Discovery of 3-(thiophen/thiazole-2-ylthio)pyridine derivatives as multitarget anticancer agents

Article abstract of DOI:10.1007/s00044-019-02400-x

A series of novel 3-(thiophen/thiazole-2-ylthio)pyridine derivatives were designed and synthesized as IGF-1R tyrosine kinase inhibitors. All the target compounds were tested for their IGF-1R kinase inhibitory activities and cytotoxicities against five cancer cell lines (K562, Hep-G2, HCT-116, WSU-DLCL2, and A549). Although all these compounds exhibited moderate to potent cancer cell proliferation inhibitory activities (the most potent compound 43 showed IC₅₀ value of 1.3 ± 0.9 μM against WSU-DLCL2 cell line), IGF-1R inhibition were not observed. In order to identify the exact target of these analogues, selected compounds were further screened for various kinases. The results indicated that this series of compounds may exert their anticancer activities through inhibiting various kinases including FGFR 3, EGFR, JAK, and RON. In addition, cell cycle analysis of compound 43 on Hep-G2 cells showed cell cycle arrest at G1/G0 phase. All the experiments validated the potential of 3-(thiophen/thiazole-2-ylthio)pyridine analogues as multi-target anticancer agents.

Full text of DOI:10.1007/s00044-019-02400-x

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02400-x
ORIGINAL RESEARCH
Discovery of 3-(thiophen/thiazole-2-ylthio)pyridine derivatives
as multitarget anticancer agents
1 ●
2 ●
2 ●
2 ●
3 ●
4 ●
2 ●
Jiankang Zhang Jianjun Xi Ruoyu He Rangxiao Zhuang Limin Kong Liping Fu Yanmei Zhao
Chong Zhang1 Linghui Zeng Jingyi Lu Rujia Tao Zhengmengtong Liu Huajian Zhu
1 ●
1 ●
1 ●
1 ●
1 ●
Shourong Liu2
●
Received: 26 April 2019 / Accepted: 16 July 2019
©
Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
A series of novel 3-(thiophen/thiazole-2-ylthio)pyridine derivatives were designed and synthesized as IGF-1R tyrosine
kinase inhibitors. All the target compounds were tested for their IGF-1R kinase inhibitory activities and cytotoxicities against
five cancer cell lines (K562, Hep-G2, HCT-116, WSU-DLCL2, and A549). Although all these compounds exhibited
moderate to potent cancer cell proliferation inhibitory activities (the most potent compound 43 showed IC50 value of 1.3 ±
0
.9 μM against WSU-DLCL2 cell line), IGF-1R inhibition were not observed. In order to identify the exact target of these
analogues, selected compounds were further screened for various kinases. The results indicated that this series of compounds
may exert their anticancer activities through inhibiting various kinases including FGFR 3, EGFR, JAK, and RON. In
addition, cell cycle analysis of compound 43 on Hep-G2 cells showed cell cycle arrest at G1/G0 phase. All the experiments
validated the potential of 3-(thiophen/thiazole-2-ylthio)pyridine analogues as multi-target anticancer agents.
●
●
●
Keywords Multitarget IGF-1R Anti-cancer Kinases
Introduction
2R, and IR), and six IGF-binding proteins (IGFBP1-6) (Sun
et al. 2017; Scagliotti and Novello 2012; Buchanan et al.
2011; Furstenberger and Senn 2002; Brahmkhatri et al.
2015). The IGF-1 is a single chain peptide, which is com-
prised of 70 amino acids in four domains (7.65 kDa). The
IGF-1 binds to at least two cell surface receptor tyrosine
kinases (RTKs): the IGF-1 receptor (IGF-1R) and the
insulin receptor. The primary action of IGF-1 is mediated
by binding to its specific receptor IGF-1R. The IGF-1R, a
transmembrane receptor tyrosine kinase, is widely expres-
sed in many cell types and tissues (LeRoith et al. 1995;
Stewart and Rotwein 1996; Sepp-Lorenzino 1998).
Through endocrine, paracrine, and autocrine mechanisms,
IGF-1R exerts its mitogenic effect on various cells by sti-
mulating proliferation and inhibiting apoptosis.
The insulin-like growth factors (IGFs) belong to a multi-
functional growth factor family, which plays a pivotal role
in the regulation of normal physiological and pathological
processes. The IGFs family consists of two ligands (IGF-1
and IGF-2), three cell-membrance receptors (IGF-1R, IGF-
These authors contributed equally: Jiankang Zhang, Jianjun Xi
*
*
Huajian Zhu
zhuhj@zucc.edu.cn
Shourong Liu
liushourong85463990@sina.com
The tyrosine kinase activity was activated after binding
of IGFs to IGF-1R, which leads to autophosphorylation.
Furthermore, this phosphorylation results activation of
various downstream signaling cascades. To date, two dis-
tinct IGF-IR signal transduction pathways have been iden-
tified: the mitogen activated protein kinase (RAS/RAF/
MAPK) pathway that predominantly stimulates cellular
proliferation, and the phosphatidylinositol-3-kinase-Akt
pathway that chiefly mediates cell survival (Sachdev and
Yee 2007; Samani et al. 2007; Kawauchi et al. 2009;
1
2
3
School of Medicine, Zhejiang University City College,
Hangzhou 310015 Zhejiang, China
Department of Pharmaceutical Preparation, Hangzhou Xixi
Hospital, Hangzhou 310023 Zhejiang, China
Department of Pharmacy, The First Affiliated Hospital, College of
Medicine, Zhejiang University, Hangzhou 310003 Zhejiang,
China
4
Department of Pharmacy, Shaoxing Hospital of Traditional
Chinese Medicine, Shaoxing 312000 Zhejiang, China

Medicinal Chemistry Research
Vanhaesebroeck and Alessi 2000). Due to its promotion
roles in both processes, IGF-1R signaling is related to
various physiological functions including differentiation,
transformation, and prevention of apoptosis. In addition,
studies indicated that IGF-1R overexpression is related to
many human cancers, such as breast (Sun et al. 2015),
prostate (Pollak et al. 1998), glioma (Trojan et al. 2007),
and lung cancer (Yeo et al. 2015). Moreover, clinical stu-
dies also showed that high levels expression of IGF-1R in
cancer was associated with tumor metastasis, resistance,
poor prognosis, and shortened survival (Sun et al. 2017).
Therefore, IGF-1R has become a potential and promising
diagnostic and therapeutic target in cancers.
lead compound. Compounds with high structural similarity
could generate similar activity. The active compound
should have suitable shape to fit the target pocket. There-
fore, 3D similarity is a potential method to find active
compounds. The designed compounds 34, 35, 43, 44, 47,
and 50 were selected in comparison with III-106 for their
3D similarity, and the similarity score of each comparison
were calculated. As shown in Table 1, compound 43
exhibited a higher similarity score of 0.67 compared with
III-106. Afterwards, the binding modes of compounds were
predicted by computational methods. Compound 43 was
prepared and docked into the active site of IGF-1R (PDB
code: 5FXS) using Glide under default setting. As shown in
Fig. 3, the pyridine core formed a hydrogen bond with the
N-H of Met84 in the hinge region, and the isopropyl ben-
zene ring extended into the P-loop region. In addition, the
thiophene ring was located into the channel to the solvent
region, while the nitro group formed a hydrogen bond with
the Arg5 of P-loop. All these hydrogen bonds formed play
an important role in the activity of the compound. These
results verify the reliability of the design strategy, and target
compound synthesis and evaluation were performed in this
study.
To date, more than two dozen of antagonistic mono-
clonal antibodies and small molecule inhibitors against IGF-
1
R have been developed. Currently, at least 12 randomized
controlled clinical trials are ongoing (NCT00887159,
NCT00799240) (Gualberto and Pollak 2009; Rodon et al.
2
008). Linsitinib (OSI-906) (Mulvihill et al. 2009), a Phase
III clinical candidate, has been evaluated for metastatic
adrenocortical carcinoma, and is also studied in combina-
tion with other anticancer agents. BMS-754807 (Carboni
et al. 2009) was developed by Bristol-Myers Squibb in a
Phase II clinical trial for breast cancer. Picropodephyllin
(
Girnita et al. 2004) is currently in Phase II/III for the
treatment of a variety of cancers, while Nordihy-
droguaiaretic acid (Hewish et al. 2009) is in a Phase II
clinical trial and evaluated for prostate cancer (Fig. 1).
III-106 (Fig. 2), an ATP competitive IGF-1R inhibitor
with a new aminopyrazoloquinazolines scaffold, was dis-
covered by Boehringer Ingelheim International GmbH with
5
2
0% inhibitory concentration (IC ) value of 5 nM (Treu
50
010). However, the poor selectivity and severe side effects
of the majority of the ATP competitive inhibitors limited its
further application (Deng et al. 2007). In order to reduce
toxicities while retain IGF-1R inhibitory activities, a novel
series of 3-(thiophen/thiazole -2-ylthio)pyridine analogues
were designed and synthesized with III-106 employed as
the lead compound.
In this study, 3D similarity analysis and docking studies
were performed to verify the design strategy. The 3D shape
similarity comparison is an important strategy for evaluat-
ing whether designed analogues were consistent with the
Fig. 1 Small-molecule IGF-1R inhibitors in clinical trials
Fig. 2 Design strategy of the
new synthesized IGF-1R
inhibitors
N
O
O
R2
Optimization
N
N
H
N
N
H
S
N
H
N
N
N
S
X
O
Y
R1
III-106
Designed compounds

