Design, synthesis and biological evaluation of type-II VEGFR-2 inhibitors based on quinoxaline scaffold

Article abstract of DOI:10.1016/j.bioorg.2014.05.010

In an effort to develop ATP-competitive VEGFR-2 selective inhibitors, a series of new quinoxaline-based derivatives was designed and synthesized. The target compounds were biologically evaluated for their inhibitory activity against VEGFR-2. The design of the target compounds was accomplished after a profound study of the structure activity relationship (SAR) of type-II VEGFR-2 inhibitors. Among the synthesized compounds, 1-(2-((4-methoxyphenyl)amino)-3- oxo-3,4 dihydroquinoxalin-6-yl)-3-phenylurea (VIIa) displayed the highest inhibitory activity against VEGFR-2. Molecular modeling study involving molecular docking and field alignment was implemented to interpret the variable inhibitory activity of the newly synthesized compounds.

Full text of DOI:10.1016/j.bioorg.2014.05.010

Bioorganic Chemistry 56 (2014) 16–26
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological evaluation of type-II VEGFR-2 inhibitors
based on quinoxaline scaffold
⇑
Mai I. Shahin, Dalal A. Abou El Ella, Nasser S.M. Ismail, Khaled A.M. Abouzid
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Abassia, Cairo 11566, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 February 2014
Available online 28 May 2014
In an effort to develop ATP-competitive VEGFR-2 selective inhibitors, a series of new quinoxaline-based
derivatives was designed and synthesized. The target compounds were biologically evaluated for their
inhibitory activity against VEGFR-2. The design of the target compounds was accomplished after a
profound study of the structure activity relationship (SAR) of type-II VEGFR-2 inhibitors. Among the synthe-
sized compounds, 1-(2-((4-methoxyphenyl)amino)-3-oxo-3,4 dihydroquinoxalin-6-yl)-3-phenylurea
(VIIa) displayed the highest inhibitory activity against VEGFR-2. Molecular modeling study involving
molecular docking and field alignment was implemented to interpret the variable inhibitory activity of
the newly synthesized compounds.
Keywords:
VEGFR-2
Kinase
Type-II
Quinoxaline
Docking study
Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
approved by FDA for treating gastrointestinal stromal tumors and
advanced renal cell carcinoma, respectively (Fig. 1).
Inhibition of receptor tyrosine kinase (RTK) signaling pathways
is an important area for the development of novel anticancer
agents. Vascular endothelial growth factor receptor-2 (VEGFR-2)
is the principal mediator of tumor angiogenesis. Angiogenesis,
which is the sprouting of new blood vessels from pre-existing ones,
is one of the most critical hallmarks of a cancerous cell. Develop-
ment of selective antiangiogenic agents required thorough study
of signal transduction pathways that hold the promise of efficacy
with minimal toxicity [1]. Tyrosine kinases were identified as
one of the efficient targets for evolving new anticancer agents
having the desired selectivity on cancerous cells [2]. Numerous
small molecule TK inhibitors were approved to treat many types
of tumors of different origins [3]. Tyrosine kinase family comprises
prominent members that can effectively induce important cell
signaling pathways [4]. Angiogenic switch is a critical element in
the growth of primary tumors and formation of metastatic sites
[5]. Proliferation and migration of endothelial cells in angiogenesis
are provoked by the imbalance between pro-angiogenic factors
and anti-angiogenic factors. VEGF was settled to be the most
important regulator of angiogenesis. This occurs by binding to
the kinase insert domain (KDR) of the receptor tyrosine kinase VEG-
FR-2 [6]. Small molecule inhibitors targeting KDR proved successful
suppression of tumor growth via blocking its signaling pathway [7].
SutentÒ (sunitinib) [8] and NexavarÒ (sorafenib tosylate) [9], were
A literature survey revealed that different scaffolds have been
reported as excellent inhibitors of VEGFR-2. Some classes are quin-
oline amides, [10] quinolinones, [11] anilinophthalazines, [12] and
quinazolines [13]. As being one of tyrosine kinases, VEGFR-2 has 2
different conformations; the active and the inactive conformation
[14]. This is determined by the movement of the DFG motif (acti-
vation loop). The DFG-in (active) conformation makes the active
site accessible for ATP binding. Type-I inhibitors can target this
active conformation and compete with ATP for binding [15]. Shift-
ing to the DFG-out (inactive) conformation occurs by movement of
the DFG motif revealing an extra hydrophobic pocket adjacent to
the binding site. Inhibiting the tyrosine kinase activity is achieved
through stabilizing this inactive conformation using type-II inhib-
itors. Type-II inhibitors provide a superior kinetic advantage over
type-I inhibitors by avoiding competition with ATP. Also they
stabilize the kinase in the DFG-out inactive conformation [16]. In
our current study, we synthesized novel quinoxaline derivatives
as type II VEGFR-2 inhibitors based on the Structure–Activity
Relationship (SAR) study of VEGFR-2 type-I and type-II inhibitors.
The newly synthesized compounds were tested for their inhibitory
activities on some kinases. The results showed good inhibition
percentages of some compounds against VEGFR-2 compared with
other tested kinases. The results were interpreted using investiga-
tional docking and field alignment studies.
Type-II tyrosine kinase inhibitors, though being highly diverse in
structure, revealed common feature pharmacophores for binding.
Two simple screening hits, phenylaminopyrimidine in case of
⇑
Corresponding author. Fax: +20 225080728.
E-mail address: Khaled.abouzid@pharma.asu.edu.eg (K.A.M. Abouzid).
http://dx.doi.org/10.1016/j.bioorg.2014.05.010
0045-2068/Ó 2014 Elsevier Inc. All rights reserved.

M.I. Shahin et al. / Bioorganic Chemistry 56 (2014) 16–26
17
O
H
N
H
N
N
N
F
N
H
H
N
HN
O
O
Cl
O
N
H
O
CF3
Sutent®(Sunitinib)
Nexavar® (Sorafenib)
Fig. 1. SutentÒ (Sunitinib) and NexavarÒ (Sorafenib).
imatinib and biaryl urea in case of both BIRB796 and Sorafenib,
were the first type II inhibitors developed by optimization of kinase
enzyme activity [17]. As illustrated in Fig. 2, GleevecÒ, NexavarÒ,
and BIRB796 were analyzed for their binding interactions as type-
II inhibitors. A flat aromatic ring system of the main scaffold adopts
the active site in the same manner as the ATP purine ring does. This
occurs along with the essential hydrogen bonding of Cys 919 in the
hinge region (presented as the left green side) [18]. Accordingly, we
determined the features necessary for a type-I inhibitor which
typically forms ꢀ1–3 hydrogen bonds with the kinase hinge
residues [15]. In case of type-II inhibitors, it would require an
additional moiety to extend into the nearby allosteric site formed
by the movement of the DFG motif in the inactive conformation
of the enzyme forming two hydrogen bonds with the allosteric site
residues: one hydrogen bond with the side chain of a conserved
glutamic acid in the C-helix (Glu 883) and the other with the
backbone amide of aspartic acid in the DFG motif (Asp 1044) (this
is presented by the core group given a yellow color).
All type-II inhibitors also has a hydrophobic moiety that is
located just after the hydrogen bond donor–acceptor pair forming
van der Waals interactions with the allosteric site (the right pink
side) [19].
Our design was based on targeting VEGFR-2 in its DFG-out con-
formation. Inspection of known type-II inhibitors, especially
Sorafenib, revealed that the conserved hydrogen bonds between
the ligand and the allosteric site residues were done using urea
or amide moieties.
linking moieties for allosteric site binding with extended alkyl or
aryl groups.
2. Results and discussion
2.1. Chemistry
Quinoxaline scaffold in compounds I and XII was synthesized
using the Hinsberg reaction [20] (Schemes 1 and 2). Their nitration
was performed using potassium nitrate in sulfuric acid [21] to give
6-nitroquinoxaline derivatives II [22] and XII [23] respectively.
Chlorination of II and XII was carried out by refluxing with POCl3
either alone in case of III [24] or combined with PCl5 in case of XIII
[23]. New intermediates IV and XIVa-b was obtained by reacting
the chloroquinoxaline derivative with the appropriate aniline
derivative in isopropanol [25] at room temperature giving IV.
While compounds XIVa-b were synthesized using ethanol [24]
under reflux with using anhydrous potassium carbonate. In case
of compound IV, substitution of the chlorine atom at the 2-position
of quinoxaline due to regioselectivity created by the electron with-
drawing effect of 6-nitro group [26]. Caution was carefully taken
by monitoring mild conditions to prevent dinucleophilic substitu-
tion of both chlorine atoms. Hydrolysis of the second chlorine atom
was accomplished by heating IV with conc. HCl to provide
V. Reduction of the nitro compounds to the corresponding amino
derivatives was achieved using 10% Pd-C in THF/MeOH due to their
poor solubility in single solvent to give compounds VI and XVa-b.
