The goal of this mechanistic study was to elucidate the influence of N-functionalization of amiridine on the biological properties of its conjugates. We synthesized conjugates of amiridine and salicylimine/amine 9, 10 with linkers containing an N-acyl group at the amiridine fragment as analogs of our previously obtained hybrids 1, 2 containing an N-aminohexamethylene spacer. A comparative study of selected biological activities of N-acylalkylene conjugates 9, 10 with conjugates 1, 2 showed substantial decreases in anti-butyrylcholinesterase (BChE) activity (35-fold for the imine and 8-fold for the amine), loss of anti-acetylcholinesterase (AChE) activity, and disappearance of the ability to block amyloid beta (1-42) (Aβ42) self-aggregation. Similar effects were observed for the model compound N-hexylamiridine 3 and its N-acyl derivative 11. However, N-acyl functionalization at the amiridine pharmacophore did not reduce the ability of the conjugates to block AChE-induced β-amyloid aggregation and only slightly decreased their antiradical activity, maintaining the higher activity of amines compared to imines. These effects were consistent with results from quantum mechanical calculations and molecular docking, showing that N-acylation of amiridine decreased the proton affinity of its endocyclic N-atom. Consequently, the amiridine pharmacophore was essentially unprotonated, thereby diminishing the anticholinesterase activity of conjugates 9 and 10 and their ability to block Aβ42 self-aggregation.

New conjugates of amiridine and salicylic derivatives (salicylamide, salicylimine, and salicylamine) with different lengths of alkylene spacers were designed, synthesized, and evaluated as potential multifunctional central nervous system therapeutic agents for Alzheimer's disease (AD). Conjugates demonstrated high acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition (IC50: AChE, 0.265-4.24 μM; BChE, 0.01-0.64 μM) but poor activity against off-target carboxylesterase (CES). Specifically, conjugates with a (CH2)8 spacer showed the highest AChE and BChE inhibition: 3-16 times more effective than amiridine. Salicylamides 7b and 7c had the maximum BChE/AChE selectivity ratios: 193 and 138, respectively. Conjugates were mixed-type reversible inhibitors of both cholinesterases and displaced propidium from the AChE peripheral anionic site (PAS) at the level of donepezil. All conjugates inhibited Aβ42 self-aggregation in the thioflavin test; inhibition increased with spacer elongation, being greatest for (CH2)8. The results agreed with molecular docking to AChE, BChE, and Aβ42. Conjugates exhibited high 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)•+-scavenging activity comparable to the standard antioxidant Trolox, and they showed the ability to bind Cu2+, Fe2+, and Zn2+. Conjugates had favorable predicted intestinal absorption and blood-brain barrier permeability. Altogether, the results indicate that the new conjugates possess potential for further development as multifunctional anti-AD drug candidates.

To assess the influence of the nature of the anticholinesterase pharmacophore on the properties of potential multitarget Alzheimer's disease (AD) agents, new conjugates of tacrine (3a, b) and amiridine (5a, b) with ethyl-2-aminomethylidene-4,4,4-trifluoro-3-oxobutanoate linked by an alkylene spacer with n = 4,6 were synthesized. All conjugates are effective cholinesterase inhibitors with predominant inhibition of butyrylcholinesterase (BChE). The inhibitory activity of tacrine conjugates 3a, b toward acetylcholinesterase (AChE) and BChE increases with spacer elongation: IC50 AChE up to 0.185 µM, IC50 BChE up to 0.0806 µM. Amiridine conjugates 5a, b are less active as AChE inhibitors and their anti-AChE activity (IC50 up to 3.09 µM) remains virtually unchanged with spacer elongation, while anti-BChE activity increases significantly (n = 6, IC50 = 0.063 µM), which leads to increased selectivity toward BChE (up to 56). The effects are consistent with the results of kinetic studies and molecular docking of 3b and 5b. Both types of conjugates displace propidium from the AChE peripheral anionic site at the level of and above that of donepezil, and are capable of blocking self-aggregation of β-amyloid (up to 49.5%). The compounds demonstrate very weak antioxidant activity (tacrine conjugates) or its absence (amiridine). Thus, new conjugates are potential multitarget anti-AD agents with high selectivity toward BChE for amiridine derivative 5b.

