Design, Synthesis of Some Novel Thiazolidin-4-one Derivatives Bearing Benzimidazole Nucleus and Biological Evaluation of their Possible in vitro Antiinflammatory as Cyclooxygenase Inhibitors and Antioxidant Activity

Some novel 1,3,4-oxdiazole derivatives bearing benz imidazole nucleus were synthesized and their invitro anti-inflammatory and antioxidant activity by inhi bit on of protein denaturation screening, 2, 2-Diph enyl-1Picryl Hydrazide screening methods respectively. Th e compound 7.XX and 7.XXIX were found to be highly active in low concentration and compounds 7.VIII, 7.IX, 7.X, 7.XIX, 7.XXII, 7. XXIV and 7.XXV were found to be moderately active at higher concentration as compar ed to ascorbic acid in 2, 2-diphenyl-1-picryl hydra zide method. In-vitro anti-inflammatory by inhibition of protein denatur ation method the compounds 7.VIII, 7.XX , 7.XXII and 7.XXIX were found to be highly active in low concentratio n and compounds 7.IX, 7.X, 7.XIX, 7. XXIV, 7.XXV and 7.XXXIII were found to be moderately active at higher conce ntration as compared to diclofenac sodium.


INTRODUCTION
The structural and therapeutic diversity coupled with commercial viability of small molecules has fascinated organic and medicinal chemists.There has been considerable interest in the chemistry of thiazolidin-4-one ring systems, which is a core structure in various synthetic pharmaceuticals displaying a broad spectrum of biological activity 1 such as anti-mycobacterial 2 , anti-fungal 3 , anti-cancers 4 , anti-convulsant 5 , antiinflammatory and analgesic 6 activities.Therefore, a general, simple and efficient method for rapid synthesis of thiazolidine-4-ones would be greatly advantageous and warrants further investigations in drug discovery.Consequently, many different protocols have been developed that allow the synthesis of thiazolidin-4-one skeletons.Their long-term clinical employment is associated with significant side effects and the steady use determines the onset of gastrointestinal lesions, bleeding and nephrotoxicity 7,8 .When the coxibs were marketed, evidence for a new side effect appeared and rofecoxibwas banned in 2004.Subsequently, some of the other coxibs have been voluntarily withdrawn from the market 9 .Some of the studies Asian Journal of Chemistry; Vol. 26, No. 24 (2014), 0000-0000 have suggested that rofecoxib's adverse cardiovascular events may not be a class effect but rather an intrinsic chemical property related to its metabolism 10 .Literature survey make known that the thiazolidinone moiety as it was an important scaffold for COX-2 inhibition [11][12][13] .In the present study whatever thiazolidinone-4-one derivatives was synthesized which may not have the irritation to the gastric mucosa also which may not cause additional damage to the gastrointestinal tract.Thiazolidin-4-ones are an interesting backbone unit in medicinal chemistry and responsible for numerous pharmacological properties and biological activity [14][15][16][17][18][19] which gives the considerable research interest in this area has been done towards the synthesis of thiazolidinone unit.In addition, several interesting investigations have been made through thiazolidin-4-ones based heterocyclic compounds and exhibited numerous biological properties such as antibacterial, antifungal, antitumor, antiarrhythmic, antithrombic, calcium antagonist, hypotensive and neuroleptic activities.An essential component of the search for new leads in drug designing program is the synthesis of molecules, which are novel still resemble known biologically active molecule by virtue of the

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presence of some pharmacophoric groups.Certain small heterocyclic molecules act as highly functionalized scaffolds and are known pharmacophores of a number of biologically active and useful molecules.Benzimidazole derivatives are well known for their antiinflammatory activity and more recently have been discovered to have anticancer effect 20,21 .Therefore, in the present paper we planned to incorporate the benzimidazole moiety with thiazolidin-4-ones to have better antioxidant and antiinflammatory activity.

L L E Y P R
O O F EXPERIMENTAL Melting points were determined by open capillary method and were uncorrected.The IR spectra (in KBr) were recorded on a Shimadzu IR Affinity-1 spectrophotometer. 1 H NMR and 13 CNMR spectra were recorded on a Perkin-Elmer EM 300 MHz spectrometer using TMS as internal standard.The mass spectra were recorded on a JEOL JMS-D 300 spectrometer operating at 70 eV.Purity of the compounds was checked by TLC silica coated plates obtained from Merck.

