Design and biological evaluation of novel triaryl pyrazoline derivatives with dioxane moiety for selective BRAFV600E inhibition

Article abstract of DOI:10.1016/j.ejmech.2018.06.043

A series of novel selective BRAF^V600E inhibitory agents (Compound 1–16) 5-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-N,3-diaryl-4,5-dihydro-1H-pyrazole-1-carbothioamides have been designed and synthesized. Their anti-proliferation and BRAF inhibito

Full text of DOI:10.1016/j.ejmech.2018.06.043

European Journal of Medicinal Chemistry 155 (2018) 725e735
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design and biological evaluation of novel triaryl pyrazoline derivatives
with dioxane moiety for selective BRAFV600E inhibition
,
,
,
, b, **
Hong-Lin Li a 1, Mi-Mi Su a 1, Yun-Jie Xu a, Chen Xu a ***, Yu-Shun Yang a
,
Hai-Liang Zhu a
, b, *
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, 210023, China
b Institute of Chemistry and BioMedical Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210023, China
a r t i c l e i n f o
a b s t r a c t
A series of novel selective BRAFV600E inhibitory agents (Compound 1e16) 5-(2,3-dihydrobenzo[b][1,4]
dioxane-6-yl)-N,3-diaryl-4,5-dihydro-1H-pyrazole-1-carbothioamides have been designed and synthe-
sized. Their anti-proliferation and BRAF inhibitory activities were evaluated. Though 15, 4 and 12 all
displayed comparable activity with the positive control Vemurafenib, only 12 indicated fine selectivity on
Article history:
Received 28 May 2018
Received in revised form
14 June 2018
Accepted 16 June 2018
Available online 19 June 2018
BRAFV600E (IC50 ¼ 0.06
m
M for BRAFV600E; GI50 ¼ 0.52
m
M for A375) over BRAFWT at both kinase and cell
levels. This result satisfied the designing concept of improving activity and introducing selectivity. Flow
cytometry analysis and western blot convinced the apoptosis induction and kinase inhibitory activity.
Docking simulation inferred the differences in binding patterns of BRAFV600E and BRAFWT, pointing out
that the future orientation might be seeking for outer space binding of BRAFV600E and avoiding in-
teractions with HIS573 of BRAFWT. These results brought potent BRAF inhibitors one step further to
selective agents, enhancing the potential for safe medication.
Keywords:
Triaryl pyrazoline
Dioxane
Antitumor
Apoptosis induction
BRAFV600E
© 2018 Elsevier Masson SAS. All rights reserved.
Selectivity
1. Introduction
was the most frequently mutated RAF kinases isoform, and
noticeably, near 90% of its activating mutations in cancers are
Risking the health of people worldwide, cancer pushed thou-
sands of researchers to develop novel agents and therapeutic
methods continuously despite the achievements made in medicinal
chemistry [1].
Classical MAPK pathway participates in vital cell procedures
such as proliferation and survival [2,3], and has been convinced
hyper-activated in up to 30% of human cancers [4e8]. All through
its RAS-RAF-MEK-ERK cascade, RAF involved activating mutations
have been most reported. In these reports, RAF showed innegligible
high relativity (50e70%) to melanoma [9], followed by thyroid
cancer [10e12] and ovarian cancer [13e15]. Inhibiting RAF was
believed to be an effective solution for treating cancer [16,17]. BRAF
substitution of VAL for GLU (V600E, formally defined as V599E)
[18,19]. Such mutations resulted in a 500-fold MEK phosphoryla-
tion basal rate increase over wild-type BRAF (BRAFWT) [20] and
consequently stimulation of tumor growth and vascular endothelial
growth factor secretion [21,22]. Since BRAF was essential for
normal cell procedure [23,24] whereas BRAFV600E was proved
momentous for tumorigenesis, inhibiting undesirable BRAF muta-
tions without interfering BRAFWT has become potential hotspot for
potent inhibition and safe medication [25e27].
At present, Vemurafenib was still the top FDA approval BRAF
inhibitor with explicit design and mechanism [28]. It was the first
agent designed using fragment-based drug discovery (FBDD). Un-
like Dabrafenib or Encorafenib which were usually used in com-
bination with MEK inhibitors, Vemurafenib was specific for
BRAFV600E with known binding pattern (Fig. 1). Seeking alternative
backbones to avoid the 7-azaindole moiety with possible side effect
led to modification of triarylimidazole derivative SB-590885
[29,30]. Considering the binding pattern of Vemurafenib into
BRAFV600E (PDB code: 3OG7) and consulting that of SB-590885
(PDB code: 2FB8), we should retain interactions with key residues
including PHE583, LYS483 and TRP531. In our previous work, we
* Corresponding author. State Key Laboratory of Pharmaceutical Biotechnology,
Nanjing University, Nanjing, 210023, China.
** Corresponding author. State Key Laboratory of Pharmaceutical Biotechnology,
Nanjing University, Nanjing, 210023, China.
*** Corresponding author.
E-mail addresses: xuchn@nju.edu.cn (C. Xu), ys_yang@nju.edu.cn (Y.-S. Yang),
zhuhl@nju.edu.cn (H.-L. Zhu).
1
Both authors contributed equally to the work.
https://doi.org/10.1016/j.ejmech.2018.06.043
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

726
H.-L. Li et al. / European Journal of Medicinal Chemistry 155 (2018) 725e735
Fig. 1. Binding patterns of Vemurafenib and SB-590885 inspired our design concept of rotating dioxane-containing group to Ring B and choosing suitable linker.
built and discussed the ABC-ring system of 1,3,5-triaryl pyrazoline
series BRAF inhibitors as shown in Fig. 1 [31e33]. Ring A and C were
almost in one plane while Ring B indicated an angle with this plane.
The selected 1,4-benzodioxane group was introduced because of its
merits on improving druggability (such as LogP and PSA) and
binding mode (enhancing interactions with LYS483 [32], TRP531
and PHE583 [33]). Previous also pointed out audacious modifica-
tion orientation of stretching the backbone to outer space or totally
reversing it [33], therefore we conducted cautious rotation of ABC
rings with fine regulation in this work. In consideration of consis-
tency and previous brought merits, we retained 1,4-benzodioxane
group and fixed it at Ring B. The ABC-ring backbone distin-
guished this series from other dioxane-containing agents and
avoided off-target effect [34,35]. As for the alternative linker, we
recognized that when we fixed Ring B as 1,4-benzodioxane, it
started to affect the steric conformation of A-C plane. Ring A was
basically fixed within the plane due to the conjugated double bond
in pryrozoline. However, Ring C could still be allowed to rotate
finitely. An ideal situation for carrying out rational design deman-
ded that Ring C should be better restricted within the same plane of
Ring A. Therefore a linker must be selected here to make the situ-
ation as close as possible to the ideal one. The linker should be short
but guiding suitable stretching orientation of Ring C. Series iv has
been chosen in consideration of Ring C orientation, as well as
ADMET properties and introduction of possible hydrogen bond.
synthesized and screened for their biological activity. All of them
were synthesized and tested for the first time. The general syn-
thesis method and the structures of compounds 1e16 were orga-
nized in Scheme 1. They were all prepared in three steps. Firstly,
different substituted aromatic ketone on treatment with 2,3-
dihydrobenzo[b][1,4]dioxane-6-carbaldehyde in presence of 50%
NaOH solution were stirred at room temperature till reactions
completed, yielding different analogues of chalcones (B). Secondly,
hydrazine hydrate was added to participate the cyclization of the
obtained powder [34]. Thirdly, phenyl isothiocyanate was added
and refluxed, leading to the corresponding target compounds 1e16.
Subsequent purification with recrystallisation was conducted and
the refined compounds were finally obtained. All of the synthetic
compounds gave satisfactory analytical and spectroscopic data,
which were in full accordance with their depicted structures.
2.2. Biological activity
Before evaluated for anti-proliferation effect and BRAF inhibi-
tory activity, ADMET properties as a preliminary evaluation for the
basic druggability were shown in Fig. 2. Although the linker was
chosen strictly, compound 6, 13, 14 and 15 might still need possible
pharmacokinetics group to improve their logP and PSA (polar sur-
face area) values. All the other compounds were in nice situation
and ready for subsequent bioassay.
With a general method, the anti-proliferation and BRAF inhib-
itory effect was evaluated. To compare the differences in inhibiting
BRAFV600E and BRAFWT, the IC50 values (the half maximal inhibitory
concentration of BRAF mediated MEK phosphorylation) were
shown together with GI50 values (the half maximal inhibitory
concentration of cell growth) of cell lines including human mela-
noma cells A375 (BRAFV600E mutated), WM2664 (BRAFV600E
2. Results and discussion
2.1. Chemistry
5-(2,3-dihydrobenzo[b]
dihydro-1H-pyrazole-1-carbothioamide derivatives 1e16 were
[1,4]dioxane-6-yl)-N,3-diaryl-4,5-

H.-L. Li et al. / European Journal of Medicinal Chemistry 155 (2018) 725e735
727
Scheme 1. General synthesis of compounds (1e16). Reagents and Conditions: i) EtOH, 50% NaOH, TLC; ii) N2H4$H2O, EtOH, NaOH, reflux, 4 h; iii) Phenyl isothiocyanate, EtOH, reflux,
4e6 h.
mutated) and WM1361 (BRAFWT), human colon cancer cells HT29
(BRAFV600E mutated) and HCT116 (BRAFWT). As presented in Table 1,
a majority of this series indicated potent BRAFV600E inhibitory ac-
tivity, with a welcome change in weakening BRAFWT inhibitory
effect. This result inferred the rotation of backbone and redesign of
linker might lead to desirable orientation.
