Process for the enzymatic racemate cleavage of racemic alcohols with/in vinyl esters by transesterification.

0001The invention relates to a process for the enzymatic resolution of racemic alcohols with or in vinyl esters by transesterification, wherein the vinyl ester is split into a ketone or an aldehyde and an "acid residue" and the remaining acid residue forms an ester enantioselectively with an added racemic alcohol and only one enantiomer of the alcohol remains unchanged. Esters and unreacted alcohol and thus both enantiomeric alcohols can be easily separated from one another. The second enantiomer of the alcohol can optionally be obtained by cleaving the ester.

0002Many optically active alcohols are important chiral precursors of biologically active substances (pharmaceuticals, natural substances, plant protection products) and an economical manufacturing process is therefore of great importance. Some pharmacological active ingredients, the presentation of which is facilitated by the process according to the invention, are, for example, NSAIDs (non-steroidal anti-inflammatory drugs) such as ibuprofen and naproxen, beta blockers such as nifenalol and penbutolol, bronchospasmolytics such as tolubuterol and bitolterol, antimycotics such as tioconazole, pyrethroids and pyrethroids such as in addition tetramisole, tetrahydrozoline, (R) - (-) - tomoxetine and (S) - (+) - fluoxetine as well as prostaglandins and carbohydrates.

0003It is known that vinyl esters can be transesterified with the addition of alcohols in the presence of solvents such as tetrahydrofuran under enzymatic catalysis (Degueil-Castaing et al., M. Tetrahedron Letters, Vol. 28, No. 9, pages 953-954, 1987). PPL (Pig Pancreatic Lipase) was used as the enzyme. Stereoselectivity was not observed in the reaction.

0004Atushi Makita et al. describe in Tetrahedron Letters, Vol. 28, No. 7, pages 805-808, 1987 the use of Lipase P for the "lactonization" of w-hydroxycarboxylic acid methyl esters in organic solvents such as cyclohexane, chloroform, hexane, heptane, benzene, toluene or dimethyl sulfoxide.

0005In German patent application P 36 24 703.0, it was proposed to present chiral compounds optically pure from prochiral diols by reaction with vinyl acetate in the presence of hydrolases. This is achieved by selective esterification of only one of the two enantiotopic primary OH groups.

0006It has now surprisingly been found that racemic alcohols can be separated enzymatically by subjecting them to a selective enzyme-catalyzed transesterification reaction with vinyl esters, it being possible to dispense with solvents. Lipases from pig liver and pig pancreas and from microorganisms such as Candida, Mucor, Rhizopus, Penicillium and in particular from Pseudomonas spec. Such as Lipase P and Lipase FP (Amano, Japan) can be used as enzymes.

0007This was all the more surprising since it was previously assumed that the carbonyl compounds (aldehydes or ketones) released react with the lipases and deactivate them.

