New N,N-dimethylcarbamate inhibitors of acetylcholinesterase: design synthesis and biological evaluation

Abstract

A series of N,N-dimethylcarbamates containing a N,N-dibenzylamino moiety was synthesized and tested to evaluate their ability to inhibit Acetylcholinesterase (AChE). The most active compounds 4 and 8, showed 85 and 69% of inhibition at 50?μM, respectively. Furthermore, some basic SAR rules were outlined: an alkyl linker of six methylene units is the best spacer between the carbamoyl and dibenzylamino moieties; electron-withdrawal substituents on aromatics rings of the dibenzylamino group reduce the inhibitory power. Compound 4 produces a slow onset inhibition of AChE and this is not due to the carbamoylation of the enzyme, as demonstrated by the time-dependent inhibition assay of AChE with compound 4 and by MALDI-TOF MS analysis of trypsinized AChE inhibited by compound 4. Instead, compound 4 could act as a slow-binding inhibitor of AChE, probably because of its high conformational freedom due to the linear alkyl chain.     

Porphyrin derivatives as inhibitors for acetylcholinesterase from Drosophila melanogaster

The cure for Alzheimer''s disease (AD) is still unknown. According to Cholinergic hypothesis, Alzheimer''s disease is caused by the reduced synthesis of the neurotransmitter, Acetylcholine. Regional cerebral blood flow can be increased in patients with Alzheimer''s disease by Acetylcholinesterase (AChE) inhibitors. In this regard, Tetraphenylporphinesulfonate (TPPS), 5,10,15,20- Tetrakis (4-sulfonatophenyl) porphyrinato Iron(III) Chloride (FeTPPS) and 5,10,15,20-Tetrakis (4-sulfonatophenyl) porphyrinatoIron(III) nitrosyl Chloride (FeNOTPPS) were investigated as candidate compounds for inhibition of Acteylcholinesterase of Drosophila melanogaster (DmAChE) by use of Molecular Docking. The results show that FeNOTPPS forms the most stable complex with DmAChE.     

alpha,beta-Dehydrophenylalanine choline esters, a new class of reversible inhibitors of human acetylcholinesterase and butyrylcholinesterase

Our goal was to design, synthesize, and evaluate new cholinesterase inhibitors. Fourteen dehydroamino acids esterified to choline and to its ternary analog were synthesized by a new method that gave a yield of 84-93%. The potency of the amino acid ester derivatives was tested by measuring K(i) values for inhibition of human red cell acetylcholinesterase and human plasma butyrylcholinesterase. The most potent compound was a choline ester of dehydrophenylalanine where the amine group of the amino acid was derivatized with a benzoyl group containing a methoxy in the 2-position, CH(3)O(C(6)H(4))CONHC(CHC(6)H(5))COOCH(2)CH(2)N(+)(CH(3))(3). This compound was a strong inhibitor of both human acetylcholinesterase and human butyrylcholinesterase, with K(i) values of 10 microM and 0.08 microM, respectively. These K(i) values are comparable to that of Rivastigmine. Docking of the most potent compound into the active site of human butyrylcholinesterase showed that the lowest energy model had two benzene rings oriented towards Trp 82 and Tyr 332 whereas the positively charged nitrogen group was stabilized by Trp 231. This orientation placed the ester group 3.89 A from the active site Ser 198, a distance too far for covalent bonding, explaining why the esters are inhibitors rather than substrates. This class of anticholinesterase agents has the potential for therapeutic utility in the treatment of disorders of the cholinergic system.     

Synthesis and biological activity of derivatives of tetrahydroacridine as acetylcholinesterase inhibitors

Current state of medical sciences does not allow to treatment neurodegenerative diseases such as Alzheimer’s disease (AD). At present treatment of AD is severely restricted. The main class of medicines which are applied in AD is acetylcholinesterase inhibitors (AChEIs) like tacrine, donepezil, galantamine and rivastigmine that do not contribute to significant and long-term improvement in cognitive and behavioural functions.In this work, we report synthesis and biological evaluation of new hybrids of tacrine-6-hydrazinonicotinamide. The synthesis was based on the condensation reaction between tacrine derivatives and the hydrazine nicotinate moiety (HYNIC). All obtained compounds present affinity for both cholinesterases and are characterized by high selectivity in relation to butyrylcholinesterase (BChE).     

