TWI488862B - Separation and Purification of Cyclohexyl Compounds and Their Salts - Google Patents

Description

Method for separating and purifying cyclohexyl peptide compounds and salts thereof

The present invention relates to a method for separating and purifying a cyclohexyl peptide compound or a pharmaceutically acceptable salt thereof, and more particularly to a method for separating and purifying caspofungin.

Caspofungin is a semi-synthetic derivative of pulmonary nystatin B ₀ , and its acetate was first marketed in the United States in February 2001. The caspofungin structure is shown in Formula I. This product has broad-spectrum antifungal activity and is suitable for esophageal candidiasis, as well as other drugs such as amphotericin B, amphotericin B liposome, itraconazole and other invasive aspergillosis.

The preparation of caspofungin acetate is described in WO 9421677, EP 620232, WO 9624613, US 5552521, WO 9747645, US 5936062, WO 02083713, EP 1785432 and J. Org. Chem., 2007, 72, 2335-2343.

The synthesized caspofungin needs to be separated and purified to achieve the purity of the medicinal standard. The method for its separation and purification is described in J. LIQ. CHROM. & REL. TECHNOL., 24(6), 781-798 (2001), using a preparative column chromatography device, C18 reverse phase chromatography packing, requiring column efficiency not lower than 15000 trays/meter, mobile phase using acetonitrile/acidic aqueous solution, preparation and separation of caspofungin synthesis intermediate and caspofungin.

US 5378804, US Pat. No. 5,525, 521, US 200929, 1996, US 2009 324 635, WO 2009 151 341, WO 2010 064 219 and US 2010 168 415 all disclose the use of reverse-phase preparative chromatography to purify caspofungin, which is eluted with an acetonitrile/acid aqueous solution, and is enriched with a card. Bofen net fraction, freeze-dried to obtain the product. The prior art has the following disadvantages: (1) using a preparative chromatography system, high equipment requirements; (2) preparing a chromatographic system mostly for a high pressure system, and amplifying production has safety hazards; (3) using a C18 reverse phase packing, which is more expensive; After separation, the lyophilization operation is required to increase the cost and power resource consumption; (5) the obtained caspofungin is sometimes not acetic acid depending on the selected acidic aqueous solution, and the pharmaceutically acceptable caspofungin is Acetate, also needs to undergo salt transfer operation through ion exchange resin, on the one hand, the operation steps are increased, on the other hand, since caspofungin itself is not very stable, excessive operation increases the possibility of impurities; (6) Some of the synthetic intermediates of Fenjing are also purified by preparative column chromatography, adding operational steps and costs.

Known methods are not the best method for industrial production in terms of yield, purity, stability and cost of separation and purification. It is known that the separation and purification of caspofungin acetate requires the use of preparative chromatography steps to purify the intermediate and final product for both the synthesis intermediate and the final product. The multiple use of preparative chromatography steps makes the cost in industrial production increase greatly, resulting in more three wastes, which also increases the difficulty of operation, and it is difficult to carry out large-scale industrial production; therefore, it is necessary to further study the caspofungin acetate suitable for industrial production. Net separation and purification methods and process conditions.

One of the objects of the present invention is to provide a method for separating and purifying a cyclohexyl peptide compound or a pharmaceutically acceptable salt thereof, which is characterized in that it is separated and purified by a macroporous adsorption resin to a purity of a pharmaceutical standard. Among them, the cyclohexyl peptide compound is preferably caspofungin.

Another object of the present invention is to provide a method for separating and purifying caspofungin or a pharmaceutically acceptable salt thereof, which comprises the steps of:

(1) taking caspofungin crude product and injecting a macroporous adsorption resin column;

(2) eluting caspofungin with an aqueous solution containing an organic solvent in an amount of 10% to 45% by volume, and concentrating the obtained eluate to obtain caspofungin or a salt thereof.

(3) Optionally, the product obtained in the step (2) is dissolved in an alcohol solvent or water, an anti-solvent is added dropwise, a solid is precipitated, and caspofungin or a salt thereof is obtained by filtration.

Another object of the present invention is to provide a method for separating and purifying caspofungin or a pharmaceutically acceptable salt thereof, which comprises the steps of:

(1) Take the crude caspofungin and inject the macroporous adsorption resin column.

