Title of Invention	PROCESS FOR PREPARATION OF CRYSTALLINE SODIUM OR POTASSIUM (2S,3S)-3-[[(1S)-1- ISOBUTOXYMETHYL-3-METHYLBUTYL] CARBAMOYL] OXIRANE-2-CARBOXYLATE
Abstract	The present invention relates a process for preparation of crystalline sodium or potassium (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate comprising the following steps (i) to (vi): (i) hydrolyzing (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methyl-butyl] carbamoyl] oxirane-2-carboxylic acid ester of an aliphatic alcohol having 1 to 6 carbon atoms, to obtain (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-car-boxylic acid, (ii) causing the carboxylic acid obtained in the preceding step react with an organic amine represented by the formula of: (R<sup>1</sup>)(R<sup>2</sup>)(R<sup>3</sup>)N wherein R<sup>1</sup>, R<sup>2</sup> and R<sup>3</sup> are as described in the description, (iii) adding an acid to the salt obtained in the preceding step, to obtain (2S,3S)-3-[[(1S)-1 -isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylic acid, (iv) causing the carboxylic acid obtained in the preceding step react with a basic sodium or potassium compound in a mixed solvent of water and an aliphatic alcohol or acetone, to obtain a sodium or potassium salt, (v) recrystallizing the sodium or potassium salt obtained in the preceding step using an aliphatic alcohol, and (vi) drying the product recrystallized in the preceding step under reduced pressure.

Title of Invention

PROCESS FOR PREPARATION OF CRYSTALLINE SODIUM OR POTASSIUM (2S,3S)-3-[[(1S)-1- ISOBUTOXYMETHYL-3-METHYLBUTYL] CARBAMOYL] OXIRANE-2-CARBOXYLATE

Abstract

The present invention relates a process for preparation of crystalline sodium or potassium (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate comprising the following steps (i) to (vi): (i) hydrolyzing (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methyl-butyl] carbamoyl] oxirane-2-carboxylic acid ester of an aliphatic alcohol having 1 to 6 carbon atoms, to obtain (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-car-boxylic acid, (ii) causing the carboxylic acid obtained in the preceding step react with an organic amine represented by the formula of: (R1)(R2)(R3)N wherein R1, R2 and R3 are as described in the description, (iii) adding an acid to the salt obtained in the preceding step, to obtain (2S,3S)-3-[[(1S)-1 -isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylic acid, (iv) causing the carboxylic acid obtained in the preceding step react with a basic sodium or potassium compound in a mixed solvent of water and an aliphatic alcohol or acetone, to obtain a sodium or potassium salt, (v) recrystallizing the sodium or potassium salt obtained in the preceding step using an aliphatic alcohol, and (vi) drying the product recrystallized in the preceding step under reduced pressure.