Background/Objectives: This study focused on synthesizing novel alkenyl derivatives of azinylferrocenes and evaluating their potential as Alzheimer’s disease (AD) therapeutics. Methods: 1-Azinyl-1′-acetylferrocenes were obtained by regioselective acetylation of azinylferrocenes, followed by the Wittig reaction or reduction of 1-azinyl-1′-acetylferrocenes and subsequent dehydration of the resulting alcohols. The synthesized compounds underwent the following biological activity testing relevant to AD: inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and off-target carboxylesterase (CES); antioxidant capacity (ABTS and FRAP assays); inhibition of Aβ42 self-aggregation (thioflavin method); blocking AChE-induced β-amyloid aggregation (propidium displacement); and cytotoxicity in SH-SY5Y and MSC-Neu cells (MTT assay). Results: Quinoline and bipyridine derivatives demonstrated effective cholinesterase inhibition, especially quinoline 7b (AChE IC50 3.32 μM; BChE IC50 3.68 μM), while acridine derivatives were poor inhibitors. Quantum chemical (QC) calculations predicted that acridine derivatives were especially prone to form stable dimers. Molecular docking into protein targets generated by an AlphaFold3 reproduction code showed that these dimers were too bulky to access enzyme active sites, yet they could bind to protein surfaces to inhibit Aβ42 self-aggregation and displace propidium from the AChE peripheral anionic site. All compounds showed high antioxidant activity in ABTS and FRAP assays, with quinoline derivatives being 2–4 times more potent than Trolox. QC calculations supported these findings. Quinoline and bipyridine derivatives also exhibited low cytotoxicity and scant CES inhibition. Conclusions: Overall, the synthesized ferrocenes, particularly the quinoline and bipyridine derivatives, appear promising for further research as multifunctional therapeutic agents targeting AD due to their anticholinesterase, antiaggregating, and antioxidant activities combined with low toxicity.

Chloroalkylene amide derivatives were obtained by acylation of known Cholinesterase inhibitors, ipidacrine and tacrine. The acylated derivatives were used in the alkylation of substituted 2-thiouracils (R = Me, CF2H, CF3, (CF2)2H)) for the synthesis of new hybrid compounds. Study of the esterase profile revealed a pronounced activity and selectivity of the obtained conjugates against butyrylcholinesterase (IC50 up to 2.03 µmol L−1), moderate displacement of propidium from the acetylcholinesterase peripheral anionic site, and moderate inhibition of the β-amyloid self-aggregation. It was found that conjugation of thiouracils with tacrine leads to a decrease in the hepatotoxicity of the hybrid compounds compared to that of tacrine, while in the case of ipidacrine derivatives, no hepatotoxicity was observed.

An approach to the synthesis of derivatives of unsymmetric 1,1′-disubstituted azinylferrocenes containing phenol fragments was proposed. It was found that the introduction of the quinoline or 2,2′-bipyridine moiety increases the E/Z selectivity of the reaction. The monosubstituted acetylferrocene produces the products of the pinacoline rearrangement due to the McMurry coupling, whereas only 1-azinyl-1′-1-[(4-hydroxyphenyl)-phenylmethylene]ethylferrocenes were formed in the reaction with 1-azinyl-1′-acetyl-ferrocenes. The high antioxidant activity of the synthesized compounds in the ABTS and FRAP assays and high inhibitory activity against self-aggregation of β-amyloid (1–42) was shown. Cytotoxic activity of these compounds was studied on human breast cancer cells (MCF7), non-small-cell lung cancer cells (A549), colorectal cancer cells (DLD-1), and normal dermal fibroblasts (DF-2).

