Synthesis and binding properties of novel selective 5-HT3 receptor ligands
Introduction
The serotonin neurotransmitter is involved in a number of different physiological functions through its interaction with 14 types of receptors.1 These are G-protein-coupled receptors, with the exception of the 5-HT3, which is a ligand gated-ion channel receptor. 5-HT3 receptor antagonists, such as ondansetron, granisetron, or tropisetron,2 are used as antiemetic drugs to prevent vomiting associated with chemotherapy or radiation-induced emesis, but literature studies3 indicate that they could possess numerous other potential therapeutic applications in the control of pain or in the treatment of psychosis, memory impairment, depression, anxiety, schizophrenia, and drugs abuse. On the other hand, little is known about the therapeutic potential of 5-HT3 receptor agonists although some potent and selective ligands with full agonistic properties4 were recently reported. It has been suggested that stimulation of the 5-HT3 receptor modulates, in the central nervous system, the release of dopamine, cholecystokinin, and acetylcholine.5 Moreover, 5-HT3 receptors are involved in the peripheral control of acethylcholine release of the distal colon.6
In recent years, we have been engaged in the preparation of piperazinyl-substituted thieno[2,3-d]pyrimidin-4(3H)-one derivatives as 5-HT3 receptor ligands.7 These compounds possess the three key pharmacophoric elements (an aromatic moiety, a hydrogen-bond acceptor and a basic amino group) required for interaction with the 5-HT3 receptor and are structurally related to quipazine (Fig. 1), a potent ligand for the 5-HT3 receptor.8 Among these thieno[2,3-d]pyrimidine derivatives, 3-amino-5,6-dimethyl-2-[4-(1-phenylmethyl)-1-piperazinyl]thieno[2,3-d]pyrimidin-4(3H)-one A (Fig. 1) exhibited the highest affinity and selectivity for the 5-HT3 receptor (5-HT3: Ki=3.92 nM; 5-HT4: not active), behaving as a full agonist in the Bezold–Jarisch reflex assay.7 This result induced us to continue research in this field with the aim to obtain more potent and selective ligands for the 5-HT3 receptor. The present work reports on the synthesis and 5-HT3 receptor-binding properties of a series of new derivatives B[9], [10] (Fig. 1), which can be regarded as structural analogues of compound A. The new molecules maintain the thieno[2,3-d]pyrimidine scaffold as in A and exhibit the following structural variations: (i) the piperazine moiety at 4-position of the pyrimidine nucleus in place of the carbonyl group; (ii) in some compounds, 5- and 6-positions of the bicyclic system bear methyl groups (as in A); in others, a trimethylene or tetramethylene chain forms a third condensed ring, which, in some cases, is substituted with an ethoxycarbonyl group; (iii) 2-position of the pyrimidine is unsubstituted or bears a methylthio group.
The groups on N-4 of the piperazine ring (e.g., benzyl, 2-methoxyphenyl, phenyl, 2-pyrimidyl, methyl, and hydrogen) were the same as in a previous work.7 They were examined, together with the other structural modifications, with the aim to obtain more information on the structure–activity relationships in this new series.
Section snippets
Chemistry
Compounds 20–45 were prepared according to Scheme 1. Derivatives 3 and 4 were obtained by refluxing in acetone the corresponding β-amino esters of 4,5-disubstituted-thiophenes 1 and 211 with benzoyl isothiocyanate, commercially available or prepared in situ. When refluxed in an ethanolic potassium hydroxide solution, 3 and 4 gave the respective monopotassium salts of the 5,6-disubstituted-2-thioxothieno[2,3-d]pyrimidinones 5 and 6. On acidification of the potassium salts 5 and 6 with
Pharmacology
The title compounds 20–45 were tested in in vitro binding assays to evaluate their affinity for the 5-HT3 and the 5-HT4 receptors, using [3H]LY 278584 or [3H]GR113808 as radioligand, respectively. Binding data, reported in Table 1, are expressed as Ki values.
Moreover, in order to evaluate the putative agonistic or antagonistic properties of new molecules, two of them (31 and 33) were tested in an in vitro functional assay on isolated guinea pig distal colon.17 Results are reported in Figure 2
Results and discussion
The results of the binding tests, presented in Table 1, demonstrate that many of the title compounds (20, 24–27, 31–33, 38, 39, and 44) possess good affinities for the 5-HT3 receptor and high selectivity over the 5-HT4 receptor. In fact, none of the new derivatives displays measurable affinity for the latter receptor.
In this new series of derivatives the presence of the third unsubstituted cyclopentane ring fused with the thieno[2,3-d]pyrimidine system leads to the compounds with the best
Experimental section
Melting points were determined in open capillary tubes on a Gallenkamp M.p. apparatus and are uncorrected. Elemental analyses for C, H, N, and S were performed on a Fisons-Carlo Erba EA1108 Elemental Analyzer and were within 0.4% of the theoretical values. The IR spectra were recorded in KBr disks on a Perkin Elmer 1600 Series FT-IR spectrometer. 1H NMR spectra were recorded in DMSO-d6 solution at 200 MHz on a Varian Inova-Unity 200 spectrometer; chemical shifts (δ) are reported in ppm, with TMS
In vitro binding assays
Male CRL:CD(SD)BR-COBS rats (weighing about 150 g, Charles River, Italy) and male CRL:(HA) BR albino guinea pigs (weighing about 300 g, Charles River, Italy) were killed by decapitation; their brains were rapidly dissected into the appropriate areas (rat cortex for 5-HT3 and guinea pig striatum for 5-HT4) and stored at −80 °C until the day of assay.
The tissues were homogenized in 50 vol of ice-cold Tris HCl, 50 mM, pH 7.4 containing 0.5 mM EDTA, and 10 mM MgSO4 for 5-HT3, or Hepes HCl, 50 mM, pH 7.4,
In vitro organ studies on isolated guinea pig colon
Animal experiments were carried out with the approval of the Hungarian Ethical Committee for Animal Research (registration number: IV/1813–1/2002).
The distal portion of the colon was removed from a Hartley guinea pig (400–500 g) starved 24 h before experiments. The colon was cleaned in Krebs-bicarbonate buffer (in mM: NaCl 118.4, KCl 4.7, CaCl2 2.5, NaHCO3 25, MgSO4 1.2, KH2PO4 1.2, glucose 11.7; pH=7.4) at room temperature and cut 2 cm segments. The segments were suspended longitudinally in an
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