Moreover, urea and thiourea derivatives were synthesized by
stirring the appropriate amine derivative with the cyclohexyl and
In this respect, we designed novel compounds based on quinox-
aline core, with urea, amide and sulfonamide pharmacophoric
Fig. 2. (a) A diagram illustrating the common features between GleevecÒ, NexavarÒ and BIRB-769 as type-II inhibitors [18]. (b) Fitness of these essential features on our
designed compounds revealing the green left side that presents the main scaffold adopting the active site. Also, the urea, amide or sulfonamide core was presented in yellow
color and finally the extra moiety occupying the allosteric site was given in pink color. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

18
M.I. Shahin et al. / Bioorganic Chemistry 56 (2014) 16–26
H
H
N
NH2
NH2
HO
HO
O
O
N
O
O
a
O2N
O
O
b
N
H
N
H
I
II
O2N
N
Cl
O2N
N
N
Cl
Cl
e
d
c
N
NH
IV
III
H
N
H
N
O2N
O
H2N
O
O
f
N
NH
N
NH
O
V
VI
O
i
g
j
h
H
N
H
N
H
N
O
R1
H
H
O
S
N
N
O
X
N
NH
O
N
NH
R2
O
H
N
H
N
O
VIIa: R1= phenyl, X=O
VIIb
O
: R1= phenyl, X=S
VIIc: R1= 3-chlorophenyl, X= O
VIId
O
VIIIa
: R2=H
VIIIb: R2= -CH3
O
OH
N
NH
: R1= 3-methylphenyl, X=O
VIIe: R1= -cyclohexyl, X=O
O
H
N
H
N
IX
O
O
N
NH
O
X
Scheme 1. 3-((4-Methoxyphenyl)amino)-7-(substituted amino)quinoxalin-2(1H)-ones. Reagents and conditions: (a) Aq. HCl, reflux, 2 h, 98%. (b) KNO3, H2SO4, rt, 96%.
(c) POCl3, reflux, 6 h, 95%. (d) P-anisidine, isopropanol, rt, 3 h, 90%. (e) Conc. HCl, 50 °C, 8 h, 86%. (f) Pd-C, THF/MeOH, 40 °C, 24 h, 95%. (g) Aryl and cyclohexyl isocyanates and
aryl isothiocyanate, THF, 2–24 h, rt, 80–85%. (h) P-substituted benzenesulfonyl chloride, pyridine, reflux, 3 h, 40–58%. (i) Phthalic anhydride, AcOH, rt, 82%. (j) Acetyl chloride,
THF, reflux, 24 h, 73%.
aryl isocyanates and aryl isothiocyanate in THF [27] to yield VIIa-e.
DCM [28] was used instead to provide XVIa-e. Refluxing the amine
derivatives with the appropriate sulfonyl chloride in pyridine [29]
gave the sulfonamide series VIIIa-b and XVIIa-c. Synthesis of dif-
ferent amide derivatives was accomplished by stirring phthalic
anhydride or acetyl chloride with VI and XVa-b in glacial acetic
acid to give IX and XVIII [30]. Refluxing acetyl chloride with VI
in THF gave the corresponding amide derivative X. Compounds
XXIa-b (Scheme 3) were synthesized by refluxing XIII with
4-aminophenyl urea derivatives in ethanol [24] and anhydrous
potassium carbonate.
2.2. Biological evaluation
Kinase inhibition assay was carried out to evaluate the effect of
the synthesized compounds on VEGFR-2 and other selected kinases
such as c-Met, EGFR, PDGFRb and B-raf kinases. The evaluation was
performed in KINEXUS Corporation where radiolabeled ATP deter-
mination method (33P-ATP) was used. The percent inhibition of the
Kinase enzymatic activity of the tested compounds was evaluated
against a reference kinase inhibitor at 10 lM. Compounds VIIa, IX,
XVIc, XXIa-b showed relatively high inhibitory enzyme activity.
Among these compounds, IX shows the highest percentage of

M.I. Shahin et al. / Bioorganic Chemistry 56 (2014) 16–26
19
N
NH2
NH2
O2N
N
O
a
b
c
O
OH
N
H
XI
O
N
H
XII
O
O2N
N
N
H2N
N
N
O2N
N
d
e
NH
R1
NH
N
Cl
XVa-b
XIVa-b
XIII
R1
h
f
g
H
N
H
H
N
H
N
O
S
N
N
N
N
N
N
R2
O
O
X
N
NH
R1
NH
R1
NH
R2
R1
XVIa
: R1= -OCH3, R2=phenyl, X=S
XVIIa: R1= -OCH3, R2= H
: R1= -OCH3, R2= -CH3
XVIIc: R1= -Cl, R2= -CH3
XVIII: R1= -Cl
XVIb: R1= -Cl, R2=phenyl, X=O
XVIIb
XVIc
: R1= -OCH3, R2= 3-methylphenyl, X= O
XVId: R1= -OCH3, R2= -cyclohexyl, X=O
XVIe
: R1= -Cl, R2= -cyclohexyl, X=O
Scheme 2. N6-Substituted-N2-(4-substituted phenyl)quinoxaline-2,6-diamines. Reagents and conditions: (a) EtOH, rt, 2 h, 98%. (b) KNO3, H2SO4, rt, 96%. (c) POCl3, PCl5,
reflux, 10 h, 86%. (d) P-anisidine, p-chloroaniline, EtOH, reflux, 5 h, 92%. (e) Pd-C, THF/MeOH, 40 °C, 24 h. (f) Aryl and cyclohexyl isocyanates and aryl isothiocyanate, DCM, rt,
2–24 h. (g) P-substituted benzenesulfonyl chloride, pyridine, reflux, 3 h. (h) Acetyl chloride, gl. Acetic acid, rt, 24 h.
NH2
H
N
H
N
H
N
H
N
b
a
O
O
O2N
H2N
NO2
R
XIXa-b
XXa-b
R
H
N
H
O2N
N
N
XIII,
c
O
N
N
H
XXIa-b
R
XXIa= H
XXIb= Cl
Scheme 3. 1-(4-((6-Nitroquinoxalin-2-yl)amino)phenyl)-3-(3-substituted phenyl)ureas. Reagents and conditions: (a) Aryl isocyanate derivatives, THF, 24 h, r.t.75.8–85.3%.
(b) SnCl2, EtOH, reflux, 16 h, 50–51.8%. (c) XIII, EtOH, anhydrous K2CO3, reflux, 6 h. 72–83.7%.
inhibition (69%) on VEGFR-2 while these compounds displayed
less significant inhibitory activities on the other tested kinases.
The rest of compounds showed weak to moderate activity with
exception of compounds VIId, XVIa, XVIb and XVId which showed
no significant effect (Table 1). The five compounds showing the
highest percentage of enzyme inhibition were selected for deter-
mination of their IC50 on VEGFR-2 (Table 2). Compound VIIa
showed highest IC50 value (10.3 lM) (Fig. 3).

20
M.I. Shahin et al. / Bioorganic Chemistry 56 (2014) 16–26
Table 1
the number of predictions. Docking of Sorafenib as a reference
compound was carried out to compare its binding mode with those
of the target compounds. Investigation of Sorafenib docking results
revealed a hydrogen bond formed with Cys 919 in the hinge region.
The urea moiety interacts with the protein through two hydrogen
bonds; the NH forms a hydrogen bond with Glu 885 while the urea
carbonyl functionality interacts with Asp 1046. The substituted
phenyl ring pushes deep into the extended hydrophobic pocket
that is formed by the movement of Phe 1047 of the ‘DFG’ motif
to induce the ‘DFG-out’ conformation. On the other hand, the com-
pounds showing highest inhibition of VEGFR-2 activity (VIIa, IX,
XVIc, XXIa-b) fulfilled the key features as done by the lead com-
pound in the ATP binding site (Cys 919, Glu 885 and Asp 1046).
For IX, extra hydrogen bonding with Glu 917 in the hinge region
was detected which may explain its high activity relative to other
compounds. Also, XVIc fulfilled all the features important for being
type-II inhibitors which explains its high activity. Compounds
showing weak to moderate activity missed one interaction of the
features essential to be acting as type-II inhibitors which is either
interaction with Glu 885 or Asp 1046 and even some lose the inter-
action with Cys 919 and rather interact with other amino acids in
the hinge region instead as Glu 917 and Thr 916 (Fig. 4).
VEGFR-2 and other kinases % inhibition achieved by the twenty targeted compounds
at 10 M.
l
Compound ID
VEGFR-2
c-Met
PDGFRb
B-raf
EGFR
VIIa
VIIb
VIIc
VIId
VIIe
VIIIa
VIIIb
IX
À62
À23
À17
3
À52
À10
À29
À69
À23
4
À43
À47
2
NT
À8
À10
À2
À1
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
0
NT
NT
À17
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
À15
1
À58
À16
À14
À18
À15
À1
X
XVIa
XVIb
XVIc
XVId
XVIe
XVIIa
XVIIb
XVIIc
XVIII
XXIa
XXIb
3
À66
À6
6
À33
À5
À11
À18
À29
À27
À5
À13
À26
À27
0
À65
À61
À34
3
NT: means not tested.
