A dual acting compound with latanoprost amide and nitric oxide releasing properties, shows ocular hypotensive effects in rabbits and dogs.

The IOP lowering effects of NCX 139, a new chemical entity comprising latanoprost amide and a NO-donating moiety, were compared to those of the respective des-nitro analog in in vitro assays and in rabbit and dog models of ocular hypertension. The NO donor, molsidomine as well as the prostamide bimatoprost (Lumigan(®)) and the prostaglandin agonist, latanoprost (Xalatan(®)) were also investigated for comparison. NCX 139 but not its des-nitro analog resulted in NO-mediated vascular relaxant effect in pre-contracted rabbit aortic rings (EC(50)=0.70±0.06 µM; E(max)=80.6±2.9%). Like bimatoprost (IC(50)=3.07±1.3 µM) or latanoprost (IC(50)=0.48±0.15 µM), NCX 139 displaced (3)H-PGF2α binding on recombinant human prostaglandin-F (FP) receptors with an estimated potency of 0.77±0.13 µM. In transient ocular hypertensive rabbits, bimatoprost and latanoprost were not effective while molsidomine elicited a dose-dependent reduction of IOP confirming the responsiveness of rabbits to NO but not to FP receptor agonists. NCX 139 tested at a therapeutically relevant dose, significantly lowered IOP while the des-nitro analog was not effective (0.03% NCX 139, Δ(max)=-12.8±2.0 mmHg). In glaucomatous dogs, 0.03% NCX 139 decreased IOP to a greater extent compared to an equimolar dose of the respective des-nitro derivative (Δ(max)=-4.6±1.0 and -2.7±1.3 mmHg, respectively for NCX 139 and its des-nitro analog). Albeit with low potency, NCX 139 also resulted effective in normotensive dogs while it did not reduce IOP in normotensive rabbits. NCX 139, a compound targeting two different and important mechanisms, is endowed with ocular hypotensive effects more evident in hypertensive conditions which may be of interest in the search of more effective treatments for hypertensive glaucoma.

Nitropravastatin stimulates reparative neovascularisation and improves recovery from limb Ischaemia in type-1 diabetic mice.

BACKGROUND AND PURPOSE: Mature endothelial cells and their progenitors are dysfunctional in diabetes, resulting in deficient neovascularisation following arterial occlusion. This study aimed to evaluate the therapeutic activity of a nitric oxide (NO) releasing statin in the setting of experimental diabetes and peripheral ischaemia. EXPERIMENTAL APPROACH: The effects of NCX 6550, an NO-releasing pravastatin derivative, on angiogenesis in ischaemic limbs was studied in normoglycaemic mice or mice made diabetic by treatment with streptozotocin (STZ). Control mice received an equimolar dosage of the parent statin compound, pravastatin. The therapeutic action of NCX 6550 was also tested in mice lacking the gene for endothelial nitric oxide synthase (eNOS). KEY RESULTS: In normoglycaemic or STZ-diabetic CD1 mice, only NCX 6550 stimulated skeletal muscle revascularisation. In addition, NCX 6550 induced greater improvement in limb reperfusion and salvage, than pravastatin. The number of circulating endothelial progenitor cells was decreased in STZ-diabetic mice, this defect being prevented by NCX 6550 and, to a lesser extent by pravastatin. In vitro, high glucose concentrations reduced the migratory capacity of endothelial progenitor EPCs, which was partly reversed by preincubation with pravastatin and completely reversed by NCX 6550. The postischaemic recovery of eNOS knockout mice was severely impaired as a consequence of depressed angiogenesis and this recovery was improved by treatment with NCX 6550, but not with pravastatin. CONCLUSIONS AND IMPLICATIONS: These findings indicate that incorporation of a bioactive NO moiety improves the therapeutic profile of statins for the treatment of peripheral vascular disease.

A topical nitric oxide-releasing dexamethasone derivative: effects on intraocular pressure and ocular haemodynamics in a rabbit glaucoma model.

