Short communication
Anticonvulsant and Toxicity Evaluation of Newly Synthesized 1-[2-(3,4-disubstituted phenyl) -3-chloro-4-oxoazetidin-1-yl]-3-(6-substituted -1,3-benzothiazol-2-yl)ureas
Nadeem Siddiqui,a'* Arpana Rana,a Suroor A. Khan,a Syed Ehtaishamul Haque'b M. Shamsher Alam'a Waquar Ahsana
and M. Faiz Arshada
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University),
New Delhi-110062, India
b Department of Pharmacology, Faculty of Pharmacy, Jamia Hamdard (Hamdard University),
New Delhi-110062, India
* Corresponding author: E-mail: nadeems_03@yahoo.co.in, nadeems_03@rediffmail.com Phone: 91-11-26059688 - Ext. 5639
Received: 30-08-2008
Abstract
A number of new 1-[2-(3,4-disubstituted phenyl)-3-chloro-4-oxoazetidin-1-yl]-3-(6-substituted-1,3-benzothiazol-2-yl)urea compounds (5a-t) were synthesized and evaluated for their anticonvulsant, hepatotoxic and neurotoxic properties. The titled compounds (5a-t) were obtained by cyclization of 3,4-disubstituted benzaldehyde-N-(6-substituted-1,3-benzothiazol-2-yl)semicarbazones (4a-t) in presence of DMF/triethylamine and chloroacetylchloride. All the newly synthesized compounds were screened for their anticonvulsant activity in i.p. Maximal Electroshock Seizure (MES) model and were compared with the standard drug phenytoin. Interestingly, compounds 5f, 5n, and 5p exhibited 100% protection in the MES test. In the neurotoxicity and hepatotoxicity screening, all the compounds were devoid of toxicity at the dose of 30 mg/kg body weight. The study showed that introduction of F, CH3 at the 6- position of benzothiazole moiety with H, OCH3 at the 3-position and OH, OCH3 at 4-position of the distant phenyl ring led to increased activity. Introduction of F, NO2, CH3, OCH3 substituents at the 6-position of the benzothiazole moiety and unsubstituted distant phenyl ring showed moderate decrease in activity.
Keywords: Azetidine, benzothiazole, maximal electroshock seizure test, anticonvulsant activity, neurotoxicity, hepato-toxicity
1. Introduction
Epilepsy is a brain function disorder characterized by recurrent seizures that has a sudden onset. It was assumed for many years that epilepsy could be treated with just one drug but it is now apparent that is not the case as more than one mechanism may be responsible for various types of seizures. Seizures remain uncontrolled in at least 30% of all epilepsies, even when adequate AED therapy is administered. During recent years, a large number of new AEDs have been marketed worldwide, but the proportion
of patients failing to respond to drug treatment has not been changed in a significant extent.
Drugs clinically active against epilepsy include derivatives with common structural characteristics such as nitrogen heterocyclic system with a carbonyl group and an aromatic or heteroaromatic nucleus linked to the hete-rocyclic system. Benzothiazole derivatives in recent years have acquired conspicuous significance due to their wide spectrum of biological activities. Although they have been known from long ago to be biologically active1-3 their varied biological features are still of great scientific interest nowadays. In the search of new anticonvulsant agents ha-
ving different substituted benzothiazole nucleus were already reported and all the compounds were found to possess significant activities.4-6 Since the benzothiazole moiety resembles with the benzisoxazole moiety present in the currently used drug zonisamide, it may have been acting through the inhibition of the sodium channel.
In our previous research we have reported7-11 several benzfused five membered heterocyclic compounds that have shown considerable anticonvulsant activity.
In the present investigation we have synthesized 1-[2-(3,4-disubstitutedphenyl)-3-chloro-4-oxoazetidin-1-yl]-3-(6-substituted-1,3-benzothiazol-2-yl)ureas (Sche-me-1). The compounds were evaluated in vivo for anticonvulsant activity by MES test and neurotoxicity by rotorod method.
2. Experimental
2. 1. Chemistry
All the solvents were of LR grade and were obtained from Merck, CDH and s. d. fine chemicals. Melting points were determined in open capillary tubes and are uncorrec-ted. Thin layer chromatography was performed on Silica gel G (Merck). The spots were developed in iodine chamber and visualized with an ultraviolet lamp. The IR spectra were recorded in KBr pellets on (BIO-RAD FTS 135) WIN-IR spectrophotometer. 1H-NMR spectra were recorded on a Bruker model DPX 300 FT- NMR spectrometer in (DMSO-rf6) using tetramethylsilane (Me4Si) TMS as an internal standard. The chemical shifts are recorded in 5 ppm scale.
