Design, synthesis and structure-activity relationship of novel quinoxaline derivatives as cancer chemopreventive agent by inhibition of tyrosine kinase receptor

Article abstract of DOI:10.1016/j.ejmech.2013.07.049

The cancer chemopreventive activity of quinoxaline derivatives 1-20 has been evaluated by studying the inhibitory effect on Epstein-Barr virus early antigen (EBV-EA) activation. The quinoxaline derivatives 1-20 showed inhibitory effect on EBV-EA activation without cytotoxicity on Raji cells. All compounds exhibited dose dependent inhibitory activities, most of them showed significant activity at 1000 mol ratio/12-O-tetradecanoylphorbol-13-acetate (TPA). Compounds 7 and 9 exhibited stronger inhibitory effects on the EBV-EA activation than that of the representative control, oleanolic acid, at the highest measured concentration. In addition, compounds 7-10 showed potent and selective inhibition of human tyrosine kinase (TRK) in liver cancer HepG2 and breast cancer MCF-7 cell lines similar to the positive control, doxorubicin.

Full text of DOI:10.1016/j.ejmech.2013.07.049

European Journal of Medicinal Chemistry 69 (2013) 115e124
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and structureeactivity relationship of novel
quinoxaline derivatives as cancer chemopreventive agent by
inhibition of tyrosine kinase receptorq
,
a
b
b,c
Shadia A. Galal a , Ahmed S. Abdelsamie , Salwa M. Soliman , Jeremie Mortier
,
*
Gerhard Wolber b, Mamdouh M. Ali d, Harukuni Tokuda e, Nobutaka Suzuki e, Akira Lida f,
Raghda A. Ramadan g, Hoda I. El Diwani a
a Department of Chemistry of Natural and Microbial Products, Division of Pharmaceutical and Drug Industries, National Research Center, Dokki,
12622 Cairo, Egypt
b Department of Pharmaceutical Chemistry, Institute of Pharmacy, Freie Universitaet Berlin, Koenigin Luise Strasse, Berlin 14195, Germany
c Department of Organic Chemistry, Free University Berlin, Takustraße 3, D-14195 Berlin, Germany
d Department Biochemistry, Division of Genetic Engineering and Biotechnology, National Research Centre, Dokki, 12622 Cairo, Egypt
e Department of Complementary and Alternative Medicine, R&D, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-machi,
Kanazawa, Japan
f Faculty of Agriculture, Kinki University, 3327-20 Naka-machi, Nara 631-8505, Japan
g Youssef Jameel Science and Technology Research Center, The American University in Cairo, New Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
The cancer chemopreventive activity of quinoxaline derivatives 1e20 has been evaluated by studying the
inhibitory effect on EpsteineBarr virus early antigen (EBV-EA) activation. The quinoxaline derivatives
1e20 showed inhibitory effect on EBV-EA activation without cytotoxicity on Raji cells. All compounds
exhibited dose dependent inhibitory activities, most of them showed significant activity at 1000 mol
ratio/12-O-tetradecanoylphorbol-13-acetate (TPA). Compounds 7 and 9 exhibited stronger inhibitory
effects on the EBV-EA activation than that of the representative control, oleanolic acid, at the highest
measured concentration. In addition, compounds 7e10 showed potent and selective inhibition of human
tyrosine kinase (TRK) in liver cancer HepG2 and breast cancer MCF-7 cell lines similar to the positive
control, doxorubicin.
Received 13 May 2013
Received in revised form
28 July 2013
Accepted 30 July 2013
Available online 13 August 2013
Keywords:
Synthesis
Quinoxalines
EpsteineBarr virus
Cancer chemopreventive activity
Docking
Ó 2013 Published by Elsevier Masson SAS.
Protein tyrosine kinase
1. Introduction
pathways that regulate cell growth, proliferation, and apoptosis.
Consequently, unregulated kinase activity can result in uncon-
The wide spectrum of biological activities including antitumor
activity of quinoxaline derivatives is known and well documented
in the literature [1e10]. The activity of quinoxalines as antitumor
agents may be due their ability of inhabitation of protein tyrosine
kinases as c-kit enzyme [11,12]. C-kit is a member of the family
protein tyrosine kinase III. Kinases are the key in cell signaling
trolled cellular growth and inappropriate regulation of apoptosis;
a key mechanism in oncogenesis. Quinoxalines are known to
display a good affinity to the ATP-binding site of c-kit tyrosine
protein [13e17].
On the other hand, the EpsteineBarr virus (EBV), also called
human herpes virus 4 (HHV-4), is a cancer-causing virus of the
herpes family which includes herpes simplex virus and cytomeg-
alovirus. On infecting the B-lymphocyte, the linear virus genome
circularizes and the virus subsequently persists within the cell as an
episome. It is also known to cause several lymphoproliferative
disorders and cancers, particularly Hodgkin’s disease, Burkitt’s
lymphoma, nasopharyngeal carcinoma, and central nervous system
lymphomas associated with HIV [18].
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-No Derivative Works License, which per-
mits non-commercial use, distribution, and reproduction in any medium, provided
the original author and source are credited.
* Corresponding author. Tel.: þ20 233371615; fax: þ20 233370931.
E-mail addresses: sh12galal@yahoo.com, sh12galal@hotmail.com (S.A. Galal).
0223-5234/$ e see front matter Ó 2013 Published by Elsevier Masson SAS.
http://dx.doi.org/10.1016/j.ejmech.2013.07.049

116
S.A. Galal et al. / European Journal of Medicinal Chemistry 69 (2013) 115e124
As a continuation to our previous work in discovering potent
cancer chemopreventive agents, the aim of the present investiga-
tion was the synthesis of new hydrazinylquinoxalinones as analogs
to compound 1 (c.f. Fig. 1) that showed a relative ratio of inhibition
of 9.2 with respect to TPA (100%) at the concentration of 1000 mol
ratio/TPA [19]. The activity of the new compounds was evaluated by
studying the inhibitory effect on EpsteineBarr virus early antigen
(EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-
acetate (TPA), which stimulates cell proliferation through rapid
activation of protein kinase C (PKC), followed by gradual degrada-
tion of the kinase [1].
2. Results and discussion
2.1. Chemistry
Quinoxalin-2,3(1H,4H)-dione (1) was synthesized by the
method of Obafemi and Pfeiferer [20]. Stirring of compound 1 with
phosphorus oxychloride in methylene chloride at room tempera-
ture gave 3-chloroquinoxalin-2(1H)-one (2) [19]. The X-ray struc-
ture of compound 2 was performed. (c.f. Fig. 2). Crystal data,
fractional atomic coordinates and equivalent isotropic thermal
parameters, anisotropic displacement parameters and geometric
parameters of 2 are given in Supplementary data [21e24].
