NEW PROCESSES FOR THE PRODUCTION OF DI-ALKYL ESTERS AND OPTICALLY ACTIVE MONO-ALKYL ESTERS OF 3-AMINOGLUTARIC ACID

Abstract: A new process is provided for the production of a di-alkyl 3-aminoglutarate which are found useful as intermediates for the synthesis of β-lactam antibiotics of carbapenem type such as thienamycin. The new process comprises reacting an ammonium salt or ammonia with a di-alkyl ester of 3-­ ketoglutaric acid to form unsaturated amine compounds and reducing the latter with an alkali metal cyanoborohydride. The N-protected derivative of di-alkyl 3-aminoglutarate pre­ pared may be converted into the optically active β-(S)-mono­ alkyl half ester by treatment with esterase to hydrolyze only one of the esters (pro-R ester), followed by conventional N-deprotection. Further, di-alkyl 3-aminoglutarate itself may immediately be converted into the optically active β-(R)-­ mono-alkyl half ester of 3-aminoglutaric acid by treatment with esterase to hydrolyze preferentially one ester-forming alkyl group (pro-S ester).

Description: BACKGROUND OF THE INVENTION Field of the Invention This invention relates to a new process for the production of a di-alkyl ester of 3-aminoglutaric acid which is known compound found useful as intermediates for the synthesis of ss-lactam antibiotics of carbapenem type. This invention further relates to processes for the production of optically active (S)-mono-alkyl ester and (R)-nonoalkyl ester of 3-aminoglutaric acid starting from the optically inactive di-alkyl ester of 3-aminoglutarnc acid by treatment with esterase for preferential, hydrolytic removal of one ester-forming alkyl group. Description of the Prior Art 3-Aminoglutaric acid is one of known ss-amino acids and is a d-basic amino acid which is important as the intermediate for the preparation of variety of physio- logically active substances. Such physiologically inactive substances include negamycin and a series of carbapenem ss-lactam type antibiotics, typically thienamycin, which are known to be highly actIve against Gram-negative bacteria. These physiologically active substances are knows as antibiotics having hi antibacterial activity against various bacteria and have been produced in extremely low yield principally by fermentation. In this situation, there is' a great demand for a highly efficient process of producing these physiologically active substances synthetically. Besides, 3-aninoglutaric acid is useful as a nutrient source in the culture medium for cultivation of the negamycin-producing microorganism, for example, Streptomyces purpeofucus H890-C2 (FERt4-P 306) (see U.S. patent No. 3,679,742). In the past, for the known method of producing a di-alkyl 3-aminoglutarate, there has been proposed the method comprising adding ammonia to the condensation product of a malonic acid ester and chloroform to produce a "Michael" type adduct (see H. Feuer and W.A. Swarts; Journal of the American Chemical Society, 77, 5427 (1955)). However, this method is comprising multiple steps and cannot be an efficient and economic one. Accordingly, we have extensively investigated in an attempt to provide a ne, easy and economic process of producing 3-aminoglutaric acid which is important as the starting material for preparation of various physiologically active substances, and now we have succeeded to develop a new process for the production of a di-alkyl ester of 3-aminoglutaric acid. On the other hand, it is well known that only one enatiomer having desired absolute configuration is physio- logically active, but the antipode is inactive. As representative examples thereof, there may be mentioned penicillins, cephalosporins and thienamycln. For instance, thienamycin is a compound containing three asymmetric carbons and represented by the following absolute structure: : EMI3.1 Thienamycin may be synthesized via (S)-4-alkoxycarbonyl- methylazetidine-2-on of the formula EMI3.2 wherein R1 denotes an alkyl group, which is prepared by multiple steps starting from a di-alkyl ester of Laspartic acid of the formula EMI3.3 (see Japanese patent application pre-publlcation "Kokai" No. 27169/80 and U.S. patent application SN. 933323 filed August 14, 1978) We have expected that if an o?tically active ss-(S)- mono-alkyl (half)ester of 3-aminoglutaric acid is produced synthetically, it will be useful as an intermediate for the production of the above-: :nentioned azetldlnone derivative having (S)-configuration which is, in turn, essential as a starting material for the synthesis of thienamycin and hence generally as a starting material for the synthesis of carbapenem antibiotics. With this expectation, we have proceeded our researches to find out that when an optically inactive B-bezyloxycarbonylaminoglutaric acid di-alkyl ester which may be prepared synthetically from the aforesaid 3-aminoglutaric acid di-alkyl ester is treated with an ester-hydrolyzlng enzyme (for example, an esterase), one ester-forming alkyl group can be removed preferentially by hydrolysis without hydrolitic removal of the other esterforming alkyl group, whereby an optically active monoalkyl ester (half ester) is yielded. When this optically active mono-ester is subjected to a conventional N-deprotecting method for removal of the amino-protecting benzyloxycarbonyl group, there is affored an optically active ss-(S)-aminoglutaric acid mono-alkyl ester. We have further found that when the ooptically inactive 3-aminoglutaric acid di-alkyl ester is immediately treated with the ester-hydrolyzing enzyme (for example, an esterase), one ester-forming alkyl group is removed preferentially by the hydrolysis without cleavage of the other ester-forming alkyl group, affording an optically active ss-(R)-aminoglutaric acid mono-alkyl ester. An object of this invention is to provide 2 new and efficient process for the production of prochiral 3-amino glutaric acid di-alkyl ester. Another object of this invention is to provide processes for the production of optically active ss-(S)-aminoglutaric acid mono-alkyl ester and optically active ss-(R)-aminoglutaric acid mono-alkyl ester starting from the prochiral 3-aminoglutaric acid di-alkyl ester. Further objects of this invention