BRPI0501179B1 - (alpha) -hydroxy esters production process - Google Patents

Abstract

"PRODUCTION PROCESS OF 244> -HYDROXY-ESTERS". Compounds with formula (X) and (XI) are produced by selective reduction of the compound with formula (XII) using reductase enzymes produced by microorganisms belonging to the Saccharomycetaceae and / or Dothioraceae families.

Description

(ALPHA) -HYDROXY-ESTER PRODUCTION PROCESS FIELD OF THE INVENTION

This invention relates to the production of α-hydroxy esters by reducing the carbonyl of compounds belonging to the class of α-keto esters or 2-keto esters. Such a reduction process has high yield and high stereoselectivity due to the fact that enzymes are used, more specifically reductases as catalysts.

BACKGROUND OF THE INVENTION

Com a utilização de 1,25 equivalente molar de ácido acético e a adição de 1,05 eq. de KF como aditivo, Huffmann et al., Tetrahedron Letters 1998, 40, 831-834, otimizaram a aminação redutiva para uma relação diastereoisomérica (SSS): (SRR) de 17:1 utilizando Ra/Ni em condições mais brandas; temperatura ambiente e uma atmosfera de H2.Reductive amination is the process used to produce angiotensin-converting enzyme (ACE) inhibitors, such as enalapril, used to treat hypertension, Harris et al, US Pat. 4,374,829 (1982). The results obtained initially showed low yields and low diastereoselectivity (SSS): (SRR), 1.06: 1.00 with the use of NaCNBH3 and 1.2: 1 with the use of Pd / C10%. The process was optimized by Blacklock et al., J. Org. Chem. 1988, 53, 836-844, using as a Ra-Ni catalyst with 40 psi H2 pressure, having obtained a diastereoisomeric ratio (SSS): (SRR) of 6.7: 1, as shown in the diagram below:

With the use of 1.25 molar equivalent of acetic acid and the addition of 1.05 eq. KF as an additive, Huffmann et al., Tetrahedron Letters 1998, 40, 831-834, optimized the reductive amination to a diastereoisomeric ratio (SSS): (SRR) of 17: 1 using Ra / Ni in milder conditions; room temperature and an H2 atmosphere.

Through this methodology, several drugs that inhibit angiotensin-converting enzyme (ACE) can be obtained and are used in the treatment of hypertension, such as linisopril, quinapril, ramipril, perindopril, cilazapril and benazepril.

The enantioselective hydrogenation of ethyl 2-oxo-4-phenylbutanoate (I) was performed on a pilot scale by Blaser et al, Jounal of Molecular Catalysis A: Chemical 1996, 107, 85-94, to obtain 2- (R) -hydroxy-4-phenylbutonoate of ethyl (II) to be used as an intermediary in the synthesis of benazepril (IV), according to the scheme below:

utilizando levedura de panificação (Saccharomyces cerevisiae) para a preparação de 2-hidroxi-ésteres com as fórmulas:

onde R=CH3CH2n: n=0, 1 ,2, 3 e 4. Foram obtidos excessos enantioméricos (ee) de 30 a 91% para o enantiômero S (VI) em meio aquoso e excessos enantioméricos (ee) de 47% e 54% para o enantiômero R (VII) para n= 3 e 4 respectivamente, quando a reação ocorreu em hexano ao invés de água. Os rendimentos químicos nos processos variaram de 29 a 47%.After a thorough study of the results obtained and the costs of using homogeneous and heterogeneous catalysis, the latter presented the best results, having been used as a 5% Pt / Al2O3 catalyst (pretreated with H2 at 400 ° C for 4 hours), dihydrocinchonidine as chiral auxiliary, H2 pressure of 70 atm, room temperature and reaction time of 3-5 hours, obtaining an enantiomeric excess of (II) of 80-92% and a yield of 98%. Bartók et al, Applied Catalysis A: General 2002, 237, 275280, optimized the process by reducing the pressure from H2 to 25 atm. By the continuous addition during the process of the chiral inducer (dihydrocinchonidine), Sun et al, J. Am. Chem. Soc. 1999, 121 (20), 4920-4921, managed to maintain the ratio Pt / dihydrocinchonidine = 1, decreasing the pressure of H2 to 5.8 atm using as catalyst 1% Pt / Al2O3. Under these conditions, the enantiomeric excesses (ee) of (II) were 80-87%.
Nakamura et al., Journal of Organic Chemistry 1988 (53), 2589-2583, reduced 2-keto esters with the formula:

using baker's yeast (Saccharomyces cerevisiae) for the preparation of 2-hydroxy esters with the formulas:

where R = CH3CH2n: n = 0, 1, 2, 3 and 4. Enantiomeric excesses (ee) of 30 to 91% were obtained for the enantiomer S (VI) in aqueous medium and enantiomeric excesses (ee) of 47% and 54 % for the R (VII) enantiomer for n = 3 and 4 respectively, when the reaction occurred in hexane instead of water. Chemical yields in the processes ranged from 29 to 47%.

Stereochemical control of the enantioselective reduction of α-ketoesters (IV) in which R = CH3, C2H5, C3H7, C4Hg, C5H11, (CH3) 2CH using baker's yeast was achieved by Nakamura et al, Bull. Chem. Soc. Jpn. 1993, 66, 2738 - 2743, using organic solvents such as: cyclohexane, hexane, t-butyl methyl ether, diisopropyl ether, p-xylene, toluene, mesitylene, benzene and t-butyl acetate, pure or containing a small amount of Water. The yields varied from 3 to 36% and the enathiomeric excesses (ee) from 55 to 85%, having obtained the product with the R configuration preferably in all cases.

Matsuyama et al., US Pat 5,371,014 (1994) attempted to produce ethyl (II) -2-hydroxy-4-phenyl-butanoate and (S) -2-hydroxy-4-phenyl- ethyl (III) butanoate by microbiological reduction of ethyl 2-oxo-4-phenylbutanoate (I) using whole cells in aqueous medium has not been successful.

136 microorganisms were tested, among them Saccharomyces cerevisiae, Pichia burtonii, Pichia farinosa, Pichia heedii, Pichia menbranaefaciens and Pichia opuntiae. The few satisfactory results obtained in relation to enantiomeric excesses showed low yields, making it impossible to increase the scale. The results obtained using the microorganisms described above are shown in Table 1 below.

Chadha et al, Tetrahedron: Asymmetry 1996, 7, 1571-1572, reported that it was not possible to reduce ethyl 2-oxo-4-phenylbutanoate (I) using Saccharomyces cerevisiae. The reduction was successfully achieved using aqueous extract of cells from Daucus carota (wild carrot) with excellent chemical yields (90%) and enatiomeric excesses (> 99%). However, the use of plant cell cultures requires a large number of cells in relation to the substrate (100: 1), which makes it difficult to scale up the process (“scale up”). In addition, another disadvantage is the long process time (10 days), making its use as an industrial process unfeasible.

Oda et al, Bioscience Biotechnology and Biochemistry 1998, 62 (9), 1762-1767, produced ethyl 2- (R) -hydroxy-4-phenylbutanoate (II) by reducing ethyl 2-oxo-4-phenylbutanoate ( I) in an interface bioreactor using the microorganisms Rhodotorula minuta IFO 0920 and Candida holmii KPY 12402. The enantiomeric excesses obtained were 95 and 94% for R. minuta and C. holmii respectively. The incubation time was 4 days and the yields around 60%.

Dao et al, Bull. Chem. Soc. Jpn. 1998, 71, 425-432, reported that Saccharomyces cerevisiae, pre-incubated in the presence of phenacyl chloride in aqueous diethyl ether enantioselectively reduces ethyl 2-oxo-4-phenylbutanoate (I) with yields of 80-90% and excesses enatiomeric around 80% providing ethyl (II) 2- (R) -hydroxy-4-phenylbutanoate.

Compounds (II) and (III) can be used as intermediates in the synthesis of angiostensin-converting enzyme (ACE) inhibitors. Enalapril (VIII), as an example, can be obtained directly through an SN2 reaction of amide (IX) with alcohol (II) or through two steps, initially making a first inversion in alcohol (III) using a modified displacement of Mitsunobu, Ponzo et al, Tetrahedron Letters 1995, 36, 91059108, and then the SN2 shift with the amide (IX).

