Synthesis and biological evaluation of a new series of 1-aryl-3-[4-(pyridin-2-ylmethoxy)phenyl]urea derivatives as new anticancer agents

Article abstract of DOI:10.1007/s00044-020-02554-z

The diaryl ureas are very important fragments in medicinal chemistry. By means of computer-aided design, 1-aryl-3-[4-(pyridin-2-ylmethoxy)phenyl]urea derivatives were designed and synthesized, and evaluated for their antiproliferative activity against A549, HCT-116, PC-3 cancer cell lines, and HL7702 human normal liver cell lines in vitro by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Most of the target compounds demonstrate significant antiproliferative effects on all the selective cancer cell lines. The calculated IC₅₀ values were reported. The target compound 1-(4-chlorophenyl)-3-{4-{[3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methoxy}phenyl}urea (7u) demonstrated the most potent inhibitory activity (IC₅₀ = 2.39 ± 0.10 μM for A549 and IC₅₀ = 3.90 ± 0.33 μM for HCT-116), comparable to the positive-control sorafenib (IC₅₀ = 2.12 ± 0.18 μM for A549 and IC₅₀ = 2.25 ± 0.71 μM for HCT-116). Conclusively, 1-aryl-3-[4-(pyridin-2-ylmethoxy)phenyl]urea derivatives as the new anticancer agents were discovered, and could be used as the potential BRAF inhibitors for further research.

Full text of DOI:10.1007/s00044-020-02554-z

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02554-z
ORIGINAL RESEARCH
Synthesis and biological evaluation of a new series of
1-aryl-3-[4-(pyridin-2-ylmethoxy)phenyl]urea derivatives
as new anticancer agents
Jian Feng1 Tai Li1 Shishao Liang1 Chuanming Zhang1 Xiaoyu Tan1 Ning Ding1 Xin Wang2 Xiaoping Liu1
Chun Hu
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Received: 12 October 2019 / Accepted: 27 April 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
The diaryl ureas are very important fragments in medicinal chemistry. By means of computer-aided design, 1-aryl-3-[4-
(pyridin-2-ylmethoxy)phenyl]urea derivatives were designed and synthesized, and evaluated for their antiproliferative
activity against A549, HCT-116, PC-3 cancer cell lines, and HL7702 human normal liver cell lines in vitro by using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Most of the target compounds demonstrate
significant antiproliferative effects on all the selective cancer cell lines. The calculated IC50 values were reported. The target
compound 1-(4-chlorophenyl)-3-{4-{[3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methoxy}phenyl}urea (7u) demon-
strated the most potent inhibitory activity (IC50 = 2.39 0.10 μM for A549 and IC50 = 3.90 0.33 μM for HCT-116),
comparable to the positive-control sorafenib (IC50 = 2.12 0.18 μM for A549 and IC50 = 2.25 0.71 μM for HCT-116).
Conclusively, 1-aryl-3-[4-(pyridin-2-ylmethoxy)phenyl]urea derivatives as the new anticancer agents were discovered, and
could be used as the potential BRAF inhibitors for further research.
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Keywords Anticancer agent BRAF Urea Synthesis Antiproliferative activity
Introduction
improve cancer cure rates and also to reduce severe adverse
actions because of the high specificity of the targeted drugs,
which are expressed and have critical roles in cancer cells,
but not in normal cells. However, the clinical effect was
found to be limited and did not last for a long period
because of the acquired resistance of the tumor cells. Fur-
thermore, these drugs often cause on-target and/or off-target
severe toxicity (Keefe and Bateman 2011). Therefore, the
development of more target-specific therapy, with minimum
toxicity, is warranted to extend disease-free survival and
improve the quality of life of cancer patients.
Sorafenib is an orally available drug that acts pre-
dominantly by targeting the Ras/Raf/MEK (mitogen-acti-
vated protein kinase (MAPK)/ERK kinase)/ERK
(extracellular signal-regulated kinase) pathway. In vitro
sorafenib inhibits several members of the RAF/MEK/ERK
signaling cascade, including wild-type BRAF (B-Raf proto-
oncogene), mutant BRAF carrying V600E, and
serine–threonine kinases CRAF (C-Raf proto-oncogene)
(Davies et al. 2002; Moreau et al. 2012; Vultur et al. 2011;
Wan et al. 2004; Wilhelm et al. 2004). BRAF is a member
of the RAF family of serine–threonine kinases, which are
Cancer is a major public health problem in developed
countries and will become the most serious life-threatening
disease worldwide in the near future (Siegel et al. 2013).
Some advances in cancer treatment by targeted drugs, such
as imatinib, gefitinib, and trastuzumab, were expected to
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02554-z) contains supplementary
material, which is available to authorized users.
* Xiaoping Liu
lxp19730107@163.com
* Chun Hu
chunhu@syphu.edu.cn
1
Key Laboratory of Structure-based Drug Design & Discovery,
Ministry of Education, Shenyang Pharmaceutical University,
Shenyang 110016, China
2
Department of Pharmacy, Liaoning Univeristy, Shenyang 110036,
China

Medicinal Chemistry Research
Fig. 1 Some anticancer drugs of
the diaryl urea moiety
Fig. 2 H-bond acceptor and donors within the diaryl urea scaffold
part of the RAS/RAF/MEK/ERK MAPK signaling pathway
(Barras 2015; Cheng et al. 2018; Schreck and Rapp 2006).
RAF is mainly activated by the G protein, RAS, in response
to mutations in cancer cells or by overexpression of dif-
ferent receptor protein tyrosine kinases such as epidermal,
platelet derived, and vascular endothelial growth factors
(Regad 2015; Sun et al. 2015).
Fig. 3 Chemical structures of proton pump inhibitors (PPIs)
As known, the diaryl urea moiety is widely used in the
design of anticancer drugs, such as sorafenib, regorafenib,
linifanib, and tivozanib (Fig. 1). And the diaryl ureas are
very important fragments in medicinal chemistry and can be
used for the synthesis of numerous heterocyclic compounds
with diversified biological activities, including antith-
rombotic (Qiao et al. 2013), antimalarial (Anderson et al.
2013), antibacterial (Keche et al. 2012), and anti-
inflammatory (Kulkarni et al. 2013) properties, and they
are characterized by their ability to form hydrogen bond
interactions with biotargets (Kim et al. 2012; Lu et al. 2014;
Xuan et al. 2013). The carbonyl oxygen atom acts as a
proton acceptor while the two amide nitrogen atoms are
proton donors (Fig. 2). This unique type of structure
endows urea derivatives with the ability to bind a variety of
enzymes and receptors in the biological systems.
lansoprazole, rabeprazole, and pantoprazole in vitro and
in vivo (Lugini et al. 2016). As shown in Fig. 3, all of the
PPIs molecules contain pyridin-2-ylmethyl fragments. It can
be predicted that these fragments should play an important
role in the antiproliferative activity.
Molecular hybridization strategy is a useful concept in
drug design and development based on the combination of
pharmacophoric moieties of different bioactive substances
to produce a new structure, the affinity and efficacy would
be improved, when compared with the parent drugs
(Viegas-Junior et al. 2007). These above interesting find-
ings and our continuous quest to identify more potent
antiproliferative agents led to the molecular hybridization
of diaryl urea and pyridin-2-ylmethyl to integrate them in
one molecular platform to generate a new hybrid, and
expected that taking this way could get the anti-
proliferative agents with highly inhibitory activity. The
molecular docking results also showed that the urea
structure and pyridine can act as a hydrogen bond donor to
generate hydrogen bond interactions. Therefore, a novel
In recent years, proton pump inhibitors (PPIs) as poten-
tial anticancer agents were intensively studied in cancer
treatment. Lugini et al. compared the antitumor efficacy of
different PPIs, including omeprazole, esomeprazole,

Medicinal Chemistry Research
series of 1-aryl-3-[4-(pyridin-2-ylmethoxy)phenyl]urea
derivatives were designed and synthesized.
bond with CYS532. This also shows the importance of
introducing a pyridine ring. Hence, we can conclude that
both the diaryl urea structure and the pyridinyl ring structure
in our designed compounds enhance the affinity of the
compound to the target BRAF kinase. In addition, it can be
seen that the amino-acid residues HIS574, LYS483,
PHE595, VAL471, GLN 530, ALA481, PHE583 formed
some weak interactions between the compound 7u and
BRAF.
