Structural Exploration of Quinazolin-4(3H)-ones as Anticonvulsants: Rational Design, Synthesis, Pharmacological Evaluation, and Molecular Docking Studies

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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–17

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Full Paper

Structural Exploration of Quinazolin-4(3H)-ones as

Anticonvulsants: Rational Design, Synthesis, Pharmacological

Evaluation, and Molecular Docking Studies

1

2

Vinod G. Ugale and Sanjay B. Bari

1

Department of Pharmaceutical Chemistry, R. C. Patel Institute of Pharmaceutical Education and

Research, Shirpur (Dhule), Maharashtra, India

Department of Pharmaceutical Chemistry, H. R. Patel Institute of Pharmaceutical Education and

Research, Shirpur (Dhule), Maharashtra, India

2

Anticonvulsants effective against multiple seizures are of wide interest as antiepileptic drugs,

especially if active against pharmaco-resistant seizures. Herein, we synthesized 16 different, rationally

designed 2-((6,7-dimethoxy-4-oxo-2-phenylquinazolin-3(4H)-yl)amino)-N-(substituted phenyl)acet-

amides and screened for anticonvulsant activities through in vivo experiments. Compound 4d

emerged as prototype with excellent anti-seizure action in mice against electroshock, chemically

induced and pharmaco-resistant 6-Hz seizure models with no symptoms of neurotoxicity and

hepatotoxicity (ED₅₀¼ 23.5 mg/kg, MES, mice, i.p.; ED ¼ 32.6 mg/kg, scPTZ, mice, i.p.; ED ¼ 45.2 mg/kg,

50

6

-Hz, mice, i.p.; TD₅₀¼ 325.9 mg/kg, mice, i.p.). In addition, investigation of compound 4l in mice for its

pharmacological proﬁle proved it as safer anticonvulsant, devoid of the side effects such as motor

dysfunction and hepatotoxicity of classical antiepileptic drugs (ED₅₀¼ 26.1 mg/kg, MES, mice, i.p.;

ED50 ¼ 79.4 mg/kg, scPTZ, mice, i.p.; TD50 ¼ 361.2 mg/kg, mice, i.p.). We also predicted physiochemical

and pharmacokinetic properties of structurally optimized quinazolin-4(3H)-ones by a computational

protocol. A combination of in vivo anticonvulsant proﬁle, ex vivo toxicity, and in silico studies suggested

that the synthesized compounds may be useful as broad-spectrum anti-seizure drug candidates with

favorable pharmacokinetic parameters.

Keywords: Anticonvulsant activity / Hepatotoxicity / In vivo studies / Neurotoxicity / Synthesis

Received: July 22, 2016; Revised: September 12, 2016; Accepted: September 14, 2016

DOI 10.1002/ardp.201600218

Additional supporting information may be found in the online version of this article at the publisher’s web-site.

:

Introduction

Correspondence: Vinod G. Ugale, Department of Pharmaceutical

Chemistry, R. C. Patel Institute of Pharmaceutical Education and

Epilepsy is a complex disorder of brain function which results

Research, Shirpur (Dhule)-425405, Maharashtra, India.

in enduring predisposition to engender seizures [1]. It is the

second most common chronic neurological condition seen by

neurologists [2]. A global campaign against epilepsy con-

ducted by World Health Organization (WHO) in partnership

with International Bureau for Epilepsy (IBE) and International

League Against Epilepsy (ILAE) suggested that around 1% of

world population at any time is afﬂicted with this neurologi-

cal disorder [3, 4]. This number increases every year by about

2.4 millions. There are several classes of AEDs protecting

against seizures through unique mechanisms: (i) drugs

E-mail: vinod.ugale@rediffmail.com

Fax: þ 91-1881263655

Abbreviations: AEDs, antiepileptic drugs; AMPA, (R,S)-2-amino-3-

(

3-hydroxy-5-methylisoxazol-4-yl)propionic acid; CNS, central

nervous system; ED50, median effective dose; EMA, European

Medicines Agency; IBE, International Bureau for Epilepsy; iGluRs,

ionotropic glutamate receptors; ILAE, International League

Against Epilepsy; NMDA, N-methyl-D-aspartic acid; TD50, median

toxic dose; USFDA, United States Food and Drug Administration;

WHO, World Health Organization.

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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–17

V. G. Ugale and S. B. Bari

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inhibiting fast current–voltage-gated sodium channels (phe-

nytoin, lamotrigine, lacosamide); (ii) high voltage-activated

calcium channel blockers (ethosuximide, pregabalin); (iii)

plausible mechanism of action for series of synthesized

compounds.

drugs increasing GABA activity at GABA

A

receptors (pheno-

Rationale and hypothesis

barbital, clobazam, lorazepam); (iv) inhibition of GABA

transaminase to increase brain GABA (vigabatrin); (v) GABA

reuptake inhibitors (tiagabine); and (vi) AMPA receptor

antagonist (perampanel) [5]. Although many new antiepilep-

tic drugs (AEDs) were also launched in last decade, the

resistance among epilepsy patients remains at constant level

of 30% and 25% of patients suffer from severe adverse

effects [6]. As a corollary, there is a need to introduce some

models of resistant seizures at early stages of AEDs develop-

ment; as an innovative idea for identiﬁcation of compounds

effective in refractory epilepsy. Considering the aforemen-

tioned facts, searching for more effective and better tolerable

AEDs still constitutes to be a great interest in the ﬁeld of

neurological drug discovery and development.

Quinazolin-4(3H)-one is an important landmark in the

ﬁeld of synthetic anticonvulsant as it is an important

skeleton of many reported anticonvulsants [7–9]. Previously

our research group has synthesized a series of fused

quinazolino-benzothiazoles and evaluated them as anti-

convulsants [10]. In our previous publication, we delineated

initial structure–activity relationship (SAR) for the series

of quinazolino-benzothiazoles and these compounds were

found effective in the maximal electroshock (MES) and

subcutaneous pentylenetetrazole (scPTZ) tests. Several pat-

ents were also reported for anticonvulsant potential of

quinazolin-4(3H)-one containing compounds [11–14]. These

patents accentuate the use of quinazolin-4(3H)-ones in

seizure disorders. Thus potency and selectivity in the

pharmacological response of quinazolin-4(3H)-ones have

attracted the attention to explore this framework as

prospective anticonvulsant agents.

Literature survey also exposed the importance of N-phenyl

amide fragment in central nervous system (CNS) active

agents [15–17]. Additionally, a hydroxyl or an oxo group in

amide fragment, acting as an electron donor, increases the

possibility of hydrogen bonding at a binding site [18]. In the

light of potential anticonvulsant activity of quinazolin-4(3H)-

ones and in continuation of our research [8, 10], structural

modiﬁcation of quinazolin-4(3H)-one moiety via fragment

fusion of a N-phenyl amide as hinge binding province through

amino methyl linker has been carried out and screened for

anticonvulsant activity. The aim of study was to identify

anticonvulsants with broad spectrum of activity in predictable

animal models of epilepsy particularly MES and scPTZ,

including 6-Hz model of pharmaco-resistant limbic seizures.

It is well known that numerous drug candidates failed during

clinical tests because of pharmacokinetics and toxicity

problems. Hence, herein, we also evaluated in silico physi-

ochemical and pharmacokinetic properties, in vivo neurotox-

icity, and ex vivo liver toxicity parameters of synthesized

compounds. Molecular docking analysis was executed to

identify the binding determinants of target and to predict

The incomplete information about etiology of epileptic

disorders and multifaceted mechanism of action of AEDs

make difﬁcult to search new antiepileptic agents [19].

Principally, ligand-based and random screening approaches

have been used by many researchers to design and synthesize

potential anticonvulsant agents [20, 21]. Random screening

approach have critical disadvantage as it is serendipitous

method focusing on diverse set of compound libraries;

requires huge time and money. On the other side, ligand-

based approaches mainly focus on utilization of pharmaco-

phore present in existing clinically effective anticonvulsants or

reported bioactive compounds. If we incorporate pharmaco-

phoric rudiments present in existing potential anticonvulsants

to design novel molecules, their appearance in the structure

may determine or boost anticonvulsant properties. Hence,

herein, we have used ligand-based approach to design and

synthesize title compounds.

Following hypothesis has been considered while designing

of outline derivatives. Methaqualone (I) is a drug bearing

quinazolin-4(3H)-one motif found as “master key” in anti-

convulsant therapy [7–9], and its 6,7-dimethoxy analog (II)

proved to possess manifest anticonvulsant potential against

both electroshock and chemically induced seizure tests [22].

Piriqualone (III) is a candidate with quinazolin-4(3H)-one

scaffold known for its signiﬁcant anticonvulsant proper-

ties [23]. If lipophilic group is inserted at second position

of quinazolin-4(3H)-one this leads to potent CNS active

agents [24].

The crucial role of N-phenyl amide side chain was also

considered while designing of novel analogs. Ralitoline (IV) is

an example of drug possessing N-phenyl amide (N-(2,6-

dimethylphenyl)acetamide) portion as a critical part of its

structure and effective in preclinical anticonvulsant evalua-

tion. It is active in both MES and kindling models of seizures

with rodents [25]. Retigabine (V) is an anticonvulsant

(ezogabine) approved by the United States Food and Drug

Administration (USFDA) and/or by the European Medicines

Agency (EMA) in 2011. This drug also has a N-phenyl amide

(N-(2-aminophenyl)acetamide) fragment that played an

important role in its anticonvulsant potency. Retigabine

has shown efﬁcacy in broad spectrum of animal models,

including electrical, chemical induction, and genetic epilepsy

models [26]. In addition, CNS drugs with multiple mecha-

nisms have a better probability of being efﬁcacious in

refractory epilepsies and other CNS disorders [20]. Figure 1

includes some examples of potent anticonvulsants contain-

ing quinazolin-4(3H)-one nucleus and N-phenyl amide

fragment. Inspired by aforementioned hypothesis, bearing

in mind the inherited anticonvulsant potency of 2-phenyl-

quinazolin-4(3H)-one, anticonvulsant activity enhancement

by N-phenyl amide fragment, and potentiating effect of

alkoxy functions to the anticonvulsant activity, combination

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Figure 1. Rational design of the synthesized

compounds 4a–p.

of all these features in one hybrid structure to synthesize

derivatives 4a–p was rationalized (Supporting Information

Fig. S1).

