Method of 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] The present invention relates to a novel purification method of tafluprost.

Background Art

[0002] Tafluprost is represented by the following formula:

[0003]

Chemical Formula

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

[0005] Since the manufacturing method described in Patent Document 1 includes a Wittig reaction step, it is difficult to avoid the contamination of the α-chain trans isomer in the final product. In the manufacturing method described in Patent Document 1, as a method for removing impurities containing the α-chain trans isomer, a method of separating and purifying by preparative HPLC (High Performance Liquid Chromatography) has been reported (Patent Document 2). However, all of the carboxylic acid compounds represented by the following formula (I):

[0006]

Chemical formula

[0007] (hereinafter referred to as "tafluprost acid") are liquid compounds having very high viscosity, so they are difficult to purify. Also, in the purification method of tafluprost described in Patent Document 2, since a large amount of organic solvent is used, it requires a great deal of cost, and it is also difficult to suppress the residual organic solvent concentration below the concentration limit value of the pharmaceutical residual solvent guideline (Non-Patent Document 2). The columns for preparative HPLC are generally expensive and are usually used repeatedly, so they have problems such as the contamination 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, a large amount of organic solvent is used for washing and its confirmation, and the separation performance of the column is confirmed. Since complicated validation is constantly required, it is not very practical as a manufacturing method for pharmaceuticals. On the other hand, methods for reducing the contamination of impurities such as the α-chain trans isomer by passing through the organic amine salt (Patent Documents 3 and 4) or metal salt (Patent Document 5) of tafluprost acid have also been reported. However, with the addition of the salt formation step and the liberation step from the salt, there is a possibility that by-products, dehydrates, and other impurities such as dimerization due to condensation with organic amines and self-condensation may increase. Also, many organic amines and metals are suspected of having toxicity and mutagenicity, etc., and there are safety problems in using them as a purification method especially at a stage close to the final process of pharmaceuticals.

[0008] ​

[0009] Furthermore, a method for producing tafluprost that prevents contamination with the α-chain trans isomer by undergoing a macro lactone ring formation step and a macro lactone ring-opening step has also been reported (Patent Document 6). However, such a production method has low practicality because the production process is long and the yield is low.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[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 which is also scalable.

Means for Solving the Problems

[0013] As a result of intensive studies to solve the above problems, the present inventors found that in a method for producing tafuprost, a crude product of tafuprost obtained in the esterification step of tafuprost acid is purified by silica gel column chromatography, and fractions containing tafuprost are collected by HPLC analysis (hereinafter, this purification method may also be referred to as "the purification method of the present invention"). By this method, tafuprost of high purity can be obtained. Further, a purification method including a step of concentrating under reduced pressure at 10 to 55°C a fraction containing tafuprost collected by HPLC analysis, 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 with a final achieved vacuum degree of 5 torr or less (hereinafter, the purification method including all of 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 concentration of residual organic solvents 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 a crude product of tafuprost by silica gel column chromatography and collecting fractions containing tafuprost by HPLC analysis. of tafuprost. [2] The method for purifying tafuprost according to [1] above, further comprising a step of concentrating under reduced pressure at 10 to 55°C a fraction containing tafuprost collected by HPLC analysis, then 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 with a final achieved vacuum degree of 5 torr or less. [3] The particle size (d50) of the silica gel used in silica gel column chromatography is 20 to 70 μm, and the purification method according to [1] or [2] above. [4] The silica gel used in silica gel column chromatography is spherical, and the purification method according to any one of [1] to [3] above. [5] The eluent of 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, and the purification method according to any one of [1] to [4] above. [6] The eluent is a mixed solvent of n-hexane and a polar solvent, and the purification method according to [5] above. [7] The polar solvent is ethyl acetate, t-butyl methyl ether, 2-propanol, or ethanol, and the purification method according to [5] or [6] above. [8] The HPLC analysis is a reverse-phase HPLC analysis, and the purification method according to any one of [1] to [7] above. [9] The fraction is a fraction containing 98% or more of tafluprost, and the purification method according to any one of [1] to [8] above.

