AbstractIn an attempt to develop potent anticancer agents, we have synthesized some substituted pyridine derivatives. Alkylation of the pyridine‐3‐carbonitrile 1a by chloroacetonitrile or methyl iodide and ethyl iodide produced the O‐alkylated nicotinonitrile derivatives (2–4), respectively. The reaction of nicotinonitrile 1a with acetic anhydride furnished the N‐acetyl derivative 5. In addition, the reaction of 1a with concentrated sulfuric acid yielded compound 6 whereas its reaction with chloroacetone gave the corresponding 2‐alkoxy‐pyridine derivative 7 which then refluxed with sodium methoxide to give compound 8. Furthermore, alkylation of 1a‐c with methyl bromoacetate and ethyl bromoacetate produced the corresponding methyl, ethyl ester derivatives 9, 10a–c, respectively. The reaction of ethyl ester 10c with a solution of sodium hydroxide in ethanol afforded the corresponding acid derivative 11. Hydrazinolysis was carried out by the reaction of ethyl ester 10a,c with hydrazine hydrate to afford the desired acetohydrazide derivative 12a,c which was used as a key intermediate for the synthesis of substituted pyridine derivatives 13–19. Chlorination of 1a with phosphorus oxychloride afforded the 2‐chloro derivative 20 which react with excess of hydrazine hydrate to yield compound 21. All synthesized compounds were characterized using IR, NMR, mass spectra, and elemental analysis. Their cytotoxic activity was evaluated against three different cancer cell lines, including breast (MCF‐7), colon (HCT‐116), and ovarian (SKOV3) cancers as well as human normal cells EA.hy 926 (human endothelial somatic cell hybrid). In general, compounds 3, 4, 5, 9, 12a, 12b, 13, 14, 15, 16, 18, 19, 21 exhibited high cytotoxicity against cancer cell lines. Besides that, the large number of synthesized compounds exhibited moderate anticancer activities.