Katarína Svrčková

On the basis of previous reports, novel 2-benzoylhydrazine-1-carboxamides were designed as potential inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Inhibitors of these enzymes have many clinical applications. 2-(Substituted benzoyl)hydrazine-1-carboxamides decorated with N-methyl or tridecyl were prepared with three methods from commercially available or self-prepared hydrazides and isocyanates. For methyl derivatives, N-succinimidyl N-methylcarbamate was used or methyl isocyanate was prepared via Curtius rearrangement. Tridecyl isocyanate was synthesized again via Curtius rearrangement or from triphosgene and tridecylamine. The compounds were evaluated for the inhibition of AChE and BChE using Ellman’s spectrophotometric method. Most of the derivatives showed the dual inhibition of both enzymes with IC₅₀ values of 44–100 µM for AChE and from 22 µM for BChE. In general, the carboxamides inhibited AChE more strongly. A large number of the compounds showed better or quite comparable inhibition of cholinesterases in vitro than that of the drug rivastigmine. Molecular docking was performed to investigate the possible conformation of the compounds and their interactions with target enzymes. In both AChE and BChE, the compounds occupied the enzyme active cavity, and, especially in the case of BChE, the compounds were placed in close proximity to the catalytic triad.

2,5-Disubstituted 1,3,4-oxadiazoles are privileged versatile scaffolds in medicinal chemistry that have exhibited diverse biological activities. Acetyl- (AChE) and butyrylcholinesterase (BChE) inhibitors are used, e.g., to treat dementias and myasthenia gravis. 5-Aryl-1,3,4-oxadiazoles decorated with dodecyl linked via nitrogen, sulfur or directly to this heterocycle have been designed as potential inhibitors of AChE and BChE. They were prepared from commercially available or in-house prepared hydrazides by reaction with dodecyl isocyanate to form hydrazine-1-carboxamides 2 (yields 67–98%) followed by cyclization using p-toluenesulfonyl chloride and triethylamine in 41–100% yields. Thiadiazole isostere was also synthesized. The derivatives were screened for inhibition of AChE and BChE using Ellman’s spectrophotometric method. The compounds showed a moderate dual inhibition with IC₅₀ values of 12.8–99.2 for AChE and from 53.1 µM for BChE. All the heterocycles were more efficient inhibitors of AChE. The most potent inhibitor, N-dodecyl-5-(pyridin-4-yl)-1,3,4-thiadiazol-2-amine 3t, was subjected to advanced reversibility and type of inhibition evaluation. Structure–activity relationships were identified. Many oxadiazoles showed lower IC₅₀ values against AChE than established drug rivastigmine. According to molecular docking, the compounds interact non-covalently with AChE and BChE and block entry into enzyme gorge and catalytic site, respectively.

A series of thirty-one hydrazones of aminoguanidine, nitroaminoguanidine, 1,3-diaminoguanidine, and (thio)semicarbazide were prepared from various aldehydes, mainly chlorobenzaldehydes, halogenated salicylaldehydes, 5-nitrofurfural, and isatin (yields of 50–99%). They were characterized by spectral methods. Primarily, they were designed and evaluated as potential broad-spectrum antimicrobial agents. The compounds were effective against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus with minimum inhibitory concentrations (MIC) from 7.8 µM, as well as Gram-negative strains with higher MIC. Antifungal evaluation against yeasts and Trichophyton mentagrophytes found MIC from 62.5 µM. We also evaluated inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The compounds inhibited both enzymes with IC₅₀ values of 17.95–54.93 µM for AChE and ≥1.69 µM for BuChE. Based on the substitution, it is possible to modify selectivity for a particular cholinesterase as we obtained selective inhibitors of either AChE or BuChE, as well as balanced inhibitors. The compounds act via mixed-type inhibition. Their interactions with enzymes were studied by molecular docking. Cytotoxicity was assessed in HepG2 cells. The hydrazones differ in their toxicity (IC₅₀ from 5.27 to >500 µM). Some of the derivatives represent promising hits for further development. Based on the substitution pattern, it is possible to modulate bioactivity to the desired one.

