Synthesis, biological activity and docking study of some new isatin Schiff base derivatives

Article abstract of DOI:10.1007/s00044-011-9896-6

A set of novel Schiff bases of isatin were synthesized and characterized by reaction of isatin with various aromatic or heterocyclic primary amines. Cytotoxic activities for some of the synthesized compounds were evaluated byMTTassay in three human cancer cell lines (HeLa, LS180 and Raji). Half of the tested compounds showed good cytotoxicity in HeLa cells. 3-(2-(4-nitrophenyl) hydrazono) indolin-2-one was found to be the most potent molecule among the studied isatin derivatives. Docking studies of 3-substituted indolin-2-one scaffolds on vascular endothelial growth factor receptor 2 (VEGFR-2) involved in cell proliferation and angiogenesis was performed. 3-(naphthalen-1-ylimino) indolin-2-one and 3-(2-(4-nitrophenyl) hydrazono) indolin-2-one exhibited higher docking binding energies with receptor. For 3-(2-(4-nitrophenyl) hydrazono) indolin-2-one, H-bond interaction with Cys917 residue of target active site was in common with reported crystallographic benzoimidazole derivative (PDB code: 2OH4). New key H-bonds involving Glu915, Asn921, and Arg1049 residues in VEGFR-2 active site could be detected for 3-(2-(4-nitrophenyl) hydrazono) indolin-2-one. Extended lipophilic rings containing H-bond acceptors on the 3 position of indoline scaffold seemed to be important factors in developing potent VEGFR-2 inhibitors virtually. Based on the ligand efficiency indices, some isoxazole or thiazole substituted isatin derivatives may be regarded as efficient candidates for further molecular developments of anticancer agents. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9896-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-011-9896-6
ORIGINAL RESEARCH
Synthesis, biological activity and docking study
of some new isatin Schiff base derivatives
•
Javad Azizian Mohammad K. Mohammadi
•
•
Omidreza Firuzi Nima Razzaghi-asl
•
Ramin Miri
Received: 30 August 2011 / Accepted: 8 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A set of novel Schiff bases of isatin were syn-
thesized and characterized by reaction of isatin with various
aromatic or heterocyclic primary amines. Cytotoxic activi-
ties for some of the synthesized compounds were evaluated
by MTT assay in three human cancer cell lines (HeLa, LS180
and Raji). Half of the tested compounds showed good
cytotoxicity in HeLa cells. 3-(2-(4-nitrophenyl) hydrazono)
indolin-2-one was found to be the most potent molecule
among the studied isatin derivatives. Docking studies of
3-substituted indolin-2-one scaffolds on vascular endothelial
growth factor receptor 2 (VEGFR-2) involved in cell pro-
liferation and angiogenesis was performed. 3-(naphthalen-1-
ylimino) indolin-2-one and 3-(2-(4-nitrophenyl) hydrazono)
indolin-2-one exhibited higher docking binding energies
with receptor. For 3-(2-(4-nitrophenyl) hydrazono) indolin-
2-one, H-bond interaction with Cys917 residue of target
active site was in common with reported crystallographic
benzoimidazole derivative (PDB code: 2OH4). New key
H-bonds involving Glu915, Asn921, and Arg1049 residues
in VEGFR-2 active site could be detected for 3-(2-(4-
nitrophenyl) hydrazono) indolin-2-one. Extended lipophilic
rings containing H-bond acceptors on the 3 position of ind-
oline scaffold seemed to be important factors in developing
potent VEGFR-2 inhibitors virtually. Based on the ligand
efficiency indices, some isoxazole or thiazole substituted
isatin derivatives may be regarded as efficient candidates for
further molecular developments of anticancer agents.
Keywords Isatin ꢀ Schiff base ꢀ Synthesis ꢀ Cytotoxicity ꢀ
Docking
Introduction
Isatin is an endogenous compound isolated in 1988 (Glover
et al., 1988) and reported to possess a wide range of central
nervous system activities (d’Ischia et al., 1988; Varma and
Nobles, 1975). It has also been found as a metabolic
derivative of adrenaline in humans (d’Ischia et al., 1988).
Isatin is a natural product found in a number of plants
including those of the genus isatis and also has been found
as a metabolic derivative of humans (d’Ischia et al., 1988).