will be clear from the following descriptions. DETAILED DESCRIPTION OF THE INVENTION According to a first aspect of this invent ion, therefore, there is provided a process for the production of a 3-aminoglutaric acid di-alkyl ester of the formula EMI5.1 wherein R is an alkyl group of 1-6 carbon atoms, which comprises the steps of: :reacting a 3-ketoglutaric acid di-alkyl ester of the formula EMI5.2 wherein R is an defIned above, with an ammonium salt or ammonia in an organic solvent at a temperature of 0 C to 1300C to form an unsaturated amine compound of the formula EMI5.3 or of the formula EMI6.1 wherein R is as defined above, and then reducing this unsaturated amine compound with an alkali metal cyanoborohydride in an organic solvent at a temperature of 0 C to 1000C. In the process of the first aspect of this Invent ion, the 3-ketoglutarlc acid di-alkyl ester (I) used as the starting compound in this process may be such a di-alkyl ester of which the alkyl group is one containing 1 to 6 carbon atoms, especially 1 to 4 carbon atoms, sunh as methyl and ethyl. The ammonium salt used as the reactant may be an ammonium salt of an organic acid, for example, a lower alkanolc acid, especially formic acid, acetic acid, propionic acid and even benzo-c acid. In stead of the ammonium salt, ammonia may be employed. The reaction of the di-alkyl 3-ketoglutarate with ammonium salt or ammonia may conveniently be carried out in an organic solvent, for example, an alkanol of 1-4 carbon atoms such as methanol and ethanol, or acetone, tetrahydrofuran and the like. The proportion of the ammonium salt to the starting compound (I) employed may be at least equimolar but it is preferred to use the ammonium salt in an excess in order to promote the rate of reaction and shift favorably the point of equilibrium of the reaction. It is preferabie to use the ammonium salt of an organic acid in a 5 to 30 molar proportion, especially in an approximately 10 molar proportion per 1 molar proportion of the di-alkyl 3-ketoglutarate. The reaction may proceed well at ambient temperature. If necessary, however, the reaction mixture may be heated or cooled. In general, the reaction temperatures may be in a range of 0 C to 1300C, for Instance. In the process of this invent ion, the first reaction step may conveniently be worked in such a way that a solution of ammonium formate or ammonium acetate in methanol is adjusted to a pH of not higher than 8.0, preferably at an acidic pH of 3.0-6.0 by addition of acetic acid and the resulting acidic solution is admixed with 3-ketoglutaric acid di-methyl ester, namely acetone dicarboxylic acid dimethyl ester. In this way, the unsaturated amine compound of the formula (II) and (II')'are produced as intermediate product from the starting di-alikyl 3-ketoglutarate employed. In the solution, tautomerlsm mag be occuring between the intermediate products of the formulae (II) and (II'). In the second reaction step of the present process, the reaction solution containing therein the intermediate unsaturated amine compounds (II) and (II') is admIxed with an alkali metal cyanoborohydride of the formula MBH3CN wherein M is an alkali metal such as sodium, potassium and lithium, to effect the reduction of said amine compounds. The reduction reaction may proceed well at ambient temperature and may be carried out under heating or cooling. In general, the reaction temperature may be in a range of 0 C to lO00C. This second step may conveniently be worked in such a way that to the reaction solution as obtained from the first step is added slowly in portions sodium cyanoborohydride or lithium cyanoborohydride at ambient temperature. The proportion of the alkali metal cyanoborohydride used may be at least equimolar and usually in a 1.5 to 2.0 molar proportion per 1 molar proportion of the intermediate unsaturated amIne compounds (II) and (II'). The reaction time required for the second step of the present process varies dependIng on the time required for addition of the alkali metal cyanoborhydride reagent and generally may be in a range of 2 to 5 hours. When the reaction Is carried out at ambient temperature3 it normally takes 10 to 30 hours to complete the reaction. The end point of reaction which takes place in the second step of the present process can be confirmed by taking out an aliquot from the reaction solution and testing it-by thin layer chromatography, where Rf values of the ketoester and the product 3-aminoglutarate show 0.50 and 0.20, respectively using a mixed eluent ethyl acetate and n-hexane (1:3 5 volume). In order to ensure the completion of the reaction, it is desirable to allow the reaction to proceed for a longer period of time than that usually required. After completion of the reaction, the reaction solution is concentrated to a smaller volume at a temperature of not more than 300C and then adjusted to a pH 9.0 by addition of aqueous saturated potassium carbonate, followed by extracting with a water-immiscible organic solvent such as methylene chloride and the like. The remaining aqueous phase is adjusted to pH 10 by addition of potassium carbonate and then thoroughly extracted with the organic solvent. The combined organic phase is dried and concentrated to give an oily material which is subsequently subjected to column chromatography with silica gel to isolate and purify the desired product. In this way, the desired 3-aminoglutaric acid di-alkyl ester of the formula (III) is obtained in a high yield. The merits obtained by the process according to the first aspect of this Invention are now described In more detail. In contrast to the known method for the production of 2 3-aminoglutaric acid alkyl ester which involves a number of reaction steps the process of this invention is able to synthesize the 3-arnoglutaric acid alkyl ester only by to reaction steps Irmediately starting from the 3-keto- glutaric acid (ie. acetone dicarboxylic acid) di-alkyl ester which may readily be prepared from citric acid. Besides, the process of tis invention is advantageously performable in an easy and economic way, because the first step of reacting the di-alkyl 3-ketoglutarate with ammonium salt or ammonia and the second