Synthesis, antiviral activity and cytotoxicity evaluation of Schiff bases of some 2-phenyl quinazoline-4(3)H-ones

Article abstract of DOI:10.1016/j.ejmech.2010.07.058

A new series of 3-(benzylideneamino)-2-phenylquinazoline-4(3H)-ones were prepared through Schiff base formation of 3-amino-2-phenyl quinazoline-4(3)H-one with various substituted carbonyl compounds. Their chemical structures were elucidated by spectral st

Full text of DOI:10.1016/j.ejmech.2010.07.058

European Journal of Medicinal Chemistry 45 (2010) 5474e5479
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis, antiviral activity and cytotoxicity evaluation of Schiff bases of some
2-phenyl quinazoline-4(3)H-ones
,
b
c
d
Krishnan Suresh Kumar a , Swastika Ganguly , Ravichandran Veerasamy , Erik De Clercq
*
a Medicinal chemistry research Laboratory, KMCH College of Pharmacy, Coimbatore, India
b Medicinal chemistry research Laboratory, Department of Pharmaceutical Sciences, Birla Institute of Technology, Ranchi, India
c Faculty of Pharmacy, AIMST University, Malaysia
d Rega Institute for Medical research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of 3-(benzylideneamino)-2-phenylquinazoline-4(3H)-ones were prepared through Schiff
base formation of 3-amino-2-phenyl quinazoline-4(3)H-one with various substituted carbonyl
compounds. Their chemical structures were elucidated by spectral studies. Cytotoxicity and antiviral
activity were evaluated against herpes simplex virus-1 (KOS), herpes simplex virus-2 (G), vaccinia virus,
vesicular stomatitis virus, herpes simplex virus-1 TK- KOS ACVr, para influenza-3 virus, reovirus-1,
Sindbis virus, Coxsackie virus B4, Punta Toro virus, feline corona virus (FIPV), feline herpes virus,
respiratory syncytial virus, influenza A H1N1 subtype, influenza A H3N2 subtype, and influenza B virus.
Compound 2a showed better antiviral activity against the entire tested virus.
Received 20 May 2010
Received in revised form
27 July 2010
Accepted 28 July 2010
Available online 6 August 2010
Keywords:
Quinazoline
Schiff bases
Crown Copyright Ó 2010 Published by Elsevier Masson SAS. All rights reserved.
3-(Benzylideneamino)-2-
phenylquinazoline-4(3H)-ones
Non-nucleoside antiviral agents
HSV
FIPV
Influenza
Vaccinia virus
Vesicular stomatitis virus
1. Introduction
quinazoline-4(3H)-ones and tested in vitro cytotoxicity and anti-
viral activity, against viruses representative of herpes simplex
Viral infections caused by the rapid emergence of antiviral drug
resistant strains have become a serious threat globally and have
fostered the search for new antiviral agents directed against
unexplored drug targets. Various nucleoside analogues have been
currently used as antiviral agents these derivatives often inhibit
viral polymerases. However, only few non-nucleoside antiviral
agents are currently marketed.
virus-1 (KOS),herpes simplex virus-2 (G), vaccinia virus, vesicular
stomatitis virus, herpes simplex virus-1 TK- KOS ACVr, para influ-
enza-3 virus, reovirus-1, Sindbis virus, Coxsackie virus B4, Punta
Toro virus, feline corona virus (FIPV), feline herpes virus, respira-
tory syncytial virus, influenza A H1N1 subtype, influenza A H3N2
subtype, influenza B and vesicular stomatitis virus.
Quinazoline is an interesting molecule and its pharmacological
activities are well documented. It has been reported as antimicro-
bial [1e7], antiviral [8], anti-HIV [9], anticonvulsant [10,11], anti-
inflammatory [12e14], antihistaminic [15], anti-tubercular [16,17],
and anticancer [18,19] activity, etc. A huge number of quinazolines
had been synthesized and evaluated for various activities however
their antiviral activity was not fully investigated to a greater extent.
