A method for purifying paricalcitol using dynamic axial compression chromatography

Abstract

The present invention relates to the technical field of purification of paricalcitol, and specifically discloses a method for purifying paricalcitol using dynamic axial compression (DAC) chromatography. The crude paricalcitol is separated by DAC chromatography, and the separated target product paricalcitol is detected from the sample at a detection wavelength of 264nm. The method has high separation, good sensitivity, fast analysis speed and low solvent consumption, and is suitable for industrial production. The purity of the pure paricalcitol obtained by the method of the present invention is greater than 98%, preferably greater than 99%, and most preferably greater than 99.9%.

Description

Technical Field

[0001] The invention relates to the field of pharmaceutical chemistry, and in particular to a method for purifying paricalcitol by utilizing dynamic axial compression chromatography. Background Art

[0002] Paricalcitol is a synthetic selective vitamin D receptor (VDR) activator, which has a stronger affinity for VDR in parathyroid cells than intestinal mucosal cells. As an active vitamin D therapeutic drug, paricalcitol can better and more efficiently inhibit the synthesis and secretion of parathyroid hormone in patients with uremia by selectively activating VDR and upregulating calcium-sensitive receptors in the thyroid gland, thereby lowering blood calcium levels, while having little effect on intestinal calcium absorption. Paricalcitol is mainly used to treat secondary hyperparathyroidism in patients with chronic renal failure receiving hemodialysis. It is a white, crystalline powder with a molecular formula of C 27 H 44 O3, the structural formula is shown in Formula I:

[0003]

[0004] The synthesis route of paricalcitol includes using 25-dihydroxyvitamin D2 as a raw material, first converting it into 1α, 25-dihydroxy-3,5-cyclovitamin D2, and then undergoing multiple steps of reaction to obtain paricalcitol; quinic acid and VD2 can also be used as raw materials to prepare intermediate fragments respectively, and then react to obtain paricalcitol. However, since paricalcitol contains isomers of various configurations and is photoinstability, diastereomeric impurities and photodegradation impurities are often produced during the preparation process, which are similar in polarity to the product and difficult to separate. These impurities may affect the purity and quality of paricalcitol, so strict control and purification steps are required during the synthesis process to ensure that the purity and quality of the final product meet pharmaceutical standards.

[0005] Patent CN103451238A discloses a method for preparing paricalcitol. Column chromatography (column chromatography silica gel, ethyl acetate: petroleum ether = 1:70) is used for separation and purification. However, this method has limited sample capacity and requires a lot of time, is cumbersome to operate, and is prone to batch differences. Patent CN107540587A discloses a method for purifying paricalcitol. This method dissolves the crude paricalcitol in a mixed solvent of acetone and water, heats and refluxes to completely dissolve the crude paricalcitol, and cools and crystallizes at room temperature to remove impurities. Cooling crystallization is a simple and economical purification method that separates impurities and purifies compounds by crystallization in an appropriate solvent. The advantages are simple operation, low cost, and no special equipment is required. However, due to the high polarity of paricalcitol, the structure of the isomeric impurities is highly similar to that of paricalcitol, and the cooling crystallization purification method is difficult to achieve the purpose of purification.

[0006] At present, there is no quality standard for the separation and analysis method of paricalcitol isomers. The existing technology mainly uses HPLC to separate isomers and purify the final compound. However, due to the high polarity of paricalcitol and the highly similar structure of the isomeric impurities in the reaction product to that of paricalcitol, it is difficult to effectively remove impurities with highly similar structures when HPLC is used for purification. Patent CN109406695A provides a high-performance liquid chromatography method for the simultaneous separation and analysis of paricalcitol and isomeric impurities. However, even if technical support can be provided for the quality control of paricalcitol, HPLC as a preparative method is generally not suitable for application on an industrial scale and is difficult to meet commercial requirements. Therefore, how to provide a purification method that can prepare high-purity paricalcitol with a simple process, a high cumulative amount of product, and can be used for commercial production has become an urgent problem to be solved. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention aims to provide a method for preparing high-purity paricalcitol by using dynamic axial compression chromatography, which has obvious impurity removal effect, strong repeatability, and uses non-toxic and harmless solvents. In addition, the method has a simple process, a high cumulative product amount, and is suitable for industrial production of paricalcitol.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] The present invention provides a method for purifying paricalcitol, wherein the method uses dynamic axial compression chromatography for purification, and the chromatographic conditions include:

[0010] Chromatographic column: C18 silica gel column;

[0011] Mobile phase: methanol as mobile phase A, 0.05% ammonia water as mobile phase B;

[0012] Column temperature: 25-45°C;

[0013] Detection wavelength: 215~300nm;

[0014] Flow rate: 300~600ml / min;

[0015] Injection volume: 100~200ml.