(2) The eluate (the percentage by volume of the organic solvent is 0% to 30%) is gradually increased by gradually increasing the ratio of the organic solvent to remove impurities in the caspofungin.

(3) The caspofungin is eluted with an aqueous solution containing an organic solvent having a volume of 10% to 45%, and the resulting eluate is concentrated to obtain caspofungin or a salt thereof.

(4) The product obtained in the step (3) may be optionally dissolved in an alcohol or water, an anti-solvent is added dropwise, a solid is precipitated, and caspofungin or a salt thereof is obtained by filtration.

The crude caspofungin described in the step (1) of the process according to the invention can be used according to the known documents WO 9421677, EP 620232, WO 9624613, US 5552521, WO 9747645, US 5936062, WO 02083713, EP 1785432 and J. Org. ., 2007, 72, 2335-2343. It is stated herein that the relevant content of these documents is incorporated into the specification of the present invention.

The macroporous adsorption resin column can be injected by wet or dry method when injecting. The macroporous adsorption resin is selected from polar or non-polar macroporous adsorption resins, preferably HP series, SP series, Amberlite XAD series or Hz series, etc., more preferably HP20SS, Hz832, Hz20SS, Hz818 or H-60 resin.

The aqueous solution as an eluent in the present invention is an acidic or neutral aqueous solution, preferably an acidic aqueous solution. The acidic aqueous solution has a pH of 2.5 to 3.0. The acidic aqueous solution is selected from the group consisting of aqueous hydrochloric acid, aqueous acetic acid, aqueous trifluoroacetic acid, aqueous perchloric acid or aqueous sulfuric acid; preferably aqueous acetic acid. The aqueous acetic acid solution is selected from aqueous solutions of acetic acid having a volume percentage of 0.1% to 5%, preferably from 0.5% to 2% by volume of aqueous acetic acid. The neutral aqueous solution is pure water.

The organic solvent in the aqueous solution as an eluent in the present invention is selected from the group consisting of methanol, ethanol, acetone, acetonitrile or isopropanol; preferably acetonitrile, ethanol or acetone; most preferably acetonitrile. The volume content of the organic solvent in the aqueous solution is from 10% to 45%, preferably from 10% to 20%, most preferably from 12% to 15%. The crude sample amount and the resin volume ratio are selected from 1 g: 10 ml to 1 g: 300 ml, preferably 1 g: 50 ml to 1 g: 150 ml.

The alcohol described in the method of the invention is a lower alcohol capable of dissolving caspofungin, mainly methanol, ethanol, isopropanol, and the anti-solvent is ethyl acetate having less solubility or insoluble to caspofungin. , solvents such as n-hexane, petroleum ether, toluene and higher alcohols.

Another object of the present invention is to provide a method for separating and purifying caspofungin or a pharmaceutically acceptable salt thereof, which comprises the following steps in more detail:

(1) Injection:

The crude caspofungin solution was dissolved in an aqueous solution, and a macroporous adsorption resin column was injected. The macroporous adsorption resin is selected from polar or non-polar macroporous adsorption resins such as HP series, SP series, Amberlite XAD series, and Hz series. The macroporous adsorption resin preferably has a particle size of 50 mesh or more, preferably from HP20SS, Hz832, Hz20SS, Hz818 or H-60 resin. The ratio (w/v) of the crude injection mass to the macroporous adsorption resin is selected from 1:10 to 1:300, preferably from 1:50 to 1:150. The aqueous solution may be an aqueous hydrochloric acid solution, an aqueous acetic acid solution, an aqueous solution of trifluoroacetic acid, an aqueous solution of perchloric acid or an aqueous solution of sulfuric acid; preferably an aqueous solution of acetic acid. The aqueous acetic acid solution is selected from aqueous solutions of acetic acid having a volume percentage of 0.1% to 5%, preferably from 0.5% to 2% by volume of aqueous acetic acid. The pH of the aqueous solution is preferably from 2.5 to 3.0.

(2) Elution:

The resin column may be first washed with an acidic or neutral aqueous solution, and then washed with an acidic or neutral aqueous solution of 5% to 30% (v/v) of an organic solvent in several times of a resin volume to remove impurities contained in the caspofungin. The acidic or neutral aqueous solution is the same as the injection. The organic solvent is selected from the group consisting of methanol, acetone, acetonitrile, ethanol or isopropanol; preferably from ethanol, acetonitrile or acetone.