We have synthesized a simple 3-(benzylideneamino)-2-phenyl
2. Chemistry
The synthetic approach for the title compounds were shown in
Scheme 1. We utilized the method reported by Anjani et al. for
synthesis of 2-phenyl– 4H-3, 1-benzoxazin-4-one [20]. Anthranilic
acid (0.02 mol) was dissolved in 30 mL of anhydrous pyridine by
stirring slowly at room temperature. The solution was cooled to
0
ꢀC and a solution of benzoyl chloride (0.02 mol) in anhydrous
pyridine 30 mL was added to this solution slowly with constant
stirring. When the addition was complete the reaction mixture was
stirred for half an hour mechanically at room temperature and set
* Corresponding author. Tel.: þ91 9486371828; fax: þ91 4222628645.
E-mail address: pharmsuki@gmail.com (K.S. Kumar).
0223-5234/$ e see front matter Crown Copyright Ó 2010 Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.058

K.S. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 5474e5479
5475
(0.05 mol) in anhydrous pyridine (25 mL), with constant stirring
[20]. When the addition was complete, the resultant reaction
mixture was stirred vigorously for 30 min at room temperature and
subsequently heated under reflux for 6 h under anhydrous reaction
conditions. It was allowed to cool at room temperature and poured
into ice cold water containing dilute hydrochloric acid on standing
for 1 h, solidification occurred which was allowed to settle down. It
was filtered off, washed repeatedly with water and dried in vacuum
and purified by HPLC.
H2N
Cl
HO
O
+
O
benzoyl chloride
Anthranilic acid
Anhydrous pyridine
Title compounds were prepared through Schiff’s reaction. An
equimolar quantity of 2-phenyl-3-amino-3(H)-quinazoline-4-one
and aldehyde/ketone were dissolved in ethanol and its pH was
adjusted to 4.0e4.5 with glacial acetic acid. The content of the
mixture were refluxed for 30e150 min and poured into ice cooled
water thus the solid obtained was filtered and purified by HPLC
(Compound 2ael, Table 1). Their structures are in agreement with
elemental and spectral data.
N
O
O
2-phenyl- 4H-3, 1-benzoxazin-4-one
3. Pharmacology
O
H2N
Anhydrous pyridine
Cytotoxicity and antiviral activity of all compounds were eval-
uated against herpes simplex virus-1 (KOS), herpes simplex virus-2
(G), vaccinia virus, vesicular stomatitis virus, herpes simplex virus-
1 TK- KOS ACVr in HEL cell culture, vero cell culture, CRFK, Hela &
MDCK cell cultures. Cytotoxic concentration, required to cause
a microscopically detectable alteration of normal cell morphology,
was measured. Cytopathogenicity concentration required to reduce
virus-induced cytopathogenicity by 50%, was determined.
H
H
NH2
hydrazine hydrate
H2N
O
N
N
2-phenyl-3-amino-3(H) quinazoline-4-one
4. Results and discussion
Anthranilic acid reaction with benzoyl chloride yielded
2-phenyl-4H-3, 1-Benzoxazin-4 one by N-acylation via dehydrative
cyclization mechanism. Subsequently which was converted to
2-phenyl-3-amino-3(H) quinazoline-4 one with hydrazine hydrate.
A series of novel 3-(benzylideneamino)-2-phenylquinazoline-4
(3H)-ones were synthesized by reaction with 2-phenyl-3-amino-3
(H) quinazoline-4 one and compounds containing aldehyde and
ketones to afford 2,3disubstituted quinazoline-4(3)H one deriva-
tives (Table 1). The spectral data of synthesized compounds were
O
R
R'
Glacial acetic acid
Table 1
R
Structure of the synthesized compounds.
O
N
C
R
N
O
C
N
R'
N
R'
N
N
Scheme 1. Synthetic scheme for the title compounds.
Compd.
R
R0
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
H
H
H
CH3
H
2-OH
aside for 1 h. The pasty mass obtained was diluted with water and
treated with aqueous sodium bicarbonate to remove the unreacted
acid. When effervescence ceased, the solid material was filtered off
and washed with water to remove the inorganic materials and the
adhered pyridine. The crude benzoxazine thus obtained was dried
and re-crystallized from diluted ethanol.
