ENZYMATIC PROCESS FOR PREPARING OPTICALLY-ACTIVE 1,2-DIHYDRO-3H-PYRROLO[1,2a]PYRROLE-1-CARBOXYLIC ACID DERIVATIVES

Abstract: Process for resolving racemic 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid esters by enantiospecific hydrolysis utilizing extracellular lipases of microbial origin (EC 3.1.1.3) or extracellular microbial proteases.

Description: Description Enzymatic Process for Preparing Optically-active 1,2 -Dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic Acid Derivatives Technical Field The present invention relates to a novel process for pro¬ ducing optically-active l,2-dihydro-3H-pyrrolo[l,2a]ρyrrole-l- carboxylic acid derivatives. Specifically, it relates to a pro¬ cess for the enzymatic enantiospedfic hydrolysis of racemic l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic acid esters to give optically-active l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l- carboxylic acids. Background Ketorolac , 5-benzoyl-l , 2-dihydro-3H-pyrrolo [ 1 , 2a] pyrrole-1- carboxylic acid (I), a structural analog of zomepirac fJ. Clin. Pharmacol .. 20. 213 (1980)], is a potent antiinflammatory and analgesic agent in animal models [W. H. Rooks et al., Agents Actions. 12, 684 (1982)]. In humans, it is essentially equiva¬ lent to morphine sulfate for the relief of postoperative pain [J. Yee et al., Clin. Pharmacol. Ther.. 35, 285 (1984)]. More recently, it was reported [A. Guzman et al., J. Wed. Chem. , 29, 589 (1986)] that the (-)-S-isomer of ketorolac (1) is about 60- 230 times more potent than the (+)-R-isomer in animal model studies . ( + )-(R)-L Rl = C00H; R 2 = H (-)-(S)-l_. R χ = H; R 2 = C00H Currently, the (-)-S-isomer is obtained via a tedious chemical resolution procedure using the expensive cinchonidine alkaloid [A. Guzman et al., J. Med. Chem.. 29, 589 (1986)]. Disclosure of the Invention Broadly, this invention comprises the use of extracellular microbial enzymes selected from the group consisting of Upases and proteases to catalyze the enantiospedfic hydrolysis of racemic l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic acid esters as hereinbelow defined. Wherein: Rl is a radical in straight chain, branched chain, or cyclic configuration selected from the class consisting of alkane radicals having from 1 to about 12 carbon atoms with or without electronegative substituents at C-2"; cycloalkane radi¬ cals having from about 5 to about 7 carbon atoms; phenyl and benzyl radicals having from about 6 to about 8 carbon atoms; (examples of electronegative substituents of the alkane radicals referred to above are radicals such as halogens, nitro groups, nitriles-i and carboxylates); R2 is an acyl radical in straight chain, branched chain or cyclic configuration having 2 to about 12 carbon atoms, cyclo¬ alkane radicals having about 5 to about 7 carbon atoms, benzoyl, naphthoyl, biphenoyl, and carbobenzoxy radicals containing nitro, halogen, methyl, or alkoxy groups in the aromatic ring. Examples of aroyl radicals which are eminently suitable for the purposes of the present invention are benzoyl and methoxyben- zoyl. It is the object of this invention to produce optically- active l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic acid derivatives in excellent yields using an enzymatic resolution process. Another object of the present invention is to provide an improved process for preparing the optically-active (-)-S.