Bisarylureas based on 1H-Pyrazolo[3,4-d]pyrimidine Scaffold as Novel Pan-RAF inhibitors with potent anti-proliferative activities: Structure-based design, synthesis, biological evaluation & molecular modelling studies

Article abstract of DOI:10.3390/molecules22040542

RAF (Ras activating factor) kinases are important and attractive targets for cancer therapy. With the aim of discovering RAF inhibitors that bind to DFG-out inactive conformation created by the movement of Asp-Phe-Gly (DFG), we conducted structure-based drug design using the X-ray cocrystal structures of BRAF (v-raf murine sarcoma viral oncogene homolog B1), starting from bisarylurea derivative based on 1H-pyrazolo[3,4-d]pyrimidine scaffold 1a. Most of the synthesized compounds showed good to excellent inhibitory activities against BRAFV600E kinase, possessed moderate to potent anti-proliferative activities against four tumor cell lines (A375, HT-29, PC-3 and A549) and good selectivity towards cancer cells rather normal cells (Madin-Darby canine kidney, MDCK). The most promising compound, 1v, exhibited potent inhibitory activity against not only BRAFV600E (half maximal inhibitory concentration, IC50 = 23.6 nM) but also wild-Type BRAF (IC50 = 51.5 nM) and C-RAF (IC50 = 8.5 nM), and effective cellular anti-proliferative activities against A375, HT-29, PC-3 and A549 cell lines as well as a very good selectivity profile. Moreover, compound 1v mainly arrested the A375 cell line in the G0/G1 stage, and showed significant suppression of MEK (mitogen-Activated protein kinase kinase) phosphorylation in A375 and HT-29 cell lines. Taken together, the optimal compound 1v showed excellent in vitro potency as a pan-RAF inhibitor. In addition, the promise of compound 1v was further confirmed by molecular dynamics simulation and binding free energy calculations.

Full text of DOI:10.3390/molecules22040542

molecules
Article
Bisarylureas Based on 1H-Pyrazolo[3,4-d]pyrimidine
Scaffold as Novel Pan-RAF Inhibitors with Potent
Anti-Proliferative Activities: Structure-Based Design,
Synthesis, Biological Evaluation and Molecular
Modelling Studies
Yu Fu, Yuanyuan Wang, Shanhe Wan, Zhonghuang Li, Guangfa Wang, Jiajie Zhang * and
Xiaoyun Wu *
Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Science, Southern
Medical University, Guangzhou 510515, China; fuyuky0312@163.com (Y.F.); 15626047153@163.com (Y.W.);
wansh@fimmu.com (S.W.); lzhuang@fimmu.com (Z.L.); wguangfa@smu.edu.cn (G.W.)
* Correspondence: zhangjj@smu.edu.cn (J.Z.); xywugz@163.com (X.W.);
Tel.: +86-20-6164-8548 (J.Z.); +86-20-6278-9416 (X.W.)
Academic Editor: Michael Gütschow
Received: 22 February 2017; Accepted: 24 March 2017; Published: 29 March 2017
Abstract: RAF (Ras activating factor) kinases are important and attractive targets for cancer therapy.
With the aim of discovering RAF inhibitors that bind to DFG-out inactive conformation created by
the movement of Asp-Phe-Gly (DFG), we conducted structure-based drug design using the X-ray
cocrystal structures of BRAF (v-raf murine sarcoma viral oncogene homolog B1), starting from
bisarylurea derivative based on 1H-pyrazolo[3,4-d]pyrimidine scaffold 1a. Most of the synthesized
compounds showed good to excellent inhibitory activities against BRAFV600E kinase, possessed
moderate to potent anti-proliferative activities against four tumor cell lines (A375, HT-29, PC-3
and A549) and good selectivity towards cancer cells rather normal cells (Madin-Darby canine
kidney, MDCK). The most promising compound, 1v, exhibited potent inhibitory activity against not
only BRAFV600E (half maximal inhibitory concentration, IC50 = 23.6 nM) but also wild-type BRAF
(IC50 = 51.5 nM) and C-RAF (IC50 = 8.5 nM), and effective cellular anti-proliferative activities against
A375, HT-29, PC-3 and A549 cell lines as well as a very good selectivity profile. Moreover, compound
1v mainly arrested the A375 cell line in the G0/G1 stage, and showed significant suppression
of MEK (mitogen-activated protein kinase kinase) phosphorylation in A375 and HT-29 cell lines.
Taken together, the optimal compound 1v showed excellent in vitro potency as a pan-RAF inhibitor.
In addition, the promise of compound 1v was further confirmed by molecular dynamics simulation
and binding free energy calculations.
Keywords: DFG-out; pan-RAF inhibitor; 1H-pyrazolo[3,4-d]pyrimidine derivatives; biological
activities; molecular docking; molecular dynamics simulation
1. Introduction
The mitogen-activated protein kinase (MAPK) cascades consist of Ras/RAF(Ras activating
factor)/MEK(mitogen-activated protein kinase kinase)/ERK(extracellular receptor kinase) signal
transduction and play an important role in the regulation of cellular activities, including proliferation,
differentiation, and survival [
viral oncogene homolog B1), and CRAF (RAF-1), which play central roles in this cascade [
them, BRAF isoform is more easily activated by Ras and has higher basal kinase activity than the
1
,2]. There are three RAF isoforms, ARAF, BRAF (v-raf murine sarcoma
3,4
]. Among
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other two isoforms, which provides a reasonable explanation for the mutational activation of BRAF
frequently observed in human tumors. The BRAF genetic mutation is present in about seven percent
of all human cancers, and the most common mutation BRAFV600E is involved in various carcinomas,
including metastatic melanoma, thyroid cancer, and colon cancer [
results in constitutive kinase activity 500-fold greater than wild type BRAF [
correlates with the increase of malignancy and the decrease of response to chemotherapy [7,8
5
]. This V600E point mutation
], which also closely
]. Hence,
6
targeting Ras/RAF/MEK/ERK signal transduction may be a legitimate approach to cancer treatment.
In the last decade, many small-molecule inhibitors targeting RAF have been developed. Sorafenib
(Figure 1) [
(FDA) for the treatment of advanced renal cell carcinoma (RCC) in 2005, unresectable hepatocellular
carcinomas (HCC) in 2007 and thyroid cancer [10]. Sorafenib is classified as a type II inhibitor [11 12],
9], as the first RAF kinase inhibitor, was approved by the Food and Drug Administration
,
which binds exclusively an inactive DFG-out conformation created by the movement of Asp-Phe-Gly
(DFG) and stabilizes its conformation. It not only binds at the adenosine triphosphate (ATP) binding
site but also extends to the hydrophobic “back pocket” of protein created by the flip of the DFG motif.
In contrast, vemurafenib [13–15] (PLX4032, Figure 1) and dabrafenib [16] (GSK-2118436, Figure 1) are
classified as type I inhibitors, which bind to the DFG-in active conformation of the ATP binding site.
However, these type I inhibitors are highly BRAF-selective. Vemurafenib and dabrafenib have been
reported to cause rapid development of squamous cell carcinoma (SCC) and keratoacanthoma, which
may be caused by paradoxical activation of the MAPK pathway by a selective inhibitor in cells bearing
wild-type (WT) BRAF [17–19]. This feedback activation was significantly suppressed by DFG-out
type pan-RAF inhibitors, but not by DFG-in type inhibitors. Therefore, we aimed at discovering
DFG-out-type pan-RAF inhibitors.
Figure 1. BRAF inhibitors.
The 1H-pyrazolo[3,4-d]pyrimidine nucleus is an important drug-like scaffold present in many
pharmacologically active compounds, and the antitumor activity was initially reported many years
ago [20]. Having similar structure of the purines, 1H-pyrazolo[3,4-d]pyrimidine, a template of tyrosine
kinase hinge-binding, was extensively chemically modified to improve the biological activity and
kinase selectivity. To find potent RAF inhibitors, encouraged by the success of Sorafenib, we designed
Sorafenib analogue 1a by replacing the pyridine ring of Sorafenib with 1H-pyrazolo[3,4-d]pyrimidine
moiety (Figure 2). To the best of our knowledge, although the inhibitory activities of 1a and its
analogues targeting VEGFR-2 (vascular endothelial growth factor receptor 2) and FLT3 (fms-related
tyrosine kinase 3) have been reported [21], the RAF inhibitory activities of these derivatives are being
reported for the first time by our group. Based on the promising activities as VEGFR-2 and FLT3
inhibitors with potent in vitro and in vivo activity against acute myeloid leukemia, we would like to
examine the RAF inhibitory activities further. Model of 1a in the active site of BRAFV600E (Protein
Data Bank (PDB) code: 1UWJ), which was reported to be in the DFG-out inactive conformation,
was constructed by Surflex–Dock according to our previous protocol [22]. As shown in Figure 3,
the model suggested that 1a overlapped well with the co-crystal ligand sorafenib in the active site
of BRAFV600E. The 1H-pyrazolo[3,4-d]pyrimidine ring in 1a occupied the ATP-binding pocket, and
formed hydrogen bonds with the hinge region of BRAFV600E. The diaryl urea portion extended into the

Molecules 2017, 22, 542
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large hydrophobic back pocket created by the rearrangement of the DFG motif, and the urea CO and
NH moiety formed a hydrogen bond with the backbone-NH of Asp 594 and side-chain carboxylate of
Glu 501 in the DFG motif, respectively. These suggested that the diaryl urea contributes significantly
to the inhibitor´s affinity. Considering the importance of the diaryl urea for the potency of kinase, we
decided to maintain the diaryl urea portion in our inhibitors and tried to focus on the replacement of
the terminal phenyl to investigate their effects on kinase inhibitory activities in this paper. In order to
illustrate the effect of the N1 hydrogen on the inhibitory activity, N1-methylated compounds were
also designed. Herein, we would like to report the synthesis and biological evaluation of these series
compounds as novel pan-BRAF inhibitors. The molecular modelling study was also discussed in
this paper.
Figure 2. The design of target compounds.
Figure 3. Overlap of the docked conformation of 1a (green) and crystal structure of Sorafenib (cyan)
bound to BRAFV600E (Protein Data Bank (PDB) code: 1UWJ). Yellow dots represent hydrogen bonds.
(BRAFV600E in the colored cartoon, with ligands in the stick model, and the key residues in the
line model).
2. Results
2.1. Chemistry
The target compounds were prepared according to the synthetic routes outlined in Scheme 1,
which was distinct from that described previously [22]. All target compounds were prepared from the
key intermediate 4-chloro-1H-pyrazolo[3,4-d]pyrimidine (
by chlorination was evaluated and determined to be unsuitable for large scale preparation due to
poor stability of the product with respect to polymerization. We therefore synthesized compound by
cycloaddition reaction of 4,6-dichloropyrimidine-5-carbaldehyde and hydrazine monohydrate [23].
2). The preparation of 2 from allopurinol
2

