JP2021151997A - Method for producing epigallocatechin gallate conjugate - Google Patents

Abstract

To provide: a method for producing an epigallocatechin gallate conjugate; and the epigallocatechin gallate conjugate and a production intermediate thereof.SOLUTION: This method for producing an epigallocatechin gallate conjugate represented by general formula (Id) comprises a step for reacting 2,2,2-trichloroethoxy-sulfuryl-1,2-dimethylimidazolium triflate with benzylated epigallocatechin gallate represented by general formula (v), or a step for reacting a glucuronic acid donor with the benzylated epigallocatechin gallate.SELECTED DRAWING: None

Description

The present invention relates to a method for producing an epigallocatechin gallate conjugate, and the epigallocatechin gallate conjugate and its production intermediate.

Epigallocatechin gallate is a representative of catechins contained in tea leaves, and is known to have various excellent bioactive functions such as cholesterol elevation inhibitory action and body fat burning action.

It is known that catechins absorbed in the body exist as glucuronic acid conjugates, sulfate conjugates, methyl conjugates and the like in addition to the free state. Therefore, it is important to verify the pharmacokinetics and biological activity of such conjugates in order to explore the usefulness of catechins.

For example, Non-Patent Document 1 verifies the biological or pharmacological effects of glucuronic acid conjugates and sulfate conjugates of epicatechin and methyl epicatechin as standard products for biological analysis. Therefore, it has been reported that these were chemically synthesized. That is, as shown by the following formula, the phenol compound 10c that protects a predetermined hydroxyl group with a benzyl group, Et ₃ the presence of N and DMAP, chlorosulfonic acid 2,2,2-trichloroethyl (TCE) ester (Cl ₃ CCH ₂ OSO ₂ Cl) was used to produce a sulfated 10f protected with a TCE group (step i), debenzylation by catalytic hydrocracking (step ii), and a TCE group using Zn-ammonium formate. (Step iii) is disclosed to produce 4'-O-methylepicatekin-7-ammonium sulfate ammonium sulfate 10S.

However, regarding the conjugate of epigallocatechin gallate, although it is speculated that it may exist as a group of compounds, there is no synthetic example, and the compound has not even been identified.

J. Nat. Prod., 2013, 76, 157-169

The present invention relates to a method for producing an epigallocatechin gallate conjugate, and to provide the epigallocatechin gallate conjugate and a production intermediate thereof.

The present inventors investigated the chemical synthesis of a conjugate of epigallocatechin gallate, and obtained it by condensing benzylated epigallocatechin (ii) with benzylated epigallocatechin gallate as shown in the following reaction formula. From benzylated epigallocatechin gallate (v), which is produced via an allylated epigallocatechin gallate compound (iv) and in which a predetermined hydroxyl group is protected with a benzyl group (Bn), an epigallocatechin gallate conjugate (sulfated product (sulfated product). It has been found that Ib) and gallocatechin oxide (Ic)) can be obtained in good yield.
Regarding the sulfate conjugate, the hydroxyl group in the gallate portion cannot be sulfated by the method using the _{chlorosulfate TCE ester (Cl 3} CCH ₂ OSO ₂ Cl) as the sulfate reagent disclosed in Non-Patent Document 1, 2 , 2,2-Trichloroethoxy-sulfryl-1,2-dimethylimidazolium trifurate (SDIS) to obtain TCE sulfate (Ia) from benzylated epigalocateringarate (v) in high yield. It was found that by subjecting the TCE sulfate to a hydrogenolysis reaction in the presence of a hydrogenation catalyst, the TCE sulfate can be converted to epigalocatecatingalate sulfate (Ib) in one step.

[In the formula, ^one of X 1 and X ² shows a benzyl group and the other shows an allyl group, R ^1a and R ^{2a correspond to X 1} and X ² , respectively, and one of R ^1a and R ^2a corresponds to X 1 and X 2, respectively. The other is a benzyl group and the other represents a hydrogen atom, R ^1b and R ^2b correspond to R ^1a and R ^2a , respectively, one of which is a benzyl group and the other of which is -SO ₃ CH ₂ CCl ₃ , and R ^1c and R ^2c. Corresponds to R ^1b and R ^2b , respectively, one of which represents a hydrogen atom and the other of -SO ₃ M (where M represents a hydrogen atom, an alkali metal atom, an alkaline earth metal atom or ammonium). R ^1d and R ^2d correspond to R ^1a and R ^2a , respectively, one of which represents a hydrogen atom and the other of which represents a glucuronosyl group. ]

That is, the present invention relates to the following 1) to 6).
1) The following general formula (v):

〔式中、Ｒ^１ａ及びＲ^２ａはいずれか一方がベンジル基で他方が水素原子を示す。〕
で表されるベンジル化エピガロカテキンガレートに２，２，２−トリクロロエトキシ−スルフリル−１，２−ジメチルイミダゾリウムトリフレートを反応させる工程、又はグルクロン酸供与体を反応させる工程を含む、一般式（Ｉｄ）：

[In the formula, one of R ^1a and R ^2a represents a benzyl group and the other represents a hydrogen atom. ]
A general formula including a step of reacting 2,2,2-trichloroethoxy-sulfuryl-1,2-dimethylimidazolium triflate with a benzylated epigallocatechin gallate represented by, or a step of reacting with a glucuronic acid donor. (Id):

〔式中、Ｒ^１ｅ及びＲ^２eはそれぞれＲ^１ａ及びＲ^２ａに対応し、いずれか一方が水素原子で他方が−ＳＯ_３Ｍ（ここで、Ｍは水素原子、アルカリ金属原子、アルカリ土類金属原子又はアンモニウムを示す）又はグルクロノシル基を示す。〕
で表されるエピガロカテキンガレート抱合体の製造方法。
２）下記一般式（ｖ）：

[In the formula, R ^1e and R ^2e correspond to R ^1a and R ^2a , respectively, and one of them is a hydrogen atom and the other is -SO ₃ M (where M is a hydrogen atom, an alkali metal atom, and an alkaline earth metal). (Indicating an atom or ammonium) or a glucuronosyl group. ]
A method for producing an epigallocatechin gallate conjugate represented by.
2) The following general formula (v):

〔式中、Ｒ^１ａ及びＲ^２ａはいずれか一方がベンジル基で他方が水素原子を示す。〕
で表されるベンジル化エピガロカテキンガレートに２，２，２−トリクロロエトキシ−スルフリル−１，２−ジメチルイミダゾリウムトリフレートを反応させる、下記一般式（Ｉａ）：

[In the formula, one of R ^1a and R ^2a represents a benzyl group and the other represents a hydrogen atom. ]
Reacting 2,2,2-trichloroethoxy-sulfuryl-1,2-dimethylimidazolium triflate with the benzylated epigallocatechin gallate represented by the following general formula (Ia):

〔式中、Ｒ^１ｂ及びＲ^２ｂはそれぞれＲ^１ａ及びＲ^２ａに対応し、いずれか一方がベンジル基で他方が−ＳＯ_３ＣＨ_２ＣＣｌ_３を示す。〕
で表されるベンジル化エピガロカテキンガレートＴＣＥ硫酸化物の製造方法。
３）２）で得られた、一般式（Ｉａ）で表されるベンジル化エピガロカテキンガレートＴＣＥ硫酸化物を、水素化触媒存在下で加水素分解する、一般式（Ｉｂ）：

[In the formula, R ^1b and R ^2b correspond to R ^1a and R ^2a , respectively, and one of them is a benzyl group and the other is −SO ₃ CH ₂ CCl ₃ . ]
A method for producing a benzylated epigallocatechin gallate TCE sulfated product represented by.
3) The benzylated epigallocatechin gallate TCE sulfate obtained by the general formula (Ia) obtained in 2) is hydrogenated in the presence of a hydrogenation catalyst, the general formula (Ib):

〔式中、Ｒ^１ｃ及びＲ^２ｃはそれぞれＲ^１ｂ及びＲ^２ｂに対応し、いずれか一方が水素原子で他方が−ＳＯ_３Ｍ（ここで、Ｍは水素原子、アルカリ金属原子、アルカリ土類金属原子又はアンモニウムを示す）を示す。〕
で表されるエピガロカテキンガレート硫酸化物の製造方法。
４）下記一般式（ｖ）：

[In the formula, R ^1c and R ^2c correspond to R ^1b and R ^2b , respectively, one of which is a hydrogen atom and the other of -SO ₃ M (where M is a hydrogen atom, an alkali metal atom, an alkaline earth metal). Indicates an atom or ammonium). ]
A method for producing epigallocatechin gallate sulfate represented by.
4) The following general formula (v):

〔式中、Ｒ^１ａ及びＲ^２ａはいずれか一方がベンジル基で他方が水素原子を示す。〕
で表されるベンジル化エピガロカテキンガレートにグルクロン酸供与体を反応させ、次いで、脱保護反応に付す、一般式（Ｉｃ）：

[In the formula, one of R ^1a and R ^2a represents a benzyl group and the other represents a hydrogen atom. ]
The benzylated epigallocatechin gallate represented by is reacted with a glucuronic acid donor and then subjected to a deprotection reaction, the general formula (Ic):

〔式中、Ｒ^１ｄ及びＲ^２ｄはそれぞれＲ^１ａ及びＲ^２ａに対応し、いずれか一方が水素原子で他方がグルクロノシル基を示す。〕
で表されるエピガロカテキンガレートグルクロン酸化物の製造方法。
５）下記一般式（Ｉ）：

[In the formula, R ^1d and R ^2d correspond to R ^1a and R ^2a , respectively, and one of them represents a hydrogen atom and the other represents a glucuronosyl group. ]
A method for producing epigallocatechin gallate glucuron oxide represented by.
5) The following general formula (I):

