New thieno[2,3-b]pyridine-fused pyrimidin-4(3H)-ones as potential thymidylate synthase inhibitors: Synthesis, SAR, in vitro and in silico study

•New thieno[2,3-b]pyridine-fused pyrimidinones were prepared.•New pyrimidinones demonstrated a wide range of in vitro cytotoxicity.•Pyrazole-linked pyrimidinones 2b and 2c demonstrated the best cytotoxicity.•Hybrids 2b and 2c demonstrated promising thymidylate synthase inhibitory activity.•Hybrids 2b and 2c had no mutagenic potential to the Salmonella strains. Cancer is the second leading cause of death, following cardiovascular disease. Thymidylate synthase (TS) has long been identified as a significant target for chemotherapy due to its critical role in DNA biosynthesis. New thieno[2,3-b]pyridine-fused pyrimidinones were prepared utilizing a three-component tandem protocol. One equivalent of 3-aminothieno[2,3-b]pyridine-2-carboxylate and 1.5 equivalents of dimethylformamide-dimethylacetal in toluene was microwave irradiated at 110 °C for 15 min. Next, the crude enamine was dissolved in dioxane and one equivalent of the respective aromatic amines or 3-aryl-1H-pyrazol-5-amines was added. The mixture was irradiated by microwaves at 100 °C for 20–45 min to produce the desired pyrimidinones in 72–89% yields. Examination of the in vitro cytotoxicity of the new products revealed a wide range of activity. Pyrazole-linked pyrimidinones 2b and 2c, attached to p-Cl and p-NO2, demonstrated the best cytotoxicity with IC50 values up to 6.59 and 3.65 µM, respectively, against MCF-7, HEPG2, and Caco2. The in vitro TS inhibitory activity of 2b and 2c was also assessed. 2b demonstrated comparable activity to the standard pemetrexed with inhibition percentages up to 86.3, whereas 2c inhibited TS the best with inhibition percentages up to 91.5. The Ames test revealed that 2b and 2c had no or weakly mutagenic potential to the Salmonella TA 98 or TA 100 strain in the presence or absence of metabolic activation. Docking study predicted the promising TS inhibitory activity of pyrimidinones 2b and 2c. According to SwissADME and Lipinski's rule of five, all new pyrimidinones could be classified as drug-like. [Display omitted]