Design, synthesis and binding properties of novel and selective 5-HT3 and 5-HT4 receptor ligands

This work reports the synthesis and the binding tests on the 5-HT3 and 5-HT4 receptors of new thienopyrimidopiperazine and piperazinylacylaminodimethylthiophene derivatives, in order to identify potent and selective ligands for each receptor. The 3-amino-2-(4-benzyl-1-piperazinyl)-5,6-dimethyl-thieno[2,3-d]pyrimidin-4(3H)-one derivative 28 showed the highest affinity and selectivity for the 5-HT3 over the 5-HT4 receptor (5-HT3 Ki=3.92 nM, 5-HT4 not active), whereas the 2-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butanoylamino]-4,5-dimethyl-3-thiophenecarboxylic acid ethyl ester (41) showed the highest affinity and selectivity for the 5-HT4 over the 5-HT3 receptor (5-HT4 Ki=81.3 nM, 5-HT3 not active). Conformational analyses were carried out on the compounds of the piperazinylacylaminodimethylthiophene series (39-42) taking compound 41 as the template.     

Design, synthesis and binding properties of novel and selective 5-HT(3) and 5-HT(4) receptor ligands

This work reports the synthesis and the binding tests on the 5-HT(3) and 5-HT(4) receptors of new thienopyrimidopiperazine and piperazinylacylaminodimethylthiophene derivatives, in order to identify potent and selective ligands for each receptor. The compound with higher affinity and selectivity for the 5-HT(3) over the 5-HT(4) receptor was the 3-amino-2-(4-benzyl-1-piperazinyl)-5,6-dimethyl-thieno[2,3-d]pyrimidin-4(3H)-one 28 (5-HT(3) K(i)=3.92 nM, 5-HT(4) not active), the compound with higher affinity and selectivity for the 5-HT(4) over the 5-HT(3) receptor was the 2-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butanoylamino]-4,5-dimethyl-3-thiophenecarboxylic acid ethyl ester 41 (5-HT(4) K(i)=81.3 nM, 5-HT(3) not active). Conformational analyses were carried out on the compounds of the piperazinylacylaminodimethylthiophene series (39-42) taking compound 41 as the template.     

Synthesis and preliminary pharmacological results on new naphthalene derivatives as 5-HT(4) receptor ligands

The indole derivative GR 113808 is currently used as the reference ligand for labelling the 5-HT(4) serotoninergic receptors. Previous works in our laboratories established the bioisosteric equivalency of the indole heterocycle and naphthalene in a series of melatonin receptor ligands. Based on this knowledge we designed new analogues of GR 113808 by introducing two bioisosteric modifications: firstly, the indole ring was replaced by a naphthalene one and secondly, the ester linkage was replaced by an amide group. Compound 8 emerged within this novel series as it displayed high and selective affinity at 5-HT(4) receptors (Ki 5-HT(4) = 6 nM, Ki 5-HT(3) = 100 nM, Ki values at other 5-HT receptors were higher than 1000 nM). Compound 8 is currently undergoing further pharmacological evaluation.     

Synthesis and structural investigation of some pyrimido[5,4-c]quinolin-4(3H)-one derivatives with a long-chain arylpiperazine moiety as potent 5-HT(1A/2A) and 5-HT(7) receptor ligands

A series of new pyrimido[5,4-c]quinolin-4(3H)-ones with variable length of the spacer between amide and 4-arylpiperazine moiety were prepared to further explore the role of a terminal portion in the serotonergic activity. The majority of compounds demonstrated high in vitro affinity for 5-HT_1A receptor, and moderate-to-low affinity for 5-HT_2A and 5-HT₇ receptors. X-ray analysis, two-dimensional NMR, conformational studies and docking into the 5-HT_1A receptor model were conducted to investigate conformational preferences of selected 5-HT_1A receptor ligands in different environments. The extended conformation of tetramethylene derivatives was found in a solid state, in DMSO (for a protonated form) and as a global energy minimum during conformational analysis in simulated water environment. Ligand geometry in top-scored complexes, obtained by docking to a set of 100 receptor models, were either fully extended or with central spacer torsion in synclinal conformation.     

