WO2020095888A1

WO2020095888A1 - Method for producing high-purity cholesterol

Info

Publication number: WO2020095888A1
Application number: PCT/JP2019/043252
Authority: WO
Inventors: 中島　秀幸; 敏哉上野
Original assignee: 日本精化株式会社
Priority date: 2018-11-05
Filing date: 2019-11-05
Publication date: 2020-05-14
Also published as: JPWO2020095888A1; JP7361715B2

Abstract

Provided is a method for producing high-purity cholesterol which does not contain a substantial amount of desmosterol, said method comprising reacting cholesterol mixed with desmosterol with a halogenating reagent and water and then separating and removing the thus formed desmosterol derivative by crystallization. According to this production method, hardly removable impurities such as desmosterol can be removed and thus high-purity cholesterol can be produced.

Description

Method for producing high-purity cholesterol

≪Cholesterol is one of the organic compounds classified into steroids, and along with proteins and carbohydrates, is an important compound involved in various life phenomena. Cholesterol is also used as a raw material for medicines, cosmetics, liquid crystals, and the like.

The purity of commercially available commercial cholesterol is 90 to 95% (based on the area% of gas chromatography (GC)). Among others, the presence of analog impurities is a cause of difficulty in obtaining higher-purity cholesterol.
Commercially available cholesterol contains several kinds of impurities similar to the structure of cholesterol, and for example, desmosterol, dihydrocholesterol, latosterol and the like are known.

As a conventionally known production method for obtaining high-purity cholesterol, a method of removing impurities of cholesterol by a reslurry method using alcohols such as methanol or a recrystallization method (Patent Documents 1 and 2) and expensive A method for converting desmosterol into cholesterol by a catalytic reduction method using a hydrogenation catalyst (Patent Document 3), or a purification method using a special device (for example, a manufacturing method using a tower-type crystallizer) (Patent Document 4). ) Can be mentioned. Therefore, no production method other than the reduction reaction is known as a production method for obtaining high-purity cholesterol by derivatizing impurities using a chemical reaction.

The above-mentioned impurities contained in cholesterol, especially desmosterol, which is a main impurity, have a structure similar to that of cholesterol, and physical properties such as solubility are also similar. It was extremely difficult to separate and remove with the purification method. Further, according to these recrystallization methods, since the operation of recrystallization is required to be repeated, the operation is complicated and the cholesterol yield is lowered. Furthermore, it has been extremely difficult to obtain high-purity cholesterol by the recrystallization method.

Further, according to the method of converting desmosterol into cholesterol by the catalytic reduction method using an expensive hydrogenation catalyst, impurities contained in cholesterol can be reduced by the reaction, but some cholesterol can be reduced by the reduction treatment. Reacts with each other to produce dihydrocholesterol, resulting in a decrease in yield.

Further, for the purpose of obtaining 25-hydroxycholesterol, desmosterol (24-dehydrocholesterol) is reacted with N-bromosuccinimide (NBS) in a THF / water mixed solution to obtain an oxybromide, and then an aluminum hydride reagent Although a method for obtaining 25-hydroxycholesterol, which is a debrominated product, is known (Patent Document 5), this production method is not intended to purify cholesterol.

Therefore, it is possible to remove impurities (especially desmosterol) that are difficult to remove by the conventional production method by crystallization, and efficiently and highly pure cholesterol can be obtained by an industrially applicable operation method. There is a need for a manufacturing method that can be manufactured.

JP, 2016-003190, A JP, 2013-184929, A Japanese Patent Laid-Open No. 06-239883 JP 2002-08049A China Patent Publication No. 1036268822

The present invention aims to provide a production method for producing high-purity cholesterol (for example, cholesterol having a purity of about 98%) that does not contain desmosterol in a substantial amount in a high yield.

As a result of diligent studies by the present inventors, cholesterol containing impurities such as desmosterol is caused to act on a halogenating reagent to selectively convert desmosterol into highly polar impurities, and the impurities are easily removed by a subsequent crystallization step. It has been found that it can be removed and that it is industrially applicable. The present invention provides the following aspects, but is not limited thereto.
(Production method)
[1] A method for producing high-purity cholesterol which does not contain desmosterol in a substantial amount, which comprises reacting cholesterol containing desmosterol with a halogenating reagent and water and fractionally removing the resulting desmosterol derivative by crystallization.
[2] A method for producing high-purity cholesterol, which comprises the following steps:
1) mixing desmosterol-containing cholesterol with a halogenating reagent in water and an organic solvent to react desmosterol with the halogenating reagent and water (step 1),
If necessary, a reducing agent is added to the obtained reaction solution to decolorize it;
2) If necessary, after the reaction, remove the aqueous layer (step 2);
3-1) Use the organic layer as it is in the next step after the reaction, or
3-2) The organic layer is partially concentrated or completely concentrated to dryness to obtain a crude reaction product (step 3);
4) An organic solvent is added to the organic layer of 3-1) or the crude reaction product of 3-2) to cause crystallization in the organic solvent (step 4),
The production method including the following (hereinafter, also referred to as “production method of the present invention”).
[2-2] A reaction of reacting cholesterol containing desmosterol with a halogenating reagent and water (step 1 of Item [2])) at a temperature of about 0 ° C. to about 100 ° C. for about 10 minutes to about 12 hours. The method according to [1] or [2], which is performed.
[2-3] A reaction of reacting cholesterol containing desmosterol with a halogenating reagent and water (step 1 of Item [2])) at a temperature of about 0 ° C. to about 60 ° C. for about 30 minutes to about 3 hours. The method according to [1] or [2], which is performed.

