CN115385793A

CN115385793A - Purification method of tafluprost

Info

Publication number: CN115385793A
Application number: CN202210974156.4A
Authority: CN
Inventors: 小西克彦; 森信明; 松村靖
Original assignee: Asahi Glass Co Ltd
Current assignee: AGC Inc
Priority date: 2021-01-27
Filing date: 2021-01-27
Publication date: 2022-11-25
Also published as: JP7192999B1; CN112851510A; CN115322094A; KR20230129019A; JPWO2022162967A1; CN112851510B; WO2022162967A1; KR102552337B1; JP2023054794A; CN115322095A; CN115448840A; KR20230012069A

Abstract

[ problem ] to provide a simple and efficient method for purifying tafluprost, which can be scaled up. [ solution ] the present invention relates to a method for purifying tafluprost, which comprises a step of purifying a crude product of tafluprost by silica gel column chromatography and collecting a fraction containing tafluprost by HPLC analysis. The present invention also relates to a method for producing tafluprost, which comprises the method for purifying tafluprost.

Description

Purification method of tafluprost

The present application is a divisional application of the invention entitled "method for purifying tafluprost" on the filing date of 2021/27, 202110111991.0.

Technical Field

The present invention relates to a novel purification process for tafluprost.

Background

Tafluprost is represented by the following formula:

the chemical name is (5Z) -7- (1R, 2R,3R, 5S) -2- [ (1E) -3, 3-difluoro-4-phenoxy-1-butenyl]-3, 5-dihydroxycyclopentyl]-isopropyl 5-heptenoate having a viscosity of 2440mPa · s at 25 ℃ and being a very viscous difluoroprostaglandin F _2α And (3) derivatives thereof. Tafluprost has an unstable chemical structure containing 2 double bonds, an unsaturated fatty acid ester site, and 4 asymmetric centers, and has a structure in which a hydroxyl group and a hydrogen atom at the C15 position present in other prostaglandin derivatives are substituted with 2 fluorine atoms, and therefore, among prostaglandin derivatives, it has a remarkable specific property of high lipid solubility, and has a property of being decomposed at high temperatures although it has high chemical stability. Further, tafluprost has a potent ocular hypotensive effect and is used as an eye drop for the treatment of glaucoma and ocular hypertension (patent document 1). Patent document 1 describes difluoroprostaglandin F containing tafluprost _2α A method for producing the derivative is also described in non-patent document 1.

The production method described in patent document 1 includes a Wittig reaction step, and thus it is difficult to avoid mixing of an α -chain trans isomer in the final product. Among the production methods described in patent document 1, a method of separation and purification by High Performance Liquid Chromatography (HPLC) for fractionation has been reported as a method of removing impurities including an α -chain trans isomer (patent document 2). However, tafluprost, and a carboxylic acid compound represented by the following formula (I) as a synthesis precursor thereof (hereinafter referred to as "tafluprost acid"):

all of them are liquid compounds having very high viscosity, and therefore, purification is difficult, and in the purification method of tafluprost described in patent document 2, a large amount of organic solvent is used, and therefore, high cost is required, and it is difficult to suppress the concentration of the residual organic solvent to be equal to or lower than the concentration limit value of the residual solvent guideline for pharmaceuticals (non-patent document 2). The column of HPLC for fractionation is generally expensive and is generally used repeatedly, and therefore, there is a problem that the number of theoretical plates is reduced due to accumulated impurities, contamination of decomposition products, and deterioration of the column, and in order to reduce the risk caused by these problems, it is often necessary to perform washing using a large amount of organic solvent, and complicated validation of validity in relation to confirmation thereof, confirmation of the separation performance of the column, and the like, and therefore, the column is not practical as a method for producing a pharmaceutical.

On the other hand, a method of reducing the incorporation of impurities such as α -chain trans-isomer through an organic amine salt of tafluprost acid (patent documents 3 and 4) or a metal salt (patent document 5) has been reported, but with the addition of a salt formation step and a step of dissociating from the salt, there is an increased possibility of byproducts, dehydrates, other impurities, and the like, which are generated by dimerization or the like by condensation with organic amine or self-condensation. In addition, many organic amines and metals are also likely to have toxicity, mutagenicity, and the like, and particularly, they have a problem in safety when used as a purification method at a stage close to the final step of a pharmaceutical product.

Further, a method for producing tafluprost has been reported in which incorporation of an α -chain trans isomer is prevented by a macrolide ring formation step and a macrolide ring opening step (patent document 6). However, the above production method has a long production process and a low yield, and therefore has low practicability.

Documents of the prior art

Patent literature

Patent document 1: description of European patent application publication No. 850926

Patent document 2: U.S. patent application publication No. 2014/0051882

Patent document 3: international publication No. 2013/118058

Patent document 4: international publication No. 2016/090461

Patent document 5: chinese patent application publication No. 108299192 specification

Patent document 6: japanese patent laid-open No. 2015-36382

Non-patent literature

Non-patent document 1: tetrahedron lett, 2004,45,1527-1529

Non-patent document 2: pharmaceutical examination No. 307, housheng province drug safety administration review management class length notice (average 10 years, 3 months and 30 days), guidelines on residual solvents of drugs

Disclosure of Invention

Problems to be solved by the invention

The purpose of the present invention is to provide a method for purifying tafluprost, which can purify tafluprost, which is a liquid compound having a very high viscosity, simply and at low cost to such a degree of purity that it can be provided directly as a pharmaceutical drug substance, and which can also be scaled up (scale up).

