Discovery of dabrafenib: A selective inhibitor of Raf Kinases with antitumor activity against B-Raf-driven tumors

Article abstract of DOI:10.1021/ml4000063

Hyperactive signaling of the MAP kinase pathway resulting from the constitutively active B-Raf^V600E mutated enzyme has been observed in a number of human tumors, including melanomas. Herein we report the discovery and biological evaluation of GSK2118436, a selective inhibitor of Raf kinases with potent in vitro activity in oncogenic B-Raf-driven melanoma and colorectal carcinoma cells and robust in vivo antitumor and pharmacodynamic activity in mouse models of B-Raf^V600E human melanoma. GSK2118436 was identified as a development candidate, and early clinical results have shown significant activity in patients with B-Raf mutant melanoma.

Full text of DOI:10.1021/ml4000063

Letter
pubs.acs.org/acsmedchemlett
Discovery of Dabrafenib: A Selective Inhibitor of Raf Kinases with
Antitumor Activity against B‑Raf-Driven Tumors
Tara R. Rheault,*,† John C. Stellwagen,† George M. Adjabeng,† Keith R. Hornberger,†
Kimberly G. Petrov,† Alex G. Waterson,† Scott H. Dickerson,∥ Robert A. Mook, Jr.,† Sylvie G. Laquerre,‡
Alastair J. King,‡ Olivia W. Rossanese,‡ Marc R. Arnone,‡ Kimberly N. Smitheman,‡
Laurie S. Kane-Carson,§ Chao Han,‡ Ganesh S. Moorthy,‡ Katherine G. Moss,‡ and David E. Uehling†
†Oncology R&D Medicinal Chemistry, GlaxoSmithKline, Research Triangle Park , North Carolina 27709, United States
‡Oncology R&D Cancer Research, GlaxoSmithKline, Collegeville Road, Collegeville, Pennsylvania 19426, United States
§Platform Technology & Science, GlaxoSmithKline, Research Triangle Park, North Carolina 27709, United States
∥Computational and Structural Chemistry, GlaxoSmithKline, Research Triangle Park, North Carolina 27709, United States
S
* Supporting Information
ABSTRACT: Hyperactive signaling of the MAP kinase
pathway resulting from the constitutively active B-RafV600E
mutated enzyme has been observed in a number of human
tumors, including melanomas. Herein we report the discovery
and biological evaluation of GSK2118436, a selective inhibitor
of Raf kinases with potent in vitro activity in oncogenic B-Raf-
driven melanoma and colorectal carcinoma cells and robust in
vivo antitumor and pharmacodynamic activity in mouse
models of B-RafV600E human melanoma. GSK2118436 was
identified as a development candidate, and early clinical results
have shown significant activity in patients with B-Raf mutant melanoma.
KEYWORDS: B-Raf, GSK2118436, dabrafenib, melanoma, MAP kinase
he MAP kinase pathway plays a central role in cellular
growth processes, and aberrant signaling through this
inhibitory activity against the B-RafV600E enzyme, and in both
cellular mechanistic (pERK) and proliferation assays in the B-
RafV600E SKMEL28 melanoma cell line (Table 1). Thiazole 1
also had low clearance and good overall oral systemic exposure
in rats. However, this compound displayed very high clearance
and poor bioavailability in nonrodent species. Additionally,
metabolite identification studies conducted in dog and monkey
liver microsomes identified several major metabolites clustered
in the tail and core regions of the molecule. We hypothesized
that drug exposure after an oral dose in the higher species was
limited by rapid compound metabolism. Medicinal chemistry
efforts were thus focused on improving pharmacokinetic
properties in dog while preserving the favorable target potency,
selectivity, and rodent pharmacokinetic properties inherent in
this template.9
T
pathway has been observed in many cancers.1−4 Notably,
activating mutations in B-Raf have been identified in more than
60% of all melanomas.1 The most common mutation, observed
in 80−90% of B-Raf mutant cancers, is the B-RafV600E mutation,
which renders the kinase constitutively active. This mutation,
which substitutes valine with glutamic acid at amino acid 600 in
the activation loop, mimics regulatory phosphorylation and
results in in vitro kinase activity of the protein being 500-fold
greater than that of wild-type B-Raf.1,5 Because tumors bearing
an activating B-RafV600E mutation show oncogenic addiction to
this hyperactivated signaling pathway, it was postulated that
small molecule inhibitors of B-RafV600E kinase activity offer a
novel, targeted approach for the treatment of B-RafV600E-driven
cancers.6 This hypothesis has since been tested clinically, and
significant responses to selective B-Raf inhibitor therapy have
been observed.7
Having established the sulfonamide as a key pharmacophore
required for potent cellular inhibition of B-RafV600E, we
performed significant structural modifications elsewhere to
lower the molecular weight and reduce the number of
metabolic sites contained within the template. Truncation of
the tail by replacing the aminopyridine in 1 with a small alkyl
Herein we describe the medicinal chemistry efforts leading to
the discovery of GSK2118436 (dabrafenib), a potent, selective,
and efficacious inhibitor of B-RafV600E. GSK2118436 is
currently in phase III clinical development for the treatment
of activating B-Raf mutant tumors.
