Identification and Biological Evaluation of Novel Type II B-RafV600E Inhibitors

Article abstract of DOI:10.1002/cmdc.201800574

The mitogen-activated protein kinase (MAPK) pathway plays a vital role in signal transduction networks. Severe diseases may be triggered if it is disturbed by mutated components, especially the kinase B-Raf^V600E. New inhibitors of the kinase ar

Full text of DOI:10.1002/cmdc.201800574

Accepted Article
Title: Design, Synthesis and Biological Evaluation of New Type II B-
RafV600E Inhibitors
Authors: Peng-Fei Wang, Ze-Feng Wang, Han-Yue Qiu, Yue Huang,
Hui-Min Hu, Zhong-Chang Wang, and Hai-Liang Zhu
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the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201800574
Link to VoR: http://dx.doi.org/10.1002/cmdc.201800574
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Identification and Biological Evaluation of New Type II B-RafV600E
Inhibitors
†
Peng-Fei Wang,†[a] Ze-Feng Wang, , Han-Yue Qiu,[a] Yue Huang,[b] Hui-Min Hu,[a] Zhong-Chang
[a]
Wang,*[a] and Hai-Liang Zhu*[a]
[a]
[b]
Dr. Peng-Fei Wang; Ze-Feng Wang; Dr. Han-Yue Qiu; Hui-Ming Hu; Dr. Zhong-Chang Wang; Prof. Hai-Liang Zhu,
State Key Laboratory of Pharmaceutical Biotechnology
Nanjing University
Nanjing 210023, People’s Republic of China
E-mail: zhuhl@nju.edu.cn
Dr. Yue Huang
College of Light Industry and Food Engineering
Nanjing Forestry University
Nanjing 210037, People’s Republic of China
†These two authors contributed equally to this paper.
Supporting information for this article is given via a link at the end of the document.
Abstract: The MAPK (Mitogen-activated protein kinase) pathway
plays a vital role in the signal transduction network. Severe diseases
may be triggered when it is disturbed by mutated components
especially the B-RafV600E kinase. New agents for the kinase are ever
needed as cases of relapse and resistance for the known drugs have
been widely reported in therapy. In the present work, we have
identified a new class of B-RafV600E inhibitors by fragment linking. In
vitro and in vivo assays were employed to depict the pharmacological
properties of the compounds, which suggest 5v (3-(3-(4-chloro-3-
(trifluoromethyl)phenyl)ureido)-N-(1-(4-methoxyphenyl)-1H-pyrazol-
4-yl)benzamide) is the most potent agent with IC50 values of 0.035 +
0.004 μm (B-RafV600E kinase) and 0.39 + 0.04 μm (A375 cells).
Meanwhile, no obvious toxicity was observed. Collectively, the results
favored our design intention and hinted that this new kind of
chemotype may worth further study in developing novel B-Raf
candidates.
By far, massive research studies have been carried out to
elucidate the underlying mechanisms and to seek for new agents
with better performance.[9] As a kinase, B-Raf may take on three
types of conformation, i.e. the type I, type II and type I/II
conformation.[10] The major differences between those types
mainly lay in the back pocket (BP) region, both in the size and
number of subpockets.[11] While the type I protein has the smallest
BP region and type I/II the intermediate, the type II protein
contains the largest BP region and concomitantly the most
subpockets. On the other side, the front pocket (FP) region for all
the types keeps almost the same. Therefore, different types of
kinase inhibitors may share similar fragment and interacting mode
in the FP region of different conformational proteins.[12]
Interestingly, type II inhibitors of kinase have the least risk to
induce the paradoxical effect in B-RafWT cells (induce the kinase
activity in low doses and decrease it in high doses). Developing
new type II inhibitors thereby is more likely to bring out paradox
breakers.[13]
In our previous work, new type I B-RafV600E inhibitors were
designed by deconstruction-reconstruction strategy.[14] The
recombinant molecules have been proved to be potent inhibitors
for B-RafV600E kinase and the in silico model helped to reveal the
putative binding mode. It is of interest to note that despite the
structural diversity, our molecules share a similar hydrogen-
bonding mode with the typical known type II B-Raf inhibitors in the
FP pocket region. Thus new hybridized inhibitors might be
developed by the recombination of these two types of molecules.
