The design of novel methoctramine-related tetraamines as muscarinic receptor subtype selective antagonists

Several novel methoctramine-related tetraamines were designed, and their biological profiles at muscarinic receptor subtypes were assessed by functional experiments in isolated guinea pig and rat atria (M2) and smooth muscle (ileum and trachea, M3) and by binding assays in rat cortex (M1), heart (M2), and submaxillary gland (M3) homogenates and NG 108-15 cells (M4). Tripitramine, a nonsymmetrical tetraamine, resulted in the most potent and the most selective muscarinic M2 receptor antagonist of the series (pA2 = 9.14-9.85; pKi = 9.54). Spirotramine (FC 15-94), a symmetrical tetraamine, was able to differentiate between muscarinic M1 receptors (pKi = 7.88) and the other subtypes (M2, pKi = 6.20; M3, pKi = 5.81; M4, pKi = 6.27). Thus, tripitramine and spirotramine could be valuable tools for the pharmacological classification and characterization of muscarinic receptor subtypes.     

Muscarinic receptor subtypes in bovine adrenal medulla

Catecholamine secretion in the bovine adrenal medulla is evoked largely by nicotinic receptor activation. However, bovine adrenal medulla also contain muscarinic receptors that mediate several cell responses. To understand the physiological role of muscarinic receptors in the bovine adrenal medulla it is important to identify the pharmacological subtypes present in this tissue. For this, we analyzed the abilities of differnt selective muscarinic antagonists in displacing the binding of the non-selective antagonist [3H] quinuclidinyl benzylate to an enriched plasma membrane fraction prepared from bovine adrenal medulla. All the selective antagonists bind at least two bindings sites with different affinities. The binding profile of the sites with high proportion is similar to the M2 subtype and those present in low proportion have a M1 profile. However, some variation in the proportion of the sites for the different ligands suggest the presence of the third pharmacological subtype (M3). We conclude that the sites in high proportion (60–80%) correspond to M2 muscarinic subtypes, and the rest is constitute by M1 plus M3 subtypes. The presence of multiplicity of subtypes in the adrenal medulla membranes suggests a diversity of functions of muscarinic receptors in the adrenal gland.Abbreviations [3H]QNB [3H]quinuclidinyl benzylate - HHSiD hexahydro-siladifenidol-hydrochloride - AF-DX 116 11-[[2-(diethylamino)methyl]]-1-piperidinyl]-5,11-dihydro-6H-pyrido[2,3,-b][1,4]benzodiazepin-6-one - 4-DAMP 4-diphenylacetoxy-N-methyl piperidine methobromide     

Structure-activity relationships of methoctramine-related polyamines as muscarinic antagonist: effect of replacing the inner polymethylene chain with cyclic moieties

The aim of the present paper was to investigate the role of the octamethylene spacer of methoctramine (1) on the biological profile. Thus, this spacer was incorporated into a dianiline or dipiperidine moiety to determine whether flexibility and the basicity of the inner nitrogen atoms are important determinants of potency with respect to muscarinic receptors. The most potent compound was 4, which displayed, in the functional assays, a comparable potency at muscarinic M(2) receptors with respect to 1, and, in the binding assays, a loss of potency and selectivity toward muscarinic M(1) and M(3) receptor subtypes. Both compounds were endowed with antinociceptive activity. Furthermore, in microdialysis tests in rat parietal cortex, they enhanced acetylcholine release, most likely by antagonizing presynaptic muscarinic receptor subtypes.     

Snake toxins with high selectivity for subtypes of muscarinic acetylcholine receptors

There are five subtypes of muscarinic acetylcholine receptors (M(1) to M(5)) which control a large number of physiological processes, such as the function of heart and smooth muscles, glandular secretion, release of neurotransmitters, gene expression and cognitive functions as learning and memory. A selective ligand is very useful for studying the function of a subtype in presence of other subtypes, which is the most common situation, since a cell or an organ usually has several subtypes. There are many non-selective muscarinic ligands, but only few selective ones. Mambas, African snakes of genus Dendroaspis have toxins, muscarinic toxins, that are selective for M(1), M(2) and M(4) receptors. They consist of 63-66 amino acids and four disulfides which form four loops. They are members of a large group of snake toxins, three-finger toxins; three loops are extended like the middle fingers of a hand and the disulfides and the shortest loop are in the palm of the hand. Some of the toxins target the allosteric site which is located in a cleft of the receptor molecule close to its extracellular part. A possible explanation to the good selectivity is that the toxins bind to the allosteric site, but because of their size they probably also bind to extracellular parts of the receptors which are rather different in the various subtypes. Some other allosteric ligands also have good selectivity, the alkaloid brucine and derivatives are selective for M(1), M(3) and M(4) receptors. Muscarinic toxins have been used in several types of experiments. For instance radioactively labeled M(1) and M(4) selective toxins were used in autoradiography of hippocampus from Alzheimer patients. One significant change in the receptor content was detected in one region of the hippocampus, dentate gyrus, where M(4) receptors were reduced by 50% in patients as compared to age-matched controls. Hippocampus is essential for memory consolidation. M(4) receptors in dentate gyrus may play a role, since they decreased in Alzheimers disease which destroys the memory. Another indication of the role of M(4) receptors for memory is that injection of the M(4) selective antagonist muscarinic toxin 3 (M(4)-toxin 1) into rat hippocampus produced amnesia.     

