Model studies on a synthetically facile series of N-substituted phenyl-N'-pyridin-3-yl ureas leading to 1-(3-pyridylcarbamoyl) indolines that are potent and selective 5-HT(2C/2B) receptor antagonists

A model series of 5-HT_2C antagonists have been prepared by rapid parallel synthesis. These N-substituted phenyl-N′-pyridin-3-yl ureas were found to have a range of 5-HT_2C receptor affinities and selectivities over the closely related 5-HT_2A receptor. Extrapolation of simple SAR, derived from this set of compounds, to the more active but synthetically more complex 1-(3-pyridyl-carbamoyl)indoline series allowed us to target optimal substitution patterns and identify potent and selective 5-HT_2C/2B antagonists.     

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.     

Does alternative exon usage contribute to serotonin receptor heterogeneity?

We have previously isolated serotonin 5-HT_1C receptor cDNA clones. In contrast to most other receptors coupled to GTP binding proteins, the 5-HT_1C receptor gene contains several introns in its coding region. A similar exon-intron distribution is found in the 5-HT₂ receptor gene. The presence of large introns tempted us to test whether exchange of exons contributes to serotonin receptor heterogeneity. Therefore, blots with RNAs from different regions of the brain and brain slices were hybridized in situ with probes representing individual exons. We did not find any evidence for the exchange of exons which should result in an unequal distribution of hybridization signals found with the exon-specific probes. With the methodology used we should be able to see differential splicing in the form where individual exons are used alternatively resulting in mRNAs coding for different serotonin receptor types. We would not, however, see if a given exon can be modified by the alternative use of several splice-junctions.     

2-(Dimethylaminomethyl)-tetrahydroisoxazolopyridobenzazepine derivatives. Synthesis of a new 5-HT(2C) antagonist with potential anxiolytic properties

Following the program started at Johnson & Johnson Pharmaceutical Research & Development searching for 5-HT_2A/2C antagonists, we now report on the synthesis of 2-(dimethylaminomethyl)-2,3,3a,8-tetrahydroisoxazolo[3,2-a]pyrido[3,4-c]-[2]benzazepine and 2-(dimethylaminomethyl)-2,3,3a,8-tetrahydroisoxazolo[3,2-a]pyrido[3,2-c]-[2]benzazepine. A new method for the synthesis of pyridobenzazepines is described as well. The affinities for several receptors as well as the mCPP antagonistic activity of the compounds synthesised are described.     

Synthesis and SAR of highly potent dual 5-HT1A and 5-HT1B antagonists as potential antidepressant drugs

Novel 5-HT₁ autoreceptor ligands based on the N-4-aryl-piperazinyl-N′-ethyl-5,6,7,8-tetrahydropyrido[4′, 3′:4,5]thieno[2,3-d]pyrimidin-4(3H)-one core are described. Aiming at antidepressants with a novel mode of action our objective was to identify potent antagonists showing balanced affinities and high selectivity for the 5-HT_1A and 5-HT_1B receptors. Strategies for the development of dual 5-HT_1A and 5-HT_1B antagonists based on 1 and 2 as leads and the corresponding results are discussed. Isoquinoline analogue 33 displayed high affinity and an antagonistic mode of action for the 5-HT_1A and the 5-HT_1B receptors and was characterized further with respect to selectivity, electrically stimulated [³H]5-HT release and in vivo efficacy.     

2-Aryl tryptamines: selective high-affinity antagonists for the h5-HT2A receptor

A series of 2-aryl tryptamines have been identified as high-affinity h5-HT_2A antagonists. Structure–activity relationship studies have shown that h5-HT_2A affinity can be attained via modifications to the tryptamine side chain and that selectivity over h5-HT_2C and hD₂ receptors can be controlled by suitable C-2 aryl groups.     

