Design of potent B-RafV600E inhibitors by multiple copy simulation search strategy

Article abstract of DOI:10.1111/cbdd.13121

B-Raf kinase is a vital intermedium in the mitogen-activated protein kinase (MAPK) signaling pathway, which transforms extracellular signals into cellular mechanisms. Mutations in this kinase, for instance, the most common V600E mutation, can lead to the ERK signaling pathologically activated and hence cause severe diseases such as somatic tumors. So far, the development of B-Raf inhibitors has made remarkable progress. However, the resistance and relapse of approved Raf drugs have been widely reported, and the optimization for old drugs and the discovery for new inhibitors still remain a significant task. In this study, we designed and evaluated a series of novel B-Raf^V600E inhibitors. A fragment library has been established before the docking simulation carried out using the MCSS strategy (multicopy simulation search). The appropriate fragments were reassembled to provide new candidate compounds, which were further screened by iterative docking simulations and molecular dynamics. Bioassays were carried out to evaluate the pharmacological profile of the compounds identified and synthesized. The result showed that compound 5n had an impressive enzyme inhibitory and antiproliferation activity, suggesting a promising potential in the future study.

Full text of DOI:10.1111/cbdd.13121

PROFESSOR HAI-LIANG ZHU (Orcid ID : 0000-0001-5254-7487)
Article type
: Research Article
Design of Potent B-RafV600E Inhibitors by MCSS Strategy
Ze-Feng Wang†, Peng-Fei Wang†, Jun-Ting Ma, Ying-Zi Chai, Hui-Min Hu, Wen-Long
Gao, Zhong-Chang Wang*, Bao-Zhong Wang*, Hai-Liang Zhu*
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210023, People’s Republic of China.
*Corresponding author:
Hai-Liang Zhu, Tel. & fax: +86-25-89682572; e-mail: zhuhl@nju.edu.cn
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13121
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†These two authors contributed equally to this paper.
Abstract
B-Raf kinase is a vital intermedium in the mitogen-activated protein kinase (MAPK)
signaling pathway, which transforms extracellular signals into cellular mechanisms.
Mutations in this kinase, for instance, the most common V600E mutation, can lead to the
ERK signaling pathologically activated and hence cause severe diseases like somatic tumors.
So far, the development of B-Raf inhibitors has made remarkable progress. However, the
resistance and relapse of approved Raf drugs have been widely reported, and the optimization
for old drugs and the discovery for new inhibitors still remain a significant task.
In this study, we designed and evaluated a series of novel B-RafV600E inhibitors. A
fragment library has been established before the docking simulation carried out using the
MCSS strategy (multi-copy simulation search). The appropriate fragments were reassembled
to provide new candidate compounds, which were further screened by iterative docking
simulations and molecular dynamics. Bioassays were carried out to evaluate the
pharmacological profile of the compounds identified and synthesized. The result showed that
compound 5n had an impressive enzyme inhibitory and anti-proliferation activity, suggesting
a promising potential in the future study.
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Key words
MCSS; B-Raf inhibitor; Docking; Molecular dynamics; Bioassays
1 Introduction
Dysfunctions of signaling pathways have long been elucidated to be closely implicated
with diverse severe diseases. For instance, when deviation appears in the MAPK
(mitogen-activated protein kinase) signaling pathways,[1] it may trigger or promote the
process of many human diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's
disease (PD), Alzheimer's disease (AD) and various types of cancers.[2-4] While other
branches are suggested to mainly mediate neuronal apoptosis in AD, PD, and ALS, the ERK
signaling is more connected with tumorigenesis,[5] including cancer cell proliferation,
migration, and invasion. In the ERK signaling, Raf is a key component whose mutation will
lead to the signaling activated abnormally.[6] The most common mutation observed is
B-RafV600E, as a substitution of glutamate for valine at amino acid 600 (V600E) occurs in the
most important subtype of Raf family.[7] This mutation mimics the phosphorylation state,
hence the B-RafV600E protein persistently performs its kinase activity without regulation. The
V600E mutation has been detected in most melanomas and some other somatic tumors
worldwide, and brings great threat to the health of humans.[8, 9]
Therefore, developing novel B-RafV600E antagonists keeps a hot topic in the medicinal
chemistry industry. With the process of structure elucidation of B-Raf kinase, it’s becoming
more feasible to design small molecular inhibitors with favorable features meeting the
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requirements to selectively and efficiently suppress the activity of B-RafV600E [10, 11]
Studies
.
