Toxicological & Environmental Chemistry Synthesis ...

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Toxicological & Environmental Chemistry

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Synthesis and anticonvulsant activity of 6,7,8,9-tetra hydro-5H-5-(2'hydroxy phenyl)-2-(4'-substituted benzylidine)-3-(4-nitrophenyl amino) thiazolo quinazoline derivatives

Theivendren Panneerselvama; Palanirajan Vijayaraj Kumarb; Chinnasamy Rajaram Prakashc; Sundararajan Rajad a Department of Biotechnology, Acharya Nagarjuna University, Guntur 522510, India b School of Pharmacy, UCSI (University College Sadaya International) University, Kuala Lumpur, Malaysia c Department of Pharmaceutical chemistry, DCRM Pharmacy College, Inkollu 523167, India d Department of Pharmaceutical Chemistry, K.P. College of Pharmacy, Thiruvannamalai 606603, India First published on: 28 February 2011 To cite this Article Panneerselvam, Theivendren , Kumar, Palanirajan Vijayaraj , Prakash, Chinnasamy Rajaram and Raja,

Sundararajan(2011) 'Synthesis and anticonvulsant activity of 6,7,8,9-tetra hydro-5H-5-(2'-hydroxy phenyl)-2-(4'substituted benzylidine)-3-(4-nitrophenyl amino) thiazolo quinazoline derivatives', Toxicological & Environmental Chemistry, 93: 4, 643 — 655, First published on: 28 February 2011 (iFirst) To link to this Article: DOI: 10.1080/02772248.2011.556636 URL: http://dx.doi.org/10.1080/02772248.2011.556636

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Toxicological & Environmental Chemistry Vol. 93, No. 4, April 2011, 643–655

Synthesis and anticonvulsant activity of 6,7,8,9-tetra hydro-5H-5-(20 hydroxy phenyl)-2-(40 -substituted benzylidine)-3-(4-nitrophenyl amino) thiazolo quinazoline derivatives Theivendren Panneerselvama*, Palanirajan Vijayaraj Kumarb, Chinnasamy Rajaram Prakashc and Sundararajan Rajad

Downloaded By: [Panneerselvam, T.] At: 04:41 19 March 2011

a

Department of Biotechnology, Acharya Nagarjuna University, Guntur 522510, India; bSchool of Pharmacy, UCSI (University College Sadaya International) University, Jalan Menara Gading, Cheras 56000, Kuala Lumpur, Malaysia; cDepartment of Pharmaceutical chemistry, DCRM Pharmacy College, Inkollu 523167, India; dDepartment of Pharmaceutical Chemistry, K.P. College of Pharmacy, Thiruvannamalai 606603, India (Received 25 October 2009; final version received 15 January 2011) In this study, a series of thiazolo quinazoline derivatives were designed and synthesized to meet the structural requirements essential for anticonvulsant activity. Anticonvulsant activity was determined after intraperitoneal administration to mice by maximal electroshock- and subcutaneous pentylenetetrazoleinduced seizure tests and minimal motor impairment was determined by rotorod test. A majority of the compounds exhibited signiEcant anticonvulsant activity after intraperitoneal administration. The most active compounds carry fluoro, bromo, or chloro substituents in the 4-position of the phenyl ring. The chemical structures of the synthesized compounds were confirmed by infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, mass spectrometry, and elemental analysis. Among the synthesized compounds, 6,7,8,9-tetra hydro-5H-5(20 -hydroxy phenyl)-2-(40 -fluoro benzylidine)-3-(4-nitrophenyl amino) thiazolo quinazoline (5d) was found to be the most potent anticonvulsant activity. Keywords: thiazolo quinazoline; benzylidine thiazolo quinazoline; anticonvulsant activity

Introduction Epilepsy is a disease characterized by paroxysmal, excessive, and hypersynchronous discharges of a large number of neurons. Being one of the world’s oldest recognized disorders, it is surrounded by fear, discrimination, and social and frightening manifestation. In fact, epilepsy is the third most frequent neurological disorder encountered in the elderly after cerebrovascular disease and dementia (Kramer 2001). In recent years, the development of novel therapeutics resulted in the availability of several newer drugs (such as pregabalin, stiripentol, zonisamide, tiagabine, lamotrigine, levetiracetam, and topiramate) as promising anticonvulsants (Donner and Snead III 2006; Pollard and French 2006; Stefan and Feuerstein 2007). Yet, these drugs are effective in only 50–80% of the patients, and also exert some undesirable side effects such as vertigo, ataxia, headache, hirsutism, hepatotoxicity, gastrointestinal, and cardiovascular side effects *Corresponding author. Email: [email protected] ISSN 0277–2248 print/ISSN 1029–0486 online ß 2011 Taylor & Francis DOI: 10.1080/02772248.2011.556636 http://www.informaworld.com


