WO2007077141A1 - Method for producing substituted phenylacetyl carbinols - Google Patents

Abstract

The invention relates to a method for producing substituted phenylacetyl carbinols by means of the biocatalytic reaction of the corresponding substituted aromatic aldehydes in the presence of pyruvate and/or acetaldehyde and a pyruvate decarboxylase. The invention also relates to (R)-1-hydroxy-1-(4-tert.-butoxyphenyl)-2-propanone.

Description

Process for the preparation of substituted phenylacetylcarbinols

description

Technical field of the invention:

The present invention relates to a process for the preparation of substituted phenylacetylcarbinols by biocatalytic reaction of the corresponding substituted aromatic aldehydes in the presence of pyruvate and / or acetaldehyde and a Pyruvatdecarboxylase. The present compound further relates to (R) -1-hydroxy-1- (4-tert-butoxyphenyl) -2-propanone.

Phenylacetylcarbinol (PAC) is an important intermediate for the preparation of the pharmaceutical active substance ephedrine and related compounds. Recently, pharmacologically interesting properties of phenyl ring-substituted derivatives of said compounds have also been found. Therefore, there is a high demand for economically advantageous and pharmacological claims sufficient synthesis routes of the compounds mentioned or their advanced intermediates.

State of the art:

WO 90/04631 describes a process for the preparation of phenylacetyl carbinol by fermentation of benzaldehyde and pyruvate in the presence of pyruvate decarboxylase producing mutants of the species Saccharomyces cerevisiae or Candida flareri. The fermentation is carried out under anaerobic conditions or under oxygen deficiency in an aqueous medium and provides phenylacetylcarbinol in a concentration of at least 1 g / l.

WO 02/02791 discloses a process for the preparation of R-phenylacetylcarbinol by biotransformation of benzaldehyde by means of filamentous fungi. The reaction is carried out in the presence of an acetaldehyde source such as acetaldehyde itself or pyruvate. Preferred filamentous fungi are called Rhizopus, Neurospora, Polyporus, Fusarium, Monilia, Paecilomyces and Mucor.

JP 10-269204 relates to a process for the preparation of optically active α-hydroxy ketones of the phenylacetylcarbinol type, wherein the phenyl ring may also be substituted, or may be replaced by an alkyl, naphthyl, furyl or thiophenyl. The method envisages reacting the aldehydes corresponding to the desired acetylcarbinols in a culture solution of a pyruvate-producing microorganism. Suitable microorganisms are Torulopsis or Candida. J. Netrval et al. describe in Eur. J. Appl. Microbiol. Biotechnol. (1982) 16: 35-37 describe the production of phenylacetylcarbinol in various yeast species. For this purpose, sucrose, acetaldehyde and benzaldehyde are added to finished culture solutions and the amount of phenylacetylcarbinol formed is determined after 30 minutes or after completion of the reaction. Particularly suitable are the strains Saccharomyces carlsbergensis and Candida and Hansenula have proven.

The preparation of acetoin and phenylacetylcarbinol by means of pyruvate decarboxylases from Zymomonas mobilis and Saccharomyces carlsbergensis are described by S. Bringer-Meyer et al. in Biocatalysis, 1988, Vol.1, 321-331 starting from benzaldehyde and acetaldehyde or benzaldehyde.

S. Bornemann et al. describe in J. Chem. Soc, Perkin Trans. 1 425 (1996) the preparation of optically active aromatic acyloins starting from the corresponding aldehydes and pyruvate or acetaldehyde in the presence of a recombinant pyruvate decarboxylase from Zymomonas mobilis. The starting materials used are especially 2-, 3- or 4-fluoro or chloro-substituted benzaldehydes. Yields of 35% were achieved with the abovementioned 4-substituted benzaldehydes.

DE 197 36 104 discloses a process for the preparation of enantiomerically pure phenylacetyl carbinols from acetaldehyde and the corresponding benzaldehyde in the presence of a pyruvate decarboxylase from Zymomonas. The substrates used are benzaldehyde itself or fluorine-, chlorine- or bromine-substituted derivatives. In the course of the biotransformation acetaldehyde is replenished continuously or discontinuously to the reaction medium.