Medicinal Chemistry Research
Results and discussion
Chemistry
transformed to the acyl chloride at the presence of (COCl)2.
Finally, acyl chloride reacted with different aryl amine to
afford the target compounds 31–50.
The synthetic route for intermediates 3, 5, and 7 were
prepared as outlined in Scheme 1. Fragments 2 and 5 were
synthesized by esterification of the corresponding acids 1
and 4 with methanol in the presence of catalytic amount of
hydrochloric acid. Afterwards, the synthesized ester 2 was
converted to fragment 5 by nitration reaction in a mixture
solution of HNO in H SO . Fragment 7 can be easily
obtained from methyl 4-cyano-5-(methylthio)thiophene-2-
carboxylate (6) with m-CPBA. And fragment 9 was syn-
thesized by 3, 5-dibromopyridine (8) and 4-methoxy-α-
toluenethiol under alkaline conditions. The target com-
pounds 31–50 were synthesized following the route
described in Scheme 2. Firstly, intermediates 11a–c were
prepared by coupling from fragment 9 with different boric
acids, and cleavage of the thioether bound provided 12a–c.
Subsequently, 12a–c coupled with fragments 3, 5 and 7 to
afford 13–18, then hydrolyzed to give 19–24, which was
IGF-1R inhibitory activities
All the target compounds (31–50) were screened for their
IGF-1R inhibitory activities in vitro by electrophoretic
mobility shift assay using Staurosporine as positive control.
The results showed that all the compounds exerted no
inhibitory activities against IGF-1R kinase with IC50 values
>30 μM. In view of this, all the target compounds were
tested for antitumor activities against different cell lines.
3
2
4
Cell proliferation assay
All the target compounds (31–50) were further screened for
their antiproliferative activities in vitro against five cancer cell
lines (K562, Hep-G2, HCT-116, WSU-DLCL2, and A549)
through standard CTG assay (Paclitaxel as positive control).
As illustrated in Table 2, all the compounds displayed various
degrees of cytotoxic activities against five cancer cell lines.
Among all of these compounds, 43 and 44 showed excellent
inhibitory activities against WSU-DLCL2 cell line with IC50
values of 1.3 ± 0.9 and 1.3 ± 1.8 μM, respectively.
Table 1 The similarity scores of target compounds
Compound
Similarity score
In vitro kinase screening
3
3
4
4
4
5
4
5
3
4
7
0
0.37
0.56
0.67
0.51
0.52
0.42
Subsequently, compound 43 was further screened for about
41 kinases inhibitory profiles on the DiscoverX’s Kino-
meScan profiling platform. Measurements were performed at
a concentration of 10 μM, and the results were illustrated in
Table 3. We defined the kinases results for primary screen
Fig. 3 a The binding mode of selected compound 43 with IGF-1R (PDB code: 5FSX). b Superposition of compound 43 (green) and compound III-
06 (pink)
1

Medicinal Chemistry Research
O
O
O
Scheme 1 Reagents and
conditions: (I) HCl/MeOH,
S
S
S
60 °C, 16 h; (II) H2SO4/HNO3,
0 °C, 3 h; (III) HCl/MeOH,
60 °C, 16 h; (IV) m-CPBA,
HO
O
O
Cl
Cl
Cl
DCM, 25 °C, 16 h. (V) 4-
Methoxy-α-toluenethiol, NaH,
DMF, 20 °C, 16 h
NO2
1
2
3
O
O
O
O
S
S
S
HO
O
Br
Br
4
5
O
S
O
O
O
S
S
CN
CN
6
7
N
N
Br
S
Br
Br
O
8
9
binding interactions reported as percent control
with compound 43 (0.5, 5, and 10 μM, respectively) caused
an increase in the percentage of apoptotic cells at G1/G0
phase (72.18%, 75.09% and 74.86%, respectively) com-
pared with control (67.08%). The percentage of cells at S
phase (21.05%, 17.80% and 17.91%, respectively) has a
concurrent reduction compared with control (25.04%), and
no significant difference was found in the percent of cells at
the G2/M phase. By contrast, there was a significant
decrease in the cells at G1/G0 phase (50.01% and 46.75%)
of Doxorubicin (0.5 and 1 μM) compared with control, and
the percentage of cells at the G2/M phase increased from
7.88% to 27.30% and 16.93%, respectively. The result
indicated that compound 43 induces cell cycle arrest at the
G1/G0 phase on Hep-G2 cells, while Doxorubicin showed a
significant arrest at the G2/M phase (Fig. 4).
(% Ctrl). The % Ctrl indicated that the dissociative kinases
(unbound to 43) were the percentages of all tested kinases,
where lower numbers indicated stronger binding to 43. The
obtained data demonstrated that compound 43 has weakly or
no % Ctrl against 41 kinases tested. Compound 43 showed
weak % Ctrl against other kinases, such as HPKE (84%),
ERK3 (86%), ERK4 (87%), MAK (79%), and MINK (86%).
In addition, the % Ctrl of 43 against AUR A, JAK3, FGFR2,
FGFR3, EGFR, and RON were tested with threefold serial
dilutions starting at concentration of 30 μM, and the results
were summarized in Table 4. Inspiringly, compound 43
showed good inhibitory activities against these kinases with
IC50 values ranging from 2 to 22 μM (except AUR A), and
especially with an IC50 value of 2.0 ± 0.3 μM against FGFR3.
Cell cycle analysis
Experimental procedures
The effect of compound 43 on Hep-G2 cell cycle was stu-
died by flow cytometric analysis. Hep-G2 cell was treated
with compound 43 (0.5, 1, 5, and 10 μM) or Doxorubicin
Chemistry
1
(
0.5 and 1 μM) for 24 h. Results showed that there was a
The H and 13C NMR spectra were recorded on Brüker
significant alterations in cell cycle phases (Fig. 2). Treatment
400 MHz spectrometer (Brüker Bioscience, Billerica, MA,

Medicinal Chemistry Research
Scheme 2 Reagents and conditions: (I) Pd(dppf)Cl2, Na2CO3, Diox-
ane/H2O, 100 °C, 16 h; (II) AlCl3, toluene, 25 °C, 3 h; (III) K2CO3,
CH CN, 82 °C, 16 h; (IV) CuI, K CO , DMSO, 80 °C, 16 h; (V)
NaOH, MeOH/H2O, 25 °C, 2 h; (VI) DMF, (COCl)2, DCM, 25 °C,
1 h; (VII) Et3N, DCM, 25 °C, 16 h
3
2
3
USA) with CDCl or d -DMSO as solvent. Chemical shifts
unless stated otherwise. Column chromatography was per-
formed using silica gel (300–400 mesh). All yields are
unoptimized and generally represent the result of a single
experiment.
3
6
(δ) were reported in parts per million relative to internal TMS,
and coupling constants (J) were reported in Hertz (Hz).
Splitting patterns were designated as singlet (s), broad singlet
(brs), doublet (d), double doublet (dd), triplet (t), quartet (q),
and multiplet (m). Mass spectral data were obtained by
Esquire-LC-00075 spectrometer (Brüker Bioscience).
Reagents and solvents were purchased from common com-
mercial suppliers and were used without further purification
Synthesis of methyl 5-chlorothiophene-2-carboxylate (2)
A solution of 5-chloro-2-thiophenecarboxylic acid (1,
90.0 g, 553.5 mmol, 1.0 eq) in HCl/MeOH (500.0 mL) was

