Synthesis and antitumor activity of novel 4-aminoquinoline derivatives

Article abstract of DOI:10.1007/s00044-012-0283-8

A series of novel 4-aminoquinoline derivatives were synthesized as antitumor agents by reacting 4-chloroquinoline with the corresponding mono/dialkyl amines. The cytotoxicity of these compounds was evaluated in vitro against HCT-116, A549, DU-145, HepG2, and LN229 cell lines. The results showed that most of the synthesized compounds displayed excellent cytotoxicity, and 5,7-dimethoxy-2-phenyl-N-propylquinoline-4-amine (6a) displayed the most potent cytotoxicity against HCT-116 cells. Furthermore, 6a could decrease VEGF protein expression.

Full text of DOI:10.1007/s00044-012-0283-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2855–2861
DOI 10.1007/s00044-012-0283-8
ORIGINAL RESEARCH
Synthesis and antitumor activity of novel 4-aminoquinoline
derivatives
•
Jie Ren Juan Zhao Yong-Sheng Zhou
•
•
•
Xian-Hua Liu Xin Chen Kun Hu
•
Received: 27 March 2012 / Accepted: 15 October 2012 / Published online: 30 October 2012
Ó Springer Science+Business Media New York 2012
Abstract A series of novel 4-aminoquinoline derivatives
were synthesized as antitumor agents by reacting 4-chloro-
quinoline with the corresponding mono/dialkyl amines. The
cytotoxicity of these compounds was evaluated in vitro
against HCT-116, A549, DU-145, HepG2, and LN229 cell
lines. The results showed that most of the synthesized
compounds displayed excellent cytotoxicity, and 5,7-dime-
thoxy-2-phenyl-N-propylquinoline-4-amine (6a) displayed
the most potent cytotoxicity against HCT-116 cells. Fur-
thermore, 6a could decrease VEGF protein expression.
antitumor drugs. The vascular endothelial growth factor
(VEGF), known as the most potent and specific angiogenetic
factor, represents an important target for the discovery of
antitumor inhibitors. Receptor tyrosine kinase (RTK)
inhibitors which target VEGF receptor are one class of the
most promising agents (Hennequin et al., 1999).
Nitrogen-containingheterocyclesarepresent inavarietyof
biologically active compounds that can be used in a wide
range of therapeutic areas (Cantin et al., 2007). 2-Phenyl-
quinoline derivatives are the effective pharmacophore in
medicinal chemistry as illustrated by its application in phar-
maceutical agents as novel antitumor promoters (Kakadiya
et al., 2010; Sirisoma et al., 2010; Zhang et al., 2008; Chen
et al., 2006; Zhao et al., 2005), antimalarial agents (Solomon
et al., 2005; Solomon et al., 2010), and DNA-intercalator
agents (Atwell et al., 1989; Atwell et al., 1988) according to
their effective binding ability with DNA. 2-Phenylquinolines
containing additional basic N-4-aminoalkyl groups would
strongly increase the biological activity of immunostimula-
tory CpG-oligodeoxynucleotides (Strekowski et al., 2003).
Tacrine, which is used for treating Alzheimer’s disease (Luo
et al., 2006), is a 4-amino-substituted quinolone derivative.
Some of the 4-aminoquinoline derivatives had also been
explored as ligands for the benzodiazepine binding site of
brain GABAA receptors (Nilsson et al., 2008).
Keywords 4-Aminoquinoline ꢀ Synthesis ꢀ
Antitumor activity ꢀ VEGF
Introduction
Theoretically, antitumor agents could be developed to target
each step in the tumor activation or inhibition process, and
numerous new antitumor agents have been developed based
on their inhibitory effects on tumor activation (Xia et al.,
2001). Inhibition of angiogenesis which is a fundamental
process of tumor growth and metastasis has become one of
the interesting areas in research and development of novel
J. Ren ꢀ J. Zhao ꢀ X.-H. Liu ꢀ X. Chen (&) ꢀ K. Hu (&)
School of Pharmaceutical Engineering & Life Science,
Changzhou University, 1 Gehu Road, Changzhou 213164,
Jiangsu, People’s Republic of China
According to the literature, 4-anilinoquinazoline could
be potent inhibitors of VEGF receptor, and the quino-
line derivative is almost equally potent as quinazoline
(Hennequin et al., 1999). The present work is a continuation
of our ongoing efforts toward developing new and effective
antitumor agents. We have designed and synthesized a series
of novel 4-aminoquinoline derivatives, and their antitumor
activity was evaluated against several tumor cell lines
in vitro. Furthermore, the effect of one active compound on
VEGF protein expression was also investigated.
