Design, synthesis and biological evaluation of novel benzo-α-pyrone containing piperazine derivatives as potential BRAFV600Einhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.09.003

The increasingly acquired resistance to vemurafenib and side effects of known inhibitors motivate the search for new and more effective anti-melanoma drugs. In this Letter, virtual screening and scaffold growth were combined together to achieve new molecu

Full text of DOI:10.1016/j.bmcl.2016.09.003

Accepted Manuscript
Design, synthesis and biological evaluation of novel Benzo-α-pyrone containing
piperazine derivatives as potential BRAFV600E inhibitors
Long-Wang Chen, Ze-Feng Wang, Bo Zhu, Ruo-Jun Man, Yan-Dong Liu,
Yuan-Heng Zhang, Bao-Zhong Wang, Zhong-Chang Wang, Hai-Liang Zhu
PII:
S0960-894X(16)30933-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.09.003
BMCL 24227
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 June 2016
13 August 2016
2 September 2016
Please cite this article as: 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, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://
dx.doi.org/10.1016/j.bmcl.2016.09.003
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Design, synthesis and biological evaluation of novel
Benzo-α-pyrone containing piperazine derivatives as
potential BRAFV600E inhibitors
Long-Wang Chena†, Ze-Feng Wanga†, Bo Zhub, Ruo-Jun Mana, Yan-Dong Liua,
Yuan-Heng Zhanga, Bao-Zhong Wanga*, Zhong-Chang Wanga*, Hai-Liang
Zhua*
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing 210023, People’s Republic of China
b Jiangsu Chia Tai Tianqing Pharmaceutical Co., Ltd. Nanjing 210000, P. R. China
*Corresponding author. Tel. & fax: +86-25-89682572; e-mail: zhuhl@nju.edu.cn
†These two authors equally contributed to this paper.

Abstract
The increasingly acquired resistance to vemurafenib and side effects of known
inhibitors motivate the search for new and more effective anti-melanoma drugs. In
this report, virtual screening and scaffold growth were combined together to achieve
new molecules as BRAFV600E inhibitors. Along with docking simulation, a primary
screen in vitro was performed to filter the modifications for the lead compound, which
was then substituted, synthesized and evaluated for their inhibitory activity against
BRAFV600E and several melanoma cell lines. Out of the obtained compounds,
derivative 3l was identified as a potent BRAFV600E inhibitor and exerted an anticancer
effect through BRAFV600E inhibition. The following biological evaluation assays
confirmed that 3l could induce cell apoptosis and marked DNA fragmentation.
Furthermore, 3l could arrest the cell cycle at the G0/G1 phase in melanoma cells. The
docking simulation displayed that 3l could tightly bind with the crystal structure of
BRAFV600E at the active site. Overall, the biological profile of 3l suggests that this
compound may be developed as a potential anticancer agent.
Key words:
Melanoma; BRAFV600E; Virtual screening; Benzo-α-pyrone; Docking simulation

The RAS/RAF/MEK/ERK (MAPK) signaling pathway which could be widely
activated in cells1 plays a crucial role in transducing extracellular signals into the
nucleus through receptor tyrosine kinases2 and it has been associated to the regulation
of cell proliferation, differentiation, survival and migration.3,4 It is commonly
accepted that RAF kinases (ARAF, BRAF and CRAF), the key components of the
MAPK signaling pathway, act downstream of RAS and are in charge of MEK and
ERK activation.5 The abnormal activation of RAF, generally BRAF, gives rise to
amplification of the MAPK pathway and is frequently observed in human cancers and
contributes to oncogenesis.6 Pathological studies have reported that activated forms of
the BRAF gene present in approximately 8% of all cancers7 and are most frequently
associated with melanoma.8 In detail, mutated BRAF is featured in 82% of cutaneous
melanocyte nevi,9 66% of primary melanomas10 and 40-68% of metastatic
melanomas.11 What is noteworthy is that more than 90% of the detected BRAF
mutations are V600E mutated, which increases the kinase activity dramatically and
concomitantly activates the signaling at relatively high levels.12 Generally, the success
of novel cancer therapies depends on the identification of functional targets that are
essential of tumor growth and survival.13 In this regard, BRAFV600E has been an
attractive target for therapeutic intervention targeting which has proved to be a major
advancement in the field of melanoma treatment.
To date, numbers of inhibitors against BRAFV600E have been developed and more
are at various stages of preclinical and clinical development.14,15 It is heartening to see
vemurafenib (PLX4032), GSK2118436 and so on, selective inhibitors of BRAFV600E
,