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In order to avoid the side reaction of 1-NH of compound
2, we designed the pyran-protected
compound
with 4-aminophenol in the present of Cs2CO3 gave the phenoxylated derivatives
of with the corresponding phenyl isocyanate in CH2Cl2 provided the urea in moderate yields.
Finally, compound was deprotected using 4 M HCl in dioxane to produce the target compounds
1a 1t in high yields. N1-methylated compounds 1u and 1v were also prepared from intermediate
. Compound was methylated using iodomethane in the presence of sodium hydride to obtain
compound , with subsequent nucleophilic substitution with 4-aminophenol in the presence of Cs2CO3
to give the phenoxylated derivatives in good yield. Finally, condensation with the corresponding
phenyl isocyanate provided the target compounds 1u and 1v in high yields.
3. Compound
3
was synthesized from compound
2
and then nucleophilic substitution
4
. Condensation
4
5
5
–
2
2
6
7
Scheme 1. Synthet◦ic route to compounds
EtOAc, 50 ◦C, overnight, 76.5%; (c) 4-Aminophenol, Cs2CO3, DMF (dimethylformamide), N2, room
temperature (r.t.), 79.5%; (d) Phenyl isocyanate, CH2Cl2, ice bath, overnight, 60.2–80.0%; (e) 4 M
1. Reagents and conditions: (a) Hydrazine monohydrate,
65 C, 2–3 h, 68.9%; (b) 3,4-Dihydro-2H-pyran, PPTs (pyridinium 4-toluenesulfonate),
Et3N, MeOH,
−
◦
HCl/1,4-dioxane, r.t., overnight, 54.2–92.6%; (f) Iodomethane, NaH, DMF, 0 C, 30 min, 69.5–70.8%.
2.2. Kinase Inhibitory Activity
The enzyme activities are summarized in Table 1. Based on the results of molecular docking,
we focused on optimization of the terminal phenyl ring to improve the kinase inhibitory activities.
The effects of ortho-, meta-, and para-substitution and disubstitution on the terminal phenyl were
investigated. Several trends can be observed from Table 1. The results displayed that the
4-chloro-3-trifluomethylphenyl group is important for potency against BRAFV600E, which is consistent
with the molecular modelling that the terminal phenyl group in 1a projects into a large hydrophobic
pocket created by the rearrangement of the DFG motif. As we expected, compound 1e, bearing no
substitution on the terminal phenyl, displayed no inhibitory activity even at 1000 nM, which clearly
indicated the importance of the hydrophobic interaction between the inhibitor and the hydrophobic
amino acid residue of the back pocket. A small group of ortho-substitution such as fluoro is tolerated,
whereas large groups are not tolerated. For example, compounds 1f and 1r, which bear 2-Cl and
2-NO2, are less potent than the parent compound 1b. A variety of substituents at the meta-position,

Molecules 2017, 22, 542
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including both electron-donating and electron-withdrawing groups are tolerated, which suggested
that the electronic interaction of the substituent had little effect on the kinase inhibitory activities.
For example, introduction of electron-donating groups methyl and ethyl in compounds 1b and 1l
increase the activities against BRAFV600E. Substitution with electron-withdrawing groups such as
Cl and Br on the phenyl ring also leads to enhancement of kinase inhibitory activities (1g and 1s).
Substitution at the para-position is also tolerated. For example, compound 1c with 4-chloro substitution
is more potent than compound 1g. In addition, di-substitution with two electron-withdrawing groups
such as F, Cl and CF3 on the phenyl ring leads to dramatic improvement of kinase inhibitory activities
(1a, 1c, 1i, 1j and 1n).
Table 1. The kinase inhibitory activities of synthesized compounds.
Structure
BRAFV600E (%) a
1000 nM 100 nM
90
Compound
R1
R2
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
3-CF3-4-Cl
3-Me
3,4-di-Cl
4-Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
37
N.T b
27
46
83
51
1
N.T
N.T
N.T
N.T
N.T
52
H
2-Cl-5-Me
3-Cl
37
54
27
83
67
42
69
62
87
69
48
32
22
71
89
93
97
2,3-di-Me
2-Cl-5-CF3
3-F-5-CF3
3-Me-4-F
3-Et
3-NO2
2-F-5-CF3
3-SMe
37
N.T
60
3
30
29
3-CN
N.T
N.T
N.T
10
3-CO2Me
2-NO2-5-Me
3-Br
3,4-di-Me
3-Cl
1s
1t
1u
1v
28
61
81
3-CF3-4-Cl
Sorafenib
94
75
a
Values are the average of two independent experiments; b N.T: Not Tested.
The modelling suggested that 1a formed hydrogen bonds with the hinge region of BRAFV600E
and elaboration at the interaction at NH position might not be tolerated. Surprisingly, the
N-methylation compounds 1u and 1v exhibited higher inhibitory activities against BRAFV600E than
the unsubstituted counterparts 1g and 1a, implying that the methyl substitution at the N1 position of
1H-pyrazolo[3,4-d]pyrimidine was beneficial for the inhibitory activity of BRAFV600E. The favorable
hydrophobic interaction imposed by the N1-methyl might be responsible for the improvement
in potency.
Subsequently, we further evaluated the inhibitory activities of compounds 1l
exhibited excellent inhibitory activities at 1000 nM and 100 nM (Table 2). As shown in Table 2, 1l
and 1v exhibited stronger inhibitory activities against BRAFV600E than the positive control Sorafenib.
,
1u and 1v, which
,
1u

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Table 2. The kinase inhibitory activities of selected compounds.
Structure
R1
Kinase Tested (IC50, nM) a
BRAFV600E
Compound
R2
1l
1u
1v
3-Et
3-Cl
3-CF3-4-Cl
H
Me
Me
51.3
38.1
23.6
52.3
Sorafenib
a
Values are the average of two independent experiments. IC50: half maximal inhibitory concentration.
2.3. In Vitro Anti-Proliferative Activity
The cell growth inhibitory activities of newly synthesized compounds were evaluated against
four human cancer cell lines, including wild-type BRAF cell lines (PC-3 and A549), and BRAFV600E cell
lines (A375, HT-29) using standard MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide) assay in vitro, with Sorafenib as the positive control (Table 3). Additionally, cytotoxic
assays were conducted using normal cell line MDCK (Madin-Darby canine kidney). As depicted in
Table 3, most compounds showed moderate to good anti-proliferative activities, while compounds
1e and 1r proved to be ineffective against all four cell lines (half maximal inhibitory concentration,
IC50 > 80
µM). Some of the synthesized compounds exhibited similar anti-proliferative activities
compared with Sorafenib against two or more cell lines. Remarkably, compounds 1c
,
1f 1h 1i 1o
,
,
,
and 1s demonstrated much higher anti-proliferative activities than the positive control Sorafenib
against the PC-3 cell line. In particular, compounds 1f and 1i represented a 12-fold improvement in
activity compared to Sorafenib with the IC50 value of 1.12 µM. Meanwhile, compound 1f exhibited a
2-fold improvement compared to Sorafenib in inhibiting A549 cell line proliferation. Compound 1n
displayed stronger anti-proliferative activities than Sorafenib against the A375 cell line. Unfortunately,
the results indicated that most of the compounds were ineffective (IC50 > 80
In addition, the results demonstrated that compounds with two electron-withdrawing substituents
on the terminal phenyl ring (1a 1c 1i, and 1n) also presented promising anti-proliferative activities.
It is worth noting that most compounds indicated no inhibitory activity against MDCK cell lines
(IC50 > 80 M), which suggested that the compounds exhibit good selectivity towards cancer cells
rather normal cells.
µM) on the HT-29 cell line.
,
,
µ
Table 3. The in vitro anti-proliferative activity (IC50, µM) a.
Compound
R1
R2
A375
HT-29
PC-3
A549
MDCK
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
3-CF3-4-Cl
3-Me
3,4-di-Cl
4-Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
29.28
>80
29.70
78.82
>80
62.27
>80
>80
26.95
30.45
>80
75.57
64.04
5.39
>80
61.78
>80
>80
>80
15.17
>80
>80
61.71
>80
19.53
>80
11.16
18.35
>80
19.75
75.60
16.18
>80
>80
5.24
17.76
>80
35.47
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
23.12
>80
>80
>80
>80
>80
>80
>80
>80
14.67
H
2-Cl-5-Me
3-Cl
1.12
>80
70.12
>80
2,3-di-Me
2-Cl-5-CF3
3-F-5-CF3
3-Me-4-F
3-Et
3-NO2
2-F-5-CF3
3-SMe
>80
11.72
1.12
45.98
22.71
>80
23.42
40.28
5.34
30.91
22.54
>80
5.52
>80
68.85
23.41
13.79
62.41
50.66
>80
71.25
31.68
27.82
>80
39.39
>80
>80
>80
>80
>80
14.72
8.12
60.83
60.32
62.10
19.93
>80
32.09
29.97
59.48
>80
>80
>80
>80
>80
3-CN
3-CO2Me
2-NO2-5-Me
3-Br
3,4-di-Me
3-Cl
>80
1s
1t
1u
1v
65.44
75.23
13.20
12.21
6.81
3-CF3-4-Cl
12.49
13.72
Sorafenib
a
IC50 values are presented as mean values of three independent experiments done in quadruplicates. Coefficients
of variation were < 10%. MDCK: Madin-Darby canine kidney.

Molecules 2017, 22, 542
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2.4. Kinase Inhibitory Profiles of Compound 1v
Compound 1v exhibited higher inhibitory activity against BRAFV600E than positive control
Sorafenib with IC50 values of 23.6 nM, effective cellular anti-proliferative potencies against four
tumor cell lines (A375, HT-29, PC-3, A549) which were comparable to the efficacy of Sorafenib, as well
as better selection to normal cells compared with Sorafenib. Therefore, we selected compound 1v
for further study. The kinase selectivity profile of compound 1v was assessed over 19 different
protein kinases (Table 4). The results exhibited that 1v had potent inhibitory activity against not only
BRAFV600E (IC50 = 23.6 nM) but also wild-type BRAF (IC50 = 51.5 nM) and C-RAF (IC50 = 8.5 nM), and
almost no significant inhibitory activity against 13 other tested protein kinases even at concentration of
1000 nM. The results revealed that compound 1v had a very good selectivity profile and was a pan-RAF
kinases inhibitor.
Table 4. Kinase selectivity of compound 1v.
Kinase Tested
Inhibition % at 1 µM
Kinase Tested
Inhibition % at 1 µM
BRAF (wt)
RAF1 (CRAF)
ABL2 (Arg)
EGFR (ErbB1)
ErbB2 (HER2)
ErbB4 (HER4)
PTK2 (FAK)
FLT1 (VEGFR1)
MET (c-Met)
VEGFR-2
105 (51.5) a
EPHA2
FLT4 (VEGFR3)
ABL1
2
58
5
102 (8.5) a
4
8
ALK
7
−7
AURKA (Aurora A)
AURKB (Aurora B)
LCK
17
42
20
3
7
13
31
6
LTK (TYK1)
MST1R (RON)
−2
56
a
IC50 values are presented as mean values of two independent experiments done in quadruplicates. Coefficients of
variation were < 10%. RAF: Ras activating factor; ABL: Abl family nonreceptor tyrosine kinase; EGFR: epidermal
growth factor receptor; HER: human epidermal growth factor receptor; FAK: focal adhesion kinase; FLT: fms-like
tyrosine kinase; MET: mesenchymal-epithelial transition; VEGFR: vascular endothelial growth factor receptor; ALK:
anaplastic lymphoma kinase; AURK: Aurora kinase; LCK: lymphocyte kinase; MST1R: macrophage-stimulating
1 receptor.
2.5. Flow Cytometric Analysis of Cell-Cycle Arrest
To study the effect of the target compounds on cell cycle progression, a flow-activated cell sorting
analysis was performed to examine the cell cycle progression upon treatment with the representative
compound 1v which exhibited most promising kinase inhibitory and anti-proliferative activities.
As shown in Figure 4 and Table 5, cell cycle assay of the A375 cell line following treatment with 1v
showed a dose-dependent cell cycle arrest in the ‘G0/G1’ phase and a decrease in ‘G2/M’ phase of cell
cycle in the treated group compared to the control.
Table 5. Effect of compound 1v on cell cycle distribution in the A375 cell line.
Concentration
Sub-G1 (%)
G0/G1 (%)
S (%)
G2/M (%)
0
1.25
4.39
2.12
5.48
39.43
42.53
47.94
52.73
38.68
38.25
38.13
36.19
19.09
10.94
7.39
6.11 µmol/L
12.21 µmol/L
24.42 µmol/L
5.48

