Integrated bioinformatics, computational and experimental methods to discover novel Raf/extracellular-signal regulated kinase (ERK) dual inhibitors against breast cancer cells

Article abstract of DOI:10.1016/j.ejmech.2016.11.009

Beginning with our previously reported ERK inhibitor BL-EI001, we found Raf1 to be an important regulator in the ERK interactive network, and then we designed and synthesized a novel series of Raf1/ERK dual inhibitors against human breast cancers through integrative computational, synthetic and biological screening methods. Moreover, we found that compound 9d suppressed the proliferation of breast cancer cell lines and induced cellular apoptosis via a mitochondrial pathway with only partial dependence on Raf1 and ERK. Our results suggest that an integrative method including in silico design, chemical synthesis, biological screening and bioinformatics analysis could be an attractive strategy for the discovery of multi-target inhibitors against breast cancer.

Full text of DOI:10.1016/j.ejmech.2016.11.009

European Journal of Medicinal Chemistry xxx (2016) 1e15
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Integrated bioinformatics, computational and experimental methods
to discover novel Raf/extracellular-signal regulated kinase (ERK) dual
inhibitors against breast cancer cells
,
,
,
b
,
, *
Yin Chen a 1, Yaxin Zheng a 1, Qinglin Jiang a 1, Feifei Qin a, Yonghui Zhang c, Leilei Fu a
,
,
**
Gu He a
a State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, West China Medical School, Sichuan University,
Chengdu 610041, China
b School of Pharmacy and Sichuan Province College Key Laboratory of Structure-Specific Small Molecule Drugs, Chengdu Medical College, Chengdu 610500,
China
c Collaborative Innovation Center for Biotherapy, School of Medicine, Tsinghua University, Beijing 100084, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Beginning with our previously reported ERK inhibitor BL-EI001, we found Raf1 to be an important
regulator in the ERK interactive network, and then we designed and synthesized a novel series of Raf1/
ERK dual inhibitors against human breast cancers through integrative computational, synthetic and
biological screening methods. Moreover, we found that compound 9d suppressed the proliferation of
breast cancer cell lines and induced cellular apoptosis via a mitochondrial pathway with only partial
dependence on Raf1 and ERK. Our results suggest that an integrative method including in silico design,
chemical synthesis, biological screening and bioinformatics analysis could be an attractive strategy for
the discovery of multi-target inhibitors against breast cancer.
Received 30 June 2016
Received in revised form
4 November 2016
Accepted 5 November 2016
Available online xxx
Keywords:
Breast cancer
Raf
© 2016 Elsevier Masson SAS. All rights reserved.
ERK
Bioinformatics
Kinase inhibitor
1. Introduction
cascades are related to the regulation of normal cell proliferation,
survival and differentiation, while the inhibitor of the extracellular
According to 2015 cancer statistics, breast cancer is expected to
account for approximately 29% of all new cancer cases among
women and is the second most common cancer worldwide after
lung cancer [1]. Breast cancers have become one of the most
challenging solid tumors to diagnose and treat due to their het-
erogeneity. Breast cancers occur with a variety of intra-tumor and
inter-tumor genetic and epigenetic alterations, so the detection and
validation of biomarkers will greatly assist targeted therapy for
breast cancer [2]. A number of studies suggest that the Raf/MEK/
ERK signaling pathway is hyperactivated in tumors, and targeting
the Raf/MEK/ERK pathway plays an important role in the treatment
of breast cancer [3e5]. Mitogen-activated protein kinase (MAPK)
signal-regulated kinase (ERK) MAPK pathway has been used for the
treatment of cancer [6e9]. Recently, the use of Raf or MEK in-
hibitors targeting ERK signaling has shown promising clinical ac-
tivity in breast cancer treatment [4,10,11]. Therefore, intrinsic and
acquired targeting resistance to the Raf/MEK/ERK signaling
pathway would be an innovative approach for breast cancer
therapy.
In our previous studies, we have designed and synthesized a
novel ERK inhibitor, named BL-EI001, that could induce ERK-
dependent mitochondrial apoptosis against breast cancer both
in vitro and in vivo [12]. Further studies suggest that apoptosis
induced by BL-EI001 is independent of the Ras/Raf/MEK pathway in
breast cancer cells. These results indicate the complexity of the
kinase signaling network. The proteomics analysis of BL-EI001-
treated breast cancer cells indicate some potential ERK interactive
proteins in breast cancer cells, such as HMGB1, BIRC6 and ATFM2,
among others. Many BL-EI001-regulated ERK interactive proteins
interact directly or indirectly with another oncogene family, the Raf
* Corresponding author.
** Corresponding author.
E-mail addresses: leilei_fu@163.com (L. Fu), hegu@scu.edu.cn (G. He).
These authors contributed equally.
1
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Please cite this article in press as: Y. Chen, et al., Integrated bioinformatics, computational and experimental methods to discover novel Raf/
extracellular-signal regulated kinase (ERK) dual inhibitors against breast cancer cells, European Journal of Medicinal Chemistry (2016),
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Y. Chen et al. / European Journal of Medicinal Chemistry xxx (2016) 1e15
protein family, which is not affected by BL-EI001. In the past few
years, some researchers have reported that the inhibition of Raf
resulted in potent suppression of different kinds of breast cancers
[13e18], so we speculate that the simultaneous inhibition of Raf
and ERK might be a feasible therapeutic approach for breast cancer.
To our knowledge, several small molecular Raf inhibitors have
been approved by the FDA for cancer therapy, e.g., sorafinib [19,20],
vemurafenib [21] and dabrafenib [22], among others. Several B-Raf
inhibitors are still in the clinical research stage, such as encorafenib
[23], CEP-32496 [24] and RO5126766 [25], among others (Fig. 1A).
Their lesser development status means that only a few ERK in-
hibitors have been registered for clinical trials. The first small
molecule ERK inhibitor developed was FR180204 [26], with only
modest inhibitory activity. Afterwards, several potent ERK in-
hibitors, such as VTX-11e [27], SCH772984 [28] and ulixertinib [29],
were discovered to inhibit ERK with IC50 values at the nanomolar
level (Fig. 1B). Most recently, Peng et al. reported the design and
synthesis of a novel pan-Raf inhibitor, LY3009120, which bound to
both the activated monomer or homodimer of B-Raf and the Ras-
dependent heterodimer of B-Raf and Raf1 [30]. Peng et al. sug-
gested that the unique binding modes of the potent Raf1 inhibitor
LY3009120 to the RAF kinase domain were responsible for its
effectiveness against both B-Raf and Ras mutant cancer cells. It is
apparent that a Raf/MEK/ERK pathway inhibitor could be more
effective than the current monomeric and dimeric B-RAF inhibitors
[31,32]. Therefore, the discovery of novel Raf/ERK dual inhibitors
and the validation of their detailed molecular mechanisms are
feasible research avenues in breast cancer therapy.
Although several compounds have been reported to possess
non-specific multiple inhibitory activities against both Raf and ERK,
to our knowledge, there have been few reports of successful Raf1/
ERK dual inhibitors for breast cancer therapy. In the current study,
we designed and synthesized a series of 4-pyrimidinyl-N-arylureas
as novel Raf1/ERK dual inhibitors. These compounds potently
suppressed the kinase activities of both Raf1 and ERK (Fig. 1C). The
IC50 values of the most potent compound, 9d, were at the sub-
micromolar level. Herein, we utilized integrated proteomics,
computational and experimental methods to discover novel Raf1/
ERK dual inhibitors as potential breast cancer therapeutics via
inducing apoptosis. The new leads show no apparent toxicity and
are suitable for further development for breast cancer therapy.
2. Results and discussion
2.1. Raf is an important regulator in the ERK interactome
Based on the online protein-protein interaction (PPI) databases,
we computationally constructed a global human PPI network
covering almost all PPIs. We derived physical proteineprotein in-
teractions from manually created PPI databases to construct the set
Fig. 1. Chemical structures of selected Raf inhibitors (A), ERK inhibitors (B) and the Ras-dependent or Ras independent mechanism of Raf-ERK pathways (C).
Please cite this article in press as: Y. Chen, et al., Integrated bioinformatics, computational and experimental methods to discover novel Raf/
extracellular-signal regulated kinase (ERK) dual inhibitors against breast cancer cells, European Journal of Medicinal Chemistry (2016),
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Y. Chen et al. / European Journal of Medicinal Chemistry xxx (2016) 1e15
3
of true-positive gene pairs, including 14,892 from Homo MINT
among 6240 proteins [33]; 39,044 from HPRD among 9619 proteins
[34]; 37,710 from BioGRID among 8982 proteins [35]; 8044 from
BOND among 4073 proteins [36]; and 34,935 from IntAct among
8849 proteins [37]. Subsequently, we modified this network into a
RAF/MEK/ERK sub-network including 812 nodes and 929 edges.
Moreover, we constructed a proteomics-based RAF/ERK-modulated
PPI network based on BL-EI001-treated MCF-7 cells, including 95
differentially expressed proteins and 132 interactions. Notably, we
found that 18 ERK1 interactors were regulated by RAF1, and 38
RAF1 interactors were regulated by ERK1 (Fig. 2). Thus, these re-
sults showed that RAF/MEK/ERK-independent pathways in breast
cancer may regulate the process of apoptosis.
superposed structures of the two proteins (Fig. S2), the four addi-
tional loops were not adjacent to the inhibitor binding pocket.
Moreover, the binding modes of Raf1 and ERK1 with their in-
hibitors were similar (Figs. S3A and B). The main difference was
that the pocket of Raf1 was more open and formed a cramped
groove for tight binding of the inhibitor, whereas the pocket of
ERK1 was constricted and only allowed smaller inhibitors.
2.3. Design and synthesis of novel Raf1/ERK dual inhibitors
To identify novel Raf1/ERK dual inhibitors for overcoming the
intrinsic or acquired resistance of breast cancer cells against the
current B-Raf inhibitors, we blended the structural features of
several pan-Raf inhibitors and ERK inhibitors based on their co-
crystallographic binding modes (Scheme 1). Compound 7a was
first discovered to inhibit both Raf1 and ERK with IC50 values at the
micromolar level (Table 1).
The synthesis route of compounds 7a-l, 8a-h, 9a-g and 10a-c is
shown in Scheme 2. In general, commercially available compound 1
was coupled with 4,6-dichloropyrimidine under base catalysis to
produce compound 2. Then, the nitrogen group of compound 2 was
reduced to an amino group by iron powder to produce compound 3.
2.2. Raf1 and ERK1 are highly homologous in the kinase domain
The sequence alignment of Raf1 and ERK1 is shown in Fig. S1.
The relatively low sequence identity (approximately 20% identity)
and sequence similarity (approximately 48% similarity) in the two
proteins were due to the four additional loops in Raf1. The sequence
identity between Raf1 and ERK1 increased from 20% to 46% if
the additional loops in Raf1 were excluded. According to the
Fig. 2. The interactors between Raf and ERK.
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extracellular-signal regulated kinase (ERK) dual inhibitors against breast cancer cells, European Journal of Medicinal Chemistry (2016),
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Y. Chen et al. / European Journal of Medicinal Chemistry xxx (2016) 1e15
bioactivities. Furthermore, some other substituents such as cyclo-
propane, 2-thiazolyl, and 2-benzothiazolyl were introduced (10a-
c), but these compounds only showed weak inhibitory potency in
both kinase and cytotoxicity assays. Finally, the 5-tBu-isoxazole
substitute was discovered as a fragment with a potent effect on Raf.
This fragment was then coupled with compound 4 (9a-g) [38e41].
The bioactivity results of compound 9 suggested that the 5-tBu-
isoxazole substituent displayed stronger activities than the 3-CF3,
4-Cl phenyl substituents for aromatic ring A.
