WO2020050342A1

WO2020050342A1 - Dicyclohexylamine salt of n,n'-dibenzylbiotin, and production method for same

Info

Publication number: WO2020050342A1
Application number: PCT/JP2019/034892
Authority: WO
Inventors: 雅彦関; 優輔高橋
Original assignee: 株式会社トクヤマ
Priority date: 2018-09-07
Filing date: 2019-09-05
Publication date: 2020-03-12
Also published as: JPWO2020050342A1

Abstract

One purpose of the present invention is to provide a dicyclohexylamine salt of N,N'-dibenzylbiotin (and, in particular, a high purity dicyclohexylamine salt of N,N'-dibenzylbiotin) and a production method for the dicyclohexylamine salt of N,N'-dibenzylbiotin. To achieve said purpose, the present invention provides a method for producing a dicyclohexylamine salt of N,N'-dibenzylbiotin. The method includes a step for bringing N,N'-dibenzylbiotin into contact with dicyclohexylamine to generate a dicyclohexylamine salt of the N,N'-dibenzylbiotin.

Description

Dicyclohexylamine salt of N, N'-dibenzylbiotin and method for producing the same

<< The present invention relates to a dicyclohexylamine salt of N, N'-dibenzylbiotin (particularly, a high-purity dicyclohexylamine salt of N, N'-dibenzylbiotin) and a method for producing the same. Further, the present invention relates to the use of N, N'-dibenzylbiotin dicyclohexylamine salt (particularly, high-purity N, N'-dibenzylbiotin dicyclohexylamine salt) to prepare N, N'-dibenzylbiotin. The present invention relates to a method for producing benzyl biotin (particularly, high-purity N, N′-dibenzylbiotin), biotin (particularly, high-purity biotin), an N, N′-dibenzylbiotin ester compound, or a biotin ester compound. Furthermore, the present invention relates to high-purity N, N'-dibenzylbiotin and high-purity biotin.

@Biotin is a compound represented by the following formula.

@Biotin is produced, for example, by the following synthetic route. Bn represents a benzyl group.

において In order to obtain biotin with high purity in the above synthesis route, it is necessary to obtain N, N'-dibenzylbiotin as a precursor (synthesis intermediate) with high purity.

Biotin is a water-soluble vitamin expected to have a diabetic preventive effect and the like, and is a useful compound as a drug, a feed additive and the like, and high-purity biotin is required. N, N'-dibenzylbiotin is a compound useful as a protein labeling substance (see Patent Document 1 and Non-patent Document 1), a viral reverse transcriptase inhibitor (see Patent Document 2), and the like. , N'-Dibenzylbiotin is needed.

International Publication WO2010 / 106347 U.S. Pat. No. 5,763,469

'N, N'-dibenzylbiotin can be crystallized, but has a low melting point and is hardly crystalline. The present inventors have confirmed that the HPLC purity of N, N'-dibenzylbiotin obtained by the above synthesis route was 75.58%.

The object of the present invention is to provide a technology that enables N, N'-dibenzylbiotin or a derivative thereof or biotin or a derivative thereof to be produced with high purity.

Means for Solving the Problems The present inventors have conducted intensive studies on a technique for producing N, N′-dibenzylbiotin or a derivative thereof or biotin or a derivative thereof with high purity in order to solve the above-described problems. By contacting N, N'-dibenzylbiotin with dicyclohexylamine to form a dicyclohexylamine salt of N, N'-dibenzylbiotin, a highly pure dicyclohexylamine of N, N'-dibenzylbiotin can be obtained. It has been found that the xylamine salt can be obtained as a solid. Further, the present inventors contact the obtained dicyclohexylamine salt of N, N′-dibenzylbiotin with at least one acid to remove the dicyclohexylamine salt of N, N′-dibenzylbiotin. High-purity N, N'-dibenzylbiotin or high-purity biotin by proceeding with decyclohexylamine salification or debenzylation of dicyclohexylamine salt or dicyclohexylamine salt of N, N'-dibenzylbiotin Was found to be able to be obtained. The present inventors have completed the present invention based on these findings.

That is, the present invention includes the following inventions.
[1] The following formula (1):

[Wherein, Bn represents a benzyl group. ]
A dicyclohexylamine salt of N, N'-dibenzylbiotin represented by the formula:
[2] The dicyclohexylamine salt of N, N'-dibenzylbiotin according to [1], wherein the HPLC purity is 96% or more.
[3] The following formula (2):

[Wherein, Bn is as defined above. ]
The N, N'-dibenzylbiotin represented by the formula, wherein the HPLC purity is 96% or more.
[4] The following formula (3):

The biotin represented by the above, wherein the HPLC purity is 96% or more.
[5] A method for producing a dicyclohexylamine salt of N, N'-dibenzylbiotin according to [1],
The following equation (2):

[Wherein, Bn is as defined above. ]
Contacting N, N'-dibenzylbiotin represented by the formula with dicyclohexylamine to form the dicyclohexylamine salt of N, N'-dibenzylbiotin.
[6] The method according to [5], wherein the dicyclohexylamine salt of N, N'-dibenzylbiotin has an HPLC purity of 96% or more.
[7] The method according to [5] or [6], wherein the N, N′-dibenzylbiotin has an HPLC purity of 95% or less.
[8] The following formula (A):

[In the formula, R ¹ represents a benzyl group or a hydrogen atom. ]
A method for producing a compound (A) represented by the formula:
The method comprising the step of contacting the dicyclohexylamine salt of N, N'-dibenzylbiotin according to [1] with at least one acid to produce the compound (A).
[9] The method according to [8], wherein the dicyclohexylamine salt of N, N'-dibenzylbiotin has a HPLC purity of 96% or more.
[10] the acid is at least one acid selected from hydrochloric acid, sulfuric acid, and hydrogen sulfate;
The compound (A) has the following formula (2):

[Wherein, Bn is as defined above. ]
The method according to [8] or [9], wherein the method is N, N'-dibenzylbiotin represented by the formula:
[11] The method according to [10], wherein the N, N′-dibenzylbiotin has an HPLC purity of 96% or more.
[12] The acid is at least one acid selected from methanesulfonic acid, sulfuric acid, and hydrobromic acid,
The compound (A) has the following formula (3):

The method according to [8] or [9], which is biotin represented by the formula:
[13] The method according to [12], wherein the HPLC purity of the biotin is 96% or more.
[14] The following formula (4):

Wherein Bn is as defined above, and R ² is an alkyl group having 1 to 30 carbon atoms, a substituted alkyl group having 1 to 30 carbon atoms, an aralkyl group having 7 to 30 carbon atoms, and a substituted group having 7 to 30 carbon atoms. Represents a group selected from aralkyl groups. ]
A method for producing an N, N′-dibenzylbiotin ester compound represented by the formula:
The dicyclohexylamine salt of N, N'-dibenzylbiotin according to [1] is converted to a compound represented by the following formula (5):

[Wherein, R ² has the same meaning as described above, and X represents a halogen atom. ]
Reacting with a halide represented by the formula to produce the N, N'-dibenzylbiotin ester compound.
[15] The method according to [14], wherein the dicyclohexylamine salt of N, N'-dibenzylbiotin has an HPLC purity of 96% or more.
[16] The following formula (6):

[Wherein, R ² has the same meaning as described above. ]
A method for producing a biotin ester compound represented by
The method according to [14] or [15], comprising a step of removing a benzyl group from the N, N'-dibenzylbiotin ester compound produced by the method according to [15] to produce the biotin ester compound.

