Synthesis and Anxiolytic Activity of 2-(Substituted)-5-[(N-Benzotriazolomethyl)-1,3, 4-Thiadiazolyl]-4-Thiazolidinone

1,2,3-Benzotriazole (BTA) is a heterocyclic compound with three nitrogen atoms. It is a polar and colourless compound which can be used for its great versatility. The enormous investigations on derivatives of benzotriazole reveal wide applicability of this molecule for tagging and delivering huge number of heterocyclic nuclei. In the present work synthesis of several derivatives of 2-(substituted)-5-[(n-benzotriazolomethyl)-1,3,4-thiadiazolyl]-4-thiazolidinone has been synthesized and are evaluated for their anxiolytic activity. The antianxiety activities of the synthesized derivatives were evaluated using EPM test and Bright and dark box test experimental models of anxiety. All results were expressed as mean± standard error mean (SEM) and analysed by one-way ANOVA. Post-hoc comparisons were performed by applying Dunnet’s test. P <0.05 was considered statistically significant

Anxiety is a normal emotional response which when chronic or severe becomes pathological and can aggravate cardiovascular and psychiatric disorders [12]. Despite the development of new molecules for pharmacotherapy of anxiety, the treatment is challenging as they produce various side effects or exhibit tolerance on continuous use.

Materials and Methods
The chemicals and reagents used in this were of AR and LR grade. They were procured from CDH, Hi-Media, Merck, Sigma Aldrich and Ranbaxy. The melting points of the synthesized compounds were determined by using Thiel's melting point apparatus (open capillary tube method) and all the compounds gave sharp melting points and are uncorrected. Purity of the compounds was ascertained by thin layer chromatography using silica gel-G as stationary phase and appropriate mixtures of the following solvents as mobile phase: n-butanol, glacial acetic acid and water. The spots resolved were visualized using iodine chamber. The IR  [13][14][15]. The Synthetic Procedure involved the following six steps as stated below.

Synthesis and Anxiolytic Activity of 2-(Substituted)-5-[(N-Benzotriazolomethyl
i. Step 1: Synthesis of Benzotriazole: In a mixture of 11.5 ml glacial acetic acid and 30 ml water, 0.1M o-phenylenediamine was dissolved and then added a solution of 0.1 M NaNO 2 in 15 ml of water, stirred continuously for 15 minutes. The temperature was maintained at 120C, chilled in ice bath and product (i) was collected by filtration. The yield obtained was 85% and M.P. was 990C. ii.
Step 2: Synthesis of N-Benzotriazolacetate: A mixture of product (i) 0.1M, ethylacetate (0.1M) and 0.3 gm of K 2 CO 3 in 60 ml of acetone was stirred of 10 hrs. The solvent was removed under reduced pressure. A solid m ass was produced which gave needle shaped crystals after recrystallization from the mixture of chloroform and ether (8:2 % V/V). The yield obtained was 70% and M.P. was 400C.

iii.
Step 3: Synthesis of N-Benzotriazol Acetyl Thiosemicarbazide: The crystals obtained from step II (0.08M) and thiosemicarbazide (0.08M) were taken in 50 ml of ethanol, stirred for 6 hrs and then refluxed for 3 hrs. The yellow coloured compound was obtained after recrystallization from the mixture of chloroform and hexane (9:1 %V/V). The yield was 60% and M.P. 1030C.
Structures of the compounds were established on the basis of C, H and N analysis reports, IR and 1H-NMR spectra ( Table 2).

Methods
Preparation of DMF (Dimethylformamide) Suspension of Synthesized Compounds: All synthesized compounds were dissolved in DMF and used as a suspension in physiological saline containing 2 drops of Tween 80 and produce a final conc. of 0.2 mg/ml. The standard drug used for this study was diazepam. Drugs were administered intraperitoneally (IP) in a constant volume of 1 ml/kg, 60 min before experiments were carried out.
Animals: Adult male Swiss albino mice weighing 25-35g obtained from our animal house. The animals were housed at 24±20C with 12: 12 h light and dark cycle. They had free access to food and water. The animals were acclimatized for a period of 7 days before the study. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Institute of Pharmacy, Bundelhand University, Jhansi (U.P.) India. The animals were used according to the CPCSEA guidelines for the use and care of experimental animals.
Experimental Design: On the day of the experiment, the animals were divided randomly into control and experimental groups (n=6). Group 1 received the vehicle, normal saline (10ml/kg) and served as the control group, group 2 received the standard drug diazepam (2mg/kg) and group 3 to 8 received DMF suspension of synthesized compounds (X 1 to X 6 ) ( Table 3). Drugs were administered to the animals 60 minutes prior to the evaluation in acute study, for chronic study once daily for a period of 10 days. Behavioural evaluation was carried out 60 minutes post drug administration on the 10th day. The antianxiety activity of the test drug was evaluated using EPM (elevated plus maze) test and Bright and dark box test experimental models of anxiety.

