Design, Synthesis and Cytotoxic Evaluation of Novel Chalcone Derivatives Bearing Triazolo[4,3-a]quinoxaline Moieties as Potent Anticancer Agents with Dual EGFR Kinase and Tubulin Polymerization Inhibitory Effects

Article abstract of DOI:10.3390/molecules23010048

A series of hybrid of triazoloquinoxaline-chalcone derivatives 7a–k were designed, synthesized, fully characterized, and evaluated for their cytotoxic activity against three target cell lines: human breast adenocarcinoma (MCF-7), human colon carcinoma (HCT-116), and human hepatocellular carcinoma (HEPG-2). The preliminary results showed that some of these chalcones like 7b–c, and 7e–g exhibited significant antiproliferative effects against most of the cell lines, with selective or non-selective behavior, indicated by IC₅₀ values in the 1.65 to 34.28 μM range. In order to investigate the mechanistic aspects of these active compounds, EGFR TK and tubulin inhibitory activities were measured as further biological assays. The EGFR TK assay results revealed that the derivatives 7a–c, 7e, and 7g could inhibit the EGFR TK in the submicromolar range (0.093 to 0.661 μM). Moreover, an antitubulin polymerization effect was noted for the active derivatives compared to the reference drug colchicine, with compounds 7e and 7g displaying 14.7 and 8.4 micromolar activity, respectively. Furthermore, a molecular docking study was carried out to explain the observed effects and the binding modes of these chalcones with the EGFR TK and tubulin targets.

Full text of DOI:10.3390/molecules23010048

molecules
Article
Design, Synthesis and Cytotoxic Evaluation of Novel
Chalcone Derivatives Bearing Triazolo[4,3-a]-
quinoxaline Moieties as Potent Anticancer Agents
with Dual EGFR Kinase and Tubulin Polymerization
Inhibitory Effects
1
1
1,2
1
3
Mohamed Alswah , Ashraf H. Bayoumi , Kamal Elgamal , Ahmed Elmorsy , Saleh Ihmaid
3
,
and Hany E. A. Ahmed *
1
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Al-Azhar University,
Cairo, Nasr City 11884, Egypt; drmohammedalswah@yahoo.com (M.A.);
bayoumi.ashraf@hotmail.com (A.H.B.); drkamalelgaml72@gmail.com (K.E.);
ahmedelmorsy232@yahoo.com (A.E.)
2
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy,
Delta University for Science and Technology, Mansoura 11152, Egypt
Pharmacognosy and Pharmaceutical Chemistry Department, Pharmacy College, Taibah University,
3
Al-Madinah Al-Munawarah 41477, Saudi Arabia; saleh_ihmaid@yahoo.com.au
*
Correspondence: heahmad@taibahu.edu.sa
Received: 6 December 2017; Accepted: 22 December 2017; Published: 27 December 2017
Abstract: A series of hybrid of triazoloquinoxaline-chalcone derivatives 7a
–
k
were designed,
synthesized, fully characterized, and evaluated for their cytotoxic activity against three target cell
lines: human breast adenocarcinoma (MCF-7), human colon carcinoma (HCT-116), and human
hepatocellular carcinoma (HEPG-2). The preliminary results showed that some of these chalcones
like 7b
–
c
, and 7e
–
g
exhibited significant antiproliferative effects against most of the cell lines,
M range.
with selective or non-selective behavior, indicated by IC50 values in the 1.65 to 34.28
µ
In order to investigate the mechanistic aspects of these active compounds, EGFR TK and tubulin
inhibitory activities were measured as further biological assays. The EGFR TK assay results
revealed that the derivatives 7a
range (0.093 to 0.661 M). Moreover, an antitubulin polymerization effect was noted for the active
derivatives compared to the reference drug colchicine, with compounds 7e and 7g displaying 14.7 and
.4 micromolar activity, respectively. Furthermore, a molecular docking study was carried out to
–c, 7e, and 7g could inhibit the EGFR TK in the submicromolar
µ
8
explain the observed effects and the binding modes of these chalcones with the EGFR TK and
tubulin targets.
Keywords: triazolo[4,3-a]quinoxaline; chalcone; EGFR; molecular modeling; anticancer; docking
1
. Introduction
Cancer ranks second of the top ten mortal diseases around the world, especially in the
industrialized countries, highlighting the imperative need to discover novel therapies and approaches
to cure this disease [ ]. Nowadays, anticancer chemotherapy is still the main method applied
in the treatment of cancer. In spite the development of new anticancer agents, the accumulation
of toxicity often limits their application as antitumor drugs [ ]. As a result, there is an urgent
need to discover and develop new classes of anticancer drugs to combat the growth of drug
resistance [ ]. Our literature survey revealed that quinoxalines and their fused analogs are attractive
candidates in medicinal chemistry as they constitute the building blocks of a wide range of compounds
1,2
3,4
5
Molecules 2018, 23, 48; doi:10.3390/molecules23010048
www.mdpi.com/journal/molecules

Molecules 2018, 23, 48
2 of 16
possessing a numerous interesting biological properties such as anticancer [6,7], antihistaminic [8],
antiviral [
effects [16]. Among the biologically active pharmacophores, amides have been associated with
fascinating anticancer [17], antitubercular [18 19], anti-inflammatory [20] and imines are reported to
have analgesic [21] properties. In addition, they have shown anti-protozoal [22], antidepressant [23],
anticonvulsant [24] and kinase inhibitor properties [25 26]. Moreover, a series of fused 1,2,4- and
,2,3-triazoles were reported as interesting molecules with dual fluorescence effects [27 29]. In addition,
,2,4-triazolo[4,3-a]quinoxalines bearing a triazole moiety have been reported as promising anticancer
39]. Their mechanism of action apparently involves binding to DNA where they
function as intercalating agents [40 41]. Chalcones (1,3-diaryl-2-propen-1-ones) are an interesting
scaffold in which the two aromatic rings are connected by a three-carbon -unsaturated carbonyl
9], antimicrobial [10–12], antifungal [13] antitubercular [14,15] and anti-inflammatory
,
,
1
1
–
agent motifs [30
–
,
α,β
system. Chalcone derivatives with diverse chemical architectures are quite significant in anticancer
drug discovery and hence are in the center of attention of drug hunters [42]. The anticancer
activity of chalcones might be due to molecular alterations such as induction of apoptosis, DNA
and mitochondrial damage, inhibition of angiogenesis, tubulin inhibition, kinase inhibition, and also
drug efflux protein activities [43,44].
Recently, the tubulin inhibitory potential of boronic acid chalcone analogs [45], cinnamic acyl
sulfonamide derivatives [46] and chalcone-based azacarbolines [47] has been reported. In view of these
facts, we herein report a facile synthesis of new series of triazoloquinoxaline-chalcone derivatives as a
privileged bioactive scaffold. The presence of the triazoloquinoxaline moiety linked to the aromatic
chalcone scaffold moiety might provide a rigid arrangement of the two aryl rings that enhances their
activity. This design strategy led to a set of novel compounds that might have cytotoxic effects via
EGFR TK and tubulin inhibitory mechanisms and looks like the reference drugs.
2
. Rationale Study
Several chalcones have been reported to act as cytotoxic agents or as microtubule destabilizing
agents, targeting the colchicine binding site [48
compounds substituted with electron donating hydroxy and/or methoxy groups at various
positions [50 51]. It was reported that the [1,2,4]triazolo[4,3-a]quinoxaline moiety was introduced as
,49]. The majority of these are naturally occurring
,
bromodomain and extra-terminal (BET) protein inhibitors for cancer treatment [52].
To establish more advanced structure–activity relationships around chalcones, the synthesis of
compounds with more diverse substitution patterns is of great importance. Our research is aimed at the
development of novel scaffold-based chalcones with incorporation of [1,2,4]triazolo[4,3-a]quinoxalines
as essential fragments for the design of anticancer agents [49]. In the present work, we unveil the
discovery of a novel scaffold with [1,2,4]triazolo[4,3-a]quinoxaline conjugated with chalcones as dual
EGFR and tubulin polymerization inhibitors potentially useful in cancer treatment (Figure 1).

