Novel Inhibitors of Acetyl- and Butyrylcholinesterase Derived from Benzohydrazides: Synthesis, Evaluation and Docking Study

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.

selective AChE inhibitors were found to be N-tridecyl/pentadecyl-2-[4-(trifluoromethyl)benzoyl]hydrazine-1-carboxamides. N-Methyl analog was identified as the third most potent AChE inhibitor along with significant inhibition of BChE. Molecular docking studies suggest that these compounds may act as non-covalent inhibitors located near the catalytic triad. Given together with the fact that AChE and/or BChE inhibitors are a cornerstone of therapy for neurodegenerative diseases, including Alzheimer's disease, myasthenia gravis [8] and other diseases, we designed and conducted a follow-up study of potential cholinesterase inhibitors based on the most potent N-methyl/tridecyl-2- [4-(trifluoromethyl)benzoyl]hydrazine-1-carboxamides. The changes performed included the type of substituent on the benzene ring and its position, as well as other types of amide linkers ( Figure 1).

Design
Based on the successful hydrazinecarboxamide scaffold (I, Figure 1; compounds P1 and P2), we decided to retain N-methyl and N-tridecyl chains. N-Tridecyl was selected and preferred over N-pentadecyl moiety. Both of these alkyls exhibit similar activity, but tridecyl derivatives are less lipophilic and, based on in silico calculations, some pentadecyl derivatives would exceed the logP value of 5. However, even among our compounds, there are tridecyl derivatives with higher logP values.
The last issue for modification was the linker connecting the 4-(trifluoromethyl)phenyl scaffold to the hydrazinecarboxamide (series 3). The C=O group was replaced by sulfonyl (3c, 3d), or one methylene group was inserted between CO and the benzene ring (3a, 3b).

Synthesis
The most common method for the preparation of 2-benzoylhydrazine-1-carboxamides and their analogs with modified linkers is the reaction of corresponding arylhydrazides with isocyanates.
The hydrazides that we used were commercially available or prepared in-house (Scheme 1; 4-iodobenzohydrazide, [1,1'-biphenyl]-4-carbohydrazide, 4- Given together with the fact that AChE and/or BChE inhibitors are a cornerstone of therapy for neurodegenerative diseases, including Alzheimer's disease, myasthenia gravis [8] and other diseases, we designed and conducted a follow-up study of potential cholinesterase inhibitors based on the most potent N-methyl/tridecyl-2- [4-(trifluoromethyl)benzoyl]hydrazine-1-carboxamides. The changes performed included the type of substituent on the benzene ring and its position, as well as other types of amide linkers ( Figure 1).

Design
Based on the successful hydrazinecarboxamide scaffold (I, Figure 1; compounds P1 and P2), we decided to retain N-methyl and N-tridecyl chains. N-Tridecyl was selected and preferred over N-pentadecyl moiety. Both of these alkyls exhibit similar activity, but tridecyl derivatives are less lipophilic and, based on in silico calculations, some pentadecyl derivatives would exceed the logP value of 5. However, even among our compounds, there are tridecyl derivatives with higher logP values.
The last issue for modification was the linker connecting the 4-(trifluoromethyl)phenyl scaffold to the hydrazinecarboxamide (series 3). The C=O group was replaced by sulfonyl (3c, 3d), or one methylene group was inserted between CO and the benzene ring (3a, 3b).

