Diazeniumdiolate ions, also known as NONOates, are extensively used in biochemical, physiological and pharmacological studies for controlled release of nitric oxide (NO.) and /or its congeneric nitroxyl (HNO) at physiological pH. They can be derivatized at the terminal oxygen to produce charge neutral derivatives that are proving useful as potential drugs, molecules that are stable in aqueous media but capable of generating nitric oxide beneficially when the protecting group is hydrolytically or enzymatically removed in the target cell. Compounds of the O2-aryl diazeniumdiolate family have shown noteworthy anticancer activity in a variety of model systems; for example, O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate or JS-K has shown antineoplastic activity in vitro and in vivo in leukemia, multiple myeloma, prostate cancer and hepatoma rodent models. In this regard, we have continued to synthesize and diversify libraries of O2-arylated diazeniumdiolates for additional screening and possible lead optimization. To this end, we prepared a series of bis-diazeniumdiolates and checked their in vitro activities towards different cancer cell lines. Initial screening was performed using the HL-60 human leukemia cell line and two human lung adenocarcinoma cell lines (H1944 and H1703). The IC50 values for bis-diazeniumdiolates analyzed were between 0.8 - 5 μM (72h). The three compounds that showed the highest potency at the initial screening were selected for further studies. They have been tested using a panel of ten cell lines derived from different human cancers (leukemia cell lines, lung adenocarcinoma cell lines, colon, ovarian, and one prostate carcinoma). All three compounds tested showed similar potency in inhibiting proliferation of cancer cells, and appear to be most active towards colorectal carcinoma and leukemia cell lines. The mechanisms of the observed cellular growth inhibition by these bis-diazeniumdiolates require further study. Nitric oxide release from bis-diazeniumdiolates may cause increase in oxidative/nitrosative stress, resulting in DNA damage and apoptosis. The irreversible arylation of glutathione and other cellular nucleophiles required to initiate NO release may also contribute to the toxicity. Furthermore, attachment of glutathione to cysteine residues in proteins (glutathionylation) is a posttranslational modification that can alter protein structure and function. Glutathionylation has been shown for the JS-K analogue PABA/NO (Townsend et al, Mol. Pharmacol. 2006, 69, 501-508). We observed rapid and massive protein glutathionylation occurring within 2 minutes of treatment with 1 µM bis-diazeniumdiolates. Our data suggest that all three mechanisms - glutathionylation, arylation and nitrosative stress - are involved in the cytotoxic activity of bis-diazeniumdiolates against cancer cells.
99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA