Development of a novel class of B-RafV600E-selective inhibitors through virtual screening and hierarchical hit optimization

Article abstract of DOI:10.1039/c2ob26081f

Oncogenic mutations in critical nodes of cellular signaling pathways have been associated with tumorigenesis and progression. The B-Raf protein kinase, a key hub in the canonical MAPK signaling cascade, is mutated in a broad range of human cancers and especially in malignant melanoma. The most prevalent B-Raf^V600E mutant exhibits elevated kinase activity and results in constitutive activation of the MAPK pathway, thus making it a promising drug target for cancer therapy. Herein, we describe the development of novel B-Raf^V600E selective inhibitors via multi-step virtual screening and hierarchical hit optimization. Nine hit compounds with low micromolar IC ₅₀ values were identified as B-Raf^V600E inhibitors through virtual screening. Subsequent scaffold-based analogue searching and medicinal chemistry efforts significantly improved both the inhibitor potency and oncogene selectivity. In particular, compounds 22f and 22q possess nanomolar IC ₅₀ values with selectivity for B-Raf^V600Ein vitro and exclusive cytotoxicity against B-Raf^V600E harboring cancer cells.

Full text of DOI:10.1039/c2ob26081f

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
Organic &
Biomolecular
Chemistry
Cite this: Org. Biomol. Chem., 2012, 10, 7402
www.rsc.org/obc
PAPER
Development of a novel class of B-RafV600E-selective inhibitors through
virtual screening and hierarchical hit optimization†
Xiangqian Kong,‡a Jie Qin,‡b Zeng Li,‡a Adina Vultur,b Linjiang Tong,a Enguang Feng,a Geena Rajan,b
Shien Liu,a Junyan Lu,a Zhongjie Liang,a Mingyue Zheng,a Weiliang Zhu,a Hualiang Jiang,a
Meenhard Herlyn,b Hong Liu,*a Ronen Marmorstein*b and Cheng Luo*a,c
Received 5th June 2012, Accepted 10th July 2012
DOI: 10.1039/c2ob26081f
Oncogenic mutations in critical nodes of cellular signaling pathways have been associated with
tumorigenesis and progression. The B-Raf protein kinase, a key hub in the canonical MAPK signaling
cascade, is mutated in a broad range of human cancers and especially in malignant melanoma. The most
prevalent B-RafV600E mutant exhibits elevated kinase activity and results in constitutive activation of the
MAPK pathway, thus making it a promising drug target for cancer therapy. Herein, we describe the
development of novel B-RafV600E selective inhibitors via multi-step virtual screening and hierarchical hit
optimization. Nine hit compounds with low micromolar IC50 values were identified as B-RafV600E
inhibitors through virtual screening. Subsequent scaffold-based analogue searching and medicinal
chemistry efforts significantly improved both the inhibitor potency and oncogene selectivity. In particular,
compounds 22f and 22q possess nanomolar IC50 values with selectivity for B-RafV600E in vitro and
exclusive cytotoxicity against B-RafV600E harboring cancer cells.
MEK/ERK signaling cascade has become one of the primary
Introduction
candidates for novel molecular targeted cancer therapies.15–17
The evolutionarily conserved Ras/Raf/MEK/ERK mitogen-
activated protein kinase (MAPK) signaling pathway, relaying the
proliferative signals generated by cell surface receptors and
cytoplasmic signaling components into the nucleus, is a critical
regulator of many cellular processes involved in cell prolifer-
ation, differentiation, and survival.1–3 Aberrant activation of the
MAPK pathway represents a common indicator for a wide
variety of proliferative diseases and especially cancer.4,5 More-
over, accumulating data demonstrates that high-frequency acti-
vating mutation6–8 or amplification9–11 at several levels in this
pathway greatly prompts multiple tumor oncogenic processes,
such as cell immortalization, angiogenesis, invasive growth,
metastases and drug resistance.12–14 Therefore, the Ras/Raf/
The Raf serine/threonine kinase family was initially identified
as a cellular homolog of the retroviral oncogene v-Raf and
consists of three isoforms named Raf-1 (C-Raf), A-Raf and
B-Raf.18–20 Distinct from other Raf paralogs, B-Raf requires
fewer post-translational modifications for optimal activation and
possesses substantially greater basal activity.21,22 In addition,
B-Raf has also been shown to be the major Raf effector in the
MAPK signaling cascade.23–26 All of these unique properties in
cellular physiology provide a molecular shortcut for B-Raf acti-
vation and greatly substantiate the potential of B-Raf to be a
bona fide oncogene.27 The importance of B-Raf in oncogenesis
is highlighted by the finding that it is mutated in approximately
7% of human cancers, with the highest incidence in malignant
melanoma (60%–70%), thyroid (30%–50%), ovarian (∼30%)
and colorectal (5%–20%) carcinomas, and with a relatively
lower frequency (1%–3%) in a wide variety of other cancers.7,28
Among more than 75 identified B-Raf mutations in human
cancers, a Glu for Val substitution at residue 600 in the kinase
domain accounts for over 90% of B-Raf mutations, which
harbors 500-fold higher intrinsic kinase activity.29 The
B-RafV600E could effectively circumvent the requirement of
phosphorylation-dependent regulation by Ras, and result in con-
stitutive activation of the MAPK signaling pathway, even in the
absence of extracellular mitogenic stimuli, facilitating the malig-
nant transformation.30,31 The presence of B-RafV600E in certain
aState Key Laboratory of Drug Research, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences, Shanghai 201203, China.
E-mail: cluo@mail.shcnc.ac.cn, hliu@mail.shcnc.ac.cn;
Fax: +86-21-50807088; Tel: +86-21-50271399
bThe Wistar Institute, Philadelphia, PA 19104, USA.
E-mail: marmor@wistar.org; Fax: +0-215-898-0381;
Tel: +0-215-898-5006
cCenter for Systems Biology, Soochow University, Jiangsu 215006,
China
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c2ob26081f
‡These authors contributed equally to this work.
7402 | Org. Biomol. Chem., 2012, 10, 7402–7417
This journal is © The Royal Society of Chemistry 2012

View Article Online
cancer cohorts also correlates with the increased risk of deterio-
ration and poor prognosis.32,33 Taken together, these findings
strongly suggested that B-Raf is a promising therapeutic target in
melanoma and other carcinomas that depend upon B-Raf
signaling.
Given the significance of B-Raf in tumorigenesis and pro-
gression, the inhibitors targeting B-Raf and especially the onco-
genic forms of B-Raf, have increasingly come into the limelight
of the drug discovery arena. To date, a number of oncogenic
B-Raf inhibitors have been reported in various developmental
stages.34–39 Some of them have entered clinical trials in recent
years and exhibit encouraging clinical efficiency.40–42 Among
them, Vemurafenib, which was approved by the FDA and EMA
(European Medicines Agency) for the treatment of unresectable
Fig. 1 Schematic representation of the virtual screening strategy
adopted in the present study.
or metastatic melanoma, is characterized as
a selective
B-RafV600E inhibitor.43 It demonstrated complete or partial
tumor regression in the majority of patients harboring the
B-RafV600E mutation and with an overall increased median sur-
vival time after the treatment, which validates the effectiveness
of targeting the oncogenic B-Raf in cancer therapy.40,44
However, the emerging data also indicates that some mutant
B-Raf tumors have intrinsic resistances to B-Raf inhibitors
accessed clinically to date, and the acquired resistance occurred
through a variety of mechanisms.45–47 Furthermore, a series of
side effects have also been observed, in particular the induction
of keratoacanthoma was frequently observed in Vemurafenib
treated patients.44 Therefore, there is clearly an unmet therapeutic
need to develop novel, potent and specific B-Raf inhibitors
possessing different properties to benefits the patients with
B-RafV600E-driven cancers.
Structure based virtual screening (SBVS) has become a
powerful tool in the medicinal chemists’ toolkit, and the incor-
poration of the characteristic information from the known active
ligands into the virtual screening campaign could further
improve the hit enrichment as well as the potential for scaffold
hopping.48,49 In the present study, a combined ligand- and struc-
ture-based virtual screening protocol was employed to discover
novel B-RafV600E inhibitors. Nine structurally novel hit com-
pounds with high ligand efficiency were successfully identified.
Subsequent scaffold-based analogue searching and structure
optimization significantly increased the inhibitory potency and
B-RafV600E selectivity in both enzymatic and cellular assays. In
particular, compound 22q possesses excellent in vitro and
ex vivo B-RafV600E inhibitory potency and exhibits cellular
selectivity towards B-RafV600E harboring cancer cell lines.
volume of the ATP binding pockets are altered upon different
ligand binding, two crystal structures of B-Raf in the active con-
formation (PDB entry: 3og7,40 2fb835) varied in binding pocket
architecture were used for conducting the structure-based virtual
screening with the GLIDE program,53 which achieved the best
performance in our pilot study (see ESI† for detailed results).
A schematic representation of the overall virtual screening pro-
cedure in this study is presented in Fig. 1. Taking the respective
bioactive conformations of Vemurafenib (Template 1) and
SB-590885 (Template 2) in complex with B-Raf as queries,35,40
SHAFTS was utilized to search the SPECS vendor databases
which contains more than 200 000 compounds. The top
1000 molecules with similarity scores >1.0 were reserved for
each template, which resulted in 581 hits for template 1 and
1000 hits for template 2. These compounds were then docked
into their corresponding structures with GLIDE using the stan-
dard precision (SP) mode, and the top 500 compounds for each
structure were submitted for further docking validation using the
GLIDE extra precision (XP) mode and visual inspection. The
candidate molecules were selected based on the following cri-
teria: (1) The molecular weight of the ligand was lower than
500 Da, rendering it suitable for further optimization. (2) Both
geometric and chemical features matching existed between the
ligand and the ATP binding site of B-Raf. (3) The binding poses
and chemical structures were reasonable. Some highly ranked
molecules that displayed either high strain energy or excessive
rotatable bonds were rejected from further evaluation. (4) At
least one H-bond was formed by the ligand with the backbone
atoms of the hinge region residues (such as Q530 and C532),
which are critical for anchoring the inhibitors in the active site.54
(5) The hydrophobic pocket surrounding the gatekeeper residue
(back pocket) was occupied by the corresponding lipophilic
groups in the ligand, which plays important roles in determining
both the kinase selectivity and inhibitor binding affinity.55,56(6)
The ligands were prioritized for selection if at least one H-bond
interaction was formed with the “DFG-motif” of B-Raf, based
on the presumption that it may stabilize the active conformation
and improve oncogenic B-Raf selectivity.38,40 Consequently, 327
compounds that met the above criteria were selected for further
scaffold diversity analysis with the Cluster Molecules component
in Pipeline Pilot 7.5. One or two candidate molecules with rela-
tively simple chemical structure and higher GLIDE score
(G-score) within each structural cluster were retained. Finally,
Results and discussion
Virtual screening
To strengthen the efficiency of scaffold hopping and hit discov-
ery rate, we designed an integrated ligand- and structure-based
virtual screening procedure aimed to counterbalance their funda-
mental limitations.49 The SHAFTS algorithm, which is a hybrid
3D molecular similarity calculation approach designed to
combine the strength of pharmacophore matching and volumetric
overlay, exhibiting satisfactory “scaffold hopping” capability
against several representative kinases,50–52 was utilized for
ligand-based virtual screening (LBVS). Since the shape and
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 7402–7417 | 7403

