Discovery of quinuclidine modulators of cellular progranulin.

Phenotypic screening of an annotated small molecule library identified the quinuclidine tetrahydroisoquinoline solifenacin (1) as a robust enhancer of progranulin secretion with single digit micromolar potency in a murine microglial (BV-2) cell line. Subsequent SAR development led to the identification of 29 with a 38-fold decrease in muscarinic receptor antagonist activity and a 10-fold improvement in BV-2 potency.

Glucosylceramide synthase inhibitors prevent replication of SARS-CoV-2 and influenza virus.

The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major threat to global health. Vaccines are ideal solutions to prevent infection, but treatments are also needed for those who have contracted the virus to limit negative outcomes, when vaccines are not applicable. Viruses must cross host cell membranes during their life cycle, creating a dependency on processes involving membrane dynamics. Thus, in this study, we examined whether the synthetic machinery for glycosphingolipids, biologically active components of cell membranes, can serve as a therapeutic target to combat SARS-CoV-2. We examined the antiviral effect of two specific inhibitors of glucosylceramide synthase (GCS): (i) Genz-123346, an analogue of the United States Food and Drug Administration-approved drug Cerdelga and (ii) GENZ-667161, an analogue of venglustat, which is currently under phase III clinical trials. We found that both GCS inhibitors inhibit replication of SARS-CoV-2. Moreover, these inhibitors also disrupt replication of influenza virus A/PR/8/34 (H1N1). Our data imply that synthesis of glycosphingolipids is necessary to support viral life cycles and suggest that GCS inhibitors should be further explored as antiviral therapies.

7-Azaindolequinuclidinones (7-AIQD): A novel class of cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor ligands.

A series of N-benzyl-7-azaindolequinuclidinone (7-AIQD) analogs have been synthesized and evaluated for affinity toward CB1 and CB2 cannabinoid receptors and identified as a novel class of cannabinoid receptor ligands. Structure-activity relationship (SAR) studies indicate that 7-AIQD analogs are dual CB1/CB2 receptor ligands exhibiting high potency with somewhat greater selectivity towards CB2 receptors compared to the previously reported indolequinuclidinone (IQD) analogs. Initial binding assays showed that 7-AIQD analogs 8b, 8d, 8f, 8g and 9b (1 µM) produced more that 50% displacement of the CB1/CB2 non-selective agonist CP-55,940 (0.1 nM). Furthermore, Ki values determined from full competition binding curves showed that analogs 8a, 8b and 8g exhibit high affinity (110, 115 and 23.7 nM, respectively) and moderate selectivity (26.3, 6.1 and 9.2-fold, respectively) for CB2 relative to CB1 receptors. Functional studies examining modulation of G-protein activity demonstrated that 8a acts as a neutral antagonist at CB1 and CB2 receptors, while 8b exhibits inverse agonist activity at these receptors. Analogs 8f and 8g exhibit different intrinsic activities, depending on the receptor examined. Molecular docking and binding free energy calculations for the most active compounds (8a, 8b, 8f, and 8g) were performed to better understand the CB2 receptor-selective mechanism at the atomic level. Compound 8g exhibited the highest predicted binding affinity at both CB1 and CB2 receptors, and all four compounds were shown to have higher predicted binding affinities with the CB2 receptor compared to their corresponding binding affinities with the CB1 receptor. Further structural optimization of 7-AIQD analogs may lead to the identification of potential clinical agents.

Discovery of a Novel, Highly Potent, and Selective Thieno[3,2-d]pyrimidinone-Based Cdc7 Inhibitor with a Quinuclidine Moiety (TAK-931) as an Orally Active Investigational Antitumor Agent.

In our pursuit of developing a novel, potent, and selective cell division cycle 7 (Cdc7) inhibitor, we optimized the previously reported thieno[3,2-d]pyrimidinone analogue I showing time-dependent Cdc7 kinase inhibition and slow dissociation kinetics. These medicinal chemistry efforts led to the identification of compound 3d, which exhibited potent cellular activity, excellent kinase selectivity, and antitumor efficacy in a COLO205 xenograft mouse model. However, the issue of formaldehyde adduct formation emerged during a detailed study of 3d, which was deemed an obstacle to further development. A structure-based approach to circumvent the adduct formation culminated in the discovery of compound 11b (TAK-931) possessing a quinuclidine moiety as a preclinical candidate. In this paper, the design, synthesis, and biological evaluation of this series of compounds will be presented.