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(Korolkovas 1988; Rang, Dale, and Rilter 1999; Kwan, Sills, and Brodie 2001; Williams and Lemke 2002; Czapinski, Blaszczyk, and Czuczwar 2005; Rogawski 2006). Recent data relevant to this problem show the efficacy of some substances with antioxidant properties in the therapy of convulsive disorders and support the hypothesis that epilepsy is mediated by oxidative stress, leading to abnormal structural alterations of cellular proteins, membrane lipids, DNA, and RNA (Bruce and Baudry 1995; Champney et al. 1996; Kabuto, Yokoi, and Ogawa 1998; Rauca, Zerbe, and Jantze 1999; Erakovic et al. 2001). There is a substantial need for the development of new, more effective, and less toxic antiepileptic drugs (AEDs) (Smith, Wilcox, and White 2007). Quinazoline and quinazolinone derivatives have continued to attract widespread interest for a long time, and literally hundreds of quinazolinone derivatives (Armarego 1963) have been synthesized and tested for central nervous system (CNS) depression and anticonvulsant activities. Quinazoline ring is an important lead moiety in medicinal chemistry which led to the discovery of a number of derivatives endowed with antitumor (Plescia et al. 1992), antiinflammatory (Mukerji et al. 1980), CNS depressant (Misra, Satsangi, and Tiwari 1982), anthelmintic (Chaurasia and Sharma 1982), muscle-relaxant (Kumar et al. 1983), hypoglycemic (Fisnerova et al. 1991), and antimicrobial activities (Saxena et al. 1992). In view of the manifold activities of quinazoline derivatives, we have synthesized a series of 6,7,8,9-tetra hydro-5H-5-(20 -hydroxy phenyl)-2-(40 -some substituted benzylidine) thiazolo (2,3-b) quinazolin-3(2H)-one (4a–4f) and 6,7,8,9-tetra hydro-5H-5-(20 -hydroxyphenyl)-2(40 -substituted benzylidine)-3-(4-nitrophenyl amino) thiazolo quinazoline (5a–5f) derivatives and have evaluated these compounds for their potential anticonvulsant properties.

Materials and methods Materials Synthetic starting material, reagents, and solvents were of analytical grade or of the highest quality commercially available. The chemicals were purchased from Aldrich Chemical Co. and Merck Chemical Co., respectively, and were dried whenever necessary.

Instrumentation The melting points were determined in open capillary tubes and are uncorrected. Infrared (IR) spectra were recorded with KBr pellets (ABB Bomem FT–IR spectrometer MB 104 ABB Limited, Bangaluru, India). Proton nuclear magnetic resonance (1H-NMR) spectra (Bruker 400 NMR spectrometer, Mumbai, India) were recorded with tetramethylsilane as internal reference. Mass spectral data were recorded with a quadrupol mass spectrometer (Shimadzu GC MS QP 5000, Chennai, India), and microanalyses were performed using a vario EL V300 elemental analyzer (Analysensysteme GmbH, Chennai, India). The purity of the compounds was checked by TLC on pre-coated SiO2 gel (HF254, 200 mesh) aluminum plates (E. Merck) using ethyl acetate:benzene (1:3) and visualized in a UV chamber. IR, 1H-NMR, mass spectral data, and elemental analyses were consistent with the assigned structures.

Synthesis procedures The synthesis strategy leading to the key intermediate and the target compounds are illustrated in Scheme 1. 6,7,8,9-Tetra hydro-5H-5-(20 -hydroxy phenyl) thiazolo (2, 3-b)

F

CH3

-

-

c

d

e

Br

Cl

O

1

CH

0

HO

N

N

N

5a - 5f

H

OH

N

H

S

C

NH

H N 2

S

OH

2

H N

2

CH

NO

Cl

S

2

NH

CH

2

NO

R

1

R

R

1

2

2

N H

R 2

NH

H

OH

S

Cl

N

S

ClCH2COOH

Cl

O

O

8

7

5„

4„

9

6

6„

3„

5

N

3

1„

2„

412

4

N

N

1

S

4a - 4f

H

OH

OHC

N

H

OH

S

3

O

O

2

CH

R1

R2

R1

Scheme 1. Synthesis of 6,7,8,9-tetra hydro-5H-5-(2 -hydroxyphenyl)-2-(40 -substituted benzylidine)-3-(4-nitrophenyl amino) thiazolo quinazoline.

