Isothiocyanatobenzyl imidazoline (IBI), the 4'-NCS analogue of tolazoline, has been used to alkylate several receptor sites in rabbit iris muscles. Because of the high affinity of tolazoline for the I2-imidazoline binding sites (Ki = 16-130 nM), this study was designed to assess whether IBI is also an alkylating agent for these sites. In competition studies, IBI displayed moderate affinity (Ki approximately 2-3 microM) against I2A-imidazoline sites in the rabbit cerebral cortex and I2B-imidazoline sites in the rat cerebral cortex labelled by [3H]2-(2-benzofuranyl)-2-imidazoline ([3H]2-BFI). However, preincubation (30 min at 25 degrees C) of rat cortical and liver membranes with IBI (10(-7) M to 10(-3) M), followed by extensive washing, markedly decreased (17% to 96%) the specific binding of [3H]2-BFI to I2B-imidazoline sites. IBI (10(-5) M to 10(-3) M) also bound irreversibly to I2A-imidazoline sites in rabbit cerebral cortex but with a lesser efficacy (27% to 83% reduction of [3H]2-BFI binding). Saturation curves of [3H]2-BFI binding in the rat cerebral cortex indicated that preincubation with 10(-6) M IBI reduced the total density (Bmax) without affecting the affinity (Kd) of I2B-imidazoline sites for IBI. Acute treatments (6 h) with IBI (10 and 30 mg/kg, i.p.) also dose-dependently reduced (26% and 41%; respectively) the total density of I2B-imidazoline sites. These results demonstrate the ability of IBI to alkylate I2-imidazoline binding sites in vitro and in vivo and provide evidence for the use of IBI as a new tool for the study of the functional implications of imidazoline binding sites.
It is known that the steric requirements for the interactions of catecholamines and catecholimidazolines with alpha 1- and alpha 2-adrenoceptors are different. New analogues of desoxycatecholimidazoline (1), desoxycatecholimidazole (3), benzylic hydroxyl substituted imidazole (4), and the aromatic fluorine substitution analogues of 1 at the 2 (5), 5 (6), and 6 (7) positions, and a set of asymmetric 4-substituted catecholimidazolines, S-8 and R-8, were prepared and tested for interaction with alpha 2-adrenoceptors in human platelets. With the exception of 3, all compounds were selective for alpha-adrenoceptor-mediated responses in human platelets. Introduction of a double bond in imidazoline 1 to give an imidazole 3 or the introduction of a benzylic hydroxyl group to 3, as in 4, reduced the inhibition of platelet aggregation with a rank order potency of 1 greater than 3 greater than 4. Fluorine atom substitution at the 2-, 5-, or 6-positions only slightly modified the inhibitory activity of 1. Each analogue (1, 3-7) produced alpha 2-mediated inhibition of platelet adenylate cyclase and can be classified as a partial agonist. The inhibition potency of S-8 and R-8 against epinephrine-induced aggregatory responses were greatly different, and only R-8 and 4 were alpha 2-agonists on human platelet function. Our studies provide further evidence for the differential interaction of catecholamines and catecholimidazolines in alpha 1- and alpha 2-adrenoceptor systems.
The synthesis and biological evaluation of 7,8-dihydroxy (2) and 7,8-methylenedioxy (3) analogues of 1-[(3,4,5-trimethyoxyphenyl)methyl]-2,3,4,5-tetradhyo-1H-2-b enzazepine on beta-adrenoceptor systems and human platelets were undertaken and compared with trimetoquinol (TMQ, 1). Whereas 1 is a potent beta-adrenoceptor agonist in guinea pig atria and trachea (pD2 = 8.2), analogue 2 was marginally effective at relaxing guinea pig tracheal smooth muscle (pD2 = 4.4) and inactive as an agonist on guinea pig atria. Analogues 2 and 3 were inhibitors of phospholipase C (PLC; from Clostridium perfringens) induced and secondary wave of ADP-induced aggregation responses and inactive against low-dose thrombin-induced or stable endoperoxide (U46619) induced human platelet aggregation. Against ADP-induced serotonin secretion, 3 was 9-fold more active than analogue 2. Further, the rank order of TMQ isomers and 3 as inhibitors of PLC-induced platelet aggregation, serotonin secretion, and phosphatidylinositol degradation was identical (3 greater than (S)-(-)-1 greater than (R)-(+)-1). The results suggest that these compounds are blocking the action of PLC by interfering with phosphatidylinositol turnover in platelet membranes. The inhibition of ADP-induced responses in human platelets by analogues 2 and 3 also suggests a site of inhibition at a level of arachidonic acid release. Thus, ring expansion of 1 as in the benzazepine analogues 2 and 3 has allowed us to develop selective inhibitors of platelet function that lack significant beta-adrenoceptor activity.
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