2-methylindole analogs as cholinesterases and glutathione S-transferase inhibitors: Synthesis, biological evaluation, molecular docking, and pharmacokinetic studies

2.1 General

All of the chemicals, solvents, and enzymes were purchased from either Merck or Sigma-Aldrich companies. Enzymes sources from different organisms were used for the inhibition studies. Acetylcholinesterase (Electric ell AChE, EC 3.1.1.7), butyrylcholinesterase (equine serum BChE, EC 3.1.1.8), and glutathione S-transferase (human placenta GST, 2.5.1.18) were purchased from Sigma-Aldrich (Sternheim, Germany). The reagents and solvents were utilized without further purification. The progress of reactions was monitored by thin-layer chromatography (TLC) analysis on Merck pre-coated silica gel 60 F254 aluminum sheets, visualized by UV light. Melting points were recorded on an Electrothermal Gallenkamp apparatus and are uncorrected. ¹H and ¹³C NMR spectra were recorded on a Varian Mercury-400 High-performance Digital FT-NMR Spectrophotometer (Varian, Fort Collins, CO, USA) at 400 MHz and 100 MHz, respectively. During the measurements, tetramethylsilane (TMS) was used as internal standard and deuterated CDCl₃ as the solvent. The chemical shifts are reported in parts per million (ppm, δ) downfield from TMS. The following abbreviations are used; singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br s). HPLC was performed on the Shimadzu LC-20 system (Shimadzu, Japan) and a diode array detector SPD-M20A (190–800 nm) was utilized. HRMS spectra were taken on a time of flight MS system coupled to an analytical ESI detector.

2.2 Synthesis of 2-chloro-1-(2-methyl-1H-indol-3-yl)ethanone (2)

The starting compounds of this study 2-methyl-1H-indole (1) (0.13 g, 1 mmol) and pyridine (0.08 mL, 1 mmol) were dissolved in anhydrous toluene (2.5 mL). The solution was heated to 60 °C under heavy stirring. Chloroacetyl chloride (0.08 mL, 1 mmol) was added dropwise over 1 h. The reaction process was stirred for an additional 4 h. The solution was allowed to warm to room temperature and water (30 mL) and MeOH (5 mL) were added under stirring. The reaction process was filtered. The solvent was evaporated and the crude product was purified by silica gel column chromatography (n-Hexane: Ethyl acetate, 5:1) as a white powder (0.14 g, 68%). R_f 0.30 (40% EtOAc in n-heptane, v/v). mp (^oC): 212–214. FTIR (cm⁻¹): 3184, 1652, 1444. ¹H NMR (400 MHz, CDCl₃): δ 9.91 (br s, 1H, NH), 7.81 (d, J = 8.0 Hz, 1H, indole-H4), 7.49 (ddd, J = 8.0, 1.2, 0.9 Hz, 1H, indole-H7), 7.25 (ddd, J = 8.2, 7.6, 1.4 Hz, 1H, indole-H6), 7.11 (ddd, J = 8.4, 7.2, 1.0 Hz, 1H, indole-H5), 4.12 (s, 2H, CH₂), 2.01 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃) δ 191.3 (C⚌O), 138.8 (indole-C7a), 131.9 (indole-C2), 128.9 (indole-C3a), 128.6 (indole-C6), 127.7 (indole-C5), 123.4 (indole-C4), 120.4 (indole-C3), 112.2 (indole-C7), 41.08 (CH₂), 21.0 (—CH₃); HPLC: R_T: 5.00 min. HRMS (ESI) Calcd. for C₁₁H₁₀ClNONa [M + Na]⁺: 230.0349; found: 230.0332.

2.3 General procedure for the synthesis of indole analogs (4a-4j)

Indole 2 (0.103 g, 0.5 mmol), NaHCO₃ (1 mmol, 0.84 mg) and NaI (0.25 g, 1 mmol) were added to a 4 mL vial. After addition of 2 mL THF and amine (0.5 mmol) was added to mixture and the resulting suspension was stirred at 60 °C for 6 h. Then 1 mL of 1 M Na₂CO₃-solution was carefully added, the mixture filtered and the filter residue washed with EtOAc. The water was added in solution. The organic phase was separated and the aqueous phase extracted with EtOAc (3 × 5 mL) and the organic phase was separated and dried over Na₂SO₄, filtered. The crude product was purified by silica gel column chromatography (n-Hexane: Ethyl acetate, 6:1) as white solid.