Subacute reserpine treatment reveals preferential coupling between the M3 muscarinic receptor subtype and phosphatidylinositol turnover.

Muscarinic receptors in the rat cerebral cortex, cardiac atria and vas deferens were identified, quantitated, and characterized relative to phosphatidylinositol (PI) turnover as the functional response to stimulation of specific receptor subtypes. Receptor densities as determined by 3H-QNB binding were ranked: cerebral cortex greater than vas deferens greater than heart. Using displacement of 3H-QNB binding by the selective M1 and M2 muscarinic receptor antagonists pirenzepine and 11[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro- 6H-pyrido [2,3-b] [1,4] benzodiazepine-6-one (AF-DX 116) respectively, heterogeneous populations were found in the cerebral cortex and vas deferens. The M1 receptor subtype predominated in the former and the M2 predominated in the latter. An homogeneous M2 receptor population was present in the heart. Methacholine-stimulated accumulation of 3H inositol-1-phosphate was greater in the vas deferens than in the cerebral cortex, whereas PI turnover was not enhanced in cardiac atria. Reserpine treatment of rats (0.5 mg kg-1 day-1 for 7 days) increased muscarinic receptor density in the vas deferens coincident with a shift in the low affinity pKi for AF-DX 116 to a value comparable to high affinity binding, and abolished the enhanced PI hydrolysis. In the cerebral cortex, reserpine treatment shifted only the early portion of the methacholine dose-response curve to the right. These results are judged to be supportive of preferential coupling between the M3 muscarinic receptor subtype and PI turnover.     

Different antagonist binding properties of rat pancreatic and cardiac muscarinic receptors

The antagonist binding properties of rat pancreatic and cardiac muscarinic receptors were compared. In both tissues pirenzepine (PZ) had a low affinity for muscarinic receptors labelled by (3H)N-methylscopolamine [3)NMS) (KD values of 140 and 280 nM, respectively, in pancreatic and cardiac homogenates). The binding properties of pancreatic and cardiac receptors were, however, markedly different. This was indicated by different affinities for dicyclomine, (11-([(2-[diethylamino)-methyl)-1-piperidinyl] acetyl)-5, 11-dihydro-6H-pyrido(2,3-b)(1,4) benzodiazepin-6-on) (AFDX-116), 4-diphenylacetoxy-N-methyl-piperidine methobromide (4-DAMP) and hexahydrosiladifenidol (HHSiD). Pancreatic and cardiac muscarinic receptors also showed different (3H)NMS association and dissociation rates. These results support the concept of M2 receptor heterogeneity and confirm that M2 receptor subtypes have different binding kinetic properties.     

Synthesis and pharmacological investigation of stereoisomeric muscarines.

The synthesis of the eight stereoisomers of muscarine has been efficiently accomplished by utilizing the two enantiomers of lactic esters as starting material. The synthetic strategy is based on a SnCl4-catalyzed addition of allyltrimethylsilane to O-protected lactic aldehydes followed by an iodocyclization process. All the final derivatives possess an enantiomeric excess higher than 98%. The four pairs of enantiomers bound to M1, M2, and M3 muscarinic receptor subtypes in membranes from cerebral cortex, heart, and salivary glands, respectively, and recognized heterogeneous states of the receptors. Of the eight isomers, only natural muscarine (+)-1 recognized three affinity states of the M2 receptor. The compound was also the only one to show selectivity in the binding study, demonstrating 37- to 44-fold higher affinity for the M2 than for the M1 or M3 receptors. In addition, the compounds were tested in functional assays on isolated guinea pig atria (M2 receptors) and ileum (mixed population of M2 and M3 receptors) and their muscarinic potencies were determined. Among the eight isomers, again only (+)-1 enantiomer was found to be very active on both tissues. Its potency was more than two orders of magnitude higher than that of its enantiomer (-)-1 as well as the other six isomers. The eudismic ratios (E.R.) deduced from the two functional tests were 324 and 331.     

