Synthesis, biological evaluation, in silico docking, and virtual ADME studies of 2-[2-Oxo-3-(arylimino)indolin-1-yl]-N-arylacetamides as potent anti-breast cancer agents

Article abstract of DOI:10.1007/s00706-015-1566-9

A series of ten novel isatin analogs have been synthesized and screened for their in vitro anti-breast cancer activity against MCF-7 cell line using sulforhodamine-B assay method. All the tested compounds showed highly potent activity against MCF-7 cell line with especially four compounds exhibited demonstrative antiproliferative effects on MCF-7 breast cancer cell line compared to reference adramycin (doxorubicin) and GI ₅₀ <0.02 μM. Docking the synthesized compounds into the epidermal growth factor receptor, which is highly expressed in breast cancer, was employed to explore the possible interactions of these compounds with the receptor. Structure activity relationship as well as virtual ADME studies were carried out and a connection between activities, electronic and physicochemical properties of the target compounds was determined.

Full text of DOI:10.1007/s00706-015-1566-9

Monatsh Chem
DOI 10.1007/s00706-015-1566-9
ORIGINAL PAPER
Synthesis, biological evaluation, in silico docking, and virtual
ADME studies of 2-[2-Oxo-3-(arylimino)indolin-1-yl]-
N-arylacetamides as potent anti-breast cancer agents
Biplab Debnath1
Swastika Ganguly1
•
Received: 5 May 2015 / Accepted: 29 August 2015
Ó Springer-Verlag Wien 2015
Abstract A series of ten novel isatin analogs have been
synthesized and screened for their in vitro anti-breast
cancer activity against MCF-7 cell line using sulforho-
damine-B assay method. All the tested compounds showed
highly potent activity against MCF-7 cell line with espe-
cially four compounds exhibited demonstrative
antiproliferative effects on MCF-7 breast cancer cell line
compared to reference adramycin (doxorubicin) and
GI50 \0.02 lM. Docking the synthesized compounds into
the epidermal growth factor receptor, which is highly
expressed in breast cancer, was employed to explore the
possible interactions of these compounds with the receptor.
Structure activity relationship as well as virtual ADME
studies were carried out and a connection between activi-
ties, electronic and physicochemical properties of the target
compounds was determined.
Keywords Antitumor agents Á Bioorganic chemistry Á
Isatin Á Molecular modeling Á Tyrosine kinase
Introduction
Globally, cancer is one of the leading causes of death. The
death rate from various types of cancer continues to grow
worldwide with an estimation of 12 million deaths in 2030
[1, 2]. One in ten of all new types of cancer diagnosed
around the world each year is breast cancer and is the most
common cancer in women, both in developing and devel-
oped countries [3]. Estrogen receptor (ER)-negative breast
cancer accounts for approximately 30 % of all breast
cancer diagnosed [4–6]. Chemotherapy has been the most
frequently used treatment for breast cancer and other can-
cers till date. However, some normal proliferating cells are
also destroyed by this method of treatment [7]. Hence,
there is an intense worldwide search for identifying new
drugs that are more effective and safe for the prevention
and treatment of cancer.
R1
NH2
N
R1
R1
O
N
N
H
O
N
NH
R2
O
A survey of the literature in this area showed that many
heterocyclic scaffolds have been investigated as anti-breast
cancer agents [8]. Among them isatin is a useful structural
motif for the development of molecules of pharmaceutical
or biological interests. Isatin is an indole derivative widely
present in human and mammalian tissues. The synthetic
versatility of isatin derived at C-2, C-3, and N positions has
led to a wide variety of pharmacological responses
including anticancer, antibacterial, antiviral, anti-HIV,
anticholinesterase, antiinflammatory, antihypertensive,
antihypoxic, antiulcer, anticonvulsant, COX-2 and car-
boxylesterase inhibitor activities [9–15]. Among these
activities, anticancer activity studies on isatin derivatives
have been accelerated after the FDA approval of C-3
2
Cl
O
HN
R2
NH2
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-015-1566-9) contains supplementary
material, which is available to authorized users.
& Biplab Debnath
biplab.d86@gmail.com
1
Department of Pharmaceutical Sciences and Technology,
Birla Institute of Technology, Mesra, Ranchi 835215,
Jharkhand, India
123

B. Debnath, S. Ganguly
2-chloroacetylchloride (2) were reacted in the presence of
glacial acetic acid in ice cold condition leading to the
formation of chloroanilides 2a–2f [23]. Then, to the
appropriate Schiff bases of isatin 1a–1d in 8–10 cm3 of
anhydrous DMF, K2CO3 was added and stirred at room
temperature for 1 h. After completion of 1 h, the solution
turned red brown in color. Appropriate cholroanilides 2a–
2f and KI were then added to this solution drop wise and
heated at 60 °C for 5.5–9 h. The reaction was monitored by
TLC and after completion of the reaction gives the entitled
compounds 3a–3j [24–26].
