Chemical modification-mediated optimisation of bronchodilatory activity of mepenzolate,a muscarinic receptor antagonist with anti-inflammatory activity

The treatment for patients with chronic obstructive pulmonary disease (COPD) usually involves a combination of anti-inflammatory and bronchodilatory drugs. We recently found that mepenzolate bromide (1) and its derivative, 3-(2-hydroxy-2, 2-diphenylacetoxy)-1-(3-phenoxypropyl)-1-azoniabicyclo[2.2.2]octane bromide (5), have both anti-inflammatory and bronchodilatory activities. We chemically modified 5 with a view to obtain derivatives with both anti-inflammatory and longer-lasting bronchodilatory activities. Among the synthesized compounds, (R)-(–)-12 ((R)-3-(2-hydroxy-2,2-diphenylacetoxy)-1-(3-phenylpropyl)-1-azoniabicyclo[2.2.2]octane bromide) showed the highest affinity in vitro for the human muscarinic M3 receptor (hM₃R). Compared to 1 and 5, (R)-(–)-12 exhibited longer-lasting bronchodilatory activity and equivalent anti-inflammatory effect in mice. The long-term intratracheal administration of (R)-(–)-12 suppressed porcine pancreatic elastase-induced pulmonary emphysema in mice, whereas the same procedure with a long-acting muscarinic antagonist used clinically (tiotropium bromide) did not. These results suggest that (R)-(–)-12 might be therapeutically beneficial for use with COPD patients given the improved effects seen against both inflammatory pulmonary emphysema and airflow limitation in this animal model.     

Novel oxotremorine-related heterocyclic derivatives: Synthesis and in vitro pharmacology at the muscarinic receptor subtypes

A set of novel heterocyclic ligands (6–27) structurally related to Oxotremorine 2 was designed, synthesized and tested at muscarinic receptor subtypes (mAChRs). In the binding experiments at cloned human receptors (hm1–5), compounds 7 and 15 evidenced a remarkable affinity and selectivity for the hm2 subtype. The in vitro functional assays, performed on a selected group of derivatives at M₁, M₂, and M₃ tissue preparations, singled out the 3-butynyloxy-5-methylisoxazole trimethylammonium salt 7 as a potent unselective muscarinic agonist [pEC₅₀: 7.40 (M₁), 8.18 (M₂), and 8.14 (M₃)], whereas its 5-phenyl analogue 12 behaved as a muscarinic antagonist, slightly selective for the M₁ subtype [pK_B: 6.88 (M₁), 5.95 (M₂), 5.53 (M₃)]. Moreover, the functional data put in evidence that the presence of the piperidine ring may generate a functional selectivity, e.g., an M₁ antagonist/M₂ partial agonist/M₃ full agonist profile (compound 21), at variance with the corresponding quaternary ammonium salt (compound 22) which behaved as a muscarinic agonist at all M_1–3 receptors, with an appreciable selectivity for the cardiac M₂ receptors.     

The striatum and cerebral cortex express different muscarinic receptor mRNAs

The existence of four distinct muscarinic acetylcholine receptor genes (m1 – m4) has recently been demonstrated. cDNAs for three of these receptors have been cloned from brain (m1, m3, m4) and one from heart (m2). To gain some understanding of the physiological role of the brain muscarinic receptors, we mapped the distribution of their mRNAs in rat brain by in situ hybridization. These mRNAs are barely detectable in the hindbrain and cerebellum. Within forebrain, each mRNA has a strikingly different pattern of distribution. The highest levels of m1 mRNA are in the cerebral cortex and hippocampus followed by the striatum. m3 mRNA is also prominent in the cerebral cortex, but has very low levels in the striatum. Conversely, the levels of m4 mRNA are highest in the striatum. Since the cognitive effects of muscarinic drugs have been localized to the cerebral cortex and hippocampus, and their psychomotor effects to the striatum, these data suggest that the muscarinic receptors which subserve these responses may be different gene products. Finally, we show that these muscarinic receptors can be distinguished pharmacologically, suggesting that it may be possible to develop drugs for the selective treatment of the psychomotor vs cognitive difficulties of Parkinson's and Alzheimer's disease, respectively.     

The development of the muscarinic acetylcholine receptor in normal and hyperphenylalaninemic rat cerebrum

The effect of hyperphenylalaninemia on the development of the muscarinic acetylcholine receptor in rat cerebrum has been studied. Rats were subjected to the hyperphenylalaninemic regimen as of 5 days of age. A gradual and steady decrease in the number of binding sites forl-[³H]quinuclidinylbenzilate was observed, with the white matter more affected than the gray matter. A return to normal blood phenylalanine levels after the age of 21 days does not lead to an increase in this number of binding sites.     

