Clonidine Derivatives Research Articles

The identification of a potent imidazoline-based vascular K(ATP) channel antagonist.

In this study the activity of a number of novel imidazoline-based compounds (IMID series) was assessed by functional and binding studies to determine their actions at K(ATP) channels. The novel compounds, which we synthesised, were methoxy-, methyl-, butyl- and fluorophenyl derivatives of clonidine. In functional studies we determined the potency (by calculating a pK(B) value) of the IMID compounds to antagonise levcromakalim responses in segments of isolated pig coronary artery. The most potent compounds identified (laboratory codes: IMID-1M, IMID-26F and IMID-4F) had apparent pK(B) values of approximately 7 which is similar to that for the sulphonylurea, glibenclamide and the lipophilic quaternary ion, tetraphenylphosphonium. This inhibitory action was specific for levcromakalim since the imidazoline antagonist IMID-1M failed to effect vasorelaxation response-curves to the non-KATP channel opener, sodium nitroprusside. In the spontaneously beating rat right atrium preparation the majority of the compounds were able to cause slowing of heart rate, but with low EC50 values (approximately 10-30 microM). In binding studies, the compounds were unable to displace binding of [3H]P1075 to bovine aortic smooth muscle preparations nor [3H]glibenclamide binding to rat cerebral cortex membranes. These studies show that some imidazoline-based compounds are potent antagonists of levcromakalim-mediated vasorelaxation responses in the pig coronary artery. The compounds displayed only minimal bradycardic activity. The site of action of the imidazoline compounds does not appear to be the same as that used by K(ATP) channel openers or sulphonylurea-based antagonists. It is likely that these compounds interact with the K(ATP) channel pore itself.

Functional and electrophysiological effects of a novel imidazoline‐based KATP channel blocker, IMID‐4F

1. The functional and electrophysiological effects of IMID-4F (2-[N-(2, 6-dichlorophenyl)-N-(4-flurorobenzyl)amino]imidazoline), a fluoro-benzyl derivative of clonidine, on vascular K(ATP) channels were investigated. In pig coronary artery, IMID-4F inhibited the vasorelaxation response to the K(ATP) channel opener levcromakalim with a pK(B) value of approximately 7.1. IMID-4F (30 microM) did not affect the vasorelaxation response to sodium nitroprusside (SNP). 2. In rat mesenteric artery smooth muscle cells IMID-4F (1 - 10 microM) caused a concentration-dependent depolarization of membrane potential. IMID-4F (10 microM) abolished the hyperpolarizing effects of levcromakalim (10 microM). 3. In patch clamp experiments using rat mesenteric artery smooth muscle cells, K(ATP) channel currents induced by levcromakalim (10 microM) were inhibited by IMID-4F (0.3 - 3 microM) in a concentration-dependent manner. The calculated IC(50) for IMID-4F inhibiting K(ATP) channel current was approximately 0.8 microM. 4. Radioligand binding studies using bovine aortic smooth muscle cell membranes showed that IMID-4F (30 microM) did not displace binding to the K(ATP) channel opener [(3)H]-P1075. However, both levcromakalim (10 microM) and glibenclamide (10 microM) caused significant displacement of [(3)H]-P1075. 5. These studies show that the imidazoline compound IMID-4F is one of the most potent antagonists of arterial K(ATP) channels identified. Vasorelaxation, hyperpolarization and K(+) currents through K(ATP) channels were all inhibited by IMID-4F at micromolar concentrations. Radioligand binding studies indicate that IMID-4F does not bind to the same site as levcromakalim or as glibenclamide. Considering other evidence, it is likely that IMID-4F acts by interacting directly with the pore of the K(IR) channel, rather than through the sulphonylurea subunit of the K(ATP) channel complex.

Prediction of distribution coefficients from structure. Comparison of calculated and experimental data for various drugs

The efficiency of the program PrologD to predict distribution coefficients (D) at any pH and pairing ion concentration has been tested using experimental logD values for various drugs measured under standard conditions of buffers and ionic strength. Clonidine derivatives, fluoroquinolones and beta-blockers were included as particular pharmacological classes within the testing data set. Calculations were performed using the three logP estimation options implemented in the program. PrologD proved to be very efficient and can be of great advantage in drug research. Prediction patterns and correlations between experimental and calculated data indicate acceptable results for more than 80% of the data. In addition, comparable studies using the different options permitted suggestions for the more suitable logP estimation method in respect of the particular classes of compounds.

