piperidines and zamifenacin

The synthesis of optically active substituted piperidines has been achieved by using four different methodologies. The first one is an intramolecular nucleophilic displacement of activated alcohol moieties that was used to build up the piperidine ring of (-)-prosophylline and (-)-slaframine, and the second one is a ring-closing metathesis of unsaturated amines which was employed in the synthesis of (+)-sedamine and 4a,5-dihydrostreptazoline. The third methodology is the alpha-functionalization of N-Boc piperidines which was particularly useful in the synthesis of argatroban, and the fourth one is a ring expansion of prolinols to 3-chloropiperidines or 3-hydroxypiperidines which was utilized to synthesize (-)-paroxetine, (-)-pseudoconhydrine, the piperidine ring of (-)-velbanamine and (+)-zamifenacin.

Topics: Alkaloids; Arginine; Chemistry; Dioxoles; Paroxetine; Pipecolic Acids; Piperidines; Pyrrolidines; Sulfonamides

Hyoscine (scopolamine), which is effective in the prophylaxis of motion sickness, shows similar binding affinities to all of the five known muscarinic receptor sub-types. The effectiveness of hyoscine was compared with zamifenacin (UK-76654), which binds selectively to the muscarinic M3 and m5 receptors.. Eighteen subjects received hyoscine hydrobromide 0.6 mg, zamifenacin 20 mg, or placebo (double-blind cross-over design). Sessions were 1 week apart and the drug (oral) was given 90 min prior to a motion sickness test. Motion sickness was elicited by cross-coupled stimulation on a turntable. The rotational velocity was incremented by 2 degrees s-1 every 30 s, and a sequence (seq) of eight head movements of 45 degrees was completed every 30 s. Motion tolerance was assessed as the number of sequences of head movement required to achieve moderate nausea. Pulse rate was recorded before and at 1 and 2 h after drug administration. Skin conductance activity in the frequency band 0.005-0.48 Hz, an index of sweat gland activity, was measured using Ag/AgCl electrodes on the palmar surfaces of fingers and across the forehead.. Both zamifenacin and hyoscine produced an increase in tolerance to the motion challenge (P < 0.01) with no significant difference between the two drugs (5.0 +/- 1.6 vs 5.7 +/- 1.6 seqs. respectively, mean +/- s.e.mean). Compared with placebo or zamifenacin, pulse rate fell following hyoscine administration (9 beats min-1, P < 0.01). Skin conductance was reduced following hyoscine compared with zamifenacin or placebo (P < 0.001).. These results suggest that compounds with selective M3 and/or m5 antagonism possess activity against motion sickness. Antagonism at these receptors may be the basis of the anti-motion sickness action of hyoscine.

Topics: Adult; Analysis of Variance; Binding, Competitive; Cross-Over Studies; Dioxoles; Double-Blind Method; Electrodes; Galvanic Skin Response; Heart Rate; Humans; Male; Middle Aged; Motion Sickness; Muscarinic Antagonists; Nausea; Piperidines; Psychomotor Performance; Receptors, Muscarinic; Rotation; Scopolamine; Sweat Glands

Zamifenacin is a new potent gut M3 selective muscarinic antagonist developed for possible use in irritable bowel syndrome.. In this multicentre, double-blind, parallel group, placebo-controlled study, the effect of a single dose of zamifenacin 10 mg or 40 mg on both fasting (30 min) and fed (60 min) colonic motor activity was assessed in 36 patients with irritable bowel syndrome (aged 25-68 years; 19 male). Colonic motility was recorded using a five-channel solid-state catheter introduced by colonoscopy to a depth of 35 cm in an unprepared colon.. Zamifenacin 40 mg profoundly reduced colonic motility, particularly after the meal (P < 0.05). This was reflected by a significant reduction in the mean amplitude of contractions, number of contractions, percentage duration of contractions, activity index and the motility index (P < 0.05). A smaller reduction in all the motility parameters was obtained with 10 mg zamifenacin, but these changes were not statistically significant. Three patients each on placebo and zamifenacin reported side-effects, but these were mild and transient.. A single 40 mg dose of zamifenacin significantly reduces colonic motility in irritable bowel syndrome patients without significant antimuscarinic effects. The results of this study confirm that the concept of developing selective antimuscarinic agents may be a promising approach to the treatment of irritable bowel syndrome. Not only would such compounds benefit from not having the usual side-effects of anticholinergics but they might also offer much more in the way of dose flexibility.

