Synthesis, biological evaluation and molecular docking of novel indole-aminoquinazoline hybrids for anticancer properties

Article abstract of DOI:10.3390/ijms19082232

A series of indole-aminoquinazolines was prepared via amination of the 2-aryl-4-chloroquinazolines with the 7-amino-2-aryl-5-bromoindoles. It was then evaluated for cytotoxicity in vitro against human lung cancer (A549), epithelial colorectal adenocarcinoma (Caco-2), hepatocellular carcinoma (C3A), breast adenocarcinoma (MCF-7), and cervical cancer (HeLa) cells. A combination on the quinazoline and indole moieties of a 2-phenyl and 2-(4-fluorophenyl) rings in compound 4b; 2-(4-fluorophenyl) and 3-chlorophenyl rings in compound 4f; or the two 2-(4-fluorophenyl) rings in compound 4g, resulted in significant and moderate activity against the Caco-2 and C3A cell lines. The indole-aminoquinazoline hybrids compounds 4f and 4g induced apoptosis in Caco-2 and C3A cells, and were also found to exhibit moderate (IC₅₀ = 52.5 nM) and significant (IC₅₀ = 40.7 nM) inhibitory activity towards epidermal growth factor receptor (EGFR) against gefitinib (IC₅₀ = 38.9 nM). Molecular docking suggests that 4a–h could bind to the ATP region of EGFR like erlotinib.

Full text of DOI:10.3390/ijms19082232

International Journal of
Molecular Sciences
Article
Synthesis, Biological Evaluation and Molecular
Docking of Novel Indole-Aminoquinazoline Hybrids
for Anticancer Properties
Malose J. Mphahlele 1,
*
ID , Mmakwena M. Mmonwa 1, Abimbola Aro 2, Lyndy J. McGaw 2 and
Yee Siew Choong 3
ID
1
Department of Chemistry, University of South Africa, Private Bag X06, Florida 1710, South Africa;
mmonwmm@unisa.ac.za
Phytomedicine Programme, Department of Paraclinical Sciences, University of Pretoria, Private Bag X04,
Onderstepoort 0110, South Africa; titotani2014@gmail.com (A.A.); lyndy.mcgaw@up.ac.za (L.J.M.)
Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 Minden, Penang
11800, Malaysia; yeesiew@usm.my
2
3
*
Correspondence: mphahmj@unisa.ac.za; Tel.: +27-11-670-9301
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 2 July 2018; Accepted: 20 July 2018; Published: 31 July 2018
Abstract:
A series of indole-aminoquinazolines was prepared via amination of the
2-aryl-4-chloroquinazolines with the 7-amino-2-aryl-5-bromoindoles. It was then evaluated for
cytotoxicity in vitro against human lung cancer (A549), epithelial colorectal adenocarcinoma (Caco-2),
hepatocellular carcinoma (C3A), breast adenocarcinoma (MCF-7), and cervical cancer (HeLa)
cells. A combination on the quinazoline and indole moieties of a 2-phenyl and 2-(4-fluorophenyl)
rings in compound 4b; 2-(4-fluorophenyl) and 3-chlorophenyl rings in compound 4f; or the two
2-(4-fluorophenyl) rings in compound 4g, resulted in significant and moderate activity against the
Caco-2 and C3A cell lines. The indole-aminoquinazoline hybrids compounds 4f and 4g induced
apoptosis in Caco-2 and C3A cells, and were also found to exhibit moderate (IC50 = 52.5 nM) and
significant (IC50 = 40.7 nM) inhibitory activity towards epidermal growth factor receptor (EGFR)
against gefitinib (IC50 = 38.9 nM). Molecular docking suggests that 4a–h could bind to the ATP region
of EGFR like erlotinib.
Keywords: indole-aminoquinazolines; cytotoxicity; apoptosis; EGFR-TK; molecular docking
1. Introduction
Nitrogen-containing heterocycles, such as quinazolines and indoles, have earned great interest
in targeted therapies as antitumor drugs. The 4-anilinoquinazolines, such as the clinical drug
gefitinib shown in Figure 1A, for example, are known to produce their anticancer activity through
inhibition of the epidermal growth factor receptor tyrosine kinase (EGFR-TK) phosphorylation—which
results from competitive binding at the ATP site [
is prevalent in many heterocycles with antimicrobial, antioxidant, anticancer and anti-tubercular
activities [ ]. A lipophilic bromine atom on the fused benzo ring of an indole framework
has been found to impart significant anti-tumour activity in both synthetic [ ] and naturally [
1–3]. The indole nucleus, on the other hand,
4–7
8
9]
occurring indoles, such as aplicyanin A (Figure 1B), shown in Figure 1. The latter exhibits increased
antiproliferative activity against the human breast cancer (MDA-MB-231), lung cancer (A549) and
colon cancer (HT-29) cell lines [9]. 4-(1-Benzyl-1H-indol-3-yl)-6,7-dimethoxyquinazoline (Figure 1C) is
an example of an indole-quinazoline hybrid that was previously found to exhibit moderate ErbB-2
activity, with little or no activity against the epidermal growth factor receptor [10]. The analogous
4-(indole-3-yl)quinazolines were found to be highly potent EGFR-TK inhibitors with excellent
Int. J. Mol. Sci. 2018, 19, 2232; doi:10.3390/ijms19082232
www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2018, 19, 2232
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cytotoxic properties against several cancer cell lines [11]. Significant anti-inflammatory properties
and dose-dependent lipopolysaccharide-induced TNF-
α and IL-6 release was observed for the
N4-(1H-indol-5-yl)quinazoline-4,6-diamines (Figure 1D) [12]. Cediranib (AZD2171) shown in Figure 1E,
on the other hand, is an example of an N-unsubstituted indole-ether quinazoline hybrid that has been
found to be a highly potent, orally bioavailable and selective vascular endothelial growth factor
receptor (VEGFR) inhibitor for the treatment of cancer [13
on the antiproliferative properties of the 2-arylindoles [15] and the 4-anilinoquinazolines [16
,
14]. We considered our previous work
17] in
,
combination with the literature analyses on bioactive compounds containing these moieties and
decided to merge the two pharmacophores to comprise the indole-aminoquinazoline analogues of
cediranib (Figure 1E).
F
H
HN
HN
N
N
HN
N
Cl
O
O
O
N
N
H3CO
H3CO
Br
N
N
H3C
N
H
(A)
(B)
(C)
H
N
R (propyl, allyl)
N
CH3
O
N
HN
N
H3CO
O
F
N
H2N
N
N
(D)
(E)
) and indole-appended quinazoline hybrids (C–E)
Figure 1. Structure of gefitinib (
A
), aplicyanin A (
B
of biological importance.
Since most of the primary 4-aminoquinazolines only differ in terms of the substituents and
side chains, we decided to replace the aniline group of the 2-arylquinazolines with a 2-arylindole
moiety to comprise the indole-appended 4-aminoquinazoline analogous of cediranib (Figure 1E).
The main aims were to evaluate the resultant hybrids for antigrowth effect against a panel of cancer
cell lines. These include; human lung cancer (A549), epithelial colorectal adenocarcinoma (Caco-2),
hepatocellular carcinoma (C3A; HepG2/C3A), breast adenocarcinoma (MCF-7), and cervical (HeLa)
cancer cell lines. Since cytotoxicity does not define the mode of cancer cell death, we evaluated the
most cytotoxic compounds for their ability to induce apoptosis. Moreover, the most active compounds
were evaluated for their capability to inhibit EGFR-TK phosphorylation complemented with molecular
docking (in silico) into the ATP binding site of EGFR.
2. Results and Discussion
2.1. Chemistry
The synthesis of the requisite indole-aminoquinazolines involved the amination of the
2-aryl-4-chloroquinaolines with the known 7-acetamido-2-aryl-5-bromoindoles, compounds 1a–d [18]
as represented in Scheme 1 and Table 1. The 1H- and 13C-NMR spectra of compounds 2a–d and 4a–h
are included as Figure S1 in the Supplementary Materials. In order to prepare the 7-amino-2-aryl-5-
bromoindoles 2a
3a or 3b, we subjected the acetamido derivatives 1a
–
d
to serve as substrates for the amination of the 4-chloroquinazolines, compounds
to hydrolysis with thionyl chloride in
–
d

