Binding of nicotine and homoazanicotine analogues at neuronal nicotinic acetylcholinergic (nACh) receptors.

A total of 20 substituted analogues of nicotine (1a) and homoazanicotine (3a) were examined in order to determine whether or not they might bind in a similar manner at alpha4beta2 nicotinic acetylcholinergic (nACh) receptors. It was found that parallel structural changes in the two series resulted in parallel shifts in affinity. Evidence suggests that the two series are binding in a comparable fashion.

Homoazanicotine: a structure-affinity study for nicotinic acetylcholine (nACh) receptor binding.

We have recently identified 3-[(1-methyl)-4,5-dihydro-1H-imidazol-2-yl)methyl]pyridine (homoazanicotine, 8) as a novel nicotinic acetylcholinergic (nACh) receptor ligand. In the present investigation, after we determined that 8 binds selectively at nicotinic (K(i) = 7.8 nM) vs muscarinic (K(i) > 10,000 nM) acetylcholinergic receptors, we examined its structure-affinity relationships for nACh receptor binding. The features investigated included the influence of (i) the composition of connector that separates the two rings, (ii) the N-methyl group, (iii) the ring opening of the imidazoline ring, (iv) the pyridine nitrogen atom, and (v) the aromatization of the imidazoline ring on nACh receptor affinity. As with nicotine, the parent structure seems optimal and most structural changes reduce nACh receptor affinity. Also, as with nicotine analogues, alteration of the spacer group influences affinity in a manner that is somewhat different than that seen with the parent structure.

Chain-lengthened and imidazoline analogues of nicotine.

Analogues of nicotine (1) and azanicotine (3) were prepared with an additional methylene group inserted between the two rings (i.e., homonicotine and homoazanicotine; 6 and 5, respectively). Although 6 (Ki = 3110 nM) and 3 (Ki = 206 nM) bind at nACh receptors with > or = 100-fold lower affinity than nicotine (Ki = 2.1 nM), 5 displays high affinity (Ki = 7.8 nM). Like nicotine (ED50 = 12 microg/mouse), both 3 and 5 (ED50 = 21 and 19 microg/mouse, respectively) produced antinociceptive activity in the tail-flick assay following intrathecal administration. The antinociceptive actions of 3 and 5, unlike those of nicotine, were not antagonized by mecamylamine. Compounds 3 and 5 might represent novel analgesic agents that act via a non-nicotinic mechanism, or via a nicotinic mechanism that is distinct from that mediating the antinociceptive actions of nicotine.

Structure-activity relationship at alpha-adrenergic receptors within a series of imidazoline analogues of cirazoline.

Several analogues of cirazoline (2), a selective alpha1-adrenoreceptor agonist, were prepared and their pharmacological profiles studied. Although at the alpha1-adrenoreceptor all the compounds displayed a significant agonist activity, at the alpha2-adrenoreceptor they showed either agonist or antagonist activity depending on the nature of the phenyl substituent. The qualitative structure-activity relationship led us to the conclusion that the oxygen atom in the side-chain is essential for alpha1-agonist activity, while the cyclopropyl ring is not, and may be replaced by several groups. Of the groups studied, isopropoxy appears to be the best. Instead, the same substitution (i.e., isopropoxy for the cyclopropyl ring) at alpha2-adrenoreceptors causes a reversal of activity. On the other hand, the cyclopropyl ring seems to be important for alpha1-selectivity. Compound 20 is the most potent alpha1-agonist of the series, being equiactive with cirazoline on rat vas deferens and in pithed rat.

Coupling of I(1) imidazoline receptors to the cAMP pathway: studies with a highly selective ligand, benazoline.

