Synthesis and Biological Evaluation of 3-Amidoquinuclidine Quaternary Ammonium Compounds as New Soft Antibacterial Agents.

Quaternary ammonium compounds (QACs) are among the most effective antimicrobial agents that have been used for more than a century. However, due to the growing trend of bacterial resistance and high toxicity of QACs, research in this field remains a pressing matter. Recent studies of the structure-activity relationship suggest that the introduction of the amide functional group into QAC structures results in soft variants that retain their antimicrobial properties while opening the possibility of fine-tuned activity regulation. Here, we report the synthesis and structure-function study of three structurally distinct series of naturally derived soft QACs. The obtained 3-amidoquinuclidine QACs showed a broad range of antibacterial activities related to the hydrophobic-hydrophilic balance of the QAC structures. All three series yielded candidates with minimal inhibitory concentrations (MIC) in the single-digit µM range. Time-resolved growth analysis revealed subtle differences in the antibacterial activity of the selected candidates. The versatile MIC values were recorded in different nutrient media, suggesting that the media composition may have a dramatic impact on the antibacterial potential. The new QACs were found to have excellent potential to suppress bacterial biofilm formation while exhibiting low ability to induce bacterial resistance. In addition, the selected candidates were found to be less toxic than commercially available QACs and proved to be potential substrates for protease degradation. These data suggest that 3-amidoquinuclidine QACs could be considered as novel antimicrobial agents that pose a low threat to ecosystems and human health.

Selective α3ß4 Nicotinic Acetylcholine Receptor Ligand as a Potential Tracer for Drug Addiction.

α3ß4 Nicotinic acetylcholine receptor (nAChR) has been recognized as an emerging biomarker for the early detection of drug addiction. Herein, α3ß4 nAChR ligands were designed and synthesized to improve the binding affinity and selectivity of two lead compounds, (S)-QND8 and (S)-T2, for the development of an α3ß4 nAChR tracer. The structural modification was achieved by retaining the key features and expanding the molecular structure with a benzyloxy group to increase the lipophilicity for blood-brain barrier penetration and to extend the ligand-receptor interaction. The preserved key features are a fluorine atom for radiotracer development and a p-hydroxyl motif for ligand-receptor binding affinity. Four (R)- and (S)-quinuclidine-triazole (AK1-AK4) were synthesized and the binding affinity, together with selectivity to α3ß4 nAChR subtype, were determined by competitive radioligand binding assay using [3H]epibatidine as a radioligand. Among all modified compounds, AK3 showed the highest binding affinity and selectivity to α3ß4 nAChR with a Ki value of 3.18 nM, comparable to (S)-QND8 and (S)-T2 and 3069-fold higher affinity to α3ß4 nAChR in comparison to α7 nAChR. The α3ß4 nAChR selectivity of AK3 was considerably higher than those of (S)-QND8 (11.8-fold) and (S)-T2 (294-fold). AK3 was shown to be a promising α3ß4 nAChR tracer for further development as a radiotracer for drug addiction.

DABCO-Customized Nanoemulsions: Characterization, Cell Viability and Genotoxicity in Retinal Pigmented Epithelium and Microglia Cells.

Quaternary derivatives of 1,4-diazabicyclo[2.2.2]octane (DABCO) and of quinuclidine surfactants were used to develop oil-in-water nanoemulsions with the purpose of selecting the best long-term stable nanoemulsion for the ocular administration of triamcinolone acetonide (TA). The combination of the best physicochemical properties (i.e., mean droplet size, polydispersity index, zeta potential, osmolality, viscoelastic properties, surface tension) was considered, together with the cell viability assays in ARPE-19 and HMC3 cell lines. Surfactants with cationic properties have been used to tailor the nanoemulsions' surface for site-specific delivery of drugs to the ocular structure for the delivery of TA. They are tailored for the eye because they have cationic properties that interact with the anionic surface of the eye.

Mono- and Dicationic DABCO/Quinuclidine Composed Nanomaterials for the Loading of Steroidal Drug: 32 Factorial Design and Physicochemical Characterization.

