Synthesis, EGFR-TK inhibition and anticancer activity of new quinoxaline derivatives

Article abstract of DOI:10.1080/00397911.2020.1787448

Ethyl 4-substituted-3-oxo-quinoxaline-2-carboxylates 3–5 were obtained via alkylation of ethyl 3-oxo-3,4-dihydroquinoxaline-2-carboxylate (1). Compound 1 was heterocyclized using hydrazines, ethylenediamine, and ethanolamine to give pyrazoloquinoxalines 6, 7, diazepinoquinoxaline 8, and oxazepinoquinoxaline 10. The quinoxaline-2-carboxamides 9, 11, 12 were prepared via condensation of compound 1 with different amines. Compound 1 was thiated using Lawesson’s reagent affording quinoxaline-3-thione 13, in fair yield. In addition, the reaction of 4-methyl-3-oxoquinoxaline 3 with some binucleophiles led to a series of new oxoquinoxaline derivatives 14–18. The molecular structure of compounds 1, 3, and 9 was confirmed by X-ray crystallography. The anti-proliferative activity showed that among all the tested compounds, compounds 3, (IC₅₀ 2.51 ± 3.0, 4.22 ± 1.6 and 2.27 ± 1.9 μM), 11 (IC₅₀ 1.32 ± 2.61, 1.41 ± 1.23 and 1.18 ± 1.91 μM) and 17 (IC₅₀ 1.72 ± 1.32, 1.85 ± 0.94 and 1.92 ± 4.83 μM) showed noteworthy anti-proliferative effects against the three cancer cell lines, HCT116, HePG2 and MCF7, respectively, compared to the reference drugs doxorubicin (IC₅₀ 1.41 ± 0.58, 0.90 ± 0.62 and 1.01 ± 3.02 μM) and erlotinib (IC₅₀ 1.63 ± 0.81, 1.57 ± 0.62 and 1.49 ± 0.54 μM). Compounds 3 (0.899 nM), 11 (0.508 nM) and 17 (0.807) showed strong EGFR inhibitory activity compared to Erlotinib (0.439 nM) and these results are in agreement with the docking study. These results suggest that compounds could probably be promising anticancer agents with EGFR inhibitory activity.

Full text of DOI:10.1080/00397911.2020.1787448

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: https://www.tandfonline.com/loi/lsyc20
Synthesis, EGFR-TK inhibition and anticancer
activity of new quinoxaline derivatives
Eman A. Ahmed, Mamdouh F. A. Mohamed, Ahmed Omran & Hanan Salah
To cite this article: Eman A. Ahmed, Mamdouh F. A. Mohamed, Ahmed Omran & Hanan Salah
(2020): Synthesis, EGFR-TK inhibition and anticancer activity of new quinoxaline derivatives,
Synthetic Communications, DOI: 10.1080/00397911.2020.1787448
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https://doi.org/10.1080/00397911.2020.1787448
Synthesis, EGFR-TK inhibition and anticancer activity of
new quinoxaline derivatives
Eman A. Ahmeda, Mamdouh F. A. Mohamedb, Ahmed Omranc, and Hanan Salaha
b
aDepartment of Chemistry, Faculty of Science, Sohag University, Sohag, Egypt; Department of
c
Pharmaceutical Chemistry, Faculty of Pharmacy, Sohag University, Sohag, Egypt; Department of
Pharmacology, Faculty of Pharmacy, Minia University, Minia, Egypt
ABSTRACT
ARTICLE HISTORY
Received 25 March 2020
Ethyl 4-substituted-3-oxo-quinoxaline-2-carboxylates 3–5 were
obtained via alkylation of ethyl 3-oxo-3,4-dihydroquinoxaline-2-carb-
oxylate (1). Compound 1 was heterocyclized using hydrazines, ethyl-
enediamine, and ethanolamine to give pyrazoloquinoxalines 6, 7,
diazepinoquinoxaline 8, and oxazepinoquinoxaline 10. The quinoxa-
line-2-carboxamides 9, 11, 12 were prepared via condensation of
compound 1 with different amines. Compound 1 was thiated using
Lawesson’s reagent affording quinoxaline-3-thione 13, in fair yield. In
addition, the reaction of 4-methyl-3-oxoquinoxaline 3 with some
binucleophiles led to a series of new oxoquinoxaline derivatives
14–18. The molecular structure of compounds 1, 3, and 9 was con-
firmed by X-ray crystallography.
KEYWORDS
Anti-proliferative activity;
heterocyclization; molecular
docking; nucleophilic
reactions; quinoxaline
derivatives; X-ray
crystallography
The anti-proliferative activity showed that among all the tested com-
pounds, compounds 3, (IC50 2.51 3.0, 4.22 1.6 and 2.27 1.9 mM),
11 (IC50 1.32 2.61, 1.41 1.23 and 1.18 1.91 mM) and 17 (IC50
1.72 1.32, 1.85 0.94 and 1.92 4.83 mM) showed noteworthy anti-
proliferative effects against the three cancer cell lines, HCT116,
HePG2 and MCF7, respectively, compared to the reference drugs
doxorubicin (IC50 1.41 0.58, 0.90 0.62 and 1.01 3.02 mM) and erlo-
tinib (IC50 1.63 0.81, 1.57 0.62 and 1.49 0.54 mM). Compounds 3
(0.899 nM), 11 (0.508 nM) and 17 (0.807) showed strong EGFR inhibi-
tory activity compared to Erlotinib (0.439nM) and these results are
in agreement with the docking study. These results suggest that
compounds could probably be promising anticancer agents with
EGFR inhibitory activity.
CONTACT Eman A. Ahmed
abdala_15@yahoo.com
Department of Chemistry, Faculty of Science, Sohag
University, Sohag 82524, Egypt.
Supplemental data for this article is available online at at publisher’s website.
ß 2020 Taylor & Francis Group, LLC