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

[11] 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 non-polar solvent, and the purification method according to any one of [2] to

[10] above.

[12] The solvent for dissolving the residue is ethyl acetate, or a mixed solvent of ethyl acetate and a non-polar solvent, and the purification method according to

[11] above.

[13] The non-polar solvent is n-hexane or n-heptane, and the purification method according to

[11] or

[12] above.

[14] The final achievable vacuum degree is 1 torr or less, and the purification method according to any one of [2] to

[13] above.

[15] After the step of distilling off the solvent of the filtrate, the residual solvent concentration of n - hexane is 290 ppm or less, and the residual solvent concentration of n - heptane, ethyl acetate, t - butyl methyl ether, 2 - propanol or ethanol is 5000 ppm or less respectively, and the purification method according to any one of the above [2] to

[14] .

[16] A method for producing taufuprost, which includes a step of subjecting the crude product of taufuprost to the purification method according to any one of the above [1] to

[15] .

[17] Taufuprost obtained by the production method according to the above

[16] .

[18] A pharmaceutical comprising the taufuprost according to the above

[17] as an active ingredient.

[19] A pharmaceutical for the prevention or treatment of eye diseases, comprising the taufuprost according to the above

[17] as an active ingredient.

[20] The pharmaceutical according to the above

[19] , wherein the eye disease is glaucoma or ocular hypertension.

Advantages of the Invention

[0015] According to the purification method of the present invention, in the final step of producing taufuprost, when separating and purifying the crude product of taufuprost by silica gel column chromatography, by collecting the fraction containing taufuprost by HPLC analysis, the mixing of impurities can be minimized. Further, by distilling off the solvent over time under reduced pressure conditions of low temperature and high vacuum degree, the residual organic solvent concentration can be suppressed below the concentration limit value of the residual solvent guideline for pharmaceuticals, and the decomposition of taufuprost, which is unstable at high temperatures, can also be suppressed. Furthermore, by incorporating a filter filtration step in the middle, fine powder of silica gel, floating particles in the air, and bacteria can be removed. Therefore, after distilling off the solvent, a high - purity taufuprost that can be used as it is as the active ingredient of a pharmaceutical can be provided simply and efficiently. The purification method of the present invention can be widely applied to the crude products of taufuprost produced by known methods and can withstand scale - up. Moreover, it is possible to suppress the decomposition of taufuprost, which is unstable at high temperatures. Furthermore, by incorporating a filter filtration step in the middle, fine powder of silica gel, floating particles in the air, and bacteria can be removed. Therefore, after distilling off the solvent, a high - purity taufuprost that can be used as it is as the active ingredient of a pharmaceutical can be provided simply and efficiently. The purification method of the present invention can be widely applied to the crude products of taufuprost produced by known methods and can withstand scale - up.

Embodiments for Carrying Out the Invention

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

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

[0018] "Silica gel column chromatography" in this specification is a normal-phase column chromatography.

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

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

[0021] In this specification, "HPLC analysis" means that when separating and purifying the crude product of taufuprost by silica gel column chromatography, the presence or absence and content ratio of taufuprost in each fraction are confirmed using high performance liquid chromatography for analysis.

[0022] In this specification, "filtration" means filter filtration. Filtration is performed for the purpose of removing fine powder of the column packing agent (silica gel), suspended particles in the air, bacteria, etc.

[0023] In this specification, the "polar solvent" means a solvent having a high dielectric constant. Specific examples of the polar solvent include, for example, 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 them, ethyl acetate, t-butyl methyl ether, 2-propanol or ethanol is preferred.

[0024] In this specification, the "non-polar solvent" means a solvent having a low dielectric constant. Specific examples of the non-polar solvent include, for example, chain hydrocarbons such as n-hexane and n-heptane. Among them, n-hexane is preferred.