Mechanism of the sympathoinhibition produced by the clonidine-like drugs rilmenidine and moxonidine.

The mechanism of the sympathoinhibition produced by two new derivatives of clonidine, rilmenidine and moxonidine, was studied. One aim was to determine the receptor responsible for the central sympathoinhibition by these drugs. Rilmenidine and moxonidine were injected into the cisterna cerebellomedullaris. They decreased blood pressure and the plasma noradrenaline concentration. After rilmenidine and moxonidine, two selective alpha 2-adrenoceptor antagonists (devoid of affinity for I1 binding sites), yohimbine and SK&F86466, were given intracisternally. They completely counteracted the hypotensive effects of rilmenidine and moxonidine, indicating that alpha 2-adrenoceptors are involved in the central sympathoinhibition produced by these drugs. The other aim was to determine if peripheral presynaptic inhibition of noradrenaline release from postganglionic sympathetic neurons contributes to the overall reduction of sympathetic tone. Rilmenidine and moxonidine were injected i.v. in pithed rabbits with electrically stimulated sympathetic outflow. They dose-dependently lowered blood pressure and the plasma noradrenaline concentration and inhibited stimulation-evoked cardioacceleration. Moreover, the doses necessary for these peripheral effects were identical to the doses that reduce the sympathetic nerve firing rate and blood pressure in conscious rabbits. These observations indicate that peripheral presynaptic inhibition of noradrenaline release from postganglionic sympathetic neurons contributes to the overall reduction of sympathetic tone produced by rilmenidine and moxonidine in intact animals.

Preparation of radiolabeled clonidine for use in radioimmunoassay

The preparation of stable and immunoreactive radioiodinated clonidine for the development of a radioimmunoassay of clonidine is described. Tyrosylmethyl ester (TME) conjugate of carboxylic acid derivative of clonidine was radioiodinated using chloramine T as the oxidizing agent. The radioiodinated clonidine was purified by thin layer chromatography, gel filtration and solvent extraction. Radiochemical purity, specific activity and immunoreactivity were determined. The stability of the labeled compound during storage was tested in various solvents at different temperatures.

Determination of clonidine in human plasma by gas chromatography—electron-impact mass spectrometry

A new and sensitive gas chromatographic—mass spectrometric (GC—MS) assay for routine analysis of clonidine in human plasma is described. Quantification is carried out with simple electron-impact ionization mass spectrometry and solid-phase cartridges for preliminary extraction from plasma. The pentafluorobenzyl derivative of clonidine yields an intense ion fragment at m/z 354, and the lower limit of detection is 0.025 ng/ml for a 1-ml plasma sample. The practical applicability of this method is demonstrated by determining plasma concentrations of clonidine in a clinical study of a new transdermal delivery system for clonidine.

Determination of moxonidine (BDF 5895) in plasma by gas chromatography—negative ion chemical ionization mass spectrometry

For the measurement of the pharmacokinetic behaviour of moxonidine, 4-chloro-5-(2-imidazolin-2-yl-amino)-6-methoxy-2-methylpyrimidine, an extremely sensitive analytical method was needed. The GC-MS method developed is specific and reliably detects moxonidine plasma levels down to 40 pg ml −1. Using negative ion chemical ionization (NICI) the M − fragment of the ditrifluoromethyl benzamide derivative of moxonidine ( m/ z 721) and the [M-HCl] − fragment of the ditrifluoromethyl benzamide derivative of clonidine (internal standard, m/ z 673) were monitored in the selected ion monitoring mode, ensuring a specific and sensitive detection of the compounds. The validation process carried out included assay precision, repeatability, linearity, accuracy, stability and estimation of the detection and determination limits. The plasma-level time-curves and pharmacokinetic parameters from two volunteers after oral administration of 0.2 mg moxonidine are presented and demonstrate the practicability of the method in, for example, clinical studies.

