A series of new 3-tropanol and 3-quinuclidinol esters of phenyl-substituted pyrrolidin-, piperidin- and azepin-2-oxocarboxylic acid were synthesized and tested for antimuscarinic activity. The compounds showed a preferential in vitro activity at M1 and M3 receptor subtypes and an interesting activity profile in vivo. A potential use as selective bronchospasmolytic agents has been suggested for selected compounds.

Alzheimer disease (AD) is a neurodegenerative disorder lacking an effective therapy. The etiology is controversial and among different drug strategies, the cholinergic approach has gained great interest owing to biochemical and pharmacological evidence of the crucial role of acetycholine in cognitive functions. Several attempts exploiting the boosting of the cholinergic system are currently under way. Inhibitors of the acetylcholinesterase enzyme sustain the availability of the natural transmitter by limiting its removal from the synapse. In a different approach, exogenous agonists may substitute acetylcholine itself. In this way the issue of the extensive cholinergic cell loss occurring in AD and leading to a reduction of cholinergic functions, could be advantageously bypassed. Moreover the discovery of different muscarinic receptor subtypes, most notably the M1 subtype as that involved in the postsynaptic transmission, has offered new opportunities to face the problem in a very specific way. In this line of research, we have now identified BIMC 182 as a new functionally selective M1 agonist. Whereas its affinity for the different receptor subtypes is almost similar (radioreceptor binding), its functional selectivity is pointed out by specific “in vitro” models. BIMC 182 behaves as a full agonist at M1 (rat superior cervical ganglion, pD2 4.8) and as a partial agonist at M2 and M3 sites (g.p. heart pD2=5.4 and g.p. ileum pD2=4.5). The agonist profile is further confirmed in hm1 transfected CHO cells where the compound stimulates PI turnover. BIMC 182 penetrates well the brain as shown by the increase in the energy of the low frequency band (theta waves) in the cortical EEG of rabbits (3 mg/kg i.v.). In the same range of doses the compound antagonises the scopolamine induced amnesia in rats (passive avoidance and Morris water maze tests).

1. This study describes the in vitro interaction of the muscarinic ligand McNeil-A-343 with two 5-hydroxytryptamine (5-HT) receptor subtypes, the 5-HT4 and 5-HT3 receptors, using functional as well as radioligand binding studies. 2. In the rat oesophageal muscularis mucosae, precontracted with carbachol, McNeil-A-343 was a competitive antagonist (pA2 6.2) of the 5-HT4 receptor which mediates the relaxation induced by 5-HT. The compound per se relaxed the oesophagus at high concentration only (> or = 10 microM), an effect unchanged by desensitization of the 5-HT4 receptor with 10 microM 5-methoxytryptamine. In the same preparation in the absence of tone, McNeil-A-343 displaced the carbachol concentration-response curve to the right, yielding an apparent affinity (pA2) of 4.9 for muscarinic receptors. 3. In the rat isolated superior cervical ganglion preparation, after blockade of muscarinic and nicotinic receptors, McNeil-A-343 caused a concentration-dependent depolarization that was unaffected by 100 nM ondansetron. The concentration-fast depolarization curve to 5-HT, mediated by the 5-HT3 receptor, was displaced to the right by McNeil-A-343, which showed an apparent affinity (pA2) of 4.8 for the 5-HT3 subtype. 4. In binding studies, McNeil-A-343 recognized a single population of 5-HT4 receptors in pig caudate nucleus, with a pKI of 5.9. The binding affinity of McNeil-A-343 for 5-HT3 receptors in NG 108-15 cells was approximately four times lower (pKI 5.3). Binding affinities (pKI) for muscarinic receptor subtypes in rat tissues were 5.3 (M1, cortex), 5.2 (M2, heart) and 4.9 (M3, submandibular glands), respectively. 5. McNeil-A-343 is an antagonist at 5-HT4 and 5-HT3 receptors; the interaction of the compound with these receptor subtypes (notably the 5-HT4) occurs in a range of concentrations which generally overlaps that relevant to the interaction with muscarinic receptors.

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.

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 H-2-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 H-2-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 H-2 receptor.

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. All the compounds studied displayed a greater affinity for muscular than for cardiac muscarinic receptors. 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. The selectivity displayed by the hexocyclium derivatives stemmed from a greater decrease in affinity towards cardiac as compared to ileal receptors.

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.

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.