Topics: Adult; Aged; Colonic Diseases, Functional; Dioxoles; Double-Blind Method; Humans; Male; Middle Aged; Muscarinic Antagonists; Piperidines

A prospective, randomized, double-blind study was undertaken to evaluate Zamifenacin 30 mg (Pfizer Ltd), a novel, orally-administered, gut-specific muscarinic receptor antagonist, as an adjuvant to the double contrast barium enema examination (DCBE). Zamifenacin was compared with placebo in terms of side-effects and colonic tone. Analysis of colonic tone was carried out by two independent observers, using a subjective grading system and also by an objective method using computerized planimetry. Interobserver variability was also assessed. Zamifenacin is safe and well tolerated but at the prescribed dose is an ineffective antispasmodic for DCBE. Subjective assessment of colonic tone was shown to be of limited value whilst the objective analysis using computerized planimetry was reliable and highly reproducible.

Topics: Adolescent; Adult; Aged; Barium Sulfate; Colon; Colonic Diseases; Dioxoles; Double-Blind Method; Enema; Humans; Image Processing, Computer-Assisted; Middle Aged; Muscarinic Antagonists; Observer Variation; Piperidines; Prospective Studies; Radiography; Spasm

Muscarinic M3 receptor antagonists have therapeutic potential for the treatment of disorders associated with altered smooth muscle contractility or tone. These include irritable bowel syndrome (IBS), chronic obstructive airways disease (COAD) and urinary incontinence. Zamifenacin is a potent muscarinic receptor antagonist on the guinea pig ileum (pA2 value 9.27) with selectivity over M2 receptors in the atria (135-fold) and M1/M4 receptors in the rabbit vas deferens (78-fold). In addition, zamifenacin had lower affinity for the M3 receptor in the salivary gland (pKi 7.97). In animals, zamifenacin potently inhibited gut motility in the absence of cardiovascular effects and with selectivity over inhibition of salivary secretion. In healthy volunteers, zamifenacin inhibited small and large bowel motility and increased the rate of gastric emptying over a dose range which was associated with minimal anticholinergic side effects. These data show that zamifenacin, a selective muscarinic M3 receptor antagonist, was well tolerated in man and was efficacious as an inhibitor of gut motility. Further studies in patients are required with muscarinic M3 receptor antagonists to confirm efficacy against symptoms in diseases associated with altered smooth muscle contractility.

Topics: Animals; Atrial Function; Atropine; Colon; Dicyclomine; Dioxoles; Dogs; Drug Evaluation, Preclinical; Gastrointestinal Motility; Guinea Pigs; Heart Atria; Humans; Ileum; Male; Muscarinic Antagonists; Muscle, Smooth; Piperidines; Rabbits; Receptor, Muscarinic M3; Receptors, Muscarinic; Salivary Glands; Trachea; Vas Deferens

An aerobic catalytic oxidation process is described for the olefin oxyamination using acids and primary amines as the sources of O and N. Our mechanistic findings point to the formation of triiodide as a critical catalytic intermediate to account for the tolerance of electron-rich nucleophiles. This dual iodide and copper catalytic system is suitable for a formal [5+1] annulation process to access valuable lactam structures and highlighted by the synthesis of the pharmaceutical Zamifenacin.

Topics: Alkenes; Amination; Amines; Carboxylic Acids; Catalysis; Copper; Dioxoles; Electrons; Iodides; Molecular Structure; Oxidation-Reduction; Piperidines; Stereoisomerism

Surfaces of pharmaceutical powders have been assessed using contact angle, inverse phase gas chromatography (IGC) and triboelectric (electrostatic) charging techniques. The suitability of the Dynamic Angle Tester (DAT), an instrument based on the sessile drop technique, in determining contact angles and then the surface energy of pharmaceutical powders was assessed. The dispersive components of the surface energy of powders determined from the DAT and IGC method ranked the powders in the same order. The dispersive component values obtained by IGC were, as expected, higher than those from the DAT, due to IGC probing the highest energy sites on the powder surface. IGC and triboelectric studies allow materials to be characterised in terms of their electron donating-accepting tendencies, so inter-relationships between the data from the two techniques were explored. Although the data set was limited, there appeared to be a correlation between the charges developed by the powders on contact with stainless steel and the ratio of the electron-donating to electron-accepting tendencies of the materials as obtained from IGC.