According to the present invention, there is provided a dicyclohexylamine salt of N, N'-dibenzylbiotin (particularly, a high-purity dicyclohexylamine salt of N, N'-dibenzylbiotin) and a method for producing the same. Further, according to the present invention, N, N ′ is prepared by using N, N′-dibenzylbiotin dicyclohexylamine salt (particularly, high-purity N, N′-dibenzylbiotin dicyclohexylamine salt). -Dibenzylbiotin (particularly high-purity N, N'-dibenzylbiotin), biotin (particularly high-purity biotin), N, N'-dibenzylbiotin ester compound or a method for producing a biotin ester compound Is done. Further, according to the present invention, high-purity N, N'-dibenzylbiotin and high-purity biotin are provided.

<< First aspect >>
A first aspect of the present invention relates to a dicyclohexylamine salt of N, N'-dibenzylbiotin.

The dicyclohexylamine salt of 'N, N'-dibenzylbiotin is a compound represented by the following formula (1). The dicyclohexylamine salt of N, N'-dibenzylbiotin is not completely purified even after purification, and contains unavoidable impurities. Therefore, "dicyclohexylamine salt of N, N'-dibenzylbiotin" is not a pure substance but means a mixture of the dicyclohexylamine salt of N, N'-dibenzylbiotin and inevitable impurities.

において In the formula (1), Bn represents a benzyl group.

The HPLC purity of the dicyclohexylamine salt of N, N'-dibenzylbiotin is preferably 96% or more, more preferably 97% or more, even more preferably 98% or more, even more preferably 98.5% or more. It is more preferably at least 99%. The HPLC purity of the dicyclohexylamine salt of N, N'-dibenzylbiotin is determined by the method described in the examples.

<< 2nd aspect >>
A second aspect of the present invention relates to high purity N, N'-dibenzylbiotin.

'N, N'-dibenzylbiotin is a compound represented by the following formula (2). N, N'-dibenzylbiotin is not completely pure even after purification, and contains unavoidable impurities. Thus, "N, N'-dibenzylbiotin" is not a pure substance but means a mixture of N, N'-dibenzylbiotin and unavoidable impurities.

において In the formula (2), Bn represents a benzyl group.

The HPLC purity of N, N'-dibenzylbiotin is preferably at least 96%, more preferably at least 97%, even more preferably at least 98%, even more preferably at least 98.5%, even more preferably at least 99%. That is all. The HPLC purity of N, N'-dibenzylbiotin is determined by the method described in the examples.

{Third aspect}
A third aspect of the present invention relates to high purity biotin.

@Biotin is a compound represented by the following formula (3). Biotin is not completely purified even after purification, and contains unavoidable impurities. Thus, "biotin" is not a pure substance but means a mixture of biotin and unavoidable impurities.

Biotin has an HPLC purity of preferably 96% or more, more preferably 97% or more, even more preferably 98% or more, even more preferably 98.5% or more, and still more preferably 99% or more. The HPLC purity of biotin is measured by the method described in the examples.

<< 4th mode >>
A fourth aspect of the present invention relates to a method for producing a dicyclohexylamine salt of N, N'-dibenzylbiotin. The dicyclohexylamine salt of N, N'-dibenzylbiotin is a compound represented by the above formula (1).

方法 The method according to the fourth aspect of the present invention comprises the step of contacting N, N'-dibenzylbiotin with dicyclohexylamine to form a dicyclohexylamine salt of N, N'-dibenzylbiotin. Note that N, N'-dibenzylbiotin is a compound represented by the above formula (2).

N, N'-dibenzylbiotin to be contacted with dicyclohexylamine can be produced by a known method. For example, it can be obtained as a synthetic intermediate in the biotin synthesis route described in the background art. Alternatively, it can be obtained by reacting biotin with a benzylating agent such as benzyl chloride, benzyl bromide, or benzyl iodide in the presence of a base. After the reaction, a reaction solution containing N, N'-dibenzylbiotin is added to ice water, acidified by adding an acid, and N, N'-dibenzylbiotin is extracted with an organic solvent such as ethyl acetate. By washing and concentrating, N, N′-dibenzylbiotin can be isolated. N, N'-dibenzylbiotin can be purified by silica gel column purification or the like. Purification can be performed using, for example, an automatic preparative purification device equipped with a silica gel column.

The HPLC purity of N, N'-dibenzylbiotin contacted with dicyclohexylamine is preferably 30-95%, more preferably 50-94%, even more preferably 70-93.5%. The HPLC purity of N, N'-dibenzylbiotin is determined by the method described in the examples. The HPLC purity of N, N'-dibenzylbiotin obtained by the biotin synthesis route described in the background art is 75.58%.

By contacting 'N, N'-dibenzylbiotin with dicyclohexylamine, a dicyclohexylamine salt of N, N'-dibenzylbiotin can be produced.

The solvent used for bringing N, N'-dibenzylbiotin into contact with dicyclohexylamine is not particularly limited as long as it is a solvent capable of dissolving N, N'-dibenzylbiotin, and is appropriately selected from known organic solvents. You can choose. Examples of the organic solvent include nitriles such as acetonitrile and propionitrile; ethers such as THF, 2-methyl-THF, 1,4-dioxane, t-butylmethyl ether, diisopropyl ether, dimethoxyethane and diglyme; acetone , Methyl ethyl ketone, diethyl ketone, etc .; ketones such as methyl acetate, ethyl acetate, butyl acetate, etc .; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, 1,2-dichloroethane, chlorobenzene; toluene, xylene And the like; and aromatic hydrocarbons such as hexane and heptane. One organic solvent may be used alone, or a mixed solvent of two or more organic solvents may be used. Among the organic solvents, ethyl acetate, butyl acetate, and methyl ethyl ketone are preferred from the viewpoints of industrial availability and easy handling, and particularly preferred are esters such as ethyl acetate and butyl acetate. Alternatively, it is preferable to use a mixed solvent of two or more of these. The amount of the solvent used when bringing N, N'-dibenzylbiotin into contact with dicyclohexylamine can be appropriately adjusted in consideration of the capacity of the reaction vessel, etc., but N, N'-dibenzylbiotin 1 mass Parts are usually in the range of 1 to 100 parts by volume. The fact that the amount of the solvent is in the range of 1 to 100 parts by volume based on 1 part by mass of N, N'-dibenzylbiotin indicates that the amount of the solvent is 1 g by mass of N, N'-dibenzylbiotin. Means in the range of 1 to 100 mL. The same applies hereinafter.

The amount of dicyclohexylamine used in contacting N, N'-dibenzylbiotin with dicyclohexylamine is not particularly limited as long as it is an amount sufficient to form a dicyclohexylamine salt of N, N'-dibenzylbiotin. However, it can be adjusted appropriately, but is usually in the range of 1.0 to 2.0 mol per 1 mol of N, N'-dibenzylbiotin.

The reaction temperature and the reaction time when N, N'-dibenzylbiotin is brought into contact with dicyclohexylamine are not particularly limited as long as the conditions are sufficient to produce a dicyclohexylamine salt of N, N'-dibenzylbiotin. The reaction temperature is usually in the range of −20 to 100 ° C., preferably in the range of −10 to 50 ° C., and the reaction time is usually in the range of 0.5 to 72 hours. .