Synthesis
The most common method for the preparation of 2-benzoylhydrazine-1-carboxamides and their analogs with modified linkers is the reaction of corresponding arylhydrazides with isocyanates.
Pharmaceuticals 2023, 16,172 3 of 20 romethyl)benzenesulfonylhydrazide). Their syntheses started from free acids, which were almost quantitatively converted to methyl ester via Fischer esterification (with sulfuric acid as a catalyst). The esters were reacted under reflux with an excess of hydrazine hydrate to give corresponding hydrazides. Alternatively, when available, substituted benzoyl chloride was converted to methyl ester using methanol in the presence of triethylamine as a tertiary base. Scheme 1. Synthesis of benzohydrazides.
Methyl isocyanate and tridecyl isocyanate are not routinely commercially available. Due to the notorious toxicity of methyl isocyanate [9], N-methylated hydrazinecarboxamides were initially prepared by reacting hydrazides with an excess of N-succinimidyl N-methylcarbamate (also known as "methyl isocyanate substitute") in the presence of Nethyl-N,N-diisopropylamine (DIPEA) as a non-nucleophilic tertiary base (method A; Scheme 2) [2]. Tridecyl isocyanate, as a compound with an odd number of carbons in the alkyl chain, was firstly prepared in situ from tridecylamine using triphosgene in the presence of triethylamine under a nitrogen atmosphere. Then, it was reacted with hydrazide to afford target N-tridecylhydrazinecarboxamides (method B; Scheme 2). N-Alkyl-2-{2- [4-(trifluoromethyl)phenyl]acetyl}hydrazine-1-carboxamides 3a and 3b were also obtained in this way.
However, both these methods gave inconsistent and generally lower yields (Table 1). Therefore, we decided to prepare both isocyanates via the Curtius rearrangement from commercially available chlorides (acetyl and myristoyl chloride), which were converted to azides via reaction with sodium azide in toluene and were then gently heated to induce rearrangement and thus the formation of isocyanates [10]. The crude isocyanate was immediately used for a subsequent reaction with hydrazides (method C; Scheme 3). This procedure led to significantly increased yields (Table 1); lower yields were in the case of nitro-and sulfamoylbenzohydrazide derivatives. Methyl isocyanate and tridecyl isocyanate are not routinely commercially available. Due to the notorious toxicity of methyl isocyanate [9], N-methylated hydrazinecarboxamides were initially prepared by reacting hydrazides with an excess of N-succinimidyl N-methylcarbamate (also known as "methyl isocyanate substitute") in the presence of N-ethyl-N,N-diisopropylamine (DIPEA) as a non-nucleophilic tertiary base (method A; Scheme 2) [2]. Tridecyl isocyanate, as a compound with an odd number of carbons in the alkyl chain, was firstly prepared in situ from tridecylamine using triphosgene in the presence of triethylamine under a nitrogen atmosphere. Then, it was reacted with hydrazide to afford target N-tridecylhydrazinecarboxamides (method B; Scheme 2). N-Alkyl-2-{2- [4-(trifluoromethyl)phenyl]acetyl}hydrazine-1-carboxamides 3a and 3b were also obtained in this way.

Inhibition of Acetylcholinesterase and Butyrylcholinesterase
Hydrazinecarboxamides 1-3 were examined in vitro for their potential to inhibit the function of AChE (obtained from electric eel) and BChE from equine serum using Ellman's spectrophotometric method ( Table 2). The activities are expressed as IC50 values, i.e., concentrations leading to 50% inhibition of enzymatic activity. Based on the IC50 values for AChE and BChE, we calculated selectivity indexes (SI) that quantified the selectivity for each cholinesterase. SI is the ratio of IC50 for BChE/IC50 for AChE. Values less than 1 indicate the preferential inhibition of BChE, whereas values above 1 mean stronger inhibition of AChE. For comparison, the clinically used drug rivastigmine, a dual inhibitor of AChE and BChE, was employed. Compounds 1-3 ( Table 2) were characterized by NMR ( 1 H, 13 C) and IR spectra and melting points. The purity was verified additionally by elemental analysis.

Inhibition of Acetylcholinesterase and Butyrylcholinesterase
Hydrazinecarboxamides 1-3 were examined in vitro for their potential to inhibit the function of AChE (obtained from electric eel) and BChE from equine serum using Ellman's spectrophotometric method ( Table 2). The activities are expressed as IC50 values, i.e., concentrations leading to 50% inhibition of enzymatic activity. Based on the IC50 values for AChE and BChE, we calculated selectivity indexes (SI) that quantified the selectivity for each cholinesterase. SI is the ratio of IC50 for BChE/IC50 for AChE. Values less than 1 indicate the preferential inhibition of BChE, whereas values above 1 mean stronger inhibition of AChE. For comparison, the clinically used drug rivastigmine, a dual inhibitor of AChE and BChE, was employed.  centrations leading to 50% inhibition of enzymatic activity. Based on the IC50 values for AChE and BChE, we calculated selectivity indexes (SI) that quantified the selectivity for each cholinesterase. SI is the ratio of IC50 for BChE/IC50 for AChE. Values less than 1 indicate the preferential inhibition of BChE, whereas values above 1 mean stronger inhibition of AChE. For comparison, the clinically used drug rivastigmine, a dual inhibitor of AChE and BChE, was employed.

Inhibition of Acetylcholinesterase and Butyrylcholinesterase
Hydrazinecarboxamides 1-3 were examined in vitro for their potential to inhibit the function of AChE (obtained from electric eel) and BChE from equine serum using Ellman's spectrophotometric method ( Table 2). The activities are expressed as IC 50 values, i.e., concentrations leading to 50% inhibition of enzymatic activity. Based on the IC 50 values for AChE and BChE, we calculated selectivity indexes (SI) that quantified the selectivity for each cholinesterase. SI is the ratio of IC 50 for BChE/IC 50 for AChE. Values less than 1 indicate the preferential inhibition of BChE, whereas values above 1 mean stronger inhibition of AChE. For comparison, the clinically used drug rivastigmine, a dual inhibitor of AChE and BChE, was employed.