N
HN
O
N
H
O
F
N
H
The IR (KBr) spectra of all the synthesized compounds
exhibited much related frequencies indicating the incidence
of amide structures for the title compounds. The IR spectra
of the synthesized compounds exhibited the acetamide
Fig. 1 Structure of sunitinib (SU11248)
derivative of isatin, sunitinib (SU11248) (Fig. 1). On the
other hand, a literature survey on anticancer activity studies
of N-alkylisatin derivatives reveals the importance of
N-substitution. In addition, structure activity relationship
(SAR) studies demonstrated that the introduction of an
aromatic ring with 1–3 carbon atom linkers at the N atom
enhanced the anticancer activity [16–18].
carbonyl stretching (C=O) band at 1731–1607 cm-1
.
Similarly, N–H stretching band of amide was seen between
3400 and 3300 cm-1. The C=N band in IR spectra of all
the compounds appeared at 1605–1550 cm-1 which is
1
similar as that of the ordinary C=N absorption. The H
NMR spectra of the title compounds 3a–3j were recorded
in DMSO-d6 solution which entirely concorded with the
predictable resonance signals in terms of chemical shifts
Thus, we describe herein the synthesis and anticancer
activity of a series of isatin analogs 3a–3j and evaluation of
their antitumor activity against breast cancer (MCF-7) cell
line [19].
1
and integrations. H NMR spectra of the title compounds
3a–3j showed a broad singlet of one proton assigned to NH
proton at d = 10.96–10.82 ppm. Depending on the nature
of the substituents and substitution patterns on the N-phe-
nyl ring, the aromatic protons of certain compounds 3a–3j
were observed in distinct chemical shifts with expected
splitting patterns as doublets, triplets, or multiplets inte-
grating more than one proton due to the close chemical
shifts ranging from 6.36 to 7.58 ppm. In the aliphatic
region, a broad singlet of two protons assigned to the
methylenic proton of N–CH2–CO at range 4.61–4.67 ppm
was observed for the compounds 3a–3j. A broad singlet of
three protons assigned to methoxy OCH3 at 3.71 and
3.72 ppm was observed for the substituents at the N -
phenyl ring of the compounds 3a and 3g, respectively. A
broad singlet of three protons assigned to methyl protons of
-CH3 at 1.99–2.28 ppm was observed for the substituents
at the N-phenyl ring of the compounds 3b, 3c, 3d, 3f, 3g,
3h, 3i, and 3j. The NMR data of the title compounds 3a–3j
are summarized in experimental sections. In 13C NMR
spectra, aromatic carbons were observed in the region of
112.88–161.28 ppm and aliphatic carbon was observed
(-CH3) in the region of 17.37–21.11 ppm and methoxy
(-OCH3) was observed in the region of 55.31–55.47 ppm.
The characteristics of carbon for C=O were observed in the
region of 164.81–164.00 ppm and -CH2 was observed
45.17–45.67 ppm. The structural confirmations of these
compounds were determined using ESI–MS. The physico-
chemical data are summarized in Table 1.
Substituted anilides have also been studied for their
cytotoxic activity and the results suggested that the activity
depends on the nature and the positions of the substituents
on the N-phenyl ring [20]. In this context, a group of N-
phenylisatin-1-acetamide derivatives bearing diverse sub-
stitutions with different electronic and hydrophobic natures
on the phenyl ring were designed and synthesized. Chem-
ical structures of the title compounds were confirmed by
IR, CHNS, 13C NMR, 1H NMR, and ESI–MS spectra. The
cytotoxic activity of the final compounds was screened
against MCF-7 cell line.
Results and discussion
Chemistry
A small library of ten isatin analogs 3a–3j was synthesized
following the reaction outlined in Scheme 1. The synthesis
of the title compounds was realized in three steps.
Firstly, 1,3-dihydroindol-2-one derivatives 1a–1d were
synthesized, following the method reported for the syn-
thesis of isatin [21, 22]. For this, isatin (1) and substituted
anilines were dissolved in warm ethanol in the presence of
1–2 drops of glacial acetic acid and refluxed for 2–3 h.