about various kinds of inhibitors have been continually reported. Some of the inhibitors have
entered different stages of preclinical or clinical study, and a few already utilized in
Raf-related cancer therapy.[12] Much progress has been made, though, the challenge is still
uphill: both resistance and relapse have been universally reported when using the known Raf
drugs.[13,14] Hence the optimization for old drugs and discovery for new agents remain an
important task.
This study presents our recent work on designing and evaluating a new series of B-RafV600E
inhibitor. To begin with, we have established a library of fragments based on the known
endogenous ligands extracted from protein-ligand complexes. On the basis of protein
structure analysis, the fragments were docked into the active pocket of B-Raf employing
MCSS strategy (multiple copy simulation search).[15-18] The fragments with top score and
suitable space distribution were recombined to afford the candidate compounds which were
redocked to validate, with the binding of the best hit tested by molecular dynamics.[19-21]
Afterwards the hit was modified to gain a chemotype diversity and synthesized.
Anti-proliferation and kinase inhibition assays in vivo were carried out to profile the potency
of these derivate entities. The most potent compound 5n, with IC50 values of 0.09 + 0.002 μM
against B-RafV600E and 0.9 + 0.17 μM against A375 cell line, was selected to perform western
blotting to verify whether the downstream signaling pathway was blocked. Altogether, the
results favored our design intention and with this effort, we hope this work will provide more
insight in the future research.
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2 Results and Discussion
2.1 Chemistry
The synthesis of target compounds followed the general pathway outlined in Scheme 1.
All of the synthetic compounds are being reported for the first time (Table S1) and gave
satisfactory analytical and spectroscopic data. 1HNMR, ESI-MS and element analysis spectra
were in full accordance with the assigned structures, which were presented in the Supporting
Information.
2.1.1 Materials
All chemicals (Analytical grade) used were purchased from Nanjing Chemical Reagent Co.
Ltd. (Nanjing, China). All the 1H NMR spectra were recorded on Bruker DPX400 or Bruker
DPX600 model spectrometer in DMSO-d6, and chemical shifts (δ) are reported as parts per
million (ppm). ESI-MS spectra were recorded by a Mariner System5304 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 a XT4 MP apparatus (Taike Corp.,
Beijing,China). Thin layer chromatography (TLC) was performed on silica gel plates (Silica
Gel60 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 (bp30 – 60 oC).
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2.1.2 Chemistry methods
2.1.2.1 General procedure for the synthesis of compounds (1)
Pyrazole (14.00 g, 205.64 mmol) was dissolved in concentrated H2SO4 (50.00 mL). The
solution was heated to 60 oC, before the dropwise addition of nitric acid (9.20 mL), which
caused the temperature rise slightly. The reaction was stirred for 1.5 h at 60 oC and poured
into 600 g of an ice/water mixture. A white precipitate formed that was filtered and washed
with water. This yielded 12.50 g (40%). The filtrate was extracted with ethyl acetate (3 × 100
mL). The combine organic phases were washed with 1% NaHCO3 (aq.100 mL), water (100
mL), and brine (100 mL). Then the ethyl acetate solution was dried over Na2SO4 and
concentrated in vacuo, which yielded another 10.5 g (45%) of the desired product.
