Design, synthesis, biological evaluation and molecular modeling of novel 1H-pyrazolo[3,4-d]pyrimidine derivatives as BRAFV600E and VEGFR-2 dual inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.05.054

Aiming to explore novel BRAF^V600E and VEGFR-2 dual inhibitors, a series of 1H-pyrazolo[3,4-d]pyrimidine derivatives were designed, synthesized and biologically evaluated in this study. Most of the synthesized 1H-pyrazolo[3,4-d]pyrimidine compounds displayed moderate to high potent activity in both enzymatic and cellular proliferation assays. Among these compounds, 9e, 9g, 9m and 9u showed remarkably high inhibitory activities against both BRAF^V600E and VEGFR-2 kinase comparable to positive control Sorafenib. Particularly, compound 9u also showed potent anti-proliferative activity against BRAF^V600E-expressing A375 (IC₅₀ = 1.74 μM) and H-29 (IC₅₀ = 6.92 μM) as well as VEGFR-2-expressing HUVEC (IC₅₀ = 5.89 μM), which was also comparable to Sorafenib. Furthermore, kinase selectivity profile showed that 9u had almost poor or no significant inhibitory activity against wild-type BRAF and 15 other tested protein kinases. Flow cytometric analysis showed that compound 9u mainly arrested the A375 and HUVEC cell lines in the G₀/G₁ stage with a concentration-dependent effect. In addition, the molecular docking and molecular dynamics simulations suggested that 9u adopted a similar binding pattern with Sorafenib at the ATP-binding sites of BRAF^V600E and VEGFR-2. Taken together, these results indicated that compound 9u may serve as novel lead compound in research on more effective BRAF^V600E and VEGFR-2 dual inhibitors.

Full text of DOI:10.1016/j.ejmech.2018.05.054

Accepted Manuscript
Design, synthesis, biological evaluation and molecular modeling of novel 1H-
V600E
pyrazolo[3,4-d]pyrimidine derivatives as BRAF
and VEGFR-2 dual inhibitors
Yuanyuan Wang, Shanhe Wan, Zhonghuang Li, Yu Fu, Guangfa Wang, Jiajie Zhang,
Xiaoyun Wu
PII:
S0223-5234(18)30479-3
10.1016/j.ejmech.2018.05.054
EJMECH 10465
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 April 2018
Revised Date: 30 May 2018
Accepted Date: 30 May 2018
Please cite this article as: Y. Wang, S. Wan, Z. Li, Y. Fu, G. Wang, J. Zhang, X. Wu, Design, synthesis,
biological evaluation and molecular modeling of novel 1H-pyrazolo[3,4-d]pyrimidine derivatives as
V600E
BRAF
and VEGFR-2 dual inhibitors, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2018.05.054.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis, biological evaluation and molecular
modeling of novel 1H-pyrazolo[3,4-d]pyrimidine derivatives
as BRAFV600E and VEGFR-2 dual inhibitors
Yuanyuan Wang1, Shanhe Wan1, Zhonghuang Li, Yu Fu, Guangfa Wang, Jiajie
Zhang*, Xiaoyun Wu*
Guangdong Provincial Key Laboratory of New Drug Screening, School of
Pharmaceutical Science, Southern Medical University, Guangzhou 510515, PR
China
*Corresponding author:
E-mail: zhangjj@smu.edu.cn (J.-J. Zhang), Tel.:+86-20-61648548;
E-mail: xywu@smu.edu.com (X.-Y. Wu), Tel.: +86-20-62789415.
1 These authors contributed equally to this work.
Abstract
Aiming to explore novel BRAFV600E and VEGFR-2 dual inhibitors,
a
series of
1H-pyrazolo[3,4-d]pyrimidine derivatives were designed, synthesized and biologically evaluated in
this study. Most of the synthesized 1H-pyrazolo[3,4-d]pyrimidine compounds displayed moderate to
high potent activity in both enzymatic and cellular proliferation assays. Among these compounds, 9e,
9g, 9m and 9u showed remarkably high inhibitory activities against both BRAFV600E and VEGFR-2
kinase comparable to positive control Sorafenib. Particularly, compound 9u also showed potent
anti-proliferative activity against BRAFV600E-expressing A375 (IC50 =1.74 µM) and H-29 (IC50 = 6.92
µM) as well as VEGFR-2-expressing HUVEC (IC50 = 5.89 µM), which was also comparable to
Sorafenib. Furthermore, kinase selectivity profile showed that 9u had almost poor or no significant
inhibitory activity against wild-type BRAF and 15 other tested protein kinases. Flow cytometric
analysis showed that compound 9u mainly arrested the A375 and HUVEC cell lines in the G0/G1
stage with a concentration-dependent effect. In addition, the molecular docking and molecular
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dynamics simulations suggested that 9u adopted a similar binding pattern with Sorafenib at the
ATP-binding sites of BRAFV600E and VEGFR-2. Taken together, these results indicated that compound
9u may serve as novel lead compound in research on more effective BRAFV600E and VEGFR-2 dual
inhibitors.
Keywords: dual inhibitor; BRAFV600E; VEGFR-2; 1H-pyrazolo[3,4-d]pyrimidine; tyrosine kinase
1. Introduction
The mitogen activated protein kinase (MAPK) signaling pathway was the first signaling pathway
elucidated from the cell surface receptors to the nucleus [1]. It consists of the RAS-RAF-MEK-ERK
signal transduction cascade which is a vital driver of a number of cellular fates, including
proliferation, differentiation and survival, making it an attractive pathway to target in several cancer
types [2-4]. The RAF includes three subtypes of ARAF, BRAF and CRAF, and related studies have
shown that the formation of complexes by these different isoforms has an important role in their
activation [5-7]. BRAF isoform is more easily activated by RAS and has higher basal kinase activity
than the other two isoforms, which provides a reasonable explanation for the mutational activation of
BRAF frequently observed in human tumors. The most prevalent mutation of BRAF, accounting for
more than 90% of the detected mutations in BRAF, is an amino acid substitution of glutamic acid for
valine at residue 600 (V600E). The BRAFV600E mutation leads to constitutive kinase activity
500-700-fold greater than wild-type BRAF, which results in amplification of the MAPK pathway, and
this mutation is most frequently associated with melanoma [8]. Besides, activating mutations are also
present in a range of other human cancers, particularly thyroid (30%), colorectal (10%), and ovarian
(35%) cancers [9]. Importantly, targeting this pathway, especially through inhibition of BRAF, is
becoming more important in many current human cancer therapeutic approaches.
However, angiogenesis plays a crucial role in solid tumor progression because the tumors require
significantly more oxygen, glucose, and other nutrients to sustain their rapid growth than do normal
tissues [10]. Many cancer tissues secrete vascular endothelial growth factor (VEGF) to promote
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angiogenesis from adjacent blood vessels [11]. The VEGFR-2 (VEGF receptor-2) is expressed on the
surface of blood vessels, and it plays an important role in tumor angiogenesis. Its activation by VEGF
initiates downstream signaling, ultimately leading to angiogenesis, vascular permeability
enhancement, tumor proliferation, and tumor migration [11]. Inhibition of the VEGF signaling
pathway has become a valuable approach in the treatment of cancers [12-16]. Therefore, targeting
VEGFR-2 has been considered a rational approach for cancer treatment.
