Novel 12-hydroxydehydroabietylamine derivatives act as potent and selective butyrylcholinesterase inhibitors

The skeleton of the diterpene dehydroabietylamine was modified, and a set of 12-hydroxy-dehydroabietylamine derivatives was obtained. 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). Additional investigations concerning the enzyme kinetics were performed and showed 12-hydroxy-N-(4-nitro-benzoyl)dehydroabietylamine (13) and 12-hydroxy-N-(isonicotinoyl)dehydroabietylamine (17) as selective BChE inhibitors holding good inhibition constants K_i = 0.72 ± 0.06 μM and K_i = 0.86 ± 0.19 μM, respectively.     

Ursolic and oleanolic acid derivatives with cholinesterase inhibiting potential

Triterpenoids are in the focus of scientific interest, and they were evaluated for many pharmacological applications among them their ability to act as inhibitors of cholinesterases. These inhibitors are still of interest as drugs that improve the life quality of patients suffering from age-related dementia illnesses especially of Alzheimer’s disease. Herein, we prepared several derivatives of ursolic and oleanolic acid and screened them in Ellman’s assays for their ability to inhibit acetylcholinesterase and/or butyrylcholinesterase, and for each of the active compounds the type of inhibition was determined. As a result, several compounds were shown as good inhibitors for acetylcholinesterase and butyrylcholinesterase even in a micromolar range. An ursolic acid derived hydroxyl-propinyl derivative 10 was a competitive inhibitor for butyrylcholinesterase with an inhibition constant of K_i = 4.29 μM, and therefore being twice as active as gold standard galantamine hydrobromide. The best inhibitor for acetylcholinesterase, however, was 2-methyl-3-oxo-methyl-ursoloate (18), acting as a mixed-type inhibitor showing K_i = 1.72 µM and K_i′ = 1.28 μM, respectively.     

New cholesterol esterase inhibitors based on rhodanine and thiazolidinedione scaffolds

We present a new class of inhibitors of pancreatic cholesterol esterase (CEase) based on ‘priviledged’ 5-benzylidenerhodanine and 5-benzylidene-2,4-thiazolidinedione structural scaffolds. The lead structures (5-benzylidenerhodanine 4a and 5-benzylidene-2,4-thiazolidinedione 4b) were identified in an in-house screening and these inhibited CEase with some selectivity over another serine hydrolase, acetylcholinesterase (AChE) (4a, CEase IC₅₀ = 1.76 μM vs AChE IC₅₀ = 5.14 μM and 4b, CEase IC₅₀ = 5.89 μM vs AChE IC₅₀ >100 μM). A small library of analogs (5a–10a) containing a core amino acid in place of the glycerol group of the lead structures, was prepared to explore other potential binding interaction with CEase. These analogs inhibited CEase with IC₅₀ values ranging from 1.44 to 85 μM, with the majority exhibiting some selectivity for CEase versus AChE. The most potent compound of the library (10a) had 17-fold selectivity over AChE. We also report molecular docking (with CEase) and detailed kinetic analysis on the amino acid analogs to further understand the associated structure–activity relationships.     

Interaction and inhibition of acetylcholinesterase from Electrophorus electricus by nitrosamines

Kinetic analysis has shown that dimethylnitrosamine, dipropylnitrosamine, dibutylnitrosamine, and diphenylnitrosamine initially act as reversible competitive inhibitors with respect to the substrate, acetylthiocholine chloride. The inhibitor constants Ki vary from 21-30 microM for the aliphatic nitrosamines to 8.2 microM for the aromatic diphenylnitrosamine. With time they act as irreversible covalent inhibitors with dimethylnitrosamine producing 82% inactivation after 40 min. Pseudo-first-order kinetics are observed with the rate constant being proportional to the concentration of the nitrosamine and the order of reaction being equal to one. Fluorometry, gel chromatography, and equilibrium dialysis have been used to study the binding of the nitrosamines with acetylcholinesterase. Scatchard analysis indicates that dimethyl-, dipropyl-, and dibutylnitrosamine have a weaker affinity for the enzyme (Kd 5.6-8.08 microM) compared to diphenylnitrosamine (Kd 2.32 microM). In all cases the number of binding sites was four.     