Int. J. Mol. Sci. 2018, 19, 2232
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methanol under reflux for 1 h. We isolated by aqueous work-up and column chromatography
on silica gel products, characterised by means of nuclear magnetic resonance (1H-NMR and
13C-NMR) and infrared (IR) spectroscopic techniques, complemented with mass spectrometry as
the requisite 7-amino-2-aryl-5-bromoindoles, 2a–d. These compounds were easily distinguished from
the corresponding precursors by the absence of signals corresponding to the acetyl group in their 1H-
and 13C-NMR spectra. Previously, the analogous 2,5- and 3,5-disubstituted 7-aminoindoles have been
prepared via a three component Wittig reaction of pyrrole-3-carboxaldehydes with fumaronitrile and
PEt3, followed by chemoselective C-6 alkylation of the incipient cis-allylic nitriles, and subsequent
cyclization through an intramolecular Houben Hoesch reaction [19]. Cacchi et al. [20] have also
−
developed an alternative method for the synthesis of the 7-aminoindole derivatives, which involves
the Buchwald-Hartwig C–N bond formation of the intermediate 7-bromoindoles with primary and
secondary amines. The 2-aryl-4-chloroquinazolines compounds, 3a and 3b, used as coupling partners,
were prepared by treatment of the known 2-arylquinazolin-4(3H)-ones with phosphoryl chloride under
reflux, following the literature procedure [21]. Amination of the electrophilic 4-chloroquinazoline
derivatives, 3a (X = H) and 3b (X = 4-F), with 7-aminoindoles, 2a
in tetrahydrofuran-isopropanol (THF-iPrOH) mixture under reflux afforded the corresponding
indole-aminoquinazoline hybrids, 4a
. Their 1H-NMR spectra revealed the presence of increased
signals in the aromatic region and two singlets integrating for single proton each around 11.4 and
12.0 ppm, which correspond to the endocyclic nitrogen and the nitrogen bridge (4-amino group),
–d, in the presence of 5% HCl
–
h
δ
respectively. The calculated m/z values for the molecular hybrids, 4a
–h, were found to be consistent
with the molecular ions of the assigned structures.
Cl
Br
H
N
N
R1
O
(3a, b)
Br
Br
NH2
R2
N
H
N
SOCl2, MeOH,
reflux, 1 h
CH3
NH
NH
R1
NH
R1
5% HCl, THF-iPrOH, reflux, 2h
N
N
R2
1a–d
2a–d
3a (X = H)
3b (X = 4-F)
4a–d (X = H)
4e–h (X = 4-F)
Scheme 1. Synthesis of indole-aminoquinazolines compounds 4a–h.
Table 1. Designation of R1 and R2 for products 4a–h.
Compound
R1
R2
4a
4b
4c
4d
4e
4f
C6H5-
4-FC6H4-
3-ClC6H4-
4-CH3OC6H4-
C6H5-
4-FC6H4-
3-ClC6H4-
4-CH3OC6H4-
C6H5-
C6H5-
C6H5-
C6H5-
4-FC6H4-
4-FC6H4-
4-FC6H4-
4-FC6H4-
4g
4h
As an introduction to indole-appended 4-aminoquinazoline hybrids with potential anticancer
activity we decided to evaluate compounds 4a for in vitro antigrowth effect against: Human
–
h
lung cancer (A549), epithelial colorectal adenocarcinoma (Caco-2), hepatocellular carcinoma
(C3A; HepG2/C3A), breast adenocarcinoma (MCF-7), and cervical (HeLa) cell lines. The structure
activity relationship (SAR) of these compounds has been rationalised with respect to the substitution
patterns on the phenyl rings of the quinazoline and indole components.

Int. J. Mol. Sci. 2018, 19, 2232
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2.2. Biological Evaluation
2.2.1. In Vitro Cytotoxicity of Indole-Aminoquinazoline Hybrids 4a–h
Compounds 4a–h were evaluated for potential antiproliferative effect against the A549,
Caco-2 and C3A (HepG2/C3A), MCF-7, and HeLa cell lines using the well-established
3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide based colorimetric cell viability (MTT)
assay. The compounds were assayed as dimethyl sulfoxide (DMSO) solutions at the following
concentrations: 5, 12.5, 25, 50, 100
as a negative control of choice since it has been found before to have no apparent effect on proliferation
of the HeLa, Caco-2, and C3A cells at 1% or less for up to 48 h [22 23]. Gefitinib used as a positive control,
on the other hand, has previously been found to induce autophagy in lung cancer A549 cells and to
enhance apoptosis and suppress cancer cell proliferation [ ]. Moreover, the Caco-2 cells have been found
to display greater sensitivity to gefitinib due to their high EGFR expression [24]. The cytotoxic activities
of the tested compounds were expressed as LC50 values (the dose that reduces survival to 50%) in
µM, against gefitinib as a reference standard. DMSO was used
,
2
µM
concentrations, using DMSO as the negative control and gefitinib as a positive control. The LC50 values
of the tested compounds (average from three independent experiments) against gefitinib as reference
drug are represented in Table 2. Gefitinib resulted in different degree of cell growth inhibition for the
A549 (modest), Caco-2 (intermediate), C3A (highly sensitive), MCF-7 (moderate) cell lines, with no
inhibition of cell growth for the HeLa cells. Within the series 4a–d, compound 4a substituted with
a phenyl ring on both the quinazoline and indole moiety was found to be inactive against all the cancer
cell lines tested. The presence of a phenyl group on the quinazoline ring and a small lipophilic fluorine
atom at the 4-position of the phenyl ring of the indole moiety in 4b resulted in significant cytotoxicity
against all the five cancer cell lines. Compound 4c substituted with a chlorine atom at the 3-position
of the phenyl ring of the indole moiety and a phenyl ring at position-2 of the quinazoline scaffold
was found to exhibit significant cytotoxicity against A549 and Caco-2 when compared to gefitinib.
Modest cytotoxicity was observed for this compound against the C3A, MCF-7, and HeLa cell lines.
The presence of a bulky 4-methoxyphenyl ring at the C-2 position of the indole framework of 4d resulted
in diminished activity against all the cancer cell lines tested. Compound 4e substituted with a 2-phenyl
ring on the indole moiety and a 4-fluorophenyl group at position-2 of the quinazoline framework
was found to be inactive against the Caco-2, C3A, MCF-7 and HeLa cells, but to exhibit cytotoxicity
against the A549 cells (LC50 = 51.64 µM) comparable to the reference standard (LC50 = 51.29 µM).
The presence of the 4-fluorophenyl group at the C-2 positions of both quinazoline and indole moieties
in compound 4f, on the other hand, also resulted in significant cytotoxicity against the four cancer cell
lines. The observed cytotoxicity of 4b and 4f is presumably due to the increased lipophilicity of the
molecule resulting from the non-polarizability of the Csp2-F bond [25]. Moreover, fluorine substitution is
known to improve the overall pharmacokinetics and pharmacodynamics of the molecule by improving
solubility, selectivity, bioavailability and metabolic stability [26]. Within the series 4e–h, compound
4g substituted with a 3-chlorophenyl group on the indole moiety and a 4-fluorophenyl group at the
2-position of the quinazoline moiety was found to be more cytotoxic against the Caco-2 (LC50 = 6.46 µM)
cell line when compared to gefitinib (LC50 = 27.91 µM). It has previously been observed for the
analogous halogenated 4-anilinoquinazolines, which were identified as non-competitive antagonists of
metabotropic glutamate receptor 5 (mGlu5) that a small lipophilic group (X = F, Cl, Br) at the 3-position
of the aniline ring resulted in up to 10-fold increase in potency [27]. Compound 4g was also found to
exhibit moderate cytotoxicity against the C3A (LC50 = 12.20 µM), MCF-7 (LC50 = 39.07
µM) and HeLa
(LC50 = 25.51 µM) cells. A combination of the bulky 4-methoxyphenyl group on the indole framework
with a 4-fluorophenyl group on the quinazoline component in 4h resulted in moderate cytotoxicity
against the A549 and Caco-2 cell lines and reduced activity against the C3A cell line. Significant
cytotoxicity for compounds 4b, 4f and 4g and moderate activity observed for 4c against the CaCo-2 and
C3A cells may suggest the presence of a halogen atom on the phenyl ring of the indole moiety to be
essential for the antiproliferative properties of these indole-aminoquinazolines.