View Article Online
Table 1 Inhibition rates (%) or IC50 values against B-RafV600E of the
hits derived from virtual screening
fact that the nitro group generally functioned as a relatively weak
H-bond acceptor in molecular interactions and has a relatively
small VDW volume,58 we then assumed that a novel hinge
region binder with more favorable H-bond and VDW interacting
capability may improve both the inhibitory potency and onco-
genic B-Raf selectivity. Therefore, a substructure-based analogue
searching with 2-phenyl-5-vinylfuran (highlighted in red in
Fig. 2) as a query template was performed to hop the potential
hinge region binders, and a focused library comprising of all of
the hit compounds was extracted from the SPECS database.
Then the focused library was docked into the active site of
B-Raf with GLIDE program and ranked by G-score. After visual
inspection, nineteen compounds with the same scaffold but with
diverse substitutions on the exocyclic methylene and phenyl
were purchased from the vendor database and their inhibitory
activities against both B-RafWT and B-RafV600E were assessed.
As shown in Table 2, 12 out of 19 compounds exhibited more
than 50% inhibition of B-RafV600E activity at 5 μM. Apparently,
independent of the distinct substitutes on the exocyclic methyl-
ene, smaller hydrophobic substitutes (e.g. halogen in compound
17–19, 24) could be well tolerated in the para position of the
phenyl group, and kinase inhibitory activity was completely
abolished in the case of ortho- and para-double substitution (e.g.
15, 20, 21). However, the broad activity spectrum for both the
ortho- and meta-substituted compounds indicated that the
potency and selectivity against B-RafV600E were remarkably
affected by the different furan-2-methylene substitutes (e.g. com-
pound 11 and 26, or compound 12, 23 and 27), which was con-
sistent with our assumption and underlined the importance of
selecting proper hinge binders for further optimization. It is also
notable that most of the inhibitors showed varying degrees of
selectivity against B-RafV600E over B-RafWT, especially for com-
pounds 11, 19 and 22, which showed about 2–3 fold selectivity.
In summary, although no significant improvement in inhibitory
potency was observed for these analogues over the original hit,
they did provide novel chemotypes with enhanced oncogene
selectivity for further evaluation. In view of the biological
potency, oncogene selectivity and chemical feasibility,
compounds 16, 17, 19 and 22, which contained the 2,3-dihydro-
1H-inden-1-one, indolin-2-one, thiazolidine-2,4-dione and
2-thiobarbituric acid moieties respectively, were selected for hit
optimization and structure–activity relationship (SAR) study.
Compound
IC50 (μM) or inhibition ratea (%)
1
2
3
4
5
6
7
8
9
3.189 (3.238)b
79%
52%
60%
3.984 (3.699)b
53%
86%
50%
67%
a Inhibition rate at 5 μM of compound. b The IC50 values for both
B-RafV600E and wild-type B-Raf (in the parentheses) were determined
for the 2 most potent compounds against B-RafV600E
.
113 compounds were designated for purchase for B-Raf in-
hibition activity assessment.
In vitro inhibition assay against B-RafWT and B-RafV600E
kinases
The 113 candidate molecules selected by virtual screening were
tested for B-RafWT and B-RafV600E inhibition as previously
described.36,39 Nine compounds with diverse chemotypes
demonstrated significant inhibition for B-RafV600E activity at
5 μM in the preliminary test, and the IC50 values towards both
B-RafWT and B-RafV600E were then determined for the two most
potent hit molecules (Table 1). Among the two hits possessing
low-micromolar IC50 values against both wild-type and
B-RafV600E, compound 1 was characterized as a structurally
novel B-Raf inhibitor with higher ligand efficiency (0.42 kcal mol−1
per heavy atom versus 0.24 kcal mol−1 per heavy atom of
hit 5)57 in inhibiting B-RafWT and B-RafV600E activity. More-
over, compound 1 also exhibited encouraging anti-proliferation
activity. It could efficiently inhibit the proliferation of both A375
(B-RafV600E mutant) metastatic melanoma and HCT-116 (Ras
mutant) colon carcinoma cells at 10 μM in which the hyperacti-
vated MAPK pathway was the main molecular driver for tumori-
genesis (Table S4†), implicating that the cytotoxicity of hit 1
may be attributed to its in vivo MAPK pathway inhibition. It is
also noteworthy that the B-RafV600E harboring A375 melanoma
cells were more sensitive to hit 1 than HCT-116 cancer cells that
contain B-RafWT. Taken together, these data suggested that hit 1
represented a novel B-RafV600E selective chemotype with high
ligand efficiency and promising anti-proliferation activity, offer-
ing a good starting point for further optimization.
Hit optimization and SAR analysis
To confirm the effectiveness of the hits identified through the
scaffold searching procedure and explore their preliminary struc-
ture–activity relationship (SAR), we initiated the hit optimization
campaign. According to isosteric replacement theory and syn-
thetic feasibility, the bivalent sulfur atom in the 2,3-dihydro-1H-
inden-1-one moiety of hit 16 was replaced by a methylene
group, for which the activity was confirmed by subsequent
experimental evaluation (16e). In total, 35 compounds were
designed and synthesized, and their chemical structures are
shown in Tables 3–5.
Scaffold-based analogue searching
Upon close examination of the binding mode of compound 1
(Fig. S2†), we noted that the nitro group attached to the exocyc-
lic methylene could form a hydrogen bond with the amide nitro-
gen of Cys532 in the hinge area, and the 5-phenylfuran moiety
exhibited significant shape complementation with the ATP-
binding site to make extensive hydrophobic interactions with the
back pocket around the gatekeeper residue. On account of the
The general synthetic route of the designed compounds
16a–g, 17a–f, 19a–k and 22a–q is depicted in Scheme 1.
5-Formyl-2-furanboronic acid was treated with various
7404 | Org. Biomol. Chem., 2012, 10, 7402–7417
This journal is © The Royal Society of Chemistry 2012

View Article Online
Fig. 2 Chemical structures of the query templates employed in SHAFTS searching and hit compounds identified from virtual screening. The struc-
tural moiety of hit 1 used in the substructure search is highlighted in red.
substituted bromobenzenes, Pd(PPh3)4 and K2CO3 to yield com-
pounds 30. Then the final products (16a–g, 17a–f, 19a–k and
22a–q) were obtained using the Knoevenagel condensation
between compound 30 and various substituted 1-indanone, 2,3-
dihydroindol-2-one, 2,4-thiazolidinedione and 2-thiobarbituric
acid, respectively. The crude products were purified by washing
with MeOH or recrystallizing from EtOH to yield pure products.
The in vitro kinase inhibition assay clearly indicated that dis-
tinct substitutes on the exocyclic methylene had a significant
effect on both the inhibitory potency and B-RafV600E selectivity.
Of the synthetic derivatives tested, 13 compounds (e.g. 16a, 16c,
16e; 22a–c, 22e–k) exhibited more than 50% inhibition against
B-RafV600E at 2 μM, wherein most of the compounds were close
analogues of hit 22, which contained a 2-thiobarbituric acid
moiety as the potential hinge binder (Tables 3–5). However, on
the whole, the majority of the derivatives of hit 16, 17 and 19
displayed relatively weak inhibition in comparison with that of
hit 22, which was more visible when the same substituents were
present on the benzene ring (e.g. 22a vs. 16a and 19a, 22f
vs. 16f and 19, 22k vs. 17d and 19k). Moreover, while the
three analogues of hit 16 (16a, 16c, 16e) exhibited encouraging
inhibitory activity at 2 μM, their activities declined at lower
concentration. In contrast, significant inhibitory potency could
still be observed for five derivatives of hit 22 at the same
concentration (22f–h, 22j, 22k) and was exemplified by the
2–5 fold improvement in their IC50 values compared with
the original hit (Table 5). Collectively, the data from the prelimi-
nary optimization highlighted the optimal performance of the
2-thiobarbituric acid analogues with respect to B-RafV600E
inhibition.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 7402–7417 | 7405

View Article Online
Table 2 Structure and potency of the compounds identified from the substructure-searching
IC50a (μM) or inhibition
IC50a (μM) or inhibition
rateb (%)
rateb (%)
ID
Structure
B-Raf
3.24c
B-RafV600E
3.19c
ID
Structure
B-Raf
4.99
B-RafV600E
2.26
1
19
10
11
12
1.38
4.04
1.96
2.78
20
21
22
n.a.
n.a.
6.60
n.a.
n.a.
2.09
43%
17%
28%
18%
13
14
20%
n.a.
56%
27%
23
24
62%
36%
75%
42%
50%
21%
40%
30%
46%
n.a.
53%
30%
15
16
n.a.
n.a.
25
26
43%
24%
72%
58%
62%
50%
78%
61%
n.a.
n.a.
17
18
2.98
3.85
27
28
30%
14%
68%
49%
40%
14%
67%
38%
32%
17%
47%
40%
a IC50 values were determined if the inhibition rate at 2 μM was larger than 65%. b For each compound, the values listed in the upper and lower panels
of the cell refer to the inhibition rate (%) at 5 μM and 2 μM, respectively. c Values listed for comparison. n.a. = not active.
7406 | Org. Biomol. Chem., 2012, 10, 7402–7417
This journal is © The Royal Society of Chemistry 2012

View Article Online
Table 3 Chemical structures and activities for the analogues of
compound 16
Table 4 Chemical structures and activities for the analogues of
compound 17
Substituent
R
Inhibition ratea (%)
Substituent
R
Inhibition ratea (%)
Compound
17a
R1
H
R2
H
B-Raf
B-RafV600E
Compound
16a
R1
H
B-Raf
B-RafV600E
3-CF3
n.a.
n.d.
n.a.
n.d.
17
n.d.
n.a.
n.d.
n.a.
n.d.
n.a.
n.d.
34
n.d.
38
n.d.
23
n.d.
23
n.d.
34
n.d.
34
2-NO2
3-CF3
3-CF3
4-NO2
4-Cl
11
64
47
22
n.d.
59
42
34
n.d.
52
41
n.a.
n.a.
n.d.
n.a.
n.a.
n.a.
n.d.
n.a.
n.a.
n.a.
n.d.
n.a.
n.d.
17b
3-CF3
H
Cl
H
16b
H
17c
3-CF3
Cl
H
16c
Cl
H
17d
2-Cl,5-CF3
2-Cl, 5-CF3
2-Cl, 5-CF3
H
16d
17e
H
Cl
H
16e
H
17f
Cl
16f
4-Br
H
42
n.d.
44
n.d.
a For each compound, the values listed in the upper and lower panels of
the cell refer to the inhibition rate (%) at 2 μM and 0.5 μM, respectively.
n.a. = not active. n.d. = not determined.
16g
2-Cl, 4-CF3
H
n.d.
a For each compound, the values listed in the upper and lower panels of
the cell refer to the inhibition rate (%) at 2 μM and 0.5 μM, respectively.
n.a. = not active. n.d. = not determined.
hand, most of the efforts were concentrated on exploring the
synergistic effects of the substitutions on the benzene ring. As
presented in Table 6, the double substitution at ortho- and para-
positions, as well as the meta- and para-positions did not show
the expected “additive” effect, albeit the corresponding mono-
substituent could be well accommodated on the benzene ring
(e.g. 22n vs. 22a and 22e, 22p vs. 22l and 22e). Considering the
observation that the presence of a H-bond acceptor at the meta-
position of the benzene improved the B-RafV600E selectivity, for
example with compound 22, and that the configurational
freedom of the methoxyl group may hinder the optimal H-bond
formation, we speculated that the introduction of a conformation-
ally constrained H-bond partner with enhanced H-bond forming
potential, as well as the relatively hydrophobic nature at both
meta- and para-positions may potentiate the B-RafV600E selec-
tivity. Encouragingly, compound 22q, with the 3,4-methylene-
dioxy substituent on the benzene ring, exhibited the expected
improvement in both potency and selectivity. It possessed nano-
molar inhibitory activity against B-RafV600E and about 4-fold
selectivity over B-RafWT, which validated the effectiveness of
our speculations and underlined the indispensable contributions
of a suitable H-bond acceptor substituent at the meta-position for
B-RafV600E selectivity.
Analyzing the data in Table 5, we found that either small or
bulky hydrophobic substituents, such as methyl, methoxyl,
halogens and tertiary butyl retained or improved the inhibition
activity to varying degrees (e.g. 22e–i), with the inhibition rate
ranging from 30–60% at 0.5 μM, and it was more evident for
compound 22f which produced a potency of nearly 5-fold over
compound 22. However, the activities substantially decreased
when introducing the more polar groups (22c and 22d). Accord-
ingly, the activity is highly sensitive to the physicochemical
properties rather than the steric size of the substituents at the
para position, indicating the existence of a relatively bulky
hydrophobic cavity. Similarly, a hydrophobic substituent was
desirable at the meta-position when comparing the activity
differences between 22b and 22. Moreover, the presence of a
cyano group at the ortho position of the benzene ring showed
reduced activity relative to the nitro substitution, which may be
attributed to the insufficient complementarity with the ATP
binding site due to its linear configuration. Nevertheless, the
B-RafV600E selectivity for these analogues did not show conco-
mitant improvement as their biological potency. For example,
compound 22f possessed nanomolar activity but exhibited rare
discrimination between B-RafV600E and B-RafWT. Consequently,
we undertook a second round of hit optimization, mainly focus-
ing on increasing B-RafV600E selectivity, which is deemed to be
critical for maximum therapeutic benefit owing to low cyto-
toxicity toward normal cells. Meanwhile, with the SAR data in
To evaluate the effects of B-Raf kinase inhibition on cellular
characteristics, the anti-proliferation activity of selected B-Raf
inhibitors was investigated. As shown in Table 7, compound
22e, 22f, 22g, 22h, 22i and 22q demonstrated potent
growth inhibition activity against the B-RafV600E harboring
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 7402–7417 | 7407