Design,synthesis and evaluation of new 4-arylthiazole-2-amine derivatives as acetylcholinesterase inhibitors

A series of new 4-arylthiazole-2-amine derivatives as acetylcholinesterase inhibitors (AChEIs) were designed and synthesized, Furthermore, their inhibitory activities against acetylcholinesterase in vitro were tested by Ellman spectrophotometry, and the results of inhibitory activity test showed that most of them had a certain acetylcholinesterase inhibitory activity in vitro. Moreover, the IC₅₀ value of compound 4f was to 0.66 μM, which was higher than that of Rivastigmine and Huperzine-A as reference compounds, and it had a weak inhibitory effect on butyrylcholinesterase. The potential binding mode of compound 4f with AChE was investigated by the molecular docking, and the results showed that 4f was strongly bound up with AChE with the optimal conformation, in addition, their binding energy reached −11.27 Kcal*mol⁻¹. At last, in silico molecular property of the synthesized compounds were predicted by using Molinspiration online servers. It can be concluded that the lead AChEIs compound 4f presented satisfactory drug-like characteristics.     

Rational design and synthesis of dihydropyrimidine based dual binding site acetylcholinesterase inhibitors

Based on the pharmacological importance of dihydropyrimidine (DHPM) scaffold, substituted DHPMs linked with acetamide linker to substituted aromatic anilines were synthesized and evaluated for their potency as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. The good AChE inhibitory activity of 4-dihydropyrimidine-2-thione (4a–h) and 2-amino-1,4-dihyropyrimidines (5a–h) series was observed with compound 4a and 4d identified as the most potent compounds with IC₅₀ values of 0.17 ± 0.01 and 0.39 ± 0.04 μM respectively. The inhibition of BChE was found in a broader range of concentrations (2.37–56.32 μM). To explore the binding insights into the enzyme, molecular docking study was carried out using GOLD software. The binding mode analysis indicated that all of these inhibitors are well accommodated in the active site and interact with the key amino acid residues of Catalytic anionic site (CAS) and peripheral anionic site (PAS). Furthermore, in silico ADMET predictions suggest that these compounds are non-AMES toxic with good blood brain barrier (BBB) penetration, human intestinal absorption.     

Design,synthesis and evaluation of phthalide alkyl tertiary amine derivatives as promising acetylcholinesterase inhibitors with high potency and selectivity against Alzheimer's disease

A series of phthalide alkyl tertiary amine derivatives were designed, synthesized and evaluated as potential multi-target agents against Alzheimer’s disease (AD). The results indicated that almost all the compounds displayed significant AChE inhibitory and selective activities. Besides, most of the derivatives exhibited increased self-induced Aβ_1-42 aggregation inhibitory activity compared to the lead compound dl-NBP, and some compounds also exerted good antioxidant activity. Specifically, compound I-8 showed the highest inhibitory potency toward AChE (IC₅₀ = 2.66 nM), which was significantly better than Donepezil (IC₅₀ = 26.4 nM). Moreover, molecular docking studies revealed that compound I-8 could bind to both the catalytic active site and peripheral anionic site of AChE. Furthermore, compound I-8 displayed excellent BBB permeability in vitro. Importantly, the step-down passive avoidance test indicated that I-8 significantly reversed scopolamine-induced memory deficit in mice. Collectively, these results suggested that I-8 might be a potent and selective AChE inhibitor for further anti-AD drug development.     

New coumarin-benzotriazole based hybrid molecules as inhibitors of acetylcholinesterase and amyloid aggregation

A novel series of triazole tethered coumarin-benzotriazole hybrids based on donepezil skeleton has been designed and synthesized as multifunctional agents for the treatment of Alzheimer’s disease (AD). Among the synthesized compounds 13b showed most potent acetylcholinesterase (AChE) inhibition (IC₅₀ = 0.059 μΜ) with mixed type inhibition scenario. Structure-activity relationship revealed that three-carbon alkyl chain connecting coumarin and triazole is well tolerable for inhibitory potential. Hybrids obtained from 4-hydroxycoumarin and 1-benzotriazole were most potent AChE inhibitors. The inhibitory potential of all compounds against butyrylcholinesterase was also evaluated but all showed negligible activity suggesting that the hybrid molecules are selective AChE inhibitors. 13b (most potent AChE inhibitor) also showed copper-induced Aβ_1-42 aggregation inhibition (34.26% at 50 μΜ) and chelating properties for metal ions (Cu²⁺, Fe^2+, and Zn²⁺) involved in AD pathogenesis along with DNA protective potential against degenerative actions of OH radicals. Molecular modelling studies confirm the potential of 13b in blocking both PAS and CAS of AChE. In addition, interactions of 13b with Aβ_1-42 monomer are also streamlined. Therefore, hybrid 13b can act as an effective hit lead molecule for further development of selective AChE inhibitors as multifunctional anti-Alzheimer’s agents.     

