Brominated oxime nucleophiles are efficiently reactivating cholinesterases inhibited by nerve agents.

Six novel brominated bis-pyridinium oximes were designed and synthesized to increase their nucleophilicity and reactivation ability of phosphorylated acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Their pKa was valuably found lower to parent non-halogenated oximes. Stability tests showed that novel brominated oximes were stable in water, but the stability of di-brominated oximes was decreased in buffer solution and their degradation products were prepared and characterized. The reactivation screening of brominated oximes was tested on AChE and BChE inhibited by organophosphorus surrogates. Two mono-brominated oximes reactivated AChE comparably to non-halogenated analogues, which was further confirmed by reactivation kinetics. The acute toxicity of two selected brominated oximes was similar to commercially available oxime reactivators and the most promising brominated oxime was tested in vivo on sarin- and VX-poisoned rats. This brominated oxime showed interesting CNS distribution and significant reactivation effectiveness in blood. The same oxime resulted with the best protective index for VX-poisoned rats.

Withanolide Profile and Acetylcholinesterase Inhibitory Activity of Two Argentinean Jaborosa Species.

Acetylcholinesterase (AChE) inhibitors are still an important option for managing symptoms of mild to moderate Alzheimer's disease. In this study, we aimed to evaluate the potential in vitro AChE inhibitory activity of two Argentinian endemic Solanaceae species, Jaborosa bergii and J. runcinata. UHPLC-DAD-HRMS metabolite profiling revealed the presence of withanolides in the active CH2Cl2 subextracts. Their fractionation led to the isolation and identification of two known spiranoid withanolides from J. runcinata and three new withanolides with a skeleton similar to that of trechonolide-type withanolides from J. bergii. The known compounds showed moderate AChE inhibitory activity, while the new ones were inactive.

Assessment of antioxidant, acetylcholinesterase, paraoxonase inhibition activities and phenolic content of Alchemilla lithophila.

In the present study, antioxidant activity and inhibition of acetylcholinesterase (AChE) and paraoxonase (hPON 1) of Alchemilla lithophila extracts were evaluated for the first time. Besides, there is no research on the contents of phenolic compounds except for fatty acids. In this context, phenolic compounds of A. lithophila were investigated by liquid chromatography/ mass spectrometry (LC-MS/MS). The methanol extract of the A. lithophila exhibited significant inhibition on the AChE (IC50 value for methanol extract 0.162 ± 0.25 mg /mL, R2:0.992). Besides, antioxidant activities of the A. lithophila extracts were examined using by the methods ABTS•+ and DPPH• free radical scavenging potentials, FRAP and CUPRAC metal-reducing activities. ABTS•+ and DPPH• scavenging activities were found for methanol extract at 70.67% and water extract at 75.38%, respectively. Also, FRAP and CUPRAC metal-reducing were determined for water extract 0.796 and hexane extract 1.570 as absorbance. According to LC-MS/MS analyses, the amounts of ellagic acid, catechin hydrate, gallic acid, fumaric acid, luteolin, quercetin, kaempferol, acetohydroxamic acid, caffeic acid, syringic acid, hydroxybenzoic acid and salicylic acid were determined by LC-MS/MS, respectively. As a consequence, this study will be a useful resource for determining bioactivity and phenolic compound profile for natural medicine research.

Covalent inhibition of hAChE by organophosphates causes homodimer dissociation through long-range allosteric effects.

