5-HT2A and 5-HT2C receptors and their atypical regulation properties

The 5-HT(2A) and 5-HT(2C) receptors belong to the G-protein-coupled receptor (GPCR) superfamily. GPCRs transduce extracellular signals to the interior of cells through their interaction with G-proteins. The 5-HT(2A) and 5-HT(2C) receptors mediate effects of a large variety of compounds affecting depression, schizophrenia, anxiety, hallucinations, dysthymia, sleep patterns, feeding behaviour and neuro-endocrine functions. Binding of such compounds to either 5-HT(2) receptor subtype induces processes that regulate receptor sensitivity. In contrast to most other receptors, chronic blockade of 5-HT(2A) and 5-HT(2C) receptors leads not to an up- but to a (paradoxical) down-regulation. This review deals with published data involving such non-classical regulation of 5-HT(2A) and 5-HT(2C) receptors obtained from in vivo and in vitro studies. The underlying regulatory processes of the agonist-induced regulation of 5-HT(2A) and 5-HT(2C) receptors, commonly thought to be desensitisation and resensitisation, are discussed. The atypical down-regulation of both 5-HT(2) receptor subtypes by antidepressants, antipsychotics and 5-HT(2) antagonists is reviewed. The possible mechanisms of this paradoxical down-regulation are discussed, and a new hypothesis on possible heterologous regulation of 5-HT(2A) receptors is proposed.     

Novel quinazolinone derivatives as 5-HT7 receptor ligands

5-HT(7) receptor antagonists generated antidepressant-like effects in animal model and the involvement of the 5-HT(7) receptor in other pathophysiological mechanisms such as thermoregulation, learning and memory, and sleep has been highlighted by various studies. As one of our efforts to discover a new type of 5-HT(7) receptor antagonists, we here report on the synthesis and binding affinities to the 5-HT(7) receptor of the quinazolinone library 1, which was designed with various substituents (X, Y, R(1), and R(2)) on the aromatic rings and different carbon chain length. Total 85 compounds of the quinazolinone library 1 were synthesized and the binding affinities of all the synthesized compounds were obtained by radioligand binding assay for the 5-HT(7) receptor. Among the 85 compounds, 24 compounds show very good binding affinities with IC(50) values below 100 nM. Mainly the compounds with IC(50) values below 100 nM have o-OMe or o-OEt as R(2) substituent. The compound with the best binding affinity is 1-68 of which the IC(50) value is 12 nM. In in vivo animal study, some synthesized compounds really have the antidepressant activity in the forced swimming test in mice.     

Relative selectivity of some conformationally constrained tryptamine analogs at 5-HT1, 5-HT1A and 5-HT2 recognition sites

In an attempt to define pharmacophoric differences between 5-HT1, 5-HT1A and 5-HT2 recognition sites, a number of rigid analogs were studied and compared to analogous free chain tryptamines. Racemic partial ergolines RU 27849 and RU 28306 showed reduced potency at all 5-HT1 sites, but were at least equipotent to analogous tryptamines at the 5-HT2 site. A nonergoline-like constrained analog of tryptamine was similar in potency to RU 27849 at all 5-HT1 sites, but showed a 4-fold enhancement in potency over RU 27849 and tryptamine at the 5-HT2 site. At all 3 sites, 3-(tetrahydropyridyl) indoles (unless substituted at the indole 2-position) were the most potent rigid analogs studied and represent the most promising class for the development of selective compounds.     

Aminotriazine 5-HT7 antagonists

The present studies have identified a series of aminotriazines as novel 5-HT(7) receptor antagonists. Compounds 10 and 17 have high affinity for the 5-HT(7) receptor and do not bind to either the 5-HT(2C) or 5-HT(6) receptors. These compounds produce no agonist effects by themselves, and shift the dose-response curve of 5-CT to the right in the manner of an antagonist.     