2.1.2.2 General procedure for the synthesis of compounds (2a-k)
To a stirred solution of 1 (1.00 g, 8.84 mmol) in DMF (6.00 mL) at 25 oC were added
K2CO3 (1.47 g, 10.60 mmol) and iodomethane (10.60 mmol, 660 μL). After stirring for 16 h
at 25 oC, the reaction mixture was treated with water (15.00 mL) and EtOAc (20.00 mL) and
transferred to a 125 mL separatory funnel. The organic layer was collected and the aqueous
layer was extracted with EtOAc (3 × 25 mL). The combined organic layers were washed with
brine (20.00 mL), dried over sodium sulfate and filtered. The filtrate was concentrated, and
the residue was purified by flash column chromatography to provide the products (for 2a-2e).
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A mixture of iodobenzene (6.82 mmol), 1 (0.70 g, 6.20 mmol), 8-hydroxyquinoline (0.09
g, 0.62 mmol), cuprous iodide (0.18 g, 0.62 mmol) and potassium carbonate (1.73 g, 12.40
mmol) in DMSO (10 mL) was heated at 135 oC for 20 h. After cooling to room temperature,
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 gave the
desired products (for 2f-2i).
2-bromo-N-(4-methoxyphenyl)acetamide (1.00 mmol, 0.25 g), 1 (1.00 mmol, 0.12 g),
sodium ethoxide (0.10 g) was dissolved DMF (50 mL). After stirring for overnight at room
temperature, the reaction mixture was treated with water (15 mL) and EtOAc (25 mL). The
organic layer was collected and the aqueous layer was extracted with EtOAc (3 × 25 mL).
The combined organic layers were washed with brine (20 mL), dried over sodium sulfate and
filtered. The filtrate was concentrated, and the residue was purified by flash column
chromatography to provide the products (for 2j-2k).
2.1.2.3 General procedure for the synthesis of compounds (3a-k)
Compound 2 (4.00 mmol) was refluxed with ethanol (4.00 mL), 80% hydrazine hydrate
(1.00 mL), and 10% palladium charcoal (0.08 g) for 10 min. The reaction was filtered by
celite. The filtrate was dried by sodium sulfate, and concentrated in vacuo to afford
compounds 3a-3k, which were used without further purification.
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2.1.2.4 General procedure for the synthesis of compounds (4a-4b)
Methyl 1-methyl-1H-indole-3-carboxylate (or Methyl 1H-indole-3-carboxylate) was added
to a solution of potassium hydroxide (1.50 equiv) in water (50 mL). Then the resulting
mixture was heated to 65 oC for 2 h. The resulting solution was cooled, neutralized with
dilute hydrochloric acid solution to pH = 5. The precipitated solid was filtered and washed
with water to give the product 4a (or 4b).
2.1.2.5 General procedure for the synthesis of compounds (5a-5p)
EDC (2.00mmol, 0.38g) was added in one portion to a mixture of 4 (2.00mmol, 1equiv)
and HOBt (2.00 mmol, 0.27) in DMF (5.00 mL) at 25 oC for 30 min. Then a crystal of
DMAP (2.00 mmol, 0.25 g), 3 (2.00 mmol), and Et3N (1 mL) was added and the reaction
mixture was stirred overnight. The resulting mixture was poured in water and was extracted
with ethyl acetate. Purification by flash chromatography gave the desired products 5a-5p.
2.2 Data preparation and analysis
Collectively, there were 45 B-Raf co-crystals deposited in the PDB database up to March,
2016 (shown in Figure S1). These complexes were downloaded and the correspondingly
endogenous ligands were extracted and revised. The receptor protein was primarily prepared
with missing amino acid residues and hydrogen atoms added before charged with force field.
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By deconstruction, a small library of fragments was established from the original ligand
molecules. Afterwards, the fragments were minimized and charged with force field.