Subsequently the crystallized material was converted to
2-phenyl-3-amino-3(H) quinazoline-4-one by adding dropwise
a solution of hydrazine hydrate (0.1 mol) in anhydrous pyridine
(25 mL) to cold solution of 2-phenyl-4H-3, 1-benzoxazin-4-one
3-NO2
4-OCH3
4-N(CH3)2
4-Cl
H
4-OH
H
4-OH
4-Cl
3-OH & 4-OCH3
2-OCH3
H
CH3
CH3
H
H
H
2j
2k
2l

5476
K.S. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 5474e5479
Table 2
Cytotoxicity and antiviral activity of compounds in HEL cell cultures.
b
Compd.
Minimum cytotoxic
EC50
(
m
g/mL)
concentrationa
(mg/mL)
Herpes simplex
virus-1 (KOS)
Herpes simplex
virus-2 (G)
Vaccinia
virus
Vesicular
stomatitis virus
Herpes simplex virus-1
TK- KOS ACVr
2a
100
1
1
0.8
15
1
2b
2c
2d
2e
2f
2g
2h
2i
ꢁ20
>20
9
>20
>20
7
>20
>100
>100
>20
>20
>20
0.04
>250
2
>20
8
>20
>20
9
>20
>100
>100
>20
>20
>20
50
>20
9
>20
>20
9
>20
>100
>20
>20
>100
>20
>100
>100
>20
>20
>20
>250
146
>20
9
>20
>20
9
>20
>100
>100
>20
>20
>20
250
>250
3
>100
100
100
>100
100
>100
>100
100
100
100
>250
>250
>250
>100
12
>100
>100
20
20
>20
4
>250
8
>100
2j
2k
2l
Brivudin (
Ribavirin (
Cidofovir (
Ganciclovir (
m
m
m
M)
M)
M)
M)
>250
2
0.07
>250
>100
m
0.05
2
Data represent mean values for three independent determinations. Variation among duplicate samples was less than 15%.
a
Required to cause a microscopically detectable alteration of normal cell morphology.
Required to reduce virus-induced cytopathogenicity by 50%.
b
consistent with the assigned structures and the compounds were
obtained in 68e78% yield.
Compounds 2c and f were found to be active against herpes
simplex virus-1 (KOS), herpes simplex virus-2 (G), vaccinia virus,
herpes simplex virus-1 TK- KOS ACVr in HEL cell culture (Table 2) at
selectivity index of 11, 13, 11, 11 and 14, 11, 11, 11 respectively, where
Title compounds were tested against representative members of
the virus includes herpes simplex virus-1 (KOS), herpes simplex
virus-2 (G), vaccinia virus, vesicular stomatitis virus, herpes
simplex virus-1 TK- KOS ACVr (Table 2), para influenza-3 virus,
reovirus-1, Sindbis virus, Coxsackie virus B4, Punta Toro virus
(Table 3), feline corona virus (FIPV), feline herpes virus (Table 4),
respiratory syncytial virus (Table 5) and influenza A H1N1 subtype,
influenza A H3N2 subtype, influenza B (Table 6).
Compound 2a exhibited antiviral activity against herpes simplex
virus-1 (KOS), herpes simplex virus-2(G), herpes simplex virus-1
(TK- KOS ACV) and vaccinia virus in HEL cell culture (Table 2) at
selectivity index of 100, 100, 100 and 125 respectively, whereas
they were not cytotoxic at 100
mg/mL none of the compounds
tested was active against influenza A H1N1, influenza A H3N2 and
influenza B in MDCK cell Cultures.
Of the compounds tested, compound 2a clearly proved the most
promising in terms of antiviral activity (potency). This lead mole-
cule 2a could be further utilized for designing newer non-nucleo-
side antiviral agents especially for activity against HSV and vaccinia
virus.
The synthetic procedure reported in this article allows a rapid
synthesis of derivative of the title compounds to be tested for their
antiviral effects.
cytotoxicity was found at 100 mg/mL. Compound 2a also inhibited
the replication of reovirus, Sindbis virus, Coxsackie virus B4 and
Punta Toro virus in Vero cell culture (Table 3) at selectivity index of
5. Experimental
10, 20, 10 and 33, whereas cytotoxicity was found 100 mg/mL. It also
showed inhibitory to vesicular stomatitis virus, respiratory syncy-
tial virus in HeLa cells (Table 5) albeit at a concentration closely
related to the cytotoxicity (4 mg/mL).
Melting points of the synthesized compounds were determined
by using an open capillary tube method and are uncorrected.
