Tröger's base. An alternate synthesis and a structural analog with thromboxane A2 synthetase inhibitory activity.

The synthesis of 2,8-dimethyl-6H,12H-5,11-methanodibenzo[b,f][1,5]diazocine (Tröger's base) from p-toluidine and of two Tröger's base analogs from other anilines by reaction with hexamethylenetetramine in trifluoroacetic acid is described. 2,3,6,7-Tetrahydro-9-methyl-2,6-di-p-tolyl-1H,5H-pyrimido[5,6,1-ij] quinazoline is formed as a secondary product in the reaction of p-toluidine and hexamethylenetetramine. One of the Tröger's base analogs, 2,8-bis(3'-pyridylmethyl)-6H,12H-5,11-methanodibenzo[b,f][1,5]d iazocine (5), is an effective inhibitor of the enzyme, thromboxane A2 (TxA2) synthase, with an ED50 of 30 ng/mL in a specified in vitro assay. Three analogs having substituents on the bridging methylene group of the bicyclic nucleus of the Tröger's base structure were prepared, but all were considerably less active than the aforementioned compound in the inhibition assay. The structures of these inhibitors of TxA2 synthase fall outside the classical structure-activity relationship that has been established for this class of enzyme inhibitors.

Triazolobenzodiazepines: a new class of stimulators of tissue-type plasminogen activator synthesis in human endothelial cells.

In our search for compounds that can stimulate endogenous fibrinolysis, we have found that certain triazolobenzodiazepines enhance the production of tissue-type plasminogen activator (t-PA) by vascular endothelial cells maintained in vitro, with no or even a lowering effect on plasminogen activator inhibitor type-1 (PAI-1) production. The most active compounds tested, U-34599, U-46195 and U-51477, were studied in more detail and showed a time- and dose-dependent increase in the production of t-PA by human umbilical vein endothelial cells. At optimal stimulatory concentrations (about 10 microM), the three compounds stimulated t-PA expression about 2-fold after 24 hr and maximally about 4-fold after 48 hr of incubation; this maximal increase in t-PA synthesis was sustained at prolonged incubations of 72 or 96 hr. The triazolobenzodiazepine effects on t-PA production were accompanied by parallel increases in t-PA mRNA levels, without marked changes in PAI-1 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA concentrations. Numerous analogues of the three lead compounds were then tested to determine the relationship between benzodiazepine structure and the ability to stimulate t-PA production. No positive correlation was found between the ability of the various triazolobenzodiazepines to stimulate t-PA production and their affinity for the benzodiazepine receptor. In agreement with this, no specific binding of [3H]flunitrazepam, a specific ligand for benzodiazepine receptors, to endothelial cell membrane preparations was observed. Thus, it is unlikely that the triazolobenzodiazepines act through central-type benzodiazepine receptors to stimulate t-PA production. Similarly, no evidence was found for the presence of peripheral-type benzodiazepine receptors on endothelial cell membranes. The ability of the benzodiazepines to stimulate t-PA production, however, appeared to be related to their platelet-activating factor (PAF) antagonist activity. Despite this finding, several non-benzodiazepine PAF antagonists did not stimulate t-PA production. While the precise mechanism of action is not yet clear, selected benzodiazepine analogues possessing PAF antagonist activity stimulate the production of t-PA by endothelial cells in vitro.

Effects of low doses of aspirin and dipyridamole on platelet aggregation in the dog coronary artery.

The circumflex coronary artery of pentobarbital-anesthetized dogs was partially obstructed with an externally applied rigid plastic band. Platelet aggregation at the site of stenosis caused a gradual decline in blood flow in the artery, which was monitored with an electromagnetic flow probe placed proximally to the obstructor. The effects of drugs on platelet aggregation were evaluated by monitoring changes in both the rate and the degree of decline in blood flow. In most dogs, aspirin inhibited intravascular platelet aggregation (ED50 = 1 mg/kg). Dipyridamole, even at doses that severely depressed blood pressure (1 mg/kg), had no effect on platelet aggregation. However, in dogs that had been pretreated with a low dose of dipyridamole (0.2 mg/kg), the antiaggregatory activity of aspirin was enhanced. This potentiation was evident only at low doses of aspirin (0.03 and 0.1 mg/kg), where the drug was 10 times more active; at high aspirin doses, which depressed vascular cyclooxygenase, no potentiation was seen. Further evidence that the mechanism of this synergism may depend on endogenous prostacyclin production at the site of the partial obstruction was seen when cyclooxygenase inhibitors applied topically on the exposed artery eliminated the antiaggregatory effect of low doses of aspirin. It is important to note that the protective effect of dipyridamole and low-dose aspirin was less than that seen at the high doses of aspirin alone, suggesting that the theoretical benefits of platelet-specific doses of aspirin may be overstated.