Full Text	SALT OF (2S,3S)-3-[[(lS)-l-ISOBUTOXYMETEIYL-3-METHYLBUTYL] CARBAMOYL] OXIRANE-2-CARBOXYLIC ACID [FIELD OF THE INVENTION] The present invention relates to a salt of (2S,3S)-3- [ [ (1S) -l-isobutoxymethyl-3-methylbutyl] carbamoyl] -oxirane-2-carboxylic acid. [BACKGROUND OF THE INVENTION] The below-illustrated sodium (2S,3S)-3-[ [ (1S)-1-iso-butoxymethyl-3-methylbutyl] carbamoyl] -oxirane-2-carboxyl-ate (hereinafter referred to as Compound A) shows a ca-thepsin-inhibitory action and is useful as a remedy for treating rheumatoid arthritis and osteoporosis (Patent reference 1: WO 99/11640 pamphlet): Patent reference 1 describes a process for producing Compound A according to the following reaction scheme (see Example 48 of Patent reference 1): The starting ester material of the above-mentioned reaction scheme is generally purified by column chroma-tography to obtain a purified Compound A. [DISCLOSURE OF THE INVENTION] Amorphous Compound A is so hygroscopic and so ther¬mally unstable that various troubles in producing the remedy are expected, and is hence difficult to treat as material for the pharmaceutical preparation. According¬ly, there is a need to provide a thermally stable crys¬tal 1 ine (2S,3S)-3-[[{lS)-l-isobutoxymethyl-3-methylbu-tyl] carbamoyl] oxirane-2-carboxylic acid salt having a long storage-life. If crystalline Compound A is prepared according to the conventional recrystallization or salt-making process in which Compound A is converted from a free form into the desired form, the product often precipitates in the form of too fine crystallites to filter or occasionally in the form of agar-like jelly. Further, since Compound A is very hygroscopic, moisture in air causes to conver¬sion of Compound A from the crystal state into the amor¬phous state and, as a result, the filterability of the product lowers. In the industrial preparation of Compound A, puri¬fication by column chromatography is a troiiblesome proce¬dure. Accordingly, it is also desired to provide a sim¬ple purification process for Compound A. When Compound A is prepared in an industrial scale, it is important to purify Conpound A in the free acid form. As described hereinafter, it is very simple and industrially advantageous to prepare Compound A in the free acid form from the after-described organic amine salt. In addition, it makes the preparation process fur¬ther simple and advantageous that Compound A in an ester form is hydrolyzed with a basic sodium or potassium com¬pound to convert directly into a sodium or potassium salt. The present invention has an object to provide a thermally stable crystalline sodium or potassium (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl] carbamoyl]-oxirane-2-carboxylate having a long storage-life. The invention also has objects to provide a simple and industrially advantageous process for preparing crystallites of the above-mentioned compound and to provide a salt of the compound with an organic amine employable in the process. The invention relates to crystalline sodium (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]-oxirane-2-carboxylate having the following characteristics: DSC: an exothermic peak observed at a temperature in the range of 170 to 175°C with weight decrease; and characteristic absorption bands of infrared absorption spectrum (KBr tablet) : 3255, 2950, 2860, 1670, 1630, 1550, 1460, 1435, 1395, 1365, 1310, 1260, 1110,890 cm"'. The invention also relates to crystalline potassium (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-carboxylate having the following characteristics: DSC: an exothermic peak observed at 177°C with weight decrease; and characteristic absorption bands of infrared absorption spectrum (KBr tablet): 3270, 3080, 2950, 2870, 1680, 1625, 1560, 1460, 1380, 1300, 1240, 1110, 895 cm"'. The present invention is directed to a process for preparation of crystalline sodium or potassium (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methyl- butyl]carbamoyl]oxirane-2-carboxylate, comprising the following steps (i) to (vi): (i) hydrolyzing (2S,3S)-3-[f(lS)-l-isobutoxymethyl-3-methyl-butyl] carbamoyl] oxirane-2-carboxylic acid ester of an aliphatic alcohol having 1 to 6 carbon atoms, to obtain (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylic acid, (ii) causing the carboxylic acid obtained in the preceding step react with an organic amine represented by the formula of: (R1)(R2)(R3)N in which R1 is hydrogen or a linear-chain, branched-chain or cyclic alkyl group having 1 to 10 carbon atoms; R is hydrogen or a linear-cham, branched-chain or cyclic alkyl or aralkyl group of 1 to 10 carbon atoms; and R is a linear-chain, branched-chain or cyclic alkyl group having 1 to 10 carbon atoms which may have a substituent selected from the group consisting of halogen atoms, nitro, hydroxyl, carboxy 1, guanidino, amino and aralkylamino groups; or otherwise R2 and R3 can be com¬bined to form a 5- to 7-membered ring comprising the nitrogen atom to which R and R are connected, the ring possibly containing additional nitrogen atom, to prepare an organic amine salt of (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylic acid, (iii) adding an acid to the salt obtained in the preceding step, to obtain (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylic acid, (iv) causing the carboxylic acid obtained in the preceding step react with a basic sodium or potassium compound in a mixed solvent of water and an aliphatic alcohol or acetone, to obtain a sodium or potassium salt, (v) recrystallizing the sodium or potassium salt obtained in the preceding step using an aliphatic alcohol, and (vi) drying the product recrystallized in the preceding step under reduced pressure. The invention still further relates to a salt of (2S,3S)-3- [ [ (1S)-l-isobutoxymethyl-3-methylbutyl]carbamo¬yl] oxirane-2-carboxylic acid with an organic amine. The invention further relates to a salt of (2S,3S)-3- [ [ (1S) -l-isobutoxynethyl-3-methylbutyl] carbamoyl] -oxirane-2-carboxylic acid with an organic amine repre¬sented by the formula of: (R1) (R2) (R3)N in which R2 is hydrogen or a linear-chain, branched-chain or cyclic allcyl group having 1 to 10 carbon atoms; R2 is hydrogen or a linear-chain, branched-chain or cyclic alJcyl or aralkyl group of 1 to 10 carbon atoms; and R2 is a linear-cliain, branched-chain or cyclic alkyl group of 1 to 10 carbon atoms which may have a substituent group selected from the group consisting of halogen atoms, nitro, hydroxyl, carboxyl, guanidino, amino and arallcyl-amino groups; or otherwise R2 and R2 can be combined to form a 5- to 7-membered ring comprising the nitrogen atom to which R2 and R2 are connected (the ring may contain additional nitrogen atom)}. The invention further relates to a salt of (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl] carbamoyl]-oxirane-2-carboxylic acid with an organic amine selected from the group consisting of piperazine, adamantane amines, cyclohexylamine, dicyclohexylamine, tris (hydroxy-methyl)aminomethane, arginine, lysine, benzathine and meglumine. [MOST PREFERRED EMBODIMENTS OF THE INVENTION] The present invention is explained below in more de¬tail. Crystalline sodium or potassium (2S,3S)-3-[ [ (1S)-1-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-carbox-ylate of the invention can be obtained according to the following flow chart. (Flow chart) Process 1: ester forro (A) - free form (B) - salt with an organ¬ic amine (C) - free form (D) - Na or K salt (E) - recrys-tallized product (F) - immediately dried crystallites (G); or otherwise Process 2: ester form (A) - Na or K salt (E) -^ recrystallized product (F) -* immediately dried crystallites (G) . Each step is explained below. (Process 1) (1) Step of ester form (A) - free form (B) In a mixed solvent of water and an aliphatic alcohol such as methanol, ethanol or isopropyl alcohol, (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]-oxirane-2-carboxylic ester (A) is hydrolyzed in the pres¬ence of a base such as sodium hydroxide or potassium hydroxide, and is then neutralized to obtain (2S,3S)-3- [ [ (1S)-l-isobutoxymethyl-3-methylbutyl] carbamoyl]oxirane-2-carboxylic acid [free form (B)]. In the above procedure, the starting (2S,3S)-3- [ [ (1S) -l-isobutoxyraethyl-3-methylbutyl] carbamoyl] oxirane-2-carboxylic ester is an ester with an aliphatic alcohol, preferably an ester with a primary, secondary or tertiary alcohol having 1 to 6 carbon atoms, more preferably an ester with ethanol, methanol, n-propyl alcohol or isopro¬pyl alcohol. For synthesizing the ester of the starting material, (2S,3S)-3-alkoxycabonyloxirane-2-carboxylic acid and (1S) -l-isobutoxymethyl-3-methylbutylamine are, for exam¬ple, condensed according to the normal condensation pro¬cess (e.g., DCC-HOSu method, acid chloride method, and acid anhydride method). (2) Step of free form (B) - salt with an organic amine (C) The free form (B) prepared in Step (1) is caused to react with an organic amine in a solvent. Examples of the solvents include aliphatic alcohols such as methanol, ethanol, n-propyl alcohol and isopropyl alcohol; aliphat¬ic ethers such as diethyl ether and diisopropyl ether; aliphatic esters such as ethyl acetate; aliphatic ketones such as acetone and ethyl methyl ketone; and water. They may be used singly or may be mixed. In consideration of flammability and toxicity, the solvent preferably is ethyl acetate, acetone, water or a mixture thereof. Thus, the salt with a desired organic amine can be ob¬tained . The organic amine is, for example, represented by the formula of: (R2) (R2) (R3)N in which R2 is hydrogen or a linear-chain, branched-chain or cyclic alkyl group having 1 to 10 carbon atoms; R2 is hydrogen or a linear-chain, branched-chain or cyclic alkyl or aralkyl group of 1 to 10 carbon atoms; and R2 is a linear-chain, branched-chain or cyclic alkyl group (including a crosslinked cyclic hydrocarbon group such as adamantyl) having 1 to 10 carbon atoms which may have a substituent selected from the group consisting of halogen atoms, nitro, hydroxyl, carboxyl, guanidino, amino and aralkylamino groups; or otherwise R2 and R2 can be com¬bined to form a 5- to 7-membered ring comprising the ni¬trogen atom to which R2 and R2 are connected (the ring may contain additional nitrogen atom). In consideration of cost, toxicity and crystallization, the organic amine preferably is piperazine, an adamantane amine, cyclohex-ylamine, dicyclohexylamine, tris (hydroxymethyl)aminometh-ane, arginine (preferably in L-form), lysine (preferably in D-form or DL-form) , benzathine or meglumine. The salts with D- or DL-lysine, benzathine, meglumine and tris(hydroxymethyl)aminomethane can be immediately used as medicaments without further processing. (3) Step of salt with an organic amine (C) -* free form (D) The salt with an organic amine (C) obtained in Step (2) is treated with an acid (e.g., hydrochloric acid) in the conventional manner, to obtain (2S,3S)-3-[ [ (1S)-1-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-carbox-ylic acid (D) of high purity. Thus obtained free form (D) gave no peak attributed to cleavage products (9.8 minutes, see Example 14) while the peak is given by the free form or the metal salt thereof obtained by hydro-lyzing the ester form (A) purified by silica-gel column chromatography. (In the case of preparing a sodium salt) (4) Step of free form (D) -> Na salt (E) The free form (D) is converted into a sodium salt using a sodium source in an organic solvent capable of dissolving the free form (D) or in a water-containing or¬ganic solvent. Examples of the sodium sources include sodium metal, sodium alkoxides such as sodium methoxide and sodium ethoxide, sodium hydroxide, sodium carbonate, and sodium organic carboxylate. The solvent preferably is a mixed solvent of water and methanol, ethanol or acetone, in consideration of flammability and toxicity. If the water content is in the range of 1 to 5%, the product is obtained in a high yield. Thus obtained solid product is well filtered so that the product is collected by conventional filtration. (5) Step of Na salt (E) - recrystallized product (F) The solid sodium salt (E) obtained in Step (4) is dissolved in an aliphatic alcohol such as methanol at a temperature of form room temperature to 60°C, and then an organic solvent is added to precipitate a crystalline product. The organic solvent preferably is a mixed sol¬vent of ethyl acetate, methanol and ethanol, in consider¬ation of flammability and toxicity. If the mixing ratio of ethyl acetate/alcohol is in the range of 10/1 to 5/1, the product is obtained in a high yield. Thus recrystallized product (F) is well filtered so that the product is collected by