[0016] According to the embodiment of the present invention, the chromatographic column is specifically DEVOTE 120A C18, with specifications of 150mm×500mm, 10um, and the column barrel material: 316L.

[0017] According to an embodiment of the present invention, the volume ratio of mobile phase A to mobile phase B is 45:5 or 50:10 or 45:15.

[0018] According to an embodiment of the present invention, the volume ratio of the mobile phase A to the mobile phase B is preferably 45:15.

[0019] According to an embodiment of the present invention, the column temperature is set to 35°C.

[0020] According to an embodiment of the present invention, the detection wavelength is 264 nm.

[0021] According to an embodiment of the present invention, the flow rate is set to 500 ml / min.

[0022] According to an embodiment of the present invention, the injection volume is 150 ml.

[0023] According to an embodiment of the present invention, the method specifically comprises the following steps:

[0024] (1) Chromatographic condition setting:

[0025] Chromatographic column: DEVOTE 120A C18, specification: 150mm×500mm, 10um, column material: 316L;

[0026] Mobile phase: methanol: 0.05% ammonia water = 45:15;

[0027] Column temperature: 35°C;

[0028] Detection wavelength: 264nm;

[0029] Flow rate: 500ml / min;

[0030] Injection volume: 150ml.

[0031] (2) Sample preparation:

[0032] Take about 150 g of crude paricalcitol, add 75 ml of methanol to dissolve, add 75 ml of acetonitrile to dilute, pass through a 0.45 μm filter membrane (polypropylene microporous filter membrane), and the filtrate is set aside.

[0033] (3) Injection: Use pump A to pump the crude paricalcitol solution into the preparative column.

[0034] (4) Elution: Pump B is used for elution. The main peak with a collection time of 18-35 min is paricalcitol, and the rest is treated as waste liquid.

[0035] (5) Detection: HPLC is used to detect the separation degree of paricalcitol and impurities in the collected solution of step (4) and the purity of paricalcitol.

[0036] According to an embodiment of the present invention, the structure of the impurity is:

[0037]

[0038]

[0039] The beneficial effects of the present invention are:

[0040] 1. The present invention provides a method for preparing high-purity paricalcitol, which for the first time utilizes the separation effect of fluid on molecules under compression in dynamic axial compression chromatography to achieve a higher paricalcitol separation efficiency, and the purity of the obtained product is greater than 98%, preferably greater than 99%, and most preferably greater than 99.9%.

[0041] 2. The present invention overcomes the shortcomings of the prior art, has a large sample capacity, does not require high pressure during the separation process, can handle samples more gently, reduces sample loss and solvent consumption, is suitable for the separation of a large number of samples, reduces enterprise costs, and can be used for the industrialization of paricalcitol. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a liquid chromatogram of paricalcitol obtained according to the method provided in Embodiment 2 of the present invention.

[0043] Figure 2 This is a liquid chromatogram of paricalcitol obtained according to the method provided in Embodiment 3 of the present invention.

[0044] Figure 3 This is a liquid chromatogram of paricalcitol obtained according to the method provided in Example 4 of the present invention.

[0045] Figure 4 This is a liquid chromatogram of paricalcitol obtained according to the method provided in Example 5 of the present invention.

[0046] Figure 5 This is a liquid chromatogram of paricalcitol obtained according to the method provided in Example 6 of the present invention.

[0047] Figure 6 This is a liquid chromatogram of paricalcitol obtained according to the method provided in Example 7 of the present invention.

[0048] Figure 7 This is a liquid chromatogram of paricalcitol obtained according to the method provided in Example 8 of the present invention.

