JP2023028473A - Method for producing everolimus - Google Patents

Abstract

To provide a method for producing highly pure everolimus including a low-cost purification step which can efficiently yield highly pure everolimus from crude everolimus with the safety confirmed.SOLUTION: According to the present invention, a step for purifying crude everolimus is executed by using a diol silica gel as a stationary phase, using a mixed solution of ethyl acetate and heptane as an eluent, and using a hydrophilic solvent as an added solvent to the eluent.SELECTED DRAWING: None

Description

The present invention relates to a method for producing everolimus of high purity (99% or higher), and more particularly to said production method, characterized by a step of purifying crude everolimus using normal phase chromatography.

Everolimus, which is used as an immunosuppressant and an anticancer drug, is a macrolide antibiotic produced by Streptomyces hygroscopicus. It is obtained by successively carrying out lithography, crystallization by adding seed crystals, and precipitation.

Novartis, the forerunner manufacturer, prepared a tert-butyldimethylsilyl (TBS)-protected triflate form of ethylene glycol with a highly sterically hindered amine, followed by a nucleophilic substitution reaction with rapamycin, followed by hydrogen fluoride. After removing the TBS group with pyridine (tetrahydrofuran solvent) to obtain crude everolimus, crude everolimus was purified by silica gel chromatography using ethyl acetate/heptane as an eluent, followed by crystallization to obtain everolimus. proposed (Patent Document 1).

In the crystallization step, crude everolimus is dissolved in ethyl acetate, heptane is added dropwise to the solution obtained by heating to 30°C, seed crystals are added, and the everolimus crystals are precipitated by cooling. there is Furthermore, the obtained everolimus crystals are dissolved in ethanol and dibutylhydroxytoluene is added. The mixture thus obtained is slowly added dropwise to water cooled to 0° C. to obtain stabilized everolimus (Patent Document 2).

However, in the everolimus manufacturing method of Novartis, a large amount of waste silica gel is generated in the chromatography process. In addition, there are problems such as a complicated crystallization step of adding seed crystals and a decrease in yield due to the crystallization step.

Chromatography in the manufacture of pharmaceuticals is generally classified roughly into normal phase chromatography using an organic solvent as an eluent and reversed phase chromatography using a water-containing eluent. Among them, after a normal phase chromatography treatment or operation using a standard silica gel having a surface of silanol groups as a carrier, the carrier is usually discarded as sludge. This is because a part of the highly polar target substance or impurities is adsorbed on the carrier, and it is expected that the separation performance is lowered.

In such a chromatographic treatment, since it is necessary to fill new silica gel every time purification is performed, a large amount of waste silica gel is discharged and the cost is increased.

In order to solve these problems, a production method has been reported in which generation of waste silica gel is suppressed by a purification method using reusable silica gel.

For example, there is a report on a method for producing high-purity everolimus, including a purification step of crude everolimus using reusable octadecylsilyl silica gel as a carrier (Patent Document 3). However, since the chromatography in this production method uses a so-called reversed-phase system, there is a problem in that the amount of water-insoluble everolimus that can be applied is smaller than that in normal-phase chromatography. There is also a report on a method for producing high-purity everolimus, which includes a purification step using reusable silica gel whose surface is capped with nitrile groups as a carrier (Patent Document 4). Since this purification step uses an organic solvent as an eluent, it has the advantage that a large amount of everolimus can be treated at one time.

Patent No. 6574173 Patent No. 5165199 CN102464668B CN102174053B

Against the above-mentioned background, the present invention provides high-purity, i.e., purity of 99% or more, from crude everolithm through a chromatographic treatment step using reusable silica gel and an inexpensive organic solvent whose safety has been confirmed. It is an object or problem to provide a method for efficiently obtaining everolimus.

In the chromatographic step, the present inventors used so-called diol silica gel as the stationary phase, in which the silanol groups on the surface of the silica gel as a carrier are moderately modified with diol groups, while ethyl acetate and heptane were used as the mobile phases. It was found that highly pure everolimus can be efficiently obtained from crude everolimus by using an eluent whose polarity is adjusted by adding a certain organic solvent in addition to the mixed solvent of .