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E. A. AHMED ET AL.
GRAPHICAL ABSTRACT
Introduction
Cancer is one of the most serious diseases for humans, and it is the second
highest leading factor in death worldwide.[1,2] Apart from the use of surgery and radio-
therapy, chemotherapy remains one of the most effective choices for suppressing
tumor growth and tumor eradication.[3] However, the use of chemotherapeutic agents is
limited due to their lack of selectivity, severe toxicity, dose-related side effects and
development of drug-resistance.[4] Notably, many patients undergoing chemotherapy
have associated severe side effects such as thrombocytopenia, anemia, nausea and vom-
iting.[5] Although, there are many research studies have reported the efficacy of new
compounds as potential chemotherapeutic agents, research is still underway in the hope
of finding new effective anticancer agents with improved efficacy and enhanced
safety profile.
Quinoxaline nucleus, a bioisoster of quinoline, quinazoline, naphthalene and benzo-
thiophene, represents an important class of heterocyclic compounds.[6] Quinoxaline
scaffold bearing compounds have proven to have a good anticancer activity and found a
great application in the discovery of novel anticancer agents. Quinoxaline derivatives
exerts their anticancer activity via different mechanisms including tyrosine kinases

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3
inhibition,[7–9] induction of apoptosis,[1,10,11] C-MET kinase inhibition,[12] tubulin poly-
merization inhibition,[13] and selective induction of tumor hypoxia.[14]
In addition to their anticancer activity, quinoxaline derivatives possess diverse bio-
logical activities such as antimicrobial,[15] anticonvulsant,[16] analgesic activities,[17] anti-
inflammatory,[18,19] antiobese,[20] and antiviral.[21,22]. Moreover, they are important
industrially due to their ability to inhibit the metal corrosion,[23–25] in the electrolumin-
escent materials,[26] and also in the preparation of the porphyrins, since their structure
is similar to the chromophores in the natural system. Due to their importance, different
synthetic strategies have been utilized for the synthesis of quinoxaline derivatives involv-
ing condensation of o-phenylenediamine derivatives with 1,2-dicarbonyl compounds,[27]
a-hydroxyketones,[28] a-halo-b-ketoester,[29] and oxidation of alkynes followed by the
condensation with 1,2-diamines.[30] Different efficient catalysts have been used for the
synthesis of quinoxalines, such as CuSO4.H2O,[31] MnO2,[32] ceric ammonium
nitrate,[33] iodine,[34] also microwave-irradiation[35] is a successfully catalyzed method.
The aforementioned findings have inspired and guided us to synthesize a new set of
heterocyclic compounds having quinoxaline moiety starting with ethyl 3-oxo-3,4-dihy-