Identification of 5HT2-Receptors on Longitudinal Muscle of the Guinea Pig Ileum

Abstract

In binding experiments with the radioligands [³H]Ketanserin (HKet) and [¹²⁵I]LSD (ILSD) 21 compounds were investigated using rat brain cortex membranes. The pK_D-values of the compounds were virtually independent of the radioligand used and their rank order was consistent with classification of the binding sites as being of the 5-HT₂-type. In contrast, in the longitudinal muscle of the guinea pig ileum in the presence of 0.3 μM cinanserin, ILSD labelled sites which were quite different to those in the cortex. In a functional test antagonism of the 5HT induced contraction of the guinea-pig ileum was measured in the presence of 1 μM atropine. The pharmacological inhibition constants (IC₅₀-values) of 8 compounds correlated well with the dissociation constants for HKet binding in the cortex and did not correlate with the data from ILSD binding in the guinea pig ileum. It is concluded that the ileum contains postjunctional 5HT₂-receptors which mediate contraction. The nature of the ILSD binding sites in the ileum remains to be elucidated.     

Protein ligand interactions:isoquinoline alkaloids as inhibitors for lactate and malate dehydrogenase

Kinetic analysis has shown that isoquinoline, papaverine and berberine act as reversible competitive inhibitors to muscle lactate dehydrogenase and mitochondrial malate dehydrogenase with respect to the coenzyme NADH. The inhibitor constants Ki vary from 7.5 microM and 12.6 microM berberine interaction with malate dehydrogenase and lactate dehydrogenase respectively to 91.4 microM and 196.4 microM with papaverine action on these two enzymes. Isoquinoline was a poor inhibitor with Ki values of 200 microM (MDH) to 425 microM (LDH). No inhibition was observed for both enzymes in terms of their respective second substrate (oxaloacetic acid - malate dehydrogenase; pyruvate - lactate dehydrogenase). A fluorimetric analysis of the binding of the three alkaloids show that the dissociation constants (Kd) for malate dehydrogenase are 2.8 microM (berberine), 46 microM (papaverine) and 86 microM (isoquinoline); the corresponding values for lactate dehydrogenase are 3.1 microM, 52 microM and 114 microM. In all cases the number of binding sites averaged at 2 (MDH) and 4 (LDH). The binding of the alkaloids takes place at sites close to the coenzyme binding site. No conformational non equivalence of subunits is evident.     

Design,synthesis and bioevaluation of tricyclic fused ring system as dual binding site acetylcholinesterase inhibitors

Due to recently discovered non-classical acetylcholinesterase (AChE) function, dual binding-site AChE inhibitors have acquired a paramount attention of drug designing researchers. The unique structural arrangements of AChE peripheral anionic site (PAS) and catalytic site (CAS) joined by a narrow gorge, prompted us to design the inhibitors that can interact with dual binding sites of AChE. Eighteen homo- and heterodimers of desloratadine and carbazole (already available tricyclic building blocks) were synthesized and tested for their inhibition potential against electric eel acetylcholinesterase (eeAChE) and equine serum butyrylcholinesterase (eqBChE). We identified a six-carbon tether heterodimer of desloratadine and indanedione based tricyclic dihydropyrimidine (4c) as potent and selective inhibitor of eeAChE with IC₅₀ value of 0.09 ± 0.003 μM and 1.04 ± 0.08 μM (for eqBChE) with selectivity index of 11.1. Binding pose analysis of potent inhibitors suggest that tricyclic ring is well accommodated into the AChE active site through hydrophobic interactions with Trp84 and Trp279. The indanone ring of most active heterodimer 4b is stabilized into the bottom of the gorge and forms hydrogen bonding interactions with the important catalytic triad residue Ser200.     