Topics: Chemistry, Pharmaceutical; Chromatography, Gas; Dioxoles; Electrochemistry; Hydrogen-Ion Concentration; Lactose; Piperidines; Powders; Starch; Thermodynamics

Cerebellar granule neurons (CGNs) possess a standing outward potassium current (IK(SO)) which shares many similarities with current through the two-pore domain potassium channel TASK-1 and which is inhibited following activation of muscarinic acetylcholine receptors. The action of muscarine on IK(SO) was unaffected by the M2 receptor antagonist methoctramine (100 nM) but was blocked by the M3 antagonist zamifenacin, which, at a concentration of 100 nM, shifted the muscarine concentration-response curve to the right by around 50-fold. Surprisingly, M3 receptor activation rarely produced a detectable increase in [Ca2+]i unless preceded by depolarization of the cells with 25 mM K+. Experiments with thapsigargin and ionomycin suggested that the endoplasmic reticulum Ca2+ stores in CGNs were depleted at rest. In contrast, cerebellar glial cells in the same fields of cells possessed substantial endoplasmic reticulum Ca2+ stores at rest. Pretreatment of the cells with BAPTA AM, thapsigargin or the phospholipase C (PLC) inhibitor U-73122 all blocked the muscarine-induced Ca2+ signal but had little or no effect on muscarinic inhibition of IK(SO). Raising [Ca2+]i directly with ionomycin caused a small but significant inhibition of IK(SO). It is concluded that muscarine acts on M3 muscarinic acetylcholine receptors both to inhibit IK(SO) and to mobilize Ca2+ from intracellular stores in CGNs. While the mobilization of Ca2+ occurs through activation of PLC, this does not seem to be the primary mechanism underlying muscarinic inhibition of IK(SO).

Topics: Animals; Animals, Newborn; Calcium; Calcium Signaling; Cells, Cultured; Cerebellum; Chelating Agents; Dioxoles; Egtazic Acid; Electric Conductivity; Estrenes; Female; Male; Membrane Potentials; Muscarinic Antagonists; Piperidines; Potassium; Potassium Channels; Protein Isoforms; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Receptor, Muscarinic M3; Receptors, Muscarinic; Thapsigargin

The muscarinic receptor subtypes that mediate cholinergic responses in cat esophageal smooth muscle were examined. Antagonist effects on carbachol-induced and nerve-evoked contractions were studied in vitro using muscle strips from the distal esophagus. Antagonists displayed similar relative selectivities in suppressing carbachol and nerve-mediated responses as follows: 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) > zamifenacin > para-fluoro-hexahydrosiladiphenidol > pirenzepine > AF-DX 116 > methoctramine, indicating that these responses are mediated by the same receptor subtype. 4-DAMP, pirenzepine and methoctramine effects on carbachol responses gave pA2 values characteristic of the M3 receptor in both the circular muscle (9.25 +/- 0.12, 6.79 +/- 0.09 and 6.04 +/- 0.11, respectively) and longitudinal muscle (9.46 +/- 0.14, 7.25 +/- 0.07 and 6.10 +/- 0.06, respectively). Reverse transcription-polymerase chain reaction analysis was done using primer sequences based on the cloned human muscarinic receptor subtypes. Messenger RNA for the m3 receptor was readily identified, whereas m2 was not detected in esophageal muscle, but was present in cardiac muscle. Sequence homology between the amplified products from cat tissue and the corresponding human m2 and m3 receptors genes were 93% and 89%, respectively. In the cat esophagus, the M3 receptor mediates functional responses and messenger RNA for the corresponding molecular form of this receptor is abundant in this tissue.

