performed using a DAC150 dynamic preparative column for preparative separation.

[0016] In some embodiments, the specific method for preparing and separating DAC in step (3) is as follows: dissolving the crude product 2 with methanol, filtering with a 0.45 μm organic filter membrane, preparing a sample solution to be prepared and then injecting the sample; mobile phase A is ethanol, mobile phase B is purified water, the flow rate is set to 700 ml / min, the detection wavelength is 265 nm, and mobile phase A / mobile phase B=4 / 3 for elution; collecting the product segment solution according to the product peak; and reducing the pressure and concentrating the target solution under the conditions of vacuum degree ≤-0.09 MPa and water bath temperature of 30±5°C until almost no liquid drips out.

[0017] In some embodiments, the specific method of recrystallization in step (3) is: using 1.0% water-acetonitrile solution to stir and dissolve at a temperature of 20-25°C, slowly cooling to 10±5°C, stirring and crystallizing for 1-2h; slowly cooling to 0±5°C, stirring and crystallizing for 1-2h; slowly cooling to -15±5°C, stirring and crystallizing for 8-10h; filtering, and drying the filter cake to obtain a crude product 3.

[0018] In some embodiments, the method includes a further refining process, specifically dissolving the crude maxacalcitol 3 obtained in step (3) in acetone, protecting with nitrogen, controlling the temperature at 10±5°C, slowly adding purified water, and after the addition, keeping the temperature at 10±5°C, stirring and crystallizing for 1 to 2 hours, then cooling to 0±5°C, stirring and crystallizing for 2 to 3 hours, filtering and drying to obtain pure maxacalcitol.

[0019] In some embodiments, the purity of the pure maxacalcitol is greater than 98%, preferably greater than 99%, and most preferably greater than 99.8%.

[0020] The beneficial effects of the present invention are:

[0021] 1. Improving product purity: The method for purifying maxacalcitol provided by the present invention can effectively remove impurities that are difficult to remove by traditional methods through supercritical fluid chromatography (SFC) and dynamic axial compression (DAC) separation technology, significantly improve the purity of maxacalcitol, and help improve the quality of drugs.

[0022] 2. Improving product stability: The stability of the purified maxacalcitol by the method of the present invention under high temperature, high humidity and light conditions is significantly better than that of the maxacalcitol produced by the traditional method.

[0023] 3. Suitable for clinical promotion: Due to the characteristics of high purity and high stability, the maxacalcitol of the present invention is more suitable for clinical application, which helps to improve the therapeutic effect and safety of patients and reduce adverse reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 HPLC spectrum for the detection of related substances of crude maxacalcitol 1.

[0025] Figure 2 HPLC spectrum for the detection of related substances of crude maxacalcitol 2.

[0026] Figure 3 This is the HPLC spectrum for detecting related substances of crude maxacalcitol 3 in Example 3.

[0027] Figure 4 This is the HPLC spectrum for related substance detection of crude maxacalcitol 3 in Example 4.

[0028] Figure 5 This is the HPLC spectrum for detecting related substances of the finished product of maxacalcitol in Example 5.

[0029] Figure 6 This is the HPLC spectrum for detecting related substances of the finished product of maxacalcitol in Comparative Example 1. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with specific examples and drawings. The following examples are only illustrative and not restrictive, and the protection scope of the present invention cannot be limited thereto. Unless otherwise specified, the raw materials used can be obtained from the market or homemade.

[0031] Example 1 Synthesis of Maxacalcitol Crude Product 1

[0032] The crude product 1 of maxacalcitol described in the present invention is prepared by chemical synthesis. The specific preparation method is obtained by referring to the method of the embodiment in patent WO2012122451A2, using (1α, 3β, 5E, 7E, 22E)-1, 3-di(tert-butyldimethylsilyloxy)-9, 10-secoergoster-5, 7, 10 (19), 22-tetraene as the starting material, and obtaining maxacalcitol by eight-step chemical synthesis. Prepare a column with a diameter of 12 cm and a height of about 120 cm, weigh 2.4 kg of 100-200 mesh silica gel to load the column, load the n-hexane solution to be used at normal pressure, after loading, use 8.0-9.0 L of n-hexane / ethyl acetate = 5 / 1 to elute the impurities, and then use n-hexane / ethyl acetate = 1 / 1 to elute the main product, TLC monitoring (developing solvent: n-hexane / ethyl acetate = 1 / 2), after elution, the main point eluate is concentrated to dryness under reduced pressure at 40-50°C to obtain 150.0 g of foamy solid, which is the crude product 1 of maxacalcitol.

[0033] The results of HPLC detection of related substances by the crude product 1 of maxacalcitol obtained after column chromatography are shown in Table 1, corresponding to the attached Figure 1 .

