Jan Gross

Evidence for muscarinic M4 receptors mediating nonadrenergic noncholinergic relaxations in rabbit anococcygeus muscle

The aim of the present study was to characterize the muscarinic receptor subtype mediating nonadrenergic noncholinergic (NANC) relaxations in the rabbit anococcygeus muscle (RAM) by the use of muscarinic receptor agonists and a battery of key muscarinic antagonists. In addition, experiments were carried out to identify the NANC neurotransmitter(s) involved in the inhibitory NANC neurotransmission. In preparations with histamine-raised tone, the non-selective muscarinic agonists (pD2 values) (+)-muscarine (5.23), cis-dioxolane (5.16), oxotremorine M (4.95) and carbachol (4.06) produced concentration-dependent relaxations corresponding to 72.6–85.0% of the histamine-induced precontraction. In contrast, the subtype-preferring (M1/M4 over M2 and M3 receptors) agonists 4-(3-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium chloride (McN-A-343), (S)-4-(dimethylamino)-1-methyl-2-butynyl-N-(3-chlorophenyl)carbamate methobromide [(S)-BN 228], (R)- and (S)-N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide [(R)- and (S)-BM 5] showed no or rather low [(S)-BN 228] muscarinic activity. The low potencies, together with the ineffectiveness of some agonists, indicated a low effective receptor reserve associated with the relaxant response. No contractile responses to (+)-muscarine were observed neither in unstimulated nor in precontracted preparations suggesting that the existence of an excitatory postjunctional muscarinic receptor may be excluded. The nicotinic antagonist hexamethonium had no influence on relaxant responses to (+)-muscarine, but abolished relaxations elicited by (–)-nicotine. This demonstrates that the RAM contains also nicotinic acetylcholine receptors (AchRs) mediating inhibitory NANC responses. Relaxations induced by the stimulation of muscarinic and nicotinic AchRs as well as by electrical field stimulation (EFS) were completely abolished by tetrodotoxin and were also sensitive to the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine (L-NOARG), indicating that NO plays an important role as an inhibitory NANC transmitter in RAM. All muscarinic antagonists investigated did not influence the histamine-induced precontraction, but shifted the concentration-response curve of (+)-muscarine to the right in a parallel fashion. Schild analysis yielded regression lines of unit slope, indicating competitive antagonism. The following rank order of antagonist potencies (pA2 values) was found: 11-({4-[4-(diethylamino)-bu-tyl]-1-piperidinyl}-acetyl-5,11-dihydro-6H-pyrido (2,3-b) (1,4)-benzodiazepine-6-one hydrochloride (AQ-RA 741; 8.08) = himbacine (8.03) ≥ tripitramine (7.69) ≥ p-fluoro-hexahydro-sila-difenidol (p-F-HHSiD; 7.48) ≥ methoctramine (7.30) ≥ pirenzepine (7.18) ≥ guanylpirenzepine (6.24). A comparison of the pA2values determined in the RAM with published data from binding studies at muscarinic M1–M4 and m5 receptors suggests that the functional muscarinic receptor mediating NANC relaxation in the RAM is of the M4 subtype. Taken together, the results obtained in the present study provide convincing evidence that relaxant responses elicited by muscarinic stimuli in RAM are nitrergic in nature and mediated by muscarinic M4 receptors located somadendritically on NANC neurons. Thus, the isolated RAM may serve as a functional muscarinic M4 receptor model.

The design and pharmacology of novel selective muscarinic agonists and antagonists

The muscarinic pharmacology of C1-methyl-substituted chiral compounds related to McN-A-343 and of (R)- and (S)-dimethindene has been studied. Among the McN-A-343 analogues, the (S)-enantiomers were more potent and had higher affinity than the (R)-isomers. The quaternary compound (S)-BN 228 was found to be the most potent M1-selective agonist known today (pEC50: M1/rabbit vas deferens = 7.83; M2/guinea-pig atria = 6.35; M3/guinea-pig ileum = 6.29). In both the atria and ileum the tertiary carbamate, (S)-4-F-MePyMcN, was a competitive antagonist (pA2 value = 7.39 and 6.82, respectively). In contrast, in rabbit vas deferens (S)-4-F-MePyMcN was a potent partial agonist (pEC50 = 7.22; apparent efficacy = 0.83). These results indicate that (S)-4-F-MePyMcN might be a useful tool to study M1 receptor-mediated effects involved in central cholinergic function. (S)-Dimethindene was a potent M2-selective antagonist (pA2 = 7.86/atria; pKi = 7.8/rat heart) with lower affinities for the M1 (pA2 = 6.36/rat duodenum; pKi = 7.1/NB-OK 1 cells), M3 (pA2 = 6.92/guinea-pig ileum; pKi = 6.7/rat pancreas) and M4 receptors (pKi = 7.0/rat striatum). It was more potent (up to 41-fold) than the (R)-isomer. In contrast, the stereoselectivity was inverse at ileal H1 receptors (pA2: (R)-isomer = 9.42; (S)-isomer = 7.48). Thus, (S)-dimethindene could be a valuable agent to test the hypothesis that M2 antagonists show beneficial effects in the treatment of cognitive disorders. It might also become the starting point for the development of diagnostic tools for quantifying M2 receptors in the CNS with PET imaging.

