TW201231475A - Method for separating and purifying cyclohexapeptide compound and salt thereof - Google Patents

Abstract

The present invention provides a method for separating and purifying a cyclic hexapeptide compound or a pharmaceutically acceptable salt thereof. The cyclohexapeptide compound is preferably caspofungin.

Description

201231475 VI. Description of the Invention: [Technical Field] 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. [Prior Art] Caspofungin (caspofungin) is a semi-synthetic derivative of pulmonary nymphin B. Its acetate was first marketed in the United States in February 2001. The caspofungin structure is as 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, etc., which are ineffective or intolerant to 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. 3 201231475 The synthesized caspofungin's need to be isolated and purified to achieve the purity of the medicinal standard. The method for its separation and purification is described in j LIQ cHR0M & REL· TECHNOL·, 24(6), 781-798 (2001), using a preparative column chromatography device, C18 reverse phase chromatography packing, requiring a column efficiency of not less than 15〇. The number of trays per meter was measured, and the mobile phase was prepared by separating the caspofungin synthesis intermediate and caspofungin using an acetonitrile/acid aqueous solution.

US 5378804, us 5552521, US 2009291990, US 2009324635, WO 2009151341, WO 2010064219, and US 2010168415 all disclose the use of reverse phase preparative chromatography to purify caspofungin 'with its acetonitrile domain aqueous solution' combined with f-containing card Bofen net part of the 'freeze dry to serve the product. The prior art has the following disadvantages: (1) using a preparative chromatography system, the equipment requirements are high; (7) the preparative chromatographic system is mostly a high pressure system, and the amplification production has a female full hazard; (3) the C18 reverse phase packing is more expensive. (4) the convection is required after separation. Carry out; Donggan operation, increase the cost and consumption of power resources; (5) the resulting Kapoo Pan Macheng corrects lL, _

, increased operational steps and costs.

A chromatographic step is performed to purify the intermediate and final product. . It is known to use a force for caspofungin acetate and the final product. More preparative chromatography steps

