JP2023097207A - Method for preparing asparagusic acid - Google Patents

Abstract

To provide a method for preparing asparagusic acid that suppresses the formation of polymers and is stable even when stored for a long time.SOLUTION: The present invention provides a method for producing asparagusic acid by oxidizing dihydro asparagusic acid using dimethyl sulfoxide, wherein, a reaction solution comprising asparagusic acid is subjected to liquid separation, recovering an organic layer, and then, a water-miscible organic solvent and water are sequentially subjected to solvent displacement before lyophilized.SELECTED DRAWING: None

Description

The present invention relates to a method for preparing asparagusic acid with excellent storage stability.

Asparagusic acid (2-dithiolane-4-carboxylic acid) is an organic sulfur compound contained in asparagus, and is known to promote the growth of asparagus. In addition, it has been reported that asparagusic acid has pharmacological actions such as an acne treatment effect (Patent Document 1) and an antidandruff action (Patent Document 2).
In addition, it has been reported that asparagusic acid forms an amide bond with an amine compound such as an amino acid or peptide to improve the uptake of the amine compound into the cell membrane (Non-Patent Document 1).

It has been reported that asparagusic acid can be chemically synthesized using halogenated isobutyric acid as a starting material (Non-Patent Document 2). That is, as shown in the following reaction formula, 3-bromo-2-(bromoethyl)propanoic acid (1) is reacted with potassium thioacetate to give 3-thioacetyl-2-(thioacetylethyl)propanoic acid (2). and then deacetylated to obtain dihydroasparagusic acid (3), which is the reduced form (dithiol) of asparagusic acid, and subjected to an oxidation reaction using dimethylsulfoxide (DMSO) to give asparagusic acid (4). can be manufactured.

However, asparagusic acid produced by oxidizing dihydroasparagusic acid with dimethyl sulfoxide has a problem that it is easily polymerized through mixed impurities.

JP-A-02-145503 JP-A-02-145508

Giulio, G. et al. Angew. Chem. Int. Ed. 2015, 54, 7328. Tirla, A. et al. Synlett 2018 29(10), 1289-1292.

The present invention relates to providing a method for preparing asparagusic acid in which the formation of polymer is suppressed and which is stable even when stored for a long period of time.

In view of the above problems, the inventors of the present invention conducted a study and found that the reaction solution containing asparagusic acid after the completion of the oxidation reaction was subjected to liquid separation treatment to recover the organic layer, and an organic solvent and water that were mixed with water were recovered. It was found that the impurities can be effectively removed and the storage stability of asparagusic acid can be enhanced by lyophilizing after sequential solvent replacement.

That is, the present invention provides a method for producing asparagusic acid by oxidizing dihydroasparagusic acid using dimethylsulfoxide, in which a reaction solution containing asparagusic acid is subjected to liquid separation treatment to recover an organic layer, and then Provided is a method for preparing asparagusic acid, comprising replacing the solvent with an organic solvent miscible with water and water in that order, followed by freeze-drying.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to efficiently remove by-products and the like produced by an oxidation reaction, and to prepare high-purity asparagusic acid with excellent storage stability.

¹ H NMR chart of asparagusic acid.

The method for preparing asparagusic acid of the present invention is a method for producing asparagusic acid by oxidizing dihydroasparagusic acid using dimethylsulfoxide, wherein a reaction solution containing asparagusic acid is subjected to liquid separation treatment to obtain an organic layer. After recovering and replacing the solvent with an organic solvent and water in that order, the product is lyophilized.

The reaction of oxidizing dihydroasparagusic acid to asparagusic acid using dimethylsulfoxide is generally known as an efficient oxidation method for obtaining asparagusic acid from dihydroasparagusic acid.
The reaction is carried out by stirring dihydroasparagusic acid in dimethylsulfoxide at 60-100° C. for 3-24 hours, preferably at 65-90° C. for 10-20 hours.
Dihydroasparagusic acid may be crudely purified, but it is highly purified by column purification filled with adsorptive resin, silica gel, ODS-modified silica gel, or the like, or by recrystallization purification, preferably by recrystallization purification. It is preferable from the viewpoint of suppression of polymer production.

In the present invention, the "reaction solution containing asparagusic acid" refers to the reaction solution after completion of the oxidation reaction. The reaction solution may contain asparagusic acid and impurities such as reaction by-products (collectively referred to as "reactants").