Effects of new clonidine derivatives on rabbit intraocular pressure

AbstractEffects of 25 topically applied clonidine derivatives on rabbit IOP recovery model using intravenous hypertonic saline were studied. Among them, 12 compounds were dissolved in aqueous medium, 11 compounds in polyethylene glycol (PEG 200) and 2 compounds in dimethyl sulfoxide (DMSO). Four compounds showed marked delay in IOP recovery and were more active than S‐timolol and clonidine. Five compounds were about equiactive to S‐timolol and more active than clonidine in suppression of IOP recovery and prolongation of the duration of drug action. There were three compounds that were less active than S‐timolol yet they were still as active or were active than clonidine. Thirteen out of 25 compounds were inactive in suppressing IOP recovery curves. Most of these active compounds (9 out of 12) suppressed IOP recovery in the same magnitude for both treated and non‐treated contralateral eyes, indicating that these effects were manifested though central actions. Three out of 12 compounds showed more effect with treated eyes than with nontreated contralateral eyes, indicating that the local effects were predomiant.

Effect of 1,4-dioxanyl substitution on the adrenergic activity of some standard α-adrenoreceptor agents

A preliminary communication reported on the pharmacology of the potent partial α 2-agonist (2-(1,4-benzodioxan-6-ylamino)-2-imidazoline, a 1,4-dioxan derivative of clonidine. Its degree of agonism/antagonism depended upon the peripheral or central α 2-adrenoreceptor system studied. It was of interest to discover whether a similar substitution of the 1,4-dioxan moiety in other standard α-adrenergic agents would similarly produce high affinity compounds of complex pharmacological profile. The same substitution when introduced into guanfacine, fenmetazole and tolazoline resulted in unpredictable changes in profile with a reduction in α-affinity.

Central α2‐Adrenergic Control of Colonic Transit in Rats

The effects of central and peripheral administration of α2‐adrenoceptor agonists on colonic propulsion were examined in conscious rats chronically fitted with a catheter inserted in the lumen of the proximal colon and a cannula placed in a cerebral lateral ventricle. The mean retention time of a marker in the colon was determined by administration of a bolus of [51Cr]sodium chromate into the proximal colon and collection of the feces at hourly intervals. In control studies the colonic mean retention time was 7.5 ± 1.6 hours. Clonidine administered intraperitonedy or intracerebroventricularly produced a dose‐dependent increase in mean retention time but was more potent when given intracerebroventricularly. St‐91, a quaternary derivative of clonidine which poorly crosses the blood‐brain barrier, delayed colonic transit after intracerebroventricular, but not after intraperitoneal, administration at the same dose. The increase in colonic mean retention time induced by intraperitonedy administered clonidine was antagonized by intracerebroventricular administration of yohimhine but not of prazosin, an α1‐adrenoceptor antagonist. Yohimbine intracerebroventricularly or intraperitonedy administered alone at the same dose accelerated the colonic transit. Such data suggest a possible role of central α2‐adrenoceptors in the regulation of colonic motility in rats.

Changes in systemic haemodynamics, regional myocardial function and perfusion induced by alinidine in pigs with and without narrowed coronary arteries.