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Table 2. Cytotoxic effects of 4a–h against the A549, Caco-2, C3A, MCF-7, and HeLa cell lines.
Br
Br
N
H
R
N
H
R
NH
NH
N
N
N
N
F
4a–d
4e–h
Cancer Cells, LC50 (µM)
Ligand
A549
Caco-2
C3A
MCF-7
HeLa
4a
4b
4c
4d
4e
4f
4g
60.99 ± 0.14
32.04 ± 0.17
36.97 ± 0.31
>100
51.64 ± 0.16
17.66 ± 0.08
86.21 ±0.14
57.51 ± 0.21
51.29 ± 0.17
41.55 ± 0.01
12.67 ± 0.00
27.32 ± 0.01
52.65 ± 0.00
37.77 ± 0.10
9.16 ± 0.01
6.45 ± 0.01
38.76 ± 0.00
27.91 ± 0.00
30.41 ± 0.02
17.65 ± 0.1
30.57 ± 0.02
59.97 ± 0.05
38.75 ± 0.05
19.92 ± 0.10
12.20 ± 0.03
32.77 ± 0.07
5.01 ± 0.04
45.99 ± 0.08
30.11 ± 1.21
41.22 ± 0.09
69.11 ± 0.12
56.67 ± 0.45
26.96 ± 2.30
39.07 ± 1.56
61.03 ± 0.39
30.74 ± 0.80
32.23 ± 1.23
24.70 ± 1.31
40.02 ± 3.23
61.23 ± 6.37
43.37 ± 2.05
34.05 ± 1.28
25.51 ± 2.94
49.13 ± 1.10
98.80 ± 0.56
4h
Gefitinib
Gefitinib has been found to inhibit tumour pathogenesis, metastasis and angiogenesis, and it
also promotes apoptosis [ ]. Since cytotoxicity does not define a specific cellular death mechanism
(apoptosis vs. necrosis) and considering the pro-apoptotic properties of the 4-anilinoquinazolines [ 28
induce cell death in these
3
3
,
]
and indole-3-carbinol [29
cancer cells.
,30], we decided to evaluate how compounds 4a–h
2.2.2. Evaluation of Cell Death Pathways
Apoptosis is one of essential mechanisms through which the body is able to get rid of unwanted
cells, and triggering this process in cancer cells would lead to automatic death and decrease cancer
proliferation. Necrosis, on the other hand, refers to changes following cell death by any mechanism
including apoptosis, and it does not necessarily tell how cell death occurred because it takes place
after the cells have already died, and reached equilibrium with their surroundings [31,32]. We selected
compound 4g to evaluate whether it induces apoptosis in the Caco-2 and C3A cell lines by means of
flow cytometry and caspase-3 activation assay. We employed the Annexin-V-Fluorescein and Proidium
iodid (PI) apoptosis assay, which is a popular method to distinguish between healthy cells (annexin
V-negative; PI-negative), early apoptotic cells (annexin V-positive; PI-negative), late apoptotic cells
(annexin V-positive; PI-positive), and necrotic cells (annexin V-negative; PI-positive). The C3A and
Caco-2 cell lines were treated with different concentrations of compound 4g (5 and 12.5 µM) for 24 h
and then stained with propidium iodide (PI) and annexin V binding buffer. The percentage of cell
populations, namely, necrotic cell (Q1), late apoptotic cells (Q2), healthy cells (Q3), and early apoptotic
cells (Q4) are represented in Figure 2. Both necrotic (Q1) and apoptotic cells (Q2) increased with the
increasing concentration, but the population of necrotic cells remained smaller at both concentrations
tested. The population of necrotic (Q1) and apoptotic cells (Q2) in the case of the Caco-2 cells is almost
equal at the highest concentration tested. Both necrotic and apoptotic cells in the case of C3A increased
with increasing concentration of 4g, but the population of necrotic cells remained higher. This is
presumably because necrosis takes place after the cells have already died and reached equilibrium
with their surroundings [31,32].

Int. J. Mol. Sci. 2018, 19, 2232
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(2a) Compound 4g against Caco-2 cell line.
(2b) Compound 4g against C3A cell line.
Figure 2. Effects of compound 4g on the induction of apoptosis in the Caco-2 (2a) and C3A cells (2b) at
5 and 12.5 M after 24 h against doxorubicin hydrochloride as determined by Annexin V/PI staining.
µ
The molecular definition of apoptosis is based on the proteolytic activity of certain caspases
(caspace-2, -3, -6, -7, -8, -9, and -10), and these enzymes are directly involved in the mediation of
apoptotic cell death [32]. Activation of caspase-3, for example, is critically important for the induction
of apoptotic pathways [33–35]. As a result, we evaluated compound EGFR for caspase-3 activation in
Caco-2 and C3A cells against gefitinib as a reference standard (Figure 3). The observed results indicate
that compound EGFR induces apoptosis in Caco-2 and C3A cells through activation of caspase that
subsequently leads to cell membrane alterations observed using the staining technique above.
Caspase-3 activity in Caco-2 cells
120
Caspase-3 activity in C3A cells
20
15
10
5
100
80
60
40
20
0
0
Geft: 12.5 25 4g: 5 12.5 25
µM µM
Geft: 12.5 25 4g: 5 12.5 25
5
5
µM
µM
Figure 3. Effects of compounds EGFR on Caspace-3 activity in Caco-2 and C3A cells compared
to gefitinib.