Effects of dopaminergic compounds on carbonic anhydrase isozymes I,II, and VI

Studies on carbonic anhydrase (CA, EC 4.2.1.1) inhibitors have increased due to several therapeutic applications while there are few investigations on activators. Here we investigated CA inhibitory and activatory capacities of a series of dopaminergic compounds on human carbonic anhydrase (hCA) isozymes I, II, and VI. 2-Amino-1,2,3,4-tetrahydronaphthalene-6,7-diol hydrobromide and 2-amino-1,2,3,4-tetrahydronaphthalene-5,6-diol hydrobromide were found to show effective inhibitory action on hCA I and II whereas 2-amino-5,6-dibromoindan hydrobromide and 2-amino-5-bromoindan hydrobromide exhibited only moderate inhibition against both isoforms, being more effective inhibitors of hCA VI. K_i values of the molecules 3–6 were in the range of 41.12–363 μM against hCA I, of 0.381–470 μM against hCA II and of 0.578–1.152 μM against hCA VI, respectively. Compound 7 behaved as a CA activator with K_A values of 27.3 μM against hCA I, of 18.4 μM against hCA II and of 8.73 μM against hCA VI, respectively.     

Carbonic Anhydrase Inhibitors: Aromatic Sulfonamides and Disulfonamides Act as Efficient Tumor Growth Inhibitors

Abstract

Aromatic/heterocyclic sulfonamides generally act as strong inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). Here we report the unexpected finding that potent aromatic sulfonamide inhibitors of CA, possessing inhibition constants in the range of 10^?-⁸-^?10^?-⁹ M (against all the isozymes), also act as efficient in vitro tumor cell growth inhibitors, with GI₅₀ (molarity of inhibitor producing a 50% inhibition of tumor cell growth) values of 10 nM-35 μM against several leukemia, non-small cell lung cancer, ovarian, melanoma, colon, CNS, renal, prostate and breast cancer cell lines. The investigated compounds were sulfanilyl-sulfanilamide-, 4-thioureido-benzenesulfonamide- and benzene-1.3-disulfonamide-derivatives. The mechanism of antitumor action with these sulfonamides is unknown, but it might involve either inhibition of several CA isozymes (such as CA IX, CA XII, CA XIV) predominantly present in tumor cells, a reduced provision of bicarbonate for the nucleotide synthesis (mediated by carbamoyl phosphate synthetase II), the acidification of the intracellular milieu as a consequence of CA inhibition or uncoupling of mitochondria and potent CA V inhibition among others. A combination of several such mechanisms is also plausible. Optimization of such derivatives from the SAR point of view, might lead to the development of effective novel types of anticancer agents/therapies.     

Inhibition of acetylcholinesterase by two arylderivatives: 3a-Acetoxy-5H-pyrrolo(1,2-a) (3,1)benzoxazin-1,5-(3aH)-dione and cis-N-p-Acetoxy-phenylisomaleimide

Two arylderivatives, 3a-Acetoxy-5H-pyrrolo(1,2-a) (3,1)benzoxazin-1,5-(3aH)-dione 3 and cis-N-p-Acetoxy-phenylisomaleimide 4, were synthesized from anthranilic acid and para-aminophenol, respectively. The inhibitory effects of these compounds on acetylcholinesterase (AChE) activity were evaluated in vitro as well as by docking simulations. Both compounds showed inhibition of AChE activity (K_i = 4.72 ± 2.3 μM for 3 and 3.6 ± 1.8 μM for 4) in in vitro studies. Moreover, they behaved as irreversible inhibitors and made π–π interaction with W84 and hydrogen bonded with S200 and Y337 according to experimental data and docking calculations. The docking calculations showed ΔG bind (kcal/mol) of ? 9.22 for 3 and ? 8.58 for 4. These two compounds that can be use as leads for a new family of anti-Alzheimer disease drugs.     

Cellular Pharmacology of the D- and L-Enantiomers of β-5-o-Carboranyl-2′-deoxyuridine

Abstract

The cellular pharmacology of the D- and L-enantiomers of β-5-o-carboranyl-2′-deoxyuridine (CDU), compounds designed for boron neutron capture therapy (BNCT), were studied using human CEM lymphoblast and U-251 glioblastoma cells, at a physiologically achievable concentration (1 μM). Accumulation of the enantiomers was rapid and indistinguishable, reaching cellular concentrations > 40-fold higher than extracellular levels, with ～5% persisting in cells after incubation in fresh medium for more than 2 hr. Uptake was not affected by nucleoside uptake inhibitors, but was inhibited by the purine base uptake inhibitor papaverine.     

