Structure-based discovery of potent and selective small-molecule inhibitors targeting signal transducer and activator of transcription 3 (STAT3)

Article abstract of DOI:10.1016/j.ejmech.2021.113525

STAT3 has been validated as an attractive anticancer target due to its important roles in cancer initiation and progression. However, discovery of potent and selective STAT3 small-molecule inhibitors with druglike properties is still challenging. In this study, two series of substituted 2-phenylquinolines and 2-arylimidazo[1,2-a]pyridines were designed through structure-based drug discovery approach by condensing the privileged structures of STX-119 and SH4-54. Our study has resulted in the discovery of a number of highly potent and selective STAT3 inhibitors, exemplified by compound 39 with the privileged structure of 2-phenylimidazo[1,2-a]pyridine, which selectively inhibits phosphorylation of STAT3 and suppresses subsequent signaling pathway. Moreover, 39 inhibits cell growth, migration and invasion of human triple negative breast cancer (TNBC) cells lines. Consistently, it achieves significant and dose-dependent tumor growth inhibition in both cell line-derived and patient-derived xenograft tumor models in mice. These results clearly indicate that 39 is a highly potent and selective STAT3 inhibitor.

Full text of DOI:10.1016/j.ejmech.2021.113525

European Journal of Medicinal Chemistry 221 (2021) 113525
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Structure-based discovery of potent and selective small-molecule
inhibitors targeting signal transducer and activator of
transcription 3 (STAT3)
,
,
,
Qiuyao Huang a 1, Yan Zhong b 1, Bingbing Li a 1, Shumin Ouyang b, Lin Deng a,
Jianshan Mo b, Shuo Shi b, Nan Lv b, Ruibo Wu b, Peiqing Liu b, Wenhao Hu a,
,
, *
Xiaolei Zhang b **, Yuanxiang Wang a
a Guangdong Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, 510006, China
b Guangdong Key Laboratory of New Drug Design and Evaluation, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, 510006, China
a r t i c l e i n f o
a b s t r a c t
Article history:
STAT3 has been validated as an attractive anticancer target due to its important roles in cancer initiation
and progression. However, discovery of potent and selective STAT3 small-molecule inhibitors with
druglike properties is still challenging. In this study, two series of substituted 2-phenylquinolines and 2-
arylimidazo[1,2-a]pyridines were designed through structure-based drug discovery approach by
condensing the privileged structures of STX-119 and SH4-54. Our study has resulted in the discovery of a
number of highly potent and selective STAT3 inhibitors, exemplified by compound 39 with the privileged
structure of 2-phenylimidazo[1,2-a]pyridine, which selectively inhibits phosphorylation of STAT3 and
suppresses subsequent signaling pathway. Moreover, 39 inhibits cell growth, migration and invasion of
human triple negative breast cancer (TNBC) cells lines. Consistently, it achieves significant and dose-
dependent tumor growth inhibition in both cell line-derived and patient-derived xenograft tumor
models in mice. These results clearly indicate that 39 is a highly potent and selective STAT3 inhibitor.
© 2021 Elsevier Masson SAS. All rights reserved.
Received 2 March 2021
Received in revised form
11 April 2021
Accepted 23 April 2021
Available online 7 May 2021
Keywords:
Structure-based drug discovery
Privileged structures
STAT3
Small-molecule inhibitors
1. Introduction
been detected in almost 70% of human solid and hematological
tumors, and is generally associated with poor clinical prognosis
As a transcription factor, STAT3 transduces extracellular signals
to the nucleus and then activates transcription of target genes.
Under physiological conditions, it mediates cell proliferation, dif-
ferentiation, apoptosis, survival, and other important cellular pro-
cesses in response to stimulation of a large number of cytokines and
growth factors [1,2]. However, constitutive STAT3 activation has
[3,4]. In view of its critical roles in cancer initiation and progression,
STAT3 has attracted considerable interest as a potential target in
cancer therapy [5e10]. On the other hand, aberrant STAT3 activa-
tion has been reported to mediate acquired resistance to a broad
spectrum of chemotherapies, radiotherapies and targeted thera-
pies, by up-regulating the expression of antiapoptotic proteins and
surviving protein (such as Bcl-2, survivin, c-Myc, cyclin-D1, and
Mcl-1) and down-regulating tumor suppressor gene (such as p53)
[11e15]. Therefore, inhibition of STAT3 represents a new advance to
reverse STAT3-addicted cancer cell resistance. Furthermore,
constitutively activated STAT3 is also reported to play critical roles
in modulation of tumor microenvironment and immune response
through up-regulation of immunosuppressive factors and down-
regulation of immune activation factors [16e18]. It has been vali-
dated that the combination of immune checkpoint inhibitors and
STAT3 specific inhibitors can enhance the efficacy of cancer
immunotherapy and demonstrates a synergistic antitumor effect
[19e22]. Accordingly, the development of potent and selective
Abbreviations: STAT3, signal transducer and activator of transcription 3; pY705,
phosphorylated Tyr705; pSTAT3, phosphorylated STAT3; PROTAC, proteolysis tar-
geting chimera; HLM, human liver microsome; EMSA, Electrophoretic mobility shift
assay; TNBC, triple negative breast cancer; PI, propidium iodide; TGI, tumor growth
inhibition; PDX, patient-derived xenograft; FITC, fluorescein isothiocyanate; HOAt,
1-hydroxy-7-azabenzotriazole; EDC, N-Ethyl-N0-(3-dimethylaminopropyl)carbodii-
mide; DIPEA, N,N-Diisopropylethylamine.
* Corresponding author.
** Corresponding author.
E-mail addresses: zhangxlei5@mail.sysu.edu.cn (X. Zhang), wangyx95@mail.
sysu.edu.cn (Y. Wang).
1
These authors contributed equally.
https://doi.org/10.1016/j.ejmech.2021.113525
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
STAT3 inhibitors has multifaceted applications in cancer therapy
and is highly desirable.
compound 39 demonstrated significant and dose-dependent tu-
mor growth inhibition in both cell line-derived and patient derived
xenograft tumor models in mice.
In the canonical STAT3 signaling pathway, STAT3 is activated by
phosphorylation of tyrosine residue 705 (Tyr705) within its Src
homology 2 (SH2) domain, followed by forming a homodimer via
reciprocal interaction between the phosphorylated Tyr705 (pY705)
within one monomer and the SH2 domain of the other. Activated
STAT3 homodimers then translocate from the cytoplasm to the
nucleus, where they bind to STAT3-specific DNA-response ele-
ments and regulate the expression of target genes. As the SH2
domain plays a pivotal role in STAT3 signaling cascade, significant
efforts have been made to discover STAT3 inhibitors targeting the
SH2 domain. These STAT3 SH2 domain inhibitors consist of small
peptidomimetics, natural products and synthetic nonpeptidic
small-molecules [1e4]. However, most of these inhibitors suffer
from unfavorable cell permeability and druglike properties, or
weak to modest binding affinity, highlighting the challenge in
developing potent and selective small-molecule STAT3 SH2 domain
inhibitors.1,2 For instance, the previously discovered peptidomi-
metic STAT3 SH2 domain inhibitors usually demonstrate high
binding affinities to STAT3 in biochemical assays, but display un-
desired cell permeability and druglike properties [1]. On the other
hand, synthetic nonpeptidic STAT3 SH2 inhibitors as exemplified by
the compounds 1-5 [23e27] in Fig. 1, which possess good cell
permeability compared to peptidomimetic inhibitors, usually
exhibit weak to moderate binding affinities to STAT3.
Recently, Wang and coworkers discovered a potent STAT3
degrader SD-36 (6) based upon the proteolysis targeting chimera
(PROTAC) concept, by employing a ligand for cereblon/cullin 4A E3
ligase and a peptidomimetic STAT3 SH2 domain inhibitor [28,29].
The degrader SD-36 is capable of effectively and selectively
degrading STAT3 protein and exhibits highly potent anticancer
activities. However, developing selective and potent nonpeptidic
STAT3 SH2 inhibitors which serve as the starting point for discov-
ering STAT3 degraders, may provide more promising clinical can-
didates with desirable druglike properties.
2. Results and discussion
2-Phenylquinoline-based STAT3 inhibitor. Our drug discovery
campaign initiated with two previously reported STAT3 inhibitors,
STX-119 [23] and SH4-54 [25], both of which are selective STAT3
inhibitors and have similar binding mode with the SH2 domain
revealed by molecular modeling. Analysis of the X-ray structure of
STAT3 SH2 domain (PDB ID: 1BG1) [30] suggested that there are
three important sub-pockets within the SH2 domain, including
pY705 binding site, Leu706 binding site and a side pocket (Fig. 2A).
The molecule docking of STX-119 with STAT3 SH2 domain revealed
that 2-phenylquinoline bound to the pY705 binding site alongside
the side pocket, and 5-(furan-2-yl)-1,3,4-oxadiazol targeted to the
Leu706 sub-pocket (Fig. 2B). Molecule modeling studies on SH4-54
showed that the salicylic acid group and tosylamide moiety
accessed to the pY705 sub-pocket and side pocket, respectively, and
cyclohexylphenyl occupied to the Leu706 sub-pocket (Fig. 2C).
While these two compounds display modest inhibitory activity
against human cancer cell lines [23,25], the privileged structures
embedded in them attracted our attentions, such as 2-
phenylquinoline in STX-119 and cyclohexylbenzene in SH4-54. 2-
Phenylquinoline is a well-known privileged structure and has
been found to be the core structure of many bioactive substances
[31e35]. Cyclohexylbenzene is also claimed to be a privileged
structure due to its common presence in various bioactive com-
pounds [36e40]. Since cyclohexylphenyl of SH4-54 was shown to
occupy an essentially similar binding pocket as compared to 5-
(furan-2-yl)-1,3,4-oxadiazol of STX-119, we surmised that fusing
cyclohexylphenyl of SH4-54 with 2-phenylquinoline of STX-119
would result in identification of novel STAT3 inhibitors with priv-
ileged structures and improved potencies. It is important to note
that C2-phenyl of STX-119 bound to the side pocket (Fig. 2B), a
specific site for STAT3 over other STAT members which provides
structure basis for the high selectivity of STX-119. Novel inhibitors
derive from 2-phenylquinoline may also display high selectivity for
STAT3. Therefore, the initial series of compounds were designed by
condensing 4-cyclohexylaniline or (4-cyclohexylphenyl)methan-
amine with 2-phenylquinoline-4-carboxylic acid and several hy-
drophilic groups were also incorporated into the C7-positon of
quinoline core to modulate their physicochemical characteristics.
As an initial screening, the 10 hybrid compounds were evaluated
for their cell growth inhibitory activities in the cell viability assays
against cancer cell lines which express constitutively active STAT3,
In this context, we performed structure-based drug design
approach to facilitate the discovery of highly potent and selective
STAT3 small-molecule inhibitors. Two series of novel STAT3 small-
molecule inhibitors were designed and synthesized with the priv-
ileged structures of 2-phenylquinoline and 2-arylimidazo[1,2-a]
pyridine, respectively. Further optimization led to the identification
of compound 39 as the most promising STAT3 small-molecule in-
hibitor. Our data demonstrated that compound 39 selectively
bound to STAT3 SH2 domain, inhibited STAT3 activation and sub-
sequent signaling pathway, and ultimately suppressed cell growth,
migration and invasion of human cancer cell lines. Moreover,
Fig. 1. Chemical structures of previously reported STAT3 inhibitors (1-5) and degrader (6).
2

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
Fig. 2. (A) Three pockets within STAT3 SH2 domain (PDB ID: 1BG1). (B) Binding mode of compound 1. (C) Binding mode of compound 3. (D) Binding mode of compound 8. (E) Our
design of new STAT3 inhibitors.
including MDA-MB-231 (breast cancer cell line), MGC-803 (gastric
cancer cell line) and A549 (non-small cell lung cancer cell line).
SH4-54 and STX-0119 were included as positive controls and the
data are summarized in Table 1, which were comparable to the
inhibition. It is interesting to note that the structural differences
between 13 and 8 are the groups that were designed to access to
the Leu706 pocket, 4-cyclohexylbenzylamine for 13 and 4-
cyclohexylaniline for 8. The huge difference in the cellular activity
of 13 and 8 revealed that 4-cyclohexylphenylamines is preferred
than 4-cyclohexylbenzylamine when 2-phenylquinoline is used as
the core structure.
reported values. Compound
7 was obtained by fusing 4-
cyclohexylaniline and 2-phenylquinoline-4-carboxylic acid
together, and showed slightly decreased inhibitory activities with
IC50 values of 10
m
M, 5.9
m
M and 5.8
m
M for MDA-MB-231, MGC-803
Upon the basis of its potent cell growth inhibitory activity and
privileged chemical structure, compound 8 was selected as a
starting point for further optimization. Before the next round of
optimization, compound 8 was first docked to the three hot spots
within the SH2 domain to determine whether it has the binding
modes as we predicted. The docking result confirmed our proposal
and demonstrated that cyclohexylphenyl fragment of compound 8
occupied to the Leu706 sub-pocket (Fig. 2D). Therefore, in an effort
to explore the structure-activity relationship (SAR) on this position
and to identify more potent STAT3 inhibitors, cyclohexyl of com-
pound 8 was replaced by a diverse of groups, including halogen (17-
19), alkyl (20, 22, 23), alkoxy (21), aryl (24-28) and heteroaryl (29-
33).
and A549, respectively. With the aim of improving the physico-
chemical characteristics of compound 7, methoxy group was
incorporated at the C7-postion of the quinoline ring and afforded
compound 8, which displayed enhanced potency with IC50 values
of 5.9 mM, 4.9 mM and 4.8 mM against the three tested cancer cells,
respectively. However, incorporation of other hydrophilic groups,
including hydroxy (9), 2-ethoxy-2-oxoethoxy (10) and carbox-
ymethoxy (11), at the quinoline C7-postion of compound 7 was not
well tolerated and led to completely abolished cell growth inhibi-
tory activity. On the other hand, it seems that the design concept of
condensation of (4-cyclohexylphenyl)methanamine with 2-
phenylquinoline scaffold was unsuccessful, and the hybrid com-
pounds 12, 13, 15 and 16 were found to be completely inactive with
As shown in Table 2, replacement of cyclohexyl group of com-
pound 8 with halogen atoms led to compounds 17-19 with signif-
icantly decreased potency against the three test cancer lines.
IC50 values greater than 100
quinoline derivative 14 displayed weak cancer cell growth
mM, even though hydroxy substituted
3

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
Table 1
Table 2
Antiproliferative activity of quinoline derivatives 7-16 against human cancer cell
Antiproliferative activity of quinoline derivatives 17-33 against human cancer cell
lines.a
lines.a
Compd
R
IC50 (mM)
MDA-MB-231
MGC-803
A549
Compd
R
n
IC50 (mM)
17
18
19
20
21
22
23
24
Br
F
Cl
Me
OMe
n-Butyl
iso-Propyl
25.5 2.48
>100
>100
>100
>100
>100
>100
5.3 0.28
15.5 2.15
>100
50.4 4.71
>100
>100
39.6 4.25
>100
21.4 2.10
>100
>100
>100
>100
40.9 6.68
>100
3.0 0.32
MDA-MB-231
MGC-803
A549
7
8
9
10
11
12
13
14
15
16
SH4-54
STX-119
H
OMe
OH
OCH2CO2Et
OCH2CO2H
H
OMe
OH
0
0
0
0
0
1
1
1
1
1
10.0 2.03
5.9 0.68
>100
>100
>100
>100
>100
50 5.16
>100
>100
5.9 0.58
4.9 0.69
>100
>100
>100
20.1 3.21
>100
14.6 2.28
>100
>100
5.6 0.14
7.43 1.74
5.8 0.72
4.9 1.12
>100
>100
>100
>100
>100
16.6 1.15
>100
>100
2.6 0.90
OCH2CO2Et
OCH2CO2H
25
26
27
28
29
>100
>100
>100
2.0 0.24
18.25 3.09
5.0 0.15
11.59 3.09
11.2 2.66
21.0 4.32
20.2 2.24
10.8 1.46
40.4 3.18
5.8 0.54
15.6 3.42
15.0 0.98
29.9 4.16
4.9 0.43
15.6 4.14
15.0 2.13
a
Data are calculated based on three independent experiments.
Compound 17 displayed very weak inhibitory activity with the IC50
values ranging from 15.5 M to 25.5 M and compounds 18,19 were
found to be completely inactive with IC50 values greater than
50 M. The similar tendency was also observed in the series of
m
m
m
simple alkyl and alkoxy derivatives 20-23, indicating that the size
of the substitution has a significant effect on inhibitory potency. In
the series of aryl substituted compounds 24-28, significant
discrepancy in the cellular inhibitory potency was observed.
Interestingly, replacement of the cyclohexyl group of 8 with phenyl
led to the compound 24, which exhibited enhanced inhibitory ef-
30
>100
64.8 5.78
>100
31
32
33
10.3 1.26
5.3 0.45
>100
2.1 0.12
4.8 0.84
>100
5.5 0.57
4.3 0.75
>100
fects with IC50 values of 5.3 mM, 2.6 mM and 3.0 mM against the three
tested cancer cells, respectively. In contrast, para-isopropylphenyl
derivative 25 did not show any antiproliferative effects in the three
type cancer cells. Para-fluorophenyl derivative 26 and para-meth-
ylthiophenyl derivative 28 displayed slight inhibitory activity
against the proliferation of the three tested cancer cells. Para-
methoxyphenyl derivative 27 exhibited moderate inhibitory ac-
tivity against MGC-803 and A549 cells with the IC50 values of 5.8
and 4.9
mM, respectively, but displayed much weaker activity
SH4-54
2.0 0.24
5.6 0.14
5.0 0.15
against MDA-MB-231 cells with the IC50 value greater than 20
mM.
a
Data are calculated based on three independent experiments.
In addition to aryl group, compounds with various heteroaryl
groups (29-33) were also examined. While thiophenyl (29), pyr-
idinyl (30), and indolyl (33), decreased the inhibitory potency,
furanyl derivative 31 showed more potent antiproliferative effects
against MGC-803 cells with an IC50 value of 2.1 mM when compared
to 8.
Further exploration the binding mode of compound 8 in a complex
with the STAT3 SH2 domain suggested that the 2-phenylquinoline
occupied both the Tyr705 and side pocket as we predicted (Fig. 2D).
However, the C2-phenyl group of 8 just located on the threshold of
the side pocket, not in the deep of the pocket mainly due to longer
distance between C2-phenyl and C4-carbamoyl of quinoline than
that between side pocket and Leu706 pocket. We surmised that
shortening the distance between C2-phenyl and C4-carbamoyl may
enhance the binding affinity with SH2 domain and translate to
increased cell growth inhibitory activity. To test this hypothesis,
compound 34 was designed and synthesized by replacing the 2-
phenylquinoline with 2-phenylimidazo[1,2-a]pyridine, which is
also defined as a privileged structure and represents one of the
most important structural fragments for new drug discovery
(Fig. 3) [41e45]. Unfortunately, compound 34 showed no inhibitory
Compound 31 also retained the antiproliferative potency against
A549 cells, but exhibited decreased inhibitory activity against the
growth of MDA-MB-231 cells, with IC50 values of 5.5
10.3 M, respectively. Benzofuranyl derivative 32 showed anti-
proliferative activity comparable to compound 8 against the three
tested cancer cell lines with the IC50 values of around 5.0 M. These
mM and
m
m
results indicate that cyclohexyl group of compound 8 can also be
replaced by phenyl, furanyl, or benzofuranyl.
Imidazo[1, 2-a]pyridine-based STAT3 inhibitor. In an effort to
identify more potent STAT3 inhibitors with privileged structure, we
turned our attention to optimize the 2-phenylquinoline moiety.
4

