Synthesis and biological profile of the enantiomers of [4-(4-amino-6,7-dimethoxyquinazolin-2-yl)-cis-octahydroquinoxalin- 1-yl]furan-2-ylmethanone (cyclazosin), a potent competitive alpha 1B- adrenoceptor antagonist.

The enantiomers of [4-(4-amino-6, 7-dimethoxyquinazolin-2-yl)-cis-octahydroquinoxalin-1-yl]-fu ran- 2-ylmethanone (cyclazosin, 1) were synthesized from the chiral furan-2-yl(cis-octahydroquinoxalin-1-yl)methanone [(+)-2 and (-)-2], which were obtained by resolution of the racemic amine with (S)-(+)- and (R)-(-)-mandelic acid. The binding profile of the enantiomers of 1 was assessed at alpha 1-, alpha 2-, D2, and 5-HT1A receptors as well as at native alpha 1A- and alpha 1B- and cloned alpha 1a-, alpha 1b-, and alpha 1d-adrenoceptor subtypes in comparison with prazosin, spiperone, and AH11110A. (+)-1 displayed a 40-90-fold selectivity for the alpha 1B(alpha 1b)-adrenoceptor relative to alpha 1A(alpha 1a) and alpha 1d subtypes. A significant enantioselectivity was observed at the alpha 1A(alpha 1a)-adrenoceptor and particularly at alpha 1d-adrenoceptors since (-)-1 was 11-14- and 47-fold, respectively, more potent than (+)-1. Furthermore the enantiomer (+)-1 displayed selectivities of 1100-, 19000-, and 12000-fold in binding to alpha 1b-adrenoceptors relative to alpha 2-adrenoceptors and 5-HT1A and D2 receptors. These results indicate that (+)-1, [(+)-cyclazosin] is the most potent and selective ligand for the alpha 1B-adrenoceptor subtype so far described and may be a valuable tool in the characterization of alpha 1-adrenoceptor subtypes.

Structure-activity relationships in 1,4-benzodioxan-related compounds. 6. Role of the dioxane unit on selectivity for alpha(1)-adrenoreceptor subtypes.

WB 4101-related benzodioxans 3-9 were synthesized, and their biological profiles at alpha(1)-adrenoreceptor subtypes and 5-HT(1A) serotoninergic receptors were assessed by binding assays in CHO and HeLa cells membranes expressing the human cloned receptors. Furthermore, receptor selectivity of selected benzodioxan derivatives was further determined in functional experiments in isolated rat vas deferens (alpha(1A)) and aorta (alpha(1D)) and guinea pig spleen (alpha(1B)), in additional receptor binding assays in rat cortex membranes containing alpha(2)-adrenoreceptors and 5-HT(2) serotoninergic receptors, and in rat striatum membranes containing D(2) dopaminergic receptors. An analysis of the results of receptor binding experiments for benzodioxan-modified derivatives 3-9 showed high affinity and selectivity toward the alpha(1a)-adrenoreceptor subtype for compounds 3-5 and 7 and a reversed selectivity profile for 9, which was a selective alpha(1d) antagonist. Furthermore, the majority of structural modifications performed on the prototype 1 (WB 4101) led to a marked decrease in the affinity for 5-HT(1A) serotoninergic receptors, which may have relevance in the design of selective alpha(1A)-adrenoreceptor antagonists. The exception to these findings was the chromene derivative 8, which exhibited a 5-HT(1A) partial agonist profile.

Design, synthesis, and biological activity of prazosin-related antagonists. Role of the piperazine and furan units of prazosin on the selectivity for alpha1-adrenoreceptor subtypes.