e-mail: xinchen@cczu.edu.cn
K. Hu
e-mail: hukun1979@163.com
Y.-S. Zhou
School of Petrochemical Engineering, Changzhou University,
1 Gehu Road, Changzhou 213164, Jiangsu,
People’s Republic of China
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Med Chem Res (2013) 22:2855–2861
Results and discussion
Chemistry
DU-145 (human prostate carcinoma cell line), HepG2
(human liver carcinoma cell line), LN229 (human glioblas-
toma cell line), and HEK293 (human embryonic kidney 293)
by the standard MTT assay, and 5-FU (5-fluorouracil) was
used as a positive control. Antitumor potency of the com-
pounds was indicated by IC50 values (Table 1).
The synthetic route used to prepare the title compounds
is outlined in Scheme 1. 3,5-Dimethoxybenzenamine 1
was acylated with benzoyl chloride to give N-(3,5-dime-
thoxyphenyl)benzamide 2, which was then treated with
acetyl chloride in the presence of tin (IV) chloride to afford
N-(2-acetyl-3,5-dimethoxyphenyl) benzamide 3. Upon
treatment with NaOH in refluxing 1,4-dioxane, compound
3 was cyclized to generate 5,7-dimethoxy-2-phenylquino-
line-4(1H)-one 4. Chlorination of compound 4 with phos-
phoryl trichloride afforded 4-chloro-5,7-dimethoxy-2-
phenylquinoline 5. In the final step, a series of 4-amino-
quinoline derivatives (6a–6i) were obtained through
reacting 4-chloroquinoline 5 with various amines at
60–90 °C over 8–36 h in the presence of Et3N. The
structures of the new compounds were confirmed based on
As shown in Table 1, most of the prepared compounds
showed potent inhibitory effects on HCT-116 cell line.
Compared to 5-FU, some compounds showed more potent
cytotoxicity to certain types of cancer cell lines, such as 6a
to HCT-116, 6a, 6b, 6g, and 6i to A549 and HepG2. The
cytotoxicity of most of the new synthesized compounds
reflected certain selectivity for HCT-116 and LN229 can-
cer cell lines except for 6g and 6i.
The cytotoxicities of the resulting 4-aminoquinoline
derivatives appeared to be related to the nature of the
substituent group at quinoline. It has been observed from
Table 1 that most of the derivatives with aliphatic amino
moieties have higher cytotoxicity than those with cyclic
amino groups and aromatic amino groups. For example, the
derivatives 6a, 6b, and 6g bearing aliphatic amino moieties
showed higher cytotoxicity than other derivatives, and
derivatives 6d, 6e, and 6f with cyclic amino groups pos-
sessed the most poor activity. For HCT-116 cell and
LN229 cell lines, the derivative with cyclohexyl amino
group (6g) had comparative activity with the derivative
with phenyl amino group (6h). For DU-145 and HepG2
1
their H NMR, 13C NMR, MS, and IR spectral data.
Biological activity
The newly synthesized 4-aminoquinoline derivatives 6a–6i
were assayed for their cytotoxicity in vitro against five
human tumor cell lines including HCT-116 (human colon
cancer cell line), A549 (human lung cancer cell line),
Scheme 1 Synthetic route for 4-aminoquinoline derivatives 6a–6i
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2857
Table 1 The cytotoxicity of 4-aminoquinoline derivatives 6a–6i
Compound Cytotoxicity (IC50, lM)a
HCT-116 A549 DU-145 HcpG2 LN229 HEK293
6a
6b
6c
0.97
3.05
13.14 9.77
19.57 4.63
[100 [100
[100 [100
[100 [100
[100 16.39
6.59 5.47
[100 22.25
12.79 10.06
[100 2.95
5.21
4.46
1.82
3.92
2.00
3.23
42.39
31.99
28.05
7.43
58.64 55.29 ND
[100 65.96 68.16
[100 [100 [100
6d
6e
6f
22.55 1.75
3.16 6.08
3.66
ND
62
6h
6i
3.82
4.28
36.39 5.90
10.91 7.15
19.20 ND
13.16
ND
11.98
1.93
5-FU
ND
ND not determined
a
Data are the mean of three independent experiments
Fig. 1 Effect of compound 6a on VEGF protein expression in HCT-
116 cells. HCT-116 cells were treated with various concentrations of 6a
for 48 h. Control cells were incubated with vehicle. a The whole-cell
lysates were analyzed by immunoblot analysis using various antibodies
against VEGF and b-actin. The results presented are representative of
three independent experiments. b The relative VEGF protein levels
were measured by scanning densitometry of the band
cell lines, 6g showed better activity than 6h. For the
derivatives with aliphatic amino moieties, the activities
appeared to be related to the size of substituent. For
example, for most of the cell lines, the derivative with a
propyl group (6a) showed more significant activity than
that with the butyl group.