S 4 201231475 The use of people makes the cost in industrial production increase greatly, resulting in more three wastes, which also increases the difficulty of operation and makes it difficult to carry out large-scale industrial production. Therefore, it is necessary to further study the separation of caspofungin acetate suitable for industrial production. Purification methods and process conditions. SUMMARY OF THE INVENTION One object of the present invention is to provide a method for separating and purifying a cyclohexyl peptide compound or a linearly acceptable salt thereof, which is characterized in that it is separated and purified by using a macroporous adsorption resin, and can be used for medicinal purposes. Standard purity. 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 following steps: (1) taking crude caspofungin and injecting a macroporous adsorption resin column (2) ^-Contains caspofungin in an aqueous solution containing an organic solvent in a volume-recognition, and concentrates the resulting eluate to give caspofungin or a salt thereof, (3) optionally ' The product obtained in the step (7) is dissolved in an alcohol solvent or water, the anti-solvent is added dropwise, and the solid is precipitated, and the caspofungin or its salt is filtered to obtain another type of caspofungin or its drug. A method for separating and purifying a salt, comprising the steps of: drying (1) taking caspofungin crude product, and injecting macroporous adsorption resin hydrazine. Use gradually increase the percentage of the product as impurities. Eluent in organic solvent ratio (% 3030% of organic solvent) is rinsed 'Removal of caspofungin (2) (3) (3) 201231475 - Water of the solvent of the machine =: Pofin, which will The obtained eluate is concentrated to obtain caspofungin (4). The product obtained in the step (3) is optionally dissolved in an alcohol or water, and an antisolvent 'precipitating solid' is added dropwise to obtain caspofungin or a salt thereof. The crude caspofungin described in the step (1) of the method of the present invention can be given as W0 9421677, EP 620232, W〇9624613, us 5 help 21, W0 9747645 ^ US 5936062 ^ W0 02083713 ^ EP 1785432 # J.Org.Chem ·, 2 thanks, 72, 2335_2343 prepared. It is hereby stated that the relevant content of the literature is incorporated into the description 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 the group consisting of polar or non-polar macroporous adsorption resins, preferably Η? series, sp series, Amberlite XAD series or Hz series, and 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 pH of the acidic aqueous solution is 2 5 to 3. The aqueous acid solution is selected from the group consisting of aqueous hydrochloric acid solution, aqueous acetic acid solution, aqueous trifluoroacetic acid solution, aqueous solution of high gas acid or aqueous sulfuric acid; The aqueous acetic acid solution is selected from aqueous solutions of acetic acid having a volume percentage of 0.1% to 5%, preferably an aqueous solution of acetic acid having a volume percentage of 0.5% to 2%. The neutral aqueous solution is pure water. The organic solvent in the aqueous solution as the eluent in the present invention is selected from the group consisting of methanol, ethanol, acetone, acetonitrile or isopropanol; preferably acetonitrile, ethanol or acetone is most preferably acetonitrile. The volume content of the organic solvent in the aqueous solution is 6 201231475 12%~15%. 1 g of crude sample: 300 ml, preferably 10% to 45%, preferably 10% to 2%, most preferably the volume and resin volume ratio is selected from 1 g: 10 ml, 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 acetic acid B which has a small or insoluble degree to Kafim. @之意,正己烧,石(四), 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: Dissolving caspofungin crude product In the aqueous solution, a large pore adsorption resin column is injected. The macroporous adsorption resin is selected from polar or non-polar macroporous adsorption resins such as Hp series, sp series, AmberHte 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-6 resin. The ratio of the crude injection mass to the amount of macroporous adsorption resin (w/v) is selected from 1: 1 〇 to 1: 300, preferably from 1: 50 to 1: 150. The aqueous solution may be an aqueous solution of hydrochloric acid, an aqueous solution of acetic acid, an aqueous solution of trifluoroacetic acid, an aqueous solution of high gas acid or an aqueous solution of sulfuric acid; preferably an aqueous solution of acetic acid. The aqueous acetic acid solution is selected from aqueous acetic acid solutions having a volume percentage of 0.1% to 5%, preferably selected from aqueous acetic acid solutions having a volume percentage of 0.5% to 2%. The pH of the aqueous solution is preferably from 2.5 to 3.0. (2) Elution: The resin column can be washed first with an acidic or neutral aqueous solution, and then several times the resin. 7 201231475

