Analysis of tretoquinol and its metabolites in human urine by liquid chromatography-tandem mass spectrometry.

Tretoquinol (trimetoquinol), a ß2-agonist, has been explicitly listed on the World Anti-Doping Agency Prohibited List 2019 since January 2019; however, it has been distributed as an antiasthmatic on the medical market. This study aimed to develop a liquid chromatography-tandem mass spectrometric method for the quantification of tretoquinol (free form plus glucuronide) in human urine for doping control purposes. An excretion study (n = 6) of tretoquinol hydrochloride hydrate (6 mg) was performed, and urine samples were collected prior to oral administration and during the first 48 h, along with spot urine samples at 7 and 14 days after administration. All the urine samples were analysed using the developed method. The limit of detection for the developed method was 0.03 ng/mL. The inter-day precision for the target analyte was excellent (2.7% to 9.2%), and the inter-day accuracy of target analyte was -0.6% to -3.6%. In all subjects, tretoquinol (free form plus glucuronide conjugate) was identified up to 48 h after administration. The maximum concentrations were in the range of 12.4-78.8 ng/mL and the mean concentration was 55.3 ng/mL. The metabolites O-methylated tretoquinol, tretoquinol sulphate and O-methylated tretoquinol sulphate could be also identified in human urine after administration. The longest-lasting urinary metabolite of tretoquinol currently known, O-methylated tretoquinol, is also likely to be a useful marker in doping controls.

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.

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.

Dopamine transporter and catechol-O-methyltransferase activities are required for the toxicity of 1-(3',4'-dihydroxybenzyl)-1,2,3, 4-tetrahydroisoquinoline.

1-(3',4'-Dihydroxybenzyl)-1,2,3,4-tetrahydroisoquinoline [3', 4'DHBnTIQ (1)] is an endogenous parkinsonism-inducing substance. It is taken up into dopaminergic neurons via the dopamine transporter, inhibits mitochondrial respiration, and induces parkinsonism in mice. We synthesized four derivatives [aromatized, N-methylated, N-methyl-aromatized, and O-methylated (2-5, respectively)] and studied the cellular uptake and cytotoxicity of 1-5, as well as the metabolism of 1. All except the O-methyl derivative (5) were specifically taken up by the dopamine transporter, but 1 was taken up most efficiently. Relative to 1, oxidation reduced v(max), N-methylation markedly increased K(m), and O-methylation eliminated the uptake activity. The cytotoxicity of 1-5 was examined in a mesencephalic cell primary culture. Compound 1 reduced cell viability by nearly 80% at 100 microM, but the other compounds had little or no effect on cell viability. In vivo and in vitro studies revealed that 1 was O-methylated by soluble catechol-O-methyltransferase (COMT). Aromatization and N-methylation of 1 were not observed. We found that dopamine transporter inhibitors and a COMT inhibitor each blocked the cytotoxicity of 1, indicating that uptake and O-methylation are both necessary for neurotoxicity. Thus, we consider that 1 is taken up into dopaminergic neurons via the dopamine transporter and then converted by COMT to 5, which has cytotoxic and parkinsonism-inducing activities.

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.

Quantitative structure-activity relationship study of iodinated analogues of trimetoquinol as highly potent beta 2-adrenoceptor ligands.

Trimetoquinol and its derivatives, reported to be highly potent beta 2-adrenoceptor (beta 2-AR) and site-selective thromboxane A2/prostaglandin H2 (TP) receptor ligands are subjected to quantitative structure-activity relationship (QSAR) study. From the significant correlation equation, obtained between the binding affinity, pKi (beta 2-AR) and the substitutional physicochemical parameters such as molar refraction, (MR), hydrophobic constant, (pi) and resonance parameter, (R), the receptor binding interactions associated with the varying sites of these compounds are discussed. The QSAR study has also explored the possibilities of having the analogous of improved binding affinities in future synthetic efforts. Likewise, the ratio of binding affinities, expressed as-log[Ki(beta 2-AR)/Ki alpha(TP)] related to two receptors are significantly correlated with MR and pi of the substituents and the relationship may, therefore, be helpful in developing the agents of greater selectivity on beta 2-AR versus TP receptor and vice versa.

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 characteristics of BDTI, a new isoquinoline-derived beta 2-adrenoceptor agonist, in canine trachea and rat heart.

