Dual mechanisms of HNO generation by a nitroxyl prodrug of the diazeniumdiolate (NONOate) class.

Here we describe a novel caged form of the highly reactive bioeffector molecule, nitroxyl (HNO). Reacting the labile nitric oxide (NO)- and HNO-generating salt of structure iPrHN-N(O)═NO(-)Na(+) (1, IPA/NO) with BrCH(2)OAc produced a stable derivative of structure iPrHN-N(O)═NO-CH(2)OAc (2, AcOM-IPA/NO), which hydrolyzed an order of magnitude more slowly than 1 at pH 7.4 and 37 °C. Hydrolysis of 2 to generate HNO proceeded by at least two mechanisms. In the presence of esterase, straightforward dissociation to acetate, formaldehyde, and 1 was the dominant path. In the absence of enzyme, free 1 was not observed as an intermediate and the ratio of NO to HNO among the products approached zero. To account for this surprising result, we propose a mechanism in which base-induced removal of the N-H proton of 2 leads to acetyl group migration from oxygen to the neighboring nitrogen, followed by cleavage of the resulting rearrangement product to isopropanediazoate ion and the known HNO precursor, CH(3)-C(O)-NO. The trappable yield of HNO from 2 was significantly enhanced over 1 at physiological pH, in part because the slower rate of hydrolysis for 2 generated a correspondingly lower steady-state concentration of HNO, thus, minimizing self-consumption and enhancing trapping by biological targets such as metmyoglobin and glutathione. Consistent with the chemical trapping efficiency data, micromolar concentrations of prodrug 2 displayed significantly more potent sarcomere shortening effects relative to 1 on ventricular myocytes isolated from wild-type mouse hearts, suggesting that 2 may be a promising lead compound for the development of heart failure therapies.

Glycosylated PROLI/NO derivatives as nitric oxide prodrugs.

GlcNAc-PROLI/NO prodrugs that are activated by N-acetylglucosaminidase to release nitric oxide (NO) are described. A classical acid-amine coupling is used to bifunctionalize these PROLI/NO prodrugs, which on activation generate up to 4 mol of NO, a peptide residue, and an N-acetylglucosamine residue. Many of the prodrugs synthesized are efficient sources of intracellular NO.

Synthesis and evaluation of piperazine and homopiperazine analogues of JS-K, an anti-cancer lead compound.

Here we report a number of novel JS-K structural analogues with sub-micromolar anti-proliferative activities against human leukemia cell lines HL-60 and U937; JS-K is the anti-cancer lead compound O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate. The ability of these compounds to generate intracellular nitric oxide correlated well with their observed anti-proliferative effects: analogues that had potent inhibitory activity against leukemia cells formed elevated levels of intracellular nitric oxide.

Aryl bis(diazeniumdiolates): potent inducers of S-glutathionylation of cellular proteins and their in vitro antiproliferative activities.

A number of bis(diazeniumdiolates) that we designed to release up to 4 mol of nitric oxide (NO) and that are structural analogues of the NO prodrug and anticancer lead compound O(2)-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino)diazen-1-ium-1,2- diolate (PABA/NO) were synthesized and studied. A majority of these compounds yielded higher levels of NO, were better inhibitors of proliferation of a number of cancer cell lines, and more rapidly induced substantially increased levels of S-glutathionylation of cellular proteins in comparison with PABA/NO. In most cases, the antiproliferative activity and extents of S-glutathionylation correlated well with levels of intracellular NO release. We report bis(diazeniumdiolates) to be a class of S-glutathionylating agents with potent antiproliferative and S-glutathionylating activity.

Synthesis, mechanistic studies, and anti-proliferative activity of glutathione/glutathione S-transferase-activated nitric oxide prodrugs.

Nitric oxide (NO) prodrugs such as O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) are a growing class of promising NO-based therapeutics. Nitric oxide release from the anti-cancer lead compound, JS-K, is proposed to occur through a nucleophilic aromatic substitution by glutathione (GSH) catalyzed by glutathione S-transferase (GST) to form a diazeniumdiolate anion that spontaneously releases NO. In this study, a number of structural analogues of JS-K were synthesized and their chemical and biological properties were compared with those of JS-K. The homopiperazine analogue of JS-K showed anti-cancer activity that is comparable with that of JS-K but with a diminished reactivity towards both GSH and GSH/GST; both the aforementioned compounds displayed no cytotoxic activity towards normal renal epithelial cell line at concentrations where they significantly diminished the proliferation of a panel of renal cancer cell lines. These properties may prove advantageous in the further development of this class of nitric oxide prodrugs as cancer therapeutic agents.

