Synthesis and anti-tumor activities of 4-anilinoquinoline derivatives

Article abstract of DOI:10.3390/molecules21010021

Twenty-two 7-fluoro (or 8-methoxy)-4-anilinoquinolines compounds were designed and synthesized as potentially potent and selective antitumor inhibitors. All the prepared compounds were evaluated for their in vitro antiproliferative activities against the HeLa and BGC823 cell lines. Ten compounds (1a-g; 2c; 2e and 2i) exhibited excellent antitumor activity superior to that of gefitinib. Among the ten compounds; seven (1a-c; 1e-1g and 2i) displayed excellent selectivity for BGC823 cells. In particular; 1f and 2i exhibited potent cytotoxic activities against HeLa cells and BGC823 cells with better IC50 values than gefitinib.

Full text of DOI:10.3390/molecules21010021

Article
Synthesis and Anti-Tumor Activities of
4-Anilinoquinoline Derivatives
Dan Liu *, Tian Luan, Jian Kong, Ying Zhang and Hai-Feng Wang
Received: 2 November 2015 ; Accepted: 2 December 2015 ; Published: 23 December 2015
Academic Editor: D. Hadjipavlou-Litina
Department of Pharmaceutical Engineering, College of Parmaceutical and Biological Engineering,
Shenyang University of Chemical Technology, Shenyang 110142, China; sapphire0614@163.com (T.L.);
kongjianwm2006@163.com (J.K.); zhingky621@126.com (Y.Z.); 38whf@163.com (H.-F.W.)
*
Correspondence: liudan20040318@163.com; Tel./Fax: +86-24-8938-3906
Abstract: Twenty-two 7-fluoro (or 8-methoxy)-4-anilinoquinolines compounds were designed and
synthesized as potentially potent and selective antitumor inhibitors. All the prepared compounds
were evaluated for their in vitro antiproliferative activities against the HeLa and BGC823 cell lines.
Ten compounds (1a–g; 2c; 2e and 2i) exhibited excellent antitumor activity superior to that of
gefitinib. Among the ten compounds; seven (1a–c; 1e–1g and 2i) displayed excellent selectivity
for BGC823 cells. In particular; 1f and 2i exhibited potent cytotoxic activities against HeLa cells and
BGC823 cells with better IC50 values than gefitinib.
Keywords: 4-anilinoquinolines; EGFR; antitumor; inhibitor
1. Introduction
The epidermal growth factor receptor (EGFR) and its closely related family member HER2 play
a critical role in mediating growth factor signaling, which make them interesting drug targets for
oncology. EGFR and HER2 have been extensively investigated, and various classes of small molecule
kinase inhibitors have emerged as promising strategies to inhibited EGFR and HER2 kinase activity.
A considerable number of 4-anilinoquinazoline-based kinase inhibitors are known, including
gefitinib (1), erlotinib (2) [1,2], lapatinib (3) [3,4], and afatinib (4) [5,6] (Figure 1). Previous SAR
studies suggest that the quinazoline core was the best scaffold for the development of EGFR inhibitors
and the quinazoline N3 interacts with the kinase domain via a water-mediated hydrogen bond to
the side chain of the gatekeeper Thr790 of EGFR [7,8]. The quinazoline-based kinase inhibitors are
widely used in medicinal chemistry and chemical biology research. For instance, Some C2 position
modification of quinazoline-based compounds resulted in potent cytotoxic activities [9,10]. On the
basis of the SAR studies of quinazolines, a series of compounds were developed where the N3 of the
quinazoline was replaced by a C-CN group, such as neratinib (5, Figure 1) [11,12]. However, Rauh
et al. have found that there was no evidence with the existence of a water molecule mediating the
binding of N3 of the quinazoline core to the side chain of Thr790 by calculating the corresponding
electron density maps. Quinolines 6 and 7 (Figure 1) were found to be highly active kinase inhibitors
in biochemical assays and were further investigated for their biological effect on EGFR-dependent
Ba/F3 cells and non-small cell lung cancer (NSCLC) cell lines [13]. Furthermore, studies have shown
that the 4-anilino group can interact with the hydrophobic pocket of EGFR, the introduction of
electron-donating groups in benzene ring of 4-anilinoquinazoline can increase the density of electron
cloud on the quinazoline N1, and follow by increase the interaction with EGFR [14].
The aforementioned findings stimulated our interest in designing and synthesizing a series of
7-fluoro or 8-methoxy 4-anilinoquinolines which were acticipated to be as potent as their quinazoline
Molecules 2016, 21, 21; doi:10.3390/molecules21010021
www.mdpi.com/journal/molecules

Molecules 2016, 21, 21
2 of 8
counterparts. The activity of the target compounds were evaluated by human cervical cancer cell line
(HeLa) and human gastric carcinoma cell line (BGC-823), and both of the cell lines had been proved
to be highly expressed cell line of EGFR [15,16].
