INTRODUCTION:

Resistance to antibiotics by bacteria is a big challenge for pharmaceutical chemistry. To fortify this challenge, a number of chemotherapeutic reagents is being discovered. Heterocyclic compounds mainly indole and their derivatives are exhibiting excellent pharmacological activities such as antidepressive, anti-inflammatory, anti-fungicidial, anti-bactericidial and anti-tuberculostatic activities^1-4. Further indole derivatives possessing azediones and thiazolidinones moietites are acting as drugs for treating a number of diseases^5-8. One of the major problems of these of these drugs is their poor solubility in polar medium which may lower their bio-accessibility. The solubility of these compounds can be enhanced significantly by forming inclusion complex with cyclodextrins, non-toxic oligosaccharides⁹. Out of all the known cyclodextrins, β -cyclodextrin is usually considered for inclusion complex formation because it is cheaper, easily available and highly stable towards heat and oxidation^10-12

In the present work an attempt has been made to synthesize some 2-[benzylidenamino]-1,3,4-thiadiazino[6,5b]indole and 2-[furfurlidenamino]-1,3,4-thiadiazino[6,5b]indole in their purest forms starting from indole-2,3-dione and corresponding aldehyde. The inclusion complexes of the compounds have been prepared with β-cyclodextrin and characterized by elemental analysis, melting point data and study of spectral characteristics.

Thermodynamics properties of the inclusion complexes are also studied to know thermodynamic stability of inclusion complexes and the type of interaction in between the host and guest. In addition antimicrobial susceptibility and antioxidant activity of these compounds and inclusion complexes are also studied. Finally, a comparison has been made in between the characteristics of both the inclusion complexes.

MATERIAL AND METHODS:

All the chemicals of acceptable standards were procured from local market. Double distilled water to be used as solvent was prepared in the laboratory. Electronic spectra were recorded on Shimadzu UV-1700 Spectrophotometer and IR spectra were recorded in KBr pellets in Shimadzu 8400 FTIR Spectrophotometer. Melting points were recorded by open capillary method.

Synthesis of 2-[benzylidenamino]-1,3,4-thiadiazino [6,5b]indole and 2-[furfurlidenamino]-1,3,4-thiadiazino [6,5b]indole

2-[benzylidenamino]-1,3,4-thiadiazino[6,5b]indole and 2-[furfurlidenamino]-1,3,4-thiadiazino[6,5b]indole were synthesized starting from indole -2,3--dione (as per the scheme-I) through the following intermediate steps⁸.

i) Synthesis of 3-Thiosemicarbazideindole-2-one: (Compound-1) :A mixture of 2gm of indole-2, 3-dione and 1.23gm of thiosemicarbazide in 50 ml of methanol was refluxed for one hour. The completion of the reaction was checked by TLC. The excess of methanol was distilled out. The content was cooled and poured into ice cold water. It was filtered, washed with water, dried and recrystallised from ethanol to obtain 3-Thiosemicarbazideindole-2-one

ii) Synthesis of 2-Amino-1, 3, 4-thiadiazino [6, 5-b] indole (Compound-2):3gm of

3-Thiosemicarbazideindole-2-one was mixed with small quantity of cold and concentrated H₂SO₄. The reaction mixture was left at room temperature for 16 hours. The reaction mixture was then poured into ice-cold water and neutralized with liquid NH₃ to obtain a solid mass. The solid mass was filtered by using Whatmann-42 filter paper. It was washed with water, dried and recrystallised from ethanol to yield 2-Amino-1, 3, 4-thiadiazino [6, 5-b] indole.

a) Synthesis of 2-[Benzylidenamino]-1, 3, 4-thiadiazino [6,5b] indole (Compound-3): 1.06gm of Benzaldehyde and 2.02gm of 2-Amino-1, 3, 4-thiadiazino [6, 5-b] indole were taken in 50ml of methanol. The mixture was refluxed for 6 hours in presence of glacial acetic acid. The completion of the reaction was checked by TLC and excess of methanol was distilled off. The refluxed mixture was poured into ice-cold water, filtered, washed with water and dried. The dried mass was recrystallized from ethanol.

