Purification method of tafluprost

Abstract

[Objective] The purpose of this invention is to provide a simple, efficient, and scalable purification method for tafluprost. [Solution] This invention relates to a purification method for tafluprost, comprising the steps of purifying crude tafluprost using silica gel column chromatography and collecting the tafluprost-containing component using HPLC analysis. Furthermore, this invention also relates to a method for manufacturing tafluprost incorporating the aforementioned purification method.

Description

[0001] This application is a divisional application of the application filed on January 27, 2021, with application number 202110111991.0 and invention title "Purification Method of Tafluprost". Technical Field

[0002] This invention relates to a novel purification method for tafluprost. Background Technology

[0003] Tafluprost is represented by the following formula:

[0004]

[0005] Its chemical name is (5Z)-7-(1R,2R,3R,5S)-2-[(1E)-3,3-difluoro-4-phenoxy-1-butenyl]-3,5-dihydroxycyclopentyl]-5-heptenoic acid isopropyl ester. It has a viscosity of 2440 mPa·s at 25°C, making it a highly viscous difluoroprostaglandin F. 2α Derivatives. Tafluprost has an unstable chemical structure containing two double bonds, an unsaturated fatty acid ester site, and four asymmetric centers. It possesses a structure found in other prostaglandin derivatives, where the C15 hydroxyl group and hydrogen atom are replaced by two fluorine atoms. Therefore, it exhibits significant high lipophilicity among prostaglandin derivatives. Although chemically stable, it is also prone to decomposition at high temperatures. Furthermore, tafluprost has a potent 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 difluoroprostaglandin F containing tafluprost. 2α The same manufacturing method for the derivative is also described in Non-Patent Document 1.

[0006] The manufacturing method described in Patent Document 1 includes a Wittig reaction step, making it difficult to avoid the contamination of the α-chain trans isomer in the final product. Patent Document 1 describes a method for separating and purifying the product using fractional HPLC (High Performance Liquid Chromatography) as a method for removing impurities containing the α-chain trans isomer (Patent Document 2). However, tafluprost and the carboxylic acid compound represented by the following formula (I) (hereinafter referred to as "tafluprost acid"), which is a precursor for its synthesis:

[0007]

[0008] All of these are highly viscous liquid compounds, making them difficult to purify. Furthermore, the purification method for tafluprost described in Patent Document 2 uses large amounts of organic solvents, resulting in high costs and difficulty in controlling the residual organic solvent concentration below the limits set by the pharmaceutical residual solvent guidelines (non-Patent Document 2). HPLC columns used for fractionation are typically expensive and frequently reused, leading to problems such as accumulated impurities, contamination by decomposition products, and decreased theoretical plate numbers due to column deterioration. To mitigate the risks associated with these problems, extensive cleaning with organic solvents and complex validation processes involving column separation performance are often required. Therefore, this method lacks practicality as a pharmaceutical manufacturing method.

[0009] On the other hand, methods to reduce the incorporation of impurities such as α-chain trans isomers have also been reported by using organic amine salts (Patent Documents 3 and 4) or metal salts (Patent Document 5) of tafluprost acid. However, with the addition of salt formation and salt liquefaction processes, there is a possibility of an increase in byproducts, dehydrated products, and other impurities generated from dimerization due to condensation and self-condensation with organic amines. Furthermore, organic amines and metals are often subject to concerns regarding toxicity and mutagenicity, particularly when used as purification methods in the final stages of pharmaceutical manufacturing, raising safety concerns.

[0010] Furthermore, a method for manufacturing tafluprost, which involves macrolide ring formation and ring-opening processes to prevent the incorporation of the α-chain trans isomer, has also been reported (Patent Document 6). However, the manufacturing process of this method is lengthy and has a low yield, thus limiting its practicality.

[0011] Existing technical documents

[0012] Patent documents

[0013] Patent Document 1: European Patent Application Publication No. 850926

[0014] Patent Document 2: U.S. Patent Application Publication No. 2014 / 0051882

[0015] Patent Document 3: International Publication No. 2013 / 118058

[0016] Patent Document 4: International Publication No. 2016 / 090461

[0017] Patent Document 5: Description of Chinese Patent Application Publication No. 108299192

[0018] Patent Document 6: Japanese Patent Application Publication No. 2015-36382

[0019] Non-patent literature

[0020] Non-patent literature 1: Tetrahedron Lett., 2004, 45, 1527-1529

[0021] Non-Patent Document 2: Pharmaceutical Examination No. 307, Notice from the Head of the Examination and Management Division of the Drug Safety Bureau, Ministry of Health, Labour and Welfare (March 30, 2010), regarding guidelines for residual solvents in pharmaceuticals. Summary of the Invention

[0022] The problem the invention aims to solve

[0023] The purpose of this invention is to provide a purification method for tafluprost, which can easily and cost-effectively purify tafluprost, a highly viscous liquid compound, to a purity suitable for direct use as a raw material for pharmaceuticals, and can also be scaled up.

