JP7361715B2 - Method for producing high purity cholesterol - Google Patents

Description

Cholesterol is a type of organic compound classified as a steroid, and, along with proteins and carbohydrates, is an important compound involved in various life phenomena. Cholesterol is also used as a raw material for pharmaceuticals, cosmetics, and liquid crystals.

The purity of commercially available cholesterol is generally 90-95% (based on gas chromatography (GC) area %). In particular, one of the reasons why it is difficult to obtain higher purity cholesterol is the presence of analog impurities.
Commercially available cholesterol contains several types of impurities with a structure similar to that of cholesterol, such as desmosterol, dihydrocholesterol, and lathosterol.

Conventionally known manufacturing methods for obtaining high-purity cholesterol include methods for removing cholesterol impurities by reslurry using alcohols such as methanol or recrystallization methods (Patent Documents 1 and 2); A method for converting desmosterol into cholesterol by a catalytic reduction method using a hydrogenation catalyst (Patent Document 3), or a purification method using special equipment (for example, a production method using a tower crystallizer) (Patent Document 4) ) can be mentioned. Therefore, other than the reduction reaction described above, there is no known production method for obtaining highly pure cholesterol by derivatizing impurities using a chemical reaction.

The above-mentioned impurities contained in cholesterol, especially desmosterol, which is a major impurity, have a similar structure and similar physical properties such as solubility to cholesterol, so it is difficult to use commonly known methods such as recrystallization. It was extremely difficult to separate and remove it using the purification methods used. Moreover, according to these recrystallization methods, it is necessary to repeat the recrystallization operation, which makes the operation complicated and reduces the yield of cholesterol. Furthermore, it is extremely difficult to obtain highly pure cholesterol using the recrystallization method.

In addition, according to the method of converting desmosterol to cholesterol using a catalytic reduction method using an expensive hydrogenation catalyst, the impurities contained in cholesterol can be reduced through the reaction, but some of the cholesterol is The reaction produces dihydrocholesterol, resulting in a decrease in yield.

In addition, for the purpose of obtaining 25-hydroxycholesterol, desmosterol (24-dehydrocholesterol) was reacted with N-bromosuccinimide (NBS) in a THF/water mixture to obtain an oxybrominated product, and then an aluminum hydride reagent was added. A method for obtaining 25-hydroxycholesterol, which is a debrominated product, is known (Patent Document 5), but this production method is not aimed at purifying cholesterol.

Therefore, it is possible to remove impurities (especially desmosterol) that are difficult to remove using conventional crystallization production methods, and it is also possible to efficiently produce high-purity cholesterol using industrially applicable operating methods. There is a need for a manufacturing method that can produce this.

JP2016-003190 JP2013-184929 JP 06-239883 JP2002-080493 China Patent Publication No. 103626822

It is an object of the present invention to provide a process for producing high-yield high-purity cholesterol (eg, cholesterol with about 98% purity) that does not contain substantial amounts of desmosterol.

As a result of intensive research by the present inventors, by allowing a halogenating reagent to act on cholesterol containing impurities such as desmosterol, desmosterol can be selectively converted into highly polar impurities, and the impurities can be easily removed through the subsequent crystallization process. It has been found that it can be removed and 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 that does not contain a substantial amount of desmosterol, which comprises reacting cholesterol containing desmosterol with a halogenating reagent and water, and separately removing the resulting desmosterol derivative by crystallization.
[2] A method for producing high purity cholesterol, comprising the following steps:
1) mixing cholesterol containing desmosterol with a halogenating reagent in water and an organic solvent to react the desmosterol with the halogenating reagent and water (step 1);
If necessary, add a reducing agent to the obtained reaction solution to decolorize it;
2) If necessary, remove the aqueous layer after the reaction (Step 2);
3-1) After the reaction, the organic layer is used as it is in the next step, or
3-2) Partially or completely condensing the organic layer to dryness to obtain a crude reaction product (Step 3);
4) Adding an organic solvent to the organic layer of 3-1) or the crude reaction product of 3-2) and crystallizing in the organic solvent (step 4),
(hereinafter also referred to as "the manufacturing method of the present invention").
[2-2] A reaction in which cholesterol containing desmosterol is reacted with a halogenating reagent and water (Step 1 of item [2])) is carried out at a temperature of about 0°C to about 100°C for about 10 minutes to about 12 hours. The manufacturing method according to [1] or [2].
[2-3] A reaction in which cholesterol containing desmosterol is reacted 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 manufacturing method according to [1] or [2].