Means for solving the problems

The present inventors have conducted extensive studies to solve the above problems and as a result have found that: in the method for producing tafluprost, high-purity tafluprost can be obtained by a purification method including a step of purifying a crude product of tafluprost obtained in the step of esterifying tafluprost acid by silica gel column chromatography and collecting a fraction containing tafluprost by HPLC analysis (hereinafter, this purification method may be referred to as "the purification method of the present invention"). The present invention has been completed by carrying out a purification method comprising a step of concentrating a fraction containing tafluprost collected by HPLC analysis under reduced pressure at 10 to 55 ℃, a step of dissolving the residue in a solvent and filtering the solution, and a step of distilling off the solvent from the filtrate under reduced pressure at 10 to 55 ℃ and a final ultimate vacuum degree of 5torr or less (hereinafter, the purification method comprising all the steps may be referred to as "the purification method of the present invention").

Namely, the present invention is as follows.

[1] A method of purifying tafluprost, comprising: a step of purifying the crude product of tafluprost by silica gel column chromatography and collecting a fraction containing tafluprost by HPLC analysis.

[2] The method for purifying tafluprost according to the above [1], further comprising: concentrating under reduced pressure the fraction containing tafluprost analyzed and collected by HPLC at 10-55 deg.C; then dissolving the residue in a solvent and filtering; and distilling off the solvent from the filtrate under reduced pressure at 10-55 ℃ and a final ultimate vacuum degree of 5torr or less.

[3] The purification method according to the above [1] or [2], wherein the silica gel used for the silica gel column chromatography has a particle size (d 50) of 20 to 70 μm.

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

[5] The purification method according to any one of the above [1] to [4], 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.

[6] The purification method according to the above [5], wherein the eluent is a mixed solvent of n-hexane and a polar solvent.

[7] The purification process according to the above [5] or [6], wherein the polar solvent is ethyl acetate, tert-butyl methyl ether, 2-propanol, or ethanol.

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

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

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

[11] The purification method according to any one of the above [2] to [10], wherein the solvent for dissolving the residue is ethyl acetate, tert-butyl methyl ether, 2-propanol or ethanol, or a mixed solvent of ethyl acetate, tert-butyl methyl ether, 2-propanol or ethanol and a nonpolar solvent.

[12] The purification process according to the above [11], wherein the solvent for dissolving the residue is ethyl acetate or a mixed solvent of ethyl acetate and a nonpolar solvent.

[13] The purification method according to the above [11] or [12], wherein the nonpolar solvent is n-hexane or n-heptane.

[14] The purification method according to any one of the above [2] to [13], wherein a final ultimate vacuum degree is 1torr or less.

[15] The purification method according to any one of the above [2] to [14], wherein the residual solvent concentration of n-hexane after the step of distilling off the solvent of the filtrate is 290ppm or less, and the residual solvent concentrations of n-heptane, ethyl acetate, tert-butyl methyl ether, 2-propanol or ethanol are 5000ppm or less, respectively.

[16] A method for producing tafluprost, which comprises subjecting a crude product of tafluprost to the purification method according to any one of the above [1] to [15 ].

[17] Tafluprost obtained by the production method according to [16 ].

[18] A pharmaceutical product comprising the tafluprost of [17] as an active ingredient.

[19] A pharmaceutical product for preventing or treating an ocular disease, which comprises the tafluprost of [17] as an active ingredient.

[20] The pharmaceutical product according to the above [19], wherein the ocular disease is glaucoma or ocular hypertension.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the purification method of the present invention, when a crude product of tafluprost is separated and purified by silica gel column chromatography in the final step of production of tafluprost, the components containing tafluprost are collected by HPLC analysis, whereby contamination of impurities can be minimized. Further, by distilling off the solvent with time under reduced pressure conditions of low temperature and high vacuum degree, the concentration of the residual organic solvent can be suppressed to a value not more than the concentration limit value of the residual solvent rule of the pharmaceutical product, and the decomposition of tafluprost which is unstable at high temperature can be suppressed. Furthermore, since the fine powder of silica gel, airborne particles in the air, and bacteria can be removed by incorporating a filter filtration step in the middle, it is possible to easily and efficiently provide high-purity tafluprost that can be used as a drug substance of a pharmaceutical product directly after the solvent is distilled off. The purification method of the present invention can be widely applied to crude products of tafluprost produced by known methods and can withstand scale-up.

Detailed Description

Embodiments of the present invention will be described in detail below.

[ definition of terms ]

The terms used in the present specification have the following meanings.

In the present specification, as a column used for the "silica gel column chromatography", either a normal pressure column or a flash column can be used.

The "silica gel column chromatography" in the present specification is a column chromatography of a positive phase system.

In the present specification, the term "crude product of tafluprost" refers to a product after post-treatment of a reaction in a final step in a known production method of tafluprost and before purification. Specifically, for example, as shown in examples described later, a product before purification of a final esterification reaction in the method for producing tafluprost described in patent document 1 can be mentioned.

