HNO and NO release from a primary amine-based diazeniumdiolate as a function of pH.

The growing evidence that nitroxyl (HNO) has a rich pharmacological potential that differs from that of nitric oxide (NO) has intensified interest in HNO donors. Recently, the diazeniumdiolate (NONOate) based on isopropylamine (IPA/NO; Na[(CH(3))(2)CHNH(N(O)NO)]) was demonstrated to function under physiological conditions as an organic analogue to the commonly used HNO donor Angeli's salt (Na(2)N(2)O(3)). The decomposition mechanism of Angeli's salt is dependent on pH, with transition from an HNO to an NO donor occurring abruptly near pH 3. Here, pH is shown to also affect product formation from IPA/NO. Chemical analysis of HNO and NO production led to refinement of an earlier, quantum mechanically based prediction of the pH-dependent decomposition mechanisms of primary amine NONOates such as IPA/NO. Under basic conditions, the amine proton of IPA/NO is able to initiate decomposition to HNO by tautomerization to the nitroso nitrogen (N(2)). At lower pH, protonation activates a competing pathway to NO production. At pH 8, the donor properties of IPA/NO and Angeli's salt are demonstrated to be comparable, suggesting that at or above this pH, IPA/NO is primarily an HNO donor. Below pH 5, NO is the major product, while IPA/NO functions as a dual donor of HNO and NO at intermediate pH. This pH-dependent variability in product formation may prove useful in examination of the chemistry of NO and HNO. Furthermore, primary amine NONOates may serve as a tunable class of nitrogen oxide donor.

Thiol activation of a model O2-aryl diazeniumdiolate prodrug in phospholipid vesicle media.

Thiolysis of the model diazeniumdiolate prodrug, O2-(2,4-dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DNP-DEA/NO), by glutathione (GSH), cysteine (CYSH) and 1-heptanethiol (heptylmercaptan, HM) has been examined in anionic (DOPG), neutral (DPPC, DOPE) and cationic (DOTAP) vesicle media and in glycine buffered aqueous solutions. DOTAP vesicles accelerate the bimolecular reaction with glutathione, cysteine and 1-heptanethiol by factors of 81, 8.2 and 4630, respectively, while reaction is inhibited 5- to 10-fold in the presence of neutral and anionic vesicles. The intrinsic nucleophilicity of the thiols has been compared through the second-order rate constants, 22.9, 5.24 and 43.1M(-1)s(-1), for nucleophilic attack on 1 by GS(-), CYS(-) and M(-), respectively, obtained in buffered aqueous media. Analysis of the catalysis by DOTAP vesicles, using pseudophase ion-exchange formalism, suggests that the rate increase is due to reactant concentration in the bilayer and interfacial region coupled with enhanced dissociation of the thiol at the vesicle surface. Some contribution from enhanced nucleophilic reactivity at the vesicle interface may also contribute to the greater catalysis by HM. Inhibition of the thiolysis reaction by phospholipid liposomes is attributed to repulsion of the thiolate anions by the negatively charged acyl phosphate of the lipid head group. DOPG=1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DPPC=1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DOPE=1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOTAP=1,2-dioleoyl-3-trimethylammonium-propane.

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.

JS-K, a glutathione/glutathione S-transferase-activated nitric oxide donor of the diazeniumdiolate class with potent antineoplastic activity.

We have previously shown that nitric oxide (NO) inhibits growth and induces differentiation and apoptosis in acute myeloid leukemia cells, with the HL-60 human myeloid leukemia line being particularly sensitive to NO-mediated cytolysis. With the goal of identifying a prodrug that can target NO to the leukemia cells without inducing NO-mediated systemic hypotension, we have screened a series of O(2)-aryl diazeniumdiolates designed to be stable at physiological pH but to release NO upon reaction with glutathione. O(2)-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) proved to be the most active antiproliferative agent among those tested in HL-60 cells, with an IC(50) of 0.2-0.5 microM. After 5 days of exposure to 0.5 micro M JS-K, HL-60 cells had differentiated and acquired some of the phenotypic features of normal monocytes. One- to 2-day treatment with JS-K at concentrations of 0.5-1 microM resulted in apoptosis induction in a concentration- and caspase-dependent manner. JS-K also inhibited the growth of solid tumor cell lines but to a lesser extent than HL-60 cells. JS-K was administered i.v. to nonobese diabetic-severe combined immune deficient mice at doses of up to 4 micromol/kg without inducing significant hypotension. The growth of s.c. implanted HL-60 cells was reduced by approximately 50% when the mice received i.v. injections three times/week with 4 micromol/kg boluses of JS-K. Histological examination of tumor explants from JS-K-treated animals revealed extensive necrosis. Similar results were seen with s.c. human prostate cancer (PPC-1) xenografts. Our data indicate that JS-K is a promising lead compound for the possible development of a novel class of antineoplastic agents.

Piperazine as a Linker for Incorporating the Nitric Oxide-Releasing Diazeniumdiolate Group into Other Biomedically Relevant Functional Molecules.

