Design, synthesis and biological evaluation of phenylpicolinamide sorafenib derivatives as antitumor agents

Article abstract of DOI:10.1007/s00044-017-2045-0

Two series of phenylpicolinamide sorafenib derivatives (14a–k, 15a–k) were designed and synthesized. They were evaluated for IC₅₀ values against three cancer cell lines (A549, Hela, and MCF-7) and VEGFR2/KDR, BRAF, and CRAF kinases. Fourteen target compounds showed moderate to excellent cytotoxicity activity against the different cancer cells with potency from the single-digit μM to nanomole range. What’s more, six of them were equal to more potent than sorafenib against one or more cell lines. Most of the compounds showed bad activity against VEGFR2/KDR, BRAF, or CRAF kinases. The most promising compound 15f showed strong antitumor activities against A549 and MCF-7 cell lines with IC₅₀ values of 5.43 ± 0.74 and 0.62 ± 0.21 μM, which were 1.29–6.79-fold more active than sorafenib (6.53 ± 0.82, 4.21 ± 0.62 μM), respectively and it exhibited moderate IC₅₀ (7.1 μM) than 14f (IC50 = 3.1 μM). Structure–activity relationships (SARs) and docking studies indicated that replacement of diarylurea of sorafenib with phenylpicolinamide moiety benefits to the activity. The position of aryl group and the substitutions of aryl group have a great influence on antitumor activity and selectivity. Small volume groups of aryl group such as (substituted) alkyl groups (–CH₃, –CF₃), halogen atoms (–F) were favorable to the cytotoxicity. Exact action mechanism of target compounds is not quite clear and further study will be carried out to identify the target in near future.

Full text of DOI:10.1007/s00044-017-2045-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2045-0
ORIGINAL RESEARCH
Design, synthesis and biological evaluation of phenylpicolinamide
sorafenib derivatives as antitumor agents
Chunjiang Wu1 Shan Xu1 Yuping Guo1 Jielian Wu1 Rong Luo2 Wenhui Wang1
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Yuanbiao Tu1 Le Chen1 Wufu Zhu1 Pengwu Zheng1
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Received: 22 October 2015 / Accepted: 18 August 2017
© Springer Science+Business Media, LLC 2017
Abstract Two series of phenylpicolinamide sorafenib
derivatives (14a–k, 15a–k) were designed and synthesized.
They were evaluated for IC50 values against three cancer
cell lines (A549, Hela, and MCF-7) and VEGFR2/KDR,
BRAF, and CRAF kinases. Fourteen target compounds
showed moderate to excellent cytotoxicity activity against
the different cancer cells with potency from the single-digit
μM to nanomole range. What’s more, six of them were
equal to more potent than sorafenib against one or more cell
lines. Most of the compounds showed bad activity against
VEGFR2/KDR, BRAF, or CRAF kinases. The most pro-
mising compound 15f showed strong antitumor activities
against A549 and MCF-7 cell lines with IC50 values of 5.43
0.74 and 0.62 0.21 μM, which were 1.29–6.79-fold
more active than sorafenib (6.53 0.82, 4.21 0.62 μM),
respectively and it exhibited moderate IC50 (7.1 μM) than
14f (IC50 = 3.1 μM). Structure–activity relationships
(SARs) and docking studies indicated that replacement of
diarylurea of sorafenib with phenylpicolinamide moiety
benefits to the activity. The position of aryl group and the
substitutions of aryl group have a great influence on
antitumor activity and selectivity. Small volume groups of
aryl group such as (substituted) alkyl groups (–CH3, –CF3),
halogen atoms (–F) were favorable to the cytotoxicity.
Exact action mechanism of target compounds is not quite
clear and further study will be carried out to identify the
target in near future.
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Keywords Synthesis Anticancer activity Breast cancer
MCF-7 Inhibitors
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Introduction
Cancer, which endangers human life, is still one of the most
common and serious diseases in the world. The develop-
ment of molecular oncology and pharmacology accelerates
the development of new anticancer drugs. However, it
remains desirable to find new antitumor agents with
improved tumor selectivity, efficiency, and safety.
In the past 15 years, many small molecules antitumor
agents targeting in different kinases were developed and
approved. vascular endothelial growth factor receptor
kinase inhibitors such as sorafenib (Strumberg 2005),
sunitinib (Motzer et al. 2006), and pazopanib (Harris et al.
2008) are one important group of tyrosine kinase inhibitors.
As the first diaryl urea multikinase inhibitor, sorafenib was
approved by FDA for the treatment of patients with
advanced renal cell carcinoma (Strumberg 2005; Wilhelm
et al. 2006) and unresectable hepatocellular carcinoma
(Keating and Santoro 2009) in December 2005 and
November 2007, respectively. It can inhibit cancer cell
growth by interrupting the RAS/RAF/MEK/ERK and
VEGFR2 signaling pathway (Wilhelm et al. 2004, 2008;
Wan et al. 2004; Lyons et al. 2001).
Chunjiang Wu and Shan Xu contribute equally to this work.
* Wufu Zhu
zhuwf@jxstnu.edu.cn
zhuwufu-1122@163.com
* Pengwu Zheng
zhengpw@126.com
1
Jiangxi Provincial Key Laboratory of Drug Design and
Evaluation, School of Pharmacy, Jiangxi Science & Technology
Normal University, Nanchang 330013, China
2
Jiangxi Province Institute of Materia Medica, Nanchang 330013,
China

Med Chem Res
In recent years, a number of new sorafenib analogs have
been reported (Fig. 1). Among those alalogs, BMS-794833
is a potent ATP competitive inhibitor of Met/VEGFR2 with
IC50 of 1.7/15 nM. The main modification of BMS-794833
focused on diarylurea and nucleus of sorafenib. The
structure–activity relationships (SARs) of BMS-794833 and
its derivatives showed that as part of 4-oxo-5-phenyl-1, 4-
dihydropyridine-3-carboxamide played an important role in
the antitumor activity. Inspired by BMS-794833, we pro-
posed phenylpicolinamide fragments instead of diarylurea
to afford several series of compounds, which showed
excellent potency as c-met inhibitor in our previous study
(Fig. 1) (Tang et al. 2013). In the same way, we replaced
diarylurea of sorafenib with phenylpicolinamide obtaining
compounds 14a–k and 15a–k in this study. Further inves-
tigations were also carried out in detail to study the effect of
different aryl ring substitutions on antitumor activity. The
design strategy for target compounds is shown in Fig. 2.
Herein, we disclosed the synthesis and activity against
A549, Hela, and MCF-7 cancer cell lines, VEGFR2/KDR,
BRAF, and CRAF kinases of phenylpicolinamide sorafenib
derivatives. Moreover, docking studies were presented in
this paper as well.
The key intermediate 4-(4-aminophenoxy)-N-methylpi-
colinamide 4 was achieved from picolinic acid 1 as shown
in Scheme 1, which has been illustrated in detail in our
previous study (Zhu et al. 2014).
4-Bromo-pyridine-carboxylic acid
5
or 5-bromo-
pyridine-carboxylic acid 6 was reacted with substituted
phenylboronic acid 7a–k through Suzuki-coupling reaction
with bis (triphenylphosphine) palladium(II) dichloride as
catalyst to get 8a–k and 9a–k, which were then chlorinated
with oxalyl chloride to obtain 10a–k and 11a–k, respec-
tively. Finally, reaction of amides 4 with acyl chlorides
10a–k or 11a–k promoted by DIPEA in dichloromethane at
room temperature yielded the target compounds 14a–k and
15a–k.
Biological evaluation
Taking sorafenib as reference compound, the target com-
pounds (14a–k and 15a–k) were evaluated by the cyto-
toxicity against three cancer cell lines A549 (human lung
cancer), Hela (human cervical cancer), and MCF-7(human
breast cancer) by 3-(4, 5-dimethylthiazolyl-2)-2, 5-
diphenyltetrazolium bromide (MTT) cell proliferation
assay. In addition, these compounds were evaluated for IC50
values against VEGFR2/KDR, B-Raf, and C-Raf kinases
in vitro by the Mobility shift assay or Lanthascreen assay
together with reference compounds sorafenib and Staur-
osporine. The results expressed as inhibition rates or IC50
values were summarized in Tables 1, 2 and the values are
the average of at least two independent experiments.
Results and discussion
Chemistry
The preparation of target compounds 14a–k and 15a–k
were described in Scheme 1.
Fig. 1 Structures of small-molecule antitumor agents
Fig. 2 Design strategies for 4-phenylpicolinamide sorafenib derivatives