step of reducing the intermediate amine product with an alkali metal cyanoborohydrlde can be effected in one and the same reaction vessel, that is to say, the present process is performable as a "one-pot11 process. Further advantages are that the reagent required in the second step of the process for conversion of the starting compound (I) into the unsaturated amine compounds (II) and (lit) may be merely ammonia or the ammonium salt of an organic acid such as formic acid and acetic acid which are very inexpensively available, and that the overall yield of the desired 3-aminoglutaric acid di-alkyl ester based on the starting 3-ketoglutarate is favorably high. Further, according to a second aspect of this inven- tion, there is provided a process for the production of an optically active ss-(S)-aminoglutaric acid mono-alkyl ester of the formula EMI10.1 wherein R is an alkyl group of 1 to 6 carbon atoms, which comprises the steps of: - reacting a ss-X-protecied aminoglutaric acid di-alkyl ester of the formula EMI11.1 wherein R is as defined above and A is an amino-protecting group removable by hydrogenolysis or mild hydrolysis. with an ester-hydrolyzing enzyme in an aqueous organic solvent at a pH and at a temperature at which the enzyme employed is enzymatically active, to remove hydrolytically and preferentially one ester-forming alkyl group, R, from the ss-N- protected aminoglutaric acid di-alkyl ester without cleavage of the other ester-forming alkyl group, and then removing the amono-protectlng group, A, from the resulting hydrolysis product in a known manner. The process according to the second aspect invention where ss-N-benzyloxycarbonylaminoglutaric acid dimethyl ester is employed as the starting material may schematically be described by the following equation:- EMI11.2 wherein Z denotes benzyloxycarbonyl group EMI12.1 and Me denotes methyl group. In the reaction shown by the above equation, it is seen that the ss-benzyloxycarbonylaminoglutaric acid dimethyl ester (V') is a prochiral compound which belongs to the compound of the "Cs-point" class having the symmetrical plane in the molecule, that this compound contains two methoxycarbonylmethyl groups which are positioning with each other in the enantiotropic relationship in the molecule, and that only one of the two methoxycarbonylmethyl groups preferentially undergoes the enzymatic hydrolysis and bring about the removal of one of the ester-forming methyl groups therefrom, affording the optically active ss-(S)-benzyloxy- carbonylaminoglutaric acid mono-methyl ester (VI). Subsequent N-detrotectlon of the compound (VI) yields the optically active ss-(S)-aminoglutaric acid mono-methyl ester (IV'). The stereochemical feature of the reaction equation as shown above is that the starting "prochiral" compound (V') is converted via the intermediate "chiral" compound (VI) into the final compound (IV') with the desired stereo chemistry. Various researches which were made in this field of ss-lactam chemistry in the past are limited to ones of academic point of view and are far away from the practical or efficient production of any commercially or therapeutically useful substances such as the antibiotics. Thus, during the research of a field of enzyme chemistry, Cohen and Khedouri reported their discovery that when ss-acetylamino- glutaric acid diethyl ester is hydrolytically treated with S-chymotrypsin, one of pancreas processes, this optically inactive di-alkyl ester is converted into optically active ss-(R)-acetylaminoglutaric acid mono-ethyl ester (S.G. Cohen and E. Khedouri; Nature 186, 75 (1960)). However, the starting ss-acetylaminoglutaric acid diethyl ester employed in the research of Cohen et al has been acetylated at the amino group thereof, and owing to this, it is very difficult or impossible to convert the resulting optically active hydrolysis product (i.e. ss-(R)-acetylaminoglutaric acid mono-ethyl ester) into a corresponding deacetylated optically active product because the acetylarilno group can hardly be converted Into te free amino group without causing hydrolytic breakdown of the ester group of said optically active hydrolysIs product. Besides, the enzymatic hydrolysis of said starting diethyl ester needed a great amount of c'-chymotrypsin or a long reaction time, as the rate o the enzymatic hydrolysis involved was very low. For these reasons, it was not considered that the research results by Cohen et al could be further developed so as to provide any method of producing physiologically active substance from the optically active ss-(R)-acetylaminoglutaric acid monoethyl ester, even if certain known procedures of present organic chemistry would be associated therewith in any ingenious way. Furthermore, the optically active 6-(R)- acetylaminoglutaric aci mono-ethyl ester so produced has the (R)-configuration and is not utilisable as a starting material for the preparation of physiologically active substances which should have (S)-conficuration at the asymmetric carbon. As a result of our extensive studies, we have now found that optically active ss-aminoglutaric acid mono-alkyl ester having the (5)-configuration can be produced even using as the substrate compound the 3-aminoglutaric acid di-alkyl ester which was previously considered to have the above-mentioned disadvantageous behaviour in the enzymatic hydrolysis thereof, if the natures of the amino-protecting group as well as the nature of the hydrolysing enzyme employed are selected appropriately in accordance with this invention. Accordingly, we have succeeded to reach the nei process of the second aspect of this InventIon as described hereinbefore. The ss-protected aminoglutaric acid di-alkyl ester (V) which is employed in the process of the second aspect invention may be prepared by introducing an appropriate knows amino-protecting group in on manner Into the 3-aminoglutaric azid di-alkyl ester which has been produced by the process of the first aspect invention as aforesaid. The amino-protecting group (A) which is incorporated into the starting di-alkyl ester (V) employed in the process of the second aspect of this invention should be removable by catalytic hydrogenolysis and may