Design, synthesis, and QSAR study of novel 2-(2,3-dioxo-2,3- dihydro-1H-indol-1-yl)-N-phenylacetamide derivatives as cytotoxic agents

Article abstract of DOI:10.1007/s00044-010-9361-y

This study deals with the synthesis of novel 2- (2,3-dioxo-2,3-dihydro-1H- indol-1-yl)-N-phenylacetamide derivatives (6a-j) from isatin (3) and 5,7-dibromoisatin (4). All newly synthesized compounds were characterized using IR, 1H NMR, MS, and elemental analysis followed by evaluation of their cytotoxic activity by XTT assay on breast cancer cell line MCF-7 and non-cancer African green monkey cell line VERO. Correlation study for QSAR and in vitro assay was performed. The outcomes indicated that electron withdrawing substitutions at para position of phenyl ring and 5, 7 positions of isatin ring and increasing lipophilicity of the compound increased the cytotoxic activity. The 2-(5,7-dibromo-2,3-dioxo-2,3-dihydro-1H-indol-1-yl)- N-(4-nitrophenyl)acetamide (6b) was found to be the most active compound in the series and demonstrated higher selectivity toward MCF-7 cell line. The IC50 values were 1.96 and 1.90 lM for test compound (6b) and vinblastin (reference drug), respectively. This indicates compound (6b) may possess equipotent cytotoxic activity to vinblastine. The compound (6b) is particularly promising, since it could kill cancer cells 19-20 times more effectively than the non-cancer cells. This property of (6b) may enable us to effectively control tumors with low side effects. Hence, we propose that 2-(5,7-dibromo-2,3-dioxo-2,3-dihydro-1Hindol- 1-yl)-N-(4-nitrophenyl)acetamide may be used as lead for further development. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s00044-010-9361-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:615–625
DOI 10.1007/s00044-010-9361-y
ORIGINAL RESEARCH
Design, synthesis, and QSAR study of novel 2-(2,3-dioxo-2,3-
dihydro-1H-indol-1-yl)-N-phenylacetamide derivatives
as cytotoxic agents
Neha R. Modi
Maulik Suthar
Laxmanbhai J. Patel
•
Ravi J. Shah
•
Manish J. Patel
•
•
•
Bhupendrasinh F. Chauhan
Received: 11 October 2009 / Accepted: 3 May 2010 / Published online: 26 May 2010
Ó Springer Science+Business Media, LLC 2010
Abstract This study deals with the synthesis of novel 2-
2,3-dioxo-2,3-dihydro-1H-indol-1-yl)-N-phenylacetamide
derivatives (6a–j) from isatin (3) and 5,7-dibromoisatin (4).
could kill cancer cells 19–20 times more effectively than the
non-cancer cells. This property of (6b) may enable us to
effectively control tumors with low side effects. Hence,
we propose that 2-(5,7-dibromo-2,3-dioxo-2,3-dihydro-1H-
indol-1-yl)-N-(4-nitrophenyl)acetamide may be used as lead
for further development.
(
All newly synthesized compounds were characterized
1
using IR, H NMR, MS, and elemental analysis followed
by evaluation of their cytotoxic activity by XTT assay on
breast cancer cell line MCF-7 and non-cancer African green
monkey cell line VERO. Correlation study for QSAR and
in vitro assay was performed. The outcomes indicated that
electron withdrawing substitutions at para position of phenyl
ring and 5, 7 positions of isatin ring and increasing lipo-
philicity of the compound increased the cytotoxic activity.
The 2-(5,7-dibromo-2,3-dioxo-2,3-dihydro-1H-indol-1-yl)-
N-(4-nitrophenyl)acetamide (6b) was found to be the most
active compound in the series and demonstrated higher
selectivity toward MCF-7 cell line. The IC50 values were
Keywords Cytotoxic assay ꢀ Isatin ꢀ QSAR ꢀ XTT assay
Introduction
The isatin molecule (1H-indole-2,3-dione) is a versatile
moiety that displays diverse biological activities, including
anticancer activity (Pandeya et al., 1999; Cane et al., 2000;
Torres et al., 2004; Pandeya et al., 2005; Abadi et al.,
2006; Vine et al., 2007a, b; Uddin et al., 2007; Hung et al.,
2008; Gonz a´ lez et al., 2009; Sirisoma et al., 2009). The
synthetic flexibility of isatin led to synthesis of a variety of
substituted derivatives; however, the susceptibility of isatin
to attack by nucleophiles at C3 resulted in the generation of
a large number of 3-substituted isatins in particular
1
.96 and 1.90 lM for test compound (6b) and vinblastin
reference drug), respectively. This indicates compound
(
(
6b) may possess equipotent cytotoxic activity to vinblas-
tine. The compound (6b) is particularly promising, since it
(Bramson et al., 2001; Andreani et al., 2005; Chen et al.,
2005). Despite the significant amount of anticancer
research being devoted to 3-substituted indolin-2-ones, it
was not proven that 3-substituted isatins are better in
bioactivity than other 1H-indole-2,3-dione derivatives.
