Design, synthesis and biological evaluation of 2-phenylquinoline-4-carboxamide derivatives as a new class of tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.09.004

A novel series of 2-phenylquinoline-4-carboxamide derivatives was synthesized, characterized and evaluated for its antiproliferative activity against five cancer cell lines, Hela, SK-OV-3, HCT116, A549 and MDA-MB-468, and a normal human fetal lung fibroblastic cell line, MRC-5. Among them, compound 7b displayed potent cytotoxic activity in vitro against SK-OV-3 and HCT116 cell lines with IC₅₀ values of 0.5 and 0.2 μM, respectively. In general, the antiproliferative activity was correlated with the binding property of the colchicine binding site and inhibitory effect on tubulin polymerization. In addition, immunofluorescence and flow cytometry analysis revealed that selected compounds caused disruption of the mitotic spindle assembly and G₂/M phase arrest of the cell cycle, which correlated with proliferation inhibitory activity. Molecular docking analysis demonstrated the interaction of 7b at the colchicine binding site of tubulin. These results indicate these compounds are promising inhibitors of tubulin polymerization for the potent treatment of cancer.

Full text of DOI:10.1016/j.bmc.2017.09.004

Accepted Manuscript
Design, synthesis and biological evaluation of 2-phenylquinoline-4-carboxa-
mide derivatives as a new class of tubulin polymerization inhibitors
Li Zhu, Kaixiu Luo, Ke Li, Yi Jin, Jun Lin
PII:
S0968-0896(17)31519-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.09.004
BMC 13964
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 July 2017
5 September 2017
6 September 2017
Please cite this article as: Zhu, L., Luo, K., Li, K., Jin, Y., Lin, J., Design, synthesis and biological evaluation of 2-
phenylquinoline-4-carboxamide derivatives as a new class of tubulin polymerization inhibitors, Bioorganic &
Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.09.004
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Please do not adjust margins
Journal Name
ARTICLE
Design, synthesis and biological evaluation of 2-phenylquinoline-
4-carboxamide derivatives as a new class of tubulin
polymerization inhibitors
Received 00th January 20xx,
Accepted 00th January 20xx
Li Zhu a‡, Kaixiu Luoa‡, Ke Li b‡, Yi Jin*a and Jun Lin*a
DOI: 10.1039/x0xx00000x
A novel series of 2-phenylquinoline-4-carboxamide derivatives was synthesized, characterized and evaluated for its
antiproliferative activity against five cancer cell lines, Hela, SK-OV-3, HCT116, A549 and MDA-MB-468, and a normal
human fetal lung fibroblastic cell line, MRC-5. Among them, compound 7b displayed potent cytotoxic activity in vitro
against SK-OV-3 and HCT116 cell lines with IC50 values of 0.5 and 0.2 μM, respectively. In general, the antiproliferative
activity was correlated with the binding property of the colchicine binding site and inhibitory effect on tubulin
polymerization. In addition, immunofluorescence and flow cytometry analysis revealed that selected compounds caused
disruption of the mitotic spindle assembly and G2/M phase arrest of the cell cycle, which correlated with proliferation
inhibitory activity. Molecular docking analysis demonstrated the interaction of 7b at the colchicine binding site of tubulin.
These results indicate these compounds are promising inhibitors of tubulin polymerization for the potent treatment of
cancer.
www.rsc.org/
widely used as first-line antitumour drugs in clinical
treatments. However, the emergence of drug resistance, high
toxicity and complex synthesis greatly limit their therapeutic
Introduction
Microtubules are key components of the cytoskeleton
which are protein polymers formed by dynamic polymerization
and depolymerization of α- and β-tubulin heterodimers, and
are involved in various important biological functions,
including cell structure maintenance, cell division, signal
transduction and intracellular transport.1-4 In the mitotic phase
of the cell cycle, microtubules are highly dynamic, and rapidly
arrange and rearrange depending on the cell’s needs.5
Disruption of the dynamic equilibrium can induce cell cycle
arrest in G2/M phase, resulting in cell death.6-8 Given their
essential role in cellular processes, microtubules have become
attractive chemotherapeutic targets for the design and
applications. Colchicine is the first colchicine site inhibitor,
which played an important role in deciphering the properties
and functions of tubulin and microtubules. Even though
colchicine is not used clinically because of its high toxicity,11
other diverse small molecules that also bind at the colchicine
site on tubulin have been discovered recently and have come
under intensive investigation. The most representative
compounds include natural products and their derivatives, as
well as synthetic small molecules, as shown in Fig. 1. Because
of their therapeutic potential, synthetic accessibility and
structural diversity, small molecular colchicine site binders
such as CA-412 and ZD612613 have been tested clinically,
synthesis of new antimitotic anticancer agents.2, A large
9
whereas ABT-751, BNC105 and ELR510444 showed promising
anticancer effects.14-16
2-Phenylquinoline-4-carboxamide derivatives have been
found to exhibit a variety of biological activities such as
number of natural products and their analogues have been
identified to act on microtubule inhibitors, and some of them
have been used clinically in the treatment of different cancers
over the past decade.2 These agents can be separated into
three groups: the taxane site for microtubule stabilizers, the
vinca site and the colchicine site for microtubule
destabilizers.10 Taxoids and vincristine alkaloids have been
antitumor,
antituberculosis and immunosuppression activity.17-19 For
example, STX-0119 Fig. 2a), a novel small-molecule STAT3
anti-inflammatory,
antibacterial,
antiviral,
(
dimerization inhibitor, displays good efficacy against human
lymphoma cells by down-regulating STAT3 target genes and
leading to apoptosis in tumours.20 HJC0123, a novel orally
bioavailable STAT3 inhibitor, has demonstrated potent
anticancer effects against a negative breast cancer MDA-MB-
231 xenograft tumour in vivo by down-regulation of phospho-
STAT3 and induction of apoptosis in breast and pancreatic
cancer cells.17 To date, no studies have reported that 2-
phenylquinoline-4-carboxamide derivatives are active as
tubulin polymerization inhibitors. Recently, we performed a
structure-based virtual screening against the colchicine binding
pocket from a screening library containing approximately
a. Key Laboratory of Medicinal Chemistry for Natural Resource (Yunnan University),
Ministry Education, School of Chemical Science and Technology, Yunnan
University, Kunming, 650091, P. R. China.
b. Department of Medical Oncology, the Third Affiliated Hospital of Kunming
Medical University, Yunnan Tumor Hospital, Kunming 650031, P. R. China.
‡
٭
the authors contributed equally to this work.
Corresponding author. Tel./fax: +86-871-65031633 (J. L.).
E-mail: jinyi@ynu.edu.cn (Y. Jin); linjun@ynu.edu.cn (J. Lin).
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1

Please do not adjust margins
ARTICLE
Journal Name
100,000 diverse drug-like compounds. The results showed that series of pyrimido[4,5-c]quinolin-1(2H)-one derivatives which
more than ten compounds are 2-phenylquinoline-4- have similar structures to 2-phenylquinoline-4-carboxamide
carboxamide derivatives in the top 100 highest scoring had weak to medium antimitotic activity.21-24
candidates. From 2007 to 2015, Metwally et al reported that a
OH
O
O
O
O
H3CO
H3CO
O
NHAc
O
O
O
H3CO
O
H3CO
OCH3
OCH3
Podophyllotoxins
SCH3
CA-4 R=H
CA-4P R=P(O)(ONa)2
Colchicine
H
N
O
S
O
O
O
H
N
O
HN
NHAc
N
O
S
N
O
OPO3H2
OH
ABT-751
nocodazole
ZD6126
Fig. 1 Representative tubulin inhibitors act through colchicine site.
O
a.
N
N
O
O
H
O
NH
O
N
O
S
HN
O
MeO
N
N
N
Neurokinin-3 (hNK-3) Receptor Antagonists
STX-0119
HJC0123
O
S
H3C
N
CH3
O
O
NH
N
O
N
H
O
F
N
H
O
N
N
F
novel chemical inhibitors of human cyclophilin A
Anti-inflammatory
Fig. 2 a).Derivative of 2-phenyl-4-quinolinecarboxylic acid with biological activity. b). Design strategy of 2-phenylquinoline-4-carboxamide
derivatives.
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins
Journal Name
ARTICLE
Therefore, 2-phenylquinoline-4-carboxamide represents a
potential lead compound to explore novel tubulin
polymerization inhibitors as new cancer agents. Meanwhile,
the trimethoxyphenyl are essential pharmacophore
contributing to the binding of colchicine.25 Specifically, the
hydroxyl and methoxy groups on the phenyl ring of CA-4 are
known to exhibit interactions with the colchicine binding site.26
On the basis of scaffold hopping of privileged scaffolds from
known bioactive compounds, our modification strategy of 2-
phenylquinoline-4-carboxamide was focused on hybridizing
two pharmacophoric units to design improved anticancer
agents to lead the development of a variety of potent, small-
molecule tubulin binder (Fig. 2b).
Herein, we would like to describe our efforts on the
biological evaluation and structural optimization of the new 2-
phenylquinoline-4-carboxamide-based tubulin inhibitors. The
synthesized compounds were evaluated for their biological
activity toward one normal human cell line and five different
human tumour cell lines. Among these derivatives, compound
7b exhibited the most potent antiproliferative activity against
Hela, SK-OV-3, HCT116, A549 and MDA-MB-468 cells (IC50
=
1.6, 0.5, 0.2, 5.7 and 10.8 μM, respectively). The binding
interactions between tubulin and compound 7b were also
investigated by using computer-based docking studies.
