Synthesis and in vitro cytotoxicity of the 4-(Halogenoanilino)-6-bromoquinazolines and their 6-(4-fluorophenyl) substituted derivatives as potential inhibitors of epidermal growth factor receptor tyrosine kinase

Article abstract of DOI:10.3390/ph10040087

Series of the 2-unsubstituted and 2-(4-chlorophenyl)–substituted 4-anilino-6-bromoquinazolines and their 6-(4-fluorophenyl)–substituted derivatives were evaluated for in vitro cytotoxicity against MCF-7 and HeLa cells. The 2-unsubstituted 4-anilino-6-brom

Full text of DOI:10.3390/ph10040087

pharmaceuticals
Article
Synthesis and In Vitro Cytotoxicity of the
4-(Halogenoanilino)-6-bromoquinazolines and Their
6-(4-Fluorophenyl) Substituted Derivatives as
Potential Inhibitors of Epidermal Growth Factor
Receptor Tyrosine Kinase
Malose Jack Mphahlele 1,
*
ID , Hugues K. Paumo 1 and Yee Siew Choong 2
ID
1
Department of Chemistry, College of Science, Engineering and Technology, University of South Africa,
Private Bag X06, Florida 1710, South Africa; Tkamdeh@unisa.ac.za
Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia,
Penang 11800, Malaysia; yeesiew@usm.my
2
*
Correspondence: mphahmj@unisa.ac.za; Tel./Fax: +27-11670-9301
Received: 21 September 2017; Accepted: 31 October 2017; Published: 20 November 2017
Abstract: Series of the 2-unsubstituted and 2-(4-chlorophenyl)–substituted 4-anilino-6-bromoquinazolines
and their 6-(4-fluorophenyl)–substituted derivatives were evaluated for in vitro cytotoxicity against
MCF-7 and HeLa cells. The 2-unsubstituted 4-anilino-6-bromoquinazolines lacked activity, whereas
most of their 2-(4-chlorophenyl) substituted derivatives were found to exhibit significant cytotoxicity
and selectivity against HeLa cells. Replacement of bromine with 4-fluorophenyl group for the
2-unsubstituted 4-anilinoquinazolines resulted in superior activity against HeLa cells compared to
Gefitinib. The presence of a 4-fluorophenyl group in the 2-(4-chlorophenyl) substituted derivatives
led to increased cytotoxicity against HeLa cells, except for the 3-chloroanilino derivative. The most
active compounds, namely, 3g, 3l, and 4l, were found to exhibit a moderate to significant inhibitory
effect against epidermal growth factor receptor tyrosine kinase (EGFR-TK). The EGFR molecular
docking model suggested that these compounds are nicely bound to the region of EGFR.
Keywords: 6-bromo-4-chloroquinazolines; amination; Suzuki–Miyaura cross-coupling; cytotoxicity;
EGFR-TK; molecular docking
1. Introduction
The quinazoline scaffold continues to draw the attention of medicinal chemists due to its
well-known anticancer properties; many substituted quinazoline derivatives have recently earned
great interest in targeted therapies as antitumor drugs. Among quinazoline-based compounds,
the 4-anilinoquinazolines assert themselves as selective inhibitors of the epidermal growth factor
receptor tyrosine kinase (EGFR-TK) phosphorylation, which results from competitive binding at
the ATP site [
(TK) are implicated in the progression of various types of cancers such as breast, ovarian, colon,
and prostate [ ]. Gefitinib ( ), shown in Figure 1, is an example of a trisubstituted 4-anilinoquinazoline
drug used for the treatment of non-small-cell lung cancer (NSCLC) with sensitive mutations of
the EGFR [ ]. Likewise, Vandetanib ( ) has been found to be a selective inhibitor that targets the
vascular endothelial growth factor receptor (VEGFR) and the EGFR signal transduction pathways for
the treatment of breast cancer [ ]. Lapatinib ( ), a 6-heteroaryl substituted 4-anilinoquinazoline
derivative, on the other hand, is an oral dual TK inhibitor that targets both EGFR and HER2 to
1,2]. The altered protein expression and activity of the receptor tyrosine kinases
3
A
4
B
5
C
inhibit the proliferation of breast cancer cells [
6
]. The 6-vinyl substituted 4-anilinoquinazoline
Pharmaceuticals 2017, 10, 87; doi:10.3390/ph10040087
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derivative (
of various types of cancer [
D
) is a selective ERBB2 angiogenesis inhibitor under investigation for the treatment
]. Structure–activity relationship (SAR) modelling studies for the
7
ability of 4-anilinoquinazoline to inhibit EGFR-TK activity revealed that both of the quinazoline
nitrogen atoms are essential for activity (as a hydrogen bond acceptor for N1 and a water-linked
hydrogen bond acceptor for N3); aniline moiety-bearing lipophilic substituents such as chloro,
bromo, and trifluoromethyl groups were also important as they occupy the lipophilic pocket [8].
Although most of the medicinally important 4-anilinoquinazolines have no substitute at the C-2
position, in a previous study on the polysubstituted 2-aryl-4-(3-fluoroanilino)quinazolines from our
laboratory, we observed that the presence of bromine in the 6-position and a 4-chlorophenyl group
at position-2 of the quinazoline moiety favour activity against the human breast cancer (MCF-7)
cell line [9]. In our quest to optimise this class of potential anti-proliferative agents, we decided to
prepare the C-2 unsubstituted and the 2-(4-chlorophenyl) substituted 4-anilino-6-bromoquinazolines
substituted with different halogen atoms on the aniline moiety and a 4-fluorophenyl group at the
6-position for further studies of anticancer properties.
(A)
(B)
(C)
(D)
Figure 1. Examples of biologically relevant 4-anilinoquinazolines. Gefitinib (
Lapatinib (C), 6-vinyl substituted 4-anilinoquinazoline derivative (D).
A), Vandetanib (B),
Our approach to the target compounds involved the use of 6-bromo-4-chloroquinazoline and
2-aryl-6-bromo-4-chloroquinazolines as substrates for amination with halogenated aniline derivatives.
The main aim was to evaluate the resultant 4-anilino-6-bromoquinazolines and their 6-(4-fluorophenyl)
substituted derivatives for anti-proliferative effect and for their ability to inhibit EGFR tyrosine
kinase phosphorylation. Their mechanism of anticancer activity is also explained theoretically
through molecular (in silico) docking into the ATP binding site of EGFR to predict the hypothetical
protein–ligand binding mode.

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2. Results and Discussion
2.1. Synthesis of 6-(4-Fluorophenyl) Substituted 4-Anilinoquinazolines 4a–l
It has previously been demonstrated that the presence of halogen atom(s) on the aniline ring
improves the biological activity of the 4-anilinoquinazoline derivatives [4,5,8,10]. The presence
of a fluorine-containing group in a molecule, on the other hand, introduces beneficial biological
and pharmacological properties such as increased metabolic stability and binding ability
as well as enhanced membrane permeability [11,12]. With these considerations in mind,
we decided to prepare 2-unsubstituted and 2-(4-chlorophenyl) substituted 4-(halogenophenylamino)-6-
bromoquinazolines and transformed them into the corresponding 6-(4-fluorophenyl)–substituted
derivatives (Schemes 1 and 2).
With the exception of the 4-(4-bromoanilino) substituted
derivatives, 4e and 4k, all other 6-(4-fluorophenyl)–substituted 4-anilinoquinazolines 4 were readily
prepared via initial amination of 6-bromo-4-chloroquinazoline 2a (R = H) or 6-bromo-4-chloro-2-
(4-chlorophenyl)quinazoline 2b (R = 4-ClC6H4–) with various halogenated aniline derivatives in
tetrahydrofuran-isopropanol (THF-iPrOH) mixture in the presence of HCl as a catalyst under reflux for
5 h (Scheme 1 and Table 1). The 4-anilino-6-bromoquinazoline derivatives 3a–l are easily distinguished
from the corresponding substrates by the presence of additional signals in the aromatic region of their
proton and carbon-13 nuclear magnetic resonance (NMR) spectra (1H- and 13C-NMR) and the NH
signal in the region δH 9.89–10.10 ppm. The presence of the aniline group is further confirmed by
) and 3413–3456 cm−1
a band in the regions νmax 3239–3280 (3a–f (3g–l) of their IR spectra. Moreover,
the molecular ion region of their mass spectra revealed the absence of the M+ and M+2 peaks in the
ratio 3:1 due to the 35Cl and 37Cl isotope observed in the spectra of the corresponding substrates,
thus confirming their aniline nature. We subjected the 4-anilino-6-bromoquinazoline derivatives
3a–l
to Suzuki–Miyaura cross-coupling with 4-fluorophenylboronic acid as a model for Csp2–Csp2
bond formation in the presence of a dichlorobis(tricyclohexylphosphine)palladium(II) pre-catalyst
◦
and K2CO3 as a base in THF-iPrOH at 70 C. We isolated by column chromatography on silica
gel the corresponding 6-arylated 4-anilinoquinazolines 4. Due to competitive Suzuki–Miyaura
cross-coupling of the Csp2–Br bond of the 4-bromoanilino–substituted derivatives 3e and 3k, we opted
for an alternative route for the synthesis of the corresponding 6-(4-fluorophenyl) substituted derivatives
4e and 4k, which is outlined in Scheme 2 and Table 2 below. The NH-4(3H)-oxo derivatives 1a and 1b
were first subjected to Suzuki–Miyaura cross-coupling with 4-fluorophenylboronic acid, followed by
aromatization of 5a and 5b under the same reaction conditions employed for the preparation of
compounds
and 4k was achieved under the same reaction conditions employed for the synthesis of compounds
The 1H- and 13C-NMR spectra of compounds 4a
are characterised by the presence of an increased
2. Amination of the corresponding products 6a and 6b with 4-bromoaniline to afford 4e
3.
–l
number of proton and carbon signals in the aromatic region, thus confirming the substitution
1
of the bromine atom. The aromatic region of their H-NMR spectra reveal the presence of two
sets of intense triplets typical of the 4-fluorophenyl group. The molecular ion region of the mass
spectra of compounds 4a–l, on the other hand, revealed the absence of M+ and M+2 peaks in the
ratio 1:1 characteristic of compounds containing the 79Br and 81Br isotopes, thus confirming their
6-carbosubstituted 4-anilinoquinazoline nature.