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Gefitinib is a selective inhibitor of the EGFR-TK [
3
] and an increased expression of this receptor
is the hallmark of ovarian cancer, breast cancer, squamous cell carcinoma of the head and neck,
and prostate cancer [36]. Inhibition of EGFR-TK activity is regarded as the most promising approach
for innovative therapeutic strategies in cancer treatment. Human lung cancer (A549), epithelial
colorectal adenocarcinoma (Caco-2) and hepatocellular carcinoma (C3A) cells have been found to
express high levels of EGFR and to be sensitive to gefitinib [25,37,38]. Breast adenocarcinoma (MCF-7)
and cervical cancer (HeLa), on the other hand, have moderate [39] and low EGFR expression [40],
respectively. In order to test whether the indole-aminoquinazolines prepared in this investigation
could inhibit the ligand binding-induced receptor phosphorylation, we performed kinase activity of
EGFR in the presence of representative compounds 4f and 4g.
2.2.3. Evaluation of Compound 4g for Potential to Inhibit EGFR-TK Phosphorylation
The inhibitory activities of compounds 4f and 4g to EGFR were tested by ELISA method
against gefitinib as a reference standard. As shown in Figure 4, compounds 4f and 4g showed
moderate (LC50 = 52.5 nM) and significant (LC50 = 40.7 nM) EGFR inhibitory activity, respectively,
when compared to gefitinib (LC50 = 38.9 nM).
Inhibition curves
100
Compound
IC50 (nM)
52.5 ± 1.17
40.7 ± 0.31
38.9 ± 0.89
80
60
40
20
0
4f
4g
Gefitinib
4f
4g
Gefitinib
0
0.1
0.2
0.3
0.4
0.5
Concentration (µM)
Figure 4. %Inhibition and IC50 values (in nM) of epidermal growth factor receptor tyrosine kinase
(EGFR-TK) by 4f and 4g against gefitinib.
We rationalised the difference in cytotoxicity and EGFR-TK phosphorylation inhibitory activity
related to the substitution pattern on the indole arm to be probably associated with the flexibility
of the amino-indole moiety, where a distinct spatial arrangement of the molecule could lead to an
improved interaction with the active site of the receptor. To try to prove this assumption, we performed
molecular docking of compounds 4a–h into EGFR-TK active site to compare the binding conformation
with gefitinib and erlotinib.
2.2.4. Molecular Docking Studies of Compounds 4a–h into the ATP Binding Site of EGFR
A crystal structure of EGRF that previously co-crystalised with erlotinib was obtained from the
protein data bank (PDB ID: 1M17). The control (gefitinib) docked on erlotinib binding site produced
root mean square deviation (RMSD) of 1.6 Å with the crystal structure (Table 3). Comparison of the
calculated binding free energy reveals that gefitinib is more favourable for the inhibition of EGFR
compared with erlotinib. The gefitinib morpholine region was positioned as anti-parallel with the
benzene ring of Phe699, but not for erlotinib. Besides, the 3-chloro-4-fluorophenyl moiety of gefitinib
is docked deeper into the binding pocket compared to erlotinib. The N-morpholine region of gefitinib
is also extended outward compared with erlotinib. The binding mode of gefitinib could be the reason
for a better binding affinity compared with erlotinib and hence inherent selectivity for the EGFR.

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Compound 4e showed the highest binding affinity among the hybrids with binding free energy of
−11.46 kcal/mol.
Table 3. Calculated binding free energy and estimated inhibition constant (Ki) of erlotinib (as control),
gefitinib and quinazoline-indole hybrids 4a–h with epidermal growth factor receptor (EGFR)
kinase domain.
Ligand
Hb Interacting Atoms
FEB (kcal/mol)
Ic, Ki (nM)
Hb Distance (Å)
Control (erlotinib)
Gefitinib
2.2
-
Met769 O–Erl HN2
-
−8.71
414.12
13.34
−10.74
2.9
2.0
2.1
-
-
3.0
-
Thr766 OG1–4a HN2
Asp831 OD1–4a HN
Met769 O–4b HN
4a
−11.03
8.22
4b
4c
4d
4e
4f
4g
4h
−9.95
−10.10
−10.31
−11.46
−9.67
50.7
39.74
27.74
4.01
81.76
28.38
25.61
-
-
Asp831 OD1–4e HN
-
-
-
-
-
−10.30
−10.36
Hb: Hydrogen bond; FEB: Free energy of binding; Ic: Estimated inhibition constant.
The binding modes of the indole-aminoquinazoline hybrids 4b and 4f are shown in Figure 5
(refer to Figure S2, Supplementary Data for the superimposed docking poses of 4a ). The compounds
4b and 4f) bind on the same binding site with erlotinib than the EGFR selective gefitinib when
–h
(
compared with other compounds, i.e., 4a and 4e. The quinazoline region and the benzene were
positioned same with the ether tails of erlotinib. Previous studies on the 4-anilinoquinazolines have
shown that the 4-anilino group can fit deep into the hydrophobic pocket of EGFR [41], and that the
introduction of electron-donating groups on the phenyl ring increase the electron density on the
quinazoline N1 thereby enhancing the interaction with EGFR [42].
Figure 5. Docked conformation of erlotinib (as control), gefitinib, indole-aminoquinazolines 4b
and 4f (stick representation) in the binding pocket of the EGFR kinase domain (surface and ribbon
representation). Blue dotted lines indicate the direct hydrogen bonding formed between the docked
ligand and EGFR. The docked conformation of other indole-aminoquinazolines can be found in
Supplementary data Figure S2.

Int. J. Mol. Sci. 2018, 19, 2232
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Compound 4b substituted with a phenyl group on the quinazoline ring and a small lipophilic
moderately pi-electron donating fluorine atom at the 4-position of the phenyl ring of the indole.
This showed moderate to significant cytotoxicity against the five cell lines tested was docked deeper
into the binding pocket compared with the other derivatives. Direct hydrogen bond formation was only
observed between EGFR, with erlotinib and quinazoline-indole hybrids 4a, 4b and 4e. The observed
hydrogen bonds in the indole-aminoquinazoline hybrids demonstrate the importance of the amine
moiety in this interaction. Compounds 4a and 4e adopted similar poses where the aryl substituent
of the quinazoline moiety extended towards the entrance of the binding site and formed T-shape
π
-stacking interaction with Phe699. Compounds 4c
conformation with their aryl/fluorophenyl group extended from quinazoline region forming parallel
displaced -stacking interactions with Phe699. The calculated binding free energies for gefitinib
10.74 kcal/mol), 4f 9.67 kcal/mol), and 4g 10.30 kcal/mol) are consistent with their inhibitory
, d, and f–h, on the other hand, have similar binding
π
(
−
(
−
(
−
effects against EGFR with IC50 values of 38.9, 52.5 and 40.7 nM, respectively. The calculated binding
free energies show that the compounds have high affinity for the EGFR than erlotinib, but relatively
less than that of most selective EGFR inhibitor, gefitinib. Besides, the calculated Ki value of the control
erlotinib that is 30
×
higher than that of gefitinib is also evidence for the EGFR selectivity for gefitinib.
3. Materials and Methods
3.1. General
The melting points of the compounds prepared in this investigation were recorded on
a Thermocouple digital melting point apparatus (Mettler Toledo LLC, Columbus, OH, USA). Their IR
spectra were recorded as powders using a Bruker VERTEX 70 FT-IR Spectrometer (Bruker Optics,
Billerica, MA, USA) equipped with a diamond attenuated total reflectance (ATR) accessory. For column
chromatography, we used the Merck kieselgel 60 (0.063–0.200 mm) (Merck KGaA, Frankfurt, Germany)
as stationary phase. The NMR spectra were obtained as DMSO-d6 solutions using Varian Mercury
300 MHz NMR spectrometer (Varian Inc., Palo Alto, CA, USA) and the chemical shifts are quoted
relative to the residual proton signal in the deuterated solvent. The mass spectra were recorded at an
ionization potential of 70 eV using Waters Synapt G2 Quadrupole Time-of-flight mass spectrometer
(Waters Corp., Milford, MA, USA) at the University of Stellenbosch. The synthesis and analytical data
of compounds 1a–d, which are known have been described before [18].
3.2. Typical Procedure for the Preparation of the 2-Aryl-4-chloroquinazolines (3a and 3b)
2-Arylquinazolin-4(3H)-one (2.00 g) was added in portions to a stirred SOCl2 (10 mL), followed by
◦
DMF (1 mL). the mixture was then refluxed for 2 h at 80 C. The reaction mixture was cooled and then
quenched slowly with saturated aqueous solution of K2CO3. The resultant precipitate was filtered and
recrystallised from acetonitrile to give pure 2-aryl-4-chloroquinazoline as solid. Compounds 3a and 3d
described below were prepared in this fashion.
3.2.1. 4-Chloro-2-phenylquinazoline (3a)
◦
◦
Solid (1.90 g, 86%), mp. 124
−
126 C (Lit. [21] 123
−
125 C); 1H-NMR (DMSO-d6) 7.56 (4H, m, ArH),
7.90 (1H, t, J = 6.9 Hz, ArH), 8.04 (4H, m, ArH).
3.2.2. 4-Chloro-2-(4-fluorophenyl)quinazoline (3b)
◦
Solid (1.81 g, 84%), Rf (60% EtOAc–hexane) = 0.81, mp. 139
−
141 C;
ν
(ATR) 1602 (C=N),
max
1645 (C=N) cm−1; H-NMR (DMSO-d6) 7.36 (2H, t, J = 8.7 Hz, ArH), 7.49 (1H, t, J = 6.9 Hz, ArH),
7.71 (1H, d, J = 8.1 Hz, ArH), 7.80 (1H, t, J = 6.9 Hz, ArH), 8.11 (1H, d, J = 8.1 Hz, ArH), 8.19 (2H, dd,
J = 5.7 and 8.7 Hz, ArH); 13C-NMR (DMSO-d6) 116.1 (d, 2JCF = 21.8 Hz), 121.2, 126.3, 127.2, 127.3, 129.2
(d, 4JCF = 2.3 Hz), 131.0 (d, 3JCF = 9.1 Hz), 135.2, 148.3, 152.2, 162.5, 164.4 (d, 1JCF = 248.4 Hz); HRMS
(ES): Found: 259.0432. C14H9N2ClF+ requires 259.0438.
1

