2-Anilino-3-Aroylquinolines as Potent Tubulin Polymerization Inhibitors

Article abstract of DOI:10.1002/cmdc.201600259

Several 2-anilino-3-aroylquinolines were designed, synthesized, and screened for their cytotoxic activity against five human cancer cell lines: HeLa, DU-145, A549, MDA-MB-231, and MCF-7. Their IC₅₀values ranged from 0.77 to 23.6 μm. Among the s

Full text of DOI:10.1002/cmdc.201600259

DOI: 10.1002/cmdc.201600259
Full Papers
2
-Anilino-3-Aroylquinolines as Potent Tubulin
Polymerization Inhibitors
[
a, c]
[a]
[a]
[a]
P. S. Srikanth,
A. Ravikumar,
V. Lakshma Nayak, Korrapati Suresh Babu, G. Bharath Kumar,
[
a, b]
[a, b, c]
and Ahmed Kamal*
Several 2-anilino-3-aroylquinolines were designed, synthesized,
and screened for their cytotoxic activity against five human
cancer cell lines: HeLa, DU-145, A549, MDA-MB-231, and MCF-
compound E7010 [N-(2-(4-hydroxyphenylamino)pyridin-3-yl)-4-
methoxybenzenesulfonamide]. Furthermore, flow cytometric
analysis revealed that these compounds arrest the cell cycle at
7. Their IC50 values ranged from 0.77 to 23.6 mm. Among the
the G /M phase, leading to apoptosis. Apoptosis was also con-
2
series, compounds 7 f [(4-fluorophenyl)(2-((4-fluorophenyl)ami-
no)quinolin-3-yl)methanone] and 7g [(4-chlorophenyl)(2-((4-flu-
orophenyl)amino)quinolin-3-yl)methanone] showed remarkable
antiproliferative activity against human lung cancer and pros-
tate cancer cell lines. The IC50 values for inhibiting tubulin poly-
merization were 2.24 and 2.10 mm for compounds 7 f and 7g,
respectively, and were much lower than that of the reference
firmed by mitochondrial membrane potential, Annexin V–FITC
assay, and intracellular ROS generation. Immunohistochemistry,
western blot, and tubulin polymerization assays showed that
these compounds disrupt tubulin polymerization. Molecular
docking studies revealed that these compounds bind efficient-
ly to b-tubulin at the colchicine binding site.
Introduction
Microtubules (MTs) are intracellular cylindrical filaments that
exhibit a distinctive phenomenon of stochastic growth and
shrinkage, and they play a crucial role in many cellular process-
E7010 (4) is a pyridine sulfonamide that exhibits profound
antiproliferative activity toward a wide range of human cancer
[11]
cell lines (Figure 1). It binds b-tubulin at the colchicine-bind-
ing site and significantly promotes microtubule dynamic insta-
bility in cells at interphase as well as purified brain tubulin. In
[
1,2]
es.
Substances that are capable of interfering with the as-
sembly of microtubules are recognized as attractive pharmaco-
logical starting points for the development of anticancer
addition, E7010 is also well known to induce apoptosis, block-
[
3,4]
[12–15]
drugs.
Natural compounds that are capable of modulating
ing the cell cycle at the G /M phase.
Structure–activity re-
2
or inhibiting microtubule assembly or disassembly can be
broadly divided into three types: Paclitaxel, a well-known mi-
crotubule assembly promoter that induces the formation of
highly stable and nonfunctional assembled microtubules, is
commonly used in the treatment of ovarian and breast can-
lationship (SAR) studies indicate that the 3-pyridyl group is cru-
[16,17]
cial for activity.
Phenstatin (3) and its derivatives were
found to exhibit substantial cytotoxic activity, similar to that of
[18]
CA-4, reported by Pettit et al. In addition, the SARs of phen-
2
statin and its derivatives demonstrate that an sp -hybridized
[
5,6]
cers.
and colchicine-binding agents such as combretastatin A-4 (CA-
, 2) and nocodazole (5) bind at the vinca and colchicine bind-
In contrast, vinca alkaloids (vinblastine and vincristine)
carbon atom between the trimethoxyphenyl ring and the
meta-hydroxy, para-methoxy ring is essential for cytotoxicity as
[19,20]
4
well as tubulin depolymerization.
The latter ring has been
ing sites of the microtubule, respectively, and these are used in
the treatment of leukemias, lymphomas, non-small-cell lung
replaced by indole, benzofuran, and benzothiazole scaffolds,
which showed significant toxic activity, including effects on
[
7–10]
[21]
cancer, and other cancers.
multidrug-resistant cancer cells. These compounds also ex-
hibit significant tubulin polymerization inhibition and bind to
the colchicine binding site of the microtubule. Moreover, these
[
a] P. S. Srikanth, V. L. Nayak, K. Suresh Babu, G. B. Kumar, A. Ravikumar,
Dr. A. Kamal
Medicinal Chemistry and Pharmacology, CSIR–Indian Institute of Chemical
Technology, Hyderabad 500007 (India)
E-mail: ahmedkamal@iict.res.in
compounds cause arrest at the cell cycle G /M phase, leading
2
[22,23]
to apoptotic cell death.
However, their clinical utility is lim-
ited owing to their high toxicity, and therefore, so is their po-
tential for use in the development of multidrug-resistance
(MDR) drug candidates. Therefore, there has been great inter-
est in identifying novel microtubule inhibitors that overcome
various modes of resistance and exhibit improved pharmaco-
logical profiles. Quinolines are a pharmacologically active class
of heterocyclic compounds, and derivatives such as (5-amino-
[b] A. Ravikumar, Dr. A. Kamal
Department of Medicinal Chemistry, National Institute of Pharmaceutical
Education and Research (NIPER), Hyderabad 500037 (India)
[
c] P. S. Srikanth, Dr. A. Kamal
Academy of Scientific and Innovative Research, CSIR–Indian Institute of
Chemical Technology, Hyderabad 500007 (India)
6
-methoxyquinolin-2-yl)(3,4,5-trimethoxyphenyl)methanone (6)
Supporting information for this article can be found under http://
dx.doi.org/10.1002/cmdc.201600259.
exhibit significant tubulin polymerization inhibition
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Results and Discussion
Chemistry
The synthesis of 2-anilino-3-aroylquinolines was carried out
with 2-nitrobenzaldehyde (8) as the starting material, which
upon Knoevenagel condensation with diethyl malonate using
potassium carbonate as a base in acetic anhydride as the sol-
[29]
vent, yielded diethyl 2-(2-nitrobenzylidene)malonate (9). Re-
duction with iron powder in acetic acid as solvent yielded di-
ethyl
2-((6-nitrocyclohexa-1,5-dien-1-yl)methylene)malonate
(
10), which upon further chlorination with phosphoryl chloride,
[30–33]
gave ethyl 2-chloroquinoline-3-carboxylate (11).
This, upon
reaction with variously substituted aromatic amines in the
presence of potassium iodide using N,N-dimethylformamide as
solvent, gave 2-anilinoquinoline ester derivatives 12a–e. Fur-
ther reaction with N,O-dimethylhydroxylamine hydrochloride in
the presence of trimethylaluminum in dry dichloromethane
provided the precursors 13a–e (Scheme 1). Subsequent reac-
tion with various phenyl-substituted Grignard reagents yielded
[34,35]
the desired 2-anilino-3-aroylquinolines 7a–o.
Figure 1. Structures of colchicine (1), combretastatin A-4 (2), phenstatin (3),
E7010 (4), nocadazole (5), (5-amino-6-methoxyquinolin-2-yl)(3,4,5-trimethox-
yphenyl)methanone (6), and 2-anilino-3-aroylquinolines 7a–o.
Biology
Antiproliferative activity
[
24,25]
(Figure 1).
These compounds shown remarkable cytotoxic
The 2-anilino-3-aroylquinolines 7a–o consisting of A, B, C and
D rings (Figure 2) were synthesized as illustrated in Scheme 1.
These compounds were prepared with different substituents at
the meta and para positions of the C and D rings (Figure 3). All
the compounds shared common A and B rings, and were eval-
uated for their cytotoxic activity against a panel of five human
tumor cell lines: lung cancer (A549), cervical cancer (HeLa),
prostate cancer (DU-145), and breast carcinoma (MCF-7 and
MDA-MB-231). Doxorubicin (Dox) and E7010 were used as ref-
erence compounds, and the results are summarized in Table 1.
The screening results revealed that compounds 7a–o pos-
sess moderate to good cytotoxic activities, with IC50 values
ranging from 0.77 to 23.6 mm. Based on the cytotoxicity data,
the SAR of these 2-anilino-3-aroylquinolines was examined.
Compounds 7 f (C and D rings 4-fluoro), 7g (D ring 4-fluoro,
C ring 4-chloro), and 7j (D ring 3,4-difluoro, C ring 4-chloro)
displayed significant cytotoxic activity with IC50 values ranging
from 0.773 to 4.89 mm against all the selected cell lines. Com-
pounds 7 f, 7g, and 7j exhibited remarkable antiproliferative
activity against A549 cells, with respective IC50 values of 0.81,
activity against various human cancer cell lines, and induce
[
26–28]
apoptosis at the G /M phase of the cell cycle.
2
Quinoline-based pharmacophores therefore offer excellent
opportunities for hybridization toward the design of improved
tubulin polymerization inhibitors. We synthesized some novel
quinoline derivatives by replacing the pyridine of E7010 with
a quinoline moiety. Moreover, the sulfonamide moiety was re-
placed with a carbonyl group, and the resulting compounds
were evaluated for cytotoxic activity (Figure 2). The interesting
cytotoxic activity profiles obtained in this study, as listed in
Table 1 below prompted us to evaluate the role of these com-
pounds in the proliferation and apoptosis of human cancer cell
lines.
0
.77, and 0.92 mm, as well as DU-145 cells with IC50 values of
.91, 0.86 and 1.11 mm. These three were found to be more
0
potent than the selected standard E7010. In contrast, removing
the 4-fluoro and 4-chloro groups from the C ring (7e and 7i)
resulted in a twofold decrease in potency. The removal of 4-
fluoro and 3,4-difluoro substituents at the D ring (7b and 7c)
caused similar effects on cytotoxic activity. Furthermore, re-
moval of all the substituents on the D and C rings of com-
pound 7a caused an approximate tenfold decrease in cytotox-
ic activity relative to 7g.
Figure 2. Strategy of the present work.
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1
Scheme 1. Reagents and conditions: a) diethyl malonate, K
DMF, 908C, 4 h, 70%; e) N,O-dimethylhydroxylamine hydrochloride, Me
2
CO
3
, Ac
2
O, 808C, 4 h, 88%; b) Fe powder, AcOH, 6 h, 80%; c) POCl
3
, 908C, 3 h, 70%; d) KI, R PhNH
2
,
2
3
2 2
Al, dry CH Cl
, 08C!RT, 12 h, 70%; f) R PhMgX, dry THF, ꢀ208C!RT, 2–8 h, 68–90%.
