Synthesis of Novel Thiazolidin-4-one Derivatives of 7-Hyroxy-4-methyl Coumarinyl Acetic Hydrazide and its Activity on Antioxidant and In Vitro Cytoxic against DLA Cells

Copyright: © 2018 Roshna S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Synthesis of Novel Thiazolidin-4-one Derivatives of 7-Hyroxy-4-methyl Coumarinyl Acetic Hydrazide and its Activity on Antioxidant and In Vitro Cytoxic against DLA Cells


Introduction
Coumarin are naturally occurring compounds present in many members of plant kingdom, containing lactone ring having 1 benzopyran-2-one ring system and possess a wide variety of biological activity [1,2]. Similarly thiazolidinone are saturated form of thiazole, that have an atom of sulfur at position 1, an atom of nitrogen at position 3 and a carbonyl group at 2, 4 or 5 [3]. Also it is well documented that coumarin with pyrazolin-5-ones, 4-thiazolidinones and 1,3,4-oxadiazoles display prounced antioxidant and antineoplastic activity [4,5]. Keeping in view of the biological importance of coumarin derivatives and thiazolidin-4 one derivatives, novel hybrid molecules of thiazolidin-4-one derivatives incorporated with coumarin nucleus and having various arylidene substituents at 5 th position of thiazolidin-4-one nucleus were designed. Then the designed hybrid molecules were synthesized and were screened for their antioxidant and in vitro cytotoxicity studies against DLA cells.

Experimental
Melting point was determined in open capillary tube and was uncorrected. IR spectra were recorded on Shimadzu FT-IR at Chemistry Lab, College of Women, Trivandrum 695036, Mass spectra were recorded on Brukerultraflextreme MALDI system at Rajiv Gandhi Centre for Biotechnology, Trivandrum and 1 H NMR spectra was recorded on Brucker Ultra shield DPX 400 at Sophisticated Test and Instrumentation Centre, Cochin.
The purity of all compounds was established by single spot on the TLC plates. The solvent system used was methanol: toluene (7:3).
The purity of all compounds was established by single spot on the TLC plates. The solvent system used was methanol: toluene (7:3).
The newly synthesized compounds were characterized [13] on the basis of physical data (Table 1).

Abstract
The main aim of the present work was to synthesis a series of novel thiazolidin-4-one derivatives of 7-hydroxy-4-methyl coumarinyl acetic hydrazide. The synthesis majorly involves six steps, where the synthesis of coumarin molecules with thiazolidin-4-one was reported with its activity study. Here ten novel derivatives of compounds were synthesized and evaluated for their antioxidant and in vitro cytotoxicity against DLA cells. Some of the compounds showed promising activity for the antioxidant and in vitro cytotoxic activity against DLA and thus these compounds represent a new class of promising lead compounds. the percentage of scavenging activity was calculated using the formula shown below. Ascorbic acid was used as the reference compound. All tests and analyses were undertaken on two duplicate and the results were averaged [14].
The antioxidant activity of the newly synthesized compounds is given in Table 2.

In Vitro Cytotoxicity Studies by Trypan Blue Exclusion Method
The synthesized compounds 3(a-j) were tested for their cytotoxicity in vitro, in comparison with cyclophosphamide as reference drug, against DLA cells. Dalton's lymphoma ascites (DLA) cells were procured from Adayar Cancer Institute, Chennai, India.
Tumour cells were aspirated from the peritoneal cavity of mice and added to test tube containing PBS. The cells were washed with PBS and centrifuged three times. The cells were diluted with PBS and the viability was checked using trypan blue stain. The cells were counted in haemocytometer. The cell count should be '100' in the four large sized quadrants. If the cell count is above or below 100, it is made to 100 by adding extra cells or diluting with PBS as required. If the cell count is 100, then number of cells in the diluted sample is 1 million/ ml. The synthesized compounds were dissolved in dimethylsulphoxide to get concentration of 20 mg/ml. After this, experiment was set up by incubating 10 μl of sample solution with 0.1 ml of diluted DLA cells. In each case, volume of the mixture was made up to 900 μl using PBS and incubated at 37°C for 3 h. After incubation, 0.1 ml of trypan blue was added and the number of dead cells was determined using haemocytometer. Cyclophosphamide was used as the standard compound [9,15]. The percentage cytotoxicity was calculated by using the under mentioned formula:

%Cytotoxicity=[No. of dead cells/(No. of viable cells+Dead cells)] × 100
The in vitro cytotoxicity studies of the newly synthesized compounds are given in Table 3.