〔式中、Ｚ^１は水素原子又はベンジル基を示し、Ｚ^１がベンジル基である場合、Ｒ^１及びＲ^２はいずれか一方がベンジル基で他方が−ＳＯ_３ＣＨ_２ＣＣｌ_３を示し、Ｚ^１が水素原子である場合、Ｒ^１及びＲ^２はいずれか一方が水素原子で他方が−ＳＯ_３Ｍ（ここで、Ｍは水素原子、アルカリ金属原子、アルカリ土類金属原子又はアンモニウムを示す）を示す。〕
で表されるエピガロカテキンガレート硫酸化物又はその誘導体。
６）下記一般式（ＩＩ）：

[In the formula, Z ¹ represents a hydrogen atom or a benzyl group, and when Z ¹ is a benzyl group, one of R ¹ and R ² represents a benzyl group and the other represents −SO ₃ CH ₂ CCl ₃ , and Z ^{When 1} is a hydrogen atom, one of R ¹ and R ² is a hydrogen atom and the other is -SO ₃ M (where M indicates a hydrogen atom, an alkali metal atom, an alkaline earth metal atom or ammonium). Is shown. ]
Epigallocatechin gallate sulfate or a derivative thereof represented by.
6) The following general formula (II):

〔式中、Ｙ^１及びＹ^２はいずれか一方がベンジル基で他方がアリル基又は水素原子を示す。〕
で表されるベンジル化エピガロカテキンガレート又はそのアリル化物。

[In the formula, ^one of Y 1 and Y ² represents a benzyl group and the other represents an allyl group or a hydrogen atom. ]
Benzylated epigallocatechin gallate represented by or an allylic product thereof.

According to the present invention, an epigallocatechin gallate conjugate can be efficiently chemically synthesized. Epigallocatechin gallate sulfate and glucuron oxide can serve as a guide for tracking the pharmacokinetics of epigallocatechin gallate conjugates formed in the body.

Quantitative results of EGCg and 4 "-sulfated EGCg in human plasma samples. Quantitative results of EGCg and 3 "-sulfated EGCg in rat plasma samples.

In the present invention, the "epigallocatechin gallate conjugate" is a metabolite of epigallocatechin gallate in vivo and means a compound in which a water-soluble molecule is introduced into epigallocatechin gallate. Specifically, it includes a sulfated product in which sulfuric acid is introduced into the gallate site of epigallocatechin gallate and a glucuron oxide in which glucuronic acid is introduced.

In the method for producing an epigallocatechin gallate conjugate of the present invention, as shown below, the benzylated epigallocatechin gallate represented by the general formula (v) is subjected to a sulfated or glucuron oxidation reaction.

[In the formula, one of R ^1a and R ^2a represents a benzyl group and the other represents a hydrogen atom, R ^1e and R ^2e correspond to R ^1a and R ^2a , respectively, and one of them is a hydrogen atom and the other is −. SO ₃ M (where M represents a hydrogen atom, an alkali metal atom, an alkaline earth metal atom or ammonium) or a glucuronosyl group. ]

In the present invention, the benzylated epigallocatechin gallate represented by the formula (v) is prepared by, for example, benzylating the phenolic hydroxyl group of the epigallocatechin represented by the formula (i) as shown in the following reaction formula. A benzylated epigallocatechin represented by (ii) is obtained, and a benzylated epigallocatechin gallate represented by the formula (iii) is condensed with this to obtain an allylated epigallocatechin gallate compound represented by the formula (iv). Then, it can be produced by removing the allyl group.

[In the formula, ^one of X 1 and X ² shows a benzyl group and the other shows an allyl group, and one of R ^1a and R ^2a shows a benzyl group and the other shows a hydrogen atom. ]

<Benzylation>
Benzylation of the phenolic hydroxyl group in epigallocatechin represented by the formula (i) can be carried out according to or according to a known method.
That is, it can be carried out by reacting epigallocatechin with benzyl halide in the presence of a base.

The solvent may be any solvent as long as it does not adversely affect the reaction. For example, a ketone solvent, an ether solvent, an ester solvent, an aprotonic polar solvent, a halogenated hydrocarbon solvent, or a mixed solvent thereof may be used. Can be mentioned. Preferred are aprotic polar solvents such as tetrahydrofuran, acetone, acetonitrile, N, N-dimethylformamide, dimethyl sulfoxide and the like, more preferably N, N-dimethylformamide.

As the base, known inorganic bases and organic bases can be used. Examples of the inorganic base include alkali metal, alkali metal hydrogen carbonate, alkali metal hydroxide, alkali metal carbonate, lower alkali metal (C _1-4 ) alkoxide, and alkali metal hydride (for example, sodium hydride and potassium hydride). Etc.) etc. Organic bases include trialkylamines (eg, trimethylamine, triethylamine, N, N-diisopropylethylamine, etc.), pyridine, quinoline, piperidine, imidazole, picolin, 4-dimethylaminopyridine, N, N-dimethylaniline, N-methyl. Morpholine, 1,5-diazabicyclo [4.3.0] nona-5-ene (DBN), 1,4-diazabicyclo [2.2.2] octane (DABCO), 1,8-diazabicyclo [5.4. 0] Undeca-7-en (DBU) and the like can be mentioned. When these bases are liquid, they can also be used as a solvent. These bases are used alone or in admixture of two or more. It is preferably an alkali metal hydride, more preferably sodium hydride.

The amount of the base used is usually 4 to 6 equivalents, preferably 4.5 to 5.5 equivalents, more preferably 4.75 to 5.4 equivalents, still more preferably 5.0 equivalents, relative to 1 equivalent of epigallocatechin. ~ 5.3 equivalents.

Examples of the benzyl halide include benzyl chloride, benzyl bromide, benzyl iodide and the like. These are used alone or in admixture of two or more. Of these, benzyl bromide is preferred.
The amount of benzyl halide used is usually 4 to 20 equivalents, preferably 4.5 to 16 equivalents, more preferably 4.75 to 15 equivalents, still more preferably 5.0 to 13 equivalents, relative to 1 equivalent of epigallocatechin. It is 5.5 equivalents.

The reaction temperature is not particularly limited, and the reaction is usually carried out under cooling, at room temperature, or under heating. The reaction is preferably carried out at a temperature condition of -50 to 20 ° C., more preferably -40 to 10 ° C., further preferably -30 to 5 ° C., still more preferably -20 to -5 ° C. for 6 to 24 hours. ..

<Condensation reaction>
To the obtained benzylated epigallocatekin (iii), an allyl ether gallic acid ester (iii) in which a hydroxyl group is protected with a benzyl group is added and subjected to a condensation reaction.
The condensation reaction can be carried out by a usual method in the presence of an appropriate solvent and condensing agent.

The solvent may be any solvent as long as it does not adversely affect the reaction. For example, aprotonic polar solvents such as acetone, acetonitrile, ethyl acetate, N, N-dimethylformamide and dimethylsulfoxide, dichloromethane, dichloroethane, trichloroethylene, tetrachloroethylene and the like. Examples thereof include chlorine-based solvents, ether-based solvents such as diethyl ether, tetrahydrofuran, and dioxane, and acetonitrile is preferable.

Examples of the condensing agent include carbodiimide compounds such as N, N-diethylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride [WSCI / HCl], and N, N-dicyclohexylcarbodiimide, which are preferable. 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride [WSCI.HCl].

The amount of the allyl ether gallic acid ester (iii) used is usually 1 to 5 equivalents, preferably 1.25 to 4.5 equivalents, and further 1 equivalent to 1 equivalent of the benzylated epigalocatechin represented by the formula (ii). It is preferably 1.35 to 4.25 equivalents, more preferably 1.5 to 3 equivalents.

The amount of the condensing agent used is usually 1 to 5 equivalents, preferably 1.25 to 4.5 equivalents, more preferably 1.5 to 1.5 equivalents, relative to 1 equivalent of the benzylated epigallocatechin represented by the formula (ii). It is 4.25 equivalents, more preferably 2 to 4.1 equivalents.

If necessary, the reaction may proceed using 1-hydroxybenzotriazole [HOBT] or the like as a reaction accelerator and N, N-diisopropylethylamine [DIPEA] or the like as an acid receptor.
The reaction is usually carried out at room temperature, or under cooling or heating, preferably under the conditions of 24-27 ° C. for 2-18 hours.

<Deallylation>
Desorption of the allylic group of the allylated epigalocateringalate compound represented by the formula (iv) involves the presence of a palladium catalyst such as palladium acetate, tetrakis (triphenylphosphine) palladium (0), and trisdibenzylideneacetonedipalladium. Below, it can be carried out by using a hydrogen source such as foric acid or an ammonium salt thereof, or a nucleophilic reagent such as morpholin.
The amount of the catalyst used is preferably 0.01 to 1 equivalent, more preferably 0.02 to 0.8 equivalent, still more preferably 0.05 to 0, relative to 1 equivalent of the compound represented by the formula (iv). 66 equivalents, more preferably 0.1 to 0.5 equivalents, and the amount of nucleophilic reagents used such as hydrogen source and morpholin is 0.1 to 5 equivalents relative to 1 equivalent of the compound represented by the formula (iv). It is preferable, more preferably 0.25 to 4 equivalents, further preferably 0.5 to 3 equivalents, even more preferably 1 to 2 equivalents.
Examples of the solvent used in this step include ether solvents such as tetrahydrofuran and 1,4-dioxane.

The reaction is carried out by stirring the allylated epigallocate catechin gallate compound represented by the formula (iv) in a solvent and adding a nucleophile such as a hydrogen source or morpholine to the catalyst, and the reaction temperature is 24. The temperature is preferably ~ 27 ° C., and the reaction time is preferably 1 to 12 hours.

The allyl ether gallic acid ester (iii) can be produced from gallic acid ester by combining known benzylation and allylation reactions of phenolic hydroxyl groups, as shown in Reference Examples 1 and 2 described later. ..