2.3.2. Field analysis
Table 2
For understanding the unexpected results of compounds VIId,
XVIa, XVIb and XVId showing no significant activity, a further
study was conducted using field analysis. The field alignment study
of compounds VIId, XVIa, XVIb and XVId in comparison to Sorafenib
as a reference showed that negative field (Hydrogen bond accep-
tor) around the NH of Sorafenib is present in these compounds
but in the totally opposite direction. This negative field represents
the interaction to Cys 919 in the hinge region which is the most
important key feature of interaction. Upon comparing active com-
pounds VIIa and XVIc with the reference, the negative field was
present in the same direction as Sorafenib. Compound XVIb
showed no negative field around that of Sorafenib. This proved
the importance of this interaction in the inhibitory activity. (Fig. 5)
IC50 values for compounds VIIa, IX, XVIc, XXIa and XXIb.
Compound ID
IC50 values
R2 values
VIIa
IX
10.3
61.8
0.9977
0.9821
0.9887
0.9664
0.9921
XVIc
XXIa
XXIb
210.4
268.2
85.4
2.3.3. ADME study
Theoretical kinetic study was performed using Discovery Studio
software to predict the ADME of the newly synthesized com-
pounds. All the compounds passed the Lipinski’s rule of five except
compound XVIa which had partition coefficient > 5. Other descrip-
tors as A logP 98 and PSA 2D were calculated to evaluate solubility
level and absorption level. Also, CYP2D inhibition was predicted
giving score 0 for non-inhibitors and 1 for inhibitors. Most of the
compounds showed good absorption levels (score = 0) with rela-
tively low solubility (score of solubility level = 1–2). Our two most
active candidates IX and VIIa showed moderate and good absorp-
tion respectively. Compound IX is expected to have good solubility,
while VIIa will be with low solubility. Finally, All the compounds
were predicted to be CYP2D non-inhibitors except XVIII. (Table 3)
Fig. 3. Graph of log compounds VIIa, IX and XVIc concentrations against % VEGFR-2
activity inhibition.
2.3. Molecular modeling study
Molecular docking study was conducted using Gold 4.1 soft-
ware in the interface of Accelry’s Discovery Studio 2.5 (Accelrys
Inc., San Diego, CA, USA). Field alignment study was done using
Field Align 2.1 software.
3. Conclusion
Quinoxaline scaffold was explored for its activity against
VEGFR-2. The compounds were designed as type-II inhibitors
based on comprehensive SAR study. Biological evaluation revealed
high inhibitory activity of compounds IX, VIIa, XVIc, XXIa, XXIb,
with highest IC50 of compound VIIa. The unexplained activity
results of inactive compounds VIId, XVIa, XIVb and XIVd was
adequately explored using field analysis. The results of this study
revealed that quinoxaline could be a promising scaffold to be
greatly considered for evolving new effective VEGFR-2 inhibitors.
2.3.1. Docking study
A deep docking study was done to investigate the possible bind-
ing modes of the newly synthesized compounds inside the ATP
binding site of VEGFR-2 using Gold 4.1 software [31]. The X-ray
crystal structure of VEGFR-2 bound to naphthamide-based com-
pound was obtained, which represents the protein in its inactive
conformation, and prepared for docking by energy minimization.
500,000 iterations were applied to get reliable results by increasing

M.I. Shahin et al. / Bioorganic Chemistry 56 (2014) 16–26
21
Fig. 4. (a) Sorafenib docking pose showing the same key interactions as reported in the literature (b) The pose adopted by compound IX showing excellent binding with
VEGFR-2 binding site in its inactive conformation. (c and d) Compounds XVIc and VIIa in their docked poses respectively revealing that all the essential features of type-II
inhibitors are fulfilled.
Fig. 5. (a and b) Showing the matching of the negative field (hydrogen bond acceptor) with Sorafenib in both compounds XVIc, VIIa. (c) Compound VIId shows the negative
field in the totally opposite directions (d). Compound XVIb shows no negative field around that of Sorafenib.
University. EI-MS spectra were recorded on Finnigan Mat SSQ
7000 (70 eV) mass spectrometer at Microanalytical Center at Cairo
University. Elemental analysis was performed at Al-Azhar Univer-
sity. Compounds I, II, III, XI, XII, XIII, XIXa-b, XXa-b were synthe-
sized according to the reported procedures [22,23,32–34].
4. Experimental
4.1. Chemistry and analysis
All chemicals either starting materials or reagents used were
purchased from Aldrich (USA) or Alfa-Aesar Organics and used
without further purification. Melting points were determined
using Stuart Scientific apparatus and were uncorrected. The reac-
tions were monitored using analytical thin layer chromatography
(TLC) purchased from Merck (Merck, Darmstadt, Germany) and
performed on 0.255 mm silica gel plates, with visualization under
U.V. light (254 nm). FT-IR spectra were recorded on a PerkineElmer
IR spectrophotometer.
4.1.1. 3-Chloro-N-(4-methoxyphenyl)-6-nitroquinoxalin-2-amine (IV)
A solution of III (2 g, 8.2 mmol) in isopropanol was stirred while
adding the p-anisidine (1.01 g, 8.2 mmol) portionwise then left
with stirring at room temperature for 6 h. Filtration of the resulted
solid and stirring it again with absolute ethanol then filtration
afforded orange powder of the required compound with a yield
of 1.6 g (59%); m.p. 174–176 °C; 1H NMR (300 MHz, DMSO-d6) d
9.56 (s, 1H), 8.59 (s, 1H), 8.37 (d, J = 8.5 Hz, 1H), 8.33
(d, J = 8.6 Hz, 1H), 7.21 (d, J = 8.6 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H),
1H spectra were run at 300 MHz spectrometer in d scale (ppm),
J (Hz) using TMS as reference at Microanalytical Center at Cairo

22
M.I. Shahin et al. / Bioorganic Chemistry 56 (2014) 16–26
Table 3
Computer aided ADME study of the targeted compounds.
A log P 98a
PSA 2Db
Absorption levelc
Solubilityd
Solubility levele
CYP 2D6f
CYP 2D6 probabilityg
VIIa
VIIb
VIIc
VIId
VIIe
VIIIa
VIIIb
IX
2.564
3.463
3.229
3.05
2.846
2.359
2.845
2.286
0.992
4.959
4.74
4.546
4.341
5.022
3.854
4.34
5.021
3.168
3.97
106.095
88.794
106.095
106.095
106.095
110.586
110.586
131.401
93.285
69.882
78.253
87.183
87.183
78.253
91.674
91.674
82.744
65.443
121.076
121.076
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
2
À4.009
À4.794
À4.921
À4.428
À4.255
À4.217
À4.635
À3.977
À2.698
À6.132
À6.066
À5.752
À5.582
À6.32
2
2
2
2
2
2
2
3
3
1
1
2
2
1
2
2
1
2
2
1
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
0
0
0.277
0.237
0.178
0.178
0.485
0.465
0.386
0.336
0.475
0.435
0.247
0.415
0.643
0.475
0.485
0.396
0.356
0.514
0.267
0.158
X
XVIa
XVIb
XVIc
XVId
XVIe
XVIIa
XVIIb
XVIIc
XVIII
XXIa
XXIb
À5.426
À5.85
À6.595
À4.68
À5.519
À6.338
4.635
a
b
c
d
e
f
Lipophilicity descriptor.
Polar surface area.
Absorption level (0 = good, 1 = moderate, 2 = low, 3 = very low).
Solubility parameter.
Solubility level (0 = extremely low, 1 = very low but soluble, 2 = low, 3 = good, 4 = optimal).
CYP2D inhibition (0 = non inhibitor, 1 = likely to inhibit).
CYP2D inhibition probability.
g
3.75 (s, 3H); FT-IR (
m
max, cmÀ1): 3320 (NH), 3032 (CH aromatic),
3-chlorophenyl isocyanate, phenyl isothiocyanate, 3-methyl
phenyl isocyanate and cyclohexyl isocyanate) and stirred for
2–24 h. The mixture was added to hexane and stirred for several
minutes. The resulting solid was filtered, washed with hexane,
recrystallized from ethanol to afford the titled compounds (VIIa-e).
2947 (CH aliphatic), 1587, 1390 (NO2); MS: (Mwt.: 330): m/z, 332
[M + 2, (2.46%)], 330 [M+, (9.66%)], 79 (100%); Anal. Calcd for
C15H11ClN4O3: C, 54.47; H, 3.35; N, 16.94; Found: C, 54.08; H,
3.04; N, 16.84.