-5- benzoyl-1,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic acid (1) using extracellular inexpensive microbial upases and pro¬ teases. These and other objects and advantages of the invention will become more apparent from the following detailed description. The process of the invention comprises subjecting said 1,2- dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic ester to the hydro- lytic action of a microbial lipase (EC 3.1.1.3) or protease and recovering the desired optically-active 1,2-dihydro-3H-pyr- rolo[l,2a]pyrrole-l-carboxylic acid derivatives. It has been found that extracellular microbial lipases and proteases are capable ,of functioning to catalyze the desired enantiospedfic hydrolysis. Particularly suitable are those lipases derived from the microorganisms of the genera Candida, Rhizopus, Hucor. Aspergillus. Penicilliu , Geotrichium, Hurni- cola, Pseudomonas and Chromobacteriu . Particularly suitable proteases are those derived from the genera Streptomyces, Bacillus, Aspergillus. Rhizopus. Extracellular microbial lipases are well known and many of these are available commercially (see M. Iwai and Y. Tsujsaka, page 443, and M. Sugiura, page 505, in "Lipases," edited by B. Borgstrδm and H. . Brockman, Elsevier, N.Y., 1984). For exam¬ ple, they are used industrially for the transesterification of fats and were incorporated in laundry detergents for removal of oily contaminants. Extracellular bacterial, mold, and yeast proteases are well documented in the literature (see H. Matsubara and J. Feder, p. 721, in "Enzymes," Vol. Ill, P. D. Boyer (ed.), Academic Press, N.Y. , 1971). One outstanding fea¬ ture of these microbial lipases and proteases that distinguishes them from intact microorganisms is that they can tolerate high substrate and product concentrations. For example, no marked substrate and product inhibition were noted. Hence, these enzy¬ matic hydrolytic reactions can be carried out in high concentra¬ tions (0.1-5 M) with an unusually high degree of enantiospeci- ficity. Moreover, they are remarkably stable under the desired reaction conditions, so that they may be reused. The l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic ester substrate may be added in solid or liquid forms at concentra¬ tions of 0.1-5 M to a suitable buffer solution containing the lipase to effect the enantiospedfic hydrolysis. Alternatively, the substrate can be dissolved in a suitable organic solvent such as carbon tetrachloride, cyclohexane, carbon disulfide, or hexane, as long as the solvent does not denature the enzyme. In addition, the substrate may be emulsified by the use of poly- vinyl alcohol or propylene glycol. Of course, the temperature and pressure conditions under which the ester substrate and the lipase are brought into contact are interdependent as will be apparent to those skilled in the art. Generally, at atmospheric pressure, the temperature can range from about 10°C to about 40 β C and the pH of the medium can range from 3 to about 8.5. Detailed Description of the Invention The following examples are presented to illustrate this invention and are not to be considered as limiting the scope of the appended claims. The esters of (+)-l,2-dihydro-3H- pyrrolo[l,2a]pyrrole-l-carboxylic esters which are to be resolved are prepared according to the procedures described by J. M. Muchowski et al., J. Med. Chem. , 28, 1037 (1985), and H. Caspio et al., Can. J. Chem., 60, 2295 (1982). The enantiomeric excess (ee) of the remaining methyl ester and the acid (after treatment with diazomethane) were determined by PMR measurements using Eu(hfc)3- The spectra were recorded on a Varian EM390 spectrometer. The procedure consisted of dissolving 15 mg of