Discovery of novel 2-substituted-4-phenoxypyridine derivatives as potential antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2017.12.063

A series of 2-substituted-4-phenoxypyridine derivatives were designed, synthesized, and evaluated for their antiproliferative activity against 4 cancer cell lines (A549, HT-29, H460, and U87MG) in vitro. Most compounds showed moderate to excellent potency. Nine tyrosine kinases (c-Met, Flt-3, ALK, VEGFR-2, VEGFR-3, PDGFR-α PDGFR-β c-Kit, and EGFR) were used to evaluate the inhibitory activities with the most promising analogue 39, which showed the Flt-3/c-Met IC₅₀ values of 2.18/2.61 nM. Structure-activity relationship studies indicated that n-Pr served as R¹ group showed a higher preference, and stronger mono-EWGs on the phenyl ring (such as R² = 4-F) was benefited to the potency.

Full text of DOI:10.1016/j.bmcl.2017.12.063

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel 2-substituted-4-phenoxypyridine derivatives as
potential antitumor agents
Yongli Duan a,c, Shan Xu , Hehua Xiong , Linxiao Wang , Bingbing Zhao , Ping Wang , Caolin Wang ,
a,c
a
a
a
a
a
a
a
b
a,
⇑
a,
⇑
Yiqing Peng , Shifan Cai , Rong Luo , Pengwu Zheng , Qidong Tang
a
Jiangxi Provincial Key Laboratory of Drug Design and Evaluation, School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang 330013, PR China
Jiangxi Province Institute of Materia Medica, Nanchang 330000, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-substituted-4-phenoxypyridine derivatives were designed, synthesized, and evaluated for
their antiproliferative activity against 4 cancer cell lines (A549, HT-29, H460, and U87MG) in vitro.
Most compounds showed moderate to excellent potency. Nine tyrosine kinases (c-Met, Flt-3, ALK,
Received 14 October 2017
Revised 25 December 2017
Accepted 28 December 2017
Available online xxxx
VEGFR-2, VEGFR-3, PDGFR-a, PDGFR-b, c-Kit, and EGFR) were used to evaluate the inhibitory activities
with the most promising analogue 39, which showed the Flt-3/c-Met IC50 values of 2.18/2.61 nM.
1
Structure-activity relationship studies indicated that n-Pr served as R group showed a higher preference,
Keywords:
Synthesis
2
and stronger mono-EWGs on the phenyl ring (such as R = 4-F) was benefited to the potency.
Ó 2017 Elsevier Ltd. All rights reserved.
4
1
-Phenoxypyridine derivatives
,8-Naphthyridinone
Antiproliferative activity
c-Met
Flt-3
Cancer is one of the leading causes of morbidity and mortality
worldwide, which is the second leading cause of death globally.
Cancer was responsible for 8.8 million deaths in 2015. Globally,
series (6).8–14 However, the main modification of these different
series of derivatives was focused on moiety C (a 5-atom linker),
which has two obvious structural characteristics. One is the ‘5
atoms regulation’, which means six chemical bonds distance
existed between moiety B and moiety D; the other is the 5-atom
linker containing hydrogen, oxygen, and nitrogen atoms which
could form hydrogen-bond donor or acceptor.
1
nearly 1 in 6 deaths is due to cancer. Despite the efforts to dis-
cover and develop small molecule anticancer drugs in the last dec-
2
–4
ades,
development of new antitumor agents with improved
tumor efficiency, selectivity, and safety remains in urgent need.
Recently, significant progress has been made in the develop-
ment of c-Met kinase inhibitors, resulting in the marketing of
cabozantinib (approved on November 2012 by the U.S. FDA for
the treatment of patients with progressive metastatic medullary
thyroid cancer, 3, Fig. 1) and more than 10 candidates under
In our previous study, we introduced 2-oxo-4-chloro-1,2-dihy-
droquinoline and pyridine fragments into the 5-atom linker based
on the two structural characteristics, and the resulting 6,7-disub-
stitutedquinoline (7, Fig. 2) and pyrrolo[2,3-b]pyridine derivatives
1
5,16
(8) showed excellent potency, respectively.
1,8-Naphthyridi-
5
–7
clinical trials.
We had analyzed the structural characteristics
none fragment was widely used as a building block in the design
of these c-Met kinase inhibitors. The general structure of small-
molecule c-Met kinases inhibitors was summarized as illustrated
in Fig. 1, which could be divided to moiety A, B, C, and D. Judging
from moiety A, many structure types of these derivatives were
included, such as substituted quinoline (1–3), thieno[2,3-b]pyri-
dine (4), pyrrolo[2,3-b]pyridine (5), and 2-amino-3-chloropyridine
of anticancer agents. For example, compounds 9 displayed a
multitude of biological activities.