Bis(het)aryl-1,2,3-triazole quinuclidines as α7 nicotinic acetylcholine receptor ligands: Synthesis, structure affinity relationships, agonism activity, [18F]-radiolabeling and PET study in rats.

In this paper we describe the design and synthesis of bis(Het)Aryl-1,2,3-triazole quinuclidine α7R ligands using an efficient three-step sequence including a Suzuki-Miyaura cross coupling reaction with commercially available and home-made boron derivatives. The exploration of SAR required the preparation of uncommon boron derivatives. Forty final drugs were tested for their ability to bind the target and nine of them exhibited Ki values below nanomolar concentrations. The best scores were always obtained when the 5-phenyl-2-thiophenyl core was attached to the triazole. The selectivity of these compounds towards the nicotinic α4ß2 and serotoninergic 5HT3 receptors was assessed and their brain penetration was quantified by the preparation and in vivo evaluation of two [18F] radiolabelled derivatives. It can be expected from our results that some of these compounds will be suitable for further developments and will have effects on cognitive disorders.

New and Potent Quinuclidine-Based Antimicrobial Agents.

Developing new antibiotics is currently very important since antibiotic resistance is one of the biggest problems of global health today. In the search for a new class of potential antimicrobial agents, ten new compounds were designed and synthesized based on the quinuclidinium heterocyclic core and the oxime functional group. The antimicrobial activity was assessed against a panel of representative gram-positive and gram-negative bacteria. All compounds demonstrated potent activity against the tested microorganisms, with the minimum inhibitory concentration (MIC) values ranging from 0.25 to 256.00 µg/mL. Among the tested compounds, two quaternary compounds, para-N-chlorobenzyl and meta-N-bromobenzyl quinuclidinium oximes, displayed the most potent and broad-spectrum activity against both gram-positive and gram-negative bacterial strains (MIC values from 0.25 to 4.00 µg/mL), with the lowest value for the important multidrug resistant gram-negative pathogen Pseudomonas aeruginosa. In the case of Klebsiella pneumoniae, activity of those compounds are 256-fold and 16-fold better than gentamicin, respectively. MTT assays showed that compounds are nontoxic for human cell lines. Multi-way analysis was used to separately reduce dimensionality of quantum chemical data and biological activity data to obtain a regression model and the required parameters for the enhancement of biological activity.

Discovery of novel quaternary ammonium compounds based on quinuclidine-3-ol as new potential antimicrobial candidates.

Quaternary ammonium compounds (QACs) are amphiphilic molecules displaying a broad-spectrum of antibacterial activity. QACs are commonly used antiseptics in industrial, home and hospital settings. Given the emergence of the QAC-resistant bacteria, there is an urgent need to design new QACs with good antimicrobial activity, able to escape the host resistance mechanism. Therefore, a series of QACs derived from quinuclidine-3-ol and an alkyl chain of variable length (QOH-C3 to -C14), was designed and synthesized. The antimicrobial potential of the new monoquaternary QACs was surveyed against seventeen strains of emerging food spoilage and pathogenic microorganisms, including clinical multidrug-resistant ESKAPE isolates. The QOH-C14 proved to have the strongest antimicrobial activity. It was highly active against all pathogens tested, particularly against the Gram-positive bacteria with minimal inhibitory concentrations (MICs) ranging from 0.06 to 3.9 µg/mL, and fungi exerting the MIC90 between 0.12 and 3.9 µg/mL. The potency of QOH-C14, confirmed that alkyl chains are an important part of the structure with their lengths playing a critical role in bioactivity of these compounds. The atomic force microscopy images show the disruption of a cell membrane upon the treatment with QOH-C14. These results were additionally confirmed by flow cytometry and fluorescence microscopy. A relatively low toxicity toward healthy human cells underline that QOH-C14 has a potential as new QAC antimicrobial candidate.

A More Sustainable Process for Preparation of the Muscarinic Acetylcholine Antagonist Umeclidinium Bromide.

A more sustainable process for the synthesis of the long-acting muscarinic acetylcholine antagonist umeclidinium bromide is described. Specifically, we report the synthesis of ethyl 1-(2-chloroethyl)-4-piperidinecarboxylate, a key intermediate in the preparation of umeclidinium bromide, in good yields using triethylamine, as well as the identification and characterization of the by-product formed in this reaction. This new method of synthesis leads to an improvement in yield over that of previously reported protocols using potassium carbonate as base (65.6 % versus 38.6 %). Moreover, in the final synthetic step of the process to obtain umeclidinium bromide, we were able to replace the use of toxic solvents (acetonitrile/chloroform) with water. The use of this green solvent allowed precipitation of the active pharmaceutical ingredient (API) from the reaction medium with high purity and in high yield. Overall, we have developed a more efficient and environmentally friendly process for the synthesis of the umeclidinium bromide API with a higher overall yield (37.8 % versus previously reported overall yield of 9.7 %).