[0049] Figure 8-10 HPLC chromatogram of paricalcitol obtained in parallel experiments according to Example 9 of the present invention. DETAILED DESCRIPTION

[0050] 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.

[0051] Example 1

[0052] This experiment uses the USP-NF2022 standard to detect the separation of impurities 1, impurity 2 and the main peak.

[0053] The specific preparation method of the crude paricalcitol of the present invention is obtained by referring to the method in patent publication number: US5086191A, using quinic acid as a raw material to prepare fragment 1 (2), oxidizing VD2 to obtain fragment 2 (1), and finally reacting fragment 1 (2) with the linked product and side chain of fragment 2 (1) to obtain crude paricalcitol. The specific route is as follows:

[0054]

[0055] The results of the peak elution time and peak area ratio of each component of the crude paricalcitol are shown in Table 1.

[0056] Table 1

[0057] Main Peak Impurity 1 Impurity 2 Peak time / min 11.904 12.11 12.55 Peak area ratio / % 83.27 4.31 7.57

[0058] The above results show that in addition to paricalcitol, there are two impurities with high content, with a total content of more than 10%. The present invention obtains these two impurities through separation and purification, and their structures are confirmed. Their structural formulas, names and sources are as follows:

[0059]

[0060] Paricalcitol is unstable to light and will produce photodegradation impurities (impurity 1) during the synthesis process. At the same time, paricalcitol contains isomers of various configurations and will produce diastereoisomerization impurities (impurity 2) during its synthesis process. From the structural comparison between impurities 1 and 2 and paricalcitol, it is not difficult to find that impurities 1 and 2 have similar polarity to the product and are difficult to separate.

[0061] The existing technology for purifying paricalcitol mainly includes column chromatography, cooling crystallization and HPLC. Even if the existing technology is optimized to separate and prepare paricalcitol, column chromatography and cooling crystallization still have the difficulty of scaling up, and HPLC as a preparative method is usually not suitable for application on an industrial scale and is difficult to meet commercial requirements. Therefore, the present invention aims to explore a method for preparing high-purity paricalcitol using dynamic axial compression chromatography through subsequent experiments, which has obvious impurity removal effect, high product accumulation, and is suitable for industrial production of paricalcitol.

[0062] Example 2

[0063] A method for purifying paricalcitol comprises the following steps:

[0064] (1) Chromatographic condition setting:

[0065] Chromatographic column: DEVOTE 120A C18, specification: 100mm×500mm, 10um;

[0066] Mobile phase: methanol: water = 45:5;

[0067] Column temperature: 35°C;

[0068] Detection wavelength: 264nm;

[0069] Flow rate: 500ml / min;

[0070] Injection volume: 150ml.

[0071] (2) Sample preparation:

[0072] Take about 150 g of crude paricalcitol (batch number: PC-C-17092501), add 75 ml of methanol to dissolve, add 75 ml of acetonitrile to dilute, pass through a 0.45 μm filter membrane (polypropylene microporous filter membrane), and the filtrate is set aside.

[0073] (3) Injection: Use pump A to pump the crude paricalcitol solution into the preparative column.

[0074] (4) Elution: Pump B is used for elution. The main peak with a collection time of 18 to 35 min is paricalcitol, and the rest is treated as waste liquid.

[0075] (5) Detection: The purity of paricalcitol in the liquid collected in step (4) was detected by HPLC.

[0076] The peak area ratios of the components after DAC purification are shown in Table 2. The detection results correspond to Figure 1 .

[0077] Table 2

[0078] Main Peak Impurity 1 Impurity 2 Peak area ratio 94.15％ 1.55％ 1.23％

[0079] The results showed that the peak area ratio of the product HPLC detection was 94.15%, which failed to achieve the purpose of removing impurities. This may be due to the poor selectivity and separation effect of the mobile phase. Therefore, the mobile phase was optimized and a mobile phase with better solubility and fluidity was selected.

[0080] Example 3

[0081] A method for purifying paricalcitol comprises the following steps:

[0082] (1) Chromatographic condition setting:

[0083] Chromatographic column: DEVOTE 120A C18, specification: 100mm×500mm, 10um;

[0084] Mobile phase: acetonitrile:methanol=50:10;

[0085] Column temperature: 35°C;

[0086] Detection wavelength: 264nm;

[0087] Flow rate: 500ml / min;

[0088] Injection volume: 150ml.

