Synthesis and pharmacological evaluation of some novel...

ORIGINAL RESEARCH

Synthesis and pharmacological evaluation of some N3-aryl/heteroaryl-substituted 2-(2-chlorostyryl)-6,7-dimethoxy-quinazolin-4(3H)-ones as potential anticonvulsant agents

Nirupam Das • Debapriya Garabadu •

Anupam G. Banerjee • Sairam Krishnamurthy •

Sushant K. Shrivastava

Received: 28 October 2013 / Accepted: 6 March 2014 / Published online: 20 March 2014

� Springer Science+Business Media New York 2014

Abstract Certain novel 3-aryl/heteroaryl-substituted

2-(2-chlorostyryl)-6,7-dimethoxy-quinazolin-4(3H)-one

(5a–5l) derivatives have been synthesized and evaluated

for their anticonvulsant activity against maximal electro-

shock (MES)-, subcutaneous pentylenetetrazole (scPTZ)-

and intracerebroventricular (icv) AMPA (a-amino-3-

hydroxy-5-methyl-4-isoxazolepropionic acid)-induced sei-

zures in mice. The acute neurotoxicity was determined

using the rotarod test, and hepatotoxicity was also assessed

by estimating the AST (alanine aminotransferase) and ALT

(alanine aminotransferase) enzyme activity. Among all the

synthesized compounds, 5g showed the most significant

anticonvulsant activity against MES (ED50: 41.3 lmol/kg)-,

scPTZ (ED50: 82.5 lmol/kg)- and AMPA (ED50:

50.3 lmol/kg)-induced seizures with a protective index of

5.1.

Keywords Quinazolin-4(3H)-ones � Synthesis �Anticonvulsant activity � AMPA-induced seizure

Introduction

Epilepsy is a chronic neurological disorder characterized

by recurrent seizures in which clusters of nerve cells signal

abnormally in the brain. It affects approximately 1 %

population (*50 million people) of the world, whereas

developing countries have 85 % of the global burden of

epilepsy (Birbeck, 2010). A number of anticonvulsant

drugs are being used in the treatment of epilepsy, but the

management of the disorder is still challenging. The inci-

dence of adverse effects such as hepatotoxicity, amnesia,

anorexia, drowsiness and headache hinders the long-term

therapy with these drugs (Das et al., 2012; Bialer et al.,

2012). Therefore, the synthesis of new and effective anti-

convulsants has become the viable platform for on-going

research. Majority of the anticonvulsant drugs act via

voltage-gated ion channels or by enhancing the effects of

GABA. The discovery of new drugs as well as receptors is

still a challenging task to medicinal chemist. Glutamate,

the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

(AMPA) receptor agonist, is one of the major endogenous

excitatory neurotransmitters involved in learning, memory

and other brain functions. Ionotropic AMPA receptors and

the quintessence of fast excitatory neurotransmission are

emerging as a promising new target for epilepsy therapy

(Rogawski, 2011). Drugs acting as AMPA receptor antag-

onist showed promising anticonvulsant activity by attenu-

ating the hyperexcitability mediated by AMPA receptors.

The prototype to be identified as AMPA receptor antagonist

was 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-

2,3-benzodiazepine (GYKI 52466) (1) (Donevan and Rog-

awski, 1993) followed by the dioxolo-benzodiazepine

derivative talampanel (2) (Gitto et al., 2003) and perampanel

that have structurally diverse 2,30-bipyridin-60-one core

(Rogawski and Hanada, 2013).

Recently, the quinazoline nucleus gains critical attention

as potential anticonvulsant agents (Kashaw et al., 2009;

Malik et al., 2013). The anticonvulsant efficacy of a

compound substituted with N3 2-chlorophenyl and

N. Das � A. G. Banerjee � S. K. Shrivastava (&)

Pharmaceutical Chemistry Research Laboratory, Department of

Pharmaceutics, Indian Institute of Technology (BHU), Varanasi

221005, India

e-mail: [email protected]

D. Garabadu � S. Krishnamurthy

Neurotherapeutics Laboratory, Department of Pharmaceutics,

Indian Institute of Technology (BHU), Varanasi 221005, India

123

Med Chem Res (2014) 23:4167–4176

DOI 10.1007/s00044-014-0990-4

MEDICINALCHEMISTRYRESEARCH

substituted (pyridin-2-yl)vinyl) at C2 position of quinazo-

lin-4(3H)-one (3) (CP-526427) showed anticonvulsant

efficacy when tested against pentylenetetrazole and

AMPA-induced seizures (Menniti et al., 2000). An analo-

gous result was achieved with another derivative CP-

465022 (4) (Welch et al., 2001). An important pharmaco-

phoric model has been put forward wherein a quinazolin-4-

one ring comprises a small C-6 substituent with orthogonal

N3 phenyl ring. Further, an aryl ring is attached to the C2

position through a two-atom spacer (Chenard et al., 2001).

However, recent investigation revealed that the presence of

small C6 substituent is not important as far as substitution

at C2 position of quinazolin-4-one nucleus is concerned

(Georgey et al., 2008).

Taking into consideration the above structural features

and in continuation of our previous work with heterocyclic

compounds exhibiting anticonvulsant activity (Dhanawat

et al., 2011; Das et al., 2012, Dhanawat et al., 2012), we

propose to synthesize certain new quinazolin-4(3H)-one

derivatives (Fig. 1). The compounds possess different N3-

substituted aryl/heteroaryl ring within a 6,7-dimethoxy-

quinazolin-4(3H)-one nucleus while a common 2-chloro-

styryl ring is present at C2 position. All the synthesized

compounds have been tested for their activity against

several seizure models such as MES (maximal electro-

shock seizure), scPTZ (subcutaneous pentylenetetrazole)

and icv AMPA. Furthermore, the rotarod test paradigm has

been used for assessment of acute neurotoxic liability and

hepatotoxicity has been determined by estimating enzyme

activity of AST (aspartate aminotransferase) and ALT

(alanine aminotransferase) followed by histopathological

study.