Thromboxane A2 synthase inhibitors. 5-(3-Pyridylmethyl)benzofuran-2-carboxylic acids.

The synthesis and screening of a series of 5-(3-pyridylmethyl)benzofuran-2-carboxylic acids as selective thromboxane A2 (TxA2) synthase inhibitors is outlined. The ability of these compounds to inhibit TxA2 biosynthesis was assayed using microsomal enzyme from human platelets. Substitution of the benzofuran ring caused small changes in potency; modification of the carboxylic acid group caused modest reductions in potency, and substitution of the pyridine ring resulted in large reductions of potency. 5-(3-Pyridylmethyl)benzofuran-2-carboxylic acid sodium salt (9b, sodium furegrelate) was chosen for further evaluation as a TxA2 synthase inhibitor.

Effect of pulmonary and renal circulations on activity of atrial natriuretic factor.

The effect of passage through the pulmonary and renal circulation on the activity of extracted and synthetic atrial natriuretic factors was determined by monitoring the amount of the vascular relaxing activity surviving passage through the organ. When crude atrial extract was infused through isolated perfused lungs of the guinea pig, approximately 75% of its activity survived. The activity of a 23-amino acid synthetic atrial peptide was decreased 21% on passage through these lungs, but this loss of activity was not significantly different from the crude extract. Analogous experiments in vivo showed that passage through the pulmonary circulation of the dog did not alter the activity of either the crude atrial extract or of synthetic atrial peptide. In contrast to the lack of effect of the pulmonary circulation, approximately 80% of the activity of crude atrial extract and synthetic atrial peptide was removed by the isolated perfused kidney of the rabbit, and in vivo the dog kidney removed approximately 80% of the activity of both these atrial substances. By surviving transit through the pulmonary circulation, these materials exhibit a necessary property of a circulating hormone stored in the right atrium, and the fact that the renal circulation extracts most of these materials is consistent with the kidney being a target organ of this putative hormone.

Prostaglandins.

A discussion of important aspects of the effects of prostaglandins on arteries must include (a) the nature of the receptors for the different humoral agents, (b) the potential modulatory role that endogenously produced prostaglandins may play when other vasoactive hormones are present, and (c) the potential for cell-to-cell transfer of substrates for formation of novel biologically active arachidonic acid metabolites. Evidence suggests that at least two vasodilator receptors for prostaglandins exist (PGE2 and PGI2) and at least one vasoconstrictor receptor (TXA2), probably more. Activation of vasodilator mechanisms by endogenously produced vasodilator prostaglandins in isolated arteries or in vivo can attenuate the vasoconstrictor activity of angiotensin II and in some cases mediate the vasodilator effects of bradykinin. Perhaps the most important source of vasoactive arachidonic acid metabolites may be the interaction of blood cells with the vessel wall. Platelets produce thromboxane (TXA2), which has vasoconstrictor effects at 10(-8) M in vitro. Diversion of the precursor from the platelet by selective inhibition of thromboxane synthetase can provide substrate for vascular PGI2 synthesis. Other interactions between leukocytes, platelets, endothelium cells, and smooth-muscle cells are likely to reveal further pathways for the formation of vasoactive arachidonic acid metabolites.

Pyrimidine and triazine 3-oxide sulfates: a new family of vasodilators.

Di- and triaminopyrimidine 3-oxides (e.g., 2,4-diamino-6-piperidinylpyrimidine 3-oxide and 2,4-diamino-6-(diallylamino)triazine 3-oxide) react with sources of sulfur trioxide, such as sulfur trioxide trimethylamine or chlorosulfuryl chloride, to yield the corresponding heterocyclic O-sulfates. These sulfates are inner salts with unusual physical properties. The structure of the O-sulfate of 2,4-diamino-6-piperidinylpyrimidine 3-oxide was confirmed by X-ray. These O-sulfates are hypotensives. They apparently act by direct vasodilation.

Inhibition of platelet thromboxane A2 synthase activity by sodium 5-(3'-pyridinylmethyl)benzofuran-2-carboxylate.

