Relationship between hyponatremia at hospital admission and cardiopulmonary profile at follow-up in patients with SARS-CoV-2 (COVID-19) infection.

PURPOSE: Hyponatremia occurs in about 30% of patients with pneumonia, including those with SARS-CoV-2 (COVID-19) infection. Hyponatremia predicts a worse outcome in several pathologic conditions and in COVID-19 has been associated with a higher risk of non-invasive ventilation, ICU transfer and death. The main objective of this study was to determine whether early hyponatremia is also a predictor of long-term sequelae at follow-up. METHODS: In this observational study, we collected 6-month follow-up data from 189 laboratory-confirmed COVID-19 patients previously admitted to a University Hospital. About 25% of the patients (n = 47) had hyponatremia at the time of hospital admission. RESULTS: Serum [Na+] was significantly increased in the whole group of 189 patients at 6 months, compared to the value at hospital admission (141.4 ± 2.2 vs 137 ± 3.5 mEq/L, p < 0.001). In addition, IL-6 levels decreased and the PaO2/FiO2 increased. Accordingly, pulmonary involvement, evaluated at the chest X-ray by the RALE score, decreased. However, in patients with hyponatremia at hospital admission, higher levels of LDH, fibrinogen, troponin T and NT-ProBNP were detected at follow-up, compared to patients with normonatremia at admission. In addition, hyponatremia at admission was associated with worse echocardiography parameters related to right ventricular function, together with a higher RALE score. CONCLUSION: These results suggest that early hyponatremia in COVID-19 patients is associated with the presence of laboratory and imaging parameters indicating a greater pulmonary and right-sided heart involvement at follow-up.

Tenderness of broiler breast fillets from carcasses treated with electrical stimulation and extended chilling times.

Postmortem electrical stimulation (ES) (450 V, 450 mA, 2 s on, 1 s off for five pulses) has been shown to decrease the toughness associated with early deboning. Most studies involving this system have been concerned with obtaining "acceptable" tenderness in fillets deboned at about 1 h postmortem, the time of carcass exit from an immersion chiller. However, the effects of ES combined with deboning at 2 h postmortem needs to be evaluated because some processors are considering extended immersion chilling and those already using air chilling require approximately 2 h for this process. Two 32-bird trials were conducted to compare tenderness in broiler breast fillets from ES-treated carcasses deboned at 1 and 2 h postmortem and fillets from control (C) carcasses deboned at 1 and 4 h postmortem. The Allo-Kramer shear value means of ES-2 h and C-4 h fillets were not different from each other and were significantly lower than that of the ES-lh fillets, which was significantly lower than the C-lh fillets. There was no significant difference among treatments associated with thaw loss or cook loss. The ES-2 h, ES-1 h, and C-4 h samples had significantly higher R-values and lower pH values than the C-1 h samples, indicating more advanced rigor development. These results indicate that deboning fillets from ES-treated carcasses at 2 h postmortem yields meat with a tenderness equivalent to the value reached with 4 h aging on the carcass. This is a 50% reduction in the time needed to achieve this level of tenderness.

Diagnosis of glucocorticoid-remediable aldosteronism in primary aldosteronism: aldosterone response to dexamethasone and long polymerase chain reaction for chimeric gene.

Aldosterone suppression by dexamethasone, and high 18-hydroxycortisol and 18-oxocortisol levels are used to differentiate glucocorticoid-remediable aldosteronism (GRA) from other forms of primary aldosteronism. These methods are time consuming, expensive, and impractical for large studies. Moreover, diagnosis of GRA requires a confirmatory genetic test. We evaluated 117 patients with primary aldosteronism referred to our centers by the use of a long PCR technique to reveal the chimeric gene of GRA. In 60 of 117 patients, the response of aldosterone to dexamethasone (2 mg/day for 4 days) was also assessed. None of our patients, including 2 pairs of siblings, was positive for the chimeric gene. The results of long PCR were confirmed by Southern blotting. Despite a negative genetic test, 6 patients (1 with aldosterone-producing adenoma and 5 with idiopathic hyperaldosteronism) had plasma aldosterone suppressed by dexamethasone (i.e. < or = 2 ng/dL). Of 117 patients, 43 were identified as having aldosterone-producing adenoma and 74 as having idiopathic hyperaldosteronism. In our experience, the long PCR technique is a reliable and simple test to at least exclude GRA in patients with primary aldosteronism. A short term dexamethasone suppression test of aldosterone can be misleading in identifying GRA. The prevalence of GRA in primary aldosteronism remains to be established.