Med Chem Res
Scheme 1 Synthetic route of
target compounds. Reagents and
conditions: aSOCl2,NaBr,
chlorobenzene, 85 °C, 20 h; b
30% MeNH2, toluene, 20, 6 h; c
t-BuOK, NaI, DMF, 6 h; d
[(C6H5)3P]2PdCl2, Na2CO3,
H2O, 1,4-dioxane, 5–8 h; e
(COCl)2, DMF, CH2Cl2, 0.5 h; f
DIPEA, CH2Cl2, 0.5 h
As illustrated in Tables 1 and 2, fourteen target com-
pounds showed moderate to excellent cytotoxicity activity
against different cancer cells with potency from the single-
digit μM to nanomole range. In general, the first series of
the target compounds (14a–k) were less active than the
second series (15a–k) against the three tested cancer cell
lines. The cytotoxicity activity against MCF-7cancer cell
line was well while it’s bad against Hela cell line. What’s
more, the two series of synthesized compounds exhibited
well selectivity against MCF-7 to the other two cell lines,
with the selectivity index (SI1 and SI2) from 0.9 to 20.5,
which was higher than reference compound sorafenib (SI
from 1.6 to 1.9). It’s indicated that the position of sub-
stituted benzene ring has a strong influence on antitumor
activity and selectivity.
and 15f–i exhibit excellent activity with the IC50 values in
single-digit μM to nanomole range. Especially, compound
15f showed superior activity against A549 and MCF-7
cancer cell lines to sorafenib, with the IC50 values of 5.43
and 0.62 μM against A549 and MCF-7 cell lines, which are
1.29 and 6.79 times more active than sorafenib (IC50 values:
6.53 and 4.21 μM), respectively. The results suggested that
replacement of sorafenib diarylurea with phenylpicolina-
mide moiety maintained even improved the potent cytotoxic
activity. Furthermore, substitutions of aryl group affected
the cytotoxicity of target compounds. But, no obvious
regularity can be found about these two series of com-
pounds. It seems that small volume aryl groups such as
substitutions alkyl groups (–CH3, –CF3), halogen atoms
(–F) may be favored by the cytotoxicity. For the first series,
small volume group in 2-position is very important and
that’s why compounds 14c (2, 4-di F), 14k (2,4-di CH3) are
more active than 14a (H), 14b (4-F) and 14f (4-CH3). For
Among fourteen active compounds, six of them (com-
pounds 14k, 15c, and 15f–i) were equal to more potent than
sorafenib against one or more cell lines. Compounds 14k,

Med Chem Res
Table 1 Structures and cytotoxicity activity of compounds 12–13, 14a–k, 15a–k
Comp. no.
R
IC50(μM)a
Selective index
A549
Hela
MCF-7
SI1c
SI2d
12
–
ND
ND
ND
–
–
13
–
ND
ND
ND
–
–
14a
14b
14c
14d
14e
14f
H
NA
NA
NA
–
–
4-F
NA
17.14 1.23e
NA
NA
–
–
2,4-di F
4-Cl
NA
11.64 1.07
NA
1.5
–
–
NA
NA
–
4-OCH3
4-CH3
3-CH3
3-F
NA
NA
NA
–
–
45.66 1.66
NA
NA
50.95 1.71
26.28 1.42
NA
0.9
–
–
14g
14h
14i
NA
–
NA
NA
–
–
4-CF3
4-CH2CH3
2,4-di CH3
H
NA
NA
NA
–
–
14j
NA
NA
NA
–
–
14k
15a
15b
15c
15d
15e
15f
13.34 1.13
29.72 1.47
27.70 1.44
9.72 0.99
NA
50.01 1.70
49.61 1.69
NA
4.49 0.65
10.37 1.02
14.89 1.17
NA
3.0
2.9
1.9
–
11.1
4.8
–
4-F
2,4-di F
4-Cl
NA
–
NA
36.73 1.56
21.33 1.33
0.62 0.21
3.41 0.53
2.69 0.43
6.06 0.78
14.38 1.16
NA
–
–
4-OCH3
4-CH3
3-CH3
3-F
NA
NA
–
–
5.43 0.74
14.69 1.17
55.26 1.74
NA
NA
8.8
4.3
20.5
–
–
15g
15h
15i
19.93 1.30
NA
5.8
–
4-CF3
4-CH2CH3
2,4-di CH3
–
NA
–
15j
18.48 1.27
23.99 1.38
6.53 0.82
36.00 1.56
14.13 1.15
8.08 0.91
1.3
–
2.5
–
15k
Sorafenibb
4.21 0.62
1.6
1.9
NA not active (IC50 > 50 μM), ND not determined
a
The values are an average of two separate determinations
b
Used as a positive controls
c
SI1 = IC50(A549)/ IC50(MCF-7)
d
SI2 = IC50(Hela)/ IC50(MCF-7)
e
Bold values means the IC50s<50μM

Med Chem Res
Table 2 Activity against VEGFR2/KDR, BRAF, and CRAF kinases
of compounds 12–13, 14a–k, and 15a–k
Shortly, the target compounds especially the second
series (15a–k) showed high cytotoxicity and selectivity
toward MCF-7 to other cancer cell lines. It is suggested that
phenylpicolinamide moiety benefits to the activity and
selectivity of target compounds. Nevertheless, almost all of
the compounds showed bad activity against VEGFR2/KDR,
BRAF, and CRAF kinases. Therefore, exact action
mechanism is not quite clear right now. Further study will
be carried out to identify the target in near future.
Comp. no.
IC50(μM)a
VEGFR2/KDR
BRAF
CRAF
12
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.31 0.62
ND
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
ND
13
>10
14a
>10
14b
14c
>10
>10
14d
14e
>10
Molecular docking study
>10
To explain why compound 15f was most active and to
explore the binding modes of target compounds with the
active site of VEGFR2/KDR, molecular docking simulation
studies were carried out by using SURFLEX-DOCK mod-
ule of SYBYL package. Based on the in vitro results, we
selected compound 15f in this study as the ligand examples,
and the structure of VEGFR2/KDR was selected as the
docking model (PDB ID code: 4ASD (Wang et al. 2014)).
The binding modes of compound 15f and lead com-
pounds were shown in Fig. 3. As depicted in Fig. 3, com-
pound 15f and Sorafenib mostly overlap in the binding
model and amide group, phenylpicolinamide group, form
three hydrogen bonds with residues ASP1046, CYS919,
respectively. According to compound 15f’s binding mode in
the active binding site, it demonstrate that the docking mode
of the 15f is similar to the lead compound sorafenib and the
three hydrogen bonds play an important role in the increa-
sement of the inhibitory potency of phenylpicolinamide
derivatives against VEGFR2/KDR kinase. Furthermore, the
docking results also give us a new direction to design new
VEGFR2 inhibitors. Those SAR analysis results and
molecular docking study reveal the possibility of rational
design of more potent VEGFR2 inhibitors
14f
3.1
14g
>10
14h
14i
>10
>10
14j
6.6
14k
15a
>10
>10
15b
15c
>10
>10
15d
15e
>10
>10
15f
7.1
15g
>10
15h
15i
>10
>10
15j
>10
15k
Sorafenibb
Staurosporineb
>10
94.9 1.1% @10 μM
97.3 2.8%
ND
ND not determined
a
The values are an average of two separate determinations
Used as a positive controls
b
the second series, substitute in 2-position is not beneficial to
the activity (15f vs. 15k, 15b vs. 15c, with 15c against
A549 as an exception). Small volume aryl groups such as
substitutions alkyl groups (–CH3, –CF3), halogen atoms
(–F) substituted in 3-position or 4-position of aryl group
remarkably improved the activity against MCF-7 cell line
(15f–i vs. 15d, 15e, 15j).
Activity against VEGFR2/KDR, BRAF, and CRAF
kinases of target compounds were further carried out in this
paper to investigate the target of these compounds. To our
disappointment, nearly all of the compounds showed no
activity against all those three kinases. Only three (14f, 14j,
and 15f) of the compounds showed IC50 less than 10 μM.
The results suggested that this series of target compounds
may target in none of VEGFR2/KDR, BRAF, and CRAF
kinases.
Conclusion
In summary, two series of phenylpicolinamide sorafenib
derivatives were designed and synthesized. All the target
compounds were evaluated against three cancer cell lines,
VEGFR2/KDR, BRAF, and CRAF kinases. Fourteen syn-
thesized compounds showed moderate to excellent cyto-
toxicity activity against different cancer cells with potencies
from the single-digit μM to nanomole range and six of them
were equal to more potent than sorafenib against one or
more cell lines. The cytotoxicity and activity against MCF-7
cancer cell line was attractive and two series compounds
exhibited well selectivity against MCF-7 to the other two
cell lines. Compound 15f showed best activity against A549
and MCF-7 cancer cell lines with the IC50 values of 5.43
and 0.62 μM against A549 and MCF-7 cell lines, which are

