Synthesis and human beta-adrenoceptor activity of 1-(3,5-diiodo-4- methoxybenzyl)-1,2,3,4-tetrahydroisoquinolin-6-ol derivatives in vitro.

Trimetoquinol (1, TMQ) is a potent nonselective beta-adrenergic receptor (AR) agonist and a thromboxane A(2)/prostaglandin endoperoxide (TP) receptor antagonist, while 3',5'-diiodo-TMQ (2) exhibits beta(3)-AR selectivity. In search of selective beta(3)-AR agonists as potential drugs for the treatment of human obesity and type II diabetes mellitus, a series of 1-(3, 5-diiodo-4-methoxybenzyl)-1,2,3,4-tetrahydroisoquinolin-6-ols has been prepared and evaluated for their biological activities at human beta(1)-, beta(2)-, and beta(3)-ARs expressed in Chinese hamster ovary (CHO) cells. The compounds have been synthesized by the Bischler-Napieralski cyclization of corresponding amides followed by NaBH(4) reduction, and the halogens in the aromatic ring A were introduced by direct halogenation of protected compound 11. Whereas halogen substitution in ring A reduced either potency or intrinsic activity on beta(3)-AR, the non-halogen-substituted compounds 8 and 10 were potent, selective, nearly full agonists for beta(3)-AR.

Beta-adrenoceptor subtype activities of trimetoquinol derivatives: biochemical studies on human beta-adrenoceptors expressed in chinese hamster ovary cells.

The beta-adrenoceptor activities of trimetoquinol (TMQ) isomers and selected derivatives were evaluated on human beta-adrenoceptor subtypes expressed in Chinese hamster ovary cells. In cAMP accumulation assays, (-)-TMQ was 214-, 281-, and 776-fold more potent than (+)-TMQ at stimulating beta(1)-, beta(2)-, and beta(3)-adrenoceptor subtypes, respectively. In radioligand binding assays, (-)-TMQ exhibited 123-, 331-, and 5-fold greater affinity than (+)-TMQ for beta(1)-, beta(2)-, and beta(3)-adrenoceptor subtypes, respectively. (-)-TMQ and (+/-)-TMQ activated the human beta(3)-adrenoceptor with an 8.2- and 3.4-fold greater efficacy, respectively, than the reference beta-adrenoceptor agonist (-)-isoproterenol (efficacy = 1). The 3',5'-diiodo analogs of TMQ were partial agonists of the beta(2)-adrenoceptor relative to (-)-isoproterenol, and their potencies were 5- to 10-fold higher at the beta(3)-adrenoceptor as compared with beta(1)-adrenoceptors. Modification of the catechol (6,7-dihydroxy) nucleus, such as replacement of the 7-hydroxy group with a chloro group (7-chloroTMQ), ring fluorination (8-fluoro and 5,8-difluoro analogs), or preparation of bioisosteric tetrahydrothiazolopyridine (THP) derivatives of TMQ yielded compounds that displayed partial agonist activity (relative to (-)-isoproterenol) or were inactive at the beta(2)-adrenoceptor and exhibited beta(3)-adrenoceptor-selective stimulation compared with the beta(1)-adrenoceptor. Furthermore, the 3',5'-diiodo-4'-methoxybenzylTHP derivative of TMQ was 65-fold more potent than the corresponding 3',4',5'-trimethoxybenzylTHP at the human beta(3)-adrenoceptor. Our results indicate that 6, 7-dihydroxy-catechol-modified and 1-benzyl halogen-substituted derivatives of TMQ represent promising leads for the development of beta(3)-adrenoceptor-selective agonists.

Direct effects of ephedrine isomers on human beta-adrenergic receptor subtypes.