Molecules 2018, 23, 48
3 of 16
Figure 1. Reference compound sets with different structures and mechanisms. The highlighted
substructures reveal the pharmacophore points related to the reported biological effect.
3
. Results and Discussion
3
.1. Chemistry
The precursor chlorotriazoloquinoxaline
with condensation of o-phenylenediamine ( ) with oxalic acid, followed by a nucleophilic substitution
reaction affording dichloroquinoxaline compound . Hydrazinolysis of this compound produced the
intermediate compound . Cyclization of compound with excess triethylortho-propionate gave the
corresponding fused chlorotriazoloquinoxaline 5 in good yield (Scheme 1).
5 was obtained after multiple reaction steps starting
1
3
4
4
Scheme 1. Synthesis of N-aryl and heteroaryl triazoloquinoxalines.
Analytically pure triazoloquinoxaline
solvent (see Experimental). Its structural proof was based on a correct elemental analyses as
well as on IR and NMR spectral data. The title compound was next prepared by dissolving
-chloro-1-ethyl[1,2,4]triazolo[4,3-a]quinoxaline ( ) freely in acetonitrile and heated under reflux with
different heteroaromatic or aromatic amines and a catalytic amount of TEA to produce a nucleophilic
5 was obtained by recrystallisation from an appropriate
6
4
5

Molecules 2018, 23, 48
4 of 16
substitution of the Cl atom giving an intermediate that was trapped by highly electronegative nitrogen
atoms and yielded the corresponding N-aryl substituted derivatives in a short time (4–6 h) with
high purity (Scheme 2). The structure of compounds
spectral data.
6 was confirmed by elemental analysis and
Substituted triazoloquinoxaline chalcone derivatives 7a
of aromatic aldehydes with compound . The structures of these novel compounds were confirmed by
their elemental analyses and spectral data. The characteristic IR band of the (C=O) group appears at
–k were finally prepared by condensation
6
−
1
1
1
734 cm . In addition, H-NMR revealed that the characteristic doublet signals of the olefinic protons
(CH=CH) occur between δ 7.4–8.4 ppm.
Compound
R1
R2
R3
R4
R5
7
a
H
OCH3
OH
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
CH3
OCH3
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
7
b
7
c
7
7
d
e
7
f
7
g
OCH3 OCH3 OCH3
7
h
H
H
H
H
NO2
Cl
Cl
H
H
H
H
7
7
i
j
7
k
Cl
H
Scheme 2. Synthesis and structures of triazoloquinoxaline chalcone derivatives 7a–k.
3
.2. Cytotoxicity Screening
The newly synthesized chalcone compounds 7a
–k were evaluated for their in vitro anticancer
activity against three cancer cell lines—HCT-116, MCF-7 and HepG-2—according to the MTT assay
method using doxorubicin as reference drug. The in vitro anticancer activity results reveal that some of
the chalcone compounds exhibited excellent activity against these tumor cells. The compounds 7b
, 7e
and 7g bearing mono- or trimethoxy-substituted benzene rings showed moderate to good anticancer
activity with IC50 values of 1.65 to 34.28
towards any particular cell line.
µM against the three cell lines and were mostly not selective
Substitution of the electron-attracting groups as in the chloro and nitro derivatives 7h
–k resulted
in no significant anticancer activity, as indicated by their IC50s more higher than 100 M, whereas the
µ
introduction of electron-releasing groups such as methyl (compound 7d) produced low activity while
substitution with hydroxy groups in 4-position on the benzene ring (compounds 7c and 7f) enhanced
the anticancer activity remarkably, selectively or non-selectively. Compound 7a without substitution
did not show significant activity against any of the tested cell lines. These results suggest that the
substitution patterns on the terminal aromatic ring of the chalcones plays a vital role in the modulation
of cytotoxicity. The results are summarized in Table 1.

Molecules 2018, 23, 48
5 of 16
Table 1. Cytotoxicity screening of selected chalcone derivatives against three different cancer cell lines.
IC50 (µM) *
Compounds
HCT-116
MCF-7
HepG-2
5
6
236 ± 0.10
411 ± 0.21
57.49 ± 0.29
25.60 ± 0.03
19.76 ± 0.23
>100
9.57 ± 0.11
18.10 ± 0.15
3.61 ± 0.18
>100
246 ± 0.13
410 ± 0.25
58.92 ± 0.32
24.04 ± 0.12
22.17 ± 0.11
93.40 ± 0.14
8.23 ± 0.25
>100
322 ± 0.15
446 ± 0.20
105.21 ± 0.50
30.72 ± 0.12
17.32 ± 0.12
>100
7
a
7
7
b
c
7
7
d
e
34.28 ± 0.14
7
f
>100
7
g
1.65 ± 0.13
8.58 ± 0.06
>100
7
h
>100
7
7
i
j
>100
>100
>100
>100
>100
>100
7
k
19.66 ± 0.25
>100
0.27 ± 0.08
>100
0.22 ± 0.01
Doxorubicin
1.55 ± 0.03
*
Cytotoxic effects of selected chalcone compounds were reported on colon, breast, and liver cell lines following
exposure to different concentrations of compounds, and cell viability was assessed using the MTT method.
3
.3. EGFR Inhibitory Assay
As the designed compounds had good antiproliferative activity, in vitro EGFR inhibition capability
was firstly evaluated for the target cytotoxic derivatives to see whether their cytotoxicity was mediated
by kinase inhibition. As shown in Table 2, the compounds 7a 7e, and 7g showed excellent
–c,
potent EGFR inhibition activity comparable or better than that of the reference drug staurosporine.
Derivative 7g exhibited the most potent activity against the kinase target which was consistent with
its cytotoxic effect. In addition, compounds 7b and 7c showed good inhibitory activity with an IC50
range of 0.127 to 0.136
µM. Moreover, chalcone 7e displayed promising inhibitory effect, indicated
by an IC50 of n0.083 against EGFR at 10
µM. Overall, most of the chalcone derivatives showed potent
EGFR TK inhibitory activity and deserve further studies and optimization as anticancer agents.
Table 2. Inhibitory data of the selected compounds to both EGFR TK and tubulin polymerization.
EGFR Inhibition (IC50 µM) a,b
Tubulin Polymerization (IC50 µM) c
Compounds
7
a
661.0 ± 0.11
127.0 ± 0.32
136.0 ± 0.05
>100
77.1 ± 0.21
18.1 ± 0.30
43.4 ± 0.01
ND
7
b
7
c
7
d
7
7
e
f
0.083 ± 0.04
14.7 ± 0.11
>50
>100
7
g
0.039 ± 0.16
>100
8.84 ± 0.02
>100
7
h
7
7
i
j
>100
>100
>100
>100
7
k
>100
-
0.054 ± 0.10
>100
Colchicine
Staurosporine
26.8 ± 0.12
-
a
Values are averages of three independent experiments; b Compounds tested at concentration of 10 M; c Values
µ
are averages of two independent experiments.
3
.4. Tubulin Assay
Tubulin is the principal component of the eukaryotic cytoskeleton and participates in several
crucial cellular processes, including cell replication. As evident from the literature, chalcones show
cytotoxic activity through the inhibition of tubulin polymerization, hence impeding microtubule

Molecules 2018, 23, 48
6 of 16
formation [48]. Compounds 7d
,
7f, and 7h
–
k
revealed no significant influence on tubulin assembly,
which suggests a different mechanism for the observed cytotoxicity than tubulin inhibition (Table 2).
However, five compounds (7a 7e, and 7g) showed significant inhibition of tubulin assembly.
As reported in Table 2, 7e and 7g efficiently inhibited tubulin polymerization with IC50 values of
4.7 and 8.84 M, respectively. These results suggest that the molecular target of these chalcone
–c,
1
µ
derivatives might be tubulin, and their strong anti-tubulin polymerization activity corresponds well
with their cytotoxicity.
3
.5. Molecular Docking Analyses
To rationalize the experimental results obtained, molecular docking studies were performed
on two representative potent compounds (7e and 7g) against the two interesting targets, EGFR TK
and tubulin by a method similar to a previously reported successful approach [53]. According to the
interaction results presented in Table 3, two compound poses showed high binding energies with EGFR
and tubulin targets, with energie s ranging from
a network of molecular interactions (H-bonds, vdw, and
−
12.8 to
−
15.5 kcal/mol. All compounds formed
-aromatic) with the active site residues of
π
EGFR when analyzed in 2D plots as shown in Figure 2. Compound 7e formed stable hydrogen bonds
with the terminal methoxy groups and linker carbonyl fragments through the Lys721 and Met769
residues. In addition, the role of triazoloquinoxaline in stabilizing the compound inside the ATP
binding pocket through the aromatic hydrophobic interaction in hydrophobic pocket produced by
the Lys704 residue. The same behavior was exhibited in case of compound 7g by forming various
interactions in the ATP pocket. It is well established that the colchicine binding site is generally
present at the interface of
compound fit well into the colchicine binding pocket present at the
with the lowest binding energy. The chalcone part occupied the hydrophobic pocket formed of Leu248,
Ala315, Ile378, Leu255 residues present in the -subunit, whereas the quinoxaline ring protruded
α
,β-tubulin heterodimers. The docking arrangement of active anti-tubulin
7
g
α
and interfaces of tubulin
β
β
towards the α subunit with the help of hydrogen bonding to the Thr179 residue (Figure 3).
Table 3. Docking scores, amino acid interactions and interacting groups of the docked compounds into
the active site of EGFR-TK and tubulin.
Compound
Target
Binding Energy (kcal/mol)
Interacting Moieties
,4 diO-CH3
C=O iminone linker
Quinoxaline
Amino Acids
3
Lys721
Met769
Lys704
Leu694
7
e
−13.51
EGFR TK
4-aminophenylcarbonyl
(
1M17)
3
,4 diO-CH3
Lys721
Met769
Lys704, Pro770, Leu768
Leu694
C=O iminone linker
Quinoxaline
4-aminophenylcarbonyl
−
−
15.5
12.8
7
g
3
,4 diO-CH3
Cys241, Thr240
Thr179
Leu248, Ala315, Ile378, Leu255
Tubulin
3E22)
Quinoxaline
Chalcone
(

