BIMT 17, a 5-HT2A receptor antagonist and 5-HT1A receptor full agonist in rat cerebral cortex.

In the search for antidepressant agents with a rapid onset of action, we have found that compound BIMT 17 (1-[2-[4-(3-trifluoromethylphenyl)piperazin-1- yl]ethyl]benzimidazol-[1H]-2-one) shows a good affinity for cerebral cortical 5-HT1A (pKi = 7.72) and 5-HT2A (pKi = 6.90) receptors, with no appreciable affinity for the other 5-HT receptor subtypes, including 5-HT2C. BIMT 17 reduced forskolin-stimulated cAMP accumulation in the cerebral cortex (pEC50 = 6.09) and in the hippocampus (pEC50 = 6.50), and antagonized 5-HT-induced phosphatidylinositol turnover (pKi = 6.96) in the cerebral cortex. The effect on cAMP accumulation was blocked by the 5-HT1A receptor antagonist tertatolol. Buspirone, 8-OH-DPAT and S 14671 (1-[2-(2-thenoylamino)ethyl]- 4[1-(7-methoxynaphtyl)]-piperazine), claimed to be 5-HT1A receptor agonists, did not reduce forskolin-stimulated cAMP formation in the cerebral cortex. On the basis of these data, it was concluded that BIMT 17 was the only compound that behaved as a full agonist with respect to the cAMP response in the cortex, while exerting concurrent agonism at 5-HT1A receptors and antagonism at 5-HT2A receptors. These characteristics might explain the peculiar behavior of BIMT 17 in mimicking the inhibitory action of 5-HT on the basal firing rate of the cortical neurons (see accompanying paper).

BIMT 17, a 5-HT1A receptor agonist/5-HT2A receptor antagonist, directly activates postsynaptic 5-HT inhibitory responses in the rat cerebral cortex.

BIMT 17 (1-[2-[4-(3-trifluoromethyl phenyl) piperazin-1-yl] ethyl] benzimidazol- [1H]-2-one), a 5-HT1A receptor agonist/5-HT2A receptor antagonist (see Borsini et al., accompanying paper), in a dose range of 1-10 mg/kg i.v., dose-dependently inhibited the electrical activity of rat medial prefronto-cortical neurons, whereas buspirone, in a dose range of 0.1-1000 micrograms/kg, increased it. 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) and 1-[2-(2-thenoylamino)ethyl]-4-[1-(7-methoxynaphthyl)] piperazine (S 14671) presented biphasic patterns of response; they increased electrical activity at doses in the range of 0.1-10 micrograms/kg and 0.1-3 micrograms/kg i.v. respectively, and reduced it at high doses, 30-300 micrograms/kg and 10-30 micrograms/kg i.v., respectively. The inhibitory effect of BIMT 17 on the firing rate of neurons in the frontal cortex was antagonized by the 5-HT1A antagonists tertatolol and WAY 100135, and was still present after destruction of serotonin (5-HT) containing neuronal endings by the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT; 150 micrograms/rat, given intraventricularly), which reduced the cortical 5-HT content by 85%. This destruction of 5-HT neurons, while suppressing the ability of 8-OH-DPAT to inhibit the firing rate at high doses, did not change the excitatory action of this compound at low doses. The addition of ritanserin, a 5-HT2A receptor antagonist, potentiated both the excitatory and inhibitory effects of 8-OH-DPAT on neuronal electrical activity. Direct microiontophoretic application (100 nA/20 s) of 5-HT and BIMT 17, but not that of 8-OH-DPAT, onto medial prefronto-cortical neurons, decreased the firing rate of these neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Gastroprokinetic properties of the benzimidazolone derivative BIMU 1, an agonist at 5-hydroxytryptamine4 and antagonist at 5-hydroxytryptamine3 receptors.

