Pharmacology of hexahydro-difenidol, hexahydro-sila-difenidol and related selective muscarinic antagonists.

A series of hexahydro-difenidol (HHD) and hexahydro-sila-difenidol (HHSiD) analogues modified in the amino group, the phenyl ring and in the alkylene chain were investigated for their binding and functional properties at muscarinic M1, M2 and M3 receptors. Novel muscarinic receptor antagonists were obtained which exhibited different receptor selectivity profiles from the parent compounds HHD and HHSiD (M1 congruent to M3 greater than M2), e.g. HHD and HHSiD methiodides, M1 greater than M2 congruent to M3; p-fluoro-HHSiD, M3 greater than M1 greater than M2; trans-hexbutenol, M1 greater than M3 greater than M2; and (s)-p-fluoro-hexbutinol, M3 greater than M2 congruent to M1. Stereoselectivity ratios [(R)/(S)] for the enantiomers of HHD, hexbutinol and p-fluoro-hexbutinol were highest at M1, intermediate at M3 and lowest at M2 receptors.

Stereoselectivity of the interaction of muscarinic antagonists with their receptors.

The stereoselectivity of the interaction with muscarinic receptors of enantiomers of a series of chiral antagonists is receptor subtype dependent. There is no overall relationship between stereoselectivity and receptor affinity. Depending on the antagonist studied, receptor stereoselectivity may indeed reflect: (1) the weakening or loss of a single interaction involving one of the four groups bound to the asymmetric carbon; (2) steric hindrance preventing optimum interaction of the low affinity steroisomer with the receptor; and/or (3) the inversion of the relative positions of two moieties of the ligand with similar structural and electronic properties i.e. comparable affinities for the two corresponding subsites in the receptor.

Crystal structure of [2-(tri-ethyl-ammonio)-eth-yl][(2,4,6-triiso-propyl-phen-yl)sulfon-yl]amide tetra-hydrate.

The zwitterionic title compound, C23H42N2O2S·4H2O, crystallized as a tetrahydrate from a solution of N-[(2,4,6-triiso-propyl-phen-yl)sulfon-yl]aziridine in tri-ethyl-amine, diethyl ether and pentane in the presence of moist air. It is formed by a nucleophillic ring-opening that is assumed to be reversible. The mol-ecular structure shows a major disorder of the triiso-propyl-phenyl group over two equally occupied locations. An inter-esting feature is the uncommon hydrate structure, exhibiting a tape-like motif which can be classified as a transition of the one-dimensional T4(2)6(2) motif into the two-dimensional L4(6)5(7)6(8) motif.

Stereoselective inhibition of muscarinic receptor subtypes by the enantiomers of hexahydro-difenidol and acetylenic analogues.

1. The affinities of the (R)- and (S)-enantiomers of hexahydro-difenidol (1) and its acetylenic analogues hexbutinol (2), hexbutinol methiodide (3) and p-fluoro-hexbutinol (4) (stereochemical purity greater than 99.8%) for muscarinic receptors in rabbit vas deferens (M1), guinea-pig atria (M2) and guinea-pig ileum (M3) were measured by dose-ratio experiments. 2. The (R)-enantiomers consistently showed higher affinities than the (S)-isomers. The stereoselectivity ratios [(R)/(S)] were greatest with the enantiomers of 1 (vas deferens: 550; ileum: 191; atria: 17) and least with those of the p-Fluoro-analogue 4 (vas deferens: 34; ileum: 8.5; atria: 1.7). 3. The enantiomeric potency ratios for compounds 1-4 were highest in rabbit vas deferens, intermediate in guinea-pig ileum and much less in guinea-pig atria. Thus, these ratios may serve as a predictor of muscarinic receptor subtype identity. 4. (S)-p-Fluoro-hexbutinol [(S)-4] showed a novel receptor selectivity profile with preference for M3 receptors: M3 greater than M2 greater than or equal to M1. 5. These results do not conform to Pfeiffer's rule that activity differences between enantiomers are greater with more potent compounds.

Binding and functional properties of hexocyclium and sila-hexocyclium derivatives to muscarinic receptor subtypes.

