Design,synthesis and structure-activity relationships of dual inhibitors of acetylcholinesterase and serotonin transporter as potential agents for Alzheimer's disease

We have designed and synthesized a dual inhibitor of acetylcholinesterase (AChE) and serotonin transporter (SERT) as a novel class of treatment drugs for Alzheimer's disease on the basis of a hypothetical model of the AChE active site. Dual inhibitions of AChE and SERT would bring about greater therapeutic effects than AChE inhibition alone and avoid adverse peripheral effects caused by excessive AChE inhibition. Compound (S)-6j exhibited potent inhibitory activities against AChE (IC(50)=101 nM) and SERT (IC(50)=42 nM). Furthermore, (S)-6j showed inhibitory activities of both AChE and SERT in mice brain following oral administration.     

Synthesis and evaluation of novel 4-[(3H,3aH,6aH)-3-phenyl)-4,6-dioxo-2-phenyldihydro-2H-pyrrolo[3,4-d]isoxazol-5(3H,6H,6aH)-yl]benzoic acid derivatives as potent acetylcholinesterase inhibitors and anti-amnestic agents

The present study was designed to synthesize and evaluate pyrrolo-isoxazole benzoic acid derivatives as potential acetylcholinesterase (AChE) inhibitors for the management of Alzheimer's disease. The synthesis of pyrrolo-isoxazole benzoic acid derivatives involved ring opening cyclization of p-aminobenzoic acid with maleic anhydride to yield maleanilic acid, which in turn afforded N-arylmaleimide via ring closed cyclization. Azomethine-N-oxides were obtained by condensation of N-arylhydroxylamine with differently substituted benzaldehydes followed by refluxing of N-arylmaleimide with differently substituted azomethine-N-oxides to pyrrolo-isoxazole benzoic acid derivatives as cis- and trans-stereoisomers. The synthesized compounds were evaluated in vitro for AChE inhibitory activity in rat brain homogenate with donepezil as standard AChE inhibitor. Thereafter, the most potent test compound was evaluated for in vitro butyrylcholinesterase inhibitory activity and in vivo memory evaluation in scopolamine (0.4mg/kg)-induced amnesia in mice by employing Morris water maze test. All pyrrolo-isoxazole benzoic acid derivatives demonstrated potent AChE inhibitory activity. Most of compounds exhibited similar activity to donepezil and four of them (7h, 7i, 8i, and 8h, IC(50)=19.1±1.9-17.5±1.5nM) displayed higher inhibitory activity as compared to donepezil (21.5±3.2nM) with compound 8ia (IC(50)=17.5±1.5nM) being the most active one. The test compound 8ia also ameliorated scopolamine-induced amnesia in mice in terms of restoration of time spent in target quadrant (TSTQ) and escape latency time (ELT). It may be concluded that pyrrolo-isoxazole benzoic acid derivatives may be employed as potential AChE inhibitors.     

Synthesis of 8-thiabicyclo[3.2.1]octanes and their binding affinity for the dopamine and serotonin transporters

Cocaine is a potent stimulant of the central nervous system. Its reinforcing and stimulant properties have been associated with inhibition of the dopamine transporter (DAT) on presynaptic neurons. In the search for medications for cocaine abuse, we have prepared 2-carbomethoxy-3-aryl-8-thiabicyclo[3.2.1]octane analogues of cocaine. We report that this class of compounds provides potent and selective inhibitors of the DAT and SERT. The selectivity resulted from reduced activity at the SERT. The 3beta-(3,4-dichlorophenyl) analogue inhibits the DAT and SERT with a potency of IC(50)=5.7 nM and 8.0 nM, respectively. The 3-(3,4-dichlorophenyl)-2,3-unsaturated analogue inhibits the DAT potently (IC(50)=4.5 nM) and selectively (>800-fold vs SERT). Biological enantioselectivity of DAT inhibition was limited for both the 3-aryl-2,3-unsaturated and the 3alpha-aryl analogues (2-fold), but more robust (>10-fold) for the 3beta-aryl analogues. The (1R)-configuration provided the eutomers.     

Design, synthesis and evaluation of flavonoid derivatives as potent AChE inhibitors

A new series of flavonoid derivatives have been designed, synthesized and evaluated as potent AChE inhibitors. Most of them showed more potent inhibitory activities to AChE than rivastigmine. The most potent inhibitor isoflavone derivative 10d inhibit AChE with a IC₅₀ of 4 nM and showed high BChE/AChE inhibition ratio (4575-fold), superior to donepezil (IC₅₀ = 12 nM, 389-fold). Molecular docking studies were also performed to explore the detailed interaction with AChE.     