BACKGROUND: Topical nitric oxide-releasing dexamethasone (NCX1021) may avoid the negative effects of dexamethasone phosphate. AIMS: To obtain more information on the role of nitric oxide in glaucoma and to compare a nitric oxide-releasing dexamethasone with dexamethasone phosphate with regard to intraocular pressure (IOP) and ocular haemodynamics in an experimental rabbit model. METHODS: Six rabbits were treated with dexamethasone phosphate 0.1% in the right eye and with NCX1021 in the left eye for 5 weeks. The parameters considered were IOP, nitric oxide marker levels in aqueous humour, ocular haemodynamics of ophthalmic artery (by means of colour Doppler imaging), expression of endothelial nitric oxide synthase (eNOS)in ciliary processes and histology of ciliary bodies. RESULTS: Dexamethasone increased IOP levels, NCX1021 did not. Nitrite and cyclic guanosine monophosphate levels in aqueous humour were lowered by dexamethasone and increased by NCX1021. Resistivity index of the ophthalmic artery was increased, eNOS expression was reduced and ciliary bodies showed histological lesions in dexamethasone-treated eyes, not in NCX1021-treated ones. CONCLUSIONS: NCX1021 may avoid the IOP increase, impairment of ocular blood flow and the morphological changes in the ciliary bodies possibly induced by corticosteroid treatment.

Dose and time effects of caffeine intake on human platelet adenosine A(2A) receptors : functional and biochemical aspects.

BACKGROUND-We determined whether repeated caffeine administration at different dosages and for different periods of time (400 or 600 mg/d for 1 week or 400 mg/d for 2 weeks) upregulates human platelet adenosine A(2A) receptors and is accompanied by increases in cAMP accumulation and decreases in aggregation and calcium levels after stimulation of A(2A) receptors by the selective agonist 2-hexynyl-5'-N-ethylcarboxamidoadenosine (HE-NECA). METHODS AND RESULTS-Platelets were obtained from peripheral venous blood of 45 healthy human volunteers at the end of 2 weeks of caffeine abstinence and at 12, 60, and 108 hours after the last dose of caffeine. The lowest dose of caffeine, when given for only 7 days, had no effect. Increasing the total dose, either by giving 400 mg/d for 14 days or giving 600 mg/d, resulted in binding assays performed with the adenosine A(2A) receptor radioligand [(3)H]SCH 58261 [5-amino-7(phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1, 5-c]pyrimidine], in the upregulation of A(2A) receptors. Moreover, the potency of HE-NECA to produce antiaggregatory effects, a rise in cAMP accumulation, and a decrease in calcium levels was significantly increased. CONCLUSIONS-Chronic caffeine intake can lead to upregulation of adenosine A(2A) receptors, which is accompanied by sensitization, in a time- and dose-dependent manner, to the actions of the agonist HE-NECA.

Pharmacology of adenosine A2A receptors.

Adenosine A2A receptors, which have been cloned from several mammalian species, are activated by physiological concentrations of adenosine to stimulate the formation of cAMP and other mediators. The A2A receptors are found on neutrophil leukocytes, platelets, blood vessels and, very abundantly, on some cells in the CNS. In the caudate nucleus they coexist with dopamine D2 receptors, and stimulation of A2A receptors causes a decrease in D2 receptor-mediated neurotransmission. Thus, drugs that act on A2A receptors can be used to modify dopaminergic neurotransmission known to be important in neurological and psychiatric disorders. In this review, Ennio Ongini and Bertil Fredholm describe how recently developed potent and selective A2A receptor antagonists can be used to delineate the physiological and pathological processes regulated by A2A receptors. Results from in vitro and in vivo pharmacology studies strengthen the notion that A2A receptors are an interesting target for drug development.

A novel nitric oxide-releasing statin derivative exerts an antiplatelet/antithrombotic activity and inhibits tissue factor expression.