conventional filtration. (6) Recrystallized product (F) -* immediately dried crys¬tallites (G) The recrystallized product (F) is collected by fil¬tration in a stream of nitrogen gas, and immediately dried to obtain, in a high yield, crystalline sodium (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]carbamo¬yl] oxirane-2-carboxylate (G) of high purity having the following characteristics: DSC (the same as that in Example 15): exothermic peak observed in the range of 170 to 175°C with weight de¬crease / and characteristic absorption bands of infrared absorp¬tion spectrum (KBr tablet): 3255, 2950, 2860, 1670, 1630, 1550, 1460, 1435, 1395, 1365, 1310, 1260, 1110, 890 cm"^. The product preferably is in the form of white crys¬talline needles. The crystalline Na salt of the invention (described in Example 9) was subjected to a comparative stability test. In the test, the Na salt was left at 80°C for three days together with both the substance obtained by con¬densing the solution to dryness and the product collected by filtration and left at room temperature after re-crystallization. As a result, it was found that the Na salt of the invention was superior to the substances for comparison in storage stability (see Exanple 15). (In the case of preparing a potassium salt) (7) Step of free form (D) - K salt (E) The free form (D) is converted into a potassium salt using a potassium source in an organic solvent capable of dissolving the free form (D) or in a water-containing or¬ganic solvent. Examples of the potassium sources include potassium metal, potassium alkoxides such as potassium methoxide and potassium ethoxide, potassium hydroxide, potassium carbonate, and potassium organic carboxylate. The solvent preferably is a mixed solvent of water and methanol, ethanol or acetone, in consideration of flammability and toxicity. If the water content is in the range of 1 to 5%, the product is obtained in a high yield. Thus obtained solid product is well filtered so that the product is collected by conventional filtration. (8) Step of K salt (E) -* recrystallized product (F) The solid potassium salt (E) obtained in Step (7) is dissolved in an aliphatic alcohol such as methanol at a temperature from room temperature to 60°C, and then an organic solvent is added to precipitate a crystalline product. The organic solvent preferably is a mixed sol¬vent of ethyl acetate, methanol and ethanol, in consider¬ation of flammability and toxicity. If the mixing ratio of ethyl acetate/alcohol is in the range of 30/1 to lO/l, the product is obtained in a high yield. Thus recrystallized product (F) is well filtered so that the product is collected by conventional filtration. (9) recrystallized product (F) - immediately dried crys¬ tallites (G) The recrystallized product (F) is collected by fil- tration in a stream of nitrogen gas, and immediately dried to obtain, in a high yield, crystalline potassium (2S,3S) -3- [ [ (1S) -l-isobutoxyT\ethyl-3-methylbutyl] carbamo¬yl] oxirane-2-carboxylate (G) of high purity having the following characteristics: DSC (the same as that in Example 15): exothermic peak observed at 177°C with weight decrease; and characteristic absorption bands of infrared absorp¬tion spectrum (KBr tablet): 3270, 3080, 2950, 2870, 1680, 1625, 1560, 1460, 1380, 1300, 1240, 1110, 895 cm'^ The product is preferably in the form of white crys¬talline needles. The crystalline K salt of the invention (described in Example 11) was subjected to a stability test in which the K salt was left at 80°C for three days. As a result, it was found that the K salt of the invention was excel¬lent in storage stability (see Example 15). (In the case of preparing a calcium or lithium salt) Crystalline calcium or lithium (2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-carbox-ylate of high purity can be prepared in a high yield through the steps similar to the above-mentioned Steps (4) to (9) . (Process 2) (1) Ester form (A) - Na or K salt (E) [Preparation of Na salt (E)] The Na salt (E) can be obtained by causing the ester form (A) described in Step (1) of Process 1 react with a basic sodium compound (e.g., sodium carbonate, sodium hydroxide) to hydrolyze, by condensing the reaction mix¬ture, and then by adding a water-conpatible organic sol¬vent such as acetone to crystallize the salt. If the water content is in the range of 1 to 5%, the product is obtained in a high yield. [Preparation of K salt (E)] The K salt (E) can be obtained by causing the ester form (A) react with a basic potassium compound (e.g., potassium carbonate, potassium hydroxide) to hydrolyze, by condensing the reaction mixture, and then by adding a water-compatible organic solvent such as acetone to crys¬tallize the salt. If the water content is in the range of 1 to 5%, the product is obtained in a high yield. (2) Na or K salt (E) - recrystallized product (F) -* imme¬diately dried crystallites (G) The steps of Na salt (E) recrystallized product (F) -* immediately dried cirystallites (G) can be the same as Steps (5) and (6) of Process 1 described above. The steps of K salt (E) recrystallized product (F) immediately dried crystallites (G) can be the same as Steps (8) and (9) of Process 1 described above. As described above, the purification process in which (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylic acid is converted to a salt with an organic amine, as well as the process in which the ester form of (2S,3S)-3-[[(1S)-1-isobutoxy-methyl-3-methylbutyl] carbamoyl] oxirane-2-carboxylic acid is hydrolyzed with a basic sodium or potassium cotrpound to prepare directly a sodium or potassium (2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate, does not need to involve the step for purifying the ester form by silica-gel column chromatog¬raphy. They are, therefore, simple and industrially advantageous processes for purifying (2S,3S)-3-[ [ (1S)-1-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-car¬boxylic acid. The crystalline sodium or potassium (2S,3S)-3- [ [ (1S) -l-isobutoxytnethyl-3-methylbutyl] carbamoyl] oxirane-2-carboxylate of the invention has a long storage life and excellent thermal stability, and accordingly is use¬ful as a material for pharmaceutical preparation. Form thus obtained crystalline sodium or potassium (2S,3S)-3- [ [ (1S)-l-isobutoxymethyl-3-methylbutyl]carbamo¬yl] oxirane-2-carboxylate, medicaments can be prepared. For preparing the medicaments, the salt can be pro¬cessed according to the conventional manner to give, for example, tablets, granules, powder, capsules, suspension, injection or suppository. In the pharmaceutical prepara¬tion, normal additives such as excipient, disintegrator, binder, lubricant, dye and diluent are used. [EXAMPLES] The invention is further described by the following examples. They by no means restrict the invention. [Exanple 1] Preparation of free form (B) Crude ethyl (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate (164.54 g) synthesized according to the process described in Patent reference 1 was dissolved in ethanol (520 mL). While the solution was cooled with ice (at a temperature of 10°C or below), 1 mol/L aqueous sodium hydroxide solution (522 mL) was dropped. After the solution was stirred for 1.5 hours while the tenperature was kept, ethanol was removed under reduced pressure. Water (340 mL) was added, and then the mixture was filtered through Celite to remove insoli±)le materials. The mixture was washed twice with ethyl acetate (300 mL). While the aqueous portion was cooled with ice, 6 mol/L aqueous hydrochloric acid (110 mL) was dropped to adjust the pH value to 1 and then the mixture was extracted twice with ethyl acetate (300 mL). The ethyl acetate portion was washed with water (300 mL) and with aqueous saturated sodium chloride solution (200 mL) , and then dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to ob¬tain (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylic acid (139.61 g, 93.1%) as orange oil. The NMR data were the same as those in Exam¬ple 6. [Example 2] Preparation 1 of salt with organic amine (C) (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylic acid (139.61 g) was dis¬solved in ethyl acetate (630 mL). The solution was cooled with ice (at a temperature of 10°C or below) and cyclohexylamine (48.19 g) dissolved in ethyl acetate (190 mL) was added. The resultant mixture was stirred over¬night at room temperature. The precipitated crystalline product was collected by filtration, washed with ethyl acetate, and dried in air to obtain white powdery cyclo¬hexylamine salt of (2S,3S)-3- [ [(1S)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylic acid (165.22 g, 88.0%). The obtained product (1.0 g) was recrystal-lized from ethanol/water-containing ethyl acetate, to prepare crystalline flakes (925 mg, 92.5%). Melting point: 128°C - 129°C; IR (KBr) cm-1 : 3310, 2950, 2850, 1660, 1620, 1570, 1540, 1460, 1445, 1380, 1360, 1340, 1295, 1250, 1220, 1120, 945, 890; and NMR (CDCI3) 6: 0.9-1.0 (12H, m) , 1.2-1.8 (9H, m) , 1.8-1.9 (3H, m), 2.0-2.1 (2H, m), 2.9-3.1 (1H, m), 3.2-3.3 (2H, m) , 3.26 (1H, d, J= 2 Hz), 3.39 (2H, d, J= 4 Hz), 3.49 (1H, d, J= 2 Hz), 4.1-4.2 (1H, m), 6.46 (1H, d, J= 9 Hz), 7.7-8.5 (1H, broad s). [Example 3] Preparation 2 of salt with organic amine (C) (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylic acid (6.59 g) was dis¬solved in diethyl ether (67 mL), and benzathine (2.75 g) dissolved in diethyl ether (18 mL) was added while the solution was stirred at room temperature. Ethanol (6 mL) was then added to dissolve the separated oily product, and diethyl ether (84 mL) was further added. While the solution was cooled with ice, it was stirred overnight. The precipitated crystalline product was collected by filtration, washed with diethyl ether, and dried at room temperature under reduced pressure to obtain white pow¬dery benzathine salt of (2S,3S)-3-[[(1S)-1-isobutoxy-methyl-3-methylbutyl] carbamoyl] oxirane-2-carboxylic acid (7.43 g, 79.6%) . The obtained product (2 g) was rec2rys-tallized from ethanol/water, to prepare the benzathine salt (1.8 g, 90.0%) in crystalline flakes. Melting point: 63°C; IR (KBr) cm-^ : 3400, 3260, 2950, 2850, 1650, 1450, ■ 1380, 1295, 1240, 1110, 890, 740; and NMR (CDCI3) 6: 0.9-1.0 (12H, m) , 1.3-1.5 (2H, m) , 1.5-1.6 (1H, m) , 1.8-1.9 (1H, m) , 3.1-3.2 (4H, m), 3.9-4.0 (2H, m), 4.1-4.2 (1H, m), 6.36 (1H, d, J= 9 Hz), 7.39 (5H, s). [Example 4] Preparation 3 of salt with organic amine (C) (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylic acid (1.31 g) was dis¬solved in acetone (6 mL) , and meglumine (0.85 g) dis¬solved in water (1.5 mL) was added while the solution was stirred at room temperature. Acetone (14 mL) was further added, and then the solution was stirred overnight at room temperature. The precipitated crystalline product was collected by filtration, washed with acetone, dried in air and further dried under reduced pressure at room temperature to obtain meglumine salt of (2S,3S)-3-[ [ (1S)-l-isobutoxymethyl-3-methylbutyl] carlDamoyl] oxirane-2-car-boxylic acid (1.28 g, 61.0%) in white crystalline plates. Melting point: 96°C - 98°C; IR (KBr) cm-^ : 3300, 2950, 1660, 1620, 1590, 1460, NMR (D2O) 6: 0.7-0.9 (12H, m), 1.2-1.4 (2H, m), 1.5-1.6 (1H, m) , 1.7-1.8 (1H, tn) , 2.69 (3H, s) , 3.1-3.6 (15H, m), 3.7-3.8 (3H, m) , 4.0-4.1 (2H, m) . [Example 5] Preparation 4 of salt with organic amine (C) (2S,3S)-3- [ [ (1S)-l-isobutoxymethyl-3-methylbutyl] -carbamoyl]oxirane-2-carboxylie acid (660 mg) was dis¬solved in ethanol (3 mL), and DL-lysine (336 mg) dis¬solved in water (1.0 mL) was added while the solution was stirred at room temperature. The solution was condensed under reduced pressure, and ethanol (10 mL) was added. The obtained solution was stirred overnight at room tem¬perature. The precipitated crystalline product was col¬lected by filtration, washed with ethanol, dried in air and further dried under reduced pressure at room terrpera-ture to obtain white powdery DL-lysine salt of (2S,3S)-3- [ [ (1S) -l-isobutoxynethyl-3-methylbutyl] carbamoyl] oxirane-2-carboxylic acid (783 mg, 78.6%). IR (KBr) cm"^ : 2950, 1640, 1460, 1380, 1100, 890; and NMR (D2O) 6: 0.7-0.9 (12H, m), 1.2-1.9 (lOH, m), 2.93 (2H, t, J= 7 Hz), 3.2-3.5 (6H, m), 3.66 (1H, t, J= 6 Hz), 4.0-4.1 (1H, m). [Example 6] Preparation 1 of free form (D) The cyclohexylamine salt of (2S,3S)-3-[[(1S)-1-iso-butoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-carboxylic acid (165.22 g) was added to a mixture of water (400 mL) and ethyl acetate (400 mL), to prepare a suspension. While the suspension was cooled with ice (at a tempera¬ture of 10°C or below) , 3 mol/L aqueous hydrochloric acid (140 mL) was dropped to adjust the pH value to approx. 