Acetylcholinesterase (EC 3.1.1.7), a key acetylcholine-hydrolyzing enzyme in cholinergic neurotransmission, is present in a variety of states in situ, including monomers, C-terminally disulfide-linked homodimers, homotetramers, and up to three tetramers covalently attached to structural subunits. Could oligomerization that ensures high local concentrations of catalytic sites necessary for efficient neurotransmission be affected by environmental factors? Using small-angle X-ray scattering (SAXS) and cryo-EM, we demonstrate that homodimerization of recombinant monomeric human acetylcholinesterase (hAChE) in solution occurs through a C-terminal four-helix bundle at micromolar concentrations. We show that diethylphosphorylation of the active serine in the catalytic gorge or isopropylmethylphosphonylation by the RP enantiomer of sarin promotes a 10-fold increase in homodimer dissociation. We also demonstrate the dissociation of organophosphate (OP)-conjugated dimers is reversed by structurally diverse oximes 2PAM, HI6, or RS194B, as demonstrated by SAXS of diethylphosphoryl-hAChE. However, binding of oximes to the native ligand-free hAChE, binding of high-affinity reversible ligands, or formation of an SP-sarin-hAChE conjugate had no effect on homodimerization. Dissociation monitored by time-resolved SAXS occurs in milliseconds, consistent with rates of hAChE covalent inhibition. OP-induced dissociation was not observed in the SAXS profiles of the double-mutant Y337A/F338A, where the active center gorge volume is larger than in wildtype hAChE. These observations suggest a key role of the tightly packed acyl pocket in allosterically triggered OP-induced dimer dissociation, with the potential for local reduction of acetylcholine-hydrolytic power in situ. Computational models predict allosteric correlated motions extending from the acyl pocket toward the four-helix bundle dimerization interface 25 Å away.

Development of versatile and potent monoquaternary reactivators of acetylcholinesterase.

To date, the only treatments developed for poisoning by organophosphorus compounds, the most toxic chemical weapons of mass destruction, have exhibited limited efficacy and versatility. The available causal antidotes are based on reactivation of the enzyme acetylcholinesterase (AChE), which is rapidly and pseudo-irreversibly inhibited by these agents. In this study, we developed a novel series of monoquaternary reactivators combining permanently charged moieties tethered to position 6- of 3-hydroxypyridine-2-aldoxime reactivating subunit. Highlighted representatives (21, 24, and 27; also coded as K1371, K1374, and K1375, respectively) that contained 1-phenylisoquinolinium, 7-amino-1-phenylisoquinolinium and 4-carbamoylpyridinium moieties as peripheral anionic site ligands, respectively, showed efficacy superior or comparable to that of the clinically used standards. More importantly, these reactivators exhibited wide-spectrum efficacy and were minutely investigated via determination of their reactivation kinetics in parallel with molecular dynamics simulations to study their mechanisms of reactivation of the tabun-inhibited AChE conjugate. To further confirm the potential applicability of these candidates, a mouse in vivo assay was conducted. While K1375 had the lowest acute toxicity and the most suitable pharmacokinetic profile, the oxime K1374 with delayed elimination half-life was the most effective in ameliorating the signs of tabun toxicity. Moreover, both in vitro and in vivo, the versatility of the agents was substantially superior to that of clinically used standards. Their high efficacy and broad-spectrum capability make K1374 and K1375 promising candidates that should be further investigated for their potential as nerve agents and insecticide antidotes.

Prediction of dose-dependent in vivo acetylcholinesterase inhibition by profenofos in rats and humans using physiologically based kinetic (PBK) modeling-facilitated reverse dosimetry.

Organophosphate pesticides (OPs) are known to inhibit acetylcholine esterase (AChE), a critical effect used to establish health-based guidance values. This study developed a combined in vitro-in silico approach to predict AChE inhibition by the OP profenofos in rats and humans. A physiologically based kinetic (PBK) model was developed for both species. Parameter values for profenofos conversion to 4-bromo-2-chlorophenol (BCP) were derived from in vitro incubations with liver microsomes, liver cytosol, and plasma from rats (catalytic efficiencies of 1.1, 2.8, and 0.19 ml/min/mg protein, respectively) and humans (catalytic efficiencies of 0.17, 0.79, and 0.063 ml/min/mg protein, respectively), whereas other chemical-related parameter values were derived using in silico calculations. The rat PBK model was evaluated against literature data on urinary excretion of conjugated BCP. Concentration-dependent inhibition of rat and human AChE was determined in vitro and these data were translated with the PBK models to predicted dose-dependent AChE inhibition in rats and humans in vivo. Comparing predicted dose-dependent AChE inhibition in rats to literature data on profenofos-induced AChE inhibition revealed an accurate prediction of in vivo effect levels. Comparison of rat predictions (BMDL10 of predicted dose-response data of 0.45 mg/kg bw) and human predictions (BMDL10 of predicted dose-response data of 0.01 mg/kg bw) suggests that humans are more sensitive than rats, being mainly due to differences in kinetics. Altogether, the results demonstrate that in vivo AChE inhibition upon acute exposure to profenofos was closely predicted in rats, indicating the potential of this novel approach method in chemical hazard assessment.