2.4 AChE, BChE, and GST enzyme activity methods

Cholinesterase inhibitions of the synthesized analogs were carried out according to Ellman’s method with some slight modifications by using AChE and BChE enzymes (Ellman et al., 1961). The tacrine compound was used as a positive standard to compare the AChE/BChE inhibition. Briefly, 100 µL buffer solution (pH 8.0, Tris-HCl, 1.0 M), 800 µL purified water, 10 µL compound solution, and 50 µL DTNB (5,5′-dithiobis 2-nitrobenzoic) were mixed with 10 µL (10 EU) AChE/BChE enzyme solutions, respectively. After the incubation of the mixtures for 20 min, 50 µL substrates (acetylthiocholine iodide or butyrylcholine iodide) were added to the mixtures. Finally, the absorbance differences for 3 min were measured at 412 nm. GST enzyme inhibitory activities of the synthesized analogs were determined according to a previous study (Aras et al., 2019). Ethacrynic acid was used as a positive standard to compare the GST inhibition. Briefly, different concentrations (20–100 µM) of the samples in phosphate buffer (pH 6.5), GST enzyme solution (20 mM), and CDNB substrate solution (25 mM) were added to the test tubes, respectively. After the incubation of the mixtures for 20 min, GST enzyme inhibitions of the compounds were analyzed by measuring the absorbance changes for 3 min at 340 nm (Habig et al., 1974).

In the enzyme studies, one enzyme unit (EU) refers to the enzyme amount that catalyzes the conversion of 1 µmol substrate to the product in one minute duration. The enzyme kinetic studies were performed using a UV/vis spectrophotometer. For this aim, the absorbance of the control sample (without adding the tested compound) was measured and accepted to be 100% activity. The absorbance of the other samples prepared with adding different concentrations of the inhibitors were measured and their activities were calculated. The reducing activity shows the inhibition potential of a sample. The enzyme activity-concentration graphs were drawn for each tested compound. Then, the IC₅₀ values (the concentration of an inhibitor to reduce 50% of enzyme activity) were calculated by using the graphic equation. The K_i values were calculated using Lineweaver-Burk graphs obtained from measurements of five different substrate concentrations.

2.5 Docking methodology

The three-dimensional X-ray crystal structures of AChE (PDB code: 4MOE), BChE (PDB code: 5NNO), and GST (PDB code: 3DK9) were retrieved in pdb format from Protein Data Bank with resolution 2.00 Å, 2.50 Å, and 0.95 Å, respectively. The structures of AChE, BChE, and GST enzymes in complex with an inhibitor were prepared by using the BIOVIA Discovery Studio software (Rao et al., 2020). The complex structures between the enzymes and co-crystallized inhibitors were illustrated in Fig. 2. The 3D structures of indole molecules synthesized were drawn with Chemdraw Ultra 8 program. The 3D structures of the indole analogs were saved in a sdf format. The 3D structures of the indole analogs were converted in a pdb format using Open Babel-2.4.0 and their energy minimizations were done by the Avogadro program (Cetin, 2021).

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Fig. 2

The complexes between AChE, BChE, and GST with their inhibitors.

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AutoDockVina software was performed between indole analogs and AChE, BChE, GST enzymes for molecular docking analyses such as binding types, binding energies, inhibition activities, ligand efficiency, distances, and possible interactions. Molecular docking scores were set as AutoDock Tools 1.5.7 of the molecular graphics laboratory software package by keeping the analog flexible. The binding pocket coordinates were identified the best pose whose the indole molecules were docked in surface of AChE, BChE, and GST enzymes. The grid box center was adjusted with dimensions for AChE, BChE and GST enzymes (X = 9.239 Y = -59.985 and Z = 25.538 for AChE), (X = 22.829, Y = 39.805 and Z = 37.569 for BChE) and (X = 16.807 Y = 17.303 and Z = 20.702 for GST). After validation of the docking protocol, binding conformations of the indole molecules 4a-4j based on virtual screening into the active site of AChE, BChE, and GST enzymes were evaluated. The ligand efficiencies, binding energies, inhibition activities, hydrogen bonds, and bond lengths of these enzyme-indole molecules’ complexes were analyzed via AutoDockVina software.

