Presence of key cholinergic enzymes in human spermatozoa and seminal fluid†.

Little is known about the non-neuronal spermic cholinergic system, which may regulate sperm motility and the acrosome reaction initiation process. We investigated the presence of the key acetylcholine (ACh)-biosynthesizing enzyme, choline acetyltransferase (ChAT), and the acetylcholine-degrading enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) and two ACh-receptors in human spermatozoa and seminal plasma. Fresh ejaculates were used for intra- and extracellular flow cytometric analysis of ChAT, AChE, BChE, and alpha-7-nicotinic and M1-muscarinic ACh-receptors in sperm. For determining the source of soluble enzymes, frozen seminal samples (n = 74) were selected on two bases: (1) from vasectomized (n = 37) and non-vasectomized (n = 37) subjects and (2) based on levels of alpha-glucosidase, fructose, or zinc to define sample subgroups with high or low fluid contribution from the epididymis and seminal vesicle, and prostate, respectively. Flow cytometric analyses revealed that ChAT was expressed intracellularly in essentially all spermatozoa. ChAT was also present in a readily membrane-detachable form at the extracellular membrane of at least 18% of the spermatozoa. These were also highly positive for intra- and extracellular BChE (>83%) and M1 (>84%) and α7 (>59%) ACh-receptors. Intriguingly, the sperm was negative for AChE. Analyses of seminal plasma revealed that spermatozoa and epididymides were major sources of soluble ChAT and BChE, whereas soluble AChE most likely originated from epididymides and seminal vesicles. Prostate had relatively minor contribution to the pool of the soluble enzymes in the seminal fluid. In conclusion, human spermatozoa exhibited a cholinergic phenotype and were one of the major sources of soluble ChAT and BChE in ejaculate. We also provide the first evidence for ChAT as an extracellularly membrane-anchored protein.

Synthesis, biological evaluation, theoretical calculations, QSAR and molecular docking studies of novel arylaminonaphthols as potent antioxidants and BChE inhibitors.

A completely green protocol was developed for the synthesis of a series of arylaminonaphthol derivatives in the presence of N-ethylethanolamine (NEEA) as a catalyst under ultrasonic irradiation and solventless conditions. The major assets of this methodology were the use of non-toxic organic medium, available catalyst, mild reaction condition, and good to excellent yield of desired products. All of the synthesized products were screened for their in vitro antioxidant activity using DPPH, ABTS, and Ferric-phenanthroline assays and it was found that most of them are potent antioxidant agents. Also, their butyrylcholinesterase inhibitory activity has been investigated in vitro. All tested compounds exhibited potential inhibitory activity toward BuChE when compared to standard reference drug galantamine, however, compounds 4r, 4u, 4 g and 4x gave higher butyrylcholinesterase inhibitory with IC50 values of 14.78 ± 0.65 µM, 16.18 ± 0.50 µM, 20.00 ± 0.50 µM, and 20.28 ± 0.08 µM respectively. On the other hand, we employed density functional theory (DFT), calculations to analyze molecular geometry and global reactivity descriptors, and MESP analysis to predict electrophilic and nucleophilic attacks. A quantitative structure-activity relationship (QSAR) investigation was conducted on the antioxidant and butyrylcholinesterase properties of 25 arylaminonaphthol derivatives, resulting in robust and satisfactory models. To evaluate their anti-Alzheimer's activity, compounds 4 g, 4q, 4r, 4u, and 4x underwent docking simulations at the active site of the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), revealing why these compounds displayed superior activity, consistent with the biological findings.

Investigation of cholinesterase and α-glucosidase enzyme activities, and molecular docking and dft studies for 1,2-disubstituted cyclopentane derivatives with phenyl and benzyl units.