[0034] Table 1

[0035] project Unknown impurity 1 Promaxacalcitol Impurity A Unknown impurity 2 Maxacalcitol Impurity C Peak time min 13.824 23.004 26.713 27.494 28.265 35.941 Peak area ratio% 0.842 1.761 3.392 0.791 87.339 2.719

[0036] The above results show that in the traditional column chromatography purification process, in addition to the traditional process impurities pro-maxacalcitol and impurity A (trans-maxacalcitol), multiple difficult-to-remove unknown impurities and impurity C (24-hydroxy-maxacalcitol, structure shown in Formula 1) were also found, and the content of each of them exceeded 0.5%.

[0037]

[0038] Example 2 Preparation of Maxacalcitol Crude Product 2

[0039] The SFC200 supercritical carbon dioxide fluid chromatography system was used for preparative separation. Chromatographic column information: Name: OJ column, specification: 50*250mm, filler particle size: 5μm, filler material: polysaccharide-coated normal phase chiral filler.

[0040] Dissolve 109.0g crude product 1 in 1635ml methanol and filter with a 0.45μm organic filter membrane for later use. Mobile phase A is CO2 and mobile phase B is methanol. Set the total flow rate to 150ml / min, column temperature to 35℃, mobile phase B injection ratio to 20%, detection wavelength to 265nm, and single injection volume to no more than 10ml. Record at least once every 5h to 6h during the preparation period and collect the target product solution. Concentrate the target solution under vacuum degree ≤-0.05MPa and water bath temperature of 30±5℃ until no liquid drips out, and then concentrate for 20 to 30min to obtain 80.3g crude product 2.

[0041] The HPLC test results of related substances of crude maxacalcitol 2 are shown in Table 2. Figure 2 .

[0042] Table 2

[0043] project Unknown impurity 1 Promaxacalcitol Impurity A Unknown impurity 2 Maxacalcitol Impurity C Peak time min 14.897 22.040 27.778 28.570 29.342 Not detected Peak area ratio% 1.647 0.792 0.064 0.040 96.762 Not detected

[0044] The above results show that the unknown impurity 2 and impurity C can be removed well after SFC supercritical fluid preparation and separation. This is because SFC has a higher density and the ability to dissolve and selectively adsorb, which enables it to better dissolve and separate complex isomeric impurities and has a good removal effect on isomeric impurities.

[0045] Example 3 Preparation of Maxacalcitol Crude Product 3

[0046] A DAC150 dynamic preparative column was used for preparative separation. The chromatographic column information was as follows: column inner diameter: 150 mm, column length: 750 mm, filler particle size: 10 μm, filler material: C18.

[0047] Dissolve 10.0g crude product 2 in 100ml methanol, filter with a 0.45μm organic filter membrane to prepare the sample solution, and take 100ml of the crude product 2 sample solution for injection. The maximum injection volume per needle is ≤10g. Mobile phase A is methanol, mobile phase B is acetonitrile, set the flow rate to 700ml / min, the detection wavelength is 265nm, and mobile phase A / mobile phase B=4 / 3 for elution. Collect the product segment solution according to the product peak. Reduce the pressure and concentrate the organic phase under the conditions of vacuum degree ≤-0.09MPa and water bath temperature of 30±5℃ until almost no liquid drips out.

[0048] Dissolve the concentrate with 1050 ml of acetonitrile solution containing 1.0% water at 20-25°C, transfer the solution to the reactor, slowly cool to 10±5°C, stir and crystallize for 1-2 hours. Slowly cool to 0±5°C, stir and crystallize for 1-2 hours. Slowly cool to -15±5°C, stir and crystallize for 8-10 hours. Filter and dry the filter cake to obtain 6.2 g of crude product 3.

[0049] The HPLC test results of the related substances of the crude product 3 of Maxacalcitol are shown in Table 3. Figure 3 .

[0050] Table 3

[0051]

[0052]

[0053] The above results combined with the attached Figure 3 It shows that after DAC dynamic preparative separation, the content of impurities other than the precursor maxacalcitol cannot be controlled below 0.1%. Considering that changing the choice of mobile phase may bring some improvements, the use of mobile phases with polarity and solubility properties different from methanol-acetonitrile may be more conducive to the separation of target compounds and impurities, thereby improving the separation effect.

[0054] Example 4 Preparation of Maxacalcitol Crude Product 3

[0055] A DAC150 dynamic preparative column was used for preparative separation. The chromatographic column information was as follows: column inner diameter: 150 mm, column length: 750 mm, filler particle size: 10 μm, filler material: C18.

