New 2-piperazinylbenzimidazole derivatives as 5-HT3 antagonists. Synthesis and pharmacological evaluation.

A series of 2-piperazinylbenzimidazole derivatives were prepared and evaluated as 5-HT3 receptor antagonists. Their 5-HT3 receptor affinities were evaluated by radioligand binding assays, and their abilities to inhibit the 5-HT-induced Bezold-Jarisch reflex in anesthetized rats were determined. Compound 7e (lerisetron, pKi = 9.2) exhibited higher affinity for the 5-HT3 receptor than did tropisetron and granisetron, while compound 7q (pKi = 7.5) had very low affinity for this receptor, showing that substitution on the N1 atom of the benzimidazole ring is essential for affinity and activity. The effect of substitution on the aromatic ring of benzimidazole by several substituents in different positions is also discussed. A strong correlation between the 5-HT3 antagonistic activity of the studied compounds and the position of substitution on the aromatic ring was established. Thus, while the 4-methoxy derivative 7m showed weak affinity for the 5-HT3 receptor (pKi = 6.7), the 7-methoxy derivative 7n exhibited the highest affinity (pKi = 9.4). Compounds 7e and 7n are now under further investigation as drugs for the treatment of nausea and emesis evoked by cancer chemotherapy and radiation.

New (2-methoxyphenyl)piperazine derivatives as 5-HT1A receptor ligands with reduced alpha 1-adrenergic activity. Synthesis and structure-affinity relationships.

New 2-(methoxyphenyl)piperazine derivatives 1 and 2 containing a terminal heteroaryl or cycloalkyl amide fragment were prepared and their 5-HT1A affinities evaluated by radioligand binding assays. The influence of the alkyl chain length or the amide group on affinity was evaluated. A four-carbon chain appears to be optimal when the amide fragment is a heteroaryl group. Derivatives with a cycloalkyl moiety displayed maximum affinity in the two methylene chain series. Electronic distribution within the amide region seems to have an influence on affinity in heteroaryl derivatives. Replacement of the heteroaryl moiety by a cycloalkyl group led to compounds with enhanced affinity. Increasing the lipophilicity of the cycloalkyl derivatives by annelation and/or saturation increased their affinity for the 5-HT1A sites. Compounds with cis-bicyclo[3.3.0]octane (2a, 2c), norbornane (2f, 2g), and norbornene (2h, 2i) groups bind at 5-HT1A sites with 2-10-fold higher affinity than NAN-190. Antagonist activity at alpha 1-adrenergic receptors was evaluated for compounds with high affinity at 5-HT1A sites. Compounds 2a, 2c, 2f, 2g, and 2h strongly bind (Ki = 0.12-0.63 nM) at 5-HT1A receptors and are devoid of antagonist activity at alpha 1-adrenergic receptors.

2,3-Dihydro-2-oxo-1H-benzimidazole-1-carboxamides with selective affinity for the 5-HT(4) receptor: synthesis and structure-affinity and structure-activity relationships of a new series of partial agonist and antagonist derivatives.

A series of 2,3-dihydro-2-oxo-1H-benzimidazole-1-carboxamide derivatives bearing a piperazine moiety was synthesized. Their in vitro 5-HT(4), 5-HT(3), and D(2) receptors affinities were evaluated by radioligand binding assay. For selected compounds functional studies at the 5-HT(4) receptor were made by using precontracted (by carbachol) preparations of rat esophageal tunica muscularis mucosae (TMM). The influence of the 3-substituent of the benzimidazole ring, the 4-substituent of the piperazine moiety, and the alkylene spacer was studied. Compounds with an ethyl or a cyclopropyl substituent in the 3-position of the benzimidazole ring showed moderate to high affinity (K(i) = 6.7-75.4 nM) for the 5-HT(4) receptor with selectivity over 5-HT(3) and D(2) receptors and moderate antagonist activity (pK(b) = 6.19-7.73). Compounds with an isopropyl substituent in the 3-benzimidazole position exhibited moderate and selective 5-HT(4) affinity (K(i) >/= 38.9 nM) and a partial agonist activity (5a, i.a. = 0.94) higher than that of the reference compound BIMU 8 (i.a. = 0.70). This reversal of the pharmacological activity due only to a small structural difference might confirm the existence of two binding sites on the 5-HT(4) receptor. In the alkylene spacer, a two-methylene chain is favorable to optimize the affinity and the antagonist or the partial agonist activity. In the ethyl and cyclopropyl series, 5-HT(4) antagonist activity seems to be unrelated to the size of the 4-substituent of the piperazine moiety, whereas a methyl group is optimal for high partial agonist activity in the isopropyl series; however, the presence of a butyl substituent is a favorable pattern for 5-HT(4) antagonism and even causes a reversal of the pharmacological profile in the isopropyl series (5h, pK(b) = 7.94). N-Butyl quaternization of 5a led to an improvement in affinity for the 5-HT(4) receptor and mantained the high partial agonist activity (5r, K(i) = 66.3 nM, i.a. = 0.93).