Design, synthesis, in silico studies and in vitro evaluation of isatin-pyridine oximes hybrids as novel acetylcholinesterase reactivators.

Organophosphorus poisoning caused by some pesticides and nerve agents is a life-threating condition that must be swiftly addressed to avoid casualties. Despite the availability of medical countermeasures, the clinically available compounds lack a broad spectrum, are not effective towards all organophosphorus toxins, and have poor pharmacokinetics properties to allow them crossing the blood-brain barrier, hampering cholinesterase reactivation at the central nervous system. In this work, we designed and synthesised novel isatin derivatives, linked to a pyridinium 4-oxime moiety by an alkyl chain with improved calculated properties, and tested their reactivation potency against paraoxon- and NEMP-inhibited acetylcholinesterase in comparison to the standard antidote pralidoxime. Our results showed that these compounds displayed comparable in vitro reactivation also pointed by the in silico studies, suggesting that they are promising compounds to tackle organophosphorus poisoning.

Cyclic Peptides from the Soft Coral-Derived Fungus Aspergillus sclerotiorum SCSIO 41031.

Three novel cyclic hexapeptides, sclerotides C-E (1-3), and a new lipodepsipeptide, scopularide I (4), together with a known cyclic hexapeptide sclerotide A (5), were isolated from fermented rice cultures of a soft coral-derived fungus: Aspergillus sclerotiorum SCSIO 41031. The structures of the new peptides were determined by 1D and 2D NMR spectroscopic analysis, Marfey's method, ESIMS/MS analysis, and single crystal X-ray diffraction analysis. Scopularide I (4) exhibited acetylcholinesterase inhibitory activity with an IC50 value of 15.6 µM, and weak cytotoxicity against the human nasopharyngeal carcinoma cell line HONE-EBV with IC50 value of 10.1 µM.

Anthraquinones, Diphenyl Ethers, and Their Derivatives from the Culture of the Marine Sponge-Associated Fungus Neosartorya spinosa KUFA 1047.

Previously unreported anthraquinone, acetylpenipurdin A (4), biphenyl ether, neospinosic acid (6), dibenzodioxepinone, and spinolactone (7) were isolated, together with (R)-6-hydroxymellein (1), penipurdin A (2), acetylquestinol (3), tenellic acid C (5), and vermixocin A (8) from the culture of a marine sponge-associated fungus Neosartorya spinosa KUFA1047. The structures of the previously unreported compounds were established based on an extensive analysis of 1D and 2D NMR spectra as well as HRMS data. The absolute configurations of the stereogenic centers of 5 and 7 were established unambiguously by comparing their calculated and experimental electronic circular dichroism (ECD) spectra. Compounds 2 and 5-8 were tested for their in vitro acetylcholinesterase and tyrosinase inhibitory activities as well as their antibacterial activity against Gram-positive and Gram-negative reference, and multidrug-resistant strains isolated from the environment. The tested compounds were also evaluated for their capacity to inhibit biofilm formation in the reference strains.

Evaluation of the acute toxic effect of azoxystrobin on non-target crayfish (Astacus leptodactylus Eschscholtz, 1823) by using oxidative stress enzymes, ATPases and cholinesterase as biomarkers.