b) Synthesis of 2-[Furfurlidenamino]-1, 3, 4-thiadiazino [6,5b] indole

(Compound-4) : 0.96 gm of Furfuraldehyde and 2.02gm of 2-Amino-1, 3, 4-thiadiazino [6, 5-b] indole were taken in 50ml of DMF. The mixture was refluxed for 6 hours in presence of glacial acetic acid. The completion of the reaction was checked by TLC and excess of methanol was distilled off. The refluxed mixture was poured into ice-cold water, filtered and washed with water and dried. The dried mass was crystallized from ethanol.

Phase Solubility Measurements:-

The aqueous phase solubility of the compound at various concentrations β -cyclodextrin (0-10mM) was studied by Higuchi-Corner method¹³. Accurately weighed sample of these compounds was shaken in rotary flash shaker at room temperature in a series of conical flask for a period of 48 hours till the attainment of equilibrium. The solutions were filtered through whatmann-42 filter paper and were analyzed in a UV-visible spectrophotometer. The various values of absorbance at λ-max were plotted against different concentrations of β –cyclodextrin.

Synthesis of inclusion complexes:-

The inclusion complexes of the compounds (3 and 4I) with β –cyclodextrin were prepared as per co-precipitation method¹⁴.The solutions of these compounds in required concentrations were added drop by drop to β –cyclodextrin solution of the required concentration. The mixtures were stirred for a period of 48 hours and filtered. The filtrate was cooled for 24 hours in refrigerators. The precipitate obtained was filtered through G-4 crucible, washed with water and dried in air for 24 hours.

Study of thermodynamic properties:-

The thermodynamic stability constant of the complexes was calculated using Benesi-Hilderband relation¹⁵. The stability constant K of each complex was calculated with increasing temperature. From the slope of the linear plot of lnK vs. 1/T, ΔH was calculated. Then ΔS was calculated from vant Hoff’s equation

ln K= ΔH/RT- ΔS/R

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

ΔG = -RT ln K

Evaluation of Antibacterial study:

The antibacterial activity of compounds is studied as per cup-plate method¹⁶ The solutions of the test compounds were prepared in dimethylsulfoxide (DMSO) at 500µg/ml. The bacterial strains are inoculated into 100ml of the sterile nutrient broth and incubated at 37±1^oC for 24 hours. The density of the bacterial suspension is standardized by McFarland method. Well of uniform diameter (6mm) are made on agar plates, after inoculating them separately with the test organisms aseptically. The drug, control and the test compounds are introduced with the help of micropipette and the plates are placed in the refrigerator at 8-10^oC for proper diffusion of drug into the media. After two hours of cold incubation, the petriplates are transferred to incubator and maintained at 37±2^oC for 18-24 hours. Then the petriplates are observed for zone of inhibition by using vernier scale. The results are reported by comparing the zone of inhibition shown by the test compounds with standard drug Tetracycline. The results are the mean value of zone of inhibition of three sets measured in millimeter.

Evaluation of Antioxidant activity

In the present study DPPH(2, 2-Diphenyl-1-picrylhydrazyl) scavenging assay method was used for screening the antioxidant activity of the synthesized compounds[17-18] The antioxidant activity using the DPPH assay was assessed by the method of Tagashira and Ohtake. Test sample solution was prepared in 100µg/ml concentration in ethanolic DPPH. After vortexing, the mixture was incubated for 10 minutes at room temperature and the absorbance at 517 nm was measured. The difference in absorbance between a test sample and a control was considered as activity. BHT (Butylated hydroxyl toluene) was used as reference substance

RESULTS AND DISCUSSION:

The synthesis of Compound-3 (2- [ Benzylidenamino]-1,3,4-thiadiazino[6,5b]indole) and Compound-4 (2-[Furfurlidenamino]-1,3,4-thiadiazino[6,5b]indole) have been confirmed from elemental analysis and IR data as shown in Table-1. The elemental composition matches with theoretical data Infra-Red data of the compound-3 show characteristic absorption at 672,1296,1611,1682 and 3141 cm^-1 indicating the presence of C-S, C-C ,N-N, C=N and benzene ring in the compound.

