Synthesis of optically active vicinal fluorocyclopentanols and fluorocyclopentanamines by enzymatic deracemization

All possible stereoisomers of cis-and trans -2-fluorocyclopentan-1-ols were obtained by kinetically controlled deracemization in the presence of lipases in organic media. High enantioselectivities and good yields of stereomers were obtained for all substrates. Optically pure 1,2-fluorocyclopentan-1-ols were converted to 2-fluoro-cyclopentan-1-amines using the Mitsunobu reaction. The absolute configurations were determined using the Kazlauskas rule and chemical correlation. The interaction of substrates with enzymes has considered using Koshland induced fit theory.


Introduction
The synthesis of fluorinated biologically active compounds of high enantiomeric purity (ee> 95%) has attracted much attention in recent years due to fluorine's ability to increase drugs' lipophilicity, selectivity and duration of action. 1 However, few methods are known for the synthesis of fluorine-containing compounds of high optical purity.For example, asymmetric metal complex catalysis only in some cases allows the achievement a high level of stereoselectivity in the preparation of chiral organofluorine compounds.Biocatalysis gives the best results.][4][5] Vicinal halocyclopentanols are synthetic blocks for a number of chiral natural and synthetic products.[8][9][10] We have previously synthesized all possible stereoisomers of cis-halocyclohexanols (Hlg = I, Br, Cl, F) using the kinetic enzymatic resolution of corresponding racemates (Scheme 1).Haufe et al. 3 and Hashimoto et al. 4 described the enzymatic deracemization of trans-2-fluorocyclohexane-1-ols, 2-fluorocycloheptan-1-ols, and 2fluorocyclooctan-1-ols using Pseudomonas Fluorescence lipase.Scheme 1. Chiral 2-fluorocyclohexan-1-ols.
In connection with our studies of the influence of stereochemistry and fluorine atoms on the biological activity of compounds, we have synthesized all four optically pure cis-and trans-stereoisomers of 2fluorocyclopentan-1-ols.These compounds are in demand as synthetic blocks for the preparation of many important biologically active products.For example, 2-substituted cyclopentanols can be converted into conformationally limited leukotriene antagonists, which can then be used in the enantioselective synthesis of (+)-Estron, Estriol, Desogestrel, Eicosanoids (V-VIII), and others (Scheme 2). 1 Scheme 2. Natural compounds containing a cyclopentane ring.
For this purpose, racemic trans-2-fluoro-substituted cyclopentanol (+/-)-6 was underwent to Swern oxidation with the formation of 2-fluorocyclopentanone 7 in good yields.Ketone 7 was reduced with various reducing reagents, that led to the formation of cis-2-fluorocyclopentan-1-ol with an admixture of trans-isomers.
The best results were obtained with K-Selectride [(i-Bu3BH)K], which gave a mixture of cis-and trans-isomers in a ratio of 75:25, reduction with sodium borohydride in methanol gave a ratio of 70:30, and the reduction with i-Bu2AlH (H-DIBAL) resulted in a 55:45 ratio.Subsequent treatment of a mixture of cis-and trans-2fluorocyclopentanols with base (DBU and others) proceeded with the preferential hydrolysis of the transisomer, that leads to the enrichment of mixture with the unreacted cis-isomer (Table 1).As a result, after additional low-temperature recrystallization in pentane or hexane, a pure cis isomer can be obtained.(Table 1).The reductions of 2-substituted fluoropentanones depended on the conformational equilibrium of the axial and equatorial forms: equatorial attack of the nucleophile leads to the formation of trans-alcohols while the axial attack provides the cis-alcohols.The cis-isomers of 2-halogencycloalkan-1-ols are more stable toward basemediated HX-elimination than the trans-isomers.Unlike trans-isomers (+/-)-6, which upon alkali treatment easily convert into epoxide 5, cis-isomers (+/-)-8 with excess DBU converted into cyclopentanone.The cis orientation of the oxygen and halogen atoms is unavailable for intramolecular SN2 reaction with the formation of the epoxide, because nucleophile cannot attack the stereocentre from the front side.In this case, an alternative anti-conformation B (via ring flip) is available for rearrangement via hydride migration and the cyclohexanone formation (Scheme 5). 12Scheme 5. Base-mediated reaction of cis-and trans-fluorocyclopentan-1-ols.a Yields for the mixture of the cis-and trans-isomers.b Yields for the purified cis-isomer (+/-)-8.
To deracemize 2-fluorocyclopentanols and obtain enantiomerically pure compounds, we used several lipases with well-known biocatalytic activity in organic solvents using vinyl acetate or isopropenylidene acetate as an acylating reagent.We tested Candida antarctica (CAL-B), Pseudomonas cepacia (PCL) and Burkholderia cepacia (BCL).The best results (highest ee) were obtained with BCL; therefore, this lipase was used.Enzymatic esterification in the presence of BCL immobilized on diatomaceous earth allowed separation of the racemic fluorocyclopentanols into enantiomerically pure optically active stereoisomers.The esterification was carried out at room temperature and stopped at 50% conversion to acetate, which was achieved by filtration of the biocatalyst from the reaction mixture.In all cases, the products were obtained with very good enantiomeric excesses (ee).