[2-4] The reducing agent is one or more selected from the group consisting of sodium sulfite, sodium bisulfite, sodium thiosulfate, oxalic acid and its salts, and formic acid and its salts, [1] or [2] ] The manufacturing method described.
[2-5] When the halogenating reagent used in the reaction of reacting cholesterol containing desmosterol with a halogenating reagent and water (Step 1 of Section [2]) is N-iodosuccinimide (NIS) or iodine The manufacturing method according to [1] or [2], which comprises adding sodium sulfite or sodium formate to decolorize the product.
[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) The cholesterol obtained in 4) or 5) above is dissolved in a solvent with high solubility for cholesterol, and then a solvent with low solubility for cholesterol is added to the solution to precipitate cholesterol crystals (step 6),
The manufacturing method according to [1] or [2], which comprises:

[3] The halogenating reagent used in the reaction in which cholesterol containing desmosterol is reacted with a halogenating reagent and water (Step 1 of item [2]) 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-undecenehydrogen tribromide, N-iodo One or more desired compounds selected from the group consisting of phthalimide, N-iodosaccharin, 1,3-diiodo-5,5-dimethylhydantoin, pyridine iodine monochloride, tetramethylammonium dichloroiodate, and benzyltrimethylammonium dichloroiodate. The manufacturing method according to [1] or [2], which is an electronic halogenation reagent.
[3-2] The halogenating reagent used in the reaction in which cholesterol containing desmosterol is reacted with a halogenating reagent and water (step 1 of item [2]) is N-iodosuccinimide (NIS), [1 ] or the manufacturing method described in [2].

[3-3] The organic solvent used in the reaction in which cholesterol containing desmosterol is reacted with a halogenating reagent and water (Step 1 of Section [2]) is ethers, alcohols, hydrocarbons, halogenated carbonized The production method according to [1] or [2], wherein the organic solvent 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. Manufacturing method described.
[3-5] 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), [1] or [2] The manufacturing method described in ].

[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 The manufacturing method according to any one of [1] to [3], wherein the amount is about 1 mol to about 10 molar equivalents.
[4-2] The halogenating reagent used in the reaction in which cholesterol containing desmosterol is reacted with a halogenating reagent and water (Step 1 of Section [2]) is The manufacturing method according to any one of [1] to [3], wherein the amount is about 1 mol to about 4 molar equivalents.

[5] The organic solvent used in crystallization (step 4 of item [2])) is ethers, ketones, esters, hydrocarbons, halogenated hydrocarbons, amides, nitriles, and alcohols. The manufacturing method according to any one of [1] to [4], wherein one or more organic solvents are selected from the group consisting of:
[5-2] The organic solvent used in crystallization (step 4 of item [2]) is one or more organic solvents selected from the group consisting of ethers, ketones, esters, and alcohols. , [1] to [4].
[5-3] Any 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. 1. The manufacturing method according to item 1.
[5-4] The production method according to any one of [1] to [4], wherein the organic solvent used in the 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 according to any one of the above.

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

[5-7] Any one of [1] to [5], wherein the crystallization (step 4 of item [2])) is performed at a temperature of about -15°C to about 60°C or less. manufacturing method.
[5-8] The production according to any one of [1] to [5], wherein the 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] Highly purified cholesterol containing desmosterol in a content of about 1% or less, obtained by the production method according to any one of [1] to [6].
[7-2] Highly purified cholesterol containing desmosterol in a content of about 0.3% or less, obtained by the production method according to any one of [1] to [6].
[8] The high-purity cholesterol according to [7], which contains a trace amount of a halogenated derivative of cholesterol.

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

By the production method of the present invention, it is possible to efficiently obtain highly pure cholesterol from which impurities (mainly desmosterol) that are difficult to remove by conventional methods (for example, crystallization) are removed.

The present invention will be explained in more detail below.
(definition)
Below, definitions of terms used in this specification and claims are provided.

The term "cholesterol" as used herein is a compound represented by the following structural formula (CAS Registration Number 57-88-5). Examples of the cholesterol include commercially available products and products synthesized by generally known organic chemical methods, such as commercially available products. Ordinary commercially available cholesterol refers to cholesterol that has a purity of 90% or more (for example, about 95%) and contains several types of impurities with a structure similar to that of cholesterol. Such impurities include desmosterol, dihydrocholesterol, lanosterol, dihydrolanosterol, or lathosterol.

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

Unless otherwise specified, the terms "purity" and "content" herein refer to common instrumental analytical methods (e.g., gas chromatography (GC)) and liquid chromatography (LC) (e.g., high performance liquid chromatography). It means the area percentage when measured by lithography (including HPLC).

The term "high purity cholesterol" as used herein refers to a purity of about 95% or more, preferably about 96% or more, more preferably about 97% or more, even more preferably about 98% purity as an area percentage during GC measurement. % or more, particularly preferably about 99% or more cholesterol.