COOH
R
1
O
O
R
R
1
1
N
O
R
2
R
1
CCI CHO,NH OH
3
4a-h
H SO
2
2
4
R
R
2
2
O
or
Na SO H O,2h,85
2
℃
N
H
or
4
2
NH
C
O
CH NOH
KOH, EtOH, 85
COOH
NH
℃,12h
2
4a: R = H,
1
R
= H
2
R
1
1
3
4b
: R = H,
2
R
2
= 4'-CH
3
1
4c
: R = H,
R
= 3'-CH
3
1
2
N
4d
: R = H,
1
4e: R = H,
R = 2'-CH
R = 4'-CF
2
2
3
R
2
4i
1
3
4f:
4g: R = F,
R = Cl, R = H
1 2
R
2
= H
1
R
4h: R = CH , R = 4'-CH
3
1
3
2
COOH
N
Method1: EDCI,HOBT/DMF, rt
Method2: a) SOCl /DMC, b)DIPEA/DMC,rt
O
NH
4i:
R = H, R = H
1 2
R
1
2
O
O
R
1
4j
:
R = H, R =
1 2
R
3
N
R
3
4a-j
5 - 9
H
N
H
N
H
N
O
O
O
R
R2
R2
R3
N
N
N
6a-f
7a-j
5a-f
7a
: R3 = 4'-CH3, R2 = 4'-OCH3,
7b: R3 = 4'-CH3, R2 = 3',4',5'-OCH3
NH
S
O
6a
: R2 = 4'-OCH3
6b: R2 = 4'-OCH2CH3
6c
R =
O
HN
O
N
7c
: R3 = 4'-CH3, R2 = 2'-CH3,4'-OCH3
7d: R3 = 3'-CH3, R2 = 4'-OCH3,
7e
: R2 = 3',4',5'-OCH3
6d: R2 = 3'-SCH3
6e
O
OCH3
c
O
b
a
: R3 = 3'-CH3, R2 = 3',4',5'-CH3
7f: R3 = 3'-CH3, R2 = 2'-CH3,4'-OCH3
: R2 = 2'-CH3, 4'-OCH3
H
N
Ph
O
Ph
S
6f: R2 = 2'-Cl, 4',5'-OCH3
7g
7h
: R3 = 2'-CH3, R2 = 4'-OCH3,
: R3 = 2'-CH3, R2 = 2'-CH3,4'-OCH3
O
f
e
d
7i: R3 = 4'-CF3, R2 =2'-CH3,4'-OCH3
H
N
O
O
7j
4'-OCH3
: R3
=
, R2 =
R2
R2
O
R1
O
NH
N
R3
8a-g
N
8a: R1 = CH3, R3 = 4'-CH3, R2 = 4'-OCH3,
8b
9a-b
: R1 = CH3, R3 = 4'-CH3, R2 = 3',4',5'-CH3
8c: R1 = CH3, R3 = 4'-CH3, R2 = 2'-CH3,4'-OCH3
9a
; R2 = 4'-OCH3
9b: R2 = 3',4',5'-OCH3
8d
: R1 = Cl, R3 = H,
8e: R1 = Cl R3 = H,
8f
R2 = 4'-OCH3,
R2 = 3',4',5'-CH3
R2 = 2'-CH3,4'-OCH3
R2 = 4'-OCH3,
: R1 = Cl R3 = H,
8g
: R1 = F
R3 = H,
Scheme 1 Synthesis of 2-phenyl-4-quinolinecarboxylic acid.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3

Please do not adjust margins
Journal Name
ARTICLE
obtained using compounds
4 and different anilines as raw
Results and discussion
materials in the presence of a condensing agent (CDI+DCC) and
NMM and DMF as solvents at room temperature. The reaction
yields were from 70% to 90%. In the second method,
Chemistry
The general synthetic approach for 2-phenyl-4-quinoline
compounds
4 were firstly converted to the corresponding acyl
carboxylic acid
substituted anilines reacted with hydroxylamine hydrochloride
and chloral hydrate to form isonitrosoacetanilide , followed
by a Sandmeyer reaction with concentrated sulfuric acid to get
desired product . Then the 2-phenylquinoline-4-carboxylic
(4) is illustrated in Scheme 1. Firstly,
chloride via reaction with a small excess of SOCl2 in THF. Then,
the desired products 5–9 were successfully obtained via the
reaction of the above-mentioned acyl chlorides with different
anilines in the presence of triethylamine (Et3N) and THF as
solvent at room temperature. We optimized these two
methods by screening the reaction conditions (see
supplementary information).
2
3
acid compounds (4a–n, Table 1) were obtained in high yields
through the Pfitzinger reaction.27
For the synthesis of compounds 5–9, two methods were
performed according to the reaction activity of the start
materials. In the first one, 4-carboxamide products were
Table 1 Cytotoxic activity (IC50 in μM) of compounds 5–9 against different cancer cell lines
HCT116
22.6
29.5
25.8
12.9
> 50
36.1
4.9
A549
48.7
> 50
37.9
32.5
> 50
> 50
20.5
> 50
17.9
> 50
23.7
> 50
18.5
5.7
MDA-MB-468
36.8
> 50
MRC-5
46.3
> 50
> 50
43.8
> 50
> 50
40.5
> 50
38.8
> 50
> 50
> 50
47.8
31.9
48.1
> 50
42.3
> 50
> 50
> 50
> 50
Entry
1
Compound
Hela
34.5
36.8
> 50
40.5
> 50
> 50
32.5
> 50
12.7
45.8
16.4
42.6
25.2
1.6
SK-OV-3
20.3
38.9
36.6
17.4
> 50
> 50
12.8
45.8
5.4
5a
5b
5c
5d
5e
5f
2
3
46.2
34.7
> 50
4
5
6
> 50
7
6a
6b
6c
6d
6e
6f
34.7
> 50
8
49.1
2.7
9
18.9
42.8
20.1
42.8
35.2
10.8
17.4
> 50
10
11
12
13
14
15
16
17
18
19
20
21
36.8
8.4
48.3
7.8
36.3
6.3
> 50
3.7
7a
7b
7c
7d
7e
7f
0.5
0.2
9.5
4.2
1.7
11.6
30.5
17.8
23.6
> 50
45.7
> 50
30.2
5.3
12.4
10.6
15.3
36.7
42.2
> 50
9.4
11.6
14.6
31.4
43.6
47.3
32.7
32.1
> 50
10.4
42.8
> 50
> 50
7g
7h
7i
41.6
> 50
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 4

Please do not adjust margins
Journal Name
ARTICLE
22
23
24
25
26
27
28
29
30
31
32
33
7j
> 50
16.4
13.5
20.4
40.2
> 50
> 50
> 50
> 50
> 50
0.5
43.4
8.3
> 50
10.4
5.2
46.3
29.7
20.3
30.1
> 50
> 50
> 50
> 50
> 50
> 50
0.02
0.09
> 50
32.4
37.1
46.1
> 50
> 50
> 50
> 50
> 50
> 50
0.06
0.2
> 50
8a
> 50
42.7
> 50
> 50
> 50
> 50
> 50
> 50
> 50
0.09
9.5
8b
6.2
8c
12.3
20.3
24.1
34.2
29.7
37.6
32.5
0.05
0.01
11.9
24.8
36.4
27.4
34.2
39.5
> 50
0.01
0.06
8d
8e
8f
8g
9a
9b
Colchicine
CA-4
0.3
groups. It was found that the position and quantity of methoxy
and methyl moieties obviously affect its biological activity.
When the 3’-methoxy group was displaced by 3’,4’,5’-
trimethoxy (6c) or 2’-methyl-3’-methoxy moieties (6e), the
antiproliferative activity was obviously improved to display IC50
values of 5.4 and 8.4 μM against the SK-OV-3 cell line and 2.7
and 7.8 μM against the HCT116 cell line, respectively. 4’-
Methoxyphenyl (6a) showed similar biological activity to 3’-
methoxyphenyl (5d). However, 4’-ethoxyphenyl (6b) lost its
inhibition potency against five cancer cell lines, which means
that no large substituted groups are suitable at this position.
Biological results and discussion
In vitro antiproliferative activity
All the newly synthesized 2-phenylquinoline-4-
carboxamide derivatives were evaluated for their
antiproliferative activities against five human cancer cell lines:
Hela (human cervical cancer), SK-OV-3 (human ovarian
cancer), HCT116 (human colon cancer), A549 (human lung
cancer), MDA-MB-468 (human breast cancer) and one normal
human cell line (MRC-5) by means of sulforhodamine B
colorimetric assay. The results are summarized as IC50 values in
Meanwhile, when a methoxy group was changed to
a
methylthio moiety, or a chlorine atom was introduced into the
phenyl ring, the antiproliferation potency of cancer cell lines
was dramatically decreased.
Table
1. Two well-known tubulin inhibitors, CA-4 and
colchicine, were used as positive compounds to validate the
screening conditions. It was found that colchicine potently
inhibited the growth of Hela, SK-OV-3, HCT116, A549 and
MDA-MB-468 cancer cells with IC50 values of 0.5, 0.05, 0.01,
0.02 and 0.06 μM, respectively, which is highly comparable to
the reported data.28-31 CA-4 also displayed similar
antiproliferative activity. We firstly screened six different 4-
carboxamide derivatives substituted with heterocycles
(thiophene 5a, isoxazole 5b and dihydropyrazine 5c), benzene
5d, N-allylethanethioamide 5e, or phenyl- propanoate 5f). The
We further investigated the effects of R3 substitution on
cytotoxic activity by fixing R2 as 4’-methoxy, 3’,4’,5’-trimethoxy
or 2’-methyl-4’-methoxy, respectively. It was found that R3 also
exhibited a great impact on the antiproliferative activity of the
resulting compounds. When the 4’-hydrogen atom in 6c was
replaced with a p-methyl group
(7b), the potency was
dramatically increased (about tenfold) with IC50 values of 0.5
and 0.2 μM against SK-OV-3 and HCT116, respectively. Further
investigation suggested that the meta- or ortho-methyl
substitution in 7d–h decreased the potency of the
antiproliferative activity against all five cancer cell lines. When
the electron-withdrawing moiety 4’-CF3 or a 1,3-dioxolane ring
was introduced into R3, compounds 7i and 7j lost their
cytotoxic activity.
heterocycles and benzene-substituted compounds
showed low to moderate cytotoxic activity with IC50 values
against Hela, SK-OV-3, HCT116, A549 and MDA-MB-468 cancer
(5a–d)
cell lines. However, aliphatic-substituted compounds (5e–f
)
were inactive against these five cancer cell lines. The most
active compound 5d exhibited better antiproliferative activity
against SK-OV-3 and HCT116 cell lines, with the IC50 values of
17.4 and 12.9 μM indicating that benzene-substituted 4-
carboxamide derivatives are a good starting point for further
structural optimization.
The effects of R1 substitution on the antiproliferation of
cancer cell lines were also investigated by fixing R2 as 4’-
methoxy, 3’,4’,5’-trimethoxy or 2’-methyl-4’-methoxy and R3
as 4’-methyl or H. When the R1 group was fixed as a methyl
moiety, it was found that 8a, 8b and 8c maintained moderately
Then we studied the potential impact of the 3’-methoxyphenyl
moiety in 5d on its cytotoxic activity by replacing it with 4’-
potent antiproliferative activity against Hela, SK-OV-3, HCT116
and A549 cell lines. However, when R1 was replaced by an
electron-withdrawing Cl or F atom, compounds 8d–g lost the
cytotoxic activity against all five cancer cell lines.
methoxyphenyl
trimethoxyphenyl (6c), 3’-methylthiophenyl (6d), 2’-methyl-4’-
(
6a), 4’-ethoxyphenyl
(6b), 3’,4’,5’-
methoxyphenyl (6e) and 2’-chloro-4’,5’-dimethoxyphenyl (6f
)
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
ARTICLE
Journal Name
spectrophotometer at 340 nm at 60 s intervals for 60 min at 37
°C.
Finally, we investigated the quinoline-[2,3]-fused cyclic
compounds 9a and 9b. It was found that the fused cyclic
structures resulted in the loss of antiproliferative activity
potency, even if the R2 substitution was fixed as a 4’-methoxy
or 3’,4’,5’-trimethoxy moiety.
Tubulin polymerization assay
To examine whether the cytotoxic activity of compound
7b was related to the interaction with tubulin, it was subjected
to evaluation by tubulin assembly assay in a cell-free in vitro
assay. To test this, different concentrations of compound 7b
were incubated with tubulin protein, using colchicine as a
positive control. The results were obtained by
spectrophotometric measurement at λ = 340 nm. As shown in
Fig. 4, compound 7b proved to be a strong inhibitor of tubulin
polymerization in different concentration (125, 500, 750 nM,
respectively), showing a trend similar to colchicine. The results
demonstrated that inhibition of tubulin polymerization of
compound 7b is correlated with growth inhibition.
Competitive colchicine binding assay
Fig. 3 SAR of the 2-phenylquinoline-4-carboxamide derivatives
Compound 7b was revealed as the parallel inhibitor
similar to that of CA-4 in the polymerization of tubulin. Thus
we explored whether this compound could bind to the
colchicine site of tubulin by using a fluorescence-based assy.32
we found an increase in the fluorescence of the tubulin-
colchicine complex in the presence of 7b, CA-4. This effect was
similar to the fact that CA-4 generates increased fluorescence
upon binding at the colchicine site (Fig. 5a).33 The result of
experiments were showed that tested compound 7b has
significant affinity toward the colchicine binding site at low
concentrations, whereas at higher concentrations 7b exhibited
a steady increase in its binding affinity. Vinblastine was used as
a negative control, which is known to bind at a different site,
and it did not show any effect on the tubulin-colchicine
complex. Therefore, this experiment suggests that compound
7b bind at the colchicine site on tubulin.
The SAR studies were concluded for these 2-
phenylquinoline-4-carboxamide derivatives
(
Fig.