Clonidine and benazoline are two structurally related imidazolines. Whereas clonidine binds both to alpha(2)-adrenoceptors (alpha(2)R) and to I(1) imidazoline receptors (I(1)R), benazoline showed a high selectivity for imidazoline receptors. Although the alpha(2)R are negatively coupled to adenylate cyclase, no effect on cAMP level by activation of I(1)R has been reported so far. We therefore aimed to compare the effects of clonidine and benazoline on forskolin-stimulated cAMP levels in cell lines expressing either I(1)R only (PC12 cells), alpha(2)R only (HT29 cells), or I(1)R and alpha(2)R together (NG10815 cells). Clonidine proved able to decrease the forskolin-stimulated cAMP level in the cells expressing alpha(2)R and this effect could be blocked by rauwolscine. In contrast, in cells lacking these adrenoceptors, clonidine had no effect. On the other hand, benazoline and other I(1) receptor-selective imidazolines decreased forskolin-stimulated cAMP level in the cells expressing I(1)R, in a rauwolscine- and pertussis toxin-insensitive manner. These effects were antagonized by clonidine. According to these results, we demonstrated that 1) alpha(2)R and I(1)R are definitely different entities because they are expressed independently in different cell lines; 2) alpha(2)R and I(1)R are both implicated in the cAMP pathway in cells (one is sensitive to pertussis toxin and the other is not); and 3) I(1)R might be coupled to more then one transduction pathway. These new data will be essential to further understand the physiological implications of the I(1)R and the functional interactions between I(1) receptors and alpha(2)-adrenoceptors.

Hyperphagic effect of novel compounds with high affinity for imidazoline I(2) binding sites.

Previous studies have suggested that imidazoline I(2) receptors play a role in feeding control in rats. The effect of subcutaneous (s.c.) injections of four novel imidazoline I(2) ligands, 2-naphthalen-2yl-4,5-dihydro-1H-imidazole hydrochloride (benazoline), 2-styryl-4,5-dihydro-1H-imidazole oxalate (tracizoline), o-nitro-tracizoline and o-methyl-tracizoline (metrazoline) on food intake during the light phase was now evaluated in freely feeding male Wistar rats. Their effect was compared to that of idazoxan, a high-affinity ligand at imidazoline I(2) binding sites, but also a potent alpha(2)-adrenoceptor antagonist. Compared to idazoxan, metrazoline exhibits a higher pK(i) for imidazoline I(2) binding sites in rat liver, while the other compounds have a slightly lower pK(i); on the other hand, the novel compounds have much lower affinity than idazoxan at alpha(2)-adrenoceptors. Idazoxan stimulated drinking at a dose as low as 1 mg/kg, and evoked feeding at a higher dose (30 mg/kg). The selective alpha(2)-adrenoceptor antagonist 2-methoxy-idazoxan (RX821002), with negligible affinity at imidazoline I(2) binding sites, significantly increased drinking but failed to stimulate feeding at doses of 10-50 mg/kg. Metrazoline induced hyperphagia and water drinking at doses of 50 mg/kg or higher. Its dipsogenic effect was secondary to the hyperphagic effect, since it was not observed in rats without access to food. Benazoline significantly increased feeding only in response to 30 mg/kg, but its effect was less pronounced than that of metrazoline. Tracizoline and o-nitro-tracizoline were inactive. Following injection into the lateral cerebroventricle at doses up to 100 microgram/rat, and into the third or fourth brain ventricle at doses up to 50 microgram/rat, neither idazoxan nor metrazoline induced hyperphagia. The present results support the idea that imidazoline I(2) ligands influence feeding in rats, and suggest that their site of action is not in the central nervous system. The finding that idazoxan elicits a more potent hyperphagic effect than metrazoline and benazoline, although its affinity for imidazoline I(2) binding sites is lower than that of metrazoline and similar to that of benazoline, raises the question whether its hyperphagic effect might also be due to interaction with other receptors.

Structure-activity relationships in 1,4-benzodioxan-related compounds. 6. Role of the dioxane unit on selectivity for alpha(1)-adrenoreceptor subtypes.