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1a (R = H)
2a (R = H)
3a–f (R = H)
4a–d & 4f (R = H)
4g–j & 4l (4-ClC6H4)
1b (R = 4-ClC6H4-)
2b (4-ClC6H4-)
3g–l (R = 4-ClC6H4-)
Scheme 1. Synthesis of 4-anilino-6-bromoquinazolines and their 6-fluorophenyl derivatives.
Reagents & conditions: (i) POCl3, Et3N, reflux, 2 h; (ii) NH2Ar, HCl, THF-iPrOH, 70 ◦C, 3 h;
◦
(iii) 4-FC6H4B(OH)2, PdCl2(PPh3)2, Cs2CO3, THF-iPrOH, 70 C, 3 h.
Table 1. Percentage yields of 3a–l, 4a–d & 4f and 4g–j & 4l.
Substrate
R
Ar
%Yield of 3
%Yield of 4
2a
2a
2a
2a
2a
2a
2b
2b
2b
2b
2b
2b
H
H
H
H
H
2-FC6H4-
3-FC6H4-
4-FC6H4-
3-ClC6H4-
4-BrC6H4-
2,4-diFC6H3-
2-FC6H4-
3-FC6H4-
4-FC6H4-
3-ClC6H4-
4-BrC6H4-
2,4-diFC6H3-
80 (3a)
76 (3b)
85 (3c)
79 (3d)
77 (3e)
79 (3f)
82 (3g)
88 (3h)
84 (3i)
87 (3j)
83 (3k)
93 (3l)
64 (4a)
72 (4b)
80 (4c)
61 (4d)
-(4e) a
77 (4f)
68 (4g)
71 (4h)
75 (4i)
59 (4j)
-(4k) a
69 (4l)
H
4-ClC6H4-
4-ClC6H4-
4-ClC6H4-
4-ClC6H4-
4-ClC6H4-
4-ClC6H4-
a
Compounds 4e and 4k were prepared following an independent route, as described in Scheme 2.
1a,b
5a,b
6a,b
4e,k
Scheme 2. Synthesis of 4-(4-bromoanilino)-6-(4-fluorophenyl)quinazoline derivatives 4e and 4k
.
Reagents & conditions: (i) 4-FC6H4B(OH)2, PdCl2(PPh3)2, K2CO3, dioxane (aq), reflux, 3 h; (ii) POCl3,
◦
Et3N, reflux, 5 h; (iii) 4-NH2C6H4Br, HCl, THF-iPrOH, 70 C, 3 h.
Table 2. Percentage yields of 5a,b; 6a,b and 4e,f.
Substrate
R
5a,b
6a,b
4e,f
1a
1b
H
81 (5a)
88 (5b)
77 (6a)
88 (6b)
81 (4a)
59 (4k)
4-ClC6H4-
2.2. Biological Activity Studies
A series of the analogous 6-substituted 4-aminoquinazoline derivatives have recently been
evaluated as potential PI3K inhibitors and were found to exhibit significant anti-proliferative activities
against the human colon cancer (HCT-116) and MCF-7 cell lines [13]. We are interested in breast and
cervical cancers, which are the most common cancers in woman of all races throughout the world [14].

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In this regard we decided to evaluate compounds 3a
–
l
and 4a
–
l
for potential anti-proliferative
activity against two cancer cell lines, namely, the human breast adenocarcinoma (MCF-7) and cervical
cancer (HeLa) cells. Since these tumours have been proven to be highly expressed cell line of EGFR,
we also evaluated the most active compounds for their ability to inhibit EGFR-TK phosphorylation
complemented with molecular docking into EGFR-TK.
2.2.1. Cytotoxicity Studies
The 4-anilinoquinazolines 3a–l and 4a–l were evaluated for growth inhibitory activity
against 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 at concentrations ranging from 0.1 to 100 µM with DMSO and clinical drug Gefitinib as
the negative and positive control, respectively. The LC50 values (the lethal concentration at which
50% of cells are killed) of the 24 compounds (the average from three independent experiments)
against Gefitinib as a reference drug are represented in Tables 3 and 4 (see Supplementary Materials
for the corresponding cell viability percentages and graphs for each compound). The LC50 values
are presented in µM concentrations, taking into account the molecular weights of the compounds.
The SAR of these compounds was studied with respect to the nature of substituent at the 2- or
6-position of the heterocyclic framework and the position and type of halogen atoms on the
aniline ring. Some of the compounds exhibited comparable or superior anti-proliferative activities
as Gefitinib against the two cancer cell lines. No activity was observed for the 2-unsubstituted
4-anilino-6-bromoquinazolines 3a
of 4-chlorophenyl group at C-2 resulted in significant cytotoxicity against both cell lines for the
corresponding 4-anilino-6-bromoquinazolines derivatives 3g Specifically, the 2-fluoroanilino
–f when compared to Gefinitib (Table 3). However, the presence
–l.
substituted derivative 3g and the 2,4-difluoroanilino analogue 3l were found to exhibit higher activity
against both MCF-7 and HeLa cell lines when compared to the reference drug. Moderate activity
against the MCF-7 cell line (LC50 = 0.73 µM), but significant cytotoxicity against the HeLa cells
(LC50 = 1.45 µM) were observed for the 3-fluoroaniline–substituted derivative 3h. The presence of
chlorine atom at position-3 of the aniline moiety in compound 3j resulted in moderate activity for
both cell lines. Diminished activity against both cell lines was observed for compounds 3i and 3k
substituted with fluorine or bromine atom at the para-position of the aniline moiety.
Replacement of the 6-bromine atom with a 4-fluorophenyl group generally resulted in significant
cytotoxicity and selectivity against the HeLa cell line for both the 2-unsubstituted 4a
2-(4-chlorophenyl) substituted derivatives 4g (Table 4). Within these series, the 4-bromoanilino
substituted derivative 4e was found to exhibit moderate (LC50 = 3.18 M) cytotoxicity against
the HeLa cell line, while the 3-chloroanilino substituted analogue 4j showed reduced activity
LC50 = 5.24 µM) against this cell line when compared to Gefitinib (LC50 = 2.37 µM). Among the
2-unsubstituted derivatives 4a , only the 3-fluoroanilino substituted derivative 4b was also found to
exhibit significant cytotoxicity against the MCF-7 cell line with LC50 value of 0.86 M compared to
Gefitinib (LC50 = 0.51 µM). Likewise, within series of the 2-(4-chlorophenyl) substituted derivatives
4g , only the 4-bromoanilino derivative 4k and the 2,4-difluoroanilino substituted derivative 4l were
found to exhibit significant cytotoxicity against the MCF-7 cell line with LC50 values 0.90 M and
0.56 M, respectively. From these preliminary in vitro cytotoxicity results and SAR, it is observed that
–f and their
–
l
µ
(
–f
µ
–l
µ
µ
the presence of a 4-chlorophenyl group at the 2-position of the 4-(arylamino)quinazolines generally
leads to increased cytotoxicity for both series of the 6-substituted 4-anilinoquinazoline derivatives.
Increased cytotoxicity against both cell lines is generally enhanced by the presence at position-4 of
a 2-fluoroanilino or 2,4-difluoroanilino group as observed for compounds 3g, 3l and 4l. This is probably
because a fluorophenyl group on heterocycles enhances biological activity and the lipophilicity of the
molecule due to the non-polarisability of the Csp2–F bond [15].

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Table 3. Cytotoxic effects of 3a–l against MCF-7 and HeLa cell lines. The results are presented as
LC50 (µM) ± standard deviation (SD) from three individual experiments.
3a–f (R = H); 3g–l (R = 4-ClC6H4-)
LC50 (µM) ± SD
Compound
R
Ar
MCF-7
HeLa
3a
3b
3c
3d
3e
3f
3g
3h
H
H
H
H
H
2-FC6H4-
3-FC6H4-
4-FC6H4-
3-ClC6H4-
4-BrC6H4-
1.60 ± 0.07
2.67 ± 0.88
3.52 ± 0.78
4.26 ± 1.45
8.85 ± 1.22
7.78 ±0.11
3.95 ± 0.44
3.24 ± 0.18
6.99 ± 0.14
11.05
±
2.38
H
2,4-diFC6H3- 1.54 ± 0.59
5.68 ± 1.04
0.57 ± 0.11
1.45 ± 0.39
4.97 ± 0.03
2.77 ± 0.15
3.76 ± 0.62
0.18 ± 0.02
2.37± 1.21
4-ClC6H4-
4-ClC6H4-
4-ClC6H4-
4-ClC6H4-
4-ClC6H4-
4-ClC6H4-
2-FC6H4-
3-FC6H4-
4-FC6H4-
3-ClC6H4-
4-BrC6H4-
0.14 ± 0.02
0.73 ± 0.66
1.44 ± 0.41
0.99 ± 0.18
2.03 ± 0.03
3i
3j
3k
3l
2,4-diFC6H3- 0.14 ± 0.04
Gefitinib
0.51 ± 0.11
Table 4. Cytotoxic effects of 4a–l against MCF-7 and HeLa cell lines.
4a–f (R = H); 4g–l (R = 4-ClC6H4-)
LC50 (µM) ± SD
Compound
R
Ar
MCF-7
HeLa
4a
4b
4c
4d
4e
4f
4g
4h
H
H
H
H
H
2-FC6H4-
3-FC6H4-
4-FC6H4-
3-ClC6H4-
4-BrC6H4-
1.60 ± 0.19
0.86 ± 0.08
2.55 ± 0.81
7.64 ± 1.88
5.65 ± 1.96
1.60 ± 0.26
1.24 ± 0.40
0.55 ± 0.47
2.15 ± 0.47
3.18 ± 0.69
2.20 ± 1.78
0.73 ± 0.34
0.87 ± 0.17
1.39 ± 0.32
5.23 ± 2.32
0.45 ± 0.29
0.47 ± 0.21
2.37± 1.21
H
2,4-diFC6H3- 2.09 ± 1.14
4-ClC6H4-
4-ClC6H4-
4-ClC6H4-
4-ClC6H4-
4-ClC6H4-
4-ClC6H4-
2-FC6H4-
3-FC6H4-
4-FC6H4-
3-ClC6H4-
4-BrC6H4-
1.32 ± 0.68
2.41 ± 1.11
2.70 ± 1.47
3.09 ± 0.83
0.90 ± 0.64
4i
4j
4k
4l
2,4-diFC6H3- 0.56 ± 0.21
Gefitinib
0.51 ± 0.11
2.2.2. Inhibition of EGFR-TK
The 4-anilinoquinazolines such as Gefitinib are known to produce their anticancer activity
through inhibition of EGFR-TK phosphorylation, which results from competitive binding at the
ATP site [
1,2]. The activity of EGFR-TK is implicated in the progression of breast, ovarian, lung,
colon, prostate, and squamous cell head and neck cancers [16]. In order to test whether the