Med Chem Res
Fig. 3 Binding poses of
compound 15f with VEGFR2.
The proteins were denoted by
green ribbon. Compound 15f
and lead compound were
denoted by orange and cyan
sticks, respectively. H-bonding
interactions between the 15f,
lead compound and VEGFR2
were denoted by dashed lines in
yellow (color figure online)
1.29 and 6.79 times more active than sorafenib. The aryl
group position and aryl group substitutions have a strong
influence on antitumor activity and selectivity. Small aryl
group volume groups such as substitutions alkyl groups
(–CH3, –CF3), halogen atoms (–F) were favorable to the
cytotoxicity. Three compounds show moderate activity
against VEGFR2/KDR kinases and the most promising
compound 15f exhibited moderate IC50 (7.1 μM) than 14f
(IC50 = 3.1 μM). Although all the target compounds
showed less activity against VEGFR2/KDR kinases than
the positive compounds sorafenib and staurosporine, it still
showed that replacement of urea with phenylpicolinamide
unit is important to the activity of this series of compounds.
More compounds of sorafenib analogs bearing a phenylpi-
colinamide may be screened by replacing the aryl groups
with heterocyclic rings in future study.
All the materials were obtained from commercial suppliers
and used without purification, unless otherwise specifica-
tion. Numbering for 13C NMR spectra was shown in Fig. 4.
General procedure for the key intermediate of 4-(4-
aminophenoxy)-N-methylpicolinamide 4
Compound 4 was synthesized from compound 1 based on
the procedures in our previous research Wilhelm et al.
(2008).
General procedure for the preparation of compounds
8a–k and 9a–k
To the mixture of an appropriate amount of 4-bromo-
pyridine-carboxylic acid 5 (0.9 mmol) or 5-bromo-pyridine-
carboxylic acid 6, substituted phenylboronic acid 7a–k
(0.18 mmol), anhydrous sodium carbonate (0.29 g, 2.7
mmol), 1,4-dioxane (30 mL), H2O (10 mL), and bis (tri-
phenylphosphine) palladium (II) dichloride (0.09 g, 0.135
mmol) were added under an atmosphere of nitrogen at room
temperature. The reaction mixture was then stirred at 100 °C
for 5–8 h and monitored by TLC. The reaction mixture was
cooled to room temperature and the resulting precipitate
was isolated by filtration to furnish the acid intermediate as
the sodium salt, and then the solution was acidified to pH 5
to furnish the desired target compounds 8a–k and 9a–k.
Experimental
Chemistry
All melting points were obtained on a Büchi Melting Point
B-540 apparatus (Büchi Labortechnik, Flawil, Switzerland)
and were unchanged. Nuclear magnetic resonance (NMR)
spectra were performed by using Bruker 400 MHz spec-
trometers (Bruker Bioscience, Billerica, MA, USA) with
tetramethylsilane as an internal standard. Mass spectra (MS)
were taken into ESI mode on Agilent 1100 LCMS (Agilent,
Palo Alto, CA, USA). All the materials were obtained from
commercial suppliers and used without purification, unless
otherwise specification. Yields were not optimized. Thin
layer chromatography (TLC) analysis was carried out on
silica gel plates GF254 (Qindao Haiyang Chemical, China).
Preparation of phenylpyridine chloride 10a–k and 11a–k
Oxalyl chloride (0.4 mmol) was added drop-wise into a
stirred mixture of compounds 8a–k or 9a–k (0.2 mmol) and
DMF(0.01 mmol) in dichloromethane (10 mL) under room

Med Chem Res
Fig. 4 Numbering for 13C
NMR spectra
temperature for 10 min, and the mixture was used in the
coming step immediately without further purification.
7.25 (d, J = 8.7 Hz, 2H), 7.18–7.14 (m, 1H), 2.78 (d, J =
4.7 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 166.63 (C, C-
15), 164.31 (C, C-22), 161.94 (C, C-16), 152.88 (C, C-6),
150.92 (C, C-4), 149.72 (C, C-8), 145.37 (CH, C-20),
136.34 (C, C-11), 131.01 (C, C-18), 129.23 (CH, C-19),
128.53 (CH, C-2), 122.30 (CH, C-17), 121.31 (CH, C-10,
C-12), 120.67 (CH, C-9, C-13), 115.45 (CH, C-1), 114.77
(CH, C-5), 26.32 (CH3, C-25). Analytical HPLC on an
Agilent (1100) using a 250 × 4.6 mm SD-C18 analytical
column, H2O/MeOH (15%H2O) eluent at 1 mL/min flow,
monitored by ultraviolet (UV) absorption at 325 nm,
revealed 98.2% purity.
General procedure for the preparation of compounds
12, 13, 14a–k, and 15a–k
A solution of an appropriate aniline 4 (0.41 mmol) and
diisopropylethylamine (0.49 mmol) in dichloromethane (10
mL) was added drop-wise into a solution of phenylpyridine
chloride (0.82 mmol, obtained in the previous step) in
dichloromethane (10 mL) in an ice bath. With the above
addition, the reaction mixture was removed from the ice
bath and placed under room temperature for 30 min and
monitored by TLC. The mixture was washed with 10%
K2CO3 (50 mL × 3) followed by brine (50 mL × 1), and the
organic phase was separated, dried, and evaporated to yield
12, 13, 14a–k, and 15a–k which was purified by
isopropanol.
4-Bromo-N-(4-(2-(methylcarbamoyl) pyridin-4-yloxy)
pheny l) picolinamide (13)
Yield: 65.52%; m.p. 172.7–173.9 °C; MS (ESI) m/z (%):
1
427.3 [M + H]+; H NMR (400 MHz, DMSO) δ 10.93 (s,
1H), 8.89 (d, J = 4.8 Hz, 1H), 8.74 (d, J = 5.2 Hz, 1H), 8.53
(d, J = 5.7 Hz, 1H), 8.17 (d, J = 1.5 Hz, 1H), 8.05 (d, J =
8.9 Hz, 2H), 7.85 (dd, J = 5.3, 2.0 Hz, 1H), 7.47 (d, J = 2.3
Hz, 1H), 7.26 (d, J = 8.9 Hz, 2H), 7.20 (dd, J = 5.7, 2.5 Hz,
1H), 2.79 (d, J = 4.8 Hz, 3H). 13C NMR (101 MHz,
DMSO) δ 167.63 (C, C-15), 164.73 (C, C-22), 160.94 (C,
C-16), 151.98 (C, C-6), 150.72 (C, C-4), 149.51 (C, C-8),
144.85 (CH, C-20), 136.25 (C, C-11), 131.31 (C, C-18),
128.93 (CH, C-19), 128.43 (CH, C-2), 122.82 (CH, C-17),
4-Chloro-N-(4-(2-(methylcarbamoyl) pyridin-4-yloxy)
phenyl) picolinamide (12)
Yield: 72.01%; m.p. 169.7–171.1 °C; MS (ESI) m/z (%):
1
383.2 [M + H]+; H NMR (400 MHz, dimethyl sulfoxide
(DMSO)) δ 10.92 (s, 1H), 8.80 (d, J = 4.6 Hz, 1H), 8.73 (d,
J = 5.3 Hz, 1H), 8.51 (d, J = 5.6 Hz, 1H), 8.17 (s, 1H), 8.04
(d, J = 8.7 Hz, 2H), 7.85 (d, J = 5.2 Hz, 1H), 7.39 (s, 1H),