Microanalyses were carried out on a Carlo Erba 1106 elemental
Table 3
Cytotoxicity and antiviral activity of compounds in Vero cell cultures.
b
Compd.
Minimum cytotoxic
EC50 (mg/mL)
concentrationa
(mg/mL)
Para influenza-3
Reovirus-1
Sindbis
virus
Coxsackie
virus B4
Punta Toro
virus
virus
2a
2b
100
20
>20
>4
10
>4
5
>4
10
>4
3
>4
2c
2d
2e
2f
100
100
100
20
>20
>20
>20
>4
>20
>20
>20
>4
>20
>20
>20
>4
>20
>20
>20
>4
>20
>20
>20
>4
2g
2h
2i
2j
100
100
>100
20
>20
>20
>100
>4
>20
>20
>100
>4
>20
>20
>100
>4
>20
>20
>100
>4
>20
>20
>100
>4
2k
2l
100
100
>100
>250
>250
>20
>20
>100
>250
146
>20
>20
>100
>250
>250
>20
>20
20
>250
>250
>20
>20
20
>250
>250
>20
>20
20
>250
146
DS-5000
(S)-DHPA (
Ribavirin (
m
M)
m
M)
a
Required to cause a microscopically detectable alteration of normal cell morphology.
Required to reduce virus-induced cytopathogenicity by 50%.
b

K.S. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 5474e5479
5477
1362 (Ar CeN Str); 1H NMR (CDCl3,
ArH), 7.66e7.92 (m, 7H, ArH), 8.68 (s, 1H, HeC]N).
d
in ppm): 7.28e7.46 (m, 6H,
Table 4
Anti-Feline Corona Virus (FIPV) and anti-Feline Herpes Virus activity and cytotox-
icity in CRFK cell cultures.
a
b
Compd.
CC50
g/mL)
EC50 (mg/mL)
5.2. 3-[(3-Nitro-benzylidine)-amino]-2-phenyl-3H-quinazolin-
4one (2b)
(
m
Feline Corona
Virus (FIPV)
Feline Herpes
Virus
Yield: 74%; m.p 246e248 ꢀC; IR (KBr, in cmꢃ1): 1678 (C]O str.),
1645.4 (C]N str.), 1502 (ring C]C str.), 1458 (Ar CeN Str), 1345
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
11.2
13.8
13.7
>100
>100
3.7
>4
>4
>4
>4
>4
>4
(N]O str. for ArNO2); 1H NMR (CDCl3,
d in ppm): 7.25e7.52 (m, 6H,
>100
>100
>0.8
>20
>0.8
>100
>20
>20
>4
>100
>100
>0.8
>20
>0.8
>100
>20
>20
>4
ArH), 7.62e7.80 (m, 4H, ArH), 8.0e8.21 (m, 3H, ArH), 8.67 (s, 1H,
HeC]N).
59.5
2.6
5.3. 3-[(4-Methoxy-benzylidine)-amino]-2-phenyl-3H-quinazolin-
4one (2c)
>100
42.2
42.7
14.4
>100
>100
>100
Yield: 68%; m.p 242e244 ꢀC; IR (KBr, in cmꢃ1): 1680 (C]O str.),
1644.6 (C]N str.), 1502 (ring C]C str.), 1446 (Ar CeN Str), 1026
HHA
UDA
Ganciclovir (mM)
2.6
11.7
>100
1.6
53.1
0.8
(CeOeC str. for eCeOeCH3); 1H NMR (CDCl3,
d in ppm): 3.73 (s, 3H,
eOCH3), 7.25e7.49 (m, 6H, ArH), 7.58e7.80 (m, 4H, ArH), 8.10e8.22
(m, 3H, ArH), 8.60 (s, 1H, HeC]N).
a
50% Cytotoxic concentration, as determined by measuring the cell viability with
the colorimetric formazan-based MTS assay.
b
50% Effective concentration, or concentration producing 50% inhibition of virus-
5.4. 3-[(4-Dimethylamino-benzylidine)-amino]-2-phenyl-3H-
quinazolin-4one (2d)
induced cytopathic effect, as determined by measuring the cell viability with the
colorimetric formazan-based MTS assay. CRFK cells: Crandell-Rees Feline Kidney
cells.
