Keith M. Davies

“NONOates” (1-substituted diazen-1-ium-1,2-diolates) as nitric oxide donors: Convenient nitric oxide dosage forms

Abstract 1-Substituted diazen-1-ium-1,2-diolates have proved useful as tools in enzymology and other pharmacological research applications in which spontaneous generation of nitric oxide according to a reasonably well-defined time course is required. This chapter summarizes relevant physicochemical data, including the NO release rates and product profiles, for a selection of these compounds. Guidelines for quality control and a systematic nomenclature scheme are also presented. It is hoped that, in summarizing this information here, our chapter will help those contemplating new applications of diazeniumdiolate technology as they seek to capitalize on what we believe are the inherent advantages of this compound type in research on the pharmacological properties of nitric oxide.

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 Angelis salt (Na(2)N(2)O(3)). The decomposition mechanism of Angelis 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 Angelis 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.

Hydrolytic Reactivity Trends among Potential Prodrugs of the O2-Glycosylated Diazeniumdiolate Family. Targeting Nitric Oxide to Macrophages for Antileishmanial Activity

Glycosylated diazeniumdiolates of structure R2NN(O)=NO−R′ (R′ = a saccharide residue) are potential prodrugs of the nitric oxide (NO)-releasing but acid-sensitive R2NN(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 R2NN(O)=NO−GlcNAc (where R2N = 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.

Conversion of a polysaccharide to nitric oxide-releasing form. dual-mechanism anticoagulant activity of diazeniumdiolated heparin

We describe heparin/diazeniumdiolate conjugates that generate nitric oxide (NO) at physiological pH. Like the heparin from which they were prepared, they inhibit thrombin-induced blood coagulation. Unlike heparin, they can also inhibit and reverse ADP-induced platelet aggregation (as expected for an NO-releasing agent), suggesting potential utility as dual-action antithrombotics.

Redox behavior of copper(II) and copper(I) complexes with tetradentate bis(pyridyl)-dithiaether and bis(pyridyl)-diaza ligands towards ruthenium ammine and bipyridyl complexes

Abstract Electron transfer reactions involving the 1,8-bis- (2-pyridyl)-3,6-dithiaoctane copper(II) complex, Cu- (pdto) 2+ , and a series of Ru(II) ammine and bipyridyl complexes have been studied kinetically in MES buffered 0.10 M LiCF 3 CO 2 . Values of the second- order rate constant, k 2 (M -1 s -1 ) are Ru(NH 3 )5 py 2+ , 3.8 × 10 4 ; Ru(NH 3 ) 5 isn 2+ , 1.4 × 10 4 ; Ru(NH 3 ) 4 - bpy 2+ , 2.0 × 10 3 ; cis -Ru(NH 3 ) 4 (isn) 2 2+ , 2.3 × 10 2 ; cis - Ru(bpy) 2 Cl 2 , 4.4 × 10 4 ; Ru(bpy) 2 C 2 O 4 , 4.5 × 10 5 ; at 25.0 °C in 50% aqueous methanol. Data for the ammine complexes, which follow a linear free energy relationship between log k 2 and E°, the Ru(III)/ Ru(II) reduction potential, suggest an outer-sphere mechanism. Inner-sphere electron transfer is indicated with Ru(bpy) 2 Cl 2 and Ru(bpy) 2 C 2 O 4 as reductants. Oxidation of coordinated oxalate occurs with Ru(bpy) 2 C 2 O 4 . The rate constant has also been determined for the oxidation of the 1,8-bis(2-pyridyl)- 3,6-dimethyl-3,6-diazaoctane copper(I) complex, Cu(pdao) + by Ru(NH 3 ) 5 py 3+ in 100% aqueous 0.10 M LiCF 3 CO 2 . A value of 1.8 × 10 4 M -1 s -1 was obtained at 25.0 °C and pH 6.1. Application of the Marcus relations has yielded estimated self-exchange electron-transfer rate constants of 0.63 and 8.1 M -1 s -1 for the Cu(pdto) 2+/+ and Cu(pdao) 2+/+ couples.