Cell-permeable esters of diazeniumdiolate-based nitric oxide prodrugs.

Although O(2)-(2,4-dinitrophenyl) derivatives of diazeniumdiolate-based nitric oxide (NO) prodrugs bearing a free carboxylic acid group were activated by glutathione to release NO, these compounds were poor sources of intracellular NO and showed diminished antiproliferative activity against human leukemia HL-60 cells. The carboxylic acid esters of these prodrugs, however, were found to be superior sources of intracellular NO and potent inhibitors of HL-60 cell proliferation.

Hydrolytic reactivity trends among potential prodrugs of the O2-glycosylated diazeniumdiolate family. Targeting nitric oxide to macrophages for antileishmanial activity.

Glycosylated diazeniumdiolates of structure R 2NN(O)NO-R' (R' = a saccharide residue) are potential prodrugs of the nitric oxide (NO)-releasing but acid-sensitive R 2NN(O)NO (-) ion. Moreover, cleaving the acid-stable glycosides under alkaline conditions provides a convenient protecting group strategy for diazeniumdiolate ions. Here, we report comparative hydrolysis rate data for five representative glycosylated diazeniumdiolates at pH 14, 7.4, and 3.8-4.6 as background for further developing both the protecting group application and the ability to target NO pharmacologically to macrophages harboring intracellular pathogens. Confirming the potential in the latter application, adding R 2NN(O)NO-GlcNAc (where R 2N = diethylamino or pyrrolidin-l-yl and GlcNAc = N-acetylglucosamin-l-yl) to cultures of infected mouse macrophages that were deficient in inducible NO synthase caused rapid death of the intracellular protozoan parasite Leishmania major with no host cell toxicity.

Second-generation aspirin and indomethacin prodrugs possessing an O(2)-(acetoxymethyl)-1-(2-carboxypyrrolidin-1-yl)diazenium-1,2-diolate nitric oxide donor moiety: design, synthesis, biological evaluation, and nitric oxide release studies.

The carboxylic acid group of the anti-inflammatory (AI) drugs aspirin and indomethacin was covalently linked to the 1-(2-carboxypyrrolidin-1-yl)diazen-1-ium-1,2-diolate ion via a one-carbon methylene spacer to obtain two new hybrid prodrugs. The aspirin prodrug ( 23) was a 2.2-fold more potent AI agent than aspirin, whereas the indomethacin prodrug ( 26) was about 1.6-fold less potent than indomethacin. Prodrugs 23 and 26 slowly released nitric oxide (NO) upon dissolution in phosphate buffer at pH 7.4 (1.1 mol of NO/mol of compound after 43 h), but the rate and the extent of NO release were higher (1.9 mol of NO/mol of compound in 3 min or less) when the compounds were incubated in the presence of porcine liver esterase. In vivo ulcer index (UI) studies showed that the aspirin prodrug 23 (UI = 0.7) and indomethacin prodrug 26 (UI = 0) were substantially less ulcerogenic than the parent drugs aspirin (UI = 51) and indomethacin (UI = 64).

Synthesis, nitric oxide release, and anti-leukemic activity of glutathione-activated nitric oxide prodrugs: Structural analogues of PABA/NO, an anti-cancer lead compound.

Diazeniumdiolate anions and their prodrug forms are reliable sources of nitric oxide (NO) that have generated interest as promising therapeutic agents. A number of structural analogues of O(2)-(2,4-dinitro-5-(4-(N-methylamino)benzoyloxy)phenyl) 1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), an anti-cancer lead compound that is designed to release NO upon activation by glutathione, were prepared. The nitric oxide release patterns of these O(2)-(2,4-dinitrophenyl) diazeniumdiolates in the presence of glutathione were tested and it was found that in the absence of competing pathways, these compounds release nearly quantitative amounts of NO. The ability of PABA/NO and its structural analogues to inhibit human leukemia cell proliferation was determined and it was found that compounds releasing elevated amounts of NO displayed superior cytotoxic effects.

Nitric oxide prodrugs: diazeniumdiolate anions of hindered secondary amines.

Nitric oxide prodrugs derived from hindered secondary amines were prepared. The decomposition patterns of these prodrugs indicate that alpha-methyl groups around the nitrogen bearing the diazeniumdiolate group prolong their half-life in aqueous buffer.

O2-acetoxymethyl-protected diazeniumdiolate-based NSAIDs (NONO-NSAIDs): synthesis, nitric oxide release, and biological evaluation studies.