[0024] Solution for solving the problem

[0025] In order to solve the above-mentioned problems, the inventors conducted in-depth research and discovered that a purification method for manufacturing tafluprost, which includes purifying the crude tafluprost product obtained by esterification of tafluprost acid using silica gel column chromatography and collecting the tafluprost-containing component by HPLC analysis (hereinafter sometimes referred to as "the purification method of the present invention"), can yield tafluprost with high purity. Furthermore, a purification method that includes concentrating the tafluprost-containing component collected by HPLC analysis under reduced pressure at 10–55°C, dissolving the residue in a solvent and filtering it, and removing the solvent from the filtrate by distillation at reduced pressure at 10–55°C and a final ultimate vacuum of 5 torr or less (hereinafter sometimes referred to as "the purification method of the present invention"), can suppress the residual organic solvent concentration below the concentration limit of the residual solvent guidelines for pharmaceuticals, and obtain tafluprost with purity suitable for direct use as a pharmaceutical raw material, thus completing the present invention.

[0026] That is, the present invention is as follows.

[0027] [1] A method for purifying tafluprost, comprising: purifying the crude tafluprost product by silica gel column chromatography and collecting the components containing tafluprost by HPLC analysis.

[0028] [2] The purification method of tafluprost described in [1] above further includes: a step of concentrating the tafluprost-containing component collected by HPLC analysis under reduced pressure at 10 to 55°C; a step of dissolving the residue in a solvent and filtering it; and a step of removing the solvent from the filtrate by distillation under reduced pressure at 10 to 55°C and a final ultimate vacuum of less than 5 torr.

[0029] [3] According to the purification method described in [1] or [2] above, the particle size (d50) of the silica gel used in the silica gel column chromatography is 20 to 70 μm.

[0030] [4] The purification method according to any one of [1] to [3] above, wherein the silica gel used in the silica gel column chromatography is spherical.

[0031] [5] The purification method according to any one of [1] to [4] above, wherein the eluent of silica gel column chromatography is a mixture of n-hexane and a polar solvent, or a mixture of n-heptane and a polar solvent.

[0032] [6] According to the purification method described in [5] above, the eluent is a mixture of n-hexane and a polar solvent.

[0033] [7] The purification method described in [5] or [6] above, wherein the polar solvent is ethyl acetate, tert-butyl methyl ether, 2-propanol, or ethanol.

[0034] [8] The purification method according to any one of [1] to [7] above, wherein the HPLC analysis is reversed-phase HPLC analysis.

[0035] [9] The purification method according to any one of [1] to [8] above, wherein the component is a component containing more than 98% tafluprost.

[0036]

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

[0037]

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

[10] above, wherein the solvent used to dissolve the residue is ethyl acetate, tert-butyl methyl ether, 2-propanol or ethanol, or a mixture of ethyl acetate, tert-butyl methyl ether, 2-propanol or ethanol and a nonpolar solvent.

[0038]

[12] According to the purification method described in

[11] above, the solvent used to dissolve the residue is ethyl acetate or a mixture of ethyl acetate and a nonpolar solvent.

[0039]

[13] According to the purification method described in

[11] or

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

[0040]

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

[13] above, wherein the final ultimate vacuum is less than 1 torr.

[0041]

[15] According to any one of [2] to

[14] above, the concentration of residual hexane after the step of removing the solvent from the filtrate by distillation is less than 290 ppm, and the concentrations of residual solvents of n-heptane, ethyl acetate, tert-butyl methyl ether, 2-propanol or ethanol are less than 5000 ppm.

[0042]

[16] A method for manufacturing tafluprost, comprising the step of supplying crude tafluprost to the purification method described in any one of [1] to

[15] above.

[0043]

[17] A tafluprost obtained by the manufacturing method described above

[16] .

[0044]

[18] A medicine that uses the tafluprost described above

[17] as its active ingredient.

[0045]

[19] A medicine that uses tafluprost as the active ingredient described above

[17] for the prevention or treatment of eye diseases.

[0046]

[20] According to the medicine described above

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

[0047] The effects of the invention

[0048] The purification method of this invention minimizes the contamination of impurities during the final stage of tafluprost production, when separating and purifying the crude tafluprost product using silica gel column chromatography and collecting the tafluprost-containing components using HPLC analysis. Furthermore, by distilling off the solvent under low-temperature, high-vacuum conditions, the concentration of residual organic solvents is suppressed below the limits set by pharmaceutical residual solvent guidelines, and the decomposition of unstable tafluprost at high temperatures is also suppressed. Moreover, by incorporating a filter in the intermediate filtration process, fine silica gel powder, airborne particles, and bacteria can be removed. Therefore, after solvent distillation removal, high-purity tafluprost that can be directly used as a pharmaceutical raw material can be provided easily and efficiently. The purification method of this invention can be widely applied to crude tafluprost products manufactured using known methods and can be scaled up proportionally. Detailed Implementation

[0049] The following provides a detailed description of how the present invention is implemented.

[0050] [Definitions of the term]

[0051] The meanings of the terms used in this instruction manual are as follows.

[0052] In this instruction manual, both atmospheric pressure columns and rapid columns can be used as columns for "silica gel column chromatography".

[0053] The "silica gel column chromatography" in this manual refers to normal phase column chromatography.

[0054] In this specification, "crude tafluprost product" refers to the product after post-processing and before purification in the final step of a known tafluprost manufacturing method. Specifically, for example, as shown in the examples described later, the product before purification after the final esterification reaction in the tafluprost manufacturing method described in Patent Document 1 can be cited.

[0055] In this specification, "impurities" include not only residual reaction reagents, residual raw material compounds, reaction byproducts, decomposition products of tafluprost, and similar substances contained in the crude tafluprost product, but also residual organic solvents, residues derived from fillers, bacteria, and all other substances other than tafluprost.