View Article Online
Table 5 Chemical structures and activities for the analogues of compound 19 and 21
IC50a or inhibition rateb (%)
IC50a or Inhibition rateb (%)
Cmpd.
19a
Substituent
2-NO2
—
B-Raf
B-RafV600E
Cmpd.
22a
Substituent
2-NO2
B-Raf
B-RafV600E
n.a.
n.d.
—
7%
n.d.
—
38%
n.a.
36%
13%
6%
61%
48%
42%
19%
63%
34%
2.09c
59%
n.a.
—
23
2-CN
19b
3-OCH3
n.a.
n.d.
—
34%
n.d.
—
48%
n.d.
n.a.
22b
3-OCH3
n.a.
—
19c
—
4-NO2
22
22c
3-NO2
4-NO2
6.60c
55%
n.a.
n.a.
n.d.
n.a.
n.d.
n.a.
n.d.
4.99c
n.a.
n.d.
3%
n.d.
17%
n.d.
n.a.
n.d.
18%
n.d.
19d
19e
4-CO2Et
4-Cl
22d
22e
4-CO2Et
4-Cl
n.a.
38%
n.d.
n.d.
n.a.
n.d.
15%
n.a.
0.60
17%
n.a.
69%
33%
0.47
59%
50%
2.69
n.d.
2.26c
41%
n.d.
41%
n.d.
34%
n.d.
51%
n.d.
37%
n.d.
19
19g
4-Br
4-Me
22f
22g
4-Br
4-Me
19h
19i
4-OCH3
4-tBu
22h
22i
4-OCH3
4-tBu
3.69
29%
n.a.
1.93
57%
36%
1.07
19j
19k
Naphthyl
2-Cl,5-CF3
22j
22k
Naphthyl
2-Cl,5-CF3
1.49
1.09
a IC50 values were determined if the inhibition rate at 0.5 μM was larger than 50%. b For each compound, the values listed in the upper and lower
panels of the cell refer to the inhibition ratio (%) at 2 μM and 0.5 μM, respectively. c IC50 values were listed for comparison. n.a. = not active. n.d. =
not determined.
Scheme 1 Reagents and conditions: (a) Pd(PPh3)4, K2CO3, PhMe, 100 °C, 4 h; (b) for the compound 16 series: substituted 1-indanone, NaOH,
EtOH, room temperature, 3–4 h; for the compound 17 series: substituted 2,3-dihydroindol-2-one, NaOH, EtOH, room temperature, 3–4 h; for the com-
pound 19 series: 2,4-thiazolidinedione, β-alanine, acetic acid, 90 °C, 2 h; for the compound 22 series: 2-thiobarbituric acid, acetic acid, 90 °C, 2 h.
A375 malignant melanoma cells at 10 μM. Notably, virtually
complete growth inhibition for A375 cell lines was achieved by
compound 22g, 22h and 22i. On the contrary, anti-proliferation
activity toward B-RafWT containing SW620 human colonic
carcinoma cells was observed to modest levels for most of the
tested inhibitors, indicating cellular selectivity for B-RafV600E
over B-RafWT by these compounds. Moreover, the negligible
anti-growth activity that was observed for the selected analogues
of compound 16 and 19 was consistent with their relatively weak
B-RafV600E inhibitory activity in vitro (Table 2), suggesting that
the selective targeting of B-RafV600E was responsible for their
cytotoxicity of the analogs of compound 22.
To further ascertain this selectivity profile, the anti-
proliferation effects of compound 22f and 22q possessing the
most potent B-RafV600E inhibition potency and selectivity at the
enzymatic level were explored in detail against four other
human-derived cells. The dose-dependent inhibition profile
exemplified in Fig. 3 clearly indicates that B-RafV600E bearing
7408 | Org. Biomol. Chem., 2012, 10, 7402–7417
This journal is © The Royal Society of Chemistry 2012

View Article Online
Table 6 Chemical structures and activities for the analogues of 22 in
the second round optimization
IC50a or inhibition rate
([I] = 1 μM)
Compound
Substituent
B-Raf
B-RafV600E
22l
3-CF3
4-COOH
2-NO2, 4-Cl
2-CF3, 4-Cl
3-CF3, 4-Cl
3,4-Methylenedioxy
30%
n.a.
20%
24%
37%
1.15
45%
n.a.
22%
49%
48%
0.30
22m
22n
22o
22p
22q
Fig. 3 Effects of the compounds 22f (A) and 22q (B) on the prolifer-
ation of human-derived cells. Cellular proliferation of melanoma cell
lines (WM3918, 1205Lu, WM983B), and human fibroblasts (FF2508)
were assessed with the MTS assay. Results are shown as the mean value
of four data sets, including standard errors, and are representative of
replicated independent experiments. Proliferation is shown as the percent
change compared to the vehicle control values.
a IC50 values were determined if the inhibition rate at 1 μM was larger
than 70%. n.a. = not active.
Table 7 In vitro anti-proliferation activity of the selected B-RafV600E
inhibitor at 10 μM
Tumor growth inhibition rate ([I] = 10 μM)
of the compounds can be well related to their capacity to selec-
tively inhibit oncogenic B-RafV600E over B-RafWT. Intriguingly,
all of the compounds possessing potent and selective anti-pro-
liferation activity contained the hydrophobic substituent at the
para-position on the benzene ring, whereas the corresponding
polar substitution essentially abolished compound potency.
Since the ability of the compounds to penetrate the cell mem-
brane is a prerequisite to cytotoxicity, the para-position of the
benzene ring may represent a functional site to orchestrate the
permeability and cellular activity for 2-thiobarbituric acid
B-RafV600E inhibitors. Taken together, the ex vivo anti-
proliferation activity data presented here indicates that the bio-
chemical selectivity of the inhibitors (e.g. 22q) against
B-RafV600E translates to the cellular selectivity, reminiscent of
the Vemurafenib and SB-590885 compounds that were utilized
as the query template during our ligand-based virtual screening
and specially targeted the active conformation of B-Raf.
Compound
A375(B-RafV600E
)
SW620 (B-Raf)
22a
22b
22c
22e
22f
22g
22h
22i
22j
22k
22q
19
n.a.
26.2
46.3
82.9
65.3
84.0
94.4
95.4
6.9
18.3
77.7
n.a.
16.5
3.2
n.a.
n.a.
n.a.
9.0
n.a.
3.6
13.0
7.6
n.a.
n.a.
47.7
n.a.
n.a.
n.a.
n.a.
16a
16c
16e
5.2
n.a. = not active.
Kinase inhibition profile
tumor cell lines (1205Lu and WM983B) were significantly
inhibited following incubation with compounds 22f or 22q,
whereas normal human primary fibroblasts (FF2508) and malig-
nant cells not expressing mutant B-Raf (WM3918) were signifi-
cantly more resistant to compound treatment. Notably, the
inhibition of tumor growth at 10 μM was more pronounced upon
treatment with compound 22q than compound 22f (Table 6 and
Fig. 3). These results in cells were consistent with the biochemi-
cal assays demonstrating that compound 22q was more potent
and selective than compound 22f against B-RafV600E (Tables 5
and 6). This correlation between the enzymatic and cellular
inhibitory potency again suggested that the anti-growth activity
To investigate the specificity and selectivity of the identified
inhibitors against B-Raf over other kinases, we performed a
selectivity profile against a panel of 16 protein kinases at both
5 μM and 1 μM for the two most potent inhibitors 22f and 22q.
As shown in Table 8, compound 22f showed only modest levels
of inhibition for most of the tested tyrosine- and serine/
threonine-kinases except for FGFR1 and IKKβ kinase, which
was significantly inhibited at 5 μM. It is noteworthy that the
most potent and selective inhibitor 22q, which was derived
from the second round optimization campaign, demonstrated
an improved selectivity profile. 22q significantly inhibited
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 7402–7417 | 7409

View Article Online
B-RafV600E at both concentrations but showed only modest
levels of inhibition for these other 16 distinct kinases. Taken
together, these data demonstrate that compound 22q is a
B-RafV600E inhibitor with more than 10-fold selectivity over
other tested protein kinases. Collectively, the promising results
from this small kinase profiling suggests that compounds 22f
and 22q, and 22q in particular, may possess specific inhibitory
activity for B-RafV600E. In addition, these studies also suggest
that synergy between in silico screening and hierarchical hit
optimization can lead to novel B-Raf inhibitors with significant
kinase selectivity.
Binding mode analysis
To understand the molecular basis for B-RafV600E inhibition by
22q and to explore the potential structural origin for the
oncogene selectivity, the molecular docking strategy was utilized
to scrutinize the binding poses of 22q. Fig. 4 illustrates the pre-
dicted binding poses of 22q in the ATP-binding site of
B-RafV600E (PDB entry: 3og740). In this model, the 2-thiobarb-
ituric acid moiety is tightly confined within the adenine-binding
site of the ATP binding pocket, intercalating between residue
W531 and F583 by forming π–π stacking interactions. The
amide portion of the 2-thiobarbituric acid group makes bidentate
hydrogen bonds with the NH group and the carbonyl oxygen of
C532 in the hinge region, forming a “handshake” interaction,
which effectively anchors the compound into the ATP binding
pocket. The 2-phenyl-5-vinylfuran portion of 22q extends
towards the activation loop (A-loop) and makes extensive hydro-
phobic interactions with the surrounding residues. In particular,
the 3,4-methylenedioxy substituted benzene ring is predicted to
occupy the hydrophobic back cavity created by the relatively
small gatekeeper residue, mainly comprised of A481, K483,
L505, L514, I527, T539 and F595. Gaining access to the kinase
back pocket results from the small gatekeeper residue may have
the dual effect of not only increasing the biological potency but
Table 8 Kinase inhibition profile of compound 22f and 22q against a
panel of 16 protein kinases
Inhibition rate (%) for compounds at 5 μM and 1 μM
Kinase
22f (5 μM)
22f (1 μM)
22q (5 μM)
22q (1 μM)
SRC
LCK
KDR
ABL
28.3
10.5
6.1
n.a.
7.7
39.7
30.7
15.6
52.1
26.4
20.4
46.1
72.2
20.3
42.2
40.3
8.6
10.5
3.9
n.a.
n.a.
11.6
14.5
9.5
27.8
16.2
7.2
12.6
15.0
10.8
34.2
18.6
15.2
0.8
1.2
n.a.
n.a.
12.6
13.7
6.8
35.7
5.2
6.2
n.a.
n.a.
n.a.
n.a.
3.3
10.0
5.6
15.0
n.a.
6.0
n.a.
4.9
4.6
18.6
18.3
P38α
CDK2
ERK1
IGF1R
IKKβ
PKCα
PDGFRα
EGFR
FGFR1
AKT1
MET
7.7
19.4
18.6
14.5
19.4
20.0
cKIT
n.a. = not active.
Fig. 4 Binding mode analysis of compound 22q with B-Raf kinase. (A) B-RafV600E kinase in the active conformation is shown in cartoon diagram.
The N-lobe and C-lobe of B-Raf kinase are shown in pink and blue, respectively. Compound 22q occupies the ATP binding site between the two
lobes represented as yellow sticks. (B) A close-up view of the key interactions between compound 22q and surrounding residues. Hydrogen bonds are
depicted as dotted red lines. (C) Structural comparison of compound 22q with Vemurafenib (in cyan sticks) in the ATP pocket of B-RafV600E. The
Raf-selective pocket is represented in surface contours.
7410 | Org. Biomol. Chem., 2012, 10, 7402–7417
This journal is © The Royal Society of Chemistry 2012