3-Hydrazinylquinoxalin-2(1H)-one (3) [25,26] can be obtained
by reaction of compound 1 or 2 with hydrazine hydrate in ethanol
(c.f. Scheme 1). Hydrazones, especially the ones containing the
azomethine (eNHN]CHe) protons, have been reported to possess
anticancer activity [27,28] and constitute an important class of
compounds for new drug development [29,30]. Scheme 2 was
performed to produce the target hydrazones.
Fig. 2. X-ray structure of compound 2.
and 1H NMR spectra of compounds 7e10 proved that alkylation took
place selectively at position 1.
As was known from the literature, annelation in heterocyclic-
fused quinoxalines was successful in causing selectivity and
strong affinity towards the receptor [5,6,19]. Prompted by the
previous findings, annelated quinoxalines were synthesized
through Scheme 3. 3-Hydrazinylquinoxalin-2(1H)-one (3) was used
as starting agent to prepare triazino[4,3-a]quinoxaline derivatives
(11e14) and triazolo[4,3-a]quinoxaline (15, 16) by treating com-
pound 3 with ethyl bromoacetate, chloroacetyl chloride, ethyl
oxamate, diethyl oxalate, ethyl chloroformate and triethyl ortho-
formate, respectively. (c.f. Scheme 3).
Spiroquinoxalines were prepared to observe the effect of such
structure on activity. Reactions of methyl isothiocyanate or benzoyl
isothiocyanate with 3-hydrazinylquinoxalin-2(1H)-one (3) were
investigated and gave the 50-thioxo-1H-spiro[quinoxaline-2,30-
[1,2,4]triazolidin]-3(4H)-one derivatives (17, 18) (Scheme 4). The
structure of compounds 17, 18 was deduced from their elemental
analyses, IR, and 1H NMR data. Their mass spectra displayed mo-
lecular ion peaks at appropriate m/z values. The formation of spiro
compounds could be explained by the mechanism involving the
imine carbon atom of quinoxaline in the reaction, with the nitrogen
becoming electron-donating amine nitrogen. The formation of
spiro-compounds was decidedly confirmed previously by single-
crystal X-ray analyses by Mamedov et al. [32].
Treatment of compound (3) with acetone, N,N-dimethylamino-
benzaldehyde or 2,6-dimethoxybenzaldehyde in ethanol yielded
hydrazinylquinoxalinones as 3-(2-(propan-2-ylidene)hydrazinyl)
quinoxalin-2(1H)-one (4) [31], 3-(2-(4-(dimethylamino)benzyli-
dene)hydrazinyl)quinoxalin-2(1H)-one(5) and 3-(2-(2,6-dimethoxy-
benzylidene)hydrazinyl)quinoxalin-2(1H)-one (6), respectively (c.f.
Scheme 2). Treatmentof compounds 5 or 6 with equimolar amountof
allyl bromide or ethyl chloroacetate yielded selectively the 1-
alkylated products 7e10 respectively (c.f. Scheme 2). This was indi-
cated by the disappearance of NH of quinoxaline in compounds 7e10
in both IR and 1H NMR spectra with respect to compound 5 and 6.
Also, the significant lower frequency shift of
n
(C]O) from 1687 cmꢀ1
in compound 5 to 1646 cmꢀ1 in compound 7 and to 1647 cmꢀ1 in
Treatment of compound 3 with diethyl malonate or diethyl
succinate in dimethyl formamide yielded quinoxalinyl pyrazolidine
derivative (19) and quinoxalinyltetrahydo pyridazine derivative
(20) respectively (c.f. Scheme 5).
compound 8 was observed. In compounds 9 and 10, the n(C]O)
signals appeared at 1649 cmꢀ1 and 1637 cmꢀ1 respectively with
remarkable lower frequency shift with respect to compound 6
(1694 cmꢀ1). The significant frequency shifts in C]O moiety in IR in
addition tothe disappearanceofNHbands of quinoxalinemoietyinIR
2.2. Inhibition of EBV-EA activation assay
A primary screening test was carried out using a short-term
in vitro synergistic assay on EBV-EA activation [33e35]. The
inhibitory effect of quinoxaline derivatives 1e20 on the EBV-EA
activation induced by TPA and the associated viability of Raji cells
was listed in Table 1. In this assay, all the tested compounds showed
inhibitory effects on EBV-EA activation and no cytotoxicity on Raji
cells. All compounds exhibited dose dependent inhibitory activities,
and the viability percentages of Raji cells treated with the tested
compounds (1e20) were 50, 60 or 70% at the highest concentration
of 1000 mol ratio/TPA. As shown in Table 1, at concentration
1000 mol ratio/TPA the inhibitory activity of compounds 7 and 9
were stronger than that of oleanolic acid, known as a representative
anti-tumor promoting agent [36]. Compound 8 had comparative
activity with oleanolic acid. The relative ratio of inhibition of
N
N
N
N
O
O
Fig. 1. Compound 1.

S.A. Galal et al. / European Journal of Medicinal Chemistry 69 (2013) 115e124
117
H
NH2
NH2
N
O
O
N
Cl
O
i
ii
N
H
N
H
1
2
iii
iii
H
N
N
NH2
N
H
O
3
Reagents and conditions:
(i) oxalic acid, 4N HCl, reflux. (ii) POCl 3, methylene chloride, stirring, 4h, room temp. (iii) Hydrazine
hydrate 98%, ethanol, stirring.
Scheme 1. Synthesis of compounds 1e3.
compound 7 with respect to TPA (100%) was 10.0, 41.6, 73.2, 100% at
the concentrations of 1000, 500, 100 and 10 mol ratio/TPA,
respectively (Table 1); meaning 90.0, 58.4, 26.8 and 0.0% inhibition
of the EBV-EA activation by TPA respectively. Compounds 9 and 8
showed 89.2, 58.0, 26.5 and 0%; and 87.3, 52.4, 21.4 and 0% inhi-
bition of the EBV-EA activation by TPA respectively, at concentra-
tions of 1000, 500, 100 and 10 mol ratio/TPA.
chemopreventive action of the quinoxaline derivatives and explore
their potential interactions with the receptor.
Enzyme inhibition study of compounds 1, 3e10, 13e16 and 18
against human tyrosine kinase was performed in two different hu-
man tumor cell lines HepG2 and MCF7 cell lines (c.f. Table 2). The
results showed that most of the tested compounds exhibited
inhibitory potencies against human TRK. Compounds 7, 9, 10 and 8
showed to possess more potent inhibition against human TRK in
HepG2 and MCF7 cell lines than that of the untreated cells (Table 2)
and of similar potency to the positive control, doxorubicin. Com-
pounds 1, 16 and 18 had no activity against TRK, while the other
compounds had low affinity and moderate activity in both cell lines.