Evaluation of 1,2,5-thiadiazoles as modulators of M1/M5 muscarinic receptor subtypes

Studies have demonstrated the presence of allosteric binding sites on each of the muscarinic acetylcholine receptor (mAChR) subtypes. Since most drugs targeting muscarinic receptors bind to the highly conserved orthosteric binding site, they fail to achieve appreciable subtype selectivity. Targeting non-conserved allosteric sites may provide a new way of enhancing selectivity for individual subtypes of muscarinic receptor. Tetra(ethyleneglycol)(3-methoxy-1,2,5-thiadiazol-4-yl)[3-(1-methyl-1,2,5,6-tetrahydropyrid-3-yl)-1,2,5-thiadiazol-4-yl] ether, CDD-0304 (10), was found to be a M_1/2/4 selective muscarinic agonist and might prove useful in treating the symptoms associated with schizophrenia (J. Med. Chem. 2003, 46, 4273). It was hypothesized that the observed subtype selectivity demonstrated by 10 may be due to its ability to function as a bitopic ligand (J. Med. Chem. 2006, 49, 7518). To further investigate this possibility, a novel series of compounds was synthesized using a 1,2,5-thiadiazole moiety along with varying lengths of a polyethylene glycol linker and terminal groups, for evaluation as potential allosteric modulators of muscarinic receptors. Preliminary biological studies were performed using carbachol to stimulate M₁ and M₅ receptors. No significant agonist activity was observed at either M₁ or M₅ receptors for any of the compounds. Compound 18, 2-(4-methoxy-1,2,5-thiadiazol-3-yloxy)-N,N-dimethylethanamine fumarate (CDD-0361F) was found to block the effects of carbachol at M₅ muscarinic receptors.     

Aceclidine and pilocarpine interact differently with muscarinic receptor in isolated rabbit iris muscle

The relationship between muscarinic receptor affinity states and the contractile response to the muscarinic agonists carbachol, aceclidine, and pilocarpine, has been examined in the isolated rabbit iris muscle. Contraction of the iris muscle by carbachol and aceclidine was more potent and/or more efficacious than the response to pilocarpine. Analysis of [³H]-Quinuclidinyl benzilate (QNB) binding showed that while both carbachol and aceclidine bound to high- and low-affinity forms of the muscarinic receptor, pilocarpine bound to one affinity state. The efficacy of carbachol and aceclidine to stimulate contraction of the iris muscle was consistent with receptor occupancy theory only when considering the low-affinity state of the muscarinic receptor, and activation of the low-affinity rather than high-affinity binding state of the receptor is likely to mediate the contraction of iris muscle. Therefore, the typical anti-glaucoma muscarinic agonists aceclidine and pilocarpine may interact differently with their target receptors in isolated rabbit iris muscle.     

Discovery of N-sulfonyl-7-azaindoline derivatives as potent,orally available and selective M4 muscarinic acetylcholine receptor agonists

We designed and synthesized novel N-sulfonyl-7-azaindoline derivatives as selective M₄ muscarinic acetylcholine receptor agonists. Modification of the N-carbethoxy piperidine moiety of compound 2, an M₄ muscarinic acetylcholine receptor (mAChR)-preferring agonist, led to compound 1, a selective M₄ mAChR agonist. Compound 1 showed a highly selective M₄ mAChR agonistic activity with weak hERG inhibition in vitro. A pharmacokinetic study of compound 1 in vivo revealed good bioavailability and brain penetration in rats. Compound 1 reversed methamphetamine-induced locomotor hyperactivity in rats (1–10 mg/kg, po).     

Fluidity of detergent micelles plays an important role in muscarinic receptor solubilization

In order to find a suitable reagent for extracting the muscarinic receptor from rat brain membranes 14 different detergents were tested. Only the plant glycoside digitonin efficiently solubilized the receptor protein in its native form. At the same time microviscosity of detergent micelles was determined by measuring the fluorescence polarization of a hydrophobic fluorescent probe diphenylhexatriene incorporated into the micelles. In the case of digitonin the polarization value was close to the corresponding value obtained for rat brain membrane fragments, while for the other detergents studied it remained considerably lower. The results obtained indicate that the fluidity of detergent micelles may play an important role in retaining the active conformation of the solubilized muscarinic receptor.     