[0089] (2) Sample preparation:

[0090] Take about 150 g of crude paricalcitol, add 75 ml of methanol to dissolve, add 75 ml of acetonitrile to dilute, pass through a 0.45 μm filter membrane (polypropylene microporous filter membrane), and the filtrate is set aside.

[0091] (3) Injection: Use pump A to pump the crude paricalcitol solution into the preparative column.

[0092] (4) Elution: Pump B is used for elution. The main peak with a collection time of 18 to 35 min is paricalcitol, and the rest is treated as waste liquid.

[0093] (5) Detection: The purity of paricalcitol in the liquid collected in step (4) was detected by HPLC.

[0094] The peak area ratios of each component are shown in Table 3. The test results correspond to Figure 2 .

[0095] Table 3

[0096] Main Peak Impurity 1 Impurity 2 Peak area ratio 94.16％ 3.04％ 2.55％

[0097] The results showed that the peak area ratio of the product HPLC detection was 94.16%, which failed to achieve the purpose of removing impurities, so the mobile phase was optimized. Since the pKa of paricalcitol is approximately between 4.8 and 5.0, appropriately increasing the pH value of the mobile phase is more suitable for the separation of paricalcitol.

[0098] Example 4

[0099] A method for purifying paricalcitol comprises the following steps:

[0100] (1) Chromatographic condition setting:

[0101] Chromatographic column: DEVOTE 120A C18, specification: 100mm×500mm, 10um;

[0102] Mobile phase: methanol: 0.05% ammonia water = 45:5;

[0103] Column temperature: 35°C;

[0104] Detection wavelength: 264nm;

[0105] Flow rate: 500ml / min;

[0106] Injection volume: 150ml.

[0107] (2) Sample preparation:

[0108] Take about 150 g of crude paricalcitol, add 75 ml of methanol to dissolve, add 75 ml of acetonitrile to dilute, pass through a 0.45 μm filter membrane (polypropylene microporous filter membrane), and the filtrate is set aside.

[0109] (3) Injection: Use pump A to pump the crude paricalcitol solution into the preparative column.

[0110] (4) Elution: Pump B is used for elution. The main peak with a collection time of 18 to 35 min is paricalcitol, and the rest is treated as waste liquid.

[0111] (5) Detection: The purity of paricalcitol in the liquid collected in step (4) was detected by HPLC.

[0112] The peak area ratios of each component are shown in Table 4. The test results correspond to Figure 3 .

[0113] Table 4

[0114] Main Peak Impurity 1 Impurity 2 Peak area ratio 98.32％ 0.42％ 1.26％

[0115] The results showed that the peak area ratio of the product HPLC detection was 98.32%, that is, increasing the pH value of the mobile phase can significantly improve the purity of the product, but it did not achieve the purpose of removing impurities. Therefore, the analysis method was further optimized and the programmed elution method was selected to improve the separation effect.

[0116] Example 5

[0117] A method for purifying paricalcitol comprises the following steps:

[0118] (1) Chromatographic condition setting:

[0119] Chromatographic column: DEVOTE 120A C18, specification: 100mm×500mm, 10um;

[0120] Mobile phase: first rinse with methanol:acetonitrile:water = 10:40:50 for 1.5 h, then rinse with methanol:water = 45:5;

[0121] Column temperature: 35°C;

[0122] Detection wavelength: 264nm;

[0123] Flow rate: 500ml / min;

[0124] Injection volume: 150ml.

[0125] (2) Sample preparation:

[0126] Take about 150 g of crude paricalcitol, add 75 ml of methanol to dissolve, add 75 ml of acetonitrile to dilute, pass through a 0.45 μm filter membrane (polypropylene microporous filter membrane), and the filtrate is set aside.

[0127] (3) Injection: Use pump A to pump the crude paricalcitol solution into the preparative column.

[0128] (4) Elution: Pump B is used for elution. The main peak with a collection time of 18 to 35 min is paricalcitol, and the rest is treated as waste liquid.

[0129] (5) Detection: The purity of paricalcitol in the liquid collected in step (4) was detected by HPLC.