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4-anilinoquinazolines prepared in this investigation inhibit the ligand binding-induced receptor
phosphorylation, we performed kinase activity of EGFR in the presence of compounds 3g 3l and 4l
The inhibitory activities of compounds 3g 3l and 4l to EGFR were tested by ELISA method against
,
.
,
Gefitinib as a reference standard. The observed results indicate that these compounds exhibit moderate
to significant inhibitory effect against EGFR-TK (Figure 2). A combination of the 2-(4-chlorophenyl)
and 4-(2,4-difluoroanilino) groups on the 6-bromoquinazoline framework of derivative 3l resulted in
significant inhibitory activity against EGFR-TK with LC50 value of 37.66 nM, which is slightly above
that of the clinical drug, Gefitinib (LC50 = 31.44 nM). This is interesting because substitution at the C-2
position of the quinazoline ring, even with a small methyl group, was previously found to diminish
activity against EGFR-TK [17]. Although a bulky group at the 6-position of the quinazoline ring can
be tolerated [18], the presence of a 4-fluorophenyl group at this position in 4l resulted in reduced
EGFR-TK inhibitory (LC50 = 59.21 nM) compared to the corresponding 6-bromo substituted precursor
3l. Previous SAR of the 4-anilinoquinazolines suggested that substantial (up to 10-fold) increase in
potency could be achieved by the introduction of small lipophilic groups at the 3-position of the aniline
ring [19,20]. We observed significantly reduced EGFR-TK inhibitory effect (LC50 = 70.05 nM) for 3g
substituted with a 4-(2-fluoroanilino) group when compared to the 4-(2,4-difluoroanilino)–substituted
derivative 3l.
Figure 2. Inhibition (LC50 in nM) of EGFR-TK by 3g, 3l and 4l against Gefitinib.
The difference in activity related to the halogen substitution pattern on the aniline ring may be
associated with the flexibility of this moiety, where a distinct spatial arrangement of the molecule could
lead to an improved interaction with the active site of the receptor. In order to prove this assumption
and further guide SAR studies, we performed molecular docking of compounds 3g, 3l, and 4l into the
ATP binding site of EGFR.
2.2.3. Molecular Docking Studies against Gefitinib
Docking simulation was conducted to study the binding affinity of the given compounds
toward EGFR-TK. The control docked on Erlotinib (an inhibitor co-crystallized with EGFR-TK in
1M17) resulted root mean square deviation (RMSD) of 1.6 Å from the crystal structure showed the
reproducibility of docking simulation (Figure 3). Compound 4l exhibits the highest binding affinity
among the docked compounds but comparable with Gefitinib, evidenced from binding free energy
calculation of
compounds 3g and 3l have a relatively lower binding free energy of
respectively. Hydrogen bonding was observed between Erlotinib, Gefitinib, compound 3g and 3l
−
10.46 and
−
10.74 kcal/mol, respectively (Table 5). On the other hand, Erlotinib and
−
8.71, 9.14, and 8.83 kcal/mol,
−
−
.
All the hydrogen bonds were within the distance of 2.2 Å, thus showed good binding affinity with
EGFR binding pocket (Figure 3). The docked conformation of Gefitinib showed the position of
the morpholine region interacts with EGRK-TK Phe699 benzene ring at antiparallel orientation.
The 2,4-difluorophenyl region of compounds 3l and 4l and the 2-fluorophenyl region of compound 3g

Pharmaceuticals 2017, 10, 87
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also showed T-shape ring stacking interactions with Phe799 benzene ring. Compound 3g and 3l have
similar binding poses. Compound 4l, which is bulkier in size, could have more interactions with the
binding pocket residues of EGFR, thus a better binding affinity compared with compounds 3g and 3l
From the binding mode, it could be assumed that the synthesised substances might act on the ATP
binding site of EGFR like Erlotinib or Gefitinib do.
.
Table 5. Calculated binding free energy and estimated inhibition constant (Ki) of Erlotinib (as control),
Gefitinib, and compounds 3g, 3l, and 4l with EGFR-TK.
Ligand
Hb Interacting Atoms
FEB (kcal/mol)
Ic, Ki (nM)
Hb Distance (Å)
Control (Erlotinib)
2.2
1.9
2.1
2.1
-
Met769 O–Erl HN2
Thr766 OG1–Gef HN
Asp831 OD2–3g HN
Asp831 OD2–3l HN
-
−8.71
−10.74
−9.14
−8.83
−10.46
414.12
13.34
200.91
337.36
21.60
Gefitinib
3g
3l
4l
Hb: Hydrogen bond; FEB: Free energy of binding; Ic: Estimated inhibition constant.
Figure 3. Docked conformation of Erlotinib (as control, red stick), Gefitinib (green stick), compound 3g
(yellow stick), 3l (blue stick), and 4l (cyan stick) in the binding pocket of epidermal growth factor
receptor (EGFR) kinase domain (surface and ribbon representation). Black dotted lines are the direct
hydrogen bonding formed between docked compound and EGFR. Crystal erlotinib is in a coloured
atom-type stick presentation.
3. Experimental Section
3.1. General
Melting points were recorded on a Thermocouple digital melting point apparatus (Mettler
Toledo LLC, Columbus, OH, USA). IR spectra were recorded as powders using a Bruker VERTEX
70 FT-IR Spectrometer (Bruker Optics, Billerica, MA, USA) with a diamond ATR (attenuated total
reflectance) accessory by using the thin-film method. For column chromatography, Merck kieselgel 60
(0.063–0.200 mm) (Merck KGaA, Frankfurt, Germany) was used as stationary phase. NMR spectra
were obtained as CDCl3 solutions using Agilent 500 MHz NMR (Agilent Technologies, Oxford, UK)
spectrometer and the chemical shifts are quoted relative to the TMS peak. Low- and high-resolution
mass spectra were recorded at an ionisation potential of 70 eV using Waters Synapt G2 Quadrupole

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Time-of-flight mass spectrometer (Waters Corp., Milford, MA, USA) at the University of Stellenbosch
Central Analytical Facility.
3.2. Typical Procedure for the Preparation of Compounds 1a and 1b
A stirred mixture of 2-amino-5-bromobenzamide (1.0 equiv.), aldehyde (1.5 equiv.) and iodine
(2.0 equiv.) in ethanol (6.0 mL/mmol of benzamide) in a round-bottomed flask equipped with a stirrer
bar and a condenser was refluxed for 8 h and then allowed to cool to room temperature. An ice-cold
saturated aqueous solution of sodium thiosulphate was added to the mixture and the resultant
precipitate was filtered and washed with cold water. The product was recrystallized from acetonitrile.
Compounds 1a and 1b were prepared in this fashion.
6-Bromoquinazolin-4(3H)-one (1a). A mixture of 2-amino-5-bromobenzamide (1.00 g, 4.65 mmol),
paraformaldehyde (0.21 g, 6.97 mmol) and iodine (2.36 g, 9.30 mmol) in ethanol (30 mL) afforded 1a as
◦
◦
a white solid (0.95 g, 91%), mp. 278–280 C (lit. [21] 273–275 C);
1271, 1316, 1387, 1462, 1603, 1688, 2809, 3445 cm−1
δH (500 MHz, DMSO-d6) 7.59 (d, J = 8.5 Hz, 1H)
7.93 (dd, J = 2.5 and 8.5 Hz, 1H), 8.12 (s, 1H), 8.16 (d, J = 2.0 Hz, 1H), 12.40 (br s, 1H).
νmax (ATR) 493, 563, 622, 824, 924, 1237,
;
,
6-Bromo-2-(4-chlorophenyl)quinazolin-4(3H)-one (1b). A mixture of 2-amino-5-bromobenzamide (1.00 g,
4.65 mmol), 4-chlorobenzaldehyde (0.97 g, 6.97 mmol) and iodine (2.36 g, 9.30 mmol) in ethanol (30 mL)
afforded 1b as a white solid (1.45 g, 93%), mp. 342–344 ◦C;
ν
(ATR) 538, 559, 649, 728, 831, 940,
max
1091, 1276, 1413, 1460, 1556, 1601, 1672, 3442 cm−1
; δH (500 MHz, DMSO-d6) 7.60 (d, J = 8.5 Hz, 2H),
7.66 (d, J = 8.5 Hz, 1H), 7.95 (dd, J = 2.5 and 8.5 Hz, 1H), 8.16 (d, J = 8.5 Hz, 2H), 8.19 (d, J = 2.0 Hz, 1H),
12.76 (brs, 1H); δC (125 MHz, DMSO-d6) 104.7, 112.5, 112.9, 119.9, 128.4, 129.2, 129.9, 130.2, 138.0, 139.1,
155.3, 159.3; m/z 335 (100, M + H); HRMS (ES): found 334.9578. C14H979Br35ClN2O+ requires 334.9587.
3.3. Typical Procedure for the Chlorination–Dehydration of 1a and 1b to Afford 2a and 2b, Respectively
Synthesis of 6-Bromo-4-chloroquinazoline (2a). Triethylamine (4 mL) was added dropwise at room
temperature to a stirred mixture of 1a (0.50 g, 2.22 mmol) and phosphoryl chloride (10 mL) in
a round-bottomed flask. The reaction mixture was stirred under reflux for 5 h and then allowed
to cool to room temperature. Ice-cold water was added to the mixture and the product was
extracted with chloroform. The combined organic layers were washed with an aqueous solution
of NaHCO3, dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude
product was recrystallized from ethanol to afford 2a as a white solid (0.46 g, 85%), mp. 164–166 ◦C
(lit. [20
1630, 2932 cm−1
8.44 (d, J = 2.0 Hz, 1H), 9.03 (s, 1H).
]
161–163 ◦C);
δH (500 MHz, DMSO-d6) 7.95 (d, J = 8.5 Hz, 1H), 8.06 (dd, J = 2.5 and 8.5 Hz, 1H),
ν
(ATR) 499, 539, 850, 1021, 1169, 1271, 1352, 1389, 1468, 1474, 1560,
max
;
Synthesis of 6-Bromo-4-chloro-3-(4-chlorophenyl)quinazoline (2b). A stirred mixture of 1b (0.50 g,
1.49 mmol), trimethylamine (4 mL), and phosphoryl chloride (10 mL) afforded 2b as a yellow solid
◦
(0.48 g, 91%), mp. 244–246 C;
ν
max (ATR) 505, 531, 732, 829, 991, 1090, 1294, 1318, 1402, 1415, 1469, 1493,
δH (500 MHz, DMSO-d6) 7.48 (d, J = 8.5 Hz, 2H), 7.93 (d, J = 8.0 Hz, 1H)
7.99 (dd, J = 2.5 and 8.5 Hz, 1H), 8.40 (d, J = 2.0 Hz, 1H), 8.51 (d, J = 8.5 Hz, 2H); δC (125 MHz,
1539, 1556, 1592, 1633 cm−1
;
,
DMSO-d6) 104.8, 112.5, 112.9, 128.5, 129.2, 129.9, 130.2, 138.0, 139.2, 149.1, 152.0 159.3; m/z (100, M + H)
;
HRMS (ES): found 334.9571. C14H979Br35Cl2+ requires 334.9587.
3.4. Typical Procedure for the Amination of 2a and 2b to Afford Compounds 3a–f and 3g–l, Respectively
6-Bromo-N-(2-fluorophenyl)quinazolin-4-amine (3a). A mixture of 2a (0.50 g, 2.05 mmol), 2-fluoroaniline
(0.25 g, 2.25 mmol) and concentrated HCl (0.01 g, 0.27 mmol) in 3:1 THF-isopropanol (v/v, 10 mL)
in a round-bottomed flask was stirred under reflux for 5 h. The reaction mixture was then allowed
to cool to room temperature and quenched with ice-cold water. The product was extracted with
ethyl acetate and the combined organic layers were washed thoroughly with an aqueous solution