Six known products (4-9) were prepared from reaction of adipoyl chloride with 1,2,3-trimethoxybenzene according to the literature. From (2,3,4-trimethoxyphenyl)(2-(2,3,4-trimethoxyphenyl)cyclopent-1-en-1-yl)methanone (4) of them, four new 1,2-disubstituted cyclopentane derivatives (10-13) with phenyl and benzyl units were synthesized by reactions such as hydrazonation, catalytic hydrogenation and bromination. The obtained compounds 4-13 were examined for their in vitro inhibitory activity against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and α-glucosidase enzymes. All compounds 4-13 showed inhibition at nanomolar level with Ki values in the range of 45.53 ± 7.35-631.96 ± 18.88 nM for AChE, 84.30 ± 9.92-622.10 ± 35.14 nM for BChE, and 25.47 ± 4.46-48.87 ± 7.33 for α-Glu. In silico molecular docking studies of the potent compounds were performed in the active sites of AChE (PDB: 1E66), BChE (PDB: 1P0I), and α-glucosidase (PDB: 5ZCC) to compare the effect of bromine atom on the inhibition mechanism. The optimized molecular structures, HOMO-LUMO energies and molecular electrostatic potential maps for the compounds were calculated by using density functional theory with B3LYP/6-31 + G(d,p).

Novel multifunctional tacrine-donepezil hybrids against Alzheimer's disease: Design synthesis and bioactivity studies.

A series of tacrine-donepezil hybrids were synthesized as potential multifunctional anti-Alzheimer's disease (AD) compounds. For this purpose, tacrine and the benzylpiperidine moiety of donepezil were fused with a hydrazone group to achieve a small library of tacrine-donepezil hybrids. In agreement with the design, all compounds showed inhibitory activity toward both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with IC50 values in the low micromolar range. Kinetic studies on the most potent cholinesterase (ChE) inhibitors within the series showed a mixed-type inhibition mechanism on both enzymes. Also, the docking studies indicated that the compounds inhibit ChEs by dual binding site (DBS) interactions. Notably, tacrine-donepezil hybrids also exhibited significant neuroprotection against H2O2-induced cell death in a differentiated human neuroblastoma (SH-SY5Y) cell line at concentrations close to their IC50 values on ChEs and showed high to medium blood-brain barrier (BBB) permeability on human cerebral microvascular endothelial cells (HBEC-5i). Besides, the compounds do not cause remarkable toxicity in a human hepatocellular carcinoma cell line (HepG2) and SH-SY5Y cells. Additionally, the compounds were predicted to also have good bioavailability. Among the tested compounds, H4, H16, H17, and H24 stand out with their biological profile. Taken together, the proposed novel tacrine-donepezil scaffold represents a promising starting point for the development of novel anti-ChE multifunctional agents against AD.

New Charged Cholinesterase Inhibitors: Design, Synthesis, and Characterization.

Triazoles and triazolium salts are very common subunits in the structures of various drugs. Medicaments with a characteristic 1,2,3-triazole core are also being developed to treat neurodegenerative disorders associated with cholinesterase enzyme activity. Several naphtho- and thienobenzo-triazoles from our previous research emerged as being particularly promising in that sense. For this reason, in this research, new naphtho- and thienobenzo-triazoles 23-34, as well as 1,2,3-triazolium salts 44-51, were synthesized and tested. Triazolium salts 44-46 showed excellent activity while salts 47 and 49 showed very good inhibition toward both butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) enzymes. In contrast, neutral photoproducts were shown to be selective towards BChE but with very good inhibition potential as molecules 24-27. The representative of newly prepared compounds, 45 and 50, were stable in aqueous solution and revealed intriguing fluorimetric properties, characterized by a strong Stokes shift of >160 nm. Despite their condensed polycyclic structure shaped similarly to well-known DNA-intercalator ethidium bromide, the studied compounds did not show any interaction with ds-DNA, likely due to the unfavorable steric hindrance of substituents. However, the studied dyes bind proteins, particularly showing very diverse inhibition properties toward AChE and BChE. In contrast, neutral photoproducts were shown to be selective towards a certain enzyme but with moderate inhibition potential. The molecular docking of the best-performing candidates to cholinesterases' active sites identified cation-π interactions as the most responsible for the stability of the enzyme-ligand complexes. As genotoxicity studies are crucial when developing new active substances and finished drug forms, in silico studies for all the compounds synthesized have been performed.

