A novel gemcitabine analog as a potential anticancer agent: synthesis and in-vitro evaluation against pancreatic cancer.

Abstract

Gemcitabine (Gem) is approved for use in pancreatic cancer chemotherapy. However, Gem undergoes rapid metabolism in the blood, producing an inactive metabolite. Due to this rapid metabolism, the effective dose of Gem is high, thereby predisposing patients to severe adverse effects. This study aimed to improve Gem's metabolic and therapeutic stability by modifying the amine group (4-NH₂) with hydroxylamine to form 4-N-hydroxylGem hydrochloride (GemAGY). Micro-elemental analysis and Nuclear Magnetic Resonance (NMR) were used to characterize GemAGY, and its anticancer activity was investigated against MiaPaCa-2, BxPC-3, and PANC-1 pancreatic cancer cell lines. The GemAGY metabolic stability was evaluated in human liver microsomal solution. In the 2D cytotoxicity assay, the IC₅₀ values of GemAGY-treated MiaPaCa-2, PANC-1, and BxPC-3 cells were significantly lower when compared to GemHCl-treated cultures. More so, in 3D spheroid assay results, GemAGY IC₅₀ values were found to be 9.5 ± 1.1 µM and 12.6 ± 1.0 µM when compared to GemHCl IC₅₀ values of 24.1 ± 1.6 µM and 30.2 ± 1.8 µM in MiaPaCa-2 and PANC-1 cells, respectively. GemAGY was stable, with 60% remaining intact after 2 hours of digestion in microsomal enzymes, compared to GemHCl, which had less than 45% remaining intact after 30 minutes. GemAGY-treated MiaPaCa-2 and PANC-1 cells at 3.12 and 6.25 μM concentrations demonstrated a significantly reduced cell migration towards the wound area compared to the GemHCl-treated cultures at the same concentrations. Further, GemAGY-treated MiaPaCa-2 cells significantly increased the expression of p53 and BAX compared to GemHCl-treated cells. GemAGY demonstrated significant anticancer activity and improved metabolic stability compared to GemHCl and is most likely to have potential anticancer activity against pancreatic cancer.