Profiling Novel Quinuclidine-Based Derivatives as Potential Anticholinesterase Drugs: Enzyme Inhibition and Effects on Cell Viability.

The cholinergic system, relying on the neurotransmitter acetylcholine (ACh), plays a significant role in muscle contraction, cognition, and autonomic nervous system regulation. The enzymes acetylcholinesterase, AChE, and butyrylcholinesterase, BChE, responsible for hydrolyzing ACh, can fine-tune the cholinergic system's activity and are, therefore, excellent pharmacological targets to address a range of medical conditions. We designed, synthesized, and profiled 14 N-alkyl quaternary quinuclidines as inhibitors of human AChE and BChE and analyzed their impact on cell viability to assess their safety in the context of application as potential therapeutics. Our results showed that all of the 14 tested quinuclidines inhibited both AChE and BChE in the micromolar range (Ki = 0.26 - 156.2 µM). The highest inhibition potency was observed for two bisquaternary derivatives, 7 (1,1'-(decano)bis(3-hydroxyquinuclidinium bromide)) and 14 (1,1'-(decano)bis(3-hydroxyiminoquinuclidinium bromide)). The cytotoxic effect within 7-200 µM was observed only for monoquaternary quinuclidine derivatives, especially those with the C12-C16 alkyl chain. Further analysis revealed a time-independent mechanism of action, significant LDH release, and a decrease in the cells' mitochondrial membrane potential. Taking all results into consideration, we can confirm that a quinuclidine core presents a good scaffold for cholinesterase binding and that two bisquaternary quinuclidine derivatives could be considered as candidates worth further investigations as drugs acting in the cholinergic system. On the other hand, specific cell-related effects probably triggered by the free long alkyl chain in monoquaternary quinuclidine derivatives should not be neglected in future N-alkyl quaternary quinuclidine derivative structure refinements. Such an effect and their potential to interact with other specific targets, as indicated by a pharmacophore model, open up a new perspective for future investigations of these compounds' scaffold in the treatment of specific conditions and diseases other than cholinergic system-linked disorders.

Polytopal Rearrangement Governing Stereochemistry of Bicyclic Oxime Ether Synthesis.

In the present study, four O-substituted oximes of quinuclidin-3-one were synthesized using appropriate O-substituted hydroxylamine hydrochlorides. In order to perform these reactions in a solvent, a mixture of (E) and (Z) products was yielded. Using mechanochemical and microwave synthesis, we then obtained pure (E) oximes. In almost all cases, the conversion to oxime ethers was completed. Reactions were monitored by ATR spectroscopy and the ratios of (E) and (Z) oxime ethers were deduced from 1H NMR data. Several reactions were very rapid (1 min) with 100% conversion and stereospecificity. To investigate the reaction mechanisms, full conformational analyses of the reaction intermediates were performed and the lowest energy conformers were determined. These conformers differed in spatial arrangement around the nitrogen atom of the amino group and were in the correct orientation for reactions to occur. Calculated standard Gibbs energies of the formation were in agreement with the experimentally obtained ratios of (E) and (Z) isomers. This work shows alternatives to the classical synthesis of O-substituted oxime ether precursors and highlights the fast reaction rate and stereoselectivity of microwave synthesis as well as the "green" aspects of mechanochemistry.

The mode of antibacterial action of quaternary N-benzylimidazole salts against emerging opportunistic pathogens.

Quaternary ammonium compounds (QACs) are antimicrobial agents displaying a broad spectrum of activity due to their mechanism of action targeting the bacterial membrane. The emergence of bacterial resistance to QACs, especially in times of pandemics, requires the continuous search for new and potent QACs structures. Here we report the synthesis and biological evaluation of QACs based on imidazole derivative, N-benzylimidazole. The antimicrobial activity was tested against a range of pathogenic bacteria and fungi, both ATCC and clinical isolates, showing varying activities ranging in minimal inhibitory concentrations (MICs) from as low as 7 ng/mL. The most promising compound, N-tetradecyl derivative (BnI-14), proved to be very potent against bacterial biofilms, even at sub-MIC doses, suggesting interference with the bacterial growth and/or division process. The BnI-14 treatment induces bacterial membrane disruption, as observed by fluorescence spectroscopy and atomic force microscopy and it also binds to DNA indicating that bacterial membrane might not be the only cellular target of QACs. Most importantly, BnI-14 exhibits low toxicity to healthy human cell lines, suggesting that N-benzylimidazolium-based QACs may be promising new antimicrobial agents.