The anti-proliferation effect showed positive correlation with
the BRAF inhibitory activity. Representatives including 15
(IC50 ¼ 0.01
(IC50 ¼ 0.05
(IC50 ¼ 0.06
m
m
m
M
for BRAFV600E
;
GI50 ¼ 0.35
mM for A375), 4
M for A375) and 12
M for BRAFV600E; GI50 ¼ 0.47
m
M for BRAFV600E; GI50 ¼ 0.52
m
M for A375) all dis-
played comparable activity with the positive controls Vemurafenib
(IC50 ¼ 0.03
m
M for BRAFV600E; GI50 ¼ 0.95
mM for A375). However,
only 12 indicated fine selectivity for BRAFV600E over BRAFWT and
this kind of selectivity retained in corresponding cell lines.
After bioassay results being compared, preliminary SAR studies on
BRAFV600E were provided. Initially, when Ring B was pushing the A-C
plane, substitutes on the A-C plane mightfacelimitof space extension
and electronic changing. For para-position on Ring A, a preliminary
tendency might be induced that substitutes with smaller steric or
electronic influence seemed better, with the corresponding order as
-Br (4, IC50 ¼ 0.05
IC50 ¼ 4.28 M) > -F(2, IC50 ¼ 11.1
NO2 (8, IC50 ¼ 57.2 M) > -SMe (7, IC50 ¼ 84.3
M) group inferred
superb potency, which might be brought by stretching or reversing
the backbone (according to the molecular overlap in Fig. 3) as
m
M) > -H (1, IC50 ¼ 0.82
M) > -CF3 (6, IC50 ¼ 29.5
M) > -Cl (3,
m
M) > -Me (5,
m
m
mM) > -
Fig. 2. ADMET properties predicted for the sixteen compounds 1e16. Compounds
located inside the innermost oval are better for this parameter. The four compounds
outside the innermost oval were 6, 13, 14 and 15.
m
m
IC50 ¼ 135
m
M). However, -OBn (15, IC50 ¼ 0.01
m
Table 1
BRAF inhibitory and anti-proliferation activity of the synthesized compounds (1e16).
Code
IC50
(m
M)
IC50
(
m
M)
GI50
A375
(mM)
GI50
WM2664
(
m
M)
GI50
WM1361
(
m
M)
GI50
HT29
(
mM)
GI50 (mM)
HCT116
BRAFV600E
BRAFWT
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
0.82 0.07
11.1 1.02
135 11.4
0.05 0.002
4.28 0.36
29.5 1.85
84.3 6.37
57.2 4.71
>200
21.3 1.96
23.1 2.03
>200
18.7 1.29
>200
35.6 3.14
156 12.7
>200
124 10.3
>200
1.85 0.15
15.7 1.28
195 18.5
0.47 0.04
6.32 0.58
43.5 3.97
159 14.2
78.1 7.32
>200
3.18 0.29
22.5 1.98
>200
0.87 0.07
10.3 1.01
52.4 4.87
197 16.8
105 9.86
>200
58.5 5.43
65.3 6.22
>200
37.5 3.31
>200
123 12.1
187 16.8
>200
>200
>200
153 14.8
>200
122 11.5
148 14.2
8.23 0.80
7.96 0.73
20.8 1.95
6.63 0.76
37.6 5.31
187 15.6
2.58 0.22
19.5 1.87
97.5 8.67
>200
153 13.8
>200
30.5 2.95
>200
2.92 0.24
95.3 9.17
133 10.5
0.86 0.08
188 17.2
1.88 0.15
57.6 5.33
61.2 5.87
>200
32.1 3.01
>200
112 10.3
193 17.2
>200
195 19.3
>200
12.1 0.95
>200
20.3 1.96
>200
25.8 2.14
>200
95.9 8.36
>200
138 12.8
>200
0.06 0.003
30.6 2.98
38.9 3.12
0.01 0.001
78.3 6.82
0.03 0.003
0.52 0.05
41.9 4.02
61.3 5.85
0.35 0.03
121 10.3
0.95 0.08
1.38 0.11
58.7 5.62
70.5 6.62
0.42 0.03
154 12.7
0.21 0.02
39.3 3.41
48.9 3.98
5.23 0.51
5.21 0.49
0.18 0.01
125 12.2
144 10.8
8.53 0.74
8.19 0.81
25.2 2.18
Vemurafenib

728
H.-L. Li et al. / European Journal of Medicinal Chemistry 155 (2018) 725e735
24 h, the percentage of apoptotic cells inferred a significant increase
in a dose-dependent manner. Meanwhile, G2-block arrest of A375
was observed in a time-dependent manner with the concentration
of 12 settled as 2 mM and in a dose-dependent manner with the
incubation time settled as 24 h. Thus, 12 could enhance the accu-
mulation of cells in G2/M phase.
BRAFV600E inhibitors should block the phosphorylated level
downstream MEK and ERK. Thus, the blocking effect of compound
12 on phosphorylation of MEK and ERK from A375 (BRAFV600E) and
WM1361 (BRAFWT) cell lines was evaluated. For BRAFV600E group,
cells were treated with different concentrations (0
mM, 0.05 mM,
0.1 M and 0.5 M) of compound 12. As shown in Fig. 5, 12 could
m
m
suppress the phosphorylation of MEK and ERK from A375 cell line
in a dose-dependent manner (Fig. 5A and B), being close to
Vemurafenib in literature. However, for BRAFWT group (Fig. 5D and
E), no such effect was observed with higher concentrations (0
mM,
0.5 M, 1 M and 5 M) of 12. In both groups, no suppression of
m
m
m
total MEK1/2 or ERK1/2 levels were observed. Compound 15, as a
contrast, also suggested blocking effect on pMEK from A375 cells
(Fig. 5C).
Fig. 3. Molecular overlap of compounds 1e16 with Vemurafenib (in pure color). Ring A
and C were almost in one plane while Ring B indicated an angle with this plane. The
backbone of 15 was stretching at Ring A and reversing at Ring B. (For interpretation of
the references to color in this figure legend, the reader is referred to the Web version of
this article.)
A375 cells were subcutaneously inoculated into right flank of
nude mice to evaluate the antitumor effect of compound 12. The
mice were randomized into 4 groups including vehicle control,
Vemurafenib (5 mg/kg) and 12 (5 and 10 mg/kg). The compounds
were intraperitoneally administered every second day for 14 days.
Body weight were monitored and recorded every two days and did
not show obvious decrease (Fig. 6A). This result supported the
safety. Tumors were taken out and weighed on day 15. This
parameter inferred evidence for the antitumor efficacy. The results
in Fig. 6B suggested the average weight of tumors in control group
(0.79 0.061 g) was 2-fold more than that of 12-treated (10 mg/kg)
group (0.38 0.029 g). Compound 12 exhibited comparable anti-
tumor efficacy with the positive control Vemurafenib (5 mg/kg,
0.41 0.031 g).
mentioned in our previous work [30]. For meta-position and ortho-
position, a clear inference was that electron-donating groups might
increase the potency and selectivity for BRAFV600E whereas electron-
withdrawing groups might ruin them. The corresponding example
for meta-position is -OMe (12, IC50 ¼ 0.06
m
M) > -Cl (11,
M) and that for ortho-position is -OMe (10,
M) > -Br (9, IC50 > 200 M). Secondly, multi substitutes
IC50 > 200
IC50 ¼ 12.1
m
m
m
on both ortho- and meta-positions suggested moderated BRAFV600E
inhibitory activity but no specific selectivity from BRAFWT. Thirdly, a
heterocyclic ring (here it was thiophene) also seemed worse than
benzene ring due to weakening BRAFV600E inhibitory effect but
increasing BRAFWT inhibiting. Thus, keeping Ring A substitutes in
low-impact status or throwing it out the pocket seemed favorable for
potent inhibition of BRAFV600E, while 12 revealed a corner of the
iceberg for introducing considerable selectivity.
2.3. Molecular docking
Molecular docking was performed to visualize the possible
binding models using CDOCKER protocol in Discovery Studio 3.5
(Discovery Studio 3.5, Accelrys, Inc. San Diego, CA). The crystal
structures of BRAFV600E (PDB Code: 1UWJ) and BRAFWT (PDB Code:
1UWH) were chosen [20]. The receptor and ligands were prepared
and the binding site was defined. The CDocker Interaction Energy
(interaction energy between the ligand and the receptor, shown in
Table 3) agreed with the BRAF inhibitory trend for all the synthe-
sized compounds. The 2D maps of compound 4, 12, 15 into
BRAFV600E and 15 into BRAFWT were depicted in Fig. 7.
