Benzofuran–appended 4-aminoquinazoline hybrids as epidermal growth factor receptor tyrosine kinase inhibitors: synthesis, biological evaluation and molecular docking studies

Article abstract of DOI:10.1080/14756366.2018.1510919

A series of 2-arylbenzo[b]furan–appended 4-aminoquinazoline hybrids were prepared and evaluated for cytotoxicity in vitro against the human lung cancer (A549), colorectal adenocarcinoma (Caco-2), hepatocellular carcinoma (C3A) and cervical cancer (HeLa) cell lines. Compounds 10d and 10j exhibited significant cytotoxicity against the C3A and Caco-2 cell lines and induced apoptosis in these cell lines. Likewise, compounds 10d and 10e exhibited significant inhibitory activity towards epidermal growth factor receptor-tyrosine kinase phosphorylation (IC₅₀ values of 29.3 nM and 31.1 nM, respectively) against Gefitinib (IC₅₀ = 33.1 nM). Molecular docking of compounds 10 into EGFR-TK active site suggests that they bind to the region of EGFR like Gefitinib does. (Figure presented.).

Full text of DOI:10.1080/14756366.2018.1510919

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Benzofuran–appended 4-aminoquinazoline
hybrids as epidermal growth factor receptor
tyrosine kinase inhibitors: synthesis, biological
evaluation and molecular docking studies
Malose J. Mphahlele, Marole M. Maluleka, Abimbola Aro, Lyndy J. McGaw &
Yee Siew Choong
To cite this article: Malose J. Mphahlele, Marole M. Maluleka, Abimbola Aro, Lyndy J. McGaw
& Yee Siew Choong (2018) Benzofuran–appended 4-aminoquinazoline hybrids as epidermal
growth factor receptor tyrosine kinase inhibitors: synthesis, biological evaluation and molecular
docking studies, Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1, 1516-1528, DOI:
10.1080/14756366.2018.1510919
To link to this article: https://doi.org/10.1080/14756366.2018.1510919
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2018, VOL. 33, NO. 1, 1516–1528
https://doi.org/10.1080/14756366.2018.1510919
RESEARCH PAPER
Benzofuran–appended 4-aminoquinazoline hybrids as epidermal growth factor
receptor tyrosine kinase inhibitors: synthesis, biological evaluation and molecular
docking studies
Malose J. Mphahlelea, Marole M. Malulekaa, Abimbola Arob, Lyndy J. McGawb and Yee Siew Choongc
b
aDepartment of Chemistry, College of Science, Engineering and Technology, University of South Africa, Florida, South Africa; Phytomedicine
c
Programme, Department of Paraclinical Sciences, University of Pretoria, Onderstepoort, South Africa; Institute for Research in Molecular
Medicine (INFORMM), Universiti Sains Malaysia, Minden, Malaysia
ABSTRACT
ARTICLE HISTORY
Received 15 June 2018
Revised 7 August 2018
Accepted 8 August 2018
A series of 2-arylbenzo[b]furan–appended 4-aminoquinazoline hybrids were prepared and evaluated for
cytotoxicity in vitro against the human lung cancer (A549), colorectal adenocarcinoma (Caco-2), hepatocel-
lular carcinoma (C3A) and cervical cancer (HeLa) cell lines. Compounds 10d and 10j exhibited significant
cytotoxicity against the C3A and Caco-2 cell lines and induced apoptosis in these cell lines. Likewise,
compounds 10d and 10e exhibited significant inhibitory activity towards epidermal growth factor recep-
tor-tyrosine kinase phosphorylation (IC50 values of 29.3 nM and 31.1 nM, respectively) against Gefitinib
(IC50 ¼ 33.1 nM). Molecular docking of compounds 10 into EGFR-TK active site suggests that they bind to
the region of EGFR like Gefitinib does.
KEYWORDS
Benzofuran-aminoquinazo-
lines; cytotoxicity; apoptosis;
EGFR-TK; molecular docking
Br
Cl
O
NH
Br
N
N
F
10d
Inhibition of EGFR
Cytotoxicity against C3A cell line
10d: IC50 = 8.96 µM
Gefitinib: IC50 = 5.01 µM
10d: IC50 = 29.26 nM
Gefitinib: IC50 = 33.07 nM
ORTEP diagram of 10f
Apoptosis (Annexin V/PI staining)
Docking pose of 10d in EGFR
1. Introduction
binding at the ATP site4,5. Gefitinib (1) shown in Figure 1, for
example, is a selective inhibitor of the EGFR-TK and it inhibits
tumor pathogenesis, metastasis and angiogenesis, and also pro-
motes apoptosis6. Structure–activity relationship studies for the
ability of the 4-anilinoquinazolines to inhibit EGFR-TK activity
revealed that both of the quinazoline nitrogen atoms are essential
for biological activity and the aniline moiety bearing lipophilic
substituents such as chloro, bromo and trifluoromethyl group was
also important as it occupies the lipophilic pocket7. In the design
of new drugs, hybridization approach has been found to provide
Epidermal growth factor receptor (EGFR) is a tyrosine kinase
receptor that has been found to play an essential role in normal
cell growth and differentiation and it is also involved in tumor
proliferation and survival1. This receptor is over expressed in vari-
ous types of tumors including colon, non-small cell lung, prostate,
breast and ovarian cancers2. EGFR continues to be an attractive
target for the design and development of compounds that can
specifically bind to it and inhibit its tyrosine kinase (TK) activity
and its signal transduction pathway in cancer cells3. The 4-amino-
quinazolines have established themselves as selective and effect-
ive inhibitors of the epidermal growth factor receptor tyrosine
a
more general method to obtain molecular hybrids with
improved biological activities capable of overcoming multi-drug
resistance. This strategy has been employed before in the synthe-
kinase (EGFR-TK) phosphorylation, which results from competitive sis of an indole-ether quinazoline hybrid, Cediranib/RecentinTM (2)
CONTACT Malose J. Mphahlele
mphahmj@unisa.ac.za
Department of Chemistry, College of Science, Engineering and Technology, University of South Africa,
Private Bag X06, Florida, 1710, South Africa
Supplemental data for this article can be accessed here.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1517
H
N
CH3
F
O
N
HO
HN
N
Cl
H3CO
O
F
O
O
N
O
O
O
N
N
O
OCH3
N
H3C
Gefitinib (1)
Cediranib/Recentin™ (2)
Egonol (3)
Figure 1. Structure of Gefitinib (1), Cediranib (2) and Egonol (3).
Scheme 1. Design of benzofuran-amino quinazolines using molecular hybridization approach.
shown in Figure 1, which is a highly potent, orally bioavailable (attenuated total reflectance) accessory. The Merck kieselgel 60
and selective vascular endothelial growth factor receptor (VEGFR)
(0.063–0.200 mm) (Merck KGaA, Frankfurt, Germany) was used as
stationary phase for column chromatography. The 1H- and 13C-
NMR spectra of the prepared compounds were obtained as CDCl3
or DMSO-d6 solutions using the Agilent 500 MHz NMR spectrom-
eter (Agilent Technologies, Oxford, UK) and the chemical shifts are
quoted relative to the TMS peak. The low- and high-resolution
mass spectra were recorded at the University of Stellenbosch
inhibitor for the treatment of cancer8,9
.
In our previous investigation we observed that a combination
of the 2–(4-fluoro/chlorophenyl) group and a bromine atom at
the 6-position of the 4-anilinoquinazoline framework leads to
increased antiproliferative activity10. Literature review revealed
that most of the EGFR-TK inhibitors have a common 4-aminoqui-
nazoline core and only differ in terms of the substituents and side
chains. Moreover, the 4-anilinoquinazolines substituted with a bro-
moaniline group have previously been found to exhibit high affin-
ity and selectivity for EGFR-TK11. A bromine atom on C-5 position
of a benzofuran ring or the analogous indole moiety has been
found to impart significant biological activity of the 2-arylbenzo-
furans12 or 2-arylindoles13. These literature precedents encouraged
us to replace the phenyl ring of the 4-anilino group of the 2-aryl-
using
a Waters Synapt G2 Quadrupole Time-of-flight mass
spectrometer (Waters Corp., Milford, MA, USA) at an ionization
potential of 70 eV. The synthesis and analytical data of the 2–(4-hal-
ogenophenyl)-4-chloroquinazolines 9a and 9 b have been described
before18. Copies of 1H- and 13C-NMR spectra of compounds 6a–e,
7a–e, 8a–e, 10a–j are included as Supplementary Material.
6-bromoquinazolines with a 2-aryl-5-bromobenzofuran moiety to 2.2. Synthesis of 5-bromo-2-hydroxy-3-iodoacetophenone (5)
comprise benzofuran-appended 4-aminoquinazoline analogs of 2
(Scheme 1). This idea was also inspired by the versatility of the
A stirred solution of hydroxyacetophenone (5.26 g, 38.7 mmol) in
naturally occurring Egonol (3) shown in Figure 1 and its deriva-
5-Bromo-2-hydroxyacetophenone
acetic acid (400 ml) was treated with N-bromosuccinimide (18.02 g,
tives, which have been found to exhibit cytotoxic, anti-inflamma-
38.7 mmol) at room temperature (RT). The mixture was stirred
tory, anti-asthma, and antioxidant properties14–16. The main aim
under reflux for 1.5 h and then quenched with an ice-cold water.
The resulting precipitate was filtered and recrystallized to afford 5-
bromo-2-hydroxyacetophenone (4.92 g, 59%); mp. 74–75 ꢀC (hex-
was to evaluate the resultant molecular hybrids for potential anti-
proliferative properties in vitro against a panel of EGFR-positive
cancer cell lines2, namely, the human lung cancer (A549), colorec-
ane); ꢀmax (ATR) 491, 516, 624, 735, 879, 1081, 1195, 1285, 1346,
tal adenocarcinoma (Caco-2) and the hepatocellular carcinoma
1473, 1559, 1640, 3280 cmꢁ1 1H NMR (CDCl3) 2.63 (3H, s, CH3),
;
(C3A; HepG2/C3A) cell lines as well as the cervical cancer (HeLa)
cell line with relatively low EGFR expression17. The ability of the
resultant benzofuran-aminoquinazoline hybrids to induce apop-
tosis and their potential to inhibit EGFR-TK phosphorylation were
evaluated experimentally complemented with molecular docking
(in silico) studies into the ATP binding site of the EGFR.