To determine the influences of substituents at the 6-position of
the pyrimidine moiety, several substituents were introduced to
replace the methylamino group. Compared to 9a and 9d, the sub-
stitution of R6 by morpholino or piperidine-1-yl groups displayed
marginal influences on the inhibitory effects against Raf1 kinase
and decreased the inhibitory activities against ERK kinase and cell
proliferation in MCF-7 and MD-MBA-231 cells. The cell prolifera-
tion assay also revealed that these compounds indeed displayed
stronger inhibition against the growth of MCF-7 cells than MD-
MBA-231 cells. Interestingly, only compound 9d strongly sup-
pressed the proliferation of MCF-7 and MD-MBA-231 breast cancer
Scheme 1. Strategy for the design of novel Raf/ERK dual inhibitors.
cells with the lowest IC50 values of 0.39 and 2.10 mM, respectively.
Compound 9d was thus used in subsequent studies.
The kinase selectivity profile of 9d was performed on a panel of
100 kinases by using the KINOMEscan assay (Discoverx Co. Ltd.,
The 6-chloro atom on the pyrimidine ring of compound 3 was
reacted with a series of secondary amines to produce compounds
4a-h. Finally, the reactions between compound 4 and different
isocyanate, isothiocyanate or in situ generated isocyanate produced
the target compounds 7a-l, 8a-h, 9a-g and 10a-c. All compounds
were fully characterized by 1H NMR, 13C NMR and high-resolution
mass spectrometry.
Fremont, CA, USA) at a constant concentration of 1.0
demonstrated that compound 9d could also potently inhibit B-Raf,
VEGFR2 and PDGFR at the submicromolar level, in addition to its
mM. The results
b
dual targets against Raf1 and ERK. Moreover, further investigation
should be performed on the safety of compound 9d in association
with its relatively broad kinase inhibitory profiles (see Table S1).
The molecular docking of compound 9d with the ATP sites of Raf1
and ERK1 was also performed (Figs. S3C and D). In Raf1, 9d formed
2.4. Novel Raf1/ERK dual inhibitors possess good kinase selectivity
and suppress breast cancer cell proliferation
stable hydrogen bonds with Glu122 and Met 125 and
teractions between Tyr53 and the pyrimidine ring. For ERK1, 9d
could formed hydrogen bonds with Lys431 and strong in-
teractions were observed between Trp423 and Phe475 and the
benzene ring.
p-p in-
Structural optimizations of the lead compounds 7a and 9a were
conducted to improve their inhibitory capacity against Raf1 and
ERK. The kinase inhibitory and cellular proliferation assay results
are shown in Table 1. The analysis of the structure-activity rela-
tionship was mainly focused on the influences of various substit-
uent groups at the following three groups (Scheme 2) on the dual
ERK and Raf1 inhibitory activities: the aromatic ring of the urea or
thiourea (Ring A), the substituents of the phenyl ring B (R4 and R5),
and the 6-substituents of the pyrimidine ring (R6).
p-p
2.5. Novel dual inhibitor induces apoptosis via the mitochondrial
pathway with partial dependence on Raf1 and ERK
Flow cytometry analysis was performed to determine the death
subroutine of compound 9d-treated MCF-7 breast cancer cells us-
ing the Annexin V/PI staining kit (Fig. 3). According to Fig. 3, the
percentage of apoptotic cells in the 9d treatment group was
41.2% 3.20%, which was significantly higher than in the BL-EI001
group (19.9% 1.8%, p < 0.05) and the NS (2.5% 0.4%, p < 0.05)
group. There were no significant differences in early apoptosis
between the 9d treatment group (5.7% 0.6%) and the BL-EI001
(3.3% 0.4%) group, but the rate of late apoptotic cells in the 9d
treatment group (35.4% 2.8%) was much higher than in the free
PTX group (16.5% 1.5%, p < 0.05). The results clearly demonstrated
that apoptotic cell death was initiated by compound 9d in a
concentration-dependent manner. We then investigated the
morphological changes of compound 9d-treated MCF-7 cells by
Hoechst 33258 staining (see Fig. 4), and apoptotic cell death was
clearly observed under fluorescence microscopy. To further validate
the dual inhibitory effects and apoptotic mechanism of compound
9d, we investigated its suppressive capacity on the activation of the
MAPK signal pathway and the apoptotic relative proteins in MCF-
7 cells bearing both Raf1 and ERK amplification (Fig. 5). As shown in
Fig. 5, treatment with compound 9d indeed decreased the phos-
phorylation of Raf1 and ERK in MCF-7 cells in a dose-dependent
manner, suggesting that compound 9d could block the MAPK
signal pathway. In addition, the total protein levels of Raf1, ERK and
First, we investigated the influence of the substitute groups in
aromatic ring B of compound 7 on its inhibitory capacity against
Raf1 and ERK kinases via the addition of a fluorine atom or methyl
group. The results suggested that the position of the substitute
groups in aromatic ring B possessed diverse effects on the inhibi-
tory activity against ERK and Raf1. Two substituents were used in
the SAR studies: a fluorine or methyl group in R4, and a methyl
group in R5. Larger substituents were limited by the steric hin-
drance of phenyl ring B. Larger substituent groups, e.g., bromide,
enthyl or trifluoromethyl group could dramatically reduce the
productivity of the target compounds. It was apparent that the
introduction of a methyl group at the R4 position led to a moderate
increase in bioactivities in both the kinase and cell proliferation
assays. Second, we explored the influences of different substituents
on aromatic ring A by setting the phenyl ring as its original group
and varying R1, R2 and R3. The kinase and cell proliferation inhibi-
tory activities of the thiourea-linked compounds (8a-h) are shown
in Table 1. It was apparent that the 3-CF3, 4-Cl phenyl substituents
had better activities than the other groups. In addition, comparison
of the urea-linked compounds (7a-l) and thiourea-linked com-
pounds (8a-h) indicated that the urea linker was slightly better
than the thiourea. In the urea-linked compounds, the 3-CF3,4-Cl
phenyl substituent in aromatic ring A also displayed preferential
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extracellular-signal regulated kinase (ERK) dual inhibitors against breast cancer cells, European Journal of Medicinal Chemistry (2016),
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Y. Chen et al. / European Journal of Medicinal Chemistry xxx (2016) 1e15
5
Table 1
Kinase inhibitory activity against Raf1 and ERK1, and cytotoxicity against MCF-7 and MDA-MB-231 of compounds 7a-l, 8a-h, 9a-g and 10 a-c.
No.
R1
R2
R3
R4
R5
R6
X
Kinase inhibitory
(IC50
Cytotoxicity (IC50, m
M)b
, m
M)a
Raf1
ERK1
MCF-7
MD-MBA-231
7a
7b
7c
7d
7e
7f
7g
7h
7i
H
CF3
H
H
H
CF3
H
H
CF3
H
H
H
Br
H
H
H
H
CF3
CF3
CF3
CF3
e
H
H
H
H
H
Cl
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
e
e
e
e
e
e
e
H
H
H
H
H
H
F
F
F
F
F
F
e
O
O
O
O
O
O
O
O
O
O
O
O
S
S
S
S
S
S
S
S
0.169
0.272
1.80
2.30
0.099
>10
1.51
0.071
>10
0.67
1.28
0.374
6.20
4.90
3.05
7.50
2.10
1.84
0.86
0.77
4.8
7.6
0.95
3.30
4.72
10.9
0.75
>20
15.3
1.56
>20
13.32
15.91
5.92
>20
>20
9.64
>20
6.84
7.26
2.19
1.79
0.56
2.70
1.89
0.39
0.72
4.24
3.96
>20
4.27
21.84
13.65
>20
6.98
>20
>20
5.73
>20
>20
>20
18.67
>20
>20
Br
MeO
Me
Cl
Cl
CF3
Cl
Cl
Cl
MeO
H
H
H
Br
H
H
e
e
>10
>10
4.7
>10
>10
3.1
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
8.3
5.2
1.62
7.3
6.9
0.79
8.7
>10
>10
>10
e
e
e
Me
Me
Me
Me
Me
Me
H
Me
Me
H
H
Me
H
Me
H
H
H
H
H
H
H
H
F
H
H
Me
F
H
F
H
F
F
F
e
e
e
7j
7k
7l
e
e
e
8a
8b
8c
8d
8e
8f
8g
8h
9a
9b
9c
9d
9e
9f
e
e
e
>20
>20
e
e
18.28
11.03
8.27
16.73
3.79
10.3
15.4
2.10
7.39
>20
H
e
Cl
Cl
e
e
e
Methylamino
Piperidine-1-yl
Morpholino
Methylamino
Morpholino
Piperidine-1-yl
Morpholino
e
e
e
e
e
e
e
0.053
0.089
0.076
0.028
0.051
0.327
0.690
>10
e
e
e
e
H
e
e
Me
Me
H
H
H
Me
Me
e
e
e
e
9g
10a
e
e
H
12.3
>20
10b
10c
>10
>10
>10
>10
>20
>20
>20
>20
a
IC50 values were determined from KinaseProfiler of Eurofins. The data represent the mean values of two independent experiments.
Each compound was tested in triplicate; the data are presented as the mean values.
b
GAPDH remained unchanged. To further assess the proteins of the
MEK/ERK pathway.
death receptor apoptotic pathway involved in 9d-treated MCF-
7 cells, the levels of Fas, FasL, Fas-Associated protein with Death
Domain (FADD) and caspase-8 were determined by Western blot
analysis. The results demonstrated that the concentrations of Fas,
FasL and FADD were not obviously changed, and nor was the
expression level of caspase-8 after compound 9d treatment (Fig. 5).
Simultaneously, the expression level of Bcl-2 declined, whereas Bax
expression was increased in MCF-7 cells after treatment with
compound 9d. Moreover, we found enhanced cleavage of caspase-9
in 9d-induced apoptosis. The total length and cleaved form of
caspase-3 were barely observed because of its expression defi-
ciency in MCF-7 cells. These results suggest that the mitochondrial
pathway, other than the death receptor, was mainly involved in 9d-
induced apoptosis. These results suggest that the crosstalk between
Raf1 and ERK might be a complicated way to follow the classic Raf/
3. Conclusions
In conclusion, a series of 4-(pyrimidin-4-yl)oxyl-phenyl-3-
arylurea derivatives were discovered as novel dual inhibitors of
Raf1 and ERK. Both compounds potently inhibited both kinases
with IC50 values at the submicromolar level, potently inhibited the
proliferation of a panel of breast cancer cells and induced mito-
chondrial apoptosis. In addition, the most potent compound 9d
demonstrated inhibitory potency of the Raf/MEK/ERK pathway in
the MCF-7 cells, suggesting that the Raf1/ERK dual inhibition might
be an attractive strategy for breast cancer chemotherapy. Primary
molecular mechanism studies of compound 9d indicated that the
individual knockdown of Raf1 and ERK could only partially inhibit
its antitumor activity, implying that the direct and indirect
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extracellular-signal regulated kinase (ERK) dual inhibitors against breast cancer cells, European Journal of Medicinal Chemistry (2016),
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Scheme 2. Reagents and conditions: (a) K2CO3, CH3CN, reflux, 2 h, 54e78%; (b) Fe, NH4Cl, EtOH, H2O, reflux, 2 h, 80e88%; (c)CH3NH2, 50 ꢀC, 2 h, 45e52%; (d)(i) Triphosgene,
Toluene, -5 ꢀC, 1 h, (ii) DCM, rt, 2 h, 54e70%; (e)(i) CS2, TEA, rt, (ii) (Boc)2O, DMAP, 0ꢀC-rt, 30 min, 48e55%; (f) CH3CN, rt, 24 h, 63e75%; (g)Morpholine or Piperidine, 50 ꢀC, 2 h,
60e75%.
interactions between Raf1 and ERK were more sophisticated than
we previously acknowledged. Taken together, our results indicated
that the integrated bioinformatics, computational and experi-
mental discovery of a synthesized Raf1/ERK dual inhibitor was
effective for the treatment of breast cancer.