Yield: 72%; m.p 175e177 ꢀC; IR (KBr, in cmꢃ1): 1676 (C]O str.),
1640 (C]N str.), 1510 (ring C]C str.), 1460 (Ar CeN Str); 1H NMR
analyzer. The results of elemental analysis were within ꢂ0.3% for C
and ꢂ0.1% for H and N of the theoretical value. 1H NMR spectra
were performed on a Varian Gemini 200 (200 MHz) spectrometer
using tetramethylsilane (TMS) as internal standard. IR spectra were
recorded on an FTIR-JASCO 4100 Spectrophotometer. GCeMS
spectra were performed with HP 6890e5973. GC parameters:
injector temperature 250 ꢀC; capillary column HP5 poly (methyl-
phenylsiloxane) 30 m, 0.35 mm, 0.25 mm; temperature program:
from 100 to 300 ꢀC at 10 ꢀC/min. MS parameters: mode SCAN
40e600 amu.
(CDCl3,
d in ppm): 3.12 (s, 6H, -NCH3), 7.23e7.42 (m, 6H, ArH),
7.62e7.80 (m, 7H, ArH), 8.56 (s, 1H, HeC]N).
5.5. 3-[1-(4-Chloro-phenyl)-ethylideneamino-]-2-phenyl-3H-
quinazolin-4one (2e)
Yield: 74%; m.p 243e245 ꢀC; IR (KBr, in cmꢃ1): 1670 (C]O str.),
1646 (C]N str.), 1515 (ring C]C str.), 1455 (Ar CeN Str); 1H NMR
(CDCl3,
d in ppm): 2.46 (s, 3H, -CH3), 7.20e7.48 (m, 6H, ArH),
7.57e7.78 (m, 7H, ArH).
5.6. 3-(Benzylidine-amino)-2-phenyl-3H-quinazolin-4one (2f)
5.1. 3-[(2-Hydroxy-benzylidine)-amino]-2-phenyl-3H-quinazolin-
4one (2a)
Yield: 75%; m.p 194e196 ꢀC; IR (KBr, in cmꢃ1): 1665 (C]O str.),
1642 (C]N str.), 1510 (ring C]C str.), 1449 (Ar CeN Str); 1H NMR
Yield: 78%; m.p 144e146 ꢀC; IR (KBr, in cmꢃ1): 3200e3100 (OeH
str. for eOH), 1683 (C]O str.), 1604 (C]N str.), 1465 (ring C]C str.),
(CDCl3,
d in ppm): 7.25e7.44 (m, 6H, ArH), 7.62e7.80 (m, 8H, ArH),
8.58 (s, 1H, HeC]N).
Table 5
5.7. 3-[(4-Hydroxy-benzylidine)-amino]-2-phenyl-3H-quinazolin-
4one (2g)
Cytotoxicity and antiviral activity of compounds in HeLa cell cultures.
b
Compd.
Minimum
EC50
(m
g/mL)
Yield: 78%; m.p 164e166 ꢀC; IR (KBr, in cmꢃ1): 3304e3212
(OeH str. for eOH), 1668 (C]O str.), 1645 (C]N str.), 1554 (ring
cytotoxic
Vesicular
stomatitis virus virus B4
Coxsackie Respiratory
concentrationa
syncytial virus
(mg/mL)
C]C str.), 1372 (Ar CeN Str); 1H NMR (DMSO-d6,
d in ppm): 5.10
2a
2b
2c
4
20
20
>0.8
>4
>0.8
>4
>0.8
>4
(s, 1H, eOH), 7.42e7.48 (m, 6H, ArH), 7.70e7.79 (m, 5H, ArH),
8.12e8.29 (m, 3H, ArH), 8.62 (s, 1H, HeC]N).
>4
>4
>4
2d
2e
2f
100
100
20
>20
>20
>4
>20
>20
>4
>20
>20
>4
5.8. 2-Phenyl-3(1-phenyl-ethylideneamino)-3H-quinazolin-4one (2h)
2g
2h
2i
2j
2k
2l
100
>20
>100
>100
>4
>4
>4
>20
>100
>100
>4
>4
>4
>20
>100
>100
>4
>4
>4
Yield: 73%; m.p 194e196 ꢀC; IR (KBr, in cmꢃ1): 1685 (C]O str.),
1614 (C]N str.), 1464.2 (ring C]C str.), 1363.8 (Ar CeN Str); 1H
NMR (CDCl3, d in ppm): 2.44 (s, 3H, CH3), 7.28e7.46 (m, 6H, ArH),
>100
>100
20
20
20
7.66e7.92 (m, 7H, ArH).