3). In
particular, the quinoline-[2,3]-fused cyclic compounds or
aliphatic substituted in section A dramatically decreased the
potency of antiproliferative activity. Specifically, methoxy
group substituted at R2 are benefit for cytotoxic activity, while
the substitution at R1 group will decrease the biological
activity, especially the electro-withdraw group, e.g. CF3,
resulted in the loss of antiproliferative activity potency.
It is worth noting that all active compounds, such as 6c
and 7b, exhibited poor cytotoxic activity against the human
normal cell line MRC-5, but the positive compounds colchicine
and CA-4 showed dramatic antiproliferative activity against
this cell line.
0.5
0.4
0.3
Effect on cellular cyclin-B1 by immunoblot analysis
Cyclin-B1 is induced at the G2/M boundary to promote
cell division. This protein is one of the important regulatory
proteins of mitosis and accumulation of cyclin-B1 is an
indication for G2/M arrest. Since compound 7b arrests the cell
at G2/M phase, we investigated its effect on cyclin-B1 protein.
Thus, we treated the HCT116 cells with the compound 7b at
0.2
Control
7b (125 nM)
0.1
0.0
different concentrations (0.06
- 1.0 µM) for 24h and
7b (500 nM)
7b (725 nM)
Colchicine (1000 nM)
performed the immunoblot analysis for cyclin-B1 and tubulin
as loading control. Therefore, the accumulation of cyclin-B1
levels suggests that these conjugates demonstrate cytotoxic
effect through cell cycle arrest at mitosis. This data suggests
that treatment by 7b induces a mitotic arrest, with the
activation of spindle checkpoint in 24h as shown in Fig. 5b.
0
10
20
30
A
40
50
60
Fig. 4 Effects of 7b on in vitro tubulin polymerization assay.
Colchicine (1 μM) was used as a reference, while ethanol (1%
v/v) was used as a vehicle control. The reaction was started by
warming the solution from 4 to 37 °C. Purified bovine tubulin
and GTP were mixed in a 96-well plate. The effect on tubulin
assembly was monitored in
a
Spectramax 340PC
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Journal Name
ARTICLE
Fig.
microtubule network. HCT116 cells treated with 7b and
colchicine were fixed and stained with -tubulin, and then
6 Effect of 7b on the organization of the cellular
α
counterstained with 4, 6-diamidino-2-phenylindole (DAPI).
Microtubules and unassembled tubulin are shown in red. DNA,
stained with DAPI, is shown in blue.
Microtubule depolymerizing agents disrupt the dynamic
equilibrium of tubulin and prevent the formation of spindles,
subsequently inducing cell cycle arrest in G2/M phase. In order
to examine the tubulin inhibition of 2-phenylquinoline-4-
carboxamide derivatives in cultured HCT116 cells, the
immunofluorescence technique was used to determine the
effects of 7b on microtubule skeletons by confocal microscopy.
In this study, DMSO was used as a blank control and colchicine
was used as a positive control. As shown in Fig. 6, we observed
a well-organized microtubule structure in control cells. In
contrast, cells treated with colchicine and 7b showed
disrupted microtubule organization at 0.5 μM. These results
support the potential inhibitory function of 7b against tubulin.
Fig. 5 a). Fluorescence-based colchicine competitive binding
assays of compound 7b. CA-4 was used as positive control, and
vinblastine was used as a negative control; b). Western blot
analysis of Cyclin-B1: Treatment of HCT116 cells with 0.06,
0.12, 0.25, 0.5 and 1.0 µM concentrations of 7b for 24 h
resulted an increase in cyclinB1 levels significantly. Tubulin was
employed as loading control and DMSO as negative control.
Cell cycle analysis
Anticancer drugs are known to interact with cells, leading
to cell growth arrest or cell death. To gain further insight into
the mode of action, the effects of compound 7b on HCT116
cell cycle progression was studied by flow cytometry against
HCT116 human cancer cells. HCT116 cells were treated with
compound 7b in comparison to colchicine for 48 h. The data
obtained clearly indicated that this compound (7b) showed
G2/M cell arrest as compared to the untreated control cells.
The tested compound 7b showed 45% and 91% of cell
accumulation in G2/M phase at concentrations of 125 and 500
nM, respectively (Fig. 7a).
Immunofluorescence staining
DNA
Merged
α-Tubulin
Annexin-V FITC assay
HCT116 cells were treated with compound 7b for 24 h at
µM concentration to examine the apoptotic effect. The
0.5
results suggested that 97.93% cells were live, 0.96% and 1.08%
cells were in early and late apoptotsis stage respectively and
0.03% cells were dead in the untreated HCT116 cells (Fig. 7b).
However, in the presence of 0.5 µM 7b, 15.23% of cells were
found to be live while 39.47 and 45.26 of cells were in the
early and late apoptosis stages and 0.04% cells were necrotic
as shown in Fig 7b. This experiment suggests that they
significantly induce apoptosis in HCT116 cells.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7

Please do not adjust margins
Journal Name
ARTICLE
Control
7b (125 nM)
7b (500 nM)
Fig. 7 a).Effects of compound 7b on HCT116 cell cycle progression. The HCT116 cancer cells were cultured without any
compounds (control) or with 7b at 125 and 500 nM, respectively; b). Flow cytometric analysis of cells stained with annexin V and
PI showing the distribution of HCT116 cells in different phases of apoptosis. Cells were incubated with 0.5 µM of 7b for 24 h.
Fig. 8 Predicted binding mode of 7b with α and β tubulin. a) Overall view of predicted binding model of compound 7b (magenta,
stick model), colchicine (green, stick model) and three binding sub-pockets (red, blue and yellow line) between α (green, ribbon
model) and β (light blue, ribbon mode) tubulin. b) Focused view of compound 7b in the context of surrounding residues (stick
model).
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 8

Please do not adjust margins
Journal Name
ARTICLE
Binding model of compound 7b with tubulin
HCT116), but low cytotoxic activity against human normal cell
line MRC-5. The current results also revealed additional SAR
information. (1) The methoxy moiety on the 4-carboxamide
phenyl ring is modifiable to enhance or maintain antitumour
Recently, some progresses were reported to understand
the binding mechanism of tubulin domain.34, 35 To help explore
the SARs observed in the in vitro antiproliferation and guide
further SAR studies, we proceeded to examine the interaction
of compound 7b with tubulin (PDB code: 4O2B). Molecular
docking was performed by simulation of synthetic compounds
into the colchicine binding site in tubulin, and the results are
shown in Fig. 8. All docking runs were applied using the
AutoDock 4.2 docking tool.36 The binding model of compound
7b and tubulin is depicted in Fig. 8. The amino acid residue
which had interaction with tubulin is labelled. The results show
that compound 7b partly shares the binding pocket of
colchicine, displaying interactions with βCys241, βLys254,
βMet259, βIle318, βAsn249, βLys352, αThr179 and αSer178 in
the α,β-tubulin, and mostly buried in the β-subunits (Fig. 8a).
The trimethoxyphenyl group occupies the hydrophobic binding
site in the tubulin with βLeu248, βLys352, βMet259 and
βCys241. The 4’-methylbenzene moiety occupies the
hydrophobic binding site in the tubulin with βLeu333, βAla354,
αGln176 and αPro175. The quinoline moiety occupies another
small hydrophobic binding site in the tubulin with βMet325,
βGln247 and βVal355. In addition, two hydrogen-bonding
interactions were observed: one formed by the hydrogen atom
of the amide in 7b and the carboxyl oxygen atom of βThr353
(bond length: C=O…H = 2.1 Å), the other by the nitrogen atom
of the quinoline ring and the amino hydrogen atom of αSer178
(bond length: N–H…N = 3.1 Å). The 3D model of the interaction
between compound 7b and the colchicine bonding site is
shown in Fig. 8b. It was found that the hydrophobic pockets of
the colchicine binding site were partly occupied by compound
7b, and two sub-hydrophobic pockets between α,β-tubulin
units were also occupied by compound 7b. Overall, the binding
model suggests that compound 7b is a potential inhibitor of
tubulin.
activity (5a, 6a, 6c, 6e), but a bulky group is unfavourable (6b).
(2) A para-methyl group on the 2-phenyl ring of quinoline is a
better moiety than meta-methyl, ortho-methyl and an
electron-withdrawing group (CF3). (3) No substituted group on
the C7 position of quinoline is favourable due to the steric
hindrance between the α,β-tubulin units. Molecular modelling
results provided additional insight into the interaction of 7b
with the tubulin colchicine site. These current results will help
us to further optimize and develop new drug candidates for
clinical studies as novel tubulin inhibitors.
Experimental
Chemistry
All chemicals and reagents used were of commercial
grade and were used without any further purification. The
reactions were monitored by thin layer chromatography (TLC)
using silica gel GF254. Column chromatography was performed
with 200–300 mesh silica gel. All yields refer to isolated
products after purification. The intermediates and products
synthesized were fully characterized by spectroscopic data.
The NMR spectra were recorded on a Bruker DRX-500 (1H: 500
MHz, 13C: 125 MHz) using CDCl3 and DMSO-d6 as solvents.
Chemical shifts (δ) are expressed in parts per million (ppm)
and J values are given in hertz (Hz). IR spectra were recorded
on an FT-IR Thermo Nicolet Avatar 360 using a KBr pellet.
HRMS was performed on an Agilent LC/MSD TOF instrument.
The melting points were measured by XT-4A melting point
apparatus without correction.
General procedure for the preparation of substitutional isatin (3a–
d)
In addition, some of ADMET properties of 7b were
calculated by using Accelry Discovery Studio software V3.5,
(supporting information Table S1). The results showed
compound 7b possible have good intestinal absorption and
aqueous solubility, but needed to improve hepatotoxicity.
Redistilled water (30 mL, 45 °C), sodium sulphate (45 g,
32.0 mmol) and chloral hydrate (4.96 g, 30.0 mmol) were
added in turn to a 250 mL three-necked bottle under stirring. A
solution of substituted aniline 1a–d (20.0 mmol) in 35 mL of
water containing concentrated hydrochloric acid (5 mL) was
then slowly added dropwise into the above mixture. When
flocculent precipitate was generated, hydroxylamine
hydrochloride (4.52 g, 65.0 mmol) was added to the reaction
mixture and stirring was continued for 2 h at 80 °C. A white
solid was afforded, and the precipitate was collected by
filtration and recrystallization to get the intermediate
compounds 2a–d. Concentrated sulfuric acid was charged into
a 150 mL round-bottom flask and slowly warmed to 60 °C
followed by addition of intermediates 2a–d (3.28 g, 20 mmol)
in time. After 30 min, the temperature rose to 90 °C and the
mixture was stirred at this temperature for 1.5–2 h. The
mixture was cooled to room temperature. Then the mixture
was poured into water (65 mL) with stirring, hydrolysed for 1
h, and the orange precipitate was collected by filtration to
Conclusions
In summary,
a
new class of 2-phenylquinoline-4-
carboxamide derivatives was prepared and tested for its
anticancer potential against five human cancer cell lines and
one human normal cell line. All the compounds exhibited
promising antiproliferation activity at micromolar (μM)
concentrations. Among the series compounds, 7b showed
significant anticancer activity in human ovarian cancer and
human colon cancer with IC50 values 0.5 and 0.2 μM,
respectively. This compound
(7b
)
arrested cell cycle
progression in G2/M phase and inhibited tubulin
polymerization. Notably, active compound 7b exhibited high
antiproliferation potency against cancer cell lines (SK-OV-3 and
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
Please do not adjust margins

Please do not adjust margins
ARTICLE
Journal Name
DMSO-d6): δ 3.76 (s, 3H, CH3), 7.38–7.53 (m, 5H, ArH), 7.68–7.71 (t,
1H, ArH), 8.06 (s, 1H, ArH), 8.11–8.13 (d, J = 7.5 Hz, 2H, ArH), 8.15–
8.17 (d, J = 8.45 Hz, 1H, ArH), 8.28–8.29 (d, J = 5.4 Hz, 2H, ArH),
10.86 (s, 1H, NH); 13C NMR (125 MHz, DMSO-d6): δ 16.94, 51.13,
66,63, 87.62, 110.58, 115.82, 121.38, 122.29, 123.93, 126.55,
127.93, 128.73, 129.29, 130.95, 135.66, 137.84, 140.66, 143.11,
148.01, 155.93, 163.59, 163.73; HRMS (TOF ES+): m/z calcd for
C22H17N2O3S [M+H]+, 389.0954; found, 389.0953.
obtain crude product. The crude product was purified by silica
gel column chromatography and eluted with a mixture of
EtOAc/petroleum ether (1ꢀ:ꢀ5, v/v) to afford 3a–3d as red
solids.