This report outlines the activity of a new thromboxane synthase inhibitor sodium, 5-(3-pyridinylmethyl)-2-benzofurancarboxylate, (U-63557A). U-63557A is a potent inhibitor of the thromboxane synthase in human platelets in vitro, as well as in rhesus monkey platelets ex vivo. A single oral dose of 3.0 mg/kg U-63557A inhibits the platelet thromboxane synthase in rhesus monkeys approximately 80% for at least 12 hrs. U-63557A has been administered to monkeys twice a day, (10 mg/kg) for 14 days, without evidence of drug tachyphylaxis or rebound. U-63557A does not inhibit thrombin-stimulated PGI2 biosynthesis in human endothelial cells, the 5-lipoxygenase in human neutrophils, or the cyclo-oxygenase in a variety of test systems. In anesthetized dogs, U-63557A injected i.v. at 0.1 to 5 mg/kg prevented the blockage of stenosed coronary arteries caused platelet aggregation. Similar effects were obtained by oral administration of 1-5 mg/kg. The thromboxane synthase inhibitor was more efficacious than cyclooxygenase inhibitors and equal to PGI2 in efficacy. Under appropriate conditions the protective effects of U-63557A could be reversed by i.v. cyclooxygenase inhibitors suggesting that its efficacy depended in part on endogenous PGI2 formation. Due to its specificity, oral activity, and extended duration of action, U-63557A is a promising compound for the evaluation of the role of thromboxane synthase in a variety of pathophysiological states.

Actions of indomethacin and prostaglandins E2 and D2 on nerve transmission in the nictitating membrane of the cat.

The effects of PGE2, PGD2 and cyclooxygenase inhibitors on sympathetic nerve transmission in the nictitating membrane of anesthetized cats were studied to further characterize the actions of these prostaglandins and to define their possible role as neuromodulators. PGD2 depressed neurotransmission throughout a broad range of stimulus free quencies, apparently by presynaptic inhibition of norepinephrine release. PGE2 enhanced the effects of both nerve stimulation and exogenous norepinephrine in intact preparations but only depressed the effects of exogenous norepinephrine in the chronically denervated nictitating membrane, suggesting that part of the effect of PGE2 on neurotransmission was presynaptic. When norepinephrine reuptake was blocked by desipramine, PGE2 still enhanced neurotransmission. Indomethacin, but not other cyclooxygenase inhibitors (aspirin, ibuprofen, flurbiprofen, meclofenamic acid), inhibited the response of the nictitating membrane to nerve stimulation without depressing the effects of exogenous norepinephine. Curiously, indomethacin, but again not other cyclooxygenase inhibitors, specifically antagonized the ability of PGE2 to enhance nerve transmission. These results further characterize the pharmacological effects of PGE2 and PGD2 at the nictitating membrane. The lack of effect of cyclooxygenase inhibitors suggests that neither endogenous PGE2 or PGD2 play a functional role at this synapse. The effects of indomethacin appear to be unrelated to inhibition of prostaglandin synthesis.

Analysis of the biological activity of azoprostanoids in human platelets.

A structure-activity analysis of seven different azoprostanoids in human platelets and rat aortas has shown that discrete changes in the prostanoid skeleton induce marked changes in biological activity. The 15S-hydroxyl group is important for agonist activity in the platelet (pro-aggregatory) and in the aorta (vasoconstriction). Removal of the 15S-hydroxyl results in a compound that inhibits the thromboxane synthetase and platelet aggregation, and is a much weaker constrictor of the aorta. The 13,14 double bond is more important for agonist activity on the aorta than the 5,6 double bond of the prostanoid skeleton. Completely saturated molecules that are 15-deoxy still retain considerable activity on the aorta, but are two orders of magnitude less active as inhibitors of the thromboxane synthetase and aggregation than the azoprostanoid with both 5,6 and 13,14 double bonds. Compounds that exhibit no agonist activity in platelets still retain considerable agonist activity on the aorta, suggesting that the thromboxane A2 receptor in platelets may be different from the thromboxane A2 receptor in blood vessels.

Effects of prostaglandin D3 on nerve transmission in nictitating membrane of cats.

