New chalcones bearing isatin scaffold: synthesis, molecular modeling and biological evaluation as anticancer agents

Article abstract of DOI:10.1007/s11164-017-3019-z

Abstract: Derivatives of isatin have been reported to possess cytotoxic effects against different human carcinoma cell lines. A series of new isatin-linked chalcones was synthesized starting from isatin. Most of the newly synthesized compounds were screened for their in vitro anticancer activity against human breast (MCF-7), liver (HepG-2), and colon (HCT-116) cancer cell lines. All the tested compounds exhibited antitumor activity, with IC₅₀ ranging from 2.88 to 62.88?μM in comparison to the reference drug used in this study, Imatinib. Compounds 2–5 were the most active, with IC₅₀ ranging from 2.88 to 18.12?μM for the three cell lines, while compound 7b also showed moderate activity against HepG-2, MCF-7 and HCT-116 with IC₅₀ 13.95, 31.66 and 11.78?μM, respectively. Furthermore, compound 7d showed high activity against HepG-2 cells with IC₅₀ 12.84?μM. Compound 4 was shown to be the most potent against both HepG-2 and HCT-116 cell lines, while compound 2 is the most potent against MCF-7. The compounds were also screened for their cytotoxic activity against normal breast cell line MCF-12A, and were found to possess mild cytotoxicity. A docking study was performed for the most active compounds in this study, 2–5, inside the active site of CDK2. All the docked compounds have shown favorable binding interactions and energy scores. Compound 4 has proved to be the best in binding interactions and energy score. These findings may explain the cytotoxic activity of the target compounds. Graphical Abstract: A novel series of isatin-linked chalcones was synthesized. The target compounds were evaluated for their cytotoxic activity towards human breast (MCF-7), liver (HepG-2), colon (HCT-116) cancer cell lines and (MCF-12A) normal breast cell line.

Full text of DOI:10.1007/s11164-017-3019-z

Res Chem Intermed
DOI 10.1007/s11164-017-3019-z
New chalcones bearing isatin scaffold: synthesis,
molecular modeling and biological evaluation
as anticancer agents
Yousry A. Ammar1 Eman A. Fayed2 Ashraf H. Bayoumi3
•
•
•
Rogy R. Ezz2 Mansour S. Alsaid4 Aiten M. Soliman5
Mostafa M. Ghorab4,5
•
•
•
Received: 8 March 2017 / Accepted: 23 June 2017
Ó Springer Science+Business Media B.V. 2017
Abstract Derivatives of isatin have been reported to possess cytotoxic effects against
different human carcinoma cell lines. A series of new isatin-linked chalcones was
synthesized starting from isatin. Most of the newly synthesized compounds were
screened for their in vitro anticancer activity against human breast (MCF-7), liver
(HepG-2), and colon (HCT-116) cancer cell lines. All the tested compounds exhibited
antitumor activity, with IC50 ranging from 2.88 to 62.88 lM in comparison to the
reference drug used in this study, Imatinib. Compounds 2–5 were the most active, with
IC50 ranging from 2.88 to 18.12 lM for the three cell lines, while compound 7b also
showed moderate activity against HepG-2, MCF-7 and HCT-116 with IC50 13.95, 31.66
Electronic supplementary material The online version of this article (doi:10.1007/s11164-017-3019-z)
contains supplementary material, which is available to authorized users.
&
&
&
Yousry A. Ammar
yossry@yahoo.com
Eman A. Fayed
alfayed_e@yahoo.com
Mostafa M. Ghorab
mmsghorab@yahoo.com; mghorab@ksu.edu.sa
Mansour S. Alsaid
msalsaid@ksu.edu.sa
1
2
Department of Chemistry, Faculty of Science (Boys), Al-Azhar University, Cairo, Egypt
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy (Girls), Al-Azhar
University, Cairo, Egypt
3
4
5
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy (Boys), Al-Azhar
University, Cairo, Egypt
Department of Pharmacognosy, College of Pharmacy, King Saud University,
P.O. Box 2457, Riyadh 11451, Saudi Arabia
Department of Drug Radiation Research, National Center for Radiation Research and
Technology, Atomic Energy Authority, Cairo, Egypt
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Y. A. Ammar et al.
and 11.78 lM, respectively. Furthermore, compound 7d showed high activity against
HepG-2 cells with IC50 12.84 lM. Compound 4 was shown to be the most potent against
both HepG-2 and HCT-116 cell lines, while compound 2 is the most potent against
MCF-7. The compounds were also screened for their cytotoxic activity against normal
breast cell line MCF-12A, and were found to possess mild cytotoxicity. A docking study
was performed for the most active compounds in this study, 2–5, inside the active site of
CDK2. All the docked compounds have shown favorable binding interactions and
energy scores. Compound 4 has proved to be the best in binding interactions and energy
score. These findings may explain the cytotoxic activity of the target compounds.
Graphical Abstract A novel series of isatin-linked chalcones was synthesized. The
target compounds were evaluated for their cytotoxic activity towards human breast
(MCF-7), liver (HepG-2), colon (HCT-116) cancer cell lines and (MCF-12A) normal
breast cell line.
70
60
50
40
IC50 (µM) MCF-7 cells
30
IC50 (µM) HepG-2 cells
20
IC50 (µM) HCT-116 cells
10
IC50 (µM) MCF-12A cells
0
Keywords Isatin Á Chalcones Á Indolines Á MCF-7 Á HepG-2 Á Anticancer
Introduction
Cancer remains a major medical challenge worldwide. It can be treated by surgery,
radiation, chemotherapy, and hormonal and biological therapy [1–3]. However, the
method of treatment depends on the location and the progression of the disease.
Chemotherapeutic agents are mainly used in cases of metastasis and hypoxic
tumors, but the major limitation to their use is toxicity [4, 5], which occurs due to
low selectivity of the current chemotherapeutic drugs [6]. Also, one of the major
side effects of chemotherapy is drug resistance, which may occur after a long period
of using the chemotherapeutic agent [7], which has led to a continuous search for
new anticancer agents offering both selectivity towards malignant cells and lowered
resistance. Isatin was first isolated from the fruits of the Cannonball tree Couroupita
guianensis [8]. This plant species has been used in folk medicine [9]. Derivatives of
isatin have been reported to possess cytotoxic effects against various human
carcinoma cell lines derived from breast, prostate [10], human acute lymphoblastic
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New chalcones bearing isatin scaffold: synthesis, molecular…
(A)
(B)
(C)
Fig. 1 Isatin-containing drugs
leukemia (MOLT-4) [11, 12], colon [13, 14] and lung [15, 16]. Significant interest
in the 2-oxoindoles derivatives as kinase inhibitors came after the disclosure of the
tyrosine kinase inhibitory properties and anti-angiogenesis properties of SU-5416
(Semaxanib, A) [17–20] and SU-11248 (Sunitinib, B) (Fig. 1); the latter molecule
has recently completed phase III clinical trials with success. On the other hand, the
structurally relevant SU-9516 (C) was reported as a potential CDK inhibitor which
induces apoptosis in colon carcinoma cells [21, 22]. Moreover, indolinones with
their dual receptor tyrosine kinase and cyclin/CDK complex inhibitory properties
have been reported to treat tumor cell proliferation [23].