Molecular Docking
The active site of AChE has been investigated thoroughly by AChE mutant studies and X-ray crystallography of the enzyme bound to various ligands [11][12][13]. The hydrolysis of acetylcholine takes place at the bottom of a deep and narrow gorge and is directly mediated by three amino acids, Ser203, His447 and Glu334, forming the so-called catalytic triad. However, several important regions, responsible for the navigation of the substrate to the gorge and its suitable orientation in the cavity, have been described. The so-called peripheral anionic site, formed mostly by aromatic residues (Trp286, Tyr124, Tyr72 and Tyr341), is important for the formation of cation-π interactions with cationic substrates, which then proceed toward a choline-binding pocket (Trp86 and Tyr337) and acyl-binding pocket (Phe295, Phe297, and Trp236), which is believed to be responsible for accommodating the acyl part of the substrate after hydrolysis takes place [14].
A comparison of the active sites of AChE and BChE revealed that most differences in the substrate's specificity originate from the fact that many aromatic residues present in the AChE active site are missing in BChE, being replaced by smaller hydrophobic residues. Out of 10 aromatic residues interacting with ligands in AChE, only 4 remain in BChE: Tyr332 (Tyr341 in AChE) in the peripheral site and Trp82, Phe329 and Trp231 (Trp86, Phe338 and Trp236 in AChE) in the active site [14]. This, among other things, allows more spacious ligands to enter the cavity.
To better understand the binding mode of the prepared compounds in AChE and BChE, a molecular modeling study was performed.
The top scoring docking pose of two most potent AChE inhibitors in the series, 1e and 2j, gave a similar orientation of these compounds in the cavity of the enzyme despite quite large differences in their structures ( Figure 2). The substituted aryl moiety showed stacking with Trp86, and the 2-acylhydrazine-1-carboxamide linker was stabilized in its position by several H-bond interactions (namely with Asp74, Tyr124, Ser125 and Tyr31). In the case of compound 2j, the long tridecyl tail was heading out toward the entrance of the cavity, where several hydrophobic amino acid residues were (Leu76, Phe338 and Trp286), which further contributed to the additional stabilization of the ligand in the enzyme.  The conformation of 1l (together with 1h, being two of the most potent inhibitors of BChE in all series) is shown in Figure 3. The acylhydrazine-1-carboxamide linker is in close proximity to the catalytic active site, able to form H-bond interactions with amino acid residues of the catalytic triad. The tridecyl moiety of both ligands was buried deeper in the cavity of BChE when compared to AChE surrounded by hydrophobic amino acids Phe329 and Ala328. This corresponded to the general assumption that the cavity of BChE is more Pharmaceuticals 2023, 16, 172 8 of 20 spacious than that of AChE. Nevertheless, both enzymes were capable of accommodateing the tridecyl moiety without any difficulties.

General
All chemicals were purchased from Merck KGaA (Darmstadt, Germany), Acros Organics B.V.B.A. (Geel, Belgium), Penta Chemicals Unlimited (Prague, Czech Republic) and Avantor (Stříbrná Skalice, Czech Republic) and were used as received. The progress of reactions was monitored via thin-layer chromatography (TLC). Plates were coated with 0.2 mm Merck 60 F254 silica gel (Merck, Darmstadt, Germany) and were visualized via UV light (254 and 366 nm). Alternatively, ALUGRAM SIL G/UV254 aluminum plates (Macherey-Nagel GmbH, Düren, Germany) coated with a 0.2 mm silica gel layer (60A, with a fluorescent indicator for 254 nm) were used (method C). Avantor Silica Gel with a particle size of 40-60 µm was used for column chromatography without prior activation. Melting

