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

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

Mitochondria and Oxidative Stress as a Link between Alzheimer's Disease and Diabetes Mellitus.

This review is devoted to the problems of the common features linking metabolic disorders and type 2 diabetes with the development of Alzheimer's disease. The pathogenesis of Alzheimer's disease closely intersects with the mechanisms of type 2 diabetes development, and an important risk factor for both pathologies is aging. Common pathological mechanisms include both factors in the development of oxidative stress, neuroinflammation, insulin resistance, and amyloidosis, as well as impaired mitochondrial dysfunctions and increasing cell death. The currently available drugs for the treatment of type 2 diabetes and Alzheimer's disease have limited therapeutic efficacy. It is important to note that drugs used to treat Alzheimer's disease, in particular acetylcholinesterase inhibitors, show a positive therapeutic potential in the treatment of type 2 diabetes, while drugs used in the treatment of type 2 diabetes can also prevent a number of pathologies characteristic for Alzheimer's disease. A promising direction in the search for a strategy for the treatment of type 2 diabetes and Alzheimer's disease may be the creation of complex multi-target drugs that have neuroprotective potential and affect specific common targets for type 2 diabetes and Alzheimer's disease.

Conjugates of Tacrine and Salicylic Acid Derivatives as New Promising Multitarget Agents for Alzheimer's Disease.

A series of previously synthesized conjugates of tacrine and salicylamide was extended by varying the structure of the salicylamide fragment and using salicylic aldehyde to synthesize salicylimine derivatives. The hybrids exhibited broad-spectrum biological activity. All new conjugates were potent inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with selectivity toward BChE. The structure of the salicylamide moiety exerted little effect on anticholinesterase activity, but AChE inhibition increased with spacer elongation. The most active conjugates were salicylimine derivatives: IC50 values of the lead compound 10c were 0.0826 µM (AChE) and 0.0156 µM (BChE), with weak inhibition of the off-target carboxylesterase. The hybrids were mixed-type reversible inhibitors of both cholinesterases and displayed dual binding to the catalytic and peripheral anionic sites of AChE in molecular docking, which, along with experimental results on propidium iodide displacement, suggested their potential to block AChE-induced ß-amyloid aggregation. All conjugates inhibited Aß42 self-aggregation in the thioflavin test, and inhibition increased with spacer elongation. Salicylimine 10c and salicylamide 5c with (CH2)8 spacers were the lead compounds for inhibiting Aß42 self-aggregation, which was corroborated by molecular docking to Aß42. ABTS•+-scavenging activity was highest for salicylamides 5a-c, intermediate for salicylimines 10a-c, low for F-containing salicylamides 7, and non-existent for methoxybenzoylamides 6 and difluoromethoxybenzoylamides 8. In the FRAP antioxidant (AO) assay, the test compounds displayed little or no activity. Quantum chemical analysis and molecular dynamics (MD) simulations with QM/MM potentials explained the AO structure-activity relationships. All conjugates were effective chelators of Cu2+, Fe2+, and Zn2+, with molar compound/metal (Cu2+) ratios of 2:1 (5b) and ~1:1 (10b). Conjugates exerted comparable or lower cytotoxicity than tacrine on mouse hepatocytes and had favorable predicted intestinal absorption and blood-brain barrier permeability. The overall results indicate that the synthesized conjugates are promising new multifunctional agents for the potential treatment of AD.

New Adamantane-Containing Edaravone Conjugates as Potential Neuroprotective Agents for ALS Treatments.

Currently, there are no effective drugs for the treatment of amyotrophic lateral sclerosis (ALS). Only two drugs-edaravone and riluzole-have been approved, but they have very limited efficacy. The aim of this work was to modify the structural core of the Edaravone-phenylpyrazolone moiety and combine it with aminoadamantane pharmacophore in order to expand the spectrum of its action to a number of processes involved in the pathogenesis of ALS. New conjugates of edaravone derivatives with 1-aminoadamantanes combined with alkylene or hydroxypropylene spacers were synthesized, and their biological activity was investigated. Compounds were found that could inhibit lipid peroxidation and calcium-related mitochondrial permeability, block fast sodium currents of CNS neurons, and reduce aggregation of the mutated form of the FUS-protein typical to ALS. So, the proposed modification of the edaravone molecule has allowed the obtaining of new original structures that combine some prospective therapeutic mechanisms against key chains of the pathogenesis of ALS. The identified lead compounds can be used for further optimization and development of new promising drugs on this basis for the treatment of ALS.