preferably be benzyl group, benzhydryl group or an aralkyloxycarbonyl group such as benzyloxycarbonyl. To this end, an alkyloxycarbonyl group of 2 to 7 carbon atoms, such as tert-butoxycarbonyl which is removable by mild hydrolysis eg. with trifluoroacetic acid may be used. The introduction of these a,nino-protecting groups may be achieved by conventional amino-protecting technique known in the synthesis of peptides. The esterforming alkyl group (R) in the starting di-alkyl ester (V) is a lower alkyl group of 1 to 4 carbon atoms, such as methyl and ethyl. The ester-hydrolyzing enzyme (esterase) employed in the present process may be chosen from those of animal origin. Liver esterase of pig is preferred, however. In the process according to the second aspect of this invention, the enzymatic reaction may conveniently be carried out in. such a manner that the starting ss-protected aminoglutaric acid di-alkyl ester (V) is dissolved in a volume of a phosphate buffer solution and the substrate solution so obtained is admixed with the esterase, followed by stirrln at ambient temperature to allow the reaction to proceed. The reaction medium may usually be water but, if necessary, be water containing therein a water-miscible organic solvent such as dImethylfoniariiide, acetone or a lower alkanol, for example, methanol at a concentration of not more than 10% or at a such a concentration that the activity of the esterase is degraded by the presence of the organic solvent. The reaction medium is preferable to have a pH of 7.0 to 9.0 and specially a pH of 7.0 to 8.0. The reaction temperature may usually be in a range of 20 to 30 C, although the enzymatic reaction may proceed at any temperature at which the enzyme employed is remaining active. The reaction time required is usually in a range of 1 to 2 hours, though it depends on the concentrations of the starting compound and the enzyme employed. The amount of the enzyme used may be catalytic but may preferably be in a range of 0.001 to 1% by weight (as protein) based on the starting substrate compound (V) in order to obtain a favourably high rate of the reaction. After the enzymatic reaction is completed, the reaction solution is adjusted to pH of not higher than 7.0 and then extracted with methanol. The meth2nolic extract is concentrated to afford a crude product as oil, which may be isolated and purified by chromatography on silica gel column developed with ethyl ether or methylene chloride. In this way, the desired S-(S)-protected aminoglutaric acid mono-alkyl ester Is afforded in a hl yield. This Nprotected Intermediate product is subjected to removal of the amino-protecting group (A) according to a conventionai N-deprotectlon technique such as catalytic hydrogenolysis or mild hydrolysis with trifluoroacetic acid, whereby the desired product, ss-(S)-aminoglutaric acid mono-alkyl ester (IV) is yielded. This final product shows an optical purity which varies depending on the reaction conditions employed but is of a negative [&alpha;]D value. The absolute stereochemistry of this product was determined to be Sconfiguration by cyclising this product into a ss-lactam compound and then comparing the optical rotation of the latter with that of an authentic sample prepared by a known route. Thus, with the process of the second aspect of this invention, it is observed that only one of the two enantropic alkoxycarbonylmethyl groups present in the starting substrate compound (V) can preferentially undergo the hydrolytic removal of the alkyl group of the "pro-S" nature therefrom, giving the ss-(S)-protected aminoglutaric acid mono-alkyl ester. This is surprising, as it is unexpectable from the fact that the opposite configuration (R) was shown by the hydrolysis product which was obtained when substrate 3aminoglutaric acid di-alkyl ester having free amino group or acetylated amIno group was enzymatically hydrolysis by the prior art method but in the same manner as in the process of the second aspect of this invention. ss-(S)-Aminoglutaric acid mono-methyl ester and monoethyl ester as obtained b this second aspect inventIon are ne compounds which exhibit antIbacterial activity against Grar.1-positlve bacteria, particularly against Staphylococcus aureus and hence are useful as antibacterial agent. Furthermore, the optically active ss-(S)-aminoglutaric acid mono-alkyl ester as obtained by this invention can readily be converted by one reaction step into a 4-alkoxyearbonyl- methylazetidine-2-on which is an important intermediate for the synthesis of carbapenen ss-lactam antibiotics, when it is processed by a method of organic chemistry comprising reacting it with triphenylphosphine and 2,21-dlpyridyl disulfide in acetonitrile (see our co-pending U.S. patent application SN. According to a third aspect of this invention, there is provided a process for the production of an optically -active ss-(R)-aminoglutaric acid mono-alkyl ester of the formula EMI18.1 wherein R is an alkyl group of 1 to 6 carbon atoms, which comprises the step of: reacting a ss-aminoalutarflc acid di-alkyl ester of the formula EMI18.2 wherein R is as defined above, with an ester-hydrolyzing enzyme in an organic solvent at a pH and at a temperature at which the enzyme employed is enzymatically active, to remove hydrolytlcally and preferentially one ester-formin alkyl group from the ss-aminoglutaric acid di-alkyl ester without cleavage of the other ester-forming alkyl group. The process according to the third aspect invention where ss-aminoglutaric acid dimethyl ester is employed as the starting material may schematically be described by the following equation: EMI19.1 <tb> <SEP> H2N\ <SEP> ,K <SEP> H <SEP> H2N <tb> <SEP> ' <SEP> Esterase <SEP> ' <tb> <SEP> C <SEP> Esterase <SEP> C <tb> <SEP> CH2 <SEP> CH2 <SEP> CH2 <SEP> CH2 <tb> <SEP> I <SEP> I <SEP> I <tb> MeOOC <SEP> COOi'4e <SEP> HOOC <SEP> COOMe <tb> <SEP> (V1111) <SEP> (VII') <tb> wherein Me denotes methyl group. With the above equatIon, it Is again seen tat the ss-aminoglutaric acid dimethyl ester (VIII') is an optically inactive compound which is belonging to the compound of the 11Cs-polnt" class having a symmetrical plan in the molecule, that this compound