Molecules 2018, 23, 48
7 of 16
Figure 2. Molecular interaction graph of target compounds 7e and 7g in the EGFR TK ATP pocket.
Figure 3. Molecular interactions graph of target compounds 7g in the tubulin pocket.
3
.6. SAR Analyses
The SAR study revealed several crucial structural requirements which enhanced the potency
of these chalcone compounds, 7a 7e, and 7g. Incorporation of the triazoloquinoxaline moiety
–c,
into an active scaffold like chalcone with reported anticancer activity resulted in enhanced potency.
This explains the importance of the molecular hybridization of active fragments.
As shown in the activity and modeling reports, the quinoxaline ring plays a crucial role in the
EGFR inhibitory effect, whereas in the case of tubulin it increases the stability of the structure in the
colchicine bind site. Moreover, the terminal aromatic ring in chalcone is essential for tubulin inhibition
and a suitable substitution pattern is a must. All compounds with OH or OCH groups exhibited
3

Molecules 2018, 23, 48
8 of 16
anti-EGFR and tubulin polymerization effects. It is noteworthy to observe that substituents like OCH3
at the 3th and 4th positions have a significant impact on the activities (Figure 4). Finally, molecular
hybrids of chalcone and triazoloquinoxaline fragments afforded potent lead compounds while
preserving the pharmacophore points essential for EGFR and tubulin polymerization inhibition.
Figure 4. Structure activity relationship of chalcone derivatives 7e and 7g.
4
. Experimental Section
4
.1. General Information
All melting points were taken on a LA 9000 SERIES digital melting point apparatus
(
Electrothermal, Staffordshire, UK) and are uncorrected. IR spectra were recorded on a SP 1000
1
13
IR spectrophotometer (Pye Unicam, Cambridge, UK). H-NMR and C-NMR spectra were recorded
1
on an AC 300 MHz NMR spectrometer (Bruker, Billerica, MA, USA) at 300 for H and 100 MHz for
1
3
1
13
C, respectively. All H- and C-NMR spectral results are recorded as chemical shift (
δ
1
) values.
Chemical shifts recorded in DMSO-d are relative to the solvent peak of 2.5 ppm for H-NMR
spectra and 39.5 ppm for 13C-NMR spectra. Mass spectra were recorded on a 5988 spectrometer
6
(Hewlett Packard, Palo Alto, CA, USA). Microanalyses were carried out at the Cairo University
Microanalytical Center. Progress of the reactions was monitored by TLC using TLC sheets precoated
with UV fluorescent silica gel (Merck 60 F254 plates, Merck & Co., Kenilworth, NJ, USA) and different
solvents as mobile phases and visualized using UV lamp. Antimicrobial and antitumor activities were
evaluated at the Regional Center for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt.
4
.2. Chemistry
4
.2.1. Synthesis of 2,3-(1H,4H)-Quinoxalinedione (2)
A solution of oxalic acid (27.1 g, 0.215 mol) in 4 N aqueous HCl (50 mL) was added to a solution
of o-phenylenediamine (20.9 g, 0.193 mol) in 4 N HCl (150 mL), and the resulting solution was heated
under reflux for 2 h. The reaction mixture was cooled to ambient temperature, and the resulting
precipitate was isolated by filtration, washed with water, dried, recrystallized from ethanol giving
3
0.5 g of 2 as dark red microcrystalline solid (30.5 g, 98%); m.p. > 360 [54].

Molecules 2018, 23, 48
9 of 16
4
0
.2.2. Synthesis of 2,3-Dichloroquinoxaline (3)
DMF (0.045 g, 0.00062 mol) was added dropwise to a slurry of 2,3-dihydroxyquinoxaline (
.012 mol) and thionyl chloride (3.7 g, 0.031 mol) in 1,2-dichloroethane (20 mL). The resulting reaction
2
, 2.0 g,
mixture was heated to reflux for 2 h then concentrated to dryness. The residue was dissolved in
1
,2-dichloroethane (25 mL) and concentrated to dryness. The resulting solid was crystallized from
◦
CH CN/H O, giving 2.3 g (95%) of 3 as fine, off-white needles. m.p. 148–150 C [55].
3
2
4
.2.3. Synthesis of 2-Chloro-3-hydrazinylquinoxaline (4)
A mixture of (10.0 g, 0.1 mol) of 2,3-dichloroquinoxaline and (10.0 g, 0.22 mol) of hydrazine
◦
hydrate in 200 mL of ethanol was stirred for 16 h at 25 C. The resulting precipitate was filtered,
and the solid was washed with ethanol and air dried to give 7.5 g (100%) of crude product as a yellow
◦
powder, m.p. 181–182 C [23].
4
.2.4. Synthesis of 4-Chloro-1-ethyl-[1,2,4]triazolo[4,3-a]quinoxaline (5)
DMF (0.045 g, 0.00062 mol) was added dropwise to a slurry of 1-ethyl-[1,2,4]triazolo[4,3-
a]quinoxalin-4(5H)-one (1.0 g, 0.1 mol) and thionyl chloride (2.77 g, 0.5 mol) in 1,2-dichloroethane
100 mL). The resulting reaction mixture was heated to reflux for 6 h filtered while hot then concentrated
(
to dryness. The precipitate purified by stirring with a mixture of diethyl ether and n-hexane (1:1) then
◦
filtered and air dried to give 1.09 g (85%) of product as a light yellow powder. m.p. 158–160 C [23].
4
.2.5. Synthesis of 1-(4-((1-ethyl-[1,2,4]triazolo[4,3-a]quinoxalin-4-yl)amino)phenyl)ethan-1-one (6)
A reaction mixture of 4-chloro-1-ethyl-[1,2,4]triazolo[4,3-a]quinoxaline ( , 0.3 g, 0.01 mol) and a
5
corresponding aromatic amine (0.011 mol) in dry CH CN (10 mL) of containing a few drops of TEA
3
was refluxed for 4–6 h. After concentrating the precipitated solid was filtered and crystallized from
◦
−1
ethanol affording the desired product
3
6
. Yield (40%); m.p. 235–237 C; IR cm (KBr); 3412 (N–H),
1
060 (C–H aromatic), 2937 (C–H aliphatic), 1629 (C=N); H-NMR (DMSO-d ): 9.99 (s, 1H, N–H
6
exchangeable with D O), 7.67–8.30 (m, 8H, Ar-H), 3.43 (q, 2H, J = 8, CH CH ), 1.49 (t, 3H, J = 8,
2
3
2
13
CH CH ); C-NMR (DMSO-d6)
δ
194.4, 161.5, 152.2, 145.1, 142.1, 135.9, 131.0, 129.6, 128.5, 125.9, 124.8,
20.7, 112.5, 111.8, 26.5, 21.5, 12.1; MS (m/z), 297 (0.97, M + 1), 296 (2.51, M+), 267 (7.53), 212 (2.06),
28 (100), 84 (2.34), 77 (16.39), 76 (78.85); Anal. Calcd. for C H N O: C, 68.87; H, 5.17; N, 21.13,
3
2
1
1
19
17
5
Found: C, 68.85; H, 5.16; N, 21.14.
4
.2.6. General Method for the preparation of (Z)-{4-[(1-Ethyl-[1,2,4]triazolo[4,3-a]quinoxalin-4-
yl)amino]phenyl}-3-substituted phenylprop-2-en-1-ones 7a–k
A mixture of the appropriate aromatic aldehyde (0.012 mol) and 1-(4-((1-ethyl-[1,2,4]triazolo[4,3-
a]quinoxalin-4-yl)amino)phenyl)ethan-1-one ( , 0.01 mol) dissolved in ethanol (70 mL) was added
6
slowly to an aqueous solution of potassium hydroxide (0.0128 mol) in water (10 mL). The reaction
◦
mixture was stirred in crushed-ice bath for half hour. Stirred at 20–25 C for 4–6 h. The mixture was
filtrated and the solid was washed with cold water and ethanol. The product was crystallized from
ethanol to give the corresponding chalcones 7a–k, respectively.
(E)-1-(4-((1-Ethyl-[1,2,4]triazolo[4,3-a]quinoxalin-4-yl)amino)phenyl)-3-phenylprop-2-en-1-one (7a)
◦
−1
Yield (82%); m.p. 260–262 C; IR cm (KBr); 3443 (N–H), 2967 (C–H aromatic), 2922 (C–H
1
aliphatic), 1732 (C=O), 1649 (C=N); H-NMR (DMSO-d ): 10.51 (s, 1H, NH exchangeable with D O),
6
2
7
.41–8.44 (m, 14H, Ar–H + 1H, C–H-arylidene), 3.45 (q, 2H, J = 7.5, CH CH ), 1.50 (t, 3H, J = 7.5,
3 2
13
CH CH ); C-NMR (DMSO-d6)
δ
189.7, 161.5, 152.2, 146.7, 145.1, 142.1, 135.9, 135.2, 132.0, 128.7, 128.6,
28.5, 127.9, 125.9, 124.8, 121.3, 120.7, 112.5, 111.8, 21.5, 12.1; MS (m/z), 420 (3.61, M + 1), 419 (11.65,
M+), 239 (39.39), 222 (2.82), 212 (2.19), 197 (1.66), 98 (58.84), 71 (82.65), 57 (100); Anal. Calcd. for
C H N O; C, 74.44; H, 5.05; N, 16.70, Found: C, 74.73; H, 5.07; N, 16.75.
3
2
1
26
21
5

