Analogue-based design, synthesis and biological evaluation of 3-substituted-(methylenehydrazono)indolin-2-ones as anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2014.03.058

The docking studies on CDK2 and GSK-3β inspired us to synthesis a series of indoline-2,3-dione hydrazones 10a-l. Treatment of indoline-2,3-dione derivatives 7a-d with hydrazine gave 3-hydrazonoindolin-2-ones 8a-d which were reacted with the appropriate aldehydes 9a-c to yield 3-substituted- (methylenehydrazono)indolin-2-ones 10a-l. Compounds 10a-l showed a significant anticancer activity against human breast cell line MCF-7. Compounds 10c, f, i exhibited the highest activity almost the same of doxorubicin (IC₅₀ = 6.10 μM) with IC₅₀ = 7.75, 6.75, 6.25 μM, respectively.

Full text of DOI:10.1016/j.ejmech.2014.03.058

European Journal of Medicinal Chemistry 78 (2014) 275e280
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Analogue-based design, synthesis and biological evaluation of
3-substituted-(methylenehydrazono)indolin-2-ones as
anticancer agents
,d,
*
Haytham E. Dweedar a, Hoda Mahrous a, Hany S. Ibrahim b, Hatem A. Abdel-Aziz c
a Industrial Biotechnology Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), Sadat City University, Egypt
b Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Egyptian Russian University, Badr City, Helwan, Egypt
c Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
d Department of Applied Organic Chemistry, National Research Center, Dokki, Cairo 12622, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
The docking studies on CDK2 and GSK-3
b inspired us to synthesis a series of indoline-2,3-dione
Received 14 December 2013
Received in revised form
15 March 2014
Accepted 17 March 2014
Available online 19 March 2014
hydrazones 10ael. Treatment of indoline-2,3-dione derivatives 7aed with hydrazine gave 3-
hydrazonoindolin-2-ones 8aed which were reacted with the appropriate aldehydes 9aec to yield 3-
substituted-(methylenehydrazono)indolin-2-ones 10ael. Compounds 10ael showed a significant anti-
cancer activity against human breast cell line MCF-7. Compounds 10c, f, i exhibited the highest activity
almost the same of doxorubicin (IC50 ¼ 6.10
m
M) with IC50 ¼ 7.75, 6.75, 6.25
mM, respectively.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Analogue-based design
Molecular docking
Cancer
Human breast cell line MCF-7
Indoline-2,3-dione hydrazones
1. Introduction
as SU5416 (semaxanibÒ, 2a) [8], SU5402 (2b) [9], SU6668 (2c) [10]
and SU14813 (2d) [11] (Fig. 1) showed a significant cytotoxic ac-
tivity. However, SU11248 (sunitinib, SutentÒ, 2) is an inhibitor of
receptor tyrosine kinase (RTK) and it is the standard first-line
treatment for the treatment of gastrointestinal stromal cancers
and renal cell carcinoma [12] (Fig. 1).
Moreover, hydrazones of 1H-indole-2,3-dione 3 (Fig. 1) have
been identified as inhibitors of the protein tyrosine phosphatase
Shp2, which plays an important role in cell signaling, cell prolifer-
ation, differentiation and migration [13]. In recent years, the cyto-
toxic activity against Artemia salina of 1H-indole-2,3-dione
thiosemicarbazones 4 (a hydrazone derivative containing a sulfur
atom) have reported [14,15] (Fig. 1). Furthermore, thio-
semicarbazones 3 have been found to display cytotoxicity against
the KB-3-1 cell line (a HeLa derivative) [16,17].
