5-Aryl-1,3,4-oxadiazol-2-amines Decorated with Long Alkyl and Their Analogues: Synthesis, Acetyl- and Butyrylcholinesterase Inhibition and Docking Study

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.


Introduction
Acetylcholine (ACh) is a potent neurotransmitter in both the central nervous system and the peripheral nervous system, that plays an important role in performing the cognitive functions. Enzyme acetylcholinesterase (AChE), which cleaves choline esters, is a serine hydrolase found mainly at neuromuscular junctions and cholinergic brain synapses. The enzyme inactivation induced by various inhibitors leads to acetylcholine accumulation, hyperstimulation of nicotinic and muscarinic receptors, and disruption of neurotransmission [1]. Uncontrolled acetylcholine can lead to a massive disturbance in the cholinergic system, respiratory arrest, and death [2].
According to the mode of action, AChE inhibitors can be divided into two groups: irreversible and reversible. Reversible competitive or non-competitive inhibitors (donepezil, rivastigmine, galantamine) are the protagonists of pharmacotherapy of Alzheimer disease symptoms. Their therapeutic effect is based on maintaining the level of ACh by slowing Considering the abovementioned facts, we designed, prepared, and evaluated N-dodecyl-5-substituted (hetero)aryl-1,3,4-oxadiazol-2-amines 3 and their isosteres 3s-3u as potential inhibitors AChE and BChE.

Synthesis
The synthetic scheme is illustrated in Figure 2. The synthesis of final oxadiazoles and their analogues 3a-3u started from aryl hydrazides that were either commercially available or prepared in-house from corresponding acid via Fisher esterification (with MeOH in the presence of a catalytic amount of sulfuric acid) and subsequent hydrazinolysis of the methyl ester. Then, hydrazide was reacted with a mild excess of dodecyl iso(thio)cyanate in acetonitrile. Crude 2-aryloylhydrazine-1-carboxamides 2 were purified by recrystallization with moderate to excellent yields of 67-98%. The lowest yields were for 2-bromopyridine derivative 2q, followed by 3,5-dinitrobenzohydrazide derivative 2k and thioamide 2t (for both 76%). Considering the abovementioned facts, we designed, prepared, and evaluated Ndodecyl-5-substituted (hetero)aryl-1,3,4-oxadiazol-2-amines 3 and their isosteres 3s-3u as potential inhibitors AChE and BChE.

Synthesis
The synthetic scheme is illustrated in Figure 2. The synthesis of final oxadiazoles and their analogues 3a-3u started from aryl hydrazides that were either commercially available or prepared in-house from corresponding acid via Fisher esterification (with MeOH in the presence of a catalytic amount of sulfuric acid) and subsequent hydrazinolysis of the methyl ester. Then, hydrazide was reacted with a mild excess of dodecyl iso(thio)cyanate in acetonitrile. Crude 2-aryloylhydrazine-1-carboxamides 2 were purified by recrystallization with moderate to excellent yields of 67-98%. The lowest yields were for 2-bromopyridine derivative 2q, followed by 3,5-dinitrobenzohydrazide derivative 2k and thioamide 2t (for both 76%).
Two analogues were prepared in a different way. Dodecylsulfanyl derivative 3s was prepared from isonicotinoylhydrazide that was heated with an excess of carbon disulfide keeping a strong basic pH (73%). The 5-(pyridin-4-yl)-1,3,4-oxadiazol-2-thiol was treated with an over-stochiometric amount of 1-bromododecane as an alkylating agent and potassium carbonate in dimethylformamide. Thioether 3s was obtained quantitatively. 5-Dodecyl derivative 3u was synthesized from isoniazid by condensation reaction with tridecanal to form hydrazide-hydrazone 2u. This intermediate was cyclized to oxadiazole using a mild excess of iodine and potassium carbonate (76%).
The prepared compounds were characterized by 1 H and 13 C NMR, IR spectra and melting point. The purity was confirmed by thin-layer chromatography and elemental analysis. In the 1 H NMR spectra, the signals of heteroaryls and substituted phenyls are clearly visible, while signals of aliphatic hydrogens from the dodecyl chain partially overlap. Here, we can distinguish signals of C1, C2 and C12, remaining hydrogen signals are merged. By analogy, the signals of most aliphatic carbons in the 13 C NMR spectra are close together. As expected, both hydrazide hydrogen signals in the spectra of 2 disappeared in the spectra of 3 due to cyclization.

