Antioxidant and Anticholinesterase Potentials of Novel 4,6-Dimethoxyindole based Unsymmetrical Azines: Synthesis, Molecular Modeling, In Silico ADME Prediction and Biological Evaluations

Abstract Chemically and biologically important -C = N-N = C- diimine linkage was used to build unsymmetrical azine systems with indole heterocyclic backbone and substituted aromatic carbaldehydes. Ten novel compounds 7a-j were synthesized by the Schiff base reaction of 4,6-dimethoxyindole-7-hydrazone 5 and a range of substituted carbaldehydes 6a-j with high yields and purities. The biological study was directed to identify the antioxidant potentials due to the expected electronic delocalization capability of designated linkage which is important for possible hydrogen or electron donation. The three different assays, namely DPPH free radical scavenging, ABTS cationic radical decolarization and CUPRAC (cupric reducing antioxidant capacity) were employed to detect the antioxidant properties. The antioxidant potentials were found to be moderate for ABTS and CUPRAC assays and the compound 7j was the most potent candidate for all the antioxidant assays. More importantly, the anticholinesterase properties were investigated by acetylcholinesterase (ACh) and butyrylcholinesterase (BCh) enzyme inhibition assays. The molecular docking studies were also carried out to determine the possible poses of ligands 7a-j in binding sites of enzymes and ligand-residue interactions. The synthesized compounds were found to be more effective for the anticholinesterase activity with three promising candidates 7d, 7h and 7j in the case of BChE inhibitions. The compound 7h was determined as the best candidate with the comparable IC50 values for AChE and better inhibition potency for BChE with 7j. A range of hydrophobic and hydrophilic interactions were detected for the designated compound 7h and 7j through different amino acid residues and the computational results were found to be compatible with the biological counterparts.


Introduction
Symmetrical and unsymmetrical azines derived from the reactions of two identical or different carbonyl compounds with hydrazines have attracted great attention due to the presence of two conjugated double bonds with the N-N dimine linkage.2][3][4][5] The particular interest has been devoted to the production of unsymmetrical azines since the opposite orientation of imine bonds provides binding capability of dissimilar groups and the literature covers a range of reports on different antibacterial, 4 antifungal, 6,7 anti-inflammation, 8 antitumor 9,10 and antiviral activities.
The polar acceptor N-N dimine bonds were determined as suitable targets for the metal ions such as iron (Fe), zinc (Zn) and copper (Cu) to produce metal complexes. 11,12It is known that the accumulation of the bioavailable transition metals Fe, Cu and Zn is responsible for Alzheimer's Disease (AD), since the self-aggregation of amyloid-b (Ab) via metal peptide chelation forms senile plaques, which are deposited outside of neurons. 135][16] The selective BChE inhibition of monoterpeneindole alkaloids nitrarine, hirsutine, rauwolscine and catharanthine was reported due to the presence of fused indole ring to the monoterpene side chain. 17The study carried out by Passos et al. displayed the interaction of indole ring with the Ser-198 and His-438 amino acids on catalytic site of the BChE via hydrogen bonds and monoterpeneindole alkaloids (MIAs) angustine, vallesiachotamine lactone and E/Z-vallesiachotamine. 15urther collaborations of the Trp-82, Trp-231, Leu286, Phe-329 and Ile-442 residues with the indole carbon atoms through the van der Waals contacts stabilized the interactions. 159][20] The indole moiety is a class of nitrogen-containing aromatic heterocyclic systems and appears in biological systems as a part of the amino acid tryptophan 21 and the neurotransmitter serotonin. 22][30][31][32] In the view of the fundamental roles of azine linker and indole heterocyclic compounds for the biological process, it was of interest to design novel azine compounds with the indole heterocyclic systems and investigate the inhibition effects toward Acethyl-(ACh) and Butyryl-(BCh) cholinesterase enzymes.The current work was designed due to the possible multiple binding patterns between the targeted compounds and different binding sites on the AChE and BChE.Anionic, esteratic and acyl binding sites are different active locations on the AChE and BChE and potential interactions would occur with the indole heterocyclic and unsymmetrical azine moieties.Additionally, it was thought that the presence of different aromatic residues on the unsymmetrical azines would be valuable source for new interactions with aminoacids on the abovementioned binding sites.Moreover, three antioxidant assays, DPPH, ABTS and CUPRAC were carried out to identify the antioxidant potency of the designated compounds.The main consideration for this purpose was the potential electron delocalization properties of N-N dimine linkers and reported free radical scavenging ability of indole heterocyclic compounds.To the best of our knowledge, anticholinesterase and antioxidant studies on such indole based azine systems are rare.