Medicinal Chemistry Research
Table 2 In vitro antiproliferative
activities of the target
a
Compound
IC50 , (μM)
compounds against various
cancer cell lines
K562
Hep-G2
HCT-116
WSU-DLCL2
A549
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
40.1 ± 3.3
24.8 ± 1.8
3.7 ± 0.9
18.2 ± 1.7
5.2 ± 0.5
29.0 ± 4.9
32.9 ± 8.7
38.7 ± 5.3
38.2 ± 3.4
27.2 ± 2.9
11.7 ± 3.1
12.9 ± 1.1
5.1 ± 2.5
5.5 ± 0.6
9.8 ± 1.7
12.2 ± 2.8
6.4 ± 1.4
26.9 ± 6.9
16.9 ± 1.1
11.2 ± 0.5
3.4 ± 0.3
23.4 ± 1.2
16.9 ± 2.6
NAb
34.7 ± 5.4
23.8 ± 2.8
2.9 ± 0.6
10.6 ± 1.1
7.4 ± 2.0
26.2 ± 2.4
27.3 ± 1.8
32.4 ± 6.4
28.9 ± 1.9
75.8 ± 15.7
7.6 ± 0.9
8.4 ± 1.5
13.2 ± 1.1
7.6 ± 2.5
7.6 ± 1.3
5.5 ± 2.2
6.1 ± 1.0
25.6 ± 6.5
10.6 ± 1.8
8.8 ± 1.6
1.2 ± 0.2
28.3 ± 2.9
27.4 ± 1.7
3.8 ± 0.4
18.1 ± 1.2
12.3 ± 3.4
35.4 ± 8.1
29.7 ± 3.6
32.5 ± 6.4
33.7 ± 0.9
40.9 ± 10.5
3.0 ± 1.1
6.0 ± 2.6
1.3 ± 0.9
1.3 ± 1.8
5.6 ± 3.1
13.4 ± 2.2
9.9 ± 1.7
22.7 ± 4.3
23.5 ± 6.1
14.9 ± 1.0
2.1 ± 0.2
38.9 ± 7.8
31.6 ± 3.5
3.2 ± 1.5
12.3 ± 2.2
5.6 ± 0.1
25.8 ± 8.4
22.7 ± 5.5
21.6 ± 6.1
20.0 ± 1.3
56.1 ± 12.1
13.9 ± 3.0
6.8 ± 1.6
5.1 ± 0.3
9.9 ± 1.9
4.8 ± 2.4
13.3 ± 1.0
5.1 ± 0.7
23.8 ± 2.5
13.9 ± 3.3
11.7 ± 2.9
10.2 ± 3.8
16.7 ± 0.7
34.8 ± 8.6
61.1 ± 16.3
39.7 ± 10.8
37.2 ± 6.5
57.5 ± 9.9
66.8 ± 2.6
35.1 ± 13.3
30.9 ± 6.6
28.1 ± 5.3
25.2 ± 2.7
10.9 ± 3.6
11.6 ± 1.1
18.9 ± 0.8
33.8 ± 9.1
27.6 ± 2.6
13.9 ± 2.9
2.0 ± 0.3
c
Paclitaxel (nM)
aThe values are an average of three separate determinations
bNot active in the highest concentration used
cUsed as positive control
stirred at 60 °C for 16 h. The reaction mixture was then
concentrated in vacuo. The residue was dissolved in EtOAc
Synthesis of methyl 5-bromothiophene-2-carboxylate (5)
(
500.0 mL), washed with NaHCO (aq.) to pH = 8–9. The
A solution of 5-bromo-2-thiophenecarboxylic acid (4,
90.0 g, 553.5 mmol, 1.0 eq) in HCl/MeOH (500.0 mL) was
stirred at 60 °C for 16 h. The reaction was concentrated in
vacuo. The residue was dissolved in EtOAc (500.0 mL) and
3
organic layer was dried over Na SO and concentrated in
vacuo to give compound 2. Yellow oil; Yield: 94.1%; H
NMR (400 MHz, CDCl ): δ = 7.58 (d, J = 4.0 Hz, 1H,
2
4
1
3
thienyl-H), 6.93 (d, J = 4.0 Hz, 1H, thienyl-H), and 3.87 (s,
washed with NaHCO (aq.) to pH = 8–9, then the organic
3
+
3
H, CH ). ESI-MS: m/z = 178.0[M + H] .
layer was dried over Na SO and concentrated in vacuo to
3
2
4
1
give compound 5. Yellow oil; Yield: 94.1%; H NMR
Synthesis of methyl 5-chloro-4-nitrothiophene-2-
carboxylate (3)
(400 MHz, CDCl ): δ = 7.55 (d, J = 4.0 Hz, 1H, thienyl-H),
3
7.07 (d, J = 4.0 Hz, 1H, thienyl-H), and 3.87 (s, 3H, CH ).
3
+
ESI-MS: m/z = 222.3[M + H] .
To a solution of compound 2 (30.0 g, 169.9 mmol, 1.0 eq) in
con.H SO (60.0 mL) was added a mixture of con.HNO
3
Synthesis of methyl 4-cyano-5-(methylsulfonyl)thiophene-
2-carboxylate (7)
2
4
(
12.8 g, 203.8 mmol, 9.2 mL, 1.2 eq) in con.H SO (12.0 mL).
2 4
The reaction was stirred at 0 °C for 3 h. The reaction was
quenched with ice-water (100.0 mL), extracted with EtOAc
To a solution of methyl 4-cyano-5-(methylthio)thiophene-2-
(100.0 mL × 3). The combined organic layer was dried over
carboxylate (6, 550.0 mg, 2.6 mmol, 1.0 eq) in CH Cl2
2
Na SO and concentrated under reduced pressure. The crude
(10.0 mL) was added m-CPBA (1.3 g, 6.5 mmol, 85.0%
purity, 2.5 eq). The reaction was stirred at 25 °C for 16 h.
The reaction was quenched with Na SO (aq., 200.00 mL),
2
4
product was purified by CombiFlash (0–10% EtOAc in PE) to
1
give compound 3 as white solid. Yield: 47.8%; H NMR
2
3
(
3
400 MHz, CDCl ) : δ = 8.19 (s, 1H, thienyl-H), and 3.94 (s,
and extracted with CH Cl (20.0 mL × 3). The combined
2 2
organic layer was washed with 1N NaOH (200.0 mL), dried
3
+
H, CH ). ESI-MS: m/z = 222.9[M + H] .
3

Medicinal Chemistry Research
Table 3 Results of compound 43 against about 50 kinases
2
0 °C, after stirring 30 min, 3,5-dibromopyridine (8, 50.0 g,
211.1 mmol, 1.0 eq) was added. The reaction was stirred at
0 °C for 16 h. The reaction was quenched with water
100.0 mL), extracted with EtOAc (100.0 mL × 3). The
combined organic layer was washed with water
200.0 mL × 3), dried over Na SO and concentrated in
Target/Gene symbol
% Ctrla
Target/Gene symbol
% Ctrla
2
(
AKT1
AKT2
AKT3
AURKB
AURKC
BTK
97
FGFR4
HCK
100
100
84
100
100
100
97
HPK1
INSR
(
2
4
98
vacuo. The crude product was purified by CombiFlash
10% EtOAc in PE) to give the compound 9. White solid;
SRC
95
(
1
100
96
TEC
100
100
94
Yield: 73.0%; H NMR (400 MHz, CDCl ): δ = 8.38 (d,
J = 2.0 Hz, 1H, pyridine-H), 8.31 (d, J = 2.0 Hz, 1H, pyr-
idine-H), 7.60 (t, J = 2.0 Hz, 1H, pyridine-H), 7.13–7.08
(m, 2H, Ar-H), 6.79–6.73 (m, 2H, Ar-H), 4.00 (s, 2H, CH ),
and 3.71 (s, 3H, CH ). ESI-MS: m/z = 311.7[M + H] .
3
DAPK1
DAPK2
DAPK3
DDR1
DDR2
DMPK
ERK1
ERK2
ERK3
ERK4
ERK5
ERK8
FAK
VEGFR2
KIT
100
95
MAK
79
2
+
96
MAP3K1
MARK1
MARK2
MINK
PAK7
PCTK3
PDPK1
QSK
100
97
3
100
100
100
96
100
86
General procedure for the synthesis of compounds 11a–c
A mixture of compounds 10a–c (53.2 mmol, 1.1 eq),
compound 9 (15.0 g, 48.4 mmol, 1.0 eq), Pd(dppf)Cl2
100
93
86
(3.5 g, 4.8 mmol, 0.1 eq), and Na CO (10.2 g, 96.7 mmol,
87
100
100
100
100
100
2 3
2
1
.0 eq) in dioxane/H O (10/1, 150.0 mL) was stirred at
2
94
00 °C for 16 h. The mixture was concentrated in vacuo,
100
100
100
95
RAF1
diluted with water (20.0 mL), and extracted with CH Cl2
2
ROCK1
ROCK2
(50.0 mL × 3). The combined organic layer was dried over
FGFR1
SLK
Na SO and concentrated under reduced pressure. The
crude product was purified by CombiFlash (20% EtOAc in
PE) to give the compounds 11a–c.
2
4
a% Ctrl was measured by method (A) at a concentration of 10 μM
Table 4 Kinase inhibitory assay of compound 43
3
-(2-isopropylphenyl)-5-((4-methoxybenzyl)thio)pyridine
1
a
Kinase
IC50 , (μM)
(11a) Yellow oil; Yield: 78.1%; H NMR (400 MHz,
CDCl ): δ = 8.52 (d, J = 2.4 Hz, 1H, pyridine-H), 8.36 (d, J =
3
FGFR2
FGFR3
EGFR
JAK3
22.6 ± 2.9
2.0 ± 0.3
8.9 ± 3.4
12.4 ± 2.1
11.6 ± 1.0
> 30
2
7
.0 Hz, 1H, pyridine-H), 7.48 (t, J = 2.0 Hz, 1H, pyridine-H),
.41–7.38 (m, 2H, Ar-H), 7.24–7.17 (m, 3H, Ar-H), 7.07 (d,
J = 7.2 Hz, 1H, Ar-H), 6.81 (d, J = 8.4 Hz, 2H, Ar-H), 4.10 (s,
H, CH ), 3.78 (s, 3H, CH ), 2.90–2.83 (m, 1H, CH), and 1.14
2
2
3
+
RON
(d, J = 6.4 Hz, 6H, 2CH ). ESI-MS: m/z = 350.9[M + H] .
3
AUR A
aThe values are an average of three separate determinations, measured
through method (B)
3-(2-ethylphenyl)-5-((4-methoxybenzyl)thio)pyridine
(11b) Yellow oil; Yield: 72.1%; H NMR (400 MHz,
1
CDCl ): δ = 8.52 (d, J = 2.0 Hz, 1H, pyridine-H), 8.38 (d,
3
J = 2.0 Hz, 1H, pyridine-H), 7.50 (t, J = 2.0 Hz, 1H, pyridine-
H), 7.37–7.17 (m, 5H, Ar-H), 7.10 (d, J = 7.6 Hz, 1H, Ar-H),
over Na SO and concentrated under reduced pressure to
6.81 (d, J = 8.8 Hz, 2H, Ar-H), 4.10 (s, 2H, CH ), 3.78 (s, 3H,
2
4
2
1
give compound 7. White solid; Yield: 93.2%; H NMR
400 MHz, CDCl ): δ = 7.92 (s, 1H, thienyl-H), 3.91 (s, 3H,
CH ), 2.50 (q, J = 7.6, 15.2 Hz, 2H, CH ), and 1.07 (t, J =
3
2
+
(
7.5 Hz, 3H, CH ). ESI-MS: m/z = 336.1[M + H] .
3
3
CH ), and 3.30 (s, 3H, CH ). ESI-MS: m/z = 245.7
3
3
+
[
M + H] .
3-((4-methoxybenzyl)thio)-5-(2-methoxyphenyl)pyridine
1
(
11c) Yellow oil; Yield: 64.5%; H NMR (400 MHz,
Synthesis of 3-bromo-5-((4-methoxybenzyl)thio)pyridine (9)
CDCl ): δ = 8.58 (d, J = 2.0 Hz, 1H, pyridine-H), 8.46 (d,
3
J = 2.0 Hz, 1H, pyridine-H), 7.75 (t, J = 2.0 Hz, 1H, pyr-
idine-H), 7.39–7.35 (m, 1H, Ar-H), 7.26–7.19 (m, 3H, Ar-
H), 7.06–6.98 (m, 2H, Ar-H), 6.84–6.82 (m, 2H, Ar-H),
4.11 (s, 2H, CH ), 3.81 (s, 3H,CH ), and 3.78 (s, 3H, CH ).
To
11.1 mmol, 29.3 mL, 1.0 eq) in DMF (500.0 mL) was
added NaH (9.3 g, 232.2 mmol, 60.0% purity, 1.1 eq) at
a
solution of 4-methoxy-α-toluenethiol (32.6 g,
2
2
3
3
+
ESI-MS: m/z = 338.2[M + H] .