The volume of 5% to 30% (v/v) of the right L machine is treated with a feat or neutral aqueous solution to remove impurities contained in caspofungin.虹 The same configuration is used for the rainbow or neutral aqueous solution and injection. The organic solution is derived from methanol, acetone, acetonitrile, ethanol isopropanol; preferably from ethanol, acetonitrile or acetone. Elution of caspofungin: The caspofungin acid or neutral aqueous solution was eluted with an acidic or neutral aqueous solution containing 10% to 45% by volume of an organic solvent. The same configuration was used for the injection. The organic solvent of the guacamole in the aqueous solution is selected from the group consisting of methanol 'acetone, acetonitrile, ethanol or isopropanol; preferably from ethanol, B guess or propyl mesh; most preferably B. The volume content of the organic solvent in the aqueous solution is H)%~45%, preferably 10%~20%, and the optimum is Illustrated. Take 12/〇~15%. The crude sample amount and the resin volume ratio are selected from the group consisting of 1.1 〜1. 300 (g: ml), preferably 1: 50 〜1 : 15 〇 (g: ml). The purity of the obtained eluate was confirmed by high-performance liquid phase, and the product obtained by concentrating the desired eluate was concentrated. The obtained product can be dissolved in an acidic or neutral aqueous solution and then purified by secondary separation of the resin. It is also possible to add a solvent such as ethanol or methanol to dissolve a small solvent such as ethyl acetate, and to obtain a caspofungin product. Table r, taking into account the yield of the separation and purification process, product quality, process cost and convenience of the amplification operation, it is preferred to carry out two macroporous adsorption resin separation operations on caspofungin. The caspofungin product having a liquid phase purity > 90% can be collected at the time of the first separation and purification, and then the resulting product is subjected to a second separation, and then the liquid phase purity > 99% of the caspofungin product is collected. The second separation and purification operation was the same as the first operation. It is also possible to directly collect the liquid phase 201231475 purity > 99°/ at the time of the first separation and purification. Caspofungin products. After the card's white fraction was eluted, the purity of the eluate was confirmed by high-performance liquid phase, and the purity was > 99% of the eluate. The solution was concentrated at a low temperature to remove the solution; Baifenjing or its salts. When the water (4) which dissolves 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 water-cooling liquids is an acidic aqueous solution, the corresponding salt of caspofungin is obtained; preferably, caspofungin acetate is obtained because it is a pharmaceutically active knife, and the other caspofungin is obtained. The net salt can be converted to caspofungin acetate by conventional methods in the art. It can be converted into acetic acid card >baifenjing by ion exchange resin, or add free caspofungin salt, and then add acetic acid to form caspofungin acetate. The obtained caspofungin or a salt thereof can be dissolved in a solvent such as an acetic acid aqueous solution of an alcohol, water or an alcohol, and then dissolved, and then the ethyl acetate is added dropwise to dissolve the solvent, and the card is removed. Or its salts. 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. After the aa operation, the caspofungin or its salt is converted into a solid, which facilitates operations such as storage, transportation, and weighing. Compared with the prior art, the invention has the advantages that: the invention has simple operation and high degree of separation, and the separated product can achieve the purity of the pharmaceutical standard, can be easily expanded in the same proportion, and is easy to be produced in jade industry. 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 significantly, resulting in more waste in 201231475, 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 intervening body 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. Due to the use of macroporous resin, the preparative chromatography step greatly simplifies operation, reduces equipment requirements, reduces the use of organic solvents, reduces costs, and increases production efficiency. Reported with J. LIQ. CHROM. & REL. TECHNOL., 24(6), 781-798 (2001), US 5378804, US 5552521, US 2009291996, US 2009324635, WO 2009151341, w〇2010064219, and US 2010168415 Compared with the preparative chromatography step, the separation and purification step of the present invention has the following advantages: since the molecular weight of caspofungin is large, about 1092, and the ordinary silica gel column and the preparative column have a fine pore size, and the adsorption capacity for a large molecular weight product. Strong, difficult to elute; and the present invention uses a macroporous adsorption resin, which has a large pore diameter and is compatible with the caspofungin or its salt to be separated, so that the use of the macroporous adsorption resin can provide a good elution effect. The caspofungin acetate isolated and purified by macroporous adsorption resin has a high yield and product purity, which is beneficial to reduce cost and quality control of the final product. The use of inexpensive macroporous adsorption resins as separation media has a significant cost advantage over the preparation of expensive fillers in the chromatography step, and macroporous adsorption resins can be 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 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 and simple operation, and rs 10 201231475 is safe and efficient. 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 EMBODIMENTS The following examples are intended to illustrate the invention, but are not intended to limit the invention. EXAMPLES 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; Hz2〇Ss 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: A column of HP20SS resin 1 〇〇 mi, 1% aqueous acetic acid solution 5 〇〇 ml equilibrium resin column. The crude product (purity 50%) obtained by caspofungin was 1 g, and dissolved in 20 ml of a 1% aqueous solution of acetic acid. 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 nU of 10% acetonitrile in 1% aqueous acetic acid. The caspofungin was eluted with 12% acetonitrile in 1% aqueous acetic acid "liquid phase detection, combined purity> 99% eluate, concentrated to dryness to give caspofungin OU g, the final purity was 99.8. %. After adding a crystallizing liquid (ethanol:water:acetic acid volume ratio: 9:1: 〇.〇5, 2 ml), the mixture was dissolved until clarification, and ethyl acetate was added dropwise to precipitated solid, which was filtered. Obtained 201231475 Purity >99%'single <Q1% of caspofungin acetate. The remaining 99% of the eluate was recovered and then applied or subjected to secondary separation. Example 2: HP20SS resin 1 〇〇 ml packed column '2% aqueous acetic acid solution 5' such as an equilibrium resin column. The crude product (purity: 5% by mass) i g obtained by the synthesis of caspofungin was dissolved in 20 ml of a 2% aqueous solution of acetic acid. Inject the resin column at a flow rate of 1 BV/h. Rinse with 2% aqueous acetic acid 4 〇〇 mi and rinse with 500 ml of 1% acetonitrile in acetic acid. Caspofungin was eluted with 2% aqueous acetic acid in 5% acetonitrile. After liquid phase detection, the eluate of purity >99% was combined and concentrated to dryness to obtain 13 g of caspofungin acetate, and the final purity was 99 7%. After adding a crystallizing liquid (ethanol:water:acetic acid volume ratio: 9:1: 〇.〇5, 2 ml), the mixture was dissolved until clarification, and ethyl acetate was added dropwise to precipitated solid, which was filtered. A purity >99% 'single impurity < 〇 1% caspofungin acetate was obtained. The remaining purity < 99% of the eluate was recovered or applied for secondary separation. Example 3: A column of Hz 832 resin ml, 500 ml of a 1% aqueous acetic acid solution was used to equilibrate the resin column. The crude product (purity 50%) obtained by caspofungin synthesis was dissolved in 20 ml of a 1% aqueous solution of acetic acid. Inject the resin column at a flow rate of 1 BV/h. Rinse with 4% aqueous solution of 1% acetic acid and rinse with 500 ml of a 1% aqueous solution of acetic acid in 20% ethanol. "Caspofungin was eluted with 2% aqueous acetic acid in 2% acetic acid. Upon liquid phase detection, the eluate of >9〇% purity was combined and concentrated to dryness to obtain cabufenacetate 0 55 g, with a final purity of 91 5%.

