INTRODUCTION:

The diversity of Thiazolidinone in the field of biological aspect draw the attention of chemists to carry out research over it. The active thiazole nucleus is explored to develop different medicinally important molecules. They have to be possessed different type of biological activities like antimicrobial^1-2, antioxidant³, anti-HIV⁴, antihistaminic⁵, anti-convulsant^6-7,anti-inflammatory ^8-10.

The present study deals with the preparation of a series of 2-(Benzothiazolyl-2’)hydrazono-3-phenyl-5-arylidene -4-thiazolidinone derivatives starting from 2-hydrazinobenzothiazoles. But these compounds do not show sufficient bio-accessibility owing to their less solubilities in aqueous medium. To eradicate the difficulties, one of the best ways to increase their bio-accessibility is to generate a supramolecule by combining with a non-toxic oligosaccharide namely β-cyclodextrin^11-13. The cavity dimension of β-cyclodextrin is regarded as suitable host which provide a conical cavities for the soluble of our synthesized compounds by the way of encapsulation. Among the different types of cyclodextrin available, β-cyclodextrin is chosen on account of its cavity size which is appropriate for the formation of inclusion complex for heterocylic compounds. So, inclusion complexes are prepared by encapsulating the synthesized (2-Benzothiazolyl-2’)hydrazono-3-phenyl-5-arylidene-4-thiazolidinone derivatives with β-cyclodextrin. The prepared inclusion complexes are analyzed through their physical parameters, thermodynamic changes and spectral data (UV, IR and NMR). The compounds and their inclusion complexes are subjected to verify antibacterial and antioxidant activities. The potential of the synthesized compounds are found to be increased through encapsulation.

MATERIAL AND METHODS:

Apparatus and Materials :

Analytical grade reagent are used for the preparation of compounds and the inclusion complex gifted from Himedia. Double distilled water is used as the solvent for dilution purpose. The electronic spectra were recorded on Shimadzu UV-1700 Spectrophotometer while IR-spectra were recorded in KBr pellets in 400-4000 cm^-1 region on a Shimadzu 8400 FTIR Spectrophotometer. ¹H NMR spectra (CDCl₃) are scanned on a DRX-300 (300MHz) spectrophotometer using TMS as internal standard and chemical shifts are expressed in δ scale. Sulphur detection is performed to check the purity of the compound and TLC method using silica gel is done to check homogeneity. Open capillary method is used to know the melting point of the compounds and their inclusions.

Synthesis of Compounds:

The compounds were synthesized as per the method describe by Garnaik et.al ¹⁴scheme-I

Procedure for synthesis of 1-(Benzothiazolyl-2’) 4-phenyl thiosemicarbazide:

2-hydrazinobenzothiazole (1.65gm, 10 mmole) is taken in a100mL round bottom flask with ethanol(10ml) as solvent and add Phenyl isothiocyanate (1.35 gm,10mmole) with stirring for 5 minutes . Reflux and stir the resulting solution with half an hour and then cool .The resulting solid filtered and recrystalised from ethanol. M.P. 179^oC, yield-2.1gm (70%), (Found S, 21.2%, C₁₄H₁₂N₄S₂ requires S, 21.4%)

Procedure for synthesis of 2-(Benzothiazolyl-2’)hydrazono-3-phenyl- 4-thiazolidinone:

Take a mixture of 1-(Benzothiazolyl-2’) 4-phenyl thiosemicarbazide (0.06 gm, 2mmole), monochloroacetic acid (0.25 gm, 2 mmole) and anhydrous sodium acetate (0.2 gm) in absolute ethanol (10 ml) in a 100mL round bootm flask and refluxed for three hour. The surplus of solvent was removed and poured into cold water. The crystals formed was filtered, washed with hot water, and recrystalised from ethanol, M.P. 166^oC, yield-0.3 gm (44%), (Found: S, 18.80% C₁₆H₁₂N₄OS₂ requires S,18.95%). IR(KBr Ʋcm^-1): 746.45 (C-S str), 1487.12 (C=C str) 1556.55 (C=N str), 1714.72(C=O str), 3030.17(Ar-H str), 3226.91(N-H str).