Quinoline- and isoquinoline-sulfonamide derivatives of LCAP as potent CNS multi-receptor-5-HT1A/5-HT2A/5-HT7 and D2/D3/D4-agents: the synthesis and pharmacological evaluation

Two series of arylpiperazinyl-alkyl quinoline-, isoquinoline-, naphthalene-sulfonamides with flexible (13-26) and semi-rigid (33-36) alkylene spacer were synthesized and evaluated for 5-HT(1A), 5-HT(2A), 5-HT(6), 5-HT(7) and selected compounds for D(2), D(3), D(4) receptors. The compounds with a mixed 5-HT and D receptors profile 16 (N-{4-[4-(3-chlorophenyl)-piperazin-1-yl]-butyl}-3-quinolinesulfonamide) and 36 (4-(4-{2-[4-(4-chloro-phenyl)-piperazin-1-yl]-ethyl}-piperidine-1-sulfonyl)-isoquinoline), displaying antagonistic activity at 5-HT(7), 5-HT(2A), D(2) postsynaptic sites, produced antidepressant-like effects in the forced swim test in mice and showed significant anxiolytic activity in the plus-maze test in rats. The lead compound 36, a multi-receptor 5-HT(2A)/5-HT(7)/D(2)/D(3)/D(4) agent, also displayed significant antipsychotic properties in the MK-801-induced hyperlocomotor activity in mice.     

3,4-Dihydro-2H-benzo[1,4]oxazine derivatives as 5-HT6 receptor antagonists

A series of novel 3,4-dihydro-2H-benzo[1,4]oxazine derivatives has been designed and synthesized as 5-HT(6) receptor antagonists. Many of the compounds displayed subnanomolar affinities for the 5-HT(6) receptor and good brain penetration in rats. The relationship of structure and lipophilicity to hERG inhibition of this series of compounds is discussed.     

Pharmacomodulation of a sulfamide 5-HT6 receptor ligand

A series of N-omega-aminoalkyl- or N-omega-amidinoalkyl-2,4,6-triisopropyl benzenesulfonamides has been synthesized and their respective affinity indices on 5-HT6 receptor determined. This evaluation clearly showed that the compounds possessing an arylpiperazine moiety or an amidine function exhibited good affinity for the model.     

Preparation of piperazine derivatives as 5-HT7 receptor antagonists

Twenty-four compounds of 4-methoxy-N-[3-(4-substituted phenyl-piperazine-1-yl)propyl] benzene sulfonamides and N-[3-(4-substituted phenyl-piperazine-1-yl)propyl] naphthyl sulfonamides were prepared and evaluated as 5-HT(7) receptor antagonists. Most of the compounds showed the IC(50) values of 12-580nM. Four methyl branched analogues were also obtained, but the activity for methyl branched analogues was almost same as its straight chain congeners. Among the synthesized compounds, 3c showed a good activity on 5-HT(7) receptors and a good selectivity on 5-HT(1a), 5-HT(2a), 5-HT(2c), and 5-HT(6) receptors.     

Cyclic guanidines as dual 5-HT5A/5-HT7 receptor ligands: optimising brain penetration

The optimisation of molecular properties within a series of 2-amino dihydroquinazoline 5-HT5A/5-HT7 receptor ligands resulted in a significantly improved brain-to-plasma ratio, enhancing the pharmacological utility of these compounds. By modulating the lipophilicity and pKa, a 20-fold increase in brain-to-plasma ratio could be achieved, leading to micromolar brain concentrations after oral administration. The enantiomers of one representative of this series of improved compounds were separated, and the configuration of the eutomer was determined by X-ray crystallography.     

Role of 5-HT in the regulation of the brain-pituitary-adrenal axis: effects of 5-HT on adrenocortical cells

Serotonin (5-HT) plays a pivotal role in the regulation of the brain-pituitary-adrenal axis. In particular, 5-HT has been shown to control the activity of hypothalamic CRF neurons and pituitary corticotrope cells through activation of 5-HT1A and (or) 5-HT(2A/2C) receptor subtypes. 5-HT, acting through 5-HT2 receptors, can also trigger the renin-angiotensin system by stimulating renin secretion and consequently can enhance aldosterone production. At the adrenal level, 5-HT produced locally stimulates the secretory activity of adrenocortical cells through a paracrine mode of communication. The presence of 5-HT in the adrenal gland has been demonstrated immunohistochemically and biochemically in various species. In the frog, rat, and pig adrenal gland, 5-HT is synthesized by chromaffin cells, while in the mouse adrenal cortex, 5-HT is contained in nerve fibers. In man, 5-HT is present in perivascular mast cells. In vivo and in vitro studies have shown that 5-HT stimulates corticosteroid secretion in various species (including human). The type of receptor involved in the mechanism of action of 5-HT differs between the various species. In frogs and humans, the stimulatory effect of 5-HT on adrenocortical cells is mediated through a 5-HT4 receptor subtype positively coupled to adenylyl cyclase and calcium influx. In the rat, the effect of 5-HT on aldosterone secretion is mediated via activation of 5-HT7 receptors. Clinical studies indicate that 5-HT4 receptor agonists stimulate aldosterone secretion in healthy volunteers and in patients with corticotropic insufficiency and primary hyperaldosteronism. Local serotonergic control of corticosteroid production may be involved in the physiological control of the activity of the adrenal cortex as well as in the pathophysiology of cortisol and aldosterone disorders.     