Chalcones are secondary metabolite precursors of flavonoids and isoflavonoids.
The major factors contributing to the increased interest in exploring their pharma-
cological activities are their good safety profile, their possibility of oral administration
[24, 25] and easy synthesis. Chalcones have been reported to possess several
biological activities such as anti-inflammatory [26], antibacterial [27], antifungal [28]
and antitumor [29–32]. The double bond of the enone system in chalcones was found
to be essential for their anticancer activity [33, 34]. The advantage of chalcones is the
low probability of interacting with DNA and the decrease in the risk of mutagenicity as
a common side effect of current chemotherapeutic agents [35]. Literature surveys
revealed that the structural modifications of chalcones mostly focused on the
replacement of the phenyl rings with heterocyclic rings and polyaromatic groups
[36, 37], the introduction of different substituents on the phenyl moieties [38] and
cyclization of the chalcone to give rigid analogs [39].
In an attempt to improve the chalcone’s anticancer activity, isatin was
introduced, generating a class of isatin–chalcone hybrids. The isatin group is fixed,
and diversity was created by introducing different substituents on the other ring. The
target compounds were evaluated against MCF-7, HepG-2 and HCT-116 cancer cell
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Y. A. Ammar et al.
lines, and the most active compounds were docked inside the active site of CDK2/
CyA [40] (cyclin-dependent kinase 2/cyclin A), to determine their possible
mechanism of action as anticancer agents.
Experimental
Chemistry
All melting points were measured on an Electrothermal LA9000 series Digital melting
point apparatus and were uncorrected. IR spectra were determined using the KBr disc
technique on a NikoletIR 200 FT-IR Spectrophotometer at the Pharmaceutical
Analytical Unit, Faculty of Pharmacy, Al-Azhar University, Egypt, and values are
1
represented in cm-1. The HNMR and 13CNMR spectra were recorded on Gemini
300 MHz, and Mercury 400 MHz NMR Spectrometers at the Main Chemical Warfare
Laboratories, Chemical Warfare Department, Ministry of Defense, Cairo, Egypt.
DMSO-d6 was used as solvent, and chemical shifts were measured in d ppm, relative to
TMS as an internal standard. Mass spectrum was recorded at 70 eV on a DI-50 unit of a
Shimadzu GC/MS-QP5050A Spectrometer at the Regional Center for Mycology and
Biotechnology (RCMB), at Al-Azhar University, represented as m/z (relative
abundance %) (formula). Element analysis (C, H, N) were also carried out at
Regional Center for Mycology and Biotechnology, and the values were found to be
within 0.4% of the theoretical ones unless otherwise indicated. The progress of the
reaction was monitored by TLC using TLC sheets pre-coated with UV fluorescent
silica gel Merck 60 F254 plates and was visualized using a UV lamp.
3-[(2-(4-Aminophenyl)-2-oxoethylidene] indolin-2-one (4)
To a solution of 3 (2.82 g, 0.01 mol) in ethanol (30 mL), conc. HCl (2 dps) was
added, the mixture was refluxed for 30 min, cooled and treated with ice-cold water,
and the obtained product was filtered and crystallized from ethanol. Dark red powder;
yield (86%); m.p.: 170–172 °C; IR: t/cm-1 = 3455, 3328, 3213 (NH2 & NH), 2940,
2833 (aliph.), 1706 (2CO).1H NMR: d/ppm = 6.38 [s, 2H, NH2], 6.60 [d, 2H,
J = 6 Hz, Ar–H], 6.84 [d,1H J = 6 Hz, indolin-7], 6.90 [t, 1H, indolin-6], 7.26 [t,
1H, indolin-5], 7.58 [s, 1H, CH=], 7.76 [d, 2H, J = 6 Hz, indolin-4], 7.85 [d, 2H,
J = 6 Hz, Ar–H], 10.69 [s, 1H, NH]. 13C NMR; 113.38, 120.70, 121.99, 126.83,
128.63, 131.84, 132.35, 134.54, 144.63, 155.29 (Ar–C ? C=C), 166.81, 188.39
(2CO). Mass spectrum exhibited a molecular ion peak at m/z 264 (M?, 23.21%) with
265 (M?1, 4.05%) with a base peak at m/z 236; Anal. Calcd. for C16H12N2O2
(264.28): C, 72.72; H, 4.58; N, 10.6; Found: C, 72.94; H, 4.63; N, 10.75.
4-(2-(2-Oxoindolin-3-ylidene)acetyl)phenyl)acetamide (5)
To a solution of 3 (2.82 g, 0.01 mol) in acetic acid (30 mL), conc. HCl (2 mL) was
added, the mixture was refluxed for 30 min. cooled and treated with ice-cold water,
and the obtained product filtered and crystallized from ethanol. Orange powder;
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New chalcones bearing isatin scaffold: synthesis, molecular…
yield (78%); m.p.: 250–252 °C; Rf = 0.60; IR: t/cm-1 = 3423, 3230 (2NH), 3080
(arom.), 2890, 2817 (aliph.), 1703 (CO), 1603 (C=C).1H NMR: d/ppm = 2.09 [s,
3H, CH3], 6.88 [d, 1H, J = 9 Hz, indolin-7], 6.95 [t, 1H, indolin-6], 7.32 [t, 1H,
indolin-5], 7.68 [s, 1H,CH=], 7.79 [d, 2H, J = 9 Hz, Ar–H], 7.96 [d, 1H, J = 9 Hz,
indolin-4], 8.05 [d, 2H, J = 9 Hz, Ar–H], 10.75, 10.36 [s, 2H, 2NH]. 13C NMR:
23.5 (CH3), 119.9 (2), 127.4 (2), 129.0 (2), 129.90 (2), 134.02 (2), 138.31 (2),
138.42, 154.04 (C=C), 159.02 (CO), 170.50 (CO), 179.73 (CO). Mass spectrum
exhibited a molecular ion peak at m/z 306 (M?, 0.91%) with a base peak at m/z: 43;
Anal. Calcd. for C18H14N2O3 (306.32): C, 70.58; H, 4.61; N, 9.15; Found: C, 70.81;
H, 4.69; N, 9.27.