Oil-in-water nanoemulsions (NEs) are considered a suitable nanotechnological approach to improve the eye-related bioavailability of lipophilic drugs. The potential of cationic NEs is prominent due to the electrostatic interaction that occurs between the positively charged droplets with the negatively charged mucins present in the tear film. This interaction offers prolonged NEs residence at the ocular surface, increasing the drug absorption. Triamcinolone acetonide (TA) is one of the first pharmacologic strategies applied as an intravitreal injection in the treatment of age-related macular degeneration (AMD). Newly synthesized quaternary derivatives of 1,4-diazabicyclo[2.2.2]octane (DABCO) and quinuclidine surfactants have been screened with the purpose to select the best compound to formulate long-term stable NEs that combine the best physicochemical properties for the loading of TA intended for ocular administration.

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.

In Silico Finding of Key Interaction Mediated α3ß4 and α7 Nicotinic Acetylcholine Receptor Ligand Selectivity of Quinuclidine-Triazole Chemotype.

The selective binding of six (S)-quinuclidine-triazoles and their (R)-enantiomers to nicotinic acetylcholine receptor (nAChR) subtypes α3ß4 and α7, respectively, were analyzed by in silico docking to provide the insight into the molecular basis for the observed stereospecific subtype discrimination. Homology modeling followed by molecular docking and molecular dynamics (MD) simulations revealed that unique amino acid residues in the complementary subunits of the nAChR subtypes are involved in subtype-specific selectivity profiles. In the complementary ß4-subunit of the α3ß4 nAChR binding pocket, non-conserved AspB173 through a salt bridge was found to be the key determinant for the α3ß4 selectivity of the quinuclidine-triazole chemotype, explaining the 47-327-fold affinity of the (S)-enantiomers as compared to their (R)-enantiomer counterparts. Regarding the α7 nAChR subtype, the amino acids promoting a however significantly lower preference for the (R)-enantiomers were the conserved TyrA93, TrpA149 and TrpB55 residues. The non-conserved amino acid residue in the complementary subunit of nAChR subtypes appeared to play a significant role for the nAChR subtype-selective binding, particularly at the heteropentameric subtype, whereas the conserved amino acid residues in both principal and complementary subunits are essential for ligand potency and efficacy.

Synergic effect of a quinuclidine benzamide complexed with borane, the LMA10233, in combination with seven pesticides.

Some quinuclidine benzamide compounds have been found to modulate nicotinic acetylcholine receptors in both mammals and insects. In particular, the quaternarization of 3-amino quinuclidine benzamide derivatives with dichloromethane gave charged N-chloromethylated quinuclidine compounds, disclosing an antagonist profile on homomeric α7 nAChRs. Here, we synthesized and studied the toxicological effect of LMA10233, a quinuclidine-borane complex analogue, the LMA10233, on the pea aphid Acyrthosiphon pisum and found that LMA10233 only exhibit proper toxicity on A. pisum larvae when applied in concentrations of over 10 µg/ml. We assessed the ability of LMA10233 to enhance the toxicity of different insecticides. When a sublethal concentration of LMA10233 was combined with the LC10 of each compound, we found a strong increase in toxicity at 24 h and 48 h of exposure for clothianidin, fipronil and chlorpyrifos, and only at 24 h for imidacloprid, acetamiprid and deltamethrin. However, when the pesticide was used at the LC50, only acetamiprid showed a synergistic effect with LMA10233. When the concentration of LMA10233 was decreased, we found that up to 80-90% of mortality was obtained due to the synergism between acetamiprid and LMA10233. No similar effect was observed with other insecticides. We conclude that such quinuclidine-borane complex compounds could increase the toxic effect of insecticides at low concentrations.

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.

Three new quinuclidine-based structures: second harmonic generation response for 1,2-bis(1-azoniabicyclo[2.2.2]octan-3-ylidene)hydrazine dichloride.