General procedure for the synthesis of 2-phenyl-4-quinoline
carboxylic acid (4a–n)
To a 100 mL round-bottom flask, isatin 3a–d (34.0 mmol),
substituted acetophenone (40.8 mmol) and KOH (102 mmol) in
ethanol (40 mL) were added with stirring. The resulting
mixture was heated to reflux at 85 °C for 3 days. After
removing the solvent under reduced pressure, the mixture was
dissolved in water and washed twice with ether. The aqueous
Methyl-3-(2-phenylquinoline-4-carboxamido)-1,4-
dihydropyrazine-2-carboxylate (5b). Brown oil; yield: 33.1%; IR
(KBr): 3436, 2981, 1722, 1590, 1242, 1194, 1062, 989, 865, 721cm−1;
1H NMR (300 MHz, DMSO-d6): δ 3.82 (s, 3H, CH3), 6.38–6.42 (m, 2H,
ArH), 7.31–7.39 (m, 3H, ArH), 7.75–7.79 (m, 1H, ArH), 7.91–7.95 (m,
1H, ArH), 8.05–8.11 (m, 2H, ArH), 8.24–8.27 (m, 2H, ArH), 8.33 (d, J
= 3 Hz, 1H, ArH); 13C NMR (75 MHz, DMSO-d6): δ 54.09, 78.96,
104.54, 111.06, 118.87, 119.79, 122.39, 125.75, 126.74, 128.29,
129.06, 129.17, 129.56, 131.20, 137.36, 142.93, 146.50, 155.44,
165.24, 169.02; HRMS (TOF ES+): m/z calcd for C22H19N4O3 [M+H]+,
387.1452; found, 387.1454.
layer was concentrated and adjusted to pH
2 with
concentrated HCl to afford an orange solid. This solid was
filtered, washed with water until neutral, dried and purified by
flash column chromatography on silica gel and eluted with a
mixture of EtOAc/petroleum ether (1ꢀ:ꢀ1, v/v) to afford 4a–n as
orange solids.
General procedure for the synthesis of the carboxamide
derivatives (5–9)
N-(5-methylisoxazol-3-yl)-2-phenylquinoline-4-carboxamide (5c).
Red solid; yield: 40.9%; mp 206.5–207.8 ℃; IR (KBr): 3207, 1688,
1617, 1549, 1475, 1426, 1275, 761 cm−1; 1H NMR (500 MHz, DMSO-
d6): δ 1.84 (s, 3H, CH3), 6.81 (s, 1H, ArH), 7.36–7.45 (m, 4H, ArH),
7.63 (t, J = 7.0 Hz, 1H, ArH), 8.06–8.08 (m, 2H, ArH), 8.13 (d, J = 8.3
Hz, 2H, ArH), 8.21 (d, J = 8.2 Hz, 1H, ArH), 11.46 (s, 1H, NH); 13C
NMR (125 MHz, DMSO-d6): δ 11.97, 96.57, 117.29, 123.31, 124.99,
127.40, 128.76, 129.68, 130.08, 138.63, 140.33, 148.78, 156.46,
158.42, 165.56, 170.36; HRMS (TOF ES+): m/z calcd for C20H16N3O2
[M+H]+, 330.1237; found, 330.1234.
Method 1: To a 25 mL round-bottom flask, 2-phenyl-4-
quinoline carboxylic acid 4a–n (1.5 mmol) and substituted
aniline (1.5 mmol) were added, dissolved in appropriate DMF
and followed by the addition of HOBT (1.5 mmol) with stirring.
After 15 min, EDCI (1.5 mmol) and TEA (1.5 mmol) were added
and the reaction mixture was stirred for 4 h at room
temperature. The reaction solution was poured into water and
the product was extracted with EtOAc three times. The
combined organic phases were washed with HCl (1 N), NaHCO3
(1 N), dried over anhydrous Na2SO4 and concentrated under
reduced pressure to give crude product. The crude compound
was then purified by recrystallization to afford compounds 5–
N-(3-methoxyphenyl)-2-phenylquinoline-4-carboxamide (5d).
White solid; yield: 40.2%; mp 191.1–192.1 ℃; IR (KBr): 3419, 1679,
9
.
Method 2: To a 25 mL round-bottom flask, 2-phenyl-4- 1589, 1446, 1033, 751 cm−1; 1H NMR (500 MHz, DMSO-d6): δ 3.74
quinoline carboxylic acid (5.0 mmol) was added, dissolved in
appropriate THF and a few drops of DMF were added. Then
the mixture was cooled in an ice bath and sulfoxide chloride
was slowly dropped into it with stirring for 30 min. The
resulting solution was warmed slowly to 45 °C, stirred at this
temperature for 6 h and the solvent was evaporated off at low
pressure to give a crude residue. Methyl 3-aminothiophene-2-
carboxylate (0.31 g, 2.0 mmol) and trimethylamine (0.10 g, 1.0
mmol) were added to a 25 mL round-bottom flask, dissolved in
appropriate THF, and the solution of the above crude residue
in THF was added dropwise with stirring in an ice bath. The
reaction mixture was stirred at room temperature for 4 h. The
solvent was evaporated under reduced pressure and the crude
product purified by flash column chromatography on silica gel
and eluted with a mixture of EtOAc/petroleum ether (1ꢀ:ꢀ8, v/v)
(s, 3H, CH3), 6.76 (d, J = 7.9 Hz, 1H, ArH), 7.30–7.41 (m, 2H, ArH),
7.54–7.69 (m, 5H, ArH), 7.84 (t, J = 7.4 Hz, 1H, ArH), 8.17 (d, J = 8.0
Hz , 2H, ArH), 8.35 (t, J = 7.5 Hz, 3H, ArH), 10.82 (s, 1H, NH); 13C
NMR (500 MHz, DMSO-d6): δ 55.47, 106.24, 110.04, 112.68, 117.19,
123.57, 125.47, 127.74, 129.30, 130.03, 130.33, 130.69, 138.55,
140.40, 143.40, 148.34, 156.23, 159.97, 165.73; HRMS (TOF ES+):
m/z calcd for C23H19N2O2 [M+H]+, 355.1441; found, 355.1442.
N-(allylcarbamothioyl)-2-phenylquinoline-4-carboxamide (5e).
Yellow solid; yield: 71.4%; mp 102.6–103.8 ℃; IR (KBr): 3417, 2922,
2855, 2234, 1709, 1595, 1546, 1453, 1346, 1247, 1199, 1116, 950,
766, 688 cm−1; 1H NMR (500 MHz, DMSO-d6): δ 4.36 (s, 2H, CH2),
5.45–5.60 (m, 2H, =CH2), 6.06 (t, J = 6.9 Hz, 1H, CH=CH2), 7.52–7.60
(m, 4H, ArH), 7.65 (t, J = 7.5 Hz, 1H, ArH), 7.83 (t, J = 7.3 Hz, 1H,
ArH), 7.92 (d, J = 8.2 Hz, 1H, ArH), 8.07 (s, 1H, NH), 8.20–8.22 (m,
2H, ArH), 8.29 (d, J = 8.4 Hz, 1H, ArH); 13C NMR (125 MHz, DMSO-
d6): δ 49.6, 109.32, 117.37, 122.31, 123.75, 127.55, 128.03, 128.57,
128.83, 128.94, 129.93, 130.60, 130.64, 137.33, 138.45, 148.72,
to afford 5–9
.
Methyl
3-(2-phenylquinoline-4-carboxamido)thiophene-2-
carboxylate (5a). Yellow solid; yield: 36.6%; mp 202.0–203.2 °C; IR
(KBr): 3432, 2316, 1674, 1576, 1442, 1286 cm−1; 1H NMR (500 MHz,
10 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Journal Name
ARTICLE
156.54, 166.75; HRMS (TOF ES+): m/z calcd for C20H18N3OS [M–HS]+, (KBr): 3793, 3434, 3264, 1645, 1587, 1518, 1412, 1282, 1222, 762
1
314.1288; found, 314.1286.
cm−1; H NMR (500 MHz, DMSO-d6): δ 2.37 (s, 3H, CH3), 3.80 (s, 3H,
OCH3), 6.87–6.89 (m, 1H, ArH), 6.93 (s, 1H, ArH), 7.47 (d, J = 8.6 Hz,
1H, ArH), 7.55 (t, J = 7.2 Hz, 1H, ArH), 7.61 (t, J = 7.1 Hz, 2H, ArH),
N-(4-methoxyphenyl)-2-phenylquinoline-4-carboxamide (6a).
Yellow solid; yield: 83.5%; mp 165.3–167.0 ℃; IR (KBr): 3277, 2916, 7.70 (t, J = 7.4 Hz, 1H, ArH), 7.86 (t, J = 7.5 Hz, 1H, ArH), 8.20 (d, J =
2873, 1565, 1550, 1499, 1493, 1410, 1349, 1224, 1078, 964 , 827, 8.4 Hz, 1H, ArH), 8.29 (d, J = 8.1 Hz, 1H, ArH), 8.39 (d, J = 9.4 Hz, 3H,
ArH), 10.24 (s, 1H, NH); 13C NMR (125 MHz, DMSO-d6): δ 18.67,
702, 639cm−1; 1H NMR (300 MHz, DMSO-d6): δ 3.80 (s, 3H, CH3),
6.91 (dt, J = 7.7 1.0 Hz, 1H), 7.34–7.36 (m, 3H, ArH), 7.58 (d, J = 3.0 55.59, 111.81, 115.88, 117.26, 123.87, 125.62, 127.74, 128.10,
128.93, 129.31, 130.01, 130.62, 135.45, 138.66, 143.58, 148.37,
156.25, 157.91, 166.06; HRMS (TOF ES+): m/z calcd for C24H21N2O2
Hz, 2H, ArH), 7.77–7.85 (m, 2H, ArH), 8.07–8.09 (m, 2H, ArH), 8.15
(d, J = 6.0 Hz, 1H, ArH), 8.30–8.32 (m, 1H, ArH), 8.53 (s, 1H, ArH); 13
C
NMR (75 MHz, DMSO-d6): δ 55.54, 114.30, 118.01, 122.32, 123.44, [M+H]+, 369.1598; found, 369.1596.
124.28, 126.80, 128.29, 129.06, 129.56, 130.37, 131.20, 131.29,
138.41, 147.70, 156.60, 156.64, 167.50; HRMS (TOF ES+): m/z calcd
N-(5-chloro-2,
4-dimethoxyphenyl)-2-phenylquinoline-4-
for C23H19N2O2 [M+H]+, 355.1441; found, 255.1445.
carboxamide (6f). Grey solid; yield: 60.9%; mp 251.8–253.6 ℃; IR
(KBr): 3386, 1666, 1593, 1523, 1454, 1391, 1201, 1025 cm−1; 1H
NMR (500 MHz, DMSO-d6): δ 3.92 (s, 6H, OCH3), 6.95 (s, 1H, ArH),
N-(4-ethoxyphenyl)-2-phenylquinoline-4-carboxamide
(6b).