4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]-trimethylammonium (McN-A-343)-related compounds. Effect of the butynyl chain inclusion into an aromatic unit on the potency for muscarinic receptors

A series of derivatives of the known M1 selective muscarinic receptor agonist McN-A-343 (1) was designed with the aim of investigating the effects of structural variations on both the butynyl chain and the phenyl ring of 1. The butynyl chain was replaced with an aromatic spacer, and the effects of such a modification on the stereoelectronic properties of the molecules were theoretically studied and considered compatible with muscarinic receptor affinity. Substituents on the phenyl ring of 1 were selected so as to vary their electronic and hydrophobic properties. This design strategy did not produce muscarinic M1 receptor agonists more potent than the prototype 1, even if some analogues displayed functional selectivity for different muscarinic receptor subtypes. Compounds 3 and 7 were selective agonists towards muscarinic M3 receptors, while compounds 14, 16 and 18 were selective muscarinic M2 receptor agonists. The most interesting derivative was 8, a full agonist at muscarinic M3 receptors devoid of activity at both muscarinic M1 and M2 subtypes. The pharmacological profile of the series was further characterized by studying the anticholinesterase and miotic activities of some representative compounds. Compounds 3-8 turned out to be weak acetylcholinesterase inhibitors, while derivatives 4, 6, 8 and 11 were able to significantly reduce the pupillary diameter in rabbit, indicating 8 as an effective miotic agent.     

毒蕈乙酰胆碱受体亚型关系的研究

利用不同类型的生物学数据, 运用生物信息学方法和策略, 从分子进化、序列相似性、表达相关性以及蛋白互作4个层面对M受体亚型之间的关系进行了比较全面的研究。分析表明, 从分子进化和序列相似性角度,毒蕈乙酰胆碱受体5种亚型可分为2个亚类,分别为M1、M3、M5亚类(第一亚类)与M2、M4亚类(第二亚类),每一亚类内部亚型之间进化距离相对较近, 序列相似性较高。在表达层面发现第一亚类中受体亚型与第二亚类中受体亚型在某些组织中正表达相关, 呈现共表达趋势。在互作层面发现两亚类之间受体亚型存在着间接互作的关系, 呈现协同作用的现象。     

M1 and M3 muscarinic antagonists inhibit human nasal glandular secretion in vitro.

Mucus glycoproteins (MGP) are high-molecular-weight glycoconjugates that are released from submucosal glands and epithelial goblet cells in the respiratory tract. Muscarinic receptors have an important role in the regulation of human nasal glandular secretion and mucus production, but it is not known which of the five muscarinic receptor subtypes are involved. The effect of nonselective and M1-, M2-, and M3-selective muscarinic antagonists on methacholine (MCh)-induced MGP secretion from human nasal mucosal explants was tested in vitro. MGP was assayed by enzyme-linked immunosorbent assay using a specific anti-MGP monoclonal antibody (7F10). MCh (100 microM) induced MGP secretion up to 127% compared with controls. MCh-induced MGP release was significantly inhibited by atropine (100 microM), the M, receptor antagonist pirenzepine (10-100 microM), and the M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; 1-100 microM). 4-DAMP significantly inhibited MCh-induced MGP release at a lower concentration (1 microM) than pirenzepine (10 microM). The M2 receptor antagonists AF-DX 116 and gallamine (both at 100 microM) had no effect. No antagonist alone had a significant effect on MGP release. These results indicate that the M1 and M3 muscarinic receptor subtypes regulate MGP secretion from human nasal mucosa and suggest that the M3 receptor has the predominant effect.     

Muscarinic receptor subtypes mediating central and peripheral antinociception studied with muscarinic receptor knockout mice: a review

To gain new insight into the physiological and pathophysiological roles of the muscarinic cholinergic system, we generated mutant mouse strains deficient in each of the five muscarinic acetylcholine receptor subtypes (M(1)-M(5)). In this chapter, we review a set of recent studies dealing with the identification of the muscarinic receptor subtypes mediating muscarinic agonist-dependent analgesic effects by central and peripheral mechanisms. Most of these studies were carried out with mutant mouse strains lacking M(2) or/and M(4) muscarinic receptors. It is well known that administration of centrally active muscarinic agonists induces pronounced analgesic effects. To identify the muscarinic receptors mediating this activity, wild-type and muscarinic receptor mutant mice were injected with the non-subtype-selective muscarinic agonist, oxotremorine (s.c., i.t., and i.c.v.), and analgesic effects were assessed in the tail-flick and hot-plate tests. These studies showed that M(2) receptors play a key role in mediating the analgesic effects of oxotremorine, both at the spinal and supraspinal level. However, studies with M(2)/M(4) receptor double KO mice indicated that M(4) receptors also contribute to this activity. Recent evidence suggests that activation of muscarinic receptors located in the skin can reduce the sensitivity of peripheral nociceptors. Electrophysiological and neurochemical studies with skin preparations from muscarinic receptor mutant mice indicated that muscarine-induced peripheral antinociception is mediated by M(2) receptors. Since acetylcholine is synthesized and released by different cell types of the skin, it is possible that non-neuronally released acetylcholine plays a role in modulating peripheral nociception. Our results highlight the usefulness of muscarinic receptor mutant mice to shed light on the functional roles of acetylcholine released from both neuronal and non-neuronal cells.     

