Synthesis and preliminary screening of novel tryptamines as 5-HT4 receptor ligands.

For the development of novel 5-HT(4) receptor ligands we have designed and synthesized two series of 5-methoxytryptamine derivatives varying the substitution on the primary amine. Their biological activities were evaluated in a receptor binding assay where a subset of compounds showed comparable potency to the agonists serotonin and 5-methoxytryptamine. Structure-activity analyses have highlighted promising avenues for further synthetic work and binding modes were proposed by docking these compounds into a homology model of the 5-HT(4) receptor.

The use of tripeptides for lead discovery of 5-HT4 receptor ligands.

A series of 30 tripeptides were synthesized and tested as novel 5-HT4 receptor ligands. Receptor binding assays showed that a subset of compounds had reasonable potency relative to the agonists serotonin and 5-methoxytryptamine. Structure-activity analyses and molecular docking have highlighted avenues for further synthetic work.

Potential antidepressants displayed combined alpha(2)-adrenoceptor antagonist and monoamine uptake inhibitor properties.

Classical antidepressants are thought to act by raising monoamine (serotonin and noradrenaline) levels in the brain. This action is generally accomplished either by inhibition of monoamine metabolism (MAO inhibitors) or by blockade of monoamine uptake (tricyclic antidepressants and selective serotonin or noradrenaline reuptake inhibitors). However, all such agents suffer from a time lag (3--6 weeks) before robust clinical efficacy can be demonstrated. This delay may reflect inhibitory actions of noradrenaline at presynaptic alpha(2A)-adrenergic auto- or heteroreceptors which gradually down-regulate upon prolonged exposure. Blockade of presynaptic alpha(2A)-adrenoceptors by an antagonist endowed with monoamine uptake inhibition properties could lead to new antidepressants with greater efficacy and a shorter time lag. In the literature, only two molecules have been described with such a pharmacological profile. Of these, napamezole (2) was chosen as a point of departure for the design of 4(5)-[(3,4-dihydro-2-naphthalenyl)methyl]-4,5-dihydroimidazole (4a), which displayed the desired profile: alpha(2A)-adrenoceptor antagonist properties and serotonin/noradrenaline uptake inhibition. From this original molecule, a series of derivatives was designed and synthesized, encompassing substituted as well as rigid analogues. Structure-activity relationships permitted the selection of 14c (4(5)-[(5-fluoroindan-2-yl)methyl]-4,5-dihydroimidazole) as a development candidate.

Pharmacological characterization of the human 5-HT(4(d)) receptor splice variant stably expressed in Chinese hamster ovary cells.

The recently identified C-terminal splice variant of the human 5-HT(4) receptor, the h5-HT(4(d)) receptor, was stably expressed in a CHO cell line at 493+/-25 fmol mg(-1) protein. We analysed its pharmacological properties by measuring binding affinities and 5-HT(4) ligand-induced cyclic AMP production. The pharmacological binding profile determined in competition studies with the specific antagonist [(3)H]-GR113808 revealed a rank order of affinity of 5-HT(4) ligands for the h5-HT(4(d)) receptor that was consistent with those previously reported for other 5-HT(4) receptor isoforms. In adenylyl cyclase functional assays, the h5-HT(4(d)) receptor displayed equipotent coupling for all 5-HT(4) agonists tested (EC(50) in the range of 1 - 6 nM). EC(50) values were lower than those previously obtained with the 5-HT(4(e)) receptor stably expressed in CHO cells indicating that the 5-HT(4(d)) receptor was more efficiently coupled to its effector than the 5-HT(4(e)) receptor isoform. Moreover, in terms of agonist efficacy (E(max)), the benzamide derivative, renzapride displayed full agonist properties at the h5-HT(4(d)) receptor (same E(max) as 5-HT) whereas it was previously shown to be a partial agonist at the h5-HT(4(e)) receptor. A constitutive activity of the h5-HT(4(d)) receptor was observed in CHO cells in the absence of any 5-HT(4) ligand. Surprisingly, two 5-HT(4) ligands, SB204070 and RS39604 which are described as highly potent antagonists in various biological models, revealed partial agonist properties at the h5-HT(4(d)) receptor. We conclude that C-terminal tails of 5-HT(4) receptor isoforms may directly influence their functional properties.

New arylpiperazine derivatives as antagonists of the human cloned 5-HT(4) receptor isoforms.