View Article Online
also improving the selectivity over other kinases that have
which potentially stabilizes the DFG-in conformation and the
active conformation of B-Raf.
a
larger gatekeeper residue and hence a smaller back
pocket,56,59,60 which may account for the selectivity profile of
22q. Notably, the 3,4-methylenedioxy substituent on the
benzene ring mediates a hydrogen bond interaction with the
backbone NH of D594 in the conserved DFG triad.
Conclusions and future prospects
The SAR data derived from the two-round structural optimiz-
ation could be rationalized by the predicted binding mode. The
ortho-nitro group on the benzene ring may form a weak H-bond
interaction with the sidechain of the gatekeeper T529, thus the
lower activity that is observed for the linear cyano substitution
may partly be attributed to the non-optimal steric complementar-
ity for H-bond formation. Considering the hydrophobic nature of
the back cavity, a lipophilic substitution may be preferred over a
polar substitution, which was validated by the aforementioned
SAR data. In particular, hydrophobic substituents such as
methyl, methoxyl and halogens on the meta- or para-position of
the benzene ring (e.g. 22f, 22h) that would be predicted to better
complement the back pocket, achieving elevated shape and
chemical feature complementarity with the ATP binding site,
greatly contributed to the improved biological potency of the
corresponding compounds. Consistent with this notion, the more
polar substitutions on the meta- or para-position of the benzene
ring, which would be predicted to be disfavorable due to a high
desolvation penalty, showed markedly less potency. For
example, nitro, carbethoxy and carboxyl substitutions in these
positions showed relatively poor potency (22c, 22d and 22m).
Furthermore, the relatively snug but length-limited back pocket
could well accommodate rigid and bulky groups (22i and 22j),
however, the more extended substitutions such as carbethoxy
group would be predicted to cause some steric clashes when pre-
sented at the para position of the benzene ring, likely contrib-
uting to the weak inhibitory potency of 22d. It is noteworthy that
the presence of a conformationally constrained H-bond acceptor
in the meta-position of the benzene ring significantly improved
B-RafV600E selectivity (22, 22q). Based on the predicted binding
mode, the H-bond acceptor at the meta-position of the benzene
ring could form a hydrogen bond interaction with the mainchain
NH group of D594, reminiscent of a similar interaction observed
by Vemurafenib when in complex with B-RafV600E. The sulf-
amide moiety in Vemurafenib was deprotonated to facilitate the
same hydrogen bond interaction with D564 which was deemed
to stabilize the DFG-in conformation and thus contributed to the
B-RafV600E selectivity.38,40 Moreover, on account of the fact that
the conformational equilibrium of B-Raf was strongly biased
toward the active state characterized by the DFG-in conformation
by the phosphorylation mimic V600E mutation, inhibitors
specially targeting the active conformation of B-Raf likely
possess higher selectivity against the oncogenic mutant owing to
the relatively small configurational entropy cost upon
binding.38–40,43,54,61 In addition, the structural comparison of the
active and inactive B-Raf conformation highlights the central
role of the mainchain conformation of D594 in directing the con-
formational switching of the A-loop between the corresponding
DFG-in and DFG-out states. Therefore, resembling the important
role of the sulfamide in Vemurafenib, the improved selectivity of
compound 22 and 22q for B-RafV600E over B-RafWT could be
rationalized by the H-bond interaction between the meta-substi-
tuted hydrogen bond acceptor and the mainchain NH of D594,
Targeted intervention of the B-RafV600E gene product represents
a promising approach for cancer therapy. In the present study, we
identified a novel class of 2-thiobarbituric acid analogues as
selective B-RafV600E inhibitors by using an integrated ligand-
and structure-based virtual screening strategy in conjunction
with hierarchical hit optimization. First, the combined ligand-
and structural-based virtual screening was utilized for the discov-
ery of structurally novel B-RafV600E inhibitors. Nine hit
compounds with low micromolar activity against B-RafV600E were
successfully identified. Among them, hit 1 was characterized
with high ligand efficiency and encouraging anti-proliferation
activity, providing an attractive starting point for a hit-to-lead
optimization campaign. The molecular modeling study of hit 1
into the ATP-binding site of B-Raf indicated that the biological
potency as well as oncogene mutant selectivity could be further
optimized by varying the potential hinge region binder and sub-
stituent on the benzene ring. The follow-up scaffold-based
searching from hit 1 led to a series of B-RafV600E inhibitors
bearing a wide variety of potency and B-RafV600E oncogene
selectivity: four of the analogues of compound 1 displayed
potent activities and synthetic feasibility (16, 17, 19 and 22), so
were then selected for hit optimization. Subsequently, the medic-
inal chemistry effort and SAR analysis was performed to explore
the activity and specificity profile of the derivatives for these
selected inhibitors, which led to the discovery of 2-thiobarbituric
acid analogues with improved inhibitory potency and
B-RafV600E selectivity. The most potent inhibitor, compound
22q, inhibited the kinase activity of B-RafV600E with an IC50
value in the nanomolar range and with 4-fold selectivity for
B-RafV600E over B-RafWT and selective cytotoxicity against
B-RafV600E harboring cancer cell lines. In addition, a small
kinase profiling study was consistent with the conclusion that the
cytotoxicity of the inhibitor was mainly related to the suppres-
sion of B-RafV600E activity.
In addition to providing a novel chemotype with significant
biological potency and oncogene selectivity for B-Raf inhibition,
the SAR studies and B-RafV600E selectivity analysis for the
2-thiobarbituric acid analogues offers valuable pharmacophore
insights for future optimizations aimed at increasing the inhibi-
tory potency as well as B-RafV600E mutant selectivity. The struc-
tural comparison of 22q with Vemurafenib in complex with
B-RafV600E reveals that the most significant difference lies in the
propyl group in Vemurafenib, which penetrates into the Raf-
selective pocket and was proposed to be the critical binding
determinant for Raf kinase specificity.38,40 In the case of com-
pound 22q, the substitution on the methylene presented in the
methylenedioxy group with a short alkyl chain was predicted to
occupy this Raf-specific pocket, which potentially augmented
the potency and specificity of the inhibitor towards the onco-
genic B-RafV600E. In addition, the structural comparison also
reveals that the lipophilic substituent at the ortho-position of the
benzene ring may further potentiate the hydrophobic interactions
with the pocket around the gatekeeper residue. However, it is
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 7402–7417 | 7411

View Article Online
noteworthy that the ortho-substitution may affect the relative
orientation between the benzene ring and the central furan
group, which in turn may impede the pivotal hydrogen bond
interaction with the beginning of the DFG region. We therefore
hypothesize that a relatively small substitution, such as fluorine,
chlorine or methyl group may be more desirable at the ortho-
position. In addition, the NH group in the 2-thiobarbituric acid
moiety that does not participate in bidentate hydrogen bonds
with the hinge area would be predicted to point towards the
solvent. Based on this, we propose that the derivatization at this
site such as introducing the solubilizing morpholine fragment
may contribute to enhanced solubility and cellular activity.
The study presented here details the optimization process from
a fragment-like hit compound derived from virtual screening to a
more drug like candidate. This underscores the importance of
selecting a proper starting point with low molecular weight and
high ligand efficiency to facilitate the subsequent hit-to-lead
optimization process. Thus, our study may provide a few
useful clues regarding the rational selection of hits for further
evaluation. Moreover, the encouraging findings obtained in our
study will lay the foundation for future development of more
potent and selective oncogenic B-Raf inhibitors for cancer
therapy.
rough positioning and scoring phase that dramatically
narrows down the search space is used to locate promising
ligand poses. The conformational energy for a few hundred sur-
viving candidate poses is then torsionally flexible minimized at
the receptor environment with the OPLS-AA force field.67
Monte Carlo simulation is finally conducted between the three to
six lowest energy poses of the previous step to explore nearby
torsional minima, which is critical for obtaining an accurate
docked pose.
GOLD 5.0. GOLD performs automated molecular docking
which employs the hydrogen bond-encoded genetic algorithm
(GA) to direct the identification of proper binding modes.62–64
A maximum of 10 000 genetic operations were performed on a
set of five islands for each of the 20 independent GA runs. The
default weights for three genetic operators (crossover, mutation
and migration) and a population size of 100 chromosomes were
applied in each docking simulation. The kinase scoring func-
tion68 implemented in GOLD were utilized to rank the docked
compounds, respectively. Early termination was allowed when
the RMSD values for the top three scoring poses was less than
1.5 Å.
AutoDock 4.2. AutoDock Tools (ADT) was used to prepare
the grid parameter files (gpf) and the docking parameter files
(dpf) for each of the docked compound. A grid spacing of
0.375 Å was applied for the energetic map calculations, which
centered on the co-crystal ligands and with the grid number of
60, 60, 60 in the x, y, and z directions. The Lamarckian genetic
algorithm (LGA) was employed to explore the optimal chemical
space in the ATP binding site for each ligand.65 The docked
compounds were subjected to 10 GA runs with a maximal
energy evaluation number of 250 000 and a maximum of
27 000 generations. Default values were used for the other para-
meters and an RMSD tolerance of 2.0 Å was defined during the
cluster analysis.
Experimental
Computational procedure. Protein preparation
Two representative B-Raf/ligand complexes (PDB entry: 3og7,40
2fb835) in the active conformation with apparently distinct shape
and volume in the ATP binding site were used during virtual
screening. The protein structures were prepared using the Protein
Preparation Wizard Workflow provided in the Maestro graphical
user interface of the Schrödinger program suite and the default
settings were used. Residues around the co-crystal ligands at a
radius of 10 Å were defined as the binding region in which the
docking grids were created. This radius was large enough to
include all of the residues that may be involved in inhibitor
binding. The crystallographic coordinates for each ligand
(Vemurafenib and SB-590885) in the complex structure were
extracted and designated as query templates in the ligand-based
virtual screening.
Virtual screening
The SHAFTS program was employed to perform the 3D mole-
cular similarity based virtual screening. It adopts a hybrid simi-
larity metric of molecular shape and pharmacophore features for
3D similarity calculation and ranking, aiming to combine the
strength of both pharmacophore matching and volumetric
overlay.50 SHAFTS had demonstrated satisfactory active com-
pounds enrichment and scaffold hopping capability against
several representative kinases in retrospective virtual screening
studies as well as recent prospective applications.51,52 Taking the
crystallographic conformation of Vemurafenib and SB-590885 in
B-Raf/ligand complex as query templates, the conformational
ensembles for each compound in the SPECS database were
searched by SHAFTS, and only the best ranked conformer was
reserved per molecule. The top 1000 molecules with similarity
score >1.0 were reserved for each template. We employed the
GLIDE program as the secondary filter to prioritize the com-
pounds for experimental assessment. All of the candidate com-
pounds derived from SHAFTS screening were docked into the
ATP binding site of B-Raf without any constraint. The hit com-
pounds were ranked based on G-score and the top ranking
Validation of the docking protocol
To evaluate the performance of various available docking
methods (GLIDE 5.5,53 GOLD 5.0,62–64 AutoDock 4.265,66
)
against B-Raf, two conventional criteria were employed during
the pilot study. One of the measurements is the root mean-square
deviation (RMSD) value between the best ranked pose predicted
by various docking programs and the corresponding experi-
mental pose. The other refers to the enrichment factors, which is
defined as the fraction of active compounds found in a certain
percentage of ranking list divided by the fraction of the screened
library.
GLIDE 5.5. GLIDE (grid-based ligand docking with ener-
getics) approximates a complete systematic search of chemical
space available to the docked ligand.53 In this search, an initial
7412 | Org. Biomol. Chem., 2012, 10, 7402–7417
This journal is © The Royal Society of Chemistry 2012

