INTRODUCTION:

Epilepsy, a ubiquitous disease characterized by recurrent seizures, inflicts more than 60 million people worldwide accordingto epidemiological studies¹. For epilepsy treatment, nearly 95% of clinically available drugs were approved before 1985 and they could provide satisfactory seizure control for 60–70% of patients. These drugs, however, also cause notable adverse side effects such as drowsiness, ataxia, gastrointestinal disturbance, hepatotoxicity and megaloblastic anemia^2-4, and even life threatening conditions. According to Jackson⁵, the origin of seizures may be intense discharge of gray matter in various regions of brain. DeRobertis⁶ list mechanisms of convulsive disorders, loss of normal inhibitory control mechanism and chemical super sensitivity hat increases excitability of neuronal elements. Involvement of motor cortex causes convulsions;involvement of the hypothalamus causes peripheral automic discharges and involvement of the reticular formation in the upper brain stem leads to loss consciousness^7,8 .

Anticonvulsant drugs acts by varities of mechanism⁹ , Research to find more effective and safer antiepileptic drugs is, therefore, imperative and challenging in medicinal chemistry.

The quinazoline nucleus has been found to possess varied pharmacological activities viz.central depressant¹⁰,central muscle relaxant¹¹,cardiovascular and respiratory depressant effect¹², antiviral action¹³, anticonvulsant,^14,15bronchodilator,^16,17anti-inflammatory,^18-20antimalarial,²¹antituberculous,²²anti-HIV,²³narcotic antagonist,²⁴anti-tumor,²⁵tyrosinekinase inhibitor,²⁶adenosine antagonist,²⁷antimicrobial,²⁸etc. Similarly various substituted benzothiazoles are known to possess pharmacological activities like anti-tumor,²⁹antimicrobials,^30,31anthelmintic,³²analgesic,³³anti-inflammatory,³⁴anticonvulsant,^35,36 and CVS agents.³⁷

The simultaneous use of several drugs to treat epileptic conditions, associated with some side effects may cause health problems. Also, from the pharmaco-economic point of view, and for better patient compliance, an anti-convulsant with minimum adverse effects and high safety margin is highly desirable. Aimed at exploring effective compounds with better anticonvulsant activity and lower neurotoxicity, 6 compounds were designed and synthesized. In view of the fact that several 4-oxoquinazoline derivatives possess useful anti-convulsant as well as antimicrobial properties,^28,38we designed and synthesized various derivatives of 2-ethyl-3-(substituted- benzothiazol-2-yl)-4(3H)-quinazolinone. Also with aim of obtaining the novel potent anti-convulsant agents with fewer side effects, we decided to combine the benzothiazole nucleus with the quinazoline molecule. Moreover, it was considered of interest to substitute various groups on the benzothiazole nucleus to investigate the influence of such structural variation on the anticipated biological activities. Thus in the present investigation, twenty six different derivatives of 6-substituted 2-ethyl-3-(substituted-benzothiazol-2-yl)-4(3H)-quinazolinone were synthesized. Their structures were characterized using IR, 1H NMR and elemental analysis techniques. In additional, their anticonvulsant activity was evaluated using MES test and reported for the first time. For explaining the possible mechanism of action, the compounds were tested in pentylenetetrazole (PTZ).

EXPERIMENTAL:

All the chemicals used for the present study were of Rankem, LOBA-chemie and Qualigens grade. Phenytoin required for anticonvulsant activity study was of Pfizer India Ltd. (Dilantin). The melting points of compounds were determined in open capillaries using a thermoink precision apparatus and are uncorrected. The purity and homogeneity of the compounds was checked by Thin Layer Chromatographic technique by using glass plate coated with silica gel G as absorbent.

The IR spectra of compounds were recorded using KBr pellets and Shimadzu Fourier Transform Infrared Spectrophotometer (FTIR-8400s). PNMR spectra were recorded using Bruker W. M. 400 spectrophotometer (Bruker AG, Fallanden, Switzerland) at 400 MHz using TMS as internal standard (Chemical shift in d ppm).