As shown in Fig. 1, both the reported molecule and control agent
sorafenib hydrogen bond with the key amino residues of the
receptor, i.e. Cys532 in the hinge region and Asp594 in the DFG
motif. In consideration that the diphenylurea is one of the most
prevalent scaffolds employed in type II kinase inhibitors which well
accommodated in the BP region, it’s more guaranteed to start
from this backbone for new agent design. Meanwhile, as the type
I and type II kinases have different distances between DFG motif
to the FP, the recombination needs a suitable linker to bridge the
distance gap. Based on this knowledge, we have designed
several linkers to accomplish the connection. The new molecules
were then investigated by consensus docking to observe whether
Introduction
It has long been a hot topic to explore new chemotypes for B-Raf
kinase inhibitors.[1] As a key component in the ERK signal
pathway, B-Raf kinase cascades the upstream signals
transduced from Ras to downstream module MEK.[2] This
cascade is precisely executed in normal cells, and when morbidly
activated by mutated B-Raf kinase (most commonly B-RafV600E),
severe diseases including malignant tumors could be triggered.[3]
Several agents targeting the hyperactive Raf kinase have been
approved into the market, and more types of potential inhibitors
are in the evaluation stage.[4] However, the pharmaceutic market
still calls for new and safe medicines as there remain
nonnegligible drawbacks for the known B-Raf inhibitors.[5] Taken
vemurafenib as an example, it’s the most successful case of
employing FBDD (Fragment-Based Drug Design) strategy and
proved to be a potent and selective inhibitor for the B-RafV600E
mutant kinase.[6] Yet it’s plagued by the relapse and resistance,
along with the side effects.[7] In addition, sorafenib and other
agents also face the kinase selectivity problems.[8]
1
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the components keep original binding interactions and to rank the
binding affinities. The lead compound was validated and further
simulated by molecular dynamics before modified and iteratively
docked. Afterward, the compounds were synthesized and tested
by a series of bioassays to establish pharmacological profiles and
a SAR model.
reported for the first time and gave satisfactory analytical and
spectroscopic data. 1HNMR, 13CNMR, ESI-MS and element
analysis spectra were in full accordance with the assigned
structures (See in the Supporting Material).
Scheme 1. The synthesis routine for target compounds 5a-5w. (a) H2SO4,
HNO3, 60 oC, 2 h; (b) K2CO3, DMF, 25 oC, 16 h; or Iodobenzenes, 8-
hydroxyquinoline, CuI, K2CO3, DMSO, 135 °C, 2 h; (c) 80% hydrazine hydrate,
10% palladium charcoal, ethanol, 80 °C, 10 min; (d) THF, rt, 60 h; (e) EDC,
HOBT, DMAP, DMF, rt, 18 h.
Lead compound validation
As demonstrated in Fig. 1, diverse linkers were employed to
connect fragment A and B to provide new chemotypes. To these
new molecules, consensus docking simulation was performed
utilizing type II B-Raf kinase (PDB code: 3IDP, V600E mutant)
which has the finest resolution to screen for the best hit. The result
suggests the hit compound as shown has the best binding energy,
hydrogen bonding with Cys532 in the hinge region, Asp594 in the
DFG motif and Glu501 in the αC helix (Fig. 2). Van der Waals and
other non-covalently interactions also contribute to the binding
affinity. Besides, the fragments keep similar interaction modes
with their sourced compounds, that is the fragment A remains in
the front region and fragment B penetrates into the deep back
pocket, fitting into our initial intention.
Figure 1. (A) The hydrogen bonding mode of the reported molecule with the
receptor; (B) The hydrogen bonding mode of sorafenib with the receptor; (C)
The construction of new Type II B-Raf kinase inhibitor
Results and Discussion
Chemistry
The synthesis of target compounds followed the general pathway
outlined in Scheme 1. All of the synthesized compounds are being
Figure 2. The binding mode of hit compound with Type II B-RafV600E kinase in 3D (A) and 2D (B) diagrams. For clarity, only key amino acids are shown.