Medicinal Chemistry Research
Fig. 4 Effect of compound 43 and Doxorubicin on flow cytometric analysis of Hep-G2 cells (a, b). The cells treated with DMSO were used as
control

Medicinal Chemistry Research
General procedure for the synthesis of compounds 12a–c
The mixture was concentrated in vacuo to give the crude
product 16 or 17, which was put into next step without
further purification.
To a mixture of AlCl (6.9 g, 51.5 mmol, 2.8 mL, 3.0 eq) in
3
toluene (50.0 mL) was added a solution of compounds 11a–c
(
17.2 mmol, 1.0 eq) in toluene (50.0 mL). The reaction was
stirred at 25 °C for 3 h. The reaction was quenched with water
500.0 mL), extracted with EtOAc (500.0 mL × 3). The
combined organic layer was dried over Na SO and con-
Methyl 5-((5-(2-isopropylphenyl)pyridin-3-yl)thio)-4-
nitrothiophene-2-carboxylate (18)
(
A mixture of compound 12a (750.0 mg, 3.3 mmol, 1.0 eq),
compound 5 (723.0 mg, 3.3 mmol, 1.0 eq), CuI (62.3 mg,
327.0 μmol, 0.1 eq), and K CO (904.0 mg, 6.5 mmol, 2.0
2
4
centrated in vacuo. The residue was diluted with EtOAc
500.0 mL), washed with Na CO (aq., 200.0 mL × 3). Then
(
2
3
2
3
the water layer was acidified to pH = 2–3 with 1N HCl. The
mixture was extracted with EtOAc (100.0 mL × 3), the com-
bined organic layer was dried over Na SO and concentrated
eq) in DMSO (5.0 mL) was stirred at 80 °C for 16 h. The
reaction was quenched with water (50.0 mL), and the
mixture was acidified to pH = 2–3 with 1N HCl, extracted
with EtOAc (100.0 mL × 3). The combined organic layer
was dried over Na SO and concentrated under reduced
2
4
under reduced pressure to give the compounds 12a–c.
2
4
5
-(2-isopropylphenyl)pyridine-3-thiol (12a) Yellow oil;
pressure to give the crude compound 18, which was put into
next step without further purification.
1
Yield: 70.9%; H NMR (400 MHz, CDCl ): δ = 8.44 (d,
3
J = 2.0 Hz, 1H, pyridine-H), 8.28 (d, J = 2.0 Hz, 1H, pyr-
idine-H), 7.48 (t, J = 2.0 Hz, 1H, pyridine-H), 7.34–7.30
General procedure for the synthesis of compounds 19–24
A solution of compounds 13–18 (1.6 mmol, 1.0 eq) and
(
m, 2H, Ar-H), 7.19–7.15 (m, 1H, Ar-H), 7.07–7.05 (m, 1H,
Ar-H), 2.92–2.82 (m, 1H, CH), and 1.10 (d, J = 6.8 Hz, 6H,
+
2
CH ). ESI-MS: m/z = 230.1[M + H] .
NaOH (129.6 mg, 3.2 mmol, 2.0 eq) in MeOH/H O (10/1,
3
2
10.0 mL) was stirred at 25 °C for 2 h. The mixture was
5
9
2
-(2-ethylphenyl)pyridine-3-thiol (12b) Yellow oil; Yield:
concentrated in vacuo. The residue was diluted with water,
extracted with methyl tert-butyl ether (50.0 mL × 2). Then
the pH of the water layer was adjusted to pH = 2 with 1N
HCl, the mixture was extracted with EtOAc (100.0 mL × 3).
The combined organic layer was dried over Na SO and
1
1.9%; H NMR (400 MHz, CDCl ): δ = 8.43 (d, J =
3
.0 Hz, 1H, pyridine-H), 8.30 (d, J = 2.0 Hz, 1H, pyridine-
H), 7.50 (t, J = 2.0 Hz, 1H, pyridine-H), 7.31–7.25 (m, 2H,
Ar-H), 7.21–7.17 (m, 1H, Ar-H), 7.09–7.01 (m, 1H, Ar-H),
2
4
2
3
.50 (q, J = 7.6, 14.8 Hz, 2H, CH ), and 1.04 (t, J = 7.6 Hz,
concentrated to give the compounds 19–24.
2
+
H, CH ). ESI-MS: m/z = 216.1[M + H] .
3
2
-((5-(2-isopropylphenyl)pyridin-3-yl)thio)thiazole-5-car-
1
5
-(2-methoxyphenyl)pyridine-3-thiol (12c) Yellow oil;
boxylic acid (19) H NMR (400 MHz, d -DMSO): δ =
6
1
Yield: 48.3%; H NMR (400 MHz, CDCl ): δ = 8.48 (d,
8.95 (d, J = 2.0 Hz, 1H, pyridine-H), 8.75 (d, J = 2.0 Hz,
1H, pyridine-H), 8.29 (s, 1H, thiazole-H), 8.23 (t, J =
2.0 Hz, 1H, pyridine-H), 7.55–7.46 (m, 2H, Ar-H),
7.38–7.34 (m, 1H, Ar-H), 7.30–7.28 (m, 1H, Ar-H),
3
J = 2.0 Hz, 1H, pyridine-H), 8.38 (d, J = 2.0 Hz, 1H, pyr-
idine-H), 7.72 (t, J = 2.0 Hz, 1H, pyridine-H), 7.33–7.28 (m,
1
H, Ar-H), 7.23–7.19 (m, 1H, Ar-H), 7.01–6.92 (m, 2H, Ar-
+
H), and 3.75 (s, 3H, CH ). ESI-MS: m/z = 218.1[M + H] .
3.00–2.93 (m, 1H, CH), and 1.18 (d, J = 6.8 Hz, 6H,
3
+
2
CH ). ESI-MS: m/z = 357.1[M + H] .
3
General procedure for the synthesis of compounds 13–15
2
-((5-(2-methoxyphenyl)pyridin-3-yl)thio)thiazole-5-car-
1
A mixture of compounds 12a–c (6.5 mmol, 1.0 eq), methyl
boxylic acid (20) H NMR (400 MHz, d -DMSO): δ =
6
2
-chlorothiazole-5-carboxylate (1.2 g, 6.5 mmol, 1.0 eq),
8.86 (d, J = 2.0 Hz, 1H, pyridine-H), 8.82 (d, J = 2.0 Hz,
1H, pyridine-H), 8.33 (t, J = 2.4 Hz, 1H, thiazole-H), 8.25
(s, 1H, pyridine-H), 7.47–7.43 (m, 2H, Ar-H), 7.19–7.17
and K CO (904.0 mg, 6.5 mmol, 1.0 eq) in CH CN
2
3
3
(
10.0 mL) was stirred at 82 °C for 16 h. The mixture was
concentrated to give the crude compounds 13–15, which
was put into next step without further purification.
(m, 1H, Ar-H), 7.11–7.07 (m, 1H, Ar-H), and 3.80 (s, 3H,
+
CH ). ESI-MS: m/z = 345.0[M + H] .
3
General procedure for the synthesis of compounds 16–17
2-((5-(2-ethylphenyl)pyridin-3-yl)thio)thiazole-5-carboxylic
1
acid (21) H NMR (400 MHz, d -DMSO): δ = 8.89 (d,
6
A mixture of compound 12a (699.5 mg, 3.0 mmol, 1.0 eq),
compound 3 or 7 (3.0 mmol, 1.0 eq), and K CO (843.1 mg,
J = 2.0 Hz, 1H, pyridine-H), 8.72 (d, J = 2.0 Hz, 1H, pyr-
idine-H), 8.24 (s, 1H, thiazole-H), 8.23 (t, J = 2.0 Hz, 1H,
pyridine-H), 7.43–7.38 (m, 2H, Ar-H), 7.34–7.30 (m, 1H,
Ar-H), 7.28–7.26 (m, 1H, Ar-H), 2.58 (q, J = 7.6, 15.2 Hz,
2
3
6
1
.1 mmol, 2.0 eq) in CH CN (10.0 mL) was stirred at
00 °C for 2 h (for compound 17, stirring 16 h at 82 °C).
3