7-Arylpiperazinylalkyl and 7-tetrahydroisoquinolinylalkyl derivatives of 8-alkoxy-purine-2,6-dione and some of their purine-2,6,8-trione analogs as 5-HT(1A), 5-HT(2A), and 5-HT(7) serotonin receptor ligands

On the basis of our earlier studies with the serotonin receptor ligands in the group of 1,3-dimethyl-3,7-dihydropurine-2,6-dione derivatives, a series of new arylpiperazinylalkyl and tetrahydroisoquinolinylalkyl analogs of 8-alkoxy-1,3-dimethyl-3,7-dihydropurine-2,6-dione (10-25) and 1,3-dimethyl-7,9-dihydro-3H-purine-2,6,8-trione (26-30) were synthesized and their 5-HT(1A), 5-HT(2A), and 5-HT(7) receptor affinities were determined. The new compounds 17, 18, 20, and 21 were found to be highly active 5-HT(1A) receptor ligands (K(i)=11-19nM) with diversified affinity for 5-HT(2A) receptors (K(i)=15-253nM). Compounds 12, 13, 15, and 19 were moderately potent 5-HT(2A) ligands (K(i)=23-57nM), whereas 17, 18, 24, and 25 showed distinct affinity for 5-HT(7) receptors (K(i)=51-83nM). Purine-2,6,8-triones showed weak affinities for 5-HT(1A) and 5-HT(7) receptors; among them, 27 and 29 were classified as 5-HT(2A) receptor ligands. The selected compounds 17 and 21 were pharmacologically evaluated to determine their functional activities at pre-(hypothermia in mice) and post-(lower lip retraction in rats) synaptic 5-HT(1A) receptors. Compound 17 showed features of a potential agonist of pre- and post-synaptic 5-HT(1A) receptors, whereas 21 was classified as a potential, weak partial agonist of postsynaptic sites. Last of all, the most interesting compound 17 tested in behavioral models showed potential anxiolytic and antidepressant activities.     

The impact of spacer structure on 5-HT7 and 5-HT1A receptor affinity in the group of long-chain arylpiperazine ligands

New cis-, trans-2-butene and 1,2-bismethylbenzene analogues of MM77 and NAN-190 (1-[4-[4-(2-methoxyphenyl)-piperazin-1-yl]-butyl]-pyrrolidine-2,5-dione and isoindole-1,3-dione, respectively) were synthesized. The differences in their in vitro affinity for serotonin 5-HT(7) and 5-HT(1A) receptors were explained using a conformational analysis. A bioactive conformation of those compounds for the 5-HT(7) receptor, different from that established for 5-HT(1A), was proposed.     

Tetrahydrothienopyridylbutyl-tetrahydrobenzindoles: new selective ligands of the 5-HT(7) receptor

The synthesis and the affinity for the 5-HT(7) receptor and other receptors of a novel series of fused-ring tetrahydropyridine derivatives are described. Some of the compounds showed high affinity for the 5-HT(7) receptor. Tetrahydrothienopyridylbutyl-tetrahydrobenzindoles and are potent ligands for the 5-HT(7) receptor, with high selectivity over the 5-HT(2) receptor and other receptors. These compounds should be useful tools for clarifying the biological role of the 5-HT(7) receptor.     

Aminoalkyl phenyl sulfones--a novel series of 5-HT7 receptor ligands

A novel series of 5-HT(7) receptor ligands has been identified and evaluated, providing compounds showing a broad spectrum of functional activities and good selectivity over selected receptors and ion channels.     

Role of essential sulfhydryl groups in drug interactions at the neuronal 5-HT transporter. Differences between amphetamines and 5-HT uptake inhibitors.

