Biochemical activities of trimetoquinol analogs at human beta(1)- and beta(3)-adrenergic receptors.

The biochemical activities of trimetoquinol (TMQ) analogs were evaluated at the human beta(1)- and beta(3)-adrenergic receptor (AR) subtypes expressed in Chinese hamster ovary cells. In radioligand binding assays, the 1-benzyl iodine-substituted analogs exhibited higher binding affinities at both beta(1)- and beta(3)-AR subtype as compared to TMQ. In cAMP accumulation assays, these analogs exhibited high potencies at both beta(1)- and beta(3)-AR. The 3',5'-diiodo-4'-amino analog of TMQ was the most potent beta(3)-AR agonist, 17-fold more potent at the beta(3)-AR versus the beta(1)-AR. Masking of the 6,7-dihydroxy group of the catechol ring of 3',5'-diiodo-4'-acetamido analog of TMQ, a potent beta(1)- and beta(3)-AR agonist, abolished activity at both beta-AR subtypes. Furthermore, substitution of a strong electron withdrawing group such as the trifluoromethyl moiety at the 1-benzyl ring of TMQ dramatically decreased potency at beta(1)- and beta(3)-AR compared to TMQ. Replacement of the 1-benzyl ring of TMQ with a naphthalene ring did not alter affinity but reduced potency of resulting 1-naphthylmethyl and 2-naphthylmethyl analogs at beta(1)- and beta(3)-AR compared to TMQ. Our results define the structural and electronic properties of substituents on TMQ necessary for potent activation of beta(1)- and beta(3)-AR and suggest that further modifications of the 1-benzyl iodine-substituted analogs may yield potent beta(3)-AR agonists.

Activation of peroxisome proliferator-activated receptor isoforms and inhibition of prostaglandin H(2) synthases by ibuprofen, naproxen, and indomethacin.

A series of nonsteroidal anti-inflammatory drugs (NSAIDs) [S(+)-naproxen, ibuprofen isomers, and indomethacin] were evaluated for their activation of peroxisome proliferator-activated receptor (PPAR) alpha and gamma isoforms in CV-1 cells co-transfected with rat PPAR alpha and gamma, and peroxisome proliferator response element (PPRE)-luciferase reporter gene plasmids, for stimulation of peroxisomal fatty acyl CoA beta-oxidase activity in H4IIEC3 cells, and for comparative inhibition of ovine prostaglandin endoperoxide H synthase (PGHS)-1 and PGHS-2 and arachidonic acid-induced human platelet activation. Each drug produced a concentration-dependent activation of the PPAR isoforms and fatty acid beta-oxidase activity, inhibition of human arachidonic acid-induced platelet aggregation and serotonin secretion, and inhibition of PGHS-1 and PGHS-2 activities. For PPARalpha activation in CV-1 and H4IIEC3 cells, and the stimulation of fatty acyl oxidase activity in H4IIEC3 cells, the rank order of stereoselectivity was S(+)- ibuprofen > R(-)-ibuprofen; S(+)-ibuprofen was more potent than indomethacin and naproxen on these parameters. On PPARgamma, the rank order was S(+)-naproxen > indomethacin > S(+)-ibuprofen > R(-)-ibuprofen. Each drug inhibited PGHS-1 activity and platelet aggregation with the same rank order of indomethacin > S(+)-ibuprofen > S(+)-naproxen > R(-)-ibuprofen. Notably, the S(+)-isomer of ibuprofen was 32-, 41-, and 96-fold more potent than the R(-)-isomer for the inhibition of PGHS-1 activity, human platelet aggregation, and serotonin secretion, respectively. On PGHS-2, the ibuprofen isomers showed no selectivity, and indomethacin, S(+)-ibuprofen, and S(+)-naproxen were 6-, 27-, and 5-fold more potent as inhibitors of PGHS-1 than PGHS-2 activity. These results demonstrate that the mechanisms of action of NSAIDs on these cell systems are different, and we propose that the pharmacological effects of NSAIDs may be related to both their profile of inhibition of PGHS enzymes and the activation of PPARalpha and/or PPARgamma isoforms.

Yohimbine dimers exhibiting binding selectivities for human alpha2a- versus alpha2b-adrenergic receptors.