Inhibition of Acetyl-and Butyrylcholinesterase
The heterocycles 3 were screened for their ability to interfere with the function of AChE from electric eel (EeAChE) and BChE from equine serum (EqBChE) using Ellman's method (Table 1). Their activities are expressed as the concentration producing 50% inhibition of enzymatic activity (IC50). In addition, we calculated selectivity indexes (SI) that quantify the selectivity for AChE as more inhibited cholinesterase. SI is the ratio of IC50 for BChE/IC50 for AChE. Clinically used AChE and BChE inhibitor rivastigmine was used as a comparator. The prepared compounds were characterized by 1 H and 13 C NMR, IR spectra and melting point. The purity was confirmed by thin-layer chromatography and elemental analysis. In the 1 H NMR spectra, the signals of heteroaryls and substituted phenyls are clearly visible, while signals of aliphatic hydrogens from the dodecyl chain partially overlap. Here, we can distinguish signals of C 1 , C 2 and C 12 , remaining hydrogen signals are merged. By analogy, the signals of most aliphatic carbons in the 13 C NMR spectra are close together. As expected, both hydrazide hydrogen signals in the spectra of 2 disappeared in the spectra of 3 due to cyclization.

Inhibition of Acetyl-and Butyrylcholinesterase
The heterocycles 3 were screened for their ability to interfere with the function of AChE from electric eel (EeAChE) and BChE from equine serum (EqBChE) using Ellman's method (Table 1). Their activities are expressed as the concentration producing 50% inhibition of enzymatic activity (IC 50 ). In addition, we calculated selectivity indexes (SI) that quantify the selectivity for AChE as more inhibited cholinesterase. SI is the ratio of IC 50 for BChE/IC 50 for AChE. Clinically used AChE and BChE inhibitor rivastigmine was used as a comparator.
In contrast, BChE enzyme was inhibited at higher concentrations, and their range was wider. The IC50 of the dodecylsulfanyl compound 3s was higher than 500 µM. In fact, only the thiadiazole 3t and 5-(p-tolyl)-oxadiazole 3b were more potent inhibitors of BChE We used the selectivity index values to describe the selectivity to particular ACh hydrolysing enzymes. All the derivatives were preferential inhibitors of AChE (SI within the range from 1.5 to 12.9). The most selective was iodinated derivative 3i; on the other hand, 4-nitro compound 3e was the most balanced inhibitor. Interestingly, an introduction of the second nitro group (3k) and increasing molecular weight of halogens (3g → 3j) resulted in a higher selectivity to AChE. Among the derivatives with a heterocycle on carbon 5, the least selective are brominated pyridine 3q and pyridazine 3r. Positional isomers (3m and 3n) of isoniazid-derived oxadiazole 3l are more selective to AChE. The  3s, and 3t vs. 3u).
Then, we tried to correlate activity with calculated lipophilicity (log P o/w ; Table 1). However, there are no clear conclusive structure-activity relationships and differences in lipophilicity cannot fully explain differences in activity. For example, compounds with close log P values (3s-3u) showed different degrees of inhibition of both enzymes. The most potent AChE inhibitors were lowly lipophilic (3t, 3k), moderately (3c) and also highly lipophilic (3j). Analogously, compounds with the same log P values (3e, 3l-3n, and 3t) inhibited BChE over a wide concentration range of 53-369 µM.
When comparing the heterocycles 3 with the drug rivastigmine, none of the new compounds had lower IC 50 values for BChE than this clinically used dual AChE/BChE inhibitor. By contrast, many of them (3a-3c, 3g-3k, 3l, 3o, and 3t) showed higher in vitro activities expressed as IC 50 (up to 4.4× for 3t) against AChE.

Reversibility of the Inhibition
Based on the promising in vitro inhibition of AChE by thiadiazole 3t, we investigated the derivative to distinguish between reversible, pseudo-irreversible and irreversible inhibition on the basis of changes in enzyme activity over time in the presence of the inhibitor. In reversible inhibition, the activity of enzyme goes down immediately, but the inhibitory molecule is bound to the target for a short period of time, after which the inhibitor-enzyme complex dissociates, and enzyme activity is restored. In pseudo-irreversible inhibition, the inhibitor binds covalently to the enzyme, but the bond is broken down more slowly, delaying the return of enzyme activity to its initial state. In the case of irreversible inhibitors, the reaction between the enzyme and the inhibitor is not rapid because there is a time-dependent drop of activity. Irreversible inhibitors decrease enzyme activity gradually. The inhibitor permanently binds to the enzyme and prevents the enzyme from restoring its activity [33].
Three different concentrations were chosen for 3t according to its IC 50 value for AChE. The first concentration was lower than IC 50 , the second was close to IC 50 and the third was higher than IC 50 (1, 10, and 100 µM, respectively). All obtained dependencies of A (expressed as residual activity on the Y-axis) versus time showed a very similar pattern ( Figure 3). First, a rapid decrease in activity (for two lower concentrations, almost maximum inhibition is reached after 5 min), then a plateau phase followed by a recovery of enzyme activity over time.
In our opinion, we can conclude that the compound 3t acts as reversible AChE inhibitor. In our opinion, we can conclude that the compound 3t acts as reversible AChE inhibitor.