6.87 (1H, d, J ¼ 8.5 Hz, H-3), 7.53 (1H, dd, J 2.4 and 8.5 Hz, H-4),
7.81 (1H, d, J ¼ 2.4 Hz, H-6), 12.14 (1H, s, OH); 13C NMR (CDCl3)
26.6, 87.6, 106.7, 118.9, 134.7, 145.7, 148.5, 203.0; HRMS (ES): MHþ,
found. C8H8O279Brþ requires: 215.9609.
5-Bromo-2-hydroxy-3-iodoacetophenone (5)
A stirred solution of 5-bromo-2-hydroxyacetophenone (3.23 g,
15.0 mmol) and N-iodosuccinimide (5.78 g, 18.0 mmol) in acetic
acid (400 ml) was refluxed for 2 h and then quenched with an ice-
cold water. The resulting precipitate was filtered and recrystallized
from EtOH to afford 5 as a brown solid (4.78 g, 93%), mp.
111–112 ꢀC (Lit19. 104–105 ꢀC); ꢀmax (ATR) 441, 539, 670, 779, 862,
2. Materials and methods
2.1. Chemistry
The melting points of the prepared compounds were recorded on
a Thermocouple digital melting point apparatus and are uncor-
rected. Their IR spectra were recorded as powders by using the
thin-film method on a Bruker VERTEX 70 FT-IR Spectrometer
1
968, 1077, 1190, 1233, 1362, 1420, 1581, 1650, 3259 cmꢁ1; H NMR
(Bruker Optics, Billerica, MA, USA) equipped with a diamond ATR (CDCl3) 2.65 (3H, s, CH3), 7.83 (1H, d, J ¼ 2.1 Hz, H-4), 8.04 (1H, d,

1518
M. J. MPHAHLELE ET AL.
J ¼ 2.1 Hz, H-6), 13.07 (1H, s, OH); 13C NMR (CDCl3) 26.4, 87.6, 2.3.4. 1–(5-Bromo-2–(3-chlorophenyl)benzofuran-7-yl)ethanone (6d)
111.1, 120.2, 133.1, 147.1, 160.2, 203.2; HRMS (ES): MHþ, found.
Solid (0.76 g, 74%), Rf ¼ 0.28, mp. 139–141 ꢀC; ꢀmax (ATR) 682, 788,
882, 1078, 1144, 1275, 1359, 1474, 1561, 1587, 1675, 3081 cmꢁ1
;
C8H7O279BrIþ requires: 341.8575.
dH (CDCl3) 2.94 (3H, s, CH3), 7.07 (1H, s, H-3), 7.41 (2H, dd, J ¼ 1.5
and 8.0 Hz, Ar), 7.78 (1H, d, J ¼ 8.0 Hz, Ar), 7.83 (1H, d, J ¼ 2.0 Hz,
H-4), 7.90 (1H, d, J ¼ 1.5 Hz, Ar), 7.97 (1H, d, J ¼ 2.0 Hz, H-6); dC
(CDCl3) 30.9, 101.6, 116.6, 123.1, 123.2, 125.2, 128.1, 128.4, 129.4,
130.4, 130.9, 132.5, 135.1, 152.1, 156.3, 194.4; HRMS (ES): found
348.9634. C16H11O235Cl79Brþ requires 348.9631. Anal calcd for
C16H10O43ClBr: C, 54.97; H, 2.88. Found: C, 54.64; H, 2.95.
2.3. Typical procedure for the synthesis of benzofuran
derivatives 6a–e
To a three necked round-bottom flask equipped with a stirrer bar,
condenser and rubber septa was added in sequence compound 5
(1 mmol), PdCl2(PPh3)2 (5 mmol % of 5), CuI (10 mmol % of 5),
Cs2CO3 (1.2 equiv. of 5) and 10:1 dioxane-water (7.0 ml/mmol of
5). The mixture was purged with nitrogen gas for 20 min. and a
balloon filled with nitrogen gas was connected to the top of the
condenser. Phenylacetylene (1.2 equiv. of 5) was added via a syr-
inge and the mixture was heated at 80 ꢀC for 2 h and then
quenched with an ice-cold water. The product was extracted with
chloroform and the combined organic layers were washed with
brine and dried over MgSO4. The salt was filtered off and the solv-
ent was evaporated under reduced pressure on a rotary evapor-
ator. The residue was purified by column chromatography on
silica gel using 30% toluene-hexane mixture as an eluent to afford
6a. The following compounds were prepared in this fashion.
2.3.5. 1–(5-Bromo-2–(4-(trifluoromethoxy)phenyl)benzofuran-7-yl)
ethanone (6e)
Solid (0.93 g, 77%), Rf ¼ 0.35, mp. 170–173 ꢀC; ꢀmax (ATR) 666, 746,
854, 987, 1033, 1199, 1267, 1363, 1405, 1502, 1614, 1676,
3082 cmꢁ1; dH (CDCl3) 2.92 (3H, s, CH3), 7.03 (1H, s, H-3), 7.34 (2H,
d, J ¼ 8.1 Hz, Ar), 7.85 (2H, d, J ¼ 9.3 Hz, Ar), 7.98 (2H, d, J ¼ 1.8 Hz,
Ar); dC (CDCl3) 30.8, 101.1, 116.6, 121.5, 123.0, 126.7, 127.7, 127.9,
128.0, 128.3, 130.2 (t, J ¼ 25.6 Hz) 132.6, 152.1, 156.4, 194.4; HRMS
þ
(ES): 398.9663. found C17H11O3F379Br
requires 398.9745. Anal
calcd for C17H10O3F3Br: C, 51.15; H, 2.53. Found: C, 51.32; H, 2.67.
2.3.1. 1–(5-Bromo-2-phenylbenzofuran-7-yl) ethanone (6a)
Solid (0.69 g, 73%), Rf ¼ 0.33, mp. 130–133 ꢀC; ꢀmax (ATR) 684, 790,
884, 989, 1095, 1179, 1279, 1360, 1490, 1567, 1586, 1677,
3064 cmꢁ1; dH (CDCl3) 2.95 (3H, s, CH3), 7.03 (1H, s, H-3), 7.42 (2H,
d, J ¼ 7.0 Hz, Ar), 7.48 (1H, t, J ¼ 7.5 Hz, Ar), 7.85 (2H, d, J ¼ 8.0 Hz,
Ar), 7.98 (1H, d, J ¼ 1.5 Hz, H-4), 7.99 (1H, d, J ¼ 1.5 Hz, H-6); dC
(CDCl3) 31.0, 100.5, 116.4, 122.9, 125.2, 127.5, 128.1, 128.6, 129.0,
129.5, 132.8, 152.1, 157.8, 194.6; HRMS (ES): found 316.9743.
C16H12O279Brþ requires 316.9726. Anal calcd for C16H11O2Br: C,
60.98; H, 3.58. Found: C, 60.96; H, 3.44.
2.4. Typical procedure for the synthesis of oxime
derivatives 7a–e
A mixture of 6 (1 equiv.) and hydroxylamine hydrochloride (1.2
equiv.) in pyridine (8.0 ml/mmol of 6) was heated at 120 ꢀC for 1 h.
The mixture was quenched with an ice-cold water and the prod-
uct was filtered. The resultant precipitate was washed thoroughly
with cold hexane and then recrystallized from ethanol to afford 7
as a solid. Compounds 7a–e were prepared in this fashion.
2.4.1. 1–(5-Bromo-2-phenylbenzofuran-7-yl)ethanone oxime (7a)
Solid (0.76 g, 93%), mp. 177–179 ꢀC; ꢀmax (ATR) 748, 777, 874,
1029, 1230, 1366, 1429, 1445, 1562, 1601, 3107, 3200 cmꢁ1; dH
(DMSO-d6) 2.42 (3H, s, CH3), 7.41 (2H, d, J ¼ 7.0 Hz, Ar), 7.47 (1H, s,
H-3), 7.53 (1H, t, J ¼ 8.5 Hz, Ar), 7.58 (2H, d, J ¼ 8.5 Hz, Ar), 7.61 (1H,
d, J ¼ 1.5 Hz, H-4), 7.84 (1H, d, J ¼ 1.5 Hz, H-6), 11.5 (1H, s, OH); dC
(DMSO-d6) 14.0, 102.2, 117.8, 122.9, 124.7, 125.3, 126.4, 129.2,
129.8, 130.8, 131.8, 151.0, 151.6, 156.9; HRMS (ES): found 330.0031.
C16H13O2N79Brþ requires 330.0051. Anal calcd for C16H12O2NBr: C,
58.20; H, 3.66; N, 4.24. Found: C, 58.33; H, 3.71; N, 4.00.
2.3.2. 1–(5-Bromo-2–(3-fluorophenyl)benzofuran-7-yl)ethanone (6b)
Solid (0.73 g, 76%), Rf ¼ 0.32, mp. 161–164 ꢀC (ATR) 667, 781, 878,
987, 1081, 1163, 1280, 1365, 1488, 1556, 1686; dH (CDCl3) 2.94 (3H,
s, CH3), 7.06 (1H, s, H-3), 7.12 (1H, t, J ¼ 8.5 Hz, Ar), 7.40 (2H, dd,
J ¼ 8.5 and 2.0 Hz, Ar), 7.64 (1H, d, J ¼ 8.5 Hz, Ar), 7.89 (1H, d, J
2.0 Hz, H-4), 8.00 (1H, d, J 2.0 Hz, H-6); dC (CDCl3) 30.9, 101.6, 112.1
2
2
(d, JCF ¼23.6 Hz) 116.4 (d, JCF ¼ 21.9 Hz), 116.5, 120.9 (d,
3
4JCF ¼ 2.8 Hz), 123.1, 127.5, 128.1, 128.4, 130.7 (d, JCF ¼ 7.5 Hz),
4
1
132.5, 152.1, 156.4 (d, JCF ¼ 2.9 Hz), 163.1 (d, JCF ¼ 249.4 Hz)
194.4; HRMS (ES): found 332.9828. C16H11O2F79Brþ requires
332.9826. Anal calcd for C16H10O2FBr: C, 57.68; H, 3.03. Found: C,
57.50; H, 2.99.