0008The invention thus relates to:<ul id="ul0001" list-style="none"><li>A process for the enzymatic resolution of racemic alcohols, using a vinyl ester of the formula I,<chemistry id="chem0001" num="0001"><img file="EP0321918B1_D0001.tif" /></chemistry>in the<ul id="ul0002" list-style="none"><li>R¹ Hydrogen, C₁-C₁₈-Alkyl, which is optionally substituted with halogen, phenyl or (C1-C₃) Alkoxy- (G -C₄) Alkyl and</li><li>R² Means hydrogen or methyl,</li><li>in the presence of lipases from pig liver, pig pancreas and from microorganisms such as Candida, Mucor, Rhizopus, Penicillium, Aspergillus and Pseudomonas reacted with an alcohol</li><li>Formula 11,<chemistry id="chem0002" num="0002"><img file="EP0321918B1_D0002.tif" /></chemistry>in the</li><li>R³ C.₁-C₁₈-Alkyl or C3-Clo-Cycloalkyl means, where these radicals can also be halogen-substituted, and</li><li>R⁴ Epoxi-C₁-C₅-Alkyl means that the epoxy group is in the β-position to the OH group in the rest of the formula II or</li><li>R⁴ C.₁-C₁₀-Alkyl, C₁-C₁₀Alkenyl, C₂-C₁₀Alkynyl, C₃-C₈-Cycloalkenyl, the alkyl, alkenyl, alkynyl and cycloalkenyl radicals optionally with COOH, halogen, N0₂, CN, C₁-C₄-Alkoxycarbonyl or phenyl are substituted, the phenyl radical in turn with halogen, N0₂ , CN or C₁-C₄-Alkoxy may be substituted, or R⁴ Aryl or heteroaryl means, the aryl or heteroaryl radicals optionally with C1-C₄-Alkyl, C1-C₄-Alkoxy, halogen, N0₂, CN or N Sgr. are substituted, with Sgr. represents an amino protecting group, or in the</li><li>R³ and R⁴ together an alkylene or alkenylene radical of the formula Illa, b<chemistry id="chem0003" num="0003"><img file="EP0321918B1_D0003.tif" /></chemistry>represent in the</li><li>n is 1, 2 or 3 and</li><li>R⁵ and R⁶ are the same or different and are hydrogen, C₂-C₄-Alkenyl, C1-C4-alkyl or</li><li>R⁵ and R⁶ together fused phenyl or fused naphthyl, the phenyl or naphthyl radical optionally being C₁-C₄-Alkyl-, C₁-C₄-Alkoxy-, N0₂-, CN- or halogen-substituted, where a methylene unit of the alkenylene chain can also be replaced by a carbonyl group, and then isolated the resulting optically pure alcohol of formula II and optionally the other enantiomer from the remaining ester.</li></ul></li></ul>

0009Halogens are understood to mean fluorine, chlorine, bromine and iodine, in particular chlorine and bromine. “Aryl” means, for example, phenyl, naphthyl, phenanthryl, anthryl and fluorenyl, in particular phenyl, naphthyl and phenanthryl. "Heteroaryl" means, for example, furyl, thienyl, pyrrolyl, pyridyl, pyrimidyl, pyrazinyl, imidazolyl, oxazolyl, thiazolyl and indolyl, in particular furyl, thienyl, pyrrolyl and pyridyl. Under the amino protection group "Sgr." are understood the amino protecting groups usually used in peptide chemistry, for example benzyloxycarbonyl (Cbz = Z), benzoyl, benzyl, butyloxycarbonyl (Boc), 9-fluorenyl-methoxycarbonyl (Fmoc), benzhydryl, allyloxycarbonyl (Aloc), tosyl, methoxymethyl (MOM), Tetrahydropyrany (THP), acetyl, but also alkyl or cycloalkyl groups, such as N-methyl, N, N-dimethyl, or piperidine or morpholine. Under "fused phenyl" or "fused naphthyl" is understood to mean a phenyl or naphthyl radical in which the CC double bond of the radical of the formula III is part of the phenyl or naphthyl radical. The optionally substituted radicals R¹, R³, R⁴, R⁵ and R⁶ are preferably monosubstituted.

0010Alkyl and alkenyl radicals with 3 and more carbon atoms and alkynyl radicals with 4 and more carbon atoms can be either straight-chain or branched. Particular preference is given to using one of the compounds listed in Table 1 as the racemic alcohol of the formula II.

0011The process according to the invention has the following advantages over conventional processes for resolving alcohols:<ul id="ul0003" list-style="none"><li>a) At least one enantiomer is always (optically) highly pure, usually even both;</li><li>b) The enzyme used can be easily recovered;</li><li>c) The separation of the "ester" formed during the cleavage from the "alcohol" is very simple, ideally the two can be separated by distillation;</li><li>d) dispensing with an additional solvent;</li><li>e) High and fast turnover when using comparatively low amounts of enzyme.</li></ul>

0012In the process according to the invention, the vinyl ester of the formula I is split into carboxylic acid and the corresponding vinyl alcohol. The vinyl alcohol immediately rearranged into a ketone or an aldehyde. A reverse reaction is therefore completely prevented. Under the catalytic action of the lipases mentioned, one enantiomer from the racemic alcohol is selectively esterified quickly and in high yields, the other enantiomer remains unchanged in the reaction mixture.