Bis(Benzofuran-1,3-N,N-heterocycle)s as Symmetric and Synthetic Drug Leads against Yellow Fever Virus.

The yellow fever virus (YFV) is an emerging RNA virus and has caused large outbreaks in Africa and Central and South America. The virus is often transmitted through infected mosquitoes and spreads from area to area because of international travel. Being an acute viral hemorrhagic disease, yellow fever can be prevented by an effective, safe, and reliable vaccine, but not be eliminated. Currently, there is no antiviral drug available for its cure. Thus, two series of novel bis(benzofuran−1,3-imidazolidin-4-one)s and bis(benzofuran−1,3-benzimidazole)s were designed and synthesized for the development of anti-YFV lead candidates. Among 23 new bis-conjugated compounds, 4 of them inhibited YFV strain 17D (Stamaril) on Huh-7 cells in the cytopathic effect reduction assays. These conjugates exhibited the most compelling efficacy and selectivity with an EC50 of <3.54 µM and SI of >15.3. The results are valuable for the development of novel antiviral drug leads against emerging diseases.

Conjugates of Methylene Blue with Cycloalkaneindoles as New Multifunctional Agents for Potential Treatment of Neurodegenerative Disease.

The development of multi-target-directed ligands (MTDLs) would provide effective therapy of neurodegenerative diseases (ND) with complex and nonclear pathogenesis. A promising method to create such potential drugs is combining neuroactive pharmacophoric groups acting on different biotargets involved in the pathogenesis of ND. We developed a synthetic algorithm for the conjugation of indole derivatives and methylene blue (MB), which are pharmacophoric ligands that act on the key stages of pathogenesis. We synthesized hybrid structures and performed a comprehensive screening for a specific set of biotargets participating in the pathogenesis of ND (i.e., cholinesterases, NMDA receptor, mitochondria, and microtubules assembly). The results of the screening study enabled us to find two lead compounds (4h and 4i) which effectively inhibited cholinesterases and bound to the AChE PAS, possessed antioxidant activity, and stimulated the assembly of microtubules. One of them (4i) exhibited activity as a ligand for the ifenprodil-specific site of the NMDA receptor. In addition, this lead compound was able to bypass the inhibition of complex I and prevent calcium-induced mitochondrial depolarization, suggesting a neuroprotective property that was confirmed using a cellular calcium overload model of neurodegeneration. Thus, these new MB-cycloalkaneindole conjugates constitute a promising class of compounds for the development of multitarget neuroprotective drugs which simultaneously act on several targets, thereby providing cognitive stimulating, neuroprotective, and disease-modifying effects.

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

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

In a search for efficient treatment for amyotrophic lateral sclerosis: Old drugs for new approaches.

Recent progress in understanding the pathological changes in the nervous system and in certain other body systems (e.g., immune system) that lead to the development and progression of amyotrophic lateral sclerosis (ALS) revealed a number of molecular and cellular processes that can potentially be used as therapeutic targets. Many of these processes are compromised not only in ALS but also in other diseases and a repertoire of drugs able to restore, at least partially, their functionality has been developed. In this review, we briefly describe current approaches to the repurposing of such "old" drugs for treatment of patients with ALS.

Mitochondria as a promising target for developing novel agents for treating Alzheimer's disease.