DS-5000
(S)-DHPA (
Ribavirin (mM) >250
>100
M) >250
>100
>250
22
7
0.8
>250
22
5.9. 3-[1-(4-Hydroxy-phenyl)-ethylideneamino]-2-phenyl-3H-
quinazolin-4one (2i)
m
>250
146
a
Required to cause
a
microscopically detectable alteration of normal cell
Yield: 68%; m.p 166e168 ꢀC; IR (KBr, in cmꢃ1): 3200e3150 (OeH
str. for eOH), 1686.4 (C]O str.), 1614 (C]N str.), 1468 (ring C]C
morphology.
b
Required to reduce virus-induced cytopathogenicity by 50%.

5478
K.S. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 5474e5479
Table 6
Anti-influenza virus activity and cytotoxicity in MDCK cell cultures.
c
Compd.
Concentration
Cytotoxicity
Antiviral EC50
a
CC50
Minimum cytotoxic
concentrationb
Influenza A
H1N1 subtype
Influenza A
H3N2 subtype
Influenza B
Visual CPE
score
MTS
Visual CPE
score
MTS
Visual CPE
score
MTS
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
m
m
m
m
m
m
m
m
m
m
m
m
m
g/mL
g/mL
g/mL
g/mL
g/mL
g/mL
g/mL
g/mL
g/mL
g/mL
g/mL
g/mL
M
2.6
>100
8.2
ꢁ0.8
20
20
100
20
20
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
0.2
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
0.1
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
4
N.A
N.A
N.A
N.A
N.A
N.A
N.A
N.A
N.A
N.A
N.A
N.A
3.8
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
2
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
1.5
>100
>100
15.0
10.2
>100
>100
76.4
47.4
12.4
>100
20
100
>100
20
20
20
Oseltamivir
carboxylate
Ribavirin
Amantadin
Rimantadin
>100
m
m
m
M
M
M
>100
>1000
472
ꢁ100
>1000
1000
9
101
68
12.9
87
58
9
117
40
7.8
115
53
9
N.A.
N.A.
6.0
N.A.
N.A.
N.A e not active at the highest concentration tested, or at subtoxic concentration.
a
50% Cytotoxic concentration, as determined by measuring the cell viability with the colorimetric formazan-based MTS assay.
Minimum compound concentration that causes a microscopically detectable alteration of normal cell morphology.
50% Effective concentration, or concentration producing 50% inhibition of virus-induced cytopathic effect, as determined by visual scoring of the CPE, or by measuring the
b
c
cell viability with the colorimetric formazan-based MTS assay. MDCK cells: Madin Darby canine kidney cells.
str.), 1372 (Ar CeN Str); 1H NMR (CDCl3,
6.84e6.92 (m, 3H, ArH), 7.29e7.51 (m, 8H, ArH), 7.64e7.82 (m, 3H,
d
in ppm): 2.47 (s, 3H, CH3),
and were allowed to form confluent monolayers by incubating
overnight in growth medium at 37 ꢀC in humidified CO2 (5%)
ArH).
atmosphere. Then, monolayers were infected with 250
virus dilutions to give 50e100 PFU/well. Following removal of
unadsorbed virus, 500 L of Dulbecco’s modified Eagle’s medium
mL of proper
5.10. 3-[(4-Chloro-benzylidine)-amino]-2-phenyl-3H-quinazolin-
m
4one (2j)
supplemented with 1% inactivated FCS and 0.75% methyl cellulose,
without or with serial dilutions of test compounds were added.
Cultures were incubated at 37 ꢀC for 2 days and then fixed with PBS
containing 50% ethanol and 0.8% crystal violet, washed and air-
dried. Plaques were then counted, and 50% effective concentrations
(EC50) values were calculated by the linear regression technique.
Cytotoxicity and antiviral activity of compounds were also
tested against vesicular stomatitis virus, Coxsackie virus B4 and
respiratory syncytical virus in HeLa cell cultures. Minimum cyto-
toxic concentration was the concentration required to cause
a microscopically detectable alteration of normal cell morphology.