F
Cl
Cl
F
O
O
HN
N
S
O
O
NH
NH
NH
O
O
O
O
N
N
O
O
N
O
N
N
F
N
Gefitinib 1
Erlotinib 2
Lapatinib 3
Cl
N
Cl
O
NH
H
N
NH
CN
H
N
N
N
N
O
O
N
O
O
N
O
Afatinib 4
Neratinib 5
Br
Br
NH
N
NH
N
H
N
H
N
N
O
O
6
7
Figure 1. Several inhibitors of EGFR tyrosine kinases.
2. Results and Discussion
2.1. Chemistry
The target compounds 1a–h and 2a–n were synthesized via a convenient six-step reaction
depicted in Scheme 1 and the respective experimental details are given in Section 3.1. Twenty-two
compounds were obtained and their MS, 1H-NMR spectroscopy data are provided in Section 3.1.
OH
EtOOC COOEt
EtOOC
COOEt
OEt
COOEt
a
b
c
+
NH2
X
X
N
H
X
N
Y
Y
Y
1III,2III
1II,2II
1I,2I
1I:X=F,Y=H
2I:X=H,Y=OCH3
R
OH
N
Cl
OH
NH
N
COOH
f
e
d
X
X
N
X
N
X
Y
Y
Y
Y
1VI,2VI
1IV,2IV
1V,2V
Scheme 1. General procedure for the synthesis of target compounds 1a–h and 2a–n. Reagents and
conditions: (a) EtOH, reflux; (b) Dowtherm A, 250–260 ˝C; (c) 10% NaOH, EtOH, reflux; (d) Dowtherm
A, 240 ˝C; (e) POCl3; (f) i-PrOH, pyridine-HCl, substituted aniline, reflux.

Molecules 2016, 21, 21
3 of 8
2.2. Biological Evaluation
The biological activities of all the target compounds 1a–h and 2a–n were evaluated in vitro
by MTT assay against HeLa and BGC-823 cell lines with gefitinib (1) as the positive control.
Their inhibition rate and IC50 values are listed in Table 1.
Table 1. Antiproliferactive activity of the target compounds on HeLa and BGC-823 cell lines.
Inhibition Rate % a
IC50 (µM) b
Compound
Substituents
HeLa
BGC-823
HeLa
BGC-823
1a
1b
1c
1d
1e
1f
1g
1h
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
X = F, Y = H, R = 31-Cl
X = F, Y = H, R = 41-Cl
46.9
40.3
37.0
20.4
40.8
46.8
41.7
36.1
35.3
31.5
31.9
18.9
30.8
37.2
30.5
21.9
59.1
32.7
32.5
35.8
32.7
28.6
42.6
77.5
63.1
65.3
49.5
56.9
67.5
59.4
35.1
19.5
13.1
48.1
37.5
48.7
20.2
31.1
32.0
78.4
24.5
27.6
36.8
25.6
16.0
46.0
17.29
20.39
19.82
43.25
36.69
10.18
18.69
55.76
66.69
ą10
3.63
7.83
9.10
11.10
8.29
8.32
X = F, Y = H, R = 31-F
X = F, Y = H, R = 41-F
X = F, Y = H, R = 31-Cl,41-Cl
X = F, Y = H, R = 31-Cl,41-F
X = F, Y = H, R = 41-CH3
7.08
X = F, Y = H, R = 41-OCH3
X = H, Y = OCH3, R = 31-Cl
X = H, Y = OCH3, R = 41-Cl
X = H, Y = OCH3, R = 31-F
X = H, Y = OCH3, R = 41-F
X = H, Y = OCH3, R = 31-Cl,41-Cl
X = H, Y = OCH3, R = 31-Cl,41-F
X = H, Y = OCH3, R = 41-CH3
X = H, Y = OCH3, R = 41-OCH3
X = H, Y = OCH3, R = 41-CH(CH3)2
X = H, Y = OCH3, R = 31-Cl,41-CH3
X = H, Y = OCH3, R = 21F, 31-F, 41-F
X = H, Y = OCH3, R = 41-NO2
X = H, Y = OCH3, R = 41-OH
X = H, Y = OCH3, R = 31-CN
21.61
ą10
ą10
11.67
46.41
11.45
ą10
61.52
74.60
4.65
ą10
ą10
ą10
ą10
ą10
19.27
75.87
ą10
45.87
45.84
99.54
ą10
7.15
71.26
57.88
13.48
39.58
30.61
17.12
2m
2n
Gefitinib
a
Inhibitory percentage of cells treated with each compound at a concentration of 10 µM for 96 h; b The agent
concentration that inhibited HeLa and BGC-823 cells growth by 50%.
