Synthesis and Antifungal Activity of β-Hydroxysulfides of 1 , 3-Dioxepane Series

Kazan (Volga Region) Federal University, Kremlyovskaya 18, Kazan 420008, Russia I.M. Sechenov First Moscow State Medical University, Trubetskaya 8, Bldg 2, Moscow 119991, Russia Kazan E. K. Zavoisky Physical-Technical Institute of the Russian Academy of Sciences, Sibirsky Tract, 10/7, Kazan 420029, Russia A.E. Arbuzov Institute of Organic and Physical Chemistry; KazSC, Russian Academy of Sciences, Arbuzova 8, Kazan 420088, Russia Kazan Scientific and Research Institute of Epidemiology and Microbiology, Bolshaya Krasnaya 67, Kazan 420015, Russia Kazan State Medical University, Butlerova 49, Kazan 420012, Russia

Various 5-and 6-substituted 1,3-dioxepanes are relatively scarce chemotype in medicinal chemistry due to synthetic difficulties and stability issues under physiological conditions.Nevertheless, in several reports, they were described as promising physiologically active compounds.us, a series of 5-hydroxy-6-sulfonamido-1,3-dioxepanes were reported as hypoglycemic agents which were active in mice with diabetes [10].
e same group of researchers reported several N-sulfonyl-tetrahydro [1,3]dioxepino [5,6] azirines which also possessed hypoglycemic activity [11].Optically active derivatives of 1,3-dioxepan-5-ol were described as flexible analogs of a known HIV-1 protease inhibitor (PI) darunavir [12].ey demonstrated excellent potency against a variety of multi-PI-resistant clinical strains, and the structure-activity studies indicated that the ring size, stereochemistry, and position of oxygens were important for the observed activity.
In attempt to combine the β-hydroxysulfide and 1,3dioxepane structural motifs, we previously synthesized a series of 2-phenyl-6-arylthio-1,3-dioxepan-5-ols, which possessed a moderate antifungal activity [13].In order to optimize structure-activity parameters of this interesting structural chemotype, in this work we have prepared a series of novel chiral β-hydroxysulfides of 1,3-dioxepane series and studied their antifungal activity against a panel of pathogenic microscopic fungi.It should be noted that microscopic fungi (micromycetes), causing mycoses, are significantly different from other infectious agents.Fungi are eukaryotic and are characterized by the presence of a rigid outer wall.Micromycetes are classified according to morphological characteristics, the degree of pathogenicity, and the ability to cause diseases of various organs and systems.In this work, we used four aggressive clinical isolates of Candida albicans, Aspergillus fumigatus, Epidermophyton floccosum, and Mucor pusillus species.Among mycelial pathogens of invasive mycoses, Aspergillus spp.are the most widespread.Dermatomycetes (Epidermophyton spp.) are one of the most common causative agents of superficial mycoses of skin, hair, and nails.Zygomycetes (Mucor spp.) cause extremely severe invasive mycoses with a very high mortality rate.And, finally, the vast majority of cases of fungal lesions of mucous membranes can be attributed to infections caused by yeast fungi of Candida species.