4.1.2. 3-((4-Methoxyphenyl)amino)-7-nitroquinoxalin-2(1H)-one (V)
This compound was obtained with a yield of 2.1 g (74%) by
heating IX (3 g, 9.07 mmol) in conc. HCl (30 ml) at 50 °C for
12hrs then filtration, washing the solid thoroughly with water
and finally triturating it with diethyl ether to give orange powder
of V; m.p. 282 °C; 1H NMR (300 MHz, DMSO-d6) d 12.70 (s, 1H),
9.87 (s, 1H), 8.92 (s, 1H), 8.06(s,1H), 8.02 (d, J = 8.5 Hz, 1H), 7.97
(d, J = 8.6 Hz, 1H), 7.58 (d, J = 8.6 Hz, 2H), 6.94 (d, J = 8.8 Hz, 2H),
4.1.4.1. 1-(2-((4-Methoxyphenyl)amino)-3-oxo-3,4 dihydroquinoxa-
lin-6-yl)-3-phenylurea (VIIa). The titled compound was separated
as buff solid 0.1 g (70%); m.p 238 °C; 1H NMR (300 MHz, DMSO-
d6) d 12.43 (s, 1H), 11.57 (s, 1H), 9.28 (s, 1H), 9.13 (s, 1H), 8.94
(s, 1H), 8.05
(d, J = 8.4 Hz, 1H), 6.89 (d, J = 8.3 Hz, 2H), 6.62 (d, J = 8.1 Hz, 2H),
3.74 (s, 3H); FT-IR (
max, cmÀ1): 3363 (NH), 3032 (CH aromatic),
– 7.94 (m, 5H), 7.32 (d, J = 8.3 Hz, 1H), 7.18
m
2910 (CH aliphatic), 1673 (C@O amide); MS: (Mwt.: 401): m/z, 402
3.75 (s, 3H); FT-IR (
m
max, cmÀ1): 3363 (NH), 3032 (CH aromatic),
[M+1, (19.80%)], 401 [M+, (1.17%)], 93 (100%); Anal. Calcd for
2910 (CH aliphatic), 1673 (C@O amide), 1580, 1383 (NO2); MS:
(Mwt.: 312): m/z, 313 [M+1, (16.5%)], 312 [M+, (100%)]; Anal. Calcd
for C15H12N4O4: C, 57.69; H, 3.87; N, 17.94; Found: C, 57.69; H,
3.58; N, 17.63.
C22H19N5O3: C, 65.83; H, 4.77; N, 17.45; Found: C, 65.97; H, 4.82;
N, 17.58.
4.1.4.2. 1-(2-((4-Methoxyphenyl)amino)-3-oxo-3,4-dihydroquinoxalin-
6-yl)-3-phenylthiourea (VIIb). The titled compound was afforded
as yellow solid 0.12 g (81%); m.p. 168 °C; 1H NMR (300 MHz,
DMSO-d6) d 12.59 (s, 1H), 10.70 (s, 1H), 9.71 (s, 1H), 9.21 (s, 1H),
8.00 (d, J = 8.9 Hz, 1H), 7.49 (d, J = 8.3 Hz, 1H), 7.45–7.30 (m, 5H),
7.28 (s, 1H), 7.13 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 8.6 Hz, 2H), 3.76
4.1.3. 7-Amino-3-((4-methoxyphenyl)amino)quinoxalin-2(1H)-one
(VI)
To a solution of X (1 g, 3.2 mmol) in THF/MeOH (50 ml) was
added 10% Pd-C wet (0.1 g) and then the mixture was stirred at
40 °C under H2. After removing the catalyst by filtration with celite,
the filtrate was concentrated in vacuo, dried giving brownish pow-
der of VI at 0.8 g (88%) yield; m.p. > 300 °C; 1H NMR (300 MHz,
DMSO-d6) d 12.11 (s, 1H), 9.86 (s, 1H), 7.98 (s, 1H), 7.95 (s,2H),
7.21 (d, J = 8.5 Hz, 1H), 7.08 (d, J = 8.6 Hz, 1H), 6.89 (d, J = 8.6 Hz,
(s, 3H); FT-IR (m
max, cmÀ1): 3395 (NH), 3084 (CH aromatic), 2984
(CH aliphatic), 1665 (C@O amide); MS: (Mwt.: 417): m/z,
418 [M+1, (27.23%)], 417 [M+, (1.98%)], 296 (100%); Anal. Calcd for
C22H19N5O2S: C, 63.29; H, 4.59; N, 16.78; Found: C, 63.17; H, 4.65;
N, 16.59.
2H), 6.54 (d, J = 8.8 Hz, 2H), 3.75 (s, 3H); FT-IR (
m
max, cmÀ1):
3440 (forked NH2), 3120 (CH aromatic), 2954 (CH aliphatic),
1620 (C@O amide); MS: (Mwt.: 282): m/z, 283 [M+1, (16.5%)],
282 [M+, (2.2%)], 154 (100%); Anal. Calcd for C15H14N4O2:
C, 63.82; H, 5.00; N, 19.85; Found: C, 63.35; H, 4.98; N, 19.42.
4.1.4.3. 1-(3-Chlorophenyl)-3-(2-((4-methoxyphenyl)amino)-3-oxo-
3,4-dihydroquinoxalin-6-yl)urea (VIIc). The titled compound was
afforded as buff solid 0.08 g (52%); m.p. 192 °C; 1H NMR
(300 MHz, DMSO-d6) d 12.30 (s, 1H), 9.13 (s, 1H), 8.92 (s, 1H),
8.87 (s, 1H), 8.03 (d, J = 8.5 Hz, 1H), 7.72 (d, J = 8.6 Hz, 1H), 7.56
(s, 1H), 7.40–7.35 (m, 3H), 7.28 (s, 1H), 7.18 (d, J = 8.6 Hz, 2H),
4.1.4. General procedure for the preparation of compounds (VIIa-e)
To a solution of VI (0.1 g, 0.35 mmol) in THF was added the
appropriate isocyanate or isothiocyanate (0.7 mmol; 2 eq.) (viz;
6.92 (d, J = 8.8 Hz, 2H), 3.75 (s, 3H); FT-IR (
(NH), 3084 (CH aromatic), 2946 (CH aliphatic), 1662 (C@O amide);
m
max, cmÀ1): 3280

M.I. Shahin et al. / Bioorganic Chemistry 56 (2014) 16–26
23
MS: (Mwt.: 435): m/z, 437 [M+2, (72.34%)], 417 [M+, (57.45%)], 361
(100%); Anal. Calcd for C22H18ClN5O3: C, 60.62; H, 4.16; N, 16.07;
Found: C, 60.71; H, 4.23; N, 16.19.
at room temperature. The formed precipitate was filtered, washed
with gl. acetic acid then water thoroughly. The solid separated was
black powder with a yield of 0.1 g (65%); m.p. > 300; 1H NMR
(300 MHz, DMSO-d6) d 12.35 (s, 1H), 10.44 (s, 1H), 9.40 (s, 1H),
9.17 (s, 1H), 8.08–7.94 (m, 4H), 7.87 (s, 1H), 7.65 (d, J = 7.4 Hz,
1H), 7.56 (d, J = 7.5 Hz, 1H), 7.38 (d, J = 8.5 Hz, 2H), 6.93
4.1.4.4. 1-(2-((3-Methoxyphenyl)amino)-3-oxo-3,4-dihydroquinoxalin-
6-yl)-3-(p-tolyl)urea (VIId). The titled compound was obtained as
buff powder 0.12 g (81%); m.p. 292 °C; 1H NMR (300 MHz,
DMSO-d6) d 12.29 (s, 1H), 9.11 (s, 1H), 8.80 (s, 1H), 8.53 (s, 1H),
8.03 (d, J = 8.6 Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.37 (d, J = 8.6 Hz,
2H), 7.23 (s, 1H), 7.18 (d, J = 8.7 Hz, 2H), 7.12 (s, 1H), 6.96–6.87
(d, J = 8.8 Hz, 2H), 3.75 (s, 3H); FT-IR (m
max, cmÀ1): 3390 (NH),
3400–2900 (OH carboxylic), 1685 (C@O acid), 1665 (C@O amide),
3032 (CH aromatic), 2929 (CH aliphatic); MS: (Mwt.: 430): m/z,
431 [M+1, (75.53%)], 430 [M+, (55.32%)], 243 (100%); Anal. Calcd
for C23H18N4O5 C, 64.18; H, 4.22; N, 13.02; Found: C, 64.03; H,
4.13; N, 12.95.
(m, 3H), 3.75 (s, 3H), 2.27 (s, 3H); FT-IR (
m
max, cmÀ1): 3275
(NH), 3054 (CH aromatic), 2936 (CH aliphatic), 1685 (C@O amide);
MS: (Mwt.: 415): m/z, 416 [M++1, (1.8%)], 415 [M+, (9.3%)], 77
(100%); Anal. Calcd for C23H21N5O3: C, 66.49; H, 5.09; N, 16.86;
Found: C, 60.63; H, 4.04; N, 16.03.