In the present specification, the term "impurities" includes all substances other than tafluprost, such as residual organic solvents, residues derived from fillers, and bacteria, in addition to residual reaction reagents, residual raw material compounds, reaction by-products, and products of decomposition of tafluprost contained in the crude product of tafluprost.

In the present specification, "HPLC analysis" refers to confirmation of the presence or absence and content ratio of tafluprost in each fraction by high performance liquid chromatography (high performance liquid chromatography) for analysis when a crude product of tafluprost is separated and purified by silica gel column chromatography.

In the present specification, "filtration" refers to filter filtration. The filtration is performed for the purpose of removing fine powder of a column packing (silica gel), floating particles in the air, bacteria, and the like.

In the present specification, the "polar solvent" refers to a solvent having a large dielectric constant. Specific examples of the polar solvent 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 them, ethyl acetate, t-butyl methyl ether, 2-propanol or ethanol is preferable.

In the present specification, the term "nonpolar solvent" refers to a solvent having a small dielectric constant. Specific examples of the nonpolar solvent include chain hydrocarbons such as n-hexane and n-heptane. Among them, n-hexane is preferable.

In the present specification, "external temperature" refers to 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 hot water bath.

In the present specification, the "concentration limit value of the criterion for the residual solvent of a pharmaceutical product" is a value which is studied as one of the subjects of the international conference on approval review of pharmaceutical products in the japanese and american EU three parties (ICH) and which defines the allowable amount of the residual solvent in the pharmaceutical product for the safety of patients, and refers to a limit value which can be tolerated in the toxicology of the residual solvent. Specific examples of the concentration limit value of the residual solvent in the pharmaceutical are, for example, 290ppm for n-hexane and 5000ppm for n-heptane, ethyl acetate, tert-butyl methyl ether, 2-propanol or ethanol, as described in non-patent document 2.

[ purification method of the present invention ]

The purification method of the present invention is characterized by comprising a step of purifying a crude tafluprost product by silica gel column chromatography and collecting a fraction containing tafluprost by HPLC analysis (step 1). In addition, in order to suppress the concentration of the residual organic solvent to be less than or equal to the limit value of the concentration of the residual solvent in the pharmaceutical, the purification method of the present invention is characterized by comprising, in addition to the step 1, a concentration step (step 2) of reducing the pressure at 10 to 55 ℃, a step (step 3) of dissolving the residue in the solvent and filtering the solution, and a step (step 4) of distilling off the solvent from the filtrate under a reduced pressure at 10 to 55 ℃ and a final ultimate vacuum degree of 5torr or less.

(step 1)

This step is a step of purifying the fraction by silica gel column chromatography and collecting the fraction containing tafluprost by HPLC analysis.

The filler used for silica gel column chromatography is not particularly limited as long as it is silica gel that can be used in a normal phase column. The silica gel may be in the form of a powder or a sphere, and a sphere is more preferable. The particle diameter (d 50) of the silica gel is not particularly limited, but is preferably 20 to 70 μm, more preferably 40 to 65 μm, and particularly preferably 45 to 60 μm. The particle diameter (d 50) is a median diameter of a particle size distribution obtained by measuring a particle size distribution by laser diffraction scattering method and preparing the particle size distribution on a volume basis.

The eluent used for the silica gel column chromatography is not particularly limited as long as it is a solvent capable of separating tafluprost from impurities in a crude product of tafluprost, and 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, and among them, 2-propanol or ethanol is preferable. The mixing ratio (volume ratio) of n-hexane to the polar solvent or n-heptane to the polar solvent can be appropriately set according to the kind, shape and/or particle size of the filler used. Preferable specific examples of the eluent include ethyl acetate, t-butyl methyl ether, 2-propanol or a mixed solvent of ethanol and a nonpolar solvent (preferably n-hexane or n-heptane), more preferably a mixed solvent of 2-propanol or ethanol and a nonpolar solvent (preferably n-hexane or n-heptane), and particularly preferably a mixed solvent of ethanol and n-hexane. The mixing ratio (volume ratio) when a mixed solvent is used as the eluent is not particularly limited, but from the viewpoint of controlling the residual solvent concentration to be equal to or less than a reference value, a mixed solvent of ethanol and n-hexane is preferably used in a range of 10: 90-1: 99 mixing ethanol: the solvent derived from n-hexane is more preferably a solvent in which the molar ratio of 6: 94-2: 98, preferably 5: 95-3: 97, particularly preferably in a ratio of 4:96 to obtain a mixed solvent.

When the presence or absence of tafluprost in the separated fractions was confirmed by silica gel column chromatography, HPLC for analysis was used. As the HPLC, both normal phase HPLC and reverse phase HPLC may be used, but reverse phase HPLC excellent in separation efficiency of impurities, detection sensitivity, quantitativity, and the like is more preferable. Specific examples of the column and analysis conditions used in the HPLC analysis include the conditions described in the following examples, but are not limited thereto.