Synthesis and anticholinesterase activities of novel 1,3,4-thiadiazole based compounds

In the present study, new (1,3,4-thiadiazol-2-yl)benzene-1,3-diol based compounds have been synthesized and their potential anticholinesterases properties have been investigated using the modified of Ellman’s spectrophotometric method. The compounds were obtained by the reaction of hydrazides or thiosemicarbazides with aryl-modified sulfinylbis[(2,4-dihydroxyphenyl)methanethione]s. Their chemical structures were elucidated by IR, ¹H-NMR, ¹³C-NMR and EI-MS spectral data and elemental analyses. Most of the compounds acted as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitors in vitro, with IC₅₀ values ranging from >500 to 0.053 μM and from >500 to 0.105 μM, respectively. The most potent compound 9 (IC₅₀ = 0.053 μM) proved to be selective toward AChE, exhibiting selectivity ratios versus BuChE of ca. 950. The kinetic studies showed that it is a mixed-type of AChE inhibitor. Another compound (2) was active against both enzymes with IC₅₀ values in the low nM range. The structure-activity relationships (SARs) of the compounds under consideration were discussed.     

Design,synthesis and biological evaluation of selected 3-[3-(amino) propoxy] benzenamines as acetylcholinesterase inhibitors

The present paper describes design, synthesis, and biological evaluation of a series of some 3-[3-(amino)propoxy]benzenamines as acetylcholinesterase inhibitors using mice as a model and piracetam as a reference drug. The structures of these compounds were confirmed by spectral analysis and compounds were tested for memory enhancing activity using elevated plus maze test and acetylcholinesterase inhibitory assay. The inhibitory range of synthesized compounds was from 8.99 to 28.31 μM. The synthesized compounds possessed higher or equivalent percent retention as compared to piracetam at 1 mg/kg with no other CNS-related activities (locomotor and muscle relaxant, analgesic and anticonvulsant activities). Compound 3-[3-(imidazolo)propoxy]benzenamine has shown significant dose-dependent (1 and 3 mg/kg) memory enhancing activity, while 3-[3-(pyrrolidino)propoxy]benzenamine also showed activity equivalent to reference drug piracetam at 1 mg/kg. Both compounds 3-[3-(pyrrolidino)propoxy]benzenamine and 3-[3-(imidazolo)propoxy]benzenamine were also found to show AChE inhibition with IC₅₀ value of 8.99 and 17.87 μM. The molecular docking, MM-GBSA and molecular dynamics simulation studies were performed in order to establish a relationship between the biological results. RMSD, root-mean-square fluctuations, and interaction patterns of 10a–AChE and Sck–AChE complexes proved that the binding affinity of 10a toward AChE was highly stable with the proposed binding orientations.     

Synthesis of α-oxycarbanilinophosphonates and their anticholinesterase activities: the most potent derivative is bound to the peripheral site of acetylcholinesterase

A novel method has been developed for the synthesis of α-oxycarbanilino phosphonates through a reaction of α-hydroxyphosphonates with isocyanate under microwave irradiation. The synthesized compounds were evaluated for their acetylcholinesterase (AChE) inhibition potency through IC₅₀determination. Molecular modelling studies suggest that the most potent inhibitor (compound 4h, IC₅₀ = 6.36 µM) is bound to the peripheral site of AChE, which suggests that it decreases the catalytic activity not through binding to the active site but through blocking the entrance of the active site gorge. This puts forward the potential of compound 4h and its derivatives to be used in the design of dual inhibitors: inhibition of the catalytic activity of AChE and of amyloid β aggregation.     

