Design, synthesis, and antitumor evaluation of quinoline-imidazole derivatives

Article abstract of DOI:10.1002/ardp.201700407

A series of compounds bearing quinoline-imidazole (8a–e, 9a–e, 10a–e, 11a–e, and 12a–e) not reported previously were designed and synthesized. The target compounds were evaluated for antitumor activity against A549, PC-3, HepG2, and MCF-7 cells by the MTT method, with NVP-BEZ235 being the positive control. Most compounds showed moderate activity and compound 12a showed the best activity against HepG2, A549, and PC-3 cells, with half-maximal inhibitory concentration (IC₅₀) values of 2.42 ± 1.02 μM, 6.29 ± 0.99 μM, and 5.11 ± 1.00 μM, respectively, which was equal to NVP-BEZ235 (0.54 ± 0.13 μM, 0.36 ± 0.06 μM, 0.20 ± 0.01 μM). Besides, the IC₅₀ value of 12a against the cell line WI-38 (human fetal lung fibroblasts) was 32.8 ± 1.23 μM, indicating that the target compounds were selective for cancer cells. So, 11a and 12a were evaluated against PI3Kα and mTOR to find out if the compounds acted through the PI3K-Akt-mTOR signal transduction pathway. The inhibition ratios to PI3Kα and mTOR were slightly lower than that of NVP-BEZ235, suggesting there may be some other mechanisms of action. The structure–activity relationships and docking study of 11a and 12a revealed that the latter was superior. Moreover, the target compounds showed better in vitro anticancer activity when the C-6 of the quinoline ring was replaced by a bromine atom.

Full text of DOI:10.1002/ardp.201700407

Received: 28 December 2017
DOI: 10.1002/ardp.201700407
Revised: 25 March 2018
Accepted: 4 April 2018
|
|
FULL PAPER
Design, synthesis, and antitumor evaluation of
quinoline-imidazole derivatives
1
1
2,3
1
1
|
|
|
|
|
Zhen Xiao
Fei Lei
Wufu Zhu
Xiuying Chen
Xiaolei Wang
Lujie Cao
1
1
1
|
|
Kejun Ye
Shan Xu
1
Jiangxi Provincial Key Laboratory of Drug
Design and Evaluation, School of Pharmacy,
Jiangxi Science and Technology Normal
University, Nanchang, China
Abstract
A series of compounds bearing quinoline-imidazole (8a–e, 9a–e, 10a–e, 11a–e, and
2a–e) not reported previously were designed and synthesized. The target compounds
1
2
College of Pharmaceutical Sciences,
Zhengzhou University, Zhengzhou, China
were evaluated for antitumor activity against A549, PC-3, HepG2, and MCF-7 cells by
the MTT method, with NVP-BEZ235 being the positive control. Most compounds
showed moderate activity and compound 12a showed the best activity against HepG2,
A549, and PC-3 cells, with half-maximal inhibitory concentration (IC50) values of
3
Key Laboratory of Advanced Drug
Preparation Technologies, Zhengzhou
University, Zhengzhou, China
Correspondence
2
.42 ± 1.02 µM, 6.29 ± 0.99 µM, and 5.11 ± 1.00 µM, respectively, which was equal to
Dr. Wufu Zhu and Shan Xu, Jiangxi Provincial
Key Laboratory of Drug Design and
Evaluation, School of Pharmacy, Jiangxi
Science and Technology Normal University,
Nanchang 330013, China.
NVP-BEZ235 (0.54 ± 0.13 µM, 0.36 ± 0.06 µM, 0.20 ± 0.01 µM). Besides, the IC50
value of 12a against the cell line WI-38 (human fetal lung fibroblasts) was
3
2.8 ± 1.23 µM, indicating that the target compounds were selective for cancer cells.
Email: zhuwf@jxstnu.edu.cn (W.Z.);
xush@jxstnu.edu.cn (S.X.)
So, 11a and 12a were evaluated against PI3Kα and mTOR to find out if the compounds
acted through the PI3K-Akt-mTOR signal transduction pathway. The inhibition ratios
to PI3Kα and mTOR were slightly lower than that of NVP-BEZ235, suggesting there
may be some other mechanisms of action. The structure–activity relationships and
docking study of 11a and 12a revealed that the latter was superior. Moreover, the
target compounds showed better in vitro anticancer activity when the C-6 of the
quinoline ring was replaced by a bromine atom.
Funding information
The National Natural Science Funds,
Grant number: 81460527; The Project
Supported by Natural Science Foundation of
Jiangxi, China, Grant numbers:
20171BAB215073, 20171BAB205101;
Natural Science Funds for Distinguished
Young Scholar of Jiangxi Province, China,
Grant number: 20171BCB23078; Key
projects of the youth fund, Natural Science
Funds of Jiangxi Province, Grant number:
K E Y W O R D S
cytotoxic activity, docking study, mTOR, PI3Kα, quinoline-imidazole
20171ACB21052; Jiangxi Provincial Key
Laboratory of Drug Design and Evaluation,
Grant number: 20171BCD40015
1
|
INTRODUCTION
number of studies have been reported on PI3K/mTOR dual inhibitors.
In contrast to single target small molecule inhibitors, the inhibitors
The signal pathway of PI3K (phosphoinositide 3-kinase)-Akt (protein
kinase B)-mTOR (rapamycin target protein) is one of the hotspots in
acting on both PI3K and mTOR targets inhibit cell proliferation and
promote cell apoptosis, and have other advantages such as containing
a small dose, high titer, and difficult to produce drug resistance.
Indeed, many compounds which contain quinoline-imidazolone
such as NVP-BBD130, NVP-BEZ235 exhibit good biological activity,
and these adduced compounds are representative ones that impede
PI3K-AKT-mTOR signaling pathway (the structures of them are shown
[1]
the study of therapeutic drugs. PI3K-Akt-mTOR signal transduction
pathway plays an important role in tumor cell cycle regulation,
apoptosis, transcriptions, translations, metabolisms and angiogenesis,
protein synthesis, and many other important biological processes. A
[
2–6]
Zhen Xiao and Fei Lei made equal contributions to this work.
in Figure 1).
In addition, it is reported that NVP-BEZ235 inhibits
|
1 of 11
Arch Pharm Chem Life Sci. 2018;e1700407.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
https://doi.org/10.1002/ardp.201700407

2
of 11
XIAO ET AL.
|
FIGURE 1 Structures of representative compounds (NVP-BEZ235, upadacitinib) containing quinoline-imidazolone moiety
mTOR (p70S6K) with an IC50 of 6 nM. And the IC50 against other
the nitrogen atom of imidazole made the fixed electron pair of the
nitrogen atom faced outwards and might not bind to the residue of
the corresponding protein. So in order to enhance the activity of the
nitrogen atom, on the one hand, we discarded the ketone and the
methyl group on nitrogen atom. On the other hand, we added methyl
at C-2 positions of imidazole to increase the electron cloud density of
the imidazole. But the new acquired compounds 9a–e, 10a–e were still
unsatisfactory in cytotoxic activity. In order to find out the reason of
affecting the activity, we tried to decrease the electron cloud density
of the quinoline ring. So the electron-donating methyl group at C-2
positions was removed and electron-withdrawing bromine atom
instead of electron-donating methoxy groups was introduced at C-6
positions to get compounds 11a–e, 12a–e. In summary, we designed
25 compounds to study the effect of quinoline-imidazole derivatives
on the anticancer activity. Synthetic routes are showed in Scheme 1.