Nowadays, cancer is one of the main reasons of death between
nations caused by different elements such as presence of mutagenic
and cancer causing materials in the environment. On the other
hand, 1H-indole-2,3-dione derivatives have aroused great attention
due to their wide variety of biological activities, relevant to appli-
cation in a broad range of drug therapies, including anticancer
drugs [1e3]. A plethora of biologically active C3-substituted indole-
2,3-dione has been generated in the literature, these due to the
susceptibility of isatin to be attacked by nucleophiles at the C3
position [3]. C3-substituted indole-2,3-dione drugs such as indir-
ubin 1a (X ¼ H, Y ¼ O) and its derivatives 5-bromoindirubin 1b
(X ¼ Br, Y ¼ O), indirubin-30-oxime 1c (X ¼ H, Y ¼ NeOH) and 5-
bromoindirubin-30-oxime 1d (X ¼ Br, Y ¼ NeOH) were found to
have potent anticancer activity with excellent inhibitory of tyrosin
Recently, we have been reported the synthesis of hydrazino
derivative 5 with selective activity against multidrug-resistant
cancer cells [18] and in vitro antiproliferative activity of synthe-
sized 2H-chromene-3-carbohydrazides 6 which exhibited good
antiproliferative profile against colon HT-29 [19] (Fig. 1).
kinases CDK2, CDK5 and GSK-3b [4e7]. Moreover, 1H-indole-2,3-
dione drugs which contain arylidene branches in position 3 such
In the light of the above data and through our docking studies
that will be mentioned later, and in a continuation of our interest in
the synthesis of hydrazone-based compounds with anticancer
* Corresponding author. Department of Pharmaceutical Chemistry, College of
Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia.
E-mail address: hatem_741@yahoo.com (H.A. Abdel-Aziz).
http://dx.doi.org/10.1016/j.ejmech.2014.03.058
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

276
H.E. Dweedar et al. / European Journal of Medicinal Chemistry 78 (2014) 275e280
R
R
R1
R2
R
S
NH
Y
NH
O
NH
O
N
NH
N
N
H
X
X
X
X
O
O
N
H
2a-e
N
H
N
N
H
H
1a-e
4
3
R
R1
N
O
Y
O
R
N
N
N
O
N
X
NH
X
N
O
O
O
X
N
N
H
O
H
N
10a-l
H
5
6
Fig. 1. Structure of 1e6 and 10ael.
activity [18e24], in this study, we have designed and synthesized
certain derivatives of indoline-2,3-dione hydrazones 10ael (Fig. 1)
that have potential utility as anticancer drugs.
compounds. In addition, variation of substitution in position 5 of
indoline-2,3-dione moiety was considered as a strategic key for
functionalization of synthesized compounds due to its importance
in the inhibition process as reported [25].
2. Results and discussion
2.2. Chemistry
2.1. Analogue-based design through molecular docking studies
Indoline-2,3-diones 7aed were reacted with hydrazine hydrate
in absolute methanol to afford the corresponding hydrazine de-
rivatives 8aed [26]. The reaction of compounds 8aed with alde-
hydes 9aec gave hydrazones 10ael (Scheme 1). The IR spectra of
10ael showed bands due to isatin NH in the region 3256e
3125 cmꢀ1 in addition to a carbonyl band in the region 1737e
Crystallographic data of CDK2 [4,5] and GSK-3b [6] in complex
with various indirubins has provided valuable information on the
active site differences along with specific interactions between
those kinases and indirubin analogues [7]. This mode of interaction
inspired us to deduce indoline-2,3-dione hydrazone derivatives
with an interaction near to indirubin analogues. During the inves-
tigation of the interaction between 5-bromoindurubin with human
cyclin dependent protein kinase 2 of the ligand gave an energy
score (S) value ¼ ꢀ21.09 kcal/mol and root of mean square devia-
1704 cmꢀ1
exchangeable signal in the region
assigned to NH isatin proton, in addition to the signal of methine
proton (eCH]Ne) in the region
8.54e8.99. However, 1H NMR
.
1H NMR spectra of these compounds revealed D2O-
d
10.59e10.97 which were
d
ꢀ
spectra showed additional NH signal appeared around 12 due to
pyrrole NH in compounds 10iel.
tion (RMSD) value ¼ 1.69 A. It was found that there are three
important hydrogen bond with Glu81 and Leu83 (Fig. 2a). After
docking simulations on same active site, it was found that our
suggested compounds 10ael gave a binding pattern near to the
original ligand as in case of 10i where the binding energy (S) was
2.3. Anticancer activity
ꢀ
Treatment of human breast cancer cell line MCF-7 with different
concentrations of synthesized compounds 10ael resulted in an
inhibition of the growth by different IC50 values comparing with
reference drug doxorubicin (Table 1). From IC50 values of 10ael, we
can deduce that the presence of pyrrole ring, as well as indoline-
2,3-dione hydrazone, gave the highest activity where compound
ꢀ18.69 kcal/mol and RMSD value was 1.55 A (Fig. 2b).