Table-1: Analytical data of Compounds with and without inclusion complex

Compound-I : 2-[Benzylidenamino]-1,3,4-thiadiazino[6,5b]indole

Compound-II : 2-[Furfurlidenamino]-1,3,4-thiadiazino[6,5b]indole

Sl. No.	Compound/ Complex	Melting Point	Colour	Elemental Analysis (First line indicates finding value and second line indicates calculated value )					λ max (A⁰)	IR( KBr ) cm-¹
1	Compound-I	224	Yellow	C 66.4 66.2	H 3.45 3.44	N 19.4 19.3	S 1.0 1.03	O --	3550	672(C-S) 1296(C-C) 1611(N-N) 1682(-C=N) 3141(Ring)
2	Compound-I- β- CD	228	Pale Yellow	-	-	-	-	-	3542	670(C-S) 1290(C-C) 1605(N-N) 1679(C=N) 3130()Ring)
3	Compound-II	201	Yellow	60.1 60.0	2.8 2.86	20.1 20.0	11.2 11.43	5.68 5.71	3659	673 (C-S) 858(C-O) 1302 (C-C) 1581(N-N) 1623(-C=N) 3174(Ring)
4	Compound-II- β- CD	205	Reddish Yellow	-	-	-	-	-	3646	671 (C-S) 856(C-O) 1299 (C-C) 1578(N-N) 1621(-C=N) 3171(Ring))

The synthesis of inclusion complexes of compound 3 and compound 4 are confirmed from changes in melting point, color and spectral characteristics (UV-Vis and IR). The melting point of compound 3 and 4 are found to be s 224ºC and 201ºC but their inclusion complexes have melting points 228ºC and 205ºC(Table-1). The color of the compound 3 and 4 are found to be yellow but their inclusion complexes have colour pale yellow and reddish yellow respectively. The absorption maximum of the compounds 3 and 4 are found at 3550 and 3659 A^obut their inclusion complexes have absorption maximum at 3542 and 3646 A^o respectively. The higher melting point of inclusion complexes than the compounds is due to the fact that extra amount of thermal energy is required for the latter to bring it out of β- cyclodextrin cavity.

It is quite interesting to note that the absorption maxima undergo a blue shift after the formation of inclusion complex.(Table-I) .This may be attributed to the transference of the compound from a more protic environment to a less protic environment within the cavity of β- cyclodextrin which may be further supported by IR data The IR stretching frequencies due to different bonds undergo a downward shift towards low energy and the peaks become broader, weaker and smoother. Such changes in spectral characteristics due to inclusion complex formation may be due to the weak interaction like H-bonding, vanderWaal’s forces, hydrophobic interactions etc. between the guest compound and the host β- cyclodextrin^19-22.

The phase solubility plots of the compounds in β- cyclodextrin solution are shown in Figure 1. In both cases, it is seen that there is a linear increase in solubility of these compounds with increasing concentration of β- cyclodextrin.Since the slopes of all the plots are less than unity the stochiometry of these complexes may be 1:1²³.

The thermodynamic stability constants (K_T) of inclusion complexes are determined by using Benesi-Hilderband relation¹⁵. Good linear correlations are obtained for a plot of 1/∆A verses [β- CD]_o for compounds.3 and 4 (Fig. 2) The values of K_Tfor all the complexes are calculated using the relation

