Synthesis and characterization of chiral 6-azaspiro[2.5]octanes as potent and selective antagonists of the M4 muscarinic acetylcholine receptor.

In this manuscript, we report a series of chiral 6-azaspiro[2.5]octanes and related spirocycles as highly potent and selective antagonists of the muscarinic acetylcholine receptor subtype 4 (mAChR4). Chiral separation and subsequent X-ray crystallographic analysis of early generation analogs revealed the R enantiomer to possess excellent human and rat M4 potency, and further structure-activity relationship (SAR) studies on this chiral scaffold led to the discovery of VU6015241 (compound 19). Compound 19 is characterized by high M4 potency and selectivity across multiple species, excellent aqueous solubility, and moderate brain exposure in rodents after intraperitoneal administration.

The Spasmolytic, Bronchodilator, and Vasodilator Activities of Parmotrema perlatum Are Explained by Anti-Muscarinic and Calcium Antagonistic Mechanisms.

Parmotremaperlatum is traditionally used in different areas of Pakistan to treat gastrointestinal, respiratory, and vascular diseases. This study evaluates the underlying mechanisms for traditional uses of P. perlatum in diarrhea, asthma, and hypertension. In vitro pharmacological studies were conducted using isolated jejunum, trachea, and aortic preparations, while the cytotoxic study was conducted in mice. Crude extract of P. perlatum(Pp.Cr), comprising appreciable quantities of alkaloids and flavonoids, relaxed spontaneously contracting jejunum preparation, K+ (80 mM)-induced, and carbachol (1 µM)-induced jejunum contractions in a concentration-dependent manner similar to dicyclomine and dantrolene. Pp.Cr showed a rightward parallel shift of concentration-response curves (CRCs) of Cch after a non-parallel shift similarto dicyclomine and shifted CRCs of Ca+2 to rightward much likeverapamil and dantrolene, demonstrating the coexistence of antimuscarinic and Ca+2 antagonistic mechanism. Furthermore, Pp.Cr, dicyclomine, and dantrolene relaxed K+ (80 mM)-induced and Cch (1 µM)-induced tracheal contractions and shifted rightward CRCs of Cch similar to dicyclomine, signifying the dual blockade. Additionally, Pp.Cr also relaxed the K+ (80 mM)-induced and phenylephrine (1 µM)-induced aortic contraction, similarly to verapamil and dantrolene, suggesting Ca+2 channel antagonism. Here, we explored for the first time thespasmolytic and bronchodilator effects of Pp.Crand whether they maybe due to the dual blockade of Ca+2 channels and muscarinic receptors, while the vasodilator effect might be owing to Ca+2 antagonism. Our results provide the pharmacological evidence that P. perlatum could be a new potential therapeutic option to treat gastrointestinal, respiratory, and vascular diseases. Hence, there is a need for further research to explore bioactive constituent of P. perlatum as well as further investigation by suitable experimental models are required to further confirm the importance and usefulness of P. perlatum in diarrhea, asthma, and hypertension treatment.

Pyrrolizidine alkaloids in tea, herbal tea and iced tea beverages- survey and transfer rates.

The transfer rate of 37 pyrrolizidine alkaloids (PA) found in ten naturally contaminated teas and herbal teas to their brews was studied in detail. Mixed herbal, peppermint, red bush, senna, black tea and green tea infusions were prepared according to the ISO guide and vendor's instructions, respectively, and parameters like herb-to-water ratio, steeping time and multiple extractions studied. In general, a transfer rate of 38-100% (median 95%) for brews following vendor's instructions was determined. The total concentration range of PA in these ten samples was 154-2412 ng/g (median 422 ng/g) in the herb and for single analytes 0.1-170 ng/g. Seven of the 37 PA occurred unexpectedly; these were tentatively identified and quantified by liquid chromatography-high resolution mass spectrometry (LC-HR-MS), since their contributions to total PA-content matter. Additionally, 46 iced tea beverages were analysed for their PA-load, determined to be in the range 0-631 ng/L (median 40 ng/L). The applied solid-phase extraction (SPE) clean-up turned out to be capable of separating PA in the free base pyrrolizidine alkaloids (PAFB) and their N-oxides (PANO) in a two-step elution, which was a valuable tool to support identification of unexpected PA. Further, atropine was found in 50% of the ten tea herb samples (range: 1-4 ng/g) and in 13% of the iced tea beverage samples (range: 2-65 ng/L).