Moreover, recent studies showed that BRAF and VEGFR-2 have a certain synergistic effect on
the occurrence and development of tumor [17-18]. Thus, the dual inhibition of BRAFV600E and
VEGFR-2 represents a promising approach to cancer treatment. In recent years, some
BRAFV600E/VEGFR-2 dual inhibitors have been approved or entered clinical trials, such as Sorafenib
and RAF265. Sorafenib (Fig. 1) was developed as an inhibitor of multi-kinases, including tyrosine
and serine/threonine kinases [19], and it has been launched for the treatment of renal cell carcinoma
(RCC) and hepatocellular carcinomas (HCC) [20]. However, its lack of activity in metastatic
melanoma phase 3 clinical trials may because it shows insufficient RAF inhibition in melanoma
tissues [21]. Another RAF inhibitor, RAF265 (Fig. 1), has also been reported as an RAF/VEGFR dual
kinase inhibitor [22]. These two compounds are classified as type II inhibitors [23,24], which bind to
and stabilize the DFG-out “inactive” conformation . They not only bind at the adenosine triphosphate
(ATP) binding site, but also extend to the hydrophobic “back pocket” of protein created by the flip of
the DFG motif. In contrast, Vemurafenib (PLX4032) [25-27] and Dabrafenib (GSK-2118436) (Fig. 1)
[28] are classified as type I inhibitors, which bind to the DFG-in active conformation of the ATP
binding site. These type I inhibitors of RAF are highly BRAF selective against other kinases,
particularly VEGFR-2. Current researches illustrated that Vemurafenib and Dabrafenib 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 [29–31]. This feedback activation was significantly suppressed by DFG-out type multi-kinases
inhibitors, but not by DFG-in type inhibitors. In addition, compared with type I inhibitors, type II
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inhibitors have higher effectiveness [32]. Therefore, we aimed at discovering DFG-out type
BRAFV600E and VEGFR-2 dual inhibitors.
Fig. 1. Structure of BRAFV600E/VEGFR-2 dual inhibitors and selective BRAFV600E inhibitors.
At the time our program began, we have reported potent DFG-out type pan-RAF inhibitors and
identified 1H-pyrazolo[3,4-d]pyrimidine 1 as an inhibitor of pan-RAF (BRAFV600E IC50, 23.6 nM;
wild-type BRAF, 51.5 nM and C-RAF IC50 = 8.5 nM) (Fig. 2) [33]. Compound 1 also showed
effective anti-proliferative activities against RAF cell lines comparable to the efficacy of the positive
control Sorafenib and significant suppression of downstream target MEK phosphorylation in
BRAFV600E cell lines (A375 and HT-29) in a concentration-dependent manner. However, compound 1
showed poor inhibition activity on VEGFR-2 (IC50 ≈ 1000 nM). These results imply that compound 1
could be exerting RAF inhibition on cancer cells rather than antiangiogenesis activity on vascular
endothelial cells. On the basis of these results, we sought to increase the antiangiogenesis activity
while retaining RAF kinase activity. As a means to understand the binding mode and generate a new
chemotype for VEGFR-2 inhibition in addition to BRAFV600E inhibition, we docked 1 into the
co-crystal structure of Sorafenib and VEGFR-2 to identify the key interactions between this molecule
and the protein. Model of 1 in the active site of VEGFR-2 (PDB code: 3WZE [34]), which was
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reported to be in the DFG-out inactive conformation, was constructed by using Glide in the XP mode
[35-37] according to literature [38]. The best docking conformation based on the Glide score (G-score)
was selected as the most probable binding conformation.
As shown in Fig. 2A, the model suggested that 1 overlapped well with the co-crystal ligand
Sorafenib in the active site of VEGFR-2. The 1H-pyrazolo[3,4-d]pyrimidine ring in 1 occupied the
ATP-binding pocket, and formed hydrogen bonds with Cys 919 in the hinge region of VEGFR-2. The
diaryl urea portion extended into the 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 1046 and side-chain carboxylate of Glu 855 in the DFG motif, respectively.
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Fig. 2. (A) Model of compound 1 (green) overlaid with co-crystal structure of Sorafenib (orange) in
the active site of VEGFR-2. (B) The design of BRAFV600E and VEGFR-2 dual inhibitors.
In order to improve inhibition activity of VEGFR-2 while retaining RAF kinase activity, on the
basis of the above binding mode analysis, we proposed the cyclization of 1 to the corresponding
2-aminobenzimidazole to enhance conformational rigidity while maintaining the key
hydrogen-bonding interactions (Fig. 2B). Reduced flexibility is expected to improve potency and
physicochemical properties [39]. Guided by the structural information, we focus on the substitution
on the terminal phenyl to investigate their effects on kinase inhibitory activities in this paper. In order
to illustrate the effect of the 2-aminobenzimidazole ring on the inhibitory activity,
2-aminobenzoxazole compounds were also designed. Herein, we would like to report the synthesis
and biological evaluation of these new compounds as BRAFV600E/VEGFR-2 dual inhibitors. The
binding modes were disclosed by docking and molecular dynamics (MD) simulation, which verified
our design strategy and displayed the structure-activity relationship.
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2. Results and Discussion
2.1 Chemistry
The 2-aminiobenzimidazole compounds were easily constructed (Schemes 1). The intermediate 3
was synthesized with starting material 4,6-dichloropyrimidine-5-carbaldehyde (2) as described in our
earlier research [33], which was under SN2 displacement reaction with iodoalkane to give 4. The
intermediate 5 was obtained by the SNAr displacement reaction of 4 with 4-amino-3-nitrophenol. The
intermediate 6 was achieved in a one-pot procedure by employing in situ protection of the amine with
the trifluoroacetate group of 5, followed by methylation with dimethyl sulfate using phase transfer
catalysis [40]. Then subsequent reduction of the nitro group of intermediate 5 or 6 using 10% Pt/C
and hydrazine hydrate afforded the desired phenylene-diamines 7. Then phenylene-diamines 7 were
treated with various isothiocyanates to afford thiourea intermediates, which were directly cyclized
employing N,N-diisopropylcarbodiimide (DIC) to give the desired products (8-11) in moderate yields.
The synthetic strategy to prepare the 2-aminobenzoxazole compounds was illustrated in Schemes
2. Intermediate 3a was treated with 4-benzyloxyphenol to yield biarylether 12, which was deprotected
using trifluoroacetic acid (TFA) to give corresponding phenol 13. Regioselective nitration of phenol
13 using nitric acid afforded compound 14. Reduction of the nitro group followed by addition of
various isothiocyanates using the similar procedure as described for the preparation of 2-aminio
benzimidazole compounds provided the target compounds 16.