The liquid separation treatment includes the steps of adding water or an aqueous solution and an organic solvent immiscible with water to the reaction solution containing asparagusic acid, separating the aqueous layer and the organic layer, and recovering the organic layer.
Organic solvents used for liquid separation include aliphatic hydrocarbon solvents (e.g., hexane, cyclohexane, pentane, cyclopentane, etc.), aromatic hydrocarbon solvents (e.g., toluene, xylene, benzene, etc.) that are immiscible with water. etc.), halogenated hydrocarbon solvents (e.g., dichloromethane, dichloroethane, chloroform, carbon tetrachloride, etc.), ether solvents (e.g., diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane, etc.), ketone solvents ( Examples thereof include methyl ethyl ketone, acetone, etc.), ester solvents (eg, ethyl acetate, butyl acetate), and the like.
You may use an organic solvent individually by 1 type or in combination of 2 or more types. Ethyl acetate is preferred as the organic solvent.

Examples of aqueous solutions include aqueous solutions of inorganic acids such as hydrochloric acid, sulfuric acid and nitric acid, and acidic to neutral aqueous buffer solutions including neutral phosphate buffers. For example, 1 to 0.001M, preferably 0.1 to 0.01M hydrochloric acid aqueous solution is suitable.

The usage ratio of water or aqueous solution and organic solvent varies depending on the type of organic solvent used. It is 10 to 70% by mass, more preferably 15 to 65% by mass, still more preferably 25 to 55% by mass with respect to water or an aqueous solution.

Separation of the aqueous layer and the organic layer is not particularly limited, and can be performed using a known liquid separation means such as decantation or liquid separation using a separatory funnel. Also, the separated organic layer can be washed with saturated saline or water.

Then, the recovered organic layer is subjected to solvent substitution in the order of a water-miscible organic solvent and water.
Solvent replacement is performed by removing the solvent under reduced pressure using a rotary evaporator, a vacuum pump, or the like, and then adding an organic solvent that is miscible with water, and then adding water. The amount of the organic solvent miscible with water to be added is preferably 1 to 5 times the amount of the organic solvent after concentration under reduced pressure. The amount of water to be added next is preferably 1 to 5 times the weight of the organic solvent solution miscible with water after concentration under reduced pressure.
The degree of pressure reduction and the temperature when removing (distilling) the solvent under reduced pressure are not particularly limited, but are preferably about 90 to 95 KPa (absolute pressure) and about 20 to 60°C.
From the viewpoint of suppressing polymerization of the reaction product, the removal of the organic solvent is preferably carried out so that the volume becomes 5% or more of the original volume, more preferably 7 to 40%. , 10 to 20%.

Examples of organic solvents that are miscible with water include nitrile solvents, alcohol solvents, ether solvents, acetone, and the like, and may be used alone or in combination of two or more.
Examples of nitrile solvents include acetonitrile and propionitrile. Examples of alcohol solvents include methanol, ethanol, propyl alcohol, isopropyl alcohol, normal butanol, isobutanol and the like. Examples of ether solvents include 1,4-dioxane and tetrahydrofuran.
As the organic solvent miscible with water, alcohol solvents and nitrile solvents are preferable, and specific examples include ethanol and acetonitrile.

Finally, the solvent is replaced with water and the aqueous solution of the reactants is lyophilized.
Here, the water used is not particularly limited, and tap water, distilled water, deionized water, ultrapure water (for example, Milli-Q water purified by an ultrapure water device Milli-Q), etc. can be mentioned.
The freeze-drying conditions are not particularly limited. For example, after freezing at -5°C to -80°C for 6 to 48 hours, the degree of vacuum inside the freeze dryer is reduced to 10 to 500 Pa. , the internal temperature is set to 10 to 40° C. and held for 15 to 50 hours.

The freeze-dried product thus obtained inhibits the formation of asparagusic acid polymers and has excellent storage stability, as shown in the test examples described later.

Reference Example 1 Synthesis of dihydroasparagusic acid

(1) Weigh 30.03 g (122.1 mmol) of 3-bromo-2-(bromoethyl) propanoic acid (Tokyo Chemical Industry Co., Ltd., B3081) in a 500 mL eggplant flask, dissolve in 143 mL of 1M NaOH aqueous solution, and obtain ultrapure 270 mL of water was added. 34.76 g of potassium thioacetate was added to this solution and stirred at room temperature for 18 hours. After cooling to 0° C., 34 mL of 6M HCl aqueous solution was added dropwise to adjust the pH to 1, followed by extraction with 200 mL, 150 mL, and 150 mL of ethyl acetate in that order. The organic layers were combined, washed with 300 mL of saturated brine containing 0.1 M hydrochloric acid, and dried over anhydrous sodium sulfate. The filtrate filtered through a cotton plug was concentrated under reduced pressure to obtain 29.83 g of crude 3-thioacetyl-2-(thioacetylethyl)propanoic acid.