Elution of caspofungin:

The caspofungin is eluted with an acidic or neutral aqueous solution containing an organic solvent in a volume of 10% to 45%, and the acidic or neutral aqueous solution is used in the same configuration as in the injection. Wherein the organic solvent in the aqueous solution is selected from the group consisting of methanol, acetone, acetonitrile, ethanol or isopropanol; preferably from ethanol, acetonitrile or acetone; most preferably acetonitrile. The volume content of the organic solvent in the aqueous solution is from 10% to 45%, preferably from 10% to 20%, most preferably from 12% to 15%. The crude sample amount and the resin volume ratio are selected from 1:10 to 1:300 (gram: milliliter), preferably from 1:50 to 1:150 (gram: milliliter).

The purity of the obtained eluate was confirmed by high-performance liquid phase, and the desired eluate was combined and concentrated to obtain a product.

The obtained product can be dissolved in an acidic or neutral aqueous solution and then purified by secondary separation of the resin. It may also be dissolved in an easy-to-capacity solvent such as ethanol or methanol, and a non-soluble solvent such as ethyl acetate may be added dropwise to form a caspofungin product.

Considering the yield, product quality, process cost and convenience of the amplification operation of the separation and purification process, it is preferred to carry out two macroporous adsorption resin separation operations on caspofungin. The caspofungin product having a liquid phase purity of >90% can be collected on the first separation and purification, and then the resulting product is subjected to a second separation, and then the caspofungin product having a liquid phase purity of >99% is collected. The second separation and purification operation was the same as the first operation. It is also possible to directly collect the caspofungin product having a liquid phase purity of >99% upon the first separation and purification.

After the elution of caspofungin, the purity of the eluate obtained by high-performance liquid phase was confirmed, and the eluate having a purity of >99% was combined, and the solution was concentrated at a low temperature to obtain caspofungin or a salt thereof. When the aqueous solution of the crude caspofungin and the aqueous solution as the eluent are pure water, the obtained caspofungin is obtained. If at least one of the above two aqueous solutions is an acidic aqueous solution, the corresponding salt of caspofungin is obtained; preferably caspofungin acetate is obtained because it is a pharmaceutically active ingredient; if other caspofungin salts are obtained It can be converted to caspofungin acetate by conventional methods in the art. It can be converted to caspofungin acetate by ion exchange resin, or added to the base free caspofungin salt, and then added with acetic acid to form caspofungin acetate.

The obtained caspofungin or a salt thereof can be dissolved in a solvent such as an aqueous solution of an alcohol, water or an alcohol in acetic acid to be clarified, and then a non-soluble solvent such as ethyl acetate is added dropwise to crystallize caspofungin or a salt thereof. . The alcohol is preferably ethanol or methanol, and the aqueous acetic acid solution of the alcohol is preferably a mixed solution of ethanol:water:acetic acid in a volume ratio of 9:1:0.05.

After the crystallization operation, caspofungin or its salts are converted into solids, which are convenient for storage, transportation, weighing and the like.

The advantages of the present invention over the prior art are:

The invention has simple operation and high degree of separation, and the separated product can reach the purity of the medicinal standard, can be conveniently expanded in the same proportion, and is easy to be industrialized. It is known that separation and purification methods for obtaining caspofungin acetate in accordance with pharmaceutical standards must use several preparative chromatography steps to purify the intermediate and final product. The multiple use of the chromatographic step makes the cost in industrial production increase greatly, resulting in more three wastes, which also increases the difficulty of operation; therefore, the preparative chromatography step is required for industrial production. In the present invention, the caspofungin acetate synthesis intermediate and the final product are not purified by the preparative chromatography step, and the macroporous adsorption resin is used to separate and purify the final product 1-2 times. Due to the use of the macroporous adsorption resin, the operation is greatly simplified compared to the preparative chromatography step, the equipment requirements are reduced, the use of the organic solvent is reduced, the cost is reduced, and the production efficiency is improved. Preparative chromatography steps reported by J. LIQ. CHROM. & REL. TECHNOL., 24(6), 781-798 (2001), US 5378804, US 5552521, US 2009291996, US 2009324635, WO 2009151341, WO 2010064219 and US 2010168415 In contrast, the separation and purification step of the present invention has the following advantages: since the molecular weight of caspofungin is large, it is about 1092, and the ordinary tannin column and the preparative column have fine pore diameters, and the adsorption capacity of the large molecular weight product is strong, and it is difficult. Elution; while the present invention employs a macroporous adsorption resin having a large pore size which is compatible with the caspofungin or its salt to be separated, the use of the macroporous adsorption resin can provide a good elution separation effect. The caspofungin acetate is separated and purified by macroporous adsorption resin, and the yield and product purity are both high, which is beneficial to reduce cost and quality control of the final product. The use of an inexpensive macroporous adsorption resin as a separation medium has a very large cost advantage over the preparation of expensive fillers in the chromatography step, and the macroporous adsorption resin can be repeatedly reused after washing activation; most of the eluting solvent used is water. Compared to the use of a large amount of chromatographically pure organic reagents in the preparative chromatography step, the cost is greatly reduced and the environmental pollution is reduced. Compared with the high-pressure stainless steel column and the high-pressure pump used in the preparative chromatography step, the equipment used for the macroporous adsorption resin is a common glass column, which has low equipment requirements, simple operation, safety and high efficiency.

Therefore, the present invention has great advantages in terms of product quality, cost, equipment requirements, and environmental pollution as compared with the prior art.

Detailed description of the preferred embodiment

The following examples are intended to illustrate the invention, but are not intended to limit the invention in any way.

Example

The solution concentrations mentioned in the present invention are all volume percent concentrations.

The HP20SS resin used in the examples was purchased from Mitsubishi Corporation of Japan; Hz20ss resin, Hz832 resin, and H-60 resin were purchased from East China University of Science and Technology Shanghai Zhenhua Technology Co., Ltd. The purity of the products in the examples were all determined by liquid chromatography.

Example 1:

Take 100 ml column of HP20SS resin and 500 ml of 1% acetic acid aqueous solution to equilibrate the resin column.

The crude product (purity 50%) obtained by caspofungin was 1 g, and dissolved in 20 ml of a 1% aqueous acetic acid solution. Inject the resin column at a flow rate of 1 BV/h.

Rinse with 400 ml of 1% aqueous acetic acid solution and rinse with 500 ml of 10% acetonitrile in 1% aqueous acetic acid. Caspofungin was eluted with 12% acetonitrile in 1% aqueous acetic acid. After the liquid phase detection, the eluates with a purity of >99% were combined and concentrated to dryness to obtain 0.12 g of caspofungin acetate, and the final purity was 99.8%.

After adding a crystallizing solution (ethanol:water:acetic acid volume ratio: 9:1:0.05, 2 ml), the mixture was dissolved until clarification, and ethyl acetate was added dropwise to precipitated solid, which was filtered. It was obtained with a purity of >99% and a single impurity <0.1% of caspofungin acetate. The remaining eluate with a purity of <99% is recovered or applied for secondary separation.

Example 2:

Take 100 ml of HP20SS resin column and 500 ml of 2% acetic acid aqueous solution to equilibrate the resin column.

The crude product (purity 50%) obtained by caspofungin was 1 g, and dissolved in 20 ml of a 2% aqueous acetic acid solution. Inject the resin column at a flow rate of 1 BV/h.

Rinse with 400 ml of 2% aqueous acetic acid solution and rinse with 500 ml of 10% acetonitrile in 2% aqueous acetic acid. Caspofungin was eluted with 15% acetonitrile in 2% aqueous acetic acid. After the liquid phase detection, the eluates with a purity of >99% were combined and concentrated to dryness to obtain 0.13 g of caspofungin acetate, and the final purity was 99.7%.

After adding a crystallizing solution (ethanol:water:acetic acid volume ratio: 9:1:0.05, 2 ml), the mixture was dissolved until clarification, and ethyl acetate was added dropwise to precipitated solid, which was filtered. It was obtained with a purity of >99% and a single impurity <0.1% of caspofungin acetate. The remaining eluate with a purity of <99% is recovered or applied for secondary separation.

Example 3:

Take Hz832 resin 100 ml column, 1% acetic acid aqueous solution 500 ml balance resin column.