General procedure for synthesis of 2-(Benzothiazolyl-2’)hydrazono-3-phenyl 5-arylidene-4-thiazolidinone (Compound-K,L and M):

Take 15mL of glacial acetic acid in 100mL round bottom flask and add a mixture of 2-(benzothiazolyl-2’)hydrazono-3-phenyl 4-thiazolidinone (0.35 gm, 2 mmole), benzaldehyde (0.22 gm, 2 mmole), fused sodium acetate (1gm) and reflux for four hour. Pale yellow solution is formed during the above step , put it into ice cold water and remove the excess water from it. Dry the compound and recrystalised from ethanol, m.p. 122^oC, yield-0.28 gm (63%). (Found: S, 14.07% C₂₃H₁₇N₄OS₂ requires S, 14.91%). In this way compound K is prepared. Similarly Compound L (O-Hydroxy benzaldehyde) and compound M(O-Nitro benzaldehyde) can be prepared by using the same procedure. The spectral and thermal data of the compound is given in the table 1 and 2.

Phase solubility study of compounds in aqueous medium:

Higuchi Connors method ¹⁵ is used for the aqueous phase solubility study of synthesized compounds at various concentrations of β-CD. Taking different concentration of β-cyclodextrin (0-10mM) in a 250 ml. conical flask and add exact amount of the synthesized compound in it. The prepared solution were shaken in a rotary flask shaker at room temperature in a conical flask for 48 hours until equilibrium established. The solutions were filtered through Whatmann-42 filter paper and were analyzed on UV-VIS spectrophotometer in the range of 200-400nm. The different values of absorbance at λ_max were plotted against different concentrations of β-cyclodextrin as given in figure 1.

Synthesis of Inclusion complexes by co-precipitation method:

The inclusion complexes of the synthesized compounds (K, L and M) with β-cyclodextrin were prepared as per co-precipitation method^16-18. Prepare the required concentration of β-cyclodextrin solution as it is found by aqueous phase solubility study, stir it for half an hour and add dropwise the solution of synthesized compound with proper concentration. The mixtures are stirred at room temperature for 48 hour and then filtered. Further, the filtrate is kept in a refrigerator for 48 hours. Finally, the precipitate is filtered through G-4 crucible, washed with distilled water and dried another for 24 hour in open places.

Preparation of solution for Antibacterial study:

The solutions of the test compounds were prepared in dimethyl sulphoxide (DMSO) at conc. of 500μg/ml .Cup-plate method ¹⁹ was used to study the antibacterial activity of the synthesised compounds and their inclusion complexes. At first the bacterial strains of Escherichia coli , Staphylococcus aureus and Proteus Vulgaris were inoculated into 100ml of the sterile nutrient broth and incubated at 37±1°C for 24 hour. Standardisation of density of bacterial suspension was performed by McFarland method. Well of uniform diameter (6mm) were made on agar plates, after inoculating them one by one with the test organisms aseptically. The drug (500μg/ml) and the test compounds (500μg/ml) were introduced with the help of micropipette and the plates were placed in the refrigerator at 8-10°C for proper diffusion of drug into the media. After two hour kept at refrigerator , the Petri plates were transferred to incubator and maintained with a temperature of 38⁰C for nearly 22 hour. Zone of inhibition of petri plates were determined by using venire scale. zone of inhibition shown by the test compounds with standard drug (Tetracycline) can be compared and results to determine. The results were the mean value of zone of inhibition of three sets measured in millimetre and the data were presented in Figure 3 to 5_.

Preparation of solution for Antioxidant study:

Tagashira and Ohtake²⁰ method of DPPH (2, 2-Diphenyl-1-picrylhydrazyl) scavenging assay is used for screening the antioxidant activity of the synthesized compounds. Test sample solution is prepared in 100µg/ml concentration in ethanolic DPPH. After vortexing, the mixture is incubated for 10 minutes at room temperature. The measurement of absorbance of the samples are taken at 517 nm. Taking the difference of absorbance between a test sample and a control, the activity of the sample is calculated. Ascorbic acid is used as reference substance (Table4).

RESULTS AND DISCUSSION:

The three pharmaceutically active compounds (K, L and M) are synthesized in their pure crystalline states. Considering the optimum conc. of β-cyclodextrin from aqueous phase solubility studies, the inclusion complexes of the compounds with β-cyclodextrin are prepared. The changes found in the melting point, colour and spectral characteristics authenticate the establishment of inclusion complex. An increased melting point of inclusion complexes of respective compounds may be featured through the fact that a supplementary thermal energy is required for deencapsulation from the β-cyclodextrin cavity. (Table 1and2).

Table 1 :Physical properties of compounds and their inclusions

COMPOUND/ COMPLEX	SUBSTITUENT	COLOUR	M.P. in ^◦C	% of yield
Compound K	Phenyl	Yellow	122	63
Compound K with β-CD		Yellowish white	145	45
Compound L	O-Hydroxy phenyl	Light yellow	180	60
Compound L with β-CD		Yellowish white	197	50
Compound M	O-Nitro phenyl	Brown	170	61
Compound M with β-CD		Brownish white	193	45

Table 2: Spectral data of synthesized compounds and their inclusions.