Structure-activity relationships in 2,2-diphenyl-2-ethylthioacetic acid esters: unexpected agonistic activity in a series of muscarinic antagonists

As a continuation of previous research on anticholinergic drugs derived from 2,2-diphenyl-2-ethylthioacetic acid, several 5,5-diphenyl-5-ethylthio-2-pentynamines (2-11) were synthetised and their antimuscarinic activity on M(1-4) receptor subtypes was evaluated by functional tests and binding experiments. One of the compounds obtained showed unexpected agonistic activity in functional experiments on M(2) receptors. Since the compound carried a phenylpiperazine moiety, other similar compounds (12-17) were prepared and found to be endowed with similar behaviour. These ligands, although possessing the bulky structure characterising muscarinic antagonists, display agonistic activity at M(2) subtypes while, as expected, behaving as antagonists on M(3) and M(4) subtypes. On M(1) subtypes, they show agonistic activity which, however, is not blocked by atropine. The peculiar pharmacological profile of these compounds is of interest for studying muscarinic receptor subtypes.     

Stereoselectivity at muscarinic receptor subtypes: observations with the enantiomers of phenglutarimide

The affinity of the enantiomers of phenglutarimide at three muscarinic receptor subtypes was examined in vitro using field-stimulated rabbit vas deferens (M1 receptors) and guinea pig atria (M2 alpha receptors) and ileum (M2 beta receptors). Extremely high stereoselectivity was observed and higher affinities (up to 6000-fold) were found for the (+)-S-enantiomer. The stereoselectivity ratios were different at the three subtypes, and the stereochemical demands made by the muscarinic receptors were most stringent at M1 receptors. (+)-(S)-Phenglutarimide was found to be a potent M1-selective antagonist (pA2 at M1 = 8.53). Its receptor selectivity profile is qualitatively similar to that of pirenzepine. (-)-(R)-Phenglutarimide showed no comparable discriminatory properties.     

Design, synthesis, and biological evaluation of pirenzepine analogs bearing a 1,2-cyclohexanediamine and perhydroquinoxaline units in exchange for the piperazine ring as antimuscarinics

Pirenzepine (2) is one of the most selective muscarinic M(1) versus M(2) receptor antagonists known. A series of 2 analogs, in which the piperazyl moiety was replaced by a cis- and trans-cyclohexane-1,2-diamine (3-6) or a trans- and cis-perhydroquinoxaline rings (7 and 8) were prepared, with the aim to investigate the role of the piperazine ring of 2 in the interaction with the muscarinic receptors. The structural change leading to compounds 3-6 abolished in binding assays the muscarinic M(1)/M(2) selectivity of 2, due to an increased M(2) affinity. Rather, compounds 3-6 displayed a reversed selectivity showing more affinity at the muscarinic M(2) receptor than at all the other subtypes tested.     

Muscarinic receptor subtypes modulating smooth muscle contractility in the urinary bladder

Normal physiological voiding as well as generation of abnormal bladder contractions in diseased states is critically dependent on acetylcholine-induced stimulation of contractile muscarinic receptors on the smooth muscle (detrusor) of the urinary bladder. Muscarinic receptor antagonists are efficacious in treating the symptoms of bladder hyperactivity, such as urge incontinence, although the usefulness of available drugs is limited by undesirable side-effects. Detrusor smooth muscle is endowed principally with M2 and M3 muscarinic receptors with the former predominating in number. M3 muscarinic receptors, coupled to stimulation of phosphoinositide turnover, mediate the direct contractile effects of acetylcholine in the detrusor. Emerging evidence suggests that M2 muscarinic receptors, via inhibition of adenylyl cyclase, cause smooth muscle contraction indirectly by inhibiting sympathetically (beta-adrenoceptor)-mediated relaxation. In certain diseased states, M2 receptors may also contribute to direct smooth muscle contraction. Other contractile mechanisms involving M2 muscarinic receptors, such as activation of a non-specific cationic channel and inactivation of potassium channels, may also be operative in the bladder and requires further investigation. From a therapeutic standpoint, combined blockade of M2 and M3 muscarinic receptors would seem to be ideal since this approach would evoke complete inhibition of cholinergically-evoked smooth muscle contractions. However, if either the M2 or M3 receptor assumes a greater pathophysiological role in disease states, then selective antagonism of only one of the two receptors may be the more rational approach. The ultimate therapeutic strategy is also influenced by the extent to which pre-junctional M1 facilitatory and M2 inhibitory muscarinic receptors regulate acetylcholine release and also which subtypes mediate the undesirable effects of muscarinic receptor blockade such as dry mouth. Finally, the consequence of muscarinic receptor blockade in the central nervous system on the micturition reflex, an issue which is poorly studied and seldom taken into consideration, should not be ignored.     