We have investigated the in vivo motor stimulating and gastroprokinetic properties of the azabicycloalkyl benzimidazolone derivative BIMU 1 (3-ethyl-2,3-dihydro-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2-oxo-1H- benzimidazole-1-carboxamide hydrochloride) and its binding profile at 5-hydroxytryptamine3 and 5-hydroxytryptamine4 receptors, in an attempt to assess the serotonergic mechanism underlying its prokinetic action. BIMU 1 dose-dependently (0.01-0.3 mg/kg i.v.) increased the motility of a denervated pouch of canine stomach. This excitatory action was sensitive to muscarinic blockade. A similar stimulatory effect was exerted by the benzamidic prokinetic agent cisapride (0.03-0.3 mg/kg i.v.) but not by the 5-HT3 receptor antagonist ondansetron (up to 1 mg/kg i.v.). The significance for propulsive efficacy of the motor stimulating activity of BIMU 1 was evaluated in a model of gastric emptying of liquids in the conscious dog. The emptying rate of a non-caloric liquid meal instilled through a gastric fistula was accelerated by both BIMU 1 (0.01-1 mg/kg i.v. and 0.1-3 mg/kg p.o.) and cisapride (0.03-1 mg/kg i.v. and 0.3-10 mg/kg p.o.). Ondansetron (1 mg/kg i.v.) did not show any effect. The activity of the 5-HT4 receptor antagonist DAU 6285 was evaluated in the gastric emptying model per se and in interaction experiments on the accelerating action of BIMU 1 (0.3 mg/kg i.v.).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of DAU 6215, a novel 5HT-3 antagonist, in animal models of anxiety.

The aim of the present study was to evaluate the effect of DAU 6215 (N-(endo-8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2, 3-dihydro-2-oxo-1H-benzimidazol-1-carboxamide, hydrochloride), which is a 5HT-3 receptor antagonist, chemically different from the other 5HT-3 antagonists, on a wide variety of animal models sensitive to anxiolytics. Nine animal models were used. DAU 6215 was active in reducing (i) aversion to a brightly lit environment in the light/dark exploratory test in mice, (ii) unpleasant properties of an aversive drug in rats (naloxone-induced place aversion), and (iii) aggressiveness in monkeys. DAU 6215 was effective at doses ranging (a) between 10 and 1000 micrograms/kg given i.p. in mice, (b) between 15 and 30 micrograms/kg given s.c. in rats and (c) between 1 and 10 micrograms/kg given orally in monkeys. DAU 6215 was inactive in (iv) the elevated plus maze, (v) conflict test and (vi) emotional hypophagia in rats and in (vii) the four plates test, (viii) staircase test and (ix) stress-induced hyperthermia in mice. Diazepam was active in all tests. In contrast to diazepam, DAU 6215 did not induce place preference, suggesting the possible lack of addictive properties.

Pharmacological properties of a novel class of 5-HT3 receptor antagonists.

The pharmacological profile of six representative members of a novel class of 5-HT3 receptor antagonists is described. The compounds are esters and amides of benzimidazolone-1-carboxylic acid with a basic azabicycloalkyl moiety (compounds 1-3) and their respective ethyl derivatives (compounds 4-6). In isolated preparations (rabbit heart and guinea pig ileum) all compounds antagonized the 5-HT3 receptor-mediated effects of serotonin, with potencies comparable with those of the reference compounds, ICS 205.930 and GR 38032F (-log IC50 9.30-11.9 and 6.8-8.20, in heart and ileum, respectively). In the anaesthetised rat, all agents potently inhibited the Bezold-Jarisch reflex whether given i.v. or i.d. I.v. administration of compounds prevented cisplatin-induced emesis in dogs (ID50 ranging from 3.7 to 147 micrograms/kg). All agents accelerated gastric emptying of solids in rats (ED50 about 10-160 micrograms/kg i.p.). In addition, compounds 4 and 5 were able to stimulate 5-HT4 receptors in the isolated guinea pig ileum, as well as enhance contractile activity in the Heidenhain gastric pouch of dogs, showing clearcut prokinetic properties.

Malonamic acid derivatives as M1 selective muscarinic receptor antagonists.

A series of malonamic acid esters with suitable amino alcohols, typical of antimuscarinic compounds, was synthesized and the affinities for the three pharmacologically defined muscarinic receptor subtypes, namely M1, M2 and M3, were evaluated by radioligand displacement experiments. It was found that the esters with 3-quinuclidinol 7b, 7f-g, 8 and 9 are ligands with intermediate to high affinity for the M1 receptors, for which they show a preferential binding. Unexpectedly, the ester 7a with tropine bound with negligible affinity to all the receptors investigated. The introduction of a phenyl group on the carboxamido moiety of 7b gave compound 9, which showed an affinity for the M1 receptor comparable with that of the reference drug Pirenzepine 1.

Substituent effect on the stereochemistry of H2-receptor antagonists of the phenylformamidine series. A conformation-dependent mode of interaction with the H2 receptor.