Med Chem Res
121.51 (CH, C-10, C-12), 119.97 (CH, C-9, C-13), 115.73
(CH, C-1), 114.51 (CH, C-5), 26.43 (CH3, C-25). Analy-
tical HPLC on an Agilent (1100) using a 250 × 4.6 mm SD-
C18 analytical column, H2O/MeOH (15%H2O) eluent at 1
mL/min flow, monitored by UV absorption at 325 nm,
revealed 98.6% purity.
4-(2,4-Difluorophenyl)-N-(4-(2-(methylcarbamoy l)
pyridin-4-yloxy) phenyl) picolinamide (14c)
Yield: 45.81%; m.p. 162.8–163.6 °C; MS (ESI) m/z (%):
1
499.1 [M + k]+; H NMR (400 MHz, DMSO) δ 10.97 (s,
1H), 8.94 (d, J = 4.4 Hz, 1H), 8.87 (d, J = 5.0 Hz, 1H), 8.56
(d, J = 5.7 Hz, 1H), 8.33 (s, 1H), 8.10 (d, J = 8.9 Hz, 2H),
7.90 (d, J = 4.9 Hz, 1H), 7.88–7.82 (m, 1H), 7.54 (s, 1H),
7.31 (dd, J = 19.0, 8.1 Hz, 3H), 7.25–7.20 (m, 1H), 2.81 (d,
J = 4.6 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 166.53 (C,
C-6), 164.68 (C, C-15), 161.67 (C, C-30), 161.45 (C, C-22),
158.02 (C, C-28), 151.72 (C, C-16), 151.17 (C, C-18),
151.00 (C, C-4), 148.01 (C, C-8), 146.47 (CH, C-2), 144.60
(CH, C-20), 133.81 (C, C-11), 130.67 (CH, C-32), 126.45
(C, C-27), 122.37 (CH, C-10, C-12), 120.93 (CH, C-19),
119.25 (CH, C-9, C-13), 114.63 (CH, C-17), 113.92 (CH,
C-1), 111.10 (CH, C-31), 109.67 (CH, C-5), 102.59 (CH,
C-29), 26.74 (CH3, C-25). Analytical HPLC on an Agilent
(1100) using a 250 × 4.6 mm SD-C18 analytical column,
H2O/MeOH (15%H2O) eluent at 1 mL/min flow, monitored
by UV absorption at 325 nm, revealed 99.1% purity.
N-(4-(2-(methylcarbamoyl) pyridin-4-yloxy) pheny l)-4-
phenylpicolinamide (14a)
Yield: 45.25%; m.p. 190.7–192.9 °C; MS (ESI) m/z (%):
1
425.3 [M + H]+; H NMR (400 MHz, DMSO) δ 10.91 (s,
1H), 8.82 (t, J = 4.5 Hz, 1H), 8.79 (d, J = 4.8 Hz, 1H), 8.52
(d, J = 5.6 Hz, 1H), 8.43 (d, J = 1.3 Hz, 1H), 8.09 (d, J =
9.0 Hz, 2H), 8.02 (dd, J = 5.1, 1.8 Hz, 1H), 7.94–7.88 (m,
2H), 7.56 (ddd, J = 10.8, 9.8, 5.4 Hz, 3H), 7.42 (d, J = 2.5
Hz, 1H), 7.26 (d, J = 9.0 Hz, 2H), 7.18 (dd, J = 5.6, 2.6 Hz,
1H), 2.79 (d, J = 4.8 Hz, 3H). 13C NMR (101 MHz,
DMSO) δ 167.91 (C, C-6), 162.76 (C, C-15), 160.92 (C, C-
22), 151.73 (C, C-16), 151.14 (C, C-18), 150.76 (C, C-4),
149.51 (C, C-8), 146.53 (CH, C-2), 144.73 (CH, C-20),
143.35 (C, C-27), 31.31 (C, C-11), 129.23 (CH, C-30),
129.10 (CH, C-31), 128.83 (CH, C-29), 128.43 (CH, C-32),
126.42 (CH, C-28), 122.82 (CH, C-10, C-12), 121.01 (CH,
C-19), 119.99 (CH, C-9, C-13), 114.82 (CH, C-17), 114.56
(CH, C-1), 109.85 (CH, C-5), 26.71 (CH3, C-25). Analy-
tical HPLC on an Agilent (1100) using a 250 × 4.6 mm SD-
C18 analytical column, H2O/MeOH (15%H2O) eluent at 1
mL/min flow, monitored by UV absorption at 325 nm,
revealed 97.9% purity.
4-(4-Chlorophenyl)-N-(4-(2-(methylcarbamoy l) pyridin-4-
yloxy) pheny l) picolinamide (14d)
Yield: 51.6%; m.p. 217.1–217.9 °C; MS (ESI) m/z (%):
1
481.1 [M+Na]+; H NMR (400 MHz, DMSO) δ 10.92 (s,
1H), 8.83 (s, 1H), 8.79 (s, 1H), 8.52 (d, J = 4.9 Hz, 1H),
8.42 (s, 1H), 8.09 (d, J = 6.9 Hz, 2H), 8.03 (s, 1H), 7.95 (d,
J = 6.7 Hz, 2H), 7.63 (d, J = 6.7 Hz, 2H), 7.42 (s, 1H), 7.26
(d, J = 6.9 Hz, 2H), 7.17 (s, 1H), 2.79 (s, 3H). 13C NMR
(101 MHz, DMSO) δ 167.83 (C, C-6), 163.51 (C, C-15),
161.78 (C, C-22), 151.83 (C, C-16), 151.22 (C, C-18),
150.87 (C, C-4), 148.74 (C, C-8), 146.62 (CH, C-2), 143.45
(CH, C-20), 140.71 (C, C-27), 134.93 (C, C-30), 132.84 (C,
C-11), 129.45 (CH, C-29, C-31), 128.10 (CH, C-28, C-32),
122.65 (CH, C-10, C-12), 120.83 (CH, C-19), 119.65 (CH,
C-9, C-13), 114.36 (CH, C-17), 113.73 (CH, C-1), 109.46
(CH, C-5), 26.82 (CH3, C-25). Analytical HPLC on an
Agilent (1100) using a 250 × 4.6 mm SD-C18 analytical
column, H2O/MeOH (15%H2O) eluent at 1 mL/min flow,
monitored by UV absorption at 325 nm, revealed 98.7%
purity.
4-(4-fluorophenyl)-N-(4-(2-(methylcarbamoy l) pyridin-4-
yloxy) phenyl) picolinamide (14b)
Yield: 58.09%; m.p. 213.6–214.2 °C; MS (ESI) m/z (%):
1
481.1 [M + k]+; H NMR (400 MHz, DMSO) δ 10.93 (s,
1H), 8.81 (d, J = 4.6 Hz, 2H), 8.52 (d, J = 5.2 Hz, 1H), 8.41
(s, 1H), 8.16–8.05 (m, 2H), 8.00 (d, J = 8.5 Hz, 2H), 7.42
(d, J = 9.2 Hz, 3H), 7.26 (d, J = 8.6 Hz, 2H), 7.18 (s, 1H),
2.79 (d, J = 4.3 Hz, 3H). 13C NMR (101 MHz, DMSO) δ
167.83 (C, C-6), 165.81 (C, C-30), 163.17 (C, C-15),
160.98 (C, C-22), 151.88 (C, C-16), 151.28 (C, C-18),
150.86 (C, C-4), 148.34 (C, C-8), 146.32 (CH, C-2), 144.62
(CH, C-20), 136.89 (C, C-27), 133.57 (C, C-11), 130.23
(CH, C-28, C-32), 122.51 (CH, C-10, C-12), 120.76 (CH,
C-19), 120.12 (CH, C-9, C-13), 115.41 (CH, C-29, C-31),
114.83 (CH, C-17), 114.23 (CH, C-1), 109.56 (CH, C-5),
26.85 (CH3, C-25). Analytical HPLC on an Agilent (1100)
using a 250 × 4.6 mm SD-C18 analytical column, H2O/
MeOH (15%H2O) eluent at 1 mL/min flow, monitored by
UV absorption at 325 nm, revealed 98.2% purity.
4-(4-Methoxyphenyl)-N-(4-(2-(methylcarbamoy l) pyridin-
4-yloxy) phenyl) picolinamide (14e)
Yield: 44.69%; m.p. 168.9–171.1 °C; MS (ESI) m/z (%):
477.3 [M + Na]+; 1H NMR (400 MHz, DMSO) δ 10.89 (s,
1H), 8.79 (d, J = 4.7 Hz, 1H), 8.76 (d, J = 5.2 Hz, 1H), 8.52
(d, J = 5.5 Hz, 1H), 8.39 (s, 1H), 8.09 (d, J = 8.7 Hz, 2H),
7.97 (d, J = 5.0 Hz, 1H), 7.89 (d, J = 8.6 Hz, 2H), 7.42 (d,
J = 2.0 Hz, 1H), 7.26 (d, J = 8.7 Hz, 2H), 7.20–7.16 (m,

