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1 We studied the effect of temperature on the binding to rat heart \(M_2\) muscarinic receptors of antagonists related to the carbon/silicon pairs pridinol/sila-pridinol and diphenidol/sila-diphenidol (including three germanium compounds) and six structurally related pairs of enantiomers [(R)- and (S)-procyclidine, (R)- and (S)-trihexyphenidyl, (R)- and (S)-tricyclamol, (R)- and (S)-trihexyphenidyl methiodide, (R)- and (S)-hexahydro-diphenidol and (R)- and (S)-hexbutinol]. Binding affinities were determined in competition experiments using \([^3H]\)-N-methyl-scopolamine chloride as radioligand. The reference drugs were scopolamine and N-methyl-scopolamine bromide.
2 The affinity of the antagonists either increased or decreased with temperature, van 't Hoff plots were linear in the 278–310°K temperature range. Binding of all antagonists was entropy driven. Enthalpy changes varied from large negative values (down to \(−29 kJ mol^{−1}\)) to large positive values (up to \(+ 30 kJ mol^{−1}\)).
3 (R)-configurated drugs had a 10 to 100 fold greater affinity for \(M_2\) receptors than the corresponding (S)-enantiomers. Enthalpy and entropy changes of the respective enantiomers were different but no consistent pattern was observed.
4 When silanols \((R_3SiOH)\) were compared to carbinols \((R_3COH)\), the affinity increase caused by C/Si exchange varied between 3 and 10 fold for achiral drugs but was negligible in the case of chiral drugs. Silanols induced more favourable enthalpy and less favourable entropy changes than the corresponding carbinols when binding. Organogermanium compounds \((R_4Ge)\) when compared to their silicon counterparts (R4Si) showed no significant difference in affinity as well as in enthalpy and entropy changes.
5 Exchange of a cyclohexyl by a phenyl moiety was associated with an increase or a decrease in drug affinity (depending on the absolute configuration in the case of chiral drugs) and generally also with a more favourable enthalpy change and a less favourable entropy change of drug binding.
6 Replacement of a pyrrolidino by a piperidino group and increasing the length of the alkylene chain bridging the amino group and the central carbon or silicon atom were associated with either an increase or a decrease of entropy and enthalpy changes of drug binding. However, there was no clear correlation between these structural variations and the thermodynamic effects.
7 Taken together, these results suggest that hydrogen bond-forming OH groups and, to a lesser extent, polarizable phenyl groups contribute significantly to the thermodynamics of interactions between these classes of muscarinic antagonists and \(M_2\) muscarinic receptors.
Wc invcstigatcd thc binding properlies of thc (R)- and (Sl-cnantiomcrs of thc muscarinic antagonists trihcxyphcnidyl, procyclidinc, hcxahydro-difcnidol. p-fluoro-hcxahydro-difcnidol. hcxbutinol, p-fluoro-hcxbutinnl. and thcir corrcsponding methiodidcs at muscarinic M\(_1\), M\(_2\)• M\(_3\) and M\(_4\) receptor subtypes. In addition. binding properlies of thc (R)- and (S)-cnantiomcrs of oxyphcncycliminc wcrc studicd. The {R)- cnantiomcrs (cutomcrs} of all the compounds had a grcatcr affinity than the (S)-isomcrs for thc four muscarinic rcccptor subtypcs. Thc binding pattcrns of thc (R)- and (S)-enantiomers wcrc gcncrally different. We did not obscrvc any gcncral corrclation hctwccn thc potcncy of thc high-affinity enantiomer and Lhc affinity ratio (cudismic ratio) of the two cnantiomcrs. Thc rcsuhs arc discusscd in tcrms of a 'four suhsitcs' binding modcl.
l In an attempt to assess the structural requirements for the musearlnie receptor selectivity of hexahydro-diphenidol (hexahydro-difenidol) and hexahydro-sila-diphenidol (hexahydro-sila-difenidol), a serles 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 musearlnie receptors mediating negative inotropic responses in guinea-pig atrla and ileal contractions has also been assessed. 3 Antagonist binding data indicated that NB-OK 1 cells (M\(_1\) type) as weil as rat heart (cardiac type) and pancreas (glandularjsmooth 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 musearlnie binding sites in rat heart and the receptors in guinea-pig atrla 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 inftuenced 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. lndeed, the tertiary analogues hexahydrodiphenidol (HHD) and hexahydro-sila-diphenidol (HHSiD) bad an M\(_1\) = glandularjsmooth muscle > cardiac selectivity profile, whereas the quaternary analogues HHD methiodide and HHSiD methiodide were M\(_1\) preferring (M\(_1\) > glandularjsmooth muscle, cardiac).