Rational Design of Photochromic Analogues of Tricyclic Drugs.

Tricyclic chemical structures are the core of many important drugs targeting all neurotransmitter pathways. These medicines enable effective therapies to treat from peptic ulcer disease to psychiatric disorders. However, when administered systemically, they cause serious adverse effects that limit their use. To obtain localized and on-demand pharmacological action using light, we have designed photoisomerizable ligands based on azobenzene that mimic the tricyclic chemical structure and display reversibly controlled activity. Pseudo-analogues of the tricyclic antagonist pirenzepine demonstrate that this is an effective strategy in muscarinic acetylcholine receptors, showing stronger inhibition upon illumination both in vitro and in cardiac atria ex vivo. Despite the applied chemical modifications to make pirenzepine derivatives sensitive to light stimuli, the most potent candidate of the set, cryptozepine-2, maintained a moderate but promising M1 vs M2 subtype selectivity. These photoswitchable "crypto-azologs" of tricyclic drugs might open a general way to spatiotemporally target their therapeutic action while reducing their systemic toxicity and adverse effects.

Synthetic approaches to FDA approved drugs for asthma and COPD from 1969 to 2020.

Respiratory infections resulting from pulmonary inflammation emerging as a leading cause of death worldwide. However, only twenty-seven new drugs were approved in the last five decades. In this review, we presented synthetic approaches for twenty-seven FDA-approved medications used to treat asthma and chronic obstructive pulmonary diseases (COPD), along with their mode of action.

A Selective and Sensitive Method Development and Validation of 1,1-Dimethyl-3-Hydroxy-Pyrrolidinium Bromide Impurity in Glycopyrrolate Oral Solution by Liquid Chromatography-Tandem Mass Spectroscopy.

OBJECTIVE: A selective and sensitive liquid chromatography-tandem mass spectrometer (LC-MS/MS) method has been developed for the quantification of 1,1-dimethyl-3-hydroxy-pyrrolidinium bromide impurity in glycopyrrolate oral solution. MATERIALS AND METHOD: The LC-MS/MS analysis was done on X Bridge HILIC (100 × 4.6 mm, 5 µm) analytical column, and the mobile phase used was10 mM ammonium formate with 0.2% formic acid as mobile phase-A and acetonitrile as mobile phase-B with a gradient programme of 5.0 min. The flow rate used was 1.2 mL/min. Triple quadrupole mass detector coupled to positive electrospray ionization operated in multiple reactions monitoring mode was used for the quantification at m/z 116.10 ± 0.5. RESULTS: Retention time of impurity was found ~3.2 min. The method was validated in terms of specificity, linearity, accuracy, precision, range, limit of detection, limit of quantitation (LOQ) and robustness. Relative standard deviation (RSD) for system suitability was found 1.3%. Calibration plot was linear over the range of 0.050-2.000 µg/mL. Limit of detection and limit of quantification were found 0.017 and 0.051 µg/mL, respectively. The intra- and inter-day precision RSD was 2.3% and the obtained recovery at LOQ to 200% was in between 86.7 and 107.4%. CONCLUSION: The low RSD values and high recoveries of the method confirm the suitability of the method.

Dibenzodiazepinone-type muscarinic receptor antagonists conjugated to basic peptides: Impact of the linker moiety and unnatural amino acids on M2R selectivity.