N-alkylated indoles have also been reported to exhibit
anticancer activity. For example, the indolyl amide
D-24851 has been found to block cell cycle progression in a
variety of malignant prostate, brain, breast, pancreas, and
colon cell lines (Bacher et al., 2001). Structurally related
other compounds have also been reported to activate casp-
ases in a cytochrome c-dependent manner and, therefore,
induce apoptosis in cancer cell lines but not in normal cells
N. R. Modi (&)
Department of Pharmaceutical Chemistry, Smt N. M. Padalia
Pharmacy College, Ahmedabad, Gujarat, India
e-mail: neha_modi2112@yahoo.co.in
R. J. Shah ꢀ B. F. Chauhan
Department of Pharmaceutical Chemistry, Kalol Institute of
Pharmacy, Kalol, Gujarat, India
M. J. Patel ꢀ M. Suthar ꢀ L. J. Patel
Department of Pharmaceutical Chemistry, S. K. Patel College of
Pharmaceutical Education and Research, Ganpat University,
Gujarat, India
123

6
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Med Chem Res (2011) 20:615–625
(
Nguyen and Wells, 2003). Furthermore, Liu et al. (2003)
Contribution of parameter PC to model is 49.34760%
Contribution of parameter BE to model is 50.65240%
identified a class of isatin O-acyl oximes that selectively
inhibited neuronal ubiquitin C-terminal hydrolase (UCH-
L1) in a H1299 lung cancer cell line, although very few
studies have described structural activity relationship
Model 2
(
SAR) attributable to modifications of benzene ring of
BA ¼ ½1:24081ðꢁ0:42753Þꢂ þ Ed½0:03967ðꢁ0:04083Þꢂ
þ VDW ½ꢃ0:06858ðꢁ0:03194Þꢂ
isatin. Moreover, Vine et al. (2007a, b) showed that the
introduction of electron-withdrawing groups to the benzene
ring of N-alkyl-substituted isatins is associated with
increased biological activity. Based on this background, the
current synthetic scheme was designed, synthesized, and
screened for their cytotoxic activity.
2
n = 22, r = 0.80813, r = 0.64580, variance = 0.02919,
2
std = 0.17084, F = 20.73510, FIT = 159.5000, q =
0.51191, Spress = 0.20293, SDEP = 0.19088
Contribution of parameter Ed to model is 25.7219%
Contribution of parameter VDW to model is 74.2781%
where BA = LogIC , is the molar logarithmic concentra-
5
0
tion of the drug leading to 50% cell death, BE = bending
energy, PC = partition coefficient (Hansch and Leo, 1995),
Ed = dipole–dipole energy, and VDW = van der waals
force. Both models are statistically significant. There is only
a slight difference in regression coefficient. Model 1 was
selected as the best model because, on the basis of the par-
tition coefficient, the drug–receptor interaction and drug
kinetics can be better explained. Ed and VDW both show
electronic property, which is why they should not be used in
one model. The calculated and predicted (LOO) activities of
the compounds by the above model are shown in Table 1,
and the variables used in the selected model have no mutual
correlation.
QSAR analysis
In order to identify the effect of substitutions on cytotoxic
activity, QSAR study using a reported data set of 32 com-
pounds of N-substituted isatin derivatives (Vine et al., 2007a,
b; Matesic et al., 2008; Solomon et al., 2009) was performed,
and outcomes are presented in Tables 1 and 2. The molar
concentration was converted to logarithmic values for
undertaking the QSAR study. The structure of all the com-
pounds were built on workpspace of Chem 3D version 8.0.3
(
Cambridge Softcorporation, http://www.cambridgesoft.
com/software/ChemOffice/), and energy minimization of
the molecules was done using MM2 force field followed by
semi empirical PM3 method available in MOPAC module
until the RMS gradient value becomes smaller than 0.01 kcal/
mol. The most stable structure for each compound was gen-
erated and used for calculating various physio-chemical
descriptors like thermodynamic, steric, and electronic values
of descriptors. The 21 descriptors were calculated by
Chem3D version 8.0.3 considered as independent variable
and biological activity as dependent variable. The total data
set was divided into training (Table 1) and test (Table 2) sets.