General procedure for the synthesis of compounds 6a–d
A mixture of isatin (1.47 g, 0.01 mol), the corresponding Schiff bases (0.01 mol)
and diethyl-amine (3 mL) in ethanol (30 mL) was stirred for 5 h., left overnight,
cooled and treated with ice-cold water, and the obtained product was filtered and
crystallized from benzene.
3-[2-(4-Benzylideneamino) phenyl)-2-oxoethyl]-3-hydroxyindolin-2-one (6a)
Pale yellow crystal; yield (83%); m.p.: 125–127 °C; Rf = 0.52; IR: t/
cm-1 = 3362–3500 (br. OH & NH), 3050 (arom.), 1715 (2CO), 1600 (CN);
1H NMR d/ppm = 3.40, 4.03 [2d, J = 12 Hz, 2H, CH2], 6.10 [s, 1H, OH],
6.60–7.92 [m, 14H, Ar–H ? N=CH], 10.20 [s, 1H, NH]. Mass spectrum exhibited a
molecular ion peak at m/z 370 (M?, 1.54%) with a base peak at m/z: 103; Anal.
Calcd. for C23H18N2O3 (370.40): C, 74.58; H, 4.90; N, 7.56; Found: C, 79.81; H,
4.98; N, 7.72.
3-Hydroxy-3-[2-(4-(2-hydroxybenzylideneamino)phenyl)-2-oxoethyl]indolin-2-one
(6b)
Orange powder; yield (83%); m.p.: 218–220 °C; Rf = 0.45; IR: t/cm-1 = 3380,
3204 (NH& 2OH), 3059 (arom.), 2900, 2808 (aliph.), 1695 (2 CO), 1611 (CN);
1H NMR: d/ppm = 3.60, 4.09 [2d, 2H J = 18 Hz, CH2], 6.03 [s, 1H, OH],
6.79–7.79 [m, 12H, Ar–H], 8.97 [s, 1H, CH=N], 10.23, 12.62 [2 s, 2H, NH &OH].
13C NMR; 46.18 (CH2), 73.49 (C3-isatin), 109.84, 117.15, 119.78, 121.56, 129.37,
129.98, 132.19, 133.05, 134.68, 134.66, 143.38, 152.99 (Ar–C), 160.75 (CN),
165.35 (C–OH), 178.77, 195.98 (2 CO). Mass spectrum exhibited a molecular ion
peak at m/z 386 (M?, 1.21%) with a base peak at m/z: 224; Anal. Calcd. for
C23H18N2O4 (386.40): C, 71.49; H, 4.70; N, 7.25; Found: C, 71.66; H, 4.81; N, 7.38.
3-Hydroxy-3-[2-(4-(4-methoxybenzylideneamino)phenyl)-2oxoethyl)indolin-2-one
(6c)
Yellowish brown powder; yield (65%); m.p.: 185–187 °C; Rf = 0.49; IR: t/
cm-1 = 3364 (br. NH & OH), 3065 (arom,), 2952, 2891 (aliph.), 1725, 1672 (2
123

Y. A. Ammar et al.
CO), 1599 (CN).1H NMR: d/ppm = 3.86 [s, 3H, OCH3], 3.58, 4.07 [2d, 2H,
J = 18 Hz, CH2], 6.02 [s, 1H, OH], 6.78–7.91 [m, 12H, Ar–H], 8.53 [s, 1H,
CH=N], 10.22 [s, 1H, NH]. 13C NMR: 46.12 (OCH3), 55.94 (CH2), 73.52 (C3-
indoline), 109.86, 114.83, 121.55, 121.58, 124.06, 129.87, 131.39, 132.27, 133.66,
143.43, 156.63, 162.14 (Ar–C), 162.81 (CN), 178.85, 195.90 (2CO). Mass spectrum
exhibited a molecular ion peak at m/z: 400 (M?, 0.85%) with a base peak at m/z 239;
Anal. Calcd. for C24H20N2O4 (400.43): C, 71.99; H, 5.03; N, 7.00; Found: C, 72.18;
H, 5.09; N, 7.13.
3-Hydroxy-3-[2-(4-nitrobenzylideneaminophenyl)-2-oxoethyl] indolin-2-one (6d)
Orange powder; yield (53%); m.p.: 220–222 °C; Rf = 0.73; IR: t/cm-1 = 3377,
3191 (NH & OH), 3067 (arom.), 2889 (aliph.), 1695 (2 CO), 1600 (CN).1H NMR:
d/ppm = 3.60, 4.09 [2d, 2H, J = 18 Hz, CH2], 6.04 [s, 1H, OH], 6.77 [d, 1H,
J = 9 Hz, indolin-7], 6.87 [t, 1H, indolin-6], 7.15 [t, 1H, indolin-5], 7.28 [d, 2H,
J = 6 Hz, Ar–H], 7.39 [d, 1H, J = 9 Hz, indolin-4], 7.97 [d, 2H, J = 6 Hz, Ar–H],
8.17 [d, 2H, J = 6 Hz, Ar–H], 8.42 [d, 2H, J = 6 Hz, Ar–H], 8.81 [s, 1H, CH=N],
10.24 [s, 1H, NH].13C NMR: 45.28 (CH2), 73.53 (C3-indoline), 109.74, 112.90,
121.41, 123.87, 124.53, 129.15, 130.75, 132.61, 140.53, 143.48, 151.09, 154.24,
155.36, 162.14 (Ar–C), 161.47 (CN), 179.03, 192.81 (2CO). Mass spectrum
exhibited a molecular ion peak at m/z: 415 (M?, 0.40%) with a base peak at m/z 150;
Anal. Calcd. for C23H17N3O3 (415.40): C, 66.50; H, 4.12; N, 10.12. Found: C,
66.73; H, 4.18; N, 10.31.
General procedure for the synthesis of compounds 7a–d
1st method: a mixture of 3 (2.82 g, 0.01 mol), and aldehyde derivatives (0.01 mol)
in ethanol (30 mL) and acetic acid (3 mL) was refluxed for 2 h. The precipitated
solid was filtered and crystallized from ethanol.
2nd method: a mixture of 4 (2.64 g, 0.01 mol), and aldehyde derivatives
(0.01 mol) in ethanol (30 mL) and acetic acid (3 mL) was refluxed for 2 h. The
precipitated formed was filtered and crystallized from ethanol.
3rd method: compound 6 (0.01 mol) in ethanol (30 mL), to which conc. HCl
(2 mL) was added, the mixture was refluxed for 30 min, cooled and the obtained
product was filtered and crystallized from ethanol.