BACKGROUND: NO-releasing statins are new chemical entities, combining HMG-CoA reductase inhibition and slow NO release, that possess stronger anti-inflammatory and antiproliferative activities than the native statins. OBJECTIVE: We evaluated the antithrombotic effects of nitropravastatin (NCX-6550) by assessing its activity on platelet activation and tissue factor (TF) expression by mononuclear cells in vitro and in vivo. METHODS AND RESULTS: In vitro, NCX-6550 inhibited (1) U46619- and collagen-induced platelet aggregation in buffer and plasma; (2) collagen-induced P-selectin expression in whole blood and (3) platelet adhesion to collagen-coated coverslips under high shear stress. These effects were displayed at concentrations of NCX-6550 ranging from 25 to 100 mum, and were totally reverted by the guanylylcyclase inhibitor ODQ (10 microm). Equimolar concentrations of pravastatin had no influence on these parameters of platelet function. LPS- and PMA-induced TF expression by blood mononuclear cells was also inhibited by NCX-6550 (IC50 13 microm), but not by pravastatin, as assessed by functional and immunological assays and by real-time PCR. In a mouse model of platelet pulmonary thromboembolism, induced by the i.v. injection of collagen plus epinephrine, pretreatment with NCX-6550 (24-48 mg kg(-1)) significantly reduced platelet consumption, lung vessel occlusion and mortality. Moreover, nitropravastatin markedly inhibited the generation of procoagulant activity by spleen mononuclear cells and peritoneal macrophages in mice treated with LPS. In these in vivo models too, pravastatin failed to affect platelet activation and monocyte/macrophage procoagulant activity. CONCLUSIONS: Our results show that nitropravastatin exerts strong antithrombotic effects in vitro and in vivo, and may represent an interesting antiatherothrombotic agent for testing in acute coronary syndromes.

Modeling hemodynamic profiles by telemetry in the rat. A study with A1 and A2a adenosine agonists.

The newly developed radiotelemetry system offers a number of advantages for the measurement of blood pressure and heart rate in laboratory animals. However, no available statistical methods permit valid use of the many data gathered with this continuous recording of hemodynamic parameters. This study describes elaboration and testing of mathematical functions as applied to the measurement of the effects of drugs on blood pressure and heart rate in spontaneously hypertensive rats. We used parametric functions analogous to those for pharmacokinetic studies. Curve fitting is in fact the only approach that provides reasonable estimates of hemodynamic kinetic constants. Nonlinear functions were assessed by analyzing telemetric hemodynamic effects induced by three adenosine receptor agonists with different selectivity for the A1 or A2a receptor. After acute administration in conscious rats, the A1 agonist 2-chloro-N6-cyclopentyladenosine induced dose-related hypotension (eg, 0.03 mg/kg; peak, -70 mm Hg; time to peak, 0.34 hour) and bradycardia (eg, 0.03 mg/kg; peak, -186 beats per minute [bpm]; time to peak, 0.38 hour). The A2a agonist 2-hexynyl-5'-N-ethylcarboxamidoadenosine induced dose-related hypotension (eg, 0.003 mg/kg; peak, -36 mm Hg; time to peak, 0.32 hour) with reflex tachycardia (eg, 0.003 mg/kg; peak, 152 bpm; time to peak, 0.35 hour). The nonselective adenosine agonist 5'-N-ethylcarboxamidoadenosine (0.1 mg/kg) induced hypotension (peak, -75 mm Hg; time to peak, 2.2 hours) and bradycardia followed by tachycardia (first peak, -131 bpm; time to peak, 1.26 hours; second peak, 123 bpm; time to peak, 13.9 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

The D1 dopamine receptor antagonist SCH 23390 enhances REM sleep in the rat.