The most effective model has approximately equal
contribution of PC and BE i.e., 49.35 and 50.65%,
respectively. Therefore, it was inferred that electronic (BE)
and lipophilic parameter (PC) have important roles in the
activity of the compounds of series. The above model is
validated by predicting the biological activities of the test
molecules, as indicated in Table 2. The plot of observed
versus predicted activities for the test compounds is rep-
resented in Fig. 1. From Table 2, it is evident that the
predicted activities of all the compounds in the test set are
in good agreement with their corresponding experimental
activities and optimal fit is obtained. The external pre-
dictability of the above QSAR model using the test set was
2
The quality of the model was assessed by cross-validated q in
the training set, and external validation was performed by
2
calculating predictive r (Pred_r ) from the test-set com-
2
pounds. Medicinal Chemistry Regression Machine, Biosoft
2
determined by Pred_r , which is 0.56688. Therefore, the
(
http://www.biosoft.com/w/downloads.html) (Sharma et al.,
008) software was used to generate QSAR model. Multiple
above results indicate that QSAR model for cytotoxic
activity generates 54.29 and 56.68% internal and external
model prediction, respectively.
2
linear regression analysis yields two statistically significant
QSAR models:
The term BE represents the energy associated with
deforming bond angles from their optimal values. The term
PC represents the lipophilicity. Its negative value in the
model indicates that cytotoxic activity increases as bending
energy and lipopholicity of the compound increase. Thus,
we conclude that, when substituents that bring about these
changes in the molecule are introduced, the biological
activity will increase. On the basis of QSAR study, the
proposed molecules were designed (6a–j) and synthesized.
Model 1
BA ¼ ½2:07992ðꢁ0:45753Þꢂ þ PC ½ꢃ0:13681ðꢁ0:07642Þꢂ
þ BE½ꢃ0:03512ðꢁ0:02604Þꢂ
2
n = 22, r = 0.81122, r = 0.65807, variance = 0.03176,
std = 0.17821,
2
q = 0.54294, Spress = 0.20605, SDEP = 0.19149
F = 18.28420,
FIT = 140.64800,
1
23

Med Chem Res (2011) 20:615–625
617
Table 1 Structure of N-substituted isatin derivatives (1–22) with observed and predicted (LOO) activities of the training-set compounds using
model 1 and physicochemical properties
O
R1
O
N
R2
R4
R3
(
1-22)
Comp.
no.
R
1
R
2
R
3
R
4
Observed
activity
PC
BE
Predicted
activity(LOO)
(LogIC50)
(
LogIC50
)
1
2
3
4
5
H
Br
H
Br
Br
I
H
1.313
1.327
1.242
1.221
1.058
2.554
2.354
3.074
3.017
2.475
11.897
12.270
11.874
12.674
19.450
1.257
1.334
1.237
1.242
1.139
Br
I
H
H
H
Br
Br
Br
H
Br
Br
H
H2C
6
7
Br
Br
H
H
Br
Br
H
2
C
O
0.873
0.840
3.981
4.292
18.862
18.581
0.872
0.839
CH
3
H C
2
8
9
Br
H
Br
0.767
4.791
18.726
0.752
H C
2
Br
Br
H
Br
H
0.816
0.758
4.011
4.211
20.362
21.239
0.827
0.808
H C
CH3
CH3
2
1
1
0
1
Br
H2C
O
Br
H
Br
0.731
5.005
18.904
0.740
H C
2
O N
2
1
1
1
2
3
4
Br
Br
Br
H
H
H
Br
Br
Br
0.705
0.669
0.688
5.155
5.415
5.175
19.067
19.061
19.476
0.704
0.673
0.676
H C
Cl
2
H C
2
Br
H C
2
I
1
1
5
6
Br
Br
H
H
Br
Br
0.835
0.709
4.035
4.261
19.717
22.421
0.808
0.661
H C
CF3
2
H
2
C
COOH
H C
3
O
O
1
1
7
8
Br
Br
H
H
Br
Br
0.529
0.935
6.118
4.917
20.317
13.442
0.548
0.943
H C
2
H C
2
1
9
Br
H
Br
0.770
6.321
12.670
0.831
H C
2
123

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Med Chem Res (2011) 20:615–625
Table 1 continued
Comp.
no.