General procedure for the preparation of 6-iodo-2-ethyl-4(3H)-3(substituted benzothiazole-2’-yl)-quinazolinones

Scheme-I Synthesis of 2-amino benzothiazole/substituted2-amino benzothiazoles

In round bottom flask, aniline (20 mL) was taken and to it concentrated hydrochloric acid (20-25 mL) was added and warmed. Saturated solution of ammonium thiocynate (20g in 50 mL) was added slowly to above solution and it was boiled until turbidity appeared. This turbid solution was poured in cold water, gave precipitate of phenylthiourea. It was filtered, washed with water and dried. The compound was recrystallised by using dilute alcohol (70% v/v). Phenylthiourea (15.2g, 0.1 mole) was taken in 250-mL beaker. To it 150 mL carbon tetrachloride was added, and then it was brominated by using 100mL of bromine solution (5% v/v) in carbon tetrachloride till orange-yellow colour persists. The slurry was kept overnight. The precipitated dibromide was filtered, washed with carbon tetrachloride until the yellow colour disappeared. The precipitate of dibromide was dissolved in rectified spirit (200 mL) and basified with concentrated ammonia solution, gave precipitate of 2-amino benzothiazole.

It was filtered, washed with water and dried. The compound was recrystallised by using dilute alcohol (70% v/v). The yield of the product was 12.46g (83.01%) and m.p. 130-132 °C (Lit. 129 °C) ^33,
34.

Substituted 2-amino benzothiazoles

Scheme 2 Synthesis of 6-iodo-2-ethyl-4(3H)-benzoxazone.

Synthesis of 2-ethyl-4(3H)-benzoxazone:

13.7 g (0.1mole) of anthranilic acid and 13.0 mL (0.1mole) of propionic anhydride were refluxed on heating mental for 30 min. Then excess of propionic anhydride was removed under vacuum. The separated solid was again refluxed with 10.2 mL (0.1mole) of acetic anhydride for 30 min. The excess of acetic anhydride was removed under vacuum. The separated solid was washed several times with petroleum ether (60:80), dried and recrystallised using dilute ethanol. The yield of the product was 12.54 g (71.73%) and m.p 84-86 °C (Lit. 85-86 °C) ³⁵.

Preparation of 5-iodo anthranilic acid: With mechanical stirring, 15g (0.184mole) of recrystallised anthranilic acid (m.p. 145°C) was dissolved in a solution of 10.94 g of potassium hydroxide (stick) in 325 mL of water contained in a two-litre beaker. A solution of 27.9 g (0.184mole) of iodine in 185 mL of water containing 14.85 g of potassium hydroxide (stick) was slowly run into the well stirred potassium anthranillate solution. After 1 minute 100 mL of glacial acetic acid was quickly added and the reaction mixture immediately diluted with 100 mL of water. A dark precipitate began to appear almost at once, stirring was continued for one hour, during which time this precipitate assumed a light brown colour. After standing undisturbed for two hours, excess iodine was removed by adding 25 mL of (15%v/v) sodium bisulphite and thoroughly agitating. The mixture was allowed to stand short while. The precipitate was collected, repeatedly washed with water and air dried. Yield of crude product was 29.23 g (78.95%). On crystallization from dilute alcohol gave (after working up the mother liquor) 17.28 g (59.11%) of product melting at 208-211°C.

(Lit.³⁶ 210-211.5°C).

Preparation of 6-iodo-2-ethyl-4(3H)-benzoxazone: It was synthesized by using 13.35 g (0.1mole) of 5-iodo anthranilic acid and 6.5mL (0.1mole) of propionic anhydride following the procedure described under the preparation of 2-ethyl-4(3H)-benzoxazone(I). The yield of recrystallised product was 11.10 g (72.78%) and melting point 178-180°C(Lit. 181°C) ³⁵.