S 12 201231475 After adding 2 ml of ethanol to dissolve for clarification, about 2 ml of ethyl acetate was added dropwise, and crystallization was carried out to obtain caspofungin acetate, and the purity in a high-performance liquid phase was 915%. Example 4: An HP20SS resin 1 〇〇 mi packed column '2% aqueous acetic acid solution 500 ml of an equilibrium resin column was taken. The crude product (purity 50%) obtained by caspofungin was 1 g, and dissolved in 20 ml of a 2% aqueous solution of acetic acid. Inject the resin column at a flow rate of 1 Bv/h. Rinse with 4% aqueous solution of 2% acetic acid and rinse with 500 ml of a 2% aqueous solution of acetic acid in 丨〇〇/〇 ethanol. Caspofungin was eluted with 20% ethanol in 2% aqueous acetic acid. The eluate was detected by liquid phase 'combined purity> 9 〇%, and concentrated to dryness to obtain caspofungin acetate. 5 〇 g, and the final purity was 90.8%. After adding 2 ml of methanol and dissolving until clarification, ethyl acetate was added dropwise to about 2 〇 ^ b. Crystallization gave caspofungin acetate, and the purity of the high-performance liquid phase showed 9 〇 8 〇 / 〇. Example 5: A column of Hz 832 resin 1 〇〇 mi, 1% aqueous acetic acid solution 5 〇〇 ml of an equilibrium resin column. The crude product (purity 50%) obtained by caspofungin was 1 g, and dissolved in 20 ml of a 1% aqueous solution of acetic acid. 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 of >9〇% purity was combined and concentrated to dryness to obtain caspofungin acetate 54 g, and the final purity was 91.8%. After adding 2 ml of ethanol to dissolve for clarification, ethyl acetate was added dropwise to about 20 nU, and crystallization was carried out to obtain caspofungin acetate, and the purity in a high-performance liquid phase was 91.8. /0. 13 201231475 Example 6: A column of 100 ml of HP20SS 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 solution of acetic acid. 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. Upon liquid phase detection, 90% of the eluate of purity > was concentrated and concentrated to dryness of 0.60 g of caspofungin acetate to a final purity of 92.5%. After adding 2 ml of ethanol to dissolve for clarification, about 20 ml of ethyl acetate was added dropwise, and the mixture was crystallized to obtain caspofungin acetate, and the purity in a high-performance liquid phase was 92.5%. Example 7: A column of 100 ml of HZ20SS 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 solution of acetic acid. 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. The caspofungin was eluted with a 1% aqueous solution of acetic acid of 12 〇/〇 acetonitrile. Upon liquid phase detection, the eluate of purity > 9% was combined and concentrated to dryness to give caspofungin acetate 0.52 g 'final purity of 92.6%. After adding 2 ml of methanol to dissolve, clarify, add about 2 〇ml of ethyl acetate, and crystallize to obtain caspofungin acetate. The purity of the HPLC is 92.6%. Example 8: Take HZ20SS resin in 100 ml column. 2% aqueous acetic acid solution 500 ml