Materials and Methods
2.1.General Information.Chromatographic purification of compounds was carried out using column chromatography on Acros silica gel (60-200 mesh).Reaction progress and purity of compounds were monitored by TLC on Sorbfil PTLC-AF-A-UF plates.
Melting points of the products were determined using a Stanford Research Systems M.P.A-100 OptiMelt apparatus. 1H and 13 C NMR spectra were recorded on a "Bruker AVANCE 400" at operating frequency 400 and 100 MHz, respectively, using TMS as an internal standard.
HPLC experiment was performed using the Chiralpak AGP column (15.0 × 0.4 sm, 5 μm).A solution of methanol and 0.01 M ammonium acetate buffer (1 : 99 v/v) was used as the eluent.e elution rate was 0.9 ml/min, and the product was detected spectrophotometrically at 250 nm.MALDI-TOF experiments were performed using an ULTRAFLEX III TOF/TOF mass spectrometer (Bruker Daltonik GmbH, Germany).
e spectra of positive ions were recorded in linear mode with ion acceleration up to 20 keV.e energy of the laser beam was attenuated down to 70% of the full laser power.Analyte and p-nitroaniline matrix compounds were dissolved in acetone (1% in case of matrix and 0.1% in case of analyte) using glassware.e samples were prepared by a "dried-droplet" method: 0.3 µl droplets of the solutions were deposited on the marked spots on the standard target plate "AnchorChip" (Bruker Daltonik GmbH, Germany) and left to dry.
Quantum-chemical calculations were performed by DFT method B3LYP/6-31G(d,p) using Gaussian-98 [14] program with full geometry optimization without restrictions on symmetry, and a matrix of second derivatives was calculated for all stationary points.All discussed structures have only positive frequencies.Scaling factors [15] were not introduced.
e heating source was removed, and a short-time (<1 minute) spontaneous boiling-up with a sharp increase of the temperature was observed.e reaction mixture was cooled, and the product was purified by column chromatography (eluent petroleum ether-ethyl acetate, 4 : 1).

3,5-Dioxa-8-thiabicyclo[5.1.0]octane
e solvent was removed on a rotary evaporator at 20 °C.Yield 68% (0.77 g), colorless crystals.e spectral data for the obtained compound completely correspond to those reported in literature [28].(20).A mixture of thiophenol (0.45 g, 4.1 mmol), thiirane 19 (0.53 g, 4.02 mmol), and a catalytic amount of L 2 SP 3 was heated until the appearance of first bubbles.e heating source was removed, and a short-time (<1 minute) spontaneous boiling-up with a sharp increase of the temperature was observed.e reaction mixture was cooled, and the product was purified by column chromatography (eluent petroleum ether-ethyl acetate, 9 : 1).
e reaction mixture was stirred at 20 °C for 48 hours.en, lipase was filtered off, the solvent was evaporated under reduced pressure, and the product was separated by column chromatography (eluent petroleum ether-ethyl acetate, 7 : 1).

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Journal of Chemistry
e spectral data completely correspond to the racemic sample.HPLC retention time 109.9min (chiral stationary phase).

Crystal Structure Determinations
e X-ray diffraction data for the crystals of 6, 7, and 9 were collected on a Smart Apex II automatic diffractometer using graphite monochromated radiation MoK α (λ 0.71073).e structures were solved by a direct method using the SHELXS [21] program and refined by full-matrix least-squares using SHELXL2014 [21] program.All the nonhydrogen atoms were refined with anisotropic atomic displacement parameters.H(C) atoms were constrained as riding atoms, with the C-H set to 0.95 Å.All calculations were performed using WinGX [22] and APEX [23] programs.Crystallographic data (excluding structure factors) for the structures 6, 7, and 9 reported in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication, the corresponding CCDC numbers are given in Table 1.ese data can be obtained free of charge from e Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif.

Antifungal Activity Studies.
e antifungal activity has been tested against 4 clinical strains, C. albicans, A. fumigatus, E. floccosum, and Mucor pusilos, that were obtained from a collection of typical and clinical cultures of the mycological laboratory of the Kazan Research Institute of Epidemiology and Microbiology.
e fungal activity was evaluated in vitro using a minimum inhibitory concentration (MIC) test according to NCCLS guidelines.e tested substances were dissolved in Sabouraud medium to obtain the solutions with concentrations two times higher than the final ones.en, 1 ml of each solution and 1 ml of fungal spore suspension (concentration of 10 6 fungal spores per 1 ml) were added to the test tube.e final concentrations of the tested compounds were (mg/ml): 10; 5; 2.5; 1.2; 0.6; 0.3; 0.15; 0.07; 0.03; 0.015; and 0.007.e fungal spore solutions or vegetative cells were obtained from a viable 48-hour old yeast-like fungal culture and 6-day old mycelial fungi.e test tubes were incubated at 28 °C-30 °C for 9 days.e culture growth was followed with a photoelectric colorimeter at 530 nm using pure medium as a blank control.