4. Experimental section
4.1. General method
NMR data was obtained for 1H at 400 MHz, and for 13C at
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Fig. 3. Apoptosis assay by FCM; (A) Control; (B) BL-EI001; (C) Compound 9d and (D) the quantification of apoptotic cells in each groups.
Fig. 4. The cellular morphology was observed without or with 9d treated under fluorescent microscopy after Hoechst 33,258 staining.
100 MHz. Chemical shifts were reported in ppm from tetrame-
thylsilane with the solvent resonance as the internal standard in
CDCl3 solution. ESI-HRMS was recorded on a Waters SYNAPT G2 Q-
TOF instrument. Column chromatography was performed on silica
gel (300e400 mesh) eluting with ethyl acetate and petroleum
ether. TLC was performed on glass-backed silica plates. UV light and
I2 were used to visualize products. Melting points were determined
on a Mel-Temp apparatus and are uncorrected. All chemicals were
used without purification as commercially available unless other-
wise noted.
4.2. Synthetic procedures
Genera procedure for the synthesis of 4-chloro-6-(substituted
phenoxy)pyrimidines 2a-c.
To a mixture of appropriate phenol (10 mmol) in Acetonitrile
(50 mL) was added K2CO3 (2.07 g, 15 mmol). The reaction mixture
was stirred at room temperature for 10 min. Then 4,6-
dichloropyrimidine (1.78 g, 12 mmol) was added to the reaction
mixture, followed by heating to reflux for 4 h. The solvent was
removed under vacuum. The resulting residue was diluted with
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Y. Chen et al. / European Journal of Medicinal Chemistry xxx (2016) 1e15
Fig. 5. Western blot analysis of Raf1, ERK and apoptosis related proteins.
water and extracted with ethyl acetate (3 ꢁ 30 mL). The combined
organic layer was dried over anhydrous sodium sulfate and
concentrated under vacuum and then recrystallized from EtOAc
and petroleum to afford the product 2a-c.
4.2.5. 4-((6-Chloropyrimidin-4-yl)oxy)-2-methylaniline (3b)
Yield: 66%, 1H NMR (Chloroform-d)
d
8.59 (d, J ¼ 0.8 Hz,1H), 6.84
(s, 1H), 6.83 (d, J ¼ 0.8 Hz, 1H), 6.80 (dd, J ¼ 8.4, 2.8 Hz, 1H), 6.70 (d,
J ¼ 8.4 Hz, 1H), 3.66 (s, 2H), 2.18 (s, 3H).HRMS (ESI) calcd. for
C
11H11ClN3Oþ [M þ H]þ, 236.0585; found, 236.0581.
4.2.1. 4-Chloro-6-(2-fluoro-4-nitrophenoxy)pyrimidine (2a)
Yield:67%, 1H NMR (Chloroform-d)
8.18e8.12 (m, 2H), 7.47e7.42 (m, 1H), 7.15 (d, J ¼ 0.8 Hz, 1H).HRMS
(ESI) calcd. for C10H5ClFN3NaOþ3 [M þ Na]þ, 291.9896; found,
291.9900.
d
8.55 (d, J ¼ 0.8 Hz, 1H),
4.2.6. 4-((6-Chloropyrimidin-4-yl)oxy)-3-methylaniline (3c)
Yield: 69%, 1H NMR (Chloroform-d)
d
8.59 (d, J ¼ 0.8 Hz, 1H),
6.84e6.82 (m, 2H), 6.60 (d, J ¼ 2.8 Hz, 1H), 6.56 (dd, J ¼ 8.4, 2.8 Hz,
1H), 3.65 (s, 2H), 2.06 (s, 3H).HRMS (ESI) calcd. for C11H11ClN3Oþ
[M þ H]þ, 236.0585; found, 236.0586.
Genera procedure for the synthesis of 6-(4-amino-substituted
phenoxy)-aromatic amine 4a-h.
4.2.2. 4-Chloro-6-(3-methyl-4-nitrophenoxy)pyrimidine (2b)
Yield: 62%, 1H NMR (Chloroform-d)
d
8.55 (d, J ¼ 0.8 Hz, 1H),
The compound 3a-c (3 mmol) was stirred in 20 mL of tetrahy-
drofuran at room temperature. To the mixture, 1 mL of 35%
methylamine ethanolic solution was added. The resulting mixture
was stirred at 45 ꢀC for 4 h. The organic solvent was removed under
vacuum and then purified by column chromatography over silica
gel to afford compound 4a-h.
8.22 (d, J ¼ 2.4 Hz, 1H), 8.16 (dd, J ¼ 8.8, 2.8 Hz, 1H), 7.24 (d,
J ¼ 8.8 Hz,1H), 7.09 (d, J ¼ 0.8 Hz,1H), 2.28 (s, 3H). HRMS (ESI) calcd.
for C11H8ClN3NaOþ3 [M þ Na]þ, 288.0146; found, 288.0141.
4.2.3. 4-Chloro-6-(2-methyl-4-nitrophenoxy)pyrimidine (2c)
Yield: 58%, 1H NMR (Chloroform-d)
d
8.55 (d, J ¼ 0.8 Hz,1H), 8.22
(d, J ¼ 2.4 Hz, 1H), 8.16 (dd, J ¼ 8.8, 2.8 Hz, 1H), 7.24 (d, J ¼ 8.8 Hz,
1H), 7.09 (d, J ¼ 0.8 Hz, 1H), 2.28 (s, 3H).HRMS (ESI) calcd. for
4.2.7. 6-(4-Amino-2-fluorophenoxy)-N-methylpyrimidin-4-amine
11H8ClN3NaOþ3 [M þ Na]þ, 288.0146; found, 288.0147.
(4a)
C
Yield: 52%, 1H NMR (Chloroform-d)
d 8.22 (s, 1H), 6.96 (t,
Genera procedure for the synthesis of 4-((6-chloropyrimidin-4-
J ¼ 8.8 Hz, 1H), 6.49 (dd, J ¼ 12.0, 2.8 Hz, 1H), 6.45e6.42 (m, 1H),
yl)oxy)-3-substituteaniline 3a-c.
5.79 (s, 1H), 5.18 (brs, 1H), 3.74 (s, 2H), 2.90 (d, J ¼ 5.2 Hz, 3H)$13C
To a suspension of 2a-c (5 mmol) in 80% EtOH (40 mL) was
added NH4Cl (1.34 g, 25 mmol) and Fe (1.4 g, 25 mmol). The reac-
tion mixture was heating to reflux for 2 h and then filtered with
diatomite. The filtrate was concentrated under vacuum, diluted
with water andextracted with ethyl acetate(3 ꢁ 30 mL).The com-
bined organic layer was dried over anhydrous sodium sulfate and
concentrated under vacuum and then recrystallized from EtOH to
afford the product 3a-c.
NMR (Chloroform-d)
d
168.98, 164.39, 157.04, 153.94 (d, J ¼ 247 Hz),
144.33 (d, J ¼ 10 Hz), 130.62 (d, J ¼ 13 Hz), 123.26 (d, J ¼ 3 Hz),
109.67 (d, J ¼ 3 Hz),102.45 (d, J ¼ 22 Hz), 27.43.HRMS (ESI) calcd. for
C
11H12FN4Oþ [M þ H]þ, 235.0990; found, 235.0989.
4.2.8. 6-(4-Amino-3-methylphenoxy)-N-methylpyrimidin-4-amine
(4b)
Yield: 48%, 1H NMR (Chloroform-d)
d 8.24 (s, 1H), 6.84 (d,
4.2.4. 4-((6-Chloropyrimidin-4-yl)oxy)-3-fluoroaniline (3a)
J ¼ 2.8 Hz, 1H), 6.80 (dd, J ¼ 8.4, 2.8 Hz, 1H), 6.67 (d, J ¼ 8.4 Hz, 1H),
Yield: 75%, 1H NMR (Chloroform-d)
d 8.57 (s, 1H), 6.98e6.94 (m,
5.65 (s, 1H), 5.24 (brs, 1H), 3.58 (brs, 2H), 2.86 (d, J ¼ 5.2 Hz, 3H),
2H), 6.51 (dd, J ¼ 12.0, 2.8 Hz, 1H), 6.46 (dd, J ¼ 8.8, 2.8 Hz, 1H), 3.81
(s, 2H).HRMS (ESI) calcd. for C10H7ClFN3NaOþ [M þ Na]þ, 262.0154;
found, 262.0157.
2.16 (s, 3H)$13C NMR (Chloroform-d)
d 171.02, 165.38, 158.28,
144.76, 142.15, 123.73, 123.36, 119.86, 115.65, 28.43, 17.59.HRMS
(ESI) calcd. for C12H15N4Oþ[M þ H]þ, 231.1240; found, 231.1234.
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9
4.2.9. 6-(4-Amino-2-methylphenoxy)-N-methylpyrimidin-4-amine
(4c)
Genera procedure for the synthesis of urea derivative com-
pounds 7a-l and 10a-c.
Yield: 62%, 1H NMR (Chloroform-d)
d
8.24 (s, 1H), 6.84 (d,
The solution of appropriate aniline (0.5 mmol) and triethyl-
amine (trace) dissolved in toluene (10 mL) was slowly dripped into
a stirred solution of triphosgene (59 mg, 0.2 mmol) in toluene
(5 mL) at 0 ꢀC by using a constant-pressure dropping funnel. The
resulting mixture was then heated to reflux for 6 h after the aniline
was added. The organic solvent was removed under vacuum and
the residue was taken up in dichloromethane (10 mL), compound
4a-h (0.5 mmol) was added directly to the solution. The reaction
mixture was stirred at room temperature for 6 h. The solvent was
removedunder vacuumand then purified by column chromatog-
raphy over silica gel toafford pure compound 7a-l and 10a-c.
J ¼ 8.4 Hz, 1H), 6.57 (d, J ¼ 2.8 Hz, 1H), 6.53 (dd, J ¼ 8.4, 2.9 Hz, 1H),
5.61 (d, J ¼ 0.8 Hz, 1H), 5.20 (brs, 1H), 3.60 (brs, 2H), 2.86 (d,
J ¼ 5.2 Hz, 3H), 2.08 (s, 3H)$13C NMR (Chloroform-d)
d 170.66,
165.44, 158.38, 144.07, 143.38, 131.46, 122.56, 117.68, 113.67, 28.42,
16.31.HRMS (ESI) calcd. for C12H15N4Oþ [M þ H]þ, 231.1240;
found,231.1237.
