Synthesis , in-vitro , in-vivo evaluation and molecular docking of 2-( 3-( 2-( 1 , 3-dioxoisoindolin-2-yl ) acetamido )-4-oxo-2-substituted thiazolidin-5-yl ) acetic acid derivatives as anti-inflammatory agents

Department of Pharmaceutical Chemistry, Y.B. Chavan College of Pharmacy, Dr. Rafiq Zakaria Campus, Rauza Bagh, P.B. No. 33, Aurangabad-431001, Maharashtra, India. Department of Pharmacology, Y.B. Chavan College of Pharmacy, Dr. Rafiq Zakaria Campus, Rauza Bagh, P.B. No. 33, Aurangabad-431001, Maharashtra, India. Department of Pharmacology, Government College of Pharmacy, Aurangabad-431001, Maharashtra, India.


INTRODUCTION
Non steroidal anti-inflammatory drugs (NSAIDs) are one of the most commonly used therapeutically important agents for the treatment of pain, fever and inflammation (Madhukar et al., 2010).The usefulness of these agents is limited due to the side effects like gastric ulceration (Lombardino, 1985), gastro intestinal (GI) bleeding (Pilotto et al., 1997) and suppression of renal function (Pirson et al., 1986), and these side effects are related to their intrinsic mechanism of action.
From the literature survey, it was observed that both phthalimide and thiazolidinone derivatives are potentially useful as anti-inflammatory agents (Pawar and Chavan, 2012;Bhalgat et al., 2011;Bosquesi et al., 2011;Pophale and Deodhar, 2010;Machado et al., 2005;Alanazi et al., 2015;Vigorita et al., 2002;Ottana et al., 2005;Bhat and Kumar, 2008;Amin et al., 2010;Unsal et al., 2012;Hu et al., 2013;Singh et al., 2014;Thomas et al., 2013;Ahmed et al., 2013).Thiazolidinone scaffold is very versatile and is present in a number of clinically used drugs.They have varied applications as antimicrobial activity (Balzarini et al., 2007), anti-HIV activity (Monforte, 2001), antimalarial activity (Solomon et al., 2013), anti cancer activity (Gududuru et al., 2004) and antiarrhythmic activity (Jackson et al., 2007).Furthermore, it was observed that aryl acetic acid and α-methyl aryl acetic acid derivatives have high degree of potency as anti-inflammatory agents.In the present work, our objective was to design and synthesize a series of novel acetic acid derivatives containing thiazolidinone ring and couple this moiety with phthalimide ring (dioxoisoindoline) through the amide linkage so as to get the coupled derivatives with enhanced bioactivity.The compounds were designed such that -CH 2 COOH group is at C5 position of thiazolidinone ring, various alkyl/aryl/heteryl groups at C2 position of thiazolidinone ring.
We report here the synthesis, docking studies, anti inflammatory activity and ulcerogenic toxicity of these novel thiazolidinone-5-yl acetic acid analogues.The antiinflammatory activity was assessed, in silico, to know the binding and affinity towards the human serum albumin (HSA), in vivo by using carrageenan induced rat paw edema model, in vitro through protein denaturation inhibition assay using albumin.

EXPERIMENTALS
All chemicals were purchased from commercial suppliers and used without further purification.Nuclear magnetic resonance (NMR) spectra were recorded on a VARIAN MERCURY YH 300 Spectrometer at 300MHz.Chemical shifts are reported in parts per million (ppm), using TMS as an internal standard and CDCl 3 as a solvent. 13C spectra were recorded on AVANCE spectrometer at 300 MHz using CDCl 3 as a solvent.Infrared (IR) spectra were recorded for the compounds on JASCO Fourier transform infrared spectroscopy (FTIR) (PS 4000) using KBr pallet, mass spectra were recorded on GC-AccuTOF GC-high resolution, EI system.The homogeneity of the compounds was monitored by ascending thin layer chromatography (TLC) on silica gel-G (Merck) coated aluminum plates, visualized by iodine vapor.Elemental analyses (C, H, and N) were undertaken with a Shimadzu's FLASHEA112 analyzer and all analyses were consistent with theoretical values (within ± 0.4%) unless indicated.Digital plethysmometer (Ugo Basil 7140, Italy) was used for evaluation of anti-inflammatory activity.

Biological activity
In our present study we have performed in vitro biological activity by protein denaturation method and in vivo activity by carrageenan induced rat paw edema method, using diclofenac as standard.

In vitro anti-inflammatory activity
The synthesized compounds were screened for anti inflammatory activity by using inhibition of albumin denaturation technique.The standard drug and test compounds were dissolved 10 mg compound in DMF and diluted with phosphate buffer saline (pH 7.4) in such a way that concentration of DMF in all solutions was less than 2.5%.Test solution (1 ml, 100 μg/ml) was mixed with 1 ml of 1% albumin solution in phosphate buffer saline and incubated at 27 ± 1°C in an incubator for 15 min.Denaturation was induced by keeping the reaction mixture at 60± 1°C in a water bath for 10 min.After cooling, the turbidity was measured at 660 nm with UV visible spectrophotometer.Percentage of inhibition of denaturation was calculated from control where no drug was added.Each experiment was done in triplicate and average is taken.The diclofenac sodium was used as standard drug.The percentage of inhibition was calculated using the following formula: Where, Vt = mean absorption of test compound, Vc = mean absorption of control.

