Method for Purifying Tafluprost

Abstract

To provide a method for purifying tafluprost which is simple, efficient and can be scaled up. The present invention provides a method for purifying tafluprost, which comprises purifying a crude tafluprost product by silica gel column chromatography and collecting fractions containing tafluprost by HPLC analysis. The present invention also relates to a method for producing tafluprost, which comprises the method for purifying tafluprost.

Description

[Technical field]

[0001] The present invention relates to a novel method for purifying tafluprost. [Background technology]

[0002] Tafluprost has the following formula:

[0003] [ka]

[0004] Its chemical name is (5Z)-7-[(1R,2R,3R,5S)-2-[(1E)-3,3-difluoro-4-phenoxy-1-butenyl]-3,5-dihydroxycyclopentyl]-5-heptenoic acid isopropyl ester, and it is a highly viscous difluoroprostaglandin F with a viscosity of 2440 mPa s at 25 °C. 2α Tafluprost is a prostaglandin derivative. Tafluprost has an unstable chemical structure with two double bonds, an unsaturated fatty acid ester moiety, and four asymmetric centers, and has a structure in which the hydroxyl group and hydrogen atom at C15, which are present in other prostaglandin derivatives, are replaced by two fluorine atoms. Therefore, it has a unique physical property of being extremely lipophilic among prostaglandin derivatives, and although it has high chemical stability as a prostaglandin derivative, it also has the property of decomposing at high temperatures. In addition, tafluprost has a strong intraocular pressure-reducing effect, and is used as an eye drop for the treatment of glaucoma and ocular hypertension (Patent Document 1). Patent Document 1 describes difluoroprostaglandin F, which includes tafluprost. 2α A method for producing the derivative is described, and a similar method is also described in Non-Patent Document 1.

[0005] The manufacturing method described in Patent Document 1 includes a Wittig reaction step, so that it is difficult to avoid contamination of the final product with α-chain trans isomers. In the manufacturing method described in Patent Document 1, a method of separating and purifying the α-chain trans isomer using preparative HPLC (High Performance Liquid Chromatography) has been reported as a method for removing impurities including the α-chain trans isomer (Patent Document 2). However, tafluprost and its synthetic precursor, the following formula (I):

[0006] [ka]

[0007] The carboxylic acid compound represented by the formula (hereinafter referred to as "tafluprost acid") is a liquid compound having a very high viscosity, and therefore is difficult to purify. In addition, the purification method of tafluprost described in Patent Document 2 requires a large amount of organic solvent, which requires a large cost, and it is also difficult to suppress the concentration of the residual organic solvent below the concentration limit value of the drug residual solvent guideline (Non-Patent Document 2). Preparative HPLC columns are generally expensive and are usually used repeatedly, so there are problems such as the inclusion of accumulated impurities and decomposition products, and the decrease in the number of theoretical plates due to column deterioration. In order to reduce the risks caused by these problems, it is necessary to regularly perform cleaning using a large amount of organic solvent and check it, and to check the separation performance of the column, etc., which makes them less practical as a manufacturing method for pharmaceuticals.

[0008] On the other hand, a method for reducing the inclusion of impurities such as α-chain trans isomers by using an organic amine salt (Patent Documents 3 and 4) or a metal salt (Patent Document 5) of tafluprost acid has been reported, but the addition of a salt formation process and a release process from the salt may increase by-products, dehydration products, and other impurities due to dimerization caused by condensation with organic amines or self-condensation. In addition, many organic amines and metals are toxic and mutagenic, and there are safety issues when used as a purification method, especially near the final process of pharmaceuticals.

[0009] Furthermore, a method for producing tafluprost has been reported in which the inclusion of α-chain trans isomers is prevented by going through a process of forming a macrolactone ring and a process of opening the macrolactone ring (Patent Document 6). However, this method is not very practical because the production process is long and the yield is low. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] European Patent Application Publication No. 850926 [Patent Document 2] US Patent Application Publication No. 2014 / 0051882 [Patent Document 3] International Publication No. 2013 / 118058 [Patent Document 4] International Publication No. 2016 / 090461 [Patent Document 5] China Patent Application Publication No. 108299192 [Patent Document 6] JP 2015-36382 A [Non-patent literature]

[0011] [Non-Patent Document 1] Tetrahedron Lett., 2004, 45, 1527-1529 [Non-Patent Document 2] Pharmaceutical Affairs and Medical Affairs Bureau, Ministry of Health, Labor and Welfare, Notification of Director of the Inspection and Management Division (March 30, 1998), Guidelines for Residual Solvents in Pharmaceuticals Summary of the Invention [Problem to be solved by the invention]

[0012] The present invention aims to provide a method for purifying tafluprost, which can easily and inexpensively purify tafluprost, a highly viscous liquid compound, to a purity sufficient for providing the compound as is as an active pharmaceutical ingredient for pharmaceuticals, and which can also be scaled up. [Means for solving the problem]

[0013] The present inventors have conducted intensive studies to solve the above problems, and have found that a high purity tafluprost can be obtained by a purification method (hereinafter, sometimes referred to as the "purification method of the present invention") comprising the steps of purifying the crude tafluprost obtained in the esterification step of tafluprost acid by silica gel column chromatography and collecting a fraction containing tafluprost by HPLC analysis in a method for producing tafluprost. In addition, a purification method (hereinafter, a purification method including all of the above steps may be referred to as the "purification method of the present invention") comprising the steps of concentrating the fraction containing tafluprost collected by HPLC analysis under reduced pressure at 10 to 55°C, dissolving the residue in a solvent and filtering, and distilling off the solvent from the filtrate under reduced pressure at 10 to 55°C with a final vacuum of 5 torr or less (hereinafter, a purification method including all of the above steps may be referred to as the "purification method of the present invention"). It has been found that the residual organic solvent concentration can be suppressed to the concentration limit value of the drug residual solvent guideline or less, and tafluprost having a purity that can be directly provided as a drug substance can be obtained, and the present invention has been completed.