The crystal structures of three quinuclidine-based compounds, namely (1-azabicyclo[2.2.2]octan-3-ylidene)hydrazine monohydrate, C7H13N3·H2O (1), 1,2-bis(1-azabicyclo[2.2.2]octan-3-ylidene)hydrazine, C14H22N4 (2), and 1,2-bis(1-azoniabicyclo[2.2.2]octan-3-ylidene)hydrazine dichloride, C14H24N42+·2Cl- (3), are reported. In the crystal structure of 1, the quinuclidine-substituted hydrazine and water molecules are linked through N-H...O and O-H...N hydrogen bonds, forming a two-dimensional array. The compound crystallizes in the centrosymmetric space group P21/c. Compound 2 was refined in the space group Pccn and exhibits no hydrogen bonding. However, its hydrochloride form 3 crystallizes in the noncentrosymmetric space group Pc. It shows a three-dimensional network structure via intermolecular hydrogen bonding (N-H...C and N/C-H...Cl). Compound 3, with its acentric structure, shows strong second harmonic 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.

Crystal structure and Hirshfeld surface analysis of 2,4,6,11-tetra-kis-(4-fluoro-phen-yl)-9-oxa-1,5-di-aza-tri-cyclo-[5.3.1.03.8]undeca-ne.

The title compound, C32H26F4N2O, crystallizes in the monoclinic space group P21/n with four mol-ecules in the unit cell. The compound was prepared by the NaBH4 reduction of 4,8,9,10-tetra-kis-(4-fluoro-phen-yl)-1,3-di-aza-adamantan-6-one in chloro-form and ethanol as solvent. The piperidine rings exhibit chair and boat conformations, and all four fluoro-phenyl groups are oriented in the equatorial direction. The crystal structure features C-H⋯F hydrogen bonds, C-H⋯π, N-H⋯π and π-π inter-actions. Hirshfeld surface and two-dimensional fingerprint analysis show that van der Waals inter-actions constitute a major contribution to the inter-molecular inter-actions, with H⋯H contacts accounting for 37.9% of the surface.

Novel 5-(quinuclidin-3-ylmethyl)-1,2,4-oxadiazoles to investigate the activation of the α7 nicotinic acetylcholine receptor subtype: Synthesis and electrophysiological evaluation.

α7 nicotinic acetylcholine receptors (nAChRs) are relevant therapeutic targets for a variety of disorders including neurodegeneration, cognitive impairment, and inflammation. Although traditionally identified as an ionotropic receptor, the α7 subtype showed metabotropic-like functions, mainly linked to the modulation of immune responses. In the present work, we investigated the structure-activity relationships in a set of novel α7 ligands incorporating the 5-(quinuclidin-3-ylmethyl)-1,2,4-oxadiazole scaffold, i.e. derivatives 21a-34a and 21b-34b, aiming to identify the structural requirements able to preferentially trigger one of the two activation modes of this receptor subtype. The new compounds were characterized as partial and silent α7 nAChR agonists in electrophysiological assays, which allowed to assess the contribution of the different groups towards the final pharmacological profile. Overall, modifications of the selected structural backbone mainly afforded partial agonists, among them tertiary bases 27a-33a, whereas additional hydrogen-bond acceptor groups in permanently charged ligands, such as 29b and 31b, favored a silent desensitizing profile at the α7 nAChR.

Characterization of structurally novel G protein biased CB1 agonists: Implications for drug development.