The mitochondria-targeting drugs can be conventionally divided into the following groups: those compensating for the energy deficit involved in neurodegeneration, including stimulants of mitochondrial bioenergetics and activators of mitochondrial biogenesis; and neuroprotectors, that are compounds increasing the resistance of mitochondria to opening of mitochondrial permeability transition (MPT) pores. Although compensating for the energy deficit and inhibition of MPT are obvious targets for drugs used in the very early stages of Alzheimer-like pathology, but their use as the monotherapy for patients with severe symptoms is unlikely to be sufficiently effective. It would be optimal to combine targets that would provide the cognitive-stimulating, the neuroprotective effects and the ability to affect specific disease-forming mechanisms. In the design of such drugs, assessment of their potential mitochondrial-targeted effects is of particular importance. The possibility of targeted drug design for simultaneous action on mitochondrial and neurotransmitter's receptors targets is, in particularly, based on the known interplay of various cellular pathways and the presence of common structural components. Of particular interest is directed search for multitarget drugs that would act simultaneously on mitochondrial calcium-dependent functions, the targets (receptors, enzymes, etc.) facilitating neurotransmission, and the molecular targets related to the action of so-called disease-modifying factors, in particular, the formation and overcoming of the toxicity of ß-amyloid or hyperphosphorylated tau protein. The examples of such approaches realized on the level of preclinical and clinical trials are presented below.

Conjugation of Aminoadamantane and γ-Carboline Pharmacophores Gives Rise to Unexpected Properties of Multifunctional Ligands.

A new series of conjugates of aminoadamantane and γ-carboline, which are basic scaffolds of the known neuroactive agents, memantine and dimebon (Latrepirdine) was synthesized and characterized. Conjugates act simultaneously on several biological structures and processes involved in the pathogenesis of Alzheimer's disease and some other neurodegenerative disorders. In particular, these compounds inhibit enzymes of the cholinesterase family, exhibiting higher inhibitory activity against butyrylcholinesterase (BChE), but having almost no effect on the activity of carboxylesterase (anti-target). The compounds serve as NMDA-subtype glutamate receptor ligands, show mitoprotective properties by preventing opening of the mitochondrial permeability transition (MPT) pore, and act as microtubule stabilizers, stimulating the polymerization of tubulin and microtubule-associated proteins. Structure-activity relationships were studied, with particular attention to the effect of the spacer on biological activity. The synthesized conjugates showed new properties compared to their prototypes (memantine and dimebon), including the ability to bind to the ifenprodil-binding site of the NMDA receptor and to occupy the peripheral anionic site of acetylcholinesterase (AChE), which indicates that these compounds can act as blockers of AChE-induced ß-amyloid aggregation. These new attributes of the conjugates represent improvements to the pharmacological profiles of the separate components by conferring the potential to act as neuroprotectants and cognition enhancers with a multifunctional mode of action.

Implications of farnesyltransferase and its inhibitors as a promising strategy for cancer therapy.

Ras proteins have been reported to play key role in oncologic diseases. Ras proteins are associated with cellular membranes for its carcinogenic activities through post-translational modifications, including farnesylation. Farnesyltransferase is responsible for a type of Ras membrane targeting, which leads to cancer origin and progression. Inhibitors of farnesyltransferase have been developed as novel anticancer agents. In this review, the role of farnesyltransferase in cancer progression and development has been discussed. Further, the current status of development of farnesyltransferase inhibitors for cancer prevention and treatment has also been reviewed.

Low Level of Expression of C-Terminally Truncated Human FUS Causes Extensive Changes in the Spinal Cord Transcriptome of Asymptomatic Transgenic Mice.

A number of mutations in a gene encoding RNA-binding protein FUS have been linked to the development of a familial form of amyotrophic lateral sclerosis known as FUS-ALS. C-terminal truncations of FUS by either nonsense or frameshift mutations lead to the development of FUS-ALS with a particularly early onset and fast progression. However, even in patients bearing these highly pathogenic mutations the function of motor neurons is not noticeably compromised for at least a couple of decades, suggesting that until cytoplasmic levels of FUS lacking its C-terminal nuclear localisation signal reaches a critical threshold, motor neurons are able to tolerate its permanent production. In order to identify how the nervous system responds to low levels of pathogenic variants of FUS we produced and characterised a mouse line, L-FUS[1-359], with a low neuronal expression level of a highly aggregation-prone and pathogenic form of C-terminally truncated FUS. In contrast to mice that express substantially higher level of the same FUS variant and develop severe early onset motor neuron pathology, L-FUS[1-359] mice do not develop any clinical or histopathological signs of motor neuron deficiency even at old age. Nevertheless, we detected substantial changes in the spinal cord transcriptome of these mice compared to their wild type littermates. We suggest that at least some of these changes reflect activation of cellular mechanisms compensating for the potentially damaging effect of pathogenic FUS production. Further studies of these mechanism might reveal effective targets for therapy of FUS-ALS and possibly, other forms of ALS.