-

CH3

-

b

f

OCH3

-

a

CH3

R2

R1

NaOH

OH

COM

O

+

CHO


R2

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quinazolin-3(2H)-one 3 prepared from equimolar quantities of cyclohexanone (0.039 mol) and salicylaldehyde (0.039 mol) was transferred into a 500-mL beaker. After adding a solution of sodium hydroxide to make the solution alkaline, the beaker was shaken and kept aside. The obtained solid was filtered, washed with water, and recrystallized from absolute ethanol. A mixture of 2-hydroxybenzylidine cyclohexanone (ring 1; 0.039 mol), thiourea (0.03 mol), and potassium hydroxide (2.5 g) in ethanol (100 mL) was heated under reflux for 3 h. The reaction mixture was concentrated to half of its volume, diluted with water, then acidified with dilute acetic acid, and kept overnight. The obtained solid was filtered, washed with water, and recrystallized from ethanol to give 4-hydroxyphenyl 3,4,5,6,7,8-hexahydro quinazolin-2-thione 2. Chloroacetic acid (0.096 mol) was melted on a water bath, and thione 2 (0.009 mol) was added to it portionwise to maintain homogeneity. The homogeneous mixture was further heated on a water bath for 30 min and kept overnight. The obtained solid was washed with water until being neutral and crystallized from ethanol to give 6,7,8,9tetra hydro-5H-5-(20 -hydroxyphenyl) thiazolo (2, 3-b) quinazolin-3(2H)-one 3 (Sharma, Kumar, and Pujari 1991). A mixture of 3 (0.002 mol), substituted benzaldehyde (0.002 mol), and anhydrous sodium acetate (0.002 mol) in glacial acetic acid (10 mL) was heated under reflux for 4 h. The reaction mixture was kept overnight and the solid, thus separated, was filtered, washed with water, and recrystallized from ethanol to furnish 6,7,8,9-tetra hydro-5H-5-(20 -hydroxyphenyl)-2-(40 -substituted benzylidine) thiazolo (2,3-b) quinazolin-3(2H)-one (4a–4f). Equimolar quantities (0.004 mol) of compounds 4a–4f were treated with thionyl chloride and dimethylformamide (DMF) to get the chloro derivative and were then coupled with p-nitroanilines in DMF at 80 C and quenched in ice water. The resulting product was separated by filtration, vaccum-dried, and recrystallized from warm ethanol to yield 6,7,8,9-tetra hydro-5H-5(20 -hydroxyphenyl)-2-(40 -substituted benzylidine)-3-(4-nitrophenyl amino) thiazolo quinazoline (5a–5f). Spectral data of (IR, 1H-NMR, and electron-impact mass spectrometry) and elemental analysis were used to ascertain the structures of the compounds. 1 H-NMR spectra were recorded for all the target compounds. The IR, 1H-NMR and EI–MS spectra of 6,7,8,9-tetra hydro-5H-5-(2-hydroxyphenyl) thiazoloquinazolin-3-one, the representative key intermediate 3 are obtained. Yield: 71%; m.p. 153–155 C; IR cm1: 3402 (phenolic OH), 3046 (Ar–CH), 1719 (C¼O), and 1462 (C¼C). 1H-NMR (CDCl3) (ppm): 6.61–6.89 (m, 4H; Ar–H), 5.71 (s, 1H; –CH), 5.30 (s, 1H; –C–OH), 3.76 (s, 2H; –CH2), and 1.6–2.42 (m, 8H; CH2, CH2, CH2, CH2). EI–MS m/z (Mþ): 300 (calcd for C16H16N2O2S; 300.38). Anal. calcd for C16H16N2O2S; C, 63.98; H, 5.37; N, 9.32; O, 10.65; and S, 10.64. Found: C, 63.92; H, 5.28; N, 9.30; O, 10.52; and S, 10.42.

6,7,8,9-Tetra hydro-5H-5-(20 -hydroxyphenyl)-2-(40 -methoxy benzylidine) thiazolo (2, 3-b) quinazolin-3(2H)-one (4a) This compound was obtained as a pale yellow solid; yield: 78%; m.p. 183–185 C; IR cm1: 3476 (O–H), 3096 (Ar–CH), 1728 (C¼O), and 1468 (C¼C) cm1. 1H-NMR (CDCl3) (ppm): 6.96–7.54 (m, 8H; Ar–H), 6.67 (s, 1H; ¼CH), 5.83 (s, 1H; H-5), 9.84 (s, 1H; Ar–OH), 3.75 (s, 3H; –OCH3), and 1.58–2.62 (m, 8H; 4 CH2); EI–MS (m/z): 418 (Mþ); (calcd for C24H22N2O3S; 418.51). Anal. calcd for C24H22N2O3S, C, 68.88; H, 5.30; N, 6.69; O, 11.47; and S, 7.66; Found: C, 68.90; H, 5.33; N, 6.72; O, 11.51; and S, 7.69.


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6,7,8,9-Tetra hydro-5H-5-(20 -hydroxyphenyl)-2-(40 -methyl benzylidine) thiazolo (2, 3-b) quinazolin-3(2H)-one (4b) This compound was obtained as a creamy solid; yield: 76%; m.p. 186–188 C; IR cm1: 3448 (O–H), 3049 (Ar–CH), 1721 (C¼O), and 1434 (C¼C) cm1; 1H-NMR (CDCl3) (ppm): 6.86–7.74 (m, 8H; Ar–H), 6.72 (s, 1H; ¼CH), 5.76 (s, 1H; H–5), 9.76 (s, 1H; Ar– OH), 2.20 (s, 3H; –CH3), and 1.62–2.32 (m, 8H; 4 CH2); EI–MS (m/z): 402 (Mþ); (calcd for C24H22N2O2S; 402.14). Anal. calcd for C24H22N2O2S; C, 71.00; H, 5.51; N, 6.96; O, 7.95; and S, 7.97; Found: C, 69.87; H, 5.32; N, 6.74; O, 7.58; and S, 7.72.