In the term "does not contain a substantial amount of desmosterol" in the present specification, "does not contain a substantial amount of desmosterol" means that the level is below a level that can be detected by normal analytical methods (for example, HPLC method). For example, the content of desmosterol is about 1.0% or less, preferably about 0.3% or less, more preferably about 0.2% or less, and even more preferably about 0.2% or less based on cholesterol. This means preferably about 0.1% or less. Alternatively, it means that the area percentage at the time of GC measurement is about 0.3% or less, preferably about 0.2% or less, more preferably about 0.1% or less.

According to one aspect of the present invention, the manufacturing method of the present invention includes:
This is a method for producing high-purity cholesterol that does not contain a substantial amount of desmosterol, which comprises reacting cholesterol containing desmosterol with a halogenating reagent and water, and separating and 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, only desmosterol is selectively reacted without reacting with cholesterol. 2) desmosterol can be converted into derivatives that have significantly different physical properties (e.g., solubility) than cholesterol; 3) using organic solvents commonly used in organic synthesis. , cholesterol and desmosterol derivatives can be easily separated by crystallization to remove desmosterol derivatives; 4) Compared with traditional purification methods (e.g. recrystallization method), cholesterol and desmosterol derivatives can be easily separated by crystallization; 5) The content of desmosterol can be reduced to an extremely small amount, and a substantial amount of desmosterol can be separated. It has the advantage that sterol-free, highly purified cholesterol can be obtained in high yield and with high efficiency.

According to one aspect of the present invention, the manufacturing method of the present invention includes:
A method for producing high purity cholesterol, the following steps:
Step 1) mixing cholesterol containing desmosterol with a halogenating reagent in water and an organic solvent to react desmosterol with the halogenating reagent and water;
If necessary, add a reducing agent 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) Partially or completely condensing the organic layer to dryness to obtain a crude reaction product;
Step 4) Adding an organic solvent to the organic layer of 3-1) or the crude reaction product of 3-2) to crystallize cholesterol.
By this, purified cholesterol is obtained.

(Step 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. As described above, desmosterol is converted into its polar compound derivative (for example, a compound in which a halogen atom and a hydroxyl group are added to the alkene moiety in desmosterol).

The term "halogenating reagent" refers to halogenating reagents commonly known in the field of organic chemistry. Preferably, a brominating reagent or an iodinating reagent is used. Specific examples of brominating reagents include bromine, N-bromosuccinimide (NBS), N-bromophthalimide, N-bromosaccharin, N-bromoacetamide, 1,3-dibromo-5,5-dimethylhydantoin, dibromoisocyanuric. acids, trimethylphenylammonium tribromide, benzyltrimethylammonium tribromide, pyridinium bromide perbromide, 4-dimethylaminopyridinium bromide perbromide, 1-butyl-3-methylimidazolium tribromide, and 1,8-diazabicyclo[5,4, Examples include, but are not limited to, one or more electrophilic brominating reagents selected from the group consisting of [0]-7-undecene hydrogen tribromide. Specific examples of iodinating reagents include iodine, N-iodosuccinimide (NIS), N-iodophthalimide, N-iodosaccharin, 1,3-diiodo-5,5-dimethylhydantoin, pyridine monochloride iodine, and dichloroiodine. Examples include, but are not limited to, one or more electrophilic iodinating reagents selected from the group consisting of tetramethylammonium acid 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 preferred in terms of the purity of the target cholesterol and handling of operations.

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 preferred. For example, ethers (e.g. diethyl ether, t-butyl methyl ether (MTBE), dimethoxyethane (DME), tetrahydrofuran (THF), 2-methyltetrahydrofuran (MeTHF)), alcohols (e.g. isopropyl alcohol (IPA), 1-butanol, 2-butanol, t-butanol), hydrocarbons (e.g. toluene, xylene, heptane), halogenated hydrocarbons (e.g. methylene chloride, chloroform, monochlorobenzene (MCB)), esters (e.g. , ethyl acetate, methyl acetate, butyl acetate), amides, nitriles, and ketones (e.g., methyl ethyl ketone (MEK), methyl isobutyl ketone (MIBK)). . Preference is given to ethers, in particular tetrahydrofuran (THF) derivatives, such as THF and 2-methyltetrahydrofuran (MeTHF). The amount of solvent is preferably small from an economical point of view, but for example, it is about 1 times to about 10 times by weight, preferably about 2 times to about 10 times by weight, relative to the crude cholesterol used in the reaction. For example, 4 times the weight.

The amount of halogenating reagent used in step 1 may vary depending on the content of desmosterol contained in cholesterol and the reactivity and type of halogenating reagent used, but the amount is sufficient to react with desmosterol. For example, the amount may be about 1 mol to about 50 mol equivalents, preferably about 1 mol to about 10 mol equivalents, per 1 mol of desmosterol contained in cholesterol. Typically, when NIS or _I2 is used, about 0.1 molar equivalent per mole of cholesterol is used, and when NBS is used, about 0.1 molar equivalent per mole of cholesterol is used. and about 0.05 molar equivalent.