後 After the reaction, the dicyclohexylamine salt of N, N'-dibenzylbiotin can be isolated from the reaction solution containing the dicyclohexylamine salt of N, N'-dibenzylbiotin by a known solid-liquid separation method. The isolated dicyclohexylamine salt of N, N'-dibenzylbiotin is sufficiently pure as it is, but methanol, ethanol, isopropanol, chloroform, methylene chloride, methyl acetate, ethyl acetate, butyl acetate, isopropyl ether , Hexane, toluene, xylene or a mixture of two or more thereof.

According to the method according to the fourth aspect of the present invention, a high-purity dicyclohexylamine salt of N, N'-dibenzylbiotin can be produced. The HPLC purity of the dicyclohexylamine salt of N, N'-dibenzylbiotin produced by the method according to the fourth aspect of the present invention is preferably 96% or higher, more preferably 97% or higher, even more preferably 98%. The above is even more preferably 98.5% or more, and still more preferably 99% or more. The HPLC purity of the dicyclohexylamine salt of N, N'-dibenzylbiotin is determined by the method described in the examples.

<< Fifth aspect >>
A fifth aspect of the present invention relates to a method for producing compound (A) using a dicyclohexylamine salt of N, N'-dibenzylbiotin.

Compound (A) is a compound represented by the following formula (A). Compound (A) does not become a completely pure substance even after purification, but contains unavoidable impurities. Therefore, "compound (A)" is not a pure substance but means a mixture of compound (A) and unavoidable impurities.

In the formula (A), R ¹ represents a benzyl group or a hydrogen atom.

In the formula (A), when R ¹ represents a benzyl group, the compound (A) is N, N′-dibenzylbiotin represented by the above formula (2).

In the formula (A), when R ¹ represents a hydrogen atom, the compound (A) is biotin represented by the above formula (3).

方法 The method according to the fifth aspect of the present invention comprises the step of contacting a dicyclohexylamine salt of N, N'-dibenzylbiotin with at least one acid to produce compound (A).

The dicyclohexylamine salt of N, N'-dibenzylbiotin contacted with at least one acid is a dicyclohexylamine salt of N, N'-dibenzylbiotin produced by the method according to the fourth aspect of the present invention. Is preferred.

The HPLC purity of the dicyclohexylamine salt of N, N'-dibenzylbiotin contacted with at least one acid is preferably at least 96%, more preferably at least 97%, even more preferably at least 98%, even more preferably. 98.5% or more, more preferably 99% or more. The HPLC purity of the dicyclohexylamine salt of N, N'-dibenzylbiotin is determined by the method described in the examples.

The at least one acid to be brought into contact with the dicyclohexylamine salt of 'N, N'-dibenzylbiotin can be appropriately selected according to the type of the compound (A) to be produced.

Hereinafter, the case where the compound (A) to be produced is N, N'-dibenzylbiotin will be described.

When the compound (A) to be produced is N, N'-dibenzylbiotin, at least one acid capable of promoting dedicyclohexylamine salification of the dicyclohexylamine salt of N, N'-dibenzylbiotin is selected. Is done. Contacting the dicyclohexylamine salt of N, N'-dibenzylbiotin with at least one acid to proceed with the dedicyclohexylamine salification of the dicyclohexylamine salt of N, N'-dibenzylbiotin, -Dibenzylbiotin can be produced.

At the time of dedicyclohexylamine salification of the dicyclohexylamine salt of N, N′-dibenzylbiotin, at least one acid to be brought into contact with the dicyclohexylamine salt of N, N′-dibenzylbiotin is, for example, hydrochloric acid, sulfuric acid, sulfuric acid It can be selected from hydrogen salts and the like. Examples of the hydrogen sulfate include potassium hydrogen sulfate and sodium hydrogen sulfate. Among these acids, it is preferable to use sulfuric acid, hydrogensulfate, particularly potassium hydrogensulfate in view of industrial availability and easy handling. The amount of the acid used is not particularly limited as long as it is an amount sufficient for the removal of dicyclohexylamine to proceed, and can be adjusted as appropriate. The amount of the acid used is preferably in the range of 1.0 to 1000 equivalents per 1 equivalent of the dicyclohexylamine salt of N, N'-dibenzylbiotin, since the dicyclohexylamine salification is efficiently performed in a short time. It is more preferably in the range of 1.0 to 100 equivalents.

A known organic solvent can be used as a solvent to be used for removing the dicyclohexylamine salt of the dicyclohexylamine salt of 'N, N'-dibenzylbiotin. Examples of the organic solvent include esters such as methyl acetate, ethyl acetate and butyl acetate; halogenated hydrocarbons such as methylene chloride and chloroform; and ethers such as tert-butyl methyl ether. One organic solvent may be used alone, or a mixed solvent of two or more organic solvents may be used. The amount of the solvent used can be appropriately adjusted in consideration of the capacity of the reaction vessel and the like, but is usually in the range of 1 to 100 parts by volume per 1 part by mass of dicyclohexylamine salt of N, N'-dibenzylbiotin It is.

The reaction temperature and the reaction temperature in the dedicyclohexylamine salification of the dicyclohexylamine salt of N, N′-dibenzylbiotin can be appropriately adjusted, but the reaction temperature is usually in the range of −20 to 120 ° C., preferably The temperature is in the range of -10 to 100 ° C, and the reaction time is usually in the range of 0.5 to 72 hours.

After the reaction, a reaction solution containing N, N'-dibenzylbiotin was added to ice water, alkali was added to make the mixture basic, and N, N'-dibenzylbiotin was extracted with an organic solvent such as ethyl acetate. , Washing and concentration, N, N'-dibenzylbiotin can be isolated. The isolated N, N'-dibenzylbiotin has sufficiently high purity and may be used for various applications as it is, but may be further purified. Purification can be performed, for example, by silica gel column purification. Silica gel column purification can be performed using, for example, an automatic preparative purification device equipped with a silica gel column.

According to the method according to the fifth aspect of the present invention, high-purity N, N'-dibenzylbiotin can be produced. The HPLC purity of N, N'-dibenzylbiotin produced by the method according to the fifth aspect of the present invention is preferably at least 96%, more preferably at least 97%, even more preferably at least 98%, even more preferably. Is 98.5% or more, more preferably 99% or more. The HPLC purity of N, N'-dibenzylbiotin is determined by the method described in the examples.

Hereinafter, the case where the compound (A) to be produced is biotin will be described.

化合物 When the compound (A) to be produced is biotin, at least one acid capable of promoting the dedicyclohexylamine salification and debenzylation of the dicyclohexylamine salt of N, N′-dibenzylbiotin is selected. Contacting the dicyclohexylamine salt of N, N'-dibenzylbiotin with at least one acid to promote dedicyclohexylamine salification and debenzylation of the dicyclohexylamine salt of N, N'-dibenzylbiotin; Biotin can be produced.

In the decyclopentadiamine salification and debenzylation of the dicyclohexylamine salt of N, N′-dibenzylbiotin, at least one acid that is brought into contact with the dicyclohexylamine salt of N, N′-dibenzylbiotin is, for example, methane. It can be selected from sulfonic acid, sulfuric acid, hydrobromic acid and the like. By using these acids, the debenzylation of the dicyclohexylamine salt of N, N'-dibenzylbiotin and the debenzylation of the dicyclohexylamine salt proceed simultaneously, or the debenzylation proceeds after the removal of the dicyclohexylamine salt and the biotin Is presumed to be obtained.