Intravenous administration of 0.2-6.0 mg kg-1 of alinidine, an N-allyl derivative of clonidine, exerted a pronounced bradycardic action and also decreased the maximum rate of rise in left ventricular pressure (max LVdP/dt), cardiac output, and mean arterial blood pressure in anaesthetized as well as in conscious pigs. When, in the anaesthetized animals, heart rate was kept constant by atrial pacing, no changes were observed with alinidine in maxLVdP/dt and cardiac output at doses up to 0.4 mg kg-1. At higher doses, however, there was a significant reduction in these variables, demonstrating a marked negative inotropic action of the drug at higher concentrations. In pigs with normal hearts alinidine homogeneously reduced myocardial perfusion, but no imbalance in the myocardial oxygen supply-demand relationship was noticed due to a simultaneous reduction of the metabolic needs of the myocardium. After constriction of the left anterior descending coronary artery to an extent that systolic myocardial wall thickening was reduced to 30% of its base-line value, transmural myocardial blood flow and the endocardial-epicardial (endo-epi) ratio in the affected myocardial segment had decreased from 1.42 +/- 0.15 to 0.68 +/- 0.11 ml min-1 g-1 and from 1.05 +/- 0.08 to 0.59 +/- 0.10, respectively. Though myocardial blood flow in the normal segments decreased, alinidine, in a dose (0.4-0.9 mg kg-1) that lowered heart rate by about 25%, caused a redistribution of blood flow to the ischaemic myocardium in favour of the endocardium as the endo-epi ratio increased to 1.08 +/- 0.12. Concomitantly, systolic wall thickening of the hypokinetic segment returned to 50% of its base-line value. Thus, alinidine ameliorated the imbalance between oxygen supply and demand in the post-stenotic myocardial segment, probably by prolongation of the diastolic perfusion time and a diminution in myocardial oxygen demand both through a reduction in heart rate. However, other factors, such as negative inotropy may also have partly contributed. Since treatment of pathological cardiovascular conditions with drugs with positive inotropic and vasodilatory properties is often accompanied by tachycardia, we also studied the effects alinidine in combination with the phosphodiesterase inhibitor sulmazole. Alinidine did not interfere with the positive inotropic and vasodilatory (systemic as well as coronary) properties of sulmazole, but prevented the tachycardia. These data suggest that combination of negative chronotropic drugs as alinidine with agents with a cardiovascular profile similar to sulmazole may be attractive, when unwanted tachycardia occurs.

The 3,4-dimethoxybenzyl group: A protective group for new 2-imino-imidazolidine derivates

The 3,4-dimethoxybenzyl group has been used to protect 2-imino-imidazolidine derivatives, so we could realize selective O-alkylation, to obtain new clonidine derivatives.

Further studies of alinidine induced bradycardia in the presence of caesium.

Alinidine, the N-allyl derivative of clonidine, reduces heart rate in animals and man by a selective action on the sinoatrial node. In this study, in the presence of caesium, which blocks the time dependent inward current activated by hyperpolarisation (ih or if), alinidine still caused a concentration related bradycardia in the rabbit sinoatrial node. Within the clinical range of concentrations of alinidine the curve relating heart rate to alinidine concentration in the presence of caesium was parallel to that observed in its absence. It is concluded that the alinidine induced bradycardia cannot be attributed to blockade of ih.

Bis(trifluoromethyl)aryl derivatives for drug analysis by gas chromatography electron capture negative ion chemical ionization mass spectrometry. Application to the measurement of low levels of clonidine in plasma.

A gas chromatographic mass spectrometric assay for clonidine in plasma with a detection limit of a few picograms per ml was required. The p-trifluoromethylbenzyl, pentafluorobenzyl and pentafluorobenzoyl derivatives of clonidine were synthesized and the electron capture negative ion chemical ionization mass spectra of these compounds show extensive fragmentation with prominent ions at m/z 35 and 37 due to the two chlorine atoms in the clonidine molecule. Selected ion monitoring of specific high mass ions in these mass spectra indicated that the required sensitivity could not be obtained with these derivatives. Several bis(trifluoromethyl)pyrimidines were synthesized and these compounds were found to give an intense negative ion current under conditions of resonance electron capture. Consequently, a derivative of clonidine containing a bis(trifluoromethyl)aryl group was synthesized by reacting the drug with 3,5-bis(trifluoromethyl)benzoyl chloride. The negative ion mass spectrum of the reaction product has a base peak at m/z 673 and, when this ion is specifically monitored, an amount of derivative equivalent to 1 picogram of clonidine can be detected. This allowed the development of an assay for clonidine in plasma with a precision of 8% (SD) at 50 pg ml-1, 22% (SD) at 20 pg ml-1 and a lower limit for quantitative determination of 10 pg ml-1. Plasma concentrations of clonidine in 10 subjects given a single 50 micrograms oral dose are reported.