2. 1. 1. Synthesis of 1-[2-(3,4-disubstitutedp-henyl)-3-chloro-4-oxoazetidin-1-yl]-3-(6-substituted-1,3-benzothiazol-2-yl)ureas (5a-t)
Step I: 6-Substituted-1,3-benzothiazol-2-amines (1a-e)
Substituted anilines (0.01 mol) and potassium thioc-yanate (0.01 mol) were dissolved in glacial acetic acid, cooled and stirred for 15 min. Cold bromine solution (0.01 mol, 3 mL in 10 mL acetic acid) was added dropwi-se. Stirring was continued for additional 3 h. Separated hydrochloride salt was filtered off, washed with acetic acid, dissolved in hot water and neutralized with aqueous ammonia solution (25%) The resulting precipitate was filtered off, washed with water and recrystallized from etha-nol to get the desired compounds (1a-e).
Step II: 1-(Substituted -1,3-benzothiazol-2-yl)ureas (2a-e)
To the solution of sodium cyanate (0.01 mol) in minimum quantity of water, glacial acetic acid (5 mL) was added. This solution was heated with 2-amino-6-substitu-ted benzothiazoles (1a-e, 0.01 mol) previously dissolved
in alcohol, till the contents of mixture become turbid and volume remained half of the original volume. The content was poured on crushed ice. The solid obtained was filtered off and dried.
Step III: ^-(substituted -1,3-benzothiazol-2-yl)hydra-zinecarboxamides (3a-e)
To the warm hydrazine hydrate solution of compounds (2a-e) in alcohol, conc. NaOH was added and ref-luxed for 6 h. Reaction mixture cooled to room temperature and was poured to crushed ice to afford a solid which was filtered and recrystallized from ethanol.
Step IV: 3,4-(Disubstituted benzaldehyde)-^-(6-sub-stituted-1,3-benzothiazol-2-yl)semicarbazo-
nes (4a-t)
The solution of compounds (3a-e, 0.1 mol) in glacial acetic acid (5 mL) and ethanol (10 mL) was heated to boiling and refluxed with appropriate aromatic aldehydes (0.1 mol) for 5 h. The reaction mixture was cooled to room temperature and kept overnight. The solid separated was collected out, washed with methanol, dried and recry-stallized from ethanol to get the pure compound.
Step V: 1-[2-(3,4-Disubstitutedphenyl)-3-chloro-4-
oxoazetidin-1-yl]-3-(6-substituted-1,3-benzot-hiazol-2-yl)ureas (5a-t) A solution of compound (4a-t, 0.1 mol) in DMF (40 mL) and triethylamine (0.1 mol) was stirred well at 0-5 °C. To this solution chloroacetyl chloride (0.2 mol) was added dropwise at the same temperature. The reaction mixture was stirred for 4 h and the separated amine hydrochloride was filtered off. The filtrate was refluxed for 2 h and the separated solid was recrystallized from methanol.
2. 2. Pharmacology
Male albino mice (Swiss, 25-30 g) were used in groups of six each as experimental animals. All the test compounds and standard drug were administered intrape-ritoneally suspended in polyethylene glycol (PEG). The animals were maintained on an adequate diet and allowed free access to food and water except during the short time they were removed from cages for testing. The animals were maintained at room temperature (25 ± 2 °C). All the experimental protocols were carried out with the permission from Institutional Animal Ethics Committee (IAEC). Animals were obtained from Central Animal House Facility, Hamdard University, New Delhi-110062. Registration number and date of registration of Animal House Facility is (173/CPCSEA, 28, JAN-2000).
2. 2. 1. Anticonvulsant Activity
Electroshock-induced seizures (MES test)12, 13
Each compound was administered as an i.p. injec-
tion at dose level of 30 mg/kg body weight and the anticonvulsant activity was assessed after 0.5 h and 4 h intervals of administration. Maximal electroshock seizures were elicited in mice by delivering 60 Hz, 50 mA electrical stimuli for 0.2 s via ear clip electrodes. The maximal seizure typically consists of a short period of tonic extension of the hind limbs and a final clonic episode. Blockade of the hind limbs tonic extensor component due to the drug treatment is taken as the end point.