The crude product (purity 50%) obtained by caspofungin was 1 g, and dissolved in 20 ml of a 1% aqueous acetic acid solution. Inject the resin column at a flow rate of 1 BV/h.

Rinse with 400 ml of 1% aqueous acetic acid solution and rinse with 500 ml of 1% aqueous acetic acid solution of 20% ethanol. Caspofungin was eluted with 2% ethanol in 1% aqueous acetic acid. After liquid phase detection, the eluates with a purity of >90% were combined and concentrated to dryness to obtain 0.55 g of caspofungin acetate, and the final purity was 91.5%.

After adding 2 ml of ethanol and dissolving until clarification, about 20 ml of ethyl acetate was added dropwise, and crystallized to obtain caspofungin acetate, and the purity in a high-performance liquid phase was 91.5%.

Example 4:

Take 100 ml of HP20SS resin column and 500 ml of 2% acetic acid aqueous solution to equilibrate the resin column.

The crude product (purity 50%) obtained by caspofungin was 1 g, and dissolved in 20 ml of a 2% aqueous acetic acid solution. Inject the resin column at a flow rate of 1 BV/h.

Rinse with 400 ml of 2% aqueous acetic acid solution and rinse with 500 ml of 2% aqueous acetic acid in 10% ethanol. Caspofungin was eluted with 20% ethanol in 2% aqueous acetic acid. After liquid phase detection, the eluate with a purity of >90% was combined and concentrated to dryness to obtain 0.50 g of caspofungin acetate, and the final purity was 90.8%.

After adding 2 ml of methanol to dissolve and clarifying, about 20 ml of ethyl acetate was added dropwise, and crystallized to obtain caspofungin acetate, and the purity in a high-performance liquid phase was 90.8%.

Example 5:

Take Hz832 resin 100 ml column, 1% acetic acid aqueous solution 500 ml balance resin column.

The crude product (purity 50%) obtained by caspofungin was 1 g, and dissolved in 20 ml of a 1% aqueous acetic acid solution. Inject the resin column at a flow rate of 1 BV/h.

Rinse with 400 ml of 1% aqueous acetic acid solution and rinse with 500 ml of 10% acetonitrile in 1% aqueous acetic acid. Caspofungin was eluted with 12% acetonitrile in 1% aqueous acetic acid. After liquid phase detection, the eluates with a purity of >90% were combined and concentrated to dryness to obtain 0.54 g of caspofungin acetate, and the final purity was 91.8%.

After adding 2 ml of ethanol to dissolve for clarification, about 20 ml of ethyl acetate was added dropwise, and crystallized to obtain caspofungin acetate, and the purity in a high-performance liquid phase was 91.8%.

Example 6

Take 100 ml column of HP20SS resin and 500 ml of 1% acetic acid aqueous solution to equilibrate the resin column.

The crude product (purity 50%) obtained by caspofungin was 1 g, and dissolved in 20 ml of a 1% aqueous acetic acid solution. Inject the resin column at a flow rate of 1 BV/h.

Rinse with 400 ml of 1% aqueous acetic acid solution and rinse with 500 ml of 10% acetonitrile in 1% aqueous acetic acid. Caspofungin was eluted with 12% acetonitrile in 1% aqueous acetic acid. After liquid phase detection, the eluate with a purity of >90% was combined and concentrated to dryness to obtain 0.60 g of caspofungin acetate, and the final purity was 92.5%.

After adding 2 ml of ethanol to dissolve for clarification, about 20 ml of ethyl acetate was added dropwise, and crystallized to obtain caspofungin acetate, and the purity in a high-performance liquid phase was 92.5%.

Example 7

Take 100 ml of HZ20SS resin column and 500 ml of 1% acetic acid aqueous solution to equilibrate the resin column.

The crude product (purity 50%) obtained by caspofungin was 1 g, and dissolved in 20 ml of a 1% aqueous acetic acid solution. Inject the resin column at a flow rate of 1 BV/h.

Rinse with 400 ml of 1% aqueous acetic acid solution and rinse with 500 ml of 10% acetonitrile in 1% aqueous acetic acid. Caspofungin was eluted with 12% acetonitrile in 1% aqueous acetic acid. After liquid phase detection, the eluate with a purity of >90% was combined and concentrated to dryness to obtain 0.52 g of caspofungin acetate, and the final purity was 92.6%.