Elevated Plus Maze Test
According to the method of Kulkarni SK et al. [16] The wooden maze consisted of two open arms (50cm×10cm) and two closed arms of (50cm×10cm×40cm). The arms of same type were opposite to each other with a central square of 10cm.The maze was elevated to a height of 50cm above the floor. Each animal was placed in the centre square of plus maze, facing one of the open arms. The number of entries into and the time spent in open and closed arms in a 5 min period was noted.

Bright and Dark
The apparatus consisted of an open top wooden box. Two distinct chambers, a black chamber (20×30×35cm) painted black and illuminated with dimmed red light and a bright chamber (30×30×35cm) painted white and brightly illuminated with 100 W white light sources, were located 17 cm above the box. The two chambers were connected through a small open doorway (7.5×5cm) situated on the floor level at the centre of the partition [17].

Behavioural Assessment
Each animal was tested initially in plus maze and, then, in bright and dark arena paradigm in a single setting. In acute study 60 min after and in chronic study 60 min after the last dose on the 10 + day of drug or vehicle administration, each animal was placed in the centre square of the plus maze, facing one of the open arms. The number of entries into and the time spent in open and closed arms and the number of rears in each arm in a five-minute period was noted. Following the elevated plus maze test, the animal was placed at the centre of the brightly lit arena in the light and dark box. The number of entries into and the time spent in the bright arena, the number of rears in the bright arenas were noted. Following each trial, the apparatus were cleaned to mask the odour left by the animal in the previous experiment. Hand operated counters and stop watches were used to score the behaviour of animals.

Statistical Analysis
All results were expressed as mean± standard error mean (SEM) and analysed by one-way ANOVA. Post-hoc comparisons were performed by applying Dunnet's test. P <0.05 was considered statistically significant.

Elevated Plus-Maze
A perusal of Table 4 shows that compared to the standard drug, the synthesized compounds X 2 and X 3 significantly increased open arm activity, increasing the duration of time spent and number of entries in open arm in EPM test compared to control in acute study but in chronic study the doses of X 2 and X 3 produced a greater increase in duration of time spent and number of entries in open arm in EPM test compared to both control and standard drug diazepam. X 2 had produced better effect than X 3 and Diazepam in chronic study (Tables 4 & 5).

Bright and Dark
Diazepam (1mg/kg) treated mice significantly increased the number of entries into the bright arena, the time spent and the rears in bright arena. In acute study, both X 2 and X 3 significantly increased the number of entries into, time spent and rears in bright arena compared to control. X 2 and X 3 both had shown significantly increased number of entries into, time spent and rears in bright arena when compared to control and diazepam in chronic study (Tables 6 & 7).

Discussion
The two experimental models of anxiety, elevated plus maze and bright and dark arena, are based on the assumption that unfamiliar, non-protective and brightly lit environmental stress provokes inhibition of normal behaviour. This normal behavioural inhibition is further augmented in the presence of fear or anxiety like state. In the elevated plus maze, the open arms are more fear provoking than the closed arms. The ratio of entries, time spent and rearing behaviour in open arms to closed arms reflects the safety of closed arms with relative fearfulness of open arms [18]. The reduction in entry, time spent, total arm entries are the indications of high level of fear or anxiety. Anxiolytic drugs increase the proportion of entries, time spent in open arms. In the bright and dark box paradigm, the brightly lit environment is a noxious environment stressor that inhibits the exploratory behaviour of rodents. Reduction in the number of entries, time spent and rearing behaviour in the bright chamber was regarded as markers of anxiety. Rearing reflects an exploratory tendency of the animal that can be reduced due to a high level of fear [19]. In the present study, the compounds X2 and X3 significantly increased the duration of time spent and number of entries in open arm, time spent in closed arm in EPM test indicating anxiolytic activity in both acute and chronic studies. They also showed an increase in the time spent and the rears in bright arena in the bright and dark arena paradigm. Anxiolytic activity of X2 was found to be greater than diazepam in chronic study.

Conclusion
The derivatives of benzotriazole (X 1 to X 6 ) were synthesized with the objective to develop better anxiolytic agents with maximum percentage of yield and optimal anxiolytic activity. The results of the present study suggest that the synthesized compounds X 2 and X 3 have anxiolytic activity better than Diazepam. It was observed that halogen substituted aromatic compounds were more active than unsubstituted aromatic compounds and aromatic compounds were more active than alkyl substituted compounds. Further investigations with appropriate structural modification of title compound may result in therapeutically useful products. Further studies are required to elucidate the possible mechanism of anxiolytic activity and its usefulness in human beings.