Various derivatives of isatin are known to possess a wide
range of pharmacological properties (Varma and Nobles,
1975; Varma and Khank, 1977). Among the important
pharmacological effects, antibacterial (Pandeya et al.,
2000, 1999a; Sarangapani and Reddy, 1994; Varma and
Nobles, 1975; Sridhar et al., 2001), antifungal (Pandeya
et al., 2000; Pandeya et al., 1999), antiviral (Varma and
Nobles, 1967; Singh et al., 1983; Logan et al., 1975), and
anti-HIV (Pandeya et al., 1999b; Pandeya et al., 2000)
activities are worth noting. Within the context of enzyme
inhibitors, isatins (also known as 2,3-dioxindoles) have
seen recent applications in the inhibition of cysteine and
serine proteases (Iyer and Hanna, 1995; Webber et al.,
J. Azizian
Science and Research Branch, Department of Chemistry,
Islamic Azad University, 11365-4435, Tehran, Iran
M. K. Mohammadi
Faculty of Sciences, Islamic Azad University, Ahvaz Branch,
Iran
O. Firuzi ꢀ R. Miri (&)
Medicinal and Natural Products Chemistry Research Center,
Shiraz University of Medical Sciences, 3288-71345, Shiraz, Iran
e-mail: mirir@sums.ac.ir
N. Razzaghi-asl
Department of Medicinal Chemistry, Faculty of Pharmacy,
Shiraz University of Medical Sciences, Shiraz, Iran
123

Med Chem Res
1996). Thus, isatin is a biologically validated starting point
for the design and synthesis of chemical libraries directed
at these targets (Shuttleworth et al., 2000).
spectra were recorded on the Shimadzu-420 infrared
1
spectrophotometer. H-NMR and 13C NMR spectra were
recorded in DMSO-d6 on Brucker 300 MHz spectrometers
(chemical shifts are given in parts per million (PPM)).
Elemental analyses (C, H, N) were performed by the
Microanalytical Unit.
Various isatin derivatives have been reported to possess
cytotoxic activity (Matesic et al., 2008; Hossain et al.,
2008; Vine et al., 2007; Pervez et al., 2011). Increasing
knowledge of the biological activities of some simple isatin
derivatives guide many researchers to the development of
antitumor agents, capable of causing apoptosis, a pro-
grammed cell death involved in many physiological and
pathological processes.
General procedure for the synthesis of isatin schiff base
derivatives
A mixture of isatin (5 mmol) and aromatic or heterocyclic
primary amines (5 mmol) are refluxed in ethanol (50 ml) in
the presence of acetic acid as the catalyst for 0.5–2 h. After
completion of the reaction (monitored by TLC), the solvent
was removed under reduced pressure and the crude product
was washed with water and recrystallized from ethanol to
give pure products (Table 1).
Due to the privileged nature of isatin, libraries designed
and synthesized around the basic structure of this scaffold
should yield medicinally active compounds with high hit
rates at significantly reduced library size compared to large
classical libraries obtained from combinatorial chemistry
efforts based on non-privileged templates. Schiff bases and
Mannich bases of isatin were reported to possess as an
extension of this study and we have now focused our atten-
tion on Schiff bases of isatin especially heterocyclic imines.
In continuation to our study on biologically active isatin
derivatives (Azizian et al., 2002; Azizian et al., 2001), here,
we report the synthesis and spectroscopic characterization of
some hydrazones and Schiff bases of isatins in fairly good
yields along with their cytotoxicity assay. Furthermore,
synthesized compounds were subjected to molecular dock-
ing simulations to preliminary find out the potential molec-
ular target and at the same moment further support the
experimental cytotoxic tests. The efficiency of Autodock 4.2
program has been well demonstrated in several studies
(Sousa et al., 2006; Sellers et al., 2010). We performed our
docking study with Autodock 4.2 program. The target was
chosen as VEGFR-2 on the basis of its involvement in cell
proliferation/angiogenesis (Phosrithong and Ungwitayatorn,
2010) and previous reports on the inhibitory activity of
3-substituted-indolin-2-ones against VEGFR-2 (Sun et al.,
1999). VEGFR-2 is a cell surface receptor for vascular
endothelial growth factors. VEGFR-2 is a key pharmaco-
logical target in angiogenesis of tumor cells (Strawn et al.,
1996). Moreover, it has been established that VEGFR-2
provokes proliferation through activation of the extracellular
signal-regulated kinases pathway (Holmes et al., 2007).
Anti-tumor drug development targeting the VEGFR-2 sig-
naling pathway is now regarded as a prominent choice in the
clinical trials (Holmes et al., 2007).
Some synthesized compounds are compared with those
reported in earlier literature (3–10) (Silva et al., 2001).
Characteristic data for new isatin Schiff base are as
follows:
(Z)-3-(6-chloro-2-methylpyrimidin-4-ylimino) indolin-
2-one (11)
Yellow powder. Yield 83%, m.p. 193–194°C. IR (KBr,
cm-1): 3412, 3110, 2923, 1702, 1667, 1450, 1130 cm-1
.