WB 4101-related benzodioxans 3-9 were synthesized, and their biological profiles at alpha(1)-adrenoreceptor subtypes and 5-HT(1A) serotoninergic receptors were assessed by binding assays in CHO and HeLa cells membranes expressing the human cloned receptors. Furthermore, receptor selectivity of selected benzodioxan derivatives was further determined in functional experiments in isolated rat vas deferens (alpha(1A)) and aorta (alpha(1D)) and guinea pig spleen (alpha(1B)), in additional receptor binding assays in rat cortex membranes containing alpha(2)-adrenoreceptors and 5-HT(2) serotoninergic receptors, and in rat striatum membranes containing D(2) dopaminergic receptors. An analysis of the results of receptor binding experiments for benzodioxan-modified derivatives 3-9 showed high affinity and selectivity toward the alpha(1a)-adrenoreceptor subtype for compounds 3-5 and 7 and a reversed selectivity profile for 9, which was a selective alpha(1d) antagonist. Furthermore, the majority of structural modifications performed on the prototype 1 (WB 4101) led to a marked decrease in the affinity for 5-HT(1A) serotoninergic receptors, which may have relevance in the design of selective alpha(1A)-adrenoreceptor antagonists. The exception to these findings was the chromene derivative 8, which exhibited a 5-HT(1A) partial agonist profile.

Ligand binding to I2 imidazoline receptor: the role of lipophilicity in quantitative structure-activity relationship models.

A series of 2-trans-styryl-imidazoline (tracizoline) congeners were designed and tested to develop 2-D and 3-D QSAR models for their binding to imidazoline (I2) receptor. The important role of lipophilicity was assessed by classical 2-D QSAR study (Hansch approach) and by comparative molecular field analysis (CoMFA) with the inclusion of the molecular lipophilicity potential (MLP), as an additional descriptor, besides standard steric and electrostatic fields. Results from these studies were compared to those obtained in a previous modeling study of I2 receptor ligands and integrated into a new, comprehensive model, based on about sixty I2 receptor ligands. This model revealed, at the three-dimensional level, the most significant steric, electrostatic, and lipophilic interactions accounting for high I2 receptor affinity.

Use of frozen sections for the pharmacological characterization of compounds active on neurotransmitter receptors.

Radioligand binding assay represents an important technique in pharmacological and pharmaceutical research for assessing the receptor profile of new drugs or of compounds under development. In this study, the pharmacological profile and the receptor specificity of compounds active on dopamine and muscarinic cholinergic receptor subtypes were evaluated using as a receptor source, membrane preparations or frozen sections. Dopamine D1-like receptors were assayed in membrane preparations or frozen sections of rat striatum and kidney with [3H]-SCH 23390 as a ligand. Rat striatum, kidney and atrium were used as a source of dopamine D2-like receptors with [3H]-spiperone as a ligand. The non-selective muscarinic cholinergic receptor antagonist [3H]-N-methyl-scopolamine was used to label muscarinic cholinergic receptors in the rat. Frontal cortex represented the source of M1 receptor subtype, heart the source of M2 receptor subtype, sub maxillary gland the source of M3 receptor subtype and striatum the source of M4 receptor subtype. With the exception of cardiac tissue, no significant differences were noticeable in the affinity of dopaminergic or muscarinic cholinergic compounds tested using membrane particles or 8 microns thick slide-mounted section. In the heart, frozen sections gave lower dissociation constant and inhibition constant values than membranes. The above findings suggest that radioligand binding assay on slide-mounted tissue sections may represent a suitable technique for assessing the receptor profile of drugs under development for the treatment of disorders characterised by dopaminergic or muscarnic cholinergic dysfunction.

Imidazoline receptors: qualitative structure-activity relationships and discovery of tracizoline and benazoline. Two ligands with high affinity and unprecedented selectivity.

The observation that all the attempts to characterize imidazoline (I) receptors have been carried out with non-selective or poorly selective ligands prompted us to undertaken research aimed at developing selective ligand(s). In previous work using, as a starting point, cirazoline I, a potent alpha 1-adrenergic receptor agonist that also binds to I receptors, we showed that removal of the cyclopropyl ring (2) retains high affinity for I2 receptors while reducing alpha 1-adrenergic agonist activity. However, it was felt that this residual, albeit modest, alpha 1-adrenergic agonist activity might diminish the usefulness of compound 2, and we now report on our continuing efforts in this field. Starting from compound 2, we first eliminated the alpha 1-agonist component by isosteric replacement and then, by means of conformational restrictions on compound 7, succeeded in discovering tracizoline (9) and benazoline (12). These two new ligands with high affinity (pKi value 8.74 and 9.07, respectively) and unprecedented selectivity with respect to both alpha 2- (I2/alpha 2 7,762 and 18,621) and alpha 1- (I2/alpha 1 2,344 and 2,691) adrenergic receptors, are valuable tools in the study of I receptor structure and function. In addition, the large number of derivatives studied has allowed us to establish congruent qualitative structure-activity relationships and identify some structural elements governing affinity and selectivity.

