A capillary electrophoresis-mass spectrometry (CE-MS) method was developed and validated for the determination of 19 amino acids and related compounds in excretions/secretions (ES) of Lucilia sericata larvae. Sample preparation consisted of dilution of ES with water, filtration and direct CE-MS analysis. Separation was performed with 50 mM formic acid as the background electrolyte, and methanol-water (1:1, v/v) containing 1% formic acid was used as sheath liquid. The total analysis time was 20 min. The method showed acceptable linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), sensitivity, apparent recovery and short-term applicability for freshly prepared samples. Correlation coefficients for the calibration curves ranged from 0.983 to 0.998. Precision was within 18% at the lowest quality control level and within 15% at medium and high levels. Accuracy ranged from 83% to 118% at the lowest level and from 85% to 115% at the other levels. In larval ES, the most abundant compounds were threonine (565 ± 7 µM), tryptophan (455 ± 6 µM), anserine (404 ± 7 µM), histidine (330 ± 14 µM) and cystine (279 ± 4 µM). The method offers a simple workflow for targeted profiling of amino acids and related compounds in larval ES.

Aromatic sulphonamides of aziridine-2-carboxylic acid have been reported by our group as PDI inhibitors displaying anti-thrombotic effects. A new compound with the acronym C-3399 was selected as a lead inhibitor of protein disulfide isomerase 1 (PDIA1) and 3 (PDIA3) with proven effectiveness. However, its mechanism of action remains unclear due to the rapid hydrolysis of its ester moiety. Here, we aimed to characterise the pharmacokinetics and pharmacodynamics of C-3399 in more detail. The main metabolite of C-3399, carboxylic acid contractually designated as C-3399-B, was identified using high resolution mass spectrometry and was later synthesised for further investigation. Although parent compound and its metabolite inhibit PDI and human platelet activation, the metabolite potency was considerably weaker (IC50 18.79 µM - PDIA1 and > 50.00 µM - PDIA3) than that of the parent compound (IC50 7.47 µM - PDIA1 and 28.00 µM – PDIA3). Metabolite C-3399-B also displayed weaker anti-platelet activity but accumulated inside platelets, in contrast to the parent molecule. The binding mechanism predicted by induced-fit docking models suggested different binding sites within ‘a’ domain for C-3399 and C-3399-B. The pharmacokinetic profiles of C-3399 and C-3399-B in rats showed immediate hydrolysis of the ester moiety and rapid elimination of the metabolite (t0.5 = 25 min). In conclusion, the results of the study indicate that the pharmacological effects of C-3399 and its active metabolite C-3399-B afford combined PDI inhibition through covalent and noncovalent binding in both extracellular and intracellular space.

Selective serotonin reuptake inhibitors (SSRIs) are commonly used to treat depression, but their chronic use is associated with side effects and residual symptoms of depression. Both effects induced by SSRIs are mediated by serotonin receptor-dependent signaling pathways, yet the molecular mechanisms underlying these effects remain unclear. Here, we investigated the impact of chronic and acute activation of the 5-HT7 receptor (5-HT7R) using the selective agonist AGH-194 in male mice. Behavioral assessment revealed that chronic AGH-194 administration induced depressive-like effects in the novelty suppressed feeding test (NSFT), female urine sniffing test (FUST), and novel object location test (NOLT). After acute injection, depressive-like effects were observed only in NSFT. At the molecular level, AGH-194 administration activated matrix metalloproteinase 9 (MMP-9) through a 5-HT7R-Gαs signaling-dependent mechanism. Acute treatment induced transient activation, while chronic treatment led to prolonged enzymatic activity, accompanied by a reduction in the expression of the GluA1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) in the hippocampus. At the cellular level, acute but not chronic AGH-194 treatment induced a shift toward more juvenile dendritic spine morphology in the CA1 and dentate gyrus (DG) regions of the hippocampus, along with an increase in dendritic spine density in DG. Electrophysiological recordings demonstrated that acute AGH-194 administration enhanced hippocampal excitability by increasing population spike amplitude in CA1. Chronic AGH-194 treatment further modulated short-term plasticity, increasing both population spike and extracellular field potential paired-pulse ratios (PS-PPR and EPSP-PPR) in CA1, while also enhancing the maximum EPSP slope amplitude. These findings provide novel evidence that chronic 5-HT7R activation can induce depressive-like behaviors in male mice, potentially through sustained MMP-9 activation and alterations in synaptic plasticity. Understanding the molecular and electrophysiological consequences of selective 5-HT7R stimulation may provide insights into receptor-specific mechanisms that could contribute to SSRI-induced side effects, thereby contributing to the development of improved antidepressant strategies.