Our early efforts aimed at discovering inhibitors of B-RafV600E
suitable for clinical evaluation led to the identification of
thiazole 1.8 This compound shows potent in vitro kinase
Received: January 12, 2013
Accepted: February 7, 2013
© XXXX American Chemical Society
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Table 1. Structure, in Vitro Data, and Pharmacokinetic Data for Compound 1 in Rat, Dog, and Monkey
a
b
c
B-RafV600E IC50 (nM)
3.8
pERK IC50 (nM)
23
SKMEL28 proliferation IC50 (nM)
48
species
intrinsic Cli
Cl
po DNAUC
% F
d
d
rat
9
9
1748
85
10
5
e
e
dog
53
38
39
42
e
e
monkey
8
23
a
b
Intrinsic clearance data from liver microsomes, reported in units of milliliters per minute per gram of liver. Intravenous blood clearance reported in
c
units of milliliters per minute per kilogram. Oral dose-normalized AUC (DNAUC) reported as nanograms hour per milliliter per milligram per
kilogram (n = 3). Dosing vehicle: 1% DMSO, 10% solutol in saline or water, at pH 5.0. Dosing vehicle: 1% DMSO, 30% PEG400 in saline or
d
e
water, at pH 3.5−4.5.
Table 2. Truncated Tail SAR
a
B-RafV600E enzymatic activity. Values are means of at least two experiments; individual values are within 2-fold of the reported mean value.
Inhibition of pERK in SKMEL28 cells. Values are means of at least two experiments; individual values are within 2-fold of the reported mean value.
b
c
Inhibition of SKMEL28 cell proliferation. IC50 values are means of at least experiments; individual values are within 3-fold of the reported mean
d
value. Intravenous clearance reported in units of milliliters per minute per kilogram. Oral dose-normalized AUC (DNAUC) reported as nanograms
hours per milliliter per milligram per kilogram (n = 3). Data from four-compound cassette dosing. Pharmacokinetic values based on cassette dosing
were found to be within 2-fold of those of discretely dosed experiments. Not tested. Dosing vehicle: 1% DMSO, 10% solutol in saline or water, at
pH 5.0. Dosing vehicle: 1% DMSO, 30% PEG400 in saline or water, at pH 3.5−4.5.
e
f
g
h
group (5) or polar functionality (6−8) largely preserved
enzyme potency, cellular mechanistic potency (pERK), and
inhibition of proliferation in B-RafV600E SKMEL28 cells but
resulted in significantly higher clearance in rat (Table 2).10
B
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ACS Medicinal Chemistry Letters
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Surprisingly, unsubstituted aminopyrimidine hinge binder (2)
maintained in vitro potency against B-Raf;11 however, this
compound also demonstrated high in vivo clearance in rat.
Core group modifications were attempted in an effort to alter
the metabolic stability of this series, leading to a tert-butyl core
analogue (3). While this change from isopropyl to tert-butyl
resulted in a noteworthy increase in potency, rat intravenous
(iv) clearance was still very high and oral exposure was
commensurately low. Since fluoro substitution at position 6 of
the phenyl ring [R1 = F (Table 2)] led to an important
improvement in rat pharmacokinetic properties in previous
SAR efforts,8 this substitution was attempted in the truncated
tail series (4). Unfortunately, in this context, fluoro substitution
at R1, when combined with the tail truncation, led to a decrease
in potency such that further progression of compound 4 was
abandoned.
pharmacokinetic profiles were observed, in contrast to those of
previously prepared analogues.