2.3. Protein kinase inhibition
The study of protein tyrosine kinase inhibition by the synthe-
sized compounds was performed in order to rationalize the
O
H
H
N
H
N
N
N
N
N
ii
iii
N
NH2
N
N
H
O
N
N
H
O
N
H
O
O
5
3
6
iv
i
iv
N
H
N
H
N
O
N
H
N
N
N
N
N
N
R
O
N
N
H
O
N
R
O
O
7, R = CH2-CH=CH2
8, R = CH2COOC2H5
4
9, R = CH2-CH=CH2
10, R = CH2COOC2H5
Reagent and conditions: i) Acetone, ethanol, reflux. ii)
N,N-dimethylbenzaldehyde, ethanol.
iii) 2,6-dimethoxybenzaldehde, ethanol, reflux. iv) Allyl bromide or, ethyl chloroacetate,
K2CO3 anhydrous, DMF, stirring.
Scheme 2. Synthesis of compounds 4e10.

118
S.A. Galal et al. / European Journal of Medicinal Chemistry 69 (2013) 115e124
O
OH
HN
NH
N
N
N
N
N
N
H
O
N
H
O
12
13
ii)
iii)
OH
HO
H
N
O
NH
N
N
N
N
i)
NH2
N
iv)
N
N
H
O
N
H
O
N
H
O
3
11
vi)
v)
14
O
NH
N
N
N
N
N
O
N
H
N
H
O
15
16
Reagents and conditions: i) Ethyl bromoacetate, DMF, reflux. ii) chloroacetyl chloride DMF,
reflux. iii) ethyl oxamate DMF, reflux. iv) diethyl oxalate DMF, reflux. v) ethyl chloroformate
DMF, reflux. vi) triethyl orthoformate DMF, reflux.
Scheme 3. Synthesis of compounds 11e16.
H
H
N
HN
O
N
O
N
N
NH2
i
S
+
RNCS
O
N
N
O
NHR
N
H
O
N
H
N
H
i)
N
H
3
19
H
N
R = CH3 or COC6H5
N
NH2
O
N
H
O
NH
N
N
O
HN
H
N
ii)
N
H
O
S
3
N
N
H
R
N
H
O
20
17, R = CH3
18, R =COC6H5
Reagents and conditions:
i)
Diethyl malonate, DMF, Et3N,reflux, 10 h. ii) Diethyl succinate, DMF, reflux,
12h.
Reagents and conditions:
i) toluene reflux, 12h.
Scheme 4. Synthesis of compounds 17 and 18.
Scheme 5. Synthesis of compounds 19 and 20.

S.A. Galal et al. / European Journal of Medicinal Chemistry 69 (2013) 115e124
119
Table 1
pocket. These residues were on one side VAL603, LEU799, PHE811
and THR670, and on the other side ILE653, LEU783, LEU647 and
ILE808. In the center of the cavity, two hydrogen bonds were
believed to stabilize the compound involving the residues GLU640
and ASP810. A complete exclusion volume coat drew boundaries
that determine the capacity of the binding site.
The crystal structures of c-kit receptor protein (PDB code: 1T46)
was prepared and used for the docking study. The docking program
GOLD was used to find plausible docking poses for four compounds
displaying the best inhibitory potencies: 7, 8, 9 and 10. The gener-
ated docking conformations were prioritized based on their ability
to fulfill the above mentioned 3D pharmacophore features. These
results were highly correlated to the chemopreventive activity of
the compound series.
Compounds 7 (Fig. 3), which displayed the highest chemo-
preventive activity, was deeply embedded into the ATP-binding
cleft of c-kit. The docking pose met the pharmacophore features
and good complementarities were observed between the docked
ligand and the hydrophobic subsites of the enzymatic cavity. In the
first subsite, the dimethylamino group of 7 was stabilized by
VAL603, LEU799, PHE811 and THR670, while the quinoxaline
moiety interacted with the residues ILE653, LEU783, LEU647 and
ILE808 of the second enzyme hydrophobic subsite. The hydrogen
bonds were identified between GLU640 and ASP810 and the
hydrazone group of the ligand (Fig. 3).
Relative ratioa of EBV-EA activation with respect to positive control (100%) in
presence of compounds 1e21 and oleanolic acid.
Compounds#
% to control (% viability) 500 mol
100 mol
10 mol
1000 mol ratio/TPAb,c
ratio/TPAb ratio/TPAb ratio/TPAb
1
2
3
4
5
6
7
8
14.2(60)
16.7(50)
14.9(60)
13.2(60)
13.9(60)
16.8(60)
10.1(60)
12.7(60)
10.8(60)
13.5(60)
13.2(60)
13.6(60)
15.0(60)
14.2(60)
15.6(60)
18.6(60)
18.7(60)
19.1(60)
17.5(60)
16.7(60)
46.8
48.5
46.5
45.0
42.2
46.6
41.6
47.6
42.0
45.7
46.0
46.3
47.1
47.0
47.2
48.2
48.3
49.1
48.0
47.7
3 0.0
78.5
80.2
78.1
77.0
71.7
81.0
73.2
78.6
73.5
75.6
77.6
78.0
79.0
78.6
79.1
84.2
84.6
85.2
83.9
82.8
80.0
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
9
10
11
12
13
14
15
16
17
18
19
20
Oleanolic acid 12.7 (70)
a
Values represent percentages relative to the positive control value (100%).
TPA concentration was 20 ng/mL (32 pmol/mL).
Values in parentheses are the viability percentages of Raji cells.
b
c
The docking conformation of compounds 8, 9 and 10, superposed
to the one of compound 7 in Fig. 4, showed the same hydrogen bond
interactions. The substitution of the dimethylamino group, in para
position of the phenyl ring (compounds 7 and 8), to two dimethoxy
groups in ortho position (compounds 9 and 10) affected the activity.
In order to decrease the steric hindrance, the dimethoxy groups
2.4. Molecular modeling: docking study
The molecular modeling study of the most potent compounds
into the protein tyrosine kinase c-kit was performed in order to
rationalize the chemopreventive action of the quinoxaline de-
rivatives and explore their potential interactions with the receptor
(c-kit) by generate plausible binding poses for the most active
compounds (7, 8, 9 and 10).
With the software LigandScout [37], a 3D interaction map was
generated from the ligand (Gleevec) co-crystallized in the protein
binding site (PDB code 1T46). The generated structure-based
pharmacophore translated the important binding features for the
inhibition of c-kit and was used to guide the selection of the more
plausible binding poses of the newly synthesized compounds. The
pharmacophore showed two main hydrophobic subsites accom-
modating the hydrophobic residues of the ligand in the binding
enforced the aromatic ring to rotate and lost the
P-stacking with
PHE811, which destabilized compounds 9 and 10. The effect of the
polar carboxylate group on compounds 8 and 10 was believed to be
unfavorable because of its orientation towards the hydrophobic
binding cleft. This molecular modeling study into the active site of c-
kit allowed to provide a rationale to the decreasing chemo-
preventive activity measured for 8, 9 and 10 compared to 7. Free NH
on the quinoxaline moiety and the presence of the dimethyl group
on the phenyl ring decreased the activity of compounds 4 and 5, but
was still close to the reference, which also emphasized the role of the
dimethyl group in hydrophobic interaction with the receptor.