Prazosin-related quinazolines 4-20 were synthesized, and their biological profiles at alpha1-adrenoreceptor subtypes were assessed by functional experiments in isolated rat vas deferens (alpha1A), spleen (alpha1B), and aorta (alpha1D) and by binding assays in CHO cells expressing human cloned alpha1-adrenoreceptor subtypes. The replacement of piperazine and furan units of prazosin (1) by 1, 6-hexanediamine and phenyl moieties, respectively, affording 3-20, markedly affected both affinity and selectivity for alpha1-adrenoreceptor subtypes in functional experiments. Cystazosin (3), bearing a cystamine moiety, was a selective alpha1D-adrenoreceptor antagonist being 1 order of magnitude more potent at alpha1D-adrenoreceptors (pA2, 8.54 +/- 0.02) than at the alpha1A- (pA2, 7.53 +/- 0.01) and alpha1B-subtypes (pA2, 7.49 +/- 0. 01). The insertion of substituents on the furan ring of 3, as in compounds 4 and 5, did not improve the selectivity profile. The simultaneous replacement of both piperazine and furan rings of 1 gave 8 which resulted in a potent, selective alpha1B-adrenoreceptor antagonist (85- and 15-fold more potent than at alpha1A- and alpha1D-subtypes, respectively). The insertion of substituents on the benzene ring of 8 affected, according to the type and the position of the substituent, affinity and selectivity for alpha1-adrenoreceptors. Consequently, the insertion of appropriate substituents in the phenyl ring of 8 may represent the basis of designing new selective ligands for alpha1-adrenoreceptor subtypes. Interestingly, the finding that polyamines 11, 16, and 20, bearing a 1,6-hexanediamine moiety, retained high affinity for alpha1-adrenoreceptor subtypes suggests that the substituent did not give rise to negative interactions with the receptor. Finally, binding assays performed with selected quinazolines (2, 3, and 14) produced affinity results, which were not in agreement with the selectivity profiles obtained from functional experiments. This rather surprising and unexpected finding may be explained by considering neutral and negative antagonism.

Phenylpiperazinylalkylamino substituted pyridazinones as potent alpha(1) adrenoceptor antagonists.

QSAR models have been used for designing a series of compounds characterized by a N-phenylpiperazinylalkylamino moiety linked to substituted pyridazinones, which have been synthesized. Measurements of the binding affinities of the new compounds toward the alpha(1a)-, alpha(1b)-, and alpha(1d)-AR cloned subtypes as well as the 5-HT(1A) receptor have been done validating, at least in part, the estimations of the theoretical models. This study provides insight into the structure activity relationships of the alpha(1)-ARs ligands and their alpha(1)-AR/5-HT(1A) selectivity.

trans-4-[4-(Methoxyphenyl)cyclohexyl]-1-arylpiperazines: a new class of potent and selective 5-HT(1A) receptor ligands as conformationally constrained analogues of 4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]-1-arylpiperazines.

The present paper concerns the influence of conformational parameters on the recognition by rat 5-HT1A receptors of derivatives 4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]-1-(2-pyridinyl)piperazine (1a) and 3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)-N-[2-(2-pyridyloxy)ethyl]propanamine (3b), two highly potent and selective 5-HT1A receptor ligands. Fifteen corresponding flexible and rigid analogues were prepared following several synthetic routes and were tested in binding assays with radioligands at 5-HT1A, D2, and alpha1 receptors from rat brain membranes. Among the new derivatives emerged trans-4-[4-(3-methoxyphenyl)cyclohexyl]-1-(2-pyridinyl)piperazine (trans-8a) and trans-N-[4-(3-methoxyphenyl)cyclohexyl]-2-(2-pyridyloxy)ethylamine (trans-8b). These compounds can be considered as conformationally constrained analogues of compounds 1a and 3a, respectively. In fact, compounds trans-8a and trans-8b showed a marked enhancement in 5-HT1A receptor affinity when compared to the corresponding cis isomers. Because compound trans-8a was a potent and selective 5-HT1A ligand (K(i), nM: 5-HT1A = 0.028, D2 = 2194, alpha1 = 767), it was chosen as a lead to prepare other analogues that were tested at 5-HT1A, D2, and alpha1 receptors from rat brain membranes, showing high affinity at the 5-HT1A and selectivity vs D2 and alpha1 receptors. Selected compounds were tested for their affinity at the human cloned 5-HT1A, alpha1a, alpha1b, alpha1d receptor subtypes. They were also submitted to the [35S]GTPgammaS binding assay stimulating the 5-HT1A receptor-mediated G-protein activation, therefore behaving as full or as partial agonists. Finally, the ability of iv administration of trans-8a to induce fore-paw treading in rats was evaluated in comparison with 8-OH-DPAT. Although the affinity (K(i)) and in vitro activity (pD'2) of trans-8a at the 5-HT1A receptor were higher than those of 8-OH-DPAT, the compound was less potent than the reference standard in inducing the symptom.

WB 4101-related compounds. 2. Role of the ethylene chain separating amine and phenoxy units on the affinity for alpha(1)-adrenoreceptor subtypes and 5-HT(1A) receptors.

