Development of certain novel N-(2-(2-(2-oxoindolin-3-ylidene)hydrazinecarbonyl)phenyl)-benzamides and 3-(2-oxoindolin-3-ylideneamino)-2-substituted quinazolin-4(3 H)-ones as CFM-1 analogs: Design, synthesis, QSAR analysis and anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2014.12.048

The reaction of N-(2-(hydrazinecarbonyl)aryl)benzamides 2a, b with indoline-2,3-diones 4ae in acidified ethanolic solution furnished the corresponding N-(2-(2-(2-oxoindolin-3-ylidene)hydrazinecarbonyl)phenyl)benzamides 5aj, respectively. Furthermore, 3-(2

Full text of DOI:10.1016/j.ejmech.2014.12.048

European Journal of Medicinal Chemistry 92 (2015) 191e201
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Development of certain novel N-(2-(2-(2-oxoindolin-3-ylidene)
hydrazinecarbonyl)phenyl)-benzamides and 3-(2-oxoindolin-3-
ylideneamino)-2-substituted quinazolin-4(3H)-ones as CFM-1
analogs: Design, synthesis, QSAR analysis and anticancer activity
Ahmed M. Alafeefy a *, Abdelkader E. Ashour b, Onkar Prasad c, Leena Sinha c,
,
,
f
,
, i, **
Shilendra Pathak c, Fatimah A. Alasmari d, Arun K. Rishi e g, Hatem A. Abdel-Aziz h
a Department of Pharmaceutical Chemistry, College of Pharmacy, Salman Bin Abdulaziz University, P.O. Box 173, Alkharj, 11942, Saudi Arabia
b Department of Pharmacology and Toxicology, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh, 11451, Saudi Arabia
c Department of Physics, University of Lucknow, 226007, Lucknow, India
d Department of Chemistry, College of Science, King Saud University, PO Box 22955, Riyadh, 11416, Saudi Arabia
e John D. Dingell VA Medical Center, Wayne State University, Detroit, MI, USA
f Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA
g Department of Oncology, Wayne State University, Detroit, MI, USA
h Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh, 11451, Saudi Arabia
i Department of Applied Organic Chemistry, National Research Center, Dokki, Cairo, 12622, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
The reaction of N-(2-(hydrazinecarbonyl)aryl)benzamides 2a, b with indoline-2,3-diones 4ae in acidified
ethanolic solution furnished the corresponding N-(2-(2-(2-oxoindolin-3-ylidene)hydrazinecarbonyl)
phenyl)benzamides 5aj, respectively. Furthermore, 3-(2-oxoindolin-3-ylideneamino)-2-substituted qui-
nazolin-4(3H)-ones 6aj were prepared by the reaction of 3-amino-2-arylquinazolin-4(3H)-one 3a, b with
4ae. Six derivatives of the twenty newly synthesized compounds showed remarkable antitumor activity
against most of the tested cell lines, Daoy, UW228-2, Huh-7, Hela and MDA-MB231. Although these six
compounds were more potent than the standard drug (CFM-1), indeed compounds 5b, 5d and 6b were
Received 1 November 2014
Received in revised form
25 December 2014
Accepted 26 December 2014
Available online 27 December 2014
Keywords:
Hydrazones
Quinazoline
2-oxoindolin-3-ylidene
CFM analogues
Anticancer agents
the best candidates with IC50 values in the range 1.866.87, 4.4210.89 and 1.468.60 mg/ml and percentage
inhibition in the range 77.188.7, 59.4184.8 and 75.488.0%, respectively. QSAR analyses on the current
series of derivatives also have been performed for all five cancer cell lines and thus 10 statistically sig-
nificant models were developed and internally cross validated.
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
attracted researchers' interest owing to their diverse biological
activities, particularly as enzyme inhibitors, receptor agonists and
Cancer is a major health problem which concerns the medical
community all over the world. In spite of the substantial progress in
many aspects of cancer research, cancer chemotherapy is highly
inadequate [1]. Quinazolin-4(3H)-one containing molecules
constitute an important category among heterocyclic compounds
of medical, technological and industrial interest [2]. They have
antagonists, as well as antiviral activities. Derivatives such as
PD153035 and gefitinib (Scheme 1) were described to selectively
inhibit EGFR, notably on cellular assays and the isolated receptors
[3], they do not directly inhibit other ErbB receptors [4]. Addi-
tionally, they were found to down-regulate the phosphorylation of
EGFR and consequently suppress the proliferation of human cancer
cell lines under conditions of overexpression of EGFR [5e7]. That is
why EGFR inhibitors are considered to be highly useful in the
treatment of several malignancies. This was supported and evi-
denced by several clinical studies which have revealed the potential
use of these small molecules in the treatment of ovarian and cer-
vical cancer, both in the frontline and in the recurrent setting [8].
* Corresponding author.
** Corresponding author. Department of Pharmaceutical Chemistry, College of
Pharmacy, King Saud University, P.O. Box 2457, Riyadh, 11451, Saudi Arabia.
E-mail addresses: a.alafeefy@sau.edu.sa (A.M. Alafeefy), hatem_741@yahoo.com
(H.A. Abdel-Aziz).
http://dx.doi.org/10.1016/j.ejmech.2014.12.048
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

192
A.M. Alafeefy et al. / European Journal of Medicinal Chemistry 92 (2015) 191e201
F
O
HN
Br
O
HN
N
Cl
F
N
N
H
O
O
N
O
N
N
NH
O
N
N
O
H
Gefitinib
PD153035
Sunitinib
H
O
N
S
O
Br
HO
S
NH
O
NH
N
O
O
N
H
H
N
H
Semaxanib
SU 6668
CFM-1
O
O
N
O
O
O
NH2
NH2
Ar
R
N
N
O
H
N
H
4a-e
NH
N
Ar
O
Ar
3a, b
1a-c
Ar
4-MeC6H4
4-NO2C6H4
thiophen-2-yl
2a, b
4
a
b
c
d
e
R
H
Cl
Me
MeO
NO2
1
a
b
c
Ar
3
a
b
2
a
b
Ar
-
-
-
4-MeC6H4
thiophen-2-yl
4-MeC6H4
4-NO2C6H4
-
-
Scheme 1. Structure of compounds PD153035, gefitinib, sunitinib, semaxanib, SU 6668, CFM-1 and 1e4.
These facts show that other quinazoline derivatives are eligible for
in vitro and in vivo antitumor testing and might be helpful for
cancer treatment [3].
oxoindolin-3-ylidene)hydrazinecarbonyl)phenyl)benzamides 5aej
and ten 3-(2-oxoindolin-3-ylideneamino)-2-substituted quinazo-
lin-4(3H)-one derivatives 6aej have been performed to develop
statistically significant models, so that in future these can be used
to predict and synthesize new derivatives having remarkable bio-
logical inhibition activity.
In addition, there are many reports on oxoindole derivatives as
potential antitumor and cytotoxic agents; these include Sunitinib
[9,10], Semaxanib [11,12], and SU 6668 [13], (Scheme 1). Recently,
certain indolin-2-one derivatives were reported as multitargeted
receptor-tyrosine kinase inhibitors which effectively inhibited
members of both the VEGFR and PDGFR families, each of which
plays a major role in angiogenesis. Some of these compounds were
low nanomolar inhibitors of the enzyme activity; the structure
activity co-relation and the virtual docking studies intensify the
importance and interest of these compounds [3].