[0014] That is, the present invention is as follows. [1] A method for purifying tafluprost, comprising the steps of purifying a crude product of tafluprost by silica gel column chromatography and collecting a fraction containing tafluprost by HPLC analysis. [2] The method for purifying tafluprost according to the above-mentioned [1], further comprising the steps of concentrating under reduced pressure at 10 to 55°C a fraction containing tafluprost collected by HPLC analysis, dissolving the residue in a solvent and filtering the resultant solution, and distilling off the solvent from the filtrate under reduced pressure at 10 to 55°C to a final vacuum of 5 torr or less. [3] The purification method according to the above [1] or [2], wherein the particle size (d50) of the silica gel used for silica gel column chromatography is 20 to 70 μm. [4] The purification method according to any one of the above [1] to [3], wherein the silica gel used for silica gel column chromatography is spherical. [5] The purification method according to any one of the above [1] to [4], wherein the eluent for silica gel column chromatography is a mixed solvent of n-hexane and a polar solvent, or a mixed solvent of n-heptane and a polar solvent. [6] The purification method according to [5] above, wherein the eluent is a mixed solvent of n-hexane and a polar solvent. [7] The purification method according to [5] or [6] above, wherein the polar solvent is ethyl acetate, t-butyl methyl ether, 2-propanol, or ethanol. [8] The purification method according to any one of the above [1] to [7], wherein the HPLC analysis is a reverse-phase HPLC analysis. [9] The purification method according to any one of the above [1] to [8], wherein the fraction contains 98% or more of tafluprost.

[10] The purification method according to any one of the above [2] to [9], wherein the filtration is carried out using a filter having a pore size of 0.5 μm or less.

[11] The purification method according to any one of the above [2] to

[10] , wherein the solvent for dissolving the residue is ethyl acetate, t-butyl methyl ether, 2-propanol or ethanol, or a mixed solvent of ethyl acetate, t-butyl methyl ether, 2-propanol or ethanol with a non-polar solvent.

[12] The purification method according to the above

[11] , wherein the solvent for dissolving the residue is ethyl acetate or a mixed solvent of ethyl acetate and a non-polar solvent.

[13] The purification method according to

[11] or

[12] above, wherein the non-polar solvent is n-hexane or n-heptane.

[14] The purification method according to any one of the above [2] to

[13] , wherein the final vacuum level is 1 torr or less.

[15] The purification method according to any one of the above [2] to

[14] , wherein after the step of distilling off the solvent from the filtrate, the residual solvent concentration of n-hexane is 290 ppm or less, and the residual solvent concentrations of n-heptane, ethyl acetate, t-butyl methyl ether, 2-propanol or ethanol are each 5,000 ppm or less.

[16] A method for producing tafluprost, comprising a step of subjecting a crude product of tafluprost to the purification method according to any one of the above-mentioned [1] to

[15] .

[17] Tafluprost obtained by the production method described in

[16] above.

[18] A medicine comprising tafluprost as described in

[17] above as an active ingredient.

[19] A pharmaceutical for preventing or treating an eye disease, comprising tafluprost as described in

[17] above as an active ingredient.

[20] The pharmaceutical composition according to

[19] above, wherein the eye disease is glaucoma or ocular hypertension. Effect of the Invention

[0015] According to the purification method of the present invention, when the crude product of tafluprost is separated and purified by silica gel column chromatography in the final step of the production of tafluprost, the fraction containing tafluprost is collected by HPLC analysis, thereby minimizing the contamination of impurities. In addition, by distilling off the solvent over a long period of time under reduced pressure conditions of low temperature and high vacuum, the concentration of residual organic solvent can be suppressed to the concentration limit value or less of the guideline for residual solvents in pharmaceuticals, and the decomposition of tafluprost, which is unstable at high temperatures, can also be suppressed. Furthermore, by incorporating a filter filtration process in the middle, silica gel fine powder, airborne particles, and bacteria can be removed, so that after distilling off the solvent, high-purity tafluprost that can be used as it is as a drug substance for pharmaceuticals can be simply and efficiently provided. The purification method of the present invention can be widely applied to crude products of tafluprost produced by known methods, and can withstand scale-up. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail below with reference to the preferred embodiments. [Terminology definition] The terms used in this specification have the following meanings:

[0017] In this specification, the column used in "silica gel column chromatography" may be either an open column or a flash column.

[0018] In this specification, "silica gel column chromatography" refers to normal phase column chromatography.

[0019] In this specification, the term "crude product of tafluprost" refers to the product before purification after post-treatment of the reaction in the final step in the known method for producing tafluprost. Specifically, for example, as shown in the examples described later, the product before purification of the final esterification reaction in the method for producing tafluprost described in Patent Document 1 can be mentioned.

[0020] In this specification, the term "impurities" refers to all substances other than tafluprost, such as residual reaction reagents, residual raw material compounds, reaction by-products, and related substances such as decomposition products of tafluprost contained in the crude product of tafluprost, as well as residual organic solvents, residues derived from fillers, and bacteria.

[0021] In this specification, "HPLC analysis" means that when a crude product of tafluprost is separated and purified by silica gel column chromatography, the presence or absence and content ratio of tafluprost in each fraction are confirmed by using analytical high performance liquid chromatography.

[0022] In this specification, the term "filtration" refers to filter filtration. Filtration is carried out for the purpose of removing fine particles of column packing material (silica gel), suspended particles in the air, bacteria, and the like.

[0023] In this specification, the term "polar solvent" refers to a solvent with a large dielectric constant. Specific examples of polar solvents include esters such as ethyl acetate and propyl acetate, ethers such as diethyl ether, t-butyl methyl ether and tetrahydrofuran, and alcohols such as 2-propanol and ethanol. Among these, ethyl acetate, t-butyl methyl ether, 2-propanol or ethanol is preferred.

