Method for purifying pirarubicin

Abstract

The invention belongs to the field of medicinal chemistry, and provides a pirarubicin purification method, which adopts a resin adsorption method and uses an aqueous solution containing acid and an organic solvent for elution. The purification method is suitable for process amplification, low in cost and simple to operate, and compared with existing silica gel purification, the purification is more thorough, and the purity and the recovery rate are higher.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and particularly relates to a purification method of the compound pirarubicin. Background Art

[0002] Pirarubicin, CAS No.: 72496-41-4, is an orange-red crystalline powder and is a broad-spectrum anti-tumor drug applicable to tumors such as head and neck cancer, breast cancer, gastric cancer, urothelial carcinoma (bladder cancer, renal pelvis / urine), fallopian tube tumor, ovarian cancer, uterine cancer, acute leukemia, malignant lymphoma, etc. Its structural formula is shown as follows:

[0003]

[0004] The pirarubicin compound has poor stability and is unstable in solutions, acidic and alkaline environments, etc. It is difficult to obtain a qualified pirarubicin sample. Xu Jianliang et al. from China Pharmaceutical University mentioned a purification method of pirarubicin in the literature "Separation and Purification of Pirarubicin" published in the Northwest Pharmaceutical Journal. A 99.6% pirarubicin sample was obtained after 2 purifications with dichloromethane-methanol as the eluent. Suzhou Sepax Technologies Co., Ltd. CN111675738A discloses a reverse-phase preparation method of a C18 packing, which can achieve a purity of 99.5%, and the requirements for equipment in large-scale production are relatively high.

[0005] Currently, there is an urgent need for an environmentally friendly and simple operation purification method to obtain high-purity and high-yield pirarubicin, which provides effective support for its production process and quality control. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to overcome the defects of the prior art and provide a purification method of pirarubicin with low cost and simple operation to obtain high purity and high yield.

[0007] The present invention provides a purification method of pirarubicin applicable to process amplification, which includes the following steps:

[0008] 1) Take the crude pirarubicin, prepare a loading solution with a dissolving solvent, and load it onto a resin column, where the dissolving solvent is an aqueous solution containing an acid and an organic solvent;

[0009] 2) Elute with an eluent, collect and combine the eluate, extract and concentrate to obtain pirarubicin, where the eluent is an aqueous solution containing an acid and an organic solvent;

[0010] Among them, the volume ratio of the organic solvent in the dissolving solvent in step 1) is different from that in the eluent in step 2).

[0011] Preferably, in the step 1), the resin is a polystyrene resin; preferably, the polystyrene resin is one of polystyrene-divinylbenzene resin or polystyrene-divinylbenzene-polyvinylpyrrolidone resin.

[0012] Preferably, the polystyrene-divinylbenzene resin is one of NanoMicro NM series resin, UniPS series resin, and UniPSA series resin; more preferably, it is one of NM series resin or UniPS series resin.

[0013] Preferably, the polystyrene-divinylbenzene-polyvinylpyrrolidone resin is UniPSN series resin.

[0014] Preferably, in the step 1), the weight-volume ratio of the acid in the dissolution solvent is 0.1‰ - 1‰, and the volume percentage of the organic solvent is 5% - 15%.

[0015] Preferably, in the step 1), the weight-volume ratio of the acid in the dissolution solvent is 0.1‰ - 0.4‰; preferably, the weight-volume ratio of the acid is 0.4‰.

[0016] Preferably, in the step 1), the volume percentage of the organic solvent in the dissolution solvent is 10% - 15%; preferably, the volume percentage of the organic solvent is 10%.

[0017] Preferably, in the step 2), the weight-volume ratio of the acid in the eluent is 0.1‰ - 1‰, and the volume percentage of the organic solvent is 20% - 40%.

[0018] Preferably, in the step 2), the weight-volume ratio of the acid in the eluent is 0.4‰ - 1‰; preferably, the weight-volume ratio of the acid is 0.4‰ - 0.5‰; preferably, the weight-volume ratio of the acid is 0.4‰.

[0019] Preferably, in the step 2), the volume percentage of the organic solvent in the eluent is 20% - 25%; preferably, the volume percentage of the organic solvent is 20%.