2. 2. 2. Neurotoxic Effects
Rota-rod test. Minimal motor impairment was measured in mice by the rotorod test.14 The mice were trained to stay on an accelerating rotorod that rotates at 10 revolu-tions/min. The rod diameter was 3.2 cm. Trained animals were given i.p. injection of the test compounds at a dose of 30 mg/kg. Unimpaired mice can easily remain on a rod rotating at this speed. Neurotoxicity was indicated by the inability of the animal to maintain equilibrium on the rod for at least 1 min in each of three concurrent trials.
destructed. It was estimated using Rietman and Frankel's method.18-20
2. 2. 6. Estimation of Serum Glutamate Pyruvate Transaminase (SGPT) or Alanine Transaminase (ALT)
It is cytosolic enzyme present abundantly in liver cells. The serum levels of ALT are elevated in liver diseases. This is considered one of the most sensitive indications of liver damage particularly in viral hepatic necrosis e.g. viral hepatitis or toxin induced liver injury. It was determined using Rietman and Frankel's method.18-20
2. 2. 7. Estimation of Alkaline Phosphatase
Alkaline phosphatases are enzymes, which catalyze the removal of phosphate group from monophosphate esters under alkaline conditions. This reaction is of considerable importance in several liver diseases. It was determined by the method reported previously.21
2. 2. 3. Histopathological Studies
The selected compounds 5f and 5n were evaluated for their histopathological study. The Luna's technique15 was used to assess the liver of mice, which were administered with test compounds at the dose level of 30 mg/kg body weight for 15 days; comparison was done with the control group. Microphotographs of section of liver were taken at the magnification of 100X and 400X. The mi-crophotographs of section of liver of mice administered with compound 5f and 5n along with control are presented in Fig. 1-3.
2. 2. 8. Estimation of total proteins and albumin
Determination of the proteins provides most useful information in chronic liver diseases. It was determined by using Biuret method.22, 23
2. 2. 9. Statistical Analysis
All the statistical analyses were carried out using the software SigmaStat 4.0 using ANOVA followed by dun-net's multiple comparison tests and the results are expressed in Mean ± SEM.
2. 2. 4. Log P Determination
The desired Log P value depends on the nature of the compounds and the testing system. Log P approximately equal to 2.0 is expected to be the best predictor for CNS activity.16 In this study, we attempted to correlate the anticonvulsant activity with 100% protection against the seizure spread in anti-MES screen, with their calculated log P values (CLOGP). The Log P values were determined for compounds 5f, 5n and 5p. The experimental log P values were determined using the octanol-water method17 and the CLOGP values were calculated from ACD free ware version 7.1.
2. 2. 5. Estimation of Serum Glutamate
Oxaloacetate Transaminase (SGOT) or Aspartate Transaminase (AST)
It is mitochondrial enzyme present in large quantities in the liver, heart, skeletal muscles and kidneys, which gets released from the damaged cells when the tissues are
3. Results and Discussion
3. 1. Chemistry
The synthesis of 1-[2-(3,4-disubstituted phenyl)-3-chloro-4-oxoazetidin-1-yl]-3-(6-substituted-1,3-benzot-hiazol-2-yl)ureas (5a-t) was accomplished as presented in Scheme-1. It involves the cyclization of 3, 4-disubstituted benzaldehyde-^-(6-substituted-1,3-benzothiazol-2-yl)se-micarbazones (4a-t) in the presence of DMF/ triethyl amine and chloroacetyl chloride by stirring and refluxing for 2 h. Synthesized compounds were characterized by elemental analysis, FT-IR, 1H-NMR and mass spectrum. The FT-IR spectrum exhibited characteristic bands for NH, CH-Ar and C=O at 3403-3059, 3091-3010 and 1681-1598 cm-1. The 1H-NMR spectrum showed singlet at 5 4.30-6.87 confirming CHCl, multiplets ranging from 5 6.60, 7.59-7.56, 8.69 confirmed aromatic protons and singlet ranges at 5 8.69-9.02, 11.00-11.57 confirmed the presence of NHN- and NHC=O respectively which were D2O exchangeable.
NH2 + KONS
Analytical and spectral data were in good agreement with the composition of synthesized compounds and the data are given in Table 1. The physicochemical properties of the titled compounds are presented in Table 2.
3. 2. Pharmacology
3. 2. 1. Anticonvulsant Activity
The pharmacological evaluation of the compounds (5a-t) was initially carried out according to the protocols of antiepileptic drug development programme (ADD), Epilepsy Branch, NIH [5, 12]. The compounds were initially screened in the mice using MES test. The MES test has become the most widely employed seizure model for the early identification and high throughput screening of investigational antiepileptic drugs. Minimal motor impairment in the form of neurotoxicity was measured by roto-rod test. The anticonvulsant activity and neurotoxicity data for the compounds are reported in Table 3. At the dose level of 30 mg/kg, all the compounds were showing average to good protection. Data is presented as % protection at time intervals of 0.5 h and 4 h. Phenytoin was used as a standard at the dose of 30 mg/kg.