Rishi et al. identified and characterized a peri-nuclear phospho-
protein, termed CARP-1/CCAR1. It was considered as a cell cycle
regulator and a key transducer of cell growth [14e16]. The function
of CARP-1 was recently exploited to recognize a number of in-
hibitors coined CARP-1 Functional Mimetics (CFMs) (Scheme 1)
which suppress the growth of a range of cancer cells in part by
stimulating apoptosis [17].
2. Results and discussion
2.1. Chemistry
Benzamide derivatives 2a, b were prepared by the reaction of 2-
methyl-4H-benzo[d][1,3]oxazin-4-ones 1a, b with hydrazine hy-
drate in ethanol while 3-amino-2-arylquinazolin-4(3H)-ones 3a, b
were obtained by the reaction of 1a, c with hydrazine hydrate using
n-butanol instead of ethanol [1] (Scheme 2).
The newly synthesized compounds N-(2-(2-(2-oxoindolin-3-
ylidene)hydrazinecarbonyl)phenyl)benzamides 5aej were pre-
pared by the reaction of N-(2-(hydrazinecarbonyl)aryl)benzamides
2a, b with indoline-2,3-dione 4aee in acidified ethanolic solution
(Scheme 2). Similarly, the new 3-(2-oxoindolin-3-ylideneamino)-
2-substituted quinazolin-4(3H)-ones 6aej were prepared by the
reaction of 3-amino-2-arylquinazolin-4(3H)-one 3a, b with indo-
line-2,3-dione 4aee (Scheme 2).
Based on the aforementioned facts, we synthesized novel 20
CFM-1 analogues 5aej and 6aej by the reaction of 4aee with 2a, b
or 3a, b, respectively, and tested their cytotoxic activities. We report
here the percentage growth inhibition, as well as the IC50 values of
these analogues along, with CFM-1, as a standard drug, against five
tumor cell lines.
2.2. Antitumor testing
Quantitative structureeactivity relationship (QSAR) is among
the most practical tool in computational chemistry. The funda-
mental idea of QSAR consists of the possibility of relationships
between a set of descriptors, which are derived from molecular
structure and a molecular response. QSAR can be regarded as a
computer-derived rule that quantitatively describes the biological
activity in terms of chemical descriptors; it has been frequently
used to predict biological activities of new compounds [18]. In the
present study, QSAR analysis on synthesized ten N-(2-(2-(2-
Thirteen compounds showed varying degrees of remarkable
antitumor activities at the micromolar range (Table 1). Three of
them namely, 5b, 5d and 6b were highly effective against the five
tumor cell lines, Daoy, UW228-2, Huh-7, Hela and MDA-MB231
with IC50 values in the range 1.86e6.87, 4.42e10.89 and
1.46e8.60 mg/ml and percentage inhibition in the range 77.1e88.7,
59.41e84.8 and 75.4e88.0%, respectively. Compounds 6d and 6i
showed good activity against four tumor cell lines, Daoy, UW228-2,

A.M. Alafeefy et al. / European Journal of Medicinal Chemistry 92 (2015) 191e201
193
R
R
O
N
O
N
N
N
NH
N
NH
i
i
3a, b + 4a-e
2a, b + 4a-e
H
O
O
Ar
NH
6a-j
O
Ar
5a-j
Ar
4-MeC6H4-
4-MeC6H4-
4-MeC6H4-
4-MeC6H4-
4-MeC6H4
4-NO2C6H4-
4-NO2C6H4-
4-NO2C6H4-
4-NO2C6H4-
4-NO2C6H4
Ar
6
a
b
c
d
e
f
g
h
i
R
H
Cl
Me
MeO
NO2
H
5
R
H
Cl
-
4-MeC6H4-
4-MeC6H4-
4-MeC6H4-
4-MeC6H4-
4-MeC6H4
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
-
a
b
c
d
e
f
g
h
i
Me
MeO
NO2
H
-
Cl
Cl
Me
MeO
NO2
Me
MeO
NO2
j
j
Scheme 2. Synthesis of the target compounds 5aej and 6aej. Reactions and conditions: (i) absolute ethanol/few drops glacial acetic acid/reflux 5e8 h.
Huh-7 and Hela. However, compounds 5a, 5f and 5h were of
interesting anti-proliferative properties against three tumor cell
lines. Compounds 5e, 6e, 6g and 6h were active only against two
tumor cell lines, whereas 6f was active only against one tumor cell
line (Daoy). On the other hand seven compounds 5c, 5g, 5i, 5j, 6a,
6c, and 6j, were totally inactive.
Daoy cell line was the most sensitive one; it was inhibited by
thirteen compounds followed by Hela cell line, the growth of which
was suppressed by nine compounds. UW228-2 and Huh-7 were
inhibited by eight compounds. The least sensitive cell line was
MDA-MB231 which was sensitive towards three compounds only.
A diverse set of molecular parameters (over 1660) including
different group and class of descriptors as constitutional, walk and
path counts, information indices, connectivity indices, topological,
radial distribution function (RDF), 2D autocorrelation edge adja-
cency indices, topological charge indices, eigenvalue-based indices,
3D-MoRSE, WHIM, GATAWAY, functional group counts, charge
molecular properties, atom centered fragment etc. were calculated
for all compounds using EDRAGON [24] software. Eight QSAR de-
scriptors (surface area approx, surface area grid, volume, hydration
energy, log P, polarizability, refractivity and mass) calculated by
Hyperchem [25] and 5 descriptors (total energy, energy of HOMO
and LUMO, HOMO-LUMO gap and molecular dipole moment) ob-
tained from DFT calculation using Gaussian 09 software, were also
included in the analysis.
2.3. The structure activity correlation
It is evident that methyl group at position-4 of aryl group in
the quinazoline moiety in addition to chlorine atom or methoxy
group in isatin moiety is essential for biological activity. Com-
pounds contain the latter substitutions were the most potent
2.4.3. Statistical analysis
For developing QSAR models, multiple linear regression
approach with stepwise selection and elimination of variables was
employed. Internal cross-validation technique with leave-one-out
(LOO) method [26] was used to verify the statistical significance
and predictive ability of the developed models. Statistical software
package SPSS 21 [27] has been used for all these propose.
compounds with average IC50 of ~4.87
mg/ml. However com-
pounds bearing thiophene moiety were less active than the
previous ones, but were more potent than those derivatives
containing 4-nitrophenyl group at position-2 of the quinazoline
nucleus. Thus, we can conclude that phenyl group at position-2
with electron donating substituent at position-4 such as CH3
has better impact on activity than electron withdrawing group
such as NO2.
2.4.4. QSAR models
After computing over 1270 structural descriptors for all syn-
thesized compounds, Pearson's correlation matrix has been per-
formed on all descriptors for selection of a set of appropriate
descriptors. Analysis of the matrix revealed 15 descriptors for the
development of multi linear regression (MLR) models. Stepwise
multilinear regression analysis on these 15 molecular descriptors
revealed the following 2 and 3 independent parametric statistically
significant QSAR equations, for biological activity of N-(2-(2-(2-
oxoindolin-3-ylidene) hydrazinecarbonyl) phenyl) benzamides
5aej and 3-(2-oxoindolin-3-ylideneamino)-2-substituted quina-
zolin-4(3H)-ones 6aej respectively, in terms of structural
descriptors:
2.4. QSAR analysis
2.4.1. Cancer cell inhibitory activity
Logarithmic value of experimentally observed percent inhibi-
tion of different cancer cell growth at a concentration of 25
mg/ml
(Log % 125 g/ml) was used as a dependent variable for the
m
development of valid QSAR models.