After adding 2 ml of methanol to dissolve for clarification, about 20 ml of ethyl acetate was added dropwise, and crystallized to obtain caspofungin acetate, and the purity in a high-performance liquid phase was 92.6%.

Example 8

Take 100 ml of HZ20SS resin column and 500 ml of 2% acetic acid aqueous solution to equilibrate the resin column.

The crude product (purity 50%) obtained by caspofungin was 1 g, and dissolved in 20 ml of a 2% aqueous acetic acid solution. Inject the resin column at a flow rate of 1 BV/h.

Rinse with 400 ml of 2% aqueous acetic acid solution and rinse with 500 ml of 10% acetone in 2% aqueous acetic acid. Caspofungin was eluted with 15% acetone in 2% aqueous acetic acid. After liquid phase detection, the eluate with a purity of >90% was combined and concentrated to dryness to obtain caspofyl acetate 0.44 g, and the final purity was 91.2%.

After adding 2 ml of ethanol to dissolve for clarification, about 20 ml of ethyl acetate was added dropwise, and crystallized to obtain caspofungin acetate, and the purity in a high-performance liquid phase was 91.2%.

Example 9

Take 100 ml column of HP20SS resin and 500 ml of 1% acetic acid aqueous solution to equilibrate the resin column.

The caspofungin obtained in any of Examples 3-8 was subjected to one-time separation and purification of the product, and 1 g of the product was dissolved in 20 ml of a 1% aqueous acetic acid solution. The resin column was injected at a flow rate of about 1 BV/h. Rinse with 400 ml of 1% aqueous acetic acid solution and rinse with 500 ml of 10% acetonitrile in 1% aqueous acetic acid.

The caspofungin was eluted with 12% acetonitrile in 1% aqueous acetic acid, and the mixture was analyzed by liquid phase, and the elution of >99% purity was combined and concentrated to dryness at low temperature to obtain caspofungin 0.4 g. The final purity was 99.8%. The remaining eluate with a purity of <99% was concentrated and concentrated after decompression.

The obtained product was dissolved in a crystallizing liquid (ethanol:water:acetic acid volume ratio: 9:1:0.05, 2 ml). After cleavage, ethyl acetate was added dropwise to precipitated solids, filtered, and the filter cake was rinsed with a small amount of ethyl acetate. It was obtained with a purity of >99% and a single impurity <0.1% of caspofungin acetate.

Example 10:

Take 100 ml of HZ20SS resin column and 500 ml of 1% acetic acid aqueous solution to equilibrate the resin column.

The caspofungin obtained in any of Examples 3-8 was subjected to one-time separation and purification of the product, and 1 g of the product was dissolved in 20 ml of a 1% aqueous acetic acid solution. The resin column was injected at a flow rate of about 1 BV/h. Rinse with 400 ml of 1% aqueous acetic acid solution and rinse with 500 ml of 10% acetonitrile in 1% aqueous acetic acid.

The caspofungin was eluted with 12% acetonitrile in 1% aqueous acetic acid solution, and the eluate was purified by liquid phase, and the elution was >99%, and concentrated to dryness at low temperature to obtain caspofyl acetate 0.45 g. The final purity was 99.8%. The remaining eluate with a purity of <99% is concentrated and recovered for reuse.

The obtained product was dissolved in a crystallizing solution (ethanol:water:acetic acid volume ratio: 9:1:0.05, 2 ml). After cleavage, ethyl acetate was added dropwise to precipitated solids, filtered, and the filter cake was rinsed with a small amount of ethyl acetate. It was obtained with a purity of >99% and a single impurity <0.1% of caspofungin acetate.

Example 11

Take 100 ml column of HP20SS resin and 500 ml of 0.5% acetic acid aqueous solution to equilibrate the resin column.

The caspofungin obtained in any of Examples 3-8 was subjected to one-time separation and purification of the product, and 1 g of the product was dissolved in 20 ml of a 0.5% aqueous acetic acid solution. The resin column was injected at a flow rate of about 1 BV/h. Rinse with 400 ml of 0.5% aqueous acetic acid solution and rinse with 500 ml of 10% acetonitrile in 0.5% aqueous acetic acid.