9(I-XXXI)]
A mixture of imine intermediate [4(I-XXXI)] (0.01 mole) and thioglycollic acid (0.01 mole) in 15 mL dioxane was refluxed at 115 °C for 24 h.The reaction mixture was triturated with 10 % sodium bicarbonate solution.The neutral solution was poured into crushed ice.The solid formed was collected by filtration, washed with water and dried.The product was crystallized from methanol to give 9(I-XXXI).The purity of the compounds was established by single spot on TLC plates.The physico-chemical characteristic data is given in Table-1 Biological evaluation: Antioxidant and free radical scavenging activity assays.
DPPH assay: This experimental procedure was adapted from 22 .In an ethanol solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, test compounds at different concentrations were added.The reaction mixtures were shaken vigorously and then kept in the dark for 0.5 h.The absorbance of the resulting solutions was measured in 1 cm cuvettes, using a UV/visible spectrophotometer at 226 nm against blank without DPPH.Decreasing of DPPH solution absorbance indicates an increase of DPPH radical scavenging activity.This activity is given as % DPPH radical scavenging that is calculated in the equation: Absorbance of control Absorbance of test Inhibition (%) 100 Absorbance of control The DPPH solution without sample solution was used as control.All tests were run in triplicate and averaged.Ascorbic acid was used as positive control.

Cyclooxygenase (COX) inhibitor screening assay:
The inhibitory activities against COX-1 and COX-2 were determined using a colourimetric COX (ovine) inhibitor assay kit (Cayman Chemical Co., Cat.No. 760111) according to the manufacturer's protocol 24,25 .The inhibitory activities of the compounds were measured by monitoring the production of oxidized N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) at 405 nm followed by incubation of either ovine COX-1 or COX-2 with arachidonic acid.The enzymes were preincubated for 5 min at 25 °C with the test compounds prior to addition of arachidonic acid (final concentration 1.1 mM) and TMPD and incubation for 5 min at 25 °C.The COX-inhibiting activity was calculated according to the equation.
COX inhibiting activity (%) = [1-(A1-A2)/A0] × 100 where A0 was the absorbance of the control (without the test compound), A1 was the absorbance in the presence of the test compound and A2 was the absorbance sample blank (without TMPD).The IC50 is the concentration of tested compounds reducing 50 % of ovine COX-1 or COX-2 under the given experimental conditions and was calculated using a calibration curve with different concentrations of samples.
All the newly synthesized compounds 9(I-XXXI) were screening for inhibitory effect on ovine COX-1 activity in vitro.It was observed that among the thiazolidinone derivatives fluro, chloro and methoxy substitution is more favourable.
All the newly synthesized compounds 9(I-XXXI) were screening for inhibitory effect on ovine COX-2 activity in vitro.It was observed that among the thiazolidinone derivatives fluro, chloro and methoxy substitution is more favourable.

Introduction
The structural and therapeutic diversity coupled with commercial capability of small molecules has enthralled organic and medicinal chemists.There has been significant interest in the chemistry of oxadiazole ring systems, which is a core structure in various synthetic pharmaceuticals displaying a broad spectrum of biological activity.4][15] Of the various human diseases, cancer has proven to be one of the most intractable diseases to which humans are subjected, and as yet no practical and generally effective drugs or methods of control are available.Therefore, identification of novel potent, selective and less toxic anti-cancer agent's remains one of the most pressing health problems. 16Targeting tubulin in rapidly dividing tumor cells has been a well validated strategy for cancer therapy. 17,18 enzimidazole derivatives are well known for their anti-inflammatory activity and more recently have been discovered to have anticancer effect. 19,20 herefore, in the present research it was planned to incorporate the Benzimidazole moiety with 1,3,4-oxadiazole to have better antioxidant and anti-inflammatory activity.