[0056] In this instruction manual, "HPLC analysis" refers to the use of high-performance liquid chromatography (HPLC) for analysis to confirm the presence and content ratio of tafluprost in each component when separating and purifying the crude tafluprost product using silica gel column chromatography.

[0057] In this manual, "filtration" refers to filter filtration. Filtration is performed to remove fine powder from the column packing material (silicone), airborne particles, bacteria, etc.

[0058] In this specification, "polar solvent" refers to 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, tert-butyl methyl ether, and tetrahydrofuran, and alcohols such as 2-propanol and ethanol. Among these, ethyl acetate, tert-butyl methyl ether, 2-propanol, or ethanol are preferred.

[0059] In this specification, "nonpolar solvent" refers to a solvent with a low dielectric constant. Specific examples of nonpolar solvents include chain hydrocarbons such as n-hexane and n-heptane. Among these, n-hexane is preferred.

[0060] In this manual, "external temperature" refers to the temperature outside the reaction vessel or concentration vessel, which is usually the ambient temperature or the temperature of a water bath or hot water bath.

[0061] In this specification, the "concentration limits for residual solvents in pharmaceuticals" are values ​​set forth as permissible levels of residual solvents in pharmaceuticals, researched as part of the International Conference on Harmonization of Drug Licensing (ICH) between Japan, the United States, and the European Union, for the safety of patients. These limits refer to the toxicologically permissible limits for residual solvents. Specific examples of concentration limits for residual solvents in pharmaceuticals, as described in Non-Patent Literature 2, include 290 ppm for n-hexane and 5000 ppm for n-heptane, ethyl acetate, tert-butyl methyl ether, 2-propanol, or ethanol.

[0062] [Purification method of the present invention]

[0063] The purification method of the present invention is characterized by including a step (step 1) of purifying the crude tafluprost product using silica gel column chromatography and collecting the component containing tafluprost using HPLC analysis. Furthermore, in order to suppress the concentration of residual organic solvents below the concentration limit value of the residual solvent guidelines for pharmaceuticals, the purification method of the present invention is characterized by, in addition to the aforementioned step 1, a concentration step (step 2) performed under reduced pressure at 10–55°C, dissolving the residue in a solvent and filtering it (step 3), and a step (step 4) of removing the solvent from the filtrate by distillation under reduced pressure at 10–55°C and a final ultimate vacuum of 5 torr or less (step 4).

[0064] (Process 1)

[0065] This process involves purifying the sample using silica gel column chromatography and then using HPLC to analyze and collect the component containing tafluprost.

[0066] As a packing material for silica gel column chromatography, there are no particular limitations as long as the silica gel can be used in a conventional normal-phase column. The shape of the silica gel can be either pulverized or spherical, with spherical shapes being more preferred. Furthermore, 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. This particle size (d50) is the median particle size of the particle size distribution when a particle size distribution is prepared on a volume basis using laser diffraction scattering particle size distribution determination.

[0067] As the eluent for silica gel column chromatography, there is no particular limitation on the solvent, as long as it can separate tafluprost from impurities in the crude tafluprost product. A mixture of n-hexane and a polar solvent, or a mixture of n-heptane and a polar solvent, is preferred, and a mixture of n-hexane and a polar solvent is even more preferred. Here, the polar solvent is selected from ethyl acetate, tert-butyl methyl ether, 2-propanol, and ethanol, with 2-propanol or ethanol being preferred. The mixing ratio (volume ratio) of n-hexane and a polar solvent, or n-heptane and a polar solvent, can be appropriately set according to the type, shape, and / or particle size of the packing material used. Preferred examples of the eluent include, for example, a mixture of ethyl acetate, tert-butyl methyl ether, 2-propanol, or ethanol with a non-polar solvent (preferably n-hexane or n-heptane), more preferably 2-propanol or ethanol with a non-polar solvent (preferably n-hexane or n-heptane), and particularly preferably a mixture of ethanol and n-hexane. As an eluent, the mixing ratio (volume ratio) when using mixed solvents 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 in which ethanol and n-hexane are mixed in a ratio of 10:90 to 1:99, more preferably a solvent in which ethanol and n-hexane are mixed in a ratio of 6:94 to 2:98, even more preferably a solvent in which ethanol and n-hexane are mixed in a ratio of 5:95 to 3:97, and particularly preferably a solvent in which ethanol and n-hexane are mixed in a ratio of 4:96.

[0068] When purifying and confirming the presence of tafluprost in the separated components using silica gel column chromatography, analytical HPLC is used. Both normal-phase and reversed-phase HPLC can be used, but reversed-phase HPLC, which offers superior separation efficiency, detection sensitivity, and quantification of impurities, is preferred. Specific examples of the column and analytical conditions used in this HPLC analysis can be given by the conditions described in the following examples, but are not limited thereto.

[0069] Typically, when performing purification by silica gel column chromatography, the presence of the target compound in the separated components is confirmed using TLC (thin-layer chromatography) (refer to Lecture 1: Basic Operations I (4th Edition), published November 5, 2003, Maruzen, 5 / 2 / 3 Column Chromatography, pp. 293-296). However, it has been found that in the purification of crude tafluprost, HPLC analysis is significantly superior to conventional TLC analysis in terms of the detection sensitivity of components containing tafluprost and impurities.