Cytotoxicity Studies. Human skin fibroblasts (HSFs)
were isolated from the skin explant according to the conventional protocol [24].HSFs cells were cultured in the minimum essential medium Eagle (a-MEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 µg/mL streptomycin, and 100 U/mL penicillin under standard conditions (37 °C, 5% CO 2 atmosphere).Adhered cells were collected from the culture flask by detaching them with trypsin-EDTA solution.Suspended cells were by centrifugation at 200g in PBS.Cytotoxic concentrations (IC 50 ) of compounds were determined with the use of MTT assay.Cells were preseeded in a 96-well plate at the density of 1,000-2,000 cells per well and then incubated with aqueous solutions of the tested compounds for 3 days under standard conditions.Culture medium in the plate was then replaced by the fresh one supplemented with 0.5 mg/mL MTT and additionally kept for 4 h to allow for reduction of MTT into colored product (formazan) by metabolically active cells.Optical absorbance of produced formazan, proportional to viable cell number, was registered on an Infinite 200 PRO analyzer at 550 nm.

Results and Discussion
In continuation of our research on reactions of 3,5,8-trioxabicyclo[5.1.0]octaneswith nucleophiles and antimycotic properties of the obtained products [13], in this work we have carried out the thiolysis reaction of epoxide 2 by thiophenol followed by functionalization of the resulting β-hydroxysulfide 3 (Scheme 1).e functionalization was performed by oxidation of the sulfur atom and/or acylation of the hydroxyl group (compounds 4-6).Our attempts to synthesize compound 4 from acetate 6 by removing the acetate group with potassium carbonate led to formation of 8 Journal of Chemistry compound 7. is result suggested that the presence of the electron-withdrawing PhSO 2 fragment increased the acidity of methine proton which led to its elimination even under weak alkali treatment.Our further attempts to obtain the corresponding 1,3-dioxolane 8 and 1,3-dioxane 9 with the reduced cycle size via acidic isomerization of compound 3 were not successful.In order to obtain compounds 8 and 9, we then tried alternative synthetic routes depicted in Scheme 2. e intermediate 12 was obtained from diol 10 via its oxidation with oxone followed by thiolysis of the resulting epoxide 11 with thiophenol in hot water in the presence of DABCO.Further condensation of triol 12 with paraformaldehyde resulted in an inseparable mixture of compounds 3, 8, and 9, as well as the products of their reaction with paraformaldehyde. 1 H NMR spectral data demonstrated that initial 1,3-dioxepane 3 was the major component of the reaction mixture in all cases.Similar results were described in a previous paper [25].
e condensation reaction of butane-1,2,4-triol with paraformaldehyde under similar conditions led to a mixture of five-and six-membered rings, where six-membered ring structure was the product of both kinetic and thermodynamic control [26,27].It was clear that the presence of thiophenyl group in triol 12 significantly changed the ratio of the obtained isomers.In general, the obtained results suggested that this method was unsuitable for the synthesis of 8 and 9.
To evaluate the relative stability of the isomeric five-, six-, and seven-membered formals, we calculated the free energies for their structures by DFT quantum-chemical B3LYP/6-31G (d,p) method, which was previously validated for solution of the similar task for stereoisomers of Journal of Chemistry oxirane 2 and related compounds [28,29].ese calculations aimed at determination of relative thermodynamic stability of cyclic acetals of di erent types-dioxepanes, dioxanes, and dioxolanes.It was found that dioxolane 8 and dioxane 9 were less stable than dioxepane 3. e di erences in the relative values of free energies for the mentioned rings (∆∆G 298 for the most stable conformers: 0.00 kcal/moldioxepan, 0.12 kcal/mol-dioxolane, and 0.69 kcal/moldioxane) allowed us to evaluate the expected "thermodynamic" ratio of products in the gas phase as 56:38:6.Accounting of solvation e ects and possible involvement of other conformers of each cyclic isomer, as well as calculations using a di erent basis, could slightly change this ratio, but the same trend was observed.us, the replacement of proton on thiophenyl group in the third position of butane-1,2,4-triol led to preferential formation of the sevenmembered cyclic product in the reaction of condensation of this triol with paraformaldehyde.It can be concluded that formation of the seven-membered ring is advantageous both in terms of thermodynamic (calculated data) and kinetic (experimental data for the condensation reaction) control.Compound 9 was obtained using a series of reactions depicted in Scheme 2. At the rst step, reaction of diol 10 with acetic anhydride (0.8 mol.equiv.)resulted in monoacetylated derivative 13.According to 1 H NMR spectral data, the ratio of mono-and diacetylated products in unseparated mixture was 3 : 1. e analytical data for the obtained monoacetate 13 completely corresponded to those described in literature [19].Compound 13 was then oxidized by oxone to previously described epoxide 14 [20].Treatment of compound 14 with thiophenol in the presence of K 2 CO 3 led to a mixture of regioisomers 15 and 16. e reaction was strongly exothermic, with the temperature increase up to 150 °C that could explain the lack of the reaction selectivity.
