WO1987006266A1 - ENZYMATIC PROCESS FOR PREPARING OPTICALLY-ACTIVE 1,2-DIHYDRO-3H-PYRROLO[1,2a]PYRROLE-1-CARBOXYLIC ACID DERIVATIVES - Google Patents

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

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WO1987006266A1
WO1987006266A1 PCT/US1987/000725 US8700725W WO8706266A1 WO 1987006266 A1 WO1987006266 A1 WO 1987006266A1 US 8700725 W US8700725 W US 8700725W WO 8706266 A1 WO8706266 A1 WO 8706266A1
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dihydro
pyrrole
pyrrolo
ester
lipase
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PCT/US1987/000725
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French (fr)
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Charles J. Sih
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Wisconsin Alumni Research Foundation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
    • C12P41/005—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of carboxylic acid groups in the enantiomers or the inverse reaction

Definitions

  • the present invention relates to a novel process for producing optically-active 1,2-dihydro-3H-pyrrolo[1,2a] ⁇ yrrole-1- carboxylic acid derivatives. Specifically, it relates to a process for the enzymatic enantiospedfic hydrolysis of racemic 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid esters to give optically-active 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1- carboxylic acids.
  • Ketorolac 5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1- carboxylic acid (1), a structural analog of zomepirac [J. 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 equivalent 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.
  • this invention comprises the use of extracellular microbial enzymes selected from the group consisting of lipases and proteases to catalyze the enantiospedfic hydrolysis of racemic 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid esters as hereinbelow defined.
  • R 1 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 radicals 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, and carboxylates);
  • R 2 is an acyl radical in straight chain, branched chain or cyclic configuration having 2 to about 12 carbon atoms, cycloalkane 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.
  • aroyl radicals which are eminently suitable for the purposes of the present invention are benzoyl and methoxybenzoyl.
  • 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[1,2a]pyrrole-1-carboxylic acid (1) using extracellular inexpensive microbial upases and proteases.
  • the process of the invention comprises subjecting said 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic ester to the hydrolytic action of a microbial lipase (EC 3.1.1.3) or protease and recovering the desired optically-active 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid derivatives.
  • a microbial lipase EC 3.1.1.3
  • protease microbial lipase
  • extracellular microbial lipases and proteases are capable ,of functioning to catalyze the desired enantiospedfic hydrolysis.
  • lipases derived from the microorganisms of the genera Candida, Rhizopus, Hucor. Aspergillus. Penicillium, Geotrichium, Hurmicola, Pseudomonas and Chromobacterium.
  • 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. Borgström and H. L. Brockman, Elsevier, N.Y., 1984). For example, 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).
  • the 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic ester substrate may be added in solid or liquid forms at concentrations of 0.1-5 M to a suitable buffer solution containing the lipase to effect the enantiospedfic hydrolysis.
  • 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.
  • the substrate may be emulsified by the use of polyvinyl alcohol or propylene glycol.
  • 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.
  • the temperature can range from about 10°C to about 40oC and the pH of the medium can range from 3 to about 8.5.
  • esters of ( ⁇ )-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-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 .
  • reaction mixture was stirred with a magnetic stirrer for 2 days at 24°C.
  • contents were then acidified with HCl and exhaustively extracted with ethyl acetate three times.
  • the combined organic extract was dried over sodium sulfate and was then evaporated to dryness.
  • Example 1 The procedure of Example 1 is repeated except that ( ⁇ )-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic methyl ester is used as the substrate to obtain optically-active 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid in good yield.
  • Example 1 The procedure of Example 1 is repeated except that ( ⁇ )-5- [4-raethoxybenzoyl]-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic methyl ester is used as the substrate to obtain (+)-5-[4-methoxybenzoyl]-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid (anirolac) in good yield.
  • EXAMPLE 4 The procedure of Example 1 is repeated except that ( ⁇ )-5- benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic chloroethyl ester is used as the substrate to obtain (+)-5-benzoyl- 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid.
  • Example 1 The procedure of Example 1 is repeated except that ( ⁇ )-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic dodecyl ester is used as the substrate to obtain (+)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid.