Compound 4, 12 and 15 were chosen as representatives for
further kinase inhibitory selectivity evaluation against CRAF (also
called RAF1), MEK, JNK1 and p38a. As shown in Table 2, only 12
provided superb selectivity for BRAFV600E against other kinases
(especially CRAF, and BRAFWT in Table 1). 4 and 15 were both active
against BRAFV600E but regrettably potent against BRAFWT and
relative kinases such as CRAF and JNK1. Meanwhile, these repre-
sentatives all suggested low cytotoxicity against HEK293T (human
embryonic kidney cell line) and LO2 (human embryonic liver cell
line) cells. Through the screening of broaden selectivity and cyto-
toxicity, 12 was chosen for further study.
The binding pattern of compound 4, 12, 15 into BRAFV600E was
similar to previous researches with PHE582 (PHE583 as previously
mentioned) between Ring A and Ring C. Especially for 4 and 12,
Flow cytometry analysis was conducted to convince that the
anti-proliferative activity was correlated to apoptosis. As shown in
Fig. 4, compound 12 could induce apoptosis of A375 cells. When the
they retained typical
TRP530 and
pep interactions between Ring A/C and
p
ecation interactions between Ring B and LYS482.
However, the new hydrogen bond (4: O/H-N, 2.13 Å, 118.806ꢀ; 12:
concentration of 12 was settled as 2
mM except control, the per-
O/H-N, 2.10 Å, 118.780ꢀ) between -NH- on linker and PHE594
centage of apoptotic cells increased as time varies (24, 48 and 72 h).
When treated with increasing concentrations of 12 (0e20 mM) for
might enhance the inhibitory activity against BRAFV600E
.
Table 2
Selectivity (against CRAF, MEK1/2, JNK1 and p38a) and Cytotoxicity (against HEK293T and LO2 cell lines) of representative compounds (4, 12 and 15).
Code
IC50
CRAF
(
mM)
IC50
MEK1/2
(
m
M)
IC50
JNK1
(
mM)
IC50
p38
(
a
mM)
GI50
HEK293T
(
m
M)
GI50
LO2
(mM)
4
12
15
17.6 1.58
>200
3.15 0.30
188 16.5
195 18.9
176 15.7
93.6 9.11
>200
88.9 8.42
>200
>200
>200
152 14.8
>200
175 15.1
188 16.2
>200
>200

H.-L. Li et al. / European Journal of Medicinal Chemistry 155 (2018) 725e735
729
Fig. 4. Flow cytometry analysis of compound 12 on A375 cell line. (A) Time-dependent cell apoptosis with 2
mM compound 12; (B) Dose-dependent cell apoptosis for 24 h; (C) Time-
dependent G2-block arrest of A375 with 2 M compound 12; (D) Dose-dependent G2-block arrest of A375 for 24 h.
m
Meanwhile, compound 15 stretched its Ring A substitute out the
pocket to interact with residues including LYS600 and SER601,
which might bring favorable effect without stick to the typical
pattern. As for binding pattern into BRAFWT, HIS573 seemed to be a
(BRAFV600E) to enhance inhibition and evading HIS573 (BRAFWT) to
introduce selectivity. This work might infer some clues for future
development of selective BRAF inhibitors and correlative safe
medication.
key residue for this series. Compound 15 suggested
pep and
p
ecation interactions here while 4 indicated weaker binding sit-
4. Experimental section
uation. Compound 12 even failed to be docked into BRAFWT. These
results agreed with their BRAFWT inhibiting performance. Besides,
the 3D binding situation and receptor surface model with 12 in
BRAFV600E in Fig. 8 all inferred that 12 was deeply occupation into
the active pocket. Herein, two new hints were found. One was
involving PHE594 of BRAFV600E to enhance inhibition, while the
other was evading HIS573 of BRAFWT to introduce selectivity.
4.1. Chemistry
4.1.1. General
All commercially available chemicals were used as received
without further purification. Melting points were determined on a
WRS-1C digital melting point apparatus and were uncorrected. 1H
NMR and 13C NMR spectra were recorded on a Bruker Avance II 600
spectrometer. Shifts are reported in parts per million based on re-
sidual solvent peaks (for 1H or 13C/CDCl3 or DMSO-d6). NMR data
were resolved with MestreNova software. Mass spectra were ob-
tained from an Agilent 6540 UHD Accurate Mass Q-TOF LC/MS.
3. Conclusions
In summary, a series of selective BRAFV600E inhibitory agents
(Compound 1e16) have been designed and synthesized. Inheriting
the ABC-ring system and 1,4-benzodioxane group, cautious rota-
tion and linker choice maintained the interactions with key resi-
dues and brought selectivity. 15, 4 and 12 all displayed comparable
activity with the positive control Vemurafenib. Remarkably, 12
indicated fine selectivity on BRAFV600E over BRAFWT at both kinase
and cell levels. Flow cytometry analysis verified the apoptosis in-
duction and western blot convinced the kinase inhibitory effect.
Docking simulation implied the binding pattern including PHE582,
LYS482 and TRP530. It provided further hints of involving PHE594
4.1.2. General method of synthesis of (E)-Chalcones (B)
Substituted aromatic ketone (1 mmol) was added to 2,3-
dihydrobenzo[b][1,4] dioxane-6-carbaldehyde (A) (1 mmol)
alcohol solution (5 mL). After dissolution, 50% NaOH (0.5 mL) was
added. After confirming the completion of the reaction by thin layer
chromatography, the sediment was filtered, washed with ethanol
and dried to obtain chalcone (B).

730
H.-L. Li et al. / European Journal of Medicinal Chemistry 155 (2018) 725e735
Fig. 5. Compound 12 blocked the ERK pathway in A375 cells but the blocking was not obvious in WM1361 cells. 12 inhibited the phosphorylation of ERK (A) and MEK (B) in
A375 cells expressing BRAFV600E; 15 inhibited the phosphorylation of MEK (C) in A375 cells expressing BRAFV600E; The inhibition of 12 with higher concentrations on the phos-
phorylation of ERK (D) and MEK (E) was not obvious in WM1361 cells expressing BRAFWT
.
Fig. 6. Compound 12 indicated low toxicity and potent antitumor effect in xenografts of A375 cell line. (A) Body weight monitored and recorded every two days; (B) Tumor weight
on day 15 of treatment.
Table 3
absolute ethanol, was added substituted phenyl isothiocyanates
(2 mmol) and refluxed for 4e6 h. The crude product was filtered,
The CDocker Interaction Energy of the synthesized compounds (1e16).
washed with petroleum ether, dried, and was recrystallized from
methanol to afford the title compounds 1e16.
Code
-CDocker Interaction
Energy (kcal/mol)
Code
-CDocker Interaction
Energy (kcal/mol)
1
2
3
4
5
6
7
8
48.2530
46.0991
40.9463
50.9873
47.3400
43.3818
41.7242
42.3797
9
33.5368
45.4921
39.2084
50.6685
44.0407
42.6499
52.9371
42.0848
10
11
12
13
14
15
16
4.1.4.1. 5-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-N,3-diphenyl-4,5-
dihydro-1H-pyrazole-1-carbothioamide (1). Orange solid; mp
135e137 ꢀC; yield: 77%; 1H NMR (400 MHz, CDCl3)
d 9.28 (s, 1H),
7.79 (d, J ¼ 7.5 Hz, 2H), 7.67 (d, J ¼ 8.0 Hz, 2H), 7.49e7.47 (m, 3H),
7.38 (t, J ¼ 7.6 Hz, 2H), 7.20 (t, J ¼ 7.4 Hz,1H), 6.86e6.78 (m, 3H), 6.12
(dd, J ¼ 11.4, 3.1 Hz, 1H), 4.25 (s, 4H), 3.85 (dd, J ¼ 17.7, 11.4 Hz, 1H),
3.24 (dd, J ¼ 17.6, 3.2 Hz, 1H); 13C NMR (151 MHz, DMSO)
d 173.81,
155.95,143.65,142.55,141.24,140.00,136.42,130.75,128.52,128.42,
128.00, 127.78, 125.27, 118.21, 117.60, 114.38, 64.51, 64.42, 63.12,
42.62; HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C24H22N3O2S
416.1354, Found 416.1352.
4.1.3. General method of synthesis of (E)-3-(2,3-dihydrobenzo[b]
[1,4]dioxane-6-yl)-1-phenylprop-2-en-1-one (C1-C16)
The solution of appropriate chalcones (B,1 mmol) and hydrazine
hydrate (2 mmol) in ethanol (5 mL) was refluxed for 4 h. The
mixture was evaporated to dryness and the residue was crystallized
from suitable solvents to receive the target compounds C1-C16.