The molecular docking results revealed that the target
compound 7u and its analogs were potential inhibitors of
BRAF. Therefore, we synthesized a novel series of 1-aryl-3-
[4-(pyridin-2-ylmethoxy)phenyl]urea derivatives, aiming to
assay their inhibitory activity to BRAF kinase.
Results and discussion
Molecular docking studies
A docking study aiming at the characterization of the
interaction between the target molecule and BRAF at
molecular level was carried out and more-effective BRAF
inhibitors were expected to be discovered.
In this study, docking model was selected from Protein
Data Bank (PDB) of Research Collaborator for Structural
Bioinformatics (RCSB), and the docking study was pre-
pared by Molegro Virtual Docker (MVD) 2008 (v3.0)
(Thomsen and Christensen 2006). Initially, the docking
studies were carried out on the BRAF—sorafenib complex
(PDB code 4R5Y) (Tang et al. 2015), using the Discovery
Studio 2016 client to remove the small-molecule ligand of
erlotinib in 4R5Y to obtain the three-dimensional crystal
structure of BRAF, and then the designed compounds were
docked to the crystal structure BRAF with MVD software.
The detailed docking data will be described below, using
compound 7u with chloro atom in the para position of the
phenyl ring, methyl group at the R3 position the tri-
fluoroethoxy group at the R4 position, and H atom at the R5
position on the pyridine ring as an example.
Synthesis
The general synthetic route is illustrated in Scheme 1. The
reaction of the commercially available 4-nitrophenol (1)
with 2-(chloromethyl)pyridine derivatives (2) in ethanol at
room temperature obtained compounds 3a–3d (Seto et al.
2005), which converted to key intermediates 4a–4d via Pd-
C catalytic hydrogenation reduction (Nguyen et al. 2014).
The aryl isocyanates 6a–6f were prepared by reaction
between aromatic amines and bis(trichloromethyl)carbonate
(BTC) (Liu et al. 2013). Finally, treatment of 4a–4d with
arylisocyanates 6a–6f in methylene dichloride yielded 1-
aryl-3-[4-(pyridin-2-ylmethoxy)phenyl]urea
derivatives
As shown as in the Table 1 and Fig. 4a, it can be seen
that compound 7u and sorafenib have similar MolDock
scores, it also was inserted inside the protein and has a high
degree of overlap in the protein cavity with sorafenib. This
implies that the compound we designed is likely to become
an effective BRAF inhibitor. From Fig. 4b, c, we can see
that the carbon-based oxygen atom in the urea structure in
the 7u structure can act as a hydrogen bond acceptor to
generate hydrogen bond interaction with the amino-acid
residue ASP594. At the same time, NH in the urea structure
close to chlorophenyl group can act as a hydrogen bond
donor to generate hydrogen bond interactions with the
amino-acid residue GLU501. All of this confirms the fea-
sibility of introducing a diphenylurea structure into the drug
design to increase the affinity of the compound to the target.
Moreover, N atom on the pyridine ring in 7u has a hydro-
gen bond with THR529, and the N atom on the pyridine
with N on amides in sorafenib can generate a hydrogen
(7a–7x) as the target compounds (Liu et al. 2013). The
structures of the target compounds and intermediates were
characterized by infrared (IR) spectra, proton nuclear
magnetic resonance spectra (1H NMR), electrospray ioni-
zation mass spectra (ESI-MS), and no target compounds
were reported in the literatures.
Bioactivity in cell line
Using sorafenib as a positive control, all of the target
compounds were evaluated for the antiproliferative activity
in vitro against cancer cell lines, we choose Raf/MEK/ERK
pathway cells, including lung cancer A549, and colorectal
cancer HCT-116 cell lines, prostate cancer PC-3 cell lines,
which Raf/MEK/ERK pathways do not affect (Lee et al.
2005), and human normal liver HL7702 cell lines by MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) assay. The antiproliferative assay results evaluated as
the half maximal inhibitory concentration (IC50) value
(Table 2) and demonstrated that several target compounds
have shown moderate to excellent potency against A549
and HCT-116 cancer cell lines, and most of the target
compounds hardly exhibit any antiproliferative activities
against PC-3 cell lines. Among the target compounds 7u
showed the similar inhibitory effect against two cancer cell
Table 1 The MolDock score of 7u and sorafenib
No.
MolDock score
7u
−141.4 kcal/mol
Sorafenib
−155.922 kcal/mol

Medicinal Chemistry Research
Fig. 4 a Interactions of 7u (blue)
and sorafenib (green) with
BRAF in a three-dimensional
diagram. b Interactions of 7u
(blue) with BRAF in a three-
dimensional diagram. c
Interactions of 7u (left) and
sorafenib (right) with BRAF in a
two-dimensional diagram (Color
figure online)
lines than positive control with IC50 values of 2.39 0.10
and 3.90 0.33 μM, respectively. All the tested compounds
exhibit weak cytotoxic activities against HL7702.
All the target compounds could be divided into three
classes according to different substituents on the pyr-
idinyl ring (Fig. 5). The analyses of the structure–activity

Medicinal Chemistry Research
Scheme 1 Synthetic route of the target compounds
Table 2 The chemical structures and inhibitory activities of the target compounds
No.
R
R3
R4
R5
IC50 (μM)a
A549
HCT-116
PC-3
HL7702
7a
7b
7c
3,5-difluoro
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–O(CH2)3OCH3
–O(CH2)3OCH3
–O(CH2)3OCH3
–O(CH2)3OCH3
–O(CH2)3OCH3
–O(CH2)3OCH3
–OCH3
H
44.80 3.18
4.38 1.03
10.24 1.48
11.44 0.77
5.74 0.44
6.19 0.45
17.28 4.66
6.65 2.16
10.38 0.21
16.72 0.76
23.27 1.0
15.40 0.67
10.16 0.21
20.21 1.55
13.72 1.46
12.94 0.34
13.20 0.89
6.63 0.28
7.67 0.77
2.65 0.17
2.39 0.10
12.46 0.79
5.37 0.14
4.35 0.09
2.12 0.18
56.61 2.94
8.81 0.46
9.50 1.90
12.66 2.17
6.44 0.71
10.10 0.19
25.70 1.20
22.88 3.71
12.85 0.12
13.14 1.02
22.29 1.78
18.13 2.18
10.76 0.39
12.51 1.51
19.34 2.76
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
n.d.
3-trifluoromethyl
4-chloro
H
>100
H
>100
7d
7e
4-methoxy
H
47.18 3.12
>100
4-fluoro
H
7f
4-chloro-3-trifluoromethyl
3,5-difluoro
H
>100
7g
7h
7i
H
>100
3-trifluoromethyl
4-chloro
–OCH3
H
75.05 1.01
>100
–OCH3
H
7j
4-methoxy
–OCH3
H
46.41 3.10
50.11 2.33
>100
7k
7l
4-fluoro
–OCH3
H
4-chloro-3-trifluoromethyl
3,5-difluoro
–OCH3
H
7m
7n
7o
7p
7q
7r
–OCH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
H
57.63 3.18
>100
3-trifluoromethyl
4-chloro
–OCH3
–OCH3
47.92 2.98
>100
4-methoxy
–OCH3
4-fluoro
–OCH3
12.68 1.20
8.17 0.34
51.96 1.22
5.43 0.28
3.90 0.33
19.25 0.95
6.35 0.07
5.60 0.15
2.25 0.71
43.32 2.21
>100
4-chloro-3-trifluoromethyl
3,5-difluoro
–OCH3
7s
–OCH2CF3
–OCH2CF3
–OCH2CF3
–OCH2CF3
–OCH2CF3
–OCH2CF3
>100
7t
3-trifluoromethyl
4-chloro
H
>100
7u
7v
7w
7x
Sorafenib
H
>100
4-methoxy
H
89.10 4.00
60.24 3.68
54.19 0.44
3.60 1.08
4-fluoro
H
4-chloro-3-trifluoromethyl
H

Medicinal Chemistry Research
Fig. 5 SARs summary for the
target compounds
relationships (SARs) were summarized as follows: (1)
the results of cell inhibitory activity assay showed, when
the 4th position (R4) of the C ring is occupied by the
−OCH2CF3 group, the inhibitory activity is optimal. (2)
The substituent on the A ring has a significant effect on
the inhibitory activity of the target compounds. When the
substituents on the C ring are the same, when the ring A of
the compound is substituted by an electron withdrawing
group such as –F, –Cl or –CF3 or the like, the inhibitory
activity is superior to the compound substituted with an
electron donating group such as –OCH3. In addition, when
electron withdrawing group coexist in the A ring, the
inhibitory activity is better than of the two electron with-
drawing groups. (3) The inhibitory effect of the compound
on the three test cells showed different selectivity. Most of
the compounds inhibited the activity of A549 cells better
than the inhibitory activities of HCT-116 cells, while the
inhibitory activity against PC-3 cells was poor.