Identification of a novel 5-HT1 binding site in rat spinal cord

High affinity, specific [³H]5-hydroxytryptamine (5-HT) binding to spinal cord synaptosomes was examined to identify the 5-HT receptor subtypes present. Computer nonlinear regression analysis of competition studies employing 8-OH-DPAT indicated that this 5-HT_1A selective agonist demonstrated high affinity competition (K_{i = 1.3 nM}) for 24.6 ± 0.7% of the total [³H]5-HT binding sites. Competition studies employing the 5-HT_1B selective agonist RU24969, in the presence of 100 nM 8-OH-DPAT, indicated that RU24969 demonstrated high affinity (K_{i = 1.1 nM}) competitive inhibition for 26.2 ± 1.4% of all [³H]5-HT binding sites. Neither 5-HT_1C, 5-HT_1D, 5-HT₂ nor 5-HT₃ selective compounds demonstrated any high affinity competition for the residual 49% of specific [³H]5-HT binding. Therefore, three major classes of [³H]5-HT binding sites could be demonstrated in spinal cord synaptosomes: 5-HT_1A, 5-HT_1B and a novel [³H]5-HT binding site which respectively represented 25, 26 and 49% of spinal cord synaptosomal [³H]5-HT binding. Further studies focusing on the function of the latter binding site are needed to determine if the presently identified novel binding site is the major 5-HT₁ receptor subtype present in spinal cord.     

Binding of beta-carbolines at 5-HT(2) serotonin receptors

A series of ring-substituted (i.e., methoxy and bromo) 3,4-dihydro- and 1,2,3,4-tetrahydro-β-carbolines was examined at 5-HT_2A and 5-HT_2C serotonin receptors. Whereas most of the methoxy-substituted derivatives typically displayed affinities similar to their unsubstituted parents, certain (particularly 8-substituted) bromo derivatives displayed enhanced affinity. A binding profile was obtained for selected β-carbolines.     

Synthesis and pharmacological evaluation of new arylpiperazines. 3-[4-[4-(3-chlorophenyl)-1-piperazinyl]butyl]-quinazolidin-4-one - a dual serotonin 5-HT(1A)/5-HT(2A) receptor ligand with an anxiolytic-like activity

On the basis of systematic studies on the structure–activity relationships in arylpiperazine group of serotonin ligands, 12 new derivatives containing quinazolidin-4(3H)-one (1–4), 2-phenyl-2,3-dihydrophthalazine-1,4-dione (5–8) or 1-phenyl-1,2-dihydropyridazine-3,6-dione (9–12) fragments were synthesized. The majority of the tested compounds (2, 4, 7, 8 and 10–12) showed a high affinity for 5-HT_1A receptors (K_i=11–54 nM) and two (1, 2) were found active at 5-HT_2A sites (16 and 68 nM, respectively). All the new 5-HT_1A ligands tested in vivo revealed an antagonistic activity at postsynaptic 5-HT_1A receptors, and three of them behaved as agonists at presynaptic ones. Additionally, both the meta-chlorophenylpiperazine derivatives containing quinazolidin-4-one fragment showed features of 5-HT_2A receptor antagonists. The dual 5-HT_1A/5-HT_2A receptor ligand (2) was further tested for its potential psychotropic activity. It showed a distinct anxiolytic-like activity in a conflict drinking test in rats and the observed effect was more potent in terms of the active dose, than that produced by diazepam (used as a reference drug).     

Central serotonin1A receptors: Respective distributions of encoding mRNA, receptor protein and binding sites by in situ hybridization histochemistry, radioimmunohistochemistry and autoradiographic mapping in the rat brain

The regional distribution of the mRNA encoding the 5-HT_1A serotonin receptor (whose selective agonists are potential anxiolytic and antidepressant drugs) was investigated in rat brain sections by in situ hybridization histochemistry using two sets of [³²P]labelled nucleoprobes, a riboprobe of 156 bases and oligoprobes of 30 bases corresponding to highly selective portions within the third intracellular loop and the N terminus domain of the amino acid sequence. These probes allowed the visualization of the 5-HT_1A mRNA mainly in the limbic regions: dentate gyrus and area CA₁ of the hippocampus, amygdala, entorhinal cortex, lateral septum and the dorsal raphe nucleus. These structures were also those which could be labelled by the specific 5-HT_1A radioligand [¹²⁵I]BH-8-MeO-N-PAT and antibodies raised against a synthetic 26 amino acid peptide whose sequence was taken from the most selective portion of the rat 5-HT_1A receptor protein. These data suggest that the 5-HT_1A receptors are not transported to a long distance from their site of synthesis, as it has been already reported for the somato-dendritic 5-HT_1A autoreceptors in the dorsal raphe nucleus. Combined autoradiographic quantification of the 5-HT_1A binding sites (labelled by a selective radioligand such as [¹²⁵I]BH-8-MeO-N-PAT, the 5-HT_1A receptor binding subunit (by radioimmunohistochemistry) and the 5-HT_1A mRNA on adjacent brain sections should be a relevant approach for assessing the molecular mechanisms responsible for the functional alterations of these receptors under various pathological and pharmacological conditions.     