Molecules 2018, 23, 48
10 of 16
(
E)-1-(4-((1-Ethyl-[1,2,4]triazolo[4,3-a]quinoxalin-4-yl)amino)phenyl)-3-(2-methoxyphenyl)prop-2-en-
1
-one (7b)
◦
−1
Yield (37%); m.p. 264–266 C; IR cm (KBr); 3446 (N–H), 3099 (C–H aromatic), 2938 (C–H
1
aliphatic), 1731 (C=O), 1649 (C=N); H-NMR (DMSO-d ): 10.54 (s, 1H, N–H exchangeable with D O),
6
2
7
.04–8.45 (m, 13H, Ar–H, 1H, C–H-arylidene), 3.91 (s, Ar–OCH ), 3.45 (q, 2H, J = 7.5, CH CH ),
3 3 2
13
1
.49 (t, 3H, J = 7.5, CH CH ); C-NMR (DMSO-d6)
δ
189.7, 161.5, 159.5, 152.2, 146.7, 142.1, 141.1, 135.2,
3
2
1
32.0, 128.9, 128.7, 127.9, 125.8, 125.7, 124.8, 121.3, 120.9, 120.7, 114.5, 112.5, 111.8, 56.4, 21.5, 12.1; MS
(m/z), 450 (0.89, M + 1), 449 (3.57, M+), 265 (100), 183 (24.52), 128 (62.61), 104 (42.53), 77 (36.27); Anal.
Calcd. for C H N O ; C, 72.14; H, 5.16; N, 15.58, Found: C, 72.42; H, 5.18; N, 15.64.
27
23
5
2
(
E)-1-(4-((1-Ethyl-[1,2,4]triazolo[4,3-a]quinoxalin-4-yl)amino)phenyl)-3-(2-hydroxyphenyl)prop-2-en-
1
-one (7c)
◦
−1
Yield (21%); m.p. 261–263 C; IR cm (KBr); 3446 (O–H), 3359 (N–H), 3062 (C–H aromatic),
1
2
933 (C–H aliphatic), 1734 (C=O), 1670 (C=N); H-NMR (DMSO-d ): 10.48 (s, 1H, N–H exchangeable
6
with D O), 9.01 (s, 1H, Ar–O–H exchangeable with D O), 7.46–8.39 (m, 13H, Ar–H, 1H, C–H-arylidene),
2
2
13
3
.47 (q, 2H, J = 7.5, CH CH ), 1.51 (t, 3H, J = 7.5, CH CH ); C-NMR (DMSO-d6) δ 189.7, 161.5, 159.5,
3 2 3 2
1
1
52.2, 146.7, 142.1, 141.0, 135.4, 132.2, 129.3, 128.9, 128.7, 127.9, 125.7, 124.8, 122.6, 121.9, 121.3, 120.7,
17.5, 112.7, 111.3, 21.5, 12.1; MS (m/z), 435 (4.79, M+), 331 (25.09), 234 (12.55), 120 (25.48), 84 (81.50),
7
7 (8.54), 57 (50.87), 44 (100); Anal. Calcd. for C H N O ; C, 71.71; H, 4.86; N, 16.08, Found: C, 71.99;
26 21 5 2
H, 4.84; N, 16.10.
(E)-1-(4-((1-Ethyl-[1,2,4]triazolo[4,3-a]quinoxalin-4-yl)amino)phenyl)-3-(p-tolyl)prop-2-en-1-one (7d)
◦
−1
Yield (43%); m.p. 247–249 C; IR cm (KBr); 3435 (N–H), 3029 (C–H aromatic), 2921 (C–H
1
aliphatic), 1733 (C=O), 1684 (C=N); H-NMR (DMSO-d ): 10.53 (s, 1H, N–H exchangeable with
6
D O), 7.26–8.45 (m, 13H, Ar–H, 1H, C–H-arylidene), 3.47 (q, 2H, J = 7.5, CH CH ), 2.36 (s, Ar–CH ),
2
3
2
3
13
1
1
4
1
.51 (t, 3H, J = 7.5, CH CH ); C-NMR (DMSO-d6)
δ
189.7, 161.5, 152.2, 145.7, 142.1, 137.4, 135.4, 132.2,
32.0, 128.9, 128.7, 128.5, 127.9, 125.7, 124.8, 121.3, 120.7, 112.8, 111.3, 21.7, 21.5, 21.3, 12.1; MS (m/z),
34 (28.46, M + 1), 433 (100, M+), 212 (3.38), 432 (59.57), 233 (30.53), 212 (6.74), 145 (26.28), 128 (3.35),
15 (46.99), 77 (7.26); Anal. Calcd. for C H N O; C, 74.81; H, 5.35; N, 16.16, Found: C, 75.02; H, 5.37;
3
2
27
23
5
N, 16.22.
(
E)-1-(4-((1-Ethyl-[1,2,4]triazolo[4,3-a]quinoxalin-4-yl)amino)phenyl)-3-(4-methoxyphenyl)prop-2-en-
1
-one (7e)
◦
−1
Yield (96%); m.p. 255–257 C; IR cm (KBr); 3438 (N–H), 2980 (C–H aromatic), 2921 (C–H
1
aliphatic), 1731 (C=O), 1613 (C=N); H-NMR (DMSO-d ): 10.45 (br s, 1H, N–H exchangeable with
6
D O), 7.01–8.44 (m, 13H, Ar–H, 1H, C–H-arylidene), 3.82 (s, 3H, OCH ), 3.43 (q, 2H, J = 6.6, CH CH ),
2
3
3
2
13
1
1
4
1
7
.51 (t, 3H, J = 6.6, CH CH ); C-NMR (DMSO-d )
δ
189.7, 161.5, 159.4, 152.2, 146.7, 145.1, 142.1, 135.3,
32.2, 130.0, 128.5, 127.9, 127.5, 125.7, 124.8, 121.3, 120.4, 114.5, 112.8, 111.3, 55.6, 21.3, 12.1; MS (m/z),
50 (5.07, M + 1), 449 (18.27, M+), 420 (2.88), 316 (6.93), 265 (100), 252 (1.21), 237 (5.60), 212 (3.38),
84 (3.94), 128 (44.19), 77 (19.10); Anal. Calcd. for C H N O ; C, 72.14; H, 5.16; N, 15.58, Found: C,
3
2
6
27
23
5
2
1.85; H, 5.18; N, 15.64.
(
E)-1-(4-((1-Ethyl-[1,2,4]triazolo[4,3-a]quinoxalin-4-yl)amino)phenyl)-3-(4-hydroxyphenyl)prop-2-en-
1
-one (7f)
◦
−1
Yield (45%); m.p. 253–255 C; IR cm (KBr); 3482 (O–H), 3374 (N–H), 3120 (C–H aromatic),
1
2
933 (C–H aliphatic), 1732 (C=O), 1662 (C=N); H-NMR (DMSO-d ): 10.45 (br s, 1H, N–H exchangeable
6
with D O), 9.02 (s, 1H, Ar–O–H exchangeable with D O), 7.40–8.33 (m, 13H, Ar–H, 1H, C–H-arylidene),
2
2
13
3
.44 (q, 2H, J = 7.2, CH CH ), 2.36 (s, Ar–CH ), 1.46 (t, 3H, J = 7.2, CH CH ); C-NMR (DMSO-d6)
3 2 3 3 2
δ 189.7, 161.5, 157.4, 152.2, 146.7, 145.1, 142.1, 135.3, 132.2, 130.0, 128.5, 127.9, 127.5, 125.7, 124.8, 121.3,
20.4, 115.5, 112.8, 111.3, 21.3, 12.1; MS (m/z), 435 (5.59, M+), 330 (40.03), 316 (77.98), 261 (63.56),
1