4.1.4.2. 5-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-3-(4-
fluorophenyl)-N-phenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide
(2). Pale yellow solid; mp 186e187 ꢀC; yield: 66%; 1H NMR
4.1.4. General method of synthesis of 5-(2,3-dihydrobenzo[b][1,4]
dioxane-6-yl)-N,3-diphenyl-4,5-dihydro-1H-pyrazole-1-
carbothioamide (1e16)
(600 MHz, DMSO-d6)
d
10.18 (s, 1H), 8.07 (dd, J ¼ 8.4, 5.6 Hz, 2H),
7.56 (d, J ¼ 7.8 Hz, 2H), 7.36e7.33 (m, 4H), 7.17 (t, J ¼ 7.3 Hz,1H), 6.81
(d, J ¼ 8.1 Hz, 1H), 6.67e6.65 (m, 2H), 5.94 (dd, J ¼ 11.2, 2.9 Hz, 1H),
4.21 (s, 4H), 3.90 (dd, J ¼ 18.1, 11.4 Hz, 1H), 3.21 (dd, J ¼ 18.1, 3.1 Hz,
To the stirred solution of compounds (C1-C16, 1 mmol) in

H.-L. Li et al. / European Journal of Medicinal Chemistry 155 (2018) 725e735
731
Fig. 7. The 2D docking models of representative compounds. (A) Compound 4 into BRAFV600E; (B) Compound 12 into BRAFV600E; (C) Compound 15 into BRAFV600E; (D) Compound 15
into BRAFWT. The H-bonds are displayed as dotted lines. The
pecation, pep and pesigma interactions are shown as orange lines. Interactions with typical PHE582, LYS482 and
TRP530 were retained. New found interactions included PHE594 of BRAFV600E and HIS573 of BRAFWT. (For interpretation of the references to color in this figure legend, the reader is
referred to the Web version of this article.)
Fig. 8. (A) The 3D binding situation of compound 12 into BRAFV600E; (B) The receptor surface model with 12 in BRAFV600E
.
1H); 13C NMR (151 MHz, DMSO)
d
174.11, 155.04, 143.65, 142.87,
DMSO-d6)
d
10.21 (s, 1H), 8.02 (d, J ¼ 8.6 Hz, 2H), 7.57e7.55 (m, 4H),
139.97, 136.22, 130.38, 130.32, 128.49, 127.94, 127.86, 127.69, 125.36,
118.68, 117.63, 116.33, 116.19, 114.48, 64.55, 64.44, 63.34, 42.55;
HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C24H21FN3O2S 434.1260,
Found 434.1262.
7.35 (t, J ¼ 7.7 Hz, 2H), 7.17 (t, J ¼ 7.4 Hz, 1H), 6.81 (d, J ¼ 8.1 Hz, 1H),
6.67e6.64 (m, 2H), 5.95 (dd, J ¼ 11.3, 3.2 Hz, 1H), 4.21 (s, 4H), 3.90
(dd, J ¼ 18.1, 11.4 Hz, 1H), 3.20 (dd, J ¼ 18.1, 3.4 Hz, 1H); 13C NMR
(151 MHz, DMSO)
d 174.02, 157.24, 143.24, 142.63, 140.01, 139.14,
136.46, 130.08, 128.88, 128.51, 127.16, 125.48, 125.32, 118.68, 117.63,
115.55, 64.55, 64.44, 63.22, 42.33; HRMS (ESI-TOF) m/z: [MþH]þ
Calcd for C24H21ClN3O2S 450.0964, Found 450.0963.
4.1.4.3. 3-(4-chlorophenyl)-5-(2,3-dihydrobenzo[b][1,4]dioxane-6-
yl)-N-phenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide
(3).
Pale yellow solid; mp 189e190 ꢀC; yield: 75%; 1H NMR (600 MHz,

732
H.-L. Li et al. / European Journal of Medicinal Chemistry 155 (2018) 725e735
4.1.4.4. 3-(4-bromophenyl)-5-(2,3-dihydrobenzo[b][1,4]dioxane-6-
yl)-N-phenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (4).
Yellow solid; mp 171e173 ꢀC; yield: 72%; 1H NMR (600 MHz,
DMSO-d6)
Pale yellow solid; mp 147e149 ꢀC; yield: 71%; 1H NMR (600 MHz,
DMSO-d6)
d
10.00 (s, 1H), 7.90 (dd, J ¼ 7.8, 1.6 Hz, 1H), 7.76 (d,
J ¼ 8.0 Hz, 1H), 7.57 (d, J ¼ 7.7 Hz, 2H), 7.51 (t, J ¼ 7.1 Hz, 1H), 7.42 (t,
J ¼ 7.7 Hz, 1H), 7.32 (t, J ¼ 7.6 Hz, 2H), 7.14 (t, J ¼ 7.4 Hz, 1H),
6.84e6.82 (m, 1H), 6.73e6.71 (m, 2H), 5.96 (dd, J ¼ 11.3, 3.2 Hz, 1H),
4.22 (s, 4H), 4.09 (dd, J ¼ 18.2, 11.5 Hz, 1H), 3.25 (dd, J ¼ 18.2, 3.4 Hz,
d
10.21 (s, 1H), 7.94 (d, J ¼ 8.6 Hz, 2H), 7.70 (d, J ¼ 8.6 Hz,
2H), 7.55 (d, J ¼ 7.6 Hz, 2H), 7.35 (t, J ¼ 7.6 Hz, 2H), 7.17 (t, J ¼ 7.4 Hz,
1H), 6.81 (d, J ¼ 8.2 Hz, 1H), 6.66e6.64 (m, 2H), 5.94 (dd, J ¼ 11.2,
3.2 Hz, 1H), 4.20 (s, 4H), 3.90 (dd, J ¼ 18.1, 11.4 Hz, 1H), 3.19 (dd,
1H); 13C NMR (151 MHz, DMSO)
d 174.55, 153.99, 143.42, 142.86,
J ¼ 18.1, 3.4 Hz, 1H); 13C NMR (151 MHz, DMSO)
d
174.17, 154.94,
139.80, 136.24, 135.32, 128.63, 128.51, 126.02, 126.00, 125.88,
125.52, 125.48, 123.53, 118.68, 117.66, 114.52, 64.42, 64.40, 63.58,
42.60; HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C24H21BrN3O2S
494.0459, Found 494.0461.
143.65, 142.89, 139.94, 136.18, 132.15, 130.59, 129.81, 128.50, 125.92,
125.37, 124.71, 118.69, 117.63, 114.48, 64.55, 64.44, 63.42, 42.29;
HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C24H21BrN3O2S 494.0459,
Found 494.0461.
4.1.4.10. 5-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-3-(2-
methoxyphenyl)-N-phenyl-4,5-dihydro-1H-pyrazole-1-
carbothioamide (10). White solid; mp 164e166 ꢀC; yield: 74%; 1H
4.1.4.5. 5-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-N-phenyl-3-(p-
tolyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (5). Pale yellow
solid; mp 144e146 ꢀC; yield: 75%; 1H NMR (600 MHz, DMSO-d6)
NMR (600 MHz, DMSO-d6)
d
10.07 (s, 1H), 8.26 (dd, J ¼ 7.8, 1.7 Hz,
d
10.12 (s, 1H), 7.89 (d, J ¼ 8.2 Hz, 2H), 7.58 (d, J ¼ 7.7 Hz, 2H), 7.34 (t,
1H), 7.57 (d, J ¼ 7.8 Hz, 2H), 7.48 (t, J ¼ 7.9, 1H), 7.33 (t, J ¼ 7.6 Hz,
2H), 7.16e7.13 (m, 2H), 7.05 (t, J ¼ 7.5 Hz,1H), 6.81 (d, J ¼ 8.2 Hz,1H),
6.67e6.65 (m, 2H), 5.89 (dd, J ¼ 11.2, 3.1 Hz, 1H), 4.21 (s, 4H), 3.96
(dd, J ¼ 18.7, 11.3 Hz, 1H), 3.82 (s, 3H), 3.22 (dd, J ¼ 18.7, 3.2 Hz, 1H);
J ¼ 7.7 Hz, 2H), 7.30 (d, J ¼ 8.0 Hz, 2H), 7.16 (t, J ¼ 7.4 Hz, 1H), 6.81 (d,
J ¼ 8.2 Hz, 1H), 6.67e6.64 (m, 2H), 5.93 (dd, J ¼ 11.1, 3.1 Hz, 1H), 4.20
(s, 4H), 3.88 (dd, J ¼ 18.0, 11.3 Hz, 1H), 3.17 (dd, J ¼ 18.1, 3.3 Hz, 1H),
2.37 (s, 3H); 13C NMR (151 MHz, DMSO)
d 173.91, 156.05, 143.64,
13C NMR (151 MHz, DMSO)
d 173.90, 158.86, 155.34, 143.63, 142.82,
142.85, 141.24, 140.01, 136.29, 129.75, 128.53, 128.46, 127.89, 125.77,
125.27, 118.67, 117.61, 114.46, 64.55, 64.44, 63.12, 42.52, 21.58;
HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C25H24N3O2S 430.1511,
Found 430.1513.