2.4.2. 1–(5-Bromo-2–(3-fluorophenyl)benzofuran-7-yl)ethanone
oxime (7b)
Solid (0.57 g, 86%), mp. 199–201 ꢀC; ꢀmax (ATR) 748, 906, 1029,
1154, 1262, 1316, 1453, 1555, 1603, 3108, 3187 cmꢁ1; dH (DMSO-
2.3.3. 1–(5-Bromo-2–(4-fluorophenyl)benzofuran-7-yl)ethanone (6c)
Solid (0.82 g, 76%), Rf ¼ 0.31, mp. 178–180 ꢀC; ꢀmax (ATR) 668, 784, d6); 2.38 (3H, s, CH3), 7.48 (1H, s, H-3), 7.49 (1H, d, J ¼ 8.0 Hz, H-6’),
898, 987, 1029, 1166, 1280, 1365, 1488, 1555, 1682 cmꢁ1; dH 7.54 (1H, t, J 7.5 Hz, H-4’), 7.59 (1H, d, J ¼ 7.5 Hz, H-2’), 7.86 (1H, t,
(CDCl3) 2.93 (3H, s, ꢁCH3), 6.98 (1H, s, H-3), 7.18 (2H, t, J ¼ 8.5 Hz, J ¼ 7.0 Hz, H-5’), 7.88 (1H, d, J ¼ 2.0 Hz, H-4), 7.94 (1H, d, J ¼ 2.0 Hz,
2
H-2’,6’), 7.83 (2H, t, J ¼ 8.7 Hz, H-3’,5’) 7.86 (1H, d, J 1.8 Hz, H-4), H-6), 11.6 (1H, s, OH); dC (DMSO-d6) 14.0, 103.3, 112.1 (d, JCF
2
4
7.97 (1H, d, J 1.8 Hz, H-6); dC (CDCl3) 30.9, 100.1, 116,3 (d, ¼23.6 Hz) 116.1 (d, JCF ¼ 21.9 Hz), 119.5, 120.9 (d, JCF ¼ 2.8 Hz),
3
3
2JCF ¼ 22.8 Hz), 125.6, 127.1, 127.6, 128.1, 128.5 (d, JCF ¼ 12.2 Hz), 123.9, 127.7, 129.6, 130.8, 131.5 (d, JCF ¼ 7.5 Hz), 132.2, 134.4,
4
1
132.2 (d, 4JCF ¼ 10.3 Hz), 132.8, 151.9, 156.9, 163.4 (d, 150.7 (d, JCF ¼ 2.9 Hz), 156.5, 163.1 (d, JCF ¼ 249.4 Hz); HRMS (ES):
1JCF ¼ 249.4 Hz) 194.5; HRMS (ES): found 332.9848. C16H11O2F79Brþ found 348.0028 C16H12NO2F79Brþ requires 348.0035. Anal calcd for
requires 333.9828. Anal calcd for C16H10O2ClBr: C, 57.68; H, 3.03. C16H11NO2FBr: C, 55.20; H, 3.18; N, 4.02. Found: C, 55.43; H, 3.33;
Found: C, 57.63; H, 3.01.
N, 3.99.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1519
2.4.3. 1–(5-Bromo-2–(4-fluorophenyl)benzofuran-7-yl)ethanone
oxime (7c)
2.5.2. 5-Bromo-2–(3-fluorophenyl)benzofuran-7-amine (8b)
Solid (0.34 g, 78%), mp. 124–126 ꢀC; ꢀmax (ATR) 721, 765, 834,
1100, 1261, 1309, 1433, 1572, 1583, 1611, 3375, 3482 cmꢁ1; dH
(DMSO-d6) 5.81 (2H, br s, NH2), 6.68 (1H, d, J ¼ 1.5 Hz, H-6), 6.95
Solid (0.61 g, 81%), mp. 216–218 ꢀC; ꢀmax (ATR) 690, 769, 852, 944,
1029, 1188, 1276, 1339, 1411, 1503, 1574, 1604 3108, 3187 cmꢁ1
;
dH (DMSO-d6) 2.39 (3H, s, CH3), 7.36 (2H, t, J ¼ 8.7 Hz, H-2’,6’), 7.43 (1H, d, J ¼ 1.5 Hz, H-4), 7.22 (1H, t, J 8.0 Hz, H-4’), 7.35 (1H, s, H-3),
(1H, s, H-3), 7.56 (1H, d, J ¼ 2.0 Hz, H-4), 7.86 (2H, d, J ¼ 2.0 Hz, H-
6), 7.97 (2H, t, J ¼ 8.7 Hz, H-3’,5’), 11.6 (1H, s, OH); dC (DMSO-d6)
7.52 (1H, dd, J 1.5 and 7.5 Hz, H-5’), 7.79 (1H, d, J ¼ 8.0 Hz, H-6’),
7.82 (1H, s, H-2’); dC (DMSO-d6) 103.5, 110.5, 111,7 (d,
2
2
3JCF ¼ 8.5 Hz), 111.9, 115.9 (d, JCF ¼ 20.9 Hz), 116.7, 121.3 (d,
(125 MHz, CDCl3) 14.0, 101.7, 115.9, 116,7 (d, JCF ¼ 21.9 Hz), 123.9,
3
3
3
4JCF ¼ 2.9 Hz), 130.8, 131.5 (d, JCF ¼ 8.5 Hz), 132.4 (d, JCF ¼ 8.6 Hz),
124.0, 125.2, 126.1, 126.2, 127.7 (d, JCF ¼ 8.5 Hz), 132.5, 150.7 (d,
1
4JCF ¼ 4.7 Hz), 156.3, 163.1 (d, JCF ¼ 245.6 Hz), HRMS (ES): found:
4
1
135.7, 142.1, 154.3 (d, JCF ¼ 2.8 Hz),163.1 (d, JCF ¼ 240.6 Hz);
HRMS (ES): found: 305.9852. C14H10NOF79Brþ requires 305.9831.
Anal calcd for C14H9NOFBr: C, 54.93; H, 2.96; N, 4.58. Found: C,
54.87; H, 3.09; N, 4.72.
348.9934. C16H12NO2F79Brþ requires 348.9937. Anal calcd for
C16H11NO2FBr: C, 55.20; H, 3.18; N, 4.02. Found: C, 55.41; H, 3.31;
N, 4.00.
2.4.4. 1–(5-Bromo-2–(3-chlorophenyl)benzofuran-7-yl)ethanone
oxime (7d)
2.5.3. 5-Bromo-2–(4-fluorophenyl)benzofuran-7-amine (8c)
Solid (0.39 g, 71%), mp. 121–123 ꢀC; ꢀmax (ATR) 688, 765, 915,
1100, 1214, 1309, 1433, 1502, 1583, 1612, 3370, 3473 cmꢁ1; dH
(DMSO-d6) 5.75 (2H, brs, NH2), 6.66 (1H, d, J ¼ 2.0 Hz, H-6), 6.93
(1H, d, J ¼ 2.0 Hz, H-4), 7.23 (1H, s, H-3), 7.34 (2H, t, J 8.7 Hz, H-
3’,5’), 7.99 (2H, t, J ¼ 8.5 Hz, H-2’,6’); dC (DMSO-d6) 102.1, 110.5,
Solid (0.39 g, 81%), mp. 192–195 ꢀC ꢀmax (ATR) 748, 849, 916, 1006,
1090, 1162, 1278, 1314, 1337, 1429, 1448, 1561, 1604, 3106,
3211 cmꢁ1; dH (DMSO-d6) 2.29 (3H, s, CH3), 7.47 (1H, s, H-3), 7.58 (1H,
dd, J¼ 2.0 and 8.5 Hz, H-6’), 7.58 (1H, t, J¼ 8.5 Hz, H-5’), 7.67 (1H, d,
J¼ 2.0 Hz, H-4), 7.85 (1H, d, J¼ 2.0 Hz, H-6), 7.67 (1H, d, J ¼ 8.0 Hz, H-
4’), 7.98 (1H, d, J¼ 2.0 Hz, H-2), 11.0 (1H, s, OH); dC (DMSO-d6) 14.0,
103.3, 116.1, 123.9, 124.1, 124.3, 124.9, 125.7, 129.6, 131.5, 131.6,
132.2, 134.4, 150.7, 150.8, 155.5; HRMS (ES): found: 364.9722.
C16H12NO235Cl79Brþ requires 364.9641. Anal calcd for C16H11NO2ClBr:
C, 52.70; H, 3.04; N, 3.84. Found: C, 52.65; H, 3.14; N, 3.88.
2
4
111.4, 116,4 (d, JCF ¼ 21.8 Hz), 116.6, 126.8 (d, JCF ¼ 2.9 Hz) 127.4
(d, 3JCF ¼ 8.5 Hz), 131.1, 135.6, 142.1, 154.8, 162.7 (d,
1JCF ¼ 244.7 Hz); HRMS (ES): found 305.9715. C14H10NOF79Brþ
requires 305.9776. Anal calcd for C14H9NOFBr: C, 54.93; H, 2.96; N,
4.58. Found: C, 54.88; H, 3.07; N, 4.70.
2.5.4. 5-Bromo-2–(3-chlorophenyl)benzofuran-7-amine (8d)
Solid (0.31 g, 65%), mp. 106–108 ꢀC; ꢀmax (ATR) 737, 854, 879,
1126, 1270, 1429, 1593, 1615, 3372, 3482 cmꢁ1; dH (DMSO-d6) 5.82
(2H, br s, NH2), 6.68 (1H, d, J ¼ 2.0 Hz, H-6), 6.94 (1H, d, J ¼ 2.0 Hz,
H-4) 7.37 (1H, s, H-3), 7.43 (1H, d, J ¼ 8.0, H-4’), 7.51 (1H, t,
J ¼ 7.5 Hz, H-5’), 7.90 (1H, d, J ¼ 7.5 Hz, H-6’), 8.05 (1H, d, J ¼ 8.0 Hz,
H-2’); dC (DMSO-d6) 103.6, 110.6, 111.7, 116.7, 123.7, 124.7, 128.9,
130.8, 131.3, 132.2, 134.4, 135.7, 142.2, 154.0; HRMS (ES): found
321.9556. C14H10NO35Cl79Brþ requires 321.9536. Anal calcd for
C14H9NOClBr: C, 52.13; H, 2.81; N, 4.34. Found: C, 52.31; H, 2.66;
N, 4.28.
2.4.5. 1–(5-Bromo-2–(4-(trifluoromethoxy)phenyl)benzofuran-7-yl)
ethanone oxime (7e)
Solid (0.61 g, 78%), mp. 167–169 ꢀC; ꢀmax (ATR) 681, 777, 908,
1029, 1161, 1279, 1337, 1449, 1460, 1510, 1583, 3074, 3208 cmꢁ1
;
dH (DMSO-d6) 2.51 (3H, s, CH3), 6.99 (1H, s, H-3), 7.30 (2H, d,
J ¼ 8.5 Hz, H-3’,5’), 7.61 (1H, d, J ¼ 1.5 Hz, H-4), 7.72 (1H, d,
J ¼ 1.5 Hz, H-6), 7.88 (2H, d, J ¼ 8.0 Hz, H-2’,6’), 8.34 (1H, s, OH); dC
(DMSO-d6) 13.6, 101.3, 116.3, 121.3, 121.3, 122.7, 124.3, 126.1,
126.8, 130.1 (t, JCF ¼ 25.6 Hz), 133.2, 149.6, 150.9, 153.1, 156.1;
HRMS (ES): found 414.9932. C17H12NO3F379Brþ requires 414.9854.