Molecules 2018, 23, 48
11 of 16
2
7
57 (100), 97 (86.70), 81 (77.95); Anal. Calcd. for C H N O ; C, 71.71; H, 4.86; N, 16.08, Found: C,
26 21 5 2
1.83; H, 4.87; N, 16.14.
(
E)-1-(4-((1-Ethyl-[1,2,4]triazolo[4,3-a]quinoxalin-4-yl)amino)phenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-
1
-one (7g)
◦
−1
Yield (11%); m.p. 247–249 C; IR cm (KBr); 3448 (N–H), 2981 (C–H aromatic), 2935 (C–H
1
aliphatic), 1732 (C=O), 1653 (C=N); H-NMR (DMSO-d ): 10.48 (s, 1H, N–H exchangeable with D O),
6
2
7
.48–8.38 (m, 11H, Ar–H, 1H, C–H-arylidene), 3.43 (q, 2H, J = 7.2, CH CH ), 2.56 (s, 9H, Ar–(OCH ) ),
3 2 3 3
13
1
.51 (t, 3H, J = 7.2, CH CH ); C-NMR (DMSO-d6)
δ
189.4, 161.3, 153.4, 152.2, 146.7, 145.1, 142.1,138.4,
3
2
135.3, 132.2, 128.5, 127.9, 126.4, 125.7, 124.8, 121.5, 120.7, 112.4, 111.3, 103.5, 61.2, 56.3, 21.3, 12.1; MS
(m/z), 510 (0.76, M + 1), 509 (1.16, M+), 330 (98.70), 261 (34.30), 233 (100), 212 (4.13), 206 (44.08),
1
58 (79.29), 128 (32.83), 77 (18.08); Anal. Calcd. for C H N O ; C, 68.36; H, 5.34; N, 13.74, Found: C,
29 27 5 4
6
8.50; H, 5.35; N, 13.78.
(
E)-1-(4-((1-Ethyl-[1,2,4]triazolo[4,3-a]quinoxalin-4-yl)amino)phenyl)-3-(4-nitrophenyl)prop-2-en-1-one (7h)
◦
−1
Yield (64%); m.p. 260–262 C; IR cm (KBr); 3368 (N–H), 3103 (C–H aromatic), 2935 (C–H
1
aliphatic), 1794 (C=O), 1656 (C=N), 1534 (NO ); H-NMR (DMSO-d ): 10.49 (bs, 1H, N–H exchangeable
2
6
with D O), 7.42–8.39 (m, 13H, Ar–H, 1H, C–H-arylidene), 3.44 (q, 2H, J = 6, CH CH ), 2.36 (s, Ar–CH ),
2
3
2
3
13
1
1
4
5
4
.51 (t, 3H, J = 6, CH CH ); C-NMR (DMSO-d6)
δ
189.4, 161.3, 152.2, 147.4, 146.7, 145.1, 142.4,
41.1,135.3, 132.2, 129.3, 128.5, 127.9, 125.7, 124.8, 123.4, 121.5, 120.7, 112.4, 111.3, 21.3, 12.1; MS (m/z),
65 (0.72, M + 1), 464 (2.37, M+), 330 (47.53), 265 (31.53), 233 (47.84), 212 (2.54), 128 (16.32), 77 (32.13),
7 (73.05), 43.09 (100); Anal. Calcd. for C H N O ; C, 67.23; H, 4.34; N, 18.09, Found: C, 67.04; H,
3
2
26
20
6
3
.35; N, 18.16.
(E)-3-(4-Chlorophenyl)-1-(4-((1-ethyl-[1,2,4]triazolo[4,3-a]quinoxalin-4-yl)amino)phenyl)prop-2-en-1-one (7i)
◦
−1
Yield (73%); m.p. 255–257 C; IR cm (KBr); 3262 (N–H), 3091 (C–H aromatic), 2932 (C–H
1
aliphatic), 1734 (C=O), 1656 (C=N), 1090 (C–Cl); H-NMR (DMSO-d ): 10.53 (s, 1H, N–H exchangeable
6
with D O), 7.38–8.68 (m, 13H, Ar–H, 1H, C–H-arylidene), 3.47 (q, 2H, J = 7.5, CH CH ), 1.52
2
3
2
13
(
1
t, 3H, J = 7.5, CH CH ); C-NMR (DMSO-d6)
32.2, 129.3, 128.5, 127.9, 125.7, 124.8, 121.5, 120.7, 112.4, 111.3, 21.3, 12.1; MS (m/z), 455 (33.18, M + 2 ,
δ
189.4, 161.3, 152.2, 146.7, 145.1, 142.4, 135.3, 133.5,133.2,
3
2
)
4
7
54 (50.31, M + 1), 453 (100, M+), 316 (42.69), 233 (74.89), 181 (63.37), 137 (48.40), 128 (9.37), 102 (83.81),
7 (18.45); Anal. Calcd. for C H ClN O; C, 68.80; H, 4.44; N, 15.43, Found: C, 68.76; H, 4.45; N, 15.49.
26
20
5
(
E)-3-(2,4-Dichlorophenyl)-1-(4-((1-ethyl-[1,2,4]triazolo[4,3-a]quinoxalin-4-yl)amino)phenyl)prop-2-en-
1
-one (7j)
◦
−1
Yield (86%); m.p. 250–252 C; IR cm (KBr); 3441 (N–H), 2925 (C–H aliphatic), 1733 (C=O),
1
1
(
682 (C=N), 1018 (C–Cl); H-NMR (DMSO-d ): 10.26 (bs, 1H, N–H exchangeable with D O), 7.43–8.48
m, 12H, Ar–H, 1H, C–H-arylidene), 3.49 (q, 2H, J = 6, CH CH ), 1.46 (t, 3H, J = 6, CH CH ); C-NMR
3 2 3 2
6
2
13
(
1
(
(
DMSO-d6)
27.4, 126.7, 125.7, 125.2, 124.8, 121.5, 120.7, 112.4, 111.3, 21.3, 12.1; MS (m/z), 490 (7.29, M + 2), 489
16.64, M + 2), 488 (21.86, M+), 487 (32.89), 316 (24.39), 265 (100), 233 (55.15), 212 (8.34), 171 (23.36), 128
57.01), 90 (45.25), 77 (38.16), 40 (81.42); Anal. Calcd. for C H Cl N O; C, 63.94; H, 3.92; N, 14.34,
δ 189.4, 161.3, 152.2, 146.7, 145.1, 142.4, 136.7, 135.3, 132.2, 131.2, 130.3, 128.5, 128.1,
26
19
2
5
Found: C, 63.85; H, 3.93; N, 14.39.
(
E)-3-(2,6-Dichlorophenyl)-1-(4-((1-ethyl-[1,2,4]triazolo[4,3-a]quinoxalin-4-yl)amino)phenyl)prop-2-en-
1
-one (7k)
◦
−1
Yield (25%); m.p. 250–252 C; IR cm
(KBr); 3384(N–H), 3070 (C–H aromatic), 2927
1
(
C–H aliphatic), 1655 (C=O), 1597 (C=N), 1014 (C–Cl); H-NMR (DMSO-d ): 10.60 (s, 1H, N–H
6
exchangeable with D O), 7.46–8.39 (m, 12H, Ar–H, 1H, C–H-arylidene), 3.45 (q, 2H, J = 7.5, CH CH ),
2
3
2
13
1
.52 (t, 3H, J = 7.5, CH CH ); C-NMR (DMSO-d6) δ 189.4, 161.3, 152.2, 146.7, 145.1, 142.4, 135.9, 135.3,
3 2