2.3 Screening for the best hits
By the MCSS strategy, 2500 ~ 5000 copies for each fragment were randomly distributed
and docked into the binding area of the receptor. Low-ranking poses for these fragments were
automatically discarded in the output stage and the rest were ordered by the MCSS score. For
each fragments, the docking modalities with top scores were collected and recorded. After
then, fragments with favorable distribution and orientation were recombined and linked to
afford a new skeleton. We used the new skeleton without substituents as a hit compound, and
the backbone compound was derivatized to obtain the compounds 5a-5p to discuss
structure-activity relationship. Finally, docking simulation was performed to the modified
compound, and the CDOCK interaction energy values were depicted in Figure S2. The best
compound 5n with highest binding energy (-57.92 kcal/mol) was shown in Figure 1. It can
be found compound 5n preferably bound to the active site of the B-RafV600E (PDB code:
4E26) by two hydrogen bonds with ILE 527 and CYS 532, and formed three Pi-Pi bonds
with TRP531 and PHE 595, along with other weak interactions. Meanwhile, we described the
interaction between compound 5n and B-RafV600E (PDB code: 4E26) by the 3D model in
Figure 2A and an overlay of vemurafinib and compound 5n with B-RafV600E (PDB code:
4E26) has been incorporated in Figure 2B. The similarities of conformation distribution
between vemurafinib and compound 5n in B-RafV600E was suggested in Figure 2B, for
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example, the distribution of their N, O atoms was similar. The molecular docking suggested
that compound 5n may be a potential B-RafV600E ligand.
In the Table S2, the molecular properties of all the target compounds and vemurafenib
have been summarized. In general, B-Raf antagonists have more N, O, S atoms and smaller
logP values than other types of antagonists. Therefore, the “Rule of 3” (known as rules for the
lead compounds) or “Rule of 5” (known as rules for drug candidates) should be carried out
more moderately in the process of designing new B-Raf inhibitors. The recombinant
compounds have a smaller molecular weight, less H-donor and acceptor, rotatable bonds and
rings than vemurafenib. The comparison is even more obvious when compared with
vemurafenib, implying an enormous potential for profile optimization.
With the rapid development of computer technology and molecular computing technology,
molecular dynamics simulation has been widely recognized and applied. Molecular dynamics
simulation, as an effective post-docking tool, plays a very important role in the verification
and optimization of molecular docking. Therefore, in this study we also use the molecular
dynamics simulation to test our predicted molecular structure. As an example, Figure 3
showed that B-Raf-ligand 5n systems settled into equilibrium state soon after the start of
simulation, with fluctuations wobbling in narrow ranges. Also, the RMSD values showed that
ligand 5n in the system was quite stable. Together the virtual simulations suggested these
compounds bear promising potential, and the best hits were thereafter synthesized and
evaluated for their biological efficiency.
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For the molecular dynamics simulation results, equilibrium state simulation of 8.0 ns - 10.0
ns were selected, and the energy composition was extracted every 100 ps. The MM/PBSA
free binding energy was analyzed for the 20 samples. MM/PBSA free binding energy is
mainly composed of van der Waals force, electrostatic force, polar solvation and non-polar
Solvation. For this system, the average energy and free binding energy of the system are
shown in Table 1. The results showed that van der Waals played a major role in the binding
process of ligand and protein (-250.43 + 4.33 kJ/mol), and the total free binding energy of
protein and ligand summed up to -219.40 + 10.41 kJ/mol. The results indicated that the
binding of small molecules to receptors was better.
2.4 Compound 5n potently inhibited B-RafV600E kinase activity and proliferation of cancer
cells
The inhibitory effects of test compounds 5a-5p on kinase B-RafWT, B-RafV600E and C-Raf
were evaluated using vemurafenib as a positive control drug. In the Table S3 and Table S4,
it can be seen that both compound 5n and vemurafenib showed impressive enzymatic
inhibition to B-RafV600E kinase (with IC50 values of 0.10 + 0.01 μM and 0.03 + 0.007 μM,
respectively). Their inhibitory effect toward wild-type B-Raf has decreased, compared to
B-RafV600E kinase (with IC50 values of 0.92 + 0.02 μM and 0.22 + 0.018 μM, respectively).
