JP2023098378A - Method for producing asparaptine - Google Patents

Abstract

To provide a method that chemically synthesizes asparaptine readily and efficiently, and an asparagus acid ester compound useful for the method.SOLUTION: The present invention provides a method for producing asparaptine (chemical name: (2S)-2-[(1,2-dithiolane-4-ylcarbonyl)amino]-5-guanidinopentanoic acid) or a salt thereof, by condensing L-arginine with asparagus acid ester represented by the formula (1) or a salt thereof in an aqueous solvent.SELECTED DRAWING: None

Description

The present invention relates to a method for producing asparaptin and novel asparagusate compounds.

Asparaptin (chemical name: ((2S)-2-[(1,2-dithiolan-4-ylcarbonyl)amino]-5-guanidinopentanoic acid) was isolated from Asparagus officinalis, It is a sulfur-containing compound represented by formula (2).

Asparaptin has an angiotensin converting enzyme (ACE) inhibitory activity and is used as an active ingredient of pharmaceuticals or foods for lowering blood pressure or preventing or treating diseases or symptoms associated with the renin-angiotensin system such as hypertension and heart failure. It has been reported that it can be used (Patent Document 1).

Asparaptin can be produced by purifying and isolating from asparagus, and chemically synthesizing by coupling arginine or its protected form with asparagusic acid. In Patent Document 1, the coupling reaction includes a protected form of arginine in which protective groups have been introduced to the C-terminal carboxyl group and side chain guanidino group, and an acid halide or active ester (for example, N-hydroxysuccinimide ester). It is disclosed that an amide bond forming reaction using (NHS)) as the carboxyl-activated form of asparagusic acid can be utilized. However, Patent Document 1 does not describe synthesis conditions, synthesis accuracy, efficiency, etc., and it is unclear from Patent Document 1 whether asparaptin can be chemically synthesized.

Japanese Patent No. 6735518

However, in conventional coupling reactions in which asparagusic acid is condensed with NHS to derivatize the active ester, asparagusic acid NHS ester, and then reacted with a protected form of arginine in a hydrophobic organic solvent, the protecting group is required to be eliminated, and there are problems such as that the product tends to decompose and is difficult to isolate.

The present invention relates to a simple and efficient method for chemically synthesizing asparaptin, and to provide an asparagusate compound useful for carrying out the method.

The present inventors have found that by using a novel asparagus acid ester compound and reacting it with L-arginine in an aqueous solvent, the L-arginine can be directly amidated without protective derivatization, resulting in asparaptin. can be produced efficiently.

That is, the present invention relates to the following 1) and 2).
1) L-arginine and the following formula (1):

Condensing an asparagus acid ester or a salt thereof represented by the following formula (2) in an aqueous solvent:

A method for producing a compound represented by or a salt thereof.
2) Formula (1) below:

Asparagus acid ester represented by or a salt thereof.

According to the present invention, L-arginine can be directly condensed with asparagusic acid without protective derivatization of L-arginine, and asparaptin or a salt thereof can be efficiently produced. In addition, the asparagusic acid ester represented by the formula (1) of the present invention or a salt thereof is useful for producing asparaptin, and is also useful for binding asparagusic acid to a water-soluble substrate having an amino group. , is useful as an asparagusic acid introduction reagent.

The method for producing the compound represented by the formula (2) (asparaptin) or a salt thereof according to the present invention comprises L-arginine and an asparagus acid ester represented by the following formula (1) or It is carried out by condensing the salt in an aqueous solvent.

The amount of the asparagus acid ester represented by formula (1) or a salt thereof to be used is generally 0.25 to 10 mol, preferably 0.25 to 10 mol, per 1 mol of the water-soluble substrate having an amino group, from the viewpoint of reaction efficiency. is 0.5 to 3 mol, more preferably 1 to 1.5 mol.