A series of 14 target benzyl [2-(arylsulfamoyl)-1-substituted-ethyl]carbamates was prepared by multi-step synthesis and characterized. All the final compounds were tested for their ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in vitro, and the selectivity index (SI) was determined. Except for three compounds, all compounds showed strong preferential inhibition of BChE, and nine compounds were even more active than the clinically used rivastigmine. Benzyl {(2S)-1-[(2-methoxybenzyl)sulfamoyl]-4-methylpentan-2-yl}carbamate (5k), benzyl {(2S)-1-[(4-chlorobenzyl)sulfamoyl]-4-methylpentan-2-yl}carbamate (5j), and benzyl [(2S)-1-(benzylsulfamoyl)-4-methylpentan-2-yl]carbamate (5c) showed the highest BChE inhibition (IC₅₀ = 4.33, 6.57, and 8.52 µM, respectively), indicating that derivatives 5c and 5j had approximately 5-fold higher inhibitory activity against BChE than rivastigmine, and 5k was even 9-fold more effective than rivastigmine. In addition, the selectivity index of 5c and 5j was approx. 10 and that of 5k was even 34. The process of carbamylation and reactivation of BChE was studied for the most active derivatives 5k, 5j. The detailed information about the mode of binding of these compounds to the active site of both BChE and AChE was obtained in a molecular modeling study. In this study, combined techniques (docking, molecular dynamic simulations, and QTAIM (quantum theory of atoms in molecules) calculations) were employed.

A series of twelve nature-inspired 3,4,5-trimethoxycinnamates were prepared and characterized. All compounds, including the starting 3,4,5-trimethoxycinnamic acid, were tested for their ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in vitro; the selectivity index (SI) was also determined. 2-Fluororophenyl (2E)-3-(3,4,5-trimethoxyphenyl)-prop-2-enoate demonstrated the highest SI (1.71) in favor of BChE inhibition. 2-Chlorophenyl (2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoate showed the highest AChE-inhibiting (IC₅₀ = 46.18 µM) as well as BChE-inhibiting (IC₅₀ = 32.46 µM) activity with an SI of 1.42. The mechanism of action of the most potent compound was determined by the Lineweaver–Burk plot as a mixed type of inhibition. An in vitro cell viability assay confirmed the insignificant cytotoxicity of the discussed compounds on the two cell lines. Trends between structure, physicochemical properties and activity were discussed.

Based on the broad spectrum of biological activity of hydrazide–hydrazones, trifluoromethyl compounds, and clinical usage of cholinesterase inhibitors, we investigated hydrazones obtained from 4-(trifluoromethyl)benzohydrazide and various benzaldehydes or aliphatic ketones as potential inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). They were evaluated using Ellman’s spectrophotometric method. The hydrazide–hydrazones produced a dual inhibition of both cholinesterase enzymes with IC₅₀ values of 46.8–137.7 µM and 19.1–881.1 µM for AChE and BuChE, respectively. The majority of the compounds were stronger inhibitors of AChE; four of them (2-bromobenzaldehyde, 3-(trifluoromethyl)benzaldehyde, cyclohexanone, and camphor-based 2o, 2p, 3c, and 3d, respectively) produced a balanced inhibition of the enzymes and only 2-chloro/trifluoromethyl benzylidene derivatives 2d and 2q were found to be more potent inhibitors of BuChE. 4-(Trifluoromethyl)-N’-[4-(trifluoromethyl)benzylidene]benzohydrazide 2l produced the strongest inhibition of AChE via mixed-type inhibition determined experimentally. Structure–activity relationships were identified. The compounds fit physicochemical space for targeting central nervous systems with no apparent cytotoxicity for eukaryotic cell line together. The study provides new insights into this CF₃-hydrazide–hydrazone scaffold.