[0024] In this specification, the term "non-polar solvent" refers to a solvent with a small dielectric constant. Specific examples of non-polar solvents include chain hydrocarbons such as n-hexane and n-heptane. Among these, n-hexane is preferred.

[0025] In this specification, "external temperature" means the temperature outside a reaction vessel or a concentration vessel, and is usually the ambient temperature or the temperature of a water bath or hot water bath.

[0026] In this specification, the "concentration limit value of the drug residual solvent guideline" refers to a toxicologically acceptable limit value of residual solvents, which is considered as one of the issues of the International Conference on Harmonization of Pharmaceuticals for Patients (ICH) and specifies the allowable amount of residual solvents in drugs for patient safety. Specific examples of the concentration limit value of residual solvents in drugs, as described in Non-Patent Document 2, are 290 ppm for n-hexane and 5000 ppm for n-heptane, ethyl acetate, t-butyl methyl ether, 2-propanol, or ethanol.

[0027] [Purification method of the present invention] The purification method of the present invention is characterized by including a step (step 1) of purifying a crude product of tafluprost by silica gel column chromatography and collecting a fraction containing tafluprost by HPLC analysis. In order to suppress the concentration of the residual organic solvent to the concentration limit value or less of the guideline for residual solvents in pharmaceuticals, the purification method of the present invention is characterized by including, in addition to the above step 1, a step (step 2) of concentrating under reduced pressure at 10 to 55°C, a step (step 3) of dissolving the residue in a solvent and filtering, and a step (step 4) of distilling off the solvent from the filtrate under reduced pressure at 10 to 55°C with a final vacuum of 5 torr or less.

[0028] (Process 1) This step involves purifying the product by silica gel column chromatography and collecting fractions containing tafluprost by HPLC analysis.

[0029] The packing material used in silica gel column chromatography is not particularly limited as long as it is a silica gel that can be used in a normal normal phase column. The shape of the silica gel may be either crushed or spherical, but spherical is more preferable. The particle size (d50) of the silica gel is not particularly limited, but is preferably 20 μm to 70 μm, more preferably 40 μm to 65 μm, and particularly preferably 45 μm to 60 μm. The particle size (d50) is the median size of the particle size distribution when the particle size distribution is created on a volume basis by laser diffraction scattering particle size distribution measurement.

[0030] The eluent used in silica gel column chromatography is not particularly limited as long as it is a solvent capable of separating tafluprost from impurities in the crude product of tafluprost, but is preferably a mixed solvent of n-hexane and a polar solvent, or a mixed solvent of n-heptane and a polar solvent, and more preferably a mixed solvent of n-hexane and a polar solvent. Here, the polar solvent is selected from ethyl acetate, t-butyl methyl ether, 2-propanol and ethanol, and among them, 2-propanol or ethanol is preferred. The mixing ratio (volume ratio) of n-hexane and a polar solvent, or n-heptane and a polar solvent can be appropriately set according to the type, shape, and / or particle size of the packing material used. Suitable specific examples of the eluent include, for example, a mixed solvent of ethyl acetate, t-butyl methyl ether, 2-propanol or ethanol and a non-polar solvent (preferably n-hexane or n-heptane), more preferably a mixed solvent of 2-propanol or ethanol and a non-polar solvent (preferably n-hexane or n-heptane), and particularly preferably a mixed solvent of ethanol and n-hexane. When a mixed solvent is used as the eluent, the mixing ratio (volume ratio) is not particularly limited. From the viewpoint of controlling the residual solvent concentration to a standard value or less, in the case of a mixed solvent of ethanol and n-hexane, it is preferable to use a solvent in which ethanol:n-hexane is mixed at a ratio of 10:90 to 1:99, more preferably a solvent in which ethanol:n-hexane is mixed at a ratio of 6:94 to 2:98, even more preferably a solvent in which 5:95 to 3:97 is mixed, and particularly preferably a solvent in which 4:96 is mixed.

[0031] Analytical HPLC is used to confirm the presence or absence of tafluprost in the fraction separated by silica gel column chromatography purification. As the HPLC, either normal phase HPLC or reversed phase HPLC can be used, but reversed phase HPLC is more preferable because it is superior in terms of impurity separation efficiency, detection sensitivity, and quantitativeness. Specific examples of the column and analytical conditions used in the HPLC analysis include, but are not limited to, the conditions described in the Examples below. Usually, when performing purification by silica gel column chromatography, the presence or absence of the target substance in the separated fraction is confirmed by TLC (thin layer chromatography) (see Experimental Chemistry Lecture 1 Basic Operations I (4th Edition), published November 5, 1990, Maruzen, 5·2·3 Column Chromatography, p.293-296). However, in the purification of the crude product of tafluprost, it was found that HPLC analysis is significantly superior in the detection sensitivity of fractions containing tafluprost and impurities compared to conventional TLC analysis. For many lots of synthesized crude tafluprost products, silica gel column chromatography was performed, and the elution pattern of impurities was analyzed by HPLC analysis, and it was confirmed that the elution pattern of impurities was always stable. By considering the elution pattern of impurities, it was found that it is preferable to collect consecutive fractions in which the HPLC area percentage of tafluprost in each fraction is 97% or more, and it is particularly preferable to collect consecutive fractions in which the HPLC area percentage of tafluprost in each fraction is 98% or more.

[0032] (Process 2) This step involves collecting fractions containing tafluprost, confirmed by HPLC analysis, and concentrating them under reduced pressure at 10 to 50°C.

[0033] The external temperature (temperature of the water bath or hot water bath) when fractions containing tafluprost confirmed by HPLC analysis are collected and concentrated under reduced pressure is preferably 10° C. to 55° C., more preferably 15° C. to 50° C., and particularly preferably 20° C. to 45° C. As shown in the test examples described later, it was confirmed that tafluprost gradually decomposes over time at temperatures of 60° C. or higher, so that it is desirable to carry out the concentration under reduced pressure or to distill off the solvent at the above temperatures.