Synthesis of new hexahydro- and octahydropyrido[1,2-c]pyrimidine derivatives with an arylpiperazine moiety as ligands for 5-HT1A and 5-HT2A receptors. Part 4

New 4-aryl-2H-pyrido[1,2-c]pyrimidine-1,3-dione derivatives of arylpiperazine (6-18) were prepared and evaluated in vitro for their affinity for 5-HT1A, 5-HT2A, and alpha1 receptors. The influence of ortho substitution in the phenyl ring, substitution at position 4 of the pyrido[1,2-c]pyrimidine system, and its unsaturation degree were explored. The tested compounds showed high affinity for the 5-HT1A receptor (Ki = 1.3-79.2 nM) and moderate to low affinity for the 5-HT2A (Ki = 51.7-1405 nM) and alpha1 receptors (Ki = 19.7-382.3 nM). Compounds 8 and 10 showed the highest 5-HT1A receptor affinity (Ki = 1.3 and 2.2 nM, respectively) and were 37- and 35.9-fold, respectively, more selective in relation to alpha1 adrenoreceptors.     

Synthesis by microwave irradiation and binding properties of novel 5-HT(1A) receptor ligands.

This work reports the synthesis by microwave irradiation and the binding tests on the 5-HT(1A), 5-HT(2A) and 5-HT(2C) receptors of new substituted piperazines in order to identify selective ligands for 5-HT(1A) subtype receptor. Conventional heating and microwave irradiation of the reactions was compared. Synthesis by microwave irradiation gave the desired compounds in better yields than those obtained by conventional heating. The overall times for the syntheses were considerably reduced. Some resulting active compounds (29 and 39) were characterised by a good selectivity profile for the 5-HT(1A) subtype receptor. The more active compounds were selected and further evaluated for their binding affinities on D(1), D(2) dopaminergic and alpha(1), alpha(2) adrenergic receptors. The compound with higher affinity and selectivity for the 5-HT(1A) over all the considered receptors was the 3-[4-[4-(1,2,3,4-tetrahydronaphthyl)-1-piperazinyl]butan]-benzotriazinone (-)29 (5-HT(1A) K(i)=36 nM, other receptors not active).     

运动和应激对海马5-羟色胺及其受体影响

目的通过观察体力运动和慢性应激对海马5-羟色胺(5-HT)水平及5-HT_1A受体mRNA表达的影响,探讨运动减缓应激性海马损伤作用在海马5-HT系统水平的可能机制。方法将24只成年雄性Wistar大鼠随机分为对照组、运动组、应激组和运动+应激组。用高效液相-电化学检测法测定海马5-HT水平,实时-荧光定量PCR法检测海马5-HT_1A受体mRNA的表达。结果与对照组比较,运动组海马5-HT水平显著升高(P<0.01),而应激组海马5-HT水平明显降低(P<0.01);与单纯应激组比较,运动+应激组5-HT水平略高于应激组,但差异无统计学意义(P>0.05)。运动组和应激组的5-HT_1A受体mRNA相对表达量分别是对照组的(4.49±1.59),(0.22±0.06)倍,且与对照比较差异有统计学意义(P<0.01)。运动+应激组5-HT_1A受体mRNA相对表达量是对照组的(0.87±1.22)倍,显著高于单纯应激组(P<0.05)。结论运动可能是通过提高海马5-HT水平和5-HT_1A受体表达来改善海马5-HT系统功能,发挥减缓慢性应激性海马损伤的作用,而且5-HT_1A受体表达的上调在此效应中作用更为明显。     

Synthesis of N-aryl-5-amino-4-cyanopyrazole derivatives as potent xanthine oxidase inhibitors