In vivo anti-inflammatory activity
The animals were procured under the CPCSEA number CPCSEA/IAEC/Pharm.Chem/19/2012-13/77 approved by Institutional Animal Ethics Committee (IAEC).Swiss Albino rats (150 to 200 g) were supplied by Wockhardt Ltd Aurangabad.The animals were housed in stainless steel cages, divided into groups of five animals each and deprived of food but not water 24 h before the experiment.The anti-inflammatory activity of the compounds under investigation was studied using carrageenan-induced rat paw oedema.
A suspension of the test compounds 5 (a-l) and standard drug diclofenac in carboxy methyl cellulose (CMC) solution (0.5% w/v in water) was administered intraperitoneally in a dose level of 10 mg/kg.Control animals were treated similarly with CMC solution (0.5% w/v in water).After 1 h, 0.1 ml of freshly prepared 1% carrageenan solution was injected into the sub plantar region of the left hind paw of rats according to the method of Winter et al. (1962).The volume was measured before and after carrageenan treatment at 1, 2, 3, 6 h with the help of digital plethysmometer (Ugo Basil 7140, Italy).Paw edema volume was compared with vehicle control group and percent reduction was calculated by formula: Paw enema = (Vc -Vt / Vc) × 100 Where Vc = paw volume of control group, Vt = paw volume of test group

Ulcerogenic toxicity study
Ulcerogenic toxicity study was performed with Wistar albino rats as per the protocol (Susan et al. 1993;Shoman et al., 2009).Adult Wistar albino rats were divided into different groups each containing five animals.Animals were deprived of food with no water 24 h before experiment.Ulcerogenic activity was evaluated after oral administration of suspension of standard drug and test compounds in carboxy methyl cellulose solution (0.5% w/v in water) in dose level of 100 mg/kg.
Control animals were treated similarly with carboxy methyl cellulose solution (0.5% w/v in water).After 5 h, rats were scarified by decapitation, the stomach were removed, collected, opened along the greater curvature, washed with water, and cleaned gently in saline solution.The stomach was stretched on a piece of foam core mat and the numbers of severity score were recorded.

Chemistry
The synthetic protocol employed for the synthesis of The title compounds 2-(3-(2-(1,3-dioxoisoindolin-2-yl) acetamido)-4-oxo-2-substituted thiazolidin-5-yl) acetic acid 5(a-l) were obtained by cyclo-condensation of Nsubstitutedbenzylidene/methylene-2-(1,3-dioxoisoindolin-2-yl)acetohydrazide with mercapto succinic acid in dimethylformamide (DMF) as solvent in presence of catalytic amount of anhydrous zinc chloride, under microwave irradiation for about 14 to 17 min (700 W) at 80°C.The reactions were carried out in microwave so as to reduce the longer reaction time of 6-8hrs refluxing in benzene, required in conventional synthesis of thiazolindinone derivatives to have better yields and to have neat and clean reactions.In the present synthesis of thiazolidinone derivatives the carcinogenic solvent benzene is replaced by DMF.The characterization data of synthesized derivatives is given in Table 1.

Biological activity
The synthesized derivatives 5(a-l) were evaluated for anti inflammatory activity using in vitro activity by protein denaturation method and in vivo activity was performed by using carageenan induced paw edema method.Diclofenac was used as the standard reference compound for both in vivo and in vitro evaluation.
The in vivo biological activity was performed according to Winter et al. (1962) and it has been observed that the new series of 2-(3-(2-(1,3-dioxoisoindolin-2-yl) acetamido)-4-oxo-2-substituted thiazolidin-5-yl) acetic acid derivatives exhibited the significant anti-inflammatory   action to all the compounds except 5b , 5k and 5l, when compared with control.Some of the synthesized derivatives have shown the enhanced anti-inflammatory activity than diclofenac as shown in Table 2.The most significant (**P < 0.01) anti-inflammatory activity is foundat 3 h and gradually reduces at subsequent hours.