Anal calcd for C17H11NO3F3Br: C, 49.30; H, 2.68; N, 3.38. Found: C,
49.19; H, 2.59; N, 3.42.
2.5.5. 5-Bromo-2–(4-(trifluoromethoxy)phenyl)benzofuran-7-amine
(8e)
2.5. Typical procedure for the one-pot beckmann rearrangement
and hydrolysis of the oxime derivatives 7a–e
Solid (0.47 g, 85%), mp. 118–119 ꢀC; ꢀmax (ATR) 685, 737, 823, 947,
1044, 1214, 1468, 1568, 1614, 3382, 3484 cmꢁ1; dH (DMSO-d6) 5.78
(2H, br s, NH2), 6.95 (1H, d, J ¼ 2.0 Hz, H-4), 6.68 (1H, d, J ¼ 2.0 Hz,
H-6), 7.32 (1H, s, H-3), 7.49 (2H, d, J ¼ 8.5 Hz, H-3’,5’), 8.05 (2H, d,
J ¼ 8.0 Hz, H-2’,6’); dC (DMSO-d6) 101.3, 116.3, 121.3, 122.7, 124.3,
126.1, 126.8, 130.1 (t, JCF ¼ 25.6 Hz), 133.2, 149.6, 150.9, 153.1,
156.1; HRMS (ES): found 371.9748. C15H10NO2F379Brþ requires
371.9769. Anal calcd for C15H9NO2F3Br: C, 48.41; H, 2.44; N, 3.76.
Found: C, 48.14; H, 2.65; N, 3.89.
A stirred mixture of 7 (1 equiv.) and triflic acid (0.4 equiv.) in aceto-
nitrile (10ml/mmol of 7) was heated at 80ꢀC for 4 h. The mixture
was cooled to room temperature and quenched with ice-cold
water. The resultant precipitate was washed thoroughly with cold
hexane and then recrystallized from ethanol to afford compound 6
as a solid. Compounds 8a–e were prepared in this fashion.
2.5.1. 5-Bromo-2-phenylbenzofuran-7-amine (8a)
Solid (0.35 g, 62%), mp. 177–179 ꢀC; ꢀmax (ATR) 736, 915, 1156,
1214, 1309, 1433, 1502, 1572, 1583, 1611, 3351, 3464 cmꢁ1; dH
(DMSO-d6) 5.70 (2H, s, NH2), 7.21 (1H, s, H-3), 7.28 (2H, d,
J ¼ 8.5 Hz, Ar), 7.41 (2H, d, J ¼ 8.0 Hz, Ar), 7.50 (1H, t, J ¼ 8.5 Hz, Ar),
7.65 (1H, d, J ¼ 1.5 Hz, H-6), 7.84 (1H, d, J ¼ 7.5 Hz, Ar); dC (DMSO-
d6) 103.3, 111.1, 125.2, 128.7, 129.3, 129.4, 130.1, 133.6, 134.2,
136.2, 145.9, 156.1; HRMS (ES): found 287.9946. C14H11NO79Brþ
requires 287.9925. Anal calcd for C14H10NOBr: C, 58.36; H, 3.50; N,
4.85. Found: C, 58.68; H, 3.20; N, 4.77.
2.6. Typical procedure for the amination of 9a and 9 b with the
7-aminobenzofurans 8a–e to afford 10a–j
A mixture of 8 (1 equiv.), 9 (1 equiv.) and HCl (5% mol equiv. of
9) in isopropanol (15.5 ml/mmol of 9) was stirred under reflux for
2 h. After completion of the reaction (tlc monitoring) the mixture
was cooled to room temperature and quenched with ice-cold
water. The resultant precipitate was washed thoroughly with hot
acetonitrile and then recrystallized from DMSO to afford 10 as a

1520
M. J. MPHAHLELE ET AL.
solid. Compounds 8a–e were prepared in this fashion. The follow- s, ¼C-H), 7.85 (1H, d, J ¼ 8.5 Hz, Ar), 7.96 (1H, dd, J ¼ 2.5 and
ing compounds were prepared in this fashion:
8.5 Hz, Ar), 8.05 (1H, dd, J ¼ 1.5 and 8.5 Hz, Ar), 8.07 (2H, d,
J ¼ 8.5 Hz, Ar), 8.15 (2H, t, J ¼ 8.5 Hz, Ar), 8.20 (1H, d, J ¼ 1.5 Hz, Ar),
8.90 (1H, d, J ¼ 1.0 Hz, Ar), 10.45 (1H, s, NH); dC (DMSO-d6) 103.6,
2
2.6.1. 6-Bromo-N-(5-bromo-2-phenylbenzofuran-7-yl)-2–(4-fluoro-
phenyl)quinazolin-4-amine (10a)
115.3, 115.5, 116.1 (d, JCF ¼ 21.9 Hz), 119.0, 121.9, 123.6, 124.9,
3
126.3, 127.3, 128.4, 128.7, 129.2, 129.4, 130.3 (d, JCF ¼ 8.7 Hz),
Solid (0.28 g, 75%) mp. > 300 ꢀC; ꢀmax (ATR) 688, 782, 873, 911,
1047, 1161, 1221, 1373, 1515, 1603, 1631, 3082, 3169 cmꢁ1; dH
(DMSO-d6) 7.29 (2H, t, J ¼ 8.5 Hz, Ar), 7.34 (2H, d, J ¼ 8.5 Hz, Ar),
7.47 (1H, s, ¼C–H), 7.78–7.79 (5H, m, Ar), 7.85 (1H, d, J ¼ 8.5 Hz,
Ar), 8.04 (1H, d, J ¼ 8.0 Hz, Ar), 8.11 (2H, d, J ¼ 8.5 Hz, Ar), 8.91 (1H,
d, J ¼ 1.5 Hz, Ar), 10.89 (1H, s, NH); dC (DMSO-d6) 103.3, 115.8,
4
132.3, 134.6, 137.0, 137.8, 147.3, 149.0 (d, JCF ¼ 3.8 Hz) 149.9,
1
155.4, 157.5, 158.8, 164.1 (d,þ JCF ¼ 246.5 Hz); HRMS (ES): found
621.9328. C28H16N3OF35Cl79Br2 requires 621.9327. Anal calcd for
C28H15N3OFClBr: C, 53.92; H, 2.42; N, 6.74. Found: C, 53.97; H, 2.35;
N, 6.848.
2
116.2 (d, JCF ¼ 21.8 Hz), 119.5, 121.1, 122.9, 125.4, 126.9, 128.4,
4
2.6.5. 6-Bromo-N-(5-bromo-2–(4-(trifluoromethoxy)phenyl)benzo-
furan-7-yl)-2–(4-fluorophenyl) quinazolin-4-amine (10e)
128.8, 129.4, 129.8, 130.2, 131.0 (d, JCF ¼ 2.8 Hz), 131.2 (d,
3JCF ¼ 9.5 Hz), 133.0, 135.7, 137.9, 147.9, 150.7, 152.5, 157.4, 158.3,
162.7 (d, 1JCF ¼ 272.1 Hz); HRMS (ES): found 589.9715.
Solid (0.36 g, 82%) mp. > 300 ꢀC; ꢀmax (ATR) 736, 856, 938, 1060,
1128, 1259, 1347, 1455, 1599, 1634, 3111, 3169 cmꢁ1, dH (DMSO-
d6) 7.09 (2H, t, J ¼ 8.0 Hz, Ar), 7.38 (2H, d, J ¼ 8.5 Hz, Ar), 7.56 (1H,
s, ¼C–H), 7.82–785 (4H, m, Ar), 8.04 (1H, d, J ¼ 8.5 Hz, Ar), 8.15
(2H, t, J ¼ 8.5 Hz, Ar), 8.20 (1H, d, J ¼ 1.5 Hz, Ar), 8.90 (1H, d,
J ¼ 1.0 Hz, Ar), 10.45 (1H, s, NH); dC (DMSO-d6) 103.6, 115.3, 115.5,
þ
C28H16N3OFBr2 requires 588.9624. Anal calcd for C28H15N3OFBr: C,
57.07; H, 2.74; N, 7.13. Found: C, 57.11; H, 2.97; N, 7.12.
2.6.2. 6-Bromo-N-(5-bromo-2–(3-fluorophenyl)benzofuran-7-yl)-2–
(4-fluorophenyl)quinazolin-4-amine (10b)
2
116.1 (d, JCF ¼ 21.9 Hz), 119.0, 121.9, 123.6, 124.9, 126.3, 127.3,
Solid (0.32 g, 82%) mp. > 300 ꢀC; ꢀmax (ATR) 683, 775, 874, 937,
1089, 1086, 1239, 1365, 1454, 1594, 1632, 3070, 3171 cmꢁ1; dH
(DMSO-d6) 7.16 (2H, t, J ¼ 9.0 Hz, Ar), 7.39 (2H, d, J ¼ 8.5 Hz, Ar),
7.60 (2H, d, J ¼ 8.5 Hz, Ar), 7.61 (1H, s, ¼C-H), 7.81 (1H, dd, J ¼ 1.5
and 9.0 Hz, Ar), 7.99 (1H, d, J ¼ 8.5 Hz, Ar), 8.12 (1H, d, J ¼ 8.5 Hz,
Ar), 8.16 (2H, t, J ¼ 8.5 Hz, Ar), 8.22 (1H, t, J ¼ 8.5 Hz, Ar), 9.03 (1H,
d, J ¼ 1.5 Hz, Ar), 11.02 (1H, s, NH); dC (DMSO-d6) 103.5, 112.0 (d,
2JCF ¼ 23.8 Hz), 115.3 (d, 3JCF ¼ 8.5 Hz), 115.6, 116.1, (d,
3
128.4, 128.7, 130.3 (d, JCF ¼ 8.7 Hz), 130.9 (t, JCF ¼ 25.6 Hz), 132.3,
4
134.6, 137.0, 137.8, 147.3, 149.0 (d, JCF ¼ 3.8 Hz), 149.9, 155.4,
1
157.5, 158.8, 164.1 (d, JCF ¼ 246.5 Hz); HRMS (ES): found 672.9227.
þ
C29H16N3O2F479Br2
requires
672.9447.