Results and discussion

Chemistry

This optimization is signiﬁcantly distinguished from

previously reported anticonvulsants. Our strategy is directed

toward designing of ligands with diverse chemical properties

hypothesizing that the potency of these molecules might

be enhanced by adding the alternative binding groups

of various N-phenyl acetamides at third position of the

The sequence of reactions has been performed to synthesize

target compounds. The steps used for synthesis of title

compounds were illustrated in Supporting Information

Scheme S1 and Scheme 1. The synthetic route used to

prepare 6,7-dimethoxy-2-phenyl-4H-benzo[d][1,3]oxazin-4-

one (1), starts from suitable anthranilic acid and involved

the sequential treatment with benzoyl chloride in pyri-

dine [27]. The compound 1 was then reﬂuxed with hydrazine

hydrate in dry pyridine to afford 3-amino-6,7-dimethoxy-2-

phenylquinazolin-4(3H)-one (2), as depicted in Supporting

Information Scheme S1 [28, 29]. The 2-chloro-N-(substituted

phenyl)acetamides 3a–p were synthesized according to

3

-amino-6,7-dimethoxyquinazolin-4(3H)-one and lipophilic

group insertion (aryl ring) at second position of 3-amino-

,7-dimethoxyquinazolin-4(3H)-one nucleus. Attesting to

6

these concerns, such substitution pattern could target

different seizure types and create differentially selective

molecules.

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were introduced into the pendent phenyl ring of fragment

acetamides with the aim of modulating afﬁnity and

intrinsic activity of 3-amino-6,7-dimethoxy-2-phenylquina-

zolin-4(3H)-one. The mechanism of reaction for the synthesis

of 3-amino-6,7-dimethoxy-2-phenylquinazolin-4(3H)-one (2)

involves, ﬁrst the ring opening of 6,7-dimethoxy-2-phenyl-

4

H-benzo[d][1,3]oxazin-4-one (1) by the attack of hydrazine

hydrate. The ring opening step is followed by removal of

water molecule to accomplish the ring closure to afford

intermediate compound (2) as shown in Supporting Infor-

mation Scheme S2. The probable mechanism for the

synthesis of ﬁnal molecules 4a–p is N-alkylation reaction.

The reaction involves an attack of primary amino group

of 3-amino-6,7-dimethoxy-2-phenylquinazolin-4(3H)-one (2)

on C–Cl bond of 2-chloro-N-(substituted phenyl)acetamides

3

a–p to yield the title compounds 4a–p. The C–Cl bond

is polarized due to high electronegativity of halogen in

substituted 2-chloro-N-phenylacetamides, and thus carbon

become electrophilic in nature. Finally, a replacement

of halogen by a nucleophilic amino group resulted into

formation of target molecules as depicted in Supporting

Information Scheme S2.

The chemical structures of synthesized compounds 4a–p

1

13

were elucidated on the basis of FTIR, H NMR, C NMR, and

mass spectra. The IR spectra of all compounds showed

ꢀ1

appearance of band at 1723–1705 cm due to the presence

of >C–O group of 6,7-dimethoxy quinazolin-4(3H)-one

nucleus. In general, a distinctive band appeared at 1634–

ꢀ

1

609 cm

for C–N, which conﬁrms the formation of

quinazolin-4(3H)-one ring. All the ﬁnal compounds showed

disappearance of bilobe due to primary amino group (3550–

ꢀ

1

3

400 cm ) of intermediate (2), owing to conversion into

1

2 6

secondary aliphatic amine (–NHCH –). The H NMR (DMSO-d )

spectrum of all derivatives displayed proton absorption

singlet at 10.20–10.96 d ppm due to –CONH– proton. In

1

2

H NMR spectrum, characteristic peak of –NH proton of

intermediate (2) has disappeared from all title compounds

indicating fusion of N-phenyl acetamides with 3-amino-6,7-

dimethoxy quinazolin-4(3H)-one. Again, all the compounds

revealed a typical proton absorption singlet at 9.03–9.18 d

ppm due to –NH– proton. Presence of two methoxy groups in

1

synthesized compounds exhibited two peaks in H NMR

spectrum around at 3.97–4.05 d ppm, which is magnetically

different. All aromatic protons appeared in the range of

1

3

6

.86–8.19 d ppm as multiplets. The C NMR spectrum of

synthesized compounds also exposed the two characteristic

Scheme 1. Synthesis of target compounds 4a–p.

methoxy peaks around at 56.37 and 56.40 d ppm. The HRMS

1

3

and C NMR spectra of all compounds 4a–p were found in

concurrence with the outlined structures.

known procedures from different substituted anilines [30].

Finally, the 2-chloro-N-(substituted phenyl)acetamides 3a–p

were conjugated with 3-amino-6,7-dimethoxy-2-phenyl-

quinazolin-4(3H)-one (2) in N,N-dimethylformamide (DMF)

at reﬂux to give the subsequent 2-((6,7-dimethoxy-4-oxo-

Pharmacological anticonvulsant evaluations

The preclinical discovery and development of novel hetero-

cyclic compounds for the treatment of epileptic seizures is

mainly depended on the use of predictable animal models.

The earlier stage of drug testing included the MES and scPTZ

in vivo screening models. Rotarod test was used to determine

2

-phenylquinazolin-3(4H)-yl)amino)-N-(substituted phenyl)

acetamides 4a–p as presented in Scheme 1. Small substituents

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the acute neurotoxicity. The results of initial in vivo

pharmacological screening are delineated in Table 1. The

title compounds 4a–p have demonstrated that diversiﬁed

bioactivities with respect to strength, dose, and time of

protection depend upon different substitutions pattern over

outlined derivatives.

The MES test identiﬁes clinical candidates that have ability

to inhibit the spread of seizures. The outcomes of preliminary

screening by MES test revealed that majority of synthesized

compounds possess moderate to good anticonvulsant activity

at both different time intervals. In MES test, compounds 4d, 4l,

mice (convulsive dose: 80 mg/kg). The scPTZ seizure model

proposed to identify the compounds that could provide

protection by elevating the seizure threshold. Exclusively,

compound 4d has shown total abolition of clonic convulsions

induced by pentylenetetrazole at both time intervals with

the lowest dose of 30 mg/kg and thus providing 100%

protection in scPTZ test. From the results of preliminary

in vivo screening, it was conﬁrmed that the compound 4d

has broad spectrum of anticonvulsant activity because

it had shown promising anticonvulsant action in both

seizure models. Compounds 4c, 4l, 4m, 4n, and 4o revealed

signiﬁcant protection at a dose of 100 mg/kg after 0.5 h

equivalent to standard drug carbamazepine. Compounds 4a,

4b, 4e, 4h, 4i, and 4j delivered unique protection at a

higher dose of 300 mg/kg and remaining compounds were

found to be inactive after 0.5 h at high dose. As shown in

Table 1, compounds 4c, 4k, 4l, 4m, and 4o also displayed

delayed anticonvulsant effect at a similar dose of 100 mg/kg

(4 h) in scPTZ seizures. Compounds 4a, 4b, 4f, 4j, and 4n

exhibited prolonged anticonvulsant effect only after a dose

of 300 mg/kg which is equivalent to standard drug carba-

mazepine. From the series of derivatives, only two com-

pounds 4g and 4p were completely devoid of anticonvulsant

action in scPTZ test.

4

m, and 4o have shown promising anticonvulsant activity

even at lowest dose of 30 mg/kg after 0.5 h as equivalent to

reference drugs. This indicates their quick onset of anticon-

vulsant effect by preventing the spread of seizures. Among

the series of compounds, 4l delivered protection against

maximal seizures at both time intervals with a same 30 mg/kg

dose, exhibiting its extended duration of action along with

high potency. The compounds 4a, 4c, 4i, and 4k has provided

protection at a dose of 100 mg/kg in both time intervals

specifying their fast onset as well as longer duration of action.

Compound 4j exhibited anti-MES effect at a dose 100 mg/kg

only after 4 h, indicating its slow onset of action for longer

duration of time. From whole series, except compound 4f all

the remaining compounds were active in MES test.

In the acute neurotoxicity screen, majority of compounds

were not showing any symptoms of neurological deﬁcits

namely, sedation, ataxia, hyperexcitability and impaired

In scPTZ test, myoclonic seizures were induced by

administration of pentylenetetrazole subcutaneously to

Table 1. Anticonvulsant evaluation (MES, scPTZ) and neurotoxicity (rotarod test) screening of synthesized compounds

4

a–p.

MES test

scPTZ test

Neurotoxicity screening

a)

Compounds

0.5 h

4 h

0.5 h

4 h

0.5 h

4 h

4

a

b

c

d

e

f

g

h

i

j

k

l

m

n

o

p

100

30

–

100

300

100

300

–

300

100

30

300

–

300

100

30

–

300

–

300

–

300

–

300

–

300

100

–

300

–

100

30

100

300

100

–

100

–

300

–

300

100

300

100

–

100

30

–

100

–

300

–

30

300

30

300

–

PHY

CBZ

30

100

–

100

300

100

300

a)

Doses of 300, 100, and 30 mg/kg were administered intraperitoneally to mice. The anticonvulsant activity and neurotoxicity

were measured after 0.5 and 4 h. The values in table represents minimum dose required for protection or neurotoxicity in half

or more number of animals in a group consisting of six mice. A dash (–) represents the absence of bioactivity and neurotoxicity

at the maximum dose given intraperitoneally to mice (300 mg/kg).

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Table 2. Anticonvulsant screening of selected compounds by psychomotor seizure test (6 Hz, current 32 mA) in mice.

a)

Intraperitoneal injection into mice

Compounds

0.25 h

0.5 h

1 h

2 h

4 h

4

b

c

d

i

k

l

n

o

2

3

2

1

2

4

2

3

2

4

2

4

2

3

4

1

4

3

2

4

3

4

1

4

3

4

3

2

4

0

1

3

4

3

Levetiracetam

a)

Each dose (100 mg/kg, mice, i.p.) of selected compounds was tested using four animals and the number of animals protected

was recorded. Data indicate number of mice protected in each group at four pretreatment times 0.25, 0.5, 1, and 2 h.

motor functions. The compound 4i showed signs of neuro-

toxicity at 100 mg/kg with increase in sedation and hyper-

excitability after delayed absorption (4 h) at higher dose of

intervals. This screen unveils the time of onset, approximate

time of peak effect (TPE), and duration of anticonvulsant

activity. Results of 6-Hz test are given in Table 2. From the

anticonvulsant data acquired, it was exposed that compound

4d offered 100% protection at four different time intervals

0.5, 1, 2, and 4 h. Compound 4l delivered anticonvulsant effect

at four different time points 0.25, 0.5, 1, and 2 h. Both of these

compounds exhibited fast onset of action span over long

period. Most of the selected compounds provoked satisfac-

tory anti-seizure effect in pharmaco-resistant seizures model.

