Developing new compounds with bioactivity, especially against tumors, targeting G-quadruplexes is of great importance. In this direction, some new phthalimide-incorporating pyridine derivatives were synthesized using the phthalimido-acetophenone derivative 1 as a key precursor. Three-component cyclocondensation of compound 1 with various aryl aldehydes and ethyl cyanoacetate or malononitrile utilizing ammonium acetate as the nitrogen source afforded 4-substituted-6-(4-phthalimidophenyl)pyridin-2-one-3-carbonitriles (2a-e) or 2-amino-4-substituted-6-(4-phthalimidophenyl)pyridine-3-carbonitriles (3a,b), respectively. Characterization of these derivatives were identified on the basis of elemental analysis and spectroscopic tools such as nuclear magnetic resonance (NMR) and infrared (IR). Additionally, the potency of the targeted molecules as anticancer agents was assessed against two carcinogenic human cell lines, namely, colon cancer (HCT-116) and human prostate cancer (PC3) cells. The synthesized derivatives exhibited promising cytotoxicity in which compounds 2a and 2b have the strongest potency towards the two cancer cell lines comparable with doxorubicin (Dox). The conducted molecular docking studies revealed that the investigated compounds possess a good correlation between their anti-cancer activities and binding effectiveness with both c-MYC and KRAS G-quadruplexes.