Accurate prediction of tissue-to-plasma partition coefficients (Kp,tissue) is critical for physiologically based pharmacokinetic (PBPK) modeling, particularly for basic compounds (pKa > 7) that display complex distribution driven by lysosomal sequestration and acidic phospholipid binding. We developed a novel in vitro method to estimate the hepatocyte partitioning coefficient (Kp,hep) using isolated, metabolically inactivated rat hepatocytes. For selected basic compounds, in vitro Kp,hep correlated strongly with in vivo liver partitioning (R² = 0.97), supporting its utility as a surrogate for hepatic distribution. Two strategies were then evaluated to extrapolate this information to other tissues. A scaling-factor approach based on the Poulin and Theil tissue composition-based model applied a liver-derived scalar across 11 tissues, improving prediction accuracy, with 60% and 73% of Kp values within 2-fold and 3-fold of observed data, respectively (AFE 0.70; AAFE 2.2). A second, regression-based approach adapted a prior muscle-derived model by estimating muscle Kp from hepatocyte Kp and extending predictions to additional tissues, yielding 63% and 83% of values within 2-fold and 3-fold, respectively (AFE 1.5; AAFE 1.8). These findings demonstrate that hepatocyte-based partitioning provides a practical, mechanistically relevant, and scalable method for predicting tissue distribution of basic compounds in PBPK modeling.

BACKGROUND:The benzodiazepine midazolam is widely used pre- and intraoperatively in intensive care units. 1´-OH-midazolam, one of the major metabolites, is pharmacologically active. Accumulation of 1´-OH-midazolam, e.g. due to hepatic dysfunction or renal insufficiency, may therefore enhance pharmacological activity. Growing evidence suggests that sex, age, drug interactions, and inflammation also have an impact on midazolam disposition and activity. Due to the complex interplay of these factors, finding the optimal midazolam dose for each critically ill patient is challenging. METHODS:We aimed to elucidate the factors that contribute significantly to pharmacokinetics of midazolam and its main metabolites in patients undergoing cardiac surgery. We collected serum and urine samples from 15 patients 1, 2, 3, 4, and 5 hours after the beginning of cardiac surgery and determined the concentrations of midazolam, 1´-OH-midazolam, 4-OH-midazolam, 1´-OH-midazolam-β-D-glucuronide, and 4-OH-midazolam-β-D-glucuronide by LC-MS/MS. RESULTS:Oxidation to 4-OH-M and subsequent glucuronidation played a role in metabolism and elimination of midazolam in our patient cohort. Patients showed relatively variable concentrations of midazolam and its metabolites, due to differences in midazolam dose and administration routes, demographic and clinical parameters. Thus, we evaluated pharmacokinetic parameters for individual patients and not for the whole patient cohort. We established a logarithmic multiple regression model linking urinary concentrations of midazolam, 1´-OH-midazolam, and 1´-OH-midazolam-β-D-glucuronide with explanatory variables. CONCLUSION:Our model linked urinary concentrations of midazolam, 1´-OH-M, and 1´-OH-MG to serum concentration, age, surgery infusion volume, creatinine concentration, and/or body temperature.

Unquestionably, pancreatic cancer belongs to one of the most aggressive cancers with a very poor survival rate. Despite the constantly developing achievements of science there is still, a strong need to develop superior delivery systems to improve the efficacy of currently available synthetic drugs. Many bioactive food-delivered substances possess great anticancer properties but their use is limited due to their poor water solubility. In this study we developed a new nanoemulsion-based delivery system for phenethyl isothiocyanate (PEITC) which is one of the most clinical tested isothiocyanates. We determined the physical parameters of the obtained nanoemulsion using dynamic light scattering (DLS). The morphology of nanoemulsion with PEITC was analyzed using cryogenic electron microscopy. The developed nanocarriers were homogeneous, with satisfactory stability. The encapsulation efficiency of PEITC was high (81.89 +/- 1.72 %) and the leakage of bioactive material was not noticed during the testing period. Further, we investigated the cytotoxic activity of the developed formulation towards two pancreatic cancer cell lines (AsPC-1 and BxPc-3) and one normal cell line (NHDF) using MTT and ATP assays. Moreover, we demonstrated that PEITC loaded nanoemulsion decreased the level of glutathione at the tested concentrations. Pharmacokinetic studies demonstrated that, after i.v. administration, compound elimination was very slow, which resulted in a very long half-life, ca. 22 h, and widespread distribution throughout the body. This study suggests that nanoemulsion formulations could be a promising system able to encapsulate and transport pharmacologically active substances to the pancreatic cancer tissue.