Scheme 3 Synthesis of 6-iodo-2-ethyl-4(3H)-3(substituted benzothiazole-2’-yl)-quinazolinones

In a 100ml round bottom flask; 2-amino benzothiazole(0.375 g) (0.025 mole) and 6-iodo-2-ethyl-4(3H)-benzoxazone (0.762 g) (0.025 mole) was added in 20 mL glacial acetic acid and 1.5 g of sodium acetate.This mixture was reflux for 25 hours on oil bath at about 115-120°C, it was cooled and poured on 100-150 g crush ice gave a precipitate. The precipitate was filtered, washes with cold water, dried and recrystallised with ethanol. The yield was (0.785 g) (71.88%) and m.p. 318-320°C.

The remaining 6-iodo-2-ethyl-4(3H)-3(substituted benzothiazole-2`yl) quinazolinones were prepared by using 0.025 moles of each of substituted 2-amino benzthiazoles and 6-iodo-2-ethyl-4(3H)-benzoxazone using above described procedure.

6-iodo-2-ethyl-4(3H)-3(substituted benzothiazole-2’-yl)-quinazolinones

Chemical data of compounds of scheme III (a-f)

Compounds	X	R₄	R₅	R₆	Formula	Yield (%)	m.p.(°C)
a	I	H	H	H	C₁₇H₁₂N₃IOS	76.36	317-320
b	I	OCH₃	H	H	C₁₈H₁₄N₃IO₂S	66.69	266-268
C	I	H	OCH₃	H	C₁₈H₁₄N₃IO₂S	63.31	267-270
d	I	CH₃	H	H	C₁₈H₁₄N₃IOS	60.43	111-112
e	I	OC₂H₅	H	H	C₁₉H₁₆N₃IO₂S	65.42	181-183
f	I	H	Cl	H	C₁₇H₁₁N₃IclOS	65.59	219-221

Elemental analyses for C, H, N are within ± 0.4% of the theoretical values.

Physicochemical studies:

Thin layer chromatography (TLC):

The solutions of the compounds were prepared in methanol. The spots were applied by the capillary tubes on the TLC plates and were chromatographed using the solvent system chloroform: toluene (3:1). After drying of the plates the spots were visualized by using iodine vapors. All the test compounds gave single spot without tailing, ascertaining the purity and homogeneity of the compounds.

Spectral data of compounds (a-f)

IR Spectral Data

Compd.	Spectral Data (cm^-1)
a	IR [KBr] 598 (C-I),1630(C=N), 1718 (C=O), 3025-3285 (C-H), 2918- 2850 (CH₂,CH₃)
b	IR [KBr] 618 (C-I), 1618 (C=N), 1720(C=O), 2850 (O-CH₃), 3025-3285 (C-H), 2918-2865 (CH₂, CH3)
c	IR [KBr] 595 (C-I), 1624 (C=N), 1725 (C=O), 2850 (O-CH₃), 3025-3275 (C-H), 2918-2850(CH₂,CH₃)
d	IR [KBr] 590 (C-I), 1635 (C=N), 1720 (C=O), 3075 -3080(C-H), 2918-2850 (CH₂,CH₃)
e	IR [KBr] 590 (C-I), 1635 (C=N), 1720 (C=O), 3075-3180 (C-H), 2918-2850 (CH₂,CH₃)
f	IR [KBr] 598 (C-I), 708 (C-Cl), 1620 (C=N), 1708 (C=O), 3108-3265 (C-H), 2918-285O (CH₂,CH₃)