Synthetic procedures have been devised to exploit the bifunctional amine piperazine (pip) as a linker capable of attaching the nitric oxide (NO)-releasing diazeniumdiolate functional group [N(O)NO]- to a diverse selection of biomedically useful molecules. One of the amino groups bears the diazeniumdiolate, which may be substituted on oxygen as necessary to control its dissociation to NO, while the other is used to provide a site suitable for covalent bonding to the molecule requiring NO donor capability. N,N'-Disubstituted piperazines of the structure R-pip-N(O)[Formula: see text]NOE were prepared either by using the nucleophilic character of the amino group or by converting it into an electrophilic moiety for reaction with nucleophilic centers in the molecules to be derivatized. Examples are reported in which E = CH3 and the R groups are bound to the N'-nitrogen: via amide linkages to the carboxyl groups of the drug ibuprofen and the amino acid derivative N-acetylmethionine; through a urea grouping to the ε-amino group of a protected lysine; via a carbamate linkage to poly(ethylene glycol); and by replacing the NH2 nitrogens of nicotinamide and adenosine. Synthesis of analogues in which E = vinyl has been facilitated by introduction of BrCH2CH2OSO2Cl as a novel, efficient bromoethylating agent. Spontaneous NO releasers in the diazeniumdiolated piperazine series include both a fluorescent anion of half-life 5.5 min in which E = Na and R = dansyl and "MOM-PIPERAZI/NO" (E = CH3OCH2, R = H), whose half-life for NO release was estimated as 17 days. The latter agent has made possible the conversion of poly(vinyl chloride) and phosphatidylethanolamine to NO-releasing derivatives. This chemistry should allow introduction of diazeniumdiolate groups into a wide variety of natural products, drugs, polymers, and other molecules whose activities could be beneficially combined with the ability to generate NO for biomedical applications.

Nucleophilic reactivity of thiolate, hydroxide and phenolate ions towards a model O-arylated diazeniumdiolate prodrug in aqueous and cationic surfactant media.

The kinetics of aromatic nucleophilic substitution of the nitric oxide generating diazeniumdiolate ion, DEA/NO, by thiols, (L-glutathione, L-cysteine, DL-homocysteine, 1-propanethiol, 2-mercaptoethanol and sodium thioglycolate) from the prodrug, DNP-DEA/NO, has been examined in aqueous solution and in solutions of cationic DOTAP vesicles. Second-order rate constants in buffered aqueous solutions (k(RS(-) ) = 3.48 - 30.9 M(-1)s(-1); 30 °C) gave a linear Brønsted plot (ß(nuc) = 0.414 ± 0.068) consistent with rate-limiting S(N)Ar nucleophilic attack by thiolate ions. Cationic DOTAP vesicles catalyze the thiolysis reactions with rate enhancements between 11 and 486-fold in Tris-HCl buffered solutions at pH 7.4. The maximum rate increase was obtained with thioglycolate ion. Thiolysis data are compared to data for nucleophilic displacement by phenolate (k(PhO(-) ) = 0.114 M(-1)s(-1)) and hydroxide (k(OH(-) ) = 1.82 × 10(-2) M(-1)s(-1), 37 °C) ions. The base hydrolysis reaction is accelerated by CTAB micelles and DODAC vesicles with vesicles being ca 3-fold more effective as catalysts. Analysis of the data using pseudophase ion-exchange formalism implies that the rate enhancement of the thiolysis and base hydrolysis reactions is due primarily to reactant concentration in the surfactant pseudophase.

Diazeniumdiolate reactivity in model membrane systems.

The effect of small unilamellar phospholipid vesicles on the acid-catalyzed dissociation of nitric oxide from diazeniumdiolate ions, R(1)R(2)N[N(O)NO](-), [1: R(1)=H(2)N(CH(2))(3)-, R(2)=H(2)N(CH(2))(3)NH(CH(2))(4)-; 2: R(1)=R(2)=H(2)N(CH(2))(3)-; 3: R(1)=n-butyl-, R(2)=n-butyl-NH2+(CH(2))(6)-; 4: R(1)=R(2)=nPr-] has been examined at pH 7.4 and 37 degrees C. NO release was catalyzed by anionic liposomes (DPPG, DOPG, DMPS, POPS and DOPA) and by mixed phosphatidylglycerol/phosphatidylcholine (DPPG/DPPC and DOPG/DPPC) covesicles, while cationic liposomes derived from 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic liposome DMPC did not significantly affect the dissociation rates of the substrates examined. Enhancement of the dissociation rate constant in DPPG liposome media (0.010M phosphate buffer, pH 7.4, 37 degrees C) at 10mM phosphoglycerol levels, ranged from 37 for 1 to 1.2 for the anionic diazeniumdiolate 4, while DOPA effected the greatest rate enhancement, achieving 49-fold rate increases with 1 under similar conditions. The observed catalysis decreases with increase in the bulk concentration of electrolytes in the reaction media. Quantitative analysis of catalytic effects has been obtained through the application of pseudo-phase kinetic models and equilibrium binding constants at different liposome interfaces are compared. The stoichiometry of nitric oxide release from 1 and 2 in DPPG/DPPC liposome media has been obtained through oxyhemoglobin assay. DPPG=1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DOPG=1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DMPS=1,2-dimyristoyl-sn-glycero-3-[phospho-l-serine], POPS=1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine], DOPA=1,2-dioleoyl-sn-glycero-3-phosphate; DPPC=1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DMPC=1,2-dimyristoyl-sn-glycero-3-phosphocholine, DOTAP=1,2-dioleoyl-3-trimethylammonium-propane.