No. Mean paw volume in ml ± SEM % inhibition 1 h 2 h 3 h 4 h 6 h 1 h 2 h 3 h 4 h 6 h
The compound with highest percent inhibition is 5a and is found to be most significant at 1 h.From the overall percent inhibition the compound 5c, 5f and 5j have shown to posses the enhanced and significant anti-inflammatory activity.Moreover, the other derivatives are also significant but less or equipotent with the standard drug, diclofenac.
The synthesized compound were subjected to in vitro anti inflammatory activity using albumin inhibition of albumin denaturation technique according to (Mizushima and Kobayashi. 1968), and with slight modification according to Bhalgat et al. 2011.Amongst all the synthesized compounds 5a, 5b and 5e have shown more inhibition as compared to diclofenac.It was observed in in-vivo activities, that lower aliphatic groups such as-CH 3 , -C 2 H 5 attached to C2 of thiazolidinone ring show the highest anti-inflammatory activity.All the compounds have resulted in decrease in rat paw edema and hence showed excellent anti-inflammatory activity.Compound 5c in which the phenyl ring is without any substituent attached at C2 of thiazolidinone ring exhibited significant and enhanced anti-inflammatory activity.Other derivatives possessing 4-nitro phenyl, 4-fluro phenyl, 4chloro phenyl, 3-hydroxy phenyl, 4-hydroxy phenyl group on C2 of thiazolidinone ring ie.5i, 5h, 5g, 5e, 5d, respectively are less active than diclofenac but show significant activity.The bulky derivatives such as indole and thiophene rings at C2 of thiazolidinone ring like 5k and 5l have exhibited very less anti-inflammatory activity when compared to the standard drug, diclofenac.The anti-inflammatory activity data is presented in Table 3.The ulcerogenic toxicity was performed for selected compounds having shown better anti-inflammatory activity, such as, compound 5a, 5b, 5c, 5f & 5j.As shown in Table 4, it was observed that all the compounds exhibited lesser ulcerogenic index than diclofenac.Thus the synthesized derivatives have shown minimum toxicity effects.

Docking methodology
Molecular docking studies were performed by using Glide, V 5.5 (Schrödinger.LLC, New York, NY 2009).The coordinates for HSA were taken from RCSB Protein Data Bank (PDB Id. 2BXQ) (Ghuman et al., 2005) and prepared for docking using protein preparation wizard.Water molecules in the structure were removed.The bond order and formal charges were added for hetero groups and the hydrogens were added to all atoms in the structure.Side chains that were not close to the binding cavity were removed.After preparation, the structures were refined to optimize the hydrogen bond network using OPLs_2005 force field which helps in the orientation of side chain hydroxyl group.The minimization was terminated when the energy converged to rootmean-square deviation (RMSD) reached a maximum cutoff of 0.30A°.Grids were then defined around refined structure by centering on ligand using default box size.The standard precision (SP) docking mode for compounds, optimized earlier by Ligprep, was performed on generated grid of protein structure.

Docking results
While performing docking study the hydrogen bonding with ARG114 was selected as constraints for the specificity of binding of compounds in activity site of enzyme as reported in literature and as detected in Ligplot (Pawar et al., 2010).The docking pose of synthesized compounds showing higher inhibition compared with that of standard.In present docking study both the standard drug Indomethacin shows the binding with Arg114 (Figure 2) and diclofenac shows the binding with Arg 117, as found in Figure 3.The compound 5a and 5b shows binding with ARG114 and ARG117, as shown in Figure 4 and Figure 5, respectively.Moreover the compound 5a showed the highest G score of -7.731, it was observed in the docking pose of 5a that -C=O of carboxyl group formed hydrogen bonding with --NH group of Arg186 and -NH of amide group formed hydrogen bonding with -C=O of Arg114.This indicates the importance of carboxyl group for anti-inflammatory activity and also confirms the importance of amide group in the structure of the synthesized derivatives.The docking results have shown that all the synthesized compounds have better anti inflammatory effect compared to Indomethacin and diclofenac.Molecular modeling helps to realize the mechanism of their actions, which could be their interactions with the same residues of ARG114 and ARG117, as shown by indomethacin and diclofenac.

Conclusion
The present study describes eco-friendly synthesis of twelve final derivatives 5(a-l) in Milestone's Microsynth microwave.All the compounds were obtained in good yield and in shorter reaction times, i.e. 14 to 17 min.The synthesized derivatives were evaluated for in vitro   and in vivo anti-inflammatory activity, using diclofenac as a reference standard.The selected compounds were studied for ulcerogenic toxicity and have shown good gastrointestinal safety profile.The compounds were also subjected to in vitro analysis by using diclofenac as standard.The compounds 5a, 5b and 5e have proved to  contains thiazolidinone ring which is also present in antidiabetic drugs such as rosiglitazone and pioglitazone.

Figure 2 .
Figure 2. Docking pose of compound indomethacin at active binding site of enzyme.Visualization of hydrogen bonding of Indomethacin with Arg114 and Arg186.

Figure 3 .
Figure 3. Docking pose of compound diclofenac at active binding site of enzyme.Visualization of hydrogen bonding of diclofenac with Arg117 (hydrogen bonding with amino acid is shown in pink dotted lines).

Figure 4 .
Figure 4. Docking pose of compound (5a) at active binding site of enzyme.Visualization of hydrogen bonding of (5a) with Arg114, Arg117 and Arg186.Hydrogen bonding with amino acid is shown in pink dotted lines.

Figure 5 .
Figure 5. Docking pose of compound (5b) at active binding site of enzyme.Visualization of hydrogen bonding of (5b) with Arg114, Arg117.Hydrogen bonding with amino acid is shown in pink dotted lines.
*Melting points are uncorrected