The compounds represented by the above formulas (iv) and (v) are all novel compounds. Therefore, in the present invention, benzylated epigallocatechin gallate represented by the following general formula (II) or an allylic product thereof is also provided. The compound represented by the formula (II) is useful as a production intermediate for synthesizing the epigallocatechin gallate conjugate of the present invention.

[In the formula, ^one of Y 1 and Y ² represents a benzyl group and the other represents an allyl group or a hydrogen atom. ]

The sulfated product of epigallocatechin gallate of the present invention or a derivative thereof can be produced from the benzylated epigallocatechin gallate represented by the formula (v) by the following steps.

[In the formula, one of R ^1a and R ^2a represents a benzyl group and the other represents a hydrogen atom, and R ^1b and R ^2b correspond to R ^1a and R ^2a , respectively, and one of them is a benzyl group and the other is −. Indicates SO ₃ CH ₂ CCl ₃ , where R ^1c and R ^2c correspond to R ^1b and R ^2b , respectively, one of which is a hydrogen atom and the other of which is -SO ₃ M (where M is a hydrogen atom and an alkali metal atom). , Indicates an alkaline earth metal atom or ammonium). ]

1) Step-1
This is a reaction in which the hydroxyl group at the 3 "position or the 4" position of the benzylated epigallocatechin gallate represented by the formula (v) in which a predetermined hydroxyl group is protected with a benzyl group is TCE sulfated. The TCE sulfate reagent used here is 2,2,2-trichloroethoxy-sulfuryl-1,2-dimethylimidazolium triflate (2,2,2-trichloroethoxy-sulfuryl-1,2-dimethylimidazolium triflate; SDIS). ) Is used.
SDIS is J.M. Org. Chem. 2009, 74, 6479. It can be manufactured according to the method described in 1. That is, 2,2,2-trichloroethylchlorosulfate (TCE-OSO ₂ Cl) was obtained from 2,2,2-trichloroethanol and sulfyl chloride, and 2-methylimidazole was reacted with this to obtain 2,2,2-trichloroethylchlorosulfate (TCE-OSO 2 Cl). SDIS can be produced by reacting with trichloroethylimidazole sulfate (TCE-OSO ₂ Im) and then with methyl trifluoromethanesulfonate (Reference Example 3).

In the reaction between the compound represented by the formula (v) and SDIS, the compound represented by the formula (v) and SDIS are stirred in the presence of a base in an ether-based, chlorine-based, aprotic or mixed solvent thereof. It is done by.

The amount of SDIS used is usually 1 to 5 equivalents, preferably 1.1 to 4 equivalents, more preferably 1.25 to 3.5 equivalents, still more preferably 1 equivalent to 1 equivalent of the compound represented by the formula (v). Is 1.5 to 3 equivalents.

Examples of the ether solvent include tetrahydrofuran, dioxane, diethylene glycol dimethyl ether (diglyme), diethyl ether and the like, and tetrahydrofuran is preferable.
Examples of the chlorine-based solvent include trichlorethylene, tetrachlorethylene, dichloromethane (methylene chloride), chloroform and the like, and dichloromethane is preferable.
Examples of the aprotic solvent include acetone, ethyl acetate, acetonitrile, N, N-dimethylformamide, N, N-dimethylacetamide, dimethyl sulfoxide and the like, and acetonitrile is preferable.
Among the above solvents, a chlorine-based solvent is more preferable, and among them, dichloromethane is further preferable.

Bases include triethylamine, N, N-diisopropylethylamine, pyridine, imidazole, quinoline, picolin, 1-methylimidazole, 1,2-dimethylimidazole, 4-dimethylaminopyridine, N, N-dimethylaniline, N-methylmorpholin. , 1,5-diazabicyclo [4.3.0] nona-5-ene (DBN), 1,4-diazabicyclo [2.2.2] octane (DABCO), 1,8-diazabicyclo [5.4.0] ] A tertiary amine such as undec-7-ene (DBU) can be mentioned, preferably 1,2-dimethylimidazole.
The amount of the base used is usually 1 to 5 equivalents, preferably 1.25 to 4 equivalents, more preferably 1.5 to 3.5 equivalents, and more preferably 1 equivalent to 1 equivalent of the compound represented by the formula (v). Is 2-3 equivalents.

The reaction temperature is usually about 23 to 27 ° C., and the reaction time is usually about 1 to 12 hours.

2) Step-2
The step is a deprotection step of removing the protecting group from the benzylated epigallocatechin gallate TCE sulfate represented by the formula (Ia).
That is, the benzylated epigalocatecatingalate TCE sulfate represented by the formula (Ia) is hydrolyzed in the presence of a hydrogenation catalyst to simultaneously eliminate the benzyl group and the TCE group to form the formula (Ib). ) Can be obtained.

The reaction is carried out by stirring the benzylated epigallocatechin gallate TCE sulfate represented by the formula (Ia) in the presence of a hydrogenation catalyst in a suitable solvent in the presence of hydrogen.
Examples of the solvent include ether solvents such as diethyl ether, tetrahydrofuran and dioxane, alcohol solvents such as methanol and ethanol, acidic solvents such as formic acid and acetic acid, and mixed solvents thereof.

Examples of the hydrogenation catalyst include palladium catalysts such as palladium (II) hydroxide / carbon (Pd (OH) 2 / C) and palladium carbon (Pd / C), and hydrogen-supporting metal catalysts such as lane nickel.

The amount of the hydrogenation catalyst used is usually 1 to 300% by mass, preferably 2 to 250% by mass, more preferably 2 to 250% by mass, based on the charged mass of the benzylated epigalocatecatingalate TCE sulfate represented by the formula (Ia). It is 3 to 200% by mass, more preferably 4 to 150% by mass, still more preferably 5 to 100% by mass.

The reaction temperature is usually about 23 to 27 ° C., and the reaction time is usually about 6 to 24 hours.

The compounds represented by the above formulas (Ia) and (Ib) are all novel compounds.
Therefore, in the present invention, epigallocatechin gallate sulfate or a derivative thereof also represented by the following general formula (I) is also provided.

[In the formula, Z ¹ represents a hydrogen atom or a benzyl group, and when Z ¹ is a benzyl group, one of R ¹ and R ² represents a benzyl group and the other represents −SO ₃ CH ₂ CCl ₃ , and Z ^{When 1} is a hydrogen atom, one of R ¹ and R ² is a hydrogen atom and the other is -SO ₃ M (where M indicates a hydrogen atom, an alkali metal atom, an alkaline earth metal atom or ammonium). Is shown. ]

In formula (I), ^one of R 1 and R ² is a benzyl group and the other is -SO ₃ CH ₂ CCl ₃ , or ^one of R ^{1 and R 2} is a hydrogen atom and the other is -SO ₃ M. However, it is preferable that R ¹ is a benzyl group or a hydrogen atom and R ² is -SO ₃ CH ₂ CCl ₃ or -SO ₃ M.
Further, examples of the alkali metal atom represented by M include potassium and sodium, but sodium is preferable. Examples of the alkaline earth metal atom represented by M include magnesium, calcium and the like, and more preferably sodium.

In the formula (I), the compound in which Z ¹ is a hydrogen atom, one of R ¹ and R ² is a hydrogen atom and the other is -SO ₃ M is an epigalocatecingalate sulfated product, which is a test example described later. As shown in, it can be used as a standard for tracking the pharmacokinetics of epigalocatecetingalate sulfate conjugates formed in the body, and also as a standard for physiologically active substances produced in vivo in in vitro tests. It is useful.
Further, Z ¹ is a benzyl group, one of R ¹ and R ² is a benzyl group, and the other is _{a sulfuric acid protected with -SO 3} CH ₂ CCl ₃ (2,2,2-trichloroethyl group (TCE)). The compound (benzylated epigalocatecetingalate TCE sulfate) is a derivative of the epigalocatecatingalate sulfate having a protective group that is easily desorbed, and can be induced to epigalocatecetingalate sulfate in one step. It is useful as a production intermediate (production precursor) for epigallocate benzylate sulfate.

The glucron oxide of epigallocatechin gallate of the present invention can be produced from the benzylated epigallocatechin gallate represented by the formula (v) by the following steps.

[In the formula, one of R ^1a and R ^2a represents a benzyl group and the other represents a hydrogen atom, R ^1d and R ^2d correspond to R ^1a and R ^2a , respectively, and one of them is a hydrogen atom and the other is a glucuronosyl. Indicates a group. ]

Glucronic acid donors used for glucuron oxidation include 2,3,4-tri-O-acetyl-α-D-methylglucuronopyranosyl-1- (N-phenyl) -2,2,2-tri. Fluoroacetoimidate, 2,3,4-tri-O-acetyl-α-D-methylglucopyranosyl-1- (N-4-methoxyphenyl) -2,2,2-trifluoroacetimidate, 2,3,4-Tri-O-acetyl-α-D-methylglucuronopyranosyl-1-O- (2,2,2-trichloroacetoimidate), acetobromo-α-D-glucuronic acid methyl ester Etc., such as ester derivatives of glucuronic acid, and methyl 2,3,4-tri-O-acetyl-1-O- (trichloroacetimideyl) -α-D-methyl glucuronate is preferably used.

The reaction between the benzylated epigallocatechin gallate represented by the formula (v) and the glucuronic acid donor is carried out by mixing and stirring both in the presence of Lewis acid in an ether-based, chlorine-based, aprotic or mixed solvent thereof. It is done by doing.

The amount of the glucuronic acid donor used is usually 1 to 10 equivalents, preferably 3 to 8 equivalents, more preferably 4 to 6 equivalents, still more preferably 4. It is 8 to 5.2 equivalents.

Examples of the ether solvent, the chlorine solvent, and the aprotic solvent include the same as those described above, and the chlorine solvent is preferable, and dichloromethane is more preferable.