Experimental section
Materials and methods
The reaction process is detected by high performance thin
layer chromatography silica gel plate. The melting point
was determined by WRX-1S type microscopic melting-
point meter, and the thermometer was not corrected. The IR
spectra were determined by Bruker IFS55 IR spectrometer
(KBr pellets), mass spectrometry data were determined by
Waters ACQUITY QDa liquid chromatography-mass
spectrometer, and nuclear magnetic resonance spectra
were determined by Bruker 400 MHz nuclear magnetic
resonance spectrometer with tetramethylsilane as internal
standard and perdeuterated dimethyl sulfoxide (DMSO-d6)
as solvent.
Preparation of 2-[(4-nitrophenoxy) methyl]pyridine
derivatives (3a–3d)
Conclusion
The reaction of the commercially available 4-nitrophenol
(0.1 mol) with 2-(chloromethyl)pyridine derivatives
(0.1 mol) in ethanol, add 3 mol/L NaOH solution (100 mL)
at r.t. and the reaction mixture was stirred for an additional
3 h. After completion of the reaction was monitored TLC,
ethanol was removed by vacuum evaporation, the residue
was added 200 mL of water, filtered off with suction after
15 min of stirring, placing the filter cake was dried in air, the
product was used directly in the next step without
purification.
Based on the structure of sorafenib, and hybridization,
and bioisosterism, combining structural optimization, a
novel series of 1-aryl-3-[4-(pyridin-2-ylmethoxy)phenyl]
urea derivatives, was designed as the target compounds.
The binding mode of 7u, a representative of the target
compounds, to BRAF was simulated by MVD software.
It was found that NH and oxygen in the urea structure,
the N atom of pyridine ring form a hydrogen bond with
the amino-acid residue in the BRAF protein. A novel
series of 1-aryl-3-[4-(pyridin-2-ylmethoxy)phenyl]urea
derivatives were evaluated for their antiproliferative
activity against A549, HCT-116, PC-3 cancer cell lines
and HL7702 human normal liver cell lines in vitro by
using the MTT colorimetric assay. Most of the target
compounds demonstrate significant antiproliferative
effects on all the selective cancer cell lines. Con-
clusively, 1-aryl-3-[4-(pyridin-2-ylmethoxy)phenyl]urea
derivatives as new anticancer agents were discovered,
and could be used as potential lead compounds for fur-
ther research.
4-(3-methoxypropoxy)-3-methyl-2-[(4-nitrophenoxy)
methyl]pyridine (3a) Yield 90.5% (white solid). ESI-MS
(m/z): 333.4 ([M + H]+).
3-methyl-2-[(4-nitrophenoxy)methyl]-4-(2,2,2-trifluor-
oethoxy)pyridine (3b) Yield 90.2% (white solid). ESI-MS
(m/z): 343.5 ([M + H]+).
4-methoxy-3,5-dimethyl-2-[(4-nitrophenoxy)methyl]pyri-
dine (3c) Yield 92.5% (white solid). ESI-MS (m/z): 289.0
([M + H]+).

Medicinal Chemistry Research
3,4-dimethoxy-2-[(4-nitrophenoxy)methyl]pyridine (3d)
7.34 (d, J = 2.2 Hz, 1H), 7.32 (d, J = 2.2 Hz, 1H), 7.18 (d,
J = 2.3 Hz, 1H), 7.16 (d, J = 2.3 Hz, 1H), 7.00 (d, J =
5.7 Hz, 1H), 6.97 (d, J = 2.3 Hz, 1H), 6.96 (d, J = 2.2 Hz,
1H), 6.79–6.73 (m, 1H), 5.11 (s, 2H), 4.11 (t, J = 6.3 Hz,
2H), 3.49 (t, J = 6.2 Hz, 2H), 3.25 (s, 3H), 2.18 (s, 3H),
2.02–1.95 (m, 2H). ESI-MS (m/z): 458.4 ([M + H]+).
Yield 95.1% (white solid). ESI-MS (m/z): 291.1 ([M + H]+).
Preparation of 4-(pyridin-2-ylmethoxy) aniline derivatives
(4a–4d)
The reaction of the commercially available 2-[(4-nitrophe-
noxy)methyl]pyridine derivatives (50.0 mmol) with 10%
Pd-C in methanol, catalytic hydrogenation reduction reac-
tion at room temperature for 4 h. After completion of the
reaction was monitored TLC, palladium carbon was to filter
by diatomaceous earth, and then distillation to remove
methanol under reduced pressure, the product was used
directly in the next step without purification.
1-{4-{[4-(3-methoxypropoxy)-3-methylpyridin-2-yl]meth-
oxy}phenyl}-3-[3-(trifluoromethyl)phenyl]urea (7b) Yield
43.6% (white solid). m.p.: 134–135 °C. IR(KBr, cm−1): υ
3424.0, 2922.3, 2852.6, 1600.8, 1560.4, 1511.1, 1384.1,
1
1339.1, 1296.0, 1164.0, 1117.7, 826.0, 617.0. H NMR
(400 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.61 (s, 1H), 8.27 (d,
J = 5.7 Hz, 1H), 8.01 (s, 1H), 7.56 (d, J = 8.5 Hz, 1H),
7.51–7.48 (m, 1H), 7.36 (d, J = 2.2 Hz, 1H), 7.34 (d, J =
2.3 Hz, 1H), 7.29 (d, J = 7.5 Hz, 1H), 7.00 (d, J = 5.8 Hz,
1H), 6.98 (d, J = 2.2 Hz, 1H), 6.96 (d, J = 2.2 Hz, 1H), 5.11
(s, 2H), 4.11 (t, J = 6.2 Hz, 2H), 3.49 (t, J = 6.2 Hz, 2H),
3.25 (s, 3H), 2.18 (s, 3H), 2.02–1.96 (m, 3H). ESI-MS
(m/z): 490.3 ([M + H]+); 512.1 ([M + Na]+).
4-{[4-(3-methoxypropoxy)-3-methylpyridin-2-yl]methoxy}
aniline (4a) Yield 90.0% (yellowish oil). ESI-MS (m/z):
329.4 ([M + H]+).
4-{[3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methoxy}
aniline (4b) Yield 91.7% (yellowish oil). ESI-MS (m/z):
313.1 ([M + H]+).
1-(4-chlorophenyl)-3-{4-{[4-(3-methoxypropoxy)-3-methyl-
pyridin-2-yl]methoxy}phenyl}urea (7c) Yield 49.1% (white
solid). m.p.: 158–160 °C. IR(KBr, cm−1): υ 3426.3, 2921.6,
2852.2, 1628.7, 1510.5, 1492.8, 1384.2, 1296.5, 1112.3,
618.3. 1H NMR (400 MHz, DMSO-d6) δ 8.72 (s, 1H), 8.49
(s, 1H), 8.26 (d, J = 5.6 Hz, 1H), 7.47 (d, J = 2.2 Hz, 1H),
7.45 (d, J = 2.2 Hz, 1H), 7.35–7.29 (m, 4H), 7.00 (d, J =
5.7 Hz, 1H), 6.96 (d, J = 2.3 Hz, 1H), 6.95 (d, J = 2.1 Hz,
1H), 5.10 (s, 2H), 4.11 (t, J = 6.2 Hz, 2H), 3.49 (t, J =
6.2 Hz, 2H), 3.25 (s, 3H), 2.18 (s, 3H), 2.02–1.95 (m, 2H).
ESI-MS (m/z): 456.3, 457.2, 458.2, 459.2 ([M + H]+).