After standing for approximately 24 h at room temperature,
product was obtained. Next, the substituted anilines and
123

Synthesis, biological evaluation, in silico docking, and virtual ADME studies…
Scheme 1
R1
NH2
O
N
R1
R1
O
O
O
N
i
N
N
H
H
1
O
O
-
1a 1d
iii
N
NH
R2
R2
O
Cl
NH2
HN
R2
Cl
Cl
3a-3j
ii
2
-
2a 2f
R1
=
=
-H, 3-Cl, 4-Cl,
2,5-(CH3)2
2-CH 3-OCH 4-OCH
,
2,4-(CH3)2 2,5-(CH3)2
3
R2
,
,
,
-H,
3
3
(i) ethanol, 1-2 drops glacial acetic acid, reflux, 2-3 h; (ii) glacial acetic acid, 0-5 °C,
stirring, 1 h; (iii) DMF, K2CO3, KI, 60 °C, 5.5-9 h
Table 1 Reaction time, melting points, and yields of the title compounds 3a–3j
R1
N
O
R2
N
H2C
C NH
O
Comp
R1
R2
Reaction time/ha
M.p./°C
Lit. m.p./°C [26]
Yield/%
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
4-OCH3
2,5-(CH3)2
2-CH3
5.5
6
212–214
180–181
216–218
231–234
214–216
260–262
226–228
262–263
213–215
230–233
–
72
72
61
69
74
67
73
74
72
65
H
–
3-Cl
9
216–218
232–233
214–216
259–261
226–228
262–263
–
3-Cl
2,5-(CH3)2
H
6
4-Cl
8
4-Cl
2-CH3
7
4-Cl
3-OCH3
2,4-(CH3)2
2-CH3
5.5
7
4-Cl
2,5-(CH3)2
2,5-(CH3)2
7
3j
a
2,5-(CH3)2
6.5
–
Completion of the reaction was tested by the use of TLC
123

B. Debnath, S. Ganguly
Biological screening
In this work, anticancer activity of the newly synthesized
isatin analogs 3a–3j was evaluated against breast (MCF-7)
cancer cell line, using sulforhodamine-B (SRB) assay method
[27]. The GI50 concentration for each compound was calcu-
lated with reference to a control sample, which represents the
concentration that results in 50 % decrease of cell growth/
proliferation after 48 h incubation in the presence of the drug.
For each compound, GI50 was calculated from dose–response
curves that were generated with data obtained from two
independent experiments carried out each in triplicate values.
GI50, LC50, and TGI values were calculated in lM and pre-
sented in Table 2. The data of adramycin (doxrubiacin) were
includedasreferencestandard. Theresultantdatashowedthat,
among ten synthesized compounds 3a–3j, four compounds
exhibited demonstrative antiproliferative effects on MCF-7
breast cancer cell line (compounds 3b, 3c, 3e, and 3f showed
the best activities compared to reference adramycin (dox-
orubicin) and GI50 \0.02 uM). Rest of the compounds 3d, 3g,
and 3h also showed moderate activity compared to reference
standard adramycin (GI50 = 0.13 uM, GI50 = 0.03 uM, and
GI50 = 0.09 uM, respectively) but the compounds 3a, 3i, and
3j have showed less anticancer activity against MCF-7 cell
line. The results also indicated that the hydrophobic group
(-CH3) with 2nd phenyl ring substituted with 3-Cl or 4-Cl
showed greater growth inhibitory potency. The plot of per-
centage control growth versus drug concentration (lg/cm3)
shows the effective drug concentration on the MCF-7 human
breast cancer cell line in Fig. 2.
It was evident that incorporation of one or two
hydrophobic methyl group/groups in the phenyl ring
attached to the isatin moiety by the linker CH2CONH
Fig. 2 The plot of percentage control growth versus drug concen-
tration (lg/cm3) shows the effective drug concentration on the MCF-7
human breast cancer cell line
Table 2 Anti-breast cancer activity against newly synthesized com-
pounds 3a–3j in human breast cancer cell line (MCF-7)
Test comp.
Concentration/lM
resulted in very high activity comparable to the standard. It
was interesting to note that a combination of the chloro group
(substituted at the para position of the second phenyl ring)
along with one methyl group in the first phenyl ring showed a
similar pattern of activity. It was also observed that absence
of any substitution in the first phenyl ring with chloro group
substituted in the second phenyl ring showed activity com-
parable to standard, as in case of compound 3e. However,
when hydrophobicity of the total molecule was increased by
incorporation of two methyl groups in the first phenyl ring
and one chloro group at para position of the second phenyl
ring, activity decreased significantly. SAR studies suggest
that, an electron withdrawing halogen group is an essential
feature for activity. It was observed that a combination of an
electron withdrawing group with a hydrophobic electron
releasing methyl group also played an important part in
contribution to activity.