Accordingly, but not by way of limitation, features or aspects of the invention disclosed herein are as follows.
Aspect 1: A step of purifying crude everolimus using normal phase chromatography, wherein the normal phase chromatography uses diol silica gel as a stationary phase, and an eluent of a mixed solvent of ethyl acetate and heptane with a hydrophilic solvent added. A method for producing everolimus, characterized in that it is carried out using

Aspect 2: The production method according to Aspect 1, wherein the stationary phase used for normal phase chromatography is diol silica gel having a particle size of 5 to 250 μm.

Aspect 3: Aspect 1, wherein the ethyl acetate/heptane mixed solvent in the eluent used for normal phase chromatography has a solvent composition in the range of 20/80 to 50/50 on a volume basis. Production method.

Aspect 4: A hydrophilic solvent selected from acetonitrile, acetone, methanol, ethanol, 1-propanol, and 2-propanol is 0.5 to 1 relative to the ethyl acetate/heptane mixed solvent in the eluent used for normal phase chromatography. .5 v/v % is added, the production method according to aspect 1.

Aspect 5: After the step of purifying crude everolimus using normal phase chromatography, the step of substituting the everolimus-containing fraction obtained in the above step with an ester solvent while concentrating under reduced pressure; to the non-polar solvent to precipitate everolimus.

Aspect 6: Aspect characterized in that the ester solvent is selected from methyl formate, ethyl formate, methyl acetate, ethyl acetate, propyl acetate and butyl acetate, and the non-polar solvent is selected from pentane, hexane, heptane and octane. 5. The manufacturing method according to 5.

According to the present invention, the diol silica gel used in the chromatographic step can be reused as stationary phase or separation carrier, and is based on a mixed solvent of ethyl acetate and heptane, with minor amounts of other organic solvents. The mobile phase or separation liquid combined with is confirmed to be safe to use, and can be easily constructed from relatively inexpensive ones. In addition, by using these stationary phases or carriers and mobile phases or eluents, highly pure everolimus can be efficiently obtained from crude everolimus.

According to the present specification, a step of purifying crude everolimus using normal phase chromatography is disclosed, which can obtain highly pure everolimus from the crude everolimus. "Crude" in "crude everolimus" is used as a concept opposite to "high purity" in "high-purity everolimus". less than 70%, 75% or more, 80% or more, 85% or more, 90% or more. Crude everolimus within these purity ranges can be obtained high-purity everolimus of interest by performing the purification step only once. Crude everolimus is not limited by its origin, but generally includes starting materials, reactants, reaction accelerators, by-products, etc., resulting from the chemical conversion reaction of rapamycin to everolimus and certain purification processing steps. It may be a product that has been subjected to an intermediate or final refining process including. Such processed products are disclosed in, for example, Patent Documents 1 to 4, WO 94/09010 A1, WO 2012/066502 A1, WO 2012/103959 A1, WO 2014/203185 A1, WO 2016/207205 A1, etc. What was done is mentioned. According to the present invention, shown in <Reference Example 1> described later, generally, rapamycin to everolimus includes the steps of introducing rapamycin-protected ethylene glycol and deprotecting the ethylene glycol protecting group. may be provided to the crude everolimus purification step using said normal phase chromatography.

Furthermore, the purity referred to above represents the peak area percentage of everolimus obtained by liquid chromatography analysis under the following conditions. That is, the purity is derived by the formula of (everolimus peak area)/(sum of all peak 20 areas) and is expressed as a % value. Liquid chromatograph conditions are two analytical systems (Impurity A, Related substances) described in the European Pharmacopoeia listed test methods for everolimus.

The normal phase chromatography referred to in the present invention means a separation system in which the polarity of the stationary phase is higher than the polarity of the eluent. Separation and purification are carried out through the process of adsorption-desorption by using an eluent having a polarity lower than that of the diol silica gel surface. As the eluent solvent for the chromatography using silica gel, heptane is used as a low-polarity solvent and a mixed solvent of ethyl acetate is used as a high-polarity solvent.