Conjugates of tacrine and 1,2,4-thiadiazole derivatives as new potential multifunctional agents for Alzheimer's disease treatment: Synthesis, quantum-chemical characterization, molecular docking, and biological evaluation.

We synthesized conjugates of tacrine with 1,2,4-thiadiazole derivatives linked by two different spacers, pentylaminopropene (compounds 4) and pentylaminopropane (compounds 5), as potential drugs for the treatment of Alzheimer's disease (AD). The conjugates effectively inhibited cholinesterases with a predominant effect on butyrylcholinesterase (BChE). They were also effective at displacing propidium from the peripheral anionic site (PAS) of acetylcholinesterase (AChE), suggesting that they could block AChE-induced ß-amyloid aggregation. In addition, the compounds exhibited high radical-scavenging capacity. Conjugates 5 had higher anti-BChE activity and greater anti-aggregant potential as well relatively lower potency against carboxylesterase than compounds 4. Quantum-mechanical (QM) characterization agreed with NMR data to identify the most stable forms of conjugates for docking studies, which showed that the compounds bind to both CAS and PAS of AChE consistent with mixed reversible inhibition. Conjugates 4 were more potent radical scavengers, in agreement with HOMO localization in the enamine-thiadiazole system. Computational studies showed that all of the conjugates were expected to have good intestinal absorption, whereas conjugates 4 and 5 were predicted to have medium and high blood-brain barrier permeability, respectively. All conjugates were predicted to have medium cardiac toxicity risks. Overall, the results indicated that the conjugates are promising candidates for further development and optimization as multifunctional therapeutic agents for the treatment of AD.

New Hybrids of 4-Amino-2,3-polymethylene-quinoline and p-Tolylsulfonamide as Dual Inhibitors of Acetyl- and Butyrylcholinesterase and Potential Multifunctional Agents for Alzheimer's Disease Treatment.

New hybrid compounds of 4-amino-2,3-polymethylene-quinoline containing different sizes of the aliphatic ring and linked to p-tolylsulfonamide with alkylene spacers of increasing length were synthesized as potential drugs for treatment of Alzheimer's disease (AD). All compounds were potent inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with selectivity toward BChE. The lead compound 4-methyl-N-(5-(1,2,3,4-tetrahydro-acridin-9-ylamino)-pentyl)-benzenesulfonamide (7h) exhibited an IC50 (AChE) = 0.131 ± 0.01 µM (five times more potent than tacrine), IC50(BChE) = 0.0680 ± 0.0014 µM, and 17.5 ± 1.5% propidium displacement at 20 µM. The compounds possessed low activity against carboxylesterase, indicating a likely absence of unwanted drug-drug interactions in clinical use. Kinetics studies were consistent with mixed-type reversible inhibition of both cholinesterases. Molecular docking demonstrated dual binding sites of the conjugates in AChE and clarified the differences in the structure-activity relationships for AChE and BChE inhibition. The conjugates could bind to the AChE peripheral anionic site and displace propidium, indicating their potential to block AChE-induced ß-amyloid aggregation, thereby exerting a disease-modifying effect. All compounds demonstrated low antioxidant activity. Computational ADMET profiles predicted that all compounds would have good intestinal absorption, medium blood-brain barrier permeability, and medium cardiac toxicity risk. Overall, the results indicate that the novel conjugates show promise for further development and optimization as multitarget anti-AD agents.

Synthesis of 2-arylhydrazinylidene-3-oxo-4,4,4-trifluorobutanoic acids as new selective carboxylesterase inhibitors and radical scavengers.