3. The ethyl acetate portion was collected, and independent¬ly the aqueous portion was extracted with ethyl acetate (200 mL). The extracted liquid portion and the above- collected ethyl acetate portion were mixed, washed with water (200 mL) and with aqueous saturated sodium chloride solution (200 mL) , and dried over anhydrous sodium sul¬fate. The solvent was distilled off under reduced pres¬sure to obtain (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylic acid (122.83 g, 99.9%) as yellow oil. NMR (CDCI3) 6: 0.9-1.0 (12H, m) , 1.3-1.6 (3H, m) , 1.8-1.9 (1H, m), 3.1-3.3 (2H, m), 3.43 (2H, d, J= 4 Hz), 3.47 (1H, d, J= 2 Hz), 3.71 (1H, d, J= 2 Hz), 4.1-4.2 (1H, m), 6.50 (1H, d, J= 9 Hz), 9.60 (1H, broad s). [Example 7] Preparation 2 of free form (D) In the same manner as Example 6, (2S,3S)-3-[ [ (1S)-1-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-car-boxylic acid was prepared as yellow oil from the amine salt such as the benzathine salt, the meglumine salt or the DL-lysine salt. The NMR data were the same as those in Example 6. [Example 8] Preparation of Na salt (E) (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylic acid (48.59 g) was dis¬solved in acetone (245 mL), and sodium carbonate (8.51 g) dissolved in water (32.9 mL) was slowly added. The solu¬tion was stirred at room temperature to 50°C for three hours until the deposited inorganic salt was dissolved. While the temperature was kept, acetone (200 mL) was dropped and a seed crystal was placed in the solution, and then the solution was stirred for 30 minutes. Ace¬tone (510 mL) was again dropped, and the solution was stirred for 1 hour while the temperature was kept. While the solution was gradually cooled to room temperature, it was stirred overnight. The precipitated crystalline product was collected by filtration, washed with a mix¬ture of 3% water-acetone (70 mL), dried in air to obtain white solid sodium (2S,3S)-3-[ [(1S)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate (46.1 g, 87.9%). The NMR data of the solid product were the same as those in Example 9. [Example 9] Preparations of recrystallized product (F) and immediately dried Na crystallites (G) The sodium (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-nethylbutyl]carbamoyl]oxirane-2-carboxylate (25.18 g) prepared in Example 8 was dissolved in methanol (85 mL) at 50°C, and ethyl acetate (100 mL) was dropped while the temperature was kept. While the temperature was still rcept, a seed crystal was placed in the solution, the solution was stirred for 1 hour, and ethyl acetate (150 nL) was again dropped. While the temperature was still kept, ethyl acetate (100 mL) was further dropped and furthermore ethyl acetate (75 mL) was dropped, and then ;he solution was stirred for 1 hour. While gradually ::ooled to room temperature, the solution was stirred Dver-night. The precipitated crystalline product was ::ollected by filtration, washed with three portions of a nixture of ethyl acetate/methanol (5/1, 40 mL, 25 mL, 25 nL) in a stream of nitrogen gas. Before completely dried Ln air, the crystalline product was dried under reduced pressure at 40°C overnight to obtain sodium (2S,3S)-3-[ [ (1S) -l-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-carboxylate (21.1 g, 83.9%) in white crystalline nee-iles. Melting point: 170°C - 175°C (dec); IR (KBr) cm-^ : 3255, 2950, 2860, 1670, 1630, 1550, L460, 1435, 1395, 1365, 1310, 1260, 1110, 890; and NMR (D2O) 6: 0.7-0.9 (12H, m) , 1.2-1.4 (2H, m), 1.5-L.6 (1H, m), 1.7-1.8 (1H, m) , 3.1-3.3 (2H, m), 3.3-3.5 (4H, m), 4.0-4.1 (1H, m). [Example 10] Preparation of K salt (E) The (2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methyl-butyl] carbamoyl] oxirane-2-carboxylic acid (4.83 g) pre¬pared in Example 6 or 1 was dissolved in acetone (23 mL), and potassium carbonate (1.11 g) dissolved in water (4.5 raL) was slowly added. The solution was stirred at room temperature until the deposited inorganic salt was dis¬solved. While the solution was heated at 50°C in a batli, acetone (100 mL) was dropped and a seed crystal was placed in the solution, and then the solution was stirred for 30 minutes. Acetone (94 mL) was again dropped, and the solution was stirred for 1 hour while the tenperature was kept. While gradually cooled to room temperature, the solution was stirred overnight. The precipitated crystalline product was collected by filtration, washed with a mixture of 2% water-acetone (20 mL), dried in air to obtain white solid potassium (2S,3S)-3-[ [ (1S)-1-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-car-boxylate (4.28 g, 81.7%). The NMR data of the solid product were the same as those in Example 11. [Example 11] Preparations of recrystallized product (F) and immediately dried K crystallites (G) The potassium (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate (500 mg) pre¬pared in Example 10 was dissolved in metlianol (3.5 mL) to prepare a solution. While the solution was heated at 50°C in a bath, ethyl acetate (12 mL) was added and a seed crystal was placed in the solution. While the tempera¬ture was kept, the solution was stirred for 1 hour and ethyl acetate (23 mL) was again dropped. The solution was stirred for 1 hour while the temperature was still Icept. While gradually cooled to room temperature, the solution was stirred overnight. The precipitated crys¬tallites were collected by filtration, washed with a mix¬ture of ethyl acetate/methanol (10/1, 10 mL) in a stream of nitrogen gas. Before completely dried in air, the crystalline product was dried under reduced pressure at 40°C overnight to obtain potassium (2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-car-boxylate (445 mg, 89.0%) in white crystalline needles. Melting point: 177°C (dec); IR (KBr) cm-^ : 3270, 3080, 2950, 2870, 1680, 1625, 1560, 1460, 1380, 1300, 1240, 1110, 895; and NMR (D2O) 5: 0.8-0.9 (12H, m), 1.2-1.4 (2H, m), 1.5-1.6 (1H, m), 1.7-1.8 (1H, m), 3.2-3.3 (2H, m), 3.3-3.4 (4H, m), 4.0-4.1 (1H, m). [Exainple 12] Preparation of Na salt (E) (Process 2) Crude ethyl (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2 -carboxylate (15.29 g) synthesized according to the process (acid chloride meth¬od) similar to that described in Patent reference 1 was dissolved in ethanol (24 mL) . While the solution was stirred at room temperature, sodium carbonate (2.44 g) dissolved in water (24 mL) was dropped. After the solu¬tion was stirred at room temperature for 1 hour, it was further stirred at 90°C to 95°C for 2 hours. The solvent was distilled off under reduced pressure, and water (50 mL) was added to the residue. The obtained solution was washed with two portions of ethyl acetate (50 mL), and water was distilled off under reduced pressure. Acetone (400 mL) was dropped to the residue, and the obtained solution was stirred overnight at room temperature. The precipitated crystalline product was collected by filtra¬tion, washed with a mixture of 3% water-acetone (30 mL), washed again with acetone (30 mL) , and dried in air to obtain white solid sodium (2S,3S)-3-[[(1S)-1-isobutoxy-methyl-3-methylbutyl] carbamoyl] oxirane-2-carboxylate (10.55 g, 70.3%). The NMR data of the solid product were the same as those in Example 9. [Example 13] Preparation of Na salt (E) (Process 2) Crude ethyl (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]carbamoyl] oxirane-2-carboxylate (7.82 g) syn¬thesized according to the process (acid chloride method) similar to that described in Patent reference 1 was dis¬solved in ethanol (24 mL). While the solution was cooled with ice and stirred, 1 mol/L aqueous solution of sodium hydroxide (23.55 mL) was dropped. After the solution was stirred for 1.5 hours while the temperature was kept, the solvent was distilled off under reduced pressure. Water (39 mL) was added to the residue, and the obtained solu¬tion was washed with two portions of ethyl acetate (39 mL), and then water was distilled off under reduced pres¬sure. Acetone (157 mL) was added to the residue, and the solution was stirred overnight at room temperature. The precipitated crystalline product was collected by filtra¬tion, washed with a mixture of 3% water-acetone (30 mL), washed again with acetone (30 mL), and dried in air to obtain white solid sodium (2S,3S)-3-[[(1S)-1-isobutoxy-methyl-3-methylbutyl] carbamoyl] oxirane-2-carboxylate (5.36 g, 69.9%). The NMR data of the solid product were the same as those in Example 9. [Example 14] Purity test on free form (D) The products in the free form (D) obtained in Exam¬ples 6 and 7 were subjected to a purity test. In the test, each free form (D) gave no peak attributable to cleavage products (9.8 minutes) under the below-described HPLC conditions, while the peak was given by the free form or the metal salt thereof obtained by hydrolyzing the ester form (A) purified by a silica-gel column chro¬matography. (HPLC conditions) column: YMC-A302 (150x4.5 mm), mobile phase: a mixture of 0.1 mol/L sodium dihydrogen phosphate reagent (pH: 3.0)/acetonitrile (5:2), flow rate: controlled so that the retention time of (2S,3S)-3- [ [(1S)-l-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylic acid would be approx. 13 minutes, and detector: UV (210 nm) . [Exarnple 15] Comparative stability test (Procedure) As a sample, 10 mg of sodium or potassium (2S,3S)-3- [ [ (1S) -1 - i sobutoxymethyl - 3 -methylbutyl ] carbamoyl ] oxirane -2-carboxylate was weighed and placed in a test tube. After the sample was kept in a thermostat at 80°C for three days under a light-shaded and airtight condition, the amount of remaining substance was measured by HPLC. (HPLC conditions) The same as those in Example 14. (Conditions of DSC: differential scanning calorimetiry) Each of the sodium or potassium (2S,3S)-3-[ [ (1S)-1-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-car-boxylate (sample) and 0.01 g of a-alumina (standard) was placed in a container, and heated at temperatures elevat¬ing from room temperature to approx. 200°C at the heating rate of 2°C/minute. (A differential scanning calorimetric thermobalance [TASIOO, TG-DCS type, Rigaku Corporation] was used.) (Results) The results are set forth in Table 1. 1) obtained by condensation to dryness; 2) recrystallized and collected by filtration, and then left at room temperature; and 3) presumed to be more in amorphous state than in crystal state. The results in Table 1 clearly indicate that the crystalline Na salt prepared in Example 9 (in which the salt was recrystallized, collected by filtration in a stream of nitrogen gas and immediately dried) was superi¬or in storage stability to the salt obtained by condensa¬tion to dryness (according to the conventional process) or obtained through recrystallization and drying in air. Further, the results in Table 1 indicate that the crys¬talline K salt prepared in Example 11 also had excellent storage stability. WE CLAIM: 1. A process for preparation of crystalline sodium or potassium (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate, compris¬ing the following steps (i) to (vi): (i) hydrolyzing (2S,3 S)-3-[[( 1S)-1 -isobutoxymethyl-3-methyl-butyl] carbamoyl]oxirane-2-carboxylic acid ester of an aliphatic alcohol having 1 to 6 carbon atoms, to obtain (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylic acid, (ii) causing the carboxylic acid obtained in the preceding step react with an organic amine represented by the formula of: (R')(R2)(R^)N in which R' is hydrogen or a linear-chain, branched-chain or cyclic alkyl group having 1 to 10 carbon atoms; R is hydrogen or a linear-chain, branched-chain or cyclic alkyl or aralkyl group of 1 to 10 carbon atoms; and R is a linear-chain, branched-chain or cyclic alkyl group having 1 to 10 carbon atoms which may have a substituent selected from the group consisting of halogen atoms, nitro, hydroxyl, carboxyl, guanidino, amino and aralkylamino groups; or otherwise R and R can be com¬bined to form a 5- to 7-membered ring comprising the nitrogen atom to which R2 and R3 are connected, the ring possibly containing additional nitrogen atom, to prepare an organic amine salt of (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylic acid, (iii) adding an acid to the salt obtained in the preceding step, to obtain (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylic acid, (iv) causing the carboxylic acid obtained in the preceding step react with a basic sodium or potassium compound in a mixed solvent of water and an aliphatic alcohol or acetone, to obtain a sodium or potassium salt, (v) recrystallizing the sodium or potassium salt obtained in the preceding step using an aliphatic alcohol, and (vi) drying the product recrystallized in the preceding step under reduced pressure.