1HNMR (300 MHz, DMSO-d6) d: 10.11 (s, 1H, OH), 7.82
(s, 1H, pyr), 6.95–7.58 (m, 4H, Ar), 2.44 (s, 3H, Me).
13CNMR (DMSO-d6) d:160–187 (pyr), 173.1(C=O),
115–152 (Ar). Anal. Calcd for C13H9ClN4O: C, 57.26; H,
3.33; N, 20.55. Found: C, 57.21; H, 3.42; N, 20.46.
(dihydrothiazol-2-ylimino) indolin-2-one (12)
Red powder. Yield 83%, m.p. 194–195°C. IR (KBr, cm-1):
3385, 3225, 2915, 1710, 1667, 1450 cm-1
.
1HNMR
(300 MHz, DMSO-d6) d: 10.34 (s, 1H, OH), 7.11–7.72 (m,
4H, Ar), 5.23 (d, 1H, heterocycl), 5.35 (s, 1H), 5.95 (d, 1H,
heterocycl). 13CNMR (DMSO-d6, 300 MHz) d: 168
(C=O), 120- 155 (Ar), 112.5, 127.1 (heterocycl), 48.4 (C–
S). Anal. Calcd for C11H9N3OS: C, 57.13; H, 3.92; N,
18.17; S, 13.86. Found: C, 57.23; H, 3.88; N, 18.25; S,
13.81.
(Z)-3-(5-chloropyridin-2-ylimino) indolin-2-one (13)
Experimental
Yield 83%, m.p. 197–199°C. IR (KBr, cm-1): 3385, 3225,
2915, 1710, 1667, 1450 cm-1
.
1HNMR (300 MHz,
Instruments
DMSO-d6) d: 10.34 (s, 1H, OH), 7.11–7.72 (m, 4H, Ar),
5.23 (d, 1H, heterocycl), 5.35 (s, 1H), 5.95 (d, 1H, het-
erocycl). 13CNMR (DMSO-d6, 300 MHz) d: 168 (C=O),
120–155 (Ar), 112.2, 127.7 (heterocycl), 48.2 (C–S). Anal.
Melting points were determined on the Electro-thermal
Melting Point apparatus and were uncorrected. Infrared
123

Med Chem Res
Table 1 Synthesized isatin Schiff base derivatives
Ar
N
O
N
H
Comp.
code
1
Ar
m.p
(°C)
Yield
(%)
84
Time
(min)
43
Compound name
170-172
3-(2-phenylhydrazono) indolin-2-one
NH
2
3
4
5
6
7
183-184
145-143
156-157
152-153
167-169
177-178
88
77
80
84
88
79
64
55
52
68
57
75
3-(2-(4-nitrophenyl)hydrazono) indolin-2-one
3-(phenylimino) indolin-2-one
O2N
NH
3-(p-tolylimino) indolin-2-one
CH3
H3C
3-(o-tolylimino) indolin-2-one
3-(4chlorophenylimino) indolin-2-one
3-(4-chlorophenylimino) indolin-2-one
Cl
OH
123

Med Chem Res
Table 1 continued
Comp.
Ar
m.p
(°C)
Yield
(%)
88
Time
(min)
75
Compound name
code
8
179-180
3-(4-nitrophenylimino) indolin-2-one
3-(4-chloro-2-methylphenylimino)indolin-2-one
3-(naphthalen-1-ylimino) indolin-2-one
NO2
Me
9
174-175
181-182
193-194
194-195
197-199
154-155
168-171
153-154
75
80
83
81
75
78
80
85
80
95
80
75
78
65
60
57
Cl
10
11
12
13
14
Cl
N
(Z)-3-(6-chloro-2-methylpyrimidin-4-ylimino)
indolin-2-one
N
Me
S
(dihydrothiazol-2-ylimino) indolin-2-one
(Z)-3-(5-chloropyridin-2-ylimino) indolin-2-one
(Z)-3-(1H-benzo[d]imidazol-2-ylimino) indolin-2-one
(Z)-3-(benzo[d]thiazol-2-ylimino) indolin-2-one
N
H
Cl
N
N
N
H
N
S
15
16
(3Z)-3-(5-methylisoxazol-2(5H)-ylimino) indolin-2-
one
N
O
123

Med Chem Res
Table 1 continued
Comp.
