[[(Arylpiperazinyl)alkyl]thio]thieno[2,3-d]pyrimidinone derivatives as high-affinity, selective 5-HT1A receptor ligands.

A series of 2-[[(4-aryl-1-piperazinyl)alkyl]thio]thieno[2,3-d]pyrimidin-4 (1H)-one and 3-substituted 2-[[(4-aryl-1-piperazinyl)alky]thio]thieno[2,3-d]pyrimidin-4 (3H)-one derivatives was prepared and evaluated for in vitro 5-HT1A receptor affinity by radioligand binding assays; the selectivity for 5-HT1A receptors rather than alpha 1-adrenoceptors was also examined (ratio of the IC50 alpha 1 to IC50 5-HT1A). The binding tests gave indications about the best features of the [(arylpiperazinyl)alkyl]thio moiety and of the substituents on the thiophene and pyrimidinone rings for efficacious and selective 5-HT1A ligands. The most effective derivative for displacing [3H]-8-OH-DPAT from rat hippocampal membranes was the 3-amino-2-[[3-[4-(2-methoxyphenyl)-1-piperazinyl] propyl]thio]-5,6-dimethylthieno[2,3-d]pyrimidin-4(3H)-one (70) (IC50 = 0.3 nM) with selectivity of 24 for the 5-HT1A over the alpha 1-adrenoceptor. Compound 73, where the 2-methoxyphenyl on the N4 piperazine ring was replaced with a pyrimidine group, showed the best selectivity, with a ratio of 74, while its affinity IC50 for 5-HT1A was 6.8 nM. These results, compared to those for compounds 46 (IC50 24 nM; selectivity 2) and 49 (IC50 226 nM; selectivity 5), N3 unsubstituted analogues of derivatives 70 and 73, show the importance of an amino group in position 3 of the thienopyrimidine system for the interaction with 5-HT1A receptor binding sites, although this fragment can affect the affinity and selectivity only if linked to the (arylpiperazinyl)alkyl moiety. The better selectivity of piperidine 74 (IC50 0.8; selectivity 45) compared to the analogous piperazine 70 is also noteworthy. Twenty of the 30 molecules used for determining the binding affinity to 5-HT1A and alpha 1-adrenergic receptors were selected for QSAR analysis using a series of molecular descriptors and calculated with the TSAR software.

In vitro affinity of piribedil for dopamine D3 receptor subtypes, an autoradiographic study.

Receptor binding autoradiography, using the selective ligand [3H]7-OH-(R)DPAT (R(+)-2-dipropylamino-7-hydroxy 1,2,3,4-tetrahydronaphthalene), showed that piribedil is a potent inhibitor at dopamine D3 receptors in limbic regions (island of Calleja), with affinity (IC50) between 30 and 60 nM. The in vitro IC50 of piribedil for inhibition of [3H]spiperone binding to receptors of the dopamine D2-like family (D2, D3 and D4), ranged between 10(-7) and 10(-6) M in different brain regions (medial and lateral caudate putamen, olfactory tubercles, and nucleus accumbens). At the highest concentration tested (10(-5 M) piribedil inhibited dopamine D1 receptor binding by < 50%. It is concluded that piribedil has 20 times higher affinity for dopamine D3 than for dopamine D2-like receptors, and very low affinity for the dopamine D1 receptor subtype in rat brain. How this pattern of receptor affinity is related to the pharmacological profile of piribedil deserves further investigation.

Effects of chronic treatment with fluoxetine and citalopram on 5-HT uptake, 5-HT1B autoreceptors, 5-HT3 and 5-HT4 receptors in rats.

The effect in rats of chronic treatment with two specific 5-HT reuptake inhibitors (SSRI) with antidepressant properties, citalopram (10 mg/kg, i.p. twice a day for 14 days, one day washout) and fluoxetine (15 mg/kg, p.o. twice a day for 21 days, 7 days washout), was evaluated on some mechanisms involved in central 5-HT neurotransmission. No adaptive modifications of brain 5-HT uptake (sites) were found by measuring functional [3H]5-HT uptake and [3H]citalopram binding in cortical and hippocampal synaptosomes, and by [3H]citalopram binding autoradiography in the raphe nuclei (5-HT cell bodies) and the ventral tegmental area (5-HT axonal pathway). Chronic treatments had no effect on presynaptic 5-HT1B autoreceptors, functionally evaluated by measuring 5-HT1B-mediated inhibition of depolarization-induced [3H]5-HT release from cortical and hippocampal synaptosomes. Chronic citalopram or fluoxetine did not significantly affect the binding of [3H]BRL-43694 to 5-HT3 receptors in the rat brain cortex. Citalopram had no effect on [125I]SB-207710 binding to 5-HT4 receptors, measured by autoradiography in the substantia nigra. Negative results, such as those reported in the present study, could be due to a number of variables including the animal species, the treatment schedule or the brain areas considered, thus explaining the differences from some previous reports of significant effects of SSRI. However, our negative data are in agreement with many other published studies, suggesting that adaptive modifications of brain 5-HT transporters, terminal 5-HT1B receptors, 5-HT3 and 5-HT4 receptors may not be a general effect induced by all SSRI.

Are 5-hydroxytryptamine7 receptors involved in [3H]5-hydroxytryptamine binding to 5-hydroxytryptamine 1nonA-nonB receptors in rat hypothalamus?

We assayed [3H]5-hydroxytryptamine ([3H]5-HT) binding in rat hypothalamic membranes to confirm the possible of measuring 5-HT7 receptors. Binding was tested in the presence of 3 microM (+/-)-pindolol, a concentration higher than previously suggested for the same purpose (0.1 micron). This higher concentration was, however, needed to fully saturate 5-HT1A and 5-HT1B receptors without interaction with 5-HT7 receptors. Under these conditions, [3H]5-HT binding could be further inhibited with methiothepin (used to determine nonspecific binding) and with 5-HT, with an IC50 of 1.4 nM and a slope of 1. The inhibition curves of (+/-)-8-hydroxy-dipropylaminotetralin, ritanserin, and mianserin were shallow (slopes, 0.35-0.58) and could be better analyzed with the two-site model, indicating that the pindolol-insensitive [3H]5-HT binding sites in rat hypothalamic membranes are heterogeneous. Although the IC50 of the compounds tested suggests that one population of sites is actually associated with 5-HT7 receptors, our data clearly indicate that this binding assay does not selectively label 5-HT7 receptors in native tissues. These results challenge a previous report and suggest that the proposed down-regulation of 5-HT7 receptors after fluoxetine treatment should be considered with caution. The development of more selective and sensitive binding assays will probably offer significant advantage.