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Cl
Cl
N
Cl
N
CHO
Cl
a
N
b
N
N
c
N
N
N
R1
N
H
N
4a R1 = Me
4b R1 = Et
4c R1 = i-Pr
2
3
R2
NH2
NH2
H
N
NH2
NO2
N
O
N
f
e
N
O
N
O
N
H
N
N
N
N
N
N
N
N
N
R1
8a R2 = H
5a R1 = Me
5b R1 = Et
7a
8b R2 = 2-Me
R2 = 3-Me
8c
R1 = i-Pr
5c
8d R2 = 3-Cl
8e R2 = 4-Cl
d
H
N
H
R2
N
N
N
N
N
N
H
NH
O
NO2
R2
e
f
O
NH2
O
N
O
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
R1
or
R1
R1
6a R1 = Me
6b R1 = Et
6c R1 = i-Pr
9a-u R1 = Me
10a-d R1 = Et
11a-d R1 = i-Pr
7b R1 = Me
9v-w
R1 = Et
7c
7d R1 = i-Pr
9a, 9v R2 = H
9b R2 = 2-Me
9c R2 = 3-Me
9d R2 = 3-Cl
9e R2 = 4-Cl
9f, 10c, 11c R2= 4-Cl-3-CF3
9g, 10b, 11b R2 = 4-Me
9h R2 = 4-CN
9i R2 = 3,4,5-tri-OMe
9j R2 = 4-t-Bu
9k R2 = 2-Cl
9l R2 = 3-Br
9m, 10d, 11d R2 = 4-Br
9n R2= 4-F
9o R2= 3,4-di-Cl
9p R2 = 2-Br-5-F
9q R2 = 4-N(CH3)2
9r R2 = 3-CF3
9s R2 = 4-CF3
9t, 9w R2 = 4-OMe
9u, 10a, 11a R2 = 4-OCF3
Scheme 1. Reagents and conditions: (a) NH2NH2·H2O, NEt3, MeOH, －65℃, 4 h; (b) iodoalkane,
Cs2CO3, DMF, rt, overnight; (c) 4-amino-3-nitrophenol, Cs2CO3, DMF, rt, overnight; (d) (i) TFAA,
DCM, 0 ℃, 45 min; (ii) Me2SO4, hexadecyl trimethyl ammonium bromide (CTMAB), 50% NaOH, rt,
overnight; (e) 10% Pt/C, NH2NH2·H2O, EtOH, rt., 2-3 h; (f) (i) phenylisothiocyanate, CH3CN, rt, 18 h;
(ii) DIC, 120 ℃, 3-6 h.
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Scheme 2. Reagents and conditions: (a) 4-(benzyloxy)phenol, Cs2CO3, DMF, rt, overnight; (b) TFA,
50 , overnight; (c) faming HNO3, AcOH, 30 , 2 h; (d) 10% Pt/C, NH2NH2·H2O, EtOH, r.t., 1 h; (e)
(i) phenylisothiocyanate, CH3CN, rt, 18 h; (ii) DIC, 120 , 3-6 h.
2.2 Biological Activities and Discussion
2.2.1. Kinase Inhibitory Activity and SARs Study
The target compounds were evaluated for their inhibitory activities against BRAFV600E and
VEGFR-2 kinase using Sorafenib as positive control (Table 1). Based on the kinase activity
evaluation, a preliminary structure-activity relationship (SAR) in this work was summarized in Fig 3.
Unfortunately, closing the urea onto the internal phenyl led to 8a with lower inhibitory activity for
both the BRAFV600E and VEGFR-2. Adding the substituent on the terminal phenyl of 8a giving 8b-e
led to a slightly increased inhibitory activity for BRAFV600E only. Guided by the molecular docking
that NH of benzimidazole filled the small hydrophobic subpocket partially defined by the “gatekeeper”
residue (Thr 529 for BRAFV600E), introduction of a methyl on the 2-aminobenzimidazole NH was
explored giving 9a-e. Compared to the unmethylated analogues 8a-e, 9a-e exhibited better inhibitory
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activities against BRAFV600E and VEGFR-2. In addition, the replacement of NH of 2-amino
benzimidazole with O gave compound 2-aminobenzoxazole compounds 16, which displayed
decreased inhibitory activities against BRAFV600E and VEGFR-2, particularly for VEGFR-2.
Apparently, the N-Me of benzimidazole turned out to be optimal for BRAFV600E and VEGFR-2
inhibition.
Then, systematic evaluation of various substituents at the terminal phenyl was conducted. Almost
all compounds with substitution at the terminal phenyl can improve the inhibitory activity compared
to unsubstituted compound 9a, indicating substitution on the aryl ring was essential for potent
BRAFV600E and VEGFR-2 inhibition. It also indicated the importance of the hydrophobic interaction
between the inhibitor and the hydrophobic amino acid residues of the back pocket created by the
rearrangement of the DFG motif. Compound 9f with the same substitution on the terminal phenyl
with Sorafenib and 1 displayed potent inhibition of VEGFR-2, while the inhibition of BRAFV600E
decreased significantly. In general, introduction of substituents on the 4-position of the terminal
phenyl led to higher inhibitory activities for BRAFV600E and VEGFR-2 than substitution on the 2- and
3-positions. For example, compound 9g exhibited higher inhibition against both the BRAFV600E and
VEGFR-2 than 9b and 9c. A variety of substituents at the para-position, including both
electron-donating and electron-withdrawing groups were tolerated, which suggested that the
electronic interaction of the substituent had little effect on the kinase inhibitory activities. For
example, introduction of the electron-donating group methyl and methyloxy in compounds 9g and 9t
increase the inhibitory activities, and substitution with electron-withdrawing groups such as Cl, Br,
and CF3 on the phenyl ring also led to enhancement of kinase inhibitory activities (9e, 9m and 9s).
Particularly, compound 9m with a 4-bromo substituent exhibited kinase inhibitory activity against
VEGFR-2 that was about 30-fold better than 1, while with slightly weaker potency for BRAFV600E
compared to that of compound 1. The necessity of the terminal aryl in retaining high potency was also
evidenced by the fact that substituting with aliphatic residue, such as benzyl (9v and 9w), led to the
decrease of inhibitory activities. This SAR was consistent with the molecular modeling that the
terminal phenyl group in 1 projected into the large hydrophobic pocket created by the rearrangement
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of the DFG motif.
A brief SAR investigation of N1-substitution of 1H-pyrazolo[3,4-d] pyrimidine ring was also
conducted. In our previous work [33], N-methylation compounds exhibited higher inhibitory
activities than the unsubstituted counterparts. In this work, introduction of larger groups led to
deterioration of the potency, with the degree of loss correlation with the steric size of the introduced
groups, implying that the methyl substitution at the N1 position of 1H-pyrazolo[3,4-d]pyrimidine was
beneficial for the inhibitory activity against BRAFV600E and VEGFR-2. For example, the inhibitory
activity of 10a and 11a gradually decreased with the increase of alkyl substituents on the N1H of the
1H-pyrazolo[3,4-d] pyrimidine.