[2-4] The reducing agent is one or more selected from the group consisting of sodium sulfite, sodium hydrogen sulfite, sodium thiosulfate, oxalic acid and its salts, and formic acid and its salts, [1] or [2] ] The manufacturing method of description.
[2-5] When the halogenating reagent used in the reaction of reacting cholesterol containing desmosterol with a halogenating reagent and water (Step 1 of [2]) is N-iodosuccinimide (NIS) or iodine The method according to [1] or [2], which further comprises adding sodium sulfite or sodium formate to decolorize.
[2-6] Furthermore, 5) the precipitate containing cholesterol crystallized in 4) above is further washed with an organic solvent to obtain purified cholesterol (step 5), and / or
6) Cholesterol crystals are precipitated by dissolving the cholesterol obtained in 4) or 5) in a solvent having a high solubility for cholesterol, and then adding a solvent having a low solubility for cholesterol to the solution (step 6),
The production method according to [1] or [2], which comprises:

[3] The halogenating reagent used in the reaction of reacting cholesterol containing desmosterol with the halogenating reagent and water (step 1 of Item [2]) is N-iodosuccinimide (NIS) or N-bromosuccinimide (NBS). ), Bromine, iodine, N-bromophthalimide, N-bromosaccharin, N-bromoacetamide, 1,3-dibromo-5,5-dimethylhydantoin, dibromoisocyanuric acid, trimethylphenylammonium tribromide, benzyltrimethylammonium tribromide, Pyridinium bromide perbromide, 4-dimethylaminopyridinium bromide perbromide, 1-butyl-3-methylimidazolium tribromide, 1,8-diazabicyclo [5,4,0] -7-undecenehydrogentribromide, -One or more selected from the group consisting of iodophthalimide, N-iodosaccharin, 1,3-diiodo-5,5-dimethylhydantoin, pyridine iodine monochloride, tetramethylammonium dichloroiodate, and benzyltrimethylammonium dichloroiodate. [1] or [2], which is the electrophilic halogenating reagent of.
[3-2] The halogenating reagent used in the reaction of reacting cholesterol containing desmosterol with a halogenating reagent and water (step 1 of Item [2]) is N-iodosuccinimide (NIS). ] Or the manufacturing method as described in [2].

[3-3] The organic solvent used in the reaction of reacting cholesterol containing desmosterol with a halogenating reagent and water (step 1 of Item [2]) is an ether, an alcohol, a hydrocarbon, a halogenated carbon. The production method according to [1] or [2], which is one or more organic solvents selected from the group consisting of hydrogens, esters, amides, nitriles, and ketones.
[3-4] In [1] or [2], the organic solvent used in the reaction of reacting cholesterol containing desmosterol with a halogenating reagent and water (step 1 of Item [2]) is an ether. The manufacturing method described.
[3-5] [1] or [2], wherein the organic solvent used in the reaction of reacting cholesterol containing desmosterol with a halogenating reagent and water (step 1 of Item [2]) is tetrahydrofuran (THF). ] The manufacturing method of description.

[4] The halogenating reagent used in the reaction of reacting cholesterol containing desmosterol with a halogenating reagent and water (step 1 of Item [2]) is based on 1 mol of desmosterol contained in cholesterol. The production method according to any one of [1] to [3], which is about 1 to about 10 molar equivalents.
[4-2] The halogenating reagent used in the reaction of reacting cholesterol containing desmosterol with a halogenating reagent and water (step 1 of item [2]) is based on 1 mol of desmosterol contained in cholesterol. And the production method according to any one of [1] to [3], which is about 1 to about 4 molar equivalents.

[5] The organic solvent used in the crystallization (step 4 of Item [2]) is an ether, a ketone, an ester, a hydrocarbon, a halogenated hydrocarbon, an amide, a nitrile, or an alcohol. The production method according to any one of [1] to [4], which is one or more kinds of organic solvents selected from the group consisting of:
[5-2] The organic solvent used in the crystallization (step 4 of Item [2]) is one or more organic solvents selected from the group consisting of ethers, ketones, esters, and alcohols. The manufacturing method according to any one of [1] to [4].
[5-3] Any one of [1] to [4], wherein the organic solvent used in crystallization (step 4 of Item [2]) is THF / methanol, methanol, butanol, ethyl acetate, or acetone. The manufacturing method according to one.
[5-4] The production method according to any one of [1] to [4], wherein the organic solvent used in crystallization (step 4 of Item [2]) is THF / methanol.
[5-5] The organic solvent used in the crystallization (step 4 of Item [2]) is a THF / methanol mixed solution in which the volume ratio of THF and methanol is 1: 3, [1] to [4]. ] The manufacturing method as described in any one of.

[5-6] Cholesterol used in the reaction of reacting cholesterol containing desmosterol with a halogenating reagent and water (step 1 of item [2])) contains one or more impurities selected from dihydrocholesterol and latosterol. The manufacturing method according to any one of [1] to [4].

[5-7] The crystallization in crystallization (step 4 of item [2])) is performed at a temperature of about −15 ° C. to about 60 ° C. or lower, according to any one of [1] to [5]. Manufacturing method.
[5-8] The production according to any one of [1] to [5], wherein crystallization in crystallization (step 4 of Item [2]) is performed at a temperature of about 0 ° C. to about 30 ° C. Method.

[6] The production method according to any one of [1] to [5], wherein the high-purity cholesterol is cholesterol having a purity of about 98% or more.
[6-2] The production method according to any one of [1] to [5], wherein the high-purity cholesterol is cholesterol having a purity of about 99% or more.

(High purity cholesterol)
[7] High-purity cholesterol containing desmosterol in a content of about 1% or less, which is obtained by the production method according to any one of [1] to [6].
[7-2] High-purity cholesterol obtained by the production method according to any one of [1] to [6], containing desmosterol in a content of about 0.3% or less.
[8] The high-purity cholesterol according to [7], which contains a trace amount of a halogenated derivative of cholesterol.

(Separation method)
[9] A method of separating cholesterol and desmosterol by reacting a mixture containing cholesterol and desmosterol with a halogenating reagent and water, and then crystallizing cholesterol.

The production method of the present invention makes it possible to efficiently obtain high-purity cholesterol from which impurities (mainly desmosterol) that were difficult to remove by conventional methods (for example, crystallization method) are removed.