Serum protein binding of lerisetron, a novel specific 5HT3 antagonist, in patients with cancer.

The aim of this study was, (1) to characterize the serum protein binding of lerisetron, a new 5-hydroxytryptamine (5-HT3) receptor antagonist under investigation as an antiemetic agent, and (2) to measure the percentage of unbound lerisetron in cancer patients. The binding parameters were determined in human serum albumin (HSA), alpha1-acid glycoprotein (AAG) and in pooled serum from six healthy volunteers. Concentrations of lerisetron ranging from 50 ng/ml to 2 microg/ml were used. The serum protein binding of 14C-lerisetron (2 microg/ml) was determined by ultrafiltration in three groups of individuals. Group I comprised healthy subjects (n = 11), group II comprised cancer patients undergoing radiotherapy (n = 9), and group III comprised cancer patients receiving chemotherapy (n = 18). The unbound concentration of lerisetron was measured in all samples by liquid scintillation counting. Concentrations of both AAG and HSA were also measured in all serum samples. The drug was extensively bound in pooled serum, involving a nonsaturated process. In HSA, lerisetron was also highly bound (4.04+/-0.8% unbound) and the protein binding was essentially unchanged within the studied concentration range of lerisetron. The extent of binding to AAG was high but significantly lower than in serum and in HSA and was also independent of lerisetron concentration. The unbound lerisetron was significantly decreased in group II cancer patients when compared with group I subjects (2.38+/-0.64% vs 3.70+/-0.70%; P < 0.001). No significant changes in lerisetron binding were observed in group III patients. HSA was diminished in both groups of patients and AAG was only significantly increased in group II. Unbound lerisetron was correlated with AAG in group II and with HSA in group III.

Determination of ipriflavone and its synthetic impurities by high-performance liquid chromatography using diode-array detection.

A simple, accurate and rapid method for the determination of ipriflavone and its synthetic impurities has been developed. It consists in RP-HPLC separation and identification of the impurities from their UV spectra using photodiode-array detection. The method has been validated and shows good specificity, accuracy, precision and sensitivity.

Synthesis and structural, conformational, and pharmacological study of some of esters derived from 3-phenethyl-3-azabicyclo[3.2.1]octan-8-beta-ol and the corresponding N-endo-methyl quaternary derivatives.

A series of 8-beta-acyloxy-3-phenethyl-3-azabicyclo[3.2.1]octane and its N-endo methiodides were synthesized and studied by 1H and 13C NMR spectroscopy, and the crystal structure of 8-beta-p-chlorobenzoyloxy-3-phenethyl-3-azabicyclo[3.2.1]octane methiodide (2c) was determined by X-ray diffraction. In CDCl3 solution, 1b-1e display the same preferred conformation. The cyclopentane and piperidine rings adopt an envelope conformation flattened at C-8 and a distorted chair conformation puckered at C-8 and flattened at N-3, respectively, with the N-substituent in the equatorial position with respect to the piperidine ring. In all cases, methylation takes place from the endo position. The ability of the title compounds to antagonize the acetylcholine-induced contraction of guinea pig ileum is also reported. An initial structure-activity relationship is proposed.

Synthesis and structural, conformational, biochemical, and pharmacological study of new compounds derived from tropane-3-spiro-4'(5')-imidazoline as potential 5-HT3 receptor antagonists.

A series of tropane-3-spiro-4'(5')-imidazolines was synthesized and studied by 1H and 13C NMR spectroscopy, and the crystal structure of 2'-(1H-indol-3-yl)tropane-3-spiro-4'(5')-imidazoline hydrochloride 5(6)f was determined by X-ray diffraction. In CD3OD solution, compounds 5(6)a-f display the same preferred conformation. The pyrrolidine and piperidine rings adopt an envelope conformation flattened at N8 and a distorted chair conformation puckered at N8 and flattened at C3, respectively, with the N-substituent in the equatorial position with respect to the piperidine ring. This conformation is similar to that observed for compound 5(6)f in the solid state. From binding studies on the compounds synthesized, compound 5(6)d demonstrated the ability to efficiently displace the binding of [3H]GR65630 to bovine brain area postrema membranes to an extent comparable to MDL 72222. In the von Bezold-Jarisch reflex, compound 5(6)d was equipotent with metoclopramide. It is, therefore, likely that the imidazoline ring may provide a useful bioisosteric replacement for the carbonyl group in 5-HT3 antagonists.

Synthesis and histamine H(1)-receptor antagonist activity of 4-(diphenylmethyl)-1-piperazine derivatives with a terminal heteroaryl or cycloalkyl amide fragment.

New 4-(diphenylmethyl)-1-piperazine derivatives with a terminal heteroaryl or cycloalkyl amide fragment were synthesized and evaluated for their antihistaminic, anticholinergic and antiallergic activities. Tested compounds were found to be moderate to potent in vitro (guinea-pig ileum) histamine H(1)-receptor antagonists. Derivatives with a four methylene chain (1e-1h) were as potent in vivo (capillary permeability in rats) as cetirizine; the heteroaryl derivatives 1e and 1h were found to be the most active agents. These compounds displayed only weak in vitro (guinea-pig ileum) muscarinic M(3)-receptor antagonist activity. Compounds 1e and 1g were about 100 times more potent than ketotifen in preventing the compound 48/80-induced histamine release from rat peritoneal mast cells. Derivatives 1e, 1f and 1h did not modify the spontaneous motor activity in rats at 100 mg/kg po. Compound 1e has been selected for further studies.