Experimental

All reagents and solvents used in the study were of ana-

lytical grade and procured from Sigma-Aldrich (India),

Merck (Germany) and SD fine Chemicals (India). The

progress of the reaction was monitored by thin layer

chromatography with ethyl acetate/hexane (2:3) as the

mobile phase on precoated Merck silica gel 60 F254 alu-

minium sheets (Merck, Germany). The products were

purified by recrystallisation. Melting points were deter-

mined in open capillaries using Stuart SMP10 (Barloworld

Scientific Ltd., UK), electrothermal melting point appara-

tus and were uncorrected. Log P values of the compounds

were experimentally determined by shake flask method

using Shimadzu UV/Visible spectrophotometer. IR spectra

were recorded on a Shimadzu 8400S FTIR spectropho-

tometer using KBr pellets, and mmax was recorded in cm-1.1H NMR (300 MHz) spectra were acquired on a JEOL

AL300 FT-NMR in CDCl3 using TMS as the internal

standard and 13C NMR using Bruker AvIII HD-300 NMR

spectrometer. The coupling constant, J is expressed in

Hertz (Hz). Elemental analyses for C, H and N were per-

formed on Exeter CE-440 elemental analyser.

Chemistry

In the present investigation, a new series of N3-aryl/

heteroaryl-substituted 2-(2-chlorostyryl)-6,7-dimethoxy-qui-

nazolin-4(3H)-one derivatives (Scheme 1, Table 1) were

synthesized using procedures mentioned in the literature

(Al-Obaid et al., 2009, Ukrainets et al., 1994). All the

compounds were synthesized by three-step methods. The

initial two steps involve amidation followed by intramole-

cular cyclization using acetic anhydride which converts the

–OH of the carboxylic acid group into a good leaving group

to form the benzo[d][1,3]oxazin-4-one ring. Further, fusion

of various substituted aryl/heteroarylamines with 2-(2-

chlorostyryl)-6,7-dimethoxy-4H-benzo[d][1,3]oxazin-4-one

(4) via formation of amidine salt followed by cyclodehy-

dration yielded the title compounds 5a–5l.

Synthesis

2-(3-(2-Chlorophenyl) acrylamido)-4,5-dimethoxybenzoic

acid (3)

2-chlorocinnamoyl chloride (2) (0.05 mol) was added to a

solution of anthranilic acid (1) (0.05 mol) in pyridine

N

N

Cl

N

F

O

CP-526, 427

NC

N

N

Cl

NCH2N

FC2H5

C2H5

O

CP-465, 022

N

N

CH3

O

O

GYKI 52466

H2N

N

N

CH3

O

O

Talampanel

H2N

COCH3

N

N

OSubstituted aryl/heterocyclic ring

Electron

donorgroup Distal Aryl Ring

H3CO

H3CO

(1) (2)

(3) (4)

(5)

Fig. 1 Chemical structure of (1) GYKI 52466, (2) talampanel, (3, 4)

reported quinazolin-4(3H)-ones as anticonvulsants, (5) Template of

titled compounds (5a–5l)

4168 Med Chem Res (2014) 23:4167–4176

123

(50 mL), and the reaction mixture was stirred at room

temperature for 3 h. The mixture was then poured in 10 %

cold dilute HCl solution (50 mL). The solid obtained was

then filtered followed by washing several times with cold

water. The product 2-(3-(2-chlorophenyl) acrylamido)-4,5-

dimethoxybenzoic acid (3) was dried and crystallized from

absolute ethanol.

2-(3-(2-Chlorophenyl) acrylamido)-4,5-dimethoxybenzoic

acid (3)

Yield: 73.86 %; m.p.: 221–223 �C; IR (KBr, cm-1): 3,437

(–NH str.); 3,074 (–OH str –COOH); 2,931 (methoxy C–H

str.); 1,732 (–C=O str –COOH); 1,685 (amide –C=O str);

1,608 (aryl-substituted –C=C str); 1,037 (–C–O–C str). 1H

NMR (300 MHz, CDCl3): d 11.34 (s, 1H, –COOH); d 8.14

(s, 1H, NHC=O, D2O exchangeable); d 7.30–7.71 (m, 4H,

Ar–H); d 7.26 (s, 1H, 1–Ar–H); d 7.06 (s, 1H, Ar–H); d6.59 (d, J = 15.6, 1H, –ethylene); d 6.46 (d, J = 14.7, 1H,

–ethylene); d 4.02 (s, 3H, –OCH3); d 3.92 (s, 3H, –OCH3).

Anal. C18H16ClNO5: C, 59.76; H, 4.46; N, 3.87; Found: C,

59.83; H, 4.47; N, 3.85.

2-(2-Chlorostyryl)-6,7-dimethoxy-4H-

benzo[d][1,3]oxazin-4-one (4)

A mixture of 2-(3-(2-chlorophenyl)acrylamido)-4,5-di-

methoxybenzoic acid (3) (0.03 mol) and acetic anhydride

(0.3 mol) was heated under reflux in an oil bath at 180 �C

for 4 h. The reaction mixture was subsequently washed

with water and then extracted with chloroform, dried over

Na2SO4 and evaporated to yield a crude product, 2-(2-

chlorostyryl)-6,7-dimethoxy-4H-benzo[d][1,3]oxazin-4-one

(4). The residue so obtained was triturated with petroleum

ether (40–60), dried and crystallized from toluene.

2-(2-Chlorostyryl)-6,7-dimethoxy-4H-benzo[d][1,3]

oxazin-4-one (4)

Yield 83.27 %; m.p.: 233–235 �C; IR (KBr, cm-1): 2,928

(methoxy C–H str.); 1,735 (–C=O str cyclic ester); 1,597

(–C=N str); 1,037 (–C–O–C str). 1H NMR (300 MHz,

CDCl3): d 7.30–7.69 (m, 4H, Ar–H); d 7.26 (s, 1H, Ar–H);

d 7.05 (s, 1H, Ar–H); d 6.76 (d, J = 15.9, 1H, –ethylene); d6.53 (d, J = 15.9, 1H, –ethylene); d 4.20 (s, 3H, –OCH3); d3.99 (s, 3H, –OCH3). Anal. C18H14ClNO4: C, 62.89; H,

4.10; N, 4.07; Found: C, 63.03; H, 4.09; N, 4.08.