Ar
m.p
(°C)
Yield
(%)
82
Time
(min)
53
Compound name
code
Cl
(Z)-3-(4-chlorothiazol-2-ylimino) indolin-2-one
172-174
N
S
17
Me
(Z)-3-(4-methylisoxazol-3-ylimino) indolin-2-one
(Z)-3-(5-methylisoxazol-3-ylimino) indolin-2-one
179-181
193-195
80
86
70
70
18
19
O
N
O
N
Calcd for C11H9N3OS: C, 57.13; H, 3.92; N, 18.17; S,
13.86. Found: C, 57.23; H, 3.88; N, 18.25; S, 13.81.
13CNMR (DMSO-d6, 300 MHz) d:170.3 (C=O), 112-147
(Ar),142.2 (isoxazole), 105.8 (isoxazole), 17.1 (Me). Anal.
Calcd for C12H11N3O2: C, 62.87; H, 4.84; N, 18.33. Found:
C, 62.82; H, 4.79; N, 18.37.
(Z)-3-(1H-benzo[d]imidazol-2-ylimino) indolin-2-one
(14)
(Z)-3-(4-chlorothiazol-2-ylimino) indolin-2-one (17)
Yield 75%, m.p. 154–155°C. IR (KBr, cm-1): 3305, 3365,
2915, 1705, 1607, 1450 cm-1
.
1HNMR (300 MHz,
Brown powder. Yield 81%, m.p. 172–174°C. IR (KBr,
cm-1): 3300, 3137, 2929, 1708, 1642, 1435 cm-1. 1HNMR
(300 MHz, DMSO-d6) d: 10.64 (s, 1H, OH), 7.34–7.70 (m,
4H, Ar), 7.31 (s, 1H, chlorothiazole). 13CNMR (DMSO-d6,
300 MHz) d: 169.7 (C=O), 120-14752 (Ar),140.6 (C–Cl),
111.8 (chlorothiazole). Anal. Calcd for C11H6ClN3OS: C,
50.10; H, 2.29; N, 15.93; S, 12.16. Found: C, 50.16; H,
2.35; N, 15.88; S, 12.11.
DMSO-d6) d: 10.57 (s, 1H, OH), 7.11–7.72 (m, 8H, Ar),
5.15 (sbr, 1H, N–H). 13CNMR (DMSO-d6, 300 MHz) d:
167 (C=O), 117–148 (Ar), 136–157 (imidazole). Anal.
Calcd for C15H10N4O: C, 68.69; H, 3.84; N, 21.36. Found:
C, 68.62; H, 3.88; N, 21.28.
(Z)-3-(benzo[d]thiazol-2-ylimino) indolin-2-one (15)
Red powder. Yield 73%, m.p. 168–171°C. IR (KBr, cm-1):
(Z)-3-(4-methylisoxazol-3-ylimino) indolin-2-one (18)
3296, 3325, 2903, 1715, 1613, 1446 cm-1
.
1HNMR
(300 MHz, DMSO-d6) d: 10.39 (s, 1H, OH), 7.15–7.66 (m,
8H, Ar). 13CNMR (DMSO-d6, 300 MHz) d: 173 (C=O),
110–152 (Ar), 130–163 (thiazole). Anal. Calcd for
C15H9N3OS: C, 64.50; H, 3.25; N, 15.04; S, 11.48. Found:
C, 64.56; H, 3.29; N, 15.11; S, 11.42.
Yellow powder. Yield 83%, m.p. 179–181°C. IR (KBr,
cm-1): 3327, 3124, 2910, 1695, 1639, 1413 cm-1. 1HNMR
(300 MHz, DMSO-d6) d: 10.62 (s, 1H, OH), 7.30–7.68 (m,
4H, Ar), 7.27 (s, 1H, isoxazole). 13CNMR (DMSO-d6,
300 MHz) d: 174.2 (C=O), 118–145 (Ar), 150.5 (C–O),
148.9 (N–C–N), 11.5 (Me). Anal. Calcd for C12H9N3O2: C,
63.43; H, 3.99; N, 18.49. Found: C, 63.48; H, 3.92; N, 18.53.
(3Z)-3-(5-methylisoxazol-2(5H)-ylimino) indolin-2-
one (16)
(Z)-3-(5-methylisoxazol-3-ylimino) indolin-2-one (19)
Yield 85%, m.p. 153–154°C. IR (KBr cm-1): 3318, 3185,
2900, 1721, 1600, 1442 cm-1
.
