Identification of a novel family of BRAFV600E inhibitors

Article abstract of DOI:10.1021/jm3004416

The BRAF oncoprotein is mutated in about half of malignant melanomas and other cancers, and a kinase activating single valine to glutamate substitution at residue 600 (BRAF^V600E) accounts for over 90% of BRAF-mediated cancers. Several BRAF^V600E inhibitors have been developed, although they harbor some liabilities, thus motivating the development of other BRAF ^V600E inhibitor options. We report here the use of an ELISA based high-throughput screen to identify a family of related quinolol/naphthol compounds that preferentially inhibit BRAF^V600E over BRAF ^WT and other kinases. We also report the X-ray crystal structure of a BRAF/quinolol complex revealing the mode of inhibition, employ structure-based medicinal chemistry efforts to prepare naphthol analogues that inhibit BRAF ^V600E in vitro with IC₅₀ values in the 80-200 nM range under saturating ATP concentrations, and demonstrate that these compounds inhibit MAPK signaling in melanoma cells. Prospects for improving the potency and selectivity of these inhibitors are discussed.

Full text of DOI:10.1021/jm3004416

Article
pubs.acs.org/jmc
Identification of a Novel Family of BRAFV600E Inhibitors
Jie Qin,† Peng Xie,†,‡ Christian Ventocilla,‡ Guoqiang Zhou,‡ Adina Vultur,† Quan Chen,† Qin Liu,†
Meenhard Herlyn,† Jeffrey Winkler,*,‡ and Ronen Marmorstein*,†,‡
‡
†The Wistar Institute and Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
S
* Supporting Information
ABSTRACT: The BRAF oncoprotein is mutated in about half of
malignant melanomas and other cancers, and a kinase activating single
valine to glutamate substitution at residue 600 (BRAFV600E) accounts for
over 90% of BRAF-mediated cancers. Several BRAFV600E inhibitors have
been developed, although they harbor some liabilities, thus motivating the
development of other BRAFV600E inhibitor options. We report here the use
of an ELISA based high-throughput screen to identify a family of related
quinolol/naphthol compounds that preferentially inhibit BRAFV600E over
BRAFWT and other kinases. We also report the X-ray crystal structure of
a BRAF/quinolol complex revealing the mode of inhibition, employ
structure-based medicinal chemistry efforts to prepare naphthol analogues
that inhibit BRAFV600E in vitro with IC50 values in the 80−200 nM range
under saturating ATP concentrations, and demonstrate that these compounds
inhibit MAPK signaling in melanoma cells. Prospects for improving the potency and selectivity of these inhibitors are discussed.
clinical trials, PLX4032 produced 2 complete responses and 24
partial responses out of 32 patients, extending life in many cases
by more than 6 months, prior to the eventual development of drug
resistance through reactivation of the MAPK pathway or through
the activation of alternative compensatory pathways, involving
receptor tyrosine kinases (RTKs), PI3K-AKT, and other
pathways.13,14 However, about half of the patients had the drug
dose reduced, and nearly two-thirds had to have their treatments
temporarily stopped because of side effects.9,10 In addition,
approximately one-quarter of patients developed cancerous or
precancerous nonmelanoma skin lesions.9,10 Given these
limitations of vemurafenib, and some other drugs that are being
evaluated to treat metastatic melanoma, it would be useful to have
additional BRAFV600E inhibitor drug options for some patients.
Here, we report the development of an ELISA-based high-
throughput assay to screen a combined diverse library of over
30,000 organic compounds for BRAFV600E inhibition. This screen,
the structure determination of BRAF bound to one of the
identified inhibitors, and the follow-up structure-based medicinal
chemistry efforts resulted in the identification of a family of
related compounds containing a quinolol or naphthol backbone
that selectively inhibit BRAFV600E over BRAFWT in vitro, display
IC50 values in the 80−200 nM range under saturation ATP
concentrations, and inhibit MAPK signaling in melanoma cells.
INTRODUCTION
■
RAF family kinases are central players in the highly conserved
MAPK signaling pathway (RAS-RAF-MEK-ERK), which relay
signals from the extracellular space through receptor tyrosine
kinases (RTKs) to the nucleus to promote the expression of
genes involved in cell proliferation and survival. RAF kinases
function by specifically phosphorylating MEK1/2 on the kinase
activation loop, leading to the subsequent activation of ERK1/
2.1 The three isoforms of the RAF kinases, ARAF, BRAF, and
CRAF, share a common three-domain structure containing the
conserved regions CR1, CR2, and CR3. The regulation of RAF
kinase activity is complex and involves a variety of different
kinases and scaffolding proteins.1 However, compared to ARAF
and CRAF, BRAF requires fewer regulation events for its
activation. Due to this property, BRAF has significantly higher
basal activity than the other RAF family isoforms and has also
been found to be a major activator of MEK1/2.2,3
BRAF is an important oncogene that is mutated in about a
half of malignant melanomas and at a lower frequency in a wide
range of other human cancers, such as thyroid, colon, ovarian,
lung, and breast cancers.4,5 Oncogenic mutations occur within
the kinase domain, with a single valine to glutamate substitution
at residues 600 (BRAFV600E) accounting for over 90% of these
mutations.1,4,6,7 Because of these findings, BRAF, and
BRAFV600E in particular, has emerged as an attractive anticancer
drug target. A number of inhibitors against BRAF have been
developed so far and more are at various stages of preclinical
and clinical development.8−11 For example, vemurafenib
(PLX4032), an azaindole compound and orally available
ATP competitive BRAF inhibitor that shows selectivity for
BRAFV600E, has received FDA approval for the treatment of
late stage metastatic melanoma.12 Encouragingly, in phase III
RESULTS
■
Identification of a Novel Family of BRAFV600E Inhibitors.
In order to screen for BRAF inhibitors in a high-throughput
Received: February 11, 2012
Published: April 26, 2012
© 2012 American Chemical Society
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format, we employed an Enzyme-Linked Immunosorbent Assay
(ELISA)-based system that was previously used by others15 and
us.16 The details of the assay were essentially as described
previously.16 Briefly, we carried out the screen in a 96-well
microtiter plate containing covalently immobilized glutathione to
enable the capture of glutathione-S-transferase (GST) fusion
protein (Pierce Biotechnology) linked to the full-length MEK
protein. A mouse monoclonal antibody recognizing BRAF-
phosphorylated residues on MEK is then added to the microtiter
plate to bind the immobilized and phosphorylated GST-MEK.
Antibody molecules that are nonspecifically bound are then
washed away, and horseradish peroxidase (HRP) linked IgG is
added to bind to the primary antibody. The amount of HRP IgG
binding is then quantified by measuring the chemilumines-
cence generated by the mixing of the HRP substrate with
specifically bound HRP IgG. As shown in Figure 1A, a Z′ factor
inhibit BRAFV600E activity to more than 90% at an inhibitor
concentration of 5 μM (Figure S1A, Supporting Information
and compounds 1−23 in Figure S1B, Supporting Information).
Encouragingly, a known CRAF inhibitor was identified in three
independent libraries (15 in Figure S1B, Supporting Informa-
tion) and scored among the top 30 inhibitor hits from this
experiment, confirming the efficacy of this assay system.
Upon close examination of the representative scaffolds with a
rough filtration according to Lipinski’s rule of five,17 23
compounds were determined to display drug-like structures
(compounds 1−23 in Figure S1B, Supporting Information).
Notably, seven of the compounds contained a quinolol,
naphthol, or related substructure (compounds 1, 2, 3, 6, 7, 16,
and 17 in Figure S1B, Supporting Information). Given that this
was the largest set of related compounds that we identified in our
screen and that these compounds were not previously identified
as kinase inhibitors, we focused on their further analysis.