Med Chem Res
1H), 7.13 (d, J = 8.6 Hz, 2H), 3.85 (s, 3H), 2.80 (d, J = 4.7
Hz, 3H). 13C NMR (101 MHz, DMSO) δ 166.92 (C, C-6),
162.57 (C, C-15), 161.32 (C, C-22), 151.58 (C, C-16),
151.13 (C, C-18), 151.07 (C, C-4), 148.09 (C, C-8), 146.74
(CH, C-2), 144.63 (CH, C-20), 139.42 (C, C-27), 133.94
(C, C-11), 132.17 (C, C-30), 129.54 (CH, C-29, C-31),
127.31 (CH, C-28, C-32), 122.47 (CH, C-10, C-12), 121.11
(CH, C-19), 119.45 (CH, C-9, C-13), 114.72 (CH, C-17),
114.02 (CH, C-1), 109.58 (CH, C-5), 55.79 (OCH3, C-33),
26.83 (CH3, C-25). Analytical HPLC on an Agilent (1100)
using a 250 × 4.6 mm SD-C18 analytical column, H2O/
MeOH (15%H2O) eluent at 1 mL/min flow, monitored by
UV absorption at 325 nm, revealed 98.8% purity.
C-19), 119.61 (CH, C-9, C-13), 114.52 (CH, C-17), 113.43
(CH, C-1), 109.46 (CH, C-5), 26.88 (CH3, C-25), 21.43
(CH3,C-33). Analytical HPLC on an Agilent (1100) using a
250 × 4.6 mm SD-C18 analytical column, H2O/MeOH
(15%H2O) eluent at 1 mL/min flow, monitored by UV
absorption at 325 nm, revealed 98.9% purity.
4-(3-Fluorophenyl)-N-(4-(2-(methylcarbamoyl) pyridin-4-
yloxy)phenyl) picolinamide (14h)
Yield: 45.31%; m.p. 198.5–199.5 °C; MS (ESI) m/z (%):
1
481.1 [M + K]+; H NMR (400 MHz, DMSO) δ 10.95 (s,
1H), 8.85 (d, J = 5.0 Hz, 1H), 8.81 (d, J = 4.4 Hz, 1H), 8.53
(d, J = 5.6 Hz, 1H), 8.45 (s, 1H), 8.08 (dd, J = 12.6, 6.9 Hz,
3H), 7.85–7.75 (m, 2H), 7.63 (dd, J = 14.3, 7.8 Hz, 1H),
7.42 (d, J = 2.0 Hz, 2H), 7.27 (d, J = 8.8 Hz, 2H),
7.21–7.16 (m, 1H), 2.80 (d, J = 4.7 Hz, 3H). 13C NMR
(101 MHz, DMSO) δ 166.87 (C, C-6), 162.44 (C, C-15),
161.99 (C, C-29), 159.61 (C, C-22), 152.37 (C, C-16),
149.97 (C, C-18), 149.71 (C, C-4), 149.06 (C, C-8), 146.72
(CH, C-2), 144.99 (CH, C-20), 143.67 (C, C-27), 133.91
(C, C-11), 124.36 (CH, C-31), 123.65 (CH, C-32), 122.85
(CH, C-10, C-12), 120.34 (CH, C-19), 119.70 (CH, C-9, C-
13), 116.01 (CH, C-30), 114.25 (CH, C-28), 114.02 (CH,
C-17), 113.23 (CH, C-1), 109.39 (CH, C-5), 26.14 (CH3, C-
25). Analytical HPLC on an Agilent (1100) using a 250 ×
4.6 mm SD-C18 analytical column, H2O/MeOH (15%H2O)
eluent at 1 mL/min flow, monitored by UV absorption at
325 nm, revealed 97.9% purity.
N-(4-(2-(methylcarbamoyl) pyridin-4-yloxy) phenyl)-4-p-
tolylpicolinamide (14f)
Yield: 57.83%; m.p. 195.0–195.8 °C; MS (ESI) m/z (%):
461.3 [M + Na]+; 1H NMR (400 MHz, DMSO) δ 10.92 (s,
1H), 8.80 (d, J = 4.9 Hz, 2H), 8.53 (d, J = 5.5 Hz, 1H), 8.42
(s, 1H), 8.10 (d, J = 8.7 Hz, 2H), 8.01 (s, 1H), 7.82 (d, J =
7.8 Hz, 2H), 7.43 (s, 1H), 7.40 (d, J = 7.6 Hz, 2H), 7.27 (d,
J = 8.6 Hz, 2H), 7.20 (s, 1H), 2.80 (d, J = 4.5 Hz, 3H), 2.40
(s, 3H). 13C NMR (101 MHz, DMSO) δ 166.56 (C, C-6),
162.78 (C, C-15), 161.53 (C-22), 151.62 (C, C-16), 151.17
(C, C-18), 151.00 (C, C-4), 148.01 (C, C-8), 146.47 (CH,
C-2), 144.60 (CH, C-20), 139.31 (C, C-27), 133.67 (C, C-
11), 132.08 (C, C-30), 129.37 (CH, C-29, C-31), 127.43
(CH, C-28, C-32), 122.67 (CH, C-10, C-12), 120.91 (CH,
C-19), 119.25(CH, C-9, C-13), 114.63 (CH, C-17), 113.92
(CH, C-1), 109.67 (CH, C-5), 26.75 (CH3, C-25), 21.68
(CH3, C-33). Analytical HPLC on an Agilent (1100) using a
250 × 4.6 mm SD-C18 analytical column, H2O/MeOH
(15%H2O) eluent at 1 mL/min flow, monitored by UV
absorption at 325 nm, revealed 97.5% purity.
N-(4-(2-(methylcarbamoyl) pyridin-4-yloxy) phenyl)-4-(4-
(trifluoromethyl) phenyl) picolinamide (14i)
Yield: 46.96%; m.p. 239.9–241.2 °C; MS (ESI) m/z (%):
1
N-(4-(2-(methylcarbamoy l) pyridin-4-yloxy) phenyl)-4-m-
tolylpicolinamide (14g)
531.2 [M + K]+; H NMR (400 MHz, DMSO) δ 10.98 (s,
1H), 8.90 (s, 1H), 8.82 (s, 1H), 8.54 (d, J = 5.6 Hz, 1H),
8.50 (s, 1H), 8.16 (d, J = 8.0 Hz, 2H), 8.11 (d, J = 7.7 Hz,
2H), 7.95 (d, J = 7.8 Hz, 2H), 7.42 (s, 1H), 7.28 (d, J = 8.6
Hz, 2H), 7.20 (s, 1H), 2.80 (s, 3H). 13C NMR (101 MHz,
DMSO) δ 166.75 (C, C-6), 163.51 (C, C-15), 161.79 (C, C-
22), 151.53 (C, C-16), 151.02 (C, C-18), 149.74 (C, C-4),
148.79 (C, C-8), 146.36 (CH, C-2), 145.67 (C, C-27),
144.60 (CH, C-20), 133.43 (C, C-11), 129.37 (C, C-30),
127.61 (CH, C-28, C-32), 125.93 (CH, C-29, C-31), 124.34
(C, C-33), 121.55 (CH, C-10, C-12), 120.88 (CH, C-19),
119.19 (CH, C-9, C-13), 114.04 (CH, C-17), 113.92 (CH,
C-1), 109.67 (CH, C-5), 26.17 (CH3, C-25). Analytical
HPLC on an Agilent (1100) using a 250 × 4.6 mm SD-C18
analytical column, H2O/MeOH (15%H2O) eluent at 1 mL/
min flow, monitored by UV absorption at 325 nm, revealed
98.6% purity.
Yield: 54.97%; m.p. 181.5–182.4 °C; MS (ESI) m/z (%):
1
477.1 [M + K]+; H NMR (400 MHz, DMSO) δ 10.94 (s,
1H), 8.81 (d, J = 4.8 Hz, 2H), 8.53 (d, J = 5.6 Hz, 1H), 8.42
(s, 1H), 8.10 (d, J = 8.9 Hz, 2H), 8.04–7.98 (m, 1H), 7.74
(s, 1H), 7.70 (d, J = 7.7 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H),
7.43–7.39 (m, 1H), 7.36 (d, J = 7.4 Hz, 1H), 7.27 (d, J =
9.0 Hz, 2H), 7.18 (dt, J = 8.3, 4.1 Hz, 1H), 2.80 (d, J = 4.8
Hz, 3H), 2.44 (s, 3H). 13C NMR (101 MHz, DMSO) δ
168.96 (C, C-6), 162.97 (C, C-15), 160.16 (C, C-22),
151.72 (C, C-16), 157.32 (C, C-18), 151.11 (C, C-4),
149.00 (C, C-8), 146.32 (CH, C-2), 144.61 (CH, C-20),
143.17 (C, C-27), 138.60 (C, C-29), 131.54 (C, C-11),
129.57 (CH, C-30), 129.13 (CH, C-31), 128.87 (CH, C-28),
124.41 (CH, C-32), 122.34 (CH, C-10, C-12), 120.62 (CH,