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3.3. Typical Procedure for the Hydrolysis of the 7-Acetamide Derivatives 1a–d
A mixture of 1 (1 equivalent) and SOCl2 (4 equivalent) in methanol (15 mL/mmol of 1) was
stirred under reflux for 1 h. The reaction mixture was cooled to room temperature (RT) and quenched
with a saturated solution of K2CO3. The product was extracted with CHCl3 (3
×
20 mL), and then
combined organic layers were dried over anhydrous MgSO4. The salt was filtered off and the solvent
was evaporated under reduced pressure on a rotary evaporator. The residue was purified by column
chromatography on silica gel using 60% EtOAc-hexane mixture as an eluent to afford 2 as a solid.
The following compounds were prepared in this fashion.
3.3.1. 5-Bromo-2-phenyl-1H-indol-7-amine (2a)
Solid (0.13 g, 78%), Rf = 0.74, mp. 222-223 ◦C;
ν
(ATR) 1578 (C=N), 3222 (NH), 3294 (NH),
max
3367 (NH) cm−1; 1H-NMR (DMSO-d6) 5.48 (2H, br s, NH2), 6.46 (1H, s, 3-H), 6.74 (1H, d, J = 1.2 Hz,
6-H), 6.91 (1H, d, J0=01.2 Hz, 4-H), 7.31 (1H, t, J = 7.5 Hz, 40-H), 7.47 (2H, t, J = 7.5 Hz, 30,50-H), 7.80
(2H, d, J = 7.5 Hz, 2 ,6 -H), 11.10 (1H, s, NH); 13C-NMR (DMSO-d6) 99.3, 107.6, 110.6, 113.6, 125.2, 125.3,
128.0, 129.4, 130.9, 132.4, 135.9, 137.8; HRMS (ES): Found 287.0187. C14H12N279Br+ requires 287.0184.
Anal calcd for C14H11N2Br: C, 58.56; H, 3.86; N, 9.76. Found: C, 58.49; H, 3.82; N, 9.76.
3.3.2. 5-Bromo-2-(4-fluorophenyl)-1H-indol-7-amine (2b)
Solid (0.14 g, 80%), Rf = 0.76, mp. 260-263 ◦C;
ν
(ATR) 1570 (C=N), 3209 (NH), 3296 (NH),
max
3368 (NH) cm−1; 1H-NMR (DMSO-d6) 6.35 (2H, br s, NH2), 6.60 (1H, s, 3-H), 6.76 (1H, d, J = 1.2 Hz,
6-H), 7.04 (1H, d, J = 1.2 Hz, 4-H), 7.34 (2H, t, J = 8.7 Hz, 30,50-H), 7.89 (2H, d, J = 8.7 Hz, 20,60-H),
11.40 (1H, s, NH); 13C NMR (DMSO-d6) 99.2, 109.2, 112.2, 113.3, 116.4 (d, 2JCF = 21.8 Hz), 126.0, 127.4
(d, 3JCF = 8.0 Hz), 128.9 (d, 4JCF = 2.3 Hz), 131.1, 133.3, 137.3, 162.1 (d, 1JCF = 243.9 Hz); HRMS (ES):
Found 305.0082. C14H11N2F79Br+ requires 305.0090. Anal calcd for C14H10N2FBr: C, 55.10; H, 3.30; N,
9.18. Found: C, 54.97; H, 3.13; N, 8.89.
3.3.3. 5-Bromo-2-(3-chlorophenyl)-1H-indol-7-amine (2c)
Solid (0.13 g, 76%), Rf = 0.82, mp. 271-274 ◦C;
νmax (ATR) 1572 (C=N), 3213 (NH), 3289 (NH), 3356
(NH) cm−1; 1H-NMR (DMSO-d6) 6.36 (2H, br s, NH2), 6.77 (1H, s, 3-H), 6.96 (1H, d, J = 1.2 Hz, 6-H), 7.24
(1H, d, J = 1.2 Hz, 4-H), 7.38-7.41 (1H, m, 40-H), 7.51 (1H, t, J = 8.4 Hz, 50-H), 7.86 (1H, d, J = 8.4 Hz, 60-H),
7.96 (1H, s, 20-H), 11.79 (1H, s, NH); 13C-NMR (DMSO-d6) 100.5, 112.1, 112.9, 115.1, 124.2, 124.9, 127.2,
127.9, 129.4, 131.3, 131.4, 134.1, 134.3, 137.2; HRMS (ES): Found 320.9778. C14H11N235Cl79Br+ requires
320.9794. Anal calcd for C14H10N2ClBr: C, 52.29; H, 3.13; N, 8.71. Found: C, 52.17; H, 2.98; N, 8.56.
3.3.4. 5-Bromo-2-(4-methoxyphenyl)-1H-indol-7-amine (2d)
Solid (0.13 g, 76%), Rf = 0.61, mp. 223-224 ◦C;
ν
(ATR) 1550 (C=N), 1609 (C=N), 3220 (NH),
max
3232 (NH), 3367 (NH) cm−1; 1H-NMR (DMSO-d6) 3.80 (3H, s, OCH3), 5.42 (2H, br s, NH2), 6.43 (1H, s,
3-H), 6.60 (1H, d, J 0= 1.2 Hz, 6-H), 6.87 (1H, d, J = 1.2 Hz, 4-H), 7.04 (2H, d, J = 8.7 Hz, 30,50-H), 7.72
0
(2H, d, J = 8.7 Hz, 2 ,6 -H), 10.97 (1H, s, NH); 13C-NMR (DMSO-d6) 55.7, 98.0, 107.3, 110.3, 113.4, 114.9,
125.0, 125.1, 126.7, 131.1, 135.7, 138.0, 159.3; HRMS (ES): Found: 317.0278. C15H14N279BrO+ requires
317.0290. Anal calcd for C15H13N2FBrO: C, 56.80; H, 4.13; N, 8.83. Found: C, 56.74; H, 4.01; N, 8.86.
3.4. Typical Procedure for the Amination of the Synthesis of 4a–h
A mixture of
2 (1 equiv.), 2-aryl-4-chloroquinazoline (1 equiv.) and HCl (5% mol equiv. of 2) in 3:1
isopropanol-THF (v/v, 30 mL/mmol of 2) was stirred under reflux for 2 h. After completion of the
reaction (tlc monitoring) the solvent was evaporated under reduced pressure and the residue obtained
was recrystallised from chloroform to obtain pure 4. The following compounds were prepared in
this fashion:

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3.4.1. N-(5-Bromo-2-phenyl-1H-indol-7-yl)-2-phenylquinazolin-4-amine (4a)
Solid (0.21 g, 81%), mp. > 300 ◦C;
ν
(ATR) 1540 (C=N), 1604 (C=N), 3052 (NH), 3197 (NH)
max
cm−1; H-NMR (DMSO-d6) 6.99 (1H, s, Ar), 7.25-7.37 (5H, m, Ar), 7.50-7.54 (2H, m, Ar), 7.74-7.79
(3H, m, Ar), 7.86 (1H, t, J = 7.5 Hz, Ar), 8.07-8.14 (3H, m, Ar), 8.48 (1H, d, J = 8.1 Hz, Ar), 9.06 (1H, d,
J = 8.1 Hz, Ar), 11.42 (1H, s, NH), 12.16 (1H, br s, NH); 13C-NMR (DMSO-d6) 99.5, 111.2, 113.8, 121.2,
121.6, 123.1, 125.5, 126.2 (2C), 128.3, 128.5, 129.1 (2C), 129.2 (2C), 129.4, 131.2, 131.8, 132.3, 133.5, 136.0,
140.4, 157.6, 160.0; HRMS (ES): Found 491.0836. C28H20N479Br+ requires 491.0871. Anal calcd for
C28H19N4Br: C, 68.44; H, 3.90; N, 11.40. Found: C, 68.43; H, 3.75; N, 11.23.
1
3.4.2. N-(5-Bromo-2-(4-fluorophenyl)-1H-indol-7-yl)-2-phenylquinazolin-4-amine (4b)
Solid (0.19 g, 79%), mp. > 300 ◦C;
ν
(ATR) 1557 (C=N), 1601 (C=N), 3052 (NH), 3198 (NH)
max
cm−1; H-NMR (DMSO-d6) 6.96 (1H, s, Ar), 7.18 (2H, t, J = 7.5 Hz, Ar), 7.25-7.34 (2H, m, Ar), 7.51
(2H, d, J = 1.5 Hz, Ar), 7.76-7.80 (3H, m, Ar), 7.88 (1H, t, J = 7.5 Hz, Ar), 8.07-8.09 (3H, m, Ar), 8.49
(1H, d, J = 8.1 Hz, Ar), 9.10 (1H, d, J = 8.1 Hz, Ar), 11.44 (1H, s, NH), 12.19 (1H, br s, NH); 13C-NMR
(DMSO-d6) 99.5, 111.2, 113.6, 116.2 (d, 2JCF = 21.8 Hz), 120.8 (d, 4JCF = 3.5 Hz), 121.3, 121.9, 122.8, 125.5,
128.3 (d, 3JCF = 8.0 Hz), 128.6, 129.2 (2C), 129.4, 131.2, 131.6, 132.3, 133.8, 136.4, 139.4, 140.8, 157.5, 160.1,
162.3 (d, 1JCF = 243.9 Hz); HRMS (ES): Found 509.0757. C28H19N4F79Br+ requires 509.0777. Anal calcd
for C28H18N4FBr: C, 66.02; H, 3.56; N, 11.00. Found: C, 66.11; H, 3.52; N, 10.79.
1
3.4.3. N-(5-Bromo-2-(3-chlorophenyl)-1H-indol-7-yl)-2-phenylquinazolin-4-amine (4c)
Solid (0.18 g, 75%), mp. 228-230 ◦C;
ν
max (ATR) 1545 (C=N), 1600 (C=N), 3060 (NH), 3186 (NH)
cm−1; H-NMR (DMSO-d6) 7.11 (1H, s, Ar), 7.30-7.41 (4H, m, 4H), 7.52-7.57 (2H, m, Ar), 7.74-7.77
(1H, m, Ar), 7.80 (1H, t, J = 1.8 Hz, Ar), 7.82 (1H, d, J = 1.5, Ar), 7.88 (1H, t, J = 7.5 Hz, Ar), 8.05-8.08
(2H, m, Ar), 8.14 (1H, t, J = 7.5 Hz, Ar), 8.45 (1H, d, J = 8.1 Hz, Ar), 9.05 (1H, d, J = 8.1 Hz, Ar), 11.46
(1H, s, NH), 12.14 (1H, br s, NH); 13C-NMR (DMSO-d6) 100.2, 111.3, 113.6, 120.7, 121.8, 122.2, 123.0,
124.9, 125.6, 128.2, 128.3, 128.6, 129.1, 129.2, 129.4, 131.0, 131.4, 131.5, 132.1, 133.8, 134.1, 136.4, 138.7,
140.7, 157.5, 160.1; HRMS (ES): Found 525.0495. C28H19N435Cl79Br+ requires 525.0482. Anal calcd for
C28H18N4ClBr: C, 63.96; H, 3.45; N, 10.66. Found: C, 63.93; H, 3.46; N, 10.59.
1
3.4.4. N-(5-Bromo-2-(4-methoxyphenyl)-1H-indol-7-yl)-2-phenylquinazolin-4-amine (4d)
Solid (0.21 g, 83%), mp. 261-264 ◦C;
νmax (ATR) 1547 (C=N), 1609 (C=N), 3060 (NH), 3134 (NH)
cm−1; 1H-NMR (DMSO-d6) 3.72 (3H, s, CH3), 6.88 (1H, s, Ar), 6.92 (2H, d, J = 8.7 Hz, Ar), 7.36 (2H, t,
J = 7.5 Hz, Ar), 7.47 (1H, d, J = 1.5 Hz, Ar), 7.52-7.57 (1H, m, Ar), 7.70 (2H, d, J = 8.7 Hz, Ar), 7.76 (1H, d,
J = 1.5 Hz, Ar), 7.88 (1H, t, J = 7.5 Hz, Ar), 8.08-8.16 (3H, m, Ar), 8.53 (1H, d, J = 8.1 Hz, Ar), 9.09 (1H, d,
J = 8.1 Hz, Ar), 11.32 (1H, s, NH), 12.21 (1H, br s, NH); 13C-NMR (DMSO-d6) 54.2, 96.9, 109.6, 112.3,
113,2 119.1, 119.5, 120.2, 121.1, 122.9, 124.2, 126.3, 127.2, 127.8, 128.2, 129.7, 130.0, 131.2, 132.6, 135.0,
139.1, 139.2, 155.0, 158.3, 158.7; HRMS (ES): Found 521.0987. C29H22N479BrO+ requires 521.0977. Anal
calcd for C29H21N4BrO: C, 66.80; H, 4.06; N, 3.07. Found: C, 66.87; H, 3.99; N, 3.01.
3.4.5. N-(5-Bromo-2-phenyl-1H-indol-7-yl)-2-(4-fluorophenyl)quinazolin-4-amine (4e)
Solid (0.21 g, 78%), mp. > 300 ◦C;
ν
(ATR) 1545 (C=N), 1603 (C=N), 3065 (NH), 3199 (NH)
max
cm−1; H-NMR (DMSO-d6) 6.99 (1H, s, Ar), 7.18-7.28 (3H, m, Ar), 7.34 (2H, t, J = 7.2 Hz, Ar), 7.51
(1H, d, J = 1.5 Hz, Ar), 7.72-7.88 (4H, m, Ar), 8.09-8.21 (3H, m, Ar), 8.55 (1H, d, J = 8.1 Hz, Ar), 9.11
(1H, d, J = 8.1 Hz, Ar), 11.40 (1H, s, NH), 12.30 (1H, br s, NH); 13C-NMR (DMSO-d6) 99.6, 111.1, 113.7,
1
116.3 (d, 2JCF = 21.8 Hz), 121.3, 121.8, 122.9, 125.5, 126.2 (2
×
C), 128.4 (d, 3JCF = 8.0 Hz), 128.5, 129.2
(2C), 131.1, 131.8, 132.1, 132.2 (d, 4JCF = 5.7 Hz), 132.3, 136.2, 140.4, 156.5, 159.9, 165.4 (d, 1JCF = 250.7 Hz;
HRMS (ES): Found 509.0769. C28H19N4F79Br+ requires 509.0777. Anal calcd for C28H18N4FBr: C, 66.02;
H, 3.56; N, 11.00. Found: C, 59.97; H, 3.46; N, 10.78.