3-[2-(4-(2-Hydroxybenzylindeneamino) phenyl)-2-oxoethylidene] indolin-2-one
(7a)
Dark orange powder; yield (78%); m.p.: 170–172 °C; Rf = 0.68; IR: t/
cm-1 = 3379, 3196 (OH & NH), 3050 (arom.), 2950 (aliph.), 1711 (2 CO), 1601
(CN); 1H NMR: d/ppm = 6.60–7.91 [m, 13H, Ar–H ? C=CH], 8.51 [s, 1H,
CH=N], 10.67, 10.24 [2 s, 2H, NH, OH].13C NMR: 110.56, 117.67, 119.83, 119.92,
120.70, 122.56, 122.73, 125.13, 126.83, 128.89, 129.59, 131.84, 132.35, 133.03,
136.85, 144.64, 155.22 (Ar–C ? C=C), 161.17 (CN), 168.81 (C–OH), 188.39,
192.11 (2CO). Mass spectrum exhibited a molecular ion peak at m/z 368 (M?,
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New chalcones bearing isatin scaffold: synthesis, molecular…
32.27%) with a base peak at m/z: 116; Anal. Calcd. for C23H16N2O3 (368.38): C,
74.99; H, 4.38; N, 7.60; Found: C, 75.12; H, 4.42; N, 7.69.
3-[2-(4-(4-Methoxybenzylideneamino) phenyl)-2-oxoethylidene] indoline-2-one
(7b)
Yellow powder; yield (69%); m.p.: 110–112 °C; Rf = 0.63; IR: t/cm-1 = 3362
(NH), 3063 (arom.), 2955, 2847 (aliph.), 1725, 1675 (2 CO), 1595 (CN). 1H NMR:
d/ppm = 3.84 [s, 3H, OCH3], 7.07–8.00 [m, 13H, Ar–H ? CH=], 8.55 [s, 1H,
N=CH], 10.20 [s, 1H, NH]. 13C NMR: 55.80 (OCH3), 112.70, 118.71, 119.81(2),
119.90 (3), 129.01 (2), 129.92 (3), 130.02 (2), 132.75 (2), 138.31, 138.51, 142.32,
162.71 (2), 170.14 (CO), 196.05 (CO). Mass spectrum exhibited a molecular ion
peak at m/z 382 (M?, 10.42%) with a base peak at m/z: 236; Anal. Calcd. for
C24H18N2O3 (382.13): C, 75.38; H, 4.74; N, 7.33. Found: C, 75.46; H, 4.81; N, 7.45.
3-[2-(4-(4-Chlorobenzylideneamino) phenyl)-2-oxoethylidine] indolin-2-one (7c)
Yellow powder; yield (64%); m.p.: 200–202 °C; Rf = 0.78; IR: t/cm-1 = 3372
(NH), 3062 (arom.), 2879 (aliph.), 1670, 1647 (2 CO), 1591 (CN), 1490 (C=C).
1H NMR: d/ppm = 7.32–8.24 [m, 13H, Ar–H ? CH=], 8.68 [s, 1H, N=CH], 10.02
[s, 1H, NH]. 13C NMR: 118.31 (2), 119.74, 128.02 (2), 129.04 (2), 129.05 (2),
130.01, 130.06 (2), 130.10 (2), 130.2, 134.65, 138.39, 138.71, 139.23, 157.13,
165.01 (CN), 170.62 (CO), 196.96 (CO). Mass spectrum exhibited a molecular ion
peak at m/z 388 (M ? 2, 2.53%), 386 (M?, 3.84%) with a base peak at m/z 97; Anal.
Calcd. for C23H15ClN2O2 (386.83): C, 68.23; H, 4.23; N, 6.92. Found: C, 68.74; H,
4.31; N, 7.08.
3-[2-(4-(4-Nitrobenzylideneamino) phenyl)-2-oxoethylidene] indoline-2-one (7d)
Yellowish powder; yield (64%); m.p.: 120–122 °C; Rf = 0.81; IR: t/cm-1 = 3385
(NH), 3050 (arom.), 2850 (aliph.), 1724 (2CO), 1597 (CN). 1H NMR d/
ppm = 7.41–8.40 [m, 13H, Ar–H ? CH=], 8.83 [s, 1H, N=CH], 10.20 [s, 1H,
NH]. 13C NMR: 111.01, 119.32, 120.00 (2), 121.98, 122.76, 128.89, 130.02 (2),
130.31 (2), 131.75 (2), 133.73, 136.41, 139.62 (2), 142.43, 151.41, 156.68, 160.31
(CN), 170.50 (CO), 189.73 (CO). Mass spectrum exhibited a molecular ion peak at
m/z 397 (M?, 2.56%) with 399 (M ? 2, 1.27%), 398 (M ? 1, 1.36%), with a base
peak at m/z: 337; Anal. Calcd. for C23H15N3O4 (397.38): C, 66.73; H, 3.80; N,
10.57; Found: C, 66.76; H, 4.15; N, 10.36.
2-[4-(2-(2-Oxoindolin-3-ylidine) acetyl) phenyl] isoindolin-1, 3-dione (8)
A mixture of 4 (2.64 g, 0.01 mol) and phthalic anhydride (0.01 mol) in acetic acid
(20 mL) was refluxed for 3 h., cooled and treated with ice-cold water, and the
obtained product was filtered and crystallized from ethanol. Dark orange powder;
yield (71%); m.p.: 215–217 °C; Rf = 0.42; IR: t/cm-1 = 3193 (NH), 3050 (arom.),
1
2827 (aliph.), 1718 (CO). H NMR: d/ppm = 6.87–8.24 [m, 13H, Ar–H ? CH=],
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Y. A. Ammar et al.
10.80 [s, 1H, NH]. 13C NMR: 113.56, 121.76, 121.94, 125.16, 125.75, 126.25,
127.56, 128.12, 128.33, 129.806, 130.48, 131.17, 132.81, 134.87, 138.85, 141.32,
146.00, 158.15 (Ar–C), 161.13, 188.20 (CO). Mass spectrum exhibited a molecular
ion peak at m/z 394 (M?, 21.92%) with a base peak at m/z: 236; Anal. Calcd. for
C24H14N2O4 (394.38): C, 73.09; H, 3.58; N, 7.10; Found: C, 73.35; H, 3.54; N, 7.21.
50-(4-Aminophenyl)-20, 40-dihydro spiro (indoline-3, 30-pyrazol)-2-one (9)
A mixture of 4 (2.64 g, 0.01 mol) and hydrazine hydrate (0.05 mol) in ethanol
(30 mL) and acetic acid (3 mL) was refluxed for 3 h., cooled and treated with ice-
cold water, and the obtained product was filtered and crystallized from ethanol.
Yellow crystal; yield (60%); m.p.: 230–232 °C; Rf = 0.39; IR: t/cm-1 = 3418,
3261, 3166 (NH2, 2NH), 3026 (arom.), 2819 (aliph.), 1650 (CO), 1596 (CN).