[0020] Preferably, the organic solvent in the step 1) or step 2) is one of methanol, ethanol, and acetonitrile; more preferably, it is one of ethanol and acetonitrile; even more preferably, it is acetonitrile.

[0021] Preferably, the acid in the step 1) or step 2) is one of phosphoric acid, acetic acid, and formic acid; preferably, the acid is one of acetic acid and formic acid; more preferably, it is acetic acid.

[0022] Preferably, the present invention provides a method for purifying pirarubicin applicable to process scale-up, which comprises the following steps:

[0023] 1) Take the crude pirarubicin and prepare a sample loading solution with a dissolving solvent, then load it onto a resin column. The dissolving solvent is an aqueous solution containing an acid and an organic solvent;

[0024] 2) Elute with an eluent, collect and combine the eluate, adjust the pH, extract, and concentrate to obtain pirarubicin. The eluent is an aqueous solution containing an acid and an organic solvent;

[0025] Among them, the volume ratio of the organic solvent in the dissolving solvent in step 1) is different from that in the eluent in step 2).

[0026] Preferably, the present invention provides a method for purifying pirarubicin applicable to process scale-up, which comprises the following steps:

[0027] 1) Take the crude pirarubicin and prepare a sample loading solution with a dissolving solvent, then load it onto a resin column. The dissolving solvent is an aqueous solution containing acetic acid and acetonitrile, where the weight / volume ratio of acetic acid is 0.1‰ - 0.4‰ and the volume percentage of acetonitrile is 10% - 15%;

[0028] 2) Elute with an eluent, collect and combine the eluate, adjust the pH, extract, and concentrate to obtain pirarubicin. The eluent is an aqueous solution containing acetic acid and acetonitrile, where the weight / volume ratio of acetic acid is 0.4‰ - 1‰ and the volume percentage of acetonitrile is 20% - 40%.

[0029] Preferably, the present invention provides a method for purifying pirarubicin applicable to process scale-up, which comprises the following steps:

[0030] 1) Take the crude pirarubicin and prepare a sample loading solution with a dissolving solvent, then load it onto a resin column. The dissolving solvent is an aqueous solution containing acetic acid and acetonitrile, where the weight / volume ratio of acetic acid is 0.4‰ and the volume percentage of acetonitrile is 10%;

[0031] 2) Elute with an eluent, collect and combine the eluate, adjust the pH, extract, and concentrate to obtain pirarubicin. The eluent is an aqueous solution containing acetic acid and acetonitrile, where the weight / volume ratio of acetic acid is 0.4‰ - 0.5‰ and the volume percentage of acetonitrile is 20% - 25%.

[0032] Preferably, the present invention provides a method for purifying pirarubicin, which comprises the following steps:

[0033] 1) Take the crude pirarubicin and prepare a sample loading solution with a dissolving solvent, then load it onto a resin column. The dissolving solvent is an aqueous solution containing acetic acid and acetonitrile, where the weight / volume ratio of acetic acid is 0.4‰ and the volume percentage of acetonitrile is 10%;

[0034] 2) Elute with an eluent, collect and combine the eluate, adjust the pH, extract, and concentrate to obtain pirarubicin, wherein the eluent is an aqueous solution containing acetic acid and acetonitrile, and the weight / volume ratio of acetic acid is 0.4‰, and the volume percentage of acetonitrile is 20%.

[0035] Preferably, in step 2), after collecting and combining the eluate, adjust the pH to 7 - 9 with a base, extract with an organic solvent, and concentrate the organic phase to obtain pirarubicin, wherein the base is one of sodium carbonate or sodium bicarbonate, and preferably, the base is sodium bicarbonate; the organic solvent is one of ethyl acetate, dichloromethane, and chloroform; preferably, the organic solvent is dichloromethane.

[0036] Preferably, in step 2), after collecting and combining the eluate, adjust the pH to 7 - 9 with sodium bicarbonate, extract with dichloromethane, and concentrate the organic phase to obtain pirarubicin.