Type of Inhibition
We also valuated the type of inhibition of 3t using AChE. In general, the reversible inhibitors can be classified as competitive, non-competitive, uncompetitive, or mixed type. The type of inhibition could be distinguished using the Lineweaver-Burk plot [34]

Type of Inhibition
We also valuated the type of inhibition of 3t using AChE. In general, the reversible inhibitors can be classified as competitive, non-competitive, uncompetitive, or mixed type. The type of inhibition could be distinguished using the Lineweaver-Burk plot [34] and the corresponding comparison of two key kinetic parameters: maximum velocity (V m ) and Michaelis constant (K M ) of the inhibited and uninhibited reactions. Based on the changes of these parameters and intercept of the lines in Lineweaver-Burk plot, the type of inhibition is uncovered.
The Lineweaver-Burk plot obtained for 3t and AChE is shown in Figure 4. Based on the plot, it can be concluded that this heterocycle causes a mixed type of inhibition. The inhibition is associated with a change in both K M and V m and also their ratio is different compared to the uninhibited reaction. In the Lineweaver-Burk plot, the intersection of the lines is in quadrant III but not on axis.
In our opinion, we can conclude that the compound 3t acts as reversible AChE inhibitor.

Type of Inhibition
We also valuated the type of inhibition of 3t using AChE. In general, the reversible inhibitors can be classified as competitive, non-competitive, uncompetitive, or mixed type. The type of inhibition could be distinguished using the Lineweaver-Burk plot [34] and the corresponding comparison of two key kinetic parameters: maximum velocity (Vm) and Michaelis constant (KM) of the inhibited and uninhibited reactions. Based on the changes of these parameters and intercept of the lines in Lineweaver-Burk plot, the type of inhibition is uncovered.
The Lineweaver-Burk plot obtained for 3t and AChE is shown in Figure 4. Based on the plot, it can be concluded that this heterocycle causes a mixed type of inhibition. The inhibition is associated with a change in both KM and Vm and also their ratio is different compared to the uninhibited reaction. In the Lineweaver-Burk plot, the intersection of the lines is in quadrant III but not on axis.