1H NMR: d/ppm = 3.38 [s, 2H, CH2], 4.72 [s, 1H, NH], 5.59 [s, 2H, NH2], 6.70 [d,
1H, J = 9 Hz, indolin-7], 7.53 [t, 1H, indolin-6], 7.74 [t, 1H, indolin-5], 8.02–8.11
[m, 4H, Ar–H], 8.14 [d, 1H, J = 9 Hz, indolin-4], 9.92 [s, 1H, NH]. 13C NMR:
44.11 (CH2), 83.71, 118.75, 119.40, 120.32, 129.14, 129.31, 130.01(2), 130.12 (2),
134.04, 138.35, 138.73, 157.54 (CN), 165.85 (CO). Mass spectrum exhibited a
molecular ion peak at m/z 278 (M?, 55.83%) with a base peak at m/z 219; Anal.
Calcd. for C16H14N4O (278.31): C, 69.05; H, 5.07; N, 20.13; Found: C, 68.99; H,
4.95; N, 19.94.
3-[2-(4-Aminophenyl)-2-(phenylhydrazanoethylidene] indolin-2-one (10)
A mixture of 4 (2.64 g, 0.01 mol) and phenyl hydrazine (0.01 mol) in ethanol
(30 mL) and acetic acid(3 mL) was refluxed for 3 h., cooled and treated with ice-
cold water, and the obtained product was filtered and crystallized from ethanol.
Orange red powder; yield (66%); m.p.: 185–187 °C; Rf = 0.36; IR: t/cm-1 = 3157
1
(br. 2NH & NH2), 3050 (arom.), 2822 (aliph.), 1680 (CO), 1598 (CN). HNMR: d/
ppm = 6.90–7.55 [m, 14H, Ar–H ? CH=], 7.36 [s, 2H, NH2], 10.99, 12.73 [2 s, 2H,
2NH]. 13CNMR: 116.19, 116.70, 117.25, 120.04, 122.52, 125.07, 125.16, 125.67,
126.41, 126.72, 127.64, 127.94, 130.51, 135.55, 136.25, 140.39, 142.92, 161.67 (Ar–
C), 161.69 (CN), 162.02 (CO). Mass spectrum exhibited a molecular ion peak at m/
z 354 (M?, 19.96%) with a base peak at m/z: 98; Anal. Calcd. for C22H18N4O
(354.40): C, 74.56; H, 5.12; N, 15.81; Found: C, 74.71; H, 5.19; N, 15.97.
Biological activity
Cell lines
Human breast (MCF-7), hepatocellular (Hep-G2), colon (HCT-116) carcinoma cells
and breast (MCF-12A) normal cell lines were obtained from the American Type
Culture Collection (ATCC, Rockville, MD, USA). The cells were grown in RPMI-
1640 medium, supplemented with 10% inactivated fetal calf serum and 50 lg/ml
gentamycin. The cells were maintained at 37 °C in a humidified atmosphere with
5% CO2.
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New chalcones bearing isatin scaffold: synthesis, molecular…
Evaluation of the antitumor activity by MTT assay
The viability of control and treated cells was evaluated using the MTT assay in
triplicate. The MTT assay is a laboratory test and a standard colorimetric assay (an
assay which measures changes in color) for measuring cellular growth, Yellow
MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide, a tetrazole)
is reduced to purple formazan in the mitochondria of living cells, and a
solubilization solution (dimethyl sulfoxide) is added to dissolve the insoluble
purple formazan product into a colored solution. Briefly, three tumor cell lines were
seeded in 96-well plates containing 100 lL of the growth medium at a density of
1 9 104 cells/well. Cells were permitted to adhere for 24 h until confluence, washed
with PBS, and then treated with different concentrations of compounds in fresh
maintenance medium from 50 to 1.56 lg/mL and incubated at 37 °C for 24 h. A
control of untreated cells was used in the absence of a test compound. Untreated
cells used as negative control. Serial two-fold dilutions of the tested compounds
were added into a 96-well tissue culture plate using a multichannel pipette
(Eppendorf, Germany). After treatment (24 h), the culture supernatant was replaced
by fresh medium. Then, the cells in each well were incubated at 37 °C with 100 ll
of MTT solution (5 mg/mL) for 4 h. After incubation, the MTT solution was
removed, then 100 lL of DMSO was added to each well. The absorbance was
detected at 570 nm using a microplate reader (Sun Rise TECAN, USA). The
absorbance of untreated cells was considered as 100%. The results were determined
by three independent experiments [41].
Data analysis
The percentage cell viability was calculated as follows = [1 - (ODt/
ODc)] 9 100%, where ODt is the mean optical density of wells treated with the
tested compound and ODc is the mean optical density of untreated cells. The tested
compounds were compared using the IC50 value, i.e., the concentration of an
individual compound leading to 50% cell death that was estimated from graphical
plots of surviving cells versus compound concentrations.
Molecular modeling and docking
The molecular model of the new isatin derivatives was built using standard bond
angles and lengths, with the Molecular Operating Environment (MOE) software
suite 10.2008. Following geometry optimization, a systematic conformational
˚
search was carried out to RMS gradient of 0.05 A with energy minimization of the
resultant conformations employing the ConfSearch module implemented in MOE.
All molecular mechanics computations were performed with the Merck Force Field
(MMFF94s). The crystallographic structure of CDK2/CyA complex was obtained
from the Protein Data Bank (PDB ID: 4BCQ). Hydrogen atoms were added to the
enzyme and partial charges were calculated. Validation followed by docking of the
compounds into the active site was carried out after removing the co-crystallized
ligand. The target protein was kept rigid, while the ligands were left free to explore
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Y. A. Ammar et al.
the conformational space inside the enzyme cavity; 50 separate docking simulations
were run using default parameters, and the conformations were chosen based on the
combination of E conformation, S score data, and appropriate fitting with the
relevant amino acids in the binding pocket.