Characterization of human A2A adenosine receptors with the antagonist radioligand [3H]-SCH 58261.

1. We have characterized the binding of the new potent and selective antagonist radioligand [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazol o[1,5- c]pyrimidine, [3H]-SCH 58261, to human cloned A2A adenosine receptors. 2. In Chinese hamster ovary (CHO) cells transfected with the human cloned A2A receptor, [3H]-SCH 58261 specific binding (about 70%) was rapid, saturable, reversible and proportional to protein concentration. The kinetic KD value was 0.75 nM. Saturation experiments showed that [3H]-SCH 58261 labelled a single class of recognition sites with high affinity (KD = 2.3 nM) and limited capacity (apparent Bmax = 526 fmol mg-1 protein). 3. Competition experiments revealed that binding of 0.5 nM [3H]-SCH 58261 was displaced by adenosine receptor agonists with the following order of potency: 2-hexynyl-5'-N-ethylcarboxamidoadenosine (2HE-NECA) > 5'-N-ethylcarboxamidoadenosine (NECA) = 2-phenylaminoadenosine (CV 1808) > 2-[4-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) > R-N6-phenylisopropyladenosine (R-PIA) > or = N6-cyclohexyladenosine (CHA) > S-N6-phenylisopropyladenosine (S-PIA). 4. Adenosine receptor antagonists inhibited [3H]-SCH 58261 binding with the following order: 5-amino-9-chloro-2-(2-furyl)-[1,2,4]-triazolo[1,5-c] quinazoline (CGS 15943) > SCH 58261 > xanthine amine congener (XAC) > (E,18%-Z,82%)7-methyl-8-(3,4-dimethoxystyryl)-1,3- dipropylxanthine (KF 17837S) > 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) > theophylline. 5. Affinity values and rank order of potency of both receptor agonists and antagonists were similar to those previously obtained in human platelet and rat striatal membranes, except for CV 1808 which was more potent than CGS 21680. SCH 58261 was a competitive antagonist at inhibiting NECA-induced adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in CHO cells transfected with human A2A receptors. Good agreement was found between binding and functional data. 6. Thus, the new antagonist radioligand is preferable to the receptor agonist radioligand [3H]-CGS 21680 hitherto used to examine the pharmacology of human cloned A2A adenosine receptors.

Fourier analysis of circadian blood pressure profile in secondary hypertension.

Different types of statistical methods have been used for circadian blood pressure (BP) rhythm analysis in secondary forms of hypertension. In the present study, we used the two-step statistical approach by Fourier analysis with four harmonics for the parametrization of the diurnal BP pattern in secondary hypertension. In 43 essential hypertensives (EH), eight patients with aldosterone producing adenoma (APA), 25 with idiopathic hyperaldosteronism (IHA), four with glucocorticoid remediable hyperaldosteronism (GRH) and seven with renovascular hypertension (RVH), 24-h ambulatory BP was measured. The diurnal BP and heart rate (HR) rhythm was present in more than 70% of patients with secondary hypertension, without significant differences with EH and despite the attenuation in the degree of the nocturnal BP fall. In conclusion, the statement that secondary hypertension is characterized by an abnormal diurnal rhythm of BP is a gross over-simplification. Our findings suggest that the two-step method with four harmonics Fourier analysis may represent a useful method and a more complete statistical approach to providing circadian parametrization of the 24-h profile in secondary hypertension.

Labeling of A2A adenosine receptors in human platelets by use of the new nonxanthine antagonist radioligand [3H]SCH 58261.