The influence of alkyl substitution on the stereoisomerism of the formamidine cation (E,E vs E,Z) of several N-substituted (imidazolylphenyl)formamidines (1-10) was investigated. As (imidazolylphenyl)formamidines having alkyl substituents of more than three carbon atoms bind to H2-receptor preparations in a pseudoirreversible mode causing unsurmountable antagonism, the four isomeric butylformamidines (5-7 and 9) having comparable lipophilic character but different E,E/E,Z composition were investigated in H2-receptor assays to determine quantitatively any difference in their pseudoirreversible inhibitory pattern. It was found that the geometry of the formamidine cation is affected by the steric bulk of the substituent on the formamidine nitrogen. A relationship between the percentage of the E,E conformation of the formamidine cation and degree of pseudoirreversible antagonism was also found. The present studies support the hypothesis that bidentate hydrogen bonding plays an important role in the interaction of (imidazolylphenyl)formamidines with the H2 receptor.

Synthesis and biological evaluation of new muscarinic receptor antagonists bearing cyclic amidines as cationic heads.

Two classes of compounds, bearing a cyclic amidino moiety instead of the tertiary amino group of the classical antimuscarinic drugs like hexahydrodifenidol 3 were synthesized. Affinities (KD) for the three pharmacologically defined M1, M2 and M3 mAChR subtypes were measured in radioligand binding assays and in functional in vitro studies (KB) in guinea pig ileum and left atrium. The results showed that the replacement of the tertiary amino group in structural analogues of 3 with a cyclic amidino moiety afforded potent antimuscarinic compounds. The selectivity shown for smooth muscle preparations suggests their usefulness as antispasmodics.

New CCK antagonists derived from the amino acid residues of the CCK-8 fragment.

The synthesis of lipophylic derivatives of the amino acid residues of the CCK-8 fragment is described. According to "in vitro" binding studies and functional test, nearly all the compounds behaves as CCK-antagonists; moreover some compounds are able to interact differentially with CCK-A and CCK-B receptor subtype. In particular, compounds 2c, 2g, and 2h possess a high affinity for the CCK-A receptor subtype coupled with a low affinity for the CCK-B subtype. This results in an interesting selectivity profile. However, the same compounds are not able to antagonize the effects exerted by CCK-itself, when tested in "in vivo" assays.

Antiemetic activity of the new 5-HT3 antagonist DAU 6215 in animal models of cancer chemotherapy and radiation.

The antiemetic activity of DAU 6215, a novel antagonist of 5-HT3 receptors, was investigated in animal models of cytotoxic treatment-evoked emesis and compared with the antiemetic activity of ondansetron and metoclopramide. In dogs, vomiting was induced by i.v. cisplatin; in ferrets, the emetic response was elicited by i.v. doxorubicin or X-ray exposure. Pretreatment with 0.1-1 mg/kg DAU 6215 given i.v. or p.o. prevented the vomiting response to the different emetic agents. In the dog, the antiemetic potency of metoclopramide was 30 times lower than that of DAU 6215. Ondansetron was less potent than DAU 6215 against cisplatin and doxorubicin but was equally effective in the radiotherapy protocol. In this model, lengthening of the pretreatment time to 2 h did not affect the antiemetic efficacy of DAU 6215, whereas it decreased that of ondansetron. The results demonstrate that DAU 6215 is a highly effective and long-lasting inhibitor of cytotoxic treatment-induced emesis in different animal species.

Synthesis and biological evaluation of new antimuscarinic compounds with amidine basic centers. A useful bioisosteric replacement of classical cationic heads.

Amidines (guanidine, formamidine, and acetamidine) were introduced as substitutes for the cationic heads present in atropine, scopolamine, and corresponding quaternary derivatives. Amidine systems are intermediate in structure between tertiary amines and quaternary compounds, at least as regards ionization and electronic properties, but differ from the latter in shape (planar not tetrahedral). They have additional binding opportunities on account of their hydrogen-bond-forming capacity. The effect of the introduction of these cationic heads on the affinity for different muscarinic acetyl choline receptor (m-AcChR) subtypes was investigated in vitro, in binding displacement studies, and in functional tests on isolated organs. All new compounds (3a,b-5a,b) showed high affinity for the m-AcChR considered, comparable or slightly inferior to that of the parent drugs (1a-e). The new amidine derivatives proved effective as spasmolytic agents, with little tendency to cause central effects. However, no separation was achieved of spasmolytic and other untoward effects, like inhibition of salivation. Thus, amidine moieties are effective bioisosteric substitutes for conventional cationic heads present in antimuscarinic agents. Their unusual physical-chemical properties make them useful tools when modulation of pharmacokinetic or pharmacodynamic effects is required.

Synthesis of a new class of 2,3-dihydro-2-oxo-1H-benzimidazole-1-carboxylic acid derivatives as highly potent 5-HT3 receptor antagonists.

