Asymmetric Bio-and Chemoreduction of 2-Benzylidenecyclopentanone Derivatives

Highly efficient asymmetric reduction of 2-benzylidenecyclopentanone derivatives to give the respective exocyclic allylic alcohols in ee’s up to 96% was performed with chiral oxazaborolidine-based catalysts. Complete enantioselectivity furnishing (S)-configured alcohol product could be achieved by bioreduction of 2-(4-chlorobenzylidene)cyclopentanone with Daucus carota root. The synthesized compounds may be used as enantiomerically enriched standards for the monitoring of the enzyme-catalyzed redox processes.


Introduction
Chiral alcohols are important building blocks and intermediates in the synthesis of pharmaceuticals, fine chemicals, agrochemicals, flavors and fragrances, as well as functional materials. 1Since ketones represent one of the most common families of unsaturated compounds, their asymmetric reduction represents the simplest and most powerful method for the preparation of enantiomerically enriched alcohols.The stereospecific reduction of carbonyl groups to the corresponding alcohols is also a functionalization reaction involved in the metabolism of endogeneous compounds and xenobiotics containing these groups.Thus, it is often catalyzed by enzymes belonging to either dehydrogenase/reductase superfamily or the aldo-keto reductase (AKR) superfamily. 2The human members of the AKR subfamily 1C are involved in the biosynthesis and inactivation of steroid hormones, and also in the biosynthesis of neurosteroids and prostaglandins. 3hese enzymes reduce carbonyl containing substrates to alcohols and also function in vivo as ketosteroid reductases, and thus regulate the activity of androgens, estrogens and progesterone in target tissues, and ligand occupancy and transactivation of their corresponding receptors. 4Aberrant expression and action of AKR1C enzymes may lead to an imbalance in the metabolism of steroid hormones, and to further development of different patho-physiological conditions. 5These enzymes thus represent promising therapeutic targets in the development of new drugs.In the literature, structurally different compounds have been evaluated as AKR1C inhibitors, for example dietary phytoestrogens, 6 benzodiazepines, 7 cinnamic acids, 8 benzofurans, and phenolphthalein derivatives, 9 Ru(II) complexes, 10 salicylic and aminobenzoic acids derivatives, as well as some nonsteroidal anti-inflammatory drugs and their analogues. 11,12In spite of a plethora of potent inhibitors of steroid metabolizing enyzmes that have emerged, the search for new and more selective ones is an important field of investigation.Štefane et al. 13 indentified compounds based on cyclopentane scaffold, which are AKR1C1 and AKR1C3 substrates active in the low micromolar range, and thus represent promising starting points in the development of potential agents for treatment of hormone-dependent forms of cancer and other diseases involving these enzymes.AKR1C inhibitors are not only interesting as potential agents for the treatment of diseases, but also as molecular tools in the study of the pathophysiological roles of these enzymes.In the recent study Beranič et al. introduced new enzymatic assays employing racemic 2-(4-chlorobenzylidene)cyclopentanol (CBCP-ol) and its ketone counterpart 2-(4-chlorobenzylidene)cyclopentanone that allow monitoring of AKR1C-catalyzed reactions in the reductive and oxidative directions. 14Since enzymes perform highly stereoselective reactions, it seems Štefane et al.: Asymmetric Bio-and Chemoreduction ... useful to know, which enantiomer of CBCP-ol is involved in the redox process.For this reason we present herein the synthesis of enantiomerically enriched cyclopentyl alcohols (CBCP-ol and its 4-methoxy analogue) via the asymmetric chemo-and bioreduction of substituted 2-benzylidenecyclopentanones, which can serve as standards in monitoring of AKR1C-catalyzed reactions.The reduction of the benzene-fused analogue, indanone-derived chalcone, to the corresponding secondary allylic alcohol is also included.