e resulting mixture of acetates 15 and 16 was puri ed from initial thiophenol and triol 12, formed in small amounts, by column chromatography on silica gel.e resulting acetates 15 and 16, which could not be separated, were treated with paraformaldehyde to obtain the corresponding mixture of compounds 8 and 9. Dioxane 9 was isolated by column chromatography on silica gel in 24% yield as white crystals.Its structure was con rmed by X-ray analysis (Figure 1).Dioxolane 8 could not be isolated in an individual form due to presence of impurities with close chromatographic mobility.
Our further synthetic e ort was focused on structural modi cations of compound 3, including their enantiomerically enriched forms (Scheme 3).Treatment of alcohol 3 with para-toluenesulfonyl chloride or methanesulfonyl chloride in the presence of triethylamine (path a) resulted in formation of chloride 17 in 87% and 76% yields, respectively.e reaction with thionyl chloride (path b) was less successful and led to the desired product in 52% yield.Methyl ester 18 was prepared by reaction of chloride 17 in boiling methanol in the presence of an excess of sodium hydroxide.
e treatment of epoxide 2 with thiourea under aqueous acidic conditions led to episul de 19. e latter was treated with thiophenol in the presence of potassium carbonate without a solvent to obtain mercaptan 20 [28].In addition, reactions of epoxide 2 with various nucleophiles, including benzylthiol, phenol, aniline, and halogen-substituted thiophenols, resulted in a series of congeneric alcohols 21-33.
Structures of all the synthesized compounds were conrmed by 1D and 2D NMR spectroscopy, mass spectroscopy, and X-ray analysis.X-ray structures of compounds 6, 7, and 9 are shown in Figure 1.
At the next stage, several racemic compounds were separated into enantiomers using enzymatic acylation by lipase PS (Scheme 4) [18].As a result of two-step synthesis, we obtained optically active alcohols 3, 29, 31, 32, and 33, which had equal but opposite values of the rotation angles.Con guration of the chiral carbon atom at the hydroxyl group was assigned using the stereospeci city pro le of reactions in the presence of lipase PS, in which only hydroxyl groups with R-con guration at the chiral center were prone to acylation.It was observed that the enzymatic reaction rate and optical purity of the resulting products did not depend on the nature and position of the halogen substituent in the aromatic rings of the studied thiophenyl moieties.
To con rm the enantiomeric purity of the obtained alcohols, NMR spectra were obtained in the presence of a shift reagent, europium(III) tris[3-(hepta uoropropylhydroxymethylene)-(+)-camphorate. Figure 2 shows fragments of 1 H NMR spectra of the studied samples in the region of signals of hydrogen atoms at the acetal carbon atom.For the optically active alcohols, there are no signals of the second enantiomer, and these data suggest that the enantiomeric excess is close to 100%.It should also be noted that a mismatch in the chemical shifts of the signals for each enantiomer with respect to racemate is explained by di erent amounts of the sample compounds loaded into a vial and the shift reagent.
is factor also a ects the shape and intensity of signals.
e high enantiomeric purity (ee > 99%) of the leading compound 32-SS was also con rmed by HPLC analysis on a chiral stationary phase.
At the nal stage of the work, the antimycotic activity of the synthesized compounds against a panel of pathogenic microscopic fungi was tested.Speci cally, we used four aggressive clinical isolates of fungi belonging to C. albicans, A. fumigatus, E. occosum, and M. pusillus species.e in vitro experiment was carried out in liquid medium (Sabouraud glucose broth) using 2-fold serial dilutions in biological test tubes.Assessment of the culture growth was performed visually by comparing the growth of microorganisms in the presence of the studied test compounds and without them.
e rst lowest concentration of the tested sample (from a series of serial dilutions), where fungi growth was not visually detected, was considered as the minimum inhibitory concentration (MIC).As the reference compounds, two antimycotics with a broad spectrum of activity frequently used in clinical practice, uconazole and terbina ne, were used.
e data presented in Table 2 indicate that several compounds had an expressed antifungal activity with MICs in the range of 15-60 μg/ml.us, the leading compound 32 in the form of a dextrorotatory SS-enantiomer inhibited the growth of the studied fungi at the level of terbina ne, one of the most powerful systemic antimycotics.Fluconazole was signi cantly less active in this experimental series.To assess potential therapeutic window, we have measured the cytotoxic activity of the obtained compounds against the normal cells, human adipose tissue broblasts.
e presented experimental data (Table 2) indicate that the leading compound 32-SS possesses a remarkable selectivity of action against the studied fungal pathogens (selectivity index is 5-10) as compared to terbina ne (selectivity index is 0.6-2 against the studied pathogens).