Medicinal Chemistry Research
2
H, CH), and 1.02 (t, J = 3.6 Hz, 3H, CH ). ESI-MS:
2H, Ar-H), 7.38 (d, J = 4.0 Hz, 2H, Ar-H), 7.28 (t, J =
7.6 Hz, 2H, Ar-H), 7.24–7.20 (m, 1H, Ar-H), 7.11–7.07 (m,
3
+
m/z = 343.1[M + H] .
2
H, Ar-H), 2.88–2.81 (m, 1H, CH), and 1.12 (d, J = 6.8 Hz,
1
3
5
-((5-(2-isopropylphenyl)pyridin-3-yl)thio)-4-nitrothio-
6H, 2CH3). C NMR (100 MHz, d -DMSO): δ = 170.07,
6
1
phene-2-carboxylic acid (22) H NMR (400 MHz, d6-
DMSO): δ = 8.94 (d, J = 2.0 Hz, 1H, pyridine-H), 8.78 (d,
J = 2.0 Hz, 1H, pyridine-H), 8.28 (t, J = 2.4 Hz, 1H, pyr-
idine-H), 8.07 (s, 1H, thienyl-H), 7.50–7.43 (m, 2H, Ar-H),
158.27, 152.12, 151.01, 146.69, 144.95, 142.90, 138.80,
138.70, 137.00, 135.80, 130.31, 129.51, 129.20, 127.60,
126.35, 126.31, 124.60, 120.89, 29.66, and 24.31. ESI-MS:
+
m/z = 431.8[M + H] .
7
.31–7.24 (m, 2H, Ar-H), 2.97–2.91 (m, 1H, CH), 1.13 (d,
+
J = 6.8 Hz, 6H, 2CH ). ESI-MS: m/z = 400.8[M + H] .
N-(4-fluorophenyl)-2-((5-(2-isopropylphenyl)pyridin-3-yl)
thio)thiazole-5-carboxamide (32) White solid; Yield:
3
1
4
-cyano-5-((5-(2-isopropylphenyl)pyridin-3-yl)thio)thio-
35.7%; Purity: 95.1%. H NMR (400 MHz, d -DMSO):
6
1
phene-2-carboxylic acid (23) H NMR (400 MHz, CDCl3):
δ = 8.72 (d, J = 2.0 Hz, 1H, pyridine-H), 8.62 (d, J =
1
H), 7.86 (s, 1H, thienyl-H), 7.43 (d, J = 3.6 Hz, 2H, Ar-H),
7
δ = 10.49 (s, 1H, NH), 8.90 (d, J = 2.0 Hz, 1H, pyridine-H),
8.70 (d, J = 2.0 Hz, 1H, pyridine-H), 8.53 (s, 1H, pyridine-
H), 8.18 (t, J = 2.0 Hz, 1H, thiazole-H), 7.70–7.66 (m, 2H,
Ar-H), 7.50–7.42 (m, 2H, Ar-H), 7.30 (td, J = 7.6, 1.2 Hz,
1H, Ar-H), 7.25–7.17 (m, 3H, Ar-H), 2.95–2.88 (m, 1H,
CH), and 1.13 (d, J = 6.8 Hz, 6H, 2CH3). C NMR
(100 MHz, d -DMSO): δ = 170.34, 160.20, 158.23, 157.81,
.6 Hz, 1H, pyridine-H), 7.87 (t, J = 2.0 Hz, 1H, pyridine-
.28–7.26 (m, 1H, Ar-H), 7.15 (d, J = 7.6 Hz, 1H, Ar-H),
1
3
2
.92–2.85 (m, 1H, CH), 1.17 (d, J = 6.8 Hz, 6H, 2CH ) .
3
+
ESI-MS: m/z = 380.8[M + H] .
6
152.44, 151.32, 146.68, 144.99, 142.67, 138.72, 136.69,
5
-((5-(2-isopropylphenyl)pyridin-3-yl)thio)thiophene-2-car-
135.88, 135.05, 135.03, 130.30, 129.48, 127.40, 126.34,
126.29, 122.82, 122.74, 115.94, 115.72, 29.66, and 24.31.
ESI-MS: m/z = 449.9[M + H] .
1
boxylic acid (24) H NMR (400 MHz, CDCl ): δ = 7.54 (s,
3
+
1
2
8
H, pyridine-H), 7.38 (s, 2H, pyridine-H), 7.19–7.16 (m,
H, thienyl-H), 7.06–7.01 (m, 2H, Ar-H), 6.90 (d, J =
.8 Hz, 2H, Ar-H), 2.67–2.56 (m, 1H, CH), and 0.90 (d, J
2-((5-(2-isopropylphenyl)pyridin-3-yl)thio)-N-(4-methoxy-
+
=
6.6 Hz, 6H, 2CH ). ESI-MS: m/z = 356.1[M + H] .
phenyl)thiazole-5-carboxamide (33) Yellow solid; Yield:
3
1
6
1.4%; Purity: 99.5%. H NMR (400 MHz, MeOD): δ =
General procedure for the synthesis of compounds 25–30
8.86 (d, J = 2.0 Hz, 1H, pyridine-H), 8.64 (d, J = 1.2 Hz,
H, pyridine-H), 8.35 (s, 1H, pyridine-H), 8.15 (t, J =
1
To a solution of compounds 19–24 (1.0 mmol, 1.0 eq) and
DMF (7.6 mg, 103.5 μmol, 8.0 uL, 0.1 eq) in CH Cl
2.0 Hz, 1H, thiazole-H), 7.53–7.42 (m, 4H, Ar-H), 7.30 (td,
J = 7.6, 1.6 Hz, 1H, Ar-H), 7.23 (dd, J = 7.6, 1.2 Hz, 1H,
Ar-H), 6.93–6.89 (m, 2H, Ar-H), 3.79 (s, 3H, CH3),
2.99–2.95 (m, 1H, CH), and 1.20 (d, J = 6.8 Hz, 6H,
2
2
(
10.0 mL), and (COCl) (131.4 mg, 1.0 mmol, 91.0 μL, 1.0
2
eq) was added. The reaction was stirred at 25 °C for 1 h.
The reaction was concentrated in vacuo to give the com-
pounds 25–30, which was put into next step without further
purification.
1
3
2CH3). C NMR (100 MHz, d -DMSO): δ = 169.80,
6
157.90, 156.37, 152.38, 151.26, 146.69, 144.59, 142.59,
138.70, 137.14, 135.89, 131.65, 130.30, 129.47, 127.49,
1
26.34, 126.30, 122.54, 114.37, 55.67, 29.66, and 24.31.
+
General procedure for the synthesis of target compounds
ESI-MS: m/z = 462.0[M + H] .
3
1–50
N-benzyl-2-((5-(2-isopropylphenyl)pyridin-3-yl)thio)thia-
A solution of compounds 25–30 (600.0 μmol, 1.0 eq),
zole-5-carboxamide (34) White solid; Yield: 64.9%; Pur-
1
corresponding amine (660.0 μmol, 1.1 eq), and Et N
ity: 99.8%. H NMR (400 MHz, CDCl ): δ = 8.80 (s, 1H,
3
3
(
121.4 mg, 1.2 mmol, 166.0 μL, 2.0 eq) in CH Cl (5.0 mL)
pyridine-H), 8.61 (s, 1H, thiazole-H), 8.01 (d, J = 6.8 Hz,
2H, pyridine-H), 7.44 (d, J = 4.0 Hz, 2H, Ar-H), 7.36–7.26
(m, 6H, Ar-H), 7.16 (d, J = 7.6 Hz, 1H, Ar-H), 6.48 (s, 1H,
2
2
was stirred at 25 °C for 16 h. The reaction was concentrated
in vacuo. The residue was purified by prep-HPLC (0.1%
HCl as additive) to give the target compounds 31–50.
NH), 4.57 (d, J = 5.6 Hz, 2H, CH ), 2.94–2.88 (m, 1H,
2
1
3
CH), and 1.17 (d, J = 6.8 Hz, 6H, 2CH3). C NMR
2
-((5-(2-isopropylphenyl)pyridin-3-yl)thio)-N-phenylthia-
(100 MHz, d -DMSO): δ = 169.08, 159.58, 152.13, 151.01,
6
zole-5-carboxamide (31) White solid; Yield: 71.8%; Pur-
146.69, 144.14, 142.65, 139.39, 138.70, 136.77, 135.84,
130.30, 129.48, 128.82, 127.77, 127.67, 127.42,
126.34, 126.30, 42.99, 29.63, and 24.30. ESI-MS: m/z =
1
ity: 95.7%. H NMR (400 MHz, CDCl ): δ = 8.79 (s, 1H,
3
pyridine-H), 8.56 (s, 1H, pyridine-H), 8.15 (s, 1H, pyridine-
H), 8.00–7.95 (m, 2H, thiazole-H, NH), 7.53 (d, J = 8.0 Hz,
+
445.9[M + H] .