As shown in Table 1, 7-fluoro-4-anilinoquinolines 1a–g displayed better cytotoxic activities
against BGC-823 cells than HeLa cells and exhibited IC50 values within the 3.63–11.10 µmol/L
range (on BGC-823 cells). It seems that substituent changes on the quinoline ring at the C7 or
C8 carbons have a great influence on the antiproliferactive activity. In general, 7-fluoro-4-anilino-
quinolines of these twenty-two compounds, were more active than those of the corresponding
8-methoxy-4-anilinoquinolines, as demonstrated by comparison of 1a–h and 2a–h. Thirdly, it was
found that substituent changes on the benzene ring had little influence on antiprolifeactive activity,
whether it was electron-donating or electron-withdrawing substituent at any position (1a vs. 1b and
1d; 2g vs. 2b and 2d; 2g and 2m vs. 2l and 2n). Lastly, most of the 8-methoxy-4-anilinoquinolines
exhibited moderate antiproliferactive activity against the HeLa and BGC-823 cell lines, except for
compound 2i. It was noteworthy that compound 2i bearing a methoxy group on the quinoline ring
and an isopropyl group on the benzene ring showed remarkable inhibitory effects on HeLa cells and
BGC-823 cells (IC50 = 7.15 µM, IC50 = 4.65 µM), which represented a 2.4 and 4.1-fold increase in
antitumor activity compared to gefitinib (IC50 = 17.12 µM against HeLa cells, IC50 = 19.27 µM against
BGC-823 cells), respectively.

Molecules 2016, 21, 21
4 of 8
3. Experimental Section
3.1. Chemistry
All reagents and solvents were commercially available and were used without further
purification. The melting points were determined on an electrically heated X-4 digital visual melting
1
point apparatus (Tech, Beijing, China) and were uncorrected. H-NMR spectra were recorded on an
ARX-300 or AV-600 spectrometer (Bruker, Fällanden, Switzerland) at room temperature, and chemical
shifts were measured in ppm downfield from TMS as internal standard. Mass spectra were recorded
on Thermo-Finnigan LCQ equipment (ThermoFinnigan, San Francisco, CA, USA) with the positive
Electron Spray Ionization (ESI) mode and are reported as m/z. Analytical thin layer chromatography
(TLC) on silica gel plates containing UV indicator (YuHua, Gongyi, China) was routinely employed
to follow the course of reactions and to check the purity of products.
3.1.1. Preparation of the Intermediates 1II–1VI
Diethyl (ethoxymethylene) malonate (6.48 g, 30.00 mmol) and m-fluroaniline (2.78 g, 25.00 mmol)
was stirred in refluxing ethanol (10 mL) for 1.5 h. The mixture was concentrated under reduced
pressure. The residue was crystallized in petroleum ether, filtered off, and air-dried to get 1II.
˝
Compound 1II (2.80 g, 9.96 mmol) was stirred in Dowtherm-A (8 mL) for 0.5 h at 260 C.
After the reaction was over by TLC, the mixture was cooled to room temperature and petroleum
ether was added to get the crude ester, which was further washed with petroleum ether to afford
1III, as a white solid. 1III was hydrolyzed in refluxing NaOH solution (10%, 20 mL) for 1.5 h to give
˝
1IV. Compound 1IV (1.24 g, 6 mmol) was stirred in Dowtherm-A (10 mL) for 1 h at 240 C. After
the reaction was over as monitored by TLC, the reaction mixture was cooled to room temperature
and the crude was washed with petroleum ether to afford 1V as a white solid. To a solution of 1V
(1.20 g, 7.36 mmol) in 1,2-dichloroethane (30 mL), POCl3 (1.35 g, 8.83 mmol) was added dropwise.
The mixture was refluxed for 1 h. Saturated NaHCO3 solution was added to neutralize the reaction
mixture, which was worked up with 1,2-dichloroethane. The organic layer was dried over anhydrous
MgSO4, filtered and concentrated in vacuo. The residue was purified by column chromatography
(silica gel) using petroleum ether/ethyl acetate as an eluent (5:1) to produce 1VI as a white solid [17].
1VI: 1H-NMR (300 MHz, CDCl3): 8.79–8.78 (d, 2-H, 1H), 8.27–8.24 (q, 5-H, 1H), 7.77–7.75 (q, 8-H, 1H),
7.47–7.46 (d, 3-H, 1H), 7.44–7.42 (q, 6-H, 1H), ESI-MS (m/z): 182.4[M + H]+.
3.1.2. Preparation of the Intermediates 2II–2VI
1
Compounds 2II–2VI were prepared in the same manner as 2II–2VI. 2VI: H-NMR (300 MHz,
CDCl3): 4.11 (3H, s, OCH3), 7.11 (1H, d, J = 7.2 Hz, Ar-H-7), 7.53–7.60 (2H, m, Ar-H-3, 6),
7.81 (1H, dd, J = 8.7 Hz, J = 1.2 Hz, Ar-H-5), 8.80 (1H, d, J = 4.8 Hz, Ar-H-2); ESI-MS (m/z):
194.4 [M + H]+.