Table 1. The kinase inhibitory activities and in vitro anti-proliferative effects of target compounds.
in vitro anti-proliferative
kinase inhibitory activities a
effects (IC50, µM)c
X
R1
R2
BRAFV600E
97(23.6) b
10.1
VEGFR-2
A375 HT-29 HUVEC
1
-
56
15.6
14.72
>100
>100
>100
>100
>100
>100
>100
>100
>100
23.41
>100
>100
>100
>100
22.87
>100
>100
87.11
99.38
5.21
>100
>100
>100
>100
>100
>100
>100
>100
79.09
NH
NH
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
8a
8b
8c
8d
8e
9a
9b
9c
9d
2-Me
3-Me
3-Cl
4-Cl
H
41.1
36.5
NH
49.6
10.4
NH
45.3
7.2
NH
82.0
14.6
NMe
NMe
NMe
NMe
49.4
78.2
2-Me
3-Me
3-Cl
62.6
77.3
90.6 (90.5) b
72.1
48.5
68.4
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NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
NMe
O
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
4-Cl
4-Cl-3-CF3
4-Me
>100
1.99
73.56
14.38
6.13
84.32
13.47
15.71
62.7
9e
9f
91.1(105.6) b
55.1
105.6 (49.8) b
97.7 (134.5) b
96.6 (17.1) b
88.7
25.36
>100
>100
1.23
9g
90.1 (85.2) b
4-CN
70.7
>100
>100
75.41
80.36
64.29
>100
79.14
81.20
>100
65.44
>100
63.21
>100
6.92
9h
3,4,5-tri-OCH3
4-t-Bu
26.8
40.7
>100
53.51
78.62
74.24
7.89
9i
50.8
9j
84.2 (140) b
2-Cl
65.4
84.2
>100
82.18
30.25
68.42
66.68
>100
5.19
9k
3-Br
65.7
73.7
9l
4-Br
9m
9n
102.3 (40.9) b
85.6 (407.2) b
68.1 (487.8) b
51.4
108.9 (29.9) b
94.0 (41.7) b
82.2 (120.4) b
48.6
4-F
12.81
>100
>100
>100
29.23
12.56
90.89
5.89
3,4-di-Cl
2-Br-5-F
4-N(CH3)2
3-CF3
9o
9p
67.2
9q
94.5 (111.5) b
88.1 (150.2) b
97.4 (63.2) b
93.6 (73.4) b
96.4 (77.9) b
9.12
10.08
4.11
9r
80.5 (476.3) b
74.4 (215.6) b
79.6
4-CF3
9s
4-OCH3
4-OCF3
H
13.72
1.74
9t
9u
83.1 (171.5) b
6.66
>100
9.31
32.48
>100
12.25
6.71
>100
20.36
54.42
5.25
9v
4-OCH3
4-OCF3
4-Me
53.6
9w
10a
10b
10c
10d
11a
11b
11c
11d
16a
16b
16c
16d
102.3 (424) b
19.6
59.6
8.52
Et
68.0
85.5
4.82
Et
4-Cl-3-CF3
4-Br
10.9
44.7
5.19
8.51
8.17
Et
69.4
81.1
7.12
8.01
10.15
5.96
i-Pr
i-Pr
i-Pr
i-Pr
Me
Me
Me
Me
4-OCF3
4-Me
13.2
15.1
7.46
9.51
36.0
35.2
33.06
15.71
12.82
>100
44.92
44.21
>100
13.89
36.85
9.01
29.58
37.64
15.86
61.62
35.37
>100
17.19
4-Cl-3-CF3
4-Br
7.5
6.1
33.3
38.5
4-OCF3
4-Me
56.1
22.3
>100
28.25
34.02
41.07
O
81.6
53.1
O
4-Cl-3-CF3
4-Br
20.0
15.6
O
60.1
18.9
94.5 (52.3) b
94.3 (17.6) b
Sorafenib
8.33
2.58
2.69
a Values indicate the kinase inhibition % at 1µM ,which are the average of two independent experiments.
b Values in the parentheses indicate IC50 ( nM ), which are the average of two independent experiments.
c IC50 values are presented as mean values of at least three independent experiments.
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NMe > NH > O
X
N
R3
n
N
H
O
N
N
para > ortho & meta
1> 0
N
N
R1
Me > Et > i-Pr
Hinge region
Fig. 3 Preliminary structure-activity relationships
2.2.2 In vitro Anti-proliferative Activities
The cell growth inhibitory activities of target compounds were evaluated against human cancer
A375 and HT-29 cell lines highly expressing the BRAFV600E protein as well as the HUVEC cell line
highly expressing the VEGFR-2 protein by the standard MTT assay, with Sorafenib as the positive
control. The anti-proliferative activities were reported in Table 1. It can be noted that most of the
target compounds displayed moderate to high potency against A375, HT-29 and HUVEC cell, some
of the compounds showed parallel anti-proliferative activities as Sorafenib. For example, compounds
9f, 9j, 9q, 9s, 9u, 9w, 10a-d, 11a and compounds 9g, 9u, 10b-d, 11a, 11d displayed significant
potency against BRAFV600E-expressing A375 and H-29 with IC50 values of 1.23-9.31 µM and
6.13-9.51 µM, respectively, which turned out to be similar to that of Sorafenib (8.33 and 2.58 µM for
A375 and HUVEC, respectively). In addition, compounds 9m, 9u, 10b-c and 11a displayed
significant potency against VEGFR-2- expressing HUVEC with IC50 values of 5.25-8.17 µM, which
was similar to that of Sorafenib (2.69 µM). Nevertheless, the cellular responses of some synthesized
compounds were not consistent with the enzymatic assays absolutely. For example, although 9e, 9g,
and 9m exhibited good enzyme potencies against BRAFV600E and VEGFR-2, they showed moderate
cell growth inhibitory activities compared to Sorafenib. This discrepancy between biochemical and
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cell potency could be explained that in the biochemical assay purified kinase domains of BRAFV600E
and VEGFR-2 were being tested compared to the cellular assay in which kinases exist as a complex
with chaperons, cytoskeleton, phosphatases and kinases [41]. In contrast, compounds 10a-d and 11a
with poor kinase inhibitory activities showed significant cell growth inhibitory activities, which may
be related to the complicated mechanism of tumor cell survival or proliferation that is regulated by
several different factors [42], or through alternate mechanism but not by targeting BRAFV600E or
VEGFR-2 [43-44].
2.2.3 Kinase Inhibitory Profile of Compound 9u
Compound 9u exhibited high inhibitory activity against BRAFV600E (IC50 = 0.171 µM) and
VEGFR-2 (IC50 = 0.779 µM), and effective anti-proliferative potencies against three cell lines (A375,
HT-29, and HUVEC), which were comparable to the efficacy of Sorafenib. Therefore, we selected 9u
for further investigation. The kinase selectivity profile of 9u was assessed over 17 different protein
kinases (Table 2). As shown in Table 2, compound 9u had moderate inhibitory activity against CRAF
(inhibition % = 87.6), almost poor or no significant inhibitory activity against wild-type (WT) BRAF
and 15 other tested protein kinases even at concentration of 1000 nM, which revealed that compound
9u had a very good selectivity profile and was a BRAFV600E/VEGFR-2 kinases dual inhibitor.
Table 2. Kinase selectivity profile of compound 9u
Kinase tested
Inhibition % at 1 µM a
Kinase tested
Inhibition % at 1 µM a
BRAF (WT)
CRAF (RAF-1)
PDGFRA (PDGFR α)
PDGFRB (PDGFR β)
FGFR3
13.7
87.6
21.9
35.6
SRC
AURKB (Aurora B)
LCK
14.0
−
11.6
5.9
ALK
−11.7
−4.9
MET (c-Met)
PTK6 (Brk)
FGR
24.9
KIT
25.4
40.5
54.7
52.0
−
6.7
6.9
CSF1R (FMS)
FLT1 (VEGFR-1)
FLT4 (VEGFR-3)
−
SRMS
7.1
a Values are the average of two independent experiments.