New derivatives of arylpiperazine 9 were designed from ML 10302, a potent 5-HT(4) receptor agonist in the gastrointestinal system. Compounds were synthesized by condensation of a number of available arylpiperazines or heteroarylpiperazines with 2-bromoethyl 4-amino-5-chloro-2-methoxybenzoate. They were evaluated in binding assays on the recently cloned human 5-HT(4(e)) isoform stably expressed in C6 glial cells with [(3)H]GR 113808 as the radioligand. The affinity values (K(i)) depended upon the substituent on the aromatic ring. A chlorine atom produced a marked drop in activity (K(i) > 100 nM), while a m-methoxy group gave a compound with nanomolar affinity (K(i) = 3 nM). The most potent compounds were the heterocyclic derivatives with pyrimidine, pyrazine, pyridazine, or pyridine moieties (compounds 9r, 9t, 9u, 9x, respectively). K(i) values for 9a and 9r were determined for the 5-HT(4(a)), 5-HT(4(b)), 5-HT(4(c)), and 5-HT(4(d)) receptor isoforms transiently expressed in COS cells. The results indicated that the compounds were not selective. They produced an inhibition of the 5-HT-stimulated cyclic AMP synthesis in the C6 glial cells stably expressing the 5-HT(4(e)) receptor and shifted the 5-HT concentration-effect curve on adenylyl cyclase activity with pK(D) values of 7.44 and 8.47, respectively. In isolated human atrial myocytes, 9r antagonized the stimulatory effect of 5-HT on the L-type calcium current (I(Ca)) with a K(D) value of 0.7 nM.

Exploration of the ligand binding site of the human 5-HT(4) receptor by site-directed mutagenesis and molecular modeling.

Among the five human 5-HT(4) (h5-HT(4)) receptor isoforms, the h5-HT(4(a)) receptor was studied with a particular emphasis on the molecular interactions involved in ligand binding. For this purpose, we used site-directed mutagenesis of the transmembrane domain. Twelve mutants were constructed with a special focus on the residue P4.53 of helix IV which substitutes in h5-HT(4) receptors the highly conserved S residue among the rhodopsin family receptors. The mutated receptors were transiently expressed in COS-7 cells. Ligand binding or competition studies with two h5-HT(4) receptor agonists, serotonin and ML10302 and two h5-HT(4) receptor antagonists, [(3)H]-GR113808 and ML10375 were performed on wild type and mutant receptors. Functional activity of the receptors was evaluated by measuring the ability of serotonin to stimulate adenylyl cyclase. Ligand binding experiments revealed that [(3)H]-GR113808 did not bind to mutants P4.53A, S5.43A, F6.51A, Y7.43A and to double mutant F6.52V/N6.55L. On the other hand mutations D3.32N, S5.43A and Y7.43A appeared to promote a dramatic decrease of h5-HT(4(a)) receptor functional activity. From these studies, S5.43 and Y7.43 clearly emerged as common anchoring sites to antagonist [(3)H]-GR113808 and to serotonin. According to these results, we propose ligand-receptor complex models with serotonin and [(3)H]-GR113808. For serotonin, three interaction points were selected including ionic interaction with D3.32, a stabilizing interaction of this ion pair by Y7.43 and a hydrogen bond with S5.43. [(3)H]-GR113808 was also docked, based on the same type of interactions with S5.43 and D3.32: the proposed model suggested a possible role of P4.53 in helix IV structure allowing the involvement of a close hydrophobic residue, W4.50, in a hydrophobic pocket for hydrophobic interactions with the indole ring of [(3)H]-GR113808.

Isolation of the serotoninergic 5-HT4(e) receptor from human heart and comparative analysis of its pharmacological profile in C6-glial and CHO cell lines.

RT - PCR technique was used to clone the human 5-HT(4(e)) receptor (h5-HT(4(e))) from heart atrium. We showed that this h5-HT(4(e)) receptor splice variant is restricted to brain and heart atrium. Recombinant h5-HT(4(e)) receptor was stably expressed in CHO and C6-glial cell lines at 347 and 88 fmol mg(-1) protein, respectively. Expression of h5-HT(4(e)) receptors at the cell membrane was confirmed by immunoblotting. The receptor binding profile, determined by competition with [(3)H]-GR113808 of a number of 5-HT(4) ligands, was consistent with that previously reported for other 5-HT(4) receptor isoforms. Surprisingly, we found that the rank order of potencies (EC(50)) of 5-HT(4) agonists obtained from adenylyl cyclase functional assays was inversely correlated to their rank order of affinities (K(i)) obtained from binding assays. Furthermore, EC(50) values for 5-HT, renzapride and cisapride were 2 fold lower in C6-glial cells than in CHO cells. ML10302 and renzapride behaved like partial agonists on the h5-HT(4(e)) receptor. These results are in agreement with the reported low efficacy of the these two compounds on L-type Ca(2+) currents and myocyte contractility in human atrium. A constitutive activity of the h5-HT(4(e)) receptor was observed in CHO cells in the absence of any 5-HT(4) ligand and two 5-HT(4) antagonists, GR113808 and ML10375, behaved as inverse agonists. These data show that the h5-HT(4(e)) receptor has a pharmacological profile which is close to the native h5-HT(4) receptor in human atrium with a functional potency which is dependent on the cellular context in which the receptor is expressed.