A purification method of desmopressin acetate and its application

Abstract

The present invention provides a purification method and application of desmopressin acetate. The crude desmopressin acetate product is dissolved in an organic solvent, and the dissolved solution is filtered through an organic membrane to obtain a crude product solution; the crude product solution is separated by supercritical fluid chromatography to obtain a high-quality product solution; and the high-quality product solution is subjected to post-processing to obtain the finished desmopressin acetate product. This solution purifies desmopressin acetate with high purity in one step, greatly reducing the production cost while shortening the production cycle, and also consuming less solvent, meeting the requirements of environmental protection development.

Description

Technical Field

[0001] The present invention relates to the field of drug processing, and particularly relates to a purification method of desmopressin acetate and its application. Background Art

[0002] Desmopressin acetate (DDAVP) is a synthetic structural analogue of natural arginine vasopressin, which is derived by deaminating cysteine at position 1 and replacing L-Arg with D-Arg at position 8. This drug has increased stability in the blood and an extended half-life. It has stronger and more persistent antidiuretic activity compared to vasopressin and lysine vasopressin, and the side effect of vasopression is reduced by 400 - 800 times compared to arginine vasopressin. It can also activate the activities of blood factor VIII and plasminogen. Desmopressin acetate has the characteristics of good hemostatic effect and no deep vein thrombosis, and is mainly used to treat hemophilia, diabetes insipidus, and for therapeutic control of bleeding and prevention of bleeding before surgery. It has good market prospects.

[0003] Currently, the purification method of desmopressin acetate mainly relies on reverse-phase high-performance chromatography. For example, patent publication number CN101372504A discloses a method for purifying desmopressin. After two-step purification, a salt conversion treatment is carried out to obtain desmopressin acetate. In the first-step purification, octadecylsilyl-bonded silica gel is used as the stationary phase, phosphate buffer solution is used as phase A, and pure acetonitrile is used as phase B for gradient elution purification. In the second-step purification, octadecylsilyl-bonded silica gel is used as the stationary phase, aqueous acetic acid solution is used as phase A, and pure acetonitrile is used as phase B for gradient elution purification; for the salt conversion treatment, an anion resin is used to convert phosphate and trifluoroacetate into acetate. The product purity is greater than 99%, and the yield is greater than 87%. Patent publication number CN114369142A also obtains pure desmopressin acetate through two-step purification (the salt conversion step is not described). In the first step, octadecylsilyl-bonded silica gel packing is used as the stationary phase, and TFA / acetonitrile is used as the mobile phase system for pretreatment to remove residual ether or other highly retained substances in the crude desmopressin acetate solution; in the second step, octadecylsilyl-bonded silica gel packing is used as the stationary phase, and phosphoric acid and sodium hydroxide are used to adjust the pH to 6.0 - 6.9 / acetonitrile as the mobile phase system for purification to obtain pure desmopressin acetate. The purity is greater than 99%, and the yield is greater than 80%.

[0004] To obtain a product with high purity, existing processes all adopt at least two-step purification steps, which means a huge consumption of solvents, a lengthened production cycle, a significant increase in production costs, and a huge environmental protection pressure in the later stage, not meeting the requirements of the national sustainable development strategy. Summary of the Invention

[0005] The object of the present invention is to provide a purification method and application of desmopressin acetate. By using supercritical fluid chromatography technology, highly pure desmopressin acetate can be obtained in one step, greatly reducing the production cost, shortening the production cycle, and consuming less solvent, meeting the requirements of environmental protection development.

[0006] In the first aspect, this solution provides a purification method of desmopressin acetate, including the following steps:

[0007] Dissolve the crude desmopressin acetate with an organic solvent, and filter the dissolved solution through an organic membrane to obtain a crude product solution;

[0008] Perform supercritical fluid chromatography separation on the crude product solution to obtain a refined product solution; the conditions for supercritical fluid chromatography separation are as follows:

[0009] Mobile phase: a mixture of carbon dioxide and a small amount of organic solvent;

[0010] Temperature: 30 - 40 °C;

[0011] Back pressure: 200 bar;

[0012] Elution method: adopt isocratic elution, and the injection interval is 8 - 12 minutes;

[0013] Perform post-processing on the refined product solution to obtain the finished product of desmopressin acetate.