2.4.2. Computational details
Three dimensional initial structures of considered compounds
were constructed by means of Gauss view 5.0.8 program [19], and
then their geometries were optimized to find out conformations
with least potential energy, using Becke's three parameter hybrid
exchange functional [20] with LeeeYangeParr correlation func-
tional (B3LYP) [21,22] of DFT and 6e311þþG(d,p) basis set with the
help of Gaussian 09 software [23].
A. QSAR equations for inhibitory activities of N-(2-(2-(2-
oxoindolin-3-ylidene) hydrazinecarbonyl) phenyl) benzamide 5aej.
Model 1 (against Daoy cancer line)
Logð%inhib:Þ ¼ ꢀ4:950 ꢀ 0:476*RDF140m þ 0:824*SPAN
(1)

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A.M. Alafeefy et al. / European Journal of Medicinal Chemistry 92 (2015) 191e201
Table 1
ꢀ
In vitro cytotoxicity of compounds 5aej, 6aej and CFM-1 against selected cancer cell
lines.
r ¼ 0:912; r2 ¼ 0:831; ra2dj ¼ 0:783; s ¼ 0:122; F ¼ 17:238; p
ꢁ
% Inhibitiona/IC50
b
Comp.
¼ 0:001975
Daoy
UW228-2
Huh-7
Hela
MDA-MB231
5a
5b
5c
5d
5e
5f
5g
5h
5i
72.8/3.94
77.1/1.86
49.1/ntc
77.2/4.42
75.8/6.61
79.1/2.72
36.6/ntc
71.7/6.06
88.7/6.87
23.2/ntc
84.8/5.82
38.5/ntc
51.8/20.0
41.6/ntc
58.7/9.62
24.5/ntc
04.8/ntc
09.3/ntc
88.0/5.70
02.1/ntc
58.9/20.8
22.8/ntc
00.0/ntc
28.4/ntc
32.1/ntc
56.4/21.4
00.0/ntc
51.4/16.4
56.4/5.00
84.1/3.20
38.0/ntc
81.6/4.6
55.2/11.6
48.6/ntc
43.1/ntc
55.8/19.3
33.2/ntc
08.2/ntc
02.9/ntc
83.3/3.76
07.2/ntc
53.6/22.8
00.0/ntc
00.0/ntc
00.0/ntc
32.9/ntc
62.7/19.93
00.0/ntc
61.8/8.18
30.4/ntc
46.8/ntc
85.0/5.95
37.7/ntc
59.41/10.89
30.2/ntc
44.9/ntc
35.5/ntc
40.8/ntc
34.8/ntc
00.0/ntc
24.7/ntc
78.1/4.80
34.3/ntc
35.36/ntc
23.6/ntc
10.7/ntc
06.1/ntc
29.5/ntc
13.6/ntc
00.0/ntc
52.5/19.7
where G(O..O) is sum of geometrical distances between O..O (3D
Atom Pairs) and EEig14d is Eigenvalue 14 from edge adj. matrix
weighted by dipole moments
86.7/6.52
46.5/ntc
80.3/8.09
26.8/ntc
Model 5 (against MDA-MB231cancer line)
55.0/16.52
31.0/ntc
Log ð%inhib:Þ ¼ 38:611 ꢀ 14:624*EEig13x ꢀ 0:629*GATS6m
62.7/11.86
46.1/ntc
11.1/ntc
16.3/ntc
(5)
5j
31.9/ntc
14.7/ntc
6a
6b
6c
6d
6e
6f
6g
6h
6i
41.4/ntc
42.3/ntc
ꢀ
r ¼ 0:993; r2 ¼ 0:985; ra2dj ¼ 0:981; s ¼ 0:073; F
¼ 235:462; p < 10ꢀ6
75.4/1.46
80.8/8.60
47.0/ntc
42.8/ntc
ꢁ
82.8/3.61
58.8/11.66
69.7/5.4
69.3/15.68
59.4/18.97
80.5/3.25
18.4/ntc
68.0/9.89
52.9/21.15
36.2/ntc
69.7/16.96
57.8/19.29
67.0/11.0
00.0/ntc
where EEig13x is Eigenvalue 13 from edge adj. matrix weighted by
edge degrees (Edge adjacency indices), GATS6m e Geary autocor-
relation of lag 6 weighted by mass (2D autocorrelations).
6j
CFM-1
B. QSAR equations for inhibitory activities of fifteen 3-(2-
64.8/8.12
57.6/13.5
oxoindolin-3-ylideneamino)-2-substituted
6aej.
quinazolin-4(3H)-one
a
Percent inhibition of cell survival at a concentration of 25
mg/ml, relative to
control.
b
c
Model 6 (for cancer line Daoy)
IC50 is expressed as
nt: Not tested.
mg/ml.
Logð% inhib:Þ ¼ 5:687 þ 1:810*GATS1v ꢀ 32:457*G2v
(6)
ꢀ
ꢀ
N ¼ 10; r ¼ 0:945; r2 ¼ 0:893; ra2dj ¼ 0:863; s ¼ 0:726; F
¼ 29:247; p < 10ꢀ3
r ¼ 0:959; r2 ¼ 0:919; ra2dj ¼ 0:753; s ¼ 0:088; F
¼ 39:839; p < 10ꢀ3
ꢁ
ꢁ
where GATS1v is Geary autocorrelation of lag 1 weighted by van der
Waals volume a 2D autocorrelations descriptor while G2v is 2nd
component symmetry directional WHIM index/weighted by van
der Waals volume (WHIM descriptors).
where RDF140m is Radial Distribution Function e 140/weighted by
mass (RDF descriptors) and SPAN is span R Geometrical descriptors.
Model 2 (against UW228-2 cancer line)
Model 7 (for cancer line UW228-2)
Logð%inhib:Þ ¼ 11:407 ꢀ 5:090*EEig12d ꢀ 19:265*G1m
(2)
Logð%inhib:Þ ¼ ꢀ5:585 þ 6:923*EEig07d ꢀ 5:568*EEig:09d
ꢀ
r ¼ 0:930; r2 ¼ 0:864; ra2dj ¼ 0:825; s ¼ 0:157; F
¼ 22:248; p < 10ꢀ3
(7)
ꢁ
ꢀ
N ¼ 10; r ¼ 0:966; r2adj ¼ 0:933; r2 ¼ 0:914; s ¼ 0:218; F
¼ 49:061; p < 10ꢀ4
ꢁ
where ESpm12d is Spectral moment 12 from edge adj. Matrix
weighted by dipole moments (Edge adjacency indices) and G1m is
1st component symmetry directional WHIM index/weighed by
mass (WHIM descriptors).
where EEig07d (Eigenvalue 07 from edge adj. matrix weighted by
dipole moments) and EEig09d (Eigenvalue 09 from edge adj. matrix
weighted by dipole moments) are Edge adjacency indices
Model 8 (for cancer line Huh-7)
Model 3 (against Huh-7 cancer line)
Logð%inhhib:Þ ¼ ꢀ14:070 þ 8:025*Ks þ 12:509*BIC4
(3)
Logð%inhib:Þ ¼ ꢀ21:933 þ 17:021*BELm5 þ 3:863*MATS5m
ꢀ
r ¼ 0:986; r2 ¼ 0:972; ra2dj ¼ 0:964; s ¼ 0:054; F
¼ 122:190; p < 10ꢀ5
(8)
ꢁ
ꢀ
N ¼ 10; r ¼ 0:950; r2 ¼ 0:902; ra2dj ¼ 0:874; s ¼ 0:297; F
¼ 32:170; p < 10ꢀ3
ꢁ
where Ks is K global shape index/weighted by I-state (WHIM de-
scriptors) and BIC4 is Bond Information Content index (neighbor-
hood symmetry of 4-order) (Information indices)
Model 4 (against Hela cancer line)
where BELm5 (lowest eigenvalue n. 5 of Burden matrix/weighted
by atomic masses) is a Burden eigenvalue descriptors and MATS5m
(Moran autocorrelation of lag 5 weighted by mass) is a 2D
autocorrelations.