From chosen derivatives, only compounds 4i and 4k offered

50% or below protection at different time intervals in this

protocol of anticonvulsant activity.

Compounds found prominent in initial qualitative differ-

ential evaluations were subjected to in vivo quantitative

determination studies. All quantitative in vivo anticonvulsant/

toxicity estimations of active compounds were performed

at compounds time of peak pharmacodynamic effect in

qualitative screening. The results of quantitative determina-

tion of the median effective dose (ED50) in the MES and

scPTZ tests as well as toxic dose (TD50) are presented in

Tables 3 and 4, respectively. Results for 6-Hz test are given in

3

00 mg/kg. The 2-ﬂuoro derivative 4n at a dose of 300 mg/kg

provoked muscle spasms, tremors, and ataxia after 0.5 h. Rest

of compounds 4c, 4e, and 4o demonstrated neurological

discrepancies at higher dose of 300 mg/kg as compared to

standard drugs after 4 h.

Although, MES and scPTZ seizure models are considered as

“gold standards” to assess the efﬁcacy of most of the novel

anticonvulsant agents, but these models would be ineffective

in therapy-resistant epileptic seizures. Recently, the 6-Hz

screening has been validated as a model of pharmaco-

resistant limbic seizures. Levetiracetam is a clinically effective

anticonvulsant against resistant seizures, even though it is not

active in MES and scPTZ seizure model but shows bioactivity in

6

-Hz test. This in vivo screening model is used to discover the

anticonvulsant clinical candidate having novel spectrum of

activity with unknown mechanism. Eight compounds from the

series were randomly chosen for 6-Hz test, tested before in the

MES and scPTZ seizures. These compounds were administered

in mice at a ﬁxed dose of 100 mg/kg (i.p.) at ﬁve different time

Table 3. Quantitative anticonvulsant screening of selected active compounds by MES test.

a)

b)

c)

d)

Compounds

TPE

ED50 (mg/kg)

TD50 (mg/kg)

PI (TD50/ED50)

4

c

0.5

4

0.5

1

123.5 (100.3–148.2)

23.5 (17.7–27.5)

128.7 (102.9–149.7)

26.1 (19.7–31.5)

35.7 (28.3–39.7)

31.5 (26.9–35.2)

25.7 (21.1–31.3)

321.2 (290.6–349.4)

325.9 (302.8–344.5)

401.6 (310.5–489.2)

361.2 (316.5–401.1)

270.6 (262.7–283.4)

310.4 (305.3–332.6)

79.1 (68.2–93.1)

2.60

13.86

3.12

13.83

7.57

9.85

3.07

d

k

l

m

o

e)

PHY

Data in parentheses are the 95% conﬁdence limits.

a)

b)

Time to peak effect (TPE) in hours. ED50: median effective dose required to elicit anticonvulsant response in 50% of animals

c)

used. TD50: median toxic dose producing minimal neurological toxicity in 50% of animals determined by rotarod test.

d)

e)

Protective index PI: TD50/ED50

.

TPE for standard was taken from literature [19].

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Table 4. Quantitative anticonvulsant screening of selected active compounds by scPTZ test.

a)

b)

c)

d)

Compounds

TPE

ED50 (mg/kg)

TD50 (mg/kg)

PI (TD50/ED50)

4

c

0.5

4

0.5

109.7 (92.7–128.3)

32.6 (26.6–41.1)

124.6 (103.1–148.7)

79.4 (64.1–101.3)

107.4 (90.1–126.2)

97.3 (80.2–112.7)

115.4 (93.5–131.4)

321.2 (290.6–349.4)

325.9 (302.8–344.5)

401.6 (310.5–489.2)

361.2 (316.5–401.1)

270.6 (262.7–283.4)

310.4 (305.3–332.6)

75.3 (52.7–126.5)

2.92

10.02

3.22

4.54

2.51

3.19

0.65

d

k

l

m

o

e)

CBZ

Data in parentheses are the 95% conﬁdence limits.

TPE in hours. ED50: median effective dose required to elicit anticonvulsant response in 50% of animals used. TD50: median

a)

b)

c)

d)

toxic dose producing minimal neurological toxicity in 50% of animals determined by rotarod test.

Protective index PI:

e)

TD50/ED50

.

TPE for standard was taken from literature [2].

Table 5. In parallel, same studies were executed for reference

drugs; phenytoin, carbamazepine, and levetiracetam which

proved potent during preclinical anticonvulsant screening.

The data obtained was used to calculate the protective

indexes (PIs), considered as a measure of beneﬁt-to-risk

ratio of clinical candidates. From the six compounds selected

for quantiﬁcation studies, compound 4d exhibited superior

anticonvulsant activity with admirable ED50 value (23.5 mg/

kg, mice, i.p.) in the MES test. The compound 4d also

showed profound anticonvulsant activity in other in vivo tests

employed during study (ED50 ¼ 32.6 mg/kg, scPTZ, mice, i.p.;

ED50 ¼ 45.2 mg/kg, 6 Hz, mice, i.p.). Remarkably, this molecule

exposed analogous efﬁciency to phenytoin (MES test),

and distinctly higher activity in comparison with scPTZ-active

carbamazepine (>3.5-fold). Additionally, this compound

displayed markedly low neurotoxicity (TD50 ¼ 325.9 mg/kg,

mice, i.p.) in rotarod test; results in more beneﬁcial PI values

for all seizure models utilized in study (PI ¼ 13.63, MES;

PI ¼ 10.02, scPTZ; PI ¼ 7.21, 6 Hz). Furthermore, the com-

pound 4l was also given a better anticonvulsant action at low

dose in both seizure models (ED50 ¼ 26.1 mg/kg, MES, mice,

i.p.; ED50 ¼ 79.4 mg/kg, scPTZ, mice, i.p.) and did not impair

the motor coordination at maximum dose of compound

though having ED50 nearly less than phenytoin but their

PI was far better than reference used (4m: 2.4-fold, MES;

4.0-fold, scPTZ; 4o: 3.2-fold, MES; 5.5-fold, scPTZ). This can be

attributed to high TD50 value for the mentioned drugs.

Unfortunately, compounds 4d and 4l were less active in 6-Hz

screening than levetiracetam but they came forward with

low neurotoxicity in rotarod test resulted in more construc-

tive PIs (Table 5). A worthy PI value indicates good margin

of safety and tolerability between anticonvulsant doses.

When compared with standard AEDs, all the chosen

compounds have good beneﬁt-to-risk ratio and provoked

hope as a future clinical candidate.

Behavioral study

All synthesized compounds were evaluated for behavioral

activity by using actophotometer at two different time points

(0.5 and 4 h). Behavioral screening was performed by

intraperitoneal administration of two doses of compounds,

i.e., 30 and 100 mg/kg. The results of locomotor activity are

presented in Table 6. Most of the synthesized compounds

displayed behavioral changes at a dose of 30 mg/kg after

0.5 h. Compounds 4d, 4k, and 4l were found to decrease

locomotor activity signiﬁcantly at the same dose after 0.5 h.

Only the compounds 4e, 4f, and 4h did not show signiﬁcant

locomotor impairment after 0.5 h at a dose 30 mg/kg.

The compounds 4e, 4f, 4h, and 4p showed no signiﬁcant

behavioral changes at 100 mg/kg (0.5 h), but all other

(

TD50 ¼ 361.2 mg/kg, mice, i.p.). In addition, compound 4l

resulted in distinctly more favorable PI in scPTZ test in

comparison to reference drug carbamazepine (> sixfold). In

case of compounds 4m and 4o, it is worth mentioning that

Table 5. Quantiﬁcation studies of selected compounds: psychomotor seizure test (6 Hz, current 32 mA).

a)

b)

c)

d)

Compounds

TPE

ED50 (mg/kg)

TD50 (mg/kg)

PI (TD50/ED50)

4

d

l

4

1

45.2 (30.2–91.1)

89.1 (52.1–106.3)

20.1 (10.3–34.6)

325.9 (302.8–344.5)

361.2 (316.5–401.1)

>500

7.21

4.05

>24

e)

Levetiracetam

a)

b)

c)

TPE in hours. ED50: median effective dose required to assure anticonvulsant protection in 50% animals. TD50: median toxic

d)

e)

dose eliciting minimal neurological toxicity in 50% animals. PI: protective index (TD50/ED50). TPE for levetiracetam was

taken from literature [6].

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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–17

V. G. Ugale and S. B. Bari

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Table 6. Locomotor activity of synthesized compounds 4a–p by using actophotometer.