Molecular Docking
For better visualization of the possible orientations of prepared compounds in enzymes, molecular modelling study was performed. Based on the obtained data, the structure-activity relationships were estimated.
The active site of AChE was studied extensively in the past and is now very well described. It is known to be at the bottom of a very narrow gorge penetrating deep under the surface of the enzyme. All the prepared most active ligands (3b, 3c, 3t) displayed similar orientation in the cavity of AChE ( Figure 5) which corresponds with the results of in vitro testing, since the obtained activities were all in relatively small range. Variable 5-aryloxadiazole moiety was heading for the bottom of the gorge (though still quite far from the active site triad). Hydrophobic 2-dodecylamino moiety was convoluted, pointing out of the cavity, and blocking the entrance to the gorge. Several hydrophobic interactions with Trp286, Val294, Phe297, and Phe338 were observed. Interestingly, an important structural element seems to be the -NH-group at the dodecylamino moiety, which is stabilized at its position by hydrogen bonds (Asp74 or Tyr124). The replacement of the nitrogen with carbon or sulfur leads to decrease in the binding affinity (and also decrease in the in vitro inhibition data). The oxadiazole part of the molecules showed hydrogen bonding with Ser125. Aromatic/heteroaromatic moieties were located at the anionic site and displayed π-π stacking with Trp86 ( Figure 6).
Similarly to AChE, the active site of BChE is located at the bottom of the gorge. However, a considerable number of aromatic residues present in active center of AChE is replaced by smaller hydrophobic residues in BChE. This allows more spacious ligands to enter the cavity. The top score docking pose of 3t (being the most potent inhibitor in the series) showed the molecule deep within the cavity in considerable proximity to active site Ser198 and His438 (Figure 7). The ligand was stabilized in its conformation by several interactions (namely, hydrogen bonds of oxadiazole moiety with Gly116 and Gly117 and hydrophobic interactions of dodecylamino moiety with Trp82, Ala328, Phe329, and Trp430). Most of the investigated compounds exhibited the same or quite similar conformation, apart from 3s. Its dodecylamino moiety was placed in the same hydrophobic part of the cavity; however, the aryloxadiazole moiety was relatively far from Ser198 and His438 and showed only one interaction with Thr120 ( Figure 8). This could stand behind its low ability to inhibit BChE. ilar orientation in the cavity of AChE ( Figure 5) which corresponds with the results of in vitro testing, since the obtained activities were all in relatively small range. Variable 5aryloxadiazole moiety was heading for the bottom of the gorge (though still quite far from the active site triad). Hydrophobic 2-dodecylamino moiety was convoluted, pointing out of the cavity, and blocking the entrance to the gorge. Several hydrophobic interactions with Trp286, Val294, Phe297, and Phe338 were observed. Interestingly, an important structural element seems to be the -NH-group at the dodecylamino moiety, which is stabilized at its position by hydrogen bonds (Asp74 or Tyr124). The replacement of the nitrogen with carbon or sulfur leads to decrease in the binding affinity (and also decrease in the in vitro inhibition data). The oxadiazole part of the molecules showed hydrogen bonding with Ser125. Aromatic/heteroaromatic moieties were located at the anionic site and displayed π-π stacking with Trp86 ( Figure 6).   ilar orientation in the cavity of AChE ( Figure 5) which corresponds with the results of in vitro testing, since the obtained activities were all in relatively small range. Variable 5aryloxadiazole moiety was heading for the bottom of the gorge (though still quite far from the active site triad). Hydrophobic 2-dodecylamino moiety was convoluted, pointing out of the cavity, and blocking the entrance to the gorge. Several hydrophobic interactions with Trp286, Val294, Phe297, and Phe338 were observed. Interestingly, an important structural element seems to be the -NH-group at the dodecylamino moiety, which is stabilized at its position by hydrogen bonds (Asp74 or Tyr124). The replacement of the nitrogen with carbon or sulfur leads to decrease in the binding affinity (and also decrease in the in vitro inhibition data). The oxadiazole part of the molecules showed hydrogen bonding with Ser125. Aromatic/heteroaromatic moieties were located at the anionic site and displayed π-π stacking with Trp86 ( Figure 6).   Similarly to AChE, the active site of BChE is located at the bottom of the gorge. However, a considerable number of aromatic residues present in active center of AChE is replaced by smaller hydrophobic residues in BChE. This allows more spacious ligands to enter the cavity. The top score docking pose of 3t (being the most potent inhibitor in the series) showed the molecule deep within the cavity in considerable proximity to active site Ser198 and His438 (Figure 7). The ligand was stabilized in its conformation by several interactions (namely, hydrogen bonds of oxadiazole moiety with Gly116 and Gly117 and hydrophobic interactions of dodecylamino moiety with Trp82, Ala328, Phe329, and Trp430). Most of the investigated compounds exhibited the same or quite similar conformation, apart from 3s. Its dodecylamino moiety was placed in the same hydrophobic part of the cavity; however, the aryloxadiazole moiety was relatively far from Ser198 and His438 and showed only one interaction with Thr120 ( Figure 8). This could stand behind its low ability to inhibit BChE.

General
All chemicals for synthesis and analysis were purchased from Merck KGaA (Darmstadt, Germany), Penta Chemicals Unlimited (Prague, Czech Republic), Avantor (Stříbrná Skalice, Czech Republic), and Lach-Ner (Neratovice, Czech Republic) and were used as received. The structures of the prepared substances were confirmed by 1 H NMR and 13 C NMR spectroscopy analysis. NMR spectra were measured in dimethylsulfoxide (DMSO-