Further, in the present invention, a solvent having a higher polarity than ethyl acetate, preferably a hydrophilic highly polar organic solvent, more preferably an aprotic and hydrophilic highly polar solvent, is added to the mixed solvent at 0.5 to 1.5 v/v%. By using the added eluent, it is possible to improve the degree of separation between everolimus and related by-products or impurities typified by Isomer C that may be generated during the chemical conversion or purification process.

The degree of separation here is the degree that indicates how much a peak is separated from adjacent peaks, and in the Japanese Pharmacopoeia and Japanese Industrial Standards General Rules for High Performance Liquid Chromatography, the degree of separation is defined by the formula shown below. It is

Here, R is the resolution, t _R1 and t _R2 (t _R1 ≦t _R2 ) are the retention times of the respective peaks, and W _0.5h1 and W _0.5h2 are the half widths of the respective peaks. The Japanese Pharmacopoeia defines complete separation of peaks as "meaning a degree of separation of 1.5 or more."

According to Patent Document 4, nitrile silica gel supporting nitrile groups is specifically used as a regenerable neutral silica gel used as a stationary phase or carrier for normal phase chromatography. However, among the eluents described in Examples using nitrile silica gel in Patent Document 4, diisopropyl ether, which is used as a non-polar solvent, has not been confirmed to be safe to use. is a solvent that should be used sparingly. After application of crude everolimus using nitrile silica gel, instead of the tetrahydrofuran/isopropyl ether solvent system described in the literature, safety has been confirmed, such as ethyl acetate/heptane/1-propanol = Elution was attempted with an eluent having a composition of 20/80/1, and it was confirmed that everolimus did not adsorb to nitrile silica gel and did not exhibit the desired separation (see Reference Example 2). From this result, it is necessary to lower the composition of ethyl acetate and hydrophilic solvent to purify everolimus using nitrile silica gel. The amount of everolimus injected into the nitrile silica gel, ie, the amount of everolimus injected into the nitrile silica gel, is relatively lower than in the case of the diol silica gel. Therefore, it can be understood that the combination of the diol silica gel used as the stationary phase in the present invention, the separation liquid, and the mixed solvent system used provides unique effects.

The diol silica gel particles used in the normal phase chromatography used in the present invention may have a size of 5 μm to 250 μm. It is convenient to use the latter because of its availability. In general, the smaller the particle size, the higher the number of theoretical plates and the higher the separation performance. Become.

It is also known that the smaller the particle size, the more expensive the diol silica gel. Therefore, it is necessary to set an appropriate particle size in consideration of the purpose of purification, the impurities to be removed, the pressure resistance of the equipment and column to be used, the amount of treatment, the treatment time, and the like.

When everolimus is purified, the particle size is preferably 5 μm to 200 μm, more preferably 20 μm to 110 μm.

Diol silica gel is an electrically neutral silica gel prepared by chemically modifying the silanol groups on the surface of ordinary silica gel with 1,2-dihydroxy-3-propoxypropyl groups. Although the modification rate of silica gel is not limited as long as the object of the present invention is met, it is preferably 100 to 600 μmol/m ² , more preferably 200 to 400 μmol/m ² .

The mixing volume ratio of the highly polar solvent ethyl acetate and the low polar solvent heptane used in the normal phase chromatography of the present invention is generally selected from the range of ethyl acetate: heptane = 5:95 to 100:0. is preferably in the range of ethyl acetate:heptane=20:80 to 50:50, more preferably ethyl acetate:heptane=20:80 to 35:65, and ethyl acetate:heptane=30:70 to 35. :65 is particularly preferred.

The hydrophilic solvent added to the ethyl acetate/heptane mixed solvent is generally selected from acetonitrile, acetone, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tetrahydrofuran, and 2-methyltetrahydrofuran. , acetone, methanol, ethanol, 1-propanol and 2-propanol are preferred, and acetonitrile and 1-propanol are particularly preferred.

