Design, synthesis, and biological evaluation of pyrazole derivatives containing acetamide bond as potential BRAFV600E inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.06.028

With the increasingly acquired resistance, relapse and side effects of known marketed BRAF^V600E inhibitors, it's significant to design the more effective and novel drugs. In this study, a series of novel pyrazole derivatives containing acetamid

Full text of DOI:10.1016/j.bmcl.2018.06.028

Accepted Manuscript
Design, synthesis, and biological evaluation of pyrazole derivatives containing
acetamide bond as potential BRAFV600E inhibitors
Chen-Ru Wang, Ze-Feng Wang, Lu Shi, Zhong-Chang Wang, Hai-Liang Zhu
PII:
S0960-894X(18)30517-1
https://doi.org/10.1016/j.bmcl.2018.06.028
BMCL 25909
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 April 2018
4 June 2018
14 June 2018
Please cite this article as: Wang, C-R., Wang, Z-F., Shi, L., Wang, Z-C., Zhu, H-L., Design, synthesis, and biological
evaluation of pyrazole derivatives containing acetamide bond as potential BRAFV600E inhibitors, Bioorganic &
Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.06.028
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphic Abstract:
Design, synthesis, and biological evaluation of pyrazole
derivatives containing acetamide bond as potential
BRAFV600E inhibitors
Chen-Ru Wang†a, Ze-Feng Wang†a, Lu Shia, Zhong-Chang Wang*a, Hai-Liang Zhu*a
aState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210023, People’s Republic of China.
*Corresponding author e-mail: zhuhl@nju.edu.cn
Figure 1. Binding mode of compound 5r with the BRAFV600E kinase (PDB code:
4XV9). (A) 2D diagram of the interaction between compound 5r and BRAFV600E
,
(B) 3D models of compound 5r binding with the active site. Green lines represent
hydrogen bonds. All of the 24 new synthesized compounds were tested using many
biological trials sequentially, such as the trail of selective inhibition of BRAFV600E
,
anti-proliferation in vitro, cell apoptosis, cell cycle arrest, and mitochondrial
membrane potential experiment. Among them, the most potent compound 5r (IC50
= 0.10 ± 0.01 μM for BRAFV600E, IC50 = 0.96 ± 0.10 μM against A375) showed the
most potent inhibitory effect, compared with the positive-controlled agents
vemurafenib (IC50 = 0.04 ± 0.004 μM for BRAFV600E, IC50 = 1.05 ± 0.10 μM against
A375). Further investigation confirmed that 5r could induce A375 cell apoptosis,
induce A375 cell death through changing mitochondrial membrane potential, and
result in cell arrest at the G1 phase of the cell cycle. Our study may contribute to the
BRAFV600E inhibitors that are promising novel cancer prevention agents.

Design, synthesis, and biological evaluation of pyrazole
derivatives containing acetamide bond as potential
BRAFV600E inhibitors
Chen-Ru Wang†a, Ze-Feng Wang†a, Lu Shia, Zhong-Chang Wang*a, Hai-Liang Zhu*a
aState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210023, People’s Republic of China.
*Corresponding author. Tel. & fax: +86-25-89682572; e-mail: zhuhl@nju.edu.cn
†These two authors contributed equally to this paper.

Abstract:
With the increasingly acquired resistance, relapse and side effects of known
marketed BRAFV600E inhibitors, it’s significant to design the more effective and novel
drugs. In this study, a series of novel pyrazole derivatives containing acetamide bond
had been designed and synthesized on the basis of analysis of the endogenous ligands
extracted from the known B-Raf co-crystals in the PDB database. Then, the
compounds were evaluated for biological activities as potential BRAFV600E inhibitors.
The bioassay results in vitro against three human tumor cell lines revealed that some
of the compounds showed very impressed antiproliferative property. Among them, the
compound 5r with IC50 values of 0.10 ± 0.01 μM against BRAFV600E and 0.96 ± 0.10
μM against A375 cell line, showed the most potent inhibitory effect, compared with
the positive-controlled agents vemurafenib (IC50 = 0.04 ± 0.004 μM for BRAFV600E
,
IC50 = 1.05 ± 0.10 μM against A375). Further investigation confirmed that the
compound 5r could induce A375 cell apoptosis, induce A375 cell death through
changing mitochondrial membrane potential, and result in A375 cell arrest at the G1
phase of the cell cycle. Docking simulations results indicated that the compound 5r
could bind tightly at the BRAFV600E active site. Meanwhile, 3D-QSAR model
suggested that these compounds may be potential anticancer inhibitors. Overall, the
article provided some new molecular scaffolds for the further BRAFV600E inhibitors.
Keywords: BRAFV600E; Inhibitors; Docking simulation; 3D-QSAR; Bioassays.