Novel muscarinic acetylcholine receptor hybrid ligands embedding quinuclidine and 1,4-dioxane fragments.

To obtain novel muscarinic acetylcholine receptor (mAChR) antagonists, the enantiomers of the hybrid compounds 3-5, in which the quinuclidin-3-yloxy fragment of solifenacin and the 6,6-diphenyl-1,4-dioxane-2-yl moiety of 2 linked by an ester or ether spacer were embedded in the same chemical entity, were prepared and evaluated for their affinity at the five mAChR subtypes (M1-M5). Stereochemistry and the nature of the linker between the quinuclidine moiety and the 1,4-dioxane nucleus play an important role on the affinities of the compounds. The presence of an ether bridge confers higher affinities for all mAChR subtypes to the ligand. Interestingly, the ether enantiomer (R,S)-5 shows the highest affinity at all mAChR subtypes with pKi values similar to that of solifenacin at M3 and higher at the other subtypes. Unlike solifenacin, it shows a preference for M1 mAChR subtype with respect to the other subtypes. This compound, lacking a permanent positive charge on the nitrogen atom, can be a useful tool for the pharmacological study of mAChRs in the central nervous system.

Identification of α7 Nicotinic Acetylcholine Receptor Silent Agonists Based on the Spirocyclic Quinuclidine-Δ2 -Isoxazoline Scaffold: Synthesis and Electrophysiological Evaluation.

Compound 11 (3-(benzyloxy)-1'-methyl-1'-azonia-4H-1'-azaspiro[isoxazole-5,3'-bicyclo[2.2.2]octane] iodide) was selected from a previous set of nicotinic ligands as a suitable model compound for the design of new silent agonists of α7 nicotinic acetylcholine receptors (nAChRs). Silent agonists evoke little or no channel activation but can induce the α7 desensitized Ds state, which is sensitive to a type II positive allosteric modulator, such as PNU-120596. Introduction of meta substituents into the benzyloxy moiety of 11 led to two sets of tertiary amines and quaternary ammonium salts based on the spirocyclic quinuclidinyl-Δ2 -isoxazoline scaffold. Electrophysiological assays performed on Xenopus laevis oocytes expressing human α7 nAChRs highlighted four compounds that are endowed with a significant silent-agonism profile. Structure-activity relationships of this group of analogues provided evidence of the crucial role of the positive charge at the quaternary quinuclidine nitrogen atom. Moreover, the present study indicates that meta substituents, in particular halogens, on the benzyloxy substructure direct specific interactions that stabilize a desensitized conformational state of the receptor and induce silent activity.

Novel quinuclidinone derivatives induced apoptosis in human breast cancer via targeting p53.

Small molecules that can target human cancers have been highly sought to increase the anticancer efficacy, the present work describes the design and synthesis of novel series of five quinuclidinone derivatives (2a-2e). Their anticancer activities were investigated against breast cancer cells MCF-7, MDA-MB-231 breast cancer cells harboring mutant p53 and normal breast counterpart MCF-12a. Derivative 2e reduced proliferation of MCF-7 and MCF-12a while it has no effect on MDA-MB-231. Derivative 2e induced apoptosis in MCF-7 cells which is further confirmed by TUNEL assay and it reduced the percentage of cell in G2/M phase as confirmed by increased expression of cyclin B and reduced expression of cyclin D1. Derivative 2e reduced expression levels of Mdm2, Akt and ERK1/2 by and increased expression level of p53. Moreover, the apoptosis induction by 2e was also inhibited by PFT-α as evidenced by non-significant induction of apoptosis after treatment of MCF-7 cells with both derivative 2e and PFT-α. In addition, docking study reveals that derivative 2e has a binding pattern close to the pattern observed in the structure of the lead fragment 5,6-dimethoxy-2-methylbenzothiazole bound to T-p53C-Y220C. The above findings demonstrate that derivative 2e induces apoptosis in MCF-7 cells via targeting p53 which merits further development.

Radiosynthesis of (S)-[18F]T1: The first PET radioligand for molecular imaging of α3ß4 nicotinic acetylcholine receptors.

