In vitro anti-Candida activity and single crystal X-ray structure of ({(1E)-[3-(1H-imidazol-1-yl)-1-phenylpropylidene]amino}oxy)(4-nitrophenyl)methanone

Materials and Methods

General: Melting points were determined on a Gallen-kamp melting point apparatus, and are uncorrected. Infrared (IR) spectra were recorded as KBr disks using the Perkin Elmer FT-IR Spectrum BX apparatus. NMR Spectra were measured in DMSO-d₆ on a Bruker NMR spectrometer operating at 500 MHz for ¹H and 125.76 MHz for ¹³C at the Research Center, College of Pharmacy, King Saud University, Saudi Arabia. Chemical shifts are expressed in δ-values (ppm) relative to TMS as an internal standard. Mass spectra were measured on Agilent Triple Quadrupole 6410 QQQ LC/MS with ESI (Electrospray ionization) source. Silica gel TLC (thin layer chromatography) cards from Merck (silica gel precoated aluminium cards with fluorescent indicator at 254 nm) were used for thin layer chromatography. Visualization was performed by illumination with UV light source (254 nm). Fluconazole was obtained from Shouguang-Fukang Pharmaceutical Ltd., Shandong, China. The antifungal discs containing 25 µg fluconazole were purchased from ROSCO (Neo-Sensitabs, Denmark). RPMI 1640 medium was purchased from Sigma-Aldrich Co., USA. Sabouraud Dextrose Agar (SDA) from Difco Laboratories, USA. Potato Dextrose Agar (PDA) was purchased from Eiken Chemical Co. Ltd., Japan. The X-ray diffraction measurements of compound 4 were performed using Bruker SMART APEXII CCD diffractometer. Crystallographic data of compound 4 have been deposited with the Cambridge Crystallographic Data Center (supplementary publication number CCDC-945902). Copies of the data may be obtained free of charge from the Director, CCDC, UK (deposit@ccdc.cam.ac.uk).

Preparation of 3-(1H-imidazol-1-yl)-1-phenylpropan-1-one (2): Acetophenone (2.4 g, 20 mmol), dimethylamine hydrochloride (2.2 g, 27 mmol) and paraformaldehyde (0.81 g, 9 mmol) were refluxed in absolute ethanol (5 mL) in the presence of catalytical amount of concentrated hydrochloric acid (0.1 mL). Reflux of the reaction mixture was continued under stirring for two hours, cooled and acetone (20 mL) was added. The formed Mannich base hydrochloride 1 (3.7 g, 17.4 mmol) was precipitated, filtered, dried and was subsequently dissolved in water (10 mL) and imidazole (2.4 g, 34.8 mmol) was added. The reaction mixture was refluxed for five hours, cooled and the precipitated solid was filtered off to give the ketone 2 (2.7 g, 77%) m.p. 95-97°C (Aboul-Enein  et al., 2001) which was pure enough to be used in the next step.

Preparation of (1E)-N-hydroxy-3-(1H-imidazol-1-yl)-1-phenylpropan-1-imine (3): A mixture of the ketone 2 (2.00 g, 10 mmol), hydroxylamine hydrochloride (1.39 g, 20 mmol), and KOH (1.12 g, 20 mmol) in ethanol (10 mL) was refluxed under stirring for 18 hrs. The reaction mixture was cooled to room temperature and the insolubles were filtered off. The filtrate was concentrated under reduced pressure and the residue was poured onto ice-cold water (15 mL). The precipitated solid was filtered, dried, and recrystallized from ethanol to give 1.51 g (70%) of the oxime 3 m.p. 155-157°C as colourless crystals (Attia et al., 2013). 