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
Fig. 3. Design of imidazo[1, 2-a]pyridine-based STAT3 inhibitor.
activity on cancer cell proliferation. We hypothesized that the
abolished activity of compound 34 could be largely attributed to its
torsion force derived from a repulsive interaction between the
oxygen of C3- carbamoyl and the hydrogen at the C-2’ position of
C2-phenyl. To address this issue, compound 35 was prepared by
adding a methylene linker between 3-carbamoyl and the imidazo
[1,2-a]pyridine core structure with the aim of eliminating the tor-
sion force of compound 34 (Fig. 3). To our delight, compound 35
recovered cancer cell growth inhibitory activity and exhibited
comparable ability as compared to 8. Next, a number of control
compounds were designed and synthesized to test the essentiality
of our new core structure. As showed in Table 3, both biphenyl
derivative 36 and 4-(furan-2-yl)phenyl derivative 37 displayed
similar tendency as in 24 and 31. Furthermore, we replaced 4-
cyclohexylphenyl of compound 35 with benzo[b]thiophene-1,1-
dioxide, which is reported to be a pharmacophore model for the
inhibition of STAT3 and predicted to bind to the SH2 domain
[46e50]. Surprisingly, the desired compound 38 showed good
inhibitory activity against the three test cancer cell lines with IC50
structural novelty and high potency, we selected compound 38 as
our lead STAT3 inhibitor for further structural optimization.
First, we fixed the phenyl group at the C-2 position of imidazo
[1,2-a]pyridine and then examined effect on the inhibitory potency
with various substituents on the pyridine scaffold. As described in
Table 4, removal of the C-7 methoxy in 38 afforded compound 39
exhibiting comparable antiproliferative activity with IC50 values of
around 1.0 mM. Replacement of C-7 methoxy with halogen atoms,
such as chlorine (40) and bromine (41) was tolerated, especially for
41 displaying submicromolar inhibitory activity against the three
cancer cell lines. The C-7 methyl derivative 42 also showed good
activity. Moving the methoxy from C7 position to C8 (43) and C6
(44) position of imidazo[1,2-a]pyridine led to slightly decreased
inhibitory activity as compared to 38. Installation of the bromo
group at the C-6 position of imidazo[1,2-a]pyridine afforded 45
displaying less potent anticancer activity against MDA-MB-231 and
MGC-803 cells than 41, but retaining potency against A549 cells.
These results implied that the C-7 position of imidazo[1,2-a]pyri-
dine may be a favorable spot for further optimization. Therefore,
larger groups such as aryl or heteroaryl group were included in
compounds 46-48, and significant discrepancy in cell growth
inhibitory activity was observed. For instance, C7-phenyl derivative
46 showed slightly weaker activity than 38. In contrast, C7-para-
methoxyphenyl derivative 47 recovered the potency against the
three test cancer cell lines. Surprisingly, pyrazol derivative 48 also
displayed good potency with submicromolar inhibitory activity
against MGC-803 and A549 cells.
values of 1.2 mM, 1.5 mM and 1.1 mM, respectively. In view of the
Table 3
Antiproliferative activity of imidazo[1, 2-a]pyridine derivatives 34-38 against hu-
man cancer cell lines.a
Afterwards, with imidazo[1,2-a]pyridine core structure
substituted with C7 methoxy group, we explored the effect of
various substitutions of the C2 phenyl ring in compound 38 on
inhibitory activity. The para-substituted phenyl derivatives (49-52)
were firstly explored. The fluoro derivative 49 exhibited good
inhibitory activity against MGC-803 cells and A549 cells, but
showed a decreased potency against MDA-MB-231 cells with an
Compd
N
R
IC50 (mM)
MDA-MB-231
MGC-803
A549
IC50 value greater than 5
trifluoromethyl demonstrated significantly increased potency
against the three tested cancer cells with IC50 values of 0.7 M,
0.4 M and 0.4 M, respectively, which is the most active com-
mM. However, compound 50 bearing a
34
35
36
37
0
1
1
1
>100
>100
>100
m
m
m
5.4 0.45
10.5 1.21
10 0.87
4.7 0.32
3.5 0.23
2.5 0.35
5.6 0.51
3.2 0.24
6.6 0.54
pound in this study, probably due to the strong hydrophobic nature
of the trifluoromethyl group. To test this hypothesis, we investi-
gated the antiproliferative effects of low hydrophobic or high hy-
drophilic groups, such as nitro (51) and methylsulfonyl (52). Nitro
derivative 51 demonstrated weaker inhibitory activity than 38. The
high hydrophilic methylsulfonyl substituted 53 showed signifi-
cantly reduced anticancer activity against all tested cancer cells
with IC50 values greater than 5 mM. Next, replacement of C-2 phenyl
with thiophenyl (53) and naphthalenyl (54) were also investigated,
and it was revealed that both compounds showed good anti-
38
1
1.2 0.29
1.0 0.17
0.6 0.14
5.0 0.15
SH4-54
2.0 0.24
5.6 0.14
proliferation activity with IC50 values of around 1.0
mM. In addition,
a
compounds 55-57 were also designed with a core structure of C6-
Data are calculated based on three independent experiments.
5

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
Table 4
Antiproliferative activity of imidazo[1, 2-a]pyridine derivative 38-61 against human cancer cell lines.a
Compd
R1
R2
R3
R4
IC50 (mM)
MDA-MB-231
1.2 0.29
MGC-803
1.0 0.17
A549
38
39
40
41
42
43
44
45
46
47
48
H
OMe
H
H
0.6 0.14
H
H
1.2 0.13
1.1 0.08
0.9 0.12
1.6 0.12
2.2 0.17
2.1 0.13
2.0 0.42
2.4 0.34
1.0 0.04
1.4 0.07
1.6 0.06
1.3 0.13
0.9 0.07
1.0 0.06
1.7 0.22
1.7 0.15
1.5 0.24
1.7 0.26
0.5 0.09
0.7 0.07
1.1 0.11
0.7 0.05
0.8 0.06
0.7 0.07
1.0 0.05
1.2 0.08
0.8 0.06
1.1 0.06
0.7 0.11
0.8 0.08
H
Cl
H
H
Br
Me
H
H
H
H
H
OMe
H
OMe
Br
H
H
H
H
H
H
H
H
H
49
50
51
52
H
H
H
H
OMe
OMe
OMe
OMe
H
H
H
H
5.1 0.56
0.7 0.11
4.4 0.32
5.8 0.78
0.9 0.06
0.4 0.12
2.6 0.14
6.4 0.34
1.3 0.16
0.4 0.10
1.7 0.11
5.8 0.48
53
54
H
H
OMe
OMe
H
H
1.4 0.04
1.4 0.05
1.3 0.05
1.5 0.24
1.1 0.06
1.0 0.15
55
56
57
58
Me
Me
Me
H
H
H
H
H
H
H
H
H
2.1 0.45
2.0 0.15
2.0 0.06
4.1 0.25
1.8 0.22
2.4 0.15
1.6 0.08
2.7 0.10
0.9 0.05
2.5 0.07
1.2 0.06
5.5 0.23
6

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
Table 4 (continued)
Compd
R1
H
R2
H
R3
H
R4
IC50 (mM)
MDA-MB-231
6.4 0.32
MGC-803
6.9 0.42
A549
59
7.5 1.12
60
H
H
H
H
H
H
6.5 0.83
20.2 2.3
5.2 0.36
5.4 0.87
61
H
22.5 1.21
23.4 2.28
a
Data are calculated based on three independent experiments.
methylated imidazo[1,2-a]pyridine, a privileged structure derived
from the drug Zolpidem.51 The three compounds demonstrated
moderate to high cell growth inhibitory activity against the tested
STAT3 phosphorylation at Ser727, which also contributes to the
transcriptional activity of STAT3. It was revealed that compound 39
slightly suppressed phosphorylation of Ser727 compared to Tyr705
three cancer cell lines with IC50 values ranging from 0.9
m
M to
in MDA-MB-231 and MDA-MB-231-4175 cells at 3
inhibit Ser727 phosphorylation in MDA-MB-468 and HCC cells at
M. These results suggest that compound 39 mainly binds to
mM, and did not
2.5 M. Removing C6-methyl from compound 55 afforded com-
m
pound 58, resulting in less potent activity. Methylsulfonyl 59 and
nitro 60 exhibited similar tendency as in compounds 52 and 51.
However, it should be noted that removal of C-2 phenyl group from
imidazo[1,2-a]pyridine was not tolerated and led to dramatically
decreased potency with IC50 values greater than 20 mM for com-
pound 61 as compared to compound 39.
3 m
STAT3 SH2 domain as we designed.
To address the selectivity of compound 39 among STAT iso-
forms, we further investigated the effects of compound 39 on
phosphorylation of STAT1 and STAT5. As shown in Fig. 5B, com-
pound 39 did not affect the total or phosphorylated levels of STAT1
and STAT5 in MDA-MB-231 and HCC cells even at the concentration
In vitro metabolic stability assessment. Based on the anti-
proliferative activity mentioned above as well as the solubility
observed in the preparation, compound 38, 39 and 50 were
selected out for in vitro metabolic stability study in human liver
microsome (HLM). As shown in Table 5, compound 38 and 50
exhibited poor stability in HLM with the T1/2 of 10.1 min and
4.4 min, respectively, while compound 39 offered an improved
microsomal stability in HLM with the T1/2 of 24.2 min. Taken
together, compound 39 showed the best combination of micro-
somal stability and cell growth inhibitory potency, and was selected
for further evaluation.
Compound 39 directly bound to STAT3 protein. To confirm a
direct interaction between compound 39 and STAT3, a surface
plasmon resonance (SPR) assay was performed, for which purified
STAT3 was immobilized on CM5 sensor chips. As shown in Fig. 4,
compound 39 exhibited high binding affinity with STAT3 with an
of 3 mM, indicating that compound 39 selectively inhibits STAT3
over STAT1 and STAT5.
Compound 39 significantly inhibited STAT3 dimerization.
Activated STAT3 proteins form a homodimer via reciprocal inter-
action between pY705 within one monomer and the SH2 domian of
the other. To validate whether compound 39 disrupts STAT3
dimerization, HEK-293T cells were transfected with Flag-tagged
STAT3 and HA-tagged STAT3 for co-localization and co-
immunoprecipitation studies. HEK-293T cells that stably co-
express HA-STAT3 and Flag-STAT3 were treated with compound
39 and DMSO as the solvent control. Then the cells were processed
for immunofluorescence staining for Flag-STAT3 (red) and HA-
STAT3 (green) and analyzed by confocal microscopy. As shown in
Fig. 6A, the co-location (yellow) of HA-STAT3 and Flag-STAT3 was
significantly reduced by compound 39 in
a dose-dependent
equilibrium dissociation constant (KD) value of 0.432
m
M, indicating
manner, suggesting that dimerization of HA-STAT3 and Flag-
STAT3 was disrupted.
that compound 39 directly binds to the STAT3 protein and might
therefore block its activation via inhibition of STAT3 phosphoryla-
tion at Tyr705.
Next, we treated the HEK-293T cells with compound 39 for
immunoprecipitation analysis and revealed that compound 39
dose-dependently inhibited dimerization of HA-STAT3 and Flag-
STAT3 after IL-6 stimulation (Fig. 6B). These results further vali-
date that compound 39 binds to the STAT3-SH2 domain and in-
hibits STAT3 dimerization in intact cells.
Compound 39 selectively inhibited STAT3 Tyr705 phosphor-
ylation. To evaluate the effect of compound 39 on inhibiting
phosphorylation of STAT3, we performed western blotting assays to
detect the protein levels of phosphorylated STAT3 (pSTAT3) and
total STAT3 in triple negative breast cancer (TNBC) cell lines with
constitutively activated STAT3, including MDA-MB-231, MDA-MB-
468, HCC70 and MDA-MB231-4175. Western blotting assays
showed that compound 39 inhibited STAT3 phosphorylation at
Tyr705 in a dose-dependent manner in these four cell lines, but had
no effect on the total STAT3 (Fig. 5A), indicating that the reduced
pSTAT3 levels did not dependent on the total level of STAT3 protein.
In addition, we also evaluated the ability of compound 39 to inhibit
Table 5
In vitro metabolic stability in human liver microsomes.
HLM
Compd
clearance (mL/min/g)
T1/2 (min)
38
39
50
208.4
87.0
478.0
10.1
24.2
4.4
Fig. 4. SPR analysis of compound 39 binding to STAT3 protein. Representative data
from three independent experiments are shown.
7

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
Fig. 5. Effects of compound 39 on phosphorylation of STAT3 (A), STAT1 and STAT5 (B) analyzed by western blotting. Representative images from three independent experiments are
shown.
Fig. 6. Effects of compound 39 on STAT3 dimerization. (A) colocalization study of compound 39 in HEK-293T cells with HA-STAT3 (green) and FLAG-STAT3 (red). DAPI nuclear was
stained in blue. (B) Coimmunoprecipitation assay of compound 39 in HEK-293T cells. Representative images from three independent experiments are shown.
Compound 39 inhibited nuclear translocation of pY705-
STAT3. To demonstrate whether compound 39 would inhibit the
nuclear translocation of pY705-STAT3, MDA-MB-231 breast cancer
cells, which contain constitutively activated STAT3, were treated
with either vehicle or compound 39 for 12 h and then were sub-
jected to immunofluorescence staining by a specific p-STAT3 pri-
mary antibody and Alexa Fluor secondary antibody. It was revealed
that pY705-STAT3 was localized predominately in the nucleus in
the absence of compound 39 (Fig. 7). In contrast, compound 39
treatment inhibited the translocation of pY705-STAT3 to the nu-
cleus in a dose-dependent manner.
Compound 39 inhibited STAT3 DNA-binding and STAT3-
dependent transcriptional activation. After translocation from
the cytoplasm to the nucleus, STAT3 dimers bind to STAT3-specific
DNA-response elements and activate their transcriptional activity.
Therefore, we next evaluated the effects of compound 39 on DNA-
8

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
Fig. 7. Effects of compound 39 on nuclear translocation of phosphorylated STAT3 in MDA-MB-231 cell line. Representative images from three independent experiments are shown.
binding ability and transcriptional activation of STAT3. Electro-
phoretic mobility shift assay (EMSA) was first performed to assess
whether compound 39 blocks STAT3 DNA-binding activity. As
shown in Fig. 8A, compound 39 remarkably decreased the DNA-
binding of STAT3 in MDA-MB-231 and MDA-MB-468 cells at the
genes. TNBC cell lines, including MDA-MB-231, MDA-MB-468,
HCC70 and MDA-MB-231-4175, were treated with compound 39
for 24 h, and then were processed for western blotting. Fig. 9 shows
that compound 39 downregulated the expression of STAT3 target
genes, including Bcl-xL, c-Myc and Mcl-1.
concentrations of 0.3
DNA-binding of STAT5 was not affected even at 3
the preferential potential of compound 39 to inhibit DNA-binding
ability of STAT3 over STAT5 in cancer cells.
We next investigated whether STAT3-dependent transcriptional
activation was disrupted by compound 39 using a luciferase re-
porter assay. HEK-293T cells were transiently co-transfected with
pGL3-STAT3 (a STAT3-responsive promoter-firefly luciferase re-
porter) and renilla reporter (as normalization control) to act as gene
reporter model to examine the effect of compound 39 on STAT3-
dependent transcriptional activation. Compared to the control
m
M and 1
m
M, respectively. However, the
Compound 39 inhibited cancer cell proliferation and colony
formation. To further evaluate the antiproliferative activities of
compound 39 in cell models, its effects on TNBC cancer cell growth
and colony survival were evaluated. MDA-MB-231-4175, MDA-MB-
468 and HCC70 containing hyperactivated STAT3 were treated with
various concentrations of compound 39 for 72 h, and subsequently
examined by Cell Counting Kit-8. As shown in Fig. 10A, compound
39 concentration-dependently reduced the proliferation of MDA-
MB-231-4175, HCC70, and MDA-MB-468 cells with the IC50
mM. This suggests
values of 2.5 mM, 1.1 mM and 2.5 mM, respectively. Interestingly, 39
showed much weaker growth inhibition in MCF-7 (breast cancer
cells without persistent activated STAT3) and normal cells (H9C2:
group, the cells treated with 1 or 3 mM compound 39 for 24 h had
lower luciferase activity (Fig. 8B), indicating that compound 39
inhibits STAT3-dependent transcriptional activity.
Compound 39 downregulated the expression of STAT3 target
genes. In an effort to further provide evidence that compound 39
inhibits STAT3-dependent transcriptional activity, we evaluated the
effect of compound 39 on regulating the expression of STAT3 target
cardiomyocyte) with IC50 values of 7.22 mM and 6.79 mM, respec-
tively, indicating its selectivity for STAT3 persistently activated
cancer cell lines. Furthermore, compound 39 also significantly
suppressed the colony formation of MDA-MB-231, MDA-MB-468,
MDA-MB-231-4175 (Fig. 10B) and HCC70 (Figure S1) at as low as
0.3 mM concentration.
9

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
To further demonstrate the specificity of compound 39 on STAT3
signaling pathway in MDA-MB-231 cells, endogenous STAT3 was
knocked down using siRNA. As shown in Figure S2A, STAT3 was
successfully knocked down by siSTAT3. Cell viability test
(Figure S2B and Figure S2C) revealed that STAT3 siRNA treatment of
MDA-MB-231 led to loss of sensitivity to inhibition of 39, indicating
that STAT3 is required for anticancer activity of compound 39.
Compound 39 induced apoptosis in TNBC cancer cells. To
assess whether the proliferative inhibition induced by compound
39 in cancer cells was attributed to apoptosis [51e55], TNBC cells
were incubated with compound 39 at different concentrations for
48 h. Annexin V-FITC/propidium iodide (PI) staining was performed
and the percentages of apoptotic cells were determined by flow
cytometry. As shown in Fig.11A, compound 39 induced apoptosis in
MDA-MB-231, MDA-MB-231-4175 and MDA-MB-468 cells in a
dose-dependent manner with the apoptotic percentages of 14.6%,
10.0% and 40.6% at 3
mM, respectively. To further elucidate the
potential mechanisms of apoptosis induced by compound 39,
several proteins as markers of apoptosis were determined by
western blotting. As shown in Fig. 11B, cleavage of PARP and cas-
pase 7 was induced after treatment of compound 39 at 1
mM in
Fig. 8. Effects of compound 39 on STAT3 DNA-binding and STAT3-dependent tran-
scriptional activation. (A) EMSA analysis of STAT3 or STAT5 DNA-binding activity in
nuclear extracts prepared from MDA-MB-231 and MDA-MB-468 cells after treatment
of compound 39. (B) Luciferase reporter assay of compound 39 in HEK-293T cells.
Representative images from three independent experiments are shown. (***)
p < 0.001; (****) p < 0.0001.
MDA-MB-231-4175 and MDA-MB-468 cells and at 3 M in MDA-
m
MB-231 cells, suggesting that compound 39 induces the apoptosis
in TNBC cells at least partially through increasing the expression of
cleaved-PARP and cleaved-caspase7.
Fig. 9. Western blotting analysis was used to examine the phosphorylation of STAT3 and expression of STAT3-regulated genes in TNBC cells treated with different doses of
compound 39 for 24 h. Representative data from three independent experiments are shown.
Fig. 10. Effects of compound 39 on proliferation and colony formation of TNBC cells. (A) Cell viability of cancer cell lines treated with compound 39 using the MTT assay. (B)
Inhibitory effects of compound 39 on colony formation of TNBC cells. Representative data from three independent experiments are shown. (*) p < 0.05; (**) p < 0.01; (***)
p < 0.001; (****) p < 0.0001.
10