Results and Discussion
The starting compounds, α-arylmethylene cyclic ketones 3 and 6 were synthesized in a base-induced aldol condensation from cyclopentanone (1) or 1-indanone ( 5) and the corresponding p-substituted benzaldehydes 2 following slightly modified literature procedure 15 (Scheme 1).The reaction of cyclopentanone with p-methoxybenzaldehyde (2b) towards benzylidenecyclopentanone 3b proceeded smothly, while using p-chlorobenzaldehyde (2a), besides the desired product 3a, symmetrical abis(benzylidene) derivative 4a was isolated as the by-product.1-Indanone reacted with p-chlorobenzaldehyde leading to the product 6 in a very low 9% isolated yield.We were not, however, interested in the optimization of these aldol condensation reactions.
With α,β-unsaturated ketones 3 and 6 in hand, we investigated different methods for the selective carbonyl reduction to obtain the highest possible enantiomeric excess of the corresponding allylic alcohol products with exocyclic C=C double bond.
The most elegant method for the asymmetric reduction of prochiral ketones is either homogeneous or heterogeneous hydrogenation or transfer hydrogenation catalyzed by chiral metal catalysts. 16Highly efficient asymmetric hydrogenation of α-arylmethylene cyclopentanones was realized by chiral tailor-made iridium-spiroaminophosphine catalysts; 17 for example, reduction of 3b gave 7b with 95% ee (enantiomeric excess).Unfortunately, in our case the use of some commercially available chiral rhodium and ruthenium catalysts C1-C4 (Figure 1) in hydrogenation of cyclopentanone 3a with molecular hydrogen (80 bars) led to very low yields and ee values of the secondary alcohol 7a; the best ee of 12% (31% isolated yield) was obtained with Noyori's bifunctional ruthenium catalyst C4.
After report by Itsuno 18 that chiral aminoalcohols together with BH 3 effected the enantioselctive reduction of prochiral ketones, Corey 19 isolated the primarily formed oxazaborolidine derivative, and developed a powerful catalytic version of an original stoichometric reduction.Con-Scheme 1. Synthesis of α-arylmethylene cyclic ketones 3, 4 and 6. sequently, enantioselective reduction of prochiral ketones with borane (or its derivatives) catalyzed by chiral oxazaborolidines has emerged as an excellent route to alcohols of high enantiomerical purity. 20Since this method has many advantages such as predictable absolute configuration and high ee of chiral secondary alcohol products, it seemed logical to investigate whether oxazaborolidine-catalysed reduction of ketones 3 and 6 could afford the desired exocyclic allylic alcohols in high enantioselectivity.Indeed, the borane reduction of chlorobenzylidenecyclopentanone 3a in the presence of 10 mol% of oxazaborolidine catalyst (S)-C5 at room temperature afforded the desired alcohol (R)-7a in 77% ee (as juged by chiral HPLC) (Table 1, entry 1).By varying different solvents, reaction temperatures, amount of reductant, and catalyst loading (Table 1, entries 2-8), the highest ee of 96% in reduction of 3a was achieved with 1.88 equiv.BH 3 × Me 2 S, 20 mol% (S)-C5 in toulene at 0 °C.Typically, reduction was carried out by slow addition of a toluene solution of the ketone to an ice-cooled toluene solution of BH 3 × Me 2 S and catalyst (stirred for 10 min prior to adding the ketone).The same protocol was used in the reduction of the methoxy-substituted analogue 3b giving (R)-7b but with significant loss of enantioselectivity (Table 1, entry 9).The opposite enantio-mers, (S)-7a and (S)-7b, were obtained by the borane reduction with the oxazaborolidine catalyst (R)-C5 (Table 1, entries 10 and 11).Interestingly, chloro-substituted alcohols (S)-7a and (R)-7a were obtained with practically identical ee values (~95%), while catalyst (R)-C5 reduced methoxy-benzylidenecyclopentanone 3b with increased enantioselectivity compared to catalyst (S)-C5 (90% vs. 82% ee).A dramatic drop in chemical yield and optical purity of the indanol alcohol 6 was observed in reduction of the indanone derivative 6 with either (S)-C5 or (R)-C5 catalyst.In spite of applying different reaction conditions (Table 1, entries 12-17), the corresponding alcohol 8 was not obtained in ee higher than 33%.Lower ee values associated with asymmetric reduction of indanone 6 as compared to cyclopentanone 3 may suggest that a fused benzene ring has a pronounced influence on the level of asymmetric induction with oxazaborolidine catalysts C5.Additionally, low isolated yield of indanol 8 might be due to its decomposition (or of parent ketone) under applied reaction conditions as was also established for reduction of analogous indanone-derived chalcones. 21he enantiomeric excess of the allylic alcohols 7 and 8 was determined by chiral stationary phase HPLC.The corresponding racemic alcohols were synthesized by che-  moselective reduction with NaBH 4 in the presence of CeCl 3 × 6H 2 O.They were used to find the optimal HPLC conditions for the separation of the pairs of the enatiomeric alcohols.Although Corey's (S)-proline-derived or stereochemically related oxazaborolidines in general delivered R-configured allylic alcohols in reduction of enones, 22 the R absolute configuration of chlorobenzylidenecyclopentanol 7a obtained from reduction with (S)-C5 was unambigously confirmed by X-ray crystallography (Figure 3).Additionally, this established also the configuration around the exocyclic C=C double bond as E. It should be made clear that stereochemical assignment for (R)-7a has not been previously made, although the absolute stereochemistry of related 2-benzylidenecyclopentanol obtained with Corey (S)-oxazaborolidine catalyst was determined to be R. 23 Thus, formation of the alcohol (R)-7a from chloro-substituted cyclopentanone 3a in the presence of oxazaborolidine catalyst (S)-C5 is also consistent with the sense of asymmetric induction predicted by the Corey mechanistic model. 24Consequently, we ascribed the R stereochemistry also to the methoxy-substituted alcohol 7b provided by oxazaborolidine catalyst (S)-C5, while for alcohols 7a,b arising from the borane reduction with catalyst (R)-C5 the S configuration was concluded.This was further supported by comparison of the sense of optical rotation and HPLC elution sequence of the enantiomeric forms of the alcohols 7a and 7b obtained with catalysts (S)-C5 and (R)-C5, respectively.Examination of the chromatogram (d) depicted in Figure 2 reveals, that for chloro-cyclopentanol 7a delivered with catalyst (S)-C5, the (+)-(R)-form of the enantiomers separated on chiral column is eluted second. .On this basis it can be speculated that catalyst (R)-C5 preferentially delivers the (S)-indanol 8 in the reduction of indanone 6, while with catalyst (S)-C5 the (R)-alcohol 8 is obtained as the major enantiomer.
Efficient asymmetric reduction of carbonyl compounds can also be achieved by means of bioreduction employing either isolated enzymes or whole cells system as mild and environmentally benign reduction systems.Fogliato et al. used baker's yeast 25 for the reduction of arylidene cyclopentanones and cyclohexanones reaching satisfactory enantioselectivity, while the secondary alcohols of excellent optical purity were obtained from Daucus carota 26 root reduction of structurally different prochiral ketones (up to 100% ee).Similarly, an α,β-unsaturated ketone trans-4-phenylbut-3-en-2-one was regio-and stereoselectively reduced using carrot, celeriac, and beetroot enzyme systems to the corresponding (S)-allylic alcohol in ee's 72-99%. 27In our case, the baker's yeast reduction of chlorobenzylidenecyclopentanone 3a gave very low isolated yield (5%) and optical purity (ee = 9%) of the corresponding alcohol 7a even after incubating the reaction mixture at 38 o C for 10 days.On the contrary, the 24-hour-bioreduction with Daucus carota root (substrate/carrot, 1/134 (w/w)) delivered alcohol (S)-7a with >99% ee as determined in the crude product (Figure 2, chromatogram (h)), the amount of which was, however, very low after removal of the biomaterial (Scheme 2).Interestingly, asymmetric induction turned out to be time-dependent, namely ee value of (S)-7a reduced to 92% after incubating reaction mixture for four days at room temperature.It is noteworthy that isolation of the desired alcohol product from bioreduction is intrinsically messy, as the aqueous media contains the cellular mass, usual metabolites, nutrients, and the starting ketone.Scheme 2. Bioreduction of benzylidenecyclopentanone 3a.