Herein, we disclosed the synthesis and antitumor activity of all the
target compounds against the A549 (human lung cancer), PC-3 (human
prostatic cancer), HepG2 (human liver cancer), and MCF-7 (human
breast cancer) cancer cell lines, as well as the action against PI3Kα and
mTOR kinases. We also took experiment against healthy cell WI-38 to
verify the selectivity for cancer cells. In addition, we introduced the
docking studies results.
[7,8]
serotonin kinases ATRs in the cells was 21 nM.
Nevertheless,
inducing apoptosis function of NVP-BEZ23 is weak, and NVP-BEZ23
can trigger self-protection autophagy of tumor cells. As a result, NVP-
BEZ235 was often used in combination with other drugs for the
[9,10]
treatment of cancer.
Another disadvantage is that NVP-BEZ235
inhibits the biological activity of other drugs in some cases. So there is
an urgent need to develop compounds that overcome these short-
comings. Moreover, upadacitinib, which has a similar structure, is a
Janus kinase (JAK) inhibitor being developed for the treatment of
[11]
rheumatoid arthritis (RA) and other inflammatory diseases.
Upadacitinib is currently being evaluated in phase III trials for the
treatment of RA and PsA, and in phase II studies in Crohn's disease,
atopic dermatitis, and ulcerative colitis. From these, we can see that
the quinoline imidazole ring is a pharmacologically effective structure.
Based on the above findings, we planned to synthesize
compounds that impede PI3K-AKT-mTOR signaling pathway, so we
retained quinoline-imidazole moiety, the valid fragment of NVP-
BEZ235 in the derivative's design. In the early stage of our experiment,
we modified the C-6 position of the core quinoline ring by substituting
larger molecular weight groups. However, the effects were poor in the
anti-tumor cell activity test due to the poor solubility of the obtained
compounds (molecular structure was too rigid). Hence, we designed
the compounds 8a–e, in which methoxy group was introduced at the
C-6 positions of the quinoline ring rather than quinoline group to
increase density of the quinoline and reduce rigidity (Figure 2).
Nevertheless, quinoline-imidazolos compounds 8a–e almost had no
inhibitory activity against all test cell lines. At the beginning, inspired by
upadacitinib, we held the view that the ketone on imidazole had an
effect on the activity of the nitrogen atom. And the methyl group on
2
2
|
RESULTS AND DISCUSSION
Chemistry
.1
|
The preparation of target compounds 8a–e, 9a–e, 10a–e, 11a–e, and
12a–e is described in Scheme 1. In the beginning, we synthesized the
key intermediates 7a–j, which were used as the starting material to yield

XIAO ET AL.
| 3 of 11
FIGURE 2 The design of target compounds
target compounds. Compounds 8a–e, 9a–e, 10a–e were synthesized
from 7a–e, and 11a–e, 12a–e from 5f–j. Compounds 7a–e were
methylated with methyl iodide under the action of sodium hydride to
give the desired compounds 8a–e. And after cyclization reaction of 7a–j
with triethylorthoformate heated at 120°C, compounds 9a–e and 12a–
e were obtained after 2.5 h. The key intermediates 7a–e as raw material
and 1,4-dioxane as the solvent, the mixture was heated at 120°C and
refluxed with triethylorthoacetate. And the reaction was carried out for
groups had a significant impact on the activity. Compounds 8a–e
almost had no inhibitory activity, probably because carbonyl ketone on
the imidazole ring can affect the activity of the nitrogen atom. And the
methyl group on the nitrogen atom of imidazole made the fixed
electron pair of the nitrogen atom faced outwards and might not bind
to the residue of the corresponding protein. Indeed, compounds 9a–e
and 10a–e obtained through discarding the ketone and adding methyl
group on imidazole to increase the electron cloud density showed
better cytotoxic activity. But the result was still not satisfactory. We
found that the electron-donating methoxy group at C-6 position and
the electron-donating methyl group at C-2 position of quinoline ring
had negative impact on activity. In addition, compound 11a–e and
2
.5 h to obtain the compounds 10a–e; using the same method we
[12]
acquired 11a–e from 7f–j.
2
.2
|
Biological evaluation
In vitro cytotoxic activity against cancer cell
1
2a–e in which introduced electron-withdrawing bromine atom
instead of electron-donating methoxy group showed better activity
than 9a–e and 10a–e, confirming this inference. In addition, the methyl
group on the imidazole ring also affected cytotoxic activity passively.
When the R group was cyano, the compounds exhibited selective
inhibition of the proliferation to different cancer cell lines. Changing
the cyano group to others like methoxy, bromine or other halogen
groups was not a wise choice. In summary, the imidazole ring in these
series of quinolino-imidazole derivatives was best kept in its original
form without substituents on it, while the quinoline ring should have
electron-withdrawing groups to increase its cytotoxic activity. At the
same time, the R groups should probably be the same as the lead
compound NVP-BEZ235, with cyano group being the best choice.
2
.2.1
|
lines HepG2, A549, PC-3, and MCF-7
The cytotoxic activities of the target compounds were evaluated in
parallel against four human cancer cell lines (HepG2, A549, PC-3, and
MCF-7) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay. Compounds NVP-BEZ235 and sorafenib were
reference compounds. Results were summarized in Table 1 and the
values were the average of at least two independent experiments.
As showed in Table 1, all the compounds showed higher inhibitory
activity on cell line HepG2 and A549 than that of cell line PC-3 and
MCF-7. Among these, the compounds 11a, 11b, 12a, and 12b showed
good inhibition to cancer cell proliferation; 11a and 12a had the better
inhibitory effect on the proliferation of human hepatoma cell line
HepG2, and had a good effect on the proliferation of cell line A549 and
cell line PC-3, but had no sensitivity to cell line MCF-7. Compound 12a
represented the best cytotoxic activity against HepG2, A549, and PC-
2
.2.2 In vitro cytotoxic activity against healthy cell
|
line WI-38
The activities against healthy cell line WI-38 of 11a, 11c, 12a–12c are
expressed in Table 2. According to Table 2, the selected five
compounds had almost no toxicity to healthy cell line WI-38. The
3
cell lines, with IC50 values of 2.42 ± 1.02 µM, 6.29 ± 0.99 µM, and
.11 ± 1.00 µM, respectively. Moreover, the different substituent
5

4
of 11
XIAO ET AL.
|
SCHEME 1 Synthetic routes to the target compounds
IC50 values of 11c, 12b, and 12c against cell line WI-38 were greater
than 50 µM, the IC50 values of 11a, 12a were 44.6 ± 2.04 µM and
seen from Table 3 that the activity of 12a was better than that of 11a,
but the inhibitions of PI3Kα and mTOR were slightly lower than that of
lead compound NVP-BEZ235, indicating that there may be other
mechanism of action.
3
2.8 ± 1.23 µM, respectively, which indicated that cytotoxicity against
cancer cell lines was not simply toxicity. And these quinoline-imidazole
derivatives had selectivity for cancer cells.
