Involvement of 5-HT(2A/2B/2C) receptors on memory formation: simple agonism,antagonism, or inverse agonism?

1. The 5-HT₂ receptors subdivision into the 5-HT_2A/2B/2C subtypes along with the advent of the selective antagonists has allowed a more detailed investigation on the role and therapeutic significance of these subtypes in cognitive functions. The present study further analyzed the 5-HT₂ receptors role on memory consolidation.2. The SB-200646 (a selective 5-HT_2B/2C receptor antagonist) and LY215840 (a nonselective 5-HT_2/7 receptor antagonist) posttraining administration had no effect on an autoshaped memory consolidation. However, both drugs significantly and differentially antagonized the memory impairments induced by 1-(3-chlorophenyl)piperazine (mCPP), 1-naphtyl-piperazine (1-NP), mesulergine, or N-(3-trifluoromethylphenyl) piperazine (TFMPP).3. In contrast, SB-200646 failed to modify the facilitatory procognitive effect produced by (±)-2,5-dimethoxy-4-iodoamphetamine (DOI) or ketanserin, which were sensitive to MDL100907 (a selective 5-HT_2A receptor antagonist) and to a LY215840 high dose.4. Finally, SB-200646 reversed the learning deficit induced by dizocilpine, but not that by scopolamine; while SB-200646 and MDL100907 coadministration reversed memory deficits induced by both drugs.5. It is suggested that 5-HT_2B/2C receptors might be involved on memory formation probably mediating a suppressive or constraining action. Whether the drug-induced memory impairments in this study are explained by simple agonism, antagonism, or inverse agonism at 5-HT₂ receptors remains unclear at this time.6. Notably, the 5-HT₂ receptor subtypes blockade may provide some benefit to reverse poor memory consolidation conditions associated with decreased cholinergic, glutamatergic, and/or serotonergic neurotransmission.     

Quinoxalin-2-carboxamides: synthesis and pharmacological evaluation as serotonin type-3 (5-HT3) receptor antagonists

A series of quinoxalin-2-carboxamides were designed as per the pharmacophoric requirements of 5-HT₃ receptor antagonists and synthesized by condensing the carboxylic group of quinoxalin-2-carboxylic acid with various amines in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole. The structures of the synthesized compounds were confirmed by physical and spectroscopic data. The carboxamides were evaluated for their 5-HT₃ receptor antagonisms in longitudinal muscle-myenteric plexus preparation from guinea pig ileum against 5-HT₃ agonist, 2-methy-5-HT. All the synthesized compounds showed 5-HT₃ receptor antagonism, (4-benzylpiperazin-1-yl)(quinoxalin-2-yl)methanone was the most potent compound among this series.     

Synthesis and structure-activity relationship of 1H-indole-3-carboxylic acid pyridine-3-ylamides: a novel series of 5-HT2C receptor antagonists

A novel series of 1H-indole-3-carboxylic acid pyridine-3-ylamides were synthesized and identified to show high affinity and selectivity for 5-HT_2C receptor. Among them, 1H-indole-3-carboxylic acid[6-(2-chloro-pyridin-3-yloxy)-pyridin-3-yl]-amide (15k) exhibits the highest affinity (IC₅₀ = 0.5 nM) with an excellent selectivity (>2000 times) over other serotonin (5-HT_1A, 5-HT_2A, and 5-HT₆) and dopamine (D₂–D₄) receptors.     

3-(4-Piperidinyl)- and 3-(8-aza-bicyclo[3.2.1]oct-3-yl)-2-phenyl-1H-indoles as bioavailable h5-HT2A antagonists

A series of 3-(4-piperidinyl)- and 3-(8-aza-bicyclo[3.2.l]oct-3-yl)-2-phenyl-1H-indoles have been prepared and evaluated as ligands for the h5-HT_2A receptor. 3-(8-Phenethyl-8-aza-bicyclo[3.2.l]oct-3-yI)-2-phenyl-1H-indole is a high-affinity (1.2 nM), selective (>800 fold over h5-HT_2C and hD₂ receptors) antagonist at the h5-HT_2A receptor with oral bioavailability in rats.     