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
Fig. 11. Effects of compound 39 on inducing apoptosis and cleavage of PARP and caspase 7 in breast cancer cells. (A) Apoptosis induced by compound 39 at different concentrations
was examined by a annexin V-FITC/PI-staining based flow cytometry. (**) p < 0.01; (***) p < 0.001; (****) p < 0.0001. (B) Western blot analysis of the effect of compound 39 on
cleaved- PARP and cleaved-caspase7. b-Action was used as the loading control. Representative data from three independent experiments are shown.
Compound 39 inhibited cancer cell migration and invasion.
Constitutively activated STAT3 is also reported to be correlated with
cancer cell migration and invasion, which contribute to cancer
metastasis and lead to poor patient survival. Therefore, compound
39 was also investigated for its effects on cancer cell migration and
invasion in wound healing and transwell assays. In the wound
healing assay, MDA-MB-231, MDA-MB-468, MDA-MB-231-4175
and HCC70 cells were treated with compound 39 at the indicated
concentrations for 12e96 h. As shown in Fig.12A, the cells migrated
to fill the scratched area in the absence of compound 39, whereas
compound 39 treatment prevented this migration dose-
dependently. Next, the ability of compound 39 to inhibit invasion
was evaluated in transwell assay. As shown in Fig. 12 B, compound
39 inhibited invasion in MDA-MB-231, MDA-MB-231-4175, HCC70
and MDA-MB-468 cells in a dose-dependent manner. Together,
these results imply that compound 39 inhibits migration and in-
vasion of cancer cells in vitro.
compound 39 inhibited tumor growth in a dose-dependent
manner as measured by tumor volume (Fig. 13A) and weight
(Fig. 13C), with tumor growth inhibition (TGI) of 20.1% at 5 mg/kg
and 40.2% at 15 mg/kg. Meanwhile, compound 39 was found to be
well tolerated and showed no apparent body weight loss during the
treatment (Fig.13B). Compound 39 was also evaluated for its effects
on the tumor mitotic index (Ki67) and the STAT3 signaling in MDA-
MB-231-4175 tumor tissues by immunohistochemistry analysis
and western blotting. It was revealed that the expression of Ki67
was significantly reduced in compound 39-treated groups
compared to control group (Fig. 13D), suggesting that the number
of the proliferating tumor cells was significantly decreased and
tumor progression was slowed down in the mice after treatment of
compound 39. Afterwards, the expression of pY705-STAT3, STAT3,
Bcl-xL and Mcl-1 in the tumor tissue was examined by western
blotting. Compound 39 apparently reduced the level of pY705-
STAT3 and subsequently downregulated the expression of STAT3
target genes Bcl-xL and Mcl-1 as shown in Fig. 13E. It is also note-
worthy that no morphological change was observed in heart, liver,
spleen, lung and kidney tissues for compound 39 treatment groups
in HE staining (Fig. 13F). These results suggested that compound 39
inhibits the growth of MDA-MB-231-4175 xenograft tumor in vivo
through inhibiting the STAT3 signaling pathway and shows no
obvious toxicity.
In vivo Pharmacokinetic Properties of compound 39. As
compound 39 exhibited high potency in vitro, the pharmacokinetic
(PK) profiles were next evaluated in male SD rats after oral
administration at 10 mg/kg (Table 6). Compound 39 exhibited a
quick oral absorption (Tmax ¼ 1.67 h) with short half-life (T1/
¼ 1.67 h). The maximum plasma concentration (Cmax) was
2
determined to be 23.8 ng/mL and small area under the curve (AUC)
values were also observed.
Next, the antitumor efficacy of compound 39 was assessed in
TNBC patient-derived xenograft (PDX) model in mice. The PDX
model was performed in NOD/SCID mice and the mice were treated
with compound 39 via intraperitoneal administration for 20 days
with 5 mg/kg or 15 mg/kg per day. As shown in Fig. 14, compound
39 exhibited enhanced antitumor effects in the PDX model
compared to that in the MDA-MB-231-4175 xenograft model
Compound 39 exhibited antitumor activity in vivo. Upon the
basis of the promising in vitro profile of the STAT3 inhibitor com-
pound 39, we then evaluated its anticancer efficacy in vivo. First, the
in vivo antitumor effect of compound 39 was performed in the
MDA-MB-231-4175 xenograft model in mice via intraperitoneal
injection every day for 20 consecutive days. As shown in Fig. 13,
11

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
Fig. 12. Effects of compound 39 on cell migration and invasion. (A) Inhibition of cell migration by compound 39 in wound healing assay. (B) Inhibition of cell invasion by compound
39 in transwell assays. Representative images from three independent experiments are shown. (***) P < 0.001, (****) p < 0.0001.
shown in Figure S3, compound 39 exhibited more potent in vivo
efficacy than STX-119 and SH4-54 (Figure S3A, B, C) with TGI of
85.1%, 63.6% and 414.8%, respectively. No apparent body weight loss
(Figure S3D) was observed during the studies. Moreover, Com-
pound 39 also showed more potent activity in reduction of the level
of pY705-STAT3 and subsequent downregulation of the expression
of STAT3 target genes c-Myc and Bcl-xL as shown in Figure S3E.
Table 6
In vivo PK Parameters for compound 39.a.
T1/2
Tmax
(h)
Cmax
AUC0-last
(h*ng/mL)
54.6
AUC0-inf
(h*ng/mL)
56.1
(h)
(ng/mL)
23.8
1.46
1.67
a
Values are the average of three runs. Vehicle: DMSO/0.5% HPMC (5/95, v/v/). T1/
2, half-life; Tmax, time of maximum concentration; Cmax, maximum concentration;
AUC, area under the plasma concentration-time curve.
2.1. Chemistry
(Fig.14A and C), with TGI of 32.7% at 5 mg/kg and 68.9% at 15 mg/kg.
The expression of Ki67 in the tumor tissues was also decreased in
the compound 39 treatment groups in immunohistochemistry
assay (Fig. 14D), which is similar to that in MDA-MB-231-4175
xenograft mice model. The immunoblotting results showed that
compound 39 reduced the level of pY705-STAT3 and STAT3 target
genes including Bcl-xL and Mcl-1 (Fig. 14E), while HE staining
analysis displayed that no morphological change was observed in
heart, liver, spleen, lung and kidney tissues in compound 39
treatment groups (Fig. 14F). Moreover, no apparent body weight
loss (Fig. 14B) or other obvious signs of toxicity, such as loss of
appetite, decreased activity, or lethargy were observed during the
studies. Taken together, compound 39 inhibited STAT3 signaling
and tumor growth in animal studies with no obvious toxicity
in vivo.
As shown in Scheme 1, condensation of acids 62a-c with 4-
cyclohexylaniline 63a or (4-cyclohexylphenyl)methanamine 63b
afforded amides 7-9 and 12-14. Among them, hydroxyl 9 and 14
were alkylated with ethyl 2-bromoacetate to provide 10 and 15,
followed by hydrolysis under alkaline condition to give carboxylic
acid 11 and 16.
As illustrated in Scheme 2, acid 62b was condensed with
substituted amines 64a-q to afford amides 17-27. On the other
hand, amide 17 underwent SuzukiꢀMiyaura coupling reaction with
suitable arylboronic acids to give amides 28-33.
As shown in Scheme 3, 2-aminepyridine 65 was condensed with
ketone 66 to yield ester 67, which was then hydrolyzed to acid 68.
Acid 68 was coupled with aniline 63a to provide amide 34.
Imidazo[1,2-a]pyridine 69 was alkylated with ethyl diazoacetate
in the presence of a Rh(II) catalyst to give ester 70 as we reported
previously [56], which was then subjected to hydrolysis and
condensation with various amine to afford amides 35-38 (Scheme
4).
To compare the in vivo antitumor efficacy between compound
39 and the positive controls, SH4-54 and STX-119, we further
evaluated these three compounds in TNBC PDX model in mice for
20 days at 15 mg/kg per day via intraperitoneal administration. As
12

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
Fig. 13. Antitumor activity of compound 39 in MDA-MB–231-4175 xenograft model in mice. Tumor volume (A), body weight (B), and tumor weight (C) of the mice with the
indicated treatment of compound 39. (D) Tumor proliferation biomarker Ki67 detection of tumor tissues by immunohistochemistry after treatment with compound 39. (E) pY705-
STAT3, STAT3, Bcl-xL and Mcl-1 immunoblot in tumor tissue lysates. (F) Morphological images of heart, liver, spleen, lung and kidney tissues from treated mice. Values, mean SEM,
n ¼ 6. (*) P < 0.05.
Fig. 14. Antitumor activity of compound 39 in breast cancer PDX model in mice. Tumor volume (A), body weight (B), and tumor weight (C) of the mice with the indicated treatment
of compound 39. (D) Tumor proliferation biomarker Ki67 detection of tumor tissues after treatment with compound 39. (E) pY705-STAT3, STAT3, Bcl-xL and Mcl-1 immunoblot in
tumor tissue lysates. (F) Morphological images of heart, liver, spleen, lung and kidney tissues from treated mice. Values, mean SEM, n ¼ 6. (*) P < 0.05.
13

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
a
Scheme 1. Synthesis of compounds 7-16a. Reagents and conditions: (a) HOAt, EDC, DIPEA, DMF, rt, 4 h, 80%; (b) ethyl 2-bromoacetate, Cs2CO3, DMF, rt, 6 h, 76%; (c) LiOH.H2O,
MeOH-H2O (2:3), rt, 1 h, 88%.
Scheme 2. Synthesis of compounds 17-33a. a Reagents and conditions: (a) HOAt, EDC, DIPEA, DMF, rt, 4 h, 60e80%; (b) suitable arylboronic acid, Pd(PPh3)4, K2CO3, 1,4-dioxane-H2O
(4:1), reflux, overnight, 75e79%.
Scheme 3. Synthesis of compound 34a. a Reagents and conditions: (a) CBr4, CH3CN, 80 ꢁC, 4 h, 85%; (b) LiOH.H2O, MeOH-H2O (2:3), rt, 1 h, 92%; (c) 63a, HATU, DIPEA, DMF, 0oC-rt,
5 h, 85%.
a
Scheme 4. Synthesis of compounds 35-38a. Reagents and conditions: (a) ethyl diazoacetate, Rh2(oct)4, CHCl3, rt, 4 h, 85%; (b) LiOH.H2O, MeOH-H2O (2:3), rt, 1 h, 80%; (c) HOAt,
EDC, DIPEA, DMF, rt, 4 h, 65e75%.
14

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
Scheme 5. Synthesis of compounds 39-61a. a Reagents and conditions: (a) ethyl diazoacetate, Rh2(oct)4, CHCl3, rt, 4 h, 70e95%; (b) LiOH.H2O, MeOH-H2O (2:3), rt, 1 h, 82e95%; (c)
HOAt, EDC, DIPEA, DMF, rt, 4 h, 65e80%.
Recently, a novel synthetic route was developed to prepare es-
ters 73a-u [51] (Scheme 5), which were then subjected to hydro-
lysis and condensation with 6-aminobenzo[b]thiophene 1,1-
dioxide to afford amides 39-61.
(doublet of doublet), m (multiple), q (quarter), s (singlet), and t
(triplet). Coupling constants (J values) were given in Hz. HRMS (ESI)
and LC-MS (ES) were recorded on SHIMADZU LCMS-IT-TOF mass
spectrometer and Thermo TSQ QUANTUM LC-MS spectrometer,
respectively. Purity of final compounds was confirmed by analytical
HPLC analysis on an Agilent HPLC system. Conditions were as fol-
lows: XSelect HSS T3 reversed-phase column (150 mm ꢂ 4.60 mm,
3. Conclusions
5
mm); flow rate 1 mL/min; UV detection at 270 and 254 nM;
In summary, we performed structure-based drug design
approach to facilitate the discovery of novel STAT3 small-molecule
inhibitors with desired druglike properties. Starting from the
privileged structures in the previously reported STAT3 small-
gradient: from 70% methanol in 30% water to 95% methanol in 5%
water (for compound 1-33) and from 5% methanol in 95% water to
95% methanol in 5% water (for compound 34-61). The purity of all
final compounds for biological evaluation was greater than 95%.
N-(4-cyclohexylphenyl)-2-phenylquinoline-4-carboxamide
(7). To a solution of 2-phenylquinoline-4-carboxylic acid 62a
(250 mg, 1 mmol) and 4-cyclohexylaniline 63a (193 mg, 1.1 mmol)
in anhydrous DMF (5 mL) were added HOAT (205 mg, 1.5 mmol),
EDC (148 mg, 1.5 mmol) and DIPEA (194 mg, 1.5 mmol). The reac-
tion mixture was stirred at room temperature for 4 h. Then, the
mixture was poured into ice water and extracted with ethyl acetate
(10 mL ꢂ 3). The combined organic layers were washed with brine
and dried over anhydrous Na2SO4. After filtration, the solvent was
removed under vacuum and the residue was purified by silica gel
column chromatography (CHCl3/MeOH ¼ 20 : 1) to afford com-
pound 7 (324 mg, 80%) as white solid, m.p. 233e235 ꢁC. 1H NMR
molecule inhibitors,
a
novel series of substituted 2-
phenylquinolines were first designed and evaluated. Then, the
core structure of 2-phenylquinoline was optimized to 2-
arylimidazo[1,2-a]pyridine based on the molecular docking
models. Further extensive optimization led to the identification of
highly potent and selective STAT3 inhibitors, exemplified by com-
pound 39 with the core structure of 2-phenylimidazo[1,2-a]pyri-
dine. Compound 39 was able to selectively inhibit phosphorylation
of STAT3, disrupt STAT3 dimerization, impair its nuclear trans-
location and DNA-binding ability, thereby downregulate tran-
scriptional activity of STAT3, and ultimately suppress the growth,
migration and invasion of human TNBC cells lines. Moreover,
compound 39 demonstrated significant and dose-dependent tu-
mor growth inhibition in both cell line-derived and patient derived
xenograft tumor models in mice and was more potent than SH4-54
and STX-119. Overall, these studies provide an example that highly
potent and selective STAT3 small-molecule inhibitors with privi-
leged structures can be successfully developed and compound 39
with favorable in vitro and in vivo pharmacological profiles deserves
further investigation.
(400 MHz, DMSO‑d6)
d
10.74 (s, 1H), 8.36 (d, J ¼ 7.1 Hz, 2H), 8.31 (s,
1H), 8.18 (d, J ¼ 8.5 Hz, 2H), 7.86 (t, J ¼ 7.7 Hz,1H), 7.73 (d, J ¼ 8.5 Hz,
2H), 7.67 (t, J ¼ 7.6 Hz, 1H), 7.63e7.52 (m, 3H), 7.26 (d, J ¼ 8.5 Hz,
2H), 1.81 (d, J ¼ 9.3 Hz, 4H), 1.72 (d, J ¼ 12.4 Hz, 1H), 1.48e1.34 (m,
4H), 1.33e1.05 (m, 2H); 13C NMR (101 MHz, DMSO‑d6)
d 165.58,
156.32, 148.40, 144.05, 143.64, 138.63, 137.08, 130.80, 130.44, 130.10,
129.42, 127.85, 127.38, 125.58, 123.72, 120.56, 117.23, 43.77, 34.55,
26.84, 26.08; MS (EI) m/z 407.21 (M þ H)þ; HRMS (ESI) calcd for
C
28H26N2O (M þ H)þ: 407.2118; found 407.2111. HPLC retention
4. Experimental section
time 6.176 min, 100.00% pure.
Compounds 8-9 and 12-14 were prepared in a similar manner as
described for compound 7.
Chemical Reagents and General Method. All commercially
available starting materials and solvents are reagent grade and used
without further purification. Column chromatography was carried
out on silica gel (200e300 mesh) manufactured by Qingdao
Haiyang Chemical Group Co., Ltd. Analytical TLC was performed on
silica gel plates and visualized under ultraviolet light (254 nm). 1H
and 13C NMR spectra were recorded on a Broker spectrometer with
N-(4-cyclohexylphenyl)-7-methoxy-2-phenylquinoline-4-
carboxamide (8). Yellow solid, 72% yield, m.p. 211e213 ꢁC. 1H NMR
(400 MHz, DMSO‑d6)
d
10.70 (s, 1H), 8.34 (d, J ¼ 7.1 Hz, 2H), 8.14 (s,
1H), 8.08 (d, J ¼ 9.1 Hz, 1H), 7.72 (d, J ¼ 8.0 Hz, 2H), 7.62e7.48 (m,
4H), 7.31 (d, J ¼ 8.8 Hz, 1H), 7.24 (d, J ¼ 8.0 Hz, 2H), 3.98 (s, 3H), 1.80
(d, J ¼ 8.8 Hz, 4H), 1.71 (d, J ¼ 12.5 Hz, 1H), 1.51e1.32 (m, 4H),
400 or 500 MHz for proton (1H NMR), and 101 MHz for carbon (13
C
1.29e1.20 (m, 1H); 13C NMR (101 MHz, DMSO‑d6)
d 165.71, 161.13,
NMR). Chemical shifts downfield from TMS were expressed in ppm,
156.56, 150.41, 143.99, 143.37, 138.80, 137.12, 130.31, 129.36, 127.74,
and the signals are described as br (broad singlet), d (doublet), dd
15