Chemicals and physical measurements
All commercially available reagents and the standards used for the biological assays were purchased from Sigma Aldrich and used without further purification.The synthetic procedures have been reported for the final compounds as general method and other procedures have been given individually with appropriate references.All reactions were monitored by thin layer chromatography using Merck TLC Silica gel 60 F 254 .Silica gel 60 (particle size: 0.040-0.063mm, 230-400 mesh ASTM) for column chromatography was obtained from Merck. 1 H and 13 C NMR spectra were recorded for all compounds either in DMSO or CDCl 3 solutions on a Bruker DPX 400 MHz spectrometer at 300 K.Chemical shifts are reported in parts per million and referenced to the residual solvent peak (DMSO-d 6 : 1 H 2.50 ppm, 13 C 39.52 ppm and CDCl 3 : 1 H 7.24 ppm, 13 C 77.23 ppm).Coupling constants (J) are reported in Hertz (Hz).Standard conventions indicating multiplicity were used: m ¼ multiplet, t ¼ triplet, d ¼ doublet, s ¼ singlet, dd ¼ doublet of doublets.Infrared spectra were recorded using a Thermo Scientific Nicolet IS10 FT-IR spectrometer between 600 and 4000 cm À1 .Melting points were measured using a Mel-Temp melting point apparatus.

Preparation of compounds 3 and 4
The preparation of methyl 4,6-dimethoxyindole-2-carboxylate 3 and methyl 7-formyl-4,6-dimethoxyindole-2-carboxylate 4 was carried out according to the synthetic procedures reported in the previous study. 332.2.The reaction of 7-Formyl-4,6-dimethoxyindole-2-carboxylate with hydrazine hydrate A mixture of the indole-7-carbaldehyde 4 and excess hydrazine hydrate were stirred overnight in ethanol in the presence of glacial acetic acid as catalyst.After completion of the reaction, the solvent was evaporated.Water was added to the residue and the resulting mixture was extracted with ethyl acetate.The organic extract was dried with anhydrous sodium sulfate and the solvent was evaporated under reduced pressure to give the methyl 7-[(E)-hydrazinylidenemethyl]-4,6dimethoxy-1H-indole-2-carboxylate 5. 25