6,7,8,9-Tetra hydro-5H-5-(20 -hydroxyphenyl)-2-(30 ,40 -dimethyl benzylidine) thiazolo (2, 3-b) quinazolin-3(2H)-one (4c) This compound was obtained as a creamy solid; yield: 79%; m.p. 181–183 C; IR cm1: 3450 (O–H), 3051 (Ar–CH), 1724 (C¼O), and 1437 (C¼C); 1H-NMR (CDCl3) (ppm): 6.89–7.76 (m, 7H; Ar–H), 6.74 (s, 1H; ¼CH), 5.78 (s, 1H; H-5), 9.84 (s, 1H; Ar–OH), 2.23 (s, 6H; –CH3), and 1.62–2.32 (m, 8H; 4 CH2); EI–MS (m/z): 416 (Mþ); (calcd for C25H24N2O2S; 416.16). Anal. calcd for C25H24N2O2S; C, 72.09; H, 5.81; N, 6.73; O, 7.68; and S, 7.70; Found: C, 72.12; H, 5.79; N, 6.75; O, 7.66; and S, 7.71. 6,7,8,9-Tetra hydro-5H-5-(20 -hydroxyphenyl)-2-(40 -fluoro benzylidine) thiazolo (2, 3-b) quinazolin-3(2H)-one (4d) This compound was obtained as a pale yellow solid; yield: 69%; m.p. 153–155 C; IR cm1: 3437 (O–H), 3026 (Ar–CH), 1734 (C¼O), 1522 (C¼C), and 826 (C–F) cm1; 1H-NMR (CDCl3) (ppm): 6.63–7.32 (m, 8H; Ar–H), 6.38 (s, 1H; ¼CH), 5.87 (s, 1H; H-5), 5.43 (s, 1H; Ar–OH), and 1.34–2.33 (m, 8H; 4 CH2); EI–MS (m/z): 406 (Mþ); (calcd for C23H19FN2O2S; 406.12). Anal. calcd for C23H19FN2O2S; C, 67.96; H, 4.71; F, 4.67; N, 6.89; O, 7.87; and S, 7.89; Found: C, 67.97; H, 4.73; F, 4.65; N, 6.87; O, 7.89; and S, 7.91. 6,7,8,9-Tetra hydro-5H-5-(20 -hydroxyphenyl)-2-(40 -bromo benzylidine) thiazolo (2, 3-b) quinazolin-3(2H)-one (4e) This compound was obtained as a yellow solid; yield: 75%; m.p. 182–184 C; IR cm1: 3447 (O–H), 3025 (Ar–CH), 1716 (C¼O), 1523 (C¼C), and 823 (C–Br) cm1; 1H-NMR (CDCl3) (ppm): 6.73–7.29 (m, 8H; Ar–H), 6.48 (s, 1H; ¼CH), 5.73 (s, 1H; H-5), 5.51 (s, 1H; Ar–OH), and 1.26–2.32 (m, 8H; 4 CH2); EI–MS (m/z): 468 (M þ 2); (calcd for C23H19BrN2O2S; 466.04). Anal. calcd for C23H19BrN2O2S; C, 59.11; H, 4.10; Br, 17.10; N, 5.99; O, 6.85; and S, 6.86; Found: C, 59.14; H, 4.13; Br, 17.12; N, 5.97; O, 6.87; and S, 6.87. 6,7,8,9-Tetra hydro-5H-5-(20 -hydroxyphenyl)-2-(40 -chloro benzylidine) thiazolo (2, 3-b) quinazolin-3(2H)-one (4f) This compound was obtained as a yellow solid; yield: 71%; m.p. 158–160 C; IR cm1: 3429 (O–H), 3030 (Ar–CH), 1729 (C¼O), 1526 (C¼C), and 846 (C–Cl) cm1; 1H-NMR (CDCl3) (ppm): 6.64–7.33 (m, 8H; Ar–H), 6.36 (s, 1H; ¼CH), 5.84 (s, 1H; H-5), 5.41 (s, 1H; Ar–OH), and 1.35–2.36 (m, 8H; 4 CH2); EI–MS (m/z): 424 (M þ 2); (calcd for C23H19ClN2O2S; 422.09). Anal. calcd for C23H19ClN2O2S; C, 65.32; H, 4.53; Cl, 8.38; N, 6.62; O, 7.57; and S, 7.58; Found: C, 65.34; H, 4.51; Cl, 8.36; N, 6.64; O, 7.51; and S, 7.53.