The reaction is carried out in an aqueous solvent. Here, water is preferably used as the aqueous solvent, but water containing an organic solvent may be used as long as the compound represented by formula (1) or a salt thereof dissolves therein. In this case, the content of the organic solvent is preferably 80% by volume or less, more preferably 60% by volume or less, even more preferably 40% by volume or less, and even more preferably 20% by volume or less, relative to the total amount of the aqueous solvent. .
Any type of organic solvent may be used as long as it is miscible with water, for example, amide solvents such as dimethylformamide, diethylformamide, dimethylacetamide, tetramethylurea, hexamethylphosphoroamide; ethylene glycol, propylene glycol, diethylene glycol , dipropylene glycol, polyhydric alcohol solvents such as glycerin; sulfoxide solvents such as dimethyl sulfoxide; nitrogen-containing cyclic compound solvents such as N-methylpyrrolidone and pyridine; nitrile solvents such as acetonitrile and propionitrile Solvents; ether solvents such as 1,4-dioxane and tetrahydrofuran; alcohol solvents such as methanol, ethanol, propyl alcohol and isopropyl alcohol; and carboxylic acid solvents such as formic acid and acetic acid. An organic solvent can be used individually by 1 type or in combination of 2 or more types.
The water containing an organic solvent is preferably water containing 1,4-dioxane. In this case, the content of 1,4-dioxane is preferably 80% by volume or less with respect to the total amount of the aqueous solvent, 50% by volume or less is more preferable, 40% by volume or less is even more preferable, and 20% by volume or less is even more preferable.
The aqueous solvent may contain other components as long as they are not involved in the condensation reaction or components that do not inhibit the condensation reaction. It may be liquid.

The reaction temperature can be any of heating, room temperature, and cooling. Usually, the temperature is such that the water-soluble substrate is not significantly denatured, for example, 0 to 45°C, and 10 to 40°C. is preferred.
The reaction time is not particularly limited, and is usually 1 hour to 72 hours, preferably 1 hour to 24 hours.

The compound represented by formula (2) thus produced may form a salt, and the salt may be either a base addition salt or an acid addition salt. Base addition salts include, for example, salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; and ammonium salts. Acid addition salts include, for example, formic acid, acetic acid, propionic acid, maleic acid, fumaric acid, succinic acid, tartaric acid, citric acid, salicylic acid, benzoic acid, p-aminobenzoic acid, benzenesulfonic acid, methanesulfonic acid, ethanesulfonic acid. Salts with acids, p-toluenesulfonic acid, phosphoric acid, nitric acid, sulfuric acid, carbonic acid, perchloric acid and the like are included.
In addition, the compound represented by formula (2) or a salt thereof can exist not only as an unsolvated form but also as a solvate.

The asparagusic acid ester represented by formula (1) used in the above reaction is also referred to as a dehydration condensate of asparagusic acid and N-hydroxysulfosuccinimide (sulfo-NHS) (“asparagusic acid sulfo-NHS ester”). ), which is a novel compound not described in the literature.

Salts of the asparagus acid ester represented by formula (1) include, for example, salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; ammonium salts; trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, cyclohexylamine, procaine, dibenzylamine, N-benzyl-β-phenethylamine, 1-ephenamine, N,N'- Examples thereof include salts with nitrogen-containing organic bases such as dibenzylethylenediamine and N-methylglucamine, among which salts with alkali metals are preferred, and sodium salts are more preferred.

The asparagus acid ester represented by formula (1) or a salt thereof can exist as a solvate as well as an unsolvated form. Therefore, the asparagus acid ester represented by formula (1) includes all crystal forms and solvates thereof. Solvates include, for example, solvates with water and alcoholic solvents (eg, ethanol, etc.). Here, hydrates include, for example, polyhydrates such as monohydrates to pentahydrates, and low hydrates such as hemihydrates.
The asparagus acid ester represented by the formula (1) or a salt thereof used in the above reaction may be a reaction mixture obtained by the condensation reaction shown below, or an isolated and purified purified product. You can use it.

The asparagus acid ester represented by formula (1) is obtained by reacting sulfo-NHS or a salt thereof and asparagus acid with N,N'-dicyclohexylcarbodiimide (DCC) or It can be produced by adding a coupling reagent (condensing agent) such as ethyl (dimethylaminopropyl) carbodiimide (EDC) and reacting.
The solvent is not particularly limited as long as it does not inhibit the reaction. Examples include halogenated hydrocarbon solvents such as dichloromethane, chloroform and carbon tetrachloride; tetrahydrofuran, 1,2-dimethoxyethane, dioxane and the like. ether solvents; aromatic hydrocarbon solvents such as benzene and toluene; amide solvents such as N,N-dimethylformamide, N,N-dimethylacetamide and N-methylpyrrolidin-2-one; In addition to these, sulfoxide solvents such as dimethyl sulfoxide and sulfolane; and ketone solvents such as acetone and methyl ethyl ketone can be used in some cases. Furthermore, these can also be mixed and used. Among these, acetonitrile and N,N-dimethylformamide are preferred.