contains two methoxycarbonylmethyl groups which are positioning with each other in an enantropic relationship in the molecule thereof, and that only one of the two methoxycarbonylmethyl groups preferentially can undergo the enzymatic hydrolysis and bring about the removal of the ester-forming methyl group therefrom, affording the optically active (R)-aminoglutaric acid methyl ester (VII'). This reaction equation is stereochemically characterized in that the starting "prochiral" compound (VII') is converted into the "chiral" compound (VII') by the above enzymatic hydrolysis with the esterase. In the above-mentioned research of Cohen et al where the optically inactive ss-acetylaminoglutaric acid di-alkyl ester is converted into the optically active ss-(R)-acetyl- aminoglutaric acid mono-alkyl ester by the enzymatic hydrolysis with &alpha;-chymotrypsin, it was found that very large amount of the enzyme was needed even with a long reaction time required. As a result of our studies, we have now found that even if the 3-aminoglutaric acid di-alkyl ester which was considered to have the above-mentioned disadvantageous behavIour zcn the enzymatic hydrolysis with &alpha; ;-chymotrypsin is used as the substate compound to be treated, this optically inactive compound can be converted into an optically active ss-aminoglutaric acid mono-alkyl ester having the (R)-conflguration without its amino group being protected, when the enzyme for use in the hydrolysis thereof is appropriately selected by careful investigation in accord- ance with the third aspect of this invention. Thus, e have succeeded to arrTe at the new process of the third aspect of this invention as stated before. The ester-hydrolyzing enzyme (esterase) used in the process of the third aspect of this invention may also be chosen from those of animal origin similarly to the second aspect of this invention. Liver esterase of pig is preferred. The process according to the third aspect of this invention may be carried out in thsame manner as in the process of the second aspect of this invention. PartIcularly, the reaction solvent, reaction pH, reaction temperature, react ion time and amount of the enzyme employed in the process of the third aspect invention are same as those employed according to the process of the second aspect Invention. After the enzymatic reaction is completed, the reaction solution is adjusted to a pH of not higher than 7.0 by added tion of diluted hydrochloric acid and the desired product ts extracted therefrom with methanol. The methanolic extract is concentrated to give a crude oily product which may be isolated and purified by cellulose column chromatography developed with n-butanol as the development solvent, afforc ing a product rich in a ss-(R)-aminoglutaric acid mono-alkyl ester (about 40 - 50% enantiomeric excess) (VII). The ss-(R)-aminoglutaric acid mono-alkyl ester so produced has an optical purity which varies depending on the reaction conditions employed but-is of a positive [&alpha;]D n ¯ range of from +2.36 to +4.72 (c 4.23, H2O). The absolute stereochemistry of this product was decided to be R-configu- ration by cyclising this product into a ss-lactam compound and then comparing the latter with an authentic sample of (S)-4-[(methoxycarbonyl)methyl]-2-azetidinone. Thus, with the process of the third aspect of this invention, it is again observed that only one of the enantropic two alkoxycarbonylmethyl groups present in the starting substrate compound (VIII) preferentially undergoes the hydrolytic removal of the alkyl group of the "pro-S" nature therefrom, giving the ss-(R)-aminoglutaric acid mono-alkyl ester (VII) preferentially. ss-(R)-Aminoglutaric acid mono-methyl ester and monoethyl ester as obtained by the third aspect invention are new compounds which exhibit an antibacterial activity against Gram-positive bacteria, paftlcularly against Staphylococcus aureus and hence are useful as antibacterial agent. Besides, this ss-(R)-aminoglutaric acid mono-alkyl ester is readily convertible into an (R)-4-alkoxycarbonylmethylazetidine-2-on. In consequence, the processes according to the second and third aspects of this invention are generically appli- cable as an enzyme-chemical process for preparing optically active ss-amino acid intermediates in the free acid form of which the center of asymmetry can be selected by option according to the desired configuration of the asymmetical carbon ate of the ultImately desired physiolo-callar active substances, when said optimally active S-amino acid is subjected to appropriate chemical conversicns at the sites of its free amino group, free carboxylic group and/or ester group. This invention is illustrated below with reference to Examples 1 - 4 (according to the first aspect of this invention) and Examples 5 - 6 (according to the second aspect of this invention) as well as Examples 7 - 10 (according to the third aspect of this invention) to which this InventIon is not limited. Example 1 A solution of 38.6 g (500 milimol) of ammonium acetate in 400 mQ of methanol was adjusted to pH 6.0 by addition of acetic acid and then admixed with 8.7 g (50 milimol) of dimethyl 3-ketoglutarate. The mixture was stirred at ambient temperature for 1 hour and 15 minutes. Into the reaction vessel containing the resulting reaction solution was added 2.5 g (40 milimol) of sodium cyanoborohydride (NaBH3CN) slowly in small port Ions over 5 hours. The reaction solution was then stirred for 22 hours at ambient temperature and subsequently adjust to pH 5.5 by addition of acetic acid, followed by further addition of 0.63 g of sodium cyanoborohydride thereto. The reaction mixture was stirred for 9 hours at ambient temperature. The reaction solution so obtained was concentrated to a smaller volume below 300 C, and the concentrated solution was adjusted to pH 9.0 by addition of aqueous saturated potassium carbonate and then extracted twice with 200 m2 portions of methylene chloride. The remaining aqueous phase was adjusted to pH 10 by addition of potassium carbonate and extracted twice with 200 mQ portions of methylene chloride. The resultant organic solution in methylene chloride (the extracts combined) was