In comparison with compound 7b, which has an electron-
withdrawing 4-fluoro substituent on the D ring, the presence
of an electron-donating 4-methoxy substituent on the D ring
and an unsubstituted C ring on compound 7d resulted in
a three- to tenfold decrease in activity. Compound 7c, with
a 4-methoxy group on the C ring and an unsubstituted D ring
showed similar results. Thus, the presence of electron-donating
substituents on either or both the C and D rings (compound
7
o) resulted in a 10–15-fold drop in activity relative to 7g.
Figure 3. Structure–activity relationships for the 2-anilino-3-aroylquinolines
a–o.
Compounds with electron-donating substituents and those
without substitution showed less antiproliferative activity than
those with electron-withdrawing substituents. Compounds
with weak electron-withdrawing substituents such as 4-fluoro
and 4-chloro on the C ring showed profound antiproliferative
7
Table 1. Cytotoxicity of 2-anilino-3-aroylquinolines 7a–o against a panel of human cancer cell lines.
1
2
[a]
Compd
R
R
IC50 [mm]
DU-145
[
b]
[c]
[d]
[e]
[f]
MCF-7
A549
HeLa
MDA-MB-231
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
g
h
i
j
H
H
H
H
4-F
4-F
4-F
4-F
H
4-F
4-Cl
4-OMe
H
15.48ꢁ0.18
6.606ꢁ0.08
3.191ꢁ0.04
17.53ꢁ3.63
6.918ꢁ0.75
2.089ꢁ0.49
1.548ꢁ0.24
10.23ꢁ2.28
5.882ꢁ1.51
2.691ꢁ0.46
7.585ꢁ0.79
19.31ꢁ3.42
3.548ꢁ0.46
15.75ꢁ1.25
23.64ꢁ0.75
1.132ꢁ0.18
1.349ꢁ0.36
7.762ꢁ2.29
4.786ꢁ0.16
1.294ꢁ0.53
11.07ꢁ2.07
2.754ꢁ1.86
0.813ꢁ0.02
0.773ꢁ0.01
7.079ꢁ1.77
1.659ꢁ0.54
0.928ꢁ0.03
6.103ꢁ0.34
7.762ꢁ0.18
1.175ꢁ0.52
6.456ꢁ0.52
23.52ꢁ0.31
1.413ꢁ0.26
1.819ꢁ0.12
9.148ꢁ0.19
5.584ꢁ0.48
1.306ꢁ1.51
13.17ꢁ1.55
3.162ꢁ0.51
0.916ꢁ0.08
0.867ꢁ0.05
8.964ꢁ0.96
1.778ꢁ0.25
1.119ꢁ0.06
7.345ꢁ0.24
9.332ꢁ2.84
1.23ꢁ0.03
20.34ꢁ4.38
10.87ꢁ1.63
5.370ꢁ0.11
13.44ꢁ0.23
7.846ꢁ3.20
3.715ꢁ0.49
2.691ꢁ0.17
16.92ꢁ1.53
5.714ꢁ4.56
4.064ꢁ0.43
5.754ꢁ0.89
21.83ꢁ0.53
4.786ꢁ0.42
17.78ꢁ9.49
12.97ꢁ0.10
1.127ꢁ0.22
1.905ꢁ0.32
18.85ꢁ2.16
8.709ꢁ0.72
7.655ꢁ0.40
12.57ꢁ2.47
9.154ꢁ0.74
4.897ꢁ0.39
2.691ꢁ0.90
16.59ꢁ1.58
6.385ꢁ0.40
4.897ꢁ0.12
9.772ꢁ0.28
21.37ꢁ2.05
6.426ꢁ2.35
19.38ꢁ4.70
11.22ꢁ1.52
1.659ꢁ0.23
1.995ꢁ0.24
4-F
4-Cl
4-OMe
H
4-Cl
4-OMe
H
4-Cl
H
4-OMe
3,4-diF
3,4-diF
3,4-diF
4-OMe
4-OMe
3,4,5-triOMe
3,4,5-triOMe
k
l
7
7
m
n
8.317ꢁ3.26
11.28ꢁ0.19
1.047ꢁ0.71
1.567ꢁ0.36
7
Dox
E7010
o
[
a] Data are the meanꢁSD of three independent experiments performed in triplicate. [b] Estrogen receptor (ER)-positive breast cancer cell line. [c] Lung
cancer cell line. [d] Prostate cancer cell line. [e] Cervical cancer cell line. [f] ER-negative breast cancer cell line.
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activities in comparison with compounds with electron-donat-
Table 2. Distribution of cells in sub-G
cell cycle.
1
, G
0
/G
1
, S, and G
2
/M phases of the
ing substituents such as methoxy groups. Compounds 7 f and
7
g were found to be the most active, and further studies were
Sample
sub-G
1
[%]
G
0
/G
1
[%]
S [%]
2
G /M [%]
carried out on these two compounds.
Control (A549)
E7010 (1 mm)
0.51
0.84
1.18
0.28
0.73
0.87
82.90
62.79
70.09
59.92
73.44
60.84
3.51
1.03
0.47
1.71
3.27
1.63
10.99
29.64
16.85
34.16
16.25
31.00
7
7
f (0.5 mm)
f (1 mm)
Cell-cycle analysis
Several anticancer compounds exert their growth inhibitory ef-
fects by arresting the cell cycle at a particular checkpoint, by
7g (0.5 mm)
g (1 mm)
7
[
36,37]
inducing apoptosis, or a combination thereof.
Regulation
of cell cycle and apoptosis are considered to be effective
[
38]
phase, respectively, whereas at 1 mm they exhibited 34.16 and
1.00% cell accumulation in G /M phase.
cancer therapeutic methods. MTT assay data showed that
the test compounds 7 f and 7g induce significant inhibition of
lung cancer cell growth, with IC50 values of 0.773 and
3
2
0
.813 mm, respectively. Cell-cycle alterations caused by these
Effects on tubulin polymerization
compounds in A549 cancer cells were investigated to under-
stand the phase distribution. A549 cells were treated with
compounds 7 f and 7g at concentrations of 0.5 and 1 mm for
G /M cell-cycle arrest is strongly associated with the inhibition
2
[39]
of tubulin polymerization. Compounds 7 f and 7g caused
48 h. The data obtained clearly indicate that these compounds
cell-cycle arrest in G /M phase; hence, their microtubule inhibi-
2
cause G /M cell-cycle arrest in comparison with untreated con-
tory function was also studied. Tubulin subunits are known to
heterodimerize, and self-assemble to form microtubules in
a time-dependent manner. The progression of tubulin poly-
merization was examined by monitoring the increase in fluo-
rescence emission at 420 nm (excitation wavelength: 360 nm)
in a 384-well microplate assay for 1 h at 378C with and without
2
trol (Figure 4, Table 2). At 0.5 mm, compounds 7 f and 7g fur-
ther showed 16.85 and 16.25% cell accumulation in G /M
2
[40,41]
the conjugates.
Compound E7010 was used as a reference
standard. The test compounds 7 f and 7g significantly inhibit-
ed tubulin polymerization by 63.91 and 67.85%, respectively,
whereas the reference compound E7010 exhibited 62.15% in-
hibition (Figure 5). Furthermore, compounds 7 f and 7g were
evaluated for their tubulin polymerization inhibition at various
concentrations. They showed potent inhibition of tubulin poly-
merization with IC50 values of 2.24, and 2.1 mm, respectively,
whereas E7010 showed an IC50 value of 2.38 mm (Table 3).
Measurement of mitochondrial membrane potential
The maintenance of mitochondrial membrane potential (DYm)
is significant for mitochondrial integrity and bioenergetic func-
[42]
tion. Mitochondrial changes, including loss of mitochondrial
Figure 4. A549 cell-cycle analysis of compounds 7 f and 7g. a) Control cells,
b) E7010 (0.5 mm), c) 7 f (0.5 mm), d) 7 f (1 mm), e) 7g (0.5 mm), and f) 7g
Figure 5. Effect of compounds on tubulin polymerization as monitored by
the increase in fluorescence at 360 nm (excitation) and 420 nm (emission)
for 1 h at 378C. E7010 was used as a positive control.
(1 mm).
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Table 3. Inhibition of tubulin polymerization by compounds 7 f and 7g.
[a]
Compound
IC50 [mm]
7
7
f
g
2.24ꢁ0.08
2.10ꢁ0.04
2.38ꢁ0.06
E7010
[a] Compound concentrations required to inhibit tubulin assembly by
5
0%; values are the meanꢁSD of two independent experiments per-
formed in triplicate.
membrane potential, are key events that take place during
drug-induced apoptosis. Mitochondrial injury by 7 f and 7g
was evaluated by detecting the decrease in mitochondrial
membrane potential. In this study we investigated the involve-
ment of mitochondria in the induction of apoptosis by these
compounds. After incubation for 48 h, they decreased the
DYm value of A549 cells, as assessed by JC-1 staining
(Figure 6).
Annexin V–FITC assays
The apoptotic effects of 7 f and 7g were further evaluated by
[
43]
Annexin V–FITC/PI (AV/PI) dual staining assay to examine the
occurrence of phosphatidylserine externalization and also to
determine whether this is due to physiological apoptosis or
nonspecific necrosis. In this study A549 cells were treated with
these compounds for 48 h at 0.5 and 1 mm to examine the
apoptotic effect. Both compounds showed significant induc-
tion of apoptosis in A549 cells (Figure 7, Table 4). The results
indicate that 7 f and 7g effect 18.20 and 12.91% apoptosis at
a concentration of 0.5 mm, whereas they exhibited 33.56 and
33.58% apoptosis, respectively, after 48 h.
Figure 6. Compounds 7 f and 7g trigger mitochondrial injury. Decreases in
membrane potential (DY ) were assessed by JC-1 staining of A549 cells
m
Immunohistochemistry studies
treated with test compounds, and samples were then subjected to flow cy-
tometry analysis to detect mitochondrial potential (see Experimental Section
for details). a) Control cells, b) E7010 (1 mm), c) 7 f (0.5 mm), d) 7 f (1 mm),
e) 7g (0.5 mm), and f) 7g (1 mm).
[
44]
Because most antimitotic agents affect microtubules, we in-
vestigated alterations in the microtubule network induced by
7
f and 7g in A549 cells by immunofluorescence microscopy.