A base can be used as necessary, and usable bases include, for example, sodium carbonate, potassium carbonate, sodium ethoxide, potassium butoxide, sodium hydroxide, potassium hydroxide, sodium hydride, potassium hydride, etc. carbonates, alkoxides, hydroxides, or hydrides of alkali metals or alkaline earth metals; alkyllithiums such as n-butyllithium, or organometallic bases typified by dialkylaminolithiums such as lithium diisopropylamide; Organometallic bases of bissilylamines such as (trimethylsilyl)amide; also pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine, N-methylmorpholine, diisopropylethylamine, diazabicyclo [5.4.0] Examples include tertiary amines such as undec-7-ene (DBU) and organic bases such as nitrogen-containing heterocyclic compounds.
The reaction temperature is generally 0 to 95°C, preferably 0 to 40°C, and the reaction time is generally 15 minutes to 24 hours, preferably 1 to 24 hours.

In addition, asparagusic acid as a raw material is synthesized from 3-bromo-2-(bromoethyl)propanoic acid by a known method (eg, Tirla, A. et al. Synlett 2018 29(10), 1289-1292.) can do.

The progress of each of the above reactions can be tracked by an ordinary method such as chromatography. After completion of the reaction, the solvent is distilled off, and if necessary, the product can be isolated and purified by a conventional method such as chromatography or recrystallization. Also, the structure of the product can be identified by elemental analysis, MS (ESI-MS) analysis, IR analysis, ¹ H-NMR, ¹³ C-NMR and the like.

Asparagusic acid binds to amino acids such as arginine as described above, and is reported to improve the uptake of amine compounds into cell membranes by forming an amide bond with the amine compounds (Giulio, G. et al. Angew. Chem. Int. Ed. 2015, 54, 7328.). Therefore, asparagusic acid can be used as a water-soluble substrate having an amino group (for example, amino acids, proteins, amino sugars, polysaccharides, nucleic acids, peptides, and other water-soluble bioactive substances having low membrane permeability). ) is thought to contribute to the improvement of biomembrane permeability. The asparagusic acid ester represented by the formula (1) of the present invention is also useful as an asparagusic acid-introducing reagent for forming an amide bond between asparagusic acid and a water-soluble substrate having an amino group.

Reference Example Synthesis of Asparagusic Acid According to a previous report (Tirla, A. et al. Synlett 2018 29(10), 1289-1292), asparagusic acid was synthesized as follows.
30.03 g of 3-bromo-2-(bromoethyl)propanoic acid was weighed into a 500 mL eggplant flask, dissolved in 143 mL of 1M NaOH aqueous solution, and 270 mL of ultrapure 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 0.1 M hydrochloric acid saturated brine, 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. 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 0.1 M hydrochloric acid saturated brine, and dried over anhydrous sodium sulfate. The filtrate filtered through a cotton plug was concentrated under reduced pressure to obtain crude 3-thio-2-(thioethyl)propanoic acid. The crude product was resin-refined using HP-20 (company) to obtain 5.97 g of dehydroasparagusic acid (2-step yield: 32%).
3.3628 g of dehydroasparagusic acid was weighed into a 50 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/0.02 M HCl aqueous solution, and the aqueous layer was further extracted with ethyl acetate (150 mL, 100 mL) successively. The organic layers were combined, washed with 150 mL of 0.02 M hydrochloric acid saturated brine, and dried over anhydrous sodium sulfate. The filtrate filtered through a cotton plug was concentrated under reduced pressure to about 50 mL. After adding 200 mL of acetonitrile and concentrating under reduced pressure to about 50 mL, adding 400 mL of ultrapure water and concentrating under reduced pressure to about 300 mL, the resulting aqueous solution was freeze-dried to obtain 3.23 g of pale yellow powder of asparagusic acid (yield rate of 97%) was obtained.

Example 1 Synthesis of Asparagusic Acid Sulfo-NHS Ester (1) Synthesis 500.3 mg (3.328 mmol) of asparagus acid was weighed into a 50 mL round-bottomed flask and treated with DMF (super dehydrated, Fujifilm Wako Pure Chemical Industries, Ltd.). Dissolved in 0 mL. After adding 759.0 mg (3.495 mg) of N-hydroxysulfosuccinimide sodium (FUJIFILM Wako Pure Chemical Industries, Ltd., 087-09371) and bringing the temperature to 0° C., N,N′-dicyclohexylcarbodiimide (Tokyo Kasei Co., Ltd., D0436) 1101.5 mg (3.495 mmol) was added and stirred for 30 minutes. Further, the reaction solution stirred at room temperature for 18 hours was allowed to stand at 4° C. for 2 hours and filtered through a cotton plug to obtain a yellow transparent solution. This solution was concentrated under reduced pressure with a vacuum pump to obtain 1.826 g of a crude product of asparagusic acid sulfo-NHS ester sodium salt as a yellow solid.