washed once with saturated solution of sodium chloride in water and dried over anhydrous sodium sulfate. The dried solution was concentrated to give 8.9 g of an oily product. This oily substance was isolated and purified by chromatography with silica gel developed with ethyl ether containing 5% methanol as the eluent, affording 7.9 g of dimethyl 3aminoglutarate as oil. In vie of the IR. and 1H-NMR. spectra, and the elemental analysis, tis product was confirmed to have the structure formula consistent with that of an authentic sample of dimethyl 3-aminoglutarate. IR. spectrum (cm-1): 3400 (amino group), 1740 (ester group), 1H-NMR. spectrum (in CDCl3): 61.92 (s, 2H, NH2), #2.24 - 2.64 (dd x 2, 4H, -CH2-), 63.54 (dd, lH, -CÚH-), 63.68 (s, 6H, CO2Me) Elemental Analysis: Caicd. for C7H1304N: C 47.99, H 7.48, g 8.00% Found: C 47.79, E 7.47, N 7.74% Example 2 A solution of 31.5 g (500 milimol) of ammonium formate in 400 mQ of methanol was adjusted to pH ó.O by addition of acetic acid and then admixed with 8.7 g (50 millmol) of dimethyl 3-ketoglutarate. The mixture was stirred at ambient temperature for 2 hours. To the resulting reaction soluion in the same reaction vessel was added 2.5 g (40 milimol) of sodium cyanoborohydride slowly in small portions over 5 hours. The reaction mixture was stirred at ambient temperature for 22 hours and then adjusted to pH 5.5 by addition of acetic acid, followed by further addition of 0.63 g of sodium cyanoborohydride. The mixture was stirred for further 9 hours at ambient temperature to complete the reductive amlnation. The reaction solution obtained was concentrated to a smaller volume at a temperature of not higher than 300C, and the concentrated solutIon was adjusted to pH 9.0 by addItion of aqueous saturated potassium carbonate, followed by extrac- tion twice with 200 mQ portions of methylene ch'oride. The remaining aqueous phase was adjusted to p 10 by addition of aqueous saturated potassium carbonate and then extracted twice with 200 mQ portions of methylene chloride. The resultant organic solution in methylene chloride (the extracts combined) was washed once with aqueous saturated sodium chloride and dried over anhydrous sodium sulfate. The dried solution was concentrated to give 7.5 g of an oil material. This oily material was subjected to column chromatography with silica gel developed with ethyl ether containing 5% methanol as the eluent, for isolation and purification. The eluate afforded 5.6 g of dimethyl 3aminoglutarate as oil after removal of the solvent. This product was identified by confirming the chemical structure with reference to its 13C-NMR and ÚH-NMR spectra data and elemental analysis. Example 3 A solution of 38.6 g (500 milimol) of ammonium acetate in 400 mS of methanol was adjusted to pH 6.0 by addition of acetic acid and then admixed with 8.7 g (50 milimol) of dimethyl 3-ketoglutarate, followed by stirring at ambient temperature for 2 hours. To the resulting reaction solution was added in the same reaction vessel 2.4 g of lithium cyanoborohydride slowly in small portions over 5 hours. The mixture was stirred at ambient temperature for 20 hours and then adjusted to pH 5.3 by addition of acetic acid, followed by further addiction of 0.60 r of lithium cyanoborohydride thereto and by stIrring at ambient temperature for 8 hours to complete the reduce reaction. The reaction solution so obtained was concentrated to a smaller volume at a temperature of not higher tan 300C, and the concentrated solution was adjusted to pH 9.0 by addition of aqueous saturated potassium carbonate and extracted twIce ih 200 mi portions of methylene chloride. The aqueous phase was adjusted to pH 1C by addition of aqueous saturated potassium carbonate and extracted with twice with 200 mZ portions of methylene chloride. The resulting organic solution in methylene chloride (the extracts combined) was washed once with aqueous saturated sodium chloride and dried over anhydrous sodium sulfate. The dried solution was concentrated to give 8.20 g of an oily material. This material was subjected to column chromatography with silica gel developed with ethyl ether containing 7.5 methanol. The eluate from the silica gel column was collected and concentrated in vacuo to give 6.0 g of dimethyl 3-aminoglutarate as oil. This product was identified by confirming the chemical structure with refer- ence to its 13C-NMR and 1H-NMR spectra data and elemental analysis. Example 4 The process of Example 1 was repeated using 10 g of dimethyl 3-ketoglutarate n stead of the dimethyl 3-ketoglutarate. Diethyl 3-aminoglutarate was afforded as oil. Yield 8.0 g. IR. spectrum (cm-1): 3400 (amino group), 1742 (ester group). Example 5 (a) A solution of ss-benzyloxycarbonylaminoglutaric acid dimethyl ester (465 mg, 1.5 milimol) in acetone (1.5 ml) was admixed with 45 ml of a phosphate buffer solution (pH 8.0). The solution obtained was admixed it 2.7 m (measured as the quantity of protein) of pig liver esterase (Esterase E-3128, a product of ShiDma Co., U.S.A.). The resultant admixture was allowed to stand at 250C for 6 hours under occasional stirring, and the resulting react Ion solution was adjusted to pH 1 by addition of 2.5 mS of 2N hydrochloric acid thereto, followed by extraction with 50 ml of methylene chloride. The organic layer was dried over anhydrous sodium sulfate and concentrated to give 419 mg of- a crude product as crystals. This crude product was purified by chromatography on silica gel developed with ethyl ether to afford Lilo mg (93%) of optically active ss-(S)-benzyloxycarbonylaminoglutaric acid mono-methyl ester which showed a specific optical rotation 25 = +0.710 (c 7.45, CHCl3). mp. 97.0 - 97.5 C. IR. spectrum (KBr): 3330, 2840 - 2960, 1740, 1725, 1700 cm H-NMR. spectrum (CDCl3): #2.69 (bd, 4H, CH2 J=6 Hz), 63.65 (s, 3H, CO2Me), 64.17-4.53 (m, 1H, CH), 65.07 (s, 2H, CE2Ph), #5.63 (bd, 1H, NH), 67.30 (s, SE, Ph). Elemental analysis Calcd. for C14H17O6N: C 56.94, H 5.80, N 4.74% Found: C 57.05, H 5.84, N 4.69 The ss-(S)-benzyloxycarbonylaminoglutaric acid monomethyl ester obtained as above is a new compound which is not described in any chemical literature. (b) The mono-methyl ester (385 mg, 1.3 milimol) obtained as above was dissolved in 20 m2 of methanol, and the resulting methanolic solution was admixed with 40 mg of 10 palladium-carbon and then stirred at ambient temperature for 30 minutes under atmosphere of hydrogen. The reaction solution was filtered and the filtrate solution was concentrated to give 205 mg (98%) of ss-(S)-aminoglutaric acid mono-methyl ester as oil which showed a specific optical rotation [&alpha;]D = -5.52 (c 3.26, H2O). This product showed the following IR. and NMR. spectra and elemental analysis. IR. spectrum: 3440, 1730, 2400 - 2900 cm-1. 