1. We have compared the binding properties of several hexocyclium and sila-hexocyclium derivatives to muscarinic M1 receptors (in rat brain, human neuroblastoma (NB-OK 1) cells and calf superior cervical ganglia), rat heart M2 receptors, rat pancreas M3 receptors and M4 receptors in rat striatum, with their functional antimuscarinic properties in rabbit vas deferens (M1/M4-like), guinea-pig atria (M2), and guinea-pig ileum (M3) muscarinic receptors. 2. Sila-substitution (C/Si exchange) of hexocyclium (-->sila-hexocyclium) and demethyl-hexocyclium (-->demethyl-sila-hexocyclium) did not significantly affect their affinities for muscarinic receptors. By contrast, sila-substitution of o-methoxy-hexocyclium increased its affinity 2 to 3 fold for all the muscarinic receptor subtypes studied. 3. The p-fluoro- and p-chloro-derivatives of sila-hexocyclium had lower affinities than the parent compound at the four receptor subtypes, in binding and pharmacological studies. 4. In binding studies, o-methoxy-sila-hexocyclium (M1 = M4 > or = M3 > or = M2) had a much lower affinity than sila-hexocyclium for the four receptor subtypes, and discriminated the receptor subtypes more poorly than sila-hexocyclium (M1 = M3 > M4 > M2). This is in marked contrast with the very clear selectivity of o-methoxy-sila-hexocyclium for the prejunctional M1/M4-like heteroreceptors in rabbit vas deferens. 5. The tertiary amines demethyl-hexocyclium, demethyl-sila-hexocyclium and demethyl-o-methoxy-sila-hexocyclium had 10 to 30 fold lower affinities than the corresponding quaternary ammonium derivatives.

Binding and functional properties of antimuscarinics of the hexocyclium/sila-hexocyclium and hexahydro-diphenidol/hexahydro-sila-diphenidol type to muscarinic receptor subtypes.

1. In an attempt to assess the structural requirements for the muscarinic receptor selectivity of hexahydro-diphenidol (hexahydro-difenidol) and hexahydro-sila-diphenidol (hexahydro-sila-difenidol), a series of structurally related C/Si pairs were investigated, along with atropine, pirenzepine and methoctramine, for their binding affinities in NB-OK 1 cells as well as in rat heart and pancreas. 2. The action of these antagonists at muscarinic receptors mediating negative inotropic responses in guinea-pig atria and ileal contractions has also been assessed. 3. Antagonist binding data indicated that NB-OK 1 cells (M1 type) as well as rat heart (cardiac type) and pancreas (glandular/smooth muscle type) possess different muscarinic receptor subtypes. 4. A highly significant correlation was found between the binding affinities of the antagonists to muscarinic receptors in rat heart and pancreas, respectively, and the affinities to muscarinic receptors in guinea-pig atria and ileum. This implies that the muscarinic binding sites in rat heart and the receptors in guinea-pig atria are essentially similar, but different from those in pancreas and ileum. 5. The antimuscarinic potency of hexahydro-diphenidol and hexahydro-sila-diphenidol at the three subtypes was influenced differently by structural modifications (e.g. quaternization). Different selectivity profiles for the antagonists were obtained, which makes these compounds useful tools to investigate further muscarinic receptor heterogeneity. Indeed, the tertiary analogues hexahydro-diphenidol (HHD) and hexahydro-sila-diphenidol (HHSiD) had an M1 = glandular/smooth muscle greater than cardiac selectivity profile, whereas the quaternary analogues HHD methiodide and HHSiD methiodide were M1 preferring (M1 greater than glandular/smooth muscle, cardiac).

Binding affinities of hexahydro-difenidol and hexahydro-sila-difenidol analogues at four muscarinic receptor subtypes: constitutional and stereochemical aspects.

Hexahydro-sila-difenidol and eight analogues behaved as simple competitive inhibitors of [3H]N-methyl-scopolamine binding to homogenates from human neuroblastoma NB-OK 1 cells (M1 sites), rat heart (M2 sites), rat pancreas (M3 sites), and rat striatum 'B' sites (M4 sites). Pyrrolidino- and hexamethyleneimino analogues showed the same selectivity profile as the parent compound. Hexahydro-sila-difenidol methiodide and the methiodide of p-fluoro-hexahydro-sila-difenidol had a higher affinity but a lower selectivity than the tertiary amines. Compounds containing a p-methoxy, p-chloro or p-fluoro substituent in the phenyl ring of hexahydro-sila-difenidol showed a qualitatively similar selectivity profile as the parent compound (i.e., M1 = M3 = M4 greater than M2), but up to 16-fold lower affinities. o-Methoxy-hexahydro-sila-difenidol has a lower affinity than hexahydro-sila-difenidol at the four binding sites. Its selectivity profile (M4 greater than M1, M3 greater than M2) was different from hexahydro-sila-difenidol. Replacement of the central silicon atom of hexahydro-sila-difenidol, p-fluoro-hexahydro-sila-difenidol and their quaternary (N-methylated) analogues by a carbon atom did not change their binding affinities significantly. The four muscarinic receptors showed a higher affinity for the (R)- than for the (S)-enantiomers of hexahydro-difenidol, p-fluorohexahydro-difenidol and their methiodides. The stereoselectivity varied depending on the receptor subtype and drug considered.