Among the tested compounds, 6a displayed the most
potent cytotoxicity against HCT-116 with IC50 values of
0.97 lM. The dose- and time-dependent curve of 6a in
HCT-116 cells was further observed (data not shown).
Thus, 6a was chose to further evaluate its anticancer
activity in human colon cancer cell line (HCT-116), and
study was carried out to determine whether or not its
anticancer mechanism is related to VEGF.
protein expression of VEGF. This study may provide valu-
able insight and clues for designing and developing more
potent antitumor agents. Further biological evaluation for the
new 4-aminoquinoline derivatives is being undertaken to
define their effects on tumor angiogenesis and action
mechanism of antiproliferative activity.
In order to explore whether the novel 4-aminoquinoline
derivatives target VEGF, we investigated the effect of 6a
on the VEGF protein expression (Fig. 1). VEGF protein
expression was assessed by western blot in cells treated
with different concentrations of 6a (2.5, 5, and 10 lM) for
48 h (Fig. 1a), and the relative protein expression levels
were measured by scanning densitometry of the band
intensities (Fig. 1b). As shown in Fig. 1, 6a significantly
inhibited the protein expression of VEGF. The results
indicated that 6a could act as a VEGF inhibitor.
Experimental protocols
General
Chemical reagents were obtained from commercial sup-
pliers, and were dried and purified by standard methods
when necessary. The progress of the reaction was moni-
tored by TLC using silica gel plates. The 1H NMR and 13
C
NMR spectra were recorded on a Bruker AVANCE III
500 Hz spectrometer using CDCl3 or DMSO-d6 as solvent.
Tetramethylsilane (d 0.0 ppm) was used as an internal
standard. IR spectra (cm-1) were recorded on a Bruker
Optics TENSOR27 infrared spectrometer using the KBr
disk technique. Mass spectra were recorded on a Shimadzu
VG-Autospec-3000 mass spectrometer.
In summary, we have designed and synthesized nine
novel 4-aminoquinoline derivatives, and evaluated their
antitumor activities against five cell lines (HCT-116, A549,
DU-145, HepG2, and LN229). Most of the new compounds
displayed the selective cytotoxicity toward the tested cell
lines. From the structure–activity relationship results we
might conclude that the introduction of substituted amino
groups at 4-position in quinoline is associated with enhanced
cytotoxicity activity. When the quinoline contained aliphatic
amino moieties, the effect was more obvious. The mecha-
nism study showed that 6a could effectively inhibit the
Synthetic procedure for N-(3,5-
dimethoxyphenyl)benzamide (2)
To a solution of 3,5-dimethoxybenzenamine 1 (765 mg,
5 mmol) in dry THF (15 mL) chilled with an ice bath,
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Med Chem Res (2013) 22:2855–2861
triethylamine (1.04 mL, 7.5 mmol) was added dropwise.
Then the mixture was stirred for 15 min, followed by
the dropwise addition of benzoyl chloride (0.87 mL,
7.5 mmol) over 20 min. After the suspension was stirred at
room temperature for 3 h, the reaction mixture was poured
into ice water. The resulting mixture was extracted with
ethyl acetate (3 9 15 mL). The organic layers were dried
over anhydrous MgSO4 and the solvent was removed under
reduced pressure. The crude product was crystallized by
appropriate solvent to give compound 2 (92 % yield) as
white needle. 1H NMR (CDCl3, 500 MHz): d 3.82 (6H, s,
–OCH3), 6.29 (1H, t, J = 2.1 Hz, –NH), 6.90 (2H, d,
J = 2.0 Hz, 20,60-2H), 7.51 (2H, d, J = 8.8 Hz, 3,5-H),
7.57 (1H, t, J = 7.4 Hz, 4-H), 7.76 (1H, s, 40-H), 7.86 (2H,
d, J = 7.6 Hz, 2,6-H); 13C NMR (CDCl3, 125 MHz): d
55.4 (–OCH3), 97.1 (40-C), 98.5 (20,60-2C), 127.0 (3,5-2C),
128.8 (2,6-2C), 131.8 (4-C), 134.9 (1-C), 139.8 (10-C),
161.1 (30,50-2C), 165.9 (–CO–); IR (KBr disk): m 3248
(–CONH–), 1653 (C=O), 1532 (C=C), 1319 (C–N), 1057
(C–O); ESI-MS: m/z 258 (M ? 1).