General procedure for the preparation of final compounds 7a-j
The methyl 7-[(E)-hydrazinylidenemethyl]-4,6-dimethoxy-1H-indole-2-carboxylate 5 (1 equiv) was reacted with the appropriate aromatic carbaldehyde 6a-j (1 equiv) in ethanol.Acetic acid or hydrochloric acid (5 drops) was added as catalyst and the solution was stirred overnight at room temperature.The resulting dark yellow solution was concentrated under vacuum and the crystallized from ethanol to give the title compounds 7a-j.
The title compound was synthesized following the general procedure using methyl 7-[(E)-hydrazinylidenemethyl]-4,6-dimethoxy-1H-indole-2-carboxylate 5 (300 mg, 1.08 mmol) and 3-ethoxy-4-hydroxybenzaldehyde (6j) (179.5 mg, 1.08 mmol) in EtOH (25 mL) with 5 drops of acetic acid.The product 7j was obtained as a yellow solid powder; yield 321.The color change from purple to yellow is measured at absorbance 517 nm.DPPH radical scavenging activity assay has been performed according to the method of Blois et al. with the minor modifications. 340.1 mM 160 lL of DPPH solution in methanol was added to 40 lL of sample solutions in DMSO at different concentrations.After 30 min.incubation, the absorbance values were read at 517 nm.Butylated Hydroxy Toluen (BHT), Butylated Hydroxy Anisole (BHA) and a-TOC (Tocopherol) were also assayed as standards for comparison.
2.3.1.2.ABTS cation radical decolorization activity.The ABTS þ (2,2 0 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) assay was carried out to determine the cationic radical reducing ability of synthesized compounds. 35BHA, BHT and a-TOC were used as standards.Freshly prepared ABTS solution (160 lL) was added to each sample solution at different concentrations from 10 to 100 mM.After 30 min incubation, the percentage inhibition at 734 nm was read for each concentration relative to a blank absorbance (DMSO).The obtained values were compared to standards.
2.3.1.3.CUPRAC cupric ion reducing antioxidant capacity.The CUPRAC 'Cupric Ion Reducing Antioxidant Capacity' method is an antioxidant capacity assay to identify the reductive efficiencies of the compounds for Cu metal. 36The oxidizing reagent bis(neocuproine) copper(II) cation (Cu(II)-Nc) is reduced to bis(neocuproine) copper(I) cation, forming the Cu(I)-chelate with antioxidants.It involves the reaction of antioxidant compound with the CuCl 2 , neocuproine and ammonium acetate at pH 7. The absorbances were measured at 450 nm after 30 min.BHA, BHT and a-TOC were used as standards.

Anticholinesterase activity determination method
ACh and BCh enzyme inhibitory activities were measured by the slightly modified spectrophotometric method developed by Ellman. 37Acetylthiocholine iodide and Butyrylthiocholine iodide were used as substrates of the reaction and DTNB (5,5 0 dithiobis nitro benzoic acid) were used for the measurement of the Anticholinesterase activity.Galanthamine was used as standard.Aliquots of 130 lL sodium phosphate buffer (pH 8.0), 10 lL of 4 mM sample solution and 20 lL AChE (or BChE) solution were mixed and incubated for 15 min at 25 C.The reaction was then initiated by the addition of 10 lL of DTNB and 10 lL Acetylthiocholine iodide (or Butyrylthiocholine iodide).Final concentration of the tested solutions was 200 lM.The hydrolysis of these substrates was monitored using microplate ELISA reader XS by the formation of yellow 5-thio-2-nitrobenzoate anion as the result of the reaction of DTNB with thiocholine, released by the enzymatic hydrolysis of acetylthiocholine iodide or butyrylthiocholine iodide, at a wavelength of 412 nm.DMSO was used as a solvent to dissolve the samples and controls.
where A 0 is the absorbance of the control and A 1 is the absorbance in the presence of the sample

Statistical analysis
The results were mean ± SD of three parallel measurements.All statistical comparisons were made by means of Student's t-test, p values < 0.05 were regarded as significant.

Receptor preparation
Acetylcholinesterase and butyrylcholinesterase crystal structures were attained from Protein Data Bank. 384EY7 (2.35 Å) 37 crystal structure was selected for AChE enzyme and 6QAA (1.897 Å) 39 crystal structure was selected for BChE enzyme.MGL Tools software was used for the preparation of the obtained structures.The amino acid residues were remained on the binding sites of the enzymes and subsequent addition of hydrogen atoms and charges was proceeded.The binding abilities of the ligands to the active sites of enzymes were assessed by the grid calculations performed by grid boxes with the dimensions of 40 Â 40 Â 40 Å.The grid box of AChE enzyme was centered to À13.99, À43.91, and 27.11, while BChE enzyme's grid box was centered to 19.04, 42.70, and 40.38 through x, y and z coordinates, respectively.

