Stereoselective Synthesis, Synthetic and Pharmacological Application of Monoterpene-Based 1,2,4- and 1,3,4-Oxadiazoles

Stereoselective synthesis of monoterpene-based 1,2,4- and 1,3,4-oxadiazole derivatives was accomplished starting from α,β-unsaturated carboxylic acids, obtained by the oxidation of (−)-2-carene-3-aldehyde and commercially available (−)-myrtenal. 1,2,4-Oxadiazoles were prepared in two steps via the corresponding O-acylamidoxime intermediates, which then underwent cyclisation induced by tetrabutylammonium fluoride (TBAF) under mild reaction conditions. Stereoselective dihydroxylation in highly stereospecific reactions with the OsO4/NMO (N-methylmorpholine N-oxide) system produced α,β-dihydroxy 1,2,4-oxadiazoles. Pinane-based 1,3,4-oxadiazoles were obtained similarly from acids by coupling with acyl hydrazines followed by POCl3-mediated dehydrative ring closure. In the case of the arane counterpart, the rearrangement of the constrained carane system occurred with the loss of chirality under the same conditions. Stereoselective dihydroxylation with OsO4/NMO produced α,β-dihydroxy 1,3,4-oxadiazoles. The prepared diols were applied as chiral catalysts in the enantioselective addition of diethylzinc to aldehydes. All compounds were screened in vitro for their antiproliferative effects against four malignant human adherent cell lines by means of the MTT assay with the O-acylated amidoxime intermediates exerting remarkable antiproliferative action.


Application of the Prepared Catalysts 9-11 and 14
The prepared potential catalysts 9-11 and 14 were used in the reaction of diethylzinc and benzaldehyde affording the formation of chiral 1-phenyl-1-propanol as a reference product (Scheme 4). They were applied in a 10% molar ratio in n-hexane at room temperature. The enantiomeric purities of 1-phenyl-1-propanols 19 and 20 obtained were determined by GC on a Chirasil-DEX CB column, according to literature methods [39,40]. Our results are presented in Table 1. Moderate enantioselectivity was observed for carane-based 1,2,4-oxadiazole 11 with S selectivity, while pinane-based 1,2,4-oxadiazoles (9 and 10) and 1,3,4-oxadiazole 14 have shown reasonable, similar S selectivity (up to 74% ee, Table 1) in the model reaction. We found that the type of the heterocyclic ring system has affected catalytic activities.

Application of the Prepared Catalysts 9-11 and 14
The prepared potential catalysts 9-11 and 14 were used in the reaction of diethylzinc and benzaldehyde affording the formation of chiral 1-phenyl-1-propanol as a reference product (Scheme 4). They were applied in a 10% molar ratio in n-hexane at room temperature. The enantiomeric purities of 1-phenyl-1-propanols 19 and 20 obtained were determined by GC on a Chirasil-DEX CB column, according to literature methods [39,40]. Our results are presented in Table 1. Moderate enantioselectivity was observed for carane-based 1,2,4-oxadiazole 11 with S selectivity, while pinane-based 1,2,4-oxadiazoles (9 and 10) and 1,3,4-oxadiazole 14 have shown reasonable, similar S selectivity (up to 74% ee, Table 1) in the model reaction. We found that the type of the heterocyclic ring system has affected catalytic activities.

Application of the Prepared Catalysts 9-11 and 14
The prepared potential catalysts 9-11 and 14 were used in the reaction of diethylzinc and benzaldehyde affording the formation of chiral 1-phenyl-1-propanol as a reference product (Scheme 4). They were applied in a 10% molar ratio in n-hexane at room temperature. The enantiomeric purities of 1-phenyl-1-propanols 19 and 20 obtained were determined by GC on a Chirasil-DEX CB column, according to literature methods [39,40]. Our results are presented in Table 1. moderate yield [25]. Dihydroxylation of 13 with OsO4/NMO afforded 14 in a highly diastereoselective reaction, similar to 1,2,4-oxadiazoles 6-8 (Scheme 2).

Application of the Prepared Catalysts 9-11 and 14
The prepared potential catalysts 9-11 and 14 were used in the reaction of diethylzinc and benzaldehyde affording the formation of chiral 1-phenyl-1-propanol as a reference product (Scheme 4). They were applied in a 10% molar ratio in n-hexane at room temperature. The enantiomeric purities of 1-phenyl-1-propanols 19 and 20 obtained were determined by GC on a Chirasil-DEX CB column, according to literature methods [39,40]. Our results are presented in Table 1. Moderate enantioselectivity was observed for carane-based 1,2,4-oxadiazole 11 with S selectivity, while pinane-based 1,2,4-oxadiazoles (9 and 10) and 1,3,4-oxadiazole 14 have shown reasonable, similar S selectivity (up to 74% ee, Table 1) in the model reaction. We found that the type of the heterocyclic ring system has affected catalytic activities. Moderate enantioselectivity was observed for carane-based 1,2,4-oxadiazole 11 with S selectivity, while pinane-based 1,2,4-oxadiazoles (9 and 10) and 1,3,4-oxadiazole 14 have shown reasonable, similar S selectivity (up to 74% ee, Table 1) in the model reaction. We found that the type of the heterocyclic ring system has affected catalytic activities.

Discussion
Starting from monoterpenic acids, dihydroxy-substituted 1,2,4-oxadiazoles and 1,3,4-oxadiazole were prepared and their catalytic activities were examined in the enantioselective synthesis of 1-phenyl-1-propanol. In pharmacological studies the O-acylamidoxime intermediates showed remarkable cytotoxic activity. Under the construction of the 1,3,4-oxadiazole system, rearrangement of the carane ring was observed with loss of chirality.