Combining Experimental and Computational Methods to Produce Conjugates of Anticholinesterase and Antioxidant Pharmacophores with Linker Chemistries Affecting Biological Activities Related to Treatment of Alzheimer's Disease.

Effective therapeutics for Alzheimer's disease (AD) are in great demand worldwide. In our previous work, we responded to this need by synthesizing novel drug candidates consisting of 4-amino-2,3-polymethylenequinolines conjugated with butylated hydroxytoluene via fixed-length alkylimine or alkylamine linkers (spacers) and studying their bioactivities pertaining to AD treatment. Here, we report significant extensions of these studies, including the use of variable-length spacers and more detailed biological characterizations. Conjugates were potent inhibitors of acetylcholinesterase (AChE, the most active was 17d IC50 15.1 ± 0.2 nM) and butyrylcholinesterase (BChE, the most active was 18d: IC50 5.96 ± 0.58 nM), with weak inhibition of off-target carboxylesterase. Conjugates with alkylamine spacers were more effective cholinesterase inhibitors than alkylimine analogs. Optimal inhibition for AChE was exhibited by cyclohexaquinoline and for BChE by cycloheptaquinoline. Increasing spacer length elevated the potency against both cholinesterases. Structure-activity relationships agreed with docking results. Mixed-type reversible AChE inhibition, dual docking to catalytic and peripheral anionic sites, and propidium iodide displacement suggested the potential of hybrids to block AChE-induced ß-amyloid (Aß) aggregation. Hybrids also exhibited the inhibition of Aß self-aggregation in the thioflavin test; those with a hexaquinoline ring and C8 spacer were the most active. Conjugates demonstrated high antioxidant activity in ABTS and FRAP assays as well as the inhibition of luminol chemiluminescence and lipid peroxidation in mouse brain homogenates. Quantum-chemical calculations explained antioxidant results. Computed ADMET profiles indicated favorable blood-brain barrier permeability, suggesting the CNS activity potential. Thus, the conjugates could be considered promising multifunctional agents for the potential treatment of AD.

The structural modification and biological evaluation of tetrahydrothienopyridine derivatives as selective BChE inhibitors.

A series of tetrahydrothienopyridine derivatives have been designed, synthesized, and evaluated as selective BChE inhibitors. Compounds were analyzed via HRMS, 1H NMR, and 13C NMR. The inhibitory effects were evaluated according to the method of Ellman et al. 6n was the most potent and selective inhibitor against BChE (eeAChE IC50 = 686.4 ± 478.6 µM, eqBChE IC50 = 10.5 ± 5.0 nM, SI = 6.5*104, hBChE IC50 = 32.5 ± 6.5 nM). Cell-based assays have confirmed the low neurotoxicity of 6a and 6n and their moderate neuroprotective effects. Compounds 6a and 6n provide novel chemical entities for the treatment of Alzheimer's disease.

Preoperative BChE serves as a prognostic marker in patients with resectable AEG after neoadjuvant chemotherapy.

BACKGROUND: Diminished systemic serum butyrylcholinesterase (BChE), a biomarker for chronic inflammation, cachexia, and advanced tumor stage, has shown to play a prognostic role in various malignancies. The aim of this study was to investigate the prognostic value of pretherapeutic BChE levels in patients with resectable adenocarcinoma of the gastroesophageal junction (AEG), treated with or without neoadjuvant therapy. METHODS: Data of a consecutive series of patients with resectable AEG at the Department for General Surgery, Medical University of Vienna, were analyzed. Preoperative serum BChE levels were correlated to clinic-pathological parameters as well as treatment response. The prognostic impact of serum BChE levels on disease-free (DFS) and overall survival (OS) was evaluated by univariate and multivariate cox regression analysis, and Kaplan-Meier curves used for illustration. RESULTS: A total of 319 patients were included in this study, with an overall mean (standard deviation, SD) pretreatment serum BChE level of 6.22 (± 1.91) IU/L. In univariate models, diminished preoperative serum BChE levels were significantly associated with shorter overall (OS, p < 0.003) and disease-free survival (DFS, p < 0.001) in patients who received neoadjuvant treatment and/or primary resection. In multivariated analysis, decreased BChE was significantly associated with shorter DFS (HR: 0.92, 95% CI: 0.84-1.00, p 0.049) and OS (HR: 0.92, 95% CI: 0.85-1.00, p < 0.49) in patients receiving neoadjuvant therapy. Backward regression identified the interaction between preoperative BChE and neoadjuvant chemotherapy as a predictive factor for DFS and OS. CONCLUSION: Diminished serum BChE serves as a strong, independent, and cost-effective prognostic biomarker for worse outcome in patients with resectable AEG who had received neoadjuvant chemotherapy.