The family of human muscarinic acetylcholine receptors (MRs) is characterized by a high sequence homology among the five subtypes (M1R-M5R), being the reason for a lack of subtype selective MR ligands. In continuation of our work on dualsteric dibenzodiazepinone-type M2R antagonists, a series of M2R ligands containing a dibenzodiazepinone pharmacophore linked to small basic peptides was synthesized (64 compounds). The linker moiety was varied with respect to length, number of basic nitrogens (0-2) and flexibility. Besides proteinogenic basic amino acids (Lys, Arg), shorter homologues of Lys and Arg, containing three and two methylene groups, respectively, as well as D-configured amino acids were incorporated. The type of linker had a marked impact on M2R affinity and also effected M2R selectivity. In contrast, the structure of the basic peptide rather determined M2R selectivity than M2R affinity. For example, the most M2R selective compound (UR-CG188, 89) with picomolar M2R affinity (pKi 9.60), exhibited a higher M2R selectivity (ratio of Ki M1R/M2R/M3R/M4R/M5R: 110:1:5200:55:2300) compared to the vast majority of reported M2R preferring MR ligands. For selected ligands, M2R antagonism was confirmed in a M2R miniG protein recruitment assay.

The optimization of a novel selective antagonist for human M2 muscarinic acetylcholine receptor.

Muscarinic acetylcholine receptors (mAChRs) comprise five distinct subtypes denoted M1 to M5. The antagonism of M2 subtype could increase the release of acetylcholine from vesicles into the synaptic cleft and improve postsynaptic functions in the hippocampus via M1 receptor activation, displaying therapeutic potentials for Alzheimer's disease. However, drug development for M2 antagonists is still challenged among different receptor subtypes. In this study, by optimizing a scaffold from virtual screening, we synthesized two focused libraries and generated up to 50 derivatives. By measuring potency and binding selectivity, we discovered a novel M2 antagonist, ligand 47, featuring submicromolar IC50, high M2/M4 selectivity (~30-fold) and suitable lipophilicity (cLogP = 4.55). Further study with these compounds also illustrates the structure-activity relationship of this novel scaffold. Our study could not only provide novel lead structure, which was easy to synthesize, but also offer valuable information for further development of selective M2 ligands.

Structure and selectivity engineering of the M1 muscarinic receptor toxin complex.

Muscarinic toxins (MTs) are natural toxins produced by mamba snakes that primarily bind to muscarinic acetylcholine receptors (MAChRs) and modulate their function. Despite their similar primary and tertiary structures, MTs show distinct binding selectivity toward different MAChRs. The molecular details of how MTs distinguish MAChRs are not well understood. Here, we present the crystal structure of M1AChR in complex with MT7, a subtype-selective anti-M1AChR snake venom toxin. The structure reveals the molecular basis of the extreme subtype specificity of MT7 for M1AChR and the mechanism by which it regulates receptor function. Through in vitro engineering of MT7 finger regions that was guided by the structure, we have converted the selectivity from M1AChR toward M2AChR, suggesting that the three-finger fold is a promising scaffold for developing G protein-coupled receptor modulators.

Carbachol dimers with primary carbamate groups as homobivalent modulators of muscarinic receptors.

Although agonists and antagonists of muscarinic receptors have been known for long time, there is renewed interest in compounds (such as allosteric or bitopic ligands, or biased agonists) able to differently and selectively modulate these receptors. As a continuation of our previous research, we designed a new series of dimers of the well-known cholinergic agonist carbachol. The new compounds were tested on the five cloned human muscarinic receptors (hM1-5) expressed in CHO cells by means of equilibrium binding experiments, showing a dependence of the binding affinity on the length and position of the linker connecting the two monomers. Kinetic binding studies revealed that some of the tested compounds were able to slow the rate of NMS dissociation, suggesting allosteric behavior, also supported by docking simulations. Assessment of ERK1/2 phosphorylation on hM1, hM2 and hM3 activation showed that the new compounds are endowed with muscarinic antagonist properties. At hM2 receptors, some compounds were able to stimulate GTPγS binding but not cAMP accumulation, suggesting a biased behavior. Classification, Molecular and cellular pharmacology.