General Methods
Commercially available compounds were used as obtained from suppliers (Molar Chemicals Ltd, Halásztelek, Hungary; Merck Ltd., Budapest, Hungary and VWR International Ltd., Debrecen, Hungary) while applied solvents were dried according to standard procedures. Optical rotations were measured in MeOH at 20 • C, with a Perkin-Elmer 341 polarimeter (PerkinElmer Inc., Shelton, CT, USA). Chromatographic separations and monitoring of reactions were carried out on Merck Kieselgel 60 (Merck Ltd., Budapest, Hungary). Elemental analyses for all prepared compounds were performed on a Perkin-Elmer 2400 Elemental Analyzer (PerkinElmer Inc., Waltham, MA, USA). GC measurements for direct separation of enantiomers was performed on a Chirasil-DEX CB column (2500 × 0.25 mm I.D.) on a Perkin-Elmer Autosystem XL GC consisting of a Flame Ionization Detector (Perkin-Elmer Corporation, Norwalk, CT, USA) and a Turbochrom Workstation data system (Perkin-Elmer Corp., Norwalk, CT, USA). Melting points were determined on a Kofler apparatus (Nagema, Dresden, Germany) and are uncorrected. 1 H and 13 C NMR spectroscopic data were recorded at room temperature on a Bruker Avance DRX 400 MHz spectrometer (Bruker Corp., Billerica, MA, USA) in CDCl 3 or in DMSO.

General
Procedure for the Preparation of 3, 4 and 5 1 or 2 (6.02 mmol) was dissolved in dry CH 2 Cl 2 (75 mL) and CDI (9.07 mmol) was added. The solution was stirred at room temperature for 2 h, then benzamid oxime (18.07 mmol) or 4-chlorobenzamide oxime (18.07 mmol) was added in one portion. The mixture was stirred overnight then evaporated to dryness and purified by column chromatography on silica gel with CH 2 Cl 2 /EtOAc 19:1.  52 mmol), was dissolved in freshly-distilled THF (35 mL) and TBAF (1 M solution in THF, 0.35 mL) was added under Ar atmosphere. The solution was stirred for 1 h then water (50 mL) was added and the mixture was extracted with CH 2 Cl 2 (3 × 80 mL). The organic phase was dried (Na 2 SO 4 ) and evaporated to dryness. The crude product was purified by column chromatography on silica gel with n-hexane/EtOAc 19:1. 1 or 2 (6.02 mmol) and CDI (9.07 mmol) were dissolved in dry CH 2 Cl 2 (75 mL). The mixture was stirred at room temperature for 1 h, then the solvent was evaporated and the residue was dissolved in anhydrous DMF (60 mL). After adding benzhydrazide (12.05 mmol), the mixture was stirred for 40 h at room temperature then evaporated to dryness. The residue was dissolved in water (50 mL) and the aqueous phase was extracted with CH 2 Cl 2 (3 × 50 mL). The organic phase was dried (Na 2 SO 4 ) and concentrated in vacuo.  8, 132.7, 131.6, 129.5, 128.4, 127.3, 28.6, 27.2, 24.0, 22.7, 21.5, 16.7, 15.6 ( Figure S14) (28 mL) at 80 • C for 3 h then the reaction mixture was cooled to room temperature, poured onto ice and made basic (pH 8) with saturated NaHCO 3 solution. The aqueous phase was extracted with CH 2 Cl 2 (3 × 100 mL), then the organic phase was dried (Na 2 SO 4 ) and evaporated to dryness. The crude product was purified by column chromatography on silica gel with n-hexane/EtOAc 4:1.

Antiproliferative Assay
The human gynecological cancer cell lines isolated from cervical adenocarcinoma (HeLa), ovarian cancer (A2780), and breast cancers (MDA-MB-231 and MCF7) were purchased from European Collection of Cell Cultures (Salisbury, UK). The cells were grown in Minimum Essential Medium (MEM) supplemented with 10% fetal calf serum (FCS), 1% non-essential amino acids, and 1% penicillin-streptomycin. All media and supplements for these experiments were obtained from Lonza Group Ltd. (Basel, Switzerland). The cells were maintained at 37 • C in humidified atmosphere containing 5% CO 2 . The antiproliferative properties of the prepared monoterpene-based oxadiazoles were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay against adherent cancer cell lines [41]. Briefly, cells were seeded into 96 well plates (5000 cells/well) and incubated with two concentrations of the tested compounds (10 and 30 µM) under cell-culturing conditions. After incubation for 72 h, MTT solution (5 mg/mL) was added to each sample which were incubated for further 4 h. The formazan crystals precipitated were dissolved in 100 µL dimethyl sulfoxide, and the absorbance was measured at 545 nm with a microplate reader (Awareness Technology, Palm City, FL, USA). Two independent experiments were performed with five wells for each condition. Cisplatin (Ebewe GmbH, Unterach, Austria), a clinically used anticancer agent, was used as a reference agent. It the case of the most effective agents, the assay was repeated with a set of concentrations (0.01-30 µM) in order to determine the IC 50 values. Calculations were done by means of the GraphPad Prism 5.01 software (GraphPad Software Inc., San Diego, CA, USA) using the non-linear regression model log (inhibitor) vs. normalized response and variable slope fit.