Ephedrine and its alkaloids are used for the treatment of asthma, nasal congestion, and obesity. Ephedrine, with two chiral centers, exists as four isomers that exhibit direct and indirect effects on both alpha- and beta-adrenergic receptors (AR). Our main goal was to study the direct effects of the ephedrine isomers on human beta1-, beta2-, and beta3-AR expressed in Chinese hamster ovary cells. Previous work indicated that the ephedrine isomers are inactive as agonists and that 1R,2S-ephedrine is more potent than the 1S,2R-isomer as an antagonist of catecholamine-induced lipolysis in rat adipose tissue (Lee et al., J Pharmacol Exp Ther 190: 249-259, 1974). Stimulation of adenylyl cyclase, associated with cyclic AMP accumulations, was measured by a luciferase reporter gene assay. On human beta1-AR, the rank order of potency (EC50 values, maximal response relative to isoproterenol = 100%) was 1R,2S-ephedrine (0.5 microM, 68%) > 1S,2R-ephedrine (72 microM, 66%) > 1S,2S-pseudoephedrine (309 microM, 53%) = 1R,2R-pseudoephedrine (1122 microM, 53%). On human beta2-AR, the rank order of potency was 1R,2S-ephedrine (0.36 microM, 78%) > 1R,2R-pseudoephedrine (7 microM, 50%) > or = 1S,2S-pseudoephedrine (10 microM, 47%) > 1S,2R-ephedrine (106 microM, 22%). Only 1R,2S-ephedrine showed significant agonist activity on human beta3-AR with an EC50 = 45 betaM and a maximal response of 31%. Our studies demonstrated that (a) stereoselective and rank order differences exist among the direct effects of ephedrine isomers; (b) 1R,2S-ephedrine is the most potent of the four ephedrine isomers on all three human beta-AR; and (c) 1R,2S- ephedrine was nearly equipotent as a beta1-/beta2-AR agonist and the only isomer possessing weak partial agonist activity on beta3-AR.

An efficient cyclic AMP assay for the functional evaluation of beta-adrenergic receptor ligands.

beta-Adrenoceptors are G-protein linked receptors that are positively coupled to adenylyl cyclase. We wanted to develop an alternative method to the detection of cAMP using radioimmunoassay for functional analysis of ligands for human beta-adrenoceptors (ARs). Chinese hamster ovary cells that stably expressed the beta 2-AR were transiently transfected with reporter plasmids containing the firefly luciferase gene under the transcriptional control of 6 or 12 cAMP response elements. The transiently transfected cells (electroporated at 150 volts, single pulse, 70 ms) were grown in 96-well microtiter plates (50,000 cells per well) for 20 hours, exposed to various ligands for 4 hours, and the luciferase activity was assayed. Stimulation of beta-ARs in these transfected cells resulted in a greater than 25-fold induction of luciferase activity. This activity was maximally increased in response to 20 microM forskolin, 1 mM 3-isobutyl-1-methylxanthine, and 10-30 nM (-)-isoproterenol. Exposure of cells to (-)-isoproterenol elicited a concentration dependent luciferase response and these effects of (-)-isoproterenol were blocked by propranolol (K(B) = 1.5 nM) and bupranolol (K(B) = 1.9 nM). The concentration of (-)-isoproterenol exhibiting half maximal response was 0.35 nM. This assay offers an excellent alternative to traditional methods of cAMP measurement and has applications to elucidate cAMP mediated signaling pathways in cells.

2-Amino-4-benzyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridines: novel selective beta3-adrenoceptor agonists.

Trimetoquinol (TMQ, 7) is a potent nonselective beta-adrenoceptor (AR) agonist. Replacement of the catechol moiety of TMQ with a 2-aminothiazole group resulted in novel thiazolopyridine derivatives 9-11 which have been synthesized and evaluated for biological activity on human beta1-, beta2-, and beta3-AR. The Bischler-Napieralski reaction has been employed as a novel approach to construct the 2-amino-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine ring system. Although in radioligand binding studies analogues 9 and 10 did not show selectivity toward beta3-AR, they exhibited a high degree of selective beta3-AR agonist activity in functional assays. Moreover, the beta3-AR agonist activity of the 2-aminothiazole derivatives is abolished by N-acetylation (analogue 11) or ring opening (analogue 25). This illustrates the importance of the intact 2-amino-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine ring for beta3-AR activity.

Toxicological evaluation of constructed wetland habitat sediments utilizing Hyalella azteca 10-day sediment toxicity test and bacterial bioluminescence.

A toxicological evaluation was conducted on wetland habitats created as a result of run-off from agricultural areas. These temporary wetlands were created by using drop pipes as a means of reducing erosional cutting in agricultural fields. Toxicity bioassays utilizing bacterial bioluminescence and Hyalella azteca were used to assess sediment pore water and whole sediment, respectively. Inhibition of bacterial bioluminescence was initially used to determine relative toxicities of pore water from ten wetland sites. Constructed wetland sites were compared to the University of Mississippi Biological Field Station, a relatively pristine reference site. The H. azteca ten day sediment toxicity test was utilized to assess sediment from four selected sites using survival and growth as toxicological endpoints. Results from the toxicological evaluation, along with extensive ecological evaluations, were used to assess the best approach for implementation of temporary wetland habitats with existing agricultural practices.