Lewis acids include aluminum chloride (III), diethylaluminum chloride, nickel (II) chloride (hexahydrate), tin (IV) chloride (pentahydrate), titanium chloride (IV), zinc chloride, and trifluorobo. A run-ether complex is mentioned, and a trifluoroboran-ether complex is preferable.
The amount of Lewis acid used is usually 1 to 10 equivalents, preferably 3 to 8 equivalents, more preferably 4 to 6 equivalents, and more preferably 4.8 to 1 equivalent, relative to 1 equivalent of the compound represented by the formula (v). 5.2 equivalents.

The reaction temperature is usually about 0 to 40 ° C., preferably about 15 to 25 ° C., and the reaction time is usually about 12 to 24 hours.

For the reaction product of the benzylated epigalocatecingalate represented by the formula (v) thus obtained and the glucuronic acid donor, the elimination of the ester residue derived from the glucuronic acid donor and the elimination of the benzyl group were subsequently performed. Glucuronic oxide (Ic) can be obtained by performing desorption (deprotection).
Elimination of ester residues can be carried out by a hydrolysis reaction, for example, by contacting the reaction product with water as appropriate in the presence of an acid or base. Examples of usable acids include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid, and organic acids such as acetic acid, trifluoroacetic acid, formic acid and p-toluenesulfonic acid. Examples of the base include metal hydroxides such as sodium hydroxide and barium hydroxide, carbonates such as sodium carbonate and potassium carbonate, and sodium acetate and the like.
As the solvent, for example, a water-miscible organic solvent such as ethanol, ethylene glycol dimethyl ether, tetrahydrofuran, or dioxane is used together with water.
The reaction is usually carried out at about 0-100 ° C., preferably room temperature to 50 ° C. for 0.5 to 3 hours, preferably 0.5 to 2 hours.

The elimination of the benzyl group can be carried out by hydrogenation decomposition in the presence of the above-mentioned hydrogenation catalyst.

The epigallocatechin gallate glucuron oxide thus obtained can serve as a standard for tracking the pharmacokinetics of the epigallocatechin gallate glucuronic acid conjugate formed in the body, and can be used in vivo in an in vitro test. It is useful as a standard for the bioactive substances produced.

Reference Example 1 Production of Compound A2 Compound A3 was synthesized from gallic acid ester by the following steps.

(1) Production of Methyl 4- (allyloxy) -3,5-dihydroxybenzoate (Compound A2) After adding methyl gallate (Compound A1) (1.00 g, 5.43 mmol) to a round-bottom flask under an argon atmosphere, A light yellow solution was obtained by adding acetonitrile (50 mL) and stirring. Subsequently, N, N-diisopropylethylamine (1.07 mL, 5.97 mmol), allyl bromide (2.31 mL, 27 mmol), and allyl iodide (catalytic amount) were sequentially added under cooling in an ice bath. Then, the temperature was raised to room temperature, and the mixture was stirred for 48 to 72 hours. After stirring, the reaction was stopped by diluting with ethyl acetate (100 mL), adding 1 mol / L hydrochloric acid under cooling in an ice bath, and acidifying the reaction solution. Subsequently, the mixture was extracted three times with ethyl acetate, the obtained organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled under reduced pressure using an evaporator. The obtained residue was purified by silica gel chromatography (eluting solvent: hexane-ethyl acetate (3: 1, v / v)) to obtain the target compound A2.

Compound A2: White solid (yield: 91%)
^{1 1} H-NMR (600 MHz, acetone-d ₆ ) δ 7.08 (s, 2H), 6.06-6.13 (m, 1H), 5.29 (ddt, J = 17, 1.6, 1 .6Hz, 1H), 5.29 (ddt, J = 10, 1.8, 1.2Hz, 1H), 5.15 (ddd, J = 6, 1.5, 1.2Hz, 1H), 3. 80 (s, 3H).
¹³ C-NMR (150 MHz, CDCl ₃ ) δ 166.88, 151.37, 138.64, 135.26, 126.38, 118.44, 109.69, 73.89, 52.15.

(2) Production of 4- (Alllyloxy) -3,5-bis (benzyloxy) bentoic acid (Compound A3) Compound A2 (4.90 g, 22 mmol) and tetrabutylammonium iodide (4.90 g, 22 mmol) and tetrabutylammonium iodide in a 200 mL round-bottom flask under an argon atmosphere. After adding 8.00 g (22 mmol), tetrahydrofuran (50 mL) was added and stirred to obtain a clear solution. Subsequently, 60% sodium hydride (1.68 g, 66 mmol as sodium hydride) and benzyl bromide (13 mL, 109 mmol) were sequentially added under cooling in an ice bath. Then, the mixture was heated to 50 ° C. and stirred for 8 hours. At this time, ethanol (32 mL), water (16 mL) and a 7 mol / L sodium hydroxide aqueous solution (16 mL) were added, and the mixture was continuously heated to 60 ° C. and stirred for 18 hours. After stirring, 1 mol / L hydrochloric acid was added under cooling in an ice bath to acidify the reaction solution, thereby stopping the reaction. The white solid obtained by distilling the solvent under reduced pressure with an evaporator was filtered, washed with water, eluted with acetone, and the filtrate was distilled under reduced pressure with an evaporator. From the obtained residue, compound A3 (6.90 g, 17 mmol, yield 81%) was obtained as a white solid.

Compound A3:
^{1 1} H-NMR (600 MHz, DMSO-d ₆ ) δ 7.46-7.47 (m, 4H), 7.39-7.41 (m, 4H), 7.32-7.36 (m, 4H) ), 5.95-6.01 (m, 1H), 5.28 (ddt, J = 17, 1.7Hz, 1H), 5.17 (s, 4H), 5.14 (ddt) , J = 10, 1.2Hz, 1H), 4.54 (ddd, J = 5.6, 1.2Hz, 1H).

Reference Example 2 Production of Compound A5 Compound A5 was synthesized from gallic acid ester by the following steps.

(1) Production of Methyl 4,5-bis (benzyloxy) -3-hydroxybenzoate (Compound A4) Methyl gallate (Compound A1) (5 g, 27 mmol) was added to a 500 mL round-bottom flask under an argon atmosphere, and then acetonitrile (5 g, 27 mmol) was added. 250 mL) was added and stirred to obtain a pale yellow solution. Subsequently, N, N-diisopropylethylamine (9.5 mL, 57 mmol) and benzyl bromide (13 mL, 114 mmol) were added sequentially under cooling in an ice bath. Then, the temperature was raised to room temperature, and the mixture was stirred for 96 hours. After stirring, the reaction was stopped by diluting with ethyl acetate (250 mL), adding 1 mol / L hydrochloric acid under cooling in an ice bath, and acidifying the reaction solution. Subsequently, the mixture was extracted three times with ethyl acetate, the obtained organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled under reduced pressure using an evaporator. The obtained residue was purified by silica gel chromatography (eluting solvent: hexane-ethyl acetate (5: 1, v / v)) to obtain compound A4 (4.8 g, 13 mmol, 49%) as a white amorphous substance.

Compound A4:
^{1 1} H-NMR (600 MHz, acetone-d ₆ ) δ 7.54-7.56 (m, 2H), 7.34-7.44 (m, 5H), 7.28-7.30 (m, 4H) ), 7.20 (d, J = 1.9Hz, 1H), 5.22 (s, 2H), 5.13 (s, 2H), 3.83 (s, 3H).

(2) Production of 3- (Alllyloxy) -4,5-bis (benzyloxy) bentoic acid (Compound A5) Compound A4 (260 mg, 0.71 mmol) and tetrabutylammonium iodide (260 mg, 0.71 mmol) and tetrabutylammonium iodide (260 mg, 0.71 mmol) in a 200 mL round-bottom flask under an argon atmosphere. After adding 230 mg (0.71 mmol), tetrahydrofuran (20 mL) was added and stirred to obtain a clear solution. Subsequently, 60% sodium hydride (81 mg, 2.1 mmol) and allyl bromide (121 μL, 1.4 mmol) were sequentially added under cooling in an ice bath. Then, the mixture was heated to 50 ° C. and stirred for 9 hours. At this time, ethanol (20 mL), water (13 mL) and a 7 mol / L sodium hydroxide aqueous solution (7 mL) were added, and the mixture was continuously heated to 60 ° C. and stirred for 9 hours. After stirring, 1 mol / L hydrochloric acid was added under cooling in an ice bath to acidify the reaction solution, thereby stopping the reaction. The white solid obtained by distilling the solvent under reduced pressure with an evaporator was filtered, washed with water, eluted with acetone, and the filtrate was distilled under reduced pressure with an evaporator. From the obtained residue, compound A5 (383 mg, 0.53 mmol, yield 76%) was obtained as a white solid.

Compound A5:
^{1 1} H-NMR (600 MHz, CD ₃ CN) δ 7.18-7.43 (m, 12H) 5.99-6.05 (m, 1H) 5.36 (ddt, J = 17, 1. 8Hz, 1H), 5.10 (ddt, J = 6.6, 1.5Hz, 1H), 5.11 (s, 2H), 5.02 (s, 2H), 4.56 (ddd, J = 17, 1.8Hz, 1H).

Reference Example 3 Production of 2,2,2-trichloroethoxy-sulfuryl-1,2-dimethylimidazolium triflate (Compound B4) Compound B4 was synthesized from 2,2,2-trichloroethanol by the following steps.

(1) Production of 2,2,2-Trichloroethyl sulfurochlorodate (Compound B2) In a 300 mL round-bottom flask under an argon atmosphere, 2,2,2-trichloroethanol (Compound B1) (5.9 mL, 61 mmol) and pyridine (4. After adding 97 mL (61 mmol), diethyl ether (100 mL) was added and stirred to obtain a clear solution. Subsequently, sulfyl chloride (5 mL, 61 mmol) was added dropwise to the reaction vessel at −78 ° C. under cooling over 1 hour. Then, the temperature was raised to room temperature, and the mixture was stirred for 3 hours. After stirring, the obtained white solid was washed twice with 20 mL of diethyl ether, and the filtrate was distilled under reduced pressure with an evaporator at 25 ° C. From the obtained residue, compound B2 (12.0 g, 48 mmol, yield 79%) was obtained as a clear liquid.