Mitogen-activated protein kinase (MAPK) signaling pathways are composed of a
family of essential kinase modules, a cascade of three intracellular protein kinases
(MKKK, MKK and MAPK) 1, 2. MAPK signaling pathways are activated by a cascade
of protein kinases phosphorylated successively and play a crucial role in the
regulation of cellular activities including cell proliferation, differentiation, survival,
death and migration 2, 3. The aberrant regulation of MAPK cascades are closely
implicated with diverse severe diseases including Alzheimer's disease (AD),
Parkinson's disease (PD), and Amyotrophic lateral sclerosis (ALS) 4, 5, 6. In particular,
the dysfunction of the Ras-Raf-MEK-ERK signaling pathway, which is one widely
activated MAPK pathway, is an important reason of human oncogenesis. Raf kinases
(A-Raf, B-Raf and C-Raf) is the major effector of Ras kinases and could lead to the
activation of downstream MEK1/2 and ERK1/2, so Raf kinases are vital central
7
players of the ERK signaling pathway . Researchers identified a high frequency of
RAF kinases mutations in cancer, thereby leading to the Raf family protein kinases as
8
a significant subject of drug discovery . Compared to A-Raf and C-Raf, B-Raf has
9
higher basal activity and is a major activator of MEK1/2 . B-Raf oncogene is
observed in 8% of all cancers, including 7% of human malignancies, 60% of
melanomas, 10% of colorectal carcinomas, 30 - 70% of papillary thyroid carcinoma 8,
10, 11. B-Raf mutations in human cancers are mainly the substitution of valine by
glutamate at residues 600 in the kinase domain (called BRAFV600E 7, 12. In this regard,
)
researchers from the academia and industry work hard on the development of
BRAFV600E target anticancer drugs and some inhibitors have been at different stages

of preclinical and clinical study 9. For example, the US Food and Drug Administration
(FDA) have approved the treatment of advanced melanoma with B-Raf inhibitor
vemurafenib for its high selection and excellent clinical activity 13. However,
increasingly acquired resistance, relapse and side effects, in particular accelerated
growth of cutaneous squamous cell carcinomas (SCC) and keratoacanthomas,
frequently occur in the therapies 14. Thus, it is significant to design the novel
BRAFV600E inhibitors to break these limitations.
In this study, we analyzed the endogenous ligands from the known B-Raf
co-crystals in the PDB database. Firstly, the endogenous ligands were extracted from
the co-crystals and then all of them were overlaid into the BRAFV600E kinase (PDB
code: 4XV9). These endogenous ligands were deconstructed into fragments to establish
a small fragments library. Afterwards, the better fragments were selected through
spatial distribution and the docking score. These selected fragments were analyzed to
reconstruct some new scaffolds. In the analysis process, some regulations were found
(as shown in Figure 1): 1) many endogenous ligands were composed of the pyrazole
ring; 2) there was a distinct hydrophobic region in the protein structure, and in the
active pocket of the protein, this region was generally occupied by benzene ring and
nitrogen-containing heterocycle of the endogenous ligands; 3) there was a certain
chemical distance between the pyrazole ring and the hydrophobic region, so a linker
was needed to combine these two chemical structures; 4) the link section generally was
rich in N, O atoms by analysis. Based on this knowledge, when the scaffolds were
designed, the pyrazole ring was kept and the acetamide bond was selected as the linker.

