Synthesis, In Vitro Biological Evaluation and In Silico Molecular Docking Studies of Indole Based Thiadiazole Derivatives as Dual Inhibitor of Acetylcholinesterase and Butyrylchloinesterase

The current study was conducted to obtain hybrid analogues of indole-based thiadiazole derivatives (1–16) in which a number of reaction steps were involved. To examine their biological activity in the presence of the reference drug Donepezil (0.21 ± 0.12 and 0.30 ± 0.32 M, respectively), the inhibitory potentials of AChE and BuChE were determined for these compounds. Different substituted derivatives showing a varied range of inhibitory profiles, when compared to the reference drug, analogue 8 was shown to have potent activity, with IC50 values for AchE 0.15 ± 0.050 M and BuChE 0.20 ± 0.10, respectively, while other substituted compounds displayed good to moderate potentials. Varied spectroscopic techniques including 1H, 13CNMR and HREI-MS were used to identify the basic skeleton of these compounds. Furthermore, all analogues have a known structure–activity relationship (SAR), and molecular docking investigations have verified the binding interactions of molecule to the active site of enzymes.


Introduction
Alzheimer's disease is associated with the acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymes, which damage the human brain. The conversion of acetylcholine into choline acetic acid by the process of hydrolysis is the primary function of these enzymes [1]. Owing to these consequences, the hippocampus and cortex of the brain, which are linked to important psychological functions, suffer from acetylcholine deficiency [2]. As a result of this ongoing process, the patient develops Alzheimer's disease

Spectral Analysis
Spectral analyses of all the synthesized compounds have been discussed, and their respective spectra's have been added in Supplementary Information as shown in Figures S1-S3.
Different substitution patterns of the compounds play a vital role to shield or deshield the proton and carbon atoms. All the synthesized compounds were found with different yields in the final products and confirmed through thin layer chromatography (TLC). After TLC confirmation, the compounds were washed with n-hexane to obtained pure products, which were then characterized through 1 H-NMR, 13C-NMR and HREI-MS techniques. The basic skeleton of the analogues were obtained and confirmed the nature of functionality attached to the aromatic ring. Spectral interpretation was done and their values were written in descending order for both proton and carbon (as shown in Supplementary Information). Furthermore, mass spectroscopic techniques confirmed the molecular weight of the synthesized compounds. Structure interpretation for compound-9 in which proton appeared at varied ranges (ppm). Using 1 HNMR (600 MHz, DMSO-d6): δ, the first proton appeared at 11.59, showing a singlet by one proton of nitrogen and 11.20 another proton of nitrogen appeared. The indole proton appeared at 8.

An Illustration of a Molecule
The comparison of identically substituted derivatives was carried out using the general structure, which divides the molecule into two distinct components, such as the chloro-indole moiety and aromatic ring with various substituents (Figure 1). These different parts of molecules play a key role in the inhibitory activity of the analogues and might

Spectral Analysis
Spectral analyses of all the synthesized compounds have been discussed, and their respective spectra's have been added in Supplementary Information as shown in Figures S1-S3.
Different substitution patterns of the compounds play a vital role to shield or deshield the proton and carbon atoms. All the synthesized compounds were found with different yields in the final products and confirmed through thin layer chromatography (TLC). After TLC confirmation, the compounds were washed with n-hexane to obtained pure products, which were then characterized through 1 H-NMR, 13C-NMR and HREI-MS techniques. The basic skeleton of the analogues were obtained and confirmed the nature of functionality attached to the aromatic ring. Spectral interpretation was done and their values were written in descending order for both proton and carbon (as shown in Supplementary Information). Furthermore, mass spectroscopic techniques confirmed the molecular weight of the synthesized compounds. Structure interpretation for compound-9 in which proton appeared at varied ranges (ppm). Using 1 HNMR (600 MHz, DMSO-d 6 ): δ, the first proton appeared at 11.59, showing a singlet by one proton of nitrogen and 11.20 another proton of nitrogen appeared. The indole proton appeared at 8.