WO 03/020942 relates to a process for the preparation of R-phenylacetylcarbinols from acetaldehyde and aromatic aldehydes in the presence of a Pyruvatdecarboxylase in a two-phase system. The aldehydes to be used may have a fluorine, chlorine or bromine substituent.

EP 0 322 973 discloses a process for preparing optically active cyanohydrins and their reaction products. The method is suitable i.a. for the preparation of acyloins, such as R - (-) 1-hydroxy-1- (4-methoxyphenyl) -2-propanone, which is prepared by reacting R - (+) - α- (trimethylsilyloxy) -4-methoxyphenylacetonitrile with MeOH thylmagnesium iodide and subsequent acid treatment is obtained.

Object of the invention:

The object of the present invention was to provide an economically and procedurally advantageous process for producing optically active, hydroxyl- or alkoxy-substituted phenylacetylcarbinols in high yield, purity and high enantiomeric excess.

DESCRIPTION OF THE INVENTION AND THE PREFERRED EMBODIMENTS

The object has been achieved according to the invention by providing a process for preparing substituted R-phenylacetylcarbinols of the formula (I)

where the radicals

R ^{1 is} hydrogen, C ¹ - to C ₂ -alkyl or phenyl,

Each R ² independently is hydroxy, C 1 to C 12 alkyl, C 1 to C 6 alkoxy, phenyl, benzyl, halogen, nitro or nitrile, and

n zero, one or two

means

by reaction of aldehydes of the formula (II)

in which R ¹ , R ² and the index n have the same meaning as in formula (I),

in the presence of pyruvate and / or acetaldehyde and in the presence of a Pyruvatde- carboxylase.

The process according to the invention is suitable for the preparation of substituted radicals

Phenylacetylcarbinols of the formula (I) in optically active form, preferably those which have an enantiomeric excess of about 90 to about 99.9% ee, particularly preferably about 94 to about 99.5% ee and particularly preferably about 97 to about 99% ee. sen. The stereogenic carbon center of the main enantiomer is in the R configuration.

The phenylacetylcarbinols of the formula (I) which are obtainable according to the invention have on the phenyl ring an R ^{1 group} bonded via an oxygen atom, where R ^{1 is} either hydrogen or a C 1 -C 12 -alkyl or phenyl radical. The term C.sub.1C-C.sub.12-alkyl is to be understood as meaning straight-chain or branched or wholly or partly cyclic alkyl radicals having 1 to 12 carbon atoms, such as, for example, methyl, ethyl, propyl, cyclopropyl, cyclopropylmethyl, 1-methylethyl, butyl, cyclobutyl, 1-methyl propyl, 2-methylpropyl, 1, 1-dimethylethyl, pentyl, cyclopentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1, 1-dimethylpropyl, 1, 2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1, 1-dimethylbutyl, 1, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1, 1, 2-trimethylpropyl, 1, 2,2-trimethylpropyl, 1-ethyl-1-methylpropyl and 1-ethyl-2-methylpropyl, cyclohexyl, heptyl, Methylcyclohexyl, octyl, dimethylcyclohexyl, nonyl, decyl, undecyl or dodecyl.

R ¹ is preferably a C 1 - to C 12 -alkyl radical as described above, particularly preferably C ¹ - to C ₆ -alkyl and very particularly preferably C ¹ - to C ₄ -alkyl, for example methyl, ethyl, propyl, isopropyl, Butyl, 2-methylpropyl or 1, 1-dimethylethyl (tert-butyl), in particular for tert. Butyl.

The phenylacetylcarbinols of the formula (I) obtainable according to the invention may additionally have one or two identical or different radicals R ² on the phenyl ring. The radical or radicals R ² may be hydroxy, as described above, C 1 - to C 12 -alkyl, preferably C 1 - to C 6 -alkyl, C 1 - to C 6 -alkoxy, phenyl, benzyl, halogen, nitro (-NO ₂ ) or nitrile (-CN).

In the context of the present invention, the index n represents the numbers zero, one or two, preferably zero or one, more preferably zero.