View Article Online
Chemistry: General methods
compounds were submitted for further docking validation with
the extra precision (XP) mode to eliminate false positives as
accomplished by more extensive sampling and advanced
scoring, resulting in even higher enrichment. Finally, after visual
inspection and chemical diversity analysis, the candidate
compounds were purchased and subject to the in vitro kinase
inhibition assay.
The reagents (chemicals) were purchased from Alfa Aesar,
Sigma, Acros and Shanghai Chemical Reagent Company, and
used without further purification. Column chromatography was
performed with CombiFlash® Companion system (Teledyne
Isco, Inc.). Nuclear magnetic resonance (NMR) spectra were per-
formed on a Brucker AMX-400 and AMX-300 NMR (IS as
TMS). Chemical shifts were reported in parts per million (ppm,
δ) downfield from tetramethylsilane. Proton coupling patterns
were described as singlet (s), doublet (d), triplet (t), quartet (q),
multiplet (m), and broad (br). Low- and high-resolution mass
spectra (LRMS and HRMS) were given with electric ionization
(EI) produced by a Finnigan MAT-95. Melting points (Mp) are
uncorrected and were measured in open capillary tubes, using a
SGW X-4 melting point apparatus (−50 to 400 °C). An Agilent
1100 series HPLC with an Agilent Eclipse XDB-C18
(4.6 × 150 mm, 5 μm) reversed phase column was used for the
Cloning, protein expression and purification
The human B-Raf kinase domain and the V600E containing
mutant (B-RafWT and B-RafV600E, residues 433–726) with an
N-terminal 6X-His tag (MDRGSH6GS) to facilitate protein
purification (in the presence of full-length mouse p50cdc37 to
facilitate the proper folding of the B-Raf kinase) was expressed
in Sf9 cells and purified to homogeneity as previously
described.39 The protein was stored at 1.5 mg mL−1 at 4 °C until
use. GST-MEK-His protein substrate for the B-Raf inhibitor
screen was over expressed at 37 °C in Escherichia coli BL21
(Gold) cells. The protein was stored at 10 mg mL−1 at −80 °C
until use.
HPLC analyses. The elution buffer was
a mixture of
H2O–MeCN = 40 : 60 (v/v) with 0.1% CF3COOH (v/v) as an
additive. The retention time of each synthesized compound is
given and the corresponding purity for each compound is proved
1
to be >95% by both HPLC test and H-NMR Spectral Scan (see
in the ESI†). The purity of compounds from the SPECS database
is more than 90% and for most compounds greater than 95%
(confirmed by SPECS, using NMR or LC−MS; data available
through the web site).
In vitro B-Raf kinase assay
For IC50 calculations of inhibitors, an ELISA-based assay was
employed as previously described39 at different inhibitor concen-
trations to generate a sigmoidal dose response curve using
B-RafV600E or B-RafWT protein. All dose response measure-
ments were carried out in duplicate or triplicate and IC50 values
were derived from fitting the data to a sigmoidal dose response
curve with a four-parameter logistic model using GraphPad
Prism.
General procedure for the synthesis of compounds
(a) General procedure for the synthesis of compound 30 via
the Suzuki reaction. To a solution of 5-formyl-2-furanboronic
acid (5.0 mmol) in a mixture (7/3) of dry toluene and ethanol
(10 mL), various substituted bromobenzene (1.0 equiv),
Pd(PPh3)4 (0.1 equiv) and K2CO3 (2.0 equiv) was added. The
mixture was stirred and heated at 100 °C for 4 h under argon pro-
tection before it was cooled to room temperature. The organic
solvents were removed under vacuum, and the residue was
extracted with CH2Cl2 (3 × 50 mL) and washed with brine. The
organic phase was finally dried over Na2SO4 and evaporated to
dryness to give a crude compound that was purified through
flash chromatography (ethyl acetate/petroleum ether) to yield
compound 30.
Melanoma proliferation assay
Human melanoma cell lines (A375, WM983B, 1205Lu,
WM3918) were isolated as previously described69 and cultured
in Dulbecco’s modified Eagle’s medium supplemented with 5%
fetal bovine serum. Normal human primary fibroblasts (FF2508)
were isolated from the human epidermis of neonatal foreskins
and cultured as described.70,71 Cells (5000/well) were seeded in
96-well plates and allowed to adhere overnight before treatment
with each compound or the DMSO vehicle control for 72 h.
Cells were then directly incubated with MTS substrate
(CellTiter-96 Aqueous One Solution Cell Proliferation Assay,
Promega). Absorbance was measured at 490 nm as per the
supplier’s instructions and percent proliferation was normalized
to the absorbance of vehicle-treated cells. For each experiment,
cell line, and treatment, the absorbance values of at least 4 wells
were used for data analysis and the experiment was conducted in
triplicate.
(b) General procedure for the synthesis of target
compound. (i) 16a–g and 17a–f: a mixture of compound 30
(1.0 mmol), substituted 1-indanone or 2,3-dihydroindol-2-one
(2.0 equiv) and NaOH (3.0 equiv) in MeOH was stirred and
heated at room temperature for 3–4 h. The precipitate was
obtained by filtration. The solid was re-crystallized with EtOH to
yield pure product.
(ii) 19a–k72: a mixture of compound 30 (1.0 mmol), 2,4-
thiazolidinedione (2.0 equiv) and β-alanine (2.0 equiv) in acetic
acid was stirred and heated at 90 °C for 2 h before it was cooled
to room temperature. The precipitate was obtained by filtration.
The solid was washed with MeOH to yield pure product without
further purification.
Kinase profiling
Kinase profiling was performed at Shanghai ChemPartner Co.,
Ltd. according to the manufacturer’s protocols. ATP concen-
trations in the kinase assay were at the Km for each enzyme.
(iii) 22a–q73: a mixture of compound 30 (1.0 mmol) and
2-thiobarbituric acid (1.0 equiv) in acetic acid was stirred and
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 7402–7417 | 7413

View Article Online
3-[5-(3-Trifluoromethyl-phenyl)-furan-2-ylmethylene]-6-chloro-
1,3-dihydro-indol-2-one (17b). 1H NMR (400 MHz, DMSO)
δ 8.32 (d, J = 8.3 Hz, 1H), 8.13 (s, 2H), 7.76 (d, J = 5.8 Hz,
2H), 7.49 (d, J = 3.7 Hz, 1H), 7.39 (d, J = 3.7 Hz, 1H), 7.30 (s,
1H), 6.96 (dd, J = 8.3, 1.9 Hz, 1H), 6.87 (d, J = 1.9 Hz, 1H).
Mp 226–228 °C. LRMS (EI) m/z 389, M+. HRMS (EI) m/z
calcd C20H11ClF3NO2 M+ 389.0430, found 389.0427.
heated at 90 °C for 2 h before it was cooled to room temperature.
The precipitate was obtained by filtration. The solid was washed
with MeOH to yield pure product without further purification.
2-[5-(2-Nitro-phenyl)-furan-2-ylmethylene]-indan-1-one (16a).
1H NMR (400 MHz, DMSO) δ 8.00 (dd, J = 10.8, 8.3 Hz, 2H),
7.83–7.75 (m, 2H), 7.72 (d, J = 7.4 Hz, 1H), 7.70–7.60 (m, 2H),
7.50 (d, J = 7.4 Hz, 1H), 7.36 (s, 1H), 7.26 (q, J = 3.7 Hz, 2H),
3.95 (s, 2H). Mp 147–148 °C. LRMS (EI) m/z 331, M+. HRMS
(EI) m/z calcd C20H13NO4 M+ 331.0845, found 331.0844.
3-[5-(3-Trifluoromethyl-phenyl)-furan-2-ylmethylene]-5-chloro-
1,3-dihydro-indol-2-one (17c). 1H NMR (400 MHz, DMSO)
δ 8.45 (dd, J = 7.7, 3.9 Hz, 1H), 8.25–8.11 (m, 2H), 7.78 (d, J =
4.1 Hz, 2H), 7.53 (dd, J = 6.3, 3.8 Hz, 1H), 7.42 (dd, J = 6.2,
2.7 Hz, 1H), 7.39–7.27 (m, 2H), 7.02 (td, J = 7.6, 3.1 Hz, 1H),
6.91 (d, J = 7.7 Hz, 1H). Mp 246–248 °C. LRMS (EI) m/z 389,
M+. HRMS (EI) m/z calcd C20H11ClF3NO2 M+ 389.0430, found
389.0432.
2-[5-(3-Trifluoromethyl-phenyl)-furan-2-ylmethylene]-indan-1-
one (16b). 1H NMR (400 MHz, DMSO) δ 8.19–8.17 (m, 2H),
7.77–7.71 (m, 5H), 7.50–7.48 (d, J = 3.7 Hz, 2H), 7.41–7.40 (s,
1H), 7.25–7.24 (d, J = 3.7 Hz, 1H), 4.17 (s, 2H). Mp
171–173 °C. LRMS (EI) m/z 354, M+. HRMS (EI) m/z calcd
C21H13F3O2 M+ 354.0868, found 354.0867.
3-[5-(2-Chloro-5-trifluoromethyl)-furan-2-ylmethylene]-1,3-
dihydro-indol-2-one (17d). 1H NMR (400 MHz, DMSO)
δ 10.64 (s, 1H), 8.37 (dd, J = 8.8, 4.4 Hz, 2H), 7.91 (d, J =
8.4 Hz, 1H), 7.85–7.77 (m, 1H), 7.56 (d, J = 3.8 Hz, 1H), 7.47
(d, J = 3.8 Hz, 1H), 7.38 (s, 1H), 7.29 (d, J = 1.0 Hz, 1H), 6.90
(d, J = 7.8 Hz, 2H), 3.33 (s, 2H). Mp 207–208 °C. LRMS (EI)
m/z 389, M+. HRMS (EI) m/z calcd C20H11ClF3NO2 M+
389.0430, found 389.0430.
2-[5-(3-Trifluoromethyl-phenyl)-furan-2-ylmethylene]-5-chloro-
indan-1-one (16c). 1H NMR (400 MHz, DMSO) δ 8.28 (s, 1H),
7.89 (d, J = 8.4 Hz, 1H), 7.83–7.76 (m, 2H), 7.71 (s, 1H),
7.59–7.47 (m, 3H), 7.31 (d, J = 3.8 Hz, 1H), 4.17 (s, 2H). Mp
220–221 °C. LRMS (EI) m/z 388, M+. HRMS (EI) m/z calcd
C21H12ClF3O2 M+ 388.0478, found 388.0475.
2-[5-(4-Nitro-phenyl)-furan-2-ylmethylene]-indan-1-one (16d).
1H NMR (400 MHz, DMSO) δ 8.36 (d, J = 8.5 Hz, 2H), 8.14
(d, J = 8.6 Hz, 2H), 7.81–7.70 (m, 3H), 7.54 (d, J = 32.1 Hz,
2H), 7.43 (s, 1H), 7.31 (s, 1H), 4.22 (s, 2H). Mp 224–225 °C.
LRMS (EI) m/z 331, M+. HRMS (EI) m/z calcd C20H13NO4 M+
331.0845, found 331.0837.
3-[5-(2-Chloro-5-trifluomethyl)-furan-2-ylmethylene]-5-chloro-
1,3-dihydro-indol-2-one (17e). 1H NMR (400 MHz, DMSO)
δ 8.31 (d, J = 2.1 Hz, 1H), 8.26 (d, J = 1.7 Hz, 1H), 7.93 (dd,
J = 12.2, 3.5 Hz, 1H), 7.85 (dd, J = 8.4, 2.2 Hz, 1H), 7.51 (dd,
J = 11.4, 3.8 Hz, 2H), 7.43 (s, 1H), 7.30 (d, J = 2.1 Hz, 1H),
6.88 (d, J = 8.3 Hz, 1H). Mp 244–245 °C. LRMS (EI) m/z 423,
M+. HRMS (EI) m/z calcd C20H10Cl2F3NO2 M+ 423.0041,
found 423.0042.
2-[5-(4-Chloro-phenyl)-furan-2-ylmethylene]-indan-1-one (16e).
1H NMR (400 MHz, DMSO) δ 7.89 (d, J = 8.6 Hz, 2H),
7.81–7.67 (m, 3H), 7.57 (d, J = 8.6 Hz, 2H), 7.53–7.46 (m, 1H),
7.38 (s, 1H), 7.29 (d, J = 3.6 Hz, 1H), 7.21 (d, J = 3.6 Hz, 1H),
4.15 (s, 2H). Mp 184–185 °C. LRMS (EI) m/z 320, M+. HRMS
(EI) m/z calcd C20H13ClO2 M+ 320.0604, found 320.0610.
3-[5-(2-Chloro-5-trifluoromethyl)-furan-2-ylmethylene]-6-chloro-
1,3-dihydro-indol-2-one (17f). 1H NMR (400 MHz, DMSO)
δ 10.80 (s, 1H), 8.32 (dd, J = 7.6, 5.1 Hz, 2H), 7.90 (t, J =
8.3 Hz, 1H), 7.85 (d, J = 2.2 Hz, 1H), 7.55 (d, J = 3.7 Hz, 1H),
7.50 (d, J = 3.8 Hz, 1H), 7.41 (s, 1H), 6.91 (d, J = 1.9 Hz, 1H),
6.87 (dd, J = 8.2, 1.9 Hz, 1H). Mp 286–287 °C. LRMS (EI) m/z
423, M+. HRMS (EI) m/z calcd C20H10Cl2F3NO2 M+ 423.0041,
found 423.0042.
2-[5-(4-Bromo-phenyl)-furan-2-ylmethylene]-indan-1-one (16f).
1H NMR (400 MHz, DMSO) δ 7.82 (d, J = 8.6 Hz, 2H), 7.76
(d, J = 7.6 Hz, 1H), 7.74–7.70 (m, 3H), 7.69 (s, 1H), 7.52–7.44
(m, 1H), 7.38 (s, 1H), 7.30 (d, J = 3.6 Hz, 1H), 7.21 (d, J =
3.6 Hz, 1H), 4.14 (s, 2H). Mp 190–192 °C. LRMS (EI) m/z 364,
M+. HRMS (EI) m/z calcd C20H13BrO2 M+ 364.0099, found
320.0124.
5-[5-(2-Nitro-phenyl)-furan-2-ylmethylene]-thiazolidine-2,4-
dione (19a). 1H NMR (400 MHz, DMSO) δ 12.51 (s, 1H),
8.00–7.98 (d, J = 8.0 Hz, 1H), 7.93–7.91 (d, J = 8.0 Hz, 1H),
7.81–7.79 (t, J = 8.0 Hz, 1H), 7.69–7.65 (t, J = 8.0 Hz, 1H),
7.61 (s, 1H), 7.25–7.24 (d, J = 4.0 Hz, 1H), 7.20–7.19 (d, J =
4.0 Hz, 1H). Mp 250–252 °C. LRMS (EI) m/z 316, M+. HRMS
(EI) m/z C14H8N2O5S M+, calcd 316.0154, found 316.0153.
2-[5-(2-Chloro-5-trifluoromethyl)-furan-2-ylmethylene]-indan-
1-one (16g). 1H NMR (400 MHz, DMSO) δ 8.28 (s, 1H), 7.87
(d, J = 8.4 Hz, 1H), 7.78 (d, J = 7.7 Hz, 2H), 7.72 (t, J = 7.4 Hz,
1H), 7.62 (d, J = 7.6 Hz, 1H), 7.50 (t, J = 5.3 Hz, 2H), 7.44 (s,
1H), 7.28 (d, J = 3.6 Hz, 1H), 4.14 (s, 2H). Mp 183–185 °C.
LRMS (EI) m/z 388, M+. HRMS (EI) m/z calcd C21H12ClF3O2
M+ 388.0478, found 388.0482.
5-[5-(3-Methoxy-phenyl)-furan-2-ylmethylene]-thiazolidine-2,4-
dione (19b). 1H NMR (400 MHz, DMSO) δ 12.50 (s, 1H), 7.65
(s, 1H), 7.47–7.43 (t, J = 7.9 Hz, 1H), 7.42–7.39 (d, J = 7.9 Hz,
1H), 7.35 (s, 1H), 7.33–7.32 (d, J = 4.0 Hz, 1H), 7.24–7.23 (d,
J = 4.0 Hz, 1H), 3.83 (s, 3H). Mp 222–223 °C. LRMS (EI) m/z
301, M+. HRMS (EI) m/z C15H11NO4S M+, calcd 301.0409,
found 301.0409.
3-[5-(3-Trifluoromethyl-phenyl)-furan-2-ylmethylene]-1,3-
dihydro-indol-2-one (17a). 1H NMR (400 MHz, DMSO)
δ 10.73 (s, 1H), 8.46 (d, J = 2.0 Hz, 1H), 8.22 (s, 2H), 7.78 (d,
J = 8.1 Hz, 2H), 7.58 (d, J = 3.7 Hz, 1H), 7.49 (d, J = 3.7 Hz,
1H), 7.40 (s, 1H), 7.32 (dd, J = 8.3, 2.1 Hz, 1H), 6.90 (d, J =
8.3 Hz, 1H). Mp 206–207 °C. LRMS (EI) m/z 355, M+. HRMS
(EI) m/z calcd C20H12F3NO2 M+ 355.0820, found 355.0820.
5-[5-(4-Nitro-phenyl)-furan-2-ylmethylene]-thiazolidine-2,4-
dione (19c). 1H NMR (400 MHz, DMSO) δ 12.53 (s, 1H),
7414 | Org. Biomol. Chem., 2012, 10, 7402–7417
This journal is © The Royal Society of Chemistry 2012

