Endogenous neurotoxic dopamine derivative covalently binds to Parkinson's disease-associated ubiquitin C-terminal hydrolase L1 and alters its structure and function.

Parkinson's disease (PD) is a common neurodegenerative disease, but its pathogenesis remains elusive. A mutation in ubiquitin C-terminal hydrolase L1 (UCH-L1) is responsible for a form of genetic PD which strongly resembles the idiopathic PD. We previously showed that 1-(3',4'-dihydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline (3',4'DHBnTIQ) is an endogenous parkinsonism-inducing dopamine derivative. Here, we investigated the interaction between 3',4'DHBnTIQ and UCH-L1 and its possible role in the pathogenesis of idiopathic PD. Our results indicate that 3',4'DHBnTIQ binds to UCH-L1 specifically at Cys152 in vitro. In addition, 3',4'DHBnTIQ treatment increased the amount of UCH-L1 in the insoluble fraction of SH-SY5Y cells and inhibited its hydrolase activity to 60%, reducing the level of ubiquitin in the soluble fraction of SH-SY5Y cells. Catechol-modified UCH-L1 as well as insoluble UCH-L1 were detected in the midbrain of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated PD model mice. Structurally as well as functionally altered UCH-L1 have been detected in the brains of patients with idiopathic PD. We suggest that conjugation of UCH-L1 by neurotoxic endogenous compounds such as 3',4'DHBnTIQ might play a key role in onset and progression of idiopathic PD. We investigated the interaction between ubiquitin C-terminal hydrolase L1 (UCH-L1) and the brain endogenous parkinsonism inducer 1-(3',4'-dihydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline (3',4'DHBnTIQ). Our results indicate that 3',4'DHBnTIQ binds to UCH-L1 specifically at cysteine 152 and induces its aggregation. 3',4'DHBnTIQ also inhibits the hydrolase activity of UCH-L1. Catechol-modified as well as insoluble UCH-L1 were detected in the midbrains of MPTP-treated Parkinson's disease (PD) model mice. Conjugation of UCH-L1 by neurotoxic endogenous compounds like 3',4'DHBnTIQ might play a key role in onset and progression of PD.

Electron transfer through the isolated mitochondrial cytochrome b-c1 complex.

(1) A kinetic analysis of electron donation into and through the cytochrome b-c1 complex isolated from bovine heart mitochondria has been undertaken, using trimethoquinol as the donor. (2) Rate constants of two routes of redox equilibration with quinols have been defined by kinetic measurements and with the use of the inhibitors antimycin A and myxothiazol. (3) A model of electron transfer based upon the original Q-cycle formulation is presented to explain these and related results.

Effect of beta and beta2 adrenoreceptor stimulants infused intrapancreatically on glucagon and insulin secretion.

L-Isoproterenol was infused at a dose of 20 pmol/kg/min for 10 min into the cranial pancreaticoduodenal artery in anesthetized dogs. Arterial plasma glucose, blood flow, and plasma concentrations of both glucagon and insulin in the cranial pancreaticoduodenal vein were significantly enhanced during the infusion, resulting in a greater increase of bihormonal output. Intrapancreatic pretreatment with propranolol abolished all of the isoproterenol-induced increases except for glucagon secretion which was suppressed only in part. Pretreatment with practolol, a specific receptor blocker of the beta1 type, did not exert any discernible inhibiting effect upon the isoproterenol-induced enhancement. Intrapancreatic infusion of trimetoquinol, a selective receptor stimulant of the beta2 type in some mammals, at an equimolar dose caused similar increases in plasma glucose, pancreatic venous blood flow, and bihormonal output when compared to those induced by isoproterenol. Pretreatment with a larger dose of propranolol totally abolished the trimetoquinol-induced enhancement of both glucagon and insulin secretion. Pretreatment with an isomolar dose of practolol, in contrast, did not show any suppressive effect on the parameters investigated. There was a dose-dependency in the bihormonal responses to trimetoquinol. Another beta2 receptor agonist, salbutamol, also significantly raised plasma glucose, pancreatic venous blood flothough to a lesser extent than did trimetoquinol. These results indicate that the adrenergic control over the function of the endocrine pancreas through beta adrenoreceptors may be mediated mainly via those of the beta2 type.

Synthesis of halogenated trimetoquinol derivatives and evaluation of their beta-agonist and thromboxane A2 (TXA2) antagonist activities.