Similarly, docking analysis of GSK-3b receptor for both indir-
ubin-30-oxime and our suggested compounds 10ael, taking 10i as
representative example, showed that they interacted through
hydrogen bonds with Val135 and Asp133 (Fig. 2c, d). Furthermore,
10i recorded an excellent binding energy score (S) ¼ ꢀ18.08 kcal/
mol when compared with that of ligand (ꢀ27.89 kcal/mol) under
10i gave IC50
(IC50 ¼ 6.10 M). Considering the substitutions in position 5 of
indoline-2,3-dione moiety, we found that furan derivative 10c, with
Cl in position 5 of indoline-2,3-dione, showed IC50 ¼ 7.75 M while
thiophene derivative 10f, with methyl substitution in position 5 of
¼
6.25 mM almost the same of doxorubicin
0
ꢀ
ꢀ
m
suitable RMSD value ¼ 0.41 A and 1.50 A for indirubin-3 -oxime and
10i, respectively. These results motivated us to synthesis a series of
indoline-2,3-dione hydrazones 10iel. Keeping in consideration the
structure of compounds 1e6, hybridization structure of compounds
10iel (Fig. 3) showed an interesting combination of the structure
features of compounds 1e6.
m
indoline-2,3-dione, exhibited IC50 ¼ 6.75
mM (Table 1).
The isosteric replacement of nitrogen in pyrrole moiety by ox-
ygen or sulfur in furan and thiophene moieties in compounds 10ae
d and 10eeh, respectively, supported the structure activity rela-
tionship (SAR) in the anticancer activity screening for targeted
3. Conclusion
Docking studies were done to predict the possible anticancer ac-
tivity of a series of 2-oxindolin-3-hydrazones 10ael. Synthesis and

H.E. Dweedar et al. / European Journal of Medicinal Chemistry 78 (2014) 275e280
277
Fig. 2. a, b. 2D representations of the binding patterns of 5-bromoindirubin and 10i with CDK2, respectively. c, d. 2D representations of the binding patterns of indirubin-30-oxime
and 10i with GSK-3 , respectively.
b
in vitro anticancer activity 10ael toward MCF-7 cell line were studied
to support our hypothesis. From the biological assay results, com-
pounds 10ael showed a significant activity and we found that 3-
(((1H-pyrrol-2-yl)methylene)hydrazono)indolin-2-one (10i) gave
the maximum activity with IC50 ¼ 6.25
mM. This result give a new
promising venue for further investigations of the targeted
compounds.
Structre 2
Structures 1, 2, 5
Structres 3-6
N
H
N
N
4. Experimental
X
O
1-6
Structures
N
H
4.1. Molecular docking studies
10i-l
Fig. 3. Structure features of compounds 10iel comparing with compounds 1e6.
The molecular modeling studies were carried out using Molec-
ular Operating Environment (MOE 2008.10; Chemical Computing

278
H.E. Dweedar et al. / European Journal of Medicinal Chemistry 78 (2014) 275e280
Y
NH2
O
N
CHO
N
N
Y
9a-c
X
X
NH2NH2
X
O
O
O
N
N
N
H
H
H
7a-d
8a-d
10a-l
X
Y
10
7, 8
a
b
c
d
X
H
Me
Cl
F
9
Y
O
S
NH
H
a
b
c
d
e
f
O
a
b
c
Me
Cl
F
O
O
O
H
S
Me
Cl
F
S
g
h
i
S
S
H
NH
NH
NH
NH
Me
Cl
F
j
k
l
Scheme 1. Synthesis of compounds 10ael.