3.1.3. Preparation of the Title Compounds 1 and 2
A mixture of compound 1VI (0.040 g, 0.22 mmol), m-chloroaniline (0.036 g, 0.31 mmol) and
pyridine hydrochloride was heated at reflux for 45 min in isopropanol (6 mL), after the reaction is
over by TLC, it was cooled to room temperature and the petroleum ether (4 mL) and NaHCO3 (10 mL)
were added into the reaction mixture. The product was filtered and recrystallised from ethanol to give
the title compound 1. Compound 2 was prepared in the same manner as 1.
1
˝
4-(3 -Chlorophenylamino)-7-fluoroquinoline (1a). Yield: 96.25%; white solid. m.p. 196–197 C; 1H-NMR
(600 MHz, CDCl3) δ: 8.62 (d, J = 4.8 Hz, 1H, ArH), 7.94 (dd, J = 9.0, 6.0 Hz, 1H, ArH ), 7.71 (dd, J = 4.2,
2.4 Hz, 1H, ArH), 7.37–7.27 (m, 3H, ArH), 7.19–7.17 (m, 2H, ArH), 7.01 (d, 1H, J = 5.4 Hz, ArH).
ESI-MS (m/z): 273.4 [M + H]+.

Molecules 2016, 21, 21
5 of 8
1
˝
1
4-(4 -Chlorophenylamino)-7-fluoroquinoline (1b). Yield: 92.93%. m.p. 194–195 C, H-NMR (600 MHz,
CDCl3) δ: 8.57 (s, 1H, ArH), 7.96 (dd, J = 8.4, 6.0 Hz, 1H, ArH), 7.70 (d, 1H, J = 10.2 Hz, ArH),
7.41–7.40 (m, 2H, ArH), 7.31 (t, J = 8.4 Hz, 1H, ArH), 7.27–7.25 (m, 2H, ArH), 6.90 (d, J = 4.8 Hz, 1H,
ArH). ESI-MS (m/z): 273.4 [M + H]+.
1
˝
1
4-(3 -Fluorophenylamino)-7-fluoroquinoline (1c). Yield: 95.37%; m.p. 173–174 C; H-NMR (600 MHz,
CDCl3) δ: 8.62 (s, 1H, ArH), 7.94 (dd, J = 9.0, 6.0 Hz, 1H, ArH), 7.70 (dd, J = 6.6, 2.1 Hz, 1H, ArH), 7.38
(dd, J = 14.7, 8.1 Hz, 1H, ArH), 7.34–7.31 (m, 1H, 1H, ArH), 7.06 (t, J = 4.5 Hz, 1H, ArH), 7.04–7.02
(m, 2H, ArH), 6.91–6.88 (m, 1H, ArH). ESI-MS (m/z): 257.4 [M + H]+.
1
˝
1
4-(4 -Fluorophenylamino)-7-fluoroquinoline (1d). Yield: 93.66%; m.p. 178–179 C; H-NMR (600 MHz,
CDCl3) δ: 8.54–8.53(s, 1H, ArH), 7.94 (d, J = 6.0 Hz, 1H, ArH), 7.68 (dd, J = 10.2, 3.0 Hz, 1H,
ArH), 7.32–7.28 (m, 3H, ArH), 7.17–7.13 (m, 2H, ArH), 6.74 (d, J = 5.4 Hz 1H, ArH). ESI-MS (m/z):
257.4 [M + H]+.
1
1
˝
1
4-(3 -Chloro-4 -chlorophenylamino)-7-fluoroquinoline (1e). Yield: 91.34%; m.p. 191–192 C; H-NMR
(600 MHz, CDCl3) δ: 8.61 (d, J = 4.8 Hz, 1H, ArH), 7.97 (dd, J = 9.0, 6.0 Hz, 1H, ArH), 7.71 (dd, J = 9.6,
2.4 Hz, 1H, ArH), 7.48 (d, J=9.0Hz, 1H, ArH), 7.41 (d, J = 2.4 Hz, 1H, ArH), 7.34–7.27 (m, 1H, ArH),
7.16 (dd, J = 8.7, 2.7 Hz, 1H, ArH), 6.97 (dd, J = 5.4 Hz, 1H, ArH). ESI-MS (m/z): 307.3 [M + H]+.
1
1
˝
4-(3 -Chloro-4 -fluoroamino)-7-fluoroquinoline (1f). Yield: 74.72%; m.p. 193–194 C; 1H-NMR (600 MHz,
CDCl3) δ: 8.58 (s, 1H, ArH), 7.93 (dd, J = 9.0, 5.4 Hz, 1H, ArH), 7.70 (dd, J = 10.2, 2.4 Hz, 1H, ArH),
7.38 (dd, J = 10.0, 2.4 Hz, 1H, ArH), 7.34–7.31 (m, 1H, ArH), 7.27–7.19 (m, 2H, ArH), 6.82 (d, J = 5.4 Hz,
1H, ArH). ESI-MS (m/z): 291.4 [M + H]+.
