Synthesis and bioevaluation of new proline prodrug of methotrexate

A prodrug of methotrexate was developed to overcome the breast cancer cell line resistance to methotrexate. Conjugation of the cytostatic agent methotrexate with the amino acid proline through imido-bond resulted in the formation of prodrug which is a good substrate for the enzyme prolidase. Prolidase is a cytosolic exopeptidase, which is specific for imido bond of dipeptide containing proline. The prodrug was synthesized by solid phase peptide synthesis method. Prodrug was examined as a substrate of pure prolidase. MDA-MB-231 cell line was selected as it is resistant to methotrexate due to compromised reduced folate carrier and MCF-7 cell line was selected as it responds to methotrexate. It has been found that Prodrug is more effectively transported into the MDA-MB-231 cells and evokes higher cytotoxicity as compared to methotrexate. While in the case of MCF-7 cells, methotrexate is more effectively transported into the MCF-7 cells and evokes higher cytotoxicity as compared to the prodrug. It was also shown that prodrug could be converted to the parent drug methotrexate in MDA-MB-231 cell homogenate faster as compared to MCF-7 cell homogenate. The results of the experiments show that the prodrug of methotrexate is more effective in methotrexate resistant breast cancer cell line as compared to the parent molecule methotrexate.