The remarkable improvement in dog pharmacokinetic
properties prompted further investigation in order to under-
stand the rationale for the finding. In an effort to understand
the high clearance observed in dog from sulfone-containing tail
compounds (Table 2), 9 was incubated in dog hepatocytes and
the major metabolites were identified by mass spectrometry
(Figure 2). Following incubation of compound 9 in dog
Given the favorable potency demonstrated with compound
8, SAR in this series was further explored. In an effort to lower
IV clearance in rats, compounds with substitution at positions
R, R1, and R2 were prepared and evaluated. Fluorination at R1
(9) decreased the enzyme and cellular potency but resulted in a
marked improvement in rat iv clearance and oral exposure.
Further SAR on this template showed that fluorination at R2
(10) improved or maintained the enzyme and cellular potency
and resulted in improved pharmacokinetic properties compared
to those of the des-fluoro analogue (8). Replacement of the
isopropyl thiazole core with a tert-butyl thiazole core (11) also
resulted in improved cellular potency. While rat pharmacoki-
netic properties could be improved by extrapolating previous
SAR findings from this series, analogous improvements in dog
pharmacokinetic properties were not realized, as compounds 9
and 10 both displayed high clearance and poor oral exposure in
dog.
Further attempts to optimize pharmacokinetic properties
while maintaining potency in this series were made by
combining the structural modifications of fluorination at R2,
replacing the isopropyl core with a tert-butyl group, and
utilizing the unsubstituted aminopyrimidine tail group from
compounds 2−4. Through this effort, compound 12
(GSK2118436) was synthesized and evaluated (Table 2 and
Figure 1).
Figure 2. Metabolite identification of 9 in dog hepatocytes.
hepatocytes, two major metabolites were identified: compound
13, showing complete dealkylation of the ethylmethylsulfone
tail, and compound 14, a product of oxidation of the isopropyl
core. In retrospect, it was surmised that removal of the tail
group and replacement of the isopropyl core group with a tert-
butyl attenuated the metabolic liabilities associated with these
positions and led to the significant improvements in the dog
pharmacokinetic properties of 12.
GSK2118436 was screened against a set of 61 kinases
representing broad coverage of the kinome and was found to be
a potent biochemical inhibitor of B-RafV600E, wild-type B-Raf,
and c-Raf, displaying subnanomolar or nanomolar potencies
(see the Supporting Information). Importantly, GSK2118436
was also found to be highly selective, exhibiting >500-fold
selectivity for B-RafV600E compared to most kinases screened.13
Significant activity (<100-fold selectivity) was observed for a
single kinase in the panel, Alk5. The impact of Alk5 enzyme
activity on cellular signaling was further investigated by
measuring the downstream phosphorylation level of SMAD2/
3 in HepG2 cells.14 GSK2118436 was significantly less effective
at inhibiting SMAD2/3 phosphorylation (IC50 = 3.7 μM)
compared with inhibiting ERK phosphorylation (IC50 = 4 nM)
in a cellular context. These data underscore the remarkable
selectivity achieved by this uniquely potent and selective
inhibitor of B-RafV600E
.
GSK2118436 was also studied in vivo and found to
dramatically reduce tumor growth in mice bearing B-RafV600E
human melanoma tumors. In this model, CD1 nu/nu mice
bearing A375P F11 (B-RafV600E) tumors were dosed orally with
GSK2118436 at doses of 0.1, 1, 10, and 100 mg/kg once daily
for 14 days (Figure 3). Dose-proportional reductions in tumor
growth were observed. Body weight was also measured, and no
significant changes were observed at any of the doses tested.
Notably, in the 100 mg/kg group, complete tumor regression
was observed in 50% of treated animals.
Additionally, GSK2118436 reduced the levels of pERK in
A375P F11 (B-RafV600E) tumor tissue in vivo in a dose-
dependent manner after a single oral dose. Tumors were
collected 2, 6, and 24 h postdose, and pERK levels in each were
Figure 1. Dabrafenib (GSK2118436).