The present study describes the binding to human platelet A2A adenosine receptors of the new potent and selective antagonist radioligand [3H]5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo [1,5-c] pyrimidine ([3H]SCH 58261). Saturation experiments revealed that [3H]SCH 58261 labels a single class of recognition sites with high affinity (Kd = 0.85 nM), limited capacity (apparent Bmax = 85 fmol/mg of protein) and good specific binding (about 60%). [3H]SCH 58261 binding was not modulated by either the divalent cation Mg(+2) or guanine nucleotides. In competition experiments, a series of both adenosine agonists and antagonists inhibited [3H]SCH 58261 binding to A2A platelet receptors with rank order of potency and affinity similar to those observed in rat striatal membranes with the same radioligand. This confirms that the platelet A2A receptor is similar to that labeled in the brain striatum. Binding data were also found to be in good agreement with the results from functional studies such as A2A agonist-induced stimulation of adenylate cyclase or platelet aggregation inhibition. The present findings indicate that [3H]SCH 58261 is the first radioligand available for the characterization of the A2A receptor subtype in platelets.

1H-pyrazolo[4,3-d]pyrimidine-7(6H)-one and 5H-pyrazolo[4,3-d] 1,2,3-triazin-4(3H)-one derivatives. Synthesis and in vitro biological activity at adenosine A1 and A2a receptors.

The affinity of newly synthesized 1H-pyrazolo[4,3-d] pyrimidine-7 (6H)- one (5a-f) and 5H-pyrazolo[4,3-d]1,2,3,-triazin-4(3H)-one (6a-i) derivatives for A1 and A2a adenosine receptors was investigated in rat cerebral membranes. The compounds showed affinities in the micromolar range for both adenosine A1 and A2a receptors. In particular 5d was the most interesting compound and showed some degree of selectivity for the A1 receptor (Ki values being 2 mumol/l; A1/A2a ratio < 0.021). The present study provides useful information for a better understanding of the structure-activity relationships of antagonists at adenosine receptors.

Binding of the radioligand [3H]-SCH 58261, a new non-xanthine A2A adenosine receptor antagonist, to rat striatal membranes.

1. The present study describes the binding to rat striatal A2A adenosine receptors of the new potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazol o [1,5-c] pyrimidine, [3H]-SCH 58261. 2. [3H]-SCH 58261 specific binding to rat striatal membranes ( > 90%) was saturable, reversible and dependent upon protein concentration. Saturation experiments revealed that [3H]-SCH 58261 labelled a single class of recognition sites with high affinity (Kd = 0.70 nM) and limited capacity (apparent Bmax = 971 fmol mg-1 of protein). The presence of 100 microM GTP in the incubation mixture did not modify [3H]-SCH 58261 binding parameters. 3. Competition experiments showed that [3H]-SCH 58261 binding is consistent with the labelling of A2A striatal receptors. Adenosine receptor agonists competed with the binding of 0.2 nM [3H]-SCH 58261 with the following order of potency: 2-hexynyl-5'-N-ethyl carboxamidoadenosine (2HE-NECA) > 5'-N-ethylcarboxamidoadenosine (NECA) > 2-[4-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) > 2-phenylaminoadenosine (CV 1808) > R-N6-phenylisopropyladenosine (R-PIA) > N6-cyclohexyladenosine (CHA) = 2-chloro-N6-cyclopentyladenosine (CCPA) > S-N6-phenylisopropyladenosine (S-PIA). 4. Adenosine antagonists inhibited [3H]-SCH 58261 binding with the following order: 5-amino-9-chloro-2-(2-furyl)-[1,2,4]-triazolo[1,5-c] quinazoline (CGS 15943) > 5-amino-8-(4-fluorobenzyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4-triazolo [1,5-c] pyrimidine (8FB-PTP) = SCH 58261 > xanthine amine congener (XAC) = (E,18%-Z,82%)7-methyl-8-(3,4-dimethoxystyryl)-1,3-dipropylxanthine (KF 17837S) > 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) > or = 8-phenyltheophylline (8-PT). 5. The Ki values for adenosine antagonists were similar to those labelled with the A2A agonist [3H]-CGS 21680. Affinities of agonists were generally lower. The A1-selective agonist, R-PIA, was found to be about 9 fold more potent than its stereoisomer, S-PIA, thus showing the stereoselectivity of [3H]-SCH 58261 binding. Except for 8-PT, the adenosine agonists and antagonists examined inhibited [3H]-SCH 58261 binding with Hill coefficients not significantly different from unity. 6. The present results indicate that [3H]-SCH 58261 is the first non-xanthine adenosine antagonist radioligand which directly labels A2A striatal receptors. High receptor affinity, good selectivity and very low non-specific binding make [3H]-SCH 58261 an excellent probe for studying the A2A adenosine receptor subtype in mammalian brain.