In anesthetized cats, intra-arterial injection of PGD3 toward the nictitating membrane caused long-lasting, dose-related decreases in the response of the nictitating membrane to sympathetic nerve stimulation. During peak depression of nerve transmission the response of the nictitating membrane to intra-arterial norepinephrine was not depressed suggesting that PGD3 suppressed the release of norepinephrine. PGD3 was as potent as PGD2 for modulating sympathetic nerve transmission but was less effective in activating a vagally mediated bradycardia. The results show that the PGD3 can modulate autonomic nerve transmission.

Angiotensin II stimulation of renal prostaglandin synthesis elevates circulating prostacyclin in the dog.

The ability of the kidney to release prostacyclin (PGI2) under direct stimulation with exogenous angiotensin II was studied in the pentobarbital-anesthetized dog. We assayed for prostacyclin-like activity in systemic arterial blood by continuously monitoring platelet aggregation in vivo and with the blood-bathed bovine coronary artery, which relaxes to prostacyclin. This parallel bioassay allows detection of small amounts of prostacyclin-like activity released into the systemic circulation. Angiotensin II, infused at a rate of 10 or 20 ng/kg/min into the renal artery inhibited latelet aggregation in 8 out of 18 dogs, relaxed the bovine coronary artery in 13 of 16 dogs, and lowered systemic arterial blood pressure 5-10 mm Hg in all but 3 animals. These effects of antiotensin II could be blocked by treating the animal with indomethacin (2 mg/kg, i.v.) and mimicked by administration of exogenous prostacyclin. Infusion of the same dose of angiotensin II (10 or 20 ng/kg/min) intravenously did not release prostacyclin; blood pressure increased during intravenous infusion, and platelet aggregation remained unchaged. These data are consistent with the hypothesis that prostacyclin, of renal origin in these experiments, can act as a circulating hormone. Prostacyclin should not be regarded only as an antiaggregatory substance, but also as a potentially important modulator of blood pressure.

Modulation of autonomic neurotransmission by PGD2: comparison with effects of other prostaglandins in anesthetized cats.

Experiments with anesthetized cats were done to study possible roles of different prostaglandins (PGs) in modulating sympathetic neuroeffector transmission. We recorded contractions of the nictitating membrane (n.m.), blood flow in the carotid artery, heart rate and blood pressure, both under control conditions and while stimulating the cut cervical sympathetic nerve. Intra-carotid arterial injection (i.a.) of PGD2 depressed sympathetic transmission to the n.m. without depressing the effects of exogenous norepinephrine (NE). In contrast, PGE2 enhanced the effects of nerve transmission or exogenous NE on the stimulated n.m. PGI2 had similar but shorter effects to PGE2. PGF2 alpha or a stable PGH2 analog, contracted the n.m. smooth muscle with no detected effect on nerve transmission. Carotid blood flow was increased by PGD2, PGE2 and PGI2. PGD2 and PGI2 caused bradycardia that could be blocked by atropine. This ability of PGD2 to modulate autonomic nerve activity is of particular interest because of recent reports that nerve tissue synthesizes PGD2.

6-keto-prostaglandin E1 is not equipotent to prostacyclin (PGI2) as an antiaggregatory agent.

A direct comparison of the relative potencies of the two antiaggregatory prostaglandins PGI2 and 6-keto-PGE1 showed PGI2 was at least 20 times more potent than 6-keto-PGE1 when tested against ADP-induced human platelet aggregation. This marked difference in potency was even more evident when the ability of PGI2 and 6-keto-PGI2 to stimulate platelet cyclic AMP levels was determined. When cyclic AMP levels were measured direct comparisons were difficult because the respective dose response curves were not parallel, but 10 ng of PGI2 was equivalent to 300 ng of 6-keto-PGE1. PGI2 was also more potent (10-20 times) than 6-keto-PGE1 as a disaggregatory agent, and the disaggregatory activity of both prostaglandins was enhanced by the phosphodiesterase inhibitor 1-methyl-3-isobutylmethylxanthine. PGI2 was also more active than 6-keto-PGE1 as an inhibitor of thrombus formation in dog coronary arteries in vivo. In vivo, 6-keto-PGE1 was at least 10 times less potent thatn PGI2, the exact difference could not be determined because 6-keto-PGE1 caused significant falls in blood pressure before anti-platelet activity could be detected. PGI2 is an intrinsically more potent anti-aggregatory molecule than 6-keto-PGE1, but these data do not rule out the possibility that some of the activities attributed to PGI2 could be the result of the conversin of PGI2 and/or 6-keto-PGF1 alpha to 6-keto-PGE1.