Building on clinical reports of symptomatic improvement in stroke patients following zolpidem administration, we investigated its neuroprotective efficacy and underlying mechanisms in a preclinical model of ischemia/reperfusion (I/R) injury. Pharmacokinetic analyses revealed that zolpidem preferentially accumulates in the hypothalamus and frontal cortex, with a notably prolonged half-life in the hypothalamus, suggesting region-specific drug retention. Early post-reperfusion administration of zolpidem (within 1.5 h) significantly reduced infarct volume, lowered glutamate levels, and improved motor recovery, underscoring a critical therapeutic window for intervention. Mechanistically, zolpidem enhanced phasic GABAergic signaling via α1 GABA-A receptors, reduced NKCC1 mRNA expression, and maintained neurochemical homeostasis without altering GABA transporter or receptor protein levels. Notably, zolpidem suppressed the frequency of cortical spreading depolarizations (CSDs), key propagators of secondary neuronal injury, without affecting wave dynamics, and modulated neurovascular coupling. Ischemic stroke outcomes are worsened by CSDs - waves of neuronal depolarization that propagate through compromised brain tissue and exacerbate secondary damage. These findings position zolpidem as a promising candidate for drug repurposing in stroke, uniquely targeting both early neuroprotection and longer-term functional recovery through selective modulation of GABAergic signaling and CSD suppression. Future clinical trials are warranted to define optimal therapeutic timing and maximize clinical benefit.

Affective disorders, the leading causes of disability and premature death worldwide, require new and effective treatment strategies. Clinically used antidepressants and second-generation antipsychotic drugs, including vortioxetine and lurasidone, act as potent 5-HT7 receptor antagonists and improve cognitive functions in the patients with mood disorders. Additionally, 5-HT7 receptor-mediated activation of matrix metalloproteinase 9 (MMP-9) induces depressive-like behavior in mice. We designed and synthesized a series of 27 arylsulfonamide derivatives of 2-[(2-aryl/2-heteroaryl)phenoxy]ethyl-piperidines and examined their in vitro and in vivo effects. These compounds are closely related to the previously reported 5-HT7 receptor ligand (PZ-1129), developed in our laboratories. Our goal was to investigate the impact of heterocyclic ring replacement on receptor selectivity and metabolic stability, because the aryloxyl moiety was postulated to determine affinity for serotonin and dopamine receptors, and interactions with metabolizing enzymes. The study identified compound 57 as a potent, selective and metabolically stable 5-HT7 receptor inverse agonist of Gs signaling pathway. Bioavailable compound 57 shortened immobility in the forced swim test in mice and reversed PCP-induced cognitive deficits in the novel object recognition test in rats suggesting antidepressant-like and pro-cognitive effects. In addition, compound 57 reduced 5-HT7 receptor-mediated MMP-9 activity in the mouse hippocampus with efficacy comparable to the reference 5-HT7 receptor antagonist, SB-269970, further suggesting its purported antidepressant-like actions. These findings support the potential therapeutic application of targeting 5-HT7 receptor/MMP-9 signaling pathway for the treatment of affective disorders.

Aging exacerbates organ injury in endotoxemia, but it is not clear whether endotoxemia is associated with a specific, age-related profile of the endothelial response. Therefore, the aim of the study was to assess the pattern of endothelial response to lipopolysaccharide (LPS) in aged mice (18-month-old) as compared to young mice (3-month-old). Our analysis was based on functional endothelial responses measured in vivo by magnetic resonance imaging (MRI) and on a comprehensive panel of biomarkers of endothelial dysfunction measured by micro-flow liquid chromatography tandem mass spectrometry (microLC-MS/MS). In aged mice, the systemic inflammatory response (serum amyloid A, IL-1β, IL-2, eotaxin), kidney injury (urea), liver injury (ALT), and endothelial dysfunction induced by a relatively low dose of LPS (3 mg/kg) were all more pronounced as compared with young mice. Interestingly, in aged mice, LPS induced a different pattern of endothelial response compared to young mice, as evidenced by glycocalyx injury biomarkers (SDC-1, ESM-1), the endothelial permeability biomarkers (Angpt-2, sTie-2) and various hemostasis-related factors (sTM, TAFI, THBS-1). In contrast, biomarkers of endothelial inflammation (sVCAM-1, sICAM-1, sE-selectin, sP-selectin) and classical hemostasis biomarkers (PAI-1, t-PA, von Willebrand factor) displayed comparable responses to LPS in aged and young mice. In conclusion, aging does not indiscriminately potentiate LPS-induced inflammatory mediator generation in the current model of endotoxemia induced by a relatively low dose of LPS, but selectively alters the endothelial response in terms of glycocalyx injury, endothelial permeability, and hemostasis. Graphical Abstract