A series of 2-arylhydrazinylidene-3-oxo acids (AHOAs) was prepared by dealkylation of alkyl-2-arylhydrazinylidene-3-oxo-3-alkanoates with AlBr3. Using X-Ray, NMR spectroscopy, and quantum mechanical calculations (QM), the existence of AHOAs in a thermodynamically favorable Z-form stabilized by two intramolecular H-bonds was established. All AHOAs had acceptable ADME parameters. The esterase profile study showed that polyfluoroalkyl-AHOAs were effective and selective carboxylesterase (CES) inhibitors, while they were inactive against acetyl- and butyrylcholinesterase. In agreement with molecular docking, the most effective CES inhibitors (IC50 as low as 42 nM) were compounds bearing long polyfluoroalkyl substituents. The acids were also active against hCES1 and hCES2, and CF3-containing acids possessed selectivity against hCES2. Non-fluorinated acids did not inhibit CES, but they exhibited potent antioxidant capability. AHOAs having unsubstituted phenyl or electron-donating groups in the arylhydrazinylidene moiety displayed high primary antioxidant activity in the ABTS, FRAP, and ORAC tests, which did not depend on the substituent in the acyl fragment in the ABTS and ORAC assays. The radical-scavenging mechanism of AHOAs was investigated using QM calculations, showing a preference for cleavage of NH rather than OH bonds. For the lead antioxidants, 4-methoxysubstituted AHOAs, protective effects on erythrocyte membranes in AAPH-induced oxidative stress conditions were shown, including membrane stabilizing activity, inhibition of AAPH-induced lipid peroxidation of erythrocyte membranes, and Fe(II)-chelating ability. Thus, a new class of potent and selective CES inhibitors with powerful antioxidant potential has been developed as promising co-drugs capable of regulating the metabolism of esterified drugs and scavenging reactive radicals that form during Phase I biotransformation.

Ipidacrine conjugates were synthesized by alkylation of 4-hydroxycoumarin, dihydrofuro[3,4-c]pyridine, bipyridine, and azolo[1,5-a]pyrimidines with 2-chloro-N-(hexahydrocyclopenta[b]quinolin-9-yl)acetamide. Their isomeric structure was determined by NMR spectroscopy. Heterocycles containing ambident N,O-nucleophilic centers underwent alkylation at the oxygen atom, while in the presence of alternative carboxy and hydroxy functions, the alkylation occurred with the formation of an ester derivative. The study of the esterase profile, inhibitory effect on β-amyloid self-aggregation, as well as antioxidant activity of the conjugates, showed that coumarin derivatives are promising for the development of butyrylcholinesterase inhibitors, whereas conjugates of azolo[1,5-a]-pyrimidines are promising in the search for β-amyloid self-aggregation inhibitors.

A large series of 2-arylchromen-4-ones containing from 1 to 3 fluorine atoms or a trifluoromethyl group in the structure was synthesized by condensation of fluorinated 2-hydroxyacetophenones with benzaldehydes in an alkaline medium and subsequent oxidative cyclization of the resulting 2’-hydroxychalcones by action of I2 in DMSO. The cytotoxicity of the obtained compounds was studied in glioblastoma cell line, SNB19, and in a monkey-derived normal kidney epithelium cell line, Vero. In addition, antiglycation activity of the obtained compounds was evaluated. The inhibitory activity of some fluorinated 2-arylchromen-4-ones against acetylcholinesterase, butyrylcholinesterase and carboxylesterase as well their primary antioxidant activity in ABTS and FRAP tests were investigated. Screening of the synthesized compounds for their inhibitory activity against influenza A virus A/Puerto Rico/8/34 (H1N1) in the MDCK cell culture revealed that fluorinated compounds 32, 31 and 39 showed manifest antiviral effects (with IS = 57, 38 and 25 correspondingly) that makes this series of new biologically attractive fluorinated heterocycles promising for further development and in-depth study. A series of new fluorine-contained 2-aryl-chromen-4-one derivatives were synthesized as potential multi-targets bioactive compounds, among which 32, 31 and 39 were found to be the most promising antiviral agents.