The reaction in step 1 is performed under an atmospheric atmosphere (preferably under a nitrogen atmosphere). The reaction temperature may vary depending on the type of halogenating reagent used, and may range, for example, from about 0°C to about 100°C, preferably from about 20°C to about 60°C. Typically, when NIS or _I2 is used, the reaction temperature is about 40°C to about 60°C, and when NBS is used, the reaction temperature is about 20°C to about 40°C. be.
Although the reaction time may vary depending on the type of halogenating reagent used and the reaction temperature, the reaction time is, for example, about 10 minutes to about 12 hours (for example, about 30 minutes to about 3 hours).

After completion of the reaction in Step 1, free halogens may be removed by adding a reducing agent to the reaction solution in Step 1 to quench the reaction solution, if necessary. The reducing agent is a reagent that removes coloring derived from the halogenated reagent used, and includes, for example, sodium sulfite, sodium bisulfite, sodium thiosulfate, oxalic acid and its salts (e.g., sodium salt, and potassium salts), formic acid and its salts (eg, sodium salts and potassium salts), but are not limited thereto. For example, when the halogenating reagent is N-iodosuccinimide (NIS) or iodine, reducing agents (eg, sodium sulfite, sodium formate) can be used. The reducing agent may be in powder form or in an aqueous solution. The amount of the reducing agent used may be such that no coloring occurs, and for example, it may be at least 1 molar equivalent per 1 molar amount of the halogenating reagent used. Further, typically, the amount of the reducing agent used is 0.1 to 0.5 molar equivalent per 1 mole of cholesterol, preferably 0.2 to 0.4 molar equivalent. .
The quenching operation can be carried out 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).

(Step 2)
If necessary, the aqueous layer is removed from the reaction solution after the reaction in step 1 (or after the decolorization operation). The aqueous layer may contain, for example, excessive amounts of the halogenating reagent, its decomposition products, and inorganic salts. Removal of the aqueous layer can be performed by a separation operation (eg, a separatory funnel).

(Step 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 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 using a desiccant commonly used in organic synthesis (e.g., magnesium sulfate, sodium sulfate) before being used in the next step 4. It's okay.
Alternatively, for step 3-2, the organic layer obtained in step 2 is partially or completely concentrated to dryness (e.g., concentrated to dryness under reduced pressure using an evaporator) to form the crude reaction product. get.

(Step 4)
An organic solvent is added to the organic layer of Step 3-1 or the crude reaction product of Step 3-2, and cholesterol is crystallized in the organic solvent.

The term "crystallization" as used herein means precipitating cholesterol crystals, and also includes maintaining the crystallization solution in a low-temperature environment for a certain period of time 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 includes, for example, about 0° C. to about 100° C. (eg, about 20° C. to about 60° C.).
Next, the crystallization solution may be cooled to promote crystallization of cholesterol.
The cooling temperature may be any low temperature at which cholesterol crystals grow, such as -15°C to about 10°C, preferably about 0°C to about 10°C (typically about 0°C). ~5°C). Therefore, in the case of 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 any period in which cholesterol crystals can sufficiently grow, and for example, about 10 minutes to about 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 a low polar organic solvent may be used in combination with a polar solvent. Examples of the organic solvent include the organic solvent used in Step 1, such as ethers (for example, diethyl ether, t-butyl methyl ether (MTBE), dimethoxyethane (DME), tetrahydrofuran (THF), 2-methyltetrahydrofuran ( MeTHF)), ketones (e.g. methyl ethyl ketone (MEK), methyl isobutyl ketone (MIBK)), esters (e.g. ethyl acetate, methyl acetate, butyl acetate), halogenated hydrocarbons (e.g. methylene chloride, chloroform, monochlorobenzene (MCB)), hydrocarbons (e.g. toluene, xylene, heptane), amides, nitriles and alcohols (e.g. isopropyl alcohol (IPA), 1-butanol, 2-butanol, t-butanol). One or more organic solvents selected from the group consisting of:
The amount of organic solvent used in step 4 does not pose any particular problem as long as it is appropriate for the target cholesterol, but if the amount is too large, the yield will decrease, while if it is too small, the yield will decrease. In this case, the fluidity of the crystallized cholesterol is poor, the operability is deteriorated, and impurities may remain.

The method may also include a step (step 5) of obtaining purified cholesterol by further washing the cholesterol-containing precipitate crystallized in step 4 with an organic solvent. Step 5 may include conventional post-treatment steps (removal of solvent, filtration, and drying), if necessary.
Typically, in step 5, the reaction solution containing the precipitate obtained in step 4 is first filtered to remove the precipitate. Next, the filtered material is washed with an organic solvent. The washed wet crystals are dried (about 50° C. to about 60° C.) to obtain purified cholesterol, that is, high-purity cholesterol.