2.3
|
Molecular docking study
2
.2.3 Activity against PI3Kα/mTOR kinase of
|
In this paper, we selected compounds 11a and 12a as ligand examples,
the docking of the compounds with the p110a protein molecule (PDB:
1E8X) and the mTOR protein molecule (PDB: 4JT6) were scored. The
results of the docking scores are shown in Table 4. In the binding mode,
compounds 11a and 12a
We further evaluated compounds 11a and 12a for the PI3K and mTOR
kinase inhibitory activity. The results are expressed in Table 3. It can be

XIAO ET AL.
| 5 of 11
TABLE 1 The structures and cytotoxicity of compounds 6a–e, 7a–e, 8a–e, 9a–e, and 10a–e
a
IC50 (μM)
Compounds
R
HepG2
A549
PC-3
MCF-7
8
8
8
8
8
9
9
9
9
9
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
a
Isobutyronitrile
-OCH3
11.31 ± 1.30
>50
39.66 ± 0.89
30.16 ± 0.89
>50
43.07 ± 1.43
>50
32.52 ± 1.51
>50
b
c
-Br
>50
>50
>50
d
-3-Cl-4-F
-4-Br-2-F
Isobutyronitrile
-OCH3
>50
>50
>50
>50
e
>50
>50
>50
>50
a
>50
>50
>50
>50
b
68.54 ± 1.05
>50
58.74 ± 0.94
>50
73.73 ± 1.16
>50
>50
c
-Br
>50
d
-3-Cl-4-F
-4-Br-2-F
Isobutyronitrile
-OCH3
>50
>50
17.60 ± 0.88
21.62 ± 3.89
>50
>50
e
11.82 ± 1.12
>50
28.03 ± 2.03
>50
43.29 ± 1.63
3.99 ± 0.60
31.03 ± 1.49
>50
0a
0b
0c
0d
0e
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
15.54 ± 2.12
>50
14.04 ± 1.18
>50
16.54 ± 1.10
11.13 ± 0.84
>50
-Br
-3-Cl-4-F
-4-Br-2-F
Isobutyronitrile
-OCH3
10.71 ± 1.14
6.08 ± 1.04
1.43 ± 1.02
5.81 ± 1.04
33.39 ± 1.43
4.83 ± 1.07
15.66 ± 1.05
2.42 ± 1.02
16.37 ± 1.11
>50
>50
26.58 ± 1.42
11.86 ± 1.07
>50
11.23 ± 1.02
13.43 ± 0.92
4.67 ± 1.44
>50
15.36 ± 1.54
6.67 ± 0.99
10.81 ± 1.21
31.68 ± 1.00
12.89 ± 0.98
31.79 ± 2.14
5.11 ± 1.00
13.20 ± 2.12
31.74 ± 0.89
32.61 ± 4.60
33.94 ± 1.02
0.20 ± 0.01
3.03 ± 0.11
6.21 ± 0.79
46.67 ± 1.66
16.17 ± 1.20
18.51 ± 1.26
>50
-Br
-3-Cl-4-F
-4-Br-2-F
Isobutyronitrile
-OCH3
3.93 ± 0.93
7.68 ± 0.88
6.29 ± 0.99
5.36 ± 1.02
>50
4.15 ± 0.62
>50
-Br
-3-Cl-4-F
-4-Br-2-F
–
13.68 ± 1.02
28.44 ± 1.44
0.54 ± 0.13
3.97 ± 0.13
5.53 ± 1.23
36.38 ± 0.98
0.36 ± 0.06
6.53 ± 0.23
19.01 ± 1.27
>50
b
BEZ235
0.14 ± 0.01
4.21 ± 0.15
b
Sorafenib
–
a
The values are an average of two separate determinations.
b
Used as a positive control.
the overall fractions of the compounds were lower than that of the lead
key H-bond interaction with the backbone of the conserved PI3Kα
Val851 (Val882 in g). The other two hydrogen bonds’ interactions
(dotted lines) involved the backbone of the conserved Asp933
(Asp964 in PI3Kα) and the conserved Ser774 (Ser806 in PI3Kα) side
chain. These H-bond interactions were complemented by favorable
van der Waals contacts with conserved hydrophobic residues of the
ATP catalytic site, including Met772 (Met804 of PI3Kα), Tyr836
compound NVP-BEZ235. Compound 12a scored slightly higher than
1
1a. The results of this docking were consistent with the antitumor
activity in vitro.
At the beginning, we have searched out the binding mode of NVP-
BEZ235 docked in the catalytic site of PI3Ka. In the structure of NVP-
BEZ235, the nitrogen atom of the core quinolone ring was involved in a

6
of 11
XIAO ET AL.
|
TABLE 2 Activity against healthy cell line WI-38 of 11a, 11c,
2a–12c
Compounds
TABLE 4 The docking scores of compounds with PI3K/mTOR
inhibitory
1
a
R
IC50 (µM)
Molecular mode score
1
1
1
1
1
1a
1c
2a
2b
2c
Isobutyronitrile
-Br
44.6 ± 2.04
>50
Compound
PI3K (p110α PDB:1e8x)
mTOR (PDB:4JT6)
1
1
1a
2a
5.20
5.07
7.87
5.51
6.23
7.05
Isobutyronitrile
-OCH3
32.8 ± 1.23
>50
NVP-BEZ235
-Br
>50
a
The values are an average of two separate determinations.
showed moderate activity, and compounds 11a and 12a with good
antitumor activity were screened out. The anticancer activity of the
compounds 11a and 12a was equal to the leading compound NVP-
BEZ235. Analysis of the test results revealed that the introduction of
the electron donor methoxy group at the C-6 position of the quinoline
ring showed a significant decrease in the antitumor activity compared
to the introduction of the electron withdrawing bromine atom. The
introduction of different substituents on the quinoline ring C-4 aniline
had a certain effect on the activity of the compounds. And the anti-
healthy cell line WI-38 experiment showed that target compounds had
almost no effect on healthy cells. These compounds were selective for
cancer cells. In order to further study its mechanism, we used NVCE-
Ultra enzyme activity evaluation method, NVP-BEZ235, PI103 as
positive control drugs, to test the inhibition activity of compounds 11a
and 12a to PI3Kα and mTOR enzyme. The inhibition of PI3Kα and
mTOR was lower than that of lead compound NVP-BEZ235 and
positive control drug PI103, indicating that there may be some other
mechanism of action. Further studies on the binding of compounds
11a and 12a to PI3K/mTOR protein kinases were carried out. The best
compound in vitro antitumor activity 12a scored higher than 11a. The
results predicted that the in vitro antitumor activity of the molecule
was related to the hydrogen bond formed by the docking of the
molecules and the type of amino acid residue.
(
Tyr837 of PI3Kα), and Met922 (Tyr953 of PI3Kα). To explore the
binding modes of target compounds with the active site of PI3K and
mTOR, molecular docking simulation studies were carried out by using
AutoDock Vina v1.02 and analyzed by Discover Studio 2.5 Visualiza-
tion. The molecular docking diagrams are shown in Figure 3.
It can be seen from the PI3K/mTOR protein kinase binding modes
of both compounds 11a and 12a that although all designed synthetic
derivatives were mated with the mTOR protein to form the same
amount of hydrogen bonds, the amino acid species associated with
their hydrogen bonds was different. The derivatives’ molecules formed
hydrogen bonds with the valine residue Val2240, and the lead
compound binds to the serine residue Ser2165 with hydrogen bonds.
At the same time, compared 11a with 12a, the amino acid residues of
hydrogen bond's binding with the PI3K ligand protein were same, but
the number of hydrogen bonds less than the lead compound. In this
paper, the overall activity of target compounds was lower than that of
the lead compound NVP-BEZ235, the reason may be related to the
number of hydrogen bonds bound to the PI3K/mTOR protein and the
type of amino acid residues. The results obtained by the above-
mentioned molecular docking and the docking pattern were consistent
with the result of in vitro antitumor activity.