Full Text

SALT OF (2S,3S)-3-[[(lS)-l-ISOBUTOXYMETEIYL-3-METHYLBUTYL] CARBAMOYL] OXIRANE-2-CARBOXYLIC ACID
[FIELD OF THE INVENTION]
The present invention relates to a salt of (2S,3S)-3- [ [ (1S) -l-isobutoxymethyl-3-methylbutyl] carbamoyl] -oxirane-2-carboxylic acid.
[BACKGROUND OF THE INVENTION]
The below-illustrated sodium (2S,3S)-3-[ [ (1S)-1-iso-butoxymethyl-3-methylbutyl] carbamoyl] -oxirane-2-carboxyl-ate (hereinafter referred to as Compound A) shows a ca-thepsin-inhibitory action and is useful as a remedy for treating rheumatoid arthritis and osteoporosis (Patent reference 1: WO 99/11640 pamphlet):

Patent reference 1 describes a process for producing Compound A according to the following reaction scheme (see Example 48 of Patent reference 1):

The starting ester material of the above-mentioned reaction scheme is generally purified by column chroma-tography to obtain a purified Compound A.
[DISCLOSURE OF THE INVENTION]

Amorphous Compound A is so hygroscopic and so ther¬mally unstable that various troubles in producing the remedy are expected, and is hence difficult to treat as material for the pharmaceutical preparation. According¬ly, there is a need to provide a thermally stable crys¬tal 1 ine (2S,3S)-3-[[{lS)-l-isobutoxymethyl-3-methylbu-tyl] carbamoyl] oxirane-2-carboxylic acid salt having a long storage-life.
If crystalline Compound A is prepared according to the conventional recrystallization or salt-making process in which Compound A is converted from a free form into the desired form, the product often precipitates in the form of too fine crystallites to filter or occasionally in the form of agar-like jelly. Further, since Compound A is very hygroscopic, moisture in air causes to conver¬sion of Compound A from the crystal state into the amor¬phous state and, as a result, the filterability of the product lowers.
In the industrial preparation of Compound A, puri¬fication by column chromatography is a troiiblesome proce¬dure. Accordingly, it is also desired to provide a sim¬ple purification process for Compound A.
When Compound A is prepared in an industrial scale, it is important to purify Conpound A in the free acid form. As described hereinafter, it is very simple and industrially advantageous to prepare Compound A in the free acid form from the after-described organic amine salt. In addition, it makes the preparation process fur¬ther simple and advantageous that Compound A in an ester form is hydrolyzed with a basic sodium or potassium com¬pound to convert directly into a sodium or potassium salt.
The present invention has an object to provide a thermally stable crystalline sodium or potassium (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl] carbamoyl]-oxirane-2-carboxylate having a long storage-life. The

invention also has objects to provide a simple and industrially advantageous process for preparing crystallites of the above-mentioned compound and to provide a salt of the compound with an organic amine employable in the process.
The invention relates to crystalline sodium (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]-oxirane-2-carboxylate having the following characteristics:
DSC: an exothermic peak observed at a temperature in the range of 170 to 175°C with weight decrease; and characteristic absorption bands of infrared absorption spectrum (KBr tablet) : 3255, 2950, 2860, 1670, 1630, 1550, 1460, 1435, 1395, 1365, 1310, 1260, 1110,890 cm"'.
The invention also relates to crystalline potassium (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-carboxylate having the following characteristics:
DSC: an exothermic peak observed at 177°C with weight decrease; and characteristic absorption bands of infrared absorption spectrum (KBr tablet): 3270, 3080, 2950, 2870, 1680, 1625, 1560, 1460, 1380, 1300, 1240, 1110, 895 cm"'.
The present invention is directed to a process for preparation of crystalline
sodium or potassium (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methyl-
butyl]carbamoyl]oxirane-2-carboxylate, comprising the following steps (i) to (vi):
(i) hydrolyzing (2S,3S)-3-[f(lS)-l-isobutoxymethyl-3-methyl-butyl]
carbamoyl] oxirane-2-carboxylic acid ester of an aliphatic alcohol having 1 to 6 carbon atoms, to obtain (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylic acid,

(ii) causing the carboxylic acid obtained in the preceding step react with an organic amine represented by the formula of:
(R1)(R2)(R3)N in which R1 is hydrogen or a linear-chain, branched-chain or cyclic alkyl group having 1 to 10 carbon atoms; R is hydrogen or a linear-cham, branched-chain or cyclic alkyl or aralkyl group of 1 to 10 carbon atoms; and R is a linear-chain, branched-chain or cyclic alkyl group having 1 to 10 carbon atoms which may have a substituent selected from the group consisting of halogen atoms, nitro, hydroxyl, carboxy 1, guanidino, amino and aralkylamino groups; or otherwise R2 and R3 can be com¬bined to form a 5- to 7-membered ring comprising the nitrogen atom to which R and R are connected, the ring possibly containing additional nitrogen atom, to prepare an organic amine salt of (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylic acid, (iii) adding an acid to the salt obtained in the preceding step, to obtain (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylic acid, (iv) causing the carboxylic acid obtained in the preceding step react with a basic sodium or potassium compound in a mixed solvent of water and an aliphatic alcohol or acetone, to obtain a sodium or potassium salt, (v) recrystallizing the sodium or potassium salt obtained in the preceding
step using an aliphatic alcohol, and (vi) drying the product recrystallized in the preceding step under reduced pressure. The invention still further relates to a salt of

(2S,3S)-3- [ [ (1S)-l-isobutoxymethyl-3-methylbutyl]carbamo¬yl] oxirane-2-carboxylic acid with an organic amine.
The invention further relates to a salt of (2S,3S)-3- [ [ (1S) -l-isobutoxynethyl-3-methylbutyl] carbamoyl] -oxirane-2-carboxylic acid with an organic amine repre¬sented by the formula of:
(R1) (R2) (R3)N
in which R2 is hydrogen or a linear-chain, branched-chain or cyclic allcyl group having 1 to 10 carbon atoms; R2 is hydrogen or a linear-chain, branched-chain or cyclic alJcyl or aralkyl group of 1 to 10 carbon atoms; and R2 is a linear-cliain, branched-chain or cyclic alkyl group of 1 to 10 carbon atoms which may have a substituent group selected from the group consisting of halogen atoms, nitro, hydroxyl, carboxyl, guanidino, amino and arallcyl-amino groups; or otherwise R2 and R2 can be combined to form a 5- to 7-membered ring comprising the nitrogen atom to which R2 and R2 are connected (the ring may contain additional nitrogen atom)}.
The invention further relates to a salt of (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl] carbamoyl]-oxirane-2-carboxylic acid with an organic amine selected from the group consisting of piperazine, adamantane amines, cyclohexylamine, dicyclohexylamine, tris (hydroxy-methyl)aminomethane, arginine, lysine, benzathine and meglumine.
[MOST PREFERRED EMBODIMENTS OF THE INVENTION]
The present invention is explained below in more de¬tail.
Crystalline sodium or potassium (2S,3S)-3-[ [ (1S)-1-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-carbox-ylate of the invention can be obtained according to the

following flow chart. (Flow chart) Process 1:
ester forro (A) - free form (B) - salt with an organ¬ic amine (C) - free form (D) - Na or K salt (E) - recrys-tallized product (F) - immediately dried crystallites (G);
or otherwise Process 2:
ester form (A) - Na or K salt (E) -^ recrystallized product (F) -* immediately dried crystallites (G) .
Each step is explained below.
(Process 1)
(1) Step of ester form (A) - free form (B)
In a mixed solvent of water and an aliphatic alcohol such as methanol, ethanol or isopropyl alcohol, (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]-oxirane-2-carboxylic ester (A) is hydrolyzed in the pres¬ence of a base such as sodium hydroxide or potassium hydroxide, and is then neutralized to obtain (2S,3S)-3-
[ [ (1S)-l-isobutoxymethyl-3-methylbutyl] carbamoyl]oxirane-2-carboxylic acid [free form (B)].
In the above procedure, the starting (2S,3S)-3-
[ [ (1S) -l-isobutoxyraethyl-3-methylbutyl] carbamoyl] oxirane-2-carboxylic ester is an ester with an aliphatic alcohol, preferably an ester with a primary, secondary or tertiary alcohol having 1 to 6 carbon atoms, more preferably an ester with ethanol, methanol, n-propyl alcohol or isopro¬pyl alcohol.
For synthesizing the ester of the starting material,
(2S,3S)-3-alkoxycabonyloxirane-2-carboxylic acid and
(1S) -l-isobutoxymethyl-3-methylbutylamine are, for exam¬ple, condensed according to the normal condensation pro¬cess (e.g., DCC-HOSu method, acid chloride method, and acid anhydride method).