A novel group of O2-acetoxymethyl-protected diazeniumdiolate-based non-steroidal anti-inflammatory prodrugs (NONO-NSAIDs) were synthesized by esterifying the carboxylate group of aspirin, ibuprofen, or indomethacin with O2-acetoxymethyl 1-[N-(2-hydroxyethyl)-N-methylamino]diazeniumdiolate. The resulting nitric oxide (*NO)-releasing prodrugs (7-9) did not exhibit in vitro cyclooxygenase (COX) inhibitory activity against the COX-1 and COX-2 isozymes (IC50s>100 microM). In contrast, prodrugs 7 and 8 significantly decreased carrageenan-induced rat paw edema showing enhanced in vivo anti-inflammatory activities (ID50's=552 and 174 micromol/kg, respectively) relative to those of the parent NSAIDs aspirin (ID50=714 micromol/kg) and ibuprofen (ID50=326 micromol/kg). The rate of porcine liver esterase-mediated *NO release from prodrugs 7-9 (2 mol of *NO/mol of test compound in 0.6-6.5 min) was substantially higher compared to that observed without enzymatic catalysis (about 1 mol of *NO/mol of test compound in 40-48 h). These incubation studies suggest that both *NO and the parent NSAID would be released upon in vivo activation (hydrolysis) by esterases. Data acquired in an in vivo ulcer index (UI) assay showed that NONO-aspirin (UI=0.8), NONO-indomethacin (UI=1.3), and particularly NONO-ibuprofen (UI=0) were significantly less ulcerogenic compared to the parent drugs aspirin (UI=57), ibuprofen (UI=46) or indomethacin (UI=34) at equimolar doses. The release of aspirin and *NO from the NONO-aspirin (7) prodrug constitutes a potentially beneficial property for the prophylactic prevention of thrombus formation and adverse cardiovascular events such as stroke and myocardial infarction.

Nitric oxide releasing polyurethanes with covalently linked diazeniumdiolated secondary amines.

Two novel strategies for synthesizing stable polyurethanes (PUs) capable of generating bioactive nitric oxide (NO) are described. The methods rely on covalently attaching diazeniumdiolate (N(2)O(2)(-)) groups onto secondary amine nitrogens at various positions within the polymer chain such that, when in contact with water or physiological fluids, only the two molecules of NO available from each diazeniumdiolate moiety are released into the surrounding medium, with potential byproducts remaining covalently bound to the matrix. Extensive analysis of the NO(x)() products released from the polymers was employed to develop appropriate strategies to better stabilize the diazeniumdiolate-based polymer structures. In one approach, diazeniumdiolate groups are attached to secondary amino nitrogens of alkane diamines inserted within the diol chain extender of a PU material. Oxidative loss of NO was minimized by blending the polymer with a biocompatible, relatively nonnucleophilic salt before exposing solutions of the polymer to NO during the diazeniumdiolation step. Fluxes of molecular NO from such materials during immersion in physiological buffer reached levels as high as 19 pmol x cm(-2) x s(-1) with a total recovery of 21 nmol of NO/mg of PU. A second general synthetic strategy involved omega-haloalkylating the urethane nitrogens and then displacing the halide from the resulting polymer with a nucleophilic polyamine to form a PU with pendent amino groups suitable for diazeniumdiolation. Commercially available Pellethane 2363-80AE that was bromobutylated and then reacted with diethylenetriamine and further exposed to gaseous NO proved stable in solid form for several months, but released NO with a total recovery of 17 nmol/mg upon immersion in physiological buffer. This material showed an initial NO flux of 14 pmol x cm(-2) x s(-1) when immersed in pH 7.4 buffer at 37 degrees C, with gradually decreasing but still observable fluxes for up to 6 days.

Metabolism of a liver-selective nitric oxide-releasing agent, V-PYRRO/NO, by human microsomal cytochromes P450.