The effect of the D1 dopamine receptor antagonist SCH 23390 on sleep patterns was studied in rats by means of the electroencephalographic (EEG) technique. Haloperidol, an established D2 antagonist neuroleptic, was used for comparison. Over a very small dose range (0.003-0.03 mg/kg, subcutaneously), SCH 23390 significantly increased the time spent in total sleep, including rapid eye movement (REM) and non-REM sleep. The magnitude of the overall change of REM sleep was considerably greater than that of non-REM sleep. Enhancement of the amount of REM was characterized by increase in number of episodes but no change of latency to the first period of REM. Haloperidol increased non-REM sleep at 0.3-3 mg/kg, orally, but did not affect other measures of REM. Considering the small dose range at which SCH 23390 was effective, and the fact that REM and non-REM sleep may be unrelated events, it is suggested that promotion of REM sleep is a specific effect induced by selective blockade of D1 receptors.

Effects of a new benzodiazepine hypnotic (quazepam--SCH 16134) on EEG synchronization and sleep-inducing mechanisms in cats.

The sleep-inducing properties of a new benzodiazepine hypnotic (quazepam--SCH 16134) were evaluated in cats with transection at different levels of the brain stem structure. Effects observed after administration of doses ranging from 0.12 and 1 mg/kg given intravenously were compared with those of pentobarbital. In the encéphale isolé small doses of quazepam induced or increased the synchronized periods. The desynchronized electroencephalogram (EEG) pattern of midpontine pretrigeminal preparations was not modified by these small doses. Only larger doses induced a fast neocortical activity of high amplitude. In midpontine pretrigeminal hemisection the synchronization occurred exclusively or predominantly in the hemisphere contralateral to the lesion. In the cerveau isolé, quazepam did not influence the typical synchronized EEG pattern. The arousal threshold after mesencephalic and physiological stimulation was raised only in encéphale isolé animals. Pentobarbital provoked sustained synchronization in all preparations with a pattern quite different from that of the benzodiazepine. These results suggest that quazepam may act through facilitation of the EEG-synchronizing mechanisms localized in the lower brain stem which are involved in physiological EEG-synchronizing and sleep-inducing processes.

Differential effects of dopamine D-1 and D-2 receptor agonists on EEG activity and behaviour in the rabbit.

SKF 38393, a selective agonist for dopamine D-1 receptors, LY 171555, a selective agonist for D-2 receptors and apomorphine, an agonist for both receptor sites, all induced activation of the electrical activity of the brain (EEG) in the rabbit. While SKF 38393 induced EEG changes without concomitant signs of stereotyped behaviour, the injection of both LY 171555 and apomorphine also elicited marked behavioural effects, mostly stereotyped mouth and head movements. The EEG effects of SKF 38393 were prevented by SCH 23390 (0.003 mg/kg i.v.), but not by (-)-sulpiride (6.2-25 mg/kg i.v.). Haloperidol attenuated the effects induced by SKF 38393 only at a dose (1 mg/kg) that induced EEG changes of its own. Similarly, effects of apomorphine on both EEG and behaviour were prevented by SCH 23390 and to a lesser extent by haloperidol, but not influenced by (-)-sulpiride. Different patterns of interactions were observed when D-2 receptors were selectively stimulated by LY 171555. Behavioural effects induced by LY 171555 were fully inhibited by both (-)-sulpiride (6.2-12.5 mg/kg i.v.) and haloperidol (0.1-0.3 mg/kg i.v.). The drug SCH 23390 attenuated some behavioural components at 0.3 mg/kg (i.v.), a dose at least 100-fold that effective on the EEG effects induced by SKF 38393. However, all these antagonists exerted weak or no effects on EEG activation induced by LY 171555 and did not restore the control patterns at any doses examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Synchronization of the EEG and sedation induced by neuroleptics depend upon blockade of both D1 and D2 dopamine receptors.