LC50
TGI
GI50
3a
3b
3c
[0.21
0.17
[0.21
0.09
[0.21
\0.02
\0.02
0.13
0.14
0.07
3d
3e
[0.19
0.16
[0.19
0.09
\0.02
\0.02
0.03
3f
0.15
0.08
3g
3h
3i
0.16
0.1
[0.19
[0.20
[0.19
0.08
0.18
0.09
[0.20
[0.19
0.02
[0.20
[0.19
\0.02
3j
ADR
GI50 value of the synthesized compounds are equal to standard
(Adrmycin). Therefore, significant antibreast cancer compounds are
indicated in bold
123

Synthesis, biological evaluation, in silico docking, and virtual ADME studies…
Docking study: binding mode analysis
binding of AQ4 in the active site of 1M17 with a docking
score of -8.74. Firstly, to validate the Glide software, we
modeled the interaction between AQ4 and PDB ID 1M17.
Superimposition of the experimental bound (co-crystal-
lized) conformation of AQ4 and that predicted by Glide is
shown in Fig. 3a. Glide successfully reproduced the
experimental binding conformations of AQ4 in the binding
pocket of 1M17 with an acceptable root-mean-square
The epidermal growth factor receptor (EGFR) is highly
expressed in many types of cancers, especially breast,
colon, and bladder cancers [28]. Targeting this receptor
represents a good strategy for the design of new anticancer
drugs [29]. Thus, molecular docking studies for all the
synthesized compounds 3a–3j into ATP-binding site of
EGFR–TKs were performed to investigate the ability of
these novel derivatives to inhibit these tumorogenic agents
and explore their binding mode in the active site of EGFR–
TKs. Considering the well-obtained in vitro results, a
profound docking study was carried out to consider the
possible binding mode of the highest active compound 3f
inside the active site of EGFR–TKs using Schrodinger
Suite software [30, 31]. In case of PDB ID 1M17 com-
plexed with the cocrystallized ligand erlotinib (AQ4), the
ligand erlotinib shows H-bond interactions with Met 769 as
depicted in Fig. 3a, b. The interactions with Leu 820, Leu
768, Leu 694, and Gly 772 are very important for stable
˚
˚
deviation (RMSD) of 1.737 A ([3A). Compound 3f with
the highest docking score in the active site of 1M17 is
visualized in its three-dimensional mode in Fig. 4a and the
residues involved in inter-atomic contact has been shown
in Fig. 4b. Analysis of the docking pose revealed that in
compound 3f the isatin scaffold is similarly oriented in the
binding site as the quinazoline moiety of erlotinib in the
active site of 1M17. Here in, the isatin moiety interacts
with multiple amino acid residues Met769, Leu820, Leu
764, Ala719, Lys721, Thr 766, Thr 830, and Gly722. The
compound also forms a strong H-bond interaction between
C=O group of the isatin ring and the C=O group present in
Fig. 4 a Molecular model of most active compound 3f in the protein
EGFR (protein data bank ID 1M17) (Glide XP Score -8.35);
b schematic (2D) representation of interactions of compound 3f in the
binding pocket of the protein EGFR
Fig. 3 a Redocked conformer of AQ4 (erlotinib) in the active site of
the protein EGFR (PDB ID 1M17); b 2D representation of the ligand
erlotinib (AQ4)
123

B. Debnath, S. Ganguly
5. Brain/blood partition coefficient (QPlogBB) (-3.0 to
Table 3 The docking score and E model score of the synthesized
compounds 3a–3j
1.2).
6. Percent human oral absorption (C80 % is
high, B25 % is poor).
Sl. no. Comp.
Docking score E model score
1
3a
3b
3c
3d
3e
3f
-5.67
-6.36
-7.29
-6.22
-6.19
-8.35
-6.05
-5.17
-4.96
-5.38
-63.08
-59.75
-54.40
-64.70
-59.52
-52.05
-56.77
-60.05
-58.34
-64.24
-51.60
All the structures showed significant values for the
properties analyzed and exhibited drug-like characteristics
based on Lipinski’s rule of 5 [32–34]. The ADME values
of newly designed compounds are given in Table 4. The
first three properties are based on Lipinski’s rule of five,
molecular weight (MW) less than 650, partition coeffi-
cient between octanol and water (logPo/w) between -2
and 6.5 and solubility (QPlogS) greater than -7. Brain/
blood partition coefficient (QPlogBB) parameter indicated
about the ability of the drug to pass through the blood–
brain barrier which is mandatory for inhibition of 1M17.
The QPPMDCK predicted apparent MDCK cell perme-
ability in nm/s. MDCK cells are considered to be a good
mimic for the blood–brain barrier. The higher the value of
MDCK cell, the higher is the cell permeability. All the
designed compounds showed ADME properties in
acceptable range.