acetate to give compound 4 (95 % yield) as pale yellow
1
powder. H NMR (DMSO-d6, 500 MHz): d 3.83 (6H, s,
–OCH3), 6.11 (1H, s, 3-H), 6.33 (1H, s, 6-H), 6.81 (1H, s,
8-H), 7.55 (3H, m, 30,40,50-H), 7.79 (2H, m, 20,60-2H),
11.24 (1H, s, –NH–); 13C NMR (DMSO-d6, 125 MHz): d
55.2 (7-OCH3), 55.7 (5-OCH3), 94.9 (6-C), 98.9 (8-C),
108.4 (3-C), 109.9 (10-C), 127.0 (30,50-2C), 128.8 (20,60-
2C), 130.0 (40-C), 145.5 (10-C), 160.3 (2-C), 160.6 (9-C),
161.8 (5-C), 164.8 (7-C), 170.3 (4-C); IR (KBr disk): m
3448 (NH), 1616 (C=O), 1507 (C=C), 1311 (C–N), 1041
(C–O); ESI-MS: m/z 282 (M ? 1).
Synthetic procedure for 4-chloro-5,7-dimethoxy-2-
phenylquinoline (5)
A solution of compound 4 (2.81 g, 10 mmol) in POCl3 was
refluxed for 3 h, and then the reaction mixture was cooled
to room temperature, and poured into ice water. The pre-
cipitate was collected and crystallized from ethyl acetate to
afford compound 5 (93 % yield) as yellow powder. 1H
NMR (CDCl3, 500 MHz): d 3.83 (6H, s, –OCH3), 6.11
(1H, s, 3-H), 6.33 (1H, s, 6-H), 6.81 (1H, s, 8-H), 7.55 (3H,
m, 30,40,50-H), 7.79 (2H, m, 20,60-H),11.24 (1H, s, -NH-);
13C NMR (CDCl3, 125 MHz): d 55.2 (7-OCH3), 55.7
(5-OCH3), 94.9 (6-C), 98.9 (8-C), 108.4 (3-C), 109.9 (10-
C), 127.0 (30,50-2C), 128.8 (20,60-2C), 130.0 (40-C), 145.5
(10-C), 160.3 (2-C), 160.6 (9-C), 161.8 (5-C), 164.8 (7-C),
170.3 (4-C); IR (KBr disk): m 1634 (C=N), 1576 (C=C),
1317 (C–N), 1050 (C–O); ESI-MS: m/z 300 (M ? 1).
Synthetic procedure for N-(2-acetyl-3,
5-dimethoxyphenyl) benzamide (3)
To a cooled solution of N-(3,5-dimethoxyphenyl)benzam-
ide 2 (645 mg, 5 mmol) in dry DCM (15 mL), SnCl4
(0.58 mL, 10 mmol) and acetyl chloride (0.25 mL,
7.5 mmol) were added dropwise. After stirring for 4 h at
room temperature, the reaction mixture was poured into ice
water (10 mL), and then extracted with ethyl acetate
(3 9 15 mL). The combined organic layer was dried over
anhydrous MgSO4 and the solvent was removed in vacuo.