Molecular dynamics simulation
overall RMSD (root mean square deviation) values for both
trajectories of ligand and system were counted and exhibited in
Fig. 3A. It indicated that the system settled into equilibrium soon
after the beginning of the simulation. Both RMSD values kept low
A systematic molecular dynamics simulation was carried out to
further explore the reliability of the binding model of the hit
compound with the receptor and to reveal more details. The
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(<0.25 nm and <0.10 nm, respectively) and smooth during the 10
ns dynamics process, hinting at a quite stable state of the binding
model. Another characteristic descriptor for the system stability is
the Rg (radius of gyration) value for the protein, which is
calculated by the distances between centroids of certain
molecules and atoms. The fluctuation range of Rg reflects the
firmness wobble of protein, and the larger the value is, the relaxer
the protein is. In this study, the total Rg and all dimension Rg
values were summarized in Fig. 3B. It can be found that the total
Rg slightly decreased and remained smooth from 2 ns. Thus the
protein kept a stable and compact state during the whole
simulation. Also, the final conformation of the complex was
overlaid with the initial, and as shown in Fig. 3C, the derivations
for both of the proteins and inhibitors were slight. Together these
results indicate that the binding system is robust and steady, and
can be used as a reliable starting point for further analysis.
dynamics simulation. The estimated number of hydrogen bonds
in dynamic was in accordance with that gained from the binding
result (4 hydrogen bonds). We also calculated the average
binding energy for the last 2 ns by MM/PBSA, which was
summarized in Fig. 3E. As shown, Van der Waal interaction,
electrostatic interaction, polar solvation and non-polar solvation
comprised the free binding energy, which summed up to 207.409
+ 18.784 kJ∙mol1, comparable to or even better than some known
drugs. The contribution of nearby amino residues for the binding
with the ligand was also explored. Important residues were
mapped in Fig. 3F, and generally those residues interacting to the
ligand (revealed by the docking simulation) contributed higher
energy values to the binding. Typically, Glu501, Cys532, and
Asp594 which hydrogen bond with the ligand and other residues
non-covalently interacting with the ligand provided more energy
than other residues, meanwhile some residues went against the
binding affinity. The analysis of residue contribution, along with
the docking simulation, gives a deeper insight into the further
medicinal chemistry optimization.
The heat map for intermolecular hydrogen bonds between the
receptor and ligand (Fig 3D) suggests an average number of
~3.98 hydrogen bonds contributed to the binding during the
Figure 3. (A) The trajectory RMSD values for both system and ligand. (B) The gyration radius values for the receptor. (C) The superimposition of binding systems
before (in grey) and after (in color) simulation. (D) The number of H-bonds between ligand and receptor during the simulation. (E) The free binding energy analysis
for the binding system. (F) The energy contribution of residues in the binding pocket.
Scaffold modification and chemical synthesis
affinity. The best substitution (4-chloro-3-(trifluoromethyl)-
benzene) contributes more as halogen and other interactions
were formed. The binding affinity decreases dramatically when
the phenyl has no or a bulky substitution.
After the synergic simulation of virtual docking and molecular
dynamics, the lead compound was validated to be capable of
binding with B-Raf kinase. Hence we primarily modified its
scaffold, mainly focusing on the substitutions on the pyrazole in
the front cleft or the phenyl ring in the back pockets. All the
derivatives along with their analog sorafenib were evaluated by
consensus docking simulation, as listed in Table 1. Many
compounds have comparable binding scores to sorafenib, with
some bearing even higher scores. Generally, the substitutions on
the phenyl ring in the back pocket played a vital role in the binding
Table 1. The information of final compounds and their docking scores.
3
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Glide
gscore
5o
5p
-CH3
H
H
9.17
8.86
Compound
R1
R2
R3
-CH2CH3
5a
5b
-CH3
-CH2CH3
H
H
H
H
9.15
9.35
5q
5r
-CH3
-CH2CH3
-Cl
-Cl
-CF3
-CF3
11.18
11.26
5c
5d
5e
H
H
H
H
H
H
11.24
8.39
8.37
5s
-Cl
-Cl
-Cl
-CF3
-CF3
-CF3
11.82
12.04
10.56
5t
5u
5f
H
H
H
H
8.45
9.09
5v
-Cl
-Cl
-CF3
-CF3
10.97
5g
5w
10.61
11.52
5h
5i
-CH3
-CH2CH3
-CF3
-CF3
H
H
10.61
10.77
sorafenib
-
5j
5k
5l
-CF3
-CF3
-CF3
H
H
H
12.02
9.32
9.59
We further estimated the drug-likeness property of all the
derivatives. As shown in Table 2, all the compounds satisfied
most criterions of the “Rules of 5”, expect the number of rotatable
bonds. However, given that many known B-Raf inhibitors also
have more rotatable bonds (sorafenib with 6 rotatable bonds and
vemurafenib with 7, etc), this rule seems to be less important in
this case.
5m
5n
-CF3
-CF3
H
H
10.68
10.65
Table 2. The molecular properties of compounds 5a-5w.