Synthesis,biological evaluation and in silico studies of novel N-substituted phthalazine sulfonamide compounds as potent carbonic anhydrase and acetylcholinesterase inhibitors

The synthesis, characterization and biological evaluation of a series of novel N-substituted phthalazine sulfonamide (5a-l) are disclosed. Phthalazines which are nitrogen-containing heterocyclic compounds are biologically preferential scaffolds, endowed with versatile pharmacological activity, such as anti-inflammatory, cardiotonic vasorelaxant, anticonvulsant, antihypertensive, antibacterial, anti-cancer action. The compounds were investigated for the inhibition against the cytosolic hCA I, II and AChE. Most screened sulfonamides showed high potency in inhibiting hCA II, widely involved in glaucoma, epilepsy, edema, and other pathologies (K_is in the ranging from 6.32 ± 0.06 to 128.93 ± 23.11 nM). hCA I was inhibited with K_is in the range of 6.80 ± 0.10–85.91 ± 7.57 nM, whereas AChE in the range of 60.79 ± 3.51–249.55 ± 7.89 nM. ADME prediction study of the designed N-substituted phthalazine sulfonamides showed that they are not only with carbonic anhydrase and acetylcholinesterase inhibitory activities but also with appropriate pharmacokinetic, physicochemical parameters and drug-likeness properties. Also, in silico docking studies were investigated the binding modes of selected compounds, to hCA I, II, and AChE.     

Effect of oxotremorine,physostigmine, and scopolamine on brain acetylcholine synthesis: A study using HPLC

The synthesis rate of brain acetylcholine (ACh) was estimated in mice following i.v. administration of [³H]choline (Ch). The measurements were performed 1 min after the tracer injection, using the [³H]ACh/[³H]Ch specific radioactivity ratio as an index of ACh synthesis rate. Endogenous and labeled Ch and ACh were quantified using HPLC methodology. Oxotremorine and physostigmine (0.5 mg/kg, i.p.) increased the steady state concentration of brain ACh by +130% and 84%, respectively and of Ch by +60% (oxotremorine); they decreased ACh synthesis by 62 and 55%, respectively. By contrast, scopolamine (0.7 mg/kg, i.p.) decreased the cerebral content of Ch by –26% and of ACh by –23% without enhancing the synthesis of ACh. The results show the utility of HPLC methodology in the investigation of ACh turnover.     

Metrifonate induces cholinesterase inhibition exclusively via slow release of dichlorvos

Metrifonate, a long-acting cholinesterase (ChE) inhibitor with very low toxicity in warm-blooded animals, inhibits rat brain and serum cholinesterase (ChE) in vitro through its hydrolytic degradation product, dichlorvos. This conclusion is based on the finding that metrifonate-induced ChE inhibition showed the same pH dependence as its reported dehydrochlorination to dichlorvos. The ChE inhibition induced by dichlorvos was not pH dependent. It was mediated by a competitive drug interaction with the catalytic site of the enzyme, which led to irreversible inhibition within several minutes of incubation. After this time, addition of further substrate to the inhibited enzyme was not able to promote drug dissociation and hence enzyme reactivation. Similar characteristics of inhibition, i.e. interaction with the substrate binding site and time-dependent switch to non-competitive inhibition were observed with the reference compound, physostigmine. However, the physostigmine-induced inhibition of ChE could be readily reversed by further substrate addition. Another reference compound, tetrahydroaminoacridine (THA), also induced a reversible inhibition of rat brain and serum cholinesterase, but with a mechanism of action different from that of both dichlorvos and physostigmine in that enzyme inhibition occurred rapidly upon drug addition at an allosteric site on the enzyme surface. It is suggested that the unique slow release plus the slow inhibition of ChE by dichlorvos is responsible for the lower toxicity of metrifonate compared to that of directly acting ChE inhibitors.     