Inhibitory effect of ursolic acid purified from Origanum majorana L on the acetylcholinesterase

We screened 139 herbal spices in search of the acetylcholinesterase (AChE) inhibitor from natural resources. AChE inhibitors, which enhance cholinergic transmission by reducing the enzymatic degradation of acetylcholine, are the only source of compound currently approved for the treatment of Alzheimer's Disease (AD). Among these herbs, edible plants and spices, the ethanol extract from Origanum majorana L. showed the highest inhibitory effect on AChE in vitro. By sequential fractionation of Origanum majorana L. the active component was finally identified as ursolic acid (3 beta-Hydroxyurs-12-en-28-oic acid). The ursolic acid of Origanum majorana L. inhibited AChE activity in a dose-dependent and competitive/non-competitive type. The Ki value (representing the affinity of the enzyme and inhibitor) of Origanum majorana L. ursolic acid was 6 pM, and that of tacrine was 0.4 nM. The concentration required for 50% enzyme inhibition of the active component (IC50 value) was 7.5 nM, and that of tacrine was 1 nM. This study demonstrated that the ursolic acid of Origanum majorana L. appeared to be a potent AChE inhibitor in Alzheimer's Disease.     

Design,synthesis and biological evaluation of coumarin alkylamines as potent and selective dual binding site inhibitors of acetylcholinesterase

Acetylcholinesterase inhibitors (AChEIs) are currently the drugs of choice, although only symptomatic and palliative, for the treatment of Alzheimer’s disease (AD). Donepezil is one of most used AChEIs in AD therapy, acting as a dual binding site, reversible inhibitor of AChE with high selectivity over butyrylcholinesterase (BChE). Through a combined target- and ligand-based approach, a series of coumarin alkylamines matching the structural determinants of donepezil were designed and prepared. 6,7-Dimethoxycoumarin derivatives carrying a protonatable benzylamino group, linked to position 3 by suitable linkers, exhibited fairly good AChE inhibitory activity and a high selectivity over BChE. The inhibitory potency was strongly influenced by the length and shape of the spacer and by the methoxy substituents on the coumarin scaffold. The inhibition mechanism, assessed for the most active compound 13 (IC₅₀ 7.6 nM) resulted in a mixed-type, thus confirming its binding at both the catalytic and peripheral binding sites of AChE.     

The new approach in development of anti-Alzheimer's disease drugs via the cholinergic hypothesis

A wide range of evidences show that cholinesterase (ChE) inhibitors can interfere with the progression of Alzheimer's disease (AD). The earliest known ChE inhibitors, namely, physostigmine and tacrine, showed modest improvement in the cognitive function of AD patients. However, clinical studies show that physostigmine has poor oral activity, brain penetration and pharmacokinetic parameters while tacrine has hepatotoxic liability. Studies were then focused on finding a new type of acetylcholinesterase (AChE) inhibitor that would overcome the disadvantages of these two compounds. During the study, by chance we found a seed compound. We then conducted a structure-activity relationship (SAR) study of this compound. After four years of exploratory research, we found donepezil hydrochloride (donepezil). Recently, acetylcholinesterase inhibitors (AChEIs) have been studied for other mechanisms of action, such as neuroprotective action and lowering of beta-amyloid (beta-amyloid). Donepezil also reduced beta-amyloid plaque in in vitro. The amyloid hypothesis is believed to be the most promising approach in the development of anti-AD drugs. We speculate the mechanism of lowering beta-amyloid by donepezil implicate alpha-secretase (alpha-secretase) enhancer.     

AChE inhibitor: a regio- and stereo-selective 1,3-dipolar cycloaddition for the synthesis of novel substituted 5,6-dimethoxy spiro[5.3']-oxindole-spiro-[6.3″]-2,3-dihydro-1H-inden-1″-one-7-(substituted aryl)-tetrahydro-1H-pyrrolo[1,2-c][1,3]thiazole

Pyrrolothiazolyloxindole analogues share vital pharmacological properties, considered useful in Alzheimer's disease (AD). The aim of this study was synthesis and evaluate pyralothiazolyloxindole analogues if possess acetyl cholinesterase (AChE) inhibitory activity. The easily accessible one-pot synthesis of these compounds resulted to be significantly less difficult and expensive than that of donepezil. Several compounds possess anti-cholinesterase activity in the order of micro and sub-micromolar. Particularly, compound was the most potent inhibitors of the series against acetyl cholinesterase enzyme with IC(50) 0.11μmol/L.     