2.6 ADMET analyses

The pharmacokinetics and toxicity properties of the indole molecules were achieved using the SwissADME which is an open online tool (http://www.swissadme.ch). The ADME properties define blood–brain barrier (BBB) permeability and passive human gastrointestinal absorption (HIA) as well as substrate or non-substrate permeability glycoprotein (P-gp) and Cytochrome P450 (CYP) (Daina et al., 2017). Furthermore, SwissADME enables predictions for risks of toxicology such as mutagen test and carcino rat.

3.1 Synthesis of indole analogs

Indoles are suitable tool molecules subject for pharmacological studies due to having more potent analogs of lead drugs (Singh Sidhu et al., 2016). For this aim, indole analogs were designed for the AChE, BChE, and GST enzymes suitable for in vitro studies by pharmacological evaluation. 2-methylindole scaffold used for the synthesis of diverse potential 3-substitued 2-methylindole molecules for the AChE, BChE, and GST. Commercially available indole 1 was utilized as starting material. That compound plays a significant role in synthesis routes based on nucleophilic substitution reactions in organic chemistry. Synthesis routes of 3-substitued 2-methylindole analogs were reported in Scheme 1. Firstly, the acylation 2-methylindole scaffold in the 3-position was synthesized using chloroacetyl chloride and pyridine according to previously published literature (Thanikachalam et al., 2019).

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Scheme 1

Synthesis route to 2-methylindole analogs.

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The synthesis approach towards analogs amine groups bearing compounds 4a-j were prepared alkylation of various amines with molecule 2 by late-stage functionalization. Commercially acquired amines were treated with molecule 2 according to reaction conditions for alkylating amines (NaI and NaHCO₃), to give indole 4a-j as shown in Scheme 1. The molecule 2 is indeed an attractive synthon. The synthesis of molecule 2 was scaled up to 5.6 g (68% yield). Application of the identified reaction conditions in THF for the indole compouns synthesis of a wide variety of primary, secondary amines including sterically hindered amine tertiary amines in yields of 24–66% as shown in Table 1. Indole 4i and 14j were synthesized at low yields probably thanks to the lower nucleophilicity of the amines used.

Table 1 Scope of the reaction for synthesis of substituted indole molecules.^a

a Reactions were performed on a 0.5 mmol scale, using 1.0 eq. of amine in THF used as solvent at 60 °C, 8 h. Yields given are of the isolated product.

Indoles 4a and 4b could be isolated in overall yields of 49 and 43%. Reaction with benzylamine afforded expected product 4c (58%). It was observed that examples the applicability of this synthesis towards more substituted 3-substitued 2-methylindole molecules. It was observed that examples the applicability of this synthesis towards more substituted 3-substitued 2-methylindole compounds in Scheme1. Indole molecules approach continue to play significant roles in the discovery of AChE, BChE, and GST enzymes (Taha et al., 2021, Kurt-Kızıldoğan et al., 2021). In this study is identified potent indole molecules based on the 2-methyl-indole scaffold were synthesized as shown Scheme 1. Organic compounds bearing amines are generally protonated at pH which increases their aqueous solubility. It was concluded that syntheses of indole molecules were obtained by aliphatic amine, aromatic amine and 2-methyl-1H-indole core structure. The compounds prepared had different characteristics in terms of basicity, hydrogen bonding abilities, geometry, and electronic properties. The primary, secondary and tertiary amines being able to be utilized for the synthesis of various indole molecules. The synthesized indole molecules exhibit different functional moieties and readily allows for the synthesis of functionalized indoles thereby influence both the ADMET and pharmacokinetic properties.

3.2 Enzyme inhibitory properties of indole compounds

The starting compound (1) and its newly synthesized molecules were evaluated for their cholinesterase and GST enzyme inhibitory activities. Remarkably, some of the compounds exhibited good inhibition activities against GST, AChE, and BChE enzymes. Ethacrynic acid was used as a positive standard inhibitor for GST whereas tacrine was used as a positive standard inhibitor for AChE and BChE to compare the results of the synthesized molecules. The enzyme inhibitions of the compounds were determined and the results were given as IC₅₀ and K_i values (Table 2). As understood from the K_i and IC₅₀ values, the synthesized molecules showed a wide range of competitive, non-competitive, and semi-competitive inhibition types against the enzymes. The lower K_i and IC₅₀ values indicate the more effective inhibition of the indole molecules.