[0070] Silica gel column chromatography was used to analyze the elution patterns of impurities in multiple batches of the synthesized crude tafluprost product. The results confirmed that the elution patterns of the impurities remained stable. Considering the elution patterns of the impurities, it is preferable to collect continuous fractions in which the HPLC area percentage of tafluprost in each component is 97% or higher, and more preferably continuous fractions with a percentage of 98% or higher.

[0071] (Process 2)

[0072] This step involves collecting the components containing tafluprost, as confirmed by HPLC analysis, and concentrating them under reduced pressure at 10–50°C.

[0073] The external temperature (temperature of water bath or hot water bath) for collecting and concentrating the tafluprost-containing fraction confirmed by HPLC analysis 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 experimental examples described later, it has been confirmed that tafluprost slowly decomposes over time at temperatures above 60°C. From this perspective, it is also ideal to perform vacuum concentration or solvent removal at the above temperatures.

[0074] (Process 3)

[0075] This step involves dissolving the residue obtained in step 2 in a solvent and then filtering it. Tafluprost is highly viscous, making it difficult to completely remove the solvent by distillation before sterilization and filtration. Therefore, the purification method of this invention is characterized by incorporating a filtration step after step 2.

[0076] As a solvent for dissolving the residue obtained in step 2, examples include the same solvent used as the eluent in the silica gel column chromatography in step 1. A solvent that sufficiently dissolves tafluprost and has a relatively low boiling point is preferred. A mixture of solvents forming an azeotropic composition with a nonpolar solvent is also acceptable. Specifically, ethyl acetate, tert-butyl methyl ether, 2-propanol, or ethanol are preferred, or a mixture of ethyl acetate, tert-butyl methyl ether, 2-propanol, or ethanol with a nonpolar solvent (preferably n-hexane or n-heptane). Ethyl acetate, or a mixture of ethyl acetate with a nonpolar solvent (preferably n-hexane or n-heptane), is more preferred. A mixture of ethyl acetate and n-hexane is particularly preferred. The mixing ratio (volume ratio) when using a mixed solvent is not particularly limited. From the viewpoint of controlling the residual solvent concentration below a reference value, in the case of a mixture of ethyl acetate and n-hexane, a solvent prepared by mixing ethyl acetate and n-hexane at a ratio of 10:1 to 1:10, preferably 4:1 to 1:4, and more preferably 2:1 to 1:2 is particularly preferred.

[0077] As for the filter used in this process, there are no particular limitations as long as it does not swell or dissolve due to the solvent and can remove fine powder of the filler (silica gel) and airborne particles. Examples include glass fiber filters, polypropylene filters, nylon filters, and fluoropolymer filters. Fluoropolymer filters such as polyvinylidene fluoride (PVDF) and polytetrafluoroethylene (PTFE) are preferred, with polytetrafluoroethylene (PTFE) filters being particularly preferred.

[0078] The filter typically has a pore size of 0.5 μm or less, preferably 0.25 μm or less, and particularly preferably 0.22 μm or less when sterilization is also required.

[0079] (Step 4)

[0080] This step involves distilling away the solvent from the filtrate obtained in step 3 above under reduced pressure at 10–55°C and a final ultimate vacuum of less than 5 torr.

[0081] Tafluprost has a very high viscosity, therefore it is necessary to maximize the surface area for solvent evaporation and slowly distill off the solvent from the filtrate while avoiding bumping. Examples of vacuum concentration devices for achieving this purpose include rotary evaporators, centrifugal evaporators, and high-vacuum thin-film evaporators.

[0082] Furthermore, as mentioned above, considering that tafluprost decomposes slowly over time at temperatures above 60°C, the external temperature (temperature of a water bath or hot water bath) for removing the solvent by distillation 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.

[0083] For the depressurization during solvent removal by distillation, the surface area for solvent evaporation should be maximized while gradually increasing the pressure over time to avoid bumping. It is preferable to control the pressure so that the final ultimate vacuum is 5 torr or less (preferably 3 torr or less, more preferably 1 torr or less, and particularly preferably 0.5 torr or less). Furthermore, when releasing the depressurization, to prevent the intrusion of airborne particles and bacteria, it is preferable to use filtered air to restore the pressure to atmospheric pressure.

[0084] The preferred time for distillation removal of the solvent is 10 to 70 hours, more preferably 15 to 60 hours, and particularly preferably 20 to 60 hours.

[0085] By using the purification method of the present invention, the concentration of residual organic solvents can be suppressed below the concentration limit value of the residual solvent guidelines for pharmaceuticals (Non-Patent Literature 2), as stated in these guidelines: "Residual solvents do not contribute to treatment, therefore all residual solvents should be reduced to a level that meets product standards, GMP or other quality benchmarks." This allows for the stable manufacture of high-quality tafluprost that can further meet various stringent quality benchmarks.

[0086] As a solvent for dissolving the eluent and residues used in the aforementioned silica gel column chromatography, it is ideal that the residual solvent concentrations of ethyl acetate, tert-butyl methyl ether, 2-propanol, ethanol, or n-heptane, as exemplified in preferred solvent form, are preferably controlled to be 1000 ppm or less, and particularly preferably 100 ppm or less. Furthermore, it is ideal that the residual solvent concentration of n-hexane is preferably controlled to be 200 ppm or less, and particularly preferably 20 ppm or less. The concentration of the residual solvent can be determined using methods such as gas chromatography (GC).