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.

Novel N6-(substituted-phenylcarbamoyl)adenosine-5'-uronamides as potent agonists for A3 adenosine receptors.

A series of adenosine-5'-uronamide derivatives bearing N6-phenylurea groups have been synthesized and tested for their affinity at A1 and A2A adenosine receptors in rat brain membranes and at cloned rat A3 receptors from stably transfected CHO cells. Some N6-arylcarbamoyl derivatives, N6-((2-chlorophenyl)carbamoyl)-, N6-((3-chlorophenyl)carbamoyl)-, and N6-((4-methoxyphenyl)carbamoyl)adenosine-5'-ethyluronamide (4l-n), were found to have affinity at A3 receptors in the low nanomolar range (Ki values < 10 nM). In CHO cells stably transfected with the rat A3 receptor, compound 4n was found to be a full agonist in inhibiting adenylate cyclase activity. The present study represents the first example of N6-acyl-substituted adenosine analogs having high affinity at adenosine receptors and, in particular, at the A3 receptor subtype.

The non-xanthine heterocyclic compound SCH 58261 is a new potent and selective A2a adenosine receptor antagonist.

We have characterized the in vitro pharmacological profile of the new potent and selective A2a adenosine receptor antagonist SCH 58261 [7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2, 4-triazolo[1,5-c]pyrimidine]. In binding studies on rat and bovine brain tissues, SCH 58261 showed affinity in the low nanomolar range at A2a adenosine striatal receptors and good A2a adenosine vs. A1 adenosine selectivity (about 50- to 100-fold in rat and bovine brain, respectively). SCH 58261 did not show affinity for either the A3 adenosine receptor or other receptors at concentrations up to 1 microM. Saturation experiments on rat A1 and A2a adenosine receptors indicated the competitive nature of the antagonism. SCH 58261 antagonized competitively the effects induced by the A2a adenosine-selective agonist CGS 21680 (2-[4-(2-carboxyethyl)-phenethyl-amino]-5'-N- ethylcarboxamidoadenosine) in two functional assays, such as inhibition of rabbit platelet aggregation and porcine coronary artery relaxation. Specifically, the compound showed pA2 values of 7.9 and 9.5, respectively. SCH 58261 (300 nM) failed to antagonize 5'-N-ethylcarboxamidoadenosine-induced vasorelaxation in the isolated guinea pig aorta, a response mediated by A2b adenosine receptors. Likewise, at the same concentration, the compound weakly inhibited the A1 adenosine-mediated negative chronotropic effect induced by 2-chloro-N6-cyclopentyladenosine in the isolated rat atria. These data show that SCH 58261 is a potent and selective non-xanthine A2a adenosine antagonist which has competitive properties in biological responses mediated by this receptor subtype. The compound is of interest for investigating the biological role of A2a adenosine receptors and deserves further attention to clarify the therapeutic potential of A2a antagonists.

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.

Pharmacology of the new selective A2a adenosine receptor agonist 2-hexynyl-5'-N-ethylcarboxamidoadenosine.