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6,7,8,9-Tetra hydro-5H-5-(20 -hydroxy phenyl)-2-(40 -methoxy benzylidine)-3-(4-nitro phenyl amino) thiazolo quinazoline (5a) This compound was obtained as a pale yellow solid; yield: 76%; m.p. 156–158 C; IR cm1: 3464 (O–H), 3027 (Ar–CH), 1494 (C¼C), 1326 (N–H bending), and 3384 (N–H stretching) cm1; 1H-NMR (CDCl3) (ppm): 6.72–7.23 (m, 12H; Ar–H), 6.36 (s, 1H; ¼CH), 5.62 (s, 1H; H-5), 9.87 (s, 1H; Ar–OH), 4.46 (s, 1H; thiazole), 3.78 (s, 3H; –OCH3), 7.26 (s, 1H; N–H), and 1.46–2.42 (m, 8H; 4 CH2); EI–MS (m/z): 540 (Mþ); (calcd for C30H28N4O4S; 540.18). Anal. calcd for C30H28N4O4S; C, 66.65; H, 5.22; N, 10.36; O, 11.84; and S, 5.93. Found: C, 66.67; H, 5.25; N, 10.38; O, 11.85; and S, 5.96.


6,7,8,9-Tetra hydro-5H-5-(20 -hydroxyphenyl)-2-(40 -methyl benzylidine)-3-(4-nitrophenyl amino) thiazolo quinazoline (5b) This compound was obtained as a creamy solid; yield: 76%; m.p. 192–193 C; IR cm1: 3438 (O–H), 3024 (Ar–CH), 1412 (C¼C), 1332 (N–H bending), and 3340 (N–H stretching) cm1; 1H-NMR (CDCl3) (ppm): 6.69–7.24 (m, 12H; Ar–H), 6.28 (s, 1H; ¼CH), 5.72 (s, 1H; H-5), 9.82 (s, 1H; Ar–OH), 4.45 (s, 1H; thiazole), 2.28 (s, 3H; –CH3), 7.69 (s, 1H; N–H), and 1.36–2.41 (m, 8H; 4 CH2); EI–MS (m/z): 524 (Mþ); (calcd for C30H28N4O3S; 524.19). Anal. calcd for C30H28N4O3S; C, 68.68; H, 5.38; N, 10.68; O, 9.15; and S, 6.11; Found: C, 68.65; H, 5.36; N, 10.70; O, 9.18; and S, 6.14. 6,7,8,9-Tetra hydro-5H-5-(20 -hydrox phenyl)-2-(30 ,40 -dimethyl benzylidine)-3-(4-nitro phenyl amino) thiazolo quinazoline (5c) This compound was obtained as a yellow solid; yield: 77%; m.p. 181–183 C; IR cm1: 3429 (O–H), 3027 (Ar–CH), 1413 (C¼C), 1334 (N–H bending), and 3313 (N–H stretching) cm1; 1H-NMR (CDCl3) (ppm): 6.79–7.24 (m, 12H; Ar–H), 6.26 (s, 1H; ¼CH), 5.74 (s, 1H; H-5), 9.93 (s, 1H; Ar–OH), 4.39 (s, 1H; thiazole), 2.34 (s, 6H; –CH3), 7.62 (s, 1H; N–H), and 1.36–2.41 (m, 8H; 4 CH2); EI–MS (m/z): 538 (Mþ); (calcd for C31H31N4O3S; 538.2). Anal. calcd for C31H31N4O3S; C, 69.12; H, 5.61; N, 10.40; O, 8.91; and S, 5.95; Found: C, 69.14; H, 5.63; N, 10.43; O, 8.92; and S, 5.97. 6,7,8,9-Tetra hydro-5H-5-(20 -hydroxy phenyl)-2-(40 -fluoro benzylidine)-3-(4-nitrophenyl amino) thiazolo quinazoline (5d) This compound was obtained as a creamy solid; yield: 89%; m.p. 184–186 C; IR cm1: 3449 (O–H), 3026 (Ar–CH), 1524 (C¼C), 1326 (N–H bending), 3319 (N–H stretching), and 821 (C–F) cm1; 1H-NMR (CDCl3) (ppm): 6.74–7.32 (m, 12H; Ar–H), 6.23 (s, 1H; ¼CH), 5.84 (s, 1H; H-5), 9.96 (s, 1H; Ar–OH), 4.42 (s, 1H; thiazole), 7.34 (s,1H; N–H), and 1.24–2.32 (m, 8H; 4 CH2); EI–MS (m/z): 528 (Mþ); (calcd for C29H25FN4O3S; 528.16). Anal. calcd for C29H25FN4O3S; C, 65.89; H, 4.77; F, 3.59; N, 10.60; O, 9.08; and S, 6.07; Found: C, 65.91; H, 4.79; F, 3.61; N, 10.62; O, 9.07; and S, 6.09. 6,7,8,9-Tetra hydro-5H-5-(20 -hydroxy phenyl)-2-(40 -bromo benzylidine)-3-(4-nitrophenyl amino) thiazolo quinazoline (5e) This compound was obtained as a pale yellow solid; yield: 82%; m.p. 183–185 C; IR cm1: 3447 (O–H), 3021 (Ar–CH), 1519 (C¼C), 1327 (N–H bending), 3319 (N–H stretching), and 818 (C–Br) cm1; 1H-NMR (CDCl3) (ppm): 6.81–7.36 (m, 12H; Ar–H), 6.49