Anti-Alzheimer's Activity of Polyphenolic Stilbene-Rich Acetone Fraction of the Oil-Removed Seeds of Passiflora edulis: in Vivo and in Silico Studies.

The stilbene-rich acetone fraction in high yield (6.6 %, PEAS) of Passiflora edulis Sims was prepared and evaluated for neuroprotective activity in murine Alzheimer's disease model induced by aluminum chloride and D-galactose. The phytochemical and HPLC-DAD-MS analysis of the polyphenolic stilbene-rich acetone fraction showed that it contained different stilbenes including trans-piceatannol, scirpusins A-B and cassigarol E. The total phenolic content (TPC) of PEAS was 413.87±1.71 mg GAE eqv/g. The neuroprotective activity of PEAS is typically presented in the Morris water maze-reference Spatial Memory test, where the Alzheimer's mice treated at 100 mg/kg (Alz-ED1) and 200 mg/kg (Alz-ED2) spent less than 47 % and 66 % of the time, respectively, than the Alzheimer's model mice (Alz). Two simple stilbenes, trans-piceatannol and trans-resveratrol, showed selectively inhibitory activity in silico against acetylcholinesterase (AChE). Two stilbene dimers, cassigarol E and scirpusin A, exhibited low nanomolar inhibitory potential against AChE and butyrylcholinesterase (BChE), significantly lower than those of the positive control, donepezil and tacrine. These findings suggest that the stilbenes from P. edulis seeds, particularly the stilbene dimers, warrant further investigation as potential neuroprotective candidates in the prevention of cognitive deficits associated with Alzheimer's disease.

Multitargeted inhibition of key enzymes associated with diabetes and Alzheimer's disease by 1,3,4-oxadiazole derivatives: Synthesis, in vitro screening, and computational studies.

A library of 22 derivatives of 1,3,4-oxadiazole-2-thiol was synthesized, structurally characterized, and assessed for its potential to inhibit α-amylase, α-glucosidase, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and antioxidant activities. Most of the tested compounds demonstrated good to moderate inhibition potential; however, their activity was lower than that of the standard acarbose. Significantly, compound 3f exhibited the highest inhibition potential against α-glucosidase and α-amylase enzymes, with IC50 values of 18.52 ± 0.09 and 20.25 ± 1.05 µM, respectively, in comparison to the standard acarbose (12.29 ± 0.26; 15.98 ± 0.14 µM). Compounds also demonstrated varying degrees of inhibitory potential against AChE (IC50 = 9.25 ± 0.19 to 36.15 ± 0.12 µM) and BChE (IC50 = 10.06 ± 0.43 to 35.13 ± 0.12 µM) enzymes compared to the standard donepezil (IC50 = 2.01 ± 0.12; 3.12 ± 0.06 µM), as well as DPPH (IC50 = 20.98 ± 0.06 to 52.83 ± 0.12 µM) and ABTS radical scavenging activities (IC50 = 22.29 ± 0.18 to 47.98 ± 0.03 µM) in comparison to the standard ascorbic acid (IC50 = 18.12 ± 0.15; 19.19 ± 0.72). The kinetic investigations have demonstrated that the compounds exhibit competitive-type inhibition for α-amylase, noncompetitive-type inhibition for α-glucosidase and AChE, and mixed-type inhibition for BChE. Additionally, a molecular docking study was performed on all synthetic oxadiazoles to explore the interaction details of these compounds with the active sites of the enzymes.

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.

