Prodrugs for nitroreductase based cancer therapy-4: Towards prostate cancer targeting: Synthesis of N-heterocyclic nitro prodrugs, Ssap-NtrB enzymatic activation and anticancer evaluation

Abstract

In this study, various N-heterocyclic nitro prodrugs (NHN1-16) containing pyrimidine, triazine and piperazine rings were designed and synthesized. The final compounds were identified using FT-IR, ¹H NMR, ¹³C NMR as well as elemental analyses. Enzymatic activities of compounds were conducted by using HPLC analysis to investigate the interaction of substrates with Ssap-NtrB nitroreductase enzyme. MTT assay was performed to evaluate the toxic effect of compounds against Hep3B and PC3 cancer cell lines and healthy HUVEC cell. It was observed that synthesized compounds NHN1-16 exhibited different cytotoxic profiles. Pyrimidine derivative NHN3 and triazine derivative NHN5 can be good drug candidates for prostate cancer with IC₅₀ values of 54.75 µM and 48.9 µM, respectively. Compounds NHN6, NHN10, NHN12, NHN14 and NHN16 were selected as prodrug candidates because of non-toxic properties against three different cell models. The NHN prodrugs and Ssap-NtrB combinations were applied to SRB assay to reveal the prodrug capabilities of these selected compounds. SRB screening results showed that the metabolites of all selected non-toxic compounds showed remarkable cytotoxicity with IC₅₀ values in the range of 1.71-4.72 nM on prostate cancer. Among the tested compounds, especially piperazine derivatives NHN12 and NHN14 showed significant toxic effect with IC₅₀ values of 1.75 nM and 1.79 nM against PC3 cell compared with standart prodrug CB1954 (IC₅₀: 1.71 nM). Novel compounds NHN12 and NHN14 can be considered as promising prodrug candidates for nitroreductase-prodrug based prostate cancer therapy.

Keywords: GDEPT; Nitro compounds; Nitroreductase; Piperazine; Prostate cancer; Pyrimidine; Triazine.