3
|
CONCLUSION
In this paper, we designed and synthesized quinoline-imidazole
derivatives, a total of 25 compounds, and the anticancer activity of
all the compounds were evaluated by MTT method. Most compounds
TABLE
3
Activity against PI3Kα/mTOR kinase of selected
compounds
a
IC50 (µM)
Compounds
PI3Kα
mTOR
1
1
1a
2a
6.8
4.6
0.9
1.4
b
NVP-BEZ235
0.004 ± 0.002
0.011 ± 0.002
0.006 ± 0.003
0.019 ± 0.002
b
FIGURE 3 Binding modes of compounds 11a, 12a with PI3K/
mTOR (A and B show the binding pattern of 11a, 12a to the PI3K
protein kinase; C and D show the pattern of binding of 11a, 12a to
the mTOR protein kinase)
PI103
a
The values are an average of two separate determinations.
b
Used as positive controls.

XIAO ET AL.
| 7 of 11
4
4
4
|
EXPERIMENTAL
Chemistry
1.644 mol) were added to a 500 mL three-necked flask and heated
to 110°C. Six drops of DMF were added dropwise and then reacted for
2 h. TLC (petroleum ether/ethyl acetate 3:1) was used to test whether
the reaction was completed. Cooled down the reaction, and
concentrated most of the phosphorus oxychloride. Then added
.1
|
.1.1 General
|
5
00 mL of ice saturated sodium bicarbonate solution and extracted
All melting points were obtained on a Büchi Melting Point B-540
apparatus (Büchi Labortechnik, Flawil, Switzerland) and were uncor-
rected. NMR spectra were performed using Bruker 400 MHz
spectrometers (Bruker Bioscience, Billerica, MA, USA) with TMS as
an internal standard. Mass spectra (MS) were taken in electrospray
ionization (ESI) mode on Agilent 1100 liquid chromatography-mass
spectrometry (LC-MS) (Agilent, Palo Alto, CA, USA). Thin layer
chromatography (TLC) analysis was carried out on silica gel plates
GF254 (Qindao Haiyang Chemical, Qingdao, China). All materials were
obtained from commercial suppliers and used without purification,
unless otherwise specified. Yields were optimized.
it three times with ethyl acetate. The extract was concentrated to dry;
finally we got yellow solid. The yield reached 50%.
Compounds 6a–j were synthesized according to the reported
[12]
procedures by our research group.
The compounds 5a,b reacted with a variety of substituted anilines
in the condition of propionic acid with room temperature; then reacted
for 2 h. The solutions were used for the next step without further
purification.
4.1.3 The general procedure for the preparation of
|
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
compounds 7a–j
Compounds 7a–j were synthesized according to the reported
[
13]
procedures by our research group.
added to hydrazine hydrate and ethanol, respectively, and heated to
0°C, using FeCl as catalyst, then we acquired 7a–j at a moderate
yield.
The solutions of 6a–j were
4
.1.2 The general procedure for the preparation of
|
8
3
compounds 3a,b, 4a,b, 5a,b, 6a–j, 7a–j
Compound 3a was synthesized in accordance with the reported
[12]
procedures by our research group.
30.05 g, 0.244 mol), ethyl acetoacetate (317.54 g, 2.44 mol), and
polyphosphoric acid (PPA) (82.45 g, 0.244 mol) was added into a
A mixture of p-methoxyaniline
4
.1.4
target compounds 8a–e, 9a–e, 10a–e, 11a–e, and
2a–e
|
General procedure for the preparation of
(
1
1
6
000 mL three-necked flask and heated to 190°C. Reaction stayed 5–
h. TLC (dichloromethane/methanol 15:1) was used to test whether
The key intermediates 7a–e, as the starting materials, were methylated
with methyl iodide under the action of sodium hydride to give the
desired compounds 8a–e. The key intermediates 7a–e and triethylor-
thoformate passed the cyclization reaction under the condition of
120°C refluxing with 1,4-dioxane being solvent. Then after 2.5 h, we
gained compounds 9a–e. The key intermediates 7a–e and triethylor-
thoformate were cyclized by refluxing at 120°C and 1,4-dioxane was
used as a solvent. The reaction was carried out for 2.5 h to obtain the
compounds 10a–e. And using the same method we acquired 11a–e
from 7f–j. Finally, compounds 12a–e were obtained by refluxing with
1,4-dioxane solvent through cyclization of key intermediate 7f–j and
triethylorthoformate at 120°C over 2.5 h.
the reaction was completed, and then cooled to 70°C (PPA will
condense into a paste when the temperature is too low, which is not
conducive to post-processing), added 1000 mL of saturated sodium
carbonate solution and stirred. After solidification, we got solid
precipitation. The residue cake was filtered and dried to give a gray
solid 2-methyl-4-hydroxy-6-methoxyquinoline 32 g in 70% yield.
Compound 3b was synthesized by using a CEM microwave
reactor, settings were T = 200°C, t = 10 min, µλ = 100 W, P = 150 PSI.
After the microwave instrument stopped running, the reaction of raw
materials was completed and no impurities were generated. The yield
reached 83.5%.
Compounds 4a,b were synthesized according to the reported
[12]
procedures by our research group.
Compound 3a or 3b (29.90 or
2-(4-(8-Methoxy-3,4-dimethyl-2-oxo-2,3-dihydro-1H-
3
5.40 g, 0.158 mol) was added to a 1000 mL three-necked flask
imidazo[4,5-c]quinolin-1-yl)phenyl)-2-methylpropanenitrile (8a)
equipped with 500 mL of propionic acid and heated to 125°C. After
stirring for 1 h, nitric acid (50.02 g, 0.794 mol) was added dropwise to
A light yellow solid; C23H22N O ; molecular weight: 386.5; m.p. 280.4–
4
+
2
1
281.5°C; ESI-MS m/z: [M+H] : 386.1; H NMR (400 MHz, DMSO-d ) δ
6
2
0 mL of propionic acid. The mixture was stirred in a flask for 2 h. TLC
7.99 (s, 1H), 7.98–7.93 (m, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.27 (dd, J = 9.2,
(
dichloromethane/methanol 15:1) tested the reaction. The reaction was
2.7 Hz, 1H), 6.21 (d, J = 2.6 Hz, 1H), 3.89 (s, 3H), 3.45 (s, 3H), 3.08 (s, 3H),
then cooled and filtered. The resulting cake was added to ice saturated
sodium bicarbonate solution (500 mL) and stirred for 1 h. The filter cake
was re-filtered and dried to give a yellow solid in 50% yield.
1.93 (s, 6H). Purity: 95.90% by HPLC (80:20 MeOH/H O).
2
8-Methoxy-1-(4-methoxyphenyl)-3,4-dimethyl-1,3-dihydro-2H-
imidazo[4,5-c]quinolin-2-one (8b)
Compounds 5a,b were synthesized according to the reported
[12]
procedures by our research group.
The compound 4a or 4b (18.03
A light yellow solid; C20H19N O ; molecular weight: 349.4; m.p.
