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.

New 1,4-dihydropyridines conjugated to furoxanyl moieties, endowed with both nitric oxide-like and calcium channel antagonist vasodilator activities.

A series of 4-phenyl-1,4-dihydropyridines substituted at the ortho and meta positions of the phenyl ring with NO-donating furoxan moieties and their non-NO-releasing furazan analogues were synthesized and pharmacologically characterized. The vasodilator activities of these compounds were evaluated on rat aorta and expressed as EC50 values or as EC50iGC values when obtained in the presence of inhibitors of guanylate cyclase methylene blue (MB) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Affinities to 1, 4-DHP receptors on Ca2+ channels, expressed as IC50 values, were determined through displacement experiments of [3H]nitrendipine on rat cortex homogenates. A linear correlation between IC50 and EC50 values was found for compounds unable to release NO. EC50calcd values for derivatives containing NO-donor moieties, expression of the Ca2+-blocking component of their vasodilator activity, were interpolated on this linear regression. They showed a good correspondence with EC50iGC values determined in the presence of soluble guanylate cyclase inhibitors. Analysis of EC50iGC/EC50 ratios provided a useful tool to distinguish well-balanced hybrids from derivatives biased toward Ca2+-blocking or NO-dependent vasodilator activity. A detrimental effect on affinity to the 1, 4-DHP receptor, due to substitution at the ortho and meta positions of the 4-phenyl ring, was observed. SAR to explain this effect is proposed.

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.

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.

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.

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.

The involvement of the release of nitric oxide in the pharmacological activity of the new furoxan derivative CHF 2363.

1. The mechanism of action and the pharmacological effects of the new furoxan derivative, CHF 2363 (4-ethoxy-3-phenylsulphonylfuroxan), were investigated. 2. Pre-incubation of CHF 2363 with human platelet-rich plasma produced a concentration-dependent inhibition of the platelet aggregation induced by collagen, adenosine diphosphate (ADP) and platelet activating factor (PAF). The test compound was about 5 times more potent than sodium nitroprusside. 3-Isobutyl-1-methyl-xanthine (IBMX) potentiated the antiaggregating effect of CHF 2363. 3. CHF 2363 was a potent inhibitor of rubbed endothelium rabbit aortic ring contraction induced by noradrenaline. Comparison of IC50 values showed that CHF 2363 was as potent as glyceryl trinitrate (GTN). 4. Increasing concentrations of CHF 2363 elevated platelet guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels. Adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels were unaffected. 5. Oxyhaemoglobin reduced all the pharmacological actions of the test compound. Moreover, CHF 2363 concentration-dependently released nitric oxide (NO) in platelet-rich plasma. The NO release was correlated to its ability to increase platelet cyclic GMP levels. 6. After exposure of rat aortic strips to supramaximal concentrations of GTN (550 microM), the vasorelaxant activity of CHF 2363 did not change, although that of GTN decreased about 55 fold. 7. It has been concluded that the new furoxan derivative CHF 2363 exerts a potent antiaggregating and vasorelaxant activity via NO release and increase of cyclic GMP levels. No in vitro cross tolerance between GTN and CHF 2363 was observed.

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.

Antisecretory and gastroprotective activities of compounds endowed with H2 antagonistic and nitric oxide (NO) donor properties.

In spite of the well recognized gastric antisecretory activity, the gastroprotective potential of histamine H2 receptor antagonists is controversial. Most clinical studies in fact indicate that these drugs do not substantially protect the gastric mucosa from aggressive factors. Nitric oxide (NO) has been recently recognized as a fundamental mediator in gastric defence mechanisms, due to its ability to increase gastric mucosal blood flow and mucus production and to inhibit neutrophils adherence to endothelial cells. The aim of this study was to investigate the gastroprotective and H2 receptor antagonistic activity of a series of lamtidine analogues which contain different NO-releasing moieties (furoxan, nitroxy and nitrosothiol). These compounds were tested, in comparison with related H2 antagonists devoid of NO-donor structures, in different H2 receptor assays and in the conscious rat against 0.6 N HCl-induced gastric lesions. All the compounds tested were able to antagonize histamine-mediated responses at cardiac and gastric H2 receptors; however, furoxan and nitroxy derivatives were 10-fold less potent than the analogues devoid of NO-donor properties. By contrast, NO-donor compounds were more active than reference H2 antagonists as gastroprotective agents against mucosal injury induced by 0.6 N HCl. Among the different NO-donor moieties, the furoxan group conferred to the H2 antagonist molecule the highest gastroprotective potential; this finding closely correlates with the characteristics of NO release. In conclusions, lamtidine-analogue H2 antagonists combined with NO-donor moieties are endowed with gastric antisecretory and protective activity and could be the prototypes of a new class of anti-ulcer drugs. Finally, the furoxan NO donor group seems to be the most favourable among the different moieties tested.