A series of 2,3-dihydro-2-oxo-1H-benzimidazole-1-carboxylic acid esters and amides containing a basic azacyclo- or azabicycloalkyl moiety has been synthesized and evaluated for 5-HT3 antagonistic activity in a radioligand binding assay ([3H]ICS 205930) and in the 5-HT-induced von Bezold-Jarisch reflex in the rat. It was found that endo-substituted azabicycloalkyl derivatives (e.g. 7a, 12a, 12b) were much more active than the corresponding exo analogues (e.g. 7b, 12h, 12i) or azacycloalkyl compounds. Amidic derivatives 12a, 12b, 12c, 12e, 13b, and 13c proved to be about 10 times more active than the corresponding ester derivatives 7a, 11a, 7c, 7d, 8a, and 8b. In particular, compound 12a (DA 6215) showed a Ki = 3.8 nM in the binding test and an ED50 = 1 nM/kg iv in the von Bezold-Jarisch reflex assay, an activity comparable to that of the reference compound 2 (ICS 205930, Ki = 2 nM, ED50 = 2.1 nM/kg). IR spectroscopy studies in the solid state and in CHCl3 solution revealed the existence of an intramolecular hydrogen bond in 13b, taken as a model compound for this class of substances. A molecular modeling study showed that 12a, in its internal hydrogen-bound conformation, well matches a recently proposed pharmacophoric model for 5-HT3 antagonist activity.

4-DAMP analogues reveal heterogeneity of M1 muscarinic receptors.

The muscarinic receptors responsible for two effects elicited by McN-A-343, i.e. the relaxation of the rat duodenum and the inhibition of the twitch contraction of rabbit vas deferens, were investigated by use of derivatives of 4-diphenyl acetoxy-N-methyl piperidine methobromide (4-DAMP). Both receptors had been previously identified as M1 on the basis of the high affinity shown towards pirenzepine. Schild analysis of antagonism revealed that the affinities of 4-DAMP and three of its analogues in the rat duodenum were significantly different from those estimated in rabbit vas deferens. These data indicate that distinct receptor subtypes mediate duodenal relaxation and vas deferens inhibition of twitch contraction and suggest that receptors classified as M1 by means of pirenzepine affinity constitute a heterogeneous population.

Hexocyclium derivatives with a high selectivity for smooth muscle muscarinic receptors.

1. The affinity of a number of derivatives of the muscarinic antagonist, hexocyclium, containing an amidine cationic head, for guinea-pig cardiac and ileal receptors was investigated. 2. All the compounds studied displayed a greater affinity for muscular than for cardiac muscarinic receptors. 3. The 5 fold ileal selectivity of hexocyclium was increased by a number of chemical substitutions. The largest discrimination between receptors (about 200 fold) was found for the formamidine derivative. 4. The selectivity displayed by the hexocyclium derivatives stemmed from a greater decrease in affinity towards cardiac as compared to ileal receptors.

Irreversible H2-antagonism of the four isomeric butyl analogues of mifentidine.

It has been hypothesized that bidentate hydrogen bonding plays an important role in the interaction of imidazolylphenylformamidines with the H2-receptor. The present study, in which the degree of pseudo-irreversible H2-antagonism of the four isomeric butyl substituted mifentidine analogues was determined on the spontaneously beating right atrium of the male guinea-pig, lends further support to this hypothesis. In solution the EE/EZ ratio is different for the four isomeric butylated mifentidine analogues. The rank order of the percentage of E,E conformation, which favors a bidentate interaction, of the formamidine moiety parallels the rank order of pseudo-irreversible H2-antagonism.

Affinity profile of the novel muscarinic antagonist, guanylpirenzepine.

The study reports the functional affinity of an amidino derivative of pirenzepine, guanylpirenzepine, for muscarinic receptors mediating relaxation of rat duodenum, inhibition of rabbit vas deferens twitch contraction (both receptors previously classified as M1), guinea pig negative inotropism (M2) and ileal contraction (M3). Unlike pirenzepine, guanylpirenzepine discriminated between duodenum and vas deferens receptors, with a 30-fold greater affinity for the former subtype. The unique selectivity pattern of guanylpirenzepine (duodenum greater than vas deferens greater than ileum greater than atrium) renders it a promising tool for the classification of muscarinic receptor subtypes.

Guanylpirenzepine distinguishes between neuronal ml and m4 muscarinic receptor subtypes.