Anal
calcd
for
C29H15N3OF4Br: C, 51.74; H, 2.25; N, 6.24. Found: C, 51.68; H, 2.11;
N, 6.35.
2
2JCF ¼ 21.9 Hz), 116.5, (d, JCF ¼ 20.9 Hz), 119.8, 121.7, 123.0, 126.8,
2.6.6. 6-Bromo-N-(5-bromo-2-phenylbenzofuran-7-yl)-2–(4-chloro-
phenyl)quinazolin-4-amine (10f)
3
2
128.4, 130.0, 130.2, 131.5 (d, JCF ¼ 9.5 Hz), 130.6 (d, JCF ¼ 8.5 Hz),
Solid (0.32 g, 83%) mp. > 300 ꢀC; ꢀmax (ATR) 683, 775, 874, 937,
1089, 1086, 1239, 1365, 1454, 1594, 1632, 3070, 3171 cmꢁ1; dH
(DMSO-d6) 7.31–7.37 (3H, m, Ar), 7.61 (1H, d, J ¼ 8.5 Hz, Ar), 7.65
(1H, d, J ¼ 7.0 Hz, Ar), 7.68 (1H, s, ¼C–H), 7.75 (1H, d, J ¼ 7.5 Hz,
Ar), 7.89 (1H, d, J ¼ 8.7 Hz, Ar), 7.96 (1H, dd, J ¼ 2.5 and 8.5 Hz, Ar),
8.07 (1H, d, J ¼ 8.7 Hz, Ar), 8.17 (1H, d, J ¼ 8.5 Hz, Ar), 8.21 (1H, d,
J ¼ 2.5 Hz, Ar), 8.26 (1H, d, J ¼ 8.5 Hz, Ar), 8.42 (1H, d, J ¼ 1.5 Hz,
Ar), 8.97 (1H, d, J ¼ 8.5 Hz, Ar), 10.71 (1H, s, NH); dC (DMSO-d6),
103.3, 115.6, 119.6, 120.9, 126.4, 127.0, 128.5, 128.7, 129.1, 129.4,
129.9, 130.2, 130.8, 131.7, 132.9, 135.8, 136.9, 137.2, 137.9, 147.9,
150.7, 152.5, 157.2,þ157.8, 158.8 161.6; HRMS (ES): found 605.9349.
131.7, 132.3, 136.5, 137.9, 147.2, 148.0, 152.5, 155.5, 158.0 (d,
1
1
4JCF ¼ 2.9 Hz), 162.7 (d, JCF ¼ 224.7 Hz) 164.7 (d, JCF ¼ 231.3 Hz);
þ
HRMS (ES): found 607.9629. C28H16N3OF279Br2 requires 607.9628.
Anal calcd for C28H15N3OF2Br: C, 55.38; H, 2.49; N, 6.97. Found: C,
55.62; H, 2.57; N, 6.85.
2.6.3. 6-Bromo-N-(5-bromo-2–(4-fluorophenyl)benzofuran-7-yl)-2–
(4-fluorophenyl)quinazolin-4-amine (10c)
Solid (0.19 g, 87%) mp. 287–289 ꢀC; ꢀmax (ATR) 737, 846, 943, 1088,
1156, 1289, 1365, 1454, 1572, 1607, 1627, 3064, 3170 cmꢁ1; dH
(DMSO-d6) 7.15 (2H, t, J ¼ 8.5 Hz, Ar), 7.22 (1H, t, J ¼ 8.5 Hz, Ar),
7.38 (1H, t, J ¼ 8.5 Hz, Ar), 7.48 (1H, s, ¼C–H) 7.66 (1H, d, J ¼ 8.5 Hz,
Ar), 7.77–7.82 (2H, m, Ar), 7.94–7.97 (2H, m, Ar), 8.08 (1H, d,
J ¼ 7.5 Hz, Ar), 8.16 (1H, t, J ¼ 8.5 Hz, Ar), 8.20–8.23 (1H, m, Ar), 8.99
(1H, d, J ¼ 1.5 Hz, Ar), 10.89 (1H, s, NH); dC (DMSO-d6) 102.1, 115.8,
C28H17N3O35Cl79Br2
requires 605.9328. Anal calcd for
C28H16N3OClBr: C, 55.52; H, 2.66; N, 6.94. Found: C, 55.65; H, 2.78;
N, 7.04.
2
2
2.6.7.
6-Bromo-N-(5-bromo-2–(3-fluorophenyl)benzofuran-7-yl)-
116.2 (d, JCF ¼ 21.8 Hz), 116.5 (d, JCF ¼ 21.8 Hz), 119.5, 121.3,
4
2–(4-chlorophenyl)quinazolin-4-amine (10g)
122.9, 123.5, 124.3, 126.0 (d, JCF ¼ 3.9 Hz), 126.6, 127.7 (d,
Solid (0.26 g, 81%) mp. > 300 ꢀC; ꢀmax (ATR) 687, 777, 850, 1108,
1217, 1366, 1464, 1519, 1520, 1627, 3102, 3170 cmꢁ1; dH (DMSO-
d6) 7.15 (1H, t, J ¼ 8.0 Hz, Ar), 7.37 (2H, d, J ¼ 8.0 Hz, Ar), 7.42 (1H,
s, ¼C–H), 7.66 (1H, d, J ¼ 8.5 Hz, Ar), 7.57–7.60 (2H, m, Ar), 7.65
(1H, d, J ¼ 8.5 Hz, Ar), 7.83 (1H, d, J ¼ 8.5 Hz, Ar), 7.97 (1H, dd,
J ¼ 1.5 and 9.0 Hz, Ar), 8.10 (2H, d, J ¼ 8.5 Hz, Ar), 8.15 (1H, d,
J ¼ 8.5 Hz, Ar), 9.03 (1H, d, J ¼ 1.5 Hz, Ar), 11.00 (1H, s, NH); dC
3JCF ¼ 8.5 Hz), 128.5, 129.4 (d, JCF ¼ 2.8 Hz), 130.2, 130.8 (d,
4
3JCF ¼ 8.5 Hz), 130.9, 132.6, 137.5, 147.9, 157.8, 161.6, 164.5 (d,
1JCF ¼ 248.8 Hz), 164.5 (dþ, JCF ¼ 267.3 Hz); HRMS (ES): found
1
605.9629. C28H16N3OF2Br2 requires 605.9628. Anal calcd for
C28H15N3OF2Br: C, 55.38; H, 2.49; N, 6.97. Found: C, 55.59; H, 2.61;
N, 7.01.
2
(DMSO-d6) 103.5, 112.0 (d, JCF ¼ 23.8 Hz), 115.4, 116.4 (d,
4
2JCF ¼ 20.9 Hz), 119.6, (d, JCF ¼ 2.8 Hz), 121.4, 123.0, 123.8, 124.5,
2.6.4. 6-Bromo-N-(5-bromo-2–(3-chlorophenyl)benzofuran-7-yl)-2–
(4-fluorophenyl)quinazolin-4-amine (10d)
3
126.6, 128.5, 129.1, 130.0, 130.2 (d, JCF ¼ 8.5 Hz), 130.5 (d,
3
Solid (0.14 g, 78%) mp. > 300 ꢀC; ꢀmax (ATR) 738, 856, 938, 1030, 4JCF ¼ 2.9 Hz), 131.7 (d, JCF ¼ 8.6 Hz), 132.3, 136.5, 137.0, 137.9,
1129, 1254, 1347, 1458, 1596, 1634, 3109, 3169 cmꢁ1; dH (DMSO- 147.1, 149.0, 152.5, 155.5, 158.8, 162.7 (d, JCF ¼ 241.7 Hz); HRMS
1
þ
d6) 7.09 (2H, t, J ¼ 8.0 Hz, Ar), 7.38 (2H, d, J ¼ 8.5 Hz, Ar), 7.58 (1H, (ES): found 621.9329. C28H16N3OF35Cl79Br2 requires 621.9333.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1521
Anal calcd for C28H15N3OFClBr: C, 53.92; H, 2.42; N, 6.74. Found: C, Highveld Biological, South Africa) supplemented with 10% fetal
calf serum (Adcock-Ingram, Midrand, South Africa) and sodium
pyruvate for C3A liver cells. Cell suspensions were prepared from
53.99; H, 2.35; N, 6.87.
confluent monolayer cultures and plated at
a density of
0.1 ꢂ 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,
2.6.8. 6-Bromo-N-(5-bromo-2–(4-fluorophenyl)benzofuran-7-yl)-2–
(4-chlorophenyl)quinazolin-4-amine (10h)
Solid (0.15 g, 78%) mp. > 300 ꢀC; ꢀmax (ATR) 736, 874, 1061, 1168,
1288, 1398, 1475, 1572, 1629, 3101, 3170 cmꢁ1; dH (DMSO-d6) 7.29
(2H, t, J ¼ 8.5 Hz, Ar), 7.34 (2H, d, J ¼ 8.5 Hz, Ar), 7.47 (1H, s, ¼C–H), added to the wells and incubated for 48 h. Doxorubicin hydro-
7.78–7.79 (4H, m, Ar), 7.85 (1H, d, J ¼ 8.5 Hz, Ar), 8.04 (1H, d, chloride (Pfizer Laboratories, Sandton, South Africa) was used as
a positive control while DMSO was the negative control. After
J ¼ 8.0 Hz, Ar), 8.11 (2H, d, J ¼ 8.5 Hz, Ar), 8.91 (1H, d, J ¼ 1.5 Hz,
incubation for 48 h, the wells were rinsed with 150 mL of phos-
phate buffered saline PBS (Sigma-Aldrich, GmBH, Schnelldorf,
Germany) and 200 mL of fresh medium was dispensed into the
wells. MTT was dissolved in PBS (30 mL) then added to each well
and the contents were incubated for 4 h at 37 ꢀC. The medium
was removed and MTT formazan crystals were dissolved in 50 mL
DMSO. The amount of MTT reduction was measured immediately
by detecting the absorbance using a BioTek microplate reader
Ar), 10.89 (1H, s, NH); dC (DMSO-d6) 102.0, 115.4, 116.5 (d,
2JCF ¼ 21.8 Hz), 119.5, 120.8, 122.9, 123.3, 124.8, 126.1, 126.3, 127.6,
3
127.7 (d, JCF ¼ 8.5 Hz), 128.8, 129.8, 130.8, 132.5, 135.8, 137.0 (d,
4JCF ¼ 2.9 Hz), 147.1, 149.9, 155.9, 157.5, 158.7, 162.9 (d,
1JCF ¼ 245.6 Hz); HRMS (ES): found 621.9333. C28H15N3OF35Cl79Br2
þ
requires 621.9333. Anal calcd for C28H14N3OFClBr: C, 53.92; H, 2.42;
N, 6.74. Found: C, 53.88; H, 2.58; N, 6.91.