(2) Production of 2', 2', 2'-Trichloroethyl 2-methyl-1H-imidazole-1-sulfonate (Compound B3) 2-Methylimidazole (18.2 g, 172 mmol) in a 300 mL round bottom flask under an argon atmosphere. Was added, and tetrahydrofuran (50 mL) was added and stirred to obtain a clear solution. Subsequently, compound B2 (12.0 g, 48 mmol) diluted with tetrahydrofuran (50 mL) was added dropwise over 1 hour under cooling in an ice bath. Then, the temperature was raised to room temperature, and the mixture was stirred for 1 hour. After stirring, the obtained white solid was washed twice with 20 mL of tetrahydrofuran, and the filtrate was distilled under reduced pressure with an evaporator at 25 ° C. The obtained residue was purified by silica gel chromatography (eluting solvent: hexane-ethyl acetate (2: 1, v / v)) to obtain compound B3 (9.70 g, 32 mmol, 68%) as a clear liquid.

(3) Production of 2,3-Dimethyl-1-((2', 2', 2'-trichloloethoxy) sulfonyl) -1H-imidazole-3-ium trifluoromethanesolufonate (Compound B4) in a 300 mL round-bottom flask under an argon atmosphere. After adding compound B3 (9.70 g, 32 mmol), diethyl ether (100 mL) was added and stirred to obtain a clear solution. Subsequently, methyl trifluoromethanesulfonate (3.8 mL, 33 mmol) was added dropwise under cooling in an ice bath, and the mixture was stirred for 3 hours. Then it was cooled to −20 ° C. The obtained white solid was washed with cooled diethyl ether to obtain compound B4 (12.9 g, 27 mmol, 85%) as a white solid.

Manufacturing example 1
4 "-sulfated epigallocatechin gallate (Compound 6) was synthesized from (-)-epigallocatechin (EGC) by the following steps.

(1) Production of (2R, 3R) -5,7-Bis (benzyloxy) -2- (3', 4', 5'-tris (benzyloxy) phenyl) chroman-3-ol (Compound 2) under an argon atmosphere , 60% sodium hydride (342 mg, 8.4 mmol) was added to a 100 mL round bottom flask, and then N, N-dimethylformamide (8 mL) was added and stirred to obtain a suspension. Subsequently, a solution prepared with (-)-epigallocatechin (Compound 1) (500 mg, 1.6 mmol), benzyl bromide (13 mL, 109 mmol), and N, N-dimethylformamide (8 mL) was cooled at -50 ° C. After that, the temperature was raised to room temperature and the mixture was stirred for 48 hours. After stirring, 1 mol / L hydrochloric acid was added under cooling in an ice bath to acidify the reaction solution, thereby stopping the reaction. Subsequently, the mixture was extracted three times with hexane-ethyl acetate (1: 1, v / v), the obtained organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled under reduced pressure using an evaporator. The obtained residue was purified by silica gel chromatography (eluting solvent: hexane-ethyl acetate (4: 1, v / v)) to obtain Compound 2 (710 mg, 4.78 mmol, yield 57%) as a white amorphous substance.

Compound 2:
^{1 1} H-NMR (600 MHz, acetone-d ₆ ) δ 7.26-7.52 (m, 25H), 7.04 (s, 2H), 6.36 (d, J = 2.2Hz, 1H), 6.23 (d, J = 2.2Hz, 1H), 5.14 (s, 4H), 5.12 (d, J = 2.8Hz, 2H), 5.08 (s, 2H), 5. 03 (s, 2H), 5.01 (m, 1H), 4.31-4.33 (m, 1H), 2.86-2.96 (m, 2H).

(2) (2R, 3R) -5,7-Biz (benzyloxy) -2- (3', 4', 5'-tris (benzyloxy) phenyl) choroman-3-yl 4 "-(alllyloxy) -3" , 5 "-Production of bis (benzyloxy) benzoate (Compound 3) Under an argon atmosphere, compound 2 (828 mg, 1.41 mmol) was placed in a round bottom flask and the benzylated pericate allyl ether (Compound A3) prepared in Reference Example 1 (Compound A3). 828 mg, 2.12 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.6 g, 8.48 mmol), and N, N-dimethyl-4-aminopyridine (172 mg, 2.12 mmol). ) Was added, acetonitrile (40 mL) was added and stirred to obtain a pale yellow solution. Subsequently, the mixture was stirred at room temperature for 1 hour. After stirring, the mixture was diluted with ethyl acetate (80 mL) and cooled in an ice bath. The reaction was stopped by adding 1 mol / L hydrochloric acid to acidify the reaction solution. Subsequently, extraction was performed three times with ethyl acetate, the obtained organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and then the reaction was stopped. The solvent was distilled under reduced pressure with an evaporator. The obtained residue was purified by silica gel chromatography (eluting solvent: hexane-ethyl acetate (4: 1, v / v)) to obtain the target compound 3.

Compound 3: White amorphous (yield: 87%)
^{1 1} H-NMR (600 MHz, acetone-d ₆ ) δ7.20-7.49 (m, 37H), 6.99 (s, 2H), 6.49 (d, J = 2.1Hz, 1H), 6 .44 (d, J = 2.3Hz, 1H), 5.93-6.00 (m, 1H), 5.73-5.75 (m, 1H), 5.31 (m, 1H), 5 .25 (ddt, J = 17, 1.6, 1.6Hz, 1H), 5.17 (s, 2H), 5.11 (s, 2H), 5.04-5.11 (m, 7H) 4.94 (d, J = 10Hz, 2H), 4.87 (d, J = 11Hz, 2H), 4.73 (d, J = 11Hz, 1H), 4.40-4.42 (m, 1H), 3.19 (dd, J = 17, 4.3Hz, 1H), 3.06 (dd, J = 18, 2.1Hz, 1H).

(3) (2R, 3R) -5,7-Bis (benzyloxy) -2- (3', 4', 5'-tris (benzyloxy) phenyl) chroman-3-yl 3 ", 5" -bis (benzyloxy) ) -4 "-Production of hydroxybenoate (Compound 4) Under an argon atmosphere, add allyl compound (Compound 3) (670 mg, 0.59 mmol) to a round-bottom flask, add tetrahydrofuran (10 mL), stir, and stir to make a clear solution. Subsequently, morpholine (106 μL, 1.18 mmol) and tetrakis (triphenylphosphine) palladium (0) (69 mg, 0.06 mmol) were added, and the mixture was stirred for 1 hour. After stirring, the mixture was stirred with ethyl acetate (20 mL). The reaction was stopped by diluting and adding 1 mol / L hydrochloric acid under cooling in an ice bath to acidify the reaction solution. Subsequently, extraction was performed 3 times with ethyl acetate, and the obtained organic layer was washed with saturated saline. After drying with anhydrous sodium sulfate, the solvent was distilled under reduced pressure with an evaporator. The obtained residue was purified by silica gel chromatography (eluting solvent: hexane-ethyl acetate (3: 1, v / v)) to obtain the target compound 4. Obtained.

Compound 4: White amorphous (yield: 99%)
^{1 1} H-NMR (600 MHz, CDCl ₃ ) δ 7.15-7.37 (m, 37H), 6.64 (s, 2H), 6.35 (d, J = 2.2 Hz, 1H), 6. 30 (d, J = 1.9Hz, 1H) 5.60-5.62 (m, 1H) 4.97-5.00 (m, 9H) 4.91 (d, J = 12Hz, 2H) ), 4.68 (d, J = 11Hz, 2H), 4.54 (d, J = 11Hz, 2H), 3.07 (dd, J = 17, 4.3Hz, 1H), 3.01 (dd) , J = 17, 2.0Hz, 1H).

(4) (2R, 3R) -5, 7-Biz (benzyloxy) -2- (3', 4', 5'-tris (benzyloxy) reagent) chroman-3-yl 3 ", 5" -bis (benzyloxy) ) -4 "-(((2,2,2-tricloroethoxy) solution) oxy) dichloromethane (Compound 5) Manufactured in a round-bottom flask with Compound 4 (660 mg, 0.60 mmol) and Reference Example 3 under an argon atmosphere. After adding the sulfated reagent (B4) (691 mg, 1.51 mmol), dichloromethane (12 mL) was added and stirred to obtain a clear solution. Subsequently, 1,2-dimethylimidazole (145 mg, 1.51 mmol) was added. ) Was added and stirred for 1 hour. After stirring, the reaction was stopped by diluting with ethyl acetate (24 mL) and adding water (24 mL). Subsequently, extraction was performed 3 times with ethyl acetate to obtain the obtained organic layer. After washing with saturated saline and drying with anhydrous sodium sulfate, the solvent was distilled under reduced pressure with an evaporator. The obtained residue was purified by silica gel chromatography (eluting solvent: hexane-ethyl acetate (4: 1, v / v)). , The target compound 5 was obtained.

Compound 5: White amorphous (yield: 91%)
^{1 1} H-NMR (600 MHz, CD ₃ CN) δ 7.13 (m, 37H), 6.63 (s, 2H), 6.31 (d, J = 2.1Hz, 1H), 6.27 (d) , J = 2.4Hz, 1H), 5.59-5.60 (m, 1H), 4.89-4.98 (m, 11H), 4.76 (d, J = 11Hz, 2H), 4 .65 (d, J = 11Hz, 2H), 4.52 (s, 2H), 2.97-3.06 (m, 2H).