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

[0026] In this specification, the "concentration limit value of residual solvents in pharmaceuticals guidelines" is one of the issues of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) in Japan, the United States, and the European Union, and defines the allowable amount of residual solvents in pharmaceuticals, and means the limit value that can be tolerated toxicologically for residual solvents. Specific examples of the concentration limit value of residual solvents in pharmaceuticals are, as described in Non-Patent Document 2, for example, n-hexane is 290 ppm, and n-heptane, ethyl acetate, t-butyl methyl ether, 2-propanol or ethanol is 5000 ppm.

[0027] [Purification method of the present invention] ​The purification method of the present invention is characterized by including a step (Step 1) of purifying the crude product of tifluprost by silica gel column chromatography and collecting the fraction containing tifluprost by HPLC analysis. Further, in order to suppress the residual organic solvent concentration below the concentration limit value of the residual solvent guideline for pharmaceuticals, the purification method of the present invention, in addition to the above Step 1, includes 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 performing filtration, and a step (Step 4) 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.

[0028] (Step 1) This step is a step of purifying by silica gel column chromatography and collecting the fraction containing tifluprost by HPLC analysis.

[0029] The filler used for silica gel column chromatography is not particularly limited as long as it is silica gel that can be used for a normal normal-phase column. The shape of the silica gel may be either crushed or spherical, but spherical is more preferable. Further, 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 diameter 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] As the eluent used for silica gel column chromatography, there is no particular limitation as long as it is a solvent capable of separating taufuprost and impurities in the crude product of taufuprost. However, 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. 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 diameter of the filler used. Specific preferred 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, it is a mixed solvent of 2-propanol or ethanol and a non-polar solvent (preferably n-hexane or n-heptane), and particularly preferably, it is 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 below the reference value, in the case of a mixed solvent of ethanol and n-hexane, it is preferable to use a solvent mixed at ethanol:n-hexane of 10:90 to 1:99, more preferably a solvent mixed at 6:94 to 2:98, even more preferably a solvent mixed at 5:95 to 3:97, and particularly preferably a solvent mixed at 4:96.

[0031] For confirming the presence or absence of taufuprost in the separated fraction by silica gel column chromatography purification, HPLC for analysis is used. As the HPLC, either normal phase HPLC or reverse phase HPLC can be used, but reverse phase HPLC, which is excellent in terms of separation efficiency of impurities, detection sensitivity, quantitativeness, etc., is more preferred. The column and specific examples of the analysis conditions include the conditions described in the examples below, but are not limited thereto. Generally, when performing silica gel column chromatography purification, the presence or absence of the target substance in the separated fractions is confirmed using TLC (thin layer chromatography) (see Experimental Chemistry Course 1 Basic Operations I (4th Edition), published on November 5, 1990, Maruzen, 5·2·3 Column Chromatography, pp. 293-296). In the purification of the crude product of taufuprost it was found that HPLC analysis was significantly superior in the detection sensitivity of fractions containing taufuprost and impurities compared to conventional TLC analysis. As a result of performing silica gel column chromatography on a number of lots of the synthesized taufuprost crude product and analyzing the elution pattern of impurities by HPLC analysis, it was confirmed that the impurity elution pattern was always stable. By considering such an impurity elution pattern, it was found that it is preferable to collect consecutive fractions in which the HPLC area percentage of taufuprost in each fraction is 97% or more, and particularly preferably to collect consecutive fractions in which it is 98% or more.

[0032] (Step 2) This step is a step of collecting the fractions containing taufuprost confirmed by HPLC analysis and concentrating them under reduced pressure at 10 to 50°C.