Locomotor activity score by using actophotometer

a)

Post-treatment

0

.5 h

4 h

Control

Compounds

(24 h before)

30 mg/kg

100 mg/kg

30 mg/kg

100 mg/kg

4

a

b

c

d

e

f

g

h

i

j

k

l

m

n

o

p

185.2 ꢁ 8.3

190.8 ꢁ 5.2

224.3 ꢁ 3.8

273.7 ꢁ 9.4

219.0 ꢁ 10.2

205.1 ꢁ 6.2

176.7 ꢁ 1.4

195.3 ꢁ 3.3

188.4 ꢁ 4.2

192.7 ꢁ 5.3

215.3 ꢁ 4.6

194.2 ꢁ 7.3

185.3 ꢁ 6.2

189.4 ꢁ 4.2

197.2 ꢁ 7.2

203.3 ꢁ 5.2

178.6 ꢁ 10.2

184.1 ꢁ 7.1

156.3 ꢁ 2.1

119.1 ꢁ 4.2

141.1 ꢁ 6.2

57.3 ꢁ 5.3

68.1 ꢁ 5.7

82.5 ꢁ 3.2

110.1 ꢁ 3.3

125.3 ꢁ 5.4

70.1 ꢁ 4.3

81.1 ꢁ 5.5

171.1 ꢁ 3.4

167.3 ꢁ 5.1

104.1 ꢁ 6.4

135.1 ꢁ 3.4

80.1 ꢁ 4.2

115.4 ꢁ 5.3

82.4 ꢁ 3.5

77.1 ꢁ 4.8

78.6 ꢁ 6.4

105.4 ꢁ 5.3

67.1 ꢁ 1.2

177.1 ꢁ 6.5

60.3 ꢁ 5.4

88.1 ꢁ 6.5

90.2 ꢁ 3.4

69.1 ꢁ 6.3

190.1 ꢁ 4.7

130.3 ꢁ 2.1

90.2 ꢁ 1.3

162.3 ꢁ 2.6

72.2 ꢁ 3.4

95.2 ꢁ 4.2

97.2 ꢁ 5.6

83.3 ꢁ 4.6

97.4 ꢁ 3.2

100.2 ꢁ 4.5

85.5 ꢁ 6.3

181.5 ꢁ 3.7

79.1 ꢁ 5.1

65.4 ꢁ 5.4

199.1 ꢁ 3.5 ns

200.5 ꢁ 4.2 ns

154.3 ꢁ 5.1

190.6 ꢁ 4.7 ns

121.2 ꢁ 4.5

175.3 ꢁ 6.4

65.3 ꢁ 5.3

195.5 ꢁ 4.4 ns

190.4 ꢁ 5.3 ns

142.1 ꢁ 3.4

190.8 ꢁ 5.7 ns

117.3 ꢁ 2.6

85.2 ꢁ 6.3

78.2 ꢁ 4.2

59.1 ꢁ 3.1

66.3 ꢁ 3.7

109.1 ꢁ 4.5

135.5 ꢁ 5.6

115.1 ꢁ 5.3

170.4 ꢁ 5.1

89.8 ꢁ 2.5

119.5 ꢁ 3.5

81.3 ꢁ 4.3

198.5 ꢁ 3.2 ns

b)

PHY

CBZ

74.4 ꢁ 5.4

48.1 ꢁ 4.7

89.1 ꢁ 4.2

65.1 ꢁ 5.3

c)

a)

ꢂ

Each value represents the mean ꢁ SEM signiﬁcantly different from the control at P < 0.05. ns, not signiﬁcant at P < 0.05

b)

c)

(

Student’s t-test). Activity score was measured for 10 min. Drug was tested at dose 25 mg/kg (i.p.). Drug was tested at dose

mg/kg (i.p.).

4

compounds shown good capability to depress central nervous

system when compared to standards used. In behavioral

study, when compared to standard drugs at 4 h (30 mg/kg);

compounds 4c, 4d, 4i, 4k, 4l, 4m, and 4o displayed signiﬁcant

reduction in locomotor activity among all the title com-

pounds. Most of the synthesized compounds provoked

signiﬁcant CNS depression at 4 h as compared to reference

drugs at relatively large dose of compounds (100 mg/kg). This

screening protocol provided evidence that these compounds

were able to depress central nervous system for prolonged

time period that is characteristic parameter of potential anti-

seizure drugs.

Histopathological evaluation

Liver toxicity is a major drawback of most of the clinically

effective anticonvulsant agents. After frequent doses for long

period, AEDs speciﬁcally carbamazepine and valproic acid are

found to demonstrate hepatotoxicity. Phenytoin is also prone

to acetaminophen hepatotoxicity [31]. With reference to

the aforesaid facts, most active compounds from series were

evaluated for hepatic toxicity and biochemical parameters

concerns with liver functions. The values of alkaline phos-

phatase, serum glutamate oxaloacetate transaminase (SGOT),

serum glutamate pyruvate transaminase (SGPT), and total

protein are depicted in Table 7. Results of serum level of liver

Table 7. Enzyme estimation of the selected most active compounds.

Alkaline

Total protein

ns

a)

ns

b)

ns

Treatment

phosphatase ꢁ SEM

SGOT ꢁ SEM

SGPT ꢁ SEM

(g/100 mL) ꢁ SEM

Control

14.67 ꢁ 0.37

15.36 ꢁ 0.18

16.43 ꢁ 0.45

14.62 ꢁ 0.17

16.04 ꢁ 0.71

16.17 ꢁ 0.16

87.83 ꢁ 1.46

89.6 ꢁ 3.02

90.7 ꢁ 1.23

88.1 ꢁ 2.17

91.6 ꢁ 1.79

92.5 ꢁ 3.37

29.23 ꢁ 2.28

31.25 ꢁ 2.16

31.29 ꢁ 2.38

30.21 ꢁ 1.49

1.90 ꢁ 0.01

1.89 ꢁ 0.17

1.80 ꢁ 0.28

1.76 ꢁ 0.05

1.79 ꢁ 0.15

1.85 ꢁ 0.34

4

c

d

l

m

29.90 ꢁ 1.28

31.37 ꢁ 2.19

PHY

a)

b)

Serum glutamate oxaloacetate transaminase. Serum glutamate pyruvate transaminase.

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Rational Design and Synthesis of Anticonvulsant Agents

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enzymes after administration of chosen compounds sug-

gested that none of the compounds had shown any

signiﬁcant variation in the aforementioned biochemical

constraints as compared to control.

Histopathological study was performed after chronic

administration of selected compounds to mice at a maximum

dose to identify the liver toxicity. This protocol conﬁrmed that

there were no major signs of liver toxicity as histopathic

images showing normal hepatic parenchyma with portal

triad, central vein, and heptocytes in comparison to a

reference carbamazepine. Histopathic pictures of mice liver

We have in silico predicted physicochemical descriptors of

molecules, such as molecular weight, hydrogen bond donors,

hydrogen bond acceptors, and were found in accordance with

the Lipinski’s rule of ﬁve (Supporting Information Table S2).

We have also determined the pharmacokinetic properties

such as absorption, distribution, metabolism, and excretion

(ADME) with the help of QikProp module [33]. For all the

molecules, the partition coefﬁcient (QPlogPo/w) and water

solubility (QPlogS) were found signiﬁcant for absorption and

distribution in the body, ranged between 3.13 to 4.85

and ꢀ5.87 to ꢀ4.70, respectively. Cell permeability (QPPCaco),

plays a pivotal role in governing drug metabolism and its

way in to the biological membrane, ranged from 171 to

1740. QikProp predicted blood/brain partition coefﬁcient

(QPlogBB) values for effective CNS activities should be in

the range of ꢀ3.0 to 1.2 [33]. If the drug is too polar, it

should not cross blood-brain barrier. QPlogBB values for

synthesized compounds ranged from ꢀ1.95 to ꢀ0.46 and

QPPMDCK values ranged from 73 to 3188. As all the

synthesized molecules fall within the acceptable range of

QPlogBB, these molecule have potential to cross blood-brain

barrier. MDCK cells are considered to be a good mimic for

the blood-brain barrier. Overall, the percentage human oral

absorption for compounds ranged from 72.67 to 100%. All of

these pharmacokinetic properties are within the suitable

range distinct for human use (see Supporting Information

Table S2 footnote), thereby signifying potential of these

compounds as drug-like molecules.

(

T.S. of liver) are given as Supporting Information Fig. S2.

Supporting Information Fig. S2A indicates the effect of

control (0.5% polyethylene glycol) showing no inﬂammation

or any signs of necrosis but only fatty changes were observed.

Section of liver for reference drug represented several signs of

hepatotoxicity such as inﬂammation and extensive necrosis

with fatty changes (Supporting Information Fig. S2B). When

compared with T.S of standard drug, active molecule (4d;

Supporting Information Fig. S2C) displayed few fatty changes

and less inﬂammation with no necrosis to liver tissue.

Regrettably, Supporting Information Fig. S2D for compound

4

l illustrates symptoms of inﬂammation and necrosis to liver

tissue at some places at the maximum dose.

Log P determination and in silico

pharmacokinetic parameters

The unfavorable physicochemical properties have been

contributed to the decline of many therapies [7]. Potential

AEDs must require favorable physicochemical parameters

and pharmaceutically relevant properties [17]. Lipophilicity

has been characterized as vital physicochemical parameter

affecting CNS activity because only appropriate partition

coefﬁcient of a molecule enables sufﬁcient penetration of

the blood-brain barrier [1, 32]. Hence, optimal lipophilic

and hydrophilic balance (log P) is a very important issue in

designing of novel antiepileptic drugs. Here, we tried to

correlate the anticonvulsant activity of synthesized com-

pounds with predicted Clog P and experimental log P.

The experimental log P and predicted Clog P values were

compared and are shown in Supporting Information Table S1.

As observed, experimental values were in good agreement

with the theoretical values predicted via computational

method (QikProp module). In particular, anti-seizure drugs

to be potent, they have to cross blood-brain barrier. Thus,

potency has been correlated with optimum lipophilicity

Molecular modeling studies

From the last decade, discovery of novel AEDs has been

extensively focused on the study of benzo-fused nitrogen

heterocyclic compounds as competitive ionotropic gluta-

mate receptors (iGluRs) antagonists [34]. There are consider-

able evidences that iGluRs are involved in many neurological

processes [35, 36]. iGluRs consist of two foremost subclasses:

N-methyl-D-aspartic acid (NMDA) and (R,S)-2-amino-3-(3-

hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) recep-

tors [37]. Thus antagonists of NMDA and/or AMPA receptors

are of great therapeutic interest for the treatment of

epilepsy, pathophysiologically linked to excessive excitatory

amino acid receptor activation. Molecular docking studies

were carried out to identify the hypothetical binding

mode of these new compounds at the NMDA and AMPA

receptors. Before docking study, available PDB structures of

both NMDA and AMPA receptors were checked to identify

the catalytic amino acids responsible for the interactions

with the co-crystallized ligand. The co-crystallized ligands

were re-docked into the active site of the receptors and

then replaced with outline derivatives in order to compare

the binding mode of both co-crystallized ligands and

compounds under examination (Supporting Information

Fig. S3). As a measure of docking reliability, the docking

results are evaluated in terms of glide dock scores by

comparison of the docked poses of the co-crystallized ligands

(Supporting Information Table S3). From the comparison of

(

log P). From the experimental log P determination, it was

found that most of the compounds are having log P values

between 2.9 and 4.4, considered optimal to cross the

blood-brain barrier. The prompt characterization of the

lipophilicity determined by experimental and computational

protocols supported the anticonvulsant potential shown

by series of synthesized compounds. Therefore, the agents

investigated appear to be promising candidates as anti-

epileptic drugs.