Importance and prospects for design of selective muscarinic agonists

There are five subtypes of muscarinic receptors that serve various important physiological functions in the central nervous system and the periphery. Mental functions like attention, learning, and memory are attributed to the muscarinic M1 subtype. These functions decline during natural aging and an early deficit is typical for Alzheimer s disease. In addition, stimulation of the M1 receptor increases non-amyloidogenic processing of the amyloid precursor protein and thus prevents accumulation of noxious beta-amyloid fragments. The selectivity of classical muscarinic agonists among receptor subtypes is very low due to the highly conserved nature of the orthosteric binding site among receptor subtypes. Herein we summarize some recent studies with the functionally-selective M1 agonist xanomeline that indicate complex pharmacological profile of this drug that includes interactions with and activation of receptor from both orthosteric and ectopic binding sites, and the time-dependent changes of ligand binding and receptor activation. These findings point to potential profitability of exploitation of ectopic ligands in the search for truly selective muscarinic receptor agonists.     

Muscarinic receptor subtypes mediate stimulatory and paradoxical inhibitory effects on an insulin-secreting beta cell line

Acetylcholine (ACh), a major neurotransmitter from the autonomic nervous system, regulates the cholinergic stimulation of insulin secretion, through interactions with muscarinic receptors. The present study has characterised the individual involvement of muscarinic receptor subtypes in ACh-induced insulin secretion, using clonal beta cells and selective muscarinic receptor antagonists. BRIN BD11 cells clearly expressed mRNA encoding m1--m4 whereas m5 was not detected by RT-PCR. Insulin release was measured from BRIN BD11 cells treated with ACh in the presence of muscarinic receptor antagonists at concentrations ranging from 3 nM to 1 microM. 300 nM of muscarinic toxin-3 (M4 antagonist) and 1 microM of methoctramine (M2 antagonist) increased ACh (100 microM) stimulated insulin secretion by 168% and 50% respectively (ANOVA, P<0.05). The antagonists alone had no effect on insulin secretion. In contrast, 300 nM of pirenzepine (M1 antagonist) and 30 nM of hexahydro-sila-difenidol p-fluorohydrochloride (M3 antagonist) inhibited ACh stimulation by 91% and 84% respectively (ANOVA, P<0.01). It is concluded that ACh acts on different receptor subtypes producing both a stimulatory and an inhibitory action on insulin release.     

Muscarinic receptor subtypes mediate stimulatory and paradoxical inhibitory effects on an insulin-secreting β cell line

Acetylcholine (ACh), a major neurotransmitter from the autonomic nervous system, regulates the cholinergic stimulation of insulin secretion, through interactions with muscarinic receptors. The present study has characterised the individual involvement of muscarinic receptor subtypes in ACh-induced insulin secretion, using clonal β cells and selective muscarinic receptor antagonists. BRIN BD11 cells clearly expressed mRNA encoding m1–m4 whereas m5 was not detected by RT-PCR. Insulin release was measured from BRIN BD11 cells treated with ACh in the presence of muscarinic receptor antagonists at concentrations ranging from 3 nM to 1 μM. 300 nM of muscarinic toxin-3 (M4 antagonist) and 1 μM of methoctramine (M2 antagonist) increased ACh (100 μM) stimulated insulin secretion by 168% and 50% respectively (ANOVA, P<0.05). The antagonists alone had no effect on insulin secretion. In contrast, 300 nM of pirenzepine (M1 antagonist) and 30 nM of hexahydro-sila-difenidol p-fluorohydrochloride (M3 antagonist) inhibited ACh stimulation by 91% and 84% respectively (ANOVA, P<0.01). It is concluded that ACh acts on different receptor subtypes producing both a stimulatory and an inhibitory action on insulin release.