Studies of the potential genotoxic effects of furoxans: the case of CAS 1609 and of the water-soluble analogue of CHF 2363.

CAS 1609 (compound 1) and CHF 2363 (compound 2) are two furoxan derivatives able to release nitric oxide (NO) under physiological conditions, and display typical NO-dependent vasodilator activity. The potential genotoxic effects of compound 1 and of the water-soluble analogue of CHF 2363 (compound 2a) were investigated. The results show that the two compounds induce genotoxic effects only at concentrations that significantly reduce cell viability. However, in the case of compound 1 this range of concentrations is one order of magnitude higher than the one leading to the beneficial effects, while in the case of compound 2a these ranges partially overlap. In both cases the release of NO plays a key role in the induction of the cytotoxic and genotoxic effects, since the non-NO-donating furazan analogues display a different toxicological profile, and since the effects were reduced in the presence of oxyhaemoglobin, a well-known NO-scavenger.

Focus on recent approaches for the development of new NO-donors.

Recent research developments in the field of NO-donor compounds have concerned conjugation of NO-donor moieties with antioxidant groups, NO-donor targeting, design of NO-donor hybrid drugs and of NO-delivery systems. These new approaches are illustrated and discussed through selected examples.

The furoxan system as a useful tool for balancing "hybrids" with mixed alpha 1-antagonist and NO-like vasodilator activities.

The design of new vasodilator derivatives in which two different pharmacophoric groups are present in a single molecule has been pursued by substitution of NO-prodrug furoxan moieties for the furanylcarbonyl function in Prazosin, a well-known alpha 1-receptor antagonist. The aim was to obtain new antihypertensive agents in which two vasodilation mechanisms, alpha 1-antagonist and NO-mediated, can operate in an appropriate balance. The alpha 1-antagonist activity was assessed on rat aortic strips in the presence and in the absence of oxyhemoglobin (HbO2), a well-known scavenger of nitric oxide. The resulting hybrids displayed different pharmacological behaviors. When the 4-furoxanylcarbonyl system, bearing an ester or an amide function at the 3-position, was present (derivatives 7a,b), hybrids with predominant alpha 1-antagonist activity were obtained. By contrast, in the derivative 7c, in which the nitrile function is linked to the 3-position of the furoxan ring, the NO-mediated vasodilating properties are predominant. Finally, the (furoxanylsulfonyl)piperidine derivatives 13a,b showed NO vasodilation and alpha 1-antagonist activities in an appropriate balance. For the furoxan derivatives, the NO-dependent vasodilating ability, assessed on the K(+)-depolarized aortic strip, and the NO release features under the action of thiol cofactors are also discussed.

Furoxans as nitric oxide donors. 4-Phenyl-3-furoxancarbonitrile: thiol-mediated nitric oxide release and biological evaluation.

4-Phenyl-3-furoxancarbonitrile (2) affords nitric oxide under the action of thiol cofactors. Two principal products were isolated in the reaction with thiophenol: the phenylcyanoglyoxime (6) and 5-amino-3-phenyl-4-(phenylthio)isoxazole (7). Mechanisms which could account for the formation of these two products are discussed. Compound 2 is an efficient activator of the rat lung soluble guanylate cyclase, displays high vasodilatory activity on strips of rat thoracic aorta precontracted with noradrenaline, and is a potent inhibitor of platelet aggregation.

4-Methyl-3-(arylsulfonyl)furoxans: a new class of potent inhibitors of platelet aggregation.

A series of 4-methyl-3-(arylthio)furoxans were synthesized by oxidation of 1-(arylthio)-2-methylglyoxymes with dinitrogen tetroxide. Reduction with trimethyl phosphite of the furoxan derivatives afforded the corresponding furazans, while oxidation with an equimolar amount of 30% hydrogen peroxide in acetic acid or with an excess of 81% hydrogen peroxide in trifluoroacetic acid afforded the corresponding arylsulfinyl and arylsulfonyl analogues, respectively. All the furoxan and furazan derivatives showed activity as inhibitors of platelet aggregation. 4-Methyl-3-(arylsulfonyl)furoxans were the most potent derivatives of the series. 4-Methyl-3-(phenylsulfonyl)furoxan (10a), one of the most active derivatives, inhibits the AA-induced increase of cytosolic free Ca2+ and production of malondialdehyde. A primary action of the compound on cyclooxygenase is excluded, as a stable epoxymethano analogue of prostaglandin H2 does not reverse the inhibitory effect of 10a. This compound produces a significant increase in cGMP which is likely to cause inhibition at an early stage of the platelet activation pathway.