Five of these quinolol/naphthol analogues, in addition to
two other closely related compounds (compounds 4 and 5)
(Figure 2A), were further characterized by measuring dose−
response curves for their inhibition against both BRAFWT and
BRAFV600E at a protein concentration of 30 nM and inhibitor
concentrations ranging from 10 μM to 5 nM to approximate
their IC50 values (Figure 2A). This analysis revealed that
the most potent inhibitor from these series, 2, inhibited
BRAFV600E with an IC50 value of about 100 nM and with about
a 3-fold selectivity for BRAFV600E over BRAFWT. Each of the
other related inhibitors also displayed strong inhibition toward
BRAFV600E with IC50 values within the submicromolar range.
All but 5 showed a 2−5-fold selectivity for BRAFV600E over
BRAFWT
.
Crystal Structure of a Quinolol Analogue in Complex
with the BRAF Kinase Domain. In order to understand the
mode of action of this family of BRAF inhibitors, cocrystalliza-
tion of the BRAF kinase domain (BRAF-KD, residues 433−
726) with several of the inhibitors was attempted. Since the
MALDI-TOF mass spectrum data of BRAF-KD expressed and
purified from Sf9 insect cells indicated that the protein is
extensively phosphorylated, lambda protein phosphatase (λPP)
treatment was used to generate homogeneous hypophosphory-
lated protein samples for cocrystallization. Extensive cocrystal-
lization efforts using the microbatch method produced
cocrystals with only 1 with the wild-type BRAF kinase domain.
Crystals of the complex were small (300 μm × 30 μm × 30 μm)
producing only low resolution data using a home X-ray source,
but data to about 2.5 Å resolution could be obtained using
a 10 μm mini-beam at the Advanced Photon Synchrotron
source. The BRAF-KD/1 structure was determined by
molecular replacement using the unliganded BRAF-KD/
sorafenib crystal structure (PDB accession code: 1UWH) as a
search model. The BRAF-KD/1 crystals contained two copies
of the BRAF-KD/1 complex in one asymmetric unit cell.
Electron density corresponding to inhibitor 1 was visible in the
ATP binding pockets of both molecules in the asymmetric unit
cell. The structure was refined with strict NCS symmetry
imposed with the inhibitor modeled only at the later stages
of refinement. The final structure was determined to 2.55 Å
resolution to an Rwork/Rfree of 0.2203/0.2659 with good
geometry (Table 1).
Figure 1. Screen for small molecule BRAFV600E inhibitors. (A) Z′
factor test for the BRAF ELISA-based enzymatic assay system. (B)
Summary of small molecule libraries that were screened. (C) Scatter plot
of the HTS in replicates against a combined library of 31,976 small
molecule compounds. Inhibitors that were analyzed further are circled.
of 0.82 was obtained indicating that this BRAF ELISA-based
assay was effective for high throughput screening.
We used the ELISA-based HTS system to screen a combined
library of 31,976 diverse compounds (at a compound con-
centration of 5 μM, a BRAFV600E concentration of 140 nM, and
a saturating ATP concentration of 100 μM) (Figure 1B and C).
From this library, the 23 top scoring compounds deemed to
have drug-like properties were purchased directly from their
corresponding vendors and were confirmed in a repeat assay to
The BRAF-KD/1 structure revealed that the inhibitor binds
within the ATP binding cleft between the N-lobe and C-lobe of
the kinase domain, and an overlay with the structure of PKA in
complex with ATP revealed significant overlap of 1 with both
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Figure 2. Family of quinolol and naphthol BRAFV600E inhibitors. (A) Structures of selected HTS inhibitors that share a common scaffold. Single
measurment dose−response inhibition curves were carried out for each of the cherry-picked compounds to approximate their IC50 values (μM) as
follows: 1 (BRAFWT= 0.8, BRAFV600E = 0.4), 2 (BRAFWT = 0.3, BRAFV600E = 0.1), 3 (BRAFWT = 3.0, BRAFV600E = 0.7), 4 (BRAFWT = 0.9,
BRAFV600E = 0.3), 5 (BRAFWT = 0.4, BRAFV600E = 0.6), 6 (BRAFWT = 4.0, BRAFV600E = 0.9), and 7 (BRAFWT = 2.0, BRAFV600E = 0.8). (B) General
scheme for the synthesis of analogues of 2.
Table 1. Crystallographic Data Collection and Refinement
Statistics for the BRAF/1 Complex
opposed to the inactive conformation that is observed with
BRAF bound to sorafenib (Figure 3C).
The R-stereoisomer of 1 appears to be bound to the BRAF
ATP binding pocket. The quinoline ring is stacked between the
N- and C-lobes and against the kinase hinge region with the
chloride atom pointing toward the DFG loop with the furan
pointing toward the P-loop and the pyridine pointing in the
opposite direction toward the D helix (Figure 4A). Specifically,
the quinolinol moiety of 1 is intercalated into the space
between residues Trp531 and Phe583 forming π−π stacking
interactions (Figure 4B). In addition, the nitrogen atom of
the quinoline ring of 1 forms hydrogen-bonding interactions
with the hinge region of the N- and C-lobes, potentially
through the bridging of a water molecule to the main chain
carbonyl of residue Cys532. There are also extensive van der
Waals contacts between 1 and other residues in the protein
pocket such as residues Ile463, Val471, Ala481, Gly464,
Thr529, Gln530, Ser535, Ser536, His539, Asn580, Asn581,
and Gly593. The furan group is pointing to the extension of the
P-loop, and the pyridine group is in proximity to His539 of the
αD helix. Similar to other BRAF/inhibitor structures with the
protein in the active conformation, an 11 Å long “Raf specificity
pocket” that is defined by the DFG motif sequence and the αC
helix is lined by residues Thr529 (the gatekeeper residue),
Leu505, Leu514, Gly593, Asp594, and Phe59518 (Figure 4C).
Taken together, structural evidence reveals extensive and
specific interactions between 1 and the ATP binding pocket
of the BRAF kinase domain, establishing 1 as a Type I inhibitor,
Data Collection
complex
BRAF−1
P41212
space group
cell dimensions
a, c (Å)
94.3, 163.5
50−2.55
0.155 (0.660)
resolution (Å)
a
Rmerge
I/σ(I)
19.6 (2.3)
completeness (%)
multiplicity
99.8% (97.8%)
13.0 (5.1)
Refinement
b
c
Rwork /Rfree
0.2203/0.2659
rmsd
bond lengths (Å)
bond angles (deg)
0.0073
1.36
a
Highest resolution shell is shown in parentheses. Rmerge = Σhkl |I(hkl) −
⟨I(hkl)⟩|/Σhkl(hkl), where ⟨I(hkl)⟩ is the mean of the symmetry-equivalent
b
c
reflections of I(hkl). Rwork = Σ||Fo| − |Fc||/|Fo|. Rfree = ΣT||Fo| −
|Fc||/ΣT|Fo| (where T is a test data set of 9.3% of the total reflections
randomly chosen and set aside before refinement).
the adenine and the ribose moieties of the ATP molecule
(Figure 3A). The activation loop of BRAF bound to 1 takes on
the active conformation that is observed with BRAF in complex
with the PLX4720 (Figure 3B)18 and CS29219 inhibitors as
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Figure 4. Details of the BRAF-1 interface. (A) Electron density
generated from a simulated annealing Fo − Fc map in which the
inhibitor was omitted from the refinement is shown contoured at 4σ.
Key active site regions of the kinase are also highlighted. (B)
Interactions between 1 and BRAF active site residues. Hydrogen
bonding interactions are represented by blue dashed lines, and
residues that mediate van der Waals interactions with inhibitor are also
highighted. (C) Electrostatic potential surface of the BRAF/1 complex
structure highlighting the inhibitor as a stick figure.
Figure 3. Crystal structure of BRAF in complex with compound 1. (A)
Overall structure of the BRAF kinase domain in complex with 1. The
N-lobe, C-lobe, P-loop, and activation loop (Act. loop) of the kinase
domain are color coded blue, red, yellow, and green, respectively.