Ligand preparation
The two-dimensional structures of compounds 7a-j were drawn by Marvinsketch and converted to three dimensional structures by Biovia DS Visualizer.Optimization procedure was performed with semi-empirical PM6 method followed by DFT approach at B3LYP level of theory with 6-31 gþ(d,p) basis set.RESP charges 40 of optimized structures were calculated by using PyRED servers. 41Optimized ligand structures 7a-j are shown in Figure 1 and computed RESP charges are given in Supplementary Materials.Ligands were prepared by using MGL Tools for Autodock4 program as pdbqt type files for docking processes.

Docking process
Autodock4 program was used for docking studies with the aid of MGL Tools.Docking protocol was performed with Lamarckian Genetic Algorithm (LGA) as explained in literature. 42

Chemistry
The synthetic pathway in this manuscript was based on the 4,6-dimethoxyindole backbone with the hydrazone functionality on C7 position.Subsequent Schiff Base reaction between the indole-hydrazone 5 and a range of aromatic carbaldehydes 6a-j provided the formation of unsymmetrical azine 7a-j system with the N-N dimine linker.

The Synthesis of dimethoxyindole-7-hydrazone 5
The heterocyclic indole backbone, methyl 4,6-dimethoxyindole-2-carboxylate 3, was prepared by the Hemetsberger reaction 33,43 via a vinyl azide which is generated by the condensation of 2,4dimethoxybenzaldehyde 1 with methyl azidoacetate in methanolic sodium methoxide (Scheme 1).The intramolecular cyclization was achieved with the high boiling solvent via the thermal decomposition of vinyl azide 2. The installation of aldehyde functionality was achieved by the Vilsmeier-Haack formylation reaction 33 at C7 position of indole moiety 4 due to the methyl ester group at C2 and the location of the methoxy groups on the benzene ring (Scheme 1).The further synthetic transformation was achieved by the reaction of the formyl group with hydrazine-hydrate to produce indole-7-hydrazone 5 under acidic condition. 29The procedures and instrumental characterization of the compounds 1-5 were reported in the previous studies. 29,33,431.2.The Synthesis of indole based unsymmetrical azines 7a-j Following preparation of the methyl 7-[(E)-hydrazinylidenemethyl]-4,6-dimethoxy-1H-indole-2carboxylate 5, the acid catalyzed condensation reaction was carried out with a range of aromatic carbaldehydes 6a-j to afford the targeted substituted indole based azine compounds 7a-j (Scheme 2).The reaction mixtures in EtOH and acetic acid were stirred at room temperature overnight and the resulting crude products were purified by recrystallization from EtOH/hexane to give the title compounds 7a-j (Table 1).
The new substituted unsymmetrical azine compounds 7a-j were characterized by physical and spectral data (IR, 1 H-NMR, and 13 C-NMR).The general features of the targeted compounds obtained from 1 H NMR spectroscopy were outlined.The most important evidence for the confirmation of final compounds 7a-j was the loss of the NH 2 peak at 6.73 ppm from indole-7-hydrazone 5 as a result of condensation with the hydrazine hydrate and the appearance of additional CH proton on the N-N linker as a singlet at the region 8.95-9.25 ppm.The azomethine CH proton at 8.23 ppm for the indole-7-hydrazone 5 was shifted to the down fielded area due to the formation of CH ¼ N-N ¼ CH linker and appeared at the region of 8.58-8.92ppm for the final compounds.The formation of the desired azines was also confirmed with the 13 C NMR spectroscopy by the assignments of additional azomethine carbon (C ¼ N) and the aromatic carbons from carbaldehyde moieties resonated at 135.0-145.0ppm and 125.0-134.0ppm, respectively.The CH ¼ N signals derived from indole-7-hydrazone shifted to down fielded area and appeared at 155-158 ppm due to the condensation reaction.The proton and carbon signals obtained from indole and corresponding substituents located on the carbaldehyde moieties were assigned at the expected region.
IR spectroscopy was also used for the confirmation of the targeted structures 7a-j and the selected bands were assigned.The intense vibrational absorptions at 1585-1595 cm À1 and 1159-1166 cm À1 were assigned to the v(C ¼ N) and v(N-N) frequencies and proved the condensation of aldehyde group with the hydrazone 5 through Schiff base reaction.The C-H stretching vibrations of hydrogen-containing aromatic systems appeared as weak bands in the region of 2940-2960 cm À1 .