Polyphenolic compounds: Synthesis, assessment of antimicrobial effect and enzymes inhibition against important medicinal enzymes with computational details.

Phenolic compounds mainly benefit human health and have many biological activities. Their activities are related to their structure, which allows them to interact with enzymes. The inhibition potencies of synthesized polyphenolic compounds (3a and 3b) were investigated on cholinesterases, α­Gly, and tyrosinase activities. The structures of 3a and 3b were determined based on spectral data (NMR, UV-vis, XRD pattern, SEM, and EDX). The compounds have effective inhibitory potential with IC50 value between 2.25 ± 0.35-5.66 ± 0.75 µM and Ki values 2.95 ± 0.37-14.86 ± 4.99 µM for AChE, BChE, and tyrosinase. It was determined that the synthesized compounds have biological activities by the MIC and cytotoxicity tests, and they have IC50 values of 16.15 µg/mL and 12.16 µg/mL for the PC-3 cell line, respectively. According to the calculated molecular docking results, these compounds showed the highest binding energy against AChE and tyrosinase enzymes (-11.3 and -10.4 kcal/mol, respectively). The compounds have synthetic accessibility scores of 2.75 and 4.55 based on the drug-likeness properties.

Carbamate-based N-Substituted tryptamine derivatives as novel pleiotropic molecules for Alzheimer's disease.

A novel series of carbamate-based N-substituted tryptamine derivatives were designed and synthesized based on functional group combination strategy, and possessed both cholinesterase inhibition and neuroprotective effects. After systematically evaluating the cholinesterase inhibitory activity of 24 synthesized compounds, compound 6H6, bearing n-heptyl residue as carbamate moiety, was highlighted due to its great BChE-selective inhibition (eeAChE IC50 > 100 µM; eqBChE IC50 = 7 nM), neuronal protection, antioxidation and anti-neuroinflammation efficacy. Cytotoxicity and acute toxicity assays confirmed the safety-efficacy profiles of compound 6H6. Besides, pharmacokinetic properties and blood-brain barrier (BBB) permeability of compound 6H6 were favorable and suitable for further study in vivo. The behavioral tests revealed that compound 6H6 could remarkably improve the scop-induced ethological changes and memory impairment, suggesting compound 6H6, as an attractive pleiotropic molecule, had great promise in treating Alzheimer's disease.

New 4-phenylpiperazine-carbodithioate-N-phenylacetamide hybrids: Synthesis, in vitro and in silico evaluations against cholinesterase and α-glucosidase enzymes.

A series of novel 4-phenylpiperazine-carbodithioate-N-phenylacetamide hybrids (6a-n) was designed, synthesized, and evaluated for their in vitro inhibitory activity against the metabolic enzymes, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and α-glucosidase. The obtained results showed that most of the synthesized compounds exhibited high to good anti-AChE and anti-BChE activity in the range of nanomolar concentrations in comparison to tacrine as a positive control. Molecular modeling of the most potent compounds 6e and 6i demonstrated that these compounds interacted with important residues of the AChE and BChE active sites. Moreover, all the newly synthesized compounds 6a-n had significant Ki values against α-glucosidase when compared with the positive control acarbose. Representatively, N-2-fluorophenylacetamide derivative 6l, with a Ki value of 0.98 nM as the most potent compound, was 126 times more potent than acarbose with a Ki value of 123.70 nM. This compound also fitted in the α-glucosidase active site and interacted with key residues. An in silico study of the druglikeness/absorption, distribution, metabolism, and excretion (ADME)/toxicity profile of the selected compounds 6e, 6i, and 6l predicts that these compounds are drug-like and have the appropriate properties in terms of ADME and toxicity.

Potential of Vitamin B6 Dioxime Analogues to Act as Cholinesterase Ligands.