(5) 4 "-sulfated epigalocatecingalate (Sodium 4"-(((((2R, 3R) -5,7-dihydroxy-2- (3', 4', 5'-trihydroxyphenyl)) chroman-3-3 production of yl) oxy) carbonyl) -3 ", 5" -dihydroxymethanol suspension; compound 6) Trichloroethyl sulfate compound (compound 5) (50 mg, 39 μmol), palladium carbon (10 mg), and formic acid in a round-bottom flask under an argon atmosphere. After adding ammonium (24 mg, 390 μmol), tetrahydrofuran-methanol (4 mL, 3: 1, v / v) was added and stirred to obtain a suspension. Subsequently, hydrogen substitution was added to the obtained suspension. After that, the mixture was stirred overnight. After stirring, unnecessary substances were filtered and washed with tetrahydrofuran-methanol (8 mL, 3: 1, v / v) and water (2 mL). Subsequently, the filtrate was distilled under reduced pressure using an evaporator. The obtained residue was purified by reverse phase preparative thin layer chromatography (eluting solvent: water-methanol (9: 1, v / v)) to obtain the target compound 6.

Compound 6: White solid (52%)
_{^{1 H-NMR (600MHz, D}} 2 O: MeOH (= 200: 1)) δ 6.93 (s, 2H), 6.51 (s, 2H), 6.09 (d, J = 2.1Hz, 1H), 6.06 (d, J = 1.9Hz, 1H), 5.56-5.56 (m, 1H), 5.04-5.04 (m, 1H), 3.00 (dd, dd, J = 17.9, 5.2, Hz, 1H) 2.89 (d, J = 17.2Hz, 1H).
_{^{13 C-NMR (150MHz, D}} 2 O: MeOH (= 200: 1)) δ 167.07,156.15,156.03,155.90,150.69,145.85,132.76,132. 42, 130.31, 128.31, 110.57, 107.03, 99.73, 96.70, 95.84, 77.73, 70.06, 25.46.
HRMS calcd. for C ₂₂ H ₁₈ O ₁₄ SNa ⁺ [M + Na] ⁺ : 561.0315; found: 561.0315

Manufacturing example 2
Compound 2 was synthesized from (-)-epigallocatekin (EGC) in the same manner as in Production Example 1 (1) by the following steps, and this and the allyl ether gallic acid ester (Compound A5) produced in Reference Example 2 were used. Compound 7 is obtained by subjecting it to a condensation reaction in the same manner as in Production Example 1 (2), and then the allyl group is eliminated to obtain Compound 8 in the same manner as in Production Example 1 (3). Then, the compound 9 was obtained by trichloroethyl sulfate esterification, and then subjected to a deprotection reaction in the same manner as in Production Example 1 (5) to synthesize a 3 ″ -sulfated epigalocatecingalate (Compound 10).

(1) (2R, 3R) -5,7-Bis (benzyloxy) -2- (3', 4', 5'-tris (benzyloxy) compound) chroman-3-yl 5 "-(alllyloxy) -3" , 4 "-Production of bis (benzyloxy) benzoate (Compound 7) Under an argon atmosphere, compound 2 (258 g, 0.34 mmol) and the allyl ether gallic acid ester (Compound A5) (200 g) prepared in Reference Example 2 were placed in a round bottom flask. , 0.51 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (196 mg, 1.00 mmol), and N, N-dimethyl-4-aminopyridine (63 mg, 0.34 mmol). After that, acetonitrile (10 mL) was added and stirred to obtain a pale yellow solution. Subsequently, the mixture was stirred at room temperature for 1 hour. After stirring, the mixture was diluted with ethyl acetate (20 mL) and cooled to 1 mol / L in an ice bath. The reaction was stopped by adding hydrochloric acid to acidify the reaction solution. Subsequently, extraction was performed three times with ethyl acetate, the obtained organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and then evaporated. The solvent was distilled under reduced pressure. The obtained residue was purified by silica gel chromatography (eluting solvent: hexane-ethyl acetate (4: 1, v / v)) to obtain the target compound 7.

Compound 7: White amorphous (yield: 100%)
^{1 1} H-NMR (600 MHz, CDCl ₃ ) δ 7.20-7.42 (m, 37H), 6.74 (s, 2H), 6.36 (d, J = 2.2 Hz, 1H), 6. 32 (d, J = 2.2Hz, 1H) 5.89-5.95 (m, 1H) 5.65-5.66 (m, 1H) 5.30 (ddt, J = 17, 1 .4Hz, 1H), 5.19 (ddt, J = 10, 1.3Hz, 1H), 5.14 (m, 1H), 4.83-5.04 (m, 12H), 4.72 (d) , J = 11Hz, 2H), 4.48-4.49 (m, 2H), 3.04-3.13 (m, 2H).

(2) (2R, 3R) -5,7-Bis (benzyloxy) -2- (3', 4', 5'-tris (benzyloxy) phenyl) chroman-3-yl 3 ", 4" -bis (benzyloxy) ) -5 "-Production of hydroxybenzoate (Compound 8) Under an argon atmosphere, add allyl compound (Compound 7) (390 mg, 0.34 mmol) to a round-bottom flask, add tetrahydrofuran (10 mL), stir, and stir to make a clear solution. Subsequently, morpholine (61 μL, 0.69 mmol) and tetrakis (triphenylphosphine) palladium (0) (40 mg, 34 μmol) were added, and the mixture was stirred for 45 minutes. After stirring, the mixture was diluted with ethyl acetate (20 mL). The reaction was stopped by adding 1 mol / L hydrochloric acid under cooling in an ice bath to acidify the reaction solution. Subsequently, extraction was performed 3 times with ethyl acetate, and the obtained organic layer was washed with saturated saline and anhydrous. After drying with sodium sulfate, the solvent was distilled under reduced pressure with an evaporator. The obtained residue was purified by silica gel chromatography (eluting solvent: hexane-ethyl acetate (3: 1, v / v)) to obtain the target compound 8. rice field.

Compound 8: White amorphous (yield: 53%)
^{1 1} H-NMR (600 MHz, CDCl ₃ ) δ 7.17-7.44 (m, 37H), 6.79 (s, 2H), 6.34 (d, J = 2.3 Hz, 1H), 6. 30 (d, J = 2.2Hz, 1H), 5.60-5.61 (m, 1H), 5.14 (m, 1H), 4.93-5.08 (m, 12H), 4. 81 (d, J = 11Hz, 2H), 3.06-3.16 (m, 2H).

(3) (2R, 3R) -5, 7-Biz (benzyloxy) -2- (3', 4', 5'-tris (benzyloxy) reagent) chroman-3-yl 4 ", 5" -bis (benzyloxy) ) -3 "-(((2,2,2-tricloroethoxy) solution) oxy) dichloromethane (Compound 9) Manufactured in a round-bottom flask with Compound 8 (190 mg, 0.17 mmol) and Reference Example 3 under an argon atmosphere. After adding the sulfated reagent (B4) (398 mg, 0.87 mmol), dichloromethane (10 mL) was added and stirred to obtain a clear solution. Subsequently, 1,2-dimethylimidazole (84 mg, 0.87 mmol) was added. ) Was added and stirred overnight. After stirring, the mixture was diluted with ethyl acetate (20 mL), and the reaction was stopped by adding water. Subsequently, extraction was performed 3 times with ethyl acetate, and the obtained organic layer was mixed with saturated saline. After washing and drying with anhydrous sodium sulfate, the solvent was distilled under reduced pressure with an evaporator. The obtained residue was purified by silica gel chromatography (eluting solvent: hexane-ethyl acetate (4: 1, v / v)), and the target compound 9 was prepared. (White amorphous) was obtained (yield: 69%).

(4) 3 ”-sulfated epigallocatekingularate (Sodium 3”-(((((2R, 3R) -5,7-dihydroxy-2- (3', 4', 5'-trihydroxyphenyl)) chroman-3-3 production of yl) oxy) carbonyl) -4 ", 5" -dihydroxymethanol sulphate; compound 10) Trichloroethyl sulfate compound (compound 9) (50 mg, 38 μmol), palladium carbon (5 mg), and formic acid in a round bottom flask under an argon atmosphere. After adding ammonium (24 mg, 380 μmol), tetrahydrofuran-methanol (4 mL, 3: 1, v / v) was added and stirred to obtain a suspension. Subsequently, the obtained suspension was subjected to hydrogen substitution and then stirred for 18 hours. After stirring, the unwanted material was filtered and washed with tetrahydrofuran-methanol (8 mL, 3: 1, v / v) and water (2 mL). Subsequently, the filtrate was distilled under reduced pressure using an evaporator. The obtained residue was purified by reverse phase preparative thin layer chromatography (eluting solvent: water-methanol (9: 1, v / v)) to obtain the target compound 10.