The azo-coupling reaction of aryldiazonium chlorides containing a sulfone fragment with ethyl (trifluoro)acetylacetates and acetylacetone led to 2-sulfonarylhydrazinylidene 1,3-dicarbonyl compounds. According to NMR spectroscopy, 2-sulfonarylhydrazinylidene 3-oxoesters exist in solutions as Z-isomers. Micromolar inhibitors of three serine esterases, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterase (CES), were found among the synthesized trifluoromethyl-substituted 3-oxoesters. A nanomolar BChE inhibitor was identified among the trifluoromethyl-substituted 1,3-diketone derivatives exhibiting selectivity towards BChE in combination with radical-scavenging activity. 3-Oxoesters with moderate activity against pathogenic fungi of the T. mentagrophytes strain were revealed.

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 β-amyloid (Aβ) aggregation. Hybrids also exhibited the inhibition of Aβ 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.

A series of new conjugates of tacrine with ethyl 2-aminomethylidene-3-oxobutanoate with a spacer length of 4, 6, and 8 methylene groups was synthesized. The synthesized conjugates were shown to be effective inhibitors of acetylcholinesterase (IC50 up to 0.143 µmol L−1) and butyrylcholinesterase (IC50 up to 0.024 µmol L−1), with the inhibitory activity increasing with the spacer elongation. The new conjugates, as their diethyl malonate analogs, demonstrated a potential ability to block the AChE-induced β-amyloid aggregation and inhibited the self-aggregation of β-amyloid (1–42) with maximum activity at the level of the standard compound myricetin for derivatives with the octamethylene spacer. For the lead compound, a conjugate of tacrine with ethyl 2-aminomethylidene-3-oxobutanoate with the (CH2)8 spacer, the ability to bind the biometal ions CuII, FeII and ZnII was also demonstrated. The obtained characteristics indicated the synthesized conjugates as promising multitarget agents for the treatment of Alzheimer’s disease.

An approach was developed for the synthesis of multitarget compounds based on a tacrine molecule conjugated with a vanillin moiety using alkylimine or alkylamine linkers with different alkyl chain lengths (C2–C4). The synthesized conjugates effectively inhibit cholinesterases, displace propidium from the peripheral anionic site of acetylcholinesterase, which implies potential antiaggregant properties, and exhibit high antioxidant activity.

4-Arylhydrazinylidene-5-(polyfluoroalkyl)pyrazol-3-ones (4-AHPs) were found to be obtained by the regiospecific cyclization of 2-arylhydrazinylidene-3-(polyfluoroalkyl)-3-oxoesters with hydrazines, by the azo coupling of 4-nonsubstituted pyrazol-5-oles with aryldiazonium chlorides or by the firstly discovered acid-promoted self-condensation of 2-arylhydrazinylidene-3-oxoesters. All the 4-AHPs had an acceptable ADME profile. Varying the substituents in 4-AHPs promoted the switching or combining of their biological activity. The polyfluoroalkyl residue in 4-AHPs led to the appearance of an anticarboxylesterase action in the micromolar range. An NH-fragment and/or methyl group instead of the polyfluoroalkyl one in the 4-AHPs promoted antioxidant properties in the ABTS, FRAP and ORAC tests, as well as anti-cancer activity against HeLa that was at the Doxorubicin level coupled with lower cytotoxicity against normal human fibroblasts. Some Ph-N-substituted 4-AHPs could inhibit the growth of N. gonorrhoeae bacteria at MIC 0.9 μg/mL. The possibility of using 4-AHPs for cell visualization was shown. Most of the 4-AHPs exhibited a pronounced analgesic effect in a hot plate test in vivo at and above the diclofenac and metamizole levels except for the ones with two chlorine atoms in the aryl group. The methylsulfonyl residue was proved to raise the anti-inflammatory effect also. A mechanism of the antinociceptive action of the 4-AHPs through blocking the TRPV1 receptor was proposed and confirmed using in vitro experiment and molecular docking.

A simple and feasible approach to the synthesis of new amine derivatives of acridine based on the methodology of direct C–H functionalization has been developed. The inhibitory effect of the synthesized compounds toward cholinesterases and carboxylesterase as well as their antioxidant activity have been studied. A high anti-BChE activity has been shown for N-methylpiperazine derivative promising for further optimization to design on its basis new series of compounds efficient for the treatment of neurodegenerative diseases.