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
127.35, 126.78, 120.56, 118.77, 115.01, 108.39, 56.08, 43.78, 34.55,
afford compound 10 (193 mg, 76%) as yellow solid, m.p.
26.85, 26.09; MS (EI) m/z 437.22 (M þ H)þ; HRMS (ESI) calcd for
246e248 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 10.71 (s, 1H), 8.33 (d,
C
29H28N2O2 (M þ H)þ: 437.2224; found 437.2228. HPLC retention
J ¼ 7.1 Hz, 2H), 8.17 (s, 1H), 8.11 (d, J ¼ 9.2 Hz, 1H), 7.72 (d, J ¼ 8.4 Hz,
2H), 7.58 (dd, J ¼ 13.9, 6.2 Hz, 3H), 7.50 (d, J ¼ 2.6 Hz, 1H), 7.38 (dd,
J ¼ 9.2, 2.6 Hz, 1H), 7.25 (d, J ¼ 8.5 Hz, 2H), 5.05 (s, 2H), 4.23 (q,
J ¼ 7.1 Hz, 2H), 1.81 (d, J ¼ 9.2 Hz, 4H), 1.72 (d, J ¼ 12.4 Hz, 1H), 1.40
(dd, J ¼ 18.4, 9.9 Hz, 4H), 1.26e1.25 (m, 2H); 13C NMR (101 MHz,
time 6.008 min, 97.58% pure.
N-(4-cyclohexylphenyl)-7-hydroxy-2-phenylquinoline-4-
carboxamide (9). Yellow solid, 78% yield, m.p. 333e335 ꢁC. 1H NMR
(400 MHz, DMSO‑d6)
d
10.31 (s, 1H), 9.45 (t, J ¼ 5.8 Hz, 1H), 8.33 (d,
J ¼ 7.3 Hz, 2H), 8.10 (d, J ¼ 9.1 Hz, 1H), 7.98 (s, 1H), 7.66 (dt, J ¼ 13.7,
6.9 Hz, 3H), 7.35 (d, J ¼ 2.2 Hz, 1H), 7.30 (d, J ¼ 7.9 Hz, 2H), 7.21 (d,
J ¼ 7.9 Hz, 2H), 7.17 (d, J ¼ 2.3 Hz,1H), 2.40 (s,1H),1.80e1.74 (m, 4H),
1.58 (d, 1H), 1.40e1.32 (m, 4H), 1.30 (s, 1H); 13C NMR (101 MHz,
DMSO‑d6) d 168.93, 165.64, 159.35, 156.74, 150.08, 144.04, 143.37,
138.70, 137.08, 130.38, 129.38, 127.76, 127.36, 127.01, 120.58, 120.35,
119.10, 115.34,109.50, 65.34, 61.30, 43.78, 34.55, 26.84, 26.08, 14.55;
MS (EI) m/z 509.24 (M þ H)þ; HRMS (ESI) calcd for C32H32N2O4
DMSO‑d6)
d
165.88, 159.53, 156.36, 150.44, 143.92, 143.40, 138.96,
(M
þ
H)þ: 509.2435; found 509.2439. HPLC retention time
137.15, 130.17, 129.30, 127.69, 127.33, 126.91, 120.51, 117.87, 114.17,
111.16, 43.77, 34.55, 26.84, 26.08; MS (EI) m/z 423.20 (M þ H)þ;
HRMS (ESI) calcd for C28H26N2O2 (M þ H)þ: 423.2067; found
423.2066. HPLC retention time 4.213 min, 100.00% pure.
5.227 min, 98.14% pure.
Ethyl
2-((4-((4-cyclohexylbenzyl)carbamoyl)-2-
phenylquinolin-7-yl)oxy)acetate (15).
Compound 15 was synthesized according to the procedure for
the preparation of compound 10.
N-(4-cyclohexylbenzyl)-2-phenylquinoline-4-carboxamide
(12). Yellow solid, 77% yield, m.p.162e164 ꢁC. 1H NMR (400 MHz,
Starting from derivative 14, compound 15 was obtained in in
DMSO‑d6)
d
9.36 (s, 1H), 8.32 (d, J ¼ 6.9 Hz, 2H), 8.24 (d, J ¼ 8.1 Hz,
72% yield, m.p. 164e166 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 9.34 (t,
1H), 8.19 (s, 1H), 8.15 (d, J ¼ 8.3 Hz, 1H), 7.83 (t, J ¼ 7.1 Hz, 1H), 7.65
(t, J ¼ 7.2 Hz, 1H), 7.61e7.52 (m, 3H), 7.35 (d, J ¼ 7.2 Hz, 2H), 7.23 (d,
J ¼ 7.3 Hz, 2H), 4.59 (s, 2H), 1.78 (d, J ¼ 9.4 Hz, 4H), 1.70 (d,
J ¼ 12.3 Hz, 1H), 1.38 (dd, J ¼ 19.3, 10.5 Hz, 4H), 1.29e1.14 (m, 2H);
J ¼ 5.6 Hz,1H), 8.28 (d, J ¼ 7.3 Hz, 2H), 8.17 (d, J ¼ 9.2 Hz,1H), 8.04 (s,
1H), 7.59e7.52 (m, 3H), 7.46 (d, J ¼ 2.1 Hz, 1H), 7.37 (d, J ¼ 2.2 Hz,
1H), 7.33 (d, J ¼ 8.0 Hz, 2H), 7.22 (d, J ¼ 7.9 Hz, 2H), 5.03 (s, 2H), 4.55
(d, J ¼ 5.6 Hz, 2H), 4.24e4.18 (m, 2H), 2.50 (s,1H),1.81e1.75 (m, 4H),
1.69 (d, J ¼ 12.4 Hz, 1H), 1.43e1.28 (m, 5H), 1.23 (d, J ¼ 7.0 Hz, 3H);
13C NMR (101 MHz, DMSO‑d6)
d 167.10, 156.29, 148.46, 146.83,
143.27, 138.76, 136.81, 130.62, 130.34, 130.05, 129.37, 127.87, 127.61,
127.20, 125.82, 123.94, 117.23, 43.98, 43.02, 34.51, 26.84, 26.08; MS
(EI) m/z 421.22 (M þ H)þ; HRMS (ESI) calcd for C29H28N2O
13C NMR (101 MHz, DMSO‑d6)
d 168.93, 167.13, 159.25, 156.71,
150.13, 146.82, 142.95, 138.82, 136.83, 130.30, 129.34, 127.92, 127.21,
120.16, 119.30, 115.35, 109.44, 65.33, 61.28, 43.98, 42.99, 34.51,
26.84, 26.08, 14.55; MS (EI) m/z 523.25 (M þ H)þ; HRMS (ESI) calcd
for C33H34N2O4 (M þ H)þ: 523.2591; found 523.2599. HPLC reten-
tion time 4.725 min, 99.66% pure.
(M
þ
H)þ: 421.2274; found 421.2268. HPLC retention time
5.463 min, 100.00% pure.
N-(4-cyclohexylbenzyl)-7-methoxy-2-phenylquinoline-4-
carboxamide (13). White solid, 80% yield, m.p. 173e175 ꢁC. 1H NMR
N-(4-cyclohexylphenyl)-7-(carboxymethoxy)-2-
(400 MHz, DMSO‑d6)
d
9.34 (t, J ¼ 5.8 Hz, 1H), 8.30 (d, J ¼ 7.1 Hz,
phenylquinoline-4-carboxamide (11). To a solution of compound
10 (152 mg, 0.3 mmol) in MeOH (4 mL) was added the solution of
LiOH.H2O (38 mg, 0.9 mmol) in H2O (6 mL). The reaction mixture
was stirred at room temperature for 1 h. Then the mixture was
concentrated in vacuum to 4 mL and was cooled to 0e4 ꢁC. The
resulting solution was acidified to pH ¼ 2 with 4 N HCl and the
precipitated solids were collected by filtration and dried to give
compound 11 (127 mg, 88%) as yellow solid, m.p. 237e239 ꢁC. 1H
2H), 8.15 (d, J ¼ 9.2 Hz, 1H), 8.02 (s, 1H), 7.56 (q, J ¼ 14.5, 6.9 Hz, 3H),
7.52 (d, J ¼ 2.3 Hz, 1H), 7.33 (d, J ¼ 8.0 Hz, 2H), 7.29 (dd, J ¼ 9.2,
2.6 Hz, 1H), 7.22 (d, J ¼ 8.1 Hz, 2H), 4.55 (d, J ¼ 5.8 Hz, 2H), 3.96 (s,
3H), 2.46 (s, 1H), 1.77 (d, J ¼ 10.7 Hz, 4H), 1.69 (d, J ¼ 12.4 Hz, 1H),
1.37 (dd, J ¼ 18.5, 9.7 Hz, 4H),1.26e1.23 (m,1H); 13C NMR (101 MHz,
DMSO‑d6)
d 167.22, 161.03, 156.52, 150.45, 146.81, 142.95, 138.90,
136.85, 130.23, 129.32, 127.81, 127.10, 120.30, 118.97, 115.00, 108.31,
56.03, 43.98, 42.98, 34.51, 26.84, 26.08; MS (EI) m/z 451.23
(M þ H)þ; HRMS (ESI) calcd for C30H30N2O2 (M þ H)þ: 451.2380;
found 451.2373. HPLC retention time 5.313 min, 100.00% pure.
N-(4-cyclohexylbenzyl)-7-hydroxy-2-phenylquinoline-4-
carboxamide (14). Yellow solid, 84% yield, m.p. 158e160 ꢁC. 1H
NMR (500 MHz, DMSO‑d6)
d
10.73 (s, 1H), 8.34 (d, J ¼ 7.4 Hz, 2H),
8.16 (s, 1H), 8.10 (d, J ¼ 9.2 Hz,1H), 7.73 (d, J ¼ 8.4 Hz, 2H), 7.59e7.55
(m, 2H), 7.46 (d, J ¼ 2.3 Hz, 1H), 7.36 (dd, J ¼ 9.2, 2.4 Hz, 1H), 7.25 (d,
J ¼ 8.4 Hz, 2H), 4.92 (s, 2H), 2.47 (s, 1H), 1.83e1.78 (m, 4H), 1.71 (d,
J ¼ 12.6 Hz, 1H), 1.47e1.33 (m, 5H); 13C NMR (101 MHz, DMSO‑d6)
NMR (400 MHz, DMSO‑d6)
d
10.32 (s, 1H), 9.27 (t, J ¼ 5.8 Hz, 1H),
d 170.38, 165.67, 159.59, 156.64, 150.12, 144.00, 143.36, 138.73,
8.25 (d, J ¼ 7.3 Hz, 2H), 8.07 (d, J ¼ 9.1 Hz, 1H), 7.91 (s, 1H), 7.54 (dt,
J ¼ 13.7, 6.9 Hz, 3H), 7.36 (d, J ¼ 2.2 Hz, 1H), 7.32 (d, J ¼ 7.9 Hz, 2H),
7.22 (d, J ¼ 7.9 Hz, 2H), 7.19 (d, J ¼ 2.3 Hz, 1H), 4.54 (d, J ¼ 5.7 Hz,
2H), 2.46 (s, 1H), 1.81e1.74 (m, 4H), 1.68 (d, 1H), 1.42e1.33 (m, 4H),
137.11, 130.34, 129.36, 127.76, 127.35, 126.90, 120.57, 118.97, 115.19,
109.34, 65.41, 43.78, 34.55, 26.85, 26.09; MS (EI) m/z 481.24
(M þ H)þ; HRMS (ESI) calcd for C30H28N2O4 (M þ H)þ: 481.2122;
found 481.2144. HPLC retention time 3.815 min, 100.00% pure.
2-((4-((4-cyclohexylbenzyl)carbamoyl)-2-phenylquinolin-7-yl)
oxy)acetic acid (16).
Compound 16 was synthesized according to the procedure for
the preparation of compound 11. Compound 15 was used to afford
compound 16 in 79% yield, m.p. 162e163 ꢁC. 1H NMR (500 MHz,
1.23 (s, 1H); 13C NMR (101 MHz, DMSO‑d6)
d 167.40, 159.42, 156.38,
150.49, 146.80, 142.99, 139.06, 136.88, 130.11, 129.29, 127.89, 127.67,
127.19, 120.23, 118.07, 114.21, 111.08, 43.97, 42.93, 34.51, 26.83,
26.07; MS (EI) m/z 437.22 (M þ H)þ; HRMS (ESI) calcd for
C
29H28N2O2 (M þ H)þ: 437.2224; found 437.2219. HPLC retention
time 3.822 min, 97.91% pure.
DMSO‑d6)
d
9.35 (t, J ¼ 5.0 Hz, 1H), 8.28 (d, J ¼ 7.3 Hz, 2H), 8.10 (d,
N-(4-cyclohexylphenyl)-7-(2-ethoxy-2-oxoethoxy)-2-
J ¼ 9.1 Hz, 1H), 7.98 (s, 1H), 7.57e7.54 (m, 2H), 7.53e7.50 (m, 1H),
7.33 (d, J ¼ 6.3 Hz, 4H), 7.28 (d, J ¼ 9.1 Hz,1H), 7.23 (d, J ¼ 7.6 Hz, 2H),
4.57 (s, 2H), 4.55 (d, J ¼ 5.3 Hz, 2H), 1.91 (s, 1H), 1.80e1.76 (m, 4H),
1.70 (d, J ¼ 12.2 Hz, 1H), 1.42e1.36 (m, 4H), 1.24 (s, 1H); 13C NMR
phenylquinoline-4-carboxamide (10). To a mixture of compound
9 (210 mg, 0.5 mmol) and Cs2CO3 (488 mg, 1.5 mmol) in anhydrous
DMF was added ethyl bromoacetate (100 mg, 0.6 mmol) dropwise.
The reaction mixture was stirred at room temperature for 4 h. Then,
the mixture was poured into ice water and extracted with ethyl
acetate (10 mL ꢂ 3). The combined organic layers were washed
with brine and dried over anhydrous Na2SO4. After filtration, the
solvent was removed under vacuum and the residue was purified
by silica gel column chromatography (CHCl3/MeOH ¼ 20 : 1) to
(101 MHz, DMSO‑d6) d 170.66,167.28,160.04,156.45,150.25,146.80,
142.90, 138.87, 136.85, 130.20, 129.32, 127.89, 127.20, 126.80, 120.50,
118.88, 114.86, 109.18, 66.73, 43.97, 42.96, 34.51, 26.83, 26.07; MS
(EI) m/z 495.22 (M þ H)þ; HRMS (ESI) calcd for C31H30N2O4
(M
þ
H)þ: 495.2278; found 495.2270. HPLC retention time
5.044 min, 98.49% pure.
16

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
Compounds 17-27 were synthesized according to the procedure
for the preparation of compound 7.
33.71, 22.15,14.27. MS (EI) m/z 411.20(M þ H)þ; HRMS (ESI) calcd for
C
27H26N2O2 (M þ H)þ: 411.2067; found 411.2060. HPLC retention
N-(4-bromophenyl)-7-methoxy-2-phenylquinoline-4-
time 17.986min, 100.00% pure.
carboxamide (17). Yellow solid, 80% yield, m.p. 212e214 ꢁC. 1H
N-(4-isopropylphenyl)-7-methoxy-2-phenylquinoline-4-
NMR (400 MHz, DMSO‑d6)
d
10.96 (s, 1H), 8.36 (d, J ¼ 7.2 Hz, 2H),
carboxamide (23). Yellow solid, 74% yield, m.p. 260e262 ꢁC. 1H
8.20 (s,1H), 8.08 (d, J ¼ 9.1 Hz,1H), 7.82 (d, J ¼ 8.3 Hz, 2H), 7.64e7.52
NMR (400 MHz, DMSO‑d6)
d
10.72 (s, 1H), 8.34 (d, J ¼ 7.1 Hz, 2H),
(m, 6H), 7.33 (d, J ¼ 8.9 Hz, 1H), 3.99 (s, 3H); 13C NMR (101 MHz,
8.15 (s,1H), 8.08 (d, J ¼ 9.2 Hz,1H), 7.73 (d, J ¼ 8.4 Hz, 2H), 7.63e7.53
(m, 4H), 7.31 (dd, J ¼ 9.2, 2.6 Hz, 1H), 7.27 (d, J ¼ 8.4 Hz, 2H), 3.98 (s,
3H), 2.90 (dt, J ¼ 13.9, 6.8 Hz, 1H), 1.22 (d, J ¼ 6.9 Hz, 6H); 13C NMR
DMSO‑d6)
d 166.04, 161.16, 156.56, 150.41, 142.88, 138.73, 132.13,
130.35, 129.35, 127.77, 126.71, 122.44, 120.60, 118.63, 116.30, 115.18,
108.41, 56.09; MS (EI) m/z 433.05 (M þ H)þ; HRMS (ESI) calcd for
(101 MHz, DMSO‑d6) d 165.73, 161.10, 150.41, 144.73, 143.24, 138.84,
C
23H17BrN2O2 (M þ H)þ: 433.0546; found 433.0544. HPLC retention
137.14, 130.29, 129.33, 127.78, 126.95, 120.64, 120.47, 118.80, 115.15,
108.38, 56.07, 33.45, 24.45; MS (EI) m/z 397.19 (M þ H)þ; HRMS
(ESI) calcd for C26H24N2O2 (M þ H)þ: 397.1911; found 397.1917.
HPLC retention time 13.625 min, 100.00% pure.
time 13.237 min, 100.00% pure.
N-(4-fluorophenyl)-7-methoxy-2-phenylquinoline-4-
carboxamide (18). Yellow solid, 76% yield, m.p. 234e236 ꢁC.1H
NMR (400 MHz, DMSO‑d6)
d
11.05 (s, 1H), 8.37 (d, J ¼ 7.2 Hz, 2H),
N-([1,1′-biphenyl]-4-yl)-7-methoxy-2-phenylquinoline-4-
8.20 (s, 1H), 8.11 (d, J ¼ 9.2 Hz, 1H), 7.90 (dd, J ¼ 8.9, 5.1 Hz, 2H),
carboxamide (24). Yellow solid, 62% yield, m.p. 213e214 ꢁC.1H
7.60e7.51 (m, 4H), 7.32 (dd, J ¼ 9.2, 2.5 Hz, 1H), 7.25 (t, J ¼ 8.8 Hz,
NMR (400 MHz, DMSO‑d6)
d 10.90 (s, 1H), 8.36 (d, 2H), 8.21 (s, 1H),
2H), 3.98 (s, 3H); 13C NMR (126 MHz, DMSO‑d6)
d
165.84, 161.10,
8.11 (d, J ¼ 9.2 Hz, 1H), 7.94 (d, J ¼ 8.6 Hz, 2H), 7.73 (dd, J ¼ 14.8,
158.96 (d, J ¼ 240 Hz), 156.57, 150.41, 142.88, 138.81, 135.81, 130.30,
129.33, 127.81, 126.80, 122.38, 120.51, 118.74, 115.85 (d, J ¼ 22.5 Hz),
115.33, 108.38, 56.08; MS (EI) m/z 373.13 (M þ H)þ; HRMS (ESI)
calcd for C23H17FN2O2 (M þ H)þ: 373.1347; found 373.1351. HPLC
retention time 10.037 min, 100.00% pure.
8.1 Hz, 4H), 7.62e7.54 (m, 4H), 7.48 (t, J ¼ 7.7 Hz, 2H), 7.38e7.32 (m,
2H), 4.00 (s, 3H); 13C NMR (101 MHz, DMSO‑d6)
d 165.97, 161.17,
156.59, 150.45, 143.21, 140.11, 138.79, 136.25, 130.34, 129.42, 127.77,
127.48, 126.83, 120.87, 120.59, 118.74, 115.10, 108.43, 56.08; MS (EI)
m/z 431.17 (M þ H)þ; HRMS (ESI) calcd for C29H22N2O2 (M þ H)þ:
431.1754; found 431.1753. HPLC retention time 14.596 min, 100.00%
pure.
N-(4-chlorophenyl)-7-methoxy-2-phenylquinoline-4-
carboxamide (19). Yellow solid, 76% yield, m.p. 210e212 ꢁC. 1H
NMR (400 MHz, DMSO‑d6)
d
10.92 (s, 1H), 8.35 (d, J ¼ 7.1 Hz, 2H),
N-(4′-isopropyl-[1,1′-biphenyl]-4-yl)-7-methoxy-2-
phenylquinoline-4-carboxamide (25). White solid, 70% yield, m.p.
8.20 (s, 1H), 8.08 (d, J ¼ 9.2 Hz, 1H), 7.86 (d, J ¼ 8.8 Hz, 2H),
7.63e7.52 (m, 4H), 7.48 (d, J ¼ 8.8 Hz, 2H), 7.32 (dd, J ¼ 9.2, 2.5 Hz,
248e250 ꢁC. 1H NMR (500 MHz, DMSO‑d6)
d 10.89 (s, 1H), 8.37 (d,
1H), 3.98 (s, 3H); 13C NMR (101 MHz, DMSO‑d6)
d
166.03, 161.17,
J ¼ 7.2 Hz, 2H), 8.20 (s, 1H), 8.11 (d, J ¼ 9.0 Hz,1H), 7.91 (d, J ¼ 7.8 Hz,
2H), 7.70 (d, J ¼ 7.9 Hz, 2H), 7.63e7.52 (m, 6H), 7.33 (d, J ¼ 7.7 Hz,
3H), 3.99 (s, 3H), 2.98e2.87 (m, 1H), 1.24 (d, J ¼ 6.6 Hz, 6H); 13C
156.57, 150.43, 142.94, 138.74, 138.30, 130.34, 129.23, 128.24, 127.76,
126.71, 122.06, 120.61, 118.65, 115.13, 108.43, 56.08; MS (EI) m/z
389.10 (M þ H)þ; HRMS (ESI) calcd for C23H17ClN2O2 (M þ H)þ:
389.1051; found 389.1059. HPLC retention time 12.555 min,
100.00% pure.
NMR (101 MHz, DMSO‑d6)
d 165.91, 161.16, 156.58, 150.43, 147.88,
143.23, 138.78, 138.55, 137.69, 136.28, 130.34, 129.37, 127.77, 127.34,
126.78, 120.84, 120.58, 118.74, 115.08, 108.42, 56.09, 33.57, 24.35;
MS (EI) m/z 473.22 (M þ H)þ; HRMS (ESI) calcd for C32H28N2O2
7-Methoxy-2-phenyl-N-(p-tolyl)quinoline-4-carboxamide
(20). Yellow solid, 68% yield, m.p. 209e211 ꢁC. 1H NMR (400 MHz,
(M
þ
H)þ: 473.2224; found 473.2224. HPLC retention time
DMSO‑d6)
d
10.71 (s, 1H), 8.36 (d, J ¼ 7.2 Hz, 2H), 8.16 (s, 1H), 8.09 (d,
15.478 min, 100.00% pure.
J ¼ 9.2 Hz, 1H), 7.71 (d, J ¼ 8.3 Hz, 2H), 7.61e7.53 (m, 4H), 7.32 (dd,
J ¼ 9.2, 2.5 Hz, 1H), 7.22 (d, J ¼ 8.3 Hz, 2H), 3.99 (s, 3H), 2.32 (s, 3H);
N-(4′-fluoro-[1,1′-biphenyl]-4-yl)-7-methoxy-2-
phenylquinoline-4-carboxamide (26). White solid, 66% yield, m.p.
13C NMR (101 MHz, DMSO‑d6)
d
171.78, 165.72, 161.12, 156.57,
255e257 ꢁC. 1H NMR (500 MHz, DMSO‑d6)
d 10.91 (s, 1H), 8.37 (d,
150.41, 143.33, 138.80, 136.85, 133.60, 130.31, 129.66, 129.35, 127.76,
126.79, 120.50, 118.77, 115.04, 108.38, 56.07, 21.03; MS (EI) m/z
369.15 (M þ H)þ; HRMS (ESI) calcd for C24H20N2O2 (M þ H)þ:
369.1598; found 369.1593. HPLC retention time 10.714 min,
100.00% pure.
J ¼ 7.4 Hz, 2H), 8.21 (s, 1H), 8.11 (d, J ¼ 9.1 Hz,1H), 7.93 (d, J ¼ 8.2 Hz,
2H), 7.73 (t, J ¼ 9.8 Hz, 4H), 7.61e7.52 (m, 4H), 7.31 (q, J ¼ 20.1,
10.9 Hz, 3H), 3.99 (s, 3H). 13C NMR (101 MHz, DMSO‑d6)
d 165.97,
163.17, 161.17, 156.58, 150.43, 143.19, 138.77, 136.61, 135.21, 130.35,
129.37, 128.80, 127.77, 127.44, 126.75, 120.86, 120.60, 118.72, 116.27,
116.10, 115.09, 108.42, 56.09; MS (EI) m/z 449.16 (M þ H)þ; HRMS
(ESI) calcd for C29H21FN2O2 (M þ H)þ: 449.1660; found 449.1660.
HPLC retention time 14.603 min, 100.00% pure.
7-Methoxy-N-(4-methoxyphenyl)-2-phenylquinoline-4-
carboxamide (21). Yellow solid, 70% yield, m.p. 208e210 ꢁC. 1H
NMR (400 MHz, DMSO‑d6)
d
10.68 (s, 1H), 8.36 (d, J ¼ 6.8 Hz, 2H),
8.16 (s, 1H), 8.11 (d, J ¼ 9.0 Hz, 1H), 7.75 (d, J ¼ 8.1 Hz, 2H), 7.57 (d,
7-Methoxy-N-(4′-methoxy-[1,1′-biphenyl]-4-yl)-2-
J ¼ 12.5 Hz, 4H), 7.32 (d, J ¼ 8.8 Hz, 1H), 6.99 (d, J ¼ 8.1 Hz, 2H), 3.99
phenylquinoline-4-carboxamide (27). Yellow solid, 72% yield,
(s, 3H), 3.78 (s, 3H); 13C NMR (101 MHz, DMSO‑d6)
d
165.49, 161.11,
m.p. 252e254 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 10.86 (s, 1H), 8.36
156.31, 150.43, 143.32, 138.84, 132.48, 130.28, 129.34, 127.77, 126.86,
122.10, 120.45, 118.83, 115.10, 114.39, 108.37, 56.06, 55.70; MS(EI) m/
z 385.15(M þ H)þ; HRMS (ESI) calcd for C24H20N2O3 (M þ H)þ:
385.1547; found 385.1550. HPLC retention time 8.678 min, 100.00%
pure.
(d, J ¼ 7.0 Hz, 2H), 8.20 (s, 1H), 8.10 (d, J ¼ 9.2 Hz, 1H), 7.89 (d,
J ¼ 8.7 Hz, 2H), 7.66 (dd, J ¼ 16.2, 8.7 Hz, 4H), 7.62e7.52 (m, 4H), 7.33
(dd, J ¼ 9.2, 2.6 Hz, 1H), 7.09e6.99 (m, 2H), 3.99 (s, 3H), 3.80 (s, 3H);
13C NMR (101 MHz, DMSO‑d6)
d 165.87, 161.16, 159.20, 156.58,
150.42, 143.25, 138.77, 138.15, 136.00, 132.51, 130.34, 129.37, 127.91,
126.83, 120.86, 120.58, 118.73, 115.07, 114.85, 108.41, 56.09, 55.66;
MS (EI) m/z 461.18 (M þ H)þ; HRMS (ESI) calcd for C30H24N2O3
N-(4-butylphenyl)-7-methoxy-2-phenylquinoline-4-
carboxamide (22). Yellow solid, 74% yield, m.p. 244e246 ꢁC. 1H
NMR (400 MHz, DMSO‑d6)
d
10.75 (s, 1H), 8.35 (d, J ¼ 6.8 Hz, 2H),
(M
þ
H)þ: 461.1860; found 461.1860. HPLC retention time
8.16 (s, 1H), 8.09 (d, J ¼ 9.1 Hz, 1H), 7.73 (d, J ¼ 7.9 Hz, 2H), 7.57 (d,
J ¼ 12.7 Hz, 4H), 7.32 (d, J ¼ 8.7 Hz, 1H), 7.22 (d, J ¼ 7.7 Hz, 2H), 3.99
(s, 3H), 2.58 (t, J ¼ 7.1 Hz, 2H), 1.61e1.52 (m, 2H), 1.38e1.29 (m, 2H),
14.045 min, 98.26% pure.
Methoxy-N-(4'-(methylthio)-[1,1′-biphenyl]-4-yl)-2-
phenylquinoline-4-carboxamide (28). To a mixture of compound
17 (215 mg, 0.5 mmol) and K2CO3 (207 mg,1.5 mmol) in 1,4-dioxane
(4 mL) and water (1 mL) was added 4-(methylthio)phenyl boronic
acid (100 mg, 0.6 mmol). The mixture was degassed before addition
0.92 (t, J ¼ 7.0 Hz, 3H); 13C NMR (101 MHz, DMSO‑d6)
d 165.73,
161.10, 156.57, 150.42, 143.23, 138.85, 138.58, 137.08, 130.27, 129.32,
128.97, 127.78, 126.82, 120.56, 118.81, 115.17, 108.39, 56.07, 34.79,
17