Chemistry
Melting points were determined by open capillary method and were uncorrected.The IR spectra (in KBr) were recorded on a Shimadzu IR Affinity-1 spectrophotometer. 1 H NMR and 13 CNMR spectra were recorded on a Perkin-Elmer EM 300 MHz spectrometer using TMS as internal standard.The mass spectra were recorded on a JEOL JMS-D 300 spectrometer operating at 70 eV.Purity of the compounds was checked by TLC silica coated plates obtained from Merck.
To a solution of imine intermediate 5 (V-XXXIII) (0.01 mole) in ethanol (15 ml) and Chloramines-T (0.01 mole) was added.The reaction mixture was exposed to microwave at 300 W intermittently at 30 sec intervals for specified time.After complete conversion as indicated by TLC, the reaction mixture was cooled and digested with cold water.

DPPH assay
This experimental procedure was adapted from. 21In an ethanol solution of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical, test compounds at different concentrations were added.The reaction mixtures were shaken vigorously and then kept in the dark for 30 min.The absorbance of the resulting solutions was measured in 1 cm cuvettes, using a UV/VIS spectrophotometer at 351nm against blank without DPPH.Decreasing of DPPH solution absorbance indicates an increase of DPPH radical scavenging activity.This activity is given as % DPPH radical scavenging that is calculated in the equation: The DPPH solution without sample solution was used as control.All tests were run in triplicate and averaged.Ascorbic acid was used as positive control in (Table 2 and Figure 2).

Inhibition of protein denaturation method
Test solution (0.5 mL) consists of 0.45 mL of BSA (5% w/v aqueous solution) and 0.05 mL of different concentration of test solutions (50, 100, 150, 200 µg/mL).Test control solution (0.5 mL) consists of 0.45 mL of BSA (5% w/v aqueous solution) and 0.05 mL of distilled water.Product control solution (0.5 mL) consists of 0.45 mL of distilled water and 0.05 mL of different concentration of test solutions (50, 100, 150, 200 µg/mL).Standard solution (0.5 mL) consists of 0.45 mL of BSA (5%w/v aqueous solution) and 0.05 mL different concentration of Diclofenac Sodium's (50, 100, 150, 200 µg/ mL).All the above solutions were adjusted to pH 6.3 using 1N hydrochloric acid.The samples were incubated at 37 0 C for 20 min and the temperature was increased to keep the samples at 57 0 C for 3 min.After cooling, 2.5 mL of phosphate buffer saline was added to the above solutions.The absorbance was measured using UV Visible spectrophotometer at 417 nm. 22The percentage inhibition of protein denaturation was calculated as, The results were compared with Diclofenac Sodium in (Table 3 and Figure 3).

Conflict of interest statement
We declare that we have no conflict of interest

INTRODUCTION
The structural and therapeutic diversity coupled with commercial capability of small molecules has enthralled organic and medicinal chemists.There has been significant interest in the chemistry of triazole ring systems, which is a core structure in various synthetic pharmaceuticals displaying a broad spectrum of biological activity.In the past few years, research for new non-steroidal anti-inflammatory agents has been reported that many compounds having a 1,2,4-triazole derivatives are known for their anti-inflammatory activity [1-6].
From the literature survey it was found that, depending on the type of substituent, the derivatives of 1, 2, 4-triazole has a high potential for biological activity, possessing a wide range of antimicrobial [7-10] and antitumor [11-14] properties.The other triazole derivatives show anti-inflammatory [15], antihypertensive [16], anticonvulsant, antiviral [17] and analgesic activities [18].Also a number of benzimidazole derivatives were identified as potent analgesic and anti-inflammatory compounds [19,20].
Therefore, in the present paper it was planned to incorporate the benzimidazole moiety with 1, 2, 4triazole to have better antioxidant and anti-inflammatory activity.

Chemistry
Melting points were determined by open capillary method and were uncorrected.The IR spectra (in KBr) were recorded on a Shimadzu IR Affinity-1 spectrophotometer. 1 H NMR and 13 CNMR spectra were recorded on a Perkin-Elmer EM 300 MHz spectrometer using TMS as internal standard.The mass spectra were recorded on a JEOL JMS-D 300 spectrometer operating at 70 eV.Purity of the compounds was checked by TLC silica coated plates obtained from Merck.All characterization data are mention in Table 1.
The mother liquor on neutralization with sodium bi-carbonate solution gave a solid, which was filtered and recrystallized from ethanol.