[0033] When collecting the fractions containing taufuprost confirmed by HPLC analysis and concentrating them under reduced pressure, the external temperature (the temperature of the water bath or steam bath) is preferably 10°C to 55°C, more preferably 15°C to 50°C, and particularly preferably 20 to 45°C. As shown in the test examples described later, since it was confirmed that taufuprost gradually decomposes over time at temperatures of 60°C or higher, it is desirable to concentrate under reduced pressure or distill off the solvent at the above temperatures.

[0034] (Step 3) This step is a step of dissolving the residue obtained in the above step 2 in a solvent and performing filtration. Since taufuprost is very viscous, it is difficult to perform sterile 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] As the solvent for dissolving the residue obtained in Step 2, solvents similar to the eluent used in the silica gel column chromatography in Step 1 can be mentioned. However, it is preferable that the solvent sufficiently dissolves taufuprost and has a relatively low boiling point, and it may be a mixed solvent with a nonpolar solvent that forms an azeotropic composition. Specifically, 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 nonpolar solvent (preferably n-hexane or n-heptane). More preferably, it is 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) in the case of using a mixed solvent is not particularly limited. However, from the viewpoint of controlling the residual solvent concentration to be below the reference value, in the case of a mixed solvent of ethyl acetate and n-hexane, it is particularly preferable to use a solvent mixed with ethyl acetate:n-hexane at 10:1 to 1:10, preferably 4:1 to 1:4, and more preferably 2:1 to 1:2.

[0036] The filter used for the filtration in this step is not particularly limited as long as it does not swell or dissolve in the solvent and can remove fine powder of the filler (silica gel), floating particles in the air, etc. For example, glass fiber filters, polypropylene filters, nylon filters, fluororesin filters, etc. can be mentioned. Filters made of fluororesins such as polyvinylidene difluoride (PVDF) and polytetrafluoroethylene (PTFE) are preferred, and among them, filters made of polytetrafluoroethylene (PTFE) are particularly preferred. Filters made of fluororesins such as polyvinylidene difluoride (PVDF) and polytetrafluoroethylene (PTFE) are preferred, and among them, filters made of polytetrafluoroethylene (PTFE) are particularly preferred. The pore size of the filter is usually 0.5 μm or less, preferably 0.25 μm or less, and when including the purpose of sterilization, 0.22 μm or less is particularly preferred.

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

[0038] Since tafuprost is very viscous, it is necessary to gradually distill off the solvent of the filtrate over time while avoiding bumping by making the surface area where the solvent evaporates as large as possible. Examples of the vacuum concentration device for achieving such an object include a rotary evaporator, a centrifugal evaporator, a high-vacuum thin-film distillation device, and the like.

[0039] Also, as described above, since tafuprost gradually decomposes over time at a temperature of 60°C or higher, the external temperature (the temperature of the water bath or the steam 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 reduced pressure when distilling off the solvent is preferably increased gradually over time while avoiding bumping by making the surface area where the solvent evaporates as large as possible, and controlled so that the final vacuum reaches 5 torr or less (preferably 3 torr or less, more preferably 1 torr or less, and particularly preferably 0.5 torr or less). Also, when releasing the reduced pressure, in order to prevent the intrusion of airborne particles and bacteria in the air, it is preferable to return to normal pressure using air passed through a filter.

[0041] The time for distilling off 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 the residual organic solvent can be suppressed below the concentration limit value of the residual solvent guideline for pharmaceuticals (Non-Patent Document 2). However, as described in this guideline, "Since residual solvents are not useful for treatment, all residual solvents should be reduced to levels that can meet product specifications, GMP, or other quality standards." As such, high-quality tafluprost that can meet various even stricter quality standards can be stably produced. As the eluent used in the silica gel column chromatography described above and the solvent for dissolving the residue, the residual solvent concentrations of ethyl acetate, t-butyl methyl ether, 2-propanol, ethanol, or n-heptane, which were exemplified as suitable solvents, are more preferably controlled to 1000 ppm or less, respectively, and particularly preferably to 100 ppm or less, respectively, for production. Also, the residual solvent concentration of n-hexane is more preferably controlled to 200 ppm or less, and particularly preferably to 20 ppm or less, for production. The concentration of the residual solvent can be measured by a method such as gas chromatography (GC).