WB 4101 (1)-related benzodioxanes were synthesized by replacing the ethylene chain separating the amine and the phenoxy units of 1 with a cyclopentanol moiety, a feature of 6, 7-dihydro-5-[[(cis-2-hydroxy-trans-3-phenoxycyclopentyl)amino]meth yl] -2-methylbenzo[b]thiophen-4(5H)-one that was reported to display an intriguing selectivity profile at alpha(1)-adrenoreceptors. This synthesis strategy led to 4 out of 16 possible stereoisomers, which were isolated in the case of (-)-3, (+)-3, (-)-4, and (+)-4 and whose absolute configuration was assigned using a chiral building block for the synthesis of (-)-3 starting from (+)-(2R)-2, 3-dihydro-1,4-benzodioxine-2-carboxylic acid ((+)-9) and (1S,2S, 5S)-2-amino-5-phenoxycyclopentan-1-ol ((+)-10). The aim of this project was to further investigate whether it is possible to differentiate between these compounds with respect to their affinity for alpha(1)-adrenoreceptor subtypes and the affinity for 5-HT(1A) receptors, as 1 binds with high affinity at both receptor systems. The biological profiles of reported compounds at alpha(1)-adrenoreceptor subtypes were assessed by functional experiments in isolated rat vas deferens (alpha(1A)), spleen (alpha(1B)), and aorta (alpha(1D)) and by binding assays in CHO and HeLa cells membranes expressing the human cloned alpha(1)-adrenoreceptor subtypes and 5-HT(1A) receptors, respectively. Furthermore, the functional activity of (-)-3, (+)-3, (-)-4, and (+)-4 toward 5-HT(1A) receptors was evaluated by determining the induced stimulation of [(35)S]GTPgammaS binding in cell membranes from HeLa cells transfected with human cloned 5-HT(1A) receptors. The configuration of the cyclopentane unit determined the affinity profile: a 1R configuration, as in (+)-3 and (-)-4, conferred higher affinity at alpha(1)-adrenoreceptors, whereas a 1S configuration, as in (-)-3 and (+)-4, produced higher affinity for 5-HT(1A) receptors. For the enantiomers (+)-4 and (-)-4 also a remarkable selectivity was achieved. Functionally, the stereoisomers displayed a similar alpha(1)-selectivity profile, that is alpha(1D) > alpha(1B) > alpha(1A), which is different from that exhibited by the reference compound 1. The epimers (-)-3 and (+)-4 proved to be agonists at the 5-HT(1A) receptors, with a potency comparable to that of 5-hydroxytryptamine.

Synthesis, pharmacological evaluation, and structure-activity relationship and quantitative structure-activity relationship studies on novel derivatives of 2,4-diamino-6,7-dimethoxyquinazoline alpha1-adrenoceptor antagonists.

A new series of novel piperazine and non-piperazine derivatives of 2, 4-diamino-6,7-dimethoxyquinazoline was synthesized and evaluated for binding affinity toward alpha1-adrenergic and other G-protein-coupled aminergic receptors. The alpha1-adrenoceptor (AR) subtype selectivity was also investigated for the most interesting compounds. Only compound 16 showed moderate selectivity toward the alpha1b-AR subtype. Selected compounds were tested in vivo in a dog model indicating activity on blood pressure and on the lower urinary tract. Compound 10 showed in vivo potency close to that of prazosin. Powerful interpretative and predictive theoretical QSAR models have been obtained. The theoretical descriptors employed in the rationalization of the alpha1-adrenergic binding affinity depict the key features for receptor binding which can be summarized in an electrostatic interaction between the protonated amine function and a primary nucleophilic site of the receptor, complemented by short-range attractive (polar and dispersive) and repulsive (steric) intermolecular interactions. Moreover, on predictive grounds, the ad hoc derived size and shape QSAR model developed in a previous paper (Rastelli, G.; et al. J. Mol. Struct. 1991, 251, 307-318) proved to be successful in predicting nanomolar alpha1-adrenergic binding affinity for compound 28.

Mediation of noradrenaline-induced contractions of rat aorta by the alpha 1B-adrenoceptor subtype.