1
˝
4-(4 -Methylphenylamino)-7-fluoroquinoline (1g). Yield: 96.56%, m.p. 171–172 C; 1H-NMR (600 MHz,
CDCl3) δ: 8.50 (d, J = 5.4 Hz, 1H, ArH), 7.96 (dd, J = 9.3, 5.7 Hz, 1H, ArH), 7.68 (dd, J = 10.2, 2.4 Hz,
1H, ArH), 7.31–7.27 (m, 1H, ArH), 7.26–7.25 (m, 2H, ArH), 7.22–7.20 (m, 2H, ArH ), 6.83 (d, J = 4.2 Hz,
1H, ArH), 2.40 (s, 3H, CH3). ESI-MS (m/z): 253.4 [M + H]+.
1
˝
4-(4 -Methoxyphenylamino)-7-fluoroquinoline (1h). Yield: 76.15%, m.p. 181–182 C; 1H-NMR (600 MHz,
CDCl3) δ: 8.46 (d, J = 5.4 Hz, 1H, ArH), 7.98 (dd, J = 9.0, 5.4 Hz, 1H, ArH), 7.68 (dd, J = 9.9, 2.7 Hz,
1H, ArH), 7.30–7.27 (m, 1H, ArH), 7.26–7.25 (m, 2H, ArH), 7.00–6.98 (m, 2H, ArH), 6.65 (d, J = 5.4 Hz,
1H, ArH). 3.88 (s, 3H, OCH3); ESI-MS (m/z): 269.3 [M + H]+.
1
˝
4-(3 -Chlorophenylamino)-8-methoxyquinoline (2a). Yield: 76.16%, m.p. 250–251 C; 1H-NMR (300 MHz,
CDCl3) δ: 8.58 (d, J = 5.4 Hz, 1H, ArH), 7.45–7.50 (m, 2H, ArH), 7.40–7.30 (m, 1H, ArH), 7.14–7.10
(m, 2H, ArH), 7.06–6.98 (m, 2H, ArH), 6.88 (d, J = 5.4 Hz, 1H, ArH), 4.08 (s, 3H, OCH3); ESI-MS (m/z):
285.5 [M + H]+.
1
˝
4-(4 -Chlorophenylamino)-8-methoxyquinoline (2b). Yield: 77.52%; m.p. 254–258 C. 1H-NMR (300 MHz,
CDCl3) δ: 8.60 (d, J = 5.1 Hz, 1H, ArH), 7.49–7.52 (m, 2H, ArH), 7.40–7.30 (m, 2H, ArH), 7.14–7.10 (m,
1H, ArH), 7.06–6.98 (m, 2H, ArH), 6.84 (d, J = 5.1 Hz, 1H, ArH), 4.08 (s, 3H, OCH3); ESI-MS (m/z):
285.5 [M + H]+.
1
˝
4-(3 -Fluorophenylamino)-8-methoxyquinoline (2c). Yield: 75.06%; m.p. 267–269 C. 1H-NMR (300 MHz,
CDCl3) δ: 8.65 (s, 1H, ArH), 7.49–7.44 (m, 2H, ArH), 7.39–7.31 (m, 1H, ArH), 7.15 (dd, J = 5.1,
2.1 Hz, 1H, ArH), 7.08–6.99 (m, 3H, ArH), 6.88–6.82 (m, 1H, ArH), 4.08 (s, 3H, OCH3); ESI-MS (m/z):
269.3 [M + H]+.
1
˝
4-(4 -Fluorophenylamino)-8-methoxyquinoline (2d). Yield: 84.59%; m.p. 256–258 C. 1H-NMR (300 MHz,
CDCl3) δ: 8.56 (d, J = 5.1 Hz, 1H, ArH), 7.52–7.41 (m, 2H, ArH), 7.30–7.27 (m, 2H, ArH), 7.15–7.05
(m, 3H, ArH), 6.82(d, J = 5.1 Hz, 1H, ArH), 4.08 (s, 3H, OCH3); ESI-MS (m/z): 269.3 [M + H]+.
1
1
˝
1
4-(3 -Chloro-4 -chlorophenylamino)-8-methoxyquinoline (2e). Yield: 79.18%; m.p. 244–246 C. H-NMR
(300 MHz, CDCl3) δ: 8.63 (d, J = 5.1 Hz, 1H, ArH), 7.44–7.49 (m, 3H, ArH), 7.41 (d, J = 2.4 Hz, 1H,

Molecules 2016, 21, 21
6 of 8
ArH), 7.16 (dd, J = 8.7, 2.4 Hz, 1H, ArH), 7.08 (d, J = 8.4 Hz, 1H, ArH), 7.03 (d, J = 5.1Hz, 1H, ArH),
4.08 (s, 3H, OCH3); ESI-MS (m/z): 319.3 [M + H]+.