Effective concentration was the concentration required to reduce
the virus-induced cytopathogenicity by 50%.
Yield: 70%; m.p 160e162 ꢀC; IR (KBr, in cmꢃ1): 1680 (C]O str.),
1587 (C]N str.), 1552 (ring C]C str.), 1374 (Ar CeN Str); 1H NMR
(CDCl3,
d in ppm): 7.42e7.58 (m, 6H, ArH), 7.66e7.82 (m, 7H, ArH),
8.76 (s, 1H, HeC]N).
5.11. 3-[(3-Hydroxy-4-methoxy-benzylidine)-amino]-2-phenyl-3H-
quinazolin-4one (2k)
Yield: 75%; m.p 180e182 ꢀC; IR (KBr, in cmꢃ1): 3206e3178 (OeH
str. for eOH), 1686 (C]O str.), 1624 (C]N str.), 1460 (ring C]C str.),
1364 (Ar CeN Str), 1025 (CeOeC str. for eCeOeCH3); 1H NMR
(DMSO-d6,
d
in ppm): 3.72 (s, 3H, eOCH3), 5.02 (s, 1H, eOH), 6.64
Compounds were also evaluated against influenza A HIN1,
influenza A H3N2 and influenza B in Madin Darby canine kidney
cells (MDCK) Cell cultures and 50% cytotoxic concentration, as
determined by measuring the cell viability with the colorimetric
formazan-based MTS assay similarly 50% effective concentration or
concentration producing 50% inhibition of virus-induced cyto-
pathic effect, as determined by visual scoring of the CPE, or by
measuring the cell viability with the colorimetric formazan-based
MTS assay protocol.
Activity of compounds against feline corona virus and feline
herpes virus was based on 50% effective concentration, or
concentration producing 50% inhibition of virus-induced cyto-
pathic effect as determined by measuring cell viability with the
colorimetric formazan-based MTS assay in Crandell-Rees Feline
Kidney cells (CRFK Cells).
(d, 1H, ArH), 7.27e7.46 (m, 8H, ArH), 7.62e7.75 (m, 3H, ArH), 8.72
(s, 1H, HeC]N).
5.12. 3-[(2-Methoxy-benzylidine)-amino]-2-phenyl-3H-
quinazolin-4one (2l)
Yield: 75%; m.p 212e214 ꢀC; IR (KBr, in cmꢃ1): 1680 (C]O str.),
1644.6 (C]N str.), 1502 (ring C]C str.), 1446 (Ar CeN Str), 1024.37
(CeOeC str. for eCeOeCH3); 1H NMR (CDCl3,
d
in ppm): 3.74 (s, 3H,
-OCH3), 6.78e6.81 (m, 2H, ArH), 7.26e7.43 (m, 7H, ArH), 7.58e7.80
(m, 4H, ArH), 8.59 (s, 1H, HeC]N).
5.13. Antiviral activity
Activity of compounds against para influenza-3 virus, reovirus-
1, Sindbis virus, Coxsackie virus B4, Punta Toro virus was measured
by plaque reduction assays in Vero cell monolayer. To this end, Vero
cells were seeded in 24-well plates at a density of 2 ꢄ 105 cells/well
Compounds which are potentially active in the earlier screening
were evaluated against herpes simplex virus-1(KOS), Herpes
simplex virus-2(G), vaccina virus and herpes simplex virus-1 TK-
KOS ACV in HEL cell cultures and analyses for their cytotoxic

K.S. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 5474e5479
[6] K. Siddappa, T. Reddy, M. Mallikarjun, C.V. Reddy, E. J. Chem.
5479
5
(2008)
concentration and effective concentration. Selective compounds
155e162.
were screened against para influenza-3 virus, reovirus-1, Sindbis
virus B4 and Punta Toro virus in Vero cell cultures.
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Acknowledgements
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We thank Mrs. Leentje Persoons for excellent technical assis-
tance with the antiviral activity assays. We also extend our thanks
to Chairman Dr. Nalla G. Palanisami & Dr Thavamani D. Palanisami
Trustee, Koval Medical Center Research and Educational Trust,
Coimbatore for their support.
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