S 14 201231475 Balance resin column. The crude product (purity 50%) obtained by caspofungin was 1 g, and dissolved in 20 ml of a 2% aqueous solution of acetic acid. Injection resin column, flow rate! BV/h. Rinse with 4% aqueous solution of 2% acetic acid and rinse with 500 ml of 2% aqueous acetic acid solution of ι〇〇/0 acetone. Caspofungin was eluted with 15% acetone in 2% aqueous acetic acid. After liquid phase detection, the eluate of purity > 9% 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 and dissolving until clarification, about 2 ml of ethyl acetate was added dropwise, and crystallization was carried out to obtain caspofungin acetate, and the purity in a high-performance liquid phase was 91 2%. Example 9: HP20SS resin 1 〇〇 mi column, 1% aqueous acetic acid solution 5 〇〇 ml equilibrium resin column. The caspofungin obtained in any of Examples 3-8 was once isolated and purified by a resin, and 1 g of the product was dissolved in 20 ml of a 1% aqueous acetic acid solution. Inject the resin column at a flow rate of approximately 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 combined with the '99% purity elution in the liquid phase, and concentrated to dryness at low temperature to obtain caspofungin 0.4 g, final purity. It is 99.8%. The remaining purity < 99% of the eluate was concentrated and concentrated after reduction under reduced pressure. The obtained product was dissolved in a crystallizing liquid (ethanol:water:acetic acid volume ratio: 9:1: 0.05) 2 ml), and ethyl acetate was added dropwise to precipitated solids, which was filtered, and the filter cake was rinsed with a small amount of ethyl acetate. A purity > 99%, a single impurity < 0.1% caspofungin acetate was obtained. 15 201231475 Example ίο : A column of 100 ml of HZ20SS resin, 5 〇〇 ml of a 1% aqueous solution of acetic acid, and an equilibrium resin column. The caspofungin obtained in any of Examples 3-8 was once isolated and purified by a resin, and 1 g of the product was dissolved in 2% aqueous solution of 1% acetic acid. Inject the resin column at a flow rate of approximately 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 'combined purity> 99% eluate, and concentrated to dryness at low temperature to obtain caspofungin 0.45 g, final purity. It is 99.8%. The remaining purity <99% of the eluate was concentrated and recovered for reuse. The obtained product was added to a crystallizing liquid (ethanol:water:acetic acid volume ratio: 9:1:0_05, 2 ml) and dissolved until clarified. Ethyl acetate was added dropwise to precipitated solids, and the mixture was filtered, and the cake was washed with a small amount of ethyl acetate. A purity > 99%, single impurity < 0.1% caspofungin acetate was obtained. Example 11: A column of 100 ml of HP20SS resin, 500 ml of a 0.5% aqueous solution of acetic acid, and an equilibration resin column were taken. The caspofungin obtained in any of Examples 3-8 was once isolated and purified by a resin, and 1 g of the product was dissolved in 20 ml of a 0.5% aqueous acetic acid solution. The injection resin column 'flow rate is 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 of the purity >99% was combined by liquid phase detection, and concentrated to dryness at a low temperature to obtain vinegar.