The human cannabinoid subtype 1 receptor (hCB1R) is highly expressed in the CNS and serves as a therapeutic target for endogenous ligands as well as plant-derived and synthetic cannabinoids. Unfortunately, acute use of hCB1R agonists produces unwanted psychotropic effects and chronic administration results in development of tolerance and dependence, limiting the potential clinical use of these ligands. Studies in ß-arrestin knockout mice suggest that interaction of certain GPCRs, including µ-, δ-, κ-opioid and hCB1Rs, with ß-arrestins might be responsible for several adverse effects produced by agonists acting at these receptors. Indeed, agonists that bias opioid receptor activation toward G-protein, relative to ß-arrestin signaling, produce less severe adverse effects. These observations indicate that therapeutic utility of agonists acting at hCB1Rs might be improved by development of G-protein biased hCB1R agonists. Our laboratory recently reported a novel class of indole quinulidinone (IQD) compounds that bind cannabinoid receptors with relatively high affinity and act with varying efficacy. The purpose of this study was to determine whether agonists in this novel cannabinoid class exhibit ligand bias at hCB1 receptors. Our studies found that a novel IQD-derived hCB1 receptor agonist PNR-4-20 elicits robust G protein-dependent signaling, with transduction ratios similar to the non-biased hCB1R agonist CP-55,940. In marked contrast to CP-55,940, PNR-4-20 produces little to no ß-arrestin 2 recruitment. Quantitative calculation of bias factors indicates that PNR-4-20 exhibits from 5.4-fold to 29.5-fold bias for G protein, relative to ß-arrestin 2 signaling (when compared to G protein activation or inhibition of forskolin-stimulated cAMP accumulation, respectively). Importantly, as expected due to reduced ß-arrestin 2 recruitment, chronic exposure of cells to PNR-4-20 results in significantly less desensitization and down-regulation of hCB1Rs compared to similar treatment with CP-55,940. PNR-4-20 (i.p.) is active in the cannabinoid tetrad in mice and chronic treatment results in development of less persistent tolerance and no significant withdrawal signs when compared to animals repeatedly exposed to the non-biased full agoinst JWH-018 or Δ9-THC. Finally, studies of a structurally similar analog PNR- 4-02 show that it is also a G protein biased hCB1R agonist. It is predicted that cannabinoid agonists that bias hCB1R activation toward G protein, relative to ß-arrestin 2 signaling, will produce fewer and less severe adverse effects both acutely and chronically.

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.

BMS-933043, a Selective α7 nAChR Partial Agonist for the Treatment of Cognitive Deficits Associated with Schizophrenia.

The therapeutic treatment of negative symptoms and cognitive dysfunction associated with schizophrenia is a significant unmet medical need. Preclinical literature indicates that α7 neuronal nicotinic acetylcholine (nACh) receptor agonists may provide an effective approach to treating cognitive dysfunction in schizophrenia. We report herein the discovery and evaluation of 1c (BMS-933043), a novel and potent α7 nACh receptor partial agonist with high selectivity against other nicotinic acetylcholine receptor subtypes (>100-fold) and the 5-HT3A receptor (>300-fold). In vivo activity was demonstrated in a preclinical model of cognitive impairment, mouse novel object recognition. BMS-933043 has completed Phase I clinical trials.

Development of 4-Heteroarylamino-1'-azaspiro[oxazole-5,3'-bicyclo[2.2.2]octanes] as α7 Nicotinic Receptor Agonists.

We describe the synthesis of quinuclidine-containing spiroimidates and their utility as α7 nicotinic acetylcholine receptor (nAChR) partial agonists. A convergent synthetic route allowed for rapid SAR investigation and provided a diverse set of fused 6,5-heteroaryl analogs. Two potent and selective α7 nAChR partial agonists, (1'S,3'R,4'S)-N-(7-bromopyrrolo[2,1-f][1,2,4]triazin-4-yl)-4H-1'-azaspiro[oxazole-5,3'-bicyclo[2.2.2]octan]-2-amine (20) and (1'S,3'R,4'S)-N-(7-chloropyrrolo[2,1-f][1,2,4]triazin-4-yl)-4H-1'-azaspiro[oxazole-5,3'-bicyclo[2.2.2]octan]-2-amine (21), were identified. Both agonists improved cognition in a preclinical rodent model of learning and memory. Additionally, 5-HT3A receptor SAR suggested the presence of a steric site that when engaged led to significant loss of affinity at that receptor.

Varying Chirality Across Nicotinic Acetylcholine Receptor Subtypes: Selective Binding of Quinuclidine Triazole Compounds.