The 5,8-difluoro (4), 5-iodo (5), 8-iodo (6), and 5-trifluoromethyl (7) derivatives of trimetoquinol (TMQ, 1) have been synthesized and evaluated for their ability to stimulate beta 1 (guinea pig atria) and beta 2 (guinea pig trachea) adrenoceptors as well as for their inhibitory activity against U46619 [a thromboxane A2 (TXA2) mimetic]-mediated contraction of rat thoracic aorta and human platelet aggregation. Both 5 and 6 were considerably less active than TMQ on both beta-adrenergic systems and gave a rank order of stimulatory potency of 1 much greater than 6 greater than or equal to 5. Similarly, iodine substitution at either position also caused a reduction in TXA2 antagonist activity with a rank order potency of 1 greater than 6 much greater than 5. Compared to 1, however, 5-iodo-TMQ (5) showed a marked selectivity for blockade of U46619 responses in rat aorta over human platelets. On beta-systems, 4 had reduced potency compared to TMQ and was similarly nonselective. Introduction of a trifluoromethyl group at the 5-position of TMQ completely abolished both beta 1- and beta 2-adrenergic agonist activities while imparting weak antagonist activity on beta 1 receptors. On TXA2 systems, both 4 and 7 possessed significantly decreased inhibitory activity compared to TMQ. The synthetic approaches to the synthesis of 8-(trifluoromethyl)-TMQ (8) are also described. The enantiomers of the 8-fluoro derivative (3) of TMQ were separated on a preparative Chiralcel OD column and evaluated on beta-adrenergic systems and TXA2 systems. On beta-adrenergic systems, (S)-(+)-8-fluoro-TMQ was at least 10-fold more potent than (R)-(-)-8-fluoro-TMQ. Conversely, (R)-(-)-8-fluoro-TMQ was approximately 14-fold more potent as an antagonist of TXA2-mediated aggregation in human platelets than (S)-(+)-8-fluoro-TMQ. In contrast to platelets, (S)-(+)-8-fluoro-TMQ was an agonist in rat aorta whereas (R)-(-)-8-fluoro-TMQ was an antagonist.

6,7-Dichloro-1-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroisoquinoline . A structurally novel beta-adrenergic receptor blocking agent.

Replacement of the catecholic hydroxyl groups of the beta-adrenergic receptor agonist 6,7-dihydroxy-1-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroisoquinoline (trimetoquinol) with chloro substituents results in a compound with marked beta-adrenoceptor antagonist properties. This, therefore, parallels the similar transformation of the beta-adrenoreceptor agonist isoproterenol into the antagonist dichloroisoproterenol. In a test for inhibition of isoproterenol-induced enhancement of the rate of contraction of spontaneously beating guinea pig atrial pairs the resultant 6,7-dichloro-1-(3,4,5-trimethoxybenzyl)-1,2,3,4-tetrahydroisoquinoline (6b) had a KB value of (6.7 +/- 2.3) X 10(-8) M. Although this is nearly 2 orders of magnitude less potent than propranolol (KB = 6.2 X 10(-10) M in this test), this compound represents the prototype of a new class of beta-adrenergic receptor blockers, and unlike dichloroisoproterenol it is not a partial agonist. It has physicochemical properties, e.g., pKa and distribution and partition coefficients, that differ from the prototypic beta-blockers. These altered properties might impart advantageous tissue distribution and altered pharmacological properties to the new molecule. This new beta-adrenoreceptor antagonist is suggested to merit further study.

Synthesis and investigation of the beta-adrenoceptor agonist and platelet antiaggregatory properties of 1,7,8-trisubstituted 2,3,4,5-tetrahydro-1H-2-benzazepine analogues of trimetoquinol.

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.

Syntheses and beta-adrenergic agonist and antiaggregatory properties of N-substituted trimetoquinol analogues.

Trimetoquinol [1-(3,4,5-trimethoxybenzyl)-6,7- dihydroxy-1,2,3,4-tetrahydroisoquinoline, TMQ] is a potent beta-adrenergic receptor agonist and inhibitor of human platelet aggregation. Selective cleavage of O-benzyl groups in the presence of an N-benzyl group using HCl and formation of a cyclic sulfite ester from the reaction of a catechol with thionyl chloride were achieved. The N-substituents included methyl, benzyl, and beta-hydroxy- and beta-chloroethyl groups. Each N-substituted TMQ caused a concentration-dependent stimulation of beta 2 (trachea) and beta 1 (atria) adrenoceptor tissues and inhibition of 15(S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13(E)-dienoic acid (U46619, a thromboxane A2 mimetic) mediated human platelet activation. TMQ remained the most potent in the series. Structure-activity results indicated that the larger the N-substituent, the lower the beta-adrenergic activity but the higher the inhibition of platelet aggregatory activity. Thus, the structural requirements of these TMQ analogues for the two types of biological activity are different.