droquinoxaline-2-carboxylate (1).
Results and discussion
Chemistry
As depicted in Scheme 1, the starting compound; ethyl 3-oxo-3,4-dihydroquinoxaline-2-
carboxylate (1) was synthesized as a major product, in addition to benzimidazole 2 as a
minor product from the reaction of o-phenylenediamine with diethyl 2-bromomalonate
in boiling diphenyl ether.
IR spectrum of compound 1 showed the existence of two strong stretching vibrations
1
at ꢀ 1729, 1656 cmꢀ1, due to carbonyl groups of ester and lactam, respectively. H
NMR spectrum revealed a singlet signal at d 12.82 assignable to exchangeable N–H pro-
ton, quartet signal due to CH2 ester at 4.40–4.35 and triplet signal for CH3 ester at
1.34–1.31. In addition, X-ray single crystal technique was employed to confirm the
structure of compound 1 (Figure 1).
Single crystals of compound 1 was grown in a mixture of diphenyl ether and petrol-
eum ether (60–80) in a ratio 1:10 at room temperature (rt, 25 ꢁC) for 48 h.
Crystal Data: C11H10N2O3, M ¼ 218.212, monoclinic, a ¼ 4.8249 (5) Å, b ¼ 19.385
(2) Å, c ¼ 11.1019 (13) Å, V ¼ 1037.9 (2) Å3 a ¼ c ¼ 90.00ꢁ, b ¼ 91.799 (4)ꢁ.
,
A Plausible reaction pathway for the formation of compound 1 is illustrated in Scheme 2.
It is clear that an initial nucleophilic substitution took place on the diethyl 2-bromomalonate
to afford diethyl 2-(2-aminophenylamino)malonate (III), which smoothly underwent intra-
molecularcyclo-condensation to form tetrahydroquinoxaline-2-carboxylate (IV), which in
turn was auto-oxidized via air oxygen affording the aromatized quinoxaline product 1.
Compound 1 was treated with some halocompounds such as methyl iodide, ethyl
chloroacetate and/or ethyl bromoacetate. Treatment of compound 1 with methyl iodide,
in presence of potassium carbonate in DMF, gave the N-methylated product; ethyl 4-
methyl-3-oxo-3,4-dihydroquinoxaline-2-carboxylate (3). Interestingly, a similar reaction
of compound 1 with ethyl chloroacetate led to the O-alkylated product; ethyl 3-(2-

4
E. A. AHMED ET AL.
O
H
N
H
N
NH2
O
OC2H5
OC2H5
Diphenyl ether
Br
refluxing 3 min.
N
NH2
N
COOC2H5
2
O
1
CH3
N
O
CH3I/K2CO3
DMF
stirring overnight
N
COOC2H5
3
N
OCH2COOC2H5
XCH2COOEt
1
DMF/K2CO3
stirring overnight at r. t.
X= Br or Cl
N
COOC2H5
4
N
OCH2COOH
BrCH2COOEt
4
+
DMF/K2CO3
N
COOC2H5
stirring overnight
at 60-65 o
C
5
Scheme 1. Synthesis of quinoxaline 1 and its reactions with methyl iodide, ethyl chloroacetate and
ethyl bromoacetate.
Figure 1. ORTEP plot of the X-ray crystallographic data determined for 1 with CCDC. 1839216.
ethoxy-2-oxoethoxy)quinoxaline-2-carboxylate (4) (Scheme 1). The reaction of com-
pound 1 with ethyl bromoacetate was found a temperature-dependent reaction. Thus,
when the reaction was carried out at room temperature, the major product is the
O-acetate ester derivative 4, whereas when the reaction was carried out at 60–65 ꢁC, the