Logð%inhib:Þ ¼ ꢀ0:023 ꢀ 0:012*GðO::OÞ þ 2:517*EEig14d
(4)
Model 9 (for cancer line Hela)

A.M. Alafeefy et al. / European Journal of Medicinal Chemistry 92 (2015) 191e201
195
consideration, from developed QSAR models along with observed
inhibition, are collected in Tables 2 and 3. The correlation plots
between experimental and the predicted data from the derived
multiple regression QSAR Eqs. (1)e(10) given in Figs. 1e10, in-
dicates that predicted values are much closer to the experimental
one. It indicates that developed models can be successfully applied
to predict percent inhibition of N-(2-(2-(2-oxoindolin-3-ylidene)
hydrazinecarbonyl)phenyl)benzamides 5aej and 3-(2-oxoindolin-
Logð%inhib:Þ ¼ 11:569 ꢀ 255:131ꢁR4eþ þ 22:995ꢁHATS7m
(9)
ꢀ
N ¼ 10; r ¼ 0:981; r2 ¼ 0:962; ra2dj ¼ 0:951; s ¼ 0:125; F
¼ 88:441; p < 10ꢀ4
ꢁ
3-ylideneamino)-2-substituted
quinazolin-4(3H)-ones
6aej
where R4eþ (R maximal autocorrelation of lag 4/weighted by
Sanderson electronegativity) and HATS7m (leverage-weighted
autocorrelation of lag 7/weighted by mass) both are GETAWAY
descriptors.
against cancer cell lines Daoy, UW228-2, Huh-7, Hela and MDA-
MB231.
Determination of predictive ability of the developed model
needs validation. To estimate the prediction ability of model by
means of internal validation procedures, cross validation approach
was conducted. In order to test the validity of the predictive power
of selected MLR models, the leave-one-out technique (LOO tech-
nique) was used. The developed models were validated by calcu-
lation of the following statistical parameters: predicted residual
sum of squares (PRESS), total sum of squares deviation (SSD) and
Model 10 (for cancer line MDA-MB231)
Logð%inhib:Þ ¼ 8:157 ꢀ 116:618*R4eþ ꢀ 0:778*GATS3m
(10)
ꢀ
N ¼ 10; r ¼ 0:985; r2 ¼ 0:970; ra2dj ¼ 0:850; s ¼ 0:961; F
¼ 111:434; p < 10ꢀ4
ꢁ
cross-validated correlation coefficients r2 (Table 4).
cv
PRESS is an important cross-validation parameter, as it is a good
approximation of the real predictive error of the models. Its value
being less than sum of squares deviation (SSD) points out that
model predicts better than chance and can be considered statically
significant. Smaller PRESS value means better model predictability.
The results depicted in Table 4, confirm that all models are statis-
tically significant.
where R4eþ (R maximal autocorrelation of lag 4/weighted by
Sanderson electronegativity) is a GETAWAY descriptors while
GATS3m (Geary autocorrelation of lag 3 weighted by mass) is a 2D
autocorrelations.
Several statistical parameters such as regression coefficient(r),
square correlation coefficient (r2), adjusted square correlation co-
efficient (ra2dj), standard error of estimate (S), value of Fischer's
value (F) and significance level (p) < 0.005 are used to check the
credibility of developed models. Large value of F, small S, very small
p-value, as well as r and r2 close to one indicates a good QSAR
model. In present study, all of developed QSAR models are statis-
tically significant with significance level being (p) < 10ꢀ3. The
values of multiple correlation coefficient (r) and square correlation
coefficient (r2) which are greater than 0.92 and 0.83 respectively,
supports the estimated ability of all QSAR models (Eqs. (1)e(10)).
Predicted activity of all compounds, against cancer lines under
The predictive ability of all models is also sustained by the leave-
one-out cross validation, square correlation coefficients (r2
)
loo-cv
which is in range 0.456e0.976 for developed models. The high and
closer value of r2 and r2 are essential criteria for the best
loo-cv
qualification of the QSAR. Cross validation parameters indicates
that QSAR models 3e10 have the most predictive ability while
models 1 and 2 have the least.
Table 2
Observed, converted and predicted inhibition activity of 5aej against Daoy, UW228-2, Huh-7, Hela and MDA-MB231cancer cell lines.
Comp. Daoy
UW228-2
Huh-7
%
%
Obs. act. Log(%Inhib.) Pred. act. Eq.
(1)
Res.
%
Obs. act. Log(%
Pred. act. Eq.
(2)
Res.
Obs. act. Log(%
Pred. act. Eq.
(3)
Res.
Inhib.
Inhib. Inhib.)
Inhib. Inhib.)
5a
5b
5c
5d
5e
5f
5g
5h
5i
72.800 1.862
77.100 1.887
1.858
0.004 71.700 1.856
ꢀ0.022 88.700 1.948
ꢀ0.147 23.200 1.365
ꢀ0.003 84.800 1.928
ꢀ0.023 38.500 1.585
0.056 51.800 1.714
0.053 41.600 1.619
0.144 58.700 1.769
ꢀ0.064 24.500 1.389
ꢀ0.008 4.800 0.681
1.882
1.811
1.503
1.671
1.774
1.776
1.502
1.754
1.514
0.670
ꢀ0.026 56.400 1.751
0.137 84.100 1.925
ꢀ0.138 38.000 1.580
0.257 81.600 1.912
ꢀ0.189 55.200 1.742
ꢀ0.062 48.600 1.687
0.117 43.100 1.634
0.015 55.800 1.747
ꢀ0.125 33.200 1.521
0.011 8.200 0.914
1.819
1.977
1.603
1.830
1.748
1.630
1.619
1.708
1.549
0.928
ꢀ0.068
ꢀ0.052
ꢀ0.023
0.082
1.909
1.838
1.891
1.903
1.842
1.510
1.653
1.109
1.512
49.100 1.691
77.200 1.888
75.800 1.880
79.100 1.898
36.600 1.563
62.700 1.797
11.100 1.045
31.900 1.504
ꢀ0.006
0.057
0.015
0.039
ꢀ0.028
5g
ꢀ0.014
Comp.
Hela
MDA-MB231
%Inhib.
Obs. act. Log(%Inhib.)
Pred. act. Eq. (4)
Res.
%Inhib.
Obs. act. Log(%Inhib.)
Pred. act. Eq. (5)
Res.