In the hydrophobic region, we tried putting some hydrophobic heterocycle structure
into this region to generate the scaffolds. By docking simulation, the better scaffolds
were selected as the lead compounds (as shown in Figure 2). The lead compounds were
substituted to obtain a library of small molecules. Using virtual screening, the small
molecules were ranked according to the docking score. The best hits were selected by
preliminarily screening in vitro.
Figure 1. The endogenous ligands were extracted from the known B-Raf co-crystals in the PDB
database and the fragmentation of these ligands provides interesting information: A is pyrazole
ring section; B is link section; C is hydrophobic region.
Figure 2. Designed the scaffolds

The synthesis of compounds 5a-5x followed the general pathway depicted in
Scheme 1. All of these compounds were reported for the first time (Table 1).
Meanwhile, satisfactory analytical and spectroscopic data were given. Experimental
methods, 1H NMR, ESI-MS and element analysis spectra were in full accordance with
the assigned structures, which are presented in the Supporting Information.
Scheme 1. The synthetic route for compounds 5a – 5x. (a) H2SO4, HNO3, 60 oC, 2 h; (b) 1.0 equiv
Iodomethane, K2CO3, DMF, 25 oC, 16 h; or 1.0 equiv, Iodobenzenes, 0.1 equiv,
o
8-Hydroxyquinoline, 0.1 equiv, CuI, 2.0 equiv, K2CO3, DMSO, 135 C, 20 h; (c) 80% Hydrazine
o
hydrate, 10% Palladium charcoal, Ethanol, 80 C, 10 min; (d) HOBt, EDC, dichloromethane, 25
oC, 16 h; (e) NaH, DMF, 25 oC, 12 h.
Table 1. Structures of compounds 5a – 5x
R1
R2
R1
R2
Compounds
5a
Compounds
-CH3
5m

5b
-CH2CH3
5n
5c
5d
5e
5o
5p
5q
-CH3
-CH2CH3
5f
5r
5s
5g
-CH3
5h
5i
-CH3
5t
5u
5v
-CH2CH3
-CH2CH3
5j
5k
5l
5w
5x
All compounds 5a-5x were evaluated for their inhibitory effects on BRAFV600E
kinase and three cancer cell lines, HT29 (BRAFV600E), A375 (BRAFV600E), WM1361
(BRAFWT). As showed in Table 2, the compound 5r and the vemurafenib showed the

best inhibition on BRAFV600E kinase activity with lower IC50 value (0.10 ± 0.01 μM
and 0.04 ± 0.004 μM, respectively). Subsequently, the MTT assays against three
cancer cell lines (HT29, A375, WM1361) were performed to test the anti-proliferative
activity of the obtained compounds. According to the IC50 values in Table 2, the
compound 5r showed better suppression compared with the vemurafenib when
exerting on the WM1361 cells for 48 h (with IC50 values of 10.15 ± 0.78 μM against
IC50 values of 16.84 ± 1.16 μM, respectively). To HT29 and A375 cancer cells, the
compound 5r and the vemurafenib showed equally remarkable anti-proliferative
activity (with IC50 values of 2.17 ± 0.18 μM and 0.96 ± 0.10 μM against IC50 values
of 1.52 ± 0.13 μM and 1.05 ± 0.10 μM, respectively). The MTT assays suggested that
the compound 5r was a potential BRAFV600E inhibitor and could efficiently inhibit the
proliferation of BRAFV600E-harbored cell lines.
When the R2 substituents were consistent, comparing the inhibitory activities of
the compounds 5a – 5g (IC50 = 4.43 ± 0.26 μM, 3.66 ± 0.34 μM, 0.50 ± 0.05 μM, 0.73
± 0.06 μM, 0.45 ± 0.03 μM, 0.41 ± 0.04 μM and 0.36 ± 0.02 μM, respectively) against
the BRAFV600E enzyme, their activity was significantly different. The activity of the
compounds substituted with methyl or ethyl was obviously weaker than others,
indicating that the large chemical substituents may be beneficial to the promotion of
activity and the aromatic substituents were better than the alkyl substituents. Based on
the experimental results, the priority of R1 substituents was obtained: methoxy phenyl
group > tolyl group > phenyl group > benzyl group > alkyl group. Meanwhile, when
the R1 substituents were consistent, in comparsion with 5f (IC50 = 0.41 ± 0.04 μM),