[0014] It is worth mentioning that supercritical fluid chromatography technology has many advantages compared with reverse-phase high-performance liquid chromatography. Supercritical fluid chromatography uses supercritical fluid as the mobile phase and relies on the solvation ability of the mobile phase for separation and analysis. It combines the characteristics of gas chromatography and liquid chromatography. It can analyze high-boiling-point and low-volatility samples that are not suitable for gas chromatography, and has a faster analysis speed and conditions than high-performance liquid chromatography. For example, as mentioned in the background technology, when purifying desmopressin acetate using the traditional high-performance liquid chromatography method, a lot of organic solvents need to be added, and the entire production cost and production cycle are very high; while in this solution, supercritical fluid chromatography is used for the purification of desmopressin acetate. It uses carbon dioxide as the mobile phase and only needs to add a small amount of organic solvent as a modifier, which can greatly reduce the use of organic solvents, and thus the organic waste generated is two-thirds less than that of reverse-phase high-performance liquid chromatography. In addition, the processing time for purifying desmopressin acetate by supercritical fluid chromatography is also two-thirds shorter than that of reverse-phase high-performance liquid chromatography.

[0015] In other words, this solution uses supercritical fluid chromatography to purify desmopressin acetate, which not only simplifies the purification process and eliminates the need for an additional salt conversion step, but also can shorten the production cycle to more than two-thirds of the traditional process, reduce the production cost by 50%, achieve a product purity greater than 99.8%, and a recovery rate greater than 92%.

[0016] In the embodiment of this solution, methanol is used to dissolve the crude desmopressin acetate product, and the dissolved solution is filtered through a 0.22-micron organic membrane to obtain a crude product solution. The reason for choosing methanol as the solution to dissolve the crude desmopressin acetate product in this solution is that desmopressin acetate exhibits good solubility in methanol, and the cost of methanol and its environmental pollution are relatively small.

[0017] In the embodiment of this solution, the crude product solution is added to a supercritical fluid chromatograph in batches, and a high-quality product solution is obtained through chromatographic separation. Supercritical fluid chromatography separates desmopressin acetate through adsorption and desorption. The conditions of supercritical fluid chromatography need to be selected in combination with the characteristics of desmopressin acetate itself to achieve the highest cost-effective separation effect.

[0018] In the embodiment of this solution, a diol-based silica gel chromatographic column is selected as the stationary phase. This is because after experimental screening, the diol-based column has the best separation effect and the shortest time consumption. Other chromatographic columns such as silica gel columns and amino columns cannot achieve the expected effect.

[0019] And in a preferred embodiment, the diol-based silica gel column preferably has a 10-micron packing, and the packing pore size is 120 Å.

[0020] In the embodiment of this solution, the mobile phase is selected as a combination of a large amount of carbon dioxide and a methanol-acetonitrile mixed solution. The methanol-acetonitrile is used as a modifier to modify its separation of desmopressin acetate. Preferably, the methanol-acetonitrile mixed solution contains a trace amount of acetic acid.

[0021] In the preferred embodiment of this solution, the mobile phase is selected as an 82% carbon dioxide - 18% methanol-acetonitrile mixed solution, and the methanol-acetonitrile mixed solution contains 0.2% acetic acid.

[0022] It is worth mentioning that the applicant combined the characteristics of desmopressin acetate itself and screened the stationary phase and mobile phase additives through a large number of experiments. Finally, an optimal condition was obtained. Experiments show that this mobile phase and stationary phase have the best performance for separating desmopressin acetate.

[0023] In the preferred embodiment of this solution, the elution method is isocratic elution, and the injection interval is 10 minutes.

[0024] In the preferred embodiment of this solution, the temperature in the chromatographic conditions is 35°C.

[0025] In an embodiment of the present solution, the fine product solution is concentrated by low-temperature rotary evaporation and freeze-dried to obtain the finished product of desmopressin acetate.

[0026] In an embodiment of the present solution, the crude desmopressin acetate is derived by deaminating cysteine at position 1 and replacing L-Arg with D-Arg at position 8.

[0027] Compared with the prior art, the present solution has the following characteristics and beneficial effects:

[0028] Different from the traditional process that requires two-step purification of desmopressin acetate using reverse-phase high-performance liquid chromatography, the present solution innovatively uses supercritical fluid chromatography to purify desmopressin acetate in one step, which can significantly reduce the processing cycle. The processing cycle of the present solution is two-thirds of that of the traditional process. In addition, the current supercritical fluid chromatography (SFC) technology on the market is mainly used in the fields of chiral compound separation and traditional Chinese medicine extraction and separation, but there is no application in the field of drug purification. The applicant has uniquely applied supercritical fluid chromatography to the purification field of desmopressin acetate and specifically designed the chromatographic conditions of the supercritical fluid chromatography for desmopressin acetate, so that the purity of the separated desmopressin acetate is as high as over 99.8%, and the recovery rate is also greater than 92%. Moreover, no additional salt conversion step and excessive addition of organic solvents are required during the purification process, which makes the consumption of organic solvents in the present solution significantly lower than that of the traditional process and reduces the production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the chromatogram of the SFC chromatography process.