2
3
4
5
6
7
3g
3h
3i
8
9
10
11
3j
Reference (adramycin) -8.45
˚
residue Met 793 (COIsatin - C=O Met793 = 1. 889 A). Thus,
it is evident that these interactions and hydrogen bond may
be responsible for the high docking score of compound 3f
(-8.35) which was found to be comparable than that of the
co-crystallized ligand AQ4 (-8.74). The docking scores of
the synthesized compounds are given in Table 3.
Virtual ADME study
Conclusion
We have analyzed 16 physical descriptors and pharma-
ceutically significant properties of isatin analogs using
Qikprop v3.0 tool of Schrodinger software, among which
major descriptors reported here are required for predicting
the drug-like properties of molecules. These properties are
A series of new isatin analogs have been synthesized and
characterized by IR, 13C NMR, 1H NMR, and mass
spectroscopy. All the newly synthesized compounds were
evaluated for their potential as antitumor lead compounds
in vitro on human breast cancer cell line MCF-7. Most
of the tested compounds showed potent antitumor
activity. Molecular docking studies for the synthesized
compounds were performed and molecular interactions
were explored. From the modeling studies, it can be
concluded that incorporation of hydrophobic and electron
withdrawing/electron releasing groups in both the aryl
1. Molecular weight (MW) (150–650).
2. Octanol/water partition coefficient (Log Po/w) (-2 to
6.5).
3. Aqueous solubility (QPlogS) (-6.5 to 0.5).
4. Apparent MDCK cell permeability (QPPMDCK) (\25
poor, [500 great).
Table 4 Prediction of ADME properties of newly designed isatin analogs using QikProp v3.0
Comp
Mol. wt.
Log Po/w
Log S
Log BB
PMDCK
HOA/%
Rule of five
3a
3b
3c
3d
3e
3f
385.421
383.449
403.867
417.894
389.840
403.867
419.866
417.894
397.476
411.502
4.047
4.592
4.310
4.616
4.155
4.155
4.248
4.787
4.591
4.728
-5.377
-6.218
-5.827
-6.394
-5.799
-5.799
-5.959
-6.760
-6.159
-6.577
-0.588
-0.49
1016
100
100
100
100
100
100
100
100
100
100
0
0
0
0
0
0
0
0
0
0
1143.2
-0.584
-0.613
-0.628
-0.584
-0.691
-0.613
-0.690
-0.739
1368.370
1369.961
1327.956
1370.082
1382.330
1554.532
604.238
635.976
3g
3h
3i
3j
ADME Absorption, distribution, metabolism and excretion, PMDCK Permeability Maden-Darby canine kidney, HOA Human oral absorption
123

Synthesis, biological evaluation, in silico docking, and virtual ADME studies…
General procedure for the synthesis of 2-[2-oxo-3-
moieties attached to the isatin pharmacophore is essen-
tial; however, hydrophobicity should be optimal for
activity. These results also support the clinical promise
of these compounds as a component of therapeutic
strategies for cancer, for which high concentrations of
chemotherapeutic agents are always a major limitation.
Moreover, the uncomplicated methodology used for the
preparation of these potent compounds allows for
obtaining sufficient amounts for more in-depth clinical
studies. The simplicity of the prepared compounds with
their potent anti-breast cancer activity highlights the
potential of elegant organic synthesis for the discovery of
new drug leads.
(arylimino)indolin-1-yl]-N-arylacetamides 3a–3j
To the appropriate Schiff bases of isatin (10 mmol) in
8–10 cm3 of anhydrous DMF, K2CO3 (15 mmol) was added
and stirred at room temperature for 1 h. After completion of
1 h, the solution turned red brown in color. Appropriate
chloroanilides (10 mmol) and KI (2 mmol) were then added
to this solution drop wise and heated at 60 °C for 5.5–9 h.
After conforming the end of reaction by TLC (ethyl acetate:
n-hexane 30:70), the mixture was poured into ice cold water.
Precipitated crude product was filtered and washed thor-
oughly with cold water (3 9 200 cm3). Compounds were
recrystallized from ethanol/water mixture (1:1). Reaction
times, melting points, and yields are depicted in Table 1.