Study of two alkylating derivatives: The p-isothiocyanato- and the p-methylisothiocyanato-clonidine

Pharmacological experiments with isolated rat aorta and radioligand binding studies in rat cerebral membranes were performed with the p-isothiocyanato (p-NCS) and p-methylisothiocyanato (p-CH 2-NCS) derivatives of clonidine in order to assess their selectivity for α 1- and α 2-adrenoceptors, and to characterie their ability to alkylate α-adrenoceptors. Preincubation of rat aortic strips with both derivatives produced non-parallel rightward shifts in the dose-response curves of noradrenaline and significantly depressed the maximum response in a manner characteristic of irreversible receptor antagonists. The p-CH 2-NCS derivative was slightly more potent than the p-NCS derivative. Further analysis of the data indicated that treatment of rat aorta with a 30 μM concentration of the p-CH 2-NCS derivative alkylated all but 2.4 percent of the α-adrenoceptors, whereas a 100 μM concentration of the p-NCS derivative was required to produce a similar degree of α-adrenoceptor alkylation. Radioligand binding studies indicate an apparent 2 fold α 2-adrenoceptor selectivity for the p-NCS derivative. In contrast, the p-CH 2-NCS derivative displayed 7 fold selectivity for α 1-adrenoceptors. Interestingly, both alkylating derivatives of clonidine produced dose-dependent contractile responses in rat aorta with pD 2 values of 6.30 and 5.56 for the p-NCS and p-CH 2-NCS derivatives, respectively, relative to a pD 2 of 7.67 for clonidine. The order of potency of the two alkylating derivatives of clonidine for producing contraction of rat aorta is the opposite of that for antagonizing the contractile effects of noradrenaline. The results suggest that the p-NCS and p-CH 2-NCS derivatives of clonidine non-competitively antagonize noradrenaline by irreversibly alkylating α-adrenoceptors.

Interaction of clonidine, its methylene-bridged analog, St 1913, and the benzylic hydroxyl-substituted derivative, St 1965, with alpha 1- and alpha 2-adrenoreceptors.

The effects of benzylic hydroxyl substitution on the activity of a close structural analog of clonidine was assessed at alpha 1- and alpha 2-adrenoreceptors both in vitro and in vivo in order to uncover possible differences that this substitution may have on the effects of imidazolines and phenethylamines at adrenoreceptors. In all test systems, the presence of the benzylic hydroxyl group was associated with a consistent and marked decrease in activity. These findings are in agreement with our previous studies with imidazolines having different pharmacological profiles and different physicochemical properties than the clonidine derivatives reported herein. We conclude, therefore, that the deleterious effects of the benzylic hydroxyl group is ubiquitous among imidazolines and, more importantly, is in marked contrast to the 100-1000 fold enhancement in activity that the benzylic hydroxyl substituent (i.e. beta-hydroxyl group) produces for the phenethylamines. The results support the concept that imidazolines and phenethylamines may interact differently with alpha-adrenoreceptors.

Hypotensive and alpha-adrenergic activities of 2-(2,3,6-trichlorophenylimino) and 2-(2,3-dichloro-6-methylphenylimino) imidazolidine, two potent derivatives of clonidine.

1 alpha-Adrenergic activities (hypotension, bradycardia, sedation) and affinities of clonidine and two newly substituted derivatives, 2-(2,3,6-trichlorophenylimino)imidazolidine (I) and 2-(2,3-dichloro-6-methylphenylimino)imidazolidine (II), were determined in various in vitro and in vitro models.2 In anaesthetized normotensive rats, the intravenous -log doses (mol/kg) of clonidine, compound I and II required to decrease mean arterial pressure by 20% were 7.96, 8.39 and 7.79, respectively, and to reduce heart rate by 20% were 7.85, 8.23 and 7.91, respectively. Comparable potencies were obtained in vagotomized rats. Following vertebral arterial infusion of clonidine, compound I and II into anaesthetized cats, the -log doses for 20% diminution in mean arterial pressure were 8.72, 9.17 and 7.92, respectively.3 In pithed normotensive rats, the intravenous -log doses (mol/kg) of clonidine, compound I and II required to elevate diastolic pressure by 50 mmHg were 7.49, 7.80 and 8.04, respectively, whereas 50% of the maximal inhibition of electrical stimulation-induced tachycardia was obtained at -log doses of 8.20, 8.25 and 8.34, respectively.4 In mice, the intraperitoneal -log doses (mol/kg) needed for a prolongation of the hexobarbitone-induced loss of the righting reflex by 100% were 6.51, 6.55 and 6.60 for clonidine, compound I and II, respectively.5 Compounds I and II displayed a higher affinity for alpha(1)- and alpha(2)-adrenoceptors than clonidine, identified in rat cerebral membranes by [(3)H]-prazosin and [(3)H]-clonidine.6 The results show that 2,3,6-trisubstituted derivatives of clonidine are potent alpha-adrenoceptor stimulants. This new class of congeners may have potential for pronounced hypotensive activity following systemic application. It is not likely that among members of this series, hypotensive potency can be separated from sedative activity.