1. The subtypes of alpha 1-adrenoceptor mediating contractions to exogenous noradrenaline (NA) in rat aorta have been examined in both biochemical and functional studies. 2. Incubation of rat aortic membranes with the irreversible alpha 1B-adrenoceptor antagonist, chloroethylclonidine (CEC: 10 microM) did not change the KD of [3H]-prazosin binding in comparison to untreated membranes, but reduced by 88% the total number of binding sites (Bmax). 3. Contractions of rat aortic strips to NA after CEC (50 microM for 30 min) incubation followed by repetitive washing, showed a marked shift in the potency of NA and a partial reduction in the maximum response. The residual contractions to NA after CEC incubation were not affected by prazosin (10 nM). 4. The competitive antagonists prazosin, terazosin, (R)-YM-12617, phentolamine, 5-methylurapidil and spiperone inhibited contractions to NA with estimated pA2 values of 9.85, 8.54, 9.34, 7.71, 7.64 and 8.41, respectively. 5. The affinity of the same antagonists for the alpha 1A- and alpha 1B- adrenoceptors was evaluated by utilizing membranes from rat hippocampus pretreated with CEC, and rat liver, respectively. 5-Methylurapidil and phentolamine were confirmed as selective for the alpha 1A-adrenoceptors, whereas spiperone was alpha 1B-selective. 6. A significant correlation was found between the pA2 values of the alpha 1-adrenoceptor antagonists tested and their affinity for the alpha 1B-adrenoceptor subtype, but not for the alpha 1A-subtype. 7. In conclusion, these findings indicate that in rat aorta most of the contraction is mediated by alpha 1B-adrenoceptors, and that the potency (pA2) of an antagonist in this tissue should be related to its antagonistic effect on this subtype of the alpha 1-adrenoceptor population.

Reduction of normal food intake in rats and dogs and inhibition of experimentally induced hyperphagia in rats by CM 57373 and fenfluramine.

The anorectic effect of CM 57373 in dogs and in rats food-deprived or with experimentally induced hyperphagia (cafeteria-diet hyperphagia and insulin hyperphagia) was compared to the effect of serotoninergic anorectic drug dl-fenfluramine. CM 57373 and dl-fenfluramine administered orally caused a dose-related reduction of food consumption by food-deprived rats (ID50 = 7.4 mg/kg and 2.5 mg/kg respectively). The oral ID50 in dogs was 2.4 mg/kg for CM 57373 and 1.1 mg/kg for dl-fenfluramine. This animal species tolerated CM 57373 better than dl-fenfluramine. The latter induced mydriasis, dyskinesia and reduced spontaneous activity. The anorectic effects of CM 57373 and dl-fenfluramine in cafeteria-diet hyperphagic rats were comparable. Tolerance to the anorectic effect developed in rats treated with both CM 57373 and dl-fenfluramine although tolerance was initially less pronounced with CM 57373 than dl-fenfluramine. The brain serotonin levels of cafeteria-fed rats were unchanged by CM 57373 throughout treatment whereas dl-fenfluramine decreased the monoamine levels starting from the 8th day. Both drugs reduced 5-hydroxyindolacetic acid levels. CM 57373 (7.4 mg/kg p.o.) and dl-fenfluramine (2.5 mg/kg p.o.) markedly reduced the overeating caused by insulin injection. These results indicate that CM 57373 shows several characteristics of drugs that act via serotonin to depress food intake in various animal species.

Characterization of alpha 1-adrenoceptor subtypes in prostate and prostatic urethra of rat, rabbit, dog and man.

The alpha 1-adrenoceptor subtypes present in the smooth muscle of urethra and prostate of different animal species, including man, were characterized by using receptor binding techniques. In prostatic urethra and prostate membranes, [3H]prazosin labelled a single population of alpha 1-adrenoceptors (Hill coefficient not different from unity) with a high affinity in the range 0.21-0.51 nM. The number of specific [3H]prazosin binding sites was partially affected by chloroethylclonidine only in human and rat prostate membranes, whereas this agent proved practically devoid of activity in rabbit and dog prostate membranes as well as in the prostatic urethra membranes of all the animal species examined. These findings indicate that in prostatic and urethral membranes the alpha 1-adrenoceptors mainly belong to the alpha 1A subtype. The binding results were confirmed by in vitro functional studies on noradrenaline-induced contractions of rabbit and dog urethral preparations. The agonist-induced contractions were practically unaffected by preincubation of both tissues with chloroethylclonidine, but were sensitive to nifedipine. We found, moreover, a good correlation between the potency of different selective and non-selective alpha 1-adrenoceptor antagonists (WB-4101, 5-methylurapidil, phentolamine, spiperone, prazosin and urapidil) tested against the noradrenaline-induced contractions of rabbit urethra and their affinity for the alpha 1A-adrenoceptor subtype, no correlation with the affinity for the alpha 1B subtype, and a lower correlation with the affinity for the alpha 1C-adrenoceptor subtype.