Hereinafter, the present invention will be described in more detail.
(Definition)
Listed below are definitions of terms used in the specification and claims.

The term "cholesterol" in the present specification is a compound represented by the following structural formula (CAS registration number 57-88-5). Examples of the cholesterol include a commercially available product and a product synthesized by a generally known organic chemical method, and for example, a commercially available product is included. Cholesterol, which is a usual commercial product, means cholesterol having a purity of 90% or more (for example, about 95%) and also containing, as impurities, several kinds of impurities similar to the structure of cholesterol. The impurities include desmosterol, dihydrocholesterol, lanosterol, dihydrolanosterol, or latosterol.

The term "desmosterol" in the present specification is a compound represented by the above structural formula (CAS registration number 313-04-2). Desmosterol is usually contained in commercially available cholesterol as an impurity in a proportion of about 1 to about 5% (for example, 2 to 4%, typically about 3%) as an area percentage at the time of GC measurement. Alternatively, it is contained in a proportion of about 5 to about 15% (eg, about 6 to 10%, typically about 8%) as an area percentage when measuring LC (eg, HPLC).

Unless otherwise specified, the terms “purity” and “content” in the present specification refer to general instrumental analysis methods (for example, gas chromatography (GC), and liquid chromatography (LC) (for example, high performance liquid chromatography). It means the area percentage at the time of measurement by graphography (HPLC).

The term “high-purity cholesterol” in the present specification means that the purity is about 95% or more, preferably about 96% or more, more preferably about 97% or more, still more preferably about 98% as an area percentage when measuring GC. % Or more, particularly preferably about 99% or more cholesterol.

As used herein, the term "not containing a substantial amount of desmosterol" in the term "not containing a substantial amount" is below a level detectable by a usual analytical method (eg, HPLC method). It means that the content of desmosterol with respect to cholesterol is about 1.0% or less, preferably about 0.3% or less, more preferably about 0.2% or less, and still more in terms of area percentage. It means preferably about 0.1% or less. Alternatively, it means that it is about 0.3% or less, preferably about 0.2% or less, and more preferably about 0.1% or less in terms of area percentage during GC measurement.

According to one aspect of the present invention, a manufacturing method of the present invention is
A method for producing high-purity cholesterol that does not contain desmosterol in a substantial amount, which comprises reacting cholesterol containing desmosterol with a halogenating reagent and water and fractionally removing the resulting desmosterol derivative by crystallization.
According to the production method of the present invention, 1) by reacting a mixture of cholesterol containing desmosterol as a main impurity with a halogenating reagent and water, cholesterol is not reacted and only desmosterol is selectively reacted. It can be derivatized; 2) Desmosterol can be converted to a derivative that has physical properties (eg, solubility) greatly different from cholesterol; and 3) using organic solvents commonly used in organic synthesis. , The derivative of cholesterol and desmosterol can be easily separated by crystallization to remove the derivative of desmosterol; 4) compared with the conventional purification method (for example, recrystallization method), Can be separated very easily and with high efficiency. The removal efficiency is significantly improved; 5) The desmosterol content can be reduced to an extremely small amount, and high-purity cholesterol containing no substantial amount of desmosterol can be obtained in a high yield and a high yield. It has the advantage that it can be obtained with efficiency.

According to one aspect of the present invention, a manufacturing method of the present invention is
A method for producing high-purity cholesterol, comprising the following steps:
Step 1) mixing desmosterol-containing cholesterol with a halogenating reagent in water and an organic solvent to react desmosterol with the halogenating reagent and water,
If necessary, a reducing agent is added to the obtained reaction solution to decolorize it;
Step 2) If necessary, remove the aqueous layer after the reaction;
Step 3-1) Use the organic layer as it is in the next step, or
Step 3-2) The organic layer is partially concentrated or completely concentrated to dryness to obtain a crude reaction product;
Step 4) An organic solvent is added to the organic layer of 3-1) or the crude reaction product of 3-2) to crystallize cholesterol.
Thus, purified cholesterol is obtained.

(Process 1)
In step 1, cholesterol containing desmosterol is suspended in water and an organic solvent in a reaction vessel, a halogenating reagent is added to the suspension, and desmosterol is selectively reacted with the halogenating reagent and water. Then, as described above, desmosterol is converted into a polar compound derivative thereof (for example, a compound in which a halogen atom and a hydroxy group are added to an alkene moiety in desmosterol).

The term “halogenating reagent” means a halogenating reagent generally known in the field of organic chemistry. Preferably, a bromination reagent or an iodination reagent is used. Specific examples of the brominating reagent include bromine, N-bromosuccinimide (NBS) N-bromophthalimide, N-bromosaccharin, N-bromoacetamide, 1,3-dibromo-5,5-dimethylhydantoin and dibromoisocyanurin. Acid, trimethylphenylammonium tribromide, benzyltrimethylammonium tribromide, pyridinium bromide perbromide, 4-dimethylaminopyridinium bromide perbromide, 1-butyl-3-methylimidazolium tribromide, and 1,8-diazabicyclo [5,4,4] Examples include, but are not limited to, one or more electrophilic brominating reagents selected from the group consisting of 0] -7-undecenehydrogen tribromide. Specific examples of the iodination reagent include iodine, N-iodosuccinimide (NIS) N-iodophthalimide, N-iodosaccharin, 1,3-diiodo-5,5-dimethylhydantoin, pyridine iodine monochloride, dichloroiodine. Examples thereof include, but are not limited to, one or more electrophilic iodination reagents selected from the group consisting of acid tetramethylammonium and benzyltrimethylammonium dichloroiodate. Examples of more preferred halogenating reagents include, but are not limited to, N-iodosuccinimide (NIS), N-bromosuccinimide (NBS), bromine, or iodine. N-iodosuccinimide (NIS) is preferable in terms of the purity of the target cholesterol and the handling of the operation.