Medicinal Chemistry Research
2
-((5-(2-isopropylphenyl)pyridin-3-yl)thio)-N-(1-methylpi-
= 7.6, 15.2 Hz, 2H, CH ), and 1.11 (t, J = 7.6 Hz, 3H,
2
1
3
peridin-4-yl)thiazole-5-carboxamide (35) White solid;
Yield: 63.5%; Purity: 97.1%. H NMR (400 MHz, CDCl ):
δ = 8.95 (d, J = 6.4 Hz, 1H, pyridine-H), 8.62 (s, 1H, pyr-
idine-H), 8.55 (s, 2H, Ar-H), 8.08 (s, 1H thiazole-H), 7.43
CH3).
C NMR (100 MHz, d -DMSO): δ = 170.08,
6
1
160.20, 158.28, 157.80, 156.73, 151.41, 150.83, 145.15,
143.37, 136.70, 135.72, 135.08, 135.06, 131.04, 130.95,
127.50, 124.94, 122.83, 122.76, 121.57, 115.91, 115.69,
3
+
(
d, J = 4.0 Hz, 2H, Ar-H), 7.28–7.24 (m, 1H, Ar-H), 7.17
112.44, and 56.17. ESI-MS: m/z = 437.9[M + H] .
(
d, J = 7.6 Hz, 1H, NH), 4.23 (s, 3H, CH , CH), 3.59 (d,
2
J = 10.8 Hz, 1H, CH ), 3.08–2.98 (m, 2H, CH ), 2.92–2.84
N-(4-fluorophenyl)-2-((5-(2-methoxyphenyl)pyridin-3-yl)
thio)thiazole-5-carboxamide (39) White solid; Yield:
24.9%; Purity: 99.7%. H NMR (400 MHz, MeOD): δ =
2
2
(
m, 3H, CH , CH), 2.49–2.40 (m, 2H, CH ), 2.18 (d, J =
2
3
1
3
1
1
2.4 Hz, 2H, CH ), and 1.17 (d, J = 6.8 Hz, 6H, 2CH ).
C
2
3
NMR (100 MHz, d -DMSO): δ = 169.07, 159.12, 151.97,
9.03 (d, J = 6.4 Hz, 2H, pyridine-H), 8.83 (s, 1H, pyridine-
H), 8.44 (s, 1H, thiazole-H), 7.68–7.64 (m, 2H, pyridine-H),
7.55–7.51 (m, 2H, Ar-H), 7.23–7.09 (m, 4H, Ar-H), and
6
1
1
4
50.88, 146.68, 144.39, 142.72, 138.74, 136.73, 135.80,
30.29, 129.51, 127.77, 126.34, 126.31, 52.85, 49.31,
1
3
4.87, 42.83, 29.64, 29.03, 26.36, and 24.31. ESI-MS:
3.91 (s, 3H, CH3). C NMR (100 MHz, d -DMSO) : δ =
6
+
m/z = 453.0[M + H] .
169.12, 159.59, 152.12, 150.86, 144.19, 142.78, 141.95,
139.40, 138.67, 136.75, 136.33, 130.44, 129.39, 129.32,
(
2-((5-(2-isopropylphenyl)pyridin-3-yl)thio)thiazol-5-yl)(4-
128.81, 127.78, 127.72, 127.42, 126.62, 43.00, 25.98, and
15.78. ESI-MS: m/z = 432.0[M + H] .
+
methylpiperazin-1-yl)methanone (36) White solid; Yield:
1
4
9
1
2
1
2
6
7.0%; Purity: 97.1%. H NMR (400 MHz, CDCl ): δ =
3
.02 (s, 1H, pyridine-H), 8.65 (s, 1H, pyridine-H), 8.25 (s,
H, pyridine-H), 8.04 (s, 1H, thiazole-H), 7.48–7.43 (m,
H, Ar-H), 7.28–7.26 (m, 1H, Ar-H), 7.17 (d, J = 7.2 Hz,
H, Ar-H), 5.86 (s, 2H, CH ), 4.48 (s, 2H, CH ), 3.59 (s,
N-benzyl-2-((5-(2-methoxyphenyl)pyridin-3-yl)thio)thiazole-
5-carboxamide (40) White solid; Yield: 36.9%; Purity:
1
98.8%. H NMR (400 MHz, MeOD): δ = 8.99 (d, J =
2.0 Hz, 1H, pyridine-H), 8.96 (d, J = 2.0 Hz, 1H, pyridine-
H), 8.75 (t, J = 2.0 Hz, 1H, pyridine-H), 8.27 (s, 1H, thia-
zole-H), 7.54–7.48 (m, 2H, Ar-H), 7.33 (d, J = 4.4 Hz, 4H,
Ar-H), 7.29–7.24 (m, 1H, Ar-H), 7.21–7.13 (m, 2H, Ar-H),
2
2
H, CH ), 3.19 (s, 2H, CH ), 2.93 (s, 3H, CH ), 2.83 (t, J =
2
2
3
1
3
.8 Hz, 1H, CH), and 1.17 (d, J = 6.4 Hz, 6H, 2CH3).
C
NMR (100 MHz, d -DMSO): δ = 168.41, 160.29, 151.98,
6
1
3
1
1
2
50.84, 146.67, 144.85, 142.76, 138.75, 135.76, 133.36,
4.52 (s, 2H, CH ), and 3.87 (s, 3H, CH ). C NMR
2 3
30.32, 129.54, 127.65, 126.37, 126.32, 52.31, 42.48,
(100 MHz, d -DMSO): δ = 169.01, 159.61, 156.72, 151.34,
6
+
9.66, and 24.31. ESI-MS: m/z = 439.0[M + H] .
150.73, 144.27, 143.21, 139.40, 136.71, 135.66, 131.02,
1
30.95, 128.81, 127.79, 127.64, 127.41, 124.95, 121.57,
+
2
-((5-(2-ethylphenyl)pyridin-3-yl)thio)-N-(4-fluorophenyl)
112.44, 56.15, and 43.00. ESI-MS: m/z = 433.9[M + H] .
thiazole-5-carboxamide (37) White solid; Yield: 48.8%;
1
Purity: 99.4%. H NMR (400 MHz, MeOD): δ = 8.96 (d,
N-(4-fluorophenyl)-5-((5-(2-isopropylphenyl)pyridin-3-yl)
thio)-4-nitrothiophene-2-carboxamide (41) White solid;
Yield: 30.4%; Purity: 99.6%. H NMR (400 MHz, d6-
J = 1.6 Hz, 1H, pyridine-H), 8.75 (d, J = 1.6 Hz, 1H, thia-
zole-H), 8.39 (s, 1H, pyridine-H), 8.38 (t, J = 2.0 Hz, 1H,
pyridine-H), 7.67–7.62 (m, 2H, Ar-H), 7.46–7.41 (m, 2H,
Ar-H), 7.36–7.31 (m, 1H, Ar-H), 7.28 (d, J = 7.6 Hz, 1H,
Ar-H), 7.13–7.07 (m, 2H, Ar-H), 2.65 (q, J = 7.6, 15.2 Hz,
1
DMSO): δ = 10.59 (s, 1H, thienyl-H), 8.94 (d, J = 2.0 Hz,
1H, pyridine-H), 8.77 (d, J = 2.0 Hz, 1H, pyridine-H), 8.70
(s, 1H, pyridine-H), 8.28 (t, J = 2.0 Hz, 1H, NH), 7.70–7.66
(m, 2H, Ar-H), 7.49–7.42 (m, 2H, Ar-H), 7.32–7.17 (m, 4H,
Ar-H), 2.98–2.91 (m, 1H, CH), and 1.13 (d, J = 6.4 Hz, 6H,
13
2
H, CH ), and 1.12 (t, J = 7.6 Hz, 3H, CH ). C NMR
2
3
(
100 MHz, d -DMSO): δ = 170.12, 160.20, 158.24, 157.80,
6
1
3
1
1
1
1
52.15, 152.07, 150.92, 150.84, 145.04, 143.04, 142.98,
41.97, 138.74, 136.79, 136.76, 136.29, 136.27, 135.06,
30.44, 129.40, 129.34, 127.60, 126.63, 122.81, 122.73,
2CH3). C NMR (100 MHz, d -DMSO): δ = 155.73,
6
155.05, 152.82, 151.93, 146.19, 143.20, 140.71, 138.50,
135.39, 134.41, 129.80, 129.00, 126.74, 125.87, 125.75,
124.57, 122.08, 122.00, 115.59, 115.36, 29.24, and 23.72.
15.91, 115.69, 26.00, and 15.79. ESI-MS: m/z = 435.9[M
+
+
+
H] .
ESI-MS: m/z = 493.9[M + H] .
N-benzyl-2-((5-(2-ethylphenyl)pyridin-3-yl)thio)thiazole-5-
N-benzyl-5-((5-(2-isopropylphenyl)pyridin-3-yl)thio)-4-
carboxamide (38) White solid; Yield: 15.1%; Purity:
nitrothiophene-2-carboxamide (42) White solid; Yield:
1
1
9
2
9.8%. H NMR (400 MHz, MeOD): δ = 8.99 (d, J =
.0 Hz, 1H, pyridine-H), 8.78 (d, J = 2.0 Hz, 1H, pyridine-
41.5%; Purity: 99.9%. H NMR (400 MHz, CDCl ): δ =
3
8.86 (s, 1H, pyridine-H), 8.75 (s, 1H, pyridine-H), 8.08 (s,
1H, pyridine-H), 7.94 (s, 1H, thienyl-H), 7.46 (d, J =
4.0 Hz, 2H, Ar-H), 7.36–7.27 (m, 6H, Ar-H), 7.20 (d, J =
7.6 Hz, 1H, Ar-H), 6.55 (s, 1H, NH), 4.56 (d, J = 5.6 Hz,
H), 8.47 (t, J = 2.0 Hz, 1H, pyridine-H), 8.25 (s, 1H, thia-
zole-H), 7.48–7.42 (m, 2H, Ar-H), 7.37–7.33 (m, 5H, Ar-
H), 7.30–7.24 (m, 2H, Ar-H), 4.52 (s, 2H, CH ), 2.63 (q, J
2