View Article Online
8.31–8.29 (d, J = 8.1 Hz, 2H), 7.95–7.93 (d, J = 8.1 Hz, 2H),
7.59 (s, 1H), 7.51–7.50 (d, J = 4.0 Hz, 1H), 7.24–7.22 (d, J =
4.0 Hz, 1H)). Mp 330–331 °C. LRMS (EI) m/z 316, M+. HRMS
(EI) m/z C14H8N2O5S M+, calcd 316.0154, found 316.0149.
4.0 Hz, 1H). Mp 302–303 °C. LRMS (EI) m/z 343, M+. HRMS
(EI) m/z C15H9N3O5S M+, calcd 343.0263, found 343.0272.
5-[5-(3-Methoxy-phenyl)-furan-2-ylmethylene]-2-thioxo-dihydro-
pyrimidine-4,6-dione (22b). 1H NMR (400 MHz, DMSO)
δ 12.43 (s, 1H), 12.37 (s, 1H), 8.62–8.61 (d, J = 4.0 Hz, 1H),
8.11 (s, 1H), 7.57–7.51 (m, 3H), 7.46–7.42 (d, J = 8.4 Hz, 1H),
7.07–7.04 (d, J = 8.4 Hz, 1H), 3.83 (s, 3H). Mp 335–336 °C.
LRMS (EI) m/z 328, M+. HRMS (EI) m/z C16H12N2O4S M+,
calcd 328.0518, found 328.0515.
4-{5-[(2,4-Dioxo-thiazolidin-5-ylidene)methyl]-furan-2-yl}benzoic
acid ethyl ester (19d). 1H NMR δ (400 MHz, DMSO) δ 12.52 (s,
1H), 8.07–8.04 (d, J = 8.6 Hz, 2H), 7.92–7.89 (d, J = 8.6 Hz,
2H), 7.64 (s, 1H), 7.43–7.42 (d, J = 3.6 Hz, 1H), 7.24–7.23 (d,
J = 3.6 Hz, 1H), 4.35–4.30 (q, J = 7.0 Hz, 2H), 1.35–1.32 (t, J =
7.0 Hz, 3H). Mp 259–261 °C. LRMS (EI) m/z 343, M+. HRMS
(EI) m/z C17H13NO5S M+, calcd 343.0514, found 343.0513.
5-[5-(4-Nitro-phenyl)-furan-2-ylmethylene]-2-thioxo-dihydro-
pyrimidine-4,6-dione (22c). 1H NMR (400 MHz, DMSO)
δ 12.50 (s, 1H), 12.44 (s, 1H), 8.60–8.58 (d, J = 4.0 Hz, 1H),
8.48–8.35 (d, J = 8.5 Hz, 2H), 8.24–8.21 (d, J = 8.6 Hz, 2H),
8.12 (s, 1H), 7.74–7.72 (d, J = 4.0 Hz, 1H). Mp 329–330 °C.
LRMS (EI) m/z 343, M+. HRMS (EI) m/z calcd C15H9N3O5S
M+ 343.0263, found 343.0272.
5-[5-(4-Chloro-phenyl)-furan-2-ylmethylene]-thiazolidine-2,4-
dione (19e). 1H NMR (400 MHz, DMSO) δ 12.49 (b, 1H),
7.83–7.80 (d, J = 8.1 Hz, 2H), 7.61–7.57 (m, 3H), 7.32–7.30 (d,
J = 3.8 Hz, 1H), 7.22–7.21 (d, J = 3.8 Hz, 1H). Mp
279–280 °C. LRMS (EI) m/z 305, M+. HRMS (EI) m/z
C14H8ClNO3S M+, calcd 304.9913, found 304.9920.
5-[5-(4-Chloro-phenyl)-furan-2-ylmethylene]-2-thioxo-dihydro-
pyrimidine-4,6-dione (22e). 1H NMR (400 MHz, DMSO)
δ 12.44 (s, 1H), 12.38 (s, 1H), 8.61–8.60 (d, J = 4.0 Hz, 1H),
8.11 (s, 1H), 8.03–7.99 (d, J = 8.6 Hz, 2H), 7.63–7.60 (d, J =
8.6 Hz, 2H), 7.55–7.53 (d, J = 4.0 Hz, 1H). Mp 317–318 °C.
LRMS (EI) m/z 332, M+. HRMS (EI) m/z C15H9ClN2O3S M+,
calcd 332.0022, found 332.0018.
5-[5-(4-Methyl-phenyl)-furan-2-ylmethylene]-thiazolidine-2,4-
dione (19g). 1H NMR (400 MHz, DMSO) δ 12.44 (s, 1H),
7.71–7.69 (d, J = 8.1 Hz, 2H), 7.61 (s, 1H), 7.34–7.32 (d, J =
8.1 Hz, 2H), 7.21–7.19 (m, 2H), 2.34 (s, 3H). Mp 262–264 °C.
LRMS (EI) m/z 285, M+. HRMS (EI) m/z C15H11NO3S M+,
calcd 285.0460, found 285.0466.
5-[5-(4-Methoxy-phenyl)-furan-2-ylmethylene]-thiazolidine-2,4-
dione (19h). 1H NMR (400 MHz, DMSO) δ 12.41 (s, 1H),
7.77–7.72 (d, J = 8.8 Hz, 2H), 7.61 (s, 1H), 7.20–7.18 (d, J =
3.6 Hz, 1H), 7.12–7.06 (m, 3H), 3.79 (s, 3H). Mp 272 °C.
LRMS (EI) m/z 301, M+. HRMS (EI) m/z C15H11NO4S M+,
calcd 301.0409, found 301.0404.
5-[5-(4-Bromo-phenyl)-furan-2-ylmethylene]-2-thioxo-dihydro-
pyrimidine-4,6-dione (22f). 1H NMR (400 MHz, DMSO)
δ 12.44 (s, 1H), 12.38 (s, 1H), 8.59–8.58 (d, J = 4.0 Hz, 1H),
8.09 (s, 1H), 7.94–7.90 (d, J = 8.6 Hz, 2H), 7.75–7.72 (d, J =
8.6 Hz, 2H), 7.54–7.53 (d, J = 4.0 Hz, 1H). Mp 327–328 °C.
LRMS (EI) m/z 376, M+. HRMS (EI) m/z C15H9BrN2O3S M+,
calcd 375.9517, found 375.9519.
5-[5-(4-tert-Butyl-phenyl)-furan-2-ylmethylene]-thiazolidine-2,4-
dione (19i). 1H NMR (400 MHz, DMSO) δ 12.44 (s, 1H),
7.75–7.72 (d, J = 8.1 Hz, 2H), 7.62 (s, 1H), 7.55–7.52 (d, J =
8.1 Hz, 2H), 7.21–7.19 (m, 2H), 1.29 (s, 3H). Mp 275–277 °C.
LRMS (EI) m/z 327, M+. HRMS (EI) m/z C18H17NO3S M+,
calcd 327.0929, found 327.0928.
5-[5-(4-Methyl-phenyl)-furan-2-ylmethylene]-2-thioxo-dihydro-
pyrimidine-4,6-dione (22g). 1H NMR (400 MHz, DMSO)
δ 12.35 (s, 1H), 12.28 (s, 1H), 8.62–8.61 (d, J = 4.1 Hz, 1H),
8.09 (s, 1H), 7.88–7.86 (d, J = 8.1 Hz, 2H), 7.43–7.42 (d, J =
4.1 Hz, 2H), 7.35–7.33 (d, J = 8.1 Hz, 1H), 2.36 (s, 3H). Mp
335 °C. LRMS (EI) m/z 312, M+. HRMS (EI) m/z C16H12N2O3S
M+, calcd 312.0569, found 312.0572.
5-[5-(2-Naphthyl)-furan-2-ylmethylene]-thiazolidine-2,4-dione
(19j). 1H NMR (400 MHz, DMSO) δ 12.51 (s, 1H), 8.32 (s,
1H), 8.06–8.03 (m, 2H), 7.96–7.94 (d, J = 8.0 Hz, 2H), 7.67 (s,
1H), 7.60–7.54 (m, 2H), 7.42–7.41 (d, J = 4.0 Hz, 1H),
7.29–7.28 (d, J = 4.0 Hz, 1H). Mp 246–247 °C. LRMS (EI) m/z
321, M+. HRMS (EI) m/z C18H11NO3S M+, calcd 321.0460,
found 321.0462.
5-[5-(4-Methoxy-phenyl)-furan-2-ylmethylene]-2-thioxo-dihydro-
pyrimidine-4,6-dione (22h). 1H NMR (400 MHz, DMSO)
δ 12.36 (s, 1H), 12.30 (s, 1H), 8.64–8.63 (d, J = 4.0 Hz, 1H),
8.07 (s, 1H), 7.95–7.90 (d, J = 8.5 Hz, 2H), 7.39–7.37 (d, J =
4.0 Hz, 1H), 7.11–7.06 (d, J = 8.5 Hz, 2H), 3.83 (s, 3H). Mp
346–347 °C. LRMS (EI) m/z 328, M+. HRMS (EI) m/z
C16H12N2O4S M+, calcd 328.0518, found 328.0517.
5-[5-(2-Chloro-5-trifluomethyl)-furan-2-ylmethylene]-thiazolidine-
2,4-dione (19k). 1H NMR (400 MHz, DMSO) δ 12.57 (s, 1H),
8.12 (s, 1H), 7.85–7.83 (d, J = 8.5 Hz, 2H), 7.77–7.75 (d, J =
8.5 Hz, 2H), 7.66 (s, 1H), 7.52–7.51 (d, J = 3.8 Hz, 1H),
7.26–7.25 (d, J = 3.8 Hz, 1H). Mp 259 °C. LRMS (EI) m/z 373,
M+. HRMS (EI) m/z C18H11NO3S M+, calcd 372.9787, found
372.9791.
5-[5-(4-tert-Butyl-phenyl)-furan-2-ylmethylene]-2-thioxo-dihydro-
pyrimidine-4,6-dione (22i). 1H NMR (400 MHz, DMSO) δ 12.40
(s, 1H), 12.35 (s, 1H), 8.64–8.63 (d, J = 4.1 Hz, 1H), 8.09 (s,
1H), 7.92–7.90 (d, J = 8.6 Hz, 2H), 7.56–7.53 (d, J = 8.6 Hz,
2H), 7.46–7.45 (d, J = 4.1 Hz, 1H), 1.29 (s, 9H). Mp
303–304 °C. LRMS (EI) m/z 354, M+. HRMS (EI) m/z
C19H18N2O3S M+, calcd 354.1038, found 354.1036.
5-[5-(2-Nitro-phenyl)-furan-2-ylmethylene]-2-thioxo-dihydro-
pyrimidine-4,6-dione (22a). 1H NMR (400 MHz, DMSO)
δ 12.51 (s, 1H), 12.45 (s, 1H), 8.61–8.60 (d, J = 4.0 Hz, 1H),
8.09–8.03 (t, J = 8.3 Hz, 2H), 7.89 (s, 1H), 7.88–7.83 (t, J =
8.3 Hz, 1H), 7.78–7.74 (t, J = 8.3 Hz, 1H), 7.42–7.40 (d, J =
5-[5-(2-Naphthyl)-furan-2-ylmethylene]-2-thioxo-dihydro-
pyrimidine-4,6-dione (22j). 1H NMR (400 MHz, DMSO) δ 12.45
(s, 1H), 12.39 (s, 1H), 8.69–8.68 (d, J = 4.0 Hz, 1H), 8.59 (s,
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 7402–7417 | 7415