R
1
R
2
R
3
R
4
Observed
activity
PC
BE
Predicted
activity(LOO)
(
LogIC50
)
(LogIC50
)
2
0
Br
H
Br
H C
0.627
5.484
20.007
0.629
2
2
2
1
2
Br
Br
H
H
Br
Br
H C
0.625
0.660
5.484
5.466
20.063
19.141
0.610
0.670
2
Br
H C
2
Br
PC partition coefficient and BE bending energy
Chemistry
Pharmacology
This synthetic strategy began with the synthesis of isatin
All the synthesized compounds were screened for their
cytotoxic activity by XTT assay (cell proliferation kit;
Roche Molecular Biochemicals, Mannheim, Germany)
(Skehan et al., 1990) on breast cancer cell line MCF-7, and
non-cancer African green monkey cell line VERO (Mar-
csek et al., 2007). MCF-7 and VERO cell cultures used in
the experiments were derived from National Centre for
Cell Science, Pune, India. Stock cells of these cell lines
were cultured in DMEM, supplemented with fetal calf
serum (FCS; 5%), gentamycin sulfate (0.004%), glucose
(0.57%), and NaHCO3 (0.12%), in a humidified atmo-
(
(
3) (Scheme 1) by the reaction of dry isonitrosoacetanilide
2) with concentrated sulfuric acid (Marvel and Hiers,
1
941). In the IR spectrum of isatin (3), the carbonyl
-
1
stretching frequency was observed at 1718 cm , whereas
-
the amide carbonyl stretching appeared at 1660 cm . In
1
the H NMR spectrum of compound (3), a singlet was
1
observed at d 8.00 ppm due to N–H proton. The aromatic
protons resonated in the range of d 7.52–7.83 ppm. The
synthesis of 5,7dibromoisatin (4) (Scheme 1) was carried
out using a reported method of Lindwall (Lindwall et al.,
1
931). In this reaction, isatin (3) reacts with bromine in
sphere with 5% CO at 37°C until confluence reached. The
2
methanol and yields 5,7dibromoisatin (4). The IR spectrum
of 5,7dibromoisatin (4) showed stretching frequencies for
carbonyl, amide carbonyl, and –C–Br groups at 1718,
cells were dissociated with 0.2% trypsin, 0.02% EDTA in
phosphate buffer saline solution. The stock cultures were
2
grown initially in 25 cm tissue culture flasks, then in
-
1
1
2
2
1
665, and 575 cm , respectively.The H NMR spectrum
of (4) exhibited a singlet which was observed at d
.00 ppm due to N–H proton. The aromatic protons reso-
75 cm , and finally in 150 cm tissue culture flask, and all
the cytotoxicity experiments were carried out in 96-well
microtiter plates. Vinblastine was used as a reference
standard for cytotoxic activity. The test compounds were
prepared with different concentrations using DMSO, and
IC50 valueswere determined. IC50 is a drug concentration
causing a 50% inhibition of cell proliferation.
8
nated in the range of d 7.86–7.95 ppm.
Final products (6a–j) were synthesized (Torres et al.,
2
004) by reactions of isatin (3) and 5,7dibromoisatin (4)
with appropriate 2-chloro-N-phenylacetamide derivatives
5a–e) (Farag et al., 2008) in the presence of K CO , KI,
(
2
3
and DMF as a solvent at temperature of 808C (Scheme 2).
The yields of synthesized products are presented in
Table 3. We hereby report the synthesis of novel 2-(2,3-
dioxo-2,3-dihydro-1H-indol-1-yl)-N-phenylacetamide
derivatives and their cytotoxic potentials.
Results and discussion
The best model 1 predicts that increasing the PC and BE
would increase the biological activity of the compound.
1
23

Med Chem Res (2011) 20:615–625
619
Table 2 Structure of N-substituted isatin derivatives (23–32) with observed and QSAR derived predicted activities of the test-set compounds
using model 1 and physicochemical properties
R1
N
O
R
1
N
CH
3
R2
R3
S
R
2
O
O
N
N
R3
R5
R5
R4
R4
(23-28)
(29-32)
Comp.
no.
R
1
R
2
R
3
R
4
R
5
Observed
activity
PC
BE
Predicted
activity
(
LogIC50
)
(LogIC50
)
2
3
H
Br
H
Br
0.365
5.466
22.345
0.547
H
2
C
CH
3
2
2
4
5
H
H
Br
Br
H
H
Br
Br
0.635
0.887
4.211
4.182
20.925
18.906
0.769
0.844
2
H C
NO2
H C
2
2
2
6
7
H
H
Br
H
H
H
Br
H
0.544
1.375
5.466
3.670
21.489
16.864
0.577
0.985
2
H C
H2C
N
2
2
8
9
Br
H
H
H
H
H
H
H
CH3
CH3
1.305
1.163
3.035
3.632
18.854
18.888
1.002
0.920
H C
N
2
H C NH
2
O
NH
3
0
Cl
H
H
H
H C NH
1.332
2.766
18.095
1.065
2
O
NH
3
3
1
2
Br
Br
H
H
H
H
H
H
CH3
CH3
1.254
1.080
3.777
3.371
17.113
17.530
0.962
1.003
H C
N
2
H C NH
2
O
NH
PC partition coefficient and BE bending energy
123

6
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Med Chem Res (2011) 20:615–625
1
.2
1
lipophilicity of the compound increased the activity as pre-
dicted by QSAR study. Graphs of predicted versus observed
activities of the synthesized compounds (6a–e) were plotted
y = 0.4751x + 0.3953
2
R = 0.9048
0
0
0
0
.8
.6
.4
.2
0
(
Fig. 3). The most active compound in the synthetic series
was compound 2-(5,7-dibromo-2,3-dioxo-2,3-dihydro-1H-
indol-1-yl)-N-(4-nitrophenyl)acetamide (6b). The IC values
50
were 1.96 and 1.90 lM for test compound (6b) and
vinblastine, respectively. Hence, the compound (6b) may be
considered almost equipotent to that of vinblastine.