5m (IC50 = 1.28 ± 0.12 μM), 5r (IC50 = 0.10 ± 0.01 μM), 5w (IC50 = 0.48 ± 0.02 μM),
the priority of R2 substituents was obtained: 3-(3-chlorophenyl)-1H-pyrazol-1-yl > 3,
4-dimethoxyphenyl > 3-(pyridin-2-yl)-1H-pyrazol-1-yl > indole ring. Therefore, the
heterocyclic rings comprising benzene ring with electron-withdrawing group may
show better inhibitory activity.
Table 2. Compounds 5a – 5x potently inhibited proliferation of three cancer cells and
BRAFV600E kinase activity.
IC50 ± SD (μM)
Compounds
HT29
A375
WM-1361
40.30 ± 3.74
31.41 ± 2.65
18.83 ± 1.37
25.62 ± 2.12
16.56 ± 1.35
17.64 ± 1.26
23.98 ± 2.13
36.54 ± 3.52
47.80 ± 3.43
26.78 ± 1.27
25.83 ± 1.18
27.36 ± 1.41
24.77 ± 1.24
BRAFV600E
4.43 ± 0.26
3.66 ± 0.34
0.50 ± 0.05
0.73 ± 0.06
0.45 ± 0.03
0.41 ± 0.04
0.36 ± 0.02
4.66 ± 0.33
1.82 ± 0.16
1.96 ± 0.17
1.23 ± 0.11
2.34 ± 0.19
1.28 ± 0.12
32.80 ± 2.75
25.62 ± 1.28
8.43 ± 0.39
11.54 ± 0.84
8.73 ± 0.21
9.33 ± 0.35
7.98 ± 0.46
35.10 ± 2.14
49.87 ± 3.32
12.35 ± 0.91
26.55 ± 1.37
24.14 ± 1.68
12.74 ± 0.59
18.63 ± 1.32
17.92 ±1.16
10.36 ± 1.02
16.23 ±1.37
2.74 ± 0.15
4.57 ± 0.29
3.49 ± 0.11
28.89 ± 1.29
27.16 ± 1.46
17.56 ± 1.24
24.23 ± 1.36
24.53 ± 2.21
19.78 ± 1.64
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m

10.13 ± 0.76
22.56 ± 1.36
12.92 ± 0.38
5.70 ± 0.36
2.17 ± 0.18
25.36 ± 1.15
26.79 ± 1.09
12.65 ± 0.65
10.82 ± 0.83
11.12 ± 0.76
6.16 ± 0.34
1.52 ± 0.13
5.67 ± 0.23
16.78 ± 1.48
19.37 ± 1.45
3.26 ± 0.31
0.96 ± 0.10
23.68 ± 1.16
23.34 ± 1.23
19.83 ± 0.71
10.60 ± 0.64
11.23 ± 0.85
3.08 ± 0.15
1.05 ± 0.10
16.47 ± 1.19
27.89 ± 2.19
26.54 ± 2.31
14.87 ± 1.05
10.15 ± 0.78
31.93 ± 2.63
29.84 ± 2.39
27.97 ± 2.23
20.74 ± 1.92
24.37 ± 1.95
15.65 ± 0.73
16.84 ± 1.16
0.46 ± 0.03
1.67 ± 0.13
1.19 ± 0.12
0.34 ± 0.02
0.10 ± 0.01
2.54 ± 0.21
3.86 ± 0.33
0.55 ± 0.06
0.53 ± 0.05
0.48 ± 0.02
0.26 ± 0.03
0.04 ± 0.004
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
5x
vemurafenib
Moreover, cells apoptosis by Annexin V-FITC/PI double staining assay was
carried out and showed that compound 5r could induce the apoptosis of A375 cells
when A375 cells were treated with various concentrations (0, 1, 3, and 10 μM) of
compound 5r for 24 h, the vemurafenib as a positive contrast. The apoptotic markers
changes were analyzed with a flow cytometer. As shown in Figure 3, the results
indicated that the percentage of apoptotic cells treated with compound 5r increased in
a dose-dependent manner (with percentage of 3.80%, 14.1%, 19.1%, and 37.5%).
Compared with the percentage of apoptotic cells treated with vemurafenib (with
percentage of 3.80%, 8.64%, 18.8%, and 33.3%), compound 5r showed better