Experimental
General.Toluene was dried with sodium and distilled.All other reagents and solvents were used as received from commercial suppliers.Melting points were deter-Štefane et al.: Asymmetric Bio-and Chemoreduction ... mined on a Kofler micro hot stage.The NMR spectra were recorded at 302 K either on a Bruker Avance DPX 300 or Avance III 500 MHz spectrometer operating at 300 MHz or 500 MHz and 75.5 MHz or 126 MHz for 1 H and 13 C nuclei.The 1 H NMR spectra in CDCl 3 are referenced with respect to TMS as the internal standard.The 13 C NMR spectra are referenced against the central line of the solvent signal (CDCl 3 triplet at δ = 77.0ppm).The coupling constants (J) are given in Hz.The multiplicities are indicated as follows: s (singlet), d (doublet), t (triplet), q (quartet), qn (quintet), m (multiplet) and br (broad).IR spectra were obtained on a Bruker FTIR Alpha Platinum ATR spectrophotometer.MS spectra were recorded with an Agilent 6224 Accurate Mass TOF LC/MS instrument.Elemental analyses (C, H, N) were performed with a Perkin-Elmer 2400 Series II CHNS/O Analyzer.TLC was carried out on Fluka silica gel TLC-cards.Column chromatography was performed on 230-400-mesh silica gel.Merck silica gel 60 PF254 containing gypsum was used to prepare chromatotron plates.Radial chromatography was performed with Harrison Research, model 7924T chromatotron.HPLC analyses were performed with Agilent Technology 1260 Infinity HPLC instrument with UV detection.The known compounds were characterized by comparison of their physical or spectrosopic data with those in the literature.