Compounds 5f, 5n, and 5p exhibited 100% protection in the anti-MES activity. Compounds 5e, 5i, 5m, 5o and 5q showed their ability to prevent seizure spread with 83% protection whereas, compounds 5a, 5b, 5d, 5l and 5t showed 66% protection indicating their ability to elevate seizure threshold. Compounds 5c, 5g, 5h, 5j, 5k, 5r and 5s showed 50% protection. Thus majority of the compounds displayed preferential MES-selectivity.
In the neurotoxicity screen all the compounds were devoid of toxicity at the dose of 30 mg/kg body weight.
Glacial acetic acid
Br.
(la-e)
Glacial acetic acid
NaCNO
Cone. (NaOH)/EtOH
NHo.NHo.HoO
■nh-nh-
(3a-e)
R,
-nh-n=ch
(4a-t)
DMF/Triethvlamine Chloroacetvl chloride
3. 2. 2. Log P Determination
Experimental Log P value of compound 5f, 5n and 5p were found to be 2.44 ± 0.21, 2.11 ± 0.74 and 1.89 ± 0.71, respectively while their calculated values were 1.58 ± 0.71, 1.99 ± 0.68 and 2.46 ± 0.69, respectively. Compound 5n was coinciding to the theoretical value. All the selected compounds were lipophilic in nature.
(5a-t)
Scheme 1
3. 2. 3. Hepatotoxicity Studies
Liver samples from control group animals and all the experimental groups were within normal histological limits, except the sample 5n that showed a moderate portal inflammation but these changes are non-specific and
insignificant in nature (Fig. 1-3). No hepatocyte necrosis or degeneration was seen in any of the samples.
Enzyme estimation was done for selected compounds (5f and 5n) and was compared to the control and no any significant changes seen (**P < 0.01). Alkaline phosphatase values (± SEM) for compounds 5f, 5n and
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Table 1:	Spectral characterization of synthesized compounds (5a-t):		
Compd.	FT-IR (KBr,Vmax cm1)	1H-NMR (DMSO-d6) 8 ppm	Mass (EI) m/z
5a	3403 (NH), 3059 (CH-Ar), 1598 (C=O), 1462 (C=N), 1254 (C-N), 1094 (N-N), 812 (C-Cl), 719 (C-S-C) .	4.48 (s, 1H, CHCl) , 6.28 (s, 1H,CH), 6.60-7.86 (m, 8H, Ar-H), 8.99 ( s, 1H, NHN-, D2O exchangeable), 11.26 (bs, IH, NHC=O, D2O exchangeable	407 (M+)
5b	3490 (OH), 3310, 3291 (NH), 3063 (CH-Ar), 1603 (C=O), 1520 (C=N), 1270 (C-N), 1073 (N-N), 823 (C-Cl), 690 (C-S-C).	4.46 (s, 1H, CHCl), 6.12 (bs, 1H, D2O exchangeable), 6.87 ( s, 1H, CH), 7.22-8.01 (m, 7H, Ar-H), 8.89 (s, 1H, NHN-, D2O exchangeable), 11.01 (bs, 1H, NHC=O, D2O exchangeable )	
5c	3430 (OH), 3315( NH), 3090 (CH-Ar), 2918, 2849 (CH-Aliph), 1600 (C=O), 1462 (C=N), 1271 (C-N), 1124 (N-N), 814 (C-Cl), 719 (C-S-C).	3.76 (s, 3H, OCH3), 4.41 (d, 1H, CHCl), 5.84 (bs, 1H, OH, D2O exchangeable), 6.25 (d, 1H, CH), 7.20-7.96 (m, 6H, Ar-H), 8.86 ( bs, 1H, NHN-, D2O exchangeable), 11.00 ( bs, 1H, NHC=O, D2O exchangeable).	
