SYNTHESIS OF SOME 2,3-DIHYDRO-1,3,4-OXADIAZOLES AND 4,5-DIHYDRO-1,2,4- TRIAZOLES AS ANTICANCER AGENTS

Methods: The 2,3-dihydro-1,3,4-oxadiazole derivatives 4a-h were synthesized by cyclization of N'-(substituted-benzylidene) isonicotinohydrazide 3a-e in refluxing acetic anhydride. The 2,3-dihydro-1,3,4-oxadiazole derivatives 4a-h were converted into the corresponding 4,5-dihydro-1,2,4triazoles 5a-h using ammonia. All the synthesized compounds were identified, depending on the physical and spectral data. Title compounds were assessed for their cytotoxic activity against human cancer cell line (MCF-7) by using Sulforhodamine B (SRB) colorimetric assay.

Inspired by these finding and in order to develop new anticancer therapeutic agents, we were encouraged to synthesize series of 2,3dihydro-1,3,4-oxadiazoles and their bioisosters, 4,5-dihydro-1,2,4triazoles, with incorporation of pyridine moiety, halogens, methoxy and methyl groups which are expected to allows simultaneous modulation of electronic, lipophilic, and steric parameters. These parameters can critically influence both the pharmacodynamic and pharmacokinetic properties of the synthesized compounds and expected to enhance their anticancer activities.

MATERIALS AND METHODS
Melting points are uncorrected and were determined on a Stuart melting point apparatus (Stuart Scientific, Redhill, UK). The FT-IR spectra (KBr) were recorded on Shimadzu FT-IR 110 spectrophotometer (Shimadzu, Koyoto, Japan) by using 1% potassium bromide discs. 1 H-NMR spectra were recorded on a Bruker proton 300 MHz (Bruker, Munuch, Germany) spectrometer using DMSO-d6 as a solvent and tetramethylsilane (TMS) as an internal standard. Chemical shift values are listed in δ scale. Mass spectra were determined using a GC/MS Mat 112 S at the 70ev spectrometer. Elemental analysis (C, H, N) were performed on a Perkin-Elmer 2400 analyzer (Perkin-Elmer, Norwalk, CT, USA) at the microanalytical laboratories of the Faculty of Science, Cairo University. Completion of the reaction was monitored by thin-layer chromatography (TLC) using precoated aluminum sheets silica gel (Merck, 60 F254). Visualization was accomplished with an ultraviolet UV lamp (Merck, Darmstadt, Germany). Synthesized compounds were purified by the re-crystallization process. The purity of the compounds was checked by a single spot in TLC and the solvent system for TLC was determined on a trial and error basis. All the chemicals and solvents used were of commercial grade.

Synthesis of N'-(substituted benzylidene) isonicotinohydrazide (3a-h)
Isonicotinohydrazide 1 (0.001 mol), appropriate aromatic aldehyde 2 (0.001 mol), ethanol (30 ml) and a catalytic amount of acetic acid were added to the round-bottomed flask (RBF) and the reaction mixture was refluxed for 3 h. The completion of the reaction was monitored by TLC. The reaction mixture was poured into crushed ice to obtain a solid product. The obtained precipitate was filtered under suction, washed thoroughly with water, dried and recrystallized from methanol.

Synthesis of 1-(5-(4-substituted phenyl)-3-(pyridin-4-yl)-4,5dihydro-1,2,4-triazol-1-yl)ethanones (5a-h)
Compounds 4a-h (0.01 mol) was added during 10 min to stirred and cooled solution of ammonia solution (10 ml) and water (10 ml). The temperature during the addition was kept below 20 °C. Then the reaction mixture was stirred for 2 h at room temperature. The reaction mixture was poured into crushed ice and acidify with hydrochloric acid to obtain precipitate, which was filtered off and, washed with water. The obtained solid was recrystallized from ethanol. IUPAC names and the molecular structures of the synthesized compounds 3a-h, 4a-h and 5a-h are shown in table 1.

Anticancer activities
The Cytotoxic activity of the synthesized compounds was measured against human mammary carcinoma cell line (MCF7) in the National Cancer Institute, Cairo University. The screening involves a calculation of the percentage growth or the surviving fraction of the drug-treated cell lines compared with untreated control using Sulforhodamine B (SRB) colorimetric assay [36]. After 48 h, cells were fixed and stained for 30 min with 0.4% (wt/vol) SRB dissolved in 1% acetic acid. Excess unbound dye was removed by four washes with 1% acetic acid and the attached stain was recovered with Tris-EDTA buffer. Color intensity was measured in an ELISA reader. The relation between surviving fraction and drug concentration is plotted to get the survival curve of the tumor cell line after the specified compound. The results were described in the table 2.
During the synthesis procedure, thin layer chromatography (TLC) analysis was carried out to verify the formation of the synthesized compounds. The structural elucidation of these compounds was confirmed through 1H NMR, IR and mas spectral data.