Blockade of dopamine (DA) receptors by neuroleptics tends to produce sedation, as shown by increased sleeping time, reduction of the arousal response to sensory stimuli and slowing of the electrical (EEG) activity of the brain. The EEG and behavioural effects of the selective compounds, SCH 23390 and raclopride, which block either D1 or D2 receptor subtypes, respectively were evaluated. Groups of rabbits were prepared for the measurement of EEG activity (neocortex and hippocampus). The EEG was analyzed visually and by spectral power analysis. Gross behaviour was also observed. The D1 antagonist, SCH 23390, by itself (0.03-0.3 mg/kg i.v.) produced small changes in the EEG and no evidence of sedation. Periods of slow waves occurred sporadically. Computerized EEG analysis showed moderate increases of total power density. The D2 receptor blocker, raclopride, alone (1-3 mg/kg i.v.) produced changes of the activity of the EEG, mostly, short periods of slow waves and slight increases of total power. No sedation was noted. Although both selective antagonists were studied at larger doses than those minimally effective, they produced slight EEG and behavioural changes which were not comparable with the marked actions produced by classical neuroleptics, such as haloperidol. However, when raclopride (1 mg/kg) was given after treatment with SCH 23390 (0.03 mg/kg) there was a marked synchronized activity in the EEG, associated with a state of sedation and diminished responsiveness to sensory stimuli. The data indicate that EEG synchronization and sedation, classically associated with treatment with neuroleptics, do not depend upon the selective blockade of either D1 or D2 receptors but, instead, require concurrent blockade of both subtypes of receptor.

Effects of the enkephalinase inhibitor SCH 34826 on the sleep-waking cycle and EEG activity in the rat.

The sedative effect of SCH 34826, an enkephalinase inhibitor, was evaluated by studying electroencephalographic (EEG) activity, behaviour and the sleep-waking cycle in the rat. The reference opioid, morphine, was used for comparison. After administration of morphine (10 mg/kg s.c.) the rats were motionless and stuporous at first and then hyperactive. An increase of slow wave sleep, at the expense of both wakefulness and REM sleep was recorded, with high-amplitude slow wave bursts appearing in the EEG tracings during the waking, albeit stuporous, phase. Relative spectral power in the 1-4 and 12-16 Hz bands was increased and there was a shift of the dominant frequency to a lower frequency. The specific opioid antagonist, naltrexone, readily reversed most of these effects. The drug SCH 34826 (10-100 mg/kg p.o.) had no effect on the parameters examined; large doses (300 and 1000 mg/kg p.o.) induced restlessness in some animals, resulting in increased waking. This effect was antagonized by naltrexone. The data indicate that SCH 34826, at doses far greater than those proposed for clinical use, is devoid of sedative liability and does not induce any of the behavioural or EEG effects typical of morphine.

Binding sites for [3H]2-oxo-quazepam in the brain of the cat: evidence for heterogeneity of benzodiazepine recognition sites.

In the present study, the distribution of benzodiazepine recognition site subtypes in the brain of the cat was investigated. To this aim, the binding properties of [3H]2-oxo-quazepam ([3H]2OXOQ) and [3H]beta-CCE, two ligands with preferential affinity for Type I benzodiazepine recognition sites, were compared to binding parameters for [3H]flunitrazepam ([3H]FNT) in different areas of the cat brain. The ratio of [3H]2OXOQ to [3H]FNT binding sites indicated that, in the cerebellum, Type I sites accounted for 90% of the total number of benzodiazepine recognition sites. The cerebral cortex, thalamus and mesencephalic reticular formation had also a high proportion of Type I sites (73-78%), whilst the two subtypes were almost equally distributed in the hippocampus, amygdala and bulbar reticular formation. A similar distribution of subtypes of benzodiazepine recognition sites was indicated by the ratio of [3H]beta CCE to [3H]FNT binding sites for different areas of the brain. These results demonstrate the existence of heterogeneity of recognition sites for benzodiazepines in the brain of the cat and support the view that [3H]2OXOQ preferentially labels Type I sites.

Rapamycin, but not FK506 and GPI-1046, increases neurite outgrowth in PC12 cells by inhibiting cell cycle progression.