A series of yohimbine dimers was prepared and evaluated at the human alpha2a- and alpha2b-adrenergic receptors (ARs) expressed in Chinese hamster ovary (CHO) cells. All dimers display higher binding selectivities for alpha2a versus alpha2b subtype than yohimbine, and four compounds (3d, 3e, 3g and 3i) represent the most potent and alpha2a versus alpha2b-AR selective ligands identified so far.

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.

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

The site of interaction for the 1-(3',4',5'-trimethoxybenzyl) group of trimetoquinol (TMQ) with beta-adrenoceptors (beta-ARs) is important for the rational design of highly potent and beta3-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 beta3-AR, and for functional activities on isolated rat tissues. Binding affinities (K1 approximately 0.055 to 1.5 microM) for the rat beta3-AR and potencies for adenylyl cyclase activation (K(act) approximately 0.43 to 2;5 nM) of the 3'-monoiodo or 3',5'-diiodo derivatives with 4'-isothiocyanato-, 4'-amino, 4'-acetamido, or 4'-alpha-haloacetamido substitutions were higher than those of (-)-isoproterenol, and comparable to those of BRL 37344 [(+/-)-(R*,R*-[4-[2-[[2-(3-chlorophenyl)-2-hydroxy-ethyl]amino]propyl]ph enoxy]-acetic acid sodium]. A similar rank order of binding affinities (K(i) approximately 0.11 to 2.5 microM) and potencies (K(act) approximately 0.45 to 9.5 nM) was obtained for TMQ analogs on the human beta3-AR. The 4'-acetamido and 4'-alpha-chloroacetamido analogs of 3',5'-diiodoTMQ were more potent than (-)-isoproterenol in rat atria (beta1-AR) and rat trachea (beta2-AR) and exhibited partial agonist activities, whereas full agonist activities were observed in rat esophageal smooth muscle (EC50 approximately 2-8 nM, beta3-AR). 4'-alpha-Chloroacetamido-3',5'-diiodoTMQ-mediated chronotropic responses in atria were sustained and resistant to washout. Further, the 4'-alpha-chloroacetamido and 4'-alpha-bromoacetamido analogs of 3',5'-diiodoTMQ demonstrated significant concentration-dependent irreversible binding to the rat beta3-AR. Reversible beta-AR agonists such as (-)-isoproterenol, BRL 37344, and 4'-acetamido-3',5'-diiodoTMQ or nucleophilic 1-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 beta-AR that may represent an exo-site or an allosteric binding site.

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.

Mechanisms of peroxisome proliferation by perfluorooctanoic acid and endogenous fatty acids.

1. The effects of endogenous fatty acids and perfluorooctanoic acid (PFOA) and its analogs on peroxisomal acyl CoA oxidase (ACO) and microsomal laurate hydroxylase (LH) activities were evaluated in primary cultures of rat hepatocytes and activation of peroxisome proliferator-activated receptor alpha (PPARalpha) in CV-1 cells. The rank order for the stimulation of ACO activity in hepatocytes for selected compounds was PFOA >> octanoic acid>octanedioic acid, perfluorooctanol (inactive). Increases in ACO activity by PFOA, like those of ciprofibrate, were associated with a marked increase in peroxisome number and cytosolic occupancy volume. Maximal effects of ciprofibrate and PFOA on the stimulation of ACO activity were not additive, suggesting that these two compounds share a common pathway of peroxisome proliferation. 2. Saturated monocarboxylic acids of C4 to C18 chain length were inactive, and, among dicarboxylic acids, only small elevations (40-45%) in ACO activity were observed with the long-chain C12 and C16 dioic acids. Of the C18 fatty acids tested, only oleic and linoleic acids, at 1 mM, produced a two- to three-fold elevation in ACO and LH activities. In comparison with endogenous fatty acids, PFOA was more potent and exhibited a different time course and greater magnitude of stimulation of ACO and LH activities in cultured hepatocytes. 3. Addition of mitochondrial beta-oxidation inhibitors (3-mercaptopropionic and 2-bromooctanoic acids) did not alter ACO activity in the presence of octanoic acid or octanedioic acid; nor did they modify the stimulation of ACO activity by PFOA. The carnitine palmitoyltransferase I inhibitor 2-bromopalmitic acid produced a 2.5-fold increase in ACO stimulatory activity and reduced both ciprofibrate- and PFOA-mediated stimulations of ACO activity. 4. Cycloheximide treatment reduced PFOA- and ciprofibrate-induced ACO activities; however, the response to oleic acid was not blocked and increased slightly. 5. In rat and human PPARalpha transactivation assays, the rank order of activation was ciprofibrate > PFOA > oleic acid > or = octanoic acid > octanedioic acid or perfluorooctanol (inactive). PFOA, ciprofibrate and oleic acid were activators of rPPARalpha at concentrations that correlated favorably with the changes in ACO activity in cell culture. Octanoic acid did not increase ACO activity and was a weak activator of PPARalpha. 6. Our findings suggest that fatty acids such as oleic acid (endogenous fatty acids) and PFOA (a stable fatty acid) act through more than one pathway to increase ACO activity in rat hepatocytes. We conclude that the potent effects of PFOA are primarily mediated by a mechanism that includes the activation of liver PPARalpha.