2-D and 3-D modeling of imidazoline receptor ligands: insights into pharmacophore.

A 3-D quantitative structure-activity relationship (3-D QSAR) study was carried out using comparative molecular field analysis (CoMFA) on both imidazoline (I2R) and alpha 2 receptor binding affinities of a large series of 2-substituted imidazolines. Significant cross-validated correlations, having promising predictive ability, were obtained along with 3-D pharmacophore models that defined the spatial regions where steric and electrostatic interactions may modulate the in vitro binding affinities and indicated possible physicochemical and structural requirements for I2/alpha 2 receptor selectivity.

Synthesis and structure-activity relationship studies in a series of 2-substituted 1,3-dioxolanes modified at the cationic head.

To develop ligands that may be useful in exploring muscarinic receptor heterogeneity, we synthesized a series of analogues of 2,2-diphenyl-[1,3]-dioxolan-4-ylmethyl-dimethylamine oxalate and methiodide bearing a modified cationic head. These compounds, when tested on tissues containing the three subtypes M1, M2, and M3, behaved as muscarinic antagonists whose results showed that different substituents on the quaternary and tertiary nitrogen affect affinity and selectivity in different ways. In particular comparison of the affinities of these ligands with those of the reference compounds points out that compounds bearing an ethyl substituent improve the affinity of the molecule at the three subtypes while compounds bearing a phenethyl substituent are more selective for the M3 sites.

Muscarinic cholinergic receptors and acetylcholinesterase activity in umbilical artery and vein in pregnancy-induced hypertension (pre-eclampsia).

The influence of pregnancy-induced hypertension (pre-eclampsia) on muscarinic cholinergic receptors and on acetylcholinesterase (AChE) activity was investigated using frozen sections of the umbilical artery and vein. Pre-eclamptic patients undergoing Caesarean delivery and normotensive pregnant control woman undergoing Caesarean delivery with similar parity, gestation length and age were examined. Muscarinic cholinergic receptors were assayed in frozen sections of the umbilical artery and vein by a radioligand binding assay technique, using [3H]-N-methyl scopolamine (NMS) as a ligand. AChE was demonstrated with a histochemical technique associated with microdensitometry. [3H]-NMS was specifically bound to sections of both umbilical artery and vein in a manner consistent with the labelling of muscarinic cholinergic receptors. The affinity of the radioligand was similar in the two vessels, whereas the maximum density of binding sites (Bmax) was higher in the umbilical vein than in the artery. A faint AChE reactivity was observed in the tunica media of both umbilical artery and vein. In pre-eclampsia, a loss of [3H]-NMS binding sites not accompanied by changes in the affinity of radioligand was found. The decrease of muscarinic cholinergic receptors involved to a greater extent the umbilical artery than the vein. No differences in AChE activity were found at the level of umbilical artery and vein between control and pre-eclamptic subjects. These findings suggest that pre-eclampsia is characterized by a loss of muscarinic cholinergic receptors in the umbilical circulation not accompanied by changes of the acetylcholine catabolizing enzyme AChE. It is possible that the decreased density of vascular muscarinic cholinergic receptors in pregnancy-induced hypertension contribute to the increased resistance of the umbilical circulation occurring in pre-eclampsia.

Muscarinic thioligands with cyclopentane nucleus.

Some thio- and the benzoyl-derivatives of deoxamuscarine were synthesized and tested as muscarinic agonists using radioligand binding assays and functional tests. In comparison with deoxamuscarine, used as reference compound, no dimension/distance modification is tolerated for correct lipophilic pocket recognition. The substitution of the ammonium group with a sulphonium group significantly decreased muscarinic potency. The so-called 'muscarinic sub-site' accepts relatively bulky functions as long as it is bound to the cyclopentane carrier by an oxygen bridge. Esterification of this moiety increases the M2 subtype selectivity, while etherification heightens that of M3.