The organic solvent used in step 1 is not particularly limited as long as it does not adversely affect the reaction, but a solvent that partially or completely dissolves cholesterol is preferable. For example, ethers (eg, diethyl ether, t-butyl methyl ether (MTBE), dimethoxyethane (DME), tetrahydrofuran (THF), 2-methyltetrahydrofuran (MeTHF)), alcohols (eg, isopropyl alcohol (IPA), 1-butanol, 2-butanol, t-butanol), hydrocarbons (eg toluene, xylene, heptane), halogenated hydrocarbons (eg methylene chloride, chloroform, monochlorobenzene (MCB)), esters (eg , Ethyl acetate, methyl acetate, butyl acetate), amides, nitriles, and ketones (for example, methyl ethyl ketone (MEK), methyl isobutyl ketone (MIBK)). . Preferred are ethers, especially tetrahydrofuran (THF) derivatives such as THF and 2-methyltetrahydrofuran (MeTHF). The amount of the solvent is preferably as small as possible from the economical viewpoint, but for example, the amount is about 1 to about 10 times by weight, and preferably about 2 to about 10 times by weight, relative to the crude cholesterol used in the reaction. 4 times the weight.

The amount of the halogenating reagent used in step 1 may vary depending on the content of desmosterol contained in cholesterol and the reactivity and type of the halogenating reagent used, but an amount sufficient to react with desmosterol It may be, for example, about 1 to about 50 molar equivalents, preferably about 1 to about 10 molar equivalents, relative to 1 mole of desmosterol contained in cholesterol. Typically, when NIS or I ₂ is used, about 0.1 molar equivalents per 1 mole of cholesterol are mentioned, and when NBS is used, relative to 1 mole of cholesterol. And about 0.05 molar equivalent.

The reaction of step 1 is carried out under an air atmosphere (preferably under a nitrogen atmosphere). The reaction temperature may vary depending on the kind of the halogenating reagent used, but is, for example, about 0 ° C. to about 100 ° C., preferably about 20 ° C. to about 60 ° C. Typically, the reaction temperature is about 40 ° C. to about 60 ° C. when NIS or I ₂ is used, and the reaction temperature is about 20 ° C. to about 40 ° C. when NBS is used. is there.
The reaction time may vary depending on the kind of the halogenating reagent used and the reaction temperature, but is, for example, about 10 minutes to about 12 hours (eg, about 30 minutes to about 3 hours).

After completion of the reaction in step 1, free halogen may be removed by quenching the reaction solution by adding a reducing agent to the reaction solution in step 1, if necessary. The reducing agent is a reagent that removes the coloring derived from the halogenating reagent to be used by a reducing action, and includes, for example, sodium sulfite, sodium hydrogen sulfite, sodium thiosulfate, oxalic acid and salts thereof (for example, sodium salt and potassium salt). Salt), formic acid and salts thereof (for example, sodium salt and potassium salt), and the like, but are not limited thereto. For example, a reducing agent (eg, sodium sulfite, sodium formate) can be used when the halogenating reagent is N-iodosuccinimide (NIS) or iodine. The reducing agent may be in the form of powder or an aqueous solution. The reducing agent may be used in such an amount that coloring is eliminated, for example, 1 molar equivalent or more relative to 1 molar amount of the halogenating reagent used. In addition, typically, the reducing agent can be used in an amount of 0.1 to 0.5 molar equivalent, preferably 0.2 to 0.4 molar equivalent, based on 1 mole of cholesterol. ..
The quenching operation can be performed at a temperature similar to the reaction temperature in Step 1, for example, about 0 ° C. to about 100 ° C. (eg, about 20 ° C. to about 60 ° C.).

(Process 2)
If necessary, the aqueous layer is removed from the reaction solution after the reaction in Step 1 (or after the decoloring operation). The aqueous layer may contain, for example, an excessive amount of halogenating reagent and its decomposition product, and an inorganic salt. The removal of the aqueous layer can be performed by a separation operation (for example, a separating funnel).

(Process 3)
The organic layer obtained by removing the aqueous layer in step 2 is used as it is in step 4 (step 3-1)), or the organic layer is partially concentrated or completely concentrated to dryness to obtain a crude reaction product ( Step 3-2).
In the case of step 3-1, the organic layer obtained in step 2 is dried with a desiccant commonly used in organic synthesis (eg magnesium sulfate, sodium sulfate) before being used in the next step 4. May be.
Alternatively, in the case of step 3-2, the organic layer obtained in step 2 is partially concentrated or completely concentrated to dryness (for example, concentrated to dryness under reduced pressure using an evaporator) to give a crude reaction product. To get

(Process 4)
An organic solvent is added to the organic layer of step 3-1 or the crude reaction product of step 3-2 to crystallize cholesterol in the organic solvent.

The term "crystallization" in the present specification means to precipitate cholesterol crystals, and also includes maintaining the crystallization liquid for a certain period of time in an environment of lower temperature to grow the precipitated crystals. obtain.

In step 4, an organic solvent is added to crystallize cholesterol. The temperature during the crystallization operation may be the reaction temperature after the reaction in step 2, and examples thereof include about 0 ° C. to about 100 ° C. (eg, about 20 ° C. to about 60 ° C.).
Then, the crystallization liquid may be cooled to promote crystallization of cholesterol.
The cooling temperature may be a low temperature at which cholesterol crystals grow, and examples thereof include -15 ° C to about 10 ° C, preferably about 0 ° C to about 10 ° C (typically about 0 ° C). Up to about 5 ° C.). Therefore, when cooling, the temperature during the crystallization operation is, for example, about -15 ° C to about 60 ° C, preferably about 0 ° C to about 30 ° C.
The cooling time may be a period during which cholesterol crystals sufficiently grow, but for example, it may be about 10 minutes to several days, preferably about 30 minutes to about 12 hours (typically about 1 hour or more).