The residual solid was purified by crystallization to give
compound 3 (90 % yield) as white needle. 1H NMR
(CDCl3, 500 MHz): d 2.64 (1H, s, –COCH3), 3.92 (6H, s,
–OCH3), 6.25 (1H, d, J = 2.25 Hz, 60-H), 7.55 (3H, m,
3,4,5-H), 8.04 (2H, m, 2,6-H), 8.27 (1H, d, J = 2.25 Hz,
40-H), 12.90 (1H, s, –NH); 13C NMR (CDCl3, 125 MHz): d
34.2 (–CH3), 55.6 (–OCH3), 55.7 (–OCH3), 94.4 (40-C),
96.8 (60-C), 108.7 (20-C), 127.5 (3,5-2C), 128.8 (2,6-2C),
131.9 (4-C), 134.9 (1-C), 143.9 (10-C), 162.7 (30-C), 164.8
(50-C), 166.3 (–CO–NH), 202.9 (–CO–); IR (KBr disk): m
3136 (–CONH–), 1672 (C=O), 1517 (C=C), 1309 (C–N),
1025 (C–O); ESI-MS: m/z 290 (M ? 1).
General procedure for preparation of 4-aminoquinoline
derivatives (6a–6i)
To a solution of compound 5 (300 mg, 1 mmol) in amine
(4 mL) Et3N (1 mmol) was added. The reaction mixture
was maintained at 60–90 °C over 8–36 h while stirring,
and then distilled water (20 mL) was added, extracted with
DCM (3 9 15 mL), and the combined DCM extracts were
dried over anhydrous MgSO4. The solvent was removed in
vacuo, and the residue was purified by flash chromatogra-
phy on silica gel to afford 6a–6i as light yellow or white
solid.
5,7-Dimethoxy-2-phenyl-N-propylquinoline-4-amine (6a)
Synthetic procedure for 5,7-dimethoxy-2-
Yield 61 %; Mp = 142 °C; 1H NMR(CDCl3, 500 MHz): d
1.09 (3H, t, J = 7.4 Hz, –CH3), 1.81 (2H, m, –CH2), 1.74
(2H, m, CH2), 3.92 (3H, s, –OCH3), 3.95 (3H, s, –OCH3),
6.37 (1H, d, J = 22.5 Hz, 6-H), 6.62 (1H, s, 3-H), 7.04
(1H, d, J = 22.5 Hz, 8-H), 7.41 (1H, m, 40-H), 7.47 (1H,
m, 30,50-H), 8.03 (2H, d, J = 21.5 Hz, 20,60-H); 13C NMR
(CDCl3, 125 MHz): d 13.9 (CH3), 20.4 (CH2), 30.9 (CH2),
42.7 (CH2), 53.3 (2CH2), 55.5 (7-OCH3), 55.9 (5-OCH3),
phenylquinoline-4(1H)-one (4)
A mixture of compound 3 (1.45 g, 5 mmol) and NaOH
(0.7 g, 17.5 mmol) in 1,4-dioxane (20 mL) was refluxed
for 12 h. After returning to room temperature, the mixture
was concentrated and then poured into 20 mL of ice water.
The precipitate was collected and crystallized from ethyl
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Med Chem Res (2013) 22:2855–2861
2859
95.3 (6-C), 96.6 (8-C), 101.3 (3-C), 104.8 (10-C), 127.5
(30,50-2C), 128.5 (20,60-2C), 128.8 (40-C), 140.7 (10-C),
152.3 (4-C), 153.0 (9-C), 157.5 (5-C), 158.4 (7-C), 160.2
(2-C); IR (KBr disk): m3432 (NH), 1630 (C=N), 1594
(C=C), 1167 (C–N), 1039 (C–O); ESI-MS: m/z 323
(M ? 1).
(7-C), 160.3 (2-C); IR (KBr disk): m1612 (C=N), 1579
(C=C), 1160 (C–N), 1044 (C–O); ESI-MS: m/z 349
(M ? 1).
5,7-Dimethoxy-4-morpholino-2-phenylquinoline (6e)
1
Yield 74 %; Mp = 169 °C; H NMR (CDCl3, 500 MHz):
N-Butyl-5,7-dimethoxy-2-phenylquinoline-4-amine (6b)
d3.28 (4H, br, –CH2), 3.96 (10H, s, 2-OCH2–, 2-OCH3),
6.50 (1H, d, J = 22.5 Hz, 6-H), 7.09 (1H, s, 3-H), 7.13
(1H, d, J = 22.5 Hz, 8-H), 7.43 (1H, m, 40-H), 7.49 (2H,
m, 30,50-H), 8.05 (2H, d, J = 21.5 Hz, 20,60-H); 13C NMR
(CDCl3, 125 MHz): d 53.3 (2CH2), 55.5 (7-OCH3), 56.1
(5-OCH3), 67.0 (2CH2), 98.9 (6-C), 101.8 (8-C), 104.5
(3-C), 109.5 (10-C), 127.5 (30,50-2C), 128.7 (20,60-2C),
129.1 (40-C), 140.1 (10-C), 153.5 (4-C), 156.6 (9-C), 157.5
(5-C), 158.3 (7-C), 160.5 (2-C); IR (KBr disk): m1613
(C=N), 1581 (C=C), 1162 (C–N), 1049 (C–O); ESI-MS:
m/z 351 (M ? 1).