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2.2.4 Flow Cytometric Analysis of Cell-cycle Arrest
To study the effect of these target compounds on cells cycle progression, flow-activated cell
sorting analysis was performed. The most promising compound 9u was tested against A375 and
HUVEC cell lines at given concentrations, respectively. Interestingly, very similar changes in cell
cycle distribution were observed. A375 and HUVEC cell lines were arrested by compound 9u in
G0/G1 phase with a concentration-dependent effect compared to untreated control cells. As shown in
Figure 4-5 and Table 3-4, the 'G0/G1' peak in A375 cell line significantly increased from 62.83% to
70.33% (0.87 µM), 75.34% (1.74 µM), 84.97% (3.48 µM), and the 'G0/G1' peak of HUVEC
significantly increased from 53.18% to 69.74% (2.95 µM), 77.70% (5.89 µM), 83.26% (11.78 µM)
after 48 h of incubation with the compound 9u.
Table 3. Effect of compound 9u on cell cycle distribution in A375 cell line.
Concentration
0
Sub-G1 (%)
2.32
G0/G1 (%)
62.83
S (%)
14.8
G2/M (%)
19.75
0.87 µmol/L
1.74 µmol/L
3.48 µmol/L
3.21
70.33
10.78
9.97
14.92
4.36
75.34
10.09
3.05
84.97
7.02
4.38
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Fig. 4. Flow cytometric analysis of cell cycle distribution in A375 cell line treated with 9u for 48 h.
(A) Control; (B) 0.87 µmol/L (1/2 IC50); (C) 1.74 µmol/L (1 IC50); (D) 3.48 µmol/L (2 IC50).
Table 4. Effect of compound 9u on cell cycle distribution in HUVEC cell line.
Concentration
0
Sub-G1 (%)
1.36
G0/G1 (%)
53.18
S (%)
24.3
G2/M (%)
20.11
2.95 µmol/L
5.89 µmol/L
11.78 µmol/L
2.68
69.74
12.54
9.24
14.92
1.94
77.70
10.34
2.63
83.26
9.89
3.85
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Fig. 5. Flow cytometric analysis of cell cycle distribution in HUVEC cell line treated with 9u for 48 h.
(A) Control; (B) 2.95 µmol/L (1/2 IC50); (C) 5.89 µmol/L (1 IC50); (D) 11.78 µmol/L (2 IC50).
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2.4 Molecular Modeling
In order to understand the interaction between the synthesized compounds and kinases, docking
experiments of the representative compounds 8a, 9u, 9w, 10a, 11a and 16a into the active site of
BRAFV600E and VEGFR-2 (PDB code: 1UWJ [45] and 3WZE [34], respectively), which were reported
to be in the DFG-out inactive conformation of kinase, was constructed using Glide (Grid-based Ligand
Docking with Energetics) in Extra-Precision (XP) mode [35-37]. The docking results indicated that all
the representative compounds could dock into the active site of BRAFV600E and KDR (Figure 6) and
adopt a similar binding mode with the compound 1 and Sorafenib except for compound 9w with benzyl
group on the terminal (Fig. S1 of supporting information). The compound 9u was taken as an example
to illustrate the binding modes. The 1H-pyrazolo[3,4-d]pyrimidine moiety inserted into the deep cleft
of ATP-binding site and formed hydrogen-bond with the backbone-NH of Cys532 and Cys919 from
hinge region of BRAFV600E and VEGFR-2, respectively, and the 2-aminobenzimidazole formed
hydrogen-bond interactions with the DFG motif of BRAFV600E and VEGFR-2, respectively. The
terminal 4-trifluoromethoxylphenyl portion extended into the large hydrophobic back pocket created by
the rearrangement of the DFG motif, which was consistent with the structure-activity relationship
(SAR) that the introduction of hydrophobic groups was beneficial for potency. The N-Me on the
2-aminobenzimidazole filled the small hydrophobic subpocket partially defined by the “gatekeeper”
residue (Thr 529 for BRAFV600E), which can partially explain the decreased inhibitory activities of
compounds 8a-e with unmethylated benzimidazole scaffold and compounds 16a-d with
2-aminobenzoxazole scaffold. The introduction of larger groups in the N1 position of
1H-pyrazolo[3,4-d]pyrimidine led to deterioration of the potency because of the steric hindrance effect
with the hinge region. The compound 9w adopted a distinct binding mode that cannot form H-bond
with the backbone-NH of Cys532 from hinge region of BRAFV600E, which gave explanation for the
decreased activity for BRAFV600E (Fig. S1 of supporting information).
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Fig. 6. (A) The docked binding modes of compound 1 (green) and 9u (cyan) overlaid with co-crystal
structure of Sorafenib (orange) in the active site of BRAFV600E (A) and VEGFR-2 (B). (BRAFV600E and
VEGFR-2 in colored cartoon, ligand in stick model and the key residues in line model. Yellow dots
represent hydrogen bonds.)
The best docking conformations of the potent compound 9u based on the Glide score (G-score)
were selected as the most probable binding conformation, which were subjected to molecular dynamics
(MD) simulation to explore in depth the binding poses. MD simulations were then carried out in
explicit aqueous solution for 50 ns. For comparison, MD simulations of positive control Sorafenib in
complex with BRAFV600E and VEGFR-2 were also performed. The stability of four systems under
simulation was evaluated by the root-mean-square deviation (RMSD) of the backbone atoms relate to
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the starting structures (Fig. 7). As can be seen in the plot, all systems were stable during the 50-ns MD
simulation. Moreover, we compared the representative snapshots of each complex taken from the last
10 ns MD trajectories with the initial structure (Fig. 8). As illustrated in Figure 8, the binding modes of
9u from MD were nearly the same as the docked structures.
The hydrogen bond plays an important role in the binding of inhibitor with kinase. As we expected,
the 1H-pyrazolo[3,4-d]pyrimidine moiety can form H-bond with the backbone-NH of Cys532 and
Cys919 from hinge region of BRAFV600E and VEGFR-2 with very high hydrogen-bond occupancies of
91.76 and 91.04 %, respectively (Table 5). Unlike the compound 9u, Sorafenib could form a pair of
H-bonds with Cys532 and Cys919 of BRAFV600E and VEGFR-2, respectively. In comparison to
Sorafenib, compound 9u formed moderate hydrogen bond interactions with Asp594 and Asp1046 of
BRAFV600E and VEGFR-2, respectively, which could partially explain that compound 9u displayed
lower inhibitory activity than Sorafenib. Besides, both compound 9u and Sorafenib form hydrogen
bond interactions with side-chain carboxylate of Glu501 and Glu885 from the DFG motif of
BRAFV600E and VEGFR-2 with very high hydrogen-bond occupancies, respectively.