Vemurafenib is a potent pan-Raf inhibitor (IC50 value of 0.19 + 0.018 μM against C-Raf), and
compound 5n has a weak inhibitory effect on C-Raf with an IC50 value of 6 times that of
vemurafenib (IC50 value of 1.09 + 0.081 μM). The other compounds also exhibited good
efficacy, even though not as effective as the positive control drug vemurafenib and 5n (Table
S3 and Table S4).
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The entities were evaluated for their anti-proliferation activities against four cancer cell
lines, HT29 (B-RafV600E), A375 (B-RafV600E), WM1361 (B-RafWT), and HCT116 (B-RafWT),
by the MTT assay, and the calculated IC50 values were shown in Table S3. To compound 5n,
it showed better suppression compared with vemurafenib when exerting on the A375 cells
and HT29 cells for 48 h (with IC50 values of 0.93 + 0.11 μM and 2.27 + 0.21 μM against IC50
values of 1.08 + 0.26 μM and 1.56 + 0.24 μM, respectively). To WM1361 and HCT116 cells
cells expressing B-RafWT, the cell viability of cells treated with compound 5n and
vemurafenib was significantly higher than that of cells carrying B-RafV600E. The MTT assays
suggested that the compound 5n is a potential B-RafV600E inhibitor and could efficiently
inhibit the proliferation of B-RafV600E-harbored cell lines.
It can be seen from the table S3 that when R1 substituent was constant, the change in R2
substituent had a crucial effect on the activity of the compounds. Comparing compound 5a
with 5l, 5c with 5m, 5g with 5n, 5h with 5o, and 5j with 5p, we can easily draw the
following conclusions: when there was the same R1 substituent, compounds with different R2
substituent groups were compared, and it was clear that the activity of the tested compounds
with different R2 substituent decreased obviously in the following order: -H > -CH3. It is
shown that N-methylated are less potent than the corresponding NH free compounds. When
R2 substituent was constant, R1 substituent biological activities of different inhibitors
declined with the following order: p-tolyl > phenyl > methoxyphenyl > the remaining other
substituents. Though other compounds are less potent, many are close to the control.
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2.5 Compound 5n induced cell apoptosis in dose- and time-dependent manner
Reports have revealed that vemurafenib could induce cell apoptosis. To verify whether
compound 5n has the similar apoptosis-inducing effect, a cell apoptotic experiment on
compound 5n was carried out. It is clear from the results that compound 5n could induce cell
apoptosis. Experimental results showed that after treating A375 cells with gradient
concentrations (0 μM, 1 μM, 3 μM and 10 μM) of compound 5n for 24 h, the percentage of
apoptotic cells was markedly elevated in a dose-dependent manner (with percentage of
3.547%, 8.27%, 56.80% and 58.50%). The results indicated that after treating A375 cells
with gradient times (0 h, 12 h, 24 h and 36 h) of compound 5n for 3 μM, the percentage of
apoptotic cells was markedly elevated in a dose-dependent manner (with percentage of
0.681%, 2.257%, 57.50% and 73.80%). In short, the results were exhibited in Figure 4A and
4B, proving that compound 5n could induce A375 cell apoptosis.
2.6 Compound 5n induced cell cycle arrest in dose- and time-dependent manner
It was found that compound 5n could induce cell cycle arrest and mitotic blocking of A375
cells by further experiments. A375 cells were treated with compound 5n at various doses (0,
1, 3, or 10 μM) for 24 h, or with compound 5n (3 μM) for various times (0, 12, 24, 36 h). The
results, as shown in Figure 5, demonstrated that compound 5n induced a gradual
accumulation of A375 cells in G1 phase in a dose- and time-dependent manner. As a result,
we could see that compound 5n could induce cell arrest in the G1 period of the cell cycle and
blocking cell mitosis.