Yield 57 %; Mp = 115 °C; 1H NMR(CDCl3, 500 MHz): d
1.01 (3H, t, J = 5.9 Hz, -CH3), 1.51 (2H, m, CH2), 1.74
(2H, m, CH2), 3.31 (2H, m, CH2), 3.92 (3H, s, –OCH3),
3.96 (3H, s, –OCH3), 6.37 (1H, d, J = 2.0 Hz, 6-H), 6.62
(1H, s, 8-H), 7.04 (1H, br, 3-H), 7.41 (1H, m, 40-H), 7.47
(2H, m, 30,50-H), 8.03 (2H, d, J = 21.5 Hz, 20,60-H); 13C
NMR (CDCl3, 125 MHz): d 13.9 (CH3), 20.4 (CH2), 30.9
(CH2), 42.7 (CH2), 55.5 (7-OCH3), 55.9 (5-OCH3), 95.3
(6-C), 96.6 (8-C), 101.3 (3-C), 104.8 (10-C), 127.5 (30,50-
2C), 128.5 (20,60-2C), 128.8 (40-C), 140.7 (10-C), 152.3 (4-
C), 153.0 (9-C), 157.5 (5-C), 158.4 (7-C), 160.2 (2-C); IR
(KBr disk): m3437 (NH), 1629 (C=N), 1594 (C=C), 1133
(C–N), 1037 (C–O); ESI-MS: m/z 337 (M ? 1).
5,7-Dimethoxy-4-(4-methylpiperazin-1-yl)-2-
phenylquinoline (6f)
Yield 64 %; Mp = 154 °C; 1H NMR(CDCl3, 500 MHz): d
2.41 (3H, s, –CH3), 2.96 (4H, br, 2CH2), 3.52 (4H, br,
2CH2), 3.95 (6H, d, 2-OCH3), 6.49 (1H, d, J = 22.5 Hz,
6-H), 7.09 (1H, s, 3-H), 7.12 (1H, d, J = 22.5 Hz, 8-H),
7.43 (1H, m, 40-H), 7.49 (2H, m, 30,50-H), 8.06 (2H, d,
J = 21.5 Hz, 20,60-H); 13C NMR (CDCl3, 125 MHz):
d46.2 (CH3), 52.7 (2CH2), 55.2 (2CH2), 55.5 (7-OCH3),
56.1 (5-OCH3), 98.6 (6-C), 101.7 (8-C), 104.7 (3-C), 109.6
(10-C), 127.5 (30,50-2C), 128.7 (20,60-2C), 129.0 (40-C),
140.3 (10-C), 153.5 (4-C), 156.7 (9-C), 157.5 (5-C), 158.3
(7-C), 160.4 (2-C); IR (KBr disk): m1613 (C=N), 1578
(C=C), 1162 (C–N), 1054 (C–O); ESI-MS: m/z 364
(M ? 1).
4-(1H-imidazole-1-yl)-5,7-dimethoxy-2-
phenylquinoline (6c)
Yield 87 %; Mp = 199 °C; 1H NMR(CDCl3, 500 MHz): d
3.64 (3H, s, -OCH3), 3.98 (3H, s, -OCH3), 6.52 (1H, d,
J = 2.0 Hz, 6-H), 7.18-7.23 (3H, m, 3,8,400-H), 7.48–7.54
(4H, m, 30,40,50,500-H), 7.69 (1H, S, 200-H), 8.06 (2H, d,
J = 21.5 Hz, 20,60-H); 13C NMR (CDCl3, 125 MHz): d
55.8 (7-OCH3), 55.9 (5-OCH3), 100.0 (6-C), 101.2 (8-C),
110.7 (3-C), 115.3 (10-C), 121.6 (500-C), 127.4 (30,50-2C),
128.6 (400-C), 129.0 (20,60-2C), 130.0 (40-C), 138.4 (200-C),
138.5 (10-C), 142.2 (4-C), 152.9 (9-C), 156.1 (5-C), 158.0
(7-C), 161.9 (2-C); IR (KBr disk): m1617 (C=N), 1590
(C=C), 1163 (C–N), 1048 (C–O); ESI-MS: m/z 332
(M ? 1).
