Synthesis, Anticonvulsant Activity and In silco Studies of Schiff Bases of 2-Aminothiophenes via Guanidine- Catalyzed Gewald Reaction

Purpose: To synthesize Schiff bases of 2-aminothiophenes and evaluate their anticonvulsant activity and in silco properties Methods: 2-Amino-N-o-tolyl-5,6-dihydro-4H-cylcopenta[b]thiophene-3-carboxamide was synthesized using 1,1,3,3-tetramethylguanidine lactate as a basic catalyst and by microwave irradiation. 2substitued-o-tolyl-5,6-dihyro-4H-cylcopenta[b]thiophene-3-carboxamide was prepared by reacting with different substituted aromatic aldehydes. The synthesized compounds were characterized by Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (H NMR) and mass spectrometry (MS) while their anticonvulsant activity was screened against maximum electroshockinduced seizure (MES), and pentylenetetrazole-induced seizure (PTZ) against phenytoin and diazepam as reference standards. Molecular docking (in silico) studies were performed using 4-aminobutyrateaminotransferase in order to predict possible protein-ligand interactions. Results: Among the 21 synthesized compounds, 2b, 2d, 2f, 2k, 2m, 2n and 2o showed good to moderate activity against MES and PTZ-induced convulsions. Compounds 2b, 2d, 2f, 2k and 2m exhibited lower activity against PTZ than against MES model while compounds 2n and 2o afforded greater protection against PTZ than against MES model. In silico results also revealed maximum binding affinity to GABA-AT protein which was higher than other compounds Conclusion: The synthesized compounds showed potent anticonvulsant activity. Molecular docking results should give an insight into how further modification of lead compound can be carried out for higher inhibitory activity.


INTRODUCTION
Thiophene scaffold have a variety of biological activities such as antitubercular [1], antioxidant [2], anti-HIV-PR inhibitors [3], antimicrobial [4], multitargeted kinase inhibition [5], A 1 adenosine receptor allosteric enhancement [6].Of Several approaches for the synthesis of 2aminothiophenes have been reported including the use of microwave assisted synthesis [7], synthesis from thiomorpholides [8], coupling reaction [9], solid support [10], base-induced inter-and intra-molecular C-S and C-C bond formation [11] and ultrasonication [12].Ionic liquids (ILs) have attracted attention for green organic synthesis because of their unique physical and chemical properties such as nonvolatility, non-flammability, thermal stability, recyclability and controlled miscibility.Synthesis using ionic liquid has great advantages including easy workup, milder reaction and high yields.Molecular docking studies have been performed on synthesized compounds with gammaaminobutryic acid aminotransferase (GABA-AT) in order to identify possible binding conformations of the ligand.[13].The aim of the present research was to study some Schiff bases for anticonvulsant activity and ligand docking with gamma-aminobutryic acid aminotransferase in order to identify possible binding conformations.By performing molecular docking studies, one can identify possible binding conformations of ligands, and the data obtained would be very helpful to achieve further modification of lead for greater inhibitory activity.

EXPERIMENTAL
Melting point was determined using an electrochemical thermal apparatus (IA 9100 Digital melting point apparatus) and were uncorrected.FT-IR spectra were recorded on a Perkin Elmer FT-IR spectrometer. 1 H NMR spectra were obtained with Bruker Spectrometer (400 MHz) in CDCl 3 ( deuterated chloroform) and DMSO (dimethyl sulfoxide) using tetramethylsilane (TMS) as an internal standard.Mass spectra were obtained with LC-MSD Trap-SL 2010 A Schimadzu.All the compounds were synthesized according to the scheme in Figure 1.

Synthesis of 2-cyano-N-o-tolylacetamide(1):
A mixture of o-toluidine (0.01 mol) and ethylcyanoacetate (0.655 mol) was heated at 180 0 C in a microwave (Raga Scientific Microwave system model RG31L) at 2450 MHZ for 5 min.The reaction mixture was left at room temperature overnight.The solid obtained was separated, washed with ethanol, dried and recrystallized from acetone:water mixture.

Synthesis of 2-amino-N-o-tolyl-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxamide under microwave irradiation(2):
A mixture of cyclopentanone (0.04 mol), 2-cyano-N-otolylacetamide (0.04 mol), sulphur(0.04mol), ionic liquid (1.5 g), ethanol (15 ml) were placed in a volumetric flask and stirred for 5 min.The reaction mixture was placed in a microwave at 100 W for 3 min.The progress of the reaction was monitored by thin layer chromatography (TLC) Water (10 ml) was added during which reddish brown crystals were separated.The preciptated product was filtered under suction.Ionic liquid was recovered by removing water under reduced pressure stored in a desicator for its reuse.The reaction mixture was mixed, allowed to adsorb on acidic alumina, dried and kept inside the alumina bath and irradtiated at 750 W for 30 s.The reaction mixture was cooled, the product extracted with ethyl acetate, concentrated and cooled.The solid product was separated filtered, washed thoroughly with water and recrystallized from dimethyl formaamide.