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Figure 4. Flow cytometry analysis of cell cycle distribution in an A375 cell line treated with 1v for 48
h. ( ) Control; ( ) 6.11 mol/L (1/2 IC50); ( ) 12.21 mol/L (IC50); ( ) 24.42 mol/L (2 IC50). PI-A:
Propidium iodide-Annexin V.
a
b
µ
c
µ
d
µ
×
2.6. Signaling Inhibition in A375 and HT-29 Cell Lines
To determine the potency of 1v as a RAF inhibitor, we preliminary investigated the in vitro
MAPK signal suppression effect of 1v in A375 and HT-29 cell lines. The effect of compound 1v on
the expression of phosphor-MEK protein (RAF’s downstream target protein) in A375 and HT-29 cell
lines was assessed by western blot analysis. As shown in Figure 5, 1v suppressed phosphor-MEK
levels in a concentration-dependent manner in two cancer cell lines, reflecting the inhibition of the
downstream molecules in MAPK signal transduction. Notably, the inhibitory potency of compound
1v on MEK phosphorylation was not so conspicuous at IC50 concentration, which may be related
to the complicated mechanism of tumor cell growth and MEK phosphorylation. On one hand, the
anti-proliferative IC50 values were determined by cell survival or proliferation, which is regulated by
several different factors [24]. On the other hand, MEK phosphorylation is regulated by not only by RAF
but also other kinases such as Mos (an upstream activator of mitogen-activated protein kinase) and
MEKK-1 (mitogen activated protein kinase kinase kinase 1) [25,26]. When compound 1v was applied
at its anti-proliferative IC50 concentration, the expression of MEK phosphorylation via RAF regulation
might have been just partly inhibited, and other kinases could still activate MEK phosphorylation,
which resulted in the unobvious inhibitory effects of MEK phosphorylation. However, when the

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concentration reached twice the IC50, MEK phosphorylation was obviously inhibited, implying that
the compound 1v can effectively inhibit the Ras/RAF/MEK/ERK pathway at higher concentrations.
Figure 5. Mitogen-activated protein kinase (MAPK) signal suppression effect by 48 h treatment of 1v
(western blotting) in two cancer cell lines. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was
used as a loading control. p-MEK: phosphorylated MAP kinase kinase.
2.7. Molecular Modelling
In order to analyze in depth the interaction of synthesized compounds with BRAF, we performed
a molecular dynamics (MD) study on 1a and N1-methylated compound 1v in complex with BRAFV600E
Firstly, compound 1v was also successfully docked into the active site of BRAFV600E (PDB code: 1UWJ)
using Surflex–Dock. MD simulations were then carried out on inhibitors 1a and 1v in complex with
BRAFV600E in explicit aqueous solution for 50 ns. The stability of the system under simulation was
evaluated by the root-mean-square deviation (RMSD) of the backbone atoms relating to the starting
structures (Figure 6). As can be seen in the plot, all systems were stable during the 50 ns MD simulation.
We compared the representative snapshots of each complex taken from the last 10 ns MD trajectories
with the initial structure (Figure 7). As illustrated in Figure 7, the binding modes of 1a and 1v from MD
simulated results were nearly the same as the docked structures. The 1H-pyrazolo[3,4-d]pyrimidine
moiety inserted into the deep cleft of ATP-binding site, and the diaryl urea portion extended into the
large hydrophobic back pocket created by the rearrangement of the DFG motif.
.
Figure 6. Root-mean-square deviations (RMSDs) of backbone atoms (C, Cα, and N) of protein, and the
heavy atoms of the ligand for the simulated systems.

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The hydrogen bond plays an important role in inhibitor binding to kinase. The major interactions
were the important H-bonding interactions between inhibitors 1a and 1v and with Glu 501, Asp594
and Cys532 (Table 6). The CO and NH motifs of the urea group of compounds 1a and 1v
formed hydrogen bond interactions with the backbone-NH of Asp594 and side-chain carboxylate
of Glu 501 in the DFG motif with very high hydrogen-bond occupancies, respectively. The N7 of
1H-pyrazolo[3,4-d]pyrimidine of compounds 1a and 1v formed moderate hydrogen bond interactions
with Cys532 in the hinge region of BRAFV600E. The 1H-pyrazolo[3,4-d]pyrimidine N1H of compound
1a established a weak hydrogen bond interaction with backbone of Cys 532.
Figure 7. Binding mode of inhibitors within the active site of BRAFV600E. Structural comparison
between docked (cyan) and molecular dynamics (MD)-simulated representative snapshots (green) of
1a in the active conformation (
a); 1v in the active conformation (b). Yellow dots represent hydrogen
bonds. (BRAFV600E in colored ribbon, ligands in stick model, the key residues in line model).
Table 6. Main hydrogen-bond interactions between inhibitors and BRAFV600E
.
1a
1v
Acceptor
Donor
◦
Occ c (%)
Ang ( )
Occ (%)
◦
Dist a (Å)
Ang b ( )
Dist (Å)
Lig@N2H
Lig@N3H
Asp 594@NH
Cys 532@NH
Lig@N1H
Glu 501@OE2
Glu 501@OE2
Lig@O
Lig@N
Cys 532@O
2.854
2.897
2.970
3.117
3.015
23.46
31.59
25.99
31.45
25.28
99.60
99.50
98.20
83.40
53.00
2.845
2.896
3.005
3.209
-
23.56
32.71
27.22
29.20
-
100.00
98.90
96.80
68.50
-
a
The average distance with standard error (SE = standard deviation/N1/2) in parentheses between
b
hydrogen-acceptor atom and hydrogen-donor atom in the investigated time period; The average angle with
standard error in parentheses for hydrogen bonds in the investigated time period; Occupancy is in unit of
c
percentage of the investigated time period.
The binding free energies were calculated by using MM-PBSA (molecular mechanics
Poisson–Boltzmann surface area) and MM-GBSA (molecular mechanics Generalized-Born surface
area) programs in AMBER (Table 7). As can been seen, MM-PBSA and MM-GBSA calculations verified
that ligand 1v showed improved potency compared to that of 1a, which was in good agreement with
the experimental data. According to the energy individual components of the binding free energies,
the favorable contributors to ligand binding were van der Waals (vdW) terms, electrostatic and
nonpolar salvation energies, whereas polar solvation and entropy terms oppose binding. The favorable
electrostatic interactions were counteracted by the unfavorable electrostatics of desolvation upon
binding. Consequently, the total electrostatic interaction contributions were unfavorable to binding in
all systems.

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Table 7. Binding free energy values for BRAFV600E complexes obtained by MM-PBSA and MM-GBSA
(kcal/mol) a.
1a
1v
∆EvdW
∆Eele
∆EMM
−56.67 (0.09)
−41.49 (0.15)
−98.16 (0.14)
48.50 (0.07)
−7.42 (0.01)
41.08 (0.07)
54.88 (0.08)
−4.14 (0.00)
50.74 (0.08)
−57.08 (0.12)
−47.42 (0.12)
−59.06 (0.10)
−39.50 (0.12)
−98.56 (0.12)
46.63 (0.07)
−7.64 (0.01)
38.99 (0.07)
55.03 (0.10)
−4.49 (0.00)
50.53 (0.09)
−59.57 (0.11)
−48.03 (0.12)
∆Gele,sol (GB)
∆Gnp,sol (GB)
∆Gsol (GB)
∆Gele,sol (PB)
∆Gnp,sol (PB)
∆Gsol (PB)
∆Gpred (GB)
∆Gpred (PB)
a
∆
Gpred: the calculated binding free energy by MM-PBSA and MM-GBSA method, ∆Gpred = ∆Gele + ∆GvdW +
∆Gnp,sol + ∆Gele,sol
.
3. Materials and Methods
3.1. Chemistry
Melting points were determined using an X-4 Melting-point Apparatus with Microscope (Yuhua
1
Instrument Co., Ltd., Henan, China) and were uncorrected. H- and 13C-NMR spectra were recorded
on a Bruker AVIII 400 MHz or Bruker AVIII 600 MHz and 100 MHz spectrometer (Bruker Co., Fällanden,
Switzerland) with tetramethylsilane (TMS) as the internal standard, the values of the chemical shifts (δ)
are given in ppm, and coupling constants (J) are given in Hz. Mass spectra (ESI-MS) were performed
on Waters ZQ4000 (Waters Co., Milford, MA, USA). All reactions were monitored using thin-layer
chromatography (TLC) on silica gel plates at 254 nm under an ultraviolet (UV) light. Flash column
chromatography separations were performed on normal phase silica gel (200–300 mesh, Merck Co.,
Elkton, VA, USA) or reverse phase silica gel by using Yamazen AI-580 flash chromatography (Yamazen
Co., Osaka, Japan) with UV detection at 254 nm. All reagents were purchased from commercial vendors
and were used without further purification. All oxygen-sensitive or moisture-sensitive reactions were
run under nitrogen atmosphere. Yields were not optimized.
4-Chloro-1H-pyrazolo[3,4-d]pyrimidine (
(1.0 g, 0.006 mol) in methanol (20 mL) at
added. A solution of hydrazine monohydrate (0.274 mL 1.0 eq.) in methanol (10 mL) was slowly
dripped into above stirred solution by using a constant-pressure dropping funnel. The mixture was
allowed to warm to room temperature and stirred for 2–3 h. The reaction mixture was concentrated
2
). To the solution of 4,6-dichloropyrimidine-5-carbaldehyde
−
65 ◦C, triethylamine (0.97 mL,1.2 equivalents (eq.)) was
in vacuo and crude product was diluted with water (20 mL), and extracted with EtOAc (60 mL
×
3).
The combined organic layer was washed with saturated solution of NaCl (60 mL
×
3), dried over
1
MgSO4 and concentrated to give compound
2. Yield: 68.9%. H-NMR (400 MHz, deuteriated dimethyl
sulfoxide (DMSO-d6)) δ 14.51 (s, 1H), 8.84 (s, 1H), 8.45 (s, 1H). ESI-MS m/z: 153.00 [M − H]−.
4-Chloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidine (3). Compound 2 (1.88 g, 0.012 mol)
was dissolved in EtOAc (50 mL) and heated to 50 ◦C. After 10 min pyridinium 4-toluenesulfonate
(PPTs) (50 mg) were added◦, followed by the addition of 3,4-dihydro-2H-pyran. The resulting reaction
mixture was at stirred 50 C. After the reaction was complete according to the TLC detection, the
mixture was cooled to room temperature, washed with water (60 mL
×
1), and a saturated solution of
NaCl (50 mL 2), and dried over MgSO4. The ethyl acetate was removed and petroleum ether (60 mL
×
×
2) was added. The mixture was heated and filtered through cotton. Removal of petroleum ether in
1
vacuo gives compound
3
as light yellow colored solid. Yield: 76.5%. H-NMR (400 MHz, DMSO-d6)
δ
8.92 (s, 1H), 8.55 (s, 1H), 6.02 (dd, J = 10.4, 2.4 Hz, 1H), 3.97 (d, J = 12.0 Hz, 1H), 3.76–3.70 (m, 1H),
2.49–2.42 (m, 1H), 2.07–2.08 (m, 1H), 1.98–1.94 (m, 1H), 1.85–1.73 (m, 1H), 1.64–1.58 (m, 2H). ESI-MS
m/z: 239.06 [M + H]+.