Results and Discussion
The IR spectrum of compound 3a showed absorption band at 3375 cm -1 due to NH stretch. The absorption band for thiazolidinone C=O stretch was observed at 1637 cm -1 . The other prominent absorption

Antioxidant Activity Studies
The compounds synthesized will be screened for antioxidant activity by DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical method and the activities will be compared with that of ascorbic acid which will be used as the standard.
1.5 ml methanolic solution of the synthesized compounds (0.2 mM) was added to 1.5 ml (0.2 mM) solution of DPPH radical in methanol (final concentration of DPPH and synthesized compounds was 0.1 mM). The mixture was shaken vigorously and allowed to stand for 30 min. After this the absorbance at 515 nm was determined and ISSN: 2161-0444 Volume 8 (7): 181-184 (2018) -183

Antioxidant Activity Studies
The antioxidant activity of the newly synthesized compounds was evaluated by their DPPH radical scavenging activity at 0.1 mM concentration. Among the ten newly synthesized compounds 3(aj) screened for DPPH free radical scavenging activity, compound 3i (p-methoxyphenylmethylidene substituent at 5th position and o-chloro phenyl at 2nd position) exhibited the highest free radical scavenging capacity of 70% with the series of newly synthesized compounds. Compounds 3b (o-chlorophenyl methylidene substituent at 5th position and phenyl substituent at 2nd position), 3a (m-nitrophenylidene substituent at 5th position and phenyl substituent at 2nd position), 3c (p-methoxy phenylmethylidene substituent at 5th position and phenyl substituent at 2nd position), 3g (p-methoxyphenylmethylidene substituent at 5th position and m-chlorophenyl substituent at 2nd position), 3e (phenylmethylidene substituent at 5th position and p-chlorophenyl substituent at 2nd position), 3j (o-nitrophenylmethyidene substituent at 5th position and p-chlorophenyl substituent at 2nd position), 3f (o-chlorophenylmethylidene substituent at 5th position and o-nitrophenyl substituent at 2nd position) and 3h (phenylmethylidene substituent at 5th position and o-chlorophenyl substituent at 2nd position) exhibiting moderate free radical scavenging activity ranging from 57-66%. Compound 3d (m-nitrophenylmethylidene substituent at 5th position and phenyl at 2nd position) showed the lowest free radical scavenging capacity of 46%. Ascorbic acid which was used as standard antioxidant in the present study showed the highest free radical scavenging activity of 95% at 0.1 mM concentration.

In Vitro Cytotoxicity Studies
Cytotoxicity studies against Dalton's Lymphoma Ascites (DLA) tumour cells were carried out with all the newly synthesized compounds 3(a-j). The results of the short term invitro cytotoxicity studies showed the compounds 3d (m-nitrophenylmethylidene substituent at 5th position and phenyl substituent at 2nd position), 3e (phenylmethylidene substituent at 5th position and o-chlorophenyl substituent at 2nd position) and compound 3i (o-methoxy phenylmethylidene substituent at 5th position and o-chlorophenyl substituent at 2nd position) exhibiting percentage of cytotoxicity of 70%, 65% and 52% respectively which was superior when compared with that of the standard cyclophosphamide which showed a percentage cytotoxicity of 45% at 200 μg/ml. Compound 3a (m-nitrophenylmethylidene substituent at 5th position and o-chlorophenyl substituent at 2nd position)showed a percentage of 45% which was comparable with the percentage cytotoxicity exhibited by standard cyclophosphamide at 200 μg/ml. Rest of the compounds showed percentage cytotoxicity ranging from 27%-40%.

Conclusion
The present work comprises the synthesis of ten novel hybrid molecules which are thiazolidin-4-one derivatives incorporated with coumarin nucleus and having various arylidene substituents at 5th position of thiazolidin-4-one nucleus.
After analysing the results following conclusions are made: • Among the synthesized compounds 3i (p-methoxyphenylmethy lidene substituent at 5th position and o-chlorophenyl at 2nd position) showed the highest antioxidant activity of 70%.
The 1 H NMR Spectrum of 3a showed a triplet at 1.639 corresponding CH 3 protons of coumarin. The two OCH 2 protons are resonated at value of 2.410. The thiazolidinone CH groups show singlet peak at value 6.221. Aromatic protons and alkenic protons resonated as multiple at 6.98-8.725.
Further evidence for the formation of 5-arylidene derivatives of thiazolidin-4-one was obtained by recording its mass spectrum. The mass spectrum of the compound 3a showed molecular ion peal at m/z 579.367(M+1) + in conformity with the molecular formula C 28 H 20 ClN 3 0 7 S. The IR spectrum of compound 3b showed absorption band at 3377 cm -1 due to NH stretch. The absorption band for thiazolidinone C=O stretch was observed at 1714 cm -1 . The other prominent absorption bands in IR spectrum were observed at 1554 cm -1 (C=C stretch) 1087 cm -1 (C-Cl stretch) and 1274 cm -1 (coumarinyl C-O-C stretch).
The 1 H NMR Spectrum of 3b showed a triplet at 2.407 corresponding CH 3 protons of coumarin. The two OCH 2 protons are resonated at value of 4.847. The thiazolidinone CH groups show singlet peak at value 5.322. Aromatic protons and alkenic protons resonated as multiple at 6.990-8.745.
Further evidence for the formation of 5-arylidene derivatives of thiazolidin-4-one was obtained by recording its mass spectrum. The mass spectrum of the compound 3b showed molecular ion peal at m/z 534.105 (M+1) + in conformity with the molecular formula C 28 H 21 ClN 3 0 7 S [16]. position and p-chlorophenyl at 2nd position) and 3i (p-methoxyphenylmethylidene substituent at 5th position and o-chlorophenyl at 2nd position) showed the percentage cytotoxicity of 70%, 65% and 52% respectively, which was more than that of the standard cyclophosphamide, that showed a percentage cytotoxicity of 45% against DLA cells.