The mixing volume ratio of ethyl acetate and heptane and the hydrophilic solvent to be added are determined under appropriate conditions depending on the column volume, treatment amount, treatment time, amount of diol silica gel used, particle size, modification rate of dihydroxypropyl groups on the silica gel surface, etc. must be selected.

Solvents used when crude everolimus to be purified by normal phase chromatography of the present invention is dissolved and applied to silica gel include halomethane solvents such as dichloromethane and chloroform, and ethyl acetate with a volume ratio of: A mixed solvent of heptane=15:85 is preferred.

The amount of diol silica gel and the amount of crude everolimus subjected to separation depend on the desired purity and recovery. For example, when eluting a diol silica gel with a particle size of 110 μm using a mixed volume ratio of ethyl acetate and heptane of 30:70 and acetonitrile as a 1 vol% additive solvent, 4.0 g ~ 4.0 g per 1 L of column head volume 7.0 g of raw material is subjected to separation. For example, 400 mg to 700 mg for a 4.5 cm inner diameter, 9.0 cm long column, and 4.0 g to 7.0 g for a 4.0 cm inner diameter, 75.0 cm long column provide the desired high purity. of everolimus can be obtained. At this time, the flow rate of the developing solvent is set within a space velocity range of 5 S.V.

The fractionated solution containing highly purified everolimus obtained by normal phase chromatography is then concentrated under reduced pressure to a concentration of 100 g/L to 600 g/L, preferably 250 g/L to 400 g/L, The solution is added dropwise to a cooled low-polarity solvent with stirring over 30 minutes, and the resulting slurry is subjected to vacuum filtration or pressure filtration to obtain everolimus powder.

Vacuum concentration of the fractionated solution containing highly pure everolimus is preferably carried out at a pressure of 50 hPa to 100 hPa and a temperature of 30°C to 40°C. Also, a highly polar solvent such as ethyl acetate may be used for rinsing and cleaning of pipes.

Examples of highly polar solvents used for rinsing include alcohol solvents, ester solutions, ether solvents, etc. Ethyl acetate is particularly preferred for everolimus. Examples of the low-polarity solvent used for precipitation include alkyl-based solvents and ether-based solvents, and heptane is particularly preferred for everolimus.

Specific examples will be given below in order to describe the present invention in more detail, but the scope of the present invention is not limited by these.

<Reference Example 1> Preparation example of crude everolimus
Toluene (63.7
mL) solution was added diisopropylethylamine (8.1 g, 11.1 mL, 62.4 mmol). The clear solution was then cooled to -20°C and trifluoromethanesulfonic anhydride (17.0 g,
9.9 mL, 60.2 mmol) was added slowly with a syringe pump at a rate of 20.0 mL/min. After washing the inner wall of the flask with toluene (5.8 mL), the temperature was raised to 0°C. After 30 minutes from heating, diisopropylethylamine (8.1 g, 11.1 mL, 62.4 mmol) was added, followed by addition of rapamycin (10.0 g, 10.9 mmol) and washing with toluene (24.8 mL). The temperature of the reaction solution was raised to 60° C., and the amount of raw material, target substance, and impurities was confirmed by HPLC at each elapsed time. After 20 hours, the reaction solution was allowed to cool to room temperature and then pyridine (3.0 g, 3.1 mL, 38.3 mmol) was added. After the reaction solution was filtered under reduced pressure, the filter cake was washed with toluene (30 mL). Subsequently, the solution was diluted with ethyl acetate (400 mL), and the organic layer was washed with 1M aqueous citric acid (400 mL) and water (400 mL). The organic layer (370 mL) was concentrated under reduced pressure to obtain 28.0 g of everolimus-TBS crude/toluene solution.