The novel quinuclidine anti-1,2,3-triazole derivatives T1-T6 were designed based on the structure of QND8. The binding studies revealed that the stereochemistry at the C3 position of the quinuclidine scaffold plays an important role in the nAChR subtype selectivity. Whereas the (R)-enantiomers are selective to α7 over α4ß2 (by factors of 44-225) and to a smaller degree over α3ß4 (3-33), their (S)-counterparts prefer α3ß4 over α4ß2 (62-237) as well as over α7 (5-294). The (R)-derivatives were highly selective to α7 over α3ß4 subtypes compared to (RS)- and (R)-QND8. The (S)-enantiomers are 5-10 times more selective to α4ß2 than their (R) forms. The overall strongest affinity is observed for the (S)-enantiomer binding to α3ß4 (Ki, 2.25-19.5 nM) followed by their (R)-counterpart binding to α7 (Ki, 22.5-117 nM), with a significantly weaker (S)-enantiomer binding to α4ß2 (Ki, 414-1980 nM) still above the very weak respective (R)-analogue affinity (Ki, 5059-10436 nM).

Design of α7 nicotinic acetylcholine receptor ligands using the (het)Aryl-1,2,3-triazole core: Synthesis, in vitro evaluation and SAR studies.

We report here the synthesis of a large library of 1,2,3-triazole derivatives which were in vitro tested as α7 nAchR ligands. The SAR study revealed that several crucial factors are involved in the affinity of these compounds for α7 nAchR such as a (R) quinuclidine configuration and a mono C-3 quinuclidine substitution. The triazole ring was substituted by a phenyl ring bearing small OMe/CH2F groups or fluorine atom and by several heterocycles such as thiophenes, furanes, benzothiophenes or benzofuranes. Among the 30 derivatives tested, the two derivatives 10 and 39 with Ki in the nanomolar range were identified (2.3 and 3 nM respectively). They exhibited a strict selectivity toward the α4ß2 nicotinic receptor (up to 1 µM) but interacted with the 5HT3 receptors with Ki around 3 nM. Synthesis, SAR studies and a full description of the derivatives are reported.

Design of α7 nicotinic acetylcholine receptor ligands in quinuclidine, tropane and quinazoline series. Chemistry, molecular modeling, radiochemistry, in vitro and in rats evaluations of a [(18)F] quinuclidine derivative.

In this report, we describe the synthesis of a novel library of α7 nAChR ligands based on the modulation of the quinuclidine, quinazoline and tropane moieties. Spirane derivatives were newly synthesized under stereo specific 1,3 dipolar cylcoadditions. Only amide derivatives bonded efficiently to the receptor with Ki measured between 14 and 133 nM. The best fluorinated candidate was selected and radiolabeled. The potent [(18)F]4 PET tracer was evaluated in rats and its brain accumulation quantified.

QM-polarized ligand docking accurately predicts the trend in binding affinity of a series of arylmethylene quinuclidine-like derivatives at the α4ß2 and α3ß4 nicotinic acetylcholine receptors (nAChRs).

Compounds containing a quinuclidine scaffold are promising drug candidates for pharmacological management of the central nervous system (CNS) pathologies implicating nAChRs. We have carried out binding affinity and in-silico docking studies of arylmethylene quinuclidine-like derivatives at the α4ß2 receptor using in-vitro receptor binding assay and comparative modeling, respectively. We found that introducing a hydrogen-bond acceptor into the 3-benzylidene quinuclidine derivative resulted in a 266-fold increase in binding affinity and confers agonism properties. By contrast, addition of a phenyl group to 3-benzylidene quinuclidine derivative only results in an 18-fold increase in binding affinity, without conferring agonism. We also found that docking into the orthosteric binding site of the α4ß2 nAChR is consistent with the fact that the basic nitrogen atom donates a hydrogen-bond to the carbonyl group of the highly conserved Trp-149, as initially observed by Dougherty and co-workers.(1) The experimentally-observed trend in binding affinity at both α4ß2 and α3ß4 nAChRs was accurately and independently confirmed by quantum mechanics (QM)-polarized docking. The reduction in binding affinity to the α3ß4 subtype primarily results from a dampening of both coulombic and cation-π interactions.