We investigated the inhibitory activities of novel 9-phosphoryl-9,10-dihydroacridines and 9-phosphorylacridines against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterase (CES). We also studied the abilities of the new compounds to interfere with the self-aggregation of β-amyloid (Aβ42) in the thioflavin test as well as their antioxidant activities in the ABTS and FRAP assays. We used molecular docking, molecular dynamics simulations, and quantum-chemical calculations to explain experimental results. All new compounds weakly inhibited AChE and off-target CES. Dihydroacridines with aryl substituents in the phosphoryl moiety inhibited BChE; the most active were the dibenzyloxy derivative 1d and its diphenethyl bioisostere 1e (IC50 = 2.90 ± 0.23 µM and 3.22 ± 0.25 µM, respectively). Only one acridine, 2d, an analog of dihydroacridine, 1d, was an effective BChE inhibitor (IC50 = 6.90 ± 0.55 μM), consistent with docking results. Dihydroacridines inhibited Aβ42 self-aggregation; 1d and 1e were the most active (58.9% ± 4.7% and 46.9% ± 4.2%, respectively). All dihydroacridines 1 demonstrated high ABTS•+-scavenging and iron-reducing activities comparable to Trolox, but acridines 2 were almost inactive. Observed features were well explained by quantum-chemical calculations. ADMET parameters calculated for all compounds predicted favorable intestinal absorption, good blood–brain barrier permeability, and low cardiac toxicity. Overall, the best results were obtained for two dihydroacridine derivatives 1d and 1e with dibenzyloxy and diphenethyl substituents in the phosphoryl moiety. These compounds displayed high inhibition of BChE activity and Aβ42 self-aggregation, high antioxidant activity, and favorable predicted ADMET profiles. Therefore, we consider 1d and 1e as lead compounds for further in-depth studies as potential anti-AD preparations.

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 β-amyloid aggregation. All conjugates inhibited Aβ42 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 Aβ42 self-aggregation, which was corroborated by molecular docking to Aβ42. 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 amiridine-thiouracil conjugates with different substituents in the pyrimidine fragment (R = CH3 , CF2 Н, CF3 , (CF2 )2 H) and different spacer lengths (n = 1-3) were synthesized. The conjugates rather weakly inhibit acetylcholinesterase (AChE) and exhibit high inhibitory activity (IC50 up to 0.752 ± 0.021 µM) and selectivity to butyrylcholinesterase (BChE), which increases with spacer elongation; the lead compounds are 11c, 12c, and 13c. The conjugates are mixed-type reversible inhibitors of both cholinesterases and practically do not inhibit the structurally related off-target enzyme carboxylesterase. The results of molecular docking to AChE and BChE are consistent with the experiment on enzyme inhibition and explain the structure-activity relationships, including the rather low anti-AChE activity and the high anti-BChE activity of long-chain conjugates. The lead compounds displace propidium from the AChE peripheral anion site (PAS) at the level of the reference compound donepezil, which agrees with the mixed-type mechanism of AChE inhibition and the main mode of binding of conjugates in the active site of AChE due to the interaction of the pyrimidine moiety with the PAS. This indicates the ability of the studied conjugates to block AChE-induced aggregation of β-amyloid, thereby exerting a disease-modifying effect. According to computer calculations, all synthesized conjugates have an ADME profile acceptable for drugs.

The synthesis of new hybrid compounds based on the domestic drug amiridine and a seven-membered cyclic homolog of tacrine combined by a thiourea-containing spacer was described. The synthesized conjugates were shown to be highly efficient inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with a nanomolar activity exceeding that of amyridine and tacrine, which was consistent with the molecular docking results. The conjugates also demonstrated a broader spectrum of biological activity uncharacteristic of the parent pharmacophores, namely, the ability to inhibit the AChE-induced and self-aggregation of β-amyloid and antioxidant properties.