After that, acetonitrile (37.4 mL) and water (3.9 mL) were added to the obtained solution, and the mixture was cooled to 0°C. Then, after adjusting the solution to pH 1.7 with 75% orthophosphoric acid, 1M hydrochloric acid was slowly added dropwise to adjust the pH to 1.8, followed by stirring for 4 hours. After separating the organic layer containing everolimus, it was washed with saturated aqueous sodium bicarbonate solution (400 mL) and water (400 mL). After that, the organic layer was concentrated under reduced pressure to obtain 13.1 g of crude everolimus.

<Reference Example 2> Example of using a comparative stationary phase After preparing a movable stopper column packed with 20 g of nitrile silica gel (CN SMB100-20/45) whose surface is capped with nitrile groups, manufactured by Fuji Silysia Ltd., the purity was 80%. 125 mg of everolimus was applied and developed with a mixed solvent of ethyl acetate/heptane/1-propanol=20/80/1 as an eluent. Everolimus did not adsorb to the nitrile silica gel.

The mobile phase when using diol silica gel as a carrier was investigated by HPLC. Everolimus containing 8% Isomer C was dissolved in acetonitrile to prepare 0.5 mg/mL in an HPLC column (Inertsil, Diol, 5 μm, 4.6×150 mm). The degree of separation was confirmed using a mixed solvent of ethyl acetate/heptane added at 1.5 v/v % as a mobile phase. The polar solvents used were methanol, ethanol, 2-propanol, acetonitrile and acetone. Table 1 shows the results.

After preparing a movable stopper column packed with 50 g of Fuji Silysia Diol silica gel (DIOL MB100-75/200), 600 mg of everolimus with a purity of 80% was dissolved in dichloromethane and applied, followed by ethyl acetate/heptane/1-propanol. = 20/80/1 mixed solvent was used as the eluent at the beginning, and ethyl acetate/heptane/1-propanol = 30/70/1 mixed solvent was used as the eluent at the end.

Each fraction was analyzed by the HPLC analysis system of the test method listed in the European Pharmacopoeia, and fractions with an everolimus purity of 99% or more were collected and concentrated under reduced pressure using an evaporator. At that time, precipitation of everolimus was suppressed by concentrating under reduced pressure while adding ethyl acetate.

After that, the solution was concentrated to an everolimus concentration of 33 wt%, and the concentrated solution was added dropwise to 600 mL of cooled heptane to obtain a precipitate. The precipitate was filtered under reduced pressure while washing with heptane, and dried under reduced pressure to obtain 100 mg of high-purity everolimus powder (yield: 21%).

After preparing a movable stopper column filled with 50 g of Fuji Silysia Diol silica gel (DIOL MB100-75/200), 600 mg of everolimus with a purity of 80% dissolved in dichloromethane was applied, and ethyl acetate/heptane/acetonitrile = 25. /75/1 mixed solvent was used as the eluent at the beginning, and ethyl acetate/heptane/acetonitrile=35/65/1 mixed solvent was used as the eluent at the end.

Each fraction was analyzed by the HPLC analysis system of the test method listed in the European Pharmacopoeia, and fractions with an everolimus purity of 99% or more were collected and concentrated under reduced pressure using an evaporator. At that time, precipitation of everolimus was suppressed by concentrating under reduced pressure while adding ethyl acetate.

After that, the solution was concentrated to an everolimus concentration of 33 wt%, and the concentrated solution was added dropwise to 600 mL of cooled heptane to obtain a precipitate. The precipitate was filtered under reduced pressure while washing with heptane, and dried under reduced pressure to obtain 102 mg of high-purity everolimus powder (yield: 21%).

After preparing a movable stopper column packed with 20 g of Fuji Silysia's diol silica gel (DIOL SMB100-20/45), 125 mg of everolimus with a purity of 80% dissolved in dichloromethane was applied, followed by ethyl acetate/heptane/1-propanol. = 20/80/1 mixed solvent was used as the eluent at the beginning, and ethyl acetate/heptane/1-propanol = 30/70/1 mixed solvent was used as the eluent at the end.