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of NaHCO3, dried over anhydrous MgSO4, filtered, a◦nd evaporated under reduced pressure to
(ATR) 493, 614, 757,
max
afford 3a as a white solid (0.52 g, 80%), mp. 174–176 C (acetonitrile);
ν
832, 926, 1188, 1253, 1404, 1454, 1563, 1619, 2849, 2916, 3259 cm−1
; δH (500 MHz, DMSO-d6)
7.23-7.32 (m, 3H)
8.49 (s, 1H)
,
7.52 (t, J = 8.0 Hz, 1H), 7.72 (d, J = 8.5 Hz, 1H), 7.97 (dd, J = 2.0 and 9.0 Hz, 1H),
2
,
8.76 (d, J = 2.0 Hz, 1H), 9.91 (s, 1H); δC (125 MHz, DMSO-d6) 116.5 (d, JCF = 20.0 Hz),
119.2, 124.9 (d, 4JCF = 2.8 Hz), 126.0, 126.3, 126.5 (d, 3JCF = 12.3 Hz), 126.5, 128.0 (d, 3JCF = 6.6 Hz), 128.8,
130.5, 136.6, 148.9, 155.5, 157.2 (d, 1JCF = 242.7 Hz); m/z 318 (100, M+H); HRMS (ES): found 318.0044.
+
C14H10BrFN3 requires 318.0042.
6-Bromo-N-(3-fluorophenyl)quinazolin-4-amine (3b). A mixture of 2a (0.50 g, 2.05 mmol), 3-fluoroaniline
(0.25 g, 2.25 mmol) and concentrated HCl (0.01 g, 0.27 mmol) in THF-isopropanol (10 mL) afforded 3b
◦
−1
as a white solid (0.49 g, 76%), mp. 214–216 C (acetonitrile);
ν
max (ATR) 494, 684, 778, 832, 843, 958, 1145,
1403, 1485, 1531, 1562, 1614, 3074, 3263 cm
;
δH (500 MHz, DMSO-d6) 6.94 (td, J = 2.0 and 8.5 Hz, 1H)
,
,
7.41 (q, J = 8.0 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.92 (dt, J = 2.0 and 11.5 Hz, 1H)
7.99 (dd, J = 2.5 and 8.5 Hz, 1H), 8.67 (s, 1H), 8.86 (d, J = 2.0 Hz, 1H), 9.94 (s, 1H); δC (125 MHz,
2
2
4
DMSO-d6) 109.1 (d, JCF = 25.5 Hz), 110.5 (d, JCF = 20.8 Hz), 116.9, 118.0 (d, JCF = 2.8 Hz),
3
3
119.4, 125.8, 130.4 (d, JCF = 9.5 Hz), 130.6, 136.6, 141.2 (d, JCF = 11.3 Hz), 149.0, 155.1, 157.1,
162.4 (d, 1JCF = 239.8 Hz); m/z 318 (100, M + H); HRMS (ES): found 318.0043. C14H10BrFN3
+
requires 318.0042.
6-Bromo-N-(4-fluorophenyl)quinazolin-4-amine (3c). A mixture of 2a (0.50 g, 2.05 mmol), 4-fluoroaniline
(0.25 g, 2.25 mmol) and concentrated HCl (0.01 g, 0.27 mmol) in THF-isopropanol (10 mL) afforded
◦
3c as a white solid (0.55 g, 85%), mp. 217–218 C (acetonitrile);
ν
(ATR) 497, 513, 830, 922,
max
1156, 1213, 1318, 1420, 1489, 1508, 1564, 1619, 2925, 3059 cm−1
(t, J = 8.5 Hz, 2H), 7.70 (d, J = 9.0 Hz, 1H), 7.82 (t, J = 8.5 Hz, 2H), 7.96 (dd, J = 2.5 and 9.0 Hz, 1H),
8.57 (s, 1H), 8.81 (d, J = 2.0 Hz, 1H), 9.87 (s, 1H); δC (125 MHz, DMSO-d6) 115.5 (d, 2JCF = 22.7 Hz)
116.8, 119.2, 124.8 (d, JCF = 7.6 Hz), 125.8, 130.5, 135.6 (d, JCF = 2.8 Hz), 136.5, 149.0, 155.3,
157.3, 159.0 (d, 1JCF = 239.8 Hz); m/z 318 (100, M + H); HRMS (ES): found 318.0027. C14H10BrFN3
; δH (500 MHz, DMSO-d6) 7.23
,
3
4
+
requires 318.0042.
6-Bromo-N-(3-chlorophenyl)quinazolin-4-amine (3d). A mixture of 2a (0.50 g, 2.05 mmol), 3-chloroaniline
(0.29 g, 2.25 mmol) and concentrated HCl (0.01 g, 0.27 mmol) in THF-isopropanol (10 mL) afforded 3d
◦
as a white solid (0.54 g, 79%), mp. 220–222 C (acetonitrile);
ν
max (ATR) 494, 683, 778, 832, 864, 958, 1145,
δH (500 MHz, DMSO-d6) 7.17 (d, J = 8.0 Hz, 1H)
7.73 (d, J = 9.0 Hz, 1H), 7.82 (d, J = 8.5 Hz, 1H), 7.98 (dd, J = 2.0 and 8.5 Hz, 1H),
1354, 1403, 1485, 1531, 1562, 1614, 2935, 3074 cm−1
;
,
7.41 (t, J = 8.0 Hz, 1H)
,
8.08 (s, 1H), 8.67 (s, 1H), 8.85 (d, J = 2.5 Hz, 1H), 9.91 (s, 1H); δC (125 MHz, DMSO-d6) 116.9, 119.4, 120.7,
121.8, 123.8, 125.8, 130.5, 130.6, 133.2, 136.6, 141.0, 149.0, 155.1, 157.1; m/z 334 (100, M+H); HRMS (ES):
found 333.9747. C14H10BrClN3+ requires 333.9747.
6-Bromo-N-(4-bromophenyl)quinazolin-4-amine (3e). A mixture of 2a (0.50 g, 2.05 mmol), 4-bromoaniline
(0.39 g, 2.25 mmol) and concentrated HCl (0.01◦g, 0.27 mmol) in THF-isopropanol (10 mL) afforded
3e as a white solid (0.60 g, 77%), mp. 220–222 C (acetonitrile);
ν
(ATR) 492, 621, 810, 820, 831,
δH (500 MHz, DMSO-d6) 7.55
max
924, 1007, 1066, 1351, 1406, 1487, 1521, 1560, 1618, 3105, 3280 cm−1
;
(d, J = 8.5 Hz, 2H), 7.71 (d, J = 9.0 Hz, 1H), 7.85 (d, J = 8.5 Hz, 2H), 7.96 (dd, J = 2.0 and 8.5 Hz, 1H),
8.62 (s, 1H), 8.83 (d, J = 2.0 Hz, 1H), 9.90 (s, 1H); δC (125 MHz, DMSO-d6) 116.0, 116.9, 119.3, 124.5,
125.9, 130.5, 131.7, 136.5, 138.8, 149.0, 155.1, 157.0; m/z 378 (100, M+H); HRMS (ES): found 377.9241.
C14H1079Br2N3+ requires 377.9241.
6-Bromo-N-(2,4-difluorophenyl)quinazolin-4-amine (3f).
A mixture of 2a (0.50 g, 2.05 mmol),
2.4-difluoroaniline (0.29 g, 2.25 mmol) and concentrated HCl (0.01 g, 0.◦27 mmol) in THF-isopropanol
(ATR)
max
(10 mL) afforded 3f as a white solid (0.55 g, 79%), mp. 198–200 C (acetonitrile);
ν
498, 536, 779, 835, 967, 1142, 1256, 1319, 1354, 1401, 1493, 1508, 1571, 3039 cm−1
; δH (500 MHz,
DMSO-d6) 7.14 (td, J = 2.0 and 9.0 Hz, 1H), 7.36 (td, J = 2.5 and 9.0 Hz, 1H), 7.53 (t, J = 9.0 Hz, 1H),

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7.72 (d, J = 8.5 Hz, 1H), 7.97 (dd, J = 2.0 and 9.0 Hz, 1H), 8.48 (s, 1H), 8.73 (d, J = 2.0 Hz, 1H),
2
9.89 (s, 1H)
;
δC (125 MHz, DMSO-d6) 105.0 (t, JCF = 26.5 Hz), 111.8 (dd, JCF = 3.7 and 21.7 Hz),
111.9 (d, 4JCF = 3.8 Hz), 116.5, 119.2, 125.9, 129.9 (d, JCF = 9.6 Hz), 130.5, 136.6, 148.9, 155.5, 158.3,
160.5 (d, 1JCF = 243.6 Hz), 160.6 (d, 1JCF = 243.6 Hz); m/z 336 (100, M + H); HRMS (ES): found 335.9952.
C14H9BrF2N3+ requires 335.9948.
3
6-Bromo-2-(4-chlorophenyl)-N-(2-fluorophenyl)quinazolin-4-amine (3g). A mixture of 2b (0.50 g, 1.41 mmol),
2-fluoroaniline (0.17 g, 1.55 mmol) and concentrated HCl (0.01 g, 0.2◦7 mmol) in THF-isopropanol
(ATR)
max
(10 mL) afforded 3g as a white solid (0.49 g, 82%), mp. 248–250 C (acetonitrile);
ν
534, 733, 782, 829, 1012, 1089, 1171, 1316, 1399, 1446, 1489, 1522, 1554, 1598, 1622, 3451 cm−1
δH (500 MHz, DMSO-d6) 7.30-7.41 (m, 3H), 7.50 (d, J = 8.5 Hz, 2H), 7.64 (td, J = 2.5 and 9.0 Hz, 1H)
7.79 (d, J = 8.5 Hz, 1H), 8.00 (dd, J = 2.0 and 8.5 Hz, 1H), 8.23 (d, J = 8.5 Hz, 2H), 8.80 (d, J = 2.0 Hz, 1H)
10.05 (s, 1H); δC (125 MHz, DMSO-d6) 115.4, 116.4 (d, 2JCF = 19.9 Hz), 119.0, 124.8 (d, 4JCF = 2.8 Hz)
;
,
,
,
126.1, 126.3 (d, 3JCF = 12.3 Hz), 127.9 (d, 3JCF = 8.5 Hz), 128.3, 128.9, 129.9, 130.7, 135.8, 136.8, 137.1,
1
149.7, 157.0 (d, JCF = 245.6 Hz), 158.2, 158.9; m/z 428 (100, M + H); HRMS (ES): found 427.9978.
+
C20H1335ClF79BrN3 requires 427.9965.
6-Bromo-2-(4-chlorophenyl)-N-(3-fluorophenyl)quinazolin-4-amine (3h). A mixture of 2b (0.50 g,
1.41 mmol), 3-fluoroaniline (0.17 g, 1.55 mmol) and concentrated HCl (0.01 g, 0.27 mmol)
in THF-isopropanol (10 mL) afforded 3h as a white solid (0.53 g, 88%), mp. 242–244 ◦C
(acetonitrile);
1488, 1522, 1555, 3431 cm−1
(q, J = 8.0 Hz, 1H) 7.59 (d, J = 8.5 Hz, 2H)
7.90 (dt, J = 2.5 and 11.5 Hz, 1H) 8.00 (dd, J = 2.5 and 8.5 Hz, 1H)
8.86 (d, J = 2.0 Hz, 1H)
ν
(ATR) 423, 487, 535, 670, 758, 824, 864, 1010, 1087, 1351, 1397, 1417,
max
;
δH (500 MHz, DMSO-d6) 7.00 (td, J = 2.0 and 8.0 Hz, 1H), 7.49
7.74 (dd, J = 2.0 and 8.0 Hz, 1H) 7.84 (d, J = 8.5 Hz, 1H),
,
,
,
,
,
8.38 (d, J = 8.5 Hz, 2H),
2
,
10.07 (s, 1H); δC (125 MHz, DMSO-d6) 109.2 (d, JCF = 26.6 Hz),
110.7 (d, 2JCF = 20.8 Hz), 115.7, 118.1 (d, 4JCF = 2.8 Hz), 119.2, 125.9, 129.0, 130.0, 130.5 (d, 3JCF = 9.3 Hz),
130.8, 135.9, 136.9, 137.2, 141.2 (d, 3JCF = 11.3 Hz), 149.7, 157.4, 158.8, 162.4 (d, 1JCF = 239.0 Hz); m/z 428
(100, M + H); HRMS (ES): found 427.9966. C20H1335ClF79BrN3+ requires 427.9965.
6-Bromo-2-(4-chlorophenyl)-N-(4-fluorophenyl)quinazolin-4-amine (3i). A mixture of 2b (0.50 g, 1.41 mmol),
4-fluoroaniline (0.17 g, 1.55 mmol) and concentrated HCl (0.01 g, 0.27 mmol) in THF-isopropanol
(10 mL) afforded 3i as a white solid (0.51 g, 84%), mp. 289–291 ◦C (acetonitrile);
ν
max (ATR) 424, 534,
669, 736, 827, 1011, 1087, 1199, 1314, 1352, 1398, 1416, 1522, 1556, 3431 cm−1
; δH (500 MHz, DMSO-d6)
7.23 (t, J = 8.5 Hz, 2H), 7.55 (d, J = 8.5 Hz, 2H), 7.77 (d, J = 9.0 Hz, 1H), 7.88 (t, J = 8.5 Hz, 2H),
7.97 (dd, J = 2.5 and 9.0 Hz, 1H), 8.35 (d, J = 9.0 Hz, 2H), 8.82 (d, J = 2.0 Hz, 1H), 10.00 (s, 1H);
δC (125 MHz, DMSO-d6) 115.6 (d, 2JCF = 21.7 Hz), 115.7, 119.0, 124.8 (d, 3JCF = 7.5 Hz), 125.9, 129.0,
130.1, 130.7, 135.6 (d, 4JCF = 2.8 Hz), 135.8, 136.7, 137.2, 149.7, 157.5, 158.9, 159.0 (d, 1JCF = 239.0 Hz);
m/z 428 (100, M + H); HRMS (ES): found 427.9951. C20H1335ClF79BrN3+ requires 427.9965.
6-Bromo-2-(4-chlorophenyl)-N-(3-chlorophenyl)quinazolin-4-amine (3j). A mixture of 2b (0.50 g, 1.41 mmol),
3-chloroaniline (0.19 g, 1.55 mmol) and concentrated HCl (0.01 g, 0.27 mmol) in THF-isopropanol
◦
(10 mL) afforded 3j as a white solid (0.55 g, 87%), mp. 261–263 C (acetonitrile);
ν
max (ATR) 484, 535, 673,
735, 765, 829, 996, 1086, 1353, 1397, 1416, 1428, 1515, 1564, 1595, 3432 cm−1
; δH (500 MHz, DMSO-d6)
7.20 (d, J = 8.5 Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.55 (d, J = 8.5 Hz, 2H), 7.77 (d, J = 8.5 Hz, 1H), 7.88
(dd, J = 2.5 and 8.5 Hz, 1H), 7.97 (dd, J = 2.0 and 8.5 Hz, 1H), 8.13 (s, 1H), 8.82 (d, J = 2.0 Hz, 1H),
10.00 (s, 1H); δC (125 MHz, DMSO-d6) 115.7, 119.3, 120.7, 122.0, 123.8, 125.9, 129.0, 130.0, 130.6, 130.7,
133.2, 135.9, 136.8, 137.1, 140.9, 149.7, 157.3, 158.7; m/z 444 (100, M + H); HRMS (ES): found 443.9684.
C20H1335Cl279BrN3+ requires 443.9670.
6-Bromo-N-(4-bromophenyl)-2-(4-chlorophenyl)quinazolin-4-amine (3k). A mixture of 2b (0.50 g, 1.41 mmol),
4-bromoaniline (0.27 g, 1.55 mmol) and concentrated HCl (0.01 g, 0.27 mmol) in THF-isopropanol
◦
(10 mL) afforded 3k as a white solid (0.57 g, 83%), mp. 263–265 C (acetonitrile);
ν
(ATR)
max
488, 541, 802, 814, 834, 875, 1011, 1083, 1351, 1400, 1489, 1520, 1554, 1564, 1596, 3434 cm−1
;