Immunophilin ligands such as rapamycin, FK506 and GPI-1046 have been reported to increase neurite outgrowth in vitro and to have neuroprotective activity in vitro and in vivo. In this study, however, FK506 and GPI-1046 (0.1-1000 nM) had little effect on neurite outgrowth in PC12 cells in either the presence or absence of nerve growth factor. In contrast, rapamycin markedly increased neurite outgrowth in PC12 cells in the presence of a low concentration of nerve growth factor (EC(50)=10 nM). Unlike FK506 and GPI-1046, rapamycin is an inhibitor of cell cycle progression. Other cell cycle inhibitors such as ciclopirox and flavopiridol also increased neurite outgrowth in PC12 cells in the presence of a low concentration of nerve growth factor (EC(50)=250 nM and 100 nM, respectively). The neuroprotective effects of FK506, rapamycin and GPI-1046 were also tested in a rodent model of permanent focal cerebral ischemia. FK506 and rapamycin decreased infarct volume by 40% and 37%, respectively, whereas GPI-1046 was ineffective. These data do not support the previous suggestion that FK506 and GPI-1046 increase neurite outgrowth of PC12 cells in vitro. Rapamycin increases neurite outgrowth of PC12 cells, an effect that can be ascribed to its ability to inhibit cell cycle progression. The neuroprotective effect of FK506 and rapamycin against cerebral ischemia is probably not due to differentiation of neuronal precursors or stimulation of neuronal regeneration.

Humoral effects of selective adenosine agonists in spontaneously hypertensive rats.

OBJECTIVE: We studied the dose-response effects of acute administration of the selective A1 adenosine receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA), the selective A2A agonists 2-hexynyl-5'-N-ethylcarboxamidoadenosine (2HE-NECA) and 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680) and the non-selective agonist N-ethylcarboxamidoadenosine (NECA) on plasma renin activity, atrial natriuretic peptide, cyclic guanosine 3',5'-monophosphate (cGMP) and endothelin-1 in spontaneously hypertensive rats. METHODS: The drugs were administered intraperitoneally in four doses to conscious rats. Systolic blood pressure and heart rate were measured by the tail-cuff technique. Both humoral and hemodynamic parameters were determined 1 h after dosing in separate sets of animals. RESULTS: All the compounds induced a dose-dependent decrease in systolic blood pressure that was associated with different changes in heart rate. Heart rate was decreased by all doses of CCPA and by the higher doses of the non-selective compound (NECA) and increased by both A2A agonists. Plasma renin activity also changed in opposite directions, being decreased by CCPA but increased dose-dependently by 2HE-NECA and CGS 21680 and only moderately by NECA. Plasma atrial natriuretic peptide and cGMP levels increased dose-dependently after CCPA and NECA, but were unaffected by the A2A agonists. None of the compounds altered plasma endothelin-1 levels. CONCLUSIONS: These results indicate that the renin-suppressive effect of the A1 agonist, which is associated with a cardiodepressant action, may be attributed either to a direct inhibition of renin release or to the concomitant increments in plasma atrial natriuretic peptide and its second messenger, cGMP. In contrast, the renin-stimulating effect of the A2A agonists may result either from direct stimulation of renin secretion or from reflex sympathetic activation secondary to the fall in blood pressure.

2-[N'-(3-arylallylidene)hydrazino]adenosines showing A2a adenosine agonist properties and vasodilation activity.

A series of 2-[N'-(3-arylallylidene)hydrazino]adenosines were prepared and studied in binding and functional assays to assess their potency for the A2a compared with the A1 adenosine receptor. These analogs possess A2a receptor affinity in the low nanomolar range associated with weak interaction with the A1 receptor. Among the compounds, in rat tissues, 2-[N'-[3-(4-nitrophenyl)allylidene] hydrazinoadenosine (5g) had the most potent affinity for the A2a receptor, the K(i) value being 23 nM. The type and position of substituents on the phenyl ring show a moderate influence on biological activity, allowing the conclusion that the latter is mostly due to the allylidenehydrazino side chain. From functional experiments 2-[N'-(2-furylmethylidene)hydrazino]adenosine (4b), 2-[N'-(3-phenylallylidene)hydrazino]adenosine (5a) and 2-[N'-[3-(2-furyl)allylidene]hydrazino]adenosine (5b) appeared to be potent in inducing vasorelaxation (an A2a-mediated response) without appreciable effects on the heart rate (an A1-mediated action). While the lack of effects on heart rate is clearly explained by the poor affinity for A1 receptors, more difficult appears the interpretation of vasorelaxant properties displayed by some compounds. Affinity for A2a has a major role, but other types of interactions, yet to be identified, may play a part.