According to the AChE inhibition studies, the compounds 4b and 4i were investigated to be the most effective inhibitors among the other newly synthesized molecules. The inhibitory potency of compound 4b was very close to the standard compound. The K_i inhibition values of the compound 4b and the standard tacrine were measured as 0.614 µM and 0.556 µM whereas, their IC₅₀ values were calculated as 0.648 µM and 0.848 µM, respectively. In terms of the structure–activity relationship point, the AChE inhibition activity order of the synthesized indole molecules with having different substituents was found to be compound 4b (-tert-butylamino group) > compound 4i (-3-methoxyphenylamino group) > standard compound (tacrine) > compound 4j (-m-toluidino group) > compound 4a (-ethylamino group).

According to the BChE inhibition studies, three of the synthesized compounds (4b, 4a, and 4c) were investigated to be more effective inhibitors than the other synthesized indole molecules and the standard compound. Interestingly, the K_i inhibition values of 4b (0.412 µM), 4a (0.564 µM), and 4c (0.772 µM) were found to be around half of the K_i values of tacrine (1.318 µM) that means these compounds showed more than two times BChE inhibitory activities compare to the standard tacrine compound. The IC₅₀ values of the compounds 4a, 4b, and 4c were found as 0.928 µM, 0.745 µM, and 1.762 µM, respectively. In terms of the structure–activity point, BChE inhibition activity order of the synthesized indole molecules with having different substituents was found to be compound 4b (-tert-butylamino group) > compound 4a (-ethylamino group) > compound 4c (-benzylamino group) > standard compound (tacrine).

Remarkably, compound 4b was found to be the most potent inhibitor of both AChE and BChE enzymes among the twelve compounds. As a comparison of the structure–activity relationship, compound 4b is a primary amine and has a butylamino group on its active site. The high inhibitory potential of this compound is mainly due to hydrophobic interactions of that alkyl (butyl) group with non-polar pockets of the enzyme. A previous study reported a similar correlation and structure–activity relationship of alkyl group with the potent AChE inhibition effects of flavonol compounds (Mughal et al., 2019).

Overall, most of the tested compounds showed potent inhibitions against BChE than AChE in comparison to the standard compound. So far, many indolic compounds have been addressed for their potent AChE and BChE inhibitions in many previously published papers. For instance, one of the former studies reported the electron-donating effects of the substituents of seventeen monoterpene indole alkaloids that showed moderate to good potential for AChE inhibitions (Zhan et al., 2020). Also, another study reported that electron-donating groups afforded good to moderate BChE enzyme inhibitory activity (Shaikh et al., 2021). The structure–activity relationship analysis could help in designing new compounds for the cholinergic pathway inhibitors based on indole molecules.

The results of in vitro and in silico studies seems to have low correlations. However, the potent inhibitor compounds according to the in vitro enzyme studies also showed low energy levels in molecular docking studies. For instance, the most potent inhibitor for both AChE and BChE, compound 4b determined to have low binding energy levels in molecular docking studies that proved the good interactions of the compound with the enzymes. Overall, the energy levels of the tested compounds were detected close to the standard compounds.

According to the GST inhibition studies, the starting compound 1 and compound 4i were investigated to be the most effective GST inhibitors among the other newly synthesized molecules. The inhibition potency of the compound 1 was higher than the standard compound (ethacrynic acid) as well. The K_i inhibition values of compound 1 and standard ethacrynic acid were 0.308 µM and 0.386 µM whereas their IC₅₀ values were determined as 0.347 µM and 0.374 µM, respectively. In terms of the structure–activity point, the GST inhibition activity order of the synthesized indole molecules with having different substituents was found to be compound 1 (2-methyl-1H-indole) > standard compound (ethacrynic acid) > compound 4e (-piperidin-1-yl group) > compound 4i (-3-methoxyphenylamino group). Similar to these results, a previous study showed the inhibition effect of an indole molecules up to a 69 % decrease in GST enzyme activity (Konus et al., 2020).