4.1.7. N-(2-((4-Methoxyphenyl)amino)-3-oxo-3,4-dihydroquinoxalin-
6-yl)acetamide (X)
The titled compound was afforded by refluxing VI (0.1 g,
0.35 mmol) with acetyl chloride (0.083 g, 1.06 mmol) in THF for
24 h. It was filtered, washed with water, dried. 0.08 g (70%) of black
solid was obtained; m.p. 176 °C; 1H NMR (300 MHz, DMSO-d6)
d 12.68 (s, 1H), 10.23 (s, 1H), 9.65 (s, 1H), 7.95 (d, J = 8.7 Hz, 1H),
7.82 (d, J = 8.6 Hz, 1H), 7.41 (d, J = 8.7 Hz, 2H), 7.31 (s, 1H), 6.95
4.1.4.5.
1-Cyclohexyl-3-(2-((4-methoxyphenyl)amino)-3-oxo-3,
4-dihydroquinoxalin-6-yl)urea (VIIe). The titled compound was
separated as yellow solid 0.12 g (81%); m.p. 237 °C; 1H NMR
(300 MHz, DMSO-d6) d 12.25 (s, 1H), 9.02 (s, 1H), 8.76 (s, 1H),
8.01 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 8.6 Hz, 1H), 7.36 (s, 1H), 7.29
(s, 1H), 7.09 (d, J = 8.7 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 3.74
(d, J = 8.7 Hz, 2H), 3.76 (s, 3H), 2.06 (s, 3H); FT-IR (m
max, cmÀ1):
(s, 3H), 2.03 – 0.89 (m, 5H); FT-IR (
m
max, cmÀ1): 3328 (NH),
3447 (NH), 3035 (CH aromatic), 2984 (CH aliphatic), 1642 (C@O
amide); MS: (Mwt.: 324): m/z, 325 [M+1, (18.7%)], 324
[M+, (53.45%)], 281 (100%); Anal. Calcd for C17H16N4O3: C, 62.95;
H, 4.97; N, 17.27; Found: C, 63.04; H, 4.99; N, 17.34.
3035 (CH aromatic), 2929 (CH aliphatic), 1627 (C@O amide); MS:
(Mwt.: 407): m/z, 408 [M+1, (24.2%)], 407 [M+, (3.8%)], 281
(100%); Anal. Calcd for C22H25N5O3: C, 64.85; H, 6.18; N, 17.19;
Found: C, 64.97; H, 6.15; N, 17.32.
4.1.8. General procedure for preparation of compounds (XIVa-b)
(2 g, 9.54 mmol) of XIII were refluxed in ethanol (30 ml) with
the appropriate amine (19.08 mol; 2 eq.) (viz: p-anisidine,
p- chloroaniline) for 5 h, allowed to cool then finally filtered. The
crude products XIVa-b were purified by recrystallization from
ethanol.
4.1.5. General procedure for preparation of compounds (VIIIa-b)
To a solution of VI (0.1 g, 0.35 mmol) in dry pyridine (10 mL)
was added the appropriate sulfonyl chloride (1.05 mmol; 3 eq.)
(viz; benzenesulfonyl chloride and 4-methylbenzenesulfonyl chlo-
ride) and heated at reflux for 2 h. The mixture was added to cold
water (30 mL) and stirred for several minutes. The resulted solid
was filtered, washed with water, dried and recrystallized from
ethanol to afford the titled compounds.
4.1.8.1. N-(4-Methoxyphenyl)-6-nitroquinoxalin-2-amine (XIVa).
The titled compound was obtained as red powder 1.8 g (63%);
m.p. 218 °C; 1H NMR (300 MHz, DMSO-d6) d 10.31 (s, 1H), 8.61
(s, 1H), 8.56 (d, J = 2.6 Hz, 1H), 8.33 (dd, J = 9.1, 2.7 Hz, 1H), 7.87
(d, J = 9.0 Hz, 1H), 7.74 (d, J = 9.1 Hz, 2H), 6.99 (d, J = 9.0 Hz, 2H),
4.1.5.1. N-(2-((4-Methoxyphenyl)amino)-3-oxo-3,4-dihydroquinoxa-
lin-6-yl)benzenesulfonamide (VIIIa). The titled compound was sep-
arated as brown solid 0.06 g (40%); m.p. 122 °C; 1H NMR (300 MHz,
DMSO-d6) d 12.29 (s, 1H), 10.35 (s, 1H), 9.27 (s, 1H), 7.94
(d, J = 8.6 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.61 – 7.50 (m, 5H),
7.29 (d, J = 8.6 Hz, 2H), 7.08 (s, 1H), 6.91 (d, J = 8.7 Hz, 2H), 3.73
3.77 (s, 3H); FT-IR (m
max, cmÀ1): 3447 (NH), 3035 (CH aromatic),
2984 (CH aliphatic), 1587, 1390 (NO2); MS: (Mwt.: 296): m/z, 297
[M+1, (17.7%)], 296 [M+, (84.64%)], 137 (100%); Anal. Calcd for
C15H12N4O3: C, 60.81; H, 4.08; N, 18.91; Found C, 60.63; H, 3.96;
N, 18.71.
(s, 3H); FT-IR (
m
max, cmÀ1): 3442 (NH), 3075 (CH aromatic),
2930 (CH aliphatic), 1625 (C@O amide), 1335 & 1160 (SO2); MS:
(Mwt.: 422): m/z, 423 [M+1, (3.16%)], 422 [M+, (9.47%)], 77
(100%); Anal. Calcd for C21H18N4O4S: C, 59.70; H, 4.29; N, 13.26;
Found: C, 59.79; H, 4.33; N, 13.41.
4.1.8.2. N-(4-Chlorophenyl)-6-nitroquinoxalin-2-amine (XIVb). The
titled compound was obtained as yellow powder 1.5 g (52%);
m.p. 142 °C; 1H NMR (300 MHz, DMSO-d6) d 10.27 (s, 1H), 8.56
(s, 1H), 8.32 (d, J = 2.6 Hz, 1H), 8.12 (dd, J = 8.6, 2.7 Hz, 1H), 7.76
(d, J = 8.5 Hz, 1H), 7.64 (d, J = 9.1 Hz, 2H), 6.84 (d, J = 8.9 Hz, 2H);
4.1.5.2. N-(2-((4-Methoxyphenyl)amino)-3-oxo-3,4-dihydroquinoxa-
lin-6-yl)-4-methylbenzene sulfonamide (VIIIb). The titled compound
was separated as brownish black solid 0.09 g (58%); m.p.116 °C; 1H
NMR (300 MHz, DMSO-d6) d 12.27 (s, 1H), 10.26 (s, 1H), 9.21
(s, 1H), 7.97 (d, J = 8.6 Hz, 2H), 7.64 (d, J = 8.5 Hz, 2H), 7.56
(d, J = 8.6 Hz, 1H), 7.34 (d, J = 8.3 Hz, 2H), 7.07 (s, 1H), 7.01
(d, J = 8.4 Hz, 1H), 6.91 (d, J = 8.2 Hz, 2H), 3.73 (s, 3H), 2.50
FT-IR (
(NO2); MS: (Mwt.: 300): m/z, 302 [M + 2, (32.2%)], 300 [M+,
(4.3%)], 141 (100%); Anal. Calcd for 14H9ClN4O2: C, 55.92;
m
max, cmÀ1): 3390 (NH), 3015 (CH aromatic), 1583, 1380
C
H, 3.02; N, 18.63; Found C, 55.75; H, 2.96; N, 18.23.
4.1.9. General procedure for preparation of compounds (XVa-b)
Reduction of XIVa-b (1 g, 3.33 mmol) was carried out by adding
(0.1 g) 10% Pd-C wet to 50 ml of their solutions in THF/MeOH then
stirring under H2 at room temperature for 8 h. After removing the
catalyst by filtration with celite and evaporation of the filtrate
under reduced pressure, we afforded the titled compounds XVa-b.
(s, 3H); FT-IR (
m
max, cmÀ1): 3404 (NH), 3062 (CH aromatic),
2926 (CH aliphatic), 1658 (C@O amide), 1335 & 1160 (SO2); MS:
(Mwt.: 436): m/z, 437 [M++1, (13.96%)], 436 [M+, (28.83%)], 281
(100%); Anal. Calcd for C21H18N4O4S: C, 59.70; H, 4.29; N, 13.26;
Found: C, 59.79; H, 4.33; N, 13.41.
4.1.6. 2-((2-((4-Methoxyphenyl)amino)-3-oxo-3,4-
dihydroquinoxalin-6-yl)carbamoyl)benzoic acid (IX)
A mixture of compound VI (0.1 g, 0.35 mmol) and phthalic
anhydride (0.1 g, 0.7 mmol) in gl. acetic acid was stirred for 48 h
4.1.9.1. N2-(4-Methoxyphenyl)quinoxaline-2,6-diamine (XVa). The
titled compound was obtained as brown solid 0.6 g (67%); m.p.