Test
Linear (Test)
0
0.5
1
1.5
Observed Activities
Conclusion
Fig. 1 Observed versus predicted activities according to model 1
In conclusion, a series of 2-(2,3-dioxo-2,3-dihydro-1H-in-
dol-1-yl)-N-phenylacetamide (6a–j) was synthesized and
screened for their in vitro cytotoxic activity. Result of
bioassay indicated that the compound (6b) possesses
equivalent cytotoxic activity to standard drug and the
remaining compounds showed good-to-moderate activity.
The compound (6b) is particularly promising, since it could
kill cancer cells 19–20 times more effectively than the non-
cancer cells. This property of (6b) may enable us to effec-
tively control tumors with low side effects. Furthermore, on
the basis of quantitative structure–activity relationship
study, it can be concluded that the presence of electron
withdrawing group substitutions at para position of phenyl
ring and 5,7 positions of isatin ring and increasing lipo-
philicity of the compound increase the cytotoxic activity.
Therefore, 2-(5,7-dibromo-2,3-dioxo-2,3-dihydro-1H-in-
dol-1-yl)-N-(4-nitrophenyl)acetamide may be further eval-
uated in other cell lines and in vivo animal models in the
line of further development, and can serve as a prototype
molecule of new class of anti-breast cancer agents.
Thus, we conclude that, when substituents that bring about
these changes in the molecule are introduced, the biologi-
cal activity will increase. On the basis of model 1, a series
of ten compounds (6a–j) were designed with the objective
of finding higher potent molecules than the existing N-
substituted isatin derivatives (Tables 1 and 2). The inde-
pendent variables were calculated and put in model 1 to
obtain predicted biological activities of newly synthesized
compounds (6a–j). The predicted activities of the newly
designed series of compounds are shown in Table 4. From
Tables 1, 2, and 4, it is observed that the introduction of
amide functional group at N-1 position into exiting
N-substituted isatin derivatives increases the PC and BE.
The predicted activities of the newly designed series of
compounds (6a–j) showed that they have predicted activ-
ities of IC50 = 1.59–3.65 lM whereas, the reported series
of N-substituted isatin derivatives have the highest IC50
value of 23.71 lM.
Based on in vitro cytotoxic assay, graphs of Log conc.
(
lM) vs. % inhibition of different compounds (6a–e) and
vinblastine were plotted (Fig. 2). IC50 values on MCF-7
and VERO cell lines for the in vitro cytotoxic activity of
the compounds (6a–e) and the standard are presented in
Table 4. Active compounds (6f–j) (with greater than 50%
survival) after an exposure time of 72 h were not further
evaluated for finding IC50 values. Based on in vitro results,
electron withdrawing substitutions at para position of
phenyl ring and 5,7 positions of isatin ring and increasing
Experimental protocol
Chemistry
Melting points of the synthesized compounds were
determined in open capillaries using Veego Melting Point
O
H
O
NH2
N
Br
O
O
a
b
c
N
HON
H
N
H
H
Br
(
1)
(
2)
(3)
(4)
Scheme 1 Synthesis of isatin (3) and 5,7dibromoisatin (4). Reagents and condition: a chloral hydrate, sodium sulfate, concentrated hydrochloric
acid, and hydroxylamine hydrochloride 40–45 min b concentrated sulfuric acid, 50–70°C and c ethanol, bromine, 70–75°C
1
23

Med Chem Res (2011) 20:615–625
621
Scheme 2 Schematic
O
representation for the synthesis
of novel 2-(2,3-dioxo-2,3-
dihydro-1H-indol-1-yl)-
R1
O
O
Nphenylacetamide derivatives
R1
(
6a–j). Reagents and condition:
a K CO , KI, anhydrous DMF,
stirred at 80°C for 5–24 h
N
a
O
2
3
O
R2
N
Cl
H
HN
R2
R3
O
NH
(
6a-j)
(
3) or (4)
R'
R4
R
(
5a-e)
Apparatus (Janki impex private limited, Ahmedabad,
India) and are uncorrected. Infrared spectra were recorded
using KBr pellets on SHIMADZU-FT-IR 8400S instru-
ment. Mass spectra were recorded on Perkin–Elmer LC–
General procedure for the preparation of compounds
Preparation of isatin (3) and 5,7dibromo isatin (4) It was
done according to the already reported procedure, men-
tioned in Vine et al. 2007a, b.