5d	3179 (NH), 3056 (CH-Ar), 2921, 2851 (CH-Aliph.), 1681 (C=O), 1476 (C=N), 1272 (C-N), 1102 (N-N), 808 (C-Cl), 664 (C-S-C).	3.70 (s, 6H, 2-OCH3), 4.30 (d, 1H, CHCl), 6.68 (d, 1H, CH), 7.17-7.59 (m, 6H, Ar-H), 8.69 ( bs, 1H, NHN-, D2O exchangeable), 11.57 ( bs, 1H, NHC=O, D2O exchangeable).	467 (M+)
5e	3230 (NH), 3070 (CH-Ar), 1610 (C=O), 1458 (C=N), 1253(C-N), 1171 (C-F), 1102 (N-N), 840 (C-Cl), 693 (C-S-C).	4.45 (d, 1H, CHCl), 6.58 (d, 1H, CH), 6.74 - 7.76 (m, 8H, Ar-H), 8.90 (s, 1H, NHN-, D2O exchangeable), 11.16 (bs, 1H, NHC=O, D2O exchangeable).	-
5f	3495 (OH), 3250 (NH), 3056 (CH-Ar), 1610 (C=O), 1460 (C=N), 1253 (C-N), 1173 (C-F), 1115 (N-N), 815 (C-Cl), 670 (C-S-C).	4.58 (d, 1H, CHCl), 6.10 (s, 1H, OH), 6.54 (d, 1H, CH), 7.21-7.64 (m, 7H, Ar-H), 8.94 (s, 1H, NHN-,D7O exchangeable), 11.12 (bs, 1H, NHC=O, D2O exchangeable).	
5g	3394 (OH), 3270 (NH), 3058 (CH-Ar), 2923 (CH- Aliph.), 1609 (C=O), 1459 (C=N), 1250 (C-N), 1179(C-F), 1103 (N-N), 815 (C-Cl).	3.81 (s, 3H, OCH3), 4.46 (d, 1H, CHCl), 5.75 (s, 1H, OH), 6.42 (d, 1H, CH), 6.91-7.62 (m, 6H, Ar-H ), 8.87 (bs, 1H, NHN-, D2O exchangeable), 11.03 (bs, 1H, NHC=O, D2O exchangeable).	
5h	3297(NH), 3090 (CH-Ar), 2922 (CH-Aliph.), 1632 (C=O), 1463 (C=N), 1258 (C-N), 1165(C-F), 1080 (N-N), 797 (C-Cl).	3,80 (s, 6H, 2-OCH3), 4.36(d, 1H, CHCl), 6.62 (d, 1H, CH), 6.95-7.41 (m, 6H, Ar-H), 8.72 (s, 1H, NHN-, D2O exchangeable), 11.54 ( bs, 1H,NHC=O, D2O exchangeable).	
5i	3396 (NH), 3060 (CH-Ar), 1650 (C=O), 1463 (C=N), 1405 (C-NO^), 1251(C-N), 1131 (N-N), 819 (C-Cl), -719 (C-S-C).	4.44 (d, 1H, CHCl), 6.55 (d, 1H, CH), 7.38-8.43 (m, 8H, Ar-H), 8.96 (bs, 1H, NHN- D2O exchangeable), 11.22 ( bs, 1H, NHC=O, D2O exchangeable).	-
5j	3493(OH), 3270, 3167 (NH), 3077 (CH-Ar), 1603 (C=O), 1464 (C=N), 1420 (C-NO2), 1248 (C-N), 1099 (N-N), 814 (C-Cl), 697 (C-S-C).	4.46 (d, 1H, CHCl), 5.79(bs, 1H, OH), 6.45 (d, 1H, CH), 7.41-8.05 ( m, 7H, Ar-H ), 8.97 (bs, 1H, NHN-, D2O exchangeable), 11.01 ( bs, 1H, NHC=O, D2O exchangeable).	
5k	3409 (OH), 3301 (NH), 3066 (CH-Ar), 2923 (CH-Aliph.), 1610 (C=O), 1465 (C=N), 1430 (C-NO2), 1238 (C-N), 1097 (N-N), 819 (C-Cl), -701(C-S-C).	3.86 (s, 3H, OCH3), 4.44 (d, 1H, CHCl), 5.38 (bs, 1H, OH), 6.40 (d, 1H, CH), 6.89-8.69 (m, 6H, Ar-H), 8.90 (s, 1H, NHN-, D2O exchangeable), 11.06 (bs, 1H, NHC=O, D2O exchangeable).	463 (M+)
5l	3310 (NH), 3067 (CH-Ar), 2920 (CH-Aliph.), 1630 (C=O), 1439 (C=N), 1382 (C-NO2), 1243 (C-N), 1044 (N-N), 853 (C-Cl), 6297 (C-S-C).	3.75 (s, 6H, 2-OCH3), 4.87 (d, 1H, CHCl), 6.58 (d, 1H, CH), 7.22-8.45 (m, 6H, Ar-H), 9.02 (bs, 1H, NHN-, DjO exchangeable), 11.19 (bs, 1H, NHC=O, DjO exchangeable).	477 (M+)
Compd.	FT-IR (KBr,Vmax cm-1)	1H-NMR (DMSO-d6) 8 ppm	Mass (EI) m/z
5m	3279 (NH), 3091 (CH-Ar), 2926 (CH-Aliph.), 1607 (C=O), 1467 (C=N), 1238 ( C-N), 1103 (N-N), 819 (C-Cl), 690 (C-S-C).	2.43 (s, 3H, CH3), 4.42 (d, 1H,.CHCl), 6.53 (d, 1H, CH), 7.25-7.75 ( m, ^H, Ar-H), 8.91 (bs, 1H, NHN-, D2O exchangeable), 11.09 (bs, 1H, NHC=O, D2O exchangeable).	