(2) Purification Dissolve 300.2 mg of asparagusic acid sulfo-NHS ester crude sodium salt in 15.0 mL of water, add 15.0 mL of ethyl acetate, stir, centrifuge (3000 rpm, 3 minutes), It was separated into a layer (upper layer, colorless and transparent), an emulsion layer (intermediate layer, white suspension), and an aqueous layer (lower phase, white suspension). The aqueous layer was lyophilized to obtain 138.5 mg (70% calculated yield) of purified asparagusic acid sulfo-NHS ester sodium salt.

(3) Spectral asparagusic acid sulfo NHS ester sodium salt: sodium 1-((1,2-dithiolane-4-carbonyl)oxy)-2,5-dioxopyrrolidine-3-sulfonate
¹ H NMR (600 MHz, DMSO-d6) δ = 4.02 (m, 1H), 3.69 (dd, J = 8.8, 2.5 Hz, 1H), 3.53 (dd, J = 12.1, 8.0 Hz, 1H), 3.43- 3.37 (m, 2H), 3.27 (dd, J = 11.3, 7.4 Hz, 1H), 2.88 (dd, J = 18.0, 8.8 Hz, 1H), 2.67 (dd, J = 18.0, 2.3 Hz, 1H).
¹³ C NMR (150 MHz, DMSO-d6) δ=176.2, 172.0, 167.9, 56.7, 54.9, 42.4, 41.7, 31.2.

Example 2 Solubility of Asparagusic Acid Sulfo-NHS Ester 10 mg of the asparagusic acid sulfo-NHS ester sodium salt product synthesized in Example 1 or asparagusic acid NHS ester (comparative) was weighed and dissolved in a phosphate buffer (0 .05 M, pH 7.7), 1,4-dioxane, and 0.10 mL (10%), 1.0 mL (1%), and 10 mL (0.1%) of a mixed solvent thereof were added to check the solubility. . Table 1 shows the results.
Asparagusic acid NHS ester was synthesized by the following method.
150.2 mg (1.000 mmol) of asparagus acid was weighed and dissolved in 3.0 mL of acetonitrile (superdehydrated). 121.0 mg (1.050 mmol) of N-hydroxysuccinimide was added, the temperature was adjusted to 0° C., and 216.0 mg (1.047 mmol) of N,N′-dicyclohexylcarbodiimide (Tokyo Kasei Co., Ltd., D0436) was dissolved in 3.0 mL of acetonitrile. ) was added and stirred for 3 hours. The white precipitate was removed by filtering the reaction solution through a cotton plug to obtain a yellow transparent solution. This solution was concentrated under reduced pressure, and the crude was purified by silica gel column chromatography to obtain 180.4 mg (0.0730 mmol, yield 73%) of asparagusic acid sulfo-NHS ester as a white solid.
From Table 1, asparagusic acid sulfo NHS ester sodium salt can be dissolved in an aqueous solvent with a 1,4-dioxane content of 80% by volume or less, and the ratio of asparagusic acid NHS ester insoluble in aqueous solvents is high. It is effective as a range of asparagus acid amidation reagents.

Example 2 Synthesis of Asparaptin 10.0 mg (0.0574 mmol) of L-arginine and 30.5 mg (0.0873 mmol) of crude asparagusic acid sulfo-NHS ester sodium salt were weighed into a 10 mL round-bottomed flask and mixed with phosphate buffer. 500 μL of liquid (0.05 M, pH 7.7) was added and stirred for 4 hours. After adding 10.0 mL of 0.02 M HCl aqueous solution and 10.0 mL of ethyl acetate to the reaction solution and stirring, centrifugation (3000 rpm, 3 minutes) was performed to effect liquid/liquid partitioning into an aqueous layer, an ethyl acetate layer, and an intermediate emulsion layer. . Lyophilization of the aqueous layer gave 42.2 mg of asparaptin-containing crude.
The asparaptin-containing crude was separated by HPLC under the following conditions, and the asparaptin-containing fraction was concentrated under reduced pressure and freeze-dried to obtain 6.8 mg (0.0222 mmol, yield 39%) of asparaptin.

HPLC preparative conditions Column: L-column ODS2, 5um, 10*250mm Cat. No. 742100
Flow rate: 6.0 mL/min, temperature: 40°C, detection: UV detection at 254 nm, eluent A: 0.1 vol% formic acid water, B: MeCN, elution conditions are listed in Table 2.
Injection: Dissolve 42 mg of crude in 1.2 mL of 20% by volume acetonitrile aqueous solution, and dispense 0.25 mL each five times.