General procedure for the synthesis of compounds 5a–5l

A mixture of 2-(2-chlorostyryl)-6,7-dimethoxy-4H-

benzo[d][1,3]oxazin-4-one (0.01 mol) (4) and an

appropriate aryl/heteroaryl amine (0.01 mol) in glacial

acetic acid (10 mL) was refluxed at 210 �C in an oil

bath for 3 h. The reaction was quenched by pouring the

mixture obtained into crushed ice and left overnight.

The solid which separated out was filtered, washed

H3CO

H3CO

COOH

NH2

+

Cl

Cl

O H3CO

H3CO NH

COOHO

Cl

H3CO

H3CO N

CO

Cl

O

H3CO

H3CO N

CN

Cl

O

R

1 2

3

45a-5l

a

b

c

Scheme 1 General scheme for

the synthesis of compounds

5a–5l. Reagents and conditions:

a Pyridine, stir, r. t., 3 h;

b Ac2O, reflux, 4 h;

c Substituted aryl/heteroaryl

amine, glacial acetic acid,

reflux, 3 h

Table 1 List of synthesized compounds

Sl. No. Code R m.p. (�C) Yield (%) Rfa

1 5a phenyl 172–174 75.45 0.57

2 5b 2,4-dimethylphenyl 158–160 74.81 0.64

3 5c m-tolyl 162–164 71.52 0.61

4 5d 2-methoxyphenyl 166–168 67.34 0.63

5 5e 3-methoxyphenyl 210–212 74.63 0.68

6 5f benzyl 178–180 65.76 0.66

7 5g 4-nitrophenyl 223–225 69.20 0.63

8 5h 2-nitrophenyl 219–221 71.23 0.69

9 5i 4-bromophenyl 175–177 66.85 0.61

10 5j pyrimidin-2-yl 226–228 73.57 0.63

11 5k pyridin-4-yl 232–234 72.41 0.59

12 5l thiazol-2-yl 229–231 68.26 0.57

a Solvent system: ethyl acetate/hexane (2:3)

Med Chem Res (2014) 23:4167–4176 4169

123

thoroughly with cold water, dried and then crystallized

from absolute ethanol to obtain the product (5a–5l).

2-(2-Chlorostyryl)-6,7-dimethoxy-3-phenylquinazolin-

4(3H)-one (5a)

Yield: 75.45 %, m.p.: 172–174 �C, IR (KBr, cm-1): 2,941

(methoxy C–H str.); 1,674 (–C=O); 1,606 (–C=N str);

1,218 (C–N str). 1H NMR (300 MHz, CDCl3): d 7.38–7.67

(m, 4H, Ar–H); d 7.15–7.30 (m, 5H, Ar–H); d 7.13 (s, 1H,

Ar–H); d 7.05 (s, 1H, Ar–H); d 6.58 (d, J = 15.3, 1H,

–ethylene); d 6.38 (d, J = 15.6, 1H, –ethylene); d 4.06 (s,

3H, –OCH3). d 3.91 (s, 3H, –OCH3). 13CNMR (75 MHz,

CDCl3): 56.41, 56.52, 107.92, 108.06, 109.75, 121.62,

126.95, 127.40, 128.74, 130.12, 133.11, 134.90, 136.85,

144.02, 156.58, 160.28. Anal. C24H19ClN2O3: C, 68.82; H,

4.57; N, 6.69; Found: C, 68.64; H, 4.55; N, 6.71.

2-(2-Chlorostyryl)-6,7-dimethoxy-3-(2,4-

dimethylphenyl)quinazolin-4(3H)-one (5b)


(methoxy C–H str.); 1,670 (–C=O str); 1,608 (–C=N str);


(m, 4H, Ar–H), d 7.25–7.63 (m, 3H, Ar–H); d 7.21 (s, 1H,

Ar–H); d 7.09 (s, 1H, Ar–H); d 6.57 (d, J = 11.7, 1H, –eth-

ylene); d 6.36 (d, J = 15.3, 1H, –ethylene); 4.06 (s, 3H,

–OCH3); d 3.90 (s, 3H, –OCH3); d d 2.11 (s, 3H, –CH3); d 2.42

(s, 3H, –CH3).13CNMR (75 MHz, CDCl3): 17.54, 21.48, 56.31,

56.54, 108.28, 114.91, 122.52, 124.91, 127.68, 130.31, 133.72

135.39, 144.41, 149.46, 155.46, 161.44. Anal. C26H23ClN2O3:

C, 69.87; H, 5.19; N, 6.27; Found: C, 69.65; H, 5.17; N, 6.29.

2-(2-Chlorostyryl)-6,7-dimethoxy-3-m-tolylquinazolin-

4(3H)-one (5c)

Yield: 71.52 %, m. p.: 162–164 �C; IR (KBr, cm-1): 2,924

(methoxy C–H str.); 1,658 (–C=O str), 1,606 (–C=N str);


(m, 4H, Ar–H); d 7.21–7.38 (m, 4H, Ar–H); d 7.16–7.17 (m,

1H, Ar–H); d 7.09 (s, 1H, Ar–H); d 6.58 (d, J = 15.6, 1H,

–ethylene); d 6.48 (d, J = 15.6, 1H, –ethylene); d 4.03 (s,

3H, –OCH3); d 3.94 (s, 3H, –OCH3), d 2.32 (s, 3H, –CH3).13CNMR (75 MHz, CDCl3): 23.49, 56.26, 56.52, 109.30,

114.26, 122.31, 124.80, 127.29, 130.45, 133.50, 135.63,

144.35, 149.53, 155.55, 161.36. Anal. C25H21ClN2O3: C,

69.36; H, 4.89; N, 6.47; Found: C, 69.45; H, 4.88; N, 6.48.