Presynaptic muscarinic receptors mediating inhibition of neurogenic contractions in rabbit vas deferens are of the ganglionic M1-type.

The present study was designed to further characterize the presynaptic muscarinic M1-receptor responsible for the inhibition of neurogenic contractions in the isolated rabbit vas deferens. Electrically induced twitch contractions of this preparation were inhibited by the M1-agonist, McN-A-343, and by some of its analogs: 4-chloro-phenyl derivative greater than McN-A-343 greater than trans-olefinic analog greater than cis-olefinic analog. The same rank order of potency was observed for these agonists to raise the blood pressure of pithed rats by stimulation of M1-receptors in sympathetic ganglia. A highly significant correlation was found between the antimuscarinic potencies of atropine, pirenzepine and a series of 9 antagonists structurally related to the ganglionic M1 beta-receptor selective compounds, hexocyclium and hexahydro-difenidol, to antagonize the McN-A-343-induced inhibition of twitch contractions in rabbit vas deferens or the muscarine-induced depolarization in rat isolated superior cervical ganglia. It is suggested that the presynaptic muscarinic receptor that mediates inhibition of neurogenic contractions in rabbit vas deferens is of the ganglionic M1 beta-type.

Selective labelling of muscarinic M1 receptors in calf superior cervical ganglia by [3H](+/-)-telenzepine.

A method was developed to determine the affinities of antimuscarinic drugs at M1 receptors. [3H](+/-)-Telenzepine served as radioligand in crude preparations of calf superior cervical ganglia and showed high affinity for a single receptor population, consisting of M1 receptors (KD = 1.12 nM). Kinetic experiments showed monophasic association (k1 = 0.017 min-1 nM-1) and dissociation (k-1 = 0.017 min-1) kinetics, the half-life of dissociation being 41 min at 37 degrees C. The kinetic KD value amounted to 1.00 nM. M1 affinities for pirenzepine, methoctramine, hexahydro-sila-difenidol and p-fluoro-hexahydro-sila-difenidol determined in competition experiments were similar to those found in functional studies with M1 receptors in rabbit isolated vas deferens. The binding assay was used to determine the affinities of the (R) and (S) enantiomers of tertiary (trihexyphenidyl, hexahydro-difenidol, hexbutinol, p-fluoro-hexbutinol) and quaternary muscarinic antagonists (trihexyphenidyl methiodide, hexbutinol methiodide). Comparison of results obtained with the rabbit vas deferens suggested that the ionic environment may influence the affinities.

Affinity profiles of hexahydro-sila-difenidol analogues at muscarinic receptor subtypes.

In an attempt to assess the structural requirements of hexahydro-sila-difenidol for potency and selectivity, a series of analogues modified in the amino group and the phenyl ring were investigated for their affinity to muscarinic M1-(rabbit vas deferens), M2- (guinea-pig atria) and M3- (guinea-pig ileum) receptors. All compounds were competitive antagonists in the three tissues. Their affinities to the three muscarinic receptor subtypes differed by more than two orders of magnitude and the observed receptor selectivities were not associated with high affinity. The pyrrolidino and hexamethyleneimino analogues, compounds substituted in the phenyl ring with a methoxy group or a chlorine atom as well as p-fluoro-hexahydro-difenidol displayed the same affinity profile as the parent compound, hexahydro-sila-difenidol: M1 approximately M3 greater than M2. A different selectivity pattern was observed for p-fluoro-hexahydro-sila-difenidol: M3 greater than M1 greater than M2. This compound exhibited its highest affinity for M3-receptors in guinea-pig ileum (pA2 = 7.84), intermediate affinity for M1-receptors in rabbit vas deferens (pA2 = 6.68) and lowest affinity for the M2-receptors in guinea-pig atria (pA2 = 6.01). This receptor selectivity profile of p-fluoro-hexahydro-sila-difenidol was confirmed in ganglia (M1), atria (M2) and ileum (M3) of the rat. Furthermore, dose ratios obtained with either pirenzepine (M1) or hexahydrosila-difenidol (M2 and M3) and the p-fluoro analogue used in combination suggested that the antagonism was additive, implying mutual competition with a single population of muscarinic receptor subtypes. These results indicate that p-fluoro-hexahydro-sila-difenidol represents a valuable tool for characterization of muscarinic receptor subtypes.