Affinity of different alpha 1-agonists and antagonists for the alpha 1-adrenoceptors of rabbit and rat liver membranes.

In membranes prepared from rabbit liver, competition with [3H]prazosin by different alpha 1-agonists and antagonists revealed different affinities in comparison to the results obtained on rat liver membranes, and showed a good correlation with the affinity of the same compounds for the cloned alpha 1c-adrenoceptor subtype. The potencies observed on rat liver membranes were well correlated with the affinity observed for the cloned alpha 1b-adrenoceptors. These results confirm that rabbit and rat liver membranes preparations can be utilized to evaluate the affinity of compounds for these alpha 1-adrenergic subtypes.

The alpha 1d-adrenoceptor subtype is involved in the noradrenaline-induced contractions of rat aorta.

The pA2 value of several alpha 1-adrenoceptor antagonists on noradrenaline-induced contractions of rat aorta, and their affinity for the cloned alpha 1a-, alpha 1b- and alpha 1d-adrenoceptor subtypes were evaluated. Selective or moderately selective alpha 1d-, partially selective alpha 1b-, selective alpha 1a- and non subtype-selective alpha 1-adrenoceptors antagonists were included in the study. The potency of these compounds on rat aorta was well correlated with the affinity observed for the alpha 1d-adrenoceptor subtype. A poor correlation was found for the alpha 1b- and alpha 1a-subtypes. These results suggest that the alpha 1d-subtype plays a determining role in rat aorta contractions induced by noradrenaline.

Antagonism to noradrenaline-induced lethality in rats is related to affinity for the alpha1A-adrenoceptor subtype.

The potency of several alpha1-adrenoceptor antagonists in preventing the noradrenaline-induced lethality in conscious rats, their binding affinity for the native alpha1A- and alpha1B-adrenoceptors, the recombinant animal alpha1a-, alpha1b- and alpha1d-adrenoceptor subtypes, as well as their functional affinity for the alpha1L-adrenoceptor subtype were evaluated. The potency of the tested compounds as antagonists of noradrenaline-induced lethality was correlated with the affinity for the alpha1A- (and alpha1a-) adrenoceptor subtype, but not with the affinity for the other subtypes. On the contrary, the hypotensive effects of the compounds, assessed in anesthetized rats, were not clearly related with the affinity for any of the alpha1-subtypes. These results suggest that the alpha1A-subtype plays a determining role in preventing lethality induced by noradrenaline in the rats, and that this activity is unrelated to the hypotensive effect of the compounds, which cannot be clearly correlated with affinity for a particular alpha1-adrenoceptor subtype.

Searching for cyclazosin analogues as alpha(1B)-adrenoceptor antagonists.

A series of quinazoline derivatives, 2-20, structurally related to the racemic alpha(1)-adrenoceptor antagonist cyclazosin (1), were synthesized and evaluated for their functional antagonism at alpha(1)- and alpha(2)-adrenoceptors and for their binding affinity at human cloned alpha(1a)-, alpha(1b)- and alpha(1d)-adrenoceptor subtypes. They displayed, like 1, preferential antagonism and selectivity for alpha(1) versus alpha(2)-adrenoceptors. Compounds 10, 13, and 18 showed high potency at alpha(1)-adrenoceptors similar to that of 1 (pK(B) values 8.47-8.89 versus 8.67), whereas 13 and 15 were endowed with the highest alpha(1)-adrenoceptor selectivity, only 3- to 4-fold lower than that of 1. In binding experiments, all of the compounds displayed an affinity practically similar to that found for 1, with the exception of 19 and 20 that were definitely less potent. The s-triazine analogue 18 was the most potent of the series with pK(i) values of 10.15 (alpha(1a)), 10.22 (alpha(1b)) and 10.40 (alpha(1d)), resulting 77-fold more potent than 1 at alpha(1a)-adrenoceptors. In addition, the majority of compounds, like prototype 1, showed the same trend of preferential affinity for alpha(1d)- and alpha(1b)-adrenoceptors that alpha(1a)-subtype. In conclusion, we identified compounds 2-5, 10, 12 and 13, bearing either an aliphatic- or an arylalkyl- or aryloxyalkyl-acyl function, with an interesting subtype-selectivity profile, which makes them suitable candidates for their resolution as enantiomers structurally related to (+)-cyclazosin.