Compound 1 is shown in a white stick and ball model. The ATP
molecule from the PKA/ATP complex structure is overlaid with
BRAF/1 to show the binding of ATP in the BRAF kinase active site.
The ATP model is shown in yellow for the adenine and ribose
fragments and in orange for the phosphates. The same color coding
scheme is preserved in all panels of this figure. (B) Overlay of active
sites of BRAF in complex with 1 (blue) and PLX4720 (orange) (PDB
ID: 3C4C) revealing conserved “active” kinase conformations. Key
residues of the active site for transition into the active conformation
(F468, D594, F595, and V600 are highlighted). (C) Overlay of active
sites of BRAF in complex with 1 (blue) and sorafenib (pink) in the
inactive conformation (PDB ID: 1UWH). The conformation of the
activation loop of the active BRAF and inactive sorafenib complexes
are shown in light and dark green, respectively. All structural
superpositions were generated by overlaying the Cα positions of
various kinase structures using secondary structure matching (SSM) in
Coot.33
in our screen, and it appeared to be most tractable for medicinal
chemistry efforts. On the basis of the BRAF-KD/1 structure
and the similarity between inhibitors 1 and 2 (Figure 2A), we
hypothesized that the naphthol heterocycle of 2 would occupy
the same space as the quinolol of 1 with the thiotriazole and the
thienylsulfonamide of 2 occupying the positions of the furan/
aminopyridine and chloride groups of 1, respectively. On the
basis of this hypothesis, we resynthesized 2 and prepared a total
of 38 compounds, varying the nature of both the nitrogen-
containing heterocycle corresponding to the thiotriazole ring
and the sulfonamide substitution in 2. The general scheme
for the synthesis of these compounds is shown in Figure 2B.
We then screened each of these analogues against BRAFV600E
and BRAFWT (at protein and ATP concentrations of 28 nM
and 100 μM, respectively) using a dose−response curve to
calculate IC50 values.
As can be seen in Figure 5 and Table 2, resynthesized 2
shows IC50 values for BRAFV600E and BRAFWT of 0.17 μM and
0.26 μM, respectively, close to the values of 0.11 μM and
0.32 μM obtained for the screen compound. To further confirm
our modeling of 2 into the ATP binding site of BRAFWT based
on the BRAFWT/1 structure, we prepared the oxidized form
of 2 (compound 24). On the basis of this modeling, we
hypothesized that the OH group of the naphthol points into
solvent, so the inhibitor potency would therefore be insensitive
to its oxidation state. Our data showed that 2 and 24 show
comparable potencies for BRAFWT and BRAFV600E (Table 2).
This is consistent with our hypothesis. Nonetheless, it is
binding to the ATP pocket in the active conformation,
confirming its inhibition properties against both BRAFWT and
BRAFV600E
.
Preparation and Characterization of a Focused BRAF
Inhibitor Library. Of the family of quinolol/naphthol BRAF
inhibitors that were identified in the screen, we carried out a
structure−activity relationship (SAR) around 2 (Figure 2A)
because it was the most potent BRAFV600E inhibitor identified
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replacement of the thiotriazole present in 2 with a hydrogen
atom reduced potency by more than 30-fold (compare 2 with
25), whereas replacement of the thiotriazole with a halogen
only reduced binding by about 3-fold (compare 2 with 26 and
27). In contrast, several different heterocycles at this position
can be accommodated (28−31), while another compound is
more poorly accommodated (32). The structure of the
BRAFWT/1 complex suggests that the thiotriazole of modeled
2 would be pointing out toward the solvent of the enzyme
where the protein substrate would bind suggesting that these
substitutions may have differential effects on substrate binding.
Substitution of the sulfonamide that presumably occupies the
RAF-specific pocket also has significant effects on inhibitor
potency. Specifically, sulfonamides that contain a functionalized
aryl group (for example, 33, 34, 40, and 41) are significantly
more potent than sulfonamides containing long aliphatic
groups (for example, 35 and 36). In addition, highly substituted
aryls in the sulfonamide moiety significantly decrease inhibitor
potency by 3- to 20-fold (for example, 37−39). We hypothesize
that the highly substituted aryls or long aliphatic groups are
too bulky or long, respectively, to be accommodated by the
BRAFWT/BRAFV600E specificity pocket. The most potent
BRAFV600E inhibitors identified were 28 (IC50 = 0.12 μM),
44 (IC50 = 0.14 μM), and 40 (IC50 = 0.08 μM) (Figure 5 and
Table 2). Each of these inhibitors exhibited about a 2-fold
Figure 5. IC50 curves for compound 2 analogues against BRAFWT and
BRAFV600E. Dose−response inhibition curves of selected compounds
are shown and plotted as normalized percent activity.
possible that the assay conditions may regenerate the oxidized
form, therefore leaving open the possibility that BRAFWT
/
BRAFV600E actually binds to the oxidized form of the naphthol
analogues prepared in this study.
A SAR of the other naphthol analogues of 2 reveals that,
as expected, other substitutions on the naphthol ring system
have significant effects on inhibitor potency for BRAFV600E
(Table 2 and Table S1, Supporting Information). Specifically,
selectivity for BRAFV600E over BRAFWT
.
a
Table 2. Structures and IC50 Values of Naphthol Inhibitor Analogues against BRAFWT and BRAFV600E
a
Compounds 3−23 are listed in Figure S1B (Supporting Information) and compounds 42−61 are listed in Table S1 (Supporting Information).
The 95% confidence interval of IC50 was calculated as the antilog of the upper and lower limits of the 95% confidence interval of Log IC50.
b
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Characterization of BRAF Inhibitor 40 in Vitro and in
Melanoma Cells. In order to survey the selectivity of the most
potent BRAFV600E inhibitor, 40, we profiled the compound
against 7 other randomly chosen kinases representing each of
the 6 major groups of serine−threonine kinases; PAK1 (STE
group), PIM1 (CAMK group), GSK3 (CMGC group), p70S6K
(AGC group), PI3Kα and PI3Kγ (CK1 group), and the BRAF
related CRAF kinase (TKL group), at an inhibitor concen-
tration of 1 μM. This analysis revealed that 40 showed signifi-
cant selectivity for BRAFV600E and BRAFWT over the other
kinases (Figure 6A). While BRAFV600E and BRAFWT both
We further investigated the effects of 40 in the context of
human-derived skin and melanoma cells. A proliferation assay
indicated that a mutant BRAF melanoma cell line (451Lu,
harboring a BRAFV600E mutation) is more sensitive to the
compound than its normal melanocyte and fibroblast counter-
parts (Figure 6B). Western blot analyses further indicated that
MAPK pathway activity is decreased, as evidenced by reduced
phospho-MEK levels in the presence of the inhibitor, compared
to the vehicle control treated cells (Figure 6C).
DISCUSSION AND CONCLUSIONS
■
In this study, we report on developing an ELISA based high-
throughput assay that is suitable for screening a large library
of small molecule compounds for inhibition of BRAF kinase
activity. We employed this assay to screen a combined diverse
library of over 30,000 organic compounds for BRAFV600E
inhibition. Of the inhibitors that were identified, we further
pursued a family of related quinolol and naphthol compounds
that appeared to represent a novel family of BRAFV600E
inhibitors. We then determined the X-ray crystal structure of
one of these inhibitors bound to the BRAF kinase domain
revealing that the compounds bind in the ATP binding cleft of
the kinase in its active conformation. This structure was used as
a scaffold to initiate a medicinal chemistry campaign that resulted
in the preparation of a family of naphthol compounds that
selectively inhibit BRAFV600E over BRAFWT in vitro with IC50
values in the 80−200 nM range in the presence of saturating levels
of ATP. We also demonstrated that one of these compounds, 40,
has significant selectivity for BRAFV600E and BRAFWT over other
kinases and inhibits MAPK signaling in melanoma cells.