The pharmacological profile of 2-hexynyl-5'-N-ethylcarboxamidoadenosine (2HE-NECA, CAS 141018-30-6), a new selective A2a adenosine receptor agonist, was characterized. In binding studies on both rat and bovine brain, 2HE-NECA was more potent on A2a receptors (Ki = 2.2 and 1.5 nmol/l, respectively) than the reference A2a agonist, 2-[4-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoaden osi ne (CPEC) or the non-selective adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA). The drug displayed a good A2a vs A1 receptor selectivity in brain tissues of both animal species (60- and 160-fold, respectively). In functional studies, 2HE-NECA showed marked vasodilating properties in rat aorta, bovine and porcine coronary arteries. The vasodilatory response in the porcine coronary preparation was greater for 2HE-NECA (EC50 = 23.3 nmol/l) than for CPEC (EC50 = 58.7 nmol/l) or NECA (EC50 = 76.6 nmol/l). In the rat Langendorff model, in which global ischemia was induced, 2HE-NECA (100 nmol/l) significantly prevented the risk of diastolic pressure and coronary perfusion pressure occurring during postischemic reperfusion. The in vitro antiaggregatory activity of 2HE-NECA (IC50 = 0.07 mumol/l), as tested in rabbit platelets, was higher than that found with NECA (IC50 = 0.2 mumol/l) or CPEC (IC50 = 2.16 mumol/l). In spontaneously beating rat atria, which are responsive to A1-receptor stimulation, 2HE-NECA did not show any negative chronotropic activity up to micromolar concentrations. In conscious spontaneously hypertensive rats, 2HE-NECA administered intraperitoneally caused a dose-dependent reduction in systolic blood pressure (ED30 = 0.005 mg/kg) with minimal reflex tachycardia.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding characteristics of the adenosine A2 receptor ligand [3H]CGS 21680 to human platelet membranes.

The binding characteristics of the selective adenosine A2 agonist [3H]CGS 21680 ([3H]2-[p-(2-carboxyethyl)-phenethyl-amino]-5'-N- ethylcarboxamidoadenosine) were determined in human platelet membranes. Specific binding was saturable, reversible and dependent upon protein concentration. Saturation experiments revealed a single class of binding sites with Kd and Bmax values of 1.4 microM and 5.9 pmol/mg of protein, respectively. Adenosine receptor agonists and antagonists competed for the binding of [3H]CGS 21680 (50 or 200 nM) to human platelet membranes showing a rank order of potency consistent with that typically found for interactions at the adenosine A2 receptor. Adenylate cyclase stimulation and platelet aggregation inhibition induced by adenosine agonists exhibited a rank order of potency close to that observed in binding experiments. However, the adenosine A1 receptor agonists, R- and S-N6-(2-phenylisopropyl)adenosine, (R-PIA) and (S-PIA), N6-cyclohexyladenosine (CHA) and 2-chloro-N6-cyclopentyladenosine (CCPA), which stimulate adenylate cyclase and inhibit platelet aggregation in the low microM range, displaced [3H]CGS 21680 only in the high microM range. In conclusion, we have found that [3H]CGS 21680, which is widely used as a specific A2 agonist in binding studies on brain tissues, is not appropriate for the characterization of the human platelet adenosine A2 receptor.

Effects of the new A2 adenosine receptor antagonist 8FB-PTP, an 8 substituted pyrazolo-triazolo-pyrimidine, on in vitro functional models.