have exhibited remarkable clinical activity in patients with melanoma. Especially,
vemurafenib, a 7-azaindole derivative, has received FDA approval for the treatment of
late stage metastatic melanoma.16 Notwithstanding these achievements, many cases of
acquired resistance to vemurafenib17,18 and high incidences (around 25%) of
keratoacanthoma and squamous cell carcinomas associated with known inhibitors19,20
have been reported in latest studies. In addition, what should be noticed is that the
incidence of melanoma has greatly increased over the past decade.21, 22 Therefore, to
break these limitations of vemurafenib and some other drugs, the development of
novel potent BRAFV600E inhibitors is significantly important.
High-throughput virtual screening, which is now widely used in pharmaceutical
research, is a screening method frequently used by medicinal chemists. The
availability of a high-resolution crystal structure of the protein target as a template for
computational screening promotes the feasibility of virtual screening.12,23 The
availability of the crystal structure of the BRAFV600E protein kinase now provides an
opportunity for utilizing the virtual screening strategy to identify BRAFV600E
inhibitors. To find more potent compounds with novel scaffolds toward BRAFV600E, a
hierarchical virtual screening process was initiated. In this study, about 20,000
theoretically lead-like small molecules were randomly selected from the ZINC
database firstly. The compounds were then docked into the ATP-binding site of the
BRAFV600E kinase (PDB code: 2FB8) using the LibDock module in Discovery Studio
(version 3.5). After virtual screening combined with scaffold growth, the new grown
molecules were ranked according to the binding modes and binding affinity. The best

ones were selected to be synthesized subsequently, whereafter the best entity 3a (Fig.
1) was picked out from the preliminary test and chosen as a lead compound.
Derivatives of this compound (3a) were thereafter synthesized and evaluated by
additional bioassays depicting their pharmacological profile.
Herein, we reported a series of BRAFV600E inhibitors bearing the benzo-α-pyrone
scaffold and emphasized the rationale behind the discovery of inhibitors, the study of
structure-activity relationship, mechanisms of inhibition and the effect of the
inhibitors on the viability of melanoma cells.
Fig. 1. Chemical structures of known BRAFV600E inhibitors and designed structures.
The synthesis of compounds 3a-3n followed the general pathway depicted in
Scheme 1a. All the target compounds were obtained in three steps as described in the
supplementary experimental section, the reactions were monitored by thin layer
chromatography (TLC), and the crude products were purified by column
chromatography. All of the synthetic compounds 3a-3n were reported for the first

1
time (Table 1) and gave satisfactory analytical and spectroscopic data, i.e. H NMR,
EI-MS, which were in accordance with the assigned structures. Furthermore, the
crystal structure of compound 3f (CCDC No. 1471892) was determined by single
crystal X-ray diffraction analysis in Fig. 2, and its crystal data, data collection and
refinement parameters were listed in Table S1.
Scheme 1a.
a: The synthetic routes of compounds 3a-3n. Reagents and conditions: (i) Phosphorus oxychloride,
°
Dimethyl formamide, 65 C, 5 h; (ii) Different substituted piperazine, K2CO3, Dichloromethane,
0 °C, 0.5 h; (iii) Sodium borohydride, anhydrous ethanol, 0 °C, 3 h.
Table 1. Structures of compounds 3a-3n.
Compounds
R
Compounds
R
3a
3h
-CH2CH3
3b
3c
3i
3j

3d
3e
3k
3l
Cl
3f
3m
3n
3g
Fig. 2. Crystal structure diagram of compound 3f.
To evaluate the antiproliferative activities of the prepared compounds, the
BRAFWT melanoma cells (WM1361), BRAFV600E melanoma cells (WM266-4) and
human skin melanoma cells (A375) were used and the results were summarized in
Table 2. Out of all the compounds, compound 3l possesses an excellent
antiproliferative activity specifically for A375 and WM266-4 (BRAFV600E) cell lines,