Design, synthesis and evaluation of bicyclic benzamides as novel 5-HT1F receptor agonists

Several fused bicyclic systems have been investigated to serve as the core structure of potent and selective 5-HT_1F receptor agonists. Replacement of the indole nucleus in 2 with indazole and ‘inverted’ indazole provided more potent and selective 5-HT_1F receptor ligands. Indoline and 1,2-benzisoxazole systems also provided potent 5-HT_1F receptor agonists, and the 5-HT_1A receptor selectivity of the indoline- and 1,2-benzisoxazole-based 5-HT_1F receptor agonists could be improved with modification of the benzoyl moiety of the benzamides. Through these studies, we found that the inherent geometries of the templates, not the nature of hybridization of the linking atom, were important for the 5-HT_1F receptor recognition.     

Autoradiographic comparison of [I]LSD-labeled 5-HT2A receptor distribution in rat and guinea pig brain

Although the density and distribution of 5-HT_2A(5-hydroxytryptamine-2A) receptors is well established for rat brain, the 5-HT_2A receptor distribution and density in guinea pig brain has not been extensively studied. In the present in vitro study, we have utilized ¹²⁵I-lysergic acid diethylamide ([¹²⁵I]LSD) to quantify and compare 5-HT_2A receptor density in coronal sections of rat and guinea pig brain. Spiperone (1 μM) and sulpiride (1 μM) were used to displace [¹²⁵I]LSD binding from 5-HT_2A and D₂ binding sites, respectively. Ligand binding was quantified by computer-aided image analysis densitometry (MCID). Similar to the rat, areas of highest specific 5-HT_2A receptor binding (fmol/mg protein) in guinea pig brain included the claustrum and Layer 4 of the cerebral cortex. Significant binding was also found in remaining neocortical layers, islands of Calleja, caudate putamen, olfactory bulb, nucleus accumbens, and choroid plexus. While the rat brain exhibited a high level of specific binding in the tenia tecta and mammillary nuclei, little binding was observed in these regions in the guinea pig. In both rat and guinea pig, low specific binding was found in amygdaloid, thalamic, or cerebellar areas. These studies indicate a general similarity between 5-HT_2A binding site distribution and relative density in guinea pig and rat brain but point to a few brain regions where significant differences exist.     

Recognition site mapping and receptor modelling: Application to 5-HT receptors

A basic pharmacophore of the 5-HT_1D agonist recognition site was defined from a conformation-activity relationship study of 11 different agonists. It consists of an aromatic ring and a nitrogen atom in well defined relative positions. The contribution of the other molecular components was also explored. Similarities and differences between the 5-HT_1D and some other G-protein coupled receptor agonist recognition sites were discussed.

Following a different approach, three-dimensional models of the 5-HT₂ and 5-HT_1A receptor transmembrane region were defined from the analysis of their primary sequences, published site directed mutagenesis and labelling data and from the experimental structure of bacteriorhodopsin. Residues likely to be involved in 5-HT binding were proposed from the models of the receptor-neurotransmitter complexes.

There is an excellent convergence between binding site models derived from ligands analysis and from receptor modelling.     