View Article Online
1H), 8.16 (s, 1H), 8.09–8.04 (m, 3H), 7.97 (s, 1H), 7.64–7.59
(m, 3H). Mp 352–354 °C. LRMS (EI) m/z 348, M+. HRMS (EI)
m/z C19H12N2O3S M+, calcd 348.0569, found 348.0569.
Acknowledgements
We gratefully acknowledge financial support from the National
Institutes of Health (NIH) to RM and MH (CA114046). We also
gratefully acknowledge financial support from the State Key
Program of Basic Research of China grant to HJ
(2009CB918502), the National Natural Science Foundation of
China grants to CL (20972174 and 91029704), HJ (21021063)
and HL (81025017), National S&T Major Projects to HL
(2012ZX09103-101-072), National High Technology Research
and Development Program of China (2012AA020302) and the
Chinese Academy of Sciences (XDA01040305) grant to CL.
5-[5-(2-Chloro-5-trifluoromethyl)-furan-2-ylmethylene]-2-thioxo-
dihydro-pyrimidine-4,6-dione (22k). 1H NMR (400 MHz,
DMSO) δ 12.49 (s, 1H), 12.44 (s, 1H), 8.59–8.58 (d, J = 4.0 Hz,
1H), 8.45 (s, 1H), 8.19 (s, 1H), 7.90–7.85 (d, J = 8.5 Hz, 1H),
7.84–7.82 (d, J = 8.5 Hz, 1H), 7.70–7.69 (d, J = 4.0 Hz, 1H).
Mp 360–362 °C. LRMS (EI) m/z 400, M+. HRMS (EI) m/z
C18H6ClF3N2O3S M+, calcd 399.9896, found 399.9857.
5-[5-(3-Trifluoromethyl-phenyl)-furan-2-ylmethylene]-2-thioxo-
dihydro-pyrimidine-4,6-dione (22l). 1H NMR (400 MHz,
DMSO) δ 12.46 (s, 1H), 12.41 (s, 1H), 8.62–8.61 (d, J = 4.0 Hz,
1H), 8.33 (s, 1H), 8.29–8.27 (d, J = 8.4 Hz, 1H), 8.16 (s, 1H),
7.83–7.85 (d, J = 8.4 Hz, 1H), 7.80–7.76 (t, J = 8.4 Hz, 1H),
7.70–7.69 (d, J = 4.0 Hz, 1H). Mp 302–303 °C. LRMS (EI) m/z
366, M+. HRMS (EI) m/z C16H19F3N2O3S M+, calcd 366.0286,
found 366.0284.
References
1 R. Seger and E. G. Krebs, FASEB J., 1995, 9, 726–735.
2 M. J. Robinson and M. H. Cobb, Curr. Opin. Cell Biol., 1997, 9,
180–186.
3 H. Rubinfeld and R. Seger, Mol. Biotechnol., 2005, 31, 151–174.
4 E. K. Kim and E. J. Choi, Biochim. Biophys. Acta, Mol. Basis Dis., 2010,
1802, 396–405.
5 A. S. Dhillon, S. Hagan, O. Rath and W. Kolch, Oncogene, 2007, 26,
3279–3290.
4-[5-(4,6-Dioxo-2-thioxo-tetrahydro-pyrimidin-5-ylidene-
methyl)furan-2-yl]benzoic acid (22m). 1H NMR (400 MHz,
DMSO) δ 12.46 (s, 1H), 12.40 (s, 1H), 8.62–8.61 (d, J = 4.1 Hz,
1H), 8.11–8.03 (m, 5H), 7.63–7.62 (d, J = 4.1 Hz, 1H). Mp
>400 °C. LRMS (EI) m/z 342, M+. HRMS (EI) m/z
C16H10N2O5S M+, calcd 342.0301, found 342.0311.
6 R. Hoshino, Y. Chatani, T. Yamori, T. Tsuruo, H. Oka, O. Yoshida,
Y. Shimada, S. Ari-i, H. Wada, J. Fujimoto and M. Kohno, Oncogene,
1999, 18, 813–822.
7 H. Davies, G. R. Bignell, C. Cox, P. Stephens, S. Edkins, S. Clegg,
J. Teague, H. Woffendin, M. J. Garnett, W. Bottomley, N. Davis,
N. Dicks, R. Ewing, Y. Floyd, K. Gray, S. Hall, R. Hawes, J. Hughes,
V. Kosmidou, A. Menzies, C. Mould, A. Parker, C. Stevens, S. Watt,
S. Hooper, R. Wilson, H. Jayatilake, B. A. Gusterson, C. Cooper,
J. Shipley, D. Hargrave, K. Pritchard-Jones, N. Maitland, G. Chenevix-
Trench, G. J. Riggins, D. D. Bigner, G. Palmieri, A. Cossu, A. Flanagan,
A. Nicholson, J. W. C. Ho, S. Y. Leung, S. T. Yuen, B. L. Weber,
H. F. Siegler, T. L. Darrow, H. Paterson, R. Marais, C. J. Marshall,
R. Wooster, M. R. Stratton and P. A. Futreal, Nature, 2002, 417,
949–954.
5-[5-(2-Nitro-4-chloro)-furan-2-ylmethylene]-2-thioxo-dihydro-
pyrimidine-4,6-dione (22n). 1H NMR (400 MHz, DMSO)
δ 12.49 (s, 1H), 12.43 (s, 1H), 8.59–8.58 (d, J = 4.0 Hz, 1H),
8.26 (s, 1H), 8.11–8.09 (d, J = 8.4 Hz, 1H), 7.94–7.92 (d, J =
8.4 Hz, 1H), 7.86 (s, 1H), 7.42–7.41 (d, J = 4.0 Hz, 1H). Mp
300–301 °C. LRMS (EI) m/z 377, M+. HRMS (EI) m/z
C15H8ClN3O5S M+, calcd 376.9873, found 376.9884.
8 H. Rajagopalan, A. Bardelli, C. Lengauer, K. W. Kinzler, B. Vogelstein
and V. E. Velculescu, Nature, 2002, 418, 934.
9 U. R. Rapp, J. Fensterle, S. Albert and R. Gotz, Adv. Enzyme Regul.,
2003, 43, 183–195.
10 R. Mukherjee, J. M. Bartlett, N. S. Krishna, M. A. Underwood and
J. Edwards, Prostate, 2005, 64, 101–107.
11 C. Damodar Reddy, S. Marwaha, R. Patti, M. Raghunath,
A. C. Duhaime, L. Sutton and P. C. Phillips, Anticancer Res., 2001, 21,
2733–2738.
12 N. Giuliani, P. Lunghi, F. Morandi, S. Colla, S. Bonomini, M. Hojden,
V. Rizzoli and A. Bonati, Leukemia, 2004, 18, 628–635.
13 J. A. McCubrey, L. S. Steelman, W. H. Chappell, S. L. Abrams,
E. W. Wong, F. Chang, B. Lehmann, D. M. Terrian, M. Milella, A. Tafuri,
F. Stivala, M. Libra, J. Basecke, C. Evangelisti, A. M. Martelli and
R. A. Franklin, Biochim. Biophys. Acta, Mol. Cell Res., 2007, 1773,
1263–1284.
14 L. S. Steelman, S. L. Abrams, J. G. Shelton, W. H. Chappell, J. Basecke,
F. Stivala, M. Donia, F. Nicoletti, M. Libra, A. M. Martelli and
J. A. McCubrey, Cell Cycle, 2010, 9, 1629–1638.
15 J. S. Sebolt-Leopold and R. Herrera, Nat. Rev. Cancer, 2004, 4, 937–947.
16 P. J. Roberts and C. J. Der, Oncogene, 2007, 26, 3291–3310.
17 J. A. Gollob, S. Wilhelm, C. Carter and S. L. Kelley, Semin. Oncol.,
2006, 33, 392–406.
18 A. Zebisch and J. Troppmair, Cell. Mol. Life Sci., 2006, 63, 1314–1330.
19 R. Roskoski Jr., Biochem. Biophys. Res. Commun., 2010, 399, 313–317.
20 D. T. Leicht, V. Balan, A. Kaplun, V. Singh-Gupta, L. Kaplun,
M. Dobson and G. Tzivion, Biochim. Biophys. Acta, Mol. Cell Res.,
2007, 1773, 1196–1212.
21 H. Chong, H. G. Vikis and K. L. Guan, Cell. Signalling, 2003, 15,
463–469.
22 K. E. Mercer and C. A. Pritchard, Biochim. Biophys. Acta, 2003, 1653,
25–40.
5-[5-(2-Trifluoromethyl-4-chloro)-furan-2-ylmethylene]-2-thioxo-
dihydro-pyrimidine-4,6-dione (22o). 1H NMR (400 MHz,
DMSO) 12.49 (s, 1H), 12.43 (s, 1H), 8.62–8.60 (d, J = 4.0 Hz,
1H), 8.08–8.06 (d, J = 8.4 Hz, 1H), 8.03 (s, 1H), 8.00 (s, 1H),
7.94–7.92 (d, J = 8.4 Hz, 1H), 7.36–7.35 (d, J = 4.0 Hz, 1H).
Mp 325–326 °C. LRMS (EI) m/z 400, M+. HRMS (EI) m/z
C16H8ClF3N2O3S M+, calcd 399.9896, found 399.9857.
5-[5-(3-Trifluoromethyl-4-chloro)-furan-2-ylmethylene]-2-thioxo-
dihydro-pyrimidine-4,6-dione (22p). 1H NMR (400 MHz,
DMSO) δ 12.47 (s, 1H), 12.43 (s, 1H), 8.57–8.56 (d, J = 4.0 Hz,
1H), 8.35 (s, 1H), 8.26–8.23 (d, J = 8.4 Hz, 1H), 8.12 (s, 1H),
7.89–7.87 (d, J = 8.4 Hz, 1H), 7.70–7.69 (d, J = 4.0 Hz, 1H).
Mp 323–325 °C. LRMS (EI) m/z 400, M+. HRMS (EI) m/z
C16H8ClF3N2O3S M+, calcd 399.9896, found 399.9869.
5-[5-(3,4-Methylenedioxy)-furan-2-ylmethylene]-2-thioxo-dihydro-
pyrimidine-4,6-dione (22q). 1H NMR (400 MHz, DMSO) δ 12.39
(s, 1H), 12.33 (s, 1H), 8.63–8.62 (d, J = 4.0 Hz, 1H), 8.10 (s,
1H), 7.59–7.56 (m, 2H), 7.42–7.41 (d, J = 4.0 Hz, 1H),
7.11–7.09 (d, J = 8.5 Hz, 1H), 6.14 (s, 2H). Mp >400 °C.
LRMS (EI) m/z 342, M+. HRMS (EI) m/z C16H10N2O5S M+,
calcd 342.0310, found 342.0302.
23 C. A. Pritchard, M. L. Samuels, E. Bosch and M. McMahon, Mol. Cell.
Biol., 1995, 15, 6430–6442.
7416 | Org. Biomol. Chem., 2012, 10, 7402–7417
This journal is © The Royal Society of Chemistry 2012