Biological studies
The novel unsymmetrical azine compounds 7a-j were subjected to Acetylcholine (ACh) and Butyrylcholine (BCh) enzyme inhibition assays to identify their anticholinesterase activity.The Table 2 illustrates the percentage of AChE and BChE inhibition values by the compounds at 200 mM concentrations and comparison with the Galanthamine as a standard.The compounds showing higher percentage of inhibitions were evaluated as potent candidates for the treatment of AD.Their antioxidant profiles against free radicals, cationic radicals and reductive efficiencies for Cu metal were also investigated using DPPH, ABTS and CUPRAC antioxidant assays (Table 3).The DPPH (2,2-diphenyl-1-picrylhydrazyl hydrate) assay was used to measure the hydrogen atom (or one-electron) donation ability of the scavenge molecule to DPPH, resulting in reduction of DPPH to DPPH 2 .The ABTS þ [2,2 0 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] assay was carried out to determine the cationic radical reducing ability of the synthesized compounds.The CUPRAC (Cupric Ion Reducing Antioxidant Capacity) method is an antioxidant capacity assay to identify the reductive efficiencies of compounds for Cu metal.It involves reaction of the antioxidant compound with CuCl 2 , neocuproine and ammonium acetate at pH 7. The mean and standard deviation (SD) values for each compound were calculated from at least three replicate experiments.

Determination of anticholinesterase activity
As expected, the potency of positive control Galanthamine for both AChE and BChE was detected as the values of 81.78% and 76.58%, respectively.In general, all the compounds demonstrated moderate inhibition for the AChE, the efficiencies of synthesized compounds were found to more potent for BChE inhibition (Table 2).More importantly, three compounds 7d, 7h and 7j demonstrated better inhibition values than the standard Galanthamine.The compound 7a with no substituent on the benzene ring showed the lowest inhibitions for both enzymes; however, the substituent addition at para position increased the inhibition values in the case of BChE.The presence of methoxy 7b, bromo 7d and nitro 7e groups resulted different AChE and BChE The administration of additional substituents on the aromatic ring derived from carbaldehyde moiety increased the inhibition values in the case of BChE.The greatest inhibition values were found to be by the treatment of compounds 7h and 7j with the values of 97.71% and 97.78%, respectively.The AChE inhibition values for the designated compounds were detected as %64.53 and 39.73%, respectively.Interestingly, the compound 7i with hydroxyl and methoxy substituents located at 4-and 3-positions demonstrated no inhibitions.
Overall, the compound 7h, di-substituted derivative with two methoxy groups located on 2 and 4 positions, were found to be the promising inhibitor for the AChE and BChE with the inhibition values of 64.53% and 97.71%, respectively.The AChE inhibition value was comparable  with the standard Galanthamine.Moreover, the obtained value for BChE inhibition was found to be better than the standard.

ADME predictions
To determine the possible poses of ligands 7a-j in enzyme binding sites and ligand-residue interactions prominent molecular docking software, Autodock4, was used.The ADME properties of ligands by SwissADME web service were also defined to identify the physicochemical and drug likeness properties of the ligands (Table 4).Drug metabolism is one of the most important steps to determine the ADME properties of potential drug candidates.The ADME predictions demonstrated variable Log P, degree of lipophilicity, values for ligands ranging from 2.91 to 4.43 and found to be acceptable rates.As a positive physicochemical property, high level of gastrointestinal absorption characteristic was determined, however none of the ligands showed blood-brain barrier permeation property (Table 5).A hemeprotein, Cytochrome P450 (CYP), plays a valuable role in the determination process 44 and CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4 enzmyes are different forms of the CYP protein estimated to be mostly active in drug metabolism. 45Analyses revealed that inhibitory potency of all the ligands was determined at sufficient level toward the CYP2C9 and CYP3A4 enzymes, however the candidates were found to be inactive against CYP2D6 (Table 5).Moreover, the ligands were also evaluated to be potential inhibitors for CYP1A2 and CYP2C19, all the compounds, except ligands 7f, 7j, 7 g and 7i showed inhibitory effect against CYP1A2 and CYP2C19 enzymes.