Group, Canada). All the minimizations were performed with MOE
ꢀꢀ1
(ppm) relative to TMS as an internal standard. Coupling constants
(J) are expressed in Hz. D2O was added to confirm the exchangeable
protons.
until a RMSD gradient of 0.05 kcal molꢀ1
forcefield and the partial charges were automatically calculated.
The X-ray crystallographic structure of GSK-3 and CDK2 com-
A
with MMFF94X
b
plexes with indirubin-30-oxime (PDB ID: 1Q41 and 2BHE respec-
tively) was obtained from the protein data bank. The interaction
mode of the ligand were investigated then the receptors were
prepared for docking studies by assigning of the hydrogen bond
state of the receptors and removing of water atoms. Molecular
docking simulations were done to predict the interactions of the
suggested compound.
4.2.2. General procedure for the synthesis of (Z)-3-
hydrazonoindolin-2-ones 8aed
A mixture of isatin 7aed (1 mmol) and hydrazine hydrate (99%,
2 mmol) in absolute methanol (25 ml) was refluxed for 1 h, and
then cooled to room temperature. The precipitate of hydrazone was
filtered, dried and then crystallized from EtOH/DMF to afford
compounds 8aed which used for next step without any further
purification [26].
4.2. Chemistry
4.2.1. General
4.2.3. General procedure for the synthesis of hydrazones 10ael
To a mixture of hydrazone 8aed (1 mmol) and aldehyde 9aec
(1 mmol) in ethanol (25 ml), 0.5 ml acetic acid was added. The
reaction mixture was refluxed for 6 h, and then cooled to room
temperature. The precipitate was filtered, dried and finally crys-
tallized from EtOH/DMF to afford hydrazones 10ael.
Melting points (ꢁC, uncorrected) were determined using a Stuart
melting point apparatus. The IR spectra (KBr) were recorded on a
PerkinElmer FT/IR spectrometer. NMR Spectra were scanned in
DMSO-d6 on a Brucker NMR spectrometer operating at 400 MHz for
1H and 75 MHz for 13C. Chemical shifts are expressed in
d-values
Table 1
In vitro anticancer screening of 10ael against human breast cell line MCF-7.
Compound
Concentration (
m
M)
IC50 (mM)
Surviving fraction (Mean ꢂ S.E.)a
10
25
50
100
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
0.637 ꢂ 0.122
0.436 ꢂ 0.032
0.384 ꢂ 0.096
0.667 ꢂ 0.035
0.352 ꢂ 0.065
0.227 ꢂ 0.162
0.619 ꢂ 0.023
0.616 ꢂ 0.035
0.465 ꢂ 0.086
0.473 ꢂ 0.056
0.510 ꢂ 0.032
0.671 ꢂ 0.098
0.257 ꢂ 0.036
0.306 ꢂ 0.089
0.213 ꢂ 0.036
0.274 ꢂ 0.069
0.429 ꢂ 0.133
0.602 ꢂ 0.045
0.132 ꢂ 0.025
0.310 ꢂ 0.123
0.505 ꢂ 0.056
0.328 ꢂ 0.032
0.351 ꢂ 0.065
0.392 ꢂ 0.096
0.274 ꢂ 0.152
0.158 ꢂ 0.068
0.158 ꢂ 0.035
0.195 ꢂ 0.069
0.259 ꢂ 0.023
0.335 ꢂ 0.086
0.522 ꢂ 0.162
0.099 ꢂ 0.065
0.204 ꢂ 0.078
0.331 ꢂ 0.032
0.395 ꢂ 0.152
0.221 ꢂ 0.138
0.238 ꢂ 0.052
0.456 ꢂ 0.121
0.098 ꢂ 0.086
0.333 ꢂ 0.086
0.102 ꢂ 0.096
0.231 ꢂ 0.169
0.257 ꢂ 0.068
0.546 ꢂ 0.108
0.137 ꢂ 0.081
0.194 ꢂ 0.037
0.308 ꢂ 0.161
0.357 ꢂ 0.059
0.206 ꢂ 0.087
0.259 ꢂ 0.098
0.528 ꢂ 0.087
0.125 ꢂ 0.075
15.8
8.75
7.75
20.3
8.25
6.75
15.3
25.8
6.25
9.25
11.8
10.8
6.10
Doxorubicin
a
Each value is the mean of three values ꢂ Standard Error.