140.02, 136.46, 132.84, 129.88,128.46,125.66,125.22,121.05,119.83,
118.60, 117.63, 114.38, 112.91, 64.55, 64.43, 63.09, 56.21, 46.09;
HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C25H24N3O3S 446.1460,
Found 446.1459.
4.1.4.6. 5-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-N-phenyl-3-(4-
(trifluoromethyl)phenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide
(6). White solid; mp 164e165 ꢀC; yield: 64%; 1H NMR (600 MHz,
4.1.4.11. 3-(3-chlorophenyl)-5-(2,3-dihydrobenzo[b][1,4]dioxane-6-
yl)-N-phenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide
Pale yellow solid; mp 142e146 ꢀC; yield: 63%; 1H NMR (600 MHz,
DMSO-d6)
(11).
DMSO-d6)
d
10.31 (s, 1H), 8.21 (d, J ¼ 8.2 Hz, 2H), 7.85 (d, J ¼ 8.3 Hz,
d
10.28 (s,1H), 8.35 (s,1H), 7.87 (d, J ¼ 6.3 Hz,1H), 7.68 (d,
2H), 7.55 (d, J ¼ 7.7 Hz, 2H), 7.36 (t, J ¼ 7.6 Hz, 2H), 7.18 (t, J ¼ 7.4 Hz,
1H), 6.81 (d, J ¼ 8.4 Hz, 1H), 6.68e6.67 (m, 2H), 5.98 (dd, J ¼ 11.7,
3.3 Hz, 1H), 4.21 (s, 4H), 3.95 (dd, J ¼ 18.2, 11.5 Hz, 1H), 3.25 (dd,
J ¼ 6.4 Hz, 1H), 7.54 (d, J ¼ 6.3 Hz, 2H), 7.44e7.18 (m, 4H), 6.82e6.65
(m, 3H), 5.95 (d, J ¼ 9.7 Hz, 1H), 4.20 (s, 4H), 3.88 (dd, J ¼ 17.2,
12.1 Hz, 1H), 3.22 (d, J ¼ 17.7, 1H); 13C NMR (151 MHz, DMSO)
J ¼ 18.2, 3.5 Hz, 1H); 13C NMR (151 MHz, DMSO)
d 174.56, 154.39,
d
174.22, 154.31, 143.46, 142.88, 140.34, 136.24, 135.52, 128.51,
143.67, 142.92, 139.91, 136.14, 135.32, 128.53, 128.51, 126.05, 126.00,
125.98, 125.54, 125.42, 123.62, 118.71, 117.65, 114.51, 64.55, 64.44,
63.59, 42.33, 40.52; HRMS (ESI-TOF) m/z: [MþH]þ Calcd for
128.06, 126.25, 126.00, 125.97, 125.54, 125.46, 123.22, 118.35, 117.55,
114.21, 64.64, 64.34, 63.58, 42.36; HRMS (ESI-TOF) m/z: [MþH]þ
Calcd for C24H21ClN3O2S 450.0964, Found 450.0966.
C
24H21F3N3O2S 484.1228, Found 484.1226.
4.1.4.12. 5-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-3-(3-
methoxyphenyl)-N-phenyl-4,5-dihydro-1H-pyrazole-1-
carbothioamide (12). Pale yellow solid; mp 136e138 ꢀC; yield: 70%;
4.1.4.7. 5-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-3-(4-(methylthio)
phenyl)-N-phenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (7).
Pale yellow solid; mp 165e167 ꢀC; yield: 75%; 1H NMR (600 MHz,
DMSO-d6)
1H NMR (600 MHz, DMSO-d6)
d 10.17 (s, 1H), 7.60 (s, 1H), 7.55 (d,
d
10.14 (s, 1H), 7.91 (d, J ¼ 8.2 Hz, 2H), 7.58 (d, J ¼ 7.8 Hz,
J ¼ 7.7 Hz, 2H), 7.51 (d, J ¼ 7.7 Hz, 1H), 7.39 (t, J ¼ 8.0 Hz, 1H), 7.35 (t,
J ¼ 7.7 Hz, 2H), 7.18 (t, J ¼ 7.4 Hz, 1H), 7.08 (dd, J ¼ 8.2, 2.4 Hz, 1H),
6.81 (d, J ¼ 8.2 Hz, 1H), 6.67e6.65 (m, 2H), 5.94 (dd, J ¼ 11.2, 3.1 Hz,
1H), 4.21 (s, 4H), 3.89 (dd, J ¼ 18.1,11.3 Hz,1H), 3.83 (s, 3H), 3.21 (dd,
2H), 7.36e7.33 (m, 4H), 7.16 (t, J ¼ 7.3 Hz, 1H), 6.81 (d, J ¼ 8.0, Hz,
1H), 6.67e6.66 (m, 2H), 5.94 (dd, J ¼ 10.9, 2.1 Hz, 1H), 4.20 (s, 4H),
3.87 (dd, J ¼ 18.0, 11.4 Hz, 1H), 3.17 (dd, J ¼ 17.9, 2.3 Hz, 1H), 2.53 (s,
3H); 13C NMR (151 MHz, DMSO)
d 173.87, 155.64, 143.65, 142.86,
J ¼ 18.1, 3.3 Hz, 1H); 13C NMR (151 MHz, DMSO)
d 174.22, 159.92,
142.56, 139.99, 136.27, 128.48, 128.27, 127.41, 125.79, 125.31, 118.68,
117.62, 114.48, 64.56, 64.44, 63.19, 42.43, 40.48; HRMS (ESI-TOF) m/
z: [MþH]þ Calcd for C25H24N3O2S2 462.1231, Found 462.1232.
155.89, 143.65, 142.86, 140.00, 136.26, 132.61, 130.28, 128.51, 126.16,
125.48, 120.53, 118.68, 117.63, 117.35, 114.46, 112.50, 64.56, 64.44,
63.27, 55.84, 42.55; HRMS (ESI-TOF) m/z: [MþH]þ Calcd for
C25H24N3O3S 446.1460, Found 446.1463.
4.1.4.8. 5-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-3-(4-nitrophenyl)-
N-phenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (8). Red solid;
mp 183e184 ꢀC; yield: 56%; 1H NMR (600 MHz, DMSO-d6)
d
10.38
4.1.4.13. 3-(3,4-dichlorophenyl)-5-(2,3-dihydrobenzo[b][1,4]dioxane-
6-yl)-N-phenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide (13).
Yellow solid; mp 169e171 ꢀC; yield: 78%; 1H NMR (600 MHz,
DMSO-d6)
(s, 1H), 8.32e8.24 (m, 4H), 7.54 (d, J ¼ 7.6 Hz, 2H), 7.36 (t, J ¼ 7.6 Hz,
2H), 7.19 (t, J ¼ 7.4 Hz, 1H), 6.82 (d, J ¼ 8.6 Hz, 1H), 6.68e6.66 (m,
2H), 5.99 (dd, J ¼ 11.5, 3.4 Hz, 1H), 4.21 (s, 4H), 3.96 (dd, J ¼ 18.2,
11.6 Hz, 1H), 3.26 (dd, J ¼ 18.1, 3.5 Hz, 1H); 13C NMR (151 MHz,
d
10.30 (s, 1H), 8.37 (d, J ¼ 2.0 Hz, 1H), 7.90 (dd, J ¼ 8.4,
2.0 Hz, 1H), 7.75 (d, J ¼ 8.4 Hz, 1H), 7.53 (d, J ¼ 7.6 Hz, 2H), 7.36 (t,
J ¼ 7.6 Hz, 2H), 7.19 (t, J ¼ 7.4 Hz, 1H), 6.81 (d, J ¼ 8.1 Hz, 1H),
6.66e6.65 (m, 2H), 5.95 (dd, J ¼ 11.3, 3.3 Hz, 1H), 4.21 (s, 4H), 3.88
(dd, J ¼ 18.2, 11.5 Hz, 1H), 3.24 (dd, J ¼ 18.2, 3.4 Hz, 1H); 13C NMR
DMSO)
d 173.99, 154.36, 143.92, 142.87, 139.93, 136.22, 135.44,
128.33, 128.31, 126.21, 126.00, 125.98, 125.26, 125.20, 123.82, 118.54,
117.26, 114.33, 64.55, 64.44, 63.29, 42.33; HRMS (ESI-TOF) m/z:
[MþH]þ Calcd for C24H21N4O4S 461.1205, Found 461.1206.
(151 MHz, DMSO)
d 174.54, 154.91, 143.42, 142.62, 139.65, 136.52,
135.59,128.45,128.05,126.06,126.02,125.88,125.67,125.18,123.48,
118.50, 117.69, 114.49, 64.55, 64.43, 63.50, 42.30; HRMS (ESI-TOF)
m/z: [MþH]þ Calcd for C24H20Cl2N3O2S 484.0575, Found 484.0574.
4.1.4.9. 3-(2-bromophenyl)-5-(2,3-dihydrobenzo[b][1,4]dioxane-6-
yl)-N-phenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide
(9).