[0087] This invention also includes a method for manufacturing tafluprost by feeding a crude product of tafluprost manufactured by a known method into the purification method of this invention (including the purification methods of steps 1 to 4 described above). In addition to the aforementioned Patent Document 1 and Non-Patent Document 1, several reported methods for manufacturing tafluprost have been described (e.g., U.S. Patent Application Publication No. 2014 / 0046086; J. Org. Chem. 2016, 81, 10832-844; Molecules, 2017, 22, 217, 1-16; Org. Lett. 2020, 22, 2991-2994, etc.), which can also be included in this invention by combining them with the purification method of this invention.

[0088] Specific examples of crude tafluprost products used in this invention include, for example, crude tafluprost products obtained by deprotection reaction of tafluprost protected by hydroxyl groups, crude tafluprost products obtained by esterification of tafluprost acid salts, and crude tafluprost products obtained by esterification of tafluprost acid. The crude tafluprost product obtained by esterification of tafluprost acid may be suitably used.

[0089] The following are specific features of the purification method of the present invention.

[0090] (A) When separating and purifying crude tafluprost using silica gel column chromatography, the fraction containing tafluprost is collected by HPLC analysis (preferably reversed-phase HPLC analysis), thereby minimizing the contamination of impurities.

[0091] (B) The solvent is removed by distillation under low temperature and high vacuum conditions, which can suppress the decomposition of tafluprost, which is unstable at high temperature. In addition, the concentration of residual organic solvent can be suppressed below the concentration limit of the residual solvent guidelines for pharmaceuticals.

[0092] (C) A filter is added midway through the filtration process, thereby providing high-purity tafluprost that can be used directly as a raw material for pharmaceuticals after the solvent is removed by distillation.

[0093] (D) The purification method of the present invention can also be applied to crude tafluprost obtained by any method in the known methods for manufacturing tafluprost. In addition, it can be easily scaled up, thus providing a simple and efficient purification method.

[0094] The purification method of the present invention is as described in (A) above. When the purpose is to improve the purity of tafluprost, it only includes step 1 above. Steps 2 to 4 can also be combined with step 1 as needed.

[0095] Example

[0096] The following examples, embodiments, and test examples illustrate the present invention in detail, but the present invention is not limited to these.

[0097] % in yields represents mol%, while in others it represents mass % unless otherwise specified. Ratios shown in mixed solvents represent volume ratios unless otherwise specified. Additionally, room temperature unless otherwise specified represents a temperature of 15–30°C. The following... 1 ¹H-NMR values ​​were determined using an ECP400 (400MHz) nuclear magnetic resonance spectrometer manufactured by NEC Corporation. HPLC was performed using a Shimadzu LC-10ADvp or LC-10A. GC was performed using a Shimadzu GC-2014ATF.

[0098] Reference Example 1: Synthesis of Tafluprost Acid

[0099]

[0100] Under a nitrogen atmosphere, tetrahydrofuran (1200 g) was added to (1S,5R,6R,7R)-6-[(1E)-3,3-difluoro-4-phenoxy-1-butenyl]-7-hydroxy-2-oxabicyclo[3.3.0]octane-3-one (280 g) and dissolved. 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. Extraction was performed with ethyl acetate. The combined organic layers were washed with water, and the filtrate was concentrated under reduced pressure to obtain the reduced product (284 g). Under a nitrogen atmosphere, tetrahydrofuran (5030 g) was added to 4-carboxybutyltriphenylphosphonium bromide (1523 g), and sodium bis(trimethylsilyl)amide solution (1 M tetrahydrofuran solution) (6684 mL) was added dropwise. The mixture was stirred for more than 1 hour. The above-mentioned reduced form (286 g) dissolved in tetrahydrofuran (970 g) was added dropwise at 0 °C and stirred for 3 hours. Water was added to the reaction solution, and the mixture was extracted with ethyl acetate. After the aqueous layer was acidified, it was extracted with ethyl acetate, concentrated under reduced pressure, and the insoluble matter was filtered off. The solution was purified by silica gel column chromatography (hexane / ethyl acetate = 1 / 1 to 1 / 3) to obtain tafluprost acid (222 g).

[0101] 1 H NMR(CDCl3)δ1.60(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),4.0 3(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).

[0102] Reference Example 2: Synthesis of crude tafluprost product

[0103]

[0104] Under a nitrogen atmosphere, 120 g of tafluprost acid obtained in Reference Example 1 was added to a 5 L flask and dissolved in acetone (600 mL) while stirring. The mixture was cooled to 5 °C, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (160 mL) was added dropwise while maintaining the temperature below 5 °C. Then, 2-iodopropane (146 mL) was added dropwise while maintaining the temperature below 5 °C. The mixture was stirred at 30 °C until the conversion reached over 95%. Ethyl acetate (1800 mL) and a 5% citric acid aqueous solution (900 mL) were added to the reaction mixture, and the mixture was separated. The organic layer was washed with a 5% citric acid aqueous solution (900 mL, once), a 5% sodium bicarbonate aqueous solution (900 mL, twice), and purified water (900 mL, once). The solvent was removed by distillation under reduced pressure at a temperature below 40°C, thereby obtaining crude tafluprost (132 g, yield 100%; HPLC purity: 95.5%, α-chain trans isomer content: 0.73%).