Bioorganogermanium Chemistry: Studies on C/Si/Ge Bioisosterism

In context with systematic investigations on C/Si/Ge bioisosterism, the following studies were carried out: (a) synthesis and pharmacological characterization of centrochiral enantiomerically pure germanium-based muscarinic antagonists; (b) synthesis and pharmacological characterization of a germanium-containing decapeptide; (c) studies on the metabolism of a germanium-based drug in the rat; (d) synthesis of centrochiral enantiomerically pure germanes using biotransformations with whole microorganisms or isolated enzymes. These investigations demonstrated that there are distinct bioisosteric relationships between the C/Si/Ge analogues studied.

Pharmacological discrimination between enantiomeric germanes by muscarinic receptors: a study on germanium/silicon bioisosterism

Abstract The (hydroxymethyl)diorgano(2-piperidinoethyl)germanes rac-Ph(c-Hex)Ge(CH2OH)CH2CH2NR2 (rac-1a), Ph2Ge(CH2OH)CH2-CH2NR2 (3a) and (c-Hex)2Ge(CH2OH)CH2CH2NR2 (5a) (NR2 = piperidino) were synthesized starting from Cl3GeCH2Cl. The (R)- and (S)-enantiomer of 1a were obtained by resolution of rac-1a using the antipodes of O,O′-di-p-toluoyltartaric acid as resolving agents (resolution by fractional crystallization of diastereomeric salts). The enantiomeric purities of the resolved antipodes of 1a were shown to be at least 98 (1H NMR) and 97% ee (13C NMR) respectively (NMR studies using a chiral shift reagent). Reaction of rac-1a, (R)-1a, (S)-1a, 3a and 5a with methyl iodide gave the corresponding methiodides rac-2a, (R)-2a, (S)-2a, 4a and 6a (1a → 2a, 3a → 4a, 5a → 6a). The absolute configuration of (R)-2a was determined by single-crystal X-ray diffraction. On the basis of the experimentally established absolute configuration of (R)-2a, the absolute configurations of all the other aforementioned optically active germanium compounds were assigned by chemical and optical correlations. The enantiomerically pure germanium compounds (R)-1a, (S)-1a, (R)-2a and (S)-2a and their achiral derivatives 3a–6a were studied for their affinities for muscarinic M1, M2, M3 and M4 receptors by functional pharmacological experiments (M1, rabbit vas deferens; M2, guinea-pig atria; M3, guinea-pig ileum) and radioligand binding experiments (M1, human NB-OK 1 cells; M2, rat heart, M3, rat pancreas; M4, rat striatum). The receptor affinities obtained in these studies were compared with those of the related silicon analogues, the (hydroxymethyl)diorgano(2-piperidinoethyl)silanes (R)- and (S)-Ph(c-Hex)Si(CH2OH)CH2CH2NR2[(R)-1b and (S)-1b], Ph2Si(CH2OH)CH2CH2NR2 (3b) and (c-Hex)2Si(CH2OH)CH2CH2NR2 (5b) (NR2 = piperidino) and their corresponding methiodides (R)-2b, (S)-2b, 4b and 6b (a → b: Ge → Si: studies on Ge Si bioisosterism). According to these studies, all the germanes and the related silicon analogues behaved as simple competitive antagonists at muscarinic M1–M4 receptors. The pKi values obtained in binding studies at M1–M3 receptors were similar to the corresponding functional affinities (pA2 values). The receptor affinities of the respective Ge Si analogues were found to be very similar, indicating a strongly pronounced Ge Si bioisosterism. The (R)-enantiomers (eutomers) of the Ge Si pairs 1 a 1 b and 2 a 2 b exhibited higher affinities (up to 26-fold) for M1–M4 receptors than their corresponding (S)-antipodes (distomers), the stereoselectivity ratios being higher at M1, M3 and M4 than at M2 receptors. In most cases, the diphenyl ( 3 a 3 b and 4 a 4 b ) and dicyclohexyl ( 5 a 5 b and 6 a 6 b ) compounds displayed lower affinities to M1–M4 receptors than the related (R)-enantiomers of 1 a 1 b and 2 a 2 b , and the sums of the respective affinity differences were very similar to the experimentally established stereoselectivity ratios [ (R) (S) ]. Thus, the stereoselective interaction of the enantiomers of 1 a 1 b and 2 a 2 b with muscarinic receptors is best explained in terms of opposite and weaker binding of the phenyl and cyclohexyl ring of the (S)-antipodes. The highest receptor selectivity was observed for compound (R)-1b at M 1 M 2 receptors (25-fold in binding studies).