The test results demonstrated a well-organized microtubular
network in control cells; however, cells treated with com-
pounds 7 f and 7g showed disrupted microtubule organization
Analysis of free vs. polymerized tubulin by western blot
Microtubules continuously undergo polymerization and depo-
lymerization, which are mediated by a- and b-tubulin, and
changing of this dynamic balance is considered to be a poten-
tial strategy for anticancer drug development (e.g., paclitaxel
or colchicines). Western blot analysis of a-tubulin in HeLa cells
was carried out to study the cellular effects of the compounds
(Figure 8), thus demonstrating the inhibition of tubulin poly-
merization.
Intracellular ROS generation
[47]
Many anticancer agents exert their cytotoxic effects by the
on tubulin polymerization, i.e., the ratio of free vs. polymer-
ized tubulin. HeLa cells were treated with nocodazole (2 mm),
paclitaxel (1 mm), or 7 f and 7g (2 mm) for 24 h. Immunoblot re-
sults showed that the cells exposed to these compounds con-
tain more tubulin in the soluble fraction than in the insoluble
fraction. Increased tubulin in the soluble fraction of the cells
treated with these compounds supports the inhibition of tubu-
lin polymerization (Figure 10, HeLa cells treated with 7 f and
7g at 2 mm for 24 h). Figure 10 shows soluble and insoluble
fractions of tubulin for 7 f and 7g. An increase in the soluble
fraction is observed for cells treated with 7g relative to those
[
45,46]
generation of reactive oxygen species (ROS).
This is consid-
ered one of the key mediators of apoptotic signaling. There-
fore, we decided to investigate the role of 7 f and 7g in induc-
ing the production of ROS that could lead to the cytotoxic
effect in A549 cells. A549 cells were treated with 7 f and 7g at
0.5 and 1 mm for 48 h. The results indicate that the level of
ROS significantly increased, and the ratios of dichlorofluores-
cein-positive cells for compounds 7 f and 7g were 10.2 and
1
0
2.7%, and 17.1 and 18.8%, respectively, at concentrations of
.5 and 1 mm for 48 h (Figure 9).
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Figure 8. Immunohistochemistry analysis of compounds on microtubulin
networks. A549 cells were independently treated with 7 f and 7g at 0.5 mm
for 48 h: a) Control cells (A549), b) E7010 (0.5 mm), c) 7 f (0.5 mm), and d) 7g
(
0.5 mm). Scale bars: 300 mm.
Figure 7. Annexin V–FITC staining. a) Control cells, b) E7010 (0.5 mm), c) 7 f
(0.5 mm), d) 7 f (1 mm), e) 7g (0.5 mm), and f) 7g (1 mm).
Table 4. Annexin V–FITC staining.
Sample
UL [%]
UR [%]
LL [%]
LR [%]
Control (A549)
E7010 (1 mm)
0.90
3.18
1.68
7.95
0.60
2.75
4.43
28.01
16.52
28.74
10.46
29.25
94.14
63.76
80.12
58.50
86.49
63.67
0.52
5.05
1.68
4.82
2.45
4.33
7
7
7
7
f (0.5 mm)
f (1 mm)
g (0.5 mm)
g (1 mm)
treated with 7 f. Nocodazole (Noc) and paclitaxel (Tax) were
used as reference standards, and untreated tubulin (UT) was
used as negative control; tubulin was detected by western
blot analysis.
Effect on cellular cyclin B1 levels by immunoblot analysis
During a normal cell cycle, the progression of cells in the G2
phase to the M phase is triggered by activation of the cyclin B1
[
48,49]
dependent kinase.
levels of cyclin B1. HeLa cells were exposed to 7 f and 7g at
mm for 24 h. Whole-cell lysates were then prepared and ana-
lyzed for cyclin B1 as evaluated by western blot (tubulin was
We further investigated the expression
Figure 9. The effect of 7 f and 7g on the ROS production in human lung
cancer cells (A549); a) Control cells (A549), b) E7010 (1 mm), c) 7 f (0.5 mm),
d) 7 f (1 mm), e) 7g (0.5 mm) and f) 7g (1 mm).
2
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[50]
3
HKC). The docking results revealed that these molecules fit
well into the E7010 binding site (Figure 12a) of the b subunit
at the a interface of tubulin. The coordinates of the crystal
structure were obtained from the RCSB Protein Data Bank. A
required correction to the protein was carried out by using
Protein Preparation Wizard from the Schrçdinger software
package. Molecules were built using ChemBio3D Ultra 12.0,
and geometries were optimized by molecular mechanics. All
docking studies were performed by using AutoDock 4.2 dock-
Figure 10. Distribution of tubulin in soluble (S) vs. insoluble (I, polymerized)
fractions as analyzed by western blotting.
used as an internal loading control; nocodazole (Noc) and pa-
clitaxel (Tax) were used as positive controls, and untreated tu-
bulin (UT) as negative control). As shown in Figure 11, there
was a marked upregulation in cyclin B1 protein levels for both
[51]
[52]
ing software, and the results were visualized by PyMOL.
Compounds 7 f and 7g showed different binding poses rela-
tive to E7010. Comparison of binding poses for these com-
pounds with E7010 (Figure 12b) revealed that rings A and B fit
exactly into the hydrophobic pocket in the b subunit, where
the 4-hydroxyphenyl moiety present in E7010 binds. The car-
bonyl group present in these compounds binds at the position
where the pyridine ring of E7010 binds. The remaining substi-
tuted aromatic rings of compounds 7 f and 7g were found to
be suspended above and below the 4-methoxybenzenesulfo-
namide moiety of E7010 toward the direction of the a subunit.
Compounds 7 f and 7g form two hydrogen bonds with the
target protein (Figure 12c,d). The carbonyl oxygen atom un-
dergoes hydrogen bonding with the thiol group of Cys241
from the b chain, and the fluorine atom of the 4-fluoroanilino
group of these compounds undergoes hydrogen bonding with
the NH group of Asn101 from the a chain. In contrast, the hy-
droxy group of E7010 shows hydrogen bonding interactions
with the phenolic oxygen present in the Tyr202 residue of the
b chain.
7
f and 7g relative to control. These results showed these com-
pounds arrest the cell cycle in the G /M phase involving the
2
cell-cycle regulator cyclin B1.
Figure 11. Immunoblot analysis for cyclin B1.
Molecular docking
The results obtained from the cell-based assays encouraged us
to carry out molecular docking studies to understand the bind-
ing poses of these compounds on tubulin. These studies were
performed at the E7010-binding site of tubulin (PDB ID:
Figure 12. a) Surface binding of compound 7g on the tubulin b subunit. b) Binding pose comparison of compound 7 f and 7g with E7010 in the binding site
present in tubulin. Compounds 7 f, 7g, and E7010 are shown in stick format, colored by atom type. Carbons: yellow (E7010), cyan (7g), and pink (7 f);
oxygen: red; hydrogen: white; nitrogen: blue; chlorine: green; fluorine: ice blue. Binding poses and interactions of compounds c) 7 f and d) 7g in the E7010
binding site present in tubulin.
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Besides this, various hydrophobic interactions were also ob-
served. Rings A and B present in 7 f and 7g show interactions
with Tyr202, VaL238, Thr239, Cys241, Leu242, Leu252, Leu255,
and Ile378 in the b chain. They also interact with VaL318 and
Asn167 respectively, in the same chain. The 4-chloro and 4-
fluoro groups of ring C show interactions with Met259, Ala316,
Lys352, and Ala354 from the b chain, and VaL181 from the
a chain, respectively. Finally, the 4-fluoro groups of ring D
show interactions with Leu248, Asn249, Ala250, Lys254, and
Asn258 from the b chain, and in the a chain with Asn101 and
Thr179. Moreover, ring D present in compound 7g also inter-
acts with Asp251 from the b chain and Ala180 from the
a chain.
mixture was heated to 908C and stirred under nitrogen. After 5 h,
the reaction mixture was poured into ice-cold water (250 mL) and
washed with EtOAc (1ꢁ300 mL). The layers were separated, and
the organic phase was washed with saturated aqueous NaHCO3
(1ꢁ250 mL) and brine (1ꢁ250 mL). The organic extract was dried
over Na SO , filtered, and concentrated under vacuum to afford
2
4
the crude compound, which was then purified by column chroma-
tography (15% EtOAc/hexanes) to obtain pure compound 9 as
1
a light-yellow solid. Yield: 8.24 g, 85%; mp: 161–1668C; H NMR
(500 MHz, CDCl
td, J=7.5, 1.2 Hz, 1H), 7.56 (ddd, J=7.6, 4.5, 1.0 Hz, 1H), 7.42 (d,
J=7.6 Hz, 1H), 4.33 (q, J=7.1 Hz, 2H), 4.07 (q, J=7.1 Hz, 2H), 1.35
): d=8.21 (dd, J=8.2, 1.2 Hz, 1H), 8.19 (s, 1H), 7.64
3
(
13
(
t, J=7.1 Hz, 3H), 1.01 ppm (t, J=7.1 Hz, 3H); C NMR (126 MHz,
CDCl ): d=188.1, 164.7, 163.2, 147.0, 141.0, 134.0, 133.7, 130.5,
3
1
30.1, 130.0, 129.5, 129.1, 124.9, 124.4, 61.9, 61.4, 14.0, 13.6 ppm;
+
m/z 294 [M+H] : HRMS (ESI) m/z for C H NO calculated m/z:
14
16
6
2
94.09721, found m/z: 294.09715.
Conclusions
Ethyl-2-oxo-1,2-dihydroquinoline-3-carboxylate (10): To a solution
of compound 9 (13.00 g, 44.06 mmol) in AcOH (100 mL) was
added iron powder (14.8 g, 264 mmol), and the mixture was stirred
at 908C for 6 h. After cooling, the mixture was filtered through
a Celite pad, and the filtrate was concentrated under vacuum. The
resultant crude was purified by column chromatography (60%
A series of 3-aroyl-2-anilinoquinolines 7a–o were synthesized
and evaluated for their antiproliferative activity against five
human cancer cell lines: lung cancer (A549), cervical cancer
(
HeLa), prostate cancer (DU-145), and breast carcinoma (MCF-7
and MDA-MB231), with IC50 values ranging from 0.77 to
3.6 mm. The lead compounds 7 f and 7g exhibited significant
EtOAc/hexanes) to obtain pure compound 10 as a white solid.