  • Mucor meihei lipase Mano, 10,000 ILu/gm, MAP
  • 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 1,2-dihydro-3H-pyrrolo-[1,2a]pyrrole-1-carboxylic acid.
  • Rhizopus oryzae lipase Mano, 750,000 ILu/gm, FAP
  • 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-1,2-dihydro-3H-pyrrolo-[1,2a]pyrrole-1-carboxylic acid.
  • Chromobacterium violaceum lipase Type XII, Sigma
  • Example 12 The procedure of Example 1 is repeated using 10 mg of purified Geotrichum candidum (ATCC 34614) lipase [Y. Tsujisaka et al., Agr. Biol. Chen., 37 1457 (1973)] as the enzyme to obtain optically-active 5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid.
  • ATCC 34614 purified Geotrichum candidum lipase
  • Example 1 The procedure of Example 1 is repeated using 200 mg of crude lipase of Penicillium cyclopium (ATCC 34613) [M. Iwai et al., Agr. Biol. Chea.. 39, 1063 (1975)] as the enzyme to obtain optically-active 5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]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 optically-active 5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid.
  • Humicola lanuginosa lipase Amano
  • Example 2 The procedure of Example 2 is repeated using 200 mg of Mucor meihei lipase (Amano) as the enzyme to obtain opticallyactive 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid.
  • Mano Mucor meihei lipase
  • Example 2 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[1,2a]pyrrole-1-carboxylic acid.
  • Example 2 The procedure of Example 2 is repeated using Aspergillus niger lipase (100 mg) (Amano K-10) as the enzyme to obtain optically-active 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-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 optically-active 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-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]-1 , 2-dihydro-3H-pyrrolo[1 , 2a]pyrrole-1-carboxylic acid.
  • Example 3 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]-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid.
  • Aspergillus niger lipase Aspergillus niger lipase (Amano AP, 120,000 Lu/gm) as the enzyme to obtain optically-active (+)-5-[4-methoxybenzoyl]-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid.
  • Example 6 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]-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid.
  • Rhizopus oryzae lipase Amano, FAP
  • EXAMPLE 24 The procedure of Example 4 is repeated using 50 mg of Mucor meihei lipase (Amano, MAP) as the enzyme to obtain opticallyactive (+)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid.
  • Mucor meihei lipase Amano, MAP
  • EXAMPLE 25 The procedure of Example 5 is repeated using 50 mg of Mucor neihei lipase (Amano, MAP) as the enzyme to obtain opticallyactive 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid.
  • Mucor neihei lipase Amano, MAP
  • Example 26 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-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic methyl ester. The incubation mixture was stirred with a magnetic stirrer for 312 hrs at 25°C.
  • 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-1,2-dihydro-3H-pyrrolo[1,2a]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.
  • Aspergillus saitoi protease Sigma type XIII, 0.3 unit per mg solid, P2143
  • (+)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrroIe-carboxylic methyl ester was incubated at 25°C for 312 hrs with stirring using the same workup procedure.
  • Example 26 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-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic methyl ester as the substrate. The reaction mixture was stirred at 25°C for 23 hrs.
  • Example 26 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-1,2-dihydro-3H-pyrrolo[1,2a]-pyrrole-1-carboxylic methyl ester as the substrate. The reaction mixture was gently stirred at 25°C for 165 hrs.
  • Example 26 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-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic methyl ester as the substrate. The reaction mixture was gently stirred at 25°C for 23 hrs.
  • 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 ( ⁇ )-)-benzoyl-1,2-dihydro-3H-pyr rolo[1,2a]pyrrole-1-carboxylic methyl ester as the substrate. The reaction mixture was gently stirred at 25°C for 74 hrs.
  • Example 26 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-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic methyl ester as the substrate.
  • the reaction mixture was gently stirred at 25oC for 74 hrs.
  • 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.
  • it may be possible to dissolve the (+)-substrate and a racemization agent in a suitable solvent so only the ester will be continuously racemized in situ without cleaving the ester grouping.
  • This not process not only facilitates product isolation but also is equivalent to second-order asymmetric transformation (Asymmetric Synthesis. Vol. 1, edited by J. D. Morris and J. W. Scott, Academic Press, Inc., N.Y., 1983, pp. 3-6). This is Illustrated by the procedure of Example 34.
  • activators and stabilizers of the lipase may be introduced to the incubation mixture or substrates 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.
  • active site directed mutagenesis or chemical modification of the enzyme may be used to prepare enzymes with improved V max /K m and/or stability.

