INTRODUCTION:

Advance in science and technology has contributed to an enormous improvement in the quality of life of humankind. Throughout history, humans have sought relief from anxiety and insomnia by using substances that depress brain activity and induce a drowsy or calming effect. Most CNS depressants activate a neurotransmitter called gamma-aminobutyric acid (GABA), which helps decrease brain activity. CNS depressants include a wide range of drugs such as alcohol, narcotics, barbiturates (Amytal, Nembutal, Seconal), benzodiazepines (Ativan, Halcion, Librium, Valium, Xanax), chloral hydrate, and methaqualone (Quaaludes), as well as newer CNS depressants developed in the 1990s, such as Buspirone (Buspar) and Zolpidem (Ambien), but these psychoneural drugs have very serious side effects like chronic use of benzodiazepines causes deterioration of cognitive function, physical dependence and tolerance. Besides addiction liabilities, benzodiazepines adversely affect the respiratory, digestive and immune system of body and chronic treatment with benzodiazepines often prove more harmful in longer run¹.

A large number of the heterocyclic compounds containing the thiazolidinone rings are known to exhibit diverse bioactivities such as antimicrobial^2,3, anti-inflamatory⁴, CNS depressant⁵, antifungal⁶, analgesic^7,8 and anti-cancer⁹ agents. Prompted by these observations, it is thought of interest to synthesis of 2-Imino-5-(4-substituted arylidene)-3-(5-aryl-1,3,4-thiadiazol-2-yl)-1,3-thiazol-4-one and screen for their possible CNS depressant and antimicrobial activity.

The condensation of the Arylaldehyde and Thiosemicarbazide in presence of ethanol to form 2-Arylaldehyde thiosemicarbazone (A). Followed by compound (A) was treated with Ferric-chloride and refluxed at 80-90 °C for 45 min. After it was filtered using hot funnel. A mixture of Citric Acid and Sodium Citrate was added to solution and stirred well for 1-hr. Then whole solution was neutralized with Ammonia (10%). Precipitate was filtered, washed with distilled water and allow to dry to form 2-Amino-5-aryl-1,3,4-thiadiazole (B). Compound (B) was refluxed with chloroacetylchloride for 6 hr. in present of alcohol to get 2-(chloro acetamido)-5-aryl-1,3,4-thiadiazole (C). Compound (C) was refluxed with KSCN for 5 hr in presence of alcohol. The resulting mixture was cooled and excess of alcohol removed by distillation gave 2-(thiocyanato acetamido)-5-aryl-1,3,4-thiadiazoles (D). Compound (D) was refluxed with DMF for 6 hr to get the 2-Imino-3-(5-aryl-1, 3, 4-thiadiazolyl)-1,3-thiazol-4-one (E). Treatment of the compound (E) with different Aryl aldehydes in present of fused sodium acetate in glacial acetic acid to form 2-Imino-5-(4-substituted arylidene)-3-(5-aryl-1,3,4-thiadiazol-2-yl) -1,3-thiazol-4-one (F1-7). The physical data of the title compound is summarized in table-1. All the synthesized compounds were characterized on basis of their analytical data’s such as IR and ¹H NMR.

EXPERIMENTAL:

The melting points of the synthesized compounds were taken in the open capillary tubes and Thiel`s melting point apparatus. The reaction were monitored by TLC (precoated-Merck) using Hexane: Ethyl acetate (1:1) and detected by UV Chamber and also using iodine as visualizing agent. The IR Spectra of the compounds were recorded on Shimadzu 1320 FT-IR spectrometer (KBr pellets method) and values of Vmax are reported in cm^-1. Nuclear magnetic resonance spectra were obtained on DMM X-200 MHz Brookfield from Astrazenica Ind. Ltd using cm^-1 and chemical shift (δ) are reported in parts per million downfield from standard reference Tetramethylsilane (TMS).

Preparation of 2-Arylaldehyde thiosemicarbazone (A)¹⁰:

Arylaldehyde (0.01 mol) and Thiosemicarbazide (0.05 mol) were dissolved in sufficient volume of ethanol (30 ml) and the mixture was refluxed for 1.5 hr. The contents were allowed to cool and the product was separated by filtration. The crude sample was recrystallized with hot ethanol.