2
1
Yield: 6.4 g, 66%; mp: 163–1688C; H NMR (300 MHz, CDCl ): d=
3
cytotoxic activity, with IC50 values ranging from 0.77 to 4.8 mm,
particularly in lung and prostate cancer cell lines. Moreover,
these lead compounds were found to arrest the cell cycle at
1
8
2.62 (s, 1H), 8.57 (s, 1H), 7.63 (dd, J=17.0, 7.7 Hz, 2H), 7.53 (d, J=
.2 Hz, 1H), 7.31–7.21 (m, 1H), 4.46 (q, J=7.1 Hz, 2H), 1.46 ppm (t,
13
J=7.1 Hz, 3H); C NMR (75 MHz, CDCl ): d=164.1, 161.1, 145.3,
3
the G /M phase. Tubulin polymerization assays revealed that
2
139.8, 132.7, 128.7, 122.7, 121.7, 118.1, 116.1, 61.0, 14.0 ppm;
+
inhibition by 7 f and 7g is similar to E7010, leading to apoptot-
ic cell death due to decreased mitochondrial membrane po-
tential, and inducing intracellular ROS generation. The tubulin
polymerization assays and immunofluorescence analyses sug-
gest that they exhibit significant inhibitory effects on tubulin
assembly. These mechanistic studies provide additional insight
for the development of newer tubulin inhibitors as potential
anticancer agents.
ESIMS: m/z 218 [M+H] : HRMS (ESI) m/z for C H NO calculated
12
12
3
m/z: 218.08117, found m/z: 218.08100.
Ethyl-2-chloroquinoline-3-carboxylate (11): Compound 10 (6.4 g,
1
1.6 mmol) in POCl (35 mL) was warmed at 1008C under nitrogen.
3
After 3 h, the reaction mixture was allowed to cool to room tem-
perature and then slowly poured into a flask containing ice cooled
to 08C. The aqueous layer was washed with EtOAc (3ꢁ100 mL).
The organic layers were separated, dried over Na SO , filtered and
2
4
concentrated under vacuum. The resultant crude was purified by
column chromatography (10% EtOAc/hexanes) to obtain pure
compound 11 as a white solid. Yield: 6.61 g, 95%; mp: 265–2708C;
Experimental Section
1
H NMR (500 MHz, CDCl ): d=8.68 (s, 1H), 8.06 (d, J=8.5 Hz, 1H),
3
Chemistry
7
.91 (d, J=8.1 Hz, 1H), 7.89–7.82 (m, 1H), 7.68–7.59 (m, 1H), 4.48
13
(q, J=7.1 Hz, 2H), 1.47 ppm (t, J=7.1 Hz, 3H); C NMR (126 MHz,
General: All chemicals and reagents were obtained from Sigma–Al-
drich (St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson Matthey
Company, Ward Hill, MA, USA), or Spectrochem Pvt., Ltd. (Mumbai,
India), and were used without further purification. Reactions were
monitored by TLC performed on glass plates coated with silica gel
CDCl ): d=164.6, 148.2, 147.5, 141.4, 132.5, 128.4, 128.3, 127.7,
3
+
1
25.8, 124.5, 62.1, 14.2 ppm; ESIMS: m/z 236 [M+H] : HRMS (ESI)
m/z for C H NO Cl calculated m/z: 236.04728, found m/z:
12
11
2
2
36.04735.
6
0 GF254, and visualization was performed by UV light or iodine in-
dicator. Column chromatography was performed with Merck 60–
20 mesh silica gel. NMR spectra were recorded with a Bruker
UXNMR/XWIN-NMR (300 MHz) or Inova Varian VXR-Unity (400,
00 MHz) instrument. Chemical shifts (d) are reported in ppm
downfield from internal (CH ) Si standard. ESIMS data were record-
Ethyl-2-(phenylamino)quinoline-3-carboxylate (12a): Compound
11 (2 g, 8.5 mmol) and aniline (0.807 mL, 8.5 mmol) were mixed
with KI (2.1 g, 12.7 mmol) and then heated at 1008C in DMF under
nitrogen. After 4 h, the reaction mixture was allowed to cool to
room temperature and then poured into a flask containing ice
cooled to 08C. The aqueous layer was washed with EtOAc (3ꢁ
100 mL). The organic layers were separated, dried over Na SO , fil-
1
5
3
4
ed with a Micromass Quattro LC instrument by using ESI+ soft-
ware, a capillary voltage of 3.98 kV, and an ESI+ mode ion trap de-
tector. HRMS data were recorded with a QSTAR XL Hybrid MS–MS
mass spectrometer. Melting points were determined with an Elec-
trothermal melting point apparatus.
2
4
tered and concentrated under vacuum. The resultant crude was
purified by column chromatography (8% EtOAc/hexanes) to obtain
pure compound 12a as a yellow solid. Yield: 2.18 g, 88%; mp:
1
163–1688C; H NMR (500 MHz, CDCl ): d=10.30 (s, 1H), 8.70 (s,
3
1
2
H), 8.03–7.94 (m, 2H), 7.81–7.72 (m, 1H), 7.62 (dd, J=12.2, 4.4 Hz,
Diethyl-2-((6-nitrocyclohexa-1,5-dien-1-yl)methylene)malonate
H), 7.43–7.31 (m, 2H), 7.31–7.20 (m, 1H), 7.13–7.00 (m, 1H), 4.41
(9): Potassium carbonate (6.8 g, 49.3 mmol) was added to a solution
1
3
(
(
dt, J=10.6, 5.0 Hz, 2H), 1.44 ppm (t, J=7.1 Hz, 3H); C NMR
126 MHz, CDCl ): d=167.1, 152.5, 149.6, 142.4, 140.2, 132.4, 128.8,
of 2-nitrobenzaldehyde (8; 5.00 g, 33.0 mmol) and diethyl malo-
nate (5.04 mL, 33.0 mmol) in acetic anhydride (12 mL). The reaction
3
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1
28.7, 126.8, 123.2, 122.3, 122.2, 120.0, 110.5, 61.6, 14.2 ppm;
mine hydrochloride (0.802 g, 8.2 mmol) were taken into a two-neck
round-bottom flask and allowed to stir in dry CH Cl under nitro-
+
ESIMS: m/z 293 [M+H] : HRMS (ESI) m/z for C H N O calculated
18
17
2
2
2
2
m/z: 293.12845, found m/z: 293.12797.
gen at RT. The components went into complete solution after 10–
1
0
5 min, at which time Me Al (1.5 equiv, 10.27 mL) was added at
8C, and the mixture was allowed to stir for 12 h at RT. After this,
3
Ethyl-2-((4-fluorophenyl)amino)quinoline-3-carboxylate
(12b):
Compound 12b was prepared according to the method described
for compound 12a by employing ethyl 2-chloroquinoline-3-carbox-
ylate (11, 2 g, 8.5 mmol), 4-fluoroaniline (0.94 g, 8.5 mmol), and KI
the reaction mixture was cooled again to 08C, and a saturated so-
lution of NH Cl was slowly added; after complete quenching of
Me Al, the mixture was washed with EtOAc (1ꢁ300 mL). The layers
were separated and the organic phase was washed with saturated
aqueous NaHCO (1ꢁ250 mL) and brine (1ꢁ250 mL). The organic
extract was dried over Na SO , filtered and concentrated under
vacuum to afford the crude compound, which was then purified
by column chromatography (40% EtOAc/hexanes) to obtain pure
compound 13a as a pale-yellow solid. Yield: 1.68 g, 80%; mp: 93–
4
3
(
2.1 g, 12.7 mmol) to obtain pure 12b as a yellow solid. Yield:
1
2
1
7
1
1
1
6
.37 g, 90%; mp: 77–828C; H NMR (300 MHz, CDCl ): d=10.25 (s,
3
3
H), 8.76 (s, 1H), 7.91 (dd, J=8.8, 4.9 Hz, 2H), 7.79–7.60 (m, 3H),
.39–7.22 (m, 1H), 7.06 (t, J=8.7 Hz, 2H), 4.45 (q, J=7.1 Hz, 2H),
2
4
13
.47 ppm (t, J=7.1 Hz, 3H); C NMR (75 MHz, CDCl ): d=167.2,
3
59.9, 156.7, 151.0 (d, J=220.6 Hz), 142.5, 136.1, 132.5, 128.9,
26.7, 123.3, 122.3, 121.6 (d, J=7.1 Hz), 115.1 (d, J=21.9 Hz), 110.4,
1
9
88C; H NMR (500 MHz, CDCl ): d=8.85 (s, 1H), 8.27 (s, 1H), 7.87
+
3
1.7, 14.3 ppm; ESIMS: m/z 311 [M+H] : HRMS (ESI) m/z for
(d, J=8.0 Hz, 2H), 7.80 (d, J=8.2 Hz, 1H), 7.62 (t, J=8.7 Hz, 2H),
C H N O F calculated m/z: 311.11903, found m/z: 311.11859.
1
8
16
2
2
7
.35 (t, J=7.7 Hz, 2H), 7.28 (t, J=7.4 Hz, 1H), 7.02 (t, J=7.2 Hz,
13
Ethyl-2-((4-methoxyphenyl)amino)quinoline-3-carboxylate (12c):
Compound 12c was prepared according to the method described
for compound 12a by employing ethyl 2-chloroquinoline-3-carbox-
ylate (11, 2 g, 8.5 mmol), 4-methoxy aniline (1.04 g, 8.5 mmol), and
1H), 3.53 (s, 3H), 3.42 ppm (s, 3H); C NMR (126 MHz, CDCl ): d=
3
168.0, 151.2, 147.8, 140.1, 138.8, 131.3, 128.6, 128.1, 126.8, 123.4,
122.2, 122.1, 119.5, 114.5, 61.4, 33.7 ppm; ESIMS: m/z 308 [M+H] :
HRMS (ESI) m/z for C18
z: 308.13910.
+
H N O calculated m/z: 308.13990, found m/
18 3 2
KI (2.1 g, 12.7 mmol) to obtain pure 12c as a yellow solid. Yield:
1
2
.33 g, 85%; mp: 161–1668C; H NMR (300 MHz, CDCl ): d=10.12
3
2
-((4-Fluorophenyl)amino)-N-methoxy-N-methylquinoline-3-car-
(
s, 1H), 8.73 (s, 1H), 7.84 (d, J=8.9 Hz, 2H), 7.73 (d, J=8.4 Hz, 1H),
boxamide (13b): Compound 13b was prepared according to the
method described for compound 13a by employing ethyl-2-((4-flu-
orophenyl)amino)quinoline-3-carboxylate (12b, 2 g, 6.4 mmol), N-
methoxy-N-methylamine hydrochloride (0.702 g, 7.55 mmol), and
Me Al (1.5 equiv, 9.6 mL) to obtain pure 13b as a yellow solid.