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[1,2a]pyrrole-1-carboxylic Acid
Derivatives
Technical Field
The present invention relates to a novel process for producing optically-active 1,2-dihydro-3H-pyrrolo[1,2a]ρyrrole-1- carboxylic acid derivatives. Specifically, it relates to a process for the enzymatic enantiospedfic hydrolysis of racemic 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid esters to give optically-active 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1- carboxylic acids.
Background
Ketorolac, 5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1- carboxylic acid (1), a structural analog of zomepirac [J. 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 equivalent 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.
Figure imgf000003_0001
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 lipases and proteases to catalyze the enantiospedfic hydrolysis of racemic 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid esters as hereinbelow defined.
Wherein:
R1 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 radicals 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, and carboxylates);
R2 is an acyl radical in straight chain, branched chain or cyclic configuration having 2 to about 12 carbon atoms, cycloalkane 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 methoxybenzoyl.
It is the object of this invention to produce opticallyactive 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-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[1,2a]pyrrole-1-carboxylic acid (1) using extracellular inexpensive microbial upases and proteases.
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[1,2a]pyrrole-1-carboxylic ester to the hydrolytic action of a microbial lipase (EC 3.1.1.3) or protease and recovering the desired optically-active 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-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. Penicillium, Geotrichium, Hurmicola, Pseudomonas and Chromobacterium. 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. Borgström and H. L. Brockman, Elsevier, N.Y., 1984). For example, 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 feature 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 enzymatic hydrolytic reactions can be carried out in high concentrations (0.1-5 M) with an unusually high degree of enantiospecificity. Moreover, they are remarkably stable under the desired reaction conditions, so that they may be reused.
The 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic ester substrate may be added in solid or liquid forms at concentrations 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 polyvinyl 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 40ºC 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 (±)-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-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-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-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 HCl 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% NaHCO3 and extracted with hexane to obtain (-)-5-benzoyl-1,2-dibydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic methyl ester (ee = 0.94). Acidification of the aqueous layer with HCl to pH 2.0, followed by extraction with dichlororaethane gives (+)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid (ee = 0.68).
EXAMPLE 2
The procedure of Example 1 is repeated except that (±)-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic methyl ester is used as the substrate to obtain optically-active 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid in good yield.
EXAMPLE 3
The procedure of Example 1 is repeated except that (±)-5- [4-raethoxybenzoyl]-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic methyl ester is used as the substrate to obtain (+)-5-[4-methoxybenzoyl]-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid (anirolac) in good yield.
EXAMPLE 4 The procedure of Example 1 is repeated except that (±)-5- benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic chloroethyl ester is used as the substrate to obtain (+)-5-benzoyl- 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid. EXAMPLE 5 The procedure of Example 1 is repeated except that (±)-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic chloroethyl ester is used as the substrate to obtain optically-active 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid.
EXAMPLE 6 The procedure of Example 1 is repeated except that (±)-5-[4-methoxybenzoyl]-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic chloroethyl ester is used as the substrate to obtain (+)-5-[4-methoxybenzoyl]-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid.
EXAMPLE 7
The procedure of Example 1 is repeated except that (±)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic dodecyl ester is used as the substrate to obtain (+)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-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-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid (ee = 0.94) and (-)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-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 1,2-dihydro-3H-pyrrolo-[1,2a]pyrrole-1-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-1,2-dihydro-3H-pyrrolo-[1,2a]pyrrole-1-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-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid (ee = 0.90) and (-)-5- benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxyllc methyl ester (ee = 0.93).
EXAMPLE 12 The procedure of Example 1 is repeated using 10 mg of purified Geotrichum candidum (ATCC 34614) lipase [Y. Tsujisaka et al., Agr. Biol. Chen., 37 1457 (1973)] as the enzyme to obtain optically-active 5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid.
EXAMPLE 13