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2.7 Western blot revealed 5n could interfere the B-RafV600E-mediated pathways
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 amount of pMEK and pERK. The effect of 5n on MEK
and ERK phosphorylation was investigated in A375 cell line expressing B-RafV600E and
WM1361 cell line expressing B-RafWT. Cells were treated with different concentrations (0
μM, 0.03 μM, 0.1 μM and 0.3 μM) of 5n, and the inhibitory activity was compared with that
of vemurafenib. As shown in Figure 6, 5n markedly suppressed the phosphorylation of MEK
and ERK in a dose-dependent manner in A375 cells, with similar inhibitory effects can be
found in vemurafenib group (Figure 7). Subsequently, the inhibition of 5n to MEK and ERK
phosphorylation were also evaluated against WM1361 cells expressing B-RafWT. While the
paradoxical effect of vemurafenib at different concentrations was reported, no such responses
of 5n were observed in the B-RafWT-bearing WM1361 cells at concentrations up to 0.3 μM,
as shown in Figure 8. Also as a control, no differences were noted for total ERK1/2 or
MEK1/2 protein levels in this experiment.
2.8 Mitochondrial membrane potential analysis
According to the experiment, with the increase of concentration of compound 5n, red
fluorescence gradually weakened, and green fluorescence gradually strengthened. The results
indicated that after treating A375 cells with gradient concentrations (0, 1, 3 or 10 μM) of
compound 5n for 12 h, the percentage of red fluorescence cells was markedly decreased in a
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dose-dependent manner (with percentage of 92.84%, 89.60%, 75.80% and 25.10%).
Meanwhile the percentage of red fluorescence cells was markedly increased in a
dose-dependent manner (with percentage of 7.16%, 10.40%, 24.20% and 74.90%) as shown
in Figure 9. Indicating that the mitochondrial membrane potential from high to low, there is a
downward trend. Decrease of mitochondrial membrane potential is an important phenomenon
in the early stage of apoptosis. It can be seen that compound 5n can lead to changes in
mitochondrial membrane potential of A375 cells, causing early apoptosis of cells.
3 Conclusion
In this study, we established a fragment library by extracting the known endogenous
ligands from the protein-ligand complexes. On the basis of the protein structure analysis, we
used the MCSS simulation to bind the copies of molecular fragment to the B-Raf activity
pocket, and the copies with best conformation and optimal orientation were selected. Finally,
the acquired lead-like compounds were screened for novelty primarily, and then docked
iteratively to estimate the binding potency with the enzyme. Above all, the results obtained
from this study suggest that our target skeleton may serve as a novel scaffold for the further
development of more potent and selective B-RAFV600E inhibitors that use as mutant B-RAF
dependent melanoma therapeutic agents. In short, the study shows that our design method
and design intent are valid, and we can use the MCSS simulation to carry out the FBDD
strategy to design a new inhibitor in other drug design campaigns. It is hoped that this study
may provide reference for the design of novel small molecule inhibitors by using MCSS
simulation to achieve FBDD strategy.
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Disclosure of potential conflicts of interest
The authors disclose that there is no potential conflict of interest.
Acknowledgement
This work was supported by National Science & Technology Supporting Project (No.
2015BAD16B07) and supported by the National Undergraduate Innovation Program and the
National Natural Science Foundation of China (No. J1210026), and supported by the
Research Innovation Program for College Graduates of Jiangsu Province in 2016 (No.
SJZZ16_0014).
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Figure Captions
Figure 1. The hydrogen bonds between compound 5n and key amino acid residues.
Figure 2. (A) The 3D binding mode of compound 5n with B-RafV600E (PDB code: 4E26). Compound
5n is shown in purple; (B) Incorporating an overlay of vemurafinib and compound 5n with
B-RafV600E. Compound 5n is shown in gray and vemurafinib is shown in cyan.
