S-Alkylated / aralkylated 2-( 1 H-indol-3-yl-methyl )-1 , 3 , 4-oxadiazole-5-thiol derivatives . 2 . Anti-bacterial , enzyme-inhibitory and hemolytic activities

Purpose: To evaluate the antibacterial, enzyme-inhibitory and hemolytic activities of Salkylated/aralkylated 2-(1H-indol-3-ylmethyl)-1,3,4-oxadiazole-5-thiol derivatives. Methods: Antibacterial activities of the compounds were evaluated using broth dilution method in 96 well plates. Enzyme inhibitory activities assays were investigated against α-glucosidase, butyrylcholinesterase (BchE) and lipoxygenase (LOX) using acarbose, eserine and baicalien as reference standards, respectively. A mixture of enzyme, test compound and the substrate was incubated and variation in absorbance noted before and after incubation. In tests for hemolytic activities, the compounds were incubated with red blood cells and variations in absorbance were used as indices their hemolytic activities. Results: The compounds were potent antibacterial agents. Five of them exhibited very good antibacterial potential similar to ciprofloxacin, and had minimum inhibitory concentrations (MIC) of at least 9.00 ± 4.12 μM against S. aureus, E.coli, and B. subtilis. One of the compounds had strong enzyme inhibitory potential against α-glucosidase, with IC50 of 17.11 ± 0.02 μg/mL which was better than that of standard acarbose (IC50 38.25 ± 0.12 μg/mL). Another compound had 1.5 % hemolytic activity. Conclusion: S-Alkylated/aralkylated 2-(1H-indol-3-ylmethyl)-1,3,4-oxadiazole-5-thiol deviratives with valuable antibacterial, anti-enzymatic and hemolytic activities have been successfully synthesized. These compounds may be useful in the development of pharmaceutical products.


INTRODUCTION
The synthesis and evaluation of 2, 5disubstituted-1,3,4-oxadiazole-2-thiol derivatives have received attention in the past decades.The medicinal values of these compounds are diverse, and include anti-edema and antiinflammatory, anti-microbial, analgesic, antimycobacterial, anti-convulsant, anti-tumor, antimalarial, anti-hepatitis B, anti-tuberculosis, insecticidal, anti-HIV and anti-Parkinsonism activities [1,2].The ability of these compounds to inhibit certain enzymes such as cholinesterases, α-glucosidase and lipoxygenase can exploited for medicinal purposes.Cholinesterases are present in cholinergic and non-cholinergic tissues, as well as plasma and other body fluids [3][4][5].α-glucosidase are used as oral antidiabetic drugs for the management of type-2 diabetes mellitus [6].Lipoxygenase are important enzymes in the biosynthesis of leukotrienes used for the treatment of inflammation and allergic diseases [7,8].

Lipoxygenase (LOX) assay
Lipoxygenase activity was assayed according to standard methods [11][12][13].The assay mixture (200 µL) contained 150 µL of sodium phosphate buffer (100 mM, pH 8.0), 10 µL of test compound and 15 µL of purified lipoxygenase.The mixture was pre-read at 234 nm and pre-incubated for 10 minutes at 25 °C.The reaction was then initiated by addition of 25 µL of substrate solution.Change in absorbance was observed after 6 min at 234 nm.All reactions were done in triplicates.Positive and negative controls were included in the assay.Quercetin (0.5 mM well -1 ) was used as positive control.Inhibition (%) was calculated as in Eq 1.
where C (i.e., control) = total enzyme activity without inhibitor, and T (i.e., test sample) = activity in the presence of test compound.IC 50 values (concentration that results in 50 % enzyme inhibition) of the derivatives were calculated using EZ-Fit Enzyme kinetics software (Perella Scientific Inc. Amherst, USA).