Design, synthesis, and evaluation of 2-phenoxy-indan-1-one derivatives as acetylcholinesterase inhibitors

A series of 2-phenoxy-indan-1-one derivatives have been designed, synthesized, and tested as acetylcholinesterase inhibitors. The most potent compound exhibited high AChE inhibitory activity (IC50 = 50 nM), and the molecular docking study indicated that it was nicely accommodated by AChE.     

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 and biological evaluation of novel flavonoid derivatives as dual binding acetylcholinesterase inhibitors

A new series of flavonoid derivatives have been designed, synthesised and evaluated as acetylcholinesterase inhibitors that could bind simultaneously to the peripheral and catalytic sites of the enzyme. Among them, fifteen derivatives were found to inhibit the enzyme in the micromolar range and isoflavone derivatives possessed more potent inhibitory activity than other flavonoid derivatives. The best compound 9a had its inhibitory activity (IC(50) = 0.093 microM) in the same range as the reference compound, donepezil (IC(50) = 0.025 microM). Preliminary structure-activity relationships and a molecular modeling study for 9a have revealed that the isoflavone moiety plays a key role in the interaction of this series of derivatives with AChE by acting as an anchor in its peripheral anionic site.     

Kinetic and structural studies on the interactions of Torpedo californica acetylcholinesterase with two donepezil-like rigid analogues

Acetylcholinesterase inhibitors were introduced for the symptomatic treatment of Alzheimer’s disease (AD). Among the currently approved inhibitors, donepezil (DNP) is one of the most preferred choices in AD therapy. The X-ray crystal structures of Torpedo californica AChE in complex with two novel rigid DNP-like analogs, compounds 1 and 2, have been determined. Kinetic studies indicated that compounds 1 and 2 show a mixed-type inhibition against TcAChE, with K_i values of 11.12?±?2.88 and 29.86?±?1.12?nM, respectively. The DNP rigidification results in a likely entropy-enthalpy compensation with solvation effects contributing primarily to AChE binding affinity. Molecular docking evidenced the molecular basis for the binding of compounds 1 and 2 to the active site of β-secretase-1. Overall, these simplified DNP derivatives may represent new structural templates for the design of lead compounds for a more effective therapeutic strategy against AD by foreseeing a dual AChE and BACE-1 inhibitory activity.     

Searching for the Multi-Target-Directed Ligands against Alzheimer's disease: discovery of quinoxaline-based hybrid compounds with AChE, H₃R and BACE 1 inhibitory activities

A novel series of quinoxaline derivatives, as Multi-Target-Directed Ligands (MTDLs) for AD treatment, were designed by lending the core structural elements required for H(3)R antagonists and hybridizing BACE 1 inhibitor 1 with AChE inhibitor BYYT-25. A virtual database consisting of quinoxaline derivatives was first screened on a pharmacophore model of BACE 1 inhibitors, and then filtered by a molecular docking model of AChE. Seventeen quinoxaline derivatives with high score values were picked out, synthesized and evaluated for their biological activities. Compound 11a, the most effective MTDL, showed the potent activity to H(3)R/AChE/BACE 1 (H(3)R antagonism, IC(50)=280.0 ± 98.0 nM; H(3)R inverse agonism, IC(50)=189.3 ± 95.7 nM; AChE, IC(50)=483 ± 5 nM; BACE 1, 46.64±2.55% inhibitory rate at 20 μM) and high selectivity over H(1)R/H(2)R/H(4)R. Furthermore, the protein binding patterns between 11a and AChE/BACE 1 showed that it makes several essential interactions with the enzymes.     