4.2.10. 3-Fluoro-4-((6-(piperidin-1-yl)pyrimidin-4-yl)oxy)aniline
(4d)
Yield: 59%, 1H NMR (Chloroform-d)
d
8.27 (d, J ¼ 0.8 Hz,1H), 6.95
(t, J ¼ 8.8 Hz, 1H), 6.48 (dd, J ¼ 12.0, 2.8 Hz, 1H), 6.43 (ddd, J ¼ 8.8,
4.2.15. 1-(3-Fluoro-4-((6-(methylamino)pyrimidin-4-yl)oxy)
2.8, 1.2 Hz, 1H), 5.96 (d, J ¼ 0.8 Hz, 1H), 3.74 (s, 2H), 3.58 (t,
J ¼ 5.2 Hz, 4H),1.71e1.59 (m, 6H)$13C NMR (Chloroform-d)
d 170.24,
phenyl)-3-(3-(trifluoromethyl)phenyl) urea (7a)
Yield: 78%, m.p.: 194.6e195.3 ꢀC. 1H NMR (DMSO-d6)
d 9.17 (s,
163.88, 157.89, 154.99 (d, J ¼ 247 Hz), 145.27 (d, J ¼ 10 Hz), 131.74 (d,
J ¼ 13 Hz), 124.32 (d, J ¼ 3 Hz), 110.70 (d, J ¼ 3 Hz), 103.48 (d,
J ¼ 21 Hz), 85.12, 45.32, 25.42, 24.60. HRMS (ESI) calcd. for
1H, CONH), 9.08 (s, 1H, CONH), 8.10 (brs, 1H, Pyrimidine Ar-H), 8.01
(s, 1H, phenyl A Ar-H), 7.63e7.59 (m, 2H, phenyl A Ar-H), 7.53 (t,
J ¼ 8.0 Hz, 1H, phenyl A Ar-H), 7.37e7.32 (m, 2H, phenyl B Ar-H),
7.24e7.16(m, 2H, Pyrimidine Ar-H, phenyl B Ar-H), 5.88 (s, 1H,
C
15H18FN4Oþ [M þ H]þ, 289.1459; found, 289.1461.
CH3NH), 2.79 (s, 3H, CH3NH)$13C NMR (DMSO-d6)
d 165.08, 153.75
4.2.11. 3-Fluoro-4-((6-morpholinopyrimidin-4-yl)oxy)aniline (4e)
Yield: 44%, 1H NMR (Chloroform-d)
8.30 (s, 1H), 6.95 (t,
(d, J ¼ 244 Hz), 152.43, 140.34, 137.83 (d, J ¼ 10 Hz), 133.96 (d,
J ¼ 13 Hz), 129.89, 129.34 (q, J ¼ 32 Hz), 125.50, 124.16 (d, J ¼ 2 Hz),
122.80, 121.91, 118.23, 114.52, 114.18 (d, J ¼ 4 Hz), 106.68 (d,
d
J ¼ 8.8 Hz, 1H), 6.49 (dd, J ¼ 12.0, 2.8 Hz, 1H), 6.43 (ddd, J ¼ 8.8, 2.8,
1.2 Hz, 1H), 5.97 (s, 1H), 3.79e3.76 (m, 4H), 3.74 (s, 2H), 3.60e3.58
J
¼
24 Hz), 27.56. HRMS (ESI) calcd. for C
19H16F4N5Oþ2
þ
(m, 4H)$13C NMR (Chloroform-d)
d 170.33, 164.39, 157.87, 154.95 (d,
[M
H]þ,422.1235; found, 422.1249. HPLC purity >99%,
J ¼ 247 Hz), 145.40 (d, J ¼ 10 Hz), 131.60 (d, J ¼ 13 Hz), 124.27 (d,
J ¼ 3 Hz), 110.69 (d, J ¼ 3 Hz), 103.47 (d, J ¼ 22 Hz), 85.58, 66.46,
44.41. HRMS (ESI) calcd. for C14H16FN4Oþ2 [M þ H]þ, 291.1252; found,
291.1253.
Rt ¼ 7.2 min.
4.2.16. 1-(4-bromophenyl)-3-(3-fluoro-4-((6-(methylamino)
pyrimidin-4-yl)oxy)phenyl)urea (7b)
Yield: 71%, m.p.: 243.1e244.0 ꢀC. 1H NMR (DMSO-d6)
d 8.96 (s,
4.2.12. 2-Methyl-4-((6-morpholinopyrimidin-4-yl)oxy)aniline (4f)
1H, CONH), 8.92 (s, 1H, CONH), 8.10 (brs, 1H, Pyrimidine Ar-H), 7.59
(dd, J ¼ 13.2, 2.4 Hz, 1H, phenyl B Ar-H), 7.48e7.42 (m, 4H, phenyl A
Ar-H), 7.35 (q, J ¼ 4.8 Hz, 1H, phenyl B Ar-H), 7.23e7.13 (m, 2H,
Pyrimidine Ar-H, phenyl B Ar-H), 5.88 (s, 1H, CH3NH), 2.78 (s, 3H,
Yield: 51%, 1H NMR (Chloroform-d)
d 8.32 (s, 1H), 6.83 (d,
J ¼ 2.8 Hz, 1H), 6.79 (dd, J ¼ 8.4, 2.8 Hz, 1H), 6.67 (d, J ¼ 8.4 Hz, 1H),
5.84 (s, 1H), 3.76 (dd, J ¼ 5.6, 4.0 Hz, 4H), 3.59 (s, 2H), 3.55 (dd,
J ¼ 5.6, 4.0 Hz, 4H), 2.16 (s, 3H)$13C NMR (Chloroform-d)
d 170.31,
CH3NH)$13C NMR (DMSO-d6)
d 164.32, 156.91, 153.67 (d,
163.37, 157.05, 143.69, 141.14, 122.70, 122.30, 118.80, 114.62, 84.72,
65.42, 43.36, 16.58.HRMS (ESI) calcd. for C15H19N4Oþ2 [M þ H]þ,
287.1503; found, 287.1505.
J ¼ 244 Hz), 152.32, 138.89, 138.20 (d, J ¼ 10 Hz), 133.59 (d,
J ¼ 13 Hz),131.50,124.11, 120.16,114.34,113.38,106.44 (d, J ¼ 24 Hz),
27.65. HRMS (ESI) calcd. for C18H16BrFN5Oþ2 [M'þ'H]þ, 432.0466;
found, 432.0464. HPLC purity >99%, Rt ¼ 6.7 min.
4.2.13. 3-Methyl-4-((6-(piperidin-1-yl)pyrimidin-4-yl)oxy)aniline
(4g)
4.2.17. 1-(3-Fluoro-4-((6-(methylamino)pyrimidin-4-yl)oxy)
phenyl)-3-(4-methoxyphenyl)urea (7c)
Yield: 39%, 1H NMR (Chloroform-d)
d 8.29 (s, 1H), 6.83 (d,
Yield: 77%, m.p.: 226.5e227.4 ꢀC. 1H NMR (DMSO-d6)
d 8.82 (s,
J ¼ 8.4 Hz, 1H), 6.57 (d, J ¼ 2.8 Hz, 1H), 6.53 (dd, J ¼ 8.4, 2.8 Hz, 1H),
5.80 (s, 1H), 3.59 (s, 2H), 3.54 (t, J ¼ 5.2 Hz, 4H), 2.08 (s, 3H),
1H, CONH), 8.55 (s, 1H, CONH), 8.10 (brs, 1H, Pyrimidine Ar-H), 7.59
(dd, J ¼ 13.2, 2.4 Hz, 1H, phenyl B Ar-H), 7.37e7.32 (m, 3H, phenyl A
Ar-H), 7.19 (t, J ¼ 8.8 Hz, 1H, phenyl B Ar-H), 7.12 (dd, J ¼ 9.2, 2.4 Hz,
1H, phenyl A Ar-H), 6.88 (d, J ¼ 8.8 Hz, 2H, phenyl B Ar-H), 5.87 (s,
1H, CH3NH), 3.72 (s, 3H, CH3O), 2.78 (s, 3H, CH3NH)$13C NMR
1.68e1.57 (m, 6H)$13C NMR (Chloroform-d)
d 169.85, 162.95, 157.16,
142.89, 142.49, 130.44, 121.54, 116.67, 112.66, 83.66, 44.23, 24.38,
23.56, 15.34.HRMS (ESI) calcd. for C16H21N4Oþ [M þ H]þ, 285.1710;
found, 285.1711.
(DMSO-d6)
d
165.13, 157.73, 154.97 (d, J ¼ 244 Hz), 154.58, 152.58,
138.39 (d, J ¼ 10 Hz), 133.51 (d, J ¼ 13 Hz), 132.37, 124.08, 120.20,
114.10 (d, J ¼ 3 Hz), 113.95, 106.26 (d, J ¼ 24 Hz), 55.12, 27.45. HRMS
(ESI) calcd. for C19H19FN5O3þ[M þ H]þ, 384.1466; found, 384.1462.
HPLC purity >98%, Rt ¼ 6.4 min.
4.2.14. 3-Methyl-4-((6-morpholinopyrimidin-4-yl)oxy)aniline (4 h)
Yield: 55%, 1H NMR (Chloroform-d)
d 8.32 (s, 1H), 6.83 (d,
J ¼ 8.4 Hz, 1H), 6.58 (d, J ¼ 2.8 Hz, 1H), 6.54 (dd, J ¼ 8.4, 2.8 Hz, 1H),
5.81 (s, 1H), 3.78e3.75 (m, 4H), 3.59 (s, 2H), 3.55 (t, J ¼ 4.8 Hz, 4H),
2.08 (s, 3H)$13C NMR (Chloroform-d)
d 169.95, 163.43, 157.14,
4.2.18. 1-(3-Fluoro-4-((6-(methylamino)pyrimidin-4-yl)oxy)
phenyl)-3-(p-tolyl)urea (7d)
143.05, 142.31, 130.39, 121.50, 116.66, 112.66, 84.10, 65.42, 43.34,
15.32.HRMS (ESI) calcd. for C15H19N4Oþ2 [M þ H]þ, 287.1503; found,
287.1504.
Yield: 69%, m.p.: 234.2e236.1 ꢀC. 1H NMR (DMSO-d6)
d 8.91 (s,
1H, CONH), 8.68 (s, 1H, CONH), 8.11 (brs, 1H, Pyrimidine Ar-H), 7.60
(dd, J ¼ 13.2, 2.4 Hz, 1H, phenyl B Ar-H), 7.40 (s, 1H, phenyl A Ar-H),
7.34 (d, J ¼ 8.4 Hz, 2H, phenyl A Ar-H), 7.20 (t, J ¼ 8.8 Hz, 1H, phenyl
A Ar-H), 7.14e7.08 (m, 3H, Pyrimidine Ar-H, phenyl B Ar-H), 5.88 (s,
1H, CH3NH), 2.79 (s, 3H, CH3NH), 2.25 (s, 3H, CH3)$13C NMR
(DMSO-d6)
d
164.74, 157.38,154.94 (d, J ¼ 245 Hz),152.46, 138.41 (d,
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Y. Chen et al. / European Journal of Medicinal Chemistry xxx (2016) 1e15
J ¼ 10 Hz), 136.82, 133.47 (d, J ¼ 13 Hz), 130.84, 129.15, 124.08,
118.38, 114.13, 106.26 (d, J ¼ 23 Hz), 27.55, 20.30. HRMS (ESI) calcd.
for C19H19FN5Oþ2 [M þ H]þ, 368.1517; found, 368.1523. HPLC purity
>98%, Rt ¼ 5.4 min.
J ¼ 6.0 Hz, 2H, CONH), 8.19 (d, J ¼ 9.2 Hz, 1H, phenyl A AreH), 8.15
(brs, 1H, Pyrimidine AreH), 7.73 (d, J ¼ 8.8 Hz, 1H, phenyl A AreH),
7.63 (d, J ¼ 2.4 Hz, 1H, phenyl A AreH), 7.38 (dd, J ¼ 9.2, 2.8 Hz, 2H,
phenyl B AreH), 7.01 (d, J ¼ 2.8 Hz, 1H, phenyl B AreH), 6.94 (dd,
J ¼ 8.8, 2.8 Hz, 1H, Pyrimidine AreH), 5.76 (s, 1H, CH3NH), 2.77 (s,
4.2.19. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(3-fluoro-4-((6-
3H, CH3NH), 2.27 (s, 3H, CH3).13C NMR (DMSO-d6)
d 164.81, 157.56,
(methylamino)pyrimidin-4-yl)oxy)phenyl)urea (7e)
152.45, 148.26, 135.39, 133.92, 130.70,128.52, 127.53, 125.97, 123.50,
122.99, 122.62, 122.39, 118.99, 27.49, 17.99. HRMS (ESI) calcd. for
C
>99%,Rt ¼ 5.5 min.