[0037] Preferably, the present invention provides a method for purifying pirarubicin, which comprises the following steps:

[0038] 1) Take the crude pirarubicin, prepare a sample loading solution with a dissolving solvent, and load it onto a resin column, wherein the dissolving solvent is an aqueous solution containing acetic acid and acetonitrile, and the weight / volume ratio of acetic acid is 0.4‰, and the volume percentage of acetonitrile is 10%;

[0039] 2) Elute with an eluent, collect and combine the eluate, adjust the pH to 7 - 9 with sodium bicarbonate, extract with dichloromethane, and concentrate to obtain pirarubicin, wherein the eluent is an aqueous solution containing acetic acid and acetonitrile, and the weight / volume ratio of acetic acid is 0.4‰, and the volume percentage of acetonitrile is 20%.

[0040] Furthermore, the purity of the crude pirarubicin is below 90%.

[0041] Furthermore, the purity of the crude pirarubicin is 80% - 90%.

[0042] Furthermore, the crude pirarubicin is a product after rough fractionation and purification.

[0043] Furthermore, the crude pirarubicin is a product after purification by silica gel.

[0044] Furthermore, the specific steps of the silica gel purification are as follows:

[0045] Dissolve pirarubicin in dichloromethane, load it onto a silica gel column, and then elute with dichloromethane - methanol, and concentrate to obtain the crude pirarubicin.

[0046] Furthermore, the crude pirarubicin is obtained by reacting doxorubicin with 3,4 - dihydropyran and then purifying by silica gel adsorption method.

[0047] The crude pirarubicin can be prepared according to the above method, or it can be the pirarubicin after rough fractionation in "Separation and Purification of Pirarubicin" by Xu Jianliang et al. (Northwest Pharmaceutical Journal, Vol. 27, No. 1, February 2012).

[0048] For the numerical values or numerical endpoints involved in the technical solution of the present invention, the scope of protection of their meanings or significances is not limited to the numbers themselves. Those skilled in the art can understand that they include the allowable error ranges widely accepted in the art, such as experimental errors, measurement errors, same-level errors, and random errors, etc., and these error ranges are all included within the scope of the present invention.

[0049] Advantages of the present invention:

[0050] 1) Resin products are used to purify pirarubicin, and the product purity is increased from 80% to 99.7%, and the unknown single impurity is less than 0.10%, meeting the quality requirements of the review agency for the product.

[0051] 2) The organic solvent is one of methanol, ethanol, and acetonitrile, which has little corrosion damage to the production equipment and its detection instruments, and can effectively save the maintenance cost of the equipment and instruments.

[0052] 3) The recovery rate is doubled: Using resin purification, the product recovery rate is greatly increased from 42.6% to 81.81%, with better efficiency. Description of the drawings

[0053] Figure 1 It is the HPLC chromatogram of pirarubicin in Example 1.

[0054] Figure 2 It is the HPLC chromatogram of the crude pirarubicin in Example 1.

[0055] Figure 3 It is the HPLC chromatogram of the refined pirarubicin in Example 2.

[0056] Figure 4 It is the HPLC chromatogram of the refined pirarubicin in Example 3.

[0057] Figure 5 It is the HPLC chromatogram of the refined pirarubicin in Example 6. Detailed implementation manners

[0058] The present invention will be described more specifically below, but the following examples do not constitute any limitation to the scope of the present invention.

[0059] Unless otherwise specified, the raw materials in the examples of this application are all purchased through commercial channels.

[0060] In the embodiments of the present application, the product purity detection method is HPLC method. Specifically, a Welch Ultimate AQ-C18 chromatographic column is used for analysis and detection, the detection wavelength is 254 nm, the sample concentration is 0.5 mg / mL. Take 0.4 g of sodium edetate, 0.2 mL of triethylamine, add 900 mL of water, adjust the pH value to 3.0 with phosphoric acid, and dilute with water to 1000 mL to obtain a sodium edetate solution; use the sodium edetate solution - acetonitrile (45:55) as phase A, and the sodium edetate solution - acetonitrile (20:80) as phase B.

[0061] In the present application, the recovery rate (%) = (mass of purified sample × purity) ÷ (mass of sample before purification × purity) × 100%;

[0062] Weight - volume ratio = weight of acid (unit: g) ÷ total volume of dissolving solvent or eluent (unit: mL) × 1000‰;

[0063] Volume percentage = volume of organic solvent ÷ total volume of dissolving solvent or eluent × 100%.