[0056] Dissolve 70.0g of crude product 2 in 800ml of methanol, filter with a 0.45μm organic filter membrane, prepare the sample solution to be prepared, and take 100ml of crude product 2 sample solution to be prepared for injection each time. The maximum injection volume per needle is ≤10g. Mobile phase A is ethanol, mobile phase B is purified water, set the flow rate to 700ml / min, the detection wavelength to 265nm, and mobile phase A / mobile phase B=4 / 3 for elution. Collect the product segment solution according to the product peak. Add 4.2kg of sodium chloride to the preparation solution, stir for 5-10min, let stand for 5-10min, separate the liquid, and collect the organic phase. Concentrate the organic phase under reduced pressure under vacuum degree ≤-0.09MPa and water bath temperature of 30±5℃ until almost no liquid drips out.

[0057] Dissolve the concentrate with 1050 ml of acetonitrile solution containing 1.0% water at 20-25°C, transfer the solution to the reactor, slowly cool to 10±5°C, stir and crystallize for 1-2 hours. Slowly cool to 0±5°C, stir and crystallize for 1-2 hours. Slowly cool to -15±5°C, stir and crystallize for 8-10 hours. Filter and dry the filter cake to obtain 55.0 g of crude product 3.

[0058] The HPLC test results of the related substances of the crude product 3 of Maxacalcitol are shown in Table 4. Figure 4 .

[0059] Table 4

[0060] project Unknown impurity 1 Promaxacalcitol Impurity A Unknown impurity 2 Maxacalcitol Impurity C Peak time min Not detected 22.065 25.193 25.905 26.672 Not detected Peak area ratio% Not detected 0.93 0.04 0.04 98.95 Not detected

[0061] The above results combined with the attached Figure 4 It shows that, compared with Example 3, the mobile phase was changed, and the content of impurities other than the precursor maxacalcitol can be well controlled below 0.1% through DAC dynamic preparation separation. This is because the ethanol-water mobile phase has a stronger affinity and the DAC dynamic preparation has different affinity and elution speed for impurities other than isomers, so it has a good removal effect on impurities other than isomers.

[0062] Example 5 Purification of Maxacalcitol

[0063] 62.0 g of the crude product 3 of maxacalcitol obtained in Example 4 was dissolved in 500 ml of acetone, protected by nitrogen, and the temperature was controlled at 10±5°C. 1000 ml of purified water was slowly added dropwise. After the addition was completed, the temperature was kept at 10±5°C, stirred and crystallized for 1 to 2 hours, then cooled to 0±5°C, stirred and crystallized for 2 to 3 hours, and filtered and dried to obtain 55.0 g of finished product of maxacalcitol.

[0064] The HPLC test results of related substances in the refined product of maxacalcitol are shown in Table 5. Figure 5 .

[0065] Table 5

[0066] project Unknown impurity 1 Promaxacalcitol Impurity A Unknown impurity 2 Maxacalcitol Impurity C Peak time min Not detected 22.197 25.327 Not detected 26.883 Not detected Peak area ratio% Not detected 0.93 0.02 Not detected 99.86 Not detected

[0067] The above results show that the purity of the purified maxacalcitol is as high as 99.86%, and unknown impurities and impurity C are effectively removed.

[0068] Comparative Example 1

[0069] Column chromatography: Take out 40.0 g of the crude product 1 obtained in Example 1, prepare a column with a diameter of 8 cm and a height of about 100 cm, weigh 0.8 kg of 100-200 mesh silica gel and load it into the column, load 2.0 L of the n-hexane solution to be used at normal pressure, and after loading, use 2.0-3.0 L of n-hexane / ethyl acetate = 5 / 1 to elute the impurities, and then use n-hexane / ethyl acetate = 2 / 1 to elute the main product, monitor by TLC (developing solvent: n-hexane / ethyl acetate = 1 / 2). After elution, concentrate the main point eluate under reduced pressure at 40-50°C to dryness to obtain 33.8 g of foamy solid, which is the crude product 2 of maxacalcitol.

[0070] Recrystallization: Take 33.0g of crude product 2, dissolve it in 265ml of acetone, protect with nitrogen, control the temperature at 10±5℃, slowly add 1000ml of purified water, keep the temperature at 10±5℃, stir and crystallize for 1~2h, then cool to 0±5℃, stir and crystallize for 2~3h, filter and dry to obtain 28.2g of finished product of maxacalcitol.

[0071] The results are attached Figure 6 As shown in the figure, the traditional column chromatography and recrystallization method is not effective in purifying maxacalcitol, and there are still many impurities in the sample. This shows that the traditional method has limited effect in removing unknown impurity 1, unknown impurity 2, impurity C and impurity A, and cannot control the impurity content within the ideal range.

[0072] Test Example 1

[0073] Stability comparison

[0074] The influencing factors comparison experiment was carried out on the maxacalcitol obtained in Example 5 and the maxacalcitol obtained in Comparative Example 1, and the influencing factors comparison experiment was carried out on the maxacalcitol obtained in Example 5 and the maxacalcitol obtained in Comparative Example 1, respectively, by placing them under high temperature conditions (25°C) for 30 days, high humidity conditions (RH75%) for 30 days and light conditions (sunlight 4500lx±500lx, ultraviolet 90μw / cm 2 ) After 12 days, the properties, related substances and contents were tested to investigate the stability of the two process samples. The results are compared in Table 6.