Background: Development of acetyl- (AChE) and butyrylcholinesterase (BuChE) inhibitors belongs to viable strategies for the treatment of dementia and other diseases related to decrease in cholinergic neurotransmission. Objective: That is why we designed twenty-two analogues of a dual AChEBuChE salicylanilide inhibitor, N-[3,5-bis(trifluoromethyl)phenyl]-5-bromo-2-hydroxybenzamide 1, to improve its potency. Methods: We prepared N,N-disubstituted (thio)carbamates via direct acylation with (thio)carbamoyl chloride, N-n-alkyl monosubstituted carbamates using isocyanates as well as its salicylanilide core analogues. The derivatives were evaluated in vitro against AChE from electric eel and BuChE from equine serum using spectrophotometric Ellman’s method. Results: The compounds showed moderate inhibition of both AChE and BuChE with IC50 from 18.2 to 196.6 μmol.L-1 and 9.2 to 196.2 μmol.L-1, respectively. Importantly, based on the substitution pattern, it is possible to modulate selectivity against AChE or BuChE and some derivatives also produced a balanced inhibition. In general, the most promising analogues were N-alkyl (C2-C6) carbamates and isomers with a changed position of phenolic hydroxyl. N-[3,5-Bis(trifluoromethyl)phenyl]-3-bromo-5- hydroxybenzamide 4a was the best inhibitor of both cholinesterases. Conclusion: A wide range of the derivatives improved the activity of the hit 1, they were superior to carbamate drug rivastigmine against AChE and some of them also against BuChE. The most promising derivatives also fit physicochemical space and structural features for CNS drugs together with an escalated lipophilicity.

Background: Hydrazide-hydrazones have been known as scaffold with various biological activities including inhibition of acetyl- (AChE) and butyrylcholinesterase (BuChE). Cholinesterase inhibitors are mainstays of dementias’ treatment. Objective: Twenty-five iodinated hydrazide-hydrazones and their analogues were designed as potential central AChE and BuChE inhibitors. Methods: Hydrazide-hydrazones were synthesized from 4-substituted benzohydrazides and 2-/4- hydroxy-3,5-diiodobenzaldehydes. The compounds were investigated in vitro for their potency to inhibit AChE from electric eel and BuChE from equine serum using Ellman’s method. We calculated also physicochemical and structural parameters for CNS delivery. Results: The derivatives exhibited a moderate dual inhibition with IC50 values ranging from 15.1-140.5 and 35.5 to 170.5 μmol.L-1 for AChE and BuChE, respectively. Generally, the compounds produced a balanced or more potent inhibition of AChE. N'-[(E)-(4-Hydroxy-3,5-diiodophenyl)methylidene]-4- nitrobenzohydrazide 2k and 4-fluoro-N'-(2-hydroxy-3,5-diiodobenzyl)benzohydrazide 3a were the most potent inhibitors of AChE and BuChE, respectively. Structure-activity relationships were established, and molecular docking studies confirmed interaction with enzymes. Conclusion: Many novel hydrazide-hydrazones showed lower IC50 values than rivastigmine against AChE and some of them were comparable for BuChE to this drug used for the treatment of dementia. They interact with cholinesterases via non-covalent binding into the active site. Based on the BOILEDEgg approach, the majority of the derivatives met the criteria for blood-brain-barrier permeability.

Based on the isosterism concept, we have designed and synthesized homologous N-alkyl-2-[4-(trifluoromethyl)benzoyl]hydrazine-1-carboxamides (from C₁ to C₁₈) as potential antimicrobial agents and enzyme inhibitors. They were obtained from 4-(trifluoromethyl)benzohydrazide by three synthetic approaches and characterized by spectral methods. The derivatives were screened for their inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) via Ellman’s method. All the hydrazinecarboxamides revealed a moderate inhibition of both AChE and BuChE, with IC₅₀ values of 27.04–106.75 µM and 58.01–277.48 µM, respectively. Some compounds exhibited lower IC₅₀ for AChE than the clinically used drug rivastigmine. N-Tridecyl/pentadecyl-2-[4-(trifluoromethyl)benzoyl]hydrazine-1-carboxamides were identified as the most potent and selective inhibitors of AChE. For inhibition of BuChE, alkyl chain lengths from C₅ to C₇ are optimal substituents. Based on molecular docking study, the compounds may work as non-covalent inhibitors that are placed in a close proximity to the active site triad. The compounds were evaluated against Mycobacterium tuberculosis H₃₇Rv and nontuberculous mycobacteria (M. avium, M. kansasii). Reflecting these results, we prepared additional analogues of the most active carboxamide (n-hexyl derivative 2f). N-Hexyl-5-[4-(trifluoromethyl)phenyl]-1,3,4-oxadiazol-2-amine (4) exhibited the lowest minimum inhibitory concentrations within this study (MIC ≥ 62.5 µM), however, this activity is mild. All the compounds avoided cytostatic properties on two eukaryotic cell lines (HepG2, MonoMac6).