3.3 Molecular docking studies

The main interactions between indole molecules and the enzymes (AChE, BChE, and GST) such as the binding affinities, hydrogen bonds, and bond lengths etc. were obtained by using AutoDock Vina software. Moreover, ethacrynic acid a standard inhibitor of GST and tacrine as a reference drug for AChE and BChE were used for comparing the molecular docking results of the indole molecules (Cetin et al., 2021). Molecular docking is a molecular modeling technique and one of the most commonly employed methods to analyze the detailed interactions between the chemical structures of the compounds and enzymes. In case of AChE and BChE, the crystal structure of Trametes ochracea and Trypanosoma brucei available at low crystallographic resolutions was selected, respectively (Turkan et al., 2019; Türkan et al., 2021). The acetylcholine transporter of Trametes ochracea was chosen due to related to the catalyze the oxidation of monoamines and acted upon amino acids. Furthermore, Trypanosoma brucei structures create electron transport chains in the occurred complexes and an enzyme that catalyzes the transfer of electrons from one molecule. For GST, the high-resolution crystallographic structure of human glutathione reductase was selected at nominal resolutions between 1.1 and 0.95 A. Since, in addition to the properties given above,these structures catalyse the transfer of specific functional groups from electrically neutral group of two or more atoms and also, have involved hundreds of different biochemical pathways and vital processes (Türkan et al., 2021). After extraction from the respective macromolecule, the co-crystallized indole molecules of AChE, BChE and GST were docked inside the active site. Also, the re-docking of co-crystallized ligands (G3F for 4MOE and 92 N for 5NNO, and FAD for 3DK9) were carried out to validate the docking studies. The crystal poses of indole molecules were docked into the binding site of AChE, BChE, and GST enzymes with identified docking search algorithms and scoring functions. All indole molecules were docked using similar optimized docking conditions (See Figures and Tables in the Supplementary Material file). The binding energies, inhibition activities, and ligand efficiency of the indole molecules with AChE, BChE, and GST enzymes were illustrated in Table 3. Initially, the drug score, Volume ų and Surface ų were determined for indole molecules-AChE, BChE, and GST 0.81, 2371.25, and 3049.62, respectively. The binding affinity values of indole molecules-AChE enzyme ranged from −9.0 to −6.9 kcal/mol and their ligand efficiencies were obtained between −0.49 and −0.37. The binding affinity values of indole molecules-BChE enzyme ranged from −8.1 to −6.8 kcal/mol and their ligand efficiencies were obtained between −0.48 and −0.33 and also, the binding affinity values of indole molecules-GST ranged from −8.7 to −6.3 kcal/mol and their ligand efficiencies were obtained between −0.45 and −0.39 using AutoDock Vina. The ligand efficient values of all indole-enzyme complexes achieved close values for the enzymes used. The binding affinities of all indole-AChE complexes were found to be higher than the binding affinity value of the standard drug-AChE complex. The binding affinity (−9.0 kcal/mol) of indole 4c-AChE complex was found to be the highest among binding affinities the all indole-AChE complexes in Fig. 3. The binding affinity of indole 4j-AChE complex was also a very close value as −8.8 kcal/mol. The binding affinities of the indole molecules 4a, 4d, 4f, and 4 g were found to be a lower value than binding affinities of the other indole molecules in the AChE activity.

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Fig. 3

The 2D and 3D interaction profiles and detailed binding mode of best-posed indole molecules into the enzymes (a) 4c-AChE, (b) 4i-BChE, and (c) 4j-GST.

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The binding affinity (−8.2 kcal/mol) of indole molecule 4i was found to be the highest value among binding affinities of the indole molecules in the BChE activities. The binding affinities of all indole molecules except compounds 4a and 4g were found to be higher values than the binding affinity of the standard drug. The binding affinities of all indole molecules except 4a, 4b for BChE activity and compound 4f for GST activity were found to be higher value than the binding affinity of the standard drug. However, the binding affinities of 4b and 4f (−7.5 kcal/mol) were achieved close values according to the binding affinity of the standard drug (−7.7 kcal/mol) for GST activity. The binding affinity of indole molecule 4j (−8.7 kcal/mol) was found to be the highest value among binding affinities of the other indole molecules in the GST activities (Fig. 3.). Moreover, the binding affinity of indole molecule 4i was a very close value as −8.6 kcal/mol for GST activity. The binding affinities of the indole molecules 4c, 4e, and 4h had values close to each other as −8.4, −8.3, and −8.3 kcal/mol in the GST activities, respectively.