2-(2-Chlorostyryl)-6,7-dimethoxy-3-(2-

methoxyphenyl)quinazolin-4(3H)-one (5d)

Yield: 67.34 %; m.p: 166–168 �C; IR (KBr, cm-1): 2,939

(methoxy C–H str.); 1,672 (–C=O); 1,604 (–C=N str);

1,211 (C–N str). 1H NMR (300 MHz, CDCl3): d7.43–7.68 (m, 4H Ar–H); d 7.04–7.25 (m, 4H, Ar–H); d6.96 (s, 1H, Ar–H); d 6.93 (s, 1H, Ar–H); d 6.60 (d,

J = 15.6, 1H, –ethylene); d 6.41 (d, J = 15.3, 1H, –eth-

ylene); d 4.06 (s, 3H, –OCH3); d 3.93 (s, 3H, –OCH3), d3.77 (s, 3H, –OCH3). 13CNMR (75 MHz, CDCl3):54.24,

55.10, 55.85, 107.11, 109.20, 113.53, 124.14, 126.05,

129.34, 130.27, 134.82, 137.26, 143.36, 152.73, 160.57,

162.43. Anal. C25H21ClN2O4: C, 66.89; H, 4.72; N, 6.24;

Found: C, 67.03; H, 4.73; N, 6.23.


methoxyphenyl)quinazolin-4(3H)-one (5e)

Yield: 74.63 %; m.p: 210–212 �C; IR (KBr, cm-1): 2,933

(methoxy C–H str); 1,606 (–C=N str); 1,656 (–C=O str);

1,213 (C–N str); 1H NMR (300 MHz, CDCl3): d7.39–7.66 (m, 4H, Ar–H); d 6.90–7.29 (m, 4H, Ar–H); d6.73 (s, 1H, Ar–H); d 6.76 (s, 1H, Ar–H); d 6.57 (d,


ylene); d 4.07 (s, 3H, –OCH3); d 4.00 (s, 3H, –OCH3);

3.84 (s, 3H, –OCH3). 13CNMR (75 MHz, CDCl3):54.64,

55.30, 55.80, 104.35, 106.21, 109.10, 112.45, 123.84,

126.25, 129.14, 129.36, 134.91, 137.89, 143.75, 152.02,

159.57, 162.65. Anal. C25H21ClN2O4: C, 66.89; H, 4.72;

N, 6.24; Found: C, 67.13; H, 4.71; N, 6.21.

2-(2-Chlorostyryl)-3-benzyl-6,7-dimethoxyquinazolin-

4(3H)-one (5f)


(methoxy C–H str); 2,835 (–CH str methylene); 1,658

(–C=O str); 1,604 (–C=N str); 1,211 (C–N str). 1H NMR

(300 MHz, CDCl3): 7.35–7.66 (m, 5H, Ar–H); 7.02–7.32

(m, 4H, Ar–H); d 6.97 (s, 1H, Ar–H); d 6.94 (s, 1H,

Ar–H); d 6.76 (d, J = 15.9, 1H, –ethylene); d 6.61

(d, J = 15.6, 1H, –ethylene); d 4.34 (s, 2H, –CH2); d 4.03

(s, 3H, –OCH3); d 3.88 (s, 3H, –OCH3).13C NMR

(75 MHz, CDCl3): 41.35, 56.24, 56.49, 112.14, 118.33,

125,52, 127.22, 130.13, 132.50, 133.49, 135.62 137.15,

138.52, 142.36, 148.73, 152.95 157.83, 163.26. Anal.

C25H21ClN2O3: C, 69.36; H, 4.89; N, 6.47; Found: C, 69.17;

H, 4.87; N, 6.48.


nitrophenyl)quinazolin-4(3H)-one (5g)



1,506, 1,379 (–N=O str); 1,212 (C–N str). 1H NMR

(300 MHz, CDCl3): d 7.55–8.22 (m, 4H, Ar–H); d7.29–7.45 (m, 4H, Ar–H); d 7.26 (s, 1H, Ar–H); d 7.04

(s, 1H, Ar–H); d 6.76 (d, J = 15.9, 1H, –ethylene); d 6.57

4170 Med Chem Res (2014) 23:4167–4176

123

(d, J = 13.2, 1H, –ethylene); d 4.02 (s, 3H, –OCH3); d 3.89

(s, 3H, –OCH3). 13C NMR (75 MHz, CDCl3): 56.63, 56.67,

108.07, 109.87, 120.24, 121.72, 125.23, 127.34, 130.45,

131.03, 133.20, 135.04, 136.91, 143.41, 150.01, 156.58,

159.26. Anal. C24H18ClN3O5: C, 62.14; H, 3.91; N, 9.06;

Found: C, 61.98; H, 3.92; N, 9.08.

2-(2-Chlorostyryl)-6,7-dimethoxy-3-(2-nitrophenyl)

quinazolin-4(3H)-one (5h)



1,504, 1,373 (–N=O str); 1,210 (C–N str). 1H NMR

(300 MHz, CDCl3): d 7.58–8.23 (m, 4H, Ar–H); d7.25–7.47 (m, 4H, Ar–H); d 7.23 (s, 1H, Ar–H); d 7.06 (s,

1H, Ar–H); d 6.76 (d, J = 15.9, 1H, –ethylene); d 6.57 (d,

J = 13.4, 1H, –ethylene); d 4.02 (s, 3H, –OCH3); d 3.88 (s,

3H, –OCH3).13C NMR (75 MHz, CDCl3): 56.62, 56.66,

108.08, 109.76, 120.27, 121.77, 126.45, 127.56, 130.69,

131.75, 133.56, 135.25, 137.85, 143.54, 150.57, 156.61,

159.72. Anal. C24H18ClN3O5: C, 62.14; H, 3.91; N, 9.06;

Found: C, 62.21; H, 3.90; N, 9.05.

2-(2-Chlorostyryl)-3-(4-bromophenyl)-6,7-

dimethoxyquinazolin-4(3H)-one (5i)


(methoxy C–H str), 1,664 (–C=O str); 1,608 (–C=N str);

1,209 (C–N str). 1H NMR (300 MHz, CDCl3): d7.55–8.22 (m, 4H, Ar–H); d 7.25–7.52 (m, 4H, Ar–H); d7.04 (s, 1H, Ar–H); d 7.02 (s, 1H, Ar–H); d 6.76 (d,

J = 16.5, 1H, –ethylene); d 6.55 (d, J = 15.6, 1H, –ethyl-

ene); d 4.06 (s, 3H, –OCH3); d 3.89 (s, 3H, –OCH3).13C

NMR (75 MHz, CDCl3): 56.31, 56.69, 106.29, 108.19,

109.99, 121.89, 122.91, 127.37, 130.51, 131.05, 133.35,

135.60, 134.75, 136.97, 137.05, 150.14, 155.67, 156.78,

159.29. Anal. C24H18BrClN2O3: C, 57.91; H, 3.64; N, 5.63;

Found: C, 58.13; H, 3.63; N, 5.65.