Cr to Cβ-alkoxy stands for a bonded via an oxygen atom d- to Cδ-alkyl radical such as methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy and 1, 1-dimethylethoxy, pentoxy, cyclopentyloxy , 1-methylbutoxy, 2-methylbutoxy, 3-methoxybutoxy, 1, 1-dimethylpropoxy, 1, 2-dimethylpropoxy, 2,2-dimethylpropoxy, 1-ethylpropoxy, hexyloxy, cyclohexyloxy, 1-methylpentoxy, 2-methylpentoxy, 3- Methylpentoxy, 4-methylpentoxy, 1, 1-dimethylbutoxy, 1, 2-dimethylbutoxy, 1, 3-dimethylbutoxy, 2,2-dimethylbutoxy, 2,3-dimethylbutoxy, 3,3-dimethylbutoxy, 1-ethylbutoxy, 2-ethylbutoxy, 1, 1, 2

Trimethylpropoxy, 1, 2,2-trimethylpropoxy, 1-ethyl-1-methylpropoxy or 1-ethyl-2-methylpropoxy. Halogen in the context of the present invention is fluorine, chlorine, bromine and iodine, preferably fluorine, chlorine, bromine, particularly preferably fluorine and chlorine.

The starting compounds used to carry out the process according to the invention are the substituted benzaldehydes of the formula (II) corresponding to the desired product compounds.

in which R ¹ , R ² and the index n have the same meanings or the same preferred meanings as in the corresponding reaction products of the formula (I).

In a preferred embodiment, the present invention relates to a process for preparing para-alkoxy-substituted R-phenylacetylcarbinols of the formula (Ia)

where the radical R ^{1 is} C ¹ - to C 12 -alkyl, preferably C 1 - to C 12 -alkyl and particularly preferably as described above is C 1 - to C 6 -alkyl. Such compounds of the formula (Ia) are obtainable by reaction according to the invention of the corresponding para-alkoxy-substituted aldehydes of the formula (Ia)

in which R ^{1 has} the same meaning as in formula (Ia).

In a particularly preferred embodiment, the present invention relates to a process for the preparation of (R) -1-hydroxy-1- (4-tert-butoxyphenyl) -2-propanone of the formula (Ib)

by inventive reaction of para-tert-butoxy-benzaldehyde.

The abovementioned starting compounds are commercially available in some cases or by synthesis methods known to the person skilled in the art, for example by deprotonation / formylation of the corresponding aromatics, for example in Tetrahedron 1978, Vol. 34, 1651 or Methods of Organic Chemistry (Houben-Weyl), 4th Ed. Vol.748 and 1 or in Topics in Current Chemistry Springer-Verlag GmbH 1988 Vol.148, 1 and especially for para-tert-butoxy-benzaldehyde in Journal of Organic Chemistry 1985, Vol. 50, 539 described readily available.

The process according to the invention is carried out in the presence of pyruvate and / or acetaldehyde and in the presence of a pyruvate decarboxylase (PDC). The pyruvate decarboxylase used in each case serves as enzyme catalyst for the reaction of the aldehyde of the formula (II) used with the pyruvate and / or the acetaldehyde. Pyruvate decarboxylases are formed in various microorganisms. For example, pyruvate decarboxylases from Zymomonas such as Zymomonas mobilis, from Aspergillus e.g. Aspergillus oryzae, Saccharomyces, e.g. Saccaromyces cerevisiae, and Candida. In the context of the process according to the invention, preference is given to using a pyruvate decarboxylase from Candida, in particular from Candida utilis.

These microorganisms are well known and readily isolated by known techniques such as those described in Evans, I.H., Yeast Protocols, Totowa / NJ: Humana Press, 1996, or obtainable from public storage locations. In addition, in the context of the process according to the invention, it is also possible to use those pyruvate decarboxylases which are or were expressed recombinantly in a host organism. Such techniques are described, for example, in Guthrie, C. Guide to Yeast Genetics and Molecular Bioiogy, Amsterdam: Academic Press, 2005; Johnston, J.R., Moiecuiar Genetics of Yeast - A Practicai Approach, Oxford / UK IRL Press, 1994; Kaiser, C. Methods in Yeast Genetics, NY CoId Spring Harbor Laboratory Press, 1994.