Isolation and functional characterization of the chick M5 muscarinic acetylcholine receptor gene

The chick is a widely used system for study of the actions of muscarinic acetylcholine receptors in the cardiovascular, visual, and nervous systems. We report the isolation and functional analysis of the gene encoding the chick M5 muscarinic receptor. RT-PCR analysis indicates that the M5 receptor is expressed at low levels in embryonic chick brain and heart. When expressed in stably transfected Chinese hamster ovary cells, the M5 receptor exhibits high-affinity binding to muscarinic antagonists and mediates robust activation of phospholipase C activity.     

Mutation screening of the muscarinic m2 and m3 receptor genes in asthmatics,outgrow subjects,and normal controls

Muscarinic receptors are important in the development of airway hyperresponsiveness. In some patients with asthma and in animal models of hyperreactivity, functional abnormalities in these receptors are suggested to contribute to disease. Here, we have screened for single nucleotide polymorphisms in the coding region of human muscarinic m2 and m3 receptor genes using direct fluorescence sequencing. DNA samples from 102 current asthmatics and 58 who had outgrown asthma ("outgrow" patients) were compared with 70 random non-asthmatic controls. A mutation characterized by a single base substitution (A1050G, Ser350Ser) was identified in the muscarinic m2 receptor gene. This polymorphism was common and was represented in all three groups studied. In contrast, in the m3 receptor coding region examined, we found a very rare nucleotide variant (C261T, Ile87Ile), identified in only one of the 230 samples genotyped. Therefore, neither A1050G in the m2 receptor nor C261T in the m3 receptor is likely to be functionally significant for airway hyperresponsiveness in asthma. Our data suggest that both the m2 and m3 receptor genes are highly conserved, and no significant genetic mutations are related to their possible functional changes in human asthma.     

Oxotremorine-M potentiates NMDA receptors by muscarinic receptor dependent and independent mechanisms

Muscarinic acetylcholine M1 receptors play an important role in synaptic plasticity in the hippocampus and cortex. Potentiation of NMDA receptors as a consequence of muscarinic acetylcholine M1 receptor activation is a crucial event mediating the cholinergic modulation of synaptic plasticity, which is a cellular mechanism for learning and memory. In Alzheimer's disease, the cholinergic input to the hippocampus and cortex is severely degenerated, and agonists or positive allosteric modulators of M1 receptors are therefore thought to be of potential use to treat the deficits in cognitive functions in Alzheimer's disease. In this study we developed a simple system in which muscarinic modulation of NMDA receptors can be studied in vitro. Human M1 receptors and NR1/2B NMDA receptors were co-expressed in Xenopus oocytes and various muscarinic agonists were assessed for their modulatory effects on NMDA receptor-mediated responses. As expected, NMDA receptor-mediated responses were potentiated by oxotremorine-M, oxotremorine or xanomeline when the drugs were applied between subsequent NMDA responses, an effect which was fully blocked by the muscarinic receptor antagonist atropine. However, in oocytes expressing NR1/2B NMDA receptors but not muscarinic M1 receptors, oxotremorine-M co-applied with NMDA also resulted in a potentiation of NMDA currents and this effect was not blocked by atropine, demonstrating that oxotremorine-M is able to directly potentiate NMDA receptors. Oxotremorine, which is a close analogue of oxotremorine-M, and xanomeline, a chemically distinct muscarinic agonist, did not potentiate NMDA receptors by this direct mechanism. Comparing the chemical structures of the three different muscarinic agonists used in this study suggests that the tri-methyl ammonium moiety present in oxotremorine-M is important for the compound's interaction with NMDA receptors.     

Identification of an Ascaris G protein-coupled acetylcholine receptor with atypical muscarinic pharmacology

Acetylcholine (ACh) is a neurotransmitter/neuromodulator in the nematode nervous system and induces its effects through interaction with both ligand-gated ion channels (LGICs) and G protein-coupled receptors (GPCRs). The structure, pharmacology and physiological importance of LGICs have been appreciably elucidated in model nematodes, including parasitic species where they are targets for anthelmintic drugs. Significantly less, however, is understood about nematode ACh GPCRs, termed GARs (G protein-linked ACh receptors). What is known comes from the free-living Caenorhabditis elegans as no GARs have been characterized from parasitic species. Here we clone a putative GAR from the pig gastrointestinal nematode Ascaris suum with high structural homology to the C. elegans receptor GAR-1. Our GPCR, dubbed AsGAR-1, is alternatively spliced and expressed in the head and tail of adult worms but not in dorsal or ventral body wall muscle, or the ovijector. ACh activated AsGAR-1 in a concentration-dependent manner but the receptor was not activated by other small neurotransmitters. The classical muscarinic agonists carbachol, arecoline, oxotremorine M and bethanechol were also AsGAR-1 agonists but pilocarpine was ineffective. AsGAR-1 activation by ACh was partially antagonized by the muscarinic blocker atropine but pirenzepine and scopolamine were largely ineffective. Certain biogenic amine GPCR antagonists were also found to block AsGAR-1. Our conclusion is that Ascaris possesses G protein-coupled ACh receptors that are homologous in structure to those present in C. elegans, and that although they have some sequence homology to vertebrate muscarinic receptors, their pharmacology is atypically muscarinic.     