MS PE Sciex API/65 Spectrophotometer (Perkin–Elmer
1
India) Pvt. Ltd., Mumbai, India). The H NMR spectra
(
was recorded on Brucker Avance-300 (300 MHz) model
General procedure for preparation of 2-chloro-N-
phenylacetamide derivatives (5a–e)
in CDCl and DMSO as solvent and TMSi as internal
3
standard with 1H resonant frequency of 300 MHz. The
chemical shifts were measured in d ppm downfield from
internal standard TMSi at d = 0. Purity of the synthe-
sized compounds was checked by thin layer chromatog-
raphy (TLC). The TLC was performed on alumina silica
gel 60 F254 (Merck). The mobile phase was ethyl acetate
and n-hexane (1:1), and detection was made using UV
light and iodine spotting. The resulting compounds were
purified by column chromatography. For column chro-
matography, Merck silica gel (0.040–0.063 mm) was
used. The elemental analyses were done on Elementar
Vario EL III Carlo Erba 1108 and were in well accor-
dance with the structures assigned to the compounds. All
the compounds gave C, H, and N analyses within ± 0.5%
of the theoretical values. Synthetic grade chemicals pro-
cured from SD fine Chemicals, Baroda, India, were used
for the synthesis of the target compounds, only after
purification. All the starting materials were prepared
according to the established procedures with some minor
modifications. General synthetic procedures used for the
preparation of the target compounds (6a–j) are as
follows:
Aromatic amines (like aniline, p-nitroaniline, p-toluedine,
o-nitroaniline, and o-toluedine (1 g)) were dissolved in a
mixture of 25 ml of glacial acetic acid and 25 ml of satu-
rated solution of sodium acetate. To this chloroacety
chloride (1 ml, 8.8 mmol) was added dropwise to avoid the
vigorous reaction. After half an hour, product (5a–e) was
filtered. The product was washed with 50% acetic acid and
finally with water. The product was crystallized from
methanol.
General procedure for preparation of final product (6a–j)
The isatin (3) or 5,7-dibromoisatin isatin (4) (1 g) (47) was
taken up in anhydrous DMF and ice cooled with stirring.
Solid K CO (1 g, 7.2 mmol) was added in one portion,
2 3
and the dark colored suspension was raised to room tem-
perature and stirred for a further 1 h. The appropriate 2-
chloro-N-phenylacetamide derivatives (5a–e) (1 g) and KI
(0.5 g, 6 mmol) were added, and the reaction mixture was
stirred at 80°C for 5–24 h, until the reaction was over
which was confirmed using TLC. The reaction mixture was
123

6
22
Med Chem Res (2011) 20:615–625
Table 3 Physical and analytical data of the novel 2-(2,3-dioxo-2,3-dihydro-1H-indol-1-yl)-N-phenylacetamide derivatives
O
R1
O
N
O
R2
HN
R3
R4
a
b
Yield (%)
c
Entry
Product
R
1
R
2
R
3
R
4
m.p. (°C)
Mol. formula/mol. wt.
