Method for purifying tafluprost

Abstract

To provide a method of purifying tafluprost which is simple and efficient and can be scaled up.SOLUTION: A method of purifying tafluprost includes the step of purifying a crude product of tafluprost by silica gel column chromatography, and collecting fractions containing tafluprost by HPLC analysis. A method of producing tafluprost including the method of purifying tafluprost is also provided.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Tafluprost is given by the following formula:

[0003] [ka]

[0004] It is represented as (5Z)-7-[(1R,2R,3R,5S)-2-[(1E [-3,3-difluoro-4-phenoxy-1-butenyl]-3,5-dihydroxycyclopentyl]-5-heptenoate isopropyl, a highly viscous difluoroprostaglandin F with a viscosity of 2440 mPa·s at 25°C. 2α It is a derivative. Tafluprost has an unstable chemical structure with two double bonds, an unsaturated fatty acid ester moiety, and four chiral centers. Because it has a structure in which the hydroxyl group and hydrogen atom at the C15 position, which are present in other prostaglandin derivatives, are replaced with two fluorine atoms, it has the unique property of being remarkably lipophilic among prostaglandin derivatives. Although it has high chemical stability as a prostaglandin derivative, it also has the property of decomposing at high temperatures. Furthermore, tafluprost has a strong intraocular pressure lowering effect and is used as an eye drop to treat glaucoma and ocular hypertension (Patent Document 1). Patent Document 1 contains difluoroprostaglandin F containing tafluprost. 2α A method for producing derivatives 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, making it unavoidable that the final product will contain α-chain trans isomers. In the manufacturing method described in Patent Document 1, a method of separation and purification by preparative HPLC (High Performance Liquid Chromatography) has been reported as a way to remove impurities containing α-chain trans isomers (Patent Document 2). However, tafluprost and its synthetic precursor, the following formula (I):

[0006] [ka]

[0007] The carboxylic acid compounds represented by (hereinafter referred to as "tafluprost acid") are all liquid compounds with very high viscosity, making them difficult to purify. Furthermore, the purification method for tafluprost described in Patent Document 2 uses a large amount of organic solvent, resulting in high costs, and it is also difficult to keep the residual organic solvent concentration below the concentration limit of the pharmaceutical residual solvent guidelines (Non-Patent Document 2). Preparative HPLC columns are generally expensive and are usually used repeatedly, leading to problems such as contamination by accumulated impurities and degradation products, and a decrease in the number of theoretical plates due to column degradation. In order to reduce the risks arising from these problems, complicated procedures such as washing with a large amount of organic solvent and confirming its performance, and confirming the separation performance of the column are necessary. Since reduction is also required on a regular basis, this method has limited practicality as a pharmaceutical manufacturing method.

[0008] On the other hand, methods have been reported to reduce the inclusion of impurities such as α-chain trans isomers by using organic amine salts (Patent Documents 3 and 4) or metal salts (Patent Document 5) of tafluprost acid. However, the addition of salt formation and liberation steps may increase the amount of by-products, dehydrated products, and other impurities resulting from condensation with organic amines or dimerization by self-condensation. Furthermore, many organic amines and metals raise concerns regarding toxicity and mutagenicity, posing safety issues when used as a purification method, especially in stages close to the final stage of pharmaceutical production.

[0009] Furthermore, a method for producing tafluprost that prevents contamination with α-chain trans isomers by going through macrolactone ring formation and macrolactone ring opening steps has also been reported (Patent Document 6). However, such a production 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] U.S. 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] Chinese Patent Application Publication No. 108299192 Specification [Patent Document 6] Japanese Patent Publication No. 2015-36382 [Non-patent literature]

[0011] [Non-Patent Document 1] Tetrahedron Lett., 2004, 45, 1527-1529 [Non-Patent Document 2] Notification No. 307 from the Director of the Review and Management Division, Pharmaceutical Safety Bureau, Ministry of Health and Welfare (March 30, 1998), regarding guidelines for residual solvents in pharmaceuticals. [Overview of the Initiative] [Problems that the invention aims to solve]

[0012] The present invention aims to provide a method for purifying tafuprost, which is a highly viscous liquid compound, to a purity level that can be directly provided as a pharmaceutical active ingredient simply and at low cost, and is also scalable.