Each fraction was analyzed by the HPLC analysis system of the test method listed in the European Pharmacopoeia, and fractions with an everolimus purity of 99% or more were collected and concentrated under reduced pressure using an evaporator. At that time, precipitation of everolimus was suppressed by concentrating under reduced pressure while adding ethyl acetate.

After that, the solution was concentrated to an everolimus concentration of 33 wt%, and the concentrated solution was added dropwise to 100 mL of cooled heptane to obtain a precipitate. The precipitate was filtered under reduced pressure while washing with heptane, and dried under reduced pressure to obtain 55 mg (yield 55%) of high-purity everolimus powder.

After preparing a movable stopper column filled with 20 g of Fuji Silysia Diol silica gel (DIOL SMB100-20), 125 mg of everolimus with a purity of 80% dissolved in dichloromethane was applied, and ethyl acetate/heptane/1-propanol = 25. /75/1 mixed solvent was used as the eluent at the beginning, and ethyl acetate/heptane/1-propanol=35/65/1 mixed solvent was used as the eluent at the end.

Each fraction was analyzed by the HPLC analysis system of the test method listed in the European Pharmacopoeia, and fractions with an everolimus purity of 99% or more were collected and concentrated under reduced pressure using an evaporator. At that time, precipitation of everolimus was suppressed by concentrating under reduced pressure while adding ethyl acetate.

After that, the solution was concentrated to an everolimus concentration of 33 wt%, and the concentrated solution was added dropwise to 100 mL of cooled heptane to obtain a precipitate. The precipitate was filtered under reduced pressure while washing with heptane, and dried under reduced pressure to obtain 80 mg of high-purity everolimus powder (80% yield).

After preparing a movable stopper column filled with 20 g of Fuji Silysia Diol silica gel (DIOL SMB100-20), 125 mg of everolimus with a purity of 80% dissolved in dichloromethane was applied, and ethyl acetate/heptane/acetonitrile = 25/75. /1 mixed solvent was used as the eluent at the beginning, and ethyl acetate/heptane/acetonitrile=35/65/1 mixed solvent was used as the eluent at the end.

Each fraction was analyzed by the HPLC analysis system of the test method listed in the European Pharmacopoeia, and fractions with an everolimus purity of 99% or more were collected and concentrated under reduced pressure using an evaporator. At that time, precipitation of everolimus was suppressed by concentrating under reduced pressure while adding ethyl acetate.

After that, the solution was concentrated to an everolimus concentration of 33 wt%, and the concentrated solution was slowly added dropwise to 100 mL of cooled heptane to obtain a precipitate. The precipitate was filtered under reduced pressure while washing with heptane, and dried under reduced pressure to obtain 79 mg of high-purity everolimus powder (79% yield).

Claims (6)

a step of purifying the crude everolimus using normal phase chromatography using diol silica gel as the stationary phase and an eluent consisting of a mixed solvent of ethyl acetate and heptane to which a hydrophilic solvent has been added; A method for producing everolimus, comprising: 2. The production method according to claim 1, wherein the stationary phase used for normal phase chromatography is silica gel having a particle size of 5 to 250 μm. The production method according to claim 1, wherein the ethyl acetate/heptane mixed solvent in the eluent used for normal phase chromatography has a solvent composition in the range of 20/80 to 50/50 on a volume basis. . A hydrophilic solvent selected from acetonitrile, acetone, methanol, ethanol, 1-propanol, and 2-propanol is added at 0.5 to 1.5 v/v per ethyl acetate/heptane mixed solvent in the eluent used for normal phase chromatography. 2. The manufacturing method according to claim 1, characterized in that v % is added. After the step of purifying crude everolimus using normal phase chromatography, the step of substituting the everolimus-containing fraction obtained in the above step with an esterified solvent while concentrating under reduced pressure; and adding to a solvent to precipitate everolimus. 6. The method according to claim 5, characterized in that the esterifying solvent is selected from methyl formate, ethyl formate, methyl acetate, ethyl acetate, propyl acetate, butyl acetate and the non-polar solvent is selected from pentane, hexane, heptane, octane. Method of manufacture as described.