General
All chemicals for synthesis and analysis were purchased from Merck KGaA (Darmstadt, Germany), Penta Chemicals Unlimited (Prague, Czech Republic), Avantor (Stříbrná Skalice, Czech Republic), and Lach-Ner (Neratovice, Czech Republic) and were used as received. The structures of the prepared substances were confirmed by 1 H NMR and 13 C NMR spectroscopy analysis. NMR spectra were measured in dimethylsulfoxide (DMSO-D 6 ) as a solvent at ambient temperature or with tempering the sample (2b-2j, 2q, 3b-3k,  3m, 3o-3q, and 3u) at 60 • C by a JNM-ECZ 600R (600 MHz for 1 H a 151 MHz for 13 C; JEOL, Tokio, Japan) or a Varian VNMR S500 instrument (Varian Comp., Palo Alto, CA, USA). The chemical shifts δ are given in ppm and were referred indirectly to tetramethylsilane via residual signals of the solvent (2.50 for 1 H and 39.51 for 13 C spectra). The coupling constants (J) are reported in Hz. Infrared spectra were recorded by a Nicolet 6700 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) in the range of 600-4000 cm −1 , ATR (Ge) technique of measuring was used. Elemental analysis was performed on Vario MICRO Cube Element Analyzer (Elementar Analysensysteme, Hanau, Germany). Calculated as well as found values are given as percentages. Melting points were recorded using a Büchi B-545 apparatus (BÜCHI Labortechnik AG, Flawil, Switzerland) without corrections. Retention factors (R f ) of all prepared compounds, as well as reaction progresses, were analyzed by thin layer chromatography (TLC); the plates were coated with 0.2 mm Merck 60 F254 silica gel (Merck Millipore, Darmstadt, Germany) and were visualized by UV irradiation (254 nm). Dichloromethane (DCM)/methanol (93:7 v/v) mixture was used as the eluent.
Lipophilicity was calculated in silico using a free web tool SwissADME (http://www. swissadme.ch/index.php, accessed on 16 March 2022). It is expressed as log P o/w (MLOGP).

Synthesis
Synthesis of 2-Aryloyl-N-Dodecylhydrazine-1-(thio)Carboxamides (2a-2r) and Other Precursors (2s-2u) Appropriate aryl hydrazide (1.0 mmol) was suspended in 10 mL of acetonitrile and heated to initiate boiling of the mixture, then dodecyl isocyanate (or dodecyl isothiocyanate for precursor of 3t; 1.05 of equivalents, 1.05 mmol) was added quickly in one portion. The reaction mixture was refluxed continuously for 2 h, then left to cool down at RT and stored for 1 h at −20 • C. Solid material was filtered off and recrystallized from methanol to provide pure hydrazides 2. The progress of the reaction was monitored by TLC.
N-Dodecyl-5-phenyl-1,3,4-oxadiazol-2-amine 3a. calculated. All experiments were carried in duplicates, and the average values of reaction rate were used for the construction of the Lineweaver-Burk plot. From obtained equations of regression curves in Lineweaver-Burk plot, the values of Michaelis constant (K M ) and maximum velocity (V m ) were calculated. Based on these results, the type of inhibition was identified.
Acetylcholinesterase was used from electric eel (Electrophorus electricus L.; EeAChE) and butyrylcholinesterase originated from equine serum (EqBChE). A drug rivastigmine was used as a reference dual enzyme inhibitor. All the enzymes, substrates and rivastigmine were purchased from Merck (Prague, Czech Republic).

Molecular Docking
Crystallographic structures of human AChE and human BChE were obtained from protein data bank (www.rcsb.org accessed on 30 January 2022; pdb codes 4PQE and 1POI, respectively). The 3D structures of ligands were prepared in Chem3D Pro 19.1 (ChemBioOffice 2019 Package, CambridgeSoft, Cambridge, MA, USA). In the preparation process, water molecules were removed from the enzymes and the structures of enzymes and ligands were optimized using UCSF Chimera software package (Amber force field) [40]. Docking was performed using Autodock Vina [41] and Autodock 4.2 [42]; a Lamarckian genetic algorithm was used. The 3D affinity grid box was designed to include the full active and peripheral site of AChE and BChE. The number of grid points in the X-, Y-and Z-axes was 20, 20 and 20, respectively, with grid points separated by 1 Å (Autodock Vina), and 40, 40 and 40, respectively, with grid points separated by 0.4 Å (Autodock 4). The graphic visualizations of the ligand-enzyme interactions were prepared in PyMOL (The PyMOL Molecular Graphics System, Version 1.5 Schrödinger, LLC, New York, NY, USA).

Conclusions
A series of twenty 1,3,4-oxadiazoles and one 1,3,4-thiadiazole were designed, prepared, and evaluated as potential inhibitors of acetylcholinesterase and butyrylcholinesterase. Almost all of them showed significant inhibition of both cholinesterases with IC 50 values in the micromolar range. Their activity toward AChE was found to be comparatively higher than that toward BChE. Many of them showed higher inhibitory effect against AChE than the drug rivastigmine. We described structure-activity relationships. The most active compound, thiadiazole decorated with 4-pyridyl and dodecylamine, was the best inhibitor of both enzymes. It represents a reversible, mixed-type AchE inhibitor. The interactions of the most potent inhibitors with target structures were also investigated using molecular docking, which showed that the binding is different for particular acetylcholine hydrolysing enzymes.
Other types of biological activities, especially antimicrobial, will be investigated in the future.