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5
O
NH2
NH2
NH2
OC2H5
OC2H5
COOC2H5
-HBr
Br
N
COOC2H5
H
I
O
II
III
-EtOH
H
N
O
H
N
O
air
oxidation
N
COOC2H5
N
H
COOC2H5
1
IV
Scheme 2. A Plausible pathway for the formation of compound 1.
Figure 2. ORTEP plot of the X-ray crystallographic data determined for 3 with CCDC. 1839218.
major product was O-acetic acid derivative (5), beside compound 4 which was separated
as a minor product. IR spectra of compounds 3, 4 showed the absence of an absorption
1
band corresponding to the N–H group. H NMR spectra showed the disappearance of
the signal corresponding to the NH group and exhibited a singlet signal at 3.72 for the
methyl protons of NCH3 in the case of compound 3, new signals corresponding to 3
methylene groups at 4.96, 4.46–4.41, 4.20–4.15 and 2CH3 at 1.52–1.35, 1.21–1.17 in the
case of compound 4. X-ray single crystal technique was employed to confirm the struc-
ture of compound 3 (Figure 2).
Single crystals of compound 3 was grown in a mixture of acetonitrile/ethanol in a
ratio 1:10 at room temperature (rt, 25 ꢁC) for 36 h.Crystal Data: C12H12N2O3,
M ¼ 232.239, triclinic, a ¼ 7.3737 (6) Å, b ¼ 7.5688 (5) Å, c ¼ 10.6435 (10) Å, V ¼ 556.52
(8) Å3 a ¼ 106.966 (3)ꢁ, b ¼ 91.561 (3)ꢁ, c ¼ 100.537 (5)ꢁ. Compound 1 was used as a
,
starting material for the synthesis of some new heterocyclic polynuclear systems

6
E. A. AHMED ET AL.
H
N
N
H2NNH2.H2O
NH
Ethanol
refluxing 5 min.
N
6
O
Ph
N
N
N
H2NNHPh
p-xylene
NH
refluxing 10 h.
7
O
1
H
N
N
H2N(CH2)2NH2
Ethanol
refluxing 15 min.
NH
N
8
O
O
H
N
N
N
O
diphenyl ether
H2N(CH2)2OH
refluxing
3 h.
Ethanol
refluxing 3 h.
NH
N
CONH
OH
9
O
10
Scheme 3. Reaction of quinoxaline 1 with hydrazine hydrate, phenyl hydrazine, ethylenediamine and
ethanol amine.
comprising quinoxaline moiety. So that, compound 1 was subjected to react with hydra-
zine hydrate, phenyl hydrazine and/or ethylenediamine, to afford 2,9-dihydro-3H-pyra-
zolo[3,4-b]quinoxalin-3-one (6), 1-phenyl-1,2-dihydro-3H-pyrzolo[3,4-b]quinoxalin-3-
one (7) and 2,3,4,11-tetrahydro-5H-[1,4]diazepino[5,6-b]quinoxalin-5-one (8), respect-
ively (Scheme 3).
The structure of the products was established on basis of their spectral and analytical data.
The IR spectra of these compounds showed the disappearance of absorption bands corre-
sponding to 2C¼ O groups and exhibited new absorption bands at ꢀ 3393–3200cmꢀ1 for
the N–H groups and at ꢀ 1675 2 cmꢀ1 due to the C ¼ O groups in compounds 6, 7 and 8,
respectively. The 1H NMR spectra showed the absence of signals due to ethoxy protons and
exhibited new signals at 4.82–4.65; 12.74; 6.69 due to new NH groups in addition to triplet
signals at 3.56–3.52, 2.97–2.94 due to 2CH2N groups in the case of compound 8. The prod-
ucts were formed via initial nucleophilic addition-elimination reaction, followed by intramo-
lecularcyclo-condensation with elimination of water molecule.
Also, when compound 1 was reacted with ethanolamine in boiling ethanol, leads to
N-(2-hydroxyethyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (9), which in turn
boiled in diphenyl ether to give 3,4-dihydro-[1,4]oxazepino[6,7-b]quinoxalin-5(2H)-
one (10).