1
1
˝
1
4-(3 -Chloro-4 -fluorophenylamino)-8-methoxyquinoline (2f). Yield: 80.14%; m.p. 247–249 C. H-NMR
(300 MHz, CDCl3) δ: 8.59 (d, J = 5.1 Hz, ArH), 7.38–7.36 (m, 1H, ArH), 7.20–7.17 (m, 2H, ArH),
7.07 (dd, J = 6.6, 2.4 Hz, 1H, ArH), 6.88 (d, J = 5.1 Hz, 1H, ArH), 4.08 (s, 3H, OCH3); ESI-MS (m/z):
303.3 [M + H]+.
1
˝
4-(4 -Methylphenylamino)-8-methoxyquinoline (2g). Yield: 78.46%; m.p. 258–260 C. 1H-NMR (300 MHz,
CDCl3) δ: 8.56 (d, J = 5.4 Hz, 1H, ArH), 7.43–7.47 (m, 2H, ArH), 7.22–7.17 (m, 4H, ArH),
7.06 (dd, J = 6.9, 1.5 Hz, 1H, ArH), 6.93 (d, J = 5.4 Hz, 1H, ArH), 4.08 (s, 3H, OCH3), 2.39 (s, 3H, CH3);
ESI-MS (m/z): 265.4 [M + H]+.
1
˝
4-(3 -Methoxyphenylamino)-8-methoxyquinoline (2h). Yield: 80.96%; m.p. 231–233 C. 1H-NMR
(300 MHz, CDCl3) δ: 8.61 (d, J = 5.1 Hz, ArH), 7.49–7.41 (m, 2H, ArH), 7.32 (t, J = 5.4 Hz, 1H, ArH),
7.13–7.10 (m, 1H, ArH), 7.06 (dd, J = 6.9, 1.8 Hz, ArH), 6.90–6.84 (m, 2H, ArH), 6.73 (dd, J = 8.1, 2.1
Hz, 1H, ArH), 4.08 (s, 3H, OCH3), 3.83 (s, 3H, OCH3); ESI-MS (m/z): 281.2 [M + H]+.
1
˝
4-(4 -Isopropylphenylamino)-8-methoxyquinoline (2i). Yield: 79.47%; m.p. 236–238 C. 1H-NMR
(300 MHz, CDCl3) δ: 8.53 (d, J = 5.1 Hz, 1H, ArH), 7.48–7.40 (m, 2H, ArH), 7.30–7.21 (m, 4H, ArH),
7.05 (dd, J = 6.9, 2.1 Hz, 1H, ArH), 6.99–6.96 (m, 1H, ArH), 4.08 (s, 3H, OCH3), 2.99–2.90 (m, 1H, CH),
1.29 (d, J = 6.9Hz, 6H, CH3) ; ESI-MS (m/z): 293.6 [M + H]+.
1
1
˝
1
4-(3 -Chloro-4 -methylphenylamino)-8-methoxyquinoline (2j). Yield: 76.48%; m.p. 270–272 C. H-NMR
(300 MHz, CDCl3) δ: 8.61 (d, J = 5.4 Hz, 1H, ArH), 7.47–7.41 (m, 2H, ArH), 7.31(d, J = 2.4 Hz, 1H,
ArH), 7.25 (d, J = 7.8 Hz, 1H, ArH), 7.12–7.04 (m, 2H, ArH), 6.99 (d, J = 5.1 Hz, 1H, ArH), 4.08 (s, 3H,
OCH3), 2.39 (s, 3H, CH3); ESI-MS (m/z): 299.3[M + H]+.
˝
4-(2,3,4-Trifluorophenylamino)-8-methoxyquinoline (2k). Yield: 70.00%; m.p. 238–239 C. 1H-NMR
(300 MHz, CDCl3) δ: 8.61 (d, J = 5.1 Hz, 1H, ArH), 7.57–7.45 (m, 2H, ArH), 7.24–7.16 (m, 1H,
ArH), 7.10–6.84 (m, 2H, ArH), 6.84 (d, J = 5.4 Hz, 1H, ArH), 4.10 (s, 3H, OCH3) ; ESI-MS (m/z):
305.2 [M + H]+.
1
˝
4-(4 -Nitrophenylamino)-8-methoxyquinoline (2l). Yield: 78.18%; m.p. 265–267 C. 1H-NMR (300 MHz,
CDCl3) δ: 8.79 (d, J = 4.8 Hz, 1H, ArH), 8.25 (d, J = 9 Hz, 2H, ArH), 7.51–7.46 (m, 2H, ArH),
7.37 (d, J = 4.8 Hz, 1H, ArH), 7.29–7.26 (m, 2H, ArH), 7.11 (t, J = 4.2 Hz, 1H, ArH), 4.10 (s, 3H, OCH3);
ESI-MS (m/z): 296.2 [M + H]+.