[0064] Example 1 Preparation and purification of pirarubicin

[0065] Add N,N - dimethylformamide to the reaction kettle, start stirring, and control the temperature at 20 - 30 °C; sequentially add doxorubicin, 3,4 - dihydro - 2H - pyran, and catalyst; after adding, stir and react at 20 - 30 °C. After the reaction is completed, dilute with dichloromethane, wash with water, add n - heptane to crystallize in the organic phase, filter by suction, and dry to obtain pirarubicin, with a purity of 40.69%, and the liquid chromatogram is shown in the appendix Figure 1 .

[0066] Purification of pirarubicin by silica gel. The silica gel is packed into the column by dry method, and the blank silica gel is rinsed with dichloromethane; dissolve the solid with dichloromethane and load the sample by wet method; elute with dichloromethane - methanol, collect the product fraction, transfer it to a rotary evaporator, concentrate to dryness to obtain the pirarubicin crude product, with a purity of 84.74%, and the liquid chromatogram is shown in the appendix Figure 2 , and the column - passing yield is 21.7%.

[0067] Example 2 Purification of pirarubicin crude product by silica gel adsorption method

[0068] Purify the pirarubicin crude product through silica gel. The silica gel is packed into the column by dry method, and the blank silica gel is rinsed with dichloromethane; dissolve the solid with dichloromethane and load the sample by wet method; elute with dichloromethane - methanol, collect the product fraction, transfer it to a rotary evaporator, concentrate to dryness to obtain the pirarubicin refined product, with a purity of 92.57%, and the liquid chromatogram is shown in the appendix Figure 3 , and the column - passing yield is 42.6%.

[0069] Example 3

[0070] After uniformly dispersing 500 mL of UniPS resin in water, load it into a column for later use. Dissolve 10.0 g of pirarubicin crude product (prepared in Example 1) in 5 L of a dissolution solvent to prepare a sample loading solution, measure the pH = 4 - 5, and directly load the sample. The dissolution solvent is an aqueous solution containing acetic acid and acetonitrile, where the weight - volume ratio of acetic acid is 0.4‰ (0.4 g of acetic acid is dissolved in 1000 mL of the dissolution solvent), and the volume percentage of acetonitrile is 10%. After the resin adsorbs the sample, use an eluent for elution. The weight - volume ratio of acetic acid in the eluent is 0.4‰, and the volume percentage of acetonitrile is 20%. Then collect the red eluate, add an aqueous sodium bicarbonate solution to adjust the system pH = 7 - 8, add 500 mL×2 of dichloromethane for extraction twice, combine the organic phases, and concentrate to obtain 6.95 g of pirarubicin refined product. The liquid chromatogram is shown in the appendix Figure 4 with a purity of 99.75% and a recovery rate of 81.81%.

[0071] Example 4

[0072] After uniformly dispersing 1000 mL of UniPSN resin in water, load it into a column for later use. Dissolve 10.0 g of pirarubicin crude product (prepared in Example 1) in 3 L of a dissolution solvent to prepare a sample loading solution, measure the pH = 3 - 4, and directly load the sample. The dissolution solvent is an aqueous solution containing formic acid and ethanol, where the weight - volume ratio of formic acid is 0.1‰ (0.1 g of formic acid is dissolved in 1000 mL of the dissolution solvent), and the volume percentage of ethanol is 15.0%. After the resin adsorbs the sample, use an eluent for elution. The weight - volume ratio of formic acid in the eluent is 1.0‰, and the volume percentage of ethanol is 20%. Then collect the red eluate, add an aqueous sodium carbonate solution to adjust the system pH = 9, add 500 mL×2 of dichloromethane for extraction twice, combine the organic phases, and concentrate to obtain 6.10 g of pirarubicin refined product.

[0073] Example 5

[0074] After uniformly dispersing 500 mL of UniPSA resin in water, load it into a column for later use. Dissolve 10.0 g of pirarubicin crude product (prepared in Example 1) in 3 L of a dissolution solvent to prepare a sample loading solution, measure the pH 3 - 4, and directly load the sample. The dissolution solvent is an aqueous solution containing phosphoric acid and methanol, where the weight - volume ratio of phosphoric acid is 0.3‰ (0.3 g of phosphoric acid is dissolved in 1000 mL of the dissolution solvent), and the volume percentage of methanol is 15.0%. After the resin adsorbs the sample, use an eluent for elution. The weight - volume ratio of phosphoric acid in the eluent is 0.5‰, and the volume percentage of methanol is 40%. Then collect the red eluate, add an aqueous sodium carbonate solution to adjust the system pH = 7, add 500 mL×2 of dichloromethane for extraction twice, combine the organic phases, and concentrate to obtain 6.10 g of pirarubicin refined product.