Endogenously generated nitric oxide (NO) mediates a host of important physiological functions, playing roles in the vascular, immunological, and neurological systems. As a result, exogenous agents that release NO have become important therapeutic interventions and research tools. O(2)-Vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) is a prodrug designed with the hypothesis that it might release nitric oxide via epoxidation of the vinyl group by cytochrome P450, followed by enzymatic and/or spontaneous epoxide hydration to release the ultimate NO-donating moiety, 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (PYRRO/NO) ion. In this study, we investigated this hypothetical activation mechanism quantitatively for V-PYRRO/NO using cDNA-expressed human cytochrome P450 (CYP)2E1. Incubation with CYP2E1 and an NADPH-regenerating system resulted in a time-dependent decomposition of V-PYRRO/NO, with a turnover rate of 2.0 nmol/min/pmol CYP2E1. Nitrate and nitrite were detected in high yield as metabolites of NO. The predicted organic metabolites pyrrolidine and glycolaldehyde were also detected in near-quantitative yields. The enzymatic decomposition of V-PYRRO/NO was also catalyzed, albeit at lower rates, by CYP2A6 and CYP2B6. We conclude that the initial step in the metabolism of V-PYRRO/NO to NO in the liver is catalyzed efficiently but not exclusively by the alcohol-inducible form of cytochrome P450 (CYP2E1). The results confirm the proposed activation mechanism involving enzymatic oxidation of the vinyl group in V-PYRRO/NO followed by epoxide hydration and hydrolytic decomposition of the resulting PYRRO/NO ion to generate nitric oxide.

PABA/NO as an anticancer lead: analogue synthesis, structure revision, solution chemistry, reactivity toward glutathione, and in vitro activity.

PABA/NO is a diazeniumdiolate of structure Me(2)NN(O)=NOAr (where Ar is a 5-substituted-2,4-dinitrophenyl ring whose 5-substituent is N-methyl-p-aminobenzoic acid). It has shown activity against human ovarian cancer xenografts in mice rivaling that of cisplatin, but it is poorly soluble and relatively unstable in water. Here we report structure-based optimization efforts resulting in three analogues with improved solubility and stability in aqueous solution. We sought to explain PABA/NO's physicochemical uniqueness among these four compounds, whose aminobenzoic acid precursors differ structurally only in the presence or absence of the N-methyl group and/or the position of the carboxyl moiety (meta or para). Studies revealed that PABA/NO's N-methyl-p-aminobenzoic acid substituent is bound to the dinitrobenzene ring via its carboxyl oxygen while the other three are linked through the aniline nitrogen. This constitutes a revision of the previously published PABA/NO structure. All four analogues reacted with GSH to produce bioactive nitric oxide (NO), but PABA/NO was the most reactive. Consistent with PABA/NO's potent suppression of A2780 human ovarian cancer xenograft growth in mice, it was the most potent of the four in the OVCAR-3 cell line.

Injectable formulation of disodium 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (PROLI/NO), an ultrafast nitric oxide donor prodrug.

PROLI/NO is an agent of structure XN(O)==NONa (X = L-prolyl) whose 2-s half-life for nitric oxide (NO) release at physiological pH makes it an excellent prodrug for localizing NO's therapeutic effects at the site of application, but a difficult one to formulate and certify as pure. Despite its extraordinary thermal and hydrolytic instability, however, PROLI/NO could be formulated as an injectable drug by dissolving it in cold 0.1 M sodium hydroxide containing 5% D-mannitol, then quickly ultrafiltering and lyophilizing it in evacuated septum vials. No evidence for decomposition was seen in the contents of these evacuated vials when stored at -20 degrees C over a 140-day observation period, as judged by quantifying NO release in simulated infusate solutions (10 mM carbonate/bicarbonate, pH 10.5). The only hydrolysis products detected were NO, nitrite ion, proline, and N-nitrosoproline, all products of normal human physiological processes.

Diazeniumdiolate ions as leaving groups in anomeric displacement reactions: a protection-deprotection strategy for ionic diazeniumdiolates.

Diazeniumdiolate ions [R2N-N(O)=N-O-] are of growing interest pharmacologically for their ability to generate up to two molar equivalents of bioactive nitric oxide (NO) spontaneously on protonating the amino nitrogen. Accordingly, their stability increases as the pH is raised. Here we show that the corresponding beta-glucosides [R2N-N(O)=N-O-Glc] decreased in stability with pH; when R2N was diethylamino, the rate equation was kobs = ko + kOH- [OH-], where ko = 7.8 x 10-7 s-1 and kOH- = 5.3 x 10-3 M-1 s-1. The primary products were 1,6-anhydroglucose and the regenerated R2N-N(O)=N-O- ion. The results were qualitatively similar to those of beta-glucosyl fluoride and p-nitrophenoxide, whose hydrolyses have been rationalized as proceeding via a glycal oxide intermediate. This chemistry offers a convenient strategy for protecting heat- and acid-sensitive diazeniumdiolate ions during manipulations that would otherwise destroy them. As an example, a poly(urethane) film that generated NO in physiological buffer at a surface flux comparable to that of the mammalian vascular endothelium was prepared by glucosylating the ionic diazeniumdiolate group attached to the diol monomer before reacting it with the bis-isocyanate, then removing the saccharide with base when the protecting group was no longer needed.