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
of tetrakis(triphenylphosphine)palladium (58 mg, 0.05 mmol) un-
der nitrogen atmosphere. The mixture was heated under reflux
overnight and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (CHCl3/
MeOH ¼ 20 : 1) to afford compound 28 (179 mg, 75%) as white
NMR (400 MHz, DMSO‑d6) d 11.15 (s, 1H), 10.85 (s, 1H), 8.38 (dd,
J ¼ 8.3, 1.3 Hz, 2H), 8.22 (s, 1H), 8.14 (d, J ¼ 9.2 Hz, 1H), 7.92 (d,
J ¼ 8.7 Hz, 2H), 7.88e7.85 (m, 1H), 7.73 (d, J ¼ 8.7 Hz, 2H), 7.62e7.52
(m, 4H), 7.50 (d, J ¼ 8.4 Hz, 1H), 7.44 (dd, J ¼ 8.5, 1.7 Hz, 1H), 7.39 (t,
1H), 7.35 (dd, J ¼ 9.2, 2.6 Hz,1H), 6.52e6.47 (m,1H), 4.00 (s, 3H); 13
C
solid, m.p. 239e241 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d
10.89 (s,
NMR (101 MHz, DMSO‑d6) d 165.83, 161.16, 156.59, 150.44, 143.33,
1H), 8.39e8.35 (m, 2H), 8.21 (s, 1H), 8.11 (d, J ¼ 9.2 Hz, 1H), 7.92 (d,
138.80, 138.17, 137.70, 135.86, 131.34, 130.34, 129.37, 128.75, 127.78,
127.32, 126.81, 126.54, 120.88, 120.64, 118.78, 118.28, 115.08, 112.27,
108.42, 101.98, 56.09; MS (EI) m/z 470.18 (M þ H)þ; HRMS (ESI)
calcd for C31H23N3O2 (M þ H)þ: 470.1863; found 470.1857. HPLC
retention time 11.307 min, 98.85% pure.
Compound 34 was synthesized according to the procedure for
the preparation of compound 7.
N-(4-cyclohexylphenyl)-7-methoxy-2-phenylimidazo[1,2-a]
pyridine-3-carboxamide (34). White solid, 82% yield, m.p.
J ¼ 8.7 Hz, 2H), 7.73 (d, J ¼ 8.7 Hz, 2H), 7.66 (d, J ¼ 8.5 Hz, 2H),
7.61e7.53 (m, 4H), 7.38e7.32 (m, 3H), 3.99 (s, 3H), 2.52 (s, 3H); 13
C
NMR (101 MHz, DMSO‑d6)
d 165.92, 161.16, 156.58, 150.42, 143.20,
138.73, 137.63, 136.60, 135.56, 135.12, 130.35, 129.37, 127.77, 127.01,
124.91, 121.04, 120.73, 118.72, 115.09, 108.42, 56.09, 15.22; MS (EI)
m/z 477.16 (M þ H)þ; HRMS (ESI) calcd for C30H24N2O2S (M þ H)þ:
477.1631; found 477.1631. HPLC retention time 14.747 min, 100.00%
pure.
Compounds 29-33 were synthesized according to the procedure
for the preparation of compound 28.
237e240 ꢁC. 1H NMR (500 MHz, CDCl3)
d
9.42 (d, J ¼ 7.6 Hz,1H), 7.74
(d, J ¼ 6.9 Hz, 2H), 7.54 (d, J ¼ 6.3 Hz, 3H), 7.48 (s, 1H), 7.16 (d,
J ¼ 7.9 Hz, 2H), 7.10 (d, J ¼ 7.8 Hz, 2H), 6.96 (s, 1H), 6.69 (d, J ¼ 7.6 Hz,
1H), 3.90 (s, 3H), 2.44 (s, 1H), 1.82 (d, J ¼ 7.4 Hz, 4H), 1.73 (d,
J ¼ 12.5 Hz, 1H), 1.36 (t, J ¼ 9.6 Hz, 4H), 1.26 (s, 1H); 13C NMR
7-Methoxy-2-phenyl-N-(4-(thiophen-2-yl)phenyl)quinoline-
4-carboxamide (29). White solid, 63% yield, m.p. 272e274 ꢁC. 1H
NMR (400 MHz, DMSO‑d6)
d
10.86 (s, 1H), 8.36 (d, J ¼ 7.0 Hz, 2H),
8.21 (s, 1H), 8.10 (d, J ¼ 9.2 Hz, 1H), 7.93e7.84 (m, 3H), 7.78 (d,
J ¼ 8.7 Hz, 2H), 7.65 (dd, J ¼ 5.0, 2.9 Hz, 1H), 7.62e7.51 (m, 5H), 7.33
(dd, J ¼ 9.2, 2.6 Hz, 1H), 3.99 (s, 3H); 13C NMR (101 MHz, DMSO‑d6)
(101 MHz, CDCl3) d 159.85, 158.89, 148.82, 148.59, 144.12, 135.38,
134.03, 129.88, 129.64, 129.16, 129.03, 127.27, 119.58, 113.99, 108.25,
94.93, 55.64, 44.00, 34.49, 26.87, 26.13; MS (EI) m/z 426.21
(M þ H)þ; HRMS (ESI) calcd for C27H27N3O2 (M þ H)þ: 426.2176;
found 426.2181. HPLC retention time 12.815min, 100.00% pure.
Ethyl 2-(7-methoxy-2-phenylimidazo[1,2-a]pyridin-3-yl)ac-
d
165.87, 161.15, 156.57, 150.42, 143.19, 141.51, 138.77, 138.37, 131.49,
130.34, 129.37, 127.77, 127.54, 126.91, 126.53, 120.76, 120.58, 118.72,
115.09, 108.41, 56.09; MS (EI) m/z 437.12 (M þ H)þ; HRMS (ESI)
calcd for C27H20N2O2S (M þ H)þ: 437.1318; found 437.1320. HPLC
retention time 13.290 min, 100.00% pure.
etate (71).
phenylimidazo[1,2-a]pyridine (450 mg,
A
dried flask was charged with 7-methoxy-2-
mmol), Ph2(oct)4
2
7-Methoxy-2-phenyl-N-(4-(pyridin-3-yl)phenyl)quinoline-4-
(78 mg, 0.1 mmol), and CHCl3 (4 mL). Diazoester (342 mg, 3 mmol)
was added into the mixture dropwise under a nitrogen atmosphere.
The reaction mixture was stirred at room temperature for 4 h and
then concentrated in vacuum. The crud product was purified by
chromatography (EtOAc: Hexane ¼ 1 : 2) to yield compound 71
carboxamide (30). White solid, 69% yield, m.p.118e120 ꢁC. 1H NMR
(400 MHz, DMSO‑d6)
d
10.96 (s, 1H), 8.95 (d, J ¼ 1.9 Hz, 1H), 8.57
(dd, J ¼ 4.7, 1.5 Hz, 1H), 8.37 (d, J ¼ 7.0 Hz, 2H), 8.22 (s, 1H),
8.15e8.07 (m, 2H), 7.97 (d, J ¼ 8.7 Hz, 2H), 7.82 (d, J ¼ 8.7 Hz, 2H),
7.64e7.54 (m, 4H), 7.50 (dd, J ¼ 7.9, 4.8 Hz, 1H), 7.34 (dd, J ¼ 9.2,
(435 mg, 70%) as white solid. 1H NMR (400 MHz, CDCl3)
d 7.99e7.94
2.6 Hz, 1H), 3.99 (s, 3H); 13C NMR (101 MHz, DMSO‑d6)
d
166.05,
(m,1H), 7.81 (d, J ¼ 7.2 Hz, 2H), 7.46 (dd, J ¼ 10.3, 4.9 Hz, 2H), 7.37 (d,
J ¼ 7.2 Hz, 1H), 6.97 (d, J ¼ 1.7 Hz, 1H), 6.58 (dd, J ¼ 7.4, 2.4 Hz, 1H),
4.21 (dd, J ¼ 7.1, 3.2 Hz, 2H), 3.98 (s, 2H), 3.86 (s, 3H),1.27 (s, 3H); MS
(EI) m/z 310.13 (M þ H)þ.
161.18, 156.59, 150.43, 148.69, 147.87, 143.13, 139.45, 138.76, 135.50,
134.17, 133.05, 130.36, 129.37, 127.74, 126.74, 124.35, 120.97, 120.62,
118.69, 115.10, 108.43, 56.10; MS (EI) m/z 432.17 (M þ H)þ; HRMS
(ESI) calcd for C28H21N3O2 (M þ H)þ: 432.1707; found 432.1709.
HPLC retention time 9,878 min, 100.00% pure.
Compound 72 was synthesized according to the procedure for
the preparation of compound 11.
N-(4-(furan-2-yl)phenyl)-7-methoxy-2-phenylquinoline-4-
2-(7-methoxy-2-phenylimidazo[1,2-a]pyridin-3-yl)acetic
carboxamide (31). White solid, 61% yield, m.p. 264e266 ꢁC. 1H
acid (72). White solid, 75% yield. 1H NMR (500 MHz, CD3OD)
d 8.64
NMR (400 MHz, DMSO‑d6)
d
10.91 (s, 1H), 8.37 (d, J ¼ 7.0 Hz, 2H),
(s, 1H), 7.69 (s, 2H), 7.64e7.56 (m, 3H), 7.33 (s, 1H), 7.19 (s, 1H), 4.19
8.22 (s, 1H), 8.12 (d, J ¼ 9.2 Hz, 1H), 7.91 (d, J ¼ 8.8 Hz, 2H), 7.76 (d,
J ¼ 8.6 Hz, 3H), 7.61e7.51 (m, 4H), 7.34 (dd, J ¼ 9.2, 2.6 Hz, 1H), 6.92
(d, J ¼ 3.0 Hz, 1H), 6.61 (dd, J ¼ 3.3,1.8 Hz, 1H), 3.99 (s, 3H); 13C NMR
(s, 2H), 4.08 (s, 3H); MS (EI) m/z 282.10 (M þ H)þ.
Compounds 35-38 were synthesized according to the procedure
for the preparation of compound 7.
(101 MHz, DMSO‑d6)
d
166.34,161.58,156.99,153.82,150.85,143.49,
N-(4-cyclohexylphenyl)-2-(7-methoxy-2-phenylimidazo[1,2-
a]pyridin-3-yl)acetamide (35). White solid, 72% yield, m.p.
139.18, 130.76, 129.78, 128.19, 127.18, 124.88, 121.15, 119.13, 115.54,
112.99, 108.83, 106.08, 56.50; MS (EI) m/z 421.15 (M þ H)þ; HRMS
(ESI) calcd for C27H20N2O3(M þ H)þ: 421.1547; found 421.1544.
HPLC retention time 12.966 min, 100.00% pure.
207e209 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 10.35 (s, 1H), 8.29 (d,
J ¼ 7.1 Hz, 1H), 7.80 (d, J ¼ 7.1 Hz, 2H), 7.57e7.44 (m, 4H), 7.35 (t,
J ¼ 6.4 Hz, 1H), 7.16 (d, J ¼ 7.6 Hz, 2H), 7.00 (s, 1H), 6.72e6.64 (m,
1H), 4.21 (s, 2H), 3.86 (s, 3H), 2.47e2.39 (m, 1H), 1.81e1.72 (m, 4H),
1.71e1.66 (m, 1H), 1.40e1.31 (m, 4H), 1.26e1.22 (m, 1H); 13C NMR
N-(4-(benzofuran-2-yl)phenyl)-7-methoxy-2-
phenylquinoline-4-carboxamide (32). Yellow solid, 77% yield,
m.p. 247e249 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d
11.00 (s, 1H), 8.37
(101 MHz, DMSO‑d6) d 167.71, 157.80, 145.77, 143.28, 142.53, 137.26,
(d, J ¼ 7.0 Hz, 2H), 8.23 (s, 1H), 8.11 (d, J ¼ 9.2 Hz, 1H), 7.98 (s, 4H),
7.68e7.62 (m, 2H), 7.61e7.52 (m, 4H), 7.40 (d, J ¼ 0.6 Hz, 1H),
7.36e7.25 (m, 3H), 3.99 (s, 3H); 13C NMR (101 MHz, DMSO‑d6)
135.16, 128.97, 128.10, 127.70, 127.29, 126.13, 119.85, 114.59, 106.86,
94.74, 56.06, 43.68, 34.53, 31.99, 26.83, 26.07; MS (EI) m/z 440.22
(M þ H)þ; HRMS (ESI) calcd for C28H29N3O2 (M þ H)þ: 440.2333;
found 440.2336. HPLC retention time 19.171 min, 100.00% pure.
([1,1′-biphenyl]-4-yl)-2-(7-methoxy-2-phenylimidazo[1,2-a]
pyridin-3-yl)acetamide (36). White solid, 76% yield, m.p.
d
166.06, 161.18, 156.58, 155.59, 154.62, 150.43, 143.02, 139.89,
138.74, 130.36, 129.37, 127.77, 126.74, 125.84, 124.89, 123.73, 121.50,
120.73, 118.67, 115.15, 111.51, 108.43, 101.76, 56.10; MS (EI) m/z
471.17 (M þ H)þ; HRMS (ESI) calcd for C31H22N2O3 (M þ H)þ:
471.1703; found 471.1701. HPLC retention time 17.385 min, 100.00%
pure.
217e218 ꢁC. 1H NMR (500 MHz, DMSO‑d6)
d 10.46 (s, 1H), 8.24 (d,
J ¼ 7.5 Hz,1H), 7.71 (d, J ¼ 7.6 Hz, 2H), 7.66 (d, J ¼ 8.1 Hz, 2H), 7.58 (d,
J ¼ 8.0 Hz, 4H), 7.39 (dt, J ¼ 14.9, 7.4 Hz, 4H), 7.28 (dt, J ¼ 11.8, 6.7 Hz,
N-(4-(1H-indol-6-yl)phenyl)-7-methoxy-2-phenylquinoline-
2H), 6.93 (s, 1H), 6.62 (d, J ¼ 7.4 Hz, 1H), 4.18 (s, 2H), 3.79 (s, 3H); 13
C
4-carboxamide (33). Yellow solid, 79% yield, m.p. 228e230 ꢁC. 1H
NMR (101 MHz, DMSO‑d6)
d 168.04, 157.76, 145.84, 142.67, 140.12,
18

