Synthesis and evaluation of proline ester prodrug of Ara-C.

摘要

Cytarabine (Ara-C), a nucleoside analogue, is one of the most effective chemotherapeutic agents used for the treatment of acute myelogenous leukemia[1] and various other hematological malignancies[2]. However, owing to its hydrophilic nature, low permeability across the intestinal membrane and its extensive first pass metabolism to its inactive form, arabinoslyuracil (Ara-U), it is often administered by continuous IV infusion or frequent administrations of high oral doses that are occasionally associated with significant side effects. [3];The intestinal peptide transporter Pept1, which transports dietary peptides as well as pharmacologically active peptidomimetic drugs, has broad substrate specificity. [2] As it is ubiquitously present in the intestinal membrane, it represents itself as a strong candidate for its use in prodrug strategies involving the improvement of the intestinal absorption of the polar drugs [4] Previous studies have shown that amino acid ester prodrugs are good substrates of PEPT1 transporter, and can be transported through the intestine using these transporters. [5];Bases on these studies, 5'-proline ester prodrug of Ara-C was synthesized and preliminarily evaluated for their in vitro capability to permeate Caco-2 cells, a surrogate for intestinal transport. Moreover, its chemical stability at various physiological pH (7.4, 5 and 2) and its enzymatic activation in Caco-2 cell homogenate were also assessed. Finally, the possibility of anti-proliferative action of 5'-proline amino acid prodrug was also tested using cancer cells such as HeLa cells and Caco2 cells.;Furthermore, as 3' ester prodrugs are not considered as a better substrate for these transporters than 5'-ester prodrugs, only 5'-proline amino acid ester of Ara-C was synthesized and tested in this study. [6];Proline-ester prodrug of Ara-C exhibited about 3-fold enhancement in permeability as compared to the parent drug in Caco2 cell monolayer. The anti-proliferative activity of Pro-Ara-C in both the cell lines was found to be significantly higher than the parent drug. IC50 of Proline-Ara-C was found to be 2.85muM and 0.27muM in HeLa cells and Caco-2 cells, respectively. Whereas, the parent drug exhibited IC50 of 392.3muM and 216muM in HeLa cells and Caco-2 cells, respectively. The conjugate was found to be the most stable in pH 7.4 (t1/2= 5015.19 min), less stable in pH 5 (t1/2= 1504 min), and the least stable in pH 2.0 (t1/2= 1003.03 min). Moreover, the activation of Pro-Ara-C was faster in Caco-2 cell homogenate (t1/2=15 min) than their hydrolysis in buffer solution, indicating enzymatic action that contributed to the hydrolysis of Pro-Ara-C to Ara-C. The chemical stability in pH 7.4 and enzymatic activation in Caco-2 cell homogenate was found to be analogous to the commercially marketed oral amino acid ester prodrugs like Valacyclovir and Valgancyclovir.[7] Conclusively, the 5' proline monoesters exhibited useful characteristics such as good solution stability and relatively fast enzymatic conversion rates.