Azoxystrobin is a broad-spectrum fungicide used worldwide. Since azoxystrobin spreads to large areas, its toxic effects on non-target organisms have aroused interest. In this study, the acute toxicity (96 h) of azoxystrobin on the crayfish (Astacus leptodactylus) was examined by using various biomarkers. The 96 h-LC50 dose (1656 mg L-) and its three sub-doses (828, 414, 207 mg L-1) were applied to crayfish. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were increased significantly compared to the control in hepatopancreas, gill and muscle tissues. The activities of acetylcholinesterase (AChE) and glutathione S-transferase (GST) increased, and glutathione reductase (GR) activity decreased significantly in hepatopancreas. Level of reduced glutathione (GSH) decreased significantly. The content of malondialdehyde (MDA) increased in a dose-dependent manner in all azoxystrobin treatments with the exception of the lowest dose (207 mg L-1)treatment. ATPases (Na+/K+ -ATPase, Mg2+ -ATPase, Ca2+ -ATPase, total ATPase) were significantly inhibited in gill and muscle tissues. The results of the present study indicate that azoxystrobin induces oxidative stress, and has adverse effects on activities of AChE and ATPases in crayfish.

Synthesis and in silico studies of triazene-substituted sulfamerazine derivatives as acetylcholinesterase and carbonic anhydrases inhibitors.

A novel series of sulfonamides, 4-(3-phenyltriaz-1-en-1-yl)-N-(4-methyl-2-pyrimidinyl)benzenesulfonamides (1-9), was designed and synthesized by the diazo reaction between sulfamerazine and substituted aromatic amines for the first time. Their chemical structures were characterized by 1 H nuclear magnetic resonance (NMR), 13 C NMR, and high-resolution mass spectra. The newly synthesized compounds were evaluated in terms of acetylcholineasterase (AChE) and human carbonic anhydrases (hCA) I and II isoenzymes inhibitory activities. According to the AChE inhibition results, the Ki values of the compounds 1-9 were in the range of 19.9 ± 1.5 to 96.5 ± 20.7 nM against AChE. Tacrine was used as the reference drug and its Ki value was 49.2 ± 2.7 nM against AChE. The Ki values of the compounds 1-9 were in the range of 10.2 ± 2.6 to 101.4 ± 27.8 nM against hCA I, whereas they were 18.3 ± 4.4 to 48.1 ± 4.5 nM against hCA II. Acetazolamide was used as a reference drug and its Ki values were 72.2 ± 5.4 and 52.2 ± 5.7 nM against hCA I and hCA II, respectively. The most active compounds, 1 (nonsubstituted) against AChE, 5 (4-ethoxy-substituted) against hCA I, and 8 (4-bromo-substituted) against hCA II, were chosen and docked at the binding sites of these enzymes to explain the inhibitory activities of the series. The newly synthesized compounds presented satisfactory pharmacokinetic properties via the estimation of ADME properties.

Hybridization-based design of novel anticholinesterase indanone-carbamates for Alzheimer's disease: Synthesis, biological evaluation, and docking studies.

Inspired by the structures of donepezil and rivastigmine, a novel series of indanone-carbamate hybrids was synthesized using the pharmacophore hybridization-based design strategy, and their biological activities toward acetylcholinesterase (AChE) and butyrylcholinesterase were evaluated. Among the synthesized compounds, 4d and 4b showed the highest AChE inhibitory activities with IC50 values in the micromolar range (compound 4d: IC50 = 3.04 µM; compound 4b: IC50 = 4.64 µM). Moreover, the results of the Aß1-40 aggregation assay revealed that compound 4b is a potent Aß1-40 aggregation inhibitor. The kinetics of AChE enzymatic activity in the presence of 4b was investigated, and the results were indicative of a reversible partial noncompetitive type of inhibition. A molecular docking study was conducted to determine the possible allosteric binding mode of 4b with the enzyme. The allosteric nature of AChE inhibition by these compounds provides the opportunity for the design of subtype-selective enzyme inhibitors. The presented indanone-carbamate scaffold can be structurally modified and optimized through medicinal chemistry-based approaches for designing novel multitargeted anti-Alzheimer agents.

Synthesis and biological evaluation of some 1-naphthol derivatives as antioxidants, acetylcholinesterase, and carbonic anhydrase inhibitors.