Recent pharmacologic data revealed the implication of α3ß4 nicotinic acetylcholine receptors (nAChRs) in nicotine and drug addiction. To image α3ß4 nAChRs in vivo, we aimed to establish the synthesis of a [18F]-labelled analog of the highly affine and selective α3ß4 ligand (S)-3-(4-(4-fluorophenyl)-1H-1,2,3-triazol-1-yl)quinuclidine ((S)-T1). (S)-[18F]T1 was synthesized from ethynyl-4-[18F]fluorobenzene ([18F]5) and (S)-azidoquinuclidine by click reaction. After a synthesis time of 130min (S)-[18F]T1 was obtained with a radiochemical yield (non-decay corrected) of 4.3±1.3%, a radiochemical purity of >99% and a molar activity of >158 GBq/µmol. The brain uptake and the brain-to-blood ratio of (S)-[18F]T1 in mice at 30min post injection were 2.02 (SUV) and 6.1, respectively. According to an ex-vivo analysis, the tracer remained intact (>99%) in brain. Only one major radiometabolite was detected in plasma and urine samples. In-vitro autoradiography on pig brain slices revealed binding of (S)-[18F]T1 to brain regions associated with the expression of α3ß4 nAChRs, which could be reduced by the α3ß4 nAChR selective drug AT-1001. These findings make (S)-[18F]T1 a potential tool for the non-invasive imaging of α3ß4 nAChRs in the brain by PET.

Synthesis and Pharmacological Evaluation of [11C]Granisetron and [18F]Fluoropalonosetron as PET Probes for 5-HT3 Receptor Imaging.

Serotonin-gated ionotropic 5-HT3 receptors are the major pharmacological targets for antiemetic compounds. Furthermore, they have become a focus for the treatment of irritable bowel syndrome (IBS) and there is some evidence that pharmacological modulation of 5-HT3 receptors might alleviate symptoms of other neurological disorders. Highly selective, high-affinity antagonists, such as granisetron (Kytril) and palonosetron (Aloxi), belong to a family of drugs (the "setrons") that are well established for clinical use. To enable us to better understand the actions of these drugs in vivo, we report the synthesis of 8-fluoropalonosetron (15) that has a binding affinity (Ki = 0.26 ± 0.05 nM) similar to the parent drug (Ki = 0.21 ± 0.03 nM). We radiolabeled 15 by nucleophilic 18F-fluorination of an unsymmetrical diaryliodonium palonosetron precursor and achieved the radiosynthesis of 1-(methyl-11C)-N-granisetron ([11C]2) through N-alkylation with [11C]CH3I, respectively. Both compounds [18F]15 (chemical and radiochemical purity >95%, specific activity 41 GBq/µmol) and [11C]2 (chemical and radiochemical purity ≥99%, specific activity 170 GBq/µmol) were evaluated for their utility as positron emission tomography (PET) probes. Using mouse and rat brain slices, in vitro autoradiography with both [18F]15 and [11C]2 revealed a heterogeneous and displaceable binding in cortical and hippocampal regions that are known to express 5-HT3 receptors at significant levels. Subsequent PET experiments suggested that [18F]15 and [11C]2 are of limited utility for the PET imaging of brain 5-HT3 receptors in vivo.

Ether and Carbamate Derivatives of 3-quinuclidinol and 3- hydroxymethylquinuclidine: Synthesis and Evaluation as Nicotinic Ligands.

BACKGROUND: A lead quinuclidine-based nicotinic ligand (EQA) served as the basis for the design of novel compounds. A new series of 3-substituted quinuclidines was designed, synthesized and evaluated as nicotinic ligands. METHODS: The goal was to improve affinity for nicotinic receptors in the CNS. Interatomic distance calculations were performed on the proposed compounds as well as, known nicotinic ligands. The proposed compounds were then synthesized, characterized and evaluated in in vitro assays as nicotinic receptor ligands. RESULTS: Compounds 9a and 9b were found to inhibit the specific binding of 3H-(S)-Nicotine with Ki values of 48 nM and 42 nM respectively, indicating high affinity interactions with the α4ß2 subtype. Data suggest that several compounds act as partial agonists at CNS receptors with an efficacy between 28 and 40% and are potent partial activators of human muscle type receptors (α1ß1γδ Emax= 80% that of 100 µM nicotine). CONCLUSIONS: Together these results indicate a partial agonism at muscle type receptors (ca. 40%) with no significant activation of rat ganglion-type receptors (α3ß4*: asterisk indicates potential additional subunit that could partner to form the ganglionic receptor). The partial agonism inducing dopamine release from striatal synaptosomes (α4ß2α6α4ß2ß3, and/or α6ß2ß3) suggest that these compounds may in addition be acting at the α4ß2 and/or the α6ß3* receptors. The partial agonists reported herein are interesting nicotinic ligands worthy of further investigation.