Molecules 2018, 23, 48
12 of 16
132.5, 132.2, 128.7, 128.0, 127.4, 126.7, 125.2, 124.8, 121.5, 120.7, 112.4, 111.3, 21.3, 12.1; MS (m/z), 490
(3.59, M + 1), 489 (9.96, M + 1), 488 (11.13, M+), 487 (16.43), 316 (75.45), 265 (8.27), 233 (100), 212 (13.18),
171 (24.14), 128 (29.10), 90 (89.94), 77 (99.74), 40 (79.75); Anal. Calcd. for C H Cl N O; C, 63.94; H,
26 19 2 5
3
.92; N, 14.34, Found: C, 64.19; H, 3.93; N, 14.28.
4
.3. Cancer Cell Antiproliferative Assay
The in vitro anticancer activities of the selected chalcone compounds against three cancer cell lines:
human colon carcinoma (HCT-116), human hepatocellular carcinoma (HEPG-2), and human breast
adenocarcinoma (MCF-7) were evaluated as described [56] with some modifications. Target tumor cells
were grown to log phase in DMEM medium supplemented with 10% fetal bovine serum. After diluting
5
−1
to 1
×
10 cells mL with the medium, 100
µ
L of the obtained cell suspension was added to each well
◦
of 96-well culture plates. Subsequently, incubation was performed at 37 C in 5% CO atmosphere for
2
4
8 h before the cytotoxicity assessment. Tested samples at preset concentrations were added to 6 wells
with doxorubicin being employed as a positive reference. After 72 h exposure period, 25 L of PBS
containing 2.5 mg mL of MTT was added to each well. After 4 h, the medium was replaced by 150
DMSO to dissolve the purple formazan crystals produced [57 58]. The absorbance at 570 nm of each
well was measured with an ELISA plate reader. The data represented the mean of three independent
experiments in triplicate and were expressed as means SD. The IC50 value was defined as the
concentration at which 50% of the cells could survive from graphic plots.
µ
−
1
µL
,
±
4
.4. EGFR Inhibition Assay
EGFR kinase activity was assessed using HTScan EGFR kinase assay kits (Cell Signaling Technology,
Danvers, MA, USA). The experiments were performed according to the manufacturer’s instructions.
In short, the GST-EGFR fusion protein was incubated with synthetic biotinylated peptide substrate
and 10 µg/mL inhibitors in the presence of 400 µM ATP. Phosphorylated substrate was captured with
strapavidin-coated 96-well plates. The level of phosphorylation was monitored by anti-phosphotyrosine
and europium-labeled secondary antibodies (DELFIA, Perkin-Elmer, Akron, OH, USA). The enhancement
solution was added at the end of the assay and enzyme activity was measured in a Victor II 1420 microplate
reader (Wallac, Boston, MA, USA) at 615 nM.
4
.5. Enzyme-Linked Immunosorbent Assay for Tubulin Beta (TUBb)
The HCT-116 cell line was obtained from the American Type Culture Collection (Manassas, VA,
USA) and cultured using DMEM (Invitrogen/Life Technologies, Carlsbad, CA, USA) supplemented
with 10% FBS (Hyclone, Logan, UT, USA), 10 mg/mL of insulin (Sigma, Mendota Heights, MN, USA),
3
and 1% penicillin-streptomycin. Plate cells (cells density 1.2–1.8
×
10 cells/well) in a volume of 100 mL
complete growth medium and 100 mL of the tested compound per well in a 96-well plate for 18–24 h
before the enzyme assay for tubulin. The microtiter plate provided in this kit has been pre-coated
with an antibody specific to TUBb. Standards or samples are then added to the appropriate microtiter
plate wells with a biotin-conjugated antibody specific to TUBb. Next, avidin conjugated to horseradish
peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is
added, only those wells that contain TUBb, biotin-conjugated antibody and enzyme-conjugated avidin
will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric
acid solution and the color change is measured spectrophotometrically at a wavelength of 450
The concentration of TUBb in the samples is then determined by comparing the O.D. of the samples to
the standard curve.
±
10 nm.
4
.6. Molecular Docking
Molecular docking simulation was done for selected active target compounds into the
three-dimensional complex of two biological targets including the crystal structures of EGFR (PDB code:
M17) at 2.6 Å resolution and tubulin in complex with DAMA-colchicine and the stathmin-like
1

Molecules 2018, 23, 48
13 of 16
domain (SLD) at 3.5 Å resolution (PDB: 3E22) [50] was carried out using the AutoDock software
package (version 4.0) as implemented through the graphical user interface AutoDockTools (ADT) [59].
Prior to the calculations, crystallographic water and ligand molecules were removed from the X-ray
structure. Hydrogen atoms were added to the structure with the Molecular Operating Environment
(MOE, 2012) [60] and atomic partial charges were calculated using AutoDock Tools. Selected active
chalcone compounds were docked into the active site of the targets to predict compound binding
modes. For flexible docking, AutoDock standard parameter settings were applied. High-scoring
binding poses were selected on the basis of visual inspection.
5
. Conclusions
A novel series of chalcone derivatives with triazoloquinoxaline-linked moiety was designed,
synthesized and evaluated as potent anticancer agents against a set of cancer cell lines. The structures
of the novel compounds were confirmed using different spectroscopic techniques and elemental
analyses. Different mechanistic investigations were done by testing the ability of the novel compounds
to inhibit EGFR TK and tubulin polymerization. Most of compounds exhibited good potency against
EGFR TK and tubulin at micromolar or submicromolar concentrations. An in-silico docking study was
done to discover their binding modes within the targets pockets and a SAR screen was performed.
Overall structural fragments contributed to the activity by different types of interactions like hydrogen
bonding, hydrophobic, and aromatic stacking interactions. Taken together, a novel scaffold was
introduced and its potent anticancer activity through dual inhibition of vital biological targets
was revealed.
Acknowledgments: Hany E. A. Ahmed gratefully acknowledges support from the Deanship of Scientific Research
at Taibah University, Al-Madinah Al-Munawarah, Saudi Arabia, (project# 6074/1435). The authors give thanks to
chemical computing group for using Molecular Operating Software (MOE), 2012.
Author Contributions: M.A. designed the chemical synthesis and their corresponding experiments. A.H.B.
and K.E. did the structures elucidation and spectroscopic data analyses. A.E. contributed by providing the
synthetic reagents and materials. H.E.A.A. and S.I. conceived and designed the biological activities and wrote the
whole manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1
.
Joshi, M.; Pal, S.K.; Drabick, J.J. Novel approaches in cancer immunotherapy—A light at the end of the
tunnel. Dis. Med. 2016, 21, 479–487.
2
.
Ali, N.A.; O’Brien, J.M., Jr.; Blum, W.; Byrd, J.C.; Klisovic, R.B.; Marcucci, G.; Phillips, G.; Marsh, C.B.;
Lemeshow, S.; Grever, M.R. Hyperglycemia in patients with acute myeloid leukemia is associated with
increased hospital mortality. Cancer 2007, 110, 96–102. [CrossRef] [PubMed]
3
.
.
Galanski, M.; Arion, V.B.; Jakupec, M.A.; Keppler, B.K. Recent developments in the field of tumor-inhibiting
metal complexes. Curr. Pharm. Des. 2003, 9, 2078–2089. [CrossRef] [PubMed]
Wang, Y.; Gu, W.; Shan, Y.; Liu, F.; Xu, X.; Yang, Y.; Zhang, Q.; Zhang, Y.; Kuang, H.; Wang, Z.;
et al. Design, synthesis and anticancer activity of novel nopinone-based thiosemicarbazone derivatives.
Bioorg. Med. Chem. Lett. 2017, 27, 2360–2363. [CrossRef] [PubMed]
4
5.
6.
7.
Utsugi, T. New challenges and inspired answers for anticancer drug discovery and development. Jpn. J.
Clin. Oncol. 2013, 43, 945–953. [CrossRef] [PubMed]
Ingle, R.; Marathe, R.; Magar, D.; Patel, H.M.; Surana, S.J. Sulphonamido-quinoxalines: Search for anticancer
agent. Eur. J. Med. Chem. 2013, 65, 168–186. [CrossRef] [PubMed]
Lee, H.; Cho, S.; Namgoong, K.; Jung, J.K.; Cho, J.; Yang, S.I. Synthesis and in vitro evaluation of
7-dialkylaminomethylbenzo[g]quinoxaline-5,10-diones. Bioorg. Med. Chem. Lett. 2004, 14, 1235–1237.
[CrossRef] [PubMed]
8
.
Sridevi, C.H.; Balaji, K.; Naidu, A.; Sudhakaran, R. Synthesis of some phenylpyrazolo benzimidazolo
quinoxaline derivatives as potent antihistaminic agents. E J. Chem. 2010, 7, 234–238. [CrossRef]