The procedure of Example 1 is repeated using 200 mg of crude lipase of Penicillium cyclopium (ATCC 34613) [M. Iwai et al., Agr. Biol. Chea.. 39, 1063 (1975)] as the enzyme to obtain optically-active 5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]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 optically-active 5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-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-1 , 2-dihydro-3H-pyrrolo[1 , 2a]pyrrole-1-carboxylic acid (ee = 0.88) and (-)-5-benzoyl-1 , 2-dihydro-3H-pyrrolo [1 , 2a]pyrrole-1-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-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid (ee = 0.80) and (-)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic methyl ester (ee = 0.08).
EXAMPLE 17
The procedure of Example 2 is repeated using 200 mg of Mucor meihei lipase (Amano) as the enzyme to obtain opticallyactive 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-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[1,2a]pyrrole-1-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 optically-active 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-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 optically-active 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-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]-1 , 2-dihydro-3H-pyrrolo[1 , 2a]pyrrole-1-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]-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-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]-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-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 opticallyactive (+)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic acid.
EXAMPLE 25 The procedure of Example 5 is repeated using 50 mg of Mucor neihei lipase (Amano, MAP) as the enzyme to obtain opticallyactive 1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-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-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic methyl ester. The reaction mixture was stirred with a magnetic stirrer for 74 hrs at 25°C. The contents were then acidified with HCl 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% NaHCO3 and extracted with hexane to obtain 40 mg of (+)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic methyl ester (ee = 0.96). Acidification of the aqueous NaHCO3 layer with HCl to pH 2.0, followed by extraction with CH2CI2 gave 39 mg of (-)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-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-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-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-1,2-dihydro-3H-pyr¬rolo[1,2a]pyrrole-1-carboxylic methyl ester (ee = 0.46) and 28 mg of (-)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic 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-1,2-dihydro-3H-pyrrolo[1,2a]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-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic methyl ester (ee = 0.80) and 40 mg of (+)-5-benzoyl-1,2-dihydro-3H-pyrroIo[1,2a]-pyrrole-1-carboxylic acid (ee = 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-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-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-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic methyl ester (ee = 0.92) and 68 mg of (-)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]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-1,2-dihydro-3H-pyrrolo[1,2a]-pyrrole-1-carboxylic methyl ester as the substrate. The reaction mixture was gently stirred at 25°C for 165 hrs. Following the same workup procedure, 90 mg of (-)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic methyl ester (ee = 0.12) and 13 mg of (+)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]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-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-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-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic methyl ester (ee = 0.92) and 72 mg of (-)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]- 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 (±)-)-benzoyl-1,2-dihydro-3H-pyr rolo[1,2a]pyrrole-1-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-1,2- dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic methyl ester (ee = 0.44) and 19 mg of (-)-5-benzoyl-1,2-dihydro-3H-pyrrolo[l12a]-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-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic methyl ester as the substrate. The reaction mixture was gently stirred at 25ºC for 74 hrs. Following the same workup procedure, 32 mg of (+)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic methyl ester (ee = 0.96) and 41 mg of (-)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]-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-1,2-dihydro-3H-pyrrolo[l,2a]pyrrole-1-carboxylic ethyl ester. The reaction mixture was stirred with a magnetic stirrer for 48 hrs at 25ºC. The contents were then acidified with HCl to pH 2.0 and exhaustively extracted with CH2Cl2 three times. The combined organic extracts were dried over sodium sulfate and were then evaporated to dryness to yield 22 mg of (-)-5-benzoyl-1,2-dihydro-3H-pyrrolo[l,2a]pyrrole-1-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 possible to dissolve the (+)-substrate and a racemization agent in a suitable solvent so only the ester will be continuously racemized in situ without cleaving the ester grouping. This not process not only facilitates product isolation but also is equivalent to second-order asymmetric transformation (Asymmetric Synthesis. Vol. 1, edited by J. D. Morris and J. W. Scott, Academic 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 substrates 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 modification of the enzyme may be used to prepare enzymes with improved Vmax/Km and/or stability.

