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The properties of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) as an antagonist ligand for A1 adenosine receptors were examined and compared with other radioligands for this receptor. DPCPX competitively antagonized both the inhibition of adenylate cyclase activity via A1 adenosine receptors and the stimulation via A2 adenosine receptors. The Ki-values of this antagonism were 0.45 nM at the A1 receptor of rat fat cells, and 330 nM at the A2 receptor of human platelets, giving a more than 700-fold A1-selectivity. A similar A1-selectivity was determined in radioligand binding studies. Even at high concentrations, DPCPX did not significantly inhibit the soluble cAMP-phosphodiesterase activity of human platelets. [3H]DPCPX (105 Ci/mmol) bound in a saturable manner with high affinity to A1 receptors in membranes of bovine brain and heart, and rat brain and fat cells (KD-values 50-190 pM). Its nonspecific binding was about 1% of total at KD, except in bovine myocardial membranes (about 10%). Binding studies with bovine myocardial membranes allowed the analysis of both the high and low agonist affinity states of this receptor in a tissue with low receptor density. The binding properties of [3H]DPCPX appear superior to those of other agonist and antagonist radioligands for the A1 receptor.

Because protection against infarction afforded by ischaemic preconditioning, adenosine, or the A3 receptor agonist APNEA could not be blocked by DPCPX and because the potent A3 receptor antagonist BW A1433 blocked protection from ischaemic preconditioning, these data indicate that the protection of preconditioning is not exclusively mediated by the adenosine A1 receptor in rabbit heart and could involve the A3 receptor.

The purpose of this investigation was to quantitatively evaluate the role of adenosine in coronary exercise hyperemia. Dogs (n = 10) were chronically instrumented with catheters in the aorta and coronary sinus, and a flow probe on the circumflex coronary artery. Cardiac interstitial adenosine concentration was estimated from arterial and coronary venous plasma concentrations using a previously tested mathematical model. Coronary blood flow, myocardial oxygen consumption, heart rate, and aortic pressure were measured at rest and during graded treadmill exercise with and without adenosine receptor blockade with either 8-phenyltheophylline (8-PT) or 8-p-sulfophenyltheophylline (8-PST). In control vehicle dogs, exercise increased myocardial oxygen consumption 4.2-fold, coronary blood flow 3.8-fold, and heart rate 2.5-fold, whereas mean aortic pressure was unchanged. Coronary venous plasma adenosine concentration was little changed with exercise, and the estimated interstitial adenosine concentration remained well below the threshold for coronary vasodilation. Adenosine receptor blockade did not significantly alter myocardial oxygen consumption or coronary blood flow at rest or during exercise. Coronary venous and estimated interstitial adenosine concentration did not increase to overcome the receptor blockade with either 8-PT or 8-PST as would be predicted if adenosine were part of a high-gain, negative-feedback, local metabolic control mechanism. These results demonstrate that adenosine is not responsible for local metabolic control of coronary blood flow in dogs during exercise.

2-Amino-3-benzoylthiophenes are allosteric enhancers (AE) of agonist activity at the A(1) adenosine receptor. The present report describes syntheses and assays of the AE activity at the human A(1)AR (hA(1)AR) of a panel of compounds consisting of nine 2-amino-3-aroylthiophenes (3a-i), eight 2-amino-3-benzoyl-4,5-dimethylthiophenes (12a-h), three 3-aroyl-2-carboxy-4,5-dimethylthiophenes (15a-c), 10 2-amino-3-benzoyl-5,6-dihydro-4H-cyclopenta[b]thiophenes (17a-j), 14 2-amino-3-benzoyl-4,5,6,7-tetrahydrobenzo[b]thiophenes (18a-n), and 15 2-amino-3-benzoyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophenes (19a-o). An in vitro assay employing the A(1)AR agonist [(125)I]ABA and membranes from CHO-K1 cells stably expressing the hA(1)AR measured, as an index of AE activity, the ability of a candidate AE to stabilize the agonist-A(1)AR-G protein ternary complex. Compounds 3a-i had little or no AE activity, and compounds 12a-h had only modest activity, evidence that AE activity depended absolutely on the presence of at least a methyl group at C-4 and C-5. Compounds 17a-c lacked AE activity, suggesting the 2-amino group is essential. Polymethylene bridges linked thiophene C-4 and C-5 of compounds 17a-j, 18a-n, and 19a-o. AE activity increased with the size of the -(CH(2))(n)()- bridge, n = 3 < n = 4 < n = 5. The 3-carbethoxy substituents of 17a, 18a, and 19a did not support AE activity, but a 3-aroyl group did. Bulky (or hydrophobic) substituents at the meta and para positions of the 3-benzoyl group and also 3-naphthoyl groups greatly enhanced activity. Thus, the hA(1)AR contains an allosteric binding site able to accommodate 3-aroyl substituents that are bulky and/or hydrophobic but not necessarily planar. A second region in the allosteric binding site interacts constructively with alkyl substituents at thiophene C-4 and/or C-5.

2-Amino-3-aroylthiophenes are agonist allosteric enhancers (AE) at the A(1) adenosine receptor (A(1)AR). Here we report the syntheses of three kinds of novel 2-aminothiophenes and assays of their AE activity at the human A(1)AR (hA(1)AR), namely, (1) 2-amino-4,5-diphenylthiophene-3-carboxylates, 3a-h, (2) 2-amino-3-benzoyl-4,5-diphenylthiophenes, 7a-p, and (3) 2-amino-5-bromo-3-benzoyl-4-phenylthiophenes, 10a-h. An in vitro assay employing the A(1)AR agonist [(125)I]ABA and membranes from CHO-K1 cells stably expressing the hA(1)AR measured an index of AE activity, the ability of a candidate AE to stabilize the agonist-A(1)AR-G protein ternary complex, scored as the percentage of ternary complex remaining after 10 min of dissociation initiated by CPX and GTPgammaS. The AE activity score of 2-amino-4,5-dimethyl-3-(3-trifluoromethylbenzoyl)thiophene (PD 81,723), which was 19%, served as a standard for comparison. Two 3-carboxythiophene 3-trifluoromethylbenzyl esters, 3d (49%) and 3f (63%), had substantial AE activity. The 3-(1-naphthoyl) substituent of 7e (52%) also supported AE activity. Compounds in series 3 tended to be more potent, 10a and 10c having scores of 91 and 80%, respectively. The activity of 2-amino-5-bromo-3-ethoxycarbonyl-4-(3-nitrophenyl)thiophene, 10h (26%), is an exception to the rule that a 3-ethoxycarbonyl substituent cannot support AE activity.

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