1
7
In this work, 1,8-naphthyridinone was introduced to the 5-atom
linker as illustrated in Fig. 3, because the carbonyl oxygen or two
nitrogen atoms in 1,8-naphthyridinone as the hydrogen-bond
acceptor have high ability to form hydrogen-bonding interactions
with c-Met. 2-substitutedpyridine was used as moiety A. Substi-
tuted phenyl ring was reserved as moiety B and moiety D. Small
1
2
substituents R and R were introduced to investigate their effects
on activity of the target compounds. Accordingly, we designed a
novel series of 2-substituted-4-phenoxypyridine derivatives
bearing the 1,8-naphthyridinone fragment.
⇑
Corresponding authors.
E-mail addresses: zhengpw@126.com (P. Zheng), tangqidongcn@126.com
(
Q. Tang).
c
These authors contribute equally to this work.
https://doi.org/10.1016/j.bmcl.2017.12.063
960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
0
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Y. Duan et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Fig. 1. The representative small-molecule c-Met kinase inhibitors and the summarized General Structure.
Fig. 2. Antiproliferative agents bearing 2-oxo-4-chloro-1,2-dihydroquinoline (7) and pyridine (8) in our previous work; potent drug bearing 1,8-naphthyridinone
fragment (9).
Fig. 3. Design strategy for the 2-substituted-4-phenoxypyridine derivatives bearing 1,8-naphthyridinone fragment.
The antiproliferative effect of the target compounds 19–45 were
phenoxypyridine derivatives, docking analysis was performed
using compound 39.
evaluated on the growth of four cell lines in vitro,1
human lung adenocarcinoma (A549), human colon cancer (HT-
9), human lung cancer (H460), and human glioblastoma
U87MG). A549, HT-29, MKN-45, and U87MG are all high express-
8,19
namely
The key intermediates 4-(4-amino-2-substitutedphenoxy)-
N-substituted picolinamide 12a–c were prepared as illustrated in
Scheme 1. Catalyzed by NaBr, chlorination of the commercially
available 2-picolinic acid with thionyl chloride resulted in the
intermediate 10. Acylation of acyl chloride 10 with amines (methy-
2
(
18,20
ing cell lines of c-Met kinase.
There is no overexpression of c-
Met in H460 cell line, we chose it to investigate whether these
compounds showed potent antiproliferative against the cell line.
Moderate to excellent growth inhibition was observed for most
of the compounds, and 11 of these compounds were more potent
than foretinib against one or more cell lines. Furthermore, seven
compounds were chosen for further evaluation of c-Met kinase
inhibitory activity in vitro. To examine the selectivity, compound
3
lamine, ethylamine, and propanamine) in the presence of Et N
proceeded smoothly to yield 11a–c. Etherification of 4-aminophe-
nol with 11a–c catalyzed by potassium t-butoxide to give the key
intermediates 12a–c.
The target compounds 19–45 were prepared as illustrated in
Scheme 2. Condensation of substituted aniline with 2-chloronico-
tinic acid in AcOH at 100 °C resulted in high yield of intermediates
3
9 was chosen to screen against 8 other tyrosine kinases. To
further elucidate the binding mode of these 2-substituted-4-
4
13a–i as white solids. 13a–i were reduced by LiAlH in THF to
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Y. Duan et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
3
2 3
Scheme 1. Reagents and conditions: (i) SOCl , NaBr, PhCl, 55 °C, 1 h, reflux, 18 h; (ii) amines, Et N, THF, 0 °C, 3 h; (iii) 4-aminophenol, potassium t-butoxide, KI, 80 °C, 4.5 h.
