Synthesis , bioactivity and Docking Study of Some New Indole-hydrazone Derivatives

Article history: Received on: 30/06/2016 Revised on: 07/09/2016 Accepted on: 20/10/2016 Available online: 28/12/2016 In the present study, a series of new indole-hydrazone derivatives were synthesized. The structures of the synthesized compounds were established on the basis of elemental and spectral (IR and 1 H-NMR) studies. All synthesized compounds were screened for their anti-inflammatory against carrageenan induced oedema in albino rats at a dose of 0.2 mmol/kg using indomethacin as a standard drug. The reduction in oedema formation was ranged between 72.3 – 89.3 % for all the synthesized compounds compared to indomethacin (46 % inhibition). Moreover, their anticancer activity against breast cancer cell line (MCF-7) at a dose of 100μg/ml was evaluated. The compounds 2f and 2j showed mild anticancer activity (61% and 68% inhibition, respectively) compared to doxorubicin. Furthermore, all molecules were docked to the active site of both cox-1 and cox-2 using the docking program Molego virtual docker. All prepared compounds showed high docking score against both cox-1 and cox-2. The dock scores and binding energy were found to be in good agreement with the pharmacological results. Finally, the drug likeness and bioactivity were predicted using Molinspiration software. The results revealed that all new compounds show good drug likeness score and bioactivity score .

Keeping in view of biological importance of the three molecular moieties viz., indole, hydrazone and azomethine group, it has been felt worthwhile to synthesize as depicted in Scheme I. We wish in the present work to generate new leads by synthesize indole-hydrazone derivatives in the hope of obtaining better antiinflammatory and anticancer agents.Further, the molecular docking were studied to help in understanding the various interactions between our derivatives and enzyme active sites (cox-1 and cox-2) as well as the correlation between docking studies and the in vivo/in vitro studies.In addition, the drug likeness and bioactivity were evaluated for all prepared compounds.

Chemistry
All melting points were determined with a Kofler Block apparatus.The progress of the reactions was monitored by thin layer chromatography (TLC) using aluminum silica gel plates 60 F 245 and further purification was performed using column chromatography (silica gel, 70-230 mesh).IR spectra were recorded on a Perking Elmer 1430 ratio recording infrared spectrophotometer with CDS data station using KBr Wafer technique at the Central Laboratory, Tanta University, and The 1 H-NMR spectra were recorded on a Varian Gemini-300 BB Spectrometer at 300 MHz with TMS as a standard, and Elemental Analysis at the Micro Analytical Center, Cairo University, Cairo, Egypt.Chemicals and solutions were purchased from Merck Co. and they were in reactive grade.
After the completion of the reaction (as monitored by TLC), the excess of solvent was removed under reduced pressure.The obtained product was recrystallized with ethanol (Lohitha, et al., 2011).
The product formed was filtered, washed with cold water, purified by silica gel column chromatography using chloroform as eluent, then the solvent was evaporated and the product was recrystallized from ethanol to yield the corresponding compounds.

Animals
Animals were obtained from the animal house of the Faculty of Pharmacy, Tanta University, Tanta Egypt; all animals were allowed free access to water and were kept on a constant standard diet.All procedures involving animals were carried out in accordance with the guidelines for the care and use of laboratory animals and were approved by the Ethics Committee of the Faculty of Pharmacy, Tanta University, Tanta, Egypt.Adult Wistar strain rats of male sex, weighing 100-140 g, were used for anti-inflammatory activity.The animals were allowed food and water ad libitum except during the experiments.They were housed in wire-mesh cages at 25 ± 2°C, with 50 ± 5% relative humidity and 12 hr light/dark cycle.The animals were randomly allocated into groups and fasted for 12-24 hours before the experimental study and used for determining the anti-inflammatory activity.All test compounds and the reference drug were administered I.P, suspended in dimethylsulfoxide (DMSO) solution.This study was done by following the procedure of Winter et al. (Winter, et al., 1962).The rats were divided into three groups (control, tested compounds and standard drug) of six animals each.A freshly prepared suspension of carrageenan (1.5% in 0.9% saline), 0.1 mL was injected S.C. into the subplantar region of the right hind paw of each rat.The test compounds and standard drug were administered I.P at the dose of 0.2 mmole/Kg to the animals of tested derivatives groups and the standard drug group, respectively, 2 hr before the carrageenan injection.The paw weight of each rat was measured after 4hr of carrageenan treatment with the help of a Plethysmometer.The percent antiinflammatory activity was calculated according to the formula given below.