Synthesis, beta-adrenergic activity, and platelet antiaggregatory activity of a positional isomer of trimetoquinol: 1-(2',4',5'-trimethoxybenzyl)-6,7-dihydroxy-1,2,3,4,-tetrahydroisoquinoline.

A positional isomer of trimetoquinol (1), 1-(2',4',5'-trimethoxybenzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (3), was synthesized and found to possess less beta-adrenergic activity than 1 in isolated guinea pig atrial and tracheal preparations. The analogue 3 was an effective antiaggregatory agent in human and rabbit platelet-rich plasma preparations, while 1 was effective only as an inhibitor of arachidonic acid induced aggregation in human platelets. These findings indicate that both qualitative and quantitative differences in biological activity have occurred as a result of changing the position of the methoxy groups on the 1-benzyl substituent of 1.

Synthesis of 1-substituted analogues of trimetoquinol possessing differential and selective beta-adrenergic properties.

The synthesis of the 1,1-disubstituted tetrahydroisoquinoline analogues, 1-methyl-1-(3,4,5-trimethoxybenzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline hydrochloride (2) and 1-benzyl-1-(3,4,5-trimethoxybenzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline hydrochloride (3), is described. The profile of beta-adrenergic activity for these analogues was determined and compared to that of trimetoquinol (1) in isolated guinea pig atrial, tracheal, and rat adipocyte preparations. Unexpected selective beta1-blocking activity in guinea pig trachea was noted with analogue 3. With the exception of 2 in guinea pig atria, 2 and 3 did not possess any beta-stimulant activity. Substitution at the 1 position of trimetoquinol (1) has revealed qualitative differences in beta-adrenergic activity.

Selective and potent beta 2-adrenoceptor agents within the tetrahydroisoquinoline class: effect of methyl substitution at the benzylic carbon of the 1-(3,4,5-trimethoxybenzyl) group of trimetoquinol.

A systematic series of methyl (2 and 3) and dimethyl (4) analogues of trimetoquinol (1) were synthesized and evaluated for their beta 1 (atria) and beta 2 (trachea) and adrenoceptor activities. Structural assignments for the erythro (2) and the threo (3) diastereoisomers of 1-(3,4,5-trimethoxy-alpha-methylbenzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline were based on NMR spectra of the 6,7-dibenzyl precursors 15 and 16, respectively, and on the synthetic derivatives of cis- and trans-13-methyl-2,3-bis(benzyloxy)-9,10,11-trimethoxytetrahydroprotoberberine (18 and 17). The rank order of beta 2-agonist activity for these compounds was 3 greater than 1 greater than 2 greater than 4. The rank order of activity as beta 1 agonists on the guinea pig atria is 1 greater than 3 greater than 2, and 4 was inactive. The methylated analogues show selectivity for beta 2 receptors in our preliminary pharmacological studies. The threo isomer 3 is the most potent and selective beta 2 stimulant reported to date in the tetrahydroisoquinoline class.

5-fluoro- and 8-fluorotrimetoquinol: selective beta 2-adrenoceptor agonists.

The 5-fluoro and 8-fluoro analogues of trimetoquinol, TMQ, have been synthesized and evaluated for beta 2- and beta 1-adrenoceptor activity in guinea pig trachea and atria, respectively. The fluoro analogues of TMQ maintained potent beta 2-adrenoceptor agonist activity but had reduced beta 1-adrenoceptor agonist activity. The changes in beta 1-activity of these compounds were correlated to differences in phenolic pKa's. The beta 1- and beta 2-adrenoceptor actions of 2 and 3 were blocked in a competitive manner by propranolol. The enhanced beta 2/beta 1 selectivity for the analogues was found to be 8-fluoro analogue 3 greater than 5-fluoro analogue 2 greater than trimetoquinol (1).

Synthesis and beta-adrenoceptor activity of the erythro and threo isomers of substituted alpha-hydroxytrimetoquinol.