(2) 29.83 g of crudely purified 3-thioacetyl-2-(thioacetylethyl)propanoic acid was weighed into a 250 mL eggplant flask, 250 mL of 2.5 M NaOH aqueous solution was added, and the mixture was stirred for 18 hours. The reaction solution was cooled to 0° C., adjusted to pH 1 by adding 115 mL of 6M HCl aqueous solution, and then extracted with 300, 150 and 150 mL of ethyl acetate. The organic layers were combined, washed with 200 mL of saturated brine containing 0.1 M hydrochloric acid, and dried over anhydrous sodium sulfate. The filtrate filtered through a cotton plug was concentrated under reduced pressure to obtain 20.56 g of crude 3-thio-2-(thioethyl)propanoic acid (dihydroasparagusic acid).
10.56 g of 1/2 of the crude purified product was resin-purified using Diaion HP-20 (Mitsubishi Chemical Co., Ltd.), and 5.97 g of dihydroasparagusic acid (39.7 mmol, 65% in terms of two-step yield) ). In the resin purification, 500 g of Diaion HP-20 was conditioned with 0.1 vol% formic acid water, and then dihydroasparagusic acid was added to 0.1 vol% formic acid water: methanol = 1: 1 mixed solution 3 times the weight. It was dissolved, charged, and eluted with 0.1% by volume aqueous formic acid:methanol=1:1. The eluted fraction was concentrated under reduced pressure to remove methanol, and then freeze-dried to obtain a white solid of dihydroasparagusic acid.

<Dihydroasparagusic acid>
¹ H NMR (600 MHz, DMSO-d ₆ ) δ = 12.56 (br s, 1H, —COOH), 3.29 (br s, 1H, —SH), 2.79-2.71 (m, 4H ), 2.65 (m, 1H), 2.38 (br s, 1H, -SH).
¹³ C NMR (150 MHz, DMSO-d ₆ ) δ=173.4, 50.5 (2C), 24.0 (2C).
HRMS (Negative) [MH] ⁻ m/Z=150.9905 (calcd 150.9893 for C ₄ H ₇ O ₂ S ₂ )

A quarter amount of 4.6553 g of the crude product was weighed out, 60 mL of n-hexane was added, and the mixture was stirred at 65°C for 5 minutes to obtain a colorless and transparent heated hexane solution of dihydroasparagusic acid and a yellow slurry-like precipitate. separated. Only the heated hexane solution was withdrawn and allowed to cool to room temperature to precipitate colorless transparent plate-like crystals. The crystals were separated by filtration with filter paper and dried under reduced pressure to give 3.0528 g (20. 32 mmol, 67% in terms of two-step yield).

Examples 1 to 3 and Comparative Example 1 Production of Asparagusic Acid Asparagusic acid was synthesized from the dihydroasparagusic acid produced in Reference Example 1 and purified by the methods shown in Examples 1 to 3 and Comparative Example 1 below. .

(1) Example 1
520 mg of recrystallized dihydroasparagusic acid was weighed into a 10 mL eggplant flask, dissolved in 5.0 mL of DMSO, and stirred at 75° C. for 19 hours. The reaction solution was subjected to liquid/liquid partitioning with ethyl acetate/0.02M hydrochloric acid aqueous solution=50 mL/50 mL, and the aqueous layer was further extracted twice with 20 mL of ethyl acetate. The organic layers were combined, washed with 20 mL of saturated brine containing 0.02 M hydrochloric acid, and dried over anhydrous sodium sulfate. The filtrate filtered through a cotton plug was concentrated under reduced pressure to about 15 mL. After adding 30 mL of acetonitrile, the solution was concentrated under reduced pressure to about 15 mL, 50 mL of ultrapure water was added, and the resulting aqueous solution was concentrated under reduced pressure to about 40 mL. yield quant., purity about 97%).

<Asparagusic acid>
¹ H NMR (600 MHz, DMSO-d ₆ ) δ=12.71 (br s, 1H, —COOH), 3.53-3.48 (m, 1H), 3.40 (dd, J=11.3 , 5.1 Hz, 2H), 3.30 (dd, J=11.3, 7.5 Hz, 2H).
¹³ C NMR (150 MHz, DMSO-d ₆ ) δ=172.8, 50.2 (2C), 41.0 (2C).
HRMS (Negative) [MH] ⁻ m/Z=148.9747 (calcd 148.9731 for C ₄ H ₅ O ₂ S ₂ )

(2) Example 2
500.2 mg of recrystallized dihydroasparagusic acid was weighed into a 10 mL eggplant flask, dissolved in 5.0 mL of DMSO, and stirred at 75° C. for 17 hours. The reaction solution was subjected to liquid/liquid partitioning with ethyl acetate/0.02M hydrochloric acid aqueous solution=50 mL/50 mL, and the aqueous layer was further extracted twice with 20 mL of ethyl acetate. The organic layers were combined, washed with 20 mL of saturated brine containing 0.02 M hydrochloric acid, and dried over anhydrous sodium sulfate. The filtrate filtered through a cotton plug was concentrated under reduced pressure to about 10 mL. After further adding 30 mL of ethanol, it was concentrated under reduced pressure to about 10 mL, further added with 50 mL of ultrapure water, and concentrated under reduced pressure to about 45 mL. (yield 93%, purity about 98%).