Topics: Amino Acid Sequence; Animals; Base Sequence; Carbachol; Cats; Dioxoles; Electric Stimulation; Esophagus; Female; Male; Molecular Sequence Data; Muscle Contraction; Muscle, Smooth; Piperidines; Polymerase Chain Reaction; Receptors, Muscarinic; RNA, Messenger

1. The pharmacological characteristics of muscarinic receptors in the rabbit iris sphincter muscle were studied and compared to M3 receptors in rabbit urinary bladder smooth muscle. 2. (+/-)-Cis-dioxolane induced concentration-dependent contractions of the iris sphincter muscle (pEC50 = 6.41+/-0.10, Emax = 181+/-17 mg, n = 38) and urinary bladder smooth muscle (pEC50 = 6.97+/-0.04, Emax = 4.28+/-0.25 g, n = 54). These contractions were competitively antagonized by a range of muscarinic receptor antagonists (pK(B) values are given for the iris sphincter muscle and the bladder smooth muscle, respectively): atropine (9.30+/-0.07 and 9.40+/-0.04), AQ-RA 741 (6.35+/-0.04 and 6.88+/-0.03), darifenacin (9.56+/-0.05 and 9.12+/-0.05), methoctramine (5.75+/-0.07 and 5.81+/-0.06), oxybutynin (8.10+/-0.09 and 8.59+/-0.06), pirenzepine (6.79+/-0.05 and 6.89+/-0.04), secoverine (7.54+/-0.05 and 7.66+/-0.05), p-F-HHSiD (7.55+/-0.09 and 7.50+/-0.05) and zamifenacin (8.69+/-0.10 and 8.36+/-0.06). A significant correlation between the pK(B) values in the bladder and the pK(B) values in the iris was obtained. 3. In both tissues, the pK(B) values correlated most favorably with pKi values for these compounds at human recombinant muscarinic m3 receptors. A reasonable correlation was also noted at human recombinant muscarinic m5 receptors given the poor discriminative ability of ligands between m3 and m5 receptors. 4. Overall, the data from this study suggest that the muscarinic receptors mediating contraction of the rabbit iris sphincter muscle and urinary bladder smooth muscle are similar and equate most closely with the pharmacologically-defined muscarinic M3 receptor.

Topics: Animals; Benzofurans; Dioxoles; Elapid Venoms; Female; Humans; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Iris; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Peptides; Piperidines; Pyrrolidines; Rabbits; Receptor, Muscarinic M3; Receptor, Muscarinic M4; Receptors, Muscarinic; Recombinant Proteins; Urinary Bladder

1. We compared the sensitivities of primary hepatocytes from rat, dog and monkey to zamifenacin and two major metabolites, the methylenedioxy ring-opened catechol, UK-80,178 and its methylated product, UK-82,201. Toxicity was determined both via neutral red uptake and enzyme leakage data. 2. Canine hepatocytes were most sensitive to the cytotoxic effects of zamifenacin during 24-h exposure. Significant decreases in medium concentrations of zamifenacin in the presence of primary hepatocytes verified cellular uptake during the initial 2-h incubation. All three cell types were much more sensitive to UK-82,201 than to the catechol metabolite or parent drug. 3. The rapid onset of cytotoxicity indicated by elevations of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and other markers in the medium after UK-82,201 exposure, the delayed but substantial cytotoxic response to the parent drug which was suggestive of biotransformation to a reactive moiety, in vivo and in vitro drug metabolism results and subacute toxicology data suggest that dog may more effectively transform zamifenacin into UK-82,201, which is relatively hepatotoxic. 4. Because the catechol was generally less toxic than the O-methylated product, species that eliminate zamifenacin primarily as the catechol or its conjugate may be less affected by the potential hepatotoxicity of the methylated product. Our studies show that dog is the most sensitive species due to metabolism of the common catechol metabolite. The low incidence of potential hepatotoxicity in the clinic points to rare but important differences in the metabolism of Zamifencin. We conclude that the findings in dog were not predictive of subsequent effects in man.