1HNMR (300 MHz,
Yellow powder. Yield 83%, m.p. 193–195°C. IR (KBr,
cm-1): 3320, 3122, 2915, 1690, 1651, 1417 cm-1. 1HNMR
(300 MHz, DMSO-d6) d: 10.60 (s, 1H, OH), 7.32–7.65
DMSO-d6) d: 10.45 (s, 1H, OH), 7.12–7.73 (m, 4H, Ar),
6.15 (d, 1H, isoxazole), 4.45 (d, 1H, isoxazole), 2.27 (Me).
123

Med Chem Res
(m, 4H, Ar), 6.12 (s, 1H, isoxazole). 13CNMR (DMSO-d6,
300 MHz) d: 170.9 (C=O), 113–138 (Ar), 152.3 (C–O),
148.1 (N–C–N), 12.9 (Me). Anal. Calcd for C12H9N3O2: C,
63.43; H, 3.99; N, 18.49. Found: C, 63.48; H, 3.92; N, 18.53.
Bio-Rad microplate reader (Model 680). The percentage of
viability compared to control wells was calculated for each
concentration of the compound and IC16 and IC50 values
were calculated with the software CurveExpert version
1.34 for Windows. Each experiment was repeated 3–4
times. Data are presented as mean SD.
Cytotoxicity assay
RPMI 1640, fetal bovine serum (FBS), trypsin and phos-
phate buffered saline (PBS) were purchased from Biosera
(Ringmer, UK). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) was obtained from Sigma
(Saint Louis, MO, USA) and penicillin/streptomycin was
purchased from Invitrogen (San Diego, CA, USA). Doxo-
rubicin and dimethyl sulphoxide were obtained from EB-
EWE Pharma (Unterach, Austria) and Merck (Darmstadt,
Germany), respectively.
Docking study
AutoDock is a free to use program (The Scripps Research
Institute, La Jolla, CA, http://autodock.scripps.edu/).
Lamarckian Genetic Algorithm of the AutoDock 4.2 pro-
gram was used to perform the flexible-ligand docking studies
(Morris et al., 2009). Receptor X-ray crystal structure
obtained from the Brookhaven protein data bank was applied
in docking studies (2OH4; http://www.pdb.org/). Optimi-
zation and pre-processing of molecules under study was done
using AM1 method and AutoDock Tools 1.5.4 program
(ADT) (Morris et al., 2008). The AM1 optimization method
was performed using Polak-Ribiere (conjugate gradient)
algorithm with termination condition as RMS gradient of 0.1
HeLa (human cervical adenocarcinoma), LS-180
(human colon adenocarcinoma), and Raji (human B lym-
phoma) cells were obtained from the National Cell Bank of
Iran, Pasteur Institute, Tehran, Iran. All cell lines were
maintained in RPMI 1640 supplemented with 10% FBS,
and 100 units/ml penicillin-G and 100 lg/ml streptomycin.
Cells were grown in monolayer cultures, except for Raji
cells, which were grown in suspension, at 37°C in
humidified air containing 5% CO2.
˚
(Kcal/A mol) and 405 up to 495 maximum cycles based on
the ligand structures.
Treatment of all molecular structures was done carefully
in a uniform and consistent manner to avoid introduction of
bias. Ligands were submitted to 100 independent genetic
algorithms (GA) runs for search. For Lamarckian GA
method, a maximum number of 2,500,000 energy evalua-
tions; 27,000 maximum generations; a gene mutation rate
of 0.02; and a crossover rate of 0.8 were used. A grid of 60,
60, and 60 points in x-, y-, and z-direction, respectively,
Cell viability following exposure to synthetic com-
pounds was estimated by using the MTT reduction assay
(Mosmann, 1983; Miri et al., 2011). HeLa, LS-180, and
Raji cells were plated in 96-well flat-bottomed microplates
at densities of 25,000, 100,000, and 50,000 cells/ml,
respectively (100 ll per well). Control wells contained no
drugs and blank wells contained only growth medium for
background correction. After overnight incubation at 37°C,
half of the growth medium was removed and 50 ll of
medium supplemented with different concentrations of
synthetic compounds dissolved in DMSO were added in
quadruplicate. Plates with Raji cells were centrifuged
before this procedure. Maximum concentration of DMSO
in the wells was 0.5%. Cells were further incubated for
72 h, except for HeLa cells, which were incubated for 96 h.
At the end of the incubation time, the medium was
removed and MTT was added to each well at a final con-
centration of 0.5 mg/ml and plates were incubated for
another 4 h at 37°C. Then, formazan crystals were solu-
bilized in 200 ll DMSO. The optical density was measured
at 570 nm with background correction at 655 nm using a
˚
with grid spacing of 0.375 A was built centered on the
center of mass of the catalytic site of considered receptor.