The sulfhydryl-selective alkylating agent, N-ethylmaleimide (NEM), has been used as a tool to discern whether different binding domains exist on the neuronal serotonin (5-HT) transporter for 5-HT and 5-HT uptake inhibitors (Reith, M. E. A., Allen, D. L., Sershen, H., and Lajtha, A. (1984) J. Neurochem. 43, 249-255; Graham, D., Esnaud, H., Habert, E., and Langer, S. Z. (1989) Biochem. Pharmacol. 38, 3819-3826). However, relatively high concentrations of NEM and long incubation times have been required for inactivation of the transporter-binding site which raises the possibility that NEM is reacting with other nucleophilic groups (Smyth, D. G., Blumenfeld, O. O., and Konigsberg, W. (1964) Biochem. J. 91, 589-595). In the present work, the reactivity and essential nature of sulfhydryl groups associated with substrate/inhibitor binding to the neuronal 5-HT transporter was assessed. [3H]Paroxetine, a potent and selective 5-HT uptake inhibitor, was used to label the 5-HT transporter. The effects of a relatively wide range of sulfhydryl reagents on [3H]paroxetine binding in digitoninsolubilized preparations of rat brain neuronal membranes and the relative abilities of different classes of drugs to protect against NEM-induced inactivation of [3H]paroxetine binding were studied. It was observed that digitonin-solubilized preparations were more sensitive than membrane preparations to the inactivating effects of NEM. The pKa of the reactive group was estimated to be 6.17, in the range expected for a reactive sulfhydryl. Sulfhydryls essential to ligand binding reacted preferentially with hydrophobic compounds (p-hydroxymercuribenzoate = dithiobisnitrobenzoate > methyl methanethiosulfonate > N-phenylmaleimide > N-ethylmaleimide) and were unreactive toward hydrophilic reagents such as iodoacetate and iodoacetamide. 5-HT, 5-HT uptake inhibitors and cocaine protected the digitonin-solubilized transporter from NEM-induced inactivation while the amphetamine-related releasing agents p-chloroamphetamine and fenfluramine were ineffective. The observation that the binding of some, but not all, ligands requires reduced sulfhydryl groups, suggests that differential mechanisms and/or different binding domains do exist for agents which interact at the neuronal 5-HT transporter.     

Novel aryloxy-8-azabicyclo[3.2.1]oct-3-enes with 5-HT transporter and 5-HT1A affinity

Joining aryl 8-azabicyclo[3.2.1]oct-3-enes with aryloxyethanes and aryloxypropanes produces novel series of compounds 11 and 12 with potent 5-HT-T affinity and moderately potent 5-HT(1A) affinity. Moreover, several of these compounds possess functional 5-HT(1A) antagonism. Optimal compounds are, 4-indolyloxyethane 21, 4-indolyloxypropanes 25, and 27, which possess potent 5-HT-T affinity (5-HT-T K(i): 21: 1.2nM, 25: 0.54nM, 27: 0.38nM) and good 5-HT(1A) affinity/antagonism (5-HT(1A)K(i), [(35)S]GTPgammaS: E(max) (%): 21: 111.1nM, 0%; 25: 173.2nM, 0%; 27: 107nM, 0%).     

1-Aryl-4-(4-succinimidobutyl)piperazines and their conformationally constrained analogues: synthesis, binding to serotonin (5-HT1A, 5-HT2A, 5-HT7), alpha1-adrenergic, and dopaminergic D2 receptors, and in vivo 5-HT1A functional characteristics