3
3
+
1
or 20.72 g, 0.077 mol) and phosphorus oxychloride (252.06 g,
160.2–161.6°C; ESI-MS m/z: [M+H] : 349.1; H NMR (400 MHz,

8
of 11
XIAO ET AL.
|
−
1
DMSO-d
6
) δ 7.95 (d, J = 9.0 Hz, 1H), 7.63 (d, J = 8.2 Hz, 2H), 7.35 (d,
100.15, 55.70, 54.71, 19.99. IR (KBr) cm : 3102, 3062, 3048, 3011,
2964, 2935, 2833, 1259, 1209.
J = 8.2 Hz, 2H), 7.28 (d, J = 7.6 Hz, 1H), 6.36 (s, 1H), 4.01 (s, 3H), 3.86 (s,
3
H), 3.53 (s, 3H), 3.07 (s, 3H). Purity: 96.10% by HPLC (80:20 MeOH/
−1
H
2
O). IR (KBr) cm : 3110, 3091, 3044, 3014, 3005, 2951, 2925, 2853,
1-(4-Bromophenyl)-8-methoxy-4-methyl-1H-imidazo[4,5-c]-
1
717, 1268, 1226.
quinoline (9c)
A light yellow solid; C18
180.4–181.3°C; ESI-MS m/z: [M+H] : 368.1; H NMR (400 MHz,
DMSO-d ) δ 8.49 (s, 1H), 7.96 (dd, J = 13.8, 8.9 Hz, 3H), 7.73 (d,
J = 8.5 Hz, 2H), 7.27 (dd, J = 9.2, 2.7 Hz, 1H), 6.63 (d, J = 2.7 Hz, 1H),
3.55 (s, 3H), 2.85 (s, 3H). Purity: 97.60% by HPLC (80:20 MeOH/H O).
3
H14BrN O; molecular weight: 368.2; m.p.
+
1
1
-(4-Bromophenyl)-8-methoxy-3,4-dimethyl-1,3-dihydro-2H-
imidazo[4,5-c]quinolin-2-one (8c)
3 2
A light yellow solid; C19 O ; molecular weight: 398.3; m.p.
6
H
16BrN
+
1
1
80.6–181.8°C; ESI-MS m/z: [M+H] : 382.1; H NMR (400 MHz,
2
6
DMSO-d ) δ 7.85 (d, J = 8.9 Hz, 1H), 7.53 (d, J = 8.4 Hz, 2H), 7.15 (d,
J = 7.1 Hz, 1H), 7.01 (d, J = 8.6 Hz, 2H), 6.64 (s, 1H), 4.21 (s, 3H), 3.82 (s,
H), 3.07 (s, 3H). Purity: 98.90% by HPLC (80:20 MeOH/H O).
1-(3-Chloro-4-fluorophenyl)-8-methoxy-4-methyl-1H-imidazo
3
2
[4,5-c]quinolone (9d)
A light yellow solid; C18
160.8–161.2°C; ESI-MS m/z: [M+H] : 341.1; H NMR (400 MHz,
DMSO-d ) δ 8.53 (s, 1H), 8.16 (dd, J = 6.5, 2.3 Hz, 1H), 8.00 (d,
3 2
H13ClFN O ; molecular weight: 357.8; m.p.
+
1
1
-(3-Chloro-4-fluorophenyl)-8-methoxy-3,4-dimethyl-1,3-
dihydro-2H-imidazo[4,5-c]quinolin-2-one (8d)
A light yellow solid; C19 ; molecular weight: 371.8; m.p.
6
H15ClFN
3
O
2
J = 9.2 Hz, 1H), 7.85–7.77 (m, 1H), 7.74 (t, J = 8.8 Hz, 1H), 7.32–7.21
(m, 1H), 6.57 (d, J = 2.5 Hz, 1H), 3.50 (s, 3H), 2.85 (s, 3H). Purity: 96.40%
+
1
1
90.3–191.6°C; ESI-MS m/z: [M+H] : 371.1; H NMR (400 MHz,
DMSO-d
6
) δ 8.17 (d, J = 9.3 Hz, 1H), 7.99 (d, J = 9.3 Hz, 1H), 7.92 (s,
2
by HPLC (80:20 MeOH/H O).
2
H), 7.34 (d, J = 8.3 Hz, 1H), 6.34 (s, 1H), 3.89 (s, 3H), 3.60 (s, 3H), 3.08
(
s, 3H). Purity: 98.70% by HPLC.
1-(4-Bromo-2-fluorophenyl)-8-methoxy-4-methyl-1H-imidazo
4,5-c]quinoline (9e)
A light yellow solid; C18
250.3–251.2°C; ESI-MS m/z: [M+H] : 386.1; H NMR (400 MHz,
DMSO-d ) δ 8.58 (s, 1H), 8.10 (d, J = 9.4 Hz, 1H), 8.01 (d, J = 9.2 Hz,
[
1
-(4-Bromo-2-fluorophenyl)-8-methoxy-3,4-dimethyl-1,3-
dihydro-2H-imidazo[4,5-c]quinolin-2-one (8e)
A light yellow solid; C19 ; molecular weight: 416.3; m.p.
3
H13BrFN O; molecular weight: 386.2; m.p.
+
1
H
15BrFN
3
O
2
6
+
1
2
70.4–271.8°C; ESI-MS m/z: [M+H] : 416.1; H NMR (400 MHz,
1H), 7.95–7.85 (m, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.32 (dd, J = 9.2, 2.3 Hz,
DMSO-d
6
) δ 8.18 (d, J = 4.4 Hz, 1H), 7.99 (d, J = 9.2 Hz, 1H), 7.89 (dd,
1H), 6.55 (s, 1H), 3.56 (s, 3H), 2.87 (s, 3H). Purity: 95.60% by HPLC
J = 17.6, 8.7 Hz, 1H), 7.34 (d, J = 9.4 Hz, 2H), 6.36 (d, J = 2). Purity:
6.60% by HPLC (80:20 MeOH/H O).
2
(80:20 MeOH/H O).
9
2
2-(4-(8-Methoxy-2,4-dimethyl-1H-imidazo[4,5-c]quinolin-1-yl)-
2
-(4-(8-Methoxy-4-methyl-1H-imidazo[4,5-c]quinolin-1-yl)-
phenyl)-2-methylpropanenitrile (9a)
A light yellow solid; C22 O; molecular weight: 356.5; m.p.
phenyl)-2-methylpropanenitrile (10a)
A light yellow solid; C23
H
22
N
4
O; molecular weight: 370.5; m.p. 197.3–
+
1
H
20
N
4
198.6°C; ESI-MS m/z: [M+H] : 370.1; H NMR (400 MHz, DMSO-d
6
) δ
+
1
2
09.4–210.7°C; ESI-MS m/z: [M+H] : 356.1; H NMR (400 MHz,
8.01 (dd, J = 8.7, 2.7 Hz, 3H), 7.89 (d, J = 8.3 Hz, 2H), 7.26 (dd, J = 9.1,
2.5 Hz, 1H), 6.27 (d, J = 2.4 Hz, 1H), 3.46 (s, 3H), 2.93 (s, 3H), 2.55 (s,
6
DMSO-d ) δ 8.67 (s, 1H), 8.12 (d, J = 9.1 Hz, 1H), 8.04 (d, J = 8.3 Hz,
2
H), 7.98 (d, J = 8.2 Hz, 2H), 7.43–7.35 (m,1H), 6.69 (s, 1H), 3.62
2
3H), 1.92 (s, 6H). Purity: 97.10% by HPLC (80:20 MeOH/H O).