Figure 3. System and ligand root mean square deviation (RMSD) gained from MD simulations.
Table 1. The contribution of different binding energy and the total binding energy calculated by
MM/PBSA.
Figure 4. (A) A375 cells treated with 0, 1, 3 and 10 μM 5n 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) A375 cells treated with 3 μM 5n for 0, 12, 24, 36 h were
collected and analyzed. The percentage of early apoptotic cells was shown in the lower right quadrant
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(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 5. Effect of compound 5n on cell-cycle progression of A375 cells was determined by flow
cytometry analysis. (A) A375 cells treated with 5n (0, 1, 3 and 10 μM) for 24 h; (B) A375 cells
treated with 5n (3 μM) for various times (0, 12, 24, 36 h). (G1 phase, green; S phase, yellow and
G2/M phase, blue).
Figure 6. 5n blocked the ERK pathway in A375 cells. (A) 5n inhibited the phosphorylation of ERK in
A375 melanoma cells bearing B-RafV600E; (B) 5n inhibited the phosphorylation of MEK in A375
melanoma cells bearing B-RafV600E
.
Figure 7. Vemurafenib blocked the ERK pathway in A375 cells. (A) The effect of vemurafenib on
the phosphorylation of ERK in A375 melanoma cells bearing B-RafV600E; (B) The effect of
vemurafenib on the phosphorylation of MEK in A375 melanoma cells bearing B-RafV600E
.
Figure 8. 5n slightly blocked the ERK pathway in WM1361 cells. (A) 5n slightly inhibited the
phosphorylation of MEK in WM1361 melanoma cells bearing B-RafWT. (B) 5n slightly inhibited the
phosphorylation of ERK in WM1361 melanoma cells bearing B-RafWT
.
Figure 9. A375 cells treated with 0, 1, 3 and 10 μM 5n for 12 h were collected and analyzed. (red
fluorescence, high mitochondrial membrane potential; green fluorescence, low mitochondrial
membrane potential).
Supporting Information
Experimental details for the compounds and biological assays.
Table S1. Structures of compounds 5a – 5p.
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Table S2. The molecular properties of target compounds, vemurafenib.
Table S3. Compounds 5a – 5p potently inhibited proliferation of four cancer cells and B-RafV600E
kinase activity.
Table S4. B-RafWT and C-Raf kinase selectivity of compound 5n and vemurafenib.
Figure S1. The revised structure of endogenous ligands extracted from B-Raf co-crystals.
Figure S2. The graph about CDOCKER INTERACTION ENERGY (-kcal/mol) of all compounds for
B-RafV600E
.
Table 1. The contribution of different binding energy and the total binding energy calculated by
MM/PBSA.
Contributing Energy
Van der Waal
Average Value (kJ/mol)
-250.43 + 4.33
-41.00 + 6.95
Electrostatic
Polar Solvation
90.01 + 5.25
Non-polar Solvation
Total Binding Energy
-17.98 + 0.89
-219.40 + 10.41
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Scheme 1. The synthetic route for compounds 5a – 5p. (a) H2SO4, HNO3, 60 °C, 2 h; (b) K2CO3,
DMF, 25 °C, 16 h; or 1.0 equiv, Iodobenzenes, 0.1 equiv, 8-Hydroxyquinoline, 0.1 equiv, CuI, 2.0
equiv, K2CO3, DMSO , 135 °C, 20 h; or Sodium methoxide, DMF, 25 °C, overnight; (c) 80%
Hydrazine hydrate, 10% Palladium charcoal, Ethanol, 80 °C, 10 min; (d) Potassium hydroxide, 65 °C,
2 h; (e) 1.0 equiv, EDC, 1.0 equiv, HOBt, 1.0 equiv, DMAP, Et3N, DMF, rt.
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.