The pyruvate decarboxylase to be used according to the invention is advantageously used in stabilized form. The stabilization can be achieved by addition of suitable stabilizers such as natural cofactors of the enzymes, buffers, salts or other compounds with stabilizing properties such as alcohols such as ethanol, isopropanol, isobutanol, glycerol, ethylene glycol, 2-butanone, dimethylformamide. mid (DMF), dimethyl sulfoxide (DMSO), dioxane, tetrahydrofuran, 1-aminopropanol, tert-butanol, methyl tert-butyl ether, preferably ethanol.

The abovementioned pyruvate decarboxylases can be used in the manner of the invention in the form of culture solutions of pre-cultivated cell cultures of said microorganisms. In this case, it is generally not important whether the microorganisms used are present in living or killed form in the respective culture solution.

Alternatively, the process according to the invention can also be carried out in the presence of a pyruvate decarboxylase, which is in the form of an extract from the cell culture producing the pyrovate decarboxylase. Such cell-free extracts include the respective pyruvate decarboxylase in soluble or solubilized form.

The pyruvate-decarboxylases to be used according to the invention can also be prepared in immobilized form, i. in a form applied to a carrier.

In the context of the present invention, in particular using a pyruvate decarboxylase from Candida utilis, it has proved advantageous to pre-cultivate the respective cell culture by methods known to the person skilled in the art. Particularly advantageous is a precultivation of Candida utilis under anaerobic conditions. The biomass thus obtained can be separated from the culture medium and then added to the reaction mixture to be reacted.

The reaction according to the invention is carried out in the presence of pyruvate and / or acetaldehyde, preferably pyruvate, as the second starting material. The term pyruvate is to be understood as meaning salts of pyruvic acid, such as, for example, sodium pyruvate or pyruvic acid itself. The pyruvate used is preferably sodium pyruvate. The chosen pyruvate is preferably used as the acetaldehyde source or acetaldehyde itself in an amount of about 1 to about 2.5 equivalents, preferably about 1 to about 2 equivalents, based on the molar amount of aldehyde used of the formula (II).

The preparation according to the invention of the phenylacetylcarbinols of the general formula (I) is advantageously carried out by initially introducing the benzaldehyde of the formula (I) to be reacted together with the chosen source of pyruvate in a suitable reaction medium and then adding the pyruvate decarboxylase in the chosen form, i. e.g. in the form of dried biomass or a culture solution.

The amount of pyruvate decarboxylase to be used is determined by the form in which it is used and by the activity which it then has. For example, about 600 g of biosolubstrate are added per mole of aldehyde to be reacted or about 200 g of dried biomass of the pyruvate decarboxylase-containing microorganism, in particular of Candida utilis.

Suitable reaction media for carrying out the reaction according to the invention are water or aqueous buffer solutions. By adding suitable organic solvents such as alcohols such as ethanol or isopropanol, the solubilities of the reactants in the aqueous reaction media can be increased and thus positively influence the reaction rate. The pH of the reaction mixture is preferably adjusted to a value of about 4 to about 8, preferably about 5 to about 7. The reaction is advantageously carried out with good mixing and with the addition of further adjuvants such as the cofactor thiamine pyrophosphate or thiaminium hydrochloride or magnesium ions, for example in the form of magnesium sulfate at a temperature in the range of usually about 10 to about 40 ° C, preferably about 20 to about 30 ° C performed. It has also proved to be advantageous to carry out the reaction under aerobic conditions, i. in the presence of oxygen or atmospheric oxygen. The reaction is usually after about 24 h, often completed after about 12 h, which is usually based on the aldehyde used of the formula (I) conversion of about 90 to about 95%.