Medicinal Chemistry Research
2
6
1
1
1
2
H, CH ), 2.97–2.90 (m, 1H, CH), and 1.20 (d, J = 6.4 Hz,
N-benzyl-4-cyano-5-((5-(2-isopropylphenyl)pyridin-3-yl)
thio)thiophene-2-carboxamide (46) White solid; Yield:
35.9%; Purity: 99.2%. H NMR (400 MHz, MeOD) : δ =
8.84 (d, J = 2.0 Hz, 1H, pyridine-H), 8.64 (d, J = 2.0 Hz, 1H,
pyridine-H), 8.07 (t, J = 2.0 Hz, 1H, pyridine-H), 7.94 (s, 1H,
thienyl-H), 7.50–7.45 (m, 2H, Ar-H), 7.35–7.22 (m, 7H, Ar-
2
1
3
H, 2CH3). C NMR (100 MHz, d -DMSO): δ = 159.67,
6
1
54.44, 153.20, 152.22, 146.74, 143.80, 141.25, 139.13,
39.04, 136.03, 135.80, 130.28, 129.54, 128.82, 127.87,
27.50, 127.38, 126.35, 126.26, 124.15, 43.14, 29.71, and
+
4.19. ESI-MS: m/z = 489.9[M + H] .
H), 4.54 (s, 2H, CH ), 2.87–2.80 (m, 1H, CH), and 1.16 (t,
2
1
3
5
-((5-(2-isopropylphenyl)pyridin-3-yl)thio)-N-(1-methylpi-
J = 6.8 Hz, 6H, 2CH3). C NMR (100 MHz, d -DMSO): δ
6
peridin-4-yl)-4-nitrothiophene-2-carboxamide (43) White
solid; Yield: 53.1%; Purity: 99.6%. H NMR (400 MHz,
MeOD): δ = 9.10 (d, J = 2.0 Hz, 1H, pyridine-H), 8.90 (d,
J = 2.0 Hz, 1H, pyridine-H), 8.56 (t, J = 2.0 Hz, 1H, pyr-
idine-H), 8.35 (s, 1H, thienyl-H), 7.54–7.48 (m, 2H, Ar-H),
= 159.45, 149.68, 149.51, 149.41, 146.64, 143.61, 139.91,
139.15, 138.61, 135.76, 130.53, 130.42, 130.26, 129.49,
128.82, 127.84, 127.49, 126.32, 113.93, 113.82, 43.14, 29.61,
1
+
and 24.29. ESI-MS: m/z = 470.0[M + H] .
7
3
2
1
2
1
1
1
.36–7.29 (m, 2H, Ar-H), 3.59 (d, J = 12.8 Hz, 2H, CH2),
.16 (t, J = 12.4 Hz, 2H, 2CH), 3.02–2.94 (m, 2H, CH2),
.89 (s, 3H, CH ), 2.21 (d, J = 13.6 Hz, 2H, CH ),
4-cyano-5-((5-(2-isopropylphenyl)pyridin-3-yl)thio)-N-(1-
methylpiperidin-4-yl)thiophene-2-carboxamide (47) White
solid; Yield: 37.5%; Purity: 95.6%. H NMR (400 MHz,
1
3
2
.98–1.88 (m, 2H, CH ), and 1.22 (d, J = 6.8 Hz, 6H,
MeOD): δ = 8.92 (s, 1H, pyridine-H), 8.73 (s, 1H, pyridine-
H), 8.22 (s, 1H, pyridine-H), 8.05–8.03 (m, 1H, thienyl-H),
7.53–7.47 (m, 2H, Ar-H), 7.35–7.24 (m, 2H, Ar-H),
4.14–4.08 (m, 1H, CH), 3.60 (d, J = 13.2 Hz, 2H, CH2),
3.17 (t, J = 13.2 Hz, 2H, CH ), 2.95–2.82 (m, 4H, CH ,
2
1
3
CH3). C NMR (100 MHz, d -DMSO) : δ = 159.78,
6
59.17, 154.31, 152.96, 151.99, 146.73, 143.99, 141.26,
41.19, 139.08, 135.94, 135.71, 130.25, 129.58, 127.49,
26.35, 126.26, 124.42, 52.75, 44.91, 42.79, 29.71, 28.94,
2
3
+
and 24.19. ESI-MS: m/z = 497.0[M + H] .
CH), 2.23 (d, J = 14.0 Hz, 2H, CH ), 1.97 (t, J = 14.0 Hz,
2
1
3
2
H, CH ), and 1.17 (d, J = 6.8, Hz, 6H, 2CH ). C NMR
2
3
(
5-((5-(2-isopropylphenyl)pyridin-3-yl)thio)-4-nitrothio-
(100 MHz, d6-DMSO): δ = 158.97, 149.75, 149.58,
149.48, 146.63, 143.46, 139.82, 138.58, 135.78, 130.77,
130.47, 130.25, 129.49, 126.32, 113.79, 113.75, 52.79,
44.98, 42.82, 29.61, 28.95, and 24.29. ESI-MS:
phen-2-yl)(4-methylpiperazin-1-yl) methanone (44) White
solid; Yield: 53.1%; Purity: 99.7%. H NMR (400 MHz,
MeOD): δ = 9.04 (d, J = 1.6 Hz, 1H, pyridine-H), 8.84 (d,
J = 1.6 Hz, 1H, pyridine-H), 8.45 (d, J = 2.0 Hz, 1H, pyr-
idine-H), 8.04 (s, 1H, thienyl-H), 7.54–7.47 (m, 2H, Ar-H),
1
+
m/z = 477.0[M + H] .
7
.39(t, J = 7.6 Hz, 1H, Ar-H), 7.28 (d, J = 7.6 Hz, 1H, Ar-
2-((5-(2-isopropylphenyl)pyridin-3-yl)thio)-5-(4-methylpi-
H), 4.56 (d, J = 14.0 Hz, 2H, CH ), 3.61–6.52 (m, 4H,
perazine-1-carbonyl)thiophene-3-carbonitrile (48) Yellow
solid; Yield: 37.5%; Purity: 97.5%. H NMR (400 MHz,
MeOD): δ = 9.00 (d, J = 2.0 Hz, 1H, pyridine-H), 8.80 (d,
J = 1.6 Hz, 1H, pyridine-H), 8.37 (t, J = 2.0 Hz, 1H, pyr-
idine-H), 7.89 (s, 1H, thienyl-H), 7.55–7.49 (m, 2H, Ar-H),
7.37–7.27 (m, 2H, Ar-H), 4.54 (d, J = 14.1 Hz, 2H, CH2),
2
1
2
1
2
1
1
1
4
CH ), 3.20 (t, J = 11.6 Hz, 2H, CH ), 3.00 (t, J = 6.8 Hz,
2
2
H, CH), 2.96(s, 3H, CH ), and 1.23 (d, J = 6.8 Hz, 6H,
3
1
3
CH3). C NMR (100 MHz, d -DMSO): δ = 160.73,
6
53.98, 153.24, 152.24, 146.71, 143.80, 140.54, 139.05,
35.75, 133.29, 130.31, 129.58, 127.30, 126.37, 126.30,
25.57, 52.19, 42.47, 29.72, and 24.24. ESI-MS: m/z =
3.61 (d, J = 12.8 Hz, 4H, 2CH ), 3.24 (t, J = 14.6 Hz, 2H,
2
+
82.9[M + H] .
CH ), 2.98(s, 3H, CH ), 2.88–2.81 (m, 1H, CH), and 1.23
2
3
1
3
(
d, J = 6.8 Hz, 6H, 2CH3). C NMR (100 MHz, d6-
4
-cyano-N-(4-fluorophenyl)-5-((5-(2-isopropylphenyl)pyri-
DMSO): δ = 160.54, 149.44, 149.24, 148.54, 146.66,
140.42, 140.05, 138.66, 135.67, 132.12, 130.56, 130.29,
129.55, 126.34, 113.90, 113.57, 52.26, 42.44, 29.63, and
din-3-yl)thio)thiophene-2-carboxamide (45) White solid;
Yield: 53.1%; Purity: 99.3%. H NMR (400 MHz, MeOD)
:
1
+
δ = 8.87 (d, J = 1.6 Hz, 1H, pyridine-H), 8.67 (d, J =
24.32. ESI-MS: m/z = 463.0[M + H] .
1
.6 Hz, 1H, pyridine-H), 8.13 (s, 2H, pyridine-H, thienyl-
H), 7.69–7.66 (m, 2H, Ar-H), 7.51–7.45 (m, 2H, Ar-H),
N-(4-fluorophenyl)-5-((5-(2-isopropylphenyl)pyridin-3-yl)
7
1
.32 (dt, J = 7.6, 1.6 Hz, 1H, Ar-H), 7.24 (d, J = 7.6 Hz,
H, Ar-H), 7.14–7.10 (m, 2H, Ar-H), 2.91–2.84 (m, 1H,
thio)thiophene-2-carboxamide (49) White solid; Yield:
1
12.6%; Purity: 99.6%. H NMR (400 MHz, CDCl ): δ =
3
13
CH), and 1.18 (d, J = 6.8 Hz, 6H, 2CH3). C NMR
100 MHz, d6-DMSO): δ = 157.55, 149.83, 149.74,
8.38 (s, 2H, thienyl-H), 8.27 (s, 1H, pyridine-H), 7.72 (s,
2H, pyridine-H), 7.61 (dd, J = 8.8, 4.8 Hz, 2H, Ar-H),
7.46–7.42 (m, 2H, Ar-H), 7.33 (s, 1H, Ar-H), 7.10 (d, J =
7.6 Hz, 1H, Ar-H), 7.03 (t, J = 8.4 Hz, 2H, Ar-H),
(
1
1
1
4
46.15, 142.44, 139.43, 138.07, 135.39, 134.28, 130.80,
29.77, 129.43, 128.96, 125.86, 122.31, 122.23, 115.59,
15.37, 113.25, 112.60, 29.16, and 23.84. ESI-MS: m/z =
2.82–2.78 (m, 1H, CH), and 1.15 (d, J = 6.8 Hz, 6H, CH ).
3
+
13
73.9[M + H] .
C NMR (100 MHz, d -DMSO): δ = 160.17, 159.24,
6