1H-NMR. spectrum (D20): 62.59 (bd, 2H, J=6 Hz), #2.84 (bd, 2H, J=6 Hz), #3.76 (s, 3H, CO2CH3), #3.85 - 4.05 (m, 1H, CH). Elemental analysis: Calcd. for C6H11O4N: C 44.71, H 6.88, N 8.69% Found: C 44.73, H 6.87, N 8.65% The product of this Example 5 (b) was tested for its antibacterial activity and its minimum concentration inhIbitory to the growth of Staphylococcus aureus was found to be 10 - 100 mcg/ml. Example 6 The procedure (a) of Example 5 as above was repeated using 500 mg of ss-benzhydrylaminoglutaric acid di-ethyl ester in stead of the ss-benzyloxycarbonylaminoglutaric acid dimethyl ester. The product from the enzymatic hydrolysis was subjected to catalytic hydrogenelysis over palladiumcarbon in the same manner as in the procedure (b) of Example 5. ss-(S)-Aminoglutaric acid mono-ethyl ester was obtained as oil in the yield of 400 mg. IR. spectrum (KBr): 3445, 1728 cm-1. This mono-ethyl ester also showed a potency value of 20 - 100 mcg/ml for its minimum concentration inhibitory to the growth or Staphylococcus aureus. Example 7 (a) A solution of dimethyl ss-t-butoxycarbonylamino- glutarate (413 mg, 1.5 milimol) in acetone (1.5 ml) was admixed with 45 ml of a phosphate buffer solution (p 8.0). The solution obtained was admixed with 2.7 mg (measured as the quantity of protein) of pig liver esterase (Esterase E-3128, a product of Shigma Co., U.S.A.). The resulting mixture was allowed to stand at 250C for 6 hours under occasional stirring, and the reaction mixture was adjusted to pH l with hydrochloric acid thereto, followed by extract tion with 50 mQ of methylene chloride. The orni layer was dried over anhydrous sodium sulfate and concentrated to give 363 mg of a crude product as crystals. This crude product was purified by chromatography on silica gel developed With 2% methanol-methylene chloride to afford 320 mg of optically active ss-(S)-t-butoxycarbonylaminoglutaric acid mono-methyl ester, showing mp. 94.1 - 97.0 C after recrystallizatioh from ether-hexane. IR. spectrum (K3r); 3330, 1735, 1720 cm 1. H-NMR. spectrum (CDCl3): #1.44 (s, 9H), #2.67 (d, J=6, 4H), 63.72 (s, 3H), 64.38 (m, 1H). Mass spectrum: 262 (M+ + 1). Elemental analysis: Calcd. for C11H19NO6: N 5.36, C 50.56, H 7.33 Found: N 5.31, C 50.32, H 7.43 The half ester, ss-(S)-t-butoxycarbonylaminoglutaric acid mono-methyl ester obtained here is a new compound which is not described in any chemical literature. Boc-aminoglutaric acid mono-methyl ester (100 m) was treated with trifluoroacetic acid (1 ml) for 15 minutes and the mixture was dried under 2 reduced pressure. The residue was dissolved in water and neutralized with potassium carbonate, affording 80 mg of ss-(S)-aminoglutaric acid monomethyl ester after usual workup. Example 8 A solution of ss-aminoglutaric acid dimethyl ester (346 mg, 2.0 milimol) in 7 ml of a phosphate buffer solution (pH 8.0) was admixed with 2.89 mg (400 units) of pig liver esterase (Esterase E-312S, a product of Shigma CQ., U.S.A.). The resultant admixture was alloyed to stand at 250C for 2.5 hours under occasional stirring, and the reaction solution was adjusted to a pH of about 7.0 by addition of 0.1 N hydrochloric acid, followed by concentration to dryness at a temperature of not higher than 200C. The oily residue was extracted with 50 mQ of methanol, and the methanol extract was concentrated to give 268 m.g of a crude product. This crude product was isolated and purified by chromatography on cellulose column (Cellulose CF-11, a product of Wattman Co., U.S.A.) developed with n-butanol saturated with water as the eluent, to afford 212 mg (66% yield) of ss-(R)-aminoglutaric acid mono-methyl ester as oil which showed a specific optical rotation [&alpha;]D22 + 2.36 (c 4.23, H2O). IR. spectrum (KBr): 3440, 1730, 2400 - 2900 cm1. 1H-NMR. (D2O): #2.59 (bd, 2H, J=6 Hz), #2.84 (bd, 2H, J=6 Hz), #3.76 (s, 3H, COOCH3), #3.85 - 4.05 (m, 1H, CH). Elemental analysis Calcd. for C6H11O4N: C 44.71, H 6.88, N 8.69,S Found: C 44.70, A- 6.86, N 8.42S. This new compound was tested for its antibacterial activity and its minimum concentration inhibitory to growth of Staphylococcus aureus was found to be 20 mcg/ml. Example 9 The process of the Example 8 was repeated using 5.7 mg (400 units) of the pig liver esterase (Esterase E-3128). The methanol extract obtained was concentrated to give 270 m of a crude product which was then purified by the cellulose column chromatography in the same way as in the Example 8 to afford 242 mg of ss-(R)-aminoglutaric acid mono-methyl ester as oil which showed a specific optical rotation [a]22 +3.630 (c 4.23, H20). Example 10 The process of the Example 8 was repeated using 10 mg (400 units) of the pig liver esterase (Esterase E-3128) and effecting the enzymatic reaction at 200C for 1.5 hours under occasional stirring. The methanollc extract obtained was concentrated to give 280 mg of a crude product, which was then purified by the cellulose column chromatography in the same manner as in the Example 8 to afford 250 mg of ss-(R)- aminoglutaric acid mono-methyl ester as oil which showed a specific optical rotation [&alpha;]D22 + 4.72 (c 4.23,H2O). Example 11 The process of the Example 10 was repeated except that 400 mg of ss-aminoglutaric acid diethyl ester was used in stead cf the dimethyl ester. ss-(R)-Aminoglutaric acid mono-ethyl ester was afforded in a yield of 320 mg as oil which showed a specific optical rotation [&alpha;]D22 + 3.84 (c 4.20, H2O). This new compound was tested for its antibacterial activity, and found t its minimum concentration inhibitory to growth of Staph'?lo coccus aureus was found to be 30 - 100 mcg/ml.