The synthesis and pharmacological activity of erythro and threo isomers of 1-(3',4',5'-trimethoxy-alpha-hydroxy-benzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, 2 and 3, are reported. The structural assignments of 2 and 3 are based upon NMR spectra of the 6,7-dibenzyl precursors, 6 and 10, and of the synthetic derivatives of 13alpha- and 13beta-hydroxy-2,3-(dibenzyloxy)-9,10,11-trimethoxytetrahydroprotoberberine, 8 and 12, respectively. The erythro isomer 2 was a more potent beta-adrenoceptor stimulant than the threo isomer 3 in guinea pig atrial, guinea pig tracheal, and rat adipocyte preparations. The differential activity of these compounds on lipolysis was favorably correlated to changes in the stimulation of adenylate cyclase activity and cAMP accumulation in rat adipocytes.

Synthesis and biological evaluation of fragmented derivatives of tetrahydroisoquinolines. 2. Trimetoquinol studies.

The synthesis of N-(3',4',5'-trimethoxyphenylethyl)-3,4-dihydroxyphenylethylamine (2) and 1-(3,4,5-trimethoxybenzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (1) is presented. Comparative pharmacological effects of the optical isomers of 1 and compound 2 are reported in guinea pig atria, rat adipose tissue, guinea pig trachea, and guinea pig aortic strip preparations. In the beta-adrenoreceptor preparations, (-)-1 was shown to be more potent than (+)-1 or 2. Racemic 1 and 2 were shown to have equal alpha-antagonist properties in the inhibition of norepinephrine-induced contractions of guinea pig aorta.

Antagonism of prostanoid-induced contractions of rat gastric fundus muscle by SC-19220, sodium meclofenamate, indomethacin or trimethoquinol.

1 The effects of SC-19220, sodium meclofenamate, indomethacin or trimethoquinol were studied on contractions of the rat stomach longitudinal muscle to prostaglandin D2 (PGD2), PGE2, PGF2 alpha, PGH2, epoxymethano PGH2 analogues, PGI2, 6-keto-PGF1 alpha, 6,15-diketo-PGF1 alpha and thromboxane B2. All the drugs reduced contractions to all the prostanoids, but the degree of reduction differed widely. Selectivity of blockade was assessed by comparison with acetylcholine (ACh). 2 With SC-19220 5 micrograms/ml the effect on thromboxane B2, PGD2 or PGH2 and its epoxymethano analogues was not significantly different from the small effect on ACh, but the other prostanoids were blocked to greater extents. 3 The effect of the cyclo-oxygenase inhibitor sodium meclofenamate, 1 or 2 micrograms/ml, on 6,15-diketo-PGF1 alpha or thromboxane B2 was similar to the small antagonism of ACh, whereas the other prostanoids were blocked to greater extents. Indomethacin, 1 microgram/ml, also reduced contractions to the prostanoids, but antagonism of the PGH2 epoxymethano analogues was considerably less than with meclofenamate. 4 The beta-adrenoceptor stimulant trimethoquinol, 50 ng/ml, was the most potent prostanoid antagonist tested; all the prostanoids except PGE2 were antagonized more than ACh.

Assessment of the selectivity of OPC-2009, a new beta2-adrenoceptor stimulatn, by the use of the blood-perfused trachea in situ and of the isolated blood-perfused papillary muscle of the dog.