[H]lysergic acid diethylamide (LSD): differential agonist and antagonist binding properties at 5-HT receptor subtypes in rat brain

[³H]Lysergic acid diethylamide (LSD) in the presence of 40 nM ketanserin labeled the 5-HT_1A receptor subtype in rat hippocampal membranes. In the presence of guanosine triphosphate (GTP), the B_max and affinity of [³H]LSD binding to the 5-HT_1A binding site were significantly decreased. [³H]LSD in the presence of 40 nM WB4101 labeled the 5-HT₂ receptor subtype in homogenates of rat frontal cortex. In contrast to the effect on [³H]LSD binding to the 5-HT_1A binding site, GTP produced no significant effect on either the B_max or the K_D of [³H]LSD binding to the 5-HT₂ binding site. Competition of 5-HT for [³H]LSD binding to the 5-HT₂ binding site was best described by a computer-derived model assuming two binding sites. In the presence of GTP, the 5-HT competition curve was shifted significantly to the right with an approx. 3-fold increase in the IC₅₀. These binding characteristics are consistent with [³H]LSD acting as an antagonist at the 5-HT₂ receptor which has multiple affinity states for agonists and is coupled to a guanine nucleotide regulatory subunit. Thus, [³H]LSD has binding characteristics consistent with it acting as an agonist at the 5-HT_1A receptor subtype but as an antagonist at the 5-HT₂ receptor subtype in rat brain.     

5-Hydroxytryptamine receptor subtypes in vertebrates and invertebrates

In the last few years, molecular biology has led to the cloning and characterization of several 5-HT receptors (serotonin receptors) in vertebrates and in invertebrates. These studies have allowed identification not only of 5-HT receptors already described but also of novel subtypes. The molecular cloning of 13 different mammalian receptor subtypes revealed an unexpected heterogeneity among 5-HT receptors. Except for the 5-HT₃ receptors which are ligand-gated ion channel receptors, all the other 5-HT receptors belong to the large family of receptors interacting with G proteins. Based on their amino acid sequence homology and coupling to second messengers these receptors can be divided into distinct families: the 5-HT₁ family contains receptors that are negatively coupled to adenylate cyclase; the 5-HT₁ family includes receptors that stimulate phospholipase C; the adenylyl cyclase stimulatory receptors are a heterogeneous group including the 5-HT₄ receptor which has not yet been cloned, the Drosophila 5-HT_drol receptor and two mammalian receptors tentatively named 5-HT₆ and 5-HT₇ receptors. The 5-HT_5A and 5-HT_5B receptors might constitute a new family of 5-HT receptors whose effectors are unknown. This review focusses on the molecular characteristics of the cloned 5-HT receptors such as their structure, their effector systems and their distribution within the central nervous system. The existence of a large number of receptors with distinct signalling properties and expression patterns might enable a single substance like 5-HT to generate simultaneously a large panel of effects in many brain structures. The availability of the genes encoding these receptors has already allowed a partial characterization of their structure-function relationship and will probably allow in the future a dissection of the contribution of each of these receptor subtypes to physiology and behaviour.     

Expression of serotonin receptor mRNAs in blood vessels

Using RT-PCR we distinguished mRNAs for all known G-protein coupled serotonin receptors expressed in various rat and porcine blood vessels. Nearly all vessels expressed 5HT1 _β, 5-HT_2A, 5-HT_2B, 5-HT₄, and 5-Ht₇ receptor mRNA to different extents. New splice variants of the porcine 5-HT₄ receptor were observed. Similar PCR assays were performed with endothelial and smooth muscle cells from human pulmonary artery, aorta, and with endothelial cells from human coronary artery and umbilical vein. All endothelial cells expressed 5-HT_{1 β}, 5-HT_2b, and 5-HT₄ receptor mRNA, whereas in smooth muscle cells 5-HT_{1 β}, 5-HT_2A, 5-HT₇, and in some experiments 5-HT_2B receptor mRNA were found. A model for the regulation of vascular tone by different 5-HT receptors is proposed.     