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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–17

V. G. Ugale and S. B. Bari

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our compounds with the co-crystallized ligands, we could

observe how our compounds might bind to the active site

based on knowledge of the structure of similar active sites.

For both receptor types, we have focused our attention on

most active compounds from the series. Docking of the most

active compounds 4d and 4l within the active site of the

NMDA receptor is illustrated in Fig. 2. Figure 2A and B displays

the binding orientation of active leads within the NMDA

receptor pocket involving hydrogen bonding with the

characteristic amino acids. A hydrophobic core and polar site

are present at the active site of receptor. The aromatic amino

acids namely PHE 92 and PHE 250 are mainly responsible for

hydrophobic interactions, whereas SER 180, GLN 144, and

ARG 131 are related to H-bond interactions. The lowest

energy docked conﬁrmation of compound 4d showed that

both oxygen and nitrogen atom of N-phenyl amide fragment

formed hydrogen bonding network with SER 180 and GLN

surrounded hydrophobic amino acids such as PHE 250 and

PHE 92 (Fig. 2B).

Binding interaction analysis of most active compounds with

AMPA receptor is depicted in Fig. 3. In the cavity of AMPA

receptor, amino acids TYR 61 and TYR 190 are responsible for

hydrophobic interaction and at polar site amino acids ARG 96

and GLU 193 formed hydrogen bonds with active compounds.

In compound 4d (Fig. 3A), it was observed that carbonyl

oxygen of cyclic amide (quinazolin-4(3H)-one) interacts with

ARG 96 through H-bonding interaction. Additional H-bond

was formed by –NH– proton donor present between

quinazolin-4(3H)-one and N-phenyl acetamide fragment of

designed compounds. N-H hydrogen donor binds with proton

acceptor site of GLU 193 indicating its pivotal role in

pharmacological activity (docking score: ꢀ7.76 kcal/mol).

Another active compound (4l; Fig. 3B) was also able to form

H-bond with both 4-carbonyl oxygen and N-H side chain

through similar amino acids, ARG 96 and GLU 193, respec-

tively. These interactions underscore the importance of both

nitrogen and oxygen atoms for binding at active site of

receptor. One of the methoxy groups of quinazolin-4(3H)-one

nucleus has shown the hydrophobic contact with side chain of

TYR 61 through methyl–phenyl interaction. From the docking

interaction, it can be postulated that the active compounds

bind in deep cavity of N-terminal province of AMPA receptor.

The deep cavity of AMPA receptor has been mostly conserved

by synthesized compounds through hydrophobic and H-bond

contacts with amino acid residues. Docking analysis identiﬁed

1

44, respectively. The 4-carbonyl group of quinazolin-4(3H)-

one was also involved in hydrogen bonding interaction with

ARG 131, indicating the importance of nucleus for binding at

receptor site (Fig. 2A). Another potential anticonvulsant

agent from series, compound 4l also ﬁts well in the active

site of NMDA receptor with acceptable docking score

(

ꢀ7.02 kcal/mol). The binding interaction analysis of com-

pound 4l exposed H-bonding interaction with >C–O of

N-phenyl amide and >C–O of quinazolin-4(3H)-one, ﬁrst with

side chain of SER 180 and second with backbone of ARG 131.

In addition, the importance of distal aryl ring was proved by

Figure 2. Binding mode of compounds into the binding pocket of NMDA receptor (PDB: 1PBQ). (A) Binding pose of most active

compound 4d; (B) binding interaction of compound 4l. Receptor–ligand hydrogen bonding interactions are depicted in yellow dash

lines. Hydrogen bonding distances are indicated by red lines.

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Rational Design and Synthesis of Anticonvulsant Agents

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Figure 3. Predicted binding interaction of compounds into the cavity of AMPA receptor (PDB: 4FTL). (A) Binding pose of highest

scoring compound 4d; (B) binding mode of active compound 4l. Receptor–ligand hydrogen bonding interactions are depicted in

yellow dash lines. Hydrogen boding distances are indicated by red lines.

imperative structural features to obtain iGluRs antagonists.

The outlined derivatives possess following pharmacophoric

elements predicted through docking protocol.

was more effective than both of these compounds. This will

support the postulate that the potency order for halogen

substitution on phenyl ring followed the outline sequence:

ortho < meta < para. Anticonvulsant potential varies signiﬁ-

cantly depending on electronegativity of substituents on

(

1) >C–O of N-phenyl amide and >C–O of quinazolin-4(3H)-

one, d-negatively charged heterocyclic atoms are able to

form interaction with positive site of the receptor.

3

N-phenyl ring; compound 4l with tri-ﬂuoro substitution (CF )

(

2) N-H proton donor engage a strong hydrogen bond

interaction with proton acceptor of the receptor site.

3) A ﬂat hydrophobic area indicated by 6,7-dimethoxy-

was signiﬁcantly more potent than compound 4o carrying

2,4-di-ﬂuoro group. When anticonvulsant activity was corre-

lated with electronegativity of substituents, it was found that

the compound 4m was less active than 4l and 4o (potency

2-phenylquinazolin-4(3H)-one enclosed by hydrophobic

amino acids.

3

order: CF > 2,4-F > 4-F). Surprisingly, compound 4k although

carrying 2,3-dichloro substitutions was found inactive at high

dose in scPTZ test after 0.5 h. The same compound was found

effective at 4 h time point, supporting its delayed absorption

and prolonged duration of anticonvulsant activity. Moreover,

derivatives carrying electron releasing groups on N-phenyl

moiety were found to be less active or inactive as anti-

convulsants. Few compounds from series having methyl,

methoxy, and ethoxy groups were much less active or inactive

at a high dose (300 mg/kg). Rational design, pharmacological

evaluation, and SAR of potential anticonvulsants propose

some crucial pharmacophoric features for anticonvulsant

activity given in Supporting Information Fig. S4.

Structure–activity relationship

In this study, a series of quinazolines were synthesized and

investigated for anticonvulsant activity. An honest effort has

been made to ﬁnd an essential pharmacophoric features

required to enhance anticonvulsant potency. As observed

from in vivo pharmacological screening, the compounds with

electron withdrawing groups such as F, Cl, and CF

3

on

N-phenyl ring showed moderate to better anticonvulsant

activity. Compound 4d with para chloro substitution greatly

enhances the anticonvulsant activity; this compound was

active in all in vivo screening models. Again, as we locate the

halogen substitution on ortho or meta position of N-phenyl

moiety; the compounds were found less potent than para-

substituted derivative. For example, compound 4b (o-chloro)

was less active than 4c (m-chloro); compound 4d (p-chloro)

Conclusion

The current study revealed that structurally explored quina-

zolin-4(3H)-ones hold a broad spectrum of anticonvulsant

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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–17

V. G. Ugale and S. B. Bari

ARC HH PHARM

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activity with less neurotoxicity and no hepatotoxicity com-

pared to standards. Two most promising derivatives 4d

(0.01 mol) were dissolved in dry pyridine and stirred

on an ice bath (0–5°C) for 3 h to afford the solid

product. The obtained solid was washed with dil. HCl to

remove excess pyridine and with sodium bicarbonate

solution to remove excess benzoyl chloride. It was then

ﬁltered, dried, and recrystallized from ethanol to get pure

compound [27].

(

ED50 ¼ 23.5 mg/kg, MES, mice, i.p.; ED50 ¼ 32.6 mg/kg, scPTZ,

mice, i.p.) and 4l (ED50 ¼ 26.1 mg/kg, MES, mice, i.p.; ED50

9.4 mg/kg, scPTZ, mice, i.p.) exhibited broad spectrum of

¼

7

anticonvulsant activity in preclinical seizure models employed

in the study. Compound 4d also displayed signiﬁcant activity

in 6-Hz pharmaco-resistant seizure test with high TD50 value

(

ED50 ¼ 45.2 mg/kg, mice, i.p., TD50 ¼ 325.9 mg/kg, mice, i.p.)

General procedure for the synthesis of 3-amino-6,7-

dimethoxy-2-phenylquinazolin-4(3H)-one (2)

signifying its safety and tolerance. Most of the synthesized

molecules were showing moderate to better anticonvulsant

activity in preclinical seizure models. Additionally, the results

of computational studies also supported the potential of

synthesized molecules as drug-like candidates with accept-

able pharmacokinetics. Molecular docking simulations also

represented the role of acetamide, secondary –NH–, and cyclic

imide; as these groups bind to the active sites on the

extracellular domain of the receptors. Lastly, bioevaluation

proﬁle and SAR studies provides chosen mark point: N-phenyl

substituted amide fragment attached through amino methyl

linker to privileged 6,7-dimethoxy-2-phenylquinazolin-4(3H)-

one could be used as a pharmacophore model for anticon-

vulsant activity. The structurally optimized molecules can

serve as preclinical candidates for the treatment of seizure

disorder and as probes for development of novel broad

spectrum AEDs.

An equimolar quantity of 6,7-dimethoxy-2-phenyl-4H-benzo-

[d][1,3]oxazin-4-one (1) (0.05 mol) and hydrazine hydrate

(99%) (0.05 mol) were reﬂuxed in dry pyridine for 24 h. The

mixture was added to crush ice and the separated solid was

ﬁltered off to afford the crude product. The product was

recrystallized from ethanol to give analytically pure com-

pound [28, 29]. Yield: 68%; mp 183–184°C; IR [KBr,

ꢀ

1

cm ] vmax: 3405 and 3349 (NH

2

), 3058 (Ar-CH), 1705 (CO),

) d ppm: 3.90, 3.90 (6H, 2s,

), 5.63 (2H, s, NH ), 7.18–7.79

: 297.1113;

1

1631 (CN); H NMR (DMSO-d

6

magnetically different, 2 ꢃ OCH

3

2

15 3 3

(7H, m, Ar-H); HRMS (EI) m/z calcd. for C16H N O

found: 297.1117.

General procedure for the synthesis of the 2-chloro-N-

(substituted phenyl)acetamides 3a–p

To a mixture of aniline (0.01 mol) and K CO (0.02 mol) in

2 3

acetone (20 mL), 2-chloroacetyl chloride (0.012 mol) was

added dropwise. The reaction mixture was stirred at 0°C

for 0.5 h and then left to warm at room temperature. The

progress of reaction was monitored by TLC till its completion.

The reaction mixture was ﬁltered and the ﬁltrate was

evaporated under reduced pressure to afford the crude

product, which was then washed with water and recrystal-

lized from ethanol to obtain pure compound. Other amides

were synthesized similarly by reacting with appropriate

anilines [30].