[0105] Example 1

[0106] (Step 1) A slurry prepared from silica gel (AGC-SITECH CO.,LTD, MSGEL D50-120A, particle size (d50): 50 μm, spherical, 50 g) and n-hexane / ethanol = 96 / 4 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 packed onto the column, eluted with n-hexane / ethanol = 96 / 4. The fractionated components were analyzed by HPLC, and the fractions containing tafluprost were collected. As the tafluprost-containing fraction, at least the fraction with an area percentage of tafluprost of 98% or more (calculated by subtracting the solvent peak) was collected.

[0107] (Step 2) The collected components containing tafluprost are concentrated under reduced pressure at 35℃~40℃.

[0108] (Step 3) Dissolve the residue in n-hexane / ethyl acetate = 3 / 2, filter with a membrane filter (pore size: 0.2μm), and wash with n-hexane / ethyl acetate = 3 / 2.

[0109] (Step 4) Under reduced pressure conditions of 35℃~40℃ and a final ultimate vacuum of less than 1 torr, the solvent of the filtrate is removed by distillation for 24 hours to obtain tafluprost (colorless to pale yellow viscous liquid, yield: 82%, HPLC purity: 99.5%, α-chain trans isomer content: 0.25%).

[0110] The residual solvent concentrations of tafluprost obtained by GC analysis were 0 ppm for hexane, 0 ppm for ethyl acetate, and 0 ppm for ethanol.

[0111] 1 1H 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), 7.3 (dd, J = 8.8, 7.3 Hz, 2H);

[0112] 19 19F NMR (CDCl3) δ -102.8 (dq, 2 J FF = 255.6 Hz), -103.6 (dq, 2 J FF = 255.6 Hz).

[0113] <HPLC (reverse phase) analysis conditions>

[0114] Column: YMC-Pack ODS-AM (5 μm, 6.0 × 150 mm)

[0115] Temperature: room temperature

[0116] Flow rate: 1 mL / minute

[0117] Detection wavelength: 220 nm

[0118] Eluent: (Solution A) 1% triethylamine-phosphate buffer (pH 6.3),

[0119] (Solution B) acetonitrile <000026*>Gradient conditions: A / B = 50 / 50 (0 - ⁴⁵ minutes), A / B = 25 / 75 (45 - 70 minutes) <00002*2><GC analysis conditions>

[0122] Note: There seems to be a formatting issue in the original text where some tags like <000026*> and <00002*2> are not complete. I've translated them as best as possible while maintaining the integrity of the existing tags. If these are incorrect, please provide the correct tags for a more accurate translation.Column: G-column G300 (1.2mm ID, 40m)

[0123] Column temperature: 50℃

[0124] Detection: Hydrogen flame ionization detector

[0125] Carrier gas: Helium

[0126] Injector temperature: 160℃

[0127] Detector temperature: 160℃

[0128] Examples 2-6

[0129] To investigate the effect of the type of silica used in silica column chromatography on the purity and yield of tafluoroprostol, the following experiments were performed in the same manner as in Example 1.

[0130] The results of silica gel column chromatography using crude tafluprost (1 g; HPLC purity: 95.5%) and silica gel (50 g) are shown in Table 1 below. Reversed-phase HPLC analysis was performed under the same conditions as described above.

[0131] [Table 1]

[0132]

[0133] Comparative Example 1

[0134] Silica gel column chromatography was performed using crude tafluprost (1 g; HPLC purity: 95.5%), silica gel (50 g) as used in Example 2, and eluent. Except for analysis of the fractionated components using TLC instead of HPLC, the fractions containing only tafluprost were visually collected, as in Example 1. The resulting tafluprost HPLC purity was 97.3%, which did not meet the quality level required for a purified pharmaceutical product (limit: 98%).

[0135] When the silica gel used had a particle size (d50) of 65 μm or less, although the shapes differed, tafluprost with a purity exceeding 98% was obtained in Examples 1-6. It was found that using spherical silica gel resulted in tafluprost with particularly high purity in high yield.

[0136] Example 7 (Scaled-up Study)

[0137] (Process 1)

[0138] Similar to step 1 of Example 1, a slurry prepared from silica gel (AGC-SITECH CO.,LTD, MSGEL 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 packed onto the column, eluted with n-hexane / ethanol = 96 / 4. The fractionated components were analyzed by HPLC, and the fractions containing tafluprost were collected. As the tafluprost-containing fraction, at least the fraction with an area percentage of tafluprost of 98% or more (calculated by subtracting the solvent peak) was collected.

[0139] (Processes 2-4)

[0140] The fraction containing tafluprost collected in step 1 was concentrated under reduced pressure at 29℃~35℃ (step 2).

[0141] Dissolve the residue in a 3 / 2 mixture of hexane and ethyl acetate, filter using a membrane filter (pore size: 0.2 μm), and wash with a 3 / 2 mixture of hexane and ethyl acetate (step 3).

[0142] The solvent in the filtrate was removed by distillation for 26 hours under reduced pressure conditions of 32℃~36℃ and a final ultimate vacuum of 0.30 torr (step 4), thereby obtaining tafluprost (colorless to pale yellow viscous liquid, yield: 86%, HPLC purity: 99.7%, α-chain trans isomer content: 0.24%, microbial content: less than 10 cfu / 0.1g).

[0143] The residual solvent concentrations of tafluprost obtained by GC analysis were 0 ppm for hexane, 0 ppm for ethyl acetate, and 0 ppm for ethanol.

[0144] Example 8

[0145] The tafluprost-containing fraction, which was purified and collected in the same manner as step 1 of Example 7, was concentrated under reduced pressure under the same conditions as step 2 of Example 7.