Activation of L-type calcium channel by tolazoline derivatives: role of isothiocyanate moiety.

Studies have investigated the pharmacologic mechanism of 2-(4'-isothiocyanatobenzyl) imidazoline (IBI) and analogs for interaction with imidazoline receptors (IRs), alpha-adrenergic receptors (alpha-ARs), and calcium channels in cardiovascular muscle systems. IBI differs from tolazoline by substitution of an electrophilic isothiocyanato (NCS) group. Unlike tolazoline, which is a partial alpha-AR agonist, IBI produced an irreversible, slow-onset, and sustained contraction of rat aorta with an median effective concentration (EC50) value of 5 microM, and a maximal contraction (116%) greater than that of phenylephrine (100%) and tolazoline (59%). The IBI-induced contractions were dependent on calcium channels and independent of alpha-ARs or IRs. Similarly, structure-activity relation studies in rat aortic smooth muscles on a series of synthesized IBI analogs indicated that NCS analogs, but not those without the NCS group, exhibited effects by a non-alpha-AR, non-IR, but a calcium channel-dependent mechanism. Thus the presence of an intact IBI ring in these analogs is not a requirement for these activities. Further, IBI inhibited dihydropyridine (DHP, [3H]PN 200-110 and [3H]Bay K 8644) binding to L-type calcium channels of T-tubule membranes in rabbit skeletal muscle. In contrast to nifedipine, IBI and NCS derivatives (nifedipine-NCS, naphazoline-NCS) only partially (50-88%) displaced specific binding of these radioligands. A single site of noncooperative interaction was observed for nifedipine (nH = 0.97), whereas tolazoline-NCS (IBI, nH = 1.46) and nifedipine-NCS (nH = 1.37) exhibited a positive cooperativity in binding to DHP sites. These receptor-binding data indicate that NCS derivatives bind to L-type calcium channels and interact allosterically with DHP-binding sites. Direct binding of the NCS group to specific nucleophilic protein sites of the calcium channel may be responsible for its activation and the subsequent contractile effects of IBI.

Medetomidine analogs as alpha 2-adrenergic ligands. 3. Synthesis and biological evaluation of a new series of medetomidine analogs and their potential binding interactions with alpha 2-adrenoceptors involving a "methyl pocket".

The synthesis and the biological evaluation of a new series of medetomidine analogs are reported. The substitution pattern at the phenyl ring of the tetralin analogs had a distinct influence on the alpha 2-adrenoceptor binding affinity. 4-Methylindan analog 6 was the most potent alpha 2-adrenoceptor binding ligand among these 4-substituted imidazoles, and its alpha 2-adrenoceptor selectivity was greater than the 5-methyl tetralin analog 4c. Ligand-pharmacophore and receptor modeling were combined to rationalize alpha 2-adrenoceptor binding data of the imidazole analogs in terms of ligand-receptor interactions. The structure-activity relationships that were apparent from this and previous studies were qualitatively rationalized by the binding site models of the alpha 2-adrenoceptor. The benzylic methyl group of medetomidine or the naphthyl analog 2a was superimposable with the alpha-methyl group of (-)-alpha-methylnorepinephrine and fit into the proposed "methyl pocket" of the alpha 2-adrenoceptor defined by the residues Leu110, Leu169, Phe391, and Thr395.

Beta-adrenergic receptor and platelet inhibitory activities of a new series of trimetoquinol and related benzazepine analogs.