Targeting organophosphorus compounds poisoning by novel quinuclidine-3 oximes: development of butyrylcholinesterase-based bioscavengers.

A library of 14 mono-oxime quinuclidinium-based compounds with alkyl or benzyl substituent were synthesized and characterized in vitro as potential antidotes for organophosphorus compounds (OP) poisoning treatment. We evaluated their potency for reversible inhibition and reactivation of OP inhibited human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) and evaluated interactions by molecular docking studies. The reactivation was notable for both AChE and BChE inhibited by VX, cyclosarin, sarin and paraoxon, if quinuclidinium compounds contained the benzyl group attached to the quinuclidinium moiety. Out of all 14, oxime Q8 [4-bromobenzyl-3-(hydroxyimino)quinuclidinium bromide] was singled out as having the highest determined overall reactivation rate of approximately 20,000 M-1 min-1 for cyclosarin-inhibited BChE. Furthermore, this oxime in combination with BChE exhibited a capability to act as a bioscavenger of cyclosarin, degrading within 2 h up to 100-fold excess of cyclosarin concentration over the enzyme. Molecular modeling revealed that the position of the cyclohexyl moiety conjugated with the active site serine of BChE directs the favorable positioning of the quinuclidinium ring and the bromophenyl moiety of Q8, which makes phosphonylated-serine easily accessible for the nucleophilic displacement by the oxime group of Q8. This result presents a novel scaffold for the development of new BChE-based bioscavengers. Furthermore, a cytotoxic effect was not observed for Q8, which also makes it promising for further in vivo reactivation studies.

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.

Novel Imidazole Aldoximes with Broad-Spectrum Antimicrobial Potency against Multidrug Resistant Gram-Negative Bacteria.

In the search for a new class of potential antimicrobial agents, five novel N-substituted imidazole 2-aldoximes and their six quaternary salts were evaluated. The antimicrobial activity was assessed against a panel of representative Gram-positive and Gram-negative bacteria, including multidrug resistant bacteria. All compounds demonstrated potent in vitro activity against the tested microorganisms, with MIC values ranging from 6.25 to 50.0 µg/mL. Among the tested compounds, two quaternary compounds (N-but-3-enyl- and meta- (10) or para- N-chlorobenzyl (11) imidazolium 2-aldoximes) displayed the most potent and broad-spectrum activity against both Gram-positive and Gram-negative bacterial strains. The broth microdilution assay was also used to investigate the antiresistance efficacy of the both most active compounds against a set of Enterobacteriaceae isolates carried a multiple extended-spectrum ß-lactamases (ESBLs) in comparison to eight clinically relevant antibiotics. N-but-3-enyl-N-meta-chlorobenzyl imidazolium 2-aldoxime was found to possess promising antiresistance efficacy against a wide range of ß-lactamases producing strains (MIC 2.0 to 16.0 µg/mL). Best results for that compound were obtained against Escherichia coli and Enterobacter cloacae producing multiple ß-lactamases form A and C molecular classes, which were 32- and 128-fold more potent than ceftazidime and cefotaxime, respectively. To visualize the results, principal component analysis was used as an additional classification tool. The mixture of ceftazidime and compound 10 (3 µg:2 µg) showed a strong activity and lower the necessary amount (up to 40-fold) of 10 against five of ESBL-producing isolates (MIC ≤ 1 µg/mL).

New Cinchona Oximes Evaluated as Reactivators of Acetylcholinesterase and Butyrylcholinesterase Inhibited by Organophosphorus Compounds.