Med Chem Res
4-(4-Ethylphenyl)-N-(4-(2-(methylcarbamoyl) pyridin-4-
yloxy) phenyl) picolinamide (14j)
= 7.3 Hz, 2H), 7.52–7.47 (m, 1H), 7.41 (s, 1H), 7.25 (d, J
= 8.8 Hz, 2H), 7.17 (d, J = 2.8 Hz, 1H), 2.79 (d, J = 4.5 Hz,
3H). 13C NMR (101 MHz, DMSO) δ 167.79 (C, C-6),
162.68 (C, C-15), 161.42 (C, C-22), 151.02 (C, C-16),
149.87 (C, C-4), 148.00 (C, C-8), 146.21 (CH, C-2), 141.97
(CH, C-20), 139.60 (C, C-19), 136.71 (C, C-27), 134.53
(CH, C-18), 133.80 (C, C-11), 129.27 (CH, C-29, C-31),
128.73 (CH, C-30), 127.65 (CH, C-28, C-32), 124.83 (CH,
C-17), 122.82 (CH, C-10, C-12), 119.10 (CH, C-9, C-13),
113.67 (CH, C-1), 109.59 (CH, C-5), 26.92 (CH3, C-25).
Analytical HPLC on an Agilent (1100) using a 250 × 4.6
mm SD-C18 analytical column, H2O/MeOH (15%H2O)
eluent at 1 mL/min flow, monitored by UV absorption at
325 nm, revealed 98.2% purity.
Yield: 43.23%; m.p. 199.1–200.5 °C; MS (ESI) m/z (%):
475.1 [M + Na]+; 1H NMR (400 MHz, DMSO) δ 10.93 (s,
1H), 8.81 (s, 2H), 8.53 (d, J = 5.5 Hz, 1H), 8.42 (s, 1H),
8.10 (d, J = 8.6 Hz, 2 H), 8.02 (s, 1H), 7.85 (d, J = 7.8 Hz,
2H), 7.43 (d, J = 7.2 Hz, 3H), 7.27 (d, J = 8.6 Hz, 2H), 7.20
(s, 1H), 2.80 (d, J = 4.5 Hz, 3H), 2.71 (dd, J = 15.1, 7.3 Hz,
2H), 1.24 (t, J = 7.6 Hz, 3H). 13C NMR (101 MHz, DMSO)
δ 166.73 (C, C-6), 163.95 (C, C-15), 161.13 (C, C-22),
151.73 (C, C-16), 150.92 (C, C-18), 150.31 (C, C-4),
148.25 (C, C-8), 146.69 (CH, C-2), 144.75 (C, C-30),
144.34 (CH, C-20), 140.11 (C, C-27), 133.59 (C, C-11),
129.43 (CH, C-29, C-31), 127.75 (CH, C-28, C-32), 122.91
(CH, C-10, C-12), 120.91 (CH, C-19), 119.25 (CH, C-9, C-
13), 114.82 (CH, C-17), 113.53 (CH, C-1), 109.79 (CH, C-
5), 28.79 (CH2CH3, C-33), 25.73 (CH3, C-25), 15.27
(CH2CH3, C-33). Analytical HPLC on an Agilent (1100)
using a 250 × 4.6 mm SD-C18 analytical column, H2O/
MeOH (15%H2O) eluent at 1 mL/min flow, monitored by
UV absorption at 325 nm, revealed 98.9% purity.
5-(4-Fluorophenyl)-N-(4-(2-(methylcarbamoyl) pyridin-4-
yloxy) phenyl) picolinamide (15b)
Yield: 46.01%; m.p. 222.5–224.4 °C; MS (ESI) m/z (%):
1
481.1 [M + K]+; H NMR (400 MHz, DMSO) δ 10.87 (s,
1H), 9.02 (s, 1H), 8.77 (s, 1H), 8.51 (d, J = 5.5 Hz, 1H),
8.34 (d, J = 8.1 Hz, 1H), 8.24 (d, J = 8.1 Hz, 1H), 8.08 (d,
J = 8.7 Hz, 2H), 7.93–7.87 (m, 2H), 7.43–7.39 (m, 2H),
7.37 (s, 1H), 7.25 (d, J = 8.8 Hz, 2H), 7.18–7.12 (m, 1H),
2.79 (d, J = 4.5 Hz, 3H). 13C NMR (101 MHz, DMSO) δ
167.12 (C, C-6), 163.74 (C, C-15), 162.61 (C, C-30),
162.14 (C, C-22), 149.72 (C, C-16), 149.17 (C, C-4),
148.30 (C, C-8), 146.32 (CH, C-2), 141.65 (CH, C-20),
139.73 (C, C-19), 134.19 (CH, C-18), 133.81 (C, C-11),
132.21 (C, C-27), 130.08 (CH, C-28, C-32), 124.17 (CH,
C-17), 122.63 (CH, C-10, C-12), 119.57 (CH, C-9, C-13),
116.25 (CH, C-29, C-31), 113.63 (CH, C-1), 109.92 (CH,
C-5), 26.68 (CH3, C-25). Analytical HPLC on an Agilent
(1100) using a 250 × 4.6 mm SD-C18 analytical column,
H2O/MeOH (15%H2O) eluent at 1 mL/min flow, monitored
by UV absorption at 325 nm, revealed 97.9% purity.
4-(2, 4-dimethylphenyl)-N-(4-(2-(methylcarbamoyl)
pyridin-4-yloxy) phenyl) picolinamide (14k)
Yield: 43.48%; m.p. 108.7–110.2 °C; MS (ESI) m/z (%):
1
491.2 [M + K]+; H NMR (400 MHz, DMSO) δ 10.89 (s,
1H), 8.78 (s, 2H), 8.51 (d, J = 5.4 Hz, 1H), 8.07 (d, J = 7.7
Hz, 3H), 7.68 (s, 1H), 7.41 (s, 1H), 7.24 (d, J = 7.5 Hz, 3H),
7.18 (d, J = 11.5 Hz, 3H), 2.79 (s, 3H), 2.34 (s, 3H), 2.26 (s,
3H). 13C NMR (101 MHz, DMSO) δ 167.18 (C, C-6),
164.68 (C, C-15), 161.33 (C, C-22), 151.92 (C, C-16),
151.62 (C, C-18), 151.23 (C, C-4), 148.15 (C, C-8), 146.41
(CH, C-2), 144.55 (CH, C-20), 138.80 (C, C-30), 133.81
(C, C-11), 132.97 (CH, C-32), 132.45 (C, C-27), 131.37
(CH, C-31), 130.53 (C, C-28), 125.95 (CH, C-29), 122.63
(CH, C-10, C-11), 120.54 (CH, C-19), 119.92 (CH, C-9, C-
13), 114.10 (CH, C-17), 113.67 (CH, C-1), 109.59 (CH, C-
5), 26.79 (CH3, C-25), 21.79 (4-CH3, C-33), 19.25 (2-CH3,
C-33). Analytical HPLC on an Agilent (1100) using a
250 × 4.6 mm SD-C18 analytical column, H2O/MeOH
(15%H2O) eluent at 1 mL/min flow, monitored by UV
absorption at 325 nm, revealed 98.0% purity.
5-(2, 4-Difluoropheny l)-N-(4-(2-(methylcarbamoyl)
pyridin-4-yloxy) phenyl) picolinamide (15c)
Yield: 57.16%; m.p. 230.9–232.2 °C; MS (ESI) m/z (%):
1
499.1 [M + K]+; H NMR (400 MHz, DMSO) δ 10.91 (s,
1H), 8.91 (s, 1H), 8.77 (d, J = 3.8 Hz, 1H), 8.52 (d, J = 5.5
Hz, 1H), 8.30–8.23 (m, 2H), 8.08 (d, J = 8.6 Hz, 2H), 7.79
(dd, J = 15.5, 8.4 Hz, 1H), 7.49 (t, J = 10.0 Hz, 1H), 7.41
(s, 1H), 7.31 (t, J = 8.8 Hz, 1H), 7.25 (d, J = 8.6 Hz, 2H),
7.17 (d, J = 2.9 Hz, 1H), 2.79 (d, J = 4.4 Hz, 3H). 13C
NMR (101 MHz, DMSO) δ 166.89 (C, C-6), 164.34 (C, C-
15), 161.78 (C, C-30), 161.22 (C, C-22), 158.47 (C, C-28),
151.43 (C, C-4), 149.01 (C, C-16), 148.47 (C, C-8), 146.95
(CH, C-2), 141.31 (CH, C-20), 139.37 (C, C-19), 134.06
(CH, C-18), 133.95 (C, C-11), 130.75 (CH, C-32), 126.83
(C, C-27), 124.07 (CH, C-17), 122.38 (CH, C-10, C-12),
N-(4-(2-(methylcarbamoyl) pyridin-4-yloxy) phenyl)-5-
phenylpicolinamide (15a)
Yield: 42.34%; m.p. 176.4–177.6 °C; MS (ESI) m/z (%):
1
425.1 [M + H]+; H NMR (400 MHz, DMSO) δ 10.88 (s,
1H), 9.04 (s, 1H), 8.77 (d, J = 4.6 Hz, 1H), 8.51 (d, J = 5.4
Hz, 1H), 8.36 (d, J = 8.1 Hz, 1H), 8.25 (d, J = 8.3 Hz, 1H),
8.08 (d, J = 8.8 Hz, 2H), 7.84 (d, J = 7.4 Hz, 2H), 7.56 (t, J