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4-((1-(Tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)aniline (
4). To the mixture of
para-aminophenol (0.28 g) in dimethylformamide (DMF, 2 mL), Cs2CO3 (1.2 eq.) was added under
the protection of nitrogen. The reaction mixture was stirred at room temperature for 1.5–2 h, and
then compound
EtOAc (50 mL) and washed with 1 M NaOH (60 mL
brine. The organic layer was dried over MgSO4 and concentrated to give compound
oily solid. Yield: 79.5%. H-NMR (400 MHz, DMSO-d6)
3
was added slowly. After stirring overnight, the reaction mixture was diluted with
1), water (80 mL 1), 5% LiCl (50 mL 2) and
as a brown
×
×
×
4
1
δ
8.57 (s, 1H), 7.75 (s, 1H), 6.99–6.95 (m, 2H),
6.67–6.31 (m, 2H), 5.96 (dd, J = 10.0, 2.4 Hz, 1H), 5.19 (s, 2H), 3.96 (d, J = 12.4 Hz, 1H), 3.73–3.67 (m, 1H),
2.48–2.40 (m, 1H), 2.06–2.00 (m, 1H), 1.92–1.88 (m, 1H), 1.79–1.71 (m, 1H), 1.61–1.56 (m, 2H). ESI-MS m/z:
310.14 [M − H]−.
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-
yl)oxy)phenyl)urea (5a). To the solution of compound 4
in CH2Cl2 at 0 ◦C 4-chloro-3-(trifluoromethyl)
phenyl isocyanate (1.0 eq.) was added. The mixture was stirred overnight at room temperature.
To the resulting suspension, petroleum ether (60 mL) was added. The solid material was collected by
1
filtration to provide the title compound as a white solid. Yield: 66.6%. H-NMR (400 MHz, DMSO-d6)
δ
9.22 (s, 1H), 8.99 (s, 1H), 8.58 (s, 1H), 8.14 (s, 2H), 7.69–7.57 (m, 4H), 7.27 (d, J = 8.8 Hz, 2H), 5.98
(d, J = 10.0 Hz, 1H), 3.97 (d, J = 11.6 Hz, 1H), 3.74–3.68 (m, 1H), 2.05 (d, J = 12.4 Hz, 1H), 1.93 (d,
J = 12.4 Hz, 1H), 1.77 (d, J = 8.0 Hz, 1H), 1.59 (s, 3H). 13C-NMR (100 MHz, DMSO-d6)
163.7, 155.7,
δ
152.9, 147.1, 139.8, 137.6, 132.4, 132.3, 127.3, 123.5, 122.7, 121.9, 120.3, 117.3, 102.7, 82.7, 67.5, 29.1, 25.0,
22.6. ESI-MS m/z: 533.12 [M + H]+.
1-(4-((1-(Tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-3-(m-tolyl)urea
(5b).
Compound 5b was prepared using the same procedure as described for the synthesis of 5a by
replacing 4-chloro-3-(trifluoromethyl)phenyl isocyanate with 3-methyl phenyl isocyanate. Yield: 80.0%.
1H-NMR (400 MHz, DMSO-d6)
δ
14.13 (s, 1H), 8.82 (s, 1H), 8.66 (s, 1H), 8.51 (s, 1H), 8.01 (s, 1H), 7.56 (d,
J = 8.9 Hz, 2H), 7.32 (s, 1H), 7.25 (d, J = 8.9 Hz, 3H), 7.17 (t, J = 7.7 Hz, 1H), 6.80 (d, J = 7.7 Hz, 1H), 2.29
(s, 3H). 13C-NMR (100 MHz, DMSO-d6)
δ 163.68, 157.13, 155.39, 153.00, 146.77, 139.99, 138.36, 138.11,
132.26, 129.04, 123.02, 122.66, 120.47, 119.63, 117.67, 117.24, 102.01. ESI-MS m/z: 445.19 [M − H]−.
1-(3,4-Dichlorophenyl)-3-(4-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)
urea (5c). Compound 5c was prepared using the same procedure as described for the synthesis of
5a by replacing 4-chloro-3-(trifluoromethyl)phenyl isocyanate with 3,4-dichlorophenyl isocyanate.
1
Yield: 67.0%. H-NMR (400 MHz, DMSO-d6)
δ 9.07 (s, 1H), 8.97 (s, 1H), 8.58 (s, 1H), 8.14 (s, 1H), 7.90 (s,
1H), 7.58–7.52 (m, 3H), 7.37 (d, J = 8.4 Hz, 1H), 7.27 (d, J = 8.8 Hz, 2H), 5.99 (d, J = 9.6 Hz, 1H), 3.97 (d,
J = 10.8 Hz, 1H), 3.71 (s, 1H), 2.05 (d, J = 12.8 Hz, 1H), 1.93 (d, J = 12.4 Hz, 1H), 1.79 (s, 1H), 1.59 (s, 2H),
1.24 (s, 1H). ESI-MS m/z: 499.10 [M + H]+.
1-(4-Chlorophenyl)-3-(4-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)urea
(
5d). Compound 5d was prepared using the same procedure as described for the synthesis of 5a by
replacing 4-chloro-3-(trifluoromethyl)phenyl isocyanate with 4-chlorophenyl isocyanate. Yield: 68.3%.
1H-NMR (400 MHz, DMSO-d6)
8.87 (s, 1H), 8.85 (s, 1H), 8.58 (s, 1H), 8.13 (s, 1H), 7.56 (d, J = 8.9 Hz,
δ
2H), 7.51 (d, J = 8.8 Hz, 2H), 7.26 (d, J = 8.8 Hz, 2H), 7.26 (d, J = 8.9 Hz, 2H), 5.98 (dd, J = 10.1, 1.9 Hz,
1H), 3.97 (d, J = 11.2 Hz, 1H), 3.77–3.65 (m, 1H), 2.49–2.41 (m, 1H), 2.05 (d, J = 12.5 Hz, 1H), 1.93 (dd,
J = 12.9, 2.3 Hz, 1H), 1.83–1.68 (m, 1H), 1.67–1.53 (m, 2H). ESI-MS m/z: 465.14 [M + H]+.
1-Phenyl-3-(4-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)urea
(5e).
Compound 5e was prepared using the same procedure as described for the synthesis of 5a by replacing
1
4-chloro-3-(trifluoromethyl)phenyl isocyanate with phenyl isocyanate. Yield: 60.2%. H-NMR (400
MHz, DMSO-d6) δ 8.81 (s, 1H), 8.71 (s, 1H), 8.58 (s, 1H), 8.12 (s, 1H), 7.57 (d, J = 7.7 Hz, 2H), 7.48 (d,
J = 7.7 Hz, 2H), 7.33–7.28 (t, 2H), 7.26 (d, J = 8.9 Hz, 2H), 6.99 (t, J = 7.3 Hz, 1H), 5.99 (d, J = 12.5 Hz,
1H), 3.97 (d, J = 11.2 Hz, 1H), 3.76–3.66 (m, 1H), 2.45 (m, 1H), 2.03 (m, 1H), 1.93 (m, 1H), 1.86–1.69 (m,
1H), 1.66–1.53 (m, 2H). ESI-MS m/z: 431.18 [M + H]+.

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1-(2-Chloro-5-methylphenyl)-3-(4-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)
phenyl)urea (5f). Compound 5f was prepared using the same procedure as described for the synthesis
of 5a by replacing 4-chloro-3-(trifluoromethyl)phenyl isocyanate with 2-chloro-5-methylphenyl
1
isocyanate. Yield: 69.4%. H-NMR (400 MHz, DMSO-d6)
δ 9.54 (s, 1H), 8.59 (s, 1H), 8.28 (s, 1H), 8.13 (s,
1H), 8.03 (s, 1H), 7.58 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 8.0 Hz, 1H), 7.28 (d, J = 8.8 Hz, 2H), 6.87 (d, J = 8.0
Hz, 1H), 5.99 (d, J = 10.0 Hz, 1H), 3.97 (d, J = 11.2 Hz, 1H), 3.75–3.68 (m, 1H), 2.30 (s, 3H), 2.08–2.00 (m,
1H), 1.93 (d, J = 11.6 Hz, 1H), 1.77 (s, 1H), 1.60 (s, 2H), 1.24 (s, 1H). ESI-MS m/z: 477.15 [M − H]−.
1-(3-Chlorophenyl)-3-(4-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)urea
(
5g). Compound 5g was prepared using the same procedure as described for the synthesis of 5a by
replacing 4-chloro-3-(trifluoromethyl)phenyl isocyanate with 3-chlorophenyl isocyanate. Yield: 72.3%.
1H-NMR (400 MHz, DMSO-d6)
8.94 (s, 1H), 8.90 (s, 1H), 8.60 (s, 1H), 8.14 (s, 1H), 7.73 (s, 1H), 7.57 (d, J =
δ
9.0 Hz, 2H), 7.30 (m, 5H), 7.03 (m, 1H), 5.99 (dd, J = 10.2, 2.3 Hz, 1H), 3.97 (d, J = 12.0 Hz, 1H), 3.76–3.66 (m,
−
1H), 2.45 (m, 1H), 2.05 (m, 1H), 1.93 (m, 1H), 1.83–1.72 (m, 1H), 1.61 (m, 2H). ESI-MS m/z: 463.14 [M
−
H] .
1-(2,3-Dimethylphenyl)-3-(4-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)
urea (5h). Compound 5h was prepared using the same procedure as described for the synthesis of
5a by replacing 4-chloro-3-(trifluoromethyl)phenyl isocyanate with 2,3-dimethylphenyl isocyanate.
1
Yield: 65.5%. H-NMR (400 MHz, DMSO-d6)
δ 9.07 (s, 1H), 8.58 (s, 1H), 8.10 (s, 1H), 8.00 (s, 1H), 7.56 (t,
J = 8.6 Hz, 3H), 7.25 (d, J = 8.4 Hz, 1H), 7.05 (t, J = 7.8 Hz, 1H), 6.92 (d, J = 7.2 Hz, 1H), 5.99 (d, J = 9.2
Hz, 1H), 3.97 (d, J = 11.2 Hz, 1H), 3.75–3.68 (m, 1H), 2.45 (d, J = 10.4 Hz, 1H), 2.27 (s, 3H), 2.16 (s, 3H),
2.05 (d, J = 10.8 Hz, 1H), 1.93 (d, J = 12.0 Hz, 1H), 1.79 (s, 1H), 1.60 (s, 2H), 1.46 (s, 1H). ESI-MS m/z:
481.21 [M + Na]+.
1-(2-Chloro-5-(trifluoromethyl)phenyl)-3-(4-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-
yl)oxy)phenyl)urea (5i). Compound 5i was prepared using the same procedure as described for
the synthesis of 5a by replacing 4-chloro-3-(trifluoromethyl)phenyl isocyanate with 2-chloro-5-
1
(trifluoromethyl)phenyl isocyanate. Yield: 74.5%. H-NMR (400 MHz, DMSO-d6)
δ 9.22 (s, 1H), 8.99 (s,
1H), 8.58 (s, 1H), 8.14 (s, 2H), 7.69–7.57 (m, 4H), 7.27 (d, J = 8.8 Hz, 2H), 5.98 (d, J = 10.0 Hz, 1H), 3.97
(d, J = 11.6 Hz, 1H), 3.74–3.68 (m, 1H), 2.05 (d, J = 12.4 Hz, 1H), 1.93 (d, J = 12.4 Hz, 1H), 1.77 (d, J = 8.0
Hz, 1H), 1.59 (s, 3H). ESI-MS m/z: 533.12 [M + H]+.
1-(3-Fluoro-5-(trifluoromethyl)phenyl)-3-(4-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-
yl)oxy)phenyl)urea (5j). Compound 5j was prepared using the same procedure as described for
the synthesis of 5a by replacing 4-chloro-3-(trifluoromethyl)phenyl isocyanate with 3-fluoro-5-
1
(trifluoromethyl)phenyl isocyanate. Yield: 60.2%. H-NMR (400 MHz, DMSO-d6)
δ
9.31 (s, 1H), 9.06
(s, 1H), 8.58 (s, 1H), 8.15 (s, 1H), 7.73 (s, 1H), 7.64 (d, J = 11.2 Hz, 1H), 7.58 (d, J = 8.8 Hz, 2H), 7.28
(d, J = 8.8 Hz, 2H), 7.24 (d, J = 8.5 Hz, 1H), 5.99 (dd, J = 10.1, 2.1 Hz, 1H), 3.97 (d, J = 11.6 Hz, 1H),
3.76–3.66 (m, 1H), 2.45 (dd, J = 12.8, 4.0 Hz, 1H), 2.05 (d, J = 12.5 Hz, 1H), 1.93 (dd, J = 13.0, 2.6 Hz, 1H),
1.84–1.70 (m, 1H), 1.61 (d, J = 10.0 Hz, 2H). ESI-MS m/z: 517.15 [M + H]+.
1-(4-Fluoro-3-methylphenyl)-3-(4-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)
phenyl)urea (5k). Compound 5k was prepared using the same procedure as described for the synthesis
of 5a by replacing 4-chloro-3-(trifluoromethyl)phenyl isocyanate with 4-fluoro-3-methylphenyl
1
isocyanate. Yield: 79.4%. H-NMR (400 MHz, DMSO-d6)
δ 8.83 (s, 1H), 8.69 (s, 1H), 8.58 (s, 1H), 8.11 (s,
1H), 7.56 (d, J = 8.8 Hz, 2H), 7.38 (dd, J = 6.8, 2.4 Hz,1H), 7.30–7.24 (m, 3H), 7.06 (t, J = 9.2 Hz, 1H),
5.98 (dd, J = 10.4, 2.4 Hz, 1H), 3.97 (d, J = 10.8 Hz, 1H), 3.74–3.66 (m, 1H), 2.22 (s, 3H), 2.05 (d, J = 11.6
Hz,1H), 1.95–1.90 (m, 1H), 1.80–1.76 (m, 1H), 1.59 (s, 2H), 1.24 (s, 1H). ESI-MS m/z: 485.15 [M + Na]+.
1-(3-Ethylphenyl)-3-(4-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)urea (5l).
Compound 5l was prepared using the same procedure as described for the synthesis of 5a by replacing
1
4-chloro-3-(trifluoromethyl)phenyl isocyanate with 3-ethylpheny isocyanate. Yield: 69.4%. H-NMR
(400 MHz, DMSO-d6)
δ 8.79 (s, 1H), 8.65 (s, 1H), 8.58 (s, 1H), 8.11 (s, 1H), 7.56 (d, J = 8.9 Hz, 2H), 7.37
(d, J = 9.2 Hz, 1H), 7.29 (s, 1H), 7.25 (d, J = 8.8 Hz, 2H), 7.06 (t, J = 9.2 Hz, 1H), 5.98 (dd, J = 9.9, 1.4 Hz,

