Development of Novel Pyrimidine Nucleosides as Potential Anticancer Agents: Synthesis, Characterization, and Invitro Evaluation against pancreatic cancer

Abstract The present study proposed modification of 5-FU by conjugation with an acyl chloride and a 5-membered heterocyclic ring to improve its invitro cytotoxicity and metabolic stability. XYZ-I-71 and XYZ-I-73 were synthesized by introducing a tetrahydrofuran ring on 5-fluorocytosine (a precursor of 5-FU) and conjugation with octanoyl chloride and lauroyl chloride, respectively. The analogs were characterized by NMR, micro-elemental analysis, HPLC, and LC-MS. The antiproliferative activity of the analogs was determined against MiaPaCa-2, PANC-1, and BxPC-3 pancreatic cancer cells. The analog’s stability in human liver microsomes was quantified by HPLC. Analysis of the 1H-NMR spectra displayed amide bonds at 7.80 ppm and 7.73 ppm, confirming the conjugation of octanoyl and lauroyl chloride to 5-fluorocutosine, respectively. The purity of both analogs was 99.6%. We found that the XYZ-I-73 (IC50 3.6 ± 0.4μM) analog was most effective against MiaPaCa-2 cells compared to XYZ-I-71(IC50 12.3±1.7μM), GemHCl (IC50 24.2 ± 1.3 μM), Irinotecan (IC50 10.1 ± 1.5 μM) and 5-FU (IC50 13.2 ± 1.1 μM). For PANC-1 cell growth inhibition, XYZ-I-73 (IC50 3.9 ± 0.5) was again observed as the most effective agent compared to XYZ-I-71(IC50 8.7±0.9 μM), GemHCl (IC50 10.07±0.9), 5-FU (IC50 20.43±1.2) and Irinotecan (IC50 11.6 ±1.1). A similar pattern of XYZ-I-73 (IC50 5.9 ± 0.7) anticancer activity against BxPC-3 cells was found to be higher than XYZ-I-71(IC50 7.7 ± 0.8), GemHCl (IC50 10.95±0.9), 5-FU (IC50 14 ± 1.1) and Irinotecan (IC50 9.5±1.0). For 24-hour MiaPaCa-2 cell migration studies, XYZ-I-73 (5μM) significantly reduced migration (# of migrated cells, 168 ± 2.9), followed by XYZ-I-71(315±2.1), Gem-HCl (762±3.1) and 5-FU (710 ± 3.2). PARP studies demonstrated significant inhibition of PARP expression of XYZ-I-73 treated cells compared to 5-FU and XYZ-I-71. Further, BAX and p53 expressions were significantly increased in cells treated with XYZ-I-73 compared to 5-FU and XYZ-I-71. In vitro, metabolic stability studies showed that 80 ± 5.9 % of XYZ-I-71 and XYZ-I-73 remained intact after 2-hour exposure in liver microsomal solution compared to 5-FU. The XYZ-I-73 analog demonstrated a remarkable cytotoxic effect and improved in vitro metabolic stability over the selected standard drugs and may have potential anticancer activity against pancreatic cancer.