A series of some naphthol derivatives 4a-f, 5a,f, 6a, and 7a,b (six novel ones: 4c,d, 5a, 6a, 7a,b) bearing F, Cl, Br, OMe, and dioxole substituents at different positions of the aromatic rings was designed, synthesized, and characterized. The naphthol derivatives were synthesized in three steps, namely the addition reaction of furan via Diels-Alder cycloaddition reaction, copper(II) trifluoromethanesulfonate (Cu(OTf)2 )-catalyzed aromatization reaction, and the bromination reaction, respectively. The structures of the newly obtained compounds (4c,d, 5a, 6a, 7a,b) were characterized by spectroscopic techniques. In addition, some biological activity studies were investigated under in vitro conditions. Inhibition studies of these compounds were performed on human carbonic anhydrase (hCA) I and II isoenzymes purified from human erythrocytes as a biological evaluation. Moreover, their potential antioxidant and antiradical activities were studied by analytical methods like ABTS•+ and DPPH• scavenging, and it was determined that some molecules showed good activity. Also, inhibition of acetylcholinesterase (AChE), which is a marker of many degenerative neurological diseases, was tested and the results were discussed. Excellent enzyme inhibition results were recorded for most of the molecules. These 1-naphthol derivatives were found as effective inhibitors for hCA I, hCA II, and AChE with K i values ranging from 0.034 ± 0.54 to 0.724 ± 0.18 µM for hCA I, 0.172 ± 0.02 to 0.562 ± 0.21 µM for hCA II, and 0.096 ± 0.01 to 0.177 ± 0.02 µM for AChE.

In silico studies, nitric oxide, and cholinesterases inhibition activities of pyrazole and pyrazoline analogs of diarylpentanoids.

A new series of pyrazole, phenylpyrazole, and pyrazoline analogs of diarylpentanoids (excluding compounds 3a, 4a, 5a, and 5b) was pan-assay interference compounds-filtered and synthesized via the reaction of diarylpentanoids with hydrazine monohydrate and phenylhydrazine. Each analog was evaluated for its anti-inflammatory ability via the suppression of nitric oxide (NO) on IFN-γ/LPS-activated RAW264.7 macrophage cells. The compounds were also investigated for their inhibitory capability toward acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), using a modification of Ellman's spectrophotometric method. The most potent NO inhibitor was found to be phenylpyrazole analog 4c, followed by 4e, when compared with curcumin. In contrast, pyrazole 3a and pyrazoline 5a were found to be the most selective and effective BChE inhibitors over AChE. The data collected from the single-crystal X-ray diffraction analysis of compound 5a were then applied in a docking simulation to determine the potential binding interactions that were responsible for the anti-BChE activity. The results obtained signify the potential of these pyrazole and pyrazoline scaffolds to be developed as therapeutic agents against inflammatory conditions and Alzheimer's disease.

Synthesis of novel sulfonamides with anti-Alzheimer and antioxidant capacities.

A series of novel dopamine analogs incorporating urea and sulfonamide functional groups was synthesized from 3,4-dimethoxyphenethylamine. The reaction of 3,4-dimethoxyphenethylamine with N,N-dimethylcarbamoyl chloride, followed by the sulfonyl chlorination of the urea derivative, gave benzene-1-sulfonyl chloride 9, which was reacted with NH3 (aq) or N-alkyl amines to give related sulfonamides. The O-demethylation reaction of the subsequent compounds with BBr3 afforded four novel phenolic dopamine analogs including sulfonamide and urea in the same structure. The anticholinergic and antioxidant effects of the synthesized compounds were examined. Compound 13 exhibited inhibition at the micromolar level for both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The IC50 value of 13 was calculated as 298 ± 43 µM for AChE and 321 ± 29 µM for BChE. The antioxidant and antiradical effects of the molecules were investigated by five different methods. Among the synthesized compounds 10-18, the best antioxidant and antiradical activities belong to the phenolic compounds 15-18. Compounds 16 and 18 have a higher reducing power than the standards used, that is, butylated hydroxytoluene, butylated hydroxyanisole, Trolox, and α-tocopherol, for Fe3+ -Fe2+ and Cu2+ -Cu+ reducing activities. For the DPPH• radical scavenging method, compounds 16-18 have a much better scavenging power than the standard molecules. In addition, it has been determined by the induced-fit docking method that compound 13 is well-fitted in the active site of the enzymes. ADME studies reveal that the pharmacokinetic and physicochemical properties of all synthesized compounds are within an acceptable range.