4-[(4-methoxy-3,5-dimethylpyridin-2-yl)methoxy]aniline
(4c) Yield 94.8% (yellowish oil). ESI-MS (m/z): 259.3
([M + H]+).
4-[(3,4-dimethoxypyridin-2-yl)methoxy]aniline (4d) Yield
91.1% (yellowish oil). ESI-MS (m/z): 261.2 ([M + H]+).
Preparation of diaryl urea derivatives (7a–7x)
To a solution of BTC (1 mmol) in CH2Cl2 (20 mL) was
added dropwise to primary aromatic amine 5 (1 mmol) in
CH2Cl2 (20 mL) followed by the dropwise addition of
triethylamine (1 mL) in CH2Cl2 (10 mL). The solvent was
evaporated. The resulting residue was dissolved in CH2Cl2
(20 mL), and intermediates (6a–6f) (1 mmol) in CH2Cl2
(10 mL) was added dropwise. The mixture was stirred for
about 3 h, monitored by TLC. After the reaction completed,
the solvent was washed with water and brine, then dried
over anhydrous magnesium sulfate. The mixture was fil-
tered, the filtrate was evaporated and purified by silica gel
chromatography (CH2Cl2/MeOH = 60/1, v/v) to obtain
target compounds.
1-(4-methoxyphenyl)-3-{4-{[4-(3-methoxypropoxy)-3-
methylpyridin-2-yl]methoxy}phenyl}urea
(7d) Yield
47.5% (white solid). m.p.: 145−146 °C. IR(KBr, cm−1): υ
3422.1, 3279.6, 2921.9, 2851.6, 1629.3, 1608.4, 1562.8,
1510.8, 1467.9, 1384.0, 1296.2, 1231.4, 1170.4, 1115.4,
1
1013.8, 826.1. H NMR (400 MHz, DMSO-d6) δ 8.38 (s,
1H), 8.38 (s, 1H), 8.26 (d, J = 5.6 Hz, 1H), 7.34 (d, J =
2.4 Hz, 1H), 7.33 (s, 1H), 7.32 (s, 1H), 7.31 (d, J = 2.2 Hz,
1H), 7.00 (d, J = 5.7 Hz, 1H), 6.95 (s, 1H), 6.93 (s, 1H),
6.86 (d, J = 2.3 Hz, 1H), 6.84 (d, J = 2.2 Hz, 1H), 5.09 (s,
2H), 4.11 (t, J = 6.2 Hz, 2H), 3.71 (s, 3H), 3.49 (t, J =
6.2 Hz, 2H), 3.25 (s, 3H), 2.18 (s, 3H), 2.02–1.95 (m, 2H).
ESI-MS (m/z): 452.3 ([M + H]+).
1-(3,5-difluorophenyl)-3-{4-{[4-(3-methoxypropoxy)-3-
methylpyridin-2-yl]methoxy}phenyl}urea
(7a) Yield
1-(4-fluorophenyl)-3-{4-{[4-(3-methoxypropoxy)-3-methyl-
pyridin-2-yl]methoxy}phenyl}urea (7e) Yield 47.9% (white
solid). m.p.: 118–119 °C. IR(KBr, cm−1): υ 3422.9, 2922.1,
2852.5, 1643.3, 1562.3, 1508.1, 1463.0, 1384.2, 1294.9,
1210.0, 1118.9, 1045.5, 1014.8, 833.3, 619.5, 521.2. H
NMR (400 MHz, DMSO-d6) δ 8.64 (s, 1H), 8.48 (s, 1H),
56.4% (white solid). m.p.: 126–127 °C. IR (KBr, cm−1): υ
3422.0, 2922.9, 1697.0, 16112, 1554.6, 1514.4, 1478.0,
1459.9, 1430.6, 1407.2, 1383.4, 1291.9, 1233.0, 1203.6,
1151.2, 1111.4, 851.8, 821.6. 1H NMR (400 MHz, DMSO-
d6) δ 9.02 (s, 1H), 8.65 (s, 1H), 8.26 (d, J = 5.6 Hz, 1H),
1

Medicinal Chemistry Research
1
8.27 (d, J = 5.6 Hz, 1H), 7.46–7.44 (m, 1H), 7.44–7.40 (m,
1H), 7.34 (d, J = 2.2 Hz, 1H), 7.32 (d, J = 2.2 Hz, 1H), 7.11
(d, J = 8.9 Hz, 1H), 7.08 (d, J = 2.2 Hz, 1H), 7.00 (d, J =
5.7 Hz, 1H), 6.96 (d, J = 2.3 Hz, 1H), 6.94 (d, J = 2.1 Hz,
1H), 5.10 (s, 2H), 4.11 (t, J = 6.2 Hz, 2H), 3.49 (t, J =
6.2 Hz, 2H), 3.25 (s, 3H), 2.18 (s, 3H), 2.02–1.95 (m, 2H).
ESI-MS (m/z): 440.2 ([M + H]+).
1103.3, 825.3, 616.4. H NMR (400 MHz, DMSO-d6) δ
8.74 (s, 1H), 8.51 (s, 1H), 8.22 (d, J = 5.5 Hz, 1H), 7.48 (d,
J = 2.2 Hz, 1H), 7.46 (d, J = 2.2 Hz, 1H), 7.35 (d, J =
2.2 Hz, 1H), 7.33 (d, J = 2.1 Hz, 1H), 7.32 (d, J = 2.0 Hz,
1H), 7.30 (d, J = 1.9 Hz, 1H), 7.15 (d, J = 5.5 Hz, 1H), 6.97
(d, J = 2.3 Hz, 1H), 6.95 (d, J = 2.2 Hz, 1H), 5.03 (s, 2H),
3.90 (s, 3H), 3.78 (s, 3H). ESI-MS (m/z): 414.2, 415.2,
416.2, 417.2 ([M + H]+).
1-[4-chloro-3-(trifluoromethyl)phenyl]-3-{4-{[4-(3-methoxy-
propoxy)-3-methylpyridin-2-yl]methoxy}phenyl}urea (7f)
Yield 37.2% (white solid). m.p.: 179–181 °C. IR(KBr, cm−1):
υ 3421.6, 2922.7, 1694.9, 1590.8, 1544.7, 1511.5, 1484.5,
1462.9, 1418.4, 1384.0, 1326.7, 1311.7, 1293.2, 1217.1,
1-{4-[(3,4-dimethoxypyridin-2-yl)methoxy]phenyl}-3-(4-
methoxyphenyl)urea (7j) Yield 38.9% (white solid). m.p.:
175–176 °C. IR(KBr, cm−1): υ 3422.8, 2922.5, 2852.4,
1599.7, 1561.1, 1509.8, 1384.2, 1299.4, 1226.4, 1110.9,
1
1
1173.4, 1129.3, 1032.4, 826.5, 662.2. H NMR (400 MHz,
1037.6, 829.2, 522.9. H NMR (400 MHz, DMSO-d6) δ
DMSO-d6) δ 9.09 (s, 1H), 8.65 (s, 1H), 8.27 (d, J = 5.6 Hz,
1H), 8.10 (d, J = 2.3 Hz, 1H), 7.64–7.61 (m, 1H), 7.59 (d,
J = 8.8 Hz, 1H), 7.36 (d, J = 2.0 Hz, 1H), 7.34 (d, J = 2.3 Hz,
1H), 7.00 (d, J = 5.7 Hz, 1H), 6.98 (d, J = 2.2 Hz, 1H), 6.96
(d, J = 2.2 Hz, 1H), 5.11 (s, 2H), 4.11 (t, J = 6.2 Hz, 2H),
3.49 (t, J = 6.2 Hz, 2H), 3.25 (s, 3H), 2.18 (s, 3H), 2.02–1.96
(m, 2H). ESI-MS (m/z): 524.3 ([M + H]+).
8.38 (s, 1H), 8.37 (s, 1H), 8.22 (d, J = 5.5 Hz, 1H), 7.35 (s,
1H), 7.34 (s, 1H), 7.32 (s, 1H), 7.31 (s, 1H), 7.15 (d, J =
5.6 Hz, 1H), 6.95 (d, J = 2.2 Hz, 1H), 6.94 (d, J = 2.2 Hz,
1H), 6.86 (d, J = 2.3 Hz, 1H), 6.84 (d, J = 2.2 Hz, 1H), 5.03
(s, 2H), 3.90 (s, 3H), 3.78 (s, 3H), 3.71 (s, 3H). ESI-MS (m/
z): 410.3 ([M + H]+); 432.2 ([M + Na]+).