In general, in the case of silica gel column chromatography, the presence or absence of a target substance in the separated fractions was confirmed by TLC (thin layer chromatography) (see basic procedure I (4 th edition) of experimental chemistry lecture 1, issued on 2 years, 11 months, 5 days, and in pellet form, 5/2/3 column chromatography, p.293-296), but it was found that HPLC analysis was significantly superior to conventional TLC analysis in the detection sensitivity of a fraction containing tafluprost and impurities in the purification of a crude tafluprost product.

Silica gel column chromatography was performed on a plurality of batches of the synthesized crude tafluprost product, and the elution pattern of impurities was analyzed by HPLC analysis, and as a result, it was confirmed that the elution pattern of impurities was always stable. As can be seen from consideration of the impurity elution pattern, it is preferable to collect a continuous fraction having an HPLC area percentage of tafluprost of 97% or more, and it is particularly preferable to collect 98% or more of the continuous fraction for each fraction.

(step 2)

This step is a step of collecting a fraction containing tafluprost which has been analyzed and confirmed by HPLC, and concentrating the collected fraction under reduced pressure at 10 to 50 ℃.

The external temperature (temperature of water bath or hot water bath) when the fraction containing tafluprost confirmed by HPLC analysis is collected and concentrated under reduced pressure is preferably 10 to 55 ℃, more preferably 15 to 50 ℃, and particularly preferably 20 to 45 ℃. As shown in the test examples described below, it was confirmed that tafluprost gradually decomposes at a temperature of 60 ℃ or higher with the passage of time, and from this point of view, it is also preferable to perform concentration under reduced pressure at the above-mentioned temperature or to distill off the solvent.

(step 3)

This step is a step of dissolving the residue obtained in the step 2 in a solvent and filtering the solution. Tafluprost is very viscous and therefore it is difficult to perform sterile filtration after complete distillation of the solvent. Therefore, the purification method of the present invention is characterized by including a filtration step after step 2.

The solvent used for dissolving the residue obtained in step 2 may be the same solvent as the eluent used in the silica gel column chromatography in step 1, preferably a solvent having a relatively low boiling point and sufficiently dissolving tafluprost, or a mixed solvent with a nonpolar solvent having an azeotropic composition. Specifically, ethyl acetate, tert-butyl methyl ether, 2-propanol or ethanol is preferable, or a mixed solvent of ethyl acetate, tert-butyl methyl ether, 2-propanol or ethanol and a nonpolar solvent (preferably n-hexane or n-heptane) is preferable, ethyl acetate is more preferable, or a mixed solvent of ethyl acetate and a nonpolar solvent (preferably n-hexane or n-heptane) is more preferable, and a mixed solvent of ethyl acetate and n-hexane is particularly preferable. The mixing ratio (volume ratio) when the mixed solvent is used is not particularly limited, and from the viewpoint of controlling the residual solvent concentration to be equal to or less than the reference value, it is particularly preferable to use a mixed solvent of ethyl acetate and n-hexane in an amount of 10: 1to 1: 10. preferably 4: 1to 1: 4. more preferably 2: 1to 1:2, mixing ethyl acetate: n-hexane.

The filter used in the filtration in this step is not particularly limited as long as it is capable of removing fine powder of the filler (silica gel), floating particles in the air, and the like without swelling and dissolving by the solvent, and examples thereof include a glass fiber filter, a polypropylene filter, a nylon filter, a fluororesin filter, and the like, and a fluororesin filter such as polyvinylidene fluoride (PVDF) and Polytetrafluoroethylene (PTFE) is preferable, and a Polytetrafluoroethylene (PTFE) filter is particularly preferable.

The pore size of the filter is usually 0.5 μm or less, preferably 0.25 μm or less, and particularly preferably 0.22 μm or less when sterilization is also included.

(step 4)

This step is a step of distilling off the solvent of the filtrate obtained in the step 3 at 10 to 55 ℃ under reduced pressure with a final ultimate vacuum degree of 5torr or less.

Tafluprost is very viscous and therefore it is necessary to maximize the surface area over which the solvent evaporates and to remove the solvent from the filtrate slowly by distillation while avoiding bumping. Examples of the vacuum concentration apparatus for achieving the above object include a rotary evaporator, a centrifugal evaporator, and a high vacuum thin film evaporator.

As described above, the external temperature (temperature of water bath or hot water bath) when the solvent is distilled off under reduced pressure is preferably 10 to 55 ℃, more preferably 15 to 50 ℃, and particularly preferably 20 to 45 ℃ in view of gradual decomposition of tafluprost at a temperature of 60 ℃ or higher over time.

The degree of reduced pressure at the time of distilling off the solvent is controlled so that the surface area of the solvent to be evaporated is as large as possible and it takes time to gradually increase while avoiding bumping, and the ultimate vacuum degree is preferably controlled to be 5torr or less (preferably 3torr or less, more preferably 1torr or less, and particularly preferably 0.5torr or less). When the reduced pressure is released, it is preferable to return to normal pressure using air passed through a filter in order to prevent the entry of floating particles and bacteria in the air.

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.

By using the purification method of the present invention, the concentration of the residual organic solvent can be suppressed to a value not more than the concentration limit value of the residual solvent guideline (non-patent document 2) for pharmaceuticals, and "residual solvent" as described in the guideline does not contribute to the treatment, and therefore, the total residual solvent should be reduced to a level not more than a level that can meet the product standard, GMP, or other quality standards. ", high-quality tafluprost which can further meet strict various quality standards can be stably produced.