Experimental

Chemistry

All chemicals and solvents were supplied by Alfa Aesar and

Sigma-Aldrich Pvt. Limited. All the solvents were distilled

and dried before use. The reactions were monitored with

the help of thin-layer chromatography using pre-coated

aluminum sheets with GF254 silica gel, 0.2 mm layer

thickness by using solvent systems, benzene/acetone (7:3),

and toluene/ethyl acetate/formic acid (5:4:1). Melting points

of the synthesized compounds were recorded on the Veego

melting point apparatus. FTIR spectrum was acquired on a

Shimadzu infrared spectrometer (model FTIR-8400S). Both

General procedure for the synthesis of the 2-((6,7-

dimethoxy-4-oxo-2-phenylquinazolin-3(4H)-yl)amino)-N-

(substituted phenyl)acetamides 4a–p

3-Amino-6,7-dimethoxy-2-phenylquinazolin-4(3H)-one (2)

(1.45 gm, 0.005 mol) and 2-chloro-N-(phenyl)acetamide

(0.85 gm, 0.005 mol) were reﬂuxed in DMF. The reaction

was monitored by TLC. After completion of reaction,

mixture was ﬁltered through celite and concentrated under

reduced pressure. The residue obtained was partitioned

between ethyl acetate and water, and the organic layer

dried above anhydrous sodium sulfate, and concentrated

under reduced pressure. The crude product was puriﬁed

through column chromatography using hexane/ethyl ace-

tate as solvent to give 2-((6,7-dimethoxy-4-oxo-2-phenyl-

quinazolin-3(4H)-yl)amino)-N-phenylacetamide (4a). The

other compounds were prepared similarly by reacting

3-amino-6,7-dimethoxy-2-phenylquinazolin-4(3H)-one (2)

with suitable 2-chloro-N-(substituted phenyl)acetamides

3a–p.

1

13

H NMR and C NMR spectra of the synthesized compounds

were performed with Bruker Avance-II 400 NMR spectro-

meter operating at 400 MHz in SAIF, Punjab University

(

Chandigarh). Tetramethylsilane was used as internal stan-

dard (chemical shifts in d, ppm). Mass spectra of the

synthesized compounds were recorded at MAT 120 in SAIF,

Punjab University.

The InChI codes of the investigated compounds together

with some biological activity data are provided as Supporting

Information.

General procedure for the synthesis of 6,7-dimethoxy-2-

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

A mixture of an equimolar quantity of 4,5-dimethoxy

anthranilic acid (A) (0.01 mol) and benzoyl chloride (B)

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Rational Design and Synthesis of Anticonvulsant Agents

ARC HH PHARM

Archiv der Pharmazie

1

2

-((6,7-Dimethoxy-4-oxo-2-phenylquinazolin-3(4H)-yl)-

amino)-N-phenylacetamide (4a)

Yield: 63%; mp 203–204°C; IR [KBr, cm ] vmax: 3285 (NH),

079 (Ar–CH), 2938 (Ali–CH), 1712 (CO quinazolinone), 1656

(CO amide), 1617 (CN); H NMR (DMSO-d

CH ), 3.61 (2H, s, N–CH –CO), 4.01, 4.05 (6H, 2s, magnetically

different, 2 ꢃ OCH ), 7.16–7.95 (11H, m, Ar–H), 9.18 (1H, s,

–N–NH), 10.79 (1H, s, NHCO); C NMR (DMSO-d

6

) d ppm: 2.25 (3H, s,

3

2

ꢀ

1

3

1

3

6

) d ppm:

1

(

CO amide), 1627 (CN); H NMR (DMSO-d

6

) d ppm: 3.97 (2H, s,

–CO), 4.00, 4.04 (6H, 2s, magnetically different, 2 ꢃ

3

), 7.20–7.80 (12H, m, Ar–H), 9.04 (1H, s, –N–NH), 10.81

19.81, 51.64, 56.36, 56.40, 108.13, 109.78, 115.81, 124.07,

125.35, 126.90, 127.06, 127.93, 128.56, 129.94, 131.62, 132.79,

132.82, 133.55, 138.90, 145.93, 151.35, 153.91, 156.01, 163.10,

N–CH

OCH

2

13

(

1H, s, NHCO); C NMR (DMSO-d

6

) d ppm: 52.04, 56.41, 56.41,

24 4 4

169.57; HRMS (EI) m/z calcd. for C25H N O : 444.1798; found:

1

05.23, 108.09, 113.48, 119.06, 119.38, 119.42, 126.75, 127.06,

27.38, 128.19, 128.19, 129.20, 130.06, 134.21, 138.09, 143.39,

49.36, 153.42, 155.14, 160.85, 167.75; HRMS (EI) m/z calcd. for

444.1802.

2-((6,7-Dimethoxy-4-oxo-2-phenylquinazolin-3(4H)-yl)-

C

H N

24 22 4

O

4

: 430.1641; found: 430.1645.

amino)-N-(m-tolyl)acetamide (4f)

Yield: 63%; mp 191–192°C; IR [KBr, cm ] vmax: 3285 (NH),

ꢀ

1

N-(2-Chlorophenyl)-2-((6,7-dimethoxy-4-oxo-2-

3072 (Ar–CH), 2952 (Ali–CH), 1708 (CO quinazolinone), 1664

1

phenylquinazolin-3(4H)-yl)amino)acetamide (4b)

(CO amide), 1611 (CN); H NMR (DMSO-d

6

) d ppm: 2.24 (3H, s,

ꢀ

1

Yield: 72%; mp211–212°C; IR [KBr, cm ] vmax: 3297 (NH), 3072

CH

different, 2 ꢃ OCH

6

–N–NH), 10.85 (1H, s, NHCO); C NMR (DMSO-d ) d ppm:

3

), 3.65 (2H, s, N–CH

2

–-CO), 4.01, 4.04 (6H, 2s, magnetically

(

Ar–CH), 2924 (Ali–CH), 1707 (CO quinazolinone), 1661 (CO

3

), 7.03–7.95 (11H, m, Ar–H), 9.10 (1H, s,

1

13

amide), 1619 (CN), 712 (CCl); H NMR (DMSO-d

2H, s, N–CH –CO), 4.01, 4.05 (6H, 2s, magnetically different,

ꢃ OCH ), 7.20–7.81 (11H, m, Ar–H), 9.02 (1H, s, –N–NH), 10.20

1H, s, NHCO); C NMR (DMSO-d

6

) d ppm: 3.66

(

2

(

1

2

23.31, 51.79, 56.30, 56.43, 109.74, 110.65, 116.85, 122.67,

124.92, 126.71, 127.14, 129.28, 130.15, 131.62, 132.90, 133.81,

139.42, 140.78, 147.55, 154.71, 155.90, 158.52, 162.65, 170.91;

HRMS (EI) m/z calcd. for C25H N O : 444.1798; found:

24 4 4

444.1801.

3

13

6

) d ppm: 53.75, 56.42, 56.42,

05.25, 108.14, 113.51, 115.14, 119.09, 119.42, 126.05, 126.31,

26.80, 128.19, 129.19, 129.19, 130.04, 134.26, 143.45, 146.75,

49.37, 154.19, 155.14, 160.87, 171.14; HRMS (EI) m/z calcd. for

24 4 4

C H21ClN O : 464.1251; found: 464.1254.

2-((6,7-Dimethoxy-4-oxo-2-phenylquinazolin-3(4H)-yl)-

amino)-N-(2-methoxyphenyl)acetamide (4g)

ꢀ

1

N-(3-Chlorophenyl)-2-((6,7-dimethoxy-4-oxo-2-

Yield: 67%; mp 218–219°C; IR [KBr, cm ] vmax: 3288 (NH),

phenylquinazolin-3(4H)-yl)amino)acetamide (4c)

Yield: 69%; mp202–203°C; IR [KBr, cm ] vmax: 3284 (NH), 3065

3102 (Ar–CH), 2926 (Ali–CH), 1714 (CO quinazolinone), 1671

ꢀ

1

(CO amide), 1623 (CN); H NMR (DMSO-d

6

) d ppm: 3.69 (2H, s,

), 3.99, 4.03 (6H, 2s, magnetically

), 6.90–7.92 (11H, m, Ar–H), 9.09 (1H, s,

(

Ar–CH), 2940 (Ali–CH), 1709 (CO quinazolinone), 1668 (CO

N–CH

different, 2 ꢃ OCH

6

–N–NH), 10.57 (1H, s, NHCO); C NMR (DMSO-d ) d ppm:

2 3

–CO), 3.93 (3H, s, OCH

1

amide), 1609 (CN), 721 (CCl); H NMR (DMSO-d

2H, s, N–CH –CO), 4.01, 4.05 (6H, 2s, magnetically different,

ꢃ OCH ), 7.19–8.02 (11H, m, Ar–H), 9.19 (1H, s, –N–NH), 10.20

1H, s, NHCO); C NMR (DMSO-d

6

) d ppm: 3.68

3

1

3

(

2

(

1

2

3

51.86, 56.35, 56.40, 57.21, 105.83, 106.92, 114.35, 116.61,

118.23, 122.15, 124.51, 126.94, 127.51, 129.76, 129.59, 130.11,

130.71, 132.96, 144.51, 151.11, 153.71, 156.23, 159.01, 162.71,

169.84; HRMS (EI) m/z calcd. for C25H N O : 460.1747; found:

24 4 5

460.1750.

13

6

) d ppm: 53.40, 56.37, 56.42,

05.24, 108.14, 113.51, 119.09, 119.38, 120.04, 126.73, 126.73,

27.06, 127.36, 128.21, 129.19, 130.04, 134.26, 143.45, 146.06,

50.75, 153.73, 155.14, 160.75, 171.23; HRMS (EI) m/z calcd. for

24 4 4

C H21ClN O : 464.1251; found: 464.1254.

