Employing a novel, rapid and sensitive method for evaluation of phospholipase C (PLC) activity, the present study characterized the actions of diverse agonists and antagonists at human (h)5-HT2C receptors expressed in Chinese Hamster Ovary (CHO) cells. In addition, affinities and efficacies at these sites were compared with those obtained at h5-HT2B receptors.5-HT elicited a robust and rapid reduction in levels of the pre-labelled, membrane-bound substrate of PLC, [3H]phosphatidylinositols ([3H]PI). The time-course of [3H]PI depletion paralleled that of [3H]inositol phosphate ([3H]IP) accumulation, as determined by conventional anion exchange chromatography. Inactivation of h5-HT2C receptors with the alkylating agent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), revealed a large receptor reserve, with half-maximal PLC activation induced by a concentration of 5-HT occupying only 5% of sites. In analogy to 5-HT ( Emax=100%), DOI, MK212 and mCPP, as well as the novel ligands, Ro600332, Ro600175 and BW723C86, showed "full" efficacy at h5-HT2C sites. Their efficacies were similar at h5-HT2B sites, with the exception of mCPP and MK212, which acted as partial agonists. Further, lisuride and Ro600869 behaved as partial agonists and antagonists at h5-HT2C and h5-HT2B receptors, respectively. As concerns functional selectivity (potency for induction of [3H]PI depletion), only Ro600175 preferentially activated h5-HT2B sites. In contrast, Ro600332 preferentially activated h5-HT2C receptors. Amongst antagonists, RS102221 and SB242084 displayed a marked preference for h5-HT2C sites, whereas LY266097, S33526 and SB204741 behaved as selective antagonists at h5-HT2B receptors. At both h5-HT2C and h5-HT2B receptors, antagonist potency (p Kb) and binding affinity (p Ki) were highly correlated. In conclusion, this rapid and innovative method for determination of PLC activity permitted characterization of an extensive range of novel ligands at h5-HT2C receptors. Although several antagonists clearly differentiated h5-HT2C from h5-HT2B receptors under these conditions, highly selective agonists remain to be identified.

Treatment of norharman with (trimethylsilyl)methyl triflate in dichloromethane gave 2-N-[(trimethylsilyl)methyl]-β-carboline triflate (10). The latter reacted with diethyl acetylenedicarboxylate (11a) or ethyl propiolate (11b) in the presence of cesium fluoride to afford, via 1,3-dipolar cycloaddition, the indolizino [8,7-b]indole derivatives 13a or 13b, respectively. In a similar manner, the (trimethylsilyl)methyl triflate salt of ethyl 9-N-(p-toluenesulfonyl)-β-carboline-3-carboxylate ( 16) reacted with 11a to give the cycloaddition product 17. The (trimethylsilyl)methyl triflates of 3,4dihydro-β-carbolines were also prepared (20a-c) and shown to be more reactive than their fully aromatic counterparts 10 and 16 in cycloaddition reactions. Thus, the 9-N-benzyl derivative 20b reacted with 11a in the presence of cesium fluoride to give the cycloaddition products 21 and 22 as well as the novel azepine derivative 23. Moreover, unlike 10 and 16, the azomethine ylides generated from 20a-c reacted with electron-deficient olefins, producing 1,2, 3,5,6,11b-hexahydroindolizino[8,7-b]indole derivatives. In the case of symmetrically substituted olefins (dimethyl maleate, dimethyl fumarate, fumaronitrile), the cycloaddition reactions were completely stereospecific. However, with unsymmetrically substituted olefins (methyl acrylate, acrylonitrile), cycloaddition reactions were generally neither regio-nor diastereoselective. In the case of 20b, both the regioand the diastereoselectivities of the cycloaddition reactions were greatly improved compared to 20a and 20c, suggesting that these two stereochemical factors can be controlled by manipulation of the protecting group at the 9-N position of the 3,4-dihydro-β-carbolines. The hexahydroindolizino-[8,7-b]indoles 33 and 37 could be selectively dehydrogenated at the 11b, 1 positions using potassium permanganate in THF to afford the tetrahydro derivatives 58 and 59, respectively. Treatment of 58 with 1 equiv of DDQ led to further selective dehydrogenation at the 2,3 positions, producing the 5,6-dihydro compound 60. Alternatively, 33 and 37 could be completely dehydrogenated using 3 equiv of DDQ to give the fully aromatic indolizino [8,7-b]indole-1,2-dicarboxylates 56 and 57, respectively.

5-HT(2) receptors mediate a large array of physiological and behavioral functions in humans via three distinct subtypes: 5-HT(2A), 5-HT(2B)and 5-HT(2C). While selective 5-HT(2A)antagonists have been known for some time, knowledge of the precise role played by the 5-HT(2B)receptor was hampered by the existence of solely 5-HT(2B)5-HT(2C) mixed antagonists. However, selective 5-HT(2B)antagonists began recently to emerge in the literature. Indeed, four structural classes belonging to the piperazine, indole, naphthylpyrimidine and tetrahydro-beta-carboline scaffolds were reported. In this paper, we will briefly review the structural and pharmacological features of selective 5-HT(2B) antagonists, including patent literature of the last five years.

The knowledge of the free energy of binding of small molecules to a macromolecular target is crucial in drug design as is the ability to predict the functional consequences of binding. We highlight how a molecular dynamics (MD)-based approach can be used to predict the free energy of small molecules, and to provide priorities for the synthesis and the validation via in vitro tests. Here, we study the dynamics and energetics of the nuclear receptor REV-ERBα with its co-repressor NCoR and 35 novel agonists. Our in silico approach combines molecular docking, molecular dynamics (MD), solvent-accessible surface area (SASA) and molecular mechanics poisson boltzmann surface area (MMPBSA) calculations. While docking yielded initial hints on the binding modes, their stability was assessed by MD. The SASA calculations revealed that the presence of the ligand led to a higher exposure of hydrophobic REV-ERB residues for NCoR recruitment. MMPBSA was very successful in ranking ligands by potency in a retrospective and prospective manner. Particularly, the prospective MMPBSA ranking-based validations for four compounds, three predicted to be active and one weakly active, were confirmed experimentally.

1H-Indolo[3',2':4,5]pyrido[3,2-b]-2-penten-5-olide (6) and 1H,5H-indolo[3',2'-c]-6,7-dihydro-2-pyridone (7), rigid analogues of methyl 4-ethyl-beta-carboline-3-carboxylate (8) and N-methyl-4-ethyl-beta-carboline-3-carboxamide (9), respectively, were synthesized and their in vitro binding affinities to the central type benzodiazepine receptors were compared. The IC50 values of 6 and 8 were approximately equivalent (42 and 27 nM, respectively). The amide derivative 9, for which theoretical energy calculations indicate that the s-trans carbonyl conformation is the preferred one, displayed very low affinity (IC50 greater than 10(4) nM). However, when the carbonyl group of 9 was forced to adopt the s-cis conformation as in lactam 7, binding to the benzodiazepine receptor was largely restored (IC50 = 150 nM), indicating that the s-cis carboxy conformation at C-3 of beta-carbolines is preferentially recognized by this receptor. In vivo, compound 6 showed neither convulsant, proconvulsant, nor anticonvulsant activity in mice. Moreover, 6 did not antagonize methyl beta-carboline-3-carboxylate induced convulsions in mice. This lack of activity of 6 was attributed to its inability to cross the blood-brain barrier since no significant displacement of [3H]Ro 15-1788 from mouse brain benzodiazepine receptors by 6 could be observed in vivo.