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
138.95, 135.59, 135.24, 129.38, 129.01, 128.07, 127.79, 127.57, 126.73,
126.16,120.11,114.43, 106.84, 94.80, 56.08, 32.10; MS (EI) m/z 434.18
(M þ H)þ; HRMS (ESI) calcd for C28H23N3O2 (M þ H)þ: 434.1865;
found 434.1863. HPLC retention time 13.844 min, 100.00% pure.
N-(4-(furan-2-yl)phenyl)-2-(7-methoxy-2-phenylimidazo
[1,2-a]pyridin-3-yl)acetamide(37). White solid, 74% yield, m.p.
yl)acetic acid (74i). White solid, 86% yield. 1H NMR
(400 MHz,CD3OD)
d
8.82 (s, 1H), 8.09 (s, 1H), 7.91 (d, J ¼ 8.2 Hz, 4H),
7.77 (d, J ¼ 6.4 Hz, 2H), 7.70e7.66 (m, 2H), 7.15 (d, J ¼ 8.2 Hz, 2H),
4.29 (s, 2H), 3.90 (s, 3H); MS (EI) m/z 358.13(M þ H)þ.
2-(7-(1-methyl-1H-pyrazol-4-yl)-2-phenylimidazo[1,2-a]pyr-
idin-3-yl)acetic acid (74j). White solid, 81% yield. 1H NMR
212e214 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d
11.00 (s, 1H), 8.34 (d,
(400 MHz, CD3OD) d 8.75 (s, 1H), 8.44 (s, 1H), 8.18 (s, 1H), 8.04 (s,
J ¼ 7.0 Hz,1H), 7.77 (d, J ¼ 6.8 Hz, 2H), 7.70 (d, J ¼ 7.6 Hz, 2H), 7.63 (s,
1H), 7.58 (d, J ¼ 7.5 Hz, 2H), 7.39 (t, J ¼ 6.5 Hz, 2H), 7.28 (t, J ¼ 6.9 Hz,
1H), 6.92 (s, 1H), 6.77 (s, 1H), 6.59 (d, J ¼ 7.1 Hz, 1H), 6.49 (s, 1H),
1H), 7.82 (s, 1H), 7.73 (d, J ¼ 6.3 Hz, 2H), 7.66 (d, J ¼ 6.0 Hz, 3H), 4.31
(s, 2H), 4.01 (s, 3H); MS (EI) m/z 332.13 (M þ H)þ.
2-(2-(4-fluorophenyl)-7-methoxyimidazo[1,2-a]pyridin-3-yl)
acetic acid (74k). White solid, 83% yield. 1H NMR (500 MHz,
4.24 (s, 2H), 3.79 (s, 3H); 13C NMR (101 MHz, DMSO‑d6)
d 168.11,
157.72, 153.48, 145.77, 142.88, 142.67, 138.99, 135.23, 128.97, 128.08,
127.66, 126.26, 126.05, 124.38, 119.97, 114.54, 112.48, 106.80, 105.30,
94.78, 56.07, 32.04; MS (EI) m/z 424.16 (M þ H)þ; HRMS (ESI) calcd
for C26H21N3O3 (M þ H)þ: 424.1656; found 424.1653. HPLC reten-
tion time 10.207 min, 100.00% pure.
CD3OD)
d
8.63 (d, J ¼ 7.5 Hz,1H), 7.74 (dd, J ¼ 8.3, 5.2 Hz, 2H), 7.38 (t,
J ¼ 8.5 Hz, 2H), 7.30 (s, 1H), 7.20 (d, J ¼ 7.3 Hz, 1H), 4.23 (s, 2H), 4.09
(s, 3H); MS (EI) m/z.300.09 (M þ H)þ.
2-(7-methoxy-2-(4-(trifluoromethyl)phenyl)imidazo[1,2-a]
pyridin-3-yl)acetic acid (74l). White solid, 78% yield. 1H NMR
N-(1,1-dioxidobenzo[b]thiophen-6-yl)-2-(7-methoxy-2-
phenylimidazo[1,2-a]pyridin-3-yl) acetamide (38). White solid,
(400 MHz, CD3OD) d 8.67 (s, 1H), 7.95 (s, 4H), 7.36 (s, 1H), 7.23 (s,
1H), 4.26 (s, 2H), 4.11 (s, 3H); MS (EI) m/z 350.09 (M þ H)þ.
2-(7-methoxy-2-(4-nitrophenyl)imidazo[1,2-a]pyridin-3-yl)
acetic acid (74m). White solid, 82% yield. 1H NMR (500 MHz,
73% yield, m.p. 167e168 ꢁC. 1H NMR (500 MHz, DMSO‑d6)
d 10.93 (s,
1H), 8.31 (d, J ¼ 7.4 Hz, 1H), 8.15 (s, 1H), 7.81e7.74 (m, 3H), 7.58 (dd,
J ¼ 16.8, 7.5 Hz, 2H), 7.48 (t, J ¼ 7.4 Hz, 2H), 7.36 (t, J ¼ 7.2 Hz, 1H),
7.29 (d, J ¼ 6.8 Hz, 1H), 7.01 (s, 1H), 6.69 (d, J ¼ 7.0 Hz, 1H), 4.29 (s,
CD3OD)
d
8.66 (d, J ¼ 6.3 Hz, 1H), 8.47 (d, J ¼ 6.7 Hz, 2H), 7.99 (d,
J ¼ 6.5 Hz, 2H), 7.34 (s, 1H), 7.22 (d, J ¼ 5.5 Hz, 1H), 4.26 (s, 2H), 4.10
2H), 3.38 (s, 3H); 13C NMR (101 MHz, DMSO‑d6)
d 168.80, 157.83,
(s, 3H); MS (EI) m/z 269.08 (M þ H)þ.
145.90, 142.80, 141.87, 137.69, 135.11, 133.29, 130.54, 129.06, 128.08,
127.77, 127.08, 126.23, 126.06, 123.79, 113.89, 111.94, 106.91, 94.77,
56.08, 32.23; MS (EI) m/z 446.11 (M þ H)þ; HRMS (ESI) calcd for
2-(7-methoxy-2-(4-(methylsulfonyl)phenyl)imidazo[1,2-a]
pyridin-3-yl)acetic acid (74n). White solid, 86% yield. 1H NMR
(500 MHz, CDCl3)
d
8.11 (d, J ¼ 7.5 Hz, 1H), 7.90 (q, J ¼ 8.5 Hz, 4H),
C
24H19N3O4S (M þ H)þ: 446.1169; found 446.1162. HPLC retention
6.80 (d, J ¼ 2.3 Hz, 1H), 6.63 (dd, J ¼ 7.5, 2.4 Hz, 1H), 3.84e3.76 (m,
time 15.632 min, 100.00% pure.
5H), 3.02 (s, 3H); MS (EI) m/z 360.08 (M þ H)þ.
Compounds 74a-u were synthesized according to the procedure
for the preparation of compound 11.
2-(7-methoxy-2-(thiophen-2-yl)imidazo[1,2-a]pyridin-3-yl)
acetic acid (74o). White solid, 82% yield. 1H NMR (400 MHz,
2-(2-phenylimidazo[1,2-a]pyridin-3-yl)acetic acid (74a).
CD3OD) d 8.64 (s, 1H), 7.79 (s, 1H), 7.65 (s, 1H), 7.31 (s, 2H), 7.18 (s,
White solid, 85% yield. 1H NMR (400.
1H), 4.33 (s, 2H), 4.09 (s, 3H); MS (EI) m/z 288.06 (M þ H)þ.
2-(7-methoxy-2-(naphthalen-2-yl)imidazo[1,2-a]pyridin-3-
yl)acetic acid (74p). White solid, 71% yield. 1H NMR (400 MHz,
MHz, CD3OD)
d
8.86 (d, J ¼ 6.9 Hz, 1H), 8.11e8.06 (m, 1H), 8.02
(d, J ¼ 8.9 Hz, 1H), 7.78e7.73 (m, 2H), 7.69e7.59 (m, 4H), 4.30 (s,
2H); MS (EI) m/z 252.15 (M þ H)þ.
MeOD)
d
8.41 (s, 1H), 7.69 (m, 7H), 7.53 (s, 3H), 4.24 (d, J ¼ 4.5 Hz,
2-(7-chloro-2-phenylimidazo[1,2-a]pyridin-3-yl)acetic acid
5H); MS (EI) m/z 333.11 (M þ H)þ.
(74b). White solid, 82% yield. 1H NMR (400 MHz, CD3OD)
d
8.82 (d,
2-(2-(4-cyanophenyl)-6-methylimidazo[1,2-a]pyridin-3-yl)
acetic acid (74q). White solid, 86% yield. 1H NMR (500 MHz,
J ¼ 7.2 Hz, 1H), 8.04 (s, 1H), 7.72 (d, J ¼ 6.0 Hz, 2H), 7.62 (dd, J ¼ 19.0,
6.4 Hz, 4H), 4.24 (s, 2H); MS (EI) m/z 286.05 (M þ H)þ.
CD3OD)
d 8.68 (s, 1H), 8.05e7.89 (m, 6H), 4.30 (s, 2H), 2.55 (s, 3H);
2-(7-bromo-2-phenylimidazo[1,2-a]pyridin-3-yl)acetic acid
MS (EI) m/z 291.10 (M þ H)þ.
(74c). White solid, 88% yield. 1H NMR (500 MHz, CD3OD)
d
8.78 (d,
2-(2-(4-chlorophenyl)-6-methylimidazo[1,2-a]pyridin-3-yl)
acetic acid (74r). White solid, 81% yield. 1H NMR (400 MHz, CD3OD)
J ¼ 6.9 Hz,1H), 8.26 (s,1H), 7.75 (d, J ¼ 7.0 Hz, 3H), 7.67 (d, J ¼ 6.6 Hz,
3H), 4.27 (s, 2H); MS (EI) m/z 330.00 (M þ H)þ.
d
8.65 (s, 1H), 7.92 (s, 2H), 7.71 (s, 2H), 7.66 (s, 2H), 4.28 (d,
2-(7-methyl-2-phenylimidazo[1,2-a]pyridin-3-yl)acetic acid
J ¼ 16.9 Hz, 5H); MS (EI) m/z 300.07(M þ H)þ.
(74d). White solid, 81% yield. 1H NMR (500 MHz,CD3OD)
d
8.69 (d,
2-(2-(p-tolyl)imidazo[1,2-a]pyridin-3-yl)acetic acid (74s).
J ¼ 6.9 Hz, 1H), 7.75 (s, 1H), 7.73e7.68 (m, 2H), 7.64 (d, J ¼ 7.0 Hz,
2H), 7.44 (d, J ¼ 6.8 Hz, 1H), 4.29 (s, 2H), 2.65 (s, 3H); MS (EI) m/z
266.11 (M þ H)þ.
White solid, 89% yield. 1H NMR (500 MHz, CD3OD)
d 8.83 (s, 1H),
8.02 (d, J ¼ 16.8 Hz, 2H), 7.60 (d, J ¼ 16.6 Hz, 3H), 7.45 (d, J ¼ 6.7 Hz,
2H), 4.27 (s, 2H), 2.45 (s, 3H); MS (EI) m/z 266.11 (M þ H)þ.
2-(2-(4-(methylsulfonyl)phenyl)imidazo[1,2-a]pyridin-3-yl)
acetic acid (74t). White solid, 80% yield. 1H NMR (400 MHz, MeOD)
2-(8-methoxy-2-phenylimidazo[1,2-a]pyridin-3-yl)acetic
acid (74e). White solid, 84% yield. 1H NMR (400 MHz, CD3OD)
d 8.37
(s, 1H), 7.70 (d, J ¼ 3.4 Hz, 2H), 7.63 (d, J ¼ 3.7 Hz, 3H), 7.49 (s, 2H),
d
8.65 (s, 1H), 7.97 (m, 4H), 7.26 (d, J ¼ 34.0 Hz, 2H), 4.15 (d,
4.20 (d, J ¼ 4.5 Hz, 5H); MS (EI) m/z 282.11 (M þ H)þ.
J ¼ 48.7 Hz, 5H); MS (EI) m/z 331.06(M þ H)þ.
2-(6-methoxy-2-phenylimidazo[1,2-a]pyridin-3-yl)acetic
2-(2-(4-cyanophenyl)imidazo[1,2-a]pyridin-3-yl)acetic acid
acid (74f). White solid, 79% yield. 1H NMR (500 MHz, CDCl3)
d
8.09
(74u). White solid, 85% yield. 1H NMR (400 MHz, MeOD)
d 8.87 (d,
(s, 1H), 7.70 (d, J ¼ 5.9 Hz, 3H), 7.50 (dd, J ¼ 18.8, 6.7 Hz, 4H), 4.06 (s,
J ¼ 6.0 Hz, 1H), 8.08 (m, 2H), 7.98 (s, 4H), 7.63 (s, 1H), 4.34 (s, 2H);
2H), 3.92 (s, 3H); MS (EI) m/z 282.12 (M þ H)þ.
MS (EI) m/z 278.08 (M þ H)þ.
2-(6-bromo-2-phenylimidazo[1,2-a]pyridin-3-yl)acetic acid
Compounds 39-61 were synthesized according to the procedure
for the preparation of compound 7.
(74g). White solid, 81% yield. 1H NMR (500 MHz, CDCl3)
d 9.08 (s,
1H), 7.95 (dd, J ¼ 48.2, 41.2 Hz, 3H), 7.61 (d, J ¼ 43.9 Hz, 4H), 4.18 (d,
N-(1,1-dioxidobenzo[b]thiophen-6-yl)-2-(7-methoxy-2-
phenylimidazo[1,2-a]pyridin-3-yl) acetamide (39). White solid,
J ¼ 16.4 Hz, 2H); MS (EI) m/z 330.00 (M þ H)þ.
2-(2,7-diphenylimidazo[1,2-a]pyridin-3-yl)acetic acid (74h).
78% yield, m.p. 201e203 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 10.99
White solid, 85% yield. 1H NMR (400 MHz, CD3OD)
d
8.85 (s, 1H),
(s, 1H), 8.46 (d, J ¼ 6.7 Hz, 1H), 8.17 (s, 1H), 7.82e7.77 (m, 3H), 7.65
(d, J ¼ 9.0 Hz, 1H), 7.59 (dd, J ¼ 11.5, 7.6 Hz, 2H), 7.50 (t, J ¼ 7.4 Hz,
2H), 7.40 (t, J ¼ 7.2 Hz,1H), 7.34e7.28 (m, 2H), 6.98 (t, J ¼ 6.7 Hz,1H),
8.15 (s, 1H), 7.91 (s, 3H), 7.77 (s, 2H), 7.66 (d, J ¼ 6.8 Hz, 3H), 7.58 (dd,
J ¼ 11.7, 6.6 Hz, 3H), 4.29 (s, 2H); MS (EI) m/z 328.12 (M þ H)þ.
2-(7-(4-methoxyphenyl)-2-phenylimidazo[1,2-a]pyridin-3-
4.36 (s, 2H); 13C NMR (101 MHz, DMSO‑d6)
d 168.60, 144.52, 143.51,
19

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
141.86, 137.71, 134.86, 133.29, 130.56, 129.12, 128.38, 128.06, 127.08,
126.11, 125.53, 125.12, 123.83, 117.10, 115.19, 112.49, 111.99, 32.31;
MS (EI) m/z 416.10 (M þ H)þ; HRMS (ESI) calcd for C24H19N3O4S
dioxidobenzo[b]thiophen-6-yl) acetamide (45). White solid, 72%
yield, m.p. 223e225 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 10.95 (s,
1H), 8.86 (s, 1H), 8.13 (s, 1H), 7.79e7.73 (m, 3H), 7.64e7.56 (m, 3H),
7.50 (t, J ¼ 7.6 Hz, 2H), 7.45e7.37 (m, 2H), 7.29 (d, J ¼ 6.9 Hz, 1H),
(M
þ
H)þ: 416.1063; found: 416.060. HPLC retention time
12.255 min, 96.70% pure.
4.37 (s, 2H); 13C NMR (101 MHz, DMSO‑d6)
d 168.46, 144.10, 143.03,
2-(7-chloro-2-phenylimidazo[1,2-a]pyridin-3-yl)-N-(1,1-
dioxidobenzo[b]thiophen-6-yl) acetamide (40). White solid, 76%
141.85, 137.70, 134.39, 133.29, 130.56, 129.21, 128.30, 128.02, 127.11,
126.08, 125.81, 123.79, 118.19, 116.13, 111.91, 106.47, 32.23; MS (EI)
m/z 494.01 (M þ H)þ; HRMS (ESI) calcd for C23H16BrN3O3S
(M þ H)þ: 494.0168; found: 446.0168. HPLC retention time
13.345min,100.00% pure.
yield, m.p. 230e232 ꢁC. 1H NMR (500 MHz, DMSO‑d6)
d 10.93 (s,
1H), 8.52 (d, J ¼ 7.4 Hz, 1H), 8.12 (s, 1H), 7.81 (d, J ¼ 2.0 Hz, 1H),
7.77e7.73 (m, 3H), 7.58 (dd, J ¼ 16.4, 7.6 Hz, 2H), 7.50 (t, J ¼ 7.7 Hz,
2H), 7.40 (t, J ¼ 7.3 Hz, 1H), 7.29 (d, J ¼ 6.9 Hz, 1H), 7.06 (dd, J ¼ 7.3,
N-(1,1-dioxidobenzo[b]thiophen-6-yl)-2-(2,7-
2.1 Hz, 1H), 4.35 (s, 2H); 13C NMR (101 MHz, DMSO‑d6)
d
168.37,
diphenylimidazo[1,2-a]pyridin-3-yl)acetamide (46). White solid,
144.23, 141.79, 137.68, 134.41, 133.29, 130.57, 130.26, 129.20, 128.33,
127.09, 126.79, 126.12, 123.83, 115.85, 113.48, 111.97, 32.21; MS (EI)
m/z 450.06 (M þ H)þ; HRMS (ESI) calcd for C23H16ClN3O3S
(M þ H)þ: 450.0674; found: 450.0676. HPLC retention time
11.189 min, 97.88% pure.
71% yield, m.p. 146e148 ꢁC. 1H NMR (500 MHz, DMSO‑d6)
d 11.11 (s,
1H), 8.55 (d, J ¼ 7.2 Hz, 1H), 8.30 (d, J ¼ 4.6 Hz, 1H), 8.16 (s, 1H), 7.96
(s, 1H), 7.87e7.81 (m, 3H), 7.59 (q, J ¼ 6.7 Hz, 2H), 7.52 (q, J ¼ 8.0 Hz,
2H), 7.41e7.38 (m, 2H), 7.29 (d, J ¼ 6.8 Hz, 1H), 7.18 (d, J ¼ 8.2 Hz,
1H), 6.88 (d,1H), 6.69 (d, J ¼ 8.2 Hz,1H), 6.10 (s,1H), 4.40 (s, 2H); 13
C
(7-bromo-2-phenylimidazo[1,2-a]pyridin-3-yl)-N-(1,1-
dioxidobenzo[b]thiophen-6-yl) acetamide (41). White solid, 71%
NMR (101 MHz, DMSO‑d6) d 168.60, 144.97, 144.26, 141.86, 138.38,
137.71, 136.63, 134.84, 133.29, 130.57, 129.58, 129.16, 128.70, 128.32,
127.05, 126.11, 125.70, 123.83, 115.17, 113.34, 111.85, 32.35; MS (EI)
m/z 492.13 (M þ H)þ; HRMS (ESI) calcd for C29H21N3O3S (M þ H)þ:
492.1376; found: 492.1378. HPLC retention time 15.083 min, 98.37%
pure.
yield, m.p. 247e249 ꢁC. 1H NMR (500 MHz, DMSO‑d6)
d 10.93 (s,
1H), 8.45 (d, J ¼ 7.3 Hz, 1H), 8.12 (s, 1H), 7.96 (s, 1H), 7.75 (d,
J ¼ 7.2 Hz, 3H), 7.58 (dd, J ¼ 16.7, 7.5 Hz, 2H), 7.50 (t, J ¼ 7.6 Hz, 2H),
7.40 (t, J ¼ 7.2 Hz, 1H), 7.29 (d, J ¼ 6.9 Hz, 1H), 7.15 (d, J ¼ 7.3 Hz, 1H),
4.34 (s, 2H); 13C NMR (101 MHz, DMSO‑d6)
d
168.36, 144.63, 144.11,
N-(1,1-dioxidobenzo[b]thiophen-6-yl)-2-(7-(4-
141.85, 137.68, 134.40, 133.29, 130.55, 129.19, 128.36, 127.07, 126.72,
126.09, 123.83, 118.99, 118.09, 115.77, 111.98, 32.22; MS (EI) m/z
494.01 (M þ H)þ; HRMS (ESI) calcd for C23H16BrN3O3S (M þ H)þ:
494.0168; found: 494.0161. HPLC retention time 11.676 min, 96.27%
pure.
methoxyphenyl)-2-phenylimidazo[1,2-a]
amide (47). White solid, 78% yield, m.p. 136e138 ꢁC. 1H NMR
(400 MHz, DMSO‑d6)
1H), 7.88 (d, J ¼ 1.0 Hz, 1H), 7.83 (s, 1H), 7.82e7.75 (m, 4H),
7.63e7.56 (m, 2H), 7.50 (t, J ¼ 7.6 Hz, 2H), 7.39 (t, J ¼ 7.4 Hz,1H), 7.34
(dd, J ¼ 7.3, 1.8 Hz, 1H), 7.29 (d, J ¼ 6.9 Hz, 1H), 7.08 (d, J ¼ 8.8 Hz,
2H), 4.37 (s, 2H), 3.83 (s, 3H); 13C NMR (101 MHz, DMSO‑d6)
pyridin-3-yl)acet-
d
10.98 (s, 1H), 8.49 (d, J ¼ 7.2 Hz, 1H), 8.15 (s,
N-(1,1-dioxidobenzo[b]thiophen-6-yl)-2-(7-methyl-2-
phenylimidazo[1,2-a]pyridin-3-yl) acetamide (42). White solid,
76% yield, m.p. 202e204 ꢁC. 1H NMR (500 MHz, DMSO‑d6)
d
10.95
d 168.64, 159.98, 145.11, 144.00, 141.86, 137.70, 136.40, 134.88,
(s, 1H), 8.29 (d, J ¼ 6.8 Hz, 1H), 8.14 (s, 1H), 7.78 (t, J ¼ 7.2 Hz, 3H),
7.61e7.56 (m, 2H), 7.49 (t, J ¼ 7.6 Hz, 2H), 7.39 (t, J ¼ 7.3 Hz, 1H), 7.29
(d, J ¼ 6.9 Hz,1H), 7.13 (d, J ¼ 6.6 Hz, 1H), 6.89 (t, J ¼ 6.8 Hz,1H), 4.31
133.30, 130.61, 129.14, 128.27, 128.06, 127.10, 126.09, 125.56, 123.81,
115.00, 112.19, 111.94, 111.56, 55.74, 32.33; MS (EI) m/z 522.14
(M þ H)þ; HRMS (ESI) calcd for C30H23N3O4S (M þ H)þ: 522.1482;
found: 522.1483. HPLC retention time 13.398 min, 99.57% pure.
N-(1,1-dioxidobenzo[b]thiophen-6-yl)-2-(7-(1-methyl-1H-
(s, 2H), 2.56 (s, 3H); 13C NMR (101 MHz, DMSO‑d6)
d 168.63, 144.86,
142.95, 141.85, 137.69, 134.96, 133.29, 130.55, 129.07, 128.43, 127.98,
127.09, 126.56, 126.08, 123.81, 123.26, 115.55, 112.51, 111.93, 32.42,
pyrazol-4-yl)-2-phenylimidazo
(48). White solid, 79% yield, m.p. 135e136 ꢁC. 1H NMR (400 MHz,
DMSO‑d6)
[1,2-a]pyridin-3-yl)acetamide
17.06; MS (EI) m/z 430.12(M
C
time 12.859 min, 100.00% pure.
þ
H)þ; HRMS (ESI) calcd for
24H19N3O3S (M þ H)þ: 430.1220; found: 430.1220. HPLC retention
d
10.94 (s, 1H), 8.44 (d, J ¼ 7.1 Hz, 1H), 8.34 (s, 1H), 8.14 (s,
1H), 8.07 (s, 1H), 7.83e7.76 (m, 4H), 7.59 (dd, J ¼ 11.2, 7.6 Hz, 2H),
7.50 (t, J ¼ 7.6 Hz, 2H), 7.39 (d, J ¼ 7.3 Hz, 1H), 7.30e7.22 (m, 2H),
4.34 (s, 2H), 3.90 (s, 3H). MS(EI) m/z 496.14 (M þ H)þ; 13C NMR
N-(1,1-dioxidobenzo[b]thiophen-6-yl)-2-(8-methoxy-2-
phenylimidazo[1,2-a]pyridin-3-yl) acetamide (43). White solid,
78% yield, m.p. 219e221 ꢁC. 1H NMR (500 MHz, DMSO‑d6)
d
11.07 (s,
(101 MHz, DMSO‑d6) d 168.66, 145.08, 143.52, 141.85, 137.70, 137.07,
1H), 8.16 (s, 1H), 8.06 (d, J ¼ 6.5 Hz, 1H), 7.79 (d, J ¼ 6.2 Hz, 3H),
7.66e7.56 (m, 2H), 7.49 (t, J ¼ 7.2 Hz, 2H), 7.37 (t, J ¼ 6.9 Hz,1H), 7.29
(d, J ¼ 6.7 Hz,1H), 6.88 (t, J ¼ 6.9 Hz,1H), 6.73 (d, J ¼ 7.3 Hz,1H), 4.33
134.84,133.29, 130.56,129.85,129.12,128.23, 128.02, 127.09, 126.09,
125.64,123.82, 120.76,114.94,111.97,111.19,110.23, 32.29,19.03; MS
(EI) m/z 496.14 (M þ H)þ; HRMS (ESI) calcd for C27H21N5O3S
(M þ H)þ: 496.1438; found: 496.1436. HPLC retention time
10.522 min, 97.28% pure.
(s, 2H), 3.98 (s, 3H); 13C NMR (101 MHz, DMSO‑d6)
d 168.57, 148.81,
142.48, 141.87, 138.84, 137.67, 134.82, 133.30, 130.54, 129.07, 128.28,
127.96, 127.07, 126.07, 123.80, 118.11, 116.08, 112.55, 111.92, 102.02,
56.23, 32.50; MS (EI) m/z 446.11 (M þ H)þ; HRMS (ESI) calcd for
N-
fluorophenyl)-7-methoxyimidazo[1,2-a]
amide (49). White solid, 73% yield, m.p. 207e209 ꢁC. 1H NMR
(500 MHz, DMSO‑d6)
(1,1-dioxidobenzo[b]thiophen-6-yl)-2-(2-(4-
Pyridin-3-yl)acet-
C
24H19N3O4S (M þ H)þ: 446.1169; found: 446.1166. HPLC retention
time 14.234 min, 98.64% pure.
d
10.95 (s, 1H), 8.31 (d, J ¼ 7.5 Hz, 1H), 8.14 (s,
N-(1,1-dioxidobenzo[b]thiophen-6-yl)-2-(6-methoxy-2-
phenylimidazo[1,2-a]pyridin-3-yl) acetamide (44). White solid,
1H), 7.78 (t, J ¼ 6.6 Hz, 2H), 7.58 (dd, J ¼ 17.1, 7.6 Hz, 2H), 7.30 (dd,
J ¼ 15.2, 7.7 Hz, 3H), 7.00 (d, J ¼ 2.0 Hz, 1H), 6.69 (dd, J ¼ 7.5, 2.3 Hz,
1H), 5.76 (s, 1H), 4.26 (s, 2H), 3.86 (s, 3H); 13C NMR (126 MHz,
71% yield, m.p. 233e234 ꢁC. 1H NMR (500 MHz, DMSO‑d6)
d 11.34 (s,
1H), 8.17 (d, J ¼ 27.5 Hz, 2H), 7.81 (dd, J ¼ 21.5, 8.0 Hz, 3H), 7.61e7.55
DMSO‑d6)
d
168.74, 162.07 (d, J ¼ 244.4 Hz), 157.88, 145.89, 141.84,
(m, 3H), 7.48 (t, J ¼ 7.5 Hz, 2H), 7.36 (t, J ¼ 7.0 Hz, 1H), 7.28 (d,
137.68, 133.29, 131.63, 130.55, 129.97, 127.06, 126.08, 123.82, 116.03,
115.86, 113.76, 111.97, 106.98, 94.77, 56.09, 32.16; MS (EI) m/z 464.10
(M þ H)þ; HRMS (ESI) calcd for C24H18FN3O4S (M þ H)þ: 464.1075;
found: 464.1074. HPLC retention time 16.149 min, 100.00% pure.
N-(1,1-dioxidobenzo[b]thiophen-6-yl)-2-(7-methoxy-2-(4-
(trifluoromethyl)phenyl)imidazo [1,2-a]pyridin-3-yl)acetamide
(50). White solid, 77% yield, m.p. 213e216 ꢁC. 1H NMR (500 MHz,
J ¼ 6.8 Hz, 1H), 7.12 (d, J ¼ 9.7 Hz, 1H), 4.39 (s, 2H), 3.84 (s, 3H); 13
C
NMR (101 MHz, DMSO‑d6)
d 168.70, 149.09, 141.97, 141.42, 137.67,
134.82, 133.31, 130.51, 129.08, 128.13, 127.91, 127.08, 126.04, 123.77,
119.99, 117.34, 116.47, 111.89, 107.59, 57.07, 32.40; MS (EI) m/z 446.11
(M þ H)þ; HRMS (ESI) calcd for C24H19N3O4S (M þ H)þ: 446.1169;
found: 446.1164. HPLC retention time 14.748 min, 98.68% pure.
2-(6-bromo-2-phenylimidazo[1,2-a]pyridin-3-yl)-N-(1,1-
DMSO‑d6)
d
10.93 (s, 1H), 8.34 (d, J ¼ 7.5 Hz, 1H), 8.14 (s, 1H), 7.98 (d,
20