Synthesis,biological activity and molecular modeling studies on 1H-benzimidazole derivatives as acetylcholinesterase inhibitors

A series of N-{2-[4-(1H-benzimidazole-2-yl)phenoxy]ethyl}substituted amine derivatives were designed to assess cholinesterase inhibitor activities. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitor activities were evaluated in vitro by using Ellman’s method. It was discovered that most of the compounds displayed AChE and/or BuChE inhibitor activity and few compounds were selective against AChE/BuChE. Compound 3c and 3e were the most active compounds in the series against eeAChE and hAChE, respectively. Molecular docking studies and molecular dynamics simulations were also carried out.     

Thermal denaturation of wild type and mutant recombinant acetylcholinesterase from amphioxus: effects of the temperature of in vitro expression and of reversible inhibitors

We have studied the thermal inactivation at 37 degrees C of wild type and mutant ChE2 (C310A, F312I, C466A, C310A/F312I, and C310A/C466A) from amphioxus (Branchiostoma floridae) expressed in vitro in COS-7 monkey cells under three sets of conditions: 30 degrees C for 48 h, 30 degrees C for 24 h and 37 degrees C for 24 h, and 37 degrees C for 48 h. We found biphasic denaturation curves for all enzymes and conditions, except wild type and C310A ChE2 expressed at 30 degrees C for 48 h. Generally, single mutants are more unstable than wild type, and the double mutants are even more unstable. We propose a model involving stable and unstable conformations of the enzymes to explain these results, and we discuss the implications of the model. We also found a correlation between the melting temperature of the ChEs and the rates at which they denature at 37 degrees C, with the denaturation of the unstable conformation dominating the relationship. Reversible cholinergic inhibitors protect the ChEs from thermal denaturation, and in some cases produce monophasic denaturation curves; we also propose a model to explain this stabilization.     

Benzene-1,2-, 1,3-, and 1,4-di-N-substituted carbamates as conformationally constrained inhibitors of acetylcholinesterase

Benzene-1,2-, 1,3-, and 1,4-di-N-substituted carbamates (1-15) are synthesized as the conformationally constrained inhibitors of acetylcholinesterase and mimic gauche, eclipsed, and anti-conformations of acetylcholine, respectively. All carbamates 1-15 are characterized as the pseudo substrate inhibitors of acetylcholinesterase. For a series of geometric isomers, the inhibitory potencies are as follows: benzene-1,4-di-N-substituted carbamate (para compound) > benzene-1,3-di-N-substituted carbamate (meta compound) > benzene-1,2-di-N-substituted carbamate (ortho compound). Therefore, benzene-1,4-di-N-substituted carbamates (para compounds), with the angle of 180 degrees between two C(benzene)-O bonds, mimic the preferable anti C-O/C-N conformers of acetylcholine for the choline ethylene backbone in the acetylcholinesterase catalysis.     

Synthesis,molecular docking,acetylcholinesterase and butyrylcholinesterase inhibitory potential of thiazole analogs as new inhibitors for Alzheimer disease

A series of thirty (30) thiazole analogs were prepared, characterized by ¹H NMR, ¹³C NMR and EI-MS and evaluated for Acetylcholinesterase and butyrylcholinesterase inhibitory potential. All analogs exhibited varied butyrylcholinesterase inhibitory activity with IC₅₀ value ranging between 1.59 ± 0.01 and 389.25 ± 1.75 μM when compared with the standard eserine (IC₅₀, 0.85 ± 0.0001 μM). Analogs 15, 7, 12, 9, 14, 1, 30 with IC₅₀ values 1.59 ± 0.01, 1.77 ± 0.01, 6.21 ± 0.01, 7.56 ± 0.01, 8.46 ± 0.01, 14.81 ± 0.32 and 16.54 ± 0.21 μM respectively showed excellent inhibitory potential. Seven analogs 15, 20, 19, 24, 28, 30 and 25 exhibited good acetylcholinesterase inhibitory potential with IC50 values 21.3 ± 0.50, 35.3 ± 0.64, 36.6 ± 0.70, 44.81 ± 0.81, 46.36 ± 0.84, 48.2 ± 0.06 and 48.72 ± 0.91 μM respectively. All other analogs also exhibited well to moderate enzyme inhibition. The binding mode of these compounds was confirmed through molecular docking.     

