Esterase-Sensitive Nitric Oxide Donors of the Diazeniumdiolate Family: In Vitro Antileukemic Activity

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Abstract
We have designed a novel prodrug class that is stable in neutral aqueous media but releases bioactive nitric oxide (NO) on metabolism by esterase. Diazeniumdiolates of structure R2N−N(O)N−OR‘, in which R‘ = Na, were reacted with BrCH2OAc to convert the spontaneously NO-releasing salts 1a (R2N = diethylamino) and 1b (R2N = pyrrolidino) to prodrugs 2a (AcOM-DEA/NO) and 2b (AcOM-PYRRO/NO), respectively, where R‘ = CH2OAc. In contrast to anions 1a and 1b (half-lives in pH 7.4 phosphate at 37 °C of 2 min and 3 s, respectively), 2a and 2b showed only minimal decomposition after 16 h under these conditions. Very rapid hydrolysis occurred in the presence of porcine liver esterase, however, with free anion 1a being observed as an intermediate in the esterase-induced generation of NO from 2a. The potential utility of this prodrug class is illustrated with a comparison of 1 and 2 as antiproliferative agents in NO-sensitive human leukemia cell lines HL-60 and U937. While the 72-h IC50's for 1a and 1b (which generate NO throughout the medium) in HL-60 cell cultures were >600 μM, those of 2a and 2b were 8.3 and 6.4 μM, respectively. This result is consistent with our hypothesis that 2 is selectively hydrolyzed to 1 and thence to NO intracellularly. For U937 cells, the 72-h IC50 for both 2a and 2b was 53 μM. By contrast, relatively high antiproliferative IC50's (>100 μM in U937 cells) were observed for analogues in which R‘ = CH2CH2SC(O)Me, from which acetyl and 2-mercaptoethyl groups must be successively cleaved to free the NO-releasing diazeniumdiolate function. Within 24 h at initial concentrations of 50 μM, 2a and 2b induced apoptosis in 50% and 57% of the HL-60 cells, respectively (35% and 40% of the U937 cells, respectively). The data reveal significant in vitro antileukemic activity on the part of these novel compounds. Moreover, their substantial ease-of-handling advantages over the anionic diazeniumdiolates from which they are derived suggest their use as convenient agents for probing the biological roles of NO.