Number of H-
acceptor
Number of H-
donor
Number of
rotatable bonds
Number of
aromatic rings
Polar surface
area
Compound
5a
AlogP
1.95
2.30
3.54
3.53
4.02
3.51
3.51
2.89
3.24
4.48
4.47
4.96
4.45
4.45
4.48
5.01
3.56
3.91
5.14
5.14
5.62
5.12
5.12
MW
335.36
349.38
411.45
397.42
411.45
427.45
427.45
403.35
417.38
479.45
465.42
479.45
495.45
495.45
427.46
441.49
437.80
451.82
513.89
499.87
513.89
529.89
529.89
3
3
3
3
3
4
4
3
3
3
3
3
4
4
4
4
3
3
3
3
3
4
4
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4
5
6
5
5
6
6
5
6
7
6
6
7
7
6
7
5
6
7
6
6
7
7
3
3
4
4
4
4
4
3
3
4
4
4
4
4
4
4
3
3
4
4
4
4
4
0.27
0.26
0.22
0.23
0.22
0.24
0.24
0.24
0.23
0.20
0.21
0.20
0.22
0.22
0.20
0.20
0.22
0.22
0.19
0.20
0.19
0.20
0.20
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
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sorafenib
4.17
5.68
464.82
489.92
4
4
3
2
6
7
3
0.22
0.23
vemurafenib
4
Inhibitory activity on kinases and cancer cells
RafV600E kinase with IC50 value of 0.035 + 0.004 μm. Subsequently,
the inhibitory activity of 5v on B-RafWT, C-Raf, p38α, VEGFR2 and
JNK1 kinases were further tested (Table 4). The results showed
the inhibitory activity of compound 5v on the other kinases was
much lower. In summary, compound 5v had stronger inhibitory
activity and higher selectivity for the B-RafV600E kinase.
With sorafenib and vemurafenib as the reference drugs, all the
new compounds were evaluated for the B-RafV600E kinase
inhibitory effects. As shown in Table 3, overall the synthesized
agents showed moderate to potent inhibitory towards the kinase.
Compounds 5v showed the most prominent inhibitory effect on B-
Table 3. In vitro antiproliferative activity and BrafV600E inhibitory activity of 5a-5w.
IC50 (μm)[a]
Compound
HT29
HCT116
A375
WM1361
B-RafV600E
5.82 + 0.44
6.17 + 0.52
3.49 + 0.23
1.56 + 0.12
2.97 + 0.19
3.09 + 0.34
1.23 + 0.09
0.95 + 0.11
1.25 + 0.15
1.64 + 0.18
1.01 + 0.13
0.92 + 0.07
1.21 + 0.08
0.09 + 0.01
4.32 + 0.52
5.11 + 0.61
0.16 + 0.02
0.25 + 0.04
0.21 + 0.03
0.10 + 0.01
0.052 + 0.007
0.035 + 0.004
0.35 + 0.05
0.031 + 0.008
0.048 + 0.006
293T
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
5a
20.15 + 1.52
19.75 + 1.83
12.68 + 1.20
10.53 + 0.87
8.82 + 0.91
10.41 + 1.03
8.06 + 0.89
2.55 + 0.81
3.08 + 0.29
9.12 + 0.68
6.01 + 0.65
4.18 + 0.54
6.65 + 0.82
3.15 + 0.57
19.72 + 1.53
21.66 + 1.58
4.65 + 0.35
5.91 + 0.56
3.58 + 0.45
1.06 + 0.11
0.77 + 0.09
1.23 + 0.16
2.38 + 0.25
1.56 + 0.24
9.78 + 0.86
35.57 + 1.96
34.22 + 1.52
25.11 + 1.12
22.26 + 1.05
20.85 + 1.39
20.41 + 2.01
18.18 + 0.73
8.66 + 0.95
11.38 + 1.01
16.87 + 1.19
13.12 +1.04
9.89 + 0.82
12.81 + 0.99
9.62 + 0.87
32.44 + 1.21
36.35 + 1.57
19.41 + 0.99
20.52 + 1.11
16.58 + 1.03
9.12 + 1.84
7.52 + 0.57
6.88 + 0.64
11.47 + 0.96
18.25 + 2.65
12.99 + 1.17
16.19 + 1.03
12.21 + 1.25
9.24 + 0.88
8.18 + 0.69
6.59 + 0.87
7.54 + 0.59
5.89 + 0.85
2.28 + 0.34
3.21 + 0.29
8.12 + 0.92
5.45 + 0.76
3.85 + 0.47
4.38 + 0.62
3.56 + 0.51
15.11 + 1.07
16.49 + 1.12
3.98 + 0.38
4.12 + 0.64
3.01 + 0.42
1.54 + 0.17
0.65 + 0.08
0.39 + 0.04
3.11 + 0.28
1.08 + 0.26
8.36 + 0.54
30.15 + 1.70
29.35 + 1.56
18.47 + 1.62
19.22 + 1.44
17.80 + 1.18
16.57 + 1.05
15.35 + 1.21
10.45 + 0.91
12.55 + 1.06
18.18 + 1.08
16.68 + 0.95
15.82 + 1.27
18.25 + 1.24
15.86 + 1.35
29.25 + 1.75
31.08 + 1.88
12.54 + 0.87
17.65 + 1.15
15.46 + 0.98
9.88 + 1.21
8.69 + 0.92
7.12 + 1.01
10.23 + 0.85
17.34 + 1.32
10.15 + 0.98
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
vemurafenib
sorafenib
[a] Data are shown as mean + SD of three independent experiments.