1
˝
4-(4 -Hydroxyphenylamino)-8-methoxyquinoline (2m). Yield: 58.17%; m.p. 236–238 C. 1H-NMR
(300 MHz, DMSO) δ: 9.44 (s, 1H, OH), 8.61 (s, 1H, ArH), 8.29 (d, J = 5.1 Hz, 1H, ArH),
7.89 (d, J = 8.7 Hz, 1H, ArH), 7.38 (t, J = 8.4 Hz, 1H, ArH), 7.12 (t, J = 8.4 Hz, 2H, ArH),
6.84 (d, J = 8.7 Hz, 2H, ArH), 6.56 (d, J = 5.1 Hz, 1H, ArH), 3.91 (s, 3H, OCH3); ESI-MS (m/z):
267.3 [M + H]+.
1
˝
4-(3 -Cyanophenylamino)-8-methoxyquinoline (2n). Yield: 91.43%; m.p. 255–256 C. 1H-NMR (300 MHz,
CDCl3) δ: 8.68 (d, J = 5.1 Hz, 1H, ArH), 7.55–7.40 (m, 5H, ArH), 7.11–7.08 (m, 2H, ArH), 6.74 (s, 1H,
ArH), 4.10 (s, 3H, OCH3); ESI-MS (m/z): 276.2 [M + H]+.
3.2. Cell Proliferative Assay
The antiproliferative activities of the prepared 4-anilinoquinolines against HeLa and BGC823 cell
lines were evaluated by MTT assay in vitro, with gefitinib as the positive control. The negative control
contains cells, culture medium, MTT and DMSO. All human tumor cells were cultured in RPMI 1640
medium supplemented with 10% fetal bovine serum (FBS). Cells were detached by trypsinisation,
˝
seeded at 1.0–2.0 ˆ 103 cells each well in a 96-well plate and incubated in 5% CO2 at 37 C overnight,
then treated with the test compounds at different concentration and incubated for 96 h. Fresh MTT

Molecules 2016, 21, 21
7 of 8
˝
solution was added to each well and incubated at 37 C for 4 h. The MTT-formazan formed by
metabolically viable cells was dissolved in 150 µL DMSO each well, and monitored by a microplate
reader at dual-wavelength of 490 nm; IC50 was defined as the drug concentrations that inhibited the
cell number to 50% after 96 h. Each test was performed three times.
4. Conclusions
In summary, two novel series of 4-anilinoquinolines were designed and synthesized as
potentially potent and selective antitumor inhibitors. All of the final compounds were generated
from aniline derivatives via six step reaction sequences including nucleophilic substitution,
cyclization, hydroxylation, decarboxylation, chlorination and nucleophilic substitution. Among the
7-fluoro-4-anilinoquinolines, all the prepared compounds displayed some cytotoxic activity against
the HeLa and BGC823 cell lines. Compounds 1a–g, 2c and 2e displayed superior cytotoxic activities
against the BGC823 cell line than gefitinib. Furthermore, compound 1f displayed good cytotoxic
activities against HeLa and BGC823 cells (IC50 value of 10.18 µM and 8.32 µM against HeLa
and BGC823 cells, respectively). In particular, compound 2i exhibited the most potent inhibitory
activity against BGC823 cells (IC50 value of 7.15 µM and 4.65 µM against HeLa and BGC823 cells,
respectively). The drug-like structural optimization based on the 4-anilinoquinoline skeleton will be
reported in the future.
Acknowledgments: This work was supported by the grant from the Scientific Research Project of the
Department of Education of Liaoning Province (Grant No. L2013171), the Natural Science Foundation
of Liaoning Province of China (Grant No. 2015020695), the Natural Science Foundation of China (Grant
No. 21372156). Additionally, we thank Weiling Wang, at the Experimental Therapeutics Center, Agency for
Science, Technology and Research of Singapore, for her English revisions and constructive criticism.
Author Contributions: T.L., J.K., Y.Z. and H.-F.W. performed the experiments. D.L. and T.L. designed the
experiments and wrote the paper. All authors took part in data analysis and discussion. All authors read and
approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Pao, W.; Miller, V.; Zakowski, M.; Doherty, J.; Politi, K.; Sarkaria, I.; Singh, B.; Heelan, R.; Rusch, V.;
Fulton, L.; et al. EGF receptor gene mutations are common in lung cancers from “never smokers” and
are associated with sensitivity of tumors to gefitinib and erlotinib. Proc. Natl. Acad. Sci. USA 2004, 101,
13306–13311. [CrossRef] [PubMed]
2.
3.
Bikker, J.A.; Brooijmans, N.; Wissner, A.; Mansour, T.S. Kinase domain mutations in cancer: Implications
for small molecule drug design strategies. J. Med. Chem. 2009, 52, 1493–1509. [CrossRef] [PubMed]
Feng, Y.F.; Zeng, C.L. Advance in research for lapatinib, a dual inhibitor of EGFR and ErbB2 tyrosine kinase
activity. Chin. New Drugs J. 2007, 16, 1990–1993.
4.
5.
Tao, L.Y.; Fu, L.W. Progress of study on lapatinib. Chin. Pharmacol. Bull. 2008, 24, 1541–1544.