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δH (500 MHz, DMSO-d6) 7.55 (d, J = 8.5 Hz, 2H), 7.63 (d, J = 8.5 Hz, 2H), 7.76 (d, J = 9.0 Hz, 1H), 7.88
(d, J = 8.5 Hz, 2H), 7.97 (dd, J = 2.0 and 8.5 Hz, 1H), 8.37 (d, J = 8.5 Hz, 2H), 8.82 (d, J = 2.0 Hz, 1H), 10.01
(s, 1H); δC (125 MHz, DMSO-d6) 115.7, 116.1, 119.1, 124.5, 125.9, 129.0, 130.1, 130.7, 131.8, 135.8, 136.8,
+
137.1, 138.8, 149.7, 157.4, 158.9; m/z 488 (100, M + H); HRMS (ES): found 487.9161. C20H13Cl79Br2N3
requires 487.9165.
6-Bromo-2-(4-chlorophenyl)-N-(2,4-difluorophenyl)quinazolin-4-amine (3l). A mixture of 2b (0.50 g,
1.41 mmol), 2.4-difluoroaniline (0.2 g, 1.55 mmol) and concentrated HCl (0.01 g, 0.27 mmol) in
◦
THF-isopropanol (10 mL) afforded 3l as a white solid (0.58 g, 93%), mp. 278–279 C (acetonitrile);
ν
(ATR) 425, 533, 735, 795, 830, 1010, 1088, 1202, 1257, 1351, 1418, 1498, 1528, 1558, 3456 cm−1
;
max
δH (500 MHz, DMSO-d6) 7.00 (td, J = 2.5 and 8.0 Hz, 1H), 7.44 (dd, J = 2.0 and 8.5 Hz, 1H),
7.51 (d, J = 8.5 Hz, 2H), 7.66 (q, J = 8.0 Hz, 1H), 7.79 (d, J = 8.5 Hz, 1H), 8.00 (dd, J = 2.0 and 8.5 Hz, 1H)
,
8.22 (d, J = 8.5 Hz, 2H), 8.78 (d, J = 2.5 Hz, 1H), 10.07 (s, 1H); δC (125 MHz, DMSO-d6) 104.9
(t, 2JCF = 26.6 Hz), 111.8 (dd, JCF = 2.8 and 21.7 Hz), 115.4, 119.0, 122.9 (d, 4JCF = 3.8 Hz), 126.1, 128.9,
129.6 (d, 3JCF = 9.5 Hz), 129.9, 130.7, 135.8, 136.9, 137.1, 149.6, 157.3 (d, 1JCF = 248.4 Hz), 158.4, 158.9,
160.5 (d, 1JCF = 243.7 Hz); m/z 446 (100, M + H); HRMS (ES): found 445.9854. C20H1235ClF279BrN3
+
requires 445.9871.
3.5. Typical Procedure for the Preparation of Compounds 4a–d, f and 4g–j, l
[6-(4-Fluorophenyl)quinazolin-4-yl]-2-fluorophenylamine (4a). A stirred mixture of 3a (0.30 g, 0.94 mmol),
PdCl2(PPh3)2 (0.033 g, 0.047 mmol) and K2CO3 (0.19 g, 1.41 mmol) in 3:1 THF-isopropanol (v/v, 10 mL)
in a two-necked round-bottomed flask was purged with argon gas for 30 min. 4-Fluorophenylboronic
acid (0.15 g, 1.13 mmol) was added to the mixture via a syringe. The reaction mixture was
◦
heated at 100 C for 3 h and then quenched with ice-cold water. The product was extracted into
chloroform and the combined organic layers were washed with water, dried over Na2SO4, filtered
and evaporated under reduced pressure. The residue was purified by column chromatography on
silica gel to afford 4a as a white solid (0.20 g, 64%), mp. 192–194 ◦C; Rf (1:1, toluene/ethyl acetate)
0.54;
ν
(ATR) 495, 592, 666, 746, 826, 1101, 1164, 1238, 1325, 1402, 1419, 1500, 1520, 1576, 1600,
max
3195 cm−1
;
δH (500 MHz, DMSO-d6) 7.24-7.34 (m, 3H), 7.38 (t, J = 8.5 Hz, 2H), 7.55 (t, J = 8.5 Hz,
1H), 7.84 (d, J = 8.5 Hz, 1H), 7.91 (t, J = 8.0 Hz, 2H), 8.19 (dd, J = 2.0 and 8.5 Hz, 1H), 8.47 (s, 1H),
2
8.78 (d, J = 2.0 Hz, 1H), 9.99 (s, 1H); δC (125 MHz, DMSO-d6) 115.5, 116.3 (d, JCF = 20.8 Hz), 116.5
3
3
(d, 2JCF = 19.0 Hz), 120.9, 124.9 (d, 4JCF = 2.8 Hz), 126.5 (d, JCF = 12.3 Hz), 127.9 (d, JCF = 7.5 Hz),
128.8 (d, 4JCF = 3.7 Hz), 128.9, 129.4 (d, 3JCF = 7.6 Hz), 132.1, 135.9 (d, 4JCF = 2.8 Hz), 137.3, 149.4, 155.2,
157.3 (d, 1JCF = 245.6 Hz), 159.2, 162.6 (d, 1JCF = 243.6 Hz); m/z 334 (100, M + H); HRMS (ES): found
334.1166. C20H14F2N3+ requires 334.1156.
[6-(4-Fluorophenyl)quinazolin-4-yl]-3-fluorophenylamine (4b). A mixture of 3b (0.30 g, 0.94 mmol),
PdCl2(PPh3)2 (0.033 g, 0.047 mmol), 4-fluorophenylboronic acid (0.15 g, 1.13 mmol) and K2CO3 (0.19 g,
1.41 mmol) in THF-isopropanol (10 mL) afforded 4b as a white solid (0.22 g, 72%), mp. 225–227 ◦C;
Rf (1:1, toluene/ethyl acetate) 0.70;
1416, 1488, 1532, 1573, 1601, 3254 cm−1
7.41 (t, J = 8.5 Hz, 2H), 7.44 (q, J = 8.5 Hz, 1H), 7.67 (d, J = 1.5 and 8.5 Hz, 1H), 7.86 (d, J = 8.5 Hz,
ν
(ATR) 506, 587, 687, 780, 827, 859, 962, 1144, 1240, 1400,
max
;
δH (500 MHz, DMSO-d6) 6.96 (td, J = 2.0 and 8.5 Hz, 1H),
1H), 7.90–7.96 (m, 3H), 818 (dd, J = 2.0 and 8.5 Hz, 1H), 8.65 (s, 1H), 8.80 (d, J = 2.0 Hz, 1H)
,
2
10.01 (s, 1H); δC (125 MHz, DMSO-d6) 109.4 (d, JCF = 25.5 Hz), 110.5 (d, 2JCF = 20.9 Hz), 115.7,
4
3
116.3 (d, 2JCF = 20.9 Hz), 118.2 (d, JCF = 2.8 Hz), 120.8, 128.9, 129.7 (d, JCF = 9.5 Hz), 130.4
4
3
(d, 3JCF = 9.5 Hz), 132.4, 136.0 (d, JCF = 3.8 Hz), 137.7, 141.3 (d, JCF = 10.4 Hz), 149.4, 154.7, 158.1,
162.4 (d, 1JCF = 239.8 Hz), 162.6 (d, 1JCF = 243.7 Hz); m/z 334 (100, M + H); HRMS (ES): found 334.1158.
C20H14F2N3+ requires 334.1156.
[6-(4-Fluorophenyl)quinazolin-4-yl]-4-fluorophenylamine (4c). A mixture of 3c (0.30 g, 0.94 mmol),
PdCl2(PPh3)2 (0.033 g, 0.047 mmol), 4-fluorophenylboronic acid (0.15 g, 1.13 mmol) and K2CO3 (0.19 g,
1.41 mmol) in THF-isopropanol (10 mL) afforded 4c as a white solid (0.25 g, 80%), mp. 215–217 ◦C;