[0075] Table 6

[0076]

[0077] The results show that the stability of the maxacalcitol sample obtained in Example 5 under high temperature, high humidity and light conditions is significantly better than that of the maxacalcitol obtained in Comparative Example 1.

Claims

1. A method for purifying maxacalcitol, characterized in that: The following steps are involved: (1) Chemical synthesis process: (1α,3β,5E,7E,22E)-1,3-di(tert-butyldimethylsilyloxy)-9,10-secoerugosta-5,7,10(19),22-tetraene is used as the starting material, and maxacalcitol is obtained by eight-step chemical synthesis, and then purified by column chromatography to obtain a crude product of maxacalcitol; (2) Supercritical fluid chromatography purification of crude product 1: The crude product 1 of maxacalcitol was dissolved in methanol, separated by SFC preparation, the preparation solution was collected, and concentrated under reduced pressure to obtain the crude product 2 of maxacalcitol; (3) Dynamic axial compression preparation and recrystallization purification of crude product 2: The crude product 2 of maxacalcitol was dissolved in methanol, filtered, separated by DAC preparation, the preparation liquid was collected, concentrated to dryness under reduced pressure, and then recrystallized from 1.0% water acetonitrile solution, filtered and dried to obtain the crude product 3 of maxacalcitol.

2. The purification method according to claim 1, characterized in that The eluent used for column chromatography separation in step (1) is ethyl acetate-n-hexane solution.

3. The purification method according to claim 1, characterized in that The specific method of step (1) is as follows: n-hexane / ethyl acetate = 5 / 1 is used to elute the impurity, and then n-hexane / ethyl acetate = 1 / 1 is used to elute the main product, and TLC monitoring is performed. After the elution is completed, the main point eluate is concentrated to dryness under reduced pressure at 40-50° C. to obtain the crude product 1 of maxacalcitol.

4. The purification method according to claim 1, characterized in that In step (2), the supercritical fluid chromatography uses a SFC200 supercritical carbon dioxide fluid chromatography system for preparative separation, and the chromatographic column is an OJ column with a specification of 50*250 mm.

5. The purification method according to claim 1, characterized in that The specific method of step (2) is as follows: the crude product 1 is dissolved in methanol, filtered through a 0.45 μm organic filter membrane and then injected; the mobile phase A is CO2, and the mobile phase B is methanol; the total flow rate is set to 150 ml / min, the column temperature is 35°C, the mobile phase B injection ratio is 20%, and the detection wavelength is 265 nm; the target solution is concentrated under the conditions of vacuum degree ≤-0.05 MPa and water bath temperature of 30±5°C until no liquid drips out, and then concentrated for 20 to 30 minutes to obtain the crude product 2.

6. The purification method according to claim 1, characterized in that In step (3), dynamic axial compression is performed using a DAC150 dynamic preparation column for preparation and separation.

7. The purification method according to claim 1, characterized in that The specific method for preparing and separating DAC in step (3) is as follows: dissolving the crude product 2 in methanol, filtering with a 0.45 μm organic filter membrane, preparing the sample solution to be prepared and then injecting the sample; mobile phase A is ethanol, mobile phase B is purified water, the flow rate is set to 700 ml / min, the detection wavelength is 265 nm, and mobile phase A / mobile phase B=4 / 3 for elution; collecting the product segment solution according to the product peak; and concentrating the target solution under reduced pressure at a vacuum degree of ≤-0.09 MPa and a water bath temperature of 30±5°C until almost no liquid drips out.

8. The purification method according to claim 1, characterized in that The specific method of recrystallization in step (3) is as follows: using 1.0% water-acetonitrile solution to stir and dissolve at a temperature of 20-25°C, slowly cooling to 10±5°C, stirring and crystallizing for 1-2h; slowly cooling to 0±5°C, stirring and crystallizing for 1-2h; slowly cooling to -15±5°C, stirring and crystallizing for 8-10h; filtering, and drying the filter cake to obtain a crude product 3.

9. The purification method according to claim 1, characterized in that The method includes a further refining process, specifically dissolving the crude product 3 of maxacalcitol obtained in step (3) in acetone, protecting with nitrogen, controlling the temperature at 10±5°C, slowly dripping purified water, and maintaining the temperature at 10±5°C after the dripping, stirring and crystallizing for 1-2h, then cooling to 0±5°C, stirring and crystallizing for 2-3h, filtering and drying to obtain the pure product of maxacalcitol.

10. The purification method according to any one of claims 1 to 9, characterized in that: The purity of the pure maxacalcitol is greater than 98%, preferably greater than 99%, and most preferably greater than 99.8%.

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