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Prodrugs for Nitroreductase Based Cancer Therapy- 1: Metabolite Profile, Cell Cytotoxicity and Molecular Modeling Interactions of Nitro Benzamides with Ssap-NtrB.

BACKGROUND
Directed Enzyme Prodrugs Therapy (DEPT) as an alternative method against conventional cancer treatments, in which the non-toxic prodrugs is converted to highly cytotoxic derivative, has attracted an ample attention in recent years for cancer therapy studies.


OBJECTIVE
The metabolite profile, cell cytotoxicity and molecular modeling interactions of a series of nitro benzamides with Ssap-NtrB were investigated in this study.


METHOD
A series of nitro-substituted benzamide prodrugs (1-4) were synthesized and firstly investigated their enzymatic reduction by Ssap-NtrB (S. saprophyticus Nitroreductase B) using HPLC analysis. Resulting metabolites were analyzed by LC-MS/MS. Molecular docking studies were performed with the aim of investigating the relationship between nitro benzamide structures (prodrugs 1-4) and Ssap-NtrB at the molecular level. Cell viability assay was conducted on two cancer cell lines, hepatoma (Hep3B) and colon (HT-29) cancer models and healthy cell model HUVEC. Upon reduction of benzamide prodrugs by Ssap-NtrB, the corresponding amine effectors were tested in a cell line panel comprising PC-3, Hep3B and HUVEC cells and were compared with the established NTR substrates, CB1954 (an aziridinyl dinitrobenzamide).


RESULTS
Cell viability assay resulted in while prodrugs 1, 2 and 3 had no remarkable cytotoxic effects, prodrug 4 showed the differential effect, showing moderate cytotoxicity with Hep3B and HUVEC. The metabolites that obtained from the reduction of nitro benzamide prodrugs (1-4) by Ssap-NtrB, showed differential cytotoxic effects, with none toxic for HUVEC cells, moderate toxic for Hep3B cells, but highly toxic for PC3 cells.


CONCLUSION
Amongst all metabolites of prodrugs after Ssap-NtrB reduction, N-(2,4- dinitrophenyl)-4-nitrobenzamide (3) was efficient and toxic in PC3 cells as comparable as CB1954. Kinetic parameters, molecular docking and HPLC results also confirm that prodrug 3 is better for Ssap-NtrB than 1, 2 and 4 or known cancer prodrugs of CB1954 and SN23862, demonstrating that prodrug 3 is an efficient candidate for NTR based cancer therapy.