Med Chem Res
119.43 (CH, C-9, C-13), 113.82 (CH, C-1), 111.62 (CH, C-
31), 109.79 (CH, C-5), 102.76 (CH,C-29), 26.83 (CH3, C-
25). Analytical HPLC on an Agilent (1100) using a 250 ×
4.6 mm SD-C18 analytical column, H2O/MeOH (15%H2O)
eluent at 1 mL/min flow, monitored by UV absorption at
325 nm, revealed 98.3% purity.
N-(4-(2-(methylcarbamoyl) pyridin-4-yloxy) phenyl)-5-p-
tolylpicolinamide (15f)
Yield: 57.48%; m.p. 189.5–190.7 °C; MS (ESI) m/z (%):
1
477.3 [M + K]+; H NMR (400 MHz, DMSO) δ 10.85 (s,
1H), 9.01 (s, 1H), 8.77 (s, 1H), 8.51 (d, J = 5.5 Hz, 1H),
8.31 (s, 1H), 8.22 (d, J = 8.1 Hz, 1H), 8.08 (d, J = 8.7 Hz,
2H), 7.73 (d, J = 7.8Hz, 2H), 7.42 (s, 1H), 7.36 (d, J = 7.8
Hz, 2H), 7.24 (d, J = 8.7 Hz, 2H), 7.16 (d, J = 3.0 Hz, 1H),
2.79 (d, J = 4.5 Hz, 3H), 2.38 (s, 3H). 13C NMR (101 MHz,
DMSO) δ 167.74 (C, C-6), 164.68 (C, C-15), 161.43 (C, C-
22), 151.30 (C, C-4), 151.17 (C, C-16), 148.32 (C, C-8),
146.65 (CH, C-2), 141.63 (CH, C-20), 139.43 (C, C-19),
134.64 (CH, C-18), 133.82 (C, C-11), 133.67 (C, C-27),
131.21 (C, C-30), 129.42 (CH, C-29, C-31), 127.53(CH, C-
28, C-32), 124.71 (CH, C-17), 122.57 (CH, C-10, C-12),
119.25 (CH, C-9, C-13), 113.92 (CH, C-1), 109.10 (CH, C-
5), 26.85 (CH3, C-25), 21.68 (CH3, C-33). Analytical
HPLC on an Agilent (1100) using a 250 × 4.6 mm SD-C18
analytical column, H2O/MeOH (15%H2O) eluent at 1 mL/
min flow, monitored by UV absorption at 325 nm, revealed
98.1% purity.
5-(4-Chlorophenyl)-N-(4-(2-(methylcarbamoy l) pyridin-4-
yloxy) phenyl) picolinamide (15d)
Yield: 64.68%; m.p. 213.2–214.7 °C; MS (ESI) m/z (%):
1
491.1 [M + K]+; H NMR (400 MHz, DMSO) δ 10.88 (s,
1H), 9.04 (s, 1H), 8.77 (s, 1H), 8.51 (d, J = 5.5 Hz, 1H),
8.37 (d, J = 8.1 Hz, 1H), 8.25 (d, J = 8.2 Hz, 1H), 8.07 (d,
J = 8.8 Hz, 2H), 7.88 (d, J = 8.4 Hz, 2H), 7.62 (d, J = 8.4
Hz, 2H), 7.41 (s, 1H), 7.25 (d, J = 8.8 Hz, 2H), 7.17 (d, J =
3.0 Hz, 1H), 2.79 (d, J = 4.5 Hz, 3H). 13C NMR (101 MHz,
DMSO) δ 166.83 (C, C-6), 163.76 (C, C-15), 162.23 (C, C-
22), 150.85 (C, C-4), 149.62 (C, C-16), 148.74 (C, C-8),
146.32 (CH, C-2), 141.43 (CH, C-20), 139.89 (C, C-19),
134.65 (C, C-27), 134.54 (C, C-30), 133.94 (CH, C-18),
133.64 (C, C-11), 130.43 (CH, C-29, C-31), 128.56 (CH,
C-28, C-32), 124.59 (CH, C-17), 122.81 (CH, C-10, C-12),
119.57 (CH, C-9, C-13), 113.51 (CH, C-1), 109.81 (CH, C-
5), 26.46 (CH3, C-25). Analytical HPLC on an Agilent
(1100) using a 250 × 4.6 mm SD-C18 analytical column,
H2O/MeOH (15%H2O) eluent at 1 mL/min flow, monitored
by UV absorption at 325 nm, revealed 98.6% purity.
N-(4-(2-(methylcarbamoyl) pyridin-4-yloxy) phenyl)-5-m-
tolylpicolinamide (15g)
Yield: 46.50%; m.p. 142.0–143.4 °C; MS (ESI) m/z (%):
1
477.1 [M + K]+; H NMR (400 MHz, DMSO) δ 10.88 (s,
1H), 9.02 (s, 1H), 8.78 (d, J = 4.2 Hz, 1H), 8.52 (d, J = 5.4
Hz, 1H), 8.34 (d, J = 8.1 Hz, 1H), 8.24 (d, J = 8.1 Hz, 1H),
8.08 (d, J = 8.7 Hz, 2H), 7.68–7.60 (m, 2H), 7.44 (dd, J =
15.3, 7.5 Hz, 2H), 7.31 (d, J = 7.2 Hz, 1H), 7.25 (d, J = 8.7
Hz, 2H), 7.20–7.15 (m, 1H), 2.79 (d, J = 4.4 Hz, 3H), 2.42
(s, 3H). 13C NMR (101 MHz, DMSO) δ 166.65 (C, C-6),
163.93 (C, C-15), 161.36 (C, C-22), 151.72 (C, C-4),
149.01 (C, C-16), 148.87 (C, C-8), 146.57 (CH, C-2),
141.31 (CH, C-20), 139.87 (C, C-19), 138.96 (C, C-29),
136.37 (C, C-27), 134.35 (CH, C-18), 133.83 (C, C-11),
129.54 (CH, C-31), 129.07 (CH, C-30), 128.25 (CH, C-28),
124.92 (CH, C-32), 124.63 (CH, C-17), 122.10 (CH, C-10,
C-12), 119.67 (CH, C-9, C-13), 113.53 (CH, C-1), 109.64
(CH, C-5), 26.87 (CH3, C-25), 21.79 (CH3, C-33). Analy-
tical HPLC on an Agilent (1100) using a 250 × 4.6 mm SD-
C18 analytical column, H2O/MeOH (15%H2O) eluent at 1
mL/min flow, monitored by UV absorption at 325 nm,
revealed 97.5% purity.
5-(4-Methoxyphenyl)-N-(4-(2-(methylcarbamoyl) pyridin-4-
yloxy)phenyl) picolinamide (15e)
Yield: 46.67%; m.p. 199.2–200.6 °C; MS (ESI) m/z (%):
1
493.3 [M + K]+; H NMR (400 MHz, DMSO) δ 10.83 (s,
1H), 9.00 (s, 1H), 8.77 (s, 1H), 8.51 (d, J = 5.5 Hz, 1H),
8.31 (d, J = 8.2 Hz, 1H), 8.21 (d, J = 8.1 Hz, 1H), 8.07 (d,
J = 8.6 Hz, 2H), 7.80 (d, J = 8.5 Hz, 2H), 7.41 (s, 1H), 7.25
(d, J = 8.6 Hz, 2H), 7.17 (d, J = 3.1 Hz, 1H), 7.11 (d, J =
8.5 Hz, 2H), 3.83 (s, 3H), 2.79 (d, J = 4.2 Hz, 3H). 13C
NMR (101 MHz, DMSO) δ 168.03 (C, C-6), 163.19 (C, C-
15), 161.94 (C, C-22), 161.22 (C, C-30), 151.03 (C, C-4),
149.08 (C, C-16), 147.02 (C, C-8), 146.79 (CH, C-2),
141.82 (CH, C-20), 140.08 (C, C-19), 134.21 (CH, C-18),
133.79 (C, C-11), 129.35 (CH, C-28, C-32), 128.34 (C, C-
27), 124.86 (CH, C-17), 122.91 (CH, C-10, C-12), 119.35
(CH, C-9, C-13), 114.28 (CH, C-29, C-31), 113.79 (CH, C-
1), 109.47 (CH, C-5), 55.34 (OCH3, C-33), 26.68 (CH3, C-
25). Analytical HPLC on an Agilent (1100) using a 250 ×
4.6 mm SD-C18 analytical column, H2O/MeOH (15%H2O)
eluent at 1 mL/min flow, monitored by UV absorption at
325 nm, revealed 98.9% purity.
5-(3-Fluorophenyl)-N-(4-(2-(methylcarbamoyl) pyridin-4-
yloxy) phenyl) picolinamide (15h)
Yield: 54.99%; m.p. 191.8–192.5 °C; MS (ESI) m/z (%):
1
481.3 [M + K]+; H NMR (400 MHz, DMSO) δ 10.89 (s,
1H), 9.07 (s, 1H), 8.78 (s, 1H), 8.52 (d, J = 5.2 Hz, 1H),