Cluster analysis was performed on the docked results using
˚
a RMS tolerance of 2 A. The best docking result in each
case was considered to be the conformation with the lowest
binding energy. Hydrogen bindings between docked potent
agents and related macromolecule were analyzed using
Autodock tools program (ADT, Version 1.5.4).
Results and discussion
Some new isatin-Schiff base compounds were obtained by
condensation of different aromatic and heterocyclic amines
and isatin, under mild conditions in ethanolic solution (see
Fig. 1 Schematic
representations of synthesized
isatin schiff bases
Ar
N
O
EtOH , reflux
Ar-NH2
O
+
O
N
N
H
H
123

Med Chem Res
Table 2 Cytotoxic activity of some isatin Schiff base derivatives
Compound no.
HeLa cells
LS-180 cells
Raji cells
IC15 (lM)
IC30 (lM)
IC15 (lM)
IC30 (lM)
IC15 (lM)
IC30 (lM)
2
3.0 0.5a
18.8 9.6
12.3 5.4
15.4 5.2
15.8 2.5
17.4 3.3
21.6 5.8
43.2 25.6
3.2 0.6
12.2 3.1
[100
5.2 2.5
[100
21.8 8.5
[100
17 9.4
15.2 5.4
64.8 10.3
35.4 7.3
48.8 11.1
[100
[100
3
[100
5
52.6 19.0
80.2 46.3
55.9 17.9
[100
[100
[100
[100
6
[100
[100
88.1 0.7
[100
10
20.8 8.3
[100
[100
11
[100
[100
13
65.1 20.9
[100
[100
[100
[100
[100
14
[100
[100
15.6 6.1
57.3 28.5
30.1 6.8
2.6 0.3
[100
17
35.9 8.4
[100
3.9 1.15
[100
[100
[100
18
49.6 12.7
2.2 0.65
[100
6.3 1.9
[100
13 3.9
[100
4.9 0.5
Cisplatin
a
Values represent the average of four experiments SEM
Fig. 1). The isolated compounds were then characterized
by elemental analyses, IR and NMR spectroscopy. Spec-
troscopic properties were compared to those of related
compounds that previously isolated and described to cor-
relate their structural features. Electronic spectra of all the
complexes were carried out in aqueous, DMSO or CH3CN
solution, depending on their solubility.
structure of VEGFR-2 (Hevener et al., 2009). In this way,
validation of the method for prediction of the known
binding poses would be supported (Cosconati et al., 2010).
˚
If the RMSD is below 2 A, it is generally considered a
successful prediction (Vyas et al., 2008). The outputs of
validation studies for different targets under study are
shown in Table 3.
The in vitro cytotoxic activities for ten synthesized
isatin Schiff base derivatives are shown in Table 2.
All the compounds under study showed superior cytotoxic
activity on the HeLa cells. Compound 2 showed higher
cytotoxicity in all of the tested cells. This indicated that a-
rylhydrazone indolin-2-one scaffold bearing electron with-
drawing/hydrogen acceptor groups is a favorable isatin
structure for designing cytotoxic agents. This rational may
also be established for arylimino indoline-2-one compounds
6, 13, and 17 in HeLa cell line. These compounds possess
electron withdrawing chlorine atom on their aryl rings.
However, the effect is more pronounced for compound 17
which contains five-member heterocyclic chlorothiazol ring
bearinganelectronegativechlorineatomatitsfourthposition.
It seemed that five-member heterocyclic rings may also
promote cytotoxic activity on HeLa cells in arylimino
indoline-2-ones since compounds 5 and 10 having electron
donating groups show higher cytotoxic activity than com-
pounds 6 and 13 which possess electron withdrawing
groups but not five-member rings bearing two hetero
atoms. However, extended cytotoxicity assays on diverse
sets of isatin molecules need to be performed to establish
more rational structure activity relationships.
3-(arylimino) indolin-2-one compounds (Table 1) were
all docked into the active site of selected receptor. Docking
results are shown in Table 4. Top ranked binding energies
(kcal/mol) in AutoDock dlg output file were considered as
response in each run.
Docking results were supported almost by high cluster
populations. This could be expected since literature evi-
dence implied that docking studies with compounds bear-
ing less active torsions can significantly promote the
docking success rates due to the limited conformational
degrees of freedom (Erickson et al., 2004).