K_T = Intercept/Slope

The K_T values of the inclusion complexes of compounds 3 and 4 with β- cyclodextrin are found to be 421 and 144.7 M^-1 respectively(Table-2) The data obtained are within 100 to 1000 M^-1(ideal values) indicating appreciable stabilities for the inclusion complexes²⁴. The lower values of stability constants for compound 4 than 3 may be due to the presence of oxygen in the heterocyclic moiety lowering the magnitude of hydrophobic interaction as in case of compound-3.The thermodynamic parameters associated with the interaction of the compound with β- cyclodextrin for 1:1 stochiometry have also been calculated by determining stability constant (K- values) at different temperatures. The K- values are to found to decrease with rise in temperature as expected for an exothermic process (deencapsulation)^25,26. The plots of ln K versus inverse absolute temperature produce linear plots(Fig. 3). From the slopes of the curves, van’tHoff’s reaction isotherm and Van’thoff equation, the values of ∆G (change in free energy), ∆H (change in enthalpy) and ∆S (change in entropy) have been calculated at 298 K (Table-2). In Table-2, it is found that ∆G values are negative for both the inclusion complexes. These data clearly demonstrates that formation of inclusion complexes of compounds 3 and 4 with β- cyclodextrin is a spontaneous process. Further it is found that in both the inclusion complexes, ∆H values are negative and ∆S values are positive (Table-2). The negative value of enthalpy change (∆H) and positive value of entropy change (∆S) indicate that both the inclusion complex formation are energy allowed and entropy allowed processes^27-28. (Table-2)

Table-2: Thermodynamic data of inclusion complexes at 298 K

Compound-I : Benzylidenamino-1,3,4-thiadiazino[6,5b]indole

Compound-II : 2-[Furfurlidenamino]-1,3,4-thiadiazino[6,5b]indole

Complexes

K(M^-1)

∆G (kJ/MOLE)

∆H (kJ/MOLE)

∆S(J/MOLE)

Compound-I- β- CD

420.9

-14.98

-12.105

9.65

Compound-II- β- CD

144.74

-12.327

-12.187

0.166

Fig. 1 : Phase solubility plot of compound 3 and 4

Fig. 2 : Plot of 1/ Abs. vs. Conc. of compound 3 and 4

The antibacterial activities of the compounds and their inclusion complexes against S.aureus and E.coli are shown in Fig. 4A and 4B. Both the compounds and their inclusion complexes are susceptible to both the bacteria. However, the inclusion complexes increase the antibacterial activity significantly as compared to their corresponding compounds.

Fig. 3 : Plot of ln K vs. 1/T of compound 3 and 4

Fig. 4A : Antimicrobial susceptibility test of compound 3 and 4 against S.aureus

Fig. 4B : Antimicrobial susceptibility test compound 3 and 4 against E.coli

Fig. 5 : Anti-Oxidant activity of compound I and II

This may be attributed to enhanced solubility of the compounds after the inclusion complex formation which becomes more available to specific tissues leading to increased antibacterial activity the higher antibacterial activity in case of compound-4 after its inclusion complex formation may be correlated with its lower stability constant. With lower value of stability constant, the compound can easily come out and interact with specific tissues^27-29. The antioxidant activities of the compounds and their inclusion complexes are shown in Fig. 5. The radical scavenging activities of the compounds increase significantly after the formation of inclusion complex. This can be correlated to the higher stability of the compounds due to inclusion complex formation thereby increasing their bioaccessibility¹⁸.

CONCLUSION:

From the above results and discussion, it is clear that the formation of inclusion complexes of compound-3 and 4 is thermodynamically allowed which can be a very good analytical tool for enhancing the bioaccessibility of the drugs. The study further reveals that the formation of inclusion complex causes a significant increase in antibacterial and antioxidant activities.

ACKNOWLEDGEMENT:

The authors thank to Dr. U.L. Narayana, (Principal, Indira Gandhi Institute of Pharmaceutical Science); Mr. Sanjay Tiwari and Mr. Dilip Kumar Pattnaik for carrying out the IR study. Also the authors are thankful to Mr. J R Panda, Department of Pharmaceutical science, Roland institute of Pharmaceutical Science, Berhampur University for carrying the antimicrobial activity.

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Received on 09.08.2011 Modified on 20.08.2011

Research J. Pharm. and Tech. 4(11): Nov. 2011; Page 1693-1698