Carbon-bound diazeniumdiolates from the reaction of nitric oxide with amidines.

[reaction: see text] The enediamine tautomer of a variety of substituted amidine free bases reacts with nitric oxide (NO) to produce compounds containing a carbon-bound diazeniumdiolate [R1R2R3C-N(O)=NO-] functional group (previously called "nitrosohydroxylamines"). The new reaction has been shown to be quite general, although the nature of the products does vary. Amidines containing more than one replaceable hydrogen produce polydiazeniumdiolates as intermolecular salts, while those in which only one diazeniumdiolation can occur provide zwitterionic salts. These diazeniumdiolated amidines are shown to be useful NO donor compounds which undergo very slow spontaneous dissociation on dissolution in pH 7.4 phosphate buffer to produce mixtures of NO and nitrous oxide containing mostly NO. The most advantageous manifestation of the new discovery is the preparation of the monodiazeniumdiolated amidine zwitterions. Reaction of the medically relevant alpha-adrenergic agonists tetrahydrozoline and idazoxan produced monodiazeniumdiolated amidine zwitterions from which NO release was observed for up to 28 days and showed little sign of ending. The reaction should be applicable to a variety of pharmaceutical agents, including NO synthase inhibitors, antitumor agents, and antibacterials.

Chemistry of the diazeniumdiolates. O- versus N-alkylation of the RNH[N(O)NO](-) ion.

Monomethylation of the potentially ambident RNH[N(O)NO](-) ion (R = isopropyl or cyclohexyl) has been shown to occur at the terminal oxygen to yield the novel diazeniumdiolate structural unit, RNHN(O)=NOMe. The NH bond of the product proved acidic, with a pK(a) of 12.3 in aqueous solution. The ultraviolet spectrum showed a large bathochromic shift on ionization (lambda(max) 244 --> 284 nm, epsilon(max) 6.9 --> 9.8 mM(-1) cm(-1)). Deprotonation led to a pH-dependent line broadening in the (1)H NMR spectrum of iPrNHN(O)=NOMe, suggesting a complex fluxionality possibly involving isomerizations around the N-N bonds. Consistent with this interpretation, evidence for extensive delocalization and associated changes in bond order on ionizing RNHN(O)=NOR' were found in density functional theory calculations using Gaussian 03 with B3LYP/6-311++G basis sets. With MeNHN(O)=NOMe as a model, all N-N and N-O bonds lengthened by 0.04-0.07 A as a result of ionization except for the MeN-N linkage, which shortened by 7%. These anions can be N-alkylated to generate R(1)R(2)NN(O)=NOR(3) derivatives that would otherwise be difficult to access synthetically. Additionally, some RNHN(O)=NOR' species may display unique and beneficial pharmacological properties. As one example, an agent with R = isopropyl and R' = beta-D-glucosyl was prepared and shown to generate nitric oxide in the presence of glucosidase at pH 5.

Tumor cell responses to a novel glutathione S-transferase-activated nitric oxide-releasing prodrug.

We have used structure-based design techniques to introduce the drug O(2)-[2,4-dinitro-5-(N-methyl-N-4-carboxyphenylamino) phenyl] 1-N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO), which is efficiently metabolized to potentially cytolytic nitric oxide by the pi isoform of glutathione S-transferase, an enzyme expressed at high levels in many tumors. We have used mouse embryo fibroblasts (MEFs) null for GSTpi (GSTpi(-/-)) to show that the absence of GSTpi results in a decreased sensitivity to PABA/NO. Cytotoxicity of PABA/NO was also examined in a mouse skin fibroblast (NIH3T3) cell line that was stably transfected with GSTpi and/or various combinations of gamma-glutamyl cysteine synthetase and the ATP-binding cassette transporter MRP1. Overexpression of MRP1 conferred the most significant degree of resistance, and in vitro transport studies confirmed that a GSTpi-activated metabolite of PABA/NO was effluxed by MRP1 in a GSH-dependent manner. Additional studies showed that in the absence of MRP1, PABA/NO activated the extracellular-regulated and stress-activated protein kinases ERK, c-Jun NH(2)-terminal kinase (JNK), and p38. Selective inhibition studies showed that the activation of JNK and p38 were critical to the cytotoxic effects of PABA/NO. Finally, PABA/NO produced antitumor effects in a human ovarian cancer model grown in SCID mice.