IR Spectrum (KBr in cm^-1): 3444 (OH), 3265 and 3163 (NH₂ and NH), 1610 (-C=N) and 1118 (C=S).

Preparation of 2-Amino-5-aryl-1, 3, 4-thiadiazole (B)¹¹:

2-Arylaldehyde thiosemicarbazone (0.01 mol) was suspended in 60 ml of water. Ferric chloride (0.03 mol in 60 ml water) was added to it. This was heated to 80-90˚C and maintained at this temp for 45 min. The solution was filtered using hot water funnel. A mixture of citric acid (0.022 mol) and sodium citrate (0.01 mol) was added to the solution and was stirred for 1 hr. After cooling the whole solution was neutralized with aqueous ammonia (10%). The precipitate so obtained was filtered, washed with distilled water and allowed to dry. It was recrystallized from ethanol.

IR Spectrum (cm^-1): 3363 (OH), 3113 (NH₂), 1612 (C=N) and 742 (C-S-C). ¹H NMR (CDCl₃): 10.8 (S, 1H, OH), 6.7- 8.4 (m, 6H, Ar-H) and 5.2 (S, 2H, NH₂).

Preparation of 2-(chloro acetamido)-5-aryl-1, 3, 4-thiadiazole (C)¹²:

Chloroacetylchloride (0.02 mol) was added to a solution of 2-amino-5-aryl-1, 3, 4-thiadiazole (0.02 mol) in dry alcohol (60 ml) at 0-5˚C under stirring, which was subsequently refluxed for 6 hr. on a water bath. The completion of the reaction was monitored by TLC using Hexane: Ethyl acetate (1:1) as solvent system. The solvent was removed by distillation. The solid obtained was recrystallized by alcohol.

IR Spectrum (cm^-1): 3441 (OH), 3261 (NH), 3161(-CH₂), 1606 (C=N), 813 (C-Cl) and 746 (C-S-C). ¹H NMR (CDCl₃): 10.8 (S, 1H, OH), 9.8 (S, 1H, NHCO), 7.2- 8.2 (m, 6H, Ar-H) and 4.2 (S, 2H, CH₂Cl).

Preparation of 2-(thiocyanato acetamido)-5-aryl -1, 3, 4-thiadiazoles (D)¹²:

A mixture of 2-(chloro acetamido)-5-aryl-1, 3, 4-thiadiazole (0.014 mol), and KSCN (0.02 mol) and dry alcohol (40 ml) was refluxed on water-bath for 5 hr. The resulting mixture was cooled, excess of alcohol was removed by distillation, and the residue was poured into crushed ice. The solid thus obtained was filtered, washed with water, dried and recrystallized from acetone.

IR Spectrum (cm^-1): 3429 (OH), 3292 (NH), 3120 (-CH₂), 1633 (C=O), 1531 (C=N) and 715 (C-S-C).

Preparation of 2-Imino-3-(5-aryl-1, 3, 4-thiadiazolyl)-1, 3-thiazol-4-one (E)¹²:

Finely powdered 2-(thiocyanato acetamido)-5-aryl -1, 3, 4-thiadiazole (0.004 mol) was refluxed in 25 ml of dimethylformamide in an oil-bath at 150-160˚C for 6 hr. The solvent was removed by distillation under vacuum and the crude product was recrystallized from ethanol to give crystals of the above compound.

IR Spectrum (cm^-1): 3443 (OH), 3286 (NH), 3084 (-CH₂), 1691 (-C=O) 1573 (C=N) and 844 (C-S-C). ¹H NMR (CDCl₃): 13.1 (S, 1H, OH), 10.8 (S, 1H, NH), 6.9- 7.8 (m, 6H, Ar-H) and 3.7 (S, 2H, CH₂).

Preparation of 2-Imino-5-(4-substituted arylidene)-3-(5-aryl-1, 3, 4-thiadiazol-2-yl) -1, 3-thiazol-4-one (F)¹²:

A mixture of 2-Imino-3-(5-aryl-1, 3, 4-thiadiazolyl)-1, 3-thiazol-4-one (0.01 mol), aryl aldehyde (0.01 mol) and fused sodium acetate (0.015 mol) in glacial acetic acid (15 ml) was refluxed for 6 hr. The resulting mixture was cooled and the residue poured into crushed ice and neutralized by adding 10% sodium bicarbonate solution. The solid thus obtained was filtered, washed with water, dried and recrystallized from DMSO to get the titled compounds.