Evaluation of cytotoxic activity of target compound
Anticancer activity evaluation using the Human breast carcinoma cell line (MCF7) was performed in the National Cancer Institute, Cairo University, Cairo, Egypt, using the method of (Skehan, et al., 1990).
Cells were plated in 96-mutiwell plate (10 cells/well) for 24 hr before treatment with the compounds to allow attachment of cell to the wall of the plate.The 100 µg/ml of each compound under test was added to the cell monolayer triplicate wells.Monolayer cells were incubated with each compound for 48 hr at 37ºC and in atmosphere of 5% CO 2 .After 48 hr, cells were fixed, washed and stained with Sulfo-Rhodamine-B stain.Excess stain was washed with acetic acid and attached stain was recovered with Tris EDTA buffer.Color intensity was measured in an ELISA reader.

Evaluation of drug likeness
The drug likeliness was evaluated using the Lipinski rule of 5 via Lipinski drug filter protocol (Lipinski, 2004) using Molinspiration software (Molinspiration, 2012).Our synthesized target compounds were passed the Lipinski rule of 5 and have properties that would make it a likely orally active drug in humans.The predicted drug likeness score of new compounds were compared with standard drugs indomethacin and doxorubicin as shown in Table 3.

Bioactivity score
Bioactivity of prepared compounds was checked by calculating the activity score toward G protein coupled receptors (GPCRs) ligand, ion channel modulator, nuclear receptor legend, kinase inhibitor, protease inhibitor and enzyme inhibitor.All the parameters were checked with the help of software Molinspiration drug-likeness score online (Verma, 2012).The predicted bioactivity score of synthesized compounds and indomethacin, doxorubicin as reference drugs were illustrated in Table 4.

Protein Preparation
The three-dimensional crystal structure of enzymes taken from Protein Data Bank (PDB) (http://www.rcsb.org/) is as follows: enzymes COX-1 (PDBID: 3N8Y) (Sidhu, et al., 2010) and COX-2 (PDB ID: 3LN1) (Wang, et al., 2010).All the PDB's were loaded in the Molegro virtual docker (MVD) (Thomsen, et al., 2009) with the removal of all water molecules and cofactors.The standard Molegro algorithm was utilized for rendering the missing charges, protonation states, and assigning of polar hydrogen to the receptor.

Ligands preparation
The Structures of ligands were drawn using marvin sketch and energy minimization was done.Energy minimization was done to help the docking program, Molegro Virtual Docker (MVD), (Thomsen, et al., 2009) to identify the bioactive conformer from the local minima.One major advantage of Molegro Virtual Docker (MVD) is that it helps in assigning the missing bond orders, charges, bonds, and hybridization states of the imported ligands.

Molecular docking
Flexible ligand models were used for docking and postdocking geometry optimizations.The post-docking geometry optimizations were carried out using Molegro Virtual Docker (MVD), Simulations the ligand was binding with site of enzymes COX-1 (PDBID: 3N8Y) and COX-2 (PDB ID: 3LN1).A docking sphere (15 Å radius) was placed on the binding sites of each protein structure in order to allow different orientations of each ligand to be searched in the binding cavities and for multiple protein-ligand poses to be returned.The Root Mean Square Deviation (RMSD) threshold for multiple cluster poses was set at <1.00 Å.
The docking algorithm was set at maximum restoration, of 1500 with a simple evolution population size of 50 and a minimum of 30 runs for each ligand.Each binding site of oligomeric structures was searched, and the lowest energy pose (based on the mol Dock Score and re-rank scores) for each ligand across enzymes COX-1 (PDBID: 3N8Y) and COX-2 (PDB ID: 3LN1) structures.The predicted mol Dock Score and re-rank scores for all prepared compounds as lowest energy pose are presented in Table 5.

RESULTS AND DISCUSSION
The synthetic route used to synthesize title compounds was outlined in Scheme 1. Hyrazone (1), the starting material was prepared according to the method reported in literature (Lohitha, et al., 2011).Hydrazone was condensed with different substituted formamides in ethanol to give the corresponding compounds in good yield.
Reaction of hydrazone with electron withdrawing group at para-position of aromatic formamide was faster and gave better yield than the corresponding meta-substituted ones.Accordingly.the derivatives with electron withdrawing group at para-position for example; p-nitro (2e) had better yield and short refluxing time than p-methoxy and p-chloro (2g and 2c).
Synthesized compounds were purified by column chromatography on silica gel.The IR spectra of synthesized compounds exhibited very similar features as the expected bands for the characteristic groups such as NH, C=N, and disappearance of NH 2 stretching vibrations.Presence of peak in the region 3191-3217 cm -1 indicates the existence of NH moiety and that of peak in the region 1619-1620 cm −1 indicates C=N, in all the compounds.
In the 1 H-NMR spectral data, all protons were seen according to the expected chemical shift and integral values, the aromatic protons appeared as a multiplet peaks within the range 6.8-7.8 δ ppm, doublet signals for Ar-NH and N-CH=N appeared at 8.361 δ ppm.Signals belonging to -NHNH 2 of hydrazone are disappeared indicating that functionalization of hydrazone with N substituted formamide.Elemental analysis data were also in line with theoretical values.

