Chiral Pharmaceutical Intermediaries Obtained by Reduction of 2-Halo-1-(4-substituted phenyl)-ethanones Mediated by Geotrichum candidum CCT 1205 and Rhodotorula glutinis CCT 2182

Enantioselective reductions of p-R1-C6H4C(O)CH2R2 (R1 = Cl, Br, CH3, OCH3, NO2 and R2 = Br, Cl) mediated by Geotrichum candidum CCT 1205 and Rhodotorula glutinis CCT 2182 afforded the corresponding halohydrins with complementary R and S configurations, respectively, in excellent yield and enantiomeric excesses. The obtained (R)- or (S)-halohydrins are important building blocks in chemical and pharmaceutical industries.


Introduction
Chiral halohydrins are important and valuable intermediates in the synthesis of fine chemicals and pharmaceuticals as optically active 1,2-aminoalcohols. The halohydrin (R)-1-aryl-2-haloethanol may be used for the preparation of (R)-1-aryl-2-aminoethanols that are used as αand β-adrenergic drugs.
An interesting chemoenzymatic synthetic route to obtain optically active 1-aryl-2-ethanolamines is from the enantioselective reduction of the correspondent α-haloacetophenones giving halohydrins that are transformed into an epoxy that reacts with the appropriate amine (Scheme 1) [1,2].
The performances of Rhodotorula glutinis CCT 2182 and Geotrichum candidum CCT 1205 in bioreduction of αhaloacetophenone have been calling our attention due to the efficiency and complementary enantioselectivity of these microorganisms giving the corresponding (R)-and (S)halohydrins in high ee, respectively [8]. Also, those microorganisms show the same efficiency in the reduction of αazido-para-substituted acetophenones [28]. In this work, we use those two microorganisms for reduction of α-bromoand α-chloroacetophenones having para-substituted groups to produce separately both enantiomers of halohydrins that can be used as chiral building blocks for preparations of the corresponding 1,2-aminoalcohols. Scheme 1: (a) reduction using chiral catalytic reagent or biocatalytic process; (b) base; (c) amine.

Materials and Methods
IR spectra were recorded on a Bomem MB Series spectrometer. 1 H and 13 C NMR spectra were recorded on a Varian Gemini 300 spectrometer in CDCl 3 . Gas chromatographic analyses were performed using a Shimadzu GC/MS Class 5000, with helium as carrier gas. The fused silica capillary columns used were either a Supelco Simplicity ITM (30 m × 0.25 mm × 0.25 μm) and a chiral GC-column CHIRASILDEX (30 m × 0.25 mm × 0.25 μm). Optical rotation was measured with a J-720, VRDM306 JASCO, 589.3 nm (25 • C) spectropolarimeter. The melting points were obtained in MQAPF-301-MicroQuímica equipment.
The racemic 2-halo-1-(4-substituted phenyl)-ethanols 2a-j, used as reference for the determination of ee in a GC provided with a chiral column, were obtained by reacting the corresponding 1a-j with NaBH 4 in water/methanol at rt. All other solvents and reagents were reagent grade.

Growth Conditions for Microorganisms
Culture. The microorganisms Geotrichum candidum CCT 1205 (isolated from industrial waste water treatment-Preston, United Kington) and Rhodotorula glutinis CCT 2182 (isolated from Psidium guajava-Atlantic Rainforest, Brazil) were stored at "André Tosello" Research Foundation (Campinas, Brazil) [30]. G. candidum was cultivated in 400 mL of nutrient broth 1 (10 g/L malt extract, 5 g/L peptone, 10 g/L glucose, 3.12 g/L K 2 HPO 4 , and 11.18 g/L KH 2 PO 4 ) at 28 • C, and R. glutinis was cultivated in 400 mL of nutrient broth 2 (3 g/L Yeast extract, 3 g/L malt extract, 5 g/L peptone, and 10 g/L glucose) at 30 • C. Both yeasts were incubated for 2 days on an orbital shaker (200 rpm) before use. All materials and medium were sterilized in an autoclave at 121 • C before use and the yeasts were manipulated in a laminar flow cabinet.