Yield: 70%, m.p.: 220.1e221.0 ꢀC. 1H NMR (DMSO-d6)
d 9.90 (s,
1H, CONH), 9.74 (s, 1H, CONH), 9.04(brs, 1H, phenyl A Ar-H), 8.35
(brs,1H, phenyl A Ar-H), 8.11 (s,1H, Pyrimidine Ar-H), 7.68e7.62 (m,
3H, phenyl A Ar-H,, phenyl B Ar-H), 7.30 (t, J ¼ 8.8 Hz, 1H, phenyl B
Ar-H), 7.20 (d, J ¼ 9.2, 2.4 Hz, 1H, Pyrimidine Ar-H), 6.07 (brs, 1H,
19H18Cl2N5Oþ2 [M þ H]þ, 418.0832; found, 418.0837. HPLC purity
4.2.24. 1-(4-chlorophenyl)-3-(2-methyl-4-((6-(methylamino)
pyrimidin-4-yl)oxy)phenyl)urea (7j)
CH3NH), 2.87 (s, 3H, CH3NH)$13C NMR (DMSO-d6)
d 161.24, 154.61
(d, J ¼ 246 Hz), 153.92, 152.42, 139.13, 138.63 (d, J ¼ 9 Hz), 133.08 (d,
J ¼ 12 Hz), 132.04, 126.71 (q, J ¼ 31 Hz), 124.09, 123.97, 122.80,
122.36, 121.38, 116.44 (d, J ¼ 6 Hz), 114.50, 106.49 (d, J ¼ 23 Hz),
28.23. HRMS (ESI) calcd. for C19H15ClF4N5Oþ2 [M þ H]þ, 456.0845;
found, 456.0851.HPLC purity >98%, Rt ¼ 9.8 min.
Yield: 69%, m.p.: 236.6e239.4 ꢀC. 1H NMR (DMSO-d6)
d 9.75 (s,
1H, CONH), 8.42 (s, 2H, CONH), 8.26 (brs, 1H, Pyrimidine Ar-H), 7.89
(d, J ¼ 8.8 Hz,1H, phenyl B Ar-H), 7.53 (d, J ¼ 8.8 Hz, 2H, phenyl A Ar-
H), 7.33 (d, J ¼ 8.8 Hz, 2H, phenyl A Ar-H), 7.07 (s, 1H, phenyl B Ar-
H), 7.03 (d,, J ¼ 8.0 Hz, 1H, phenyl B Ar-H), 5.81 (s, 1H, CH3NH), 2.85
(s, 3H, CH3NH), 2.29 (s, 3H, CH3)$13C NMR (DMSO-d6)
d 162.80,
4.2.20. 1-(2,4-dichlorophenyl)-3-(3-fluoro-4-((6-(methylamino)
154.38, 152.72, 146.83, 138.96, 135.31, 130.15, 128.59, 125.03, 122.70,
122.48, 119.29, 118.75, 27.97, 18.11. HRMS (ESI) calcd. for
C
>96%, Rt ¼ 4.4 min.
pyrimidin-4-yl)oxy)phenyl)urea (7f)
Yield: 66%, m.p.: 231.1e232.6 ꢀC. 1H NMR (DMSO-d6)
d
9.69 (s,
19H19ClN5Oþ2 [M þ H]þ, 384.1222; found, 384.1219. HPLC purity
1H, CONH), 8.47 (s, 1H, CONH), 8.19 (d, J ¼ 9.2 Hz, 1H, phenyl A
AreH), 8.11 (brs, 1H, Pyrimidine AreH), 7.65e7.60 (m, 2H, phenyl A
AreH), 7.42e7.38 (m, 2H, phenyl B AreH), 7.23 (t, J ¼ 8.8 Hz, 1H,
Pyrimidine AreH), 7.15e7.12 (m, 1H, phenyl B AreH), 5.89 (s, 1H,
4.2.25. 1-(4-methoxyphenyl)-3-(2-methyl-4-((6-(methylamino)
pyrimidin-4-yl)oxy)phenyl)urea (7k)
CH3NH), 2.79 (s, 3H, CH3NH).13C NMR (DMSO-d6)
d
164.93, 157.57,
Yield: 66%, m.p.: 237.9e238.6 ꢀC. 1H NMR (DMSO-d6)
d 8.86 (s,
153.78 (d, J ¼ 244 Hz),151.92,137.69 (d, J ¼ 10 Hz),134.94,134.03 (d,
1H, CONH), 8.15 (brs, 1H, Pyrimidine Ar-H), 7.90 (s, 1H, CONH), 7.81
(d, J ¼ 8.8 Hz, 1H, phenyl A Ar-H), 7.39e7.34(m, 3H, phenyl A Ar-H,
phenyl B Ar-H), 6.98 (d, J ¼ 2.8 Hz, 1H, phenyl A Ar-H), 6.92 (dd,
J ¼ 8.8, 2.8 Hz, 1H, phenyl A Ar-H), 6.89e6.85 (m, 2H, phenyl B Ar-H,
Pyrimidine Ar-H), 5.73 (s, 1H, CH3NH), 3.72 (s, 3H, CH3O), 2.77 (s,
J ¼ 13 Hz), 128.56, 127.63, 126.35, 124.29, 122.85, 122.25, 114.34,
106.50 (d,
C
J
¼
24 Hz), 27.52. HRMS (ESI) calcd. for
18H15Cl2FN5Oþ2 [M þ H]þ, 422.0581; found, 422.0581. HPLC purity
>98%, Rt ¼ 6.3 min.
3H, CH3NH), 2.24 (s, 3H, CH3)$13C NMR (DMSO-d6)
d 164.85, 157.59,
4.2.21. 1-(2-Methyl-4-((6-(methylamino)pyrimidin-4-yl)oxy)
154.29, 152.89, 147.55, 134.76, 132.89, 129.55, 122.90, 122.31, 119.66,
phenyl)-3-(3-(trifluoromethyl)phenyl) urea (7g)
118.96, 113.99, 55.12, 27.47, 17.86. HRMS (ESI) calcd. for C20H22N5O3þ
Yield: 72%, m.p.: 190.2e192.3 ꢀC. 1H NMR (DMSO-d6)
d
9.34 (s,
[M
þ
H]þ, 380.1717; found, 380.1725.HPLC purity >97%,
1H, CONH), 8.13 (s, 1H, CONH), 8.07e8.04 (m, 2H, Pyrimidine AreH,
phenyl A AreH), 7.76 (d, J ¼ 8.8 Hz, 1H, phenyl A AreH), 7.55 (dt,
J ¼ 15.6, 8.4 Hz, 2H, phenyl B AreH), 7.31 (d, J ¼ 7.6 Hz, 1H, phenyl A
AreH), 7.28 (q, J ¼ 4.8 Hz, 1H, phenyl A AreH), 7.00 (d, J ¼ 2.8 Hz,
1H, phenyl B AreH), 6.94 (dd, J ¼ 8.8, 2.8 Hz, 1H, Pyrimidine AreH),
5.75 (s, 1H, CH3NH), 2.77 (s, 3H, CH3NH), 2.25 (s, 3H, CH3).13C NMR
Rt ¼ 5.3 min.
4.2.26. 1-(3-bromophenyl)-3-(2-methyl-4-((6-(methylamino)
pyrimidin-4-yl)oxy)phenyl)urea (7l)
Yield: 67%, m.p.: 205.4e207.4 ꢀC. 1H NMR (DMSO-d6)
d 9.55 (s,
1H, CONH), 8.28 (s, 2H, CONH, Pyrimidine Ar-H), 7.89 (s, 1H, phenyl
A Ar-H), 7.82 (d, J ¼ 8.8 Hz, 2H, phenyl A Ar-H), 7.32e7.30 (m, 1H,
phenyl A Ar-H), 7.24 (t, J ¼ 8.0 Hz, 1H, phenyl B Ar-H), 7.14 (dt,
J ¼ 8.0, 1.2 Hz, 1H, phenyl B Ar-H), 7.04 (s, 1H, phenyl B Ar-H), 6.98
(d, J ¼ 8.8, 2.8 Hz, 1H, Pyrimidine Ar-H), 5.78 (d, J ¼ 6.8 Hz, 1H,
CH3NH), 2.81 (s, 3H, CH3NH), 2.27 (s, 3H, CH3)$13C NMR (DMSO-d6)
(DMSO-d6)
d 163.62, 155.61, 152.81, 147.45, 140.79, 134.70, 130.45,
129.89, 129.50 (q, J ¼ 32 Hz), 125.54, 122.89, 122.82, 121.38, 118.86,
117.81, 113.70 (d, J ¼ 4 Hz), 27.77, 17.99. HRMS (ESI) calcd. for
C
20H19F3N5Oþ2 [M þ H]þ, 418.1485; found, 418.1493. HPLC purity
>98%, Rt ¼ 6.7 min.
d
163.93, 156.09, 152.64, 147.52, 141.60, 134.66, 130.69, 130.30,
4.2.22. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(2-methyl-4-((6-
124.11, 122.84, 122.76, 121.73, 120.06, 118.89, 116.66, 27.71, 17.97.
HRMS (ESI) calcd. for C19H19BrN5Oþ2 [M þ H]þ, 428.0717; found,
428.0717. HPLC purity >97%, Rt ¼ 4.6 min.
(methylamino)pyrimidin-4-yl)oxy)phenyl)urea (7h)
Yield: 68%, m.p.: 202.2e203.8 ꢀC. 1H NMR (DMSO-d6)
d 9.45 (s,
1H, CONH), 8.12 (s, 3H, CONH,, Pyrimidine AreH, phenyl A AreH),
7.72 (d, J ¼ 8.8 Hz, 1H, phenyl A AreH), 7.66e7.60 (m, 2H, phenyl B
AreH), 7.29 (q, J ¼ 4.8 Hz,1H, phenyl A AreH), 7.01 (d, J ¼ 2.8 Hz,1H,
phenyl B AreH), 6.94 (dd, J ¼ 8.8, 2.8 Hz,1H, Pyrimidine AreH), 5.75
(s, 1H, CH3NH), 2.77 (s, 3H, CH3NH), 2.24 (s, 3H, CH3).13C NMR
Genera procedure for the synthesis of thiourea derivative
compounds 8a-h.
The solution of appropriate aniline (2 mmol) was stirred in
10 mL of carbon disulphide at room temperature. To the mixture,
triethylamine (606 mg, 6 mmol) was added. After 3 h, the resulting
(DMSO-d6)
d
165.17, 157.93, 152.65, 148.51, 139.47, 133.76, 131.99,
mixture was cooled to 0 A solution of di-tert-butyl
ꢀC.
130.85, 126.69 (q, J ¼ 31 Hz), 124.14, 123.46, 123.01, 122.70, 122.06,
dicarbonate(436 mg, 2 mmol) in EtOH (5 mL) was added into the
resulting mixture, followed by adding 4-dimethylaminopyridine
(24 mg, 0.2 mmol).The resulting mixture was stirred at room
temperature for another 1 h. The organic solvent was removed-
under vacuumand theresidue was taken up inacetonitrile (10 mL).
Compound 4a-c (2 mmol) was added directly to the solution. The
reaction mixturewas stirred at room temperature for 12 h. The
solvent was removedunder vacuumand then purified by column
121.42, 119.07, 116.46 (d, J ¼ 6 Hz), 27.41, 17.75. HRMS (ESI) calcd. for
C
20H18ClF3N5Oþ2 [M þ H]þ, 452.1096; found, 452.1104. HPLC purity
>99%, Rt ¼ 5.5 min.