Radical scavenging assays by the DPPH
This experimental procedure was adapted from [21] .In an ethanol solution of 2, 2-diphenyl-1picrylhydrazyl (DPPH) radical, test compounds at different concentrations were added.The reaction mixtures were shaken vigorously and then kept in the dark for 30 min.The absorbance of the resulting solutions was measured in 1 cm cuvettes, using a UV/VIS spectrophotometer at 328nmagainst blank without DPPH.Decreasing of DPPH solution absorbance indicates an increase of DPPH radical scavenging activity.This activity is given as % DPPH radical scavenging that is calculated in the equation:

Percentage inhibition =
Absorbance of control -Absorbance of test Absorbance of control X 100 .
The DPPH solution without sample solution was used as control.All tests were run in triplicate and averaged.Ascorbic acid was used as positive control in Table 2 and Figure 2.

In vitro anti-inflammatory activity by inhibition of protein denaturation method
Test solution (0.5 mL) consists of 0.45 mL of BSA (5% w/v aqueous solution) and 0.05 mL of different concentration of test solutions (50, 100, 150, 200 µg/mL).Test control solution (0.5 mL) consists of 0.45 mL of BSA (5% w/v aqueous solution) and 0.05 mL of distilled water.Product control solution (0.5 mL) consists of 0.45 mL of distilled water and 0.05 mL of different concentration of test solutions (50, 100, 150, 200 µg/mL).Standard solution (0.5 mL) consists of 0.45 mL of BSA (5%w/v aqueous solution) and 0.05 mL different concentration of Diclofenac Sodium's (50, 100, 150, 200 µg/ mL).All the above solutions were adjusted to pH 6.3 using 1N hydrochloric acid.The samples were incubated at 37 0 C for 20 min and the temperature was increased to keep the samples at 57 0 C for 3 min.After cooling, 2.5 mL of phosphate buffer saline was added to the above solutions.The absorbance was measured using UV Visible spectrophotometer at 416 nm [22] .The percentage inhibition of protein denaturation was calculated as, The results were compared with Diclofenac Sodium in Table 3 and Figure 3.

2 Figure 1 :
Figure 1: Scheme I Synthetic route for the preparation of the compounds.

Figure 3 :
Figure 3: The IC 50 values of compounds (6.I to 7.XXXIII) and diclofenac sodium (standard) in inhibition of protein denaturation screening of In-Vitro Anti-Inflammatory activity.The results are (mean±SEM) of three experiments, performed in triplicate.

Figure 2 :
Figure 2: Comparison of IC 50 values of compounds and ascorbic acid The IC 50 values of compounds and ascorbic acid (standard) in 2, 2-Diphenyl-1-Picryl Hydrazide screening of antioxidant activity, the results are (mean±SEM) of three experiments, performed in triplicate.

Figure 3 :
Figure 3: Comparison of IC 50 values of compounds and diclofenac sodium The IC 50 values of compounds and diclofenac sodium (standard) in inhibition of protein denaturation screening of In Vitro Anti-Inflammatory activity, the results are (mean±SEM) of three experiments, performed in triplicate.

TABLE - 2
IC 50 ± SEM (STANDARD ERROR OF MEAN) VALUES OF ASCORBIC ACID AND COMPOUNDS 9(I-XXXI)

TABLE - 1
IC 50 ± SEM (STANDARD ERROR OF MEAN) VALUES OF DICLOFENAC SODIUM AND COMPOUNDS 9(I-XXXI)
a Other compounds were considered to be inactive (IC 50 >100 µg/mL) in both COX-1 and COX-2 b Each value is expressed as the mean of triplicate experiments c

Table 1 :
Characterization Data Scheme I

Table 2 :
The IC 50 ±SEM (standard error of mean) Values of Ascorbic Acid and Compounds (6.I to 7.XXXIII)

Table 3 :
The IC 50 ±SEM (standard error of mean) Values of Diclofenac Sodium and Compounds (6.I to 7.XXXIII)