Topics: Alanine Transaminase; Alkaline Phosphatase; Animals; Aspartate Aminotransferases; Catechols; Cells, Cultured; Dioxoles; Dogs; Half-Life; Kinetics; L-Lactate Dehydrogenase; Liver; Macaca fascicularis; Male; Methylation; Muscarinic Antagonists; Neutral Red; Piperidines; Rats

Expression of muscarinic receptor subtypes in rat gastric smooth muscle was examined with reverse transcriptase-polymerase chain reaction (RT-PCR). Under the condition for detecting the messages of m1-m4 subtypes in brain, atrium, and gastric mucosa, only the fragments of m2 and m3 subtypes were amplified with RNA prepared from rat gastric smooth muscles. Furthermore, the amplified fragments were digested by restriction enzymes, reconfirming that the predicted size products of m2 and m3 contain the partial DNA sequences of m2 and m3 subtypes, respectively. We measured gastric motility in rats with a pressure transducer system under the continuous venous infusion of the muscarinic antagonists atropine and butylscopolamine (nonselective), AF-DX 116 (M2), zamifenacine (M3), and glucagon. Heart rate was monitored simultaneously in the tail. Gastric motility was inhibited in the presence of glucagon and zamifenacine without alteration of heart rate, whereas there was no inhibition in the presence of AF-DX 116 even after the augmentation of heart rate was observed. Gastric emptying was also suppressed in the presence of zamifenacine, which had an effect comparable with that of atropine, butylscopolamine, and glucagon. These results indicate that the activation of the M3 subtype in gastric smooth muscle causes its contraction, and the M3 selective antagonist could be a potentially useful drug without an adverse effect on the heart for radiological and endoscopic examination in the upper gastrointestinal tract.

Topics: Animals; Atropine; Butylscopolammonium Bromide; Dioxoles; Gastric Emptying; Gastrointestinal Motility; Glucagon; Male; Muscarinic Antagonists; Muscle, Smooth; Piperidines; Pirenzepine; Polymerase Chain Reaction; Rats; Receptors, Muscarinic; Stomach

1. The effects of muscarinic antagonists on cationic current evoked by activating muscarinic receptors with the stable agonist carbachol were studied by use of patch-clamp recording techniques in guinea-pig single ileal smooth muscle cells. 2. Ascending concentrations of carbachol (3-300 microM) activated the cationic conductance in a concentration-dependent manner with conductance at a maximally effective carbachol concentration (Gmax) of 27.4+/-1.4 nS and a mean -log EC50 of 5.12+/-0.03 (mean+/-s.e.mean) (n=114). 3. Muscarinic antagonists with higher affinity for the M2 receptor, methoctramine, himbacine and tripitramine, produced a parallel shift of the carbachol concentration-effect curve to the right in a concentration-dependent manner with pA2 values of 8.1, 8.0 and 9.1, respectively. 4. All M3 selective muscarinic antagonists tested, 4-DAMP, p-F-HHSiD and zamifenacin, reduced the maximal response in a concentration-dependent and non-competitive manner. This effect could be observed even at concentrations which did not produce any increase in the EC50 for carbachol. At higher concentrations M3 antagonists shifted the agonist curve to the right, increasing the EC50, and depressed the maximum conductance response. Atropine, a non-selective antagonist, produced both reduction in Gmax (M3 effect) and significant increase in the EC50 (M2 effect) in the same concentration range. 5. The depression of the conductance by 4-DAMP, zamifenacin and atropine could not be explained by channel block as cationic current evoked by adding GTPgammaS to the pipette (without application of carbachol) was unaffected. 6. The results support the hypothesis that carbachol activates M2 muscarinic receptors so initiating the opening of cationic channels which cause depolarization; this effect is potentiated by an unknown mechanism when carbachol activates M3 receptors. As an increasing fraction of M3 receptors are blocked by an antagonist, the effects on cationic current of an increasing proportion of activated M2 receptors are disabled.

Topics: Animals; Carbachol; Dioxoles; Dose-Response Relationship, Drug; Electric Conductivity; Guinea Pigs; Ileum; In Vitro Techniques; Ion Channels; Male; Muscarinic Agonists; Muscarinic Antagonists; Muscle, Smooth; Patch-Clamp Techniques; Piperidines; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Muscarinic

1. Zamifenacin was rapidly metabolized in vitro by liver microsomes from rat, dog, and man. 2. Zamifenacin exhibited extensive plasma protein binding with human plasma showing 20 and 10-fold higher binding that that in rat and dog respectively. 3. Following oral administration to animals, metabolic clearance resulted in decreased bioavailability due to first-pass metabolism in rat and mouse. Oral clearance in man was low as a result of increased metabolic stability and increased plasma protein binding compared with animals. 4. Metabolism was the major route of clearance of zamifenacin with the primary metabolic step resulting in opening of the methylenedioxy ring to yield the catechol. In man, this metabolite was excreted as the glucuronide conjugate, whereas in the animal species it was further metabolized by mono-methylation of the catechol.