inhibition activity. Meanwhile, A375 cells were treated with 3 μM of the compoumd
5r and the vemurafenib for 0, 12, 24, and 36 h, respectively. The results showed that
A375 cells treated with compound 5r were induced to apoptosis in a time-dependent
manner (with percentage of 4.61%, 19.7%, 29.2%, and 59.7%). Compared with the
percentage of apoptotic cells treated with vemurafenib (with percentage of 4.61%,
8.06%, 27.7%, and 44.5%), compound 5r showed better inhibition activity. Thus, it
could be concluded that compound 5r could effectively induce apoptosis of A375
cells in a dose- and time-dependent manner and was better than the vemurafenib.
To ascertain whether A375 cells could be blocked in mitosis by compound 5r,
the bioassay of cell-cycle arrest was implemented by flow cytometry assays, and its
cell cycle distribution was assessed. A375 cells were treated with compound 5r at
various doses (0, 1, 3, 10 μM) for 24 h and with the compound 5r (3 μM) for various
times (0, 12, 24, 36 h), the vemurafenib as a positive contrast. As illustrated in Figure
4, the compound 5r same as the vemurafenib induced a gradual decrease of cells in
the G1 phase in a dose- and time-dependent manner. The results showed that
compound 5r could induce cell arrest in the G1 period of the cell cycle and blocked
cell mitosis.
Studies have shown that mitochondria are closely related to cells apoptosis, and
the destruction of mitochondrial transmembrane potential is considered to be one of
the causes of cells apoptosis cascade reaction. It occurs in the early stage of apoptosis.
Once the mitochondrial transmembrane potential collapse, then cells apoptosis cannot
be reversed. We measured the mitochondrial transmembrane potential (ΔΨm) using

JC-1 staining to test whether compound 5r could destroy the mitochondrial membrane
potential to induce apoptosis of A375. A375 cells were treated with 0, 2, 4, 8 μM of
compound 5r for 12 h. The results in Figure 5 indicated that compound 5r might
induce cell death through changing mitochondrial membrane potential.
Figure 3. (A) (a) A375 cells treated with 0, 1, 3 and 10 μM compound 5r for 24 h were collected
and analyzed; (b) A375 cells treated with 0, 1, 3 and 10 μM vemurafenib for 24 h were collected
and analyzed. The percentage of early apoptotic cells was shown in the lower right quadrant
(Annexin V-FITC positive/PI negative cells), and late apoptotic cells are located in the upper right
quadrant (Annexin V-FITC positive/PI positive cells); (B) (a) A375 cells treated with 3 μM
compound 5r for 0, 12, 24, 36 h were collected and analyzed; (b) A375 cells treated with 3 μM

vemurafenib for 0, 12, 24, 36 h were collected and analyzed. The percentage of early apoptotic
cells was shown in the lower right quadrant (Annexin V-FITC positive/PI negative cells), and late
apoptotic cells are located in the upper right quadrant (Annexin V-FITC positive/PI positive cells).
Figure 4. Effect of compound 5r and vemurafenib on cell-cycle progression of A375 cells was
determined by flow cytometry analysis. (A) (a) A375 cells treated with compound 5r (0, 1, 3 and
10 μM) for 24 h; (b) A375 cells treated with vemurafenib (0, 1, 3 and 10 μM) for 24 h;(B) (a)
A375 cells treated with compound 5r (3 μM) for various times (0, 12, 24, 36 h); (b) A375 cells
treated with vemurafenib (3 μM) for various times (0, 12, 24, 36 h). (G1 phase, green; S phase,
yellow and G2/M phase, blue).

Figure 5. Effects of 5r on mitochondrial transmembrane potential (ΔΨm) was analyzed using
JC-1 staining. The A375 cells were treated with the designed concentration of compound 5r (0, 2,
4, 8 μM) at 37 °C for 12 h. A375 cells were incubated with JC-1 staining (1 μM) for 30 min and
then harvested for analysis by flow cytometry. (A) reflected the reduction of ΔΨm. (B) presented
the offset of the peaks.
The high-throughput virtual screening was carried out to rank the compounds
which were obtained through scaffold modifications. The top score compounds were
selected. Meanwhile, docking simulations were also performed to explore the
probable binding modes of these compounds. The docking results, summarized in
Figure 6, showed that the change trend of the actual inhibition effect of the
compounds on BRAFV600E protein was basically consistent with the variation trend of
the CDOCKER INTERNATIONAL ENERGY. The activities of compounds with high
CDOCKER INTERNATIONAL ENERGY were better than that of compounds with
low CDOCKER INTERNATIONAL ENERGY. We may draw the conclusion that the
bioactive effect of the compound was related to the binding of the compound to

BRAFV600E protein. Moreover, compound 5r had the highest CDOCKER
INTERNATIONAL ENERGY (-54.963 kcal/mol). Compound 5r docked favorably
into the active site of BRAFV600E through two hydrogen bonds with THR 529 and LYS
483 (Figure 7A). There were one important section participating to form hydrogen
bonds: nitrogen atom and oxygen atom in acetamide bond were responded to Threonine
and Lysine, respectively. The receptor surface model shown in Figure 7B revealed that
compound 5r had a good shape complementarity with the ATP-binding pocket of
BRAF kinase and the above mentioned hydrogen bonds may contribute to afford an
explanation for its nice activity.
Figure 6. Interaction energy of compounds 5a – 5x.