The tracheal relaxing effects and beta 2-selectivity of BDTI (1-benzyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline HBr) were investigated in canine trachea and rat heart by radioligand binding assay and pharmacological experiments in comparison with those of other beta-adrenoceptor agonists, salbutamol and isoprenaline. The potency of relaxing effect on carbachol-induced contraction in isolated canine trachea was in the order of isoprenaline (pD2 = 6.70 +/- 0.08) > BDTI (6.11 +/- 0.06) approximately salbutamol (6.14 +/- 0.08). ICI-118,551 (a selective beta 2-antagonist) and atenolol (a selective beta 1-antagonist) inhibited the relaxant action of BDTI with pKB values of 8.4 and 5.3, respectively, corresponding to high affinity for ICI-118,551 and low affinity for atenolol in antagonizing this response. The Kd values of radioligand ([3H]-CGP12177) were 453.3 +/- 30.8 and 563.4 +/- 96.7 pmol/l in cultured canine tracheal smooth muscle cells (TSMCs) and rat cardiomyocytes, respectively, and the Bmax values were 64.6 +/- 10.7 and 245.7 +/- 44.5 fmol/mg protein, respectively. BDTI, salbutamol and isoprenaline inhibited the binding of [3H]-CGP12177 in a concentration-dependent manner in cultured canine TSMCs (Ki 0.73 +/- 0.15, 0.75 +/- 0.21 and 0.24 +/- 0.05 mumol/l, respectively) and rat cardiomyocytes (Ki 2.76 +/- 0.36, 2.31 +/- 0.26 and 0.22 +/- 0.03 mumol/l, respectively). These results demonstrated that BDTI possessed moderate selectivity (3.8-fold) to beta 2-adrenoceptors as judged from the Ki (heart)/Ki (trachea) value (salbutamol 3.1-fold, isoprenaline 0.92-fold). BDTI and salbutamol also stimulated cAMP formation in a concentration-dependent manner in cultured canine TSMCs (EC50 0.5 +/- 0.2 and 0.4 +/- 0.1 mumol/l, respectively) and rat cardiomyocytes (EC50 6.2 +/- 0.5 and 5.7 +/- 0.6 mumol/l, respectively). The selectivity of BDTI and salbutamol for beta 2-adrenoceptors on the cAMP response were 12.4 and 14.3 times, respectively. It is concluded that BDTI is a beta 2-selective adrenoceptor agonist.

Biochemical and pharmacological characterization of high-affinity trimetoquinol analogs on guinea pig and human beta adrenergic receptor subtypes: evidence for partial agonism.

Radioligand binding assays were used to characterize the interaction of a series of trimetoquinol [1-(3',4',5'-trimethoxybenzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoqui nol ine; TMQ] analogs with beta adrenergic receptors (beta-AR). The results indicated that TMQ analogs bound with similar affinities to guinea pig (heart, lung and skeletal muscle) and human (beta-AR in Escherichia coli) beta-1- and beta-2-AR subtypes. However, the isomers of TMQ and 8-fluoro-TMQ bound stereoselectively to beta-AR with the S-isomers having affinities at least 112- and 8-fold greater, respectively, than their corresponding R-isomers. In general, a direct relationship existed between TMQ analog binding to guinea pig beta-AR and functional activity on guinea pig right atria (beta-1) and trachea (beta-2). For selected halogenated TMQ analogs (3',5'-diiodo-TMQ, 3'-iodo-TMQ, 5,8-difluoro-TMQ and 5-iodo-TMQ) which had higher beta-AR affinities than TMQ, but were less potent beta-AR agonists than TMQ, this relationship was not seen. To explain this, the function of the TMQ analogs was analyzed at the level of the beta-AR-associated effector mechanism (i.e., G-protein and adenylyl cyclase). In Chinese hamster ovary cells expressing human beta-2-AR, TMQ and halogenated analogs bound to the receptor with high affinity (nanomolar range); however, they failed to effectively couple with beta-AR-associated G-protein and only partially activated receptor-associated adenylyl cyclase. Receptor occupancies of 0.14, 2 and 23% were required for (-)-isoproterenol, S-(-)-TMQ and 3'5'-diiodo-TMQ to produce equivalent cyclic AMP accumulations in human beta-2-AR Chinese hamster ovary cells. Thus, TMQ and halogenated TMQ derivatives bind stereoselectively to beta-AR with high affinity, and may be classified as partial beta-AR agonists.

Interactions of nonprostanoid trimetoquinol analogs with thromboxane A2/prostaglandin H2 receptors in human platelets, rat vascular endothelial cells and rat vascular smooth muscle cells.