N-(4-Methoxyphenyl)-2-[2-oxo-3-(phenylimino)indolin-1-
yl]acetamide (3a, C23H19N3O3)
Experimental
Brownish yellow powder; IR (KBr): m = 3286.81 (N–H
stretching), 3074, 2939.61 (Ar–CH stretching), 1733.10,
1722 (C=O stretching), 1603.86 (C=N stretching) cm-1; 1H
NMR (400 MHz, DMSO-d6): d = 3.717 (s, 3H, -OCH3),
4.613 (s, 2H, -CH2-), 6.42 (d, 1H, J = 7.6 Hz, Ar–H),
6.79–6.83 (t, 1H, J = 7.6 Hz, Ar–H), 6.908–6.865 (dd, 2H,
J = 8.4, 8.8 Hz, Ar–H), 7.02–7.00 (d, 2H, J = 7.6 Hz,
Ar–H), 7.16–7.09 (m, 1H, Ar–H), 7.27 (d, J = 7.6 Hz, 1H,
Ar–H), 7.521–7.403 (m, 5H, Ar–H), 10.228 (s, 1H,
-NH-) ppm; 13C NMR (100 MHz, CDCl3): d = 45.08,
55.47, 110.35, 114.14, 115.88, 117.78, 121.99, 123.45,
125.64, 126.29, 129.52, 130.31, 134.46, 146.58, 149.97,
153.77, 156.74, 163.91, 164.42 ppm; MS (ESI):
m/z = 386.0 ([M ? 1)?], calc for C23H19N3O3 385.42.
All the chemicals were purchased from the following
companies: Sigma-Aldrich Chemicals, Rankem, and
Spectrochem PVT LTD. Precursors 1a–1d [21, 22] and
2a–2f [23] were synthesized according to published
methods. The purity and homogeneity of the compounds
were assessed by TLC performed on Merck silica gel 60
F254 aluminum sheets using ethyl acetate:n-hexane (3:7)
as eluents. Iodine chamber and Shimadzu (UV-254)
spectrometer were used for visualization of TLC spots.
Ashless Whatmann No. 1 filter paper was used for vac-
uum filtration. Melting points were determined on an
OPTIMELT automated system apparatus. Infrared spectra
were determined as KBr solid disks or pellets, recorded
on SHIAMADZU FT/IR 8400 and were reported in cm-1
.
13C NMR spectra were recorded on a Bruker Avance
100 MHz spectrometer using CDCl3 as solvent. Chemical
shifts were reported in ppm using TMS as the internal
standard. 1H NMR spectra were recorded on a Varian
spectrometer 400 MHz using DMSO-d6 as solvent.
Chemical shifts are expressed in d values (ppm) relative
to tetramethylsilane (TMS) as an internal standard. The
peak multiplicity is reported as singlet (s), doublet (d),
triplet (t), and multiplet (m). Mass spectra were recorded
using (ESI?) MS. Elemental analysis was performed on a
Vario Micro cube CHNS analyzer of Elementar and
values were within the acceptable 0.3 % limit of the
calculated values.
N-(2,5-Dimethylphenyl)-2-[2-oxo-3-(phenylimino)indolin-
1-yl]acetamide (3b, C24H21N3O2)
Pale yellow powder; IR (KBr): m = 3282.95 (N–H stretch-
ing), 3019.66, 2922.25 (Ar–CH stretching), 1728.10,
1688.12 (C=O stretching), 1605.79 (C=N stretching)
;
cm-1 1H NMR (400 MHz, DMSO-d6): d = 2.153 (s,
3H, CH3), 2.236 (s, 3H, CH3), 4.659 (s, 2H, -CH2-),
6.426 (d, 1H, J = 7.6 Hz, Ar–H), 6.800–6.838 (dd, 1H,
J = 8.0, 7.2 Hz, Ar–H), 6.924 (d, 1H, J = 7.2 Hz, Ar–H),
7.089–7.120 (m, 2H, Ar–H), 7.007 (d, 2H, J = 7.6 Hz, Ar–
H), 7.187 (s, 1H, Ar–H), 7.265–7.337 (m, 1H, Ar–H),
7.426–7.520 (m, 3H, Ar–H), 9.668 (s, 1H, -NH-) ppm;
13C NMR (100 MHz, CDCl3): d = 17.37, 21.11, 45.17,
110.38, 116.37, 117.04, 120.26, 122.99, 123.80, 124.85,
126.10, 126.32, 129.09, 131.01, 134.46, 136.68, 137.45,
146.45, 148.91, 153.82, 164.07, 164.81 ppm; MS (ESI):
m/z = 384.0 ([M ? 1)?], calc for C24H21N3O2 383.44.
All the novel isatin analogs were screened on MCF-7
breast cancer cell line by the Advanced Centre for Treat-
ment Research and Education in Cancer (ACTREC)
Mumbai, India. The cell viability was measured using SRB
assay. All the environmental conditions were maintained
throughout the experiment for all the groups. The assay
was performed in triplicate for each of the compounds. The
growth curve was plotted against lg/cm3 drug concentra-
tion of compounds and % control growth.