Inhibition of Acetylcholinesterase by three New Pyridinium Compounds and their Effect on Phosphonylation of the Enzyme

Abstract

Three new mono-pyridinium compounds were prepared: 1-phenacyl-2-methylpyridinium chloride (1), 1-benzoylethylpyridinium chloride (2) and 1-benzoylethylpyridinium-4-aldoxime chloride (3) and assayed in vitro for their inhibitory effect on human blood acetylcholinesterase (EC 3.1.1.7, AChE). All the three compounds inhibited AChE reversibly; their binding affinity for the enzyme was compared with their protective effect (PI) on AChE phosphonylation by soman and VX. Compound 1 was found to bind to both the catalytic and the allosteric (substrate inhibition) sites of the enzyme with estimated dissociation constants of 6.9 μM (K_cat) and 27 μM (K_all), respectively. Compound 2 bound to the catalytic site with K_cat= 59 μM and compound 3 only to the allosteric site with K_all = 328 μM. PI was evaluated from phosphonylation measured in the absence and in presence of the compounds applied in a concentration corresponding to their K_cat or K_all value, and was also calculated from theoretical equations deduced from the reversible inhibition of the enzyme. Compounds 1 and 3 protected the enzyme from phosphonylation by soman and VX, whereas no protection was observed in the presence of compound 2 under the same conditions. Irrespective of the binding sites to AChE, PI for compounds 1 and 3 evaluated from phosphonylation agreed with PI calculated from reversible inhibition. Compound 3 was found to be a weak reactivator of methylphosphonylated AChE with k_r = 1.1 × 10²Lmol^-1 min^-1.     

Synthesis of new isoxazoline derivatives from harmine and evaluation of their anti-Alzheimer,anti-cancer and anti-inflammatory activities

Abstract

In our study, a series of new harmine derivatives has been prepared by cycloaddition reaction using various arylnitrile oxides and evaluated in vitro against acetylcholinesterase and 5-lipoxygenase enzymes, MCF7 and HCT116 cancer cell lines. Some of these molecules have been shown to be potent inhibitors of acetylcholinesterase and MCF7 cell line. The greatest activity against acetylcholinesterase (IC₅₀?=?10.4?µM) was obtained for harmine 1 and cytotoxic activities (IC₅₀?=?0.2?µM) for compound 3a. Two derivatives 3e and 3f with the thiophene and furan systems, respectively, showed good activity against 5- lipoxygenase enzyme (IC₅₀?=?29.2 and 55.5?µM, respectively).     

The Mechanisms of Action and Inhibition of Pancreatic Lipase and Acetylcholinesterase: A Comparative Modeling Study

Abstract

Pancreatic lipase and acetylcholinesterase are both serine esterases. Their X-ray structures reveal a similar overall fold, but no sequence homology can be detected. A catalytic triad like in the trypsin family of serine proteases consisting of serine, histidine and aspartate (glutamate in acetylcholinesterase) suggests mechanistic similarities. Models of the transition states of the substrate cleavage have been built and possible catalytic pathways were examined. The model that could produce a consistent pathway throughout the reactions had a transition state of the opposite handedness compared to trypsin. These models could be used to rationalise binding modes of inhibitors of both enzymes. The lipase inhibitor tetrahydrolipstatin (THL) contains a gamma-lactone which is opened by the catalytic serine; the alcohol leaving group prohibits deacylation by locking the pathway for incoming water and thus inactivates the enzyme. Carbamate inhibitors of acetylcholinesterase transfer a carbamoyl group to the serine-OH which deacylates slowly. These observations can be used as a starting point for the discovery of new classes of inhibitors.     

Kinetic,thermodynamic and statistical studies on the inhibition of adenosine deaminase by aspirin and diclofenac

The kinetic and thermodynamic effects of aspirin and diclofenac on the activity of adenosine deaminase (ADA) were studied in 50 mM phosphate buffer pH = 7.5 at 27 and 37°C, using UV-Vis spectrophotometry and isothermal titration calorimetry (ITC). Aspirin exhibits competitive inhibition at 27 and 37°C and the inhibition constants are 42.8 and 96.8 μM respectively, using spectrophotometry. Diclofenac shows competitive behavior at 27°C and uncompetitive at 37°C with inhibition constants of 56.4 and 30.0 μM, at respectively. The binding constant and enthalpy of binding, at 27°C are 45 μM, ? 64.5 kJ/mol and 61 μM, ? 34.5 kJ/mol for aspirin and diclofenac. Thermodynamic data revealed that the binding process for these ADA inhibitors is enthalpy driven. QSAR studies by principal component analysis implemented in SPSS show that the large, polar, planar, and aromatic nucleoside and small, aromatic and polar non-nucleoside molecules have lower inhibition constants.     