View Article Online
24 C. S. Mason, C. J. Springer, R. G. Cooper, G. Superti-Furga,
C. J. Marshall and R. Marais, EMBO J., 1999, 18, 2137–2148.
25 S. A. Moodie, M. J. Paris, W. Kolch and A. Wolfman, Mol. Cell. Biol.,
1994, 14, 7153–7162.
26 L. Wojnowski, L. F. Stancato, A. C. Larner, U. R. Rapp and A. Zimmer,
Mech. Dev., 2000, 91, 97–104.
27 M. Garnett and R. Marais, Cancer Cell, 2004, 6, 313.
28 C. Wellbrock and A. Hurlstone, Biochem. Pharmacol., 2010, 80,
561–567.
29 P. T. Wan, M. J. Garnett, S. M. Roe, S. Lee, D. Niculescu-Duvaz,
V. M. Good, C. M. Jones, C. J. Marshall, C. J. Springer, D. Barford and
R. Marais, Cell, 2004, 116, 855–867.
30 C. A. Pratilas, B. S. Taylor, Q. Ye, A. Viale, C. Sander, D. B. Solit and
N. Rosen, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 4519–4524.
31 C. Wellbrock, M. Karasarides and R. Marais, Nat. Rev. Mol. Cell Biol.,
2004, 5, 875–885.
47 R. Nazarian, H. Shi, Q. Wang, X. Kong, R. C. Koya, H. Lee, Z. Chen,
M. K. Lee, N. Attar, H. Sazegar, T. Chodon, S. F. Nelson, G. McArthur,
J. A. Sosman, A. Ribas and R. S. Lo, Nature, 2010, 468, 973–977.
48 Q. Zhang and I. Muegge, J. Med. Chem., 2006, 49, 1536–1548.
49 A. Nicholls, G. B. McGaughey, R. P. Sheridan, A. C. Good, G. Warren,
M. Mathieu, S. W. Muchmore, S. P. Brown, J. A. Grant, J. A. Haigh,
N. Nevins, A. N. Jain and B. Kelley, J. Med. Chem., 2010, 53,
3862–3886.
50 X. F. Liu, H. L. Jiang and H. L. Li, J. Chem. Inf. Model., 2011, 51,
2372–2385.
51 W. Q. Lu, X. F. Liu, X. W. Cao, M. Z. Xue, K. D. Liu, Z. J. Zhao,
X. Shen, H. L. Jiang, Y. F. Xu, J. Huang and H. L. Li, J. Med. Chem.,
2011, 54, 3564–3574.
52 F. Bai, H. Y. Liu, L. J. Tong, W. Zhou, L. Liu, Z. J. Zhao, X. F. Liu,
H. L. Jiang, X. C. Wang, H. Xie and H. L. Li, Bioorg. Med. Chem. Lett.,
2012, 22, 1365–1370.
32 R. Houben, J. C. Becker, A. Kappel, P. Terheyden, E. B. Brocker,
R. Goetz and U. R. Rapp, J. Carcinog., 2004, 3, 6.
33 W. S. Samowitz, C. Sweeney, J. Herrick, H. Albertsen, T. R. Levin,
M. A. Murtaugh, R. K. Wolff and M. L. Slattery, Cancer Res., 2005, 65,
6063–6069.
34 L. Liu, Y. Cao, C. Chen, X. Zhang, A. McNabola, D. Wilkie, S. Wilhelm,
M. Lynch and C. Carter, Cancer Res., 2006, 66, 11851–11858.
35 A. J. King, D. R. Patrick, R. S. Batorsky, M. L. Ho, H. T. Do,
S. Y. Zhang, R. Kumar, D. W. Rusnak, A. K. Takle, D. M. Wilson,
E. Hugger, L. Wang, F. Karreth, J. C. Lougheed, J. Lee, D. Chau,
T. J. Stout, E. W. May, C. M. Rominger, M. D. Schaber, L. Luo,
A. S. Lakdawala, J. L. Adams, R. G. Contractor, K. S. Smalley,
M. Herlyn, M. M. Morrissey, D. A. Tuveson and P. S. Huang, Cancer
Res., 2006, 66, 11100–11105.
36 C. Luo, P. Xie and R. Marmorstein, J. Med. Chem., 2008, 51,
6121–6127.
37 S. Ramurthy, S. Subramanian, M. Aikawa, P. Amiri, A. Costales, J. Dove,
S. Fong, J. M. Jansen, B. Levine, S. Ma, C. M. McBride, J. Michaelian,
T. Pick, D. J. Poon, S. Girish, C. M. Shafer, D. Stuart, L. Sung and
P. A. Renhowe, J. Med. Chem., 2008, 51, 7049–7052.
53 Glide, version 5.5, Schrödinger, LLC, New York, NY, 2009.
54 J. D. Hansen, J. Grina, B. Newhouse, M. Welch, G. Topalov, N. Littman,
M. Callejo, S. Gloor, M. Martinson, E. Laird, B. J. Brandhuber,
G. Vigers, T. Morales, R. Woessner, N. Randolph, J. Lyssikatos and
A. Olivero, Bioorg. Med. Chem. Lett., 2008, 18, 4692–4695.
55 S. Wenglowsky, L. Ren, K. A. Ahrendt, E. R. Laird, I. Aliagas,
B. Alicke, A. J. Buckmelter, E. F. Choo, V. Dinkel, B. N. A. Feng,
S. L. Gloor, S. E. Gould, S. Gross, J. Gunzner-Toste, J. D. Hansen,
G. Hatzivassiliou, B. N. Liu, K. Malesky, S. Mathieu, B. Newhouse,
N. J. Raddatz, Y. Q. Ran, S. Rana, N. Randolph, T. Risom, J. Rudolph,
S. Savage, L. T. Selby, M. Shrag, K. Song, H. L. Sturgis, W. C. Voegtli,
Z. Y. Wen, B. S. Willis, R. D. Woessner, W. I. Wu, W. B. Young and
J. Grina, ACS Med. Chem. Lett., 2011, 2, 342–347.
56 F. Zuccotto, E. Ardini, E. Casale and M. Angiolini, J. Med. Chem., 2010,
53, 2681–2694.
57 A. L. Hopkins, C. R. Groom and A. Alex, Drug Discovery Today, 2004,
9, 430–431.
58 H. E. Ungnade, E. M. Roberts and L. W. Kissinger, J. Phys. Chem.,
1964, 68, 3225–3228.
59 R. Morphy, J. Med. Chem., 2010, 53, 1413–1437.
38 J. Tsai, J. T. Lee, W. Wang, J. Zhang, H. Cho, S. Mamo, R. Bremer,
S. Gillette, J. Kong, N. K. Haass, K. Sproesser, L. Li, K. S. Smalley,
D. Fong, Y. L. Zhu, A. Marimuthu, H. Nguyen, B. Lam, J. Liu,
I. Cheung, J. Rice, Y. Suzuki, C. Luu, C. Settachatgul, R. Shellooe,
J. Cantwell, S. H. Kim, J. Schlessinger, K. Y. Zhang, B. L. West,
B. Powell, G. Habets, C. Zhang, P. N. Ibrahim, P. Hirth, D. R. Artis,
M. Herlyn and G. Bollag, Proc. Natl. Acad. Sci. U. S. A., 2008, 105,
3041–3046.
60 M. S. Cohen, C. Zhang, K. M. Shokat and J. Taunton, Science, 2005,
308, 1318–1321.
61 R. Kefford, H. Arkenau, M. Brown, M. Millward, J. Infante, G. Long,
D. Ouellet, M. Curtis, P. Lebowitz and G. Falchook, J. Clin. Oncol.,
2010, 28, 8503.
62 G. Jones, P. Willett and R. C. Glen, J. Mol. Biol., 1995, 245, 43–53.
63 G. Jones, P. Willett, R. C. Glen, A. R. Leach and R. Taylor, J. Mol. Biol.,
1997, 267, 727–748.
39 P. Xie, C. Streu, J. Qin, H. Bregman, N. Pagano, E. Meggers and
R. Marmorstein, Biochemistry, 2009, 48, 5187–5198.
64 M. L. Verdonk, J. C. Cole, M. J. Hartshorn, C. W. Murray and
R. D. Taylor, Proteins: Struct., Funct., Genet., 2003, 52, 609–623.
65 G. M. Morris, D. S. Goodsell, R. S. Halliday, R. Huey, W. E. Hart,
R. K. Belew and A. J. Olson, J. Comput. Chem., 1998, 19, 1639–1662.
66 R. Huey, G. M. Morris, A. J. Olson and D. S. Goodsell, J. Comput.
Chem., 2007, 28, 1145–1152.
40 G. Bollag, P. Hirth, J. Tsai, J. Z. Zhang, P. N. Ibrahim, H. N. Cho,
W. Spevak, C. Zhang, Y. Zhang, G. Habets, E. Burton, B. Wong,
G. Tsang, B. L. West, B. Powell, R. Shellooe, A. Marimuthu, H. Nguyen,
K. Y. J. Zhang, D. R. Artis, J. Schlessinger, F. Su, B. Higgins, R. Iyer,
K. D’Andrea, A. Koehler, M. Stumm, P. S. Lin, R. J. Lee, J. Grippo,
I. Puzanov, K. B. Kim, A. Ribas, G. A. McArthur, J. A. Sosman,
P. B. Chapman, K. T. Flaherty, X. W. Xu, K. L. Nathanson and K. Nolop,
Nature, 2010, 467, 596–599.
41 A. M. Alcala and K. T. Flaherty, Clin. Cancer Res., 2012, 18, 33–39.
42 A. Zambon, I. Niculescu-Duvaz, D. Niculescu-Duvaz, R. Marais and
C. J. Springer, Bioorg. Med. Chem. Lett., 2012, 22, 789–792.
43 H. Yang, B. Higgins, K. Kolinsky, K. Packman, Z. Go, R. Iyer, S. Kolis,
S. Zhao, R. Lee, J. F. Grippo, K. Schostack, M. E. Simcox,
D. Heimbrook, G. Bollag and F. Su, Cancer Res., 2010, 70, 5518–5527.
44 K. T. Flaherty, I. Puzanov, K. B. Kim, A. Ribas, G. A. McArthur,
J. A. Sosman, P. J. O’Dwyer, R. J. Lee, J. F. Grippo, K. Nolop and
P. B. Chapman, N. Engl. J. Med., 2010, 363, 809–819.
67 W. L. Jorgensen, D. S. Maxwell and J. TiradoRives, J. Am. Chem. Soc.,
1996, 118, 11225–11236.
68 M. L. Verdonk, V. Berdini, M. J. Hartshorn, W. T. Mooij, C. W. Murray,
R. D. Taylor and P. Watson, J. Chem. Inf. Model., 2004, 44, 793–806.
69 D. Fang, T. K. Nguyen, K. Leishear, R. Finko, A. N. Kulp, S. Hotz,
P. A. Van Belle, X. Xu, D. E. Elder and M. Herlyn, Cancer Res., 2005,
65, 9328–9337.
70 M. Y. Hsu, D. T. Shih, F. E. Meier, P. Van Belle, J. Y. Hsu, D. E. Elder,
C. A. Buck and M. Herlyn, Am. J. Pathol., 1998, 153, 1435–1442.
71 M. Fukunaga-Kalabis, G. Martinez, Z. J. Liu, J. Kalabis, P. Mrass,
W. Weninger, S. M. Firth, N. Planque, B. Perbal and M. Herlyn, J. Cell
Biol., 2006, 175, 563–569.
72 S. L. Johnson, D. Jung, M. Forino, Y. Chen, A. Satterthwait,
D. V. Rozanov, A. Y. Strongin and M. Pellecchia, J. Med. Chem., 2006,
49, 27–30.
73 V. Pomel, J. Klicic, D. Covini, D. D. Church, J. P. Shaw, K. Roulin,
F. Burgat-Charvillon, D. Valognes, M. Camps, C. Chabert, C. Gillieron,
B. Francon, D. Perrin, D. Leroy, D. Gretener, A. Nichols, P. A. Vitte,
S. Carboni, C. Rommel, M. K. Schwarz and T. Ruckle, J. Med. Chem.,
2006, 49, 3857–3871.
45 P. B. Chapman, A. Hauschild, C. Robert, J. B. Haanen, P. Ascierto,
J. Larkin, R. Dummer, C. Garbe, A. Testori, M. Maio, D. Hogg,
P. Lorigan, C. Lebbe, T. Jouary, D. Schadendorf, A. Ribas, S. J. O’Day,
J. A. Sosman, J. M. Kirkwood, A. M. Eggermont, B. Dreno, K. Nolop,
J. Li, B. Nelson, J. Hou, R. J. Lee, K. T. Flaherty and G. A. McArthur,
N. Engl. J. Med., 2011, 364, 2507–2516.
46 D. B. Solit and N. Rosen, N. Engl. J. Med., 2011, 364, 772–774.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 7402–7417 | 7417