For the last six decades, researchers have been focused on finding efficient reactivators of organophosphorus compound (OP)-inhibited acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). In this study, we have focused our research on a new oxime scaffold based on the Cinchona structure since it was proven to fit the cholinesterases active site and reversibly inhibit their activity. Three Cinchona oximes (C1, C2, and C3), derivatives of the 9-oxocinchonidine, were synthesized and investigated in reactivation of various OP-inhibited AChE and BChE. As the results showed, the tested oximes were more efficient in the reactivation of BChE and they reactivated enzyme activity to up to 70% with reactivation rates similar to known pyridinium oximes used as antidotes in medical practice today. Furthermore, the oximes showed selectivity towards binding to the BChE active site and the determined enzyme-oxime dissociation constants supported work on the future development of inhibitors in other targeted studies (e.g., in treatment of neurodegenerative disease). Also, we monitored the cytotoxic effect of Cinchona oximes on two cell lines Hep G2 and SH-SY5Y to determine the possible limits for in vivo application. The cytotoxicity results support future studies of these compounds as long as their biological activity is targeted in the lower micromolar range.

Spectroscopic and Chemometric Analysis of Binary and Ternary Edible Oil Mixtures: Qualitative and Quantitative Study.

The aim of this study was to investigate the feasibility of FTIR-ATR spectroscopy coupled with the multivariate numerical methodology for qualitative and quantitative analysis of binary and ternary edible oil mixtures. Four pure oils (extra virgin olive oil, high oleic sunflower oil, rapeseed oil, and sunflower oil), as well as their 54 binary and 108 ternary mixtures, were analyzed using FTIR-ATR spectroscopy in combination with principal component and discriminant analysis, partial least-squares, and principal component regression. It was found that the composition of all 166 samples can be excellently represented using only the first three principal components describing 98.29% of total variance in the selected spectral range (3035-2989, 1170-1140, 1120-1100, 1093-1047, and 930-890 cm(-1)). Factor scores in 3D space spanned by these three principal components form a tetrahedral-like arrangement: pure oils being at the vertices, binary mixtures at the edges, and ternary mixtures on the faces of a tetrahedron. To confirm the validity of results, we applied several cross-validation methods. Quantitative analysis was performed by minimization of root-mean-square error of cross-validation values regarding the spectral range, derivative order, and choice of method (partial least-squares or principal component regression), which resulted in excellent predictions for test sets (R(2) > 0.99 in all cases). Additionally, experimentally more demanding gas chromatography analysis of fatty acid content was carried out for all specimens, confirming the results obtained by FTIR-ATR coupled with principal component analysis. However, FTIR-ATR provided a considerably better model for prediction of mixture composition than gas chromatography, especially for high oleic sunflower oil.

A comprehensive evaluation of novel oximes in creation of butyrylcholinesterase-based nerve agent bioscavengers.

A well-considered treatment of acute nerve agents poisoning involves the exogenous administration of butyrylcholinesterase (BChE, EC 3.1.1.8) as a stoichiometric bioscavenger efficient in preventing cholinergic crises caused by acetylcholinesterase (AChE, EC 3.1.1.7) inhibition. An additional improvement in medical countermeasures would be to use oximes that could reactivate BChE as well to upgrade bioscavenging from stoichiometric to oxime-assisted catalytic. Therefore, in this paper we investigated the potency of 39 imidazolium and benzimidazolium oximes (36 compounds synthesized for the first time) to be considered as the reactivators specifically designed for reactivation of phosphylated human BChE. Their efficiency in the reactivation of paraoxon-, VX-, and tabun-inhibited human BChE, as well as human AChE was tested and compared with the efficiencies of HI-6 and obidoxime, used in medical practice today. A comprehensive analysis was performed for the most promising oximes defining kinetic parameters of reactivation as well as interactions with uninhibited BChE. Furthermore, experimental data were compared with computational studies (docking, QSAR analysis) as a starting point in future oxime structure refinement. Considering the strict criteria set for in vivo applications, we determined the cytotoxicity of lead oximes on two cell lines. Among the tested oxime library, one imidazolium compound was selected for preliminary in vivo antidotal study in mice. The obtained protection in VX poisoning outlines its potential in development oxime-assisted OP-bioscavenging with BChE.