Scheme 2. Reagents and conditions: (i) 2-chloronicotinic acid, AcOH, 100 °C, 3–6 h; (ii) LiAlH
4
, N
2
, THF, r.t. 3.5 h; (iii) Pyridinium dichromate, CH
2
Cl
2
, r.t. 5–7 h; (iv) Dimethyl
Cl , 0 °C, 1
malonate, piperidine, EtOH, reflux, 30–35 h; (v) K
2
CO
3
, 1,4-dioxane/H
2
O, 80 °C, 4–5 h (vi) SOCl , reflux, 6 h; (vii) Appropriate aniline, carbonyl chloride, DIPEA, CH
2
2
2
h, r.t., 5–8 h.
afford intermediates 14a–i as white solids, which were oxidized by
pyridinium dichromate to get intermediates 15a–i. Acylation of the
into the phenyl ring (moiety D). Compound 19, with no substituent
on the phenyl ring, displayed strong antiproliferative activity with
1
5a–i with dimethyl malonate in the presence of piperidine in
an IC50 of 0.20
lM against A549 cells. The introduction of stronger
refluxing EtOH yielded ethyl 2-oxo-1-substitutedphenyl-1,2-
dihydro-1,8-naphthyridine-3-carboxylates 16a–i. Simple proce-
dures such as hydrolysis and acyl chlorination were used to
convert ethyl 16a–i to the corresponding acyl chloride 18a–i,
mono-electron-withdrawing groups (mono-EWGs) at 4-position of
the phenyl ring (21, R = 4-F, IC50 = 0.11 lM) led to an improve-
ment on antiproliferative activity, which could be further con-
2
firmed by compounds 30 and 39. However, the introduction of
2
proceeded with K
2
CO
3
and thionyl chloride, respectively. Reaction
mono-EWGs (20, R = 2-F, IC50 = 0.61
l
M) or mono-electron-
2
of anilines 12a–c with acyl chloride 18a–i promoted by DIPEA in
donating groups (mono-EDGs, 24, R = 4-OCH
3
, IC50 = 3.11 M) at
l
dichloromethane at room temperature yielded the target
compounds 19–45.
other position reduced the activities. Moreover, double electron-
withdrawing groups (double-EWGs) could decrease the potency
of the compounds. For example, the inhibitory efficacy of 34
2
1,22
The antiproliferative activity of these novel 2-substituted-4-
phenoxypyridine derivatives bearing 1,8-naphthyridinone moiety
have been evaluated against the H460 and HT-29 cell lines using
2
2
(R = 2-F-4-Br, IC50 = 1.82
l
M) and 35 (R = 2-Cl-4-CF
3
, IC50 = 3.29
2
lM) are 11.4 times and 20.1 times lower than 28 (R = H,
2
3
the MTT assay. Some potent compounds were further evaluated
against the A549 and U87MG cell lines. The results expressed as
IC50 values are shown in Table 1 as the mean values of triplicate
experiments.
IC50 = 0.16 lM), respectively.
The c-Met enzymatic assays of seven 2-substituted-4-phe-
noxypyridine derivatives were evaluated using homogeneous
2
5
time-resolved fluorescence (HTRF) assay. The results suggested
that the inhibition of c-Met may be one mechanism of the antipro-
liferative effect of these derivatives (Table 2). Compound 39
showed the most potent activity with an IC50 value of 2.61 nM,
which was comparable to that of the positive control foretinib
(IC50 = 1.93 nM), and this compound should be studied further.
As shown in Table 3, compound 39 was chosen for further eval-
uation of the selectivity on c-Met over other tyrosine kinases. Com-
pared with its high potency against c-Met (IC50 = 2.61 nM), 39 also
exhibited high inhibitory effects against Flt-3 (IC50 = 2.18 nM) and
VEGFR-3 (IC50 = 23.6 nM). While, 39 showed weak potency on ALK,
As illustrated in Table 1, all target compounds 19–45 were
found to be active against different cancer cells with potencies in
the single-digit
lM range. 11 of these compounds were more
potent than foretinib against one or more cell lines. The IC50 values
of the most promising compound 3924 were 0.062, 0.084, 0.12, and
0
.96 lM against the A549, HT29, H460, and U87MG cell lines,
respectively. The data indicated that it’s a good design strategy
to use 2-substitutedpyridine as moiety A and introduce 1,8-naph-
thyridinone fragment to moiety C to form the 5-atom linker.
According to the data shown in Table 1, the cell lines data
revealed a preference for activity when the R1 group was n-Pr
instead of Me or Et, indicating that introduction of proper flexible
terminal chain on moiety A had a positive effect. For example, the
VEGFR-2, PDGFR-b, c-Kit, PDGFR-a, and EGFR. These data sug-
gested that compound 39 is a promising multitarget kinase
inhibitor.
IC50 value of compound 37, 0.095
l
M, was lower than that of 19
M, respectively.
To further elucidate the binding mode of the target compounds,
three-dimensional structure of the c-Met (PDB code: 3LQ8) and Flt-
3 (PDB code: 4XUF) was obtained from RCSB Protein Data Bank.