A series of 2-arylhydrazinylidene-3-oxo-4,4,4-trifluorobutanoic acids was synthesized via dealkylation of ethyl 2-arylhydrazinylidene-3-oxo-4,4,4-trifluorobutanoates under the action of a Lewis acid. Under the same conditions, ethyl 2-arylhydrazinylidene-3-oxobutanoates were also found to undergo dealkylation rather than the previously described cyclization into cinnolones. Study of the esterase profile of these compounds showed that trifluoromethyl-containing acids, in contrast to non-fluorinated analogs, were effective and selective inhibitors of carboxylesterase (CES), without substantially inhibiting structurally related cholinesterases (acetylcholinesterase and butyrylcholinesterase). Moreover, both 3-oxo-4,4,4-trifluorobutanoic and 3-oxobutanoic acids having methyl or methoxy substituent in the arylhydrazinylidene fragment showed high antioxidant activity in the ABTS test. Thus, 2-arylhydrazinylidene-3-oxo-4,4,4-trifluorobutanoic acids were found to constitute a new class of effective and selective CES inhibitors that also possess high radical-scavenging activity.

Synthesis, molecular docking, and biological evaluation of 3-oxo-2-tolylhydrazinylidene-4,4,4-trifluorobutanoates bearing higher and natural alcohol moieties as new selective carboxylesterase inhibitors.

To search for effective and selective inhibitors of carboxylesterase (CES), a series of 3-oxo-2-tolylhydrazinylidene-4,4,4-trifluorobutanoates bearing higher or natural alcohol moieties was synthesized via pre-transesterification of ethyl trifluoroacetylacetate with alcohols to isolate transesterificated oxoesters as lithium salts, which were then subjected to azo coupling with tolyldiazonium chloride. Inhibitory activity against porcine liver CES, along with two structurally related serine hydrolases, acetylcholinesterase and butyrylcholinesterase, were investigated using enzyme kinetics and molecular docking. Kinetics studies demonstrated that the tested keto-esters are reversible and selective mixed-type CES inhibitors. Analysis of X-ray crystallographic data together with our IR and NMR spectra and QM calculations indicated that the Z-isomers were the most stable. The kinetic data were well explained by the molecular docking results of the Z-isomers, which showed specific binding of the compounds in the CES catalytic active site with carbonyl oxygen atoms in the oxyanion hole and non-specific binding outside it. Some compounds were studied as inhibitors of the main human isozymes involved in biotransformation of ester-containing drugs, hCES1 and hCES2. Esters of geraniol (3d) and adamantol (3e) proved to be highly active and selective inhibitors of hCES2, inhibiting the enzyme in the nanomolar range, whereas esters of borneol (3f) and isoborneol (3g) were more active and selective against hCES1. Computational ADMET studies revealed that all test compounds had excellent intestinal absorption, medium blood-brain barrier permeability, and low hERG liability risks. Moreover, all test compounds possessed radical-scavenging properties and low acute toxicity. Overall, the results indicate that members of this novel series of esters have the potential to be good candidates as hCES1 or hCES2 inhibitors for biomedicinal applications.

Mild cognitive impairment due to Alzheimer disease: Contemporary approaches to diagnostics and pharmacological intervention.

Alzheimer disease (AD) and related forms of dementia are among the main medical and social problems in the economically developed countries. It is connected with significant increase in human life span in these regions and with the absence of efficient medicines for treatment and prevention of such diseases. Lack of positive results in the developing of novel drugs for AD treatment stimulates special attention on problem of early diagnosis and drug discovery for pharmacotherapy on the very early stages of dementia, in particular, on mild cognitive impairments (MCI) due to AD. Here we review the state of art in the field of MCI diagnostics and analyze the data on the pharmacological agents developed for MCI treatment, which currently are in preclinical and clinical trials. The conclusion was made that only the agents that act on the very early pathogenetic stages of the disease, when the damage of cholinergic neurons is not observed, can be efficient for pharmacotherapeutic intervention of MCI. Therefore, the focused search and design of "disease-modifying" medicines should be accepted as the most (and may be the only) efficient strategy for treatment and prevention of MCI.

Drugs in Clinical Trials for Alzheimer's Disease: The Major Trends.