[0130] The peak area ratios of each component are shown in Table 5. The test results correspond to Figure 4 .

[0131] Table 5

[0132] Main Peak Impurity 1 Impurity 2 Peak area ratio 99.14％ 0.73％ 0.13％

[0133] The results showed that the peak area ratio of the product HPLC detection was 99.14%, and the separation effect was significantly improved, but the purpose of removing impurities was still not achieved. In view of the large sample volume of this method, the inner diameter of the chromatographic column was increased to continue to observe the separation effect.

[0134] Example 6

[0135] A method for purifying paricalcitol comprises the following steps:

[0136] (1) Chromatographic condition setting:

[0137] Chromatographic column: DEVOTE 120A C18, specification: 150mm×500mm, 10um, column material: 316L;

[0138] Mobile phase: first rinse with methanol: acetonitrile: water = 10:40:50 for 1.5 h. Then rinse with methanol: water = 45:5;

[0139] Column temperature: 35 °C;

[0140] Detection wavelength: 264nm;

[0141] Flow rate: 500ml / min;

[0142] Injection volume: 150ml.

[0143] (2) Sample preparation:

[0144] Take about 150 g of crude paricalcitol, add 75 ml of methanol to dissolve, add 75 ml of acetonitrile to dilute, pass through a 0.45 μm filter membrane (polypropylene microporous filter membrane), and the filtrate is set aside.

[0145] (3) Injection: Use pump A to pump the crude paricalcitol solution into the preparative column.

[0146] (4) Elution: Pump B is used for elution. The main peak with a collection time of 18 to 35 min is paricalcitol, and the rest is treated as waste liquid.

[0147] (5) Detection: The purity of paricalcitol in the liquid collected in step (4) was detected by HPLC.

[0148] The peak area ratios of each component are shown in Table 6. The test results correspond to Figure 5 .

[0149] Table 6

[0150] Main Peak Impurity 1 Impurity 2 Peak area ratio 99.81％ 0.14％ Not detected

[0151] The results showed that the peak area ratio of the product HPLC detection was 99.81%. The increase in the inner diameter of the chromatographic column can improve the separation effect of paricalcitol, but it still fails to achieve the purpose of removing impurities. Considering the simplicity of operation required for the industrialization of paricalcitol purification, and the small difference in affinity between paricalcitol and impurities 1 and 2, isocratic elution was still selected as the elution method. The above experiments confirmed that the separation effect was better when the mobile phase was methanol-0.05% ammonia water, so the ratio was further optimized.

[0152] Example 7

[0153] A method for purifying paricalcitol comprises the following steps:

[0154] (1) Chromatographic condition setting:

[0155] Chromatographic column: DEVOTE 120A C18, specification: 150mm×500mm, 10um, column material: 316L;

[0156] Mobile phase: methanol: 0.05% ammonia water = 50:10;

[0157] Column temperature: 35 °C;

[0158] Detection wavelength: 264nm;

[0159] Flow rate: 500ml / min;

[0160] Injection volume: 150ml.

[0161] (2) Sample preparation:

[0162] Take about 150 g of crude paricalcitol, add 75 ml of methanol to dissolve, add 75 ml of acetonitrile to dilute, pass through a 0.45 μm filter membrane (polypropylene microporous filter membrane), and the filtrate is set aside.

[0163] (3) Injection: Use pump A to pump the crude paricalcitol solution into the preparative column.

[0164] (4) Elution: Pump B is used for elution. The main peak with a collection time of 18 to 35 min is paricalcitol, and the rest is treated as waste liquid.

[0165] (5) Detection: The purity of paricalcitol in the liquid collected in step (4) was detected by HPLC.

[0166] The peak area ratios of each component are shown in Table 7. The test results correspond to Figure 6 .

[0167] Table 7

[0168] Main Peak Impurity 1 Impurity 2 Peak area ratio 98.95％ 0.08％ 0.83％

[0169] The results showed that the peak area ratio of the product HPLC detection was 98.95%, which failed to achieve the purpose of removing impurities, and the mobile phase ratio was further optimized.