1. A series of dimethoxy and methylenedioxy analogs of trimetoquinol (TMQ) and structurally related 7-membered ring benzazepines (BA) were evaluated for their pharmacological effects in beta-adrenergic (atria, trachea) and thromboxane A2/prostaglandin H2 receptor systems (aorta, platelets). 2. Results show that both the 6,7-dihydroxy (catechol) moiety of trimetoquinol and an intact tetrahydroisoquinoline nucleus are essential for maintaining potent beta-stimulating and antithromboxane A2 activities. 3. By contrast, ring enlargement, as in the BA analogs, or masking of the catechol with dimethoxy or methylenedioxy functional groups enhanced the potency of inhibitors on thromboxane A2-independent activation of human platelets induced by bacterial phospholipase C (PLC). 4. The selective blockade of this pathway by these compounds suggests that they may represent a new and novel class of antiplatelet drugs.

Synthesis and evaluation of trimetoquinol derivatives: novel thromboxane A2/prostaglandin H2 antagonists with diminished beta-adrenergic agonist activity.

Trimetoquinol (TMQ, 1) is a unique catecholamine with a strong stereodependence for agonism at beta-adrenergic (S > > R) and antagonism at thromboxane A2/prostaglandin H2 (TP; R > > S) receptors. Our laboratory has reported the effects of N-alkylation and modification of the trisubstituted benzyl group in these receptor systems. For iodinated derivative 5, maintaining potency in TP receptor systems (112%) was coupled with maintaining limited potency in beta-adrenergic receptor systems (34% for beta 1 and 47% for beta 2). In this study, several diverse TMQ derivatives were prepared to probe for binding interactions specific to a particular receptor system. Planar amidine 2, which was designed to explore the importance of TMQ's chiral center, showed a dramatic loss of potency (< 1%) in each receptor system. Likewise, the homologation of a previously described N-benzyl derivative (3) to the N-phenylethyl derivative 4 also showed reduced potency (< 3%) in both receptor systems. However, modification of the trimethoxybenzyl group of TMQ to a 4-hydroxy-3-nitrobenzyl group (7) provided a unique lead for TMQ derivatives with significant potency in TP receptor systems (91%) and reduced potency in beta-adrenergic receptor systems (4% for beta 1 and 19% for beta 2).

Stereoselective effects of chiral clofibric acid analogs on rat peroxisome proliferator-activated receptor alpha (rPPAR alpha) activation and peroxisomal fatty acid beta-oxidation.

Enantiomers of a series of substituted analogs of 2-(4-chlorophenoxy) -acetic acid (CPAA) were synthesized and used to examine the influence of steric and structural parameters on peroxisome proliferation. The effects of these compounds were studied on the activation of the peroxisome proliferator-activated receptor alpha (PPAR alpha) in CV-1 cells using an in vitro co-transfection assay. Selected sets of isomers were tested for their ability to increase peroxisomal fatty acyl-CoA oxidase (ACO) activity in H4IIEC3 (rat Reuber hepatoma) cells. Of the series of 2-substituted analogs studied, the isomers of the nu-propyl and phenyl derivatives of CPAA showed a high degree of stereoselectivity [(S)-isomer >> (R)-isomer]. In general, the potency of the compound to activate the receptor increased with the size of the 2-alkyl substituent. Among the 4-chlorobenzyloxy- and 4-(4'-chlorophenyl)benzyloxy- analogs studied, 2-[4-(4'-chlorophenyl)-benzyloxy]-propanoic acid exhibited a high degree of stereoselectivity in both the biological systems studied [(R) >> (S)]. The congeners of 2-methyl substituted CPAA showed a reverse stereoselectivity (R) > (S)] as compared to the other 2-substituted analogs [(S) > (R)]. Our results indicate that (1) both structural and steric characteristics of CPAA analogs play an important role in the activation of rPPAR alpha and on stimulation of peroxisomal ACO activities, and (2) clofibric acid and analogs exert their peroxisome proliferative effects by interaction with a specific site on a protein. The enantiomers of the 2-nu-propyl and the 2-phenyl CPAA analogs may be useful as mechanistic probes in elucidating the nature of this binding site.

Iodinated analogs of trimetoquinol as highly potent and selective beta 2-adrenoceptor ligands.