Yield: 1.78 mg, 85%; mp: 100–1058C; H NMR (300 MHz, CDCl3):
d=8.85 (s, 1H), 8.32 (s, 1H), 7.84–7.80 (m, 2H), 7.78 (d, J=8.4 Hz,
1
(s, 3H), 3.46 ppm (s, 3H); C NMR (75 MHz, CDCl ): d=168.0, 159.8,
3
156.6, 149.6 (d, J=263.5 Hz), 139.1, 136.2 (d, J=2.5 Hz), 131.4,
128.3, 126.8, 123.5, 122.2, 121.2 (d, J=7.6 Hz), 115.2 (d, J=22.2 Hz),
7
8
7
1
1
3
3
.63 (dd, J=13.3, 7.9 Hz, 2H), 7.24 (t, J=7.3 Hz, 1H), 6.93 (d, J=
.9 Hz, 2H), 4.44 (d, J=7.1 Hz, 2H), 3.82 (s, 3H), 1.46 ppm (t, J=
1
3
.1 Hz, 3H); C NMR (75 MHz, CDCl ): d=167.19, 155.17, 152.74,
3
49.83, 142.42, 133.36, 132.38, 128.82, 126.66, 122.94, 122.15,
3
21.82, 113.95, 110.34, 61.59, 57.97, 55.51, 14.25 ppm; ESIMS: m/z
1
+
23 [M+H] : HRMS (ESI) m/z for C H N O calculated m/z:
19
19
2
3
23.13902, found m/z: 323.13817.
H), 7.67–7.62 (m, 2H), 7.34–7.28 (m, 1H), 7.08–7.02 (m, 2H), 3.59
13
Ethyl-2-((3,4-difluorophenyl)amino)quinoline-3-carboxylate
12d): Compound 12d was prepared according to the method de-
scribed for compound 12a by employing ethyl 2-chloroquinoline-
(
+
3
8
-carboxylate (11, 2 g, 8.5 mmol), 3,4-difluoro aniline (1.09 g,
.5 mmol), and KI (2.1 g, 12.7 mmol) to obtain pure 12d as
114.2, 61.5, 33.8 ppm; ESIMS: m/z 326 [M+H] : HRMS (ESI) m/z for
C H N O calculated m/z: 326.12638, found m/z: 326.12939.
18 17 3 2
1
a yellow solid. Yield: 2.289 g, 82%; mp: 134–1398C; H NMR
N-Methoxy-2-((4-methoxyphenyl)amino)-N-methylquinoline-3-
carboxamide (13c): Compound 13c was prepared according to
the method described for compound 13a by employing ethyl 2-(4-
methoxyphenylamino)quinoline-3-carboxylate (12c, 2 g, 6.2 mmol),
N-methoxy-N-methylamine hydrochloride (0.726 g, 7.4 mmol), and
Me Al (1.5 equiv, 9.3 mL) to obtain pure 13c as a pale-yellow solid.
Yield: 1.77 g, 85%; mp: 201–2068C; H NMR (500 MHz, CDCl ): d=
8
7
3
1
(
500 MHz, CDCl ): d=10.36 (s, 1H), 8.77 (s, 1H), 8.30 (ddd, J=13.3,
3
7
2
.4, 2.6 Hz, 1H), 7.79 (d, J=8.4 Hz, 1H), 7.68 (ddd, J=12.3, 6.8,
.5 Hz, 2H), 7.39–7.28 (m, 2H), 7.11 (dd, J=18.8, 9.1 Hz, 1H), 4.45
13
(
q, J=7.1 Hz, 2H), 1.48 ppm (dd, J=9.1, 5.1 Hz, 3H); C NMR
(
126 MHz, CDCl ): d=166.9, 151.9, 150.8, 149.8 (dd, J=244.8,
3
3
1
1
2.7 Hz), 149.0, 145.4 (dd, J=242.5, 12.7 Hz), 142.2 (d, J=5.4 Hz),
36.8 (dd, J=9.0, 1.8 Hz), 132.4, 128.7, 126.6, 123.5, 122.2, 116.5 (d,
1
3
.66 (s, 1H), 8.24 (s, 1H), 7.75 (d, J=8.2 Hz, 3H), 7.60 (dd, J=13.7,
J=18.2 Hz), 114.97, 110.13, 108.8 (dd, J=22.7, 4.5 Hz), 61.7,
.6 Hz, 2H), 7.26 (t, J=7.1 Hz, 1H), 6.91 (d, J=8.4 Hz, 2H), 3.80 (s,
+
1
4.1 ppm; ESIMS: m/z 329 [M+H] : HRMS (ESI) m/z for
13
H), 3.56 (s, 3H), 3.43 ppm (s, 3H); C NMR (126 MHz, CDCl ): d=
3
C H N O F calculated m/z: 329.10961, found m/z: 329.10897.
1
8
15
2
2 2
68.0, 155.0, 151.5, 148.0, 138.7, 133.3, 131.2, 128.1, 126.7, 123.0,
Ethyl-2-((3,4,5-trimethoxyphenyl)amino)quinoline-3-carboxylate
12e): Compound 12e was prepared according to the method de-
scribed for compound 12a by employing ethyl 2-chloroquinoline-
122.0, 121.4, 114.3, 113.8, 61.4, 55.4, 33.7 ppm; ESIMS: m/z 338
+
(
[M+H] : HRMS (ESI) m/z for C19
found m/z: 338.14812.
H N O calculated m/z: 338.14992,
20 3 3
3
8
-carboxylate (11, 2 g, 8.5 mmol), 3,4,5-trimethoxy aniline (3.2 g,
2
-((3,4-Difluorophenyl)amino)-N-methoxy-N-methylquinoline-3-
.5 mmol), and KI (2.1 g, 12.7 mmol) to obtain pure 12b as
carboxamide (13d): Compound 13d was prepared according to
the method described for compound 13a by employing ethyl-2-
1
a yellow solid. Yield: 2.60 g, 80%; mp: 172–1778C; H NMR
(
500 MHz, CDCl ): d=10.28 (s, 1H), 8.76 (s, 1H), 7.67 (dt, J=15.2,
3
(
6
7
3,4-difluorophenylamino)quinoline-3-carboxylate
(12d,
.1 mmol), N-methoxy-N-methylamine hydrochloride (0.684 g,
.3 mmol), and Me Al (1.5 equiv, 9.1 mL) to obtain pure 13d as
2 g,
7
.9 Hz, 3H), 7.35 (s, 2H), 7.28 (dd, J=12.6, 4.8 Hz, 1H), 4.45 (d, J=
.1 Hz, 2H), 3.94 (s, 6H), 3.86 (s, 3H), 1.48 ppm (t, J=7.1 Hz, 3H);
7
1
3
3
C NMR (126 MHz, CDCl ): d=167.3, 153.1, 152.6, 149.5, 142.5,
1
3
a yellow solid. Yield: 1.78 g, 85%; mp: 116–1218C; H NMR
400 MHz, CDCl ): d=9.03 (s, 1H), 8.37 (s, 1H), 8.19 (ddd, J=13.2,
1
5
36.3, 133.2, 132.6, 129.0, 126.7, 123.4, 122.3, 110.5, 97.5, 61.8, 61.1,
(
+
3
6.0, 14.3 ppm; ESIMS: m/z 383 [M+H] : HRMS (ESI) m/z for
7
.3, 2.4 Hz, 1H), 7.83 (d, J=8.7 Hz, 1H), 7.67 (t, J=7.1 Hz, 2H), 7.34
C H N O calculated m/z: 383.16015, found m/z: 383.16018.
2
1
23
2
5
(t, J=7.3 Hz, 1H), 7.25 (d, J=4.5 Hz, 1H), 7.10 (dd, J=18.7, 9.1 Hz,
13
N-Methoxy-N-methyl-2-(phenylamino)quinoline-3-carboxamide
13a): Compound 12a (2 g, 6.8 mmol) and N-methoxy-N-methyla-
1H), 3.58 (s, 3H), 3.47 ppm (s, 3H); C NMR (101 MHz, CDCl ): d=
3
167.9, 151.0, 149.9 (dd, J=244.3, 13.2 Hz), 147.7, 145.5 (dd, J=
(
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Full Papers
2
1
1
42.3, 13.1 Hz), 139.3, 136.9 (d, J=8.6 Hz), 131.6, 128.3, 126.9,
a
1.0m THF solution of 4-chorophenylmagnesium bromide
23.8, 122.3, 116.70 (d, J=18.0 Hz), 115.0 (dd, J=5.2, 3.6 Hz), 113.9,
(0.65 mL, 0.65 mmol) to obtain pure 7c as a pale-yellow solid.
Yield: 101.2 mg, 87%; mp: 172–1778C; H NMR (500 MHz, CDCl ):
+
1
08.91 (d, J=22.2 Hz), 61.5, 33.8 ppm; ESIMS: m/z 344 [M+H] :
3
HRMS (ESI) m/z for C H N O calculated m/z: 344.11945, found m/
z: 344.11903.
d=10.30 (s, 1H), 8.24 (s, 1H), 7.98 (d, J=7.7 Hz, 2H), 7.82 (t, J=
13.5 Hz, 1H), 7.69 (ddt, J=8.8, 4.3, 2.3 Hz, 3H), 7.58 (d, J=7.9 Hz,
1
8
16
3
2
1
7
1
1
H), 7.51 (t, J=5.3 Hz, 2H), 7.40–7.37 (m, 2H), 7.31–7.25 (m, 1H),
N-Methoxy-N-methyl-2-((3,4,5-trimethoxyphenyl)amino)quino-
line-3-carboxamide (13e): Compound 13e was prepared accord-
ing to the method described for compound 13a by employing
ethyl-2-(3,4,5-trimethoxyphenylamino)quinoline-3-carboxylate (12e,
13
.08 ppm (t, J=7.4 Hz, 1H); C NMR (126 MHz, CDCl ): d=197.1,
3
52.6, 149.7, 145.5, 139.9, 138.9, 137.2, 133.2, 132.5, 131.1, 129.1,
28.9, 128.2, 127.0, 123.6, 122.8, 121.8, 120.3, 116.9 ppm; ESIMS: m/
+
z 359 [M+H] : HRMS (ESI) m/z for C H N Cl calculated m/z:
22
16
2
2
6
g, 5.2 mmol), N-methoxy-N-methylamine hydrochloride (0.587 g,
3
59.09457, found m/z: 359.09356.