Ketorolac methyl ester in 0.5 ml of CDCI3, to which was added 50 mg of Eu(hfc)3- EXAMPLE 1 To a suspension of Candida cylindracea lipase (Meito Sangyo OF 360,000 u/g) (100 mg) in 1 ml of 0.2 M phosphate buffer, pH 8.0, was added 268 mg of (+)-5-benzoyl-l,2-dihydro-3H-pyr- rolo[l,2a]pyrrole-l-carboxylic methyl ester (1 M) . The reaction mixture was stirred with a magnetic stirrer for 2 days at 24°C. The contents were then acidified with HC1 and exhaustively extracted with ethyl acetate three times. The combined organic extract was dried over sodium sulfate and was then evaporated to dryness. The residue was suspended in 5% NaHCθ3 and extracted with hexane to obtain (-)-5-benzoyl-l,2-dibydro-3H-pyrrolo- [l,2a]pyrrole-l-carboxylic methyl ester (ee_= 0.94). Acidifica¬ tion of the aqueous layer with HC1 to pH 2.0, followed by extraction with dichlororaethane gives (+)-5-benzoyl-l,2-dihydro- 3H-pyrrolo[l,2a]pyrrole-l-carboxylic acid (ee_= 0.68). EXAMPLE 2 The procedure of Example 1 is repeated except that (+)- l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxy!ic methyl ester is used as the substrate to obtain optically-active 1,2-dihydro-3H- pyrrolo[l,2a]pyrrole-l-carboxylic acid in good yield. EXAMPLE 3 The procedure of Example 1 is repeated except that (+)-5- [4-raethoxybenzoyl]-l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carbox- ylic methyl ester is used as the substrate to obtain (+)-5-[4- methoxybenzoyl]-l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic acid (anirolac) in good yield. EXAMPLE 4 The procedure of Example 1 is repeated except that (+)-δ- benzoyl-l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic chloro- ethyl ester is used as the substrate to obtain (+)-5-benzoyl- l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic acid. EXAMPLE 5 The procedure of Example 1 is repeated except that (+)- l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic chloroethyl ester is used as the substrate to obtain optically-active 1,2- dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic acid. EXAMPLE 6 The procedure of Example 1 is repeated except that (+)-5- [4-methoxybenzoyl]-l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carbox- ylic chloroethyl ester is used as the substrate to obtain (+) * -•_•- [4-methoxybenzoyl]-l,2-dihydro-3H-pyrrolo[1,2a]pyrrole-l-carbox- ylic acid. EXAMPLE 7 The procedure of Example 1 is repeated except that (+_)-5- benzoyl-1,2-dihydro-3H-pyrrolo[l, a]pyrrole-l-carboxylic dodecyl ester is used as the substrate to obtain (+)-5-benzoyl-l,2- dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic acid. EXAMPLE 8 The procedure of Example 1 was repeated except that 30 mg of Mucor meihei lipase (Amano, 10,000 ILu/gm, MAP) was used instead as the enzyme to obtain (+)-5-benzoyl-l,2-dihydro-3H- pyrrolo[l,2a]pyrrole-l-carboxylic acid (ee_= 0.94) and (-)-5- benzoyl-1,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic methyl ester (ee_= 0.90). EXAMPLE 9 The procedure of Example 2 is repeated except that 20 mg of Rhizopus oryzae lipase (Amano, 750,000 ILu/gm, FAP), is used as the enzyme to obtain optically-active l,2-dihydro-3H-pyrrolo- [l,2a]pyrrole-l-carboxylic acid. EXAMPLE 10 The procedure of Example 1 is repeated using 2500 units of Chromobacterium violaceum lipase (Type XII, Sigma) as the enzyme to obtain optically-active 5-benzoyl-l,2-dihydro-3H-pyrrolo- [l,2a]pyrrole-l-carboxylic acid. EXAMPLE 11 The procedure of Example 1 was repeated using 10 mg of Pseudomonas lipo-protein lipase 80 (Amano, 800 u/gm) as the enzyme to obtain optically-active (+)-5-benzoyl-l,2-dihydro-3H- pyrrolo[l,2a]pyrrole-l-carboxylic acid (ee_= 0.90) and (-)-5- benzoyl-l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxyllc methyl ester (ee_= 0.93). EXAMPLE 12 The procedure of Example 1 is repeated using 10 mg of puri¬ fied Geotrichum candidum (ATCC 34614) lipase [Y. Tsujisaka et al., Agr. Biol. Chen.. 37_ 1457 (1973)] as the enzyme to obtain optically-active 5-benzoyl-l,2-dihydro-3H-pyrrolo[l,2a]pyrrole- 1-carboxylic acid. EXAMPLE 13 The procedure of Example 1 is repeated using 200 mg of crude lipase of Penicillium cyclopiu (ATCC 34613) [M. Iwai et al., Agr. Biol. Chea.. 39, 1063 (1975)] as the enzyme to obtain optically-active 5-benzoyl-l,2-dihydro-3H-pyrrolo[l,2a]pyrrole- 1-carboxylic acid. EXAMPLE 14 The procedure of Example 1 is repeated using 200 mg of Humicola lanuginosa lipase (Amano) as the enzyme to obtain opti¬ cally-active 5-benzoyl-l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l- carboxylic acid. EXAMPLE 15 The procedure of Example 1 was repeated using 100 mg of Aspergillus niger lipase (Amano K-10, 10,000 ILu/gm) as the enzyme to obtain optically-active (+) -5-benzoyl-l , 2-dihydro-3H- pyrrolo[l , 2a]pyrrole-l-carboxylic acid (ee_ = 0.88) and (-)-5- benzoyl-1 , 2-dihydro-3H-pyrrolo [l , 2a]pyrrole-l-carboxylic methyl ester (ee_ = 0.30) . EXAMPLE 16 The procedure of Example 1 was repeated using 50 mg of Rhizopus niveus lipase (Amano, 45,000 ILu/gm, N) as the enzyme to obtain optically-active (+)-5-benzoyl-l,2-dihydro-3H-pyr- rolo[l,2a]pyrrole-l-carboxylic acid (ee_= 0.80) and (-)-5-ben- zoyl-l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic methyl ester (ee_= 0.08). EXAMPLE 17 The procedure of Example 2 is repeated using 200 mg of Mucor eihei lipase (Amano) as the enzyme to obtain optically- active l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic acid. EXAMPLE 18 The procedure of Example 2 is repeated using 2,000 units of Rhizopus delemar lipase (Chemical Dynamics Corp., 5,000 units/ mg) as the enzyme to obtain optically-active 1,2-dihydro-3H- pyrrolo[l,2a]pyrrole-l-carboxylic acid. EXAMPLE 19 The procedure of Example 2 is repeated using Aspergillus niger lipase (100 mg) (Amano K-10) as the enzyme to obtain opti¬ cally-active l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic acid. EXAMPLE 20 The procedure of Example 2 is repeated using 30 mg of Pseudomonas lipase (Amano LPL-80) as the enzyme to obtain opti¬ cally-active 1,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic acid. EXAMPLE 21 The procedure of Example 3 is repeated using 100 mg of Rhizopus niveus lipase (Amano , N) as the enzyme to obtain optically-active 5-[4-methoxybenzoyl]-l , 2-dihydro-3H-pyr- rolo[l , 2a]pyrrole-l-carboxylic acid. EXAMPLE 22 The procedure of Example 3 is repeated using Aspergillus niger lipase (Amano AP, 120,000 Lu/gm) as the enzyme to obtain optically-active (+)-5-[4-methoxybenzoyl]-l,2-dihydro-3H-pyr- rolo[l,2a]pyrrole-l-carboxylic acid. EXAMPLE 23 The procedure of Example 6 is repeated using 30 mg of Rhizopus oryzae lipase (Amano, FAP) as the enzyme to obtain optically-active (+)-5-[4-methoxybenzoyl]-l,2-dihydro-3H-pyr- rolo[l,2a]pyrrole-l-carboxylic acid. EXAMPLE 24 The procedure of Example 4 is repeated using 50 mg of Mucor meihei lipase (Amano, MAP) as the enzyme to obtain optically- active (+)-5-benzoyl-l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-car- boxylic acid. EXAMPLE 25 The procedure of Example 5 is repeated using 50 mg of Mucor neihei lipase (Amano, MAP) as the enzyme to obtain optically- active l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic acid. EXAMPLE 26 To a suspension of Streptomyces griseus protease (Sigma type XXI 15-20 units per mg solid, P0652) (6 mg) in 2 ml of 0.2 M potassium phosphate buffer, pH 8.0, was added 96 mg of (+)-5-benzoyl-l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic methyl ester. The reaction mixture was stirred with a magnetic stirrer for 74 hrs at 25°C. The contents were then acidified with HC1 and exhaustively extracted with CH2CI2 three times. The combined organic extract was dried over sodium sulfate and was then evaporated to dryness. The residue was suspended in 5% NaHC03 and extracted with hexane to obtain 40 mg of (+)-5- benzoyl-1,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic methyl ester (ee_= 0.96). Acidification of the aqueous NaHC0 3 layer with HC1 to pH 2.0, followed by extraction with CH2CI2 gave 39 mg of (-)-5-benzoyl-l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l- carboxylic acid (ee_= 0.96). EXAMPLE 27 The procedure of Example 26 was repeated using 24 mg of Streptomyces griseus protease (Sigma type XIV, pronase E, P5147) and 110 mg of (+)-5-benzoyl-l,2-dihydro-3H-pyrrolo[l,2a]pyrrσle- l-carboxylic methyl ester. The incubation mixture was stirred with a magnetic stirrer for 312 hrs at 25°C. Using the same workup procedure, 58 mg of (+)-5-benzoyl-l,2-dihydro-3H-pyr- rolo[l,2a]pyrrole-l-carboxylic methyl ester (ee_= 0.46) and 28 mg of (-)-5-benzoyl-l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-car- boxylic acid (ee_= 0.96) were obtained. EXAMPLE 28 The procedure of Example 26 was repeated using 88 mg of Aspergillus saitoi protease (Sigma type XIII, 0.3 unit per mg solid, P2143) and 114 mg of (+)-5-benzoyl-l,2-dihydro-3H-pyr- rolo[l,2a]pyrroIe-carboxylic methyl ester as the substrate. The reaction mixture was incubated at 25°C for 312 hrs with stirring using the same workup procedure. 52 mg of (-)-5-benzoyl-l,2- dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic methyl ester (ee = 0.80) and 40 mg of (+)-5-benzoyl-l,2-dihydro-3H-pyrroIo[l,2a]- pyrrole-1-carboxylic acid (e_= 0.96) were obtained. EXAMPLE 29 The procedure of Example 26 was repeated using 49 mg of Aspergillus sojae protease (Sigma Type XIX, 0.4 units per mg solid, P7026) and 147 mg of (+)-5-benzoyl-l,2-dihydro-3H-pyr- rolo[l,2a]pyrrole-l-carboxylic methyl ester as the substrate. The reaction mixture was stirred at 25°C for 23 hrs. Following the same workup procedure, 60 mg of (+)-5-benzoyl-l,2-dihydro- 3H-pyrrolo[l,2a]pyrrole-l-carboxylic methyl ester (ee_= 0.92) and 68 mg of (-)-5-benzoyl-l,2-dihydro-3H-pyrrolo[l,2a]pyrrole- 1-carboxylic acid (ee_= 0.76) were obtained. EXAMPLE 30 The procedure of Example 26 was repeated using 62 mg of Rhizopus sp. protease (Sigma type XVIII, 0.5 unit per mg solid, P5027) and 117 mg of (+)-5-benzoyl-l,2-dihydro-3H-pyrrolo[l,2a]- pyrrole-1-carboxylic methyl ester as the substrate. The reac¬ tion mixture was gently stirred at 25°C for 165 hrs. Following the same workup procedure, 90 mg of (-)-5-benzoyl-l,2-dihydro- 3H-pyrrolo[l,2a]pyrrole-l-carboxylic methyl ester (ee_= 0.12) and 13 mg of (+)-5-benzoyl-l,2-dihydro-3H-pyrrolo[l,2a]pyrrole- 1-carboxylic acid (ee_= 0.62) were obtained. EXAMPLE 31 The procedure of Example 26 was repeated using 75 mg of Aspergillus oryzae protease (Sigma type XXIII, 4 units per mg solid, P-4032) and 87 mg of (+)-5-benzoyl-l,2-dihydro-3H-pyr- rolo[l,2a]pyrrole-l-carboxylic methyl ester as the substrate. The reaction mixture was gently stirred at 25°C for 23 hrs. Following the same workup procedure, 5 mg of (+)-5-benzoyl-l,2- dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic methyl ester (ee = 0.92) and 72 mg of (-)-5-benzoyl-l,2-dihydro-3H-pyrrolo[l,2a]- pyrrole-1-carboxylic acid (ee_= 0.08) were obtained. EXAMPLE 32 The procedure of Example 26 was repeated using 19 mg of Bacillus subtilis protease (Amano protease N, 1800 northrop units per gram) and 77 mg of ( -_)-5-benzoyl-l,2-dihydro-3H-pyr- rolo[l,2a]pyrrole-l-carboxylic methyl ester as the substrate. The reaction mixture was gently stirred at 25°C for 74 hrs. Following the same workup procedure, 48 mg of (+)-5-benzoyl-l,2- dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic methyl ester (ee = 0.44) and 19 mg of (-)-5-benzoyl-l,2-dihydro-3H-pyrrolo[l,2a]- pyrrole-1-carboxylic acid (ee_= 0.96) were obtained. EXAMPLE 33 The procedure of Example 26 was repeated using 30 mg of Aspergillus oryzae protease [Amano 2A fungal protease (neutral), 20,000 units/gm] and 85 mg of (+)-5-benzoyl-l,2-dihydro-3H-pyr- rolo[l,2a]pyrrole-l-carboxylic methyl ester as the substrate. The reaction mixture was gently stirred at 25 β C for 74 hrs. Following the same workup procedure, 32 mg of (+)-5-benzoyl-l,2- dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic methyl ester (ee - 0.96) and 41 mg of (-)-5-benzoyl-l,2-dihydro-3H-pyrrolo[l,2a]- pyrrole-1-carboxylic acid (ee_= 0.70) were obtained. EXAMPLE 34 To a suspension of Streptomyces griseus protease (Sigma type XXI, 15-20 units per mg solid P-0652) (3 mg) in 1 ml of 0.3 M potassium borate buffer, pH 9.8, was added 30 mg of (+)-5- benzoyl-l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic ethyl ester. The reaction mixture was stirred with a magnetic stirrer for 48 hrs at 25 β C. The contents were then acidified with HCl to pH 2.0 and exhaustively extracted with CH 2 Cl2 three times. The combined organic extracts were dried over sodium sulfate and were then evaporated to dryness to yield 22 mg of (-)-δ-benzoyl- l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic acid (ee - 0.80). The enantiomeric excess (ee) of the acid was raised to 0.96 by recrystallization of the acid from hexane-ethyl acetate, m.p. 170°C. It will be obvious to those skilled in the art that the process of this invention as set forth hereinbefore can be modified and perhaps improved by various means. For example, the process may be made continuous wherein the enzyme is immobilized and recycled several times to reduce cost; the (+)-ester can be recovered, racemized, and reused; or the substrate can be exposed to the enzyme as a microcrystalline powder to obtain better dispersion. Furthermore, it may be pos¬ sible to dissolve the (+)-substrate and a racemization agent in a suitable solvent so only the ester will be continuously race¬ mized in situ without cleaving the ester grouping. This not process not only facilitates product isolation but also is equivalent to second-order asymmetric transformation tAsymmetric Synthesis. Vol. 1, edited by J. D. Morris and J. W. Scott, Aca¬ demic Press, Inc., N.Y., 1983, pp. 3-6). This is Illustrated by the procedure of Example 34. Also, activators and stabilizers of the lipase [N. Tomizuka et al., Agr. Biol. Chem., 30, 576 (1966)] may be introduced to the incubation mixture or sub¬ strates possessing many different types of activated esters (Bodanszky et al., Peptide Synthesis, Second Ed.., Wiley, 1976, pp. 99-108) may be used to enhance the rate of conversion. In addition, active site directed mutagenesis or chemical modifica¬ tion of the enzyme may be used to prepare enzymes with improved V maχ /K a and/or stability.