Conclusions
In conclusion, in this work, we have developed e cient synthetic approaches to a series of novel 6-(arylthio)-1,3dioxepan-5-ols starting from 1,3-dioxacyclohept-5-ene through its oxidation followed by thiolysis with various thiophenols.Depending on the reaction conditions, this process can theoretically lead to the corresponding isomeric five-, six-, and seven-membered cyclic acetals: 1,3-dioxolanes, 1,3-dioxanes, and 1,3-dioxepanes, respectively.However, our experimental data supported by quantumchemical calculations using B3LYP/6-31G (d,p) method demonstrated that cyclic formals of 1,3-dioxepane series are the major products of this reaction.For such structures, the isomerization reaction is impossible due to instability of the intermediate carbocation.We have demonstrated that the corresponding 1,3-dioxolanes and 1,3-dioxanes can be obtained via alternative synthetic route based on condensation of monoacetylated butanetriols with paraformaldehyde.
Several racemic compounds of 1,3-dioxepane series have been separated into enantiomers using enzymatic acylation by lipase PS.For the optically active alcohols, our experimental suggest that the developed approach leads to enantiomerically pure compounds with the enantiomeric excess close to 100%.
Biological screening experiments have demonstrated that several compounds possess promising antifungal activity.us, the leading compound 32 with S-configuration of both stereocenters has expressed antifungal activity against four highly aggressive clinical isolates of fungi belonging to C. albicans, A. fumigatus, E. floccosum, and M. pusillus species, which is comparable with that of terbinafine and higher than activity of fluconazole.Of note, the synthesized 1,3-dioxanes were inactive against the studied fungal strains.e obtained chemotype represents a promising starting point for the development of viable antifungal drug candidates.

Table 1 :
Parameters of crystals of compounds 6, 7, and 9 and conditions of X-ray diffraction experiments.Scheme 1: Synthesis of hydroxysulfide of 1,3-dioxepane series and its further functionalization.

Table 2 :
Minimum inhibitory concentrations (μg/ml) of the synthesized compounds against the studied fungal strains.