The medicinal chemistry campaign revealed that substitutions
on the naphthol ring system had significant effects on inhibitor
potency for BRAFV600E. Most significantly, substitutions in the
naphthol ring system corresponding to the thienylsulfonamide
present in 2 (the most potent compound obtained from the
primary screen) had the most dramatic effects on inhibitor
potency (Table 2 and Table S1, Supporting Information).
Modeling of 2 onto the X-ray structure of the BRAF-KD/1
complex (where the thienylsulfonamide of 2 overlays with the
chloride group of 1) suggests that this sensitive position of the
inhibitor points into a pocket that is unique to BRAF kinase
(Figure 4C). Indeed, the superposition of the BRAF complexes
with 1 and PLX4720 confirms this, as it reveals that the chloride
group of 1 points toward the sulfonamide group of PLX4720
that sits in the BRAF specificity pocket (Figure 7). In light of
this, it is not a surprise that different sulfonamides at this
position on the backbone of 2 have dramatic effects on inhibitor
potency, with sulfonamides that contain a functionalized aryl
group yielding more potent inhibitors than sulfonamides
containing long aliphatic groups. Clearly, the preparation of
other sulfonamide analogues of 2 might yield more potent
inhibitors, and hybrid molecules containing the 2 backbone with
the sulfonamide derivative of PLX4720 could provide
particularly potent and selective BRAFV600E inhibitors.
Figure 6. In vitro kinase selectivity and melanoma cell response to
compound 40. (A) Activity of 40 against 7 other randomly chosen
kinases (indicated) at an inhibitor concentration of 1 μM. Data is
shown as the mean
inhibition curves of human melanocytes (FOM), fibroblasts (FF2508),
SEM (n = 3). (B) Dose−response growth
and a melanoma cell line (451Lu) in the presence of increasing doses
of 40. Data are shown as the mean
SD (n = 4) and are
representative of three independent experiments. (C) 451Lu
melanoma cells were treated with increasing concentrations of 40.
Cell lysates were analyzed by immunoblotting with the indicated
antibodies.
Initial in vitro and cell-based assays indicate that our current
approach yielded a BRAF/BRAFV600E selective inhibitor (40)
that reduces melanoma cell proliferation preferentially over
primary fibroblasts and melanocytes. However, the downstream
effector of BRAF (MEK) appears to be inhibited regardless of
the cells’ transformation or BRAF status. It is therefore possible
that these inhibitors might be hitting other pathways in cells
that are important for melanoma cell proliferation, and broader
kinase profiling and cell based studies would have to be carried
showed less than 10% remaining activity, CRAF showed about
60% remaining activity, while the other kinases showed 75% or
greater remaining activity. Taken together, these data
demonstrates that 40 has significant selectivity for BRAFV600E
and BRAFWT over representative kinases from the other major
groups of serine−threonine kinases.
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(Ser222/226) monoclonal antibody (Millipore) in TTBS buffer was
subsequently added to the wells and incubated for 1 h with shaking.
Goat antirabbit IgG (H+L)-HRP conjugate (Bio-Rad Laboratories) in
a 1:5000 dilution was added to the wells for incubation at room
temperature with shaking. Finally, the SuperSignal ELISA Pico
chemiluminescent substrate (Pierce Biotechnology) was added to
the wells. The luminescence signal was recorded with a luminescence
filter using a Wallac 1420 luminometer (PerkinElmer).
High throughput inhibitor screening was performed at the Broad
Institute of Harvard and MIT Screening Center. Assays were
conducted in glutathione coated 384-well plates and followed the
procedures essentially as described above but using a 50 μL reaction
volume instead of a 100 μL reaction volume to fit the 384-well plate
format. Specifically, GST-MEK protein diluted in TTBS to 50 μg/mL
was dispensed into the wells of the glutathione coated 384-well plate
to a final volume of 50 μL/well using a Matrix Wellmate Dispanser
with a microplate stacker (Thermo Scientific). Each plate was agitated
using an orbital shaker at 2,500 rpm for 1 min and incubated at room
temperature for 1 h. Plates were aspirated and washed once using a
wash program with vigorous agitation using an automated microplate
washer (Biotek). Then, 3.5 pmol of BRAFV600E kinase domain diluted
in 25 μL of 50 mM HEPES at pH 7.5 buffer was added into each well
of the plate using the Matrix Wellmate Dispanser, and 25 nL of
individual compound (10 mM in 100% DMSO) was transferred into
the solution using a Cybi-Well pin-transfer station (Cybio). Plates
were agitated using an orbital shaker for 1 min and incubated at room
temperature for 1 h. Twenty-five microliters of phosphorylation buffer
was then added into the wells to start the kinase reaction (controls
were tested to ensure the precise timing for the start of the kinase
reaction). Kinase reactions were conducted at room temperature for
30 min and stopped by washing using the microplate washer. A
1:5,000 dilution of Antiphospho-MEK1 (Ser218/222)/MEK2
(Ser222/226) monoclonal antibody (Millipore) in TTBS buffer was
subsequently dispensed into the wells to a final volume of 50 μL and
incubated for 1 h with shaking. Goat antirabbit IgG (H+L)-HRP
conjugate (BioRad Laboratories) in a 1:5,000 dilution was then
dispensed into the wells to a final volume of 50 μL to incubate at
room temperature with agitation. Finally, 50 μL of the SuperSignal
ELISA Pico chemiluminescent substrate (Pierce Biotechnology) was
dispensed into the wells to generate the chemiluminescence signal,
which was detected using a 700 nm luminescence filter by an Envision
chemiluminescence detector (PerkinElmer). A total of 31,976
compounds were screened in duplicate including libraries of a
diversity oriented synthesis (DOS), commercially available drug-like
compounds, bioactive compounds, natural products, and compounds
collected from academic organic synthesis laboratories and a
ChemBridge Kinase inhibitor biased library. Compounds were ranked
based on a composite Z-score of both duplicates, and the top 100
compounds were cherry-picked from the compound plates at the
Broad Institute, and they were reanalyzed by the same assay to confirm
their inhibitory activities. According to the results, the top 23 compounds
that were deemed to have drug-like properties were confirmed by
reordering the compounds from their source vendors and confirming
their inhibitor activities.
Figure 7. Superposition of compound 1 and PLX4720 bound to
BRAF. The inhibitors are shown as stick figures with the protein
shown as an electrostatic surface highlighting the RAF specificity
pocket that is accessible off the chlorine atom of 1.
out to address this possibility. In addition, our cell based studies
reveal that MEK appears to be activated at higher inhibitor
concentrations (Figure 6C), suggesting that high inhibitor
concentration may lead to the transactivation of BRAF/CRAF
dimers as has been noted elsewhere.20,21 Given this observation,
the further development of these inhibitors could take two
paths. In one scenario, these inhibitors could be further
optimizated toward increased BRAFV600E potency and specificity
to develop an alternative BRAFV600E inhibitor that might have
more favorable properties than other BRAFV600E selective
inhibitors. Greater BRAFV600E potency and specificity could be
achieved by further filling the BRAF/BRAFV600E specificity
pocket (Figure 7). Alternatively, these inhibitors could be
optimized to also inhibit CRAF, as our kinase profiling studies
do show some inhibition of 40 against CRAF. Such compounds
might be particularly effective against the reactivation of MAPK
pathway activity through the transactivation of BRAF/CRAF
dimers. Taken together, this study has resulted in the
identification of a new lead series of BRAF inhibitors with the
potential for further preclinical development for therapeutic use
in melanoma.
EXPERIMENTAL SECTION
■
Protein Expression and Purification. The human BRAF kinase
domain and the V600E containing mutant (BRAFWT and BRAFV600E
,
residues 433−726) with an N-terminal 6X-His tag (MDRGSH6GS) to
facilitate protein purification (in the presence of full-length mouse
p50cdc37 to faciliate the proper folding of the BRAF kinase) was
expressed in Sf9 cells and purified to homogeneity as previously
described.19 The protein was stored at 1.5 mg/mL at 4 °C until use.