AIM:Protein disulfide isomerases (PDIs) are involved in platelet aggregation and intravascular thrombosis, but their role in regulating endothelial function is unclear. Here, we characterized the involvement of vascular PDIA1 in angiotensin II (Ang II)-induced endothelial dysfunction in mice. METHODS:Endothelial dysfunction was induced in C57BL/6JCmd male mice via Ang II subcutaneous infusion, and PDIA1 was inhibited with bepristat. Endothelial function was assessed in vivo with magnetic resonance imaging and ex vivo with a myography, while arterial stiffness was measured as pulse wave velocity. Nitric oxide (NO) bioavailability was measured in the aorta (spin-trapping electron paramagnetic resonance) and plasma (NO2 - and NO3 - levels). Oxidative stress, eNOS uncoupling (DHE-based aorta staining), and thrombin activity (thrombin-antithrombin complex; calibrated automated thrombography) were evaluated. RESULTS:The inhibition of PDIA1 by bepristat in Ang II-treated mice prevented the impairment of NO-dependent vasodilation in the aorta as evidenced by the response to acetylcholine in vivo, increased systemic NO bioavailability and the aortic NO production, and decreased vascular stiffness. Bepristat's effect on NO-dependent function was recapitulated ex vivo in Ang II-induced endothelial dysfunction in isolated aorta. Furthermore, bepristat diminished the Ang II-induced eNOS uncoupling and overproduction of ROS without affecting thrombin activity. CONCLUSION:In Ang II-treated mice, the inhibition of PDIA1 normalized the NO-ROS balance, prevented endothelial eNOS uncoupling, and, thereby, improved vascular function. These results indicate the importance of vascular PDIA1 in regulating endothelial function, but further studies are needed to elucidate the details of the mechanisms involved.

Substances derived from insects can serve therapeutic functions due to their diverse biological properties. This article focuses on the species Lucilia sericata and the benefits of larval therapy in patients who, due to hospitalization, have developed pressure ulcers and other difficult-to-heal wounds. Larval therapy, also known as maggot debridement therapy, employs sterile fly larvae to treat chronic, non-healing wounds by enzymatically degrading necrotic tissue and decreasing bacterial colonization. The larvae are applied to the wound for a period of 48-72 h, during which they effectively clean the wound and stimulate tissue regeneration. This therapeutic approach is particularly efficacious for recalcitrant wounds, such as diabetic foot ulcers and pressure sores, which have not responded to conventional treatments. Larvae may also constitute an alternative material in entomotoxicological studies to detect substances ingested at not only toxic but also therapeutic doses. The present work describes a method for assaying ciprofloxacin in L. sericata larvae using capillary electrophoresis coupled to mass spectrometry. The developed method features high sensitivity with a limit of quantification of 100 ± 0.018 ng/mL, as well as accuracy and precision estimated within 87%-103% and 1%-4%, respectively. An application of a simple and fast precipitation of proteins procedure for sample cleaning resulted in a highly satisfactory recovery of the analyte (90%-104%). The method was linear in a range of 100-1000 ng/mL with a determination coefficient higher than 0.9973. The method was used to determine ciprofloxacin in larval homogenate after antibiotic administration to the patient at a dose of 500 mg twice daily per os during application of the larvae dressing. Ciprofloxacin was shown to distribute from the patient's circulation to the larvae at a concentration of 150 ng/mL (750 ng/g).