Results and discussion
Chemistry
The synthetic routes of the proposed compounds are outlined in Schemes 1, 2 and 3.
p-amino-acetophenone has two nucleophilic centers, so its reaction with isatin
depends on the reaction conditions. Condensation of p-aminoacetophenone with
isatin in acetic acid afforded 3-[4-acetylphenyl) imino] indolin-2-one 2 [42]. On the
other hand, crossed aldol condensation of p-aminoacetophenone with isatin in
absolute ethanol, in the presence of 3 mL N,N-diethylamine (DEA) as a catalyst,
gave 3-[2-(4-aminophenyl)-2-oxoethyl]-3-hydroxyindolin-2-one 3 [43]. This reac-
tion undergoes aldol condensation through the reaction of the acetyl group of p-
aminoacetophenone with the carbonyl function of isatin. Dehydration of compound
3 using dilute alcoholic hydrochloric acid yielded the expected a,b-unsaturated
carbonyl compound, 3-[(2-(4-aminophenyl)-2-oxoethyl-idene]indolin-2-one 4. The
IR of 4 displayed absorption bands at 3455, 3328, and 3213 cm-1 due to the NH2
and NH groups, and another at 1706 cm-1 due to the CO group. 1HNMR exhibited
the lack of the CH2 and OH groups and the presence of signals due to NH2 and
aromatic protons. On repeating this reaction using a mixture of hydrochloric and
Scheme 1 Synthesis of the isatin derivatives 2–5
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New chalcones bearing isatin scaffold: synthesis, molecular…
Scheme 2 Synthesis of isatin-chalcone derivatives 6, 7
acetic acids (1:3), dehydration and acetylation of the amino group furnished the N-
(4-(2-(oxoindolino-3-ylidene) acetamide 5 (Scheme 1). The IR of 5 revealed the
lack of bands corresponding to the OH and NH2 groups and showed broad bands
around 3423 and 3230 cm-1 corresponding to the two NH groups and a band at
1703 cm-1 due to three CO groups. 1H NMR showed the lack of the CH2, OH and
NH2 groups and the presence of a singlet at 2.09 ppm for the CH3, besides two
singlet signals at 10.36 and 10.75 ppm corresponding to the two NH protons.
In the present work, a novel series of various substituted Schiff bases bearing the
indolinone scaffold in their molecules were synthesized using three pathways; (1)
condensation of compound 4 with some aromatic aldehydes afforded the
corresponding Schiff bases 7a–d; (2) condensation of the hydroxyl ethyl derivative
3 with aromatic aldehydes in acetic acid; and (3) the reaction of Schiff bases derived
of p-aminoacetophenone with isatin furnished the hydroxyl ethyl derivatives 6a–
d which were subjected to dehydration with ethanolic hydrochloric acid to yield the
same Schiff bases 7a–d (Scheme 2). The IR of 6c revealed the presence of OH and
NH as a broad band around 3364 cm-1and two carbonyl groups at 1725 and
1672 cm-1. 1H NMR exhibited two doublets at 3.58 and 4.07 ppm assigned to CH2
protons, two singlets at 3.86 and 8.53 ppm specific for OCH3 and azomethine
protons, respectively, an other two singlets at 6.02 and 10.22 ppm due to OH and
NH protons. 13C NMR exhibited three signals at 46.12, 55.94 and 73.52 ppm due to
OCH3, CH2 and C3-indoline, besides two CO groups at 178.85 and 195.90 ppm.
The mass spectrum showed a molecular ion peak at m/z = 400, corresponding to a
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Y. A. Ammar et al.
Scheme 3 Synthesis of the isatin derivatives 8–10
molecular formula C24H20N2O4. The IR of 7b showed the disappearance of
absorption band corresponding to the NH2 group and the presence of another at
3362 cm-1 due to the NH group, and 1725 and 1675 cm-1 due to the two CO
groups. 1H NMR exhibited two singlets at 3.84 and 8.55 ppm specific for the
methoxy and azomethine protons, in addition to a singlet at 10.20 ppm exchange-
able with D2O due to the NH proton. The mass spectrum showed a molecular ion
peak at m/z = 382 corresponding to the molecular formula C24H18N2O3.
In an attempt to collect indolinone and isoindolinone in one structure,
condensation of the hydroxyethyl derivative 3 with phthalic anhydride in acetic
acid furnished 2-[4-(2-(2-oxoindolin-3-ylidene)acetyl)phenyl]isoindoline-1,3-dione
8. The IR of 8 is characterized by the absence of the amino and hydroxyl groups
found in the starting material and the presence of absorption bands at 3193 and
1718 cm-1 corresponding to the NH and CO groups. 13C NMR revealed aromatic
carbons in the region 113.56–158.15 and four carbonyl carbons at 161.13 and
188.20 ppm. The mass spectrum showed a molecular ion peak at m/z = 394,
corresponding to the molecular formula C24H14N2O4.
Finally, cyclo-condensation of a, b-unsaturated ketone 3 with hydrazine in
ethanol containing acetic acid gave the spiro-pyrazoline derivative 9. The IR of 9
showed bands at 3418, 3261, and 3166 cm-1 due to NH2 and two NH groups, and a
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New chalcones bearing isatin scaffold: synthesis, molecular…
123

Y. A. Ammar et al.
1
band around 1650 cm-1 attributed to CO. H NMR showed a doublet at 3.38 ppm
due to the CH2 and a singlet at 4.72 ppm attributed to the NH. On the other hand,
the phenyl hydrazono derivative 10 was obtained by the treatment of hydroxyl ethyl
derivative 3 or the unsaturated ketone 4 with hydrazine hydrate in ethanol
containing acetic acid. The IR of 10 showed stretching frequencies around
1
3157 cm-1 due to NH2 and two NH groups. H NMR showed the absence of CH2
protons and the presence of three singlet signals at 7.37, 10.99 and 12.73 ppm for
the NH2 and NH protons. 13C NMR exhibited two signals at 161.69 and 162.67 ppm
due to spiro pyrazoline derivative CN and CO groups.
Biological activity
Most of the newly synthesized compounds were screened for their in vitro
anticancer activity against human breast (MCF-7), liver (HepG-2), colon (HCT-116)
cancer cell lines and breast (MCF-12A) normal cell line. The tested compounds
showed favorable activity against MCF-7 cells with IC50 ranging from 2.88 to
51.08 lM. Compounds 2, 3 and 5 revealed higher antitumor activity (IC50 2.88,
9.95 and 7.93 lM) against the MCF-7 cell line when compared with the standard
drug Imatinib (IC50 12.29 lM), as shown in Table 1 and Fig. 2. For the HepG-2 cell
line, compounds 2–5 also showed higher activity (IC50 9.23, 10.20, 5.22 and
7.70 lM), in comparison to Imatinib (IC50 11.16 lM) (Table 2; Fig. 2). The same
compounds, 2–5, showed potent activity against the HCT-116 cell line in which the
IC50 were 9.38, 11.4, 2.95 and 8.65 lM, respectively (Table 3; Fig. 2). In addition,
compounds 7b and 7d showed anticancer activity against the HepG-2 cell line with
IC50 of 13.95 and 12.84 lM, respectively. However, all the compounds show mild
cytotoxicity against the normal breast cell line (MCF-12A) (Table 4). Furthermore,
the most active compound against the HCT-116 cell line was compound 4 with IC50
of 2.95 lM followed by 5, 2 and 3 as shown in Table 3 and Fig. 2. Also,
compounds 7b and 7d showed moderate activity against the same cell line with IC50
of 11.78 and 15.04 lM, respectively.