1. We have characterized the in vitro pharmacological profile of putative A2 adenosine antagonists, two non-xanthine compounds, 5-amino-8-(4-fluorobenzyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine (8FB-PTP) and 5-amino-9-chloro-2-(2-furyl 1,2,4-triazolo [1,5-c] quinazoline (CGS 15943), and the xanthine derivative (E)7-methyl-8-(3,4-dimethoxystyryl)-1,3-dipropyl- xanthine (KF 17837). 2. In binding studies on bovine brain, 8FB-PTP was the most potent (Ki = 0.074 nM) and selective (28 fold) drug on A2 receptors, whereas CGS 15943 and KF 17837 exhibited affinity in the low and high nanomolar range, respectively, and showed little selectivity. 3. In functional studies, 8FB-PTP antagonized 5'-N-ethyl-carboxamidoadenosine (NECA)-induced vasorelaxation of bovine coronary artery (pA2 = 7.98) and NECA-induced inhibition of rabbit platelet aggregation (pA2 = 8.20). CGS 15943 showed weak activity in the platelet aggregation model (pA2 = 7.43) and failed to antagonize NECA-induced vasodilatation. KF 17837 was ineffective in both models up to micromolar concentrations. 4. Antagonism of A1-mediated responses was tested versus 2-chloro-N6-cyclopentyladenosine (CCPA) in rat atria. 8FB-PTP and CGS 15943 also antagonized competitively the negative chronotropic response induced by CCPA. Conversely, KF 17837 was unable to reverse A1-mediated responses. 5. 8FB-PTP is a potent and competitive antagonist of responses mediated by A2 adenosine receptors. The data provided a basis to reduce, by further chemical modifications, the affinity at A1 receptor and therefore enhance A2 receptor selectivity.

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.

Repeated administration of selective adenosine A1 and A2 receptor agonists in the spontaneously hypertensive rat: tolerance develops to A1-mediated hemodynamic effects.

We investigated the effects of the selective A1 adenosine receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA), the selective A2 adenosine agonists 2-hexynyl-5'-N-ethyl-carboxamidoadenosine(2-hexynyl-NECA) and 2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosi ne (CGS 21680), and the nonselective adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) on systolic blood pressure (SBP), heart rate (HR) and receptor binding characteristics. The drugs were studied after acute and repeated i.p. administration in conscious, spontaneously hypertensive rats (SHRs). SBP and HR were recorded by the tail-cuff method. In acute studies the drugs induced a dose-dependent antihypertensive effect with the following order of potency: 2-hexynyl-NECA > NECA > CCPA = CGS 21680. As expected, the A1 agonist CCPA induced a dose-dependent bradycardia, whereas the A2 agonists had minimal influence on HR, and the nonselective agonist NECA induced bradycardia only at the two highest doses. In chronic experiments, the compounds were administered twice daily at equihypotensive doses. 2-Hexynyl-NECA, CGS 21680 and NECA maintained their antihypertensive effects throughout the experimental period. After 21 days, SBP levels were -32, -38 and -28% vs. baseline, respectively. HR was slightly affected. Conversely, tolerance developed to both hypotensive and bradycardic effects of CCPA: at day 21 SBP regained the pretreatment value (+2% vs. baseline), and HR also recovered (-14% vs. baseline). Binding studies were performed on cerebral tissues: no differences were observed between treated and control rats in number and affinity of either A1 and A2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of adenosine derivatives on human and rabbit platelet aggregation. Correlation of adenosine receptor affinities and antiaggregatory activity.

The inhibitory effects of several adenosine analogues, including the new A2-selective agonists 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamido-adenosi ne (CGS 21680) and 2-hexynyl-5'-N-ethylcarbox-amidoadenosine (2-hexynyl-NECA), were investigated in vitro on human and rabbit platelet aggregation. The compounds examined inhibited ADP-induced platelet aggregation over a wide range of potency. The rank order of activity was similar between the two species thus showing that the rabbit is a useful animal model for studying the effects of adenosine derivatives on platelet aggregation. 2-Hexynyl-NECA was found to be the most potent adenosine compound of those currently available, having IC50 values of 0.10 and 0.07 microM in human and rabbit platelets, respectively. Conversely, the A1 agonists R(-)-N6-(2-phenylisopropyl) adenosine (R-PIA), S(+)-N6-(2-phenylisopropyl) adenosine (S-PIA) and 2-chloro-N6-cyclopentyladenosine (CCPA) were the least potent compounds with IC50 values in the micromolar range. The potency of the compounds in inhibiting platelet aggregation was found to be highly correlated with their affinity for A2 receptors as measured using 3H-CGS 21680 binding in rat brain striatum.