(
s, 3H), 3.02 (s, 3H), 1.96 (s, 6H). Purity: 97.20% by HPLC (80:20
13
MeOH/H
2
O). C NMR (101 MHz, DMSO-d
6
) δ 156.02, 150.45,
8-Methoxy-1-(4-methoxyphenyl)-2,4-dimethyl-1H-imidazo[4,5-
1
43.58, 143.39, 139.30, 136.42, 135.73, 131.92, 130.80, 128.13
c]quinoline (10b)
(
(
2C), 126.76(2C), 124.27, 118.09, 117.06, 99.60, 54.50, 36.75, 28.15
A light yellow solid; C20
200.2–201.5°C; ESI-MS m/z: [M+H] : 333.1; H NMR (400 MHz,
DMSO-d ) δ 7.91 (d, J = 9.1 Hz, 1H), 7.59 (d, J = 8.8 Hz, 2H), 7.26 (d,
19 3 2
H N O ; molecular weight: 333.4; m.p.
−
1
+
1
2C), 20.08. IR (KBr) cm : 3067, 3051, 3028, 3001, 2981, 2957,
2
937, 2916, 2836, 2229, 1228.
6
J = 8.8 Hz, 2H), 7.18 (dd, J = 9.1, 2.7 Hz, 1H), 6.35 (d, J = 2.7 Hz, 1H),
8
-Methoxy-1-(4-methoxyphenyl)-4-methyl-1H-imidazo[4,5-c]-
3.90 (s, 3H), 3.45 (s, 3H), 2.81 (s, 3H), 2.40 (s, 3H). Purity: 97.80% by
quinoline (9b)
2
HPLC (80:20 MeOH/H O).
17 3 2
A light yellow solid; C19H N O ; molecular weight: 319.4; m.p.
+
1
2
16.2–217.8°C; ESI-MS m/z: [M+H] : 319.1; H NMR (400 MHz,
1-(4-Bromophenyl)-8-methoxy-2,4-dimethyl-1H-imidazo[4,5-c]-
DMSO-d
6
) δ7.87 (d, J = 8.4 Hz, 2H), 7.74 (t, J = 8.5 Hz, 1H), 7.26
quinoline (10c)
(
d, J = 9.4 Hz, 2H), 7.15 (dd, J = 9.2, 2.7 Hz, 1H), 7.0 (dd, J = 3.2 Hz, 1H),
A light yellow solid; C19
140.5–141.3°C; ESI-MS m/z: [M+H] : 382.1; H NMR (400 MHz,
DMSO-d ) δ 8.10 (dd, J = 15.8, 8.8 Hz, 3H), 7.85 (d, J = 8.5 Hz, 2H), 7.36
(dd, J = 9.2, 2.7 Hz, 1H), 6.44 (d, J = 2.7 Hz, 1H), 3.63 (s, 3H), 2.98 (s,
3H), 2.57 (s, 3H). Purity: 95.60% by HPLC (80:20 MeOH/H O).
3
H16BrN O; molecular weight: 382.3; m.p.
+
1
6
.23 (d, J = 2.5 Hz, 1H), 3.44 (s, 3H), 2.65 (s, 3H), 2.52 (d, J = 11.1 Hz,
1
3
3
H). Purity: 97.70% by HPLC (80:20 MeOH/H2O).
) δ 160.14, 155.97, 150.44, 144.02, 139.00,
36.22, 132.14, 130.59, 128.79 (3C), 117.53, 117.24, 114.86(2C),
C NMR
6
(
101 MHz, DMSO-d
6
1
2

XIAO ET AL.
| 9 of 11
1
-(3-Chloro-4-fluorophenyl)-8-methoxy-2,4-dimethyl-1H-
imidazo[4,5-c]quinoline (10d)
A light yellow solid; C19
8-Bromo-1-(4-bromo-2-fluorophenyl)-2-methyl-1H-imidazo
[4,5-c]quinoline (11e)
H
15ClFN
3
O; molecular weight: 355.8; m.p.
2 3
A light yellow solid; C17H10Br FN ; molecular weight: 435.1; m.p.
+
1
+
1
1
60.6–161.8°C; ESI-MS m/z: [M+H] : 355.0; H NMR (400 MHz,
165.7–166.2°C; ESI-MS m/z: [M+H] : 435.1; H NMR (400 MHz,
DMSO-d ) δ 9.27 (s, 1H), 8.19 (d, J = 11.2 Hz, 1H), 8.07 (d, J = 10.2 Hz,
1H), 7.92 (d, J = 15.2 Hz, 2H), 7.78 (d, J = 9.8 Hz, 1H), 7.18 (s, 1H), 2.43
(s, 3H). Purity: 95.90% by HPLC (80:20 MeOH/H O).
DMSO-d
6
) δ 8.20 (d, J = 6.5 Hz, 1H), 7.98 (d, J = 9.2 Hz, 1H), 7.84 (d,
6
J = 6.9 Hz, 2H), 7.25 (dd, J = 9.2, 2.5 Hz, 1H), 6.31 (d, J = 2.6 Hz, 1H),
.50 (s, 3H), 2.85 (s, 3H), 2.44 (s, 3H). Purity: 96.30% by HPLC (80:20
MeOH/H O).
3
2
2
2-(4-(8-Bromo-1H-imidazo[4,5-c]quinolin-1-yl)phenyl)-2-
1
-(4-Bromo-2-fluorophenyl)-8-methoxy-2,4-dimethyl-1H-
imidazo[4,5-c]quinoline (10e)
A light yellow solid; C19
methylpropanenitrile (12a)
A light yellow solid; C20
H15BrN
4
; molecular weight: 391.3; m.p. 202.3–
+
1
H
15BrFN
3
O; molecular weight: 400.3; m.p.
203.5°C; ESI-MS m/z: [M+H] : 391.1; H NMR (400 MHz, DMSO-d
6
) δ
+
1
1
68.1–169.5°C; ESI-MS m/z: [M+H] : 399.0; H NMR (400 MHz,
9.49 (s, 1H), 8.76 (d, J = 9.5 Hz, 1H), 8.23 (d, J = 8.9 Hz, 1H), 8.04 (d,
J = 8.4 Hz, 2H), 7.99 (d, J = 8.4 Hz, 2H), 7.96–7.88 (m, 1H), 7.49 (d,
J = 13.8 Hz, 1H), 1.96 (s, 6H). Purity: 96.80% by HPLC (80:20 MeOH/
6
DMSO-d ) δ 8.22 (d, J = 9.3 Hz, 1H), 8.07–7.97 (m, 2H), 7.95 (d,
J = 8.4 Hz, 1H), 7.33 (d, J = 9.1 Hz, 1H), 6.41 (s, 1H), 3.61 (s, 3H), 2.92 (s,
H), 2.53 (s, 3H). Purity: 96.90% by HPLC (80:20 MeOH/H O).
1
3
3
2
H
2
O). C NMR (101 MHz, DMSO-d
44.28, 140.26, 136.60, 132.83, 131.72, 129.08 (2C), 127.69 (2C),
125.26, 119.04, 117.94, 100.51, 55.43, 37.69, 29.08 (2C). IR (KBr)
6
) δ 156.90, 151.41, 144.53,
1
2
-(4-(8-Bromo-2-methyl-1H-imidazo[4,5-c]quinolin-1-yl)-
−1
phenyl)-2-methylpropanenitrile (11a)
cm : 3108, 3074, 2974, 2956, 2918, 2827, 2238, 1624, 1481, 1459,
1401.