S 16 201231475 Acid caspofungin 0.42 g 'final purity is 99.8%. The remaining purity < 99% of the eluate was concentrated and recovered for reuse. The obtained product was added to a crystallizing solution (ethanol:water: vinegar·acid volume ratio: 9:1:0.05 '2 ml), and dissolved to clarification. Ethyl acetate was added dropwise to precipitated solid, and the filtered cake was washed with a small amount of ethyl acetate. . A purity > 99%, a single impurity < 〇 · 1% of caspofungin acetate was obtained. Example 12: A 100 ml column of HP20SS resin, a 0.5% aqueous acetic acid solution of 5 〇〇 ml of an equilibrium resin column was taken. The obtained product was subjected to one-time separation and purification of the caspofungin obtained by any of the examples 3 to 8 and dissolved in 20 ml of a 2% aqueous acetic acid solution. Inject the resin column at a flow rate of approximately 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 the liquid phase detection, the eluate of purity >99% was combined and concentrated to dryness at a low temperature to obtain caspofungin acetate 0.35 g, and the final purity was 99.8%. The remaining purity <99% of the eluate' was concentrated under reduced pressure and recovered for reuse. The obtained product was added to a crystallizing liquid (ethanol·water:acetic acid volume ratio: 9:1: 0.05 Å 2 ml) and dissolved until clarified, and ethyl acetate was added dropwise to precipitate a solid, which was filtered and washed with a small amount of ethyl acetate. A purity > 99%, a single impurity < 0.1% caspofungin acetate was obtained. Example 13: HP20SS resin 1 〇〇 mi column, 2% aqueous acetic acid solution 5 〇〇 ml balance resin column. 17 201231475 Take the crude product (purity 50%) obtained from Kafim. 1 g, dissolved in 20 ml of 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 liquid phase detection, the eluate of purity >99% was combined and concentrated to dryness to obtain caspofungin acetate 〇8 g, and the final purity was 99.8%. After adding a crystallizing liquid (ethanol:water:acetic acid volume ratio: 9:1: 〇.〇5, 2 ml), the mixture was dissolved until clarification, and ethyl acetate was added dropwise to precipitated solid, which was filtered. A purity > 99%, single impurity < 〇 1% caspofungin acetate was obtained. The remaining 99% of the eluate was recovered and then applied or subjected to secondary separation. Example 14: A column of Hz 832 resin 1 〇〇 ml, a 1% aqueous solution of acetic acid 500 ml of an equilibrium resin column was taken. The crude product (purity 50%) obtained by caspofungin was 1 g, and dissolved in 20 ml of a 1% aqueous solution of acetic acid. Inject the resin column at a flow rate of 1 BV/h. Rinse with 400 ml of 10/〇 aqueous acetic acid solution and elute caspofungin with 45% acetonitrile in 1% aqueous acetic acid. After liquid phase detection, the purity > 90% of the eluate was combined and concentrated to dryness to obtain caspofungin acetate 61 g, and the final purity was 92.8%. After adding 2 ml of ethanol to dissolve for clarification, about 2 ml of ethyl acetate was added dropwise, and crystallization was carried out to obtain caspofungin acetate. The purity of the high-performance liquid phase was about 92.8%. Example 15: A column of 100 ml of HP20SS resin and 500 ml of a 1% aqueous solution of acetic acid were used to equilibrate the resin column.