The organic solvent used in step 4 is preferably a polar organic solvent, but in the case of a low polar organic solvent, it may be used in combination with a polar solvent. Examples of the organic solvent include organic solvents used in Step 1, for example, ethers (eg, diethyl ether, t-butylmethyl ether (MTBE), dimethoxyethane (DME), tetrahydrofuran (THF), 2-methyltetrahydrofuran ( MeTHF)), ketones (eg methyl ethyl ketone (MEK), methyl isobutyl ketone (MIBK)), esters (eg ethyl acetate, methyl acetate, butyl acetate), halogenated hydrocarbons (eg methylene chloride, chloroform, Monochlorobenzene (MCB)), hydrocarbons (eg toluene, xylene, heptane), amides, nitriles and alcohols (eg isopropyl alcohol (IPA), 1-butanol, 2-butanol, t-butanol). One or more organic solvents selected from Ranaru group and the like.
The amount of the organic solvent used in step 4 is not particularly problematic as long as it is an appropriate amount with respect to cholesterol as a target product, but if the amount is too large, the yield decreases, while if it is too small, However, there is a possibility that the crystallized cholesterol has poor fluidity, the operability is deteriorated, and impurities remain.

Further, the method may further include a step (step 5) of washing the precipitate containing cholesterol crystallized in step 4 with an organic solvent to obtain purified cholesterol. In step 5, a usual post-treatment step (evaporation of solvent, filtration, and drying) can be included, if necessary.
Typically, in step 5, the reaction liquid containing the precipitate obtained in step 4 is filtered first. Then, the collected material is washed with an organic solvent. The washed wet crystals are dried (about 50 ° C. to about 60 ° C.) to obtain purified cholesterol as a target, that is, high-purity cholesterol.

The organic solvent for washing used in step 5 is preferably an organic solvent having low solubility in cholesterol in order to suppress cholesterol loss during washing. For example, alcohols (for example, methanol), acetone, acetonitrile are mentioned.

Further, the precipitate containing the cholesterol crystallized in the step 4 or the cholesterol obtained after the washing in the step 5 is further purified by a known purification method (for example, suspension washing method, recrystallization method, chromatography). It is also possible to purify by combining the method). In particular, the production method of the present invention may include a step of recrystallizing (step 6).

In step 6, if necessary, the cholesterol obtained in step 4 or step 5 is dissolved in a solvent having a high solubility for cholesterol (that is, a good solvent), and then the solubility of cholesterol in the solution is low. Cholesterol crystals are precipitated by adding a solvent (that is, a poor solvent). The formed slurry is cooled (5 ° C or lower), and the precipitate is filtered. Then, the collected material is washed with a poor solvent. The washed precipitate is dried (about 50 ° C. to about 60 ° C.) to obtain purified cholesterol as a target, that is, high-purity cholesterol.

Examples of the good solvent used in step 6 include, but are not limited to, a toluene / methanol mixed solution, a heptane / methanol mixed solution, THF and the like. When a toluene / methanol mixture or a heptane / methanol mixture is used, examples include a mixture of toluene or heptane and methanol in a volume ratio of about 50: 1 to about 2: 1. Further, examples of the poor solvent include, but are not limited to, methanol (may be hydrous methanol), heptane, and the like. The good solvent and the poor solvent may be used in an amount of about 10 mL to about 50 mL (for example, about 10 mL to about 30 mL is preferable) of the good solvent with respect to 12.00 g of the cholesterol raw material to be purified. Further, the amount of the poor solvent used can be about 3 times to about 20 times (for example, about 4 times to about 15 times) the volume ratio of the good solvent.

In the production method of the present invention, by the crystallization of step 4 (further, the washing of step 5), the desmosterol derivative (for example, a polar compound) generated in step 1 is separated from the crystallized cholesterol and removed. It is considered that purified cholesterol can be easily obtained.

(Purified cholesterol)
The purified (purified) cholesterol produced by the production of the present invention contains desmosterol in a content of about 1% or less, and is appropriately derived from the reaction with a halogenating reagent used at the time of production, It is high-purity cholesterol containing a halogenated derivative of cholesterol. Further, the purified cholesterol includes only desmosterol having an area percentage of about 0.3% or less at the time of GC measurement and about 1.0% or less as an area percentage at the time of LC measurement. Cholesterol having a purity of about 98% or greater, which may include derivatives, is preferred. Here, the minute amount of the halogenated derivative means a detectable limit amount, for example, an amount of about 1% or less, about 0.5% or less, or about 0.1% or less as an area percentage during GC measurement. Means

According to one embodiment of the invention,
The halogenating reagent used in step 1 is NIS or I ₂ , the organic solvent used in step 1 is THF, step 3 is step 3-1 and the organic solvent used in step 4 is THF / Provided is a manufacturing method which is methanol.

Includes adding sodium sulfite or sodium formate to the reaction solution of step 1 for decolorization after completion of the reaction of step 1.

According to one embodiment of the invention,
The halogenating reagent used in step 1 is NBS, the organic solvent used in step 1 is THF, step 3 is step 3-1 and the organic solvent used in step 4 is THF / methanol. , A manufacturing method is provided.

According to one embodiment of the present invention, there is provided a production method, wherein the organic solvent used in Step 5 is methanol.

According to one embodiment of the present invention, there is provided a method for producing cholesterol of higher purity, which further comprises recrystallizing the purified cholesterol obtained in step 4 or 5 in step 6.

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not specifically limited to these Examples.
Commercial cholesterol (Nippon Seika Co., Ltd. product: trade name CHOLESTEROL) (cholesterol purity is about 89% as an area percentage at the time of HPLC measurement, 96% as an area percentage at the time of GC measurement, and desmosterol content is The area percentage at the time of HPLC measurement is about 8%, and the area percentage at the time of GC measurement is about 3%)) was used in the following Examples and Comparative Examples.

First, the presence or absence of a halogenating reagent in the production method of the present invention and the influence of the crystallization solvent were examined.