Orange solid; yield: 83.2%; mp 202.5–203.7 ℃; IR (KBr): 3296, 2970, 7.54 (t, J = 7.0 Hz, 1H, ArH), 7.60 (t, J = 7.2 Hz, 2H, ArH), 7.68 (t, J =
1644, 1588, 1514, 1397, 1229 cm−1; 1H NMR (500 MHz, DMSO-d6): δ 7.5 Hz, 1H, ArH), 7.85 (t, J = 7.3 Hz, 1H, ArH), 7.94 (s, 1H, ArH), 8.17
(d, J = 8.4 Hz, 1H, ArH), 8.24 (d, J = 8.3 Hz, 1H, ArH), 8.31 (s, 1H,
ArH), 8.36 (d, J = 7.6 Hz, 2H, ArH), 10.14 (s, 1H, NH); 13C NMR (125
1.31–1.37 (m, 3H, CH3), 4.02–4.07 (m, 2H, CH2), 6.99 (d, J = 9.0 Hz,
2H, ArH), 7.54–7.62 (m, 3H, ArH), 7.67–7.70 (m, 2H, ArH), 7.76 (d, J
= 8.8 Hz, 2H, ArH), 7.85–7.88 (m, 1H, ArH), 8.19 (t, J = 9.5 Hz, 2H, MHz, DMSO-d6): δ 56.76, 56.86, 98.55, 111.46, 117.33, 119.88,
ArH), 8.31–8.40 (m, 4H, ArH), 10.73 (s, 1H, NH); 13C NMR (125 MHz,
123.77, 125.66, 126.31, 127.63, 127.70, 129.29, 129.93, 130.27,
130.58, 138.64, 143.34, 148.26, 152.56, 153.33, 156.13, 166.04;
DMSO-d6): δ 15.56, 64.06, 115.37, 117.68, 122.48, 126.08, 128.24,
128.89, 129.80, 130.51, 130.81, 131.15, 132.78, 139.09, 144.08, HRMS (TOF ES+): m/z calcd for C24H20N2O3Cl [M+H]+, 419.1157;
148.85, 156.02, 156.74, 165.74; HRMS (TOF ES+): m/z calcd for
C24H20N2O2Na [M+Na]+, 391.1417; found, 391.1419.
found, 419.1154.
N-(4-methoxyphenyl)-2-p-tolylquinoline-4-carboxamide
(7a).
2-phenyl-N-(3, 4, 5-trimethoxyphenyl)quinoline-4-carboxamide
(6c). Yellow solid; yield: 78.1%; mp 213.4–214.3 ℃; IR (KBr): 3411,
3070, 2956, 2880, 1601, 1532, 1440, 1367, 1271, 1159, 1116, 1051,
Yellow solid; yield: 82.1%; mp 232.1–233.5 ℃; IR (KBr): 3256, 1642,
1580, 1520, 1413, 1229, 1025 cm−1; 1H NMR (500 MHz, DMSO-d6): δ
2.40 (s, 3H, CH3), 3.77 (s, 3H, OCH3), 6.98–7.00 (m, 2H, ArH), 7.38 (d,
826, 766, 716 cm−1; 1H NMR (300 MHz, DMSO-d6): δ 3.88 (s, 9H, J = 8.1 Hz, 2H, ArH), 7.63–7.66 (m, 1H, ArH), 7.75 (d, J = 9.1 Hz, 2H,
ArH), 7.81–7.85 (m, 1H, ArH), 8.14–8.19 (m, 2H, ArH), 8.27 (t, J = 8.1
Hz, 3H, ArH), 10.70 (s, 1H, NH); 13C NMR (125 MHz, DMSO-d6): δ
OCH3), 6.58 (s, 2H, ArH), 7.31–7.39 (m, 3H, ArH), 7.75–7.86 (m, 2H,
ArH), 8.05–8.10 (m, 2H, ArH), 8.15 (ddd, J = 7.7, 1.5, 0.8 Hz, 1H,
ArH), 8.30–8.33 (m, 1H, ArH), 8.53 (s, 1H, ArH); 13C NMR (75 MHz, 21.30, 55.61, 114.32, 116.92, 121.96, 123.59, 125.55, 127.47,
127.63, 129.91, 130.57, 132.39, 135.81, 140.06, 143.49, 148.33,
156.15, 156.23, 165.29; HRMS (TOF ES+): m/z calcd for C24H20N2O2
DMSO-d6): δ 56.30, 61.40, 102.69, 118.01, 123.44, 124.28, 126.80,
128.29, 129.06, 129.56, 130.37, 131.20, 133.92, 138.41, 140.64,
147.70, 152.28, 156.64, 167.22; HRMS (TOF ES+): m/z calcd for [M+Na]+, 391.1417; found, 391.1415.
C23H19N2OS [M+H]+, 371.1213; found, 371.1214.
2-(4-methoxyphenyl)-N-(3, 4, 5-trimethoxyphenyl)quinoline-4-
N-(3-(methylthio)phenyl)-2-phenylquinoline-4-carboxamide
carboxamide(7b). Yellow solid; yield: 79.9%; mp 235.1–235.9 ℃; IR
(KBr): 3267, 1649, 1596, 1511, 1452, 1411, 1347, 1234, 1130 cm−1;
1H NMR (500 MHz, DMSO-d6): δ 2.42 (s, 3H, CH3), 3.69 (s, 3H, OCH3),
3.82 (s, 6H, OCH3), 7.28 (s, 2H, ArH), 7.40 (d, J = 7.9 Hz, 2H, ArH),
7.66 (t, J = 7.0 Hz, 1H, ArH), 7.84–7.87 (m, 1H, ArH), 8.17–8.21 (m,
2H, ArH), 8.29 (t, J = 7.6 Hz, 3H, ArH), 10.79 (s, 1H, NH); 13C NMR
(125 MHz, DMSO-d6): δ 21.30, 56.18, 60.56, 62.30, 98.21, 116.95,
123.47, 125.50, 127.62, 129.93, 130.61, 134.54, 135.42, 135.78,
140.10, 143.25, 148.34, 153.18, 156.14, 165.57; HRMS (TOF ES+):
m/z calcd for C26H25N2O4 [M+H]+, 429.1809; found, 429.1805.
(6d). Red solid; yield: 72.6%; mp 189.1–190.3 ℃; IR (KBr): 3233,
1
3053, 1675, 1588, 1533, 1412, 1348, 1254, 764, 697 cm−1; H NMR
(500 MHz, DMSO-d6): δ 2.49 (s, 3H, CH3), 7.02 (s, 1H, ArH), 7.28 (s,
1H, ArH), 7.50 (s, 1H, ArH), 7.55 (s, 2H, ArH), 7.61 (s, 2H, ArH), 7.79
(s, 1H, ArH), 7.83 (s, 1H, ArH), 8.16 (t, J = 9.7 Hz, 2H, ArH), 8.25 (d, J
= 8.0 Hz, 1H, ArH), 8.30 (s, 1H, ArH), 10.71 (s, 1H, NH); 13C NMR (125
MHz, DMSO-d6): δ 15.33, 78.55, 78.82, 79.01, 116.84, 117.12,
117.71, 121.88, 123.58, 125.42, 127.30, 127.54, 128.99, 129.25,
129.96, 130.20, 138.67, 139.13, 139.64, 142.98, 148.49, 156.22,
165.76; HRMS (TOF ES+): m/z calcd for C23H19N2OS [M+H]+,
371.1213; found, 371.1214.
N-(4-methoxy-2-methylphenyl)-2-p-tolylquinoline-4-
carboxamide (7c). Purple solid; yield: 90.2%; mp 234.4–236.0 ℃; IR
(KBr): 3729, 3433, 3275, 2921, 1648, 1593, 1518, 1416, 1349, 1281,
1224, 1112, 1047, 755 cm−1; 1H NMR (500 MHz, DMSO-d6): δ 2.36
N-(4-methoxy-2-methylphenyl)-2-phenylquinoline-4-
carboxamide (6e). Grey solid; yield: 91.2%; mp 207.6–208.9 ℃; IR
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
Please do not adjust margins

Please do not adjust margins
ARTICLE
Journal Name
(s, 3H, CH3), 2.43 (s, 3H, CH3), 3.80 (s, 3H, OCH3), 6.86–6.93 (m, 2H, 8.13 (d, J = 8.3 Hz, 1H, ArH), 8.22 (d, J = 8.2 Hz, 1H, ArH); 13C NMR
ArH), 7.41 (dd, J = 7.9, 8.6 Hz, 2H, ArH), 7.67 (t, J = 7.7 Hz, 1H, ArH), (125 MHz, DMSO-d6): δ 20.65, 55.60, 114.31, 120.43, 121.97,
7.84 (t, J = 7.5 Hz, 1H, ArH), 8.16 (d, J = 8.4 Hz, 1H, ArH), 8.26–8.31 123.09, 125.54, 126.41, 127.81, 129.13, 129.87, 130.32, 130.54,
(m, 3H, ArH), 8.35 (s, 1H, ArH), 10.22 (s, 1H, NH); 13C NMR (125 131.29, 132.31, 136.34, 139.88, 142.84, 147.96, 156.24, 159.52,
MHz, DMSO-d6): δ 18.67, 21.31, 55.59, 111.79, 115.86, 117.00, 165.12; HRMS (TOF ES+): m/z calcd for C24H21N2O2 [M+H]+,
123.76, 125.59, 127.46, 127.63, 128.11, 128.93, 129.92, 130.55, 369.1598; found, 369.1596.
135.45, 140.04, 143.50, 148.35, 156.15, 157.89, 166.09; HRMS (TOF
ES+): m/z calcd for C25H23N2O2 [M+H]+, 383.1754; found, 397.1754.
N-(4-methoxy-2-methylphenyl)-2-m-tolylquinoline-4-
carboxamide (7h). Red solid; yield: 56.4%; mp 130.8–131.8 ℃; IR
N-(4-methoxyphenyl)-2-m-tolylquinoline-4-carboxamide (7d).
(KBr): 3793, 3729, 3439, 2351, 1646, 1597, 1504, 1452, 1238, 1110,
Red solid; yield: 68.2%; mp 205.0–207.6 ℃; IR (KBr): 3729, 3455, 1041, 757 cm−1; 1H NMR (500 MHz, DMSO-d6): δ 2.31 (s, 3H, CH3),
3318, 2352, 1646, 1593, 1520, 1239, 1102, 1024 cm−1; 1H NMR (500 2.47 (s, 3H, CH3), 3.77 (s, 3H, OCH3), 6.83–6.85 (m, 1H, ArH), 6.90 (d,
MHz, DMSO-d6): δ 2.45 (s, 3H, CH3), 3.77 (s, 3H, OCH3), 6.98–7.00 J = 2.6 Hz, 1H, ArH), 7.37–7.43 (m, 4H, ArH), 7.63–7.64 (m, 1H, ArH),
(m, 2H, ArH), 7.34 (d, J = 7.4 Hz, 1H, ArH), 7.45 (t, J = 7.5 Hz, 1H, 7.71 (t, J = 7.5 Hz, 1H, ArH), 7.85–7.88 (m, 1H, ArH), 7.91 (s, 1H,
ArH), 7.64–7.68 (m, 1H, ArH), 7.75 (d, J = 8.9 Hz, 2H, ArH), 7.83–7.86 ArH), 8.13 (d, J = 8.3 Hz, 1H, ArH), 8.29 (d, J = 8.3 Hz, 1H, ArH), 10.20
(m, 1H, ArH), 8.16–8.21 (m, 4H, ArH), 8.32 (s, 1H, ArH), 10.70 (s, 1H, (s, 1H, NH); 13C NMR (125 MHz, DMSO-d6): δ 18.61, 20.64, 55.57,
NH); 13C NMR (125 MHz, DMSO-d6): δ 21.50, 55.62, 114.32, 117.19, 111.78, 115.83, 120.55, 123.27, 125.59, 126.42, 127.80, 128.12,
121.97, 123.67, 124.92, 125.55, 127.61, 128.23, 129.19, 129.97, 128.83, 129.13, 129.86, 130.29, 130.54, 131.28, 135.47, 136.32,
130.61, 130.96, 132.37, 138.51, 143.53, 148.33, 156.24, 156.31, 139.96, 142.88, 147.97, 157.89, 159.52, 165.96; HRMS (TOF ES+):
165.27; HRMS (TOF ES+): m/z calcd for C24H21N2O2 [M+H]+, m/z calcd for C25H23N2O2 [M+H]+, 383.1754; found, 383.1756.