MIF-1 fails to modify agonist-induced oral activity in neonatal 6-OHDA-treated rats

-Prolyl- -leucyl-glycinamide (MIF-1) is known to attenuate apomorphine-induced stereotypies in adult rats that are lesioned as neonates with 6-hydroxydopamine (6-OHDA). To test whether MIF-1 would affect dopamine (DA) agonist-induced and serotonin (5-HT) agonist-induced oral activity, both intact and neonatal 6-OHDA-treated rats were studied. Rats at 3 days from birth were injected with desipramine (20 mg/kg, IP), 1 h before 6-OHDA HBr (100 μg, salt form, in each lateral ventricle) or its vehicle, saline-ascorbic acid (0.1%). At approximately 6 months rats were treated with MIF-1 (0.1, 1.0, or 10.0 mg/kg, IP), 10 min before SKF 39393 HCl (1.0 mg/kg, IP) or m-chlorophenylpiperazine 2HCl (m-CPP 2HCl; 0.5 mg/kg, IP), DA D₁ and 5-HT_1C,2 receptor agonists, respectively. Although both agonists increased oral activity in control and neonatal 6-OHDA-treated rats, MIF-1 did not modify the response. In rats that received either of the three doses of MIF-1 for 21 consecutive days, there was still no observed effect of MIF-1 on the oral response of control and 6-OHDA-lesioned rats to SKF 38393 and m-CPP. These findings indicate that MIF-1 does not modify the oral activity response of supersensitized D₁ and 5-HT_1C receptors in adult rats that are lesioned neonatally with 6-OHDA.     

5-HT1a receptors positively coupled to C-AMP formation in the rabbit retina

Serotonin, 8-OH-DPAT, buspirone, ipsapirone, RU-24969, dopamine and isoproterenol all stimulate C-AMP production in rabbit retinal homogenates. The dopamine and isoproterenol responses are specifically antagonized by haloperidol and propranolol, respectively. In contrast the effects produced by the other substances are specifically nullified by spiroxatrine, a known 5-HT_1a antagonist. Since 5-HT₂ antagonists (methysergide, kentanserin), 5-HT_1b antagonist (propranolol), 5-HT₄ antagonist (ICS-205930), 5-HT₃ antagonist (MDL-72222, ICS-205930) or the primarily ₂-antagonist mianserin, had no influence on the stimulation of C-AMP caused by serotonin or 8-OH-DPAT, it is concluded that 5-HT_1a receptors, positively coupled to C-AMP production exist in the retina.     

Guinea Pig Terminal 5-HT1D Autoreceptors Do Not Display a Circadian Variation in Their Responsiveness to Serotonin

Many of the components involved in the synthesis and release of serotonin (5-HT) display a circadian variation in their activity. Autoreceptors located on nerve terminals were recently suggested to underlie some of these circadian variations. The aim of this study was to examine whether terminal 5-HT_1D autoreceptors in the cerebral cortex of the guinea pig exhibit a circadian variation in their responsiveness. The responsiveness of these autoreceptors was assessed by the ability of exogenously applied 5-HT to inhibit the potassium-evoked release of [³H]5-HT from slices of guinea pig cortex. Identical experiments were conducted at four different, equally spaced time points during the light:dark cycle of the guinea pig. The results presented here demonstrate that terminal 5-HT_1D autoreceptors do not exhibit a circadian variation in their responsiveness. Therefore, terminal 5-HT_1D autoreceptors bear similarity to terminal 5-HT_1B autoreceptors identified in rat brain in being devoid of a significant rhythm in their responsiveness.     

Universal polyethylene glycol linkers for attaching receptor ligands to quantum dots

Biologically active small molecule derivatives that can be conjugated to quantum dots have the promise of revolutionizing fluorescent imaging in biology. In order to achieve this several technical hurdles have to be surmounted, one of which is non-specific adsorption of quantum dots to cell membranes. Pegylating quantum dots has been shown to eliminate non-specific binding. Consequently it is necessary to develop a universal synthetic methodology to attach small molecule ligands to polyethylene glycol. These pegylated small molecules may then be conjugated to the surfaces of quantum dots. Ideally this universal strategy should be adaptable and be applicable to PEG chains of varying lengths. This paper describes the development of one such methodology and the synthesis of a pegylated derivative of the known 5HT₂ agonist 1-(2-aminopropyl)-2,5-dimethoxy benzene. This compound was tested and found to be an agonist for the 5HT_2A and 5HT_2C receptor having EC₅₀ values of 250 and 50 nM, respectively.