0013The process according to the invention is particularly suitable for cleaving alcohols which have a CC double or triple bond in the β position to the OH group, or which have an epoxide or a double bond equivalent such as a C in this position₃- own ring.

0014The process according to the invention is best carried out by introducing the vinyl ester of the formula I, preferably vinyl acetate or vinyl chloroacetate, and adding both the enzyme and the alcohol to be split to this solution. A lipoprotein lipase from Pseudomonas spec., In particular Lipase P or Lipase FP, which are commercially available (Amano Pharmaceuticals, Nagoya, Japan), is preferably used as the enzyme. In addition, the enzyme can also be used in immobilized form, with all common immobilization methods and carriers being suitable for this. Immobilized enzyme and free enzyme can also be used in the column process. The amount of enzyme is chosen depending on the size of the batch, the reactivity of the alcohol, the desired reaction time and the type of enzyme (free or fixed) and can easily be determined in individual cases by simple preliminary tests. The alcohol to be split is used in concentrations of 1% to 200%, preferably from 10% to 40%, based on the volume of the vinyl or methyl vinyl ester used. In some cases it is even possible to present vinyl esters only in stoichiometric amounts, based on an enantiomer.

0015The reaction temperature is -10_°C to 100_°C, preferably 15_°C to 50 ° C. It is advisable to stir the solution during the reaction. The reaction times vary depending on the racemic alcohol used and depending on the amount of enzyme between a few hours and up to 4 weeks; however, the majority of cases are between 3 hours and 3 days.

0016Unless commercially available, the vinyl or methyl vinyl esters of the formula I can be prepared in a simple manner, for example by transesterification of vinyl acetate with the corresponding carboxylic acids.

0017The racemic alcohols of the formula 11 are obtained if they are not commercially available, for example by reduction from the corresponding ketones, which are usually commercially available, or by a-bromination of corresponding ketones with subsequent reduction to alcohol. Other non-commercially available alcohols or ketones can be easily prepared using processes known from the literature, for example using Grignard or other common addition reactions.

0018The products formed in the process according to the invention, acetaldehyde or acetone, carboxylic acid ester and alcohol, can be separated in a known manner. Ideally, the unreacted alcohol is obtained by separation by distillation. If separation by distillation is not possible, the separation is carried out chromatographically or by extraction or crystallization. One way of increasing the boiling point difference between the enzymatically formed lower ester and the unreacted alcohol is to use the corresponding vinyl a-halo-carboxylic acid ester instead of vinyl ester (e.g. vinyl a-chloroacetic acid ester).

0019If the other enantiomer is also to be shown in pure form, the ester is first isolated and this is split into acid and alcohol in a known manner.

0020This gives alcohols with enantiomeric purities of over 95% (ee value).

0021The enzyme used for the reactions can easily be recovered by filtration.

0022The invention is explained in detail in the following examples.

example

General working instructions

0023The racemic alcohol is dissolved or suspended in vinyl ester. Lipase P (or Lipase FP) is added in free or fixed form and the mixture is stirred at the temperature given in Table 1. When the reaction has ended, the enzyme is filtered off (both the free and the fixed enzyme can be reused), the remaining solution is evaporated to dryness in vacuo and the alcohol / ester mixture which remains as a residue is separated by column chromatography on silica gel or by extraction, crystallization or distillation.

0024Table 1 shows the products obtained, the variable process parameters (amount of enzyme, amount of alcohol, amounts of vinyl ester, reaction temperature, reaction time) as well as product characteristics and chemical yield.<tables id="tabl0001" num="0001"><img file="EP0321918B1_D0004.tif" /></tables><tables id="tabl0002" num="0002"><img file="EP0321918B1_D0005.tif" /></tables><tables id="tabl0003" num="0003"><img file="EP0321918B1_D0006.tif" /></tables><tables id="tabl0004" num="0004"><img file="EP0321918B1_D0007.tif" /></tables><tables id="tabl0005" num="0005"><img file="EP0321918B1_D0008.tif" /></tables>