with IC50 values of 2.24 μM and 1.35 μM, compared to the positive control
vemurafenib which showed better inhibitory potential for A375 and WM266-4 (IC50 =
0.21 μM and IC50 = 0.07 μM), while compound 3l exhibited relatively worse
inhibitory activity against WM1361 (wild type BRAF) cell line (IC50 = 21.08 μM).
Moreover, for most compounds, there was no or negligible effects were observed for
the WM1361 (wild type BRAF) cell line even when treated with doses as high as 50
μM although they could mildly inhibit BRAFV600E cell lines to some extent. These
data demonstrated that the compounds were able to induce cytotoxicity selectively in
the BRAF mutated melanoma cancerous cells.
Table 2 In vitro inhibitory effects of compounds 3 (3a-3n) against human tumor cell lines, normal
cell line.
IC50 ± SD (μM)
WM-266-4a
16.55 ± 0.57
> 50
CC50 ± SD (mM)
293Tb
> 50
Compounds
A375a
WM1361a
> 50
17.25 ± 0.33
> 50
3a
> 100
> 50
3b
32.09 ± 0.28
24.72 ± 0.34
40.45 + 0.53
19.96 ± 0.47
7.23 + 0.21
6.51 ± 0.36
31.94 ± 0.31
8.45 ± 0.25
7.66 ± 0.63
2.24 ± 0.15
3.16 ± 0.32
3.89 ± 0.17
0.21 ± 0.02
37.64 ± 0.19
23.35 ± 0.25
46.33 ± 0.36
18.34 ± 0.45
5.61 ± 0.17
5.33 ± 0.22
30.04 ± 0.43
6.56 ± 0.16
6.04 ± 0.42
1.35 ± 0.17
2.57 ± 0.14
2.74 ± 0.21
0.07 ± 0.01
> 50
> 50
3c
> 50
> 50
3d
> 50
> 50
3e
> 50
> 50
3f
40.36 ± 0.24
38.09 ± 0.38
> 50
> 50
3g
> 50
3h
> 50
3i
45.88 ± 0.44
42.72 ± 0.33
21.08 ± 0.54
29.41 ± 0.26
30.15 ± 0.24
1.86 ± 0.12
> 50
3j
> 50
3k
> 50
3l
> 50
3m
3n
> 50
Vemurafenibc
> 50
a
Inhibition of the growth of tumor cell lines
b Inhibition of the growth of nomal cell line
c Used as positive control
Subsequently, all the synthetic compounds 3a-3n were evaluated for inhibitory

activity against the BRAFV600E enzyme to obtain a further understanding of the
structure-activity relationships (SAR) and the results were presented in Table 3. From
the data listed, it can be found that compound 3b of which piperazine connected to an
alkane substituent showed the worst inhibitory activity (IC50 > 50 μM). In comparison
with 3b, aromatic-substituted compounds were more potent, indicating that the short
chain group was not conducive to BRAFV600E inhibition and the modification on the
aryl rings seems to be beneficial to enzyme inhibition. When it comes to the
phenylpiperazine, we introduced a variety of groups to enrich the diversity.
Halo-substituted compounds 3c-3e and compound 3i with a trifluoromethyl displayed
a dramatic loss in activity compared to unsubstituted analog (3a, IC50 = 8.93 μM),
while compounds 3g and 3h afforded about 5-fold improvement over the compound
3a, which suggested that substituent groups can affect the inhibitory activities and
compounds with electron-donating groups exhibited better inhibitory activities than
those with electron-withdrawing groups.
Additionally, the level of BRAF inhibition was almost not altered when we
changed the position of the fluoro atom from the ortho-position (3c) to the
para-position (3d), and the exchange of the methoxy group from the meta- (3h) to the
para-position (3g) resulted in an equal potency too. All the results demonstrated that
the change of substituent position may not have much effect on the inhibitory activity.
In the case of compounds (3e, 3j and 3k) with chlorine groups on phenyl ring,
compounds 3j and 3k which were introduced two chlorine atoms at 2,3-position or
3,4-positon on phenyl ring, exhibited over 10-fold greater advantages than compound