[0034] (Step 3) This step is a step of dissolving the residue obtained in step 2 in a solvent and filtering it. Tafluprost is very viscous, so it is difficult to perform sterilization filtration after completely distilling off the solvent. Therefore, the purification method of the present invention is characterized by incorporating a filtration step after step 2.

[0035] Examples of the solvent for dissolving the residue obtained in step 2 include the same solvent as the eluent used in the silica gel column chromatography in step 1, but it is preferable that the solvent is a solvent that sufficiently dissolves tafluprost and has a relatively low boiling point, and may be a mixed solvent with a non-polar solvent that forms an azeotropic composition. Specifically, it is preferably ethyl acetate, t-butyl methyl ether, 2-propanol or ethanol, or a mixed solvent of ethyl acetate, t-butyl methyl ether, 2-propanol or ethanol and a non-polar solvent (preferably n-hexane or n-heptane), more preferably ethyl acetate, or a mixed solvent of ethyl acetate and a non-polar solvent (preferably n-hexane or n-heptane), and particularly preferably a mixed solvent of ethyl acetate and n-hexane. The mixing ratio (volume ratio) when using a mixed solvent is not particularly limited, but from the viewpoint of controlling the residual solvent concentration to a standard value or less, in the case of a mixed solvent of ethyl acetate and n-hexane, it is particularly preferable to use a solvent in which ethyl acetate:n-hexane is mixed at 10:1 to 1:10, preferably 4:1 to 1:4, more preferably 2:1 to 1:2.

[0036] The filter used for filtration in this step is not particularly limited as long as it is not swollen or dissolved by the solvent and can remove fine powder of the filler (silica gel), airborne particles, and the like. Examples of the filter include glass fiber filters, polypropylene filters, nylon filters, and fluororesin filters. Fluororesin filters such as polyvinylidene difluoride (PVDF) and polytetrafluoroethylene (PTFE) are preferred, and polytetrafluoroethylene (PTFE) filters are particularly preferred. The pore size of the filter is usually 0.5 μm or less, preferably 0.25 μm or less, and when sterilization purposes are also included, 0.22 μm or less is particularly preferable.

[0037] (Step 4) This step is a step of distilling off the solvent from the filtrate obtained in the above step 3 under reduced pressure at 10 to 55° C. and a final vacuum of 5 torr or less.

[0038] Tafluprost has a very high viscosity, so it is necessary to evaporate the solvent from the filtrate slowly over time while increasing the surface area for the solvent to evaporate and avoiding bumping. Examples of vacuum concentration devices for achieving this purpose include rotary evaporators, centrifugal evaporators, and high vacuum thin film distillation devices.

[0039] As described above, tafluprost gradually decomposes over time at temperatures of 60°C or higher. Therefore, the external temperature (temperature of the water bath or hot water bath) when distilling off the solvent under reduced pressure is preferably 10°C to 55°C, more preferably 15°C to 50°C, and particularly preferably 20°C to 45°C.

[0040] The degree of vacuum when distilling off the solvent is preferably controlled so that the surface area for evaporating the solvent is maximized, and gradually increased over time while avoiding bumping, so that the final vacuum is 5 torr or less (preferably 3 torr or less, more preferably 1 torr or less, and particularly preferably 0.5 torr or less). When releasing the reduced pressure, it is preferable to return the pressure to normal pressure using filtered air to prevent the intrusion of airborne particles and bacteria.

[0041] The time for distilling off the solvent is preferably from 10 to 70 hours, more preferably from 15 to 60 hours, and particularly preferably from 20 to 60 hours.

[0042] By using the purification method of the present invention, the concentration of residual organic solvents can be reduced to below the concentration limit value of the Pharmaceutical Residual Solvents Guideline (Non-Patent Document 2). As described in the guideline, "Since residual solvents are of no therapeutic value, all residual solvents should be reduced to a level that complies with product specifications, GMP, or other quality standards," it is possible to stably produce high-quality tafluprost that can comply with various even stricter quality standards. As the eluent or the solvent for dissolving the residue used in the above-mentioned silica gel column chromatography, it is preferable that the residual solvent concentration of ethyl acetate, t-butyl methyl ether, 2-propanol, ethanol, or n-heptane, which are given as examples of suitable solvents, is controlled to 1000 ppm or less, more preferably 100 ppm or less, and the residual solvent concentration of n-hexane is controlled to 200 ppm or less, more preferably 20 ppm or less. The residual solvent concentration can be measured by a method such as gas chromatography (GC).

[0043] The present invention also encompasses a method for producing tafluprost, which includes a step of subjecting a crude product of tafluprost produced by a known method to the purification method of the present invention (the purification method including the above steps 1 to 4). In addition to the above-mentioned Patent Document 1 and Non-Patent Document 1, there are several other known methods for producing tafluprost (e.g., U.S. Patent Application Publication No. 2014 / 0046086; J. Org. Chem. 2016, 81, 10832-844; Molecules, 2017, 22, 217, 1-16; Org. Lett. 2020, 22, 2991-2994, etc.), which are also included in the present invention by combining them with the purification method of the present invention.

[0044] Specific examples of the crude product of tafluprost used in the present invention include, for example, a crude product of tafluprost obtained by deprotection reaction from tafluprost in which hydroxyl groups, etc. are protected, a crude product of tafluprost obtained by esterification of a salt of tafluprost acid, and a crude product of tafluprost obtained by esterification of tafluprost acid, etc. Among them, the crude product of tafluprost obtained by esterification of tafluprost acid is preferably used.

[0045] Specific features of the purification method of the present invention include the following.