Means for Solving the Problems

[0013] As a result of intensive studies to solve the above problems, the inventors of the present invention found that in the production method of tafuprost, the crude product of tafuprost obtained in the esterification step of tafuprost acid is purified by silica gel column chromatography, and a fraction containing tafuprost is collected by HPLC analysis (hereinafter, this purification method may also be referred to as "the purification method of the present invention"). By this method, highly pure tafuprost can be obtained. Further, a purification method including a step of concentrating the fraction containing tafuprost collected by HPLC analysis under reduced pressure at 10 to 55°C, a step of dissolving the residue in a solvent and performing filtration, and a step of distilling off the solvent of the filtrate under reduced pressure at 10 to 55°C until the final vacuum reaches 5 torr or less (hereinafter, the purification method including all the above steps may also be referred to as "the purification method of the present invention") was carried out, and it was found that the residual organic solvent concentration can be suppressed below the concentration limit value of the pharmaceutical residual solvent guideline, and tafuprost having a purity that can be directly provided as a pharmaceutical active ingredient can be obtained, thus completing the present invention.

[0014] That is, the present invention is as follows. [1] A method for purifying tafuprost, comprising a step of purifying the crude product of tafuprost by silica gel column chromatography and collecting a fraction containing tafuprost by HPLC analysis. Purification method. ​[3] The purification method according to [1] or [2] above, 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 of [1] to [3] above, wherein the silica gel used for silica gel column chromatography is spherical. [5] The purification method according to any one of [1] to [4] above, 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 of [1] to [7] above, wherein the HPLC analysis is reverse-phase HPLC analysis. [9] The purification method according to any of [1] to [8] above, wherein the fraction is a fraction containing 98% or more tafluprost.

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

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

[10] above, 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 and a nonpolar solvent.

[12] The purification method according to

[11] above, wherein the solvent for dissolving the residue is ethyl acetate or a mixed solvent of ethyl acetate and a nonpolar solvent.

[13] The purification method according to

[11] or

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

[14] A purification method according to any of [2] to

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

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

[14] above, wherein the residual solvent concentration of n-hexane after the step of distilling off the solvent of the filtrate 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 5000 ppm or less.

[16] A method for producing tafluprost, comprising the step of subjecting the crude product of tafluprost to the purification method described in any of [1] to

[15] above.

[17] Tafluprost obtained by the manufacturing method described in

[16] above.

[18] A pharmaceutical product containing tafluprost as the active ingredient described in

[17] above.

[19] A pharmaceutical product for the prevention or treatment of eye diseases, comprising tafluprost as described in

[17] above as the active ingredient.

[20] The medicine described in

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

[0015] According to the purification method of the present invention, in the final step of tafluprost production, when separating and purifying the crude tafluprost product by silica gel column chromatography, the contamination of impurities can be minimized by collecting the fraction containing tafluprost by HPLC analysis. Furthermore, by removing the solvent over time under low temperature and high vacuum conditions, the residual organic solvent concentration can be reduced to below the concentration limit of the pharmaceutical residual solvent guideline. This method not only enables the decomposition of tafluprost, which is unstable at high temperatures, but also suppresses its degradation. Furthermore, by incorporating a filter filtration step, fine silica gel particles, airborne particles, and bacteria can be removed, so that after the solvent is removed, high-purity tafluprost that can be used directly as a pharmaceutical active ingredient can be provided simply and efficiently. The purification method of the present invention can be widely applied to crude tafluprost produced by known methods and is also scalable. [Modes for carrying out the invention]

[0016] Embodiments of the present invention will be described in detail below. [Definition of Terms] The meanings of the terms used in this specification are as follows:

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

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

[0019] In this specification, "crude tafluprost product" means the product after post-treatment of the final reaction step in a known method for producing tafluprost, but before purification. Specifically, examples include the product before purification of the final esterification reaction in the method for producing tafluprost described in Patent Document 1, as shown in the examples described later.

[0020] In this specification, "impurities" include all substances other than tafluprost, such as residual reaction reagents, residual raw material compounds, reaction by-products, and tafluprost decomposition products 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 confirming the presence and content ratio of tafluprost in each fraction during the separation and purification of the crude tafluprost product by silica gel column chromatography, using high-performance liquid chromatography for analysis.