In formula (I) R is an alkyl group, preferably having 1 to 6 carbon atoms and more preferably an ethyl group.

Not only in the synthesis of precursors of ACE-inhibiting substances do α-hydroxy esters have applicability: US 4,332,952 reports the use of antihyperglycemic molecules belonging to the class of 5-substituted oxazolidine-2,3-diones that use as an immediate precursor to -hydroxy-esters. EP 1055664, on the other hand, describes the production of a-hydroxy-esters, especially β-amino-a-hydroxy-esters, which are important intermediates for pharmaceutical and agrochemical products. WO 04/069835 describes muscarinic agonists derived from hexane azabicycles whose production involves the reduction of α-keto esters to α-hydroxy esters. However, none of these documents describes the preparation of α-hydroxy esters from enzymatic reduction of α-keto esters.

However, no document or patent describes the preparation of a-hydroxy-esters with high yield and stereoselectivity from the use of fungi reductases and a-keto-esters. It has been found that α-hydroxy esters can be produced by reducing α-keto esters with moderate to high selectivities, at a convenient temperature, using a ketone reductase commonly found in fungi belonging to the Saccharomycetaceae family, especially in the genera Kluyveromyes, Pichia, Saccharomyces, Hansenula, Dekkera and Candida, as well as fungi belonging to the family Dothioraceae, especially the genus Aureobasidium.

OBJECT OF THE INVENTION

onde:
R1 corresponde a um radical alquil; e
R2 corresponde a um radical arilalquil;
pela reação de uma enzima com um composto de fórmula geral (XII):

onde:
R1 corresponde a um radical alquil; e
R2 corresponde a um radical arilalquil;
Mais especificamente, tal produção envolve uma etapa de redução enzimática, especificamente o uso de uma redutase, mais especificamente o uso de uma carbonila redutase, e de um a-ceto-ester.It is an object of the present invention to produce an a-hydroxy ester of the general formula (X) or (XI)

Where:
R1 corresponds to an alkyl radical; and
R2 corresponds to an arylalkyl radical;
by the reaction of an enzyme with a compound of general formula (XII):

Where:
R1 corresponds to an alkyl radical; and
R2 corresponds to an arylalkyl radical;
More specifically, such production involves an enzymatic reduction step, specifically the use of a reductase, more specifically the use of a carbonyl reductase, and an α-keto-ester.

It is an additional object of the present invention to produce a-hydroxy esters with high yields and high stereoselectivity.

It is an additional object of the present invention to use enzymes for the reduction of α-keto-ester. In particular, enzymes can be used from whole cells of the microorganism itself or in solution, together with a regenerative system.

It is an additional object of the present invention to use microorganisms belonging to the Saccharomycetaceae family, especially in the genera Kluyveromyes, Pichia, Saccharomyces, Hansenula, Dekkera and Candida, as well as those belonging to the Dothioraceae family, especially the genus Aureobasidium. In particular the microorganisms Kluyveromyces marxianus, Hansenula sp, Pichia pastoris, Pichia angusta, Pichia anomala, Aureobasidium pullulans and / or Candida quilliermondii for the production of a stereoisomer and Saccharomyces cerevisiae or Dekkera sp for the production of the other stereoisomer.

DETAILED DESCRIPTION OF THE INVENTION

All the species mentioned above can be used to obtain the desired products, however, high conversions and high selectivities are preferably obtained using enzymes or whole cells of Pichia pastoris and more preferably Pichia angusta to obtain the compound (XI) by reducing the compound (XII ) and enzymes or whole cells of Saccharomices cerevisiae or Dekkera sp to obtain the compound (X) by reducing the compound (XII).