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Rf (1:1, toluene/ethyl acetate) 0.64;
ν
(ATR) 466, 502, 535, 660, 824, 1156, 1210, 1222, 1357, 1421,
max
1498, 1534, 1574, 1613, 1630, 3278 cm−1
; δH (500 MHz, DMSO-d6) 7.24 (t, J = 8.5 Hz, 2H), 7.38
(t, J = 8.5 Hz, 2H), 7.80–7.85 (m, 3H), 7.90 (t, J = 8.5 Hz, 2H), 8.15 (dd, J = 2.0 and 8.5 Hz, 1H), 8.55
(s, 1H), 8.78 (d, J = 2.0 Hz, 1H), 9.94 (s, 1H); δC (125 MHz, DMSO-d6) 115.5 (d, 2JCF = 21.8 Hz), 115.6,
2
3
3
116.3 (d, JCF = 20.9 Hz), 120.7, 125.2 (d, JCF = 7.5 Hz), 128.8, 129.5 (d, JCF = 8.5 Hz), 132.2, 135.6
(d, 4JCF = 2.9 Hz), 136.0 (d, 4JCF = 3.7 Hz), 137.4, 149.3, 154.9, 158.4, 159.1 (d, 1JCF = 239.9 Hz), 162.6
(d, 1JCF = 243.7 Hz); m/z 334 (100, M + H); HRMS (ES): found 334.1166. C20H14F2N3+ requires 334.1156.
[6-(4-Fluorophenyl)quinazolin-4-yl]-3-chlorophenylamine (4d). A mixture of 3d (0.30 g, 0.89 mmol),
PdCl2(PPh3)2 (0.033 g, 0.044 mmol), 4-fluorophenylboronic acid (0.15 g, 1.07 mmol) and K2CO3 (0.18 g,
1.33 mmol) in THF-isopropanol (10 mL) afforded 4d as a white solid (0.19 g, 61%), mp. 207–209 ◦C;
Rf (1:1, toluene/ethyl acetate) 0.71;
ν
(ATR) 510, 587, 683, 777, 827, 933, 1236, 1356, 1395, 1417, 1478,
max
1498, 1529, 1562, 1597, 2849, 2918, 3245 cm−1
; δH (500 MHz, DMSO-d6) 7.18 (dd, J = 2.0 and 8.5 Hz,
1H), 7.36-7.45 (m, 3H), 7.85 (d, J = 8.5 Hz, 1H), 7.86 (d, J = 8.5 Hz, 1H), 7.91 (t, J = 8.5 Hz, 2H), 8.08
(s, 1H), 8.18 (dd, J = 2.0 and 8.5 Hz, 1H), 8.65 (s, 1H), 8.79 (d, J = 2.5 Hz, 1H), 9.98 (s, 1H); δC (125 MHz,
DMSO-d6) 115.4, 116.3 (d, 2JCF = 20.7 Hz), 116.4, 116.5, 120.8, 125.0, 128.1, 128.2, 128.6, 128.8, 129.5
(d, 3JCF = 8.5 Hz), 135.8 (d, 4JCF = 2.8 Hz), 137.5, 149.1, 155.1, 156.3, 159.3, 162.6 (d, 1JCF = 244.6 Hz);
m/z 350 (100, M + H); HRMS (ES): found 350.0853. C20H1435ClFN3+ requires 350.0860.
[6-(4-Fluorophenyl)quinazolin-4-yl]-2, 4-difluorophenylamine (4f). A mixture of 3f (0.30 g, 0.89 mmol),
PdCl2(PPh3)2 (0.031 g, 0.044 mmol), 4-fluorophenylboronic acid (0.15 g, 1.07 mmol) and K2CO3 (0.18 g,
1.33 mmol) in THF-isopropanol (10 mL) afforded a white solid (0.24 g, 77%), mp. 228–231 ◦C; Rf (1:1,
toluene/ethyl acetate) 0−.610;
νmax (ATR) 470, 496, 513, 665, 784, 824, 965, 1141, 1241, 1325, 1401, 1499,
1578, 2852, 2922, 3159 cm
;
δH (500 MHz, DMSO-d6) 7.17 (td, J = 2.0 and 8.5 Hz, 1H), 7.35–7.43 (m, 3H)
,
,
7.56 (q, J = 8.0 Hz, 1H), 7.84 (d, J = 8.5 Hz, 1H), 7.91 (t, J = 8.0 Hz, 2H), 8.19 (dd, J = 2.0 and 8.5 Hz, 1H)
8.46 (s, 1H), 8.76 (d, J = 2.0 Hz, 1H), 9.96 (s, 1H); δC (125 MHz, DMSO-d6) 105.0 (t, 2JCF = 26.5 Hz), 112.0
(d, 2JCF = 21.8 Hz), 115.2, 116.4 (d, 2JCF = 20.8 Hz), 120.7, 125.8, 128.5, 129.4 (d, 3JCF = 7.6 Hz), 130.0
(d, 4JCF = 2.8 Hz), 130.1, 132.5, 135.7, 137.7, 148.7 (d, 4JCF = 2.8 Hz), 155.0, 157.5 (d, 1JCF = 248.5 Hz)
,
1
+
159.4, 162.6 (d, JCF = 243.6 Hz); m/z 352 (100, M + H); HRMS (ES): found 352.1074. C20H13F3N3
requires 352.1062.
[2-(4-Chlorophenyl)-6-(4-fluorophenyl)quinazolin-4-yl]-2-fluorophenylamine (4g). A mixture of 3g (0.30 g,
0.70 mmol), PdCl2(PPh3)2 (0.024 g, 0.035 mmol), 4-fluorophenylboronic acid (0.12 g, 0.84 mmol)
and K2CO3 (0.14 g, ◦1.05 mmol) in THF-isopropanol (10 mL) afforded 4g as a white solid (0.21 g,
68%), mp. 195–197 C; Rf (toluene) 0.53;
ν
(ATR) 503, 516, 741, 815, 1013, 1086, 1231, 1420,
max
1454, 1494, 1514, 1538, 1559, 1597, 3045, 3446 cm−1
; δH (500 MHz, DMSO-d6) 7.31-7.42 (m, 5H),
7.49 (d, J = 8.5 Hz, 2H), 7.67 (td, J = 2.5 and 8.5 Hz, 1H), 7.90-7.96 (m, 3H), 8.20 (dd, J = 2.5 and 8.5 Hz,
1H), 8.25 (d, J = 8.5 Hz, 2H), 8.82 (d, J = 2.0 Hz, 1H), 10.11 (s, 1H); δC (125 MHz, DMSO-d6) 114.4, 116.3
(d, 2JCF = 20.9 Hz), 116.4 (d, 2JCF = 19.9 Hz), 120.9, 124.9 (d, 4JCF = 2.8 Hz), 126.5 (d, 3JCF = 11.4 Hz)
127.8 (d, JCF = 8.5 Hz), 128.4, 128.9, 129.1, 129.4 (d, JCF = 8.5 Hz), 129.9, 132.4, 135.6, 135.9
(d, 4JCF = 2.8 Hz), 137.2, 137.4, 150.1, 157.1 (d, 1JCF = 245.6 Hz), 158.5, 159.2, 162.6 (d, 1JCF = 243.7 Hz);
m/z 444 (100, M + H); HRMS (ES): found 444.1068. C26H1735ClF2N3+ requires 444.1079.
,
3
3
[2-(4-Chlorophenyl)-6-(4-fluorophenyl)quinazolin-4-yl]-3-fluorophenylamine (4h). A mixture of 3h (0.30 g,
0.70 mmol), PdCl2(PPh3)2 (0.024 g, 0.035 mmol), 4-fluorophenylboronic acid (0.12 g, 0.84 mmol) and
K2CO3 (0.14 g, 1.05 mmol) in THF-isopropanol (10 mL) afforded 4h as a white solid (0.22 g, 71%),
mp. 226–228 ◦C; Rf (toluene) 0.38;
ν
(ATR) 431, 522, 670, 762, 820, 969, 1084, 1138, 1159, 1232, 1428,
δH (500 MHz, DMSO-d6) 7.00 (td, J = 2.0 and 8.5 Hz, 1H), 7.40
max
1488, 1560, 1604, 2857, 2926, 3446 cm−1
;
(t, J = 8.5 Hz, 2H), 7.49 (q, J = 8.0 Hz, 1H), 7.58 (d, J = 8.5 Hz, 2H), 7.75 (dd, J = 2.0 and 8.5 Hz, 1H),
7.88–7.97 (m, 4H), 8.20 (dd, J = 2.0 and 8.5 Hz, 1H), 8.42 (d, J = 8.5 Hz, 2H), 8.83 (d, J = 2.0 Hz, 1H),
10.15 (s, 1H); δC (125 MHz, DMSO-d6) 109.4 (d, 2JCF = 25.6 Hz), 110.6 (d, 2JCF = 20.9 Hz), 114.6, 116.3
4
3
(d, 2JCF = 21.9 Hz), 118.3 (d, JCF = 2.8 Hz), 120.9, 129.1, 129.2, 129.4, 129.6 (d, JCF = 7.6 Hz), 129.9,