The Validation of Molecular docking
The accuracy of molecular modeling process was determined by the docking validation of the desired enzymes AChE and BChE through the re-docking of existed reference ligands in the crystal structures (Figure 2).In the case of AChE, re-docking of the reference ligand in the binding site of enzyme provided 0.684 Å RMSD value and the value of 0.737 Å RMSD was recorded in the presence of BChE ligand.The settings obtained from the validated docking process were used for the novel compounds synthesized in the current study.

Molecular docking of the ligands 7a-j
The molecular modeling study was designed with respect to the results obtained from the biological assay and the compounds with the dual inhibition activity against the designated enzymes were chosen for the computational analysis.The ligand-residue interactions between the novel compounds and amino-acids located on the binding site of the enzymes were outlined.The anionic, esteratic and the acyl binding sites of the designated enzymes provided a range of important interactions namely, conventional and carbon hydrogen bonds, p-p, p-alkyl, alkyl-alkyl and Vander Waals with the functional groups located on the novel compounds (Figures 3 and 4).The general interaction patterns were briefly discussed via the common structural motifs for all the compounds, and the detailed interactions were given in Table 6.
In the case of AChE (Figure 3), the benzene ring on the indole moiety demonstrated the main interaction with the Try337 and Trp86 amino acids through the p-p T shaped hydrophobic attachments.The presence of the carbonyl oxygen on the methyl ester located at C2 position of the indole backbone provided conventional hydrogen bonds with Gly121, Gly122, and Ala204 residues on the esteratic binding site of the enzyme.In addition to that, the methyl residue on the methyl ester showed p-alkyl interactions with Phe295, Phe297 and Trp236.The substituted benzene rings administered by the aromatic carbaldehydes provided additional p-p-T shaped and p-alkyl hydrophobic interactions with Trp286 and Val294, respectively.The N,N-dimine linker acted as hydrogen bond donor for the carbon atoms in Tyr124 residue.The computational study revealed that the common feature for the potent compounds is the presence of oxygen containing substituent administered with the aromatic carbaldehydes.Specifically, methoxy (7h), hydroxy (7 g) and 3ethoxy,4-hydroxy (7j) fragments provide important conventional hydrogen bonds with the Phe295, Tyr341 and Ser293 and Arg296 to increase the binding strength at the catalytic side of AChE.
The general binding pattern in the presence of BChE (Figure 4) revealed different ligand residue interactions from those determined residues in the binding site of AChE.The benzene and pyrrole rings cooperated for the p-p T shaped, carbon hydrogen bonds and p-sigma interactions with the Thr120, Trp82, Pro285 and His438 amino acids.The esteratic site of the enyzme provided a range of interactions, conventional hydrogen bonds, carbon hydrogen bonds, alkyl and Van der Waals, with the methyl ester fragments on the C2 position of indole through the Tyr128, His438, Trp82, Gly115, Val 288, Leu286 and Gly121, Ser198, Thr120, Gly117, Gly439, Tyr440, Trp82 and Tyr430 aminoacids.The methoxy groups located at 4 and 6 positions of the indole were found to be crucial for additional p-alkyl, conventional and carbon hydrogen bonds bindings with the Ile69, Asp83, Pro84, Tyr114, Tyr128, Leu125, Tyr332 and Thr284 residues.The hydrophobic interactions between the residues Trp231, Phe329, Leu286, Tyr332, Ala328, Phe329 and Ser287 and the substituted benzene rings at the end of N,N-dimine linker occured as p-p T shaped, alkyl and p-lone pair attachments.Interestingly, N,N-dimine linker participated for the conventional hydrogen bonds with Thr120 residue in the presence of compound 7d.In the case of BChE, not only the oxygen containing substituents but also the chloro substituted compound (7c) showed reasonable alkyl interactions with the Met437, Tyr440, Trp430, Trp82 and Ala328.The compounds with 2,4-dimethoxy (7h) and 3-ethoxy,4-hydroxy (7j) substituents revealed high number of alkyl carbon and conventional hydrogen bond interactions with the Ala328, Phe329, Tyr332, Pro285 and His438, Trp82 and Gly439, respectively.
The calculated binding free energies of ligands were correlated with experimental binding free energies of ligands obtained from Equation 1.The analyses were performed with the Pearson correlation test.The analyses showed that calculated binding energies of AChE and BChE ligands don't have significant correlation with experimental binding energies where the p values were found to be 0.9315 and 0.1446 values, respectively (p > 0.05).Since the inhibition activities of the ligands were experimentally defined as inhibition% by using 200 mM of ligands, we have proportioned these values to obtain IC 50 values.It should be considered that the usage of derived IC 50 values from inhibition% may lower the correlation of calculated and experimental binding energies.The obtained IC 50 values were then used in equation 1 to acquire binding free energies of ligands.The plots of calculated binding energies of AChE and BChE ligands versus their experimental values are given in Figure 5 and 6.
3.4.Identification of antioxidant potency 3.4.1.DPPH free radical scavenging assay Table 3 illustrates the DPPH (2,2-diphenyl-1-picrylhydrazyl hydrate) radical scavenging activity of the final indole based azines 7a-j and the positive controls, BHT, BHA and a-TOC.According to the results, the synthesized compounds have been detected as not suitable targets for the free radical scavenging activity.The compounds with no or mono substitutions on the benzene ring demonstrated no scavenging activity.The di-substituted analogues 7i and 7j demonstrated lower activity with the IC 50 values of 143.02 and 142.39 mM concentrations.The results were found to be incomparable with the values obtained by the standards.