As a solvent for dissolving the eluent and the residue used in the silica gel column chromatography, preferred are: the residual solvent concentrations of ethyl acetate, t-butyl methyl ether, 2-propanol, ethanol, or n-heptane, which are exemplified as preferred solvents, can be produced more preferably by controlling the respective residual solvent concentrations to 1000ppm or less, particularly preferably to 100ppm or less, and further preferably: the residual solvent concentration of n-hexane can be controlled more preferably to 200ppm or less, particularly preferably to 20ppm or less. The concentration of the residual solvent can be measured by a method such as Gas Chromatography (GC).

The present invention also includes a method for producing tafluprost, in which a crude product of tafluprost produced by a known method is subjected to the step of the purification method of the present invention (the purification method including the above steps 1to 4). As a known production method of tafluprost, there are several reports (for example, U.S. patent application publication nos. 2014/0046086; j. Org. Chem.2016,81,10832-844, molecules,2017,22,217,1-16, org. Lett.2020,22,2991-2994, etc.) in addition to the aforementioned patent document 1 and non-patent document 1, and these can be included in the present invention by combining with the purification method of the present invention.

Specific examples of the crude product of tafluprost used in the present invention include, for example, a crude product of tafluprost obtained by deprotection reaction of tafluprost protected with a hydroxyl group or the like, a crude product of tafluprost obtained by esterification of a salt of tafluprost acid, a crude product of tafluprost obtained by esterification of tafluprost acid, and the like. Among them, a crude product of tafluoroprostane obtained by esterification of tafluoroprostane acid can be suitably used.

Specific features of the purification method of the present invention include the following.

(A) When a crude product of tafluprost is separated and purified by silica gel column chromatography, components containing tafluprost are collected by HPLC analysis (preferably reverse phase HPLC analysis), whereby contamination of impurities can be suppressed to a minimum.

(B) By removing the solvent by distillation over a long period of time under reduced pressure conditions of low temperature and high vacuum, the decomposition of tafluprost which is unstable at high temperature can be suppressed, and the concentration of the residual organic solvent can be suppressed to a value equal to or lower than the concentration limit of the residual solvent rule of the pharmaceutical product.

(C) By incorporating a filter filtration step in the middle, it is possible to provide high-purity tafluprost that can be used as a drug substance of a pharmaceutical as it is after the solvent is distilled off.

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

The purification method of the present invention may be carried out for the purpose of improving the purity of tafluprost as described in (a) above, and may include only step 1, or may be combined with steps 2to 4, if necessary, in step 1.

Examples

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

% represents mol% in the yield, and% represents mass% in the rest unless otherwise specified. The ratio shown in the mixed solvent represents a volume ratio unless otherwise specified. Unless otherwise specified, room temperature means a temperature of 15 to 30 ℃. The following are mentioned ¹ The H-NMR value was measured by using ECP400 (400 MHz) manufactured by Nippon electronics Co., ltd. The HPLC apparatus used Shimadzu LC-10ADvp or LC-10A. The GC apparatus used Shimadzu GC-2014ATF.

Reference example 1: synthesis of tafluprost acid

Tetrahydrofuran (1200 g) was added to and dissolved in (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) under a nitrogen atmosphere, and diisobutylaluminum hydride (1M toluene solution) (2160 mL) was added dropwise at-70 ℃. After the completion of the dropwise addition, the mixture was stirred for 30 minutes, and 1N hydrochloric acid was added thereto to conduct extraction with ethyl acetate. The organic layers were combined, washed with water, and the filtrate was concentrated under reduced pressure to obtain a reduced sample (284 g). Tetrahydrofuran (5030 g) was added to 4-carboxybutyltriphenylphosphonium bromide (1523 g) under a nitrogen atmosphere, and a sodium bis (trimethylsilyl) amide solution (1M tetrahydrofuran solution) (6684 mL) was added dropwise and stirred for 1 hour or more. The above-mentioned reducing agent (286 g) dissolved in tetrahydrofuran (970 g) was added dropwise thereto at 0 ℃ and stirred for 3 hours. Water was added to the reaction mixture, followed by extraction with ethyl acetate. After the aqueous layer was acidified, it was extracted with ethyl acetate, concentrated under reduced pressure, and insoluble matter was filtered off and purified by silica gel column chromatography (hexane/ethyl acetate =1/1 to 1/3), whereby tafluprost acid (222 g) was obtained.

¹ H NMR(CDCl ₃ )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),4.03(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).

Reference example 2: synthesis of crude Tafluprost product

Tafluoroprostanoic acid (120 g) obtained in reference example 1 was put into a 5L flask under nitrogen atmosphere, and dissolved in acetone (600 mL) with stirring. After cooling to 5 ℃ and dropping 1, 8-diazabicyclo [5.4.0] undec-7-ene (DBU) (160 mL) while maintaining the temperature at or below 5 ℃ and dropping 2-iodopropane (146 mL) while maintaining the temperature at or below 5 ℃, the mixture was stirred at 30 ℃ until the conversion of the reaction became 95% or more. To the reaction mixture were added ethyl acetate (1800 mL) and a 5% aqueous citric acid solution (900 mL) and the mixture was separated, and the organic layer was washed with a 5% aqueous citric acid solution (900 mL, 1 time), a 5% aqueous sodium bicarbonate solution (900 mL, 2 times), and purified water (900 mL, 1 time). The solvent was distilled off under reduced pressure at 40 ℃ or lower, whereby a crude product of tafluprost (132 g, yield 100%; HPLC purity: 95.5%, alpha-chain trans-isomer content: 0.73%) was obtained.