Some pyrazolo[3,4-d]pyrimidines, structurally related with allopurinol, a well known xanthine oxidase inhibitor, clinically used in the therapy of gout, have also been reported as potent inhibitors of xanthine oxidase and the growth of several human tumour cell lines. Considering the potential interest of this family of compounds, the aim of the present study was to synthesise and provide a full chemical characterization of new N-aryl-5-amino-4-cyanopyrazole derivatives and their corresponding pyrazolo[3,4-d]pyrimidines. Their biological activity pertaining to the xanthine oxidase inhibition and effect on the growth of three tumour cell lines (MCF-7, NCI-H460, and SF-268) are also provided. With only one exception, the synthesised compounds showed no effect on the growth of the three tumour cell lines. However, a strong xanthine oxidase inhibitory activity was observed for almost all pyrazolo[3,4-d]pyrimidines tested, revealing some of them IC(50) values below 1 microM. The results of the molecular docking studies of these compounds, against xanthine oxidoreductase are also described, providing an atomistic explanation of the differences in the inhibitory efficiency. MEP calculations were used to explain different inhibitory efficiency of similar inhibitors.     

Synthesis and 5-HT(1A), 5-HT(2A) receptor activity of new beta-tetralonohydantoins

A series of new 3-[4-(4-arylpiperazinyl)-butyl]-beta-tetralonohydantoins (8a-13a) were synthesized. The compounds exhibited high affinity for 5-HT(1A) receptors (K(i)=6 to 55 nM) combined with moderate-to-high 5-HT(2A) receptor affinities (K(i)=45 to 213 nM). The results of in vivo studies indicated that of the compounds tested, 3-[4-(4-phenylpiperazinyl)-butyl-beta-tetralonohydantoin (8a) showed features of full (pre- and postsynaptic) 5-HT(1A) receptor agonists, whereas compounds 9a-13a behaved like antagonists of postsynaptic 5-HT(1A) receptors; additionally, compound 13a produced an effect characteristic of presynaptic 5-HT(1A) receptor agonists. Moreover, compounds 8a and 10a-13a exhibited properties of 5-HT(2A) receptor antagonists. Due to the most interesting 5-HT(1A)/5-HT(2A) functional profile compounds 8a and 13a were further tested for their potential psychotropic activity. In fact, compound 8a (but not 13a) showed diazepam-like anxiolytic activity and behaved like a weak antidepressant.     

Optimization of a pharmacophore model for 5-HT4 agonists using CoMFA and receptor based alignment

Twenty two 5-HT4 agonists obtained from our laboratory and the recent literature were used to develop a CoMFA model to predict 5-HT4 agonist activity. Two models were produced and compared for predictivity, the first by alignments based on atom overlapping (model A) and the second by adding agonist binding site interacting points of the 5-HT4 receptor (model B). Comparison of the two models showed that the q2 value for model A was 0.564 vs. 0.582 for model B. Model B indicated that the predictive power model stems from far lower steric contributions, 0.270 compared to model A's 0.502. The dominant defining features were the electrostatic contributions for model B, 0.664 up from 0.477 in model A. The contributions from the LogP factor were minimal, 0.085 in both models. The synthesized compounds showed agonist activity at mumol level.     

Synthesis and pharmacological evaluation of carboxamide derivatives as selective serotoninergic 5-HT(4) receptor agonists

A number of new carboxamide derivatives were synthesized. The affinity of these compounds for the serotoninergic 5-HT(4) receptor was evaluated by use of radioligand-binding techniques. The agonistic activity was evaluated as the contractile effect of the ascending colon isolated from guinea-pigs. Among these compounds, 4-amino-5-chloro-2-methoxy-N-[1-[2-[(methylsulfonyl)amino]ethly]-4-piperidinylmethyl]benzamide (24) showed a high affinity for the 5-HT(4) receptor (Ki = 9.6 nM). Compound 24 displayed a higher affinity for 5-HT(4) receptors than the other receptors, including, 5-HT(3) and dopamine D(2) receptors. In addition, compound 24 was confirmed to be a potent 5-HT(4) receptor agonist (ED(50) = 7.0 nM). An interaction model between compound 24 and 5-HT(4) receptor was proposed.     