[0146] The resulting residue was dissolved in ethyl acetate, filtered through a membrane filter (pore size: 0.2 μm), and washed with ethyl acetate (step 3).

[0147] The solvent in the filtrate was removed by distillation for 27 hours under reduced pressure conditions of 23℃~37℃ and a final ultimate vacuum of 0.26 torr (step 4), thereby obtaining tafluprost (colorless to pale yellow viscous liquid, yield: 85%, HPLC purity: 99.7%, α-chain trans isomer content: 0.27%, microbial content: less than 10 cfu / 0.1g).

[0148] The residual solvent concentrations of tafluprost obtained by GC analysis were 0 ppm for hexane, 0 ppm for ethyl acetate, and 0 ppm for ethanol.

[0149] Example 9

[0150] The tafluprost-containing fraction, purified and collected in the same manner as step 1 of Example 7, was concentrated under reduced pressure under the same conditions as 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).

[0151] The solvent in the filtrate was removed by distillation for 3 hours under reduced pressure conditions of 20℃~36℃ and a final ultimate vacuum of 2.6 torr (step 4), thereby obtaining tafluprost (colorless to pale yellow viscous liquid, yield: 75%, HPLC purity: 99.4%, α-chain trans isomer content: 0.30%, microbial content: less than 10 cfu / 0.1g).

[0152] The residual solvent concentrations of tafluprost obtained by GC analysis were 36 ppm for hexane, 4803 ppm for ethyl acetate, and 66 ppm for ethanol.

[0153] Example 10

[0154] The tafluprost-containing fraction, purified and collected in the same manner as step 1 of Example 7, was concentrated under reduced pressure under the same conditions as 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).

[0155] The solvent in the filtrate was removed by distillation for 5 hours under reduced pressure conditions of 34℃~37℃ and a final ultimate vacuum of 2.1 torr (step 4), thereby obtaining tafluprost (colorless to pale yellow viscous liquid, yield: 78%, HPLC purity: 99.5%, α-chain trans isomer content: 0.32%, microbial content: less than 10 cfu / 0.1g).

[0156] The residual solvent concentrations of tafluprost obtained by GC analysis were 2 ppm for hexane, 785 ppm for ethyl acetate, and 0 ppm for ethanol.

[0157] Example 11

[0158] The tafluprost-containing fraction, purified and collected in the same manner as step 1 of Example 7, was concentrated under reduced pressure under the same conditions as 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).

[0159] The solvent in the filtrate was removed by distillation for 8 hours under reduced pressure conditions of 32℃~36℃ and a final ultimate vacuum of 0.92 torr (step 4), thereby obtaining tafluprost (colorless to pale yellow viscous liquid, yield: 82%, HPLC purity: 99.6%, α-chain trans isomer content: 0.26%, microbial content: less than 10 cfu / 0.1g).

[0160] The residual solvent concentrations of tafluprost obtained by GC analysis were 0 ppm for hexane, 86 ppm for ethyl acetate, and 0 ppm for ethanol.

[0161] Example 12

[0162] The tafluprost-containing fraction, purified and collected in the same manner as step 1 of Example 7, was concentrated under reduced pressure under the same conditions as 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).

[0163] The solvent in the filtrate was removed by distillation for 50 hours under reduced pressure conditions of 35℃~39℃ and a final ultimate vacuum of 0.09 torr (step 4), thereby obtaining tafluprost (colorless to pale yellow viscous liquid, yield: 80%, HPLC purity: 99.5%, α-chain trans isomer content: 0.26%, microbial content: less than 10 cfu / 0.1g).

[0164] The residual solvent concentrations of tafluprost obtained by GC analysis were 0 ppm for hexane, 0 ppm for ethyl acetate, and 0 ppm for ethanol.

[0165] Example 13

[0166] The tafluprost-containing fraction, purified and collected in the same manner as step 1 of Example 7, was concentrated under reduced pressure under the same conditions as 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).

[0167] Under reduced pressure conditions of 36°C to 45°C and a final ultimate vacuum of 0.24 torr, the solvent of the filtrate was distilled off for 60 hours (Step 4), whereby tafluprost (colorless to pale yellow viscous liquid, yield: 79%, HPLC purity: 99.5%, content of α-chain trans isomer: 0.26%, microbial content: 10 cfu / 0.1 g or less) was obtained.

[0168] The residual solvent concentrations of tafluprost obtained by GC analysis were 0 ppm for n-hexane, 0 ppm for ethyl acetate, and 0 ppm for ethanol.

[0169] The results of the conditions for distilling off the solvent in Examples 7 to 13 above, the yield, purity, and content of α-chain trans isomer of tafluprost, and the residual solvent concentrations are shown in Table 2 below.

[0170] [Table 2]

[0171]

[0172] From Table 2, it can be seen that in Examples 7 to 13, tafluprost was obtained with good purity and high yield, and the concentration of residual organic solvents could be suppressed below the concentration limit value of the residual solvent standard for pharmaceuticals. The purification method of the present invention is a highly versatile purification method that can withstand scale-up. On the other hand, it was confirmed that in the comparative example where concentration under reduced pressure was carried out with a final ultimate vacuum of 8 torr, the concentration of residual organic solvents exceeded the concentration limit value of the residual solvent standard for pharmaceuticals.