N-Cyclohexyl-5,7-dimethoxy-2-phenylquinoline-4-
amine (6g)
Yield 55 %; Mp = 123 °C; 1H NMR(CDCl3, 500 MHz): d
1.24 (2H, m, CH2), 1.44 (2H, m, CH2),1.50 (2H, m, CH2),
1.66 (2H, m, CH2), 1.77 (2H, m, CH2), 2.10 (1H, m, CH),
3.56 (1H, br, NH), 3.92 (3H, s, –OCH3), 3.96 (3H, s,
–OCH3), 6.36 (1H, d, J = 22.5 Hz, 6-H), 6.62 (1H, s, 3-H),
7.00 (1H, s, 8-H), 7.40 (1H, t, 40-H), 7.47 (2H, t, 30,50-H),
8.00 (2H, d, J = 21.5 Hz, 20,60-H); 13C NMR (CDCl3,
125 MHz): d 24.6 (400-C), 25.9 (300,500-C), 32.4 (200,600-C),
55.5 (7-OCH3), 56.1 (5-OCH3), 67.0 (2CH2), 95.8 (6-C),
96.5 (8-C), 101.5 (3-C), 104.9 (10-C), 127.5 (30,50-2C),
128.5 (20,60-2C), 128.7 (40-C), 141.2 (10-C), 151.9 (4-C),
152.9 (9-C), 158.5 (5-C), 158.7 (7-C), 160.1 (2-C); IR (KBr
disk): m3396 (NH), 1618 (C=N), 1590 (C=C), 1132 (C–N),
1041 (C–O); ESI–MS: m/z 363 (M ? 1).
5,7-Dimethoxy-2-phenyl-4-(piperidin-1-yl)quinoline
(6d)
Yield 56 %; Mp = 154 °C; 1H NMR (CDCl3, 500 MHz):d
1.82 (2H, s, –CH2), 2.74 (4H, br, 2CH2), 3.55 (4H, br,
CH2), 3.94 (6H, d, –OCH3), 6.48 (1H, d, J = 22.5 Hz,
6-H), 7.10 (1H, s, 3-H), 7.11 (1H, d, J = 22.5 Hz, 8-H),
7.42 (1H, m, 40-H), 7.48 (2H, m, 30,50-H), 8.06 (2H, d,
J = 21.5 Hz, 20,60-H); 13C NMR (CDCl3, 125 MHz): d
24.5 (CH2), 26.1 (2CH2), 54.3 (2CH2), 55.5 (7-OCH3),
56.1 (5-OCH3), 98.3 (6-C), 101.6 (8-C), 104.7 (3-C), 109.8
(10-C), 127.5 (30,50-2C), 128.6 (20,60-2C), 128.9 (40-C),
140.4 (10-C), 153.5 (4-C), 156.9 (9-C), 158.2 (5-C), 158.6
123

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Med Chem Res (2013) 22:2855–2861
5,7-Dimethoxy-N,2-diphenylquinoline-4-amine (6h)
in culture media after cells were treated with various
concentrations of drugs for 72 h, and cells were incubated
for further 4 h at 37 °C. The purple formazan crystals were
dissolved in 100 lL DMSO. After 10 min, the plates were
read on an automated microplate spectrophotometer (Bio-
Tek Instruments, Winooski, VT, USA) at 570 and 630 nm.
Assays were performed in triplicate on three independent
experiments. The concentration required for 50 % inhibi-
tion of cell viability (IC50) was calculated using the soft-
ware ‘‘Dose–Effect Analysis with Microcomputers.’’ The
tumor cell lines panel consisted of HCT-116, A549,
DU-145, HepG2, and LN229. In all of these experiments,
three replicate wells were used to determine each point.