The acylation of 2-fluorocyclopentanols (+/-)-6 biocatalyzed by BCL proceeded with the highest enantioselectivity to provide (1R,2R)-4 with 96% ee (Scheme 6).Acylation in the presence of the CALB biocatalyst proceeded faster, then with BCL, but the enantioselectivity of reaction was lower.In turn, the transesterification of the corresponding fluorocyclopentanols with vinyl acetate proceeded faster than with propenyl acetate.However, the enantioselectivity in this case was also slightly lower.Usually, esterification with vinyl acetate proceeds relatively slowly and within 15-18 hours leads to a 50% conversion of (+/-)-6 to acetate (R,R)-9.Scheme 6. Enzymatic deracemization of 2-cis-and trans-fluorocyclopentanols 6,8.
Similarly, acylation of racemic trans-cyclopentanol with vinyl acetate in the presence of BCL under kinetically controlled conditions (50% conversion of the starting alcohol) led to the formation of alcohols (1R,2S)-2 and acetates (1R,2R)-9 that were isolated by column chromatography.Hydrolysis of (1R,2R)-9 acetates in phosphate buffer at pH 7.2 gave the second stereoisomer of (1R, 2R)-fluorocyclopentanol with high enantiomeric and diastereomeric purity.
Hydrolysis of the obtained acetates 9, 10 with BCL lipase in phosphate buffer at pH 7.2 led to the formation of the second stereoisomer of alcohols 2, 4 with high enantiomeric purity (98-99% ee).Acetates 9, 10 were also hydrolyzed with K2CO3 in methanol to afford the hydrolyzed alcohols 2, 4 with the same ee and yields (Table 2).Additional low-temperature (-20 0 C) crystallization of alcohols in pentane made it possible to obtain enantiomerically pure alcohols (1R, 2S)-2 and (1R, 2R)-4 with de and ee >97-98%, that was determined by derivatization of the compounds with Mosher's acid.Enantiomerically pure 2-fluorocyclopentan-2-ols are colorless low-melting compounds or liquids that are stable at room temperature or in a refrigerator.No racemization was observed during operation with 2-fluorocyclopentan-1-ols or during storage.The nature of the solvents used had no significant effect on the enantioselectivity of the kinetic resolution.However, diisopropyl ether (DIPE) and methyl-tert-butyl ether (MTBE) or solvent-free vinyl acetate showed the highest ee, while the reaction proceeded faster in cyclohexane.In the case of trans-2-fluorocyclopentanol (+/-) -6, the reaction in the above-mentioned solvents took much longer and the enantioselectivity was slightly lower.The rate of acylation of 2-fluorocyclopentanols with vinyl acetate and PCL in DIPE was slightly higher compared to the corresponding reactions with isopropenylidene acetate.In all cases, (R)-selectivity was observed in accordance with the Kazlauskas' rule.Better solubility of most substrates in organic solvents and easy separation of immobilized lipases promote reactions in organic media.In accordance with the theory of Koshland ("induced fit model"), it is implied the flexibility of the active site of the deracemization reaction.The attachment of the substrate to the active center of the enzyme causes a change in the configuration of the catalytic center, as a result of which its shape matches the shape of the substrate ("hand-glove").If this transformation contributes to better complementarity of the substrate and the active center, then this gives a gain in the reaction rate, i.e. provides a significant catalytic effect.Due to their mutual influence, when the substrate approaches the active center of the enzyme, the structures of both reacting molecules are transformed.[15] Scheme 7. Conformational change in the active centre of enzyme according to the "induced fit model".
The structure and purity of the reaction products were confirmed by physical-chemical methods.The 13  To confirm the structures and absolute configuration of 2-fluorocyclopropanols, we converted the (1S,2S)and (1R,2R)-2-fluorocyclopentanols into enantiomers of (1R,2S)-and (1S,2R)-2-fluoro-1aminocyclopentanols 16,17 using the Mitsunobu reaction.A solution of alcohol in THF was reacted with phthalimide in the presence of triphenylphosphine and diethyl (DEAD).The reaction proceeded with the inversion of the absolute configuration at the carbon atom containing the hydroxyl group.After the reaction was completed, the products were isolated by column chromatography 12,14.The products were then treated with hydrochloric acid to form aminofluorocyclopentane hydrochloride.As a result, trans-1,2-fluorocyclopentanol was converted to cis-1,2-aminofluorocyclopentanes.Pure products were obtained with an enantiomeric excess of 95% ee (Scheme 8).2-Fluorocyclopentyl-1-amines are of interest as intermediates for the synthesis of a number of important biologically active compounds: For example, they can be used in the synthesis of potent and selective opioid receptor-like antagonists. 15,16he resulting products 12,14 were separated by column chromatography.The yields of diones 11,13 and amines 12,14 were 70 and 90% correspondingly (Scheme 8).Scheme 8. Synthesis of chiral 2-fluorocyclopentan-1-amine hydrochloride using Mitsunobu reaction.