2-(2-Chlorostyryl)-6,7-dimethoxy-3-(pyrimidin-2-

yl)quinazolin-4(3H)-one (5j)


(methoxy C–H str); 1,629 (–C=O str); 1,597 (–C=N str);


(m, 3H, pyrimidine); d 7.29–7.55 (m, 4H, Ar–H); d 7.25

(s, 1H, Ar–H); d 7.04 (s, 1H, Ar–H); d 6.76 (d, J = 16.2,

1H, –ethylene); d 6.56 (d, J = 16.5, 1H, –ethylene); d 4.02

(s, 3H, –OCH3); d 4.00 (s, 3H, –OCH3). 13C NMR

(75 MHz, CDCl3): 56.51, 56.64, 106.26, 108.14, 109.81,

121.78, 122.51, 125.08, 127.60, 129.29, 130.46,133.30,

135.09, 136.97, 137.84, 156.64, 161.84. Anal.

C22H17ClN4O3: C, 62.79; H, 4.07; N, 13.31; Found: C,

62.64; H, 4.06; N, 13.33.

2-(2-Chlorostyryl)-6,7-dimethoxy-3-(pyridin-4-

yl)quinazolin-4(3H)-one (5k)

Yield: 72.41 %; m.p.: 232–234 �C, IR (KBr, cm-1): 2,939


1,200 (C–N str). 1H NMR (300 MHz, CDCl3): d7.55–7.69 (m, 4H, pyridine); d 7.29–7.45 (m, 4H, Ar–H);

d 7.26 (s, 1H, Ar–H); d 7.04 (s, 1H, Ar–H); d 6.76 (d,


ylene); d 4.02 (s, 3H, –OCH3), d 4.00 (s, 3H, –OCH3).13C NMR (75 MHz, CDCl3): 56.21, 56.69, 107.99,

108.96, 109.98, 121.86, 127.37, 130.50, 131.04, 133.33,

135.09, 137.00, 143.49, 150.10, 156.66, 159.26. Anal.

C23H18ClN3O3: C, 65.79; H, 4.32; N, 10.01; Found: C,

66.01; H, 4.33; N, 10.04.

2-(2-Chlorostyryl)-6,7-dimethoxy-3-(thiazol-2-

yl)quinazolin-4(3H)-one (5l)

Yield 68.26 %; m.p.: 229–231 �C; IR (KBr, cm-1): 2,939


1,212 (C–N str); 1,138 (C–S). 1H NMR (300 MHz,

CDCl3): d 7.68 (s, 1H, thiazole methine); d 7.66 (s, 1H,

thiazole methine); d 7.29–7.55 (m, 4H, Ar–H); d 7.25

(s, 1H, Ar–H); 7.04 d (s, 1H, Ar–H); d 6.76 (d, J = 15.9,

1H, –ethylene); d 6.58 (d, J = 15.3, 1H, –ethylene); d4.02 (s, 3H, –OCH3); d 4.00 (s, 3H, –OCH3). 13C NMR

(75 MHz, CDCl3): 56.32, 56.71, 108.02, 110.00, 121.89,

127.38, 130.52, 131.06, 133.36, 135.11, 137.04, 143.52,

150.12, 156.68, 159.29. Anal. C21H16ClN3O3S: C, 59.22;

H, 3.79; N, 9.87; Found: C, 58.99; H, 3.80; N, 9.89.

Solubility and partition coefficient

The solubility of all the compounds was assessed in water,

ethanol, ethylacetate, chloroform and hexane. The lipo-

philic constant of all the compounds (5a–5l) was deter-

mined in n-octanol and buffer (pH 7.4) by shake flask

method (Podunavac-kuzmanovic et al., 2008). The Log P

was calculated by correlating the absorbance with the

concentration in standard plot (Table 2).

Pharmacological evaluation

Animals

Swiss albino mice (20–25 g) and rats (200–220 g) of either

sex were procured from Central Animals House, Institute

of Medical Sciences, Banaras Hindu University. The

Med Chem Res (2014) 23:4167–4176 4171

123

animals were housed in polypropylene cages and were kept

under controlled environmental conditions at a temperature

of 25 ± 1 �C and 45–55 % relative humidity and a 12:12 h

light/dark cycle. The animals had free access to commer-

cial rodent feed (Doodh Dhara Pashu Ahar, India) and

water ad libitum. Animals were acclimatized for at least

1 week before using them for in vivo screening. Experi-

ments on animals were approved by the Central Animal

Ethical Committee of BHU, Varanasi, India (Protocol No:

Dean/10-11/282).

Anticonvulsant activity

The anticonvulsant activity of the title compounds was

evaluated on Swiss albino mice (20–25 g) of either sex.

The animals were kept under standard conditions and

allowed free access to standard pellet and water ad libitum.

Food was withdrawn 12–15 h before commencing the

experiment, while water was withdrawn immediately

before the experiment. All the newly synthesized com-

pounds (5a–5l) were tested for their anticonvulsant activity

against MES-induced seizure. The compounds exhibiting

protection against MES-induced seizure were selected for

further evaluation against subcutaneous scPTZ-induced

seizure model in Swiss albino mice. Subsequently, all the

compounds were evaluated for their possible activity in

AMPA-induced seizure model. The rotarod test was per-

formed to assess any probable changes in motor coordi-

nation induced by the test compounds. Selected compounds

were also subjected to liver function test to assess the

serum AST and ALT enzyme activity. Phenytoin, GYKI

52,466 and talampanel were selected as standard drugs.

The synthesized compounds and the standards were sus-

pended in 30 % of aqueous solution of poly(ethylene

glycol) (PEG 400).