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
J ¼ 8.1 Hz, 2H), 7.84 (d, J ¼ 8.2 Hz, 2H), 7.76 (s, 1H), 7.58 (dd, J ¼ 16.3,
7.5 Hz, 2H), 7.29 (d, J ¼ 6.9 Hz, 1H), 7.04 (d, J ¼ 2.0 Hz, 1H), 6.72 (d,
J ¼ 7.4 Hz, 1H), 4.32 (s, 2H), 3.87 (s, 3H); 13C NMR (101 MHz,
141.90, 137.69, 137.17, 133.31, 132.22, 130.53, 129.67, 128.12, 127.83,
127.10, 126.04, 123.77, 122.81, 121.49, 116.46, 114.47, 111.89, 32.32,
21.28, 18.28; MS (EI) m/z 444.13 (M þ H)þ; HRMS (ESI) calcd for
DMSO‑d6)
d
168.52, 158.13, 146.17, 141.79, 141.26, 139.17, 137.68,
C
25H21N3O3S (M þ H)þ: 444.1376; found: 444.1376. HPLC retention
133.28, 130.58, 128.54, 127.81, 127.06, 126.35, 126.03, 123.88, 115.21,
112.03, 107.42, 94.84, 56.16, 32.18; MS (EI) m/z 514.10 (M þ H)þ;
HRMS (ESI) calcd for C25H18F3N3O4S (M þ H)þ: 514.1043; found:
514.1042. HPLC retention time 14.887 min, 97.07% pure.
time 8.613 min, 100.00% pure.
2-(2-(4-cyanophenyl)-6-methylimidazo[1,2-a]pyridin-3-yl)-
N-(1,1-dioxidobenzo[b]thiophen-6-yl)acetamide (56). White
solid, 71% yield, m.p. 229e231 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
N-(1,1-dioxidobenzo[b]thiophen-6-yl)-2-(7-methoxy-2-(4-
nitrophenyl)imidazo[1,2-a] pyridin-3-yl)acetamide (51). White
solid, 80% yield, m.p. 245e247 ꢁC. For compound 52:1H NMR
d 11.01 (s, 1H), 8.32 (s, 1H), 8.15 (s, 1H), 8.00e7.92 (m, 4H), 7.79 (d,
J ¼ 8.2 Hz, 1H), 7.59 (dd, J ¼ 11.7, 7.8 Hz, 3H), 7.29 (d, J ¼ 6.9 Hz, 1H),
7.22 (d, J ¼ 9.1 Hz, 1H), 4.35 (s, 2H), 2.33 (s, 3H); 13C NMR (101 MHz,
(400 MHz, DMSO‑d6)
d
11.01 (s, 1H), 8.35 (dd, J ¼ 14.0, 8.3 Hz, 3H),
DMSO‑d6) d 168.29, 143.84, 141.81, 141.34, 139.65, 137.66, 133.30,
8.14 (s, 1H), 8.07 (d, J ¼ 8.9 Hz, 2H), 7.79 (dd, J ¼ 8.2, 1.9 Hz, 1H), 7.58
(dd, J ¼ 11.3, 7.6 Hz, 2H), 7.28 (d, J ¼ 6.9 Hz, 1H), 7.04 (d, J ¼ 2.4 Hz,
1H), 6.74 (dd, J ¼ 7.5, 2.5 Hz, 1H), 4.37 (s, 2H), 3.88 (s, 3H); 13C NMR
133.07, 130.56, 128.67, 127.07, 126.13, 123.87, 122.94, 122.27, 119.45,
116.79, 116.45, 112.01, 110.17, 32.28, 18.27; MS (EI) m/z 455.11
(M þ H)þ; HRMS (ESI) calcd for C25H18N4O3S (M þ H)þ: 455.1172;
found: 455.1177. HPLC retention time 8.357 min, 98.80% pure.
2-(2-(4-chlorophenyl)-6-methylimidazo[1,2-a]pyridin-3-yl)-
N-(1,1-dioxidobenzo [b]thiophen -6-yl)acetamide (57). White
solid, 73% yield, m.p. 217e219 ꢁC. 1H NMR (500 MHz, DMSO‑d6)
(101 MHz, DMSO‑d6) d 168.36, 158.31, 146.64, 146.35, 141.82, 140.52,
137.64, 133.29, 130.56, 128.66, 127.03, 126.40, 126.13, 124.37, 123.91,
116.31, 112.06, 107.72, 94.82, 56.18, 32.22; MS (EI) m/z 491.10
(M þ H)þ; HRMS (ESI) calcd for C24H18N4O6S (M þ H)þ: 491.1020;
found: 491.1017. HPLC retention time 13.263 min, 97.66% pure.
N-(1,1-dioxidobenzo[b]thiophen-6-yl)-2-(7-methoxy-2-(4-
(methylsulfonyl)phenyl)imidazo [1,2-a]pyridin-3-yl)acetamide
(52). White solid, 69% yield, m.p. 153e155 ꢁC. 1H NMR (500 MHz,
d
10.94 (s, 1H), 8.29 (s, 1H), 8.15 (s, 1H), 7.79e7.75 (m, 3H), 7.61e7.53
(m, 5H), 7.29 (d, J ¼ 6.9 Hz, 1H), 7.20 (d, J ¼ 9.2 Hz, 1H), 4.29 (s, 2H),
2.33 (s, 3H); 13C NMR (101 MHz, DMSO‑d6)
168.44, 143.49, 141.82,
d
137.69, 133.67, 133.28, 132.73, 130.56, 129.87, 129.15, 128.53, 127.07,
126.12, 123.87, 122.92, 122.05, 116.44, 115.21, 112.01, 32.22, 18.26;
MS (EI) m/z 464.08 (M þ H)þ; HRMS (ESI) calcd for C24H18ClN3O3S
(M þ H)þ: 464.0830; found: 464.0837. HPLC retention time
11.831 min, 100.00% pure.
DMSO‑d6)
d
11.00 (s, 1H), 8.36 (d, J ¼ 7.6 Hz, 1H), 8.14 (s, 1H),
8.09e7.97 (m, 4H), 7.81e7.76 (m, 1H), 7.58 (dd, J ¼ 14.8, 7.5 Hz, 2H),
7.29 (d, J ¼ 6.7 Hz, 1H), 7.04 (d, J ¼ 2.3 Hz, 1H), 6.73 (dd, J ¼ 7.5,
1.3 Hz, 1H), 4.34 (s, 2H), 3.87 (s, 3H), 3.25 (s, 3H); 13C NMR
(101 MHz, DMSO‑d6)
d
168.44, 158.18, 146.24, 141.77, 141.03, 140.09,
N-(1,1-dioxidobenzo[b]thiophen-6-yl)-2-(2-(p-tolyl)imidazo
[1,2-a]pyridin-3-yl)acetamide (58). White solid, 71% yield, m.p.
139.62, 137.67, 133.25, 130.57, 128.50, 127.80, 127.03, 126.33, 126.14,
123.91, 115.60, 112.07, 107.50, 94.85, 56.16, 44.07, 32.21; MS (EI) m/z
524.09 (M þ H)þ; HRMS (ESI) calcd for C25H21N3O6S2 (M þ H)þ:
524.0945; found: 524.0951. HPLC retention time 10.781 min,
100.00% pure.
225e227 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 10.92 (s, 1H), 8.46 (d,
J ¼ 6.9 Hz, 1H), 8.13 (s, 1H), 7.77 (dd, J ¼ 8.2, 1.8 Hz, 1H), 7.61 (m, 5H),
7.31 (m, 4H), 7.00 (t, J ¼ 6.5 Hz, 1H), 4.33 (s, 2H), 2.36 (s, 3H); 13C
NMR (101 MHz, DMSO‑d6)
d 168.52, 141.80, 137.54, 133.25, 130.56,
N-(1,1-dioxidobenzo[b]thiophen-6-yl)-2-(7-methoxy-2-(thi-
ophen-2-yl)imidazo[1,2-a] pyridin-3-yl)acetamide (53). White
solid, 80% yield, m.p. 146e148 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
129.73, 128.29, 127.06, 126.10, 125.58, 123.81, 116.72, 114.98, 112.66,
111.95, 32.24, 21.29; MS (EI) m/z 430.12 (M þ H)þ; HRMS (ESI) calcd
for C24H19N3O3S (M þ H)þ: 430.1220; found: 430.1219. HPLC
retention time 10.558 min, 100.00% pure.
d
10.95 (s, 1H), 8.33 (d, J ¼ 7.4 Hz, 1H), 8.11 (s, 1H), 7.78 (d, J ¼ 8.1 Hz,
1H), 7.56 (dd, J ¼ 14.2, 4.7 Hz, 3H), 7.40 (s, 1H), 7.27 (d, J ¼ 5.9 Hz,
1H), 7.15 (s,1H), 6.99 (s,1H), 6.67 (d, J ¼ 7.4 Hz,1H), 4.36 (s, 2H), 3.86
N-(1,1-dioxidobenzo[b]thiophen-6-yl)-2-(2-(4-(methyl-
sulfonyl)phenyl)imidazo[1,2-a]
White solid, 62% yield, m.p. 156e159 ꢁC. 1H NMR (500 MHz,
DMSO‑d6)
11.34 (s, 1H), 8.55 (s, 1H), 8.07 (d, J ¼ 31.7 Hz, 5H), 7.84
(s, 1H), 7.63 (d, J ¼ 34.5 Hz, 3H), 7.32 (d, J ¼ 38.3 Hz, 2H), 7.01 (s, 1H),
4.45 (s, 2H), 3.26 (s, 3H); 13C NMR (101 MHz, DMSO‑d6)
168.38,
pyridine-3-yl)acetamide(59).
(s, 3H); 13C NMR (101 MHz, DMSO‑d6)
d 168.46, 158.03, 145.89,
141.84, 138.48, 137.69, 137.48, 133.28, 130.54, 128.44, 127.09, 126.14,
124.01, 123.74, 113.12, 111.86, 106.97, 94.56, 56.11, 31.99; MS (EI) m/z
452.07 (M þ H)þ; HRMS (ESI) calcd for C22H17N3O4S2 (M þ H)þ:
452.0733; found: 452.0742. HPLC retention time 12.357 min,
98.68% pure.
N-(1,1-dioxidobenzo[b]thiophen-6-yl)-2-(7-methoxy-2-
(naphthalen-2-yl)imidazo[1,2-a] pyridin-3-yl)acetamide (54).
White solid, 78% yield, m.p. 136e138 ꢁC. 1H NMR (500 MHz,
d
d
144.76, 141.89, 141.61, 139.92, 137.67, 133.30, 130.57, 128.87, 127.86,
127.04, 126.12, 125.76, 123.88, 117.38, 116.82, 112.96, 112.04, 44.08,
32.20; MS (EI) m/z 494.08 (M þ H)þ; HRMS (ESI) calcd for
C
24H19N3O5S2 (M þ H)þ: 494.0839; found: 494.0835. HPLC reten-
tion time 9.322 min, 100.00% pure.
DMSO‑d6)
d
11.01 (s, 1H), 8.36 (d, J ¼ 7.5 Hz, 1H), 8.24 (s, 1H), 8.17 (s,
2-(2-(4-cyanophenyl)imidazo[1,2-a]pyridin-3-yl)-N-(1,1-
dioxidobenzo[b]thiophen-6-yl) acetamide (60). White solid, 66%
1H), 8.01 (d, J ¼ 8.6 Hz, 1H), 7.99e7.91 (m, 3H), 7.81 (dd, J ¼ 8.2,
1.7 Hz, 1H), 7.59 (dd, J ¼ 12.6, 7.6 Hz, 2H), 7.56e7.50 (m, 2H), 7.29 (d,
J ¼ 6.9 Hz, 1H), 7.04 (d, J ¼ 2.4 Hz, 1H), 6.71 (dd, J ¼ 7.5, 2.4 Hz, 1H),
4.41 (d, J ¼ 25.1 Hz, 2H), 3.88 (s, 3H); 13C NMR (101 MHz, DMSO‑d6)
yield, m.p. 243e244 ꢁC. 1H NMR (500 MHz, DMSO‑d6)
d 10.99 (s,
1H), 8.48 (d, J ¼ 6.9 Hz, 1H), 8.13 (s, 1H), 8.01 (d, J ¼ 8.2 Hz, 2H), 7.95
(d, J ¼ 8.2 Hz, 2H), 7.78 (d, J ¼ 8.1 Hz,1H), 7.67 (d, J ¼ 9.1 Hz,1H), 7.59
(dd, J ¼ 15.4, 7.5 Hz, 2H), 7.36 (m, 1H), 7.29 (d, J ¼ 6.8 Hz, 1H), 7.02 (t,
d
168.89, 157.91, 146.05, 142.62, 141.90, 137.71, 133.30, 132.66,
130.56, 128.61, 128.04, 127.11, 126.82, 126.52, 126.28, 126.11, 123.84,
114.43, 111.97, 106.99, 94.78, 56.11, 32.36; MS (EI) m/z 496.13
(M þ H)þ; HRMS (ESI) calcd for C28H21N3O4S (M þ H)þ: 496.1326;
found: 496.1324. HPLC retention time 12.366 min, 96.04% pure.
N-(1,1-dioxidobenzo[b]thiophen-6-yl)-2-(6-methyl-2-(p-
tolyl)imidazo[1,2-a]pyridin-3-yl) acetamide (55). White solid,
J ¼ 6.7 Hz, 1H), 4.39 (s, 2H); 13C NMR (101 MHz, DMSO‑d6)
d 168.23,
144.75, 141.74, 141.56, 139.50, 137.68, 133.27, 133.11, 130.61, 128.86,
127.08, 126.18, 125.84, 125.67, 123.90, 119.42, 117.39, 116.79, 113.01,
112.04,110.37, 32.27; MS (EI) m/z 441.10 (M þ H)þ; HRMS (ESI) calcd
for C24H18ClN3O3S (M þ H)þ: 441.1016; found:441.1017. HPLC
retention time 9.322 min, 98.00% pure.
75% yield, m.p. 252e254 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d
10.92
N-(1,1-dioxidobenzo[b]thiophen-6-yl)-2-(imidazo[1,2-a]pyr-
(s, 1H), 8.26 (s, 1H), 8.14 (s, 1H), 7.78 (dd, J ¼ 8.2, 1.9 Hz, 1H), 7.63 (d,
J ¼ 8.1 Hz, 2H), 7.61e7.56 (m, 2H), 7.52 (d, J ¼ 9.1 Hz, 1H), 7.28 (d,
J ¼ 7.0 Hz, 3H), 7.16 (dd, J ¼ 9.2, 1.5 Hz, 1H), 4.28 (s, 2H), 2.34 (d,
idin-3-yl)acetamide (61). White solid, 82% yield, m.p. 222e224 ꢁC.
1H NMR (500 MHz, DMSO‑d6)
d
10.87 (s, 1H), 8.41 (d, J ¼ 6.8 Hz,1H),
8.12 (s, 1H), 7.74 (d, J ¼ 8.2 Hz, 1H), 7.55 (m, 4H), 7.25 (dd, J ¼ 13.3,
J ¼ 8.6 Hz, 6H); 13C NMR (101 MHz, DMSO‑d6)
d 168.70, 143.52,
7.3 Hz, 2H), 6.93 (t, J ¼ 6.7 Hz, 1H), 4.19 (s, 2H); 13C NMR (101 Hz,
21