When the dicyclohexylamine salt and the debenzylation of the dicyclohexylamine salt of N, N'-dibenzylbiotin are carried out, the amount of the acid to be used is an amount that is sufficient to allow the dedicyclohexylamine chloride and the debenzylation to proceed. Although not particularly limited, from the viewpoint that dedicyclohexylamine salification and debenzylation are efficiently performed in a short time, the amount is preferably in the range of 10 to 1000 equivalents per 1 equivalent of dicyclohexylamine salt of N, N'-dibenzylbiotin. It is.

A known organic solvent can be used as a solvent to be used for dechlorination and debenzylation of dicyclohexylamine salt of 'N, N'-dibenzylbiotin. Examples of the organic solvent include halogenated hydrocarbons such as chloroform and methylene chloride; esters such as methyl acetate, ethyl acetate and butyl acetate; ethers such as isopropyl ether; aliphatic hydrocarbons such as hexane; Examples include aromatic hydrocarbons such as xylene, mesitylene, anisole, and chlorobenzene. Above all, in order to further efficiently purify the obtained N, N'-dibenzylbiotin and biotin, the decyclohexylamine salification and debenzylation of the dicyclohexylamine salt of N, N'-dibenzylbiotin proceed efficiently. It is preferable to use aromatic hydrocarbons, particularly mesitylene. One organic solvent may be used alone, or a mixed solvent of two or more organic solvents may be used. The amount of the solvent used can be appropriately adjusted in consideration of the capacity of the reaction vessel and the like, but is usually in the range of 1 to 100 parts by volume per 1 part by mass of the dicyclohexylamine salt of N, N'-dibenzylbiotin. It is.

The reaction temperature and reaction time for the decyclohexylamine salification and debenzylation of the dicyclohexylamine salt of N, N'-dibenzylbiotin can be appropriately adjusted, but the reaction temperature is usually 20 to 200 ° C. The temperature is preferably in the range of 40 to 150 ° C., more preferably 80 to 150 ° C., and still more preferably 100 to 150 ° C., and the reaction time is usually in the range of 0.5 to 17 hours.

After the reaction, biotin can be isolated by pouring the reaction solution containing biotin into water and filtering the precipitated solid. The isolated biotin is sufficiently pure and may be used for various applications as it is, but may be further purified. Purification can be performed, for example, by silica gel column purification. Silica gel column purification can be performed using, for example, an automatic preparative purification device equipped with a silica gel column.

According to the method according to the fifth aspect of the present invention, high-purity biotin can be produced. The HPLC purity of biotin produced by the method according to the fifth aspect of the present invention is preferably at least 96%, more preferably at least 97%, even more preferably at least 98%, even more preferably at least 98.5%, It is even more preferably at least 99%. The HPLC purity of biotin is measured by the method described in the examples.

When sulfuric acid is used as the acid, the reaction temperature is adjusted to 40 ° C. or lower, whereby the dicyclohexylamine salt of N, N′-dibenzylbiotin is removed from the dicyclohexylamine salt, and the N, N′-dibenzyl While biotin can be obtained, by adjusting the reaction temperature to more than 40 ° C., dedicyclohexylamine salification and debenzylation of the dicyclohexylamine salt of N, N′-dibenzylbiotin progress, and biotin can be obtained. it can.

<< Sixth embodiment >>
A sixth aspect of the present invention relates to a method for producing an N, N'-dibenzylbiotin ester compound using a dicyclohexylamine salt of N, N'-dibenzylbiotin.

The 'N, N'-dibenzylbiotin ester compound is a compound represented by the following formula (4).

In the formula (4), Bn represents a benzyl group, R ² represents an alkyl group, a substituted alkyl group, a group selected from aralkyl and substituted aralkyl groups.

"Alkyl group" means a linear or branched aliphatic saturated hydrocarbon group.

The number of carbon atoms of the alkyl group is usually 1 to 30, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 8, still more preferably 1 to 6, and still more preferably 1 to 4. . In addition, the carbon number of a linear alkyl group is 1 or more, and the carbon number of a branched alkyl group is 3 or more.

Examples of the alkyl group include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, isohexyl, heptyl, , 4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl and the like.

"Substituted alkyl group" means an alkyl group having one or more substituents. The above description regarding “alkyl group” also applies to the alkyl group from which the substituted alkyl group is derived, unless otherwise specified.

In the substituted alkyl group, one or more substituents are each substituted with a hydrogen atom of the alkyl group. The number of substituents in the substituted alkyl group is preferably 1 to 3, more preferably 1 or 2. When the number of substituents is two or more, the two or more substituents may be the same or different. One or more substituents of the substituted alkyl group can be independently selected from a substituent group α described below.

The number of carbon atoms of the substituted alkyl group (when the substituted alkyl group has one or more substituents containing carbon atoms, the total number of carbon atoms of the substituted alkyl group) is usually 30 or less, preferably 20 or less, more preferably It is 18 or less, still more preferably 16 or less, even more preferably 14 or less, even more preferably 12 or less, and still more preferably 10 or less.

Examples of the substituted alkyl group include a cycloalkyloxycarbonyloxyalkyl group such as a cilexetil group.

<< "Aralkyl group" means an alkyl group having one or more aryl groups. The above description regarding the “alkyl group” also applies to the alkyl group included in the aralkyl group, unless otherwise specified. The following description of the “aryl group” also applies to the aryl group included in the aralkyl group, unless otherwise specified.

“Aryl group” means a group formed by removing one hydrogen atom of an aromatic ring from an aromatic hydrocarbon having one or more monocyclic or fused polycyclic aromatic rings. .

The carbon number of the aryl group is usually 6 to 30, preferably 6 to 20, more preferably 6 to 18, even more preferably 6 to 16, even more preferably 6 to 14, and still more preferably 6 to 12, More preferably, it is 6 to 10.

Examples of the aryl group include a monocyclic or condensed polycyclic aromatic ring group. The condensed polycyclic aromatic ring group is usually bicyclic to tetracyclic, preferably bicyclic or tricyclic, more preferably bicyclic. The number of ring-constituting carbon atoms in the monocyclic or condensed polycyclic aromatic ring group is usually 6 to 18, preferably 6 to 14, and more preferably 6 to 10. Examples of the monocyclic aromatic ring group include a phenyl group. Examples of the condensed polycyclic aromatic ring group include a naphthyl group, an anthracenyl group, and a phenanthrenyl group. The fused polycyclic aromatic ring group may be partially saturated (that is, some of the bonds constituting the aromatic ring may be hydrogenated). Examples of the partially saturated condensed polycyclic aromatic ring group include a dihydronaphthyl group, an indanyl group, an acenaphthenyl group and the like.

The aryl group also includes a monocyclic or condensed polycyclic aromatic ring group having one or more alkyl groups. The above description of the “alkyl group” also applies to the alkyl group of the monocyclic or condensed polycyclic aromatic ring group, unless otherwise specified. The alkyl group of the monocyclic or condensed polycyclic aromatic ring group preferably has 1 to 10, more preferably 1 to 8, still more preferably 1 to 6, and still more preferably 1 to 4 carbon atoms. , Still more preferably 1 to 3, and still more preferably 1 or 2. The number of alkyl groups that the monocyclic or condensed polycyclic aromatic ring group may have is preferably 1 to 4, more preferably 1 to 3, and still more preferably 1 or 2. When the number of alkyl groups is two or more, the two or more alkyl groups may be the same or different. Examples of the monocyclic aromatic ring group having one or more alkyl groups include a tolyl group and a xylyl group.