Novel Potent Muscarinic Receptor Antagonists: Investigation on the Nature of Lipophilic Substituents in the 5- and/or 6-Positions of the 1,4-Dioxane Nucleus.

A series of novel 1,4-dioxane analogues of the muscarinic acetylcholine receptor (mAChR) antagonist 2 was synthesized and studied for their affinity at M1-M5 mAChRs. The 6-cyclohexyl-6-phenyl derivative 3b, with a cis configuration between the CH2N+(CH3)3 chain in the 2-position and the cyclohexyl moiety in the 6-position, showed pKi values for mAChRs higher than those of 2 and a selectivity profile analogous to that of the clinically approved drug oxybutynin. The study of the enantiomers of 3b and the corresponding tertiary amine 33b revealed that the eutomers are (2S,6S)-(-)-3b and (2S,6S)-(-)-33b, respectively. Docking simulations on the M3 mAChR-resolved structure rationalized the experimental observations. The quaternary ammonium function, which should prevent the crossing of the blood-brain barrier, and the high M3/M2 selectivity, which might limit cardiovascular side effects, make 3b a valuable starting point for the design of novel antagonists potentially useful in peripheral diseases in which M3 receptors are involved.

Red-Emitting Dibenzodiazepinone Derivatives as Fluorescent Dualsteric Probes for the Muscarinic Acetylcholine M2 Receptor.

Fluorescently labeled dibenzodiazepinone-type muscarinic acetylcholine receptor (MR) antagonists, including dimeric ligands, were prepared using red-emitting cyanine dyes. Probes containing a fluorophore with negative charge showed high M2R affinities (pKi (radioligand competition binding): 9.10-9.59). Binding studies at M1 and M3-M5 receptors indicated a M2R preference. Flow cytometric and high-content imaging saturation and competition binding (M1R, M2R, and M4R) confirmed occupation of the orthosteric site. Confocal microscopy revealed that fluorescence was located mainly at the cell membrane (CHO-hM2R cells). Results from dissociation and saturation binding experiments (M2R) in the presence of allosteric M2R modulators (dissociation: W84, LY2119620, and alcuronium; saturation binding: W84) were consistent with a competitive mode of action between the fluorescent probes and the allosteric ligands. Taken together, these lines of evidence indicate that these ligands are useful fluorescent molecular tools to label the M2R in imaging and binding studies and suggest that they have a dualsteric mode of action.

Regiospecific Introduction of Halogens on the 2-Aminobiphenyl Subunit Leading to Highly Potent and Selective M3 Muscarinic Acetylcholine Receptor Antagonists and Weak Inverse Agonists.

Muscarinic M3 receptor antagonists and inverse agonists displaying high affinity and subtype selectivity over the antitarget M2 are valuable pharmacological tools and may enable improved treatment of chronic obstructive pulmonary disease (COPD), asthma, or urinary incontinence. On the basis of known M3 antagonists comprising a piperidine or quinuclidine unit attached to a biphenyl carbamate, 5-fluoro substitution was responsible for M3 subtype selectivity over M2, while 3'-chloro substitution substantially increased affinity through a σ-hole interaction. Resultantly, two piperidinyl- and two quinuclidinium-substituted biphenyl carbamates OFH243 (13n), OFH244 (13m), OFH3911 (14n), and OFH3912 (14m) were discovered, which display two-digit picomolar affinities with Ki values from 0.069 to 0.084 nM, as well as high selectivity over the M2 subtype (46- to 68-fold). While weak inverse agonistic properties were determined for the biphenyl carbamates 13m and 13n, neutral antagonism was observed for 14m and 14n and tiotropium under identical assay conditions.