.2 mmol), and Me Al (1.5 equiv, 7.8 mL) to obtain pure 13e as
3
1
a yellow solid. Yield: 1.767 g, 85%; mp: 105–1108C; H NMR
(4-Methoxyphenyl)(2-(phenylamino)quinolin-3-yl)methanone
(7d): Compound 7d was prepared according to the method de-
scribed for compound 7a by employing N-methoxy-N-methyl-2-
(phenylamino)quinoline-3-carboxamide (13a, 100 mg, 0.325 mmol),
and a 0.5m THF solution of 4-methoxyphenylmagnesium bromide
(
300 MHz, CDCl ): d=8.87 (s, 1H), 8.31 (s, 1H), 7.74 (t, J=7.3 Hz,
3
1
2
H), 7.65 (dd, J=11.7, 6.3 Hz, 2H), 7.31 (t, J=7.4 Hz, 1H), 7.25 (s,
H), 3.92 (s, 6H), 3.84 (s, 3H), 3.59 (s, 3H), 3.47 ppm (s, 3H);
1
3
C NMR (75 MHz, CDCl +DMSO): d=206.5, 170.7, 167.8, 152.8,
3
1
51.1, 147.5, 138.7, 136.1, 132.8, 131.2, 128.0, 126.5, 123.2, 121.9,
(1.3 mL, 1.3 mmol) to obtain pure 7d as a yellow solid. Yield:
1
114.2, 97.0, 61.2, 60.6, 60.0, 55.7, 33.6, 30.5, 20.6, 13.8 ppm; ESIMS:
92.2 mg, 80%; mp: 132–1378C; H NMR (500 MHz, CDCl ): d=10.14
3
+
m/z 398 [M+H] : HRMS (ESI) m/z for C H N O calculated m/z:
3
(s, 1H), 8.27 (s, 1H), 7.98 (d, J=7.9 Hz, 2H), 7.82 (d, J=8.4 Hz, 1H),
7.74 (t, J=24.4 Hz, 2H), 7.63 (dd, J=32.0, 7.6 Hz, 2H), 7.39 (s, 1H),
21
24
3
5
98.17160, found m/z: 398.17028.
1
3
7
.39–7.16 (m, 3H), 7.16–6.94 (m, 3H), 3.90 ppm (s, 3H); C NMR
(2-(Phenylamino)quinolin-3-yl)methanone (7a): A solution of N-
(
126 MHz, CDCl ): d=196.9, 163.4, 152.6, 149.3, 144.5, 140.1, 132.6,
3
methoxy-N-methyl-2-(phenylamino)quinoline-3-carboxamide (13a,
00 mg, 0.325 mmol) in dry THF (10 mL) was cooled to ꢀ208C. A
solution of phenylmagnesium bromide in THF (1.0m, 0.65 mL,
.65 mmol) was added dropwise by syringe. After stirring at room
1
32.3, 131.2, 128.9, 128.8, 127.0, 123.5, 122.5, 122.0, 120.1, 117.9,
1
+
113.9, 55.6 ppm; ESIMS: m/z 355 [M+H] : HRMS (ESI) m/z for
C H N O calculated m/z: 355.14410, found m/z: 355.14414.
23
19
2
2
0
temperature for 2 h, a solution of saturated aqueous NH Cl was
(2-((4-Fluorophenyl)amino)quinolin-3-yl)(phenyl)methanone
(7e): Compound 7e was prepared according to the method de-
scribed for compound 7a by employing 2-((4-fluorophenyl)amino)-
4
added slowly dropwise to the reaction mixture, and the aqueous
layer was extracted with EtOAc (3ꢁ40 mL), washed with water and
saturated aqueous NaCl solution and dried over Na SO . The filtrate
N-methoxy-N-methylquinoline-3-carboxamide
(13b,
100 mg,
2
4
was concentrated under vacuum. The residue was purified by
column chromatography (EtOAc/hexane 5%) to give phenyl(2-
0.307 mmol), and a 1m THF solution of phenylmagnesium bromide
(0.615 mL, 0.615 mmol) to obtain pure 7e as an orange solid.
Yield: 92.6 mg, 88%; mp: 168–1738C; H NMR (500 MHz, CDCl3):
d=10.37 (s, 1H), 8.32 (s, 1H), 7.93 (dd, J=7.1, 4.3 Hz, 2H), 7.79 (d,
J=8.4 Hz, 1H), 7.74 (d, J=7.3 Hz, 2H), 7.69 (d, J=7.1 Hz, 1H), 7.65
(dd, J=13.0, 5.5 Hz, 1H), 7.59 (d, J=7.9 Hz, 1H), 7.54 (t, J=7.6 Hz,
1
(
phenylamino)quinolin-3-yl)methanone (7a) as a light-orange solid.
1
Yield: 98.7 mg, 85%; mp: 125–1308C; H NMR (500 MHz, CDCl3):
d=10.40 (s, 1H), 8.30 (s, 1H), 8.00 (d, J=7.7 Hz, 2H), 7.81 (d, J=
8
7
.4 Hz, 1H), 7.78–7.71 (m, 2H), 7.70–7.60 (m, 2H), 7.55 (dt, J=15.3,
.7 Hz, 3H), 7.39 (t, J=7.9 Hz, 2H), 7.33–7.20 (m, 1H), 7.07 ppm (t,
2H), 7.27 (dd, J=13.3, 5.6 Hz, 1H), 7.08 ppm (t, J=8.6 Hz, 2H);
1
3
13
J=7.4 Hz, 1H); C NMR (126 MHz, CDCl ): d=198.5, 152.7, 149.6,
C NMR (75 MHz, CDCl ): d=198.4, 160.0, 156.8, 151.0 (d, J=
3
3
1
1
45.7, 140.1, 138.9, 133.0, 132.4, 129.7, 129.1, 128.9, 128.6, 127.0,
23.5, 122.7, 122.0, 120.3, 117.2 ppm; ESIMS: m/z 325 [M+H] :
241.5 Hz), 145.8, 138.7, 136.0, 133.1, 132.4, 129.6, 129.1, 128.5,
+
126.7, 123.5, 121.7 (d, J=8.8 Hz), 116.9, 115.4 ppm (d, J=21.9 Hz);
+
HRMS (ESI) m/z for C1 H N O calculated m/z: 325.13354, found m/
ESIMS: m/z 343 [M+H] : HRMS (ESI) m/z for C H N OF calculated
2
17
2
22 16
2
z: 325.13327.
m/z: 343.12412, found m/z: 343.12414.
(
4-Fluorophenyl)(2-(phenylamino)quinolin-3-yl)methanone (7b):
(4-Fluorophenyl)(2-((4-fluorophenyl)amino)quinolin-3-yl)metha-
none (7 f): Compound 7 f was prepared according to the method
described for compound 7a by employing 2-((4-fluorophenyl)ami-
no)-N-methoxy-N-methylquinoline-3-carboxamide (13b, 100 mg,
0.307 mmol), and a 1m THF solution of 4-fluorophenylmagnesium
bromide (0.615 mL, 0.615 mmol) to obtain pure 7 f as a light-
Compound 7b was prepared according to the method described
for compound 7a by employing N-methoxy-N-methyl-2-(phenyla-
mino)quinoline-3-carboxamide (13a, 100 mg, 0.325 mmol), and
a
1.0m THF solution of 4-fluorophenylmagnesium bromide
(
0.65 mL, 0.65 mmol) to obtain pure 7b as a light-orange solid.
1
1
Yield: 90.2 mg, 81%; mp: 161–1668C; H NMR (500 MHz, CDCl3):
d=10.25 (s, 1H), 8.26 (s, 1H), 7.98 (d, J=7.8 Hz, 2H), 7.90–7.74 (m,
orange solid. Yield: 97.5, 88%; mp: 154–1598C; H NMR (500 MHz,
CDCl ): d=10.22 (s, 1H), 8.28 (s, 1H), 8.00–7.88 (m, 2H), 7.79 (dd,
3
3
2
7
1
H), 7.74–7.65 (m, 1H), 7.60 (d, J=7.7 Hz, 1H), 7.39 (t, J=7.9 Hz,
H), 7.28 (dd, J=13.4, 6.2 Hz, 1H), 7.23 (dd, J=15.4, 6.8 Hz, 2H),
J=8.5, 5.0 Hz, 3H), 7.70 (t, J=7.2 Hz, 1H), 7.60 (d, J=7.9 Hz, 1H),
7.29 (t, J=7.4 Hz, 1H), 7.24 (dd, J=14.6, 6.0 Hz, 2H), 7.08 ppm (t,
13
13
.08 ppm (t, J=7.3 Hz, 1H); C NMR (126 MHz, CDCl ): d=196.9,
J=8.7 Hz, 2H); C NMR (126 MHz, CDCl ): d=196.9, 165.3 (d, J=
3
3
65.2 (d, J=255.2, Hz), 152.6, 149.6, 145.3, 140.0, 135.0 (d, J=2.7,
254.2 Hz), 158.4 (d, J=241.5 Hz), 152.6, 149.5, 145.3, 136.0 (d, J=
1.6 Hz), 135.0 (d, J=2.2 Hz), 133.2, 132.2 (d, J=8.8 Hz), 129.1,
126.9, 123.6, 121.9 (d, J=7.7, Hz), 121.8 (d, J=2.7 Hz), 116.9, 115.6
Hz), 133.1, 132.2 (d, J=9.1, Hz), 128.7 (d, J=25.4 Hz), 127.43,
1
2
27.01, 123.62, 122.73, 121.87, 120.30, 117.15, 115.8 ppm (d, J=
+
+
1.8, Hz); ESIMS: m/z 343 [M+H] : HRMS (ESI) m/z for C H N OF
(dd, J=36.3, 22.0 Hz), 115.2 ppm; ESIMS: m/z 361 [M+H] : HRMS
2
2
16
2
calculated m/z: 343.12412, found m/z: 343.12306.
(ESI) m/z for C H N OF calculated m/z: 361.11470, found m/z:
22
15
2
2
3
61.11471.