Aryl groups also include groups having two or more monocyclic or fused polycyclic aromatic rings covalently linked by a single bond. Examples of the group having two or more monocyclic aromatic rings covalently bonded by a single bond include, for example, biphenyl, terphenyl and the like.

The aryl group is preferably a phenyl group or a phenyl group having one or more alkyl groups.

The number of carbon atoms of the aralkyl group is usually 7 to 30, preferably 7 to 20, more preferably 7 to 18, even more preferably 7 to 16, even more preferably 7 to 14, and still more preferably 7 to 12, More preferably, it is 7 to 10.

The carbon number of the alkyl group contained in the aralkyl group is preferably 1 to 10, more preferably 1 to 8, even more preferably 1 to 6, still more preferably 1 to 4, and still more preferably 1 to 3. .

The number of aryl groups possessed by the aralkyl group is preferably from 1 to 3, more preferably 1 or 2, and even more preferably 1. When the number of aryl groups in the aralkyl group is two or more, the two or more aryl groups may be the same or different.

Examples of the aralkyl group include benzyl, phenethyl, α-methylbenzyl, diphenylmethyl, trityl, biphenylmethyl, terphenylmethyl and the like.

<< A "substituted aralkyl group" means an aralkyl group having one or more substituents. The above description of the “aralkyl group” also applies to the aralkyl group from which the substituted aralkyl group is based, unless otherwise specified.

{In the substituted aralkyl group, one or more substituents are each substituted with a hydrogen atom of an aryl moiety and / or an alkyl moiety. The hydrogen atom to be substituted may be a hydrogen atom in the aryl moiety or a hydrogen atom in the alkyl moiety, but is preferably a hydrogen atom in the aryl moiety. The number of substituents of the substituted aryl group is preferably from 1 to 3, more preferably 1 or 2. When the number of substituents is two or more, the two or more substituents may be the same or different. One or more substituents of the substituted aryl group can be independently selected from a substituent group α described below. The number of carbon atoms of the substituted aryl group (when the substituted aryl group has one or more substituents containing carbon atoms, the total number of carbon atoms of the substituted aryl group) is usually 30 or less, preferably 20 or less, more preferably It is 18 or less, still more preferably 16 or less, even more preferably 14 or less, even more preferably 12 or less, and still more preferably 10 or less.

Substituent group α is composed of the following substituents.
(Α-1) halogen atom (α-2) nitro group (α-3) alkyloxy group (α-4) alkylcarbonyl group (α-5) alkylcarbonyloxy group (α-6) alkyloxycarbonyl group (α -7) alkyloxycarbonyloxy group (α-8) cycloalkyl group (α-9) cycloalkyloxy group (α-10) cycloalkylcarbonyl group (α-11) cycloalkylcarbonyloxy group (α-12) cyclo Alkyloxycarbonyl group (α-13) cycloalkyloxycarbonyloxy group (α-14) aryloxy group (α-15) arylcarbonyl group (α-16) arylcarbonyloxy group (α-17) aryloxycarbonyl group ( α-18) aryloxycarbonyloxy group (α-19) aralkyloxy group (α-20) aralkylcal Bonyl group (α-21) aralkylcarbonyloxy group (α-22) aralkyloxycarbonyl group (α-23) aralkyloxycarbonyloxy group

Substituent group α is a halogen atom, nitro group, alkyloxy group, alkylcarbonyloxy group, alkyloxycarbonyloxy group, cycloalkyl group, cycloalkyloxy group, cycloalkylcarbonyloxy group, cycloalkyloxycarbonyloxy group, aryl An oxy group, an arylcarbonyloxy group, an aryloxycarbonyloxy group, an aralkyloxy group, an aralkylcarbonyloxy group and an aralkyloxycarbonyloxy group are preferable, and a halogen atom, a nitro group, an alkyloxy group, an alkyloxycarbonyloxy More preferably, it is composed of a group, a cycloalkyl group, a cycloalkyloxy group, a cycloalkyloxycarbonyloxy group and an aryloxy group.

"Halogen atom" means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

"Alkyloxy group" means a group represented by an alkyl group -O-.

"Alkylcarbonyl group" means a group represented by an alkyl group -CO-.

"Alkylcarbonyloxy group" means a group represented by an alkyl group -CO-O-.

“Alkyloxycarbonyl group” means a group represented by an alkyl group —O—CO—.
“Alkyloxycarbonyloxy group” means a group represented by an alkyl group —O—CO—O—.

The above description regarding the “alkyl group” also applies to the alkyl group included in the alkyloxy group, the alkylcarbonyl group, the alkylcarbonyloxy group, the alkyloxycarbonyl group, or the alkyloxycarbonyloxy group, unless otherwise specified. . The number of carbon atoms of the alkyloxy group, alkylcarbonyl group, alkylcarbonyloxy group, alkyloxycarbonyl group or alkyloxycarbonyloxy group is preferably 1 to 10, more preferably 1 to 8, even more preferably 1 to 6, It is more preferably 1 to 4, still more preferably 1 to 3, and still more preferably 1 or 2.

"Cycloalkyl group" means a cyclic aliphatic saturated hydrocarbon group. The carbon number of the cycloalkyl group is usually 3 to 10, preferably 3 to 8, and more preferably 3 to 6. Examples of the cycloalkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, and the like.

"Cycloalkyloxy group" means a group represented by cycloalkyl group -O-.

"Cycloalkylcarbonyl group" means a group represented by cycloalkyl group -CO-.

"Cycloalkylcarbonyloxy group" means a group represented by cycloalkyl group -CO-O-.

"Cycloalkyloxycarbonyl group" means a group represented by cycloalkyl group -O-CO-.

"Cycloalkyloxycarbonyloxy group" means a group represented by cycloalkyl group -O-CO-O-.

The above description regarding the “cycloalkyl group” also applies to a cycloalkyloxy group, a cycloalkylcarbonyl group, a cycloalkylcarbonyloxy group, a cycloalkyloxycarbonyl group, or a cycloalkyl group contained in a cycloalkyloxycarbonyloxy group.

"Aryloxy group" means a group represented by an aryl group -O-.

"Arylcarbonyl group" means a group represented by an aryl group -CO-.

"Arylcarbonyloxy group" means a group represented by an aryl group -CO-O-.

"Aryloxycarbonyl group" means a group represented by an aryl group -O-CO-.

"Aryloxycarbonyloxy group" means a group represented by an aryl group -O-CO-O-.

The above description regarding “aryl group” also applies to an aryloxy group, an arylcarbonyl group, an arylcarbonyloxy group, an aryloxycarbonyl group, or an aryl group contained in an aryloxycarbonyloxy group, unless otherwise specified. . The number of carbon atoms of the aryl group contained in the aryloxy group, the arylcarbonyl group, the arylcarbonyloxy group, the aryloxycarbonyl group or the aryloxycarbonyloxy group is preferably 6 to 18, more preferably 6 to 16, and still more preferably. 6-14, still more preferably 6-12, even more preferably 6-10.

"Aralkyloxy group" means a group represented by an aralkyl group -O-.

"Aralkylcarbonyl group" means a group represented by an aralkyl group -CO-.