(2) Step of free form (B) - salt with an organic amine (C)
The free form (B) prepared in Step (1) is caused to react with an organic amine in a solvent. Examples of the solvents include aliphatic alcohols such as methanol, ethanol, n-propyl alcohol and isopropyl alcohol; aliphat¬ic ethers such as diethyl ether and diisopropyl ether; aliphatic esters such as ethyl acetate; aliphatic ketones such as acetone and ethyl methyl ketone; and water. They may be used singly or may be mixed. In consideration of flammability and toxicity, the solvent preferably is ethyl acetate, acetone, water or a mixture thereof. Thus, the salt with a desired organic amine can be ob¬tained .
The organic amine is, for example, represented by the formula of:
(R2) (R2) (R3)N
in which R2 is hydrogen or a linear-chain, branched-chain or cyclic alkyl group having 1 to 10 carbon atoms; R2 is hydrogen or a linear-chain, branched-chain or cyclic alkyl or aralkyl group of 1 to 10 carbon atoms; and R2 is a linear-chain, branched-chain or cyclic alkyl group (including a crosslinked cyclic hydrocarbon group such as adamantyl) having 1 to 10 carbon atoms which may have a substituent selected from the group consisting of halogen atoms, nitro, hydroxyl, carboxyl, guanidino, amino and aralkylamino groups; or otherwise R2 and R2 can be com¬bined to form a 5- to 7-membered ring comprising the ni¬trogen atom to which R2 and R2 are connected (the ring may contain additional nitrogen atom). In consideration of cost, toxicity and crystallization, the organic amine preferably is piperazine, an adamantane amine, cyclohex-ylamine, dicyclohexylamine, tris (hydroxymethyl)aminometh-ane, arginine (preferably in L-form), lysine (preferably

in D-form or DL-form) , benzathine or meglumine. The salts with D- or DL-lysine, benzathine, meglumine and tris(hydroxymethyl)aminomethane can be immediately used as medicaments without further processing.
(3) Step of salt with an organic amine (C) -* free form
(D)
The salt with an organic amine (C) obtained in Step (2) is treated with an acid (e.g., hydrochloric acid) in the conventional manner, to obtain (2S,3S)-3-[ [ (1S)-1-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-carbox-ylic acid (D) of high purity. Thus obtained free form (D) gave no peak attributed to cleavage products (9.8 minutes, see Example 14) while the peak is given by the free form or the metal salt thereof obtained by hydro-lyzing the ester form (A) purified by silica-gel column chromatography.
(In the case of preparing a sodium salt)
(4) Step of free form (D) -> Na salt (E)
The free form (D) is converted into a sodium salt using a sodium source in an organic solvent capable of dissolving the free form (D) or in a water-containing or¬ganic solvent. Examples of the sodium sources include sodium metal, sodium alkoxides such as sodium methoxide and sodium ethoxide, sodium hydroxide, sodium carbonate, and sodium organic carboxylate.
The solvent preferably is a mixed solvent of water and methanol, ethanol or acetone, in consideration of flammability and toxicity. If the water content is in the range of 1 to 5%, the product is obtained in a high yield.
Thus obtained solid product is well filtered so that the product is collected by conventional filtration.
(5) Step of Na salt (E) - recrystallized product (F)

The solid sodium salt (E) obtained in Step (4) is dissolved in an aliphatic alcohol such as methanol at a temperature of form room temperature to 60°C, and then an organic solvent is added to precipitate a crystalline product. The organic solvent preferably is a mixed sol¬vent of ethyl acetate, methanol and ethanol, in consider¬ation of flammability and toxicity. If the mixing ratio of ethyl acetate/alcohol is in the range of 10/1 to 5/1, the product is obtained in a high yield.
Thus recrystallized product (F) is well filtered so that the product is collected by conventional filtration.
(6) Recrystallized product (F) -* immediately dried crys¬tallites (G)
The recrystallized product (F) is collected by fil¬tration in a stream of nitrogen gas, and immediately dried to obtain, in a high yield, crystalline sodium (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]carbamo¬yl] oxirane-2-carboxylate (G) of high purity having the following characteristics:
DSC (the same as that in Example 15): exothermic peak observed in the range of 170 to 175°C with weight de¬crease / and
characteristic absorption bands of infrared absorp¬tion spectrum (KBr tablet): 3255, 2950, 2860, 1670, 1630, 1550, 1460, 1435, 1395, 1365, 1310, 1260, 1110, 890 cm"^.
The product preferably is in the form of white crys¬talline needles.
The crystalline Na salt of the invention (described in Example 9) was subjected to a comparative stability test. In the test, the Na salt was left at 80°C for three days together with both the substance obtained by con¬densing the solution to dryness and the product collected by filtration and left at room temperature after re-crystallization. As a result, it was found that the Na

salt of the invention was superior to the substances for comparison in storage stability (see Exanple 15).
(In the case of preparing a potassium salt)
(7) Step of free form (D) - K salt (E)
The free form (D) is converted into a potassium salt using a potassium source in an organic solvent capable of dissolving the free form (D) or in a water-containing or¬ganic solvent. Examples of the potassium sources include potassium metal, potassium alkoxides such as potassium methoxide and potassium ethoxide, potassium hydroxide, potassium carbonate, and potassium organic carboxylate.
The solvent preferably is a mixed solvent of water and methanol, ethanol or acetone, in consideration of flammability and toxicity. If the water content is in the range of 1 to 5%, the product is obtained in a high yield.
Thus obtained solid product is well filtered so that the product is collected by conventional filtration.
(8) Step of K salt (E) -* recrystallized product (F)
The solid potassium salt (E) obtained in Step (7) is dissolved in an aliphatic alcohol such as methanol at a temperature from room temperature to 60°C, and then an organic solvent is added to precipitate a crystalline product. The organic solvent preferably is a mixed sol¬vent of ethyl acetate, methanol and ethanol, in consider¬ation of flammability and toxicity. If the mixing ratio of ethyl acetate/alcohol is in the range of 30/1 to lO/l, the product is obtained in a high yield.
Thus recrystallized product (F) is well filtered so that the product is collected by conventional filtration.
(9) recrystallized product (F) - immediately dried crys¬
tallites (G)
The recrystallized product (F) is collected by fil-

tration in a stream of nitrogen gas, and immediately dried to obtain, in a high yield, crystalline potassium (2S,3S) -3- [ [ (1S) -l-isobutoxyT\ethyl-3-methylbutyl] carbamo¬yl] oxirane-2-carboxylate (G) of high purity having the following characteristics:
DSC (the same as that in Example 15): exothermic peak observed at 177°C with weight decrease; and
characteristic absorption bands of infrared absorp¬tion spectrum (KBr tablet): 3270, 3080, 2950, 2870, 1680, 1625, 1560, 1460, 1380, 1300, 1240, 1110, 895 cm'^
The product is preferably in the form of white crys¬talline needles.
The crystalline K salt of the invention (described in Example 11) was subjected to a stability test in which the K salt was left at 80°C for three days. As a result, it was found that the K salt of the invention was excel¬lent in storage stability (see Example 15).
(In the case of preparing a calcium or lithium salt)
Crystalline calcium or lithium (2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-carbox-ylate of high purity can be prepared in a high yield through the steps similar to the above-mentioned Steps (4) to (9) .
(Process 2)
(1) Ester form (A) - Na or K salt (E)
[Preparation of Na salt (E)]
The Na salt (E) can be obtained by causing the ester form (A) described in Step (1) of Process 1 react with a basic sodium compound (e.g., sodium carbonate, sodium hydroxide) to hydrolyze, by condensing the reaction mix¬ture, and then by adding a water-conpatible organic sol¬vent such as acetone to crystallize the salt. If the water content is in the range of 1 to 5%, the product is

obtained in a high yield.
[Preparation of K salt (E)]
The K salt (E) can be obtained by causing the ester form (A) react with a basic potassium compound (e.g., potassium carbonate, potassium hydroxide) to hydrolyze, by condensing the reaction mixture, and then by adding a water-compatible organic solvent such as acetone to crys¬tallize the salt. If the water content is in the range of 1 to 5%, the product is obtained in a high yield.
(2) Na or K salt (E) - recrystallized product (F) -* imme¬diately dried crystallites (G)
The steps of Na salt (E) recrystallized product (F) -* immediately dried cirystallites (G) can be the same as Steps (5) and (6) of Process 1 described above.
The steps of K salt (E) recrystallized product (F) immediately dried crystallites (G) can be the same as Steps (8) and (9) of Process 1 described above.
As described above, the purification process in which (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylic acid is converted to a salt with an organic amine, as well as the process in which the ester form of (2S,3S)-3-[[(1S)-1-isobutoxy-methyl-3-methylbutyl] carbamoyl] oxirane-2-carboxylic acid is hydrolyzed with a basic sodium or potassium cotrpound to prepare directly a sodium or potassium (2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate, does not need to involve the step for purifying the ester form by silica-gel column chromatog¬raphy. They are, therefore, simple and industrially advantageous processes for purifying (2S,3S)-3-[ [ (1S)-1-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-car¬boxylic acid.
The crystalline sodium or potassium (2S,3S)-3-

[ [ (1S) -l-isobutoxytnethyl-3-methylbutyl] carbamoyl] oxirane-2-carboxylate of the invention has a long storage life and excellent thermal stability, and accordingly is use¬ful as a material for pharmaceutical preparation.
Form thus obtained crystalline sodium or potassium
(2S,3S)-3- [ [ (1S)-l-isobutoxymethyl-3-methylbutyl]carbamo¬yl] oxirane-2-carboxylate, medicaments can be prepared.
For preparing the medicaments, the salt can be pro¬cessed according to the conventional manner to give, for example, tablets, granules, powder, capsules, suspension, injection or suppository. In the pharmaceutical prepara¬tion, normal additives such as excipient, disintegrator, binder, lubricant, dye and diluent are used.
[EXAMPLES]
The invention is further described by the following examples. They by no means restrict the invention.
[Exanple 1] Preparation of free form (B)
Crude ethyl (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate (164.54 g) synthesized according to the process described in Patent reference 1 was dissolved in ethanol (520 mL). While the solution was cooled with ice (at a temperature of 10°C or below), 1 mol/L aqueous sodium hydroxide solution (522 mL) was dropped. After the solution was stirred for 1.5 hours while the tenperature was kept, ethanol was removed under reduced pressure. Water (340 mL) was added, and then the mixture was filtered through Celite to remove insoli±)le materials. The mixture was washed twice with ethyl acetate (300 mL). While the aqueous portion was cooled with ice, 6 mol/L aqueous hydrochloric acid (110 mL) was dropped to adjust the pH value to 1 and then the mixture was extracted twice with ethyl acetate (300 mL). The ethyl acetate portion was washed with water (300 mL) and with aqueous saturated sodium chloride solution (200