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(s, 1H; ¼CH), 5.84 (s, 1H; H-5), 9.81 (s, 1H; Ar–OH), 4.46 (s, 1H; thiazole), 7.79 (s, 1H; N–H), and 1.29–2.34 (m, 8H; 4 CH2); EI–MS (m/z): 560 (M þ 2); (calcd for C29H25BrN4O3S; 588.08). Anal. calcd for C29H25BrN4O3S; C, 59.09; H, 4.27; Br, 13.55; N, 9.50; O, 8.14; and S, 5.44; Found: C, 59.11; H, 4.29; Br, 13.54; N, 9.51; O, 8.13; and S, 5.45. 6,7,8,9-Tetra hydro-5H-5-(20 -hydroxyphenyl)-2-(40 -chloro benzylidine)-3-(4-nitrophenyl amino) thiazolo quinazoline (5f) This compound was obtained as a yellow solid; yield: 82%; m.p. 183–184 C; IR cm1: 3446 (O–H), 3028 (Ar–CH), 1526 (C¼C), 1348 (N–H bending), 3336 (N–H stretching), and 824 (C–Cl) cm1; 1H-NMR (CDCl3) (ppm): 6.71–7.33 (m, 12H; Ar–H), 6.24 (s, 1H; ¼CH), 5.86 (s, 1H; H-5), 5.54 (s, 1H; Ar–OH), 4.47 (s, 1H; thiazole), 7.31 (s,1H; N–H), and 1.21–2.31 (m, 8H; 4 CH2); EI–MS (m/z): 546 (M þ 2); (calcd for C29H25ClN4O3S; 544.13). Anal. calcd for C29H25ClN4O3S; C, 63.90; H, 4.62; Cl, 6.50; N, 10.28; O, 8.81; and S, 5.88; Found: C, 63.92; H, 4.61; Cl, 6.52; N, 10.25; O, 8.79; and S, 5.85. The series of heterocycles 4a–4f and 5a–5f was synthesized by reaction of 3 with appropriate aromatic aldehydes and p-nitrophenylamine in the presence of anhydrous sodium acetate and DMF as presented in Scheme 1. IR, 1H-NMR, mass spectroscopy, and elemental analysis data were in accordance with the assigned structures. The IR spectra of compounds 4a–4f showed stretching band of the keto group at 1715–1740 cm1. In 5a–5f, stretching and bending NH bands of thiazolo quinazoline moiety appear at 3300– 3400 cm1 and at 1300–1350 cm1, respectively. Keto group bands were missing in the IR spectra of compounds 5a–5f. This clearly suggested that the keto group of 4a–4f was converted into a secondary amino group (NH). The proton magnetic resonance spectra of thiazolo quinazoline and its corresponding derivatives (5a–5f) have been recorded in CDCl3. In these spectra, the NH signals of 5a–5f 3-(4-nitro phenyl) amino thiazolo quinazoline moiety appear at 7.26 (s), 7.69 (s), 7.62 (s), 7.89 (s), 7.79 (s), and 7.34 (s) ppm, respectively. The position and presence of the NH signal in the 1H-NMR spectra of final compounds confirm the secondary NH proton in the thiazolo quinazoline moiety. This clearly indicates that the thiazole-3-one moiety is involved in the 3-(4-nitrophenyl) amine formation. All these observed facts clearly demonstrate that the 3rd position of the keto group in the thiazole ring is converted into a secondary amino group, as indicated in Scheme 1 and confirm the proposed structure (5a–5f).

Pharmacology Male albino mice (CF-1 strain, 18–25 g) and male albino rats (Sprague–Dawley/Wistar, 100–150 g) were used as experimental animals. The animals were housed in metabolic cages and allowed free access to food and water. The evaluation of anticonvulsant activity in the maximal electroshock-induced seizure (MES) model, the subcutaneous pentylenetetrazole tests as well as the determination of toxicity in the rotarod test, and positional stence tests were performed at the NIH Epilepsy Branch as a part of the Anticonvulsant Drug Development Program according to the protocols described in White et al. (2002). All compounds for testing were prepared either by dissolving or suspending in 0.5% polyethylene glycol 200. The tested compounds were given in a concentration that permits optimal accuracy of dosage without the volume contributing excessively to total body fluid. Thus, the volume used in mice was 0.01 mL per gram body weight, and in rats 0.04 mL per 10 g body weight. Results are shown in Table 1.