An Illustration of a Molecule
The comparison of identically substituted derivatives was carried out using the general structure, which divides the molecule into two distinct components, such as the chloroindole moiety and aromatic ring with various substituents (Figure 1). These different parts of molecules play a key role in the inhibitory activity of the analogues and might be the presence of attached substituents upon it. The inhibitory profile depends on nature, number and position of the substituents.
doublet of doublet with a coupling constant 7.1, 1.9 Hz for an aromatic proton. Multiple were shown in the range between 7.65-7.64 and 7.45-7.41 by two aromatic protons, 7.20 showing a doublet for the indole proton with a coupling constant 8.3 Hz, while the other indole proton also appeared at 6.45 showing a doublet with a coupling constant 6.7 Hz and 6.47 showing a singlet for one proton. Additionally, the CH2 proton appeared at 3.20 showing a singlet for two protons. Carbon NMR also represents the following values appearing in different ranges due to the attached substituent. Using 13 C-NMR (150 MHz, DMSO-d6) δ, the carbons appeared in the range from 150.6-45.6 and the High Resolution Electron Impact Mass Spectroscopy were conducted to figure out m/z 358.0274; [M+1] + Calcd for C17H12ClFN4S;358.0292.

An Illustration of a Molecule
The comparison of identically substituted derivatives was carried out using the general structure, which divides the molecule into two distinct components, such as the chloro-indole moiety and aromatic ring with various substituents (Figure 1). These different parts of molecules play a key role in the inhibitory activity of the analogues and might be the presence of attached substituents upon it. The inhibitory profile depends on nature, number and position of the substituents.

Inhibitory Profile of Synthesized Compound against AchE and BuChE
The synthetic scaffolds' AchE and BuChE inhibitory profiles were evaluated in the presence of the reference medication Donepezil. Due to the presence of the electron-donating group, the majority of the compounds were discovered to have higher potential.

Bio-Activity of Indole Based Thiadiazole Derivatives Inhibitory Profile of Synthesized Compound against AchE and BuChE
The synthetic scaffolds' AchE and BuChE inhibitory profiles were evaluated in the presence of the reference medication Donepezil. Due to the presence of the electrondonating group, the majority of the compounds were discovered to have higher potential.
Moreover, other substituted analogues such as compounds having a methyl group on varied positions of the ring containing analog 5 (5.80 ± 0.20 and 9.30 ± 0.10 µM) 6 (8.70 ± 0.20 and 10.90 ± 0.10 µM), and 7 (9.60 ± 0.20 and 13.60 ± 0.30 µM), are composed of two methyl groups in different positions, such as para, ortho, and meta. The para > ortho > meta substituted scaffold was confirmed by the IC50 values of the enzyme inhibition pattern. It was readily recognized that the methyl group's orientation on the aromatic ring caused a difference in the inhibition activity. Analog 8 was a candidate in this comparative study of methyl-substituted compounds, however due to the location of the substituent on the aromatic ring, it demonstrated insufficient activity. The flouro-group-containing scaffolds 8 (0.15 ± 0.050 and 0.20 ± 0.10 µM), 9 (0.35 ± 0.050 and 0.50 ± 0.050 µM), and 10 (1.10 ± 0.10 and 2.80 ± 0.10 µM) displayed varying levels of inhibition against AChE and BuChE. The inhibitory potential of the compound was shown to be increased by the flourogroup in the para-position. Due to the strong hydrogen bond interaction with the enzyme active site, analogue 8 was thus regarded as the most active molecule in the tested series and demonstrated high activity when compared to the standard drug. Due to the presence of the flouro-group in the ortho-position, analogue 9 got the second highest ranking in the inhibitory profile. Methoxy substituted analogues 12 (14.70 ± 0.30 and 19.20 ± 0.30 µM) and 13 (19.10 ± 0.30 and 25.30 ± 0.40 µM) bearing nitro-and methoxy-groups at different position of aromatic ring -OMe substituted analogues 12 (14.70 ± 0.30 and 19.20 ± 0.30 µM) and 13 (19.10 ± 0.30 and 25.30 ± 0.40 µM), bearing -NO 2 and -OMe groups at different position of aromatic ring were contrasted in order to investigate their efficacy to inhibit AChE and BuChE, respectively. Comparing these analogues to the reference drug Donepezil, they demonstrated moderate to poor inhibitions (IC50 = 0.21 ± 0.12 and 0.30 ± 0.32 µM, respectively).
The AChE and BuChE inhibitory action may slightly increase or decrease depending on the type, number, and orientation of the substituents. It was discovered that analogues 8 and 9 were slightly more capable than the reference drug Donepezil. The better interactions of the molecule that were found might be due to the attached functionality of the compounds, which make hydrogen bond in order to inhibit the negative effect of enzymes; thus, the reduction of enzymatic activity was observed and confirmed the potency of the molecules. In this study, compound 8 and 9 were found with very significant characters and considered as potent analogs when we compared their inhibitory profile with the standard drug Donepezil.