Example 1

Step 1 a column was filled with a slurry prepared from silica gel (AGC-SITECH co., LTD, m.s.gel D50-120A, particle size (D50): 50 μm, spherical, 50 g) and n-hexane/ethanol =96/4, and the crude tafluprost (1 g) obtained in reference example 2 was dissolved in n-hexane/ethyl acetate =1/1, packed onto the column, and eluted with n-hexane/ethanol = 96/4. The fractions were analyzed by HPLC and fractions containing tafluprost were collected. As the component containing tafluprost, at least a component in which the area percentage of tafluprost is 98% or more (calculated by subtracting a solvent peak) is collected.

(step 2) the collected fraction containing tafluprost is concentrated under reduced pressure at 35 ℃ to 40 ℃.

(step 3) the residue was dissolved in n-hexane/ethyl acetate =3/2, filtered through a membrane filter (pore size: 0.2 μm), and washed with n-hexane/ethyl acetate = 3/2.

(step 4) the solvent in the filtrate was distilled off overnight under reduced pressure at 35 to 40 ℃ and a final ultimate vacuum degree of 1torr or less, whereby tafluprost (colorless to pale yellow viscous liquid, yield: 82%, HPLC purity: 99.5%, content of. Alpha. -chain trans-isomer: 0.25%) was obtained.

The residual solvent concentration of the obtained tafluprost was analyzed by GC, and as a result, n-hexane was 0ppm, ethyl acetate was 0ppm, and ethanol was 0ppm.

¹ H NMR(CDCl ₃ )δ1.22(d,J＝6.2Hz,3H),1.22(d,J＝6.2Hz,3H),1.58-1.63(m,1H),1.63-1.69(m,2H),1.84(d,J＝14.7Hz,1H),2.02-2.08(m,1H),2.10-2.16(m,3H),2.25(t,J＝7.3Hz,1H),2.26(t,J＝7.1Hz,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＝6.2Hz,1H),5.35-5.42(m,2H),5.80(dt,J＝15.8,11.2Hz,1H),6.10(dd,J＝15.8,8.8Hz,1H),6.91(d,J＝8.8Hz,2H),7.00(t,J＝7.3Hz,1H),7.30(dd,J＝8.8,7.3Hz,2H)；

¹⁹ F NMR(CDCl ₃ )δ-102.8(dq, ² J _FF ＝255.6Hz),-103.6(dq, ² J _FF ＝255.6Hz).

< conditions for HPLC (reverse phase) analysis >

Column: YMC-Pack ODS-AM (5 μm, 6.0X 150 mm)

Temperature: at room temperature

Flow rate: 1 mL/min

Detection wavelength: 220nm

Eluent: (solution A) 1% triethylamine-phosphate buffer solution (pH 6.3),

(solution B) acetonitrile

Gradient conditions: A/B =50/50 (0 to 45 minutes), A/B =25/75 (45 to 70 minutes)

< GC analysis conditions >

Column: g-column G300 (1.2mm I.D.,40 m)

Column temperature: 50 deg.C

And (3) detection: hydrogen flame ionization detector

Carrier gas: helium

Injector temperature: 160 ℃ C

Detector temperature: 160 deg.C

Examples 2to 6

In order to examine the influence of the type of silica used for silica gel column chromatography on the purity and yield of tafluprost, the following experiment was performed in the same manner as in example 1.

The results of silica gel column chromatography using a crude product of tafluprost (1g, HPLC purity: 95.5%) and silica gel (50 g) are shown in Table 1 below. Reverse phase HPLC analysis was performed under the same conditions as described above.

[ Table 1]

Comparative example 1

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

When the particle size (d 50) of the silica gel used was 65 μm or less, tafluprost having a purity of more than 98% was obtained in all of examples 1to 6, although the shape was different. Among them, it was found that when spherical silica gel is used, tafluprost having particularly high purity can be obtained in high yield.

Example 7 (scale-up study)

(step 1)

In the same manner as in step 1 of example 1, a slurry prepared from silica gel (AGC-SITECH co., LTD, m.s.gel D50-120A, particle size (D50): 50 μm, spherical, 6.0 kg) and n-hexane/ethanol =96/4 was packed in a column, and the crude tafluprost (120 g) obtained in reference example 2 was dissolved in n-hexane/ethyl acetate =1/1, packed in the column, and eluted with n-hexane/ethanol = 96/4. The fractionated fractions were analyzed by HPLC and fractions containing tafluprost were collected. As the component containing tafluprost, at least a component in which the area percentage of tafluprost is 98% or more (calculated by subtracting the solvent peak) is collected.