Example 1
Production method using N-iodosuccinimide (NIS) After dissolving 10 g (25.9 mmol) of commercially available cholesterol (product of Nippon Seika Co., Ltd .: trade name CHOLESTEROL) in 34 mL of THF, 20 g of water (1.1 mol) and NIS 0. 0.6 g (2.6 mmol) was added, and the mixture was reacted with stirring at 50 ° C. for 1 hour.
0.5 g of sodium sulfite was added to the reaction solution to decolorize the organic layer, and then the aqueous layer was removed. After adding 76 mL of methanol at 50 to 60 ° C. to the organic layer, the mixture was cooled to 5 ° C. or lower and then kept at 0 to 5 ° C. for 1 hour or more to precipitate crystals. The precipitate was filtered, and the separated precipitate was washed with 13 mL of methanol. The precipitate was dried under reduced pressure to obtain 8.5 g of purified cholesterol (yield 85%).
The purity of the obtained purified cholesterol and the content of desmosterol were measured by gas chromatography (GC) analysis. The results are shown in Table 1.

Example 2
Production Method Using N-Bromosuccinimide (NBS) 10 g (25.9 mmol) of commercially available cholesterol (product of Nippon Seika Co., Ltd .: trade name CHOLESTEROL) was dissolved in 34 mL of THF, and then 20 g (1.1 mol) of water and NBS 0 0.2 g (1.3 mmol) was added, and the mixture was reacted with stirring at 20 ° C. for 1 hour.
After the reaction, the aqueous layer was removed. After adding 76 mL of methanol at 50 to 60 ° C. to the organic layer, the mixture was cooled to 5 ° C. or lower and then kept at 0 to 5 ° C. for 1 hour or more to precipitate crystals. The precipitate was filtered, and the separated precipitate was washed with 13 mL of methanol. The precipitate was dried under reduced pressure to obtain 8.3 g of purified cholesterol (yield 83%).
The purity of the obtained purified cholesterol and the content of desmosterol were measured by gas chromatography (GC) analysis. The results are shown in Table 1.

Example 3
Production method using iodine (I2) 10 g (25.9 mmol) of commercially available cholesterol (product of Nippon Seika Co., Ltd .: trade name CHOLESTEROL) was dissolved in 34 mL of THF, and then 20 g (1.1 mol) of water and 0.7 g of iodine ( 2.6 mmol) was added and the mixture was reacted at 50 ° C. for 2 hours with stirring.
0.5 g of sodium sulfite was added to the reaction solution to decolorize the organic layer, and then the aqueous layer was removed. After adding 76 mL of methanol at 50 to 60 ° C. to the organic layer, the mixture was cooled to 5 ° C. or lower and then kept at 0 to 5 ° C. for 1 hour or more to precipitate crystals. The precipitate was filtered, and the separated precipitate was washed with 13 mL of methanol. The precipitate was dried under reduced pressure to obtain 8.3 g of purified cholesterol (yield 83%).
The purity of the obtained purified cholesterol and the content of desmosterol were measured by gas chromatography (GC) analysis. The results are shown in Table 1.

Example 4
Production method using N-iodosuccinimide (NIS) 1.5 kg (25.9 mmol) of commercially available cholesterol (product of Nippon Seika Co., Ltd .: trade name CHOLESTEROL) is dissolved in 4.5 kg of THF, and then 3.0 kg of water (1 0.1 mol) and NIS 87.3 g (2.6 mmol) were added, and the mixture was reacted with stirring at 50 ° C. for 2.5 hours.
750 g of 10% aqueous sodium sulfite solution was added to the reaction solution to decolorize the organic layer, and then the aqueous layer was removed. The organic layer was washed and separated with 1.5 kg of 20% saline and then cooled, and 9.0 kg of 5% hydrous methanol was added at room temperature, and the mixture was cooled to 5 ° C or lower. After that, the precipitate was filtered after being kept at 0 to 5 ° C. for 1 hour or more, and the separated precipitate was washed with 1.5 kg of methanol. The precipitate was dried under reduced pressure to obtain 1.28 kg of purified cholesterol (yield 85%).
As a result of measuring the obtained purified cholesterol by gas chromatography (GC) analysis, the purity was 99.3% and the desmosterol content was 0.04%.
Further, the purified cholesterol was measured by high performance liquid chromatography (HPLC) analysis. As a result, the purity was 99.0% and the desmosterol content was 0.2%.

Example 5
Manufacturing method using N-iodosuccinimide (NIS) 50 g (12.9 mmol) of commercially available cholesterol (product of Nippon Seika Co., Ltd .: trade name CHOLESTEROL) is dissolved in a mixed solvent of 100 g of THF and 150 g of water, and 2.9 g of NIS ( 13 mmol) was added, and the mixture was reacted with stirring at 50 ° C. for 4 hours.
To the reaction solution was added 17.5 g of a 20% sodium formate aqueous solution to decolorize the organic layer, and then the aqueous layer was separated and removed. The organic layer was washed with a small amount of water and then 370 mL of 5% hydrous methanol was added dropwise for crystallization. The obtained slurry was cooled to 5 ° C. or lower and then kept at 0 to 5 ° C. for 1 hour or more, and then the precipitate was filtered. The obtained precipitate was washed with 60 mL of methanol. The precipitate was dried under reduced pressure to obtain 45 g of purified cholesterol (yield: 90%).
As a result of measuring the obtained purified cholesterol by high performance liquid chromatography (HPLC) analysis, the purity was 99.0% and the desmosterol content was 0.2%.

Comparative Example 1
Crystallization-only manufacturing method (that is, manufacturing method without halogenating reagent)
6 g (15.5 mmol) of commercially available cholesterol (product of Nippon Seika Co., Ltd .: trade name CHOLESTEROL) was dissolved in 20 mL of THF, 46 mL of methanol was added at 50 to 60 ° C., and the mixture was cooled to 5 ° C. or lower. Then, the mixture was kept at 0 to 5 ° C. for 1 hour or more to precipitate crystals. The precipitate was filtered, and the separated precipitate was washed with 8 mL of methanol. The precipitate was dried under reduced pressure to obtain 4.5 g of purified cholesterol (yield 75%).
The purity of the obtained purified cholesterol and the content of desmosterol were measured by gas chromatography (GC) analysis. The results are shown in Table 1.