Evaluation of anticonvulsant activity
Male albino Swiss mice, weighing 20 -25 g, were used to study the effect of the synthesized compounds on MES-and PTZ-induced seizures.Female animals were excluded because of the fact that estrous cycle could influence their activity threshold.They were procured from National Institution of Nutrition; Hyderabad and had free access to balanced food and water ad libitium Prior to experimentation, the animals were allowed a week time to acclimatize to laboratory conditions.However, food, but not water, was withdrawn 8 h before and during the experiments.Animal experiments were performed in accordance with the institutional guidelines for animal experimentation [14].Institutional animal ethical permission was obtained from Malla Reddy College of Pharmacy (ref no.1217/a/08/CPCSEA) which is affiliated to Osmania University, prior to the animal studies.All the protocols and the experiments were conducted in strict compliance according to ethical principles and guidelines the animals received due care according to the criteria outlined in the "Guide for the Care and Supervision of Experiments on Animals" prepared of the National Academy of Sciences and published by National Institutes of Health, USA.

Maximal electroshock-induced seizure test
Seizure was induced by maximal electroshock in albino mice (n=6)with the aid of an electroconvulsiometer (EC 01,Orchid Scientifics) by passing a current of 45 mA for 0.2 s using ear clip electrodes.The duration of different phases of seizures was noted [15].

Pentylenetetrazole-induced seizure test
Albino mice (n = 6) were treated with test prior to subcutaneous injection of PTZ (80 mg/kg), The mice were observed for clonic and tonic convulsions and 24 hr mortality.The mice were observed for onset of convulsion up to 30 min after PTZ administration.

In silco studies
A set of 21 different ligands were drawn using Chemsketch, (11.0 ACD labs) while geometrical optimization was carried out with LigPrep.2.5 (Schrodinger Suite 2011).Using this tool, a single low energy 3D structure was obtained for each ligand and different conformers obtained during ionization of the ligands using EPIK module which generates ionization states in the pH range of 7 ± 2. The molecular properties of the minimized ligands were studied using QIKPROP module 2.5 Schrodinger suite 2011 which predict absorption, distribution, metabolism and excretion (ADME, Quiprop version 3.4, Schrodinger Suite 2011) [16].
Ligand protein interaction of 2-aminosubstituted thiophenes with GABA-AT was studied by docking GLIDE-XP.The protein structure was retrieved from the protein data bank server (PDB entry 1OHV, (http://www.rcsb.org/pdb/home/home.do).The structure of the protein was corrected by adding hydrogen and removing water moieties with force field OPLS-2005, i.e., optimized potentials for liquid simulations.The active site was identified by SITEMAP tool.Receptor grid generation was carried out using glide; the ligands were docked by employing XP mode and they were docked flexibly.Best pose was selected based on E-model energy and docking score from glide (glide score) was entirely based on Chem score.It also includes a steric-clash term, and adds polar terms featured by Schrödinger to correct electrostatic mismatches [17,18].

Statistical analysis
The data are expressed as mean ± standard error of mean (SEM).One-way analysis of variance (ANOVA) followed by Tukey multiple comparison test was carried out with Graph Pad Prism software (version 3.0).Differences between means were considered significant at p < 0.01.