(BioTek
Synergy,
Analytical
and
Diagnostic
Products,
2.6.9. 6-Bromo-N-(5-bromo-2–(3-chlorophenyl)benzofuran-7-yl)-2–
(4-chlorophenyl)quinazolin-4-amine (10i)
Johannesburg, South Africa) at a wavelength of 570 nm. Each
dilution of the test sample was assayed in quadruplicate and
the experiments were repeated three times. The percentage of
cell viability and the LC50 values for each compound tested
were calculated as described before10.
Solid (0.29 g, 67%) mp. 282–285 ꢀC; ꢀmax (ATR) 683, 736, 829, 940,
1012, 1159, 1372, 1501, 1530, 1516, 3048, 3170 cmꢁ1; dH (DMSO-
d6) 7.35 (1H, d, J ¼ 8.5 Hz, Ar), 7.38 (1H, d, J ¼ 7.0 Hz, Ar), 7.60 (2H,
d, J ¼ 8.5 Hz, 2H), 7.67 (2H, d, J ¼ 8.5 Hz, Ar), 7.81 (1H, s, C-H), 7.85
(1H, d, J ¼ 8.5 Hz), 7.96 (1H, dd, J ¼ 2.5 and 8.5 Hz, Ar), 8.05 (1H,
dd, J ¼ 1.5 and 8.5 Hz, Ar), 8.13 (1H, d, J ¼ 8.0 Hz, Ar), 8.17 (1H, d,
J ¼ 8.5 Hz, Ar), 8.20 (1H, d, J ¼ 2.5 Hz, Ar), 8.93 (1H, d, J ¼ 1.5 Hz,
Ar), 10.51 (1H, s, NH); dC (DMSO-d6) 103.6, 115.6, 119.5, 121.0,
123.0, 124.8, 126.4, 128.4, 129.2, 129.4, 129.8, 130.2, 130.5, 131.3,
131.4, 131.7, 132.2, 134.2, 135.9, 136.9, 137.9, 147.0, 152.4, 155.2,
2.8. Apoptosis assay
2.8.1. Annexin V-FITC staining assay on 10d and 10j against C3A
and caco-2, respectively
Apoptotic cells were quantified using flow cytometry. The C3A or
Caco-2 cells were cultured in 24 well plates and each was then
treated with compound 10d or 10j at concentrations 5, 12.5 and
25 mM against doxorubicin hydrochloride (0.20 mM) as a reference
standard, respectively. 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 x 106 cells/
sample. Cells were harvested and transferred into plastic flow
tubes (BD Biosciences, South Africa). The cells were, in turn,
stained with Annexin-V-FUOS staining kit (Roche, Mannheim,
Germany) according to the manufacturer’s instructions. The cells
were analyzed using Becton, Dickinson and Company FACS Accuri
flow cytometer.
þ
157.5, 158.6; HRMS: found 637.9041. C28H16N3O35Cl279Br2 requires
637.9037. Anal calcd for C28H15N3OCl2Br: C, 52.53; H, 2.36; N, 6.56.
Found: C, 52.68; H, 2.50; N, 6.45.
2.6.10. 6-Bromo-N-(5-bromo-2–(4-(trifluoromethoxy)phenyl)benzo-
furan-7-yl)-2–(4-chlorophenyl) quinazoline-4-amine (10j)
Solid (0.17 g, 82%) mp. > 300 ꢀC; ꢀmax (ATR) 682, 781, 858, 938,
1091, 1199, 1259, 1372, 1455, 1553, 1615, 3082, 3170 cmꢁ1; dH
(DMSO-d6) 7.33 (1H, d, J ¼ 8.0 Hz, Ar), 7.36 (1H, d, J ¼ 8.0 Hz, Ar),
7.59 (1H, s, ¼C-H), 7.61 (1H, d, J ¼ 8.5 Hz, Ar), 7.67 (1H, d,
J ¼ 8.0 Hz, Ar), 7.81 (1H, d, J ¼ 9.0 Hz, Ar), 7.85 (2H, d, J ¼ 8.5 Hz,
Ar), 7.96 (1H, d, J ¼ 9.0 Hz, Ar), 8.04 (1H, d, J ¼ 9.0 Hz, Ar), 8.11 (1H,
d, J ¼ 8.5 Hz, Ar), 8.17 (1H, d, J ¼ 8.5 Hz, Ar), 8.20 (1H, d, J ¼ 1.5 Hz,
Ar), 8.91 (1H, d, J ¼ 1.5 Hz, Ar), 10.48 (1H, s, NH); dC (DMSO-d6)
103.6, 115.3, 119.6, 121.9, 123.0, 124.8, 126.3, 127.2, 128.4, 128.7,
129.2, 129.8, 130.7, 131.7, 132.3, 135.8, 136.9 (t, JCF ¼ 25.6 Hz),
137.9, 147.2, 149.0, 149.8, 152.4, 155.4þ, 157.5, 158.6; HRMS (ES):
found 687.9236. C29H16N3O2F335Cl79Br2 requires 687.9250. Anal
calcd for C28H15N3O2F3ClBr: C, 50.50; H, 2.19; N, 6.09. Found: C,
50.29; H, 2.32; N, 6.02.
2.8.2. Caspase-3 analysis on 10d and 10j against C3A and caco-2,
respectively
Caspase-3 activity was detected by means of Caspase-3
Colorimetric Assay Kit (Abcam, Cambridge, MA, USA). The cells
were cultured in 24 well plates and treated for 24 h. The cells
were then washed with PBS buffer and lysed on ice. The experi-
ments were carried out according to the manufacturer’s instruc-
tions. Optical density was measured at absorbance of 450 nm
using the BioTek microplate reader. The concentration of active
Caspase-3 (Asp 175) were measured in duplicates and interpolated
from the active caspase-3 (Asp 175) standard curve and corrected
for sample dilution.
2.7. Materials and methods for the in vitro cytotoxicity assay
The cytotoxic activities of the compounds were screened against
the C3A/HepG2 human liver cells, human colorectal tumor
(Caco-2) cells, the human lung carcinoma (A549) and the cervical
cancer (HeLa) cells. The lethal concentration was determined
using the 3–(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium brom-
2.9. Inhibition for EGFR-TK
The inhibitory activities of compound 10d or 10j and Gefitinib
ide (MTT) assay previously developed by Mosmann20. The cells towards EGFR-TK were tested using enzyme-linked immunosorb-
were maintained in Dubelsco minimal essential medium (DMEM, ent assay (ELISA) technique with purified epidermal growth factor

1522
M. J. MPHAHLELE ET AL.
O
O
NOH
R
O
Br
Br
Br
R
Br
NH2
CH3
OH
(i)
(iv)
(iii)
CH3
(v)
(ii)
O
OH
O
O
I
Ar
Ar
Ar
4
5
6a_e
7a_e
8a_e
Cl
N
Br
N
(vi)
C6H4X
Reagents & conditions:
(i) NBS, AcOH, reflux, 1.5 h; (ii) NIS, AcOH, reflux, 1 h
ArC CH, PdCl (PPh ) CuI., Cs CO , DMF(aq), reflux, 3 h;
9a (X = F); b (X = Cl)
Br
(iii)
2
3 2,
2
3
A r
(iv) NH2OH.HCl, pyridine, reflux, 1 h; (v) TfOH, CH3CN, reflux, 1 h
(vi) 5% HCl, iPrOH, reflux, 3 h
O
NH
Br
N
N
X
10a_e (X = F); 10f_j (X = Cl)
Scheme 2. Reaction sequence for the synthesis of 10a–j.
3. Results and discussion
Table 1. Percentage yields of compounds 10a–j and the corresponding
intermediates.
3.1. Chemistry
Ar
%Yield 6 %Yield 7 %Yield 8 %Yield 10a–e %Yield 10f–j
The benzofuran-aminoquinazolines 10a–j and the corresponding
intermediates were prepared following the reaction sequence out-
lined in Scheme 2 and their yields are listed in Table 1. The initial
task of this investigation involved the preparation of 5-bromo-2-
hydroxy-3-iodoacetophenone 5 to serve as a substrate for the tan-
dem palladium catalyzed Sonogashira cross-coupling with arylace-
C6H5-
73 (6a)
76 (6b)
76 (6c)
74 (6d)
77 (6e)
93 (7a)
86 (7b)
72 (7c)
81 (7d)
78 (7e)
62 (8a)
78 (8b)
71 (8c)
65 (8d)
85 (8e)
75 (10a)
82 (10b)
87 (10c)
78 (10d)
82 (10e)
83 (10f)
81 (10g)
78 (10h)
67 (10i)
84 (10j)
3-FC6H4-
4-FC6H4-
3-ClC6H4-
4-CF3OC6H4-
receptor (Sigma-Aldrich, Bradford, UK). The procedure was carried tylenes and subsequent endo-dig Csp–O cyclization to afford
out according to the manufacturer’s protocol as described in our the requisite 7-acetyl–substituted 2-aryl-5-bromobenzofurans.
previous investigation10.
Recourse to the literature revealed that 5-bromo-2-hydroxy-3-
iodoacetophenone has been prepared before by treatment of the
commercially available 5-bromo-2-hydroxyacetophenone with pyri-
dinium iodochloride (1 equiv.) in methanol under reflux for 2 h19.
2.10. Molecular docking studies
We opted for the use of commercially available 2-hydroxyaceto-
phenone 4 (purchased from Sigma-Aldrich) as a substrate for ini-
tial halogenation with 1 equivalent of N-bromosuccinimide (NBS)
in acetic acid under reflux for 1.5 h to afford 5-bromo-2-hydroxya-
cetophenone in 59% yield (Scheme 2). The latter was, in turn, sub-
jected to iodination with N-iodosuccinimide (NIS) in acetic acid
under reflux for 1 h to afford 5-bromo-2-hydroxy-3-iodoacetophe-
none 5. Sonogashira cross-coupling of 5 with terminal acetylenes
afforded the corresponding 1–(5-bromo-2-arylbenzofuran-7-yl)e-
thanones 6a–e in appreciable yields. Oximation of compounds
6a–e with hydroxylamine hydrochloride in pyridine under reflux
for 1 h followed by aqueous work-up and recrystallization afforded
the corresponding oximes 7a–e. The Beckmann rearrangement of
these oximes with 20% mol equivalent of trifluoromethanesulfonic
acid (triflic acid, TfOH) in acetonitrile under reflux for 4 h afforded
compounds characterized using a combination of NMR, IR and
mass spectrometric techniques as the 7-aminobenzofuran deriva-
tives 8a–e. The latter are the result of the initial Beckmann
rearrangement via aryl carbon migration followed by an in situ
acid-mediated hydrolysis of the intermediate 7-acetamido-2-aryl-5-
The structure of epidermal growth factor receptor (EGFR) kinase
domain was obtained from RCSB PDB (id: 1M17)21 where all heter-
oatoms and water molecules were first removed. Polar hydrogen
atoms, Kollman-Amber united atom partial charges and solvation
parameters were then added by utilizing AutoDockTools22.