Green Roccella phycopsis Ach. mediated silver nanoparticles: synthesis, characterization, phenolic content, antioxidant, antibacterial and anti-acetylcholinesterase capacities.

In this study, we develop here for the first time an easy, eco-friendly method for synthesizing silver nanoparticles (AgNPs) using the lichen Roccella phycopsis. AgNPs formation was first determined by a color change of the lichen filtrate to brown, subsequent to addition of AgNO3 solution, and confirmed by a maximum absorbance peak at 425 nm in UV-vis spectrum. Scanning electron microscope images showed a spherical shape with a size distribution between 11 and 18 nm, while the elemental composition was elucidated by the energy dispersive X-ray spectroscopy. The chemical compounds responsible for reduction and stabilization of silver nanoparticles were detected by Frourier transform infrared spectroscopy analysis. The synthesized R. phycopsis silver nanoparticles displayed a strong antioxidant activity. Further, the antibacterial activity was more effective against Gram-negative than Gram-positive bacteria. Besides, the R. phycopsis-AgNPs were potent in inhibiting acetylcholinesterase enzyme with IC50 value of 1.65 ± 0.07 mg/mL.

Synthesis and Evaluation of the Acetylcholinesterase Inhibitory Activities of Some Flavonoids Derived from Naringenin.

Alzheimer's disease (AD) is an irreversible neurodegenerative disease that affects many older people adversely. AD has been putting a huge socioeconomic burden on the healthcare systems of many developed countries with aging populations. The need for new therapies that can halt or reverse the progression of the disease is now extremely great. A research approach in the finding new treatment for AD that has attracted much interest from scientists for a long time is the reestablishment of cholinergic transmission through inhibition of acetylcholinesterase (AChE). Naringenin is a flavonoid with the potential inhibitory activity against AChE. From naringenin, many other flavonoid derivatives, such as flavanones and chalcones, can be synthesized. In this study, by applying the Williamson method, nine flavonoid derivatives were synthesized, including four flavanones and five chalcones. The evaluation of AChE inhibitory activity by the Ellman method showed that there were four substances (2, 4, 5, and 7) with relatively good biological activities (IC50 < 100 µM), and these biological activities were better than that of naringenin. The molecular docking revealed that strong interactions with amino acid residue Ser200 of the catalytic triad and those of the peripheral region of the enzyme were crucial for strong effects against AChE. Compound 7 had the strongest AChE inhibitory activity (IC50 13.0 ± 1.9 µM). This substance could be used for further studies.

The long-term effects of Δ9-tetrahydrocannabinol on microtubule dynamicity in rats.

Studies reported that Δ9-tetrahydrocannabinol (Δ9-THC) is an essential drug as an anti-cancer, neuroprotective, anti-inflammatory, and immune-modulatory agent. However, the mechanism by which Δ9-THC causes these events remains to be elucidated. We attempted to investigate the in vivo studies of Δ9-THC on brain microtubule dynamicity, and acetylcholinesterase (AChE) activity. The microtubule polymerization, secondary and tertiary structures of α/ß-tubulins, as well as the AChE activity, were evaluated in the experimental groups. The significantly lowest optical density and initial rate of polymerization was observed in THC 3 mg/kg, THC 9 mg/kg, and THC 18 mg/kg treated groups. The content of secondary and tertiary structures of α/ß-tubulins was significantly affected in treated groups. The AChE activity was significantly lower in treated groups in a dose-dependent manner. These data highlight the microtubule dynamicity as a molecular target for Δ9-THC, which affects memory dysfunction. However, Δ9-THC can be inhibited the AChE activity and provide an improved therapeutics for neurodegenerative diseases.

Inhibition of acetylcholinesterase attenuated retinal inflammation via suppressing NF-κB activation.