Si. No.	Compound/ complexes	UV_λmax	IR(KBr) cm^-1	¹H NMR
1	Compound –K	275	742.59(C-Sstr),1492.60(C=C Str),1589.34(C=N str),1645.28(C=O str),3194.12(N-H str)	¹H NMR (CDCl₃) : d 6.81-8.23 (d, 6H, Ar-H), 4.23(s,1H,C-NH),7.58(s,1H,C-H),7.34-7.61 (m,8H, Ar-H)
2	Compound-K with β-CD	278	746.45(C-Sstr),1494.83(C=C str),1581.83(C=N str),1714.12(C=O str),3224.34(N-H str)	¹H NMR (CDCl₃): d 6.12-7.81 (d, 6H, Ar-H), 3.83 (s,1H,C-NH),7.11(s,1H,C-H),6.82-7.24 (m,8H, Ar-H)
3	Compound –L	271	692.44(C-Cl str),744.52(C-S str),1487.12(C=C Str),1583.56(C=N str),2920.23(Ar-Hstr),1699.29(C=O str),2358.94,1373.32,1153.43,1010.70	¹H NMR (CDCl₃) : d 6.62 -7.81 (d, 6H, Ar-H), 4.53(s,1H,C-NH),7.71(s,1H,C-H),6.91-7.62 (m,8H, Ar-H) 5.13 (s, 1H, OH)
4	Compound -L with β-CD	275	692.44,748.38(C-S str),1494.83(C=C Str),153 9.20(C=Nstr),1697.36(C=O str),3062.96(Ar-Hstr),1423.47,1317.38,1159.22.	¹H NMR (CDCl₃): d 6.41-7.52 (d, 6H, Ar-H), 4.13 (s,1H,C-NH),7.23(s,1H,C-H),6.44-7.12 (m,8H, Ar-H) 5.11 (s,1H, OH)
5	Compound –M	276	742.59(C-Sstr),850.61,1338.60(N=Ostr) ,1616.35(C=C Str),1450.26(C=N str),1696.36 (C=Ostr),2916.37(Ar-HStr),3196.50 (N-Hstr)	¹H NMR (CDCl₃) : d 6.72-7.44 (d, 6H, Ar-H), 4.34(s,1H,C-NH),7.91(s,1H,C-H),7.51-7.92(m,8H, Ar-H)
6	Compound –M with β-CD	281	690.52(C-Cl str),748.38(C-S str),1157.92(C-N str),1494.83(C=C Str),1597.60(C=N str),2922.16(Ar-Hstr),	¹H NMR (CDCl₃): d 6.33-7.12 (d, 6H, Ar-H), 4.14 (s,1H,C-NH),7.33(s,1H,C-H),7.11-7.54 (m,8H, Ar-H)

The IR absorption energies of Compound K are found to be formed at 742.59(C-S str),1492.60(C=C str),1589.34(C=N str),1645.28(C=O str),3194.12(N-H str) indicating the presence of C-S, C=C, C=N, C=O and N-H in the compound which resembles theoretical data and similarly the IR data of inclusion complex of Compound K shows characteristics absorption at 746.45(C-S str), 1494.83(C=C str), 1581.83 (C=N str)1714.12(C=O str),3224.34(N-H str) indicates the absorption energies of C-S,C=C,C=N,CO and N-H in the complex move towards higher end which may be due to different interactions in the hydrophobic core of the host (β-cyclodextrin). Similarly the IR data of compounds L, M and their inclusion complexes are found to be absorbed at the suitable characteristic frequency as shown in the Table 2. In case of IR data for all compounds, it is found that the IR frequencies shift towards higher wave number side with the broader and smoother peaks after inclusion complex formation which ascertain the interactions like H-bonding, van der Waals forces, in the cavity of β-cyclodextrin ²¹. The comparison between the δ values of compounds and their inclusion complexes revealed that the δ values of PMR signals of compounds are shifted towards up field in the inclusion complex which could be explained on the caging based shielding phenomenon within the interior cavity of β-cyclodextrin. The graphs are drawn with a particular conc. of the synthesized Thiazolidinone derivatives and different conc. (0-10mM) of β-CD. The graphs demonstrate that solubility of the synthesized compounds in aqueous medium enhance linearly as a function of β-cyclodextrin concentration up to a particular point followed by a visible decline (Figure 1). Better correlation coefficients are obtained which have values close to unity, assume the stoichiometry of these complexes may be 1:1. By using Benesi-Hilderband relation, thermodynamic stability constants (K_T) of host-guest complexes were determined ²².