α-Glucosidase inhibitory assay
α-Glucosidase inhibitory activity was determined according to the method of Pierre [14].The assay mixture (100 µL) contained 70 µL of 50 mM phosphate buffered saline (pH 6.8); 10 µl (0.5 mM) of test compound and 10 µl (0.057 units) enzyme.The contents were mixed, preincubated for 10 min at 37 ºC and pre-read at 400 nm.Then 10 µl of 0.5 mM substrate (pnitrophenyl glucopyranoside) was added to initiate the reaction.Acarbose was used as positive control.After 30 min of incubation at 37 ºC, absorbance was measured at 400 nm using Synergy HT microplate reader.All experiments were carried out in triplicate.Inhibition (%) was calculated as in Eq 2.
where C (i.e., control) = total enzyme activity without inhibitor, and T (i.e., test sample) = activity in the presence of test compound.IC 50 values (concentration that causes 50 % inhibition of enzyme catalyzed reaction) of compounds were calculated using EZ-Fit Enzyme Kinetics Software (Perrella Scientific Inc. Amherst, USA).
where C (i.e., control) = total enzyme activity without inhibitor, and T (i.e., test sample) = activity in the presence of test compound.IC 50 values (concentration at which there is 50 % enzyme inhibition) of compounds were calculated using EZ-Fit Enzyme kinetics software (Perella Scientific Inc. Amherst, USA).
Results are expressed as mean of triplicate ± SEM).Ciprofloxacin was used as standard.

Antibacterial assay
This was performed in sterile 96-wells micro plates under aseptic environments.The method is based on the principle that microbial cell number increases as the microbial growth proceeds in a log phase, which results in increased absorbance of the broth medium [16,17].Three gram-negative (Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi) and two gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus) maintained on agar medium were tested against the compounds.Test samples in suitable solvents and dilutions were pipetted into wells (20 µg/well) along with 180 µL of freshly maintained overnight bacterial culture, after suitable dilution with fresh nutrient broth and incubated at 37 o C for 16-24 h.Absorbance was measured at 540 nm using microplate reader, before and after incubation, and the difference was noted as an index of bacterial growth.The percent inhibition was calculated as in Eq 4.
where X is the absorbance in control with bacterial culture and Y is the absorbance of test sample.

Hemolytic activity assay
Hemolytic activities of the compounds were studied by the reported method [18,19].Freshly obtained heparinized human blood (3 mL) was collected from volunteers after consent and counseling.The blood was centrifuged for 5 min at 1000 g and the plasma was discarded.The cells were then washed three times with 5 mL of chilled (4 o C) sterile isotonic phosphate-buffered saline (PBS, pH 7.4).The concentration of erythrocytes was maintained at 10 8 cells per mL for each assay.Hundred μL of each compound was separately mixed with erythrocytes (10 8 cells/mL), and incubated for 35 min at 37 o C, followed by agitation after 10 min.Thereafter the samples were placed on ice for 5 min and then centrifuged for 5 min at 1000 x g.Supernatant samples (100 μL) were taken from each tube and diluted 10 times with chilled (4 o C) PBS.Triton X-100 (0.1 % v/v) was used as positive control, while phosphate buffered saline (PBS) was used as negative control.Absorbance was read at 576 nm using μQuant (Bioteck, USA).The % of lysed RBCs for each sample was calculated.

Docking methodology
To predict the bioactive conformations, various compounds (ligands) were docked into the binding pockets of the selected proteins (enzymes) by using the default parameters of MOE-Dock program.Prior to docking, the protein molecules of α-glucosidase, LOX and BChE were retrieved from Protein Data Bank having PDB ID codes of 3NO4 (Resolution: 2.02Å), 1IK3 (Resolution: 2.0Å) and 1POP (Resolution: 2.0Å) respectively.All the water molecules were removed from receptor proteins and 3D protonation was carried out by using the Protonate 3D Option.The energies of protein molecules were minimized by using the default parameters of MOE 2009-10 energy minimization algorithm (gradient: 0.05, Force Field: MMFF94X).Then all the ligands were docked into the binding pockets (selective residues/amino acids as shown in the docking images) of the above mentioned proteins using Triangular Matching docking method.Re-docking procedure was also applied to validate the docking protocol.Each complex was analyzed for the type of interactions; bond distances and their 3D images were taken.