369.1598; found, 369.1599.
N-(4-methoxy-2-methylphenyl)-2-(4-(trifluoromethyl)phenyl)-
2-M-tolyl-N-(3, 4, 5-trimethoxyphenyl)quinoline-4-carboxamide quinoline-4-carboxamide (7i). Brown solid; yield: 33.6%; mp 239.0–
(7e). Yellow solid; yield: 69.4%; mp 234.8–236.4 ℃; IR (KBr): 3793, 239.8 ℃; IR (KBr): 3381, 3261, 2971, 2315, 1637, 1524, 1416, 1325,
3729, 3436, 3266, 2939, 1649, 1598, 1512, 1453, 1409, 1233, 1130, 1171, 1103, 1051 cm−1; 1H NMR (500 MHz, DMSO-d6): δ 2.34 (s, 3H,
995 cm−1; 1H NMR (500 MHz, DMSO-d6): δ 2.45 (s, 3H, CH3), 3.68 (s, CH3), 3.75 (s, 3H, OCH3), 6.85–6.91 (m, 2H, ArH), 7.45 (d, J = 8.6 Hz,
3H, OCH3), 3.77 (s, 6H, OCH3), 7.26 (s, 2H, ArH), 7.35 (d, J = 7.5 Hz, 1H, ArH), 7.72 (t, J = 7.5 Hz, 1H, ArH), 7.87 (t, J = 7.2 Hz, 1H, ArH),
1H, ArH), 7.46 (t, J = 7.6 Hz, 1H, ArH), 7.66 (t, J = 7.5 Hz, 1H, ArH), 7.96 (d, J = 8.0 Hz, 2H, ArH), 8.20 (d, J = 8.4 Hz, 1H, ArH), 8.28 (d, J =
7.84 (t, J = 7.4 Hz, 1H, ArH), 8.15–8.21 (m, 4H, ArH), 8.32 (s, 1H, 8.3 Hz, 1H, ArH), 8.45 (s, 1H, ArH), 8.58 (d, J = 8.1 Hz, 2H, ArH),
ArH), 10.76 (s, 1H, NH); 13C NMR (125 MHz, DMSO-d6): δ 21.50, 10.23 (s, 1H, NH); 13C NMR (125 MHz, DMSO-d6): δ 14.44, 18.67,
56.17, 60.56, 98.19, 117.21, 123.54, 124.92, 125.50, 127.68, 128.21, 55.57, 111.79, 115.86, 117.47, 124.16, 125.66, 126.21, 128.04,
129.21, 129.99, 130.65, 130.99, 134.48, 135.40, 138.47, 138.54, 128.29, 128.50, 128.84, 130.15, 130.92, 135.40, 142.38, 143.93,
143.30, 148.32, 153.17, 156.29, 165.54; HRMS (TOF ES+): m/z calcd 148.28, 154.67, 157.89, 165.84; HRMS (TOF ES+): m/z calcd for
for C26H25N2O4 [M+H]+, 429.1809; found, 429.1808.
C25H20F3N2O2 [M+H]+, 437.1471; found, 437.1473.
N-(4-methoxy-2-methylphenyl)-2-m-tolylquinoline-4-
2-(Benzo[d][1,3]dioxol-5-yl)-N-(4-methoxyphenyl)quinoline-4-
carboxamide (7f). White solid; yield: 70.6%; mp 193.5–195.5 ℃; IR carboxamide(7j). Yellow solid; yield: 62.1%; mp 263.1–264.5 ℃; IR
(KBr): 3793, 3729, 3437, 3276, 1643, 1593, 1520, 1400, 1220, 1036 (KBr): 3265, 1651, 1590, 1506, 1449, 1245, 1243, 1036 cm−1; 1H
1
cm−1; H NMR (500 MHz, DMSO-d6): δ 2.34 (s, 3H, CH3), 2.46 (s, 3H, NMR (500 MHz, DMSO-d6): δ 3.78 (s, 3H, OCH3), 6.15 (s, 2H, CH2),
CH3), 3.78 (s, 3H, OCH3), 6.84–6.91 (m, 2H, ArH), 7.35–7.49 (m, 3H, 6.99–7.01 (m, 2H, ArH), 7.11–7.13 (m, 1H, ArH), 7.63 (t, J = 7.1 Hz,
ArH), 7.67 (t, J = 7.4 Hz, 1H, ArH), 7.83 (t, J = 7.7 Hz, 1H, ArH), 8.15– 1H, ArH), 7.76 (d, J = 7.4 Hz, 2H, ArH), 7.81 (t, J = 7.0 Hz, 1H, ArH),
8.21 (m, 3H, ArH), 8.25 (d, J = 8.3 Hz, 1H, ArH), 8.34 (s, 1H, ArH), 7.95–7.97 (m, 2H, ArH), 8.12–8.18 (m, 2H, ArH), 8.29 (s, 1H, ArH),
10.20 (s, 1H, NH); 13C NMR (125 MHz, DMSO-d6): δ 18.69, 21.54, 10.73 (s, 1H, NH); 13C NMR (125 MHz, DMSO-d6): δ 55.62, 101.93,
55.59, 111.79, 115.86, 117.26, 123.84, 124.96, 125.60, 127.61, 107.59, 108.93, 114.30, 116.92, 122.01, 122.36, 123.47, 125.50,
128.11, 128.21, 128.92, 129.21, 129.97, 130.59, 130.96, 135.44, 127.34, 129.80, 130.54, 132.37, 132.92, 143.34, 148.20, 148.53,
138.52, 143.55, 148.34, 156.33, 157.88, 166.06; HRMS (TOF ES+): 149.33, 155.61, 156.23, 165.27; HRMS (TOF ES+): m/z calcd for
m/z calcd for C25H26N2O2 [M+H]+, 383.1754; found, 383.1750.
C24H18N2O4Na [M+Na]+, 421.1159; found,421.1158.
N-(4-methoxyphenyl)-2-o-tolylquinoline-4-carboxamide
(7g).
N-(4-methoxyphenyl)-6-methyl-2-p-tolylquinoline-4-
Red solid; yield: 60.1%; mp 193.0–197.1 ℃; IR (KBr): 3233, 3063, carboxamide (8a). Red solid; yield: 81.2%; mp 255.5–255.9 ℃; IR
1
2313, 1732, 1648, 1594, 1511, 1457, 1301, 1239, 1175, 1032, 824, (KBr): 3433, 3278, 1646, 1588, 1518, 1233, 1025, 824 cm−1; H NMR
769 cm−1; 1H NMR (500 MHz, DMSO-d6): δ 2.47 (s, 3H, CH3), 3.76 (s, (500 MHz, DMSO-d6): δ 2.39 (s, 3H, CH3), 2.50 (s, 3H, CH3), 3.77 (s,
3H, OCH3), 6.96–6.99 (m, 2H, ArH), 7.36–7.42 (m, 3H, ArH), 7.62 (d, 3H, OCH3), 6.98 (d, J = 8.9 Hz, 2H, ArH), 7.36 (d, J = 8.0 Hz, 2H, ArH),
J = 6.7 Hz, 1H, ArH), 7.69–7.75 (m, 3H, ArH), 7.84–7.87 (m, 1H, ArH), 7.65 (d, J = 8.7 Hz, 1H, ArH), 7.75 (d, J = 8.9 Hz, 2H, ArH), 7.94 (s, 1H,
12 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Journal Name
ARTICLE
ArH), 8.04 (d, J = 8.6 Hz, 1H, ArH), 8.24 (t, J = 8.2 Hz, 3H, ArH), 10.68 135.28, 138.28, 142.11, 147.00, 153.30, 156.89, 166.01; HRMS (TOF
(s, 1H, NH); 13C NMR (125 MHz, DMSO-d6): δ 21.28, 21.77, 55.61, ES+): m/z calcd for C25H22N2O4Cl [M+H]+, 449.1263; found, 449.1259.
114.30, 116.87, 121.95, 123.54, 124.20, 127.47, 129.69, 129.87,
6-Chloro-N-(4-methoxy-2-methylphenyl)-2-phenylquinoline-4-
132,44, 132.68, 135.90, 137.06, 139.80, 142.81, 146.97, 155.21,
156.21, 165.43; HRMS (TOF ES+): m/z calcd for C25H22N2O2Na carboxamide (8f). Purple solid; yield: 56.9%; mp 232.6–233.1 ℃; IR
[M+Na]+, 405.1573; found, 405.1577.
(KBr): 3395, 2972, 2312, 1640, 1524, 1049 cm−1; H NMR (500 MHz,
DMSO-d6): δ 2.35 (s, 3H, CH3), 3.79 (s, 3H, OCH3), 6.86 (t, J = 6.5 Hz,
1H, ArH), 6.92 (s, 1H, ArH), 7.45 (d, J = 8.5 Hz, 1H, ArH), 7.56–7.64
1
6-Methyl-2-p-tolyl-N-(3, 4, 5-trimethoxyphenyl)quinoline-4-
carboxamide (8b). White solid; yield: 80.1%; mp 236.1–237.6 ℃; IR (m, 3H, ArH), 7.88–7.90 (m, 1H, ArH), 8.20 (d, J = 8.9 Hz, 1H, ArH),
(KBr): 3432, 3266, 1648, 1593, 1510, 1411, 1234, 1131 cm−1; 1H 8.33 (d, J = 2.0 Hz, 1H, ArH), 8.38 (d, J = 7.3 Hz, 2H, ArH), 8.47 (s, 1H,
NMR (500 MHz, DMSO-d6): δ 2.41 (s, 3H, CH3), 2.51 (s, 3H, CH3), ArH), 10.31 (s, 1H, ArH); 13C NMR (125 MHz, DMSO-d6): δ 18.60,
3.69 (s, 3H, OCH3), 3.79 (s, 6H, OCH3), 7.28 (s, 2H, ArH), 7.39 (d, J = 55.60, 111.84, 115.88, 118.57, 124.44, 124.54, 127.82, 128.11,
8.0 Hz, 2H, ArH), 7.68 (d, J = 8.6 Hz, 1H, ArH), 7.95 (s, 1H, ArH), 8.05 128.76, 129.35, 130.57, 131.13, 132.15, 135.42, 138.24, 142.10,
(d, J = 8.6 Hz, 1H, ArH), 8.25 (d, J = 8.4 Hz, 3H, ArH), 10.76 (s, 1H, 146.90, 156.81, 157.96, 165.45; HRMS (TOF ES+): m/z calcd for
NH); 13C NMR (125 MHz, DMSO-d6): δ 21.28, 21.75, 56.18, 60.56, C24H20N2O2Cl [M+H]+, 403.1208; found, 403.1212.
