Abstract
BACKGROUND: Theophylline, which is biologically important and found in tea, coffee, and cocoa beans, can be synthesized chemically or by direct extraction and concentration from natural sources. Theophylline derivatives have garnered attention in recent years for their potential therapeutic effects on Mycobacterium tuberculosis, antihistaminic, anti-inflammatory, and anticancer. Also, trifluoromethyl (CF(3)) group has also been widely used in drug and agrochemical design. METHODS: In this study, a series of new theophylline derivatives containing substituted trifluoromethyl and trifluoromethoxy groups were synthesized. The structures of these new compounds were confirmed by NMR, FT-IR, and elemental analyses. Additionally, the anticancer activities of the molecules were analyzed against VEGFR-2, CYP P450, and estrogen receptor by molecular docking method. Furthermore, in vitro biological effects of the compounds were comprehensively evaluated in cancer (A549 and HeLa) and normal (BEAS-2B) cells. Cell viability was assessed by MTT assay, and selectivity index (SI) values were calculated to determine tumor-specific toxicity. RESULTS: N(7)-substituted theophyllines were prepared by the reaction of 1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione (theophylline) and trifluoromethyl substituted benzyl halide compounds. The synthesized N(7)-substituted theophyllines were obtained as white powder in high yield. The structure of synthesized compounds was confirmed by various spectroscopic techniques such as (1)H, (13)C, (19)F NMR, and FT-IR spectroscopy, and elemental analysis. The highest interaction was recorded as -5.69 kcal/mol for 3-CF(3) substituted against VEGFR-2 structure while the best binding affinity was determined for 4-OCF(3) substituted with -6.69 kcal/mol against Human Cytochrome P450 with in silico analysis. The in vitro anticancer activities of the molecules were also evaluated against A549 and HeLa cells, and displayed considerably higher cytotoxicity with 2-CF(3), 3-CF(3,) and 4-CF(3) substituted molecules in Hela and A549 cell line. To elucidate the molecular mechanism, apoptosis-related gene expression changes were analyzed by RT-qPCR in A549 and HeLa cells treated with compound 2-CF(3). Significant upregulation of pro-apoptotic markers and downregulation of anti-apoptotic genes were observed. Consistently, ELISA-based quantification confirmed increased protein levels of Caspase-3, BAX, and Cytochrome C, and decreased BCL-2, validating the apoptotic mechanism at the protein level. Also, the antibacterial and antibiofilm activity details of the molecules were evaluated against DNA Gyrase, and SarA crystal structures by molecular docking method. The highest interaction was recorded as -5.56 kcal/mol for 2-CF(3) substituted with H-bonds with Asn46, Val71, Asp73, and Thr165 against DNA Gyrase crystal structure while 3-CF(3) substituted has the best binding affinity against SarA. The in vitro antimicrobial effects of the molecules were also evaluated. CONCLUSIONS: The synthesized molecules may provide insight into the development of potential therapeutic agents to the increasing antimicrobial resistance and biofilm-forming capacity of microorganisms. Additionally, compound 2-CF(3) substituted exhibited promising and selective anticancer activity through apoptosis induction, supported by gene and protein level evidence.