PNMR Spectral Data

Compd.	PNMR data 1H (CD₃OD,ppm)^a
a	8.42(d,J=8.41 Hz,1H), 7.98(d,J=7.94 Hz,1H), 7.38(d,J=8.00 Hz,1H) , 7.34(t,J=7.90 Hz,1H), 2.57(q,J=7.44 Hz,2H), 2.87(s,3H), 1.21(t,J=7.43Hz,3H).
b	8.40 (d,J=7.93 Hz, 1H), 7.93 (t,J=7.87 Hz, 1H), 7.72 (d, J =7.42 Hz , 1H), 7.34 (dd, J =7.30 Hz,1H), 7.07 (t, J =7.42Hz,2H), 3.80 (s, 3H), 2.62(q,J=7.4 Hz,2H),1.27(t,J=4.2 Hz,3H).
c	8.80(d,J=8.48Hz,1H),8.00(d,J=7.94Hz,1H),7.62(t,J=7.12Hz,1H), 7.62(t,J=7.12,1H),2.43(s,3H),2.71(q,J=2.80Hz,2H),2.75(q,J=2.84,2H),
d	8.38(d,J=7.9Hz,1H),7.90(t,J=8.1,1H),7.35(d,J=8.00Hz,2H) 7.30(d,J=7.43Hz,1H),7.13(t,J=7.4Hz,2H),2.63(q,J=7.44Hz,2H), 2.52(q,J=7.4Hz,2H),1.27(t,J=7.34Hz,3H).
e	8.80(d,J=8.48Hz,1H),8.00(d,J=7.94Hz,1H),7.62(t,J=7.12Hz,1H), 7.62 (t,J=7.12,1H),2.43(s,3H),2.71(q,J=2.80Hz,2H),2.75(q,J=2.84,2H).
f	8.42(d,J=8.41 Hz,1H), 7.98(d,J=7.94 Hz,1H), 7.38(d,J=8.00 Hz,1H) , 7.34(t,J=7.90 Hz,1H), 2.57(q,J=7.44 Hz,2H), 2.87(s,3H), 1.21(t,J=7.43Hz,3H).

s, singlet ; dd , doublet ; m, multiplet.

Pharmacology:

Animals: Swiss albino mice of either sex weighing between 25-30 g were used. Animals were housed in perpex cages under standard conditions of temperature (26±2ºC) and 12:12 hr light: dark cycle (lights on at 7.00 am). Animals were allowed to acclimatize to laboratory conditions with free access to food and water for a period of 24hr before testing. All the experimental protocols were approved by the Institutional Animal Ethical Committee (IAEC) and experiments were conducted in accordance with the guidelines provided by the Committee for the Purpose of Control Supervision of Experimental Animals (CPCSEA).

Drugs Used:

The following drugs were used.

1: Pentylenetetrazole(Sigma USA)

2: Phenytoin injection (Eptoin, Sun Pharmaceuticals India Ltd.)

3: Diazepam injection (Dilantin, Pfizer India Ltd.)

Suspensions of all the test compounds were prepared by using aqueous solution of 30% PEG 400. The above injections were diluted with saline for administration.

EXPERIMENTAL:

The anticonvulsant activity was assessed by using MES and PTZ induced seizures models described by Swinyard.⁴²

Maximal Electroshock Method:

The animals were randomly divided into different groups (control, standard and test) consisting of five animals each. The animals were treated with Phenytoin (25mg/kg i.p). The test compounds (A-1 to A-6) or vehicle were given intraperitoneally; 30 min prior to subjecting them to the Maximal electroshock test (MES) using electro convulsiometer (Techno Instruments India). The seizures were induced by shock (45mA/0.2sec) via corneal electrodes and the animals were observed for the presence or absence of tonic convulsions. The reduction in the duration of tonus or abolition of tonic extensor phase was considered as anticonvulsant activity of test compounds.

Statistical analysis:

The data was analyzed by One Way ANOVA followed by Dunnett’s test using Graph Pad Prism software. A value of P<0.05 was considered as statistically significant. All values are expressed as mean ± SEM.

Anticonvulsant profile of test compounds (3a-3f)

Treatment	Mean duration of tonic		Protection (%)
Treatment	On set time	hind leg extension ± SEM	Protection (%)
Vehicle + shock	3±0.246	15.40 (± 0.50)	100
Phenytoin + shock (25mg/kg)	nil	nil	100
3a + shock	9.5±0.745	7.40 (± 0.50)**	100
3b + shock	8.6±0.647	8.60 (± 0.67) **	100
3c + shock	10.4±0.790	10.60 (± 0.44)**	100
3d + shock	9.8±0.493	11.20 (± 0.86)**	100
3e + shock	10.6±0.897	11.80(± 0.86)**	100
3f + shock	8.7±0.986	12.00 (± 0.70)**	100

Compounds were tested at 20 mg/ kg dose level, i.p.