[0043] The present invention also includes 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). Known methods for producing tafluprost include, in addition to Patent Document 1 and Non-Patent Document 1 described above, several reported examples (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 and are thus included in the present invention.

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

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

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

[0047] (B) By distilling off the solvent over time under reduced pressure conditions of low temperature and high vacuum, decomposition of tifluprost, which is unstable at high temperatures, can be suppressed, and the concentration of residual organic solvents can be suppressed below the concentration limit value of the residual solvent guideline for pharmaceuticals.

[0048] (C) By incorporating a filter filtration step in the middle, after distilling off the solvent, high-purity tifluprost that can be used as it is as the active ingredient of a pharmaceutical can be provided.

[0049] (D) The purification method of the present invention can be applied to crude products of tifluprost obtained by any method of known manufacturing methods of tifluprost, and scale-up can also be easily performed, so a simple and efficient purification method can be provided.

[0050] When the purification method of the present invention is carried out for the purpose of increasing the purity of tifluprost as described in (A) above, it only needs to include only the above step 1, and steps 2 to 4 can also be carried out in combination with step 1 as needed.

Example

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

[0052] % represents mol% for the yield, and mass% for others unless otherwise specified. The ratio shown in the mixed solvent represents the volume ratio unless otherwise specified. Also, room temperature represents a temperature of 15 to 30°C unless otherwise specified. The following 1 The 1H-NMR values were measured with a nuclear magnetic resonance apparatus ECP400 (400 MHz) manufactured by JEOL Ltd. The HPLC apparatus used was Shimadzu LC-10ADvp or LC-10A. The GC apparatus used was Shimadzu GC-2014ATF.

[0053] Reference Example 1: Synthesis of Tafuprostic Acid

[0054] [Chemical formula]

[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 by adding tetrahydrofuran (1200 g) thereto under a nitrogen atmosphere, and diisobutylaluminum hydride (1 M toluene solution) (2160 mL) was added dropwise thereto at -70°C. After completion of the dropwise addition, the mixture was stirred for 30 minutes, 1 N hydrochloric acid was added thereto, and the mixture was extracted with ethyl acetate. The combined organic layers were washed with water, and the filtrate was concentrated under reduced pressure to obtain a reduced product (284 g). 4-Carboxybutyltriphenylphosphonium bromide (1523 g) was added with tetrahydrofuran (5030 g) thereto under a nitrogen atmosphere, and a sodium bis(trimethylsilyl)amide solution (1 M tetrahydrofuran solution) (6684 mL) was added dropwise thereto and stirred for 1 hour or more. The above reduced product (286 g) dissolved in tetrahydrofuran (970 g) was added dropwise thereto at 0°C and stirred for 3 hours. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. After the aqueous layer was acidified, the mixture was extracted with ethyl acetate, concentrated under reduced pressure, insolubles were filtered off, and the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 1 / 1 to 1 / 3) to obtain taufuprost acid (222 g). 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 Taufuprost Crude Product

[0057]