The goals of the present study were: (1) to investigate thc binding properlies oi (R)- and (S)-procyclidine and two aehiral derivatives of muscarinic M\(_1\)• M\(_2\) and M\(_4\) receptor subtypes and (2) to identify the interaetions which allow these receptors to diseriminate between the two stereoisomers. (R)-Procyclidine showed a higher affinity for human neuroblastoma NB-OK 1 muscarinie M\(_1\) and rat striatum musearinie M\(_4\) receptors. a~ compared to rat cardiac M\(_2\) receptors. (S)-Procyclidine had a 130-iold lower affinity than (R)-procyclidine for M\(_1\) and M\(_4\) receptors. and a 40-fold lower affinity for M\(_2\) receptors. Pyrrinol. the aehiral diphenyl derivative with the eyclohexyl g.roup of (S}-procyclidine replaeed by a phenyl group, has an eight-fold lower affinity for M\(_1\) and M\(_4\) receptors. as eompared to (R)-procyclidine, and a three-fold lower affinity for M\(_2\) receptors. Hexahydro-procyclidine. the eorresponding achiral dicyclohexyl compound, had a 10- to 20-fold lower affinity than (R)-procyclidine for the three reeeptors. The inerease in binding free energy, which is observed when the phenyl and eyclohexyl groups of procyelidine are separately replaeed by cyclohexyJ and phenyl groups, respectively. was additive in the ease of M\(_1\)• M\(_2\) and M\(_4\) receptcrs. This indicates that the musearinic reeeptor s!ereoseleetivity was based on the eoexistence of two binding sites, one preferring a phenylrather than eyclohexyl group and the seeond preferring a cyclohexyl rather than a phenyl group. In addition. there were aiso binding sites for the hydroxy moiety and the protonated amino group of the ligands. The greater affinity and stereoselectivity of M\(_1\) and M\(_4\) muscarinic receptors for (R)-procyelidine reflected the better fit of the eyclohexyl group of (R)-procyclidine to the subsite of M\(_1\) and M\(_4\) as compared to M\(_2\) receptors.
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), Mr (guinea-pig atria) and Mr (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 phenylring with a methoxy group or a chlorine atom as weil as p-fluoro-hexahydro-difenidol displayed the same affinity profile as the parent compound, hexahydro-sila-difenidol: M1 = M3 > M2 • A different selectivity patternwas observed for p-fluoro-hexahydro-sila-difenidol: M3 > M1 > M2 • This compound exhibited its highest affinity for M3-receptors in guinea-pig ileum (pA 2 = 7.84), intermediate affinity for M1-receptors in rabbit vas deferens (pA 2 = 6.68) and lowest affinity for the Mrreceptors in guinea-pig atria (pA 2 = 6.01). This receptor selectivity profile of p-fluoro-hexahydro-sila-difenidol was confirmed in ganglia (M1), atria (M2 ) and ileum (M 3 ) of the rat. Furthermore, dose ratios obtained with either pirenzepine (Mt) 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.
1 Tbc affinities of the (R)- and (S)-enantiomers of hexahydro-difenidol (1) and its acetylenie analogues hexbutinol (2), hexbutinol methiodide (3) and p-fluoro-hexbutinol (4) (stereochemieal purity > 99.8%) for musearlnie 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 aßinities than the (S)-isomers. The stereosclectivity ratios [(R)/(S)] wcrc greatest with thc enantiomers of 1 (vas deferens: 550; ilcum: 191; atria: 17) and least with thosc ofthc p-Fluoro-analogue 4 (vas defercns: 34; ileum: 8.5; atria: 1.7). 3 The enantiomerie 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 predietor of muscarinic receptor subtype identity. 4 (S)-p-Fluoro-hexbutinol [(S)-4] showed a novel receptor selectivity profile with preference for M\(_3\) receptors: M\(_3\) > M\(_2\) \(\geq\) M\(_1\)• 5 These results do not conform to Pfeiffer's rule that aetivity differences between enantiomers are greater with more potent compounds.
Hexahydro-sila-difenidoJ and eight analogues behaved as simple cumpetitive inhibitors of eHJN·methyl·scopoJamine binding to homogenates frorn human neuroblastoma NB-OK 1 cells (M\(_1\) sites), rat heart (M\(_2\) sites), rat pancreas (M\(_3\) sites), and rat striatum 'B' sites (M\(_4\) sites). Pyrrolidino- and hexamethyleneimino analogues showed the same sekctivity profile as the parent compound. Hexahydro-sila-difenidol methiodide and the methiodide of p-fluoro-hexahydro·sila-difenidol had a fügher affinity but a lower selectivity than the tertiary amines. Compounds containing a p·methoxy, p-chJoro or p-fluoro substituent in the phenyl ring of hexahydro-sila-difenidol showed a qualitative)y similar selectivity profile as the parent compound (i.e., M\(_1\)= M\(_3\) = M\(_4\) >M\(_2\) ), but up to 16-fold lower affinities. o-Methoxy-hexahydro-sila-difenidol has a lower affinity than hexahydro-sila-difeni.:!o! at the four binding sites. lts selectivity profile (M\(_4\) > M\(_1\), M\(_3\) > M\(_2\) ) was different from hexahydro-sila-difenidol. Replacement of the centrat silicon atom of hexahydro-sila-difenidol, p-fluoro-hexahydro-sila-difenidol and thdr quatemary (N-methylated) analogues by a carbon atom did not change their binding affinities significantly. The iour 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.