Pharmacological activities of clobazam and diazepam in the rat: relation to drug brain levels.

Brain distribution and various pharmacological effects of clobazam and diazepam were studied in rats. When given at 10 mg/kg i.p. the compounds reached peak brain levels 15 min after injection, and showed similar half lives. At peak time brain levels were proportional to the dose administered. Very little of the N-desmethylmetabolite of each compound was found in the brain. Clobazam was less effective than diazepam in protecting rats from pentetrazol convulsions. Disrupting rota-rod performance and increasing punished responses in a "conflict" test, the relative potencies ranging from 4 to 8 in the various tests. The results are discussed in relation to the importance of animal species selection for predicting favourable therapeutic effects in humans.

Effects of some antidepressants on the volume-induced reflex contractions of the rat urinary bladder: lack of correlation with muscarinic receptors affinity.

It has been suggested that tricyclic antidepressants such as imipramine, might exert their anti-enuretic action by a blockade of muscarinic receptors in the detrusor muscle of the urinary bladder. We have therefore investigated the effects of two tricyclic (imipramine and nortriptyline) and three atypical (citalopram, amineptine and mianserin) antidepressants on the micturition reflex and muscarinic receptors in rats. The micturition reflex pathway was monitored indirectly by recording the rhythmic intravesical pressure waves which occurred when the bladder was distended and maintained under constant saline-volume. The activity of the antidepressants was correlated to their potencies as antagonists of [3H]QNB binding to rat brain (mainly M1 receptors) and bladder (mainly M2 receptors) membranes, as well as antagonists of carbachol-induced contractions of rat bladder strips. Only imipramine and citalopram dose dependently inhibited the voiding contractions, whereas nortriptyline, imipramine and mianserin (in order of potency) were active both in binding studies and as competitive antagonists of carbachol-induced bladder contractions, but were inactive in inhibiting the micturition reflex. The present data seem to suggest that affinities for muscarinic receptors are unrelated to the inhibition of micturition reflex.

Effects of terflavoxate on stimulated contractions of urinary bladder in vitro.

The antispasmodic activity of terflavoxate (CAS 86433-39-8), a flavone derivative with spasmolytic properties on the urinary tract, has been studied in vitro, in comparison to the most common drugs utilized in the therapy of overactive detrusor, namely flavoxate, oxybutynin, and terodiline. Terflavoxate showed affinity for bladder (and brain) muscarinic receptors at micromolar level, however, its activity on carbachol-induced contractions of rat bladder was clearly non competitive, indicating that the compound is devoid of functional antimuscarinic property. Moreover, the observation that unlike antimuscarinic drugs, terflavoxate inhibited by more than 50% field stimulation-induced contractions of rabbit bladder strips, indicates that mechanisms other than the anticholinergic one should be responsible for its smooth muscle relaxant properties. Terflavoxate, flavoxate, oxybutynin, and terodiline were equally effective in inhibiting the two components of K(+)-induced contractions, while nifedipine and nicardipine were more potent than the other compounds, and more effective in inhibiting tonic than phasic contractions. In addition, while nifedipine and nicardipine antagonized in a competitive manner calcium-induced contractions of potassium-depolarized bladder strips, the other spasmolytics behaved as mixed antagonists. Differences in calcium antagonistic properties between nifedipine and nicardipine on one side, and terflavoxate on the other, are further demonstrated by the data on binding experiments. Nevertheless, present results suggest that Ca(++)-antagonistic effects are mainly responsible for terflavoxate smooth muscle relaxant properties.

Effect of different 5-hydroxytryptamine receptor subtype antagonists on the micturition reflex in rats.