Rosiglitazone is an activator of nuclear peroxisome proliferator-activated (PPAR) receptor gamma used in the treatment of type 2 diabetes mellitus. The elimination of rosiglitazone occurs mainly via metabolism, with major contribution by enzyme cytochrome P450 (CYP) 2C8. Primary routes of rosiglitazone metabolism are N-demethylation and hydroxylation. Modulation of CYP2C8 activity by co-administered drugs lead to prominent changes in the exposure of rosiglitazone and its metabolites. Here, we attempt to develop mechanistic parent-metabolite physiologically based pharmacokinetic (PBPK) model for rosiglitazone. Our goal is to predict potential drug-drug interaction (DDI) and consequent changes in metabolite N-desmethyl rosiglitazone exposure. The PBPK modeling was performed in the PKSim® software using clinical pharmacokinetics data from literature. The contribution to N-desmethyl rosiglitazone formation by CYP2C8 was delineated using vitro metabolite formation rates from recombinant enzyme system. Developed model was verified for prediction of rosiglitazone DDI potential and its metabolite exposure based on observed clinical DDI studies. Developed model exhibited good predictive performance both for rosiglitazone and N-desmethyl rosiglitazone respectively, evaluated based on commonly acceptable criteria. In conclusion, developed model helps with prediction of CYP2C8 DDI using rosiglitazone as a substrate, as well as changes in metabolite exposure. In vitro data for metabolite formation can be successfully utilized to translate to in vivo conditions.

Selective enhancement of synaptic GABA signaling mediated by GABA-A receptors has been previously reported to promote functional recovery after ischemic stroke, while tonic GABA signaling has been detrimental. To identify agents that enhance synaptic signaling, we synthesized GABA-A ligands based on three chemotypes with affinity values pK i= 6.44-8.32. Representative compounds showed a preference in functional responses toward synaptic type of GABA-A receptors, compared to the extrasynaptic ones. In a cellular ischemia model (OGD), selected compounds showed the potential to improve neuronal recovery. The selected lead, compound 4, demonstrated the ability to reduce mitochondrial dysfunction, regulate intracellular calcium levels, decrease caspase 3 levels, and promote neurite outgrowth in in vitro assays. In an animal model, compound 4 enhanced motor recovery and showed neuroprotective activity by reducing infarct volume and decreasing poststroke acidosis. These findings underscore the value of selective ligands modulating synaptic GABA-A receptors in promoting recovery from ischemic stroke.

There is clear evidence that the presence of inflammatory factors and impaired GABA-ergic neurotransmission in depressed patients is associated with poor clinical outcome. We designed hybrid molecules, bearing the GABA molecule assembled with chemical fragments that interact with the serotonin 5-HT6 receptor. Such a combination aimed to curb neuroinflammation, remodel GABA-ergic signaling, and provide antidepressant-like activity. The most promising hybrid 3B exerted nanomolar affinity for 5-HT6 receptors and exerted agonistic properties on GABA-A receptors. Developability studies conferred that 3B exerted favorable drug-like properties and optimal brain penetration. In in vivo studies, 3B exerted robust antidepressant-like activity and proved to be highly effective in reducing levels of oxidative stress markers and the pro-inflammatory cytokine IL-6. The inetersting pharmacological profile of 3B makes it a promising candidate for further development for depression associated with neuroinflammation.

The multifactorial origin and neurochemistry of Alzheimer's disease (AD) call for the development of multitarget treatment strategies. We report a first-in-class triple acting compound that targets serotonin type 6 and 3 receptors (5-HT-Rs) and monoamine oxidase type B (MAO-B) as an approach for treating AD. The key structural features required for MAO-B inhibition and 5-HT6R antagonism and interaction with 5-HT3R were determined using molecular dynamic simulations and cryo-electron microscopy, respectively. Bioavailable PZ-1922 reversed scopolamine-induced cognitive deficits in the novel object recognition test. Furthermore, it displayed superior pro-cognitive properties compared to intepirdine (a 5-HT6R antagonist) in the AD model, which involved intracerebroventricular injection of an oligomeric solution of amyloid-β peptide (oAβ) in the T-maze test in rats. PZ-1922, but not intepirdine, restored levels of biomarkers characteristic of the debilitating effects of oAβ. These data support the potential of a multitarget approach involving the joint modulation of 5-HT6R/5-HT3R/MAO-B in AD.