1-{4-[(3,4-dimethoxypyridin-2-yl)methoxy]phenyl}-3-(4-
fluorophenyl)urea (7k) Yield 48.3% (yellow solid). m.p.:
161–162 °C. IR(KBr, cm−1): υ 3424.9, 2922.8, 2852.8,
1664.8, 1595.7, 1550.6, 1507.0, 1452.7, 1403.4, 1383.8,
1299.9, 1205.9, 1113.4, 1083.5, 1015.5, 832.6, 800.7,
518.3. 1H NMR (400 MHz, DMSO-d6) δ 8.60 (s, 1H), 8.44
(s, 1H), 8.22 (d, J = 5.5 Hz, 1H), 7.46–7.44 (m, 1H), 7.44–
7.41 (m, 1H), 7.34 (d, J = 2.2 Hz, 1H), 7.32 (d, J = 2.2 Hz,
1H), 7.14 (d, J = 5.5 Hz, 1H), 7.11 (d, J = 8.9 Hz, 1H), 7.08
(d, J = 2.1 Hz, 1H), 6.96 (d, J = 2.2 Hz, 1H), 6.95 (d, J =
2.1 Hz, 1H), 5.03 (s, 2H), 3.90 (s, 3H), 3.78 (s, 3H). ESI-
MS (m/z): 398.2, 399.2 ([M + H]+).
1-(3,5-difluorophenyl)-3-{4-[(3,4-dimethoxypyridin-2-yl)
methoxy]phenyl}urea (7g) Yield 46.1% (white solid). m.
p.: 162–164 °C. IR(KBr, cm−1): υ 3422.3, 2921.9, 2852.1,
1609.0, 1559.6, 1511.8, 1490.0, 1434.0, 1384.0, 1301.7,
1
1226.0, 1197.0, 1114.4, 1081.7, 1015.7, 832.6, 672.2. H
NMR (400 MHz, DMSO-d6) δ 9.03 (s, 1H), 8.65 (s, 1H),
8.22 (d, J = 5.5 Hz, 1H), 7.35 (d, J = 2.2 Hz, 1H), 7.33 (d,
J = 2.2 Hz, 1H), 7.18 (d, J = 2.3 Hz, 1H), 7.16 (d, J =
2.4 Hz, 1H), 7.14 (d, J = 5.5 Hz, 1H), 6.98 (d, J = 2.3 Hz,
1H), 6.96 (d, J = 2.1 Hz, 1H), 6.79–6.73 (m, 1H), 5.04 (s,
2H), 3.90 (s, 3H), 3.78 (s, 3H). ESI-MS (m/z): 416.3 ([M +
H]+); 438.3 ([M + Na]+).
1-[4-chloro-3-(trifluoromethyl)phenyl]-3-{4-[(3,4-dimethoxy-
pyridin-2-yl)methoxy]phenyl}urea (7l) Yield 40.1% (yel-
lowish solid). m.p.: 106–107 °C. IR(KBr, cm−1): υ 3447.1,
3329.3, 2922.5, 2851.4, 1694.1, 1610.3, 1559.8, 1511.6,
1486.1, 1318.1, 1256.4, 1228.6, 1170.3, 1141.1, 1111.3,
1-{4-[(3,4-dimethoxypyridin-2-yl)methoxy]phenyl}-3-[3-(tri-
fluoromethyl)phenyl]urea (7h) Yield 55.5% (white solid).
m.p.: 149–150 °C. IR(KBr, cm−1): υ 3422.1, 2922.2,
2852.1, 1709.7, 1593.6, 1549.4, 1511.4, 1492.4, 1447.2,
1383.7, 1340.2, 1301.0, 1230.8, 1201.7, 1172.2, 1110.6,
1040.4, 818.4, 795.5, 699.6, 666.6, 617.6. 1H NMR
(400 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.60 (s, 1H), 8.22 (d,
J = 5.5 Hz, 1H), 8.01 (d, J = 2.1 Hz, 1H), 7.59–7.53 (m,
1H), 7.52–7.48 (m, 1H), 7.36 (d, J = 2.3 Hz, 1H), 7.35 (d,
J = 2.1 Hz, 1H), 7.29 (d, J = 7.4 Hz, 1H), 7.15 (d, J =
5.5 Hz, 1H), 6.98 (d, J = 2.2 Hz, 1H), 6.96 (d, J = 2.1 Hz,
1H), 5.04 (s, 2H), 3.90 (s, 3H), 3.78 (s, 3H). ESI-MS (m/z):
448.3 ([M + H]+); 470.2 ([M + Na]+).
1
1040.5, 999.7, 874.3, 819.3, 745.2, 686.2, 658.9. H NMR
(400 MHz, DMSO-d6) δ 9.08 (s, 1H), 8.63 (s, 1H), 8.22 (d,
J = 5.5 Hz, 1H), 8.09 (d, J = 2.4 Hz, 1H), 7.64–7.61 (m,
1H), 7.59 (d, J = 8.8 Hz, 1H), 7.36 (d, J = 2.1 Hz, 1H), 7.34
(d, J = 2.4 Hz, 1H), 7.14 (d, J = 5.6 Hz, 1H), 6.98 (d, J =
2.0 Hz, 1H), 6.96 (d, J = 2.4 Hz, 1H), 5.04 (s, 2H), 3.90 (s,
3H), 3.78 (s, 3H). ESI-MS (m/z): 482.1, 484.1 ([M + H]+).
1-(3,5-difluorophenyl)-3-{4-[(4-methoxy-3,5-dimethylpyri-
din-2-yl)methoxy]phenyl}urea (7m) Yield 46.6% (white
solid). m.p.: 195–196 °C. IR(KBr, cm−1): υ 3421.1, 3298.0,
2922.4, 2852.2, 1642.2, 1606.0, 1563.1, 1510.0, 1477.3,
1444.9, 1383.8, 1292.2, 1234.4, 1202.5, 1159.0, 1118.3,
1102.0, 1002.1, 822.0, 671.3. 1H NMR (400 MHz,
1-(4-chlorophenyl)-3-{4-[(3,4-dimethoxypyridin-2-yl)meth-
oxy]phenyl}urea (7i) Yield 42.1% (yellow solid). m.p.:
197–198 °C. IR(KBr, cm−1): υ 3422.2, 2921.8, 2852.4,
1636.1, 1563.1, 1509.5, 1492.6, 1462.8, 1399.6, 1384.3,

Medicinal Chemistry Research
DMSO-d6) δ 9.03 (s, 1H), 8.66 (s, 1H), 8.21 (s, 1H), 7.34
(d, J = 2.1 Hz, 1H), 7.33 (d, J = 2.3 Hz, 1H), 7.18 (d, J =
2.3 Hz, 1H), 7.16 (d, J = 2.4 Hz, 1H), 6.99 (d, J = 2.1 Hz,
1H), 6.97 (d, J = 2.3 Hz, 1H), 6.79–6.73 (m, 1H), 5.09 (s,
2H), 3.74 (s, 3H), 2.25 (s, 3H), 2.22 (s, 3H). ESI-MS (m/z):
414.4 ([M + H]+).
(d, J = 2.3 Hz, 1H), 7.32 (d, J = 2.1 Hz, 1H), 7.12 (d, J =
5.5 Hz, 1H), 7.09 (d, J = 8.9 Hz, 1H), 6.97 (d, J = 2.3 Hz,
1H), 6.95 (d, J = 2.1 Hz, 1H), 5.08 (s, 2H), 3.74 (s, 3H),
2.25 (s, 3H), 2.22 (s, 3H). ESI-MS (m/z): 396.3 ([M + H]+);
418.2 ([M + Na]+).