[0022] In this specification, "filtration" means filter filtration. Filtration is performed to remove fine particles of the column packing material (silica gel), airborne particles, bacteria, etc.

[0023] In this specification, "polar solvent" means a solvent with a high 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 are preferred.

[0024] In this specification, "nonpolar solvent" means a solvent with a low dielectric constant. Specific examples of nonpolar solvents include, for example, 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 the reaction vessel or concentration vessel, and is usually the ambient temperature or the temperature of the water bath or hot water bath.

[0026] In this specification, "concentration limits of residual solvents in pharmaceuticals" refers to the three standards of Japan, the United States, and the EU. This was considered as one of the issues of the International Conference on Harmonization of Pharmaceuticals (ICH), and it defines the permissible amount of residual solvent in pharmaceuticals for the safety of patients. It refers to the toxicologically acceptable limit for residual solvents. Specific examples of concentration limits for residual solvents in pharmaceuticals, as described in Non-Patent Document 2, include, for example, 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 present invention provides a purification method characterized by comprising the step of purifying the crude tafluprost product by silica gel column chromatography and collecting the fraction containing tafluprost by HPLC analysis (Step 1). Furthermore, in order to reduce the residual organic solvent concentration to below the concentration limit value of the residual solvent guidelines for pharmaceuticals, the present invention provides a purification method characterized by comprising, in addition to Step 1, a step of concentrating under reduced pressure at 10 to 55°C (Step 2), a step of dissolving the residue in a solvent and filtering it (Step 3), and a step of distilling off the solvent from the filtrate under reduced pressure at 10 to 55°C to achieve a final vacuum of 5 torr or less (Step 4).

[0028] (Process 1) This process involves purification by silica gel column chromatography and collection of the fraction containing tafluprost by HPLC analysis.

[0029] The packing material used in silica gel column chromatography is not particularly limited as long as it is silica gel that can be used in a normal-phase column. The silica gel may be in the form of crushed or spherical particles, but spherical particles are more preferred. 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 most preferably 45 μm to 60 μm. The particle size (d50) is the median diameter of the particle size distribution when the particle size distribution is prepared 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 can separate tafluprost from impurities in the crude tafluprost product, but a mixed solvent of n-hexane and a polar solvent, or a mixed solvent of n-heptane and a polar solvent is preferred, and a mixed solvent of n-hexane and a polar solvent is more preferred. Here, the polar solvent is selected from ethyl acetate, t-butyl methyl ether, 2-propanol, and ethanol, with 2-propanol or ethanol being preferred. The mixing ratio (volume ratio) of n-hexane and the polar solvent, or n-heptane and the polar solvent, can be appropriately set depending on 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 using a mixed solvent as the eluent, the mixing ratio (volume ratio) is not particularly limited. However, from the viewpoint of controlling the residual solvent concentration to below a standard value, in the case of a mixed solvent of ethanol and n-hexane, it is preferable to use a solvent in which ethanol and n-hexane are mixed in a ratio of 10:90 to 1:99, more preferably a solvent mixed in a ratio of 6:94 to 2:98, even more preferably a solvent mixed in a ratio of 5:95 to 3:97, and particularly preferably a solvent mixed in a ratio of 4:96.

[0031] To confirm the presence or absence of tafluprost in the fraction separated by silica gel column chromatography, analytical HPLC is used. Both normal-phase and reverse-phase HPLC can be used, but reverse-phase HPLC is more preferred due to its superior impurity separation efficiency, detection sensitivity, and quantitative accuracy. The column used for this HPLC analysis is... Examples of analytical conditions include, but are not limited to, the conditions described in the examples below. Normally, when performing silica gel column chromatography purification, the presence or absence of the target product in the separated fraction is confirmed using TLC (thin-layer chromatography) (see Experimental Chemistry Course 1 Basic Operations I (4th edition), published November 5, 1990, Maruzen, 5.2.3 Column Chromatography, pp. 293-296), but in the purification of the crude product of tafluprost Compared to conventional TLC analysis, HPLC analysis was found to be significantly superior in terms of detection sensitivity for fractions containing tafluprost and other impurities. Numerous lots of the synthesized crude tafluprost product were subjected to silica gel column chromatography, and the elution patterns of impurities were analyzed by HPLC. The results confirmed that the impurity elution patterns were consistently stable. Considering these impurity elution patterns, 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 higher, and it is particularly preferable to collect consecutive fractions in which it is 98% or higher.