Possible key hydrogen bonds between synthesized
docked compounds and VEGFR-2 active site were estimated
and gathered in Table 5. A key hydrogen bonding has been
reported between carbamate NH and Cys917 in a crystallo-
graphic benzoimidazole-urea derivative (Hasegawa et al.,
2007). Compound 2 exhibited a similar electrostatic
Table 3 Docking validation results for cognate cytotoxic ligand
docked into VEGFR-2
PDB code
2OH4
No. of active torsions in ligand
No. of runs
6
100
No. of evaluations
2,500,000
85
Docking studies
No. of conformations in optimum cluster (Out of 100)
˚
RMSD (A) from reference structure
0.61
Docking validation step was performed by re-docking of
the co-crystallized conformation of cognate ligands into 3D
Estimated binding energy (kcal/mol)
-12.19
123

Med Chem Res
Table 4 Docking results of 3-(arylimino) indolin-2-ones docked into
cell proliferation dependent target (VEGFR-2, PDB ID: 2OH4)
Table 6 Different interactions between docked isatin compounds
and residues of VEGFR-2 active site
Comp. No. of
code
Estimated
conformations in binding
optimum cluster energya
Estimated Ligand
Ki (lM)
PDB Docked
code compound
Interacted residues
H-bond
efficiencyb
Non-bonded
(LE) (kcal/
mol.atom)
(out of 100)
(kcal/mol)
2OH4 2
Glu915, Cys917, Phe1045, Leu1033, Leu838,
Val914
Asn921,
Arg1049
2
67
100
100
100
100
100
100
100
100
100
-7.89
-7.19
-7.70
-7.58
-9.20
-7.56
-7.33
-7.85
-7.02
-7.66
1.63
5.33
2.28
2.77
0.18
2.85
4.23
1.75
7.16
2.44
0.375
0.423
0.428
0.421
0.438
0.398
0.407
0.393
0.413
0.451
3
3
Glu915, Cys917
Val914, Leu1033, Asp1044,
Phe1045
5
6
5
Glu915
Phe1045, Val914, Asp1044,
Phe916, Leu1033, Leu838
10
11
13
14
17
18
a
6
Glu915
Phe1045, Leu1033, Phe916,
Val914, Asp1044
10
11
13
14
17
18
Glu915
Phe1045, Leu1033, Ala864,
Val914, Cys1043
Glu915, Cys917
Glu915
Val864, Ala864, Val914,
phe1045, Leu1033
Results are the average of three replicates for each datum
Val914, Phe916, Leu1033,
Cys1043, Phe1045
b
The least optimum amount for LE is 0.3
Glu915
Asp1044, Cys1043, Leu1033,
Cys1043, Phe1045
interaction via its indolinone carbonyl oxygen with Cys917
NH (Table 5). However, the crystallographic data revealed a
shorter distance of H-bond in comparison to our results.
Asp1044 has been reported to make H-bond with urea oxy-
gen in a cognate benzoimidazole-urea ligand and this inter-
action could not be observed in our studies. However, the
spatial direction of compound 2 with regard to Asp1044
revealed a requirement of para-hydrogen acceptor substit-
uents in the phenyl ring of indolinone scaffold. Possible
interaction profiles for other docked compounds are also
provided in Table 6.
Glu915
Val914, Phe916, Leu1033,
Phe1045
Glu915
Phe1045, Asp1044, Cys1043,
Leu1033, Phe916, Val914
provide additional H-bonds with the active sites of the
receptor. This additional H-bond acceptor site would sup-
port two key H-bond interactions with Arg1049 and
Asn921 residues through oxygen atoms of a nitro sub-
stituent (Fig. 2a). Further, H-bond may also be considered
between indoline NH and Glu915 (Fig. 2a). Our experi-
mental cytotoxicity data were in good agreement with
docking binding energies since compound 2 was the most
potent compound in all the tested cell lines (Table 2).
Docking data revealed that 3-(naphthalen-1-ylimino)
indolin-2-one (compound 10) was the top-ranked docking
result in terms of its binding free energy to the receptor
(Table 4). The most characteristic feature of this compound
may be attributed to its naphthalene scaffold. This may
emphasize on the importance of hydrophobic interactions
in the active site of VEGFR-2 (Fig. 2b). In our study, the
geometric orientation of this ligand in the target active site
provided a key H-bond interaction with Glu915 while no
key H-bonds with other residues responsible for interac-
tions of compound 2 could be detected. As was mentioned
above, possible key hydrophobic interactions with active
site residues may play an important role in interaction
profile of compound 10 (Ala864, Val914, Phe1045,
Cys1043, and Leu1033; Fig. 2b).