Chemical Structure

[0058] In a 5 L flask, taufuprostic acid (120 g) obtained in Reference Example 1 was charged under a nitrogen atmosphere and dissolved in acetone (600 mL) with stirring. The solution 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 lower. Then, 2-iodopropane (146 mL) was added dropwise while maintaining the temperature at 5°C or lower. After that, the mixture was stirred at 30°C until the conversion rate of the reaction reached 95% or more. Ethyl acetate (1800 mL) and a 5% aqueous citric acid solution (900 mL) were added to the reaction mixture, and the layers were separated. The organic layer was washed with a 5% aqueous citric acid solution (900 mL, once), a 5% aqueous sodium hydrogen carbonate solution (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 taufuprost crude product (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 (manufactured by AGC Si-Tech Co., Ltd., M.S.GEL D50-120A, particle size (d50): 50 μm, spherical, 50 g) and n-hexane / ethanol = 96 / 4 was packed into a column. The taufuprost crude product (1 g) obtained in Reference Example 2 was dissolved in n-hexane / ethyl acetate = 1 / 1 and charged onto the column, and then eluted with n-hexane / ethanol = 96 / 4. Each fraction collected was analyzed by HPLC, and the fractions containing taufuprost were collected. As the fractions containing taufuprost, fractions with an area percentage of at least 98% of taufuprost (calculated excluding the solvent peak) were collected. (Step 2) The fractions containing taufuprost collected 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. (Process 4) At 35°C to 40°C, under reduced pressure conditions where the final achievable vacuum is 1 torr or less, the solvent of the filtrate was distilled off over 24 hours 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 influence of the type of silica used for silica gel column chromatography on the purity and yield of taufuprost, the following experiment was conducted in the same manner as in Example 1. Using the taufuprost crude product (1 g; HPLC purity: 95.5%) and silica gel (50 g), the results of silica gel column chromatography 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 the taufuprost crude product (1 g; HPLC purity: 95.5%), the silica gel (50 g) used in Example 2, and the 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, tafuprost having a purity exceeding 98% was obtained in any of Examples 1 to 6 regardless of the difference in shape. Among them, it was found that when spherical silica gel was used, tafuprost with particularly high purity was obtained in high yield.

[0066] Example 7 (Investigation of scale-up) (Step 1) In the same manner as in Step 1 of Example 1, silica gel (manufactured by AGC Inc., M.S.GEL D50-120A, particle size (d50): 50 μm, spherical, 6.0 kg) and a slurry prepared from n-hexane / ethanol = 96 / 4 were packed into a column, and the tafuprost 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 collected was analyzed by HPLC, and the fractions containing tafuprost were collected. As the fractions containing tafuprost, fractions having an area percentage of at least tafuprost of 98% or more (calculated excluding the solvent peak) were collected.

[0067] (Steps 2 to 4) The fraction containing tafuprost 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). At 32°C to 36°C, under reduced pressure conditions where the final ultimate vacuum reached 0.30 torr, the solvent of the filtrate was distilled off for 26 hours (Step 4), whereby tafuprost (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) was obtained. As a result of analyzing the residual solvent concentration of the obtained tafuprost by GC, n-hexane was 0 ppm, ethyl acetate was 0 ppm, and ethanol was 0 ppm.

[0068] Example 8 The fraction containing taufuprost 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). At 23°C to 37°C, the solvent of the filtrate was distilled off under reduced pressure conditions where the final achieved vacuum degree was 0.26 torr for 27 hours (Step 4), thereby obtaining taufuprost (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). 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.

[0069] Example 9 The fraction containing taufuprost 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 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). At 20°C to 36°C, the solvent of the filtrate was distilled off under reduced pressure conditions where the final achieved vacuum degree was 2.6 torr for 3 hours (Step 4), thereby obtaining taufuprost (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). As a result of analyzing the residual solvent concentration of the obtained taufuprost by GC, n-hexane was 36 ppm, ethyl acetate was 4803 ppm, and ethanol was 66 ppm.

[0070] Example 10 The fraction containing taufuprost 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). The solvent of the filtrate was distilled off for 5 hours under reduced pressure conditions where the final achieved vacuum degree was 2.1 torr at 34°C to 37°C (Step 4), thereby obtaining taufuprost (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). As a result of analyzing the residual solvent concentration of the obtained taufuprost by GC, n-hexane was 2 ppm, ethyl acetate was 785 ppm, and ethanol was 0 ppm.