In general, a cofactor, usually NADPH (nicotinamide adenine dinucleotide phosphate) or NADH (nicotinamide adenine dinucleotide) and a system for regenerating the reduced form of the cofactor, involving a carbon / energy source, for example, glucose and enzymes of metabolic pathways that oxidize the carbon source, for example, glucose dehydrogenase, are used together with the enzyme to promote the reaction. The co-factors and the necessary mechanism to promote the reduction are already present in the healthy cells, being preferable the use of the healthy cells in a medium containing nutrients, preferably containing an appropriate carbon source that can include one or more of the following substances: a sugar, as an example: maltose, sucrose or preferably glucose; a polyol, as an example: glycerol or sorbitol; citric acid, or a low molecular weight alcohol, such as methanol or ethanol. Thus, for the purpose of this invention, the term "regenerating system" means a set of substances that comprise a cofactor for the enzyme, an energy source and additional enzymes capable of effectively promoting the reduction of the enzyme reductase.

If growth of intact cells occurs during the reaction, sources or traces of nitrogen and phosphorus may be present in the medium. These sources are normally used in the culture of microorganisms.

The process can be carried out by adding the compound of formula (XII) to a culture of the growing microorganism, in an environment conducive to this, or to a suspension of living cells in a medium containing a carbon source, but lacking a or more nutrients needed for cell growth. Dead cells can be used if provided with enzymatic systems and substrates necessary for the regeneration of reduced cofactors, so that the reaction can take place.

The process can be carried out with cells immobilized on a support, ensuring, however, the contact of the cells with the compound with formula (XII), preferably in the presence of an appropriate carbon source, previously described.

The appropriate pH is in the range of 3.5 to 9, more preferably 4 to 6. The process is preferably carried out at a temperature in the range of 10 to 50 ° C, preferably 20 to 40 ° C, and more preferably 25 to 30 ° Ç. It is preferable to use aerobic conditions if healthy cells from the aforementioned microorganisms are present. An equivalent aeration speed of 0.01 to 1 volume of oxygen, measured under normal conditions of temperature and pressure, per volume of the culture medium per minute is used appropriately for the aforementioned conditions of pH and temperature and considerable variations may occur. The oxygen used can come from atmospheric air. Similar pH, temperature and aeration conditions can be used during the growth of the microorganism if it is done separately from the process.

The reactions can be carried out with enzymes isolated from these microorganisms. Isolated enzymes can be purified using known methods, such as: centrifuging a suspension of disintegrated cells, separating the clear solution from cell fragments, separating the desired enzyme from the solution, for example, by means of an appropriate ion exchange chromatography or by selective precipitation by adding an appropriate ionic substance, for example, ammonium sulfate. The operations can be repeated to increase the purity of the isolated enzymes.

Example 1 Preparation of the compound of formula (II) (R = Et)

The microorganisms were kept in a Petri dish containing Agar, prepared by dissolving 0.8 g of yeast extract, 2.0 g of malt extract, 0.8 g of glucose, 4.0 g of agar in 200 ml of water and later sterilization in autoclave.

Cell growth was carried out by aseptically inoculating the microorganism from the Petri dish into a 250ml conical flask containing a sterile solution with 1% glucose, 0.5% yeast extract, 0.5% peptone, 0.1% (NH4) 2SO4 (ammonium sulfate), 0.1% MgSO4. 7H20 (magnesium sulfate hepta hydrate) in 100ml of water.
The microorganisms were cultured at 24 ° C in an orbital shaker at 150 rpm for 24-48 hours.

The microorganisms were collected by centrifugation at 3070 rpm for 15 min at room temperature (25 ° C). The cells were resuspended in 100 ml of an aqueous solution containing 5% glucose, 0.1% MgCl2 (Magnesium chloride), 1% ethanol. After adding 150mg of the compound of formula (I), the cells were incubated in a rotary shaker at 24 ° C at a rotation of 150 rpm for 18-24 hours. After this period, the reaction mixture was centrifuged at 3070 rpm for 15 min at room temperature (25 ° C), extracted with ethyl acetate, the organic phase was dried with anhydrous sodium sulfate and the solvent was removed by vacuum distillation. providing a slightly yellowish oil.