General
All chemicals were purchased from Merck KGaA (Darmstadt, Germany), Acros Organics B.V.B.A. (Geel, Belgium), Penta Chemicals Unlimited (Prague, Czech Republic) and Avantor (Stříbrná Skalice, Czech Republic) and were used as received. The progress of reactions was monitored via thin-layer chromatography (TLC). Plates were coated with 0.2 mm Merck 60 F254 silica gel (Merck, Darmstadt, Germany) and were visualized via UV light (254 and 366 nm). Alternatively, ALUGRAM SIL G/UV 254 aluminum plates (Macherey-Nagel GmbH, Düren, Germany) coated with a 0.2 mm silica gel layer (60A, with a fluorescent indicator for 254 nm) were used (method C). Avantor Silica Gel with a particle size of 40-60 µm was used for column chromatography without prior activation. Melting points were recorded using a Büchi B-560 apparatus (BÜCHI Labortechnik AG, Flawil, Switzerland), and they were without correction.
The structures of both known and new compounds were determined via 1 H NMR and 13 C NMR spectroscopy. NMR spectra were measured in a dimethyl sulfoxide (DMSO-d 6 ) solution at an ambient temperature with a JNM-ECZ 600R (600 MHz for 1 H and 151 MHz for 13 C; JEOL, Tokio, Japan) or a Varian VNMR S500 instrument (500 MHz for 1 H and 126 MHz for 13 C; Varian Comp. Palo Alto, CA, USA). The chemical shifts (δ) are reported in parts per million (ppm) and are indirectly related to tetramethylsilane via residual signals of the solvent (2.50 for 1 H and 39.51 for 13 C spectra). Coupling constants (J) are given in Hz. Infrared spectra were recorded via a Nicolet 6700 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) in the range of 600-4000 cm −1 . The attenuated total reflectance technique (ATR) on a germanium crystal was used. Elemental analysis was performed on a Vario MICRO Cube Elemental Analyzer (Elementar Analysensysteme, Hanau, Germany). Calculated and found values are given as percentages; all the calculated and experimental values were within ±0.4%.

Synthesis Synthesis of Parent Hydrazides
Hydrazide precursors for final compounds 1-3 were obtained commercially or prepared in-house. Their synthesis involved the hydrazinolysis of methyl esters in boiling ethanol. The esters were prepared from commercially available acyl chlorides and methanol in the presence of an excess of triethylamine. Alternatively, free acid was esterified with methanol using a catalytic amount of sulfuric acid under reflux.
The spectral and physical characterizations (NMR, IR and melting points) of these precursor compounds were in accordance with literature. Their purity was checked via elemental analysis.

Synthesis of Hydrazine-1-carboxamides
Method A Substituted benzohydrazide (1.0 mmol) was dissolved in acetonitrile (5 mL) and mixed with N,N-diisopropylethylamine (DIPEA; 2.0 mmol, 348 µL) followed by N-succinimidyl N-methylcarbamate (1.5 mmol; 258.2 mg). The reaction mixture was stirred at room temperature for 24 h, the formed precipitate was filtered off, it was washed with cold water and diethyl ether, and it was crystallized from ethyl acetate.
Method B Triphosgene (0.4 mol, 118.7 mg) was dissolved in anhydrous dichloromethane (DCM; 5 mL) under a nitrogen atmosphere. Then, tridecylamine (1.01 mmol; 201.4 mg) dissolved in anhydrous DCM (5 mL) was added dropwise. After 30 min of stirring at room temperature, triethylamine (2.1 mmol, 293 µL) was added. After an additional 30 min, substituted benzohydrazide (1.0 mmol) was added. The reaction mixture was stirred for 10 h at room temperature and was then evaporated to dryness, treated with water (10 mL) and extracted with ethyl acetate (3 × 15 mL). The combined organic phase was dried over anhydrous sodium sulfate, filtered off and evaporated to dryness to give the final product, which was crystallized from ethyl acetate.
Method C Both alkyl isocyanates were prepared via the Curtius rearrangement from commercially available acyl chlorides according to the known method [10]. The crude isocyanate was used immediately for a subsequent reaction.
interactions were prepared in PyMOL (The PyMOL Molecular Graphics System, Version 1.5 Schrödinger, LLC).

Conclusions
A series of forty-eight N-methyl/tridecyl-2-aryloylhydrazine-1-carboxamides and their analogs were designed, synthesized, characterized and investigated as potential inhibitors of acetylcholinesterase and butyrylcholinesterase. Almost all of them exhibited the dual inhibition of both cholinesterases, with IC 50 values in the micromolar range. Their activity against AChE was generally higher than that against BChE, with a narrower range of IC 50 values. Some of them achieved better or fairly comparable IC 50 values to the drug rivastigmine. Molecular docking showed details of the molecular interactions of inhibitors with enzymes that differed for AChE and BChE. 2-(4-Nitrobenzoyl)-N-tridecylhydrazine-1carboxamide was identified as the most promising molecule in terms of the inhibition of both enzymes at low concentrations and lipophilicity.
Further biological activity (especially antimicrobial and anticancer) will be investigated in the near future.