Molecules 2017, 22, 542
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1H), 3.97 (d, J = 11.4 Hz, 1H), 3.72 (m, 1H), 2.50–2.41 (m, 1H), 2.23 (s, 3H), 2.05 (m, 1H), 1.92 (m, 1H),
1.85–1.70 (m, 1H), 1.59 (m, 2H). ESI-MS m/z: 459.21 [M + H]+.
1-(3-Nitrophenyl)-3-(4-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)urea
(
5m). Compound 5m was s prepared using the same procedure as described for the synthesis of 5a by
replacing 4-chloro-3-(trifluoromethyl)phenyl isocyanate with 3-nitrophenyl isocyanate. Yield: 62.3%.
1H-NMR (400 MHz, DMSO-d6)
9.41 (s, 1H), 9.11 (s, 1H), 8.59 (s, 2H), 8.16 (s, 1H), 7.85 (s, 1H), 7.74 (s,
1H), 7.59 (s, 2H), 7.29–7.00 (m, 2H), 5.99 (d, J = 12.4 Hz, 1H), 5.31 (s, 1H), 3.96 (s, 2H), 3.72 (s, 2H), 2.02
(s, 2H), 1.77 (s, 1H), 1.59 (s, 1H). 13C-NMR (100 MHz, DMSO-d6)
167.36, 163.69, 155.70, 152.91, 148.58,
δ
δ
147.07, 141.45, 137.66, 132.23, 131.95, 130.46, 129.04, 124.77, 122.64, 120.27, 116.72, 112.61, 102.74, 82.72,
67.82, 28.77, 22.79. ESI-MS m/z: 474.16 [M − H]−.
1-(2-Fluoro-5-(trifluoromethyl)phenyl)-3-(4-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-
yl)oxy)phenyl)urea (5n). Compound 5n was prepared using the same procedure as described
for the synthesis of 5a by replacing 4-chloro-3-(trifluoromethyl)phenyl isocyanate with 2-fluoro-
1
5-(trifluoromethyl)phenyl isocyanate. Yield: 70.2%. H-NMR (400 MHz, DMSO-d6)
δ 9.35 (s, 1H), 8.96
(s, 1H), 8.61 (d, J = 22.8 Hz, 2H), 8.16 (s, 1H), 7.55 (d, J = 26.4 Hz, 3H), 7.41 (s, 1H), 7.29 (d, J = 5.6 Hz,
2H), 5.98 (d, J = 8.8 Hz, 1H), 3.95 (s, 1H), 3.71 (s, 1H), 2.03 (s, 1H), 1.92 (d, J = 12.4 Hz, 2H), 1.78 (s, 1H),
1.59 (s, 2H). ESI-MS m/z: 517.15 [M + H]+.
1-(3-(Methylthio)phenyl)-3-(4-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)
urea (5o). Compound 5o was prepared using the same procedure as described for the synthesis of 5a
by replacing 4-chloro-3-(trifluoromethyl)phenyl isocyanate with 3-(methylthio) phenyl isocyanate.
1
Yield: 67.8%. H-NMR (400 MHz, DMSO-d6)
δ
8.83 (s, 1H), 8.77 (s, 1H), 8.58 (s, 1H), 8.12 (s, 1H), 7.57
(d, J = 9.0 Hz, 2H), 7.49 (t, J = 1.7 Hz, 1H), 7.26 (d, J = 8.9 Hz, 2H), 7.22 (d, J = 7.8 Hz, 1H), 7.17 (d,
J = 8.8 Hz, 1H), 6.88 (d, J = 7.8 Hz, 1H), 5.98 (dd, J = 10.2, 2.2 Hz, 2H), 2.51 (d, J = 1.8 Hz, 2H), 2.47 (s,
2H), 1.99 (dd, J = 42.9, 17.3 Hz, 2H), 1.59 (s, 3H). ESI-MS m/z: 477.16 [M + H]+.
1-(3-Cyanophenyl)-3-(4-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)urea
(
5p). Compound 5p was prepared using the same procedure as described for the synthesis of 5a by
replacing 4-chloro-3-(trifluoromethyl)phenyl isocyanate with 3-cyanophenyl isocyanate. Yield: 69.8%.
1H-NMR (400 MHz, DMSO-d6)
9.10 (s, 1H), 9.00 (s, 1H), 8.58 (s, 1H), 8.13 (s, 1H), 8.00 (t, J = 1.8
δ
Hz, 1H), 7.71 (ddd, J = 3.2, 2.0, 1.2 Hz, 1H), 7.59 (d, J = 2.4 Hz, 1H), 7.57 (d, J = 2.0 Hz, 1H), 7.51 (t,
J = 8.0 Hz, 1H), 7.43 (dt, J = 7.6, 1.2 Hz, 1H), 7.29–7.27 (m, 2H), 5.99 (dd, J = 10.0, 2.4 Hz, 1H), 3.18 (d,
J = 5.2 Hz, 2H), 1.99 (s, 6H). ESI-MS m/z: 456.17 [M + H]+.
Methyl 3-(3-(4-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)ureido)benzoate
(5q). Compound 5q was prepared using the same procedure as described for the synthesis of 5a by
replacing 4-chloro-3-(trifluoromethyl)phenyl isocyanate with methyl 3-isocyanatobenzoate. Yield:
1
74.5%. H-NMR (400 MHz, DMSO-d6)
δ 9.03 (s, 1H), 8.89 (s, 1H), 8.58 (s, 1H), 8.23 (s, 1H), 8.13 (s, 1H),
7.65 (d, J = 7.9 Hz, 1H), 7.58 (d, J = 8.9 Hz, 3H), 7.44 (t, J = 7.9 Hz, 1H), 7.27 (d, J = 8.9 Hz, 2H), 5.98 (dd,
J = 10.2, 2.1 Hz, 1H), 4.03–3.93 (m, 1H), 3.87 (s, 3H), 3.68–3.71 (m, 1H), 2.44–2.51 (m, 1H), 1.99–2.03 (m,
1H), 1.91–1.95 (m, 1H), 1.78 (m, 1H), 1.59 (m, 3H). ESI-MS m/z: 489.18 [M + H]+.
1-(5-Methyl-2-nitrophenyl)-3-(4-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)
phenyl)urea (5r). Compound 5r was prepared using the same procedure as described for the synthesis of
5a by replacing 4-chloro-3-(trifluoromethyl)phenyl isocyanate with 5-methyl-2-nitrophenylisocyanate.
1
Yield: 66.8%. H-NMR (400 MHz, DMSO-d6)
δ 10.04 (s, 1H), 9.71 (s, 1H), 8.57 (s, 1H), 8.23 (s, 1H), 8.13
(s, 1H), 8.03 (d, J = 7.7 Hz, 1H), 7.61 (d, J = 6.9 Hz, 2H), 7.28 (d, J = 6.7 Hz, 2H), 7.02 (d, J = 7.3 Hz, 1H),
5.98 (d, J = 9.1 Hz, 1H), 3.96 (d, J = 10.6 Hz, 1H), 3.69 (d, J = 9.5 Hz, 1H), 2.39 (s, 3H), 1.98 (dd, J = 50.4,
13.4 Hz, 2H), 1.42 (d, J = 31.1 Hz, 2H), 0.84 (s, 2H). ESI-MS m/z: 490.18 [M + H]+.
1-(3-Bromophenyl)-3-(4-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)urea
(5s). Compound 5s was prepared using the same procedure as described for the synthesis of 5a by