"Aralkylcarbonyloxy group" means a group represented by an aralkyl group -CO-O-.

"Aralkyloxycarbonyl group" means a group represented by an aralkyl group -O-CO-.

"Aralkyloxycarbonyloxy group" means a group represented by an aralkyl group -O-CO-O-.

The above description regarding the “aralkyl group” also applies to the aralkyl group included in the aralkyloxy group, the aralkylcarbonyl group, the aralkylcarbonyloxy group, the aralkyloxycarbonyl group, or the aralkyloxycarbonyloxy group, unless otherwise specified. . The number of carbon atoms of the aralkyloxy group, the aralkylcarbonyl group, the aralkylcarbonyloxy group, the aralkyloxycarbonyl group or the aralkyl group contained in the aralkyloxycarbonyloxy group is preferably 7 to 18, more preferably 7 to 16, and still more preferably 7-14, more preferably 7-12, even more preferably 7-10.

In a method according to a sixth aspect of the present invention, a dicyclohexylamine salt of N, N'-dibenzylbiotin is converted into a compound represented by the following formula (5):

Reacting with a halide represented by the formula (I) to form an N, N'-dibenzylbiotin ester compound.

The dicyclohexylamine salt of N, N'-dibenzylbiotin to be reacted with the halide is preferably a dicyclohexylamine salt of N, N'-dibenzylbiotin produced by the method according to the fourth aspect of the present invention.

The HPLC purity of the dicyclohexylamine salt of N, N'-dibenzylbiotin reacted with the halide is preferably 96% or more, more preferably 97% or more, even more preferably 98% or more, and even more preferably 98.5. % Or more, more preferably 99% or more. The HPLC purity of the dicyclohexylamine salt of N, N'-dibenzylbiotin is determined by the method described in the examples.

In the formula (5), R ² has the same meaning as in the formula (4), and X represents a halogen atom. The halogen atom can be selected from a fluorine atom, a chlorine atom, a bromine atom and an iodine atom, but is preferably selected from a chlorine atom, a bromine atom and an iodine atom.

Examples of the halide include iodomethane, ethyl bromide, propyl bromide, allyl bromide, benzyl chloride, benzyl bromide, cilexetil chloride and the like.

The amount of the halide to be used is not particularly limited as long as it is an amount sufficient for the reaction to proceed, and can be adjusted as appropriate. Preferably, it is in the range of 1.0 to 10 mol, more preferably, 1.0 to 3.0 mol.

As the base used when reacting the dicyclohexylamine salt of 'N, N'-dibenzylbiotin with a halide, for example, a base used for an esterification reaction can be suitably used. Examples of such a base include potassium carbonate, potassium hydrogen carbonate, sodium carbonate, baking soda, triethylamine, diisopropylethylamine, DBU and the like. Among these bases, potassium carbonate and triethylamine are preferred because they can be produced under mild conditions.

The amount of the base to be used is not particularly limited as long as it is an amount sufficient for the reaction to proceed, and can be appropriately adjusted, but is preferably based on 1 mol of the dicyclohexylamine salt of N, N'-dibenzylbiotin. Is in the range of 1 to 10 mol, more preferably in the range of 1 to 3 mol.

As a solvent used when reacting the dicyclohexylamine salt of 'N, N'-dibenzylbiotin with a halide, for example, a solvent used for an esterification reaction can be suitably used. Examples of such a solvent include nitriles such as acetonitrile and propionitrile; amides such as NN′-dimethylformamide (DMF), NN′-dimethylacetamide (DMA) and N-methylpyrrolidone (NMP) Ethers such as tetrahydrofuran (THF), 2-methyl-THF, 1,4-dioxane, t-butyl-methyl ether, diisopropyl ether, dimethoxyethane and diglyme; ketones such as acetone, methyl ethyl ketone and diethyl ketone; methyl acetate Esters such as acetic acid, ethyl acetate and butyl acetate; halogenated hydrocarbons such as methylene chloride, chloroform, carbon tetrachloride, 1,2-dichloroethane and chlorobenzene; aromatic hydrocarbons such as toluene and xylene; hexane, heptane and the like Of aliphatic hydrocarbons It is. One type of solvent may be used alone, or a mixed solvent of two or more types of solvents may be used. Among such solvents, amides such as DMF, DMA, and NMP, or a mixed solvent of two or more of these are preferred.

The amount of the solvent used can be appropriately adjusted in consideration of the capacity of the reaction vessel and the like, and is usually 0.5 to 100 parts by volume per 1 part by mass of dicyclohexylamine salt of N, N'-dibenzylbiotin. Parts by volume, preferably 2.0 to 20 parts by volume.

The reaction temperature and reaction time when reacting the dicyclohexylamine salt of N, N'-dibenzylbiotin with a halide can be appropriately adjusted, but the reaction temperature is usually in the range of -20 to 100 ° C, preferably It is in the range of 0 to 50 ° C., and the reaction time is usually in the range of 0.5 to 48 hours, preferably in the range of 1.0 to 17 hours.

After the reaction, the reaction solution containing the N, N'-dibenzylbiotin ester compound is neutralized by adding water and an acid, and the N, N'-dibenzylbiotin ester compound is extracted with an organic solvent such as ethyl acetate. Then, by washing, and concentrating, the N, N'-dibenzylbiotin ester compound can be isolated. The isolated N, N'-dibenzylbiotin ester compound may be further purified. The N, N'-dibenzylbiotin ester compound can be purified by silica gel column purification or the like. Purification can be performed using, for example, an automatic preparative purification device equipped with a silica gel column.

７Seventh aspect≫
A seventh aspect of the present invention relates to a method for producing a biotin ester compound.

The biotin ester compound is a compound represented by the following formula (6).

In the formula (6), R ² has the same meaning as in the formula (4).

The method according to the sixth aspect of the present invention is represented by the formula (6), wherein a benzyl group is eliminated from the N, N′-dibenzylbiotin ester compound produced by the method according to the fifth aspect of the present invention. Generating a biotin ester compound.

Elimination of the benzyl group from the 'N, N'-dibenzylbiotin ester compound can be carried out according to a conventional method.

Hereinafter, examples of the present invention will be described, but the present invention is not limited to these examples.

Measurement of HPLC Purity In the following Production Examples and Examples, the HPLC purity values of N, N′-dibenzylbiotin, the dicyclohexylamine salt of N, N′-dibenzylbiotin, and each compound of biotin were measured under the following measurement conditions. HPLC (High Performance Liquid Chromatography) is performed in accordance with the ratio of the peak area value of each compound measured by the HPLC to the sum of the area values of all peaks (excluding solvent-derived peaks) measured by the HPLC. This is the required value.

<HPLC measurement conditions>
(1) HPLC measurement conditions for N, N′-dibenzylbiotin dicyclohexylamine salt and N, N′-dibenzylbiotin ・ Column: L-coulmn ODS 5 μm (4.6 × 150 mm)
Mobile phase: 50 mM KH ₂ PO ₄ (pH 3.0) / CH ₃ CN = 60: 40
・ Flow rate: 1 mL / min
-Column temperature: 40 ° C
-Measurement wavelength: 254 nm
・ Analysis time: 40 min

Under the above HPLC measurement conditions, a peak is observed at about 20.7 min for N, N′-dibenzylbiotin dicyclohexylamine salt and about 20.7 min for N, N′-dibenzylbiotin.