Docking simulation was conducted using SURFLEX-DOCK module
and 28 against A549 cells, 0.20 M and 0.16
l
l
Further analysis clearly revealed that different antiproliferative
activities were observed when various R groups were introduced
2
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Y. Duan et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Table 1
Antiproliferative activities of the target compounds 19–45 against the A549, HT-29, H460, and U87MG cancer cell lines in vitro.
Compd.
R1
R2
IC50
(
lM)
A549
HT-29
H460
U87MG
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
Et
Et
Et
Et
Et
Et
Et
Et
n-Pr
n-Pr
n-Pr
n-Pr
n-Pr
n-Pr
n-Pr
n-Pr
n-Pr
H
2-F
4-F
4-Cl
4-Br
4-OCH
2-F-4-Br
2-Cl-4-CF
3-Cl-4-F
H
0.20 ± 0.053 a
0.61 ± 0.050
0.11 ± 0.026
0.37 ± 0.016
1.05 ± 0.10
3.11 ± 0.12
1.42 ± 0.15
2.09 ± 0.21
1.56 ± 0.17
0.16 ± 0.040
0.49 ± 0.061
0.12 ± 0.047
0.25 ± 0.039
0.91 ± 0.032
2.01 ± 0.29
1.82 ± 0.18
3.29 ± 0.41
1.92 ± 0.22
0.095 ± 0.0035
0.15 ± 0.065
0.062 ± 0.0051
0.12 ± 0.052
0.15 ± 0.041
0.82 ± 0.050
1.31 ± 0.26
1.25 ± 0.28
0.59 ± 0.025
0.26 ± 0.029
0.25 ± 0.036
0.26 ± 0.016
0.16 ± 0.018
0.39 ± 0.012
1.18 ± 0.28
0.92 ± 0.013
1.95 ± 0.22
3.19 ± 0.45
1.81 ± 0.24
0.22 ± 0.027
0.26 ± 0.032
0.19 ± 0.042
0.33 ± 0.024
1.16 ± 0.40
3.35 ± 0.16
2.07 ± 0.23
4.77 ± 0.56
2.60 ± 0.21
0.098 ± 0.0061
0.20 ± 0.082
0.084 ± 0.0069
0.39 ± 0.038
0.53 ± 0.084
1.56 ± 0.64
2.16 ± 0.37
1.96 ± 0.14
0.92 ± 0.070
0.29 ± 0.018
0.32 ± 0.049
0.59 ± 0.012
0.21 ± 0.032
ND b
3.71 ± 0.30
ND
ND
2.11 ± 0.19
ND
1.12 ± 0.21
1.23 ± 0.15
0.22 ± 0.025
ND
1.82 ± 0.27
3.36 ± 0.30
2.91 ± 0.52
ND
15.03 ± 1.31
ND
ND
19.82 ± 1.63
ND
2.65 ± 0.30
3.25 ± 0.63
1.13 ± 0.11
ND
3
3
3
3
2-F
4-F
4-Cl
4-Br
ND
ND
4-OCH
3
1.72 ± 0.33
1.79 ± 0.19
ND
7.21 ± 0.39
10.26 ± 1.01
ND
2-F-4-Br
2-Cl-4-CF
3-Cl-4-F
H
2-F
4-F
4-Cl
4-Br
4-OCH
ND
ND
0.11 ± 0.039
0.39 ± 0.043
0.12 ± 0.021
ND
0.62 ± 0.036
1.73 ± 0.58
ND
3.17 ± 0.64
1.60 ± 0.51
0.36 ± 0.021
1.51 ± 0.12
2.32 ± 0.19
0.96 ± 0.036
ND
5.19 ± 0.54
8.21 ± 0.61
ND
10.92 ± 1.06
3.59 ± 0.34
1.35 ± 0.10
3
2-F-4-Br
2-Cl-4-CF
3-Cl-4-F
c
Foretinib
a
b
c
Bold values show the IC50 values of the target compounds lower than the values of the positive control.
ND: Not determined.
Used as the positive control.
of SYBYL 8.1 package version. The shape and properties of the
receptor are represented on a grid by several different sets of fields
that provide more accurate scoring of the ligand poses. The binding
model was exemplified by the interaction of compound 39 with
c-Met (Fig. 4A) and Flt-3 (Fig. 4B). As shown in Fig. 4A, the nitrogen
atom of pyridine ring and the adjacent oxygen atom of amide bond
in compound 39 formed two hydrogen-bonding interactions with
MET1160. In the 5-atom linker, hydrogen/oxygen atom of amide
bond and oxygen atom of 1,8-naphthyridin-2-one formed three
hydrogen bonds with ASP1222 and LYS1110. As we can see, five
H-bonds were formed. In the c-Met enzymatic assays, compound
39 showed the most potent activity with an IC50 value of
Table 2
c-Met kinase activity of selected compounds 19, 21, 28,
3
0, 37, 38, 39 and foretinib in vitro.