mL) , and then dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to ob¬tain (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylic acid (139.61 g, 93.1%) as orange oil. The NMR data were the same as those in Exam¬ple 6.
[Example 2] Preparation 1 of salt with organic amine (C)
(2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylic acid (139.61 g) was dis¬solved in ethyl acetate (630 mL). The solution was cooled with ice (at a temperature of 10°C or below) and cyclohexylamine (48.19 g) dissolved in ethyl acetate (190 mL) was added. The resultant mixture was stirred over¬night at room temperature. The precipitated crystalline product was collected by filtration, washed with ethyl acetate, and dried in air to obtain white powdery cyclo¬hexylamine salt of (2S,3S)-3- [ [(1S)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylic acid (165.22 g, 88.0%). The obtained product (1.0 g) was recrystal-lized from ethanol/water-containing ethyl acetate, to prepare crystalline flakes (925 mg, 92.5%).
Melting point: 128°C - 129°C;
IR (KBr) cm-1 : 3310, 2950, 2850, 1660, 1620, 1570, 1540, 1460, 1445, 1380, 1360, 1340, 1295, 1250, 1220, 1120, 945, 890; and
NMR (CDCI3) 6: 0.9-1.0 (12H, m) , 1.2-1.8 (9H, m) , 1.8-1.9 (3H, m), 2.0-2.1 (2H, m), 2.9-3.1 (1H, m), 3.2-3.3 (2H, m) , 3.26 (1H, d, J= 2 Hz), 3.39 (2H, d, J= 4 Hz), 3.49 (1H, d, J= 2 Hz), 4.1-4.2 (1H, m), 6.46 (1H, d, J= 9 Hz), 7.7-8.5 (1H, broad s).
[Example 3] Preparation 2 of salt with organic amine (C)
(2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylic acid (6.59 g) was dis¬solved in diethyl ether (67 mL), and benzathine (2.75 g)

dissolved in diethyl ether (18 mL) was added while the solution was stirred at room temperature. Ethanol (6 mL) was then added to dissolve the separated oily product, and diethyl ether (84 mL) was further added. While the solution was cooled with ice, it was stirred overnight. The precipitated crystalline product was collected by filtration, washed with diethyl ether, and dried at room temperature under reduced pressure to obtain white pow¬dery benzathine salt of (2S,3S)-3-[[(1S)-1-isobutoxy-methyl-3-methylbutyl] carbamoyl] oxirane-2-carboxylic acid (7.43 g, 79.6%) . The obtained product (2 g) was rec2rys-tallized from ethanol/water, to prepare the benzathine salt (1.8 g, 90.0%) in crystalline flakes.
Melting point: 63°C;
IR (KBr) cm-^ : 3400, 3260, 2950, 2850, 1650, 1450, ■ 1380, 1295, 1240, 1110, 890, 740; and
NMR (CDCI3) 6: 0.9-1.0 (12H, m) , 1.3-1.5 (2H, m) , 1.5-1.6 (1H, m) , 1.8-1.9 (1H, m) , 3.1-3.2 (4H, m), 3.9-4.0 (2H, m), 4.1-4.2 (1H, m), 6.36 (1H, d, J= 9 Hz), 7.39 (5H, s).
[Example 4] Preparation 3 of salt with organic amine (C)
(2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylic acid (1.31 g) was dis¬solved in acetone (6 mL) , and meglumine (0.85 g) dis¬solved in water (1.5 mL) was added while the solution was stirred at room temperature. Acetone (14 mL) was further added, and then the solution was stirred overnight at room temperature. The precipitated crystalline product was collected by filtration, washed with acetone, dried in air and further dried under reduced pressure at room temperature to obtain meglumine salt of (2S,3S)-3-[ [ (1S)-l-isobutoxymethyl-3-methylbutyl] carlDamoyl] oxirane-2-car-boxylic acid (1.28 g, 61.0%) in white crystalline plates. Melting point: 96°C - 98°C; IR (KBr) cm-^ : 3300, 2950, 1660, 1620, 1590, 1460,

NMR (D2O) 6: 0.7-0.9 (12H, m), 1.2-1.4 (2H, m), 1.5-1.6 (1H, m) , 1.7-1.8 (1H, tn) , 2.69 (3H, s) , 3.1-3.6 (15H, m), 3.7-3.8 (3H, m) , 4.0-4.1 (2H, m) .
[Example 5] Preparation 4 of salt with organic amine (C)
(2S,3S)-3- [ [ (1S)-l-isobutoxymethyl-3-methylbutyl] -carbamoyl]oxirane-2-carboxylie acid (660 mg) was dis¬solved in ethanol (3 mL), and DL-lysine (336 mg) dis¬solved in water (1.0 mL) was added while the solution was stirred at room temperature. The solution was condensed under reduced pressure, and ethanol (10 mL) was added. The obtained solution was stirred overnight at room tem¬perature. The precipitated crystalline product was col¬lected by filtration, washed with ethanol, dried in air and further dried under reduced pressure at room terrpera-ture to obtain white powdery DL-lysine salt of (2S,3S)-3-
[ [ (1S) -l-isobutoxynethyl-3-methylbutyl] carbamoyl] oxirane-2-carboxylic acid (783 mg, 78.6%).
IR (KBr) cm"^ : 2950, 1640, 1460, 1380, 1100, 890; and
NMR (D2O) 6: 0.7-0.9 (12H, m), 1.2-1.9 (lOH, m), 2.93
(2H, t, J= 7 Hz), 3.2-3.5 (6H, m), 3.66 (1H, t, J= 6 Hz), 4.0-4.1 (1H, m).
[Example 6] Preparation 1 of free form (D)
The cyclohexylamine salt of (2S,3S)-3-[[(1S)-1-iso-butoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-carboxylic acid (165.22 g) was added to a mixture of water (400 mL) and ethyl acetate (400 mL), to prepare a suspension. While the suspension was cooled with ice (at a tempera¬ture of 10°C or below) , 3 mol/L aqueous hydrochloric acid
(140 mL) was dropped to adjust the pH value to approx. 3. The ethyl acetate portion was collected, and independent¬ly the aqueous portion was extracted with ethyl acetate
(200 mL). The extracted liquid portion and the above-

collected ethyl acetate portion were mixed, washed with water (200 mL) and with aqueous saturated sodium chloride solution (200 mL) , and dried over anhydrous sodium sul¬fate. The solvent was distilled off under reduced pres¬sure to obtain (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylic acid (122.83 g, 99.9%) as yellow oil.
NMR (CDCI3) 6: 0.9-1.0 (12H, m) , 1.3-1.6 (3H, m) , 1.8-1.9 (1H, m), 3.1-3.3 (2H, m), 3.43 (2H, d, J= 4 Hz), 3.47 (1H, d, J= 2 Hz), 3.71 (1H, d, J= 2 Hz), 4.1-4.2 (1H, m), 6.50 (1H, d, J= 9 Hz), 9.60 (1H, broad s).
[Example 7] Preparation 2 of free form (D)
In the same manner as Example 6, (2S,3S)-3-[ [ (1S)-1-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-car-boxylic acid was prepared as yellow oil from the amine salt such as the benzathine salt, the meglumine salt or the DL-lysine salt. The NMR data were the same as those in Example 6.
[Example 8] Preparation of Na salt (E)
(2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylic acid (48.59 g) was dis¬solved in acetone (245 mL), and sodium carbonate (8.51 g) dissolved in water (32.9 mL) was slowly added. The solu¬tion was stirred at room temperature to 50°C for three hours until the deposited inorganic salt was dissolved. While the temperature was kept, acetone (200 mL) was dropped and a seed crystal was placed in the solution, and then the solution was stirred for 30 minutes. Ace¬tone (510 mL) was again dropped, and the solution was stirred for 1 hour while the temperature was kept. While the solution was gradually cooled to room temperature, it was stirred overnight. The precipitated crystalline product was collected by filtration, washed with a mix¬ture of 3% water-acetone (70 mL), dried in air to obtain

white solid sodium (2S,3S)-3-[ [(1S)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate (46.1 g, 87.9%). The NMR data of the solid product were the same as those in Example 9.
[Example 9] Preparations of recrystallized product (F) and immediately dried Na crystallites (G)
The sodium (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-nethylbutyl]carbamoyl]oxirane-2-carboxylate (25.18 g) prepared in Example 8 was dissolved in methanol (85 mL) at 50°C, and ethyl acetate (100 mL) was dropped while the temperature was kept. While the temperature was still rcept, a seed crystal was placed in the solution, the solution was stirred for 1 hour, and ethyl acetate (150 nL) was again dropped. While the temperature was still kept, ethyl acetate (100 mL) was further dropped and furthermore ethyl acetate (75 mL) was dropped, and then ;he solution was stirred for 1 hour. While gradually ::ooled to room temperature, the solution was stirred Dver-night. The precipitated crystalline product was ::ollected by filtration, washed with three portions of a nixture of ethyl acetate/methanol (5/1, 40 mL, 25 mL, 25 nL) in a stream of nitrogen gas. Before completely dried Ln air, the crystalline product was dried under reduced pressure at 40°C overnight to obtain sodium (2S,3S)-3-[ [ (1S) -l-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-carboxylate (21.1 g, 83.9%) in white crystalline nee-iles.
Melting point: 170°C - 175°C (dec);
IR (KBr) cm-^ : 3255, 2950, 2860, 1670, 1630, 1550, L460, 1435, 1395, 1365, 1310, 1260, 1110, 890; and
NMR (D2O) 6: 0.7-0.9 (12H, m) , 1.2-1.4 (2H, m), 1.5-L.6 (1H, m), 1.7-1.8 (1H, m) , 3.1-3.3 (2H, m), 3.3-3.5 (4H, m), 4.0-4.1 (1H, m).
[Example 10] Preparation of K salt (E)