Synthesis and pharmacological properties of novel benzamide derivatives acting as ligands to the 5-hydroxytryptamine 4 (5-HT4) receptor

Aseries of 4-amino-5-chloro-2-methoxy-N-(1-substituted piperidin-4-ylmethyl)benzamides was synthesized as novel gastroprokinetic agents. The affinity of these compounds for the 5-hydroxytryptamine 4 (5-HT₄) receptor was evaluated. Among these compounds, 4-amino-5-chloro-2-methoxy-N-[1-[5-(1-methylindol-3-ylcarbonylamino)pentyl]piperidin-4-ylmethyl]benzamide (3f, Y-34959) showed a higher affinity for the 5-HT₄ receptor (Ki = 0.30 nmol/L) than for other receptors, and was confirmed to be a potent 5-HT₄ receptor agonist having contractile effects in the isolated guinea-pig ascending colon (EC₅₀ = 1.2 nmol/L). In dogs, compound 3f increased gastroprokinetic motility of both the gastric antrum and the ascending colon. In addition, this effect on the colon was inhibited by azasetron, a selective 5-HT₃ receptor antagonist, demonstrating that the effect of gastroprokinetic agents having 5-HT₃ receptor antagonism on the colon were reduced compared with that of selective 5-HT₄ receptor agonists.     

Synthesis, binding properties and receptor docking of 4-halo-6-[2-(4-arylpiperazin-1-yl)ethyl]-1H-benzimidazoles, mixed ligands of D2 and 5-HT1A receptors

In this publication we are describing synthesis, binding properties, and receptor docking of 4-halo-6-[2-(4-arylpiperazin-1-yl)ethyl]-1H-benzimidazoles, a new compounds with potential antipsychotics properties. Affinity towards the dopamine D(1)-like and D(2)-like, and serotonin 5-HT(1A) receptors was evaluated using the radioligand binding assays. All compounds tested had affinity for the D(2)-like and 5-HT(1A) receptors, but were inactive towards the D(1)-like receptor. Halogenated 6-[2-(4-arylpiperazin-1-yl)ethyl]-1H-benzimidazoles showed higher affinity compared to their nonhalogenated congeners. In silico docking analysis of selected ligands was performed in order to explain the results of binding assays. Our analysis suggests that stabilizing interactions between the halogen atom at the benzimidazole ring and the Ser-122 of the D(2)-like and Trp-358 of the 5-HT(1A) receptor. Energy contributions for these interactions were calculated using the ab initio method.     

硫柳汞对早产大鼠前额叶5-HT2 AR 和 DRD4表达的研究

目的探讨硫柳汞对早产大鼠前额叶（PFC）皮层5羟色胺受体2A（5-HT2AR）和多巴胺受体D4（DRD4）的研究。方法以人用疫苗中的常用硫柳汞含量（12．5μg/剂）为参照,分别按1、2、3、4倍剂量,根据体表面积转换法得出大鼠肌注计量为32．80μg/kg、65．60μg/kg、98．4μg/kg和131．20μg/kg,在出生第1天,分别一次性臀部注射给孕20天剖腹出生的早产大鼠,同时选注射生理盐水组作为对照,并采用Western blot 技术测量出生后第49天PFC中5-HT2AR和DRD4的表达。结果5组间5-HT2AR和DRD4的表达有显著性差异（F＝27．089,P＜0．01;F＝91．034,P＜0．01）。与生理盐水组比较,131．20μg/kg组PFC的5-HT2AR和DRD4的表达减退（q＝7．824,P＜0．01;q＝8．311,P＜0．01）,余组未发现显著变化。结论高浓度硫柳汞暴露后PFC的5-HT2AR和DRD4的表达减低,提示注意回避同时或短时间内连续接种以硫柳汞为防腐成分的疫苗。     