Elevated inflammatory cytokines contribute to the pathogenesis of various retinal diseases such as diabetic retinopathy, retinal vasculitis and retinitis. However, the underlying mechanism of retinal inflammation remains largely unknown. Recent studies demonstrated that acetylcholinesterase (ACHE) is an inflammatory indicator in central neural system. This study was aimed to dissect the role of ACHE in retinal inflammation, and its mechanism of action. Retinal inflammation was induced by intravitreal injection of tumor necrosis factor-α (TNF-α) in heterozygous ACHE knockdown mice (ACHE+/-) and wild type mice (ACHE+/+). Donepezil, a well-known ACHE inhibitor, was administrated by daily gavage. Expression of ACHE and intercellular adherent molecule-1 (ICAM-1), infiltration of CD11b+ inflammatory cells, retinal leukostasis and vascular leakage was determined in both ACHE+/- and ACHE+/+ mice. ARPE-19 cells, a human retinal pigment epithelial cell line, were cultured for in vitro assay. Knockdown of ACHE was achieved by lipofectamine-mediated siRNA transfection and pharmaceutical suppression of ACHE was manipulated by donepezil. Cellular expression and distribution of ACHE, ICAM-1, and phosphorylation of NF-κB, IκB and IKKα/ß were detected by western-blot analysis or immunocytochemistry. Retinal expression of ACHE was dramatically upregulated, in parallel with increased ICAM-1 expression, enhanced leukostasis and augmented CD11b+ inflammatory cell infiltration as well as vascular hyperpermeability in ACHE+/+ mice injected with TNF-α. However, TNF-α-injected ACHE+/- mice showed lower level of ICAM-1, less leukostasis and fewer infiltrated CD11b+ cells. Moreover, TNF-α-induced retinal vascular leakage was significantly reduced in ACHE+/- mice. Similarly, TNF-α-induced retinal inflammatory response were also attenuated by donepezil intervention. In addition, TNF-α treatment resulted in significant induction of ACHE, upregulation of ICAM-1 and nuclear translocation of NF-κB, phosphorylation of IκB and IKKα/ß in ARPE-19 cells. However, inhibition of ACHE reduced TNF-α-induced phosphorylation of NF-κB, IκB and IKKα/ß in ARPE-19 cells. The present study reveals a pivotal role of ACHE in retinal inflammation. Inhibition of ACHE attenuates retinal inflammation and retinal leakage likely through suppressing NF-κB signaling activation.

Synthesis and cholinesterase inhibiting potential of A-ring azepano- and 3-amino-3,4-seco-triterpenoids.

In this study, a series of A-ring azepano- and 3-amino-3,4-seco-derivatives were synthesized from betulin, oleanolic, ursolic and glycyrrhetinic acids aiming to develop new cholinesterase inhibitors. Azepanobetulin, azepanoerythrodiol and azepanouvaol were modified to give amide and tosyl derivatives, while azepano-anhydrobetulines and azepano-glycyrrhetols were obtained for the first time. Oleanane and ursane type 3-amino-3,4-seco-4(23)-en triterpenic alcohols were synthesized by reducing the corresponding 2-cyano-derivatives accessible from Beckmann type 2 rearrangements. The compounds were screened in colorimetric Ellman's assays to determine their ability to act as inhibitors for the enzymes acetylcholinesterase (AChE, from electric eel) and butyrylcholinesterase (BChE, from equine serum). While most of these compounds were only moderate inhibitors for AChE, several of them were shown to be inhibitors for BChE acting as mixed-type inhibitors. Azepanobetulin 1, its C28-amide derivatives 7 and 8, azepano-11-deoxo-glycyrrhetol 12 and azepanouvaol 18 held inhibition constants Ki ranging between 0.21 ± 0.06 to 0.68 ± 0.19 µM. Thus, they were approximately 4 to 10 times more active than standard galantamine hydrobromide. For all of the compounds reasonably high docking scores for BChE were obtained being in good agreement with the experimental results from the enzymatic studies. As a result, A-ring azepano-triterpenoids were found to be new scaffolds for the development of BChE inhibitors.