Medicinal Chemistry Research
1
1
1
1
57.78, 147.32, 146.59, 146.39, 145.19, 138.32, 137.41,
37.19, 135.82, 135.54, 135.22, 135.20, 134.24, 130.66,
30.23, 129.46, 126.37, 126.33, 122.91, 122.83, 115.88,
with ATP concentration at 75 μM. Briefly, the compound was
prepared as 50× of the final desired concentration stocks in
100% DMSO and directly diluted at five different con-
centrations (10 nM, 50 nM, 0.25 μM, 1.25 μM, and 6.25 μM)
into the assay. The substrate solution contains 3 μM Peptide
FAM-P13 (5-FAM-KKSRGDYMTMQIG-CONH2), 75 μM
ATP, 50 mM HEPES (pH 7.5), 0.0015% Brij-35, and 10 mM
+
15.65, 29.58, and 24.35. ESI-MS: m/z = 448.9[M + H] .
N-benzyl-5-((5-(2-isopropylphenyl)pyridin-3-yl)thio)thio-
phene-2-carboxamide (50) White solid; Yield: 15.6%;
Purity: 99.6%. H NMR (400 MHz, CDCl ): δ = 8.38 (s,
1
MgCl . The kinase buffer contains 4 nM IGF-1R, 50 mM
3
2
2
H, pyridine-H), 7.71 (s, 1H, pyridine-H), 7.51 (s, 1H, Ar-
H), 7.47–7.41 (m, 2H, thienyl-H), 7.35–7.26 (m, 7H, Ar-H),
.10 (d, J = 7.6 Hz, 1H, Ar-H), 6.53 (s, 1H, NH), 4.61 (d,
J = 1.6 Hz, 2H, CH ), 2.80–2.73 (m, 1H, CH), and 1.13 (d,
HEPES (pH 7.5), 0.0015% Brij-35, and 2 mM DTT. The stop
buffer contains 100 mM HEPES (pH 7.5), 0.015% Brij-35,
and 0.2% Coating Reagent #3 and 50 mM EDTA. Transfer
10 µL of compound to a new 96-well plate as the intermediate
plate, add 90 µL kinase buffer to each well. Transfer 5 µL of
each well from the 96-well intermediate plate to a 384-well
plate in duplicates. Then 10 μL kinases solution was added to
the 384-well plate. And then 10 μL substrate solution was
added to each well. The plate was then incubated at 28 °C for
7
2
13
J = 6.4 Hz, 6H, 2CH3). C NMR (100 MHz, d -DMSO):
δ = 160.58, 147.11, 146.59, 146.08, 145.56, 139.62,
1
1
2
6
38.29, 137.54, 137.20, 135.79, 134.20, 130.23, 129.59,
29.46, 128.78, 127.73, 127.36, 126.38, 126.33, 42.99,
+
9.54, and 24.35. ESI-MS: m/z = 445.0[M + H] .
60 min. After incubation, then 25 μL stop buffer was added to
Similarity search and molecular docking
stop the reaction. A reaction that contained no active enzyme
and without compound was used as the DMSO control, and
the staurosporine for the positive control. Read the conversion
data on Caliper EZ reader II, convert conversion values to
inhibition values. The inhibition rate (%) was calculated using
the following equation = (max-conversion)/(max − min) ×
100, “max” stands for the DMSO control and “min” stands
for the low control. Fit the data in XLFit excel add-in version
5.4.0.8 to obtain the IC50 values.
The 3D shape similarity to the reference molecule com-
pound III-106 was calculated by using Phase (Schrödinger,
LLC, New York, NY, 2018. All the software used for
similarity search and docking study belong to the same
company.). Each molecule was prepared by LigPrep to
generate their 3D structure, and their conformers were
generated by using ConfGen. Each conformer from a given
molecule is aligned to the reference molecule, and a simi-
larity is computed based on overlapping hard-sphere
volumes. MacroModel types were chosen as atom typing
in the shape similarity calculation.
Cell proliferation assay
The antiproliferative activities in vitro of the compounds
were measured by the standard CTG assay. K562 (Leuke-
mia), Hep-G2 (Human liver cancer), HCT-116 (Colon
cancer), WSU-DLCL2 (Lymphoma), and A549 (Non-small
cell lung cancer) cell lines were cultured in EMEM sup-
plement with 10% fetal bovine serum (FBS). Briefly, cells
The molecular docking procedure was performed by using
Glide with the default option. The cocrystal structure of IGF-
1R (PDB ID: 5FXS) was selected as the docking template. For
the preparation of protein, the hydrogen atoms were added by
using protein preparation Wizard module of Maestro, and the
OPLS3 force field was employed. For the preparation of
ligands, the 3D structures were generated and their energy
minimization was performed by using LigPrep. Conformers
were generated by using ConfGen. A 30 Å docking grid was
generated using centroid of ligand in the 5FXS crystal struc-
ture. Then the ligand was removed and compound was placed
during the molecular docking procedure. Types of interaction
of the docked IGF-1R with ligand were analyzed and then the
docking conformations were selected and saved based on cal-
culated glide docking energy score.
3
were plated at a plating density of 4 × 10 cells per well, and
incubated in 5% CO at 37 °C for 24 h. The cells were
2
treated with all the tested compounds at the indicated final
concentrations, respectively (DMSO as the negative, and
Paclitaxel as the positive), and then the cell cultures were
continued to incubate for 72 h. Cell titer-Glo reagent was
added to each well, and shake the plates for 10 min to
induce cell lysis. Allow the plate incubates at room tem-
perature for 2 min to stabilize luminescent signal, then the
luminescence was read with Envision. The IC50 values were
obtained by using the GraphPad Prism 5.0. All the target
compounds were tested in each of cell lines, and the inhi-
bition rates of proliferation were calculated with the fol-
lowing equation: Inhibition (%) = (Max signal −
Compound signal)/(Max signal − Min signal) × 100. Max
signal was obtained from the action of DMSO. Min signal
was obtained from the action of medium only.
Biological evaluation
IGF-1R kinase activity assay
Effects of the tested compound on the activities of IGF1R
kinases were evaluated by electrophoretic mobility shift assay

Medicinal Chemistry Research
Enzymatic inhibitory assay
Reagent #3, and 50 mM EDTA) was added to stop the
reaction. A reaction that contained no active enzyme and
without compound was used as the DMSO control, and the
staurosporine for the positive control. Read the conversion
data on Caliper EZ reader II, convert conversion values to
inhibition values. The inhibition rate (%) was calculated using
the following equation = (max-conversion)/(max − min) ×
100, “max” stands for the DMSO control and “min” stands
for the low control. Fit the data in XLFit excel add-in version
5.4.0.8 to obtain the IC50 values.
Method (A): For most assays, kinase-tagged T7 phage
strains were grown in parallel in 24-well blocks in an E. coli
host derived from the BL21 strain. E. coli were grown to log
phase and infected with T7 phage from a frozen stock
(
multiplicity of infection = 0.4) and incubated with shaking
at 32 °C until lysis (90–150 min). The lysates were cen-
trifuged (6000 × g) and filtered (0.2 µm) to remove cell
debris. The remaining kinases were produced in HEK-293
cells and subsequently tagged with DNA for qPCR detec-
tion. Streptavidin-coated magnetic beads were treated with
biotinylated small molecule ligands for 30 min at room
temperature to generate affinity resins for kinase assays. The
liganded beads were blocked with excess biotin and washed
with blocking buffer (SeaBlock (Pierce), 1% BSA, and
Flow cytometry analysis for cell cycle assay
Cell cycle arrest of Hep-G2 cells induced by the tested
compound was measured by FACS. Hep-G2 cells at a
6
density of 1 × 10 cells were treated with the tested com-
0
.05% Tween 20, 1 mM DTT) to remove unbound ligand
pound (0.5, 1, 5, and 10 μM), Doxorubicin (0.5 μM, 1 μM)
or DMSO (control) for 24 h. The cells were harvested by
digestion with Trypsin-EDTA and centrifugation at
1500 rpm for 15 min. Fix the cells with 75% EtOH, and
vortex the tube at half speed while adding EtOH to prevent
clustering of cells during the fixation. Incubate on ice for
15 min and then stored at −20 °C overnight. Afterwards,
PBS was added and centrifugation at 1500 rpm for 5 min,
discard the supernatant. Then the cells were washed once by
PBS (including 1%FBS), and incubated with PI/RNase
Staining Buffer at room temperature for 20 min in the dark.
Cells were analyzed by CytoFLEX at the lowest speed and
results were analyzed by ModFit software.
and to reduce nonspecific phage binding. Binding reactions
were assembled by combining kinases, liganded affinity
beads, and the tested compound in 1× binding buffer (20%
SeaBlock, 0.17× PBS, 0.05% Tween 20, and 6 mM DTT).
The tested compound was prepared as 40× stocks in 100%
DMSO and directly diluted into the assay. All reactions
were performed in polypropylene 384-well plates in a final
volume of 20 µL. The assay plates were incubated at room
temperature with shaking for 1 h and the affinity beads were
washed with wash buffer (1× PBS, 0.05% Tween 20). The
beads were then resuspended in elution buffer (1× PBS,
0
.05% Tween 20, and 0.5 µM nonbiotinylated affinity
ligand) and incubated at room temperature with shaking for
0 min. The kinase concentration in the eluates was mea-
3
sured by qPCR. The results for primary screen binding
interactions are reported as “% Ctrl”, which were calculated
with the following equation: “% Ctrl” = (compound signal
Conclusions
In conclusion, a series of 3-(thiophen/thiazole-2-ylthio)pyr-
idine derivatives were synthesized and evaluated. Different
from what was envisaged, all the target compounds exhib-
ited no inhibitory activities against IGF-1R. However, most
analogues showed moderate to potent anticancer activities
against various cancer cell lines. Representative compound
43 was further screened against more than 50 kinases, and
potent activities were observed against kinases such as
FGFR 3, FGFR 2, EGFR, JAK, and RON. Moreover,
according to cell cycle assay, compound 43 induced G1/G0
phase arrest of the cell cycle in Hep-G2 cells. In the light of
this study, 3-(thiophen/thiazole-2-ylthio)pyridine derivatives
were considered to be multi-target anticancer agents, which
are worth for further optimization for cancer treatment.
−
positive control signal)/(negative control signal − posi-
tive control signal) × 100. Negative control as the DMSO
control (100% Ctrl) and positive control stands for the
control compound (0% Ctrl).
Method (B): Further evaluation of the enzymatic selectivity
of the compounds against six kinases (AURA, JAK3,
FGFR2, FGFR3, EGFR, and RON) with ATP concentration
at Km was carried out by mobility shift assay. Dilute the
tested compounds to 50× of the final desired highest inhibitor
concentration in reaction by 100% DMSO. Then 5 μL of the
tested compound solution was added to a 384-well plate. And
then 10 μL kinases solution (4 nM kinases, 50 mM HEPES
[pH 7.5], 0.0015% Brij-35, and 2 mM DTT) was added to the
3
84-well plate. Then 10 μL substrate solution (3 μM Peptide
Acknowledgements The authors thank the project supported by the
key Development Program of the Hangzhou Science and Technology
Committee (Grant No. 20152013A03), the Research project on
application of public welfare technology supported by Science Tech-
nology Department of Zhejiang Province (Grant No. 2017C33233 and
LGF18H300001).
FAM-P13(5-FAM-KKSRGDYMTMQIG-CONH2), 75 μM
ATP, 50 mM HEPES [pH 7.5], 0.0015% Brij-35, and 10 mM
MgCl ) was added to each well. The plate was then incubated
at 28 °C for 60 min. After incubation, then 25 μL stop buffer
2
(100 mM HEPES [pH 7.5], 0.015% Brij-35, 0.2% Coating

Medicinal Chemistry Research
Compliance with ethical standards
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