Synthesis and Biological Evaluation of tert-Butyl Ester and Ethyl Ester Prodrugs of L-γ-Methyleneglutamic Acid Amides for Cancer

In cancer cells, glutaminolysis is the primary source of biosynthetic precursors, and recent efforts to develop amino acid analogs to inhibit glutamine metabolism in cancer have been extensive. Our lab recently discovered many L-γ-methyleneglutamic acid amides that were shown to be as efficacious as tamoxifen or olaparib in inhibiting the cell growth of MCF-7, SK-BR-3, and MDA-MB-231 breast cancer cells after 24 or 72 h of treatment. None of the compounds inhibited the cell growth of non-malignant MCF-10A breast cells. These compounds hold promise as novel therapeutics for the treatment of multiple subtypes of breast cancer. Herein we report our syntheses and evaluation of two series of tert-butyl ester and ethyl ester prodrugs of these L-γ-methyleneglutamic acid amides and the cyclic metabolite and its tert-butyl ester and ethyl ester prodrugs on the three breast cancer cell lines MCF-7, SK-BR-3, and MDA-MB-231 and the non-malignant MCF-10A breast cell line. These prodrugs were also found to suppress the growth of the breast cancer cells, but less potent compared to their parent L-γ-methyleneglutamic acid amides. Therefore, pharmacokinetic (PK) studies were carried out on the lead L-γ-methyleneglutamic acid amide (compound 5) to establish the tissue-specific distribution and other PK parameters. Notably, 5 showed moderate exposure to the brain with a half-life of 0.71 h and a good tissue distribution in the kidney and liver. The L-γ-methyleneglutamic acid amides were then tested on head and neck cancer cell lines HN30 and HN31 and glioblastoma cell lines BNC3 and BNC6 and were found to effectively suppress the growth of these cancer cells after 24 or 72 h of treatment in a concentration-dependent manner. These results suggest broad applications of these L-γ-methyleneglutamic acid amides in anticancer therapy.