Statistical analysis: The data was analyzed by One Way ANOVA followed by Dunnett’s test using Graph Pad Prism software. A value of P<0.05 was considered as statistically significant. All values are expressed as mean ± SEM.

Anticonvulsant profile of test compounds (3a-3f)

Treatment	Clonic convulsion duration ± SEM (sec)		Protection (%)
Treatment	On set time	Total duration	Protection (%)
Vehicle + shock	76.80±2.41	660(±20.89)	100
Diazepam + shock (1mg/kg)	111.6±2.015	223.0(±20.45) **	100
3a + PTZ	91.60±2.31	330.0(± 19.80)**	100
3b + PTZ	93.00±1.18	380.0(± 20.80) **	100
3c + PTZ	89.80±1.020	350.0(± 16.50)**	100
3d + PTZ	91.6±1.280	328.0(± 25.70)**	100
3e + PTZ	93.20±1.028	395.0(± 20.86)**	100
3f + PTZ	91.00±1.51	365 (± 25.70)**	100

Dose of PTZ; 80mg/kg, s.c.

Compounds were tested at 20 mg/ kg dose level, i.p

CONCLUSION:

In general, most of the compounds showed significant anticonvulsant activity in mice at 20 mg/kg dose level. The anticonvulsant activity was assessed by using MES and PTZ induced seizures models described by Swinyard. It was found that the compounds possessing electron releasing groups such as methyl, methoxy, ethoxy, halogen substituent on benzthiazoles ring at position 4 and 5 of 6-iodo-2-ethyl-4(3H)-3(substituted benzothiazole-2’-yl)-quinazolinones considerably enhanced the anticonvulsant activity when compared to the 6-iodo-2-ethyl-4(3H)-3(benzothiazole-2’-yl)-quinazolinones having no substituent’s on the rings.

REFERENCES:

1. Goodman and Gilman, “The Pharmacological basis of therapeutics”, Vol.X, McGraw Hill publication Division, NY, (2001) p. 521-522.

2. Wilson and Gisvold, “Textbook of Organic Medicinal and Pharmaceutical Chemistry”, Vol. XI, Lippincott Williams and Wilkins, A Wolter Kluwer company, 530 Walnut Street Philadelphia, (2004) p.1-2.

3. Bertram G. Katzung, “Basic and Clinical Pharmacology” Vol.X, McGraw Hill publication, NY, (2004) p.548-551.

4. J.A.Donald, Burger’s, In “Medicinal Chemistry and Drug Discovery”, Vol.VI, A John Will and Sons, Inc., Publication Hoboken New Jersey, (2003) p. 264-311.

5. J. H. Jackson, “Selected Writings of John Hughlings Jackson”, Hodder and Stroughton, 1, (1931).

6. E. De Roberties, G. De Loves-Amuiz and M. Alberici, “Basic Mechanism of Epilepsies”, Little Brown and Co., Boston,(1969) p. 137-158.

7. Vogels Drug Discovery and Evaluation, “Pharmacological assays” Springer-Verlag Publication,Berlin Heidelberg, Vol.II, (2007) p. 504-510.

8. F. S. K. Barar, “Essentials of Pharmacotherapeutics”, S. Chand and Co. Ltd. Ram nagar, New Delhi (2004) p. 95-103.

9. H. P. Rang, M. M. Dale, J. M. Ritter and P. K. Moore, “Antiepeleptic Drug”, Vol. VI, Elsevier Science, London (2003)p. 552-553.