Use of a sympathetic-cholinergic system in the analysis of sympatho-inhibition produced by clonidine and some congeneric derivatives of clonidine

The effects of clonidine and five analogs of clonidine were tested with regard to their ability to depress centrally and peripherally evoked electrodermal responses (EDR) in control cats, as well as in animals pretreated with yohimbine hydrochloride. With the exception of St-91, all of the clonidine-like substances selectively reduced the amplitude of centrally (hypothalamic) evoked responses in a dose-dependent fashion. Clonidine was found to have no significant inhibitory effect at the level of the sympathetic ganglion. The order of central nervous system sympatho-inhibitory potency of these compounds was clonidine ( St-155) > St-375 > St-606 > St-600 > St-608 ⋙ St-91 . Prior treatment with yohimbine hydrochloride (0.5 mg/kg i.v.) antagonized the depressant effect of all of these drugs. These results indicate that clonidine and the clonidine congeners tested (with the exception of St-91) all produce sympatho-inhibition by an action on a CNS α-adrenergic mechanism and demonstrate the usefulness of this electrodermal model system for the analysis of drugs affecting central sympathetic reactivity.

Effects of alinidine (ST 567) on barorecetor-heart rate reflexes and its interactions with clonidine on the baroreflex and on the sympathetic terminals of the isolated atrium

Alinidine (ST 567), an N-allyl derivative of clonidine, slowed the heart rate of conscious rabbits by 41 ± 2.3 (S.E.D.) b/min and reduced mean arterial pressure (MAP) by 6.4±1.4 mmHg (P<0.001). The cardiac slowing was considered to be a direct effect in agreement with previous findings by others, since it was present in rabbits without functioning autonomic nerves, but the fall in blood pressure did not occur in these animals. Alinidine produced no significant changes in the reflex tachycardia response evoked by infusing nitroprusside, or in the pressure-related parameters of the MAP-heart period (HP) curve of the baroreceptor-heart rate reflex (i.e. HP range, gain, or median blood pressure BP 50). Intravenous (i.v.) clonidine produced characteristic rises in baroreflex HP range and gain, which were due to vagal facilitation, and also produced falls in BP 50 and resting MAP. I.v. alinidine suppressed the clonidine-induced vagal facilitation, but had no effect on the blood pressure changes. Intracisternal alinidine could be given in only relatively low dose, but reduced the clonidine-induced rise in vagal component of HP range. The main site of antagonism between i.v. alinidine and clonidine was probably in the CNS. Westudied the nature of the antagonism at the sympathetic nerve terminal of the isolated left guinea pig atrium. Clonidine depressed the inotropic response to field stimulation of the sympathetic nerves and this was competitively antagonised by phentolamine>yohimbine>alinidine at potencies of about 1200:80:1. Alinidine was considered to be a weak but specific α 2-antagonist; it has no α 1-antagonist properties since it was without effect on the contractile response to noradrenaline of the guinea pig aorta. The α 2-antagonist property explains the suppression by alinidine of the clonidine-induced facilitation of the vagal component of the baroreceptor-heart rate reflex.

The measurement of clonidine in human plasma and urine by combined gas chromatography mass spectrometry with ammonia chemical ionization.

The dimethyl derivatives of clonidine and [2H4]clonidine give good chemical ionization mass spectra when ammonia is used as reagent gas. A gas chromatographic mass spectrometric selected ion monitoring assay for the estimation of clonidine in plasma and urine using ammonia chemical ionization is described.