[0046] (A) When a crude product of tafluprost is separated and purified by silica gel column chromatography, the inclusion of impurities can be minimized by collecting fractions containing tafluprost through HPLC analysis (preferably, reverse-phase HPLC analysis).

[0047] (B) By distilling off the solvent over a long period of time under reduced pressure conditions at low temperature and high vacuum, it is possible to suppress the decomposition of tafluprost, which is unstable at high temperatures, and also to suppress the concentration of residual organic solvents to below the concentration limit set out in the guidelines for residual solvents in pharmaceuticals.

[0048] (C) By incorporating a filter filtration step midway, after the solvent is distilled off, it is possible to provide high-purity tafluprost that can be used as is as an active pharmaceutical ingredient for pharmaceuticals.

[0049] (D) The purification method of the present invention can be applied to a crude product of tafluprost obtained by any of the known methods for producing tafluprost, and can be easily scaled up, so that a simple and efficient purification method can be provided.

[0050] As described above in (A), when the purification method of the present invention is carried out for the purpose of increasing the purity of tafluprost, it is sufficient to include only the above step 1, and steps 2 to 4 can each be performed in combination with step 1, as necessary. EXAMPLES

[0051] The present invention will be described in detail below with reference to Reference Examples, Examples and Test Examples, but the present invention is not limited to these.

[0052] The percentage indicates mol% for yield, and mass% for other values ​​unless otherwise specified. The ratio of the mixed solvent is a volume ratio unless otherwise specified. Furthermore, room temperature means a temperature of 15 to 30°C unless otherwise specified. 1H-NMR values ​​were measured using a nuclear magnetic resonance device, ECP400 (400 MHz), manufactured by JEOL Ltd. The HPLC device used was Shimadzu LC-10ADvp or LC-10A. The GC device used was Shimadzu GC-2014ATF.

[0053] Reference Example 1: Synthesis of Tafluprost Acid

[0054] [ka]

[0055] (1S,5R,6R,7R)-6-[(1E)-3,3-difluoro-4-phenoxy-1-butenyl]-7-hydroxy-2-oxabicyclo[3.3.0]octan-3-one (280g) was dissolved in tetrahydrofuran (1200g) under a nitrogen atmosphere, and diisobutylaluminum hydride (1M toluene solution) (2160mL) was added dropwise at -70°C. After the dropwise addition, the mixture was stirred for 30 minutes, 1N hydrochloric acid was added, and the mixture was extracted with ethyl acetate. The organic layers were combined and washed with water, and the filtrate was concentrated under reduced pressure to obtain a reduced form (284g). Tetrahydrofuran (5030g) was added to 4-carboxybutyltriphenylphosphonium bromide (1523g) under a nitrogen atmosphere, and sodium bis(trimethylsilyl)amide solution (1M tetrahydrofuran solution) (6684mL) was added dropwise and stirred for more than 1 hour. The above-mentioned reduced product (286 g) dissolved in tetrahydrofuran (970 g) was added dropwise at 0° C. and stirred for 3 hours. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The aqueous layer was acidified and then extracted with ethyl acetate. After concentrating under reduced pressure, insoluble matters were filtered off, and the mixture was purified by silica gel column chromatography (hexane / ethyl acetate=1 / 1 to 1 / 3) to obtain tafluprost acid (222 g). 1H NMR (CDCl3) δ 1.60 (m, 1H), 1.67 (m, 2H), 1.84 (m, 1H), 2.02-2.16 (m, 4H), 2.25-2.35 (m, 3H), 2.47 (m, 1H), 4.03 (m, 1H), 4.18 (m, 3H), 5.35-5.42 (m, 2H), 5.80 (m, 1H), 6.10 (m, 1H), 6.91 (m, 2H), 7.00 (m, 1H), 7.30 (m, 2H).

[0056] Reference Example 2: Synthesis of crude tafluprost

[0057] [ka]

[0058] In a 5L flask, under a nitrogen atmosphere, tafluprost acid (120 g) obtained in Reference Example 1 was charged and dissolved in acetone (600 mL) while stirring. The mixture was cooled to 5°C, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (160 mL) was added dropwise while maintaining the temperature at 5°C or less, and 2-iodopropane (146 mL) was added dropwise while maintaining the temperature at 5°C or less, and then the mixture was stirred at 30°C until the conversion rate of the reaction reached 95% or more. Ethyl acetate (1800 mL) and 5% aqueous citric acid (900 mL) were added to the reaction mixture, which was then separated, and the organic layer was washed with 5% aqueous citric acid (900 mL, once), 5% aqueous sodium bicarbonate (900 mL, twice), and purified water (900 mL, once). The solvent was distilled off under reduced pressure at 40° C. or lower to obtain a crude product of tafluprost (132 g, yield 100%; HPLC purity: 95.5%, α-chain trans isomer content: 0.73%).