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7
Figure 3. ORTEP plot of the X-ray crystallographic data determined for compound 9 with
CCDC. 1839221.
The IR spectrum of compound 9 showed new absorption bands at ꢀ 3324 and
3228 cmꢀ1 corresponding to O–H and N–H groups, beside a new absorption band at ꢀ
1
1637 cmꢀ1 for the new C¼O group. Its H NMR spectrum showed the disappearance of
the CH3 and CH2 signals and exhibited a new singlet signal at 9.09 for the N–H proton;
singlet signal at 4.71 for the O–H group beside two triplet signals at 3.59–3.54 and
3.44–3.38 due to CH2O and CH2N groups, respectively. Also, the IR spectrum of com-
pound 10 showed the disappearance of absorption bands corresponding to O–H, C ¼ O
1
and N–H groups. HNMR spectrum showed the absence of signals corresponding to
O–H and N–H protons. Structure of compound 9 was confirmed via the X-ray crystal-
lographic analysis (Figure 3).
Single crystal of compound 9 was grown in a dimethylsulfoxide at room temperature
(rt, 25 ꢁC) for 7 days. Crystal Data: C12H11N3O3, M ¼ 233.227, orthorhombic, a ¼ 5.0698
(4) Å, b ¼ 12.8607 (12) Å, c ¼ 16.241 (2) Å, V ¼ 1059.0 (2) Å3 a ¼ b¼c ¼ 90.00ꢁ
,
On the other hand, when compound 1 was allowed to react with cysteamine hydro-
chloride in refluxing ethanol and triethylamine as a catalyst, compound N,N0-(dithio-
ethane-2,1-diyl)bis(3-oxo-3,4-dihydroquinoxaline-2-carboxamide (11) was afforded
(Scheme 4). The structure of the product was established on the basis of its elemental
and spectral analyses. Obviously, the product was formed through initial N-nucleophilic
addition-elimination reaction, followed by air oxidation. Also, when compound 1 was
reacted with ethyl glycinate hydrochloride in refluxing ethanol and triethylamine as a
catalyst, the corresponding ethyl 2-(3-oxo-3,4-dihydroquinoxaline-2-carboxamido)ace-
tate (12) was obtained (Scheme 4). The IR spectrum displayed new absorption bands at
3299 cmꢀ1 for the new N–H group and at 1747, 1687 cmꢀ1 due to the new C ¼ O
1
groups. The H NMR spectrum showed a new signal at d 9.35 for the new NH group
and at d 3.95–3.90 for two methylene groups.
Treatment of compound 1 with Lawesson’s reagent (2,4-bis(4-methoxy-phenyl)-1,3-
dithia-2,4diphosphetane-2,4-disulphide; LR) gave ethyl 3-thioxo-3,4-dihydroquinoxaline-2-
carboxylate (13),[36] in fair yield (62%). The IR spectrum of compound 13 showed the
disappearance of absorption band corresponding to amidic C¼O group and displayed a
new absorption band at ꢀ 1107cmꢀ1 for C¼S group.
Also, compound 3 was used as a starting material to react with hydrazine hydrate,
phenylhydrazine, ethylenediamine, ethanol amine and cystamine hydrochloride to give

8
E. A. AHMED ET AL.
H
N
O
H2N(CH2)2SH.HCl
TEA/Ethanol
refluxing 6 h.
N
CONH
SH
O
air oxidation
H
N
H
N
O
N
N
CONH
S
S
NHC
O
11
H
N
O
H2NCH2COOEt.HCl
1
TEA/Ethanol
refluxing 6 h.
N
CONH
OEt
12
O
H
N
S
LR
chloroform/toluene
refluxing overnight
N
COOC2H5
13
Scheme 4. Reaction of quinoxaline 1 with cysteamine hydrochloride ethyl glycinate hydrochloride
and Lawesson’s reagent (LR).
the corresponding compounds 4-methyl-3-oxo-3,4-dihydroquinoxaline-2-carbohydrazide
(14), 4-methyl-3-oxo-N0-phenyl-3,4-dihydroquinoxaline-2-carbohydrazide (15), 11-
methyl-2,3,4,11-tetrahydro-5H-[1,4]diazepino[5,6-b]quinoxalin-5-one(16), N-(2-hydrox-
yethyl)-4-methyl-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (17) and N-(2-mercap-
toethyl)-4-methyl-3-oxo-3,4-dihydroquinoxaline-2-carboxamide
(18),
respectively
(Scheme 5). The structure of compounds 14–18 was established on basis of their spec-
tral and analytical data.

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9
N
N
O
HN
O
NH2
H2NNH2.H2O
Ethanol
refluxing 30 min.
14
N
O
O
H2NNHPh
H
N
1,4-dioxane
refluxing 10 h.
N
N
H
15
N
N
N
H2N(CH2)2NH2
3
Ethanol
refluxing 40 min.
NH
16
O
N
N
O
H2N(CH2)2OH
Ethanol
refluxing 3 h.
CONH
OH
17
N
O
H2N(CH2)2SH.HCl
TEA/Ethanol
refluxing 6 h.
N
CONH
SH
18
Scheme 5. Reaction of N-methyl quinoxaline 3 with hydrazine hydrate, phenyl hydrazine, ethylenedi-
amine, ethanol amine and cysteamine hydrochloride.