2-[3-(2,5-Dimethylphenylimino)-2-oxoindolin-1-yl]-N-(o-
tolyl)acetamide (3i, C25H23N3O2)
Yellow powder; IR (KBr): m = 3212.35 (N–H), 3048.84,
2920.32 (Ar–CH), 1602.90 (C=N), 1708.10, 1658 (C=O)
123

B. Debnath, S. Ganguly
1
cm-1; H NMR (400 MHz, DMSO-d6): d = 1.993 (s, 3H,
drugs. After 24 h, one plate of every cell line was fixed
in situ with TCA, to represent a measurement of the cell
population for every cell line at the time of drug addiction.
Experimental drugs were solubilized in suitable solvent at
400-fold the preferred final maximum test concentration
and stored frozen prior to use. At the time of drug addition,
an aliquot of frozen concentrate was thawed and diluted to
ten times the preferred final maximum test concentration
with complete medium containing test article at a con-
centration of 10-3. Additional three, tenfold serial dilutions
were made to provide a total of four drug concentrations
plus control. Aliquots of 10 mm3 of these different drug
dilutions were added to the appropriate microtiter wells
already containing 90 mm3 of medium, resulting in the
necessary final drug concentrations.
-CH3), 2.218 (s, 3H, -CH3), 2.282 (s, 3H, -CH3), 4.686
(s, 2H, -CH2-), 6.365 (d, 1H, J = 7.6 Hz, Ar–H), 6.666
(s, 1H, Ar–H), 6.843 (dd, 1H, J = 7.2, 7.6 Hz, Ar–H),
6.999 (d, 1H, J = 7.6 Hz, Ar–H), 7.12 (d, 2H, J = 7.6 Hz,
Ar–H), 7.158 (dd, 1H, J = 7.2, 1.6 Hz, Ar–H), 7.239 (d,
2H, J = 7.6 Hz, Ar–H), 7.375 (d, 1H, J = 7.6 Hz, Ar–H),
7.451 (dd, 1H, J = 8.0, 7.2 Hz, Ar–H), 9.759 (s, 1H,
-NH-) ppm; 13C NMR (100 MHz, CDCl3): d = 17.23,
17.72, 21.03, 45.63, 110.10, 116.29, 116.83, 122.55,
122.74, 123.93, 125.57, 126.13, 126.25, 126.80, 128.92,
130.62, 130.91, 134.37, 135.05, 136.58, 145.86, 148.84,
153.40, 163.82, 164.68 ppm; MS (ESI): m/z = 398.1
([M ? 1)?], calc for C25H23N3O2 397.47.
N-(2,5-Dimethylphenyl)-2-[3-(2,5-Dimethylphenylimino)-
2-oxoindolin-1-yl]acetamide (3j, C26H25N3O2)
After compound addition, the cells were then incubated
for 48 h with the test compounds. The experiment was
completed by adding 30 % chilled TCA to the wells. Then,
cells were fixed in situ by the gentle addition of 50 mm3 of
cold 30 % (w/v) TCA (final concentration, 10 % TCA) and
incubated for 60 min at 4 °C. The supernatant was dis-
carded, the plates were washed five times with tap water
and air dried. Sulforhodamine B (SRB) solution (50 mm3)
at 0.4 % (w/v) in 1 % acetic acid was added to each of the
wells, and plates were incubated for 20 min at room tem-
perature. After staining, unbound dye was recovered and
the residual dye was removed by washing five times with
1 % acetic acid. The plates were air dried. Bound stain was
afterward eluted with 10 mM trizma base, and the absor-
bance was read on an Elisa plate reader at a wavelength of
540 with 690 nm reference wavelength [38].
Shiny yellow crystals; IR (KBr): m = 3281.99 (N–H),
3031.23, 2912.29 (Ar–CH), 1606.79 (C=N), 1730.10,
;
1665.90 (C=O) cm-1 1H NMR (400 MHz, DMSO-d6):
d = 1.991 (s, 3H, -CH3), 2.164 (s, 3H, -CH3), 2.237 (s,
3H, -CH3), 2.281 (s, 3H, -CH3), 4.671 (s, 2H, -CH2-),
6.362 (d, 1H, J = 7.2 Hz, Ar–H), 6.662 (s, 1H, Ar–H),
6.821–6.859 (t, 1H, J = 7.6 Hz, Ar–H), 6.925 (d, 1H,
J = 7.6 Hz, Ar–H), 6.996 (d, 1H, J = 7.6 Hz, Ar–H),
7.095–7.120 (m, 2H, Ar–H), 7.205–7.249 (dd, 2H, J = 9.6,
8.0 Hz, Ar–H), 7.432–7.468 (dd, 1H, J = 8.0, 6.4 Hz Ar–
H), 9.6904 (s, 1H, -NH-) ppm; 13C NMR (100 MHz,
CDCl3): d = 17.42, 19.34, 19.96, 21.16, 45.67, 110.33,
116.44, 117.03, 117.75, 121.59, 123.01, 123.91, 126.20,
126.34, 130.15, 131.02, 133.48, 134.47, 134.91, 136.69,
137.51, 146.26, 153.65, 164.00, 164.52 ppm; MS (ESI): m/
z = 412.2 ([M ? 1)?], calc for C26H25N3O2 411.19.