H.E. Dweedar et al. / European Journal of Medicinal Chemistry 78 (2014) 275e280
279
4.2.3.1. 3-((Furan-2-ylmethylene)hydrazono)indolin-2-one
Yield (55%); mp: 209 ꢁC; IR (KBr) v 3196 (NH), 1729 (C]O), 1622
(C]N) cmꢀ1 1H NMR (300 MHz, DMSO-d6)
: 6.77e8.09 (m, 7H,
Ar-H), 8.55 (s, 1H, eCH]), 10.81 (s, D2O exch., 1H, NH); 13C NMR
(75 MHz, DMSO-d6) : 110.73, 113.08, 116.67, 119.67, 127.32, 129.00,
(10a).
DMSO-d6)
eCH]), 10.59 (s, D2O exch., 1H, NH), 11.96 (s, D2O exch., 1H, NH);
13C NMR (75 MHz, DMSO-d6)
: 20.50 (CH3), 109.99, 111.00, 116.98,
119.28, 126.62, 127.18, 129.66, 131.02, 133,18, 142.03, 150.21, 155.86,
165.25 (C]O).
d: 2.32 (s, 3H, CH3), 6.26e8.22 (m, 6H, Ar-H), 8.55 (s, 1H,
;
d
d
d
133.64, 144.87, 148.04, 149.03, 150.76, 151.31, 164.62 (C]O).
4.2.3.11. 3-(((1H-Pyrrol-2-yl)methylene)hydrazono)-5-chloroindolin-
4.2.3.2. 3-((Furan-2-ylmethylene)hydrazono)-5-methylindolin-2-one
2-one (10k). Yield (45%); mp: 248 ꢁC; IR (KBr) v 3303, 3125 (2NH),
(10b). Yield (52%); mp: 194 ꢁC; IR (KBr) v 3245 (NH), 1719 (C]O),
1704 (C]O), 1613 (C]N) cmꢀ1 1H NMR (300 MHz, DMSO-d6)
; d:
1618 (C]N) cmꢀ1
;
1H NMR (300 MHz, DMSO-d6)
d
: 2.26 (s, 3H,
CH3), 6.78e8.01 (m, 6H, Ar-H), 8.54 (s, 1H, eCH]), 10.7 (s, D2O
exch., 1H, NH); 13C NMR (75 MHz, DMSO-d6)
: 20.65 (CH3), 110.45,
6.35e8.43 (m, 6H, Ar-H), 8.59 (s, 1H, eCH]),10.83 (s, D2O exch.,1H,
NH), 12.18 (s, D2O exch., 1H, NH); 13C NMR (75 MHz, DMSO-d6)
d
:
d
111.30, 111.73, 117.95, 120.31, 126.00, 127.08, 127.48, 128.29, 132.25,
142.97, 148.85, 157.27, 164.89 (C]O).
113.09, 116.72, 119.47, 129.40, 131.01, 133.98, 142.5, 148.01, 149.07,
150.82, 151.44, 164.68 (C]O).
4.2.3.12. 3-(((1H-Pyrrol-2-yl)methylene)hydrazono)-5-fluoroindolin-
2-one (10l). Yield (37%); mp: 195 ꢁC; IR (KBr) v 3176 (2NH), 1706
4.2.3.3. 5-Chloro-3-((furan-2-ylmethylene)hydrazono)indolin-2-one
(10c) [27]. Yield (47%); mp: 265 ꢁC; IR (KBr) v 3145 (NH), 1737 (C]
(C]O), 1620 (C]N) cmꢀ1
;
1H NMR (300 MHz, DMSO-d6)
d
: 6.35e
8.39 (m, 6H, Ar-H), 8.61 (s, 1H, eCH]), 10.73 (s, D2O exch., 1H, NH),
12.13 (s, D2O exch., 1H, NH); 13C NMR (75 MHz, DMSO-d6)
: 109.65,
O), 1616 (C]N) cmꢀ1; 1H NMR (300 MHz, DMSO-d6)
d
: 6.81e8.15 (m,
6H, Ar-H), 8.62 (s, 1H, eCH]), 10.97 (s, D2O exch., 1H, NH); 13C NMR
(75 MHz, DMSO-d6) : 112.27, 113.36, 117.83, 120.91, 125.80, 128.27,
d
d
111.31, 116.31, 117.39, 119.16, 120.76, 127.02, 127.49, 140.54, 141.84,
149.60, 150.54, 156.23, 157.56, 159, 37, 165.24 (C]O).