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
DMSO‑d6)
d
168.46, 145.36, 141.92, 137.70, 133.27, 133.22, 130.51,
docking parameter, XP precision can calculate the binding free
energy of ligand and receptor. The greater the negative absolute
value is, the greater the affinity between ligand and receptor is. In
addition, we also used the “Protein Surface Analyzer” module to
analyze the STAT3 protein structure based on electronegativity and
marked with different colors.
In vitro Metabolic Stability Study. Microsomes in 0.1 M TRIS
buffer pH 7.4 (final concentration 0.33 mg/mL), co-factor MgCl2
(final concentration 5 mM) and tested compound (final concen-
127.03, 125.99, 125.50, 124.30, 123.69, 118.96, 117.41, 112.20, 111.81,
32.23; MS (EI) m/z 340.07 (M þ H)þ; HRMS (ESI) calcd for
C
17H13N3O3S (M þ H)þ: 340.0750; found: 340.0743. HPLC retention
time 6.732 min, 100.00% pure.
Cell lines and cell culture. HEK-293T cells, triple-negative
breast cancer MDA-MB-231 and MDA-MB231-4175 cell lines were
obtained from American Type Culture Collection and cultured in
Dulbecco Modified Eagle Medium (DMEM, Gibco, USA) plus 10% (v/
v) fetal bovine serum (FBS, Gibco, USA), 50
m
g/mL penicillin and
tration 0.1 mM, co-solvent (0.01% DMSO) and 0.005% Bovin serum
50 g/mL streptomycin (Gibco). A549, MGC-803, HCC70 and MDA-
m
albumin (BSA)) were incubated at 37 ꢁC for 10 min. The reaction
was started by the addition of NADPH (final concentration 1 mM).
Aliquots were sampled at 0, 7, 17, 30 and 60 min respectively and
methanol (cold in 4 ꢁC) was added to terminate the reaction. After
centrifugation (4000 rpm,5 min), samples were then analyzed by
LC-MS/MS.
MB-468 cell lines were obtained from American Type Culture
Collection and cultured in RPMI 1640 medium (Gibco, USA) with
10% (v/v) FBS, 50
mg/mL penicillin and 50 mg/mL streptomycin
(Gibco, USA). All cells were incubated at 37 ꢁC in an incubator
containing 5% CO2.
Reagents and antibodies. The compound 39 was dissolved in
sterile dimethyl sulfoxide (DMSO; MP, 196055) and stored
at ꢀ20 ꢁC. For animal studies, 39 was dissolved in PBS (0.01 M;
8.1 mM Na2HPO4$12H2O,1.9 mM NaH2PO4$2H2O and 145 mM NaCl,
pH 7.2e7.4; cat# G0002, Servicebio, China) containing 15% Cre-
mophor EL (Millipore, 238470-1SET). The primary antibodies for
phosphorylated (p-)STAT3 (Y705, #9145; S727, #9134), phosphor-
ylated (p-)STAT1 (Y701, #9167), phosphorylated (p-)STAT5 (Y694,
Surface Plasmon Resonance (SPR) analysis. SPR assay was
performed using Biacore 8K (GE Healthcare, USA). Purified STAT3
protein (0.1 mg/mL) was dissolved in PBS and immobilized onto the
CM5 chip (GE Healthcare, USA). Different concentrations of 39 were
dissolved in PBS (0.1% DMSO) and passed over the chip to produce
diverse response signals. The binding affinity (KD) was evaluated
using the Biacore Insight Evaluation Software.
Cell viability assay. Cell viability assay was performed using Cell
Counting Kit-8 (Bimake, B34302) to measure anti-proliferative ac-
tivity of designed compounds. The cells were seeded in 96-well
plates with 1000e8000 cells per well and allowed to grow over-
night. Subsequently, cells were treated with vehicle (0.1% DMSO, as
the solvent control) or different concentrations of compounds for
72 h. Before testing, the cells were added to 1/10 volume of CCK-8
each well and incubated for 0.5e4 h at 37 ꢁC until the OD values of
vehicle are about 0.8e1.5. Then, the absorbance (also called optical
density, OD) of each hole at 480 nm was determined by microplate
reader (FLUOstar Omega ACU, Germany). Cell viability (%) ¼
(ODcompounds-ODblank)/(ODvehicle-ODblank) X 100%. “Blank” repre-
sents medium only. Finally, the IC50 values were evaluated by
GraphPad Prism 7. The concentration range used for this assay was
#9351), STAT5 (#25656), b-actin (#3700), c-myc (#5605), Mcl-1
(#4572), HA-tag (#3742), PARP (#9542), cleaved-caspase7
(#9491), second-fluorescence antibodies Alexa Fluor® 488 Conju-
ate2 (#4412) and Alexa Fluor® 555 Conjuate2 (#4409) were pur-
chased from Cell Signaling Technology, Inc. The antibodies against
STAT3 (10253-2-AP) and STAT1 (10144-2-AP) were purchased from
ProteinTech Group, Inc. Bcl-xL antibody (sc-8392) was from Santa
Cruz Biotechnology. Monoclonal ANTI-FLAG® M2 antibody was
from Sigma-Aldrich. The second antibodies Goat Anti-Rabbit IgG
H&L (HRP) (ab6721) and Goat Anti-Mouse IgG H&L (HRP) (ab6789)
antibodies were purchased from Abcam.
Plasmids. pGL3-STAT3 is a STAT3-dependent luciferase reporter
with seven copies of the Stat3-specific binding sequence (AATCC-
CAGAA) in the C-reactive protein gene promoter. This new
construct was verified by sequencing. STAT3C (or CSTAT3) is a an
engineered constitutively dimerizeable STAT3, through substitut-
ing cysteine residues for specific amino acids within the C-terminal
loop in the SH2 domain. Flag-STAT3 was purchased from FulenGen
(#EX-Z2385-M35, Guangzhou, China) and STAT3 sequence was
confirmed by sequence. HA-STAT3 in pCDNA3.1 backbone was
purchased from PPL (#BC00627, Nanjing, China). STAT3 protein
expression plasmid was constructed by inserting full length STAT3
into plasmid pET28a. The insert was confirmed by DNA sequencing.
Molecular docking. In this study, the STAT3 crystal structure
(PDB ID: 1BG1) was obtained from the PDB database, and the
Maestro 11.1 (Schrodinger, Inc.) was used for molecular docking.
Firstly, the “Protein Preparation Wizard” module in Schrodinger
software is used to optimize the structure of STAT3 protein,
including removing water molecules, hydrogenation, energy
minimization and treatment of metal ions and disulfide bonds. The
small molecule was imported into the software and then optimized
by the “Lig Prep” module, including the formation of 3D structure
and low energy conformation. Next, the SH2 domain of the protein
was extracted as the active pocket center, and the “Receptor Grid
Generation” module in the software was applied to generate the
pocket Grid points. Finally, we dock STAT3 protein and small
molecule by using the “Ligand Docking” module in Schrodinger
software. During docking, the STAT3 protein is considered rigid,
while the small molecule is considered flexible. The Glide Score
scoring function was applied to calculate the interaction and
binding capacity of small molecule and STAT3 protein. As the main
from 0.1 mM to 100 mM.
Colony formation assay. Colony formation assay was per-
formed to examine the effect of 39 on cell colony survival. MDA-
MB-231, MDA-MB-468, MDA-MB231-4175 and HCC70 cells were
seeded in 6-well plates with 0.5 ꢂ 103e1 ꢂ 103 cells per well. The
second day, various concentrations of 39 were added into the cells.
After that, the cell culture medium was changed and the corre-
sponding concentration of 39 was added every 2e3 days until the
colonies visible. About 10e15 days later, the cells were fixed using
4% paraformaldehyde fix solution (Beyotime, China) and stained
with crystal violet (Beyotime, China). The results of colonies were
scanned by printer, processed with image J and analyzed with
GraphPad Prism 7. All experiments were set in triplicate.
Wounding healing assay. Wounding healing assay was per-
formed to measure whether 39 inhibit TNBC cells migration. MDA-
MB-231, MDA-MB-468, MDA-MB231-4175 and HCC70 cells were
seeded in 6-well plates with 0.8 ꢂ 106e1.2 ꢂ 106 cells per well.
When the cell fusion degree reaches 100%, the cells were scratched
with pipette tips. Then, the cells were treated with 0e3 mM 39 until
the vehicle cells migrate into the scratched area. About 12e96 h
later, pictures of the cells were taken with microscope. All experi-
ments were set in triplicate.
Transwell assay for cell invasion. Invasion assay was per-
formed in 24-well Transwell® plates with 8.0
mm pore poly-
carbonate filters (Corning, USA). MDA-MB-231, MDA-MB-468,
MDA-MB231-4175 and HCC70 cells were seeded in the top cham-
ber of insert at 1.5 ꢂ 104e3 ꢂ 104 cells/well. Then, the cells were
treated with different concentrations of 39. After the treatment
22

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
with 39 for 12 h, the cells in the top chamber were carefully
removed by cotton and then washed with PBS twice, and the
invaded cells in the bottom chamber were fixed with 4% para-
formaldehyde and stained with crystal violet. Finally, the invaded
cells were photographed and calculated. All experiments were set
in triplicate.
STAT3 and HA-STAT3 detection, respectively, for 1 h at room tem-
perature in the dark. Then, the cells were rinsed with DAPI (Yeasen,
China) for 10 min in the dark followed by washed three times with
PBS. Finally, the specimens were examined by Laser Scanning
Confocal Microscope FV3000 (Olympus, Japan) and imaged using
Olympus software.
Apoptosis assay. Annexin V-fluorescein isothiocyanate (FITC)/
propidium iodide (PI) Cell Death Detection Kit (BestBio, China) was
applied in cell apoptosis assay. The MDA-MB-231, MDA-MB-468
and MDA-MB231-4175 cells were seeded in 6-well plates and
treated with vehicle (0.1% DMSO, as the solvent control) or 39 for
48 h. Then, the cells were digested with trypsin without EDTA, then,
Co-immunoprecipitation. HEK-293T cells were seeded into 6-
well plates with the concentration of 5 ꢂ 105 cells per well and
allowed to grow overnight. Then, the cells were transiently trans-
fected with 2500 ng HA-STAT3 and 2500 ng Flag-STAT3. After 24 h,
HEK-293T cells, which stably express HA- and Flag-tagged STAT3,
were treated with vehicle (0.1% DMSO, as the solvent control) and
39 for 3 h. Then, the cells were added to 100 ng/mL IL-6 to stimulate
the interaction between HA-STAT3 and Flag-STAT3 proteins. Sub-
sequently, the cell lysates were extracted for IP by cell lysis buffer
(Beyotime, China) containing 1 mM PMSF and 1% phosphatase in-
hibitor cocktail. Anti-Flag Affinity Gel (Bimake, B23101) was
washed three times with PBST (PBS with 0.2% Tween) and incu-
washed with cold PBS twice and resuspended in 400
buffer solution. Subsequently, the cells were incubated with
Annexin V-FITC (5 l) for 5 min and PI (10
l) for 15 min at 4 ꢁC in
mL 1X binding
m
m
the dark. Finally, the samples were detected within 1 h by flow
cytometry (Millipore, USA) and processed by FlowJo 7.6 software.
All experiments were set in triplicate.
RNA interference. MDA-MB-231 cells were seeded into 6-well
or 96-well plates. After incubating for 18e24 h, cells grew to
60e70%. According to the manufacturer's protocol of DharmaFECT
(T-2001-03, Dharmacon, USA), cells were transfected with negative
control or siRNA targeting STAT3. For STAT3 interference, the
sequence of siRNA targeting STAT3:
bated with 600e800
tures were washed three times with PBST to remove unbound
proteins and boiled in 50
m
g lysates at 4 ꢁC overnight. Then, the mix-
m
L 1 ꢂ SDS-PAGE loading buffer for 5 min.
Finally, the samples were run on SDS-PAGE gel, transferred to
polyvinylidene difluoride membranes and then blotted for HA-tag
and Flag-tag antibodies.
STAT3-F: 50-UCCAGUUUCUUAAUUUGUUGACGGGUC-3’;
Reporter gene assay for STAT3 transcriptional activity. HEK-
293T cells were plated into 96-well plates with 2.5 ꢂ 104 cells per
well and incubated overnight. Subsequently, the cells were tran-
siently transfected with 50 ng pGL3-STAT3 (a STAT3-dependent
luciferase reporter, containing seven copies of the Stat3-specific
binding sequence (AATCCCAGAA), 50 ng STAT3C and 40 ng TK-
Renilla as control. After 24 h, HEK-293T cells were treated with
vehicle and 39 for 24 h. Samples were processed with Dual Lucif-
erase Reporter Gene Assay Kit (Beyotime, China) following the
manufacturer's protocol. The luciferase activity was measured in
the luminometer (Promega, GloMax® Navigator, USA).
Electrophoretic Mobility Shift Assay. MDA-MB-231 and MDA-
MB-468 cells were treated with vehicle and 39 for 3 h. Then, nu-
clear extract preparations of the cells were collected with Nuclear
and Cytoplasmic Protein Extraction Kit (Beyotime, China) following
the manufacturer's protocol and quantified with Pierce™ BCA
STAT3-R: 50-GACCCGUCAACAAAUUAAGAAACUGGA-3’;
A nonspecific oligonucleotide without complementary to any
human gene was used as negative control. All above siRNAs were
synthesized by Sangon Biotech (Guangdong, China).
Western blotting analysis. The human breast cancer cells were
treated with vehicle (0.1% DMSO, as the solvent control) and
various concentrations of 39 for 3, 24 or 48 h. Subsequently, the
cells were lysed with RIPA buffer(50 mM Tris (pH 7.4), 150 mM
NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS sodium
orthovanadate, sodium fluoride, EDTA, leupeptin containing 1 mM
PMSF (Beyotime, China) and 1% phosphatase inhibitor cocktail
(Bimake, China) and the specimen were quantified with Pierce™
BCA Protein Assay Kit (Thermo, 23225). The cell lysates containing
20e50 mg of proteins were denatured by boiling and then analyzed
by electrophoresis on 8 or 12% SDS-PAGE gels and transferred onto
polyvinylidene difluoride membranes (Millipore, USA). The mem-
branes were blocked with 5% Bovine Serum Albumin (Beyotime,
China) for 1 h at room temperature, and then, incubated with pri-
mary antibodies (pY705-STAT3, pS727-STAT3, STAT3, Mcl-1, Bcl-xL,
Protein Assay Kit (Thermo, USA). 8e10 mg nuclear proteins were
incubated with biotin-STAT3 (Beyotime, China) or biotin-STAT5
probe (Beyotime, China) prior to EMSA. The STAT3 or STAT5 DNA-
protein complexes were separated in 6% native polyacrylamide
gels and transferred to a positively charged nylon membrane. The
membrane was detected by EMSA/gel-shift Kit (Beyotime, China)
following the manufacturer's instructions.
Pharmacokinetic study. Compound 39 was dissolved in DMSO/
0.5% HPMC ¼ (5/95, v/v) and was given to SD rats by oral admin-
istration. Animal procedures were performed according to insti-
tutional ethical guidelines of animal care. The blood samples were
collected at 0.25, 0.5, 1, 2, 4, 8, 24h after administration, and then
c-Myc, b
-actin, PARP and cleaved-caspase7) overnight at 4 ꢁC. After
washed three times with TBST (TBS with 0.1% Tween-20) buffer, the
membranes were incubated with corresponding anti-rabbit
(Abcam, ab6721) or anti-mouse (Abcam, ab6789) HRP-conjugated
secondary antibodies for 1 h at room temperature. Finally, the
membranes were washed with TBST buffer three times for 10 min
each time and then developed with ECL kit (Tanon, China)
following the manufacturer's protocol.
Immunofluorescence for colocalization. HEK-293T cells were
seeded in confocal dishes and incubated overnight. Then, the cells
were transfected with HA-STAT3 and Flag-STAT3. 24 h later, HEK-
293T cells were treated with vehicle (0.1% DMSO) and 39 for
24 h. The cells were added to 100 ng/mL IL-6 (PeproTech, China) for
30 min before imaged. Subsequently, HEK-293T cells were fixed
with 4% paraformaldehyde for 15 min and permeabilized with 0.3%
Triton X-100 for 10 min. The samples were blocked with goat serum
(Boster, China) for 1 h at room temperature, and then, incubated
with HA-tag and Flag-tag antibody at 1: 500 dilution (in goat
serum) at 4 ꢁC overnight. After washed three times with PBS, the
cells were incubated with two Alexa Fluor secondary antibodies,
Alexa Fluor 555 Conjugate and Alexa Fluor 488 Conjugate for Flag-
plasma samples were harvested. 20
mL plasma samples were
precipitated by 200 L acetonitrile/methanol (1/1, v/v), and the
m
supernatant was mixed with equal volume of water before analysis.
The analysis was performed on a LC-MS/MS system consisted of
triple quadrupole mass spectrometer (TQ-S; Waters; America) and
liquid chromatography (ACQUITY I-Class; Waters; USA). The chro-
matographic separation was performed on an ACQUITY UPLCr BEH
Phenyl 1.7
m
m column (50 mm ꢂ 2.1 mm I.D.,1.7
mm) (Waters; USA).
The mobile phase consisted of 0.1% formic acid in water and 0.1%
formic in acetonitrile.
Animal studies. In human breast carcinoma cells xenograft
tumor model, 2 ꢂ 106e3 ꢂ 106 MDA-MB231-4175 cells were sub-
cutaneously injected into the flank of 4-5-week-old NOD-SCID
23

Q. Huang, Y. Zhong, B. Li et al.
European Journal of Medicinal Chemistry 221 (2021) 113525
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female mice (SCXK 2018-0008). In human triple-negative breast
cancer patient-derived tumor xenograft (TNBC PDX) model, we
used the tumor tissues from the TNBC patient (No. J00099327,
Jackson Lab, USA) in serum-free DMEM, which was then cut into
pieces. Approximately 2 mm3 patient tumor tissue fragments were
subcutaneously implanted into the flank of female NOD-SCID mice
(SCXK 2018-0008) using a trocar. For these two models, when tu-
mor volume reached about 100 mm3, the mice were randomly
divided into three groups (n ¼ 6/group) including vehicle (treated
with PBS containing 15% Cremophor EL), 5 mg/kg and 15 mg/kg 39
(diluted in PBS containing 15% Cremophor EL). Mice were admin-
istered with different doses of 39 by intraperitoneal injection every
day. The volume (V) of the tumor was measured every 2e3 days
and calculated using the formula, V ¼
p
/6 ꢂ S2 ꢂ L, where S is the
minor axis and L is the major axis. At day 20, the mice were
sacrificed and their tumor tissues were harvested and weighed. The
tumor tissues were detected by immunohistochemistry assay using
Ki67 antibody. The hearts, livers, spleen, lungs, kidneys of the mice
were also collected for hematological analysis to study the toxi-
cology of 39. For immunoblot assay, we gained the protein samples
of tumor lysed with RIPA buffer (Beyotime, China) subjecting to
western blot analysis as described above. All animal procedures
were approved by Animal Care and Use Committee of Sun Yat-Sen
Univeristy, Guangzhou, China.
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Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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D. Huang, S. Nagarajan, B.K. Chia, D. Cheah, Y. Liu, F. Zhang, H. Rao, T. Tang,
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[23] K. Matsuno, Y. Masuda, Y. Uehara, H. Sato, A. Muroya, O. Takahashi,
T. Yokotagawa, T. Furuya, T. Okawara, M. Otsuka, N. Ogo, T. Ashizawa,
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Acknowledgments
This work was supported by grants from National Natural Sci-
ence Foundation of China (Grant 21977128 and 81973359) and
Guangzhou Science and Technology Planning Programme
(202103000097 and 202002030408). Natural Science Foundation
of Guangdong (Grant 2018A030313300 and 2019A1515011215) and
Fundamental Research Funds for the Central Universities
(20ykpy117 and 19ykpy125) were also highly appreciated.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113525.
STAT3 inhibitors by virtual screening, ACS Med. Chem. Lett.
1 (2010)
371e375.
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