A light yellow solid; C21
H
17BrN
+
4
; molecular weight: 405.3; m.p. 150.5–
1
1
8
7
2
51.6°C; ESI-MS m/z: [M+H] : 405.1; H NMR (400 MHz, DMSO-d
6
) δ
.63 (d, J = 9.6 Hz, 1H), 8.10 (d, J = 8.9 Hz, 1H), 7.90 (t, J = 9.0 Hz, 2H),
.87–7.81 (m, 2H), 7.78 (d, J = 7.3 Hz, 1H), 7.36 (d, J = 13.8 Hz, 1H),
8-Bromo-1-(4-methoxyphenyl)-1H-imidazo[4,5-c]quinoline
(12b)
.63 (S, 3H), 1.83 (s, 6H). Purity: 96.60% by HPLC (80:20 MeOH/H
2
O).
A light yellow solid; C17
H
3
12BrN O; molecular weight: 354.2; m.p.
+
1
1
43.2–144.9°C; ESI-MS m/z: [M+H] : 354.1; H NMR (400 MHz,
8
-Bromo-1-(4-methoxyphenyl)-2-methyl-1H-imidazo[4,5-c]-
6
DMSO-d ) δ 9.49 (s, 1H), 8.70 (s, 1H), 8.24 (d, J = 8.8 Hz, 1H), 7.93 (d,
quinoline (11b)
J = 7.6 Hz, 1H), 7.83 (d, J = 8.5 Hz, 2H), 7.61 (s, 1H), 7.41 (d, J = 8.2 Hz,
A light yellow solid; C18
H14BrN
3
O; molecular weight: 368.3; m.p.
2
2H), 4.06 (s, 3H). Purity: 98.20% by HPLC (80:20 MeOH/H O).
+
1
13
1
74.3–175.9°C; ESI-MS m/z: [M+H] : 368.1; H NMR (400 MHz,
6
C NMR (101 MHz, DMSO-d ) δ 161.31, 146.61, 146.48, 138.44,
DMSO-d
6
) δ 9.11 (s, 1H), 7.93 (d, J = 8.8 Hz, 1H), 7.60 (d, J = 8.4 Hz,
133.24, 131.12, 129.58 (2C), 129.45, 123.20, 120.02, 119.64, 119.55,
−1
1
H), 7.54 (d, J = 8.3 Hz, 2H), 7.21 (d, J = 8.3 Hz, 2H), 7.06 (d, J = 13.3 Hz,
H), 3.84 (s, 3H), 2.30 (s, 3H). Purity: 98.80% by HPLC (80:20 MeOH/
O).
116.09 (2C), 67.28, 56.70. IR (KBr) cm : 3098, 3071, 3056, 3010,
2970, 2934, 2854, 2842, 1612, 1250.
1
H
2
8
-Bromo-1-(4-bromophenyl)-1H-imidazo[4,5-c]quinoline (12c)
8
-Bromo-1-(4-bromophenyl)-2-methyl-1H-imidazo[4,5-c]-
A light yellow solid; C16
H
9
Br
2
N
3
; molecular weight: 403.1; m.p. 210.3–
quinoline (11c)
+
1
2
9
1
11.5°C; ESI-MS m/z: [M+H] : 403.1; H NMR (400 MHz, DMSO-d
6
) δ
2 3
A light yellow solid; C17H11Br N ; molecular weight: 417.1; m.p.
.52 (s, 1H), 8.77 (s, 1H), 8.27 (s, 1H), 8.13 (s, 3H), 7.95 (s, 4H), 7.65 (s,
+
1
1
80.1–181.4°C; ESI-MS m/z: [M+H] : 417.1; H NMR (400 MHz,
13
H). Purity: 97.40% by HPLC (80:20 MeOH/H
2
O).
C NMR
6
DMSO-d ) δ 9.37 (s, 1H), 8.16 (dd, J = 22.5, 8.6 Hz, 3H), 7.87 (d,
(
101 MHz, DMSO-d
6
) δ 146.43, 146.18, 143.71, 138.51, 136.31,
J = 8.5 Hz, 3H), 7.25 (d, J = 14.5 Hz, 1H), 2.54 (s, 3H). Purity: 97.90% by
1
1
33.95 (2C), 133.26, 131.17, 130.35 (2C), 124.21, 123.21, 122.20,
20.05, 119.38.
1
3
HPLC (80:20 MeOH/H
2
O). C NMR (101 MHz, DMSO-d
45.17, 143.95, 143.12, 137.30, 136.36, 134.45 (2C), 134.38, 132.84,
31.25 (2C), 130.86, 124.75, 122.60, 120.09, 14.72.
6
) δ 153.96,
1
1
8-Bromo-1-(3-chloro-4-fluorophenyl)-1H-imidazo[4,5-c]-
8
-Bromo-1-(3-chloro-4-fluorophenyl)-2-methyl-1H-
imidazo[4,5-c]quinoline (11d)
A light yellow solid; C17
quinoline (12d)
8 3
A light yellow solid; C16H BrClFN ; molecular weight: 376.6; m.p.
+
1
H10BrClFN
3
; molecular weight: 390.6; m.p.
170.1–171,2°C; ESI-MS m/z: [M+H] : 376.1; H NMR (400 MHz,
DMSO-d ) δ 9.51 (s, 1H), 8.78 (s, 1H), 8.42 (d, J = 5.0 Hz, 1H), 8.26 (d,
J = 8.9 Hz, 1H), 8.05 (s, 1H), 8.00–7.90 (m, 2H), 7.62 (s, 1H). Purity:
+
1
2
50.9–251.2°C; ESI-MS m/z: [M+H] : 390.1; H NMR (400 MHz,
6
6
DMSO-d ) δ 9.40 (s, 1H), 8.39 (s, 1H), 8.22 (d, J = 8.0 Hz, 1H), 8.02 (s,
1
3
1
H), 7.90 (d, J = 7.9 Hz, 1H), 7.28 (s, 1H), 2.58 (s, 1H). Purity: 98.60% by
96.60% by HPLC (80:20 MeOH/H
2
O). C NMR (101 MHz, DMSO-
1
3
HPLC (80:20 MeOH/H
2
O). C NMR (101 MHz, DMSO-d
6
) δ 144.52,
d
6
) δ 156.85, 145.51, 142.76, 137.56, 133.08, 132.39, 131.92,
1
1
42.63, 136.31, 133.42, 133.28, 132.36, 130.84, 129.81, 129.55,
29.48, 121.61, 121.35, 118.97, 118.70, 118.64, 118.15, 13.81.
130.36, 130.00, 128.66, 128.56, 122.25, 119.17, 118.46, 118.33,
117.98.

10 of 11
XIAO ET AL.
|
8
-Bromo-1-(4-bromo-2-fluorophenyl)-1H-imidazo[4,5-c]-
previous orifice plate dosing arrangement, added 2.5 µL kinase
solution to each well, added 2.5 µL reaction solution to the control
wells, shook the plate to mix the added solution; added 2.5 µL to
each well according to the plate dosing setting. Then 2.5 µL test
compounds or positive control drug solution was added to each well
according to the plate dosing pattern. Add 5 µL basal solution to
each well and shake the plate to mix the solution. The well plates
were placed at room temperature for 1 h. Then, 10 µL of Eu-anti-
phospho-4E-BP1 and EDTA reaction solution was added, centri-
fuged, and left at room temperature for 1 h. Envision was used to
read the test data. The 665 nM Lance signal was read and then
converted to inhibition (%) according to the formula: (Lance signal
value − min)/(max−min) × 100. The IC50 value was obtained by MS
Excel and Graphpa 5.0 analysis.
quinoline (12e)
8 2 3
A light yellow solid; C16H Br FN ; molecular weight: 421.1; m.p.