Total Synthesis and Characterization of 7-Hypoquinuclidonium Tetrafluoroborate and 7-Hypoquinuclidone BF3 Complex.

Derivatives of the fully twisted bicyclic amide 7-hypoquinuclidone are synthesized using a Schmidt-Aubé reaction. Their structures were unambiguously confirmed by X-ray diffraction analysis and extensive spectroscopic characterization. Furthermore, the stability and chemical reactivity of these anti-Bredt amides are investigated. 7-Hypoquinuclidonium tetrafluoroborate is shown to decompose to a unique nitrogen bound amide-BF3 complex of 7-hypoquinuclidone under anhydrous conditions and to react instantaneously with water making it one of the most reactive amides known to date.

Synthesis of iboga-like isoquinuclidines: Dual opioid receptors agonists having antinociceptive properties.

Some novel iboga-analogues consisting of benzofuran moiety and dehydroisoquinuclidine ring connected by -CH2-, (CH2)2 and (CH2)3 linkers have been synthesized with the view to develop potential antinociceptive drugs. The compounds 14 and 21 showed binding at the µ-opioid receptor (MOR), while the compound 11a exhibited dual affinities at both MOR and κ-opioid receptor (KOR). MAP kinase activation indicated all three compounds have opioid agonistic properties. The presence of a double bond and endo-methylcarboxylate group in the dehydroisoquinuclidine ring and the benzofuran and methylene spacer appeared to be essential for opioid receptor binding. Further studies demonstrated 11a caused significant antinociception in mice in the hot-plate test which was comparable to that produced by morphine. The compound 11a was also found to be nontremorigenic unlike various iboga congeners. This study identifies a new pharmacophore which may lead to the development of suitable substitute of morphine in the treatment of pain.

9-Amino-(9-deoxy)cinchona alkaloid-derived new chiral phase-transfer catalysts.

A new class of 9-amino-(9-deoxy)cinchona alkaloid-derived chiral phase-transfer catalysts bearing amino groups was developed by using known cinchona alkaloids as the starting materials. Due to the transformation of the 9-hydroxyl group into a 9-amino functional group, the catalytic performances were significantly improved in comparison with the corresponding first generation phase-transfer catalysts, and excellent yields (92-99%) and high enantioselectivities (87-96% ee) were achieved in the benchmark asymmetric α-alkylation of glycine Schiff base. Based on the special contribution of the amino group to the high yield and enantioselectivity, the possible catalytic mechanism was conjectured.

Novel quinuclidinyl heteroarylcarbamate derivatives as muscarinic receptor antagonists.

Herein, we describe the synthesis and pharmacological profiles of novel quinuclidinyl heteroarylcarbamate derivatives. Among them, the quinuclidin-4-yl thiazolylcarbamate derivative ASP9133 was identified as a promising long-acting muscarinic antagonist (LAMA) showing more selective inhibition of bronchoconstriction against salivation and more rapid onset of action in a rat model than tiotropium bromide.

Stereoselective synthesis of isoquinuclidines through an aza-[4 + 2] cycloaddition of chiral cyclic 2-amidodienes.

A highly stereoselective aza-[4 + 2] cycloaddition of chiral cyclic 2-amidodienes with N-sulfonyl aldimines is described. While this Lewis acid promoted heterocycloaddition provides an efficient strategy for constructing optically enriched isoquinuclidines, it is mechanistically intriguing. The cycloaddition favored the endo-II pathway in the absence of a viable bidentate coordination. This represents an unexpected switch from the anticipated endo-I selectivity obtained in the all-carbon cycloaddition.

Highly efficient synthesis of (R)-3-quinuclidinol in a space-time yield of 916 g L(-1) d(-1) using a new bacterial reductase ArQR.

A new keto reductase (ArQR), identified from Agrobacterium radiobacter ECU2556, can efficiently reduce 3-quinuclidinone in excellent enantioselectivity and high space-time yield for the synthesis of (R)-3-quinuclidinol, a chiral building block of many antimuscarinic agents. This is the first time that a high yield of (R)-3-quinuclidinol up to 916 g L(-1) d(-1) using a bioreduction approach is reported.