GST-MEK-His protein was overexpressed at 37 °C in Escherichia coli
BL21 (Gold) cells as previously described.19 The protein was stored at
10 mg/mL at −80 °C until use.
In Vitro ELISA-Based Kinase Assay. Recombinantly expressed
GST MEK-His, diluted in TTBS buffer [20 mM Tris (pH 7.5), 150 mM
NaCl, and 0.05% Tween 20] to 50 μg/mL in a volume of 100 μL,
was bound to the wells of a 96-well glutathione-coated plate (Pierce
Biotechnology). One microliter of compound (as racemic mixtures)
with 2 serial dilutions in a 100% DMSO stock solution was added to a
mixture of 50 μL of a buffer containing 50 mMHEPES (pH7.0) with
0.7 pmol of BRAFV600E kinase. This mixture was incubated at room
temperature for 1 h before it was added to the GST-MEK-His-bound
wells of the 96-well plate. An additional 50 μL of phosphorylation
buffer [50 mM HEPES (pH 7.0), 200 mM NaCl, 10 mM MgCl2, and
200 μM ATP] was added to the well mixture to start the kinase
reaction at 37 °C for 30 min with intermittent shaking. The kinase
reaction was stopped by extensive washing with TTBS buffer,
and a 1:5000 dilution of antiphospho-MEK1 (Ser218/222)/MEK2
IC50 Value Determination. For IC50 calculations of the related
quinolol and naphthol inhibitors, the same assay described above was
used at different inhibitor concentrations to generate a sigmoidal
dose−response curve using BRAFV600E or BRAFWT protein. All dose−
response measurements 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.
Crystallization, Data Collection, and Structure Determina-
tion of the Kinase/Inhibitor Complex. Compound 1 dissolved in
100% DMSO was added to 1.5 mg/mL BRAFWT solution in 4-fold
molar excess, and this mixture was incubated at room temperature for
1 h before the precipitate was removed by high speed centrifugation.
The supernatant was used in the crystallization trials. Crystals were
obtained by mixing 1.35 μL of the BRAFWT/1 complex with 1.35 μL
of crystallization reservoir solution containing 200 mM magnesium
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Article
acetate tetrahydrate, 100 mM sodium cacodylate trihydrate, pH 6.5,
and 20% polyethylene glycol (PEG) 8,000) (Hampton Research)
supplemented with 0.3 μL of 100 mM nicotinamide adenine
dinucleotide (Hampton Research) as an additive by the microbatch
method underneath 6 mL of light mineral oil (Sigma) in a 12 × 6
microbatch plate (Hampton Research). Crystals reached a maximum
size of 30 μm × 30 μm × 300 μm after about 1 week. Crystals were
washed and harvested in cryoprotecting harvest solution containing
200 mM magnesium acetate tetrahydrate, 100 mM sodium cacodylate
trihydrate, pH 6.5, 25% PEG 8,000, and 15% glycerol, and flash frozen
in liquid propane. Data was collected at beamline GM/CA-CAT 23ID-
B at the Advanced Photon Synchrotron Source (Argonne National
Laboratory). We discovered that although the crystals showed
diffraction to 3.5 Å using a typical X-ray beam (300 μm), they
suffered very badly from radiation decay. We were not able to collect
more than 5 frames of diffraction data using this size X-ray beam. In
contrast, we found that by using an X-ray beam of 10 by 10 μm
(minibeam setup at APS beamline 23ID-B), we were able to collect
about 20−30 images per crystal position, and both the resolution and
mosaicity were significantly improved. Also, importantly, the minibeam
setup allowed us to move the beam center to new, unexposed parts
of the crystal, allowing us to collect a complete data set from a single
crystal. Diffraction data was indexed, integrated, and scaled using the
HKL2000 package (HKL Research) and was further processed using
the CCP4 program suite.22
The structure was determined by molecular replacement using the
program Molrep23 from the CCP4 suite using a previously determined
BRAF structure (PDB ID: 1UWH) as a search model. The spacegroup
was determined to be P41212, and each asymmetric unit contained two
molecules. Electron density corresponding to the organic BRAF
inhibitor 1 was well resolved in both molecules of the asymmetric unit,
and the ligand model was placed into the electron density from the
calculated Fo − Fc map and adjusted in Coot.24 Parameter and
topology files for 1 used in the refinements were generated from the
HIC-UP XDICT server (http://alpha2.bmc.uu.se/hicup/xdict.html).
This was followed by additional refinement using CNS,25 and the final
model was checked for errors using an CNS composite omit map for
the protein model and a simulated annealing omit map for inhibitor.
The final model was refined with excellent geometry and refinement
statistics (Table 1).
Compound 24: Spectroscopic data was identical to that reported
previously (WO Patent 2010/005534, January 14, 2010).26
Compound 25: To a mixture of 4-amino-1-naphthol·HCl (0.196 g,
1.00 mmol), pyridine (0.17 mL, 2.1 mmol), and DMAP (0.005 g, 0.04
mmol) in CH2Cl2 was added 2-thiopenesulfonyl chloride (0.194 g,
1.06 mmol). The mixture was heated at reflux for 18 h. The mixture
was allowed to cool to 25 °C, and the solvent was removed in vacuo.
The resulting residue was diluted with EtOAc (20 mL), and the
solution was washed with water (2 × 10 mL), washed with brine, dried
over Na2SO4, filtered, and concentrated to afford a brown residue. The
residue was purified by flash chromatography (5:95 MeOH/CH2Cl2)
1
to afford the product as a tan solid (0.240 g, 79%). H NMR (500
MHz, DMSO-d6) δ 10.32 (s, 1H), 9.99 (s, 1H), 8.10−8.08 (m, 1H),
7.84 (dd, J = 5.0, 1.4 Hz, 2H), 7.43−7.37 (m, 2H), 7.35 (dd, J = 3.7,
1.4 Hz, 1H), 7.06 (dd, J = 5.0, 3.7 Hz, 1H), 6.97 (d, J = 8.1 Hz, 1H),
6.76 (d, J = 8.1 Hz, 1H); 13C NMR (125 MHz, DMSO-d6) δ 153.3,
141.3, 133.5, 132.6, 132.2, 128.1, 126.8, 126.6, 125.5, 125.4, 123.6,
123.4, 122.7, 107.9; IR (thin film) 3268, 3115, 1699, 1584, 1317, 1153,
1016, 774 cm−1; HRMS (ESI) m/z calcd for C14H11NO3NaS2 (M +
Na)+ 328.0078; found, 328.0078.
Compound 26: To a solution of 2-chloro-1,4-naphthoquinone27
(0.119 g, 0.50 mmol) and 2-thiophene sulfonamide (0.098 g, 0.60
mmol) cooled to 0 °C in THF (1.8 mL) was added the titanium(IV)
chloride tetrahydrofuran complex (0.200 g, 0.60 mmol), followed by
the addition of triethylamine (0.18 mL, 1.3 mmol). The mixture was
allowed to warm to 25 °C and was heated to 60 °C using a microwave
(80 W) for 1 h. The mixture was cooled to 25 °C and added to EtOAc
(20 mL). The mixture was filtered though a pad of Celite with EtOAc.
The filtrate was concentrated to dryness in vacuo. The resultant
residue was suspended in CH2Cl2 (10 mL) and filtered though a pad
of Celite. The filtrate was concentrated in vacuo to dryness, and the
resulting residue was suspended in EtOAc (10 mL). To the suspension
was added Na2S2O4 (0.436 g, 2.50 mmol) followed by H2O (5 mL).