(2) HPLC measurement conditions for biotin / column: X bridge O18 5 μm (4.6 × 150 mm)
Mobile phase: mobile phase _{_{A] 50mM KH 2 PO 4 (}} pH3.0), [ Mobile phase B] _CH 3 CN
・ Flow rate: 1 mL / min
-Column temperature: 40 ° C
-Measurement wavelength: 210 nm
・ Analysis time: 50 min

The concentration gradient was controlled by changing the mixing ratio of mobile phases A and B as shown in Table 1 below.

Under the above HPLC measurement conditions, biotin has a peak at about 17.3 min.

Production Example: Production of N, N′-dibenzylbiotin Biotin (1.0 g, 4.09 mmol, HPLC purity: 99.98%) was dissolved in dimethyl sulfoxide (DMSO, 20 mL), and potassium hydroxide was added at 25 ° C. (758 mg, 13.5 mmol) was added. At the same temperature, benzyl chloride (1.55 mL, 13.5 mmol) was added, and the mixture was stirred for 15 hours.

終了 After the reaction was completed, 10% hydrochloric acid was added at 0 ° C. to adjust the pH to 1. Ethyl acetate (80 mL) was added thereto to carry out liquid separation, and the obtained aqueous layer was extracted again with ethyl acetate (50 mL). The organic layers were combined, washed with water (3 × 100 mL), washed with saturated saline (50 mL), and dried over magnesium sulfate. Methanol (8 mL) and water (4 mL) were added to and dissolved in the concentrated residue obtained by concentrating the organic layer. Sodium hydroxide (0.41 g, 10.2 mmol) was added at 0 ° C. Stirred for hours.

終了 After the reaction was completed, the reaction solution was concentrated. Water (140 mL) was added to the concentrated residue to dissolve it, and the aqueous layer was washed with tert-butyl methyl ether (40 mL). The aqueous layer was cooled to 0 ° C. and adjusted to pH 1 with 10% hydrochloric acid. Ethyl acetate (80 mL) was added thereto, and the mixture was separated. The obtained aqueous layer was extracted again with ethyl acetate (20 mL). The organic layers were combined, washed with water (3 × 60 mL), washed with saturated saline (20 mL), dried over magnesium sulfate, concentrated under reduced pressure, and then with Isolera (Biotage) (elution solvent: hexane / ethyl acetate = 1/2). Purification gave N, N'-dibenzylbiotin (1.23 g, 71.0%, HPLC purity: 93.01%). The obtained N, N'-dibenzylbiotin was used in the following examples.

Example 1: Preparation of dicyclohexylamine salt of N, N'-dibenzylbiotin N, N'-dibenzylbiotin (80 mg, 0.188 mmol, HPLC purity: 93.01%, oily) in ethyl acetate (1. 6 mL) solution was added with dicyclohexylamine (37.4 μL, 0.188 mmol) at 25 ° C., and the mixture was stirred at the same temperature for 3.5 hours and at 0 ° C. for 1 hour. After washing with ethyl and drying under reduced pressure, dicyclohexylamine salt of N, N'-dibenzylbiotin (73.6 mg, yield: 64.5%, HPLC purity: 99.25%) was obtained.

Physical property data of the dicyclohexylamine salt of N, N'-dibenzylbiotin obtained in Example 1 is as follows.
Melting point: 126 ° C
IR: 2940, 1697, 1447 cm ^-1
¹ H-NMR: δ7.30-7.18 (m, 10H), 4.76 (d, 1H), 4.48 (d, 1H), 4.10 (d, 1H), 3.99 (d , 1H), 3.94 (m, 1H), 3.79 (m, 1H), 3.14 (m, 1H), 2.78 (dd, 1H), 2.61 (m, 3H), 2 .05 (t, 2H), 1.79-0.98 (m, 26H)
¹³ C-NMR: δ 175.6, 161.6, 138.2, 137.9, 129.0, 128.4, 128.3, 127.8, 127.7, 62.7, 61.6, 55 .9, 52.6, 48.2, 45.6, 35.4, 35.2, 32.8, 29.5, 28.9, 26.2, 25.5, 25.0

In the HPLC measurement conditions, the peak derived from the dicyclohexylamine salt of N, N'-dibenzylbiotin and the peak derived from N, N'-dibenzylbiotin are detected at the same position (time). This is presumed to be because the dicyclohexylamine salt of N, N'-dibenzylbiotin is dissociated into N, N'-dibenzylbiotin and dicyclohexylamine in the HPLC column. Therefore, the measurement of the dicyclohexylamine salt of N, N'-dibenzylbiotin was performed indirectly by measuring the dissociated N, N'-dibenzylbiotin, and the purity was evaluated.

Example 2: Preparation of dicyclohexylamine salt of N, N'-dibenzylbiotin N, N'-dibenzylbiotin (5.0 g, 11.7 mmol, HPLC purity: 93.01%) solution in butyl acetate (50 mL) To the mixture was added dicyclohexylamine (2.34 mL, 11.7 mmol) at 25 ° C, and the mixture was stirred at the same temperature for 2 hours and at 0 ° C for 1 hour. The precipitated crystals were collected by filtration and washed with cold butyl acetate. By drying under reduced pressure, dicyclohexylamine salt of N, N'-dibenzylbiotin (6.05 g, yield: 84.8%, HPLC purity: 98.93%) was obtained.

Physical property data of the dicyclohexylamine salt of N, N'-dibenzylbiotin obtained in Example 2 is as follows.
Melting point: 126 ° C
IR: 2940, 1697, 1447 cm ^-1
¹ H-NMR: δ7.30-7.18 (m, 10H), 4.76 (d, 1H), 4.48 (d, 1H), 4.10 (d, 1H), 3.99 (d , 1H), 3.94 (m, 1H), 3.79 (m, 1H), 3.14 (m, 1H), 2.78 (dd, 1H), 2.61 (m, 3H), 2 .05 (t, 2H), 1.79-0.98 (m, 26H)
¹³ C-NMR: δ 175.6, 161.6, 138.2, 137.9, 129.0, 128.4, 128.3, 127.8, 127.7, 62.7, 61.6, 55 .9, 52.6, 48.2, 45.6, 35.4, 35.2, 32.8, 29.5, 28.9, 26.2, 25.5, 25.0

Example 3: Preparation of dicyclohexylamine salt of N, N'-dibenzylbiotin In a solution of N, N'-dibenzylbiotin (80 mg, 0.188 mmol, HPLC purity: 93.01%) in acetone (1.6 mL). After adding dicyclohexylamine (37.4 μL, 0.188 mmol) at 25 ° C. and stirring at the same temperature for 2 hours and at 0 ° C. for 1 hour, the precipitated crystals were collected by filtration, washed with cold butyl acetate, and dried under reduced pressure. By drying, dicyclohexylamine salt of N, N'-dibenzylbiotin (40.0 mg, yield: 35.1%, HPLC purity: 98.66%) was obtained.