4.2.23. 1-(2,4-dichlorophenyl)-3-(2-methyl-4-((6-(methylamino)
pyrimidin-4-yl)oxy)phenyl)urea (7i)
Yield: 65%, m.p.: 220.4e223.4 ꢀC. 1H NMR (DMSO-d6)
d 8.74 (d,
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Y. Chen et al. / European Journal of Medicinal Chemistry xxx (2016) 1e15
11
chromatography over silica gel to afford compound 8a-h.
found, 460.0832.HPLC purity >99%, Rt ¼ 9.1 min.
4.2.27. 1-(3-Fluoro-4-((6-(methylamino)pyrimidin-4-yl)oxy)
phenyl)-3-phenylthiourea (8a)
4.2.32. 1-(2-Methyl-4-((6-(methylamino)pyrimidin-4-yl)oxy)
phenyl)-3-(3-(trifluoromethyl)phenyl) thiourea (8f)
Yield: 53%, m.p.: 171.4e172.1 ꢀC$1H NMR (DMSO-d6)
d
9.94 (d,
Yield: 41%, m.p.: 128.6e130.3 ꢀC$1H NMR (DMSO-d6)
d
9.89 (s,
J ¼ 10.0 Hz, 2H, CONH), 8.10 (brs, 1H, Pyrimidine Ar-H), 7.65 (d,
J ¼ 10.0 Hz, 1H, phenyl B Ar-H), 7.49 (d, 2H, phenyl A Ar-H),
7.41e7.33 (m, 3H, phenyl A Ar-H), 7.29e7.22 (m, 2H, phenyl B Ar-H),
7.17e7.13 (m, 1H, Pyrimidine Ar-H), 5.93 (s, 1H, CH3NH), 2.79 (s, 3H,
1H, CONH), 9.58 (s, 1H, CONH), 8.14 (brs, 1H, Pyrimidine Ar-H), 7.97
(s, 1H, phenyl A Ar-H), 7.80e7.78 (m, 1H, phenyl A Ar-H), 7.56 (t,
J ¼ 8.0 Hz,1H, phenyl A Ar-H), 7.46 (d, J ¼ 7.6 Hz,1H, phenyl A Ar-H),
7.34 (q, J ¼ 4.8 Hz, 1H, phenyl B Ar-H), 7.29 (d, J ¼ 8.4 Hz, 1H, phenyl
B Ar-H), 7.05(m, 1H, phenyl B Ar-H), 6.97 (dd, J ¼ 8.4, 2.8 Hz, 1H,
Pyrimidine Ar-H), 5.84 (s, 1H, CH3NH), 2.78 (s, 3H, CH3NH), 2.25 (s,
CH3NH).13CNMR (100 MHz, DMSO-d6)
d 179.55, 165.22, 157.71,
153.23 (d, J ¼ 245 Hz), 139.17, 137.74 (d, J ¼ 10 Hz), 135.91 (d,
J ¼ 13 Hz), 128.50, 124.63, 123.72, 123.66, 119.66 (d, J ¼ 3 Hz), 111.76
(d, J ¼ 22 Hz), 27.51. HRMS (ESI) calcd. for C18H17FN5OSþ [M þ H]þ,
370.1132; found, 370.1142.HPLC purity >99%, Rt ¼ 13.1min.
3H, CH3)$13C NMR (DMSO-d6)
d 180.69, 165.18, 157.85, 151.21,
140.52, 136.66, 134.14, 129.38, 129.14,128.88 (q, J ¼ 32 Hz), 127.38,
125.41, 122.95, 122.70, 120.51, 119.83, 119.09, 27.47, 17.82. HRMS
(ESI) calcd. for
434.1261.HPLC purity >99%, Rt ¼ 7.5 min.
C
20H19F3N5OSþ [M
þ
H]þ, 434.1257; found,
4.2.28. 1-(2-Methyl-4-((6-(methylamino)pyrimidin-4-yl)oxy)
phenyl)-3-phenylthiourea (8b)
Yield: 50%, m.p.: 145.7e146.5 ꢀC. 1H NMR (DMSO-d6)
d
9.71 (s,
4.2.33. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(3-fluoro-4-((6-
1H, CONH), 9.31 (s, 1H, CONH), 8.14 (brs, 1H, Pyrimidine Ar-H),
7.50e7.48 (m, 2H, phenyl A Ar-H), 7.36e7.31 (m, 3H, phenyl A Ar-H),
7.28 (d, J ¼ 8.4 Hz,1H, phenyl B Ar-H), 7.15e7.11 (m, 1H, phenyl B Ar-
H), 7.03 (d, J ¼ 2.8 Hz, 1H, phenyl B Ar-H), 6.95 (dd, J ¼ 8.4, 2.8 Hz,
1H, Pyrimidine Ar-H), 5.82 (s, 1H, CH3NH), 2.77 (s, 3H, CH3NH), 2.24
(methylamino)pyrimidin-4-yl)oxy)phenyl)thiourea (8g)
Yield: 49%, m.p.: 157.6e158.9 ꢀC. 1H NMR (DMSO-d6)
d 10.21 (d,
J ¼ 12.0 Hz, 2H, CONH), 8.08 (d, J ¼ 2.4 Hz, 2H, Pyrimidine Ar-H,
phenyl A Ar-H), 7.80 (dd, J ¼ 8.8, 2.4 Hz, 1H, phenyl A Ar-H), 7.69 (d,
J ¼ 8.8 Hz,1H, phenyl A Ar-H), 7.60 (dd, J ¼ 12.0, 2.4 Hz,1H, phenyl B
Ar-H), 7.40 (q, J ¼ 4.8 Hz, 1H, phenyl B Ar-H), 7.31e7.24 (m, 2H,
phenyl B Ar-H, Pyrimidine Ar-H), 5.95 (s, 1H, CH3NH), 2.79 (s, 3H,
(s, 3H, CH3)$13C NMR (DMSO-d6)
d 180.50, 165.15, 157.85, 150.97,
139.44, 136.62, 134.60, 129.20, 128.41, 124.41, 123.76, 122.84, 118.93,
27.48,17.90. HRMS (ESI) calcd. for C19H20N5OSþ [M þ H]þ, 366.1383;
found, 366.1389.HPLC purity >99%, Rt ¼ 10.2 min.
CH3NH)$13C NMR (DMSO-d6)
d 179.80, 165.15, 157.72, 153.37 (d,
J ¼ 245 Hz), 138.97, 137.09 (d, J ¼ 9 Hz), 136.43 (d, J ¼ 13 Hz), 131.55,
128.65, 126.23 (q, J ¼ 31 Hz), 125.35, 124.01, 123.91, 122.56 (d,
J ¼ 6 Hz), 121.30, 120.01 (d, J ¼ 3 Hz), 112.14 (d, J ¼ 22 Hz), 27.47.
HRMS (ESI) calcd. for C19H14ClF4N5NaOSþ [M þ Na]þ, 494.0436;
found, 494.0440.HPLC purity >99%,Rt ¼ 13.4 min.
4.2.29. 1-(4-bromophenyl)-3-(2-methyl-4-((6-(methylamino)
pyrimidin-4-yl)oxy)phenyl)thiourea (8c)
Yield: 48%, m.p.: 182.4e182.7 ꢀC. 1H NMR (DMSO-d6)
d 9.76 (s,
1H, CONH), 9.42 (s, 1H, CONH), 8.13 (brs, 1H, Pyrimidine Ar-H),
7.52e7.47 (m, 4H, phenyl A Ar-H), 7.34 (q, J ¼ 4.7 Hz, 1H, phenyl B
Ar-H), 7.26 (d, J ¼ 8.4 Hz, 1H, phenyl B Ar-H), 7.03 (d, J ¼ 2.8 Hz, 1H,
phenyl B Ar-H), 6.95 (dd, J ¼ 8.4, 2.8 Hz, 1H, Pyrimidine Ar-H), 5.83
(s, 1H, CH3NH), 2.78 (s, 3H, CH3NH), 2.23 (s, 3H, CH3)$13C NMR
4.2.34. 1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(2-methyl-4-((6-
(methylamino)pyrimidin-4-yl)oxy)phenyl)thiourea (8h)
Yield: 43%, m.p.: 132.4e134.1 ꢀC. 1H NMR (DMSO-d6)
d 9.95 (s,
1H, CONH), 9.66 (s,1H, CONH), 8.13 (s, 1H, Pyrimidine Ar-H), 8.10 (d,
J ¼ 2.8 Hz, 1H, phenyl A Ar-H), 7.83 (dd, J ¼ 8.8, 2.8 Hz, 1H, phenyl A
Ar-H), 7.66 (d, J ¼ 8.8 Hz, 1H, phenyl A Ar-H), 7.35 (q, J ¼ 4.8 Hz, 1H,
phenyl B Ar-H), 7.27 (d, J ¼ 8.4 Hz, 1H, phenyl B Ar-H), 7.06 (d,
J ¼ 2.8 Hz, 1H, phenyl B Ar-H), 6.97 (dd, J ¼ 8.4, 2.8 Hz, 1H, Py-
rimidine Ar-H), 5.84 (s, 1H, CH3NH), 2.78 (s, 3H, CH3NH), 2.24 (s, 3H,
(DMSO-d6)
131.13,129.15,125.70,122.88,118.99,116.35, 27.47,17.87. HRMS (ESI)
calcd. for
19H19BrN5OSþ [M H]þ, 444.0488; found,
d 180.53, 165.14, 157.87, 151.08, 138.97, 136.63, 134.38,
C
þ
444.0496.HPLC purity >99%, Rt ¼ 9.5 min.
4.2.30. 1-(3-Methyl-4-((6-(methylamino)pyrimidin-4-yl)oxy)
phenyl)-3-phenylthiourea (8d)
CH3)$13C NMR (DMSO-d6)
d 180.63, 165.10, 157.91, 151.30, 139.27,
136.67, 134.00, 131.81, 131.41, 129.12, 128.52, 126.11 (q, J ¼ 31 Hz),
125.01, 124.05, 122.98, 122.39, 121.33, 119.14, 26.31, 17.80. HRMS
(ESI) calcd. for C20H18ClF3N5OSþ [M þ H]þ, 468.0867; found,
468.0862.HPLC purity >99%, Rt ¼ 7.7 min.
Yield: 45%, m.p.: 164.6e165.2 ꢀC. 1H NMR (DMSO-d6)
d 9.78 (s,
1H, CONH), 9.74 (s, 1H, CONH), 8.11 (brs, 1H, Pyrimidine Ar-H),
7.49e7.47 (m, 2H, phenyl B Ar-H), 7.39e7.29 (m, 5H, phenyl A Ar-H),
7.15e7.11 (m, 1H, phenyl B Ar-H), 7.01 (d, J ¼ 8.4 Hz, 1H, Pyrimidine
Ar-H), 5.76 (s,1H, CH3NH), 2.77 (s, 3H, CH3NH), 2.06 (s, 3H, CH3)$13C
NMR (DMSO-d6)
d 179.64, 165.13, 157.96, 147.74, 139.41, 136.41,
130.14, 128.41, 126.40, 124.41, 123.67, 122.77, 121.90, 27.46, 15.97.
HRMS (ESI) calcd. for C19H19N5NaOSþ[M þ Na]þ, 388.1203; found,
388.1209.HPLC purity >99%, Rt ¼ 4.3 min.
Genera procedure for the synthesis of thiourea derivative
compounds 9a-g.
4.2.31. 1-(3-Fluoro-4-((6-(methylamino)pyrimidin-4-yl)oxy)
phenyl)-3-(3-(trifluoromethyl)phenyl) thiourea (8e)
5-(tert-butyl)-3-isocyanatoisoxazole (166 mg, 1 mmol) was
stirred in 10 mL of dichloromethane at room temperature. To the
mixture, compound 4a-b, 4d-h (1 mmol) was added. The resulting
mixture was stirred at room temperature for 1 h. The solvent was
removed under vacuum and then purified by column chromatog-
raphy over silica gel to afford compound 9a-g.