Claims

Claims
1. A process for preparing optically-active 1,2-dihydro-3H- pyrroIo[1,2a]pyrroIe-1-carboxylic acids which comprises subjecting esters of racemic 1,2-dihydro-3H-pyrrolo[1,2a]- pyrrole-1-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 opticallyactive compounds.
2. The process of Clain 1 wherein the racemic esters have the formula
Where R1 is a radical in straight chain, branched chain or cyclic configuration selected from the class consisting 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 radicals 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 R2 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.
5. 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 (±)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1- carboxylic chloroethyl ester.
8. The process of Claim 1 wherein the substrate racemic ester is (±)-5-benzoyl-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1- carboxylic methyl ester.
9. The process of Claim 1 wherein the substrate racemic ester is (±)-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic chloroethyl ester.
10. The process of Claim 1 wherein the substrate racemic ester is (±)-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic methyl ester.
11. The process of Claim 1 wherein the substrate racemic ester is (±)-5-[4-methoxybenzoyl]-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic chloroethyl ester.
12. The process of Claim 1 wherein the substrate racemic ester is (±)-5-[4-methoxybenzoyl]-1,2-dihydro-3H-pyrrolo[1,2a]pyrrole-1-carboxylic methyl ester.
13. The process of Claim 1 wherein the lipase is an extracellular lipase derived from microorganisms selected from the genera consisting of Candida, Rhizopus, Mucor, Aspergillus, Penicillium, 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 15 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 extracellular 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.
PCT/US1987/000725 1986-04-16 1987-04-02 ENZYMATIC PROCESS FOR PREPARING OPTICALLY-ACTIVE 1,2-DIHYDRO-3H-PYRROLO[1,2a]PYRROLE-1-CARBOXYLIC ACID DERIVATIVES WO1987006266A1 (en)

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WO1994013283A1 (en) * 1992-12-17 1994-06-23 Sepracor Inc. Antipyretic and analgesic methods and compositions containing optically pure r-ketorolac
US5464609A (en) * 1990-03-16 1995-11-07 The Procter & Gamble Company Use of ketorolac for treatment of oral diseases and conditions
GB2295148A (en) * 1994-11-16 1996-05-22 Dong Kook Pharm Co Ltd Pyrrolizine derivatives
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CN115181105A (en) * 2022-08-26 2022-10-14 长春亿诺科医药科技有限责任公司 Preparation method and application of R-type ketorolac

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US5464609A (en) * 1990-03-16 1995-11-07 The Procter & Gamble Company Use of ketorolac for treatment of oral diseases and conditions
US5646174A (en) * 1990-03-16 1997-07-08 Kelm; Gary Robert Use of ketorolac for treatment of oral diseases and conditions
US5785951A (en) * 1990-03-16 1998-07-28 The Procter & Gamble Company Use of ketorolac for treatment of oral diseases and conditions
WO1994013283A1 (en) * 1992-12-17 1994-06-23 Sepracor Inc. Antipyretic and analgesic methods and compositions containing optically pure r-ketorolac
GB2295148A (en) * 1994-11-16 1996-05-22 Dong Kook Pharm Co Ltd Pyrrolizine derivatives
ES2101653A1 (en) * 1995-07-10 1997-07-01 Astupaharma S A (+)-6-(5-chloropyrid-2-yl)-7-oxo-vinyloxycarbonyloxy-5,6- dihydropyrrole (3,4b) pyrazine and its use in a process for the preparation of (+)-6-(5-chloropyrid-2-yl)-5-(4- methylpiperazin-1-yl)-carbonyloxy-7-oxo-5,6- dihydropyrrole (3,4b) pyrazine

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