Percent growth was calculated on a plate-by-plate basis
for test wells relative to control wells. Percent growth was
expressed as the ratio of average absorbance of the test well
to the average absorbance of the control wells 9 100:
[(Ti - Tz)/(C - Tz)] 9 100.
In vitro anticancer activity evaluation by SRB assay
Anticancer activity of the newly synthesized isatin analogs
was evaluated against MCF-7 breast cancer cell line using
sulforhodamine-B (SRB) assay method [35–38]. Human
breast cancer cell line (MCF-7) evaluation was performed
at the Advanced Centre for Treatment, Research and
Education in Cancer (ACTREC), Mumbai, India and
adriamycin (doxorubicin) was used as a reference drug.
Reagents and chemicals were purchased from Sigma-
Aldrich Chemical Company. The cell line was grown in
RPMI 1640 medium containing 10 % fetal bovine serum
and 2 mM L-glutamine. For present screening experiment,
cells were inoculated into 96-well microtiter plates in
90 mm3 at plating densities, depending on the doubling
time of the individual cell line [37, 38]. The microtiter
plates were incubated at 37 °C in a carbon dioxide (CO2)
incubator at 5 % CO2, 95 % air, and 100 % relative
humidity for 24 h prior to the addition of experimental
Using the six absorbance measurements [time zero (Tz),
control growth (C), and test growth in the presence of drug
at the four concentration levels (Ti)], the percentage growth
was calculated at each of the drug concentration levels
[39].
For concentrations for which Ti C Tz (Ti - Tz) positive
or zero = [(Ti - Tz)/(C - Tz)] 9 100.
For concentrations for which Ti \ Tz (Ti - Tz) nega-
tive = [(Ti - Tz)/Tz] 9 100.
Growth inhibition of 50 %, GI50 = [(Ti - Tz)/
(C - Tz)] 9 100.
GI50 is that value of the drug concentration resulting in a
50 % reduction in the net protein increase (as measured by
SRB staining) in control cells during the drug incubation.
The drug concentration resulting in total growth inhibition
(TGI) was calculated from Ti = Tz. The LC50 is the drug
concentration resulting in a 50 % reduction in the
123

Synthesis, biological evaluation, in silico docking, and virtual ADME studies…
In silico ADME evaluation
measured protein at the end of the drug treatment as
compared to that at the beginning. During this, there is a
net loss of 50 % cells following treatment and is calculated
from [(Ti - Tz)/Tz] 9 100 = -50 [40].
In silico ADME evaluation of compounds was performed
by ADME software Qikprop v 3.0 [33, 34]. Structures of
the compounds were saved in the mol format using
Chem Office software. Then, mol files of compounds
were uploaded into the ADME predictor software for
further evaluation. All descriptors were estimated at pH
7.4.
Statistical analysis
Values were calculated for each of these three parameters if
the level of activity was reached; however, if the effect was
not reached or was exceeded, the values for that parameter
were expressed as greater or less than the maximum or
minimum concentration tested. The experiment data were
estimated using linear regression method of plots of the cell
viability against the lg/cm3 drug concentration of tested
compounds.
Acknowledgments The authors acknowledge the University Grants
Commission for providing financial support in the form of a Major
Research Project. The authors are also grateful to Dr. Reddy’s
Institute of Life sciences, Hyderabad for spectral characterization and
ACTREC, Mumbai for anticancer screening facility. One of the
authors (BD) gratefully acknowledges the University Grants Com-
mission-Basic Science Research (UGC-BSR) for the award of
fellowship during the work.
In silico molecular docking studies
Carcinogenesis is usually accompanied by over activation
of receptor tyrosine kinase (RTK) signaling pathways, so
inhibitors which block these receptors have a significant
therapeutic potential in cancer treatment [41]. On this
basis, RTK was selected as the target receptor for docking
studies of the synthesized compounds. EGFR kinase
domain in complex with 4-anilinoquinazoline inhibitor
(AQ4) (PDB ID: IM17) [42] was used. For docking studies,
all water molecules and ligands were removed from the
protein for docking studies, and the protein was minimized
by the protein preparation wizard. Partial atomic charges
were assigned according to the OPLS_AA force field [43,
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