Deep reinforcement learning classification of sparkling wines based on ICP-MS and DOSY NMR spectra.

An approach that combines NMR spectroscopy and inductively coupled plasma mass spectrometry (ICP-MS) and advanced tensor decomposition algorithms with state-of-the-art deep learning procedures was applied for the classification of Croatian continental sparkling wines by their geographical origin. It has been demonstrated that complex high-dimensional NMR or ICP-MS data cannot be classified by higher-order tensor decomposition alone. Extension of the procedure by deep reinforcement learning resulted in an exquisite neural network predictive model for the classification of sparkling wines according to their geographical origin. A network trained on half of the sample set was able to classify even 94% of all samples. The model can particularly be useful in cases where the number of samples is limited and when simpler statistical methods fail to produce reliable data. The model can further be exploited for the identification and differentiation of sparkling wines including a high potential for authenticity or quality control.

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.

Further Study of the Polar Group's Influence on the Antibacterial Activity of the 3-Substituted Quinuclidine Salts with Long Alkyl Chains.

Quaternary ammonium compounds (QACs) are among the most potent antimicrobial agents increasingly used by humans as disinfectants, antiseptics, surfactants, and biological dyes. As reports of bacterial co- and cross-resistance to QACs and their toxicity have emerged in recent years, new attempts are being made to develop soft QACs by introducing hydrolyzable groups that allow their controlled degradation. However, the development of such compounds has been hindered by the structural features that affect the bioactivity of QACs, one of them being polarity of the substituent near the quaternary center. To further investigate the influence of the polar group on the bioactivity of QACs, we synthesized 3-aminoquinuclidine salts for comparison with their structural analogues, 3-acetamidoquinuclidines. We found that the less polar amino-substituted compounds exhibited improved antibacterial activity over their more polar amide analogues. In addition to their better minimum inhibitory concentrations, the candidates were excellent at suppressing Staphylococcus aureus biofilm formation and killing bacteria almost immediately, as shown by the flow cytometry measurements. In addition, two candidates, namely QNH2-C14 and QNH2-C16, effectively suppressed bacterial growth even at concentrations below the MIC. QNH2-C14 was particularly effective at subinhibitory concentrations, inhibiting bacterial growth for up to 6 h. In addition, we found that the compounds targeted the bacterial membrane, leading to its perforation and subsequent cell death. Their low toxicity to human cells and low potential to develop bacterial resistance suggest that these compounds could serve as a basis for the development of new QACs.

Catalytic activity of halohydrin dehalogenases towards spiroepoxides.

A novel activity of halohydrin dehalogenases towards spiroepoxides has been found. The enzyme from Arthrobacter sp. (HheA) catalysed highly regioselective azidolysis of spiroepoxides containing 5, 6 and 7-membered cycloalkane rings, while the enzyme from Agrobacterium radiobacter (HheC), besides high regioselectivity, also displayed moderate to high enantioselectivity (E up to >200) that can be applied for the kinetic resolution of chiral spiroepoxides. The orientations of spiroepoxides in the active site of halohydrin dehalogenases were studied by quantum-chemical calculations and docking simulations. Analyses of the complexes obtained revealed the origins of diastereoselectivity and enantioselectivity of the investigated biotransformations.

Adamantane-substituted guanylhydrazones: novel inhibitors of butyrylcholinesterase.