5n	3399 (OH), 3278, 3109 (NH), 3058 (CH-Ar), 1615 (C=O), 1464 (C=N), 1253 (C-N), 1107 (N-N), 817 (C-Cl), 691 (C-S-C).	2.46 (s, 3H, CH3), 4.49 (d, 1H, CHCl), 5.60 (bs, 1H, OH), 6.49 (d, 1H, CH), 6.70-7.65 (m, 7H, Ar-H), 8.92 s, 1H, NHN-, D2O exchangeable), 11.12 (bs, 1H, NHC=O, D2O ex2changeable).	
5o	3388 (OH), 3107 (NH), 3059 (CH-Ar), 2923 (CH-Aliph.), 1630 (C=O), 1452 (C=N), 1267 (C-N), 1114 (N-N), 814 (C-Cl), 691 (C-S-C).	2.48 (s, 3H, CH3), 3.92 (s, 3H, OCH3), 4.52 (d, 1H, CHCl), 5.50 ( bs, 1H, OH), 6.44 (d, 1H, CH), 7.32-7.67 (m, 6H, Ar-H), 8.92 (s, 1H, NHN-, D2O exchangeable), 11.08 ( bs, 1H, NHC=O, D2O exchangeable ).	
5p	3310 (NH), 3058 (CH-Ar), 2923 (CH-Aliph.), 1632 (C=O), 1486 ( C=N), 1265 (C-N), 111O (N-N), 813 (C-Cl), 691 (C-S-C).	2.17 (s, 3H, CH3), 3.69 (s, 3H, OCH3), 3.75 (s, 3H, OCH3),4.52 ( d, 1H, CHCl), 6.38 (d, 1H, CH), 6.79-7.86 (m, 7H, Ar-H), 9.02 (bs, 1H, NHN-, D2O exchangeable, 11.00 (bs, 1H, NHC=O, D2O exchangeable)	
5q	3241 (NH), 3056 (CH-Ar), 2919 (CH-Aliph.), 1604 (C=O), 1466 (C=N), 1242 (C-N), 1111 (N-N), 815 (C-Cl), 701 (C-S-C).	3.79 (s, 3H, OCH3), 4.61 (d, 1H, CHCl), 6.61 (d, 1H, CH), 7.19-7.51 ( m, 8H, Ar-H), 8.90 (s, 1H, NHN-, D2O exchangeable, 11.11 (bs, 1H, NHC=O, D2O exchangeable).	
5r	3490 (OH), 3287, 3167 (NH), 3088 (CH-Ar), 2933 (CH-Aliph.), 1610 (C=O), 1490 (C=N), 1247 (C-N), 1097 (N-N), 819 (C-Cl), 692 (C-S-C).	3.75 ( s, 3H, OCH3), 4.48 (d, 1H, CHCl), 6.09 (bs, 1H, OH), 6.68 (d, 1H, CH), 7.56-7.99 (m, 7H, Ar-H), 9.01 (s, 1H, NHN-, D2O exchangeable), 11.17 ( bs, 1H, NHC=O, D2O ex2changeable).	