The compounds were also tested by MTT assay for anti-
proliferative activity against two melanoma cancer cell lines, A375
(B-RafV600E) and WM1361 (B-RafWT), along with two colon cancer
cell lines, HT29 (B-RafV600E) and HCT116 (B-RafWT). Besides,
human kidney epithelial cell 293T was employed as the non-
cancer cell line to evaluate the cytotoxicity of all the compounds,
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as summarized in Table 3. A similar inhibitory effect can be
observed for the compounds against A375 cell. Toward another
B-RafV600E harbored cell line HT29, the inhibitory effects were also
significant compared to B-RafWT cell line HCT116. The cytotoxicity
values of all tested compounds against 293T were all above 50
μm, indicating a high selectivity over the normal cell lines.
Especially, compound 5v showed the most potent anti-
proliferative activity to A375 and HT29 cells expressing B-RafV600E
with lower IC50 values (0.39 + 0.04 μm and 1.23 + 0.16 μm)
compared with vemurafenib (1.08 + 0.26 μm and 1.56 + 0.24 μm).
In addition, against WM1361 and HCT116 cells expressing B-
RafWT, compound 5v showed a decreased anti-proliferative effect
on the contrary. In short, 5v is a potential B-RafV600E inhibitor and
efficiently inhibit the proliferation of tumor cells expressing B-
the favorable (in green) or unfavorable (in yellow) steric effects
while information of favorable (in blue) or unfavorable (in red)
electrostatic field regions is also given in a contour plot. A good
compliance is observed between the model and the actual
situation for the tested compounds. Hence, optimized agents may
be developed utilizing this model in the future study.
RafV600E
.
Table 4. Inhibition of kinases activity by compound 5v.
Kinase
B-RafV600E
B-RafWT
C-Raf
IC50 (μm)[a]
0.035 + 0.004
0.22 + 0.04
0.62 + 0.08
26.2 + 2.12
13.4 + 1.87
10.7 + 1.24
Figure 4. Plot of actual vs. predicted pIC50 of the training set (dots) and test set
(triangles).
p38α
VEGFR2
JNK1
[a] Data are shown as mean + SD of three independent experiments.
SAR analysis and 3D-QSAR model
Based on the in vitro B-RafV600E kinase assay and cellular
antiproliferation assay, it could be found that to a certain extent,
the potency of the compounds keeps up with their virtual binding
affinity. Compounds with an oxydibenzene group (5o, 5p) in the
BP region have low potency, for the bulky substitution may hinder
the formation of hydrogen bonds between the residues with urea
group. Meanwhile, halogen and trifluoromethyl substitutions on
the same phenyl improve the potency, compared to those non-
substituted (e.g., 5v>5m>5f). This result is in accordance with the
suggestion by virtual simulation that halogen and trifluoromethyl
groups provide additional interactions favoring the binding. As to
the investigated groups in the front cleft, substituted phenyl
groups tend to be more favorable than alkanes and especially
compounds with 4-methoxyl phenyl exhibited better inhibitory
activity.
Figure 5. (A) 3D QSAR model coefficients on van der Waals grids. Green
represents positive coefficients; yellow represents negative coefficients (B) 3D
QSAR model coefficients on electrostatic potential grids. Blue represents
positive coefficients; red represents negative coefficients.