[0032] (Process 2) This process involves collecting the fraction containing tafluprost, as confirmed by HPLC analysis, and concentrating it under reduced pressure at 10-50°C.

[0033] When collecting the fraction containing tafluprost, as confirmed by HPLC analysis, and concentrating it under reduced pressure, the ambient temperature (temperature of the water bath or hot water bath) 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 has been confirmed that tafluprost gradually decomposes over time at temperatures above 60°C, so it is desirable to concentrate under reduced pressure or remove the solvent by distillation at the above temperatures.

[0034] (Step 3) This step involves dissolving the residue obtained in step 2 in a solvent and then filtering it. Because tafluprost is extremely viscous, it is difficult to completely remove the solvent before sterile filtration. Therefore, the purification method of the present invention is characterized by incorporating a filtration step after step 2.

[0035] The solvent used to dissolve the residue obtained in step 2 may be the same solvent as the eluent used in silica gel column chromatography in step 1, but it is preferable that the solvent sufficiently dissolves tafluprost and has a relatively low boiling point, and may be a mixed solvent with a nonpolar solvent that forms an azeotropic composition. Specifically, preferably, it 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 and a nonpolar solvent (preferably n-hexane or n-heptane), more preferably ethyl acetate, or a mixed solvent of ethyl acetate and a nonpolar 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 be below a reference value, 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 in a ratio of 10:1 to 1:10, preferably 4:1 to 1:4, and more preferably 2:1 to 1:2.

[0036] The filters used for filtration in this process are not particularly limited, as long as they do not swell or dissolve in the solvent and can remove fine particles of the packing material (silica gel) and airborne particles, etc. Examples include glass fiber filters, polypropylene filters, nylon filters, fluororesin filters, etc. Fluoropolymer filters such as PVDF and polytetrafluoroethylene (PTFE) are preferred, and among these, polytetrafluoroethylene (PTFE) filters are particularly preferred. The pore size of the filter is typically 0.5 μm or less, preferably 0.25 μm or less, and particularly preferably 0.22 μm or less if sterilization is also required.

[0037] (Step 4) This step involves removing the solvent from the filtrate obtained in step 3 by distillation under reduced pressure at 10-55°C, with a final vacuum level of 5 torr or less.

[0038] Because tafluprost is extremely viscous, it is necessary to maximize the surface area on which the solvent evaporates, avoid bumping, and gradually remove the solvent from the filtrate over time. Examples of vacuum concentration apparatuses that can achieve this objective include rotary evaporators, centrifugal evaporators, and high-vacuum thin-film distillation apparatuses.

[0039] Furthermore, as mentioned above, since tafluprost gradually decomposes over time at temperatures above 60°C, the ambient temperature (temperature of the water bath or hot water bath) when removing 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] When removing the solvent by distillation, it is preferable to control the degree of reduced pressure so that the surface area on which the solvent evaporates is as large as possible, and the pressure is gradually increased over time while avoiding bumping, so that the final vacuum level is 5 torr or less (preferably 3 torr or less, more preferably 1 torr or less, and particularly preferably 0.5 torr or less). Furthermore, when releasing the reduced pressure, it is preferable to return to atmospheric pressure using filtered air to prevent the intrusion of airborne particles and bacteria.

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

[0042] By using the purification method of the present invention, the concentration of residual organic solvent can be reduced to below the concentration limit values ​​of the residual solvent guidelines for pharmaceuticals (Non-Patent Literature 2). Furthermore, as the guidelines state, "Since residual solvents do not serve a therapeutic purpose, all residual solvents should be reduced to a level that can comply with product specifications, GMP, or other quality standards." Therefore, it is possible to stably produce high-quality tafluprost that can comply with various even stricter quality standards. As solvents for dissolving the eluent and residue used in the silica gel column chromatography described above, it is desirable that the residual solvent concentration of ethyl acetate, t-butyl methyl ether, 2-propanol, ethanol, or n-heptane, which were exemplified as suitable solvents, be controlled to 1000 ppm or less, more preferably 100 ppm or less, for production. Furthermore, it is desirable that the residual solvent concentration of n-hexane be controlled to 200 ppm or less, more preferably 20 ppm or less, for production. The concentration of the residual solvent can be measured by methods such as gas chromatography (GC).