H.-L. Li et al. / European Journal of Medicinal Chemistry 155 (2018) 725e735
733
4.1.4.14. 5-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-3-(3,4-
dimethylphenyl)-N-phenyl-4,5-dihydro-1H-pyrazole-1-
carbothioamide (14). Pale yellow solid; mp 162e164 ꢀC; yield: 72%;
performed at 37 ꢀC, 5% CO2 atmosphere for 24 h to allow reat-
taching. Subsequently, cells were treated with the target com-
pounds at 0.001
mM, 0.01 mM, 0.1 mM, 1 mM, 10 mM, 100 mM and
1H NMR (600 MHz, DMSO-d6)
d
10.09 (s, 1H), 7.79 (s, 1H), 7.69 (dd,
500 M in the presence of 10% FBS for 48 h. Afterwards, cell viability
m
J ¼ 7.7, 1.4 Hz, 1H), 7.57 (d, J ¼ 7.5 Hz, 2H), 7.34 (t, J ¼ 7.6 Hz, 2H), 7.25
(d, J ¼ 7.9 Hz, 1H), 7.16 (t, J ¼ 7.4 Hz, 1H), 6.81 (d, J ¼ 8.2 Hz, 1H),
6.66e6.64 (m, 2H), 5.93 (dd, J ¼ 11.1, 3.0 Hz, 1H), 4.20 (s, 4H), 3.87
(dd, J ¼ 17.9, 11.3 Hz, 1H), 3.17 (dd, J ¼ 18.0, 3.2 Hz, 1H), 2.28 (s, 6H);
was assessed by the conventional 3-(4, 5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide (MTT) reduction assay carried out
strictly according to the manufacturer's instructions (Sigma-
Aldrich). The absorbance (OD570) was read on LX300 Epson Diag-
nostic micro-plate reader. Three replicate wells were used for each
concentration and each assay was measured three times, after
which the average of GI50 was calculated. The GI50 was calculated
using SPSS 13.0 software.
13C NMR (151 MHz, DMSO)
d 174.56, 154.39, 143.67, 142.92, 139.91,
136.14,135.32, 128.53,128.51, 126.05,126.00,125.98,125.54,125.42,
123.62, 118.71, 117.65, 114.51, 64.55, 64.44, 63.59, 42.33, 40.52,
40.49; HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C26H26N3O2S
444.1667, Found 444.1665.
4.2.2. Kinase inhibitory assay
4.1.4.15. 3-(4-(benzyloxy)phenyl)-5-(2,3-dihydrobenzo[b][1,4]
dioxane-6-yl)-N-phenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide
(15). Pale yellow solid; mp 203e205 ꢀC; yield: 56%; 1H NMR
This V600E mutant BRAF kinase assay was performed in tripli-
cate for each tested compound in this study. Briefly, 7.5 ng Mouse
Full-Length GST-tagged BRAFV600E (Invitrogen, PV3849) was pre-
(600 MHz, DMSO-d6)
d
10.11 (s, 1H), 7.95 (d, J ¼ 8.7 Hz, 2H), 7.61 (d,
incubated at room temperature for 1 h with 1
assay dilution buffer. The kinase assay was initiated when 5
solution containing 200 ng recombinant human full length, N-ter-
minal His-tagged MEK1 (Invitrogen, PV3093), 200 M ATP, and
30 mM MgCl2 in assay dilution buffer was added. The kinase re-
action was allowed to continue at room temperature for 25 min and
m
L drug and 4
mL
J ¼ 8.0 Hz, 2H), 7.47 (d, J ¼ 7.3 Hz, 2H), 7.40 (t, J ¼ 7.4 Hz, 2H), 7.34 (t,
J ¼ 7.6 Hz, 3H), 7.16 (t, J ¼ 7.4 Hz, 1H), 7.12 (d, J ¼ 8.8 Hz, 2H),
6.82e6.81 (m, 1H), 6.68e6.67 (m, 2H), 5.94 (dd, J ¼ 11.0, 2.5 Hz, 1H),
5.19 (s, 2H), 4.19 (s, 4H), 3.85 (dd, J ¼ 17.9, 11.3 Hz, 1H), 3.16 (dd,
m
L of a
m
J ¼ 17.9, 2.9 Hz, 1H); 13C NMR (151 MHz, DMSO)
d 173.92, 159.32,
157.00, 151.72, 148.73, 148.52, 138.55, 133.12, 129.56, 129.01, 128.99,
128.42, 128.40, 127.96, 127.41, 127.39, 126.52, 126.48, 121.80, 118.93,
118.60, 117.81, 117.62, 117.52, 111.98, 109.54, 64.56, 64.43, 63.42,
42.33; HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C30H28N3O3S
522.1851, Found 522.1847.
was then quenched with 5
solution. Protein was further denatured by heating for 5 min at
70 ꢀC. 10
L of each reaction was loaded into a 15-well, 4e12%
mL 5 ꢁ protein denaturing buffer (LDS)
m
precast NuPage gel (Invitrogen) and run at 200 V, and upon
completion, the front, which contained excess hot ATP, was cut
from the gel and discarded. The gel was then dried and developed
onto a phosphor screen. A reaction that contained no active enzyme
was used as a negative control, and a reaction without inhibitor was
used as the positive control.
4.1.4.16. 5-(2,3-dihydrobenzo[b][1,4]dioxane-6-yl)-N-phenyl-3-(thi-
ophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide
Pale yellow solid; mp 183e184 ꢀC; yield: 76%; 1H NMR (600 MHz,
DMSO-d6)
(16).
d
9.92 (s, 1H), 7.82 (d, J ¼ 4.9 Hz, 1H), 7.58e7.55 (m, 3H),
Detection of the effect of compounds on cell based pERK1/2
activity in WM2664 cells was performed using ELISA kits (Invi-
trogen) and strictly according to the manufacturer instructions.
The wild type BRAF kinase (Invitrogen, PV3848) and CRAF ki-
nase (Invitrogen, PV3805) were evaluated use the same method as
V600E mutant BRAF kinase.
7.34e7.30 (m, 2H), 7.18 (dd, J ¼ 4.9, 3.8 Hz, 1H), 7.15 (t, J ¼ 7.4 Hz,
1H), 6.82 (d, J ¼ 8.1 Hz, 1H), 6.68e6.66 (m, 2H), 5.98 (dd, J ¼ 11.2,
3.0 Hz, 1H), 4.21 (s, 4H), 3.94 (dd, J ¼ 17.8, 11.2 Hz, 1H), 3.19 (dd,
J ¼ 17.8, 3.1 Hz, 1H); 13C NMR (151 MHz, DMSO)
d 174.12, 155.62,
148.74, 148.54, 135.50, 133.12, 129.01, 128.48, 128.42, 127.23, 126.99,
126.52, 125.24, 123.80, 125.62, 117.82, 115.93, 109.52, 65.23, 64.44,
62.20, 42.66; HRMS (ESI-TOF) m/z: [MþH]þ Calcd for C22H20N3O2S2
438.0946, Found 438.0942.
Assays on p38
a
and JNK1 using radio labeled [
g
-
32P] ATP (Hal-
and
ingBio, Shanghai) were performed in 96 well plates. The p38
a
JNK1 were expressed as N-terminal FLAG-tagged proteins using a
baculovirus expression system. The reaction conditions were
4.2. Biological assay
optimized for each kinase: p38
per well of MBP (Wako Pure Chemical Ind., Japan), 0.1
of [
32P] ATP, 60 min reaction at 30 ꢀC; JNK1 (10 ng per well of
enzyme, 1 mg per well of c-Jun, 0.1 mCi per well of [g-
32P] ATP,
a
(100 ng per well of enzyme, 1
mg
m
Ci per well
4.2.1. Anti-proliferation assay
g-
Human melanoma cells A375 (BRAFV600E mutated, ATCC CRL-
1619), WM2664 (BRAFV600E mutated, from Nanjing University, P.
R. China) and WM1361 (BRAFWT, from Nanjing University, P. R.
China), human colon cancer cells HT29 (BRAFV600E mutated, ATCC
HTB-38) and HCT116 (BRAFWT, ATCC CCL-247) together with hu-
man embryonic cells HEK293T (kidney, ATCC CRL-3216) and LO2
(liver, from Nanjing University, P. R. China) were used in this work.
A375 was maintained in Dulbecco's modified Eagle's medium
(DMEM) 12430 (Invitrogen) with Sodium Pyruvate (Invitrogen,
11360-070) added. The other cell lines grew in DMEM (Hyclone). All
media was supplemented with 10% foetal bovine serum (FBS, BI),
60 min reaction at 30 ꢀC. The reactions were performed in 25 mM
HEPES, pH 7.5, 10 mM magnesium acetate, 1 mM dithiothreitol and
0.5
m
M ATP containing enzyme, substrate and radio labeled ATP as
L. Prior to the kinase re-
described above in a total volume of 50
m
action, compound and enzyme were incubated for 5 min at reaction
temperature as described above. The kinase reactions were initi-
ated by adding ATP. After the reaction period as described above,
the reactions were terminated by the addition of 10% (final con-
centration) trichloroacetic acid. The [
teins were filtrated in Harvest Plate (STEMCELL Technologies Inc.