3e. Besides, compounds (3l-3n) which have a benzhydryl group exhibited a
significant increment in activity comparing to other compounds. Among them,
compound 3l, possessed the most potent BRAFV600E inhibitory activity with IC50 of
0.37 μM. In a word, the new class of compounds furnished several antagonists of
BRAFV600E which have potential development value for further exploration.
Table 3. Inhibition activity of all tested compounds against BRAFV600E
.
IC50 ± SD (mM)
BRAFV600E
8.93 ± 0.21
> 50
IC50 ± SD (mM)
BRAFV600E
24.23 ± 0.21
3.27 ± 0.25
2.56 ± 0.14
0.37 ± 0.12
0.68 ± 0.06
0.92 ± 0.13
0.04 ± 0.02
Compounds
Compounds
3a
3i
3b
3j
22.01 ± 0.14
17.81 ± 0.21
33.49 ± 0.18
10.12 ± 0.16
1.84 ± 0.13
1.76 ± 0.19
3c
3k
3d
3l
3e
3m
3f
3n
Vemurafeniba
3g
3h
a Used as positive control
To test whether the inhibition of cell growth of WM266-4 was related to cell
apoptosis and verify further the selective cytotoxicity between WM266-4 and
WM1361, two kinds of cell lines apoptosis induced by compound 3l was determined
using flow cytometry. WM266-4 cells were treated with varying concentrations (0 μM,
2 μM, 4 μM and 8 μM) of 3l for 24 h. As shown in Fig. 3, the percentage of apoptotic
cells was markedly increased in a dose-dependent manner after treatment with
different doses of 3l. Meanwhile, we treated WM1361 cells with 20 μM, 40 μM and
60 μM of 3l for 24 h. However, the results presented in Fig. 4 showed that cells
remained unaffected even after 24 h treatment at high doses. Combing these results
together we concluded that compound 3l can selectively cause cell apoptosis in

WM266-4 cells while wild type BRAF melanoma cells will be unaffected by this
treatment.
Fig. 3. Annexin V-FITC/PI dual-immuno-fluorescence staining after treatment with different
concentrations of 3l on WM266-4 cells revealed significantly increased number of apoptotic cells.
(A) The apoptosis rate of WM266-4 cells treated with 0, 2, 4 and 8 mM 3l for 24 h. (B) The
percentage of apoptotic cells were calculated after the treatment of 3l. Images are representative of

three independent experiments.
Fig. 4. Annexin V-FITC/PI dual-immuno-fluorescence staining after treatment with different
concentrations of 3l on WM1361 cells revealed significantly increased number of apoptotic cells.
(A) The apoptosis rate of WM1361 cells treated with 0, 20, 40 and 60 mM 3l for 24 h. (B) The
percentage of apoptotic cells were calculated after the treatment of 3l. Images are representative of
three independent experiments.

Generally speaking, when apoptosis occurs, DNA is cleaved into
internucleosomal fragments which is a late marker of apoptosis. To further confirm
cell programed death mediated by 3l in WM266-4, a DNA fragmentation assay was
implemented. WM266-4 cells were cultured in the presence of the various
concentrations of compound 3l (0 μM, 2 μM, 4 μM and 8 μM) for 24 h. As depicted in
Fig. 5, compound 3l could result in marked DNA fragmentation in WM266-4,
whereas the control (untreated with 3l) did not exhibit any DNA ladder.
Fig. 5. The gel electrophoresis image obtained after DNA fragmentation assay for apoptosis
detection. WM266-4 cells were cultured at 37 °C for 24 h in the presence or absence of the various
concentrations of compound 3l. DNA was isolated and visualized on a 2.0% agarose gel. From left
lanes are: Lane M: Marker (2000bp); Lane 1: 8 μM; Lane 2: 4 μM; Lane 3: 2 μM; Lane 4:0 μM
(CON); Lane M: Marker (15000bp).
We next assessed the cell cycle distribution of WM266-4 cells by flow cytometry.
Cells treated with compound 3l of different concentrations (0, 2, 4, and 8 μM) for 24 h
(Fig. 6) and cells treated with 8 μM 3l for different times (0, 12, 24, and 36 h) (Fig. 7)
could both arrest a significant number of cells in G0/G1 phase. In summary, as shown
in Fig. 6, the accumulation of cells in G0/G1 phase increases with increased drug

concentration. Furthermore, Fig. 7 demonstrated that the maximum accumulation of
cells in G0/G1 phase was observed after treatment with 8 μM 3l for 36 h.
Fig. 6. Effect of compound 3l on the cell cycle distribution of WM266-4 cells in a dose-dependent
manner (0, 2, 4 and 8 mM). Images are representative of three independent experiments. (G1 phase,
green; S phase, yellow and G2/M phase, blue).