Compound 10: White solid (yield 78%)
^{1 1} H-NMR (600 MHz, D ₂ O: MeOH (= 200: 1)) δ 7.44 (d, J = 1.4 Hz, 1 H), 7.20 (d, J = 1.5 Hz, 1 H), 6.54 (s, 2H), 6.11 (d, J = 1.9Hz, 1H), 6.06 (d, J = 1.5Hz, 1H), 5.53-5.53 (m, 1H) ), 5.06-5.06 (m, 1H), 3.00 (dd, J = 17.6, 3.9, Hz, 1H), 2.89 (d, J = 17.3Hz, 1H) ..
_{^{13 C-NMR (150MHz, D}} 2 O: MeOH (= 200: 1)) δ 167.14,156.14,156.03,155.94,145.81,145.78,143.36,139. 54, 132.73, 120.73, 117.17, 115.21, 107.12, 99.73, 96.70, 95.87, 77.81, 69.88, 25.55.
HRMS calcd. for C ₂₂ H ₁₉ O ₁₄ S ⁺ [M + H] ⁺ : 539.0496; found: 539.0483

Comparative Example 1
In the production of Compound 5 of Production Example 1 (4), by the method using _{chlorosulfate 2,2,2-trichloroethyl (Cl 3} CCH ₂ OSO _{2 Cl) described in Non-Patent Document 1 as a sulfate reagent.} An attempt was made to sulfate compound 4.
That is, under an argon atmosphere, 2,2,2-trichloroethanol (Compound B1) (9 μL, 92 μmol) and pyridine (8 μL, 92 μmol) were added to a 10 mL round-bottom flask, and then diethyl ether (1 mL) was added and stirred. A clear solution was obtained. Subsequently, sulfuryl chloride (8 μL, 61 mmol) was added dropwise at −78 ° C. under cooling, and the mixture was stirred for 1 hour. After stirring, the obtained white solid was washed twice with 1 mL of diethyl ether, and the filtrate was distilled under reduced pressure with an evaporator at 25 ° C. Dichloromethane (1 mL) was cooled to the obtained residue in an ice bath, and phenol compound (Compound 4) (10 mg, 9.2 μmol) and 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) were added. (2 μL, 9.2 μmol) was added, and the mixture was stirred for 1 to 24 hours. After stirring, the reaction was stopped by diluting with ethyl acetate (2 mL) and adding water. Subsequently, the mixture was extracted three times with ethyl acetate, the obtained organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled under reduced pressure using an evaporator. When the obtained residue was confirmed by thin layer chromatography, the result was that the trichloroethyl sulfate compound (Compound 5) was not obtained, but only the phenol compound (Compound 4) was obtained.

Manufacturing example 3
From the compound 4 produced in Production Example 1 or the compound 8 produced in Production Example 2 by the following steps, 4 "-glucuron-oxidized epigalocatecating gallate (Compound 11) and 3" -glucuron-oxidized epigalocatecingalate (compound), respectively. 12) was synthesized.

(1) Under an argon atmosphere, compound 4 or compound 8 (1 equivalent), 2,3,4-tri-O-acetyl-1-O- (trichloroacetimideyl) -α-D-glucron in a 50 mL round bottom flask. Methyl acid (5 eq) and 4 Å (5 parts by weight) of molecular flask were taken, dehydrated dichloromethane (0.2 M dilution) was added and stirred to obtain a clear solution. Subsequently, a trifluoroboran-ether complex (5 equivalents) was added at 0 ° C., the temperature was raised to room temperature, and the mixture was stirred for 24 hours. After the reaction, the reaction was stopped by diluting with ethyl acetate (20 mL) and then adding water. Subsequently, the mixture was extracted three times with ethyl acetate, the obtained organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled under reduced pressure using an evaporator. The obtained residue was crudely purified by silica gel chromatography (eluting solvent: hexane-ethyl acetate (2: 1, v / v)) to obtain a mixture as a white amorphous substance.

(2) The mixture obtained in (1) was placed in a 100 mL round-bottom flask, tetrahydrofuran-ethanol-purified aqueous solution (2: 2: 1, 50-fold dilution) was added and stirred to obtain a clear solution. Subsequently, a 1N aqueous sodium hydroxide solution (diluted 1-fold) was added at room temperature, and the mixture was stirred for 30 minutes. After the reaction, Amberlyst (registered trademark) 15 (H) and acetic acid were added to acidify the reaction solution to terminate the reaction. Subsequently, the filtrate was filtered, and the solvent was distilled under reduced pressure using an evaporator. The obtained residue was added with a tetrahydrofuran-methanol-acetic acid solution (30:10: 2, diluted 52 times) under an argon atmosphere and stirred to obtain a clear liquid. Subsequently, palladium carbon (0.2 parts by mass) was added at room temperature, the inside of the flask was replaced with hydrogen, and the mixture was vigorously stirred for 5 hours. After the reaction, palladium carbon was filtered, washed with a tetrahydrofuran-methanol solution, and the obtained organic layer was distilled under reduced pressure using an evaporator. The obtained residue was subjected to preparative HPLC and developed under the following conditions to obtain compound 11 or compound 12 as a white solid.

[Prescription conditions]
Preparative column: L-colum ODS, size 20 mm x 259 mm 5 μm
Eluent: A (0.1% formic acid water), B (methanol)
Flow velocity: 20 mL / min
Injection volume: 500 μL
Temperature: 40 ° C
Detection wavelength: 280 nm
Gradient condition B (%): 3 → 20% (5 minutes), 20 → 30% (10 minutes), 30 → 97 (0.1 minutes), 97% (2.9 minutes), 97 → 3% (2) Minutes)
Sorting time: 8-9 minutes (3 ″ -β-glucuronate EGCg: compound 12)
8.5-9.5 minutes (4 ″ -β-glucuronate EGCg: Compound 11)

Compound 11: (2'''R, 3'''R, 4'''R, 5'''S, 6'''R) -6''''-(4''-((((2R) , 3R) -5,7-Dihydroxy-2- (3', 4', 5'-trihydroxyphenyl) chroman-3-yl) oxy) carbonyl) -2'', 6''-dihydroxyphenoxy) -3''' , 4''', 5'''-trihydroxyterrahydro-2H-pyran-2'''-carboxylic acid
^{1 1} H NMR (600 MHz, D ₂ O (1% acetic acid)) δ6.93 (s, 2H), 6.52 (s, 2H), 6.10 (d, J = 2.1Hz, 1H), 6 .07 (d, J = 1.8Hz, 1H), 5.55-5.55 (m, 1H), 5.07-5.07 (m, 1H), 4.97 (d, J = 7. 8Hz, 1H), 3.65 (dd, = 5.0, 4.1Hz, 1H), 3.58 (dd, = 9.6,8.2Hz, 1H), 3.51-3.49 (m) , 2H), 3.01 (dd, J = 18.3, 4.9, Hz, 1H), 2.89 (d, J = 17.2Hz, 1H)
_{^{13 C-NMR (150MHz, D}} 2 O (1% acetic acid)) δ165.78,165.76,154.75,154.73,148.52,144.41,135.76,131.33,128 .90, 125.61, 109.10, 105.61, 102.49, 98.30, 95.27, 94.42, 76.30, 75.82, 74.47, 72.32, 70.89 , 68.57, 67.26, 24.03
HRMS calcd. for C ₂₈ H ₂₇ O ₁₇ ⁺ [M + H] ⁺ : 635.1248; found: 635.1261.

Compound 12: (2'''R, 3'''R, 4'''R, 5'''S, 6'''R) -6'''-(5''-((((2R) , 3R) -5,7-Dihydroxy-2- (3', 4', 5'-trihydroxyphenyl) chroman-3-yl) oxy) carbonyl) -2'', 3''-dihydroxyphenoxy) -3''' , 4''', 5'''-trihydroxyterrahydro-2H-pyran-2'''-carboxylic acid
^{1 1} H NMR (600 MHz, D ₂ O (1% acetic acid)) δ 7.08 (d, J = 1.5 Hz, 1 H), 7.07 (d, J = 1.5 Hz, 1 H), 6.51 (S, 2H), 6.13 (d, J = 1.9Hz, 1H), 6.07 (d, J = 1.8Hz, 1H), 5.56-5.56 (m, 1H), 5 .11-5.1 (m, 1H), 4.93 (d, J = 6.4Hz, 1H), 3.71 (d, = 9.4Hz, 1H), 3.57 (d, = 7. 8Hz, 1H), 3.50-3.53 (m, 2H), 2.99 (dd, J = 17.4,3.9, Hz, 1H), 2.90 (dd, J = 17.7) , 2.2Hz, 1H)
¹³ C-NMR (150 MHz, D ₂ O (1% acetone-d ₆ )) δ166.96, 155.82, 155.78, 155.73, 155.64, 155.60, 145.61, 145.39 , 145.17, 140.52, 132.45, 130.30, 120.76, 112.85, 110.44, 106.60, 101.59, 99.14, 96.45, 95.64, 77 .10, 75.47, 72.93, 72.16, 69.87, 25.29
HRMS calcd, for C ₂₈ H ₂₇ O ₁₇ ⁺ [M + H] +: 635.1248; found: 635.1261.

Test Example 1 Catechin-rich green tea human oral intake test using compound 6 (4 "-sulfated EGCg) as a standard 1. Human test content This human test was conducted the day before after obtaining the approval of the in-house ethics committee. Ten healthy men who refrained from ingesting catechin and its relatives (average age: 33.7 ± 2.7 years, average height: 171.1 ± 2.3 cm, average weight: 60.1 ± 2. 1 kg) ingested 350 mL of green tea beverage (containing 540 mg of catechin and its relatives and 150 mg of EGCg), and blood was collected over time. Blood was collected 0.5, 1, 1.5, 2, 3 after ingestion. And at 6 hours, the collected blood sample was immediately centrifuged (3000 × g, 10 minutes) to separate the plasma. The plasma sample was 20w in an amount corresponding to 10% of its volume. After adding 0.4 mol / L phosphate buffer (pH 3.6) containing / v% ascorbic acid and 0.1 w / v% ethylenediamine tetraacetic acid disodium salt, the mixture was stored at −80 ° C.