Shanghai) with a Cell Harvester (PerkinElmer) and then free [
32P]
ATP was washed out with 3% phosphoric acid. The plates were
dried, followed by the addition of 40 L of MicroScint0 (Perki-
g-
32P]-phosphorylated pro-
2 mmol/L of
L-glutamine, 100 units/mL of penicillin-streptomycin
g-
(Sigma-Aldrich), 100 mg/mL streptomycin (Hyclone) and incu-
bated at 37 ꢀC in a water saturated atmosphere containing 5% CO2.
The anti-proliferative activities of the prepared compounds
against the cultured cells were evaluated using a standard MTT-
based colorimetric assay with some modification. Cell lines grew
to log phase in DMEM supplemented with 10% FBS (BI). Cell sus-
m
nElmer). The radioactivity was counted by a Top Count scintillation
counter (PerkinElmer).
4.2.3. Cell apoptosis and cell cycle analysis
pensions were prepared and 100
mL/well dispensed into 96-well
Approximately 105 per well cells were cultured in a 24-well
plate and allowed to adhere. Subsequently, the medium was
plates to give 104 per well. The subsequent incubation was

734
H.-L. Li et al. / European Journal of Medicinal Chemistry 155 (2018) 725e735
replaced with fresh culture medium containing compound 12 at
final concentrations of 0, 2.5, 5, 10 and 20 M. Non-treated wells
tool.
m
received an equivalent volume of ethanol (<0.1%). After 24 h, cells
4.4. Experimental protocol of docking study
were trypsinized, washed in PBS and centrifuged at 2000 rpm for
5 min. The pellet was resuspended in 500
m
L staining solution
The three-dimensional structures of the aforementioned com-
pounds were constructed using Chem. 3D ultra 14.0 software
[Chemical Structure Drawing Standard; Cambridge Soft corpora-
tion, USA (2015)]. The crystal structures of BRAFV600E (PDB Code:
1UWJ) and BRAFWT (PDB Code: 1UWH) were retrieved from the
RCSB Protein Data Bank (http://www.rcsb.org). All bound waters
and ligands were eliminated from the protein and the polar
hydrogen was added to the proteins. Molecular docking of all
compounds including preliminary designed ones was then carried
out using the Discovery Studio (version 3.5) as implemented
through the graphical user interface CDOCKER protocol.
CDOCKER is an implementation of a CHARMm based molecular
docking tool using a half-flexible receptor, including the following
steps:
(containing 5 L AnnexinV-FITC and 5 L PI (5 mg/mL) in Binding
m
m
Buffer), mixed gently and incubated for 15 min at room tempera-
ture assay dilution buffer in dark. The samples were then analyzed
by a FACSCalibur flow cytometer (Becton Dickinson, US). Time-
dependent protocol was similar to the process above with con-
centration of 12 set as 2 mM and incubation time varying (0, 24, 48
and 72 h).
Cells were plated in 6-well plates (106 per well) and incubated at
37 ꢀC or 24 h. Exponentially growing cells were then incubated
with compound 12 at different concentrations (0, 2.5, 5, 10 and
20 m
M). After 24 h, cells were centrifuged at 1500 rpm at 4 ꢀC for
5 min, fixed in 70% ethanol at 4 ꢀC for at least 12 h and subsequently
resuspended in phosphate buffered saline (PBS) containing 0.1 mg/
mL RNase A and 5 mg/mL propidium iodide (PI). The cellular DNA
content was measured by flow cytometry for cell cycle distribution
analysis, plotting at least 10,000 events per sample. The percentage
of cells in the subG0/G1, G0/G1, S and G2/M phases of the cell cycle
were determined using Flowjo 7.6.1 software.
(1) A series of ligands conformations are generated using high
temperature molecular dynamics with different random
seeds.
(2) Random orientations of the conformations are generated by
translating the center of the ligand to a specified position
within the receptor active site, and making a series of
random rotations. A softened energy is calculated and the
orientation is kept when it is less than a specified limit. This
process repeats until either the desired number of low-
energy orientations is obtained, or the test times of bad
orientations reached the maximum number.
(3) Each orientation is subjected to simulated annealing mo-
lecular dynamics. The temperature is heated up to a high
temperature then cooled to the target temperature. A final
energy minimization of the ligand in the rigid receptor using
non-softened potential is performed.
(4) For each of the final pose, the CHARMm energy (interaction
energy plus ligand strain) and the interaction energy alone
are figured out. The poses are sorted according to CHARMm
energy and the top scoring (most negative, thus favorable to
binding) poses are retained. The whole kinase domain
defined as a receptor and the site sphere was selected based
on the original ligand binding location, then the original
ligand was removed and the ligands prepared by us were
placed during the molecular docking procedure. CHARMm
was selected as the force field. The molecular docking was
performed with a simulated annealing method. The heating
steps were 2000 with 700 of heating target temperature. The
cooling steps were 5000 with 300 cooling target tempera-
ture. Ten molecular docking poses saved for each ligand were
ranked according to their dock score function. The pose with
the highest -CDOCKER energy was chosen as the most suit-
able pose.
4.2.4. Western blot
A375 cells on 6-well plates were rinsed twice with cold PBS and
lysed in RIPA lysis buffer containing a protease inhibitor mixture at
1:100 dilution on ice for 30 min. The insoluble components of cell
lysates were removed by centrifugation (4 ꢀC, 12,000 g, 10 min).
Protein concentrations were measured using a Pierce BCA protein
assay kit. Proteins were separated by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) and transferred
onto polyvinylidene difluoride (PVDF) membranes. Membranes
were blocked using skim milk and then incubated with diluted
indicated primary antibodies (1:500 dilution) at 4 ꢀC with gentle
shaking overnight. After washing five times, membranes were
incubated with secondary antibody (1:5000 dilution) for 1 h at
room temperature.
4.2.5. Xenograft model
Cultured A375 cells were washed and resuspended in cold PBS.
Portions of the suspension (3 ꢁ 106 cells in 0.1 mL) were injected
into the right flank of nude mice. The treatment was initiated when
tumor volume reached approximately 100 mm3 and mice were
randomized into groups (5 mice in each group). Compound 12 and
positive control vemurafenib were suspended at the desired con-
centration as needed for each dose group in olive oil. Then they
were administered every second day for 14 days by intraperitoneal
injection. Body weight were monitored and recorded every two
days. Tumor tissues were excised and weighted on day 15. Animal
welfare and experimental procedures were followed in accordance
with the Guide for Care and Use of Laboratory Animals (National
Institutes of Health, the United States) and the related ethical reg-
ulations of Nanjing University.
Ethical statement
4.3. Experimental protocol of ADMET study
Animal welfare and experimental procedures were followed in
accordance with the Guide for Care and Use of Laboratory Animals
(National Institutes of Health, the United States) and the related
ethical regulations of Nanjing University.
The three-dimensional structures of the aforementioned com-
pounds were constructed using Chem. 3D ultra 14.0 software
[Chemical Structure Drawing Standard; Cambridge Soft corpora-
tion, USA (2015)]. Then they were minimized a CHARMm based
force field the same as in docking study. The ADMET study was
conducted using the Calculate Molecular Properties in Small Mol-
ecules module of the Discovery Studio (version 3.5). The ADMET
properties map and data were provided by the ADMET Descriptors
Acknowledgment
This work is supported by the Public Science and Technology
Research Funds Projects of Ocean (No. 201505023), the Funda-
mental Research Funds for the Central Universities (No.

H.-L. Li et al. / European Journal of Medicinal Chemistry 155 (2018) 725e735
735
[19] A.S. Dhillon, S. Hagan, O. Rath, W. Kolch, MAP kinase signalling pathways in
cancer, Oncogene 26 (2007) 3279e3290.
[20] P.T. Wan, M.J. Garnett, S.M. Roe, S. Lee, D. Niculescu-Duvaz, V.M. Good,
C.M. Jones, C.J. Marshall, C.J. Springer, D. Barford, R. Marais, C.G. Project,
Mechanism of activation of the RAF-ERK signaling pathway by oncogenic
mutations of B-RAF, Cell 116 (2004) 855e867.
020814380063) and Jiangsu Planned Projects for Postdoctoral
Research Funds (No. 1701091C).
Appendix A. Supplementary data
[21] A.P. Algazi, E. Cha, S.M. Ortiz-Urda, T. McCalmont, B.C. Bastian, J. Hwang,
M.H. Pampaloni, S. Behr, K. Chong, B. Cortez, A. Quiroz, F. Coakley, S. Liu,
A.I. Daud, The combination of axitinib followed by paclitaxel/carboplatin
yields extended survival in advanced BRAF wild-type melanoma: results of a
clinical/correlative prospective phase II clinical trial, Br. J. Canc. 112 (2015)
1326e1331.
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2018.06.043.