[0030] Figure 2 It is the typical chromatogram of the finished product of desmopressin acetate prepared in Example 1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art shall fall within the protection scope of the present invention.

[0032] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and a single point value, and between single point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0033] To verify the feasibility and effectiveness of the purification method provided in this solution, the following examples are provided as illustrations in this solution.

[0034] Example 1: Purifying desmopressin acetate using SFC

[0035] (1) Take 1.01 g of the crude desmopressin acetate product obtained by solid-phase synthesis (purity 91.2%), add about 15 ml of methanol, and ultrasonically dissolve it until completely dissolved. Then filter it through a 0.22-μm organic membrane to obtain a crude product solution.

[0036] (2) The stationary phase of SFC is selected as: diol-based silica gel chromatographic column; set the SFC column temperature to 35 °C, the back pressure to 200 bar, the total system flow rate to 70 ml / min, the carbon dioxide flow rate to 82% of the total flow rate, and the flow rate ratio of the organic solvent (methanol to acetonitrile 3:1, containing 0.2% acetic acid) to 18%. Turn on the flow rate balance system and balance for 8 minutes.

[0037] (3) Inject the crude product solution into a 1.5-ml injection system, collect the main peak, determine the peak elution time for the first injection, and after observing the elution of impurity peaks, the remaining crude products can be set for automatic loop injection, and set the parameters to automatically collect the main peak. This is the desmopressin acetate refined product solution.

[0038] (4) Concentrate the obtained refined product solution by low-temperature rotary evaporation and freeze-dry it to obtain the desmopressin acetate finished product.

[0039] The purity is detected by reverse-phase HPLC, and the recovery rate is directly calculated by weight. The purity and recovery rate of the desmopressin acetate finished product obtained in Example 1 are measured to obtain the data: purity 99.91%, recovery rate 92.9%.

[0040] Example 2: Purifying desmopressin acetate using SFC

[0041] (1) Take 10.02 g of the crude desmopressin acetate product obtained by solid-phase synthesis (purity 91.2%), add about 120 ml of methanol, and ultrasonically dissolve it until completely dissolved. Then filter it through a 0.22-μm organic membrane to obtain a crude product solution.

[0042] (2) The stationary phase of SFC is selected as: diol-based silica gel chromatographic column; set the SFC column temperature to 35 °C, the back pressure to 200 bar, the total system flow rate to 550 ml / min, the carbon dioxide flow rate to 82% of the total flow rate, and the flow rate ratio of the organic solvent (methanol to acetonitrile 2:1, containing 0.2% acetic acid) to 18%. Turn on the flow rate balance system and balance for 8 minutes.

[0043] (3) Inject the crude solution into the 10 ml injection system, collect the main peak, determine the peak time of the first injection, and set the remaining crude solution to automatic cycle injection after the impurity peak has appeared. Set the parameters to automatically collect the main peak. This is the desmopressin acetate fine solution.

[0044] (4) The obtained fine solution is concentrated by low-temperature rotary evaporation and freeze-dried to obtain the finished product of desmopressin acetate.

[0045] The purity and recovery rate of the finished product of desmopressin acetate were obtained in Example 2 using the same measuring method as in Example 1, and the data were as follows: purity 99.93% and yield 92.7%.

[0046] Example 3 Purification of desmopressin acetate using SFC

[0047] (1) Take 1.02 g (purity 91.2%) of crude desmopressin acetate obtained by solid phase synthesis, add about 15 ml of methanol and perform ultrasonic treatment until it is completely dissolved, then filter with a 0.22 μm organic membrane to obtain a crude product solution.