Molecules 2018, 23, 48
14 of 16
1
9
1
.
Patel, N.; Bergman, J.; Graslund, A. H-NMR studies of the interaction between a self-complementary
deoxyoligonucleotide duplex and indolo[2,3-b]quinoxaline derivatives active against herpes virus.
Eur. J. Biochem. 1991, 197, 597–604. [CrossRef] [PubMed]
0. Ahmed, H.E.A.; Ihmaid, S.K.; Omar, A.M.; Shehata, A.M.; Rateb, H.S.; Zayed, M.F.; Ahmed, S.; Elaasser, M.M.
Design, synthesis, molecular docking of new lipophilic acetamide derivatives affording potential anticancer
and antimicrobial agents. Bioorg. Chem. 2017. [CrossRef] [PubMed]
1
1. Refaat, H.M.; Moneer, A.A.; Khalil, O.M. Synthesis and antimicrobial activity of certain novel quinoxalines.
Arch. Pharm. Res. 2004, 27, 1093–1098. [CrossRef] [PubMed]
1
2. Tandon, V.K.; Yadav, D.B.; Maurya, H.K.; Chaturvedi, A.K.; Shukla, P.K. Design, synthesis, and biological
evaluation of 1,2,3-trisubstituted-1,4-dihydrobenzo[g]quinoxaline-5,10-diones and related compounds as
antifungal and antibacterial agents. Bioorg. Med. Chem. 2006, 14, 6120–6126. [CrossRef] [PubMed]
3. Carta, A.; Sanna, P.; Gherardini, L.; Usai, D.; Zanetti, S. Novel functionalized pyrido[2,3-g]quinoxalinones as
antibacterial, antifungal and anticancer agents. Farmaco 2001, 56, 933–938. [CrossRef]
1
1
1
4. Vicente, E.; Duchowicz, P.R.; Castro, E.A.; Monge, A. Qsar analysis for quinoxaline-2-carboxylate
1
,4-di-N-oxides as anti-mycobacterial agents. J. Mol. Graph. Model. 2009, 28, 28–36. [CrossRef] [PubMed]
5. Jaso, A.; Zarranz, B.; Aldana, I.; Monge, A. Synthesis of new 2-acetyl and 2-benzoyl quinoxaline
,4-di-N-oxide derivatives as anti-mycobacterium tuberculosis agents. Eur. J. Med. Chem. 2003, 38, 791–800.
CrossRef]
1
[
1
6. Burguete, A.; Pontiki, E.; Hadjipavlou-Litina, D.; Villar, R.; Vicente, E.; Solano, B.; Ancizu, S.;
Perez-Silanes, S.; Aldana, I.; Monge, A. Synthesis and anti-inflammatory/antioxidant activities of some
new ring substituted 3-phenyl-1-(1,4-di-N-oxide quinoxalin-2-yl)-2-propen-1-one derivatives and of their
4
,5-dihydro-(1H)-pyrazole analogues. Bioorg. Med. Chem. Lett. 2007, 17, 6439–6443. [CrossRef] [PubMed]
1
7. Mathew, B.; Hobrath, J.V.; Connelly, M.C.; Kiplin Guy, R.; Reynolds, R.C. Diverse amide analogs of sulindac
for cancer treatment and prevention. Bioorg. Med. Chem. Lett. 2017, 27, 4614–4621. [CrossRef] [PubMed]
8. Jaso, A.; Zarranz, B.; Aldana, I.; Monge, A. Synthesis of new quinoxaline-2-carboxylate 1,4-dioxide
derivatives as anti-mycobacterium tuberculosis agents. J. Med. Chem. 2005, 48, 2019–2025. [CrossRef]
1
[PubMed]
1
2
2
9. Hearn, M.J.; Cynamon, M.H. Design and synthesis of antituberculars: Preparation and evaluation against
mycobacterium tuberculosis of an isoniazid schiff base. J. Antimicrob. Chemother. 2004, 53, 185–191. [CrossRef]
[PubMed]
0. Ismail, M.M.; Ammar, Y.A.; Ibrahim, M.K.; El-Zahaby, H.S.; Mahmoud, S.S. Synthesis and pharmacological
evaluation of novel quinoxalines as potential nonulcerogenic anti-inflammatory and analgesic agents.
Arzneimittelforschung 2005, 55, 738–743. [CrossRef] [PubMed]
1. Bourgeois, C.; Werfel, E.; Galla, F.; Lehmkuhl, K.; Torres-Gomez, H.; Schepmann, D.; Kogel, B.;
Christoph, T.; Strassburger, W.; Englberger, W.; et al. Synthesis and pharmacological evaluation of
5-pyrrolidinylquinoxalines as a novel class of peripherally restricted kappa-opioid receptor agonists.
J. Med. Chem. 2014, 57, 6845–6860. [CrossRef] [PubMed]
2
2
2
2
2
2. Hui, X.; Desrivot, J.; Bories, C.; Loiseau, P.M.; Franck, X.; Hocquemiller, R.; Figadere, B. Synthesis and
antiprotozoal activity of some new synthetic substituted quinoxalines. Bioorg. Med. Chem. Lett. 2006
6, 815–820. [CrossRef] [PubMed]
,
1
3. Sarges, R.; Howard, H.R.; Browne, R.G.; Lebel, L.A.; Seymour, P.A.; Koe, B.K. 4-amino[1,2,4]triazolo[4,3-
a]quinoxalines. A novel class of potent adenosine receptor antagonists and potential rapid-onset
antidepressants. J. Med. Chem. 1990, 33, 2240–2254. [CrossRef] [PubMed]
4. Alswah, M.; Ghiaty, A.; El-Morsy, A.; El-Gamal, K. Synthesis and biological evaluation of some [1,2,4]
triazole[4,3-a]quinoxaline derivatives as novel anticonvulsant agents. ISRN Org. Chem. 2013, 2013, 587054.
[CrossRef] [PubMed]
5. Mielcke, T.R.; Mascarello, A.; Filippi-Chiela, E.; Zanin, R.F.; Lenz, G.; Leal, P.C.; Chiaradia, L.D.; Yunes, R.A.;
Nunes, R.J.; Battastini, A.M.; et al. Activity of novel quinoxaline-derived chalcones on in vitro glioma cell
proliferation. Eur. J. Med. Chem. 2012, 48, 255–264. [CrossRef] [PubMed]
6. Gururaja, T.L.; Yung, S.; Ding, R.; Huang, J.; Zhou, X.; McLaughlin, J.; Daniel-Issakani, S.; Singh, R.;
Cooper, R.D.; Payan, D.G.; et al. A class of small molecules that inhibit tnfalpha-induced survival and death
pathways via prevention of interactions between tnfalphari, tradd, and rip1. Chem. Biol. 2007, 14, 1105–1118.
[CrossRef] [PubMed]