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3.4.6. N-(5-Bromo-2-(4-fluorophenyl)-1H-indol-7-yl)-2-(4-fluorophenyl)quinazolin-4-amine (4f)
Solid (0.20 g, 80%), mp. > 300 ◦C;
ν
(ATR) 1557 (C=N), 1605 (C=N), 3039 (NH), 3259 (NH)
max
cm−1; H-NMR (DMSO-d6) 6.97 (1H, s, Ar), 7.21 (2H, t, J = 8.7 Hz, Ar), 7.24 (2H, t, J = 8.7 Hz, Ar),
7.49 (1H, d, J = 1.5 Hz, Ar), 7.80-7.90 (4H, m, Ar), 8.10-8.15 (3H, m, Ar), 8.47 (1H, d, J = 8.1 Hz, Ar),
9.03 (1H, d, J = 8.1 Hz, Ar), 11.40 (1H, s, NH), 12.13 (1H, br s, NH); 13C-NMR (DMSO-d6) 99.6, 111.2
(2C), 116.2 (d, 2JCF = 21.8 Hz), 116.4 (d, 2JCF = 22.9 Hz), 121.0 (d, 4JCF = 3.5 Hz), 121.3, 121.9, 122.8,
125.5, 128.3 (d, 3JCF = 9.1 Hz), 128.4 (2C), 128.6, 131.2, 132.2 (d, 3JCF = 9.1 Hz), 132.3 (d, 4JCF 4.5 Hz),
136.3, 139.4 (2C), 156.5, 160.0, 162.3 (d, 1JCF = 243.9 Hz), 165.5 (d, 1JCF = 249.6 Hz); HRMS (ES): Found
527.0689. C28H18N4F279Br+ requires 527.0683. Anal calcd for C28H17N4FBr: C, 63.77; H, 3.25; N, 10.62.
Found: C, 63.80; H, 3.22; N, 10.53.
1
3.4.7. N-(5-Bromo-2-(4-chlorophenyl)-1H-indol-7-yl)-2-(4-fluorophenyl)quinazolin-4-amine (4g)
Solid (0.20 g, 78%), mp. 266-269 ◦C;
νmax (ATR) 1557 (C=N), 1605 (C=N), 3038 (NH), 3136 (NH)
cm−1; 1H-NMR (DMSO-d6) 7.09 (1H, s, Ar), 7.28 9 (2H, t, J = 8.7 Hz, Ar), 7.30-7.41 (2H, m, Ar), 7.54
(1H, d, J = 1.5 Hz, Ar), 7.72-7.77 (2H, m, Ar), 7.82 (1H, d, J = 1.5 Hz, Ar), 7.87 (1H, t, J = 7.8 Hz, Ar),
8.10-8.20 (3H, m, Ar), 8.47 (1H, d, J = 8.1 Hz, Ar), 9.05 (1H, d, J = 8.1 Hz, Ar), 11.44 (1H, s, NH), 12.23
(1H, br s, NH); 13C-NMR (DMSO-d6) 100.8, 111.3, 113.6, 116.4 (d, 2JCF = 21.8 Hz), 121.8, 122.1, 123.1,
124.9, 125.5, 125.6, 127.1, 128.0, 128.2, 128.5, 131.0, 131.1, 131.3, 132.0, 132.1 (d, 3JCF = 8.0 Hz), 133.8,
134.1, 136.3 (d, 4JCF = 3.4 Hz), 138.6, 156.6, 160.0, 165.5 (d, 1JCF = 250.7 Hz); HRMS (ES): Found 543.0377.
C28H18N4F35Cl79Br + requires 543.0387. Anal calcd for C28H17N4FClBr: C, 61.84; H, 3.15; N, 10.30.
Found: C, 61.79; H, 3.00; N, 10.12.
3.4.8. N-(5-Bromo-2-(4-methoxyphenyl)-1H-indol-7-yl)-2-(4-fluorophenyl)quinazolin-4-amine (4h)
Solid (0.17 g, 67%), mp. 250-253 ◦C;
νmax (ATR) 1553 (C=N), 1607 (C=N), 3037 (NH), 3187 (NH)
cm−1; 1H-NMR (DMSO-d6) 3.68 (3H, s, CH3), 6.82 (1H, s, Ar), 6.88 (2H, d, J = 8.7 Hz, Ar), 7.20 (2H, t,
J = 8.7 Hz, Ar), 7.41 (1H, d, J = 1.5 Hz, Ar), 7.64 (2H, d, J = 8.1 Hz, Ar), 7.71 (1H, d, J = 1.5 Hz, Ar),
7.82 (1H, t, J = 7.8 Hz, Ar), 8.05 8.14 (3H, m, Ar), 8.40 (1H, d, J = 8.1 Hz, Ar), 8.95 (1H, d, J = 8.1 Hz,
−
Ar), 11.24 (1H, s, NH), 11.97 (1H, br s, NH); 13C-NMR (DMSO-d6) 55.6, 98.3, 111.0, 113.6, 114.6 (2C),
116.3 (d, 2JCF = 21.8 Hz), 120.7, 121.3, 122.0, 124.3, 125.3, 127.7 (2C), 128.4, 131.0, 132.0 (d, 3JCF = 9.1 Hz),
132.5, 136.2 6 (d, 4JCF = 3.5 Hz), 140.5 (2C), 156.6, 159.7, 159.9, 165.4 (d, 1JCF = 250.7 Hz); HRMS (ES):
Found 539.0878. C29H21N4F79BrO+ requires 539.0883. Anal calcd for C29H20N4FBrO: C, 64.57; H, 3.74;
N, 10.39. Found: C, 64.53; H, 3.70; N, 10.41.
3.5. Materials and Methods for In Vitro Cytotoxicity Assay of 4a–h
The cytotoxicity of the compounds were screened against four different cell lines: C3A/HepG2
human liver cells (derivatives of Hep G2 (ATCC HB8065)) (ATCC® CRL10741), Caco2
(Human colorectal tumor cell), A549 (Human lung carcinoma), MCF-7 (breast adenocarcinoma),
and HeLa (cervical cancer) cells. The lethal concentration was determined using the
3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide (MTT) assay [43]. The cells were maintained
in Dubellsco minimal essential medium (DMEM, Highveld Biological, Sandton, South Africa)
supplemented with 10% foetal calf serum (Adcock-Ingram, Midrand, South Africa) and sodium
pyruvate for C3A liver cells, while Vero cells were supplemented with 5% foetal calf serum and
0.1% gentamicin (Virbac, Centurion, South Africa). Cell suspensions were prepared from confluent
monolayer cultures and plated at a density of 0.1
×
106 cells into each well of 96-well microtitre
plates. For cell attachment, plates were incubated for 24 h at 37 ◦C in a 5% CO2 incubator prior to
the exposure. The compounds were dissolved in DMSO (5 mg/mL), and appropriate dilutions were
prepared, then added to the wells and incubated for 48h. Doxorubicin (Pfizer Laboratories, Sandton,
South Africa) was used as a positive control while acetone was the negative control. After incubation
for 48 h, the wells were rinsed with 150 µL of phosphate buffered saline PBS (Sigma-Aldrich, GmBH,