The mixture was stirred at 25 °C for 1 h. The layers were separated,
and an aqueous layer was extracted with EtOAc (3 × 10 mL). The
combined organic layers were washed with brine (25 mL), dried over
Na2SO4, filtered, and concentrated to afford a brown residue. The
residue was purified by flash chromatography (40:60 EtOAc/hexanes)
1
to afford the product as a tan solid (0.081 g, 47%). H NMR (500
MHz, DMSO-d6) δ 10.27 (s, 2H), 8.17 (d, J = 8.3 Hz, 1H), 7.87 (d,
J = 8.4 Hz, 1H), 7.83 (d, J = 4.4 Hz, 1H), 7.50 (d, J = 7.6 Hz, 1H),
7.46−7.41 (m, 2H), 7.07 (s, 1H), 7.05 (s, 1H);); 13C NMR (125
MHz, DMSO-d6) δ 148.4, 140.5, 133.9, 133.1, 130.7, 128.3, 127.3,
127.0, 126.54, 126.47, 125.0, 123.8, 122.8, 113.7; IR (thin film) 3276,
1581, 1458, 1401, 1315, 1153, 849, 759 cm−1; HRMS (ESI) m/z calcd
for C14H9NO3S2Cl (M − H)− 337.9712; found, 337.9714.
Compounds and Chemical Synthesis. General. Solvents used
for extraction and purification were HPLC grade from Fisher. Unless
otherwise indicated, all reactions were run under an inert atmosphere
of argon. Anhydrous tetrahydrofuran, ethyl ether, and toluene were
obtained via passage through an activated alumina column. Merck
precoated silica gel plates (250 mm, 60 F254) were used for analytical
TLC. Spots were visualized using 254 nm ultraviolet light, with either
anisaldehyde or potassium permanganate stains as visualizing agents.
Chromatographic purifications were performed on Sorbent Tech-
nologies silica gel (particle size 32−63 μm). 1H and 13C NMR spectra
were recorded at 500 and 125 MHz, or 360 and 90 MHz, respectively,
in CDCl3, DMSO-d6, or CD3OD on a Bruker AM-500, a DRX-500, or
a DMX-360 spectrometer. Chemical shifts are reported relative to
internal chloroform (d 7.26 for 1H, d 77.0 for 13C), DMSO-d6 (d 2.50
Compound 27: To a solution of 2-bromo-1,4-naphthoquinone28
(0.119 g, 0.50 mmol) and 2-thiophene sulfonamide (0.098 g, 0.60
mmol) cooled to 0 °C in THF (1.8 mL) was added the titanium(IV)
chloride tetrahydrofuran complex (0.200 g, 0.60 mmol), followed by
the addition of triethylamine (0.18 mL, 1.3 mmol). The mixture was
allowed to warm to 25 °C and was heated to 60 °C using a microwave
(80 W) for 1 h. The mixture was cooled to 25 °C and added to EtOAc
(20 mL). The mixture was filtered though a pad of Celite with EtOAc.
The filtrate was concentrated to dryness in vacuo. The resultant
residue was suspended in CH2Cl2 (10 mL) and filtered though a pad
of Celite. The filtrate was concentrated in vacuo to dryness, and the
resulting residue was suspended in EtOAc (10 mL). To the suspension
was added Na2S2O4 (0.436 g, 2.50 mmol) followed by H2O (5 mL).
The mixture was stirred at 25 °C for 1 h. The layers were separated,
and the aqueous layer was extracted with EtOAc (3 × 10 mL). The
combined organic layers were washed with brine (25 mL), dried over
Na2SO4, filtered, and concentrated to afford a brown residue. The
residue was purified by flash chromatography (40:60 EtOAc/hexanes)
to afford the product as a brown solid (0.036 g, 19%). 1H NMR (500
MHz, DMSO-d6) δ 10.26 (s, 1H), 10.24 (s, 1H), 8.18 (d, J = 8.4 Hz,
1H), 7.88−7.86 (m, 2H), 7.51 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.45
(ddd, J = 8.4, 6.9, 1.3 Hz, 1H), 7.40 (dd, J = 3.7, 1.3 Hz, 1H), 7.08 (dd,
J = 5.0, 3.7 Hz, 1H), 7.06 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ
147.8, 140.1, 133.4, 132.5, 130.1, 127.7, 126.6, 126.3, 126.0, 125.9,
1
1
for H, d 39.5 for 13C), or CD3OD (d 3.31 for H, d 49.0 for 13C).
Infrared spectra were recorded on a NaCl plate using a Perkin-Elmer
1600 series Fourier transform spectrometer. High resolution mass
spectra were obtained by Dr. Rakesh Kohli at the University of
Pennsylvania Mass Spectrometry Service Center on an Autospec high
resolution double-focusing electrospray ionization/chemical ionization
spectrometer with either the DEC 11/73 or OPUS software data
system. Melting points were obtained on a Thomas-Hoover capillary
melting point apparatus and are uncorrected. On the basis of the
analytical methods described above, all compounds were judged to
have purities of ≥95%.
General procedure for the synthesis of naphthol derivatives was
adapted from WO 2010/005534.26 Unless otherwise specified,
analogues were synthesized using the general procedure.
Compound 2: Spectroscopic data was identical to that reported
previously (WO Patent 2010/005534, January 14, 2010).26
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1
124.5, 123.3, 122.2, 113.2; IR (thin film) 3479, 3098, 1582, 1459, 1415,
1315, 1154, 759 cm−1; HRMS (ESI) m/z calcd for C14H9NO3S2Cl
(M − HBr + Cl)− 337.9712; found, 337.9707.
Compound 36: H NMR (500 MHz, DMSO-d6) δ 10.23 (s, 1H),
9.44 (s, 1H), 8.62 (s, 1H), 8.24 (d, J = 8.3 Hz, 1H), 8.18 (d, J = 8.1
Hz, 1H), 7.62−7.55 (m, 2H), 7.31 (s, 1H), 3.01 (t, J = 7.8 Hz, 2H),
1.71−1.65 (m, 2H), 1.30−1.19 (m, 10H), 0.84 (t, J = 7.0 Hz, 3H); 13C
NMR (125 MHz, DMSO-d6) δ 145.1, 140.5, 134.4, 131.6, 127.2,
127.1, 126.1, 125.8, 123.9, 122.6, 50.7, 31.2, 28.5, 28.4, 27.5, 23.2, 22.1,
14.0; IR (thin film) 3253, 2925, 2855, 1568, 1454, 1279, 1141, 766
cm−1; HRMS (ESI) m/z calcd for C20H26N4O3NaS2 (M + Na)+
457.1344; found, 457.1325.
1
28: H NMR (500 MHz, DMSO-d6) δ 10.09 (s, 1H), 8.20 (d, J =
8.0 Hz, 1H), 7.88−7.82 (m, 2H), 7.53−7.47 (m, 1H), 7.47−7.41 (m,
1H), 7.31 (dd, J = 1.5, 3.5 Hz, 1H), 7.21 (s, 2H), 7.05 (dd, J = 3.5, 5.0
Hz, 1H), 6.83 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 152.2,
145.1, 137.4, 131.4, 131.0, 129.8, 129.2, 127.2, 126.1, 125.7, 123.9,
123.4, 123.1, 122.6; IR (KBr disk) 3262, 3105, 1709, 1567, 1506, 1451,
1403, 1377, 1325, 1157, 1016, 911, 854, 766, 723, 669 cm−1; HRMS
(ESI) m/z calcd for C17H14N3O3S3 (M + H)+ 404.0197; found,
404.0204.