Molecules 2018, 23, 48
15 of 16
2
7. Matwijczuk, A.; Janik, E.; Luchowski, R.; Niewiadomy, A.; Gruszecki, W.I.; Gago s´ , M. Spectroscopic studies
of the molecular organization of 4-([1,2,4] triazolo [4,3-a] pyridin-3-yl)-6-methylbenzene-1,3-diol in selected
solvents. J. Lumin. 2018, 194, 208–218. [CrossRef]
2
8. Yuan, C.; Li, S.; Wu, Y.; Lu, L.; Zhu, M. Zn(ii)-selective and sensitive fluorescent chemosensor based on steric
constrains and inhibition of esipt. Sens. Actuators B Chem. 2017, 242, 1035–1042. [CrossRef]
9. Padalkar, V.S.; Chemate, S.B.; Lanke, S.K.; Sekar, N. N-2-aryl-1,2,3-triazoles: A novel class of blue–green
2
emitting fluorophores-synthesis, photophysical properties study and dft computations. J. Lumin. 2015
68, 114–123. [CrossRef]
,
1
3
0. Habib, N.S.; El-Hawash, S.A. Synthesis and antimicrobial testing of thiazolinyl-, thiazolidinonyl-quinoxalines
and 1,2,4-triazolo[4,3-a]quinoxalines. Die Pharm. 1997, 52, 594–598.
3
1. El-Hawash, S.A.; Habib, N.S.; Fanaki, N.H. Quinoxaline derivatives. Part ii: Synthesis and antimicrobial
testing of 1,2,4-triazolo[4,3-a]quinoxalines, 1,2,4-triazino[4,3-a]quinoxalines and 2-pyrazolylquinoxalines.
Pharmazie 1999, 54, 808–813. [PubMed]
3
3
3
3
3
2. Suresh, M.; Lavanya, P.; Sudhakar, D.; Vasu, K.; Rao, C.V. Synthesis and biological activity of 8-chloro-
[
1,2,4]triazolo[4,3-a]quinoxalines. J. Chem. Pharm. Res. 2010, 2, 497–504.
3. Johnson, L.B.; Kauffman, C.A. Voriconazole: A new triazole antifungal agent. Clin. Infect. Dis. 2003, 36, 630–637.
CrossRef] [PubMed]
[
4. Torres, H.A.; Hachem, R.Y.; Chemaly, R.F.; Kontoyiannis, D.P.; Raad, I.I. Posaconazole: A broad-spectrum
triazole antifungal. Lancet Infect. Dis. 2005, 5, 775–785. [CrossRef]
5. Kharb, R.; Sharma, P.C.; Yar, M.S. Pharmacological significance of triazole scaffold. J. Enzyme Inhib. Med. Chem.
2
011, 26, 1–21. [CrossRef] [PubMed]
6. Issa, D.A.E.; Habib, N.S.; Abdel Wahab, A.E. Design, synthesis and biological evaluation of novel
,2,4- triazolo and 1,2,4-triazino[4,3-a]quinoxalines as potential anticancer and antimicrobial agents.
1
Med. Chem. Comm. 2015, 6, 202–211. [CrossRef]
3
7. Ali, I.; Lee, J.; Go, A.; Choi, G.; Lee, K. Discovery of novel [1,2,4]triazolo[4,3-a]quinoxaline aminophenyl
derivatives as bet inhibitors for cancer treatment. Bioorg. Med. Chem. Lett. 2017, 27, 4606–4613. [CrossRef]
[PubMed]
3
3
8. Lu, Y.; Chen, J.; Xiao, M.; Li, W.; Miller, D.D. An overview of tubulin inhibitors that interact with the
colchicine binding site. Pharm. Res. 2012, 29, 2943–2971. [CrossRef] [PubMed]
9. El-Hawash, S.A.M.; Habib, N.S.; Kassem, M.A. Synthesis of some new quinoxalines and 1,2,4-triazolo[4,3-
a]-quinoxalines for evaluation of in vitro antitumor and antimicrobial activities. Arch. Der Pharm. 2006
39, 564–571. [CrossRef] [PubMed]
,
3
4
4
4
4
0. Dell, A.; Williams, D.H.; Morris, H.R.; Smith, G.A.; Feeney, J.; Roberts, G.C. Structure revision of the antibiotic
echinomycin. J. Am. Chem. Soc. 1975, 97, 2497–2502. [CrossRef] [PubMed]
1. Wakelin, S.P.; Waring, M.J. The binding of echinomycin to deoxyribonucleic acid. Biochem. J. 1976
57, 721–740. [CrossRef] [PubMed]
2. Lieva, D.B.; Todorova, I.T. Trends in utilization of the pharmacological potential of chalcones. Curr. Clin. Pharmacol.
010, 5, 1–29.
,
1
2
3. Mielcke, T.R.; Muradas, T.C.; Filippi-Chiela, E.C.; Amaral, M.E.A.; Kist, L.W.; Bogo, M.R.; Mascarello, A.;
Neuenfeldt, P.D.; Nunes, R.J.; Campos, M.M. Mechanisms underlying the antiproliferative effects of a series
of quinoxaline-derived chalcones. Sci. Rep. 2017, 7, 15850. [CrossRef] [PubMed]
4
4. Sahu, N.K.; Balbhadra, S.S.; Choudhary, J.; Kohli, D.V. Exploring pharmacological significance of chalcone
scaffold: A review. Curr. Med. Chem. 2012, 19, 209–225. [CrossRef] [PubMed]
4
5. Kong, Y.; Wang, K.; Edler, M.C.; Hamel, E.; Mooberry, S.L.; Paige, M.A.; Brown, M.L. A boronic acid chalcone
analog of combretastatin a-4 as a potent anti-proliferation agent. Bioorg. Med. Chem. 2010, 18, 971–977.
[CrossRef] [PubMed]
4
4
6. Luo, Y.; Qiu, K.M.; Lu, X.; Liu, K.; Fu, J.; Zhu, H.L. Synthesis, biological evaluation, and molecular modeling
of cinnamic acyl sulfonamide derivatives as novel antitubulin agents. Bioorg. Med. Chem. 2011, 19, 4730–4738.
[CrossRef] [PubMed]
7. Sharma, S.; Kaur, C.; Budhiraja, A.; Nepali, K.; Gupta, M.K.; Saxena, A.K.; Bedi, P.M.S. Chalcone based
azacarboline analogues as novel antitubulin agents: Design, synthesis, biological evaluation and molecular
modelling studies. Eur. J. Med. Chem. 2014, 85, 648–660. [CrossRef] [PubMed]

Molecules 2018, 23, 48
16 of 16
4
8. Dyrager, C.; Wickstrom, M.; Friden-Saxin, M.; Friberg, A.; Dahlen, K.; Wallen, E.A.; Gullbo, J.; Grotli, M.;
Luthman, K. Inhibitors and promoters of tubulin polymerization: Synthesis and biological evaluation of
chalcones and related dienones as potential anticancer agents. Bioorg. Med. Chem. 2011, 19, 2659–2665.
[CrossRef] [PubMed]
4
5
5
5
5
9. Kamal, A.; Kumar, G.B.; Vishnuvardhan, M.V.; Shaik, A.B.; Reddy, V.S.; Mahesh, R.; Sayeeda, I.B.; Kapure, J.S.
Synthesis of phenstatin/isocombretastatin-chalcone conjugates as potent tubulin polymerization inhibitors
and mitochondrial apoptotic inducers. Org. Biomol. Chem. 2015, 13, 3963–3981. [CrossRef] [PubMed]
0. Ducki, S.; Rennison, D.; Woo, M.; Kendall, A.; Chabert, J.F.; McGown, A.T.; Lawrence, N.J.
Combretastatin-like chalcones as inhibitors of microtubule polymerization. Part 1: Synthesis and biological
evaluation of antivascular activity. Bioorg. Med. Chem. 2009, 17, 7698–7710. [CrossRef] [PubMed]
1. Boumendjel, A.; Boccard, J.; Carrupt, P.A.; Nicolle, E.; Blanc, M.; Geze, A.; Choisnard, L.;
Wouessidjewe, D.; Matera, E.L.; Dumontet, C. Antimitotic and antiproliferative activities of chalcones:
Forward structure-activity relationship. J. Med. Chem. 2008, 51, 2307–2310. [CrossRef] [PubMed]
2. Chaidos, A.; Caputo, V.; Karadimitris, A. Inhibition of bromodomain and extra-terminal proteins (bet) as a
potential therapeutic approach in haematological malignancies: Emerging preclinical and clinical evidence.
Ther. Adv. Hematol. 2015, 6, 128–141. [CrossRef] [PubMed]
3. Tiwari, S.V.; Siddiqui, S.; Seijas, J.A.; Vazquez-Tato, M.P.; Sarkate, A.P.; Lokwani, D.K.; Nikalje, A.P.G.
Microwave-assisted facile synthesis, anticancer evaluation and docking study of n-((5-(substituted methylene
amino)-1,3,4-thiadiazol-2-yl)methyl) benzamide derivatives. Molecules 2017, 22, 995. [CrossRef] [PubMed]
4. Berman, H.M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T.N.; Weissig, H.; Shindyalov, I.N.; Bourne, P.E.
The protein data bank. Nucleic Acids Res. 2000, 28, 235–242. [CrossRef] [PubMed]
5
5
5
5
5
5
5. Romer, D.R. Synthesis of 2,3-dichloroquinoxalines via vilsmeier reagent chlorination. J. Heterocycl. Chem.
2
009, 46, 317–319. [CrossRef]
6. Klancnik, A.; Piskernik, S.; Jersek, B.; Mozina, S.S. Evaluation of diffusion and dilution methods to determine
the antibacterial activity of plant extracts. J. Microbiol. Methods 2010, 81, 121–126. [CrossRef] [PubMed]
7. Gangadevi, V.J.M. Preliminary studies on cytotoxic effect of fungal taxol on cancer cell lines. Afr. J. Biotechnol.
2
007, 6, 1382–1386.
8. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and
cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. [CrossRef]
9. Morris, G.M.; Goodsell, D.S.; Halliday, R.S.; Huey, R.; Hart, W.E.; Belew, R.K.; Olson, A.J. Automated docking
using a lamarckian genetic algorithm and an empirical binding free energy function. J. Comput. Chem. 1998
9, 1639–1662. [CrossRef]
,
1
6
0. Chemical Computing Group. Molecular Operating Environment (MOE); Chemical Computing Group: Montreal,
QC, Canada, 2012; Available online: http://www.chemcomp.com (accessed on 30 February 2013).
Sample Availability: Samples of the compounds are currently not available from the authors.
©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(