The organic solvent for washing used in step 5 is preferably an organic solvent with low solubility for cholesterol in order to suppress loss of cholesterol during washing. Examples include alcohols (eg, methanol), acetone, and acetonitrile.

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

In step 6, if necessary, the cholesterol obtained in step 4 or step 5 above is dissolved in a solvent with high solubility for cholesterol (that is, a good solvent), and then in the solution, the cholesterol obtained in step 4 or step 5 is dissolved in a solvent with low solubility for cholesterol. Cholesterol crystals are precipitated by adding a solvent (ie, a poor solvent). The resulting slurry is cooled (below 5° C.) and the precipitate is filtered. Next, the filtered product is washed with a poor solvent. The washed precipitate is dried (about 50° C. to about 60° C.) to obtain purified cholesterol, that is, high-purity cholesterol.

Examples of the good solvent used in Step 6 include, but are not limited to, a toluene/methanol mixture, a heptane/methanol mixture, THF, and the like. When a toluene/methanol mixture or a heptane/methanol mixture is used, a mixture in which the volume ratio of toluene or heptane to methanol is about 50:1 to about 2:1 is included. Furthermore, examples of poor solvents include methanol (which may be water-containing methanol), heptane, etc., but are not limited thereto. As for the amount of the good solvent and the poor solvent, for example, about 10 mL to about 50 mL (for example, about 10 mL to about 30 mL is preferable) of the good solvent can be used for 12.00 g of the cholesterol raw material to be purified. Further, the amount of the poor solvent to be used can be about 3 times to about 20 times (for example, preferably about 4 times to about 15 times) the volume of the good solvent.

In the production method of the present invention, the desmosterol derivatives (e.g., polar compounds) produced in Step 1 are separated from the crystallized cholesterol by the crystallization in Step 4 (further, washing in Step 5) and removed. It is believed that this makes it easier to obtain purified cholesterol.

(purified cholesterol)
The purified (purified) cholesterol produced by the production of the present invention includes desmosterol with a content of about 1% or less, and is derived from a reaction with a trace amount of the halogenating reagent used during production, as appropriate. Highly purified cholesterol, including halogenated derivatives of cholesterol. In addition, the purified cholesterol only contains desmosterol in an area percentage of about 0.3% or less when measured by GC, and about 1.0% or less as an area percentage when measured by LC, and a trace amount of cholesterol is appropriately halogenated. Preferably, the cholesterol has a purity of about 98% or greater, which may include derivatives. Here, the trace amount of the halogenated derivative means an amount within the detectable limit, for example, an amount of about 1% or less, about 0.5% or less, about 0.1% or less as an area percentage at the time of GC measurement. means.

According to one embodiment of the invention:
The halogenating reagent used in step 1 is NIS or _I2 , 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/ A method for producing methanol is provided.

After the reaction in Step 1 is completed, the reaction solution in Step 1 is decolorized by adding sodium sulfite or sodium formate.

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. , provides a manufacturing method.

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

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

The present invention will be described in more detail below with reference to Examples, but the present invention is not specifically limited by these Examples.
Commercially available cholesterol (Nippon Fine Chemical Co., Ltd. product: trade name CHOLESTEROL) (cholesterol purity is approximately 89% as an area percentage when measured by HPLC, and 96% as an area percentage when measured by GC, and desmosterol content is The area percentage in HPLC measurement was about 8%, and the area percentage in GC measurement was about 3%)) was used in the following Examples and Comparative Examples.

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

Example 1
Production method using N-iodosuccinimide (NIS) After dissolving 10 g (25.9 mmol) of commercially available cholesterol (manufactured by Nippon Fine Chemical Co., Ltd.: trade name CHOLESTEROL) in 34 mL of THF, 20 g (1.1 mol) of water and NIS 0 .6 g (2.6 mmol) was added, and the mixture was stirred and reacted at 50° C. for 1 hour.
After decolorizing the organic layer by adding 0.5 g of sodium sulfite to the reaction solution, the aqueous layer was removed. After adding 76 mL of methanol to the organic layer at 50 to 60°C, the mixture was cooled to below 5°C and then kept at 0 to 5°C for over 1 hour 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 (yield: 85%) of purified cholesterol.
The purity and desmosterol content of the purified cholesterol obtained was determined by gas chromatography (GC) analysis. The results are shown in Table 1.