Stereoselective interaction of procyclidine, hexahydro-difenidol, hexbutinol and oxyphencyclimine, and of related antagonists, with four muscarinic receptors.

We investigated the binding properties of the (R)- and (S)-enantiomers of the muscarinic antagonists trihexyphenidyl, procyclidine, hexahydro-difenidol, p-fluoro-hexahydro-difenidol, hexbutinol, p-fluoro-hexbutinol, and their corresponding methiodides at muscarinic M1, M2, M3 and M4 receptor subtypes. In addition, binding properties of the (R)- and (S)-enantiomers of oxyphencyclimine were studied. The (R)- enantiomers (eutomers) of all the compounds had a greater affinity than the (S)-isomers for the four muscarinic receptor subtypes. The binding patterns of the (R)- and (S)-enantiomers were generally different. We did not observe any general correlation between the potency of the high-affinity enantiomer and the affinity ratio (eudismic ratio) of the two enantiomers. The results are discussed in terms of a 'four subsites' binding model.

Pharmacokinetic properties of the antimuscarinic drug [3H]-hexahydro-sila-difenidol in the rat.

The pharmacokinetics of tritiated hexahydro-sila-difenidol [( 3H]-HHSiD) were examined in rats. Furthermore, the distribution of radioactivity was studied by means of whole body autoradiography. After i.v. administration of 2.9 mg/kg HHSiD plus [3H]-HHSiD to anaesthetized rats bearing a catheter implanted in the ductus choledochus and receiving a mannitol infusion, HHSiD was rapidly distributed and metabolized. Only 5% of the radioactivity was recovered in blood after 23 s and 0.4% after 2.5 h. 64% of the plasma radioactivity could be extracted with hexane from the samples taken 23 s after administration. 52% of the radioactivity was eliminated within 2.5 h, 13% by urinary and 39% by biliary excretion. Following oral administration of 8.6 mg/kg HHSiD plus [3H]-HHSiD there was an absorption of approximately one fourth of the administered radioactivity within 4 h. By means of whole body autoradiography (i.v. injection) as well as by tissue distribution measurement the highest levels of radioactivity were found in bile, urine, lung, kidney, adrenals, liver and pancreas. Thus, after i.v. administration to rats HHSiD is rather quickly distributed, metabolized and excreted. This explains its low antimuscarinic potency in vivo.

Stereoselectivity of (R)- and (S)-hexahydro-difenidol binding to neuroblastoma M1, cardiac M2, pancreatic M3, and striatum M4 muscarinic receptors.

(R)-Hexahydro-difenidol has a higher affinity for M1 receptors in NB-OK 1 cells, pancreas M3 and striatum M4 receptors (pKi 7.9 to 8.3) than for cardiac M2 receptors (pKi 7.0). (S)-Hexahydro-difenidol, by contrast, is nonselective (pKi 5.8 to 6.1). Our goal in the present study was to evaluate the importance of the hydrophobic phenyl, and cyclohexyl rings of hexahydro-difenidol for the stereoselectivity and receptor selectivity of hexahydro-difenidol binding to the four muscarinic receptors. Our results indicated that replacement of the phenyl ring of hexahydro-difenidol by a cyclohexyl group (----dicyclidol) and of the cyclohexyl ring by a phenyl moiety (----difenidol) induced a large (4- to 80-fold) decrease in binding affinity for all muscarinic receptors. Difenidol had a significant preference for M1, M3, and M4 over M2 receptors; dicyclidol, by contrast, had a greater affinity for M1 and M4 than for M2 and M3 receptors. The binding free energy decrease due to replacement of the phenyl and the cyclohexyl groups of (R)-hexahydro-difenidol by, respectively, a cyclohexyl and a phenyl moiety was almost additive in the case of M4 (striatum) binding sites. In the case of the cardiac M2, pancreatic M3, or NB-OK 1 M1 receptors the respective binding free energies were not completely additive. These results suggest that the four (R)-hexahydro-difenidol "binding moieties" (phenyl, cyclohexyl, hydroxy, and protonated amino group) cannot simultaneously form optimal interactions with the M1, M2, and M3 muscarinic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)