Antifungals and acetylcholinesterase inhibitors from the stem bark of Croton heliotropiifolius

The ethanolic extract of the stem bark of Croton heliotropiifolius Kunth (Euphorbiaceae) showed significant in vitro inhibition of acetylcholinesterase using a dilution spectrophotometric assay and antifungal activity against Candida albicans with a thin layer chromatography (TLC) bioautographic assay. In order to isolate the active compounds, bioassay-guided fractionation was undertaken using HPLC to localize the active compounds. Different zones of the HPLC-UV chromatogram were linked to acetylcholinesterase inhibition or to antifungal activities. In parallel to this HPLC-based activity profiling, HPLC-PDA-ESI-MS and HPLC-TOF-HRMS were used for the early identification of some of the compounds present. The targeted isolation of the active compounds was performed by medium pressure liquid chromatography (MPLC-UV) and further semi-preparative HPLC. Using this approach, nine compounds were isolated, one of them being a new indole alkaloid derivative. The structures of the isolated compounds were elucidated by spectroscopic methods including UV, NMR, MS and HRMS.     

Design,synthesis and biological evaluation of novel carbamates as potential inhibitors of acetylcholinesterase and butyrylcholinesterase

Rivastigmine, a dual inhibitor of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), has been approved by U.S. Food and Drug Administration to treat Alzheimer’s disease (AD) and Parkinson’s disease (PD) dementia. In the current work, a bambuterol derivative lacking one of the carbamoyloxy groups on the benzene ring (BMC-1) and its analogues were synthesized using 1-(3-hydroxyphenyl) ethan-1-one and 1-(4-hydroxyphenyl) ethan-1-one as starting materials. In-vitro cholinesterase assay established that nine compounds were more potent to inhibit both electric eel AChE and equine serum BChE than rivastigmine under the same experimental conditions. Further study confirmed that among the nine carbamates, BMC-3 (IC_50(AChE) = 792 nM, IC_50(BChE) = 2.2 nM) and BMC-16 (IC_50(AChE) = 266 nM, IC_50(BChE) = 10.6 nM) were excellent cholinesterase inhibitors with potential of permeating through the blood-brain barrier. These carbamates could be used as potential dual inhibitors of AChE and BChE and to discover novel drugs for the treatment of AD and PD dementia.     

Donepezil-tacrine hybrid related derivatives as new dual binding site inhibitors of AChE

A new series of donepezil–tacrine hybrid related derivatives have been synthesised as dual acetylcholinesterase inhibitors that could bind simultaneously to the peripheral and catalytic sites of the enzyme. These new hybrids combined a tacrine, 6-chlorotacrine or acridine unit as catalytic binding site and indanone (the heterocycle present in donepezil) or phthalimide moiety as peripheral binding site of the enzyme, connected through a different linker tether length. One of the synthesised compounds emerged as a potent and selective AChE inhibitor, which is able to displace propidium in a competition assay. These results seem to confirm the ability of this inhibitor to bind simultaneously to both sites of the enzyme and make it a promising lead for developing disease-modifying drugs for the future treatment of Alzheimer’s disease. To gain insight into the molecular determinants that modulate the inhibitory activity of these compounds, a molecular modelling study was performed to explore their binding to the enzyme.     

The effect of pH and reversible inhibitors on decarbamylation of acetylcholinesterase

Decarbamylation rate of membrane-bound methyl- and dimethyl-carbamylated acetylcholinesterase of human erythrocytes and bovine brain is reliably 1.1-1.6 times lower than that of the soluble enzyme. Such reversible inhibitors as tacrine (of non-competition action), ambenonium (mixed action) and galanthamine (competitive type of action) decelerate the decarbamylation rate of acetylcholinesterase. At pH 6 tacrine inhibits the reduction rate of soluble acetylcholinesterase activity of human erythrocytes more intensively than that of membrane-bound acetylcholinesterase. No differences in decarbamylation rate were found for the both forms of the enzyme at pH 8. Tacrine, a non-competitive inhibitor in concentrations below the inhibition constant (Ki = 1.4 x 10(-7) M) exerts the most intensive effect on the decarbamylation rate of methyl- and dimethylcarbamylated acetylcholinesterase of the mouse brain, while ambenonium and galanthamine in concentrations much (tens times) exceeding their Ki (3.1 x 10(-10) M and 4.4 x 10(-7) M, respectively) provide a decrease of the decarbamylation rate.