Cell apoptosis analysis
To determine whether compound 5v induced cell apoptosis, an
Annexin V/PI double staining assay was conducted. A375 cells
were treated with increasing concentrations (0, 2, 4 and 8 µm) of
5v or vemurafenib for 24 h, followed by Annexin V/PI double
staining and flow cytometric measurement. The increased
apoptotic rate was detected after cells being treated with
escalating doses of 5v. As shown in Fig. 6A, the percentages of
cell apoptosis 10.22%, 30.3%, 48.5%, 75.5% correspond to the
concentration of 5v 0, 2, 4 and 8 μm, respectively, including both
the early and the late apoptotic cells. Clearly, 5v can cause
apoptosis more effectively than vemurafenib. Moreover, an
extend time-course apoptosis analysis of A375 cells treated with
4 μm 5v was carried out. The results indicated that longer
treatment periods showed an increase in the percentage of
We also established a 3D-QASR model to gain more analysis and
have better understanding. All the molecules were converted to
the active conformation and attributed with corresponding pIC50
(mm) values before randomly partitioned into the training set and
test set. CHARMm force field and PLS regression were employed
in building the model.
A scatter plot was obtained with
conventional R2 of 0.9189 (Fig. 4), hinting a high compatibility
between the predicted and actual pIC50 values.
Critical regions (steric or electrostatic) which affect the binding of
compounds and enzyme were marked in Fig. 5: compounds were
aligned with the iso-surfaces of the 3D-QSAR model coefficients
on van der Waals grids (Fig. 5A) and electrostatic potential grids
(Fig. 5B). The steric map shows energy grids corresponding to
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apoptotic cells (Fig. 6B). Taken together, compound 5v could
induce A375 cells apoptosis in a dose- and time-dependent
manner.
Figure 7. (A) A375 cells were treated with 0, 2.0, 4.0 and 8.0 μm 5v and
vemurafenib for 24 h. (B) A375 cells were treated with 4 μm 5v and vemurafenib
for 0, 12, 24, and 36 h. Effect of compound 5v on the cell-cycle progression of
A375 cells was determined by flow cytometry analysis (G0/G1 phase, green; S
phase, yellow and G2/M phase, blue).
Figure 6. (A) A375 cells treated with 0, 2.0, 4.0 and 8.0 μm 5v and vemurafenib
for 24 h were collected and analyzed. (B) A375 cells treated with 4.0 μm 5v and
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).
Western blot analysis
While the abnormally activated ERK pathway caused by B-
RafV600E results in the phosphorylated level of MEK and ERK
increased, effective B-RafV600E inhibitors shall block the pathway
and down-regulate the expression of pERK and pMEK. The ability
of 5v inhibits ERK and MEK phosphorylation was investigated in
A375 cells, with vemurafenib as a positive control drug. As shown
in Fig. 8(A) and 8(B), compound 5v and vemurafenib both
reduced the expression of the phosphorylation of ERK and MEK
in A375 cells in a dose-dependent manner and had no effect on
total expression of ERK and MEK. By comparison, we found that
the inhibitory effect of 5v was more potent than vemurafenib.
Subsequently, the abilities of 5v and vemurafenib inhibiting the
phosphorylation of ERK and MEK in WM1361 cells were also
evaluated. While the paradoxical effects of vemurafenib at
different concentrations were reported, the treatment to WM1361
cells with 5v showed no significant effect on phosphorylation of
ERK and MEK, as shown in Fig. 9.
Cell cycle analysis
We further assessed the cell cycle distribution of A375 cells by
flow cytometry. A375 cells were treated with compound 5v at
various doses (0, 2, 4, and 8 μm) for 24 h. As illustrated in Fig. 7A,
the accumulation of cells in G0/G1 phase increased with
increasing drug concentration, and 69.31% of cells were arrested
in the G0/G1 phase upon exposure to 4 μm compound 5v for 24
h. When the concentration of compound 5v increased to 8 μm,
78.73% of cells were arrested in the G0/G1 phase. Meanwhile,
A375 cells were treated with 4 μm compound 5v at gradient times
(0, 12, 24 and 36 h). The accumulation of cells in G0/G1 phase
was markedly elevated in a time-dependent manner (with the
percentage of 53.11%, 59.42%, 67.85% and 78.42%, Fig. 7B). In
summary, 5v could induce cell cycle arrest in the G0/G1 phase,
and the effect was more potent than vemurafenib.
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10.1002/cmdc.201800574
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become a promising lead compound for the development of novel
antitumor drugs.