ABTS Cation radical Decolorization assay
Generally, the inhibition values for the di-substituted compounds 7h, 7i and 7j were found to be greater than those of analogues 7b-g with mono substitutions (Table 3).Compound 7a with no subtitution exhibited lower cationic radical scavenging activity with the IC 50 value of 162.10 mM.The highest inhibition for the mono substituted compounds has been obtained by the 4-OMe substituted compound 7b with the value of 198.40 mM.The hydroxyl functionality at 2-position on the benzene ring 7 g increased the inhibition potency more than 2-fold compared to the best candidate for the compounds with the substitution located at 4-position.In the case of double substituted compounds, the inhibition efficiency was considered as promising target for the cationic radical scavenging activity.Although, the compound 7h with double methoxy groups located at 2 and 4 positions demonstrated lower inhibition, the replacement of one methoxy group with hydroxyl group 7i provided a great increase for the inhibition with the IC 50 values of 20.01 mM.The compound 7j with the hydroxyl and ethoxy functionalities was found to be the best candidate for cationic radical scavenging activity with the value of 18.11 mM.

Cupric reducing antioxidant Capacity (CUPRAC)
Table 3 shows the absorbance values obtained from the cupric reducing antioxidant capacity assay for the synthesized compounds 7a-j and the standards BHT and BHA.The obtained values determine the compound concentrations for the 0.5 absorbance.As in the cationic radical scavenging activity, the reducing potency of indole-based azine derivatives with the di-substituted benzene ring 7h, 7i and 7j was found to be more promising than the analogues of mono substituted compounds 7b-g.The methoxy derivative 7b was determined as the most potent analogue among the mono substituted compounds with the value of 46.21 mM.The compounds with no substitution 7a and 2-hydroxyl functional group 7 g demonstrated similar potency with the values of 101.95 and 109.15mM concentrations, respectively.The compound 7j was identified as the most promising candidate for Cupric reducing antioxidant capacity with the value of 44.92 mM.