98.20, 116.91, 123.42, 124.11, 127.47, 129.71, 129.89, 132.73,
6-Fluoro-N-(4-methoxyphenyl)-2-phenylquinoline-4-
155.20, 165.71; HRMS (TOF ES+): m/z calcd for C27H26N2O4Na carboxamide (8g). White solid; yield: 57.5%; mp 207.8–209.6 ℃; IR
134.51, 135.47, 135.87, 137.16, 139.84, 142.58, 146.97, 153.17,
[M+Na]+, 465.1785; found, 465.1792.
(KBr): 3434, 3277, 1640, 1519, 1454, 1402, 1242, 1034, 833, 694
cm−1; 1H NMR (500 MHz, DMSO-d6): δ 3.79 (s, 3H, OCH3), 7.00 (d, J =
N-(4-methoxy-2-methylphenyl)-6-methyl-2-p-tolylquinoline-4-
8.8 Hz, 2H, ArH), 7.50 (t, J = 7.2 Hz, 2H, ArH), 7.56 (t, J = 8.5 Hz, 3H,
carboxamide (8c). White solid; yield: 91.1%; mp 217.1–218.0 ℃; IR ArH), 7.76 (d, J = 8.6 Hz, 2H, ArH), 8.08 (d, J = 9.1 Hz, 1H, ArH), 8.30–
(KBr): 3793, 3729, 3433, 3247, 1649, 1594, 1516, 1448, 1286, 1237, 8.34 (m, 3H, ArH), 10.68 (s, 1H,NH); 13C NMR (125 MHz, DMSO-d6):
1042, 820 cm−1; 1H NMR (500 MHz, DMSO-d6): δ 2.36 (s, 3H, CH3), δ 14.87, 55.63, 63.96, 104.28, 114.32, 117.67, 122.09, 123.07,
2.42 (s, 3H, CH3), 2.55 (s, 3H, CH3), 3.80 (s, 3H, OCH3), 6.87–6.92 (m, 124.88, 127.39, 129.22, 129.83, 131.59, 132.39, 138.74, 141.68,
2H, ArH), 7.40 (dd, J = 7.8, 8.5 Hz, 3H, ArH), 7.68 (d, J = 8.3 Hz, 1H, 144.47, 153.64, 156.26, 157.49, 165.45; HRMS (TOF ES+): m/z calcd
ArH), 8.04–8.30 (m, 5H, ArH), 10.19 (s, 1H, NH); 13C NMR (125 MHz, for C23H18N2O2F [M+H]+, 373.1347; found, 373.1345.
DMSO-d6): δ 18.65, 21.29, 21.83, 55.59, 111.78, 115.86, 116.98,
9-Chloro-N-(4-methoxyphenyl)-5,6-dihydrobenzo[c]acridine-7-
carboxamide (9a)
123.73, 124.32, 127.48, 128.10, 128.98, 129.69, 129.88, 132.64,
135.35, 135.99, 136.98, 139.78, 142.79, 147.01, 155.24, 157.85,
166.20; HRMS (TOF ES+): m/z calcd for C26H25N2O2 [M+H]+,
397.1911; found, 397.1903.
Yellow solid; yield: 40.2%; mp 267.0–269.1
℃; IR (KBr): 3437,
3241, 1645, 1521, 1116, 624 cm−1; H NMR (400 MHz, DMSO-
d6): δ 3.02 (dd, J = 4.4 6.4 Hz, 4H, CH2), 3.77 (s, 3H, OCH3), 6.98
(d, J = 8.8 Hz, 2H, ArH), 7.38–7.48 (m, 3H, ArH), 7.70–7.82 (m,
4H, ArH), 8.15 (d, J = 9.2 Hz, 1H, ArH), 8.45–8.48 (m, 1H, ArH),
10.76 (s, 1H, NH); 13C NMR (101 MHz, DMSO-d6): δ 26.11,
27.28, 55.76, 114.60, 121.88, 123.64, 124.93, 126.16, 127.71,
127.82, 128.72, 130.46, 130.95, 131.95, 133.86, 139.86,
140.67, 145.42, 153.67, 156.52, 164.30; HRMS (TOF ES+): m/z
calcd for C25H20ClN2O2 [M+H]+, 415.1208; found, 415.1205.
1
6-Chloro-N-(4-methoxyphenyl)-2-phenylquinoline-4-
carboxamide (8d). Yellow solid; yield: 61.6%; mp 275.1–278.9 ℃; IR
(KBr): 3290, 1647, 1589, 1520, 1519, 1250, 1120 cm−1; 1H NMR (500
MHz, DMSO-d6): δ 3.79 (s, 3H, OCH3), 7.00 (d, J = 8.9 Hz, 2H, ArH),
7.55–7.62 (m, 3H, ArH), 7.78 (d, J = 8.7 Hz, 2H, ArH), 7.86–7.89 (m,
1H, ArH), 8.19 (d, J = 9.0 Hz,1H, ArH), 8.27 (d, J = 2.1 Hz, 1H, ArH),
8.38 (d, J = 7.3 Hz, 2H, ArH), 8.45 (s, 1H, ArH), 10.88 (s, 1H, NH); 13C
NMR (125 MHz, DMSO-d6): δ 55.63, 114.30, 118.61, 122.20, 124.41,
127.84, 129.32, 130.56, 131.11, 132.14, 132.22, 138.22, 142.08,
146.87, 156.35, 156.78, 164.65; HRMS (TOF ES+): m/z calcd for
C23H18N2O2Cl [M+H]+, 389.1051; found, 389.1053.
9-Chloro-N-(4-methoxy-2-methylphenyl)-5,6-dihydrobenzo[c]-
acridine-7-carboxamide (9b). Purple solid; yield: 49.9%; mp 272.9–
274.5 ℃; IR (KBr): 3790, 3439, 3237, 1645, 1527, 1036, 746 cm−1; 1H
NMR (500 MHz, DMSO-d6): δ 2.33 (s, 2H, CH2), 2.51 (s, 2H, CH2),
3.06 (s, 3H, CH3), 3.79 (s, 3H, OCH3), 6.92 (s, 2H, ArH), 7.41 (d, J =
35.5 Hz, 5H, ArH), 7.86 (s, 3H, ArH), 8.16 (s, 1H, ArH), 8.47 (s, 1H,
ArH), 10.23 (s, 1H, NH); 13C NMR (125 MHz, DMSO-d6): δ 18.68,
26.09, 27.27, 55.64, 60.11, 112.03, 116.03, 123.61, 125.03, 126.08,
127.61, 128.43, 130.28, 130.82, 131.86, 133.83, 134.69, 139.78,
140.86, 145.53, 153.54, 157.87, 165.14; HRMS (TOF ES+): m/z calcd
for C26H22ClN2O2 [M+H]+, 429.1364; found, 429.1364.
6-Chloro-2-phenyl-N-(3, 4, 5-trimethoxyphenyl)quinoline-4-
carboxamide (8e). Red solid; yield: 60.8%; mp 256.3–260.1 ℃; IR
(KBr): 3440, 2929, 1644, 1546, 1509, 1453, 1415, 1384, 1346, 1233,
1131, 1048, 777 cm−1; 1H NMR (500 MHz, DMSO-d6): δ 3.69 (s, 3H,
OCH3), 3.81 (s, 6H, OCH3), 7.25 (s, 2H, ArH), 7.56–7.62 (m, 3H, ArH),
7.86 (d, J = 9.0 Hz, 1H, ArH), 8.19 (d, J = 9.0 Hz, 1H, ArH), 8.27 (s, 1H,
ArH), 8.36 (d, J = 7.8 Hz, 2H, ArH), 8.41 (s, 1H, ArH), 10.77 (s, 1H,
NH); 13C NMR (125 MHz, DMSO-d6): δ 56.39, 60.67, 98.70, 118.62,
124.40, 127.90, 129.44, 130.69, 131.28, 132.26, 132.36, 134.94,
Biological evaluations
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 13
Please do not adjust margins

Please do not adjust margins
ARTICLE
Journal Name
×
105 cells per
Sulforhodamine B (SRB) assays
Cells were seeded in 12-well plates at 1
well in complete growth medium. Following treatment of cells
with respective compound 7b for a duration of 24 h, cells were
washed with PBS and subsequently soluble and insoluble
tubulin fractions were collected. To collect the soluble tubulin
Target compounds’ inhibition of human cancer cell
growth was assessed using the sulforhodamine B assay, as
previously described. The panel of cell lines included HCT116
(colon cancer), A549 (lung cancer), and MDA-MB-468 (breast
cancer).The growth inhibitory rate of treated cells was
calculated by the following formula: [1–(A515treated/A515
control)] × 100%. The results were also expressed as IC50 (the
compound concentration required for 50% growth inhibition
of cancer cells), which was calculated by the Logit method. The
mean IC50 was determined from the results of three
independent tests.
fractions, cells were permeabilized with 200 µL of pre-warmed
lysis buffer [80 mM Pipes-KOH (pH 6.8), 1 mM MgCl2, 1 mM
EGTA, 0.2% Triton X-100, 10% glycerol, 0.1% protease inhibitor
cocktail (Sigma-Aldrich)] and mixed with 100 µL of 3
×
Laemmli’s sample buffer (180 mM Tris-Cl pH 6.8, 6% SDS, 15
glycerol, 7.5% β-mercaptoethanol and 0.01% bromophenol
blue). Samples were immediately heated to 95 ℃ for 3 min.
To collect the insoluble tubulin fraction, 300 µL of 1 ×
Laemmli’s samples buffer was added to the remaining cells
in each cell, and the samples were heated to 95 ℃ for 3 min.
Equal volumes of samples were run on an SDS-10%
polyacrylamide gel and were transferred to a nitrocellulose
membrane employing semidry transfer at 50 mA for 1 h.
Blots were probed with mouse anti-human α-tubulin
diluted 1:2000 ml (Sigma) and stained with rabbit anti-
mouse secondary antibody coupled with horseradish
peroxidase, diluted 1:5000 ml (Sigma). Bands were
visualized using an enhanced Chemiluminescence protocol
(Pierce) and radiographic film (Kodak).
Immunofluorenscence staining
A549 cells (1
and incubated for 12 h at 37
×
104) were seeded in 96-well culture plate
and 5% CO2. Cells were
℃
treatment with DMSO, colchicine, and 7b for 24 h. The cells
were fixed with methanol and then permeabilized with 0.5%
Triton X-100/PBS. The cells were incubated overnight with
primary antibody (α-tubulin) at 4 °C. After washing three times
with PBS, cells were incubated with corresponding
fluorescence-conjugated secondary antibody for 1 h. The
nuclei of cells were labeled with DAPI. Cells were visualized
using a fluorescence microscopy.
In vitro tubulin polymerization
Flow cytometric evaluation of apoptosis
HCT116 were seeded in six-well plates and allowed to
grow overnight. The medium was then replaced with
A fluorescence based tubulin polymerization assay was
performed accoding to the manufacturers protocol (BK011,
Cytoskeleton, Inc.). Briefly, the reaction mixture was taken
within a total volume of 10 µL contained PEM buffer, GTP (1
µM) in the presence or absence of the test compound 7b at a
final concentration. In vitro tubulin polymerization was
followed by a time dependent increase in fluorescence due to
the incorporation of a fluorescene reporter intomicrotubules
as polymerization proceeds. Fluorescence emission at 420 nm
(excitation wavelength is 360 nm) was measured by using a
Biotec multimode plate reader. Polymerization was finally
monitored by increase in the fluorescence mentioned above at
37 °C. The IC50 value was defined as the drug concentration
required for inhibiting 50% of tubulin assembly compared to
the control.
complete medium containing 0.5
µM concentration of
compound 7b for 24 h along with vehicle alone (0.001%
DMSO) as the control. After 24 h of drug treatment, the cells
from the supernatant and adherent monolayer cells were
harvested by trypsinization, and washed with PBS at 3000
rpm. Then the cells were stained with Annexin V-FITC and
propidium iodide using the Annexin-V-PI apoptosis
detection kit (Sigma Aldrich). Flow cytometry was
performed using a FACScan (Becton Dickinson) equipped
with a single 488 nm argon laser as described earlier.