IR Spectrum (cm^-1): 3423 (OH), 3281 (NH), 3169 (Ar-CH), 2924 (-CH), 1689 (C=O) 1566 (-C=N) and 813 (C-S-C). ¹H NMR (CDCl₃): 10.5 (S, 1H, OH), 9.8 (S, 1H, NH), 9.0 (S, 1H, CH=CH-R'), 7.6- 8.2 (m, 6H, Ar-H) and 1.5 (S, 3H, OCH₃).

CNS DEPRESSANT ACTIVITY:

Acute toxicity studies (LD₅₀)¹³:

The acute toxicity of 2-Imino-5-(4-substituted arylidene)-3-(5-aryl-1, 3, 4-thiadiazol-2-yl) -1, 3-thiazol-4-one was determined by using male Wistar rats (200-250 gm). The animals were fasted for 24 hr prior to the experiment and up and down procedure (OECD guideline no. 425) method of CPCSEA was adopted for acute toxicity studies⁴². Newly synthesized compounds suspended in gum acacia was administered to groups of rats (n=3) upto a dose level of 1000mg/kg b.w p.o. Animals were placed individually in plastic cages and observed atleast once daily for the first 30 mins and periodically for 24 hr to observe signs of toxicity.

SCHEME OF WORK:

Table-1: Physicochemical Properties of the synthesized thiazolidinone derivatives

Compound No.	Substituents		Molecular Formula	Melting Point (˚C)	Yield (%)	TLC Mobile Phase (Hexane: Ethyl acetate)
Compound No.	R	R'	Molecular Formula	Melting Point (˚C)	Yield (%)	TLC Mobile Phase (Hexane: Ethyl acetate)
F1	C₈H₈O₂	C₁₁H₈O₂	C₂₃H₁₆N₄O₃S₂	148-150	55	1:1
F2	C₇H₅NO₃	C₁₁H₈O₂	C₂₂H₂₀N₅O₄S₂	140-143	57	1:1
F3	C₁₁H₈O₂	C₇H₅ClO	C₂₂H₁₃ClN₄O₂S₂	156-159	46	1:1
F4	C₁₁H₈O₂	C₈H₈O₂	C₂₃H₁₆N₄O₃S₂	126-128	56	1:1
F5	C₁₁H₈O₂	C₇H₄Cl₂O	C₂₂H₁₂Cl₂N₄O₂S₂	166-168	52	1:1
F6	C₁₁H₈O₂	C₆H₆O₂	C₂₂H₁₄N₄O₃S₂	125-128	48	1:1
F7	C₁₁H₈O₂	C₈H₈O	C₂₃H₁₆N₄O₂S₂	142-146	54	1:1

METHODS:

Pentobarbitone induced sleeping time¹⁴:

CNS depressant activities of the newly synthesized compounds were carried out using pentobarbitone sleeping time method. The healthy mice of the minimum body weight 25g were selected and kept for 8 hr fasting. These fasted animals were devided in to 7 groups randomly, each of 5 animals. Group 1 animals received standard drug pentobarbitone 40 mg/kg intraperitoneally and all other groups of animals were received test compounds 40 mg/kg along with pentobarbitone. The time of onset of action was noted as the animal loses its righting reflux, i.e. falls sleep. The time of recovery from sleep is noted as it turns to recover its normal posture. The onset and duration of action was calculated and tabulated in table-2.

Table-2: CNS depressant activity data of synthesized compounds

Pentobarbitone (40 mg/kg, i.p) post treatment of the vehicle and compounds	Onset of action (min)	Duration of action (min)	% sleeping time
Vehicle	3.98 ± 0.75	91.6 ± 4.17	100
Compound 1	1.48 ± 0.28	115.4 ± 4.52**	125.98
Compound 2	1.16 ± 0.14	140.2 ± 4.07***	153
Compound 3	2.48 ± 0.13	85.6 ± 4.37^ns	93.44
Compound 4	1.9 ± 0.34	131.6 ± 4.53***	143.66
Compound 5	3.16 ± 0.09	89.9 ± 3.61^ns	98
Compound 6	2.43 ± 0.21	90 ± 4.43^ns	98.25
Compound 7	1.6 ± 0.26	110.4 ± 2.79*	120.52

All values are Mean ± SEM. n = 5. *P<0.05, **P<0.01, ***P<0.001 when compare to control. Values were analyzed by Tukey multiple column comparison test.