Figure 7. A) The hydrogen bonds between the compound 5r and key amino acid residues. B)
The binding mode of compound 5r.
For further exploration, all the molecules with definite IC50 values against
BRAFV600E were selected as the model dataset by using the Create 3D-QSAR
protocol of Discover Studio 3.5. Firstly, all of them were converted to the active
conformation and corresponding pIC50 (μM) values obtained from the IC50 (μM)
values of BRAFV600E inhibition. By default, the alignment conformation of each
molecule possessed the lowest CDOCKER_INTERACTION_ENENGY among all
of the docked poses. This 3D-QSAR model gained the critical regions (steric or
electrostatic) which affect the binding affinity. Exerting CHARMM force filed and
PLS regression, the model was set up with the correlation coefficient R2 is 0.94856.

These details indicated that this model possessed pretty good predicting capability.
The relationship between experimental and predicted pIC50 values had been shown in
Figure 8. The molecules aligned with the iso-surfaces of the 3D-QSAR model
coefficients on electrostatic potential grids and Van der Waals grids were listed in
Figure 9. Electrostatic map indicates that high electron density (negative charge) in
red contours around regions can increase activity, while the opposite of blue contours.
Steric map indicates that increasing steric bulk in green contours can increase activity,
while the opposite of yellow contours. For compounds based on the 3D-QSAR
model, big steric bulk substituents were fit in the green areas and high negative
charged group in the red areas can achieve potent bioactivity. This model was
accordant with the actual situation for compounds. Accordingly, this promising
model would provide a guideline to design and optimize more effective BRAFV600E
inhibitors and pave the way for us in the further study.
Figure 8. Plot for experimental vs. predicted BRAFV600E inhibitory activities of training set and
test set. The model was set up with correlation coefficient R2 of 0.94856, which indicated that this
model possessed pretty good predicting capability.

Figure 9. A) Iso-surface of the 3D-QSAR model coefficients on electrostatic potential grids. Blue
represents positive coefficients; red represents negative coefficients. B) Iso-surface of the
3D-QSAR model coefficients on Van der Waals grids. Green represents positive coefficients;
yellow represents negative coefficients
According to previous studies, aberrant regulation of BRAFV600E are
significantly related to various types of cancers. However, due to many
marketed-BRAFV600E inhibitors increasingly acquired resistance, relapse and side
effects, it’s significant to design the more effective and novel drugs. In the study, a
library of pyrazole derivatives containing acetamide bond was built on the basis of
analysis of the endogenous ligands extracted from the known B-Raf co-crystals in the
PDB database. Then, they were evaluated for biological activities as potential
BRAFV600E inhibitors. The results revealed that the compound 5r may be promising
BRAFV600E inhibitor in the future. The compound 5r was the most impressive for a
higher binding affinity to BRAFV600E. To HT29 and A375 cancer cells, the compound
5r showed equally remarkable anti-proliferative activity with vemurafenib. Further
investigation confirmed that compound 5r could induce A375 cell apoptosis, induce
cell death through changing mitochondrial membrane potential, and cause cell arrest

at the G1 phase of the cell cycle in a dose- and time-dependent manner. In addition, a
reliable 3D-QSAR model was obtained to analyze the structure-activity relationship.
In summary, a series of pyrazole derivatives containing acetamide bond we designed
may be very promising BRAFV600E inhibitors.
Acknowledgements
We thank Mr. Yun-Bo Zhou in Nanjing Foreign Languages School for the
synthesis of compounds 5a and 5b of this paper.