10
E. A. AHMED ET AL.
Table 1. Anti-proliferative activity (IC50 mM) of compounds 3, 4, 6, 8, 9, 11, 13, 14, 16 and 17.
Compound
HCT-116
HepG2
MCF-7
3
4
6
8
9
11
13
14
2.51 3.0
57.32 1.27
12.56 0.91
57.28 2.4
74.76 1.12
1.32 2.61
38.24 0.74
26.16 1.72
49.71 2.03
1.72 1.32
1.41 0.58
1.63 0.81
4.22 1.6
63.41 0.82
25.48 0.67
71.32 0.28
66.45 0.27
1.41 1.23
41.43 2.18
32.46 0.59
32.33 2.32
1.85 0.94
0.90 0.62
1.57 0.62
2.27 1.9
59.75 1.7
13.50 1.31
64.47 0.92
57.16 2.32
1.18 1.91
27.85 0.63
44.92 2.07
25.91 0.76
1.92 4.83
1.01 3.02
1.49 0.54
16
17
Doxorubicin
Erlotinib
Biology
Anti-proliferative activity
Anti-proliferative properties of the synthesized compounds 3, 4, 6, 8, 9, 11, 13, 14,
16 and 17 were evaluated against HCT116 (colon), HePG2 (liver) and MCF7
(breast) cancer cell lines by the standard MTT bioassay using doxorubicin and erlo-
tinibas reference standards.[37–40] GraphPad Prism software (GraphPad Software, San
Diego, CA, USA) was used to calculate the median inhibition concentration (IC50)
for all compounds.
From the results have been displayed in Table 1, it is clear that compounds 3, (IC50
2.51 3.0, 4.22 1.6 and 2.27 1.9 mM), 11 (IC50 1.32 2.61, 1.41 1.23 and
1.18 1.91 mM) and 17 (IC50 1.72 1.32, 1.85 0.94 and 1.92 4.83 mM) showed note-
worthy anti-proliferative effects against the three cancer cell lines, HCT116, HePG2 and
MCF7, respectively, compared to the reference drugs doxorubicin (IC50 1.41 0.58,
0.90 0.62 and 1.01 3.02 mM) and erlotinib (IC50 1.63 0.81, 1.57 0.62
and 1.49 0.54 mM).
Moreover, compounds 6 exhibited strong anti-proliferative effects against HCT116
(IC50 12.56 0.91) and MCF7 (IC50 13.50 1.31) and moderate activity against HePG2
(IC50 25.48 0.67). Additionally, compounds 13, 14 and 16 showed moderate anti-pro-
liferative activity against the three cancer cell line with IC50 values ranging
from25.91 0.76 to 49.71 2.03 mM. Finally, compounds 4, 8 and 9 exhibited the lowest
anti-proliferative activity with IC50 values more than 50 mM.
EGFR TK inhibitory activity. To investigate the mechanism at molecular level of the
most potent anti-proliferative compounds 3, 6, 11 and 17 were assessed on EGFR TK
kinase[41] (Table 2). The findings of the cancer cell-based assays have been powerfully
complemented by the results from the EGFR assay.
Compounds 3, 6, 11 and 17 showed potent EGFR inhibition with IC50 0.899, 0.508
and 0.807 nM, respectively while that of erlotinib was 0.439 nM. Compound 6 displayed
the least potent activity (IC50 ¼ 1.071 nM) among all the tested compounds. The inhibi-
tory effect against EGFR TK kinase is consistent with their anti-proliferative effects.
This study demonstrates that compounds 3, 11 and 17 are strong inhibitors of EGFR
and could probably be promising lead as anticancer agents.