Acetylcholinesterase inhibitors increase ADAM10 activity by promoting its trafficking in neuroblastoma cell lines

Acetylcholinesterase inhibitors (AChEIs) are the only currently available drugs for treating Alzheimer's Disease (AD). Some authors have suggested a function of AChEIs not only in the induction of AChE overproduction and alternative splicing shifts but also a possible role of these drugs in amyloid metabolism beyond their well-known symptomatic effect. Here, we investigate the mechanisms of action of the AChEI donepezil on APP (amyloid precursor protein) metabolism and on the activity/trafficking of the alpha-secretase candidate ADAM 10, in differentiated human neuroblastoma cells (SH-SY5Y). In these cells, the activity of AChE is significantly decreased after 2 h of donepezil treatment. Further, SH-SY5Y cells released significantly more sAPPalpha into the medium, whereas total APP levels in cell lysates were unchanged. Interestingly, treated cells showed increased ADAM 10 levels in membrane compartments. This effect was prevented by pretreatment with tunicamycin or brefeldin, suggesting that donepezil affects trafficking and/or maturation of ADAM 10; additionally, this pretreatment significantly decreased sAPPalpha levels. Pre-incubation with atropine decreased release of sAPPalpha significantly but did not revert ADAM 10 activity to control levels further suggesting that donepezil acts not solely through a purely receptor mediated pathway. These findings indicate that donepezil exerts multiple mechanisms involving processing and trafficking of key proteins involved in AD pathogenesis.     

No interaction of memantine with acetylcholinesterase inhibitors approved for clinical use

The loss of cholinergic neurons within the basal forebrain of patients with Alzheimer's disease (AD) may underlie aspects of the dementia. Excessive activation of N-methyl-D-aspartate (NMDA) receptors may underlie the degeneration of cholinergic cells. New drug therapies have been designed to either enhance cholinergic function by inhibition acetylcholinesterase (AChE), e.g. galanthamine, tetrahydroaminoacridine or donepezil, or by attenuation of NMDA receptor function, e.g. memantine. A combination of these two therapeutic approaches may be more beneficial at slowing the progression of the AD. The current study investigated whether memantine would attenuate the inhibition of AChE produced by these three drugs. The results indicate that these AChE inhibitors do not lose their therapeutic efficacy in combination with memantine. Our in vitro data suggest that the clinical combination of memantine with a reversible AChE inhibitor should be a valuable pharmacotherapeutic approach to dementia.     

Kinetic analysis of the inhibition of human butyrylcholinesterase with cymserine

Accompanying the gradual rise in the average age of the population of most industrialized countries is a regrettable progressive rise in the number of individuals afflicted with age-related neurodegenerative disorders, epitomized by Alzheimer's disease (AD) but, additionally, including Parkinson's disease (PD) and stroke. The primary therapeutic strategy, to date, involves the use of cholinesterases inhibitors (ChEIs) to amplify residual cholinergic activity. The enzyme, acetylcholinesterase (AChE), along with other elements of the cholinergic system is depleted in the AD brain. In contrast, however, its sister enzyme, butyrylcholinesterase (BuChE), that likewise cleaves acetylcholine (ACh), is elevated and both AChE and BuChE co-localize in high amounts with the classical pathological hallmarks of AD. The mismatch between increased brain BuChE and depleted levels of both ACh and AChE, particularly late in the disease, has supported the design and development of new ChEIs with a preference for BuChE; exemplified by the novel agent, cymserine, whose binding kinetics are characterized for the first time. Specifically, as assessed by the Ellman method, cymserine demonstrated potent concentration-dependent binding with human BuChE. The IC50 was determined as 63 to 100 nM at the substrate concentration range of 25 to 800 microM BuSCh. In addition, the following new binding constants were investigated for human BuChE inhibition by cymserine: T(FPnubeta), K(nubeta), K(Bs), K(MIBA), M(IC50), D(Sc), R(f), (O)K(m), OIC100, K(sl), theta(max) and R(i). These new kinetic constants may open new avenues for the kinetic study of the inhibition of a broad array of other enzymes by a wide variety of inhibitors. In synopsis, cymserine proved to be a potent inhibitor of human BuChE in comparison to its structural analogue, phenserine.     