1-[4-chloro-3-(trifluoromethyl)phenyl]-3-{4-[(4-methoxy-
3,5-dimethylpyridin-2-yl)methoxy]phenyl}urea (7r) Yield
30.1% (white solid). m.p.: 151–152 °C. IR(KBr, cm−1): υ
3423.6, 2292.3, 2852.1, 1608.9, 1551.8, 1511.7, 1480.6,
1417.3, 1383.9, 1323.7, 1258.1, 1220.0, 1171.8, 1131.9,
1-{4-[(4-methoxy-3,5-dimethylpyridin-2-yl)methoxy]phe-
nyl}-3-[3-(trifluoromethyl)phenyl]urea (7n) Yield 41.5%
(yellowish solid). m.p.: 158–159 °C. IR(KBr, cm−1): υ
3423.4, 2922.0, 2852.6, 1637.8, 1562.9, 1511.3, 1446.9,
1384.2, 1340.0, 1222.3, 1169.9, 1124.2, 1071.2, 699.6,
617.5. 1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.62
(s, 1H), 8.22 (s, 1H), 8.01 (d, J = 2.0 Hz, 1H), 7.56 (d, J =
8.5 Hz, 1H), 7.50 (t, J = 7.9 Hz, 1H), 7.36 (d, J = 2.2 Hz,
1H), 7.34 (d, J = 2.2 Hz, 1H), 7.29 (d, J = 7.6 Hz, 1H), 6.99
(d, J = 2.3 Hz, 1H), 6.97 (d, J = 2.1 Hz, 1H), 5.09 (s, 2H),
3.74 (s, 3H), 2.26 (s, 3H), 2.22 (s, 3H). ESI-MS (m/z):446.3
([M + H]+); 468.3 ([M + Na]+).
1
1112.3, 1032.8, 1004.9, 823.1, 660.0. H NMR (400 MHz,
DMSO-d6) δ 9.10 (s, 1H), 8.65 (s, 1H), 8.22 (s, 1H), 8.10
(d, J = 2.3 Hz, 1H), 7.64–7.61 (m, 1H), 7.59 (d, J = 8.8 Hz,
1H), 7.36 (d, J = 2.0 Hz, 1H), 7.34 (d, J = 2.2 Hz, 1H), 6.99
(d, J = 2.0 Hz, 1H), 6.97 (d, J = 2.2 Hz, 1H), 5.10 (s, 2H),
3.74 (s, 3H), 2.26 (s, 3H), 2.22 (s, 3H). ESI-MS (m/z): 480.2
([M + H]+).
1-(3,5-difluorophenyl)-3-{4-{[3-methyl-4-(2,2,2-trifluor-
1-(4-chlorophenyl)-3-{4-[(4-methoxy-3,5-dimethylpyridin-2-
yl)methoxy]phenyl}urea (7o) Yield 48.3% (yellowish
solid). m.p.: 202–204 °C. IR(KBr, cm−1): υ 3373.3, 2922.9,
2851.7, 1716.0, 1592.8, 1546.9, 1510.8, 1490.2, 1458.3,
1376.4, 1301.3, 1230.0, 1192.1, 1043.5, 999.8, 828.1,
803.9, 681.3, 507.5. 1H NMR (400 MHz, DMSO-d6) δ 8.76
(s, 1H), 8.53 (s, 1H), 8.22 (s, 1H), 7.48 (d, J = 2.1 Hz, 1H),
7.46 (d, J = 2.2 Hz, 1H), 7.34 (d, J = 2.2 Hz, 1H), 7.33 (d,
J = 2.1 Hz, 1H), 7.32 (d, J = 2.2 Hz, 1H), 7.30 (d, J =
2.1 Hz, 1H), 6.98 (d, J = 2.2 Hz, 1H), 6.96 (d, J = 2.2 Hz,
1H), 5.09 (s, 2H), 3.74 (s, 3H), 2.25 (s, 3H), 2.22 (s, 3H).
ESI-MS (m/z): 412.2, 413.2, 414.2, 415.2 ([M + H]+).
oethoxy)pyridin-2-yl]methoxy}phenyl}urea
(7s) Yield
45.2% (white solid). m.p.: 185–187 °C. IR(KBr, cm−1): υ
3423.3, 2922.2, 2852.5, 2651.0, 1607.4, 1560.6, 1511.2,
1478.5, 1384.3, 1309.9, 1258.1, 1170.2, 1135.0, 1115.9,
1
1006.6, 834.2, 669.3, 620.0, 580.5. H NMR (400 MHz,
DMSO-d6) δ 9.07 (s, 1H), 8.69 (s, 1H), 8.35 (d, J = 5.7 Hz,
1H), 7.34 (d, J = 2.1 Hz, 1H), 7.33 (d, J = 2.3 Hz, 1H), 7.18
(d, J = 2.3 Hz, 1H), 7.16 (d, J = 2.4 Hz, 1H), 7.14 (d, J =
5.7 Hz, 1H), 6.98 (d, J = 2.2 Hz, 1H), 6.96 (d, J = 2.2 Hz,
1H), 6.78–6.73 (m, 1H), 5.14 (s, 2H), 4.91 (q, J = 8.7 Hz,
2H), 2.21 (s, 3H). ESI-MS (m/z): 468.3 ([M + H]+); 490.2
([M + Na]+).
1-{4-[(4-methoxy-3,5-dimethylpyridin-2-yl)methoxy]phe-
nyl}-3-(4-methoxyphenyl)urea (7p) Yield 37.4% (white
solid). m.p.: 181–182 °C. IR(KBr, cm−1): υ 3420.6, 3286.5,
2923.1, 2852.3, 1632.9, 1597.2, 1557.6, 1503.6, 1464.7,
1383.8, 1297.2, 1259.7, 1219.2, 1170.2, 1106.5, 1032.1,
1-{4-{[3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]meth-
oxy}phenyl}-3-[3-(trifluoromethyl)phenyl]urea (7t) Yield
61.4% (white solid). m.p.: 162–163 °C. IR(KBr, cm−1): υ
3443.4, 2922.3, 2852.2, 1691.1, 1592.4, 1550.8, 1534.8,
1488.0, 1397.6, 1330.3, 1315.5, 1287.7, 1269.3, 1214.3,
1172.5, 1144.5, 1115.8, 1090.4, 1075.0, 1031.6, 995.3,
916.5, 878.9, 814.8, 747.7, 663.3, 618.1. 1H NMR
(400 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.61 (s, 1H), 8.35 (d,
J = 5.7 Hz, 1H), 8.00 (s, 1H), 7.55 (d, J = 8.1 Hz, 1H),
7.51–7.48 (m, 1H), 7.36 (s, 1H), 7.34 (d, J = 2.1 Hz, 1H),
7.29 (d, J = 7.6 Hz, 1H), 7.14 (d, J = 5.7 Hz, 1H), 6.99 (d,
J = 3.6 Hz, 1H), 6.96 (d, J = 2.0 Hz, 1H), 5.15 (s, 2H), 4.92
(q, J = 8.8 Hz, 2H), 2.21 (s, 3H). ESI-MS (m/z): 500.2 ([M
+ H]+); 522.1 ([M + Na]+).
1
1003.1, 826.5, 679.8, 560.9. H NMR (400 MHz, DMSO-
d6) δ 8.38 (s, 1H), 8.38 (s, 1H), 8.22 (s, 1H), 7.34 (s, 1H),
7.33 (s, 1H), 7.32 (s, 1H), 7.31 (s, 1H), 6.96 (d, J = 2.2 Hz,
1H), 6.94 (d, J = 2.2 Hz, 1H), 6.86 (d, J = 2.3 Hz, 1H), 6.84
(d, J = 2.1 Hz, 1H), 5.08 (s, 2H), 3.74 (s, 3H), 3.71 (s, 3H),
2.25 (s, 3H), 2.22 (s, 3H). ESI-MS (m/z): 408.3 ([M + H]+);
430.2 ([M + Na]+).