Maximal electroshock (MES) test

The synthesized compounds and the standards were

administered to the Swiss albino mice intraperitoneally (ip)

in a standard volume of 0.5 mL per 20 g body mass at a

dose of 20–500 lmol/kg body weight. Control animals

received 30 % aqueous PEG 400. After 0.5 h following the

drug administration, seizure was induced by application of

an electrical stimulus 50 mA at 60 Hz of 0.2 s in duration

transmitted via ear clip electrode across the brain. After

applying the shock, the animals were observed for the type

of convulsion produced and the hind limb extensor

response was taken as the end point. The reduction in time

or the absence of hind limb tonic extension of seizure was

taken as protection against seizure. Median effective dose

(ED50) was calculated at different doses of test compounds

(5a–5l) and GYKI 52,466 using probit analysis at 95 %

confidence limit as per the reported procedure (Castel-

Branco et al., 2009). ED50 values, the dose at which tonic

hind limb seizure was prevented in 50 % of animals with

95 % confidence limit were calculated (Table 3).

Subcutaneous pentylenetetrazole (scPTZ)-induced

seizure

PTZ dissolved in 0.9 % w/v NaCl solution at a dose of

70 mg/kg was injected in mice subcutaneously, and the

onset and severity of convulsion were noted for the control

group. The test (5c, 5g, 5i and 5k) and the standard groups

were administered ip 0.5 h prior to the administration of

PTZ, and the activity was calculated in terms of ED50 at

95 % confidence interval (Table 3) at a dose of

20–500 lmol/kg body weight in 30 % aqueous PEG 400.

AMPA-induced seizure

Mice were anaesthetized with pentobarbital sodium

(2.5–3.0 mg/kg ip) and placed in a stereotaxic instrument

(Quintessential stereotaxic injector, Stoelting Co., USA).

For the intracerebroventricular (icv) injection of AMPA, a

24-gauge cannula was implanted at 0.9 mm lateral and

0.7 mm posterior to bregma, at a depth 3.0 mm below the

surface of the skull. It was held in place with dental cement

applied to the exposed skull surface. Post-operative, mice

were housed individually to avoid damage to the injection

apparatus. After a recovery period of 1 week after

implantation, test compounds (5a–5l) and the standards

were given ip at a dose of 20–500 lmol/kg body weight in

30 % aqueous PEG 400. AMPA at a dose of 1 lgm/mouse

was injected after 60 min at a volume of 4 ll, and wild

running, tonic and clonic seizures were monitored for

10 min. ED50 was calculated using Environmental Pro-

tection Agency, USA probit analysis program (version

Table 2 Partition coefficient and solubility of synthesized

compounds

Code Log P Water Ethanol Ethylacetate Chloroform Hexane

5a 2.14 ?? ?? ?? ??? -

5b 2.68 - ?? ?? ??? ??

5c 2.43 - ?? ?? ??? -

5d 2.61 - ?? ?? ??? -

5e 2.50 - ?? ?? ??? ??

5f 2.23 - - - ??? -

5g 2.75 - ?? ?? ??? ??

5h 2.56 ?? ?? ??? ??? -

5i 2.78 ?? ?? ?? ??? -

5j 2.54 - ?? ?? ??? -

5k 2.81 - - ?? ??? ??

5l 2.47 - - ?? ??? ??

?? sparingly soluble, ??? soluble, - insoluble

4172 Med Chem Res (2014) 23:4167–4176

123

1.5). (n = 8/group, where n represents the number of mice

in a group) (Table 3) (Yamashita et al., 2004).

Acute neurotoxicity study

The rotarod test (Vogel, 2002) was carried out to assess the

impairment of motor performance, ataxia, loss of skeletal

muscular strength and acute neurotoxicity produced by the

synthesized compounds. Swiss albino mice weighing

between 20 and 25 g (n = 8), were trained to balance on

the knurled wooden rod (3.2 cm diameter) rotating at

6 rpm. Trained animals were treated with the test com-

pounds (5a–5l) and the standards at a dose of

20–500 lmol/kg body weight in 30 % aqueous PEG 400

administered ip. The mice were placed onto the rotating

rod for 1 min after 30 min of dose treatment. Neurological

impairment was determined as the inability of the animal to

remain on the rod for 1 min. The number of mice having

motor impairment was counted, and the TD50 (the dose

which induced motor toxicity in 50 % of mice) at 95 %

confidence intervals were calculated using probit analysis

(Table 3).

Hepatotoxicity study

The rats were divided into groups of six, and the control

group received standard diet and vehicle. The other

groups were administered the selected test drug (5g and

5i) in dose of 100 lmol/kg/day (in PEG 400) for 14 days.

After the period, blood samples were collected and ana-

lysed for serum AST and ALT activity using a photo-

metric auto analyser (ERBA Chem Pro, Transasia Bio-

Medicals, Mumbai, India). After collection of blood

samples, animals were sacrificed for isolation of liver

tissues to observe histopathological changes, if any. The

samples were dissected out and were fixed in 10 % for-

malin solution. Paraffin sections were made and stained

with haematoxylin and eosin for the study using Nikon

(Tokyo, Japan) digital microscope (Eclipse E200)

(Table 4; Fig. 2).

Statistical analysis

Statistical comparisons between groups of control and

drug-treated groups were made using Fisher’s exact prob-

ability test (incidence of the seizure phases) or one-way

ANOVA followed by post-hoc Turkey’s test (AST and

ALT activity) using InStat Graph Pad Software (San

Diego, CA, USA). The ED50 values of each phase of the

seizures induced in all the models and TD50 values of ro-

tarod test were calculated using Environmental Protection

Agency, USA probit analysis program (version 1.5).

P \ 0.05 was considered to be statistically significant.