132.99, 143.61, 148.56, 148.84, 150.81, 152.21, 152.26, 164.33 (C]O).
4.2.3.4. 5-Fluoro-3-((furan-2-ylmethylene)hydrazono)indolin-2-one
(10d). Yield (48%); mp: 236 ꢁC; IR (KBr) v 3256 (NH), 1732 (C]O),
4.3. Anticancer activity
1619 (C]N) cmꢀ1 1H NMR (300 MHz, DMSO-d6)
; d: 6.82e8.14 (m,
6H, Ar-H), 8.62 (s, 1H, eCH]), 10.86 (s, D2O exch., 1H, NH).
4.3.1. Materials and methods
4.3.1.1. Chemicals. Dimethylsulfoxide (DMSO), Doxorubicin and
Sulfo-Rhodamine-B stain (SRB) were purchased from Merck
(Darmstadt, Germany). All other chemicals and reagents used in
this study were of analytical grade and purchased from Sigmae
Aldrich chemical Co. (St. Louis, MO, USA).
4.2.3.5. 3-((Thiophen-2-ylmethylene)hydrazono)indolin-2-one (10e).
Yield (67%); mp: 206 ꢁC; IR (KBr) v 3249 (NH), 1733 (C]O), 1605
(C]N) cmꢀ1
;
1H NMR (300 MHz, DMSO-d6)
d
: 6.89e8.85 (m, 7H,
Ar-H), 8.90 (s, 1H, eCH]), 10.76 (s, D2O exch., 1H, NH); 13C NMR
(75 MHz, DMSO-d6) : 110.78, 116.63, 122.22, 127.82, 128.81, 128.91,
d
133.11, 133.64, 135.16, 138.42, 144.00, 157.04, 164.64 (C]O).
4.3.1.2. Cell culture. The MCF-7 cancer cell line was obtained from
the American Type Culture Collection (Rockville, MD, USA). The
tumor cells were maintained in Dulbecco’s modified Eagle’s me-
dium (DMEM) supplemented with 10% heat inactivated fetal calf
4.2.3.6. 5-Methyl-3-((thiophen-2-ylmethylene)hydrazono)indolin-2-
one (10f). Yield (63%); mp: 228 ꢁC; IR (KBr) v 3163 (NH), 1735 (C]
O), 1617 (C]N) cmꢀ1; 1H NMR (300 MHz, DMSO-d6)
d
: 2.26 (s, 3H,
CH3), 6.78e8.46 (m, 6H, Ar-H), 8.91 (s, 1H, eCH]), 10.71 (s, D2O
exch., 1H, NH); 13C NMR (75 MHz, DMSO-d6)
: 20.67 (CH3), 110.51,
serum (GIBCO), penicillin (100 U/ml) and streptomycin (100 mg/ml)
at 37 ꢁC in humidified atmosphere containing 5% CO2. MCF-7 cells
at a concentration of 0.50 ꢃ 106 were grown in a 25 cm2 flask in
5 ml of complete culture medium.
d
116.69, 128.9, 129.56, 130.98, 133.14, 133.90, 135.10, 138.51, 142.71,
151.60, 156.92, 164.72 (C]O).