Comparative example 2
Manufacturing method only by crystallization 6 g (15.5 mmol) of commercially available cholesterol (product of Nippon Seika Co., Ltd .: trade name CHOLESTEROL) was put into 91 mL of methanol, and the mixture was kept at 60 ° C. for 1 hour. Then, the mixture was cooled to 30 ° C. or lower and kept at 20 to 30 ° C. for 1 hour or more to precipitate crystals. The precipitate was filtered, and the separated precipitate was washed with 4 mL of methanol. The precipitate was dried under reduced pressure to obtain 5.7 g (yield 95%) of purified cholesterol.
The purity of the obtained purified cholesterol and the content of desmosterol were measured by gas chromatography (GC) analysis. The results are shown in Table 1.

Comparative Example 3
Production method only by crystallization 3 g (7.8 mmol) of commercially available cholesterol (product of Nippon Seika Co., Ltd .: trade name CHOLESTEROL) is dissolved in 15 mL of 1-butanol by heating and then cooled to 20 to 25 ° C. Was held for 1 hour or more to precipitate crystals. The precipitate was filtered and the separated precipitate was washed with 1 mL of 1-butanol and dried under reduced pressure to obtain 2.1 g of purified cholesterol (yield 70%).

Comparative Example 4
Manufacturing method only by crystallization After dissolving 12 g (31 mmol) of commercially available cholesterol (product of Nippon Seika Co., Ltd .: trade name CHOLESTEROL) in a mixed solvent of 28 mL of toluene and 10 mL of methanol, 180 mL of methanol was added for crystallization. It was cooled to 0-5 ° C. and kept at this temperature for 1 hour or more. The precipitate was filtered, and the separated precipitate was washed with about 7 mL of methanol and then dried under reduced pressure to obtain 9.8 g of purified cholesterol (yield 82%).
As a result of measuring this purified cholesterol by high performance liquid chromatography (HPLC) analysis, the purity was 93.1% and the desmosterol content was 6.0%.

The results of Examples 1 to 3 and Comparative Examples 1 to 3 are shown in Table 1.

According to the production method of the present invention using a halogenating reagent, in any of Examples 1 to 5, a high purity of about 98% or more, particularly about 99% or more, is higher than that of cholesterol before purification. Cholesterol was obtained. In addition, the obtained purified cholesterol had a very small amount of desmosterol or was not contained at a detectable level as compared with the cholesterol before purification.
On the other hand, when the halogenating reagent was not used as a comparative example, the purity of the obtained cholesterol was about 98% or less, and the content of desmosterol in cholesterol was a significant amount.

Examples 6 to 17
Next, in the production method of the present invention, the influence of the crystallization solvent during the crystallization treatment of the reaction mixture reacted with the halogenating reagent was examined. Specifically, various solvents shown in Table 2 below were used as crystallization solvents, and the purity of the obtained purified cholesterol and the content of desmosterol were examined.

After dissolving 70 g (181 mmol) of commercially available cholesterol (Nippon Seika Chemicals Co., Ltd .: trade name CHOLESTEROL) in 236 mL of THF, 140 g (7.8 mol) of water and 4.1 g (18.1 mmol) of NIS were added, and the mixture was mixed with 50 The mixture was reacted with stirring at 1 ° C for 1 hour.
After adding 3.5 g of sodium sulfite to the reaction solution to decolorize the organic layer, the aqueous layer was removed. The organic layer was concentrated to dryness to obtain crude cholesterol (purity 96.19%, desmosterol not detected (ND)). Subsequently, the crude cholesterol was crystallized in various solvents. Examples using each solvent are shown as Examples 6 to 17.
To 5.5 g (13.0 mmol) of crude cholesterol, 2 times the amount of THF and 6 times the amount of methanol relative to the pure cholesterol were added and dissolved by heating. The mixture was cooled to 5 ° C. or lower and kept at 0 to 5 ° C. for 1 hour or more to precipitate crystals. The precipitate was filtered and the separated precipitate was dried under reduced pressure to obtain purified cholesterol (Example 16).
The purity of the obtained purified cholesterol and the content of desmosterol were measured by gas chromatography (GC) analysis.
Thereafter, the purity of each crystallization solvent was confirmed by the same method. The experimental results are shown in Table 2.

As a crystallization solvent, t-butyl methyl ether (Example 8), methylene chloride (Example 9), ethyl acetate (Example 11), acetone (Example 12), butanol (Example 14), methanol (Example) 15), THF / methanol (Example 16), and heptane / methanol (Example 17) were used, respectively, high-purity (for example, about 98% pure by GC area percentage) cholesterol was obtained. ..

Example 18
In the production method using N-iodosuccinimide (NIS), the solvent of THF / water used in Example 1 and the like is changed to various organic solvents to improve the purity of cholesterol and the content of desmosterol. I investigated the effect of.
5 g (12.9 mmol) of commercially available cholesterol (product of Nippon Seika Co., Ltd .: trade name CHOLESTEROL) was suspended in a mixed solvent of 20 g of organic solvent and 10 g of water, and 0.3 g (1.3 mmol) of NIS was added to the mixture to form a mixture. Was stirred and reacted at 50 ° C. for 3 hours.
To the reaction solution was added 1.75 g of 20% aqueous sodium formate to decolorize the organic layer, and then the aqueous layer was removed if the aqueous layer separated. The reaction solution (organic layer) was diluted with methanol to 50 mL, the mixture was cooled to 5 ° C. or lower, and then kept at 0 to 5 ° C. for 1 hour or more to precipitate crystals. The precipitate was filtered, and the separated precipitate was washed with 7 mL of methanol. The precipitate was dried under reduced pressure to obtain purified cholesterol.
The purity of the obtained purified cholesterol and the content of desmosterol were measured by high performance liquid chromatography (HPLC) analysis. The results are shown in Table 3.