[0071] Example 11 The fraction containing taufuprost 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). The solvent of the filtrate was distilled off for 8 hours under reduced pressure conditions where the final achieved vacuum degree was 0.92 torr at 32°C to 36°C (Step 4), thereby obtaining taufuprost (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). As a result of analyzing the residual solvent concentration of the obtained taufuprost by GC, n-hexane was 0p pm, ethyl acetate was 86 ppm, and ethanol was 0 ppm.

[0072] Example 12 A fraction containing taufuprost 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). The solvent of the filtrate was distilled off for 50 hours under reduced pressure conditions where the final vacuum reached 0.09 torr at 35°C to 39°C (Step 4), whereby taufuprost (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. 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.

[0073] Example 13 A fraction containing taufuprost 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). The solvent of the filtrate was distilled off for 60 hours under reduced pressure conditions where the final vacuum reached 0.24 torr at 36°C to 45°C (Step 4), whereby taufuprost (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. 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.

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

[0075]

Table 2

[0076] According to Table 2, in any of Examples 7 to 13, tifluprost 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 a final ultimate 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 Tifluprost Approximately 120 mg of tifluprost obtained in Example 1 was weighed into a glass container and stored in a constant temperature bath at 40 °C, and quantified by reversed-phase HPLC analysis to examine the change in the tifluprost content over time. Similarly, approximately 20 mg of tifluprost was weighed into a glass container and stored in a constant temperature bath at 60 °C or 80 °C, and the change in the tifluprost content over time was examined.

[0078] <HPLC (Reversed-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 to 45 minutes), A / B = 25 / 75 (45 to 70 minutes)

[0079] The results of examining the change over time in the tifluprost 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 taufuprost gradually decomposes over time even during a storage period of several days to about two weeks at temperatures of 60°C or higher, and particularly decomposes significantly at 80°C, but was found to be stable even after 6 months at 40°C. It was found. From the above results, in the purification method of the present invention, by performing concentration under reduced pressure or distillation of the solvent at a temperature of 55°C or lower (particularly preferably 45°C or lower), it is possible to suppress the incorporation of impurities (related substances) derived from the decomposition of taufuprost.

Industrial Applicability

[0084] According to the purification method of the present invention, in the final step of manufacturing taufuprost, when separating and purifying the crude product of taufuprost by silica gel column chromatography, by collecting the fraction containing taufuprost by HPLC analysis, the incorporation of impurities can be minimized. Further, by distilling off the solvent over time under reduced pressure conditions of low temperature and high vacuum degree, the concentration of the residual organic solvent can be suppressed below the concentration limit value of the residual solvent guideline for pharmaceuticals, and the decomposition of taufuprost, which is unstable at high temperatures, can be suppressed. Furthermore, by incorporating a filter filtration step in the middle, fine silica gel powder, airborne particulate matter, and bacteria can be removed, so that after distilling off the solvent, it is possible to simply and efficiently provide high-purity taufuprost that can be used as it is as the active ingredient of a pharmaceutical. In addition, the purification method of the present invention is a highly versatile method that can be widely applied to the crude products of taufuprost 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 content of which is incorporated herein in its entirety.

Claims

1. A process of purifying a crude product of tifluprost by silica gel column chromatography using spherical silica gel and collecting the fraction containing tifluprost by HPLC analysis, wherein the HPLC analysis is a reverse-phase HPLC analysis, the step of collecting the fraction is a step of collecting consecutive divisions where the HPLC area percentage of tifluprost in each fraction is 97% or more, further comprising the step of filtering the fraction containing tifluprost collected by the HPLC analysis and the step of distilling off the solvent of the filtrate under reduced pressure at 10 to 55 °C with a final achieved vacuum of 5 torr or less, and the pore size of the filter used for the filtration is 0.25 μm or less. A method for purifying tifluprost.

2. The method for purification 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 method for purification according to claim 1, wherein the eluent for the 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 method for purification according to claim 3, wherein the eluent is a mixed solvent of n-hexane and a polar solvent.

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

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

7. After the step of distilling off the solvent of 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. The method for purification according to claim 5.

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

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