1
Yield 75 %; Mp = 190 °C; H NMR (CDCl3, 500 MHz):
d 3.95 (3H, s, –OCH3), 4.01 (3H, s, –OCH3), 6.49 (1H, d,
J = 22.5 Hz, 6-H), 7.06 (1H, s, 3-H), 7.19 (1H, m, 8-H),
7.22 (1H, m, Ar–H), 7.40–7.45 (7H, m, 30,40, 50-H, Ar–H),
7.94 (2H, d, J = 21.5 Hz, 20,60-H), 9.05 (1H, s, NH); 13C
NMR (CDCl3, 125 MHz):
d 55.6 (7-OCH3), 56.2
(5-OCH3), 97.2 (6-C), 97.7 (8-C), 101.7 (3-C), 105.3 (10-
C), 124.0 (2Ar–C), 124.8 (1Ar–C), 127.4 (30,50-2C), 128.5
(20,60-2C), 129.1 (40-C), 130.9 (2Ar–C), 139.9 (1Ar–C),
140.4 (10-C), 150.7 (4-C), 153.1 (9-C), 158.0 (5-C), 158.4
(7-C), 160.3 (2-C); IR (KBr disk): m3385 (NH), 1608
(C=N), 1584 (C=C), 1156 (C–N), 1043 (C–O); ESI-MS:
m/z 357 (M ? 1).
Western blot analysis
N-Benzyl-5,7-dimethoxy-2-phenylquinoline-4-amine (6i)
HCT-116 cells were treated with compound 6a (2.5, 5, and
10 lM) for 48 h. Proteins were extracted with cell lysis
buffer for Western and IP (Beyotime Institute of Biotech-
nology, Haimen, China). Equal amounts (40 lg/lane) of
protein were separated on 12 % SDS-polyacrylamide gel
electrophoresis, transferred to polyvinylidene fluoride
(PVDF) membranes (Millipore Corporation, Bedford, MA,
USA), and blocked at room temperature for 1 h in 3 %
(w/v) non-fat milk in TBST. The blots were incubated
overnight at 4 °C with the VEGF and b-actin primary
antibodies (1:500 or 1,000) diluted in TBST buffer. After
washing with TBST, the membranes were incubated with
horseradish peroxidase-conjugated goat anti-rabbit or goat
anti-mouse secondary antibodies (1:5,000), and visualized
with the ECL detection kit (Thermo, USA) according to the
manufacturer’s instructions.
1
Yield 63 %; Mp = 160 °C; H NMR (CDCl3, 500 MHz):
d 3.91 (3H, s, –OCH3), 3.92 (3H, s, –OCH3), 6.39 (1H, d,
J = 2.5 Hz, 6-H), 7.64 (1H, s, 3-H), 7.04 (1H,
d, J = 2.5 Hz, 8-H), 7.30 (1H, m, Ar–H), 7.36–7.44 (7H,
m, 30,40,50-H, Ar–H), 7.94 (2H, d, J = 1.5 Hz, 20,60-H); 13C
NMR (CDCl3, 125 MHz): d 47.3 (CH2), 55.5 (7-OCH3),
56.0 (5-OCH3), 96.0 (6-C), 96.8 (8-C), 101.6 (3-C), 104.9
(10-C), 127.2 (2Ar–C), 127.4 (1Ar–C), 127.5 (30,50-2C),
128.5 (20,60-2C), 128.7 (40-C), 128.9 (2Ar–C), 138.2 (1Ar–C),
140.8 (10-C), 152.5 (4-C), 152.7 (9-C), 158.4 (5-C), 158.6 (7-
C), 160.2 (2-C); IR (KBr disk): m 3431 (NH), 1614 (C=N),
1589 (C=C), 1165 (C–N), 1055 (C–O); ESI-MS:
m/z 371 (M ? 1).
Cell lines and culture conditions
Acknowledgments We sincerely thank the financial supports from
the National Natural Science Foundation of China (Grant No.
21202012), 2010 Industry for Attracting Ph.D. Scientists Program of
Jiangsu Province and Changzhou Key Technology R&D Program
(Society Development).
HCT-116 (human colon cancer cell line), A549 (human
lung cancer cell line), DU-145 (human prostate carcinoma
cell line), HepG2 (human hepatoma cell line), LN229
(human glioblastoma cell line), and HEK293 (human
embryonic kidney 293) were kindly provided by Shanghai
Jiao-Tong University. The cell was routinely cultured in
RPMI-1640 medium or DMEM medium, supplemented
with 10 % neonatal bovine serum (NBS) or 10 % fetal
bovine serum (FBS). The culture was maintained at 37 °C
with a gas mixture of 5 % CO2/95 % air. All media were
supplemented with 100 U/mL penicillin and 100 lg/mL
streptomycin.
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