Building up a tricyclic system, except for compounds 11 and 12, or
spiro compounds, had a negative effect on the activity.
Table 2
IC50 values of human TRK of the tested compounds in human liver cancer HepG2 and
breast cancer MCF-7 cell lines.
3. Conclusion
The cancer chemopreventive activity of the newly synthesized
quinoxaline derivatives 1e20 was estimated by studying their
inhibitory effects on EpsteineBarr virus early antigen (EBV-EA)
activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA).
The synthesized quinoxaline derivatives 1e20 showed dose
dependent inhibitory activities, most of them showed promising
activity at the highest concentration of 1000 mol ratio/TPA. Com-
pounds 7 and 9 showed stronger effect than that of oleanolic acid,
the reference inhibitor. Allyl or acetate substitution on the qui-
noxaline nitrogen, as well as the presence of N(CH3)2 at position 4
of the phenyl ring, showed a great effect on potency. The results of
TRK inhibition showed that compounds 7, 8, 9 and 10 had potent
activity in both HepG2 and MCF-7 cancer cell lines similar to
doxorubicin. Then, a molecular docking study of the four best in-
hibitors revealed potential binding mode into the c-kit receptor,
and explained the main structureeactivity relationships. Finally,
compound 7 was discovered to be highly potent and will be sub-
jected for further investigations as in vivo study.
Compound#
IC50 of TRK (pmol/ml)a
HepG2
MCF-7
1
3
4
5
6
7
8
9
10
13
14
15
16
18
NA
NA
9.00 ꢁ 0.07
15.00 ꢁ 1.38
9.80 ꢁ 0.08
7.50 ꢁ 0.06
2.65 ꢁ 0.03
4.50 ꢁ 0.04
3.50 ꢁ 0.03
4.10 ꢁ 0.04
18.00 ꢁ 1.60
20.00 ꢁ 2.00
25.00 ꢁ 1.40
NA
10.20 ꢁ 1.10
17.20 ꢁ 1.60
11.80 ꢁ 1.20
8.75 ꢁ 0.07
2.90 ꢁ 0.04
4.85 ꢁ 0.05
4.15 ꢁ 0.04
4.60 ꢁ 0.05
20.20 ꢁ 2.00
27.80 ꢁ 2.85
30.00 ꢁ 2.50
NA
NA
2.50 ꢁ 0.01
NA
3.20 ꢁ 0.02
Doxorubicin
Data were expressed as mean ꢁ S.E. of triplicate independent experiments.
a
Indicate the concentration of the tested compounds required to inhibit human
TRK by 50%. NA stands for no activity.

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S.A. Galal et al. / European Journal of Medicinal Chemistry 69 (2013) 115e124
Fig. 3. Plausible binding mode for compound 7 in 2D (up) and 3D (down) showing favorable hydrophobic interactions (yellow spheres) and two hydrogen bonds to GLU640 (green)
and ASP810 (red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
4. Experimental
mat SSQ 7000 (Thermo. Inst. Sys. Inc., USA) spectroscopy at 70 eV.
Dimethyl sulfoxide (DMSO), doxorubicin, penicillin, and strepto-
mycin were obtained from Sigma Chemical Company (Saint Louis,
MO, USA). Human Tyrosine kinase (TRK) ELISA kit was purchased
from Glory Science Co., Ltd (Del Rio, TX 78840, USA).
4.1. Physical measurements
Microanalyses, spectral data and X-ray structures of the com-
pounds were performed in the Microanalytical and X-ray Labora-
tories, National research centre, Cairo, Egypt. The IR spectra (4000e
400 cmꢀ1) were recorded using KBr pellets in a Jasco FT/IR 300E
Fourier transform infrared spectrophotometer on a Perkin Elmer
FT-IR 1650 (spectrophotometer). The 1H and 13C NMR spectra were
recorded using Joel EX-270 MHz and 500 MHz NMR spectropho-
tometers. Chemical shifts are reported in parts per million (ppm)
from the tetramethylsilane resonance in the indicated solvent.
Coupling constants are reported in Hertz (Hz); spectral splitting
partners are designed as follows: singlet (s); doublet (d); triplet (t);
multiplet (m). The mass spectra were carried out using Finnigan
4.1.1. Preparation of compounds 4e6
A mixture of compound 3 (50 mmol) in absolute ethanol
(100 mL) and acetone, N,N-dimethylaminobenzaldehyde or 2,6-
dimethoxybenzaldehyde, respectively (50 mmol) was refluxed for
7e9 h. After reaction completion, the solvent was evaporated under
vacuum and the solid residue obtained was washed with water,
filtered off and recrystallized from ethanol.
4.1.1.1. 3-[2-(Propan-2-ylidene)hydrazinyl]quinoxalin-2(1H)-one (4)
[31]. Reflux time 8 h, Rf ¼ 0.15 (petroleum ether/ethyl acetate, 1:3),

S.A. Galal et al. / European Journal of Medicinal Chemistry 69 (2013) 115e124
121
Fig. 4. Plausible binding modes for compounds 7, 8, 9 and 10 superposed in the binding site of c-kit. Yellow spheres indicate hydrophobic features, red and green arrows hydrogen
bond acceptor and donor respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
yield 78.7%, m.p. 256e258 ꢂC (lit 256 ꢂC) IR (cmꢀ1): 3451 (NH
quinoxaline), 3274 (NH, hydrazinyl), 1674(C]O), 1617(C]N), 1570
(C]N and C]C). 1H NMR (270 MHz, DMSO-d6): 1.99 (t, 3H, CH3),
2.09 (t, 3H, CH3), 6.95 (m, 1H), 7.18 (m, 2H), 7.42 (m, 1H), 9.28 (s, 1H,
NH hydrazinyl,D2O exchangeable), 12.41 (s, 1H, NH quinoxaline,
D2O exchangeable). MS: m/z: 216 (Mþ, 15%), 176 (m*, 100). Anal.
Calcd for C11H12N4O (FW: 216.10): C, 61.10; H, 5.59; N, 25.91. Found:
C, 61.15; H, 5.65; N, 25.85.
(4.2 mmol) in 10 mL of DMF was stirred for 10e12 h. The inorganic
salt was filtered off and the solution was evaporated under reduced
pressure. The precipitate obtained was washed with water and
crystallized from acetone.