247 °C; 1H NMR (300 MHz, DMSO-d6) d 10.27 (s, 1H), 8.56
(s, 1H), 8.32 (d, J = 2.6 Hz, 1H), 8.12 (dd, J = 7.5, 2.7 Hz, 1H), 7.76

24
M.I. Shahin et al. / Bioorganic Chemistry 56 (2014) 16–26
(d, J = 8.5 Hz, 1H), 7.64 (d, J = 9.1 Hz, 2H), 6.84 (d, J = 7.6 Hz, 2H),
5.37 (s, 2H), 3.80 (s, 3H); FT-IR (
max, cmÀ1): 3420 (forked
NH2), 3115 (CH aromatic), 2932 (CH aliphatic); MS: (Mwt.: 266):
m/z, 267 [M+1, (16.4%)], 266 [M+, (1.7%)], 145 (100%); Anal. Calcd
for C15H14N4O: C, 67.65; H, 5.30; N, 21.04; Found: C, 67.36;
H, 5.12; N, 20.93.
(m
max, cmÀ1): 3380 (NH), 3020 (CH aromatic), 2926 (CH aliphatic),
1680 (C@O amide); MS: (Mwt.: 391): m/z, 392 [M+1, (25.6%)], 391
[M+, (3.2%)], 265 (100%); Anal. Calcd for C22H25N5O2: C, 67.50; H,
6.44; N, 17.89; Found: C, 67.61; H, 6.48; N, 18.02.
m
4.1.10.5.
1-(2-((4-Chlorophenyl)amino)quinoxalin-6-yl)-3-cyclo-
hexylurea (XVIe). The titled compound was separated as yellow
solid; m.p. 194 °C; 1H NMR (300 MHz, DMSO-d6) d 9.55 (s,1H),
8.51 (s,1H), 8.40 (s, 1H), 7.40 (d, J = 8.5 Hz, 1H), 7.38(d, J = 8.6 Hz,
1H), 7.36 (s, 1H), 7.24 (d, J = 8.9 Hz, 2H), 6.07 (d, J = 7.8 Hz, 2H),
4.1.9.2. N2-(4-Chlorophenyl)quinoxaline-2,6-diamine (XVb). The
titled compound was separated as brown solid 0.75 g (83%);
m.p.159 °C; 1H NMR (300 MHz, DMSO-d6) d 10.13 (s, 1H), 8.23
(s, 1H), 8.06 (d, J = 2.6 Hz, 1H), 7.89 (dd, J = 7.5, 2.7 Hz, 1H), 7.67
(d, J = 8.5 Hz, 1H), 7.52 (d, J = 9.1 Hz, 2H), 6.54 (d, J = 7.6 Hz, 2H),
5.75 (s, 1H), 1.91–1.00 (m, 5H); FT-IR (m
max, cmÀ1): 3430 (NH),
3016 (CH aromatic), 2920 (CH aliphatic), 1670 (C@O amide); MS:
(Mwt.: 395): m/z, 397 [M+2, (32.4%)], 395 [M+, (1.6%)], 269
(100%); Anal. Calcd for C21H22ClN5O: C, 63.71; H, 5.60; N, 17.69;
Found: C, 63.61; H, 5.48; N, 17.52.
4.96 (s, 2H); FT-IR (m
max, cmÀ1): 3420 (forked NH2), 3115 (CH
aromatic); MS: (Mwt.: 270): m/z, 272 [M + 2, (15.3%)], 270
[M+, (32%)], 141 (100%); Anal. Calcd for C14H11ClN4: C, 62.11;
H, 4.10; N, 20.70; Found: C, 62.01; H, 4.00; N, 20.52.
4.1.11. General procedure for preparation of compounds (XVIIa-c)
To a solution of the appropriate amine XVa-b (0.1 g, 0.37 mmol)
in dry pyridine (10 mL) the appropriate sulfonyl chloride was
added (1.05 mmol; 3 eq.) (viz; benzenesulfonyl chloride and
4- methylbenzenesulfonyl chloride) and heated at reflux for 2 h.
The mixture was added to cold water (30 mL) and stirred for several
minutes. The resulted solid was filtered, washed with water, dried
and recrystallized from ethanol to afford the titled compounds.
4.1.10. General procedure for preparation of compounds (XVIa-e)
To a solution of XVa-b (0.1 g, 0.37 mmol) in methylene chloride
was added the appropriate isocyanate or isothiocyanate
(0.7 mmol; 2 eq.) (viz; phenyl isothiocyanate, phenyl isocyanate,
3-methyl phenyl isocyanate, and cyclohexyl isocyanate) and stir-
red overnight. The resulted solid was filtered, triturated with
diethyl ether and recrystallized from ethanol to afford the titled
compounds. (XVIa-e)
4.1.11.1. N-(2-((4-Methoxyphenyl)amino)quinoxalin-6-yl)benzene-
sulfonamide (XVIIa). The titled compound was separated as black
solid; m.p. 218 °C; 1H NMR (300 MHz, DMSO-d6) d 10.58 (s, 1H),
8.94 (d, J = 3.4 Hz, 1H), 8.63 (s, 1H), 8.53 (s, 1H), 7.78
(d, J = 8.3 Hz, 1H), 7.60–7.46 (m, 5H), 7.41 (dd, J = 8.7, 2.5 Hz, 1H),
7.31 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 8.5 Hz, 2H), 3.74 (s, 3H); FT-IR
4.1.10.1. 1-(2-((4-Methoxyphenyl)amino)quinoxalin-6-yl)-3-phenyl-
thiourea (XVIa). The titled compound was separated as yellow
solid; m.p. 249 °C; 1H NMR (300 MHz, DMSO-d6) d 10.01 (s, 1H),
9.92 (s, 1H), 9.72 (s, 1H), 8.48 (s, 1H), 7.98 (d, J = 2.4 Hz, 1H), 7.86
(d, J = 8.0 Hz, 1H), 7.73 (dd, J = 8.9, 2.4 Hz, 1H), 7.61 (d, J = 8.9 Hz,
2H), 7.54–7.45 (m, 2H), 7.38–7.29 (m, 1H), 7.15 (d, J = 8.6 Hz,
(
m
max, cmÀ1): 3390 (NH), 3045 (CH aromatic), 2926 (CH
2H), 6.97 (d, J = 9.1 Hz, 2H), 3.76 (s, 3H); FT-IR (
m
max, cmÀ1):
aliphatic),1330 & 1150 (SO2); MS: (Mwt.: 406): m/z, 407 [M+1,
(25.1%)], 406 [M+, (5.7%)], 251 (100%); Anal. Calcd for C21H18N4O3S:
C, 62.05; H, 4.46; N, 13.78; Found: C, 62.13; H, 4.49; N, 13.86.
3383 (NH), 3054 (CH aromatic), 2952 (CH aliphatic), 1660 (C@O
amide),; MS: (Mwt.: 401): m/z, 402 [M++1, (26.4%)], 401 [M+,
(5.2%)], 166 (100%); Anal. Calcd for
C22H19N5OS: C, 65.81;
H, 4.77; N, 17.44; Found: C, 65.63; H, 4.54; N, 17.21.
4.1.11.2. N-(2-((4-Methoxyphenyl)amino)quinoxalin-6-yl)-4-methyl-
benzenesulfonamide (XVIIb). The titled compound was obtained as
brownish solid; m.p. 166 °C; 1H NMR (300 MHz, DMSO-d6) d 10.40
(s, 1H), 9.74 (s, 1H), 8.75 (d, J = 3.2 Hz, 1H), 8.44 (s, 1H), 7.81
(d, J = 8.9 Hz, 2H), 7.66 (d, J = 8.1 Hz, 1H), 7.53 (d, J = 8.8 Hz, 2H),
7.38 (dd, J = 8.9, 2.5 Hz, 1H), 7.33 (d, J = 8.6 Hz, 2H), 6.93
4.1.10.2. 1-(2-((4-Chlorophenyl)amino)quinoxalin-6-yl)-3-phenylu-
rea (XVIb). The titled compound was obtained as yellow powder;
m.p. 134 °C; 1H NMR (300 MHz, DMSO-d6) d 8.63 (s, 1H), 8.59
(s, 1H), 8.52 (s, 1H), 8.37 (s, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.44
(d, J = 2.4 Hz, 1H), 7.27 (dd, J = 8.6, 2.3 Hz, 1H), 7.06–6.88 (m, 5H),
(d, J = 9.0 Hz, 2H), 3.74 (s, 3H), 2.30 (s, 3H); FT-IR (m
max, cmÀ1):
6.56 (d, J = 8.6 Hz, 2H), 6.48 (d, J = 8.3 Hz, 2H); FT-IR (
m
max,
3350 (NH), 3092 (CH aromatic), 2910 (CH aliphatic),1350 & 1165
cmÀ1): 3383 (NH), 3054 (CH aromatic), 1660 (C = O amide),; MS:
(Mwt.: 389): m/z, 391 [M + 2, (32.4%)], 389 [M+, (22.9%)], 269
(100%); Anal. Calcd for C21H16ClN5O: C, 64.70; H, 4.14; N, 17.96;
Found: C, 64.52; H, 4.03; N, 17.64.