General
Procedure for Bioreduction of 2-Halo-1-(4-substituted phenyl)-ethanones. The yeasts were incubated for two days (400 mL nutrient broth in Erlenmeyer of 1 L). After that, the ketone 1 (2 mmol) dissolved in 1.5 mL of ethanol was added directly to the suspension where the yeasts grew. The resulting suspension was stirred in an orbital shaker (200 rpm) at 28 • C for G. candidum and at 30 • C for R. glutinis until the full conversion of 1 (18 h). The product was extracted with CH 2 Cl 2 and purified by flash silica gel column chromatography using hexane/ethyl acetate (7 : 3).    [31], giving an optical purity of >99% determined by GC using a chiral column; 1 H and 13 C NMR and IR spectra and MS analysis were identical to those observed with its (S) enantiomer.  (2), 121 (7), 115 (6) [32], giving an optical purity of >99% determined by GC using a chiral column; 1 H and 13 C NMR and IR spectra and MS analysis were identical to those observed with its (S) enantiomer.    [32], giving an optical purity of >99% determined by GC using a chiral column; 1 H and 13 C NMR and IR spectra and MS analysis were identical to those observed with its (S) enantiomer.      [33,36], giving an optical purity of >99% determined by GC using a chiral column; 1 H and 13 C NMR and IR spectra and MS analysis were identical to those observed with its (S) enantiomer.    [14], giving an optical purity of >99% determined by GC using a chiral column; 1 H and 13 C NMR and IR spectra and MS analysis were identical to those observed with its (S) enantiomer.    [36], giving an optical purity of >99% determined by GC using a chiral column; 1 H and 13 C NMR and IR spectra and MS analysis were identical to those observed with its (S) enantiomer.    [32], giving an optical purity of >99% determined by GC using a chiral column; 1 H and 13 C NMR and IR spectra and MS analysis were identical to those observed with its (S) enantiomer.

(S)-(+)-2-Chloro-1-(4-methoxyphenyl)ethanol (S)-2i.
The bioreduction of ketone 1i (0.369 g, 2 mmol) by   [36], giving an optical purity of >99% determined by GC using a chiral column; 1 H and 13 C NMR and IR spectra and MS analysis were identical to those observed with its (S) enantiomer. Enzyme Research 5     [33], giving an optical purity of >99% determined by GC using a chiral column; 1 H and 13 C NMR and IR spectra and MS analysis were identical to those observed with its (S) enantiomer.

Results and Discussion
The reduction of ethanones 1a-j was carried out in 5 mmol/L in a slurry of growing yeast of Rhodotorula glutinis CCT 2182 and Geotrichum candidum CCT 1205. These ethanones having substituted groups (electron withdrawing groups-EWG: -NO 2 , -Br, -Cl; electron donating groups-EDG: -CH 3 , -OCH 3 ) attached to position 4 of benzene ring were studied in order to investigate the influence of these groups in the bioreduction performed by these two microorganisms.  The reaction progress was monitored by GC analysis, and the yields and enatiomeric excesses are shown in Table 1.
α-Haloacetophenones have been used as mechanistic probe in the reduction reactions of NADH-dependent horse liver alcohol dehydrogenase [37][38][39][40], for identification of reductants in sediments [41] and even in the whole cells [42]. This probe enables the differentiation between reduction processes which proceed through hydride transfer (H − ) or by a multistep electron transfer (e − , H • or e − , H + , e − as has been suggested). Acetophenone is the reduction product obtained by electron transfer, while optically active halohydrin is obtained when an enzyme mediates a hydride transfer process. In this work, the reductions of 1a-e proceed via hydride transfer mediated by an oxireductase, since halohydrins were obtained in high ee and no 4-substituted acetophenone was detected.
Rhodotorula glutinis gives products following the Prelog rule [43], which predicts that, in general, hydrogen transfer from NAD(P)H to the prochiral ethanones 1a-j occurs to the face of carbonylic group shown in Figure 1, taking into account that the aryl group is larger than the -CH 2 Br and -CH 2 Cl groups. On the contrary, the Geotrichum candidum gives anti-Prelog halohydrins.
The excellent results and complementary enantioselectivities of the produced halohydrins obtained by using Rhodotorula glutinis CCT 2182 and Geotrichum candidum CCT 1205 in reduction of ethanones 1a-j are remarkable and highlight the potential of such approach to obtain separately the two isomers of the 1,2-aminoalcohols, by reaction of the easily obtainable epoxy with the appropriated amine (Scheme 2), as an alternative to the approach using the reduction of α-azido-para-substituted acetophenones mediated by those microorganisms [28]. The separate synthesis of two enantiomers is important since the FDA Guidance for Development of New Stereoisomeric Drugs [44] says that "to evaluate the pharmacokinetics of a single enantiomer or mixture of enantiomers, manufacturers should develop quantitative assays for individual enantiomers in in vivo samples early in drug development." However, the products of biotransformation of 1b-e and 1g-j using Rhodotorula glutinis CCT 2182 may be used as important starting material for the preparation of the known pharmaceuticals products with (R) configuration: Eliprodil from halohydrins 2b and 2g; Tembamide from halohydrins 2c and 2h; Aegeline from halohydrins 2d and 2i; Nifenalol from halohydrins 2e and 2j (Figure 2).

Conclusions
The use of Rhodotorula glutinis CCT 2182 and Geotrichum candidum CCT 1205 in bioreduction reaction of 2-halo-1-(4substituted phenyl)-ethanones results in an important chiral halohydrins in high ee, excellent yield, and complementary enantioselectivity. These halohydrins may be used as intermediates in the synthesis of optically active substituted styrene oxides and aminoalcohols which have numerous industrial applications.