The optical purities of the (R,S)-and (S,R)-2-fluorocyclopentan-1-amines 12, 14 were on the order of 96% determined by derivatization with Mosher's acid.The structures of amines were confirmed by NMR and massspectra.In the NMR spectra of the hydrochlorides the signals of NH3 + protons at 8.5 ppm, signals of CHF at 5.1 ppm, a doublet with 2 JHF constant 55 Hz and CHN at 3.4 ppm, doublet, 2 JHF 26 Hz, as well as the CH2 protons were observed. 17e definition of absolute configuration.Kazlauskas rule 18 was used for determination of the absolute stereochemistry of the resolved 2-fluorocyclopentanols.According to the Kazlauskas rule, the enantioselectivity should be proportional to the size difference between the large (L) and medium-size (M) substituents in the substrate.The physical essence of the Kazlauskas rule is determined by structure of lipase active center, which has two pockets -one is larger and the other is smaller.In accordance with the structure of the active center, the orientation of the secondary alcohol occurs and the esterification/hydrolysis of the corresponding esters proceeds.According to the Kazlauskas rule, the biocatalytic acetylation of 2-fluorocyclopentan-1-ols should be (R)-selective, leading to the formation of (1R,2S)-acetates and (1S,2R)-unreacted alcohols in the case of the cisfluorocyclopentanols and correspondingly to the formation of (1R,2R)-acetates and (1S,2S)-unreacted alcohols in case of trans-2-fluorocyclopentanols (Scheme 9). 19Empirically, as shown earlier on a very large number of examples, trans-esterification of secondary alcohols in the presence of BCL, and as well as of many other lipases, always leads to the formation of (R)-acetate and unreacted (S)-alcohol.Therefore we have obtained the cis-(1R,2S)-10 acetate and the cis-(1S,2R)-3 alcohol, as well as trans-(1R,2R)-9 acetate and trans-(1S,2S)-2 alcohol.1][22] In addition, we confirmed the results obtained on the basis of the Kazlauskas rule by independent synthesis of (1S, 2S) -fluorophosphonate 2 whose configuration is known. 2The determination of absolute configuration of compound 2, attained by two different methods was identical.

Experimental Section
General. 1 H (500MHz) and 13 C (125 MHz) NMR spectra were recorded on Bruker Avance DRX 500 spectrometer in dimethylsulfoxide (DMSO-d6) solution with tetramethylsilane (TMS) as an internal standard.Unless otherwise specified NMR spectra have been made in CDCl3 Chemical shifts (δ) of 1 H and 13 C are reported in ppm relative to CHCl3 (δ = 7.26 for 1 H and δ = 77.0for 13C).J values are given in Hz.Proton ( 1 H) NMR information is given in the following format: multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; sept; septet; m, multiplet, b, broad), coupling constant J, number of protons).Melting points were measured with a Büchi melting-point apparatus and are uncorrected.The chromatomass spectra were recorded on an Agilent 1100 Series high-performance liquid chromatograph equipped with a diode matrix with an Agilent LCnMSD SL mass selective detector, allowing fast switching of the ionization modes.The GC-MS studies were performed using an Agilent Technologies 1200 device.The HPLC was performed with Chiralcel OJ-H chiral column (250*4,6 mm, 5 mkm with Selector Celulose tris(4-methylbenzoate) coated on 5 mµ silica gel.The reaction progress was monitored by thin-layer chromatography (TLC) on silica gel 60F254 Merck and visualized under ultraviolet light (254 and 366 nm), or through spraying with 5% phosphomolybdic acid in EtOH, H2SO4 acidified by Anise aldehyde solution in EtOH or by placing in iodine vapor.Flash chromatography was performed with Merck silica gel 60 (230-400 mesh).Most part of the reactants were obtained from a commercially available source (Aldrich) and used without further purification.All solvents were purified by standard procedures or obtained from a Solvent Purification System (Braun SPS 800).Unless otherwise mentioned, all reactions were carried out under an atmosphere of dry argon.
C NMR spectra of 2-fluorocyclopentanols show signals from all five carbon atoms split at the fluorine atom [ 1 JCF 177 Hz (C-F)].In the proton resonance spectra, there are doublets of the signals of CHF and CHOH groups at 4.3 and 4.8 ppm, respectively.The signal of the fluorine atom was discovered at -185 ppm.The mass spectra contain signals of mass peaks.The purity of the compounds was confirmed by HPLC using a chiral column OJ-H".

Table 2 .
Enzymatic resolution of 2-fluorocyclopentan-1-ols 1-4 by Burkholderia cepacia lipase-catalyzed esterification using vinyl acetate as acyl donor 19Calculated by the acylated conversion and% ee of the product.E = ln[1c(1 + eep)]/ln[(1c)(1eep)], where p = product.bDeterminedfrom the19F NMR spectra of the respective MTPA esters.c Obtained from the NMR spectra of the MTPA esters of the recovered alcohols.d [α]D and ee were defined for isolated and purified products.