Regarding the data obtained for binding energies with
docked target (Table 4), docking outputs supported that
higher binding energy of 3-(2-(4-nitrophenyl) hydrazono)
indolin-2-one (compound 2) can be interpreted by addi-
tional para-nitro substituent on phenyl ring which may
Table 5 Possible key hydrogen bonds between most potent synthe-
sized isatin derivative; (3-(2-(4-nitrophenyl) hydrazono) indolin-2-
one (compound 2) and VEGFR-2 target
PDB Ligand
code
Receptor Bond
Distance of similar
distance Distance of H-bond in
˚
(A)
˚
crystallographic file (A)
2OH4 Indolinone Cys917
C=Oa
3.77
(Urea oxygen-Asn163
NH)
NH
2.16
–
Indolinone Glu915
C=O
3.57
NH
–NO2
Asn921 2.12
NH
–
–
–NO2
Arg1049 2.20
NH
It is worth noting that compounds such as 14 (Table 4)
or 15 possessing additional fused phenyl ring to their
a
The atom participated in H-bonding is characterized by bold style
123

Med Chem Res
Fig. 2 Best docked pose of a
3-(2-(4-nitrophenyl) hydrazono)
indolin-2-one (compound 2) as
the most experimentally potent
cytotoxic agent and b 3-
(naphthalen-1-ylimino) indolin-
2-one (compound 10) as the
most potent docked agent in the
VEGFR-2 active site
heterocyclic moieties may also be noticeable examples in
this regard. Generally speaking, our results showed that
designing indoline-2-ones including extended hydrophobic
rings and appropriate H-bond acceptors on the third posi-
tion of indoline scaffold may provide potent VEGFR-2
inhibitors for further drug development strategies.
the least point of view; heterocycles bearing 3-(arylimino)
indolin-2-one or 3-(arylhydrazono) indolin-2-one moieties
may be regarded as promising candidates for further
molecular extensions to develop potent cytotoxic agents in
future.
Ligand efficiency indices
Conclusion
Another criterion which has recently absorbed much
attention in binding studies is the ligand efficiency (LE)
parameter. The concept of analyzing ligand binding in
terms of the free energy per heavy atom was first proposed
by Andrews (Andrews et al., 1984). Concept of the binding
energy per atom or binding efficiency of a ligand could be a
useful parameter in the selection of a lead compound,
considering the real potency of a compound and hence
optimizing fragments (Hopkins et al., 2004). Molecules
that achieve a given potency with fewer heavy atoms are by
definition more efficient. All of the synthesized compounds
could pass the generally accepted filter for ligand efficiency
as being efficient binding molecules themselves or as
fragments to develop efficient biological molecules (Abad-
Zapatero and Metz, 2005).
Versatile new isatin derivatives (3-(arylimino) indolin-2-
ones) bearing heterocyclic substituents were synthesized
and identified. All the tested compounds showed higher
cytotoxic effect on HeLa cancer cell lines compared to
LS180 and Raji cancer cells. Electron withdrawing groups
along with heterocyclic rings bearing more hetero atoms
seemed to be necessary factors in providing higher cyto-
toxic activities in HeLa cell lines. Docking simulation
technique with VEGFR-2 revealed that similar binding
patterns could be observed for all studied isatin compounds
and VEGFR-2 may be a potential molecular target for
further drug development strategies based on 3-substituted-
indoline-2-ones, confirming previous results. Biological
and computational results revealed that arylhydrazono in-
dolin-2-ones would be more potent cytotoxic agents than
arylimino indolin-2-ones. Based on the obtained data,
extended lipophilic rings bearing H-bond acceptors on the
third position of indoline scaffold provide potent VEGFR-2
inhibitors for further drug development strategies. Isoxaz-
ole/thiazole-based compounds demonstrated to be appro-
priate scaffolds extendable to highly efficient cytotoxic
agents due to their higher ligand efficiency indices in
binding to the receptor.
Putting LE concept in mind, one may realize that
compounds 18 and 10 (Table 4) which contain 5-methy-
lisoxazolyl and naphthyl moieties, respectively, are the
most efficient molecules among our evaluated scaffolds.
Compound 18 contains appropriate hydrogen bond donor
and acceptor sites and compound 10 possess extendable
hydrophobic skeleton to provide desirable hydrophobic
interactions with VEGFR-2 active residues (Fig. 2b). From
123

Med Chem Res
Acknowledgments Financial supports of this project by research
council of Shiraz University of Medical Sciences are acknowledged.
We gratefully acknowledge the financial support from the Medicinal
and Natural Products Chemistry Research Centre, Shiraz University
of Medical Sciences.
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DS, Olson AJ (2009) AutoDock4 and AutoDockTools4: auto-
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Chem 30(16):2785–2791
Mosmann T (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays.
J Immunol Methods 65(1–2):55–63
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