Physical property data of the dicyclohexylamine salt of N, N'-dibenzylbiotin obtained in Example 3 is as follows.
Melting point: 126 ° C
IR: 2940, 1697, 1447 cm ^-1
¹ H-NMR: δ7.30-7.18 (m, 10H), 4.76 (d, 1H), 4.48 (d, 1H), 4.10 (d, 1H), 3.99 (d , 1H), 3.94 (m, 1H), 3.79 (m, 1H), 3.14 (m, 1H), 2.78 (dd, 1H), 2.61 (m, 3H), 2 .05 (t, 2H), 1.79-0.98 (m, 26H)
¹³ C-NMR: δ 175.6, 161.6, 138.2, 137.9, 129.0, 128.4, 128.3, 127.8, 127.7, 62.7, 61.6, 55 .9, 52.6, 48.2, 45.6, 35.4, 35.2, 32.8, 29.5, 28.9, 26.2, 25.5, 25.0

Example 4: Preparation of N, N'-dibenzylbiotin by dedicyclohexylamine salification of dicyclohexylamine salt of N, N'-dibenzylbiotin Preparation of N, N'-dibenzylbiotin obtained in Example 2 Dicyclohexylamine salt (200 mg, 0.330 mmol) was suspended in ethyl acetate (1.5 mL), and a solution of potassium hydrogen sulfate (58 mg, 0.429 mmol) in water (500 μL) was added at 25 ° C. Stirred for hours. After completion of the reaction, ethyl acetate (20 mL) was added to the reaction solution to carry out liquid separation, and the obtained aqueous layer was extracted again with ethyl acetate (10 mL). The organic layers were combined, washed with water (30 mL), washed with saturated saline (30 mL), and dried over magnesium sulfate. The organic layer was concentrated to give N, N′-dibenzylbiotin (140 mg, yield: quant., HPLC purity: 98.66%, colorless and transparent solid, melting point: 94 ° C.).

Example 5: Preparation of biotin by decyclohexylamine chloride and debenzylation of dicyclohexylamine salt of N, N'-dibenzylbiotin Dicyclohexylamine salt of N, N'-dibenzylbiotin obtained in Example 2 (2.0 g, 3.30 mmol) was added and dissolved in a two-phase solvent of mesitylene (5.0 mL) and methanesulfonic acid (3.17 g, 33.0 mmol), and the mixture was stirred at 135 ° C. for 2.5 hours. Further, methanesulfonic acid (3.17 g, 33.0 mmol) was added, and the mixture was stirred at the same temperature for 4.5 hours. After completion of the reaction, acetic acid (580 μL) was added at 80 ° C. to separate into two layers, and mesitylene was removed. Distilled water (40 mL) was added to the remaining reaction solution at 0 ° C., and the mixture was stirred at the same temperature for 1 hour. The precipitated crystals were collected by filtration, washed with water and acetone, and dried under reduced pressure to obtain biotin (625 mg, yield: 77.6%, HPLC purity: 99.00%).

Example 6: Preparation of N, N'-dibenzylbiotin ester compound by esterification of dicyclohexylamine salt of N, N'-dibenzylbiotin As shown in the following reaction formula, N, N'-dibenzylbiotin Lexetil ester was synthesized.

ジ Dicyclohexylamine salt of N, N'-dibenzylbiotin (1.0 g, 1.65 mmol) obtained in Example 2 was added to N, N-dimethylformamide (DMF, 10 mL) and dissolved. After potassium carbonate (342 mg, 2.47 mmol) was added thereto at 25 ° C., cilexetil chloride (452 μL, 2.47 mmol) was added at the same temperature, and the mixture was stirred at 60 ° C. for 6 hours. After completion of the reaction, water (10 mL) was added at 25 ° C., and the pH was adjusted to 7 with 5% hydrochloric acid. Ethyl acetate (80 mL) was added thereto and the mixture was separated, and the obtained aqueous layer was extracted again with ethyl acetate (30 mL). The organic layers were combined, washed with water (3 × 50 mL), washed with saturated saline (50 mL), and dried over magnesium sulfate. After concentrating the organic layer, it was purified with Isolera (Biotage) (elution solvent: hexane / ethyl acetate = 2/1) to give cilexetil (3aS, 4S, 6aR) -5- (1,3-dibenzyl-2,3). , 3a, 4,6,6a-Hexahydro-2-oxo-1H-thiene [3,4-d] imidazol-5-yl) pentanoate (1.46 g, yield: quant.) Was obtained.

Claims

The following equation (1):

[Wherein, Bn represents a benzyl group. ]
A dicyclohexylamine salt of N, N'-dibenzylbiotin represented by the formula:
The dicyclohexylamine salt of N, N'-dibenzylbiotin according to claim 1, wherein the HPLC purity is 96% or more.
The following equation (2):

[Wherein, Bn is as defined above. ]
The N, N'-dibenzylbiotin represented by the formula, wherein the HPLC purity is 96% or more.
The following equation (3):

The biotin represented by the above, wherein the HPLC purity is 96% or more.
A method for producing a dicyclohexylamine salt of N, N'-dibenzylbiotin according to claim 1, wherein
The following equation (2):

[Wherein, Bn is as defined above. ]
Contacting N, N'-dibenzylbiotin represented by the formula with dicyclohexylamine to form the dicyclohexylamine salt of N, N'-dibenzylbiotin.
The method according to claim 5, wherein the HPLC purity of the dicyclohexylamine salt of the N, N'-dibenzylbiotin is 96% or more.
7. The method according to claim 5, wherein the HPLC purity of the N, N'-dibenzylbiotin is 95% or less.
The following formula (A):

[In the formula, R 1 represents a benzyl group or a hydrogen atom. ]
A method for producing a compound (A) represented by the formula:
2. A method comprising the step of contacting the dicyclohexylamine salt of N, N'-dibenzylbiotin according to claim 1 with at least one acid to form said compound (A).
The method according to claim 8, wherein the HPLC purity of the dicyclohexylamine salt of the N, N'-dibenzylbiotin is 96% or more.
The acid is at least one acid selected from hydrochloric acid, sulfuric acid, and hydrogen sulfate;
The compound (A) has the following formula (2):

[Wherein, Bn is as defined above. ]
10. The method according to claim 8 or 9, which is N, N'-dibenzylbiotin represented by the formula:
The method according to claim 10, wherein the HPLC purity of the ＮN, N'-dibenzylbiotin is 96% or more.
The acid is at least one acid selected from methanesulfonic acid, sulfuric acid and hydrobromic acid,
The compound (A) has the following formula (3):

The method according to claim 8 or 9, which is biotin represented by the following formula:
The method according to claim 12, wherein the biotin has an HPLC purity of 96% or more.
The following equation (4):

Wherein Bn is as defined above, and R 2 is an alkyl group having 1 to 30 carbon atoms, a substituted alkyl group having 1 to 30 carbon atoms, an aralkyl group having 7 to 30 carbon atoms, and a substituted group having 7 to 30 carbon atoms. Represents a group selected from aralkyl groups. ]
A method for producing an N, N′-dibenzylbiotin ester compound represented by the formula:
The dicyclohexylamine salt of N, N′-dibenzylbiotin according to claim 1 in the presence of a base in the following formula (5):

[Wherein, R 2 has the same meaning as described above, and X represents a halogen atom. ]
Reacting with a halide represented by the formula to produce the N, N'-dibenzylbiotin ester compound.
15. The method according to claim 14, wherein the HPLC purity of the dicyclohexylamine salt of the N, N'-dibenzylbiotin is 96% or more.
The following equation (6):

[Wherein, R 2 has the same meaning as described above. ]
A method for producing a biotin ester compound represented by
A method for producing the biotin ester compound by removing a benzyl group from the N, N'-dibenzylbiotin ester compound produced by the method according to claim 14 or 15.