Trimetoquinol (TMQ), a nonprostanoid compound, inhibits thromboxane A2 agonist-induced responses in platelets and vascular smooth muscle. Sixteen TMQ analogs were used to examine the stereochemical requirements of the interaction with thromboxane A2/prostaglandin H2 (TP) receptor sites in human platelets (HP), and cultured rat vascular endothelial (RVEC) and smooth muscle (RVSMC) cells. [3H]SQ 29548 was used as the ligand for TP receptors. The receptor binding affinities of these TMQ analogs for TP receptors in HP, RVEC and RVSMC were highly correlated with each other, and to their reported inhibitory potency values against U46619-induced HP aggregation and serotonin secretion, and contraction of rat aorta. TP receptor binding affinities of TMQ and 8-fluoro TMQ isomers were highly stereoselective (R-isomer > S-isomer), and only the 8-fluoro TMQ isomers gave qualitatively different functional responses in rat aorta. The affinity of TMQ for TP receptors was increased by addition of iodine and fluorine atoms at the 5- and 8-positions of the catechol ring or by replacement of the methoxy groups with iodine atoms on the 1-benzyl ring system. The results indicate that: 1) the stereochemical requirements of TMQ analogs for interaction with TP receptors in these cell systems are the same; 2) although TMQ analogs act as TP receptor antagonists, differences in functional responses by 8-fluoro TMQ isomers in platelets and aorta are not explained by their relative binding affinities to TP receptors; and 3) asymmetric halogenated TMQ analogs should be useful as affinity probes for further characterization of TP receptors.

Use of [3H]trimetoquinol as a radioligand in human platelets: interaction with putative endoperoxide/thromboxane A2 receptor sites.

We have recently shown that trimetoquinol [1-(3,4,5-trimethoxybenzyl)-6,7-dihydroxy-1,2,3,4- tetrahydroisoquinoline; TMQ] is a potent and stereoselective [R(+)-isomer greater than S(-)-isomer] antagonist of aggregation induced by thromboxane A2 and stable epoxymethano PGH2 analogues (U46619; U44069) in human platelets. The present study was undertaken to characterize the pharmacological specificity of binding sites for racemic- [3H]TMQ in washed human platelets. Specific binding of [3H]TMQ, determined by addition of 100 microM R(+)-TMQ, accounted for 12-26% of the total binding at 1.0 microM. Saturation data suggested two binding sites with apparent dissociation constants of 2.8 nM and 1.4 microM for the high and low affinity binding sites, respectively. Maximal binding densities (pmol/mg protein) were 2.3 and 42.9 for the respective high and low affinity sites. The optical isomers of TMQ, cis and trans-isomers of 13-azaprostanoic acid (13-APA), and U46619 inhibited [3H]TMQ specific binding to the low affinity site. R(+)-TMQ was 32-fold more potent than S(-)-TMQ at inhibiting [3H]TMQ binding, and the stereoselective potency difference and concentrations needed to inhibit binding were similar to those required for inhibition of U46619-induced platelet aggregation and serotonin secretion. Similarly, trans-13-APA was 90-fold more potent than cis-13-APA as a competitor of [3H]TMQ binding. U46619 also inhibited TMQ binding at concentrations (IC50 = 0.4 microM) similar to those required for aggregation (EC50 = 0.16 microM) and secretion (EC50 = 0.23 microM). At 1 mM, arachidonic acid inhibited [3H] TMQ binding by only 25%. Our studies indicate that [3H]TMQ interacts with specific binding sites with characteristics indicative of putative endoperoxide/thromboxane A2 receptors in human platelets.

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).

[Determination of trimethoquinol in plasma by electrochemical detection after HPLC separation]. / Bestimmung von Trimethoquinol in Plasma mittels elektrochemischer Detektion nach HPLC-Trennung.

A high pressure liquid chromatographic method with internal analogue standardization for the determination of trimethoquinol (1-1-(3,4,5-trimethoxybenzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoq uinoline hydrochloride) in plasma is described. The method comprises extraction from plasma at pH 9 with ethyl acetate, back-extraction into phosphoric acid and quantification after HPLC separation using a reverse phase by means of electrochemical (reductive) detection after electrochemical preoxidation. When using 5 ml plasma the lower limit of detection is 40 pg/ml. Under routine conditions the limit of determination is determined to be 0.1 ng/ml. The variation coefficients drop from about 7% in the low range to about 4% at 5 ng/ml. The determination of trimethoquinol is not impaired by its monomethylated metabolites.