Annexin V-FITC was analysed using excitation and emission
settings of 488 nm and 535 nm (FL-1 channel); PI, 488 nm
and 610 (Fl-2 channel).
Molecular modeling
Colchicine competition assay
To investigate the binding mode of compound to the
tubulin, the advanced docking program Autodock 4.2 tool was
used to automatically dock the inhibitor to the enzyme. The
crystal structure of tubulin in a complex with its inhibitor
colchicine (PDB entry code 4O2B) was recovered from
Brookhaven Protein Database (PDB). The missing atoms and
residues were modeled in Sybyl 6.8.17. Atomic charges were
taken as Kollman-united-atom26 for the macromolecule and
Gasteiger–Marsili27 for the inhibitor. To explore the binding
mode of compounds to the colchicine binding domain of
tubulin, the advanced docking program Autodock 4.2 tool was
used to automatically dock the inhibitor to the protein. The
Lamarckian genetic algorithm (LGA) was applied to analyze
inhibitor–protein interactions. A Solis and Wets local search
Compound 7b with different concentration (0.1, 0.5, 1, 5
and 10 µM) was incubated with 3 µM tubulin in the presence
and absence of 3µM colchicine in 30µM Tris buffer for 1hr at
37
was used as
℃
. CA-4 was used as a positive control, whereas Vinblastine
negative control. After incubation, the
a
fluorescence of the tubulin-colchicine complex was
determined by using a Tecan multimode reader at an exitation
wavelength of 350 nm and an emission wavelength of 435 nm.
Tris buffer (30 mM) was used as blank, which was subtracted
from all the samples, and the fluorescence values were
normalized to DMSO (control).
Western blot analysis of soluble versus polymerized tubulin
14 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Journal Name
ARTICLE
J. Goossens, P. Berthelot and N. Lebegue, Eur. J. Med.
Chem., 2013, 59, 39–47.
performed the energy minimization on
a user-specified
proportion of the population. The docked conformations of
the inhibitor were generated after a reasonable number of
evaluations. The whole docking operation in this study can be
summarized follows. First, the colchicine binding domain of
tubulin was checked for polar hydrogens and assigned for
partial atomic charges, then a PDBQ file was created, and the
atomic solvation parameters were assigned for the
macromolecule. Meanwhile, some of the torsion angles of the
inhibitor that would be explored during molecular docking
stage were defined, allowing the conformation search for the
ligand during the docking process. Second, the grid map with
60 × 60 × 60 points and a spacing of 0.375 Å was calculated
using the AutoGrid program in order to evaluate the binding
energies between the inhibitor and the macromolecule. Third,
some important parameters for LGA calculations were
reasonably set (see Box 1), not only the atom types, but also
the generations and the number of runs for the LGA algorithm
were edited and properly assigned according to the
requirements of the Amber force field. The maximum number
of generations, energy evaluations and docking runs were set
to 3.0 × 105, 1.5 × 107, and 30, respectively. Finally, the
docked complex of the inhibitor-enzyme for the inhibitor was
selected according to the criterion of interaction energy
combined with geometrical matching quality.
7. K. Odlo, J. Hentzen, J. F. Dit Chabert, S. Ducki, O. A. B.
S. Gani, I. Sylte, M. Skrede, V. A. Florenes and T. V.
Hansen, Bioorg. Med. Chem., 2008, 16, 4829–4838.
8. E. Pasquier, N. Andre and D. Braguerl, Curr. Cancer
Drug Tar., 2007, 7, 566–581.
9. A. Jordan, J. A. Hadfield, N. J. Lawrence and A. T.
McGown, Med. Res. Rev., 1998, 18, 259–296.
10. C. Dumontet and M. A. Jordan, Nat. Rev. Drug Discov.,
2010, 9, 790–803.
11. Y. Li, J. Liu, N. Liu, D. Shi, X. Zhou, J. Lv, J. Zhu, C.
Zheng and Y. Zhou, Bioorg. Med. Chem., 2011, 19,
3579–3584.
12. M. M. Cooney, T. Radivoyevitch, A. Dowlati, B.
Overmoyer, N. Levitan, K. Robertson, S. L. Levine, K.
DeCaro, C. Buchter, A. Taylor, B. S. Stambler and S. C.
Remick, Clin. Cancer Res., 2004, 10, 96–100.
13. L. V. Beerepoot, S. A. Radema, E. O. Witteveen, T.
Thomas, C. Wheeler, S. Kempin and E. E. Voest, J. Clin.
Oncol., 2006, 24, 1491–1498.
14. A. L. Risinger, C. D. Westbrook, A. Encinas, M.
Muelbaier, C. M. Schultes, S. Wawro, J. D. Lewis, B.
Janssen, F. J. Giles and S. L. Mooberry, J. Pharmacol.
Exp. Ther., 2011, 336, 652–660.
15. B. L. Flynn, G. S. Gill, D. W. Grobelny, J. H. Chaplin, D.
Paul, A. F. Leske, T. C. Lavranos, D. K. Chalmers, S. A.
Charman, E. Kostewicz, D. M. Shackleford, J. Morizzi, E.
Hamel, M. K. Jung and G. Kremmidiotis, J. Med. Chem.,
2011, 54, 6014–6027.
Docking simulations were performed using the
Lamarckian genetic algorithm (LGA) and the Solis and Wets
local search method. Each run of the docking operation was
terminated after a maximum of 2500000 energy evaluations.
16. T. Owa, A. Yokoi, K. Yamazaki, K. Yoshimatsu, T.
Yamori and T. Nagasu, J. Med. Chem., 2002, 45, 4913–
4922.
17. T. Ashizawa, H. Miyata, H. Ishii, C. Oshita, K. Matsuno,
Y. Masuda, T. Furuya, T. Okawara, M. Otsuka, N. Ogo,
A. Asai and Y. Akiyama, Int. J. Oncol., 2011, 38, 1245–
1252.
18. K. Matsuno, Y. Masuda, Y. Uehara, H. Sato, A. Muroya,
O. Takahashi, T. Yokotagawa, T. Furuya, T. Okawara, M.
Otsuka, N. Ogo, T. Ashizawa, C. Oshita, S. Tai, H. Ishii,
Y. Akiyama and A. Asai, ACS Med. Chem. Lett., 2010, 1,
371–375.
Acknowledgements
The authors gratefully acknowledge the Program for
Changjiang Scholars and Innovative Rsearch Team in University
(IRT17R94), the NSFC (Nos. 21662044, 21262043, 81760621
and U1202221), the Foundation of “Yunling Scholar” Program
of Yunnan Province for financial support, and thank the High
Performance Computing Center at Yunnan University for use
of the high performance computing platform.
19. J. Li, J. Chen, C. S. Gui, L. Zhang, Y. Qin, Q. Xu, J.
Zhang, H. Liu, X. Shen and H. L. Jiang, Bioorg. Med.
Chem., 2006, 14, 2209–2224.
Notes
The authors declare no competing financial interest.
20. A. V. Zimichev, M. N. Zemtsova, A. G. Kashaev and Y.
N. Klimochkin, Pharm. Chem. J, 2011, 45, 217–219.
21. K. Metwally, A. Khalil, A. Sallam, H. Pratsinis, D.
Kletsas and K. El Sayed, Med. Chem. Res., 2013, 22,
4481–4491.
References
1. K. J. Verhey and J. Gaertig, Cell Cycle, 2007, 6, 2152–
2160.
2. M. A. Jordan and L. Wilson, Nat. Rev. Cancer, 2004, 4,
253–265.
3. O. Valiron, N. Caudron and D. Job, Cell. Mol. Life Sci.,
2001, 58, 2069–2084.
4. E. Nogales, Annu. Rev. Bioph. Biom., 2001, 30, 397–420.
5. K. H. Downing, Annu. Rev. Cell Dev. Bi., 2000, 16, 89–
111.
22. K. Metwally, H. Pratsinis and D. Kletsas, Med. Chem.
Res., 2015, 24, 2604–2611.
23. K. Metwally, H. Pratsinis and D. Kletsas, Planta Med.,
2008, 74, 1147.
24. K. Metwally, H. Pratsinis and D. Kletsas, Eur. J. Med.
Chem., 2007, 42, 344–350.
25. J. M. Andreu, B. Perez-Ramirez, M. J. Gorbunoff, D.
Ayala and S. N. Timasheff, Biochemistry-Us, 1998, 37,
8356–8368.
6. V. Verones, N. Flouquet, M. Lecoeur, A. Lemoine, A.
Farce, B. Baldeyrou, C. Mahieu, N. Wattez, A. Lansiaux,
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 15
Please do not adjust margins

Please do not adjust margins
ARTICLE
Journal Name
26. B. Perez-Ramirez, M. J. Gorbunoff and S. N. Timasheff,
Biochemistry-Us, 1998, 37, 1646–1661.
27. R. Islam, M. I. Hossain, Y. Okamoto, T. Nagamatsu, K.
Anraku and T. Okawara, Heterocycles, 2014, 89, 693–
708.
28. C. A. Herdman, L. Devkota, C. Lin, H. Niu, T. E.
Strecker, R. Lopez, L. Liu, C. S. George, R. P. Tanpure,
E. Hamel, D. J. Chaplin, R. P. Mason, M. L. Trawick and
K. G. Pinney, Bioorg. Med. Chem., 2015, 23, 7497–7520.
29. Y. Yin, F. Qiao, L. Jiang, S. Wang, S. Sha, X. Wu, P. Lv
and H. Zhu, Bioorg. Med. Chem., 2014, 22, 4285–4292.
30. S. Wang, X. Wang, B. Qin, E. Ohkoshi, K. Hsieh, E.
Hamel, M. Cui, D. Zhu, M. Goto, S. L. Morris-Natschke,
K. Lee and L. Xie, Bioorg. Med. Chem., 2015, 23, 5740–
5747.
31. M. Yang, Y. Qin, C. Chen, Y. Zhang, B. Li, T. Liu, H.
Gong, B. Wang and H. Zhu, RSC Adv., 2016, 6, 30412–
30424.
32. R. Mahesh, V. L. Nayak, K. S. Babu, S. Riyaz, T. B.
Shaik, G. B. Kumar, P. L. Mallipeddi, C. R. Reddy, K. C.
Shekar, J. Jose, N. Nagesh and A. Kamal,
ChemMedChem, 2017, 12, 678–700.
33. C. Kanthou, O. Greco, A. Stratford, I. Cook, R. Knight
and O. Benzakour, Am. J. Pathol., 2004, 165, 1401–1411.
34. M. Athar, M. Yousuf Lone, M. K. V, A. Radadiya, A.
Shah and C. J. P, Comb. Chem. High Throughput Screen,
2017.
35. P. Ranjan, S. P. Kumar, V. Kari and P. C. Jha, Comput.
Biol. Chem., 2017, 68, 78–91.
36. G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner, R.
K. Belew, D. S. Goodsell and A. J. Olson, J. Comput.
Chem., 2009, 30, 2785–2791.
16 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Journal Name
ARTICLE
Abstract
Design, synthesis and biological evaluation of 2-
phenylquinoline-4-carboxamide derivatives as a
new class of tubulin polymerization inhibitors
Kaixiu Luoa‡, Li Zhua‡, Ke Lib‡, Yi Jin*a and Jun
Lin*a
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 17