Int. J. Mol. Sci. 2018, 19, 2232
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Schnelldorf, Germany) and 200
dissolved in PBS (30
removed and MTT formazan crystals were dissolved in 50
µ
L of fresh medium was dispensed into the wells. MTT (Sigma)
µ
L) was added to each well and incubated for 4 h at 37 ◦C. The medium was
µL DMSO. Absorbance was measured on
a BioTek microplate reader (BioTek Synergy, Analytical and Diagnostic Products, Johannesburg, South
Africa) at a wavelength of 570 nm. Each concentration was tested in quadruplicate and the assay was
repeated three times. The percentage of cell viability and the LC50 values for each compound tested
were calculated as described before [15,17].
3.6. Apoptosis Assay
3.6.1. Annexin V-FLUOS Staining Assay on 4g against Caco-2 and C3A Cells
Apoptotic cells were quantified using flow cytometry. The Caco-2 or C3A cells were cultured in
6 well plates and each was then treated with compound 4g at concentrations of 5, 12.5, and 25
against doxorubicin hydrochloride (0.20 M) as a reference standard. After the cells were incubated
for 24 h, both treated and untreated cells were harvested, washed two times with ice PBS and then
adjusted at a density of 1
106 cells/sample. The cells were, in turn, stained with Annexin-V-FLUOS
µM,
µ
×
staining kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. The cells
were analysed using Becton, Dicknson and Company FACS Accuri Flow Cytometer (Woodmead,
South Africa).
3.6.2. Caspace-3 Analysis on 4g against C3A and Caco-2 Cells
Caspace-3 activity was detected by means of Caspace-3 Colorimetric Assay Kit (Abcam;
Cambridge, MA, USA). The cells were cultured in 96 well plates and treated for 24 h. The cells
were then washed with PBS buffer and lysed on ice the experiments were carried out according to the
manufacturer’s instructions. Optical density was measured at absorbance of 405 nm using the BioTek
microplate reader. The concentration of active Caspace-3 (Asp 175) were measured in duplicates and
interpolated from the active caspase-3 (Asp 175) standard curve and corrected for sample dilution.
3.7. EGFR-TK Inhibition Assays
The inhibitory activities of compounds 4f
, 4g and gefitinib towards EGFR-TK were
tested using enzyme-linked immunosorbent assay (ELISA) technique with purified Epidermal
Growth Factor Receptor (Sigma-Aldrich, Bradford, UK), following a procedure described in our
previous investigation [17].
3.8. Molecular Docking of Compounds 4a–h into EGFR-TK Active Site
The starting structure of EGFR kinase domain was retrieved from Research Collaboratory for
Structural Bioinformatics Protein Data Bank (RCSB PDB) with the identity 1M17 [44]. All heteroatoms
and water molecules were first removed. Polar hydrogen atoms, Kollman-Amber united atom partial
charges and solvation parameters were then added by utilising AutoDockTools (The Scripps Research
Institute, La Jolla, California, USA) [45]. The coordinates for control docking (erlotinib) was retrieved
from the ligand of EGRF (PDB id: 1M17), while the coordinates for compounds 4a–h were generated
using ChemDraw Professional 15.0 (PerkinElmer Informatics, Waltham, MA, USA) and minimised with
Chem3D module in ChemOffice Professional 15.0 (PerkinElmer Informatics). Only polar hydrogen
remained. AutoDockTools was then used to assign Gasteiger charges and torsional angles to all ligands.
Hydrated docking was applied in this work with standard protocol from AutoDock4.2 [45] where water
atoms were added to the ligand. Docking was performed by AutoDock4.2.6 [45] with Lamarckian
genetic algorithm of 1,000,000 energy evaluations per run and a maximum number of 27,000 generation.
The corresponding docked conformations were clustered with root mean square of 2.0 Å.

Int. J. Mol. Sci. 2018, 19, 2232
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4. Conclusions
We have demonstrated that compound 4g, which exhibited significant cytotoxicity against several
cancer cell lines tested was able to trigger apoptosis in Caco-2 and C3A cells. This demonstrates that
the indole-aminoquinazoline hybrids prepared in this investigation possess in vitro antiproliferative
and pro-apoptotic activity. Moreover, compound 4g not only exhibited significant cytotoxicity against
most of the cancer cell lines, but also showed significant inhibitory activity (IC50 = 40.7 nM) towards
EGFR comparable to that of the medicinally important EGFR inhibitor, gefitinib (IC50 = 38.9 nM). It has
been reported in the literature that bioisosteric replacement of hydrogen by fluorine in position 4
of the phenyl ring makes electronic modulation to reinforce and enhance the binding interaction
process [46]. This probably accounts for the observed increased cytotoxicity of 4-fluorophenyl
substituted derivatives, compounds 4b, 4f and 4g, as well as their inhibitory effect against EGFR-TK
phosphorylation. We attribute the difference in activity related to the halogen substitution pattern
on the aminoindole arm to be associated with the flexibility of this moiety, where a distinct
spatial arrangement of the molecule leads to an improved interaction with the active site of the
receptor. Molecular docking (in silico) studies of compounds 4a
bonding between the indole–NH and the peptide backbone of the EGFR, which would account for
the improved inhibitory activity of these compounds. Besides, the -stacking interactions of the
–h confirm the presence of hydrogen
π
indole-aminoquinazoline hybrids with Phe699 could contribute to the favourable binding affinity with
EGFR. The docking studies showed that these title compounds bind more like erlotinib than gefitinib,
which is a selective inhibitor for the EGFR. This thus suggests that the title compounds may also target
proteins other than EGFR. This in our view forms a basis for extension of this study to other types of
protein kinases to explore the mechanism of their action and selectivity for specific type of protein
kinase. These preliminary in vitro cytotoxicity results and SAR analysis form a basis for the design and
synthesis of derivatives substituted on the 4-position of the phenyl ring with other lipophilic atoms or
groups, such as the chlorine atom or a methyl group. This establishes the effect of lipophilicity of the
substituents on the biological activity of these indole-aminoquinazoline hybrids.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/8/
2232/s1.
Author Contributions: M.J.M. reviewed the literature, interpreted the data and results, and wrote the manuscript.
M.M.M. performed the synthesis as part of a PhD project. A.A. and L.J.M. evaluated the compounds for cytotoxicity
and mode of cancer cell death. Molecular docking studies were performed by Y.S.C. who also contributed in the
discussion of the corresponding data.
Acknowledgments: We are grateful to the University of South Africa, University of Pretoria and the National
Research Foundation for financial assistance. Part of the work was also supported by Higher Institution Center of
Excellence (311/CIPPM/44001005) from Malaysia Ministry of Higher Education. We also thank the University of
Stellenbosch Central Analytical Facility (CAF) for mass spectrometric and elemental analyses. The authors also
thank Marole M Maluleka for valuable input and criticism.
Conflicts of Interest: The authors declare no conflict of interest.
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