α2-adrenoceptor ligands have been investigated as potential therapeutic agents for the treatment of obesity. Our previous studies have shown that guanabenz reduces the body weight of obese rats, presumably through its anorectic action. This demonstrates an additional beneficial effect on selected metabolic parameters, including glucose levels. The purpose of this present research was to determine the activity of guanabenz's metabolite—4-hydroxy guanabenz hydrochloride (4-OH-Guanabenz). We performed in silico analyses, involving molecular docking to targets of specific interest as well as other potential biological targets. In vitro investigations were conducted to assess the selectivity profile of 4-OH-Guanabenz binding to α-adrenoceptors, along with intrinsic activity studies involving α2-adrenoceptors and trace amine-associated receptor 1 (TAAR1). Additionally, the effects of 4-OH-Guanabenz on the body weight of rats and selected metabolic parameters were evaluated using the diet-induced obesity model. Basic safety and pharmacokinetic parameters were also examined. 4-OH-guanabenz is a partial agonist of α2A-adrenoceptor. The calculated EC50 value for it is 316.3 nM. It shows weak agonistic activity at TAAR1 too. The EC50 value for 4-OH-Guanabenz calculated after computer simulation is 330.6 µM. Its primary mode of action is peripheral. The penetration of 4-OH-Guanabenz into the brain is fast (tmax = 15 min), however, with a low maximum concentration of 64.5 ng/g. 4-OH-Guanabenz administered ip at a dose of 5 mg/kg b.w. to rats fed a high-fat diet causes a significant decrease in body weight (approximately 14.8

The need for physical distancing due to COVID-19 mitigation efforts forced prolonged social isolation, which may affect sleep and lead to mental health problems. Previous research has shown that young adults are particularly vulnerable to psychological stress caused by social isolation, the negative psychological impact of the pandemic, and greater frequency and severity of sleep problems. Therefore, the main goal of the present study was to examine whether insomnia could constitute a mediation mechanism that explains the relationship between social isolation experienced during the COVID-19 pandemic and mental health outcomes (depression and anxiety) reported up to 1.5 years later. The study was conducted among young (M±SD; 24.08±3.75) men (N = 1025) in Poland. Data were collected by means of self-report questionnaires, including The Social Isolation Index, The Athens Insomnia Scale, The State-Trait Anxiety Inventory (STAI-S) and Beck's Depression Inventory (BDI-II). The results show that insomnia mediates the relationships between social isolation and both anxiety and depression. The current findings emphasize the role of insomnia in the relationships between social isolation experienced during COVID-19 and negative emotional states. From a clinical perspective, the results suggest that implementing therapeutic components that address social isolation in insomnia treatment programs may prevent the development of depression and anxiety symptoms among young men.

AbstractIn this study, we report a series of newly synthesised sulphonamides of aziridine-2-carboxylic acid (Az-COOH) ester and amide analogues as potent protein disulphide isomerase (PDI, EC 5.3.4.1) inhibitors. The inhibitory activity on PDI was determined against recombinant human PDIA1 and PDIA3 proteins using an insulin reduction assay. These compounds in low micromolar to low nanomolar concentrations showed the effective in vitro inhibitory properties of PDIA1 with weaker effects on PDIA3. Complexes of 15N- and 15N,13C- uniformly labelled recombinant human PDIA1a with two PDIA1 inhibitors were produced and investigated by a protein nuclear magnetic resonance (NMR) spectroscopy. It was found that both C53 and C56 of the PDIA1 enzyme were involved in covalent binding. Finally, in a range of pharmacological studies, we demonstrated that investigated compounds displayed anti-cancer and anti-thrombotic activity. These findings demonstrate that sulphonamides of Az-COOH derivatives are promising candidates for the development of novel anti-cancer and anti-thrombotic agents.

The symptomatic and disease-modifying effects of butyrylcholinesterase (BuChE) inhibitors provide an encouraging premise for researching effective treatments for Alzheimer's disease. Here, we examined a series of compounds with a new chemical scaffold based on 3-(cyclohexylmethyl)amino-2-hydroxypropyl, and we identified a highly selective hBuChE inhibitor (29). Based on extensive in vitro and in vivo evaluations of the compound and its enantiomers, (R)-29 was identified as a promising candidate for further development. Compound (R)-29 is a potent hBuChE inhibitor (IC50 = 40 nM) with selectivity over AChE and relevant off-targets, including H1, M1, α1A and β1 receptors. The compound displays high metabolic stability on human liver microsomes (90% of the parent compound after 2 h of incubation), and its safety was confirmed through examining the cytotoxicity on the HepG2 cell line (LC50 = 2.85 μM) and hERG inhibition (less than 50% at 10 μM). While (rac)-29 lacked an effect in vivo and showed limited penetration to the CNS in pharmacokinetics studies, compound (R)-29 exhibited a procognitive effect at 15 mg/kg in the passive avoidance task in scopolamine-treated mice.