Anti- anxiety activity:

Elevated plus maze test¹⁵:

The apparatus consist of two open arms (5 × 10 cm) and two closed arms (5 × 10 × 15 cm) radiating from a platform (5 × 5 cm) to form a plus-sign figure. The apparatus was situated 40 cm above the floor. The open arms edges were 0.5 cm in height to keep the mice from falling and the closed arms edges were 15 cm in height. The drugs and treatment were shown in table-3.

Table-3: Effect of diazepam and synthesized compounds in elevated plus maze test

Groups	No. of entries		Time spent in open arm (min)
Groups	Closed arm	Open arm	Time spent in open arm (min)
Control (vehicle 5 ml/kg, p.o)	12.0 ± 1.0	8.0 ± 0.71	78 ± 3.79
Diazepam(4 mg/kg, p.o)	7.2 ± 0.37***	4.8 ± 0.37*	158.2 ± 4.95***
Compound 1	7.2 ± 0.66***	4.6 ± 0.51**	178.4 ± 13.64***
Compound 2	7.0 ± 0.89***	5.0 ± 0.316*	160.8 ± 7.96***
Compound 3	11.6 ± 0.51^ns	6.6 ± 0.51^ns	85.6 ± 4.06^ns
Compound 4	6.4 ± 0.51***	5.2 ± 0.58*	174.8 ± 5.8***
Compound 5	12.4 ± 0.68^ns	6.6 ± 0.51^ns	73.6 ± 3.11^ns
Compound 6	10.6 ± 0.68^ns	5.8 ± 0.58^ns	78.8 ± 2.56^ns
Compound 7	6.6 ± 0.51***	6.0 ± 1.0^ns	158.6 ± 4.67***

All values are Mean ± SEM. n = 5. *P<0.05, **P<0.01, ***P<0.001 when compare to control. Values were analyzed by Tukey multiple column comparison test.

The animal was placed at the center of the maze, facing one of the closed arms.

During 5 min test period the following measures are taken.

· The no of entries into open arms.

· The no of entries into closed arms.

· Time spent in open arms.

Arm entry was counted when the animal had placed all of its four paws on it. The procedure was conducted in a sound attenuated room, with observations made from in adjacent room.

Test for locomotor activity using actophotometer on rat¹⁶:

CNS depressant activity of the compounds studied using actophotometer. Animals of either sex weighing 120-130g were divided into group of six each. The actophotometer counts for 10 min were recorded by, placing the animals in actophotometer. Control gives the initial readings. Compounds were administered orally in form of suspension prepared in 1% tween-80 at a dose of 35 mg/kg body weight. Diazepam was administered as standard drug to one of the group at a dose of 4 mg/kg body weight. After 30 min and 60 min of administration of test compounds, the actophotometer counts were noted for 10 min decreasing the no. of counts for each group was recorded and finally the percentage CNS depressant activity was determine. Results are tabulated in table-4.

ANTIMICROBIAL STUDIES¹⁷:

The synthesized compounds were screened for antibacterial activity against Escherichia coli (Gram negative), Pseudomonas aeruginosa (Gram negative), Staphylococcus aureus (Gram positive) Streptococcus pneumococci (Gram positive) and Klebsiella pneumoniae (Gram negative) by the filter disk method. Here responses of microorganisms to the synthesized compounds were measured in mm and compared with the response of the standard reference drug. The standard reference drugs used in the present work was Amoxycillin trihydrate. Each test compound and the standard were dissolved in DMSO to get a concentration of 5mg/ml and 10 mg/ml. This concentration was used for testing antibacterial activity. The petridishes were thoroughly washed and sterilized. About 0.1ml of the innoculum was added to sterile petridishes, afterwards the cooled medium was poured into petridishes and spreaded for the distribution of innoculum over the medium and kept for solidifying. Nutrient agar media was used for antibacterial screening. The Petri plates containing bacterial organisms were incubated at 37ºC for 24hrs. Here both high and low strength disks are applied for each antibiotic to be tested. The zone of inhibition was calculated by measuring the diameter of the inhibition zone and tabulated in Table-5.