5a
5b
5c
5d
5e
5f
5g
5h
5i
30.400
86.700
46.500
80.300
26.800
55.000
31.000
46.100
16.300
14.700
1.483
1.938
1.667
1.905
1.428
1.740
1.491
1.664
1.212
1.167
1.566
1.847
1.618
1.963
1.610
1.607
1.608
1.607
1.370
1.147
ꢀ0.083
0.091
0.049
ꢀ0.058
ꢀ0.182
0.133
46.800
85.000
37.700
59.400
30.200
44.900
35.500
40.800
34.800
1.000
1.670
1.929
1.576
1.774
1.480
1.652
1.550
1.611
1.542
0.000
1.720
1.881
1.601
1.756
1.509
1.562
1.641
1.511
1.611
0.003
ꢀ0.050
0.048
ꢀ0.025
0.018
ꢀ0.029
0.090
ꢀ0.117
ꢀ0.091
0.057
0.100
ꢀ0.158
0.020
ꢀ0.069
ꢀ0.003
5g
%Inhib.-Percent inhibition of cell survival at a concentration of 25 mg/ml, relative to control.

196
A.M. Alafeefy et al. / European Journal of Medicinal Chemistry 92 (2015) 191e201
Table 3
Observed, converted and predicted inhibition activity of 6aej against Daoy, UW228-2, Huh-7, Hela and MDA-MB231cancer cell lines.
Comp. Daoy
UW228-2
Huh-7
%
%
Obs. act. Log(%Inhib.) Pred. act. Eq.
(6)
Res.
%
Obs. act. Log(%
Pred. act. Eq.
(7)
Res.
Obs. act. Log(%
Pred. act. Eq.
(8)
Res.
Inhib.
Inhib. Inhib.)
Inhib. Inhib.)
6a
6b
6c
6d
6e
6f
6g
6h
6i
41.400 1.617
75.400 1.877
1.600
0.017 9.300 0.968
ꢀ0.077 88.000 1.944
ꢀ0.022 2.100 0.322
0.000 58.900 1.770
0.083 22.800 1.358
0.062 1.000 0.000
0.064 28.400 1.453
ꢀ0.100 32.100 1.507
0.034 56.400 1.751
ꢀ0.064 1.000 0.000
1.175
1.859
0.620
1.798
1.446
0.096
1.534
1.263
1.534
ꢀ0.250
ꢀ0.207 2.900 0.462
0.085 83.300 1.921
ꢀ0.298 7.200 0.857
ꢀ0.028 53.600 1.729
ꢀ0.088 1.000 0.000
ꢀ0.096 1.000 0.000
ꢀ0.081 1.000 0.000
0.244 32.900 1.517
0.217 62.700 1.797
0.362
1.633
0.551
1.522
0.567
0.024
ꢀ0.025
1.687
1.988
ꢀ0.027
0.100
0.287
0.306
1.954
1.694
1.918
1.686
1.781
1.777
1.874
1.872
1.329
47.000 1.672
82.800 1.918
58.800 1.769
69.700 1.843
69.300 1.841
59.400 1.774
80.500 1.906
18.400 1.265
0.207
ꢀ0.567
ꢀ0.024
0.025
ꢀ0.170
ꢀ0.191
0.027
6j
0.250
1.000 0.000
Comp.
Hela
MDA-MB231
%Inhib.
Obs. act. Log(%Inhib.)
Pred. act. Eq. (9)
Res.
%Inhib.
Obs. act. Log(%Inhib.)
Pred. act. Eq. (10)
Res.
6a
6b
6c
6d
6e
6f
6g
6h
6i
42.300
80.800
42.800
68.000
52.900
36.200
69.700
57.800
67.000
1.000
1.626
1.907
1.631
1.833
1.723
1.559
1.843
1.762
1.826
0.000
1.524
2.078
1.666
1.896
1.634
1.508
1.922
1.770
1.628
0.088
0.102
24.700
78.100
34.300
35.360
23.600
10.700
6.100
29.500
13.600
1.000
1.393
1.893
1.535
1.549
1.373
1.029
0.785
1.470
1.134
0.000
1.358
1.837
1.601
1.662
1.208
0.956
0.918
1.426
1.185
0.005
0.034
0.056
ꢀ0.066
ꢀ0.113
0.165
ꢀ0.171
ꢀ0.035
ꢀ0.063
0.089
0.051
0.073
ꢀ0.079
ꢀ0.008
0.198
ꢀ0.088
ꢀ0.133
0.044
ꢀ0.051
ꢀ0.005
6j
%Inhib.-Percent inhibition of cell survival at a concentration of 25
m
g/ml, relative to control.
Fig. 3. Observed versus predicted activity for model 3.
Fig. 1. Observed versus predicted activity for model 1.
Fig. 4. Observed versus predicted activity for model 4.
Fig. 2. Observed versus predicted activity for model 2.

A.M. Alafeefy et al. / European Journal of Medicinal Chemistry 92 (2015) 191e201
197
was refluxed for 5e8 h, and then cooled to room temperature. The
precipitate was filtered and dried. The crude product was crystal-
lized from EtOH/DMF to obtain the target.
4.1.2.1. (Z)-4-Methyl-N-(2-(2-(2-oxoindolin-3-ylidene)hydrazine-
carbonyl)phenyl)benzamide (5a). Yield (63%); m.p. 258e260 ꢂC; IR
n
3342e3301 (3NH), 1692e1668 (3C]O) cmꢀ1 1H NMR (DSMO-d6)
;
d
2.33 (s, 3H, CH3), 7.0e8.36 (m, 12H, ArH); 13C NMR (DSMO-d6)
d
23.17 (CH3), 119.59, 123.60, 127.85, 128.27, 129.29, 129.51, 130.04,
130.16, 130.26, 130.39, 132.13, 138.11, 139.89, 145.23, 149.24, 155.09,
157.32, 170.16; MS m/z (Rel. Int.) (Mþ, 398, 7.0); Anal. (C23H18N4O3,
398) C, 69.34 (69.05); H, 4.55 (4.78); N, 14.06 (13.75).
4.1.2.2. (Z)-N-(2-(2-(5-Chloro-2-oxoindolin-3-ylidene)hydrazine-
carbonyl)phenyl)-4-methyl
benzamide
(5b). Yield
(66%);
m.p. > 300 ꢂC; IR
NMR (DSMO-d6)
NMR (DSMO-d6)
n
3347e3302 (3NH), 1695e1667 (3C]O) cmꢀ1; 1H
2.32 (s, 3H, CH3), 7.01e8.35 (m, 11H, ArH); 13C
23.5 (CH3), 119.7, 121.6, 123.6, 123.9, 125.2, 128.3,
Fig. 5. Observed versus predicted activity for model 5.
d
d
128.5,129.5,129.9,130.4,131.4,131.7,133.0,133.4,138.1,142.4,145.7,
164.2, 164.7, 167.1; MS m/z (Rel. Int.) 432 (Mþ, 71). Anal.
(C23H17ClN4O3, 432.86) C, 63.82 (63.63); H, 3.96 (4.17); Cl, 8.19
(7.89); N, 12.94 (13.08).
3. Conclusion
In conclusion, we prepared twenty novel quinazoline de-
rivatives structurally similar to CFM-1 and tested them against five
tumor cell lines. Six compounds showed interesting activity better
than the standard drug against most of the tested cancer cell lines.
Compound 5b, 5d and 6b were the most potent with IC50 values in
4.1.2.3. (Z)-4-Methyl-N-(2-(2-(5-methyl-2-oxoindolin-3-ylidene)
hydrazinecarbonyl)phenyl) benzamide (5c). Yield (55%); m.p.
