|Abstract||The International Agency for Research on Cancer indicated that cancer burden was raised in 2012 to 14.1 million incidence cases and 8.2 mortality cases. Consequently, novel approaches are urgently required for further improvement in existing cancer therapies. Synthesis of the pyridine ring system and its derivatives occupy an important place in the realm of synthetic organic chemistry, due to their therapeutic and pharmacological properties. The current study investigated the anti-cancer activity of novel pyridine derivatives. Twenty novel Pyridine Derivatives were screened using WST-1 assay on several cell lines to assess their toxicity and to determine whether the effect is tumor or cell type specific. Dose response curves were obtained for compound 9a for further investigations. Detection of apoptosis and cell cycle checkpoint analysis was done using flow cytometry and expression of several apoptotic and anti-apoptotic proteins was carried out by Western Blot. Initial screening revealed that 100μM treatment with pyridine derivatives for the above mentioned cell lines for 24 hours suppressed the viability as follows: compound 9a reduced the viability by 40% in MCF-7 cells and by 45% in HCT-116, compound 7b reduced the viability in SKOV-3 by 35% and finally compound 11 suppressed the viability of SKOV-3 by 44%. Compound 9a induced growth inhibition in MCF-7 cells and resulted in the induction of apoptosis with an IC50 of 20μM. It upregulated the expression of p53, Bax and Caspase-3 in MCF-7 cells. In addition, it caused significant down-regulation of Bcl-2, Mdm-2 and Akt. Compound 9a exhibited less toxicity on non-tumorigenic breast epithelial cell line MCF-12a. Findings of the study indicated that compound 9a possesses potent anti-proliferative activity against MCF-7 cells and could be a promising chemotherapeutic agent with less toxicity on non-tumorigenic cells. Further screening and investigating molecular mechanisms of this potent agent on other cancer cell lines is highly required.