(3) Example 3
3.383 g of crude dihydroasparagusic acid (purity 75%) was weighed into a 100 mL eggplant flask, dissolved in 33.6 mL of DMSO, and stirred at 75° C. for 19 hours. The reaction solution was subjected to liquid/liquid partitioning with ethyl acetate/0.02M hydrochloric acid aqueous solution=300 mL/300 mL, and the aqueous layer was further extracted twice with 150 mL and 100 mL of ethyl acetate. The organic layers were combined, washed with 150 mL of saturated brine containing 0.02 M hydrochloric acid, and dried over anhydrous sodium sulfate. The filtrate filtered through a cotton plug was concentrated under reduced pressure to about 50 mL. After further adding 200 mL of acetonitrile, it was concentrated under reduced pressure to about 60 mL, 400 mL of ultrapure water was further added, and the aqueous solution obtained by concentrating under reduced pressure to about 350 mL was freeze-dried to obtain 3.230 g of pale yellow powder of asparagusic acid. (purity 71%, yield 93%).

(4) Comparative Example 1
102 mg of the dihydroasparagusic acid resin column-purified product was weighed into a 100 mL eggplant flask, dissolved in 10 mL of DMSO, and stirred at 75° C. for 15 hours. The reaction solution was subjected to liquid/liquid partitioning with ethyl acetate/0.02M HCl aqueous solution=30 mL/100 mL, and the aqueous layer was further extracted twice with 30 mL and 30 mL of ethyl acetate. The organic layers were combined, washed with 40 mL of saturated brine containing 0.02 M hydrochloric acid, and dried over anhydrous sodium sulfate. The filtrate filtered through a cotton plug was concentrated under reduced pressure to about 50 mL, the solvent was dried and dried under reduced pressure with a vacuum pump to obtain 101 mg of a yellow rubber-like solid (mixture of asparagusic acid and its polymer).
The resulting rubber-like solid consisting of a mixture of asparagusic acid and its polymer did not dissolve in water, chloroform or ethyl acetate, but dissolved in DMSO.

Test Example Storage Stability of Asparagusic Acid The asparagusic acid prepared in Example 1 was stored at −20° C. for 1 month, then dispensed into brown sample bottles under argon enclosure, and stored at −20° C., 5° C. and room temperature. 90 days or 230 days under each temperature condition. 5.0 mg of the sample after storage was dissolved in 0.60 mL of DMSO-d ₆ (internal standard 1,4-bis(trimethylsilyl)benzene 503.1 mg/L added), and 1H NMR (600 MHz, DMSO-d ₆ ) was measured. Asparagusic acid purity and storage stability in the samples were evaluated. In addition, 1.0 mg of the rubber-like solid (mixture of asparagusic acid and its polymer) prepared in Comparative Example 1 was dissolved in 0.60 mL of DMSO-d ₆ and the asparagusic acid polymer (δ = 3.02 ppm broad signal) was confirmed. A ¹ H NMR chart is shown in FIG.
From FIG. 1, the purity of asparagusic acid described in Example 1 after storage is 95% (-20°C, after storage for 90 days), 91% (-20°C, after storage for 230 days), 83% (5°C, After storage for 230 days), 81% (after storage at room temperature for 230 days), and in Comparative Example 1 (immediately after preparation), a wide signal of δ = 3.02 ppm derived from asparagusic acid polymer was confirmed.

Claims (6)

In a method for producing asparagusic acid by oxidizing dihydroasparagusic acid with dimethylsulfoxide, a reaction solution containing asparagusic acid is subjected to liquid separation treatment to recover an organic layer, and then an organic solvent miscible with water and A method for preparing asparagusic acid, comprising lyophilizing after solvent substitution in order of water. 2. The method according to claim 1, wherein the water-miscible organic solvent is one or more organic solvents selected from nitrile solvents, alcohol solvents, and ether solvents. 2. The method according to claim 1, wherein the water-miscible organic solvent is a nitrile solvent or an alcohol solvent. 4. The method according to claim 3, wherein the nitrile solvent is acetonitrile. 4. The method of claim 3, wherein the alcoholic solvent is ethanol. The method according to any one of claims 1 to 5, wherein the liquid separation treatment is liquid-liquid partitioning using ethyl acetate and an aqueous hydrochloric acid solution.

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