From the reaction mixtures thus obtained, the (R) -phenylacetylcarbinols of the formula (I) prepared can be isolated by methods known to those skilled in the art, for example by filtration, subsequent extraction and finally distillation. The process according to the invention provides efficient access to the desired (R) -phenylacetylcarbinols of the formula (I) in high yield, purity and high enantiomeric excess. In particular, the process according to the invention is suitable for preparing para-alkoxy-substituted (R) -phenylacetylcarbinols Formula (Ia), especially for the preparation of (R) -1-hydroxy-1- (4-tert-butoxyphenyl) -2-propanone of the formula (Ib), in a particularly high chemical yield and a particularly high enantiomeric excess of usually 97th % and above. The tert-butyl group is an advantageous protective group for the para-phenolic hydroxy group, which can be cleaved under very mild acidic conditions but is stable against a variety of other conditions. (R) -1-Hydroxy-1- (4-tert-butoxyphenyl) -2-propanone thus represents a versatile synthetic building block for a wide variety of possible synthesis products of para-hydroxy-phenylacetylcarbinol. The present invention therefore relates in one another aspect also (R) -1-hydroxy-1- (4-tert-butoxyphenyl) -2-propanone.

The following examples are illustrative of the invention without in any way limiting it: Examples:

ODδoo values were determined by a standard method using a Pharmacia Biotech Ultraspec 300 UVA / isible spectrometer at 600 nm.

Example 1 :

A seed culture of the Candida utilis strain was inoculated from agar plates into five separate 200 ml shake flasks filled with MES buffer trace element solution and glucose and incubated at 30 ° C for 24 h.

The inoculations were transferred to a 10 l fermenter (together with phosphate buffer, glucose, ammonium and magnesium phosphate and trace elements) and adjusted to pH 6.5 by addition of sodium hydroxide solution. The fermentation was carried out at an oxygen partial pressure of 0 and a stirring speed of 300 min- ¹ and a constant air flow of 1, 0 l / min for 24 h until an ODδoo value of 35 was reached.

The reaction mixture thus obtained was transferred to a 200 l fermenter, made up with 160 l of phosphate buffer and additionally made up with 4 kg (2.5% w / v) of yeast extract. The pH of the mixture was adjusted to 6.0 by addition of acetic acid or sodium hydroxide solution. The mixture was further vented at 1 m ³ / h with a pressure of 0.1 bar and stirred at a stirring speed of 100 min ^-1, thus maintaining the oxygen partial pressure at zero. After 27 h, the ODδoo value of the fermentation broth was constant at 29. Subsequently, the biomass was separated by centrifugation (centrifuge MS-01, Carl Padberg GmbH, type 61 G). This gave 5.75 kg of wet cell mass (cww) and, after drying, 1.9 kg of dried cell mass (cdw). The biomass thus obtained exhibited a pyruvate decarboxylase activity of 50 units / g cdw.

Example 2:

1 kg (5.6 mol) of 4-tert-butoxybenzaldehyde was dissolved in 13.5 l of ethanol and to a buffer solution of 1, 2 kg (1 1, 2 mol) of sodium pyruvate, 20 g (0.04 mol) of thiamine pyrophosphate , 0.1 kg (0.4 mol) MgSO ₄ , 1, 24 kg NaSO ₄ and 0.45 kg citric acid in 82 l water. The pH of the resulting mixture was adjusted to 6.5 by addition of sodium hydroxide solution and then 1 kg (cdw) of Candida utilis was added. The pH was kept constant during the course of the reaction. After 4 h, a solution of another 0.46 kg (4.2 mol) of sodium pyruvate, 0.03 kg (0.13 mol) of MgSO ₄ and 7 g (0.013 mol) of thiamine pyrophosphate in 5 L of water at a rate of 0.8 kg / hr. After 6 h, a conversion of 4-tert-butoxybenzaldehyde of 95% to (R) -1-hydroxy-1- (4-tert-butoxyphenyl) -2-propanone was determined by HPLC analysis. Subsequently, the fermentation broth was freed from the biomass by membrane filtration and the aqueous product solution (138 l ^{) was} extracted with 100 kg of methyl tert-butyl ether for 30 min at a stirrer speed of 100 min -1. Subsequently, 4 l of ethanol were added and the phases were separated. The organic phase was separated and freed from the solvent at a pressure of 500 mbar and a temperature of 55 ° C. This gave 0.8 kg (3.6 mol) of (R) -1-hydroxy-1- (4-tert-butoxyphenyl) -2-propanone corresponding to a yield of 65% in the form of a yellow oil. The enantiomeric excess (ee) was 94%. It was determined by HPLC as described below:

Method for quantitative, chiral determination of tert-butoxy-phenyl-2-propanone Meth. Short name: tB-PAC-C

Column: Chiracel OD-RH, 150 ^* 4, 6mm (Daicel)

Guard column: C18 ODS

Temperature: 40 ^° C

Flow rate: 0.50 ml / min

Injection volume: 5.0 μl

Deletion: UV 210nm

Stop time: 45.0 minutes

Follow-up time: 0.0 minutes

Maximum pressure: 75 bar

Eluent A: 1 OmM KH 2 PO 4, pH 2.5

Eluent B: acetonitrile

Gradient: Time [min] A [%] B [%] Flow [ml / min]

0.0 75.0 25.0 0.50

25.0 73.0 27.0 0.50

27.0 40.0 60.0 0.50

37.0 40.0 60.0 0.50

40.0 73.0 27.0 0.50

45.0 75.0 25.0 0.50

Matrix: fermentation broths, enzyme batches. Sample is filtered through 0.22 μm

Comment: Calibration: no pure standard material available Analytes: retention times [min]

(-) - Tert-butoxy-phenylacetcarbinol 23,85

(+) - tert -butoxy-phenylacetcarbinol 24.77

(R) -1 -Hydroxy-1 - (4-tert-butoxyphenyl) -2-propanone:

1-H-NMR (400MHz, CDCl3, δ in ppm): 1.35 ppm (s, 9H), 2.1 (s, 3H), 4.25 (br, 1H), 5.1 (s , 1H), 7.0 (d, 2H), 7.2 (d, 2H).

Claims

claims 1 . Process for the preparation of substituted R-phenylacetylcarbinols of the formula (I)

where the radicals R ^{1 is} hydrogen, C ¹ - to C ₂ -alkyl or phenyl, Each R ² independently is hydroxy, C 1 to C 12 alkyl, C 1 to C 6 alkoxy, phenyl, benzyl, halogen, nitro or nitrile, and n zero, one or two means by reaction of aldehydes of the formula (II)

in which R ¹ , R ² and the index n have the same meaning as in formula (I), in the presence of pyruvate and / or acetaldehyde and in the presence of a pyruvate decarboxylase. 2. Process according to Claim 1 for the preparation of substituted R-phenylacetylcarbinols of the formula (Ia)

the rest R ¹ Cr to Ci2-alkyl means by reaction of aldehydes of the formula (Ia)

in which R ^{1 has} the same meaning as in formula (Ia). 3. The method according to claim 1 or 2, characterized in that the radical R ^{1 is} tert-butyl. 4. The method according to any one of claims 1 to 3, characterized in that the Pyruvatdecarboxylase is a Pyruvatdecarboxylase a yeast of the genus Candida. 5. The method according to any one of claims 1 to 4, characterized in that the Pyruvatdecarboxylase is a Pyruvatdecarboxylase from Candida utilis. 6. The method according to any one of claims 1 to 5, characterized in that one uses a Pyruvatdecarboxylase, which is expressed recombinantly in a host organism. 7. The method according to any one of claims 1 to 6, characterized in that one uses the Pyruvatdecarboxylase in stabilized form. 8. The method according to any one of claims 1 to 7, characterized in that one uses the Pyruvatdecarboxylase in the form of an extract from the pyrovatde carboxylase-producing cell culture. 9. The method according to any one of claims 1 to 8, characterized in that one uses the Pyruvatdecarboxylase in immobilized form. 10. The method according to any one of claims 1 to 7, characterized in that one uses the Pyruvatdecarboxylase in the form of a pre-cultured cell culture. 1 1. A method according to claim 10, characterized in that the cell culture is precultured under anaerobic conditions. 12. The method according to any one of claims 1 to 1 1, characterized in that one carries out the reaction under aerobic conditions. 13. The method according to any one of claims 1 to 12, characterized in that one uses pyruvate and / or acetaldehyde in a relative to the molar amount of aldehyde used of the formula (II) amount of 1 to 2.5 equivalents. 14. The method according to any one of claims 1 to 13, characterized in that one carries out the reaction at a pH in the range of 4 to 8. 15. (R) -1 -Hydroxy-1- (4-tert-butoxyphenyl) -2-propanone.