[0173] Test Example

[0174] Study on the thermal stability of tafluprost

[0175] Approximately 120 mg of tafluprost obtained in Example 1 was separately measured in a glass container and stored in a constant temperature bath at 40°C, and quantified by reversed-phase HPLC analysis to study the change in the content of tafluprost over time. Similarly, approximately 20 mg of tafluprost was separately measured in a glass container and stored in a constant temperature bath at 60°C or 80°C to study the change in the content of tafluprost over time.

[0176] <HPLC (reversed-phase) analysis conditions>

[0177] Column: YMC-Pack ProC18 AS-303 (5 μm, 4.6 × 250 mm)

[0178] Temperature: 50°C

[0179] Flow rate: 1 mL / min

[0180] Detection wavelength: 220 nm

[0181] Elution buffer: (Solution A) 10 mmol / L sodium phosphate buffer (pH 6.9),

[0182] (Liquid B) Acetonitrile

[0183] Gradient conditions: A / B = 50 / 50 (0–45 minutes), A / B = 25 / 75 (45–70 minutes)

[0184] The results of the study on the time-dependent changes in tafluprost content at various temperatures are shown in Tables 3-5 below.

[0185] [Table 3]

[0186] Stability of tafluprost at 40°C

[0187] Time (month) 0 3 6 Tafluprost content (%) 101.3 99.8 99.4

[0188] [Table 4]

[0189] Stability of tafluprost at 60°C

[0190] Time (days) 0 3 7 14 Tafluprost content (%) 99.0 98.9 98.5 95.4

[0191] [Table 5]

[0192] Stability of tafluprost at 80°C

[0193] Time (days) 0 1 3 7 Tafluprost content (%) 99.5 99.1 94.4 86.7

[0194] Based on the results in Tables 3-5, it can be confirmed that tafluprost will slowly decompose over time at temperatures above 60°C, even during storage periods of several days to about two weeks, especially at 80°C. However, it was found that it remained stable at 40°C even after 6 months.

[0195] Based on the above results, it can be seen that in the purification method of the present invention, the concentration under reduced pressure and the removal of solvent by distillation are carried out at a temperature below 55°C (particularly preferably below 45°C), thereby suppressing the mixing of impurities (similar substances) derived from the decomposition of tafluprost.

[0196] Industrial availability

[0197] The purification method of this invention, in the final step of the manufacture of tafluprost, when separating and purifying the crude tafluprost product using silica gel column chromatography, collects the tafluprost-containing components using HPLC analysis, thereby minimizing the contamination of impurities. Furthermore, under low-temperature and high-vacuum reduced-pressure conditions, the solvent is removed by distillation over a period of time, thereby suppressing the concentration of residual organic solvents below the concentration limits of pharmaceutical residual solvent guidelines and inhibiting the decomposition of tafluprost, which is unstable at high temperatures. Moreover, a filtration step is incorporated midway through the process, thereby removing fine silica gel powder, airborne particles, and bacteria. Therefore, it has the advantage of providing high-purity tafluprost that can be directly used as a pharmaceutical raw material, simply and efficiently after solvent distillation removal. In addition, the purification method of this invention can be widely applied to crude tafluprost products manufactured by known methods and is a highly versatile method that can withstand scale-up.

Claims

1. A method for purifying tafluprost, comprising: The process of purifying the crude tafluprost product using spherical silica gel column chromatography and collecting the fraction containing tafluprost by HPLC analysis was described. The HPLC analysis was reversed-phase HPLC analysis. The purification method further includes: a step of concentrating the tafluprost-containing fraction collected by HPLC analysis under reduced pressure at 10–55°C; a step of dissolving the residue in a solvent and filtering it; and a step of removing the solvent from the filtrate by distillation at 10–55°C and a final ultimate vacuum of less than 5 torr. The silica gel used in silica gel column chromatography has a particle size d50 of 20–65 μm. The eluent for silica gel column chromatography is a mixture of n-hexane and a polar solvent, or a mixture of n-heptane and a polar solvent. The polar solvents are ethyl acetate, tert-butyl methyl ether, 2-propanol, or ethanol. The purified product remained stable for 6 months at 40°C.

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

3. The purification method according to claim 1 or 2, wherein, The components contain more than 98% tafluprost.

4. The purification method according to claim 1 or 2, wherein, Filtration is performed using a filter with a pore size of less than 0.5 μm.

5. The purification method according to claim 1 or 2, wherein, The solvent used to dissolve the residue is ethyl acetate, tert-butyl methyl ether, 2-propanol or ethanol, or a mixture of ethyl acetate, tert-butyl methyl ether, 2-propanol or ethanol and a nonpolar solvent.

6. The purification method according to claim 5, wherein, The solvent used to dissolve the residue is ethyl acetate, or a mixture of ethyl acetate and a nonpolar solvent.

7. The purification method according to claim 5, wherein, The nonpolar solvent is n-hexane or n-heptane.

8. The purification method according to claim 6, wherein, The nonpolar solvent is n-hexane or n-heptane.

9. The purification method according to claim 1 or 2, wherein, The ultimate vacuum level is below 1 torr.

10. The purification method according to claim 1 or 2, wherein, After the solvent removal process by distillation, the residual solvent concentration of n-hexane in the filtrate is below 290 ppm, and the residual solvent concentrations of n-heptane, ethyl acetate, tert-butyl methyl ether, 2-propanol, or ethanol are below 5000 ppm.

11. A method for manufacturing tafluprost, comprising the step of feeding crude tafluprost into the purification method according to any one of claims 1 to 10.

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