[0059] Example 1 (Step 1) A slurry prepared from silica gel (AGC Si-Tech, MSGEL D50-120A, particle size (d50): 50 μm, spherical, 50 g) and n-hexane / ethanol = 96 / 4 was filled into a column, and the tafluprost crude product (1 g) obtained in Reference Example 2 was dissolved in n-hexane / ethyl acetate = 1 / 1 and charged onto the column, and eluted with n-hexane / ethanol = 96 / 4. Each fraction was analyzed by HPLC, and fractions containing tafluprost were collected. As fractions containing tafluprost, fractions with an area percentage of at least 98% of tafluprost (calculated excluding the solvent peak) were collected. (Step 2) The collected fractions containing tafluprost were concentrated under reduced pressure at 35°C to 40°C. (Step 3) The residue was dissolved in n-hexane / ethyl acetate=3 / 2, filtered through a membrane filter (pore size: 0.2 μm), and washed with n-hexane / ethyl acetate=3 / 2. (Step 4) The solvent of the filtrate was distilled off overnight at 35°C to 40°C under reduced pressure conditions with a final vacuum of 1 torr or less to obtain tafluprost (colorless to pale yellow viscous liquid, yield: 82%, HPLC purity: 99.5%, α-chain trans isomer content: 0.25%). The residual solvent concentrations in the obtained tafluprost were analyzed by GC, and as a result, n-hexane was 0 ppm, ethyl acetate was 0 ppm, and ethanol was 0 ppm. 11H NMR (CDCl3) δ 1.22 (d, J = 6.2 Hz, 3H), 1.22 (d, J = 6.2 Hz, 3H), 1.58 - 1.63 (m, 1H), 1.63 - 1.69 (m, 2H), 1.84 (d, J = 14.7 Hz, 1H), 2.02 - 2.08 (m, 1H), 2.10 - 2.16 (m, 3H), 2.25 (t, J = 7.3 Hz, 1H), 2.26 (t, J = 7.1 Hz, 1H), 2.30 - 2.35 (m, 1H), 2.46 - 2.49 (m, 2H), 2.61 - 2.63 (m, 1H), 4.02 - 4.03 (m, 1H), 4.18 - 4.21 (m, 3H), 5.00 (heptet, J = 6.2 Hz, 1H), 5.35 - 5.42 (m, 2H), 5.80 (dt, J = 15.8, 11.2 Hz, 1H), 6.10 (dd, J = 15.8, 8.8 Hz, 1H), 6.91 (d, J = 8.8 Hz, 2H), 7.00 (t, J = 7.3 Hz, 1H), 7.30 (dd, J = 8.8, 7.3 Hz, 2H); 19 19F NMR (CDCl3) δ -102.8 (dq, 2 J FF = 255.6 Hz), -103.6 (dq, 2 J FF = 255.6 Hz).

[0060] <HPLC (Reverse Phase) Analysis Conditions> Column: YMC - Pack ODS - AM (5μm, 6.0×150mm) Temperature: Room temperature Flow rate: 1 mL / min Detection wavelength: 220 nm Eluent: (Solution A) 1% Triethylamine - Phosphate buffer (pH 6.3), (Solution B) Acetonitrile Gradient conditions: A / B = 50 / 50 (0 - 45 minutes), A / B = 25 / 75 (45 - 70 minutes)

[0061] <GC Analysis Conditions> Column: G-column G300 (1.2mm ID, 40m) Column temperature: 50℃ Detection: Hydrogen flame ionization detector Carrier: Helium Injector temperature: 160℃ Detector temperature: 160℃

[0062] Examples 2 to 6 In order to examine the influence of the type of silica used in silica gel column chromatography on the purity and yield of tafluprost, the following experiment was carried out in the same manner as in Example 1. The results of silica gel column chromatography performed using the crude tafluprost product (1 g; HPLC purity: 95.5%) and silica gel (50 g) are shown in the following Table 1. The reversed-phase HPLC analysis was performed under the same conditions as above.

[0063] [Table 1]

[0064] Comparative Example 1 Silica gel column chromatography was performed using the crude tafluprost product (1 g; HPLC purity: 95.5%), the silica gel (50 g) used in Example 2, and the eluent. The fractions containing only tafluprost were collected by visual observation in the same manner as in Example 1, except that each fraction was analyzed by TLC instead of HPLC. As a result, the HPLC purity of the obtained tafluprost was 97.3%, which did not reach the quality level (limit value: 98%) required for a purified pharmaceutical product.

[0065] When the particle size (d50) of the silica gel used was 65 μm or less, regardless of the shape, tafluprost having a purity of more than 98% was obtained in all of Examples 1 to 6. Among them, it was found that when spherical silica gel was used, tafluprost with particularly high purity was obtained in high yield.

[0066] Example 7 (Scale-up study) (Process 1) In the same manner as in step 1 of Example 1, a slurry prepared from silica gel (AGC Si-Tech, MSGEL D50-120A, particle size (d50): 50 μm, spherical, 6.0 kg) and n-hexane / ethanol = 96 / 4 was packed into a column, and the tafluprost crude product (120 g) obtained in Reference Example 2 was dissolved in n-hexane / ethyl acetate = 1 / 1 and charged onto the column, and eluted with n-hexane / ethanol = 96 / 4. Each fraction was analyzed by HPLC, and fractions containing tafluprost were collected. As the fractions containing tafluprost, fractions having an area percentage of at least 98% of tafluprost (calculated excluding the solvent peak) were collected.

[0067] (Steps 2~4) The fractions containing tafluprost collected in step 1 were concentrated under reduced pressure at 29° C. to 35° C. (step 2). The residue was dissolved in n-hexane / ethyl acetate=3 / 2, filtered through a membrane filter (pore size: 0.2 μm), and washed with n-hexane / ethyl acetate=3 / 2 (Step 3). The solvent of the filtrate was distilled off for 26 hours at 32°C to 36°C under reduced pressure conditions with a final vacuum of 0.30 torr (step 4), to obtain tafluprost (colorless to pale yellow viscous liquid, yield: 86%, HPLC purity: 99.7%, α-chain trans isomer content: 0.24%, microbial content: 10 cfu / 0.1 g or less). The residual solvent concentrations in the obtained tafluprost were analyzed by GC, and as a result, n-hexane was 0 ppm, ethyl acetate was 0 ppm, and ethanol was 0 ppm.

[0068] Example 8 The fractions containing tafluprost, which had been purified and collected in the same manner as in step 1 of Example 7, were concentrated under reduced pressure under the same conditions as in step 2 of Example 7. The resulting residue was dissolved in ethyl acetate, filtered through a membrane filter (pore size: 0.2 μm), and washed with ethyl acetate (Step 3). The solvent of the filtrate was distilled off for 27 hours under reduced pressure conditions at 23°C to 37°C with a final vacuum of 0.26 torr (step 4) to obtain tafluprost (colorless to pale yellow viscous liquid, yield: 85%, HPLC purity: 99.7%, α-chain trans isomer content: 0.27%, microbial content: 10 cfu / 0.1 g or less). The residual solvent concentrations in the obtained tafluprost were analyzed by GC, and as a result, n-hexane was 0 ppm, ethyl acetate was 0 ppm, and ethanol was 0 ppm.