[0048] (2) The stationary phase of SFC was selected as a silica gel column; the SFC column temperature was set to 35°C, the back pressure was set to 200 bar, the total system flow rate was set to 70 ml / min, the carbon dioxide flow rate was set to 82% of the total flow rate, the organic solvent (methanol to acetonitrile 3:1, containing 0.2% acetic acid) flow rate ratio was 18%, and the flow rate balance system was turned on and balanced for 8 minutes;

[0049] (3) Inject the crude solution into a 1.5 ml injection system and collect the main peak. After determining the peak time and the peak status of the impurity peak in the first injection, set the remaining crude solution to automatic cycle injection and set the parameters to automatically collect the main peak. This is the desmopressin acetate fine solution.

[0050] (4) The obtained fine solution is concentrated by low-temperature rotary evaporation and freeze-dried to obtain the finished product of desmopressin acetate.

[0051] The purity and recovery rate of the desmopressin acetate product obtained in Example 3 were measured in the same manner as in Example 1 to obtain data: purity 98.05%, yield 90.4%. It can be seen that the performance of the chromatographic column with the glycol-based silica gel chromatographic column as the stationary phase is better.

[0052] Example 4 Purification of Desmopressin Acetate by SFC

[0053] (1) Take 1.03 g (purity 91.2%) of crude desmopressin acetate obtained by solid phase synthesis, add about 15 ml of methanol and perform ultrasonic treatment until it is completely dissolved, then filter with a 0.22 μm organic membrane to obtain a crude product solution.

[0054] (2) The stationary phase of SFC is selected as: diol-based silica gel chromatographic column; the SFC column temperature is set at 35 °C, the back pressure is set at 200 bar, the total system flow rate is set at 70 ml / min, the carbon dioxide flow rate is set at 82% of the total flow rate, the organic solvent is methanol, and the flow rate ratio is 18%. Turn on the flow rate balancing system and balance for 8 minutes;

[0055] (3) Inject the crude product solution into a 1.5 ml injection system, collect the main peak, determine the peak elution time in the first injection, and after observing the elution of impurity peaks, the remaining crude product can be set for automatic loop injection, and the parameters can be set to automatically collect the main peak. This is the purified solution of desmopressin acetate.

[0056] (4) Concentrate the obtained purified solution by low-temperature rotary evaporation and freeze-dry it to obtain the finished product of desmopressin acetate.

[0057] Use the same measurement method as in Example 1 to measure the purity and recovery rate of the desmopressin acetate finished product obtained in Example 4, and the data obtained are: purity 97.50%, recovery rate 90.0%. It can be seen that the separation performance of the mobile phase selected in this solution is better.

[0058] The data statistical tables of each example are shown as follows:

[0059]

[0060] The present invention is not limited to the above best implementation mode. Anyone can obtain other various forms of products under the inspiration of the present invention. However, no matter what changes are made in its shape or structure, as long as it has the same or similar technical solutions as this application, they all fall within the protection scope of the present invention.

Claims

1. A purification method of desmopressin acetate, characterized in that, Comprising the following steps: Dissolve the crude desmopressin acetate product with an organic solvent, and filter the dissolved solution with an organic membrane to obtain a crude product solution; Perform supercritical fluid chromatography separation on the crude product solution to obtain a fine product solution; wherein the conditions for supercritical fluid chromatography separation are: Mobile phase: a mixture of carbon dioxide and a small amount of organic solvent; Temperature: 30 - 40 °C; Back pressure: 200 bar; Elution mode: isocratic elution is adopted, and the injection interval is 8 - 12 minutes; Perform post-processing on the fine product solution to obtain the finished product of desmopressin acetate; the stationary phase is selected as a diol-based silica gel chromatographic column, the diol-based silica gel column has a 10-micron packing, the packing pore size is 120 Å, the mobile phase is selected as an 82% carbon dioxide - 18% methanol - acetonitrile mixed solution, and the methanol - acetonitrile mixed solution contains 0.2% acetic acid.

2. The purification method of desmopressin acetate according to claim 1, wherein Dissolve the crude desmopressin acetate product with methanol.

3. The purification method of desmopressin acetate according to claim 1, characterized in that, Filter the dissolved solution with a 0.22-micron organic membrane to obtain a crude product solution.

4. The purification method of desmopressin acetate according to claim 1, wherein Perform low-temperature rotary evaporation concentration and freeze-drying on the fine product solution to obtain the finished product of desmopressin acetate.

5. The purification method of desmopressin acetate according to claim 1, wherein The crude desmopressin acetate product is derived by deaminating cysteine at position 1 and replacing L-Arg with D-Arg at position 8.

6. The purification method of desmopressin acetate according to claim 1, wherein The elution mode is isocratic elution, and the injection interval is 10 minutes.

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