Figure 8. (A) 5v inhibited the phosphorylation of ERK and MEK in A375
melanoma cells bearing B-RafV600E at different doses; (B) Vemurafenib inhibited
the phosphorylation of ERK and MEK in A375 melanoma cells bearing B-Raf
V600E at different doses.
Figure 10. (A) Tumor volumes. A375 cells were injected subcutaneously into
the right flank of nude mice. When the treatment began, the tumor volumes were
monitored and recorded every two days; (B) Tumor weight on day 15 of
treatment; (C) Body weight. When the treatment began, the body weight was
monitored and recorded every two days; (D) Weight of liver and spleen on day
15. Data are shown as mean ± SD.*P < 0.05, **P < 0.01.
Conclusions
In light of the drawbacks of the known B-Raf inhibitors, great
efforts have been made to optimize the existing agents and to
explore new chemical space. On the whole, the massive studies
both depicting the biological mechanisms of Raf kinase and
revealing the interaction mode between inhibitors and Raf kinase
have pointed out the direction for better-performance agents. Yet
attempts to do so still need to take many factors into consideration,
such as breaking the paradoxical effects in kinases. Our previous
and current efforts mainly focused on the recombination of high-
efficient fragments from different sources into a new molecule.
The synergic simulation was carried out in this work: while the
consensus docking by various applications eliminated the
deviations between algorithms to a certain extent, the molecular
dynamics as a validated post-docking tool further tested the
credibility of the binding model. The analyses of MD were in high
accordance with the result of docking simulation, and it also
provided more details which may benefit future study. The
molecular properties were investigated to evaluate the drug-
likeness of the new compounds. Overall, the analyses gave
interesting results and thereby we synthesized all the compounds.
By in vitro assays of anti-proliferation and kinases inhibition, the
best compound 5v was selected and underwent further bioassays
to depict its pharmacological profile and give SAR suggestions.
Nude mice xenograft models were also established to
demonstrate the in vivo performance of 5v. All results together
suggest we have identified a new series of B-RafV600E inhibitors
and we hope such efforts shall provide new insight into the related
studies.
Figure 9. (A) 5v slightly inhibited the phosphorylation of ERK and MEK in
WT
WM1361 melanoma cells bearing B-Raf
when dose increased; (B)
Vemurafenib paradoxically inhibited the phosphorylation of ERK and MEK in
WM1361 melanoma cells bearing B-RafWT
Xenografts models
We utilized xenograft models of A375 cells to determine whether
compound 5v inhibited tumor growth in vivo. When the tumors
began to grow (50~80 mm3), the mice were randomly divided into
6 groups (six mice/group) and administrated intraperitoneally with
vector, 2.5 mg/kg, 5 mg/kg, 10 mg/kg compound 5v or 5 mg/kg
vemurafenib as a positive control every second day for 15 days.
The average tumor volume recorded every other day was shown
in Fig. 10A. While the vehicle control group was observed with a
rapid development in tumor size, the other groups had distinctly
different shapes in the growth curve. At the end of the experiment,
tumors were excised and weighed. The average weight of the
control group tumors was more than 3-fold to the 5v-treated mice
at the 10 mg/kg dose. When exerted on the same dose of 5 mg/kg,
5v exhibited equivalent antitumor efficacy in comparison with the
positive control drug vemurafenib (Fig. 10B). Briefly, the
administration of 5v inhibited tumor growth in a dose-dependent
manner. Meanwhile, 5v did not show a significant effect on the
body weight (Fig. 10C), as well as the weight of the liver or spleen
(Fig. 10D). It can be concluded that the adminstration of 5v up to
10 mg/kg didn’t cause weight loss, liver harm or spleen swelling,
hinting a considerable safety in vivo. Together, these in vivo data
indicate that 5v exhibits excellent antitumor ability and may
8
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10.1002/cmdc.201800574
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Synthesis of compounds 5a-5w
Experimental Section
EDC (1.5 mmol, 0.28 g) was added in one portion to a mixture of 4a-4b
(1.5 mmol) and HOBt (1.5 mmol, 0.2 g) in DMF (4.00 mL) at 25 oC for 30
min. Then DMAP (1.5 mmol, 0.19 g), 3a-3g (1.5 mmol), and Et3N (1 mL)
were added and the reaction mixture was stirred overnight. The resulting
mixture was poured into water and was extracted with ethyl acetate.
Purification by flash chromatography (petroleum ether : ethyl acetate = 6 :
1) gave the desired products 5a-5w.