2
.61 nM, which is consistent with the docked model depicted in
Compd. IC50 on c-Met (nM)
Fig. 4A. In Fig. 4B, oxygen atom of amide bond connected with
pyridine ring of compound 39 formed hydrogen-bonding interac-
tions with CYS694. In the 5-atom linker, hydrogen and oxygen
atom of amide bond formed two hydrogen bonds with GLU661
and ASP829, respectively. Three hydrogen-bonding interactions
were formed, which is consistent with the IC50 value of 2.18 nM
against Flt-3. Therefore, Compound 39 was bound well with
c-Met and Flt-3.
19
21
28
30
37
38
39
7.06
16.29
12.53
8.24
9.89
8.32
2.61
1.93
Foretinib
In summary,
a total of 27 novel 2-substituted-4-phe-
noxypyridine derivatives bearing 1,8-naphthyridinone fragment
were designed and synthesized. 4 human cancer cell lines were
used to evaluate the antiproliferative potency of the synthesized
compounds. 11 of these compounds were more potent than fore-
tinib against one or more cell lines. Compound 39 (Flt-3/c-Met
Table 3
Inhibition of tyrosine kinases by compound 39.
Kinase
Enzyme IC50 (nM)
IC50 = 2.18/2.61 nM,
a multi-target tyrosine kinase inhibitor)
Flt-3
2.18
showed the strongest antiproliferative activities against A549,
HT29, H460, and U87MG cell lines (IC50 values: 0.062, 0.084,
VEGFR-3
ALK
VEGFR-2
PDGFR-b
c-Kit
23.6
186.2
291.3
482.1
529.3
691.5
>100,000
0.12, and 0.96 lM, respectively). Analysis of SARs indicated that
1
n-Pr served as R group showed a higher preference, and stronger
2
mono-EWGs at 4-position (such as R = 4-F) of the phenyl ring
PDGFR-
a
(
moiety D) was benefit to improve the inhibitory activity of the
EGFR
target compounds.
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Y. Duan et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
5
Fig. 4. Binding poses of compound 39 with c-Met (A) and Flt-3 (B). The proteins were displayed by grid ribbon. Compound 39 was displayed by multicolor sticks. H-bonding
interactions between compound 39 and c-Met/Flt-3 were indicated with dashed yellow lines.
3
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23. Cytotoxicity assay: A standard MTT assay was used to measure cell growth.
Cancer cell lines were cultured in minimum essential medium (MEM)
3
supplemented with 10% fetal bovine serum (FBS). Approximate 4 Â 10 cells,
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6
Y. Duan et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
suspended in MEM medium, were plated onto each well of a 96-well plate and
incubated in 5% CO at 37 °C for 24 h. The compounds tested at the indicated
final concentrations were added to the culture medium and the cell cultures
were continued for 72 h. Fresh MTT was added to each well at a terminal
pressure to give 4-(4-aminophenoxy)-N-propylpicolinamide as
viscous oil. 2-oxo-1-(4-fluorophenyl)-1,2-dihydro-1,8-naphthyridine-3-
carbonyl chloride were synthesized according our previously reported
procedures in Ref. 20. The crude product was purified by chromatography on
a yellow
2
concentration of 5
l
g/mL, and incubated with cells at 37 °C for 4 h. The
silica gel using MeOH/CH
2
Cl
2
to afford the target compounds 39 as white
) d 11.80 (s, 1H), 9.17 (s, 1H),
formazan crystals were dissolved in 100 mL DMSO of each well, and the
absorbency at 492 nm (for absorbance of MTT formazan) and 630 nm (for the
reference wavelength) was measured with an ELISA reader. All of the
compounds were tested in triplicate in each cell line. The results expressed
as IC50 were the average of three determinations and calculated by using the
Bacus Laboratories Incorporated Slide Scanner software.