v5s	3488 (OH), 3323 (NH), 3058 (CH-Ar), 2923 (CH-Aliph.), 1598 (C=O), 1462 (C=N), 1238 (C-N), 1104 (N-N), 813 (C-Cl), 691 (C-S-C).	3.69 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 4.75 (d, 1H, CHCl), 5.57 ( bs, 1H, OH), 6.23 (d, 1H, CH), 7.08-7.51 (m, 6H, Ar-H), 8.93 (s, 1H, NHN-, D2O exchangeable), 11.13 ( bs, 1H, NHC=O, D2O exchangealble).	448 (M+)
5t	3251 (NH), 3010 (CH-Ar), 2918 (CH-Aliph.), 1604 (C=O), 1479 (C=N), 1253 (C-N), 1103 (N-N), 813 (C-Cl), 703 (C-S-C).	3.77 (s, 9H, 3-OCH3), 4.51 (d, 1H, CHCl), 6.22 (d, 1H, CH), 7.37-7.44 (m, 6H, A^r-H), 8.88 (bs, 1H, NHN-, D2O exchangeable), 11.08 ( bs, 1H, NHC=O, D2O excliangeable).	462 (M+)
Fig: 1 (100X)
Fig: 2 (100X)
Fig: 3 (400X)
Fig. 1-3. Low power (HE x 100x) and high power (HE x 400x) photomicrographs of liver from control (Fig. 1) and compound 5f (Fig. 2) showing a normal hepatic parenchyma with Portal Triad (PT), Central Vein (CV) and the hepatocytes. While in case of compound 5n (Fig. 3), liver showing normal Portal triad and Bile duct (BD) structures except here is mild but insignificant periportal inflammatory cell infiltration.
Table 2: Physicochemical properties of compounds (5a-t)
Compd No.	R	R1	R2	aMol. Formula	Yield (%)	bM.P. (oC)	cElemental analysis (% N) Calcd.(Found)	dRr (Rm)e
5a	Cl	H	H	ClvHl3Cl3N4O3S	50	286	13.70 (13.76)	0.96 (-1.38)
5b	Cl	OH	H	CJ7HJ3Cl3N4O3S	49	240	13.28 (13.24)	0.98 (-1.69)
5c	Cl	OH	OCH3	Cl8Hl4Cl3N4O4S	55	270	13.40 (12.36)	0.85 (-0.75)
5d	Cl	OCH3	OCH33	C19H16Cl3N4O4S	52	296	12.03 (11.99)	0.99 (-1.99)
5e	F	H	H	Cj7Hj3ClFN4O3S	54	230	14.40 (14.34)	0.84 (-0.72)
5f	F	OH	H	Cj7Hj3ClFN4O3S	53	242	13.81 (13.77)	0.90 (-0.95)
5g	F	OH	OCH3	C18H14ClFN4O4S	50	260	12.80 (12.83)	0.92 (-1.06)
5h	F	OCH3	OCH33	C19H16ClFN4O4S	51	188	12.47 (12.43)	0.87 (-0.82)
5i	NO2	H	H	C1vH13ClN5O4S	54	265	16.70 (16.76)	0.83 (-0.68)
5j	NO2	OH	H	Cj7Hj3ClN5O5S	56	290	16.18 (16.14)	0.91 (-1.00)
5k	NO2	OH	OCH3	C18H12ClN5O5S	45	280	15.14 (15.10)	0.89 (-0.90)
5l	NO2	OCH3	OCH33	C19H16ClN5O6S	53	238	14.70 (14.65)	0.82 (-0.65)
5m	CH3	H	H	C18H15ClN4O3S	53	268	14.44 (14.48)	0.87 (-0.82)
5n	CH3	OH	H	C18H15ClN4O3S	52	250	13.96 (13.91)	0.93 (-1.12)
5o	CH3	OH	OCH3	C19H17ClN4O4S	50	300	13.00 (12.94)	0.88 (-1.18)
5p	CH3	OCH3	OCH33	C3„Hj9ClN4O4S	55	250	12.56 (12.54)	0.94 (-1.19)
5q	OCH3	H	H	C18H15ClN4O3S	56	220	13.95 (13.91)	0.86 (-1.18)
5r	OCH3	OH	H	C18H15ClN4O4S	54	254	13.42 (13.38)	0.97 (-1.50)
5s	OCH33	OH	OCH3	C19H17ClN4O5S	58	288	12.54 (12.48)	0.95 (-1.27)
5t	OCH33	OCH3	OCH33	C30H19ClN4O5S	54	270	12.16 (12.10)	0.81 (-0.62)
"Solvent of crystallization : Ethanol; bMelting point of the compounds at their decomposition; cThe elemental analysis data were in agreement to the calculated values (in the range of ± 0.4%) ^Solvent system Benzene: Acetone (8:2); eA logarithmic function of Rf value was also calculated ;
Table 3: Anticonvulsant and neurotoxicity data of the titled compounds (5a-t)
Compd. No.	MES screena (% Protection)		Neurotoxicity screena	
	0.5 h	4 h	0.5 h	4 h
5a	66	66	X	X
5b	66	66	X	X
5c	50	50	X	X
5d	66	66	X	X
5e	83	83	(-)	(-)
5f	100	100	(-)	(-)
5g	50	50	X	X
5h	50	33	X	X
5i	83	66	(-)	(-)
5j	50	50	X	X
5k	50	50	X	X
5l	66	66	X	X
5m	83	83	(-)	(-)
5n	100	100	(-)	(-)
5o	83	83	(-)	(-)
5p	100	100	(-)	(-)
5q	83	83	(-)	(-)
5r	50	50	X	X
5s	50	50	X	X
5t	66	66	X	X
Phenytoin	100	100	(-)	(-)
aIntraperitoneal dose of 30 mg/kg was administered and the animals were examined 0.5 and 4 h after administration. The dash (-) indicates an absence of activity. x denotes not tested.