Fig. 7. Effect of compound 3l on the cell cycle distribution of WM266-4 cells in a time-dependent
manner (0, 12, 24 and 36 h). Images are representative of three independent experiments. (G1
phase, green; S phase, yellow and G2/M phase, blue).
In order to explore potential anti-tumor agents, all the compounds were evaluated
by the MTT assay for their toxicity against 293T which was defined by the median
cytotoxic concentration (CC50) data. As shown in Table 2, most of the analogues
possessed low cytotoxicity.
In this study, the docking simulation was employed repetitiously and the
screened skeleton A (Fig. 1) as springboard was firstly calculated to predict the
binding mode (Fig. S1A). As showed in picture there was still adequate margin of
space to be utilized. From the perspective of strong potency, the inner side groove
should be occupied to offer additional interactions and the binding potency could be
enhanced if the screened skeleton can be optimized by favorable fragments.

Meanwhile, modification of the benzene ring is so limited that we removed the
skeleton artificially to give structure B (Fig. 1). Skeleton B was then used as a
template to grow scaffold to acquire new molecules as BRAFV600E inhibitors. In this
work this process was performed automatically by the Scaffold Grow module in
Discovery Studio 3.5, complemented by the docking screening. After calculating and
scoring, series of new molecules were constructed and ranked and they were
synthesized and initially tested. The lead compound 3a was then validated.
Modification was made to lead compound to provide chemical diversity and each of
the derivatives was evaluated by bioassays.
In addition, for the purpose of giving a better understanding of the excellent
activity observed, we examined the interaction of the most potent inhibitor 3l with the
BRAFV600E. The binding modes of compound 3l and BRAF were depicted in Fig. 8
and in this binding mode, compound 3l binded into BRAFV600E via two hydrogen
bonds, one π-sulfur interaction and one π-π stacked interaction. The oxygen atom of
hydroxyl group connected with benzo-α-pyrone skeleton formed two hydrogen bonds
with Asn581 and Asp594 residues respectively. The π-sulfur interaction was formed
between a benzene ring of benzhydryl group and Cys532. Moreover, the benzene ring
moiety of benzhydryl group is intercalated into the space to form a π-π stacked
interaction with residue Phe583. The receptor surface model shown in Fig. 8B
revealed that 3l with benzhydryl has a good shape complementarity with the
ATP-binding pocket of BRAF and the above-mentioned three kinds of forces may
contribute to afford an explanation for its nice activity. Besides, the higher binding

affinity of 3l might also be accounted for by some weak interactions, such as π-alykl
interaction, Van der Waals forces and others. All in all, this molecular docking result,
along with the enzyme assay data, suggests that the modification suited our intention
and 3l was a potential inhibitor of BRAFV600E
.
Fig. 8. Binding mode of compound 3l with BRAFV600E (PDB code: 2FB8). (A) 2D diagram of the
interaction between compound 4d and amino acid residues of the nearby active site. (B) 3D
diagram of compound 3l inserted in the BRAFV600E binding site: for clarity, only interacting
residues are displayed.
The BRAF mutant, BRAFV600E, an important and interesting therapeutic target in

human melanoma and other kinds of cancer, has attracted increasing attentions of the
researchers and vemurafenib was developed under this situation. However, recent
clinical studies have reported the acquired resistance to vemurafenib and side effects
of some other drugs in many cases. Therefore, to further explore the anti-proliferative
potential of BRAFV600E inhibitors, we utilized virtual screening combined with
scaffold growth to design a sequence of novel harmfulless benzo-α-pyrone-piperazine
derivatives and decent activities which were tested in a series of experiments, and the
results indicated that some compounds may be promising in drug development.
Among them, 3l was impressive and it showed better selective cytotoxicity towards
three cell lines (A375, WM266-4 and WM1361) in MTT assay in which 3l displayed
better anti-proliferative activity against WM266-4 and A375 cell lines than WM1361.
The BRAFV600E inhibitory assay further revealed that 3l possessed superior inhibitory
potency with an IC50 of 0.37 μM. Apoptosis results confirmed that compound 3l can
selectively cause cell apoptosis in WM266-4 cells while wild type BRAF cells will be
unaffected by this treatment. The following DNA fragmentation assay showed 3l
could be a potential pro-apoptotic agent. Additionally, compound 3l could induce cell
cycle arrest in G0/G1 phase in a dose- and time-dependent manner. Molecular
docking studies afforded a good understanding and explanation of 3l impressive
performance. In summary, we hope these findings will benefit the study of cancer
treatment through BRAFV600E inhibition.
Acknowledgements
The work was financed by the Projects (Nos. CXY1409 & CG1305) from the