2-((6,7-Dimethoxy-4-oxo-2-phenylquinazolin-3(4H)-yl)-

N-(4-Chlorophenyl)-2-((6,7-dimethoxy-4-oxo-2-

amino)-N-(3-methoxyphenyl)acetamide (4h)

Yield: 58%; mp 199–200°C; IR [KBr, cm ] vmax: 3293 (NH),

ꢀ

1

phenylquinazolin-3(4H)-yl)amino)acetamide (4d)

ꢀ

1

Yield: 61%; mp207–208°C; IR [KBr, cm ] vmax: 3289 (NH), 3086

3075 (Ar–CH), 2967 (Ali–CH), 1723 (CO quinazolinone), 1657

1

(

Ar–CH), 2947 (Ali–CH), 1721 (CO quinazolinone), 1659 (CO

(CO amide), 1617 (CN); H NMR (DMSO-d

6

) d ppm: 3.58 (2H, s,

), 3.99, 4.03 (6H, 2s, magnetically

), 6.86–7.97 (11H, m, Ar–H), 9.14 (1H, s,

1

amide), 1624 (CN), 732 (CCl); H NMR (DMSO-d

2H, s, N–CH –CO), 3.99, 4.05 (6H, 2s, magnetically different,

ꢃ OCH ), 7.15–8.15 (11H, m, Ar–H), 9.12 (1H, s, –N–NH), 10.76

1H, s, NHCO); C NMR (DMSO-d

6

) d ppm: 3.58

N–CH

different, 2 ꢃ OCH

6

–N–NH), 10.95 (1H, s, NHCO); C NMR (DMSO-d ) d ppm: 51.2,

2 3

–CO), 3.90 (3H, s, OCH

(

2

(

1

2

3

1

3

13

6

) d ppm: 53.10, 56.32, 56.40,

07.81, 109.24, 114.34, 118.71, 120.92, 125.16, 126.01, 126.72,

56.3, 56.4, 57.5, 106.4, 107.2, 110.5, 112.5, 114.5, 118.6, 126.7,

128.0, 129.7, 130.0, 130.7, 131.5, 132.5, 137.8, 146.2, 154.8,

155.3, 157.6, 161.9, 164.5, 171.5; HRMS (EI) m/z calcd. for

26.95, 127.41, 127.95, 129.84, 131.07, 132.46, 135.67, 138.12,

42.94, 150.31, 152.92, 156.81, 161.04, 170.92; HRMS (EI) m/z

C

H N

25 24 4

O

5

: 460.1747; found: 460.1744.

4 4

calcd. for C24H21ClN O : 464.1251; found: 464.1255.

2-((6,7-Dimethoxy-4-oxo-2-phenylquinazolin-3(4H)-yl)-

2

-((6,7-Dimethoxy-4-oxo-2-phenylquinazolin-3(4H)-yl)-

amino)-N-(o-tolyl)acetamide (4e)

Yield: 56%; mp 196–197°C; IR [KBr, cm ] vmax: 3287 (NH),

058 (Ar–CH), 2913 (Ali–CH), 1719 (CO quinazolinone), 1675

amino)-N-(4-nitrophenyl)acetamide (4i)

Yield: 63%; mp 228–229°C; IR [KBr, cm ] vmax: 3285 (NH),

3081 (Ar–CH), 2953 (Ali–CH), 1715 (CO quinazolinone),

1668 (CO amide), 1634 (CN), 1551 and 1343 (NO

ꢀ

1

ꢀ

1

3

2

); H NMR

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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–17

V. G. Ugale and S. B. Bari

ARC HH PHARM

Archiv der Pharmazie

(

DMSO-d

magnetically different, 2 ꢃ OCH

.08 (1H, s, –N–NH), 10.77 (1H, s, NHCO); C NMR (DMSO-d

d ppm: 51.54, 56.37, 56.40, 107.72, 108.36, 116.31, 120.52,

21.40, 122.53, 125.37, 126.61, 127.04, 128.95, 129.23, 129.67,

32.21, 144.91, 146.11, 147.51, 151.41, 151.53, 156.57, 161.21,

6

) d ppm: 3.64 (2H, s, N–CH

2

–CO), 4.01, 4.05 (6H, 2s,

different, 2 ꢃ OCH

3

), 7.19–7.90 (11H, m, Ar–H), 9.06 (1H, s,

NH), 10.67 (1H, s, NHCO); C NMR (DMSO-d ) d ppm: 51.92,

6

1

3

), 7.17–8.19 (11H, m, Ar–H),

13

9

6

)

56.33, 56.41, 107.21, 108.52, 113.91, 115.21, 115.93, 121.01,

122.12, 127.11, 128.13, 128.97, 129.31, 129.91, 132.34, 136.21,

142.25, 154.21, 154.10, 158.41, 161.35, 165.27, 171.15; HRMS

1

4

4 4

(EI) m/z calcd. for C24H21FN O : 448.1547; found: 448.1551.

21 5 6

69.58; HRMS (EI) m/z calcd. for C24H N O : 475.1492; found:

75.1496.

2-((6,7-Dimethoxy-4-oxo-2-phenylquinazolin-3(4H)-yl)-

amino)-N-(2-ﬂuorophenyl)acetamide (4n)

ꢀ

1

N-(2-Aminophenyl)-2-((6,7-dimethoxy-4-oxo-2-

Yield: 69%; mp 215–216°C; IR [KBr, cm ] vmax: 3299 (NH),

phenylquinazolin-3(4H)-yl)amino)acetamide (4j)

3107 (Ar–CH), 2963 (Ali–CH), 1705 (CO quinazolinone), 1687

ꢀ

1

Yield: 67%; mp 217–218°C; IR [KBr, cm ] vmax: 3398 and 3345

NH ), 3295 (NH), 3043 (Ar–CH), 2947 (Ali–CH), 1708 (CO

quinazolinone), 1651 (CO amide), 1619 (CN); H NMR (DMSO-

) d ppm: 3.59 (2H, s, N–CH –CO), 3.97, 4.01 (6H, 2s,

magnetically different, 2 ꢃ OCH ), 6.29 (2H, br s, NH ), 6.95–

.92 (11H, m, Ar–H), 9.09 (1H, s, –N–NH), 10.89 (1H, s, NHCO);

(CO amide), 1637 (CN), 1225 (CF); H NMR (DMSO-d

6

) d ppm:

(

2

3.70 (2H, s, N–CH

different, 2 ꢃ OCH

6

–N–NH), 10.58 (1H, s, NHCO); C NMR (DMSO-d ) d ppm:

2

–CO), 3.97, 4.01 (6H, 2s, magnetically

1

3

), 7.13–7.96 (11H, m, Ar–H), 9.17 (1H, s,

1

3

d

6

2

3

2

51.20, 56.34, 56.43, 108.72, 109.94, 113.21, 115.77, 122.21,

124.51, 126.91, 127.90, 128.12, 128.57, 128.71, 129.39, 129.90,

132.15, 145.21, 151.90, 152.17, 155.19, 159.20, 161.23, 170.57;

7

13

6

C NMR (DMSO-d ) d ppm: 51.90, 56.31, 56.40, 105.16, 106.45,

1

4

12.11, 113.76, 119.62, 122.75, 124.31, 126.06, 126.81, 127.53,

28.14, 129.91, 130.24, 133.57, 145.81, 148.94, 150.55, 152.31,

58.19, 162.36, 170.11; HRMS (EI) m/z calcd. for C24H N O :

23 5 4

4 4

HRMS (EI) m/z calcd. for C24H21FN O : 448.1547; found:

448.1550.

45.1750; found: 445.1754.

N-(2,4-Diﬂuorophenyl)-2-((6,7-dimethoxy-4-oxo-2-

phenylquinazolin-3(4H)-yl)amino)acetamide (4o)

ꢀ

1

N-(2,3-Dichlorophenyl)-2-((6,7-dimethoxy-4-oxo-2-

Yield: 57%; mp 203–204°C; IR [KBr, cm ] vmax: 3283 (NH),

phenylquinazolin-3(4H)-yl)amino)acetamide (4k)

Yield: 64%; mp 195–196°C; IR [KBr, cm ] vmax: 3291 (NH),

3078 (Ar–CH), 2912 (Ali–CH), 1719 (CO quinazolinone), 1680

ꢀ

1

(CO amide), 1621 (CN), 1218, 1183 (CF); H NMR (DMSO-d

6

)

3

079 (Ar–CH), 2927 (Ali–CH), 1717 (CO quinazolinone), 1648

d ppm: 3.62 (2H, s, N–CH

different, 2 ꢃ OCH ), 6.90–7.98 (10H, m, Ar–H), 9.11 (1H, s,

6

–N–NH), 10.82 (1H, s, NHCO); C NMR (DMSO-d ) d ppm:

2

–CO), 4.00, 4.04 (6H, 2s, magnetically

1

(

CO amide), 1625 (CN), 755, 737 (CCl); H NMR (DMSO-d

6

)

3

1

3

d ppm: 3.84 (2H, s, N–CH

2

–CO), 3.99, 4.03 (6H, 2s, magnetically

different, 2 ꢃ OCH

3

), 7.18–8.04 (11H, m, Ar–H), 9.16 (1H, s,

N–NH), 10.96 (1H, s, NHCO); C NMR (DMSO-d ) d ppm:

6

51.39, 56.37, 56.47, 101.29, 107.26, 108.51, 110.53, 114.91,

119.13, 125.91, 127.85, 128.41, 128.92, 129.56, 129.92, 131.11,

143.92, 152.26, 152.90, 155.81, 161.37, 161.50, 166.14, 170.93;

HRMS (EI) m/z calcd. for C24H F N O : 466.1453; found:

20 2 4 4

466.1457.

13

–

5

1

2.26, 56.31, 56.37, 105.26, 108.07, 113.46, 118.21, 119.05,

26.77, 127.02, 127.28, 128.14, 129.20, 129.20, 130.01, 133.73,

34.19, 139.02, 143.34, 149.39, 153.37, 155.15, 160.78, 171.09;

2 4 4

HRMS (EI) m/z calcd. for C24H20Cl N O : 498.0862; found:

4

98.0865.