Starting with the structure of potent 5-HT(1A) ligands, that is, MM77 [1-(2-methoxyphenyl)-4-(4-succinimidobutyl)piperazine, 4] and its constrained version 5 (MP349), previously obtained in our laboratory, a series of their direct analogues with differently substituted aromatic ring (R=H, m-Cl, m-CF(3), m-OCH(3), p-OCH(3)) were synthesized. The flexible and the corresponding 1e,4e-disubstituted cyclohexane derivatives were designed in order to investigate the influence of rigidification on 5-HT(1A) affinity, selectivity for 5-HT(2A), 5-HT(7), D(1), and D(2) binding sites and functional profile at pre- and postsynaptic 5-HT(1A) receptors. The new compounds 19-25 were found to be highly active 5-HT(1A) receptor ligands (K(i)=4-44 nM) whereas their affinity for other receptors was: either significantly decreased after rigidification (5-HT(7)), or controlled by substituents in the aromatic ring (alpha(1)), or influenced by both those structural modifications (5-HT(2A)), or very low (D(2), K(i)=5.3-31 microM). Since a distinct disfavor towards rigid compounds was observed for 5-HT(7) receptors only, it seems that the bioactive conformation of chain derivatives at those sites should differ from the extended one. Several in vivo models were used to asses functional activity of 19-25 at pre- (hypothermia in mice) and postsynaptic 5-HT(1A) receptors (lower lip retraction in rats and serotonin syndrome in reserpinized rats). Unlike the parent antagonists 4 and 5, all the new derivatives tested were classified as partial agonists with different potency, however, similar effects were observed within pairs (flexible and rigid) of the analogues. The obtained results indicated that substitution in the aromatic ring, but not spacer rigidification, controls the 5-HT(1A) functional activity of the investigated compounds. Moreover, an o-methoxy substituent in the structure of 5 seems to be necessary for its full antagonistic properties. Of all the new compounds studied, trans-4-(4-succinimidocyclohexyl)-1-(3-trifluoromethylphenyl)piperazine 24 was the most potent 5-HT(1A) receptor ligand in vitro (K(i)=4 nM) and in vivo, with at least 100-fold selectivity for the other receptors tested.     

Synthesis of potent and selective serotonin 5-HT1B receptor ligands

A series of serotonin 5-HT1B ligands were synthesized and evaluated for their potency and selectivity against other 5-HT receptor subtypes. Many of these new compounds displayed high affinity and selectivity for the 5-HT1B receptor and compound 6c was found to have the in vitro binding profile necessary for development as a PET radioligand.     

Hypothalamic 5-HT functional regulation through 5-HT1A and 5-HT2C receptors during pancreatic regeneration

5-HT receptors are predominantly located in the brain and are involved in pancreatic function and cell proliferation through sympathetic nervous system. The objective of this study was to investigate the role of hypothalamic 5-HT, 5-HT1A and 5-HT2C receptor binding and gene expression in rat model of pancreatic regeneration using 60% pancreatectomy. The pancreatic regeneration was evaluated by 5-HT content, 5-HT1A and 5-HT2C receptor gene expression in the hypothalamus of sham operated, 72 h and 7 days pancreatectomised rats. 5-HT content was quantified by HPLC. 5-HT1A receptor assay was done by using specific agonist [3H]8-OH DPAT. 5-HT2C receptor assay was done by using specific antagonist [3H]mesulergine. The expression of 5-HT1A and 5-HT2C receptor gene was analyzed by RT-PCR. 5-HT content was higher in the hypothalamus of 72 h pancreatectomised rats. 5-HT1A and 5-HT2C receptors were down-regulated in the hypothalamus. RT-PCR analysis revealed decreased 5-HT1A and 5-HT2C receptor mRNA expression. The 5-HT1A and 5-HT2C receptors gene expression in the 7 days pancreatectomised rats reversed to near sham level. This study is the first to identify 5-HT1A and 5-HT2C receptor gene expression in the hypothalamus during pancreatic regeneration in rats. Our results suggest the hypothalamic serotonergic receptor functional regulation during pancreatic regeneration.     

Implication of 5-HT2A receptors in the genetic mechanisms of the brain 5-HT system autoregulation

Brain serotonin (5-HT) system has been implicated in pathophysiology of anxiety, depression, drug addiction, and schizophrenia. 5-HT2A receptor is involved in the mechanisms of stress-induced psychopathology and impulsive behavior. Here, we investigated the role of 5-HT2A receptor in the autoregulation of the brain 5-HT system. The chronic treatment with agonist of 5-HT2A receptor DOI (1.0 mg/kg, i.p./14 days) produced considerable decrease of 5-HT2A receptor-mediated "head-twitches" in AKR/J mice indicating desensitization of 5-HT2A receptors. Chronic DOI treatment failed to alter 5-HT2A receptor gene expression in the midbrain, hippocampus and frontal cortex. At the same time, the increase in the expression of the gene encoding key enzyme of 5-HT synthesis, tryptophan hydroxylase 2 (TPH2), the increase in TPH2 activity and 5-HT levels and decreased expression of serotonin transporter (5-HTT) gene was found in the midbrain of DOI-treated mice. The results provide new evidence of receptor-gene cross-talk in the brain 5-HT system and the implication of 5-HT2A receptor in the autoregulation of the brain 5-HT system.