(4-Chlorophenyl)(2-(phenylamino)quinolin-3-yl)methanone (7c):
Compound 7b was prepared according to the method described
for compound 7a by employing N-methoxy-N-methyl-2-(phenyla-
mino)quinoline-3-carboxamide (13a, 100 mg, 0.325 mmol), and
(4-Chlorophenyl)(2-((4-fluorophenyl)amino)quinolin-3-yl)metha-
none (7g): Compound 7g was prepared according to the method
described for compound 7a by employing 2-((4-fluorophenyl)ami-
&
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www.chemmedchem.org
10
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Papers
no)-N-methoxy-N-methylquinoline-3-carboxamide (13b, 100 mg,
.307 mmol), and a 1m THF solution of 4-chlorophenylmagnesium
(2-((3,4-Difluorophenyl)amino)quinolin-3-yl)-(4-methoxyphenyl)-
methanone (7k): Compound 7k was prepared according to the
method described for compound 7a by employing 2-(3,4-difluoro-
phenylamino)-N-methoxy-N-methylquinoline-3-carboxamide (13d,
100 mg, 0.291 mmol), and a 0.5m THF solution of 4-methoxyphe-
nylmagnesium bromide (1.16 mL, 1.16 mmol) to obtain pure 7k as
0
bromide (0.615 mL, 0.615 mmol) to obtain pure 7g as a light-
1
orange solid. Yield: 101.8 mg, 88%; mp: 133–1388C; H NMR
(
500 MHz, CDCl ): d=10.32 (d, J=50.0 Hz, 1H), 8.29 (d, J=25.0 Hz,
3
1
H), 7.92 (dd, J=8.5, 4.8 Hz, 2H), 7.77 (t, J=10.6 Hz, 1H), 7.69 (d,
J=8.0 Hz, 3H), 7.58 (t, J=9.8 Hz, 1H), 7.53 (d, J=8.2 Hz, 2H), 7.28
a light-orange solid. Yield: 101.9 mg, 89%; mp: 163–1688C;
13
1
(
(
dd, J=15.0, 7.7 Hz, 1H), 7.08 ppm (t, J=8.5 Hz, 2H); C NMR
H NMR (500 MHz, CDCl ): d=10.22 (s, 1H), 8.40–8.25 (m, 2H), 7.84
3
126 MHz, CDCl ): d=197.1, 158.4 (d, J=241.6 Hz), 152.6, 149.6,
(d, J=8.4 Hz, 1H), 7.77 (d, J=8.7 Hz, 2H), 7.70 (dd, J=11.3, 4.1 Hz,
1H), 7.63 (d, J=7.8 Hz, 1H), 7.32 (dd, J=14.0, 7.1 Hz, 2H), 7.12 (dd,
3
1
45.6, 138.9, 137.1, 135.9, 133.3, 131.0, 129.1, 128.9, 126.9, 123.6,
1
2
21.9 (d, J=7.3 Hz), 121.8 (d, J=4.5 Hz), 116.7, 115.4 ppm (d, J=
J=18.8, 9.1 Hz, 1H), 7.02 (d, J=8.7 Hz, 2H), 3.92 ppm (s, 3H);
+
13
1.7 Hz); ESIMS: m/z 377 [M+H] : HRMS (ESI) m/z for C H N ClF
C NMR (126 MHz, CDCl ): d=196.9, 163.5, 152.2, 151.0 (dd, J=
22
15
2
3
calculated m/z: 377.08515, found m/z: 377.08470.
244.3, 13.6 Hz), 148.8, 145.7 (dd, J=242.5, 12.7 Hz), 144.6, 136.7
(
1
1
dd, J=9.1, 1.8 Hz), 132.8, 132.3, 130.9, 128.0, 126.9, 123.8, 122.0,
(
2-((4-Fluorophenyl)amino)quinolin-3-yl)-(4-methoxyphenyl)me-
17.6, 116.8 (d, J=18.2 Hz), 115.4 (dd, J=3.6, 1.8 Hz), 113.9, 109.4,
thanone (7h): Compound 7h was prepared according to the
method described for compound 7a by employing 2-((4-fluorophe-
nyl)amino)-N-methoxy-N-methylquinoline-3-carboxamide
+
09.3 (d, J=22.7 Hz), 55.6 ppm; ESIMS: m/z 391 [M+H] : HRMS
(ESI) m/z for C H N O calculated m/z: 391.11798, found m/z:
27
27
2
5
(13b,
3
91.12469.
1
00 mg, 0.307 mmol), and a 0.5m THF solution of 4-methoxyphe-
nylmagnesium bromide (1.23 mL, 1.230 mmol) to obtain pure 7h
(2-((4-Methoxyphenyl)amino)quinolin-3-yl)(phenyl)methanone
(7l): Compound 7l was prepared according to the method de-
scribed for compound 7a by employing N-methoxy-2-(4-methoxy-
phenylamino)-N-methylquinoline-3-carboxamide (13c, 100 mg,
0.297 mmol), and a 1m THF solution of phenylmagnesium bromide
(0.593 mL, 0.593 mmol) to obtain pure 7l as a light-yellow solid.
as a light-orange solid. Yield: 91.5 mg, 80%; mp: 140–1458C;
1
H NMR (300 MHz, CDCl ): d=10.10 (s, 1H), 8.28 (s, 1H), 7.92 (dd,
3
J=8.8, 4.8 Hz, 2H), 7.78 (dd, J=7.8, 5.6 Hz, 3H), 7.73–7.56 (m, 2H),
7
.33–7.22 (m, 1H), 7.05 (dd, J=19.6, 8.7 Hz, 4H), 3.92 ppm (s, 3H);
1
3
C NMR (75 MHz, CDCl ): d=196.8, 163.4, 158.6 (d, J=240.5 Hz),
3
1
1
1
52.6, 149.2, 144.6, 136.2, 132.7, 132.3, 131.1, 129.0, 126.8, 123.5,
Yield: 89.2 mg, 85%; mp: 161–1668C; H NMR (400 MHz, CDCl3):
22.0, 121.5 (d, J=7.1 Hz), 117.6, 116.1, 115.3 (d, J=22.0 Hz), 114.8,
d=10.25 (s, 1H), 8.27 (s, 1H), 7.87 (d, J=8.7 Hz, 2H), 7.80–7.70 (m,
3H), 7.64 (dd, J=14.6, 7.2 Hz, 2H), 7.54 (dd, J=13.8, 7.0 Hz, 3H),
7.28–7.18 (m, 1H), 6.95 (d, J=8.8 Hz, 2H), 3.83 ppm (s, 3H);
+
113.9, 55.6 ppm; ESIMS: m/z 373 [M+H] : HRMS (ESI) m/z for
C H N O F calculated m/z: 373.13468, found m/z: 373.13450.
2
3
18
2
2
13
C NMR (101 MHz, CDCl ): d=198.5, 155.4, 153.0, 149.8, 145.9,
3
(
(
2-((3,4-Difluorophenyl)amino)quinolin-3-yl)(phenyl)methanone
7i): Compound 7i was prepared according to the method de-
1
1
39.0, 133.3, 133.0, 132.3, 129.6, 129.1, 128.6, 126.8, 123.2, 122.1,
21.8, 117.0, 114.1, 55.6 ppm; ESIMS: m/z 355 [M+H] : HRMS (ESI)
+
scribed for compound 7a by employing 2-(3,4-difluorophenylami-
no)-N-methoxy-N-methylquinoline-3-carboxamide (13d, 100 mg,
m/z for C H N O calculated m/z: 355.14410, found m/z:
23
19
2
2
3
55.14425.
0
.291 mmol), and a 1m THF solution of phenylmagnesium bromide
(
0.583 mL, 0.583 mmol) to obtain pure 7i as a pale-yellow solid.
(4-Chlorophenyl)(2-((4-methoxyphenyl)amino)quinolin-3-yl)me-
thanone (7m): Compound 7m was prepared according to the
method described for compound 7a by employing N-methoxy-2-
(4-methoxyphenylamino)-N-methylquinoline-3-carboxamide (13c,
100 mg, 0.297 mmol), and a 1m THF solution of 4-chlorophenyl-
magnesium bromide (0.593 mL, 0.593 mmol) to obtain pure 7m as
1
Yield: 88.2 mg, 84%; mp: 180–1858C; H NMR (500 MHz, CDCl3):
d=10.46 (s, 1H), 8.38–8.26 (m, 2H), 7.89–7.80 (m, 1H), 7.80–7.69
(
7
1
2
1
1
m, 3H), 7.69–7.63 (m, 1H), 7.64–7.60 (m, 1H), 7.58–7.52 (m, 2H),
.39–7.34 (m, 1H), 7.34–7.28 (m, 1H), 7.13 ppm (dt, J=9.9, 8.9 Hz,
1
3
H); C NMR (126 MHz, CDCl ): d=198.5, 152.4, 150.0 (dd, J=
3
1
45.2, 12.7, Hz),149.2, 145.9, 145.8 (dd, J=242.5, 12.7, Hz), 138.7,
36.7, 136.6 (dd, J=9.0, 2.7, Hz), 133.3, 132.5, 129.6, 129.2, 128.7,
a pale-yellow solid. Yield: 94.4 mg, 82%; mp: 157–1628C; H NMR
(400 MHz, CDCl ): d=10.14 (s, 1H), 8.17 (s, 1H), 7.83 (d, J=8.3 Hz,
3
26.9, 123.9, 122.1, 116.8 (dd, J=19.1, Hz), 115.7 (dd, J=5.4, 2.7,
2H), 7.72 (d, J=8.2 Hz, 1H), 7.64 (d, J=7.4 Hz, 3H), 7.49 (dd, J=
22.5, 7.6 Hz, 3H), 7.20 (t, J=7.0 Hz, 1H), 6.92 (d, J=8.3 Hz, 2H),
+
Hz), 109.5 ppm (dd, J=21.8, Hz); ESIMS: m/z 361 [M+H] : HRMS
(
13
ESI) m/z for C H N F calculated m/z: 361.11470, found m/z:
3.80 ppm (s, 3H); C NMR (101 MHz, CDCl ): d=197.1, 155.5, 152.8,
2
2
15
2
2
3
3
61.11402.
149.9, 145.5, 138.7, 137.2, 133.2, 131.1, 129.2, 128.9, 126.8, 123.3,
+
1
22.1, 121.7, 116.6, 114.1, 55.6 ppm; ESIMS: m/z 389 [M+H] :
(4-Chlorophenyl)-(2-((3,4-difluorophenyl)amino)quinolin-3-yl)me-
HRMS (ESI) m/z for C H N O Cl calculated m/z: 389.10513, found
23
18
2
2
thanone (7j): Compound 7j was prepared according to the
method described for compound 7a by employing 2-(3,4-difluoro-
phenylamino)-N-methoxy-N-methylquinoline-3-carboxamide (13d,
m/z: 389.10531.
Phenyl-(2-((3,4,5-trimethoxyphenyl)amino)quinolin-3-yl)metha-
none (7n): Compound 7n was prepared according to the method
described for compound 7a by employing N-methoxy-N-methyl-2-
1
00 mg, 0.291 mmol), and 1m THF solution of 4-chlorophenylmag-
nesium bromide (0.583 mL, 0.583 mmol) to obtain pure 7j as
a pale-yellow solid. Yield: 93.0 mg, 81%; mp: 145–1508C; H NMR
1
(3,4,5-trimethoxyphenylamino)quinoline-3-carboxamide
(13e,
(
1
2
500 MHz, CDCl ): d=10.36 (s, 1H), 8.29 (s, 2H), 7.84 (d, J=8.4 Hz,
100 mg, 0.251 mmol), and a 1m THF solution of phenylmagnesium
bromide (0.503 mL, 0.503 mmol) to obtain pure 7m as a pale-
yellow solid. Yield: 93.85 mg, 90%; mp: 169–1748C; H NMR
3
H), 7.78–7.67 (m, 3H), 7.62 (d, J=7.9 Hz, 1H), 7.53 (d, J=8.4 Hz,
13
1
H), 7.37–7.30 (m, 2H), 7.14 ppm (dd, J=18.8, 9.0 Hz, 1H); C NMR
(
126 MHz, CDCl ): d=197.14, 152.1, 150.0 (dd, J=244.3, 12.7, Hz),
(300 MHz, CDCl ): d=10.38 (s, 1H), 8.33 (s, 1H), 7.73 (dd, J=12.8,
3
3
1
49.3, 145.9 (dd, J=243.4, 12.7, Hz), 145.6, 139.0, 136.9, 136.5 (dd,
7.6 Hz, 4H), 7.64 (dd, J=12.5, 7.8 Hz, 2H), 7.56 (t, J=7.5 Hz, 2H),
7.38 (s, 2H), 7.31 (d, J=7.8 Hz, 1H), 3.95 (s, 6H), 3.87 ppm (s, 3H);
C NMR (75 MHz, CDCl ): d=198.5, 153.1, 152.6, 149.4, 145.7,
3
J=9.0, 2.7 Hz), 133.4, 131.0, 129.0 (d, J=18.1 Hz), 126.9, 124.0,
1
2
13
21.9, 116.8 (t, J=18.2, Hz), 115.6 (t, J=4.5, Hz), 109.5 ppm (d, J=
+
2.7, Hz); m/z 395 [M+H] : HRMS (ESI) m/z for C H N ClF calcu-
138.7, 136.0, 133.4, 133.0, 132.4, 129.5, 129.1, 128.5, 126.7, 123.5,
22
14
2
2
+
lated m/z: 395.07572, found m/z: 395.07504.