The caspofungin was eluted with a 0.5% aqueous solution of acetonitrile in 0.5% acetic acid, and the eluate with a purity of >99% was combined and concentrated to dryness at a low temperature to obtain 0.42 g of caspofungin acetate. The final purity was 99.8%. The remaining eluate with a purity of <99% is concentrated and recovered for reuse.

The obtained product was dissolved in a crystallizing liquid (ethanol:water:acetic acid volume ratio: 9:1:0.05, 2 ml). After cleavage, ethyl acetate was added dropwise to precipitated solids, filtered, and the filter cake was rinsed with a small amount of ethyl acetate. It was obtained with a purity of >99% and a single impurity <0.1% of caspofungin acetate.

Example 12

Take 100 ml column of HP20SS resin and 500 ml of 0.5% acetic acid aqueous solution to equilibrate the resin column.

The caspofungin obtained in any of Examples 3-8 was subjected to one-time separation and purification of the product, and 1 g of the product was dissolved in 20 ml of a 2% aqueous acetic acid solution. The resin column was injected at a flow rate of about 1 BV/h. Rinse with 400 ml of 2% aqueous acetic acid solution and rinse with 500 ml of 10% acetone in 2% aqueous acetic acid.

Caspofungin was eluted with 15% acetone in 2% aqueous acetic acid. After liquid phase detection, the eluates with a purity of >99% were combined and concentrated to dryness at a low temperature to obtain 0.35 g of caspofungin acetate, and the final purity was 99.8%. The remaining eluate with a purity of <99% was concentrated and concentrated after decompression.

The obtained product was dissolved in a crystallizing liquid (ethanol:water:acetic acid volume ratio: 9:1:0.05, 2 ml). After cleavage, ethyl acetate was added dropwise to precipitated solids, filtered, and the filter cake was rinsed with a small amount of ethyl acetate. It was obtained with a purity of >99% and a single impurity <0.1% of caspofungin acetate.

Example 13

Take 100 ml of HP20SS resin column and 500 ml of 2% acetic acid aqueous solution to equilibrate the resin column.

The crude product (purity 50%) obtained by caspofungin was 1 g, and dissolved in 20 ml of a 1% aqueous acetic acid solution. Inject the resin column at a flow rate of 1 BV/h.

Rinse with 400 ml of 1% aqueous acetic acid solution and elute caspofungin with 30% ethanol in 1% aqueous acetic acid. After the liquid phase detection, the eluates with a purity of >99% were combined and concentrated to dryness to obtain 0.08 g of caspofungin acetate, and the final purity was 99.8%.

After adding a crystallizing solution (ethanol:water:acetic acid volume ratio: 9:1:0.05, 2 ml), the mixture was dissolved until clarification, and ethyl acetate was added dropwise to precipitated solid, which was filtered. It was obtained with a purity of >99% and a single impurity <0.1% of caspofungin acetate. The remaining eluate with a purity of <99% is recovered or applied for secondary separation.

Example 14

Take Hz832 resin 100 ml column, 1% acetic acid aqueous solution 500 ml balance resin column.

The crude product (purity 50%) obtained by caspofungin was 1 g, and dissolved in 20 ml of a 1% aqueous acetic acid solution. Inject the resin column at a flow rate of 1 BV/h.

Rinse with 400 ml of 1% aqueous acetic acid and elute caspofungin with 45% acetonitrile in 1% aqueous acetic acid. After liquid phase detection, the eluate with a purity of >90% was combined and concentrated to dryness to obtain 0.61 g of caspofungin acetate, and the final purity was 92.8%.

After adding 2 ml of ethanol to dissolve for clarification, about 20 ml of ethyl acetate was added dropwise, and crystallized to obtain caspofungin acetate, and the purity of the high-performance liquid phase was about 92.8%.

Example 15

Take 100 ml column of HP20SS resin and 500 ml of 1% acetic acid aqueous solution to equilibrate the resin column.

The crude product (purity 50%) obtained by caspofungin was 1 g, and dissolved in 20 ml of a 1% aqueous acetic acid solution. Inject the resin column at a flow rate of 1 BV/h.

Rinse with 400 ml of 1% aqueous acetic acid solution and rinse with 500 ml of 10% acetonitrile in 1% aqueous acetic acid. Caspofungin was eluted with 12% acetonitrile in 1% aqueous acetic acid. After the liquid phase detection, the eluates with a purity of >99% were combined and concentrated to dryness to obtain 0.13 g of caspofungin acetate, and the final purity was 99.8%.