Seven pyridoxal dioxime quaternary salts (1-7) were synthesized with the aim of studying their interactions with human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The synthesis was achieved by the quaternization of pyridoxal monooxime with substituted 2-bromoacetophenone oximes (phenacyl bromide oximes). All compounds, prepared in good yields (43-76%) and characterized by 1D and 2D NMR spectroscopy, were evaluated as reversible inhibitors of cholinesterase and/or reactivators of enzymes inhibited by toxic organophosphorus compounds. Their potency was compared with that of their monooxime analogues and medically approved oxime HI-6. The obtained pyridoxal dioximes were relatively weak inhibitors for both enzymes (Ki = 100-400 µM). The second oxime group in the structure did not improve the binding compared to the monooxime analogues. The same was observed for reactivation of VX-, tabun-, and paraoxon-inhibited AChE and BChE, where no significant efficiency burst was noted. In silico analysis and molecular docking studies connected the kinetic data to the structural features of the tested compound, showing that the low binding affinity and reactivation efficacy may be a consequence of a bulk structure hindering important reactive groups. The tested dioximes were non-toxic to human neuroblastoma cells (SH-SY5Y) and human embryonal kidney cells (HEK293).

In Vitro and In Silico Investigation of Diterpenoid Alkaloids Isolated from Delphinium chitralense.

This study reports the isolation of three new C20 diterpenoid alkaloids, Chitralinine A-C (1-3) from the aerial parts of Delphinium chitralense. Their structures were established on the basis of latest spectral techniques and single crystal X-rays crystallographic studies of chitralinine A described basic skeleton of these compounds. All the isolated Compounds (1-3) showed strong, competitive type inhibition against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in comparison to standard allanzanthane and galanthamine however, chitralinine-C remained the most potent with IC50 value of 11.64 ± 0.08 µM against AChE, and 24.31 ± 0.33 µM against BChE, respectively. The molecular docking reflected a binding free energy of -16.400 K Cal-mol-1 for chitralinine-C, having strong interactions with active site residues, TYR334, ASP72, SER122, and SER200. The overall findings suggest that these new diterpenoid alkaloids could serve as lead drugs against dementia-related diseases including Alzheimer's disease.

Screening of Big Pharma's Library against Various in-house Biological Targets.

Open innovation initiatives provide opportunities for collaboration and sharing of knowledge and experience between industry, academia, and government institutions. Through open innovation, Merck is offering a Mini Library of 80 carefully selected compounds from previous research and development projects to a broader scientific community for testing in academic drug discovery projects. These compounds are predominantly drug-like and cover a broad range of molecular targets. They could potentially interact with other enzymes, receptors, transporters, and ion channels of interest. The Mini Library was tested on seven in-house enzymes (bacterial MurA, MurC ligase, and DdlB enzyme, human MAO-A/B, human BChE, and murine AChE), and several hits were identified. A follow-up series of structural analogues provided by Merck gave a more detailed insight into the accessibility and the quality of the hit compounds. For example, sartan derivatives were moderate inhibitors of MurC, whereas bisarylureas were potent, selective, nanomolar inhibitors of hMAO-B. Importantly, 3-n-butyl-substituted indoles were identified as low nanomolar selective inhibitors of hBChE. All in all, the hit derivatives provide new starting points for the further exploration of the chemical space of high-quality enzyme inhibitors.

Bis-Amiridines as Acetylcholinesterase and Butyrylcholinesterase Inhibitors: N-Functionalization Determines the Multitarget Anti-Alzheimer's Activity Profile.