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11
Table 2. Effects of compounds 3, 6, 11, 17 and erlotinib
on EGFR.
Compound
IC50 nM
3
6
11
17
0.899
1.071
0.508
0.807
0.439
Erlotinib
Figure 4. Interaction of compound 11 with EGFR enzyme.
Docking study
The present docking simulation study was carried out in order to examine whether
these compounds could inhibit EGFR kinase as an EGFR kinase inhibitor. This study
was performed using CDOCKER docking protocol, embedded in the Discovery Studio
software 2.5 (San Diego, USA). The quinoxaline derivatives 3, 11 and 17 were docked
into the ATP-active sites of EGFR using the 3 D protein structure (PDB ID: 1M17)
retrieved from protein data bank for EGFR[42]. Interestingly, from the analysis of the
docking results, it was clear that the docking studies were in agreement with the EGFR
kinase inhibitory assay. The docking results indicated that compound 11, the most
active one, nicely bound to the substrate binding pocket of 1M17 (Figure 4) via incorp-
oration of five hydrogen bonds and five hydrophobic interactions with Glu738, Met742,
Met769, Thr830 and Asp8321. In addition to many hydrophobic interactions and this
may explain the high inhibitory activity of compound 11 on 1 M17. Compound 17
engaged in three hydrogen bonds, one with Thr766 and two hydrogen bonds with
Met769. Additionally, compound 17 forms many hydrophobic interactions with differ-
ent amino acids residues as shown in Figure 5. Moreover, docking analysis of com-
pound 3 showed that it forms two hydrogen bonds with Thr830 and Met769 and many
other hydrophobic interactions (Figure 6).
Conclusion
This study reports a successful synthesis of some new fused heterocycles containing a
quinoxaline moiety. The anti-proliferative activity of the tested compounds showed
noteworthy anti-proliferative effects against HCT116, MCF7 and HePG2 cancer cell

12
E. A. AHMED ET AL.
Figure 5. Interaction of compound 17 with EGFR enzyme.
Figure 6. Interaction of compound 3 with EGFR enzyme.
lines. Mechanistically, compounds 3, 11 and 17 showed strong EGFR inhibitory activity
which was in agreement with the docking study. These results suggest that compounds
3, 11 and 17 could probably be promising lead as anticancer agents with EGFR inhibi-
tory activity.
Experimental
Chemistry
Ethyl 3-oxo-3,4-dihydroquinoxaline-2-carboxylate (1) and 1H-benzo[d]imidazole (2)
A mixture of o-phenylenediamine (1.08 g, 0.01 mol) and diethyl 2-bromomalonate
(2.39 g, 0.01 mol) in anhydrous diphenyl ether (1 mL) was refluxed for 3 min, the reac-
tion mixture was dissolved then precipitated on hot. The solid residue was collected by
decantation, triturated with petroleum ether (60–80) several times, the product was
boiled in chloroform, filtered on hot, the precipitate was recrystallized from ethanol to
give yellow crystals of compound 2 (0.035 g, 30%) m.p. 170–171 ꢁC (Lit. 170–172 ꢁC).[43]