Elem. analysis (cal./found)
C
H
N
1
2
3
4
5
6
7
8
9
6a
6b
6c
6d
6e
6f
Br
Br
Br
Br
Br
H
Br
Br
Br
Br
Br
H
H
H
67.85
61.05
59.00
54.00
59.87
62.35
56.87
57.60
51.64
47.90
230–234
212–218
225–228
234–238
214–218
215–219
222–225
225–228
215–221
222–226
b
C
16
H
10Br
2
N
2
O
3
3
3
43.87
43.92
39.78
39.75
45.16
45.20
39.78
39.74
45.16
45.18
68.56
68.60
59.08
59.12
69.38
69.35
59.08
59.06
69.38
69.33
2.30
2.25
1.88
1.83
2.68
2.73
1.88
1.92
2.68
2.72
4.32
4.28
3.41
3.45
4.79
4.83
3.41
3.38
4.79
4.83
6.39
4
38
6.43
8.70
8.74
6.20
6.18
8.70
8.73
6.20
6.18
9.99
9.95
12.92
12.98
9.52
9.55
12.92
12.88
9.52
9.55
NO
2
H
16 9 2 3 5
C H Br N O
4
83
CH
H
3
H
17 2 2
C H12Br N O
4
52
NO
2
16 9 2 3 5
C H Br N O
4
83
H
CH
H
3
17 2 2
C H12Br N O
4
52
H
C
16
H
12
N
11
N
14
N
11
N
14
N
2
3
2
3
2
O
O
O
O
O
3
2
80
6g
6h
6i
H
H
NO
2
H
C
16
H
5
3
5
3
3
25
H
H
CH
H
3
H
17
C H
2
94
H
H
NO
2
16
C H
3
25
1
0
6j
H
H
H
CH
3
17
C H
2
94
a
1
c
Products were characterized by IR, H NMR, MS, and elemental analyses, isolated yields, melting points are uncorrected
poured into HCl (50 ml) and extracted with ethyl acetate
2-(5,7-dibromo-2,3-dioxo-2,3-dihydro-1H-indol-1-yl)-N-
(4-nitrophenyl)acetamide (6b) Light red crystals from
(
50 ml). The ethyl acetate layer was washed with brine and
-
1
dried over MgSO . The solvent was removed under vac-
4
DMF: Yield 61.05%; m.p. 212–2188C; IR (KBr, t cm ):
uum to yield products (6a–j).
1730 ([C=O of isatin), 1655 (–NH–CO), 558 (–C–Br),
1
1355 (NO2 of phenyl); H NMR (300 MHz, d ppm,
2
-(5,7-dibromo-2,3-dioxo-2,3-dihydro-1H-indol-1-yl)-N-
phenylacetamide (6a) Colorless crystals from DMF:
DMSO-d ): 3.90–3.95 (m, 2H, H C–CO), 8.00 (s, H, NH),
6 2
7.20–7.85 (m, 6H, Ar–H) ppm; and MS: m/z 484 (M ? 1),
485 (M ? 2), 487 (M ? 4).
-
1
Yield 67.85%; m.p. 230–2348C; IR (KBr, t cm ): 1726
1
([C=O of isatin), 1650 (–NH–CO), 552 (–C–Br);
H
NMR (300 MHz, d ppm, DMSO-d ): 3.85–3.95 (m, 2H,
H C–CO), 8.00 (s, H, NH), 7.00–7.90 (m, 7H, Ar–H)
2
2-(5,7-dibromo-2,3-dioxo-2,3-dihydro-1H-indol-1-yl)-N-
(4-methylphenyl)acetamide (6c) Lightbrowncrystalsfrom
6
-1
ppm; and MS: m/z 439 (M ? 1), 440 (M ? 2), 442
DMF: Yield 59.00%; m.p. 225–2288C; IR (KBr, t cm ):
1734 ([C=O of isatin), 1640 (–NH–CO), 550 (–C–Br);
(
M ? 4).
1
23

Med Chem Res (2011) 20:615–625
623
Table 4 Predicted and observed activities of the synthesized compounds (6a–j) with physicochemical properties
Comp. no. PC
BE
Predicted activity Log Predicted activity IC50 Observed activity MCF-7 IC50 Observed activity VERO IC50
a
IC50
(lM)
(lM)
(lM)
6
6
6
6
6
6
6
6
6
6
a
b
c
d
e
f
6.128 20.417 0.538
8.130 22.437 0.202
7.258 21.318 0.356
7.568 21.215 0.319
6.538 20.851 0.468
6.115 20.217 0.547
6.121 20.316 0.542
5.998 20.225 0.562
6.105 19.998 0.556
6.155 20.338 0.537
3.45
1.59
2.27
2.08
2.94
3.52
3.48
3.65
3.60
3.44
3.91
1.96
2.43
2.04
3.85
–
28.06
38.09
24.98
25.99
29.87
–
g
h
i
–
–
–
–
–
–
j
–
–
Vinblastine
1.90
27.06
MCF-7: human mammary gland adenocarcinoma (non-metastatic) cell line, VERO: African green monkey cell line (non-cancer cells)
‘–’’: compounds (with greater than 50% survival) after an exposure time of 72 h were not further evaluated for finding IC50 values
‘
PC partition coefficient and BE bending energy
a
Values are the mean of triplicates of at least two independent experiments
2
-(5,7-dibromo-2,3-dioxo-2,3-dihydro-1H-indol-1-yl)-N-
2-nitrophenyl)acetamide (6d) Colorless crystals from
80
70
60
50
40
30
20
10
0
(
6a
6b
-
1
DMF: Yield 54.00%; m.p. 234–2388C; IR (KBr, t cm ):
1730 ([C=O of isatin), 1650 (–NH–CO), 555 (–C–Br),
1360 (NO2 of phenyl); H NMR (300 MHz, d ppm,
DMSO-d ): 3.85–3.95 (m, 2H, H C–CO), 7.90 (s, H, NH),
6
6
6
c
d
e
1
6
2
Vinblastin
7.00–7.90 (m, 6H, Ar–H) ppm; and MS: m/z 484 (M ? 1),
4
85 (M ? 2), 487 (M ? 4).