A series of novel adamantane-substituted guanylhydrazones was synthesized and used in a study of inhibitory potential toward butyrylcholinesterase. The experimental results were further supported by using docking studies to examine the behavior of the inhibitors within the active site regions of the enzyme. The enzyme-inhibitor dissociation constants K(i) were determined from Hunter-Downs diagrams using Ellman's method for cholinesterase activity determination. Compounds 2-(N-guanidino)iminoadamantane hydrochloride (1) and 2,4-bis(N,N'-guanidino)iminoadamantane dihydrochloride (2) were found to be the best BChE inhibitors and their affinities for the enzyme active site were about five times higher compared to the enzyme peripheral site. The strongest interaction observed in complexes obtained by docking studies was the H-bond between the guanidine and the carboxylate of Glu199 and the second guanidine group in bisguanidine compounds was stabilized with additional H-bonds.

Preparation of novel meta- and para-substituted N-benzyl protected quinuclidine esters and their resolution with butyrylcholinesterase.

Since the optically active quinuclidin-3-ol is an important intermediate in the preparation of physiologically or pharmacologically active compounds, a new biocatalytic method for the production of chiral quinuclidin-3-ols was examined. Butyrylcholinesterase (BChE; EC 3.1.1.8) was chosen as a biocatalyst in a preparative kinetic resolution of enantiomers. A series of racemic, (R)- and (S)-esters of quinuclidin-3-ol and acetic, benzoic, phthalic and isonicotinic acids were synthesized, as well as their racemic quaternary N-benzyl, meta- and para-N-bromo and N-methylbenzyl derivatives. After the resolution, all N-benzyl protected groups were successfully removed by catalytic transfer hydrogenation with ammonium formate (10% Pd-C). Hydrolyses studies with BChE confirmed that (R)-enantiomers of the prepared esters are much better substrates for the enzyme than (S)-enantiomers. Introduction of bromine atom or methyl group in the meta or para position of the benzyl moiety resulted in a considerable improvement of the stereoselectivity compared to the non-substituted compounds. Optically pure quinuclidin-3-ols were prepared in high yields and enantiopurity by the usage of various N-benzyl protected groups and BChE as a biocatalyst.

Synthesis, Biological Evaluation and Machine Learning Prediction Model for Fluorinated Cinchona Alkaloid-Based Derivatives as Cholinesterase Inhibitors.

A series of 46 Cinchona alkaloid derivatives that differ in positions of fluorine atom(s) in the molecule were synthesized and tested as human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. All tested compounds reversibly inhibited AChE and BChE in the nanomolar to micromolar range; for AChE, the determined enzyme-inhibitor dissociation constants (Ki) ranged from 3.9-80 µM, and 0.075-19 µM for BChE. The most potent AChE inhibitor was N-(para-fluorobenzyl)cinchoninium bromide, while N-(meta-fluorobenzyl)cinchonidinium bromide was the most potent BChE inhibitor with Ki constant in the nanomolar range. Generally, compounds were non-selective or BChE selective cholinesterase inhibitors, where N-(meta-fluorobenzyl)cinchonidinium bromide was the most selective showing 533 times higher preference for BChE. In silico study revealed that twenty-six compounds should be able to cross the blood-brain barrier by passive transport. An extensive machine learning procedure was utilized for the creation of multivariate linear regression models of AChE and BChE inhibition. The best possible models with predicted R2 (CD-derivatives) of 0.9932 and R2(CN-derivatives) of 0.9879 were calculated and cross-validated. From these data, a smart guided search for new potential leads can be performed. These results pointed out that quaternary Cinchona alkaloids are the promising structural base for further development as selective BChE inhibitors which can be used in the central nervous system.

New Membrane Active Antibacterial and Antiviral Amphiphiles Derived from Heterocyclic Backbone of Pyridinium-4-Aldoxime.