4.1.2.1. 1-Allyl-3-(2-(4-(dimethylamino)benzylidene)hydrazinyl)qui-
noxalin-2(1H)-one (7). Reflux time ¼ 12 h, Rf ¼ 0.85 (petroleum
ether/ethyl acetate, 1:3), yield, 69.7%, m.p. 219e221 ꢂC. IR (cmꢀ1):
3255 (NH, hydrazide), 3071 (CH, aromatic), 3034 (CH]CH2),
2852(CH, aliphatic), 1646 (C]O), 1607(C]N), 1569 (C]N, C]C). 1H
NMR (500 MHz, DMSO-d6): 3.27 (s, 6H, N(CH3)2), 4.87 (d, 2H,
J ¼ 2 Hz, CH2), 5.06 (dd, 1H), 5.16 (dd, 1H), 5.92 (m, 1H), 6.72 (d, 2H,
J ¼ 5 Hz), 7.19 (d, 2H, J ¼ 5 Hz), 7.55 (m, 1H); 7.61 (m, 3H); 8.39 (s,
1H); 10.94 (br., NH, D2O exchangeable). 13C NMR (270 MHz, DMSO-
d6): 40.5, 53.01, 111.8, 113.6, 118.2, 123.7, 124.7, 128.7, 129.4, 130.6,
131.2, 132.4, 142.8, 146.4, 150.7, 152.8, 153.6. MS: m/z: 334 (Mþ, 8%),
176 (m*, 100). Anal. Calcd for C20H21N5O (FW: 347.17): C, 69.14; H,
6.09; N, 20.16. Found: C, 69.11; H, 6.14; N, 20.18.
4.1.1.2. 3-(2-(4-(Dimethylamino)benzylidene)hydrazinyl)quinoxalin-
2(1H)-one (5). Reflux time 7 h, Rf ¼ 0.25 (petroleum ether/ethyl
acetate, 1:3), yield 71.1%, m.p. 250e253 ꢂC IR (cmꢀ1): 3423 (NH
quinoxaline), 3182 (NH hydrazide), 3056 (CH, aromatic), 2852 (CH,
aliphatic), 1687 (C]O), 1605 (C]N), 1571 (C]N, C]C). 1H NMR
(270 MHz, DMSO-d6): 2.97 (s, 6H, N(CH3)2), 6.77 (d, 2H, J ¼ 7.6 Hz),
7.15 (m, 2H); 7.6 (m, 4H); 8.49 (s, 1H); 10.91 (br., NH, D2O
exchangeable), 12.35 (s, NH, D2O exchangeable). 13C NMR
(270 MHz, DMSO-d6): 40.5, 111.5, 115.2, 123.4, 128.4, 129.1, 131.6,
142.6, 147.4, 150.89, 153.4, 154.5 MS: m/z 307 (Mþ, 5%), 176 (m*,
100). Anal. Calcd for C17H17N5O (FW: 307.14): C, 66.43; H, 5.58; N,
22.79. Found: C, 66.49; H, 5.61; N, 22.74.
4.1.2.2. Ethyl 2-(3-(2-(4-(dimethylamino)benzylidene)hydrazinyl)-2-
oxoquinoxalin-1(2H)-yl)acetate (8). Reflux time ¼ 10 h, Rf ¼ 0.5
(petroleum ether/ethyl acetate, 1:3), yield 81.3%, m.p. 210e213 ꢂC.
IR (cmꢀ1): 3264 (NH, hydrazide), 3036 (CH, aromatic), 2811 (CH,
aliphatic), 1752 (C]O), 1647 (C]O), 1608 (C]N), 1571 (C]N and
C]C). 1H NMR (500 MHz, DMSO-d6): 1.19 (t, 3H, CH3), 3.2 (s, 6H,
N(CH3)2), 3.84 (s, 2H, CH2), 4.16 (q, 2H, CH2), 7.02 (m, 2H), 7.25 (m,
2H), 7.33 (m, 2H), 7.57 (m, 1H), 7.91 (m, 1H), 8.88 (s, 1H), 11.36 (s, 1H,
NH, D2O exchangeable). 13C NMR (500 MHz, DMSO-d6): 14.23,
40.44, 49.2, 62.5, 111.8, 113.8, 122.3, 125.5, 127.7, 128.4, 129.3, 126.1,
130.33,132.63, 142.55,146.4,150.89,153.2,169.2. MS: m/z: 393 (Mþ,
10%), 176 (m*, 100). Anal. Calcd for C21H23N5O3 (FW: 393.18): C,
64.11; H, 5.89; N, 17.80. Found: C, 64.17; H, 5.87; N, 17.74.
4.1.1.3. 3-(2-(2,6-Dimethoxybenzylidene)hydrazinyl)quinoxalin-
2(1H)-one (6). Reflux time 9 h, Rf ¼ 0.7 (petroleum ether/ethyl
acetate, 1:3), yield, 67.9%, m.p. 220e222 ꢂC. IR (cmꢀ1): 3316 (NH
quinoxaline), 3205 (NH), 3046, 2999 (CH, aromatic), 2894, 2832
(CH, aliphatic), 1694 (C]O), 1612 (C]N), 1567 (C]N, C]C). 1H
NMR (270 MHz, DMSO-d6): 3.68 (s, 3H, OCH3), 3.71 (s, 3H, OCH3),
7.03e733 (m, 5H); 7.49 (m, 2H); 8.89 (s, 1H); 11.39 (s, NH, D2O
exchangeable), 12.43 (s., NH, D2O exchangeable). 13C NMR
(270 MHz, DMSO-d6): 56.4, 106.4, 110.0, 114.9, 122.6, 123.5, 125.8,
128.76, 131.13, 132.96, 133.7, 142.51, 146.2, 150.89, 157.6, 161.2 MS:
m/z 324 (Mþ, 17%), 176 (m*, 100). Anal. Calcd for C17H16N4O3 (FW:
324.12): C, 62.95; H, 4.97; N, 17.27. Found: C, 62.89; H, 5.03; N, 17.25.
4.1.2.3. 1-Allyl-3-(2-(2,6-dimethoxybenzylidene)hydrazinyl)quinox-
alin-2(1H)-one (9). Reflux time ¼ 11 h, Rf ¼ 0.25 (petroleum ether/
ethyl acetate, 1:3), yield 67%, m.p. 155e157 ꢂC. IR (cmꢀ1): 3264
(NH), 3062 (CH, aromatic), 2832 (CH, aliphatic), 1649 (C]O), 1606
(C]N), 1569 (C]N, C]C). 1H NMR (270 MHz, DMSO-d6): 3.80
4.1.2. Preparation of compounds 7e10
General procedure: A mixture of compound 5 or 6 (4 mmol),
K2CO3 (4.2 mmol) and allyl bromide or ethyl chloroacetate

122
S.A. Galal et al. / European Journal of Medicinal Chemistry 69 (2013) 115e124
(s, 3H, CH3), 3.82 (s, 3H, CH3), 4.20 (m, 2H, CH2), 5.16 (m, 2H, CH2),
6.37 (m, 1H, CH]), 7.04 (m, 3H), 7.38 (m, 3H), 7.60 (m, 1H); 8.91 (s,
1H); 11.43 (s, 1H, NH, D2O exchangeable). 13C NMR (270 MHz,
DMSO-d6): 52.5, 55.9, 110.3, 107.8, 113.6, 125.4, 128.1, 129.7, 130.61,
132.7, 134.13, 142.51, 143.5, 150.12, 153.4, 159.8. MS: m/z: 364 (Mþ,
40%), 176 (m*, 100). Anal. Calcd for C20H20N4O3 (FW: 364.15): C,
65.92; H, 5.53; N, 15.38. Found: C, 65.96; H, 5.50; N, 15.45.