(Steps 2to 4)

The fraction containing tafluprost collected in step 1 is concentrated under reduced pressure at 29 to 35 ℃ (step 2).

The residue was dissolved in n-hexane/ethyl acetate =3/2, filtered through a membrane filter (pore size: 0.2 μm), and washed with n-hexane/ethyl acetate =3/2 (step 3).

The solvent in the filtrate was distilled off under reduced pressure at 32 to 36 ℃ and a final ultimate vacuum of 0.30torr for 26 hours (step 4), whereby tafluprost (colorless to pale yellow viscous liquid, yield: 86%, HPLC purity: 99.7%, a.chain trans-isomer content: 0.24%, and microorganism content: 10cfu/0.1g or less) was obtained.

Example 8

The fraction containing tafluprost purified and collected in the same manner as in step 1 of example 7 was concentrated under reduced pressure in the same manner 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).

The solvent in the filtrate was distilled off under reduced pressure at 23 to 37 ℃ and a final ultimate vacuum degree of 0.26torr for 27 hours (step 4), whereby tafluprost (colorless to pale yellow viscous liquid, yield: 85%, HPLC purity: 99.7%, content of. Alpha. -chain trans-isomer: 0.27%, content of microorganism: 10cfu/0.1g or less) was obtained.

Example 9

The fraction containing tafluprost purified and collected in the same manner as in step 1 of example 7 was concentrated under reduced pressure in the same manner as in step 2 of example 7, and the residue was dissolved in n-hexane/ethyl acetate =3/2, filtered through a membrane filter (pore size: 0.2 μm), and washed with n-hexane/ethyl acetate =3/2 (step 3).

The solvent in the filtrate was distilled off under reduced pressure at 20 to 36 ℃ and a final ultimate vacuum degree of 2.6torr for 3 hours (step 4), whereby tafluprost (colorless to pale yellow viscous liquid, yield: 75%, HPLC purity: 99.4%, content of. Alpha. -chain trans-isomer: 0.30%, content of microorganism: 10cfu/0.1g or less) was obtained.

The residual solvent concentration of the obtained tafluprost was analyzed by GC, and as a result, n-hexane was 36ppm, ethyl acetate was 4803ppm, and ethanol was 66ppm.

Example 10

The solvent in the filtrate was distilled off under reduced pressure at 34 to 37 ℃ and a final ultimate vacuum degree of 2.1torr for 5 hours (step 4), whereby tafluprost (colorless to pale yellow viscous liquid, yield: 78%, HPLC purity: 99.5%, content of. Alpha. -chain trans-isomer: 0.32%, content of microorganism: 10cfu/0.1g or less) was obtained.

The residual solvent concentration of the obtained tafluprost was analyzed by GC, and as a result, n-hexane was 2ppm, ethyl acetate was 785ppm, and ethanol was 0ppm.

Example 11

The solvent in the filtrate was distilled off under reduced pressure at 32 to 36 ℃ and a final ultimate vacuum degree of 0.92torr for 8 hours (step 4), whereby tafluprost (colorless to pale yellow viscous liquid, yield: 82%, HPLC purity: 99.6%, content of. Alpha. -chain trans-isomer: 0.26%, content of microorganism: 10cfu/0.1g or less) was obtained.

The residual solvent concentration of the obtained tafluprost was analyzed by GC, and as a result, n-hexane was 0ppm, ethyl acetate was 86ppm, and ethanol was 0ppm.

Example 12

The solvent in the filtrate was distilled off under reduced pressure at 35 to 39 ℃ and a final ultimate vacuum degree of 0.09torr for 50 hours (step 4), whereby tafluprost (colorless to pale yellow viscous liquid, yield: 80%, HPLC purity: 99.5%, a.chain trans-isomer content: 0.26%, and microorganism content: 10cfu/0.1g or less) was obtained.

The residual solvent concentration of the obtained tafluprost was analyzed by GC, and as a result, n-hexane was 0ppm, ethyl acetate was 0ppm and ethanol was 0ppm.

Example 13

The solvent in the filtrate was distilled off under reduced pressure at 36 to 45 ℃ and a final ultimate vacuum degree of 0.24torr for 60 hours (step 4), whereby tafluprost (colorless to pale yellow viscous liquid, yield: 79%, HPLC purity: 99.5%, content of. Alpha. -chain trans-isomer: 0.26%, content of microorganism: 10cfu/0.1g or less) was obtained.

The conditions for distilling off the solvent, the yield, purity and α -chain trans-isomer content of tafluoroprostatin, and the residual solvent concentration in examples 7 to 13 are shown in table 2 below.

[ Table 2]

As is clear from table 2, examples 7 to 13 all obtained tafluprost with good purity and high yield, and the residual organic solvent concentration could be suppressed to be less than the concentration limit value of the residual solvent criterion of the pharmaceutical products, and the purification method of the present invention was a highly versatile purification method that could withstand scale-up. On the other hand, it was confirmed that: in the comparative example in which concentration was performed under reduced pressure at a final ultimate vacuum of 8torr, the residual organic solvent concentration exceeded the concentration limit value of the residual solvent criterion for the pharmaceutical.