Design,synthesis and biological activity of novel tacrine-isatin Schiff base hybrid derivatives

A series of novel tacrine-isatin Schiff base hybrid derivatives (7a-p) were designed, synthesized and evaluated as multi-target candidates against Alzheimer’s disease (AD). The biological assays indicated that most of these compounds displayed potent inhibitory activity toward acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) and specific selectivity for AChE over BuChE. It was also found that they act as excellent metal chelators. The compounds 7k and 7m were found to be good inhibitors of AChE-induced amyloid-beta (Aβ) aggregation. Most of the compounds inhibited AChE with the IC₅₀ values, ranging from 0.42 nM to 79.66 nM. Amongst them, 7k, 7m and 7p, all with a 6 carbon linker between tacrine and isatin Schiff base exhibited the strongest inhibitory activity against AChE with IC₅₀ values of 0.42 nM, 0.62 nM and 0.95 nM, respectively. They were 92-, 62- and 41-fold more active than tacrine (IC₅₀ = 38.72 nM) toward AChE. Most of the compounds also showed a potent BuChE inhibition among which 7d with an IC₅₀ value of 0.11 nM for BuChE is the most potent one (56-fold more potent than that of tacrine (IC₅₀ = 6.21 nM)). In addition, most compounds exhibited the highest metal chelating property. Kinetic and molecular modeling studies revealed that 7k is a mixed-type inhibitor, capable of binding to catalytic and peripheral site of AChE. Our findings make this hybrid scaffold an excellent candidate to modify current drugs in treating Alzheimer’s disease (AD).     

Potencies and selectivities of inhibitors of acetylcholinesterase and its molecular forms in normal and Alzheimer's disease brain

Eight inhibitors of acetylcholinesterase (AChE), tacrine, bis-tacrine, donepezil, rivastigmine, galantamine, heptyl-physostigmine, TAK-147 and metrifonate, were compared with regard to their effects on AChE and butyrylcholinesterase (BuChE) in normal human brain cortex. Additionally, the IC50 values of different molecular forms of AChE (monomeric, G1, and tetrameric, G4) were determined in the cerebral cortex in both normal and Alzheimer's human brains. The most selective AChE inhibitors, in decreasing sequence, were in order: TAK-147, donepezil and galantamine. For BuChE, the most specific was rivastigmine. However, none of these inhibitors was absolutely specific for AChE or BuChE. Among these inhibitors, tacrine, bis-tacrine, TAK-147, metrifonate and galantamine inhibited both the G1 and G4 AChE forms equally well. Interestingly, the AChE molecular forms in Alzheimer samples were more sensitive to some of the inhibitors as compared with the normal samples. Only one inhibitor, rivastigmine, displayed preferential inhibition for the G1 form of AChE. We conclude that a molecular form-specific inhibitor may have therapeutic applications in inhibiting the G1 form, which is relatively unchanged in Alzheimer's brain.     

Design,synthesis and biological evaluation of dual acetylcholinesterase and phosphodiesterase 5A inhibitors in treatment for Alzheimer’s disease

With the recent research advances in molecular biology and technology, multiple credible hypotheses about the progress of Alzheimer’s disease (AD) have been proposed; multi-target drugs have emerged as an innovative therapeutic approach for AD. Current clinical therapy for AD patients is mainly palliative treatment targeting acetylcholinesterase (AChE). Inhibition of phosphodiesterase 5A (PDE5A) has recently been validated as a potentially novel therapeutic approach for Alzheimer’s disease (AD). In this work, series of new compounds were designed, synthesized and evaluated as dual cholinesterase and PDE5A inhibitor. Biological results revealed that some of these compounds display good biological activities against AChE with IC₅₀ values about 44.67–169.80 nM (donepezil IC₅₀ 50.12 nM). Notably, compound 12 presented potent activities against PDE5A with IC₅₀ values about 50 μM (sildenafil IC₅₀ 12.59 μM), and some of these compounds showed low cell toxicity to A549 cells in vitro.     

Homo- and hetero-bivalent edrophonium-like ammonium salts as highly potent, dual binding site AChE inhibitors

A number of mono- and bis-quaternary ammonium salts, containing edrophonium-like and coumarin moieties tethered by an appropriate linker, proved to be highly potent and selective dual binding site acetylcholinesterase inhibitors with good selectivity over butyrylcholinesterase. Homobivalent bis-quaternary inhibitors 11 and 12, differing by only one methylene unit in the linker, were the most potent and selective inhibitors exhibiting a sub-nanomolar affinity (IC(50)=0.49 and 0.17 nM, respectively) and a high butyryl-/acetylcholinesterase affinity ratio (SI=1465 and 4165, respectively). The corresponding hetero-bivalent coumarinic inhibitors 13 and 14 were also endowed with excellent inhibitory potency but a lower AChE selectivity (IC(50)=2.1 and 1.0 nM, and SI=505 and 708, respectively). Docking simulations enabled clear interpretation of the structure-affinity relationships and detection of key binding interactions at the primary and peripheral AChE binding sites.