S 201231475 Take the crude product (purity 50%) obtained from caspofungin 1 g, dissolved in 20 ml of 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. The caspofungin was eluted with 12% acetonitrile in 1% aqueous acetic acid. After liquid phase detection, the eluate of purity >99% was 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 mixture was filtered, and ethyl acetate was added dropwise to precipitated solid. A purity > 99%, a single impurity < 0.1% caspofungin acetate was obtained. The remaining purity < 99% of the eluate was recovered and applied or subjected to secondary separation. Example 16: A column of H-60 resin 1 〇〇 mi was packed, and a 1% aqueous solution of acetic acid was 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 solution of acetic acid. Inject the resin column at a flow rate of 1 BV/h. Rinse with 400 ml of 1% aqueous acetic acid solution and use 1% acetonitrile of 10% acetonitrile. Rinse with 500 ml of aqueous acetic acid solution. Caspofungin was eluted with 12% acetonitrile in 1% aqueous acetic acid. After liquid phase detection, the 99% purity of the eluate was combined and concentrated to dryness to obtain 12 g of caspofungin acetate, and the final purity was 99.8%. After adding 3 ml of ethanol to dissolve, the mixture was filtered, and ethyl acetate was added dropwise to precipitated solids. A purity > 99%, a single impurity < 0.1% caspofungin acetate was obtained. The remaining purity < 99% of the eluate was recovered and applied or subjected to secondary separation. [Simple description of the diagram] 19 201231475 (none) [Explanation of main component symbols] (none)

Claims (1)

201231475 VII. Patent application scope: 1. A method for purifying a cyclohexyl peptide compound or a pharmaceutically acceptable salt thereof, which is characterized in that it is separated and purified by macroporous adsorption resin. 2. The method of claim 1, wherein the cycloheximide compound is caspofungin. 3. According to the method of claim 2, which comprises the following steps (1) taking caspofungin crude product and injecting a macroporous adsorption resin column; (2) using a volume containing 10% to 45 An aqueous solution of an organic solvent is eluted with caspofungin' to concentrate the resulting eluate to give caspofungin or a salt thereof; and (3) optionally 'dissolving the product obtained in step (2) in an alcohol solvent Or in water, an anti-solvent is added dropwise to precipitate a solid, which is filtered to obtain caspofungin or a salt thereof. 4. The method according to any one of claims 3 to 3, which is characterized in that it can be purified by using 丨_2 macroporous adsorption resin, preferably by using two macroporous adsorption resins. 5. The method according to any one of claims 3 to 3, wherein the macroporous adsorption resin is selected from the group consisting of HP series, SP series, Amberlite XAD series, Hz series, etc., polar or non-polar macroporous adsorption resin, preferably HP20SS , Hz832, Hz20SS, Hz818 resin. 6. The method of claim 3, wherein the aqueous solution is an acidic or neutral aqueous solution, preferably an acidic aqueous solution. 7. The method according to claim 6, wherein the acidic aqueous solution 21 201231475 is selected from the group consisting of aqueous hydrochloric acid, aqueous acetic acid, aqueous trifluoroacetic acid, aqueous high-oxygen acid, aqueous sulfuric acid; preferably aqueous acetic acid. 8. The method of claim 7, wherein the aqueous acetic acid solution is selected from the group consisting of 0.1% to 5% aqueous acetic acid, preferably from 5% to 2% aqueous acetic acid. 9. According to the method described in item 6 of the special (4), the pH of the acidic aqueous solution is 2.5 to 3.0. The method of claim 3, wherein the aqueous solution contains an organic solvent selected from the group consisting of methanol, ethanol, acetone, acetonitrile, isopropanol; preferably acetonitrile, ethanol, acetone; most preferably acetonitrile . The method according to claim 3, wherein the aqueous solution contains an organic solvent in a volume content of from 1% to 45%, and most preferably from 12% to _ 12. according to the third item of the patent scope The method wherein the crude sample amount and the resin volume ratio are selected from! Gram: 10 ml ~ i g: 3 ml ml preferred 1 g: 50 ml ~ 1 g: 150 ml. S 22 201231475 IV. Designated representative map: (1) The representative representative of the case is: ( ). (No picture) (2) A brief description of the symbol of the representative figure: (none) 5. If there is a chemical formula in this case, please disclose the chemical formula that best shows the characteristics of the invention: S 2