Water soluble furoxan derivatives as NO prodrugs.

The synthesis, characterization, NO donor properties, and in vitro vasodilating activity of a series of water soluble furoxans (5-14a,b) are described. All of the compounds released NO when treated with a large excess of cysteine under physiological conditions (pH 7.4; 37 degrees C). The amount of NO produced after 1 h of incubation was evaluated by detecting nitrites, via the Griess reaction. Derivatives 5b, 7b, and 14b were able to release nitric oxide also in the absence of the thiol cofactor. The initial rates of NO release were determined at different concentrations, using a spectrophotometric technique based on the NO-induced oxidation of oxyhemoglobin (HbO2) to methemoglobin (MetHb). The initial rates of NO release were linearly dependent on the concentrations of the single compounds. The vasodilating potency (EC50) of all the derivatives was assessed on rat aortic strips precontracted with noradrenaline. Correlation between potency and initial NO release rate is discussed.

Synthesis and voltage-clamp studies of methyl 1,4-dihydro-2, 6-dimethyl-5-nitro-4-(benzofurazanyl)pyridine-3-carboxylate racemates and enantiomers and of their benzofuroxanyl analogues.

Racemic methyl 1,4-dihydro-2, 6-dimethyl-5-nitro-4-(benzofurazanyl)pyridine-3-carboxylates (+/-)-10 and (+/-)-11 and their benzofuroxanyl analogues (+/-)-12 and (+/-)-13 were prepared using a modified Hantzsch reaction that involved the condensation of nitroacetone with methyl 3-aminocrotonate and the appropriate aldehydes. The racemic mixtures were resolved into the corresponding enantiomers. Whole-cell voltage-clamp studies on L-type Ca2+ channels expressed in a rat insulinoma cell line (RINm5F) showed that all the dextrorotatory antipodes were effective agonists of L-type Ca2+ currents, while the levorotatory ones were weak Ca2+ entry blockers. The (+)-enantiomer of benzofurazan-5'-yl derivative 11 demonstrated unusual activity in that, in addition to producing a potentiation of L-type currents, it interfered with the voltage-dependent gating of L-type channels by producing a net delay of their activation at low voltages. This compound represents an interesting tool to probe L-type Ca2+ channel structure and function.

A new class of ibuprofen derivatives with reduced gastrotoxicity.

A new series of nonsteroidal antiinflammatory drugs (NSAIDs) obtained by linking ibuprofen to selected furoxan moieties and to related furazans were synthesized and tested for their antiinflammatory, antiaggregatory, and ulcerogenic properties. All the derivatives are endowed with antiinflammatory activity comparable to that of ibuprofen, but, unlike this drug, they display reduced acute gastrotoxicity. The masking of the ibuprofen-free carboxylic group seems to be principally at the basis of this reduced topical irritant action. The two furoxan derivatives 8 and 9 also trigger potent antiaggregatory effects, principally as a consequence of their NO-donor ability.

A new class of furoxan derivatives as NO donors: mechanism of action and biological activity.