Yield: 44%, m.p.: 154.4e155.1 ꢀC. 1H NMR (DMSO-d6)
d 10.15 (d,
J ¼ 4.4 Hz, 2H, CONH), 8.10 (brs, 1H, Pyrimidine Ar-H), 7.95 (s, 1H,
phenyl A Ar-H), 7.76 (d, J ¼ 8.4 Hz,1H, phenyl A Ar-H), 7.64e7.56 (m,
2H, phenyl A Ar-H), 7.49 (d, J ¼ 7.6 Hz, 1H, phenyl B Ar-H), 7.40 (q,
J ¼ 4.8 Hz, 1H, Pyrimidine Ar-H), 7.27 (d, J ¼ 6.0 Hz, 2H, phenyl B Ar-
H), 5.94 (s, 1H, CH3NH), 2.79 (s, 3H, CH3NH)$13C NMR (DMSO-d6)
d
179.83, 165.18, 157.85, 153.34 (d, J ¼ 245 Hz), 140.22, 137.28 (d,
4.2.35. 1-(5-(tert-butyl)isoxazol-3-yl)-3-(3-fluoro-4-((6-
J ¼ 10 Hz), 136.28 (d, J ¼ 13 Hz), 129.53, 129.01 (q, J ¼ 32 Hz), 127.46,
125.37, 123.85, 122.66, 120.80, 119.92, 112.02 (d, J ¼ 22 Hz), 27.51.
HRMS (ESI) calcd. for C19H15F4N5NaOSþ [M þ Na]þ, 460.0826;
(methylamino)pyrimidin-4-yl)oxy)phenyl) urea (9a)
Yield: 56%, m.p.: 208.6e209.2 ꢀC$1H NMR (DMSO-d6)
d
9.60 (s,
1H, CONH), 9.02 (s, 1H, CONH), 8.09 (brs, 1H, Pyrimidine Ar-H), 7.60
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12
Y. Chen et al. / European Journal of Medicinal Chemistry xxx (2016) 1e15
(dd, J ¼ 12.8, 2.4 Hz, 1H, phenyl Ar-H), 7.35 (q, J ¼ 4.8 Hz, 1H, phenyl
Ar-H), 7.22 (t, J ¼ 8.8 Hz, 1H, phenyl Ar-H), 7.15 (dd, J ¼ 9.2, 2.4 Hz,
1H, Pyrimidine Ar-H), 6.50 (s, 1H, Isoxazole Ar-H), 5.88 (s, 1H,
CH3NH), 2.78 (s, 3H, CH3NH), 1.29 (s, 9H, CH3)$13C NMR (DMSO-d6)
C
23H29N6Oþ4 [M þ H]þ, 453.2245; found, 453.2239. HPLC purity
>99%, Rt ¼ 7.6 min.
4.2.40. 1-(5-(tert-butyl)isoxazol-3-yl)-3-(3-methyl-4-((6-
(piperidin-1-yl)pyrimidin-4-yl)oxy)phenyl) urea (9f)
Yield: 55%, m.p.: 223.1e223.7 ꢀC. 1H NMR (DMSO-d6)
1H, CONH), 8.77 (s, 1H, CONH), 8.11 (s, 1H, Pyrimidine AreH), 7.38
(d, J ¼ 2.4 Hz, 1H, phenyl AreH), 7.27 (dd, J ¼ 8.8, 2.8 Hz, 1H, phenyl
AreH), 6.96 (d, J ¼ 8.8 Hz, 1H, phenyl AreH), 6.50 (s, 1H, Isoxazole
AreH), 6.19 (s, 1H, Pyrimidine AreH), 3.58 (t, J ¼ 5.6 Hz, 4H,
Piperidine CH2), 2.05 (s, 3H, CH3), 1.64e1.60 (m, 2H, Piperidine
CH2), 1.54e1.51 (m, 4H, Piperidine CH2), 1.29 (s, 9H, CH3).13C NMR
d
180.23, 165.19, 158.20, 157.80, 153.75 (d, J ¼ 244 Hz), 151.30, 137.31
(d, J ¼ 10 Hz), 134.29 (d, J ¼ 13 Hz), 124.23 (d, J ¼ 2 Hz), 114.66,
106.81 (d, J ¼ 24 Hz), 92.47, 32.44, 28.30, 27.48. HRMS (ESI) calcd.
for C19H22FN6Oþ3 [M þ H]þ, 401.1732; found, 401.1737. HPLC pu-
rity>99%, Rt ¼ 11.7 min.
d 9.50 (s,
4.2.36. 1-(5-(tert-butyl)isoxazol-3-yl)-3-(3-fluoro-4-((6-
(piperidin-1-yl)pyrimidin-4-yl)oxy)phenyl) urea (9b)
Yield: 53%, m.p.: 222.2e223.1 ꢀC. 1H NMR (DMSO-d6)
d
9.59 (s,
(DMSO-d6) d 180.11, 169.76, 163.41, 158.36, 157.47, 151.33, 146.22,
1H, CONH), 9.02 (s,1H, CONH), 8.13 (d, J ¼ 0.8 Hz,1H, Pyrimidine Ar-
H), 7.59 (dd, J ¼ 12.8, 2.4 Hz, 1H, phenyl Ar-H), 7.21 (t, J ¼ 8.8 Hz, 1H,
phenyl Ar-H), 7.16e7.13 (m, 1H, phenyl Ar-H), 6.51 (s, 1H, Isoxazole
Ar-H), 6.34 (d, J ¼ 0.8 Hz, 1H, Pyrimidine Ar-H), 3.61 (t, J ¼ 5.2 Hz,
4H, Piperidine CH2), 1.63 (q, J ¼ 6.4 Hz, 2H, Piperidine CH2),
1.55e1.52 (m, 4H, Piperidine CH2), 1.30 (s, 9H, CH3)$13C NMR
135.89, 130.55, 122.29, 120.92, 117.25, 92.39, 84.99, 44.61, 32.43,
28.31, 24.99, 24.07, 16.11. HRMS (ESI) calcd. for C24H31N6O3þ
[M
þ
H]þ, 451.2452; found, 451.2446. HPLC purity >98%,
Rt ¼ 15.6 min.
4.2.41. 1-(5-(tert-butyl)isoxazol-3-yl)-3-(3-methyl-4-((6-
morpholinopyrimidin-4-yl)oxy)phenyl) urea (9g)
(DMSO-d6)
d 180.23, 169.19, 163.37, 158.20, 157.31, 153.77 (d,
J ¼ 244.2 Hz), 151.31, 137.21 (d, J ¼ 10.0 Hz), 134.37 (d, J ¼ 12.7 Hz),
124.19, 114.57 (d, J ¼ 3.0 Hz), 106.76 (d, J ¼ 23.5 Hz), 92.46, 85.13,
44.70, 32.44, 28.31, 25.01, 24.09. HRMS (ESI) calcd. for C23H28FN6O3þ
Yield: 51%, m.p.: 217.3e218.2 ꢀC. 1H NMR (DMSO-d6)
d 9.50 (s,
1H, CONH), 8.78 (s, 1H, CONH), 8.17 (s, 1H, Pyrimidine AreH), 7.39
(d, J ¼ 2.4 Hz, 1H, phenyl AreH), 7.28 (dd, J ¼ 8.8, 2.8 Hz, 1H, phenyl
AreH), 6.97 (d, J ¼ 8.8 Hz, 1H, phenyl AreH), 6.50 (s, 1H, Isoxazole
AreH), 6.24 (s, 1H, Pyrimidine AreH), 3.67e3.65 (m, 4H, Morpholin
CH2), 3.57e3.55 (m, 4H, Morpholin CH2), 2.06 (s, 3H, CH3), 1.30 (s,
[M
þ
H]þ, 455.2201; found, 455.2199. HPLC purity >99%,
Rt ¼ 16.1 min.
4.2.37. 1-(5-(tert-butyl)isoxazol-3-yl)-3-(3-fluoro-4-((6-
morpholinopyrimidin-4-yl)oxy)phenyl) urea (9c)
Yield: 59%, m.p.: 219.7e221.2 ꢀC. 1H NMR (DMSO-d6)
9H, CH3)$13C NMR (DMSO-d6)
d 180.12, 169.75, 164.01, 158.36,
157.43, 151.33, 146.11, 135.99, 130.53, 122.29, 120.94, 117.27, 92.39,
85.54, 65.70, 43.97, 32.43, 28.31, 16.10. HRMS (ESI) calcd. for
d
9.60 (s,
1H, CONH), 9.03 (s,1H, CONH), 8.18 (d, J ¼ 0.8 Hz,1H, Pyrimidine Ar-
H), 7.60 (dd, J ¼ 12.8, 2.4 Hz, 1H, phenyl Ar-H), 7.22 (t, J ¼ 8.8 Hz, 1H,
phenyl Ar-H), 7.17e7.14 (m, 1H, phenyl Ar-H), 6.50 (s, 1H, Isoxazole
Ar-H), 6.39 (s, 1H, Pyrimidine Ar-H), 3.68e3.66 (m, 4H, Morpholin
CH2), 3.59 (q, J ¼ 4.8, 4.0 Hz, 4H, Morpholin CH2), 1.30 (s, 9H,
C
23H29N6Oþ4 [MþH]þ, 453.2245; found, 453.2239. HPLC purity
>99%, Rt ¼ 7.7 min.
4.2.42. 1-Cyclopropyl-3-(2-methyl-4-((6-(methylamino)pyrimidin-
4-yl)oxy)phenyl)urea (10a)
CH3)$13C NMR (DMSO-d6)
d
180.24, 169.17, 163.97, 158.19, 157.29,
Yield: 40%, m.p.: 208.6e209.4 ꢀC. 1H NMR (DMSO-d6)
d 8.11 (brs,
153.72 (d, J ¼ 244 Hz), 151.30, 137.32 (d, J ¼ 10 Hz), 134.25 (d,
J ¼ 13 Hz), 124.17, 114.59 (d, J ¼ 3 Hz), 106.76 (d, J ¼ 24 Hz), 92.47,
85.67, 65.70, 44.04, 32.45, 28.31. HRMS (ESI) calcd. for C22H26FN6O4þ
1H, Pyrimidine Ar-H), 7.75 (d, J ¼ 8.8 Hz, 1H, phenyl Ar-H), 7.54 (s,
1H, CONH), 7.25 (q, J ¼ 4.8 Hz, 1H, CONH), 6.92 (d, J ¼ 2.8 Hz, 1H,
phenyl Ar-H), 6.87 (dd, J ¼ 8.8, 2.8 Hz, 1H, phenyl Ar-H), 6.69 (d,
J ¼ 2.8 Hz, 1H, Pyrimidine Ar-H), 5.70 (s, 1H, CH3NH), 2.75 (s, 3H,
CH3NH), 2.55 (dq, J ¼ 7.2, 3.2 Hz, 1H, Cyclopropyl CH), 2.17 (s, 3H,
CH3), 0.64 (td, J ¼ 6.8, 4.8 Hz, 2H, Cyclopropyl CH2), 0.43e0.39 (m,
[M
þ
H]þ, 457.1994; found, 457.1991. HPLC purity >99%,
Rt ¼ 8.0 min.