2-((6,7-Dimethoxy-4-oxo-2-phenylquinazolin-3(4H)-yl)-

amino)-N-(2-ethoxyphenyl)acetamide (4p)

ꢀ

1

2

-((6,7-Dimethoxy-4-oxo-2-phenylquinazolin-3(4H)-yl)-

Yield: 62%; mp 221–222°C; IR [KBr, cm ] vmax: 3292 (NH),

amino)-N-(4-(triﬂuoromethyl)phenyl)acetamide (4l)

Yield: 71%; mp 197–198°C; IR [KBr, cm ] vmax: 3281 (NH),

3051 (Ar–CH), 2926 (Ali–CH), 1722 (CO quinazolinone), 1689

ꢀ

1

(CO amide), 1613 (CN); H NMR (DMSO-d

6

) d ppm: 1.49 (3H, t,

3

068 (Ar–CH), 2950 (Ali–CH), 1710 (CO quinazolinone), 1657

CH

different, 2 ꢃ OCH

Ar–H), 9.14 (1H, s, –N–NH), 10.70 (1H, s, NHCO); C NMR

(DMSO-d ) d ppm: 16.23, 51.41, 56.31, 56.45, 66.11, 106.17,

107.34, 111.25, 113.71, 117.15, 121.76, 125.31, 126.10, 128.29,

128.54, 128.73, 129.50, 129.79, 132.21, 142.93, 151.18, 151.93,

155.34, 157.27, 161.31, 169.19; HRMS (EI) m/z calcd. for

3

), 3.57 (2H, s, N–CH

2

–CO), 4.01, 4.05 (6H, 2s, magnetically

1

(

CO amide), 1633 (CN); H NMR (DMSO-d

6

) d ppm: 3.65 (2H, s,

–CO), 3.99, 4.03 (6H, 2s, magnetically different, 2 ꢃ

), 7.11–7.92 (11H, m, Ar–H), 9.12 (1H, s, –N–NH), 10.86

3

), 4.05 (2H, q, OCH

2

), 6.97–8.02 (11H, m,

13

N–CH

OCH

2

3

6

13

(

1H, s, NHCO); C NMR (DMSO-d

6

) d ppm: 52.71, 56.36, 56.41,

1

07.25, 108.44, 115.01, 118.85, 119.71, 123.73, 125.01, 125.75,

28.34, 128.51, 128.77, 129.10, 129.51, 131.55, 133.57, 142.91,

45.20, 154.11, 154.90, 158.19, 162.47, 170.21; HRMS (EI) m/z

26 26 4 5

C H N O : 474.1903; found: 474.1907.

21 3 4 4

calcd. for C25H F N O : 498.1515; found: 498.1511.

Pharmacological studies

2

-((6,7-Dimethoxy-4-oxo-2-phenylquinazolin-3(4H)-yl)-

Male Swiss Albino mice (CF-1 strain, 20–30 g) were used for

pharmacological evaluation studies. Animals were housed in

wire-mesh cages under the laboratory conditions (26 ꢁ 2°C),

12–12 h light/dark; allowed free access to water and food for

24 h before testing. The animals were divided into three

groups (control, standard, and test) and each group consisted

amino)-N-(4-ﬂuorophenyl)acetamide (4m)

ꢀ

1

Yield: 67%; mp 211–212°C; IR [KBr, cm ] vmax: 3293 (NH),

095 (Ar–CH), 2935 (Ali–CH), 1720 (CO quinazolinone), 1679

3

1

(

CO amide), 1640 (CN), 1231 (CF); H NMR (DMSO-d

6

) d ppm:

3

.55 (2H, s, N–CH –CO), 4.01, 4.05 (6H, 2s, magnetically

2

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14

Arch. Pharm. Chem. Life Sci. 2016, 349, 1–17

Rational Design and Synthesis of Anticonvulsant Agents

ARC HH PHARM

Archiv der Pharmazie

of six mice. The anticonvulsant activities of the tested

compounds were evaluated by three different in vivo models,

namely, MES, scPTZ, and 6-Hz tests by reported procedures [38,

and Straub-tail. If the animal resumed its normal exploratory

behavior within 10 s after stimulation this was considered

as evidence for protection.

3

9]. All the test compounds 4a–p and standard drugs were

injected intraperitoneally to mice as a suspension of 0.5%

polyethylene glycol in normal saline at doses of 300, 100, and

Quantiﬁcation studies

In quantiﬁcation studies, the median effective dose (ED50);

the dose which gives biological responses in 50% of animals,

and median toxic dose (TD50); the dose that showed

neurological deﬁcits in 50% of animals were determined.

Both these values were investigated for selected compounds

from preliminary in vivo evaluation. For determination of

ED50 and TD50, six groups of animals were injected with

various doses of the tested compounds until three points were

found in the range of 10–90% seizure protection or minimal

neurotoxicity. To calculate both ED50 and TD50 values with

95% conﬁdence limits, GraphPad Prism 5 software was used.

The protective indexes (PI) for tested compounds were

3

0 mg/kg for MES and scPTZ tests. Pharmacological evaluation

was done at two different time intervals (0.5 and 4 h).

Phenytoin and carbamazepine were used as standard drugs

for the comparison. The 6-Hz psychomotor seizure test was

performed at ﬁve different time points i.e., 0.25, 0.5, 1.0, 2.0,

and 4.0 h at a ﬁxed dose of 100 mg/kg. The neurological

toxicity was detected by the rotarod test. The data were

analyzed by one-way ANOVA followed by Dunnett’s test

using GraphPad Prism 5 software. A value of P < 0.05 was

considered as statistical signiﬁcance. All pharmacological tests

were performed in accordance with guidelines laid out by

the Institutional Animal Ethics Committee. The approval of

the Institutional Animal Ethics Committee (CPCSEA/IAEC/

RCPIPER/2014-17/14) of R. C. Patel Institute of Pharmaceutical

Education and Research, Shirpur, Maharashtra, India, was

taken prior to the start of the experiments.

calculated by following formula: TD50/ED50

.

Neurotoxicity screening

Toxicity induced by tested compounds was detected in mice

using the standardized rotarod test [40]. Control group

mice, when placed on 3.2 cm diameter knurled rod rotating at

6 rpm can maintain their equilibrium for a prolonged period

of time. Neurological impairment was demonstrated by

the failure of a mouse to maintain equilibrium for 1 min in

each of three consecutive trials.

MES model

MES test was preformed according to the reported method.

In the MES test, an electrical stimulus of 0.2 s (50 mA)

was given via corneal electrodes primed with an electrolyte

solution containing an anesthetic agent. The maximal

seizure characteristically consists of a short period of initial

toxic ﬂexioni and a prolonged period of tonic hind limb

extension. Protection against electrical seizures was deﬁned

as the abolition of hind leg and tonic maximal extension

component of the seizure.

Behavioral screening

The title compounds were screened for their behavioral effect

using actophotometer at 0.5 and 4 h after drug (30 and

100 mg/kg) administration. The activity was measured on

digital actophotometer [41]. Carbamazepine was used as

standard. The mice were placed in the box and behavior was

noted for 10 min and the basal activity scores for all the

animals were recorded. The results of test compounds were

compared with the standard drug. The data were analyzed

by one-way ANOVA followed by Student’s t-test using

GraphPad Prism 5 software. All the values were expressed

as mean ꢁ SEM. A value of P < 0.05 was considered as

statistical signiﬁcance.

scPTZ induced seizure model

For the chemically induced convulsant test, PTZ dissolved in

0

.9% sodium chloride solution was administered in the

posterior midline of mice and the onset of convulsion was

noted for the control group. The test compounds were

administered intraperitoneally, 30 min prior to subcutaneous

injection of PTZ (80 mg/kg). The absence of clonic seizures

was deﬁned as protection. The time of clonic and tonic

seizures as well as the number of deaths was noted. The

results of test compounds were compared with the standard

drugs.

Assessment of liver function parameters

Animals were divided into groups of six, and the control

group received a basal diet and vehicle. Selected test

compounds were given in a dose of 100 mg/kg/day for

2 weeks in other group of animals. At the end of the

experimental period, animals were sacriﬁced by cervical

decapitation under mild anesthesia, blood was collected,

and the serum was separated by centrifuging at 300 rpm for

10 min. The collected serum was used to estimate the

biochemical parameters such as serum glutamate oxaloace-

tate transaminase (SGOT), serum glutamate pyruvate trans-

aminase (SGPT), alkaline phosphate, total protein, and total

albumin. These parameters were measured according to

reported methods [31, 42].

6

-Hz seizure model

The 6-Hz test was performed according to reported protocol.

The animals were subjected to low-frequency (6 Hz, 32 mA)

stimulation for 3 s delivered via corneal electrodes. Animals

were manually restrained and released immediately after

stimulation and observed for the presence or absence of

seizures. Untreated animals exhibit limbic seizures after such

electrical stimulation. Seizures were characterized by a

minimal clonic phase that was followed by stereotyped,

automatistic behaviors, including twitching of the vibrissae,

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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–17

V. G. Ugale and S. B. Bari

ARC HH PHARM

Archiv der Pharmazie

Ex vivo liver toxicity

study was performed along with the binding energy

calculation to understand the effectiveness of binding.

The histopathological evaluation of liver was carried out by

a reported method [43]. Selected test compounds were

administered to mice at a ﬁxed dose of 300 mg/kg. Carba-

mazepine was used as standard. The mice were sacriﬁced

under light anesthesia after 24 h of last dosage; the liver was

removed and washed with Kreb’s solution. The liver was

stored in 10% formalin and specimen was subjected to

histopathological studies.

Authors are thankful to the Principal Dr. S. J. Surana, R. C.

Patel Institute of Pharmaceutical Education and Research,

Shirpur, Maharashtra (India) for providing necessary

facilities. V.G.U. is thankful to North Maharashtra University,

Jalgaon, Maharashtra (India) for providing ﬁnancial assis-

tance through Vice-Chancellor Research Motivation Scheme

(

NMU/11A/VCRMS/2014-15/Pharmacy-4/170) to carry out the

Log P determination

study.

The partition coefﬁcient between octanol and phosphate

buffer was determined at room temperature using a reported

procedure [44]. A total of 10 mL of octanol and 10 mL of

phosphate buffer were taken in glass-stoppered graduated

tube and 5 mg of accurately weight test drug was added.

The mixture was then shaken with mechanical shaker for 24 h

at room temperature and then transferred to a separating

funnel and allowed to equilibrate for 6 h. The aqueous and

octanol phase were separated; the phases were ﬁltered

though membrane ﬁlter and drug content in aqueous phase

was analyzed by UV spectroscopy. Partition coefﬁcient [45]

was calculated by using the formula PC ¼ (Ct ꢀ Ca)/Ca. PC,

partition coefﬁcient; Ct, concentration of total drug; Ca,

concentration of drug in aqueous phase.

The authors have declared no conﬂicts of interest.

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Article Doi

Structural Exploration of Quinazolin-4(3H)-ones as Anticonvulsants: Rational Design, Synthesis, Pharmacological Evaluation, and Molecular Docking Studies

DOI: 10.1002/ardp.201600218

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