1. The mechanism of action and biological activity of a series of R-substituted and di-R-substituted phenylfuroxans is reported. 2. Maximal potency as vasodilators on rabbit aortic rings, precontracted with noradrenaline (1 microM), was shown by phenyl-cyano isomers and by the 3,4-dicyanofuroxan, characterized by a potency ratio 3-10 fold higher than glyceryl trinitrate (GTN). This effect was reduced upon coincubation with methylene blue or oxyhaemoglobin (10 microM). 3. The furoxan derivatives showing maximal potency as vasodilators were also able to inhibit collagen-induced platelet aggregation, with IC50 values in the sub-micromolar range. 4. The furoxan derivatives were able to stimulate partially purified, rat lung soluble guanylate cyclase; among the most active compounds, the 3-R-substituted isomers displayed a higher level of stimulatory effect than the 4-R analogues. 5. Solutions (0.1 mM) of all the tested furoxans, prepared using 50 mM phosphate buffer, pH 7.4, (diluting 10 mM DMSO stock solutions) did not release nitric oxide (NO) spontaneously; however in presence of 5 mM L-cysteine, a significant NO-releasing capacity was observed, which correlated significantly with their stimulation of the guanylate cyclase activity.

N-Substituted analogues of S-nitroso-N-acetyl-D,L-penicillamine: chemical stability and prolonged nitric oxide mediated vasodilatation in isolated rat femoral arteries.

Previous studies show that linking acetylated glucosamine to S-nitroso-N-acetyl-D,L-penicillamine (SNAP) stabilizes the molecule and causes it to elicit unusually prolonged vasodilator effects in endothelium-denuded, isolated rat femoral arteries. Here we studied the propanoyl (SNPP; 3 carbon side-chain), valeryl (SNVP; 5C) and heptanoyl (SNHP; 7C) N-substituted analogues of SNAP (2C), to further investigate other molecular characteristics that might influence chemical stability and duration of vascular action of S-nitrosothiols. Spectrophotometric analysis revealed that SNVP was the most stable analogue in solution. Decomposition of all four compounds was accelerated by Cu(II) and cysteine, and neocuproine, a specific Cu(I) chelator, slowed decomposition of SNHP. Generation of NO from the compounds was confirmed by electrochemical detection at 37 degrees C. Bolus injections of SNAP (10 microl; 10(-8)-10(-3) M) into the perfusate of precontracted, isolated rat femoral arteries taken from adult male Wistar rats (400-500 g), caused concentration-dependent, transient vasodilatations irrespective of endothelial integrity. Equivalent vasodilatations induced by SNVP and SNHP were transient in endothelium-intact vessels but failed to recover to pre-injection pressures at moderate and high concentrations (10(-6)-10(-3) M) in those denuded of endothelium. This sustained effect (> 1 h) was most prevalent with SNHP and was largely reversed by the NO scavenger, haemoglobin. We suggest that increased lipophilicity of SNAP analogues with longer sidechains facilitates their retention by endothelium-denuded vessels; subsequent slow decomposition within the tissue generates sufficient NO to cause prolonged vasodilatation. This is a potentially useful characteristic for targeting NO delivery to areas of endothelial damage.

Characterization of a new compound, S35b, as a guanylate cyclase activator in human platelets.

The effects of S35b (4-methyl-3-phenyl sulfonylfuroxan), a new phenyl sulfonylfuroxan compound, were investigated on human platelets activated by different agonists. Platelet aggregation evoked by arachidonic acid (AA), collagen, ADP and thrombin was inhibited by the drug in a dose-dependent manner. S35b inhibited the AA-induced increase of cytosolic free Ca2+ ([Ca2+]i) and production of malondialdehyde. A primary action of the compound on cyclooxygenase is unlikely since: (1) U-46619 (15s-hydroxy-11,9-[epoxymethano]-prosta-5Z,13E-dienoic acid, a stable epoxymethano analog of prostaglandin H2) could not reverse the inhibitory effect of S35b on AA-induced aggregation and [Ca2+]i increase; (2) U-46619-induced aggregation and [Ca2+]i rise were inhibited by S35b; and (3) at high collagen concentrations platelet aggregation (which is unresponsive to aspirin under such conditions) was blocked by S35b as well. Thus the drug action is likely to be exerted at an early step of the platelet activation pathway. The elevation in the platelet cGMP level evoked by S35b in a time- and concentration-dependent manner can account for the inhibitory effect: increased cGMP levels could interfere, for instance, with G protein-phospholipase C coupling and subsequent phosphoinositide hydrolysis.