[0043] The present invention also encompasses a method for producing tafluprost, which includes the step of subjecting a crude product of tafluprost produced by a known method to the purification method of the present invention (a purification method including steps 1 to 4 described above). In addition to the aforementioned Patent Document 1 and Non-Patent Document 1, there are several other reported examples of known methods for producing tafluprost (for example, 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.), and these can also be combined with the purification method of the present invention. This is therefore included in the present invention.

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

[0045] The following are specific features of the purification method of the present invention.

[0046] (A) When separating and purifying the crude tafluprost product by silica gel column chromatography, the contamination of impurities can be minimized by collecting the fraction containing tafluprost by HPLC analysis (preferably reverse-phase HPLC analysis).

[0047] (B) By removing the solvent over time under low temperature and high vacuum conditions, the decomposition of tafluprost, which is unstable at high temperatures, can be suppressed, and the concentration of residual organic solvent can be kept below the concentration limit of the pharmaceutical residual solvent guidelines.

[0048] (C) By incorporating a filter filtration process, high-purity tafluprost can be provided that can be used directly as a pharmaceutical active ingredient after the solvent has been removed.

[0049] (D) The purification method of the present invention can be applied to crude tafluprost products obtained using any known method for producing tafluprost, and can also be easily scaled up, thus providing a simple and efficient purification method.

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

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

[0052] The percentages indicate mol% for yields, and mass% for all other values ​​unless otherwise specified. Ratios shown for mixed solvents are volume ratios unless otherwise specified. Room temperature refers to a temperature of 15-30°C unless otherwise specified. 1 ¹H-NMR values ​​were measured using a JEOL ECP400 (400MHz) nuclear magnetic resonance spectrometer. A Shimadzu LC-10ADvp or LC-10A HPLC system was used. A Shimadzu GC-2014ATF GC system was used.

[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 (280 g) was dissolved in tetrahydrofuran (1200 g) under a nitrogen atmosphere, and diisobutylaluminum hydride (1 M toluene solution) (2160 mL) was added dropwise at -70°C. After the addition was complete, the mixture was stirred for 30 minutes, and 1 N hydrochloric acid was added and the mixture was extracted with ethyl acetate. The organic layers were washed with water, and the filtrate was concentrated under reduced pressure to obtain the reduced product (284 g). 4-carboxybutyltriphenylphosphonium bromide (1523 g) was dissolved in tetrahydrofuran (5030 g) under a nitrogen atmosphere, and sodium bis(trimethylsilyl)amide solution (1 M tetrahydrofuran solution) (6684 mL) was added dropwise and the mixture was stirred for more than 1 hour. The above 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 mixture and extracted with ethyl acetate. After acidifying the aqueous layer, it was extracted with ethyl acetate, concentrated under reduced pressure, and the insoluble material was filtered off. Tafluprost acid (222 g) was obtained by purifying the mixture by silica gel column chromatography (hexane / ethyl acetate = 1 / 1 to 1 / 3). 1 H 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 Tafluprost Crude Product

[0057] [ka]

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

[0059] Example 1 (Step 1) Silica gel (manufactured by AGC SI-TEC, MSGEL D50-120A) A slurry prepared from n-hexane / ethanol = 96 / 4 (particle size (d50): 50 μm, spherical, 50 g) was packed into a column. The crude tafluprost 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. The fractions containing tafluprost were those in which the area percentage of tafluprost was at least 98% (calculated excluding the solvent peak). (Step 2) The collected fraction containing tafluprost was 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. (Process 4) At 35°C to 40°C, under a reduced pressure condition where the final achieved vacuum degree is 1 torr or less, the solvent of the filtrate was distilled off over one day and night to obtain taufuprost (colorless to pale yellow viscous liquid, yield: 82%, HPLC purity: 99.5%, α-chain trans isomer content: 0.25%). As a result of analyzing the residual solvent concentration of the obtained taufuprost by GC, n-hexane was 0 ppm, ethyl acetate was 0 ppm, and ethanol was 0 ppm. 1 H 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 F 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 condition: A / B = 50 / 50 (0 - 45 minutes), A / B = 25 / 75 (45 - 70 minutes)

[0061] <GC analysis conditions> Column: G-column G300 (1.2 mm I.D., 40 m) Column temperature: 50 °C Detection: flame ionization detector Carrier: helium Injector temperature: 160 °C Detector temperature: 160 °C

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

[0063]

Table 1

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

[0065] When the particle size (d50) of the silica gel used was 65 μm or less, tafluprost with a purity exceeding 98% was obtained in all of Examples 1 to 6, regardless of the shape. In particular, it was found that tafluprost with a very high purity could be obtained in high yield when spherical silica gel was used.