After adding 5 ml of methanol to dissolve, the ethyl acetate was added dropwise to precipitate a solid, which was filtered. It was obtained with a purity of >99% and a single impurity <0.1% of caspofungin acetate. The remaining eluate with a purity of <99% is recovered or applied for secondary separation.

Example 16:

A column of 100 ml of H-60 resin and 500 ml of a 1% aqueous solution of acetic acid were used to equilibrate the resin column.

The crude product (purity 50%) obtained by caspofungin was 1 g, and dissolved in 20 ml of a 1% aqueous acetic acid solution. Inject the resin column at a flow rate of 1 BV/h.

Rinse with 400 ml of 1% aqueous acetic acid solution and rinse with 500 ml of 10% acetonitrile in 1% aqueous acetic acid. Caspofungin was eluted with 12% acetonitrile in 1% aqueous acetic acid. After the liquid phase detection, the eluates with a purity of >99% were combined and concentrated to dryness to obtain 0.12 g of caspofungin acetate, and the final purity was 99.8%.

After adding 3 ml of ethanol to dissolve until clarification, ethyl acetate was added dropwise to precipitate a solid, which was filtered. It was obtained with a purity of >99% and a single impurity <0.1% of caspofungin acetate. The remaining eluate with a purity of <99% is recovered or applied for secondary separation.

Claims (16)

A method for purifying caspofungin or a pharmaceutically acceptable salt thereof, which is characterized in that it is separated and purified by macroporous adsorption resin, and comprises the following steps: (1) taking crude caspofungin and injecting macroporous adsorption a resin column; (2) eluting caspofungin with an aqueous solution containing a volume of 10% to 45% of an organic solvent, and concentrating the resulting eluate to obtain caspofungin or a salt thereof, wherein The aqueous solution is an acidic aqueous solution having a pH of 2.5 to 3.0; and (3) optionally, the product obtained in the step (2) is dissolved in an alcohol solvent or water, and the anti-solvent is added dropwise to precipitate a solid, which is filtered to obtain caspofungin or Its salts. The method according to claim 1, wherein the method can be purified by using 1-2 times of macroporous adsorption resin. The method according to claim 2, characterized in that it can be purified by using two macroporous adsorption resins. The method according to any one of claims 1 to 3, wherein the macroporous adsorption resin is selected from the group consisting of a polar or non-polar macroporous adsorption resin such as HP series, SP series, Amberlite XAD series, Hz series. The method of claim 4, wherein the macroporous adsorption resin is selected from the group consisting of HP20SS, Hz832, Hz20SS, and Hz818 resin. The method according to claim 1, wherein the acidic aqueous solution is selected from the group consisting of aqueous hydrochloric acid, aqueous acetic acid, aqueous trifluoroacetic acid, aqueous perchloric acid, and aqueous sulfuric acid. The method of claim 6, wherein the acidic aqueous solution is selected from the group consisting of aqueous acetic acid. The method according to claim 6 or 7, wherein the aqueous acetic acid solution is selected from the group consisting of 0.1% to 5% aqueous acetic acid. The method of claim 8, wherein the aqueous acetic acid solution is selected from the group consisting of 0.5% to 2% aqueous acetic acid. The method of claim 1, wherein the aqueous solution contains an organic solvent selected from the group consisting of methanol, ethanol, acetone, acetonitrile, and isopropanol. The method of claim 10, wherein the aqueous solution contains an organic solvent selected from the group consisting of acetonitrile, ethanol, and acetone. The method according to claim 1, wherein the aqueous solution contains the organic solvent in a volume content of 10% to 45%. The method according to claim 12, wherein the aqueous solution contains the organic solvent in a volume content of 10% to 20%. The method according to claim 13, wherein the aqueous solution contains an organic solvent in a volume content of 12% to 15%. The method of claim 1, wherein the crude sample amount and the resin volume ratio are selected from the group consisting of 1 gram: 10 ml to 1 gram: 300 ml. The method of claim 15, wherein the crude sample amount and the resin volume ratio are selected from the group consisting of 1 gram: 50 ml to 1 gram: 150 ml.