Cytotoxic activity of aryl and heteroaroxylazoxy derivatives.

Synthesis of aryl and heteroarylazocyanides, azoxyesters and azoxysulphones, was carried out by the action of appropriate reagents on the corresponding nitroso derivatives and arylazoxyamides were obtained by hydrolysis of the corresponding azoxycyanides. Cytotoxic activity was assessed utilizing cultured P388 leukemia cells. Most of the compounds tested showed a marked cytotoxicity at concentrations below 1 micrograms/ml.

Cytotoxic activity of a series of heteroaryl-ONN-azoxy-sulphones and aryl sulphonylhydrazones.

Synthesis of heteroaryl-ONN-azoxysulphones and pyrazolyl-ONN-azoxycyanides was carried out by the action of the appropriate reagents on the corresponding nitroso derivatives. Pyrazolyl-ONN-azoxyamides were obtained by hydrolysis of the corresponding cyanides. Synthesis of the arylsulphonylhydrazones was carried out by reacting R-substituted phenyl-sulphonylhydrazines on the isomers of methylfuroxancarbaldehyde. Cytotoxic activity was assessed on HeLa cells. Some of the compounds tested inhibit the colony-forming ability of the tumor cells at low concentrations.

Effect of the leaving group on the electrodic reduction mechanism of anti-Helicobacter pylori metronidazole derivatives, in aprotic and protic media.

Because redox properties are central to bioreductive drug activity and selectivity, six 2-methyl-5-nitroimidazole, substituted at the N1-ethyl side chain with I, Br, Cl, OAc, OMs and NH(3)(+) were synthesized and submitted to cyclic voltammetry and electrolyses, in order to define their electrodic reduction mechanism, in aprotic [dimethylsulphoxide (DMSO)+0.1 mol l(-1) tetrabuthylammonium perchlorate (TBAP)] and phosphate-buffered media, on glassy carbon electrode, in comparison with metronidazole. Three of these compounds, namely, the iodo, bromo and ammonium salt derivatives showed significant anti-Helicobacter pylori (strain resistant to metronidazole) activity. All the cyclic voltammograms (CV), in aprotic medium, are similar to the one for metronidazole, except for -I, -Br and -NH(3)(+) derivatives. The CV of the N1-ethylhalide (-I, -Br) 5-nitroimidazole showed more intense and irreversible first waves, even at faster sweep rates (nu<2 V s(-1)). The absence of the first wave anodic counterpart, along with analysis of the dependence of E(p), I(p) and other parameters with nu, and results from electrolysis (consumption of two electrons) showed the process to be an ECE system, with halide release, after uptake of two electrons. This behaviour represents a case of dissociative electron transfer (ET). For the ammonium salt, self-protonation mechanism was evident. The facility of reduction represented by the first wave potential and concerning the substituents is NH(3)(+)>Br>I>Cl>OMs>OH>OAc. In aqueous phosphate-buffered medium, the electrochemical behaviour of all the compounds is similar to the one of metronidazole, represented by a unique and irreversible 4e(-)/4H(+) wave. The order of reduction ease is NH(3)(+)>Br approximately OMs>I>OH>OAc. Aprotic medium allows a better discrimination between the substituents. Concerning biological activity, despite the impossibility of establishing a correlation, it has been observed that the more electrophilic compounds showed better anti-H. pylori activity.

Pyrazole sulfanilamides: nitroderivatives of 1-phenyl-3-sulfanilamidopyrazole. Note XVI.

Synthesis and structural characterization of 3 sulfanilamido-1-phenylpyrazoles bearing on 1-phenyl group nitro substituent o-, m-, p-positioned are reported. All derivatives are analysed through 1H and 13C NMR spectroscopy. The MIC values obtained against Escherichia coli are briefly discussed in terms of structure-activity relationship.

Nitroanilines are the reduction products of benzofuroxan system by oxyhemoglobin (HbO2 2+).