4.2.38. 1-(5-(tert-butyl)isoxazol-3-yl)-3-(2-methyl-4-((6-
(methylamino)pyrimidin-4-yl)oxy)phenyl) urea (9d)
Yield: 50%, m.p.: 131.5e134.5 ꢀC. 1H NMR (DMSO-d6)
2H, Cyclopropyl CH2)$13C NMR (DMSO-d6)
d 169.27, 165.05, 158.00,
156.13, 147.37, 135.10, 129.27, 122.80, 122.19, 118.87, 27.40, 22.39,
17.74, 6.36. HRMS (ESI) calcd. for C16H20N5Oþ2 [M þ H]þ, 314.1612;
found, 314.1615. HPLC purity >99%, Rt ¼ 4.5 min.
d
10.05 (s,
1H, CONH), 8.62 (s, 1H, CONH), 8.21 (m, 1H, Pyrimidine AreH), 7.83
(d, J ¼ 8.8 Hz, 1H, phenyl AreH), 7.60 (s, 1H, phenyl AreH), 7.02 (d,
J ¼ 2.4 Hz, 1H, phenyl AreH), 6.96 (dd, J ¼ 8.8, 2.8 Hz, 1H, Isoxazole
AreH), 6.47 (s, 1H, Pyrimidine AreH), 5.78 (s, 1H, CH3NH), 2.79 (s,
3H, CH3NH), 2.27 (s, 3H, CH3), 1.30 (s, 9H, CH3).13C NMR (DMSO-d6)
4.2.43. 1-(2-Methyl-4-((6-(methylamino)pyrimidin-4-yl)oxy)
phenyl)-3-(thiazol-2-yl)urea (10b)
Yield: 38%, m.p.: 203.1e205.0 ꢀC. 1H NMR (DMSO-d6)
d 10.83 (s,
d
180.02, 164.35, 158.45, 156.85, 151.70, 147.93, 134.12, 130.14,
1H, CONH), 8.41 (s, 1H, CONH), 8.13 (brs, 1H, Pyrimidine Ar-H), 7.83
(d, J ¼ 8.8 Hz, 1H, phenyl Ar-H), 7.39 (d, J ¼ 3.6 Hz, 1H, Thiazole Ar-
H), 7.29 (q, J ¼ 4.8 Hz, 1H, phenyl Ar-H), 7.12 (d, J ¼ 3.6 Hz, 1H,
Thiazole Ar-H), 7.02 (d, J ¼ 2.8 Hz,1H, phenyl Ar-H), 6.96 (dd, J ¼ 8.8,
2.8 Hz, 1H, Pyrimidine Ar-H), 5.75 (s, 1H, CH3NH), 2.77 (s, 3H,
122.94, 122.54, 118.97, 92.35, 32.41, 28.32, 27.59, 17.88. HRMS (ESI)
calcd. for C20H25N6Oþ3 [M þ H]þ, 397.1983; found, 397.1985. HPLC
purity >99%, Rt ¼ 6.5 min.
4.2.39. 1-(5-(tert-butyl)isoxazol-3-yl)-3-(2-methyl-4-((6-
morpholinopyrimidin-4-yl)oxy)phenyl) urea (9e)
CH3NH), 2.25 (s, 3H, CH3)$13C NMR (DMSO-d6)
d 165.09, 159.50,
157.96, 151.70, 148.44, 137.51, 133.44, 130.09, 123.10, 122.51, 119.17,
112.36, 27.43, 17.67. HRMS (ESI) calcd. for C16H17N6O2Sþ [M þ H]þ,
357.1128; found, 357.1131. HPLC purity >99%, Rt ¼ 4.3 min.
Yield: 51%, m.p.: 205.5e208.5 ꢀC. 1H NMR (DMSO-d6)
d 9.85 (s,
1H, CONH), 8.29 (s, 1H, CONH), 8.20 (s, 1H, Pyrimidine AreH), 7.80
(d, J ¼ 8.8 Hz, 1H, phenyl AreH), 6.99 (d, J ¼ 2.8 Hz, 1H, phenyl
AreH), 6.92 (dd, J ¼ 8.8, 2.8 Hz, 1H, phenyl AreH), 6.45 (s, 1H,
Isoxazole AreH), 6.26 (s, 1H, Pyrimidine AreH), 3.67e3.65 (m, 4H,
Morpholin CH2), 3.57e3.55 (m, 4H, Morpholin CH2), 2.24 (s, 3H,
4.2.44. 1-(benzo[d]thiazol-2-yl)-3-(2-methyl-4-((6-(methylamino)
pyrimidin-4-yl)oxy)phenyl)urea (10c)
Yield: 41%, m.p.: 257.9e260.2 ꢀC. 1H NMR (DMSO-d6)
d 11.23
CH3), 1.29 (s, 9H, CH3).13C NMR (DMSO-d6)
d
180.00, 168.38, 163.25,
(brs, 1H, CONH), 8.86 (s, 1H, CONH), 8.15 (brs, 1H, Pyrimidine Ar-H),
7.92 (d, J ¼ 8.0 Hz, 1H, benzothiazole Ar-H), 7.83 (d, J ¼ 8.8 Hz, 1H,
benzothiazole Ar-H), 7.69 (d, J ¼ 8.0 Hz, 1H, benzothiazole Ar-H),
158.44, 155.75, 151.72, 147.65, 134.27, 130.04, 122.80, 122.38, 118.82,
92.36, 86.00, 65.62, 44.37, 32.41, 28.32, 17.97. HRMS (ESI) calcd. for
Please cite this article in press as: Y. Chen, et al., Integrated bioinformatics, computational and experimental methods to discover novel Raf/
extracellular-signal regulated kinase (ERK) dual inhibitors against breast cancer cells, European Journal of Medicinal Chemistry (2016),
http://dx.doi.org/10.1016/j.ejmech.2016.11.009

Y. Chen et al. / European Journal of Medicinal Chemistry xxx (2016) 1e15
13
7.42e7.38 (m, 1H, benzothiazole Ar-H), 7.31 (q, J ¼ 4.8 Hz, 1H,
5 min and resuspended in 500
m
L of binding buffer (1x), containing
phenyl Ar-H), 7.25 (t, J ¼ 7.2 Hz, 1H, phenyl Ar-H), 7.05 (d, J ¼ 2.8 Hz,
1H, phenyl Ar-H), 6.99 (dd, J ¼ 8.8, 2.8 Hz, 1H, Pyrimidine Ar-H),
5.77 (d, J ¼ 4.8 Hz, 1H, CH3NH), 2.78 (s, 3H, CH3NH), 2.31 (s, 3H,
5
mL of Annexin V-FITC and 5 mL of propidium iodide (PI), and
incubated for 15 min at room temperature. The samples were then
measured by FCM (BD FACS Calibur, BD, USA) using the FL1 channel
for Annexin V-FITC and the FL2 channel for PI. Both early apoptotic
(Annexin Vþ/PI-) and late apoptotic (Annexin Vþ/PIþ) cells were
included in cell apoptosis determinations.
CH3)$13C NMR (DMSO-d6)
d 169.57, 165.62, 159.90, 158.47, 152.29,
149.25, 133.70, 131.83, 131.15, 126.40, 123.65, 123.49, 123.36, 121.94,
120.36, 119.70, 27.92, 18.30. HRMS (ESI) calcd. for C20H19N6O2Sþ
[M
þ
H]þ, 407.1285; found, 407.1283. HPLC purity >99%,
Rt ¼ 5.4 min.
4.8. Western blot analysis
4.3. Proteomics-based PPI network construction
All the primary antibodies were purchased from Cell Signaling
Technology or Santa Cruz Biotechnology. After various treatments
as indicated in the figure legends, MCF-7 cells were harvested by
trypsinization and then washed with cold PBS. The cells were lysed
in RIPA buffer (Invitrogen, CA, USA) on ice for 30 min followed by
sonication denaturation. Cell lysates were then centrifuged at
13,000 g for 30 min at 4 ꢀC. Supernatant was collected, and protein
concentration was determined using a bicinchoninic acid protein
assay kit (Thermo, USA). The protein was applied to a 10e15% SDS-
polyacrylamide gel, transferred to a nitrocellulose membrane, and
then detected by the proper primary and secondary antibodies
before visualization by chemiluminescence Kit (Millpore, USA).
To build the RAF/MEK/ERK kinase proteineprotein interaction
(PPI) network, we collected diverse sets of biological evidence from
seven online databases. Different PPIs were collected fromthe
Database of Interacting Proteins (DIP), Biomolecular Object
Network Databank (BOND), Human Protein Reference Database
(HPRD), HomoMINT, IntAct, BioGRID and PrePPI. Moreover, we
extract the RAF/MEK/ERK subnetwork from the PPI network based
on the ITRAQ-based proteomics results.
4.4. Sequence alignment and molecular docking
The sequence alignment between Raf1 and ERK1 were perform
by the MODELER module embedded in the Accelrys Discovery
Studio 2.5 software packages (BIOVIA, San Diego, CA, USA). The
initial coordinates of the X-ray crystal structure of Raf1 (PDB No.
3OMV) and ERK1 (PDB No. 4QTB) were downloaded from the
Protein Data Bank (http://www.rcsb.org). Moreover, the ligandfit
module embedded in the Accelrys Discovery Studio was used for
the generation of binding conformations of compound 9d into Raf1
and ERK1, respectively.
Competing interests
The authors declare no competing interests.
Acknowledgements
We are grateful for financial support from the National
Natural Science Foundation of China (81273471), China Post-
doctoral
Science
Foundation
(No.
2016M602696
and
2016M592679) and the Fundamental Research Funds for the Cen-
tral Universities and Distinguished Young Scholars of Sichuan
University (2015SCU04A13). The sequence alignment, structure
superposed results of Raf1 and ERK1, NMR spectrum and KINO-
MEscan profiling results were supplied as Supporting Information.
This material is available free of charge via the Internet.
4.5. Kinase inhibition assay and KINOMEscan profiling
Kinase inhibition assays were using KinaseProfiler services
provided by Eurofins, and ATP concentrations used are the ATP Km
of corresponding kinases. The KINOMEScan selectivity profiling
data of compound 9d was determined by DiscoveRx Co. Ltd., which
was obtained from manufacturer's protocol. The results were
shown in Table S1.
List of abbreviations
Raf
ERK
RAF proto-oncogene serine/threonine-protein kinase
Extracellular signaleregulated kinases
Mitogen-activated protein kinase kinase
Mitogen-activated protein kinase
isobaric tags for relative and absolute quantitation
Protein-protein interaction
4.6. Cell proliferation assay
MEK
MAPK
iTRAQ
PPI
FasL
FADD
Bax
The cellular proliferation activities of compounds were evalu-
ated in MCF-7 and MD-MBA-231 breast cancer cells. In general,
cancer cells seeded in 96-well plates were treated by a series of
concentration for 48 h. The mean percentage of cell survival rates
were determined from data of three individual experiments, and all
the data were expressed as mean value. The cells viability was
evaluated by adding the MTT solution (5 mg/ml) to the cells and
incubating the cells for 3 h. After that, the MTT solution was
removed and the formazan crystals were dissolved in DMSO. The
absorbance of the solution was measured at 570 nm. The control
group consisted of untreated cells.
Fas ligand
Fas-Associated protein with Death Domain
Bcl-2-associated X protein
B-cell lymphoma 2
Bcl-2
TEA
Triethylamine
DMAP 4-dimethylaminopyridine.
Appendix A. Supplementary data
4.7. Apoptosis assay
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.11.009.
Apoptosis induction assay of compound 9d and BL-EI001 were
carried out on MCF-7 cells. The flow cytometric (FCM) analysis was
used to confirm the apoptotic induction effect of different micelles.
Apoptosis of MCF-7 cells treated with compound 9d, BL-EI001 or
saline were determined were gently trypsinized without EDTA and
centrifuged at 2000g for five minutes. Then, the harvested cells
were washed with 1.0 mL cold PBS by centrifugation at 2000g for
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http://dx.doi.org/10.1016/j.ejmech.2016.11.009

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