[0075] Example 6 Scale - up Process

[0076] Disperse 27 L of the Navi Technology NM series resin (polystyrene resin) evenly with water, load it into a column, and set aside. Dissolve 300 g of pirarubicin crude product in 150 L of a dissolution solvent to prepare a sample loading solution, measure the pH = 4 - 5, and directly load the sample. The dissolution solvent is an aqueous solution containing acetic acid and acetonitrile, where the weight - volume ratio of acetic acid is 0.4‰ and the volume percentage of acetonitrile is 10%. After the resin adsorbs the sample, use an eluent for elution. In the eluent, the weight - volume ratio of acetic acid is 0.4‰ and the volume percentage of acetonitrile is 20%. Then collect the red eluate, transfer it to a 1000 L extraction kettle, add an aqueous sodium bicarbonate solution to adjust the system pH = 7 - 8, extract three times with dichloromethane. After combining the organic phases, transfer them to a 1000 L concentration kettle, concentrate, and finally transfer them to a 20 L rotary evaporator, concentrate (≤40 °C, ≤ - 0.06 MPa) to dryness, weigh 210 g, and the liquid chromatogram is shown in the appendix Figure 5 , and the purity is 99.14%.

[0077] Replacing the resin with the PMM series resin (polymethacrylate) also has good separation and purification effects.

[0078] Example 7

[0079] Adopt the same second - stage purification step as in Example 3, the difference is that the volume ratio of the organic solvent, the type of acid, and the weight - volume ratio of the acid in the dissolution solvent of the pirarubicin crude product are changed. The specific results are as follows in the table:

[0080]

[0081]

[0082] Note: v / m represents the volume - mass ratio, and 500 v / m means 1 g of the sample is dissolved in 500 mL of the solvent.

[0083] For the convenience of sample loading, the organic solvent / water mixed solution needs to dissolve the sample. If the proportion of the organic solvent is too high (such as more than 15%), the product will be eluted, making it difficult to achieve the purpose of purification. If the proportion is too low, a large amount of water is required for dilution, resulting in a large sample - loading volume.

[0084] Example 8

[0085] Adopt the same purification step as in Example 3, the difference is that the type of the organic solvent, the volume ratio of the organic solvent, the type of acid, and the weight - volume ratio of the acid in the elution solvent are changed. The specific results are as follows in the table:

[0086]

[0087] Note: The successful elution of the product can be judged from the color of the eluate (the color of the product component is darker).

[0088] The result monitored by TLC shows that there are relatively many impurities eluted by hydrochloric acid.

[0089] As can be seen from the above table, it is very difficult to elute the product without the presence of acid, and it is also very difficult to elute the product when the proportion of organic solvent is too low.

Claims

1. A purification method of pirarubicin suitable for process scale-up, characterized in that, It includes the following steps: 1) Take the crude pirarubicin, prepare a sample loading solution with a dissolving solvent, and load it onto a resin column. The dissolving solvent is an aqueous solution containing an acid and an organic solvent; 2) Elute with an eluent, collect and combine the eluate, extract and concentrate to obtain pirarubicin. The eluent is an aqueous solution containing an acid and an organic solvent; Among them, the volume ratio of the organic solvent in the dissolving solvent in step 1) is different from that in the eluent in step 2).

2. The method according to claim 1, characterized in that, The resin is a polystyrene resin; preferably, the polystyrene resin is one of polystyrene-divinylbenzene resin or polystyrene-divinylbenzene-polyvinylpyrrolidone resin; preferably, the polystyrene-divinylbenzene resin is one of the NM series resins, UniPS series resins, and UniPSA series resins of Navi Technology; more preferably, it is one of the NM series resins or UniPS series resins; preferably, the polystyrene-divinylbenzene-polyvinylpyrrolidone resin is the UniPSN series resin.