The (2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methyl-butyl] carbamoyl] oxirane-2-carboxylic acid (4.83 g) pre¬pared in Example 6 or 1 was dissolved in acetone (23 mL), and potassium carbonate (1.11 g) dissolved in water (4.5 raL) was slowly added. The solution was stirred at room temperature until the deposited inorganic salt was dis¬solved. While the solution was heated at 50°C in a batli, acetone (100 mL) was dropped and a seed crystal was placed in the solution, and then the solution was stirred for 30 minutes. Acetone (94 mL) was again dropped, and the solution was stirred for 1 hour while the tenperature was kept. While gradually cooled to room temperature, the solution was stirred overnight. The precipitated crystalline product was collected by filtration, washed with a mixture of 2% water-acetone (20 mL), dried in air to obtain white solid potassium (2S,3S)-3-[ [ (1S)-1-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-car-boxylate (4.28 g, 81.7%). The NMR data of the solid product were the same as those in Example 11.
[Example 11] Preparations of recrystallized product (F) and immediately dried K crystallites (G)
The potassium (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate (500 mg) pre¬pared in Example 10 was dissolved in metlianol (3.5 mL) to prepare a solution. While the solution was heated at 50°C in a bath, ethyl acetate (12 mL) was added and a seed crystal was placed in the solution. While the tempera¬ture was kept, the solution was stirred for 1 hour and ethyl acetate (23 mL) was again dropped. The solution was stirred for 1 hour while the temperature was still Icept. While gradually cooled to room temperature, the solution was stirred overnight. The precipitated crys¬tallites were collected by filtration, washed with a mix¬ture of ethyl acetate/methanol (10/1, 10 mL) in a stream of nitrogen gas. Before completely dried in air, the

crystalline product was dried under reduced pressure at 40°C overnight to obtain potassium (2S,3S)-3-[[(1S)-1-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-car-boxylate (445 mg, 89.0%) in white crystalline needles.
Melting point: 177°C (dec);
IR (KBr) cm-^ : 3270, 3080, 2950, 2870, 1680, 1625, 1560, 1460, 1380, 1300, 1240, 1110, 895; and
NMR (D2O) 5: 0.8-0.9 (12H, m), 1.2-1.4 (2H, m), 1.5-1.6 (1H, m), 1.7-1.8 (1H, m), 3.2-3.3 (2H, m), 3.3-3.4 (4H, m), 4.0-4.1 (1H, m).
[Exainple 12] Preparation of Na salt (E) (Process 2)
Crude ethyl (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2 -carboxylate (15.29 g) synthesized according to the process (acid chloride meth¬od) similar to that described in Patent reference 1 was dissolved in ethanol (24 mL) . While the solution was stirred at room temperature, sodium carbonate (2.44 g) dissolved in water (24 mL) was dropped. After the solu¬tion was stirred at room temperature for 1 hour, it was further stirred at 90°C to 95°C for 2 hours. The solvent was distilled off under reduced pressure, and water (50 mL) was added to the residue. The obtained solution was washed with two portions of ethyl acetate (50 mL), and water was distilled off under reduced pressure. Acetone
(400 mL) was dropped to the residue, and the obtained solution was stirred overnight at room temperature. The precipitated crystalline product was collected by filtra¬tion, washed with a mixture of 3% water-acetone (30 mL), washed again with acetone (30 mL) , and dried in air to obtain white solid sodium (2S,3S)-3-[[(1S)-1-isobutoxy-methyl-3-methylbutyl] carbamoyl] oxirane-2-carboxylate
(10.55 g, 70.3%). The NMR data of the solid product were the same as those in Example 9.
[Example 13] Preparation of Na salt (E) (Process 2)

Crude ethyl (2S,3S)-3-[[(1S)-l-isobutoxymethyl-3-methylbutyl]carbamoyl] oxirane-2-carboxylate (7.82 g) syn¬thesized according to the process (acid chloride method) similar to that described in Patent reference 1 was dis¬solved in ethanol (24 mL). While the solution was cooled with ice and stirred, 1 mol/L aqueous solution of sodium hydroxide (23.55 mL) was dropped. After the solution was stirred for 1.5 hours while the temperature was kept, the solvent was distilled off under reduced pressure. Water
(39 mL) was added to the residue, and the obtained solu¬tion was washed with two portions of ethyl acetate (39 mL), and then water was distilled off under reduced pres¬sure. Acetone (157 mL) was added to the residue, and the solution was stirred overnight at room temperature. The precipitated crystalline product was collected by filtra¬tion, washed with a mixture of 3% water-acetone (30 mL), washed again with acetone (30 mL), and dried in air to obtain white solid sodium (2S,3S)-3-[[(1S)-1-isobutoxy-methyl-3-methylbutyl] carbamoyl] oxirane-2-carboxylate
(5.36 g, 69.9%). The NMR data of the solid product were the same as those in Example 9.
[Example 14] Purity test on free form (D)
The products in the free form (D) obtained in Exam¬ples 6 and 7 were subjected to a purity test. In the test, each free form (D) gave no peak attributable to cleavage products (9.8 minutes) under the below-described HPLC conditions, while the peak was given by the free form or the metal salt thereof obtained by hydrolyzing the ester form (A) purified by a silica-gel column chro¬matography.
(HPLC conditions)
column: YMC-A302 (150x4.5 mm),
mobile phase: a mixture of 0.1 mol/L sodium
dihydrogen phosphate reagent (pH: 3.0)/acetonitrile

(5:2),
flow rate: controlled so that the retention time of (2S,3S)-3- [ [(1S)-l-isobutoxymethyl-3-methylbutyl]-carbamoyl]oxirane-2-carboxylic acid would be approx. 13 minutes, and
detector: UV (210 nm) .
[Exarnple 15] Comparative stability test
(Procedure)
As a sample, 10 mg of sodium or potassium (2S,3S)-3-
[ [ (1S) -1 - i sobutoxymethyl - 3 -methylbutyl ] carbamoyl ] oxirane -2-carboxylate was weighed and placed in a test tube. After the sample was kept in a thermostat at 80°C for three days under a light-shaded and airtight condition, the amount of remaining substance was measured by HPLC.
(HPLC conditions)
The same as those in Example 14.
(Conditions of DSC: differential scanning calorimetiry)
Each of the sodium or potassium (2S,3S)-3-[ [ (1S)-1-isobutoxymethyl-3-methylbutyl] carbamoyl] oxirane-2-car-boxylate (sample) and 0.01 g of a-alumina (standard) was placed in a container, and heated at temperatures elevat¬ing from room temperature to approx. 200°C at the heating rate of 2°C/minute. (A differential scanning calorimetric thermobalance [TASIOO, TG-DCS type, Rigaku Corporation] was used.)
(Results)
The results are set forth in Table 1.

1) obtained by condensation to dryness;
2) recrystallized and collected by filtration, and then left at room temperature; and
3) presumed to be more in amorphous state than in crystal state.
The results in Table 1 clearly indicate that the crystalline Na salt prepared in Example 9 (in which the salt was recrystallized, collected by filtration in a stream of nitrogen gas and immediately dried) was superi¬or in storage stability to the salt obtained by condensa¬tion to dryness (according to the conventional process) or obtained through recrystallization and drying in air. Further, the results in Table 1 indicate that the crys¬talline K salt prepared in Example 11 also had excellent storage stability.

WE CLAIM:
1. A process for preparation of crystalline sodium or potassium (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylate, compris¬ing the following steps (i) to (vi):
(i) hydrolyzing (2S,3 S)-3-[[( 1S)-1 -isobutoxymethyl-3-methyl-butyl]
carbamoyl]oxirane-2-carboxylic acid ester of an aliphatic alcohol having 1 to 6 carbon atoms, to obtain (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylic acid, (ii) causing the carboxylic acid obtained in the preceding step react with an organic amine represented by the formula of:
(R')(R2)(R^)N in which R' is hydrogen or a linear-chain, branched-chain or cyclic alkyl group having 1 to 10 carbon atoms; R is hydrogen or a linear-chain, branched-chain or cyclic alkyl or aralkyl group of 1 to 10 carbon atoms; and R is a linear-chain, branched-chain or cyclic alkyl group having 1 to 10 carbon atoms which may have a substituent selected from the group consisting of halogen atoms, nitro, hydroxyl, carboxyl, guanidino, amino and aralkylamino groups; or otherwise R and R can be com¬bined to form a 5- to 7-membered ring comprising the nitrogen atom to which R2 and R3 are connected, the ring possibly containing additional nitrogen atom, to prepare an organic amine salt of (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylic acid, (iii) adding an acid to the salt obtained in the preceding step, to obtain (2S,3S)-3-[[(lS)-l-isobutoxymethyl-3-methylbutyl]carbamoyl]oxirane-2-carboxylic acid,

(iv) causing the carboxylic acid obtained in the preceding step react with a basic sodium or potassium compound in a mixed solvent of water and an aliphatic alcohol or acetone, to obtain a sodium or potassium salt,
(v) recrystallizing the sodium or potassium salt obtained in the preceding step using an aliphatic alcohol, and
(vi) drying the product recrystallized in the preceding step under reduced pressure.

Documents:

2749-chenp-2005 abstract duplicate.pdf

2749-chenp-2005 abstract.pdf

2749-chenp-2005 claims duplicate.pdf

2749-chenp-2005 claims.pdf

2749-chenp-2005 correspondence-others.pdf

2749-chenp-2005 correspondence-po.pdf

2749-chenp-2005 descripition completed duplicate.pdf

2749-chenp-2005 description(complete).pdf

2749-chenp-2005 form-1.pdf

2749-chenp-2005 form-18.pdf

2749-chenp-2005 form-26.pdf

2749-chenp-2005 form-3.pdf

2749-chenp-2005 form-5.pdf

2749-chenp-2005 pct search report.pdf

2749-chenp-2005 pct.pdf

2749-chenp-2005 petition.pdf

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Patent Number

229436

Indian Patent Application Number

2749/CHENP/2005

PG Journal Number

12/2009

Publication Date

20-Mar-2009

Grant Date

17-Feb-2009

Date of Filing

24-Oct-2005

Name of Patentee

NIPPON CHEMIPHAR CO., LTD.

Applicant Address

2-3, Iwamoto-cho 2-chome, Chiyoda-ku, Tokyo 101-8678, l

Inventors:

#	Inventor's Name	Inventor's Address
1	TENDO, Atsushi	299-1, Hibori, Kasukabe-shi, Saitama 344-0005,
2	TAKAHASHI, Toshihiro	4-1-6-505, Hikonari, Misato-shi, Saitama 341-0003,
3	YAMAKAWA, Tomio	2-23-2, Midoridai, Kashiwa-shi, Chiba 277-0884,
4	OKAI, Kazuki	1-6-62, Minami-koshigaya, Koshigaya-shi, Saitama 343-0845,
5	NIHASHI, Susumu	1778-66, Kamishizu, Sakura-shi, Chiba 285-0846,

PCT International Classification Number

C07D303/48

PCT International Application Number

PCT/JP04/05767

PCT International Filing date

2004-04-22

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	2003-121103	2003-04-25	Japan