Statistical analysis
All the measurements were carried out in triplicate and presented as mean ± SEM.Statistical analysis was performed using Microsoft Excel 2010.

Enzyme inhibitory activity
In vitro screening of the synthesized compounds against α-glucosidase, BChE and LOX revealed that these molecules exhibited variable inhibitory potential as shown by their IC 50 values (Table -1

Molecular docking
Molecular docking studies confirmed the antienzymatic activities of all the compounds Figure-1.2D and 3D docked pictures of some more potent compounds are given here.

DISCUSSION
Compounds bearing oxadiazole moieties have been previously synthesized by our research group and their biological activities were shown to be related to structural modifications in the molecules [20,21].The synthesis of these 5-(1Hindol-3yl-methyl)-1,3,4-oxadiazole-2-thiols (6a-u) in the present study is a part of our continued efforts to generate new molecules with biological activities.Our results revealed that the 5-(1Hindol-3yl-methyl)-1,3,4-oxadiazole-2-thiol derivatives (6a-6u) had variable inhibitory potencies against α-glucosidase, BChE and LOX.As expected, the compounds with comparatively more inhibitory activities were those that fitted more closely into the active sites of the enzymes inhibited.Compound 3-{[5-(pentylsulfanyl)-1,3,4-oxadiazol-2-yl]methyl}-1Hindole (6g) showed excellent inhibitory potential against α-glucosidase, which was even better than the reference standard, acarbose.This molecule contains n-pentyl group substituted at the thiol position of the oxadiazole ring along with the indole moiety.These structural features probably conferred on the compound the best fit into the active site of the enzyme, which might be responsible for its high inhibitory potency against α-glucosidase.
All the synthesized compounds exhibited variable antibacterial potential.It has been reported earlier that slight modifications in structure may affect antibacterial potential [24].In this study we made changes at the thiol position of the parent indole-containing oxadiazole molecule (4) by substituting different alkyl/aralkyl groups to evaluate their antibacterial profile [25].On the basis of these results, it seems reasonable to assume that these synthesized thiol derivative compounds are either non-toxic or at most, only moderately so.Thus they may be suitable drug candidates for further investigations.
In order to test the validity of our enzyme inhibition results, the co-crystallized ligands of the enzymes were extracted and then re-docked into the binding pockets of the receptors.In all cases, RMSD values between docked and cocrystallized ligands were less than 2A° which indicated the reliability of docking method.Thus our protocol can be used for further studies.Almost all the synthesized derivatives were computationally docked against α-glucosidase, BChE and lipoxygenase to explore the binding modes of all the ligands.The results were very much in favor of the experimental data.For example, 3-{[5-(pentylsulfanyl)-1,3,4-oxadiazol-2-yl]methyl}-1Hindole (6g) showed interactions with two active site amino acid residues of the target protein.Amino acid Asp B307 showed a hydrogen bond acceptor (polar) interaction with -NH group of indole moiety with a bond distance of 2.10 angstroms, while Trp307 showed arene-arene interaction with oxadiazole ring having a bond distance of 3.44 angstroms.The other residues present in the nearby vicinity of the ligand were Trp B269, Arg B404 and IleB198.

CONCLUSION
All the compounds exhibited variable antienzymatic potential which were also supported by the molecular docking results.Compound 3-{[5-(pentylsulfanyl)-1,3,4-oxadiazol-2-yl]methyl}-1H-indole (6g) showed excellent anti-enzymatic activity against α-glucosidase, which was superior to that of the standard Baicalein.Their anti-bacterial activities revealed that the synthesized derivatives displayed moderate to very good to activities.These compounds also showed least to moderate % hemolytic activities.These results suggest that the 2-(1H-indol-3-ylmethyl)-1,3,4oxadiazole-5-thiol derivatives, due to their multiple functional groups, possess remarkable anti-enzymatic and antibacterial potential with acceptable cytotoxicity levels.Thus they may be useful agents for development of new antidiabetic drugs and anti-inflammatory agents.