Bis-Hydrazonoyl chloride as precursors for synthesis of novel polysubstituted bis-azoles

2.1.1 Synthesis of bis-heterocycles 4a,b, 7, 10a,b, 13 and 14

General method:

To sodium ethoxide solution, [prepared from sodium metal (0.12 g, 5 mmol) and absolute ethanol (15 mL)] was added the appropriate active methylene compounds (malononitrile or cyanoacetamide or pentane-2,4-dione or ethyl 3-oxo-3-phenylpropanoate or 3-oxo-3-phenylpropanenitrile or 6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-one (11) or 4-hydroxy-2H-chromen-2-one (12) (5 mmol of each). The mixture was stirred for 10 min. To the resulting solution was added bis-hydrazonoyl chloride 1 (2.5 mmol) and the reaction mixture was left over night at room temperature, while being stirred. The solid, that precipitated, was filtered off, washed with water, dried and finally crystallized from the appropriate solvent to give the respective products 4a,b, 7, 10a,b, 13 and 14, respectively.

2.1.2 1,1′-(1,1′-(Biphenyl-4,4′-diyl)-bis(4-benzoyl-1H-pyrazole-3,1-diyl))diethanone (18)

To a mixture of the bis-hydrazonoyl chloride 1 (0.391 g, 1 mmol) and 3-(dimethylamino)-1-phenylprop-2-en-1-one (16) (0.350 g, 2 mmol) in dry benzene (20 mL), triethylamine (0.2 mL) was added. The reaction mixture was refluxed for 6 h then cooled to room temperature. The triethylamine hydrochloride salt was filtered off and the filtrate was evaporated under reduced pressure. The residue was treated with methanol to give a yellow solid product which was filtered off, washed with ethanol, and recrystallized from DMF to afford bis-pyrazole 18 in 90% yield, mp > 300 °C; IR (KBr) ν 1685, 1647 (2C⚌O) cm⁻¹; ¹H NMR (DMSO-d₆) δ 2.61 (s, 6H, 2CH₃), 7.51–7.86 (m, 10H, ArH), 7.99 (d, J = 8 Hz, 4H, Ar-H), 8.12 (d, J = 8 Hz, 4H, Ar-H), 9.11 (s, 2H, pyrazole-H) ppm; MS, m/z (%) 578 (M⁺, 62), 577 (35), 105 (100), 77 (82). Anal. Calcd. for C₃₆H₂₆N₄O₄ (578.62): C, 74.73; H, 4.53; N, 9.68. Found: C, 74.50; H, 4.36; N, 9.48%.

2.1.3 Reaction of bis-hydrazonoyl chloride 1 with 3-amino-1,2,4-triazole 19 or 2-aminobenzimidazole 22

Triethylamine (2 mmol) was added drop wise at room temperature to a mixture of a bis-hydrazonoyl halide 1 (1 mmol) and the appropriate heterocyclic amine (2 mmol) in ethanol (40 mL). The reaction mixture was heated under reflux condition for 6 h. The resulting solid was collected by filtration, washed with ethanol, dried and finally crystallized from DMF to afford the corresponding bis-heterocycles 21 and 24. The physical and spectral data for obtained products are listed below.

2.2.1 Cytotoxic activity

The anticancer activity of the synthesized compounds 4a, 7, 10b, 13, 14, 18 and 21 was determined against the liver carcinoma cell line (HEPG-2), using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and Doxorubicin was used as a reference drug. Data generated were used to plot a dose response curve of which the concentration of test compounds required to kill 50% of cell population (IC₅₀) was determined. Cytotoxic activity was expressed as the mean IC₅₀ of three independent experiments (Table 1). The method applied is similar to that reported by Vijayan et al. (Vijayan et al., 2004) using Crystal violet stain (1%). Cells were seeded in 96-well plate at a cell concentration 1 × 10⁴ cells per well in 100 μl of growth medium. Fresh medium containing different concentrations of the test sample was added after 24 h of seeding. Serial twofold dilutions of the tested chemical compound were added to confluent cell monolayers dispensed into 96-well, flat-bottomed microtiter plates using a multichannel pipette. The microtiter plates were incubated at 37 °C in a humidified incubator with 5% CO₂ for 48 h. Three wells were used for each concentration of the test sample. Control cells were incubated without test sample with DMSO. After incubation of the cells for 24 h at 37 °C, various concentrations of sample (50, 25, 12.5, 6.25, 3.125, and 1.56 μg) were added, and the incubation was continued for 48 h and the viable cells yield was determined by a colorimetric method. After the end of incubation period, media were aspirated and the crystal violet solution was added to each well for at least 30 min. The stain was removed and the plates were rinsed using tap water until all excess stain is removed. Acetic acid (30%) was then added to all wells and mixed thoroughly, and then the absorbance of the plates was measured after gently shaking on a Microplate reader, using a test wavelength of 490 nm. All results were corrected for background absorbance detected in wells without added stain. Treated samples were compared with the cell control in the absence of the tested compounds. All experiments were carried out in triplicate.

3.2.1 Anticancer activity

The anticancer activity of the synthesized compounds (4a, 7, 10b, 13, 14, 18 and 21) was determined against a human liver cancer cell line (HEPG2) in the Regional Center for Mycology and Biotechnology at Al-Azhar University. Data generated were used to plot a dose^_ response curve of which the concentration of test compounds required to kill 50% of cell population (IC₅₀) was determined.

The results revealed that compounds 14, 13, 7 and 4a have promising anticancer activity against HEPG-2 cell line with IC₅₀ = 14.4, 15.3, 16.4 and 16.6 μg/mL respectively. On the other hand, compounds 10b, 18 and 21 have moderate activities. The most reactive derivative is the compound with coumarin moiety.

3.2.2 Antimicrobial evaluation

The antibacterial activity of the newly synthesized compounds (4a,b 7, 10b, 13, 14, 18) was evaluated in vitro against Staphylococcus aureus (RCMB 010028) (SA) and Bacillus subtilis (RCMB 010067) (BS) as examples of Gram-positive bacteria and Pseudomonas aeruginosa (RCMB 010043) (PA) and Escherichia coli (RCMB 010052) (EC) as examples of Gram-negative bacteria. The in vitro antifungal potential of these compounds against Aspergillus fumigatus (RCMB 02568) (AF), Pseudomonas italicum (RCMB 03924) (PI), Candida albicans (RCMB 05031) (CA) and Geotrichum candidum (RCMBB 05097) (GC) fungal trains was also evaluated. The inhibition zone diameter (IZD) in millimeters was used as a criterion for the antimicrobial activity using the agar diffusion method. Ampicillin and Gentamicin were used as reference drugs for antibacterial activity and Amphotericin B was used as reference drug for antifungal activity. The results are depicted in Tables 2 and 3.

The results indicated that compound 14 have high degree of antibacterial activity against SA and BS, relative to the standard drug ampicillin. Also, it exhibited high activity against EC, relative to standard drug gentamicin and exhibited a high inhibition effect against AF and CA, relative to standard drug amphotericin B. Compound 4b showed a high degree of activity against SA and BS and fungi species GC and CA. All the tested compounds exhibited no inhibition of growth against PA and PI. Compound 18 exhibited moderate activity against AF, CA, and GC) and no inhibition of growth against SA, BS, PA, EC and PI. Compounds 7, 10b and 13 exhibited no activity against all the fungal strains. The structure antimicrobial activity relationship of the synthesized compounds revealed that the maximum activity was attained with compound 14 having coumarin ring as substituent and 4b which has pyrazole ring with CONH₂ group at position 4.