Neuronal soma-dendritic and prejunctional M1-M4 receptors in gastrointestinal and genitourinary smooth muscle.

A variety of neurons in gastrointestinal and genitourinary smooth muscle express muscarinic auto- as well as heteroreceptors. These receptors are found on the soma and dendrites of many cholinergic, sympathetic and NANC neurons and on axon terminals. A given neuron may contain both excitatory and inhibitory presynaptic muscarinic receptors. The subtypes involved are species- and tissue-dependent, and neuronal M1 to M4 receptors have been shown to be expressed in smooth muscle tissues. In this study, the ability of several selective muscarinic receptor antagonists to inhibit the effect of arecaidine propargyl ester (APE) on prejunctional muscarinic receptors on sympathetic nerve endings in the rabbit anococcygeus muscle (RAM) was investigated to characterise the receptor subtype involved. Electrical field stimulation (EFS) resulted in a release of noradrenaline (NA) eliciting monophasic contractions due to stimulation of postjunctional alpha1-adrenoceptors. The selective muscarinic agonist APE did not reduce contractions to exogenous NA, but caused a concentration-related and N-methylatropine-sensitive inhibition of neurogenic responses. All muscarinic antagonists investigated failed to affect the EFS-induced contractions, but shifted the concentration-response curve of APE to the right in a parallel and surmountable fashion. Schild analysis yielded regression lines of unit slope, indicating competitive antagonism. The following rank order of antagonist potencies (pA2 values) was found: tripitramine (9.10) > AQ-RA 741 (8.26) > or = himbacine (8.04) > or = (S)-dimethindene (7.69) > pirenzepine (6.46) > or = p-F-HHSiD (6.27). A comparison of the pA2 values determined in the present study with literature binding and functional affinities obtained at native or recombinant M1 to M5 receptors strongly suggests that NA release from sympathetic nerve endings in RAM is inhibited by activation of prejunctional muscarinic M2 receptors.

Fluorine-containing derivatives of the muscarinic antagonists sila-pridinol and sila-difenidol: Syntheses and antimuscarinic properties

Abstract The fluorine-containing sila-pridinol and sila-difenidol derivatives p -fluoro-sila-pridinol ( 5a ), p,p ′-difluoro-sila-pridinol ( 6a ), p -fluoro-sila-difenidol ( 7a ), p,p ′-difluoro-sila-difenidol ( 8a ), p -fluoro-sila-difenidol methiodide ( 9a ) and p,p ′-difluoro-sila-difenidol methiodide ( l0a ) were synthesized, starting from the silanes Cl 3 SiCHCH 2 ( 5a and 6a ) and (CH 3 O) 3 Si(CH 2 ) 3 Cl ( 7a–10a ) respectively. The chiral compounds 5a, 7a and 9a were obtained as racemic mixtures. The muscarinic pharmacology of the silanols 5a–10a was studied and compared with that of their carbon analogues, the carbinols 5b–10b (studies on silicon-carbon bioisosterism). The affinities and receptor selectivities (Ml–M4 receptors) of the SiC pairs 5a/5b–l0a/l0b were found to depend on the following structural parameters: length of the carbon chain El-(CH 2 ) n -N (El  Si or C; n = 2, 3), N -methylation, fluorine substitution of the phenyl rings and the nature of the central atom (silicon or carbon). Most interestingly, replacement of the central carbinol carbon atom in p -fluoro-difenidol methiodide ( 9b ) by a silicon atom (→ 9a ) leads to an increase in affinity for muscarinic receptor subtypes by factors of 32–81. Such a high increase in biological activity by sila-substitution (CSi exchange) has not yet been reported.