3. The method according to claim 1, characterized in that, In step 1), the weight-volume ratio of the acid in the dissolving solvent is 0.1‰ - 1‰, and the volume percentage of the organic solvent is 5% - 15%; preferably, the weight-volume ratio of the acid is 0.1‰ - 0.4‰; preferably, the weight-volume ratio of the acid is 0.4‰; preferably, the volume percentage of the organic solvent is 10% - 15%; preferably, the volume percentage of the organic solvent is 10%.

4. The method according to claim 1, characterized in that, In step 2), the weight-volume ratio of the acid in the eluent is 0.1‰ - 1‰, and the volume percentage of the organic solvent is 20% - 40%; preferably, the weight-volume ratio of the acid is 0.4‰ - 1‰; preferably, the weight-volume ratio of the acid is 0.4‰ - 0.5‰; preferably, the weight-volume ratio of the acid is 0.4‰; preferably, the volume percentage of the organic solvent is 20% - 25%; preferably, the volume percentage of the organic solvent is 20%.

5. The method according to claim 1, characterized in that, The organic solvent in step 1) or step 2) is one of methanol, ethanol, and acetonitrile; more preferably, it is one of ethanol and acetonitrile; even more preferably, it is acetonitrile; the acid is one of phosphoric acid, acetic acid, and formic acid; preferably, the acid is one of acetic acid and formic acid; even more preferably, it is acetic acid.

6. The method according to claim 1, characterized in that, It includes the following steps: 1) Take the crude pirarubicin, prepare a sample loading solution with a dissolving solvent, and load it onto a resin column. The dissolving solvent is an aqueous solution containing an acid and an organic solvent; 2) Elute with an eluent, collect and combine the eluate, adjust the pH, extract and concentrate to obtain pirarubicin. The eluent is an aqueous solution containing an acid and an organic solvent; Among them, the volume ratio of the organic solvent in the dissolving solvent in step 1) is different from that in the eluent in step 2).

7. The method according to claim 1, characterized in that, It includes the following steps: 1) Take the crude pirarubicin, prepare a sample loading solution with a dissolving solvent, and load it onto a resin column. The dissolving solvent is an aqueous solution containing acetic acid and acetonitrile, where the weight-volume ratio of acetic acid is 0.4‰ and the volume percentage of acetonitrile is 10%; 2) Elute with an eluent, collect and combine the eluate, adjust the pH, extract and concentrate to obtain pirarubicin. The eluent is an aqueous solution containing acetic acid and acetonitrile, where the weight-volume ratio of acetic acid is 0.4‰ and the volume percentage of acetonitrile is 20%.

8. The method according to claim 1, characterized in that, In step 2), after collecting and combining the eluate, adjust the pH to 7-9 with an alkali, extract with an organic solvent, and concentrate the organic phase to obtain pirarubicin, where the alkali is one of sodium carbonate or sodium bicarbonate. Preferably, the alkali is sodium bicarbonate; the organic solvent is one of ethyl acetate, dichloromethane, and chloroform; preferably, the organic solvent is dichloromethane.

9. The method according to claim 1, characterized in that, The method comprises the following steps: 1) Take the crude pirarubicin product, prepare a sample loading solution with a dissolving solvent, and load it onto a resin column. The dissolving solvent is an aqueous solution containing acetic acid and acetonitrile, where the weight-volume ratio of acetic acid is 0.4‰ and the volume percentage of acetonitrile is 10%. 2) Elute with an eluent, collect and combine the eluate, adjust the pH to 7-9 with sodium bicarbonate, extract with dichloromethane, and concentrate to obtain pirarubicin. The eluent is an aqueous solution containing acetic acid and acetonitrile, where the weight-volume ratio of acetic acid is 0.4‰ and the volume percentage of acetonitrile is 20%.

10. The method according to any one of claims 1-9, characterized in that, The crude pirarubicin product is a purified product after rough fractionation; further, the crude pirarubicin product is a product purified by silica gel; further, the specific steps of silica gel purification are as follows: dissolve pirarubicin in dichloromethane, load it onto a silica gel column, and then elute with dichloromethane-methanol, and concentrate to obtain the crude pirarubicin product.

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