title: Esterase-sensitive nitric oxide donors of the diazeniumdiolate family: in vitro antileukemic activity.
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 R(2)N-N(O)=N-OR', in which R' = Na, were reacted with BrCH(2)OAc to convert the spontaneously NO-releasing salts 1a (R(2)N = diethylamino) and 1b (R(2)N = pyrrolidino) to prodrugs 2a (AcOM-DEA/NO) and 2b (AcOM-PYRRO/NO), respectively, where R' = CH(2)OAc. In contrast to anions 1a and 1b (half-lives in pH 7.4 phosphate at 37 degrees 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 IC(50)'s for 1a and 1b (which generate NO throughout the medium) in HL-60 cell cultures were >600 microM, those of 2a and 2b were 8.3 and 6.4 microM, 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 IC(50) for both 2a and 2b was 53 microM. By contrast, relatively high antiproliferative IC(50)'s (>100 microM in U937 cells) were observed for analogues in which R' = CH(2)CH(2)SC(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 microM, 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.
authors.affiliation: Intramural Research Support Program, SAIC Frederick, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland 21702, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Saavedra
authors.forename: P J
authors.initials: PJ
authors.lastname: Shami
authors.forename: L Y
authors.initials: LY
authors.lastname: Wang
authors.forename: K M
authors.initials: KM
authors.lastname: Davies
authors.forename: M N
authors.initials: MN
authors.lastname: Booth
authors.forename: M L
authors.initials: ML
authors.lastname: Citro
authors.forename: L K
authors.initials: LK
authors.lastname: Keefer
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D004950:Esterases / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D006868:Hydrolysis; D007094:Imides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D007938:Leukemia / Q000473:pathology*; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D051381:Rats; D020298:U937 Cells
chemical_list: D007094:Imides; D020030:Nitric Oxide Donors; D004950:Esterases