寻呼作业女工血清5-羟色胺水平的探讨

目的研究寻呼台作业人员血清5-羟色胺(5-HT)的变化规律。方法采用荧光分光光度测定法对76名寻呼台作业女工进行了血清5-HT含量测定。结果观察组工前血清5-HT含量为(72.7±12.8)μg/L,工后为(79.4±16.3)μg/L,对照组为(68.5±10.8)μg/L,三组间差异有显著性(P＜0.01);经q检验发现观察组工后高于工前(P＜0.05),且明显高于对照组(P＜0.01)。结论寻呼台作业人员血清5-HT含量可在一定程度上反映其职业应激状态。     

2-Substituted 5,6-dimethyl-3-phenylsulfonyl-pyrazolo[1,5-a]pyrimidines: new series of highly potent and specific serotonin 5-HT6 receptor antagonists

Syntheses, biological evaluation, and structure-activity relationships for a series of novel 2-substituted 3-benzenesulfonyl-5,6-dimethyl-pyrazolo[1,5-a]pyrimidines are disclosed. In spite of a wide, four orders of magnitude, SAR range (K_i varied from 260 pM to 2.96 μM), no significant correlation of 5-HT₆R antagonistic potency was observed with major physiochemical characteristics, such as molecular weight, surface polar area, cLogP, or number of rotatable bonds. Statistically significant trend was only observed for size of substitute group, which was not enough to explain the deep SAR trend. Besides with the substitute group size, another factor that presumably plays a role in defining the compound potencies is a relative position of the heterocycle and sulfophenyl moieties. Among all synthesized derivatives, (3-benzenesulfonyl-5,7-dimethyl-pyrazolo[1,5-a]pyrimidin-2-yl)-methyl-amine 18 is the most potent (K_i = 260 pM) and extremely selective, 5000 to >50,000-fold relative to 55 therapeutic targets, antagonist of the 5-HT₆ receptor.     

5-羟色胺2A受体基因、转运体基因与注意缺陷多动障碍冲动的关联分析

【目的】探讨5-羟色胺2 A受体基因(5-HT2A)T102C、5-羟色胺转运体基因(5-HTTLPR)启动子区多态性与伴有或不伴有冲动的注意缺陷多动障碍(attention deficit hyperactivity disorder,ADHD)的关系。【方法】抽取147例ADHD患儿,采用儿童Barratt冲动性量表(Barratt Impulsiveness Scale,BIS)父母版,将BIS总分≥划界分者为冲动组,共72例;BIS总分<划界分者为非冲动组,共75例;采用聚合酶链式反应(polymerase chain reaction PCR)、聚丙烯酰胺凝胶电泳结合银染技术,检测冲动组和不冲动组5-HT2A和5-HTTLPR基因型和等位基因的频率。【结果】冲动组5-HT2AT102C和5-HTTLPR的基因型和等位基因的频率与对非冲动组相比差异无显著性(P>0.05)。【结论】本研究结果不支持5-HT2AT102C、5-HTTLPR与冲动存在关联。     

Simultaneous localization of two linked disease susceptibility genes

For diseases with complex genetic etiology, more than one susceptibility gene may exist in a single chromosomal region. Extending the work of Liang et al. ([2001] Hum. Hered. 51:64-78), we developed a method for simultaneous localization of two susceptibility genes in one region. We derived an expression for expected allele sharing of an affected sib pair (ASP) at each point across a chromosomal segment containing two susceptibility genes. Using generalized estimating equations (GEE), we developed an algorithm that uses marker identical-by-descent (IBD) sharing in affected sib pairs to simultaneously estimate the locations of the two genes and the mean IBD sharing in ASPs at these two disease loci. Confidence intervals for gene locations can be constructed based on large sample approximations. Application of the described methods to data from a genome scan for type 1 diabetes (Mein et al. [1998] Nat. Genet. 19:297-300) yielded estimates of two putative disease gene locations on chromosome 6, approximately 20 cM apart. Properties of the estimators, including bias, precision, and confidence interval coverage, were studied by simulation for a range of genetic models. The simulations demonstrated that the proposed method can improve disease gene localization and aid in resolving large peaks when two disease genes are present in one chromosomal region. Joint localization of two disease genes improves with increased excess allele sharing at the disease gene loci, increased distance between the disease genes, and increased number of affected sib pairs in the sample.