1 The potency and selectivity of 5-(1-hydroxy-2-isopropylamino)butyl-8-hydroxy carbostyril hydrochloride hemihydrate (OPC-2009), a new beta(2)-adrenoceptor stimulant, was compared with those of isoprenaline, trimetoquinol and salbutamol by the use of blood-perfused tracheal preparations in situ and of blood-perfused papillary muscle preparations of the dog. All drugs were injected intra-arterially.2 All the four drugs decreased tracheal intraluminal pressure (tracheal relaxation) and increased tracheal blood flow in a dose-dependent manner. The four drugs produced a dose-dependent increase in developed tension of papillary muscles. In both preparations the duration of action of isoprenaline and salbutamol was short, whereas that of OPC-2009 and trimetoquinol was long. These effects were antagonized by propranolol.3 Dose-response curves to the four drugs for tracheal relaxation were almost parallel. OPC-2009 was 2.4 times more potent, and trimetoquinol and salbutamol were 2.2 and 6.2 times less potent than isoprenaline in causing tracheal relaxation.4 Dose-response curves to the four drugs for tracheal vasodilatation were also parallel. OPC-2009, trimetoquinol and salbutamol were 3.9, 6.7 and 23 times less potent than isoprenaline.5 Slopes of the dose-response curves to the four drugs for increased developed tension were not parallel; that of OPC-2009 was the least steep, whereas that of isoprenaline was the steepest. Trimetoquinol, salbutamol and OPC-2009 were about 18, 570 and 2400 times less potent than isoprenaline.6 Selectivity calculated from relative potencies indicate that OPC-2009 was about 6000 times, salbutamol about 92 times and trimetoquinol about 8.2 times more selective than isoprenaline for tracheal smooth muscle as compared to ventricular muscle.7 The high potency and selectivity of OPC-2009 for tracheal smooth muscle and its long duration of action suggest its potential usefulness for treatment of bronchial asthma.8 The present results are also compatible with the concept that beta(1)-adrenoceptors in cardiac muscle and beta(2)-adrenoceptors in tracheal and vascular smooth muscle can be distinguished. Furthermore, the results revealed that the beta-adrenoceptors mediating tracheal relaxation and vasodilatation may also be different.

Characterization of the inhibition of U46619-mediated human platelet activation by the trimetoquinol isomers. Evidence for endoperoxide/thromboxane A2 receptor blockade.

Sites of inhibition for the trimetoquinol (TMQ) isomers on 15S-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619)-, 12-O-tetradecanoylphorbol 13-acetate (TPA)- and A23187-induced human platelet activation were investigated. Experiments using washed human platelets were designed to characterize relationships among functional (aggregation, secretion) and biochemical (protein phosphorylation, metabolism of inositol phospholipids and radioligand displacement analysis) processes of platelet activation by U46619 and the specificity of inhibition by the TMQ isomers. Thromboxane A2 receptor stimulation by U46619 in human platelets resulted in a time- and concentration-dependent breakdown of inositol phospholipids [phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol (PI)], phosphatidic acid (PA) accumulation, phosphorylation of 20 and 45 kD proteins, aggregation and serotonin secretion. The TMQ isomers stereoselectively inhibited all U46619-mediated platelet activation processes. R(+)-TMQ was 40- and 22-fold more potent than S(-)-TMQ as an inhibitor of U46619-induced platelet aggregation and serotonin secretion respectively. In addition, R(+)-TMQ blocked U46619-induced 20 kD protein phosphorylation, 45 kD protein phosphorylation, PIP2, PIP and PI breakdown, and PA accumulation with a potency which was 8-, 13-, 45-, 37-, 33- and 33-fold greater than the S(-)-isomer respectively. In contrast to S(-)-TMQ, R(+)-TMQ produced a concentration-dependent inhibition of specific [3H]U46619 binding to endoperoxide/thromboxane A2 receptor sites in washed platelets. In other experiments, S(-)-TMQ was more potent than R(+)-TMQ as an inhibitor of TPA- and A23187-induced platelet aggregation and serotonin secretion, and of TPA-induced phosphorylation of 45 and 20 kD proteins. The inhibitory potencies of S(-)-TMQ against TPA- or A23187-induced responses were similar to those needed for antagonism of U46619-mediated platelet activation. In contrast, much higher concentrations of R(+)-TMQ were required for blockade of TPA or A23187 versus U46619-mediated responses in human platelets. Taken collectively, the data show that the TMQ isomers interfered with the endoperoxide/thromboxane A2 receptor-mediated phospholipase C-signal cascade of inositol phospholipid hydrolysis, calcium mobilization, and protein phosphorylation leading to platelet aggregation and secretion. R(+)-TMQ acted as a pharmacologically selective and highly stereospecific [R(+)-TMQ much greater than S(-)-TMQ] antagonist of endoperoxide/thromboxane A2 receptor sites in platelets.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Halogen-substituted trimetoquinol analogs as thromboxane A2 receptor antagonists in platelets and aorta.