New Nocobactin Derivatives with Antimuscarinic Activity, Terpenibactins A-C, Revealed by Genome Mining of Nocardia terpenica IFM 0406.

We report a genomics-guided exploration of the metabolic potential of the brasilicardin producer strain Nocardia terpenica IFM 0406. Bioinformatics analysis of the whole genome sequence revealed the presence of a biosynthetic gene cluster presumably responsible for the generation of formerly unknown nocobactin derivatives. Mass spectrometry-assisted isolation led to the identification of three new siderophores, terpenibactins A (1), B (2) and C (3), which belong to the class of nocobactins. Their structures were elucidated by employing spectroscopic techniques. Compounds 1-3 demonstrated inhibitory activity towards the muscarinic M3 receptor, while exhibiting only a low cytotoxicity.

Ligand biological activity predicted by cleaning positive and negative chemical correlations.

Predicting ligand biological activity is a key challenge in drug discovery. Ligand-based statistical approaches are often hampered by noise due to undersampling: The number of molecules known to be active or inactive is vastly less than the number of possible chemical features that might determine binding. We derive a statistical framework inspired by random matrix theory and combine the framework with high-quality negative data to discover important chemical differences between active and inactive molecules by disentangling undersampling noise. Our model outperforms standard benchmarks when tested against a set of challenging retrospective tests. We prospectively apply our model to the human muscarinic acetylcholine receptor M1, finding four experimentally confirmed agonists that are chemically dissimilar to all known ligands. The hit rate of our model is significantly higher than the state of the art. Our model can be interpreted and visualized to offer chemical insights about the molecular motifs that are synergistic or antagonistic to M1 agonism, which we have prospectively experimentally verified.

Structural insights into the subtype-selective antagonist binding to the M2 muscarinic receptor.

Human muscarinic receptor M2 is one of the five subtypes of muscarinic receptors belonging to the family of G-protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype-selective ligands against one of the five muscarinic receptors. We report high-resolution structures of a thermostabilized mutant M2 receptor bound to a subtype-selective antagonist AF-DX 384 and a nonselective antagonist NMS. The thermostabilizing mutation S110R in M2 was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M2 receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, which is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular dynamics simulations reveal that tightening of the ligand-residue contacts in M2 receptors compared to M3 receptors leads to subtype selectivity of AF-DX 384.

Structure-guided development of selective M3 muscarinic acetylcholine receptor antagonists.

Drugs that treat chronic obstructive pulmonary disease by antagonizing the M3 muscarinic acetylcholine receptor (M3R) have had a significant effect on health, but can suffer from their lack of selectivity against the M2R subtype, which modulates heart rate. Beginning with the crystal structures of M2R and M3R, we exploited a single amino acid difference in their orthosteric binding pockets using molecular docking and structure-based design. The resulting M3R antagonists had up to 100-fold selectivity over M2R in affinity and over 1,000-fold selectivity in vivo. The crystal structure of the M3R-selective antagonist in complex with M3R corresponded closely to the docking-predicted geometry, providing a template for further optimization.

New developments in optimizing bronchodilator treatment of COPD: a focus on glycopyrrolate/formoterol combination formulated by co-suspension delivery technology.

COPD causes considerable health and economic burden worldwide, with incidence of the disease expected to continue to rise. Inhaled bronchodilators, such as long-acting muscarinic antagonists (LAMAs) and long-acting ß2-agonists (LABAs), are central to the maintenance treatment of patients with COPD. Clinical studies have demonstrated that combined LAMA + LABA therapies improve efficacy while retaining a safety profile similar to LAMA or LABA alone. This has led to the development of several LAMA/LABA fixed-dose combination (FDC) therapies, which provide patients with the convenience of two active compounds in a single inhaler. GFF MDI (Bevespi Aerosphere®) is an FDC of glycopyrrolate/formoterol fumarate 18/9.6 µg formulated using innovative co-suspension delivery technology for administration via metered dose inhaler (MDI). GFF MDI was developed to make a treatment option available for patients who have a requirement or preference to use an MDI, rather than a dry powder or soft mist inhaler. Now that several LAMA/LABA FDCs have been approved for use in COPD, we review the impact of dual-bronchodilator treatment on COPD therapy and discuss recent clinical studies that are helping to develop a more comprehensive understanding of how LAMA/LABA FDCs can improve patient outcomes.