Conclusion
Ten novel indole-based unsymmetrical azine compounds 7a-j have been synthesized by the Schiff base reaction of methyl 4,6-dimethoxy-7-hydrazone-2-carboxylate 5 and the corresponding aromatic carbaldehydes 6a-j in high yields.The method describes the preparation of methyl 4,6dimethoxyindole-2-carboxylate 3 via Hemetsberger reaction followed by Vilsmeier-Haack reaction to afford the carbaldehyde 4. The reaction of 7-formyl-dimethoxyindole structure with the hydrazine hydrate provides the targeted indole hydrazone 5 with a high yield and purity.The biological potencies of the targeted compounds 7a-j were investigated by the evaluation of antioxidant and anticholinesterase studies due to their possible characteristics in terms of metal complex formation and hydrogen or electron donation.The biological data revealed that the compounds 7b-g derived from di-substituted carbaldehydes were found to be more promising targets than the derivatives formed with no 7a or mono substitutions 7h, 7i and 7j for the antioxidant study.Although, the compounds showed less or no inhibition for DPPH antioxidant inhibition, the best antioxidant activity was demonstrated by the compound 7j, as an example of a di-substituted derivatives, in the case of ABTS and CUPRAC assays with the reasonable IC 50 values compared to the standards.Most importantly, the anticholinesterase enzyme inhibitions were found to be more sensitive for the synthesized compounds.Although, the moderate inhibition was determined for ACh enzyme, the three compounds 7d, 7h and 7j demonstrated better BChE inhibitions compare to the standard Galanthamine with the over 90% inhibition values.Molecular modeling study also confirmed the efficiencies of double substituted azine compounds 7h and 7j with the important hydrophobic and hydrophilic interactions on the binding sites of the designated enzymes.From a synthetic aspect, our work has provided ten new unsymmetrical indole-based azine ligands suitable for future studies in the area of metal complexes synthesis and the biological evaluation.

Figure 3 .
Figure 3. Ligand-residue interactions for the compounds and the amino acids on the AChE binding site.

Figure 4 .
Figure 4. Ligand-residue interactions for the compounds and the amino acids on the BChE binding site.

Figure 5 .
Figure 5.Comparison of the calculated and experimental binding free energies estimated from IC 50 for AChE ligands.

Figure 6 .
Figure 6.Comparison of the calculated and experimental binding free energies estimated from IC50 for BChE ligands.

Table 1 .
Ten novel 4,6-dimethoxyindole based unsymmetrical azines 7a-j.Although, the compound 7e demonstrated no inhibition for both enzymes, moderate BChE inhibition potency was detected with the value of %45.36 in the case of compound 7b.The highest inhibition value among the mono substituted derivatives was obtained by the compound 7d with the value of 93.59% toward the BChE.The inhibitions patterns for the chloro 7c and tert-butyl substituted 7f derivatives were found to be moderate for both AChE and BChE with the values of 49.33% and 51.90% and 47.75% and 62.34%, respectively.The other mono substituted derivative 7 g with a hydroxyl group at ortho position displayed better potency than the compound 7c and 7f, the inhibition values were detected as 58.30% and 68.46% for BChE and AChE, respectively.

Table 2 .
Acetylcholinesterase and Butyrylcholinesterase enzyme inhibitory activities of the final Indole-based Azines 7a-j a,b .All enzyme inhibition acitivities values were given as inhibition % at 200 mM.b Standard compound for AChE and BChE.NA Not Active. a

Table 3 .
The antioxidant activities of final Indole-based Azines 7a-j a .

Table 4 .
Physicochemical properties of ligands obtained from SwissADME.

Table 5 .
Predicted ADME properties of the ligands.

Table 6 .
Interactions of ligand atoms with AChE and BChE residue atoms.