2-Aralkynyl and 2-heteroalkynyl derivatives of adenosine-5'-N-ethyluronamide as selective A2a adenosine receptor agonists.

A series of new 2-aralkynyl and 2-heteroaralkynyl derivatives of NECA were synthesized and studied in binding and functional assays to assess their potency for the A2a compared to A1 adenosine receptors. Compounds bearing an aromatic or heteroaromatic ring, conjugated to the triple bond, showed generally weaker activity at the A2a receptor and lower selectivity (A2a vs A1) than the alkylakynyl derivatives previously reported. However, the (4-formylphenyl)-ethynyl derivative 17 showed affinity in the low nanomolar range and high selectivity (about 160-fold) for the A2a receptor. The presence of heteroatoms improved vasorelaxant activity, the 2-thiazolylethynyl derivative 30 being the most potent in the series. Introduction of methylene groups between the triple bond and the phenyl ring favored the A2a binding affinity, and the 5-phenyl-1-pentynyl derivative 24 was found to be highly potent and selective (about 180-fold) at A2a receptors. With regard to antiplatelet activity, the presence of aromatic or heteroaromatic rings decreased potency in comparison with that of NECA and of N-ethyl-1'-deoxy-1'-(6-amino-2-hexynyl-9H-purin-9-yl)-beta-D-ribofura nuronamide (HENECA). Introduction of a methylene group was effective in increasing antiaggregatory potency only when this group is linked to a heteroatom (31-35). From these data and those previously reported, the structure-activity relationships derived for the 2-alkynyl-substituted ribose uronamides would indicate that potentiation of A2a receptor affinity could be obtained by aromatic rings not conjugated with the triple bond or by heteroaromatic groups. As for A2a receptors on platelets, the presence of aromatic rings, either conjugated or unconjugated to the triple bond, is detrimental for the antiaggregatory activity. However, the introduction of polar groups alpha to the triple bond strongly increases the potency when steric hindrance is avoided. Some of the compounds included in this series retain interesting vasodilating properties and merit further investigation for their potential in the treatment of cardiovascular disorders.

Pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine derivatives: potent and selective A(2A) adenosine antagonists.

A series of pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine derivatives (10a-o,q,r), bearing alkyl and aralkyl chains on positions 7 and 8, were synthesized in the attempt to obtain potent and selective antagonists for the A(2A) adenosine receptor subtype. The compounds were tested in binding and functional assays to evaluate their potency for the A(2A) compared with the A1 adenosine receptor subtype. In binding studies in rat brain membranes, most of the compounds showed affinity for A(2A) receptors in the low nanomolar range with a different degree of A(2A) versus A1 selectivity. Comparison of N(7) (10a-d,h-o)- and N(8) (10e-g)-substituted pyrazolo derivatives indicates that N(7) substitution decreases the A1 affinity with the concomitant increase of A(2A) selectivity. Specifically, the introduction of a 3-phenylpropyl group at pyrazolo nitrogen in position 7 (101) increased significantly the A(2A) selectivity, being 210-fold, while the A(2A) receptor affinity remained high (Ki=2.4 nM). With regards to the affinity for A(2A) receptors, also the compound 10n, bearing in the 7-position a beta-morpholin-4-ylethyl group, deserves attention (Ki=5.6 nM) even though the A2A selectivity (84-fold) was not as high as that of 101. Conversely, the compound 10m (N(7)-4-phenylbutyl derivative) showed a remarkable selectivity (A1/a(2A) ratio = 129) associated with lower A(2A) affinity (Ki = 21 nM). In functional studies, most of the compounds examined reversed 5'-(N-ethylcarbamoyl) adenosine-induced inhibition of rabbit platelet aggregation inhibition which is a biological response mediated by the A2A receptor subtype. The compounds are potent and selective A2A antagonists which can be useful to elucidate the pathophysiological role of this adenosine receptor subtype. These compounds deserve to be further developed to assess their potential for treatment of neurodegenerative disorders such as Parkinson's disease.