Solca, F.; Dahl, G.; Zoephel, A.; Bader, G.; Sanderson, M.; Klein, C.; Kraemer, O.; Himmelsbach, F.;
Haaksma, E.; Adolf, G.R. Target binding properties and cellular activity of afatinib (BIBW 2992), an
irreversible ErbB family blocker. J. Pharmacol. Exp. Ther. 2012, 343, 342–350. [CrossRef] [PubMed]
Chen, X.; Zhu, Q.; Zhu, L.; Pei, D.; Liu, Y.; Yin, Y.; Schuler, M.; Shu, Y. Clinical perspective of afatinib in
non-small cell lung cancer. Lung Cancer. 2013, 81, 155–161. [CrossRef] [PubMed]
Rewcastle, G.W.; Denny, W.A.; Bridges, A.J.; Zhou, H.R.; Cody, D.R.; McMichael, A.; Fry, D.W. Tyrosine
kinase inhibitors. 5. Synthesis and structure-activity relationships for 4-[(phenylmethyl)amino]- and
4-(phenylamino) quinazolines as potent adenosine 51-triphosphate binding site inhibitors of the tyrosine
kinase domain of the epidermal growth factor receptor. J. Med. Chem. 1995, 38, 3482–3487. [PubMed]
Wissner, A.; Berger, D.M.; Boschelli, D.H.; Floyd, M.B.; Greenberger, L.M.; Gruber, B.C.; Johnson, B.D.;
Mamuya, N.; Nilakantan, R.; Reich, M.F.; et al. 4-Anilino-6,7-dialkoxyquinolime-3-carbonitrile inhibitors
of epidermal growth factor receptor kinase and their bioisosteric relationship to the 4-anilino-6,7-
dialkoxyquinazoline inhibitors. J. Med. Chem. 2000, 3, 3244–3256. [CrossRef]
6.
7.
8.

Molecules 2016, 21, 21
8 of 8
9.
Barbosa, M.L.D.C.; Lima, L.M.; Tesch, R.; Sant’Anna, C.M.R.; Totzke, F.; Kubbutat, M.H.G.; Schächtele, C.;
Laufer, S.A.; Barreiro, E.J. Novel 2-chloro-4-anilino-quinazoline derivatives as EGFR and VEGFR-2 dual
inhibitors. Eur. J. Med. Chem. 2014, 71, 1–14. [CrossRef] [PubMed]
10. Jiang, N.; Zhai, X.; Zhao, Y.F.; Liu, Y.J.; Qi, B.H.; Tao, H.Y.; Gong, P. Synthesis and biological evaluation
of novel 2-(2-arylmethylene) hydrazinyl-4-aminoquinazoline derivatives as potent antitumor agents.
Eur. J. Med. Chem. 2012, 54, 534–541. [CrossRef] [PubMed]
11. Minami, Y.; Shimamura, T.; Shah, K.; LaFramboise, T.; Glatt, K.A.; Liniker, E.; Borgman, C.L.;
Haringsma, H.J.; Feng, W.; Weir, B.A.; et al. The major lung cancer-derived mutants of ERBB2 are oncogenic
and are associated with sensitivity to the irreversible EGFR/ERBB2 inhibitor HKI-272. Oncogene 2007, 26,
5023–5027. [CrossRef] [PubMed]
12. Wissner, A.; Mansour, T.S. The development of HKI-272 andrelated compounds for the treatment of cancer.
Arch. Pharm. 2008, 341, 465–477. [CrossRef] [PubMed]
13. Pawar, V.G.; Sos, M.L.; Rode, H.B.; Rabiller, M.; Heynck, S.; Otterlo, W.A.L.V.; Thomas, R.K.; Rauh, D.
Synthesis and biological evaluation of 4-anilinoquinolines as potent inhibitors of epidermal growth factor
receptor. J. Med. Chem. 2010, 53, 2892–2901. [CrossRef] [PubMed]
14. Roskoski, R., Jr. The ErbB/HER family of protein-tyrosine kinases and cancer. Pharmacol. Res. 2014, 79,
34–74. [CrossRef] [PubMed]
15. Zhou, S.Z.; Li, X.H.; Zhang, Y.X. Methylation patterns of the human EGFR gene promoter region.
Chin. J. Biochem. Mol. Biol. 2010, 26, 568–574.
16. Long, H.; Li, H.; Wu, Q.M. RNA interference targeting EGFR on proliferation and apoptosis of human
gastric carcinama cell line BCG823. China J. Mod. Med. 2012, 22, 59–62.
17. Ray, A.W.; Joseph, C.M.; Victor, K.E.; Phil, J.G.; George, S.R. Quinoline, Quinazoline, and Cinnoline
Fungicides. E.P. Patent 0,326,330(A2), 2 August 1989.
Sample Availability: Not available.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open
access article distributed under the terms and conditions of the Creative Commons by
Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).