Fig. 7. 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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Fig.8. Structural comparison between docked (green) and molecular dynamics simulated
representative snapshots (cyan) of 9u in the active site of BRAFV600E (A) and VEGFR-2 (B).
(BRAFV600E and VEGFR-2 in colored cartoon, ligand in stick model and the key residues in line
model. Yellow dots represent hydrogen bonds.)
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Table 5. Main hydrogen-bond interactions between inhibitors and BRAFV600E and VEGFR-2.
BRAFV600E
VEGFR-2
Occupancy
(%)a
Occupancy
Acceptor
Donor
Acceptor
Donor
(%)a
91.04
57.11
56.69
37.63
Lig @N3
Lig @N4
Cys532@NH
Asp594@NH
Lig @N6H
91.76
Lig @N3
Lig @N4
Cys919@NH
Asp1046@NH
Lig @N6H
66.12
9u
Glu501@OE1
Glu501@OE2
59.05
Glu885 @OE1
Glu885 @OE2
Lig @N6H
39.34
Lig @N6H
Lig @O2
Asp594@NH
Lig @N2H
99.07
63.16
Lig @O2
Asp1046@NH
Lig @N2H
96.83
54.34
Glu501@OE1
Glu885 @OE1
Glu501@OE2
Glu501@OE1
Glu501@OE2
Lig @N4
Lig @N3H
Lig @N3H
Lig @N2H
Cys532@NH
Lig @N1H
62.73
37.07
36.90
27.88
42.63
Glu885 @OE2
Glu885 @OE1
Glu885 @OE2
Lig @N4
Lig @N3H
Lig @N3H
Lig @N2H
Cys919@NH
Lig @N1H
53.52
45.69
46.69
79.28
61.10
Sorafenib
Cys532@O
Cys919@O
a Occupancy is in unit of 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 6). As can been seen, MM-PBSA and MM-GBSA calculations
verified that 9u showed decreased potency ligand compared to that of Sorafenib, 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.
Table 6. Binding-free energies for 9u and Sorafenib in complex with BRAFV600E and VEGFR-2
obtained by MM-PBSA and MM-GBSA (kcal/mol) a
Sorafenib
BRAFV600E
9u
BRAFV600E
VEGFR-2
−68.80 (2.55)
−21.35 (2.79)
− 90.15 (3.26)
39.21 (1.76)
− 7.77 (0.15)
VEGFR-2
−59.92 (2.63)
−36.33 (3.35)
− 96.25 (3.68)
44.67 (2.18)
− 7.93 (0.17)
∆EvdW
∆Eele
−62.51 (2.72)
−25.57 (3.48)
−88.08 (3.82)
42.20 (2.34)
−7.51 (0.17)
− 57.10 (2.75)
− 35.27 (3.72)
− 92.37 (4.11)
45.71 (2.66)
∆EMM
∆Gele,sol (GB)
∆Gnp,sol (GB)
− 7.78 (0.18)
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∆Gsol (GB)
∆Gele,sol (PB)
∆Gnp,sol (PB)
∆Gsol (PB)
∆Hpred (GB)
∆Hpred (PB)
T∆S
34.69 (2.24)
47.84 (2.51)
−4.52 (0.09)
43.32 (2.51)
−53.38 (3.01)
−47.75 (3.25)
− 27.73 (5.66)
− 25.65
31.45 (1.80)
43.77 (2.14)
− 4.39 (0.08)
39.38 (2.12)
− 58.70 (2.83)
− 50.77 (3.10)
− 27.46 (2.64)
− 31.24
37.93 (2.63)
36.74 (2.20)
47.64 (2.54)
−4.55 (0.08)
43.08 (2.52)
− 59.52 (3.10)
− 53.17 (3.24)
− 25.20 (2.31)
− 34.32
49.93 (3.01)
− 4.57 (0.09)
45.36 (2.99)
− 54.44 (2.98)
− 47.01 (3.60)
−25.39 (6.27)
− 29.05
∆Gpred (GB)
∆Gpred (PB)
− 20.02
− 23.31
− 21.62
− 27.97
a ∆Gpred: the calculated binding-free energy by MM-PBSA and MM-GBSA method
∆Hpred = ∆Gele + ∆GvdW + ∆Gnp,sol + ∆Gele,sol
∆Gpred = ∆Gele + ∆GvdW + ∆Gnp,sol + ∆Gele,sol− T∆S
3. Conclusion
In conclusion, a series of 1H-pyrazolo[3,4-d]pyrimidine derivatives were designed, synthesized
and evaluated for their BRAFV600E and VEGFR-2 inhibitory activity as well as their anti-proliferative
activity. The preliminary investigation showed most of the synthesized 1H-pyrazolo[3,4-d]pyrimidine
compounds displayed moderate to high potent activity in both enzymatic and cellular proliferation
assays. Among these compounds, 9e, 9g, 9m and 9u showed remarkably high inhibitory activities
against both BRAFV600E and VEGFR-2 kinase comparable to positive control Sorafenib. Particularly,
compound 9u also showed potent anti-proliferative activity against BRAFV600E-expressing A375 and
H-29 as well as VEGFR-2-expressing HUVEC, which was also comparable to Sorafenib. Furthermore,
9u showed almost poor or no significant inhibitory activity against wild-type BRAF and 15 other tested
protein kinases, implying a very good selectivity profile. Flow cytometric analysis showed that
compound 9u mainly arrested the A375 and HUVEC cell lines in the G0/G1 stage with a
concentration-dependent effect. In addition, the molecular docking and MD simulations suggested a
similar binding pattern with Sorafenib at the ATP-binding sites of BRAFV600E and VEGFR-2. Taken
together, these results indicated that compound 9u may serve as novel lead compounds in research on
more effective BRAFV600E and VEGFR-2 dual inhibitors.
4. Experimental
4.1. Chemistry
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All starting materials, reagents, and solvents 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. Reactions were monitored by 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) or
reverse phase silica gel by using Yamazen AI-580 flash chromatography (Yamazen Co., Osaka, Japan)
1
with UV detection at 254 nm. H and 13C NMR spectra were recorded on a BRUKER AVIII 400 MHz
and 101 MHz spectrometer 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). High resolution mass spectra
(HRMS) were recorded on a Thermo Fisher Scientific Orbitrap Fusion Tribrid (Q-OT-qIT) mass
spectrometer (Thermo Fisher Scientific, Bremen, German). FT-IR spectra were recorded on a Thermo
Scientific Nicolet 6700 spectrometer. Melting points were determined using an X-4 melting-point
apparatus with microscope (Gongyi Yuhua Instrument Co., Ltd., Henan, China) and were uncorrected.
4.1.1. 4-chloro-1H-pyrazolo[3,4-d]pyrimidine (3)
To the solution of 4,6-dichloropyrimidine-5-carbaldehyde 2 (1.0 g, 5.6 mmol) in methanol (20 mL)
at −65 °C, triethylamine (0.97 mL) was 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 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 MgSO4 and concentrated to give compound 3 (0.602 g, yield: 68.9%. )
1H 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]−.
