Biochemical and functional characterization of 1-benzyl substituted trimetoquinol affinity analogs on rat and human β-adrenoceptors

Abstract

The site of interaction for the 1-(3′,4′,5′-trimethoxybenzyl) group of trimetoquinol (TMQ) with β-adrenoceptors (β-ARs) is important for the rational design of highly potent and β 3-AR-selective analogs. 1-Benzyl ring-substituted TMQ analogs were evaluated for binding affinities and biochemical activities (cyclic AMP accumulations) in Chinese hamster ovary (CHO) cells expressing the rat and human β 3-AR, and for functional activities on isolated rat tissues. Binding affinities ( K i ≈ 0.055 to 1.5 μM) for the rat β 3-AR and potencies for adenylyl cyclase activation ( K act ≈ 0.43 to 2.5 nM) of the 3′-monoiodo or 3′,5′-diiodo derivatives with 4′-isothiocyanato-, 4′-amino, 4′-acetamido, or 4′-α-haloacetamido substitutions were higher than those of (-)-isoproterenol, and comparable to those of BRL 37344 [(±)-( R∗, R∗-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]-acetic acid sodium]. A similar rank order of binding affinities ( K i ≈ 0.11 to 2.5 μM) and potencies ( K act ≈ 0.45 to 9.5 nM) was obtained for TMQ analogs on the human β 3-AR. The 4′-acetamido and 4′-α-chloroacetamido analogs of 3′,5′-diiodoTMQ were more potent than (-)-isoproterenol in rat atria (β 1-AR) and rat trachea (β 2-AR) and exhibited partial agonist activities, whereas full agonist activities were observed in rat esophageal smooth muscle ( ec 50 ≈ 2–8 nM, β 3-AR). 4′-α-Chloroacetamido-3′,5′-diiodoTMQ-mediated chronotropic responses in atria were sustained and resistant to washout. Further, the 4′-α-chloroacetamido and 4′-α-bromoacetamido analogs of 3′,5′-diiodoTMQ demonstrated significant concentration-dependent irreversible binding to the rat β 3-AR. Reversible β-AR agonists such as (-)-isoproterenol, BRL 37344, and 4′-acetamido-3′,5′-diiodoTMQ or nucleophilic l-amino acids (lysine, glutathione, cysteine) did not protect against this irreversible binding. Thus, the lipophilic 1-benzyl ring of TMQ analogs interacts with a hydrophobic region of the β-AR that may represent an exo-site or an allosteric binding site.