The coordinates for erlotinib (control) docking were retrieved
from the ligand co-crystallised with EGRF (PDB id: 1M17) while the
coordinates for compounds 10a–j were generated using
ChemDraw Professional 15.0 (PerkinElmer Informatics, Waltham,
MA, USA). Minimization of compounds 10 was then performed with
Chem3D module in ChemOffice Professional 15.0 (PerkinElmer
Informatics). Ligands were in united atom format where only polar
hydrogen atoms remained. Gasteiger charges and torsional angles
of all ligands were assigned by AutoDockTools.
Hydrated docking was performed with standard protocol from
AutoDock4.2 where random water atoms were added around the
ligand. A total of 200 runs were performed by AutoDock4.2.622
with semi-empirical free energy scoring function, Lamarckian gen-
etic algorithm of 1,000,000 energy evaluations each run and the
maximum number of 27,000 generations. The number of individu-
als in population was set as 350 and the rate of crossover was 0.8. bromobenzofuran derivatives. Hitherto, Cacchi et al.23 had
All docked conformations were clustered with root mean square reported a method for the synthesis of the 7-aminobenzofuran
of 2.0 Å.
derivatives which makes use of the Buchwald-Hartwig C–N bond

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1523
Figure 2. Oak Ridge Thermal Ellipsoid Plot (ORTEP) diagram of 10f. Displacement ellipsoids are drawn at the 50% probability level and H atoms are shown as small
spheres of arbitrary radii.
formation of the intermediate 7-bromobenzofurans with the pri- viability (MTT) assay. The compounds were screened as DMSO solu-
mary and secondary amines. Despite what looks like a simple tions at concentrations ranging from 5–100 mM and their cytotoxic
activities were expressed as LC50 values (the dose that reduces sur-
vival to 50%) in mM concentrations using DMSO as the negative
control and Gefitinib as a reference standard. We used DMSO as a
negative control because at 1% or less this substance has been
found to exhibit no apparent effect on proliferation of the HeLa
and Caco-2 cells up to 48h25,26. Likewise, Gefitinib was used as a
positive control because it has previously been found to enhance
apoptosis and suppress proliferation of lung cancer (A549) cells27.
Moreover, the Caco-2 cells have also been found to display greater
sensitivity to this drug due to their high EGFR expression26. The
corresponding LC50 values of the tested compounds against
Gefitinib are represented in Table 2. We observed variable inhib-
ition of cell growth of the A549 (modest), Caco-2 (intermediate),
C3A (highly sensitive) and HeLa (resistant) cells against Gefitinib,
which seem not to correlate the level of EGFR expression. The sen-
sitivity to growth inhibition by Gefitinib has been associated with
dependence on protein kinase B/AKT (Akt) and extracellular
signal-regulated kinase 1/2 (ERK1/2) activation in response to EGFR
signaling for survival and proliferation and extent of EGF-induced
down-regulation of cell surface EGFR28. The presence in 10a of a
phenyl group on the benzofuran ring and 2–(4-fluorophenyl) sub-
stituent on the quinazoline framework led to diminished cytotox-
icity against the A549, Caco-2 or C3A cell lines, but significant
cytotoxicity against the HeLa cells with LC50 value of 17.6mM.
Compound 10b substituted with a 3-fluorophenyl group on the
benzofuran ring and a 4-fluorophenyl group on the quinazoline
moiety resulted in moderate cytotoxicity against the A549 cells
(LC50 ¼ 54.3mM) and no activity against the other three cells. No
cytotoxicity was observed against the Caco-2 and C3A cell lines for
compound 10c substituted with the 4-fluorophenyl group at the 2-
molecular framework, we found that no attempts have been
made before towards the synthesis of benzofuran-quinazoline
hybrids in which the two pharmacophores are linked through a
heteroatom bridge. Consequently, we reacted the nucleophilic 7-
aminobenzofurans 8a–e with the electrophilic 6-bromo-4-chloro-
2–(4-halogenophenyl)quinazoline 9a (X ¼ F) or 9 b (X ¼ Cl) in the
presence of 5% HCl in isopropanol (iPrOH) under reflux for 4 h to
afford the corresponding benzofuran-aminoquinazoline hybrids
10a–e or 10f–j, respectively. Dechloroamination was confirmed by
the presence of increased number of proton and carbon signals in
the aromatic region of the 1H- and 13C-NMR spectra of com-
pounds 10a–j.
The structure of these compounds was distinctly confirmed by
single crystal X-ray diffraction (XRD) analysis of compound 10f
(Figure 2)24. The crystal structure reveals the presence of an intra-
molecular hydrogen bonding between the amine proton and
endocyclic oxygen atom.
Since the molecular construct of compounds 10a–j resembles
that of the EGFR-TK inhibitor, Gefitinib, we decided to evaluate
them for antiproliferative effect against a panel of EGFR-positive
cancer cell lines, namely, the A549, Caco-2, C3A (HepG2/C3A) and
HeLa cell lines. The structure activity relationship (SAR) of these
compounds was studied with respect to the substitution pattern
on the phenyl rings of the quinazoline and benzofuran scaffolds.
3.2. Biological evaluation
3.2.1. In vitro cytotoxicity of quinazoline-benzofuran hybrids
10a–j
Compounds 10a–j were evaluated for potential antiproliferative
properties in vitro using the well-established 3–(4,5-dimethylthia- positions of the benzofuran and quinazoline moieties. However,
zole-2-yl)-2,5-diphenyltetrazoliumbromide based colorimetric cell this compound was found to exhibit significant cytotoxicity against

1524
M. J. MPHAHLELE ET AL.
Table 2. Cytotoxic effects of 10a–j and Gefitinib against A549, Caco-2, C3A and
HeLa cell lines.
quinazoline framework seems to be more desirable for cytotoxicity
than with a 2–(4-fluorophenyl) ring, which resulted in diminished
cytotoxicity for 10e.
Br
We took into consideration the pro-apoptotic properties of the
R
O
4-anilinoquinazolines6,30
,
and benzofuran derivatives31 and
NH
decided to verify whether the observed inhibition of cell growth
by compounds 10 was related to cell apoptosis. Apoptosis is an
essential mechanism by the body to get rid of unwanted cells and
induction of this process in cancer cells would lead to automatic
death and decrease cancer proliferation.
Br
N
N
X
10a–e (X = F) & 10f–j (X = Cl)
3.2.2. Evaluation of cell death pathways
Cancer cells LC50 (mM)
We selected compounds 10d and 10j to evaluate whether they
induce apoptosis in the C3A and Caco-2 cell lines against doxo-
rubicin hydrochloride as a reference standard, respectively. The
population of apoptotic cells was determined by means of
Annexin-V and propium iodide (PI) double staining by flow cytom-
etry, which is a useful tool for evaluation of molecular and mor-
phological events that take place during cell death and cell
proliferation. The Annexin V-FITC/PI apoptosis assay is a popular
method to distinguish between viable cells (Annexin V-negative;
PI-negative), early apoptotic cells (Annexin V-positive; PI-negative),
late apoptotic cells (Annexin V-positive; PI-positive) and unviable
or necrotic cells (Annexin V-negative; PI-positive) through differen-
ces in plasma membrane integrity and permeability. After treat-
ment with different concentrations of compound 10d and 10j (5
and 12.5 mM) for 24 h, the C3A and Caco-2 cell lines were stained
with propidium iodide (PI) and Annexin V binding buffer, and
then analyzed by the flow cytometry. The percentage of cell pop-
ulations, i.e., necrotic cell (Q1), late apoptotic cells (Q2), healthy
cells (Q3) and early apoptotic cells (Q4) are represented in
Figure 3. As shown in Figure (3(a)) and (3(b)), compounds 10d
and 10j induced apoptosis in the C3A and Caco-2 cell lines,
respectively. Both necrotic (Q1) and apoptotic cells (Q2) seem to
increase with the increasing concentration, but the population of
necrotic cells remained higher at the highest concentration tested.
This is presumably because necrosis takes place after the cells
have already died by any mechanism including apoptosis and
Compound
R
A549
Caco-2
C3A
HeLa
10a
10b
10c
10d
10e
10f
10g
10h
10i
H
3-F
4-F
3-Cl
4-OCF3
H
3-F
4-F
3-Cl
4-OCF3
64.7 0.11
54.3 0.19
52.6 0.20
65.1 0.12
97.6 0.07
48.0 0.10
78.5 0.15
74.1 0.21
86.6 0.16
47.4 0.07
51.3 0.17
96.3 0.10
83.9 0.07
98.8 0.02
47.7 0.01
65.2 0.10
75.0 0.02
94.4 0.02
49.1 0.01
33.5 0.10
18.4 0.07
27.9 1.05
81.0 0.03 17.6 0.39
90.1 0.02 47.0 0.38
>100
9.0 0.01 22.1 1.10
>100 23.4 0.49
73.9 0.02 15.4 0.45
79.1 0.01 53.5 4.46
>100
68.2 0.02 75.1 5.25
>100 28.1 0.79
5.0 0.04 98.8 0.56
11.4 0.56
28.6 1.12
10j
Gefitinib
the A549 (LC50 ¼ 52.6mM) and the HeLa (LC50 ¼ 11.4mM) cell lines
when compared to Gefitinib. A combination of 3-chlorophenyl on
the benzofuran ring and 4-fluorophenyl group on the quinazoline
framework in 10d was found to result in reduced cytotoxicity
against the A549 and Caco-2 cell lines, but increased anti-growth
effect against the C3A (LC50 ¼ 9.0 mM) and the HeLa
(LC50 ¼ 22.1mM) cell lines when compared to Gefitinib. Compound
10e substituted with a 4-trifluoromethoxy group on the benzofuran
ring and a 4-fluorophenyl group on the quinazoline framework
exhibits moderate cytotoxicity against the HeLa cells
(LC50 ¼ 23.4mM) and no activity against the other three cell lines.