Molecular Docking
The primary linkage between ligands and the targeted enzymes has been identified using molecular docking studies. Several software, such Auto Dock Vina and Discovery Studio Visualizer, were used. [29][30][31][32][33][34]. Proteins were retrieved for this work by searching for their codes, such as 1Acl and 1p0p, in the online protein data bank (RCSB PDB).
Ligand and protein preparation steps were done through a stepwise mechanism. In the initial stage, the removing of water molecules and preserving the protein and ligand in PDB format, the recovered protein was initially opened in auto dock vina. By the addition of polar hydrogen and charges like kollman and gasteiger, the docking procedure was continued. After protein preparation, the ligand was introduced. Automatic charges were applied to the ligand molecule, and torsion tree selection was made to find the root. After generating the configuration and saving the X, Y, and Z axis files in PDBQT format, the configuration was finally created. In order to create the many ligands poses needed to study the binding interaction between the ligand and active sites due to the abundance of protein interaction sites, a command prompt was employed. Figures 2-7 illustrate the analysis of the binding interaction of the ligand with the protein's active sites using nine distinct poses that were gathered in PDBQT format and analyzed in DSV.

Docking Results
To investigate the mechanism in which synthesized scaffolds attach to the specified enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), molecular docking was carried out. From www.rcsb.org, (28 January 2022) both enzyme crystallographic coordinates were obtained. When tested against the targeted enzymes, the docking strategy shows that the chosen compounds had a wide range of potentials. Among the examined series, several substituted scaffolds interact with various amino acids, but in this case, scaffolds 8 and 9 were found to have great interactions because they both had a flouro-group in the para-or ortho-position of an aromatic ring. Higher hydrogen bond counts result in greater interactions with enzyme active sites. As a result, the flouro-substituted scaffolds 8 and 9 were discovered to have strong interactions, as illustrated in Figures 2-7 with a superposed surface complex.
In particular, the protein-ligand interaction (PLI) profile in the case of effective compounds 8 and 9 not only had the highest potential (in silico), but also displayed greater potency during an in vitro investigation. The potential of the molecule was found to be better as compared to the standard drug Donepezil, perhaps due to the presence of a flouro-group on the aromatic ring. This flouro-group is involved in making a hydrogen bond, which reduces the enzyme potential. Figure 2 describes the protein ligand interaction profile (PLI) for compound 8 with flouro-groups in para-position revealed a variety of interacting residues for AChE, including GLU-199 (Hydrogen bonding), TRP-84 (Hydrogen bonding), TYR-121 (Hydrogen bonding), TYR-334 (Hydrogen bonding), PHE-330 (Hydrogen bonding) and GLU-199 (πanion). As illustrated in Figure 2, the interacting residues for BuChE include TYR-282 (T-shaped), GLU-238 (H-F), VAL-280 (π-R), ARG-242 (π-cation), ASN-241 (Hydrogen bond with flourine), VAL-279 (Hydrogen bond with flourine), and PRO-281 (π-Alkyl).
The attached substituents and PLI profiles are the only differences between compounds 8 and 9. Both cases showed that the indole and thiadiazole moieties had a strong interface with the enzyme active site. Additionally, the nucleophilic nature of aromatic rings is enhanced by flouro-groups linked to the para and ortho sites.

Conclusions
Overall studies of the present work were achieved against AChE and BuChE, which represent scaffolds 8 (0.15 ± 0.050 and 0.20 ± 0.10 µM, respectively) and 9 (0.35 ± 0.050 and 0.50 ± 0.050 µM, respectively), which were found to be most potent among the tested series when compared to Donepezil, which was used as the standard drug (IC 50 = 0.21 ± 0.12 and 0.30 ± 0.32 µM, respectively). Due to strong hydrogen interactions with enzyme active sites, the flouro-group at para-and ortho-positions on the aromatic ring clearly displays the inhibitory profiles of scaffolds. The interactions were further confirmed through molecular docking studies in which both scaffolds were identified through a varied range of interactions and their PLI profile was much better than other substituent compounds. Hydrogen bonds, pi-pi interactions, pi-sulfur interactions, and pi-cation interactions were employed to study how the scaffolds interacted. Due to attached rings and their heteroatoms, the scaffolds demonstrated all the specified interactions. Other substituted analogues were also found with good or moderate activity, which might be due to the presence of different groups among the series, as some compounds possess bulky groups like bromine, by which activity profiles were reduced and compounds were considered as poor inhibitors due to a much lower number of interactions with enzymes active sites.