(SO2); MS: (Mwt.: 420): m/z, 421 [M+1, (24.1%)], 420 [M+,
(4.5%)], 185 (100%); Anal. Calcd for
C22H20N4O3S: C, 62.84;
H, 4.79; N, 13.32; Found: C, 62.97; H, 4.84; N, 13.39.
4.1.11.3.
N-(2-((4-Chlorophenyl)amino)quinoxalin-6-yl)-4-methyl-
4.1.10.3. 1-(2-((4-Methoxyphenyl)amino)quinoxalin-6-yl)-3-(m-toly-
l)urea (XVIc). The titled compound was separated as buff solid;
m.p.216 °C; 1H NMR (300 MHz, DMSO-d6) d 9.61 (s, 1H), 8.89
(s, 1H), 8.66 (s, 1H), 8.46 (s, 1H), 7.85 (d, J = 8.6 Hz, 1H), 7.62
(s, 1H), 7.33 (s, 1H), 7.27 (d, J = 8.1 Hz, 1H), 7.22–7.12 (m, 3H),
6.95 (d, J = 8.6 Hz, 2H), 6.80 (d, J = 7.4 Hz, 2H), 3.75 (s, 3H), 2.29
benzenesulfonamide (XVIIc). The product was separated as brown-
ish black solid; m.p. 268 °C; 1H NMR (300 MHz, DMSO-d6) d 10.52
(s, 1H), 10.23 (s, 1H), 8.55 (s, 1H), 7.98 (d, J = 2.3 Hz, 1H), 7.95
(d, J = 8.3 Hz, 1H), 7.68 (d, J = 8.2 Hz, 2H), 7.59 (d, J = 8.6 Hz, 2H),
7.43 (dd, J = 8.9, 2.6 Hz, 1H), 7.36 (d, J = 8.1 Hz, 2H), 7.34
(d, J = 8.3 Hz, 2H), 2.29 (s, 3H); FT-IR (m
max, cmÀ1): 3420 (NH),
(s, 3H); FT-IR (
m
max, cmÀ1): 3383 (NH), 3054 (CH aromatic), 2935
3055 (CH aromatic), 2905 (CH aliphatic),1400 & 1160 (SO2); MS:
(Mwt.: 424): m/z, 426 [M+2, (36.5%)], 424 [M+, (23.8%)], 127
(100%); Anal. Calcd for C21H17ClN4O2S: C, 59.36; H, 4.03; N,
13.19; Found: C, 59.25; H, 4.01; N, 13.08.
(CH aliphatic), 1685 (C@O amide); MS: (Mwt.: 399): m/z, 400 [M+1,
(26.8%)], 399 [M+, (2.3%)], 164 (100%); Anal. Calcd for C23H21N5O2:
C, 69.16; H, 5.30; N, 17.53; Found: C, 69.28; H, 5.28; N, 17.69.
4.1.10.4. 1-Cyclohexyl-3-(2-((4-methoxyphenyl)amino)quinoxalin-6-
yl)urea (XVId). The titled compound was separated as yellowish
solid; m.p. 258 °C; 1H NMR (300 MHz, DMSO-d6) d 9.55 (s, 1H),
8.51 (d, J = 8.8 Hz 1H), 8.43 (s, 1H), 7.96 (s, 1H), 7.83 (d,
J = 8.8 Hz, 1H), 7.54 (s, 1H), 6.94 (d, J = 8.8 Hz, 2H), 6.12 (d,
J = 7.8 Hz, 2H), 3.81 (s, 1H), 3.75 (s, 3H), 1.95–1.05 (m, 5H); FT-IR
4.1.12. N-(2-((4-Chlorophenyl)amino)quinoxalin-6-yl)acetamide (XVIII)
The titled compound was afforded by stirring XVb (0.1 g,
0.37 mmol) with acetyl chloride (0.083 g, 1.06 mmol) in glacial
acetic acid (10 ml) overnight. It was filtered, washed with water
thoroughly and dried, giving brownish black solid with a yield of
0.06 g (52%); m.p. 176 °C; 1H NMR (300 MHz, DMSO-d6) d 10.17

M.I. Shahin et al. / Bioorganic Chemistry 56 (2014) 16–26
25
(s, 1H), 9.95 (s, 1H), 8.51 (s, 1H), 8.24 (d, J = 2.3 Hz, 1H), 7.98
(d, J = 8.9 Hz, 1H), 7.77 (dd, J = 8.9, 2.4 Hz, 1H), 7.68 (d, J = 8.9 Hz,
2H), 7.40 (d, J = 8.9 Hz, 2H), 2.10 (s, 3H); FT-IR (
3380 (NH), 3032 (CH aromatic), 2932 (CH aliphatic), 1665 (C@O
amide); MS: (Mwt.: 312): m/z, 314 [M++2, (17.5%)], 312 [M+,
(5.6%)], 255 (100%); Anal. Calcd for C16H13ClN4O: C, 61.44; H,
4.19; N, 17.91; Found: C, 61.36; H, 4.02; N, 17.85.
CHARMm force field. Water molecules were preserved because of
their importance in ligand interaction to VEGFR-2 enzyme. The
protein structure was minimized using steepest descent minimiza-
tion algorithm. The integrated naphthyl-based compound was
removed from the binding site. Our ligands were prepared using
Accelry’s discovery studio prepare ligands protocol. This adds
hydrogen atoms and minimizes them. Docking was accomplished
using GOLD 4.1 software in the interface of Accelry’s discovery stu-
dio 2.5. The default values of GOLD were used but with enabling
early termination and allowing generating diverse solutions to
get more possible docking solutions. Also the number of docking
iterations was raised to 500,000 ones so high number of predic-
tions was obtained. 10 Different docking solutions were generated
and were inspected thoroughly for getting the best binding mode.
m
max, cmÀ1):
4.1.13. General procedure for preparation of compounds (XXIa-b)
The final compounds XXIa-b were synthesized by refluxing XIII
(0.5 g, 2.39 mmol) with the 4-aminophenyl urea derivatives XXa-b
(1 g, 4.4 mmol) in ethanol (20 ml) and in presence of anhydrous
K2CO3 (0.6 g, 4.4 mmol) for 6 h. The reaction mixture is allowed
to cool, filtered and the resulted solid product is washed
thoroughly with water, left to dry.
Acknowledgments
4.1.13.1. 1-(4-((6-Nitroquinoxalin-2-yl)amino)phenyl)-3-phenylurea
(XXIa). The titled compound was obtained as red powder, 0.8 g
The authors are grateful for the Science and Technology Devel-
opment Fund (STDF) for funding the center for drug discovery
and development research at the Faculty of Pharmacy Ain Shams
University (Project No. 5251) and for financial support of this study.
(83.7%); m.p. 258 °C; 1H NMR (300 MHz, DMSO-d6)
d 8.97
(s, 1H), 8.61 (s, 1H), 8.57 (s, 1H), 8.33 (d, J = 8.4 Hz, 1H), 7.88
(d, J = 8.2 Hz, 1H), 7.77 (d, J = 8.3 Hz, 2H),7.52 (s,1H), 7.49
(d, J = 8.1 Hz, 2H), 7.40–7.18 (m, 5H), 6.95 (s, 1H); FT-IR (m max,
cmÀ1): 3410 (NH), 3015 (CH aromatic), 1660 (C@O amide), 1540,
1350 (NO2); MS: (Mwt.: 400): m/z, 401 [M+1, (25.2%)], 400 [M+,
(2.5%)], 226 (100%); Anal. Calcd for C21H16N6O3: C, 62.99; H, 4.03;
N, 20.99; Found: C, 63.10; H, 4.07; N, 21.13.
References
[1] A. Levitzki, S. Klein, Signal transduction therapy of cancer, Mol. Aspects Med.
31 (4) (2010) 287–329.
[2] A. Bennasroune, A. Gardin, D. Aunis, G. Cremel, P. Hubert, Crit. Rev. Oncol.
Hematol. 50 (1) (2004) 23–38.
4.1.13.2. 1-(3-Chlorophenyl)-3-(4-((6-nitroquinoxalin-2-yl)amino)-
phenyl)urea (XXIb). Compound XXIb was obtained as red–orange
powder in 0.75 g (72%); m.p. 280 °C; 1H NMR (300 MHz, DMSO-
d6) d 9.85 (s,1H), 8.68 (s, 1H), 8.3 (s,1H,8.19 (s, 1H), 7.69 (s, 1H),
7.51 (d, J = 8.5 Hz, 1H), 7.34–7.18 (m, 3H), 7.06 (d, J = 8.3 Hz, 1H),
6.97 (d, J = 8.3 Hz, 2H), 6.51 (d, J = 8.2 Hz, 2H), 4.77 (s, 1H); FT-IR
[3] S. Madhusudan, T.S. Ganesan, Clin. Biochem. 37 (7) (2004) 618–635.