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130.5 (d, 3JCF = 9.5 Hz), 132.6, 135.7, 136.0 (d, 4JCF = 2.8 Hz), 137.4, 141.4 (d, 3JCF = 10.5 Hz), 150.2, 158.3,
158.4, 162.5 (d, 1JCF = 239.9 Hz), 162.6 (d, 1JCF = 243.6 Hz); m/z 444 (100, M + H); HRMS (ES): found
444.1081. C26H1735ClF2N3+ requires 444.1079.
[2-(4-Chlorophenyl)-6-(4-fluorophenyl)quinazolin-4-yl]-4-fluorophenylamine (4i). A mixture of 3i (0.30 g,
0.70 mmol), PdCl2(PPh3)2 (0.024 g, 0.035 mmol), 4-fluorophenylboronic acid (0.12 g, 0.84 mmol) and
K2CO3 (0.14 g, 1.05 mmol) in THF-isopropanol (10 mL) afforded 4i as a white solid (0.23 g, 76%),
mp. 221–222 ◦C; Rf (toluene) 0.31;
ν
(ATR) 435, 487, 506, 527, 822, 945, 1009, 1082, 1153, 1205, 1420,
max
1503, 1533, 1560, 3451 cm−1
; δH (500 MHz, DMSO-d6) 7.31 (t, J = 8.5 Hz, 2H), 7.39 (t, J = 8.5 Hz, 2H),
7.55 (d, J = 8.5 Hz, 2H), 7.86–7.96 (m, 5H), 8.17 (dd, J = 2.0 and 8.5 Hz, 1H), 8.38 (d, J = 8.5 Hz,
2H), 8.80 (d, J = 2.0 Hz, 1H), 10.06 (s, 1H); δC (125 MHz, DMSO-d6) 114.5, 115.6 (d, 2JCF = 21.7 Hz),
3
3
116.3 (d, 2JCF = 21.7 Hz), 120.8, 124.9 (d, JCF = 8.0 Hz), 128.9, 129.1, 129.4 (d, JCF = 8.0 Hz), 130.0,
4
4
132.3, 135.5, 135.7 (d, JCF = 2.8 Hz), 136.0 (d, JCF = 3.7 Hz), 137.2, 137.5, 150.0, 158.4, 158.5, 158.9
(d, 1JCF = 239.4 Hz), 162.6 (d, JCF = 243.9 Hz); m/z 444 (100, M + H); HRMS (ES): found 444.1078.
1
C26H1735ClF2N3+ requires 444.1079.
[2-(4-Chlorophenyl)-6-(4-fluorophenyl)quinazolin-4-yl]-3-chlorophenylamine (4j). A mixture of 3j (0.30 g,
0.67 mmol), PdCl2(PPh3)2 (0.023 g, 0.034 mmol), 4-fluorophenylboronic acid (0.11 g, 0.80 mmol) and
K2CO3 (0.14 g, 1.00 mmol) in THF-isopropanol (10 mL) afforded 4j as a white solid (0.19 g, 61%),
◦
mp. 244–246 C; Rf (toluene) 0.40;
ν
(ATR) 530, 673, 765, 818, 828, 1086, 1156, 1206, 1351, 1403,
max
1425, 1474, 1494, 1508, 1526, 1556, 1598, 3450 cm−1
; δH (500 MHz, DMSO-d6) 7.23 (dd, J = 2.5 and
8.5 Hz, 1H), 7.39 (t, J = 8.5 Hz, 2H), 7.49 (t, J = 8.5 Hz, 1H), 7.57 (d, J = 8.5 Hz, 2H), 7.90-7.95 (m, 4H)
,
8.13 (s, 1H), 8.18 (dd, J = 2.0 and 8.5 Hz, 1H), 8.41 (d, J = 8.5 Hz, 2H), 8.80 (d, J = 2.5 Hz, 1H), 10.09
(s, 1H)
;
δC (125 MHz, DMSO-d6) 114.6, 116.3 (d, 2JCF = 21.8 Hz), 120.8, 120.9, 122.2, 123.8, 129.0, 129.2,
4
129.5 (d, 3JCF = 8.5 Hz), 129.9, 130.6, 133.6, 133.2, 135.7, 135.9 (d, JCF = 2.8 Hz), 137.4, 137.5, 141.1,
1
150.2, 158.2, 158.3, 162.6 (d, JCF = 243.7 Hz); m/z 460 (100, M + H); HRMS (ES): found 460.0780.
C26H1735Cl2FN3+ requires 460.0784.
[2-(4-Chlorophenyl)-6-(4-fluorophenyl)quinazolin-4-yl]-2,4-difluorophenylamine (4l). A mixture of 3l (0.30 g,
0.67 mmol), PdCl2(PPh3)2 (0.023 g, 0.034 mmol), 4-fluorophenylboronic acid (0.11 g, 0.80 mmol) and
K2CO3 (0.14 g, 1.00 mmol) in THF-isopropanol (10 mL) afforded 4l as a white solid (0.21 g, 69%),
mp. 210–212 ◦C; Rf (toluene) 0.48;
ν
(ATR) 425, 515, 737, 817, 838, 964, 1012, 1089, 1161, 1234, 1415,
max
1502, 1530, 1558, 1585, 3450 cm−1
; δH (500 MHz, DMSO-d6) 7.22 (td, J = 2.5 and 8.0 Hz, 1H), 7.36–7.43
(m, 3H), 7.45–7.52 (m, 3H), 7.68 (q, J = 8.0 Hz, 1H), 7.91–7.96 (m, 3H), 8.21 (dd, J = 2.0 and 8.5 Hz,
1H), 8.25 (d, J = 8.5 Hz, 2H), 8.80 (d, J = 2.0 Hz, 1H), 10.09 (s, 1H); δC (125 MHz, DMSO-d6) 105.0
(t, 2JCF = 25.2 Hz), 112.0 (d, 2JCF = 21.7 Hz), 114.3, 116.3 (d, 2JCF = 21.7 Hz), 120.9, 123.0, 123.3, 128.9,
129.4 (d, 3JCF = 8.0 Hz), 129.5, 129.7 (d, 4JCF = 2.8 Hz), 129.8, 132.4, 135.6, 135.8 (d, 4JCF = 3.5 Hz), 137.2,
137.3, 150.0, 157.2 (d, 1JCF = 248.5 Hz), 158.4, 158.5, 162.6 (d, 1JCF = 243.9 Hz); m/z 462 (100, M + H);
HRMS (ES): found 462.0982. C26H1635ClF3N3+ requires 462.0985.
3.6. Reaction Sequence for the Preparation of 4e and 4k from 1a and 1b, Respectively
3.6.1. Typical Procedure for the Preparation of 5a and 5b
6-(4-Fluorophenyl)quinazolin-4(3H)-one (5a). A mixture of 6-bromoquinazolin-4(3H)-one 1a (0.50 g,
2.22 mmol) PdCl2(PPh3)2 (0.078 g, 0.11 mmol) and K2CO3 (0.46 g, 3.33 mmol) in dioxane-water
(20 mL) in a two-necked round-bottomed flask was purged with argon gas for 30 minutes.
4-Fluorophenylboronic acid (0.37 g, 2.66 mmol) in dioxane (10 mL) was added via a syringe.
The reaction mixture was stirred at 70 ◦C for 3 h until the starting material was completely consumed
(TLC monitoring). After cooling the mixture, the product was extracted with chloroform and the
combined organic layers were washed with water, dried over Na2SO4, filtered, and evaporated under
reduced pressure. The residue was purified by recrystallization from acetonitrile to afford 5a as a white
solid (0.43 g, 81%), mp. 228–230 ◦C;
ν
(ATR) 496, 698, 746, 821, 923, 1160, 1239, 1275, 1333, 1384,
max

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1477, 1603, 1687, 2673, 3046 cm−1
;
δH (500 MHz, DMSO-d6) 7.31 (t, J = 9.0 Hz, 2H), 7.73 (d, J = 8.5 Hz,
1H), 7.79 (t, J = 8.5 Hz, 2H), 8.09 (dd, J = 2.0 and 8.5 Hz, 1H), 8.10 (s, 1H), 8.28 (d, J = 2.0 Hz, 1H), 12.30
(br.s, 1H); δC (125 MHz, DMSO-d6) 116.4 (d, 2JCF = 21.7 Hz), 123.2, 123.6, 127.8, 129.4 (d, 3JCF = 7.9 Hz),
133.4 (d, 4JCF = 2.8 Hz), 134.0, 135.5, 135.6, 138.0, 146.4, 162.6 (d, 1JCF = 240.5 Hz); m/z 241 (100, M + H);
HRMS (ES): found 241.0780. C14H10FN2O+ requires 241.0777.
2-(4-Chlorophenyl)-6-(4-fluorophenyl)quinazolin-4(3H)-one (5b). A mixture of 6-bromo-2-(4-chlorophenyl)
quinazolin-4(3H)-one 1b (0.5 g, 1.49 mmol) PdCl2(PPh3)2 (0.052 g, 0.074 mmol), 4-fluorophenylboronic
acid (0.25 g, 1.78 mmol) and K2CO3 (0.31 g, 2.23 mmol) in dioxane-water (30 mL) afforded 5b as
a white solid (0.46 g, 88%), mp. 151–153 ◦C;
1308, 1436, 1483, 1603, 1674, 2926, 3056 cm−1
ν
max (ATR) 501, 539, 693, 719, 752, 825, 1118, 1180, 1190,
δH (500 MHz, DMSO-d6) 7.32 (t, J = 8.0 Hz, 2H), 7.61
;
(d, J = 8.5 Hz, 2H), 7.79–7.85 (m, 3H), 8.13 (dd, J = 2.0 and 8.5 Hz, 1H), 8.20 (d, J = 8.5 Hz, 2H), 8.33
(d, J = 2.5 Hz, 1H), 12.67 (br.s, 1H); δC (125 MHz, DMSO-d6) 116.4 (d, 2JCF = 21.8 Hz), 122.6, 128.3, 128.5,
3
4
128.9, 129.1, 129.2 (d, JCF = 8.6 Hz), 130.1, 131.8, 132.4 (d, JCF = 2.8 Hz), 136.4, 144.8, 146.3, 148.9,
161.2, 162.5 (d, 1JCF = 243.5 Hz); m/z 351 (100, M + H); HRMS (ES): found 351.0702. C20H1335ClFN2O
requires 351.0700.
3.6.2. Typical Procedure for the Preparation of 6a and 6b
4-Chloro-6-(4-fluorophenyl)quinazoline (6a). Triethylamine (4 mL) was added dropwise at room
temperature to a stirred mixture of 5a (0.40 g, 1.66 mmol) and phosphoryl chloride (10 mL) in
a round-bottomed flask. The reaction mixture was stirred under reflux for 5 h and then allowed
to cool to room temperature. Ice-cold water was added to the mixture and the product was extracted
with chloroform. The combined organic layers were washed with an aqueous solution of NaHCO3,
dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude product was
◦
recrystallized from ethanol to afford 6a as a white solid (0.33 g, 77%), mp. 154–156 C (EtOH);
ν
(ATR) 478, 513, 523, 697, 825, 991, 1096, 1146, 1225, 1335, 1484, 1511, 1545, 1565, 1600 cm−1
;
max
δH (500 MHz, DMSO-d6) 7.16 (t, J = 8.5 Hz, 2H), 7.47 (d, J = 8.5 Hz, 1H), 7.80 (t, J = 8.5 Hz, 2H), 8.12
(dd, J = 2.0 and 8.5 Hz, 1H), 8.33 (s, 1H), 8.37 (d, J = 2.0 Hz, 1H); δC (125 MHz, DMSO-d6) 116.3
3
4
(d, 2JCF = 21.8 Hz), 122.8, 123.1, 128.8, 129.2 (d, JCF = 8.3 Hz), 132.3, 134.1 (d, JCF = 3.2 Hz), 136.7,
1
149.8, 157.7, 159.0, 162.4 (d, JCF = 240.2 Hz); m/z 258 (100, M + H); HRMS (ES): found 257.8652.
C14H835ClFN2 requires 257.8652.
4-Chloro-2-(4-chlorophenyl)-6-(4-fluorophenyl)quinazoline (6b). A mixture of 5b (0.40 g, 1.14 mmol),
trimethylamine (4 mL) and phosphoryl chloride (10 mL) afforded 6b as a white solid (0.35 g, 83%),
◦
mp. 216–218 C (EtOH);
ν
(ATR) 514, 553, 700, 735, 778, 821, 988, 1010, 1088, 1162, 1237, 1360,
max
1401, 1431, 1492, 1514, 1554, 1604 cm−1
; δH (500 MHz, DMSO-d6) 7.31 (t, J = 8.5 Hz, 2H), 7.61
(d, J = 8.5 Hz, 2H), 7.79–7.84 (m, 3H), 8.13 (dd, J = 2.0 and 8.5 Hz, 1H), 8.19 (d, J = 8.5 Hz, 2H),
8.31 (d, J = 2.0 Hz, 1H)
;
δC (125 MHz, DMSO-d6) 116.2 (d, 2JCF = 20.4 Hz), 121.3, 123.4, 125.6, 128.7,
4
128.9, 129.1 (d, 3JCF = 8.3 Hz), 130.2, 131.9, 134.6 (d, JCF = 3.2 Hz), 138.4, 139.8, 148.6, 157.8, 158.2,
162.6 (d, 1JCF = 245.3 Hz); m/z 368 (100, M + H); HRMS (ES): found 368.0435. C20H1235Cl2FN2
requires 368.0273.
3.6.3. Typical Procedure for the Preparation of 4e and 4k
[6-(4-Fluorophenyl)quinazolin-4-yl]-4-bromophenylamine (4e). A mixture of 6a (0.30 g, 1.16 mmol),
4-bromaniline (0.22 g, 1.28 mmol) and concentrated HCl (0.01 g, 0.27 mmol) in 3:1 THF-isopropanol
(v/v, 10 mL) in a round-bottomed flask was stirred under reflux for 5 h. The mixture was then allowed
to cool to room temperature and quenched with ice-cold water. The product was extracted with
ethyl acetate and the combined organic layers were washed with an aqueous solution of NaHCO3,
dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The crude product
was purified by column chromatography on silica gel to afford 4e as a white solid (0.37 g, 81%),