4.1.2. General procedure A for the synthesis of compounds (4)
A suspension of compound 3 (6.5 mmol) and Cs2CO3 (13.0 mol) in dry DMF (25 mL) was stirred
at 0 for 30 min, and then iodide alkane (7.8 mmol) was added. After stirring overnight, the reaction
mixture was extracted with EtOAc (60 mL × 3), washed with water, dried over Na2SO4, and
concentrated in vacuo to give compound 4.
4.1.2.1. 4-chloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine (4a)
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Compound 3 (1.0 g, 6.5 mmol) was reacted with iodomethane (1.1 g, 7.8 mmol) to give
compound 4a (0.42 g, yield: 38.5%). 1H NMR (400 MHz, DMSO-d6) δ 8.83(s, 1H), 8.41(s, 1H), 4.06(s,
3H). ESI-MS, m/z: 169.3[M+H]+.
4.1.2.2. 4-chloro-1-ethyl-1H-pyrazolo[3,4-d]pyrimidine (4b)
Compound 3 (1.0 g, 6.5 mmol) was reacted with iodoethane (1.3 g, 8.1 mmol) to give compound
1
4b (0.40 g, yield: 33.6%). H NMR (400 MHz, CDCl3) δ 8.77(s, 1H), 8.17(s, 1H), 4.57(q, J = 7.2 Hz,
2H), 1.56(t, J = 7.2 Hz, 3H). ESI-MS, m/z: 183.4 [M+H]+.
4.1.2.3. 4-chloro-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidine (4c)
Compound 3 (1.0 g, 6.5 mmol) was reacted with 2-iodopropane (1.5 g, 8.8 mmol) to give
compound 4b (0.28 g, yield: 21.7%). 1H NMR (400 MHz, CDCl3) δ 8.77(s, 1H), 8.17(s, 1H), 4.57(q, J
1
= 7.2 Hz, 2H), 1.56(t, J = 7.2 Hz, 3H). ESI-MS, m/z: 183.4 [M+H]+. H NMR (400 MHz, CDCl3) δ
8.69(s, 1H), 8.10(s, 1H), 5.19-5.29(m, 1H), 1.56(d, J = 6.8 Hz, 6H). ESI-MS, m/z: 197.3 [M+H]+.
4.1.3. General procedure B for the synthesis of compounds (5)
To the mixture of 4-amino-3-nitrophenol (1.0 g, 6.4 mmol) in DMF (50 mL), Cs2CO3 (3.12 g, 9.6
mmol) was added under the protection of nitrogen. The reaction mixture was stirred at r.t. for 1.5–2 h,
and then compound 4 (6.4 mmol) was added slowly. After stirring overnight, the reaction mixture was
diluted with EtOAc (50 mL), washed with water and brine, dried over Na2SO4 and concentrated to give
compound 5 as an orange solid.
4.1.3.1. 4-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)-2-nitroaniline (5a)
Compound 4a (1.08 g, 6.4 mmol) was reacted with 4-amino-3-nitrophenol (0.99 g, 6.4 mmol) to
1
give compound 5a as an orange solid (1.51 g, yield: 82.3%). H NMR (400 MHz, CDCl3) δ 8.55 (s,
1H), 8.09 (s, 1H), 8.07 (d, J = 2.4 Hz, 1H), 7.32-7.35 (m, 1H), 6.94 (d, J = 8.8 Hz, 1H), 6.20 (s, 2H),
4.16 (s, 3H). ESI-MS, m/z: 287.5 [M+H]+.
4.1.3.2. 4-((1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)-2-nitroaniline (5b)
Compound 4b (1.05 g, 5.7 mmol) was reacted with 4-amino-3-nitrophenol (0.88 g, 5.7 mmol) to
give compound 5b as an orange solid (1.36 g, yield: 79.7%). 1H NMR (400 MHz, CDCl3) δ 8.54(s, 1H),
8.09(s, 1H), 8.07(d, J = 2.8 Hz, 1H), 7.34(dd, J = 2.8, 9.2 Hz, 1H), 6.93(d, J = 9.2 Hz, 1H), 6.17(s,
25

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2H), 4.57(q, J = 7.2 Hz, 2H), 1.57(t, J = 7.2 Hz, 3H). ESI-MS, m/z: 301.4 [M+H]+.
4.1.3.3. 4-((1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)-2-nitroaniline (5c)
Compound 4c (1.25 g, 6.4 mmol) was reacted with 4-amino-3-nitrophenol (0.99 g, 6.4 mmol) to
give compound 5c as an orange solid (1.49 g, yield: 74.3%). 1H NMR (400 MHz, CDCl3) δ 8.53(s, 1H),
8.09(s, 1H), 8.07(d, J = 2.4 Hz, 1H), 7.34(dd, J = 2.4, 9.2 Hz, 1H), 6.93(d, J = 9.2 Hz, 1H), 6.16(s,
2H), 5.19-5.29(m, 1H), 1.63(d, J = 6.8 Hz, 6H). ESI-MS, m/z: 315.7[M+H]+.
4.1.4. General procedure C for the synthesis of compounds (6)
To a cooled (0 ) flask charged with CH2Cl2 (18 mL) and compound 5 (3.49 mmol),
trifluoroacetic anhydride (0.93 mL, 6.59 mmol) was added dropwise over 15 min and the mixture was
stirred for 45 min. After that, hexadecyl trimethyl ammonium bromide (0.63 g, 1.73 mmol), dimethyl
sulfate (0.66 mL, 6.94 mmol), and 50% NaOH aqueous (14 mL) were added. The mixture was allowed
to warm to rt and stirred for 16 h. Then the mixture was diluted with CH2Cl2, washed with water, dried
over Na2SO4, filtered, and concentrated in vacuo. The crude residue was purified by recrystallization
from ethanol-water to give 5 as fine red needles.
4.1.4.1. N-methyl-4-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)-2 -nitroaniline (6a).
Compound 5a (1.0 g, 3.49 mmol) was reacted with dimethyl sulfate (0.66 mL, 6.94 mmol) to give
6a as fine red needles (0.88 g, yield: 84.3%). 1H NMR (400 MHz, CDCl3) δ 8.56 (s, 1H), 8.13 (d, J =
2.8 Hz, 2H), 8.09 (s, 1H), 7.46 (dd, J = 9.2, 2.8 Hz, 1H), 6.98 (d, J = 9.2 Hz, 1H), 4.16 (s, 3H), 3.10 (s,
3H). ESI-MS m/z: 301.5 [M+H]+.
4.1.4.2. 4-((1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)-N-methyl-2-nitroaniline (6b)
Compound 5b (1.0 g, 3.33 mmol) was reacted with dimethyl sulfate (0.32 mL, 3.33 mmol) to give
1
6b as fine red needles (0.84 g, yield: 80.6%). H NMR (400 MHz, CDCl3) δ 8.54(s, 1H), 8.13(d, J =
2.8 Hz, 2H), 8.08(s, 1H), 7.45(dd, J = 2.8, 9.2 Hz, 1H), 6.98(d, J = 9.2 Hz, 1H), 4.57(q, J = 7.2 Hz,
2H), 3.11(d, J = 5.2 Hz, 3H), 1.57(t, J = 7.2 Hz, 3H). ESI-MS, m/z: 315.3 [M+H]+.