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.

Effect of hydrophobic structure on the catalysis of nitric oxide release from zwitterionic diazeniumdiolates in surfactant and liposome media.

The effect of phospholipid liposomes and surfactant micelles on the rate of nitric oxide release from zwitterionic diazeniumdiolates, R1R2N[N(O)NO]-, with significant hydrophobic structure, has been explored. The acid-catalyzed dissociation of NO has been examined in phosphate-buffered solutions of sodium dodecylsulfate (SDS) micelles and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-[phospho-(1-glycerol)] sodium salt (DPPG) phospholipid liposomes. The reaction behavior of dibenzylamine-, monobenzylamine-, and dibutylamine-derived substrates [1]: R1 = C6H5CH2, R2 = C6H5CH2 NH2+(CH2)2, 2: R1 = C6H5CH2, R2 = NH3+(CH2)2, and 3: R1 = n-butyl, R2 = n-butyl-NH2+(CH2)6] has been compared with that of SPER/NO, 4: R1 = H2N(CH2)3, R2 = H2N(CH2) 3NH2+(CH2)4]. Catalysis of NO release is observed in both micellar and liposome media. Hydrophobic interactions contribute to micellar binding for 1-3 and appear to be the main factor facilitating catalysis by charge neutral DPPC liposomes. Binding constants for the association of 1 and 3 with SDS micelles were 3-fold larger than those previously obtained with comparable zwitterionic substrates lacking their hydrophobic structure. Anionic DPPG liposomes were much more effective in catalyzing NO release than either DPPC liposomes or SDS micelles. DPPG liposomes (at 10 mM total lipid) induced a 30-fold increase in the NO dissociation rate of SPER/NO compared to 12- and 14-fold increases in that of 1 and 3.

Chemistry of the diazeniumdiolates: Z right harpoon over left harpoon E isomerism.

Here, we explore the chemistry of the previously undocumented E form of diazeniumdiolates having the structure R(1)R(2)NN(O)=NOR(3). Reported crystallographic studies have uniformly revealed the Z configuration, and our attempts to observe a Z --> E conversion through thermal equilibration or photochemical means have, until now, consistently failed to reveal a significant amount of a second conformer. As a typical example, the NMR spectrum of trimethyl derivative Me(2)NN(O)=NOMe revealed no evidence for a second configuration. Electronic structure calculations attribute this finding to a prohibitively high interconversion barrier of approximately 40 kcal/mol. A similar result was obtained when we considered the case of R(1) = Me = R(3) and R(2) = H at the same levels of theory. However, when MeHNN(O)=NOMe was ionized by dissociating the N-H bond, the barrier was calculated to be lower by approximately 20 kcal/mol, with the E form of the anion being favored over Z. This circumstance suggested that an E isomer might be isolable if a Z anion were formed and given sufficient time to assume the E configuration, then quenched by reaction with an electrophile to trap and neutralize the E form and restore the putatively high interconversion barrier. Consistent with this prediction, basifying iPrHNN(O)=NOCH(2)CH(2)Br rapidly led to a six-membered heterocycle that was crystallographically characterized as containing the -N(O)=NO- functional group in the E configuration. The results suggest an approach for generating pairs of Z and E diazeniumdiolates for systematic comparison of the rates at which the individual isomers release bioactive NO and of other physicochemical determinants of their biomedical utility.

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.

Mechanistic insight into exclusive nitric oxide recovery from a carbon-bound diazeniumdiolate.

We report that NaON=N(O)-X-N(O)=NONa (1), where X is para-disubstituted benzene, hydrolyzes to 2 mol of nitric oxide (NO) with concurrent production of 1 mol of p-benzoquinone dioxime at physiological pH. The reaction is acid catalyzed, with a rate that slows as the substrate concentration is increased. The results demonstrate that a carbon-bound diazeniumdiolate can be quantitatively hydrolyzed to produce NO as the only gaseous nitrogen-containing product. The data also suggest that N-N bond cleavage is the rate-determining step in NO release, since C-N cleavage followed by dissociation of O=N-N=O to two NO molecules cannot be operative in this case. The finding that this oxime can absorb NO in organic media and regenerate it quantitatively at physiological pHs extends the potential pharmacological implications of the carbon-bound diazeniumdiolates.

Preparation and reactivity of O2-sulfonated diazeniumdiolates.

We report the facile preparation of O2-sulfonated diazeniumdiolates and mechanistic investigation of their reactions with representative nucleophiles. This new class of compounds extends the range of O2-substituted diazeniumdiolates available for potential applications in research and medicine.