Guanylpirenzepine, a polar, non-quaternary analog of pirenzepine, exhibited a novel binding behavior in rat brain regions: in competition binding experiments against [3H]pirenzepine labeling the M1 receptor in membranes from cerebral cortex, hippocampus and striatum, the compound, differently from pirenzepine, displayed heterogeneous binding curves. Computer assisted analysis of these curves, evidenced the existence of two populations of binding sites: a large proportion (84-89%) of high affinity receptors (KH = 64-92 nM) and a remainder with very low affinity (KL = 19-28 microM). Like pirenzepine, guanylpirenzepine showed low affinity for the glandular M3 and the cardiac M2 receptors when [3H]N-methylscopolamine was used to label the receptors in membranes from these two tissues; affinity values for guanylpirenzepine were 1336 and 5790 nM respectively, vs 323 and 683 nM for pirenzepine. We conclude that guanylpirenzepine is able to discriminate between m1 and m4 receptor subtypes and may represent a new tool for deeper studies on muscarinic receptors classification.

N-(fluoroethyl)(imidazolylphenyl)formamidines. The issue of the active species of mifentidine.

Three N-fluoroethyl-substituted (imidazolylphenyl)formamidine derivatives, namely, 2-fluoroethyl (3b), 2,2-difluoroethyl (3c), and 2,2,2-trifluoroethyl (3d), were prepared to test the effect of fluorine substitution on basicity and, then, on H2-antagonist affinity in comparison with the unsubstituted N-ethyl derivative (3a), taken as a model of mifentidine. Imidazolylphenyl isothiocyanate (1), obtained by reaction of 4-(aminophenyl)imidazole with carbon disulfide and ethyl chloroformate, was condensed with the requisite 2-fluoro-substituted ethylamines to give the intermediate thioureas (2b-d). Desulfurization of these thioureas by Raney nickel furnished the desired formamidines (3b-d). Increasing fluorine substitution was found to decrease basicity of the formamidino group substantially (3a, pKa = 8.65; 3b, pKa = 8.12; 3c, pKa = 6.60; 3d, pKa = 6.14), while having a modest effect on the imidazole portion. Affinity at the H2 receptors, evaluated from antagonism of histamine-stimulated chronotropic response on guinea pig atria, increased following fluorine substitution (3a, KB = 177; 3b, KB = 61; 3c, KB = 21; 3d, KB = 7.6). It is concluded that H2-receptor antagonist affinity in the mifentidine series is mostly dependent on the availability of the neutral species. These data support the hypothesis that mifentidine, like cimetidine, acts through the neutral species.

Pharmacological profile of mifentidine: a novel H2-receptor antagonist.

Mifentidine, a representative compound of a novel class of H2-antagonists, has been investigated for its ability to interact with H2-receptors and to inhibit gastric acid secretion. Affinity estimates (KB) of mifentidine obtained from in vitro studies on cardiac and gastric mucosal histamine (H2) receptors were in the 20-50 nM range. Mifentidine appeared to be endowed with strong anti-secretory properties against histamine-stimulated secretion in the anaesthetized rat and in the conscious dog. Distinct features of mifentidine were considerable bioavailability and duration of anti-secretory effect.

Conformational studies of two histamine H2-receptor antagonistic phenylformamidines: mifentidine and its guanidinothiazole analogue DA 4643.

Two histamine H2-receptor antagonists of the phenylformamidine type, mifentidine (N-isopropyl-N'-(4-1H-imidazol-4-yl-phenyl) formamidine dihydrochloride; I) and DA 4643 (N-methyl-N'-(3-(2-guanidinothiazol-4-yl)-phenyl) formamidine dihydrochloride; II), have been investigated by experimental physico-chemical studies and theoretical conformational analysis. PKa determinations on the two molecules I and II show that these substances exist at physiological pH (7.4) predominantly as their monoprotonated forms at the formamidine moiety. Semiempirical quantum mechanical (MNDO, CNDO/2) and molecular mechanics (MMPI) calculations show a preference of the nearly planar conformations for I and of different low energy rotamers for II. The energy of these conformers is a function of two important torsion angles, one around the bond joining the imidazole, or the guanidinothiazole, and the phenyl ring and the other around the bond joining the phenyl ring and the formamidinium cation. When the distances between crucial parts present in I and II are considered, it results that the relatively higher flexibility of II allows accommodation of amidine pairs present in the latter at a distance similar to that found for correspondent pairs in the conformationally more restricted I. Conformational aspects of I and II are discussed with reference to a recently described conformation of cimetidine determined by X-ray method. A hypothesis of binding of H2-receptor antagonists of the phenylformamidine type is advanced with reference to electrostatic potential maps calculated for crucial part structures of I, II and cimetidine. The present work supports the hypothesis that both mifentidine and DA 4643 interact with the histamine H2-receptor at the same site, utilizing in the binding process the same, or closely similar, receptor structural features.