When the organic solvent used in Step 1 in Example 1 and the like was replaced with THF / water and various organic solvents were used to examine the purity of cholesterol and the content of desmosterol, ethers, ketones, esters, In both cases of halogenated hydrocarbons and alcohols, the purity of the starting material cholesterol was remarkably improved, and the content of desmosterol was remarkably reduced. In particular, ethers, esters, alcohols are preferred, ethers are more preferred, and tetrahydrofuran derivatives (eg, tetrahydrofuran and 2-methyltetrahydrofuran) have been found to be particularly preferred.

Example 19
Furthermore, in order to obtain high-purity cholesterol, the purified cholesterol prepared in Example 4 was used for an additional purification operation.
12 g of purified cholesterol (purified cholesterol of Example 4) was dissolved in several types of good solvents, and a poor solvent was added to precipitate crystals. The purified slurry was cooled to 5 ° C. or lower and then kept at 0 to 5 ° C. for 1 hour or more, and then the precipitate was filtered. The obtained precipitate was washed with 10 mL of a poor solvent. The precipitate was dried under reduced pressure to obtain highly purified purified cholesterol.
The purity of the obtained purified cholesterol and the content of desmosterol were measured by high performance liquid chromatography (HPLC) analysis. The results are shown in Table 4.

In the above table, entries 18 to 23 were subjected to repurification treatment using cholesterol obtained by iodination treatment using NIS or the like as a raw material. On the other hand, entry 24 was subjected to repurification treatment using cholesterol obtained without iodination treatment (prepared in Comparative Example 4) as a raw material.
The cholesterol purity and the desmosterol content of each cholesterol raw material before the additional purification were as follows (the purity and the content are the area percentage values at the time of HPLC measurement).
Cholesterol raw material obtained by iodination (used in entries 18-22):
Purity 99.04%, desmosterol content 0.19%
Cholesterol raw material (used in entry 23) obtained by iodizing low purity cholesterol (cholesterol purity is 78.5% and desmosterol content is 12.3%):
Purity 98.0%, desmosterol content 0.6%
Cholesterol raw material obtained without iodination (used in entry 24):
Purity 93.1%, desmosterol content 6.0%
As a result of the above, in the case of entries 18 to 22, when the cholesterol obtained by the iodination treatment obtained in Example 4 was used as the raw material and the additional purification was performed, the high level of about 99.5% was obtained. It was possible to obtain pure cholesterol. In addition, in the case of entry 23, even if a low-purity cholesterol raw material is used, it is possible to obtain cholesterol having a significantly high degree of purity and a significantly low degree of desmosterol content by the iodination treatment. When purified, cholesterol with a purity close to about 99.0% could be obtained.
On the other hand, as entry 24, when cholesterol obtained without iodination was used as a raw material, 95% or more high-purity cholesterol could not be obtained even if additional purification was performed.
From this result, when the halogenation (eg, iodination) treatment in the production method of the present invention can significantly reduce the content of desmosterol contained in cholesterol as a starting material, the present Example It was found that the additional purification shown in 1) can further improve the purity of cholesterol. On the other hand, in the case of using cholesterol obtained without iodination treatment, the content of desmosterol contained in the starting material cholesterol cannot be reduced, and thus the additional purification of this example is performed. However, it was difficult to further improve the cholesterol purity, and it was not possible to obtain cholesterol having a purity of 95% or more.

By the production method of the present invention, high-purity cholesterol with extremely low content of impurities can be produced by an industrially efficient method.

Claims

A method for producing high-purity cholesterol containing substantially no desmosterol, which comprises reacting desmosterol-containing cholesterol with a halogenating reagent and water and fractionally removing the resulting desmosterol derivative by crystallization.
A method for producing high-purity cholesterol, comprising the following steps:
Cholesterol containing desmosterol is mixed with a halogenating reagent in water and an organic solvent to allow desmosterol to react with the halogenating reagent and water,
If necessary, a reducing agent is added to the obtained reaction solution to decolorize it. 2) If necessary, after the reaction, the aqueous layer is removed;
3-1) Use the organic layer as it is in the next step after the reaction, or
3-2) The organic layer is partially concentrated or completely concentrated to dryness to obtain a crude reaction product;
4) An organic solvent is added to the organic layer of 3-1) or the crude reaction product of 3-2) to cause crystallization in the organic solvent.
The manufacturing method comprising:
The halogenating reagent used in the reaction of reacting cholesterol including desmosterol with a halogenating reagent and water is N-iodosuccinimide (NIS), N-bromosuccinimide (NBS), bromine, iodine, N-bromophthalimide, N- Bromosaccharin, N-bromoacetamide, 1,3-dibromo-5,5-dimethylhydantoin, dibromoisocyanuric acid, trimethylphenylammonium tribromide, benzyltrimethylammonium tribromide, pyridinium bromide perbromide, 4-dimethylaminopyridinium bromide perbromide , 1-Butyl-3-methylimidazolium tribromide, 1,8-diazabicyclo [5,4,0] -7-undecenehydrogentribromide, N-iodophthalimide, N- One or more electrophilic halogenations selected from the group consisting of iodosaccharin, 1,3-diiodo-5,5-dimethylhydantoin, pyridine iodine monochloride, tetramethylammonium dichloroiodate, and benzyltrimethylammonium dichloroiodate. The production method according to claim 1, which is a reagent.
2. The halogenating reagent used in the reaction of reacting cholesterol containing desmosterol with a halogenating reagent and water is 1 to 10 molar equivalents relative to 1 mol of desmosterol contained in cholesterol. The manufacturing method according to any one of 3 above.
The organic solvent used in the crystallization is at least one organic solvent selected from the group consisting of ethers, ketones, esters, halogenated carbons, hydrocarbons, amides, nitriles, and alcohols. The manufacturing method according to any one of claims 1 to 4.
The production method according to any one of claims 1 to 5, wherein the high-purity cholesterol is cholesterol having a purity of 98% or more.