Example 2
Production method using N-bromosuccinimide (NBS) After dissolving 10 g (25.9 mmol) of commercially available cholesterol (manufactured by Nippon Fine Chemical Co., Ltd.: trade name CHOLESTEROL) in 34 mL of THF, 20 g (1.1 mol) of water and NBS 0 .2 g (1.3 mmol) was added, and the mixture was stirred and reacted at 20° C. for 1 hour.
After the reaction, the aqueous layer was removed. After adding 76 mL of methanol to the organic layer at 50 to 60°C, the mixture was cooled to below 5°C and then kept at 0 to 5°C for over 1 hour 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 (yield: 83%) of purified cholesterol.
The purity and desmosterol content of the purified cholesterol obtained was determined by gas chromatography (GC) analysis. The results are shown in Table 1.

Example 3
Production method using iodine (I2) After dissolving 10 g (25.9 mmol) of commercially available cholesterol (manufactured by Nippon Fine Chemical Co., Ltd.: trade name CHOLESTEROL) in 34 mL of THF, 20 g (1.1 mol) of water and 0.7 g of iodine ( 2.6 mmol) was added thereto, and the mixture was stirred and reacted at 50° C. for 2 hours.
After decolorizing the organic layer by adding 0.5 g of sodium sulfite to the reaction solution, the aqueous layer was removed. After adding 76 mL of methanol to the organic layer at 50 to 60°C, the mixture was cooled to below 5°C and then kept at 0 to 5°C for over 1 hour 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 (yield: 83%) of purified cholesterol.
The purity and desmosterol content of the purified cholesterol obtained was determined 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 (manufactured by Nippon Fine Chemical Co., Ltd.: trade name CHOLESTEROL) was dissolved in 4.5 kg of THF, and then dissolved in 3.0 kg (1 mol) of water. .1 mol) and NIS 87.3 g (2.6 mmol) were added, and the mixture was stirred and reacted at 50° C. for 2.5 hours.
After decolorizing the organic layer by adding 750 g of a 10% aqueous sodium sulfite solution to the reaction solution, the aqueous layer was removed. The organic layer was washed and separated with 1.5 kg of 20% brine, cooled, and 9.0 kg of 5% aqueous methanol was added at room temperature, and the mixture was cooled to below 5°C. Thereafter, the precipitate was kept at 0 to 5°C for 1 hour or more, and the precipitate was filtered, and the separated precipitate was washed with 1.5 kg of methanol. The precipitate was dried under reduced pressure to obtain 1.28 kg (yield: 85%) of purified cholesterol.
The obtained purified cholesterol was measured by gas chromatography (GC) analysis, and 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, and the purity was 99.0% and the desmosterol content was 0.2%.

Example 5
Production method using N-iodosuccinimide (NIS) 50 g (12.9 mmol) of commercially available cholesterol (manufactured by Nippon Fine Chemical Co., Ltd.: trade name CHOLESTEROL) was 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 thereto, and the mixture was stirred and reacted at 50° C. for 4 hours.
After decolorizing the organic layer by adding 17.5 g of a 20% aqueous sodium formate solution to the reaction solution, the aqueous layer was separated and removed. After washing the organic layer with a small amount of water, 370 mL of 5% aqueous methanol was added dropwise to crystallize it. 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%).
The obtained purified cholesterol was measured by high performance liquid chromatography (HPLC) analysis, and the purity was 99.0% and the desmosterol content was 0.2%.

Comparative example 1
Manufacturing method using only crystallization (i.e., manufacturing method that does not use a halogenating reagent)
After dissolving 6 g (15.5 mmol) of commercially available cholesterol (manufactured by Nippon Fine Chemical Co., Ltd., trade name: CHOLESTEROL) in 20 mL of THF, 46 mL of methanol was added at 50 to 60°C, and the mixture was cooled to below 5°C. Thereafter, the mixture was kept at 0 to 5°C for over 1 hour 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 (yield: 75%) of purified cholesterol.
The purity and desmosterol content of the purified cholesterol obtained was determined by gas chromatography (GC) analysis. The results are shown in Table 1.

Comparative example 2
Production method using only crystallization 6 g (15.5 mmol) of commercially available cholesterol (manufactured by Nippon Fine Chemical Co., Ltd.: trade name CHOLESTEROL) was added to 91 mL of methanol, and the mixture was maintained at 60° C. for 1 hour. Thereafter, the mixture was cooled to below 30°C 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 and desmosterol content of the purified cholesterol obtained was determined by gas chromatography (GC) analysis. The results are shown in Table 1.

Comparative example 3
Production method using only crystallization: 3 g (7.8 mmol) of commercially available cholesterol (manufactured by Nippon Fine Chemical Co., Ltd.: trade name CHOLESTEROL) was heated and dissolved in 15 mL of 1-butanol, and then cooled to 20 to 25°C. was maintained for over 1 hour to precipitate crystals. The precipitate was filtered, and the separated precipitate was washed with 2 mL of 1-butanol and dried under reduced pressure to obtain 2.1 g (yield: 70%) of purified cholesterol.