Compound 37:1H NMR (500 MHz, CD3OD) δ 8.25 (dt, J = 4.4,
2.4 Hz, 1H), 8.19 (s, 1H), 8.06 (dt, J = 4.3, 2.4 Hz, 1H), 7.42−7.41
(m, 2H), 7.38 (s, 1H), 7.18−7.08 (m, 10H), 6.90 (dt, J = 8.8, 4.4 Hz,
1H), 6.63 (t, J = 9.4 Hz, 1H), 4.02 (s, 4H); 13C NMR (125 MHz,
CD3OD) δ 156.2, 155.7, 155.3, 154.2, 153.3, 138.6, 137.1, 133.5,
130.8, 129.3, 128.9, 128.6, 128.5, 128.2, 127.2, 127.1, 125.2, 124.3,
123.8, 119.1, 112.8, 112.6, 68.8, 57.0, 26.4; IR (thin film) 3087, 2924,
1705, 1570, 1485, 1364, 1171, 745 cm−1; HRMS (ESI) m/z calcd for
C32H25N5O3F2NaS2 (M + Na)+ 652.1265; found, 652.1271.
Compound 29: 1H NMR (500 MHz, CDCl3) δ 10.85 (s, 1H), 8.44
(d, J = 5.0 Hz, 1H), 8.39 (d, J = 7.5 Hz, 1H), 7.76 (d, J = 7.5 Hz, 1H),
7.61 (t, J = 7.5 Hz, 1H), 7.53−7.43 (m, 3H), 7.41 (s, 1H), 7.36 (d, J =
4.0 Hz, 1H), 7.24 (s, 1H), 7.16 (dd, J = 4.0, 7.5 Hz, 1H), 6.89 (dd, J =
3.5, 5.0 Hz, 1H), 6.61 (br s, 1H); 13C NMR (125 MHz, CDCl3) δ
159.8, 156.4, 149.1, 139.8, 138.1, 133.2, 132.6, 131.1, 128.5, 127.5,
126.4, 126.3, 124.0, 123.5, 123.1, 121.9, 121.6, 110.1; IR (KBr disk)
3235, 3062, 2920, 2848, 1564, 1451, 1414, 1374, 1322, 1153, 1070,
1015, 762, 719 cm−1; HRMS (ESI) m/z calcd for C19H14N2O3S3 (M +
H)+ 415.0245; found, 415.0257.
1
Compound 38: H NMR (500 MHz, DMSO-d6) δ 10.98 (s, 1H),
10.38 (s, 1H), 8.59 (s, 1H), 8.24−8.22 (m, 1H), 7.99 (s, 1H), 7.58 (s,
2H), 6.97 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 157.1, 152.4,
145.5, 143.2, 138.8, 136.8, 131.8, 128.8, 127.7, 126.7, 126.1, 123.5,
123.0, 115.6, 112.1; IR (thin film) 3129, 2928, 1704, 1520, 1502, 1176,
1101, 992 cm−1; HRMS (ESI) m/z calcd for C18H10N4O3F5S2 (M +
H)+ 489.0115; found, 489.0110.
Compound 30: Spectroscopic data was identical to that reported
previously (WO Patent 2010/005534, January 14, 2010).26
Compound 31: 1H NMR (500 MHz, CDCl3) δ 8.31 (d, J = 8.0 Hz,
1H), 7.98 (d, J = 8.0 Hz, 1H), 7.62−7.53 (m, 2H), 7.42 (dd, J = 1.0,
5.0 Hz, 1H), 7.35 (dd, J = 1.0, 3.5 Hz, 1H), 7.30−7.16 (m, 5H), 7.10−
7.06 (m, 2H), 6.83 (dd, J = 4.0, 5.0 Hz, 1H), 6.62 (br s, 1H); 13C
NMR (125 MHz, DMSO-d6) δ 154.9, 139.4, 135.5, 133.3, 133.0,
132.6, 131.5, 129.4, 128.8, 127.6, 127.4, 126.7, 124.3, 124.0, 123.8,
122.6, 108.9; IR (KBr disk) 3391, 3272, 2920, 2851, 1568, 1435, 1373,
1346, 1311, 1266, 1220, 1156, 1070, 907, 720 cm−1; HRMS (ESI) m/z
calcd for C20H15NNaO3S3 (M + H)+ 436.0112; found, 436.0120.
Compound 32: 1H NMR (500 MHz, DMSO-d6) δ 10.50−9.50 (br,
2H, contains two s), 8.16 (d, J = 8.5 Hz, 1H), 8.15−7.75 (m, 3H,
contains one br s; one d, J = 8.5 Hz; one dd, J = 1.5, 5.0 Hz), 7.54−
7.47 (m, 1H), 7.47−7.40 (m, 1H), 7.26 (dd, J = 1.5, 3.5 Hz, 1H), 7.02
(dd, J = 3.5, 5.0 Hz, 1H), 6.68 (s, 1H); 13C NMR (125 MHz, DMSO-
d6) δ 140.1, 133.0, 132.1, 130.7, 127.5, 126.4, 126.0, 125.5, 124.6,
123.5, 122.1; IR (KBr disk) 3101, 2991, 2917, 1568, 1506, 1452, 1402,
1376, 1323, 1263, 1156, 1072, 1020, 912, 854, 764, 723, 670 cm−1;
HRMS (ESI) m/z calcd for C16H13N4O3S3 (M + H)+ 405.0150; found,
405.0166.
1
Compound 39: H NMR (500 MHz, DMSO-d6) δ 10.76 (s, 1H),
10.31 (s, 1H), 8.57 (s, 1H), 8.23−8.21 (m, 1H), 7.93 (dd, J = 6.6, 2.9
Hz, 1H), 7.69−7.65 (m, 1H), 7.53 (dd, J = 6.4, 3.2 Hz, 2H), 7.32 (t,
J = 9.0 Hz, 1H), 7.16 (s, 1H), 2.90−2.82 (m, 4H); 13C NMR (125
MHz, DMSO-d6) δ 176.2, 159.6, 157.5, 155.4, 153.3, 145.5, 135.6,
131.8, 129.8, 127.7, 126.4, 126.0, 123.4, 123.0, 119.0, 118.4, 114.0,
113.7, 29.1; IR (thin film) 2920, 2848, 1719, 1592, 1562, 1492, 1298,
1172 cm−1; HRMS (ESI) m/z calcd for C22H15N5O5NaS2F2 (M +
Na)+ 554.0380; found, 554.0386.
Compound 40: To a solution of 2-chloro-1,4-naphthoquinone27
(0.096 g, 0.50 mmol) and biphenyl-4-sulfonamide (0.140 g, 0.60 mmol)
cooled to 0 °C in THF (1.8 mL) was added the titanium(IV) chloride
tetrahydrofuran complex (0.200 g, 0.60 mmol), followed by the addition
of triethylamine (0.18 mL, 1.3 mmol). The mixture was allowed to
warm to 25 °C and was heated to 60 °C using a microwave (80 W) for
1 h. The mixture was cooled to 25 °C and added to EtOAc (20 mL).
The mixture was filtered though a pad of Celite with EtOAc. The filtrate
was concentrated to dryness in vacuo. The resultant residue was
suspended in CH2Cl2 (10 mL) and filtered though a pad of Celite.
The filtrate was concentrated to dryness and then dissolved in THF
(10 mL). To the solution was added 1H-1,2,4-triazole-3-thiol (0.051 g,
0.50 mmol), and the mixture was stirred at 25 °C for 4 h. The mixture
was concentrated in vacuo to dryness, and the resultant residue was
suspended in EtOAc (10 mL). To the suspension was added Na2S2O4
(0.436 g, 2.50 mmol) followed by H2O (5 mL). The mixture was stirred
at 25 °C for 1 h. The layers were separated, and the aqueous layer
was extracted with EtOAc (3 × 10 mL). The combined organic layers
were washed with brine (25 mL), dried over Na2SO4, filtered, and
concentrated to afford a brown residue. The residue was purified by flash
chromatography (3:97 MeOH/CH2Cl2) to afford the product as a
colorless solid (0.111 g, 47%): 1H NMR (500 MHz, DMSO-d6) δ 10.22
(s, 0.5H), 9.98 (s, 1H), 8.54 (s, 0.5H), 8.19 (d, J = 7.9 Hz, 1H), 7.97 (d,
J = 8.0 Hz, 1H), 7.74 (d, J = 8.5 Hz, 2H), 7.68 − 7.66 (m, 5H), 7.51−
7.41 (m, 6H), 6.96 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 144.1,
138.6, 138.4, 129.2, 128.5, 127.5, 127.2, 127.1, 127.0, 126.0, 125.7, 123.5,
122.6; IR (thin film) 3249, 2923, 1704, 1593, 1452, 1320, 1160,
763 cm−1; HRMS (ESI) m/z calcd for C24H18N4O3NaS2 (M + Na)+
497.0718; found, 497.0711.