Anti-inflammatory activity
All the newly synthesized compounds 2a-2l were evaluated for their anti-inflammatory activity against carrageenin-induced paw edema method in rats, using indomethacin as reference drug at a dose of 0.2 mmol/kg I.P. Percent of the edema inhibition was calculated after 4 hr of carrageenin treatment.The tested compounds showed good anti-inflammatory activity ranging between 72.3 -89.3%.The results were tabulated in Table1.
Table1: Anti-inflammatory activity of the tested compounds.On comparison, standard drug indomethacin showed 46% inhibition after 4 hr of treatment.Among the tested compounds, 2d (m-NO 2 ), 2e (p-NO 2 ) and 2g (p-OCH 3 ) may be considered as potent anti-inflammatory agents (edema reduction percent: 89.3%, 87.7% and 80.7% respectively), while indomethacin was 46%.On the other hand, the derivative with electron withdrawing group at para-position such as 2e (p-NO 2 ) showed somewhat high anti-inflammatory activity (89.3%) compared with the other position such as meta-positions (87.7%) e.g.2d (m-NO 2 ).These gave an indication that, the nature and the position of substituent effect on the activities of the prepared compounds.

Evaluation of cytotoxic activity of target compounds
The synthesized target compounds were evaluated for their cytotoxic activity in vitro against Human breast carcinoma cell line (MCF7) using doxorubicin as a reference compound.The results of single dose experiment (100µg/ml) of synthesized compounds performed on Human breast carcinoma cell line (MCF7) were represented in Table 2.
From the results found that the compounds 2f and 2j exhibited the percent of inhibition 61.5 and 68 % respectively at the dose 100 µg/ml.While, the rest of compounds showed less activity (the percent of inhibition were ranged between 0 -20.5 %).Also, the derivatives at meta-position had higher activity than derivatives at para-position.For example, the m-methoxy derivative (2f) has inhibition percent at 100 µg/ml equal 61.5 %; while the p-methoxy derivative (2g) has inhibition percent = 0 % at the same dose.

Evaluation of drug likeliness
Lipinski's rule of five or simply the Rule of five (RO5) is a rule of survey and to evaluate drug likeness (physical-chemical properties) or determine if a chemical compound will be orally bioavailable (Lipinski, 2004).The drug likeness was calculated and discussed on the basis of Lipinski's rule and its component for all prepared compounds using Molinspiration software "on-line test".The results were summarized in Table 3.The physicalchemical properties including: an octanol-water partition coefficient (Milog P) < 5 that means these shows good permeability across cell membrane, polar surface area (TPSA) < 160 Ǻ2 which shown to be a very good descriptor characterizing drug absorption, number of violation (n violations) =1 or < 0 it means compound easily bind to receptor, molecular weight (MW) < 500, number of rotatable bonds (n rotb) < 10 this measures molecular flexibility (Veber, et al., 2002), number hydrogen bond donors (n OHNH) ≤ 5 (The sum of OHs and NHs), number hydrogen bond acceptor (n ON ) ≤ 10 (the sum of Os and Ns) and also, the total number of atoms between (n Atoms) 20 and 70 (dProperties user`s manual, 2011).The results from Table 3 reveal that all synthesized compounds (2a -2i) obeyed the Lipinski's rule and showed good drug likeness score.Octanol-water partition coefficient (MiLog P) values were found below 5 which indicated good permeability of these compounds.The new compounds were found to have polar surface area (TPSA) in the range of 52.55 to 98.37 that is well below 160A o2 and their molecular weights (MW) less than 500.Number of hydrogen bond donors (nONH) < 5 and hydrogen bond acceptors (nON) < 7; rotatable bonds (nrotb) < 10 and found to have n violations (NV) =0.Comparable with reference drugs, indomethacin and doxorubicin, they exhibited n violations (NV) = 0 and = 3 respectively.In the case of doxorubicin obeyed Rule of five (RO5) except molecular weight (MW) > 500, total molecular polar surface area (TPSA) > 160A o2 and hydrogen bond acceptors (nON) > 7, so doxorubicin has n violations (NV) = 3, as shown in Table 3. From the results reveal that these compounds are orally bioactive because they possess groups which act as substrate for transporter.