282e284 ꢂC; IR
NMR (DSMO-d6)
NMR (DSMO-d6)
n
3341e3305 (3NH), 1696e1664 (3C]O) cmꢀ1; 1H
the range 1.86e6.87, 4.42e10.89 and 1.46e8.60
mg/ml, and per-
d
2.37 (s, 6H, 2CH3), 6.99e8.34 (m, 11H, ArH); 13
23.9 (2CH3), 118.5, 121.2, 121.6, 123.3,124.7, 127.5,
C
centage inhibition in the range 77.1e88.7, 59.41e84.8 and
75.4e88.0%, respectively. By QSAR analysis of the target com-
pounds, 10 statistically significant models were developed and
internally cross validated.
d
127.9, 129.4,129.9,131.3, 131.8,132.3,133.8, 135.1, 138.6,141.9, 145.2,
162.8, 163.5, 166.9; MS m/z (Rel. Int.) 412 (Mþ, 38). Anal.
(C24H20N4O3, 412.44) C, 69.89 (70.09); H, 4.89 (5.13); N, 13.58
(13.29).
4. Experimental
4.1.2.4. (Z)-N-(2-(2-(5-Methoxy-2-oxoindolin-3-ylidene)hydrazine-
4.1. Chemistry
carbonyl)phenyl)-4-methyl benzamide (5d). Yield (57%); m.p.
254e256 ꢂC; IR 3352e3307 (3NH), 1698e1661 (3C]O) cmꢀ1; 1H
n
4.1.1. General
NMR (DSMO-d6)
d
2.35 (s, 3H, CH3), 3.82 (s, 3H, -OCH3), 7.0e8.36
24.2 (CH3), 55.5 (OCH3), 114.1,
Melting points (ꢂC, uncorrected) were determined in open
capillaries on a Gallenkamp melting point apparatus (Sanyo Gal-
lenkamp, Southborough, UK) and were uncorrected. Precoated
silica gel plates (silica gel 0.25 mm, 60G F254; Merck, Germany)
were used for thin layer chromatography, dichloromethane/meth-
anol (9.5:0.5) mixture was used as a developing solvent system and
the spots were visualized by ultraviolet light and/or iodine. Infra-
red spectra were recorded in KBr discs using IR-470 Shimadzu
spectrometer (Shimadzu, Tokyo, Japan). 1H NMR spectra were
(m, 11H, ArH); 13C NMR (DSMO-d6)
d
116.4,119.0,121.5, 122.8,123.5,124.9, 127.2, 127.7, 129.5,131.2, 132.5,
132.9, 137.8, 139.4, 142.1, 155.7, 163.0, 164.3, 167.5; MS m/z (Rel. Int.)
428 (Mþ, 46). Anal. (C24H20N4O4, 428.44) C, 67.28 (67.02); H, 4.71
(4.85); N, 13.08 (12.82).
4.1.2.5. (Z)-4-Methyl-N-(2-(2-(5-nitro-2-oxoindolin-3-ylidene)
hydrazinecarbonyl)phenyl)
benzamide
(5e). Yield
(61%);
m.p. > 300 ꢂC; IR
NMR (DSMO-d6)
NMR (DSMO-d6)
n
3365e3312 (3NH),1698e1661 (3C]O) cmꢀ1; 1H
2.33 (s, 3H, CH3), 7.19e8.37 (m, 11H, ArH); 13C
recorded on Bruker AC-300 Ultra Shield NMR spectrometer (d ppm;
d
Bruker, Flawil, Switzerland) at 300 MHz for 1H and 75 MHz for 13C,
using TMS as internal standard and peak multiplicities were
designed as follows: s, singlet; d, doublet; t, triplet; m, multiplet.
Electron Impact Mass Spectra were recorded on a Shimadzu GC-
MS-QP 5000 instrument (Shimadzu, Tokyo, Japan), and the purity
of compounds was >95%. Elemental analyses were performed, on
Carlo Erba 1108 Elemental Analyzer (Heraeus, Hanau, Germany), at
the Micro-analytical Unit, Faculty of Science, Cairo University, Cairo,
Egypt, and the found results were within 0.4% of the theoretical
d
24.7 (CH3), 119.3, 121.4, 122.7, 123.1, 123.6, 124.4,
124.8, 127.2, 127.6, 129.9, 130.9, 132.3, 132.8, 138.3, 142.0, 145.5,
153.1, 162.7, 163.4, 167.1; MS m/z (Rel. Int.) 443 (Mþ, 17). Anal.
(C23H17N5O5, 443.41) C, 62.30 (62.11); H, 3.86 (4.04); N, 15.79
(15.58).
4.1.2.6. (Z)-4-Nitro-N-(2-(2-(2-oxoindolin-3-ylidene)hydrazine-
carbonyl)phenyl)benzamide (5f). Yield (65%); m.p. > 300 ꢂC; IR
n
3345e3296 (3NH), 1699e1662 (3C]O) (3NH) cmꢀ1
;
1H NMR
118.0,
7.16e8.37 (m, 12H, ArH); 13C NMR (DSMO-d6)
d
values. N-(2-(Hydrazinecarbonyl)aryl)benzamide 2a,
amino-2-arylquinazolin-4(3H)-one 3a, b were prepared according
to the reported method [2].
b and 3-
(DSMO-d6)
d
121.2, 121.7, 123.5, 124.7, 127.4, 128.3, 129.7, 131.5, 132.4, 132.9, 137.6,
141.0, 146.5, 152.4, 162.5, 163.7, 167.4; MS m/z (Rel. Int.) 429 (Mþ,
36). Anal. (C22H15N5O5, 429.39) C, 62.30 (62.02); H, 3.86 (4.05); N,
15.79 (14.57).
4.1.2. Synthesis of N-(2-(2-(2-oxoindolin-3-ylidene)
hydrazinecarbonyl)phenyl)benzamides 5aej
To a mixture of N-(2-(hydrazinecarbonyl)aryl)benzamide 2a, b
(1 mmol) and indoline-2,3-dione 4aee (1 mmol) in ethanol (25 ml),
few drops of glacial acetic acid were added. The reaction mixture
4.1.2.7. (Z)-N-(2-(2-(5-Chloro-2-oxoindolin-3-ylidene)hydrazine-
carbonyl)phenyl)-4-nitro
benzamide
(5g). Yield
(64%);
m.p. > 300 ꢂC; IR 3348e3294 (3NH), 1699e1661 cmꢀ1
n
;
1H NMR

198
A.M. Alafeefy et al. / European Journal of Medicinal Chemistry 92 (2015) 191e201
Fig. 9. Observed versus predicted activity for model 9.
Fig. 6. Observed versus predicted activity for model 6.
Fig. 10. Observed versus predicted activity for model 10.
Fig. 7. Observed versus predicted activity for model 7.
Table 4
Cross-validation parameters.
Model no.
PRESS
SSY
PRESS/SSY
rꢀ2 cvꢀloo
1
2
3
4
5
6
7
8
9
10
0.277
0.564
0.051
0.186
0.062
0.119
0.963
1.163
0.174
0.127
0.663
1.278
0.736
0.619
2.580
0.346
4.985
6.297
2.855
2.496
0.417
0.441
0.069
0.301
0.024
0.344
0.193
0.185
0.061
0.051
0.583
0.559
0.931
0.699
0.976
0.656
0.807
0.815
0.939
0.949
4.1.2.8. (Z)-N-(2-(2-(5-Methyl-2-oxoindolin-3-ylidene)hydrazine-
carbonyl)phenyl)-4-nitro benzamide (5h). Yield (59%); m.p.