Synthesis and SAR of potent and selective tetrahydropyrazinoisoquinolinone 5-HT2C receptor agonists

The 5-HT_2C receptor has been implicated as a critical regulator of appetite. Small molecule activation of the 5-HT_2C receptor has been shown to affect food intake and regulate body weight gain in rodent models and more recently in human clinical trials. Therefore, 5-HT_2C is a well validated target for anti-obesity therapy. The synthesis and structure–activity relationships of a series of novel tetrahydropyrazinoisoquinolinone 5-HT_2C receptor agonists are presented. Several members of this series were identified as potent 5-HT_2C receptor agonists with high functional selectivity against the 5-HT_2A and 5-HT_2B receptors and reduced food intake in an acute rat feeding model upon oral dosing.     

Design,synthesis and preliminary screening of novel 3-(2-N,N-dimethylaminoethylthio) indole derivatives as potential 5-HT6 receptor ligands

The synthesis and potential 5-hydroxytryptamine₆ receptor (5-HT₆R) antagonist activity of a novel series of N-arylsulfonyl-3-(2-N,N-dimethylaminoethylthio) indoles has been reported. The molecular modeling, synthesis and in-vitro radioligand binding data of this series are discussed. The present article describes 37 derivatives of the title series. It was observed that the increased side-chain length with the insertion of a sulfur atom did not lead to the loss of binding affinity of these compounds, although the affinities were reduced. The compounds exhibited moderate affinity and selectivity to human 5-HT₆ receptors.     

Rapid Desensitization of Serotonin 5-HT2C Receptor-Stimulated Intracellular Calcium Mobilization in CHO Cells Transfected with Cloned Human 5-HT2C Receptors

Abstract: Serotonin 5-HT_2C receptor-mediated intracellular Ca²⁺ mobilization was investigated in Chinese hamster ovary (CHO) cells transfected with 5-HT_2C receptors. Fura-2 acetoxymethyl ester was used to investigate the regulation of 5-HT_2C receptor function. CHO cells, transfected with a cDNA clone for the 5-HT_2C receptor, expressed 287 fmol/mg of the receptor protein as determined by mianserin-sensitive [³H]mesulergine binding (K_D = 0.49 nM). The addition of 5-HT mobilized intracellular Ca²⁺ in a dose-dependent fashion, ranging from a basal level of 99 ± 1.8 up to 379 ± 18 nM, with an EC₅₀ value for 5-HT of 0.029 µM. Exposure to 5-HT, 1-(3-chlorophenyl)piperazine dihydrochloride (a 5-HT_2C agonist), and 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (a 5-HT_2C and 5-HT_2A agonist) resulted in increased intracellular Ca²⁺ levels. Mianserin, mesulergine, ritanserin, and ketanserin each blocked 5-HT-mediated intracellular Ca²⁺ mobilization more effectively than spiperone. The receptor was rapidly desensitized by preexposure to 5-HT in a time- and concentration-dependent manner. Mezerein and phorbol 12-myristate 13-acetate, protein kinase C activators, weakly inhibited the intracellular Ca²⁺ mobilization induced by 10 µM 5-HT. Furthermore, the protein kinase C inhibitor H-7 partially prevented the protein kinase C activator-induced inhibition of the 5-HT-mediated increase in intracellular Ca²⁺ concentration. The desensitization induced by pretreatment with 5-HT was blocked by W-7, added in conjunction with 5-HT, and partially inhibited by W-5, a nonselective inhibitor of protein kinases and weak analogue of W-7. Therefore, the 5-HT_2C receptor may be connected with protein kinase C and calcium/calmodulin turnover. These results suggest that 5-HT_2C receptor activation mobilizes Ca²⁺ in CHO cells and that the acute desensitization of the receptor may be due to calmodulin kinase-mediated feedback.     