Design, synthesis, and biological evaluation of a second generation of pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines as potent and selective A2A adenosine receptor antagonists.

New A2A adenosine receptor antagonists in the series of pyrazolo[4, 3-e]-1,2,4-triazolo[1,5-c]pyrimidines, bearing oxygenated substituents on the phenylalkyl chains on the 7-position, have been synthesized. The compounds were tested in binding and functional assays to evaluate affinity, potency, and selectivity for rat A2A compared to rat A1 and human A3 receptor subtypes. The most interesting compounds (5d,e,h) were tested also in binding to human A1 and A2A adenosine receptors. They showed very good affinity (Ki = 0.94 nM for compound 5h) and interesting selectivity with respect to both rA1 and hA3 (compound 5h: rA1/rA2A = 787, hA3/rA2A > 10 000). These important findings make this new series of compounds the first really selective for A2A adenosine receptors. Thermodynamic parameters were evaluated; all the tested compounds displayed an enthalpy-driven binding as expected for antagonists. Moreover, compound 5h showed a negative entropy value. The highly negative enthalpic and entropic contributions could mean that 5h fits very well in the binding site where, probably, an electrostatic interaction is present associated to a scarce solvent reorganization around the receptor binding site. These compounds deserve to be further developed to assess their potential for treatment of neurodegenerative disorders such as Parkinson's disease.

2-Alkynyl derivatives of adenosine-5'-N-ethyluronamide: selective A2 adenosine receptor agonists with potent inhibitory activity on platelet aggregation.

A series of new 2-alkynyl and 2-cycloalkynyl derivatives of adenosine-5'-N-ethyluronamide (NECA) and of N-ethyl-1'-deoxy-1'-(6-amino-2-hexynyl-9H-purin-9-yl)-beta-D- ribofuranuronamide (1, HE-NECA), bearing hydroxy, amino, chloro, and cyano groups in the side chain, were synthesized. The compounds were studied in binding and functional assays to assess their potency for the A2 compared to A1 adenosine receptor. The presence of an alpha-hydroxyl group in the alkynyl chain of NECA derivatives accounts for the A2 agonist potency, leading to compounds endowed with sub-nanomolar affinity in binding studies. However, these analogues also possess good A1 receptor affinity resulting in low A2 selectivity. From functional experiments the 4-hydroxy-1-butynyl (6) and the 4-(2-tetrahydro-2H-pyranyloxy)-1-butynyl (16) derivatives appear to be very potent in inducing vasorelaxation without appreciable effect on heart rate. The new compounds were also tested as inhibitors of platelet aggregation induced by ADP. Introduction of an alpha-hydroxyl group in the alkynyl side chain caused a greater increase in antiaggregatory activity than either NECA or HE-NECA, resulting in the most potent inhibitors of platelet aggregation so far known in the nucleoside series. The presence of an alpha-quaternary carbon such as the 3-hydroxy-3,5-dimethyl-1-hexynyl (12) and the 3-hydroxy-3-phenyl-1-butynyl (15) derivatives markedly reduced the antiaggregatory potency without affecting the A2 affinity. The hydrophobicity index (k') of the new nucleosides barely correlated with the binding data, whereas high k' values were associated with increased A2 vs A1 selectivity but with reduced activity in all functional assays. Some of the compounds synthesized possess interesting pharmacological properties. Compounds having an appropriate balance between vasorelaxation and antiplatelet activity, if confirmed in vivo, deserve further development for the treatments of cardiovascular disorders.