m.p. 234e237 ꢂC IR (cmꢀ1): 3440 (OH), 3400 (NH quinoxaline), 3018
(CH, aromatic), 1671 (C]O), 1611 (C]N), 1581 (C]C). 1H NMR
(270 MHz, DMSO-d6): 7.28 (m, 3H), 7.36 (m, 1H), 8.7 (br., OH, D2O
exchangeable),12.18 (br., NH, D2O exchangeable).13C NMR (500 MHz,
DMSO-d6): 116.9, 121.3, 122.8, 125.5, 129.5, 133.9, 152.7, 153.4, 155.1,
157.5. MS: m/z: 230 (Mþ, 26%). Anal. Calcd for C10H6N4O3 (FW:
230.04): C, 52.18; H, 2.63; N, 24.34. Found: C, 52.16; H, 2.55; N, 24.39.
4.1.2.4. Ethyl 2-(3-(2-(2,6-dimethoxybenzylidene)hydrazinyl)-2-
oxoquinoxalin-1(2H)-yl)acetate (10). Reflux time ¼ 12 h, Rf ¼ 0.6
(petroleum ether/ethyl acetate, 1:3), yield 68.4%, m.p. 190e192 ꢂC.
IR (cmꢀ1): 3280 (NH, hydrazide), 3061 (CH, aromatic), 2826 (CH,
aliphatic),1748 (C]O),1637 (C]O),1602 (C]N),1570 (C]N, C]C).
1H NMR (270 MHz, DMSO-d6): 1.43 (t, 3H, CH3), 3.79 (s, 3H, CH3),
3.82 (s, 3H, CH3), 4.93 (s, 2H, CH2), 5.18 (q, 2H, CH2), 7.07 (m, 2H),
7.30 (m, 2H), 7.60 (m, 3H), 8.89 (s, 1H), 11.43 (s, 1H, NH hydrazide,
D2O exchangeable). 13C NMR (270 MHz, DMSO-d6): 14.7, 48.5, 60.8,
106.9, 110.9, 113.6, 124.9, 126.1, 128.6, 129.4, 131.21, 132.9, 142.55,
143.3, 146.9, 151.27, 153.1, 160.5, 167.22. MS: m/z: 350 (Mþ, 14%), 176
(m*,100). Anal. Calcd for C21H22N4O5 (FW: 410.16): C, 61.45; H, 5.40;
N, 13.65. Found: C, 61.41; H, 5.42; N, 13.70.
4.1.3.5. [1,2,4]Triazolo[4,3-a]quinoxaline-1,4 (2H,5H)-dione (15).
Rf ¼ 0.32 (petroleum ether/ethyl acetate, 1:3), yield 69%, m.p. 247e
249 ꢂC. IR (cmꢀ1): 3423 (NH), 3200 (NH), 3041 (CH, aromatic), 1682
(C]O), 1650 (C]O), 1608 (C]N), 1581 (C]C). 1H NMR (270 MHz,
DMSO-d6): 7.12 (m, 3H); 7.3 (m, 1H), 12.0 (br., NH(s), D2O
exchangeable). 13C NMR (270 MHz, DMSO-d6): 114.1, 124.9, 129.5,
132.1, 145.8, 151.3, 154.5. MS: m/z: 202 (Mþ, 26%). Anal. Calcd for
C9H6N4O2 (FW: 202.05): C, 53.47; H, 2.99; N, 27.71. Found: C, 53.39;
H, 2.84; N, 27.55.
4.1.3.6. [1,2,4]Triazolo[4,3-a]quinoxalin-4(5H)-one (16). Rf
¼
0.17
(petroleum ether/ethyl acetate, 1:3), yield 78%, m.p. 241e243 ꢂC. IR
(cmꢀ1): 3402 (NH), 3055 (CH, aromatic), 1681 (C]O), 1615 (C]N),
1524 (C]C). 1H NMR (270 MHz, DMSO-d6): 7.2 (m, 1H), 7.3 (m, 3H);
8.1 (m, 1H), 12.0 (br., NH, D2O exchangeable). 13C NMR (270 MHz,
DMSO-d6): 125.6, 128.2, 129.4, 132.3, 137.4, 144.5, 148.3, 157.4, 163.4.
MS: m/z: 186 (Mþ, 100%). Anal. Calcd for C9H6N4O (FW: 186.05): C,
58.06; H, 3.25; N, 30.09. Found: C, 58.10; H, 3.33; N, 30.05.
4.1.3. Preparation of compounds 11e16
A mixture of compound 3 (5 mmol) and ethyl bromoacetate,
chloroacetyl chloride, ethyl oxamate, diethyl oxalate, ethyl chlor-
oformate or triethyl orthoformate (5 mmol) was dissolved in DMF
(30 mL). The reaction mixture was stirred at 90 ꢂC for 8 h. After
reaction completion, the formed solid was filtered off. The crude
product obtained was washed with methanol and purified by
recrystallization from DMF/methanol (1:2) mixture.
4.1.4. Preparation of compounds 17 and 18
A solution of methyl isothiocyanate or benzoyl isothiocyanate
(5 mmol) in toluene (15 mL) was added to a solution of compound 3
(5 mmol) in toluene (15 mL). The reaction mixture was refluxed for
12 h. The solvent was evaporated under reduced pressure. The solid
was washed with cold water and recrystallized from ethanol.
4.1.3.1. 1-Hydroxy-3H-[1,2,4]triazino[4,3-a]quinoxalin-5
(6H)-one
(11). Rf ¼ 0.3 (petroleum ether/ethyl acetate, 1:3), yield 88.3%, m.p.
184e186 ꢂC. IR (cmꢀ1): 3567 (OH), 3438 (NH quinoxaline), 3220
(NH), 3032 (CH, aromatic), 1673 (C]O), 1606 (C]N), 1580 (C]C).
1H NMR (500 MHz, DMSO-d6): 7.09 (m, 4H); 7.33 (m, 1H), 12.06 (br.,
NH(s), OH, D2O exchangeable). 13C NMR (500 MHz, DMSO-d6): 82.5,
110.3, 118.6, 122.8, 125.98, 126.5, 142.22, 151.5, 155.23, 163.7. MS: m/
z: 216 (Mþ, 40%). Anal. Calcd for C10H8N4O2 (FW: 216.06): C, 55.55;
H, 3.73; N, 25.91. Found: C, 55.41; H, 3.81; N, 25.88.