The indole 4j and 4i had good binding affinities for the enzymes used. This is thought to be due to the substituents attached to the phenyl ring at the meta position of the 4j and 4i indole compounds (Konus et al., 2020). The molecular docking results of the indole-enzyme complexes were revealed that, all indole-enzyme complexes showed π-alkyl interactions with Lys332, Val429, Tyr510, Arg525, Ala397, Leu394, Val330 residues in the active site of the AChE, Phe329, Leu286, Ala328, Trp82, His438, Pro84 residues in the active sites in the BChE, and Ala342, Cys58, Cys63, Ala130, Leu298, Ile26, Val49 residues in the active sites in the GST, respectively (Table 4 and Supp. Mater. Tables S1-S3) The indole molecules 4d and 4f showed Van der Waals in the hydrophobic bonding types with Thr383 residue in the active site of the AChE. The indole molecules 4h, 4i, and 4j showed Van der Waals in the hydrophobic bonding type with Gly117 residue in the active site of the BChE enzyme. The indole molecules 4a, 4h, and 4i also showed Van der Waals with Asn294 and Gly157 residues in the active site of the GST enzyme. In addition, all of the indole-enzyme complexes showed alkyl interactions with Ile26, Leu298, Val49, Ala130, Lys66, Ile198 residues, and Val408, Arg525, Val330, Arg393, Arg397, Lys332, Leu394 Arg383, Phe359 Ala526 residues in the active sites of the AChE and GST enzymes, respectively.

The indole compounds 4a, 4b, and 4c only showed alkyl interactions with Tyr440, Met437, Trp82, Leu442, Leu286, Ile69, and Ala328 residues in the active sites of the BChE enzyme. The complex structures between the indole compounds and the AChE, BChE, and GST enzymes were stabilized by hydrogen bonds containing oxygen (-O), hydrogen (-H), and carbonyl (—C⚌O) moieties simultaneously as donor and acceptor with Arg521, Tyr510, Lys332, Gly523, Asp400, Arg356, Pro285, His438, Gly116, Asp70, Tyr332, Gln67, Cys58, Ser30, Asp331, Glu50, Ser51, Ala130, Gly27, Glu201, and Gly62 residues. The indole compounds except indole 4d showed π donor hydrogen bond with Arg525 residue in the active site of the AChE. The indole compounds 4h and 4i showed π donor hydrogen bond with Trp82 residue in the active site of the BChE. The indole compound 4g showed π donor hydrogen bond with Asp331 residue in the active site of the GST (Türkan et al., 2021). The indole-enzyme complexes had electronic interactions with the AChE amino acids Arg525, Val330, Val429, Val329, Ala397, and Tyr382, with the BChE amino acids Phe329, Trp231, His438, Trp430, Trp82, Gly116, and Trp231, and with the GST amino acids Asp331, His129, Thr156, Gly62, Ile198, and Gly28. The molecular docking simulations of indole molecules-enzyme complexes are given detailly in the Supplemental Material files.

3.4 Pharmacokinetic studies

Physicochemical, drug-likeness, and pharmacokinetic properties of the indole molecules 4a-j were illustrated by the online website of SwissADME server as shown in Table 5 (Daina et al., 2017). Furthermore, drug metabolism, pharmacokinetics (ADME), and drug-induced toxicity properties of the indole compounds 4a-j were calculated using SwissADME database. All indole compounds 4a-j obey the Lipinski’s rule of five. The indole compounds 4a-j have a bioavailability score of 0.55 and are moderately soluble in water. Also, the mutagenicity and carcinogenicity of the indole compounds 4a-j were evaluated using the SwissADME server (Mpiana et al., 2020). The pharmacokinetics and toxicity properties of the indole compounds 4c, 4h, 4i, and 4j with high binding energies were evaluated the potential of these compounds as drug candidates. As seen in Table 5, the transporter class P-glycoprotein (P-gp) and Cytochrome P450 (CYP) metabolic enzymes, which are important in drug metabolism, were assessed in this study. The indole 4c, 4h, and 4j were found to be effective inhibitors of all CYPs except CYP2C9. The indole 4i was found to be an inhibitor of all CYPs. The CYP inhibitors of these indole compounds are significant to explore the pharmacokinetics for preventing undesired drug-drug interactions. Indole 4c having P-gp and CYP3A4 can be a barrier in the transmembrane transportation of drugs since both are good transporters in the intestine (Daina et al., 2017).

It was observed that the indole compounds achieved good results in terms of their BBB permeant and GI absorption properties from the SwissADME database. Since, the indole compounds 4a-j had BBB permeant and GI absorption properties. The BBB permeant and GI absorption of the indole compounds can protect the brain and the central nervous system as well as allows molecules to enter the cell according to the size and lipid solubility of the molecules (Ramesh, et al., 2020).