solids. Yield: 59%. 1H NMR (400 MHz, DMSO-d
6
8.79 (s, 1H), 8.60 (d, J = 6.0 Hz, 2H), 8.52 (d, J = 5.6 Hz, 1H), 7.89 (d, J = 8.7 Hz,
2H), 7.50–7.43 (m, 4H), 7.41 (d, J = 3.8 Hz, 2H), 7.25 (d, J = 8.6 Hz, 2H), 7.17 (d, J
= 2.9 Hz, 1H), 3.21 (dd, J = 13.3, 6.6 Hz, 2H), 1.51 (dd, J = 14.3, 7.2 Hz, 2H), 0.84
(t, J = 7.3 Hz, 3H); 13C NMR (100 MHz, DMSO-d
) d 166.32, 163.75, 163.60,
6
163.31, 161.16, 161.10, 153.17, 151.48, 150.99, 149.90, 144.37, 139.98, 136.39,
133.66, 131.80, 131.71, 123.33, 122.44 (2C), 122.15 (2C), 120.42, 116.78,
2
4. The procedure for preparation of compound 39: To the mixture of picolinic acid
(
5.00 g, 0.04 mol), sodium bromide (0.42 g, 0.004 mol), and chlorobenzene (40
116.55, 115.34, 114.81, 109.62, 41.21, 22.95, 11.90; MS m/z (ESI): 538.2 [M
+
mL), dilute thionyl chloride was added slowly with stirring maintaining the
temperature at 55 °C. The temperature was raise to 85 °C to stir for 18 h.
Reaction was complete by TLC analysis. Filtration was used to remove solids.
The filtrate was concentrated under reduced pressure to afford 4-
chloropicolinoyl chloride as a yellow viscous oil, which was used for next
step without further purification. 4-chloropicolinic acid chloride (3.00 g, 0.017
mol) and n-propylamine were added sequentially to tetrahydrofuran (30 mL).
The reaction was stirred at 0 °C for 3 h. The reaction was completed by TLC
analysis. The reaction mixture was added to a suitable amount of water and
extracted 2–3 times with ethyl acetate. The organic phase was concentrated
under reduced pressure to give 4-chloro-N-propylpicolinamide as a yellow
viscous oil. 4-Aminophenol (2.50 g, 0.023 mol) and potassium tert-butoxide
+H] ; Anal. calcd. for C30
H
5
24FN O
4
(%): C, 67.04; H, 4.50; N, 13.03. Found (%): C,
67.01; H, 4.47; N, 12.98.
25. In vitro kinase assay: The kinase assays were performed by homogeneous time-
resolved fluorescence (HTRF) assay as previously reported protocol. Briefly, 20
l
g/mL poly (Glu, Tyr) 4:1 (Sigma) was preloaded as a substrate in 384-well
plates. Then 50 L of 10 mM ATP (Invitrogen) solution diluted in kinase
reaction buffer (50 mM HEPES, pH 7.0, 1 M DTT, 1 M MgCl , 1 M MnCl , and
0.1% NaN ) was added to each well. Various concentrations of compounds
diluted in 10 L of 1% DMSO (v/v) were used as the negative control. The kinase
reaction was initiated by the addition of purified tyrosine kinase proteins
diluted in 39 L of kinase reaction buffer solution. The incubation time for the
reactions was 30 min at 25 °C, and the reactions were stopped by the addition
of 5 L of Streptavidin-XL665 and 5 L Tk Antibody Cryptate working solution
l
2
2
3
l
l
(
3.39 g, 0.03 mol) were added to dimethyl sulfoxide (DMSO), and stirred at
l
l
room temperature under nitrogen atmosphere for 1 h. 4-Chloro-N-
a
to all of wells. The plates were read using Envision (PerkinElmer) at 320 nm
and 615 nm. The inhibition rate (%) was calculated using the following
equation: % inhibition = 100 À [(Activity of enzyme with tested compounds
À Min)/(Max À Min)] Â 100 (Max: the observed enzyme activity measured in
the presence of enzyme, substrates, and cofactors; Min: the observed enzyme
activity in the presence of substrates, cofactors and in the absence of enzyme).
IC50 values were calculated from the inhibition curves.
propylpicolinamide (3.00 g, 0.015 mol) and potassium iodide (0.33 g, 0.002
mol) was dissolved in DMSO (30 mL) and stirred at room temperature for 30
min, which was added drop wise to the former mixture and stirred at 80 °C for
4
.5 h. The reaction was completed by TLC analysis. The reaction solution was
added to sodium chloride and extracted with ethyl acetate. The organic layer
was dried over anhydrous sodium sulfate and concentrated under reduced
Please cite this article in press as: Duan Y., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2017.12.063