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SYNTHETIC COMMUNICATIONSV
13
The chloroform layer was cooled, evaporated to give pale yellow crystals of compound
1, which was collected by filtration and recrystallized from ethanol or a mixture of
diphenyl ether and petroleum ether (60–80) in a ratio 1:10 afforded 1 (0.131 g, 60%)
m.p. 180–181 ꢁC; (Lit. 175.5–176.5 ꢁC);[44] 1H NMR (400 MHz, DMSO-d6) d: 12.82 (s,
1H, NH disappeared on addition of D2O), 7.84–7.82 (d, 1H, Harom.), 7.66–7.62 (t, 1H,
Harom.), 7.37–7.35 (dd, 2H, Harom.), 4.40–4.35 (q, 2H, CH2), 1.34–1.31 (t, 3H, CH3); IR
(KBr) ꢀ: 3161 (NH), 1729, 1656 (2C¼O) cmꢀ1. Anal. Calcd. For C11H10N2O3 (218.21):
C, 60.55; H, 4.62; N, 12.84. Found: C, 60.7; H, 4.75; N, 12.71. Crystallographic Analysis
for (1): all diagrams and calculations were performed using maXus (BrukerNonius,
Delft &MacScience, Japan) Crystal Data: C11H10N2O3, M ¼ 218.212, monoclinic,
a ¼ 4.8249 (5) Å, b ¼ 19.385 (2) Å, c ¼ 11.1019 (13) Å, V ¼ 1037.9 (2) Å3 a ¼ c ¼ 90.00ꢁ,
,
b ¼ 91.799 (4)ꢁ, space group: P21/c, Z ¼ 4, Dx ¼ 1.397 Mg mꢀ3, No. of reflections meas-
ured 2222, hmax ¼ 27.47ꢁ, Rint ¼ 0.095.
In vitro cytotoxicity assay
Materials and methods
Cell lines
Four human tumor cell lines namely; Hepatocellular carcinoma (HePG-2), Mammary
gland (MCF-7) and Colorectal carcinoma (HCT-116). The cell lines were obtained from
ATCC via Holding company for biological products and vaccines (VACSERA), Cairo,
Egypt. Doxorubicin and erlotinibwere used as
for comparison.
a
standard anticancer drug
Chemical reagents
The reagents RPMI-1640 medium, MTT and DMSO (sigma co., St. Louis, USA), Fetal
Bovine serum (GIBCO, UK).
MTT assay
The cell lines mentioned above were used to determine the inhibitory effects of com-
pounds on cell growth using the MTT assay. This colorimetric assay is based on the
conversion of the yellow tetrazolium bromide (MTT) to a purple formazan derivative
by mitochondrial succinate dehydrogenase in viable cells. Cell lines were cultured in
RPMI-1640 medium with 10% fetal bovine serum. Antibiotics added were 100 units/ml
penicillin and 100 mg/ml streptomycin at 37 ꢁC in a 5% CO2 incubator. The cell lines
were seeded in a 96-well plate at a density of 1.0 ꢂ 104 cells/well. At 37 oC for 48 h
under 5% CO2. After incubation the cells were treated with different concentration of
compounds and incubated for 24 h. After 24 h of drug treatment, 20 ml of MTT solution
at 5 mg/mL was added and incubated for 4 h. Dimethyl sulfoxide (DMSO) in volume of
100 ml is added into each well to dissolve the purple formazan formed. The colorimetric
assay is measured and recorded at absorbance of 570 nm using a plate reader (EXL 800,
USA). The relative cell viability in percentage was calculated as (A570 of treated sam-
ples/A570 of untreated sample) ꢂ 100.[37–40]

14
E. A. AHMED ET AL.
EGFR inhibitory assay
A cell-free assay was used to investigate the mechanism of inhibition of EGFR kinase
according to the reported method.[41] Kit used for immune-assay was cloud clone
SEA757Hu 96 Tests. 200 lM (EGFR) was used. From the following equation: E(%) ¼
Emax/(1 þ [I]/ID50), where E (%) is the fraction of the enzyme activity measured in the
presence of the inhibitor, Emax is the activity in the absence of the inhibitor, [I] is the
inhibitor concentration and ID50 is the inhibitor concentration at which E(%) ¼ 0.5
Emax, a dose–response curve was generated. Mean values of two independent replicates
for each experiment were used for the interpolation.
Docking methodology
Discovery Studio 2.5 software (Accelrys Inc., San Diego, CA, USA) was used for dock-
ing analysis. Fully automated docking tool using “Dock ligands (CDOCKER)” protocol
R
running on IntelV core (TM) i32370 CPU @ 2.4 GHz 2.4 GHz, RAM Memory 2 GB
under the Windows 7.0 system. The crystal structure of EGFR (PDB ID: 1M17) was
downloaded from protein data bank.[42] The docked compounds were built using
Chem. 3 D ultra 12.0 software [Chemical Structure Drawing Standard; Cambridge Soft
corporation, USA (2010)], and copied to Discovery Studio 2.5 software. Automatic pro-
tein preparation module was used applying MMFF94 force field. The binding site
sphere has been defined automatically by the software. Now the above prepared recep-
tor is given as input for “input receptor molecule” parameter in the CDOCKER proto-
col parameter explorer. Force fields are applied on compounds 3, 11 and 17 to get the
minimum lowest energy structure. The obtained poses were studied and the poses
showing best ligand–EGFR interactions were selected and used for CDOCKER energy
(protein-ligand interaction energies) calculations. Receptor–ligand interactions of the
complexes were examined in 2 D and 3 D styles.
Acknowledgements
We are thankful to Sohag University and Faculty of Science in Egypt for the support of
this work.
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