(R)-Hexahydro-difenidol has a higher affinity for M\(_1\) receptors in NB-OK 1 cells, pancreas M\(_3\) and striatum M\(_4\) receptors (pKi 7.9 to 8.3) than for cardiac M2 receptors (pKi 7 .0). (8)-Hexahydro-difenidol, by contrast, is nonselective (pKi 5.8 to 6.1). Our goal in the present study was to evaluate the importance ofthe hydrophobic phenyl, and cyclohexyl rings of hexahydro-difenidol for the stereoselectivity and reeeptor selectivity of hexahydro-difenidol binding to the four muscarinic receptors. Our results indieated that replacement of the phenyl ring of hexahydro-difenidol by a cyclohexyl group <~ dicyclidol) and ofthe cyclohexyl ring by a phenyl moiety <~ difenidol) indueed a !arge (4- to 80-fold) decrease in binding affinity for all musearlnie receptors. Difenidol had a signifieant preference for M\(_1\) , M\(_3\) , and M\(_4\) over M\(_2\) receptors; dicyclidol, by eontrast, had a greater affinity for M\(_1\) and M\(_4\) than for M\(_2\) and M\(_3\) receptors. The binding free energy deerease due to replacement ofthe phenyl and the cyelohexyl groups of(R)-hexahydro-difenidol by, respectively, a eyclohexyl and a phenyl moiety was almostadditive in the ease of M\(_4\) (striatum) binding sites. In the ease ofthe cardiac M\(_2\), pancreatic M\(_3\) , or NB-OK 1 M\(_1\) receptors the respective binding free energies were not eompletely additive. These results suggest that the four (R)-hexahydro-difenidol ''binding moieties" (phenyl, cyclohexyl, hydroxy, and protonated amino group) cannot simultaneously form optimal interaetions with the M\(_1\), M\(_2\), and M\(_3\) muscarinic receptors. When eaeh of the hydrophobic groups is modified, the position of the whole molecule, relative to the four subsites, was changed to allow an optimal overall interaction with the musearlnie receptor.
1 We have compared the binding properties of several hexocyclium and sila-hexocyclium derivatives to muscarinic Ml receptors (in rat brain, human neuroblastoma (NB-OK I) 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 (Ml/M4-like), guinea-pig atria (M2), and guinea-pig ileum (M3) muscarinic receptors.
2 Si la-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 demethoxy-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 (Ml = M4 ~ M3 ~ M2) had a much lower affinity than sila-hexocyclium for the four receptor subtypes, and discriminated the receptor subtypes more poorly than sila-hexocyclium (Ml = M3> M4> M2)' This is in marked contrast with the very clear selectivity of demethoxy-sila-hexocyclium for the prejunctional MtlM4-like heteroreceptors in rabbit vas deferens.
5 The tertiary amines demethyl-hexocyclium, demethyl-sila-hexocyclium and demethyl-o-methoxy-silahexocyclium had 10 to 30 fold lower affinities than the corresponding quaternary ammonium derivatives.
Starting from trichloro(vinyl)silane (Cl\(_3\)SiCH=CH\(_2\)), the musearinic antagonists sila-biperiden [rac-(SiRS,C2SR>-ao-2] and endosila- biperiden [rac-(SiRS,C2SR)-endo-2] were prepared by a seven-step synthesis. Both silanols are configurationally stableininert organic solvents but undergo slow epimerization in aqueous solution (pH 7.4, 32°C) by inversion of the configuration at the silicon atom. The relative configurations of sila-biperiden and endo-sila-biperiden were detennined by single-crystal X-ray diffraction. Both compounds form intennolecular 0-H · · · N hydrogen bonds in the crystal leading to the fonnation of centrosymmetric dimers (sila-biperiden) and infinite chains (endo-sila-biperiden), respectively. Sila-biperiden is a silicon analogue (C/Si exchange) of the antiparkinsonian drug biperiden [rac-(CRS/C2SR}-exo-1]. In functional phannacological experiments, as well as in radioligand competition studies, biperiden, sila-biperiden and endo-sila-biperiden behaved as simple competitive antagonists at muscarinic Ml-, M2-, M3- and M4-receptors. The three compounds displayed the highest affinity for Ml-receptors (pA\(_2\) values: 8.72-8.80; pK\(_i\) values: 8.8-9.1), intermediate affinity for M4- and M3-receptors, and lowest affinity for M2-receptors (pA\(_2\) values: 7.57-7.79; pK\(_i\) values: 7.7-7.8). The affinity profile (Ml >. M4 > M3 > M2) of biperiden, sila-biperiden and endo-sila-biperiden is qualitatively similar to that of the M1-selective muscarinic antagonist pirenzepine. The antimuscarinic properlies of the C/Si analogues biperiden and sila-biperiden are almost identical.