1
Compound 33: H NMR (500 MHz, DMSO-d6) δ 10.13 (s, 1H),
9.73 (s, 1H), 8.54 (s, 1H), 8.19−8.17 (m, 1H), 8.02−7.99 (m, 2H),
7.52−7.47 (m, 2H), 7.44−7.42 (m, 1H), 7.40 (d, J = 0.3 Hz, 1H), 6.89
(s, 1H), 4.11 (t, J = 8.5 Hz, 2H), 3.06 (t, J = 8.4 Hz, 2H), 2.18 (s, 3H);
13C NMR (125 MHz, DMSO-d6) δ 169.9, 135.1, 134.7, 134.1, 133.1,
127.3, 127.2, 126.5, 126.3, 126.2, 126.0, 124.1, 123.9, 123.8, 122.8,
115.4, 113.6, 111.3, 49.0, 27.2, 24.5; IR (thin film) 3121, 2924, 1708,
1660, 1481, 1397, 1149, 1072 cm−1; HRMS (ESI) m/z calcd for
C22H19N5O4NaS2 (M + Na)+ 504.0776; found, 504.0788.
1
Compound 34: H NMR (500 MHz, DMSO-d6) δ 14.00 (br s,
1H), 10.80−9.60 (br s, 2H), 8.46 (br s, 1H), 8.18 (d, J = 8.0 Hz, 1H),
7.93−7.73 (m, 5H), 7.55−7.37 (m, 2H), 6.88 (br s, 1H); 13C NMR
(125 MHz, DMSO-d6) δ 152.2, 145.1, 143.5, 132.7, 132.4, 132.2,
131.3, 129.1, 127.7, 126.9, 126.2, 126.1, 125.9, 125.6, 124.5, 123.8,
123.2, 122.6, 122.3; 19F NMR (470 MHz, DMSO-d6) δ −61.6; IR
(KBr disk) 3102, 2989, 2928, 1730, 1568, 1504, 1453, 1403, 1376,
1322, 1267, 1168, 1062, 762, 711 cm−1; HRMS (ESI) m/z calcd for
C19H14F3N4O3S2 (M + H)+ 467.0459, found: 467.0471.
1
Compound 35: H NMR (500 MHz, DMSO-d6) δ 9.42 (s, 1H),
8.49 (s, 1H), 8.23 (d, J = 8.1 Hz, 1H), 8.17 (d, J = 8.5 Hz, 1H), 7.62−
7.55 (m, 2H), 7.29 (s, 1H), 3.00 (dd, J = 7.9, 7.7 Hz, 2H), 1.70−1.64
(m, 2H), 1.30−1.17 (m, 14H), 0.85 (t, J = 6.9 Hz, 3H); 13C NMR
(125 MHz, DMSO-d6) δ 151.9, 133.1, 132.1, 131.6, 130.2, 128.7
127.1, 126.0, 125.0, 124.5, 123.8, 122.6, 50.7, 31.3, 28.87, 28.68, 28.48,
27.5, 23.1, 22.1, 14.0; IR (thin film) 2923, 2853, 1567, 1454, 1279,
1141, 1076, 767 cm−1; HRMS (ESI) m/z calcd for C22H30N4O3NaS2
(M + Na)+ 485.1657; found, 485.1656.
1
Compound 41: H NMR (360 MHz, DMSO-d6) δ 14.11 (br s,
1H), 10.60−9.70 (br s, 2H), 8.32 (br s, 1H), 8.21 (s, 1H), 8.15 (d, J =
8.0 Hz, 1H), 8.15−7.75 (m, 4H), 7.80−7.55 (m, 3H), 7.55−7.30 (m,
2H), 6.92 (br s, 1H); 13C NMR (125 MHz, DMSO-d6) δ 151.8, 145.0,
136.9, 134.1, 131.5, 131.4, 129.2, 129.1, 128.7, 128.6, 127.8, 127.7,
127.5, 126.8, 125.9, 125.6, 124.4, 123.5, 122.4, 122.3; IR (KBr disk)
3259, 3053, 2851, 1564, 1508, 1468, 1450, 1347, 1318, 1285, 1156,
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Journal of Medicinal Chemistry
Article
1026, 945, 766, 666 cm−1; HRMS (ESI) m/z calcd for C22H17N4O3S2
(M + H)+ 449.0742; found, 449.0753.
Kinase Specificity Profile. The kinase assays for PAK1, PIM1,
GS3Kα, PI3Kα, and PI3Kγ were carried out as described previously,29
and CRAF was assayed as described above for BRAF using between 20
and 30 nM enzyme and 100 μM ATP .
ACKNOWLEDGMENTS
■
We thank Jessie Villanueva for useful discussions. This work
was supported by NIH grant CA114046 awarded to M.H.,
R.M. and J.W. This project was also funded in part with Federal
funds from the National Cancer Institute’s Initiative for
Chemical Genetics, National Institutes of Health, under
Contract No. N01-CO-12400 with some experiments per-
formed with the assistance of the Chemical Biology Platform of
the Broad Institute of Harvard and MIT. We acknowledge
support of the Protein Expression and Libraries Core Facility
at the Wistar Institute, which is supported by NIH grant
CA010815.
The p70S6K kinase assay was performed using labeled γ-P32 ATP,
and the incorporation of labeled phosphate onto substrate (S6 kinase/
Rsk2 peptide) was monitored. Various concentrations of inhibitor
were incubated at room temperature in 20 mM MOPS, 30 mM MgCl2,
0.8 μg/μL BSA, and 5% DMSO (resulting from the inhibitor stock
solution), pH 7.0, in the presence of substrate (S6 kinase/Rsk2
substrate peptide 2: 3.6 μM) and p70S6K kinase (20nM). After 15 min,
the reaction was initiated by adding ATP to a final concentration of
100 μM, including approximately 0.4 μCi/μL γ-P32 ATP. Reactions
were performed in a total volume of 25 μL. After 30 min, the reaction
was terminated by spotting 17.5 μL on a circular P81 phosphocellulose
paper (diameter 2.1 cm, Whatman) followed by washing four times
(five minutes each wash) with 0.75% phosphoric acid and once with
acetone. The dried P81 papers were transferred to a scintillation vial
with 5 mL of scintillation cocktail and the counts per minute (CPM)
determined with a Beckmann 6000 scintillation counter. IC50 values
were defined to be the concentration of inhibitor at which the CPM
was 50% of the control sample, corrected by the background.
Melanoma Proliferation Assay. The human melanoma cell line
(451Lu) was isolated as previously described30 and cultured in
Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine
serum. Normal human primary melanocytes (FOM) and fibroblasts (FF)
were isolated from the human epidermis of neonatal foreskins and
cultured as described.31 Cells (5000/well) were seeded in 96-well plates
and allowed to adhere overnight before treatment with 40 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 DMSO-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.
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ASSOCIATED CONTENT
■
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* Supporting Information
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AUTHOR INFORMATION
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Corresponding Author
*(J.W.) Department of Chemistry, University of Pennsylvania,
Philadelphia, PA 19104. Phone: 215-898-0052. Fax: 215-573-
2112. E-mail: winkler@sas.upenn.edu. (R.M.) The Wistar Institute,
3601 Spruce Street, Philadelphia, PA 19104. Phone: 215-898-
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Notes
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