121.8, 117.0, 97.8, 61.0, 56.0 ppm; ESIMS: m/z 415 [M+H] : HRMS
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ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Full Papers
(
4
ESI) m/z for C H N O calculated m/z: 415.16523, found m/z:
15.16455.
were collected by trypsinization and washed with PBS followed by
2
5
23
2
4
ꢀ1
resuspension in JC-1 (5 mgmL ) and incubated at 378C for 15 min.
Cells were rinsed three times with medium and suspended in pre-
warmed medium. The cells were then subjected to flow cytometric
analysis on a flow cytometer (Becton Dickinson) in the FL1, FL2
(
3
4-Methoxyphenyl)-(2-((3,4,5-trimethoxyphenyl)amino)quinolin-
-yl)methanone (7o): Compound 7o was prepared according to
the method described for compound 7a by employing N-me-
thoxy-N-methyl-2-(3,4,5-trimethoxyphenylamino)quinoline-3-car-
boxamide (13e, 100 mg, 0.251 mmol), and a 0.5m THF solution of
[54]
channel to detect mitochondrial potential.
6
Annexin staining assay for apoptosis: A549 cells (1ꢁ10 ) were
4
-methoxyphenylmagnesium bromide (1.004 mL, 1.004 mmol) to
seeded in six-well plates and allowed to grow overnight. The
medium was then replaced with complete medium containing
compounds 7 f and 7g at 0.5 and 1 mm. After 48 h of drug treat-
ment, cells from the supernatant and adherent monolayer cells
were harvested by trypsinization, washed with PBS, and centri-
fuged at 5000 rpm, 2655 g (CGF), 5 min, 48C. Then the cells were
stained with Annexin V–FITC and propidium iodide using the An-
nexin V–FITC apoptosis detection kit (Sigma–Aldrich). Flow cytome-
obtain pure 7o as a yellow solid. Yield: 102.9 mg, 92%; mp: 158–
1
1
7
1
6
3
1
1
4
4
638C; H NMR (500 MHz, CDCl ): d=10.12 (s, 1H), 8.30 (s, 1H),
3
.81–7.78 (m, 2H), 7.76 (d, J=8.5 Hz, 1H), 7.69 (ddd, J=8.4, 6.9,
.4 Hz, 1H), 7.63 (d, J=8.0 Hz, 1H), 7.36 (s, 2H), 7.30 (ddd, J=8.0,
.9, 1.1 Hz, 1H), 7.10–7.00 (m, 2H), 3.95 (s, 6H), 3.93 (s, 3H),
1
3
.86 ppm (s, 3H); C NMR (126 MHz, CDCl ): d=197.0, 163.5, 153.2,
3
52.6, 149.1, 144.5, 136.2, 133.4, 132.7, 132.3, 131.1, 129.0, 126.8,
23.5, 121.9, 117.9, 113.9, 97.7, 61.1, 56.1, 55.6 ppm; ESIMS: m/z
45 [M+H] : HRMS (ESI) m/z for C H N O calculated m/z:
[55]
try was performed for this study as described earlier.
+
26
25
2
5
ROS generation: The production of reactive oxygen species was
45.17580, found m/z: 445.17559.
measured by flow cytometry using 2’,7’-dichlorofluorescein diace-
[56]
tate (DCFDA) as previously described. In this study A549 cells
were treated with compounds 7 f and 7g at 0.5 and 1 mm for 48 h.
After treatment, cells were incubated with DCFDA (2 mm) at 378C
for 30 min and then measured by flow cytometry (FACS).
Biology
MTT assays: The cytotoxic activity of the compounds was deter-
mined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
Western blot analysis of soluble vs. polymerized tubulin and cy-
clin B1: Cells were seeded in six-well plates at 1ꢁ10 cells per well
[13]
6
6
lium bromide (MTT) reduction assay. 1ꢁ10 cells per well were
seeded in 100 mL Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) in each well of
in complete growth medium. Following treatment of cells with
compounds 7 f and 7g for 24 h, cells were washed with PBS, and
subsequently soluble and insoluble tubulin fractions were collect-
ed. To collect the soluble tubulin fractions, cells were permeabi-
lized with 200 mL of pre-warmed lysis buffer [80 mm PIPES-KOH
9
6-well microculture plates and incubated for 24 h at 378C in a hu-
midified incubator (5% CO ). Compounds, diluted to the desired
2
concentrations in culture medium, were added to the wells with
respective vehicle control. After 48 h incubation, MTT (10 mL,
(
pH 6.8), 1 mm MgCl , 1 mm EGTA, 0.2% Triton X-100, 10% glycerol,
2
ꢀ1
5
mgmL ) was added to each well, and the plates were further in-
0
3
1
.1% protease inhibitor cocktail (Sigma–Aldrich)] and incubated for
min at 308C. Lysis buffer was gently removed, and mixed with
00 mL of 3ꢁ Laemmli’s sample buffer (180 mm Tris·HCl pH 6.8, 6%
cubated for 4 h. The supernatant from each well was carefully re-
moved, formazan crystals were dissolved in DMSO (100 mL), and
absorbance at was recorded at 540 nm.
SDS, 15% glycerol, 7.5% b-mercaptoethanol and 0.01% bromophe-
nol blue). Samples were immediately heated at 958C for 3 min. To
collect the insoluble tubulin fraction, 300 mL of 1ꢁ Laemmli’s
sample buffer was added to the remaining cells in each well, and
the samples were heated at 958C for 3 min. Equal volumes of sam-
ples were separated by 10% SDS-PAGE and transferred to a nitro-
cellulose membrane by semidry transfer at 50 mA for 1 h. Blots
were probed with mouse anti-human a-tubulin diluted 1:3000 mL
Cell-cycle analysis: Flow cytometric analysis (FACS) was performed
to evaluate the distribution of the cells through the cell-cycle
phases. Lung cancer (A549) cells were incubated for 48 h with
compounds 7 f and 7g at concentrations 0.5 and 1 mm. Untreated
and treated cells were harvested, washed with phosphate-buffered
saline (PBS), fixed in ice-cold 70% EtOH, and stained with propidi-
um iodide (Sigma–Aldrich). Cell-cycle analysis was performed by
flow cytometry (Becton Dickinson FACSCaliber instrument) as de-
(Sigma) and stained with a anti-mouse secondary antibody cou-
[53]
pled with horseradish peroxidase, diluted 1:5000 mL (Sigma).
Bands were visualized using an enhanced chemiluminescence pro-
tocol (Pierce) and radiographic film (Kodak). For cyclin B1 immuno-
scribed earlier.
Tubulin polymerization assay: A fluorescence-based in vitro tubu-
lin polymerization assay was performed according to the manufac-
turer’s protocol (BK011, Cytoskeleton Inc.). Briefly, the reaction mix-
ture in a total volume of 10 mL contained PEM buffer and GTP
6
blots, cells were seeded in 12-well plates at 1ꢁ10 cells per well in
complete medium and treated with various concentrations of 7 f
and 7g for 24 h. After treatment, cells were washed twice with PBS
and lysed in 1ꢁ SDS sample buffer. Proteins were separated, trans-
ferred, probed, and analyzed similar to tubulin. The primary anti-
cyclin B1 antibody was used at 1:1500 (Sigma) and horseradish per-
oxidase (HRP)-coupled goat anti-rabbit secondary antibody diluted
1:5000 (Sigma).
(
1 mm) in the presence or absence of test compounds. Tubulin
polymerization was followed by a time-dependent increase in fluo-
rescence due to the incorporation of a fluorescence reporter into
microtubules as polymerization proceeds. Fluorescence emission at
4
20 nm (excitation wavelength: 360 nm) was measured by using
a Varioscan multimode plate reader (Thermo Scientific Inc.). E7010
was used as a reference compound in this study. To determine the
IC50 values of compounds 7 f and 7g against tubulin polymeri-
zation, the compounds were pre-incubated with tubulin at varying
concentrations. Assays were performed under similar conditions as Acknowledgements
[40,41]
used for polymerization assays as described above.
6
P.S.S., K.S.B., and G.B.K. acknowledge the Council of Scientific and
Industrial Research (CSIR), New Delhi (India) for the award of
a senior research fellowship. The authors also acknowledge CSIR
Mitochondrial membrane potential: A549 cells (1ꢁ10 cells per
well) were cultured in six-well plates after treatment with com-
pounds 7 f and 7g at 0.5 and 1 mm for 48 h. After treatment, cells
&
ChemMedChem 2016, 11, 1 – 14
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12
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ÝÝ These are not the final page numbers!

Full Papers
for financial support under the 12th Five-Year Plan Project ‘Af-
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Published online on && &&, 0000
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
13
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

FULL PAPERS
P. S. Srikanth, V. L. Nayak, K. Suresh Babu,
G. B. Kumar, A. Ravikumar, A. Kamal*
A red light for cell division: A new
series of 2-anilino-3-aroylquinolines
were synthesized and evaluated for
their anticancer activity. Tubulin poly-
merization assays, western blot analyses,
Annexin V–FITC assays, analysis of intra-
cellular ROS generation, and immuno-
histochemistry data indicate that these
compounds effectively disrupt tubulin
polymerization by interacting with the
colchicine binding site of tubulin.
&& – &&
2-Anilino-3-Aroylquinolines as Potent
Tubulin Polymerization Inhibitors
&
ChemMedChem 2016, 11, 1 – 14
www.chemmedchem.org
14
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!