+
1
1
40.3–141.9°C; ESI-MS m/z: [M+H] : 418.9; H
NMR (400 MHz,
DMSO-d
6
) δ 8.66 (s, 1H), 8.29 (dd, J = 6.5, 2.3 Hz, 1H), 8.13 (d,
J = 9.2 Hz, 1H), 7.99–7.90 (m, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.44–7.32
(
m, 1H), 7.0 (s, 1H), 6.70 (d, J = 2.5 Hz, 1H). Purity: 98.60% by HPLC
13
(
80:20 MeOH/H
2
O).
C
6
NMR (101 MHz, DMSO-d ) δ 158.74,
1
1
56.23, 146.21, 146.08, 143.48, 138.09, 133.06, 131.99, 131.28,
29.88, 129.85, 122.17, 121.40, 121.17, 120.27, 118.91.
4
.2
|
Biological evaluation
Cytotoxicity assay in vitro
4
.2.1
|
The target compounds (8a–e, 9a–e, 10a–e, 11a–e, and 12a–e) were
evaluated for cytotoxic activity against A549 (human lung cancer cell),
PC-3 (human prostate cell), HepG2 (human hepatocellular carcinoma
cell) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
ACKNOWLEDGMENTS
We gratefully acknowledge the generous support provided by The
National Natural Science Funds (No. 81460527), The Project
Supported by Natural Science Foundation of Jiangxi, China
(
MTT) method, with NVP-BEZ235 being the positive control according
[13]
to the reported procedures.
(
20171BAB215073, 20171BAB205101); Natural Science Funds
for Distinguished Young Scholar of Jiangxi Province, China
20171BCB23078); Key projects of the youth fund, Natural Science
The cells were seeded evenly in 96-well plates and 24 h later
5 target compounds were added to 96-well plates at five
2
(
concentrations (1000, 333, 111, 37, 1 µg/mL). Plates were
incubated at 37°C in a 5% CO incubator for 72 h. After 72 h,
Funds of Jiangxi Province (20171ACB21052); Jiangxi Provincial Key
Laboratory of Drug Design and Evaluation (20171BCD40015)
Graduate Students’ Science and Technology Innovation Project of
Jiangxi Science and Technology Normal University.
2
the culture medium in the well plate was removed and the prepared
MTT solution (MTT powder was dissolved in PBS at pH 7 to a
concentration of 5 mg/mL, filtered through a 0.22 µm membrane
filter) was added to 20 µL per well. After 3.5 h, the fluid was
removed and 150 µL DMSO was added per well. Placed plates in a
magnetic shaker shock for 3 min using dual wavelength (492 and
CONFLICT OF INTEREST
The authors declare no conflict of interest.
6
30 nm) crystals completely dissolved in the enzyme immunoassay.
Measured the absorbance of each well. The IC50 value for
each drug was calculated using the Bliss method based on
absorbance.
ORCID
Wufu Zhu
http://orcid.org/0000-0001-6075-0450
À
Á
IR% ¼ ODcontrol ꢀ ODsample =ðODcontrol ꢀ ODblankÞ ꢁ 100%
REFERENCES
[
1] J. LoPiccolo, G. M. Blumenthal, W. B. Bernstein, P. A. Dennis, Drug
Resist. Updat. 2008, 11(1), 32.
4
.2.2 PI3K and mTOR kinase assay
|
[
2] X. Q. Wang, L. X. Liu, Y. Li, C. J. Sun, W. Chen, L. Li, H. B. Zhang, X. D.
Yang, Eur. J. Med. Chem. 2013, 62, 111.
The inhibitory activities against PI3K and mTOR kinase assay were
evaluated through the mobility shift assay or Lantha screen assay
together with reference compound NVP-BEZ235b according to the
[3] B. Zhou, Z. F. Liu, G. G. Deng, W. Chen, M. Y. Li, L. J. Yang, Y. Li, X. D.
Yang, H. B. Zhang, Org. Biomol. Chem. 2016, 14(39), 9423.
[4] A. Kim, S. Park, J. E. Lee, W. S. Jang, S. J. Lee, H. J. Kang, S. S. Lee,
Leukemia Res. 2012, 36(7), 912.
[11]
reported procedures.
PI3K alpha and mTOR enzymes were
purchased from Millipore and Lance Ultra evaluation kit was
purchased from PE. The PI3Kα and mTOR enzymes were dissolved
in kinase buffer to a concentration of 2.5 nM; the Ulight-4E-BP1
peptide and ATP were dissolved in kinase buffer; the EDTA was
dissolved in kinase buffer to a concentration of 8 nM. The Eu-anti-
phospho-4E-BP1 antibody was dissolved in kinase buffer and
adjusted to a concentration of 2 nM. In accordance with the
[
5] N. Awasthi, P. L. Yen, M. A. Schwarz, R. E. Schwarz, J. Cell. Biochem.
012, 113(3), 784.
2
[6] D. Sarker, J. E. Ang, R. Baird, R. Kristeleit, K. Shah, V. Moreno, P. A.
Clarke, F. I. Raynaud, G. Levy, J. A. Ware, K. Mazina, R. Lin, J. Wu, J.
Fredrickson, J. M. Spoerke, M. R. Lackner, Y. Yan, L. S. Friedman, S. B.
Kaye, M. K. Derynck, Clin. Cancer Res. 2015, 21(1), 77
[
7] D. Roulin, L. Waselle, A. Dormond-Meuwly, M. Dufour, N.
Demartines, O. Dormond, Mol. Cancer 2011, 10(2), 90.

XIAO ET AL.
11 of 11
|
[
8] M. C. Manara, G. Nicoletti, D. Zambelli, S. Ventura, C. Guerzoni, L.
Landuzzi, P.-L. Lollini, S.-M. Maira, C. García-Echeverría, M. Mercuri,
Clin. Cancer Res. 2010, 16(2), 531.
SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
[
9] J. A. Sznol, L. B. Jilaveanu, H. M. Kluger, Curr. Cancer Drug Targets
2
012, 13(2), 165.
10] E. Jifu, J. Xing, H. Gong, J. He, W. Zhang, Tumour Biol. 2015, 36(2),
091.
11] B. Klünder, M. E. F. Mohamed, A. A. Othman, Clin. Pharmacokinet.
017, (6937), 1.
[
[
[
[
1
How to cite this article: Xiao Z, Lei F, Chen X, et al. Design,
synthesis, and antitumor evaluation of quinoline-imidazole
derivatives. Arch Pharm Chem Life Sci. 2018;1–11.
https://doi.org/10.1002/ardp.201700407
2
12] T. S. Xu, F. Lei, H. Tao, J. Q. Li, Z. W. Zhang, L. X. Wang, S. Xu, P. W.
Zheng, Basic Clin. Pharmacol. Toxicol. 2016, 119, 31.
13] F. Lei, C. Sun, S. Xu, Q. Wang, Y. OuYang, C. Chen, H. Xia, L. Wang, P.
Zheng, W. Zhu, Eur. J. Med. Chem. 2016, 116, 27.