Design, Synthesis, and Cytotoxic Evaluation of N-Phenyl-3-(Thiophen-2-Yl)acrylamide Derivatives.

A series of N-phenyl-3-(thiophen-2-yl)acrylamide derivatives were designed and synthesized to investigate the influence of electronic and steric modifications on cytotoxic activity. Although related thiophene-containing acrylamides have been reported, the specific compounds studied in this study have not been previously described. Structural variations were introduced at the para position of the N-phenyl ring (H, CF3, and OCF3) and at the 5-position of the thiophene ring (H and CH3) to enable preliminary structure-activity relationship (SAR) analysis. The target compounds were synthesized via Knoevenagel-Doebner condensation, followed by carbodiimide-mediated amide coupling. The compounds were characterized using nuclear magnetic resonance (NMR) spectroscopy, high-resolution mass spectrometry (HRMS), Fourier-transform infrared (FTIR) spectroscopy, and high-performance liquid chromatography (HPLC). Computational evaluation indicated compliance with Lipinski's rule of five and the absence of predicted mutagenic, tumorigenic, irritant, and reproductive risks. Cytotoxic activity was determined using the sulforhodamine B (SRB) assay against human breast adenocarcinoma (MCF-7), gastric adenocarcinoma (AGS), colorectal adenocarcinoma (Caco-2), oral squamous cell carcinoma (HSC-3), hepatocellular carcinoma (HepG2) cell lines and non-cancerous human dermal fibroblasts (HDF). Compounds 11 and 16 displayed the most consistent activity, with half-maximal inhibitory concentration (IC50) values of 379, 420, and 265 μM and 272, 256, and 242 μM against AGS, Caco-2, and MCF-7 cells, respectively. MCF-7 cells were the most sensitive model. The most active derivative was compound 16, suggesting that the combination of a 5-methylthiophene moiety and a para-CF3 substituent may be beneficial within this limited compound series. These findings provide a preliminary evaluation of this scaffold and will help guide future studies on structural modifications.