1-(4-fluorophenyl)-3-{4-[(4-methoxy-3,5-dimethylpyridin-2-
yl)methoxy]phenyl}urea (7q) Yield 46.4% (white solid).
m.p.: 195–196 °C. IR(KBr, cm−1): υ 3425.2, 2922.9,
2852.8, 1638.2, 1607.4, 1561.6, 1508.6, 1042.4, 1384.0,
1-(4-chlorophenyl)-3-{4-{[3-methyl-4-(2,2,2-trifluoroethoxy)
pyridin-2-yl]methoxy}phenyl}urea
(7u) Yield
23.0%
1
1293.6, 1262.1, 1215.7, 1106.0, 1004.0, 830.1, 792.1. H
(white solid). m.p.: 143–144 °C. IR(KBr, cm−1): υ 4324.2,
2922.0, 2852.7, 1596.1, 1548.9, 1509.9, 1493.0, 1399.6,
1384.1, 1308.7, 1260.4, 1211.0, 1170.2, 1133.5, 828.3,
NMR (400 MHz, DMSO-d6) δ 8.62 (s, 1H), 8.47 (s, 1H),
8.22 (s, 1H), 7.46–7.44 (m, 1H), 7.44–7.41 (m, 1H), 7.34

Medicinal Chemistry Research
578.0. 1H NMR (400 MHz, DMSO-d6) δ 8.74 (s, 1H), 8.51
(s, 1H), 8.35 (d, J = 5.6 Hz, 1H), 7.47 (d, J = 2.1 Hz, 1H),
7.46 (d, J = 2.3 Hz, 1H), 7.34 (d, J = 2.2 Hz, 1H), 7.32 (s,
1H), 7.31 (s, 1H), 7.30 (d, J = 2.2 Hz, 1H), 7.14 (d, J =
5.7 Hz, 1H), 6.97 (d, J = 2.1 Hz, 1H), 6.95 (d, J = 2.3 Hz,
1H), 5.14 (s, 2H), 4.92 (q, J = 8.7 Hz, 2H), 2.21 (s, 3H).
ESI-MS (m/z): 466.2, 467.2, 468.1, 469.2 ([M + H]+).
after eliminating the inhibitor (5-{{(1R,1aS,6bR)-1-[5-(tri-
fluoromethyl)-1H-benzimidazol-2-yl]-1a,6b-dihydro-1H-
cyclopropa[b][1]benzofuran-5-yl}oxy}-3,4-dihydro-1,8-
naphthyridin-2(1H)-one). The missing residues were built
and the polar hydrogen atoms in amino-acid residues were
added. Molecular docking was carried out using MVD. The
docking was set at 1500 maximum iterations, with a sim-
plex evolution population size of 50 and a minimum of 10
runs. The schematic diagrams of interactions between 4R5Y
and the small molecules were analyzed by PyMOL 1.7.
1-(4-methoxyphenyl)-3-{4-{[3-methyl-4-(2,2,2-trifluor-
oethoxy)pyridin-2-yl]methoxy}phenyl}urea
(7v) Yield
19.9% (yellow solid). m.p.: 174–175 °C. IR(KBr, cm−1): υ
3424.7, 2921.8, 2852.5, 1627.5, 1559.2, 1509.3, 1462.0,
1384.2, 1293.8, 1261.3, 1208.2, 1168.8, 1108.7, 828.7,
619.2. 1H NMR (400 MHz, DMSO-d6) δ 8.38 (s, 1H), 8.38
(s, 1H), 8.35 (d, J = 5.6 Hz, 1H), 7.34 (d, J = 2.1 Hz, 1H),
7.33 (d, J = 1.9 Hz, 1H), 7.32 (s, 1H), 7.31 (d, J = 2.1 Hz,
1H), 7.14 (d, J = 5.7 Hz, 1H), 6.95 (d, J = 2.2 Hz, 1H), 6.94
(d, J = 2.2 Hz, 1H), 6.86 (d, J = 2.2 Hz, 1H), 6.84 (d, J =
2.3 Hz, 1H), 5.13 (s, 2H), 4.92 (q, J = 8.7 Hz, 2H), 3.71 (s,
3H), 2.21 (s, 3H). ESI-MS (m/z): 462.2, 463.2 ([M + H]+);
484.2, 485.2 ([M + Na]+).
Bioactivity in cell line
The antiproliferative activities of target compounds were
determined using a standard MTT assay (Mosmann
1983; Zhang et al. 2017). Exponentially growing cells
A549, HCT-116, PC-3, and HL7702 were seeded into
96-well plates and incubated for 24 h to allow the cells to
attach. After 24 h of incubation, the culture medium was
removed and fresh medium containing various con-
centrations of the candidate compounds was added to
each well. The cells were then incubated for 72 h,
thereafter MTT assays were performed and cell viability
was assessed at 570 nm by a microplate reader (Ther-
moFisher Scientific (Shanghai) Instrument Co., Ltd,
Shanghai, China). Inhibitory effects were determined as
IC50 value.
1-(4-fluorophenyl)-3-{4-{[3-methyl-4-(2,2,2-trifluoroethoxy)
pyridin-2-yl]methoxy}phenyl}urea (7w) Yield 41.4% (yel-
lowish solid). m.p.: 173–175 °C. IR(KBr, cm−1): υ 3425.8,
2922.0, 2852.4, 1630.2, 1561.5, 1508.3, 1402.9, 1384.3,
1261.4, 1206.8, 1170.0, 1108.8, 831.4. 1H NMR (400 MHz,
DMSO-d6) δ 8.64 (s, 1H), 8.48 (s, 1H), 8.35 (d, J = 5.7 Hz,
1H), 7.45 (d, J = 5.0 Hz, 1H), 7.43 (d, J = 5.0 Hz, 1H), 7.34
(d, J = 2.2 Hz, 1H), 7.32 (d, J = 2.2 Hz, 1H), 7.14 (d, J =
5.7Hz, 1H), 7.11 (d, J = 8.8 Hz, 1H), 7.08 (d, J = 2.2 Hz,
1H), 6.96 (d, J = 2.2 Hz, 1H), 6.95 (d, J = 2.2 Hz, 1H), 5.14
(s, 2H), 4.92 (q, J = 8.7 Hz, 2H), 2.21 (s, 3H). ESI-MS
(m/z): 450.2 ([M + H]+).
Acknowledgements This work was supported by the National Science
Foundation of China (NSFC) for the grant No. 21342006 and the
Program for Innovative Research Team of the Ministry of Education of
China for the grant No. IRT_14R36. The authors would like to thank
Molegro ApS for kindly providing a free evaluation copy of their
software package and Schrödinger, LLC for allowing the use of
PyMOL Molecular Graphics System.
Compliance with ethical standards
1-[4-chloro-3-(trifluoromethyl)phenyl]-3-{4-{[3-methyl-4-
(2,2,2-trifluoroethoxy)pyridin-2-yl]methoxy}phenyl}urea
(7x) Yield 21.1% (yellow solid). m.p.: 122–123 °C. IR
(KBr, cm−1): υ 3383.8, 2922.6, 2852.7, 1705.5, 1658.6,
1592.0, 1551.4, 1510.9, 1482.0, 1456.7, 1419.2,1383.3,
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with animals
performed by any of the authors.
1
1259.1, 1172.3, 1133.1, 1032.3, 974.7, 828.2, 664.9. H
NMR (400 MHz, DMSO-d6) δ 9.08 (s, 1H), 8.63 (s, 1H),
8.22 (d, J = 5.5 Hz, 1H), 8.09 (d, J = 2.4 Hz, 1H),
7.64–7.61 (m, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.36 (d, J =
2.1 Hz, 1H), 7.34 (d, J = 2.4 Hz, 1H), 7.14 (d, J = 5.6 Hz,
1H), 6.98 (d, J = 2.0 Hz, 1H), 6.96 (d, J = 2.4 Hz, 1H), 5.04
(s, 2H), 3.90 (s, 3H), 3.78 (s, 3H). ESI-MS (m/z): 534.2
([M + H]+).
Informed consent Informed consent was obtained from all individual
participants included in the study.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
References
Anderson JW, Sarantakis D, Terpinski J, Kumar TR, Tsai HC, Kuo M,
Ager AL, Jacobs Jr WR, Schiehser GA, Ekins S, Sacchettini JC,
Jacobus DP, Fidock DA, Freundlich JS (2013) Novel diaryl ureas
with efficacy in a mouse model of malaria. Bioorg Med Chem
Lett 23:1022–1025
Molecular docking
The complex for the enzyme was deposited in the RCSB
PDB for the BRAF (PDB code: 4R5Y) (Tang et al. 2015)

Medicinal Chemistry Research
Barras D (2015) BRAF mutation in colorectal cancer: an update. Bio
Cancer 7:9–12
Cheng L, Lopez-Beltran A, Massari F, MacLennan GT, Montironi R
(2018) Molecular testing for BRAF mutations to inform mela-
noma treatment decisions: a move toward precision medicine.