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Table 1. Anticonvulsant and NT screening of 6,7,8,9-tetra hydro-5H-5-(20 -hydroxy phenyl)-2(substituted benzylidine)-3-(4-nitrophenyl amino) thiazolo quinazoline. Intraperitoneal injection in micea (h)


MES screen

ScPTZ

NT screen

Compounds

0.5 h

4h

0.5 h

4h

0.5 h

4h

4a 4b 4c 4d 4e 4f 5a 5b 5c 5d 5e 5f Phenytoin Ethosuximide

300 300 300 100 100 100 100 100 100 100 100 100 30 –

– – – – – – – – – 100 100 100 30 –

– –b –

– – – 100 100 100 300 300 300 300 300 300 – 300

– – 100 – – 100 300 – – – – – 100 –

300 300 100 300 – 100 300 100 100 – – – 100 –

100 100 100 100 100 100 – 100

Notes: aDoses of 30, 100 and 300 mg kg1 were administered. The figures in the table indicate the minimum dose whereby bioactivity was demonstrated in half or more of the mice. The animals were examined 0.5 and 4 h after administration. The dash (–) indicates an absence of activity at maximum dose administered (300 mg kg1). b Death following tonic extension.

Behavioral testing The title compounds 4a–4f and 5a–5f (100 mg kg1) were screened for their behavioral effect using an actophotometer (Boisser and Simon 1965) at 30 min and 1 h after drug administration. The behavior of animals inside the photocell was recorded as a digital score. Increased scores suggested good behavioral activity. The percentage decrease in locomotor activity is calculated with the help of the activity score of the control (24 h before) and the score 1 h after drug treatment. Mean values were taken for the calculations. Polyethylene glycol 200 was administered to the animal control group. The recorded scores are listed in Table 2.

CNS depressant activity The forced swimming method was employed to test CNS depressant activity (Porsolt et al. 1978). Wistar rats were placed in a chamber (diameter 45 cm, height 20 cm) containing water up to a height of 15 cm at 25 2 C. Two swim sessions were conducted. An initial 15-min pretest was followed by a 5-min test session 24 h later. The test compound (100 mg kg1) was administered orally to the animals 30 min before the test session. The period of immobility (passive floating without struggling, making only those movements which are necessary to keep its head above the surface of water) during the 5-min test period was measured. The results are presented in Table 3.

Toxicological & Environmental Chemistry Table 2. Behavioral study of 6,7,8,9-tetra hydro-5H-5-(20 -hydroxy phenyl)-2-(substituted benzylidine)-3-(4-nitrophenyl amino) thiazolo quinazoline. Activity scoreb Post treatment


Compoundsa

Control (24 h prior)

After 0.5 h

After 1 h

Inhibition (%)

396 9 383 9 406 2 317 10 421 9 362 25 461 11 385 7 239 12 450 15 239 16 358 27 456 31

304 10 310 17 301 27 281 10 372 22 286 26 360 9 242 7 181 15* 466 14NS 188 15 245 20 161 12

172 3 177 22 137 10 104 5 149 15 182 22NS 140 18 137 13 46 10 343 7 86 110 15 107 30

45 42 53 51 52 38 56 51 59 48 49 55 64

4a 4b 4c 4d 4e 4f 5a 5b 5c 5d 5e 5f Phenytoinc

Notes: aThe compounds were tested at a dose of 100 mg kg1 i.p. b Each score represents the means SEM of six mice. c Tested at 30 mg kg1 (p.o). *SigniEcantly different from the control score at p 5 0.05 and NS denotes nonsigniEcance at p 4 0.05 (Student’s t-test).

Table 3. CNS study on 6,7,8,9-tetra hydro-5H-5-(20 -hydroxyphenyl)-2-(substituted benzylidine)-3-(4-nitrophenyl amino) thiazolo quinazoline by forced swim pool test. Immobility time (s)b Compoundsa PEG 4a 4b 4c 4d 4e 4f 5a 5b 5c 5d 5e 5f Carbamazepined

Control (24 h prior)

Post treatment (after 1 h)

110 8 48 17 132 21 56 17 126 13 154 16 137 12 81 9 62 18 75 8 117 20 134 19 113 8 139 15

128 2NS 83 3 168 8 103 11 177 17 150 22NS 142 23NS 127 14 74 5NS 118 14 133 7 163 12* 164 20* 240 14

Notes: aThe compounds were tested at a dose of 100 mg kg1 (oral). b Each value represents the means SEM of six rats. c Tested at 30 mg kg1 (i.p). *SigniEcantly different from the control at p 5 0.05 and NS denotes nonsigniEcance at p 5 0.05 (Student’s t-test).