Claims: Patent claims: 1. A process for the production of a 3-aminoglutaric acid di-alkyl ester of the formula EMI34.1 wherein R is an alkyl group of 1 to 6 carbon atoms, which comprises the steps of: reacting a 3-ketoglutaric acid di-alkyl ester of the formula EMI34.2 wherein R is as defined above, with an ammonium salt or ammonia in an organIc solvent at a temperature of 00C to 1300C to rorm an unsaturated amine compound of the formula EMI34.3 or of the formula EMI34.4 wherein R is as defined above, and then reducing this unsaturated amine compound with an alkali metal cyanoborohydride in an organic solvent at a temperature of 0 C to 1000C. 2. The method of Claim 1 In which the ammonium salt is the ammonium salt of formic acid, acetic acid, propionic acid or benzoic acid. 3. The method of Claim 1 in which the reaction of 3-ketoglutaric acid di-alkyl ester with ammonium salt is conducted in methanol or ethanol under a pH of not higher than 8.0 and particularly at an acidific pH of 3.0 to 6.0. 4. The method of Claim 1 In which the alkali metal cyanoborohydride is sodium, potassium or lithium cyanoborohydride. 5. The method of Claim 1 in which the steps of reacting 3-ketoglutaric acid di-alkyl ester with ammonium salt and of reducing te unsaturated amine product with an alkali metal cyanoboroh;;dride are effected in one and the same reaction vessel. 6. A process for the production of an optically active ss-(S)-aminoglutaric acid mono-alkyl ester of the formula EMI35.1 wherein R is an alkyl group of 1 to 6 carbon atoms, which comprises the steps of: reacting a ss-N-protected aminoglutaric acid di-alkyl ester of the formula EMI36.1 wherein F is as defined above and A is an amino-protecting group removable by hydrogenolysis or mild hydrolysis, With an ester-hydrolyzing enzyme in an organic solvent at a pH and at a temperature at which the enzyme employed is enzymatically active, to remove hydrolytically and pre ferentially one ester-forming alkyl group (R) from the ss-N-protected aminoglutaric acid di-alkyl ester without cleavage of the other ester-forming alkyl group, and then removing the residual amino-protecting group (A) from the resulting hydrolysis product in a known manner. -7. The process of Claim 6 in which the ss-N-protected aminoglutaric acid di-alkyl ester is ss-N-benzyloxycarbonyl aminoglutaric acid dimethyl or diethyl ester. 8. The process of Claim 6 in which the ester-hydrolyzlng enzyme is pig liver esterase and reacted with the ss-N- protected aminoglutaric acid di-alkyl ester at a pH of 7.0 to 9.0, and at a temperature of 20 to 300C. 9. A process for the production of an optically active ss-(R)-aminoglutaric acid mono-alkyl ester of the formula EMI37.1 wherein R is an alkyl group of 1 to 6 carbon atoms, which comprises the steps of: reacting a S-aminoglutaric acid di-alkyl ester of the formula EMI37.2 wherein R is as defined above, with an ester-hydrolyzing enzvme in an organic solvent at a pH and at 2 temperature at which the enzyme employed is enzymatically active, to remove hydrolytically and preferentially one ester-forming alkyl group from the B-aminoglubaric acid di-alkyl ester without cleavage of the other ester-forming alkyl group. 10. The process of Claim m 9 in which the ester-hydrolyzing enzyme is pig liver esterase and reacted with a S-amino- glutaric acid di-alkyl ester at a pH of 7.0 to 9.0 and at a temperature of 20 to 300C. 11. The new compound which is ss-(S)-benzyloxycarbonyl aminoglutaric acid monomethyl ester; B-(S)-aminoglutaric acid monomethyl ester; and ss-(S)-aminoglutaric acid monoethyl ester. 12. The new compound which is ss-(R)-aminoglutaric acid monomethyl ester and ss-(R)-aminoglutaric acid mono-ethyl ester.

Inventors: OHNO, MASAJI, UMEZAWA, HAMAO
Applicants: MICROBIAL CHEM RES FOUND
Application year: 1981
Main classification: C07C101
IPC codes: C12P41, C07C227