Synthesis, chiral chromatographic separation, and biological activities of the enantiomers of 10,10-dimethylhuperzine A

(+/-)-10,10-Dimethylhuperzine A (2, DMHA) has been synthesized, and its enantiomers have been separated using chiral HPLC. (-)-DMHA inhibits AChE with a Ki value approaching that of (-)-huperzine A, whereas (+)-DMHA shows no AChE inhibitory activity. On the other hand, both enantiomers are equally potent against glutamate-induced neurotoxicity when tested in neurons.     

Pilocarpine-induced seizures in adult rats: monoamine content and muscarinic and dopaminergic receptor changes in the striatum

High doses of the muscarinic cholinergic agonist pilocarpine are a useful model for investigation of the essential mechanisms for seizure generation and spread in rodents. Pilocarpine (400 mg/kg; subcutaneously) was administered in 2-month-old female rats, and the content of striatum monoamines and (M(1)+M(2)) muscarinic and D(2) dopaminergic receptors was measured in the acute period. All treated animals showed peripheral cholinergic signs, stereotyped and clonic movements, tremors, seizures and the percentage mortality was approximately 63%. High performance liquid chromatography determinations, performed 24 h later, showed a decrease of striatal levels of dopamine, dihydroxyphenylacetic acid, 4-hydroxy-3-methoxy-phenylacetic acid and 5-hydroxytryptamine. Pilocarpine treatment induced downregulation of (M(1)+M(2)) muscarinic receptors and reduced the dissociation constants of (M(1)+M(2)) muscarinic and D(2) dopaminergic receptors, suggesting that these systems exert opposite effects on the regulation of convulsive activity. These and other important neurochemical changes found in the course of establishment of an epileptic focus can be observed after status epilepticus induced by pilocarpine.     

Aclidinium bromide, a novel long-acting muscarinic antagonist for COPD with improved preclinical renal and urinary safety profile

AimsAclidinium bromide is a novel, long-acting, inhaled muscarinic antagonist currently in registration phase for the treatment of chronic obstructive pulmonary disease. Since urinary difficulty and retention have been reported for anticholinergic agents such as tiotropium and ipratropium, it is important to examine the preclinical urinary and renal safety profile of aclidinium.Main methodsThe effect of aclidinium on urine and electrolyte excretion, renal function and voiding cystometry was analysed in conscious water-loaded Wistar rats (10–1000 μg/kg, s.c.), anaesthetised Beagle dogs (1000 μg/kg, i.v.) and anaesthetised guinea pigs (3–100 μg/kg, intratracheally), respectively. Aclidinium plasma levels were determined in an independent study. Active comparators were tiotropium (all studies) and ipratropium (cystometry only).Key findingsAclidinium 1000 μg/kg had no effect on urine excretion in rats, in contrast to tiotropium 100 μg/kg which significantly decreased this parameter (p < 0.05). Aclidinium 1000 μg/kg also had no effect on renal function in Beagle dogs. In guinea pigs, aclidinium 3–100 μg/kg had no effect on urinary bladder function, whereas tiotropium and ipratropium 100 μg/kg decreased the peak micturition pressure (p < 0.05), increased the volume of urine retained in the bladder (p < 0.01) and showed a trend to decrease the volume of urine excreted.SignificanceAclidinium had no significant effect on urinary and renal function in the animal models studied. These results, together with the rapid plasma clearance of aclidinium reported previously, suggest a lower propensity to induce urinary retention in humans than tiotropium and ipratropium.     