Table 3 Anticonvulsant activity of compounds 5a–5l against MES-, scPTZ- and AMPA-induced seizure in Swiss albino mice. TD50 assessed by

rotarod test

Compd ED50 (lmol/kg)

MES scPTZ AMPA TD50 (lmol/kg) PI

Rotarod

5a 135.1 (97.4–189.7) ND 150.9 (76.8–269.6) 229.6 (186.3–276.2) 1.7

5b 109.4 (66.8–148.8) ND 119.8 (87.4–160.1) 306.3 (225.5–321.3) 2.8

5c 59.7 (20.0–99.1) 90.2 (54.2–132.7) 69.8 (42.5–97.2) 160.0 (109.0–265.7) 2.6

5d 103.0 (60.8–161.1) ND 120.8 (53.3–226.1) 136.2 (94.3–202.9) 1.3

5e 134.8 (98.3–186.6) ND 146.3 (107.5–205.9) 213.3 (151.2 –382.7) 1.5

5f 88.0 (65.4–106.9) ND 95.8 (70.5–120.2) 155.9 (110.2–238.9) 1.7

5g 41.3 (26.1–63.4) 82.5 (60.9–99.6) 50.3 (25.9–73.1) 212.8 (148.6–396.7) 5.1

5h 98.2 (59.2–148.1) ND 105 (77.6–136.3) 201.9 (144.4–344.7) 2.0

5i 52.3 (26.2–76.9) 72.7 (51.9–90.9) 66.3 (44.1– 87.4) 228.7 (150.6–312.9) 4.3

5j 94.6 (44.4–132.0) ND 114.3 (86.1–145.3) 160.1 (87.6–265.7) 1.6

5k 66.4 (34.0–99.2) 75.3 (40.3–112.9) 72.7 (51.9–90.9) 135.1 (98.5–186.6) 2.0

5l 93.9 (54.9–142.5) ND 118.6 (88.3–154.1) 111.3 (77.7–144.7) 1.1

GYKI 52466 35.9 (27.1– 63.3) 68.7 (41.7–95.8) 41.5 (14.8–66.1) 76.0 (34.4–122.6) 2.1

Phenytoin 15.7 (7.2–28.3) 23.7 (12.2–42.4) – 85.2 (50.2–126.0) 5.4

Talampanela 28.8 56.3 40.5 – –

ED50 and TD50 values were calculated by probit method (95 % confidence limits are given in parentheses)

PI protective index indicates TD50 of rotarod/ED50 of MES, ND activity not determineda Data from De Sarro et al. (2003)

Med Chem Res (2014) 23:4167–4176 4173

123

In silico ADME prediction

The compounds (5g, 5i and 5k) were subjected to evalua-

tion by the QikProp� (version 3.2) module of the Maestro

Schrodinger software for prediction of their pharmacoki-

netic properties. All the compounds were neutralized

before being subjected to QikProp� analysis, and signifi-

cant pharmacokinetic properties consisting of principal

descriptors such as log P (octanol/water), Lipinski’s rule of

five violation, % human oral absorption, CNS activity,

detection of reactive functional groups and predicted brain/

blood partition coefficient are reported (Tables 5, 6).

Results and discussion

Chemistry

The proposed quinazolin-4(3H)-one derivatives were syn-

thesized as illustrated in Scheme 1. Elemental analysis and

the spectral data are in agreement with the structures of the

synthesized compounds. The spectra of intermediate (3)

displayed the characteristic –C=O stretch of amide and

–COOH group at 1,685 and 1,732 cm-1, respectively, and

a broad peak at 3,437 cm-1 due to –NH stretch. In par-

ticular, the –C=O stretch of cyclic ester of intermediate (4)

was observed at 1,735 cm-1. All the derivatives showed

infrared absorptions at 1,595–1,608 cm-1 due to C=N

stretch. Further, the –C=O stretching of the quinazolin-

4(3H)-one nucleus was observed at 1,629–1,674 cm-1. The1H NMR spectra of 5a–5l exhibited common spectral

ranges each appearing as a doublet due to the presence of

non-magnetically equivalent ethylene protons. The cou-

pling constant between the ethylene protons is in the range

of 11.7–16.5 Hz and indicates Jtrans coupling. The methoxy

protons displayed signals at d 3.89–4.00 ppm, and all the

other protons belonging to the methyl and aromatic ring

were seen according to the expected chemical shift. The13C NMR depicted the peaks of quinazoline nucleus cor-

responding to d 159.26–163.26 ppm (quinazoline –C=O),

and the –OCH3 peak appears at around d 56 ppm. The

aromatic carbons appear at around d 121–134 ppm, and all

other 13C NMR spectra were seen according to the

expected chemical shift. The elemental analysis results

were within ±0.4 % of the theoretical values.

In silico ADME prediction and partition coefficient

In silico study predicted the compounds (5g, 5i and 5k)

were active for CNS and also cross blood–brain barrier.

The experimentally determined log P values were found to

be in the range (2.14–2.81), and the values were lower than

computationally calculated values (Tables 5, 6). The

parameter was unable to affect the activity as no substantial

correlation could be drawn between lipophilicity and

activity. Nevertheless, log P conforms to the suggested

value of B5 and the mean value of all experimental log

P values is 2.54. This indicates that the compounds are

suitable as CNS drugs (Pajouhesh and Lenz, 2005).

Anticonvulsant activity

The synthesized compounds (5a–5l) were evaluated for

their anticonvulsant activity in various physicochemically

induced seizure models. Preclinical assessment of potential

anticonvulsant drugs depends on these acute models. The

MES test involves transauricular or transcorneal electrical

induction, whereas scPTZ and icv AMPA involve chemical

induction of generalized seizure. The ED50 values are listed

in Table 3, and the results were compared with those of

phenytoin, GYKI 52466 and talampanel. GYKI 52466 and

talampanel were included in the study to correlate the

anticonvulsant activity of the compounds with standard

AMPA receptor antagonist. The effect on motor coordi-

nation was examined in the rotarod test. Few of the com-

pounds displayed their ability to prevent seizure spread in

the various seizure models used for the in vivo screening.