Bioactivity score
Similarly, all compounds were taken for calculation of bioactivity score towards G protein-coupled receptors (GPCR) ligands, ion channel modulator, kinase inhibitors, nuclear receptor inhibitors and other enzyme targets based on Molinspiration software "on-line test".These bioactivity results were summarized in Table 4.The scores allowed adequate identification of active, moderately active or inactive molecules.If the bioactivity scores is (≥ 0.00) may refer to considerable biological activities, if the bioactivity scores (-5.0 to 0.0) it is moderately active and finally if the bioactivity scores (< -5.0) it is inactive.Consequently, the results showed the following observations: a) GPCR ligand: all our compounds were found to be moderately bioactive, the bioactivity scores (-0.0119 to -0.2783) comparing with indomethacin and doxorubicin (0.30 and 0.20) respectively.b) ion channel modulator: all our compounds were found to be better bioactive, the bioactivity scores ( -0.3124 to -0.5696) comparing with indomethacin and doxorubicin (-0.25 and -0.20) respectively.c) kinase inhibitors: all our compounds were found to be moderately bioactive the bioactivity scores (-0.1174 to -0.3451) comparing with indomethacin and doxorubicin (-0.11 and -0.07) respectively.d) nuclear receptor inhibitors: in comparison with indomethacin and doxorubicin the bioactivity scores were -0.44 and 0.32 respectively whereas our compounds were found to be moderately bioactive with the bioactivity scores (-0.2695 to -0.4252).e) Protease inhibitor: all our compounds were found to be moderately bioactive, the bioactivity scores (-0.2607 to -0.5089), while indomethacin and doxorubicin were -0.07 and 0.67 respectively.f) Enzyme inhibitor: all our compounds were found to be better bioactive; the bioactivity scores were ranged between 0.024 to -0.1772, whereas indomethacin and doxorubicin were 0.36 and 0.66 respectively.
The results herein are well below the limits of bioactivity score (-0.5089 to 0.024) (Table 4).In addition, the designed molecules obeyed the Lipinski rule of five.So the designed molecules may be useful as a lead compound for various diseases like depression, anti inflammatory, cancer and others diseases.

Molecular docking studies
Molecular docking study was performed to investigate the binding affinities and interaction modes between our compounds and the target enzymes using the Molegro Virtual Docker (MVD).All prepared compounds were incorporated in the active site of the enzymes isoform COX-1 (PDBID: 3N8Y) and isoform COX-2 (PDB ID: 3LN1).The docking scores were expressed in negative energy terms; the lower binding free energy is the better binding affinity.In addition, the docking scoring function of Mol Dock is an extension of the piecewise linear potential (PLP) including new hydrogen bonding and electrostatic terms.To further improve docking accuracy, a re-ranking scoring function is introduced.Subsequently, Mol Dock score and re-rank score were indicated in Table 5.The docking study displayed that most of the prepared compounds showed promising affinity to inhibit both cox-1 and cox-2.Thus, the synthesized compounds showed good docking scores ranged from -129.601 to -148.276 with cox-1 and from -92.797 to -108.757 with cox-2.From Table 5 appears that the compounds 2d and 2e are considered as good anti-inflammatory agents because they have good mol dock scores of -146.71 and -148.276 with the active site of cox-1 and -104.223, and -108.757 with cox-2 enzymes respectively.While indomethacin showed dock score of -131.578 and 92.5925 with cox-1 and cox-2 respectively.According to docking analysis the most active ligand showed good binding interactions in terms of hydrogen bond and hydrophobic interactions with the residues of proteins amino acids.From the results, compound 2d indicated hydrogen bond interaction with Arg-374, Arg-376, Asp-375 and Phe-142 and compound 2e displayed hydrogen bond interaction with Arg-374, Arg-376, Arg-375 and Gly-225 with cox-1, whereas, the reference compounds (indomethacin) showed hydrogen bond with Arg-374, Arg-376 and electrostatic with Arg-374, Arg-376 as represented in Fig. 1 (a).While with cox-2, compound 2d showed hydrogen bond interaction with Lys-239, Ser-549, and His-228 and Lys-346, while compound 2e showed hydrogen bond interaction with Ser-549 and His-228 and Lys-239, whereas, the reference compounds (indomethacin) showed hydrogen bond with Lys-346, Lys-328, Phe-347 and Asn-546 as indicated in Fig. 1 (b).These results are further supported to the anti-inflammatory activity in vivo as illustrated in Table1 which revealed that the most active compounds (2d and 2e) showed edema reduction percent of 89.3%, 87.7%.So, all the compounds exhibited a good docking score which were agreed and supported to the in-vivo anti inflammatory activity of these compounds.

Table 3 :
Drug likeness score for the tested compounds compared with indomethacin and doxorubicin.Total molecular polar surface area; n.rotb:number of rotatable bonds; N A:number of atoms; Milog P: partition coefficient; n ON: number of hydrogen acceptor; nONH: number of hydrogen donor, NV: number of violation of five Lipinsky rules; MW: molecular weight and DLMS: Drug likness model score.

Table 4 :
Bioactivity score of the tested compounds compared with indomethacin and doxorubicin.

Table 5 :
Docking parameters in active site of the enzymes COX-1 and COX-2.