Identification,biological characterization and pharmacophoric analysis of a new potent and selective NK1 receptor antagonist clinical candidate

The last two decades have provided a large weight of preclinical data implicating the neurokinin-1 receptor (NK₁) and its cognate ligand substance P (SP) in a broad range of both central and peripheral disease conditions. However, to date, only the NK₁ receptor antagonist aprepitant has been approved as a therapeutic and this is to prevent chemotherapy-induced nausea & vomiting (CINV). The belief remained that the full therapeutic potential of NK₁ receptor antagonists had yet to be realized; therefore clinical evidence that NK₁ receptor antagonists may be effective in major depression disorder, resulted in a significant further investment in discovering novel CNS penetrant druggable NK₁ receptor antagonists to address this condition. At GlaxoSmithKline after the discovery of casopitant, that went on to demonstrate efficacy as a novel antidepressant in the clinic, additional novel analogues of this NK₁ receptor antagonist were designed to further enhance its drug developability characteristics. Herein, we therefore describe the discovery process and the vivo pharmacological and pharmacokinetic profile of the new NK₁ receptor antagonist 3a (also called orvepitant), selected as clinical candidate and further progressed into clinical studies for major depressive disorder. Moreover, molecular modeling studies enabled us to improve the pharmacophore model of the NK₁ receptor antagonists with the identification of a region able to accommodate a variety of heterocycle moieties.     

The design of a novel series of muscarinic receptor antagonists leading to AZD8683, a potential inhaled treatment for COPD

A novel series of muscarinic receptor antagonists was developed, with the aim of identifying a compound with high M₃ receptor potency and a reduced risk of dose-limiting side effects with potential for the treatment of COPD.Initial compound modifications led to a novel cycloheptyl series, which was improved by focusing on a quinuclidine sub-series. A wide range of N-substituents was evaluated to determine the optimal substituent providing a high M₃ receptor potency, high intrinsic clearance and high human plasma protein binding. Compounds achieving in vitro study criteria were selected for in vivo evaluation. Pharmacokinetic half-lives, inhibition of bronchoconstriction and duration of action, as well as systemic side effects, induced by the compounds were assessed in guinea-pig models.Compounds with a long duration of action and good therapeutic index were identified and AZD8683 was selected for progression to the clinic.     

Synthesis of benzopolycyclic cage amines: NMDA receptor antagonist, trypanocidal and antiviral activities

The synthesis of several 6,7,8,9,10,11-hexahydro-9-methyl-5,7:9,11-dimethano-5H-benzocyclononen-7-amines is reported. Several of them display low micromolar NMDA receptor antagonist and/or trypanocidal activities. Two compounds are endowed with micromolar anti vesicular stomatitis virus activity, while only one compound shows micromolar anti-influenza activity. The anti-influenza activity of this compound does not seem to be mediated by blocking of the M2 protein.     

Truncation of the peptide sequence in bifunctional ligands with mu and delta opioid receptor agonist and neurokinin 1 receptor antagonist activities

The optimization and truncation of our lead peptide-derived ligand TY005 possessing eight amino-acid residues was performed. Among the synthesized derivatives, NP30 (Tyr¹-DAla²-Gly³-Phe⁴-Gly⁵-Trp⁶-O-[3′,5′-Bzl(CF₃)₂]) showed balanced and potent opioid agonist as well as substance P antagonist activities in isolated tissue-based assays, together with significant antinociceptive and antiallodynic activities in vivo.     

Stereoselective inhibition of human, mouse, and horse cholinesterases by bambuterol enantiomers

Bambuterol is a chiral carbamate and a selective inhibitor of butyrylcholinesterase (BChE, EC 3.1.1.8). In order to relate bambuterol selectivity and stereoselectivity of BChE and acetylcholinesterase (AChE, EC 3.1.1.7) of different species, we studied the inhibition of human, mouse, and horse BChE, as well as AChE of human and mouse by (R)- and (S)-bambuterol. AChE and BChE of all studied species were progressively inhibited by both bambuterol enantiomers, with a preference for the (R)-bambuterol whose inhibition rate constants were about five times higher than that of (S)-bambuterol. We observed no significant difference between human and mouse in bambuterol enantiomer BChE inhibition. However, (R)-bambuterol inhibited horse BChE about 14 times slower than human and mouse BChE, and the inhibition rate for (S)-bambuterol was about 18 times slower. Although the primary structure of horse BChE differs from the other two species in 15 amino acids, we presumed that differences in inhibition rates could be attributed to threonine at position 69 located close to the peripheral site of BChE. Since BChE inhibition by bambuterol enantiomers was at least 8000 times faster than that of AChE, both bambuterol enantiomers proved to be selective BChE inhibitors, as was previously shown for racemate.