The conversions of the compound of the formula (I) to form the compounds of the formulas (II) and (III) and the respective enatiomeric excesses were determined by high-performance chiral gas chromatography. The conditions used are described below:
Chromatograph: Variant Star 3400 CX.
Column: Capillary Cyclodex B (30m x 0.25, id) provided by J & W Scientific (112-2532).
Carrier gas: Helium.
Split: 40: 1
Injector temperature: 230 ° C.
Detector temperature: 230 ° C.
Initial temperature: 120 ° C (30 min).
Ramp: 2 ° C / min.
Final temperature: 200 ° C.

The compound (III) and a racemic mixture of (II) + (III), obtained by reducing (I) using BH3, were used as a chromatographic standard. The retention times of the compounds with formulas (I), (II) and (III) were respectively: 49.5 minutes, 50.3 minutes and 50.9 minutes. All compounds were isolated and characterized by hydrogen nuclear magnetic resonance spectrometry (1H NMR) and infrared (IR) spectrometry.

The results obtained, such as enantiomeric excesses (ee), conversions and yields, are shown in Table 2.

Example 2 Preparation of the compound with the formula (III) enantiomer of the compound with the formula (II) (R = Et).

The microorganisms were conserved and grown as described in Example 1. Biotransformation and analyzes were performed exactly as described in Example 1.
The results obtained are shown in Table 3.

Deposit data for biological material

The microbial strains mentioned below were supplied in August 2004 by the Collection of Fungi from the Collection of Reference Microorganisms in Sanitary Surveillance (Fiocruz / CMRVS) of the National Institute for Quality Control in Health of the Oswaldo Cruz Foundation (Fiocruz / INCQS) which , in turn, acquired them from the American Type Culture Collection (ATCC), a depository institution for cell lines in the United States.
Aureobasidium pullulans ATCC 9348 (INCQS 40069)
Candida guilliermondii ATCC 6260 (INCQS 40037)
Pichia angusta ATCC 34438 (INCQS 40116)
Pichia anomala ATCC 16763 (INCQS 40101)

Abbreviations:

ATCC - American Type Culture Collection, 123031 Parklawn Drive, Rockville, Maryland 20852 USA.
IQ-DBQ - Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro
EQ-UFRJ - School of Chemistry, Federal University of Rio de Janeiro ee - Enantiomeric excess
IV - Infrared
NADPH - Nicotinamide adenine dinucleotide phosphate
NAD - Nicotinamide adenine dinucleotide
1H NMR - Hydrogen-1 nuclear magnetic resonance

Claims (5)

Process for the production of an alpha-hydroxy ester of the general formula (X) or (XI):

Where:
R1 corresponds to an alkyl radical; and
R2 corresponds to an arylalkyl radical:
through the reaction of an enzyme with a compound of general formula (XII):

Where:
R1 corresponds to an alkyl radical; and
R2 corresponds to an aralkyl radical
characterized by the use of microorganisms of the species Pichia angusta (ATCC 34438), Pichia anomala (ATCC 16763), Aureobasidium pullulans (ATCC 9348) and Candida guilliermondii (ATCC 6260), in a bioconversion medium consisting of 5% glucose , 0.1% MgCl2, 1% ethanol, 150 mg% ethyl 2-oxo-4-phenylbutanoate in an aqueous solution, incubating at 24 ° C, at a speed of 150 rpm, for 18 to 48 hours hours. Process for the production of an alpha-hydroxy ester according to claim 1, characterized in that the species are Pichia angusta (ATCC 34438) and Pichia anomala (ATCC 16763). Production process of an alpha-hydroxy ester according to claim 1, characterized in that the species are Aureobasidium pullulans (ATCC 9348) and Candida guilliermondii (ATCC 6260). Process for the production of an alpha-hydroxy ester according to any one of claims 1 to 3, characterized in that the cell growth culture medium comprises 1% glucose, 0.5% yeast extract, 0.5% peptone, 0.1% (NH4) 2SO4 and 0.1% MgSO4.7H2O in an aqueous solution. Process for the production of an alpha-hydroxy ester according to claim 4, characterized in that the incubation of the growth of the microorganism is carried out at 24 ° C, at a rotation of 150 rpm, for 24 to 48 hours.