[4] T. Mikalsen, N. Gerits, U. Moens, Biotechnol. Annu. Rev. 12 (2006) 153–223.
[5] G. Bergers, L.E. Benjamin, Nat. Rev. Cancer 3 (6) (2003) 401–410.
[6] S. Guo, L.S. Colbert, M. Fuller, Y. Zhang, R.R. Gonzalez-Perez, Vascular
endothelial growth factor receptor-2 in breast cancer, Biochim. Biophys. Acta
1806 (1) (2010) 108–121.
[7] S. Tugues, S. Koch, L. Gualandi, X. Li, L. Claesson-Welsh, Mol. Aspects Med. 32
(2) (2011) 88–111.
[8] K.M. Sakamoto, Curr. Opin. Investig. Drugs 5 (12) (2004) 1329–1339.
[9] T. Ahmad, T. Eisen, Clin. Cancer Res. 10 (18 Pt 2) (2004) 6388S–6392S.
[10] Y. Yang, L. Shi, Y. Zhou, H.Q. Li, Z.W. Zhu, H.L. Zhu, Bioorg. Med. Chem. Lett. 20
(22) (2010) 6653–6656.
(m
max, cmÀ1): 3400 (NH), 3010 (CH aromatic), 1650 (C@O amide),
1530, 1320 (NO2); MS: (Mwt.: 434): m/z, 436 [M + 2, (33.1%)], 434
[M+, (25%)], 175 (100%); Anal. Calcd for C21H15ClN6O3: C, 58.00;
H, 3.48; N, 19.33; Found C, 58.09; H, 3.46; N, 19.51.
[11] M.E. Fraley, K.L. Arrington, C.A. Buser, P.A. Ciecko, K.E. Coll, C. Fernandes, G.D.
Hartman, W.F. Hoffman, J.J. Lynch, R.C. McFall, K. Rickert, R. Singh, S. Smith,
K.A. Thomas, B.K. Wong, Bioorg. Med. Chem. Lett. 14 (2) (2004) 351–355.
[12] G. Bold, K.H. Altmann, J. Frei, M. Lang, P.W. Manley, P. Traxler, B. Wietfeld,
J. Bruggen, E. Buchdunger, R. Cozens, S. Ferrari, P. Furet, F. Hofmann, G.
Martiny-Baron, J. Mestan, J. Rosel, M. Sills, D. Stover, F. Acemoglu, E. Boss, R.
Emmenegger, L. Lasser, E. Masso, R. Roth, C. Schlachter, W. Vetterli, J. Med.
Chem. 43 (12) (2000) 2310–2323.
[13] L.F. Hennequin, E.S. Stokes, A.P. Thomas, C. Johnstone, P.A. Ple, D.J. Ogilvie,
M. Dukes, S.R. Wedge, J. Kendrew, J.O. Curwen, J. Med. Chem. 45 (6) (2002)
1300–1312.
[14] M. McTigue, B.W. Murray, J.H. Chen, Y.L. Deng, J. Solowiej, R.S. Kania, Proc. Natl.
Acad. Sci. USA 109 (45) (2012) 18281–18289.
[15] P. Traxler, P. Furet, Pharmacol. Ther. 82 (2–3) (1999) 195–206.
[16] R.R. Kroe, J. Regan, A. Proto, G.W. Peet, T. Roy, L.D. Landro, N.G. Fuschetto, C.A.
Pargellis, R.H. Ingraham, J. Med. Chem. 46 (22) (2003) 4669–4675.
[17] (a) P.W. Manley, S.W. Cowan-Jacob, E. Buchdunger, D. Fabbro, G. Fendrich,
P. Furet, T. Meyer, J. Zimmermann, Eur. J. Cancer 38 (Suppl 5) (2002) S19–S27;
(b) T.B. Lowinger, B. Riedl, J. Dumas, R.A. Smith, Curr. Pharm. Des. 8 (25) (2002)
2269–2278.
[18] J. Dietrich, C. Hulme, L.H. Hurley, Bioorg. Med. Chem. 18 (15) (2010) 5738–
5748.
[19] Y. Liu, N.S. Gray, Nat. Chem. Biol. 2 (7) (2006) 358–364.
[20] M.I. Abasolo, C.H. Gaozza, B.M. Fernández, J. Heterocycl. Chem. 24 (6) (1987)
1771–1775.
[21] G. Cheeseman, J. Chem. Soc. (Resumed) (1962) 1170–1176.
[22] D.R. Romer, J. Heterocycl. Chem. 46 (2) (2009) 317–319.
[23] J. Deng, E. Feng, S. Ma, Y. Zhang, X. Liu, H. Li, H. Huang, J. Zhu, W. Zhu, X. Shen,
L. Miao, H. Liu, H. Jiang, J. Li, J. Med. Chem. 54 (13) (2011) 4508–4522.
[24] S.A. Galal, A.S. Abdelsamie, H. Tokuda, N. Suzuki, A. Lida, M.M. Elhefnawi, R.A.
Ramadan, M.H. Atta, H.I. El Diwani, Eur. J. Med. Chem. 46 (1) (2011) 327–340.
[25] P. Corona, M. Loriga, M.P. Costi, S. Ferrari, G. Paglietti, Eur. J. Med. Chem. 43 (1)
(2008) 189–203.
[26] E. Ford, A. Brewster, G. Jones, J. Bailey, N. Sumner, Tetrahedron Lett. 41 (17)
(2000) 3197–3198.
[27] K. Aoki, T. Obata, Y. Yamazaki, Y. Mori, H. Hirokawa, J. Koseki, T. Hattori, K.
Niitsu, S. Takeda, M. Aburada, K. Miyamoto, Chem. Pharm. Bull. (Tokyo) 55 (2)
(2007) 255–267.
4.2. In vitro enzyme inhibition assay
In vitro kinase inhibition assay of the twenty final compounds
was performed by KINEXUS Corporation, Vancouver, British
Columbia, Canada. In a designated radioactive working area, a
radioactive assay format for profiling evaluation of protein kinase
targets is performed. Assays are performed for 15 min duration,
at 30 °C, with 50
assays are terminated by spotting 20
l
M of 33P-ATP in a final volume of 25
l of the reaction mixture
ll. The
l
onto a phosphocellulose P81 plate. The P81 plate was washed 3
times for approximately 15 min each in a 1% phosphoric acid solu-
tion to remove excess unreacted 33P-ATP. The radioactivity on the
33P-labeled peptide/protein in the P81 plate was counted in the
presence of scintillation fluid in a Trilux scintillation counter. Blank
control was set up for each protein kinase target which included all
the assay components except the addition of appropriate substrate
in which we replace with equal volume of kinase assay buffer. The
corrected activity for the target was determined by removing the
blank control value.
4.3. Molecular docking
The X-ray crystal structure of VEGFR-2 kinase was obtained
from Protein Data Bank (accession code is 3B8Q). The protein
structure was prepared according to the standard protein prepara-
tion procedure integrated in Accelry’s discovery studio 2.5. This
was accomplished by adding hydrogen atoms, completing the
missing loops and applying force field parameters by using

26
M.I. Shahin et al. / Bioorganic Chemistry 56 (2014) 16–26
[28] G. Kettschau, H. Briem, I. Hartung, U. Luecking, M. Schaefer, K.-H. Thierauch,
W. Schwede, M. Husemann, Sulfonamido-macrocycles as Tie2 inhibitors and
the salts thereof, a pharmaceutical composition comprising these compounds,
the method of preparing and the use thereof. Google Patents, 2005.
[31] G. Jones, P. Willett, R.C. Glen, A.R. Leach, R. Taylor, J. Mol. Biol. 267 (3) (1997)
727–748.
[32] J. Houben, T. Weyl, E. Müller, Methoden der organischen Chemie (Houben-
Weyl), 1952.
[29] M. Gonzalez-Alvarez, G. Alzuet, J. Borras, L. del Castillo Agudo, S. Garcia-
Granda, J.M. Montejo Bernardo, J. Inorg. Biochem. 98 (2) (2004) 189–198.
[30] E. Valeur, M. Bradley, Chem. Soc. Rev. 38 (2) (2009) 606–631.
[33] P. Sah, Recl. Trav. Chim. Pays-B. 59 (3) (1940) 231–237.
[34] F. Rodriguez, I. Rozas, M. Kaiser, R. Brun, B. Nguyen, W.D. Wilson, R.N. Garcia,
C. Dardonville, J Med. Chem. 51 (4) (2008) 909–923.