A series of trimetoquinol (1, TMQ) analogs were designed and synthesized based on the lead compound 2, a diiodinated analog of trimetoquinol which exhibits improved selectivity for beta 2-versus beta 1-adrenoceptors (AR). To determine the influence of 1-benzyl substituents of trimetoquinol on beta 2-AR binding affinity and selectivity, we replaced and/or removed the 3'-, 4'-, and 5'-methoxy substituents of trimetoquinol. Replacement of the 4'-methoxy group of 2 with an amino (21c) or acetamido (15) moiety did not significantly alter beta 2-AR and thromboxane A2/prostaglandin H2 (TP) receptor affinity. Substitution with a 4'-hydroxy (18) or -iodo (21b) group did not significantly alter beta 2-AR affinity, but greatly reduced TP receptor affinity (380- and 1200-fold, respectively). Further, the beta 2-AR can accommodate larger substituents such as a benzamide at the 4'-position (26b). Other monoiodo derivatives (24, 26a) have similar or slightly lower affinity to both beta 2-AR and TP receptor compared to their diiodo analogs. Interestingly, removal of the 4'-substituent of 3',5'-diiodo analogs increased beta 2-AR affinity with little or no effect on beta 1-AR and TP binding. Thus, analog 21a displayed highly potent (pKi 9.52) and selective (beta 2/beta 1 = 600) binding affinity for beta 2-AR. On the other hand, trifluoromethyl substituents at the 3'- and 5'-positions (27) essentially abolished binding affinity at beta 2-AR and TP receptors. The differential binding effects of the aforementioned trimetoquinol modifications on the receptor systems may reflect differences in the binding pocket that interacts with the benzyl portion of trimetoquinol analogs. Thus, manipulation of the 1-benzyl moiety of trimetoquinol (1) has resulted in analogs that exhibit potent beta 2-AR binding affinity and significantly lower beta 1-AR and TP receptor affinities.

Pharmacological study of atypical beta-adrenoceptors in rat esophageal smooth muscle.

The chemical specificity for the beta-adrenoceptor mediated relaxation of rat esophageal smooth muscle was evaluated using selective and non-selective beta-adrenoceptor agonists and antagonists. Pindolol, ICI 89,406, ICI 118551 [erythro-1-(7-methylindan-4-yloxy)-3-(isopropylamine)-but an-2-ol] and the beta-adrenoceptor alkylating agent, pindobind, produced only small rightward shifts in the concentration-response curves of (-)-isoprenaline and (-)-trimetoquinol in this preparation. Rank order potency (pD2 values) of agonists was: (+/-)-trimetoquinol [1-(3',4',5'-trimethoxybenzyl)-6,7-dihydroxy-1,2,3, 4-tetrahydroisoquinoline] (8.34) = (-)-trimetoquinol (8.26) = BRL 37344 [(R* R*)-(+/-)-4-[2'-2-hydroxy 2-(3-chlorophenyl)ethylamino]propyl] phenoxyacetic acid] (8.16) = ICID7114 [(S)-4-(2-hydroxy- 3-phenoxy-propylamino-ethoxy)-N-(2-methoxyethyl) phenoxyacetamide] (8.03) > or = (-)-isoprenaline (7.82) > 3',5'-diiodotrimetoquinol [1-(3',5'-diiodo-4'-methoxybenzyl)-6, 7-dihydroxy-1,2,3,4-tetrahydroisoquinoline] (7.28) > 3'-iodotrimetoquinol [1-(3'-iodo-4',5'-dimethoxybenzyl)-6, 7-dihydroxy-1,2,3,4-tetrahydroisoquinoline] (7.04) > ractopamine (6.84) = 5,8-difluorotrimetoquinol [5,8-difluoro-6,7-dihydroxy-1- (3',4',5'-trimethoxybenzyl)-1,2,3,4-tetrahydroisoquinoline] (6.82) > 8-fluorotrimetoquinol [6,7-dihydroxy-8-fluoro-1-(3',4',5'- trimethoxybenzyl)-1,2,3,4-tetrahydroisoquinoline] (6.56) > or = (-)-noradrenaline (6.46) > or = (-)-adrenaline (6.36) > (+/-)-noradrenaline (6.24) > (+/-)-adrenaline (6.00) > clenbuterol (5.83) > (-)-1-benzyl-6, 7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (5.75). Isomeric activity ratios of trimetoquinol isomers [(-)-(S)- > > (+)-(R)-] in esophageal smooth muscle in the presence and absence of 1 microM pindolol were 1995- and 2951-fold, respectively; and were much greater than those in rat atria (282-fold) and rat trachea (107-fold). The atypical beta/beta 3-adrenoceptor partial agonist, ICI D7114, produced concentration-dependent rightward shifts of the concentration-response curves of (-)-isoprenaline, (-)-trimetoquinol and the reference atypical beta/beta 3-adrenoceptor agonist, BRL 37344. Schild plot analysis of ICI D7114 against trimetoquinol gave slope and pA2 values of 0.91 and of 7.9, respectively. These results clearly demonstrate that the relaxant effects of these agonists in rat esophageal smooth muscle are primarily mediated through the activation of atypical beta/beta 3-adrenoceptors. (-)-Trimetoquinol was as potent as (-)- isoprenaline and BRL 37344, and was the most stereoselective agonist evaluated in this tissue system.