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replacing 4-chloro-3-(trifluoromethyl)phenyl isocyanate with 3-bromophenylisocyanate. Yield: 79.0%.
1H-NMR (400 MHz, DMSO-d6)
8.92 (s, 1H), 8.89 (s, 1H), 8.58 (s, 1H), 8.13 (s, 1H), 7.87 (s, 1H), 7.57 (d,
δ
J = 8.8 Hz, 2H), 7.34 (d, J = 8.1 Hz, 1H), 7.28 (s, 1H), 7.24 (d, J = 8.7 Hz, 2H), 7.16 (d, J = 7.7 Hz, 1H), 5.98
(d, J = 8.8 Hz, 1H), 3.97 (d, J = 11.2 Hz, 1H), 3.71 (t, J = 12.6 Hz, 1H), 1.99 (dd, J = 47.7, 13.1 Hz, 2H),
1.82–1.71 (m, 1H), 1.59 (d, J = 3.0 Hz, 2H), 1.45–1.35 (m, 1H). ESI-MS m/z: 509.09 [M + H]+.
1-(3,4-Dimethylphenyl)-3-(4-((1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyri-midin-4-yl)oxy)
phenyl)urea (5t). Compound 5t was prepared using the same procedure as described for the synthesis
of 5a by replacing 4-chloro-3-(trifluoromethyl)phenyl isocyanate with 3,4-dimethylphenylisocyanate.
1
Yield: 61.8%. H-NMR (400 MHz, DMSO-d6)
δ
8.74 (s, 1H), 8.58 (s, 1H), 8.52 (s, 1H), 8.10 (s, 1H), 7.55
(d, J = 8.8 Hz, 2H), 7.25 (s, 2H), 7.23 (s, 1H), 7.19 (d, J = 8.1 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H), 5.98 (d,
J = 9.7 Hz, 1H), 3.97 (dd, J = 10.9, 1.0 Hz, 1H), 3.75–3.66 (m, 1H), 2.44 (dd, J = 12.8, 3.9 Hz, 1H), 2.20 (s,
3H), 2.16 (s, 3H), 2.08–2.01 (m, 1H), 1.92 (d, J = 14.7 Hz, 1H), 1.83–1.73 (m, 1H), 1.59 (t, J = 8.3 Hz, 2H).
ESI-MS m/z: 459.21 [M + H]+.
1-(4-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-3-(4-chloro-3-(trifluoromet-hyl)phenyl)urea (1a). A mixture
of 1a (0.1 mmol) in 4 M HCl/1,4-dioxane (5 mL) was stirred at room temperature for 30–70 min. To the
resulting suspension, ether (60 mL) was added. The solid material was filtered, washed twice with
ether and further purified by flash column chromatography to provide the title compound. Yield:
70.2%; m.p. 222.6–225.8 ◦C; 1H-NMR (400 MHz, DMSO-d6)
8.51 (s, 1H), 8.13 (s, 1H), 8.04 (s, 1H), 7.71–7.60 (m, 2H), 7.58 (d, J = 8.8 Hz, 2H), 7.27 (d, J = 8.8 Hz, 2H).
δ 14.13 (s, 1H), 9.25 (s, 1H), 9.01 (s, 1H),
13C-NMR (100 MHz, DMSO-d6)
δ 163.61, 157.13, 155.36, 152.91 147.18, 139.77, 137.54, 132.41, 132.25,
127.28, 126.98, 124.76, 123.48, 122.71, 120.31, 117.23, 101.76. ESI-MS m/z: 449.40 [M + H]+.
1-(4-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-3-(m-tolyl)urea (1b). Compound 1b was prepared
◦
using the same procedure as described for the synthesis of 1a. Yield: 71.8%; m.p. 227.4–230.0 C;
1H-NMR (400 MHz, DMSO-d6)
δ
14.13 (s, 1H), 8.82 (s, 1H), 8.66 (s, 1H), 8.51 (s, 1H), 8.01 (s, 1H), 7.56 (d,
J = 8.9 Hz, 2H), 7.32 (s, 1H), 7.25 (d, J = 8.9 Hz, 3H), 7.17 (t, J = 7.7 Hz, 1H), 6.80 (d, J = 7.7 Hz, 1H), 2.29 (s,
3H). 13C-NMR (100 MHz, DMSO-d6)
δ 163.76, 157.15, 155.33, 153.15, 148.13, 146.61, 140.15, 138.32, 138.29,
132.05, 128.99, 122.83, 122.62, 119.42, 118.90, 115.59, 101.75, 66.74, 21.63. ESI-MS m/z: 361.60 [M + H]+.
1-(4-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-3-(3,4-dichlorophenyl)urea (1c). Compound 1c was
prepared using the same procedure as described for the synthesis of 1a
.
Yield: 92.6%; m.p.
1
245.0–247.2 ◦C; H-NMR (400 MHz, DMSO-d6)
δ 14.12 (s, 1H), 9.08 (s, 1H), 8.97 (s, 1H), 8.51 (s,
1H), 8.03 (s, 1H), 7.90 (d, J = 2.4 Hz, 1H), 7.54 (dd, J = 27.2, 8.8 Hz, 3H), 7.36 (dd, J = 8.8, 2.4 Hz, 1H), 7.26
(d, J = 8.8 Hz, 2H). 13C-NMR (100 MHz, DMSO-d6)
δ 163.59, 157.10, 155.34, 152.79, 147.08, 140.35, 137.60,
132.23, 131.43, 130.95, 123.53, 122.69, 120.14, 119.71, 118.76, 101.75. ESI-MS m/z: 415.04 [M + H]+.
1-(4-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-3-(4-chlorophenyl)urea (1d). Compound 1d was
prepared using the same procedure as described for the synthesis of 1a
.
Yield: 80.5%; m.p.
1
260.8–262.0 ◦C; H-NMR (400 MHz, DMSO-d6)
δ 14.13 (s, 1H), 8.86 (d, J = 10.2 Hz, 2H), 8.51 (s,
1H), 8.03 (s, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 8.8 Hz, 2H), 7.26 (d,
J = 8.4 Hz, 2H). 13C-NMR (100 MHz, DMSO-d6) δ 157.13, 155.38, 152.92, 146.92, 139.10, 137.88, 132.26,
129.04, 125.79, 122.69, 120.18, 119.94, 101.76. ESI-MS m/z: 391.50 [M + H]+.
1-(4-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-3-phenylurea (1e). Compound 1e was prepared using
the same procedure as described for the synthesis of 1a. Yield: 81.2%; m.p. 247.3–249.2 ◦C; 1H-NMR
(400 MHz, DMSO-d6)
δ 14.13 (s, 1H), 8.80 (s, 1H), 8.71 (s, 1H), 8.52 (s, 1H), 8.02 (s, 1H), 7.56 (d, J = 8.9 Hz,
2H), 7.48 (d, J = 7.7 Hz, 2H), 7.30 (t, J = 7.7 Hz, 2H), 7.25 (d, J = 8.9 Hz, 2H), 6.98 (t, J = 7.3 Hz, 1H).
13C-NMR (100 MHz, DMSO-d6)
δ 163.68, 157.13, 155.39, 153.01, 146.80, 140.07, 138.07, 132.27, 129.20,
122.68, 122.28, 119.79, 118.66, 101.76, 0.51. ESI-MS m/z: 347.40 [M + H]+.
1-(4-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-3-(2-chloro-5-methylphenyl)urea (1f). Compound 1f
was prepared using the same procedure as described for the synthesis of 1a. Yield: 79.5%; m.p.

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230.2–232.4 ◦C; 1H-NMR (400 MHz, DMSO-d6)
δ
14.13 (s, 1H), 9.53 (s, 1H), 8.52 (s, 1H), 8.28 (s, 1H),
8.03 (s, 1H), 7.58 (d, J = 9.2 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.27 (d, J = 8.8 Hz, 2H), 6.87 (d, J = 9.2 Hz,
1H), 2.90 (s, 1H), 2.74 (s, 1H), 2.30 (s, 3H). 13C-NMR (100 MHz, DMSO-d6)
δ
163.64, 157.14, 155.39,
152.60, 147.00, 137.81, 137.48, 135.95, 132.27, 129.22, 124.46, 122.79, 122.20, 119.75, 119.44, 101.76, 21.33,
0.51. ESI-MS m/z: 395.30 [M + H]+.
1-(4-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-3-(3-chlorophenyl)urea (1g). Compound 1g was
prepared using the same procedure as described for the synthesis of 1a
δ 14.13 (s, 1H), 8.94 (s, 1H), 8.89 (s, 1H), 8.51 (s,
1H), 8.03 (s, 1H), 7.73 (s, 1H), 7.57 (d, J = 8.8 Hz, 2H), 7.34–7.26 (m, 4H), 7.06–7.00 (m, 1H). 13C-NMR
.
Yield: 75.4%; m.p.
1
234.8–236.0 ◦C; H-NMR (400 MHz, DMSO-d6)
(100 MHz, DMSO-d6)
δ 163.64, 157.13, 155.37, 152.87, 147.01, 141.66, 137.75, 133.61, 132.26, 130.80,
122.70, 121.89, 120.04, 118.02, 117.10, 101.76, 99.93. ESI-MS m/z: 381.08 [M + H]+.
1-(4-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-3-(2,3-dimethylphenyl)urea (1h). Compound 1h was
prepared using the same procedure as described for the synthesis of 1a. Yield: 81.4%; m.p. 250.4–252.3 ◦C
;
1H-NMR (400 MHz, DMSO-d6)
3H), 7.25 (d, J = 7.4 Hz, 2H), 7.06–7.02 (m, 1H), 6.91 (d, J = 6.6 Hz, 1H), 2.27 (s, 3H), 2.16 (s, 3H).
δ 14.13 (s, 1H), 9.06 (s, 1H), 8.51 (s, 1H), 8.00 (s, 2H), 7.57 (d, J = 6.8 Hz,
13C-NMR (100 MHz, DMSO-d6)
δ 163.82, 157.13, 155.39, 153.40, 146.63, 138.40, 137.34, 137.00, 132.28,
128.05, 125.65, 125.41, 122.67, 120.92, 119.51, 101.75, 20.75, 14.05. ESI-MS m/z: 375.15 [M + H]+.
1-(4-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-3-(2-chloro-5-(trifluoromethyl)phenyl)urea
(1i).
Compound 1i was prepared using the same procedure as described for the synthesis of 1a. Yield:
◦
1
82.3%; m.p. 239.2–240.3 C; H-NMR (400 MHz, DMSO-d6)
J = 3.2 Hz, 2H), 8.52 (s, 1H), 8.05 (s, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 8.8 Hz, 2H), 7.39 (d,
J = 8.4 Hz, 1H), 7.29 (d, J = 8.8 Hz, 2H). 13C-NMR (100 MHz, DMSO-d6)
163.29, 157.14, 155.68, 152.91,
δ 14.13 (s, 1H), 9.70 (s, 1H), 8.66 (d,
δ
147.56, 137.32, 137.25, 132.22, 130.54, 128.80, 128.48, 125.49, 123.25, 120.47, 119.63, 117.67, 117.24, 102.01.
ESI-MS m/z: 449.07 [M + H]+.
1-(4-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-3-(3-fluoro-5-(trifluoromethyl)phenyl)urea (1j). Compound
1j was prepared using the same procedure as described for the synthesis of 1a. Yield: 88.0%; m.p.
249.2–250.6 ◦C; 1H-NMR (400 MHz, DMSO-d6)
δ
14.13 (s, 1H), 9.30 (s, 1H), 9.04 (s, 1H), 8.51 (s, 1H),
8.04 (s, 1H), 7.73 (s, 1H), 7.64 (d, J = 11.3 Hz, 1H), 7.58 (d, J = 8.6 Hz, 2H), 7.27 (d, J = 8.6 Hz, 2H), 7.22
(d, J = 8.2 Hz, 1H). 13C-NMR (100 MHz, DMSO-d6)
δ 163.87, 163.58, 161.39, 157.11, 155.33, 152.81, 147.27,
143.05, 137.41, 132.20, 122.69, 120.40, 110.95, 108.89, 105.75, 105.54, 101.76. ESI-MS m/z: 433.10 [M + H]+.
1-(4-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-3-(3-fluoro-5-methylphenyl)urea (1k). Compound 1k
was prepared using the same procedure as described for the synthesis of 1a. Yield: 81.6%; m.p.
◦
242.8–245.2 C; 1H-NMR (400 MHz, DMSO-d6)
δ
9.06 (s, 1H), 8.45 (s, 1H), 8.28 (s, 1H), 7.33 (dd, J = 6.8
,
2.4 Hz, 1H), 7.25–18 (m, 3H), 7.02 (t, J = 9.2 Hz, 1H), 6.69 (d, J = 8.8 Hz, 2H). 13C-NMR (100 MHz,
DMSO-d6) δ 163.71, 157.48, 155.37, 155.13, 153.21, 146.68, 138.21, 136.15, 132.24, 124.65, 124.48, 122.62,
121.44, 119.57, 117.63, 115.40, 115.17, 101.73, 14.77. ESI-MS m/z: 379.12 [M + H]+.
1-(4-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-3-(3-ethylphenyl)urea (1l). Compound 1l was
prepared using the same procedure as described for the synthesis of 1a
.
Yield: 61.2%; m.p.
1
219.5–221.5 ◦C; H-NMR (400 MHz, DMSO-d6)
δ 14.13 (s, 1H), 8.78 (s, 1H), 8.65 (s, 1H), 8.52 (s,
1H), 8.01 (s, 1H), 7.57 (d, J = 8.7 Hz, 2H), 7.34 (s, 1H), 7.24–7.20 (m, 4H), 6.83 (d, J = 7.3 Hz, 1H), 2.58
(d, J = 7.2 Hz, 2H), 1.24–1.17 (m, 3H). 13C-NMR (100 MHz, DMSO-d6)
δ
157.18, 155.39, 153.05, 146.75,
144.75, 140.08, 138.15, 129.09, 129.02, 122.66, 121.80, 120.74, 119.71, 117.99, 117.74, 116.09, 115.58, 101.76,
28.71, 15.96. ESI-MS m/z: 375.15 [M + H]+.
1-(4-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-3-(3-nitrophenyl)urea (1m). Compound 1m was
prepared using the same procedure as described for the synthesis of 1a
.
Yield: 70.3%; m.p.
1
229.2–231.4 ◦C; H-NMR (400 MHz, DMSO-d6)
δ 14.13 (s, 1H), 9.27 (s, 1H), 8.97 (s, 1H), 8.58 (s,
1H), 8.51 (s, 1H), 8.04 (s, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 8.1 Hz, 1H), 7.60 (d, J = 8.9 Hz, 2H),