Sila-biperiden und endo-Sila-biperiden: Synthesen, Kristallstrukturen und antimuscarinische Eigenschaften

Summary Starting from trichloro(vinyl)silane (Cl 3 SiCH=CH 2 ), the muscarinic antagonists sila- biperiden [ rac -(Si RS ,C2 SR ) -exo - 2 ] and endo -sila-biperiden [ rac -(Si RS ,C2 SR )- endo - 2 ] were prepared by a seven-step synthesis. Both silanols are configurationally stable in inert 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 determined by single-crystal X-ray diffraction. Both compounds form intermolecular O-H ⋯ N hydrogen bonds in the crystal leading to the formation 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 -(C RS /C2 SR ) -exo - 1 ]. In functional pharmacological experiments, as well as in radioligand competition studies, biperiden, sila-biperiden and endo -sila-biperiden behaved as simple competitive antagonists at muscarinic M1-, M2-, M3- and M4-receptors. The three compounds displayed the highest affinity for M1-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 (M1 ≫ 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 properties of the C/Si analogues biperiden and sila-biperiden are almost identical.

Unsaturated derivatives of the muscarinic antagonists hexahydro-sila-difenidol (HHSiD) and p-fluoro-hexahydro-sila-difenidol (p-F-HHSiD) with an (E)-SiCHCHCH2rmN moiety: Syntheses and binding affinities at muscarinic receptor subtypes

Abstract The unsaturated HHSiD ( 1 ) and p -F-HHSiD ( 2 ) derivatives ( E )-cyclohexyl(phenyl)(3-piperidino-1-propen-1-yl)silanol ( 5 , isolated as 5 · HCl) and ( E )-cyclohexyl(4-fluorophenyl)(3-piperidino-1-propen-1-yl)silanol ( 6 , isolated as 6 · HCl) were synthesized in four steps, starting from (CH 3 O) 3 SiH. Reaction of 5 and 6 with CH 3 Cl gave the corresponding methochlorides 7 and 8 , respectively. All compounds were obtained as racemic mixtures. The binding affinities at muscarinic receptor subtypes (M1–M4) of the silanols 5–8 were determined and compared with those of the selective muscarinic antagonists 1 and 2 and their methiodides 3 and 4 . These studies demonstrated that the ammonium compounds 3, 4, 7 and 8 display similar binding affinities at M1–M4 receptors and comparable receptor subtype selectivities. On the other hand, the conformationally restricted amines 5 and 6 (( E )SiCHCHCH 2 N moiety) exhibit higher affinities but lower receptor subtype selectivities than the more flexible parent compounds 1 and 2 SiCH 2 CH 2 CH 2 N moiety).

Muscarinic receptor subtypes - search for selective agonists and antagonists

Since the late eighties five muscarinic receptor subtypes (m1 – m5) have been cloned and four of them (M1 – M4) have also been pharmacologically characterized. However, there is still a lack of potent muscarinic agonists and antagonists, which are highly selective for one muscarinic receptor subtype over all other subtypes. For the treatment of Alzheimers disease, M1-selective agonists capable of penetrating into the CNS are needed. It is hypothezised that such substances would not only improve memory and cognitive ability, but also delay the progression of the disease. In our laboratory, the functionally M1-selective quaternary ammonium compound McN-A-343 has been used as a starting point for the design of such CNS active muscarinic ligands. Structure-activity relationship studies led to the tertiary amine 4-(4-fluorophenylcarbamoyloxy)-2-butynylpyrrolidine (4-F-PyMcN), which was found to stimulate M1 receptors with some functional selectivity. In order to increase the potency and selectivity of 4-F-PyMcN several new derivatives were synthezised and pharmacologically characterized in different functional assays as well as in binding and biochemical (PI turnover) studies. The most promising results were obtained with (S)-4-(4-fluorophenylcarbamoyloxy)-1-methyl-2-butynylpyrrolidine (4-F-MePyMcN). Due to its potent partial agonistic activity at M1 receptors and its M2-antagonistic properties leading to an increase of acetylcholine release by blockade of M2 autoreceptors, this compound may be considered as an important tool for future drug research of cognitive disorders. M2 receptor antagonists may also be used for the treatment of Alzheimers disease, furthermore in the therapy of supraventricular bradycardia and for quantifying M2 receptors in the CNS with PET imaging. In the search for antagonists which clearly differentiate M2 from other muscarinic receptors, we investigated the two enantiomers of the widely used H1-antihistaminic drug dimethindene. (S)-Dimethindene proved to be a potent M2-selective antagonist with lower affinities for the M1, M3 and M4 receptors. In addition, the (S)-enantiomer was more potent than the (R)-enantiomer in all muscarinic assays. Interestingly, the stereoselectivity was inverse at histamine H1 receptors, the (R)-enantiomer being the eutomer. M3 receptor antagonists may be useful in the treatment of spastic disorders of the gastrointestinal, urogenital and respiratory tract as well as for the relief of glandular hypersecretion. In previous studies, hexahydro-difenidol (HHD) and its sila-analogue, hexahydro-sila-difenidol (HHSiD), as well as the antiparkinsonian drug trihexyphenidyl (THP) were found to be valuable tools for the discrimination of M3 and M2 receptors. In order to further assess the structural requirements (including stereochemical aspects) of the above-mentioned compounds for potency and selectivity, a series of HHD and THP analogues as well as of the corresponding silicon and germanium derivatives (sila- and germa-substitution) were studied. The (R)-enantiomers displayed higher affinities and selectivities than the corresponding (S)-isomers. The enantioselectivity of some of these analogues is best explained by the concept of the four-binding-subsite model suggesting that the differences in affinity of the (R)- and (S)-enantiomers at muscarinic receptors are due to opposite binding of the phenyl and the cyclohexyl ring to the preferring subsites. Surprisingly, there was no significant difference between the Si and Ge analogues indicating a strongly pronounced Si/Ge bioisosterism in this series of compounds. The related carbon derivatives, however, showed higher receptor affinities as well as greater stereoselectivities at all muscarinic receptors studied compared with the silicon and germanium analogues.