291e293 ꢂC; IR
NMR (DSMO-d6)
NMR (DSMO-d6)
n
3347e3290 (3NH), 1705e1662 (3C]O) cmꢀ1; 1H
d
2.31 (s, 3H, CH3), 7.02e8.37 (m, 11H, ArH); 13C
Fig. 8. Observed versus predicted activity for model 8.
d
24.3 (CH3), 117.5, 121.3, 121.6, 121.9, 123.5, 124.7,
127.6,128.3,129.7,131.5,132.3,132.7,134.6,137.3,141.0,144.6,152.5,
(DSMO-d6)
d d 118.9,
7.17e8.35 (m, 11H, ArH); 13C NMR (DSMO-d6)
163.1, 164.9, 167.6; MS m/z (Rel. Int.) 443 (Mþ, 37).
121.2, 121.6, 123.4, 123.7, 124.5, 127.3, 128.5, 129.3, 130.6, 131.5,
132.3, 132.8, 137.4, 141.7, 144.5, 151.9, 162.8, 163.5, 167.1; MS m/z
(Rel. Int.) 465 (Mþ þ 2, 13), 463 (Mþ, 42). Anal. (C22H14ClN5O5,
463.83) C, 56.97 (57.16); H, 3.04 (3.25); Cl, 7.64 (7.39); N, 15.10
(14.94).
4.1.2.9. (Z)-N-(2-(2-(5-Methoxy-2-oxoindolin-3-ylidene)hydrazine-
carbonyl)phenyl)-4-nitro benzamide (5i). Yield (54%); m.p.
285e287 ꢂC; IR
NMR (DSMO-d6)
NMR (DSMO-d6)
n
3353e3291 (3NH), 1703e1665 (3C]O) cmꢀ1; 1H
d
3.95 (s, 3H, OCH3), 7.0e8.40 (m, 11H, ArH); 13C
57.7 (OCH3), 117.5, 121.3, 121.6, 121.9, 123.5, 124.7,
d
127.6,128.3,129.7,131.5,132.3,132.7,134.6,137.3,141.0,144.6,152.5,

A.M. Alafeefy et al. / European Journal of Medicinal Chemistry 92 (2015) 191e201
199
163.1, 164.9, 167.6; MS m/z (Rel. Int.) 459 (Mþ, 46). Anal.
(C23H17N5O6, 459.41) C, 60.13 (59.97); H, 3.73 (3.95); N, 15.24
(14.96).
4.1.3.6. (Z)-3-(2-Oxoindolin-3-ylideneamino)-2-(thiophen-2-yl)qui-
nazolin-4(3H)-one (6f). Yield (64%); m.p. 283e285 ꢂC; IR
3286
(NH), 1701, 1675 (2C]O) cmꢀ1; 1H NMR (DSMO-d6)
7.02e8.14 (m,
11H, ArH), (s, D2O exchangeable, 1H, NH); 13C NMR (DSMO-d6)
118.0, 120.4, 121.7, 122.5, 124.2, 125.3, 126.8, 127.5, 128.3, 129.6,
131.0, 132.2, 133.6, 146.5, 147.1, 159.5, 162.7, 167.2; MS m/z (Rel. Int.)
372 (Mþ, 26). Anal. (C20H12N4O2S, 372.40) C, 64.50 (64.27); H, 3.25
(3.49); N, 15.04 (14.85); S, 8.61 (8.34).
n
d
4.1.2.10. (Z)-4-Nitro-N-(2-(2-(5-nitro-2-oxoindolin-3-ylidene)hydra-
d
zinecarbonyl)phenyl) benzamide (5j). Yield (53%); m.p. > 300 ꢂC; IR
n
3357e3290 (3NH), 1701e1664 (3C]O) cmꢀ1; 1H NMR (DSMO-d6)
d
7.02e8.35 (m, 11H, ArH); 13C NMR (DSMO-d6)
d 119.1, 121.4, 121.9,
122.5, 123.3, 123.8, 124.5, 124.7, 127.4, 128.1, 132.6, 132.9, 137.6,
140.8, 144.9, 151.6, 153.0, 162.7, 164.2, 167.5; MS m/z (Rel. Int.) 474
(Mþ, 28). Anal. (C22H14N6O7, 474.38) C, 55.70 (55.93); H, 2.97 (3.18);
N, 17.72 (17.95).
4.1.3.7. (Z)-3-(5-Chloro-2-oxoindolin-3-ylideneamino)-2-(thiophen-
2-yl)quinazolin-4(3H)-one (6g). Yield (62%); m.p. > 300 ꢂC; IR
n
3306 (NH), 1708, 1681 (2C]O) cmꢀ1
;
1H NMR (DSMO-d6)
119.3, 120.6, 122.5,
d
7.01e8.11 (m, 10H, ArH); 13C NMR (DSMO-d6)
d
4.1.3. Synthesis of 3-(2-oxoindolin-3-ylideneamino)-2-substituted
quinazolin-4(3H)-ones 6aej
These compounds were prepared following the same procedure
for synthesis of compounds 5aej by using 3-amino-2-
arylquinazolin-4(3H)-one 3a, b (1 mmol) instead of hydrazides
2a, b.
123.3, 125.4, 126.3, 127.1, 127.6, 128.3, 129.5, 130.2, 131.6, 132.3,
133.5, 144.7, 148.1, 159.6, 161.8, 166.5; MS m/z (Rel. Int.) 406 (Mþ,
29). Anal. (C20H11ClN4O2S, 406.4) C, 59.04 (58.83); H, 2.73 (2.57); N,
13.77 (13.52); S, 7.88 (7.61).
4.1.3.8. (Z)-3-(5-Methyl-2-oxoindolin-3-ylideneamino)-2-(thiophen-
2-yl)quinazolin-4(3H)-one (6h). Yield (52%); m.p. 269e271 ꢂC; IR
n
4.1.3.1. (Z)-3-(2-Oxoindolin-3-ylideneamino)-2-p-tolylquinazolin-
3301 (NH), 1700, 1689 (2C]O) cmꢀ1; 1H NMR (DSMO-d6)
d 2.37 (s,
4(3H)-one (6a). Yield (68%); m.p. > 300 ꢂC; IR
n
3289 (NH), 1695,
2.38 (s, 3H, CH3),
24.6 (CH3), 118.1,
3H, CH3), 7.0e8.16 (m, 10H, ArH); 13C NMR (DSMO-d6)
d
25.1, 117.5,
1675 (2C]O) cmꢀ1
;
1H NMR (DSMO-d6)
d
120.7, 121.3, 122.6,125.2,126.8,127.3,127.7,128.2,129.8,131.5, 132.1,
133.4, 134.7, 143.1, 147.4, 159.8, 162.0, 166.9; MS m/z (Rel. Int.) 386
(Mþ, 35). Anal. (C21H14N4O2S, 386.43) C, 65.27 (64.99); H, 3.65
(3.34); N, 14.50 (14.68); S, 8.30 (8.52).
7.08e8.08 (m, 12H, ArH); 13C NMR (DSMO-d6)
d
120.0, 121.6, 123.5, 124.2, 125.2, 126.1, 127.2, 128.6, 129.0, 129.4,
131.3, 132.5, 133.4,139.7, 146.4, 151.6, 157.1, 161.2, 167.8; MS m/z (Rel.