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7.57 (t, J = 8.3 Hz, 1H), 7.28 (d, J = 8.9 Hz, 2H). 13C-NMR (100 MHz, DMSO-d6)
δ
155.35, 152.91, 148.52,
147.11, 141.43, 137.55, 130.46, 124.74, 122.73, 120.23, 116.71, 112.55, 101.74, 40.50, 40.29, 40.08, 39.87,
39.67, 39.46, 39.25. ESI-MS m/z: 392.10 [M + H]+.
1-(4-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-3-(2-fluoro-5-(trifluoromethyl)phenyl)urea
(1n).
Compound 1n was prepa◦red using the same procedure as described for the synthesis of 1a. Yield:
1
78.6%; m.p. 178.4–179.0 C; H-NMR (400 MHz, DMSO-d6)
δ 14.13 (s, 1H), 9.70 (s, 1H), 8.66 (d,
J = 3.2 Hz, 2H), 8.52 (s, 1H), 8.05 (s, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.39 (d,
J = 8.4 Hz, 1H), 7.29 (d, J = 8.8 Hz, 2H). 13C-NMR (100 MHz, DMSO-d6)
δ
155.34, 153.38, 152.64, 152.56,
152.44, 147.24, 137.30, 130.76, 129.47, 125.66, 122.82, 120.88, 119.96, 119.19, 116.72, 116.42, 116.21, 115.69,
101.76. ESI-MS m/z: 406.12 [M + H]+.
1-(4-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-3-(3-(methylthio)phenyl)urea (1o). Compound 1o
was prepared using the same procedure as described for the synthesis of 1a. Yield: 99.6%; m.p.
227.3–228.4 ◦C; 1H-NMR (400 MHz, DMSO-d6)
δ
14.13 (s, 1H), 8.80 (d, J = 23.2 Hz, 2H), 8.51 (s, 1H), 8.02
(s, 1H), 7.56 (d, J = 8.8 Hz, 2H), 7.49 (s, 1H), 7.26–7.21 (m, 3H), 7.17 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 7.6 Hz
,
1H), 2.51 (s, 3H). 13C-NMR (100 MHz, DMSO-d6)
δ 163.64, 157.12, 155.36, 152.95, 146.89, 140.61, 139.00,
137.92, 132.24, 129.66, 122.65, 119.92, 119.70, 115.68, 115.26, 101.75, 15.04. ESI-MS m/z: 393.11 [M + H]+.
1-(4-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-3-(3-cyanophenyl)urea (1p). Compound 1p was
prepared using the same procedure as described for the synthesis of 1a
235.2–237.4 ◦C; 1H-NMR (400 MHz, DMSO-d6)
14.15 (s, 1H), 9.05 (d, J = 39.2 Hz, 2H), 8.51 (s, 1H),
8.03 (d, J = 20.4 Hz, 2H), 7.70 (s, 1H), 7.57–7.44 (m, 4H), 7.27 (s, 2H). 13C-NMR (100 MHz, DMSO-d6)
.
Yield: 86.7%; m.p.
δ
δ
163.60, 157.08, 155.36, 152.89, 147.05, 141.01, 137.61, 132.25, 130.59, 125.74, 123.32, 122.74, 121.19, 120.11,
111.99, 101.74. ESI-MS m/z: 372.11 [M + H]+.
Methyl-3-(3-(4-((1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)ureido)benzoate (1q). Compound 1q was
prepared using the same procedure as described for the synthesis of 1a
.
Yield: 66.0%; m.p.
14.15 (s, 1H), 9.30 (s, 1H), 9.01 (s, 1H), 8.59 (s,
1H), 8.51 (s, 1H), 8.06 (s, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 8.4 Hz, 3H),
7.28 (d, J = 7.6 Hz, 2H), 3.39 (s, 3H). 13C-NMR (100 MHz, DMSO-d6)
166.61, 163.63, 157.09, 155.37,
◦
1
217.3–220.1 C; H-NMR (400 MHz, DMSO-d6)
δ
δ
152.98, 146.92, 140.53, 137.83, 132.26, 130.56, 129.61, 123.19, 122.91, 122.69, 120.01, 119.02, 101.74, 52.58.
ESI-MS m/z: 405.12 [M + H]+.
1-(4-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-3-(5-methyl-2-nitrophenyl)urea (1r). Compound 1r
was prepared using the same procedure as described for the synthesis of 1a. Yield: 54.2%; m.p.
◦
359.7–360.2 C; 1H-NMR (400 MHz, DMSO-d6)
δ
14.12 (s, 1H), 9.53 (s, 1H), 8.50 (d, J = 6.8 Hz, 1H), 8.26
(d, J = 6.0 Hz, 1H), 8.03 (s, 2H), 7.57 (s, 2H), 7.33–7.26 (m, 3H), 6.85 (s, 1H), 2.28 (d, J = 6.0 Hz, 3H).
13C-NMR (100 MHz, DMSO-d6)
δ 163.61, 157.11, 155.36, 152.58, 146.98, 137.80, 137.45, 135.93, 132.24,
129.18, 124.42, 122.76, 122.18, 119.74, 119.42, 101.75, 21.32. ESI-MS m/z: 406.12 [M + H]+.
1-(4-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-3-(3-bromophenyl)urea (1s). Compound 1s was
prepared using the same procedure as described for the synthesis of 1a
.
Yield: 85.5%; m.p.
1
234.5–237.0 ◦C; H-NMR (400 MHz, DMSO-d6)
δ 14.16 (s, 1H), 8.95 (s, 1H), 8.91 (s, 1H), 8.51 (s,
1H), 8.06 (s, 1H), 7.88 (s, 1H), 7.56 (d, J = 7.2 Hz, 2H), 7.34 (d, J = 8.0 Hz, 1H), 7.26 (d, J = 6.8 Hz, 3H),
7.16 (d, J = 7.6 Hz, 1H). 13C-NMR (100 MHz, DMSO-d6)
δ 155.38, 152.84, 146.95, 141.79, 137.73, 131.13,
124.78, 122.72, 122.13, 120.82, 120.02, 117.47, 101.74. ESI-MS m/z: 425.03 [M + H]+.
1-(4-((1H-Pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)-3-(3,4-dimethylphenyl)urea (1t). Compound 1t was
prepared using the same procedure as described for the synthesis of 1a. Yield: 87.7%; m.p. 249.9–250.6 ◦C;
1H-NMR (400 MHz, DMSO-d6)
δ
14.12 (s, 1H), 8.75 (s, 1H), 8.52 (d, J = 5.6 Hz, 2H), 8.00 (s, 1H), 7.55 (d,
J = 8.8 Hz, 2H), 7.24 (d, J = 8.8 Hz, 3H), 7.19 (d, J = 8.0 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 2.20 (s, 3H), 2.16
(s, 3H). 13C-NMR (101 MHz, DMSO-d6)
163.67, 157.11, 155.37, 153.02, 146.69, 138.19, 137.69, 136.73,
132.25, 130.04, 129.93, 122.62, 120.02, 119.67, 116.25, 101.74, 20.05, 19.07. ESI-MS m/z: 375.15 [M + H]+.
δ