Test examples

Study of thermal stability of Tafluprost

Approximately 120mg of tafluprost obtained in example 1 was weighed out into glass containers, stored in a thermostatic chamber at 40 ℃ and quantified by reverse phase HPLC analysis, and the change in the content of tafluprost over time was examined. Similarly, approximately 20mg of tafluprost was measured in glass containers, stored in a thermostatic bath at 60 ℃ or 80 ℃, and the change in tafluprost content over time was examined.

< conditions for HPLC (reverse phase) analysis >

Column: YMC-Pack ProC18 AS-303 (5 μm, 4.6X 250 mm)

Temperature: 50 deg.C

Flow rate: 1 mL/min

Detection wavelength: 220nm

Eluent: (solution A) 10mmol/L phosphate (sodium) buffer solution (pH6.9),

(solution B) acetonitrile

Gradient conditions: A/B =50/50 (0-45 minutes), A/B =25/75 (45-70 minutes)

The results of examining the change with time of the content of tafluprost at each temperature are shown in tables 3to 5 below.

[ Table 3]

Stability of Tafluprost at 40 ℃

Time (moon)	0	3	6
				Content of Tafluprost (%)	101.3	99.8	99.4

[ Table 4]

Stability of Tafluprost at 60 ℃

Time (sky)	0	3	7	14
					Content of Tafluprost (%)	99.0	98.9	98.5	95.4

[ Table 5]

Stability of Tafluprost at 80 ℃

Time (sky)	0	1	3	7
					Content of Tafluprost (%)	99.5	99.1	94.4	86.7

From the results of tables 3to 5, it was confirmed that: tafluprost slowly decomposes over time, particularly significantly decomposes at 80 ℃ at a temperature of 60 ℃ or higher even during storage for a period of about several days to 2 weeks, but it is found that tafluprost stably exists at 40 ℃ even after 6 months.

From the above results, it can be seen that: in the purification method of the present invention, the concentration under reduced pressure and the distillation removal of the solvent are performed at a temperature of 55 ℃ or lower (particularly preferably 45 ℃ or lower), whereby the contamination of impurities (analogous substances) derived from the decomposition of tafluprost can be suppressed.

Industrial applicability

According to the purification method of the present invention, when a crude product of tafluprost is separated and purified by silica gel column chromatography in the final step in the production of tafluprost, the contamination of impurities can be minimized by collecting fractions containing tafluprost by HPLC analysis. Further, by distilling off the solvent with time under reduced pressure conditions of low temperature and high vacuum degree, the concentration of the residual organic solvent can be suppressed to be not more than the limit value of the residual solvent criterion of the pharmaceutical, and the decomposition of tafluprost unstable at high temperature can be suppressed. Further, since the fine powder of silica gel, floating particles in the air, and bacteria can be removed by incorporating a filter filtration step in the middle of the process, there is an advantage that high-purity tafluprost which can be directly used as a drug substance of a pharmaceutical can be easily and efficiently provided after the solvent is distilled off. The purification method of the present invention can be widely applied to a crude product of tafluprost produced by a known method, and is a highly versatile method that can also withstand scale-up.

Claims

1. A method of purifying tafluprost, comprising: a step of purifying the crude product of tafluprost by silica gel column chromatography and collecting a fraction containing tafluprost by HPLC analysis, wherein the HPLC analysis is reverse phase HPLC analysis;

it still includes: a step of filtering a fraction containing tafluprost collected by HPLC analysis; and distilling off the solvent from the filtrate under reduced pressure at 10 to 55 ℃ and a final ultimate vacuum degree of 5torr or less.

2. The purification process of claim 1, comprising: a step of concentrating the fraction containing tafluprost collected by HPLC analysis under reduced pressure at 10 to 55 ℃ and a subsequent step of dissolving the residue in a solvent.

3. The purification method according to claim 1 or 2, wherein the crude product of tafluoroprostatin is a crude product of tafluoroprostatin obtained from protected tafluoroprostatin by a deprotection reaction, a crude product of tafluoroprostatin obtained by esterification of a salt of tafluoroprostatin acid, or a crude product of tafluoroprostatin obtained by esterification of tafluoroprostatin acid.

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

5. The purification method according to claim 1 or 2, wherein the pore size of the filter used for the filtration is 0.25 μm or less.

6. The purification method according to claim 1 or 2, wherein the silica gel used in the silica gel column chromatography has a particle size d50 of 20 to 70 μm.

7. The purification process according to claim 1 or 2, wherein the silica gel used in the silica gel column chromatography is spherical.

8. The purification method according to claim 1 or 2, 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.

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

10. The purification process according to claim 8, wherein the polar solvent is ethyl acetate, tert-butyl methyl ether, 2-propanol, or ethanol.

11. The purification method according to claim 1 or 2, wherein the component is a component containing 98% or more of tafluprost.

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

13. The purification method according to claim 12, wherein the solvent for dissolving the residue is ethyl acetate or a mixed solvent of ethyl acetate and a nonpolar solvent.

14. The purification process according to claim 12, wherein the non-polar solvent is n-hexane or n-heptane.

15. The purification method according to claim 1 or 2, wherein the final ultimate vacuum degree is 1torr or less.

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

17. A method for producing tafluprost, which comprises subjecting a crude product of tafluprost to the purification method according to any one of claims 1to 16.