Comparative example 4
Production method using crystallization only: 12 g (31 mmol) of commercially available cholesterol (manufactured by Nippon Fine Chemical Co., Ltd.: trade name CHOLESTEROL) was dissolved in a mixed solvent of 28 mL of toluene and 10 mL of methanol, and then 180 mL of methanol was added for crystallization. , cooled to 0-5°C and held at this temperature for more than 1 hour. 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 (yield: 82%) of purified cholesterol.
The purified cholesterol was measured by high performance liquid chromatography (HPLC) analysis, and 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 all cases of Examples 1 to 5, the purity is about 98% or more, especially about 99% or more, as compared to the purity of cholesterol before purification. of cholesterol was obtained. Moreover, the obtained purified cholesterol contained very little desmosterol or no detectable level of desmosterol compared to the cholesterol before purification.
On the other hand, when no halogenating reagent was used as a comparative example, the purity of the obtained cholesterol was about 98% or less, and the content of desmosterol in the cholesterol was significant.

Examples 6-17
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 investigated. Specifically, the purity of the purified cholesterol obtained and the content of desmosterol were examined using various solvents shown in Table 2 below as crystallization solvents.

After dissolving 70 g (181 mmol) of commercially available cholesterol (Nippon Fine Chemical Co., Ltd. product: 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 The reaction was stirred at ℃ for 1 hour.
3.5 g of sodium sulfite was added to the reaction solution to decolorize the organic layer, and then the aqueous layer was removed. The organic layer was concentrated to dryness to obtain crude cholesterol (purity 96.19%, no desmosterol detected (ND)). Subsequently, the crude cholesterol was subjected to crystallization using various solvents. Examples using each solvent are shown as Examples 6 to 17.
Two times the amount of THF and six times the amount of methanol relative to the pure cholesterol were added to 5.5 g (13.0 mmol) of crude cholesterol and dissolved by heating. After the mixture was cooled to 5°C or lower, it was kept at 0 to 5°C for over 1 hour to precipitate crystals. The precipitate was filtered and the separated precipitate was dried under reduced pressure to obtain purified cholesterol (Example 16).
The purity and desmosterol content of the purified cholesterol obtained was determined by gas chromatography (GC) analysis.
Thereafter, the purity of each crystallization solvent was confirmed in the same manner. The experimental results are shown in Table 2.

As crystallization solvents, 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), high purity (e.g., approximately 98% purity by GC area percentage) cholesterol was obtained. .

Example 18
In the production method using N-iodosuccinimide (NIS), the THF/water solvent used in Example 1 etc. was replaced with various organic solvents to improve the purity of cholesterol and reduce the content of desmosterol. We investigated the influence of
5 g (12.9 mmol) of commercially available cholesterol (manufactured by Nippon Fine Chemical 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. were stirred and reacted at 50°C for 3 hours.
After decolorizing the organic layer by adding 1.75 g of a 20% aqueous sodium formate solution to the reaction solution, the aqueous layer was removed if it separated. The reaction solution (organic layer) was diluted to 50 mL with methanol, the mixture was cooled to below 5°C, and then kept at 0 to 5°C for over 1 hour 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 and desmosterol content of the purified cholesterol obtained was determined by high performance liquid chromatography (HPLC) analysis. The results are shown in Table 3.

前記実施例１等における工程１で用いる有機溶媒を、ＴＨＦ／水に代えて、各種有機溶媒を用いて、コレステロールの純度およびデスモステロールの含量について調べたところ、エーテル類、ケトン類、エステル類、ハロゲン化炭化水素類、アルコール類のいずれの場合についても、原料のコレステロールの純度と比べて著しく向上し、また、デスモステロールの含量が著しく低下した。特に、エーテル類、エステル類、アルコール類が好ましく、エーテル類がより好ましく、テトラヒドロフラン誘導体（例えば、テトラヒドロフランおよび２－メチルテトラヒドロフラン）が特に好ましいことが分かった。

When the purity of cholesterol and the content of desmosterol were investigated using various organic solvents instead of THF/water as the organic solvent used in step 1 in Example 1 etc., it was found that ethers, ketones, esters, In both cases of halogenated hydrocarbons and alcohols, the purity of the raw material cholesterol was significantly improved, and the content of desmosterol was significantly reduced. In particular, ethers, esters, and alcohols are preferred, ethers are more preferred, and tetrahydrofuran derivatives (eg, tetrahydrofuran and 2-methyltetrahydrofuran) are particularly preferred.

Example 19
Furthermore, in order to obtain highly pure cholesterol, an additional purification operation was performed using the purified cholesterol prepared in Example 4.
12 g of purified cholesterol (purified cholesterol of Example 4) was dissolved in several kinds 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 the precipitate was filtered. The obtained precipitate was washed with 10 mL of poor solvent. The precipitate was dried under reduced pressure to obtain purified cholesterol of high purity.
The purity and desmosterol content of the purified cholesterol obtained was determined by high performance liquid chromatography (HPLC) analysis. The results are shown in Table 4.