Evidence for a calcium-binding site on the eggshell of Globodera rostochiensis with a role in hatching

Two inhibitors of hatching in Globodera rostochiensis, ruthenium red and lanthanum, have been shown to bind to the eggshell using the techniques of microdensitometry for ruthenium red and X-ray microanalysis for lanthanum. Neither inhibitor penetrated or adhered to unhatched or hatched viable juveniles. Scatchard analysis for binding of lanthanum and ruthenium red to eggshells gave dissociation constants (K) of K_La 32.5 ± 14.0 μM and K_Rured 33.5 ± 5.0 μM respectively. Both values are within the 95% fiducial limits of those shown to cause 50% inhibition of hatch in previous work. Pretreatment with sodium hypochlorite separated an outer part of the eggshell from an inner region which exclusively bound ruthenium red. It is the inner lipoprotein layer that is believed to include the membranes controlling the permeability of the tylenchid eggshell. The rate of binding of ruthenium red was similar for intact and isolated eggshells with 50% binding occurring after 6.11 ± 0.91 min and 4.95 ± 2.38 min but the latter gave a significantly higher maximum binding suggesting that rupture of the eggshells made available additional binding sites on their inner surface. The binding of ruthenium red to the eggshells was pH dependant over most of the range pH 2.8–8.5 with 50% binding, given with its standard deviation, occurring at pH 5.75 ± 0.85. Competitive binding of lanthanum influenced the binding of ruthenium red to the eggshells from which Scatchard analysis gave K_la of 176 ± 79 μM. Similarly, calcium influenced the binding but this caused a biphasic plot with high and low affinity binding sites of K“_ca of 0.423 ± 1.16 μm and K‘_ca of 1078 ± 462 μM. The existence of a high affinity site for calcium that also binds ruthenium red, suggests that the eggshell membrane includes a calcium binding glycoprotein as found in some other receptor mechanisms.     

Structural insight into the inhibition of acetylcholinesterase by 2,3,4, 5-tetrahydro-1, 5-benzothiazepines

Benzothiazepines 1–3 inhibited acetylcholinesterase (AChE; EC 3.1.1.7) enzyme in a concentration-dependent fashion with IC₅₀ values of 1.0 ± 0.002, 1.2 ± 0.005 and 1.3 ± 0.001 μM, respectively. By using linear-regression equations, Lineweaver-Burk, Dixon plots and their secondary replots were constructed which indicated that compounds 1–3 are non-competitive inhibitors of AChE with K_i values of 0.8 ± 0.04, 1.1 ± 0.002, and 1.5 ± 0.001 μM, respectively. Molecular docking studies revealed that all the compounds are completely buried inside the aromatic gorge of AChE, extending deep into the gorge of AChE. A comparison of the docking results of compounds 1–3 displayed that these compounds generally adopt the same binding mode in the active site of AChE. The superposition of the docked structures demonstrated that the non-flexible benzothiazepine always penetrate into the aromatic gorge through the six-membered ring A, which allowed the ligands to interact simultaneously with more than one subsites of the active center of AChE. The higher AChE inhibitory potential of compounds 1–3 was found to be the cumulative effect of hydrophobic contacts and π-π interactions between the ligands and AChE. The relatively high affinity of benzothiazepine 1 with AChE was found to be due to additional hydrogen bond in benzothiazepine 1-AChE complex. The results indicated that substitution of halogen and methyl groups by hydrogen at aromatic ring of the benzothiazepine decreased the affinity of these molecules towards enzyme that may be due to the polar non-polar repulsions of these moieties with the amino acid residues in the active site of AChE. The observed binding modes of benzothiazepines 1–3 in the active site of AChE explain the affinities of benzothiazepines and provide a rational basis for the structure-based drug design of benzothiazepines with improved pharmacological properties.