CHF 2206, a new potent vasodilating and antiaggregating drug as potential nitric oxide donor.

The effects of the new 3,4-disubstituted furoxan, CHF 2206, on vascular tone, platelet aggregation and platelet cyclic 3',5'-guanosine monophosphate (cGMP) levels were investigated. The compound was a potent inhibitor of rabbit aortic ring contraction induced by norepinephrine, the stable prostaglandin F2 alpha analogue, U-46619, and KCl; this activity was independent of endothelium integrity. When pre-incubated with platelet-rich plasma, CHF 2206 potently reduced in a dose-dependent manner the aggregation induced by the threshold aggregating concentration of collagen, ADP or platelet activating factor (PAF). In the same experimental conditions, the test compound increased the platelet cGMP levels and this rise was clearly associated with the inhibition of platelet aggregation. Moreover, 3-isobutyl-1-methyl-xanthine (IBMX) 5 microM potentiated the antiaggregating activity of CHF 2206. These results indicate that the increase in cGMP plays a key role in the CHF 2206-elicited responses. Oxyhemoglobin reduced all the pharmacological actions of the test compound. This evidence, along with the capacity of CHF 2206 to cause inorganic nitrite production in the presence of platelet-rich plasma, strongly suggests that nitric oxide (NO) may be a common reactive intermediate responsible for the effects induced by the drug. In conclusion, the furoxan derivative CHF 2206 exerts a potent antiaggregating and vasodilating activity with a pharmacological profile similar to the one described for NO-donating pro-drugs.

Resolution of some 4-benzofurazanyl and 4-benzofuroxanyl 1, 4-dihydropyridine derivatives by chiral HPLC on whelk-o1 and some polysaccharide chiral stationary phases

The chromatographic chiral resolution of racemic methyl 1, 4-dihydro-2,6-dimethyl-5-nitro-4-benzofurazanyl-3-carboxylates 1 and 2 and their benzofuroxanyl analogues 3 and 4 were studied on Whelk-O1, Chiralcel OD-H, Chiralcel OJ, and Chiralpak AD and AS. These CSPs were selected on the basis of the results of structural searches in Chirbase. Examination of the data and cluster analysis pointed out the influence of benzofurazane-benzofuroxane change versus alpha-beta connection change on retention and enantioselectivity, respectively. The major contribution to the retention change arose from the type of heterocycle, whereas the major contribution to the enantioselectivity change came from the mode of connection (alpha or beta) almost irrespective of the nature of the heterocycle. It resulted in a similarity of behaviour between 1 and 2 on one hand and 3 and 4 on the other as far as capacity factors were concerned, and in a similarity of behaviour between 1 and 3 on the one hand and 2 and 4 on the other as far as enantioselectivities were concerned. Chiralpak AS was selected for semipreparative resolution of the enantiomers. The study of several CSPs allowed us to obtain correlations of structure with retention and enantioselectivity as well as the choice of a semipreparative support to provide the quantities for biological tests. Copyright 1999 Wiley-Liss, Inc.

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 H2-antagonist properties of some 1,2,5-thiadiazole-1-oxide derivatives.

A series of 1,2,5-thiadiazole-1-oxide derivatives has been synthesized and studied for its H2-antagonist properties. These derivatives can be considered derived from classical H2-antagonists in which the structure was deeply modified in order to evidence the minimal structural requirements for the activity. It was found that it is sufficient to have the 1,2,5-thiadiazole-1-oxide ring substituted with an alkylamino moiety and with an aliphatic chain linked to the hydroxy or ether group to achieve compounds as active as cimetidine. A few considerations on the binding on guinea-pig cerebral cortex of a series of H2-antagonists with more and more simplified structures are also reported.

Pyrazole analogues of prazosin.

A series of analogues of prazosin, in which 1-methyl or 1-phenylpyrazole moieties were substituted for the furan ring, were synthesized and studied for their alpha 1-adrenoceptor antagonist activity. The role of the five member heterocyclic substructures in determining the affinity for the alpha 1-receptor is briefly discussed.

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.

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.