Within the series 10f–j, compound 10f substituted with a phenyl
ring on the benzofuran ring and a 4-chlorophenyl group at C-2
position of the quinazoline was found to exhibit significant cytotox-
icity against the A549 (LC50 ¼ 48.0mM) and the HeLa
(LC50 ¼ 15.4mM) cell lines when compared to Gefitinib. Compound
10g substituted with a 4-chlorophenyl group at the C-2 position of
the quinazoline framework and a 3-fluorophenyl ring on the benzo-
furan moiety, on the other hand, exhibited no activity against all
the four cell lines. Likewise, a combination of 4-chlorophenyl group
on the quinazoline ring and a 4-fluorophenyl group on the benzo-
furan arm resulted in loss of activity for 10h against the A549,
Caco-2 and C3A cell lines. However, this compound exhibited sig-
nificant cytotoxicity against the HeLa cell lines (LC50 ¼ 28.6mM).
Compound 10i substituted with a 4-chlorophenyl ring on position
2 of the quinazoline and 3-chlorophenyl group on the 2-position of
the benzofuran moiety was found to exhibit significant cytotoxicity
against the Caco-2 cell line with the LC50 value of 33.5mM, but no
activity against the other three cell lines. The presence of a 4-tri-
fluoromethoxyphenyl group on the benzofuran framework in com-
pound 10j led to significant cytotoxicity against the A549
have reached equilibrium with their surroundings32,33
.
A major biochemical feature of apoptosis is the activation of
caspases (caspase-2, -3, -6, -7, -8, -9, and -10), which play a central
role in the morphological changes associated with apoptosis34.
Activation of caspase-3, for example, is critically important for the
induction of apoptotic pathways34. Consequently, we evaluated
compounds 10d and 10j for potential to induce caspase-3 activa-
tion in C3A and Caco-2 cells using Gefitinib as a reference stand-
ard (Figure 4). The results of this study indicate that these
compounds induce apoptosis through activation of caspase-3 that
subsequently leads to cell membrane alterations.
The hepatocellular carcinoma and the colorectal adenocarcin-
oma cell lines have been found to express high levels of EGFR
and sensitivity to Gefitinib27,35,36. Inhibition of EGFR-TK activity is
regarded as the most promising approach for innovative thera-
peutic strategies in cancer treatment37.
(LC50 ¼ 47.4mM) and HeLa (LC50 ¼ 28.1mM) and more so against the 3.2.3. Inhibition of EGFR-TK by compounds 10a–j
Caco-2 cell line (LC50 ¼ 18.4mM) with no activity against the C3A
In order to test whether the benzofuran-appended 4-aminoquina-
cells when compared with Gefitinib. Compounds substituted with zolines prepared in this investigation inhibit the ligand binding-
the strong electron-withdrawing trifluoromethyl group have been induced receptor phosphorylation, we performed kinase activity of
found to exhibit superior metabolic stability and improved activity the human EGFR in the presence of compounds 10a–j against
profile compared to the corresponding methyl analogs29. A com- Gefitinib as the reference standard (Table 3). Within the series
bination of this moiety with a 4-chlorophenyl group on the 10a–e, compounds 10a and 10b substituted with
a 2–(4-

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1525
Figure 3. Effects of compounds 10d and 10j on the induction of apoptosis in C3A (3a) and Caco-2 cells (3b) as determined by Annexin V/PI staining. Data represent
the percentage of apoptotic cells for the control and compounds 10d and 10j at 5 and 12.5 mM after 24 h.
40
Caspase-3 activity in C3A cells
Caspase-3 activity in Caco-2 cells
120
100
80
60
40
20
0
35
30
25
20
15
10
5
0
10j: 5
12.5 25 µM Gef: 5
12.5 25 µM
10d: 5
12.5
25 µM
Gef: 5
12.5
25 µM
Figure 4. Effects of compounds 10d and 10j on Human Caspase-3 (Asp175) activity in C3A and Caco-2 cells against Gefinitib (Gef), respectively.

1526
M. J. MPHAHLELE ET AL.
fluorophenyl) ring on the quinazoline framework and a phenyl or relationship (SAR) studies, we performed molecular docking of
3-fluorophenyl ring on the benzofuran arm were found to exhibit
reduced inhibitory effect against the EGFR when compared to the
reference standard. A combination of 4-fluorophenyl group at the
2-positons of the quinazoline and benzofuran moieties in 10c, on
the other hand, resulted in moderate inhibitory effect with an IC50
value of 52.2 nM. The most cytotoxic compound 10d against the
C3A cell line was found to inhibit the EGFR (IC50 ¼ 29.3 nM)
slightly higher than the reference drug. The presence of a 3–(4-flu-
oromethoxyphenyl) group on the benzofuran arm of 10e also
resulted in increased inhibitory effect (IC50 ¼ 31.1 nM) when com-
pared to Gefitinib. Derivatives 10f–j substituted with a 2–(4-chlor-
ophenyl) group on the quinazoline framework were generally less
active against the EGFR. Only compound 10j found to exhibit sig-
nificant cytotoxicity against the A549, Caco-2 and Hela cells
showed moderate EGFR inhibitory activity (IC50 ¼ 61.5 nM) com-
pared to the reference standard. The hepatocellular carcinoma
cells have been found to display greater sensitivity to Gefitinib
due to high EGFR expression38,39. Based on the observed results,
we can conclude that the EGFR-TK inhibitory activity of the benzo-
furan-aminoquinazolines 10 is correlated to their antiprolifera-
tive activities.
compounds 10 into EGFR-TK active site against Gefitinib.
3.2.4. Molecular docking of compounds 10a–j into the ATP
binding site of EGFR
In order to explore probable interaction model of inhibitors and
enzyme active site, molecular docking of 10d and 10j into ATP
binding site of EGFR kinase was performed on the binding
model based on the EGFR complex structure (Figure 5). A crys-
tal structure of EGFR that previously co-crystalized with
Erlotinib (an inhibitor of EGFR) 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. The morpholine
region of Gefitinib was positioned as anti-parallel with Phe699
side chain, but no such orientation was observed in Erlotinib. In
addition, the 3-chloro-4-fluorophenyl moiety of Gefitinib also
docked deeper into the EGFR binding pocket compared with
Erlotinib. The binding conformations of the 2-arylbenzofura-
n–appended 4-aminoquinazoline hybrids 10a–j are depicted in
Figure 5 and these were similar for all of the compounds except
for compound 10f, which docked at the entrance of the bind-
ing pocket.
EGFR tyrosine kinase is often used as a model in molecular
docking (in silico) studies to predict the hypothetical protein-
ligand binding mode. To help us understand the anticancer
activity of compounds 10 and guide further structure activity
Direct hydrogen bond formation between the ligand and the
receptor was only observed with Erlotinib (Hb distance ¼ 2.2 Å)
despite the fact that the 2-arylbenzofuran–appended 4-aminoqui-
nazoline hybrids 10 with the exception of 10f bind on the same
site as this reference drug. The aminoquinazoline and chloro/fluo-
rophenyl moieties of compounds 10a–e and 10g–j, on the other
hand, extended towards the entrance of the binding pocket. The
chloro/fluorophenyl rings of these compounds (with the exception
of 10f) formed T-shape p-stacking interaction with Phe699 that
might contribute towards the favorable binding affinity with EGFR.
Compared to the docking mode, it could be assumed that the
synthesized compounds might act on the ATP binding site of
EGFR like Gefitinib does.
Table 3. IC50 values (in nM) of 10a–j and Gefitinib against
EGFR-TK.
Compound
IC50 (nM)
10a
10b
10c
10d
10e
10f
10g
10h
10i
111.3 0.42
122.5 0.50
52.2 0.18
29.3 0.02
31.1 0.12
40.4 0.16
132.9 0.55
125.7 0.52
90.2 0.34
61.5 0.01
33.1 0.02
10j
Gefitinib
Figure 5. Docked conformation of Erlotinib (as docking control), Gefitinib and 4-aminoquinazoline–appended 2-arylbenzofurans (compound 10; stick representation) in
the binding pocket of epidermal growth factor receptor (EGFR) kinase domain (surface and ribbon representation). Blue dotted line is the hydrogen bonding formed
between erlotinib and EGFR. T-shape p-stacking interaction between the chlorophenyl/fluorophenyl of 10a–e and 10g–j with EGFR Phe699 was observed.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1527
4. Conclusions
References
In summary, we have developed a simple method for linking the
2-arylbenzofuran and quinazoline scaffolds through an amino
bridge to comprise analogs of the medicinally important 4-anilino-
quinazolines or Cediranib (2). Some of the prepared benzofuran-
aminoquinazoline hybrids exhibited moderate to significant
cytotoxicity in vitro against the A549 and HeLa cancer cell lines.
Although most of these compounds are less active against the
Caco-2 and C3A cell lines, a combination of a 2–(3-chlorophenyl)
group on the benzofuran moiety and a 2–(4-fluorophenyl) group
on the quinazoline framework of 10d resulted in significant cyto-
toxicity against the C3A cell line (LC50 ¼ 9.0 mM) when compared
to Gefitinib (LC50 ¼ 5.01 mM). Likewise, compound 10j substituted
with a 4-chlorophenyl group at the 2-position of the quinazoline
moiety and 4-trifluoromethoxyphenyl group at the 2-position of
the benzofuran framework was found to exhibit increased cytotox-
icity against the Caco-2 cells (LC50 ¼ 18.4 mM) more so than
Gefitinib (LC50 ¼ 27.9 mM). Compounds 10a–j were found to
exhibit moderate to significant cytotoxicity against the HeLa cells
with the exception of 10b, 10g and 10i substituted with a fluor-
ine or chlorine atom on the 3-position of the phenyl ring on the
benzofuran arm. Compounds 10d and 10j, which were selected
for further evaluation of the mode of cell death were able to trig-
ger apoptosis in C3A & Caco-2 cells, respectively. This demon-
strates that the benzofuran-aminoquinazoline hybrids prepared in
this investigation exhibit cytotoxicity and pro-apoptotic properties.
Compounds 10d and 10j not only demonstrated strong antiprolif-
eration activities against some of the tested cancer cell lines, but
also showed significant inhibitory activity towards EGFR
(IC50 ¼ 29.3 nM and 61.5 nM, respectively) compared to the medi-
cinally important EGFR inhibitor, Gefitinib (IC50 ¼ 33.1 nM).
Molecular docking studies into the EGFR also demonstrated that
the benzofuran-aminoquinazoline hybrids prepared in this investi-
gation have potential to inhibit EGFR-TK phosphorylation. Since
the title compounds and their derivatives may also target protein
kinases other than EGFR, future studies will be extended to other
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