References and notes
1.
Wang, P. F.; Qiu, H. Y.; Wang, Z. F.; Zhang, Y. J.; Wang, Z. C.; Li, D. D.; Zhu, H. L.,
Identification of novel B-RafV600E inhibitors employing FBDD strategy. Biochem Pharmacol
2017, 132, 63-76.
2.
3.
4.
5.
6.
Brunet, A.; Pouyssã©Gur, J., Mammalian MAP kinase modules: how to transduce specific
signals. Essays Biochem 1997, 32 (32), 1-16.
Kim, E. K.; Choi, E. J., Pathological roles of MAPK signaling pathways in human diseases.
BBA-Mol Basis Dis . 2010, 1802 (4), 396-405.
Roberts, P. J.; Der, C. J., Targeting the Raf-MEK-ERK mitogen-activated protein kinase
cascade for the treatment of cancer. Oncogene 2007, 26 (22), 3291-3310.
Sekine, Y.; Takeda, K.; Ichijo, H., The ASK1-MAP kinase signaling in ER stress and
neurodegenerative diseases. Curr Mol Med 2006, 6 (1), 87-97.
Wang, Z. F.; Wang, P. F.; Ma, J. T.; Chai, Y. Z.; Hu, H. M.; Gao, W. L.; Wang, Z. C.; Wang, B.
Z.; Zhu, H. L., Design of Potent B-RafV600E Inhibitors by MCSS Strategy. Chem Biol Drug
Des 2017, 91 (1), 567-574.
7.
Yang, H.; Higgins, B.; Kolinsky, K.; Packman, K.; Go, Z.; Iyer, R.; Kolis, S.; Zhao, S.; Lee, R.;
Grippo, J. F., RG7204 (PLX4032), a selective BRAFV600E inhibitor, displays potent antitumor
activity in preclinical melanoma models. Cancer Res 2010, 70 (13), 5518-5527.
Holderfield, M.; Deuker, M. M.; Mccormick, F.; Mcmahon, M., Targeting RAF kinases for
cancer therapy: BRAF-mutated melanoma and beyond. Nat Rev Cancer 2014, 14 (7), 455-467.
Qin, J.; Xie, P.; Ventocilla, C.; Zhou, G.; Vultur, A.; Chen, Q.; Liu, Q.; Herlyn, M.; Winkler, J.;
Marmorstein, R., Identification of a Novel Family of BRAFV600E Inhibitors. J Med Chem
2012, 55 (11), 5220-5230.
8.
9.
10.
11.
12.
Nazarian, R.; Shi, H.; Wang, Q.; Kong, X.; Koya, R. C.; Lee, H.; Chen, Z.; Lee, M. K.; Attar,
N.; Sazegar, H., Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS
upregulation. Nature 2010, 468 (7326), 973-977.
Prahallad, A.; Sun, C.; Huang, S.; Di, N. F.; Salazar, R.; Zecchin, D.; Beijersbergen, R. L.;
Bardelli, A.; Bernards, R., Unresponsiveness of colon cancer to BRAF(V600E) inhibition
through feedback activation of EGFR. Nature 2012, 483 (7387), 100-103.
Schindler, G.; Capper, D.; Meyer, J.; Janzarik, W.; Omran, H.; Herold-Mende, C.; Schmieder,
K.; Wesseling, P.; Mawrin, C.; Hasselblatt, M., Analysis of BRAF V600E mutation in 1,320
nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma,
ganglioglioma and extra-cerebellar pilocytic astrocytoma. Acta Neuropathol 2011, 121 (3),
397-405.
13.
14.
Finn, L.; Markovic, S. N.; Joseph, R. W., Therapy for metastatic melanoma: the past, present,
and future. BMC Med, 10, 1(2012-03-02) 2012, 10 (1), 1-10.
Chen, L. W.; Wang, Z. F.; Zhu, B.; Man, R. J.; Liu, Y. D.; Zhang, Y. H.; Wang, B. Z.; Wang, Z.
C.; Zhu, H. L., Design, synthesis and biological evaluation of novel benzo-α-pyrone containing
piperazine .derivatives as potential BRAFV600E inhibitors. Bioorg Med Chem Lett 2016, 26
(20), 4983-4991.

Highlights
● Analyzing and screening the fragments to reconstruct the new scaffolds.
● Compound 5r could bind into the active pocket of the BRAFV600E kinase tightly.
● Compound 5r showed the better antiproliferative and BRAFV600E inhibition
activity.
● Compound 5r could induce the cell apoptosis and cell cycle arrest of A375 cells.
15.