[0069] Example 9 The fractions containing tafluprost, which were purified and collected in the same manner as in step 1 of Example 7, were concentrated under reduced pressure under the same conditions as in step 2 of Example 7, and the residue was dissolved in n-hexane / ethyl acetate=3 / 2, filtered through a membrane filter (pore size: 0.2 μm), and washed with n-hexane / ethyl acetate=3 / 2 (step 3). The solvent of the filtrate was distilled off for 3 hours at 20°C to 36°C under reduced pressure conditions with a final vacuum of 2.6 torr (step 4) to obtain tafluprost (colorless to pale yellow viscous liquid, yield: 75%, HPLC purity: 99.4%, α-chain trans isomer content: 0.30%, microbial content: 10 cfu / 0.1 g or less). The residual solvent concentrations in the obtained tafluprost were analyzed by GC, and as a result, n-hexane was 36 ppm, ethyl acetate was 4803 ppm, and ethanol was 66 ppm.

[0070] Example 10 The fractions containing tafluprost, which were purified and collected in the same manner as in step 1 of Example 7, were concentrated under reduced pressure under the same conditions as in step 2 of Example 7, and the residue was dissolved in n-hexane / ethyl acetate=3 / 2, filtered through a membrane filter (pore size: 0.2 μm), and washed with n-hexane / ethyl acetate=3 / 2 (step 3). The solvent of the filtrate was distilled off for 5 hours at 34°C to 37°C under reduced pressure conditions with a final vacuum of 2.1 torr (step 4) to obtain tafluprost (colorless to pale yellow viscous liquid, yield: 78%, HPLC purity: 99.5%, α-chain trans isomer content: 0.32%, microbial content: 10 cfu / 0.1 g or less). The residual solvent concentrations in the obtained tafluprost were analyzed by GC, and as a result, n-hexane was 2 ppm, ethyl acetate was 785 ppm, and ethanol was 0 ppm.

[0071] Example 11 The fractions containing tafluprost, which were purified and collected in the same manner as in step 1 of Example 7, were concentrated under reduced pressure under the same conditions as in step 2 of Example 7, and the residue was dissolved in n-hexane / ethyl acetate=3 / 2, filtered through a membrane filter (pore size: 0.2 μm), and washed with n-hexane / ethyl acetate=3 / 2 (step 3). The solvent of the filtrate was distilled off for 8 hours at 32°C to 36°C under reduced pressure conditions with a final vacuum of 0.92 torr (step 4), to obtain tafluprost (colorless to pale yellow viscous liquid, yield: 82%, HPLC purity: 99.6%, α-chain trans isomer content: 0.26%, microbial content: 10 cfu / 0.1 g or less). The residual solvent concentrations in the obtained tafluprost were analyzed by GC, and as a result, n-hexane was 0 ppm, ethyl acetate was 86 ppm, and ethanol was 0 ppm.

[0072] Example 12 The fractions containing tafluprost, which were purified and collected in the same manner as in step 1 of Example 7, were concentrated under reduced pressure under the same conditions as in step 2 of Example 7, and the residue was dissolved in n-hexane / ethyl acetate=3 / 2, filtered through a membrane filter (pore size: 0.2 μm), and washed with n-hexane / ethyl acetate=3 / 2 (step 3). The solvent of the filtrate was distilled off for 50 hours under reduced pressure conditions at 35°C to 39°C with a final vacuum of 0.09 torr (step 4) to obtain tafluprost (colorless to pale yellow viscous liquid, yield: 80%, HPLC purity: 99.5%, α-chain trans isomer content: 0.26%, microbial content: 10 cfu / 0.1 g or less). The residual solvent concentrations in the obtained tafluprost were analyzed by GC, and as a result, n-hexane was 0 ppm, ethyl acetate was 0 ppm, and ethanol was 0 ppm.

[0073] Example 13 The fractions containing tafluprost, which were purified and collected in the same manner as in step 1 of Example 7, were concentrated under reduced pressure under the same conditions as in step 2 of Example 7, and the residue was dissolved in n-hexane / ethyl acetate=3 / 2, filtered through a membrane filter (pore size: 0.2 μm), and washed with n-hexane / ethyl acetate=3 / 2 (step 3). The solvent of the filtrate was distilled off for 60 hours at 36°C to 45°C under reduced pressure conditions with a final vacuum of 0.24 torr (step 4), to obtain tafluprost (colorless to pale yellow viscous liquid, yield: 79%, HPLC purity: 99.5%, α-chain trans isomer content: 0.26%, microbial content: 10 cfu / 0.1 g or less). The residual solvent concentrations in the obtained tafluprost were analyzed by GC, and as a result, n-hexane was 0 ppm, ethyl acetate was 0 ppm, and ethanol was 0 ppm.

[0074] The conditions for distilling off the solvent in the above Examples 7 to 13, the yield, purity and α-chain trans isomer content of Tafluprost, and the results of the residual solvent concentration are shown in Table 2 below.

[0075] [Table 2]

[0076] According to Table 2, in any of Examples 7 to 13, tafurprostone can be obtained with good purity and high yield, and the concentration of the residual organic solvent is suppressed below the concentration limit value of the residual solvent guideline for pharmaceuticals. It was found that the purification method of the present invention is a highly versatile purification method that can withstand scale-up. On the other hand, in the comparative example where concentration under reduced pressure was performed with a final achievable vacuum degree of 8 torr, it was confirmed that the concentration of the residual organic solvent exceeded the concentration limit value of the residual solvent guideline for pharmaceuticals.