RESULTS AND DISCUSSION:

CNS depressant activity:-

Pentobarbitone induced sleeping time:

Table-2: Indicates the test compounds were screened for CNS depressant activity by pentobarbitone sleeping time method using pentobarbitone as standard, and the following activity was observed.

Table-4: Effect of diazepam and synthesized compounds in locomotor activity using actophotometer

Compd.	Before administration of drugs (mean ± SEM)	After administration of drugs
		30 min		60 min
		(mean ± SEM)	% decrease in locomotor activity	(mean ± SEM)	% decrease in locomotor activity
1	155.3 ± 40.44^ns	72 ± 2.31*	53.64	48.67 ± 2.4**	68.67
2	149 ± 4.04^ns	31.33 ± 8.29***	78.97	20 ± 5.51***	86.58
3	117.33 ± 3.71^ns	89.3 ± 3.48^ns	23.89	79 ± 1.53^ns	32.67
4	123 ± 6.81^ns	63 ± 2.65**	48.78	44.33 ± 6.33***	63.96
5	117.66 ± 6.77^ns	66.67 ± 8.26**	43.46	72.33 ± 2.6^ns	38.52
6	109 ± 3.61^ns	79 ± 6.81^ns	27.52	62.67 ± 7.05^ns	42.50
7	126.33 ± 9.33^ns	79.33 ± 4.48^ns	37.20	31 ± 2.52***	75.46
Control	121.67 ± 14.4^ns	108.33 ± 7.7	10.96	82.67 ± 3.38	32.05
Diazepam	126.33 ± 8.11^ns	34 ± 10.44***	73.08	32.33 ± 6.98***	74.46

n=5. *P<0.05, **P<0.01, ***P<0.001 when compare to control. Values were analyzed by Tukey multiple column comparison test.

Table-5: Antibacterial activity of the synthesized compounds.

Compd.	Zone of Inhibition (mm)
Compd.	*S. aureus*	*Strep. Pneumococci*	*E.coli*	*P. aeruginosa*	*Klebsiella pneumoniae*
F1	12	11	8	8	10
F2	20	12	27	11	12
F3	-	-	-	-	-
F4	17	12	11	13	15
F5	-	-	-	-	-
F6	16	14	9	11	12
F7	-	-	-	-	-
Amoxycillin trihydrate	28	28	30	29	27

Compounds 2 and 4 showed extremely significant CNS depressant activity (p<0.001) of all the compounds tested, where as Compound 1 showed highly significant CNS depressant activity (p<0.01) and Compound 7 showed significant CNS depressant activity (p<0.05).

Elevated plus maze test:

Table-3: Indicates the test compounds were screened for CNS depressant activity by elevated plus maze test using diazepam (4 mg/kg p.o) as standard. And the following activity was observed. Of all the synthesized compounds 1, 2, 4 and 7 showed extremely significantly increased time spent in open arms (p<0.001). But compounds 3, 5 and 6 did not show any significant activity.

Test for locomotor activity using actophotometer on rat:

Table-4: Indicates among the synthesized compounds the compound 2, 4, 7 and diazepam treated (STD) were found to show highly significantly decreased locomotor activity (p<0.001), while the compound 1 was found to show significantly decreased locomotor activity (p<0.01). Compounds 3, 5 and 6 did not show significant decreased locomotor activity.

Among the synthesized compounds, only compounds 1, 2, 4 and 6 showed some of the antimicrobial activity as compared to standard drug Amoxycillin trihydrate and rest of the compounds were found inactive. In case of E.coli and S. aureus, compound 2 exhibited good activity, while compounds 1, 4 and 6 showed moderate activity. In case of Streptococcus pneumococci, compounds 1, 2, 4 and 6 was found to show moderate activity, while all of the synthesized compounds had mild antibacterial effect against Pseudomonas aeruginosa.

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Received on 18.03.2010 Modified on 08.04.2010

Asian J. Research Chem. 3(3): July- Sept. 2010; Page 698-702