[0066] Example 7 (Scaling-up study) (Process 1) In the same manner as in Step 1 of Example 1, silica gel (MSGEL, manufactured by AGC SI-TEC) A slurry prepared from D50-120A (particle size (d50): 50 μm, spherical, 6.0 kg) and n-hexane / ethanol = 96 / 4 was packed into a column. The crude tafluprost 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. The fractions containing tafluprost were those in which the area percentage of tafluprost was at least 98% (calculated excluding the solvent peak).

[0067] (Steps 2~4) The fraction containing tafluprost collected in step 1 was 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). By distilling off the solvent from the filtrate at 32°C to 36°C under reduced pressure conditions to achieve a final vacuum of 0.30 torr for 26 hours (step 4), tafluprost (colorless to pale yellow viscous liquid, yield: 86%, HPLC purity: 99.7%, α-chain trans isomer content: 0.24%) is obtained. A microbial content of 10 cfu / 0.1 g or less was obtained. GC analysis of the residual solvent concentrations of the obtained tafluprost revealed that n-hexane was 0 ppm, ethyl acetate was 0 ppm, and ethanol was 0 ppm.

[0068] Example 8 The fraction containing tafluprost, which was purified and collected in the same manner as in Step 1 of Example 7, was concentrated under reduced pressure under the same conditions as in Step 2 of Example 7. The obtained residue was dissolved in ethyl acetate, filtered through a membrane filter (pore size: 0.2 μm), and washed with ethyl acetate (step 3). 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) was obtained by distilling off the solvent from the filtrate under reduced pressure conditions at 23°C to 37°C for 27 hours at a final vacuum level of 0.26 torr (step 4). GC analysis of the residual solvent concentrations of the obtained tafluprost revealed that n-hexane was 0 ppm, ethyl acetate was 0 ppm, and ethanol was 0 ppm.

[0069] Example 9 The fraction containing tafluprost, collected by purification in the same manner as in Step 1 of Example 7, was concentrated under reduced pressure under the same conditions as in Step 2 of Example 7. 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). 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) was obtained by distilling off the solvent from the filtrate for 3 hours under reduced pressure conditions at 20°C to 36°C, with a final vacuum level of 2.6 torr (step 4). GC analysis of the residual solvent concentrations of the obtained tafluprost revealed that n-hexane was 36 ppm, ethyl acetate was 4803 ppm, and ethanol was 66 ppm.

[0070] Example 10 The fraction containing tafluprost, collected by purification in the same manner as in Step 1 of Example 7, was concentrated under reduced pressure under the same conditions as in Step 2 of Example 7. 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). 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) was obtained by distilling off the solvent from the filtrate for 5 hours under reduced pressure conditions at 34°C to 37°C, with a final vacuum of 2.1 torr (step 4). GC analysis of the residual solvent concentrations of the obtained tafluprost revealed that n-hexane was 2 ppm, ethyl acetate was 785 ppm, and ethanol was 0 ppm.

[0071] Example 11 The fraction containing tafluprost, collected by purification in the same manner as in Step 1 of Example 7, was concentrated under reduced pressure under the same conditions as in Step 2 of Example 7. 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). 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) was obtained by distilling off the solvent from the filtrate for 8 hours under reduced pressure conditions at 32°C to 36°C, with a final vacuum of 0.92 torr (step 4). The residual solvent concentration of the obtained tafluprost was analyzed by GC, and the result showed that n-hexane was 0p The concentrations of ethyl acetate were 86 ppm, and ethanol was 0 ppm.