GSK2118436 (12) displayed compelling inhibitory activity in
enzyme and cellular mechanistic assays, and in cell proliferation
assays in B-RafV600E-driven melanoma lines, SKMEL28 and
A375P F11 (IC50 = 3 and 8 nM, respectively), and colorectal
carcinoma line Colo205 (IC50 = 7 nM). Gratifyingly,
GSK2118436 was found to have a minimal effect in vitro on
cells with wild-type B-Raf (HFF IC50 = 3.0 μM) and in tumor
cells not harboring the activating B-RafV600E mutation.12
Additionally, acceptable rat and dramatically improved dog
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ACS Medicinal Chemistry Letters
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clinical studies. Additionally, vast improvements in higher-
species PK allowed for the achievement of exposures necessary
to fully explore the safety of the molecule preclinically. Finally,
a >20% decrease in molecular weight compared to that of 1
resulted in improved physicochemical properties, expediting the
identification of a salt version (mesylate) and form suitable for
early clinical studies.
The efforts described herein ultimately led to the discovery
of GSK2118436 (dabrafenib), which is currently in advanced
clinical studies in B-Raf mutant tumors. GSK2118436 has
shown remarkable efficacy in melanoma patients with activating
B-Raf mutations, including patients with brain metastases, and
has further shown enhanced clinical activity in combination
with the MEK inhibitor, trametinib.15−17 Details of these
studies will be reported in due course.
Figure 3. In vivo efficacy of GSK2118436 in CD1 nu/nu mice bearing
A375P F11 (B-RafV600E) tumors (n = 8 per group).
ASSOCIATED CONTENT
* Supporting Information
■
S
Biochemical and cellular assays, metabolite identification
experimental procedures, kinase selectivity data, A375P F11
melanoma xenograft, PK analysis, pharmacodynamic measure-
ment of pERK in tissues, and experimental procedures for the
compounds and characterization of dabrafenib. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: tara.r.rheault@gsk.com.
■
Notes
The authors declare the following competing financial interests:
T.R.R., S.H.D., K.G.P., S.G.L., M.R.A., K.N.S., L.S.K.-C., and
K.G.M. are current employees and stockholders of Glax-
oSmithKline.
Figure 4. In vivo pharmacodynamic response of a single dose of
GSK2118436 in CD1 nu/nu mice bearing A375P F11 (B-RafV600E
tumors (n = 4 per group).
)
All studies involving the use of animals were conducted after
review by the GlaxoSmithKline (GSK) Institutional Animal
Care and Use Committee and in accordance with the GSK
Policy on the Care, Welfare and Treatment of Laboratory
Animals.
measured and normalized to the total ERK (tERK) present.
The MEK inhibitor PD0325901 was used as a control. Figure 4
shows pERK/tERK levels compared to those of vehicle-treated
animals and pharmacokinetic data in a composite of two
separate studies. Levels of pERK/tERK were substantially
reduced 2 and 6 h postdose, with a notable pharmacodynamic
effect (>50% inhibition) at doses of ≥3 mg/kg in this single-
dose study, which returned to untreated levels by 24 h at doses
of ≤30 mg/kg. The concentration of drug measured in the
blood and tumor during the course of the study correlated with
the observed pharmacodynamic effects. These data suggest that
under the conditions tested, a single oral dose of ≥3 mg/kg
GSK2118436 can maintain ≥50% B-Raf inhibition for at least 6
h.
ACKNOWLEDGMENTS
■
The authors thank Erich Baum, Neil Bifulco, Ronda Davis-
Ward, Kelly Donaldson, Kyle Emmitte, Phil Harris, Kevin
Kuntz, Kristen Nailor, Jim Salovich, Doug Sammond, Greg
Schaaf, Stephon Smith, and Brian Wilson for scientific support
and Dash Dhanak and Dirk Heerding for critically reviewing
the manuscript and support throughout the duration of the
program.
In summary, lead molecule 1, with high potency and poor
pharmacokinetic properties in higher species, was rapidly
evolved into clinical development candidate GSK2118436
(12). Identification of the key B-RafV600E potency contributors
(sulfonamide head, R2 fluorination, and tert-butyl thiazole core)
through detailed and iterative SAR studies allowed for the
elimination of nonessential portions of the lead structure and
high selectivity to be achieved across the kinome. With high
enzyme and cellular potency engineered into the original lead,
pharmacokinetic issues were addressed by combining multiple
changes in the template, resulting in the improved multispecies
PK properties observed for 12. With high rodent exposure
realized in GSK2118436, we were able to demonstrate target-
related pharmacology in two in vivo models, gaining a high
level of confidence in the progression of this molecule into
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