OBJECTIVE: To evaluate the effects of antagonists of different subfamilies of 5-hydroxytryptamine (5-HT) receptors on bladder function in anaesthetized and conscious rats. MATEERIALS AND METHODS: The urinary bladder of female anaesthetized rats was catheterized urethrally and filled with physiological saline until spontaneous bladder contractions occurred. Infravesical pressure was measured by a pressure transducer and displayed continuously on a chart recorder. The time of bladder quiescence (to the disappearance of rhythmic contractions) after injection with different compounds tested was recorded. Conscious rats underwent cystometry with chronically (infravesical) implanted catheters to continuously record bladder capacity (evaluated as amount of saline infused between voiding cycles) and maximal voiding pressure. The affinity for the human recombinant serotoninergic 5-HT1A subtype (inhibition of specific binding of [3H]8-hydroxy-2-(di-n-propylamino) tetralin) and the effects on the [35S]guanosine 5'-(gamma-thio) triphosphate (GTPgammaS) binding in HeLa cells was also evaluated. RESULTS: Among the compounds tested, only 4-(2'-methoxy-phenyl)-1-[2'-(n-2"-pyridinyl)-p-iodobenzamido]-ethyl-piperazine (p-MPPI) and methiothepin showed nanomolar affinity for the 5-HT1A receptors, the former being a neutral antagonist and the latter an inverse agonist in the [35S]GTPgammaS binding model. Intravenous injection of low doses of p-MPPI and methiothepin induced a dose-dependent disappearance of isovolumic bladder contractions in anaesthetized rats (> 10 min). At the highest doses, the dose-response curves were bell-shaped. The amplitude of bladder contractions was not markedly altered. The tested antagonists of 5-HT2, 5-HT3, 5-HT4, and 5-HT6 serotoninergic subtypes were poorly active or inactive in the model. Similarly, these compounds were inactive on cystometry in conscious rats, whereas p-MPPI and methiothepin induced a consistent increase in bladder capacity. Methiothepin also decreased the voiding pressure, whereas p-MPPI did not affect this variable. CONCLUSIONS: These findings confirm that only selective 5-HT1A receptor antagonists have favourable effects on the bladder, inducing an increase in bladder capacity with no derangement of bladder contractility.

N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-nitrophenyl) cyclohexanecarboxamide: a novel pre- and postsynaptic 5-hydroxytryptamine(1A) receptor antagonist active on the lower urinary tract.

N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-nitrophenyl) cyclohexanecarboxamide (Rec 15/3079) was synthesized with the aim of obtaining a novel compound with 5-hydroxytryptamine (5-HT)(1A) antagonistic properties and activity in controlling bladder function at the level of the central nervous system. Rec 15/3079 showed a selective high affinity for the 5-HT(1A) receptor (K(i) = 0.2 nM). At the human recombinant 5-HT(1A) receptor, Rec 15/3079 acted as a competitive, neutral antagonist in that it did not modify basal [(35)S]guanosine-5'-O-(3-thio)triphosphate binding to HeLa cell membranes but shifted the activation isotherm to 5-HT to the right, in a parallel manner, with a pK(b) value of 10.5. Accordingly, Rec 15/3079 (i.v.) potently antagonized 8-hydroxy-2-dipropylaminotetralin (8-OH-DPAT)-induced hypothermia in mice (ID(50) = 20 microg/kg) and 8-OH-DPAT-induced forepaw treading in rats (ID(50) = 36 microg/kg). In vitro Rec 15/3079 was poorly active in antagonizing carbachol-induced bladder (pD'(2) = 5.03) and norepinephrine-induced urethral (apparent pK(b) = 6) contractions. However, in anesthetized rats, Rec 15/3079 (10-100 microg/kg i.v.) blocked isovolumic bladder contractions with no effect on their amplitude. In conscious rats and guinea pigs with bladders filled with saline, Rec 15/3079 (300-1000 microg/kg i.v.) increased bladder volume capacity (BVC) without affecting bladder contractility. In conscious rats with bladders filled with dilute acetic acid, Rec 15/3079 (300 microg/kg i.v.) reversed the decrease of BVC induced by the acid. To evaluate apparent selective effect on lower urinary tract reflexes, Rec 15/3079 was tested in experimental models for sedative, analgesic, anxiolytic, and antidepressant activity. Rec 15/3079 showed only a slight decrease in the duration of immobility in the behavioral despair test (antidepressant activity) at 1 mg/kg i.v. No anxiolytic activity was observed at 10 mg/kg i.v. No effect was observed in the hot plate test, but Rec 15/3079 increased tail-flick latencies after 3 to 10 mg/kg i.v. In conclusion, these studies demonstrate that Rec 15/3079 is endowed with favorable effects on bladder function, and it is devoid of unwanted side effects at the level of central nervous system at doses at least 10-fold higher than those active on the bladder.