Esterase-sensitive nitric oxide donors of the diazeniumdiolate family: In vitro antileukemic activity

We have designed a novel prodrug class that is stable in neutral aqueous me dia but releases bioactive nitric oxide (NO) on metabolism by esterase. Dia zeniumdiolates of structure R2N-N(O)=N-OR', in which R' = Na, were reacted with BrCH2OAc to convert the spontaneously NO-releasing salts 1a (R2N = die thylamino) and 1b (R2N = pyrrolidino) to prodrugs 2a (AcOM-DEA/NO) and 2b ( AcOM-PYRRO/NO), respectively, where R' = CH2OAc. In contrast to anions la a nd Ib (half-lives in pH 7.4 phosphate at 37 degrees C of 2 min and 3 s, res pectively), 2a and 2b showed only minimal decomposition after 16 h under th ese conditions. Very rapid hydrolysis occurred in the presence of porcine l iver esterase, however, with free anion 1a being observed as an intermediat e in the esterase-induced generation of NO from 2a. The potential utility o f this prodrug class is illustrated with a comparison of 1 and 2 as antipro liferative 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 medi um) in HL-60 cell cultures were >600 mu M, those of 2a and 2b were 8.3 and 6.4 mu M, 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 mu M. By contrast, relative ly high antiproliferative IC50's (>100 mu M in U937 cells) were observed fo r analogues in which R' = CH2CH2SC(O)Me, from which acetyl and 2-mercaptoet hyl groups must be successively cleaved to free the NO-releasing diazeniumd iolate function. Within 24 h at initial concentrations of 50 mu M, 2a and 2 b induced apoptosis in 50% and 57% of the HL-60 cells, respectively (35% an d 40% of the U937 cells, respectively). The data reveal significant in vitr o antileukemic activity on the part of these novel compounds. Moreover, the ir substantial ease-of-handling advantages over the anionic diazeniumdiolat es from which they are derived suggest their use as convenient agents for p robing the biological roles of NO.