Compound
2b with 4-hydroxy-3-methoxy substitution showed good activity compared to control and standard, while compounds 2m, 2n and 2o with heterocyclic ring system such as furan, indole and isatin, showed moderate activity against maximum electroshock-induced seizure (MES).In the case of pentylene tetrazole-induced seizure, compounds 2b, 2e, 2f, 2o and 2q with substitutions 4-Hydroxy-3methoxybenzylideneamino,3-nitrobenzylideneamino, 4-nitrobenzylideneamino, -(2-oxoindolin-3-ylideneamino, 1-methylpyrrolidin-2-ylideneamino substitution at 2-amino-N-o-tolyl-5,6dihydro-4H-Cyclopenta[b]thiophene-3-carboxamide produced greater reduction in the duration of convulsion than control but lower than the reference standard.All the synthesized compounds showed lower protection against PTZ-induced seizure than diazepam (reference standard).Flexible docking of all the synthesized compounds was carried out at the active site of GABA-AT.The results reveal that eleven compounds are capable of binding to the active site of the receptor amino acid, Tyr 348; this predicts favorable interaction with protein residues that exhibit hydrophobic and hydrophilic interactions.Protein ligand interaction results indicate that protein residue, such as TYR 348, is the common amino acid that interacted with most of the ligands and other residues such as GLY 104, LEU 103, THR 132, PRO 344, and ILE 350, and that they also participate in the formation of hydrogen bonds.Compounds 2b, 2e, 2f, 2o and 2q with 4-hydroxy-3-methoxy substitution showed the highest docked score of 7.01, and were more tightly bound to the active site of GABA-AT than other compounds.The phenyl ring and carbonyl moiety of amide in compound 2b displayed good interactions at the active site.The best ligand protein interactions are shown in Figure 2.These observations provide a good basis for the estimation of the inhibitor activity of these compounds.Compounds 2c, 2g, 2m, 2n and 2s with 4-methylbenzylideneamino , 4isopropylbenzylideneamino, furan-2-ylmethyleneamino, 1H-indol-3-yl-methyleneamino, 4oxopentan-2-ylideneamino at 2-amino-N-o-tolyl-5,6-dihydro-4H-Cyclopenta[b]thiophene-3-carboxamide did not demonstrate any hydrogen bond interactions may be responsible for the low docking score.

DISCUSSION
The compounds synthesized by both microwave and conventional method.2-Amino-N-o-tolyl-5,6dihydro-4H-cyclopenta[b]thiophene-3-carboxamide was prepared by the reaction of 2-cyano-No-tolylacetamide with cyclohexanone, sulphur and ionic liquid.The IR spectrum of 2-cyano-N-otolylacetamide showed the presence of CN peak at 2260 cm -1 (C-N), disappearance of CN peak and formation of NH 2 peak at 3429 cm -1 , and thus confirmed the formation of 2aminothiophenes when basic ionic liquids are used for synthesis.Ionic liquids are non-volatile, recylable, non-explosive, thermally robust.2-Amino-N-tolyl-5,6-dihydro-4H-cyclopenta(b)   Inhibition of MES test indicates activity against generalized tonic-clonic (grand mal) and cortical focal seizures [19].It permits evaluation of the capacity of a substance to prevent seizure spread through neural tissue.MES test is not suitable for the testing of drugs acting on Na + channels; the model is suitable for the evaluation of the majority of standard and newly developed anticonvulsant drugs, despite the fact that these drugs interact with other drug targets.In MESinduced convulsion model, reference drug, phenytoin (25 mg/kg) prevented the important tonic extensor phase of the tonic-clonc seizures, offered 100 % protection and produced tonic flexion convulsions, whereas compounds 2b, 2e, 2f, 2m, 2n, 2o, 2q altered extensor phase duration and onset of clonus phase; probably due to the inhibition of high frequency action potential firing but the compounds did not totally abolish the various phases at 30mg/kg dose.PTZ is a most frequently used substance as well as an acute experimental model in the preliminary screening to test potential anticonvulsant drugs.PTZ is a selective blocker of the chloride ionophore complexation to the GABA A receptor, and after repeated or singledose administration, leads to a decrease in GABAergic function and to the stimulation and modification of density or sensitivity of different glutamate receptor subtypes in many brain regions [19].The precise mechanism of the synthesized compounds is not clear, but in silico studies revealed that the action of the test substances was on the GABA mediated chloride channel.

CONCLUSION
A simple rapid, efficient synthesis of 2aminothiophenes by introduction of amide linkage at 3 rd position has been achieved by onepot synthesis utilizing cost-effective guanidinebased ionic liquid.Recent advances in the synthesis of 2-aminothiophenes reveal imidazoles-based ILs is replaced by guanidinebased ionic liquid.Isolation is simple, without the need for chromatographic purification, and there is no loss of catalytic activity.The anticonvulsant activity of the synthesized compounds is moderate against screening models.The results of in silco studies reveal significant role of topological parameters in inhibiting GABA-AT while docking results reveal significant role of phenyl ring and terminal carboxamide in drugreceptor interaction.

Figure 2 :
Figure 2: Interaction of compound 2b with the receptor.Docking score of compound 2b was -7.01 interacting with the protein residues Ile 350 and Tyr 348

Table 4 :
Docking1which means that the compound is derivatized.thiophene-3-carboxamide was treated with different aldehydes to form Schiff bases, and the IR spectra showed the dissappearance of NH 2 .
'-' indicates compound did not interacting with protein residues that had not hydrogen bond peak and formation of N=CH at 1569 Cm -