Materials
All chemicals (reagent grade) used were purchased from Nanjing
Chemical Reagent Co. Ltd. (Nanjing, China). Vemurafenib was purchased
from Sigma-Aldrich (St. Louis, MO). All the 1H NMR spectra were recorded
on a Bruker DPX 600 model spectrometer in DMSO-d6, and chemical shifts
(δ) are reported as parts per million (ppm). ESI-MS spectra were recorded
by a Mariner System 5304 Mass spectrometer. Elemental analyses were
performed on a CHN-O-Rapid instrument and were within 0.4% of the
theoretical values. Melting points were determined on an XT4 MP
apparatus (Taike Corp, Beijing, China). Purity of compounds were tested
by HPLC (purity > 95%). Thin layer chromatography (TLC) was performed
on silica gel plates (Silica Gel 60 GF254) and visualized in UV light (254
nm and 365 nm). Column chromatography was performed using silica gel
(200 - 300 mesh) and eluting with ethyl acetate and petroleum ether (bp.
30 - 60 oC). A375 cells were kindly provided by Stem Cell Bank, Chinese
Academy of Sciences. The other cell lines (WM1361, HT29 and HCT116)
were preserved in the State Key Laboratory of Pharmaceutical
Biotechnology of Nanjing University. The AnnexinV-FITC cell apoptosis
assay kit was purchased from Vazyme Biotechco., Ltd (Nanjing, China).
The Raf kinases were purchased from Invitrogen (US). All antibodies were
obtained from WanleiBio (Shenyang, China). The balb/c nude mice
(female, 6 - 8 weeks, 20 - 21 g) were purchased from Nanjing University
Animal Center (Nanjing, China).
Virtual simulation
The consensus docking simulation and graphics processing work were
carried out following the procedure described previously[15] Molecular
dynamics simulations were carried out using the GROMACS package
version 5.1.2 with GPU-accelerating supported. The parameter setting and
operating procedure can be seen in previous reports.[16] A ligand-based
3D-QSAR model was established by the QSAR module of DS 3.5,
following the same protocol reported in the previous work.[17]
Biological assays
All the biological assays were carried out following the protocols from
product manufacturers or reference literature.[14-16, 18] 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.
Chemistry
Statistical analysis
Synthesis of compound 1
All assays, with established cell lines, were repeated three times. All data
are expressed as mean + SD. The statistical analysis was performed by
the Student’s t-test, and if appropriate, by a one-way ANOVA test, using
the statistical software OriginPro 2015.
Pyrazole (2 g, 29.4 mmol) was dissolved in concentrated sulfuric acid (6
mL). Afterward the solution was heated to 60 oC, before the dropwise
addition of nitric acid (1.2 mL). The reaction was stirred for 2 h and poured
into ice water. A white precipitate (1) formed which was filtered and
washed with water. The filtrate was neutralized using sodium carbonate
and extracted with ethyl acetate (3 × 20 mL). The combined organic
phases were washed by brine solution, dried over sodium sulfate and
concentrated in vacuo to give the rest product 1.
Acknowledgements
The work was financed by the Public Science and Technology
Research Funds Projects of Ocean (No. 201505023), the
International Postdoctoral Exchange Fellowship Program 2017
(No. 20170090) and Nanjing University Undergraduate Innovation
Program and the National Natural Science Foundation of China
(No. J1210026).
Synthesis of compounds 2a-2g
A mixture of alkane or aryl iodide (6.82 mmol), 1 (0.7 g, 6.2 mmol), 8-
hydroxyquinoline (0.09 g, 0.62 mmol), cuprous iodide (0.18 g, 0.62 mmol)
and potassium carbonate (1.73 g, 12.4 mmol) in DMSO (10 mL) was
heated at 135 oC overnight. After cooling to rt, the reaction mixture was
diluted with 20 mL of water and extracted with ethyl acetate. The organic
layer was washed with aqueous saturated sodium bicarbonate, dried by
sodium sulfate, filtered and concentrated in vacuo. Purification by flash
chromatography (petroleum ether : ethyl acetate = 6 : 1) gave the desired
products 2a-2g.
Keywords: B-Raf inhibitors; molecular optimization; in silico;
rational design; mice xenograft
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Based on our previously reported B-Raf inhibitors and known drugs, a new series of type II B-Raf inhibitors were designed by
fragment linking strategy. The new compounds were validated by synergic simulations, acquired by synthesis and profiled by in vitro
and in vivo pharmacological assays. Several agents showed excellent performance which worth further study in the future research.
11
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