Characterisation of Human 5-Hydroxytryptamine2A and 5-Hydroxytryptamine2C Receptors Expressed in the Human Neuroblastoma Cell Line SH-SY5Y: Comparative Stimulation by Hallucinogenic Drugs

Abstract: Stable transfection of the human neuroblastoma cell line SH-SY5Y with the human 5-hydroxytryptamine_2A (5-HT_2A) or 5-HT_2C receptor cDNA produced cell lines demonstrating ligand affinities that correlated closely with those for the corresponding endogenous receptors in human frontal cortex and choroid plexus, respectively. Stimulation of the recombinant receptors by 5-HT induced phosphoinositide hydrolysis with higher potency but lower efficacy at the 5-HT_2C receptor (pEC₅₀ = 7.80 ± 0.06) compared with the 5-HT_2A receptor (pEC₅₀ = 7.30 ± 0.08). Activation of the 5-HT_2A receptor caused a transient fourfold increase in intracellular Ca²⁺ concentration. Whole-cell recordings of cells clamped at ?50 mV demonstrated a small inward current (2 pA) in response to 10 µM 5-HT for both receptors. There were no differences in potency or efficacy of phosphoinositide hydrolysis among four hallucinogenic [d-lysergic acid diethylamide (LSD), 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (DOI), 5-methoxy-N,N-dimethyltryptamine, and mescaline] and three nonhallucinogenic drugs (m-chlorophenylpiperazine, quipazine, and ergotamine). Comparison of equipotent doses producing 20% of the maximal response induced by 5-HT revealed selective activation of the 5-HT_2A receptor by LSD and to a lesser degree by DOI, mescaline, and ergotamine. Quipazine and 5-methoxy-N,N-dimethyltryptamine were relatively nonselective, whereas m-chlorophenylpiperazine selectively activated the 5-HT_2C receptor. It is unlikely therefore that hallucinosis is mediated primarily by activity at the 5-HT_2C receptor, whereas activity at the 5-HT_2A receptor may represent an important but not unique mechanism associated with hallucinogenic drug action.     

The Serotonin 5-HT2C Receptor Is a Prominent Serotonin Receptor in Basal Ganglia: Evidence from Functional Studies on Serotonin-Mediated Phosphoinositide Hydrolysis

Abstract: Serotonin (5-hydroxytryptamine; 5-HT) 5-HT_2A and 5-HT_2C receptors belong to the class of phosphoinositide-specific phospholipase C (PLC)-linked receptors. Conditions were established for measuring 5-HT_2A-linked and 5-HT_2C-linked PLC activity in membranes prepared from previously frozen rat frontal cortex and caudate. In the presence of Ca²⁺ (300 nM) and GTPγS (1 µM), 5-HT increased PLC activity in caudate membranes. Pharmacological analysis using the selective 5-HT_2A antagonist, spiperone, and the nonselective 5-HT_2A/2C antagonist, mianserin, demonstrated that over half of the 5-HT-stimulated PLC activity was due to stimulation of 5-HT_2C receptors as opposed to 5-HT_2A receptors. Radioligand binding assays with [³H]RP 62203 and [³H]-mesulergine were used to quantify 5-HT_2A and 5-HT_2C sites, respectively, in caudate. From these data, the B_max for caudate 5-HT_2A sites and 5-HT_2C sites was 165.4 ± 9.7 fmol/mg of protein and 49.7 ± 3.3 fmol/mg of protein, respectively. In contrast to that in caudate, PLC activity in frontal cortex was stimulated by 5-HT in a manner that was inhibited by the 5-HT_2A-selective antagonists, spiperone and ketanserin. Taken together, the results indicate that 5-HT_2A- and 5-HT_2C-linked PLC activity can be discerned in brain regions possessing both receptor subtypes using membranes prepared from previously frozen tissue. More importantly, significant 5-HT_2C-mediated phosphoinositide hydrolysis was observed in caudate, despite the relatively low density of 5-HT_2C sites. The significance of these observations with respect to the physiological function of 5-HT_2C receptors is discussed.     