[0170] Example 8

[0171] A method for purifying paricalcitol comprises the following steps:

[0172] (1) Chromatographic condition setting:

[0173] Chromatographic column: DEVOTE 120A C18, specification: 150mm×500mm, 10um, column material: 316L;

[0174] Mobile phase: methanol: 0.05% ammonia water = 45:15;

[0175] Column temperature: 35 °C;

[0176] Detection wavelength: 264nm;

[0177] Flow rate: 500ml / min;

[0178] Injection volume: 150ml.

[0179] (2) Sample preparation:

[0180] Take about 150 g of crude paricalcitol, add 75 ml of methanol to dissolve, add 75 ml of acetonitrile to dilute, pass through a 0.45 μm filter membrane (polypropylene microporous filter membrane), and the filtrate is set aside.

[0181] (3) Injection: Use pump A to pump the crude paricalcitol solution into the preparative column.

[0182] (4) Elution: Pump B is used for elution. The main peak with a collection time of 18 to 35 min is paricalcitol, and the rest is treated as waste liquid.

[0183] (5) Detection: The purity of paricalcitol in the liquid collected in step (4) was detected by HPLC.

[0184] The peak area ratios of each component are shown in Table 8. The test results correspond to Figure 7 .

[0185] Table 8

[0186] Main Peak Impurity 1 Impurity 2 Peak area ratio 99.99％ Not detected Not detected

[0187] The peak area ratio of the product HPLC detection is 99.99%. Under this condition, it has a good separation and removal effect on the impurity 1 and impurity 2 of the paricalcitol raw material drug.

[0188] Example 9

[0189] Parallel test data

[0190] The crude product of Paricalcitol obtained by synthesis was used as raw material. The inner diameter of the dynamic axial compression preparation column was 500 mm. The filler was DEVOTE 120A C18. The amount of crude product loaded at a single time was 150 ml. The separation conditions of the parallel experiment were referred to Example 8.

[0191] The HPLC test results of the separated samples are shown in the table below, corresponding to Figure 8 , Fig. 9 and Fig.10 :

[0192] Table 9

[0193] Main Peak Impurity 1 Impurity 2 Parallel test 1 99.99％ Not detected Not detected Parallel experiment 2 99.97％ Not detected Not detected Parallel test three 99.98％ Not detected Not detected

Claims

1. A method for purifying paricalcitol, characterized in that: The method uses dynamic axial compression chromatography for purification, and the chromatographic conditions include: Chromatographic column: DEVOTE 120A C18, specification: 150mm×500mm, 10um; Mobile phase: methanol as mobile phase A, 0.05% ammonia water as mobile phase B, the volume ratio of mobile phase A to mobile phase B was 45:15; Column temperature: 25~45℃; Detection wavelength: 215~300nm; Flow rate: 300~600ml / min; Injection volume: 100~200ml.

2. The method for purifying paricalcitol according to claim 1, characterized in that: The column temperature was set at 35°C.

3. The method for purifying paricalcitol according to claim 1, characterized in that: The detection wavelength is 264 nm.

4. The method for purifying paricalcitol according to claim 1, characterized in that: The flow rate was set to 500 ml / min.

5. The method for purifying paricalcitol according to claim 1, characterized in that: The injection volume was 150 ml.

6. The method for purifying paricalcitol according to claim 1, characterized in that: The following steps are involved: (1) Chromatographic condition setting: Chromatographic column: DEVOTE 120A C18, specification: 150mm×500mm, 10um, column material: 316L; Mobile phase: methanol: 0.05% ammonia water = 45:15; Column temperature: 35°C; Detection wavelength: 264nm; Flow rate: 500ml / min; Injection volume: 150ml; (2) Sample preparation: Take 150 g of crude paricalcitol, add 75 ml of methanol to dissolve, add 75 ml of acetonitrile to dilute, filter through a 0.45 μm polypropylene microporous filter membrane, and set aside the filtrate; (3) Injection: Use pump A to pump the crude paricalcitol solution into the preparative column; (4) Elution: Pump B is used for elution. The main peak with a collection time of 18 to 35 min is paricalcitol, and the rest is treated as waste liquid. (5) Detection: HPLC is used to detect the separation degree of paricalcitol and impurities in the collected solution of step (4) and the purity of paricalcitol.

7. The method for purifying paricalcitol according to claim 6, characterized in that: The structure of the impurity is: 。

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