Int.) 380 (Mþ, 24). Anal. (C23H16N4O2, 380.40) C, 72.62 (72.47); H,
4.24 (3.98); N, 14.73 (14.55).
4.1.3.9. (Z)-3-(5-Methoxy-2-oxoindolin-3-ylideneamino)-2-(thio-
phen-2-yl)quinazolin-4(3H)-one (6i). Yield (47%); m.p. 285e287 ꢂC;
4.1.3.2. (Z)-3-(5-Chloro-2-oxoindolin-3-ylideneamino)-2-p-tolylqui-
IR
n
3317 (NH), 1702, 1684 (2C]O) cmꢀ1; 1H NMR (DSMO-d6)
d
3.81
nazolin-4(3H)-one (6b). Yield (53%); m.p. > 300 ꢂC; IR
n
3288 (NH),
(s, 3H, OCH3), 7.01e8.15 (m, 10H, ArH); 13C NMR (DSMO-d6)
d
55.3,
1694, 1667 (2C]O) cmꢀ1
;
1H NMR (DSMO-d6)
d
2.37 (s, 3H, CH3),
114.0, 116.3, 119.1, 120.4, 122.5, 122.7, 125.3, 126.1, 127.2, 127.6, 128.1,
132.5, 133.1, 139.4, 147.1, 157.0, 159.7, 162.3, 167.5; MS m/z (Rel. Int.)
402 (Mþ, 38). Anal. (C21H14N4O3S, 402.43) C, 62.68 (62.41); H, 3.51
(3.35); N, 13.92 (14.22); S, 7.97 (8.15).
7.03e8.12 (m, 11H, ArH); 13C NMR (DSMO-d6)
d
24.6 (CH3), 119.4,
121.1, 122.1, 123.5, 125.2, 126.1, 127.6, 128.2, 129.3, 129.7, 130.3, 131.5,
132.4, 133.1, 139.5, 145.1, 152.7, 162.0, 166.7; MS m/z (Rel. Int.) 414
(Mþ, 32). Anal. (C23H15ClN4O2, 414.84) C, 66.59 (66.75); H, 3.64
(3.46); N, 13.51 (13.27).
4.1.3.10. (Z)-3-(5-Nitro-2-oxoindolin-3-ylideneamino)-2-(thiophen-
2-yl)quinazolin-4(3H)-one (6j). Yield (51%); m.p. 294e296 ꢂC; IR
n
4.1.3.3. (Z)-3-(5-Methyl-2-oxoindolin-3-ylideneamino)-2-p-tolylqui-
3309 (NH), 1705, 1687 (2C]O) cmꢀ1; 1H NMR (DSMO-d6)
nazolin-4(3H)-one (6c). Yield (55%); m.p. 284e286 ꢂC; IR
n
3279
d d 118.3, 120.7, 122.1,
6.97e8.15 (m, 10H, ArH); 13C NMR (DSMO-d6)
(NH), 1701, 1685 (2C]O) cmꢀ1
;
1H NMR (DSMO-d6)
d
2.35 (s, 6H,
24.7 (CH3),
122.5, 123.2, 124.5, 125.4, 126.1, 127.0, 127.5, 128.3, 132.6, 133.5,
145.1, 147.5, 153.1, 159.6, 161.6, 167.2; MS m/z (Rel. Int.) 417 (Mþ, 36).
Anal. (C20H11N5O4S, 417.40) C, 57.55 (57.37); H, 2.66 (2.45); N, 16.78
(16.55); S, 7.68 (7.83).
2CH3), 7.01e8.14 (m, 11H, ArH); 13C NMR (DSMO-d6)
d
117.9,120.6,121.9,122.7,125.4,126.3,127.7,128.4,129.5,129.9,131.4,
132.6,133.7,134.3,140.1,144.1,151.5,159.3,162.7,167.3; MS m/z (Rel.
Int.) 394 (Mþ, 13). Anal. (C24H18N4O2, 394.43) C, 73.08 (72.89); H,
4.60 (4.83); N, 14.20 (13.99).
4.2. Anti-proliferative activity
4.1.3.4. (Z)-3-(5-Methoxy-2-oxoindolin-3-ylideneamino)-2-p-tol-
4.2.1. Materials
ylquinazolin-4(3H)-one (6d). Yield (57%); m.p. 277e79 ꢂC; IR
n
3307
2.34 (s, 3H,
CH3), 3.81 (s, 3H, OCH3), 6.98e8.16 (m, 11H, ArH); 13C NMR (DSMO-
d6) 24.7 (CH3), 57.3 (OCH3), 114.0, 116.3, 118.7, 120.5, 122.3, 122.7,
4.2.1.1. Chemicals and supplies. MTT (3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyltetrazolium bromide) was purchased from Sigma
Aldrich (St Louis, MO, USA). DMEM/high glucose, FBS and peni-
cillin/streptomycin were obtained from Gibco (Grand Island, NY,
USA).
(NH), 1703, 1677 (2C]O) cmꢀ1 1H NMR (DSMO-d6)
; d
d
125.3,126.4,127.5,128.3,129.6,132.7,133.9,139.5,139.7,151.7,157.0,
157.9, 162.1, 167.5; MS m/z (Rel. Int.) 410 (Mþ, 34). Anal.
(C24H18N4O3, 410.42) C, 70.23 (70.05); H, 4.42 (4.16); N, 13.65
(13.41).
4.2.1.2. Cell lines. Five human tumor cell lines were utilized in this
study, namely medulloblastoma (Daoy and UW228-2), hepatocel-
lular carcinoma (Huh-7), cervical carcinoma (Hela) and breast
carcinoma (MDA-MB231). The routine maintenance and culture
conditions for Daoy and UW228-2, HeLa and MDA-MB-231 cells
have been previously described [17,28,29]. The hepatic cancer Huh-
7 cells were kindly provided by Dr. Kezhong Zhang, Center for
Cellular and Molecular Genetics, Wayne State University, Detroit,
MI, and were maintained essentially as described [30]. Daoy and
UW228-2 were cultured in DMEM/F12 supplemented with 10% FBS,
4.1.3.5. (Z)-3-(5-Nitro-2-oxoindolin-3-ylideneamino)-2-p-tolylqui-
nazolin-4(3H)-one (6e). Yield (60%); m.p. > 300 ꢂC; IR
n 3291 (NH),
1706, 1675 (2C]O) cmꢀ1
;
1H NMR (DSMO-d6)
d
2.33 (s, 3H, CH3),
24.3 (CH3), 118.3,
7.06e8.17 (m, 11H, ArH); 13C NMR (DSMO-d6)
d
120.6, 122.1, 122.8, 123.6, 124.2, 125.4, 126.3, 127.4, 128.1, 129.5,
132.3, 133.5, 139.6, 144.0, 151.6, 153.2, 159.7, 161.4, 166.7; MS m/z
(Rel. Int.) 425 (Mþ, 33). Anal. (C23H15N5O4, 425.40) C, 64.94 (65.17);
H, 3.55 (3.29); N, 16.46 (16.70).
2 mM L-glutamine and 1% penicillin/streptomycin. Huh-7, Hela and

200
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Acknowledgment
This work was supported by a grant from the National Plan of
Science, Technology and Innovation (Grant No.10-MED1188-02)
King Saud University, Riyadh, Saudi Arabia., and the US Department
of Veterans review grant (AKR).
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