10. E.F. Domino, R. J. Peterson and K. R. Unna, J. Pharmacol. Exp Ther., 103, 342 (1951).

11. W.H. Funderburk, E. E. King, E. F. Domino and K. R. Unna, J. Pharmacol. Exp Ther., 107, 356 (1952).

12. J.Domino, E. F. Unna and K. R. Kerwin, J. Pharmacol. Exp Ther., 105, 486 (1952).

13. Akerfellt Sting, J. Med. Chem., 13, 1012 (1970).

14. K. P. Bhusari, P. B. Khedekar, S. N. Umathe, R. H. Bahekar, A. Raghu Ram Rao, Indian Journal of Heterocyclic Chemistry, 275-278 (2000).

15. R. Kailash Raj and A. Kurup, J. Pharmacol. Exp Ther., 74, 64 (1962).

16. Bullowitzkurt, Arch. Expt. Pathol. Pharmacol., 163, 687 (1931).

17. R. A. Webster, Brit. Pharmacol., 17, 507 (1961).

18. K. P. Bhargava and R. K. Srivastava, Brit. Pharmacol., 25, 74 (1965).

19. H. Fujimura, Bull. Inst. Chem., Res. Kyoto Univ., 20, 69 (1950).

20. K. P. Bhusari, P.B. Khedekar, S. N. Umathe, R. H. Bahekar, A. R. R. Rao, Indian Journal of Heterocyclic Chemistry, 10, 231-232 (2001).

21. J.F. Wolfe, T. L. Rathman, M. C. Sleevi, J. A. Campbell and T. D. Greenwood, J. Med. Chem., 33, 161-166 (1990).

22. Archana, V. K. Srivastava, Ramesh Chandra and Ashok Kumar, Indian Journal of Chemistry, 41B, 2371-2375 (2001).

23. U.S. Pathak, I. S. Rathod, M. H. Patel, V. S. Shirsath and K. S. Jain, Indian Journal of Chemistry, 42B, 617-623 (1995).

24. D.Raffa, G. Daidone, D. Schillaci, B. Maggio and F. Plescia, Pharmazie, 4, 54 (1999).

25. Mirdulla Tyagi and Ashok Kumar, Indian Journal of Chemistry, 41B, 2381-2385 (2001).

26. A.R. Bhat, G. Goutham Shenoy, Mohan Kotian, Indian Journal of Heterocyclic Chemistry, 9, 319-320 (2000).

27. S.D. Singh, M. B. Raju, R. H. Bahekar, K. S. Rajan and R. R. Rao, Indian Journal of Chemistry, 49D, 813-816 (2001).

28. M.A. Aziza, M. W. Nasser, S. G. Abdul Hamide, A. E. El-Hakim and A. S. El-Azab, Indian Journal of Heterocyclic Chemistry, 6, 25-30 (1996).

29. S.El-Meligie, A. K. El-Ansari, M. M. Said and M. M. M. Ansari, Indian Journal of Chemistry, 40B, 62-69 (2001).

30. R.H.Bahekar and A. R. R. Rao, Arzneimittel-Forschung/ Drug Research, 50(1), 8, 248-292 (2001).

31. J.C. Cartillo and De Beer, J. Pharmacol. Exp Ther., 90, 104 (1947).

32. Van Arman and J. C. Cartillo, J. Pharmacol. Exp Ther., 133 (1960).

33. B.R. Shah, J. J. Bhat, H. H. Patel, N. K. Undavia, P. B. Trivedi and N. C. Desai., Indian Journal of Chemistry, 34B, 201-208 (1995).

34. Sachin S. Laddha, Sudhir G. Wadodkar, and Sharad K. Meghal, ARKIVOC, (xi), 1-20 (2006).

35. P.H. Bhargava, K. A. Ghosh, J. Ind Chem Soc., 35, 314 (1960).

36. D.T. Zentmyer, E. C. Wagner, J. Org. Chem, 19, 967-980 (1949).

37. Cohen, J. Chem. Soc., 85, 770-773 (1904).

38. R.A. Turner, “Screening Methods of Pharmacology”, Academic Press Inc., 111 Fifth Avenue, NY, (1965) p. 34-35, 162.

Received on 05.09.2011 Modified on 11.09.2011

Asian J. Research Chem. 4(11): Nov., 2011; Page 1717-1721