Benzofuroxans are interesting compounds which display several biochemical and pharmacological properties. Recent studies from our laboratory demonstrate that they are reduced by ferrous salts at room temperature and that the principal reaction products are o-nitroanilines. This paper shows that simple benzofuroxan derivatives are also able to oxidise HbO2 2+ to methemoglobin (MetHb3+) (UV detection) and to form o-nitroanilines (HPLC detection). From a toxicological point of view this reaction is interesting, since it indicates that the blood is a site for metabolism of these compounds with consequent methemoglobinemia and formation of toxic compounds.

Pharmacochemistry of the furoxan ring: recent developments.

In the present work recent results obtained in the pharmacochemistry of the furoxan system are reported. In particular, after a brief description of the salient points of the furoxan chemistry, the synthesis and the properties of a series of Nifedipine and Prazosin analogues, containing this heterocyclic system, are described. Since we observed that a few furoxan derivatives are able to elicit both a dose-dependent rise in platelet cGMP levels and to promote a dose-dependent inhibition of AA-induced [Ca++] rise, and that many substituted furoxans show potent vasodilating and antiaggregatory activity, the possibility of using the furoxan system as a lead in the design of new vasodilators is also discussed.

Synthesis and vasodilating properties of N-alkylamide derivatives of 4-amino-3-furoxancarboxylic acid and related azo derivatives.

A series of N-alkylamide derivatives of 4-amino-3-furoxancarboxylic acids 5a-11a and their oxidation products, the azo derivatives 5b-11b, were synthesised and studied for their vasodilating properties. All the products were able to release rat aorta strips precontracted with (-)noradrenaline. Azo derivatives proved to be 20-200 times more potent than the parent amines. The large variation of lipophilicity within the two series does not seem to influence significantly the activity. Experiments carried out in the presence of oxyhaemoglobin (HbO(2)) suggest the involvement of nitric oxide (NO*) in the vasodilation.

Inhibition of bovine beta-trypsin, human alpha-thrombin and porcine pancreatic beta-kallikrein-B by benzamidine and its bis-, tris- and tetra-derivatives: thermodynamic and molecular modeling study.

The inhibitory effect of bis-, tris- and tetra-benzamidine derivatives (DAPP, TAPB and TAPP, respectively) on the catalytic properties of bovine beta-trypsin (beta-trypsin), human alpha-thrombin (alpha-thrombin) and porcine pancreatic beta-kallikrein-B (beta-kallikrein-B) was investigated (between pH 2.0 and 7.0, I = 0.1 M; T = 37.0 +/- 0.5 degrees C), and analyzed in parallel with that of benzamidine, commonly taken as a molecular inhibitor model of serine proteinases. Over the whole pH range explored, benzamidine, DAPP, TAPB and TAPP, show the same value of the association inhibition constant (Ki, M-1) for beta-trypsin; at variance, the affinity of DAPP, TAPB and TAPP for alpha-thrombin and beta-kallikrein-B is higher than that found for benzamidine association around neutrality, but tends to converge in the acidic pH limb. On lowering the pH from 5.5 to 3.0, the decrease in affinity for benzamidine binding to beta-trypsin, alpha-thrombin and beta-kallikrein-B as well as for DAPP, TAPB and TAPP association to beta-trypsin reflects the acidic-pK shift, upon inhibitor binding, of a single ionizing group. Over the same pH range, values of Ki for DAPP, TAPB and TAPP binding to alpha-thrombin and beta-kallikrein-B appear to be modulated by the acidic-pK shift, upon inhibitor association, of two equivalent proton-binding residues. Considering the X-ray three dimensional structures and the computer-generated molecular models of the serine proteinase inhibitor complexes, the observed binding behaviour of benzamidine, DAPP, TAPB and TAPP to beta-trypsin, alpha-thrombin and beta-kallikrein-B has been related to the inferred stereochemistry of the enzyme:inhibitor contact region(s).

In vitro cytotoxic activity of aryl and heteroaryl-ONN-azoxycyanides.

Some aryl-N(O) = N-X and heteroaryl-N(O) = N-CN compounds were synthetized and tested against a culture of HeLa cells. The results obtained show that the - N(O) = N beta CN function, a new cytostatic group, is useful in the design of potential antitumoral compounds.