Mod Pathol 31:24–38
Qiao JX, Wang TC, Ruel R, Thibeault C, L’Heureux A, Schumacher
WA, Spronk SA, Hiebert S, Bouthillier G, Lloyd J, Pi Z, Schnur
DM, Abell LM, Hua J, Price LA, Liu E, Wu Q, Steinbacher TE,
Bostwick JS, Chang M, Zheng J, Gao Q, Ma B, McDonnell PA,
Huang CS, Rehfuss R, Wexler RR, Lam PY (2013) Con-
formationally constrained ortho-anilino diaryl ureas: discovery of
1-(2-(1’-neopentylspiro[indoline-3,4’-piperidine]-1-yl)phenyl)-3-
(4-(trifluoromethoxy)phenyl)urea, a potent, selective, and bioa-
vailable. P2Y1 J Med Chem 56:9275–9295
Regad T (2015) Targeting RTK signaling pathways in cancer. Cancers
7:1758–1784
Schreck R, Rapp UR (2006) Raf kinases: oncogenesis and drug dis-
covery. Int J Cancer 119:2261–2271
Seto M, Miyamoto N, AikawaK, Aramaki Y, Kanzaki N, Iizawa Y,
Baba M, Shiraishia M (2005) Orallyactive CCR5 antagonists as
anti-HIV-1 agents. Part 3: synthesis and biological activities of 1-
benzazepine derivatives containing a sulfoxide moiety. Bioorg
Med Chem 13:363–386
Siegel R, Naishadham D, Jemal A (2013) Cancer statistics, 2013. CA
Cancer J Clin 63:11–30
Sun Y, Liu WZ, Liu T, Feng X, Yang N, Zhou HF (2015) Signaling
pathway of MAPK/ERK in cell proliferation, differentiation,
migration, senescence and apoptosis. J Recept Signal Transduct
Res 35:600–604
Tang Z, Yuan X, Du R, Cheung SH, Zhang G, Wei J, Zhao Y, Feng Y,
Peng H, Zhang Y, Du Y, Hu X, Gong W, Liu Y, Gao Y, Liu Y,
Hao R, Li S, Wang S, Ji J, Zhang L, Li S, Sutton D, Wei M, Zhou
C, Wang L, Luo L (2015) BGB-283, a novel RAF kinase and
EGFR Inhibitor, displays potent antitumor activity in BRAF-
mutated colorectal cancers. Mol Cancer Ther 14:2187–2197
Thomsen R, Christensen MH (2006) MolDock: a new technique for
high-accuracy molecular docking. J Med Chem 49:3315–3321
Viegas-Junior C, Danuello A, da Silva Bolzani V, Barreiro EJ, Fraga
CA (2007) Molecular hybridization: a useful tool in the design of
new drug prototypes. Curr Med Chem 14:1829–1852
Vultur A, Villanueva J, Herlyn M (2011) Targeting BRAF in
advanced melanoma: a first step toward manageable disease. Clin
Cancer Res 17:1658–1663
Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM,
Project CG, Jones CM, Marshall CJ, Springer CJ, Barford D,
Marais R (2004) Mechanism of activation of the RAF-ERK
signaling pathway by oncogenic mutations of B-RAF. Cell
116:855–867
Wilhelm SM, Carter C, Tang L, Wilkie D, McNabola A, Rong H,
Chen C, Zhang X, Vincent P, McHugh M, Cao Y, Shujath J,
Gawlak S, Eveleigh D, Rowley B, Liu L, Adnane L, Lynch M,
Auclair D, Taylor I, Gedrich R, Voznesensky A, Riedl B, Post
LE, Bollag G, Trail PA (2004) BAY 43-9006 exhibits broad
spectrum oral antitum or activity and targets the RAF/MEK/ERK
pathway and receptor tyrosine kinases involved in tumor pro-
gression and angiogenesis. Cancer Res 64:7099–7109
Xuan W, Ding W, Hui HX, Zhang SQ (2013) Synthesis and cytotoxic
activity of diaryl urea derivatives with a 4-methylpiperazinylcarbonyl
moiety. Med Chem Res 22:3857–3862
Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague
J, Woffendin H, Garnett MJ, Bottomley W, Davis N, Dicks E,
Ewing R, Floyd Y, Gray K, Hall S, Hawes R, Hughes J, Kos-
midou V, Menzies A, Mould C, Parker A, Stevens C, Watt S,
Hooper S, Wilson R, Jayatilake H, Gusterson BA, Cooper C,
Shipley J, Hargrave D, Pritchard-Jones K, Maitland N, Chenevix-
Trench G, Riggins GJ, Bigner DD, Palmieri G, Cossu A, Fla-
nagan A, Nicholson A, Ho JW, Leung SY, Yuen ST, Weber BL,
Seigler HF, Darrow TL, Paterson H, Marais R, Marshall CJ,
Wooster R, Stratton MR, Futreal PA (2002) Mutations of the
BRAF gene in human cancer. Nature 417:949–954
Keche AP, Hatnapure GD, Tale RH, Rodge AH, Birajdar SS, Kamble
VM (2012) A novel pyrimidine derivatives with aryl urea,
thiourea and sulfonamide moieties, synthesis, anti-inflammatory
and antimicrobial evaluation. Bioorg Med Chem Lett
22:3445–3448
Keefe DM, Bateman EH (2011) Tumor control versus adverse events
with targeted anticancer therapies. Nat Rev Clin Oncol 9:98–109
Kim HJ, Cho HJ, Kim H, El-Gamal MI, Oh CH, Lee SH, Sim T, Hah
JM, Yoo KH (2012) New diarylureas and diarylamides posses-
sing acet(benz)amidophenyl scaffold, design, synthesis, and
antiproliferative activity against melanoma cell line. Bioorg Med
Chem Lett 22:3269–3273
Kulkarni RG, Laufer S, Mangannavar C, Garlapati A (2013) Design,
synthesis and characterization of N’,N”-diaryl ureas as p38 kinase
inhibitors. Med Chem 9:213–221
Lee Jr JT, Steelman LS, McCubrey JA (2005) Modulation of Raf/MEK/
ERK kinase activity does not affect the chemoresistance profile of
advanced prostate cancer cells. Int J Oncol 26:1637–1644
Liu DZ, Tian Z, Yan ZH, WuL X, Ma Y, Wang Q, Liu W, Zhou HG,
Yang C (2013) Design, synthesis and evaluation of 1,2-benzi-
sothiazol-3-one derivatives as potent caspase-3 inhibitors. Bioorg
Med Chem 21:2960–2967
Lu C, Tang K, Li Y, Li P, Lin Z, Yin D, Chen X, Huang H (2014)
Design, synthesis and evaluation of novel diaryl urea derivative
as potential antitumor agents. Eur J Med Chem 77:351–360
Lugini L, Federici C, Borghi M, Azzarito T, Marino ML, Cesolini A,
Spugnini EP, Fais S (2016) Proton pump inhibitors while
belonging to the same family of generic drugs show different
anti-tumor effect. J Enzyme Inhib Med Chem 31:538–545
Moreau S, Saiag P, Aegerter P, Bosset D, Longvert C, Helias-
Rodzewicz Z, Marin C, Peschaud F, Chagnon S, Zimmermann U,
Clerici T, Emile J (2012) Prognostic value of BRAF V600
mutations in melanoma patients after resection of metastatic
lymph nodes. Ann Surg Oncol 19:4314–4321
Mosmann T (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays. J
Immunol Methods 65:55–63
Nguyen T, Yang TM, Go ML (2014) Functionalized acridin-9-yl
phenylamines protected neuronal HT22 cellsfrom glutamate-
induced cell death by reducing intracellular levels of free radical
species. Bioorg Med Chem Lett 24:1830–1838
Zhang L, Deng XS, Zhang C, Meng GP, Wu JF, Li XS, Zhao QC, Hu
C (2017) Design, synthesis and cytotoxic evaluation of a novel
series of benzo[d]thiazole-2-carboxamide derivatives as potential
EGFR inhibitors. Med Chem Res 26:2180–2189