70
60
50
40
IC50 (µM) MCF-7 cells
30
IC50 (µM) HepG-2 cells
20
IC50 (µM) HCT-116 cells
10
IC50 (µM) MCF-12A cells
0
Fig. 2 IC50 of the tested compounds against human MCF-7, HepG-2, HCT-116 and MCF-12A cell lines.
(Color figure online)
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New chalcones bearing isatin scaffold: synthesis, molecular…
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Y. A. Ammar et al.
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New chalcones bearing isatin scaffold: synthesis, molecular…
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Y. A. Ammar et al.
Molecular docking
CDKs (cyclin-dependent kinases) are a group of serine-threonine protein kinases
which play a significant role in cell cycle progression [44–46]. CDKs together with
cyclins (Cy) manage cell division, cycle progression, neuronal function, differenti-
ation, and apoptosis. These CDKs control cell division and survival through
phosphorylation of proteins. Malignant tissues suffer from either overexpression of
cyclins or CDK inhibitory proteins (CDKIs) suppression, resulting in tumor
progression. From the literature review, it was found that indolinones posses cyclin/
CDK complex inhibitory properties [23], which make them useful targets for
chemotherapy [40].
In our present study to determine the possible mechanism of action of the target
compounds, molecular docking of compounds 2–5 was performed in the active site
of CDK2/CyA to explore their binding interactions with the amino acids inside the
active site. The protein data bank file (PDB: 4BCQ) was selected for this purpose.
The file contains CDK2/CyA enzyme co-crystallized with N,1,4,4-tetramethyl-
8-((4-(4-methylpiperazin-1-yl)phenyl)amino)-4,5-dihydro-1H-pyrazolo
(4,3-H)
quinazoline-3-carboxamide (Pha-848125). All docking procedures were carried
out using MOE software 10.2008. The docking protocol was verified by re-docking
of the co-crystallized ligand in the vicinity of the active site of the enzyme with the
energy score (S) = -10.82 kcal mol-1and root mean standard deviation = 0.90.
The co-crystallized ligand interacts with the active site of CDK2 by two
interactions: Leu 83 with a hydrogen bond of 2.90 A° and Lys 33 with a hydrogen
bond of 2.79 A°. All the docked compounds were fitted in the active site of the
enzyme and the results of energy scores (S), as well as amino acids interactions, are
listed in Figs. 3, 4, 5, 6 and Table 5. It is obvious that compound 4 shows the best
Fig. 3 2D ligand interaction of compound 4 with the active site amino acids of 4BCQ
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New chalcones bearing isatin scaffold: synthesis, molecular…
Fig. 4 3D Docking of compound 4 (S = -10.04 kcal mol-1) in the active site of 4BCQ
Fig. 5 2D ligand interaction of compound 5 with the active site amino acids of 4BCQ
docking score (-10.04 kcal mol-1) and binding interactions, as it binds to the
active site of CDK2 by three interactions: Leu 83 with a hydrogen bond of 1.94,
2.56 A° and Phe 80 with a hydrophobic bond of 2.47 A°.
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Y. A. Ammar et al.
Fig. 6 3D Docking of compound 5 (red) (S = -8.91 kcal mol-1) in the active site of 4BCQ showing
the surface of the binding pocket to give an estimation of any possible steric hindrance. (Color
figure online)
Table 5 Docking results of the most active compounds within CDK2/CyA active site
˚
Length (A)
Compound
Ligand
Energy score (S) (Kcal/mol)
Amino acids
Interacting groups
-10.82
Leu 83
Lys 33
Leu 83
Lys 33
Tyr 15
Leu 83
Lys 33
Leu 83
Leu 83
Phe 80
Leu 83
Lys 33
NH
CO
NH
CO
NH
NH
CO
NH
CO
Phe
NH
CO
2.90
2.79
2.37
2.94
2.03
2.19
2.65
1.94
2.56
2.47
1.64
2.09
2
-9.15
3
4
-10.03
-10.04
5
-8.91
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New chalcones bearing isatin scaffold: synthesis, molecular…
Conclusion
A new series of isatin-linked chalcones were synthesized. The target compounds
were designed and synthesized as potential cyclin-dependent kinase 2 inhibitors
(CDKI) and evaluated for their cytotoxic activity against three human carcinoma
cell lines, MCF-7, HepG-2, and HCT-116, and MCF-12A normal breast cell line.
Compounds 2–5 were found to be the most potent in this study compared to
Imatinib, the reference drug. The acetyl derivative 2 showed the highest activity
against MCF-7 with IC50 2.88 lM followed by the acetamide derivative 5, then, for
compounds 3 and 4, the activity was diminished but still stronger than the standard
compound. The IC50 was 9.95 lM for the saturated compound 3 and 18.12 lM for
the unsaturated compound 4. On the other hand, the most active compound against
the HepG-2 cell line was 4 with IC50 5.22 lM followed by 5, 2 and 3. Compound 2
was found to be the most potent against MCF-7, while compound 4 was the most
potent for both the HepG-2 and HCT-116 cell lines. The docking study showed that
all the docked compounds exhibited similar binding interactions as those previously
reported with the ligand when docked into the active site of CDK2/CyA. Compound
4 showed the best energy score and binding interactions, which suggests that these
compounds may possibly act as CDK inhibitors and thus may participate in
anticancer activity.
Acknowledgements The authors are thankful to the Deanship of the Scientific Research and Research
Center, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia.
References
1. S. Kameshwaran, V. Suresh, G. Arunachalam, S. Kanthlal, M. Mohanraj, Int. Res. J. Pharm. 3, 246
(2012)