Alzheimer's disease (AD) is characterized by a chronic and progressive neurodegenerative process resulting from the intracellular and extracellular accumulation of fibrillary proteins: beta-amyloid and hyperphosphorylated Tau. Overaccumulation of these aggregates leads to synaptic dysfunction and subsequent neuronal loss. The precise molecular mechanisms of AD are still not fully understood but it is clear that AD is a multifactorial disorder and that advanced age is the main risk factor. Over the last decade, more than 50 drug candidates have successfully passed phase II clinical trials, but none has passed phase III. Here, we summarize data on current "anti-Alzheimer's" agents currently in clinical trials based on findings available in the Thomson Reuters «Integrity¼ database, on the public website www.clinicaltrials.gov, and on database of the website Alzforum.org. As a result, it was possible to outline some major trends in AD drug discovery: (i) the development of compounds acting on the main stages of the pathogenesis of the disease (the so-called "disease-modifying agents") - these drugs could potentially slow the development of structural and functional abnormalities in the central nervous system providing sustainable improvements of cognitive functions, which persist even after drug withdrawal; (ii) focused design of multitargeted drugs acting on multiple molecular targets involved in the pathogenesis of the disease; (3) finally, the repositioning of old drugs for new (anti-Alzheimer's) application offers a very attractive approach to facilitate the completion of clinical trials.

9-Substituted acridine derivatives as acetylcholinesterase and butyrylcholinesterase inhibitors possessing antioxidant activity for Alzheimer's disease treatment.

We investigated the inhibitory activity of 4 groups of novel acridine derivatives against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and carboxylesterase (CaE) using the methods of enzyme kinetics and molecular docking. Antioxidant activity of the compounds was determined using the 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) radical decolorization assay as their ability to scavenge free radicals. Analysis of the esterase profiles and antiradical activities of the acridine derivatives showed that 9-aryl(heteroaryl)-N-methyl-9,10-dihydroacridines have a high radical-scavenging activity but low potency as AChE and BChE inhibitors, whereas 9-aryl(heteroaryl)-N-methyl-acridinium tetrafluoroborates effectively inhibit cholinesterases but do not exhibit antiradical activity. In contrast, a group of derivatives of 9-heterocyclic amino-N-methyl-9,10-dihydroacridine has been found that combine effective inhibition of AChE and BChE with rather high radical-scavenging activity. The results of molecular docking well explain the observed features in the efficacy, selectivity, and mechanism of cholinesterase inhibition by the acridine derivatives. Thus, in a series of acridine derivatives we have found compounds possessing dual properties of effective and selective cholinesterase inhibition together with free radical scavenging, which makes promising the use of the acridine scaffold to create multifunctional drugs for the therapy of neurodegenerative diseases.

Synthesis, molecular docking, and biological activity of polyfluoroalkyl dihydroazolo[5,1-c][1,2,4]triazines as selective carboxylesterase inhibitors.

To search for effective and selective inhibitors of carboxylesterase (CaE), a series of 7-hydroxy-7-polyfluoroalkyl-4,7-dihydroazolo[5,1-c][1,2,4]triazines has been synthesized. Their inhibitory activity against acetylcholinesterase, butyrylcholinesterase, and CaE were investigated using the methods of enzyme kinetics and molecular docking. It was shown that the tested compounds are reversible selective CaE inhibitors of mixed type. Elongation of the polyfluoroalkyl substituent and the presence of an ester, preferably the ethoxycarbonyl group, enhance inhibitory activity toward CaE. Furthermore, the compounds with a tetrazole ring are more active against CaE than their triazole analogues. The obtained kinetic data are well explained by the results of molecular docking, according to which there is a similar orientation of triazolo- and tetrazolotriazines in the active site of CaE and the opposite one for pyrazolotriazines. In the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay, all of the studied tetrazolotriazines and some pyrazolotriazines demonstrated good antiradical activity comparable with a standard antioxidant, Trolox. The leading compounds were nonafluorobutyl substituted tetrazolo- and 7-phenylpyrazolotriazines, which possess effective and selective CaE inhibitory activity as well as additional useful radical-scavenging properties.