4.1.4.3. 4-((1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)-N-methyl-2-nitroaniline (6c)
Compound 5c (1.0 g, 3.18 mmol) was reacted with dimethyl sulfate (0.31 mL, 3.18 mmol) to give
1
6c as fine red needles (0.81 g, yield: 77.8%). H NMR (400 MHz, CDCl3) δ 8.53(s, 1H), 8.13(d, J =
26

ACCEPTED MANUSCRIPT
2.4 Hz, 3H), 8.07(s, 1H), 7.45(dd, J = 2.8, 9.2 Hz, 1H), 6.98(d, J = 9.2 Hz, 1H), 5.19-5.29(m, 1H),
3.10(d, J = 5.2 Hz, 3H), 1.63(d, J = 6.8 Hz, 6H). ESI-MS, m/z: 329.7[M+H]+.
4.1.5. General procedure D for the synthesis of compounds (7)
To a flask charged with compound 5 or 6 (17.4 mmol) in EtOH (300 mL), 10% Pt/C (1.56 g) was
added, then a solution of hydrazine hydrate (4.22 mL, 87 mmol) in EtOH (70 mL) was added dropwise
over 30 min, and the mixture was stirred for 3 h at rt. The mixture was filtered, diluted with EtOAc
(100 mL), washed with water and brine, dried over Na2SO4 and concentrated to give compound 7 as a
brown solid.
4.1.5.1 4-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)benzene-1,2-diamine (7a)
Compound 5a (5.0 g, 17.4 mmol) was reacted with hydrazine hydrate (4.22 mL, 87 mmol) to give
compound 7a as a brown solid (3.42 g, yield: 76.7 %). 1H NMR (400 MHz, DMSO-d6) δ 8.54 (s, 1H),
7.42 (s, 1H), 6.57 (d, J = 8.4 Hz, 1H), 6.42 (d, J = 2.4 Hz, 1H), 6.30 (dd, J = 2.4, 8.4 Hz, 1H), 4.75 (s,
2H), 4.56 (s, 2H), 4.00 (s, 3H). ESI-MS, m/z: 257.3 [M+H]+.
4.1.5.2 N1-methyl-4-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)benzene-1,2-diamine (7b)
Compound 6a (5.0 g, 16.7 mmol) was reacted with hydrazine hydrate (4.02 mL, 83 mmol) to give
1
compound 7b as a brown solid (3.38 g, yield: 74.8%). H NMR (400 MHz, DMSO-d6) δ 8.53 (s, 1H),
7.45 (s, 1H), 6.40-6.48 (m, 3H), 4.82 (s, 3H), 3.99 (s, 3H), 2.76 (s, 3H). ESI-MS, m/z: 271.6 [M+H]+.
4.1.5.3 4-((1-ethyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)-N1-methylbenzene-1,2-diamine (7c)
Compound 6b (5.0 g, 15.9 mmol) was reacted with hydrazine hydrate (3.83 mL, 79 mmol) to give
1
compound 7c as a brown solid (3.22 g, yield: 71.4%). H NMR (400 MHz, CDCl3) δ 8.53(s, 1H),
7.44(s, 1H), 6.41-6.47(m, 3H), 4.79(s, 3H), 4.42(q, J = 7.2 Hz, 2H), 2.76(s, 3H), 1.57(t, J = 7.2 Hz,
3H). ESI-MS, m/z: 285.4 [M+H]+.
4.1.5.4 4-((1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)-N1-methylbenzene-1,2-diamine (7d)
Compound 6c (5.0 g, 15.2 mmol) was reacted with hydrazine hydrate (3.64 mL, 75 mmol) to give
1
compound 7d as a brown solid (3.14 g, yield: 71.4%). H NMR (400 MHz, DMSO-d6) δ 8.53(s, 1H),
7.42(s, 1H), 6.46-6.47(m, 1H), 6.43-6.44(m, 2H), 5.07-5.14(m, 1H), 4.78(s, 3H), 2.76(s, 3H), 1.47(d, J
27

ACCEPTED MANUSCRIPT
= 6.4 Hz, 6H). ESI-MS, m/z: 299.7[M+H]+.
4.1.6. General procedure E for the synthesis of compounds (8, 9, 10
，
11)
To a solution of compound 7 (0.78 mmol) in CH3CN (15 mL), a solution of isothiocyanate (0.78
mmol) in CH3CN (1 mL) was added dropwise over 5 min. The reaction mixture was stirred 18 h at rt.
After that, the reaction was diluted with additional CH3CN (10 mL), followed by addition of DIC
(0.147 g, 1.17 mmol). Then, the reaction was heated at 120 ℃for 6 h, cooled to rt, and concentrated in
vacuo. The crude mixture was then partitioned between EtOAc and water. The combined organic layer
was washed with brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by
silica gelcolumn chromatography using petroleum ether-EtOAc to yield the title compound as a white
solid.
4.1.6.1. 5-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)-N-phenyl-1H-benzo[d]imidazol-2-amine
(8a)
Compound 7a (0.2 g, 0.78 mmol) was reacted with phenyl isothiocyanate (0.105 g, 0.78 mmol) to
1
give 8a as a white solid (0.086 g ,yield: 31.2%). mp: 128-131 ℃. H NMR (400 MHz, DMSO-d6) δ
11.06 (s, 1H), 9.53 (s, 1H), 8.55 (s, 1H), 7.70-7.77 (m, 3H), 7.24-7.40 (m, 4H), 6.93-6.96 (m, 2H), 4.03
(s, 3H).13C NMR (101 MHz, DMSO-d6) δ 164.5, 155.5, 155.3, 140.9, 131.4, 129.2, 121.3, 117.6, 102.1,
34.4. FT-IR (ύmax, cm-1): 3649 (NH), 3629 (NH), 3064 (CH, aromatic). ESI-MS m/z: 358.6 [M+H]+.
HRMS (ESI, m/z) calcd. for C19H15N7O [M+H]+ 358.1411; found, 358.1412.
4.1.6.2. 5-((1-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy)-N-(o-tolyl)-1H-benzo[d]imidazol-2-amine
(8b)
Compound 7a (0.2 g, 0.78 mmol) was reacted with 2-methylphenyl isothiocyanate (0.12 g, 0.78
1
mmol) to give 8b as a white solid (0.10 mg, yield: 35.3%). mp: 124-136 ℃. H NMR (400 MHz,
DMSO-d6) δ 11.00 (s, 1H), 8.47-8.54 (m, 2H), 8.11-8.23 (m, 1H), 7.69-7.72 (m, 1H), 7.36 (d, J = 7.6
Hz, 1H), 7.21-7.22 (m, 3H), 6.90-6.98 (m, 2H), 4.03 (s, 3H), 2.31 (s, 3H). 13C NMR (101 MHz,
DMSO-d6) δ 164.5, 155.5, 155.2, 138.6, 131.4, 130.8, 126.9, 123.0, 120.6, 102.1, 34.4, 18.2. FT-IR
(ύmax, cm-1): 3650 (NH), 3563 (NH), 3050 (CH, aromatic). ESI-MS m/z: 372.5 [M+H]+. HRMS (ESI,
m/z) calcd. for C20H17N7O [M+H]+ 372.1368; found,372.1570 .
4.1.6.3.
28