1/ΔA = 1/ ΔЄ + 1/ K_T [ Guest ]_oΔЄ. [β-CD]_o

Where ΔA is change in absorbance, ΔЄ is change in molar extension coefficient, [Guest]_o is concentration of compound in inclusion complex and [β-CD]_o is molar concentration of β-CD.

Figure 1 :Variation of absorption with β- CD concentration

Figure 2: Variation of 1/absorption with 1/ β- CD concentration

Good linear correlations were obtained for a plot of 1/ΔA verses 1/[β- CD]_ofor compounds as shown compounds in figure 2 . The values of K_T for all the complexes were calculated using the relation. K_T = Intercept/Slope. The K_T values of the inclusion complexes of compounds with β- Cyclodextrin were found to be 526.13, 835.971 and 268.359 M^-1 respectively (Table 3). The data obtained were within a standard range (100 to 1000 M^-1)explains the appreciable stabilities of the inclusion complexes through host-guest interaction like van der Waal’s force, hydrophobic interaction etc.^23-25

The value of ΔG was calculated at 298 K using the equation:

ΔG = -2.303RT log K

The value of free energy of activation has been calculated and found to be -15.627, -16.782 and -13.948 kJ/mole (Table 3) for the inclusion complexes of Compound K, L and M respectively. The negative value of free energy change indicates that the inclusion complex formation is a thermodynamically allowed process.

Table 3:Equlibrium constant and free energy change of inclusion complexes

SI. No	Inclusion complex of Compound	Equilibrium Constant(K_T)	∆G (kJ/mole)	Correlation coefficient(r)
1	I.C._K	526.123	-15.627	0.9897
2	I.C._L	835.971	-16.782	0.9899
3	I.C._M	268.359	-13.948	0.9965

The data obtained from the antibacterial studies concludes that inclusion complexes of the respective compounds (K, L and M) show a very good result against three bacterial pathogens E. coli, S. aureus and P. vulgaris (Figure 3, 4and 5) with respect to their compounds. This can be explained on the basis of solubility induced bio accessibility after encapsulation within the host cavity. Among the tested substances the inclusion complex of compound-M exhibited maximum activity against S. aureus than that of other complexes whereas compound-L shows maximum activity against E. coli and P. vulgaris with respect to other complexes. This increase of bactericidal activity of the inclusion complexes may be due to the enhanced solubility of the synthesized medicinally active molecules which makes them more bioactive to specific infected tissues leading to increased drug activity. The free radical entrapping activity of the compound increases significantly after caging in the core of β-CD as shown in Table 4. This can be correlated to the higher solubility of the compounds due to inclusion complex formation there by increasing the bio accessibility. Higher the bio accessibility of the compounds, more is the entrapping reactive oxygen species or free radicals character, thereby escalating antioxidant character of the compounds ²⁶.

Figure 3: Antibacterial activity of the compound and inclusion complex against S.aureus

Figure 4 : Antibacterial activity of the tested compounds and their inclusions against E.coli

Figure 5:Antibacterial activity of the compound and inclusion complex against P.vulgaris

Table 4 : Antioxidant activity of synthesized compounds and their inclusions

Compound/Complex	Conc.(500µg/ml) % of inhibition	Conc.(100µg/ml) % of inhibition
Compound-K	34.6	28.8
Inclusion with β-CD	47.8	35..87
Compound-L	41.5	27.8
Inclusion with β-CD	52.8	33.9
Compound-M	43.4	29.4
Inclusion with β-CD	54.6	36.7
Ascorbic acid	90.00	74.30

CONCLUSION:

In conclusion, present result suggest that the solubility and bio-accessibility, Thermodynamically stability, antibacterial and antioxidant activities of the synthesized compounds of 2-(Benzothiazolyl-2’)hydrazono-3-phenyl 5-arylidene-4-Thiazolidinone are increased considerably by the formation of inclusion complexes with β-cyclodextrin. Future work will include to prepare some more derivatives of 4-thiazolidinone by using the Schiff’s base and study their activities.

ACKNOWLEDGEMENT:

The authors are thankful to Dr. J.R. Panda, Department of Pharmaceutical Science, Roland Institute of Pharmaceutical Sciences, Berhampur, Odisha, India for doing antibacterial and antioxidant study.

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Received on 19.10.2016 Modified on 30.10.2016

Research J. Pharm. and Tech 2016; 9(12):2265-2270

DOI: 10.5958/0974-360X.2016.00457.1