References
[22] K. Kurata, N. Onoda, S. Noda, S. Kashiwagi, Y. Asano, K. Hirakawa, M. Ohira,
Growth arrest by activated BRAF and MEK inhibition in human anaplastic
thyroid cancer cells, Int. J. Oncol. 49 (2016) 2303e2308.
[23] Z.Y. Abd Elmageed, R.F. Moore, K. Tsumagari, M.M. Lee, A.B. Sholl,
P. Friedlander, Z. Al-Qurayshi, M. Hassan, A.R. Wang, H.A. Boulares, E. Kandil,
Prognostic Role of BRAFV600E Cellular Localization in Melanoma, vol 226, 2017,
pp. 526e537.
[1] J. Ferlay, I. Seorjomataram, R. Dikshit, S. Eser, C. Mathers, M. Rebelo,
D.M. Parkin, D. Forman, F. Bray, Cancer incidence and mortality worldwide:
sources, methods and major patterns in GLOBOCAN 2012, Int. J. Canc. 136
(2015) E359eE386.
[2] C. Wellbrock, M. Karasarides, R. Marais, The RAF proteins take centre stage,
Nat. Rev. Mol. Cell Biol. 5 (2004) 875e885.
[3] P. Marchetti, A. Trinh, R. Khamari, J. Kluza, Melanoma metabolism contributes
to the cellular responses to MAPK/ERK pathway inhibitors, BBA-Gen. Subjects
1862 (2018) 999e1005.
[4] H. Davies, G.R. Bignell, C. Cox, P. Stephens, S. Edkins, et al., Mutations of the
BRAF gene in human cancer, Nature 417 (2002) 949e954.
[24] J. Chesney, Y. Imbert-Fernandez, S. Telang, M. Baum, S. Ranjan, M. Fraig,
N. Batty, Potential clinical and immunotherapeutic utility of talimogene
laherparepvec for patients with melanoma after disease progression on im-
mune checkpoint inhibitors and BRAF inhibitors, Melanoma Res. 28 (2018)
250e255.
[5] N. Li, D. Batt, M. Warmuth, B-Raf kinase inhibitors for cancer treatment, Curr.
Opin. Invest. Drugs 8 (2007) 452e456.
[6] G. Maurer, B. Tarkowski, M. Baccarini, Raf kinases in cancer-roles and thera-
peutic opportunities, Oncogene 30 (2011) 3477e3488.
[7] M. Burotto, V.L. Chiou, J.M. Lee, E.C. Kohn, The MAPK pathway across different
malignancies: a new perspective, Cancer 120 (2014) 3446e3456.
[8] B. Rodenak-Kladniew, A. Castro, P. Starkel, C. De Saeger, M.G. De Bravo,
R. Crespo, Linalool induces cell cycle arrest and apoptosis in HepG2 cells
through oxidative stress generation and modulation of Ras/MAPK and Akt/
mTOR pathways, Life Sci. 199 (2018) 48e59.
[9] S.J. Heidorn, Combined BRAF and MEK inhibition versus BRAF inhibition alone
in melanoma, N. Engl. J. Med. 371 (2014) 1877e1888.
[10] Y. Cohen, M. Xing, E. Mambo, Z. Guo, G. Wu, B. Trink, U. Beller, W.H. Westra,
P.W. Ladenson, D. Sidransky, BRAF mutation in papillary thyroid carcinoma,
J. Natl. Cancer Inst. 95 (2004) 625e627.
[11] M. Xing, BRAF mutation in papillary thyroid cancer: pathogenic role, molec-
ular bases, and clinical implications, Endocr. Rev. 28 (2016) 742e762.
[12] X. Xu, R.M. Quiros, P. Gattuso, K.B. Ain, R.A. Prinz, High prevalence of BRAF
gene mutation in papillary thyroid carcinomas and thyroid tumor cell lines,
Canc. Res. 63 (2003) 4561e4567.
[13] G. Singer, R. Oldt, Y. Cohen, B.G. Wang, D. Sidransky, R.J. Kurman, I.M. Shih,
Mutations in BRAF and KRAS characterize the development of low-grade
ovarian serous carcinoma, J. Natl. Cancer Inst. 95 (2003) 484e486.
[14] K.T. Kuo, T.L. Mao, S. Jones, E. Veras, A. Ayhan, T.L. Wang, R. Glas, D. Slamon,
V.E. Velculescu, R.J. Kuman, I.M. Shih, Frequent activating mutations of PIK3CA
in ovarian clear cell carcinoma, Am. J. Pathol. 174 (2009) 1597e1601.
[15] H.R. Harris, M.S. Rice, A.L. Shafrir, E.M. Poole, M. Gupta, J.L. Hecht, K.L. Terry,
S.S. Tworoger, Lifestyle and reproductive factors and ovarian cancer risk by
p53 and MAPK expression, Cancer Epidem. Biomar 27 (2018) 96e102.
[16] L. Si, X.S. Zhang, Z. Xu, Q.D. Jiang, L.L. Bu, X. Wang, L.L. Mao, W.J. Zhang,
N. Richie, J. Guo, Vemurafenib in Chinese patients with BRAFV600 mutation-
positive unresectable or metastatic melanoma: an open-label, multicenter
phase I study, BMC Canc. 18 (2018), 520.
[25] Y. Cui, T.M. Guadagno, B-Raf(V600E) signaling deregulates the mitotic spindle
checkpoint through stabilizing Mps1 levels in melanoma cells, Oncogene 27
(2008) 3122e3133.
[26] P. Koelblinger, O. Thuerigen, R. Dummer, Development of encorafenib for
BRAF-mutated advanced melanoma, Curr. Opin. Oncol. 30 (2018) 125e133.
[27] B. Agianian, E. Gavathiotis, Current insights of BRAF inhibitors in Cancer, J.
Med. Chem. https://doi.org/10.1021/acs.jmedchem.7b01306.
[28] S. Mathieu, S.N. Gradl, L. Ren, Z. Wen, I. Aliagas, et al., Potent and selective
aminopyrimidine-based B-Raf inhibitors with favorable physicochemical and
pharmacokinetic properties, J. Med. Chem. 55 (2012) 2869e2881.
[29] A.J. King, D.R. Patrick, R.S. Batorsky, M.L. Ho, H.T. Do, et al., Demonstration of a
genetic therapeutic index for tumors expressing oncogenic BRAF by the ki-
nase inhibitor SB-590885, Canc. Res. 66 (2006) 11100e11105.
[30] C. Blackburn, M.O. Duffey, A.E. Gould, B. Kulkarni, J.X. Liu, S. Menon,
M. Nagayoshi, T.J. Vos, J. Williams, Discovery and optimization of N-acyl and
N-aroylpyrazolines as B-Raf kinase inhibitors, Bioorg. Med. Chem. Lett 20
(2010) 4795e4799.
[31] C.Y. Li, Q.S. Li, L. Yan, X.G. Sun, R. Wei, H.B. Gong, H.L. Zhu, Synthesis, biological
evaluation and 3D-QSAR studies of novel 4,5-dihydro-1H-pyrazole niacin-
amide derivatives as BRAF inhibitors, Bioorg. Med. Chem. 20 (2012)
3746e3755.
[32] Y.S. Yang, Q.S. Li, S. Sun, Y.B. Zhang, X.L. Wang, F. Zhang, J.F. Tang, H.L. Zhu,
Design, modification and 3D QSAR studies of novel 2,3-dihydrobenzo[b][1,4]
dioxin-containing 4,5-dihydro-1H-pyrazole derivatives as inhibitors of B-Raf
kinase, Bioorg. Med. Chem. 20 (2012) 6048e6058.
[33] Y.S. Yang, B. Yang, Y. Zou, G. Li, H.L. Zhu, Design, Biological Evaluation and 3D
QSAR Studies of Novel Dioxin-containing Triaryl Pyrazoline Derivatives as
Potential B-Raf Inhibitors, vol 24, 2016, pp. 3052e3061.
[34] H. Khalilullah, S. Khan, M.J. Ahsan, B. Ahmed, Synthesis and antihepatotoxic
activity of 5-(2,3-dihydro-1,4-benzodioxane-6-yl)-3-substituted-phenyl-4,5
-dihydro-1H-pyrazole derivatives, Bioorg. Med. Chem. Lett 21 (2011)
7251e7254.
[35] H.H. Wang, K.M. Qiu, H.E. Cui, Y.S. Yang, Y. Luo, M. Xing, X.Y. Qiu, L.F. Bai,
H.L. Zhu, Synthesis, molecular docking and evaluation of thiazolyl-pyrazoline
derivatives containing benzodioxole as potential anticancer agents, Bioorg.
Med. Chem. 21 (2013) 448e455.
[17] J.J. Grob, Is there any interest in a new BRAF-MEK inhibitor combination in
melanoma? Lancet Oncol. 19 (2018) 580e581.
[18] K. Kalinsky, F.G. Haluska, Novel inhibitors in the treatment of metastatic
melanoma, Expert Rev. Anticancer Ther. 7 (2007) 715e724.