Med Chem Res
8.40 (d, J = 6.8 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 8.08 (d,
J = 8.5 Hz, 2H), 7.73 (dd, J = 17.1, 9.0 Hz, 2H), 7.61 (dd,
J = 14.0, 7.4 Hz, 1H), 7.41 (s, 1H), 7.33 (t, J = 7.5 Hz, 1H),
7.25 (d, J = 8.5 Hz, 2H), 7.17 (d, J = 2.6 Hz, 1H), 2.79 (d,
J = 3.9 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 167.27 (C,
C-6), 163.14 (C, C-15), 162.68 (C, C-29), 161.10 (C, C-22),
151.72 (C, C-4), 149.17 (C, C-16), 148.07 (C, C-8), 146.31
(CH, C-2), 141.87 (CH, C-20), 139.60 (C, C-19), 138.31
(C, C-27), 134.07 (CH, C-18), 133.87 (C, C-11), 127.43
(CH, C-31), 124.47 (CH, C-17), 123.11 (CH, C-32), 122.65
(CH, C-10, C-12), 119.63 (CH, C-9, C-13), 115.92 (CH, C-
28), 115.64 (CH, C-30), 113.67 (CH, C-1), 109.86 (CH, C-
5), 26.88 (CH3, C-25). Analytical HPLC on an Agilent
(1100) using a 250 × 4.6 mm SD-C18 analytical column,
H2O/MeOH (15%H2O) eluent at 1 mL/min flow, monitored
by UV absorption at 325 nm, revealed 98.2% purity.
133.84 (C, C-11), 133.69 (C, C-27), 129.34 (CH, C-29, C-
31), 127.69 (CH, C-28, C-32), 124.11 (CH, C-17), 122.83
(CH, C-10, C-12), 119.38 (CH, C-9, C-13), 113.85 (CH, C-
1), 109.09 (CH, C-5), 28.79 (CH2CH3, C-33), 26.84 (CH3,
C-25), 14.62 (CH2CH3, C-33). Analytical HPLC on an
Agilent (1100) using a 250 × 4.6 mm SD-C18 analytical
column, H2O/MeOH (15%H2O) eluent at 1 mL/min flow,
monitored by UV absorption at 325 nm, revealed 98.4%
purity.
5-(2, 4-Dimethylphenyl)-N-(4-(2-(methylcarbamoyl)
pyridin-4-yloxy) phenyl) picolinamide (15k)
Yield: 45.81%; m.p. 172.1–173.4 °C; MS (ESI) m/z (%):
1
491.2 [M + K]+; H NMR (400 MHz, DMSO) δ 10.89 (s,
1H), 8.77 (d, J = 4.3 Hz, 1H), 8.70 (s, 1H), 8.52 (d, J = 5.4
Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.07 (t, J = 8.1 Hz, 3H),
7.41 (s, 1H), 7.20 (m, 6H), 2.79 (d, J = 4.4 Hz, 3H), 2.34 (s,
3H), 2.26 (s, 3H). 13C NMR (101 MHz, DMSO) δ 167.78
(C, C-6), 164.82 (C, C-15), 161.08 (C, C-22), 151.83 (C, C-
4), 149.61 (C, C-16), 148.47 (C, C-8), 146.26 (CH, C-2),
141.81 (CH, C-20), 139.56 (C, C-19), 138.37 (C, C-30),
134.07 (CH, C-18), 133.95 (C, C-11), 133.03 (CH, C-32),
131.56 (CH, C-31), 130.59 (C, C-27), 130.47 (C, C-28),
125.95 (CH, C-29), 124.63 (CH, C-17), 122.75 (CH, C-10,
C-12), 119.54 (CH, C-9, C-13), 113.93 (CH, C-1), 109.73
(CH, C-5), 26.76 (CH3, C-25), 21.79 (4-CH3, C-33), 19.27
(2-CH3, C-33). Analytical HPLC on an Agilent (1100)
using a 250 × 4.6 mm SD-C18 analytical column, H2O/
MeOH (15%H2O) eluent at 1 mL/min flow, monitored by
UV absorption at 325 nm, revealed 98.6% purity.
N-(4-(2-(methylcarbamoyl) pyridin-4-yloxy) phenyl)-5-(4-
(trifluoromethyl) phenyl)picolinamide (15i)
Yield: 51.57%; m.p. 240.5–241.7 °C; MS (ESI) m/z (%):
1
531.2 [M + K]+; H NMR (400 MHz, DMSO) δ 10.91 (s,
1H), 9.10 (s, 1H), 8.77 (d, J = 4.2 Hz, 1H), 8.52 (d, J = 5.4
Hz, 1H), 8.44 (d, J = 8.0 Hz, 1H), 8.29 (d, J = 8.1 Hz, 1H),
8.05 (t, J = 17.1 Hz, 4H), 7.92 (d, J = 7.9 Hz, 2H), 7.41 (s,
1H), 7.25 (d, J = 8.6 Hz, 2H), 7.17 (d, J = 2.9 Hz, 1H), 2.79
(d, J = 4.3 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 167.37
(C, C-6), 164.58 (C, C-15), 161.10 (C, C-22), 151.00 (C, C-
4), 149.62 (C, C-16), 148.15 (C, C-8), 146.38 (CH, C-2),
141.56 (CH, C-20), 139.72 (C, C-27), 139.35 (C, C-19),
134.05 (CH, C-18), 133.97 (C, C-11), 131.21 (C, C-30),
127.43 (CH, C-28, C-32), 125.67 (CH, C-29, C-31), 124.52
(CH, C-17), 124.11 (C, C-33), 122.67 (CH, C-10, C-12),
119.25 (CH, C-9, C-13), 113.75 (CH, C-1), 109.10 (CH, C-
5), 26.78 (CH3, C-25). Analytical HPLC on an Agilent
(1100) using a 250 × 4.6 mm SD-C18 analytical column,
H2O/MeOH (15%H2O) eluent at 1 mL/min flow, monitored
by UV absorption at 325 nm, revealed 98.6% purity.
VEGFR2/KDR kinase assay
The inhibitory activity against VEGFR2/KDR at 10 μM
level in vitro was evaluated through the mobility shift assay
together with reference compounds sorafenib and Staur-
osporine (Roper et al. 2011). All kinase assays were per-
formed on 384-well plates in a 50 μL reaction volume. The
kinase base buffer contains 50 mM HEPES, pH 7.5, 10 mM
MgCl2, 0.0015% Brij-35 and 2 mM DTT. The stop buffer
contains 100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2%
Coating Reagent #3 and 50 mM EDTA. Dilute the com-
pounds to 500 μM by 100% DMSO, then 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 of the intermediate plate to 384-well plates. The fol-
lowing amounts of enzyme and substrate were used per
well: kinase base buffer, FAM-labeled peptide, ATP and
enzyme solution. Wells containing the substrate, enzyme,
DMSO without compound were used as DMSO control.
Wells containing substrate without enzyme were used as
low control. Incubate at room temper-ature for 10 min. Add
5-(4-Ethylphenyl)-N-(4-(2-(methylcarbamoy l) pyridin-4-
yloxy) pheny l) picolinamide (15j)
Yield: 45.67%; m.p. 164.5–165.6 °C; MS (ESI) m/z (%):
1
491.1 [M + K]+; H NMR (400 MHz, DMSO) δ 9.02 (s,
1H), 8.76 (s, 1H), 8.51 (d, J = 5.6 Hz, 1H), 8.33 (d, J = 8.3
Hz, 1H), 8.23 (d, J = 8.1 Hz, 1H), 8.07 (d, J = 8.8 Hz, 2H),
7.76 (d, J = 7.9 Hz, 2H), 7.41 (s, 2H), 7.39 (s, 1H), 7.25 (d,
J = 8.6 Hz, 2H), 7.17 (d, J = 2.9 Hz, 1H), 2.79 (s, 3H), 2.68
(q, J = 7.5 Hz, 2H), 1.22 (t, J = 7.5 Hz, 3H). 13C NMR
(101 MHz, DMSO) δ 166.74 (C, C-6), 163.85 (C, C-15),
162.91 (C, C-22), 151.43 (C, C-4), 149.72 (C, C-16),
148.79 (C, C-8), 146.58 (CH, C-2), 144.28 (C, C-30),
140.04 (CH, C-20), 139.96 (C, C-19), 134.71 (CH, C-18),

Med Chem Res
10 μL peptide solution to each well. Incubate at 28 °C for
specified period of time and stop reaction by 25 μL stop
buffer. At last, collect data on Caliper program and convert
conversion values to inhibition values. Percent inhibition =
(max-conversion)/ (max-min)*100. “max” denoted DMSO
control; “min” denoted low control.
reference wavelength) was measured by the ELISA reader.
All the compounds were tested three times in each of the
cell lines. The results expressed as inhibition rates or IC50
(half-maximal inhibitory concentration) were the averages
of two determinations and calculated by using the Bacus
Laboratories Incorporated Slide Scanner (Bliss) software.
Acknowledgements This work is supported by National Natural
Science Foundation of China (No.81460527, 21662014), Project
supported by the Natural Science Foundation of Jiangxi, China
BRAF and CRAF kinases assay
The kinase activity of BRAF and CRAF was measured by
Lanthascreen assay (Roper et al. 2011). All kinase assays
were performed on 384-well plates in a 100 μL reaction
volume. The kinase base buffer contains 50 mM HEPES,
pH 7.5, 10 mM MgCl2, 1 mM EGTA and 0.01% Brij-35.
The stop buffer contains 2-fold of final concentration in
Antibody Dilution Buffer. The final concentration: Anti-
body 2 nM and EDTA 10 mM. Dilute the compound to
100× of the final desired highest inhibitor concentration in
reaction by 100%DMSO. Then transfer 4 μl of compound
from source plate to a new 96-well plate as the intermediate
plate. Add 96 μL of 1x kinase buffer to each well of the
intermediate plate. Transfer 2.5 μL of each well from the
96-well intermediate plate to a 384-well plate in duplicates.
The following amounts of enzyme and substrate were used
per well: kinase base buffer, FAM-labeled peptide, ATP and
enzyme solution. Wells containing the substrate, enzyme,
DMSO without compound and enzyme were used as
DMSO control. Add 2.5 μL of substrate solution to each
well of the assay plate to start reaction. Cover the assay
plate and incubate under room temperature for 1 h. Add 10
μL of detection solution to each well of the assay plate to
stop the reaction. At last, collect data on Caliper program
and convert conversion values to inhibition values. Percent
(20161BAB215216,20171ACB21052,20171BCB23078),
Doctoral
Scientific Research Foundation of Jiangxi Science & Technology
Normal University and Program of Key Laboratory of Drug Design
and Optimization, Jiangxi Science & Technology Normal University
(300098010306) and College Students’ science and Technology
Innovation Project of Jiangxi Province (201411318033)
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
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