Electrolyte effects in electron transfer reactions of 1,8-bis(2-pyridyl)-3,6-dithiaoctane copper(II) with ferrocene in acetonitrile. Specific rate enhancement by tetrafluoroborate ion

Abstract The kinetics of electron transfer between 1,8-bis(2-pyridyl)-3,6-dithiaoctane copper(II), Cu(pdto) 2+ , and ferrocene, FeCp 2 , in acetonitrile have been studied by stopped-flow spectrophotometry, as a function of ferrocene concentration and the concentration of added electrolytes, n-Bu 4 NPF 6 , n-Bu 4 NBF 4 and NaCF 3 CO 2 . The reactions exhibit different rate dependencies with the three salts. The second-order rate constant, k 2 =(2.1±0.2)x10 4 M −1 S −1 , measured in CH 3 CN at 25 °C with excess ferrocene between 1.2x10 −3 and 7.5x10 −3 M is unaffected by n-Bu 4 NPF 6 added up to 0.10 M. A lowered second-order rate constant, k 2 =(1.56±0.15)x10 3 M −1 S −1 is obtained with [NaCF 3 CO 2 ] between 0.01 and 0.10 M. Inner-sphere association of CF 3 CO 2 − with the Cu(II) center is proposed to explain the rate inhibition in trifluoroacetate solutions. Values of 15 and 0.25 M −1 S −1 have been estimated for the Cu(II)/Cu(I) self-exchange electron transfer rate constant in n-Bu 4 NPF 6 and NaCF 3 CO 2 media, using the Marcus cross relations. With added n-Bu 4 NBF 4 , the measured second order rate constant, k 2 , increases with [BF 4 − ] reaching 1.75x10 5 M −1 S −1 at 0.050 M salt concentration. The rate data in BF 4 − solutions is fitted to a rate law involving ion-pair formation between BF 4 − and Cu(pdto) 2+ , with the ion-paired species showing enhanced electron transfer reactivity. Spectral changes that accompany the addition of BF 4 − to Cu(pdto) 2+ solutions suggest that the association of BF 4 − facilitates electron transfer by modifying the energy of the electron acceptor orbital in the redox change.

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.

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.

Unexpected Incorporation of Bromine at a Non-anomeric Position during the Synthesis of an O2-Glycosylated Diazeniumdiolate

We recently reported that the novel NO-releasing O2-glycosylated diazeniumdiolates of structure 1 showed promising anti-parasitic activity against Leishmania major.1 While preparing some 2-deoxyglucose analogs for lead optimization, we have observed the facile displacement of acetate by bromide from the 4-position of peracetylated 2-deoxyglucose 2, as shown in Scheme 1, providing a convenient synthesis of 4-brominated 2,4-dideoxyglucose derivatives that would otherwise be difficult to access by currently preferred, directed synthetic routes. Scheme 1 In an effort to prepare compound 4a, peracetylated 2-deoxyglucose 2 was treated with HBr in glacial acetic acid. A tar assumed to be bromide 3 was formed and immediately reacted with diazeniumdiolate salt 5 to generate a product expected to be 4a. However, this easily crystallized, sharp-melting product was characterized by elemental analysis values that were vastly different from our expectation. Further examination revealed the presence of only two methyl singlets in the proton NMR, and mass spectrometry pointed to the presence of a bromine atom in the molecular ion isotopic cluster. Detailed analysis of the 1H NMR spectral properties showed that the halide had replaced the C4 acetoxy group with retention of configuration. Further work-up afforded 7a in 38% yield. The product 7a was deacetylated with methoxide in methanol to produce 7b. An alternate bromination procedure using BiBr3 and trimethylsilyl bromide gave 4-α-bromo-2,4-dideoxyglucosyl bromide diacetate 6, presumably the same glycosylating agent produced in the HBr/HOAc reaction. The reaction with BiBr3 was rapid, efficient, and gave a higher yield of 6 than the HBr procedure. Diazeniumdiolates generate up to two moles of NO upon hydrolysis under various conditions. Replacement of the C4 acetoxy group of compound 4b by bromine had little effect (only about three-fold) on hydrolysis rates at pH values of 14, 7.4, and 3.8–4.6 (Table 1), a key predictor of anti-leishmanial activity.1 Table 1 Half-lives of Hydrolysis at 37°C for Diazeniumdiolated Glycosides of Structure Et2NN(O)=NOR In conclusion, we have discovered a novel and simple BiBr3/TMS bromide-mediated preparation of compound 6, an extremely useful intermediate in the preparation of the 2-deoxy sugars primed for further functionality at C4. In particular, compounds 7a and 7b offer an electrophilic center at C4, making it potentially useful for further reaction with nucleophiles, including peptide chains or additional saccharides.

Reaction between hexacyanoferrate(III) ion and nitric and nitrous acids. Part I. Kinetics

Hexacyanoferrate(III) ion, [Fe(CN)6]3–, reacts with 6 mol dm–3 nitric acid, in the presence of nitrous acid, to form an iron cyanonitrosyl complex according to the rate law v=k[Fe(CN)63–][HNO2]½. The reaction has been carried out in sulphuric acid, with low concentrations of nitric and nitrous acids, and k varies with h–½, an acidity function for ionisation of HNO3, and [HNO3]½. Reaction occurs by attack of an equilibrium concentrations of nitrogen dioxide on [Fe(CN)6]3– ion. The intermediate species so formed undergoes a further slow reaction, following first-order kinetics.