Investigation of molecular mobility of pressure-sensitive-adhesive in oxybutynin patch in vitro and in vivo: Effect of sorbitan monooleate on drug release and patch mechanical property.

The aim of present study was to develop an oxybutynin (OXY) transdermal patch with good permeation behavior and mechanical property. Special attention was paid to the effect of chemical enhancer on the molecular mobility of pressure sensitive adhesive (PSA) at molecular level. PSAs and permeation enhancers were investigated through in vitro experiment using rat skin. The optimized formulation was evaluated through pharmacokinetic study using rat. In addition, the molecular mechanism of sorbitan monooleate (Span® 80) in the improvement of PSA molecular mobility was investigated using FT-IR, molecular dynamics simulation, DSC and rheological study. As a result, the optimized formulation using amide PSA demonstrated good adhesion property. And the AUC0-t and Cmax of optimized patch were 6435.8 ±â€¯747.8 h ∗ ng/mL and 127.8 ±â€¯18.0 ng/mL, respectively, which had no significant difference with commercial product. Furthermore, the improvement of the PSA mobility by Span® 80 rather than the decrease of interaction between drug and PSA was the main factor that enhanced the release of OXY from patch. In conclusion, a drug-in-adhesive OXY patch was developed, and the effect of PSA molecular mobility increase on the enhancement of drug skin permeation was proposed at molecular level.

Dose-response of an extrafine dry powder inhaler formulation of glycopyrronium bromide: randomized, double-blind, placebo-controlled, dose-ranging study (GlycoNEXT).

Introduction: An extrafine formulation of the long-acting muscarinic antagonist, glycopyrronium bromide (GB), has been developed for delivery via the NEXThaler dry powder inhaler (DPI). This study assessed the bronchodilator efficacy and safety of different doses of this formulation in patients with COPD to identify the optimal dose for further development. Patients and methods: This was a multicenter, randomized, double-blind, placebo-controlled, incomplete block, three-way crossover study, including three 28-day treatment periods, each separated by a 21-day washout period. Eligible patients had a diagnosis of COPD and post-bronchodilator forced expiratory volume in 1 s (FEV1) 40%-70% predicted. Treatments administered were GB 6.25, 12.5, 25 and 50 µg or matched placebo; all were given twice daily (BID) via DPI, with spirometry assessed on Days 1 and 28 of each treatment period. The primary end point was FEV1 area under the curve from 0 to 12 h (AUC0-12 h) on Day 28. Results: A total of 202 patients were randomized (61% male, mean age 62.6 years), with 178 (88%) completing all the three treatment periods. For the primary end point, all the four GB doses were superior to placebo (p<0.001) with mean differences (95% CI) of 114 (74, 154), 125 (85, 166), 143 (104, 183) and 187 (147, 228) mL for GB 6.25, 12.5, 25 and 50 µg BID, respectively. All four GB doses were also statistically superior to placebo for all secondary efficacy end points, showing clear dose-response relationships for most of the endpoints. Accordingly, GB 25 µg BID met the criteria for the minimally acceptable dose. Adverse events were reported by 15.5, 16.2, 10.9 and 14.3% of patients receiving GB 6.25, 12.5, 25 and 50 µg BID, respectively, and 14.8% receiving placebo. Conclusion: This study supports the selection of GB 25 µg BID as the minimal effective dose for patients with COPD when delivered with this extrafine DPI formulation.