Serotonin 5-HT2C Receptor Stimulates Cyclic GMP Formation in Choroid Plexus

Abstract: The serotonin 5-HT_2C receptor (formerly designated the 5-HT_1C receptor) of the choroid plexus triggers phosphoinositide turnover. In the present study, we demonstrate that receptor activation also triggers the formation of cyclic GMP (cGMP). Application of 1 µM 5-HT to porcine choroid plexus tissue slices resulted in stimulation of cGMP formation to a maximum of five-fold basal level, with an EC₅₀ of 11 nM. This response was not inhibited by muscarinic or β-adrenergic receptor antagonists. Serotonin receptor antagonists inhibited cGMP formation with apparent K_i values of 1.3 (mianserin), 200 (ketanserin), and 5,500 (spiperone) nM, respectively. Neither serotonin-stimulated cGMP formation nor PI turnover was inhibited by pertussis toxin pretreatment. Preliminary biochemical studies suggested that serotonin-stimulated cGMP formation was calcium, phospholipase A₂, and lipoxygenase dependent, as incubation in low calcium buffers or inclusion of the phospholipase A₂ or lipoxygenase inhibitors p-bromophenacyl bromide or BW 755c resulted in significant reduction of cGMP formation. The present results suggest that in addition to triggering phosphoinositide turnover, choroid plexus serotonin 5-HT_2C receptors trigger cGMP formation in a calcium-sensitive manner.     

Up-Regulation of 5-HT2B Receptor Density and Receptor-Mediated Glycogenolysis in Mouse Astrocytes by Long-Term Fluoxetine Administration

The effects were studied of short-term (1 week) versus long-term (2-3 weeks) fluoxetine treatment of primary cultures of mouse astrocytes, differentiated by treatment with dibutyryl cyclic AMP. From previous experiments it is known that acute treatment with fluoxetine stimulates glycogenolysis and increases free cytosolic Ca²⁺ concentration ([Ca²⁺]_i]) in these cultures, whereas short-term (one week) treatment with 10 M down-regulates the effects on glycogen and [Ca²⁺]_i, when fluoxetine administration is renewed (or when serotonin is administered). Moreover, antagonist studies have shown that these responses are evoked by activation of a 5-HT₂ receptor that is different from the 5-HT_2A receptor and therefore at that time tentatively were interpreted as being exerted on 5-HT_2C receptors. In the present study the cultures were found by RT-PCR to express mRNA for 5-HT_2A and 5-HT_2B receptors, but not for the 5-HT_2C receptor, identifying the 5-HT₂ receptor activated by fluoxetine as the 5-HT_2B receptor, the most recently cloned 5-HT₂ receptor and a 5-HT receptor known to be more abundant in human, than in rodent, brain. Both short-term and long-term treatment with fluoxetine increased the specific binding of [³H]mesulergine, a ligand for all three 5-HT₂ receptors. Long-term treatment with fluoxetine caused an agonist-induced up-regulation of the glycogenolytic response to renewed administration of fluoxetine, whereas short-term treatment abolished the fluoxetine-induced hydrolysis of glycogen. Thus, during a treatment period similar to that required for fluoxetine's clinical response to occur, 5-HT_2B-mediated effects are initially down-regulated and subsequently up-regulated.     

Decreased 5-HT2C receptor binding in the cerebral cortex and brain stem during pancreatic regeneration in rats

The purpose of this study was to investigate the role of central 5-HT_2C receptor binding in rat model of pancreatic regeneration using 60–70% pancreatectomy. The 5-HT and 5-HT_2C receptor kinetics were studied in cerebral cortex and brain stem of sham operated, 72 h pancreatectomised and 7 days pancreatectomised rats. Scatchard analysis with [³H] mesulergine in cerebral cortex showed a significant decrease (p < 0.05) in maximal binding (B_max) without any change in K_d in 72 h pancreatectomised rats compared with sham. The decreased B_max reversed to sham level by 7 days after pancreatectomy. In brain stem, Scatchard analysis showed a significant decrease (p < 0.01) in B_max with a significant increase (p < 0.01) in K_d. Competition analysis in brain stem showed a shift in affinity towards a low affinity. These parameters were reversed to sham level by 7 days after pancreatectomy. Thus the results suggest that 5-HT through the 5-HT_2C receptor in the brain has a functional regulatory role in the pancreatic regeneration.     

Novel N-biphenyl-2-ylmethyl 2-methoxyphenylpiperazinylalkanamides as 5-HT7R antagonists for the treatment of depression

5-HT₇ receptor (5-HT₇R) is a promising target for the treatment of depression and neuropathic pain. 5-HT₇R antagonists exhibited antidepressant effects, while the agonists produced strong anti-hyperalgesic effects. In our efforts to discover selective 5-HT₇R antagonists or agonists, N-biphenylylmethyl 2-methoxyphenylpiperazinylalkanamides 1 were designed, synthesized, and biologically evaluated against 5-HT₇R. Among the synthesized compounds, N-2′-chlorobiphenylylmethyl 2-methoxyphenylpiperazinylpentanamide 1–8 showed the best binding affinity with a K_i value of 8.69 nM and it was verified as a novel antagonist according to functional assays. The compound 1–8 was very selective over 5-HT_1DR, 5-HT_2AR, 5-HT₃R, 5-HT_5AR and 5-HT₆R and moderately selective over 5-HT_1AR, 5-HT_1BR and 5-HT_2CR. The novel 5-HT₇R antagonist 1–8 exhibited an antidepressant effect at a dose of 25 mg/kg in the forced swimming test in mice and showed a U-shaped dose–response curve which typically appears in 5-HT₇R antagonists such as SB-269970 and lurasidone.     

Arylpiperazide derivatives of phenylpiperazines as a new class of potent and selective 5-HT1B receptor antagonists

A new series of arylpiperazide derivatives of phenylpiperazines of general formula 4 has been prepared and evaluated as 5-HT_1B receptor antagonists. In vitro experiments at human cloned 5-HT_1B receptors show that these derivatives are potent and selective 5-HT_1B receptor antagonists. Among them, compound 4f was found to be orally active, to gain access to the CNS and more importantly to induce an increase in extracellular brain 5-HT upon systemic administration.     

Tricyclic dihydroquinazolinones as novel 5-HT2C selective and orally efficacious anti-obesity agents

Agonists of the 5-HT_2C receptor have been shown to suppress appetite and reduce body weight in animal models as well as in humans. However, agonism of the related 5-HT_2B receptor has been associated with valvular heart disease. Synthesis and biological evaluation of a series of novel and highly selective dihydroquinazolinone-derived 5-HT_2C agonists with no detectable agonism of the 5-HT_2B receptor is described. Among these, compounds (+)-2a and (+)-3c were identified as potent and highly selective agonists which exhibited weight loss in a rat model upon oral dosing.     

Identification of 3-sulfonylindazole derivatives as potent and selective 5-HT(6) antagonists

As part of our efforts to develop agents for cognitive enhancement, we have been focused on the 5-HT₆ receptor in order to identify potent and selective ligands for this purpose. Herein we report the identification of a novel series of 3-sulfonylindazole derivatives with acyclic amino side chains as potent and selective 5-HT₆ antagonists. The synthesis and detailed SAR of this class of compounds are reported.     

Rigidized 1-aryl sulfonyl tryptamines: synthesis and pharmacological evaluation as 5-HT6 receptor ligands

A series of N₁-arylsulfonyl-3-(pyrrolidin-3-yl)-1H-indole and N₁-arylsulfonyl-3-(4-chloro-2,5-dihydro-1H-pyrrol-3-yl)-1H-indole derivatives (tryptamine derivatives with rigidized side chain) have been prepared and tested for their binding affinity to 5-HT₆ receptor. Several compounds displayed potent binding affinity for the 5-HT₆ receptor when tested in in vitro binding assay. The primary SAR indicates that rigidification of dimethylamino alkyl chain at C₃ of indole carbon maintains the binding affinity to 5-HT₆R. The lead compound N₁-benzenesulfonyl-3-(4-chloro-1-methyl-2,5-dihydro-1H-pyrrol-3-yl)-1H-indole, 10a (K_b = 0.1 nM) has shown excellent in vitro affinity and was active in animal models of cognition like NORT and water maze.     

Functional expression of 5-HT2A receptor in osteoblastic MC3T3-E1 cells

In the previous study, we reported the gene expression for proteins related to the function of 5-hydroxytryptamine (5-HT, serotonin) and elucidated the expression patterns of 5-HT₂ receptor subtypes in mouse osteoblasts. In the present study, we evaluated the possible involvement of 5-HT receptor subtypes and its inactivation system in MC3T3-E1 cells, an osteoblast cell line. DOI, a 5-HT_2A and 5-HT_2C receptor selective agonist, as well as 5-HT concentration-dependently increased proliferative activities of MC3T3-E1 cells in their premature period. This effect of 5-HT on cell proliferation were inhibited by ketanserin, a 5-HT_2A receptor specific antagonist. Moreover, both DOI-induced cell proliferation and phosphorylation of ERK1 and 2 proteins were inhibited by PD98059 and U0126, selective inhibitors of MEK in a concentration-dependent manner. Furthermore, treatment with fluoxetine, a 5-HT specific re-uptake inhibitor which inactivate the function of extracellular 5-HT, significantly increased the proliferative activities of MC3T3-E1 cells in a concentration-dependent manner. Our data indicate that 5-HT fill the role for proliferation of osteoblast cells in their premature period. Notably, 5-HT_2A receptor may be functionally expressed to regulate mechanisms underlying osteoblast cell proliferation, at least in part, through activation of ERK/MAPK pathways in MC3T3-E1 cells.     

Novel serotonin type 3 receptor partial agonists for the potential treatment of irritable bowel syndrome

Serotonin type 3 (5-HT₃) receptor partial agonists are being targeted as potential new drugs for the treatment of irritable bowel syndrome (IBS). Two new chemical series bearing indazole and indole cores have exhibited nanomolar binding affinity for the h5-HT₃A receptor. A range of partial agonist activities in HEK cells heterologously expressing the h5-HT₃A receptor were measured for the indazole series. Excellent 5-HT₃ receptor selectivity, favorable in vitro metabolic stability and CYP inhibition properties, and good oral in vivo potency in the murine von Bezold-Jarisch reflex model is exemplified thereby indicating the series to have potential utility as improved IBS agents.     

Synthesis of novel 5-substituted-2-aminotetralin analogs: 5-HT1A and 5-HT7 G protein-coupled receptor affinity, 3D-QSAR and molecular modeling

The serotonin 5-HT₇ G protein-coupled receptor (GPCR) is a proposed pharmacotherapeutic target for a variety of central and peripheral indications, albeit, there are no approved drugs selective for binding 5-HT₇. We previously reported that a lead analog based on the 5-substituted-N,N-disubstituted-1,2,3,4-tetrahydronaphthalen-2-amine (5-substituted-2-aminotetralin, 5-SAT) scaffold binds with high affinity at the 5-HT₇ GPCR, and can treat symptoms of autism in mouse models; subsequently, the lead was found to have high affinity at the 5-HT_1A GPCR. Herein, we report the synthesis of novel 5-SAT analogs to develop a 3-dimensional quantitative structure—affinity relationship (3D-QSAR) at the human 5-HT₇ receptor for comparison with similar studies at the highly homologous 5-HT_1A receptor. We report 35 new 5-SAT ligands, some with very high affinity (K_i ≤ 1 nM) and stereoselectivity at 5-HT₇ + or 5-HT_1A receptors, several with modest selectivity (up to 12-fold) for binding at 5-HT₇, and, several ligands with high selectivity (up to 40-fold) at the 5-HT_1A receptor. 3D-QSAR results indicate that steric extensions at the C(5)-position improve selectivity for the 5-HT₇ over 5-HT_1A receptor, while steric and hydrophobic extensions at the chiral C(2)-amino position impart 5-HT_1A selectivity. In silico receptor homology modeling studies, supplemented with molecular dynamics simulations and binding free energy calculations, were used to rationalize experimentally-determined receptor selectivity and stereoselective affinity results. The data from these studies indicate that the 5-SAT chemotype, previously shown to be safe and efficacious in rodent paradigms of neurodevelopmental and neuropsychiatric disorders, is amenable to structural modification to optimize affinity at serotonin 5-HT₇ vs. 5-HT_1A GPCRs, as may be required for successful clinical translation.