control were found to be 19.46 ± 0.80, 23.70 ± 0.30 and 13.06 ± 0.25, respectively. SGOT ± SEM values for compounds 5f, 5n and control were found to be 150.0 ± 1.50, 176.1 ± 1.96 and 148.67 ± 1.50, respectively. SGPT ± SEM values for compounds 5f, 5n and control were found to be 19.50 ± 0.85, 39.17 ± 1.83 and 27.67 ± 0.84, respectively.
Protein estimation was also done for the compounds (5f and 5n) and was compared to the control and no any significant changes seen (**P < 0.01). Albumin (g/100 mL) ± SEM values for compounds 5f, 5n and control were found to be 1.70 ± 0.27, 1.98 ± 0.03 and 1.67 ± 0.009, respectively. Globulin (g/100 mL) ± SEM values for compounds 5f, 5n and control were found to be 0.74 ± 0.02, 0.12 ± 0.007 and 0.13 ± 0.01 respectively. Total Protein (g/100 mL) ± SEM values for compounds 5f, 5n and control were found to be 2.45 ± 0.02, 2.11 ± 0.02 and 1.80 ± 0.01, respectively. Albumin / Globulin (g/100 mL) ± SEM values for compounds 5f, 5n and control were found to be 2.30 ± 0.09, 16.00 ± 1.16 and 13.84 ± 1.57, respectively.
4. Conclusions
In this study it was concluded that the basic structure of the compounds have all the pharmacophoric elements necessary for the anticonvulsant activity. In general, compounds bearing F, CH3 at the 6-position of benzot-
Rm = log (1-1/Rf).
hiazole moiety with H, OCH3 at the 3-position and OH, OCH3 at 4-position of the distant phenyl ring showed a highly potent activity. Whereas, replacements with Cl, NO2, OCH3 at the 6-position of benzothiazole ring with H, OCH3 at the 3-position and OH at 4-position of distant phenyl ring resulted in 50% decrease in potency. Compounds with F, NO2, CH3, OCH3 substituents at the 6-po-sition of the benzothiazole moiety and unsubstituted distant phenyl ring showed moderate decrease in activity. A compound substituted with OCH3 at the 6-position of the benzothiazole moiety and 3, 4-positions of distant phenyl ring displayed significant activity.
5. Acknowledgement
The Financial Assistance by the University Grants Commission (UGC), New Delhi is gratefully acknowledged.
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Povzetek
Sintetizirali smo številni nove spojine 1-[2-(3,4-disubstituirani fenil)-3-kloro-4-oksoazetidin-1-il]-3-(6-substituirani-1,3-benzotiazol-2-il) uree poimenovane (5a-t), ovrednotili smo njihovo antikonvulzivno delovanje, hepatotoksicnost in nevrotoksicnost. Spojine (5a-t) smo pridobili s ciklizacijo 3,4-disubstituirani benzaldehid-N-(6-substituirani-1,3-benzo-tiazol-2-il) semikarbazonov (4a-t) v prisotnosti DMF/trietilamina in kloracetilklorida. Vse novo sintetizirane spojine smo testirali za antikonvulzivno delovanje s testom z elektrošokom izzvanega epilepticnega napada (model MES i.p.) in jih primerjali z delovanjem standardnega zdravila fenitoina. Zanimivo, spojine 5f, 5n in 5p so 100 % zaščitile pred epi-lepticnimi napadi. Pri odmerku 30 mg/kg telesne teže nobena izmed substanc ni imela hepatotoksičnih ali nevrotoksič-nih učinkov. Naša študija je pokazala, da uvedba F, zamenjava CHj na položaju 6 benzotiazolne strukture z H, OCHj na položaju 3 in OH, OCHj na položaju 4 oddaljenega fenilnega obroča povzroči povečano aktivnost. Uvedba F, NO^, CH3, OCH3 na položaju 6 benzotiazola in nesbstituiranega oddaljenega fenilnega obroča povzroči zmerno zmanjšanje aktivnosti.