2. D.A.A. El Ella, M.M. Ghorab, H.I. Heiba, A.M. Soliman, Med. Chem. Res. 21, 2395 (2012)
3. M.M. Ghorab, F.A. Ragab, H.I. Heiba, A.M. Soliman, Eur. J. Med. Chem. 117, 8 (2016)
4. C. Peitzsch, R. Perrin, R.P. Hill, A. Dubrovska, I. Kurth, Int. J. Radiat. Biol. 90, 636 (2014)
5. T. Sugrue, N.F. Lowndes, R. Ceredig, Stem Cells 32, 2188 (2014)
6. K.G. Daniel, P. Gupta, R.H. Harbach, W.C. Guida, Q.P. Dou, Biochem. Pharmacol. 67, 1139 (2004)
7. Y. Luqmani, Med. Princ. Pract. 14, 35 (2005)
¨
8. J. Bergman, J.O. Lindstrom, U. Tilstam, Tetrahedron 41, 2879 (1985)
9. K. Kirtikar, B.B.I.M. Indian Medicinal Plants, Vols. 8, 2nd edn. (Delhi, 1918)
10. L. Matesic, J.M. Locke, J.B. Bremner, S.G. Pyne, D. Skropeta, M. Ranson, K.L. Vine, Bioorg. Med.
Chem. 16, 3118 (2008)
11. N. Gao, L. Kramer, M. Rahmani, P. Dent, S. Grant, Mol. Pharmacol. 70, 645 (2006)
12. F. Wang, Y. Fang, T. Zhu, M. Zhang, A. Lin, Q. Gu, W. Zhu, Tetrahedron 64, 7986 (2008)
13. M.E. Lane, B. Yu, A. Rice, K.E. Lipson, C. Liang, L. Sun, C. Tang, G. McMahon, R.G. Pestell, S.
Wadler, Cancer Res. 61, 6170 (2001)
14. S. Patyna, A.D. Laird, D.B. Mendel, A.M. O’Farrell, C. Liang, H. Guan, T. Vojkovsky, S. Vasile, X.
Wang, J. Chen, Mol. Cancer Ther. 5, 1774 (2006)
15. H.-H. Li, X.-H. Zhang, J.-Z. Tan, L.-L. Chen, H. Liu, X.-M. Luo, X. Shen, L.-P. Lin, K.-X. Chen, J.
Ding, Acta Pharmacol. Sin. 28, 140 (2007)
16. W.M. Eldehna, A. Altoukhy, H. Mahrous, H.A. Abdel-Aziz, Eur. J. Med. Chem. 90, 684 (2015)
17. R.D. Connell, Expert Opin. Ther. Pat. 13, 737 (2003)
18. J. Ma, S. Li, K. Reed, P. Guo, J.M. Gallo, J. Pharmacol. Exp. Ther. 305, 833 (2003)
123

Y. A. Ammar et al.
19. P. Marzola, A. Degrassi, L. Calderan, P. Farace, C. Crescimanno, E. Nicolato, A. Giusti, E. Pesenti,
A. Terron, A. Sbarbati, Clin. Cancer Res. 10, 739 (2004)
20. J.J. Haddad, Saudi Pharm. J. 20, 103 (2012)
21. C. Munoz, F. Adasme, J.H. Alzate-Morales, A. Vergara-Jaque, T. Kniess, J. Caballero, J. Mol. Graph.
Model. 32, 39 (2012)
22. A.K. Abdel-Aziz, E.M. Mantawy, R.S. Said, R. Helwa, Exp. Neurol. 283, 129 (2016)
23. X. Li, P. Huang, J.J. Cui, J. Zhang, C. Tang, Bioorg. Med. Chem. Lett. 13, 1939 (2003)
24. C.S. Mizuno, S. Paul, N. Suh, A.M. Rimando, Bioorg. Med. Chem. Lett. 20, 7385 (2010)
25. B.W. Vanhoecke, F. Delporte, E. Van Braeckel, A. Heyerick, H.T. Depypere, M. Nuytinck, D. De
Keukeleire, M.E. Bracke, In Vivo 19, 103 (2005)
26. J.-H. Cheng, C.-F. Hung, S.-C. Yang, J.-P. Wang, S.-J. Won, C.-N. Lin, Bioorg. Med. Chem. 16,
7270 (2008)
27. Y.-T. Liu, X.-M. Sun, D.-W. Yin, F. Yuan, Res. Chem. Intermed. 39, 1037 (2013)
´
´
28. M. Sortino, P. Delgado, S. Juarez, J. Quiroga, R. Abonıa, B. Insuasty, M. Nogueras, L. Rodero, F.M.
Garibotto, R.D. Enriz, Bioorg. Med. Chem. 15, 484 (2007)
29. A.-M. Katsori, D. Hadjipavlou-Litina, Curr. Med. Chem. 16, 1062 (2009)
30. G. Achanta, A. Modzelewska, L. Feng, S.R. Khan, P. Huang, Mol. Pharmacol. 70, 426 (2006)
31. A. Modzelewska, C. Pettit, G. Achanta, N.E. Davidson, P. Huang, S.R. Khan, Bioorg. Med. Chem.
14, 3491 (2006)
32. K. Kundu, M. Tyagi, B.S. Patro, S. Chattopadhyay, S.K. Nayak, Org. Comm. 7, 85 (2014)
33. B. Letafat, R. Shakeri, S. Emami, S. Noushini, N. Mohammadhosseini, N. Shirkavand, S.K.
Ardestani, M. Safavi, M. Samadizadeh, A. Letafat, Iran. J. Basic Med. Sci. 16, 1155 (2013)
34. P. Hinnen, F. Eskens, Br. J. Cancer 96, 1159 (2007)
35. D. Elias, M. Beazely, N. Kandepu, Curr. Med. Chem. 6, 1125 (1999)
36. H.-B. Shi, S.-J. Zhang, Q.-F. Ge, D.-W. Guo, C.-M. Cai, W.-X. Hu, Bioorg. Med. Chem. Lett. 20,
6555 (2010)
37. L. Firoozpour, N. Edraki, M. Nakhjiri, S. Emami, M. Safavi, S.K. Ardestani, M. Khoshneviszadeh,
A. Shafiee, A. Foroumadi, Arch. Pharm. Res. 35, 2117 (2012)
38. H. Aryapour, G.H. Riazi, A. Foroumadi, S. Ahmadian, A. Shafiee, O. Karima, M. Mahdavi, S.
Emami, M. Sorkhi, S. Khodadady, Med. Chem. Res. 20, 503 (2011)
39. Y.S. Shan, J. Zhang, Z. Liu, M. Wang, Y. Dong, Curr. Med. Chem. 18, 523 (2011)
40. P. Lan, W.-N. Chen, G.-K. Xiao, P.-H. Sun, W.-M. Chen, Bioorg. Med. Chem. Lett. 20, 6764 (2010)
41. A. Wilson and J. Masters, (Oxford University Press, Oxford, 2000)
42. M. Gupta, S. Raman, S. Srivastava, Asian J. Chem. 16, 779 (2004)
43. S. Pandeya, S. Smitha, S. Singh, A. Kumar, Acta Ciencia Indica Chem. 33, 549 (2007)
44. Y.M. Ahn, L. Vogeti, C.-J. Liu, H.K. Santhapuram, J.M. White, V. Vasandani, L.A. Mitscher, G.H.
Lushington, P.R. Hanson, D.R. Powell, Bioorg. Med. Chem. 15, 702 (2007)
45. G. Traquandi, M. Ciomei, D. Ballinari, E. Casale, N. Colombo, V. Croci, F. Fiorentini, A. Isacchi, A.
Longo, C. Mercurio, J. Med. Chem. 53, 2171 (2010)
46. M.G. Brasca, N. Amboldi, D. Ballinari, A. Cameron, E. Casale, G. Cervi, M. Colombo, F. Colotta, V.
Croci, R. D’Alessio, J. Med. Chem. 52, 5152 (2009)
123