Using two ways of functionalizing amiridine-acylation with chloroacetic acid chloride and reaction with thiophosgene-we have synthesized new homobivalent bis-amiridines joined by two different spacers-bis-N-acyl-alkylene (3) and bis-N-thiourea-alkylene (5) -as potential multifunctional agents for the treatment of Alzheimer's disease (AD). All compounds exhibited high inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with selectivity for BChE. These new agents displayed negligible carboxylesterase inhibition, suggesting a probable lack of untoward drug-drug interactions arising from hydrolytic biotransformation. Compounds 3 with bis-N-acyl-alkylene spacers were more potent inhibitors of both cholinesterases compared to compounds 5 and the parent amiridine. The lead compounds 3a-c exhibited an IC50(AChE) = 2.9-1.4 µM, IC50(BChE) = 0.13-0.067 µM, and 14-18% propidium displacement at 20 µM. Kinetic studies of compounds 3a and 5d indicated mixed-type reversible inhibition. Molecular docking revealed favorable poses in both catalytic and peripheral AChE sites. Propidium displacement from the peripheral site by the hybrids suggests their potential to hinder AChE-assisted Aß42 aggregation. Conjugates 3 had no effect on Aß42 self-aggregation, whereas compounds 5c-e (m = 4, 5, 6) showed mild (13-17%) inhibition. The greatest difference between conjugates 3 and 5 was their antioxidant activity. Bis-amiridines 3 with N-acylalkylene spacers were nearly inactive in ABTS and FRAP tests, whereas compounds 5 with thiourea in the spacers demonstrated high antioxidant activity, especially in the ABTS test (TEAC = 1.2-2.1), in agreement with their significantly lower HOMO-LUMO gap values. Calculated ADMET parameters for all conjugates predicted favorable blood-brain barrier permeability and intestinal absorption, as well as a low propensity for cardiac toxicity. Thus, it was possible to obtain amiridine derivatives whose potencies against AChE and BChE equaled (5) or exceeded (3) that of the parent compound, amiridine. Overall, based on their expanded and balanced pharmacological profiles, conjugates 5c-e appear promising for future optimization and development as multitarget anti-AD agents.

Evaluation of γ-carboline-phenothiazine conjugates as simultaneous NMDA receptor blockers and cholinesterase inhibitors.

Alzheimer's disease (AD) is the most common form of dementia. It is associated with the impairment of memory and other cognitive functions that are mainly caused by progressive defects in cholinergic and glutamatergic signaling in the central nervous system. Inhibitors of acetylcholinesterase (AChE) and ionotropic glutamate receptors of the N-methyl-d-aspartate (NMDA) receptor family are currently approved as AD therapeutics. We previously showed using a cell-based assay of NMDA receptor-mediated glutamate-induced excitotoxicity that bis-γ-carbolinium conjugates are useful NMDA receptor blockers. However, these compounds also act as subnanomolar AChE inhibitors, which may cause serious anticholinergic side effects when applied in vivo. Here, we evaluated new structures containing γ-carbolines linked to phenothiazine via a propionyl spacer. These compounds were superior to the previously characterized bis-γ-carbolinium conjugates because they blocked NMDA receptors without requiring a quaternary pyridine N-atom and inhibited AChE with moderate IC50 values of 0.54-5.3 µM. In addition, these new compounds displayed considerable selectivity for the inhibition of butyrylcholinesterase (BChE; IC50 = 0.008-0.041 µM), which may be favorable for AD treatment. Inhibitory activities towards the NMDA receptors and AChE were in the same micromolar range, which may be beneficial for equal dosing against multiple targets in AD patients.

Inducing new bioactive metabolites production from coculture of Pestalotiopsis sp. and Penicillium bialowiezense.

Coculturing two or more fungi is a useful strategy to awaken the silent genes to produce structurally diverse and bioactive natural products. Through the coculture of Pestalotiopsis sp. and Penicillium bialowiezense, six new isoprenylated chromane derivatives, including two pairs of enantiomeric ones (1a/1b-2a/2b) and two optical pure ones (3-4), two new isoprenylated phenol glucoside derivatives (6-7), as well as eight known structural analogues (5 and 8-14), were obtained. The structures of these new compounds were characterized by NMR spectroscopy, single-crystal X-ray crystallography, and ECD calculation. The Δ10,11 double bond of pestaloficin D (5) was revised to E-configurated based on the extensive spectroscopic analyses. Compounds 1a/1b and 2a/2b were the first examples of enantiomeric isoprenylated chromane derivatives, which were successfully separated by chiral HPLC. Additionally, all the isolated compounds were evaluated for the in vitro ß-glucuronidase (GUS) and butyrylcholinesterase (BChE) inhibitory activities. Compounds 1a and 1b showed significant ß-glucuronidase inhibitory potency with IC50 values of 7.6 and 10.3 µM, respectively. Compound 14 exhibited moderate BChE inhibitory activity with an IC50 value of 21.3 µM. In addition, the structure-enzyme inhibitory activity relationship of compounds 1-14 is discussed.