Evaluation of bronchospasmolytic, antiallergic, anti-inflammatory, mucolytic and antitussive activities of decasilate in experimental models.

The bronchospasmolytic, antiallergic, anti-inflammatory, mucolytic and antitussive activities of 8-(2-phenylethyl)-1-oxa-diazaspiro[4,5]decan-2-one 2-tiophenecarboxylate (decasilate, CAS 76652-72-7) have been evaluated using different experimental models. 1. Decasilate showed a remarkable spasmolytic activity against histamine-induced contractions in the isolated guinea-pig tracheal preparation with an IC50 of 2.7 x 10(-6) mol/l. In addition, the oral administration of decasilate (5-30 mg.kg-1) significantly reduced the histamine aerosol-induced bronchospasm in guinea-pigs. 2. Decasilate had a preventive effect against antigen-induced contractions of ileum segments from sensitized guinea-pigs (EC50 8.0 x 10(-6) mol/l) and relaxed them when added after the antigen challenge (IC50 9.5 x 10(-7) mol/l). 3. Both carrageenin- and dextran-induced rat hind paw oedemas were significantly reduced by the oral administration of decasilate with ED50 values of 169.5 and 34.5 mg.kg-1, respectively. However, it was ineffective against the cotton pellet-induced granuloma in the rat. 4. Furthermore, decasilate had a significant mucolytic activity in rabbits and reduced the number of tussive seizures induced by an aerosol of citric acid in guinea-pigs. The pharmacological profile of decasilate suggests that it might be useful in the management of chronic bronchitis.

Semi-automated solid-phase extraction procedure for the high-performance liquid chromatographic determination of alinastine in biological fluids.

A solid-phase extraction (SPE) method for sample clean-up followed by a reversed-phase HPLC procedure for the assay of alinastina (pINN) in biological fluids is reported. The effects of the sample pH, composition of the washing and elution solvents and the nature of the SPE cartridge on recovery were evaluated. The selectivity of SPE was examined using spiked rat urine and plasma samples and the CH and PH cartridges gave rise to the cleanest extracts. The recoveries obtained in spiked rat urine and plasma samples were 91.2+/-2.7 and 99.9+/-2.8%, respectively. The proposed SPE method coupled off-line with a reserved-phase HPLC system with fluorimetric detection was applied to the quantitation of alinastine in real rat urine samples. The analytical method was also applied and validated for the determination of alinastine in dog plasma. The recovery from spiked dog plasma samples using the PH cartridge was around 65%. The within-day and between-day precisions were 7 and 12%, respectively. The detection and quantitation limits in dog plasma were 0.024 and 0.078 microg/ml, respectively.

Synthesis and 5-HT3 receptor affinity of new quinolinecarboxylic acid derivatives.

A series of quinolinecarboxylic acid amides and an ester with a quinuclidine moiety were synthesized and their in vitro affinities at 5-HT3, 5-HT4, and D2 receptors evaluated by radioligand binding assays. Highest affinity at 5-HT3 receptor corresponded to derivative 5 with Ki = 9.9 nM and with selectivity over 5-HT4 and D2 receptors. Compounds displayed moderate 5-HT3 antagonist activity (ED50 = 10.5-21.5 microg/kg i.v.). The obtained data suggest that the 5-HT3 receptor sites can accommodate the acyl group of the 2-quinoline derivatives. The results indicate the existence of an optimal distance between the lone electron pair of the quinoline nitrogen atom and the azabicyclic nitrogen atom, and a no-pharmacophoric pocket in the 5-HT3 receptor which would hold the fragment at the position 4 of the quinoline ring.

Matrix solid-phase dispersion technique for the determination of a new antiallergic drug, bilastine, in rat faeces.

A matrix solid-phase dispersion (MSPD) procedure for the isolation and HPLC determination of a new antiallergic agent, bilastine, in rat faeces is presented. The effect on recovery of empirical variables such as nature, pH and volume of the washing and elution liquids and nature of the adsorbent has been tested. The best recoveries were attained using an octadecylsilyl sorbent, 10 ml of a 0.1 M NaHCO3-Na2CO3 aqueous buffer of pH 10.0 as washing solvent and 10 ml of methanol as elution solvent. The extracts were evaporated to dryness and reconstituted in mobile phase before their injection into a HPLC system, equipped with a Discovery RP-amide C16 column and a fluorescence detector. The method allows one to reach recoveries of 95.0% within the concentration range 0.05-10 microg/g, with within-day repeatabilities of less than 5% and between-day repeatabilities of less than 9% within this range. This method has been successfully applied to the excretion studies of bilastine in the rat.