Preparation of ({(E)-[3-(1H-imidazol-1-yl)-1-phenylpropylidene]amino}oxy)(4-nitrophenyl)methanone (4): Ethyl chloroformate (0.67 mL, 0.97 g, 9.00 mmol) was added dropwise to an ice cold stirred solution of triethylamine (1.45 mL, 1.62 g, 16.00 mmol) and 4-nitrobenzoic acid (1.34 g, 8.00 mmol) in dichloromethane (20 mL). The reaction mixture was stirred at room temperature for half an hour, then oxime 3 (1.72 g, 8.00 mmol) was added to the reaction mixture and stirring was continued at room temperature for 18 hours. The organic phase was washed with water (2 x 15 mL), sodium bicarbonate solution (2 x 10 mL) and water (2 x 10 mL). The organic phase was separated, dried (Na₂SO₄) and evaporated under vacuum to give pale yellow powder of 4. The crude 4 was recrystallized from dimethyl sulfoxide to yield 1.02 g (35%) of the title compound 4 as pale yellow crystals m.p. 130-132°C. IR (KBr): d (cm^-1) 3115, 2713, 1743 (C=O), 1645 (C=N), 1520, 1257, 749; ¹H NMR (DMSO-d₆): d (ppm) = 3.55 (t, J = 6.7 Hz, 2H, -CH₂-CH₂-N), 4.32 (t, J = 6.7 Hz, 2H, -CH₂-CH₂-N), 6.78 (s, 1H, -N-CH=CH-N=), 7.17 (s, 1H, -N-CH=CH-N=), 7.50-7.59 (m, 4H, -N-CH=N-, Ar-H), 7.78 (d, J = 7.3 Hz, 2H, Ar-H_.), 8.27 (d, J = 8.8 Hz, 2H, Ar-H), 8.40 (d, J = 8.8 Hz, 2H, Ar-H); ¹³C NMR (DMSO-d₆): d 30.1 (-CH₂-CH₂-N), 43.0 (-CH₂-CH₂-N), 119.4 (-N-CH=CH-N=), 123.9, 127.3, 128.5, 128.8, 130.9, 131.2, 132.8 (-N-CH=CH-N=, Ar-CH, Ar-C), 133.8, 137.2 (-N-CH=N-, Ar-C), 150.4 (Ar-C_.), 161.4 (C=N), 165.1 (C=O); MS m/z (ESI): 365.1 [M + 1]⁺.

Crystal structure determination: Slow evaporation of the pure ester 4 in dimethyl sulfoxide yielded its pale yellow single crystals. A yellow block-shaped single crystal of suitable size, 0.40 mm X 0.54 mm X 0.78 mm, was selected for X-ray diffraction analysis. Data were collected on a Bruker APEX-II CCD area diffractometer equipped with graphite monochromatic CuK\α radia tion (λ=1.54178 beta) at 296 K. Cell refinement and data reduction were done by Bruker SAINT (Brucker, 2009); program used to solve structure and refine structure is SHELXTL (Sheldrick, 2008). The final refinement was performed by full-matrix least-squares techniques with anisotropic thermal data for non hydrogen atoms on F². All the hydrogen atoms were placed in calculated positions and constrained to ride on their parent atoms. Multi-scan absorption correction was applied by use of SADABS software (Brucker, 2009). The crystallographic data and refinement information are summarized in Table I.

Table I: Crystallographic data and refinement information

Anti-Candida agents: Stock solution (1,000 µg/mL) of test compound 4 as well as fluconazole were prepared in 100% dimethylsulfoxide and were diluted with sterile distilled water. Fluconazole antifungal discs were stored at -80°C until used.

Organisms: Two clinical isolates of Candida species were kindly provided by King Khaled Hospital, Riyadh, Saudi Arabia, one identified as C. albicans and the other as C. tropicalis. The isolates were preserved in glycerol broth at -80°C until used.

Media: PDA and SDA were used for routine subculturing of the Candida strains while RPMI 1640 medium supplemented with L-glutamine was buffered to pH 7.0 with 0.165 M 3-(N-morpholino)propane sulfonic acid (MOPS) was used for broth microdilution assay.

Broth microdilution testing: Preparation of inocula was performed in accordance with CLSI documents M27-A2 (CLSI, 2002) with RPMI 1640 medium. Isolates of Candida species were subcultured at 35°C for 48 hours on PDA plates. Yeast cells were recovered and suspended in 5 mL of sterile saline.  The turbidity of each Candida cell suspension was adjusted to a 0.5 McFarland standard (1.3 x 10⁶ to 5.3 x 10⁶ CFU/mL) at a wavelength of 530 nm according to the reported method (CLSI, 2002). Each suspension was diluted with RPMI 1640 medium (1:1000) to give a final inoculum of 1.3 x 10³ to 5.3 x 10³ CFU/mL.

Disk diffusion assay: As reported previously (CLSI, 2005), cell suspensions of the Candida isolates under test were adjusted to 5 x 10⁶ CFU/mL (0.5 McFarland standard). A 100 µL suspension of each tested strain was uniformly plated onto SDA plates. Whatman filter paper disks with a diameter of 6 mm were impregnated with 1,000 µg of the test compound 4. The disks were allowed to dry then they were placed onto the surface of the inoculated agar plates together with fluconazole antifungal discs. The plates were then incubated at 35°C and diameters of inhibition zones were measured at 24 hours.

Antifungal susceptibility studies: 100 µL Aliquots of the prepared Candida inocula were added to each well of the 96-well microdilution plates; each well contained 100 µL of twofold serial dilutions of fluconazole or test compound 4 (1 µg/mL to 500 µg/mL)  in RPMI 1640 medium. The plates was incubated at 35 °C for 48 hours then the turbidity of each well was measured at 490 nm with a microplate ELISA reader. The MICs of the Candida species were recorded as the lowest concentration at which a prominent decrease (50%-80%) in turbidity relative to the turbidity of the growth control.