Trimetoquinol (TMQ) is a non-prostanoid compound that blocks prostaglandin H2/thromboxane A2 (TXA2) receptor-mediated responses initiated by a prostaglandin (PG) H2 analog, U46619, in human platelets and rat aorta. Ring fluorine-substituted TMQ analogs selectively antagonized PG-dependent human platelet activation induced by U46619, arachidonic acid, collagen, ADP or epinephrine; and were about 300-fold less potent as inhibitors of PG-independent responses mediated by thrombin or bacterial phospholipase C. For each inducer of the PG-dependent pathway, the rank order of inhibitory potency was identical (TMQ > 8-fluoro-TMQ > 5-fluoro- TMQ). Iodine substitution yielded a similar rank order of antagonism against U46619-induced platelet activation (TMQ > 8-iodo-TMQ > 5-iodo-TMQ), and all TMQ analogs inhibited platelet aggregation in whole blood as well as in platelet-rich plasma. Inhibition of specific [3H]SQ 29,548 binding by TMQ analogs was highly correlated with inhibition of functional responses to U46619. Radioligand binding experiments using TMQ analogs with rat platelets showed no interspecies difference in comparison with human platelets. The rank order of inhibitory potencies for the fluorinated (but not iodinated) TMQ analogs changed in rat thoracic aorta with 8-fluoro-TMQ > TMQ > or = 5-fluoro-TMQ as antagonists of U46619-induced vascular contraction. These findings demonstrate that the primary mechanism of antiplatelet action of TMQ analogs is related to a blockade of TXA2 receptor sites, and ring-halogenated TMQ analogs distinguish between TXA2-mediated functional responses in vascular smooth muscle and platelets.

Pharmacological evaluation of the beta-adrenoceptor agonist properties of N-benzyl substituted trimetoquinol analogues.

The beta 1- and beta 2-adrenoceptor agonist properties of trimetoquinol (TMQ, I) and N-benzyl ring substituted TMQ analogues (II, 4'-methylbenzylTMQ; III, 4'-chloro-benzylTMQ; IV, 4'-methoxybenzylTMQ; V, 4'-nitrobenzylTMQ; VI, 3',4'-dichlorobenzylTMQ; and VII, 4'-aminobenzylTMQ) were studied in guinea pig atria and trachea. All compounds gave concentration-dependent responses in atria and trachea, and the rank order of beta-adrenoceptor agonist potency was I greater than VII greater than II greater than V greater than IV greater than VI greater than III and I greater than VII greater than IV = VI greater than V greater than III greater than II, respectively. Whereas the N-benzyl substitution reduced potency for beta-agonist activity, the beta 2/beta 1-selectivity ratio was enhanced by addition of groups to the N-benzyl ring, and the rank order of beta 2-selectivity was VI (10-fold) greater than III (8-fold) = IV (8-fold) greater than VII (3-fold) greater than V = I greater than II. The results show that varying the nature of substituents on the N-benzyl ring of TMQ produces compounds which retain greater beta 2-selectivity.

Pharmacological evaluation of the beta-adrenoceptor agonist and thromboxane antagonist properties of N-substituted trimetoquinol analogues.

The beta 1- and beta 2-adrenoceptor agonist and U46619 (a thromboxane A2 agonist) antagonist properties of trimetoquinol (TMQ, I) and its optical isomers, and N-substituted TMQ analogues (methyl, II; 2-hydroxyethyl, III; cyclic sulfite N-2-chloroethyl, IV; 2-chloroethyl, V; benzyl, VI) were studied in guinea pig atria and trachea and rat aorta, respectively. All compounds gave concentration-dependent responses in atria and trachea, and the rank order of beta-adrenoceptor agonist potency was I greater than II greater than III greater than IV greater than V greater than VI. Whereas N-substitution reduced potency for beta-agonism, the beta 2/beta 1-selectivity ratio was enhanced by increasing the size of the N-substituent. All analogues possessed equal or greater (up to 41-fold more) beta 2-selectivity than I. Propranolol was a competitive antagonist of selected TMQ analogues in guinea pig trachea and atria, thus confirming the beta-adrenoceptor actions of these drugs. The optical isomers of TMQ gave a rank order of agonist potency of S(-)-TMQ greater than R(+)-TMQ, and a beta 2/beta 1-selectivity equal to or greater than racemic-TMQ. Each TMQ analogue also blocked the contractile responses of U46619 in rat aorta in a competitive manner, and the rank order of inhibition of U46619-induced contraction in rat aorta was I greater than VI greater than II = III greater than IV greater than V. N-Benzyl TMQ (VI) possessed the greatest potency for U46619 blockade and beta 2/beta 1-selectivity ratio of the N-substituted analogues. The results show that varying the N-substituents on TMQ produces compounds which retain beta 2-selectivity and give a different activity profile for beta-adrenoceptor activation vs. endoperoxide/thromboxane A2 antagonism.