4.1.4.1. 40-Methyl-50-thioxo-1H-spiro[quinoxaline-2,30-[1,2,4]tri-
azolidin]-3(4H)-one (17). Rf ¼ 0.42 (petroleum ether/ethyl acetate,
1:3), yield 58%, m.p. 173e175 ꢂC. IR (cmꢀ1): 3400e3219 (NH(s)),
2923 (CH, aromatic), 2875 (CH, aliph.), 1680 (C]O), 1647 (C]N,
thiadiazole moiety), 1609 (C]N), 1536 (C]C). 1H NMR (270 MHz,
DMSO-d6): 2.78 (s, 3H, CH3), 3.9 (s, 1H, NH, D2O exchangeable), 6.99
(m, 1H), 7.28 (m, 1H), 7.56 (br., NH, D2O exchangeable), 7.6 (m, 1H),
8.0 (m, 1H), 9.5 (br., NH, D2O exchangeable). 13C NMR (270 MHz,
DMSO-d6): 25.9, 83.8, 114.9, 118.2, 121.2, 125.9, 137.4, 145.1, 155.6,
167.2. MS: m/z: 249 (Mþ, 20%). Anal. Calcd for C10H11N5OS (FW:
249.07): C, 48.18; H, 4.45; N, 28.09; S, 12.86. Found: C, 48.22; H,
4.31; N, 28.22; S, 12.92.
4.1.3.2. 1H-[1,2,4]Triazino[4,3-a]quinoxaline-2,5 (3H,6H)-dione (12).
Rf ¼ 0.36 (petroleum ether/ethyl acetate, 1:3), yield 81%, m.p. 197e
199 ꢂC. IR (cmꢀ1): 3429 (NH quinoxaline), 3284 (NH), 3053 (CH,
aromatic), 1671 (C]O), 1596 (C]N), 1500 (C]C). 1H NMR
(500 MHz, DMSO-d6): 3.9 (s, 2H, CH2), 7.26 (m, 3H), 7.62 (m,1H), 9.5
(br., NH, D2O exchangeable), 11.9 (br., NH, D2O exchangeable). 13
C
NMR (500 MHz, DMSO-d6): 48.3, 116.9, 118.3, 121.3, 122.8, 125.5,
4.1.4.2. 40-Benzoyl-50-thioxo-1H-spiro[quinoxaline-2,30-[1,2,4]tri-
azolidin]-3(4H)-one (18). Rf ¼ 0.38 (petroleum ether/ethyl acetate,
1:3), yield 55%, m.p. 213e215 ꢂC. IR (cmꢀ1): 3432 (NH, quinoxaline),
3220 (NH), 2950 (CH, aromatic), 2820 (CH, aliph.), 1683 (C]O),
1669 (C]O), 1642 (C]N, thiadiazole moiety), 1605 (C]N), 1576
(C]C). 1H NMR (270 MHz, DMSO-d6): 7.05 (m, 1H), 7.11 (m, 1H),
7.24 (m, 1H), 7.35 (m, 1H), 7.5 (m, 2H), 7.7 (br., NH, D2O exchange-
able), 8.06 (m, 3H), 11.05 (br., NH, D2O exchangeable), 12.7 (br., NH,
D2O exchangeable). 13C NMR (270 MHz, DMSO-d6): 84.9, 114.1,
117.3, 121.1, 129.7, 132.1, 133.4, 134.6, 145.5, 151.8, 158.4, 165.2. MS:
m/z: 339.08 (Mþ, 15%). Anal. Calcd for C16H13N5O2S (FW: 339.08): C,
56.63; H, 3.86; N, 20.64; S, 9.45. Found: C, 56.58; H, 3.84; N, 20.59;
S, 9.46.
138.9, 152.1, 159.23, 161.0. MS: m/z: 216 (Mþ, 21%). Anal. Calcd for
C
10H8N4O2 (FW: 216.06): C, 55.55; H, 3.73; N, 25.91. Found: C,
55.39; H, 3.84; N, 25.85.
4.1.3.3. 2-Hydroxy-1-imino-1H-[1,2,4]triazino[4,3-a]quinoxalin-
5(6H)-one (13). Rf ¼ 0.27 (petroleum ether/ethyl acetate, 1:3), yield
79%, m.p. 223e225 ꢂC. IR (cmꢀ1): 3443 (OH), 3400 (NH quinoxaline),
3298 (NH), 3053 (CH, aromatic),1681 (C]O),1511 (C]N),1580 (C]
C). 1H NMR (500 MHz, DMSO-d6): 4.54 (s, NH, D2O exchangeable),
7.09 (m, 3H); 7.33 (m, 1H), 8.7 (s, OH, D2O exchangeable), 10.9 (br.,
NH, D2O exchangeable). 13C NMR (500 MHz, DMSO-d6): 109.1, 116.9,
118.3, 121.3, 125.2, 125.5, 138.9, 152.1, 159.23, 161.0. MS: m/z: 228
(Mþ ꢀ 1, 26%). Anal. Calcd for C10H7N5O2 (FW: 229.06): C, 52.40; H,
3.08; N, 30.56. Found: C, 52.38; H, 3.12; N, 30.44.
4.1.5. Preparation of compounds 19 and 20
A mixture of compound 3 (5 mmol) and diethyl malonate or
diethyl succinate (5 mmol) was dissolved in DMF (30 mL) and few
drops of triethyl amine. The reaction mixture was stirred at 90 ꢂC
4.1.3.4. 2-Hydroxy-1H-[1,2,4]triazino[4,3-a]quinoxaline-1,5(6H)-
dione (14). Rf ¼ 0.31 (petroleum ether/ethyl acetate, 1:3), yield 73%,

S.A. Galal et al. / European Journal of Medicinal Chemistry 69 (2013) 115e124
123
for 12 h. After reaction completion, the formed solid was filtered off.
The crude product obtained was washed with methanol and finally
purified by recrystallization from DMF/methanol (1:2) mixture.
human tumor cell lines including liver cancer HepG2 and breast
cancer MCF-7 cell lines obtained from the American Type Culture
Collection (Rockville, MD, USA). The tumor cells were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10% heat inactivated fetal calf serum (GIBCO), penicillin (100 U/ml)
4.1.5.1. 1-(3-Oxo-3,4-dihydroquinoxalin-2-yl)pyrazolidine-3,5-dione
(19). Rf ¼ 0.39, Yield: 75%, m.p. 266e268. IR (cmꢀ1): 3400 (NH,
quinoxaline), 3299 (NH), 3016 (CH, aromatic), 2898 (CH, pyr-
azolidine), 1680 (C]O), 1615 (C]N), 1576 (C]C). 1H NMR
(270 MHz, DMSO-d6): 4.15 (s, 2H, CH2), 4.54 (br., NH, D2O
exchangeable), 7.12 (m, 3H), 7.36 (m, 1H), 8.78 (br., NH, D2O
exchangeable). 13C NMR (270 MHz, DMSO-d6): 47.9, 116.1, 123.2,
125.7, 129.2, 132.4, 142.8, 155.2, 159.4, 169.4. MS: m/z: 244 (Mþ,
20%). Anal. Calcd for C11H8N4O3 (FW: 244.06): C, 54.10; H, 3.30; N,
22.94. Found: C, 54.12; H, 3.35; N, 22.86.
and streptomycin (100 m
g/ml) at 37 ꢂC in humidified atmosphere
containing 5% CO2. Cells at a concentration of 0.50 ꢃ 106 were
grown in a 25 cm2 flask in 5 mL of complete culture medium.