Quaternary ammonium salts (QAS) are irreplaceable membrane-active antimicrobial agents that have been widely used for nearly a century. Cetylpyridinium chloride (CPC) is one of the most potent QAS. However, recent data from the literature indicate that CPC activity against resistant bacterial strains is decreasing. The major QAS resistance pathway involves the QacR dimer, which regulates efflux pump expression. A plausible approach to address this issue is to structurally modify the CPC structure by adding other biologically active functional groups. Here, a series of QAS based on pyridine-4-aldoxime were synthesized, characterized, and tested for antimicrobial activity in vitro. Although we obtained several potent antiviral candidates, these candidates had lower antibacterial activity than CPC and were not toxic to human cell lines. We found that the addition of an oxime group to the pyridine backbone resulted in derivatives with large topological polar surfaces and with unfavorable cLog P values. Investigation of the antibacterial mode of action, involving the cell membrane, revealed altered cell morphologies in terms of corrugated and/or disrupted surface, while 87% of the cells studied exhibited a permeabilized membrane after 3 h of treatment at 4 × minimum inhibitory concentration (MIC). Molecular dynamic (MD) simulations of the interaction of QacR with a representative candidate showed rapid dimer disruption, whereas this was not observed for QacR and QacR bound to the structural analog CPC. This might explain the lower bioactivity of our compounds, as they are likely to cause premature expression of efflux pumps and thus activation of resistance.

Quinuclidine-Based Carbamates as Potential CNS Active Compounds.

The treatment of central nervous system (CNS) diseases related to the decrease of neurotransmitter acetylcholine in neurons is based on compounds that prevent or disrupt the action of acetylcholinesterase and butyrylcholinesterase. A series of thirteen quinuclidine carbamates were designed using quinuclidine as the structural base and a carbamate group to ensure the covalent binding to the cholinesterase, which were synthesized and tested as potential human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. The synthesized compounds differed in the substituents on the amino and carbamoyl parts of the molecule. All of the prepared carbamates displayed a time-dependent inhibition with overall inhibition rate constants in the 103 M-1 min-1 range. None of the compounds showed pronounced selectivity for any of the cholinesterases. The in silico determined ability of compounds to cross the blood-brain barrier (BBB) revealed that six compounds should be able to pass the BBB by passive transport. In addition, the compounds did not show toxicity toward cells that represented the main models of individual organs. By machine learning, the most optimal regression models for the prediction of bioactivity were established and validated. Models for AChE and BChE described 89 and 90% of the total variations among the data, respectively. These models facilitated the prediction and design of new and more potent inhibitors. Altogether, our study confirmed that quinuclidinium carbamates are promising candidates for further development as CNS-active drugs, particularly for Alzheimer's disease treatment.

Antimicrobial Activity of Quasi-Enantiomeric Cinchona Alkaloid Derivatives and Prediction Model Developed by Machine Learning.

Bacterial infections that do not respond to current treatments are increasing, thus there is a need for the development of new antibiotics. Series of 20 N-substituted quaternary salts of cinchonidine (CD) and their quasi-enantiomer cinchonine (CN) were prepared and their antimicrobial activity was assessed against a diverse panel of Gram-positive and Gram-negative bacteria. All tested compounds showed good antimicrobial potential (minimum inhibitory concentration (MIC) values 1.56 to 125.00 µg/mL), proved to be nontoxic to different human cell lines, and did not influence the production of reactive oxygen species (ROS). Seven compounds showed very strong bioactivity against some of the tested Gram-negative bacteria (MIC for E. coli and K. pneumoniae 6.25 µg/mL; MIC for P. aeruginosa 1.56 µg/mL). To establish a connection between antimicrobial data and potential energy surfaces (PES) of the compounds, activity/PES models using principal components of the disc diffusion assay and MIC and data towards PES data were built. An extensive machine learning procedure for the generation and cross-validation of multivariate linear regression models with a linear combination of original variables as well as their higher-order polynomial terms was performed. The best possible models with predicted R2(CD derivatives) = 0.9979 and R2(CN derivatives) = 0.9873 were established and presented. This activity/PES model can be used for accurate prediction of activities for new compounds based solely on their potential energy surfaces, which will enable wider screening and guided search for new potential leads. Based on the obtained results, N-quaternary derivatives of Cinchona alkaloids proved to be an excellent scaffold for further optimization of novel antibiotic species.

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.