Medetomidine analogs as alpha 2-adrenergic ligands. 2. Design, synthesis, and biological activity of conformationally restricted naphthalene derivatives of medetomidine.

A new series of naphthalene analogs of medetomidine have been prepared and evaluated for their alpha-adrenergic activities. The methylnaphthyl analog 5a showed significant selectivity for alpha 2-adrenoceptors and behaved as a partial alpha 1-agonist in rat aorta preparations. In contrast, the Z-ethylene analog 8c was alpha 1-selective and behaved as a potent alpha 1-antagonist. Two rigid analogs (6 and 7) exhibited large differences in binding affinities at alpha 1-VS alpha 2-receptors, indicating that the conformational flexibility of 5a is important for the fulfillment of the alpha-adrenergic activities. Molecular modeling studies began with conformational analysis of classical phenethylamines and medetomidine analogs. Superimposition of medetomidine conformations with those of phenethylamines provided a tentative explanation for the alpha 2-adrenergic activity of the new imidazoles. A common binding mode for phenethylamines and imidazoles with alpha 2-adrenoceptors is proposed. Knowledge of the biological properties of the 4-substituted imidazoles, integrated with the information derived from computer-assisted molecular modeling, has provided new insights for the structural and conformational requirements of this class as new adrenergic drugs.

Pharmacological activities of trimetoquinol and 1-benzyl halogen-substituted analogues on rat beta-adrenoceptor subtypes.

The beta-adrenoceptor activity profile of trimetoquinol and its 1-benzyl halogen-substituted analogues was studied in rat tissues containing primarily beta 1 (atria)-, beta 2 (trachea)- and atypical beta/beta 3 (distal colon and brown adipose tissue)-adrenoceptors. Functional biological activity resided in the (-)-isomer of trimetoquinol which was 112-, 275-, 372- and 513-fold more potent than (+)-trimetoquinol in trachea, right atria, distal colon and brown adipose tissue, respectively. (+/-)-Trimetoquinol was equally or slightly less active than (-)-trimetoquinol. The 1-benzyl halogen-substituted analogues of trimetoquinol exhibited differential activation of beta-adrenoceptor subtypes. In functional assays, 3'-iodotrimetoquinol was a potent activator of all beta-adrenoceptor subtypes. 3',5'-Diiodotrimetoquinol was 10-fold more potent as an agonist in tissues containing atypical beta/beta 3-adrenoceptors than those tissues containing beta 1- and beta 2-adrenoceptor sites. Furthermore, this drug was a partial agonist as compared to (+/-)-trimetoquinol and 3'-iodotrimetoquinol on beta 1-adrenoceptors. Pharmacological properties of the compounds on rat beta 3-adrenoceptors expressed in Chinese hamster ovary (CHO) cells were consistent with results observed in functional assays. 3',5'-Diiodotrimetoquinol possessed the greatest potency for activation of adenylyl cyclase. Rank order of affinity for rat beta 3-adrenoceptor was 3'-iodotrimetoquinol = 3',5'-diiodotrimetoquinol > (+/-)-trimetoquinol > (-)-isoprenaline. These results suggest that 3',5'-diiodotrimetoquinol is a promising drug for further chemical modification in the development of selective beta 3-adrenoceptor ligands.