Topics: Animals; Biological Availability; Blood Proteins; Dioxoles; Dogs; Feces; Glucuronates; Humans; Hydroxylation; Mice; Microsomes, Liver; Muscarinic Antagonists; Piperidines; Protein Binding; Rats; Urine

The interaction of zamifenacin ((3R)-(+)-diphenylmethoxy-1-(3,4)-methylenedioxyphenethyl)pi peridine) at muscarinic receptor subtypes was studied using radioligand binding and functional techniques, in vitro. In radioligand binding studies, zamifenacin acted as a competitive antagonist, with the following pKi values; rat cerebral cortex (M1) 7.90 +/- 0.08, myocardium (M2) 7.93 +/- 0.13, submaxillary gland (M3) 8.52 +/- 0.04 and rabbit lung (M4) 7.78 +/- 0.04. In functional studies zamifenacin acted as a surmountable antagonist, exhibiting the following apparent affinity values; canine saphenous vein (putative M1) 7.93 +/- 0.09, guinea-pig left atria (M2) 6.60 +/- 0.04, guinea-pig ileum (M3) 9.31 +/- 0.06, guinea-pig oesophageal muscularis mucosae (M3) 8.84 +/- 0.04, guinea-pig trachea (M3) 8.16 +/- 0.04, and guinea-pig urinary bladder (M3) 7.57 +/- 0.15. Therefore, zamifenacin is selective for muscarinic M3 receptors in guinea-pig ileum, oesophageal muscularis mucosae, trachea and bladder over muscarinic M2 receptors in atria. The degree of muscarinic M3/M2 receptor selectivity depends upon the muscarinic M3 receptor preparation studied.

Topics: Animals; Dioxolanes; Dioxoles; Dogs; Female; Guinea Pigs; In Vitro Techniques; Kinetics; Male; Muscarinic Agonists; Muscarinic Antagonists; Muscle, Smooth; Muscle, Smooth, Vascular; Piperidines; Rabbits; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic

1. 4-Diphenylacetoxy-N-cyclohexylmethyl-piperidine HCl (hexahydro-benz-4DAP) is more active as an antagonist of carbachol at receptors in guinea-pig isolated ileum, log K (pA2) = 6.64 +/- 0.14 (s.e. 7 results), than at receptors in guinea-pig isolated atria, log K = 5.43 +/- 0.14 (7). In the presence of neostigmine bromide (0.2 microM) the value for atria was 5.62 +/- 0.19 (4), so the lower activity on atria cannot be attributed to hydrolysis of the compound by cholinesterases present in this tissue. 2. The limit of solubility of the free base in Krebs solution (pH 7.6) is about 50 microM for both hexahydrobenz-4DAP and benzyl-fourdapine (benz-4DAP). 3. In experiments on guinea-pig isolated ileum with hexahydro-benz-4DAP given together with 4-DAMP methobromide, the combined dose-ratio was consistent with competition: similar results were obtained with benz-4DAP. 4. In rats anaesthetized with pentobarbitone, hexahydro-benz-4DAP antagonized the effects of bethanechol on blood-pressure in doses that had little effect on heart rate or airflow. There was a limit to the effect which could be obtained, however, and the slopes of the Schild plots were less than one. The effects on rat blood-pressure had a half-life of at least 30 min. 5. In similar experiments with zamifenacin the slopes of the Schild plots were close to 1 and the compound was 10 to 20 times as active on blood-pressure at it was on heart-rate. 6. The limited solubility of the base probably accounts for the flat Schild plots obtained with hexahydro-benz-4DAP, which had about 10 fold selectivity for effects on blood-pressure and was more active than expected from tests on isolated ileum.

Topics: Animals; Artifacts; Blood Pressure; Dioxoles; Diphenylacetic Acids; Guinea Pigs; Heart Atria; Heart Rate; Hirudins; Ileum; In Vitro Techniques; Molecular Structure; Muscarinic Antagonists; Piperidines; Rats; Recombinant Proteins