[0072] Example 12 The fraction containing tafluprost, collected by purification in the same manner as in Step 1 of Example 7, was concentrated under reduced pressure under the same conditions as in Step 2 of Example 7. 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). 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) was obtained by distilling off the solvent from the filtrate for 50 hours under reduced pressure conditions at 35°C to 39°C, with a final vacuum level of 0.09 torr (step 4). GC analysis of the residual solvent concentrations of the obtained tafluprost revealed that n-hexane was 0 ppm, ethyl acetate was 0 ppm, and ethanol was 0 ppm.

[0073] Example 13 The fraction containing tafluprost, collected by purification in the same manner as in Step 1 of Example 7, was concentrated under reduced pressure under the same conditions as in Step 2 of Example 7. 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). 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) was obtained by distilling off the solvent from the filtrate for 60 hours under reduced pressure conditions at 36°C to 45°C, with a final vacuum of 0.24 torr (step 4). GC analysis of the residual solvent concentrations of the obtained tafluprost revealed that n-hexane was 0 ppm, ethyl acetate was 0 ppm, and ethanol was 0 ppm.

[0074] The conditions for solvent removal, yield, purity, and α-chain trans-isomer content of tafluprost in Examples 7-13, as well as the results for residual solvent concentration, are shown in Table 2 below.

[0075] [Table 2]

[0076] According to Table 2, in any of Examples 7 to 13, taufuprost 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 carried out with the final achievable degree of vacuum being 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 Taufuprost Approximately 120 mg of the taufuprost 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 taufuprost content over time. Similarly, approximately 20 mg of taufuprost was weighed into a glass container and stored in a constant temperature bath at 60°C or 80°C, and the change in the taufuprost content over time was examined.

[0078] <HPLC (Reverse Phase) Analysis Conditions> Column: YMC-Pack ProC18 AS-303 (5 μm, 4.6 × 250 mm) Temperature: 5 °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 taufuprost 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 3-5, tafluprost gradually decomposes over time at temperatures above 60°C, even during storage periods of several days to two weeks, and decomposition is particularly pronounced at 80°C. However, it was found to remain stable at 40°C even after 6 months. Ta. Based on the above results, the purification method of the present invention can suppress the inclusion of impurities (related substances) derived from the decomposition of tafluprost by performing reduced-pressure concentration and solvent removal at a temperature of 55°C or lower (particularly preferably 45°C or lower). [Industrial applicability]

[0084] According to the purification method of the present invention, in the final step of tafluprost production, when separating and purifying the crude tafluprost product by silica gel column chromatography, the contamination of impurities can be minimized by collecting the fraction containing tafluprost by HPLC analysis. Furthermore, by removing the solvent over time under low temperature and high vacuum conditions, the residual organic solvent concentration can be kept below the concentration limit of the pharmaceutical residual solvent guideline, and the decomposition of tafluprost, which is unstable at high temperatures, can be suppressed. In addition, by incorporating a filter filtration step, fine silica gel powder, airborne particles, and bacteria can be removed, so that after solvent removal, high-purity tafluprost that can be used directly as a pharmaceutical active pharmaceutical ingredient can be provided simply and efficiently. Moreover, the purification method of the present invention can be widely applied to crude tafluprost products produced by known methods and is a highly versatile method that can withstand scale-up.

[0085] This application is based on patent application 202110111991.0 filed in China on January 27, 2021, the contents of which are fully incorporated herein.

Claims

1. The process includes purifying the crude tafluprost product by silica gel column chromatography using spherical silica gel, and collecting the fraction containing tafluprost by HPLC analysis. The aforementioned HPLC analysis is reversed-phase HPLC analysis. The process of collecting the aforementioned fractions is a process of collecting consecutive fractions in which the HPLC area percentage of tafluprost in each fraction is 97% or more. A method for purifying tafluprost, further comprising the steps of filtering a fraction containing tafluprost collected by the HPLC analysis, 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, wherein the pore size of the filter used for filtration is 0.25 μm or less.

2. 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.

3. The purification method according to claim 1, 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.

4. The purification method according to claim 3, wherein the eluent is a mixed solvent of n-hexane and a polar solvent.

5. The purification method according to claim 3, wherein the polar solvent is ethyl acetate, t-butyl methyl ether, 2-propanol, or ethanol.

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

7. The purification method according to claim 5, wherein, after the step of removing 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 5000 ppm or less.

8. A method for producing tafluprost, comprising the step of subjecting a crude product of tafluprost to the purification method described in any one of claims 1 to 7.

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