2. Analysis of plasma samples EGCg and its metabolites in human plasma samples are quantified by ultrafast, high-separation liquid chromatography equipped with a quadrupole orbitrap-type high-resolution mass spectrometer (HRAM, Q-Exactive Focus, Thermo Fisher Scientific). A graphing device (Vanquish UHPLC, manufactured by Thermo Fisher Scientific) was used and performed in full scan mode with a mass range of 150-1000 m / z. The parameters of the mass spectrometer were sheath gas pressure: 40 psi, auxiliary gas pressure: 10 psi, spray voltage: 2.0 kV, and S-lens RF level: 50.0. The UPLC analysis column is Poroshell 120 EC-C18 (manufactured by Agilent Technologies Inc., particle size: 2.7 μm, column diameter: 4.6 mm, column length: 50 mm), and the guard column is Poroshell 120 EC-C18 guard volume. (Manufactured by Agilent Technologies Inc.) was used, and the column temperature was set to 40 ° C. The injection volume is 2 μL, the autosampler temperature is 4 ° C., solution A: 0.1% by volume forgic acid aqueous solution, solution B: 0.1% by volume formic acid-containing acetonitrile is used for the mobile phase, and the flow rate: 0.4 mL / min. The volume was analyzed by the gradient program shown in Table 1. EGCg and 4 "-sulfated EGCg in plasma are designated as high-purity samples ((-)-Epigallocatechin gallate of Nagara Science Co., Ltd. for EGCg, compound 6 synthesized according to Production Example 1 for 4" -sulfated EGCg). A calibration curve (calibration curve range: 0, 5, 10, 20, 50, 100, 250, 500, 1000 ng / mL) was prepared and quantified.

3. 3. Preparation of plasma sample for quantification 0.4 mL / L phosphate buffer (pH 3.6) 16 μL, 0.4 mL / L phosphate buffer (pH 7.4) 16 μL, water 24 μL, internal standard in 100 μL of human plasma sample sample 100 μL of the solution (ethyl gallate 40 ng / mL) and 0.6 mL of a 0.2 mL / L aqueous acetic acid solution were added, and the mixture was stirred. Human plasma samples Samples were placed in solid phase cartridges (Waters Oasis HLB 10 mg / 1 cc) conditioned with 1 mL of water and 0.1 mL of 0.1 vol% DMF acetate and treated by suction. The solid phase cartridge treated with the sample was washed with 1 mL of water and 1 mL of 30% methanol, and then eluted with 0.1 mL of 0.1% DMF acetate.

The quantification results of EGCg and 4 "-sulfated EGCg in plasma samples are shown in FIG. 1, and the pharmacokinetic parameters of each are shown in Table 2 (C _max : maximum blood concentration, T _max : maximum blood concentration after ingestion). Time, AUC _0-6hr : Time-lower area of blood concentration curve, T _1/2 : Blood concentration half-life).

By using the compound 6 of the present invention, the presence in the biological sample could be confirmed and the content could be quantified.

Test Example 2 EGCg rat oral ingestion test using compound 11 (3 "-sulfated EGCg) as a standard 1. Rat test contents (animal)
SD rats (8 weeks old, male) were purchased from Charles River Laboratories, Japan. After acclimation to the environment for one week or more, it was subjected to the test. The number of animals per test group was set to 7 which was considered to be the minimum necessary to obtain a statistical advantage in this test system.
(Preparation of test solution)
EGCg, which is the administration sample of this test, was administered as an aqueous solution of 10 mL / kg.
(Oral administration and plasma preparation)
The test was conducted using rats bred at the facility for one week under the free feeding of a solid diet (MF, Oriental Yeast Co., Ltd.). The rats were fasted for 16 hours or more before administration, and under isoflurane anesthesia, an EGCg aqueous solution was intragastrically administered using a sonde at a dose of 15 mg / kg body weight per rat. Blood was collected from the jugular vein at 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours, 4 hours and 8 hours after administration of the solution. The obtained blood was immediately centrifuged (8000 × g, 4 ° C., 10 minutes), and the obtained plasma was added to the obtained plasma in an amount corresponding to 10% of its volume, 2 w / v% ascorbic acid, 0.1 w / v%. After adding 0.4 mol / L phosphate buffer containing ethylenediaminetetraacetic acid disodium salt and 25% methanol, the mixture was stored at −80 ° C.

2. Analysis of plasma samples EGCg and its metabolites in rat plasma samples are quantified by an ultrafast, high-separation liquid chromatograph device (ExionLC) equipped with a triple quadrupole tandem high-resolution mass spectrometer (QTRAP5000, manufactured by ABSciex). , ABScine), and performed in the multiple reaction monitoring mode. The parameters of the mass analyzer are curtain gas pressure: 25 psi (nitrogen), ion source 1 gas pressure: 50 psi, ion source 2 gas pressure: 75 psi, collision gas: Medium, ion spray voltage: -4500 V, ion source temperature: 600 ° C. Met. The UPLC analysis column is Poroshell 120 EC-C18 (manufactured by Agilent Technologies Inc., particle size: 2.7 μm, column diameter: 4.6 mm, column length: 50 mm), and the guard column is Poroshell 120 EC-C18 guard volume. (Manufactured by Agilent Technologies Inc.) was used, and the column temperature was set to 40 ° C. The injection volume is 5 μL, the autosampler temperature is 4 ° C., solution A: 0.1% forgic acid aqueous solution, solution B: 0.1% forgic acid-containing acetonitrile is used for the mobile phase, and the flow rate is 0.5 mL / min. It was analyzed by the gradient program shown in. EGCg and 3 "-sulfated EGCg in plasma were quantified after preparing a calibration curve using a high-purity sample as a standard and using ethyl gallate as an internal standard substance. Each analysis target was in negative mode. Detected, EGCg quantified 457.0 → 160.9 ions, 3 “-sulfated EGCg quantified 537.0 → 169.3 ions, and ethyl gallate quantified 197.0 → 123.9 ions. Using.

3. Preparation of plasma sample for quantification For 80 μL of plasma collected from rats, 30 μL of 0.4 mol / L phosphate buffer, 80 μL of analytical standard solution, aqueous ethyl gallate solution (40 ng / mL, internal standard substance) and acetonitrile 300 μL was added sequentially and mixed. The mixed solution was centrifuged (20000 × g, 4 ° C., 10 minutes), and the supernatant was collected.
Then, 30 μL of 0.4 mol / L phosphate buffer and 300 μL of acetonitrile are sequentially added to the residue, centrifuged (20000 × g, 4 ° C., 10 minutes), and the supernatant is collected. The procedure is repeated three times. After mixing all the obtained supernatants, the solvent was dried by centrifugation. Then, 220 μL of 0.15 mmol / L ascorbic acid-containing acetonitrile and 100 μL of 0.15 mmol / L ascorbic acid aqueous solution were added, and after mixing, centrifugation (20000 × g, 4 ° C., 10 minutes) was carried out, and in the obtained supernatant. EGCg and 3 "-sulfated EGCg concentrations were measured.

The quantitative results of EGCg and 3 "-sulfated EGCg in rat plasma samples are shown in FIG. 2, and the pharmacokinetic parameters of each are shown in Table 4 (C _max : maximum blood concentration, T _max : intake giving maximum blood concentration). Later time, AUC _{0-8 hr} : Time-lower area of plasma concentration curve, T _1/2 : Blood concentration half-life). Synthetic 3 "-sulfated EGCg (Compound 11) in biological sample The presence of plasma was confirmed, and the content could be quantified.

Claims (6)

The following general formula (v):

[In the formula, one of R ^1a and R ^2a represents a benzyl group and the other represents a hydrogen atom. ]
A general formula including a step of reacting 2,2,2-trichloroethoxy-sulfuryl-1,2-dimethylimidazolium triflate with a benzylated epigallocatechin gallate represented by, or a step of reacting with a glucuronic acid donor. (Id):

[In the formula, R ^1e and R ^2e correspond to R ^1a and R ^2a , respectively, and one of them is a hydrogen atom and the other is -SO ₃ M (where M is a hydrogen atom, an alkali metal atom, and an alkaline earth metal). (Indicating an atom or ammonium) or a glucuronosyl group. ]
A method for producing an epigallocatechin gallate conjugate represented by. The following general formula (v):

[In the formula, one of R ^1a and R ^2a represents a benzyl group and the other represents a hydrogen atom. ]
Reacting 2,2,2-trichloroethoxy-sulfuryl-1,2-dimethylimidazolium triflate with the benzylated epigallocatechin gallate represented by the following general formula (Ia):

[In the formula, R ^1b and R ^2b correspond to R ^1a and R ^2a , respectively, and one of them is a benzyl group and the other is −SO ₃ CH ₂ CCl ₃ . ]
A method for producing a benzylated epigallocatechin gallate TCE sulfated product represented by. The benzylated epigallocatechin gallate TCE sulfate obtained by the general formula (Ia) obtained in claim 2 is hydrogenated in the presence of a hydrogenation catalyst, the general formula (Ib):

[In the formula, R ^1c and R ^2c correspond to R ^1b and R ^2b , respectively, one of which is a hydrogen atom and the other of -SO ₃ M (where M is a hydrogen atom, an alkali metal atom, an alkaline earth metal). Indicates an atom or ammonium). ]
A method for producing epigallocatechin gallate sulfate represented by. The following general formula (v):

[In the formula, one of R ^1a and R ^2a represents a benzyl group and the other represents a hydrogen atom. ]
The benzylated epigallocatechin gallate represented by is reacted with a glucuronic acid donor and then subjected to a deprotection reaction, the general formula (Ic):

[In the formula, R ^1d and R ^2d correspond to R ^1a and R ^2a , respectively, and one of them represents a hydrogen atom and the other represents a glucuronosyl group. ]
A method for producing epigallocatechin gallate glucuron oxide represented by. The following general formula (I):

[In the formula, Z ¹ represents a hydrogen atom or a benzyl group, and when Z ¹ is a benzyl group, one of R ¹ and R ² represents a benzyl group and the other represents −SO ₃ CH ₂ CCl ₃ , and Z ^{When 1} is a hydrogen atom, one of R ¹ and R ² is a hydrogen atom and the other is -SO ₃ M (where M indicates a hydrogen atom, an alkali metal atom, an alkaline earth metal atom or ammonium). Is shown. ]
Epigallocatechin gallate sulfate or a derivative thereof represented by. The following general formula (II):

[In the formula, ^one of Y 1 and Y ² represents a benzyl group and the other represents an allyl group or a hydrogen atom. ]
Benzylated epigallocatechin gallate represented by or an allylic product thereof.

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