651

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Results and discussion The anticonvulsant efficacy was evaluated by the application of the MES model and the subcutaneous pentylenetetrazole-induced seizure threshold tests. Neurotoxicity (NT) in mice was measured by the rotarod test. Initial anticonvulsant activity and NT data for the thiazolo quinazoline derivatives (4a–f and 5a–f) are given in Table 1 along with literature data on phenytoin and ethosuximide (Porter et al. 1984; Dimmock et al. 1996; Flaherty et al. 1996). The derivatives were injected intraperitoneally into mice using doses of 30, 100, and 300 mg kg1, and the tests to evaluate anticonvulsant efficacy were carried out 0.5 and 4 h after dosage. The MES model has served to identify antiepileptic drugs that are functionally similar to phenytoin, and most of these compounds display, in common, the ability of these antiepileptics to inactivate voltage-dependent Naþ channels. Activity in this model seems highly predictive of the ability of those antiepileptic drugs to protect against partial and secondarily generalized tonic–clonic seizures. The subcutaneous pentylenetetrazole model has proven to be a good predictor of clinical efficacy against generalized spike-wave epilepsies of the absence type. Thus, the MES model and the subcutaneous pentylenetetrazole-induced seizure threshold test have become the most widely employed seizure models for early identification of candidate anticonvulsants. Introduction of the fluoro or trifluoromethyl substituents into the aryl moiety enhanced the anticonvulsant efficacy in comparison to respective chloro, methoxy, or methyl analogs (Obniska et al. 2005). Furthermore, introduction of the p-nitrophenyl substituent exhibited best activity in the subcutaneous pentylenetetrazole test (Patel et al. 2006). The importance of the 4-bromophenyl substituent for the anticonvulsant activity is documented in the literature (Pandeya, Yogeeswari, and Stables 2000). Our results are in accordance with these findings. All the compounds exhibited anticonvulsant activity. The majority of the compounds, except 4a, 4b, and 4c, were active in both the MES and subcutaneous pentylenetetrazole tests. Compounds 5d–5f) and (4d–4f) with fluoro, bromo, and chloro substituents, respectively, showed activity at 100 mg kg1 after 0.5 h in the MES model. Compounds 5d, 5e, and 5f were found to be active at the same dose after 4 h. Compounds 4a, 4b, and 4c were less active and inhibited the seizures at a dose of 300 mg kg1 in the MES model after 0.5 h. All compounds except 4a, 4b, and 4c were found to be active in the subcutaneous pentylenetetrazole test. Compounds 4d–4f showed activity at a dose of 100 mg kg1 after 4 h. Compounds 5a–5f are active at the doses 100 and 300 mg kg1 after 0.5 and 4 h, respectively, in the subcutaneous pentylenetetrazole test, comparable to ethosuximide. In a neurological toxicity screening test, compounds 4a, 4b, 4d and 5a were found to be toxic at the maximum administered dose (300 mg kg1). Compounds 4c, 4f, 5b, and 5c were toxic at a dose of 100 mg kg1. The observations are tabulated in Table 1. The compounds were tested at a dose of 50 mg kg1 and compared with the standard drug ethosuximide. Compounds 5d, 5e, and 5f showed better protection than the standard drug ethosuximide. These compounds did not exhibit NT. All compounds were also screened for behavioral effects and CNS depressant activity. In the behavioral study using the actophotometer scoring technique, all synthesized compounds showed a decrease in locomotor activity; 38% was the lowest and 59% was the maximal decrease in locomotor activity when compared to phenytoin as reported in Table 2. All compounds except 4a, 4b, and 4f exhibited more than a 50% decrease in locomotor activity (p 5 0.05) after 1 h. Compound 4f was the least potent compound and 5c the most potent one in the prepared series with a 38% and 59% decrease in locomotor activity, respectively. In a similar study with the forced swimming test, the immobility time



653

after administration of the test compounds was compared with that of carbamazepine (Table 3). Readings of the control groups were taken individually for each compound 24 h prior to compound administration. Experimental results indicate that our compounds exhibited better sedative-hypnotic and CNS depressant activity. Biological activity was also ascertained for polyethylene glycol 200 because it was used as a vehicle for the synthesized compounds. Except 4f, other tested compounds were found to exhibit potent CNS depressant activity, as indicated by the increased immobility time. Generally, compounds possessing a higher log P value showed a higher decrease in locomotor activity. Bulkier compounds are more lipophilic and can cross blood–brain barrier to exert their effect on CNS. The present study revealed that substitution of p-nitroaniline at the third position and of aromatic aldehydes at the second position of thiazolo quinazoline leads to the development of new chemical entities with potent sedative-hypnotic, CNS depressant, and anticonvulsant activities. Structure–activity relationship studies indicated that different substitutions on the thiazolo quinazoline nucleus exerted varied biological activity. The electronic nature of the substituent groups at the second and third positions in thiazole nucleus led to signiEcant variation in anti-convulsant activity. Electron-withdrawing groups enhanced the biological activity, whereas electron-releasing groups made the compounds less active. In addition, 4-nitrophenyl amino substitution in the second positions of the thiazolo quinazoline nucleus further enhanced the activity. Anticonvulsant activity increases when electronwithdrawing groups are substituted on the thiazolo quinazoline nucleus, whereas an electron-releasing group decreases the activity of title compounds. Among the series, compounds substituted by electron-withdrawing groups (nitro, fluoro, bromo, and chloro) and of high lipophilic nature due to nitrophenyl amino substitution, (5d–5f) exert a higher activity than compounds without these features (4d–4f). Compounds substituted by the electron-releasing methoxy, methyl, and dimethyl groups (4a, 4b, 4c) exhibit a lesser anticonvulsant activity.

Acknowledgments The authors thank the management of DCRM College of Pharmacy, Inkollu, Andhra Pradesh, India, for providing the facilities necessary to carry out the research work.

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