The cells in culture medium were treated with 20
ml of deferent
concentration (2.5, 5, 10, 20 and 40 g/ml) of the tested compounds
m
or the standard reference drug, doxorubicin dissolved in DMSO, then
incubated for 48 h at 37 ꢂC, in a humidified 5% CO2 atmosphere. The
cells were harvested and homogenates were prepared in saline
using a tight pestle homogenizer until complete cell disruption.
To determine the level of TRK, A double-antibody sandwich
enzyme-linked immunosorbent assay (ELISA) was used. This assay
is based on, add TRK to monoclonal antibody enzyme well which is
pre-coated with human TRK monoclonal antibody, incubation;
then, add TRK antibodies labeled with biotin, and combined with
Streptavidin-HRP to form immune complex; then carry out incu-
bation and washing again to remove the uncombined enzyme.
Then add Chromogen solution A, B, the color of the liquid changes
into the blue, and at the effect of acid, the color finally becomes
yellow. The chroma of color and the concentration of the human
TRK of sample were positively correlated and the optical density
was determined at 450 nm. The level of Human Tyrosine kinase
(TRK) in samples was calculated as triplicate determinations from
the standard curve and the IC50 of TRK for each compound was
calculated as the 50% inhibition in the level of TRK as compared
with control untreated cancer cells.
4.1.5.2. 1-(3-Oxo-3,4-dihydro-quinoxalin-2-yl)-tetrahydro-pyr-
idazine-3,6-dione (20). Rf ¼ 0.41, Yield: 78%, m.p. 254e256 ꢂC. IR
(cmꢀ1): 3400 (NH, quinoxaline), 3298 (NH), 3015 (CH, aromatic),
2898 (CH, pyrazolidine), 1680 (C]O), 1615 (C]N), 1576 (C]C). 1H
NMR (270 MHz, DMSO-d6): 4.15 (s, 2H, CH2), 4.54 (s, 1H, NH, D2O
exchangeable), 7.12 (m, 3H), 7.36 (m, 1H), 8.78 (br., NH, D2O
exchangeable). 13C NMR (270 MHz, DMSO-d6): 25.8, 30.1, 115.6,
124.6, 126.3, 129.2, 132.4, 142.4, 154.8, 158.9, 165.2. MS: m/z: 258
(Mþ, 20%). Anal. Calcd for C12H10N4O3 (FW: 258.08): C, 55.81; H,
3.90; N, 21.70. Found: C, 55.77; H, 3.82; N, 21.77.
4.1.6. Cells
EBV genome-carrying lymphoblastoid cells (Raji cells derived
Burkitt’s lymphoma) were cultured in 10% fetal bovine serum (FBS)
in RPMI-1640 under the conditions described previously [33].
Spontaneous activation of EBV-EA in our subline of Raji cells was
less than 0.1%.
4.3. Molecular docking study
4.1.7. Inhibition of EBV-EA activation assay
4.3.1. Docking study
Inhibition of EBV-EA activation was assayed using Raji cells
(Virus non producer type), an EBV genome-carrying human lym-
phoblastoid cell, which were cultivated in 10% fetal bovine serum
The studied ligands were protonated using Protonate 3D [38] and
minimized in MOE (Chemical Computing Group, Montreal, CA) until
the root-mean-square gradient of 0.000001 was reached using a
MMFF94x force field The crystal structures of c-kit receptor protein-
tyrosine kinase in complex with STI-571 (Imatinib or Gleevec) was
obtained from the Protein Data Bank (PDB code: 1T46). Hydrogen
atoms were added in GOLD, V 5.1 (CCDC, Cambridge, UK) and default
parameters were used unless otherwise stated. Before the docking
study, the co-crystallized substrate STI-571 was removed from the
structure. No structural water molecules were observed in the active
site, and hence all water molecules were deleted. The docking
program GOLD, V 5.1 (CCDC, Cambridge, UK) was used to perform
the docking of compounds 7, 8, 9 and 10 into the crystal structure of
c-kit. Based on the position of the co-crystalized ligand, the active
site was defined choosing all residues around the original ligand in a
(FBS) RPMI 1640 medium. The indicator cells (Raji, 1
were incubated at 37 ꢂC for 48 h in 1 mL of medium containing n-
butyric acid (4 mM as trigger), TPA (32 pM ¼ 20 ng in 2 L of DMSO
as inducer), and various amounts of the test compounds dissolved
in 5 L of DMSO (ca. 0.7% DMSO). Smears were made from the cell
m 106/mL)
m
m
suspension. The EBV-EA inducing cells were stained with high titer
EBV-EA positive serum from NPC patients and detected by an in-
direct immunofluorescence technique. In each assay, at least 500
cells were counted, and the number of stained cells (positive cells)
was recorded. Triplicate assays were performed for each data point.
as a relative ratio to the positive control experiment (100%), which
was carried out within-butyric acid (4 mM) plus TPA (32 pM). In the
experiments, the EBV-EA induction was normally around 35%, and
this value was taken as the positive control (100%). n-Butyric acid
(4 mM) alone induced 0.1% EA-positive cells. The viability of treated
Raji cells was assayed by the trypan blue staining method. The cell
viability of the TPA positive control was greater than 80%. There-
fore, only the compounds that induced less than 80% (% of control)
of the EBV-activated cells (those with a cell viability of more than
60%) were considered able to inhibit the activation caused by
promoter substances. Student’s t-test was used for all statistical
analyses [33e35].
ꢀ
circle of 7 A. All docking runs were performed three times using
standard default variables and GoldScore. The docking protocol
utilized the study of 10 best docking poses per ligand. The top ten
docked solutions for each ligand were retained for analysis. Software
Ligandscout [37] was used to generate a 3D pharmacophore from
the PDB structure 1T46 using default options. This pharmacophore
was used to rank the best docking poses of the studied compounds.
Docking poses visualization was done by LigandScout. For each
ligand, the most plausible conformation was selected based on its
ability to fulfill highest amount of chemical features described in the
3D pharmacophore.
4.2. Protein kinase inhibition
Appendix A. Supplementary data
4.2.1. Cell lines and culturing
The effect of tested compounds on the inhibition of human
tyrosine kinase (TRK) was determined utilizing two different
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.07.049.

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