Asparaptin: (2S)-2-[(1,2-dithiolane-4-ylcarbonyl)amino]-5-guanidinopentanoic acid (1,2-dithiolane-4-carbonyl)-L-arginine
¹ H NMR (600 MHz, D ₂ O) δ = 4.21 (dd, J = 8.2, 5.0 Hz, 1H), 3.48-3.42 (m, 3H), 3.36 (m, 1H), 3.29 (m, 1H), 3.22 (dd, J = 6.8, 6.8 Hz, 2H), 1.88 (m, 1H), 1.73 (m, 1H), 1.64-1.59 (m, 2H).
¹³ C NMR (150 MHz, D ₂ O) δ=181.4, 176.9, 159.6, 57.7, 54.1, 45.6, 44.7, 43.4, 31.6, 27.3.
TOF-MS m/Z _{= 307.0894} (calculated 307.0899 [ _{C10H18N4O3S2 +} H] ₊ ⁾

Comparative Example 1 Synthesis of Asparaptin 1: Reaction Using Arginine and Asparagusic Acid NHS Ester A homogeneous reaction was not possible due to the different ranges.
86.6 mg of asparagusic acid NHS ester and 12.0 mg of 4-dimethylaminopyridine were weighed and dissolved in 1.0 mL of 1,4-dioxane (solution A). 50.0 mg of L-arginine and 36.5 mg of sodium hydrogen carbonate were dissolved in 1.0 mL of water (Solution B). Solution A was added to solution B and stirred at room temperature, but the solution became cloudy after 15 minutes, and a gummy white solid precipitated after 30 minutes, and the reaction did not proceed.

Comparative Example 2 Synthesis of Asparaptin 2: Reaction Using Arginine Methyl Ester and Asparagusic Acid NHS Ester Arginine is highly soluble in water, but has low solubility in organic solvents. On the other hand, asparagusic acid NHS ester dissolves well in organic solvents, but has low solubility in water. Therefore, arginine methyl ester, which is soluble in an organic solvent, was used in order to carry out the condensation reaction in an organic solvent. That is, after condensing arginine methyl ester and asparagusic acid, an attempt was made to synthesize asparaptin by hydrolyzing the methyl ester. Asparagus acid NHS ester soluble in a mixed solution of 1,4-dioxane: phosphate buffer = 67/33 (vol%) and arginine methyl ester were subjected to asparagus acid amidation, and then in a basic aqueous solution. investigated the deprotection of the methyl ester of
50.2 mg of L-arginine methyl ester was weighed into a 10 mL eggplant flask and dissolved in 1.5 mL of a mixed solution of 1,4-dioxane:phosphate buffer=67/33 (vol %). 52.1 mg of asparagusic acid NHS ester and 12.5 mg of 4-dimethylaminopyridine were added to this solution and stirred for 1 hour. The reaction solution was diluted with 10 mL of saturated sodium bicarbonate aqueous solution, extracted three times with 10 mL of ethyl acetate to remove the organic layer, and the aqueous layer was lyophilized to obtain 155 mg of crude. The crude was suspended in 10 mL of methanol, filtered through a cotton plug, and the filtrate was concentrated under reduced pressure, followed by purification using silica gel column chromatography to obtain 47.1 mg of asparaptin methyl ester (yield: 76%).
45.0 mg of asparaptin methyl ester was weighed into a 10 mL eggplant flask, 2.0 mL of 1 M NaOH aqueous solution was added, and the mixture was stirred for 2 hours. After cooling to 0° C., 1.85 mL of 1 M HCl aqueous solution was added to adjust the pH to 6, and 25 mL of water was added to freeze-dry. After adding 13 mL of methanol to the freeze-dried product and suspending it, it was centrifuged to obtain a supernatant methanol solution. As a result of analyzing this methanol solution using LC-TOFMS and ¹ H NMR, asparaptin was not confirmed as complicated decomposition products.

Claims (5)

L-arginine and the following formula (1):

Condensing an asparagus acid ester or a salt thereof represented by the following formula (2) in an aqueous solvent:

A method for producing a compound represented by or a salt thereof. 2. The method of claim 1, wherein the aqueous solvent is water or water containing an organic solvent. 3. The method according to claim 2, wherein the water containing the organic solvent contains the organic solvent in an amount of 80% by volume or less with respect to the total amount of the aqueous solvent. A method according to claim 2 or 3, wherein the organic solvent is 1,4-dioxane. Formula (1) below:

Asparagus acid ester represented by or a salt thereof.