上記表中、エントリー１８～２３は、ＮＩＳ等を用いてヨウ素化処理して得られたコレステロールを原料として用いて、再精製処理を行った。一方で、エントリー２４は、ヨウ素化処理なしで得られたコレステロール（比較例４で調製したもの）を原料として用いて、再精製処理を行った。
追加精製前の各コレステロール原料の、コレステロール純度およびデスモステロール含量は、以下のとおりであった（以下、純度と含量は、ＨＰＬＣ測定時の面積百分率の値を記載した）。
ヨウ素化処理して得られたコレステロール原料（エントリー１８～２２で使用）：
純度99.04 ％、デスモステロール含量 0.19 ％
低純度品のコレステロール（コレステロール純度が78.5％であり、そして、デスモステロール含量が12.3％である）をヨウ素化処理して得られたコレステロール原料（エントリー２３で使用）：
純度98.0 ％、デスモステロール含量 0.6 ％
ヨウ素化処理なしで得られたコレステロール原料（エントリー２４で使用）：
純度93.1 ％、デスモステロール含量 6.0 ％
上記の結果、エントリー１８～２２の場合、実施例４で得られたヨウ素化処理して得られたコレステロールを原料として用いて、追加精製を行った場合には、約９９．５％程度の高純度コレステロールを得ることができた。また、エントリー２３の場合、低純度のコレステロール原料を用いても、ヨウ素化処理することによって、有意な程度の高い純度および有意な程度の低いデスモステロール含量を有するコレステロールを得ることができるため、追加精製を行った場合には、約９９．０％近い純度のコレステロールを得ることができた。
一方で、エントリー２４として、ヨウ素化処理をせずに得られたコレステロールを原料に用いた場合には、追加精製を行っても９５％以上の高純度のコレステロールを得ることはできなかった。
この結果より、本発明の製法におけるハロゲン化（例えば、ヨウ素化）処理した場合には、出発原料のコレステロール中に含まれるデスモステロールの含量を有意に低下させることができるため、続く、本実施例に示す追加精製により更にコレステロールの純度を向上することができることが分かった。一方で、ヨウ素化処理をせずに得られたコレステロールを用いた場合には、出発原料のコレステロール中に含まれているデスモステロールの含量を低下することができないために、本実施例の追加精製を行ってもコレステロールの純度を更に向上することが難しく、９５％以上の純度を有するコレステロールを得ることはできなかった。

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

According to the production method of the present invention, highly pure cholesterol with extremely low content of impurities can be produced in an industrially efficient manner.

Claims (5)

A method for producing high-purity cholesterol that does not contain a substantial amount of desmosterol, the method comprising reacting cholesterol containing desmosterol with a halogenating reagent and water and separately removing the resulting desmosterol derivative by crystallization ;
High-purity cholesterol means cholesterol with a purity of 98% or more as an area percentage when measured by gas chromatography (GC) method, and
Not containing a substantial amount means that it is 1.0% or less as an area percentage when measured by high performance liquid chromatography (HPLC) method.
The manufacturing method. A method for producing high purity cholesterol, the following steps:
mixing cholesterol containing desmosterol with a halogenating reagent in water and an organic solvent to react the desmosterol with the halogenating reagent and water;
If necessary, add a reducing agent to the obtained reaction solution to decolorize it. 2) If necessary, remove the aqueous layer after the reaction;
3-1) After the reaction, the organic layer is used as it is in the next step, or
3-2) Partially or completely condensing the organic layer to dryness to obtain a crude reaction product;
4) Add an organic solvent to the organic layer of 3-1) or the crude reaction product of 3-2) and crystallize in the organic solvent;
including that
High-purity cholesterol means cholesterol with a purity of 98% or more as an area percentage when measured by gas chromatography (GC) method.
The manufacturing method. The halogenating reagents used in the reaction in which cholesterol containing desmosterol is reacted with a halogenating reagent and water include 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-undecenehydrogen tribromide, N-iodophthalimide, N-iodosaccharin, 1,3-diiodo- 2. The electrophilic halogenating reagent of claim 1 or 2 is one or more electrophilic halogenating reagents selected from the group consisting of 5,5-dimethylhydantoin, pyridine iodine monochloride, tetramethylammonium dichloroiodate, and benzyltrimethylammonium dichloroiodate. The manufacturing method described in. Claims 1 to 3, wherein the halogenating reagent used in the reaction of reacting cholesterol containing desmosterol with a halogenating reagent and water is 1 mol to 10 molar equivalents per 1 mol of desmosterol contained in cholesterol. 3. The manufacturing method according to any one of 3. The organic solvent used in crystallization is one or more organic solvents 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.