Reduction of 3a with baker's yeast:
To a stirred solution of D-glucose (10.0 g, 55.5 mmol) and baker's yeast (56.0 g) in water (200 mL) at 38 °C, 2-(4-chlorobenzylidene)cyclopentanone (3a) (1.0 g, 4.84 mmol) dissolved in the minimum amount of EtOH (5 mL) was added; the reaction mixture was stirred for 10 days.Then EtOAc (100 mL) was added and the crude reaction mixture was filtered through a pad of Celite.The filtrate was extracted with EtOAc (3 × 100 mL), the organic phase was dried over anhydrous Na-24 h, then carrot root was filtered off and washed with water.Filtrate was extracted with EtOAc (3 × 50 mL).The organic phase was dried over anhydrous Na 2 SO 4 and the solvent was evaporated under reduced pressure to give 10 mg (10%) of crude red oily product 7a; ee >99%.

Conclusion
In summary, we synthesized enantiomerically enriched exocyclic allylic alcohols by asymmetric reduction of cyclic α-arylmethylene cyclic ketones.Highly enantioselective chemoreduction of 2-benzylidenecyclopentanone derivatives was achieved by applying chiral oxazaborolidine-derived catalysts under mild reaction conditions.The sense of asymmetric induction was in accordance with Corey mechanistic model, thus (S)-catalyst delivered (R)-alcohols, while (R)-catalyst gave (S)-alcohol products with ee values of up to 96%.The indanone-derived chalcone was much less efficiently reduced regarding the chemical yield and optical purity (33% ee).Bioreduction of 2-(4-chlorobenzylidene)cyclopentanone with baker's yeast gave very low ee of the corresponding allylic alcohol, while reduction with Daucus carota root turned out to be completely enantioselective.The synthesized allylic alcohols can serve as enantioenriched probes for the monitoring of oxidation-reduction processes catalyzed by AKR1C enzymes; these studies are currently under progress.

Figure 1 .
Figure 1.Chiral catalysts employed in the asymmetric reduction of cyclic ketones 3 and 6.
Prepared by the above procedure from 6 (125 mg, 0.49 mmol

. entry ketone catalyst solvent BH 3 × Me 2 S T (°C) product yield a (%) ee b (%) (mol%) (equiv.)
a Isolated yield is given.bDeterminedbychiralHPLC.cFirst solution of 6 added to a solution of (R)-C5, then BH 3 × Me 2 S. d Ketone dissolved in CH 2 Cl 2 , and catalyst in toluene.e Reaction quenched with MeOH.f The desired alcohol was not isolated.

Table 2
Crystallographic data, structure refinement summary, selected bond lengths, bond angles, and torsion angles for compound (R)-7a.