Claims: Claims 1. A process for preparing optically-active 1,2-dihydro-3H- pyrroIo[l,2a]pyrroIe-l-carboxylic acids which comprises subjecting esters of racemic l,2-dihydro-3H-pyrrolo[l,2a]- pyrrole-l-carboxylic acids to the hydrolytic enzymatic action of an extracellular microbial enzyme selected from the group consisting of microbial lipase (EC 3.1.1.3) and microbial protease and recovering the desired optically- active compounds. 2. The process of Clain 1 wherein the racemic esters have the formula Where R j is a radical in straight chain, branched chain or cyclic configuration selected from the class con¬ sisting of alkane radicals having 1 to about 12 carbon atoms with or without electronegative substituents at C-2' ; cycloalkane radicals having from about 5 to about 7 carbon atoms; and phenyl and benzyl radicals having from about 6 to about 8 carbons; and . R2 is a hydrogen or an acyl radical in straight chain, branched chain or cyclic configuration having about 2 to about 12 carbon atoms, substituted or unsubstituted radi¬ cals comprised of cycloalkane radicals having about 5 to about 7 carbon atoms, benzoyl, naphthoyl, biphenoyl and carbobenzoxyl radicals wherein substituents on the aromatic ring(s) are comprised of as nitro, halogen, methyl, and alkoxyl groups. 3. The process of Claim 2 wherein R 2 is selected from the group consisting of hydrogen, benzoyl, and methoxybenzoyl groups. 4. The process of Claim 1 wherein the racemic ester is an activated ester. δ. The process of Claim 1 wherein an activator for the lipase is present. 6. The process of Claim 1 wherein the enzyme is immobilized. 7. The process of Claim 1 wherein the substrate racemic ester is (+J-5-benzoyl-l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l- carboxylic chloroethyl ester. 8. The process of Claim 1 wherein the substrate racemic ester is (+)-5-benzoyl-l,2-dihydro-3H-pyrrσlo[l,2a]pyrrole-l- carboxylic methyl ester. 9. The process of Claim 1 wherein the substrate racemic ester is (+_)-l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic chloroethyl ester. 10. The process of Claim 1 wherein the substrate racemic ester is ( )-l,2-dihydro-3H-pyrrolo[l,2a]pyrrole-l-carboxylic methyl ester. 11. The process of Claim 1 wherein the substrate racemic ester is (+)-5-[4-methoxybenzoyl]-l,2-dihydro-3H-pyr- rolo[l,2a]pyrrole-l-carboxylic chloroethyl ester. 12. The process of Claim 1 wherein the substrate racemic ester is (+)-δ-[4-methoxybenzoyl]-l,2-dihydro-3H-pyr- rolo[l,2a]pyrrole-l-carboxylic methyl ester. 13. The process of Claim 1 wherein the lipase is an extracellu¬ lar lipase derived from microorganisms selected from the genera consisting of Candida, Rhizopus, Mucor, Aspergillus, Penicilliu , Pseudomonas, Chromobacterium. and Geotrichium. 14. The process of Claim 8 wherein the lipase is derived from Mucor meihei. 15. The process of Claim 9 wherein the lipase is derived from Candida cylindracea. 16. The process of Claim lδ wherein the lipase is Meito Sangyo OF-360,000 u/gm. 17. The process of Claim 10 wherein the lipase is derived from Rhizopus delemar. 18. The process of Claim 1 wherein the protease is an extracel¬ lular protease derived from a microorganism selected from the genera Streptomyces, Bacillus, Aspergillus, and Rhizopus. 19. The process of Claim 8 wherein the protease is derived from Streptomyces griseus. 20. The process of Claim 8 wherein the protease is derived from Aspergillus sojae. 21. The process of Claim 8 wherein the protease is derived from Bacillus subtilis. 22. The process of Claim 8 wherein the protease is derived from Aspergillus oryzae.

Inventors: SIH CHARLES J, SIH, CHARLES, J.
Applicants: WISCONSIN ALUMNI RES FOUND, WISCONSIN ALUMNI RESEARCH FOUNDATION
Application year: 1987
Main classification: C12P17
IPC codes: C07D487, C12P17, C12P41