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4-Chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine (
6
). A suspension of compound
2
(1.88 g, 0.012 mol) and
NaH (0.864 g, 0.036 mol) in dry DMF (10 mL) was stirred at 0 ◦C for 30 min. Iodomethane (2.56 g,
0.018 mol) was added, and the resulting mixture was stirred overnight. The reaction mixture was
treated with water (100 mL) and extracted with EtOAc (60 mL
×
3). The combined organic layers were
removed in vacuo to give compound
8.44 (s, 1H), 4.08 (s, 3H).
6
. Yield: 70.8%; 1H-NMR (400 MHz, DMSO-d6)
δ
8.85 (s, 1H),
4-(1-Methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)aniline (
in DMF (2 mL), Cs2CO3 (1.2 eq.) was added under the protection of nitrogen. The reaction mixture
was stirred at room temperature for 1.5–2 h, and then compound was added slowly. After stirring
overnight, the reaction mixture was diluted with EtOAc (50 mL) and was washed with 1 M NaOH
(60 mL × 1), water (80 mL 1), 5% LiCl (50 mL 2), and brine. The organic layer was dried over
MgSO4 and concentrated to give compound 8.5 (s,
. Yield: 69.5%; 1H-NMR (400 MHz, DMSO-d6)
1H), 7.66 (s, 1H), 6.97 (d, J = 8.8 Hz, 2H), 6.67 (d, J = 8.8 Hz, 2H), 5.37 (s, 2H), 4.01 (s, 3H).
7). To the mixture of para-aminophenol (0.28 g)
6
×
×
7
δ
1-(3-Chlorophenyl)-3-(4-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)urea (1u). Compound 1u
was prepared from compound and 3-chlorophenyl isocyanate using the same procedure as described
for the synthesis of 1a. Yield: 83.4%; m.p. 309.8–312.0 C; 1H-NMR (400 MHz, DMSO-d6)
8.89 (s, 1H), 8.55 (s, 1H), 8.04 (s, 1H), 7.73 (s, 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.30–7.25 (m, 4H), 7.03 (s,
7
◦
δ
8.94 (s, 1H),
1H), 4.05 (s, 3H). 13C-NMR (100 MHz, DMSO-d6)
δ 163.70, 155.39, 155.27, 152.86, 146.96, 141.65, 137.81,
133.61, 131.32, 130.80, 122.65, 121.90, 120.04, 118.02, 117.10, 102.19. ESI-MS m/z: 395.09 [M + H]+.
1-(4-Chloro-3-(trifluoromethyl)phenyl)-3-(4-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)phenyl)urea
(1v). Compound 1v was prepared using the same procedure as described for the synthesis of 1u by
replacing 3-chlorophenyl isocyanate with 4-chloro-3-(trifluoromethyl)phenyl isocyanate. Yield: 89.3%;
◦
m.p. 212.1–213.0 C; 1H-NMR (600 MHz, DMSO-d6)
δ 9.21 (s, 1H), 8.98 (s, 1H), 8.54 (s, 1H), 8.12 (s, 1H),
8.04 (s, 1H), 7.62-7.68 (m, 2H), 758 (d,J = 9.0 Hz, 2H), 7.26 (d,J = 9.0 Hz, 2H), 4.04 (s, 3H). 13C-NMR
(100 MHz, DMSO-d6)
δ 163.65, 155.35, 155.24, 152.88, 147.10, 139.73, 137.57, 132.37, 131.29, 127.26,
126.95, 123.47, 122.63, 120.29, 117.17, 102.17, 34.43. ESI-MS m/z: 461.81 [M − H]−.
3.2. Kinase Inhibitory Activity
The BRAFV600E inhibitory activity was evaluated by using the Z’-LYTE technology platform
(Life Technologies, Carlsbad, CA, USA), and Sorafenib was employed as the positive control.
The experiments were performed according to the instructions of the manufacturer, and single point
concentration testing with two independent data points (n = 2). According to the anti-proliferative
activities and percent inhibition values of compounds, we chose compounds 1l, 1u and 1v to evaluate
kinase activity at ten concentration gradients from 1000 to 0.0508 nM (3-fold dilution). The IC50 values
were calculated from the inhibition curves from two separate experiments.
3.3. Cell proliferation Inhibition Assay
The anti-proliferative activities of compounds were evaluated against A375, HT-29, PC-3, A549
and MDCK cell lines by the standard MTT assay in vitro, with Sorafenib as the positive control. All cell
lines were purchased from Cell Bank of China Science Academy (Shanghai, China). All chemicals
and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Gibco (Gibco BRL, Grand
Island, NY, USA). The PC-3 and A375 cell lines was maintained in Dulbecco's modified eagle (DEME)
medium supplement with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. The others
were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplement with 10% fetal
bovine serum (FBS) and 1% penicillin–streptomycin. Approximatel◦y 5
×
103 cells, suspended in
medium, were plated into each well of a 96-well plate and grown at 37 C in a humidified atmosphere
with 5% CO2 for 24 h. The following day, various concentrations of tested compounds were added
to the culture medium and incubated for 48 h. Fresh MTT was added to each well at the terminal
◦
concentration of 5 mg/mL in phosphate buffered saline (PBS), and incubated with cells at 37 C for 4 h.

Molecules 2017, 22, 542
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The formazan crystals in each well were dissolved in 150
µL DMSO, and the absorbency at 570 nm
was measured with an enzyme-linked immunosorbent assay plate reader. All of the compounds were
tested three times in each of the cell lines.
3.4. Flow-Activating Cell Sorting Analysis
The effect of compound 1v on cell cycle phase distribution of human malignant melanoma A375 was
assessed using flow cytometry. When the cells grew to about 70% confluence in 6-well microplates, they
were treated with compound 1v at given concentrations (6.11
µ
mol/L, 12.21
µ
mol/L, 24.42
µ
mol/L). After
◦
48 h, cells were harvested by trypsinization, washed with PBS, and fixed in 70% ice cold (4 C) ethanol
overnight. They were then washed with PBS, incubated with Ribonuclease (RNase) (50 mg/mL final
concentration) at 37 ◦C for 30 min, stained with propidium iodide (50 mg/mL final concentration),
and analyzed by flow cytometry (BD FACS Canto II, BD Biosciences, San Jose, CA, USA).
3.5. Western Blot Analysis
Total cells were harvested after treatment, washed twice with cold PBS, and lysed with RIPA lysis
buffer (KeyGEN BioTECH, Nanjing, China) including 1% cocktail (Selleck Chemicals, Houston, TX,
USA). Lysates were centrifuged at 12,000
×
g at 4 ◦C for 15 min, and the supernatant was collected.
Total protein concentration was quantified using the bicinchoninic acid (BCA; Beyotime Institute
of Biotechnology, Nanjing, China) protein assay kit (Sigma Chemical Co., St. Louis, MO, USA).
The proteins were separated electrophoretically using 10% sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) and then the gel was transferred onto polyvinylidene fluoride
(PVDF; Millipore, Billerica, MA, USA) membranes. The membranes were subsequently blocked
with the use of 5% milk in tris-buffered saline Tween (TBST) for 1 h and incubated overnight
with antibodies against p217/p221-MEK (Pmek, Cell Signaling Technology, Beverly, MA, USA),
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Santa Cruz Biotechnology, Inc., Santa cruz,
◦
CA, USA) in TBST containing 5% defatted milk at 4 C followed by incubation with horseradish
peroxidase-linked secondary antibodies (Beyotime Institute of Biotechnology, Nanjing, China) at
4 ◦C for additional 1 h. The immunobands were detected with an enhanced chemiluminescence kit
(Beyotime Institute of Biotechnology, Nanjing, China).
3.6. Molecular Modelling
Molecular docking was performed with Surflex–Dock program that is interfaced with Sybyl
7.3 [27]. The crystal structure of BRAFV600E kinase in complex with Sorafenib (PDB ID: 1UWJ [28]) was
obtained from the PDB. All the water and ligands were removed and the random hydrogen atoms
were added. The structures of the synthesized compounds were generated and minimized using tripos
force fields. All the other default parameters were used. The highest-scored conformation based on
the Surflex–Dock scoring functions, was selected as the final bioactive conformation.
The MD simulations were performed using AMBER 12 software package (San Francisco, CA,
USA) [29,30] according to our previously published protocol [31]. The missing residues, which were
not solved in the crystal structures, were modeled using the loop building routine in Modeler [32].
Geometry optimization and the electrostatic potential calculations for 1a and 1v were carried out at
the HF/6-31G* level of the Gaussian03 suite [33]. The atomic partial charge was obtained by using
the restrained electrostatic potential (RESP) fitting technique [34] implemented in AmberTools [35].
The force field parameters for 1a and 1v were generated by the general amber force field (GAFF) [36]
using the Antechamber program. The AMBER 99SB force field [37] was used to simulate the protein
structure and the ionization states of amino acid residues were set according to the standard protocol.
The model was neutralized by adding suitable counterions and were solvated in a truncated octahedron
box of the transferable interaction potential (TIP3P) [38] water molecules with a margin distance of
12 Å. The fully solvated and neutralized system was subjected to energy minimization. Following
minimization, the system was gradually heated from 0 to 300 K in 50 ps using a Langevin thermostat

Molecules 2017, 22, 542
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with a coupling coefficient of 1.0/ps with a force constant 2.0 kcal
·
mol−1
·
Å
−2 on the complex. Then,
−1 −2
on the complex was performed.
50 ps of density equilibration with a force constant 2.0 kcal
·
mol
·
Å
Subsequently the systems were again equilibrated for 500 ps by releasing all the restraints. Finally, the
50-ns MD production was performed at 300 K with 1.0 atm pressure. During the MD simulations, the
long-range Coulombic interactions were handled using the particle mesh Ewald (PME) method [39].
The cutoff distance for the long-range vdW energy term was set at 10.0 Å. Periodic boundary conditions
were applied to avoid edge effects in all calculations. The SHAKE algorithm [40] was employed on all
atoms covalently bond to hydrogen atoms, allowing for an integration time step of 2 fs. Coordinate
trajectories were recorded every 10 ps throughout all equilibration and production runs. The binding
free energies were calculated by using the MM-PBSA and MM-GBSA [41] procedure in AMBER12.
An average of 1000 snapshots were extracted from the last 10 ns MD trajectory for the calculations.
The calculations of entropic terms are time-consuming and have low prediction accuracy, thus the
conformational entropy change was omitted.
4. Conclusions
We designed and synthesized pan-RAF bisarylurea inhibitors bearing 1H-pyrazolo[3,4-
d]pyrimidine scaffold targeting DFG-out conformation on the basis of structure-based drug design
using docking of compound 1a into the active site of X-ray cocrystal structures of BRAF. Molecular
docking indicated that these compounds bind to the ATP-binding site of DFG-out BRAF conformation,
with the urea portion forming hydrogen bonds with Asp594 and Glu501, and the terminal-substituted
phenyl projecting into the large hydrophobic pocket. By optimizing the substituents at both terminal
phenyl ring and 1-NH, a series of compounds were obtained. In particular, the most promising
compound, 1v, exhibited potent inhibitory activity against not only BRAFV600E (IC50 = 23.6 nM) but also
wild-type BRAF (IC50 = 51.5 nM) and C-RAF (IC50 = 8.5 nM), and effective cellular anti-proliferative
potencies against A375, HT-29, PC-3 and A549 cell lines, as well as a very good selectivity profile.
Furthermore, flow-activated cell sorting analysis revealed that compound 1v arrested the cell cycle of
the A375 cell line in the G0/G1 phase with a concentration-dependent effect. Moreover, compound 1v
showed significant suppression of MEK phosphorylation in A375 and HT-29 cell lines. Taken together,
the optimal compound 1v showed excellent in vitro potency as a pan-RAF inhibitor. In addition,
molecular dynamics simulation and binding free energy calculations were conducted to analysis the
interaction of synthesized compounds with BRAF, and the results suggested that ligand 1v showed
improved potency compared to that of 1a.
Acknowledgments: This work was supported by grants from the National Natural Science Foundation of China
(No. 81202413 and 81573263), Natural Science Foundation of Guangdong Province, China (No. 2015A030313285)
and the Science and Technology Planning Project of Guangdong Province, China (No. 2014A020210012). We are
thankful to the scientific computing grid (ScGrid) of Supercomputing Center of the Chinese Academy of Science.
Author Contributions: Xiaoyun Wu and Jiajie Zhang conceived and designed the experiments; Yu Fu,
Yuanyuan Wang, Shanhe Wan, and Zhonghuang Li synthesized the compounds; Yu Fu, Shanhe Wan, and
Guangfa Wang performed the biological experimental studies; Xiaoyun Wu performed the molecular dynamics
analysis and analyzed the data; Xiaoyun Wu, Jiajie Zhang, and Yu Fu wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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