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◦
mp. 227–229 C; Rf (1:1, toluene/ethyl acetate) 0.69;
ν
max (ATR) 477, 507, 662, 823, 925, 1006, 1160, 1232,
1358, 1416, 1487, 1498, 1523, 1560, 1603, 3280 cm−1
;
δH (500 MHz, DMSO-d6) 7.38 (t, J = 8.5 Hz, 2H),
7.58 (d, J = 8.5 Hz, 2H), 7.84 (d, J = 8.5 Hz, 1H), 7.85 (d, J = 8.5 Hz, 2H), 7.90 (t, J = 8.5 Hz, 2H), 8.16
(dd, J = 2.0 and 8.5 Hz, 1H), 8.59 (s, 1H), 8.78 (d, J = 8.0 Hz, 1H), 9.96 (s, 1H); δC (125 MHz, DMSO-d6)
115.8, 115.8, 116.3 (d, 2JCF = 21.7 Hz), 120.8, 124.7, 128.9, 129.6 (d, 3JCF = 8.0 Hz), 131.7, 132.3, 136.0
1
(d, 4JCF = 2.8 Hz), 137.5, 138.9, 149.4, 154.8, 158.1, 162.6 (d, JCF = 242.8 Hz); m/z 394 (100, M + H);
HRMS (ES): found 394.0356. C20H1479BrFN3+ requires 394.0356.
[2-(4-Chlorophenyl)-6-(4-fluorophenyl)quinazolin-4-yl]-4-bromophenylamine (4k). A mixture of 6b (0.30 g,
0.81 mmol), 4-bromoaniline (0.15 g, 0.89 mmol) and concentrated HCl (0.01 g, 0.27 mmol) in 3:1
THF-isopropanol (v/v, 10 mL) afforded 4k as a white solid (0.32 g, 79%), mp. 257–260 ◦C; Rf (toluene)
0.36;
ν
(ATR) 499, 521, 666, 740, 817, 1012, 1090, 1161, 1230, 1354, 1398, 1417, 1489, 1511, 1525,
max
1557, 1595, 3428 cm-1; δH (500 MHz, DMSO-d6) 7.39 (t, J = 8.5 Hz, 2H), 7.58 (d, J = 8.5 Hz, 2H), 7.65
(d, J = 8.5 Hz, 2H), 7.87–7.96 (m, 5H), 8.20 (dd, J = 2.5 and 8.5 Hz, 1H), 8.40 (d, J = 9.0 Hz, 2H), 8.82
(d, J = 2.0 Hz, 1H), 10.19 (s, 1H); δC (125 MHz, DMSO-d6) 114.5, 116.2, 116.3 (d, 2JCF = 20.7 Hz), 120.9,
124.9, 129.1, 129.2, 129.3, 129.5 (d, 3JCF = 8.5 Hz), 130.1, 131.8, 131.9, 132.7, 135.8, 135.9 (d, 4JCF = 2.8 Hz),
137.5, 138.8, 158.3, 158.4, 162.7 (d, 1JCF = 243.7 Hz); m/z 504 (100, M + H); HRMS (ES): found 504.0278.
C26H1779Br35ClFN3 requires 504.0282.
+
3.7. Materials and Methods for the In Vitro Cytotoxicity Assay
The 4-anilinoquinazolines 3a
–l and 4a–l were evaluated for growth inhibitory activity against the
MCF-7 and HeLa cell lines against Gefitnib as a reference standard using the MTT assay developed by
Mosmann [22] with slight modification as described in our previous investigation [
of cell viability was calculated using the following formula:
9]. The percentage
Mean Absorbance of sample × 100
%Cell viability =
(1)
Mean Absorbance of control
The LC50 values (the lethal concentration at which 50% of the cells are killed) were calculated
as the concentration of the test sample that resulted in 50% reduction of absorbance compared to
untreated cells. The intensity of the MTT formazan produced by living metabolically active cells is
directly proportional to the number of live cells present.
3.8. Inhibition of EGFR-TK
The inhibitory activities of compounds 3g, 3l, 4l and Gefitinib towards EGFR-TK were tested
using enzyme-linked immunosorbent assay (ELISA) technique with purified Epidermal Growth
Factor Receptor (Sigma-Aldrich, Bradford, UK). The procedure was carried out according to the
manufacturer’s protocol. Ninety-six-well plates were pre-coated with 100
µ
L of a 4:1 poly(Glu,Tyr)
◦
solution (5 mg/mL) at 37 C overnight. The enzyme reaction was conducted in freshly prepared
kinase reaction buffer (25 mmol/L HEPES, pH 7.4, 5 mmol/L MgCl2, 2 mmol/L MnCl2, 100 µmol/L
Na3VO4 and 1 mmol/L dithiothreitol). Ten microlitres of the test samples at six different concentrations
(0.005–0.2
µM) and 20
µL of dilute solution of purified recombinant EGFR tyrosine kinase proteins were
added to each reaction well. The kinase reaction was initiated by the addition of 50
µL of a solution of
adenosine-50-triphosphate, disodium salt (5 mmol/L). DMSO and EGFR (without any test sample)
were also included in this experiment as negative and positive controls, respectively. The plates
were then incubated at 37 ◦C for 1 h and washed three times with phosphate-buffered saline (PBS).
Phosphorylated proteins were probed with monoclonal anti-phosphotyrosine antibody produced in
mouse (Sigma-Aldrich, St. Louis, MO, USA). After another 1 h incubation at 37 ◦C, the plates were
again washed three times with PBS, followed by the addition 100
substrate solution. After incubation in the dark for extra 10 min, the reaction was terminated by adding
100 L of 2 M H2SO4 solution. The plates were analysed using the Thermo Varioskan Flash Spectral
µL of freshly prepared peroxidase
µ

Pharmaceuticals 2017, 10, 87
17 of 19
Scanning Multimode Reader (Thermo Fisher Scientific, Waltham, MA, USA) at 492 nm. The assay was
performed in triplicate. The EGFR-TK inhibition percentage was determined as follows:
(ODsamples − ODpositive
)
)
% Inhibition =
where OD is the optical density.
(2)
(ODnegative − ODpositive
The half-maximal inhibitory concentration value (LC50) was obtained from the curves of
percentage inhibition.
3.9. Molecular Docking Studies
3.9.1. Protein Structure
The starting structure of EFGR-TK was obtained from RCSB PDB (PDB id: 1M17) [23] and all
water molecules and heteroatoms were removed. The polar hydrogen atoms, Kollman–Amber united
atom charges and solvation parameters were then added using AutoDockTools [24].
3.9.2. Ligand Structure
The starting coordinates of the ligand for control docking (erlotinib) was obtained from the
heteroatoms in the EFGR-TK crystal (PDB id: 1M17) and the coordinates for Gefitinib and compounds
3g
,
3l and 4l were generated using ChemDraw Professional 15.0 (PerkinElmer Informatics, Waltham,
3l and 4l were minimised with Chem3D module in
MA, USA). Gefitinib and compounds 3g
,
ChemOffice Professional 15.0 (PerkinElmer Informatics). All polar hydrogen atoms for the ligands
were left intact. The Gasteiger chargers and torsional angles of the ligands were assigned with
AutoDockTools [24]. Standard protocol from AutoDock4.2 [24] for hydrated docking was applied
where water molecules were added randomly around the ligands.
3.9.3. Molecular Docking Simulation
Grid maps were centred at the ligand binding site in EGFR-TK crystal (PDB id: 1M17) with 90
90 points with 0.375 Å spacing. Lamarckian genetic algorithm of 1,000,000 energy evaluations
×
90
×
per run and a maximum number of 27,000 generation was applied. The number of individuals in
population was 350 and the crossover rate was 0.8. The docked conformations were clustered with
root mean square of 2.0 Å from 200 docking runs.
4. Conclusions
In summary, a series of novel 4-(halogenophenylamino)-6-bromoquinazolines and their
6-(4-fluorophenyl)–substituted derivatives was designed, synthesised, and evaluated for cytotoxicity
in vitro. Most of the synthesised compounds exhibited inhibitory activity against MCF-7 and HeLa
cells. SAR based on these in vitro cytotoxicity results revealed that the presence of a 4-fluorophenyl- or
2,4-difluorophenylaniline is more desirable for cytotoxicity against both cell lines. A combination of
2-(4-chlorophenyl) and 6-(4-fluorophenyl) substituents favours activity against HeLa cells more so than
the MCF-5 cell line. Compound 3l not only demonstrated strong anti-proliferation activities against
the two cancer cell lines, but also showed significant inhibitory activity (LC50 = 37.66 nM) towards
EGFR, comparable to that of the medicinally important EGFR inhibitor Gefitinib (LC50 = 31.44 nM)
Molecular docking (in silico) studies suggested that the title compounds bind nicely to the region of
EGFR, with comparable binding affinity with Gefitinib. The observed increased in vitro cytotoxicity
and inhibitory activity against EGFR-TK due to the presence of a 2-(4-halogenophenyl) substituent
encourages further elaboration of this position to lead to derivatives with anticancer properties.
.

Pharmaceuticals 2017, 10, 87
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Supplementary Materials: The following are available online at www.mdpi.com/1424-8247/10/4/87/s1.
Acknowledgments: The authors thank the University of South Africa and the National Research Foundation
(SA) for financial assistance. The Higher Institution Centre of Excellence (311/CIPPM/44001005) of the Malaysia
Ministry of Higher Education has also supported part of this work. We are also grateful to T.J. Makhafola for
cytotoxicity assays and the University of Stellenbosch Central Analytical Facility for mass spectrometric data.
Author Contributions: Hugues K. Paumo executed the synthesis and characterisation part as well as the EGFR-TK
inhibition assays under the supervision of Malose Jack Mphahlele (M.J.M.), who conceptualised the project and
the paper. Yee Siew Choong conducted the molecular docking studies. All the authors contributed in the design
and writing of the paper, with M.J.M. as the lead author.
Conflicts of Interest: The authors declare no conflict of interest.
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