(Hydroxymethyl)diphenyl(piperidinoalkyl)silane des Typs (HOCH2)(C6H5)2Si(CH2)nNC5H10(n=2,3) und deren Methoiodide: Synthese, Struktur und antimuscarinische Eigenschaften / (Hydroxymethyl)diphenyl(piperidinoalkyl)silanes of the Type (HOCH2)(C6H5)2Si(CH2)nNC5H10(n = 2,3) and their Methiodides: Synthesis, Structure and Antimuscarinic Properties

Abstract Starting from (MeO)3SiCH2Cl (10) and Ph2(H)SiCH2OH (16), respectively, the (hydroxymethyl)diphenyl(piperidinoalkyl)silanes (HOCH2)Ph2Si(CH2)2NC5H10 (6) and (HOCH2)Ph2Si(CH2)3NC5H10 (8) have been synthesized [10→Ph2(MeO)SiCH2Cl (11)→Ph2(CH2=CH)SiCH2Cl (12)→Ph2(CH2=CH)SiCH2OAc (13)→Ph2(CH2=CH)SiCH2OH (14)→Ph2(CH2=CH)SiCH2OSiMe3 (15)→6; 16→Ph2(H)SiCH2OSiMe3 (17)→8; NC5H10 = piperidino]. N-Quaternization of 6 and 8 with MeI gave the corresponding methiodides 7 and 9, respectively. As shown by IR-spectroscopic studies, compounds 6 and 8 form intramolecular O-H···N hydrogen bonds in solution (CCl4). In the crystal, 6 (space group Pna21; two crystallographically independent molecules) also forms intramolecular O-H···N hydrogen bonds whereas 8 (space group P1̅) forms intermolecular O-H···N hydrogen bonds leading to the formation of centrosymmetric dimers (single-crystal X-ray diffraction studies). The (hydroxymethyl) silanes 6-9 and the related silanols (HO)Ph2Si(CH2)2NC5H 10 (sila-pridinol; 1), sila-pridinol methiodide (2), (HO)Ph2Si(CH2)3NC5H10 (sila-difenidol; 3) and sila-difenidol methiodide (4) were investigated for their antimuscarinic properties. In functional pharmacological experiments as well as in radioligand competition studies, all compounds behaved as simple competitive antagonists at muscarinic M1-, M2-, M3- and M4-receptors. In general, the silanols 1-4 displayed higher receptor affinities (up to 100-fold) than the corresponding (hydroxymethyl) silanes 6-9 . In the (hydroxymethyl)silane series, compound 7 was found to be the most potent muscarinic antagonist [pA2/pKi= 8,71/8,6 (M1), 8,23/7,8 (M2), 8,19/7,8 (M3); pKi = 8,2 (M4)]. In the silanol series, the related compound 2 showed the most interesting antimuscarinic properties [pA2/pKi = 10,37/9,6 (M1), 8,97/8,8 (M2), 9,08/8,8 (M3); pKi = 9,4 (M4)].