[0077] Test Example Examination of the Thermal Stability of Tafurprostone Approximately 120 mg of tafurprostone obtained in Example 1 was weighed into a glass container and stored in a constant temperature bath at 40°C, and quantified by reverse-phase HPLC analysis to examine the change in the tafurprostone content over time. Similarly, approximately 20 mg of tafurprostone was weighed into a glass container and stored in a constant temperature bath at 60°C or 80°C, and the change in the tafurprostone content over time was examined.

[0078] <HPLC (Reverse Phase) Analysis Conditions> Column: YMC-Pack ProC18 AS-303 (5 μm, 4.6 × 250 mm) Temperature: 50°C Flow Rate: 1 mL / min Detection Wavelength: 220 nm Eluent: (Solution A) 10 mmol / L Phosphate (Sodium) Buffer (pH 6.9), (Solution B) Acetonitrile Gradient Conditions: A / B = 50 / 50 (0 - 45 minutes), A / B = 25 / 75 (45 - 70 minutes)

[0079] The results of examining the change over time in the tafurprostone content at each temperature are shown in Tables 3 to 5 below.

[0080]

Table 3

[0081]

Table 4

[0082] [Table 5]

[0083] According to the results in Tables 3 to 5, it was confirmed that tafluprost gradually decomposes over time at temperatures of 60°C or higher, even when stored for a storage period of several days to about 2 weeks, and that the decomposition is particularly notable at 80°C. However, it was found that at 40°C, tafluprost remains stable even after 6 months. From the above results, in the purification method of the present invention, by performing vacuum concentration or solvent distillation at a temperature of 55°C or less (particularly preferably 45°C or less), the inclusion of impurities (related substances) resulting from the decomposition of tafluprost can be suppressed. [Industrial Applicability]

[0084] According to the purification method of the present invention, when the crude product of tafluprost is separated and purified by silica gel column chromatography in the final step of the production of tafluprost, the fraction containing tafluprost is collected by HPLC analysis, thereby minimizing the contamination of impurities. In addition, by distilling off the solvent over a long period of time under reduced pressure conditions of low temperature and high vacuum, the concentration of residual organic solvent can be suppressed to the concentration limit value or less of the guideline for residual solvents in pharmaceuticals, and the decomposition of tafluprost, which is unstable at high temperatures, can be suppressed. Furthermore, by incorporating a filter filtration process in the middle, silica gel fine powder, airborne particles, and bacteria can be removed, so that after distilling off the solvent, it is possible to easily and efficiently provide high-purity tafluprost that can be used as it is as a pharmaceutical raw material. In addition, the purification method of the present invention is a highly versatile method that can be widely applied to crude products of tafluprost produced by known methods and can withstand scale-up.

[0085] This application is based on patent application No. 202110111991.0 filed in China on January 27, 2021, the contents of which are incorporated in full herein.

Claims

1. The method includes purifying the crude product of tafluprost by silica gel column chromatography and collecting fractions containing tafluprost by HPLC analysis; the HPLC analysis is a reverse phase HPLC analysis; The method for purifying tafluprost further comprises the steps of filtering a fraction containing tafluprost collected by HPLC analysis, and distilling off a solvent from the filtrate under reduced pressure at 10 to 55°C with a final vacuum of 5 torr or less, wherein the pore size of the filter used for the filtration is 0.25 μm or less.

2. The method for purifying tafluprost according to claim 1, comprising the steps of concentrating the fractions containing tafluprost collected by the HPLC analysis under reduced pressure at 10 to 55°C, and then dissolving the residue in a solvent.

3. The purification method according to claim 1, wherein the crude product of tafluprost is a crude product of tafluprost obtained by deprotection reaction from protected tafluprost, a crude product of tafluprost obtained by esterification of a salt of tafluprost acid, or a crude product of tafluprost obtained by esterification of tafluprost acid.

4. 2. The purification method according to claim 1, wherein a polytetrafluoroethylene filter is used as the filter used for the filtration.

5. The purification method according to claim 1, wherein the particle size (d50) of the silica gel used in the silica gel column chromatography is 20 to 70 μm.

6. 2. The purification method according to claim 1, wherein the silica gel used in the silica gel column chromatography is spherical.

7. 2. The purification method according to claim 1, wherein an eluent for silica gel column chromatography is a mixed solvent of n-hexane and a polar solvent, or a mixed solvent of n-heptane and a polar solvent.

8. The purification method according to claim 7, wherein the eluent is a mixed solvent of n-hexane and a polar solvent.

9. The purification method according to claim 7 or 8, wherein the polar solvent is ethyl acetate, t-butyl methyl ether, 2-propanol, or ethanol.

10. The purification method according to claim 1, wherein the fraction contains 98% or more of tafluprost.

11. 2. The purification method according to claim 1, wherein the solvent for dissolving the residue is ethyl acetate, t-butyl methyl ether, 2-propanol, or ethanol, or a mixed solvent of ethyl acetate, t-butyl methyl ether, 2-propanol, or ethanol with a non-polar solvent.

12. The purification method according to claim 11, wherein the solvent for dissolving the residue is ethyl acetate or a mixed solvent of ethyl acetate and a non-polar solvent.

13. The purification method according to claim 11, wherein the non-polar solvent is n-hexane or n-heptane.

14. 2. The purification method according to claim 1, wherein the final vacuum level is 1 torr or less.

15. The purification method according to claim 1, wherein after the step of distilling off the solvent from the filtrate, the residual solvent concentration of n-hexane is 290 ppm or less, and the residual solvent concentrations of n-heptane, ethyl acetate, t-butyl methyl ether, 2-propanol, and ethanol are each 5000 ppm or less.

16. A method for producing tafluprost, comprising subjecting a crude product of tafluprost to the purification method according to claim 1.

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