4.2.3.7. 5-Chloro-3-((thiophen-2-ylmethylene)hydrazono)indolin-2-
4.3.2. In vitro cytotoxic assay
one (10g). Yield (57%); mp: 274 ꢁC; IR (KBr) v 3142 (NH), 1732 (C]
The cytotoxic activity was determined in vitro for all the syn-
thesized compounds and the reference drug using the Sulfo-
Rhodamine-B stain (SRB) colorimetric assay according to Skehan
et al. [28] This assay was performed in National Cancer Institute,
Cairo, Egypt. Cells were inoculated in 96-well microliter plate
(104 cells/well) for 24 h before treatment with the compounds to
allow attachment of cell to the wall of the plate. Test compounds
were dissolved in DMSO and diluted with saline to the appropriate
volume. Different concentrations of the compound under test (10,
O), 1667 (C]N) cmꢀ1
;
1H NMR (300 MHz, DMSO-d6)
d
: 6.91e8.07
(m, 6H, Ar-H), 8.99 (s, 1H, eCH]), 10.96 (s, D2O exch., 1H, NH); 13
NMR (75 MHz, DMSO-d6) : 112.31, 117.80, 125.80, 128.39, 128.98,
C
d
132.94, 133.84, 136.12, 138.25, 143.68, 150.86, 158.59, 164.35 (C]O).
4.2.3.8. 5-Fluoro-3-((thiophen-2-ylmethylene)hydrazono)indolin-2-
one (10h). Yield (50%); mp: 244 ꢁC; IR (KBr) v 3155 (NH), 1735 (C]
O), 1619 (C]N) cmꢀ1 1H NMR (300 MHz, DMSO-d6)
; d: 6.89e8.02
(m, 6H, Ar-H), 8.99 (s, 1H, eCH]), 10.88 (s, D2O exch., 1H, NH).
25, 50, and 100 mM) were added to the cell monolayer. Triplicate
wells were prepared for each individual concentration. Monolayer
cells were incubated with the compound(s) for 48 h at 37 ꢁC and in
atmosphere of 5% CO2. After 48 h, cells were fixed, washed and
stained for 30 min with 0.4% (wt/vol) SRB dissolved in 1% acetic
acid. Excess unbound dye was removed by four washes with 1%
acetic acid and attached stain was recovered with TriseEDTA buffer.
Color intensity was measured in an ELISA reader. The relation be-
tween surviving fraction and drug concentration is plotted to get
the survival curve for breast tumor cell line after the specified time.
The molar concentration required for 50% inhibition of cell viability
(IC50) was calculated and compared with the reference drug
doxorubicin (CAS, 25316-40-9).
4.2.3.9. 3-(((1H-Pyrrol-2-yl)methylene)hydrazono)indolin-2-one
(10i). Yield (44%); mp: 280 ꢁC; IR (KBr) v 3242 (2NH), 1714 (C]O),
1606 (C]N) cmꢀ1; 1H NMR (300 MHz, DMSO-d6)
d
: 6.91e8.45 (m,
6H, Ar-H), 8.56 (s, 1H, eCH]), 10.95 (s, D2O exch., 1H, NH), 11.91 (s,
D2O exch., 1H, NH); 13C NMR (75 MHz, DMSO-d6)
: 111.04, 115.75,
d
122.52, 126.81, 128.16, 129.59, 132.79, 133.53, 134.36, 144.32, 144.74,
145.74, 145.16, 168.38 (C]O).
4.2.3.10. 3-(((1H-Pyrrol-2-yl)methylene)hydrazono)-5-
methylindolin-2-one (10j). Yield (42%); mp: 241 ꢁC; IR (KBr) v 3300,
3144 (2NH), 1706 (C]O), 1616 (C]N) cmꢀ1 1H NMR (300 MHz,
;

280
H.E. Dweedar et al. / European Journal of Medicinal Chemistry 78 (2014) 275e280
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Acknowledgments
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This work was supported by the Industrial Biotechnology
Department, Genetic Engineering and Biotechnology Research
Institute (GEBRI), Sadat City University, Egypt. The authors would
like to extend their sincere appreciation to the Deanship of Scien-
tific Research at King Saud University for its funding of this research
through the Research Group Project no. RGP-VPP-321.
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Appendix A. Supplementary data
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