-3
-2
-1
0
1
2
3
Log Conc. [µM]
2-(5,7-dibromo-2,3-dioxo-2,3-dihydro-1H-indol-1-yl)-N-
(2-methylphenyl)acetamide (6e) Colorless crystals from
Fig. 2 Graph of Log conc. (lM) versus % inhibition of different
compounds (6a–e) and vinblastine
-
1
DMF: Yield 59.87%; m.p. 214–2188C; IR (KBr, t cm ):
1
1
734 ([C=O of isatin), 1640 (–NH–CO), 550 (–C–Br); H
NMR (300 MHz, d ppm, DMSO-d ): 3.90–3.95 (m, 2H,
4
6
H C–CO), 8.00 (s, H, NH), 2.35–2.40 (m. 3H, Ar–CH ),
3
2
3
2
1
0
.5
3
7
.00–7.90 (m, 6H, Ar–H) ppm; and MS: m/z 453 (M ? 1),
54 (M ? 2), 456 (M ? 4).
4
.5
2
y = 0.7183x + 0.4276
2
2-(2,3-dioxo-2,3-dihydro-1H-indol-1-yl)-N-phenylacetamide
6f) Light red crystals from DMF: Yield 62.35%; m.p.
-1
R = 0.9002
.5
1
(
Observed
2
1
15–2198C; IR (KBr, t cm ): 1726 ([C=O of isatin),
Linear (Observed)
1
.5
0
650 (–NH–CO); H NMR (300 MHz, d ppm, DMSO-d ):
6
3.85–3.95 (m, 2H, H C–CO), 8.00 (s, H, NH), 7.00–7.70
2
0
1
2
3
4
5
(m, 9H, Ar–H) ppm; and MS: m/z 281 (M ? 1).
Observed activities
2-(2,3-dioxo-2,3-dihydro-1H-indol-1-yl)-N-(4-nitrophenyl)
acetamide (6g) Colorless crystals from DMF: Yield
Fig. 3 Graph of observed versus predicted activities of synthesized
compounds (6a–e)
-
1
5
6.87%; m.p. 222–225 8C; IR (KBr, t cm ): 1730 ([C=O
1
1
H NMR (300 MHz, d ppm, DMSO-d ): 3.90–3.95 (m, 2H,
of isatin), 1655 (–NH–CO), 1355 (NO
NMR (300 MHz, d ppm, DMSO-d ): 3.90–3.95 (m, 2H,
C–CO), 8.00 (s, H, NH), 7.20–8.20 (m, 8H, Ar–H) ppm;
and MS: m/z 326 (M ? 1).
2
of phenyl); H
6
H C–CO), 8.00 (s, H, NH), 2.35–2.40 (m. 3H, Ar–CH ),
2
6
3
7
.00–7.90 (m, 6H, Ar–H) ppm; MS: m/z 453 (M ? 1), 454
H
2
(
M ? 2), 456 (M ? 4).
123

6
24
Med Chem Res (2011) 20:615–625
2-(2,3-dioxo-2,3-dihydro-1H-indol-1-yl)-N-(4-methylphenyl)
acetamide (6h) Colorless crystals from DMF: Yield
VERO cell line was used to find out cytotoxic effects of
synthesized compound on non-cancer cells.
-
1
5
7.60%; m.p. 225–2288C; IR (KBr, t cm ): 1734 ([C=O
1
Acknowledgments The authors are grateful to Cadila Pharmaceu-
tical Limited and Zydus Research Center, Ahmedabad, India for
1
providing assistance for H NMR, mass spectral, and elemental
analyses, and Ganpat University, Mehsana, India for providing
financial support.
of isatin), 1640 (–NH–CO); H NMR (300 MHz, d ppm,
DMSO-d ): 3.90–3.95 (m, 2H, H C–CO), 8.00 (s, H, NH),
6
2
2
.35–2.40 (m. 3H, Ar–CH ), 7.00–7.90 (m, 8H, Ar–H)
3
ppm; and MS: m/z 295 (M ? 1).
2-(2,3-dioxo-2,3-dihydro-1H-indol-1-yl)-N-(2-nitrophenyl)
acetamide (6i) Light brown crystals from DMF: Yield
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