Science & Technology Bureau of Lianyungang City of Jiangsu Province.
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Figure Captions
Table 1. Structures of compounds 3a-3n.
Table 2 In vitro inhibitory effects of compounds 3 (3a-3n) against human tumor cell lines, normal
cell line.
Table 3. Inhibition activity of all tested compounds against BRAFV600E
.
Fig. 1. Chemical structures of known BRAFV600E inhibitors and designed structures.
Fig. 2. Crystal structure diagram of compound 3f.
Fig. 3. Annexin V-FITC/PI dual-immuno-fluorescence staining after treatment with different
concentrations of 3l on WM266-4 cells revealed significantly increased number of apoptotic cells.
(A) The apoptosis rate of WM266-4 cells treated with 0, 2, 4 and 8 mM 3l for 24 h. (B) The
percentage of apoptotic cells were calculated after the treatment of 3l. Images are representative of
three independent experiments.
Fig. 4. Annexin V-FITC/PI dual-immuno-fluorescence staining after treatment with different
concentrations of 3l on WM1361 cells revealed significantly increased number of apoptotic cells.
(A) The apoptosis rate of WM1361 cells treated with 0, 20, 40 and 60 mM 3l for 24 h. (B) The
percentage of apoptotic cells were calculated after the treatment of 3l. Images are representative of
three independent experiments.
Fig. 5. The gel electrophoresis image obtained after DNA fragmentation assay for apoptosis
detection. WM266-4 cells were cultured at 37 °C for 24 h in the presence or absence of the various
concentrations of compound 3l. DNA was isolated and visualized on a 2.0% agarose gel. From left
lanes are: Lane M: Marker (2000bp); Lane 1: 8 μM; Lane 2: 4 μM; Lane 3: 2 μM; Lane 4:0 μM
(CON); Lane M: Marker (15000bp).
Fig. 6. Effect of compound 3l on the cell cycle distribution of WM266-4 cells in a dose-dependent
manner (0, 2, 4 and 8 mM). Images are representative of three independent experiments. (G1 phase,
green; S phase, yellow and G2/M phase, blue).
Fig. 7. Effect of compound 3l on the cell cycle distribution of WM266-4 cells in a time-dependent
manner (0, 12, 24 and 36 h). Images are representative of three independent experiments. (G1
phase, green; S phase, yellow and G2/M phase, blue).
Fig. 8. Binding mode of compound 3l with BRAFV600E (PDB code: 2FB8). (A) 2D diagram of the
interaction between compound 4d and amino acid residues of the nearby active site. (B) 3D
diagram of compound 3l inserted in the BRAFV600E binding site: for clarity, only interacting
residues are displayed.
Scheme 1a.

*Graphical Abstract (for review)
Design, synthesis and biological evaluation of novel
Benzo-α-pyrone containing piperazine derivatives as
potential BRAFV600E inhibitors
Long-Wang Chena†, Ze-Feng Wanga†, Bo Zhub, Ruo-Jun Mana, Yan-Dong Liua,
a
Yuan-Heng Zhanga, Bao-Zhong Wang *, Zhong-Chang Wanga*, Hai-Liang
Zhua*
a State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing 210023, People’s Republic of China
b Jiangsu Chia Tai Tianqing Pharmaceutical Co., Ltd. Nanjing 210000, P. R. China
*Corresponding author. Tel. & fax: +86-25-89682572;
E-mail: zhuhl@nju.edu.cn
†These two authors equally contributed to this paper.

Virtual screening and scaffold growth were combined together to achieve a
series of novel compounds (3a-3n) bearing the benzo-α-pyrone scaffold. Among them,
compound 3l showed most powerful antiproliferative activity (IC50 = 2.24 μM for
A375 and IC50 = 1.35 μM for WM266-4) and enzyme inhibition activity (IC50 = 0.37
μM). 3l could effectively cause melanoma cells apoptosis in a dose-dependent manner,
significantly induce DNA fragmentation and arrest cell cycle at the G0/G1 phase.
Docking simulation was performed to explore the binding model of compound 3l with
BRAFV600E
.