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 > vas deferens > ileum > atrium) renders it a promising tool for the classification of muscarinic receptor subtypes.

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.

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.

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.

AbstractThe MNDO semiempirical SCF‐MO method has been used to calculate charge distributions of a series of histamine H2‐receptor antagonists. The investigated compounds concern mifentidine (N‐isopropyl‐N′‐(4‐1H‐imidazol‐4‐yl‐phenyl)formamidine) analogues, in which the imidazole moiety has been substituted or replaced by various aromatic heterocyclic groups. A correlation has been found between H2‐antagonistic activity as determined by inhibition of specific [3H]‐tiotidine binding to the H2 receptors of a guinea pig cortex preparation, and the net atomic charges of two of the atoms (X‐H bond in which X is a nitrogen or carbon atom) in the heterocycle. The binding capacity of the ligand molecule to the receptor is enhanced when the electronic population of atom X and the positive charge on the proton are higher. Assuming that the difference between both charges might effectively serve as a valuable substitute for the hydrogen bond forming capacity of the X‐H group, the conclusion can be drawn that this property of the heterocycle plays an important role in the interaction process of the ligand with its H2‐receptor. The obtained regression equation(s) suggest that the monocation (formamidine moiety protonated) is the species related to pharmacological activity.

A series of substituted sulfinyl benzimidazoles were prepared and tested for gastric anti-secretory activity. Following initial screening, two compounds were tested for anti-ulcer activity. The new compounds showed pharmacological properties different from those of omeprazole 1, since they proved to be weak anti-secretory agents displaying non-specific anti-ulcer activity. Some structural requirements for optimum activity were elucidated.

AbstractDie Umsetzung der aromatischen Amine (I) mit N‐ Cyanformimidoethylester (II) führt zu N2‐Aryl‐N1‐cyanformamidinen (III), die mit aliphatischen Aminen (IV) die Aryl‐alkylformamidine (V) bilden.

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.

N1-[(4-Imidazolyl)-phenyl]-N2-isopropylformamidine (mifentidine, DA 4577 is a potent and selective H2 antagonist, representative of a new class of compounds, the imidazolylphenyl-formamidines, characterized by a semi-rigid structural conformation. Mifentidine appeared to be a specific and competitive antagonist of several histamine-mediated responses. Thus, in isolated guinea pig atria and ventricles it antagonized histamine chronotropic and dimaprit inotropic effects in a competitive manner providing affinity estimates (pA2) of 7.66 and 7.74, respectively. Mifentidine exerted potent antisecretory effects in: the isolated mouse stomach where it antagonized the acid promoting activity of histamine (EC50 3.28 mumol/l) but not that of bethanechol or db-cAMP (adenosine 3',5'-monophosphate); the lumen perfused stomach of the anaesthetized rat, inhibiting histamine (ED50 0.1 mumol/kg i.v.) and pentagastrin (ED50 0.2 mumol/kg i.v.) stimulated secretion; the pylorus ligated rat (ED50 1.35 mumol/kg i.v.); the gastric fistula dog, reducing the secretagogue effect of pentagastrin (ED50 96 nmol/kg i.v.); the conscious dog equipped with the Heidenhain pouch, where it was effective both following intravenous (ED50 119.7 nmol/kg) and oral administration (ED50 323.8 nmol/kg) in antagonizing histamine action. Mifentidine antisecretory effect, examined in the dog, appeared to last for a significantly longer time than that of ranitidine. Mifentidine was free of cardiovascular effects (on aortic blood pressure and heart rate) when administered repeatedly to the conscious dog at doses far above those needed to suppress acid secretion.

The protective effect of cimetidine, ranitidine and a newer H2-receptor antagonist, mifentidine (proposed INN), on models of gastric and duodenal damage, caused by activation of H2 receptors, was studied. Gastric erosions were induced in rats by intravenous dimaprit (100 mg/kg) while duodenal damage was investigated in guinea pigs following subcutaneous administration of dimaprit (2 mg/kg, 6 doses). All the compounds reduced or abolished gastric and duodenal damage in rats and guinea pigs, mifentidine being more potent than both cimetidine and ranitidine. The antiulcer effect of the H2-receptor antagonists was related to the dose and to their ability to inhibit dimaprit-induced gastric acid secretion. The duration of action proved to be different for the three compounds. According to the two dosing schedules adopted to evaluate the duration of action, mifentidine, compared to cimetidine and ranitidine, required considerably lower oral dosages to display its protective effect.