The N3 phenyl and 3-methoxyphenyl derivative of qui-

nazolin-4(3H)-one (5a and 5e) were found to be least

active against all seizure models compared to the other

derivatives. In MES test, it was revealed that a methylene

linkage (5f) in between the phenyl ring and quinazolin-

4(3H)-one nucleus tends to moderately increase the

potency. Mono substitution of methyl group at the meta

position of N3 aryl moiety (5c) showed potent activity,

while disubstitution of methyl group (5b) results in marked

decrease in activity. Considering the compounds with N3

heteroaryl substitution, the pyridin-4-yl derivative (5k)

showed good activity (ED50 66.4 lmol/kg) against MES-

induced seizure. Among all the compounds, 5g and 5i

exhibited promising activity. In particular, 5g with N3 para

nitrophenyl substitution with an median effective dose

(ED50) of 41.3 lmol/kg demonstrated comparable potency

as that of GYKI 52466. Unlike 5g, striking difference in

Table 4 Effects of compounds on serum ALT and AST of rats

Treatment AST (U/mL) ALT (U/mL)

Control 94.53 ± 2.53 37.97 ± 2.13

5g 92.17 ± 2.48 38.26 ± 2.51

5i 95.76 ± 2.53 42.10 ± 2.06

GYKI 52466 94.69 ± 2.98 37.47 ± 2.04

Values are Mean ± SD. [One-way analysis of variance (ANOVA)

followed by post hoc Tukey’s test]. Values were not significant

(P [ 0.05) indicating the non–hepatotoxic nature of the synthesized

compounds

4174 Med Chem Res (2014) 23:4167–4176

123

activity was observed with the N3 ortho nitrophenyl (5h)

derivative (ED50 98.2 lmol/kg). Nevertheless, considering

the quantitative parameters (ED50 and TD50) obtained from

MES test, all the derivatives were found to be relatively

less efficacious than phenytoin. Investigational anticon-

vulsant drugs are initially screened for efficacy in both

MES and scPTZ tests. Therefore, based on the level of

activity obtained in MES test, few compounds (5c, 5g, 5i

and 5k) were selected for activity assessment against

scPTZ-induced seizure. In contrary to the anticonvulsant

activity against MES-induced seizure, the N3 para

bromophenyl derivative (5i) elicited highest potency

against scPTZ-induced seizure. Rest of them showed sim-

ilar pattern of efficacy as obtained from MES test, and the

activity is dose-dependent as increase in dose was required

to protect against scPTZ-induced seizure.

To explain the possible mechanism for anticonvulsant

action, the synthesized compounds were subjected to AMPA-

induced seizure test. The order of potency against AMPA-

induced seizure was found to follow similar pattern as those

observed against MES-induced seizure. However, the poten-

cies of 5k in scPTZ- and AMPA-induced seizure test were

found to be similar. In AMPA-induced seizure, the ED50 value

of 5g was 50.3 lmol/kg and under the present experimental

condition, the potency of 5g was comparable with talampanel.

These observations indicate that substitution of electron

withdrawing group at the para position of N3 aryl ring dem-

onstrated better anticonvulsant activity with significantly low

impairment of rotarod performance in mice compared to the

N3 aryl derivatives substituted with electron donor groups (5d

and 5e). From these findings, it can also be assumed that the

substituted aryl/heteroaryl ring at the N3 position of a 6,7-

dimethoxy-quinazolin-4(3H)-one nucleus has crucial modu-

latory effects on anticonvulsant activity. The present study

showed that 5g exerts promising anticonvulsant actions in

various seizure models and can be used as lead for the

development of more potent anticonvulsant agents.

Liver function test

Compounds showed no significant change in the activities

of enzymes (AST and ALT) as compared to the control

animals. Histopathological examination of control and

representative compounds (5g and 5i) showed no focal or

diffuse necrosis of hepatocytes. Further, infiltrations of

chronic inflammatory cells were not observed (Fig. 2)

compared to the control and GYKI 52466. Thus, it can be

Fig. 2 Photomicrograph of liver of control (a), compound 5g (b), 5i(c) and GYKI 52466 (d)-treated groups at 409

Table 5 Pharmacokinetic prediction of selected compounds by

QikProp� 3.2

Compd. QPlog

Po/w

Rule

of five

#rtvFG CNS QPlogBB % Human oral

absorption

5g 4.21 0 0 -2 -1.22 100

5i 4.54 1 0 1 0.00 100

5k 4.27 0 0 0 -0.35 100

Table 6 Qikprop� properties and descriptors

S.No Descriptor Description Recommended

Range

1 QPlogP

(o/w)

Predicted octanol/water

coefficient

-2.0 to 6.5

2 Lipinski’s

rule of

five

Lipinski’s rules of five are:

mol_MW \ 500, QPlog

Po/w \ 5, donorHB B 5,

accptHB B 10. Compounds

that satisfy these rules are

considered drug like. (The

‘five’ refers to the limits,

which are multiples of 5

Maximum is 4

3 #rtvFG This particular descriptor

indicates the number of

reactive functional groups.

The presence of these groups

can lead to decomposition,

reactivity or toxicity problems

in vivo

0 to 2

4 CNS Predictive central nervous

activity on a -2 (inactive) to

?2 (active) scale

-2 to ?2

5 QPlogBB Predicted brain/blood partition

coefficient. Predictions are for

orally delivered drugs

-3.0 to 1.2

6 % Human

oral

absorption

It predicts human oral

absorption on 0 to 100 %

scale. The prediction is based

on a quantitative multiple

linear regression model. This

property usually correlates

well with human oral

absorption

[80 % is high

\25 % is

poor

Med Chem Res (2014) 23:4167–4176 4175

123

assumed that the selected compounds are devoid of hepa-

totoxicity which is one of the major adverse effects of

prolonged use of some anticonvulsant drugs (Bjornsson,

2008).

Conclusion

From the in vivo assays, we herein report a new class of

3-aryl/heteroaryl-substituted 2-(2-chlorostyryl)-6,7-dime-

thoxy-quinazolin-4(3H)-ones as anticonvulsant agents.

Among all the synthesized compounds, few have exhibited

(5c, 5g, 5i and 5k) capabilities to protect against seizure

induced by MES, scPTZ and icv AMPA. The compound 5g

showed better pharmacological profile and might be a

useful lead for future design, modification and investiga-

tion in developing therapeutically promising anticonvul-

sant agent.

Acknowledgments The authors are grateful to the Head, Depart-

ment of Chemistry, Faculty of Science, Banaras Hindu University,

Varanasi, India for 1H NMR and SAIF-Central Drug Research

Institute, Lucknow, India for 13C NMR. We gratefully acknowledge

the financial assistance provided by University Grants Commission,

New Delhi for the grant of RGNF (SRF) to Mr. Nirupam Das.

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